the first report of the dangerous scorpion hemiscorpius acanthocercus from hormoz island in the persian gulf http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 347–348 m shahi and h sanaei-zadeh.: the first... letter to the editor the first report of the dangerous scorpion hemiscorpius acanthocercus from hormoz island in the persian gulf *mehran shahi1; hossein sanaei-zadeh2 1department of medical entomology and vector control, school of public health and infectious and tropical diseases research center, hormozgan university of medical sciences, bandar abbas, iran 2emergency room, division of medical toxicology, hazrat ali-asghar hospital, shiraz university of medical sciences, shiraz, iran *corresponding author: dr mehran shahi, e-mail: mshahi1346@gmail.com (received 21 sep 2020; accepted 11 aug 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. dear editor the genus hemiscorpius of scorpions belonging to the family hemscorpiidae is among the most dangerous scorpions in iran. different species of this genus were reported from the western and southern regions of iran (1, 2). so far, death cases due to sting of two species of hemiscorpius, including h. lepturus and h. acanthocercus, have been reported in iran (3, 4). the venom of these scorpions causes severe hemoglobinuria and acute renal failure in the victims (5). the body coloration of h. acanthocercus is bright to dark yellow. pedipalps are bulky and stout. the tip of fingers is brownish. the metazoma segment in males is elongated and slender than in females (figs. 1, 2). the maximum size of females is 53.0 and males is 60.0 millimeter. these specifications help doctors to identify this dangerous scorpion. keywords: hemiscorpius; scorpion sting; renal failure; hemoglobinuria; iran figs. 1. dorsal view of hemiscorpius acanthocercus, female (left) and male (right), scale in mm. figs. 1. dorsal view of hemiscorpius acanthocercus, female (left) and male (right), scale in mm. 348 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 347–348 m shahi and h sanaei-zadeh.: the first... it is considered as one of the main agents of scorpion sting in patients with acute renal failure and severe hemoglobinuria (results in red to brown urine color) in these regions. the sting of this scorpion is painless. this leads to the patient’s lack of attention, and as a result, the patient does not usually go to health care centers immediately. generally, signs and symptoms of envenomation in these patients appear after several hours to days (up to 10 days). the delay in visiting health care centers causes severe complications and even death in victims. also, due to the absence of clinical symptoms and signs (laboratory evidences) during the first hours of sting, if the physician does not know about the recognition of the scorpion type, the discharge of the patient is a correct decision that will have irreparable consequences for the patient. therefore, physicians should consult with a toxicologist or scorpion experts in order to ensure the correct implementation of the treatment process of scorpion sting victims. the island of hormoz with a population of 7000 is about 42 square kilometers. its climate is warm and humid. during the summer months the temperature can rise to over 43 °c. hormoz island is one of the most popular tourist destinations in the province of hormozgan in the south of iran. in recent years, tourism has boomed on this island. so far, the scorpion studies have not been carried out on this island. this is the first report of the identification of h. acanthocercus danger from the island of hormoz. considering the importance of the mentioned issue, the establishment and introduction of high risk areas for the presence of dangerous species of scorpions will be great help for the attention of the health care personnel to the scorpionstung patients in these areas. authors’ contribution all the work has been done by mehran shahi and hossein sanaei-zadeh. conflict of interests the author has no conflict of interest funding/support none references 1. dehghani r, kassiri h (2018) a checklist of scor pions in iran (by 2017). asian j pharm. 12(3): 880–887. 2. salari m, sampour m (2017) first two records of hemiscorpius species (scorpiones: hemiscorpi idae) from kerman province, southeast of iran. asian j appl sci. 5(1): 101–108. 3. jalali a, bavarsad-omidian n, babaei m, najafzadeh h, rezaei s (2012) the pharmacokinetics of hemiscorpius lepturus scorpion venom and razi antivenom following intramuscular administration in rat. j venom res. 3:1-6. 4. shahi m, rafinejad j, az-khosravi l, moosavy s h (2015) first report of death due to hemiscorpius acanthocercus envenomation in iran: case report. electron physician. 7(5):1234-8. 5. sanaei-zadeh h, marashi s m, dehghani r (2017) epidemiological and clinical characteristics of scorpionism in shiraz (2012–2016); development of a clinical severity grading for iranian scorpion envenomation. med j islam repub iran. 31:27. the first report of the dangerous scorpion hemiscorpius acanthocercus from hormoz island in the pers dear editor keywords authors’ contribution conflict of interests funding/support references j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 79 original article pediculosis capitis among primary school children and related risk factors in urmia, the main city of west azarbaijan, iran k hazrati tappeh 1, *ar chavshin 2, h mohammadzadeh hajipirloo 1, s khashaveh 1, h hanifian 1, a bozorgomid 1, m mohammadi 2, d jabbari gharabag 1, h azizi 1 1department of medical parasitology, school of medicine, urmia university of medical sciences, urmia, iran 2department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran (received 26 apr 2011; accepted 18 sep 2011) abstract background: pediculosis capitis is cosmopolitan health problem. in addition to its physical problems, its psychological effects especially on pupils are more important. this study was conducted to determine the pediculosis capitis among primary school pupils and also find out the role of probable related risk factors in urmia city, iran 2010. methods: 35 primary schools of urmia city according to the defined clusters randomly have been selected during 2010. 2040 pupils (866 boys and 1174 girls) were included and examined individually and privately by experts. presence of adult or immature lice or having nits less than 1 cm from the hair basis were defined as positive. data about demographic features and factors which their effect should be determined were recorded in standard questionnaire. data were analyzed by spss software with proper statistical test. results: infestation was determined around 4%. girls show significantly greater infestation. the availability of suitable warm water for bathing and hair length (separately in girls and boys) are significantly related to infestation load as well as infestation among different age groups. there was no significant relation between parent’s education and job and infestation as well as bathing repetition per week and the kind of energy source which they have. also there is no significant correlation between educational grades and head lice infestation. conclusion: the head louse pediculosis is a health problem and remains a health threatening for school children.effective risk factors should be determined carefully and regionally. proper training plays a great role in order to prevent and control the problem. keywords: pediculus capitis, primary schools, urmia, head louse infestation introduction pediculus capitis as one of the most important human obligate ectoparasite which infests human beings called head lice. it is a common health problem mainly in children below 15 years old and infests the hair and scalp (nutanson et al. 2008). except for the common cold, the head louse infestation among elementary-school-age children is the most prevalent communicable disease rather than all other communicable diseases combined (hensel 2000). some of patients with louse infestation are asymptomatic but pruritus is most common symptom which is because of sensitization to louse salivary and fecal antigens. excoriations and secondary bacterial infections could be occurred (malcolm et al. 2006). in addition, in hypersensitive infested persons, cervical lymphadenopathy and conjunctivitis could be occurred (ko et al. 2004, scott et al. 2005). allergic reactions such as nasal obstruction, rhinorrhea, and nightly whistles which could be *corresponding author: dr ali reza chavshin, e-mail: chavshin@umsu.ac.ir j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 80 seen in chronic infestation (fernandez et al. 2006). the patients show not only physical symptoms but also psychological distress occurred and this could be more important point in head louse infestation especially in children (oh et al. 2010). infestation also can affect concentration or could be a reason for stigmatization which detrimentally influences children’s learning quality. during the last decades a significant increase has been reported in pediculosis “infestation with louse” worldwide as a result of louse resistance to routine insecticides, improper application, formulation changes, and misdiagnosis (burgess 2004). another concern in head louse infestation is misguided use of caustic or toxic substances to eliminate the lice (pollack et al. 2000). head louse still remains an important health problem and is endemic in both developing and developed countries. several studies worldwide showed various prevalence from 0.7% to 29.7% (cazorla et al. 2007, jahnke et al. 2008, soultana et al. 2009, toloza et al. 2009). in iran the prevalence of head louse infestation has been reported frequently from several parts of the country. in two recent studies the prevalence ranged between 0.2% to 0.49% in fars province (davarpanah et al. 2009) to 4.8% in khajeh city in east azarbaijan province (shayeghi et al. 2010) whereas another study report a higher prevalence (27.1%) in iranshahr, sistan and baluchistan province (alempour-salemi et al. 2003). despite the previous thoughts, pediculosis affects school children of all socioeconomic strata and not just the poors, uneducated or those living in unhygienic conditions (speare et al. 1999, downs et al. 2000). current study was conducted to determine the prevalence of head lice infestation in primary school children in the main city of west azarbaijan (urmia) and some probable effective factors. materials and methods study area and design the main city of west azarbaijan “urmia” has been chosen and divided in two regions according to official and geographical conditions. several clusters have been determined. 35 pre-primary and primary schools were selected randomly according to defined clusters. the study was conducted during october–december 2010. finally 2040 pupils (866 boys and 1174 girls) were included and examined in this study. study procedure at the first the urmia city was divided into two regions and several clusters were defined in each region. according to the randomized sample selection method, in all defined clusters and determined sample size, 1000 pupils should be observed and examined but in order to increase the border of study and getting better picture, more than 2040 pupils were observed and examined. all selected pupils were examined individually and privately by an expert person. girls were examined by female experts for all stages of lice or their nits. presence of adult lice or immature stages of them or having nits less than 1 cm from the hair basis were defined as positive. infestation and its load were documented in specific forms. data collection and analysis a standard questionnaire were designed to record data about sex, parents’ education and their job, school grade, bathing repetition per week, availability of suitable energy source and other related factors. all pupils were asked to fulfill in the standard questionnaire containing questions to find out their living style and probable risk factors. obtained data were deposited in to the spss data sheets and based on study questions were analyzed using proper statistical tests and parameters. j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 81 results observation on 2040 pupils by experts showed that the total prevalence of head louse infestation among them is 4%. girls have a significantly greater infestation with 5.5% in comparison to the boys infestation 1.8 (p< 0.05) (table1). analyzing the correlation between some socio-economic factors and head louse infestation revealed that some of them such as length of hair and availability of suitable warm water for bathing and hygienic use could affect head louse infestation significantly (p< 0.05) (table 2) but other factors such as parent’s education level and job, bathing repetition per week and the kind of energy source which they have did not show significant correlation with head louse infestation (p> 0.05). table 2 represents the details properly. the data also showed that the majority of infested pupils have had more than 10 nits on their hair (table 3). the additional analysis of probable relationship between class grade and infestation load revealed no significant difference between different grades and infestation load (p= 0.443) (table 4). to determine the statistical difference between age groups, all observed pupils were classified into 4 age groups (table 5) and statistical analysis showed that there is significant difference between age groups (p< 0.000). table 1. observed pupils according to sex and the prevalence of head louse infestation among them in primary schools, urmia, iran, 2010 sex observed pupils n of infested pupils prevalence (%) exact sig. n % male 866 42.5 16 1.8 (p< 0.05)female 1174 57.5 65 5.5 total 2040 100.0 81 4.0 table 2. head louse infestation in primary school pupils according to some socio-economic factors in urmia, iran, 2010 characteristics n ofobserved pupils n of infested pupils p value. mother education un-educated 351 7 p> 0.05under-high school 984 41finished high school 375 15 university degree 314 17 mother job housewife 1692 65 p> 0.05works outside 337 16 father education deceased 29 1 p> 0.05 un-educated 187 7 under high school 932 33 finished high school 476 21 university degree 405 19 father job un-employed 25 1 p> 0.05 worker 440 9 official employee 585 24 private section 924 46 sick and old 25 0 deceased 31 1 j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 82 length of hair girls shaved completely 0 0 p< 0.05less than 1 cm 30 1until ear 68 4 less than shoulder 385 23 until shoulder and more 689 31 boys shaved completely 25 1 p< 0.05less than 1 cm 261 9until ear 420 3 less than shoulder 159 4 until shoulder and more 1 0 gas plumbing yes 1734 64 p> 0.05no 305 17 having water boiler at home yes 1982 73 p< 0.05no 57 8 weekly bath number once per week 900 29 p> 0.05twice per week 824 39 three and more 315 13 table 3. infestation load amon/ g pupils of primary schools of urmia, iran, 2010 infestation load average infestation with nits average infestation with adults n % n % less than 5 21 1.0 2 0.1 between 5–10 13 0.6 2 0.1 more than 10 42 2.1 2 0.1 table 4. infestation load of head lice among observed pupils according to their educational grade, urmia, iran, 2010 school grade average number of nits total negative less than 5 between 5–10 more than 10 pre-school 35 1 0 2 38 grade 1 403 7 3 10 423 grade 2 404 2 3 12 421 grade 3 360 2 1 5 368 grade 4 399 4 6 5 414 grade 5 363 5 0 8 376 total 1964 21 13 42 2040 table 5. infestation load of head lice among observed pupils according to their age groups, urmia, iran, 2010 age groups average number of nits total negative less than 5 between 5–10 more than 10 pre-school [5–6] 43 2 0 2 47 [7–9] 1108 10 6 27 1151 [10–12] 797 9 7 13 826 [13–16] 14 0 0 0 14 total 1962 21 13 42 2038 table 2. countinued ... ….. j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 83 discussion this study showed that 4% of examined pupils of primary schools were infested with head louse. other studies conducted around the world revealed different prevalences. in australia the prevalence has been reported 33% (speare et al. 1999), 35% in brazil (borges et al. 2002), 48.7% in france (courtiade et al. 1993), 4.1% in korea, throughout the country (oh et al. 2010), and between 0.48 to 1.59% in boys, 29.7% in buenos aires, argentina (toloza et al. 2009) and more than 50% in girls of lublin province, eastern poland (buczek et al. 2004). in iran, a study reported the infestation prevalence 27.1 % in iranshahr, south-east of iran (alempour-salemi et al. 2003), in a recent study in khajeh city in east azarbaijan the total prevalence was reported 4.8% and in fars province the prevalence ranged between 0.2% to 0.49% (davarpanah et al. 2009). the statistical analysis showed that girls have significantly greater infestation prevalence (p< 0.05). this difference could be because of the different behaviors between girls and boys and their living and playing patterns. also maybe some cultural aspects such as girls clothing plays effective role. analysis of infestation among age groups showed significant correlation between head lice infestation and some of age groups (p= 0.000) especially the lower age group (7–9 years old). results revealed higher significance in mentioned age group in comparison with other age groups which could reflect the ability of older pupils to act hygienic and using their personal equipments properly. there is no significant relationship between infestation and parent’s education as well as their job (p> 0.05). father’s and mother’s education and job were analyzed separately and no significant differences have been seen. parent’s education and job could be assumed as a representative of their socio-economic level and having no significant differences between the socio-economic level and head louse infestation could approve the hypothesis that head louse infestation is not limited to uneducated or poor strata (speare et al. 1999, downs et al. 2000). the educational system should clarify that all parents from all families “not only the poors” should play a more effective role then more communication between school and families is necessary. on the other hand simple and effective health training courses related to the head lice will be useful for parents to examine their children periodically and carry out preventive programs and also train their children properly about the infestation routs and prevention measures. analysis of relationship between hair length and head lice infestation has been done separately between girls and boys. results showed significant relationship between infestation and hair length in both groups (p< 0.05) longer hairs harbor heavier head louse infestation. bathing carefully and with longer hairs could be difficult for children as well as inspection for infestation by parents and health officers. also pupils from families with water boiler which produce warm water for bathing significantly have lesser infestation (p< 0.05). bathing with warm and suitable water would be gracious and could increase bathing quality. similarly the bathing repetition per week has no significant role in infestation and it could be assumed that the quality of bathing is more important than its quantity. all studied families have suitable energy source (gas, oil and electricity) for preparation of warm water and the kind of energy could not affect the infestation significantly (p> 0.05). the results of this study and similar studies show that the head louse pediculosis is a cosmopolitan health problem with various prevalences regionally but it remains a j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 84 noticeable pediatric problem. the main factors which significantly are related to the head louse infestation are cultural and conceptual. according to acquired results it seems that training courses in order to help school staffs and parents to know the symptoms and transmission routs of head louse infestation are necessary. this knowledge will help them to discover the infestation and design simple and effective preventive and control programs. management of infested cases without psychological side effects is very important and in all cases delicate facts and unique personality of each infested pupil should be minded. despite of the simple face of the head lice problem, several concerns should be calculated and based on them, proper training, surveillance and control programs should be designed and carried out. acknowledgements this study was financially supported by the urmia university of medical sciences (project no: 730–1389). the authors would like to thank dr motezaker, dr shahabi and the staffs of the selected schools for their collaboration, kindness and their positive appetite. references alempour-salemi j, shayeghi m, zeraati h, akbarzadeh k, basseri h, ebrahimi b rafinejad j (2003) some aspects of head lice infestation in iranshahr area (southeast of iran). iranian j of pub health. 32(3): 60–63. borges r, mendes j (2002) epidemiological aspects of head lice in children attending day care centres, urban and rural schools in uberlandia, central brazil. mem inst oswaldo cruz. 97 (2): 189–192. buczek a, markowska-gosik d, widomska d, kawa im (2004) pediculosis capitis among schoolchildren in urban and rural areas of eastern poland. eur j epidemiol. 19(5): 491–495. burgess if (2004) human lice and their control. annu rev entomol. 49: 457– 81. cazorla d, ruiz a, acosta m (2007) clinical and epidemiological study of pediculosis capitis in schoolchildren from coro, venezuela. invest clin. 48 (4): 445–457. courtiade c, labrèze c, fontan i, taïeb a, maleville j (1993) pediculosis capitis: a questionnaire survey in 4 schools of the bordeaux academy 1990–1991. ann dermatol venereol. 120(5): 363– 368. davarpanah m, mehrabani d, khademolhosseini f, mokhtari a, bakhtiari h, neirami r (2009) the prevalence of pediculus capitis among school children in fars province, southern iran. iranian j parasitol. 4(2): 48–53. downs am, stafford ka, stewart gh,coles gc (2000) factors that may be influencing the prevalence of head lice in british school children. pediatr dermatol. 17(1): 72–74. fernandez s, fernandez a, armentia a, pineda f (2006) allergy due to head lice (pediculus humanus capitis). allergy. 61(11): 1372. hensel p (2000) the challenge of choosing a pediculicide. public health nurs. 17 (4): 300–304. jahnke c, bauer e , feldmeier h (2008) pediculosis capita in childhood: epidemiological and socio-medical results from screening of school beginners. gesundheitswesen. 70(11): 667–673. ko cj, elston dm (2004) pediculosis. j am acad dermatol. 50(1): 1–12, quiz 13– 4. j arthropod-borne dis, 2012, (6)1: 79–85 k hazrati tappeh et al.: pediculosis capitis among … 85 malcolm ce, bergman jn (2006) trying to keep ahead of lice: a therapeutic challenge. skin therapy lett. 11(10): 1–6. nutanson i, steen cj, schwartz ra, janniger ck (2008) pediculus humanus capitis: an update. acta dermatovenerol alp panonica adriat. 17(4): 147–154, 156– 157, 159. oh jm, lee iy, lee wj, seo m, park sa, lee sh, seo jh, yong ts, park sj, shin mh, pai ks, yu jr, sim s (2010) prevalence of pediculosis capitis among korean children.parasitol res. 107(6): 1415–1419. pollack rj, kiszewski ae, spielman a (2000) overdiagnosis and consequent mismanagement of head louse infestations in north america. pediatr infect dis j. 19(8): 689–693, discussion 694. scott p, middlefell ls, fabbroni g, mitchell da (2005) interesting case: cervical lymphadenopathy, induced by head lice. br j oral maxillofac surg. 43(6): 515. shayeghi m, paksa a, salim abadi y, dehkoordi ar s, ahmadi a, eshaghi a , bazrafkan s (2010)epidemiology of head lice infestation in primary school pupils, in khajeh city, east azerbaijan province, iran. iran j arthropod-borne dis. 4(1): 42–46. soultana v, euthumia p, antonios m, angeliki rs (2009) prevalence of pediculosis capitis among schoolchildren in greece and risk factors: a questionnaire survey. pediatr dermatol. 26(6): 701–705. speare r, buettner pg (1999) head lice in pupils of a primary school in australia and implications for control. int j dermatol. 38(4): 285–290. toloza a, vassena c, gallardo a, gonzálezaudino p, picollo mi (2009) epidemiology of pediculosis capitis in elementary schools of buenos aires, argentina. parasitol res. 104(6): 1295–1298. j arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 185 http://jad.tums.ac.ir published online: june 30, 2020 original article assessment the changing trend of susceptibility to two insecticides among field-population culex quinquefasciatus compared with the same population undergoing to multiple colonization atieh shemshadian 1 ; *mohammad reza abai 1,2 ; *hassan vatandoost 1,2 ; navid dinparastdjadid 3 ; mohammad ali oshaghi 1 ; abdolrasoul mojahedi 1,4 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3 malaria and vector research group, biotechnology research center (brc), pasteur institute of iran, tehran, iran 4 provincial health center, bandar abbas university of medical sciences, bandar abbas, iran (received 25 apr 2019; accepted 08 june 2020) abstract background: during the past decade, rapid development of insecticide resistance have been reported among many species of mosquito vectors against four main categories of insecticides worldwide. the aim of the research was to assess the variation trend of susceptibility levels of culex quinquefasciatus to two insecticides separately for the field population compared with subsequent generations of the same sample after multiple colonization. methods: larvae and pupae of cx. quinquefasciatus were collected from house sewages and reared to adult which blood-fed on roosters. ten percent sucrose fed female mosquitoes aged 2–3 days were used for susceptibility tests with ddt and deltamethrin. susceptibility levels was assessed in the adult stage of field stran cx. quinquefasciatus against ddt 4.0% and deltamethrin 0.05% and continued up to next six generations undergoing multiple rearing at insectary condition. results: the susceptibility levels to ddt 4.0% did not change compared to the field with the lab population to six generations. regarding deltamethrin 0.05%, no significant difference was shown between field strain (58.3%) and 3 rd generation (52.7%) compared to the 6 th one (33.8%). conclusion: this finding may reflect the role of the kdr gene in resistance to organochlorine which has cross-resistance with pyrethroid insecticides. the results of this study clearly showed the irreversible trend of p yrethroid resistance among colonized mosquitoes. this is the first study of the resistance status of cx. quinquefasciatus in iran. keywords: culex quinquefasciatus; variation; susceptibility; insecticides multiple generations introduction culex quinquefasciatus say, commonly known as the southern house mosquito, is widely distributed in the tropical and subtropical zones (1). it is also found at high densities in similar climates southern iran. the biologic behavior of this species brings mosquitoes to the adjacent premises, providing conditions for easy transmission of arboviral diseases to humans as well as to domestic and wild animals, including west nile fever, saint louis encephalitis, western equine encephalitis, rift valley fever, avian malaria and lymphatic filariasis. this mosquito also plays an important role in the transmission of the nocturnal periodic form of wuchereria bancrofti (2) and is a major cause of acute and chronic morbidity, affecting all ages and both sexes throughout the tropical and sub-tropical areas of the world (3). moreover, cx. quinquefasciatus is iden*corresponding authors: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir, dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com https://en.wikipedia.org/wiki/avian_malaria mailto:abaimr@tums.ac.ir j arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 186 http://jad.tums.ac.ir published online: june 30, 2020 tified as a potential vector of zika virus (4). the major strategy for controlling this mosquito is application of insecticides. additionally, this species can become quickly resistant to most insecticides compared to other mosquitoes (5) and resistant to different ones in most countries in the world (6). resistance to ddt in cx. quinquefasciatus was reported in west africa for the first time (7). there are several reports of resistance to different groups of insecticides from west african regions including coˆte d’ivoire, burkina faso, benin and ghana (8-10). according to these reports, resistance to the organochlorine insecticides has developed by releasing pesticide residues into wastewater in major cities (11). resistance of cx. quinquefasciatus to pyrethroid was first reported from california (12). resistance to organophosphates was reported in a strain of this vector in sri lanka due to increased esterase activity (13). there are also reports on resistance to a variety of insecticides from thailand, pakistan, france, and saudi arabia (14-16). however, there is no report of the resistance status of cx. quinquefasciatus to insecticides in southern iran. the aim of the research was to assess variation trend of susceptibility levels of culex quinquefasciatus to two insecticides separately for field population compared with subsequent generations of the same sample achieved after multiple colonization. materials and methods mosquito collection the larval and pupae sampling of cx. quinquefasciatus were done in suru county, bandar-abbas district, hormozgan province in 27.168142° n and 56.252175° e at an elevation of 3m above sea level from march to april 2016 (fig. 1). in order to provide laboratory colonies of mosquitoes, the larvae and pupae of cx. quinquefasciatus were collected from breeding places in the wastewater of the houses running outside near the seaside using the dipping method (fig. 1). moreover, immature mosquitoes were collected from abandoned boats where the polluted water could penetrate. the eggs, larvae and pupae stages of cx. quinquefasciatus along with their breeding places, water, and herbs were transported to an insectary located in bandarabbas training and research station. the mosquitoes were reared at 30±5 °c with 65– 80% relative humidity. female mosquitoes aged 2–3 days were fed on 10% sucrose solution and used for susceptibility tests. insecticides susceptibility tests was done using with both ddt 4.0% impregnated-papers with impregnation date july 2013, expiry date july 2018 with batch no under code: dd 188 and deltamethrin impregnated-papers with impregnation date july 2016, expiry date july 2017 with batch no under code: de 332. the impregnated-papers to insecticides was purchased from the vector control research unit, school of biological sciences, 11800 minden, penang, malaysia. susceptibility tests adult susceptibility tests were conducted using who standard kits provided by who (17). twenty-five female mosquitoes aged 2–3 days fed on 10% sucrose solution during past night were exposed to ddt 4.0% and deltamethrin 0.05% impregnated papers for 60 minutes. the solvent-impregnated papers were used for the control group. there were 10 replicates for the treatment group and two replicates for the control one. after ending the exposure time, both mosquito groups were allowed to recover in holding tubes with a piece of cotton containing 10% sucrose solution on the top of tube for 24 hours and then the number of dead and live mosquitoes was counted. the susceptibility tests were separately repeated in the 3 rd and 6 th generations of cx. quinquefasciatus in the insectary. j arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 187 http://jad.tums.ac.ir published online: june 30, 2020 mosquito colonization for the first time, a success was gained for establishment and colonization of cx. quinquefasciatus in the water containing intact bermuda grass “cynodon dactylon” a perennial plant with spreading rhizomes and stolon. the plant was used directly in the immature breeding pans at natural state and without any chopping (fig. 2). the adult mosquitoes were easily mated in a wooden netted cages dimensioned 30x 30x 30cm. the mosquitoes were blood-fed on a rooster, why the protein sources needed maturing the eggs. statistical analysis the mortality rate of cx. quinquefasciatus after 24 hours recovery was calculated for each insecticide and generation and corrected using the abbott’s formula if the mortality rate was 5–20% in the control group. the interpretation of the resistance level in cx. quinquefasciatus was adopted from the who criteria and the terms resistant, suggestive of the existence of resistance and further investigation required, and susceptible were used for mortality rates <90%, 90–98% and >98%, respectively (17). univariate analysis of variance (anova) was applied after arcsin transformation of the mortality rate to determine differences in mosquito mortality rates among generations. significant differences of the mean of mortality rate were compared using tukey or games-howell tests depending on the post hoc test result. the significant difference in the mortality rate of cx. quinquefasciatus between different generations was calculated using following formula: se= √pq/n, where: “p” is the mortality rate, “q” is equal to 1 minus the mortality rate, and “n” is the sample size. results mass colonization for the first time, a success was gained for establishment and colonization of cx. quinquefasciatus in the water containing intact bermuda grass “cynodon dactylon” a perennial plant with spreading rhizomes and stolon. the plant was used directly in the immature breeding pans at natural state and without any chopping (fig. 2). the adult mosquitoes were easily mated in a wooden netted cage dimensioned 30x 30cm. the mosquitoes were blood-fed on a rooster why the protein sources needed maturing the eggs. bioassays two-three days old female mosquitoes was exposed to ddt 4.0% and mortality rate of the field strain was 12.1%, compared to 9.5% and 10.5% in the 3rd (f3) and 6th (f6) generations. the mortalities between different generations exposed to ddt was not differed with each other, but with significant difference compared with control group (f= 2.036, p< 0.05) (fig. 3). the cx. quinquefasciatus were revealed 58.3%, 52.7%, and 33.8% of mortality, when exposed 60min to deltamethrin 0.05% respectively for field strain, f3, and f6 generations. it was shown significant differences f3 compared to field strain and f6 mosquitoes and the mortality in control group showed significant differences with the treatment group (fig. 4). the evidence showed that the subsequent mass colonization of a field strain, the susceptibility level will be unchangeable compared with the lab-colonized of the same population which undergone several masscolonization which established the pyrethroid resistance. j arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 188 http://jad.tums.ac.ir published online: june 30, 2020 fig. 1. a. satellite map of suru location, bandar-abbas port, hormozgan province, persian gulf littoral, iran b. ground landscapes of the breeding places of culex quinequefaciatus in study area fig. 2. collection of immature culex quinquefasciatus from natural breeding places with floating bermuda grass and subsequent establishment in the insectary of bandar-abbas health training and research station, persian gulf littoral, iran fig. 3. comparison of susceptibility level of culex quinquefasciatus exposed to ddt 4.0% among field population and subsequent generations gained from same population a b j arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 189 http://jad.tums.ac.ir published online: june 30, 2020 fig. 4. comparison of susceptibility level of culex quinquefasciatus to deltamethrin 0.05% on field population compared with lab strain achieved after mass colonization up to the six generations discussion despite the confirmed role of cx. quinquefasciatus as an arboviral vector in the subtropical climate, little attention has been paid to its bioecological characteristics in iran. this is while huge densities of this species at larval and adult stages were observed at the urban area and extensive larval habitats could be found around the houses which its water originated from houses sewage. moreover, pools and superficial wells at agricultural lands are potential larval habitats in the south and southeast iran. the susceptibility levels of female cx. quinquefasciatus were tested according to a method recommended by the who. the results showed that this species was highly resistant to ddt and deltamethrin at diagnostic dose and 60min of exposure time. after the mass colonization of cx. quinquefasciatus, the results of susceptibility tests were compared between field population and lab generations up to six generations achieved from the same field population to shown stability of resistance both for ddt and deltamethrin despite mass colonization up to six generations in the insectary condition. in this study, the susceptibility testing was followed from the latest who’s instruction (17), which indicated a 60min exposure time for all mosquito species and for specified diagnostic concentrations of insecticides, but according to the newly published who’s guidelines, the exposure time for ddt and culex species were changed to 240min at the same discriminating concentration (18). resistance-related studies of cx. quinquefasciatus have been carried out for numerous insecticides around the world, from asia to europe, west africa, and america. a high level of resistance to pyrethroids was found in coˆte d’ivoire and burkina faso, west africa (8). there is a report of the resistance of cx. quinquefasciatus to ddt and deltamethrin in thailand caused by continuous use of insecticides for dengue vector control (19). in northeastern india, a study showed resistance to ddt and deltamethrin in cx. quinquefasciatus collected from army cantonments and neighboring villages (20). there is a report of the resistance of cx. quinquefasciatus to organochlorines, organj arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 190 http://jad.tums.ac.ir published online: june 30, 2020 ophosphates, and pyrethroids in la réunion island, france (21). according to recent findings, resistance to ddt and pyrethroid in this species subsequently may lead to the inefficacy of the long-used insecticide-treated nets (10). this high resistance can be caused by several factors in the study area, one of which could be long-term use of ddt for combatting malaria vectors, especially in the south of iran that goes back to half a century ago. another factor may be the tendency of female gravid adults to lay eggs at sewage enriched with organic materials. because of the bloodfeeding tendency of cx. quinquefasciatus towards the poultries and wild birds, which are considered as potential reservoirs of arboviral agents, the risk of the transmission of these diseases to humans increases in the subsequent blood-feeding sessions. this vector is exposed to the chemicals that may be present in the urban wastewaters. cross-resistance should also be considered as another factor for inactivation of pyrethroid insecticides. the target of organochlorines (ddt) and synthetic pyrethroids is the sodium channels of the nerve sheath (22). therefore, due to the similar target site, cross-resistance may develop and resistance to one of these insecticides may make mosquitoes resistant to the other one. the findings of present study may reflect the role of the kdr gene in resistance to organochlorine which has cross-resistance with pyrethroid insecticides. furthermore, another finding is irreversible and progressive trends of resistance intensity to the pyrethroids among the colonized mosquitoes reared for several generations. this is the first study of the resistance status of cx. quinquefasciatus in iran. conclusion the findings indicated the stability of ddt resistance in the field population of cx. quiquefasciatus as well as among lab-bread of same population. the deltamethrin resistance showed progressive trend in the lab compared with wild population after multiple colonization in the insectary. hence, precise control management should be spotted for efficacy of control operations against culex vectors. acknowledgements the authors wish to thank dr k holakouienaieni, head of bandar-abbas research and training station (brts), school of public health, tehran university of medical sciences for his sincere cooperation during field works. moreover, the authors are grateful to hosein shabkhiz, the internal manager of brts for his continuous assistance during this research. this study was funded and supported by tehran university of medical sciences. this study was a part of msc dissertation funded and supported by tehran university of medical sciences; grant no 921126016. the authors claim no conflicts of interest. references 1. harbach re (1988) the mosquitoes of the subgenus culex in southwestern asia and egypt (diptera: culicidae). contributions of the american entomological institute (gainesville). 24(1): 1–240. 2. manimegalai k, sukanya s (2014) biology of the filarial vector, culex quinquefasciatus (diptera:culicidae). int j curr microbiol app sci. 3(4): 718–724. 3. njenga 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https://www.ncbi.nlm.nih.gov/pubmed/?term=bhattacharyya%20ik%5bauthor%5d&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=borkotoki%20a%5bauthor%5d&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=borkotoki%20a%5bauthor%5d&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=goswami%20d%5bauthor%5d&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=goswami%20d%5bauthor%5d&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=baruah%20i%5bauthor%5d&cauthor=true&cauthor_uid=19493193 https://www.ncbi.nlm.nih.gov/pubmed/?term=srivastava%20rb%5bauthor%5d&cauthor=true&cauthor_uid=19493193 j arthropod-borne dis, june 2020, 14(2): 185–192 a shemshadian et al.: assessment the … 192 http://jad.tums.ac.ir published online: june 30, 2020 sistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, culex quinquefasciatus, in northeastern india. med 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http://www.sciencedirect.com/science/article/pii/s0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/s0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/s0965174810000342#aff1 http://www.sciencedirect.com/science/article/pii/s0965174810000342#aff1 j arthropod-borne dis, march 2019, 13(1): 1–8 v contreras et al.: infection by rickettsia … 1 http://jad.tums.ac.ir published online: april 27, 2019 original article infection by rickettsia felis in ctenocephalides felis felis fleas from north of colombia verónica contreras 1, andrés f. londoño 2, jorge miranda 1, *salim mattar 1, leidy y. acevedo-gutiérrez 2, francisco j. diaz 3, juan d. rodas 2 1instituto de investigaciones biológicas del trópico, universidad de córdoba, colombia 2grupo de investigación en ciencias veterinarias, centauro, universidad de antioquia, colombia 3grupo de inmunovirología, universidad de antioquia, colombia (received 22 apr 2015; accepted 11 july 2018) abstract background: rickettsia felis is an emergent rickettsial agent whose main vector is ctenocephalides felis, but ticks, mites and lice are also infected. we aimed to search for molecular evidence of rickettsia spp. in fleas collected from dogs and wild rodents (heteromys anomalous) from three villages of córdoba and antioquia provinces (northern of colombia), where outbreaks of rickettsioses have occurred, and discuss the possible role of fleas on endemic/enzootic regions for rickettsia. methods: during 2010 and 2012, 649 ctenocephalides felis felis and 24 pulex irritans fleas were removed from dogs and wild rodents (heteromys anomalous), respectively, in 3 locations from córdoba and antioquia provinces (colombia). these fleas were tested into pools for rickettsial infection by pcr, targeting glta, ompb, and ompa rickettsial genes. results: almost 20% (30/153) of c. felis felis pools contained rickettsial dna. the fragments of ompb gene showed high identity values between sequences from necocli and los cordobas with r. felis strain from senegal (100% and 99.7% respectively) and all were highly related by phylogenetic analyses. rickettsial dna in pools of p. irritans was not detected. conclusion: our findings highlighted the endemicity of the infection by r. felis in fleas from northern of colombia and showed the likely importance of dogs as hosts of c. felis felis fleas and their potential role as reservoirs of r. felis. keywords: rickettsia felis; fleas; dogs; rodents; ctenocephalides felis introduction the genus rickettsia comprised arthropodassociated intracellular and gram-negative bacteria. it is divided into 4 groups based on their genotypic characteristics: spotted fever group (r. rickettsii, r. conorii, r. parkeri, and several others), typhus group (r. prowazekii and r. typhi), transitional group (r. felis, r. akari, and r. australis), and the nonpathogenic ancestral group (r. bellii and r. canadensis) (1). rickettsia felis is globally distributed and is the etiological agent of flea-borne spotted fever. the main vector is the flea ctenocephalides felis, but ticks, mites and lice have also been found infected (2). rickettsia felis in c. felis populations is principally maintained by transstadial and transovarial transmission (3). in colonized c. felis fleas, vertical transmission of r. felis is thought to be the primary route of maintenance, since the reported prevalence of r. felis in c. felis colonies ranged from 43–100% (4-6). in nature, fleas feeding on r. felis-infected mammalian hosts likely amplify the prevalence of r. felis in a flea´s population. studies on the ecology of r. felis identified a role for opossums in the transmission cycle (7-9). furthermore, a role for companion animals, rodents, and, specifically, their fleas as the potential source of human *corresponding author: dr salim máttar, e-mail: mattarsalim@gmail.com, smattar@correo.unicordoba.edu.co j arthropod-borne dis, march 2019, 13(1): 1–8 v contreras et al.: infection by rickettsia … 2 http://jad.tums.ac.ir published online: april 27, 2019 exposure has been suggested (4, 9, 10). infecting fleas have been reported in many american countries and human cases of spotted fever by r. felis have been recently described in the united states, mexico, and brazil (11). clinical manifestations of flea-borne spotted fever are variable and similar to other rickettsial diseases. in colombia, a transversal serological study was performed in seven municipalities of caldas province, and a human seroprevalence of 25.2% and 17.8% against r. typhi and r. felis, respectively was found (12). additionally, the infection by r. felis in c. felis, c. canis, and p. irritans fleas was reported in the province of caldas (13). three important spotted fever group (sfg) rickettsiosis outbreaks occurred in colombia, in the municipalities of turbo y necoclí (antioquia province) and los cordobas (córdoba province), between 2006 and 2008 (14, 15). consequently, these areas have been described as endemic for rickettsioses in this country. the purpose of this study was to search for molecular evidence of rickettsia spp. in fleas collected from dogs and wild rodents (heteromys anomalous) from the three zones where outbreaks of rickettsioses occurred and discuss the likely role of fleas in the epidemiology of rickettsia spp. in this region of colombia. materials and methods study area and sampling the study was conducted in 3 neighboring municipalities: turbo, (8°8.272´n, 76°33.009´ w) located at 400 m above sea level (masl), and necocli, (8°32.892´n, 76°34.429´w), at 182 m above sea level. both are located in the antioquia province, and los cordobas, (8°50.195´n, 76°20.252´w) located at eight meters above sea level, in the cordoba province (fig. 1). all of these municipalities are placed on the colombian atlantic coast. these three sites comprise part of the natural caribbean region, and have a tropical humid climate characterized by a dry period from jan to mar and a rainy season from apr to dec, with an annual average temperature of 28 °c and relative humidity of 85% (fig. 1). during 2010 and 2012, a total of 649 fleas were removed from 92 dogs of all studied locations (194 from turbo, 225 from necocli and 230 from los cordobas) and 24 fleas from three heteromys anomalus rodents captured in turbo and necocli. they were obtained using tweezers or by combing wild and domestic animals, and care was taken to avoid damaging structures essential for taxonomic classification. the fleas were collected from each animal in one or various vials with alcohol 95% (depending on the number collected per animal) and were transported to the laboratory. because the population of dogs in the study zones was unknown, a sample size was not determined. however, we were able to estimate the number of animals that lived with people at each site. ethical, technical, scientific and administrative standards to perform research in animals were taken into consideration according to national regulations for the procedures of collection, management and conservation of samples (resolution no. 008430 of 1993 and law 84 of dec 27th from 1989). molecular detection of rickettsia spp. fleas were classified according to morphological keys (16-18). they were grouped in maximum “pools” of 10 individuals, according to host and sampling site: 153 pools of c. felis felis were collected from dogs and six pools of pulex irritans fleas were collected from rodents. dna from pools was extracted by using qiaamp dna mini-kit (qiagen®, valencia, ca, usa), according to manufacturer conditions. samples were stored at -20 °c until they were used for pcr assays. j arthropod-borne dis, march 2019, 13(1): 1–8 v contreras et al.: infection by rickettsia … 3 http://jad.tums.ac.ir published online: april 27, 2019 samples were tested by pcr assay with primers cs-78 (forward gcaagtatcggt gaggatgtaat) and cs323 (reverse gc ttccttaaaattcaataaatcaggat), which amplify a 401bp fragment of the citrate synthase gene (glta), previously reported as appropriate for the screening of rickettsia spp. (19). samples that came up positive for glta were tested with the primers rr190.70p (forward: 5´atggcgaa tatttctccaaaa)-rr190.701 (reverse: 3´gttccgttaatggcagcatct), that amplify a 632bp fragment of ompa genes (20); primers 120.m59 (forward 5´aaacaa taatcaaggtactgt)-120.807 (reverse 3´tacttccggttacagcaaagt) that amplify an 812bp fragment of ompb gene, previously described (21). negative (molecular grade water) and positive controls (dna r. amblyomii) were included for each reaction. positive products were purified by using a quick gel extraction kit (purelinktm, invitrogen) and subsequently these were sequenced by a commercial facility (macrogen). the sequences were assembled and edited with the seqman program from the dnastar packet (lasergene®, madison wi, usa), and phylogenetic analysis was performed with the mega 6 (22) and mrbayes 3.2 programs (23). results of 153 pools of c. felis felis (54 were from turbo, 65 from necocli and 34 from los cordobas), rickettsial dna was detected in 30 (19%) pools by glta gene. four pools amplified for ompb gene and none amplified for ompa. pulex irritans pools were negative by pcr. the prevalence of rickettsia in fleas expressed as percentage and minimum infection rate (mir) of fleas were calculated. we made this assessment on the assumption that a pcr-positive pool contains only one positive specimen. the overall mir of infected fleas was 4.45 (30/673). of these, 4.6% (9/194) of c. felis was from turbo, 5.7% (13/225) from necocli and 3.5% (8/230) from los cordobas (table 1). nucleotide sequences of the ompb gene from necocli and los cordobas were 99.9% identical to each other (fig. 2). sequence homology obtained from necocli and los cordobas were 100 and 99.7% with r. felis strain senegal, respectively. evolutionary history of glta gene was inferred by using the neighbor-joining method (not shown) and the bayesian method was used for ompb gene (fig. 2). the sequences generated in this study have been submitted to genbank under the accessions kp870106 to kp870109. table 1. prevalence of rickettsia felis in fleas expressed as the minimum percentage of fleas in a pool with detectable rickettsiae and minimum infection rate (mir) (ic 95%) location species of fleas number fleas number pools positive / number tested pools % positives pools minimum infection rate (mir) turbo c. felis felis 194 9/54 16 9/194 (4.6%) pulex irritans 5 0/2 0 0/5 (0%) necoclí c. felis felis 225 13/65 20 13/225 (5.7%) pulex irritans 19 0/4 0 0/19 (0%) los córdobas c. felis felis 230 8/34 23 8/230 (3.5%) total fleas pools 673 30/159 19 30/673 (4.4%) j arthropod-borne dis, march 2019, 13(1): 1–8 v contreras et al.: infection by rickettsia … 4 http://jad.tums.ac.ir published online: april 27, 2019 fig. 1. geographic location of the study areas in northwestern of colombia. square corresponds to the municipality of los cordobas (cordoba province), the star and circle corresponds to the municipalities of turbo and necoclí (antioquia province), respectively fig. 2. bayesian phylogenetic tree of rickettsial ompb gene. samples from this study are shown on bold font. two parallel searches were runned by 1,000,000 generations sampling every 1,000 states. average standard deviations of split frequencies were <0.01 at the end of the runs. substitution model used was the general time reversible with a discrete gamma distribution of the variation of the evolutionary rate (gtr+g), previously found as the best model by the bayesian information criterion. the analysis was performed in mrbayes 3.2.0. there were a total of 723 positions in the final dataset. the tree was drawn in the software figtree, 1.4 and it was rooted with r. prowazekii. j arthropod-borne dis, march 2019, 13(1): 1–8 v contreras et al.: infection by rickettsia … 5 http://jad.tums.ac.ir published online: april 27, 2019 discussion we reported the infection by r. felis in c. felis felis fleas collected from dogs from endemic areas of rickettsioses in cordoba and antioquia provinces, northern of colombia. values of minimum infection rates (mir) reported herein for c. felis are similar to the one previously reported in the province of caldas, colombia (5.3% mir) (13). however, there are lower than mirs shown in other countries, such as brazil (14.3%) (24), the united states (13.3%) (25) and taiwan (8.2%) (26). the proportion of c. felis felis positive pools in our study was 19% (30/153) and the proportion obtained in caldas (colombia) was 41% (54/132) (13). the rates of mirs of these studies were calculated based on the assumption that only one flea from each positive pool was positive for the rickettsia gene evaluated. it may underestimate the frequency of r. felis in pools, possibly because of the greater amount of dna in pools or other contaminants that may inhibit pcr assays (27). otherwise, in brazil, differences in the percentage of infection between regions were related to the environmental and climatic conditions (28). higher rates of r. felis infection in fleas were significantly related with regions with temperate climates, and lower rates were linked with dry climates. several studies highlight the broad distribution of infection by r. felis in c. felis. different proportions of infection have been reported in other american countries. for example, in mexico, 20% of 54 pools of c. felis collected from dogs were reported infected (29). sixty-four percent (55/86) and 58% (47/81) of pools of c. felis removed from cats and dogs were infected in guatemala and costa rica, respectively (30); and 41% of infected pools (25/62 c. felis and 2/4 c. canis) collected from 15 cats and dogs were reported in uruguay (31). in our study, r. felis was detected in 30/153 (19%) c. felis pools removed from dogs, which is very similar to the mexican report which, by the way, suggests the likely relevance of this host in maintaining c. felis and possibly r. felis in studied areas. moreover, some studies have detected r. felis by pcr in blood of dogs, suggesting that dogs may have the potential to act as an important reservoir of infection (32, 33). in the present study, the sequences obtained from necocli and los cordobas were identical to each other and they showed extremely high sequence homology to a r. felis strain from senegal (100 and 99.7% respectively, fig. 2). in province of caldas (colombia), authors have described a high homology (>98%), between several r. felis sequences obtained from c. felis and the r. felis urrwxcal2 (genbank accession cp 000053). likewise, they showed a very close monophyletic relationship of these sequences with the r. akari group (13). sequences of r. felis from necoclí (kp 870109) and los cordobas (kp870106) obtained in the present study were compared with the sequences obtained in caldas (colombia), called colombia5 and colombia7 (fig. 2) (13). phylogenetic relationship between the sequences of our results and caldas (colombia), showed identity values of 95.9% of necoclí vs colombia5; and 92.7% of los cordobas vs colombia7. variations could exist between the sequences of strains of r. felis from two different areas of colombia. moreover, rickettsial dna was not detected in p. irritans pools of our study, in contrast, 3/10 pools of this species infected with r. felis in caldas were reported (colombia) (13). r. felis infection in p. irritans, has also been reported in studies from democratic republic of the congo (34) and the united states (4), that shows a likely wide j arthropod-borne dis, march 2019, 13(1): 1–8 v contreras et al.: infection by rickettsia … 6 http://jad.tums.ac.ir published online: april 27, 2019 distribution of r. felis in this fleas species around the world. human infection with r. felis and its clinical implications have been controversial. this microorganism may be an emerging human pathogen; meanwhile, other authors consider that their casual appearance in human samples and vector is a proof of endosymbiosis (11, 35, 36). before we determine whether human beings of this region of colombia could be at real risk of getting ill by r. felis, further studies are necessary to show the seroprevalence in humans and animals and demonstrate its presence in other human cases compatible with rickettsiosis. conclusion in the present study, we reported the infection by rickettsia felis in c. felis felis fleas collected from dogs from endemic areas of rickettsioses in cordoba and antioquia provinces (colombia). almost 20% (30/153) of c. felis felis pools contained rickettsial dna. our findings highlighted the endemicity of the infection by r. felis in fleas from northern of colombia and suggest the importance of dogs as host of c. felis felis fleas and their potential as reservoirs of r. felis. human infection with r. felis and its clinical implications have been controversial. may before we determine whether human beings of this region of colombia could be at real risk of getting ill by r. felis, further studies are necessary to show the seroprevalence in humans and animals and demonstrate its presence in other human cases compatible with rickettsiosis. acknowledgements we thank colciencias for the financial support through grant no. 111549326228 (“estudio ecológico de endemicidad por rickettsia en colombia”). we thank also the universidad de antioquia program “sostenibilidad 2013/2014”. to university of cordoba, vicerector of investigation, sustainability program of research groups, 2017-2018. the authors declare that the disclosure of this paper will not generate or constitute any conflict of interest. references 1. mansueto p, vitale g, cascio a, seidita a, pepe i, carroccio a, di rosa s, rinni gb, cillari, walker dh (2012) new insight into immunity and immunopathology of rickettsial diseases. clin dev immunol. 2012: 967852. 2. labruna mb, mattar s, nava s, bermudez s, venzal jm, dolz 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democratic republic of congo. emerg infect dis. 14: 1972–1974. 35. faccini-martínez aa, forero-becerra e, cortés-vecino j, polo-teran l, jácome j, vargas j, valbuena g, hidalgo m (2013) caso probable de fiebre manchada (rickettsia felis) transmitida por pulgas. biomédica. 33(1): 9–13. 36. labruna mb, walker d (2014) rickettsia felis and changing paradigms about pathogenic rickettsiae. eid. 20(10): 1768–1769. j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 116 short communication homology modeling and conformational epitope prediction of envelope protein of alkhumra haemorrhagic fever virus naghmeh poorinmohammad, *hassan mohabatkar department of biotechnology, faculty of advanced sciences and technologies, university of isfahan, isfahan, iran (received 3 july 2013; accepted 1 july 2014) abstract background: the aim of this study was to generate in silico 3d-structure of the envelope protein of ahfv using homology modeling method to further predict its conformational epitopes and help other studies to investigate its structural features using the model. methods: a 3d-structure prediction was developed for the envelope protein of alkhumra haemorrhagic fever virus (ahfv), an emerging tick-borne flavivirus, based on a homology modeling method using m4t and modweb servers, as the 3d-structure of the protein is not available yet. modeled proteins were validated using modfold 4 server and their accuracies were calculated based on their rsmds. having the 3d predicted model with high quality, conformational epitopes were predicted using discotope 2.0. results: model generated by m4t was more acceptable than the modweb-generated model. the global score and pvalue calculated by modfold 4 ensured that a certifiable model was generated by m4t, since its global score was almost near 1 which is the score for a high resolution x-ray crystallography structure. furthermore, itsthe p-value was much lower than 0.001 which means that the model is completely acceptable. having 0.46 å rmsd, this model was shown to be highly accurate. results from discotope 2.0 showed 26 residues as epitopes, forming conformational epitopes of the modeled protein. conclusion: the predicted model and epitopes for envelope protein of ahfv can be used in several therapeutic and diagnostic approaches including peptide vaccine development, structure based drug design or diagnostic kit development in order to facilitate the time consuming experimental epitope mapping process. keywords: 3d structure, homology modeling, conformational epitope, alkhumra haemorrhagic fever virus, envelope protein. introduction alkhumra haemorrhagic fever virus (ahfv), a tick-borne flavivirus, was firstly discovered in 1995 from a butcher who had lately slaughtered a sheep in the city of alkhumra in makkah province. ahfv is now an emerging virus causing febrile reactions with fatal haemorrhagic and neurological manifestations (alzahrani et al. 2010, mohabatkar 2011, memish et al. 2012). ahfv was misnamed as alkhurma virus for many times in scientific papers and publications but has been corrected recently by the international committee on taxonomy of viruses (ictv) (madani 2005, king et al. 2011, madani et al. 2011, madani et al. 2012). ahfv is the first tick-borne haemorrhagic virus whose whole genome has been sequenced and based on the sequence analysis, it has 89 % homology with kyasanur forest disease virus (kfdv) which belongs to the genus flavivirus of the flaviviridae family (mohabatkar 2011). this genus includes nearly over 70 viruses, mostly arthropodeborne, which infect humans and animals (pastorino et al. 2006). kfdv and its genetic variants, ahfv and seven other species *corresponding author: dr hassan mohabatkar, email: h.mohabatkar@ast.ui.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 117 including langat, louping ill, omsk haemorrhagic fever, powassan, royal farm ,gadgets gully and tick-borne encephalitis are grouped in the same subclassification (memish et al. 2010). however, ahfv among all these viruses makes a major concern in the local health authorities as it can be exported to other countries by the muslims returning from yearly hajj rites. since it is suggested to be one of the most deadly flavivirus infections, with case fatality rates >30 % (charrel et al. 2005), it can be one of our major concerns in iran as a muslim country. generally, incidence of many life-threatening viruses in humans has increased during the last two decades (mohabatkar 2011) and there are many others which threaten to increase in the near future. these urgent situations greatly force us to accelerate our investigations and studies. undoubtedly in vitro and in vivo studies are of major importance in discovering the therapeutic and prophylactic solutions, but the fact is that in silico studies and simulations can accelerate these experiments due to the timesaving manner they have. high-accuracy and knowledge-based in silico predictions will result in a better and faster handling of the disease being studied since it can highlights the most important parts to be studied (mohabatkar et al. 2012). during recent years, useful data have been computationally produced for many viruses such as human papillomavirus type 16 (hpv16) (mohabatkar 2007), influenza a h3n2 virus (a/hong kong/1/68) (liang et al. 2012), isfahan virus (isfv) (mohabatkar and mohsenzadeh 2009) and many others parallel to that of in vivo studies, showing the complementary and necessary role of computational investigations. a comprehensive in silico study on medically important structural properties of protein e of ahfv was done by mohabatkar 2011, including the prediction of its secondary structure, glycosylation sites, tetraspan membrane protein of hair cell stereocilia (tmh), disulfide connectivity, subcellular localization, evolutionary distance and t-cell epitope prediction, but was not including conformational epitope prediction while lacking a good 3d model (mohabatkar 2011). it should be noted that, protein e plays a central role in the biology of flaviviruses. it is the dominant antigen in eliciting neutralizing antibodies and plays a main role in inducing immunologic responses in the infected host (wu et al. 2003). the aim of this study was to generate in silico 3d-structure of the envelope protein of ahfv using homology modeling method to further predict its conformational epitopes and help other studies to investigate its structural features using the model. however, better models can be generated in the future due to the fact that bioinformatics servers and tools are progressing rapidly. materials and methods retrieval of target amino acid sequence the amino acid sequence of the envelope protein of ahfv was obtained from the protein database of ncbi (accession number np_775470.1). after ensuring that there is no 3d-structure of the protein in protein data bank (pdb) (http://www.rcsb.org/pdb/), the present work was held. homology modeling and quality assessment modeling was done using two homology modeling programs, m4t (fernandez-fuentes et al. 2007) and modweb (http://salilab.org/ modweb) which are both automated webservers. since unfavorable distortions are exerted on the side chains of amino acids during homology modeling process, energy minimization was performed on both models to move them to their optimal condition where the protein is in its most stable state. here energy minimization process was performed using gromos96 force-field implehttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 118 mentation in swiss pdbviewer software (version 4.0.1) (kaplan et al. 2001). refined models were then evaluated via quality assessment methods. the most common method is root-mean-square deviation (rmsd) metric which shows the mean distance between the corresponding atoms in the two structures (maiorov 1994). rmsds of the modeled structures from the selected template were calculated using rmsd calculator tool in swiss-pdbviewer (version 4.0.1) based on the backbone atoms (n, cα and c). the other method to evaluate the generated protein models used here was based on the use of modfold4 web-based server. the server allows user to make quantitative judgments about the credibility of models using different quantitative parameters like global score and p-value (mcguffin et al. 2013). finally, the overall stereochemical property of the more accurate model was further assessed by ramachandran plot analysis using rampage server. (http://mordred.bioc.cam.ac. uk/~rapper/rampage.php). conformational epitope prediction the best selected model was subjected to predict its possible conformational epitopes with discotope 2.0 server (haste andersen et al. 2006), which uses a combination of amino acid statistics, spatial information, and surface exposure. the server is trained on a compiled data set of discontinuous epitopes from 76 x-ray structures of antibody-antigen protein complexes (haste andersen et al. 2006) and is one of the most known and reliable epitope prediction servers. according to the server constructors study, discotope detects 15.5 % of residues located in discontinuous epitopes with a specificity of 95 %. this level of specificity is higher than several similar servers (haste andersen et al. 2006). results homology modeling, quality assessment and streochemical quality evaluation homology modeling was performed using two programs: m4t and modweb which are servers for automated comparative protein structure modeling that calculate their models based on the best available template structures from pdb without the need of manually identifying a template and doing pairwise alignment. however, results of the alignment and the choice of template can be observed in the servers’ output (eswar et al. 2003). here, the template by which the two modeling programs generated their models was the crystal structure of chain a of envelope glycoprotein from tick-borne encephalitis virus (pdb: 1svb_a) at 2 å resolution (rey et al. 1995). alignment results performed automatically by the server revealed that the selected template has almost 82 % identity with the query sequence. although the accuracy of the generated model is highly dependent on its sequence identity with the template sequence, by having the results of rmsd metric and modfold4 server the quality of the model can be reliably assessed. the results of rmsd and modfold4 for the final energy-minimized models are provided in table 1. during energy minimization process which was perfomed for 5 runs, the final calculated energy of m4t and modweb generated models were reduced to -15335.445 kj/mol and -14327.418 kj/mol respectively. before energy minimization process, these values were -4808.651 kj/mol and -3147.817 kj/mol for m4t and modweb generated models respectively. in case of rmsd, m4t generated model showed 0.46 å rmsd with the template and is therefore more accurate than the model generated by modweb with 3.45 å rmsd. using modfold 4, the global model quality score is calculated and from this feature a p-value is calculated which represents the probability that a model is incorrect. the global model quality scores range between 0 and 1 and scores less than 0.2 indicates an incorrectly modeled proteins while scores http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 119 greater than 0.4 are models with higher quality and indicate that the models are highly similar to the native structures. the other output parameter of the server is p-value. in general, p-values more than 0.05, represent low-quality models. when p-value reaches 0.01–0.05, it can be concluded that the model quality is medium. high quality models show 0.001–0.1 p-values and if having absolutely certifiable models, the p-values of less than 0.001 are expected. here both the global model quality score and p-value are acceptable especially for the model generated by m4t, where p-value is less than 0.001 and the global model quality score is near 1, at about 0.77. the p-value and global model quality of 2.92e–4 and 0.773, respectively, shows that the model scores are both in the range required for a high quality model. therefore the model generated by m4t was then visualized by pymol (fig. 1). the evaluation of m4t generated model for its stereochemical quality was performed by ramachandran plot analysis using rampage server and the output plot is shown in fig. 2. it showed 98 % of residues in favored region, 1.5 % in allowed region and just one residue (0.3 %) in outlier region which highly indicates a good streochemical quality of the predicted model. the detailed result from rampage is given in table 2. b-cell conformational epitope prediction having an acceptable model, the prediction of envelope protein of ahfv was undertaken using discotope 2.0. the input is the pdb file format of the model. disco tope 2.0 utilizes calculation of surface accessibility -estimated in terms of contact numbersand a novel epitope propensity amino acid score. the final scores are calculated by combining the propensity scores of residues in spatial proximity and the contact numbers. in the most recent version of discotope which is version 2.0 used in this paper, novel definition of the spatial neighborhood is used to sum propensity scores and half-sphere exposure as a surface measure (kringelum et al. 2012a). results from discotope 2.0 showed 26 residues out of the whole 395 total residues of the model as epitopes (table 3). these residues can then interact as the consequence of protein folding and form conformational epitopes. fig. 1. 3d predicted model visualized by pymol fig. 2. ramachandran plot for the model http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 120 table 1. quality assessment of models using different parameters modeling program confidence p-value global model quality score rmsd (å) m4t certifiable 2.92e-4 0.7730 0.46 modweb medium 1.399e-2 0.4408 3.45 table 2. ramachandran plot analaysis results using rampage %(, )positionresidueanalysis result 1.5 (-106.51, -157.54) (-81.16, -79.63) (-34.43, 122.46) (43.99, -123.34) (82.85, -42.90) (21.20, 69.10) 56 202 223 249 351 361 thr leu leu ala asn asn in allowed region 0.3(90.29, 127.61)54alain outlier region 98.2-all other positionsall other residuesin favored region table 3. predicted conformational epitiope residues by discotope 2 epitope residue residue position discotope score glu asn ala asn asp glu gly ser met thr ser lys ala his gly glu pro asn thr thr pro gly asp gln phe lys 51 52 54 154 178 277 278 279 302 303 335 336 346 347 348 349 350 351 366 367 378 379 380 391 393 395 -2.368 -0.729 -3.180 -3.107 -3.487 -3.678 -0.777 -1.855 -3.086 -0.787 -3.399 -3.567 -3.463 -3.001 -1.157 -2.459 -3.312 -3.003 -1.287 -1.642 -0.886 -1.292 -1.495 -3.275 -1.356 -1.260 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 121 discussion interaction between b-cell receptors or soluble antibodies and protein antigens is one of the major mechanisms of immune system to overcome infectious pathogens. identification of the epitopes which antigens can bind to antibodies is undoubtedly essential in many biomedical applications including vaccine design, immunotherapeutics studies and also for developing and designing of diagnostic kits. experimental epitope mapping is difficult and expensive in term of time and cost, making in silico methods a good complementary accelerant (kringelum et al. 2012a). although most b-cell epitopes are conformational, computational methods and studies often concentrate on mapping linear epitopes (yasser and honavar 2010). in recent years interests are increasing for mapping conformational epitopes and this assertion can be confirmed by the fact that different tools potential for predicting conformational epitopes are increasingly developing, these are including ellipro (ponomarenko et al. 2008), pepito (sweredoski and baldi 2008), seppa (sun et al. 2009), epsvr (liang et al. 2010), discotope 1.0 and 2.0 and many others. however due to the computational complexity and lack of sufficient knowledge about antibody-antigen complex structures, there are still a limited number of high accuracy web-based servers and tools. linear b-cell and t-cell epitopes of envelope protein of ahfv have already been predicted and both have essential roles in the immune response to pathogens (mohabatkar 2011). in this paper, predicting the conformational epitope of the envelope protein of ahfv using a structure-based prediction method was held after generating a certifiable 3d model. there are sequence-based prediction methods available for conformational epitope mapping which does not require the 3d structure of the protein being studied. however, the structure-based prediction can be more accurate if equipping with strong algorithms and therefore, the latter was used in the present study. although we can more conveniently gain information about protein 3d structures in silico, x-ray crystallography and nmr are undoubtedly the first and best choices to get such information. homology modeling of proteins is an alternative way when there is no information about their experimentallyprepared 3d structures. the critical step in homology modeling is the identification of a 3d template chosen by virtue of having the highest sequence identity with the target sequence, if indeed any are available. sometimes there is no suitable template structure available or there are templates with mediumlow sequence identities (less than 50–70 % identity). when there are template structures available but with low sequence identity to our target protein sequence, it is not recommended to perform homology modeling. here, the selected template structure (1svb) showed 82 % sequence identity to our target sequence. having 82 % sequence identity, it was ensured that homology modeling is an appropriate method to predict the 3d structure of our target protein. furthermore, model accuracy was described based on its rmsd with the template structure and also by modfold4. according to related literatures and to provide a frame of reference for rmsd values, up to 0.5 å rmsd occurs in independent determinations of relatively the same protein, meaning that this range of rmsd value for any modeled protein shows high modeling accuracy (chothia and lesk 1986). rmsds of m4t and modweb generated models were 0.46 and 3.45 å respectively and it is obvious that m4t-generated model is more accurate. according to previous studies of homology modeling cases, highly accurate models were reported to have http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 116–124 n poorinmohammad and h mohabatkar: homology modeling … 122 0.4–1.5 å rmsd (lindin et al. 2013, zhu et al. 2014). thus, our predicted protein structure has excellent backbone rmsd and can be referred to as highly accurate. it is important to note that, there is a defect in the use of rmsd as a quality assessment method: rmsd metric is sensitive to outlier regions created by poor modeling of individual loop regions in a structure that its core is accurately modeled and for this reason there are cases when as many as one in ten highly accurate homology models (60%–95% identity), have an rmsd value of more than 5 å vs. the empirical structure (guex et al. 2009). due to this defect, modfold4 quality assessment server was also used along with rmsd-based accuracy assessment. using the scores given by modfold4, the similarity between the template and target structures which are identical in amino acid sequences but different in tertiary structures can be described. both models were regarded as accurate due to the results of modfold4, however, again these scores were better for the m4t generated model (0.7730 and 2.92e-4 for global model quality score and p-value, respectively). using this server, the model is ranked by global model quality score, and a p-value is calculated for it, which relates to the likelihood that the global model is incorrect (mcguffin et al. 2013). our m4t-generated model has gained a high global model quality score along with a very low p-value, meaning that the model is highly certifiable. ramachandran plot analysis also verified this fact that the model is accurate. here, the choice of epitope prediction server was relied on a study showed that discotope 2.0 server has the highest predictive performance among some other similar servers (kringelum et al. 2012b). results from discotope 2.0 showed 26 residues as conformational epitopes. according to ramachandran plot analysis, alanine 54 which was one of the predicted epitope residues, was shown to be in outlier region of ramachandran plot. therefore, it seems to be unacceptable to name this residue as an epitope compartment. however, it should be also noted that it is not bizzare when a residue or more adopt a disallowed stereochemical angle. it can be explained by the fact that they do so to maintain the stability of the whole protein structure. due to this reason, alanine can adopt disallowed conformations to stabilize the whole protein tertiary structure with high rate of occurrence (pal and chakrabarti 2002). thus, this residue can be also accepted as an epitope compartment regardless of being in the outlier region of ramachandran plot. conclusion the predicted model for envelope protein of ahfv is of an acceptable quality. since no therapeutic or prophylactic vaccine is available for ahflv, such data on its potential conformational epitopes might be helpful for scientists in future drug and vaccine development. acknowledgements support of this study by university of isfahan is highly acknowledged. the authors declare that there is no conflict of interests. references alzahrani ag, al shaiban hm, al mazroa ma, al-hayani o, macneil a, rollin pe, memish za (2010) alkhurma hemorrhagic fever in humans, najran, saudi arabia. emerg infect dis. 16: 1882–1888. charrel rn, zaki am, fakeeh m, yousef ai, de chesse r, attoui h, de lamballerie x (2005) low diversity of alkhurma hemorrhagic fever virus, saudi arabia, 1994–1999. emerg infect dis. 11: 683– 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bioinformatics. 24: 1459–1460 wu kp, wu cw, tsao yp, kuo tw, lou yc, lin cw, cheng jw (2003) structural basis of a flavivirus recognized by its neutralizing antibody solution structure of the domain iii of the japanese encephalitis virus envelope protein. j biol chem. 278: 46007– 46013. yasser e-m, honavar v (2010) recent advances in b-cell epitope prediction methods. immunome res. 6: s2. zhu k, day t, warshaviak d, murrett c, friesner r, pearlman d (2014) antibody structure determination using a combin’ation of homology modeling, energy‐based refinement and loop prediction. proteins: struct, funct, bioinf. in press. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 70 original article a survey on ectoparasite infestations in companion dogs of ahvaz district, south-west of iran b mosallanejad 1, *ar alborzi 2, n katvandi 2 1department of clinical sciences, faculty of veterinary medicine, shahid chamran university of ahvaz, ahvaz, iran 2department of pathobiology, faculty of veterinary medicine, shahid chamran university of ahvaz, ahvaz, iran (received 4 nov 2011; accepted 7 dec 2011) abstract background: the objective was to determine the prevalence of ectoparasite infestations in referred companion dogs to veterinary hospital of shahid chamran university of ahvaz, from 2009 to 2010. methods: a total of 126 dogs were sampled for ectoparasites and examined by parasitological methods. the studied animals were grouped based on the age (<1 year, 1–3 years and >3 years), sex, breed and region results: thirty six out of 126 referred dogs (28.57%) were positive for external ectoparasites. the most common ectoparasites were heterodoxus spinigera, which were recorded on 11 dogs (8.73%). rhipicephalus sanguineus, sarcoptes scabiei, otodectes cynotis, xenopsylla cheopis, cetenocephalides canis, cetenocephalides felis, hippobosca sp. and myiasis (l3 of lucilia sp.) were identified on 9 (7.14%), 7 (5.56%), 6 (4.76%), 3 (2.38%), 3 (2.38%), 2 (1.59%), 2 (1.59%) and one (0.79%) of the studied dogs respectively. mixed infestation with two species of ectoparasites was recorded on 8 (6.35%). prevalence was higher in male dogs (35.82%; 24 out of 67) than females (20.34%; 12 out of 59), age above 3 years (31.81%; 7 out of 22) and in the season of winter (30.95%; 13 out of 42), but the difference was not significant regarding to host gender, age and season (p> 0.05). conclusion: apparently this is the first study conducted in companion dogs of ahvaz district, south-west of iran. our results indicated that lice and ticks were the most common ectoparasites in dogs of this area. the zoonotic nature of some ectoparasites can be regard as a public health alert. keywords: dogs, ectoparasite, infestation, prevalence, iran introduction ectoparasites are a common and important cause of skin diseases in dogs and cats. they have a worldwide distribution and are capable of disease transmission. ectoparasites cause life-threatening anemia and occasionally hypersensitivity disorders in young and debilitated animals (araujo et al. 1998). some ectoparasites of pet animals, notably fleas, can infest humans and may lead to the development of dermatitis and transmit vector-borne diseases (scott et al. 2001). human infestation with otodectes cynotis (hering, 1838) has also been reported (hewitt et al. 1971). ticks, after mosquitoes, are the second most important arthropods that may transmit pathogens like viruses (crimean-congo hemorrhagic fever, colorado tick fever and tick-borne encephalitis), bacteria including rickettsiae (rocky mountain spotted fever, tularemia, q fever, ehrlichiosis and lyme diseases), protozoa (babesiosis) and filarial nematodes (onchocerciasis) to other animals and humans. cases of human parasitism (such as astrakhan fever) have been reported by rhipicephalus sanguineus from southeastern europe too, i.e., *corresponding author: dr alireza alborzi, e-mail: alirezaalborzi@yahoo.com j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 71 from bosnia and greece (fournier et al. 2003, chaligiannis et al. 2009). ticks also cause paralysis, the condition caused by toxins found in the saliva of ticks (xhaxhiu et al. 2009). canine demodiciosis caused by demodex canis is considered to be a part of the normal fauna of the canine skin and is present in small number of most healthy dogs. diagnostic procedures, such as trichography or skin biopsy may be required to obtain an accurate estimation of the mite infestation (scott et al. 2001). sarcoptic mange is a highly contagious non-seasonal and pruritic skin condition caused by infestation with sarcoptes scabiei var. canis, a burrowing mite, which is transmitted by direct contact between dogs. otoacariosis, caused by o. cynotis, is characterized by irritation, rubbing, ear twitching, scratching and head shaking particular in cats. other parasites such as trichodectes canis are highly host specific, but they can act as an intermediate host for the tapeworm dipylidium caninum that may affect humans, especially children (scott et al. 2001). because older animals may acquire immunity, puppies appear to be most susceptible to o. cynotis (curtis 2004). various studies have found that ctenocephalides felis, c. canis and pulex irritans, are the 3 most common flea species on dogs. different species of ticks and fleas may infest dogs and cats in different geographical regions. c. felis was the most prevalent species in london (beresford-jones 1981), egypt (amin 1966), and denmark (kristensen et al. 1978), while c. canis was the dominant species in dogs in rural parts of the united kingdom (chesney 1995), dublin (baker and hatch 1972), and australia (coman et al. 1981). pulex irritans was the prevalent species in dogs of the southern part of usa (kalkofen and greenberg 1974), and this species was also commonly found in dogs in hawaii (haas and wilson 1967). methods of deep and superficial skin scraping, acetate tape preparation, combing the entire body surfaces, otic swabs and clinical trials are usually used for the detection of ectoparasites (scott et al. 2001). in urban or suburban areas, people traditionally raise dogs as pets. health check-ups protect pets from infestation by ectoparasites. thus, knowledge of types of species, density and prevalence of ectoparasites is needed to effectively control them (scott et al. 2001, nuchjangreed and somprasong 2007). scarce information is available on the parasites of dogs from iran. c. canis was reported as the most common ectoparasite in shiraz, southern iran (jafari shoorijeh et al. 2008). to the best of our knowledge, this is the first report examining the prevalence of various ectoparasites infesting companion dogs in ahvaz district, south-west of iran. we conducted this study in order to determine the prevalence of ectoparasite infestations in companion dogs of this area. materials and methods in the present investigation, ectoparasites were collected from 126 companion dogs (53 terriers, 42 mixed breed, 11 german shepherd, 5 spitz, 5 pekingense, 4 great dane, 3 doberman pinscher, 2 lahasa apso and one shitzu) with age range 2 months– 8 years. they were referred to veterinary hospital of shahid chamran university of ahvaz district, in southwestern iran. this area is located at an elevation of 12 meters above sea level and the climate is warm-humid. weather parameters were obtained of weather bureau of khuzestan province. the studied animals were divided into three groups based on the age (<1 year, 1–3 years and >3 years). classification was made by sex, breed and region also. most of the studied dogs were referred for a healthy check up and vaccination. age was estimated by dental formulary and owner information's. the dogs were examined for ectoparasite infestation by a complete examination of the j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 72 skin, clinical trials, skin scraping, acetate tape preparation, otic swabs, and combing of the whole body with a stainless steel finetoothed comb (zakson et al. 1995). the skins of all dogs were palpated and visually inspected thoroughly for the presence of ticks. all ticks were manually removed carefully to ensure that the mouthparts remained intact and collected together with any fleas and lice in the comb. the ticks and lice removed from the animals were stored in 70% ethanol. all dogs were assessed for fleas by combing for 5 minute exactly (zakson et al. 1995). the combing samples were collected in 70% ethanol also. all fleas were counted at 40× and identified at 400× microscopically. bilateral otoscopic examination was carried out for clinical signs of erythema, inflammation, excess debris or exudates. the visual presence of mite movement or black ceruminous exudates was indicative of o. cynotis. deep ear swab specimens were obtained from both ears from all dogs. all ear swab specimens were examined microscopically (at 40× for detection and 400× for species identification) within 24 h with mineral oil for the presence of mites. deep and superficial skin scraping was performed with mineral oil in suspected dogs to mites. skin scrapings and ear swabs were placed in 10% potassium hydroxide and gently heated to scales, crusts and hair or aural material. thereafter, the material was centrifuged, and the sediment was microscopically examined for mites. a 2 × 2 inch section of hair was clipped from the ventral abdomen on each dog. the clipped hair was collected in 70% ethanol. all hair samples were thoroughly examined with aid of microscope for the presence of nits, lice, mites, fleas and other arthropods. the specimens were mounted on a glass slide with mineral oil preparation. each slide was completely and carefully examined microscopically for superficial ectoparasites. finally all arthropods were identified microscopically at 40× to species using the diagnostic keys (anon 1966, estradapena et al. 2007). the study was approved by ethic committee by shahid chamran university of ahvaz. statistics analysis dogs were grouped by age, sex, breed and season to determine whether these factors were associated with ectoparasite infestation, using chi-square analysis and fisher’s exact test. statistical comparisons were carried out using spss 16.0 statistical software. differences were considered significant at p< 0.05 level. results a total of 126 companion dogs were randomly selected from approximately 550 dogs referred during the four time periods. samples were collected from 25, 32, 27 and 42 dogs in spring, summer, autumn and winter seasons respectively. thirty six out of 126 referred dogs (28.57%) were positive for external ectoparasites. the most common ectoparasites were lice and ticks followed by mites and fleas. heterodoxus spinigera, rhipicephalus sanguineus, sarcoptes scabiei, otodectes cynotis, xenopsylla cheopis, cetenocephalides canis, c. felis, hippobosca sp. and myiasis (l3 of fly lucilia sp.) were identified on 11 (8.73%), 9 (7.14%), 7 (5.56%), 6 (4.76%), 3 (2.38%), 3 (2.38%), 2 (1.59%), 2 (1.59%) and one (0.79%) of the studied dogs respectively (fig. 1”a-j”). mixed infestation with two species of ectoparasites was recorded on 6.35% (8 out of 126) of the dogs. r. sanguineus plus o. cynotis infestation were the most common combination (table 1). most of infestations were in mixed breeds (45.23%, 19 out of 42) and terriers (16.98%, 9 out of 53). prevalence was higher in male dogs (35.82%, 24 out of 67) than females (20.33%; 12 out of 59), age above 3 years (31.81%, 7 out of 22) j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 73 (table 2) and in the season of winter (30.95%, 13 out of 42) (table 3), but the difference was not significant relative to host gender, age, breed and season (p> 0.05). heterodoxus spinigera lice were recorded on 1, 9 and 1 of the dogs examined in seasons of autumn, winter and spring respectively, but they were absent in summer. otoscopic examination of both ears revealed mite movement and black ceruminous exudates. they were typically indicative of the presence of o. cynotis in (4.76%, 6 out of 126) dogs. the affected dogs to s. scabies had the typical signs of mange mite infestation, such as pruritis, papules, and alopecia. the average temperature for spring–winter was 29.83, 37.26, 23.2, and 17.86° c, respectively. the average relative humidity for spring–winter was 31.0, 23.66, 52 and 61.66% as well. table 1. occurrence of mixed ectoparasite infestations in companion dogs in ahvaz district, south-west of iran, 2009–2010 (n=126) number (prevalence %)mixed-species ectoparasite infestation (two species) 1 (0.79)ctenocephalides felis + heterodoxus spinigera 1 (0.79)xenopsylla cheopis + heterodoxus spinigera 3 (2.38)rhipicephalus sanguineus + otodectes cynotis 1 (0.79)heterodoxus spinigera + sarcoptes scabiei 2 (1.59)ctenocephalides canis + hippobosca sp. 8 (6.35)total table 2. number of ectoparasite infestations in companion dogs based on different ages in ahvaz district, southwest of iran, 2009–2010 season species age<1 years age 1-3 years age >3 years total h. spinigera 9 2 0 11 r. sanguineus 3 3 3 9 s. scabiei 5 2 0 7 o. cynotis 3 1 2 6 x. cheopis 3 0 0 3 c. canis 1 0 2 3 c. felis 2 0 0 2 hippobosca 0 0 2 2 myiasis 0 0 1 1 total 26 8 10 44 table 3. number of ectoparasite infestations in companion dogs based on different seasons in ahvaz district, southwest of iran, 2009–2010 season species autumn winter spring summer total h. spinigera 1 9 1 0 11 r. sanguineus 3 1 2 3 9 s. scabiei 0 3 3 1 7 o. cynotis 1 0 3 2 6 x. cheopis 2 1 0 0 3 c. canis 1 0 0 2 3 c. felis 0 1 1 0 2 hippobosca 0 0 0 2 2 myiasis 0 0 1 0 1 total 8 15 11 10 44 j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 74 fig. 1. the original pictures of ectoparasites found in companion dogs of ahvaz district including (a) s. scabies, (b) o. cynotis, (c) hyppobosca sp., (d) h. spiniger, (e) the affected dog to myiasis (l3 of fly lucilia sp.), (f) r. sanguinus (female), (g) r. sanguinus (male and female), (h) x. cheopis, (i) c. felis, (j) c. canis . the pictures were taken by a digital camera in the parasitology lab of the faculty of veterinary medicine, shahid chamran university of ahvaz. j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 75 discussion the present study is the first reach on the prevalence of infestations in companion dogs in ahvaz district. it was revealed that 28.57% of the dogs were infested to ectoparasites. these results indicate that ectoparasites are relatively common in companion dogs of this area, as many parts of the world. according to our study, h. spinigera (8.73%) and r. sanguineus (7.14%) were the most abundant ectoparasites, followed by mites and fleas. the canine sarcoptic mange and the ear mite (o. cycotis) can infest humans and cause pruritic skin lesions (hewitt et al. 1971). fleas not only act as intermediate hosts of tapeworms but also directly infest human. humans, if parasitized, may be exposed to babesiosis, rocky mountain spotted fever, ehrlichiosis, borreliosis or tick paralysis (scott et al. 2001). several reports (anvar et al. 1972, jafari shoorijeh et al. 2008,) have documented ectoparasite infestations among dogs in iran. ctenocephalides canis and c. felis have been reported on various host species from different parts of iran, but their prevalence was not noted (anvar et al. 1972). in research of jafari shoorijeh, the most common ectoparasite was c. canis, which infested 22 of the 160 dogs. in their study, pulex irritans was identified on 2 of the dogs and 142 r. sanguineus ticks were found on 13 dogs. trichodectes canis was observed on 2 dogs and 8 dogs had hippobosca flies, which were seen mostly in spring. all superficial skin scrapes for mite detection were negative (jafari shoorijeh et al. 2008). the ticks feed on a wide range of hosts; however, all stages of r. sanguineus are primarily associated with dogs (dantastorres 2008). lice are reportedly the most common ectoparasites on dogs of the northern hemisphere (christensson et al. 1998). heterodoxus spiniger was found on 11 of 126 dogs infested with ectoparasites in our study. it is a major ectoparasite of the dog population in ahvaz district. in several studies, d. canis has been reported as the most frequent infestation (nayak et al. 1997), but the current study was not consistent with previous studies. these differences can not be easily explained, but it may be attributed to epidemiological factors, such as weather, seasonal variations, geographical location, intrinsic resistance, and particularly the age of the animals examined (nayak et al. 1997). studies from various parts of the world have indicated that c. felis, c. canis, and p. irritans are the most commonly occurring flea species in dogs. it is reported that c. canis is the dominant species in dublin, regions of england and australia (linardi et al. 1973, zygner et al. 2006). however, harman et al. (1987) reported that c. canis was not identified among the dogs examined for fleas in florida. the greatest prevalence infestation of ectoparasites belonged to fleas which followed by ticks, 89% and 30% infestations, respectively (estares et al. 1999). in our study, the most common ectoparasites were lice and ticks followed by mites and fleas. although utilization of an otoscope or cotton-tipped swab for the diagnosis of ear mite and examination of vellus hair for the sarcoptic mange might have increased the detection rates of those mites, but only the skin scraping technique is definitive for diagnosis (foreyt 2001). a comparative study of ectoparasite infestation of different breeds of dogs was performed in four veterinary clinics in nigeria. of a total of 820 dogs examined, 30.0% were infected by ticks, 27.6% by lice, 25.8% by fleas and 13.3% by mites. the species of ectoparasites identified and their prevalence rates were: r. sanguineus (19.5%), o. megnini (10.5%), c. canis (25.8%), demodex canis (13.3%) (ugochukwu et al. 1985). a total of j arthropod-borne dis, 2011, 6(1): 70–78 b mosallanejad et al.: a survey on ectoparasite ... 76 344 dogs belonging to people in north west province, south africa, were examined for ectoparasites, accordingly the dogs harbored 14,724 ixodid ticks belonging to 6 species, 1,028 fleas belonging to 2 species, and 26 lice. pure infestations of h. leachi were present in 14 dogs and r. sanguineus in 172 dogs (bryson et al. 2000). in western romania, the most common flea in dogs was c. felis, followed by c. canis and p. irritans (morariu et al. 2006). hippobosca flies were found on 2 of 126 (1.59%) dogs infested with ectoparasites in our study. in kabul, afghanistan, 25 of 105 dogs had hippobosca capensis (le riche et al. 1988). it is stated that dogs less than 1 year old are more susceptible to ectoparasite infestations (kwochka 1987, nayak et al. 1997, chee et al. 2008). in contrast, in the present study, the higher prevalence was seen in the dogs of above 3 years old, compared with younger 3 years old. because older animals may acquire immunity, puppies appear to be most susceptible to ectoparasites; of course the difference was not significant. in the present survey, we found that the prevalence of ectoparasites was more frequent in male dogs (35.82%) than females (20.33%) without significant difference. this is agree to the results of other researchers (nayak et al. 1997, rodriguez-vivas et al. 2003), who suggested that both sexes are susceptible to the infestation of ectoparasites. mean (±sd) of temperature and relative humidity of ahvaz district were noted during the collection period in our study. among the 36 ectoparasite positive dogs, the highest involvement was recorded in winter season (30.95%), but the difference was not significant. dipeolu (1975) reported that the highest number of parasites occurred in summer in nigeria, whereas alcaino et al. (1999) established that they are predominant in spring in chile, showing a distinct decrease since the beginning of summer, until completely disappearing in autumn. for c. canis, horak (1982) considered that the most favorable months for adults are summer-autumn, maybe due to the higher temperature and humidity, whereas the most unfavorable period is that from winter-spring, due to low temperature and humidity. to prevent the possibility of continued transmission of ectoparasites from pet animals, practicing veterinarians should advice pet owners to pay attention to and be aware of ectoparasites of zoonotic importance. to the best of our knowledge this is the first study conducted in ahvaz district, south-west of iran that examined the prevalence of ectoparasites in companion dogs population, however, this survey was limited to the ahvaz area and additional studies are required to complement these findings and help veterinarians prepare a complete program for the control of these 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vector control, school of public health, kerman university of medical sciences, kerman, iran 3department of biostatistics and epidemiology, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran (received 16 aug 2018; accepted 07 mar 2020) abstract background: the common bed bug, a nocturnal bloodsucking insect, is known as a human parasite and public health problem in the world. the prevalence and geographical dispersion of bed bug in ahvaz city, southwest of iran was measured. methods: spatial distribution of cimex lectularius was determined by surveying 520 houses in 62 areas of ahvaz city in 2017. some information like as infested points, the concern level of the residents and allergic reaction to the insect bite were registered in a form using the secondhand instrument. results: according to the spatial distribution map, of 62 areas, 27 of them are infested with bed bugs. infestation is scattered throughout the city, but its focus is on the east of the karun river. the most bed bug infestation was in asiabad followed by manbaab areas. prevalence of bed bug infestation estimated 9.61% in ahvaz city. it was 5.4% and 11.6% in apartments and single houses, respectively. the lowest and highest infestation rates based on its source were 1.35% and 9.03% in wallpaper and cracks and crevices, respectively. bedroom and sitting room were the main harborages for bed bug in the houses. the majority of residents who had bitten by a bed bug showed various allergic reactions like redness skin, papules, vesicles, pustules and blisters. most of the people in the infested houses (62%) were very concern about bed bug infestation. conclusion: public education and increasing the knowledge of people can lead to successful management, prevention and elimination of this nuisance pest. keywords: bed bug; infestation; prevalence; distribution; cimex lectularius introduction bed bug as mainly active insect at night is belonging to the family cimicidae. two effective species in human infestations are cimex lectularius and c. hemipterus (1). bed bugs used to live close to where they feed. they were established and found a variety places of inside dwellings like furniture, loosened wallpaper, cracks of plaster, window and door cosigns (2). gerardo (2014) indicated that %70 of bed bug infestations may not be detected in private homes due to the cryptic lifestyle of the insect and lack of resident’s knowledge (3). this bloodsucking insect is considered as vector for 45 pathogens due to vectorial capacity and competence in the wild and laboratory conditions (2). although bed bugs have not been introduced as vector of human diseases, but they are considered because of the bites following skin symptoms. cutaneous reaction may be varied from mild, asymptomatic to intense lesions (2). depression, anxiety and sleep disturbance can be counted as mental effects of bed bug infestation (4). it appears that c. lectularius problem ap *corresponding author: dr elham jahanifard, e-mail: elham.jahani56@gmail.com http://jad.tums.ac.ir/ mailto:elham.jahani56@gmail.com j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 30 http://jad.tums.ac.ir published online: march 31, 2020 peared in the hotels of developed countries and this species had fast and cheap transmission by air transport (1). also, the increase in bed bug infestation may be facilitated by poor knowledge of people regarding the identification, prevention, and treatment of the infestation. furthermore, resistance to commonly used pesticides seems to be another notable factor in a resurgence of cimex spp (5). bed bugs have been known the cosmopolitan insects due to active and passive dispersal. there are some documents on the resurgence, spatial and temporal patterns, status of the infestation and case reports in different countries (6–8). the prevalence of cimex lectularius was reported 6.7% to 90.7% in previous studies (9–12). in despite the medical impact of this urban pest, the prevalence and infestation of cimex spp. have been rarely studied in iran. only, scattered researches have been reported from kohgiloyeh-boyerahmad, mazandaran, khorasan razavi, and isfahan provinces. geographic information systems (gis) are fundamental computer-based systems that enable show data as a map and help with decision-making system. furthermore, gis application analysis information based on places and environment data can predict the distribution of the urban pests. although, it uses to mapping, analysis and prediction vector-borne disease distribution of epidemiological information (13). this survey was conducted to prepare the spatial dispersal map of bed bug infestation in the southwest of iran, with a view to determining the prevalence of the insect among all areas of ahvaz city. materials and methods study area ahvaz (31° 20ˈ n, 48° 40ˈ e), the center of khuzestan province, in the southwest of iran is situated at an altitude of 12m above the sea level. karun river passes by the middle of the city. from past to now, ahvaz has been wellknown due to industries as well as environmental pollution. in the last decade, an anthropogenic source of air pollution (dust storm) has joined to other environmental problems. ahvaz ranked as air-polluted city due to its oil industry and environmental pollution (14). the study area has a hot semi humid climate (15) and it is also located in a plain section, but ahvaz introduced the warmest regions of iran regarding the severe shortage of vegetation. temperature varies the range of -5 °c to 50 °c in the winter to summer. data collection and analysis as there were no registered reports about bed bug infestations, so all study sites were considered on the same chance of the presence of insect as inclusion criteria. ahvaz city divided into two main parts in the east and west of karun river. this city has 8 regions and 127 areas. all areas were coded then %50 fraction of them selected randomly by using a table of random number. about 520 houses (353 single houses and 167 apartments) were randomly selected in these two parts based on their population to determine the prevalence of bed bug infestations (fig. 1). this one month survey was conducted in january 2017. sampling sites of ahvaz city including golestan, sadi, padad, padad faze1, padad phase2, nehzatabad, shaykh bhaei, kampolo jonobi, kampolo shomali, alavi, zibashahr, aryashahr, yousefi, kyanpars, kyanpars phase1, kyanpars phase 2, kyanabad. ameri-cymetri, 24metri, koyeabozr, gavmishabad, akhar asfalt, baghe shaykh, goldasht, malashyeh, karyshan, razmandegan, chonaybeh, mahdis, modares, koy bahonar, fuladshahr, koy isar, hasirabad, zayton kargari, soltanmanesh, manbaeab, koy ramazan, koy shahed, koy peirozei, koy abedi, zonyeh, koy farhangian, kourosh, asiaabad, farhangshar, shahrak daneshgah, pardis, baharestan, shahrak bargh, koy police, sepidar, nabovat, resalat, sanayefolad, manabetabeiei, melihafari, koy naft, koy niro, zayton karmandi and melirah. alive and dead http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 31 http://jad.tums.ac.ir published online: march 31, 2020 bedbugs, nymphs and eggs were searched in bedding including matters, bed sheet, bed frame and pillows, wooden furniture, electric boxes, frame of the door and window, light switches, vacuum cleaner, picture frame, wallpaper, cracks and crevices (fig. 2). observed bedbugs collected by forceps and flashlight. the samples transferred to the department of medical entomology and vector control in ahvaz jundishapur university of medical sciences. bed bugs preserved in 70% alcohol and identified by a reliable key (16). the infestation rate inside the houses was calculated by dividing the total number of infested houses by all visited houses. some information like the source and infested points, the number of families that use secondhand instrument, the level concern of residents to bed bug infestation and allergic reaction to bed bug bite were registered in the forms. in this study, all places were inspected by an entomologist and pointed by gps. all data of gps transferred to arcgis 9.3 and arcmap used to prepare the dispersal map of bed bugs in ahvaz city, the center of khuzestan province. results totally, 520 residential houses, including 353 and 167 single houses and apartments were inspected in this survey, of which 41 and 9 were infested with cimex lectularius, respectively. the bed bug infestation rate was estimated 9.61% in ahvaz city. it was 5.4% and 11.6% in apartments and single-houses, respectively. a chi-square test showed that a type of houses is associated with bed bug infestation (p< 0.001). the prevalence rate of bed bug in the east and west of karun river was 10.3% and 8.5%, respectively. although, there was socioeconomic difference between the residents of two sides of the river, but based on the result, the chi-square test did not show significant difference between bed bug infestations in the east and west of karun river (p= 0.3). of the 62 studied areas, 27 areas were infested by bed bug. the most bed bug infestation was in asiabbad followed by manbaab area with 60% and 30% rate of infestation. also, in the areas of padad, nehzatabad, alavi, malashiye, kerishan, modares, soltanmanesh, farhangshahr, farhangian and fulad the same level of bed bug infestation was observed (fig. 3). the geographical distribution of bed bug in ahvaz city is shown in figure 4. moreover, infested and non-infested areas determined with stars and circles signs in the map, respectively. the east of ahvaz was more infested than the west. bedding, furniture, cracks and crevices were infested in 28% of houses. furthermore, 4% of houses reported the infestation in all harborage sites (table 1). bed room and sitting rooms were the main harborages in the houses and no bed bugs were found in a lavatory and kitchen. based on the result of mann-whitney test, there was no significant difference between bedroom and sitting room (p= 0.16). residents of the infested homes referred to various ways of transmitting the bed bug infestation to human environments, including second-hand instrument, infested bag of children at school, infested hotel and transmission by a bag and suitcase, introducing the infestation via going to the relatives. only 6% of residents of the infested houses used second-hand equipment. about 127 persons have bitten by bed bugs with various allergic reactions like redness skin, papules, vesicles, pustules and blisters. moreover, the cutaneous reactions were severed in children. a total of 50 individuals responded to the question about their concern level toward bed bug infestation. about 62% of respondents were very concern about bed bug infestation and they search how to get rid of the bed bug. only 12% of respondents were not concerned regarding the increasing bed bug population. the results showed 94% of the families refused to buy second hand instruments. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 32 http://jad.tums.ac.ir published online: march 31, 2020 table 1. infested harborage sites with bed bugs, ahvaz city, khuzestan province, iran infested harborage sites infested harborage 1 3 1 , 2 1 , 5 1 , 2 , 3 1 , 2 , 4 1 , 4 , 5 1 , 2 , 3 , 4 1 , 2 , 4 , 5 1 , 2 , 3 , 5 1 , 2 , 3 , 4 , 5 t o ta l n 3 1 10 4 14 1 3 3 4 5 2 50 % 6 2 20 8 28 2 6 6 8 10 4 100 1= cracks and crevices, 2= bedding (including mattress, bed sheet, bed frame and pillow), 3= wooden furniture, 4= wall paper, 5= others (including electric box, vacuum cleaner) fig. 1. study areas in ahvaz city, khuzestan province, iran, 2017 fig. 2. a door-to-door investigation of bed bug in ahvaz city, southwest of iran (a–d) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 33 http://jad.tums.ac.ir published online: march 31, 2020 fig. 3. bed bugs infestation rate in different areas in ahvaz city, khuzestan province, iran, 2017 fig. 4. spatial distribution of cimex lectularius in ahvaz city, khuzestan province, iran, 2017 discussion our results showed a widespread bed bug infestation in ahvaz city as an urban area. the epidemiology of this urban pest is rarely studied in iran. there are only a few published data on the bed bug infestation and most of the information is restricted to individual reports to the health center. it is worth nothing that psychological and social problems observed due to bed bug infestations and it also can cause financial losses on hospitality and poultry inhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 34 http://jad.tums.ac.ir published online: march 31, 2020 dustries (17). the lack of sufficient data on the true prevalence of bed bug infestation in different areas can be for some reasons like as poor knowledge and awareness of public, absence effective relation between scientific research centers and private pest companies and no transmission serious illness by common bed bugs. our finding revealed the amount of bed bug infestation equal to 9.61% in ahvaz city. motevalli-haghi et al. (11) reported 2.8% the prevalence of bed bug in bahnamir city in mazandaran province. the presence of bed bugs has been documented in kashan (6.7%). a meta-analysis study of the bed bug infestation (cimex lectularius) in iran during 1995–2019 reported the weighted mean of this infestation was estimated 0.28 (18). a door-to-door survey showed 11.1% of cimex lectularius infestations in row homes in philadelphia, pennsylvania (19). the bed bug infestation was reported 21.8% in benue state in nigeria (20). also, the prevalence of bed bug infestations was demonstrated 69.9% in kampala capital city of uganda (21) that the high infestations may be attributed to the individual and health system elements. the resurgence of bed bugs as an important health problem had happened in some european countries, france, usa, canada and australia (22). early reports about bed bug infestation indicates this urban pest has been adopted to various human environments such as dormitory, houses, hotels, guesthouses and construction sites (9–12). our study confirmed the widespread dispersion of bed bugs in human buildings. severe infestation in some areas of the study has changed the bed bug into a serious health problem in those areas. the bed bug can transmit passively by infested luggage, clothing and other equipment (9, 23). also, the trade of used furniture and second hand goods facilitates transmission of egg, nymph and adult bed bug in many sub-saharan countries (10, 24, 25, 26). in our study, 94% of participants believed that second hand instruments are the cause of house infestation. el-azazy et al. (2013) suggested two main reasons for bed bug transmission, including increase in immigrants and using second hand furniture (27). these findings are comparable with our study result. the bed bugs can follow their host and move actively from bedroom to sitting room and even though to adjoining house (28). our results also revealed a bedroom and sitting room are preferable harborage for bed bug. although a similar finding has been reported by motevali-haghi et al. (11), but we did not observe the insect in the kitchen and lavatory. this finding clears the point that this bloodsucking insect tends to habit in close to the human host. our data indicated that cracks and crevices and bedding were more infested harborage sites. how and lee (28) reported bedding, headboard, and cracks and crevices as three most common c. hemipterus harborages. the bedding like bed frame, mattress and sheets introduced as frequently infested areas by hwang et al. (29). it seems that bed bugs move from the places with the highest frequency to the other harborage sites. the bedding is probably is the start point of infestation. the low knowledge of people is the main cause of the bed bug resurgence and increase bed bug population (3). the public usually confuses bed bug with other insects (30). we limited identifying bias via investigation the buildings by medical entomologist. then, bed bug prevalence supposed to estimate the real value. seidal and reinhardt (31) found the previous contact with bed bugs increased recognition rate among respondents. although, the kind of educational interventions should be tested as former factors, but the level of knowledge public may increase via seminars, workshops, or even face to face training by health staffs (3) as a principle key to raising self-reporting bed bug infestation in participants and to making effort for eradication the bed bugs. the people should be educated about bed bug controls via integrated pest management (ipm) approach. this is the collection of physical methods like killing and eliming the insects using heating, freezing, http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 35 http://jad.tums.ac.ir published online: march 31, 2020 steaming, vacuuming, washing and drying and chemical tactics based on recommended insecticide dosage (32). although bed bug infestations are due to various reasons, the resident’s socio-economic status effects the prevalence of bed bug. socio-economic factors can influence bedbug infestation. while senabulya et al. (21) revealed that the very weak economically individuals had more chances to be infested with bed bugs. wang et al. (30) reported low-income communities were more susceptible to burden bed bug infestation due to financial situation. poverty, household size and neighborhood regarded as a risk factor of bed bug infestations (33). despite of these studies, we encountered the bed bug problem in asiabad, golestan and kyanpars areas where their residents have low, middle and high incomes but the infestation rate was various. the existence of bed bugs in all social issues may be are due to various reasons like poverty, increased international travel, lack of awareness or transmission by second hand equipment. in our survey, various allergic reactions like redness skin, papules, vesicles, pustules and blisters have appeared in residents who had bitten by bed bug which the cutaneous reactions were more severe in the children. the high reaction of children to bed bug bite and its severity is related to the immunological status of individual (8). also, our findings are confirmed by alizadeh et al. (34), as they reported severe cutaneous allergic reactions with itching that appeared one hour after bed bug biting in forearm of 28-years old men. a lesion that included papules, blisters, and nodules had appeared at 48h after bed bug bite. the investigation found that 62% of residents were concerned about bed bug infestation which is similar to the reported concern level in public health officials by kaylor et al. (35). alizadeh et al. (36) demonstrated that more than 65% of the residents were very concerned about the insect infestation. it can be assumed that the concern toward the bed bug prevalence and resurgence can increase people's motivation to raise their awareness. moreover, 50% of the participants in the study were unaware about the nuisance insect in their apartments (37). conclusion in conclusion, we reported c. lectularius in an urban environment that can affect people health and life situation. temporal and spatial distribution of bed bug has little been known. the spatial dispersal maps of bed bug infestation may provide a valuable source for preparing control program of bed bugs. we recommend further studies in ahvaz city, including determining the effect of the season on the insect mobility within households, surveying the role of education of people and increasing their awareness about bed bug, studying insect infestation in dormitories, hotels and public accommodations and investigating the relation between self-reporting the infestation with reducing the bed bug prevalence. acknowledgements this study is financially supported by social determinants of health research center,ahvaz jundishapur university of medical sciences, ahvaz, iran with project no. sdh-9510 and ethical number, ir.ajums.rec.1395.443. we would like to thank all the residents who participated in our study and helped us to complete the research. the authors declare that there is no conflict of interest. references 1. reinhardt k, siva-jothy mt (2007) biology of the bed bugs (cimicidae). annu rev entomol. 52: 351–374. 2. delaunay p, blanc v, del giudice p, levybencheton a, chosidow o, marty p, brouqui p (2011) bedbugs and infectious diseases. clin infect dis. 52(2): 200–210. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 36 http://jad.tums.ac.ir published online: march 31, 2020 3. gerardo em (2014) increasing awareness of and education about bed bugs (cimex lectularius) as a public health issue in hawai. 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222–227. 21. senabulya gw, alege jb, atuhairwe c, taremwa im (2019) prevalence and associated factors of bed bug infestations (cimex lectularius) in lubaga division, kampala capital city, uganda. prev med commun health. 2: 1–5. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 29–37 m sharififard et al.: prevalence and … 37 http://jad.tums.ac.ir published online: march 31, 2020 22. levy bencheton a, berenger jm, del giudice p, delaunay p, pages f, morand jj (2011) resurgence of bedbugs in southern france: a local problem or the tip of the iceberg?. j eur acad dermatol venereol. 25(5): 599–602. 23. cooper r (2006) bed bugs-still more questions than answers: a need for research and public awareness. am entomol. 52 (2): 111–112. 24. boase cj (2004) bedbugs: reclaiming our cities. biologists. 51(1): 9–12. 25. emmanuel oi, cyprian a, agbo oe (2014) a survey of bedbug (cimex lectularius) infestation in some homes and hostels in gboko, benue state, nigeria. psyche. 76(1): 28–44. 26. richards l, boase cj, gezan s, cameron mm (2009) are bed bug infestations on the increase within greater london? j environ health res. 51: 3–11. 27. el-azazy om, al-behbehani b, abdou nm (2013) increasing bedbug, cimex lectularius infestations in kuwait. j egypt soc parasitol. 43(2): 415–418. 28. how yf, lee cy (2010) survey of bed bugs in infested premises in malaysia and singapore. j vector ecol. 35(1): 89–94. 29. hwang sw, svoboda tj, de jong ij, kabasele kj, gogosis e (2005) bed bug infestations in an urban environment. emerg infect dis. 11: 533–588. 30. wang c, singh n, zha c, cooper r (2016) bed bugs: prevalence in low-income communities, resident’s reactions, and implementation of a low-cost inspection protocol. j med entomol. 53(3): 639–646. 31. seidel c, reinhardt k (2013) bugging forecast: unknown, disliked, occasionally intimate. bed bugs in germany meet unprepared people. plos one. 8(1): e51083. 32. romero a, sutherland am, gouge dh, spafford h, nair s, lewis v, choe dh, li s, young d (2017) pest management strategies for bed bugs (hemiptera: cimicidae) in multiunit housing: a literature review on field studies. j integr pest manag. 8(1): 1–10. 33. ralph n, jones he, thorpe le (2013) selfreported bed bug infestation among new york city residents: prevalence and risk factors. j environ health. 76(1): 38–45. 34. alizadeh i, jahanifard e, sharififard m (2017) allergic reactions and dermatitis to common bed bug bites: a case report from ahvaz, southwest iran. iran j dermatol. 20: 65–67. 35. kaylor m, wenning d, eddy c (2015) prevalence, knowledge, and concern about bed bugs. j environ health. 78(1): 20–24. 36. alizadeh i, sharififard m, jahanifard e, rajaei f, mousavian g, mehraghaei m (2018) identification, knowledge, and awareness of people regarding public health nuisance insect (cimex lectularius) in southwest of iran. jundishapur j health sci. 10 (3): 1–7. 37. wang c, saltzmann k, chin e, bennett gw, gibb t (2010) characteristics of cimex lectularius (hemiptera: cimicidae), infestation and dispersal in a high-rise apartment building. j econ entomol. 103(1): 172–177. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 334 http://jad.tums.ac.ir published online: december 25, 2018 original article spironucleus muris and eperythrozoon coccoides in rodents from northwestern iran: rare infections soudabeh heidari 1 , mehdi mohebali 1, 2 , zabihollah zarei 1 , mehdi nateghpour 1 , *afsaneh motevalli-haghi 1 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran (received 9 apr 2017; accepted 1 july 2018) abstract background: rodents perform a crucial role in dispersal of zoonosis causes globally. we aimed to investigation about infection levels of parasitic agents in rodents’ population in meshkinshahr areas, northwest of iran from apr to sep 2014. methods: two hundred four rodents were trapped and anaesthetized. a sample of blood was collected via cardiopuncture from each one. thin and thick blood smears were prepared and stained with giemsa. all stained smear were examined under light microscopy with high magnification by two expert microscopists. every suspected unicellular observed were measured microscopically and compared with key references to diagnose. results: captured rodents were identified as three genera including meriones persicus, mus musculus, cricetulus migraturius. protozoa identified in this study were included of spironucleus muris and eperythrozoon coccoides, these parasites were observed in blood smear of 0.98% of rodents. s. muris and e. coccoides were seen in m. musculus and c. migraturius, respectively. conclusion: the present study increases awareness about eperythrozoonosis in rodents and its potential transmission to domestic animals and even to human in rural districts in iran. moreover, the attack of spironucleus on the mucus of colon and its systemic risk was confirmed. keywords: spironucleus muris, eperythrozoon coccoides, rodents, iran introduction rodentia are the largest group of mammals worldwide. they are found in vast numbers on all continents except antarctica. rodents play important roles as reservoirs and carriers of diseases agent such as leishmaniasis, plague, leptospirosis, salmonellosis, rat bite fever, dermatophytosis, sporotrichosis, murine typhus, trichinellosis, cestodes and trematodes infections, toxoplasmosis, relapsing fever (1). blood parasites in rodent such as trypanosoma lewisi, leishmania spp., plasmodium berghei, babesia microti, eperythrozoon coccoides, haemobartonella muris are important because they are transmitted to humans by ectoparasites of rodents (1). spironucleus muris (formerly hexamita muris) is an opportunistic pathogen of several rodent species including rat, mice, golden hamster and european hamster. this flagellated protozoan usually inhabits in the crypts of lieberkuhn in the small intestine after ingesting parasitic cysts and may cause an acute or choronic form of disease. the organisms invade the lamina propria of the intestinal villi in immunocompromised animals and can disseminate systemically through the lymphatics or vasculature. circulating parasite is visible in the peripheral blood of an infected animal (2-4). eperythrozoon coccoides is blood parasite *corresponding author: dr afsaneh motevallihaghi, e-mail: a-motevalli@tums.ac.ir, amh.mot@gmail.com http://www.ucmp.berkeley.edu/mammal/mammal.html j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 335 http://jad.tums.ac.ir published online: december 25, 2018 in laboratory and wild mice that causes a mild haemolytic anaemia. eperythrozoon coccoides was first identified in laboratory mice in germany in 1928. wild mice are natural hosts and rats and rabbits have been experimentally infected. eperythrozoon coccoides was classified into the group of haemotropic mycoplasmas (haemoplasmas) (5-7). eperythrozoonosis is a zoonotic disease (transmissible from animals to humans). the first recognized human case of eperythrozoonosis was reported in 1986 worldwide (8). the disease may manifest with fever, hemolytic anemia, lethargy, jaundice, swollen lymph nodes of the neck, leucopenia, neutropenia, thrombocytopenia, splenomegaly and lymphadenopathy, acidosis (9-11). the most important way for the transmission of e. coccoides is blood-feeding arthropod vector such as adults and nymphs of the lice polypax spinulosa and p. serrate, this transmission is mechanical (12). we aimed to investigate the diversity and infection levels of parasitaemia in rodent population from meshkinshahr district. materials and methods study area meshkinshahr city located in ardebil province in the northwest of iran (38°23′56″n 47°40′55″e) (fig. 1), is situated at an altitude of 1830m above sea level and the average temperature of city is between 22.4 and 2.4 °c. the weather of the city and the district is moderate mountainous. it is limited from the north to the moghan city and from the west to the ahar city and from south to the sabalan high mountain and from the east to ardebil province and from the northeast to the republic of azerbaijan. sample collection and parasitological study two hundred four rodents (117 meriones persicus, 63 mus musculus, 24 cricetulus migraturius or grey hamsters) were trapped alive from meshkinshahr ardebil province, iran, between apr to sep 2014. trapped rodents were anaesthetized by placing cotton wool soaked with chloroform. blood was collected from the heart using a needle and syringe. thin and thick blood smears were prepared with a drop of blood. thin blood smear was fixed with methanol. slides were stained with giemsa stain and examined under light microscopy at 400x magnification for parasites screening and 1000x magnification under oil immersion for identification. analysis protozoa were microscopically measured and compared with key references (13, 14). ethical consideration this study was approved by the research ethical review committee of tehran university of medical sciences, tehran, iran (no: 22943). results two hundred four rodents include 3 genera (117 m. persicus, 63 m. musculus, 24 c. migraturius (grey hamsters) were collected from meshkinshahr, ardabil province. protozoa identified in this study were included of s. muris and e. coccoides, these parasites were observed in blood smear of 0.98% of rodents. spironucleus muris was observed in blood smear of one rodent (1.58% of m. musculus) (fig. 2). this organism was ovoid shape approximately 2–3×7–9µm and was bilaterally symmetrical with two nuclei, 6 anterior and 2 posterior flagella (13, 15). in addition, this organism existed in the feces of this rodent. moreover, e. coccoides was microscopically detected in one rodent (4.16% of hamsters). cocci-shaped objects in large numbers on the surface of red blood cells and red-purple color with size 0.5–3µm (14) were observed in blood smear of this rodent (fig. 3). https://maps.google.com/maps?hl=en-ir&gbv=2&um=1&ie=utf-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=meshgin+shahr,+ardabil&sa=x&ei=2agwvdfxlmg3sqgfnksica&ved=0cbuq8geoataa https://en.wikipedia.org/wiki/ardabil_province https://en.wikipedia.org/wiki/ardabil_province https://tools.wmflabs.org/geohack/geohack.php?pagename=meshginshahr&params=38_23_56_n_47_40_55_e_type:city%2880000%29_region:ir https://tools.wmflabs.org/geohack/geohack.php?pagename=meshginshahr&params=38_23_56_n_47_40_55_e_type:city%2880000%29_region:ir https://maps.google.com/maps?hl=en-ir&gbv=2&um=1&ie=utf-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=meshgin+shahr,+ardabil&sa=x&ei=2agwvdfxlmg3sqgfnksica&ved=0cbuq8geoataa https://maps.google.com/maps?hl=en-ir&gbv=2&um=1&ie=utf-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=meshgin+shahr,+ardabil&sa=x&ei=2agwvdfxlmg3sqgfnksica&ved=0cbuq8geoataa j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 336 http://jad.tums.ac.ir published online: december 25, 2018 fig. 1. map of ardebil province in iran (up). the geographical location of collected samples meshkinshahr in ardebil (down) fig. 2. spironucleus muris in a blood smear of mus musculus. giemsa stain (a and b). magnification, 1000x (original) j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 337 http://jad.tums.ac.ir published online: december 25, 2018 fig. 3. eperythrozoon coccoides in thick blood smear of hamester. giemsa stain. magnification, 1000x (original) discussion we aimed to determine the diversity of blood parasitaemia in rodent population from meshkinshahr ardabil. our findings showed presence of two kinds of protozoan parasite including s. muris and e. coccoides. previously, s. muris has been commonly seen in laboratory rats and mice (16). the current prevalence is unknown and probably is still high. in a study in brazil conducted on 344 mice and 111 laboratory rats, 46.2% colonies of mice and 85.7% of rat’s colonies were infected with s. muris (17). in another study, 15 mice colonies and 10 rat colonies were collected from 18 laboratories and showed that 90% of rats and 80% of mice were infected with s. muris (18). moreover, in iraq, 8% of rats were positive for s. muris infection (19). furthermore, a 6.3% of the infection was found in bandar abbas, iran (20). spironucleus muris were detected in fecal samples, indicating the presence of infection in the intestines. in addition, infection in immunocompetent and adult mice is usually subclinical. however, infection in athymic (nu/nu), young and immunocompromised mice characterized clinically by weight loss, enteritis, hunched posture, rough hair coat, hair lacks shine, lethargy, distention of the abdomen, depression, diarrhea and death (3). trophozoites can cause degeneration enterocytes and shortening of microvilli on the crypt epithelium and increase in crypt depth (2, 21). histologically, the formation of cysts was lead to dilation lieberkuhn glands with an inflammatory reaction in the lamina propria and the sloughing of the epithelium. the main pathological changes had happened in the duodenum, and then in the jejunum and ileum. in acute infections, the lumen of the small intestine has a large number of flagellated organisms that tend to invade the lamina propria of intestinal villi and filling glands. in chronic infection, dilated lymph glands were seen which were similar to the cyst and were filled with inflammatory cells, cellular debris, mucoid material and cystic organisms. such clinical symptoms has been previously explained (22, 23). associated pathological findings with the presence of parasite such as pyogranulomatous pneumonia, colitis, lymphadenitis and multifocal abdominal abscess has been previously reported in two immunosuppressed monkeys suffering from systematic spironucleus infection (24). s. muris is an opportunistic pathogen that feeds the intestinal bacteria. some stress or debilitating factors such as lack of the thymus is necessary for disease (13, 25). spironucleus muris increase the mortality of treated mice with cadmium, as well as is associated with changes in immune function of macrophages in mice and immune responses. young animals are more sensitive, older animals are not at risk of disease and spontaneously recover mice with history of disease may be resistant to re-infection (26-28). co-infection with b. microti, p. berghei, and p. yoelii, has been reported with reducing of the trophozoite production in s. muris (29). https://maps.google.com/maps?hl=en-ir&gbv=2&um=1&ie=utf-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=meshgin+shahr,+ardabil&sa=x&ei=2agwvdfxlmg3sqgfnksica&ved=0cbuq8geoataa https://maps.google.com/maps?hl=en-ir&gbv=2&um=1&ie=utf-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=meshgin+shahr,+ardabil&sa=x&ei=2agwvdfxlmg3sqgfnksica&ved=0cbuq8geoataa j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 338 http://jad.tums.ac.ir published online: december 25, 2018 furthermore, microvilli damage, loss of height of microvilli, increase in crypt depth, hyperplasia of crypt, abscesses in crypts and atrophy of villis have been reported in mice with s. muris (21, 28). this organism is likely to be systemic through the attack to the mucus of colon. large lesions in the lymph nodes are most likely to indicate the diffusion of parasites from the liver through lymphatic vessels as compared to blood circulation. however, the vessels diffusion cannot be disproved. after enterocytes destruction and necrosis, trophozoites of s. muris pass from the intestinal barrier and enter the blood stream in these areas (24, 30). in the present study, one out of 204 (0.49%) rodents was infected with s. muris in the intestinal contents and blood. it may be interpreted that the parasites had passed from the intestinal barrier and entered to the bloodstream. numerous parasites were observed in peripheral blood. in this study, e. coccoides was another organism detected and is a parasitic bacteria that attach to the surface of erythrocytes in mouse which can induce erythrocytic damage. this organism was previously classified as protozoa but in 2005, based on phylogenetic evidence and 16s rrna gene sequences was classified in haemotropic mycoplasmas and family of mycoplasmataceae (7). eperythrozoon coccoides is mechanically transmitted to humans by arthropods and it is important because human infection have been reported. infection is more common in farmers and veterinarians who are in close contact with domestic animals. congenital transmission has also been reported (31). infected people may be asymptomatic or symptoms, including fever, severe hemolytic anemia and jaundice, especially in infants. pregnant women and infants are more vulnerable than others and show more severe symptoms. in iran, there is no report of e. coccoides in animals and humans. in this study, one out of 204 rodents (0.49%) was infected with e. coccoides. a molecular and microscopic study of blood in china indicated that e. coccoides were causative agent of anemia in a person who has a long history of anemia (9). in a meta-analysis and systematic study, the rate of infection with eperythrozoon species in the population of chinese has been investigated. overall, 14951 out of 52433 cases were infected. according to the seasons and the geographical areas, the infection rate varied from 0% to 97.29% (32). conclusion the present study increases awareness about eperythrozoonosis in rodents and its potential transmission to domestic animals and even to human in rural districts in iran. moreover, information obtained from this study confirmed the attack of spironucleus on the mucus of colon and its systemic risk. acknowledgements the authors would like to thanks prof mj gharagozlou from faculty of veterinary medicine university of tehran. this study was financially supported by tehran university of medical sciences, iran. (grant no: 92-0227-22943). the authors declare that there is no conflict of interest. references 1. meerburg bg, singleton gr, kijlstra a (2009) rodent-borne diseases and their risks for public health. crit rev microbiol. 35(3): 221–270. 2. wanger je, doyle re, ronald nc, garrison rg, schmitz ja (1974) hexamitiasis in laboratory mice, hamsters, and rats. lab anim sci. 24: 349–354. 3. flatt re, halvorsen ja, kemp rl (1978) hexamitiasis in a laboratory mouse colony. lab anim sci. 28: 62–65. j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 339 http://jad.tums.ac.ir published online: december 25, 2018 4. letvin nl, o'connell mj, blake bj, king nw (1984) hematogenous hexamitiasis in a macaque monkey with an immunodeficiency syndrome. j infect dis. 149 (5): 828. 5. schilling v (1928) eperythrozoon coccoides, eine neue durch splenektomie aktivierbare dauerinfektion der weissen maus. mol med. 7(39): 1853–1855. 6. eliot cp, ford ww (1930) eperythrozoon coccoides in mice. am j epidemiol. 12 (3): 677–680. 7. neimark h, peters w, robinson bl, stewart lb (2005) phylogenetic analysis and description of eperythrozoon coccoides, proposal to transfer to the genus mycoplasma as mycoplasma coccoides comb. nov. and request for an opinion. int j syst evol microbiol. 55(3): 1385–1391. 8. puntaric v, borčić d, vukelic d, jeren t, burek v, wikerhauser t, richter b (1986) eperythrozoonosis in man. lancet. 328 (8511): 868–869. 9. yuan c, liang a, yu f, yang z, li z, zhu j, cui l, han y, hua x (2007) eperythrozoon infection identified in an unknown aetiology anaemia patient. ann microbiol. 57(3): 467–469. 10. tai x, yang d (1991) human eperythrozoonosis. chin inner mong med j. 11: 122. 11. henry s (1979) clinical observations on eperythrozoonosis. j am vet med assoc. 174(6): 601–603. 12. eliot cp (1936) the insect vector for the natural transmission of eperythrozoon coccoides in mice. science. 84: 397. 13. brugerolle g, kunstyr i, senarud j, friedhoff k (1980) fine structure of trophozoites and cysts of the pathogenic diplomonad spironucleus muris. z parasitenkd. 62: 47–61. 14. kreier jp, ristic m (1968) haemobartonellosis, eperythrozoonosis, grahamellosis and ehrlichiosis. in: weinman d and ristic m (eds) infectious blood diseases of man and animals, vol. 2. academic press, new york, pp. 387–472. 15. baker dg (2008) flynn's parasites of laboratory animals. john wiley and sons. blackwell publishing, australia. 16. casebolt d, lindsey j, cassell g (1988) prevalence rates of infectious agents among commercial breeding populations of rats and mice. lab anim sci. 38(3): 327–329. 17. bicalho ka, araújo ftm, rocha r, carvalho ods (2007) sanitary profile in mice and rat colonies in laboratory animal houses in minas gerais: i-endo and ectoparasites. arq bras med vet zoo. 59(6): 1478–1484. 18. gilioli r, andrade l, passos l, silva f, rodrigues d, guaraldo a (2000) parasite survey in mouse and rat colonies of brazilian laboratory animal houses kept under differents sanitary barrier conditions. arq bras med vet zoo. 52(1): 33–37. 19. rahemo z, ahmed r, koyee q (2012) intestinal parasites of experimental rodents with testing the efficacy of diagnostic methods. int res j of pharmaceuticals. 2(3): 77–81. 20. hamedi y, heidari m, soleimani-ahmadi m (2003) intestinal and blood parasites of brown rats in bandar abbas. hormozgan med j. 7(3): 123–127. 21. whitehouse a, france mp, pope se, lloyd je, ratcliffe rc (1993) spironucleus muris in laboratory mice. aust vet j. 70(5): 193. 22. lussier g, loew f (1970) an outbreak of hexamitiasis in laboratory mice. can j comp med. 34(4): 350–353. 23. meshorer a (1969) hexamitiasis in laboratory mice. lab anim care. 19: 33–37. 24. bailey c, kramer j, mejia a, mackey j, mansfield k, miller a (2010) systemic spironucleosis in 2 immunodeficient rhesus macaques (macaca mulatta). vet pathol. 47(3): 488–494. j arthropod-borne dis, december 2018, 12(4): 334–340 s heidari et al.: spironucleus muris and … 340 http://jad.tums.ac.ir published online: december 25, 2018 25. boorman g, lina p, zurcher c, nieuwerkerk h (1973) hexamita and giardia as a cause of mortality in congenitally thymus-less (nude) mice. clin exp immunol. 15: 623–627. 26. sebesteny a (1969) pathogenicity of intestinal flagellates in mice. lab anim. 3(1): 71–77. 27. kunstyr i, ammerpohl e, meyer b (1977) experimental spironucleosis (hexamitiasis) in the nude mouse as a model for immunologic and pharmacologic studies. lab animl sci. 24: 782–788. 28. brett sj, cox f (1982) immunological aspects of giardia muris and spironucleus muris infections in inbred and outbred strains of laboratory mice: a comparative study. parasitology. 85(1): 85–99. 29. brett sj, cox f (1982) interactions between the intestinal flagellates giardia muris and spironucleus muris and the blood parasites babesia microti, plasmodium yoelii and plasmodium berghei in mice. parasitology. 85(1): 101–110. 30. hofmeister k (1993) spironukleose des feldhamsters. lichtund elektronenmikroskopische. [phd dissertation]. tierärztliche hochschule hannover. 31. yang d, tai x, qiu y, yun s (2000) prevalence of eperythrozoon spp. infection and congenital eperythrozoonosis in humans in inner mongolia, china. epidemiol infect. 125(2): 421–426. 32. huang ds, guan p, wu w, shen tf, liu hl, cao s, zhou h (2012) infection rate of eperythrozoon spp. in chinese population: a systematic review and metaanalysis since the first chinese case reported in 1991. bmc infect dis. 12: 171. j arthropod-borne dis, december 2018, 12(4): 426–431 m fatemi et al.: designing and introducing … 426 http://jad.tums.ac.ir published online: december 25, 2018 short communication designing and introducing a new artificial feeding apparatus for sand fly rearing mahboubeh fatemi 1, zahra saeidi 1, parviz noruzian 2, *amir ahmad akhavan 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2tajhiz gostar omid iranian co, tehran, iran (received 26 june 2018; accepted 13 july 2018) abstract background: due to strict ethical rules, the risk of accidental disease transmission and the most importantly, inconvenience regarding using of live animals, artificial feeding apparatus has been developed for colonization of haematophagous insects. rearing of sandfly is more difficult than other haematophagous insects. methods: in the current study, a new apparatus for membrane feeding of phlebotomus papatasi was designed, made and compared with available apparatus in sand fly insectary, tehran university of medical sciences, tehran, iran, in 2014. results: in comparison to other apparatus designed for artificial feeding of other arthropods, our designed apparatus had the highest performance which after up to 1h, the majority of sand flies landed and took blood and among tested membranes, chicken skin was proved the most efficient membrane. conclusion: sand fly artificial feeding apparatus can be used at least for rearing of ph. papatasi. keywords: phlebotomus papatasi, artificial feeding apparatus, blood-feeding insects, sandfly introduction insect colonization has been intensified by increasing of interest in understanding of their life cycle, physiology, anatomy, genetics, and study on arthropod-borne diseases, insecticide and repellency tests to control the diseases (1). blood-feeding is one of the most critical and difficult steps in rearing process of haematophagous insects. animal maintenance for blood feeding of the colony, not only is costly and time-consuming but also requires ethical considerations. due to these restrictions, risk of accidental disease transmission and inconvenience of using live animals, artificial feeding apparatus has been developed. artificial feeding is a useful technique in nutritional and behavioral studies on insects such as feeding stimulants and interaction between parasite and invertebrate host (2). moreover, one of the most advantages of this technique in the parasite and vector interaction studies is capability of adding a certain concentration of parasite to feeding mixture (3). precise control over the test conditions is considered as another advantage of this method compared to feeding on live hosts. based on advantages mentioned above, membrane feeding is preferred to feeding on animals. the earliest attempt at feeding tsetse flies through rat skin was made by rodhain et al. (4). subsequently, this technique has been used successfully to feed various haematophagous insects including vectors and their transmittable pathogens to study developmental stages and transmission cycle of micro-organism inside their body (5-15). establishment and maintenance of sandflies are far more difficult than other haematophagous insects (16-17). the aim of the current study was to design and introduce a new apparatus for artificial feeding of phlebotomus papatasi in the insectary condition. *corresponding author: dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir j arthropod-borne dis, december 2018, 12(4): 426–431 m fatemi et al.: designing and introducing … 427 http://jad.tums.ac.ir published online: december 25, 2018 materials and methods in order to membrane feeding of ph. papatasi four artificial feeding apparatus including: tick artificial feeding apparatus, mosquito artificial feeding apparatus based on the idea of cosgrove et al. (18), modified cosgrove et al. apparatus and a new artificial feeding apparatus which was designed and made in this study in sand fly insectary, tehran university of medical sciences, tehran, iran, in 2014, (the patent number: 86836, islamic republic of iran) were compared. tick artificial feeding apparatus has a chamber to hold the blood, 5.5cm in diameter and 12.5cm deep. the minimum required volume of blood is 5ml. it was connected to a water bath that circulates warm water inside the apparatus. a glass tube was oriented vertically in the chamber that is 4.5×7cm. membranes were stretched over one end of the glass tube which is in contact with blood mixture. fine mesh gauze is fitted on another end of the apparatus to transfer sand flies through it (fig. 1). the second apparatus consisted of an 8×7 cm aluminum chamber with a volume of 2.8 ml heated by electrical elements. a thermostat has been connected to adjust the desired temperature (fig. 2). chamber in modified cosgrove et al. apparatus has a volume of 9ml and the temperature is displayed on a digital screen that is adjustable with buttons (fig. 3). the idea of the design of sand flies artificial feeding apparatus was inspired by the two previously available equipment (19, 20). the apparatus contains a stainless steel container in a volume of 6l and a steel plate embedded on the top of the container. the steel plate is used as a container for a water bath if needed. to assure a constant temperature in the blood feeders, a pair of water outlet and inlet pipes was designed for each blood feeder of the apparatus. in order to offer blood supply for the insects, 7.5×2.5cm glass blood-feeders were designed (fig. 4). a cavity at the top was designed to hold the blood mixture (up to 2ml) and a pair of water outlet and inlet pipes for circulating warm water inside the blood-feeder. in each experiment, up to four blood-feeders can be connected to apparatus at the same or different angles, simultaneously. all the pipes were considered 120cm in length in order to make the same condition. a temperature sensor was placed inside the outlet, and a built-in display installed to monitor experiment process. the temperature and time were adjustable with buttons on the screen. the alarm sound could be heard after finishing the time given to apparatus (fig. 5). to mimic natural condition of blood feeding, the membrane temperature was adjusted to 35±1 °c. the bottom of feeding containers (5.5cm height, 4cm diameter) was moistened with distilled water before the introduction of the sand flies and placed in close contact with the membrane by the clamp. therefore, the membrane is accessible to the sand flies for blood-feeding. in four mentioned artificial feeding apparatus, parafilm, nescofilm and skin of threeweek-old chicken as membrane were tested and compared. membranes were stretched over the blood mixtures in one end of each feeder and fastened with elastic band. in each case, a new membrane and fresh blood were used and ph. papatasi were starved for 2h before the blood meal. moreover, three sources of defibrinated or heparinized blood of human, balb/c mice and rhombomys opimus were used to achieve the highest blood feeding. all experiments were carried out under the same condition at 26±2 °c temperature and 80 % relative humidity. animal protocol was approved by the ethics committee of tehran university of medical sciences, tehran, iran. results in tick and cosgrove's artificial feeding apparatus, no sand fly landed to blood chamber. j arthropod-borne dis, december 2018, 12(4): 426–431 m fatemi et al.: designing and introducing … 428 http://jad.tums.ac.ir published online: december 25, 2018 in modified cosgrove's artificial feeding apparatus, the sand flies landed on the membranes but no ph. papatasi took blood. in three above-mentioned apparatuses, because of presence of only one blood feeder, each blood sample was tested separately. in sand fly artificial feeding apparatus made in the current study, after up to 1h the majority of sand flies landed and took blood. moreover, three blood sources were checked simultaneously. among the tested membranes, the chicken skin was the best and the heparinized blood had the most efficient because it was not clotted even after 4h. no preference among the tested blood was observed. fig. 1. tick artificial feeding apparatus fig. 2. mosquito artificial feeding apparatus of based on the idea of cosgrove et al. fig. 3. modified cosgrove’s apparatus fig. 4. glass blood feeder fig. 5. phlebotomus papatasi artificial feeding apparatus j arthropod-borne dis, december 2018, 12(4): 426–431 m fatemi et al.: designing and introducing … 429 http://jad.tums.ac.ir published online: december 25, 2018 discussion feeding through membrane has been utilized for various hematophagous arthropods to purpose of mass rearing of insects or to artificially infect vectors with parasites (3, 5, 18, 20-22). there were disadvantages in the available artificial feeding equipment such as the temperature which could only be measured in the water tank with no temperature sensor in the vicinity of membrane, and also the time that was not adjustable. the current new designed feeding apparatus does not have the above-mentioned weakness points. perhaps the lack of circulation of warm water and the moisture, that is critical for sand fly feeding, were responsible for unattractiveness of blood chamber for ph. papatasi in modified and cosgrove's artificial feeding apparatus. each blood-sucking arthropod has a preference host to take blood, determining the most appropriate blood lead to the highest blood feeding that has direct effect on egg production. because of capability of supporting several blood feeder by this apparatus various blood sources can be tested simultaneously to achieve the best result. results of artificial-feeding of ph. papatasi showed no significant differences in bloodfeeding proportion of sand flies between human, balb/c mice, and rh. opimus. in agreement with our results, there was no preference between human blood and blood from different types of host (cow, dog, guineapig, hamster, horse, pig, rabbit) in ph. papatasi blood feeding (23). angle of bloodfeeder can affect on percent of lutzomyia shannoni blood feeding (20). therefore, ability to change the angle on this device has been provided. besides, different kinds of membrane including natural or synthetic such as guinea pig mesentery and large bowel, batwing, human peritoneum, human kidney capsule, ox liver capsule, baudruche, chick skin, parafilm, chitosan, dialyzing bag and so on have been tested to stimulate insects to taking a blood-meal through membrane (8, 19, 24-26). however, different studies, same to our study, have shown that chicken skin is more effective than other membranes (25, 27, 22, 28). conclusion new designed artificial feeding apparatus could be used for rearing of ph. papatasi instead of live animals. acknowledgements this study was supported financially by research deputy of tehran university of medical sciences (tums) and the center for research and training in skin diseases and leprosy, tehran, iran, (project no. 93-03-27-26140). we are grateful to staff of leishmaniasis unit, department of medical entomology and vector control, school of public health, tums, for their kind assistance. the authors declare that there is no conflict of interests. references 1. endris rg, perkins pv, young dg, johnson rn (1982) techniques for laboratory rearing of sand flies (diptera: psychodidae). mosq news. 42: 400–407. 2. allen jr, west as (1962) collection of oral secretion from mosquitoes. mosq news. 22: 157–159. 3. adler s, theodor o (1927) the behaviour of cultures of leishmania tropica, l. infantum and l. braziliensis in the sandfly phlebotomus papatasi. nature. 119: 48– 49. 4. rodhain j, pons c, vandenbranden j, bequaert j (1912) contribution au mecanisme de la transmission des trypanosomes par les glussinen. arch schiffus trop hyg. 16: 732–739. j arthropod-borne dis, december 2018, 12(4): 426–431 m fatemi et al.: designing and introducing … 430 http://jad.tums.ac.ir published online: december 25, 2018 5. galun r (1967) feeding stimuli and artificial feeding. bull world health organ. 36: 590–593. 6. langley pa, maly h (1969) membrane feeding technique for tsetse flies (glossina spp.). nature. 221: 855–856. 7. lauer dm, sonenshine de (1978) adaptions of membrane feeding techniques for feeding the squirrel flea, orchopeas howardi, and the squirrel louse, neohaematopinus sciuropteri, with notes on the feeding of the human body louse, pediculus humanus var. corporis. j med entomol. 14: 595–596. 8. owens l (1981) a method for membrane feeding blood to culicoides. aust vet j. 57: 396–397. 9. tesh rb, modi gb (1984) a simple method for experimental infection of phlebotomine sand flies with leishmania. am j trop med hyg. 33: 41–46. 10. bernardo mj, cupp ew (1986) rearing black flies (diptera: simuliidae) in the laboratory: mass-scale in vitro membrane feeding and its application to collection of saliva and parasitological and repellent studies. j med entomol. 23: 666–679. 11. hagen he, grunewald j (1990) routine blood-feeding of aedes aegypti via a new membrane. j am mosq control assoc. 6: 535–536. 12. burg jg, knapp fw, silapanuntakul s (1993) feeding haematobia irritans (diptera: muscidae) adults through a nylonreinforced silicone membrane. j med entomol. 30: 462–466. 13. blackwell a, mellor ps, mordue w (1994) laboratory feeding of culicoides impunctatus (diptera: ceratopogonidae) through natural and artificial membranes. j med entomol. 31: 302–305. 14. samish m, kozlowska a, maramorosch k (1995) factors affecting membrane feeding of anopheles stephensi. j am mosq control assoc. 11: 408–415. 15. ghosh kn, mukhopadhyay j (1998) the effect of anti-sand fly saliva antibodies on phlebotomus argentipes and leishmania donovani. int j parasitol. 28: 275– 281. 16. killick-kendrick r (1978) recent advances and outstanding problems in the biology of phlebotomine sandflies. a review. acta trop. 35: 297–313. 17. killick-kendrick m, killick-kendrick r (1991) the initial establishment of sand fly colonies. parassitologia. 33: 315–320. 18. cosgrove jb, wood rj, petric d, evans dt, abbott rh (1994) a convenient mosquito membrane feeding system. j am mosq control assoc. 10: 434–436. 19. ward rd, lainson r, shaw jj (1978) some method for membrane feeding of laboratory reared, neotropical sandflies (diptera: psychodidae). ann trop med parasitol. 72(3): 269–276. 20. mann rs, kaufman pe (2010) colonization of lutzomyia shannoni (diptera: psychodidae) utilizing an artificial blood feeding technique. j vector ecol. 35(2): 286–294. 21. adler s, ber m (1941) the transmission of leishmania tropica by the bite of phlebotomus papatasi. indian j med res. 29: 803–809. 22. ghosh kn (1994) a modified artificial membrane feeding method for the study of the transmission dynamics of leishmaniasis. trans roy soc trop med hyg. 88 (4): 488–489. 23. harre jg, dorsey km, armstrong kl, burge jr, kinnmon ke (2001) comparative fecundity and survival rates of phlebotomus papatasi sand flies membrane fed on blood from eight mammal species. med vet entomol. 15: 189–196. 24. hertig m, mcconnell e (1963) experimental infection of panamanian phlebotomus sandflies with leishmania. exp parasitol. 14: 92–106. 25. behin r (1967) artificial feeding apparatus for mosquitoes. mosq news. 27(1): 7–90. 26. tabart j, colin m, carayon j, tene n, payre j arthropod-borne dis, december 2018, 12(4): 426–431 m fatemi et al.: designing and introducing … 431 http://jad.tums.ac.ir published online: december 25, 2018 b, vetillard a (2013) artificial feeding of varroa destructor through a chitosan membrane: a tool for studying the hostmicroparasite relationship. exp appl acarol. 61: 107–118. 27. gerberg ej, kutz fw (1971) a large-scale artificial feeding technique for infecting mosquitoes and its application to screening antimalarial chemicals. j med entomol. 8(5): 610–612. 28. probst rj, wellde bt, lawyer pg, stiteler js, rowton ed (2001) rhesus monkey model for leishmania major transmitted by phlebotomus papatasi sandfly bites. med vet entomol. 15: 12–21. j arthropod-borne dis, september 2020, 14(3): 293–301 s sukumaran and r maheswaran: larvicidal activity of … 293 http://jad.tums.ac.ir published online: september 30, 2020 short communication larvicidal activity of elytraria acaulis against culex quinquefasciatus and aedes aegypti (diptera: culicidae) soorya sukumaran, *rajan maheswaran department of zoology, entomology laboratory, school of life sciences, periyar university, salem, tamil nadu, india *corresponding author: dr rajan maheswaran, e-mail: mahes1380@gmail.com (received 05 feb 2016; accepted 18 jul 2020) abstract background: mosquitoes are blood sucking arthropods and serve as vectors of many diseases causing serious health problems to human beings. culex quinquefasciatus and aedes aegypti were responsible for filariasis and dengue. synthetic pesticides were effective against mosquitoes as well as main sources of environmental pollution and most of them are immunosuppressant. botanicals were widely used as insecticides, growth disruptors, repellents, etc. the aim of this research was to determine larvicidal properties of powdered leaf, elytraria acaulis against late third or early fourth instar larvae of cx. quinquefasciatus and ae. aegypti. methods: larvae of cx. quinquefasciatus and ae. aegypti were tested at various concentrations of 100, 120, 140, 160, 180 and 200mg/100ml and mortality was recorded after 24h. the lc50 values of the e. acaulis leaf powder were calculated by probit analysis. results: the plant powder exhibited strong larvicidal activity against cx. quinquefasciatus with lc50 value of 116.07mg/100ml against ae. aegypti 124.25mg/100ml respectively. the result indicated that the plant powder of e. acaulis showed potential larvicidal activity against cx. quinquefasciatus and ae. aegypti. conclusion: the overall findings of the present investigation suggested that the e. acaulis highly effective against cx. quinquefasciatus and ae. aegypti larvae. elytraria acaulis may be used as an alternative to synthetic chemical pesticides for control of vectors to reduce vector borne diseases and did not harm to total environment. keywords: elytraria acaulis; larvicidal activity; culex quinquefasciatus; aedes aegypti introduction man suffers extensively due to the nuisance of vector mosquito population in public health manner. mosquitoes directly transmit diseases such as filarial, malaria and dengue fever. mosquitoes are blood sucking insects and serve as vectors for spreading human diseases and therefore, they continue to pose a serious health problem throughout the world. these are not only the most important vector for the transmission of diseases (1) but also an important pest to humans, causing allergic responses that include local skin reaction and systemic reaction such angio-edema and urticaria (2). culex quinquefasciatus, a vector of lymphatic filariasis, is widely distributed in tropical zones with around 120 million people infected worldwide and 44 million people having common chronic mani festation (3). despite its debilitating effects, lymphatic filariasis is given a very low control priority (4). in most of its range the females intensely anthropophilic, fed actively only at night and it causes nuisance (5) and are vectors of japanese encephalitis, west nile virus st. louis encephalitis and avian malaria. aedes aegypti, the principal vector of dengue, chikungunya, zika and yellow fever viruses, is an anthropophilic species adapted to urban environments, particularly to housing (6). dengue hemorrhagic fever (dhf) and chikungunya are the major mosquito-borne diseases in india. the first dengue hemorrhagic fever was reported in thailand and philippines in 1950s. dengue infections are reported throughout the world including india, where the first dengue outbreak was re copyright © 2020 iranian scientific society of biology & control of diseases vectors, and tehran university of medical sciences. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/by-nc/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:mahes1380@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 293–301 s sukumaran and r maheswaran: larvicidal activity of … 294 http://jad.tums.ac.ir published online: september 30, 2020 ported in delhi in 1988 (7). now the dengue infection has been reported from all over the country (8-13) with major outbreaks reported from tamil nadu (14-17). chikungunya which is endemic to south asia, the pacific island area, africa, and the americas and has infected millions of people mainly in developing countries (18). the lack of a commercial vaccine and the failure of vector control strategies to limit the expansion of chikungunya have prompted the need for further options to prevent the spread of this disease. nowadays synthetic insecticides are at the fore front of mosquito-controlling agents. the continual usage of the synthetic chemical insecticides possess various environmental hazards such as development of resistance in vectors mosquitoes to these chemicals, disruption of natural biological control systems in mosquito populations (19). hence, the necessity of plant derived insecticides especially target specific, eco-friendly, readily biodegradable and cost-effective (20). in general, plant essential oil has been recognized as important natural resources of insecticides (21). many researchers have reported the control of mosquito larvae using the plant extract and the essential oils obtained from the different parts of the plants (22-24). natural insecticides meet the needs for alternatives to controlling resistant populations of different species of mosquitoes. they can affect different stages of development through a variety of mechanisms. in this study, we have chosen elytraria acaulis belongs to the family acanthaceae is a small shrub, which grows in shady dry places and it is commonly known as asian scalystem. it is a stem less perennial herb with one to several unbranched flowering stems; up to 30cm. stems are covered with overlapping bracts. leaves occur in a rosette at the base. they are obovate, 4–10 centimeter long. flowers are white, lower lip and lateral lobes spreading, 2-lobed. this plant is frequently found on rocky or sandy soils. the whole shrub is used for medicinal purposes (25). the decoction of e. acaulis leaves prescribed for fever, venereal diseases and root is used in mammary tumor, abscesses, pneumonia, antidiabetic effects, antibacterial activity, treating wounds infected with worms and infantile diarrhea as well as traditional medicine for long days (26-27). the sub-acute toxicity of methanolic extract of e. acaulis was tested against female wistar rats with the concentrations of 50 to 2000mg/kg by oral administration (28). they observed that no significant alteration on any of the biological parameters. the present study was aimed to investigate the larvicidal properties of powdered leaf of e. acaulis against late third or early fourth instar larvae of cx. quinquefasciatus and ae. aegypti. materials and methods selected medicinal plant fresh and matured leaves of e. acaulis was used for the research work. the selected plant was collected from kattukollai, kanchipuram district, tamil nadu based on their abundance, availability, medicinal and insecticidal properties. the plant specimen was identified by dr p paramasivam, department of botany, pachaiyappa’s college for men, kanchipuram, india. the specimen plant was preserved at herbarium of department of botany, pachaiyappa’s college for men, kanchipuram, india for further reference. the collected plant material were washed with tap water to remove all the unwanted impurities and shade dried at laboratory temperature (27±2 °c) and macerated with electric blender and stored at 4 °c for larvicidal bioassay. maintenance of mosquito larvae culex quinquefasciatus and ae. aegypti mosquito larvae were collected from stagnant water in and around of kattukollai, kanchipuram district, tamil nadu, india. all the larvae were kept in plastic trays containing tap water and were maintained in the laboratory. the larvae were fed with dog biscuits and yeast powder in the 3:1 ratio. all the experiments were carried out at 27±2 °c and 75–85% relative huhttp://jad.tums.ac.ir published online: september 30, 2020 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 293–301 s sukumaran and r maheswaran: larvicidal activity of … 295 http://jad.tums.ac.ir published online: september 30, 2020 midity under a photoperiod of 14:10h (light/ dark) cycles. they were maintained until the larvicidal bioassay. larvicidal activity larvicidal activity was evaluated by using the standard method (29). twenty five individuals of late third or early fourth instar larvae of cx. quinquefasciatus and ae. aegypti were released in a 250ml glass beaker containing 100 ml of dechlorinated tap water mixed with desired e. acaulis plant powder at different concentrations (mg), an equal number of controls were set up simultaneously using tap water. five replicates of each concentration were run at a time. the experimental concentrations were 100, 120, 140, 160, 180 and 200mg/100ml respectively. negative control (water) was run simultaneously. mortality and survival rate were recorded after 24 hours. based on the who protocol no food was offered to avoid the difference in mortality. the moribund and dead larvae in five replicates were combined and expressed as a percentage of larval mortality for each concentration. dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical region. moribund larvae were those incapables of rising to the surface (within reasonable period of time) or the water was disturbed the characteristic diving reaction was not seen. the lc50 value was calculated by epa probit analysis software. results in the present investigation the toxic effect of powdered leaf of e. acaulis tested at six different concentrations such as 200, 180, 160, 140, 120 and 100mg/100ml to evaluate the larvicidal activity against the larvae of cx. quinquefasciatus and ae. aegypti. besides, the control set up also compared with different concentrations of plant powder. the highest concentration (200mg/100ml) of powdered leaf of e. acaulis showed 100% mortality against the larvae of cx. quinquefasciatus and ae. aegypti. however, 180 and 160 and 140mg/100ml of e. acaulis leaf powder inflicted moderate larval mortality. the least concentrations of 120 and 100mg/100ml exhibited least larvicidal activity. in comparison with the control, all the concentrations of e. acaulis leaf powder contributed potential larvicidal activity. the lc50 and lc90 value of e. acaulis leaf powder exhibited 116.07 and 190.38mg/100ml against cx. quinquefasciatus and 124.25 and 198.21 mg/100ml against ae. aegypti (table 1). no larval mortality of observed in control. after 96hrs 100% mortality was observed in all the tested concentrations e. acaulis. symptomatological observations were carried out through the exposure period at laboratory temperature among the two species of mosquitoes revealed that immediately after exposure to e. acaulis. all larvae were active and exhibited a normal appearance with the siphon pointed up and head hung down. after 5 minutes of treatment, some of the larvae became restless and frequently sank down and floated up quickly at 200mg/100mlconcentration. at 30th minute, the restlessness persisted; tremor and convulsion at the bottom of the container were observed approximately in 1 to 2 larvae. similar evidences of restlessness, tremors, and convulsions followed by paralysis were clearly seen after an hour approximately in 4 to 5 larvae. at 12h, approximately 1 to 2 moribund and dead larvae were found. after 24h of treatment, approximately one-third of the larvae was paralyzed and sank to the bottom of the bowl. more and more larvae exhibited toxic symptoms during 12h. subsequently, all of them died within 24 h in the 200mg/100ml treatment. the powdered leaf of e. acaulis caused rapid mortality, suggesting larvicidal property. the symptoms observed in treated larvae were similar to those caused by nerve poisons, such as excitation, convulsion, paralysis and death. dead larvae were observed under the light microscope after 24h of exposure, where the body attained a dark brown color; length of the larvae was shrunk. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 293–301 s sukumaran and r maheswaran: larvicidal activity of … 296 http://jad.tums.ac.ir published online: september 30, 2020 table 1. larvicidal activity of elytraria acaulis against the larvae of culex quinquefasciatus and aedes aegypti culex quinquefasciatus replication concentration (mg/100ml)/ number of dead out of 25 tested lc50 95% confidence limit lc90 95% confidence limit control 200mg 180mg 160mg 140mg 120mg 100mg lcl ucl lcl ucl 1 0 25 20 19 15 13 10 116.07 90.05 131.08 190.38 164.26 279.40 2 0 25 20 20 15 13 9 3 0 25 21 20 16 13 9 4 0 25 20 20 18 14 11 5 0 25 20 19 16 12 11 total dead 0/125 125/125 101/125 98/125 80/125 65/125 50/125 s.d. 0 0 0.44 0.54 1.22 0.70 1 % of mortality 0 100 80.8 78.4 64 52 40 aedes aegypti 1 0 25 18 19 16 12 8 124.25 102.97 138.73 198.21 171.24 284.61 2 0 25 17 18 15 11 7 3 0 25 20 18 14 14 8 4 0 24 22 17 15 12 8 5 0 25 20 16 16 12 6 total dead 0/125 124/125 97/125 88/125 76/125 61/125 37/125 s.d. 0 0.44 1.94 1.14 0.83 1.09 0.89 % of mortality 0 99.2 77.6 70.4 60.8 48.8 29.6 values are mean ±sd of five replicates. in each concentration 25 larvae were used http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 293–301 s sukumaran and r maheswaran: larvicidal activity of … 297 http://jad.tums.ac.ir published online: september 30, 2020 discussions world health organization has estimated globally the contribution of commercial pesticides to health (30). naturally occurring pesticides thus appear to have a prominent role in the development of future safety of the environment and public health (31). there are different methods for controlling mosquitoes. the plant crude formulation was responsible for larvicidal activity. the pool of plants processing insecticidal substance is enormous. the use of plant essential extract for the pest and diseases management has recently been reversed. the preliminary screening is a good means of evaluating the potential larvicidal activity of plants popularly used for this purpose. in the present investigation e. acaulis inflicted potential larvicidal activity with the lc50 value of 116.07 mg/100ml against cx. quinquefasciatus and 124.25mg/100ml against ae. aegypti. like ways, the sustained toxicity test of some medicinal plants such as nerium oleander, calotropis procera and ricinus communis powders against anopheles arabiensis, cx. quinquefasciatus (32). they reported that the after 6 days 100% mortality was observed in an. arabiensis, whereas, cx. quinquefasciatus 60% mortality was observed. the larvicidal activity due to the presence of alkaloids, flavonoids, protein, amino acid, glycosides, carbohydrates, phenol, steroids, saponins and tannins from e. acaulis (33). the results were coincides with earlier findings in which the leaf powder of croton sparsiflorus had lc50 value of 122.73mg/100ml and lc90 value of 180.04mg/100ml followed by bauhinia variegata with lc50 value of 142.47mg/100ml and lc90 value of 210.16mg/ 100ml, respectively (34). in another study the solvent extracts of e. acaulis showed moderate effect on cx. quinquefasciatus and ae. aegypti (35). similarly, 1mg/ml of ethanolic extracts of the leaves of lantana camara exhibited 84% larval mortality while treated with methanolic extract caused 48% mortality on fourth instar larvae of ae. aegypti (36). the ethanolic extract from leaves of cassia occidentalis caused larval mortality against malarial vector mosquito an. stephensi at a dose equivalent to lc50 of 70.56% for fourth instar larvae (37). ethanolic extract from bulbs of allium sativum inflicted remarkable insecticidal activity against larvae of aedes albopictus with lc50 value of 4.48g/l (38). the toxic effect of ricinus communis crude extract was tested against immatures of cx. quinquefasciatus and an. arabiensis (39). they recorded lc50 values as 403.65, 445.66, and 498.88ppm against second, third, and fourth instar larvae of an. arabiensis and 1091.44, 1364.58, and 1445.44 ppm against second, third, and fourth instar larvae of cx. quinquefasciatus, respectively several plants were evaluated against main malaria vector, an. stephensi, and cx. pipiens including mentha spicata, cymbopogon olivieri, azadirachta indica, melia azedarach, lagetes minuta, calotropis procera, eucalyptus camaldulensis, cupressus arizonica, thymus vulgaris, lawsonia inermis, cedrus deodara, cionura erecta, bunium persicum, carum carvi, artemisia dracunculus, rosmarinus officinalis. mentha spicata and eucalyptus camaldulensis, had the lowest and highest lc50 respectively (40-55). our results clearly indicated the e. acaulis highly effective against cx. quinquefasciatus and ae. aegypti. powdered leaves of e. acaulis may be a good source to develop newer mosquitocidal biopesticide. conclusion the overall findings of the present investigation suggested that the e. acaulis highly effective against cx. quinquefasciatus and ae. aegypti larvae. however solvent extractions are time consuming and costlier technique. elytraria acaulis may be used as an alternative for synthetic chemical pesticides to control vechttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 293–301 s sukumaran and r maheswaran: larvicidal activity of … 298 http://jad.tums.ac.ir published online: september 30, 2020 tors mosquitoes and reduce vector borne diseases. according to our knowledge it seems not harmful to the natural environment and it needs more study to understand the level of toxicity. acknowledgements the authors are thankful to periyar university, salem, tamil nadu, india for the financial support. all authors declare that there is no conflict of interest. references 1. james aa (1992) mosquito molecular genetics: the hands that feed bite back. science. 257(5066): 37–38. 2. peng z, yang j, wang h, simons fer (1999) production and characterization of monoclonal antibodies to two new mosquito aedes aegypti salivary proteins. 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421–425 pg de mendonça: impact of flag … 421 http://jad.tums.ac.ir published online: december 25, 2018 original article impact of flag texture on tick sampling efficiency philippe gil de mendonça institute of comparative tropical medicine and parasitology, ludwig maximilian university, munich, germany (received 17 jan 2017; accepted 13 nov 2018) abstract background: there is a strong interest in tick-borne diseases worldwide due to their negative impact on both human and animal health. epidemiological studies of tick-borne diseases depend on reliable data on tick population dynamics and activity patterns. such data are essentially based on tick sampling in the field. this study aimed to evaluate the impact of cloth type on the efficiency of field sampling by the flagging technique. methods: the impact of cloth type on the efficiency of field sampling by the flagging technique was investigated by comparing tick sampling yields of two different fabrics, the munich type (muc) vs. the oxford type (ox), based on 30 pairs of transect lines. data analysis included classical statistics and computer modelling. results: the muc flag yielded nearly five times more larval ticks than the ox flag, whereas the differences in yields for nymphs and adult ticks were not statistically significant based on classical statistics. conclusion: the flag made of muc type fabric, thanks to its tight and relatively flat texture, facilitates detection and collection of ticks from its surface. the ox flag, due to its loose texture, is unsuitable for the quantitative sampling of larval ixodes ricinus. keywords: ixodes ricinus, larval ticks, nymphal ticks, flagging introduction there is an increasing interest in tick-borne diseases worldwide, as it is believed several of these diseases are emerging and spreading (1, 2). consequently, various research projects were launched to investigate ticks and tickborne diseases at regional, national, and international levels. the eu-funded eden project focused specifically on investigating emerging zoonoses (3). eden actually stands for 'emerging diseases in a changing european environment'. within this context, ticks were routinely sampled and screened for a palette of zoonotic pathogens throughout most of europe. to ensure comparability of results, standardized protocols were issued to all participants. a present from prof se randolph (oxford university) provided the opportunity to compare the standard flannel flag routinely used by the german eden team with a totally different type of material, used in other tick studies abroad. preliminary testing revealed that users experienced difficulties in retrieving ticks embedded in the fibres of this fleecy cloth (fig. 1). this raised the question of the potential impact of such a different cloth type on tick sampling efficiency. indeed cloth consistency is known to be a factor influencing flagging efficiency (4), as are flagging distance and pace. this last point introduces an additional factor, the human factor, which includes many components and plays an important part in sampling efficiency. the test campaign described here therefore aimed to evaluate the impact of cloth type on flagging efficiency by comparing the standard 'munich cloth' with the 'oxford cloth' while limiting the impact of the human factor by removing inter-individual variability. materials and methods questing ticks were sampled in the morning *corresponding author: dr philippe gil de mendonça, e-mail: pgm@cantab.net j arthropod-borne dis, december 2018, 12(4): 421–425 pg de mendonça: impact of flag … 422 http://jad.tums.ac.ir published online: december 25, 2018 hours by dragging a 1m2 flag over soil and veg etation in mixed woodlands including both coniferous and broad-leaved trees with moderate herb and moss layers. the flag was made of a 1m2 removable cloth strung over the terminal section of a long bamboo cane. two cloth types were available. the munich type (muc) was made of a thin and simple white flannel fabric, whereas the oxford type (ox) was made of a heavier and thicker fabric with a complex structure (fig. 2). two transect lines, close to each other, were surveyed on each sampling session. one transect line was surveyed using the muc flag, while the other was surveyed using the ox flag. in order to mitigate the effect of timing on sampling efficiency, 15 sampling sessions started with the muc transect line and ended with the ox transect line, while the other 15 sampling sessions followed the reverse order. sampling order was based on a computer-generated table of random numbers containing 15 odd numbers and 15 even numbers. overall, 30 pairs of transect lines were surveyed by one and the same person at four field sites in the bavarian districts of amberg-sulzbach (site am1), landshut (sites la1 and la2) and passau (site pa1) in 2007–2008. transect lines were divided into 25 segments of 2m2 each, thus covering a 50m2 surface. at the end of each segment, the flag was inspected and ticks were removed and preserved in 80% ethanol for taxonomic and molecular investigations. tick identification was based on morphological criteria (5-7). before leaving any field sampling site, each cloth was bagged separately in a tight zip locking plastic pouch. between each of the field sampling trips, each cloth was frozen overnight at -70 °c to kill any tick that might have remained hidden inside the fabric. to avoid condensation on the cloth fibres, no cloth was removed from its plastic pouch until it had regained ambient temperature. the mann-whitney u test, which is suitable for skewed distributions, was applied to statistical comparisons between transect lines. ad ditionally, computer modelling was performed using raw data from each transect segment rather than cumulative values from transect lines. a computer program was written to perform 10000 monte carlo simulations based on a null model. results overall, 3957 ticks were collected during this test campaign. of these, 2587 (i.e. 65.38%) were collected with the muc flag, while 1370 (i.e. 34.62%) were collected with the ox flag. all ticks were ixodes ricinus. tick distributions were skewed (s2 >m) due to the occurrence of 'tick nests', i.e. local concentrations of larvae near their hatching site. the muc flag yielded 1704 out of 2059 larvae (i.e. 82.76%) while only 355 larvae (i.e. 17.24%) were collected from the ox flag. between 1 and 455 larvae were collected per transect line using the muc flag, while transect lines surveyed with the ox flag yielded between 0 and 112 larvae. this difference in yield is very highly significant (p< 0.001). yields for each of the transect lines, ranked by tick stage, are provided in table 1. a total of 829 nymphs (out of 1782, i.e. 46.52%) were collected with the muc flag, while the ox flag yielded 953 nymphs (i.e. 53.48%). yields per transect line ranged between 2 and 70 nymphs for the muc flag, and between 7 and 109 nymphs for the ox flag. the differences in yields for nymphs are not statistically significant (p> 0.05) according to the mann-whitney u test. however, computer modelling revealed that nymphal yield from the muc flag is lower than expected under a null model based on similar sampling conditions from similar virtual tick populations (p< 0.05). twenty-five adult females and 29 adult males were recovered from the muc flag, versus 36 adult females and 26 adult males for the ox flag. the differences in yields for adult ticks are not statistically significant (p> 0.05). j arthropod-borne dis, december 2018, 12(4): 421–425 pg de mendonça: impact of flag … 423 http://jad.tums.ac.ir published online: december 25, 2018 table 1. tick yields for each pair of transect lines, ranked by tick stage and sex larvae (muc) larvae (ox) nymphs (muc) nymphs (ox) females (muc) females (ox) males (muc) males (ox) 1 0 2 8 0 0 0 0 1 1 5 8 0 0 0 0 4 0 6 10 0 0 0 0 5 0 7 13 0 0 0 0 5 1 8 7 0 0 0 0 7 0 9 12 0 0 0 0 7 2 10 24 0 0 0 0 8 2 15 12 0 0 0 0 10 1 16 24 0 0 0 1 11 2 17 24 0 1 0 1 13 0 20 24 0 1 0 1 18 5 21 37 0 1 0 1 19 0 24 13 0 1 0 1 21 22 24 29 0 1 0 1 26 9 25 18 0 2 1 0 26 20 26 16 0 2 1 0 26 41 27 12 0 4 1 0 27 0 27 18 1 0 1 0 27 3 28 35 1 0 1 1 28 7 31 39 1 1 1 2 30 0 35 33 1 2 1 2 33 1 35 47 1 2 1 4 47 1 36 70 1 6 2 0 63 0 39 30 2 1 2 0 74 5 42 41 2 1 2 1 136 2 45 56 2 1 2 2 178 4 58 58 2 2 3 1 185 112 58 109 2 5 3 2 213 95 63 45 4 0 3 3 455 19 70 81 5 2 4 2 fig. 1. photograph of the oxford fabric showing its fleecy texture. the coin used as a size marker is 23.3mm in diameter j arthropod-borne dis, december 2018, 12(4): 421–425 pg de mendonça: impact of flag … 424 http://jad.tums.ac.ir published online: december 25, 2018 fig. 2. scanning electron micrographs of the munich (muc) and oxford (ox) fabrics. scale bars: 500µm fig. 3. numerous larval ixodes ricinus ticks are clearly visible to the naked eye on this photograph of a portion of a muc type flag discussion the muc flag yielded nearly five times more larvae than the ox flag. the causes (or at least parts of the causes) for such a dramatic difference were actually observed. the ox cloth is relatively thick and has a very loose and complex texture. larval ticks, which are small enough, manage to crawl between the fibres of the ox flag. therefore, although larvae were actually sampled by the flag, many of them were not found (and thus not collected) by the observer because these larvae had become invisible and unreachable deep inside the fabric. on several occasions, larvae were observed crawling out of the cloth on the reverse side, indicating that larvae were able to cross through the cloth to eventually escape. these observations raise a health and safety issue. indeed, ticks hidden inside the cloth may inadvertently be transported from their original sampling site to other locations, thus possibly transferring tick-borne pathogens to new areas or closer to humans. in stark contrast to the ox fabric, the muc fabric presents a relatively smooth and flat surface. the fibres are tight enough to prevent larval ticks from crawling through them. larvae have nowhere to hide and cannot escape through the cloth. thanks to the white and flat surface, larvae are easily detected visually (fig. 3) and easily collected. a trend suggesting there might be a difference in efficiency at sampling nymphs between the muc and ox flags was observed. based on classical statistics, this trend was not statistically significant. however, computer modelling suggests that the muc flag might be marginally less efficient at sampling nymphs than the ox flag, which is consistent with the above-mentioned trend. the aforementioned differences in sampling efficiency for larvae and (to a much lesser extent) nymphs clearly have an impact on estimates of questing activity for these two j arthropod-borne dis, december 2018, 12(4): 421–425 pg de mendonça: impact of flag … 425 http://jad.tums.ac.ir published online: december 25, 2018 developmental stages. studies relying heavily on such estimates, e.g. studies of cofeeding potential (and its implications for disease transmission) between larvae and nymphs, will undoubtedly suffer biases due to heavily underestimated larval activity when using the ox cloth. the validity of comparisons between various studies needs to be questioned when no indications of relative sampling efficiency are provided, particularly when larvae are considered. the nearly five times difference in yield observed here for larval ticks using two different cloth types is an extreme example. however, this extreme example serves well to illustrate an important point: in large research projects spread over wide geographic areas where equipment is purchased from local providers, it is wise to perform some preliminary comparative testing as part of internal quality control prior to engaging in large-scale sampling. indeed, locally purchased material may vary to a non-negligible extent from place to place, thus potentially introducing biases in the study. computer modelling based on preliminary test results should help highlight potential biases. indeed, monte carlo methods are known to have as much power as (and often more power than) classical statistical tests, while offering a wider flexibility of use (8). conclusion the flag made of muc type fabric, thanks to its tight and relatively flat texture, facilitates detection and collection of ticks from its surface. the ox flag, due to its loose texture, is unsuitable for the quantitative sampling of larval i. ricinus, as these can crawl between the fibres to hide inside the fabric itself and eventually escape. consequently, the ox flag also presents a potential health and safety issue due to the risk of transporting ticks and transferring tick-borne pathogens to new areas or closer to humans. acknowledgements this publication was partially funded by eu grant goce-2003-010284 eden. the contents of this publication are the sole responsibility of the author and do not necessarily reflect the views of the european commission. it is a pleasure to thank ms h schöl for technical assistance with the preparation of the sem illustrations. the blanket from which pieces of fabric were cut to make the ox flags was kindly provided by prof se randolph (university of oxford, uk). the author declares no conflict of interests. references 1. fritz cl (2009) emerging tick-borne diseases. vet clin small anim. 39: 265–278. 2. parola p, raoult d (2001) ticks and tickborne bacterial diseases in humans: an emerging infectious threat. clin infect dis. 32(6): 897–928. 3. de mendonça pg (2008) the eden project. parasitol res. 103(suppl.1): 158–159. 4. wilson ml (1994) population ecology of tick vectors: interaction, measurement, and analysis. in: sonenshine de, mather tn (eds): ecological dynamics of tickborne zoonoses, oxford university press, new york, pp. 20–44. 5. arthur dr (1963) british ticks. butterworths, london. 6. hillyard pd (1996) ticks of north-west europe. field studies council, shrewsbury. 7. snow kr (1979) identification of larval ticks found on small mammals in britain. the mammal society, reading. 8. manly bfj (2007) randomization, bootstrap and monte carlo methods in biology (3rd ed). chapman and hall, boca raton. j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 71 original article resistance mechanisms of anopheles stephensi (diptera: culicidae) to temephos aboozar soltani 1, 3, *hassan vatandoost 3, *mohammad ali oshaghi 3, naseh maleki ravasan 3, ahmad ali enayati 2, fatemeh asgarian 2 1department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran 2school of public health and health research centre, mazandaran university of medical sciences, sari, iran 3department of medical entomology and vector control, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran (received 9 apr 2014; accepted 24 may 2014) abstract background: anopheles stephensi is a sub-tropical species and has been considered as one of the most important vector of human malaria throughout the middle east and south asian region including the malarious areas of southern iran. current reports confirmed an. stephensi resistance to temephos in oman and india. however, there is no comprehensive research on mechanisms of temephos resistance in an. stephensi in the literature. this study was designed in order to clarify the enzymatic and molecular mechanisms of temephos resistance in this species. methods: profile activities of αand ß-esterases, mixed function oxidase (mfo), glutathione-s-transferase (gst), insensitive acetylcholinesterase, and para-nitrophenyl acetate (pnpa)-esterase enzymes were tested for an. stephensi strain with resistance ratio of 15.82 to temephos in comparison with susceptible strain. results: results showed that the mean activity of α-est, gst and ache enzymes were classified as altered indicating metabolic mechanisms have considerable role in resistance of an. stephensi to temephos. molecular study using pcr-rflp method to trace the g119s mutation in ace-1 gene showed lack of the mutation responsible for organophosphate insecticide resistance in the temephos-selected strain of an. stephensi. conclusion: this study showed that the altered enzymes but not targets site insensitivity of ace-1 are responsible for temephos resistance in an. stephensi in south of iran. keywords: anopheles stephensi, temephos, mechanisms of resistance, acetylcholinesterase gene, malaria introduction malaria still remains as a public health problem in the world. southern parts of iran are involved with this problem (vatandoost et al. 2010). anopheles stephensi is a sub-tropical species and also an important vector of human malaria throughout the middle east and south asian region, including the indo-pakistan subcontinent, with a westward extension through iran and iraq into the middle east and arabian peninsula. this species is considered to be the main malaria vector in the persian gulf area (oshaghi et al. 2006a and 2006b). previous studies have shown an. stephensi to be the most prevalent anopheline species in the malarious areas of southern iran (vatandoost et al. 2004, hanafi-bojd et al. 2012). temephos, a most widely used organophosphorus insecticide, has been included in the list of world health organization (who) as a suitable and safe mosquito larvicide that can be used even in drinking water for con*corresponding author: prof hassan vatandoost, email: hvatandoost1@yahoo.com, dr mohammad ali oshaghi, e-mail: oshaghima@yahoo.com http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 72 trolling of the most mosquito vectors .the toxicity of this insecticide is low and unlikely to present acute hazard for human (who 2006). temephos (ec 50%) has been used for some years for larval control program of malaria in southern iran (vatandoost et al. 2006). many studies on the susceptibility level of an. stephensi to various pesticides have been done in iran and other countries. resistance of an. stephensi to different insecticide was reported from around the world (vatandoost et al. 1996). different levels of resistance to larvicides were reported in anopheline malaria vectors worldwide. anopheles stephensi has an extensive resistance comparing to other species and is resistant or tolerant to fenitrothion, temephos and fenthion in india, fenitrothion and pirimiphos-methyl in iraq, fenitrothion, pirimiphos-methyl, chlorfoxim and foxim in iran and fenitrothion in pakistan (vatandoost and hanafi-bojd 2005a). resistance of other anopheline mosquito such as an. dthali to temephos also was reported (hanafi-bojd et al. 2006). in 2006 for the first time in the middle east, resistance to temephos was confirmed in an. stephensi breeding in water storage tanks in the al-dhahira region of oman (anderasen 2006). the level of resistance was 2.5 times higher than that of the who diagnostic dose (0. 25 mg/l). however, there was no confirmed report of resistance of an. stephensi to temephos in iran. previous studies in iran showed that this species was completely susceptible to temephos at the who diagnostic dose (vatandoost et al. 2004, vatandoost and hanafi-bojd 2005a, vatandoost et al. 2005b, vatandoost et al. 2006). one of the most important molecular mechanisms of resistance to organophosphate insecticide in mosquitoes is structural mutations that occur in acetylcholinesterase gene. in mosquitoes two cholinesterase genes are existed (ace-2 and ace-1). ace genes have been cloned from the mosquitoes aedes aegypti and an. stephensi, both of these genes are also sex linked (hemingway and ranson 2000). the existence of both ace genes in an. stephensi is approved by other researchers (malcolm and hall 1990, weill et al. 2002,). but as yet, there is no recorded acebased resistance mechanism in an. stephensi (hemingway and ranson 2000). it is known that insensitive acetylcholinesterase (ache) due to a g119s mutation is associated with tolerance to carbamate and organophosphate insecticides in anopheles gambiae and the mutation can be detected using a pcr-rflp assay (weill et al. 2004a). as yet there is no comprehensive research about mechanisms of temephos resistance in an. stephensi in the literature. the current study was designed in order to clarify the enzymatic and molecular mechanisms of temephos resistance in this species. materials and methods study area eight different areas in two most important malarious provinces of iran were considered to collect live wild specimens of an. stephensi including: bandar abbas port, minab county and hormoodar village in hormozgan province, and chabahar port, villages of bampoor and abtar from iranshahr county, villages of angoori and machkor from sarbaz county in sistan and baluchistan province (fig. 1). mosquito strains the field collected strains of an. stephensi were reared in the insectarum for further tests. a susceptible laboratory strain of an. stephensi (beech-lab from insectarium of department of medical entomology and vector control group, school of public health, tehran university of medical sciences) was used to compare the susceptibility status of the field strains. this strain has been mainhttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 73 tained in the laboratory without exposure to insecticides for 28 years. insecticide technical grade insecticide used in the present study was temephos 90 % (batch no: tem/136-229) which was obtained from levant overseas development ltd., argenteuil, france. based on pre-tests, five concentrations of the larvicide (0.25, 0.0625, 0.0156, 0.0039 and 0.00195 mg/l) were considered for susceptibility assays. bioassay consisted of five concentrations resulting 10–90% mortality. butanone 2% in absolute ethanol was used as a control. larval bioassays susceptibility assays was carried out according to the method described by world health organization (who 2012). the toxicity of temephos to an. stephensi, from field-collected population was determined and compared with laboratory reared susceptible beech-lab strain. abbott’s formula was used to correct the observed mortality of larvae. all the data were corrected if the control mortality is between 5 and 20 % (abbott 1965). data were analyzed using probit analysis to determine the 50% lethal concentration values (lc50) and 90% lethal concentration values (lc90) of the field and beech-lab strains (finney 1971). selection process the strain which showed the highest resistance ratio (rr) to temephos was preceded for selection pressure. this strain was selected for 5 generations by exposing late third or early fourth instars to the concentrations which produced 50–70 % mortality (paeporn et al. 2004). selection was continued as long as a homogenous resistant population with resistance ratio more than 10fold was achieved. biochemical assays thirty mosquito larvae from each susceptible and resistant strain were assayed for αand ß-esterases, mixed function oxidase (mfo) and glutathione-s-transferase (gst), insensitive acetylcholinesterase and pnpaesterase enzymes. each larva was homogenized in 100 µ l of potassium phosphate (kpo4) buffer (6.6 g dibasic potassium phosphate/1.7g mono basic potassium phosphate/1000ml distilled water (dh2o), ph 7.2) and then diluted to 2 ml with the same buffer. each mosquito was analyzed in duplicate with 100 µ l of mosquito homogenate transferred to two wells on a 96 well flatbottomed microtitration plate. absorbance levels were measured spectrophotometrically with a microplate reader (elx808 ultra microplate reader bio-tek ®), at wave lengths indicated for each enzyme, and the mean absorbance calculated based on data for the two replicate wells per mosquito. procedures were followed based on slight modifications of a protocol from the centers of disease control (polson et al. 2011). the activities of all enzymes were evaluated according to this protocol. the details of procedures were described completely in this research (polson et al. 2011). reagents and substrates for biochemical assays were provided by sigma. data analyses of biochemical assays absorbance values which were obtained for mosquito replicates were corrected in relation to the volume of mosquito homogenates, the enzyme activity unit and the total protein content of each mosquito (polson et al. 2011). the means of enzyme activities for each an. stephensi larval strain were compared with the susceptible (beech-lab strain) by unpaired t-test, mann-whitney test (p< 0.05). the beech-lab 99th percentile was calculated for each enzyme and the percentage of specimens with enzymatic activity above http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 74 that of the beech-lab 99th percentile was calculated. enzyme activities were then classified as “altered”, “incipiently altered” or “unaltered” if the rate was >50 %, between 15 % and 50 % and <15 %, respectively (montella et al. 2007). molecular study of resistance mosquito genomic dna was extracted from triplex homogenate of mosquitoes by qiaamp dna mini kit. dna was then pcr amplified with the degenerated primers moustdir1 5′ccgggngcsacyatgtggaa3′ and moustrev1 5′acgatmacgttctcytccga3′ according to the conditions and thermal cycles already introduced by weill et al. (2004). the pcr products were digested with alui restriction enzyme according to the manufacturer’s instructions and fractionated on a 2% agarose ethidium bromide gel (weill et al. 2004a). the primers created a 194 bp amplicon in both temephos resistance and susceptible strains, however after restriction enzyme digestion, homozygous resistant individuals cut to 120 bp and 74 bp fragments if the g119s mutation was existed. representative pcr products of both temephos resistance and susceptible strains of an. stephensi were sent for sequencing in order to confirm the pcr-rflp assays as well as to find other possible mutation on ace-1 gene except for the g119s mutation. bioinformatic softwares such as clustal w2, blast, and mega 5 were used for sequence alignment, homology, and phylogenetic analysis. we also used transeq software for translation nucleic acids to amino acids. results larval bioassays considerable variation in temephos resistance ratio of filed strains in comparison with susceptible strain was noticed from all the locations studied. a low level of resistance ratio was observed in the populations of an. stephensi except in chabahar strain. (rr= 4.27 folds) compared to beechlab strain (p< 0.05) (fig. 2). according to our findings, chabahar strain of an. stephensi with resistance ratio more than 4folds was chosen for selection process as the most tolerant strain. selection process after larval bioassays, chabahar strain was established into the insectary for selection process. selection process was continued for 15 months. after 5th selection a resistant population of an. stephensi was achieved with 15.82 and 35.34-folds resistance ratio at lc50 and lc90 level, respectively. biochemical assays analyses were conducted through comparing the median value for beech-lab strain (s) with those of the temephos selected strain (r), for each enzyme. by unpaired t-test and mann-whitney test, the median activity for all enzymes differed significantly (p< 0.05). according to the classification scheme detailed in method, for each respective enzyme, activities were classified as “unaltered”, “incipiently altered” or “altered” if the values were <15%, between15 and 50% and >50%, respectively. tables 1 and 2 show the number of mosquitoes assessed in each assay, along with the median values and percentage of strains with enzymatic activities in relation to beech-lab strain (s). α-esterase in relation to beech-lab strain (s), temephos resistant strain (r) was significantly different in α-est activity levels (p< 0.0001). r strain showed altered activity with >50 % (95%) of individuals recording http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 75 activity above that of the 99th percentile of the beech-lab reference strain (table 1). ß-esterase the median activity levels of ß-est seen in the resistant strain were significantly different from the beech-lab strain (p< 0.0001). based on the classification of activity profiles, r strain showed unaltered activity with <15 % (8.33%) of individuals recording activity above that of the 99th percentile of the beech-lab reference strain (table 1). pnpa-esterase there were significant differences observed between the median pnpa-est activities of the beech-lab strain and temephos resistant strain (p= 0.0096). an unaltered profile of pnpa-est (6.67%) was found in r strain of an. stephensi (table 1). mixed function oxidase (mfo) in relation to beech-lab strain (s), temephos resistant strain (r) was significantly different in mfo activity levels (p= 0.0002). r strain showed unaltered activity with <15 % (1.67%) of individuals recording activity above that of the 99th percentile of the beech-lab reference strain (table 2). glutathione-s-transferase (gst) the gst activity in the beech-lab strain was significantly different from that of the temephos resistant strain (p< 0.0001). based on the classification of activity profiles, r strain showed altered activity with >50 % (86.67%) of individuals recording activity above that of the 99th percentile of the beech-lab reference strain (table 2). insensitive acetylcholinestersase (iache) the rate of activity of ache in the presence of propoxur for beech-lab strain was significantly different from that observed in the temephos resistant strain (p< 0.0001). an altered profile of ache (90%) was found in the r strain of an. stephensi (table 2). the activity levels of the enzymes in both strains (r and s) are graphically displayed in scatter plots (figs. 3–8). molecular study on ace-1 resistance in anopheles stephensi with moustdir1 and moustrev1 primers, a 194 bp amplicon was amplified by pcr. the result of pcr-rflp with alui showed that pcr products of both temephos resistance and susceptible strains of an. stephensi were remain intact (fig. 9) indicating lack of the g119s mutation in ace-1 of resistance strain. three specimens from each resistant and susceptible strain of an. stephensi to temephos, were sent for sequencing. the sequences were deposited in the european nucleotide archive (ena) with accession numbers (hg38032024). the results of 5 sequencing that were trustable, analyzed with blast, and clustal w2 softwares. the blast analysis revealed that there was no counterpart sequence data of the ace-1 gene of an. stephensi in the genbank database. the most similar sequence data available in genbank database were anopheles albimanus s (accession number: aj566402), an. albimanus r (an: aj566403) and anopheles funestus r (an: dq534435) (fig. 10). the results showed sequences of ace-1 for both resistant and susceptible strains were identical and no g119s mutation was observed in resistance strainthat equenced. the blast analysis of this region of ace-1 for an. stephensi, showed sequence of ace1 was more similar to an. albimanus than an. funestus (table 3). in comparison with available data in genbank, 2 indels, and 34 substitutions were observed (fig. 10). construction of phylogram was done using mega 5 for ace-1 sequences of this study (an. stephensi r/s) and other available data in genbank (an. albimanus s, an. albimanus r and an. funestus r) (fig. 11). amino acids sequences of an. stephensi ace-1 gene were compared with other similar amino acid sequences of mosquitoes were available in the genbank (fig. 12). the results showed lack of glycine to serine substituhttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 76 tion at position 119 in the ace-1 gene that confers high levels of resistance to organophosphate in the resistant (r) strain of an. stephensi of this study. this substitution only observed in r strain of an. albimanus. two species specific amino acid sequences in ace-1 gene of an. stephensi were observed in analogy with other sequences. in this species two arginine have been substituted with glutamic acid and aspartic acid. these differences seem to be structural and not related to insecticide resistance property of this species (fig. 12). fig. 1. location of anopheles stephensi collection sites from malarious areas of iran, 2011 fig. 2. temephos resistance ratio pattern in anopheles stephensi field strains from malarious area of southern iran data 1 mosquito strains nm ol /m g pt n/ m in be ec h r 0.00000 0.00005 0.00010 0.00015 0.00020 fig. 3. activity profile of α-esterase enzymes data 1 mosquito strains nm ol /m g pt n/ m in be ec h r 0.00000 0.00005 0.00010 0.00015 0.00020 0.00025 fig. 4. activity profile of ß-esterase enzymes data 1 mosquito strains  a bs /m g pt n/ m in be ec h r -0.05 0.00 0.05 0.10 0.15 fig. 5. activity profile of pnpa-esterase enzymes http://jad.tums.ac.ir published online: july 16, 2014 mosquito strains mosquito strains mosquito strains resisrance ratio ab s/ m g p tn /m in n m ol /m g p tn /m in n m o/ m g p tn /m in j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 77 table 1. quantification of enzymatic activity of esterases in two strains (resistant and susceptible) of anopheles stephensi pnpa-est(abs/mg ptn/min)ß-est (nmol/mg ptn/min)α-est (nmol/mg ptn/min)strains p99mediannp99mediannp99 cmedian bn a 0.079692780.04560881300.000209710.00014011300.000104110.0000688130beech-lab %>p99mediann%>p99mediann%>p99 dmediann 6.670.05132526308.330.0001772530950.0001365430chabahar (selected with temephos) a number of mosquitoes tested. b median value for each enzymatic activity. c 99th percentile for beech-lab reference strain. d percentage of mosquito specimen with activity above 99th percentile for beech-lab reference strain. table 2. quantification of enzymatic activity of mfo, gst and iache in two strains (resistant and susceptible) of anopheles stephensi ache (% activity)gst (nmol/mg ptn/min)mfo (nmol cyt/mg ptn)strains p99mediannp99mediannp99 cmedian bn a 5.365172821.40618583300.00028138-.00085944300.000081080.0000326430beech-lab %>p99mediann%>p99mediann%>p99 dmediann 9014.168171183086.670.00033844301.670.0000227930chabahar (selected with temephos) a number of mosquitoes tested. b median value for each enzymatic activity. c 99th percentile for beech-lab reference strain. d percentage of mosquito specimen with activity above 99th percentile for beech-lab reference strain. table 3. blast analyze of ace-1 region sequence for temephos-resistant anopheles stephensi r/s (this study) and other species of mosquitoes (anopheles albimanus s, anopheles albimanus r and anopheles funestus r) seqa name length seqb name length score 1 an. stephensi r/s 160 2 aj566402 an.albimanus s 162 88.75 1 an. stephensi r/s 160 3 aj566403 an.albimanus r 162 88.12 1 an. stephensi r/s 160 4 dq534435 an.funestus r 162 83.12 2 aj566402 an.albimanus s 162 3 aj566403 an.albimanus r 162 99.38 2 aj566402 an.albimanus s 162 4 dq534435 an.funestus r 162 82.72 3 aj566403 an.albimanus r 162 4 dq534435 an.funestus r 162 82.1 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 78 data 1 mosquito strains nm ol c yt /m g pt n be ec h r 0.00000 0.00002 0.00004 0.00006 0.00008 0.00010 fig. 6. activity profile of mfo enzymes data 1 mosquito strains nm ol /m g pt n/ m in be ec h r -0.003 -0.002 -0.001 0.000 0.001 fig. 7. activity profile of gst enzymes data 1 mosquito strains % r em ai ni ng a ct iv it y be ec h r -20 0 20 40 60 fig. 8. percent remaining activity acetylcholinesterase (ache) fig. 9. diagnostic pcr-rflp to identify g119s mutation in amplified region of ace-1 (194 bp) in individuals of anopheles stephensi. m: 50 bp ladder (fermentas), lane 1–6: temephos-resistant strain (lane 1–2: 4th generation of selected strain with temephos, lane 3–4: 5th generation of selected strain with temephos, lane 5–6: 6th generation of selected strain with temephos) clustal 2.1 multiple sequence alignment table 4. summary of some biochemical studies were done in order to characterizing the mechanisms of temephos resistance in different vector species referencemain mechanism of resistanceinsecticidecountryyearspecies peiris and hemingway 1990 general esterase (α and ß)temephossrilanka1990culex quinquefasciatus penilla et al. 1998pnpa-estrase and mfoorganophosphatemexico1998anopheles albimanus paeporn et al. 2003general esterase (α and ßtemephosthailand2003aedes aegypti saelim et al. 2005general esterase (α and ß)temephosthailand2005aedes aegypti montella et al. 2007general esterase (α and ß), pnpa estrase and gst (only in north-east strain of brazil) temephosbrazil2007aedes aegypti melo-santosa et al. 2010general esterase (α and ß) and gsttemephosbrazil2010aedes aegypti polson 2011general esterase (α and ß) , gst, mfo and ache temephostrinidad2011aedes aegypti this studyα-est, gst and achetemephosiran2013anopheles stephensi http://jad.tums.ac.ir published online: july 16, 2014 mosquito strains mosquito strains mosquito strains n m ol /m g p tn /m in n m ol cy t/ m g p tn % r em ai n in g ac ti vi ty j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 79 clustal 2.1 multiple sequence alignment aj566402 an.albimanus s tctcggaggactgcctgtacatcaacgtggtggcgccgaggccacggccc 50 aj566403 an.albimanus r tctcggaggactgcctgtacatcaacgtggtggcgccgaggccacggccc 50 seq1 tctcggag--atgtctgtacatcaacgtggtagcaccacgaccccgtccc 48 seq2 tctcggag--atgtctgtacatcaacgtggtagcaccacgaccccgtccc 48 seq3 tctcggag--atgtctgtacatcaacgtggtagcaccacgaccccgtccc 48 seq4 tctcggag--atgtctgtacatcaacgtggtagcaccacgaccccgtccc 48 seq5 tctcggag--atgtctgtacatcaacgtggtagcaccacgaccccgtccc 48 dq534435 an.funestus r tgtcggaggactgtctgtacattaatgtggtagcaccacgaccgcgaccg * ****** .** ******** ** *****.**.**..*.** ** ** 50 aj566402 an.albimanus s aagaatgctgccgtcatgctgtggatcttcggcggtggcttctactccgg 100 aj566403 an.albimanus r aagaatgctgccgtcatgctgtggatcttcggcggtagcttctactccgg 100 seq1 aagaatgctgccgttatgctgtggatctttggtggaggattctactccgg 98 seq2 aagaatgctgccgttatgctgtggatctttggtggaggattctactccgg 98 seq3 aagaatgctgccgttatgctgtggatctttggtggaggattctactccgg 98 seq4 aagaatgctgccgttatgctgtggatctttggtggaggattctactccgg 98 seq5 aagaatgctgccgttatgctgtggatctttggtggaggattctactccgg 98 dq534435 an.funestus r aagaatgctgccgttatgctgtggatctttggcggtggattttactccgg ************** ************** ** **:.*.** ******** 100 aj566402 an.albimanus s tacggccacactggacgtgtacgatcaccgggcgctcgcctcggaagaga 150 aj566403 an.albimanus r tacggccacactggacgtgtacgatcaccgggcgctcgcctcggaagaga 150 seq1 tacggccacactggacgtgtacgatcatcgggcgcttgcctcggaggaga 148 seq2 tacggccacactggacgtgtacgatcatcgggcgcttgcctcggaggaga 148 seq3 tacggccacactggacgtgtacgatcatcgggcgcttgcctcggaggaga 148 seq4 tacggccacactggacgtgtacgatcatcgggcgcttgcctcggaggaga 148 seq5 tacggccacactggacgtgtacgatcatcgggcgcttgcctcggaggaga 148 dq534435 an.funestus r taccactacgctcgacgtgtacgatcaccgtgcgctcgcatcggaggaga *** .* **.** ************** ** ***** **.*****.**** 150 aj566402 an.albimanus s acgttatcgtac 162 aj566403 an.albimanus r acgttatcgtac 162 seq1 acgttatcgtaa 160 seq2 acgttatcgtaa 160 seq3 acgttatcgtaa 160 seq4 acgttatcgtaa 160 seq5 acgttatcgtaa 160 dq534435 an.funestus r atgtgatcgtcg * ** *****. 162 seq1 an. stephensi chabahar strain with 9.67 resistant ratio, f5 selected with temephos, ace-1 gene, partial cds seq2 an. stephensi chabahar strain with 15.82 resistant ratio,, f6 selected with temephos, ace-1 gene, partial cds seq3 an. stephensi chabahar strain with 6.06 resistant ratio,, f4 selected with temephos, ace-1 gene, partial cds seq4 an. stephensi beech-lab susceptible strain 5, ace-1 gene, partial cds seq5 an. stephensi beech-lab susceptible strain 9, ace-1 gene, partial cds fig. 10. comparison of sequencing results of this study (seq1, seq2, seq3, seq4, seq5*) with other three registered genes in gene bank http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 80 fig. 11. phylogram a part of ace-1 for sequences of this study (anopheles stephensi r/s) and other similar registered genes in gene bank (anopheles albimanus s, anopheles albimanus r and anopheles funestus r) clustal 2.1 multiple sequence alignment an. albimanus r sedclyinvvaprprpknaavmlwifggsfysgtatldvydhralaseenviv 53 an. funestus r sedclyinvvaprprpknaavmlwifgggfysgtttldvydhralaseenviv 53 an. albimanus s sedclyinvvaprprpknaavmlwifgggfysgtatldvydhralaseenviv 53 an. stephensi r/s srrclyinvvaprprpknaavmlwifgggfysgtatldvydhralaseenviv *. *************************.*****:****************** 53  arginine (r)  glutamic acid (e)  aspartic acid (d)  glycine (g)  serine (s)  alanine (a)  threonine (t) fig. 12. comparison of translated ace-1 region amino acids sequence of anopheles stephensi r/s (this study) with other similar registered genes in gene bank (anopheles albimanus s, anopheles albimanus r and anopheles funestus r) discussion in this study, it was found that an. stephensi in southern part of iran would normally be susceptible to insecticides but become resistant to temephos under insecticide pressure in laboratory condition. on the other hand, in field condition, temephos resistance of this species has been reported from otherneighboring malarious countries such as india and oman (vatandoost and hanafi-bojd 2005a, anderasen 2006). this warrants precautions and insecticide vector management (ivm) before the resistance becomes widespread in iran and neighboring countries. biochemical assays were carried out by several researchers in order to characterizing the mechanisms of temephos resistance in different vector species. some of them are mentioned in table 4. biochemical assays were done in temephos selected strain of an. stephensi for the first time in the literature. profile of enzyme activity in temephos-resistant an. stephensi showed that the mean enzymatic activity of location of g119s mutation http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 81 α-est, gst and ache were classified as altered. these results clarified that metabolic mechanisms have considerable role in resistance of an. stephensi to temephos. and the most important mechanisms of resistance are α-est, gst and ache. it seems ß-est, mfo and pnpa-esterase are not important in resistance of an. stephensi to this insecticide. vector control programs have, for a long time, utilized bioassays to monitor insecticide resistance in field mosquito populations. these assays only inform the susceptibility level of a certain population to a specific insecticide (polson et al. 2011). biochemical assays also should be included in routine activities of surveillance programs in order to finding incipiently altered enzyme activity of field populations. these are more informative than the bioassays in that they provide some information on the resistance mechanisms involved. with this information we can prevent the development of insecticide resistance in whole population by proper and timely interventions. biochemical assays should be simultaneously carried out with routine bioassays in order to improve the surveillance of resistance and monitoring of the efficacy of insecticides in malarious area. considering that the mechanisms and molecular basis of resistance are very diverse, these mechanisms (metabolic and molecular) should be identified as well for each insecticide which will be used. in this case, the efficient monitoring strategies shall be applicable and finally management of insecticide resistance in vectors can be obtained. high insecticide resistance resulting from insensitive acetylcholinesterase has emerged in mosquitoes. a single mutation (g119s of the ace-1 gene) explains this high resistance in culex pipiens and in an. gambiae (weill et al. 2004 a,b). it has been recently shown that the high insensitivity of acetylcholinesterase displayed by cx. pipiens and an. gambiae is due to the same glycine to serine substitution (g119s mutation), resulting from a single point mutation ggc to agc in the gene ace-1 (weill et al. 2002). the results of weill et al. study (2004) showed that, the gly 119 codon was found serine immutable in 31 vector species including an. stephensi. molecular and biochemical assays were carried out to identify ace-1 mutation in an. gambiae and culex quinquefasciatus. in this study less than 1 % of mosquitoes showed the presence of the ace-1 mutation (corbel et al. 2007). another study was surveyed acetyl cholinesterase sequencing in ae. aegypti. in all individuals, a pcr product of 507 bp was amplified. sequences were aligned and no mutations were observed within this region of ace. resistant and susceptible individuals presented the same nucleotide and amino acid sequence, with 100 % homology to the sequence (melo-santosa et al. 2010). these studies clarified that the frequency of ace-1 mutation into the field population of mosquitoes are very low. the mechanisms of temephos resistance based on the existence of g119s mutation on ace-1 gene for one of the most important malaria vector an. stephensi were studied. in this study pcr-rflp showed no g119s mutation was existed in this part of gene of the an. stephensi strains. resistant and susceptible individuals presented the same nucleotide and amino acid sequence, with 100 % homology to the sequence. these results are completely similar to other researchers' results and approved the immutable characteristic of this region of ace-1 gene in an. stephensi. finally we can conclude based on molecular studies of temephos resistance, there is no mechanisms of temephos resistance in relation to studied region of acetylcholinesterase 1 gene in an. stephensi. probably mechanisms of temephos http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 71–83 a soltani et al.: resistance mechanisms of … 82 resistance in an. stephensi are more enzymatic or are belong to other parts of the mosquito genome that we didn't studied in this research. conclusion the results of this study will provide information about mechanisms of temephos resistance in the main malaria vector in iran. this finding is very crucial for management of malaria vector control. acknowledgements this article is a part of the first author’s dissertation for fulfillment of a phd degree in medical entomology and vector control from department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. the authors are very grateful to mr m yarian from hormozgan university of medical sciences and mr a pakari and mr shahbakhsh technicians of the national institute of health research, bandar abbas and iranshahr research stations, for their kind collaboration during this study. this study was financially supported by the deputy for research, tehran university of medical sciences. the authors declare that there is no conflict of interest. references abbott ws (1965) a 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focus of malaria, southern iran. j vector borne dis. 43(1): 34–38. hemingway j, ranson h (2000) insecticide resistance in insect vectors of human disease. ann rev entomol. 45: 371–391. malcolm c a, hall lmc (1990) cloning and characterization of a mosquito acetylcholinesterase gene, in molecular insect science, ed. by hagedorn hh, hildebrand jg, kindwell mg and lawet jh, new york: plenum, pp. 57–65. melo-santosa mav, varjal-meloa jjm, arajoa ap, gomesa tcs, paivaa mhs, regisa ln, furtadoa af, magalhaesa t, macorisd mlg, andrighettid mtm, ayresa cfj (2010) resistance to the organophosphate temephos: mechanisms, evolution and reversion in an aedes aegypti laboratory strain from brazil. actatrop. 113: 180–189. montella ir, martins aj, viana-medeiros pf, lima jb, braga ia, valle d (2007) insecticide resistance mechanisms of brazilian aedes aegypti populations from 2001 to 2004. am j trop med hyg. 77: 467–477. oshaghi ma, yaghoobi f, vatandoost h, abai mr, akbarzadeh k (2006a) anopheles 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vectors and pests of public health importance (sixth edition). who, geneva, switzerland, who/ cds/ntd/whopes/gcdpp/2006.1. world health organization (2012) malaria entomology and vector control (learner's guide). who, geneva, switzerland. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, september 2020, 14(3): 270–276 l ganushkina et al.: detection of the … 270 http://jad.tums.ac.ir published online: september 30, 2020 original article detection of the invasive mosquito species aedes (stegomyia) aegypti and aedes (hulecoeteomyia) koreicus on the southern coast of the crimean peninsula lyudmila ganushkina1; alexander lukashev1; *ivan patraman1; vladimir razumeyko2; еlena shaikevich1,3 1martsinovsky institute of medical parasitology, tropical and vector-borne diseases, sechenov first moscow state меdical university, moscow, russia 2department of ecology and zoology taurida academia, vernadsky cremian federal university, simferopol, republic of crimea 3vavilov institute of general genetics russian academy of sciences, moscow, russia *corresponding author: dr ivan patraman, e-mail: ivanpatraman@gmail.com (received 24 sep 2018; accepted 05 sep 2020) abstract background: the incidence and area of arbovirus infections is increasing around the world. it is largely linked to the spread of the main arbovirus vectors, invasive mosquito of the genus aedes. previously, it has been reported that aedes aegypti reemerged in russia after a 50-year absence. moreover, in 2011, ae. albopictus was registered in the city of sochi (south russia, black sea coast) for the first time. in 2013, asian ae. koreicus was found in sochi for the first time. methods: mosquitoes were collected using the following methods: larvae with a dip net, imago on volunteers and using bait traps. the mosquitoes were identified using both morphology and sequencing of the second internal transcribed spacer of the nuclear ribosomal rna gene cluster. results: in august 2016, ae. koreicus larvae and imago and a single male of ae. aegypti were found on the southern coast of the crimean peninsula, where they were not registered before. newly obtained dna sequences were registered in genbank with the accession numbers mf072936 and mf072937. conclusion: detection of invasive mosquito species (ae. aegypti and ae. koreicus) implies the possibility of their area expansion. intensive surveillance is required at the crimean peninsula to evaluate the potential for the introduction of vector-borne diseases. keywords: mosquito monitoring; arbovirus vectors; aedes aegypti; aedes koreicus; dna introduction at present, the invasive species aedes (stegomyia) aegypti, aedes (stegomyia) albopictus and aedes (hulecoeteomyia) koreicus are spreading across the world and have been registered in caucasian coast of the black sea in southern russia since 2011 (1-4). all of them are known as vectors of human pathogens (4). invasive aedes species have adapted its habitat preferences to human made containers, such as water storage tanks, discarded tires and jars, and water-filled pots (4). due to the changing climate and the introduction of artificial hatching habitats, it was expected that these mosquito species would also spread to the southern coast of crimea, which has a climate very similar to caucasus. in 2010, an entomological survey in the southern coast of crimea found 15 bloodsucking mosquito species, namely three species of the genus anopheles (an. claviger, an. maculipennis, an. plumbeus), 3 species of the genus culex (cx. hortensis, cх. pipiens, cх. territans), five species of the genus aedes (ae. refiki, ae. cantans, ae. dorsalis, ae. cataphylla, ae. geniculatus), three species of the genus culiseta (cs. annulata, cs. longiareolata, cs. morsitans) and uranotaenia unguiculata (5). the coastal area of crimea, especially the southern coast, is densely populated and attracts ap copyright © 2020 iranian scientific society of biology & control of diseases vectors, and tehran university of medical sciences. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/by-nc/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 270–276 l ganushkina et al.: detection of the … 271 http://jad.tums.ac.ir published online: september 30, 2020 proximately five million tourists each year. the goal of the present study was to survey the southern coast of crimea on the presence of invasive mosquito species. materials and methods mosquito collection was carried out in august, 2016. mosquito sampling sites are shown in figure 1. the larvae were collected with a dip net, while the imago were collected either on volunteers using sucking tubes or “electrofrog” bait traps (lmd-komplekt plus, russia). mosquito sampling sites had different conditions. priboy vacation resort (number 1 in table 1, fig. 1) is located 300–500 meters to the northwest of sakskoe lake, one kilometer to the east is the resort city saki, a town with well-developed residential areas. frunzenets vacation resort (number 2 in table 1, fig. 1) is located one kilometer away from a village on the shores of lake bagaily (number 7 in table 1, fig. 1), which were overgrown with reeds and the inshore depth reached 1 meter. mosquito larvae were collected in the thickets of the reeds. departure of imago from the lake was registered. imagoes were collected in the peak mosquito activity period from 8:30pm to 9:30pm. the mosquitoes were identified by morphological key (7) and dna sequencing. the most reliable characteristics for ae. koreicus larvae were the form and number of сomb scales on the lateral view of larval abdominal segment (fig. 2a), position of the siphonal tuft and the number of the pectin teech (fig. 2b). imago ae. koreicus show a black and white pattern due to the presence of white scale patches on a black background and have clear longitudinal lines on the scutum (fig. 2c). two narrow white dorsocentral stripes are characteristic for ae. aegypti (fig. 2d). dna extraction was performed using diatoms dna prep kit (isogen, moscow). the encyclo pcr kit (evrogen, moscow) and primers complementary to 5.8s and 28s rrna genes (8) were used for amplification of the second internal transcribed spacer (its2) region of rrna genes. amplification products were visualized in 1% agarose gel and purified from the gel using a clean-up kit (evrogen, moscow). sequencing was performed using abi prism® 310 genetic analyzer with bigdye® terminator kit (applied biosystems, usa). the obtained dna sequences were registered in genbank (accession numbers mf072936 and mf072937). the internet resource https://www.gismeteo.ru/diary/ was used for climate analysis. results imago and larvae of eight mosquito species, 661 individuals in total, were collected on the crimean black sea coast and in simferopol (fig. 1, table 1). in simferopol, cx. pipiens was collected in water storage tanks. in lesnoy village (№6 in table 1, fig. 1), located close to the town of sevastopol, mosquitoes were collected in an artificial concrete basin with an area of 23m2 and a depth of 0.5m. in this artificial basin, the larval density per 1m2 was the highest: ae. koreicus – 238 individuals; cs. longiareolata – 256; and cx. pipiens – 17. among them, one male ae. aegypti was discovered at priboy vacation resort. morphological identification was confirmed by dna sequencing. this ae. aegypti its2 dna sequence was deposited to genbank (accession number mf072936). the its2 nucleotide sequence of the crimean ae. aegypti was identical to the one from abkhazia, 2012 (mh142316) and adler (russia), 2013 (mh142319). there was one nucleotide substitution in its2 (99% identity) between ae. aegypti found in crimea (mf072936) and ae. aegypti sequences registered in genbank: rockefeller strain kf471581kf471583; jx423805 and jx423807 from saudi arabia; ku497616 from china; and kf471579 from new caledonia. larvae of the species ae. koreicus were found http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 270–276 l ganushkina et al.: detection of the … 272 http://jad.tums.ac.ir published online: september 30, 2020 close to sevastopol in lesnoy village (table 1), in an artificial concrete basin. aedes koreicus imago were found next to this pond. the sequence of the its2 genome region of ae. koreicus larvae found in crimea was identical to the dna sequence of ae. koreicus from belgium (kf471636 and jf430391) and sochi (hg763830). the crimean ae. koreicus dna sequence was deposited in genbank with the accession number mf072937. in addition to these two invasive species, representatives of six autochthonous mosquito species were registered in the studied area. imagoes sampled at all locations were mainly represented by the species cx. pipiens, cx. modestus, and a few individual coquillettidia richiardii and ae. caspius (table 1). culiseta longiareolata and cx. pipiens larvae were found in lesnoy village (table 1). fig. 1. map of crimean peninsula with the mosquito sampling locations indicated as in table 1. the map of europe with the current known established populations of aedes koreicus and ae. aegypti in europe (ecdc; 2020. available from: https://ecdc.europa.eu/en/disease-vectors/surveillance-and-disease-data/mosquito-maps), crimean peninsula indicated with a circle. locations of ae. aegypti collection are shown with a square, ae. koreicus with a star. fig. 2. diagnostic characters of aedes koreicus larva (a, b) and female (c), ae. aegypti male (d) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 270–276 l ganushkina et al.: detection of the … 273 http://jad.tums.ac.ir published online: september 30, 2020 table 1. composition of adults and larvae of 661 collected mosquitoes number on the map sample site coordinates mosquito species ae. aegypti ae. koreicus ae. caspius cx. pipiens cx. modestus cx. hortensis cq. richiardii cs. longiareolata 1 priboy 45°13'05″n 33°52'22″ e 1♂ 3♀ 154♀ 5♂ 157♀4♂ 11♀ 2 frunzenets 45°02′37″n 33°63′28″е 1♀ 52♀3♂ 90♀3♂ 2♀ 3 evpatoria 44°12'00″n 33°21'30″e 26♀ 21♀ 4 koktebel 44°57'35″n 35°14'52″e 3♀ 3♀ 5 simferopol 44°56'53″n 34°06'15″e 12♀10l 6 sevastopol (lesnoy village) 44°43′55″n 33°34′55″e 3♀29l 2l 33l 7 lake bagaily 45°02′37″n 33°63′28″е 8l 16l 9l total number of individuals 1 32 4 275 294 9 13 33 discussion screening of mosquitos on the south coast of crimean peninsula found, among others, ae. aegypti and ae. koreicus, which have not been registered in crimea before 2016 (5). in 2018, kovalenko and tikhonov (9) examined eight regions of the crimea and collected seven ae. koreicus females in sevastopol and three in simferopol. although no ae. koreicus larvae or breeding sites were found in this study, this data implied the spread of mosquitoes to the center of the peninsula. so, additional finding of ae. koreicus imagoes in crimea in 2018 confirms the establishment of ae. koreicus population in crimea. native areas of ae. koreicus are korean peninsula, china, japan, and the russian far east. recently, ae. koreicus has been expanding to new areas due to exobiological plasticity. the species ae. koreicus was first found in the european part of russia in 2013 in the center of the town of sochi (4, 9, 10). at present, ae. koreicus has been registered in many european countries including belgium, northern italy, hungary, slovenia and germany (1118). it is known that ae. koreicus can withstand lower winter temperatures and is drawn to populated areas located near woods. its larvae, as well as those of ae. aegypti and ae. аlbopictus, develop in artificial basins. females usually attack in the open air, occasionally indoors. mosquitoes overwinter in the egg stage. aedes koreicus has shown vector competence in the field and in laboratory experiments for a number of pathogens including dengue, west nile fever, japanese encephalitis viruses and dirofilaria (12, 19). therefore, discovery of ae. koreicus in crimea marks a significant threat of the introduction and spread of vector-borne diseases. detection of a single ae. aegypti male can be attributed to either breeding in the close-by residential area, which provides artificial reservoirs serving as breeding habitats, or an isolated introduction, possibly from the black sea coast of the caucasian region, where these mosquitoes were recently registered (2, 4, 6). the latter possibility has been exemplified previously by discovery of a single ae. aegypti male in northwest england (53°30′42″n, 2°59′01″w) in 2014, where climate, temperature and humidity were generally unsuitable for this new invasive mosquito (20). in crihttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 270–276 l ganushkina et al.: detection of the … 274 http://jad.tums.ac.ir published online: september 30, 2020 mea, where the ae. aegypti male was discovered in the present study, the highest temperature during the summer season was registered in august 2016. at the time of mosquito collection, the temperature reached 36 °c during daylight hours and 28–31 °c at night. the relative humidity in july and august (67–68%) was uncomfortable for these mosquitoes. during may–august, it rained for only 2 days and the total amount of precipitation over the year was approximately 400mm whereas in sochi, where ae. aegypti mosquitoes were previously registered, the annual rainfall exceeds 1500 mm. it is believed that ae. aegypti distribution is limited to areas with a january isotherm of 10 °c and mean annual temperatures of 15 °c, whereas in crimea in the winter 2015–2016 the lowest winter temperature reached -15 °c, and in subsequent years it was between -6 °c and -7 °c. unfavorable conditions may indicate that ae. aegypti was an occasional introduction to the peninsula. to our knowledge, there is no published data on findings of ae. aegypti in crimea (https://ecdc.europa.eu, 2018). at the same time, ae. aegypti is well established on the black sea coast in caucasus (4), and is now spreading along the black sea coast of turkey and georgia (6, 21). in 2014, we suggested that ae. albopictus mosquitoes were likely to appear in crimea (22), but neither larvae nor imago of this species were discovered during the present study. nevertheless, climate change and the detection of the more cold sensitive ae. aegypti highlights the risk of ae. albopictus rooting in crimea and calls for detailed entomological monitoring on the peninsula. the predominance of сх. pipiens and cx. modestus imagoes can be explained by the proximity of the sampling areas frunzenets and priboy to lakes (fig. 1), which are a favorable environment for the development of the genus сulex larvae, as evidenced by the presence of culex hortensis larvae at bagaily lake (table 1). the absence of cx. hortensis imago among the collected samples (as compared with the larvae samples) is due to the fact that these species are reluctant to attack humans and spend most of the time in caves and similar sheltered environments. conclusion in the current study, larvae breeding habitats for ae. koreicus and ae. aegypti imago have been found on the southern coast of the crimean peninsula for the first time. the ae. aegypti species was represented by a single male specimen; therefore, it is not clear if it has yet been established in crimea. our study indicates that ae. koreicus is locally established in crimea and its northward expansion is expected, highlighting the challenges for vector surveillance and control programs. acknowledgements this work was supported by the rfbr grants no.16-04-00091 and no.19-04-00739. we are grateful to “lmd-komplekt plus” company, namely to a.s. poznyshev, s.i. kozhurin and r.a. mingaliev, for providing electrofrog traps for mosquito collection. we declare that we have no conflict of interest. references 1. ganushkina la, tanygina eyu, bezzhonova ov, sergiev vp (2012) detection of aedes stegomyia) albopictus skuse mosquitoes in the russian federation. med parazitol (mosk). 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(4): 8–11. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=kotsakiozi%20p%5bauthor%5d&cauthor=true&cauthor_uid=29980229 https://www.ncbi.nlm.nih.gov/pubmed/?term=schaffner%20f%5bauthor%5d&cauthor=true&cauthor_uid=29980229 https://www.ncbi.nlm.nih.gov/pubmed/?term=schaffner%20f%5bauthor%5d&cauthor=true&cauthor_uid=29980229 https://www.ncbi.nlm.nih.gov/pubmed/?term=robert%20v%5bauthor%5d&cauthor=true&cauthor_uid=29980229 https://www.ncbi.nlm.nih.gov/pubmed/?term=powell%20jr%5bauthor%5d&cauthor=true&cauthor_uid=29980229 https://www.ncbi.nlm.nih.gov/pubmed/29980229 j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 39 http://jad.tums.ac.ir published online: april 27, 2019 original article effect of chlorine and temperature on larvicidal activity of cuban bacillus thuringiensis isolates *aileen gonzález-rizo1; camilo e castañet2; ariamys companioni1; zulema menéndez1; hilda hernández3; m magdalena-rodríguez1; rene gato1 1departamento control de vectores, centro de investigación diagnóstico y referencia, instituto de medicina tropical ¨pedro kourí¨, la habana, cuba 2facultad de biología, universidad de la habana, la habana, cuba 3departamento de parasitología, centro de investigación diagnóstico y referencia, instituto de medicina tropical ¨pedro kourí¨, la habana, cuba (received 23 jan 2018; accepted 8 jan 2019) abstract background: the efficacy of biolarvicides may be influenced by species of mosquito, larval age and density, temperature, water quality, bacterial formulation, and others. the aim of this study was to evaluate the influence of temperature and chlorine on larvicidal activity of bacillus thuringiensis cuban isolates against aedes aegypti. methods: the influence of temperature (25, 30, 35 °c) and chlorine (2.25mg/l) on the larvicidal activity of eleven b. thuringiensis cuban isolates (collected between 2007 and 2009) were tested under laboratory conditions following who protocols. bioassay data were analyzed by probit program. the effect of chlorine and temperature (25, 30, 35 and 40 °c) on the cry and cyt proteins of these isolates was determined by sds-page polyacrylamide gel electrophoresis. results: the pathogenicity of the isolates u81, x48 was affected at 35 °c. however, a21, a51, l910, and r89 isolates increase their entomopathogen activity at 35 °c. no differences were observed in toxicity of m29, r84, r85 and r87 isolates at different temperatures. the cry 4, cry 10 and cry 11 proteins were reduced in a21, x48, r85 isolates at 35 and 40 °c. the cyt proteins were reduced at 35 and 40 °c in a21, x48, r85, and a51 isolates. in l910 and r84 isolates, the cyt toxin was degraded only at 40 °c. in chlorinated water, the lethal concentrations 50 and 90 in a21, a51, m29, r84, u81, and x48 isolates were increase. conclusion: a21, a51, l910, r85, and x48 isolates have a strong larvicidal activity for the treatment of ae. aegypti breeding’s sites exposed to high temperature and chlorine. keywords: mosquitoes; biological control; bacillus thuringiensis; bioassays; chlorine introduction mosquitos from the genus aedes, specifically ae. aegypti (linnaeus) and ae. albopictus (skuse) are responsible to the arbovirus transmission and are closely related to urbanized environments. for this reason, this genus has become the main vector of arboviruses in the world (1). vector control is the best way to reduce the arbovirus’s incidence. the ae. aegypti control should be directed to the elimination of immature and adults’ stages near to the urbanizing sites (2). for many years, bacillus thuringiensis for mulations have been the most important biopesticide used for agriculture and health (3). they have the advantages of specificity, high efficiency and environmentally safe (4). bacillus thuringiensis is a gram-positive sporulated bacterium. this bacterium in limited nutrient conditions sporulates and produces parasporal crystals with natural insecticidal properties. these crystals showed specific toxicity against invertebrates of the orders: lepidoptera, diptera, coleoptera, some nematodes, mites, and protozoa (5). *corresponding author: dr aileen gonzález-rizo, e-mail: aileen@ipk.sld.cu, ailen@infomed.sld.cu mailto:ailen@infomed.sld.cu j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 40 http://jad.tums.ac.ir published online: april 27, 2019 thousands of b. thuringiensis strains have shown considerable variability in their insecticidal toxicity. however, only highly toxic strains are used for biopesticide production (3). different factors like temperature and water quality affect the larvicidal effect of b. thuringiensis suspension (6, 7). this lack of residual activity is due to the low stability of its cry and cyt toxins and the poor recycling of spores under field conditions (6, 7). therefore the present study assessed the influence of temperature and water chlorination on the larvicidal activity of eleven b. thuringiensis cuban native isolates (8, 9) in order to select the better isolates to control ae. aegypti larvae in breeding sites. materials and methods reference strains bacillus thuringiensis serotype h-14: ips 82 from the international entomopathogenic bacillus centre, institute pasteur; paris, france. mosquitoes: ae. aegypti (rockefeller strain). mosquitoes were reared in 18x 18x 18 inch collapsible cages (bio quip, usa) maintained at 26 ºc±0.5 ºc in 80–85% relative humidity (rh) with a photoperiod of 12: 12h (light/ dark). a continuous supply of sucrose solution was provided. female mosquitoes were given access to an anesthetized mouse and allowed to blood feed for 30min weekly. the larvae were fed finely powdered fish food. bacillus thuringiensis isolates a21, a51, l95, l910, m29, r84, r85, r87, r89, u81, x48 cuban native isolates from soil samples of cuba collected between 2007 and 2009 (8, 9). these isolates belongs to the entomopathogenic bacteria collection from the biological control laboratory of the tropical medicine institute, ¨pedro kourí¨, ipk, cuba. bacterial formulations the bacterial isolates and reference strain were inoculated in a fermentation medium consisted of sucrose (2g/l), bacteriological peptone (2g/l), yeast extract (1g/l), and inorganic salts (12.5mmol/l mgso4, 0.05mmol/l mnso4, 1.2mmol/l feso4, 1.2mm znso4, 25mmol/l cacl2), incubated at 30 °c 48–72h at 150rpm (retomed, cuba). the bacterial sporulation was monitored through microscope. when more than 90% of cells lysed, the sporulated broth culture was transferred to 4 °c considered the final product (fp). concentrations were expressed in mg/l (dry weight). larvicidal efficacy quantitative bioassays were conducted following who protocol (10). twenty-five larvae (iii-iv instar) were introduced into 120ml cups with 100ml of dechlorinating water. four replicates per dose were included and the experiments were repeated at least three times. five concentrations of fp that cause mortalities between 10% and 90% were accepted for validating the bioassay. mortality data were recorded after 24h of exposure and were used to calculate the lethal concentrations for 50% and 90% of exposed individuals (lc50 and lc90, respectively) through log-probit analysis (11). abbott formula was used and the survival percentages were corrected if necessary (12). these values were compared to those obtained for the reference strain in order to estimate the efficacy of each isolate. the means of larval mortality caused by each isolate against ae. aegypti were calculated. a value of p< 0.05 was considered statistically significant. once the lethal doses were calculated, the cl95/cl50 ratio was performed to determine how many times it is necessary to increase the lc50 in order to obtain higher mortality. a lower ratio is indicative of better formulation efficiency (13). effect of temperature on larvicida activity: taking into account the temperatures average in cuba (14). larvicidal efficacy bioassays were performed at temperatures of 25, 30 and 35 ºc. the effect of chlorine on larvicide efficacy: j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 41 http://jad.tums.ac.ir published online: april 27, 2019 the water for larvicidal efficacy bioassays was treated with sodium dichloroisocyanurate resulting in chlorine concentration of 2.25 mg/l, ph 6.8. the who guidelines on drinking water quality recommended not exceed the value of 5mg/l for free chlorine as sodium dichloroisocyanurate (15). the bioassays performed in dechlorinate water at 25 °c were used as control. the effect of abiotic factors on bacillus thuringiensis cry and cyt proteins temperature treatment: in order to determine how the temperature affected the main virulence factors of b. thuringiensis native isolates. the fp of isolates was exposed to a range of temperatures 25, 30, 35 and 40 °c for 72h. chlorine treatment: the fp was treated with sodium dichloroisocyanurate resulting in chlorine concentration of 2.25mg/l, ph 6.0 and incubated at 25 °c for 24h. protein profiles: after each treatment (temperature and chlorine), the fps were centrifuged (10,000 rpm for 20min) and the pellets were washed twice with 1mol/l nacl and then with distilled water. the pellet was re-suspended in 100μl of distilled water and 100 of sample buffer (500mmol/l tris-hcl ph 6.8, 10% sds, 4% 2-mercaptoethanol, 8% glycerol, 0.1 % bromophenol blue), and boiled at water bath for 5min (16). the protein profiles of the crystal components were determined by sds-polyacrylamide gel electrophoresis (page) (17) with 10% acrylamide separating gels. tenμl of each sample was loaded onto a gel immediately before electrophoresis. fiveμl of a molecular weight marker (broad range protein molecular weight markers, promega, usa) was added to each gel. the molecular weight of each protein was calculated with gelquant software version 2.7.0 (bio-imaging systems, israel). statistical analysis data from the evaluation of temperature were subjected to analysis of variance (anova) and means were separated at the 5% significance level by using the tukey hsd posttest. a log-transformation was used to calculate the slope values. the analysis of chlorine data was performed by t-student test. ethical approval this study was carried out according to the principles expressed in the declaration of helsinki. the protocols were approved by the institutional research ethical committee at the institute of tropical medicine ¨pedro kourí¨. results influence of temperature on the toxicity of native isolates of bacillus thuringiensis on ae. aegypti larvae the pathogenicity of the isolates u81 and ips-82 were affected at 35 °c. however, the isolates a21, a51, l910, r84, r85, and r89 showed a significant improvement of their concentration lethal 90 (cl90) at 35 °c compared to the cl90 obtained at 25 °c (p< 0.05). however, only the r89 and x48 isolates improve their efficiency at high temperature. no differences were observed in toxicity of m29 and r87 isolates when bioassays were performed at different temperatures (table 1). effect of temperature on protein profile the stability of these proteins at different temperatures (25, 30, 35 and 40 °c) was tested by sds-page (fig. 1). all four bands corresponding with cry and cyt proteins were observed after treatment at 25 °c. after treatment at 35 and 40 °c the bands corresponding to cry 4, cry 10 and cry11 proteins were observed reduced in a21, x48, r85 isolates. by another hand, cyt protein reduction by temperature treatment was observed in a21, x48, r85 and a51 isolates at 35 and 40 °c. in l910 and r84 isolates, the cyt toxin was degraded only by 40 °c treatment (fig. 1). j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 42 http://jad.tums.ac.ir published online: april 27, 2019 the effect of chlorine on the larvicidal activity of native isolates chlorine had negative effects on the larvicidal activity of b. thuringiensis cuban isolates. lethal concentrations 50 and 90 were increased significantly in a21, a51, m29, r84, u81 and x48 isolates (table 2). however, a21 efficiency was improved in chlorinate water. the a51 and m29 were in the same range at both con ditions (chlorinated and dechlorinated water). there were not differences in lethal concentrations 50 and 90 of l95 and l910 isolates. the lc90 of r89 was reducing significantly in chlorinated water (table 2). the protein analysis by sds-page does not reveal visible reduction of the major virulence factors of these isolates (fig. 2). table 1. entomopathogenic activity [lethal concentration (lc50 and lc90)] and efficiency of isolates of b. thuringiensis on aedes aegypti at 25, 30 and 35 °c isolates and strains variable 25 °c 30 °c 35 °c a21 lc50 (mg/l) (cl) 0.00374 (0.00329–0.00422) 0.00133 (0.00121–0.00145) 0.00070 (0.00062–0.00079)* lc90 (mg/l) (cl) 0.01278 (0.0106–0.01609) 0.00411 (0.00352–0.00508)* 0.00464 (0.0037–0.00606)* lc95 (mg/l) (cl) 0.01810 (0.01455–0.02400) 0.005704 (0.00487–0.00790)* 0.00790 (0.00603–0.01105)* efficiency 4.84 4.29 11.2 a51 lc50 (mg/l) (cl) 0.00153 (0.00135–0.00173) 0.00070 (0.00061–0.00080)* 0.00002 (0.000001–0.00007)* lc90 (mg/l) (cl) 0.00455 (0.00374–0.00593) 0.001602 (0.00132–0.00211)* 0.00012 (0.00007–0.00030)* lc95 (mg/l) (cl) 0.00621 (0.00491–0.00855) 0.00226 (0.00202–0.03230)* 0.00035 (0.00023–0.00093)* efficiency 4.05 3.74 13.78 l95 lc50 (mg/l) (cl) 0.09819 (0.08325–0.11700) 0.07864 (0.06237–0.10706)* 0.06140 (0.05490–0.069680)* lc90 (mg/l) (cl) 0.21750 (0.17275–0.30899) 0.38221 (0.23742–0.82760) 0.20240 (0.15830–0.28520) lc95 (mg/l) (cl) 0.2724 (0.23088–0.3247) 0.5983 (0.34174–1.5000) 0.9345 (0.83600–1.05940) efficiency 2.77 7.6 15.22 l910 lc50 (mg/l) (cl) 0.01019 (0.00941–0.01093) 0.01010 (0.00808–0.01300) 0.00199 (0.00179–0.00219)* lc90 (mg/l) (cl) 0.01920 (0.01757–0.02185) 0.02119 (0.01577–0.03689) 0.00593 (0.00520–0.00695)* lc95 (mg/l) (cl) 0.02316 (0.0206–0.02706) 0.02614 (0.01864–0.0508) 0.00808 (0.00689–0.00989)* efficiency 2.55 2.59 4 m29 lc50 (mg/l) (cl) 0.06569 (0.05833–0.07440) 0.05458 (0.04310–0.07440) 0.02447 (0.02111–0.03178) lc90 (mg/l) (cl) 0.13652 (0.11449–0.17510) 0.32005 (0.18700–0.83119) 0.06622 (0.05640–0.15280) lc95 (mg/l) (cl) 0.1678 (0.1368–0.2250) 0.5284 (0.2779–0.71686) 0.08746 (0.07544–0.11357) efficiency 2.56 9.62 3.57 j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 43 http://jad.tums.ac.ir published online: april 27, 2019 r84 lc50 (mg/l) (cl) 0.00954 (0.00862–0.10358) 0.00293 (0.00254–0.00337) 0.00284 (0.00244–0.00339) lc90 (mg/l) (cl) 0.01913 (0.01741–0.02162) 0.00717 (0.00588–0.00958)* 0.00803 (0.00610–0.01230)* lc95 (mg/l) (cl) 0.02573 (0.0235–0.0298) 0.00924 (0.00800–0.01219) 0.01080 (0.00779–0.01794) efficiency 2.72 3.15 3.8 r85 lc50 (mg/l) (cl) 0.00793 (0.00697–0.00901) 0.00189 (0.00153–0.00220) 0.00107 (0.00091–0.00126) lc90 (mg/l) (cl) 0.01700 (0.01478–0.02176) 0.00496 (0.00410–0.00665)* 0.00507 (0.00359–0.00855)* lc95 (mg/l) (cl) 0.02170 (0.01792–0.02847) 0.00652 (0.00516–0.00959)* 0.00788 (0.00518–0.01498)* efficiency 2.77 3.45 7.37 r87 lc50 (mg/l) (cl) 0.02897 (0.02616–0.03232) 0.00565 (0.00500–0.00643) 0.00253 (0.00199–0.00325) lc90 (mg/l) (cl) 0.07831 (0.06713–0.09530) 0.0105 (0.00886–0.01351) 0.01242 (0.00821–0.02462) lc95 (mg/l) (cl) 0.10400 (0.0866–0.13102) 0.0125 (0.0109–0.0151) 0.01950 (0.01182–0.0.04533) efficiency 3.58 2.21 7.7 r89 lc50 (mg/l) (cl) 0.07146 (0.06444–0.07903) 0.01252 (0.00527–0.01637) 0.00529 (0.00302–0.00649) lc90 (mg/l) (cl) 0.17308 (0.14962–0.20820) 0.05094 (0.03350–0.27200)* 0.01191 (0.01020–0.04760)* lc95 (mg/l) (cl) 0.2223 (0.1873–0.27823) 0.07584 (0.043416– 0.78746)* 0.01499 (0.00799–0.09271)* efficiency 3.14 6.06 2.83 u81 lc50 (mg/l) (cl) 0.00461 (0.00424–0.00491) 0.00163 (0.00145–0.00181)* 0.00466 (0.00346–0.00683) lc90 (mg/l) (cl) 0.00905 (0.00810–0.01043) 0.00332 (0.00285–0.00409)* 0.04605 (0.02259–0.15460)* lc95 (mg/l) (cl) 0.01095 (0.0096v0.01304) 0.00406 (0.00339–0.00523)* 0.08895 (0.03814–0.3774)* efficiency 2.37 2.48 19.08 x48 lc50 (mg/l) (cl) 0.00213 (0.00192–0.00236) 0.00079 (0.00060–0.00090)* 0.00222 (0.00200–0.00248) lc90 (mg/l) (cl) 0.00527 (0.00450–0.00643) 0.00094 (0.00082–0.00125)* 0.00585 (0.00486–0.00746) lc95 (mg/l) (cl) 0.00945 (0.00825–0.01127) 0.00108 (0.009212– 0.01576)* 0.007677 (0.00619–0.01028) efficiency 4.44 1.36 3.46 ips-82 lc50 (mg/l) (cl) 0.00224 (0.00170–0.00273) 0.00067 (0.00061–0.00073)* 0.00567 (0.00490–0.00680)* lc90 (mg/l) (cl) 0.00567 (0.00457–0.00749) 0.00177 (0.00157–0.00207)* 0.02417 (0.01740–0.03865)* lc95 (mg/l) (cl) 0.00892 (0.00731–0.01174) 0.002128 (0.001805–0.00262) 0.0364 (0.0246–0.06371)* efficiency 3.3 3.17 6.41 *p≤ 0.05 cl: 95% confidence limits efficiency: lc95/lc50 table 1. continued … j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 44 http://jad.tums.ac.ir published online: april 27, 2019 table 2. entomopathogenic activity (lethal concentration (lc) 50 and 90) and efficiency of isolates of bacillus thuringiensis on aedes aegypti in dechlorinated water and chlorinated water isolates and strains variable dechlorinate water 25 °c chlorinate water 25 °c a21 lc50 (mg/l) (cl) 0.00374 (0.00329–0.00422) 0.02342 (0.02052–0.02707)* lc90 (mg/l) (cl) 0.01278 (0.0106–0.01609) 0.05545 (0.04470–0.07637)* lc95 (mg/l) (cl) 0.01810 (0.01455–0.02400) 0.0689 (0.05568–0.10375)* efficiency 4.84 2.79 a51 lc50 (mg/l) (cl) 0.00153 (0.00135–0.00173) 0.01132 (0.00901–0.01404)* lc90 (mg/l) (cl) 0.00455 (0.00374–0.00593) 0.04702 (0.03150–0.10350)* lc95 (mg/l) (cl) 0.00621 (0.00491–0.00855) 0.04700 (0.03730–0.05800)* efficiency 4.05 4.14 l95 lc50 (mg/l) (cl) 0.09819 (0.08325–0.11700) 0.12627 (0.11321–0.14025) lc90 (mg/l) (cl) 0.21750 (0.17275–0.30899) 0.23140 (0.19980–0.28970) lc95 (mg/l) (cl) 0.2724 (0.23088–0.3247) 0.27534 (0.23099–0.3615) efficiency 2.77 2.14 l910 lc50 (mg/l) (cl) 0.01019 (0.00941–0.01093) 0.01730 (0.01460–0.02122) lc90 (mg/l) (cl) 0.01920 (0.01757–0.02185) 0.03460 (0.02680–0.05490) lc95 (mg/l) (cl) 0.02316 (0.0206–0.02706) 0.04157 (0.01849–0.24528) efficiency 2.55 2.42 m29 lc50 (mg/l) (cl) 0.06569 (0.05833–0.07440) 0.10393 (0.09322–0.11521)* lc90 (mg/l) (cl) 0.13652 (0.11449–0.17510) 0.21480 (0.18660–0.25910)* lc95 (mg/l) (cl) 0.1678 (0.1368–0.2250) 0.2639 (0.2237–0.33110)* efficiency 2.56 2.53 r84 lc50 (mg/l) (cl) 0.00954 (0.00862–0.10358) 0.03412 (0.02797–0.04334)* lc90 (mg/l) (cl) 0.01913 (0.01741–0.02162) 0.14650 (0.09805–0.28373)* lc95 (mg/l) (cl) 0.02573 (0.0235–0.0298) 0.16262 (0.14396–0.22360)* efficiency 2.72 5.16 r85 lc50 (mg/l) (cl) 0.00793 (0.00697–0.00901) 0.01244 (0.01097–0.01425) lc90 (mg/l) (cl) 0.01700 (0.01478–0.02176) 0.02232 (0.01925–0.03167)* lc95 (mg/l) (cl) 0.02170 (0.01792–0.02847) 0.02772 (0.02223–0.04034)* efficiency 2.77 2.15 r87 lc50 (mg/l) (cl) 0.02897 (0.02616–0.03232) 0.03800 (0.03280–0.04600) lc90 (mg/l) (cl) 0.07831 (0.06713–0.09530) 0.10720 (0.08040–0.16730) lc95 (mg/l) (cl) 0.10400 (0.0866–0.13102) 0.14250 (0.12460–0.18640) efficiency 3.58 3.75 r89 lc50 (mg/l) (cl) 0.07146 (0.06444–0.07903) 0.04899 (0.04208–0.05907) lc90 (mg/l) (cl) 0.17308 (0.14962–0.20820) 0.13740 (0.10310–0.21440)* lc95 (mg/l) (cl) 0.2223 (0.1873–0.27823) 0.1841 (0.1313–0.31279) efficiency 3.14 3.76 u81 lc50 (mg/l) (cl) 0.00461 (0.00424–0.00491) 0.01682 (0.01250–0.03050)* lc90 (mg/l) (cl) 0.00905 (0.00810–0.01043) 0.06720 (0.03501–0.03630)* lc95 (mg/l) (cl) 0.01095 (0.0096–0.01304) 0.0996 (0.04610–0.74310)* efficiency 2.37 5.9 x48 lc50 (mg/l) (cl) 0.00213 (0.00192–0.00236) 0.01140 (0.01140–0.01860)* lc90 (mg/l) (cl) 0.00527 (0.00450–0.00643) 0.05950 (0.03850–0.12500)* lc95 (mg/l) (cl) 0.00945 (0.00825–0.01127) 0.07182 (0.03585–0.19502)* efficiency 4.44 6.3 ips-82 lc50 (mg/l) (cl) 0.00224 (0.00170–0.00273) 0.01803 (0.01440–0.02332)* lc90 (mg/l) (cl) 0.00567 (0.00457–0.00749) 0.07023 (0.04757–0.13180)* lc95 (mg/l) (cl) 0.00892 (0.00731–0.01174) 0.12100 (0.08250–0.23779)* efficiency 3.3 6.73 *p≤ 0.05 cl: 95% confidence limits efficiency: lc95/lc50 j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 45 http://jad.tums.ac.ir published online: april 27, 2019 fig. 1. analysis of cry and cyt protein of a51, a21, r85 and r84 isolates by sds-page (coomassie brilliant blue-stained gel) lane mm: molecular weight marker, lanes-1: proteins profiles of fp treated at 25 °c, lanes-2: proteins profiles of fp treated at 30 °c, lanes-3: proteins profiles of fp treated at 35 °c, lanes-4: proteins profiles of fp treated at 40 °c fig. 2. analysis of cry and cyt protein by sdspage (coomassie brilliant blue-stained gel) lanemm: molecular weight marker, and lanes-1: proteins profiles of fp without chlorine, lanes-2: fp exposed to chlorine (2.25mg/l, ph 6.0) discussion bacillus thuringiensis is a viable alternative for insect control due to its specific toxicity against insect larvae. nevertheless, parasporal crystals activity can be affected by abiotic factors, such as high temperature (18). in cuba, the annual temperature average has increased since 1951, and in 1997 and 1998 reached the highest values throughout its history. overall, the temperature average of the years after 2000 was the warmest of all available climate records (14, 19) increased in dry season, +2.0 °c, and in jun–aug: +0.8 °c with higher percentage of days with maximum temperature ≥ 30 °c (20). climate variability influences vector population dynamics, distribution and disease transmission (21). dengue transmission is associated in space and time with local climate effects on survival of its vector ae. aegypti. thus, more rain and higher temperature generates more transmission (22, 23). obviously, the biolarj arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 46 http://jad.tums.ac.ir published online: april 27, 2019 vicides should keep high entomopathogen activity at temperatures values over annual average. most of our isolates increased their toxicity when temperature raise from 25 to 30 °c and five isolates from 30 to 35 °c. similar results were obtained with b. thuringiensis var. israelensis on chironomus kiiensis larvae, results showed changes of lc50 doses with a temperatures variation from 15 ºc to 30 ºc (24). however, no detected differences in larvicidal activity of b. thuringiensis (vectobac as12, abg-6164, and ac-513695) against culex quinquesfasciatus with water temperature variations from 15 to 30 °c. the reduction in lc90 in the a21, a51, l910, r84, r85, r89, u81, and x48 isolates, when the temperature increase from 25 to 30 °c may be due to the influence of this factor on the ae. aegypti larvae behavior. the growth of larvae is accelerated with temperature raise, so they begin to feed faster and more toxins cry and cyt are ingested (24, 26). in previous studies, cry4, cry10, cry11 and cyt proteins were established as the principal virulence factors of cuban native b. thuringiensis isolates (8, 9). nevertheless we associated the reduction of the cry and cyt toxins under then effect of temperature with lack of toxicity of u81 isolate (25–35 °c) and worse efficiency of a21 and a51 isolates. taking into account that cyt proteins potentiate the action of cry (7), the loss of these proteins may be the cause of the significant reduction in the toxicity and efficiency. we also demonstrated reduction of cry and cyt proteins at 40 °c but no bioassays were performed. we speculate that in natural breeding sites the toxicity of biolarvicides fail by the loss of these proteins at temperatures over 40 °c. this result should be taken into account for the preservation of b. thuringiensis aqueous formulations. the influence of temperature over cry proteins has been demonstrated by different authors. the cry1ac protein was degraded by exposure to temperature of 35 °c (27). a reduction in cry protein after 50 °c treatment was demonstrated by sds-page. they achieved temperature protection of the internal crystals by micro-capsulation (18). according to our results the isolates, a21, a51, l910, r84, r85 and x48 keep acceptable larvicidal activity at high temperatures, so they would be excellent candidates for the development of formulations better adapted to the climate change effects. the variability of temperature average, increase the incidence of water and foodborne diseases (20). chlorine is the most used domestic water disinfectant in the world to prevent these diseases (28), and the mainly breeding sites of ae. aegypti are domestic water containers (29). therefore, an ideal biolarvicide should keep its activity in chlorinated water. a biologist from the cuban vector control programme in matanzas and santiago de cuba provinces referred to the reduction of operational effectiveness of biolarvicides based in b. thuringiensis in chlorinated water (personal communications). in our study, the bioassays showed a significantly increase in cl50 in chlorinated water but there are not visible reduction of cry and cyt toxins by treatment with chlorine in sds-page analysis. chlorine is a non-selective oxidant with a number of effects over the living systems: reacts with a variety of cellular components (30), deactivates enzymatic active sites, decreases the biological functions of proteins and produces deleterious effects on dna (31). in addition chlorinated waters affect the permeability of the cytoplasmic membrane (32), which could lead to cell death and inhibit the growth of b. thuringiensis (33). different layers of proteinaceous present in the spore act as a protection from chemical attacks, including oxidizing agents like hydrogen peroxide, sodium hypochlorite, chlorine dioxide, or ozone (34). in case of the chlorinereleasing products produce the lost of refractivity, separation of the spore coats from the cortex, extensive discharge of ca+, dipicolinic j arthropod-borne dis, march 2019, 13(1): 39–49 a gonzález-rizo et al.: effect of chlorine … 47 http://jad.tums.ac.ir published online: april 27, 2019 acid, and dna and finally lysis occurred (35). however, b. thuringiensis subs israelensis spores are more resistant to chlorine than other spores of either b. anthracis or b. cereus (33). it may be that b. thuringiensis spores resistance to the chlorine effect allows the larvicidal effect in our native isolates when the doses were increases and explain why there were not a significant dose variation in a l95, l910 isolates and the increase of the toxicity 90 in r89 isolate. in spite of the significantly doses variations under chlorine treatment, a21, a51, l910, r85 and x48 isolates had a better larvicidal activity than ips 82 control strain. for this reason these isolates will be good candidate for ae. aegypti breeding site treatment. our results have shown the possibility to treat the chlorinated ae. aegypti breeding sites with b. thuringiensis based products. conclusion our results showed that a21, a51, l910, r85, and x48 isolates have a strong larvicidal activity at 25, 30, 35 °c and chlorinated water. the use of these isolates as biolarvicides can reduce the operational problems in ae. aegypti breeding´s sites. acknowledgements we are thankful to our colleagues jorge anaya and israel garcia who provided expertise in the maintened and breeding of ae. aegypti (rockefeller strain) in the laboratory conditions. the authors would like to thank to the public cuban ministry of health (minsap) for its supports to this research as a task of the project 1701041. the authors declare that there is no conflict of interest. references 1. patterson j, sammon m, garg m (2016) dengue, zika and chikungunya: emerging arboviruses in the new world. western j emerg med. 17(6): 671–679. 2. who (2009) dengue guidelines for diagnosis, treatment, prevention and control. world 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prabhakara rao avs (1977) mechanism of disinfection: effect of chlorine on cell membrane functions. water res. 11(8): 727–729. 33. rice e, adcock n, sivaganesan m, rose l (2005) inactivation of spores of bacillus anthracis sterne, bacillus cereus, and bacillus thuringiensis subsp. israelensis by chlorination. appl environ microbiol. 71(9): 5587–5589. 34. setlow p (2006) spores of bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. j appl microbiol. 101(3): 514–525. 35. stockel s, schumacher w, meisel s, elschner m, rosch p, popp j (2010) raman spectroscopy-compatible inactivation method for pathogenic endospores. appl environ microbiol. 76(9): 2895– 2907. j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 334 http://jad.tums.ac.ir published online: september 30, 2019 short communication molecular detection of leishmania major in hemiechinus auritus, a potential reservoir of zoonotic cutaneous leishmaniasis in damghan, iran *behrad pourmohammadi1, 2; sadegh mohammadi-azni3 1department of parasitology, school of medicine, semnan university of medical sciences, semnan, iran 2department of health education and promotion, school of health, semnan university of medical sciences, semnan, iran 3damghan health center, semnan university of medical sciences, semnan, iran (received 1 jan 2018; accepted 9 jul 2019) abstract background: zoonotic cutaneous leishmaniasis caused by leishmania major is endemic in 17 of 31 iranian provinces. various species of rodents have been introduced as the main reservoirs of the disease. this study was conducted to determine the natural infection of hedgehogs with leishmania spp. in an endemic area of the disease, northern iran. methods: fifteen long-eared hedgehogs were captured alive during 18 months study period, from apr 2015 to sep 2016, in damghan city, semnan province, iran. the animals were identified using apparent characteristics and to determine the leishmania infection, impression smears were prepared from their ear lobes, hind feet, livers, and spleens. microscopic examination and semi-nested pcr were applied to determine the infection and to identify the parasites species respectively. results: all examined animals were identified as hemiechinus auritus (family: erinaceidae). in microscopic examination, 8 (53.3%) samples were shown to be infected with leishmania parasites. the higher and lower rate of the infection was observed in the ears as well as the feet and in the liver specimens, 53.3%, and 33.3% respectively. forty percent (6/ 15) of the samples were molecularly positive and all were identified as l. major parasites. all the examined animals in autumn and 50% of them in summer were shown to be infected with leishmania parasites. conclusion: this study demonstrated the natural infection of h. auritus with l. major for the first time in damghan city and introduced these mammals as new potential reservoirs of zcl in the study area. keywords: hemiechinus auritus; potential reservoir; leishmania major; semi-nested pcr; iran introduction leishmanioses, a significant group of protozoan parasitic diseases caused by around 20 of 30 obligate intracellular parasite species of genus leishmania in humans, are endemic and occur in more than 100 countries of four continents of the world including; asia, africa, europe, and america. the disease represents four types of clinical manifestations from the selfhealing cutaneous to the life-threatening, visceral form, if untreated (1-3). so far, more than 50 species of genus leishmania have been identified, of which 31 species can infect mammals and 20 species can cause the various types of leishmaniasis in humans with either zoonotic or anthroponotic origins (1, 4). no more than 70 species of approximately 900 sand fly species have been implicated in the transmission of leishmaniasis (5). leishmaniasis is still one of the most neglected diseases in the world which affecting largely the poorest of the poor, mainly in developing countries (3). overall, 350 million people are at risk of all types of disease and an estimated 1.6 million cases occur annually all over the world. there is also an estimation of 0.2–0.4 million cases of visceral leishmaniasis (vl) and 0.7–1.2 million cases of cutaneous leishmaniasis (cl) in the endemic areas of the world (6). cutaneous leishmaniasis is an endemic disease in 88 countries worldwide (7). approxi*corresponding author: dr behrad pourmohammadi, e-mail: behrad89@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 335 http://jad.tums.ac.ir published online: september 30, 2019 mately, 90% of cl cases have been reported from iran, algeria, afghanistan, saudi arabia, syria, brazil and peru (8). iran is one of the ten countries that around 70–75% of new cl cases occur in it (6). cutaneous leishmaniasis (cl), is one of the two clinical forms of the disease in iran, is more prevalent with a prevalence rate of 1.8–37.9% and around 30000 new cases annually (9). globally, it is estimated to be the ninth cause of human infectious diseases' burden (10). the zoonotic form of the disease (zcl) due to l. major is the most prevalent type of the cl mainly in rural areas of 17 out of 31 provinces of the country (11, 12). according to the geographical distribution and various types of reservoirs and vectors of zcl, three epidemiological types of the disease have been classified in iran (13). various species of rodents including rhombomys opimus, nesokia indica, meriones libycus, tatera indica, m. hurrianae, rattus norvegicus, m. persicus, and mus musculus have been identified as reservoir and or potential reservoir hosts of the parasite in the country (11, 14-19). furthermore, the parasite species have recently been isolated and reported in gorillas (20) and hedgehogs in several parts of the world (21-24). phlebotomus papatasi, the most prevalent species of phlebotomus genus, is the predominant and the only proven vector of zcl (13, 25). hedgehogs are distributed in various parts of iran. these mammals are found in the wild and in a lower amount of pet animals. despite wide distribution in iran, these animal species have not been studied enough about their parasites (26). hedgehogs are omnivorous. they feed mostly on invertebrates such as slugs, beetles, caterpillars, earthworms, and some other insects. furthermore, grass snakes, frogs, vipers, fish, young birds, birds′ eggs and small rodents can be fed by those small mammals (27). the long-eared hedgehog lives in burrows that it makes or finds. it is recognized by its long ears. they inhabit a few various types of dry steppes, deserts and semi-deserts. they often inhabit in oasis and around human settlements. the long-eared hedgehogs live in burrows that they dig under bushes with a length of 45cm long with one opening. they may live in abandoned burrows of other small mammals. they are nocturnal solitary hedgehogs. during the day, they are found resting under rocks, hollows or rock piles (28). in a study to determine the reservoirs and vectors of leishmania parasites in damghan city, an endemic focus of zcl in iran, we accidentally encountered with a hedgehog captured in wire live trap set out for rodents in the southern part of the city, where the rodents infection with l. major parasites have been proven more recently (11). the existence of some recent reports about the infection of the hedgehogs with leishmania parasites in other parts of the world drew our interest in assessing leishmania spp. in these animals. therefore, the present study was conducted to investigate the possible leishmania infection of these small mammals in the city in an eighteen month period, from apr 2015 to sep 2016. materials and methods study area and period the present study was carried out during 4 apr 2015 to 22 sep 2016 in damghan, a wellknown endemic region of zcl in iran. zoonotic cutaneous leishmaniasis have been reported in an epidemic form in damghan district (at about 36°10′ n, 54°20ʹ e) with around 1000 cases in 1999. since 1999 until now the disease remains in endemic form with around 100 cases every year (29, 30). damghan is a city situated between semnan and shahroud cities of semnan province with an altitude of 1170m and arid climate. the temperature and annual rainfall averages of the city are about 16.3 °c and 120mm respectively. the city is bounded on the plain areas in the south with 23.5 °c and the mountainous areas in the north with 9.8 °c average http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 336 http://jad.tums.ac.ir published online: september 30, 2019 temperature. however, it is classified as a desert region with an arid climate (31, 32). animal capturing the first animal was captured in a wire live trap (sherman) laid at the entrance of the rodent burrow in farmland of wheat close to houses (fig. 1) and the rest of the animals were found in their daily resorts, holes in the old brick walls, and were caught by hand. the captured animals were put in a carton and were transferred to the laboratory. the ethical code (ir.semums.rec.1397. 136) was obtained from the ethics committee of semnan university of medical sciences. measurements and sampling after transferring to the laboratory and taking photographs, the animals were slightly anesthetized using chloroform for recording the apparent characteristics; sex, probable ulcers in each part of the body, and external measures of the ear, the hind foot, the head, the body and the tail of each animal. impression smears were made from the hindfoot and the ear lobe on clean glass slides. then, the sampling of spleen and liver were done after euthanizing the animals following deep anesthesia and autopsy. the animals were identified according to morphologic characteristics and using valid identification key (33). microscopic investigation all in absolute methanol fixed smears were stained using 5% giemsa stain for 25min and were carefully examined under a light microscope at 1000× magnification. at least two slides of each sampled organs (ear lobe, hind foot, spleen, and liver) were checked to make the final decision about parasitological results. molecular identification for parasite species identification, the ears and feet smears of all fifteen samples were checked by a sensitive molecular technique, semi-nested pcr assay. dna extraction the dry giemsa-stained smears of the ears and feet of each animal were scratched off the slides using lysis buffer and leishmania parasites total dna was extracted by the yta genomic dna extraction mini kit (yekta tajhiz azma, iran) following the manufacturer's training. semi-nested pcr amplification the semi-nested pcr was performed, as formerly described and applied by other researchers (34-36), for detection of kinetoplast dna (kdna) of l. major in the specimens prepared from ear lobes and hind feet of the hedgehogs. the applied primers were the forward linr4 (5′ggg gtt ggt gta aaa tag gg-3′), the first-round reverse lin17 (5′ttt gaa cgg gat ttc tg-3′), and the secondround reverse lin19 (5'-cag aac gcc cct acc cg-3'). in the first-round amplification reaction totally 10μl mixture containing 1μm 10x buffer, 200μm of each dntp, 1.5mm mgci2, 1μm primer linr4, 0.2μm primer lin17, 1 unit taq polymerase (cinnagen, tehran, iran) and 1.5μl of target kdna from each hedgehog’s specimens were prepared. the mixture was overlaid with mineral oil and then incubated in a cg1-96 thermocycler (corbett research, sydney, australia) at 94 °c for 5min succeeded by 17 cycles, each consisting of 30sec at 94 °c, 30 sec at 52 °c and 30sec at 72 °c. after the last cycle, the extension was continued for a further 10min then held at 4 °c. the second-round amplification was accomplished following the addition of 90μl of buffer comprising mgcl2, dntps and taq polymerase, as described for the first round, and primer linl9 (final concentration 1μm) for 33 cycles (94 °c for 30sec, 58 °c for 30sec and 72 °c for 1min). kdna of reference strain of l. major (mhom/ir/54/lv39) and distilled water were used as positive and negative controls respectively. 15μl of each pcr products were resolved in 5μl loading buffer and then electrophoresed in a 1.5% agarose gel in tbe buffer containing 0.75% ethidium bromide and visualized under ultraviolet transilluminator. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 337 http://jad.tums.ac.ir published online: september 30, 2019 leishmania parasites were identified by comparison of pcr products of specimens with the reference strains and molecular weight markers. results animal measurements and descriptions during an 18-month period of the study, 4 apr 2015 to 22 sep 2016, 15 (8 male and 7 female) porcupines were caught from different locations in the city. the samples were caught in three seasons of the year. ten of 15 examined animals were captured in the summer. none of the animals were caught in winter. all animals were morphologically identified as long-eared hedgehog, hemiechinus auritus (gmelin, 1770) belong into family; erinaceidae (33). the animals' dorsal spines were white on the peak with darker banding inferior. they had a light-colored underside along with long and highly visible ears (length, 41– 49mm) on the head with whitish hairs on the tips of them. the tops and heels of their feet (hind feet length, 37–45mm) were covered with hair but the soles were bare (fig. 2). the length of the heads, the bodies, the tails and the ears were 50–65mm, 205–230mm, 21–26mm and 41–50 mm respectively (table 1). none of the animals had any skin ulcers. microscopic and molecular investigation in the microscopic examination of the prepared smears from four organs (ear lobes, hind feet, liver, and spleen) of each 15 animals, eight (53.3%) of samples were shown to be infected with leishmania parasites. the higher rates of infection were seen in the samples of ears the same as feet with 53.3% for each. the lower rates of infections (33.3%) were observed in liver specimens (table 1). all the captured and the examined animals in autumn (3/3, 100%) and 50% (5/10) of the examined animals in summer were shown to be infected with leishmania parasites. in the molecular assay, six of the specimens (40%) were identified as l. major with sharp bands (fig. 3). two of the parasitological positive samples were negative in the molecular assay. table 1. morphologic characteristics, measurements, parasitological and molecular results of the hedgehogs captured from damghan city, semnan province, iran, 2015 to 2016 animals g cd measurements (mm) parasitological result molecular result h f b t e e l s f h1 f 22 may 2015 63 44 225 24 47 + + + l. major h2 m 28 may 2015 59 43 213 24 49 + + + h3 m 25 jun 2015 65 45 222 25 50 h4 f 3 jul 2015 64 41 230 26 45 h5 m 4 sep 2015 51 40 212 23 41 + + + l. major h6 f 14 sep 2015 50 38 205 21 46 + + + + l. major h7 m 27 sep 2015 62 42 225 25 45 h8 m 1 nov 2015 53 40 215 23 43 h9 f 3 dec 2015 53 37 210 23 42 + + + + l. major h10 f 1 jul 2016 60 42 224 24 44 h11 m 8 jul 2016 52 41 215 22 42 + + + h12 m 22 jul 2016 54 39 217 23 43 l. major h13 f 5 aug 2016 62 41 224 25 47 h14 m 19 aug 2016 57 42 218 24 48 + + + h15 f 9 sep 2016 61 43 225 25 50 + + + + l. major total 15 8 5 6 8 6 (40%) 8 (53.3%) abbreviations: g= gender, cd= capture date, h= head, f= foot, b= body, t= tail, e= ear, l= liver, s= spleen http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 338 http://jad.tums.ac.ir published online: september 30, 2019 fig. 1. the hedgehog captured in wire trap set up at rodent’s burrow entrance and the animal found in a resort in the radix of an ancient wall surrounding the city fig. 2. the examined hedgehogs in dorsal and ventrolateral view fig. 3. the pcr-based productsʹ gel electrophoresis of the samples prepared from the hemiechinus auritus samples captured from damghan city. the bands correspond to reference strain of leishmania major (lane 1), hind feet smears (lanes 2 and 5), ear lobes samples (lanes 6, 7 and 8), ladder marker (lane 4) and negative control (lane 3) discussion four species of hedgehogs including h. auritus (long-eared hedgehog), p. hypomelas (brandtʹs hedgehog), p. aethiopicus (desert hedgehog), and e. concolar (european hedgehog) have been identified and reported in different parts of iran (26, 37, 38). all the captured animals in the present study were belonging to hemiechinus auritus (longeared hedgehog). these spiny-coated small mammals are widely distributed throughout the country particularly in provinces surrounding the studied area, damghan city, semnan province. they have been reported from kurdistan, khuzestan, golestan, khorasan, qazvin, tehran, sistan and baluchistan and some other iranian provinces (37, 38). the long-eared hedgehogs like better to stay in middle climates, avoiding the hot desert and the colder mountain areas. moreover, these species of animals prefer moderate areas with average rainfall of 100–400mm. regarding arid climate of damghan city, with an average of 120mm annual rainfall, these species of mammals seem to be well adopted in the study region. however, environmental modifications induced by humans can lead to the imhttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 339 http://jad.tums.ac.ir published online: september 30, 2019 migration of some wild animals such as hedgehogs from their common geographical zones to humansʹ inhabited areas and subsequently being infected with new pathogens in new habitat (39). similar modifications increasingly occur recently in the study area. leishmania parasites were observed in 53.3 % (8/15) of the examined animals. in microscopic positive samples, the parasites were seen in at least three of five examined organs. all the infected animals were positive for leishmania species in ear lobes and hind feet smears. using the semi-nested pcr method, 40% (6/15) of the samples were identified as l. major. molecular methods have already been used as useful and sensitive methods for identifying leishmania parasites in potential reservoir hosts without the need to parasites isolation and culture (40). leishmania major parasite has also been identified and reported in one of the three (33.3 %) examined h. auritus from turkmen sahra, golestan province, a northern neighboring of the semnan province, using molecular and conventional methods (21). natural infection of hedgehogs with leishmania parasites have also been demonstrated in some other countries of the world such as tunisia and an endemic area of zcl in algeria. the examined hedgehogs have been identified as atelerix algirus, infected with l. major and l. infantum, in tunisia and both paraechinus aethiopicus and atelerix algirus, infected with l. major, in algeria (23, 24). moreover, leishmania dna has been detected in the hair of another species of hedgehog, erinaceus europaneus, in spain (22). these findings have been suggested that hedgehogs comprise potential reservoir hosts for leishmania parasites. the parasite species were not identified in two of the eight microscopic positive samples with applied specific primers. a similar result has been found in a survey among rodents, main reservoir hosts of zcl in the study area (11). however, some leishmania species other than l. major may exist and circulate between sand fly vectors and animal reservoirs in the region. leishmania gerbilli and l. turanica have already been reported in rodents from the other endemic areas of iran (41, 42). in the present study, all the hedgehogs were caught during three seasons; spring, summer, and autumn. the most examined animals (10/15) were caught in the summer. five of ten (50%) animals caught in summer and all captured in autumn were shown to be infected with leishmania parasites. any animal was observed and caught in winter, possibly due to the cold weather and the animals’ hibernation state. the longeared hedgehog spends the daytime in a burrow and may hibernate for up to 3.5 months over winter. in warmer areas, there is no extended winter hibernation but during periods of food scarcity, there may be aestivation (43). leishmania species were observed and identified in the samples obtained from the hedgehogs captured in each three above mentioned seasons of the year, from may to december. the activity period of the main vector of zcl, phlebotomus papatasi, in iran has been reported from early apr to mid-oct (44). in damghan district, two peaks of activity, in jun and sep, have been reported for sand flies, but most of the sand fly infection has been occurred in sep (29). regarding the leishmania infection of two examined animals (h1 and h2) captured in may, the animals were probably infected in the last peaks of sand flies activity and they have acted as the "maintenance hosts" of leishmania parasites in the region. the nests of captured animals were quite close to the rodents barrows. this condition could prepare a good chance of being bitten the hedgehogs by infected sand flies in resting and activity place of the animals. the presence of rodents (nesokia indica) and sand flies infected with l. major have been demonstrated in the studied locations (11). none of the examined mammals were shown to have any apparent skin lesions. other studies on hedgehogs (23) and rodents as reservoir hosts of leishmania species have shown similar results in a different focus of zcl. leishmania http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 334–343 b pourmohammadi and s mohammadi-azni: molecular … 340 http://jad.tums.ac.ir published online: september 30, 2019 major may create no or just slight skin lesions in rodents. therefore, it is not always simple to recognize the leishmania infection in wild animal reservoirs such as rodents (11, 17). regarding the infection of the h. auritus with l. major parasites in three studied seasons (late spring to late autumn), they can be incriminated as potential reservoir hosts of the leishmania spp. particularly in endemic areas of zcl. moreover, they can be considered as a “maintenance hosts” or even “amplifier hosts”. the “maintenance hosts” were defined as mammals infected and maintain the infection and the “amplifier hosts” were defined as the mammals that can display a characteristic that favors transmission the infection in addition to maintaining it (45). therefore, to confirm these characteristics, more field and also experimental studies are needed in the study area. conclusion the present study demonstrated the natural infection of h. auritus with l. major parasites for the first time in damghan city, an endemic area of zcl in iran. close contact between infected rodents mainly nesokia indica, hedgehogs, sensitive human population and also the presence of suitable sand fly vectors, principally phlebotomus papatasi, can lead to easily exposure of h. auritus, a potential reservoir of zcl, in the transmission cycle of the disease in the studied area. besides performing the fighting programs against rodents as the main reservoir hosts of the disease in endemic areas of zcl, infection of other susceptible mammals with leishmania parasites and possibly reservoir alteration must be seriously paid attention. acknowledgements the authors would like to thank the deputy of research and technology of semnan university of medical sciences for monetary support. the authors declare that there is no conflict of interests. references 1. ashford rw (2000) the leishmaniasis as emerging and reemerging zoonoses. int j parasitol. 30: 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molecular screening of mosquitoes (diptera: culicidae) for flaviviruses in a focus of west nile virus infection in northern iran vahideh moin-vaziri1; remi n charrel2; mehdi badakhshan3; xavier de lamballerie2; nourina rahbarian1; mulood mohammadi bavani3; *shahyad azari-hamidian4 1department of parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 2unité des virus emergents (uve: aix marseille univ, ird 190, inserm 1207, ihu méditerranée infection), marseille, france 3department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran 4department of health education, research center of health and environment, school of health, guilan university of medical sciences, rasht, iran (received 05 dec 2018; accepted 05 dec 2019) abstract background: mosquito-borne arboviruses such as west nile (wn), dengue, rift valley fever, and sindbis viruses are reported in iran, but large-scale studies have not been performed on mosquitoes to find their vectors. a molecular study of the adult mosquitoes (diptera: culicidae) for flaviviruses was carried out in a focus of wn infection, guilan province, northern iran. methods: mosquito collections were carried out in five stations of two counties (anzali and rasht) using light traps, hand catch by manual aspirators and night landing catch during august–september 2013 and 2014. molecular screening of wn virus and more widely for flavivirus rna was carried out using a specific pcr technique. results: in total, 1015 adult mosquitoes were collected including eight species representing four genera. the most prevalent species were aedes vexans (33.2%), culex tritaeniorhynchus (22%), cx. pipiens (20.7%), and anopheles maculipennis s.l. (15.6%). molecular screening was carried out on the 1015 mosquitoes after they were organized as 38 pools according to sex, species and trapping location. none of the pools were positive. conclusion: surveillance should be continued while increasing the sampling campaigns due to the presence of wetlands in the region and abundant species which are considered as vectors, feeding on both birds and humans. keywords: aedes; anopheles; coquilletidia; culex; flavivirus introduction based on the latest classification, mosquitoes (diptera: culicidae) include two subfamilies, 11 tribes, 41 or 113 genera (depending on the generic classification of the tribe aedini) and 3563 species (1). mosquitoes are involved in the transmission of several arboviruses belonging to different families such as flaviviridae, phenuiviridae and togaviridae (2). the family flaviviridae comprises 58 viruses that are of greatest concerns for human health. the genus flavivirus includes 53 virus species, of which 39 are transmitted by mosquitoes or ticks (3). west nile virus (wnv) (flaviviridae: flavivirus) is distributed in eurasia, africa, north and central america and australia. mosquitoes are the principal vectors of the virus and some virus isolations have been reported from soft and hard ticks (arachnida: ixodida). wild birds, especially wetland species, are the principal vertebrate hosts and also the virus has been isolated from mammals and frogs (4). the most recent checklist of iranian mosquitoes includes seven genera and 64 species (5, 6). subsequently, anopheles superpictus was *corresponding author: dr shahyad azarihamidian, e-mail: azari@gums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 391–398 v moin-vaziri et al.: a molecular screening of … 392 http://jad.tums.ac.ir published online: december 31, 2019 found to be a complex including two species in iran based on the internal transcribed spacer 2 (its2) sequences of rdna (7) which later were listed as species a and b (8). a new species of the an. hyrcanus group (an. hyrcanus spir) was recognized from southwestern iran, also based on its2 sequence data (9). more recently, the occurrence of aedes albopictus [stegomyia albopicta] and ae. unilineatus [stegomyia unilineata] in southeastern iran was reported (10, 11). finally, orthopodomyia pulcripalpis was reported in northern iran (12). several pathogens, which are known to be transmitted by mosquitoes, are reported in iran such as dirofilaria immitis and d. repens (13), different plasmodium spp. (9, 14), west nile, dengue viruses (denv) (flaviviridae: flavivirus), sindbis virus (sinv) (togaviridae: alphavirus) and rift valley fever virus (rvfv) (phenuiviridae: phlebovirus) (15–20). it is noteworthy that there is the possibility of some other mosquito-borne arboviral outbreaks including japanese encephalitis virus (jev) (flaviviridae: flavivirus) in the world health organization eastern mediterranean region (21). recently, infection to wnv has been confirmed by the polymerase chain reaction (pcr) technique in ae. caspius [ochlerotatus caspius] and culex pipiens in northwestern and northern iran, respectively (22, 23). west nile virus is reported in 26 provinces (out of 31) of iran in horses (17, 24, 25), humans (15–17, 26–30) and birds (31). guilan province in caspian sea littoral, northern iran, with vast wetlands is probably one of the foci of wnv where the infection is found in humans (1.4–10%) (16, 17, 26), horses (2.2–25%) (17, 24) and birds (especially common coot as a main reservoir) (62.7%) (31). surprisingly, despite the obvious importance in the emergence of viral diseases caused by flaviviruses such as wnv and denv, little data is published about their mosquito vectors in iran and large-scale studies have not been performed. also, there is no official vector surveillance for wnv. the aim of the present investigation was to inventory mosquitoes in guilan province and to screen these mosquitoes for wnv and more widely for flaviviruses using both virus-specific real-time rt-pcr and a realtime pan-flavivirus rt-pcr, respectively. materials and methods study areas the study took place in guilan province (36o33'–38o27' n and 48o32'–50o36' e) during august–september 2013 and 2014. the province is located along the caspian sea and surrounded by mazandaran province in the east, ardebil in the west and zanjan and qazvin in the south. it also borders the republic of azerbaijan in the north, as well as russia across the caspian sea. the province is humid with mean annual rainfall ranging 1000–2000mm. collections were carried out in five sites including saghalaksar of rasht, dehboneh of sangar, saravan park and chonchenan of zibakenar (all in rasht county) and ghazian alongside anzali wetland, which is one of the few international iranian wetlands (anzali county) (fig. 1). the collection sites are shown over the layers of minimum and maximum degree of temperature and annual rainfall of guilan province in fig. 2. mosquito sampling and processing adult mosquitoes were collected by using cdc miniature light traps, operating overnight from sunset to sunrise, i.e. from 18:00pm to 6:00am, hand catch by manual aspirators from hen shelters, barns and bathrooms, and night landing catch from human bate. specimens were transferred alive to the laboratory where identified using morphological-based keys (6) after anaesthetizing using an ice bag then transferred to the portable nitrogen tank. pooling of mosquitoes for viral rna testing the mosquitoes were grouped into the pools according to sex, species and trapping location. pools were homogenized in a final volume of 600μl as previously described (32). a 200μl http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 391–398 v moin-vaziri et al.: a molecular screening of … 393 http://jad.tums.ac.ir published online: december 31, 2019 volume was used for viral nucleic acid (na) extraction using the biorobot ez1-xl advanced system (virus extraction mini kit, qiagen). real-time rt-pcr for specific detection of west nile virus rna fiveμl of na was used for rt-pcr. sense (proc-f1: cctgtgtgagctgacaaactt agt) and reverse (proc-r: gcgttttagc atattgacagcc) primers were combined with the fluorogenic taqman probe (proc-tm: 6fam-cctggtttcttagacatcgagat cttcgtgc tamra), and used with the go taq probe 1-step rt-qpcr (promega) as previously reported (33). this assay was developed and is routinely used by the french reference centre for arboviruses. molecular detection of flavivirus rna total nucleic acid extraction was conducted by using a biorobot ez1, with virus mini kit v2.o (qiagen). another 5µl aliquot of na was used in a one-step real-time quanti-tec syber-green rt-pcr assay (qiagen) as previously described (34). this assay located in a highly conserved region of the polymerase gene allows detection of all recognized flaviviruses, species identification is then achieved by sequencing the pcr product and comparing the sequence using blast ncbi program software and database. results a total of 1015 adult mosquitoes were collected from guilan province including eight species representing four genera as follow: three species within subfamily anophelinae (an. maculipennis s.l., an. pseudopictus, an. sacharovi) and five species within subfamily culicinae (ae. vexans [aedimorphus vexans], coquillettidia richiardii, cx. pipiens, cx. theileri, cx. tritaeniorhyncus) (table 1). the largest species diversity was observed in ghazian, alongside anzali wetland, where all eight species were recorded. the most prevalent species were ae. vexans (33.2%), cx. tritaeniorhynchus (22 %), cx. pipiens (20.7%), and an. maculipennis s.l. (15.6%), respectively. culex pipiens was collected from all five sites followed by ae. vexans and cx. tritaeniorhynchus (four sites) and an. maculipennis s.l. (three sites) (table 1). the composition percentages of species based on the collection methods were as follow: an. maculipennis s.l. [91.8% by night landing catch (nlc), 8.2% by aspirator (as), an. pseudopictus (90.9% by nlc, 9.1% by as), an. sacharovi (100% by as), ae. vexans (96.4% by nlc, 3.6% by as), cq. richiardii (100% by nlc), cx. pipiens [55.7% by as, 37.6% by nlc, 6.7% by light trap (lt)], cx. theileri (100% by as), cx. tritaeniorhynchus (78.1% by as, 21.9% by nlc). the 1015 mosquitoes were grouped into 38 pools according to sex, species and trapping location. none of the 38 pools tested by real-time rt-pcr either for flaviviruses or more specifically for west nile virus was positive. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 391–398 v moin-vaziri et al.: a molecular screening of … 394 http://jad.tums.ac.ir published online: december 31, 2019 table 1. details of collected specimens based on study areas and collection methods species n collected sites collection methods year total an. maculipennis s.l. 11 rasht-saghalaksar asa 2013 158 (15.6%) 1 anzali wetland nlcb 2013 2 sangar-dehboneh as 2013 144 anzali wetland nlc 2014 an. pseudopictus 4 anzali wetland as and nlc 2013 22 (2.2%) 4 anzali wetland nlc 2014 14 saravan park nlc 2014 an. sacharovi 18 anzali wetland as 2014 18 (1.8%) ae. vexans 1 rasht-saghalaksar as 2013 330 (32.5%) 8 zibakenar-chonchenan nlc 2013 57 anzali wetland as and nlc 2013 255 anzali wetland nlc 2014 9 saravan park nlc 2014 cq. richiardii 11 anzali wetland nlc 2013 35 (3.4%) 24 anzali wetland nlc 2014 cx. pipiens 84 rasht-saghalaksar as 2013 210 (20.7%) 30 zibakenar-chonchenan ltc and as 2013 9 anzali wetland as 2013 8 sangar-dehboneh as 2013 51 saravan park nlc 2013 26 anzali wetland nlc 2014 2 saravan park nlc 2014 cx. theileri 18 anzali wetland as 2014 18 (1.8%) cx. tritaeniorhyncus 30 rasht-saghalaksar as 2013 224 (22.0%) 143 anzali wetland as 2013 2 sangar-dehboneh as 2013 29 anzali wetland nlc 2014 20 saravan park nlc 2014 total 1015 (100%) aas = aspirator, bnlc = night landing catch, clt = light trap) fig. 1. map showing the sampling sites (marked by signs) and the geographical location of guilan province in iran http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 391–398 v moin-vaziri et al.: a molecular screening of … 395 http://jad.tums.ac.ir published online: december 31, 2019 fig. 2. sampling sites over the layers of different climatic condition of guilan province (a: average maximum degree, b: average minimum degree c: annual rainfall) discussion in total, eight different species of mosquitoes were collected during this study, all found in guilan province before (9, 35). the most prevalent and widespread species of the present study i.e. ae. vexans, cx. tritaeniorhynchus, cx. pipiens and an. maculipennis s.l. were also found frequently as larvae in the previous investigations in the province (35–37). aedes vexans was the most abundant species (table 1) mostly captured in the second year of study (2014) adjoining anzali wetland. this species is important as a vector of wnv, snowshoe hare virus (sshv) and tahyna virus (tahv) (bunyaviridae: orthobunyavirus) (2, 4). the second prevalent species which collected in the current study was cx. tritaniorhynchus (table 1) is an important vector of wnv and jev (4, 21). culex pipiens was found in all collected sites (table 1). this species is a domestic mosquito which has a role in the transmission of some arboviruses including wnv, tahv and sinv (2, 4). culex theileri was collected only in 2014 from anzali wetland (table 1). the species is known the vector of wnv (4). some insect-specific flaviviruses (isfs) were isolated from this species in portugal (38) and turkey (39) and designated culex theileri flavivirus (ctfv). coquilletidia richiardii was also found (table 1), which is considered among the vectors of wnv, sinv and batai virus (batv) (bunyaviridae: orthobunyavirus) (2, 4). among the aforementioned species, cq. richiardii and cx. pipiens in europe and cx. tritaeniorhynchus in asia are the main vectors of wnv (4). among anopheline mosquitoes there are some reports of isolation of wnv and batv from an. maculipennis s.l. (2, 4), which also was collected during this study. favorable climate of the region, including high precipitations, provides conditions that can lead to the emergence or re-emergence of mosquito-borne diseases (fig. 2). the presence of rice fields, wetlands and lagoons used by migratory birds may cause new viral outbreaks (31). the area also provides suitable larval habitats for many mosquito species. guilan province is a well-known touristic place with thousands of visitors annually from different parts of the country. that can increase probable imported cases of mosquito-borne infections. also the important vectors of wnv, cx. pipiens and cx. tritaeniorhynchus, are among the most abundant and widespread species in the province (37). as far as the authors know this study is the first one with the aim of flavivirus screening in mosquitoes in guilan province. most of the available data about the mosquitoes of the province are based on larval collections (35–37). sampling adult mosquitoes in guilan province, using light traps and hand catch, can be another advantage of the present investigation. the http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 391–398 v moin-vaziri et al.: a molecular screening of … 396 http://jad.tums.ac.ir published online: december 31, 2019 collection, anaesthetizing, identification, and preparing of collected specimens based on molecular protocols are time-consuming. that effected sample size which could be one of the limitations of the present study to detect the virus. using sentinel birds as bait may increase the possibility of sampling ornithophilic species which are wnv vectors and detect the virus. that was not among the goals of this study, however that may be used in forthcoming surveys. in view of the absence of official vector surveillance for wnv and many other mosquito-borne viruses in iran, such investigations should be continued. conclusion although, the mosquito species found in this survey are among proven or potential flavivirus vectors worldwide, our screening by rt-pcr did not reveal any flavivirus products. the total sample size could effect the outcomes. despite this fact surveillance should be continued due to the presence of wnv infections in humans, horses, birds and known vectors in the region. the probable forthcoming finding of wnv in mosquitoes will make a chance to compare the relativeness of detected virus strains in vectors and vertebrate hosts in the region. also screening for other arboviruses other than flaviviridae, which were not investigated in the present study, should be considered for future studies. acknowledgements this work was financially supported partially by unite des virus emergents (umr190), faculte de medecine, aix-marseille univ, marseille, france and research department of school of medicine shahid beheshti university of medical sciences, tehran, iran (grant no: 5543) as a joint project. the authors thank the administrative support of the health 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(persian). 37. azari-hamidian s (2007) larval habitat characteristics of mosquitoes of the genus culex (diptera: culicidae) in guilan province, iran. iran j arthropod borne dis. 1: 9–20. 38. parreira r, cook s, lopes â, de matos ap, de almeida apg, piedade j, esteves a (2012) genetic characterization of an insect-specific flavivirus isolated from culex theileri mosquitoes collected in southern portugal. virus res. 167: 152–161. 39. ergünay k, litzba n, brinkmann a, günay f, kar s, öter k, örsten s, sarikaya y, alten b, nitsche a, linton y-m (2016) isolation and genomic characterization of culex theileri flaviviruses in field-collected mosquitoes from turkey. infect genet evol. 46: 138–147. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2015, 9(1): 98–103 n hajipour et al.: hedgehogs (erinaceus europaeus) … 98 original article hedgehogs (erinaceus europaeus) as a source of ectoparasites in urban-suburban areas of northwest of iran nasser hajipour 1, mousa tavassoli 1, *tahmineh gorgani-firouzjaee 1, soraya naem 1, behzad pourreza 2, kia bahramnejad 1, jafar arjmand 1 1department of pathobiology, faculty of veterinary medicine, urmia university, urmia, iran 2department of clinical science, faculty of veterinary medicine, tehran university, tehran, iran (received 29 dec 2012; accepted 28 june 2014) abstract background: hedgehogs are small, nocturnal mammals which become popular in the world and have important role in transmission of zoonotic agents. thus, the present study aimed to survey ectoparasite infestation from april 2010 to december 2011 in urban and suburban parts of urmia and tabriz cities, northwest of iran. methods: a total number of 84 hedgehogs (40 females and 44 males) were examined. they have been carefully inspected for ectoparasites and collected arthropods were stored in 70% ethanol solution. the identification of arthropods was carried out using morphological diagnostic keys. results: the occurrence of ticks on hedgehogs was 23 (67.7%) with rhipicephalus turanicus in urmia and 11 (22%) as well as 1(2%) with rh. turanicus and hyalomma anatolicum anatolicum in tabriz, respectively. one flea species, archaeopsylla erinacei, was found with prevalence of 19 (55.9%) and 27 (54%) in urmia and tabriz cities, respectively. prevalence of infestation with rh. turanicus and a. erinacei were not different (p> 0.05) between sexes of hedgehogs in two study areas. highest prevalence of tick and flea infestation was in june in urmia, whereas it was observed in august in tabriz. both tick and flea parasitizing hedgehogs showed seasonal difference in prevalence (p< 0.05) in urmia, but it was not detected in tabriz (p> 0.05). conclusion: the result showed the high occurrence of ectoparasites in hedgehog population and according to the zoonotic potential of these animals as vector of some agents further studies are needed to investigate in different parts of iran. keywords: ectoparasites, hedgehog, erinaceus europeus, tick, flea introduction the european hedgehogs erinaceus europaeus are widespread in rural, suburban and urban environment and have become popular as exotic pets. these animals belong to order erinaceomorpha, family erinaceidae and originally arrived from europe, asia and africa (rilley and chomel 2005). hedgehogs are considered as host of a wide variety of different parasites and pathogens (keymer et al. 1991, riley and chomel 2005). besides, these animals can be a reservoir host for leishmaniasis and trichinellosis in some parts of the world (yaghoobi-ershadi and javadian 1996, pozio 2007). hedgehogs can carry several tick and flea species (beck 2007). arthropod ectoparasites are a highly adapted group of invertebrates that live on the external body surfaces of animals and humans. severe infestation with ectoparasites especially ticks can lead to anemia and weight loss (pfaffle et al. 2009). moreover, these arthropods can act as vectors of pathogenic agents, such as borreliasis, rickettsiasis and piroplasmosis, which cause serious diseases in humans and animals (skuballa et al. 2007, skuballa et al. 2010, marie et al. 2012). these mammals are pre*corresponding author: dr tahmineh gorganifirouzjaee, e-mail: t.gorgani@gmail.com http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 98–103 n hajipour et al.: hedgehogs (erinaceus europaeus) … 99 sented with six species in three genera throughout parts of iran (etemad 1984). several studies have been conducted on parasites of hedgehogs from different part of the world that reveal the high occurrence of parasite including the ectoparasites and endoparasite helminthes (dziemian et al. 2010, gaglio et al. 2010, foldvari et al. 2011, pfaffle et al. 2011). few studies have focused on prevalence of hedgehog infestation in the most parts of iran (youssefi et al. 2011). consequently, the present work was performed in order to determine and compare prevalence and intensity of infestation on hedgehogs in northwest of iran. materials and methods the study was carried out in two cities in northwestern region in iran. urmia has semihumid climate, yearly mean rainfall is of about 350 mm with the maximum mean temperature of 28.3 °c in august and the minimum mean temperature of -5 °c in january. tabriz is arid, its mean rain fall is 330 mm, maximum temperature of 22 32.7 in july and the minimum mean temperature of 2.8 in december. a total number of 84 hedgehogs (44 males and 40 females) were examined from april 2010 to december 2011 from urmia (17 females and 17 males) and tabriz (23 females and 27 males) cities. for sampling, two study regions were divided to five parts (north, south, west, east and center) and animals were searched by torchlight during night through parts of study regions especially in grassland, parks and garden areas. then, animals were captured by hands and maintained in plastic baskets and transferred to parasitology laboratory, faculty of veterinary medicine, urmia university, urmia. about 22 hedgehogs were found dead following road accidents and referred to parasitology laboratory. all data about sex and weight were recorded. each individual was visually inspected for ectoparasites, then arthropods collected and stored in 70% ethanol solution. fleas were cleared in 10% potassium hydroxide solution and mounted using routine technique. the ectoprasites morphologically were examined under the stereo microscope and identified using morphological taxonomic keys (beaucournu and launay 1999, walker et al. 2007). statistics analysis the following parasitological parameters were evaluated: percentage prevalence, the mean intensity and the mean abundance (bush et al. 1997). chi-square and fisher’s exact tests were used to test for differences in parasite prevalence in the hedgehog population. statistical comparisons were carried out using spss 16.0 statistical software. differences were considered significant at p< 0.05 level. results a total number of 37 ticks (23 males and 14 females) and 241 ticks (127 males and 114 females) were collected from hedgehogs in urmia and tabriz cities, respectively. the occurrence of ticks in urmia on hedgehogs was 23 (67.7%) with rhipicephalus turanicus and tick infestation in tabriz was 11 (22%) and 1 (2%) with rh. turanicus and hyalomma anatolicun anatolocum, respectively. other ectoparasite that found was archaeosylla erinacei with prevalence of 19 (55.9%) and 27 (54%) in urmia and tabriz cities, respectively. there were significant differences in female and male ticks and fleas (p< 0.05) that collected from tabriz but significant differences was observed only in flea in urmia. all information about number of infestation with ticks and fleas are shown in table 1. prevalence with rh. turanicus and a. erinacei were not different (p> 0.05) between sexes of animals in two study areas (table 2). http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 98–103 n hajipour et al.: hedgehogs (erinaceus europaeus) … 100 highest occurrence of infestation in both tick and flea was in june in urmia. the significant variations were observed among seasons in both ectoparasite species (p< 0.05). in region two, highest prevalence of two parasite species was in august (fig. 1). the significant differences were not seen among season in flea and tick population (p> 0.05). overall, the intensity of ectoparasite was higher in urmia than tabriz in hedgehog population. table 1. prevalence and intensity of ectoparasite species on hedgehogs study area parasite species prevalence (%) mean intensity (±sd) mean abundance (±sd) range sex ratio urmia rhipicephalus turanicus archaeosylla erinacei 23(67.7) 19(55.9) 10.47(±7) 7.78(±7.2) 4.35(±6.6) 7.08(±7.6) 1–25 2–34 1/1.15 1/ 2.52 tabriz rhipicephalus turanicus hyalomma anatolicum anatolicum 11(22) 1(2) 27(54) 3.3(±1.3) 1(±0) 3.6(±1.7) 0.72(±1.5) 0.02(±0.1) 2(±0.2) 1–5 0–1 1–8 1/1.57 1/7.6archaeosylla erinacei table 2. prevalence of ectoparasite infestation in relation to sex of the hedgehogs (n= 84) city species sex of animals significance female male urmia (n=17) (n=17) p> 0.05rhipicephalus turanicus archaeopsylla erinacei 11(64.7) 12(70.6) 10(58.8) 9(52.9) tabriz (n=23) (n=27) p> 0.05 rhipicephalus turanicus archaeopsylla erinacei 5(21.7) 6(26.08) 15(55.5) 12(44.5) fig. 1. infestation rate of hedgehog fleas and ticks collected from urmia and tabriz cities, iran discussion in the present study, the ectoparasite prevalence was observed in european hedgehogs (erinaceus europaeus) in northwest of iran. a total number of 37 ticks with the species of rh. turanicus (67.7%) were identified from urmia. of the hedgehogs sampled in tabriz, 22% and 2% were infested with rh. turanicus and h. anatolicum anatolicum, respectively. the result revealed that infestation rate with ticks was higher than in urmia. youssefi et al. (2011) found prevalence of 75% for rh. turanicus in hedgehogs (erinaceus concolor) in north of iran. at the investigation on identification of tick species, the presence of rh. turanicus among ruminants confirmed in two ecologic regions in iran (rahbari et al. 2008). besides in another study rh. turanicus was found in west and east azerbaijan (nabian and rahbari 2008). http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 98–103 n hajipour et al.: hedgehogs (erinaceus europaeus) … 101 seasonal variation was observed in hedgehog population in urmia, but not in tabriz. the possible reason could be associated with climatic conditions. rainfall was considered as the most important climatic factor that influenced the seasonal variation in parasite population. according to previous study by nabian and rahbari (2008), seasonal activity of ticks has been during the hot and humid months in the western and northwestern regions of iran, which is resulted from several factors including increase of rainfall, temperatures and relative humidity. the results of this study are similar to study carried out by pfaffle et al. (2011) on tick populations in hedgehogs. moreover, seasonal variation in tick infestation on hedgehog had been shown in urban ecosystem of the city of poznan, poland (dziemian et al. 2010). we observed no significant differences in the abundance of any arthropod between hedgehog sexes. the findings of other studies were in accordance with our study (gaglio et al. 2010, pfaffle et al. 2011). different molecular investigations were conducted on hedgehog ectoparasites which suggested the possibility of transmission of rickettsial agents by arthropods (matsumoto et al. 2005, harrus et al. 2010). marie et al. (2011) showed that european hedgehogs (erinaceus europeaus) were infested with rh. sanguineus and rickettsiae masssiliae was identified from ticks. this pathogen is a causative agent of spotted fever disease in humans. in addition, similar study on african hedgehog (atelerix algirus) and desert hedgehog (paraechinus aethiopicus) in algeria indicated that examined ticks including rh. sanguineus and haemaphysalis erinacei were positive for r. masssiliae (khaldi et al. 2012). therefore, it was concluded that horizontal transmission of rickettsia between hedgehog and tick results in hedgehog become potential reservoir host for these emergent pathogens and at this point of view, in environments which there are close communication between human and these animals, the risk factor of infection will increased for humans. furthermore, rh. turanicus can act as a vector for rickettsia, anaplasma, theileria, babesia, and arboviruses (psaroulaki et al. 2006). other arthropod species found in our study was a. erinacei with infestation rate of 55.8% and 54% in urmia and tabriz cities, respectively. highest prevalence was in may to june in urmia but it was from august to september in tabriz. there was no data about flea infestation on hedgehogs in iran. in the northern white-breasted hedgehog (erinaceus roumanicus) in urban park of central budapest, hungary, 99.4% were infected with a. erinacei flea (foldvari et al. 2011). in an epidemiological survey, which was done on european hedgehogs (erinaceus europaeus) in britain, two ectoparasites species a. erinace (8%) and ixode s hexagonus (59%) were reported (gaglio et al. 2010). according to thamm et al. (2009), investigated hedgehogs had infestation with a. aerinacei and i. hexagonus which suggested there were association between ectoparasite infestation and urban environment. mixed infestation with a. erinacei and dog flea reported from pet animals and hedgehog in germany (visser et al. 2001). similar to tick that mentioned above, flea have the ability of transmission of rickettsial agents (bitam et al. 2006). there was one report about human infestation with a. erinacei in the world (pomykal et al. 1985). based on the results, it was concluded that the occurrence of ectoparasites in hedgehog population was high in urmia and tabriz cities and their ability in transmission of infectious agents should be noted. for these reasons, further studies are needed to investigate hedgehog ectoparasites in other parts of iran. conclusion the result of this investigation showed the http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 98–103 n hajipour et al.: hedgehogs (erinaceus europaeus) … 102 high prevalence ectoparasites in hedgehog population and according to the zoonotic potential of these animals as vector of some agents further studies are needed to investigate in different parts of iran. acknowledgements the authors would like to appreciate the staffs of department of parasitology, faculty of veterinary medicine, urmia university, for sincere assistance and support of this research. the authors declare that there is no conflict of interests. references beck w (2007) endoparasitismbeimigel. wien klin wochenscher. 119(3): 40–44. beaucournu jc, launay f (1999) fleas (siphonaptera) of france and western mediterranean. paris federation of companies sciences, france. bitam i, parola p, dela-cruz kd, matsumoto k, baziz b, rolin jm, belkaid m, raoult d (2006) first molecular detection of rrickettsia felis in fleas from algeria. am j trop med hyg. 74(4): 532–535. bush ao, lafferty kd, lotz jm, shostak aw (1997) parasitology meets 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431–442. youssefi mr, rahimimt, hosseini sm, darvishi mm (2011) first report of rhipicephalus turanicus from hedgehog (erinaceus concolor) in north of iran. world j zool. 6(4): 401–403. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, december 2015, 9(2): 246–252 b esmaeilnejad et al.: determination of prevalence … 246 original article determination of prevalence and risk factors of infection with babesia ovis in small ruminants from west azerbaijan province, iran by polymerase chain reaction *bijan esmaeilnejad 1, mousa tavassoli 1, siamak asri-rezaei 2, bahram dalir-naghadeh 2, karim mardani 3, mostafa golabi 1, jafar arjmand 1, ali kazemnia 4, ghader jalilzadeh 2 1department of pathobiology, faculty of veterinary medicine, urmia university, urmia, iran 2departments of clinical sciences, faculty of veterinary medicine, urmia university, urmia, iran 3department of food hygiene and quality control, faculty of veterinary medicine, urmia university, urmia, iran 4departments of microbiology, faculty of veterinary medicine, urmia university, urmia, iran (received 20 jan 2014; accepted 28 june 2014) abstract background: small ruminants’ babesiosis caused by babesia ovis, is transmitted during blood feeding by infected ticks and is the most economically important tick-borne disease in tropical and subtropical areas. this study was carried out to to estimate the infection rate of b. ovis in sheep and goats by pcr. we have analysed risk factors that might influence infection of sheep and goats with b. ovis. methods: a total 402 blood samples were examined microscopically for the presence of babesia infection. all samples were tested by pcr. during sampling, whole body of each animal and farm dogs was examined for the presence of ticks. results: forty-two animals (10.4%) were positive for babesia spp. upon microscopic examination, whereas 67 animals (16.7%) yielded the specific dna for b. ovis of which 52 animals were sheep and 15 animals were goats. twenty-nine farms (72.5%) were found positive for b. ovis. the percentage of positive animals in each location varied from 13 % to 20 %. the relative risk of the presence of ticks in sheep and goats (p< 0.01) and farm dogs (p< 0.01) for pcrpositive results forb. ovis in sheep and goats was found 3.8 and 2.9, respectively. a total of 747 ticks identified as rhipicephalus bursa, r. sanguineus and r. turanicus on the basis of morphological features. conclusion: other animal species besides dogs may also be risk factors for babesiosis in sheep and goats. also, r. bursa may play an important role as a vector of the parasite in iran. keywords: babesia, rhipicephalus, small ruminant, iran, pcr introduction the genus babesia contains tick-borne hemoprotozoan parasites that infect a wide variety of vertebrate hosts. the economic losses in small ruminant production due to babesiosis are significant in tropical and subtropical regions of the world (theodoropoulos et al. 2006). babesia spp. are transmitted by ixidid ticks and causes, anemia, jaundice, haemoglobinuria and in some cases mortality may occur (sevinc et al. 2007). the disease is caused by b. ovis, b. motasi and b. crassa (hashemi-fesharki 1997, razmi et al. 2003, uilenberg 2006). babesia ovis, a small babesia (< 2.5 µ m), is the most common species that causes sheep babesiosis in iran (rahbari et al. 2008, shayan et al. 2008). the pathogenicity of b. motasi is not high and appears to be moderately virulent (soulsby 1982). whereas, b. crassa is considered as being non-pathogenic to samall ruminants (uilenberg 2006). the diagnosis of babesiosis can be achieved by microscopic examination of giemsa-stained blood smears and clinical *corresponding author: dr bijan esmaeilnejad, e-mail: b_esmaeilnejad@yahoo.com http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 246–252 b esmaeilnejad et al.: determination of prevalence … 247 signs in acute phase of the disease, but after acute or primary infections, recovered animals frequently sustain subclinical infections, which are microscopically undetectable (aktas et al. 2005). this carrier state serves as reservoir for infection in the herds, since animals that are not clinically ill may continue it infect the tick vector (aktas et al. 2007). diagnostic tests which depend on serology for detecting this carrier state, but serological methods are not specific for any babesia spp. due to cross-reactivity with other babesia spp. and false positive and negative results are commonly observed in these tests (aktas et al. 2005). the use of alternative techniques, such as dna amplification methods, has become necessary to detect and identify babesia infections effectively and has been reported in numerous recent studies (jefferies et al. 2003). molecular techniques are more sensitive and specific than other traditional diagnostic methods (aktas et al. 2007). studies on small ruminants’ babesiosis in iran are very limited. previous microscopic and serological studies state that the disease caused by b. ovis is endemic in the country (tavassoli and rahbari 1998, razmi et al. 2003). taking into account the limitation of serological studies, the objective of the present study was to determine the prevalence of the infection in northwest of iran by polymerase chain reaction (pcr). pcr was compared with the examination of thin blood smear. we have identified risk factors that favour infection of sheep and goats with b. ovis. we have also identified tick species found on sheep, goats and farm dogs in the region. materials and methods surveyed-area (west azerbaijan province, north-western iran) was divided into three different geographical areas including north, center and south. blood samples were collected from 40 randomly selected flocks located in four important livestock production regions of west azerbaijan province iran (maku, khoy, urmia, piranshahr). ecologically, this area is classified as a semi-arid zone. small ruminants rising are economically a very important occupation in this province. a total of 402 blood samples in edta tubes were collected from 280 sheep and 122 goats that belonged to twelve flocks with sheep only, four flocks with goats only, and 24 flocks with sheep and goats together. data on the characteristics of the animals (species, gender and tick burden) and the flock (size, species of animals, dogs associated with the flocks and tick burden of dogs associated with the flocks) were collected through questionnaires completed by the investigators on location during sample collection. the whole body of each sampled small ruminant and farm dog was inspected for the presence of ticks by palpation, mainly on their ears, along their nape, perineum, and udder/scrotum, between thigh, shoulder region and tail base. the ticks were manually removed and transferred to the parasitology laboratory in tubes containing 70 % ethanol solution. thin blood smears were prepared immediately after blood sampling. the thin blood smears were fixed in methanol for 5 min and stained in 10 % giemsa solution in phosphate buffer solution (pbs), ph 7.2, for 20 min and examined under an oil-immersion objective of a magnification of 1000 x for the presence of intracellular forms of the parasite with morphology compatible with b. ovis (aktas et al. 2007). parasitemia was expressed as the log number of red blood cells infected with babesia parasites per 105 erythrocytes (schetters et al. 2009). the smears were recorded as negative for babesia spp. if no parasites were detected in observed oilimmersion fields. the collected adult ticks from animals were counted. tick species were identified using standard taxonomic keys (walker et al. 2003, estrada-pena et al. 2004). http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 246–252 b esmaeilnejad et al.: determination of prevalence … 248 dna was extracted using a dna purification kit (fementas, germany) according to the manufacturer's instruction. a pair of primers, bbo-f 5'-tgggcaggaccttgg ttcttc t-3' and bbo-r 5'-ccgcgtag cgccggctaaata-3' were used to amplify a 549 bp fragment of the ssu rrna gene of b. ovis. the primers’ specificity and sensitivity was assessed by aktas et al. (2005). pcr was carried out in 50 µ l total reaction volume containing 5 µ l of 10 x pcr buffer, 2 mm mgcl2, 250 µ m of each of the four deoxynucleotide triphosphate, 1.25 u taq dna polymerase (fermentas, germany), 50 pmol of each primer and 50 ng of extracted dna. amplification of parasite dna was done in a cp2-003 thermocycler (corbett research, australia). cycling conditions for b. ovis were 95 °c for 5 min, followed by 45 cycles at 94 °c for 45 sec, 63 °c for 45 sec and 72 °c for 1 min with a final extension step of 72 °c for 10 min. the pcr products were separated by electrophoresis on 1.5 % agarose gel in tris-borate-edta (tbe) buffer and visualized using ethidium bromide (1 µ g/ml) and uv transilluminator (bts-20m, japan). the positive control for b. ovis was obtained from sheep with clinical babesiosis (diagnosis was done on the basis of clinical signs and light microscopic examination giemsa-stained thin blood smear). venous blood sample, taken from healthy lamb without contact with ticks, served as negative control in the study. the fisher’s exact test and mantelhaenszel test was used to express association between the presence (positive and negative blood samples) of babesia and the various parameters ie flock size, species, gender and age of animal, tick infestation of sheep and goats, presence of ticks in the flock and flock composition. software spss version 17.0 was used to compare the data of blood smears with blood pcr method. results were displayed as p values as well as relative risk values (with 95 % confidence intervals). p< 0.05 was accepted to be statistically significant. results microscopic examination of thin blood smears showed parasitaemia in infected animals ranging from 0.01 to 3 %. piroplasms, detected inside the red blood cells, were polymorphous. the shapes of the parasites were pyriform and single ring. all of these forms classified as babesia spp. of the 402 blood samples examined, 42/402 (10.4%) were positive for piroplasms upon microscopic examination, whereas, 67/ 402 (16.7%) were positive for presence of b. ovis by pcr. all of forty-two positive by microscopic examination were also positive by pcr (fig. 1). compared to microscopic examination results, pcr showed a significantly higher efficacy of detection of babesia spp. (p< 0.05). out of 40 examined farms, twentynine (72.5%) were found positive for b. ovis. the percentage of positive animals in each farm varied from 13 % to 20 %. the prevalence of b. ovis in sheep and goats in relation to the parameters describing the characteristics of the animals and the flock is shown in table 1. out of the 280 sheep and 122 goats examined, 18.5 % (52/ 280) and 12.2 % (15/122) were infected with b. ovis. the difference between the prevalence of b. ovis infection in sheep and goats were statistically significant (p< 0.05). the prevalence of b. ovis infection in age groups and different gender were not significantly different (p> 0.05). frequency of b. ovis infection was significantly higher in flocks with tick burden (p< 0.05). the statistical analysis of the data showed that the relative risk of the presence of ticks in sheep and goats (p< 0.01) and farm dogs (p< 0.01) for pcr-positive results for b. ovis in sheep and goats was found 3.8 and 2.9, respectively. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 246–252 b esmaeilnejad et al.: determination of prevalence … 249 during this survey, a total of 747 adult ixodidae ticks were collected from different body areas, i.e. external ear, perineum, between thigh and udder/scrotum of sampled small ruminants and farm dogs. the following ticks were identified, rhipicephalus bursa 49.9 % (373/747), r. sanguineus 43.9 % (328/747) and r. turanicus 6.2 % (46/747). the main attachment site of ticks was the perineum region. table 1. association between the presence (pcr-positive and negative blood samples) of babesia ovis infection in sheep and goats and the studied parameters describing animal and flock characteristics total sheep and goats flock location flock size species of animal maku khoy urmia pirans hahr 100–250 animals >250ani mals sheep goats number 402 101 84 97 120 277 125 280 122 negative 335 (83.4%) 88 (87.2%) 64 (76.2%) 80 (82.5%) 103 (85.9%) 230 (83%) 105 (84%) 228 (81.4%) 107 (87.8%) positive 67 (16.6%) 13 (12.8%) 20 (23.8%) 17 (17.5%) 17 (14.1%) 47 (17%) 20 (16%) 52 (18.6%) 15 (12.2%) p(f), p(mh) p(f)=0. 52(ns) p(f)=0.1 9(s) p(f)=0.005 rr gender of animal age of animal flock composition male female <1year 1year sheep only goats only sheep and goats together number 170 232 114 288 52 30 320 negative 141 (83.0%) 194 (83.6%) 92 (80.8%) 243 (84.3%) 41 (78.8%) 25 (83.4%) 269 (84.1%) positive 29 (17.0%) 38 (16.3%) 22 (19.2%) 45 (15.7%) 11 (21.2%) 5 (16.6%) 51 (15.9%) p(f),p(mh) p(f)=0.45 (ns) p(f)=0.09 (ns) p(mh)=0.08 (ns) rr tick burden of sheep and goats presence of dogs in the flock tick burden of dogs in the flock no ticks more than one tick yes no no tick more than one tick number 312 90 310 92 74 121 negative 280 (89.8%) 55 (61.1%) 255 (82.2%) 80 (86.9%) 69 (93.3%) 97 (80.2%) positive 32 (10.2%) 35 (38.9%) 55 (17.8%) 12 (13.1%) 5 (6.7%) 24 (19.8%) p(f),p(mh) p(f)=0.005 p(f)=0.06 (ns) p(f)=0.003 rr 3.8 2.9 http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 246–252 b esmaeilnejad et al.: determination of prevalence … 250 fig. 1. pcr products amplified using babesia ovisspecific primers lane m50bp dna ladder (fermentas, germany), lanes 1, 2positive control, lane 3negative control, lanes 4, 5infected sheep blood, lanes 6, 7infected goats blood, lanes 8, 9, 10infected ticks discussion microscopic examination, such as giemsastained blood smears is mostly used as a confirmatory diagnosis of vertebrate host suffering of piroplasm infections. however, the method requires expertise because these parasites have similar morphological features and therefore, may confuse the examiner when mixed infections occur. serological tests were also used, but there are some difficulties with specificity and sensitivity (passos et al. 1998). an exact differentiation between these parasites is crucial to understanding their epidemiology. the detection of babesia infection in carrier animals by dna amplification has been a powerful tool for epidemiological investigation, since these animals represent an important source of alimentary infection of ixodid ticks (aktas et al. 2005, altay et al. 2008). the present study is the first molecular diagnostic technique that was employed to determine the epidemiology of small ruminant’s babesiosis in iran. the results showed more than 16 % of small ruminants in northwest of iran were infected with b. ovis. in present study, as expected, the prevalence of b. ovis infection in small ruminants detected by pcr (16.7%) was significantly higher than obtained in microscopic examination of thin blood smears (10.4%). due to main drawbacks for microscopic detection of babesia spp. in the early stage of infection and the long-term carrier status are difficult to detect of the parasites in very low parasitemia, therefore pcr method has higher efficiency than microscopic examination for detection of b. ovis. the results agree with a previous report about b. bovis (calder et al. 1996). in the microscopic examination of blood smears, we found that parasitaemia ranges from 0.01 to 3 %, in sheep and goats. in similar studies, razmi et al. (2003) and papadopoulos et al. (1996) has observed that sheep infected with b. ovis commonly had low parasitemia. in previous studies in iran, serological tests employing indirect fluorescent antibody test (ifat) was used and the prevalence of seropositive animals varied from 12 % to 58.8 % in different regions of the country (tavassoli and rahbari 1998). in the present study, covering four different regions of west-azerbaijan province, iran, the prevalence ranged from 13 % to 20 % on the farms that were examined. although the results of the present and previous study cannot be compared due to the different methods employed, they both indicate that ovine babesiosis is seen in almost each region of iran and prevalence of the disease shows difference among the provinces located in regions with different endemic features. the finding that the prevalence of ovine babesiosis was higher in herds with tick burden indicates the presence of a positive correlation between the prevalence of the disease and the presence of vector ticks. the http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 246–252 b esmaeilnejad et al.: determination of prevalence … 251 result is accordance with the find of theodorpoulos et al. (2005). among the factors examined in the present study, the presence of ticks in sheep, goats and farm dogs were associated with pcr-positive results, which indicate a high risk of infection with b. ovis in sheep and goats. ticks suitable for transmission of babesia have been reported in iran (razmi et al. 2003). concerning the species of animals (sheep or goats), it is stated that in the field, b. ovis causes disease exclusively in sheep, rarely in goats (papadopoulos et al. 1996). in regard to the role of farm dogs as a risk factor for babesiosis in sheep and goats, ticks can transmit babesia even after feeding on other hosts (yeruham et al. 1996) and r. sanguineus that usually infests dogs has been found also on ruminants (bouattour et al. 1999). however, the role of dogs in ovine or caprine babesiosis needs to be further studies. on the other hand, according to criadofornelio et al. (2003) the same babesia species can infect a wide variety of animal hosts. this finding indicates that other animal species besides dogs may also be risk factors for babesiosis in sheep and goats. conclusion the epidemiology of ovine babesiosis due to b. ovis is closely related to the ecology of vector ticks. the disease occurs yearly in the rhipicephalus spp. infested areas, during the activity period of the adult tick stage (yeruham et al. 1995). in the present study, r. bursa exhibited the highest (49.9%) frequency of infection. according to tavassoli and haji-ghahremani (2004) in a tick’s survey carried out in northwester iran, the majority of the ticks found on sheep and goats were identified as r. bursa. thus, the tick may play an important role as a vector of the parasite in iran. work is currently underway to provide data on the tick vector competency of b. ovis. acknowledgements this project was supported by urmia university (no.1-67). the authors would like to express their 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walker ar, bouattour a, camicas jl, estrada-pena a, horak ig, lati a, pefram rg , preston pm (2003) ticks of domestic animals in africa, a guide to identification of species. bioscience reports, uk. yeruham i, hadani a,galker f (1996) effect of passage of babesia ovis in the gerbil (acomys cahirinus) on the course of infection in splenectomized lambs. vet parasitol. 65: 157–161. yeruham i, hadani a, galker f, rosen s (1995) a study of an enzootic focus of sheep babesiosis (babesia ovis, babes, 1892). vet parasitol. 60: 349–354. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2020, 14(4): 408–415 y rassi et al.: efficiency of two … 408 http://jad.tums.ac.ir published online: december 31, 2020 original article efficiency of two capture methods providing live sand flies and assessment the susceptibility status of phlebotomus papatasi (diptera: psychodidae) in the foci of cutaneous leishmaniasis, lorestan province, western iran yavar rassi1; hamed asadollahi1; *mohammad reza abai1,2; mohammad hassan kayedi3; *hassan vatandoost1,2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3faculty of medicine, department of parasitology, lorestan university of medical sciences, khorramabad, iran *corresponding authors: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir, prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com (received 24 june 2020; accepted 22 dec 2020) abstract background: the aims of this study were to evaluate the efficiency of two capture methods for providing live sandflies used for determining the susceptibility level of phlebotomus papatasi, the main vector of zoonotic cutaneous leishmaniasis in lorestan province, west of iran. methods: the sand flies were collected from indoor and outdoor by hand-catch and baited traps during the peak of seasonal activity. the susceptibility level of sand flies was assessed using insecticide-impregnated papers against ddt 4%, bendiocarb 0.1%, permethrin 0.75%, deltamethrin 0.05%, and cyfluthrin 0.15%. results: a total of 2486 live sandflies were caught from both indoor and outdoor places. totally 849 sand flies were caught from outdoors with a sex ratio(sr) 0.1 versus 1637 sand flies collected from indoor using the hand-catch method with sr= 0.6. the dominant species of sand flies was ph. papatasi in the study area. mortality rates of outdoor-collected sand flies were exposed to ddt 4%, deltamethrin 0.05%, permethrin 0.75%, and bendiocarb 0.1%, and mortality rate ranged from 92.0–97.9% and for indoor-collected sand flies were 87.7–96.8%. both outdoor and indoor collected sand flies were susceptible to cyfluthrin 0.15% that caused 100% mortality. conclusion: based on the findings, the most appropriate method for collecting the live female ph. papatasi is the baited traps due to providing enough females is necessary for conducting the susceptibility tests. the finding indicated that ph. papatasi was resistant to ddt, under ’verification required’ status to deltamethrin, permethrin, bendiocarb, and susceptible to cyfluthrin. keywords: phlebotomus papatasi; insecticides; resistance; baited traps; hand-catch introduction phlebotomus papatasi was the main vector of zoonotic cutaneous leishmaniasis (zcl) and papatasi fever in the past. this species is prevalent in many areas in thewestern palaearctic region including europe (france, spain, greece, poland, czech republic, and italy), russia, and central asia as well as all countries in the eastern mediterranean region, the western and northern ethiopian region (saudi arabia, yemen, ethiopia, and sudan) and the oriental region (india and bangladesh). phleboto mus papatasi is found almost in all provinces of iran (1). it prefers the climatical conditions with hot summers, temperate winters, and monsoon rains and the minimum temperature should not less than -6 ˚c. this species prefers the environmental moisture and warmth and is frequently caught in areas where the water has a high level (2). numerous biological observations on ph. papatasi was indicative of the higher abundance in the plains compared to high mountainous regions. it has been recurrently copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:abaimr@tums.ac.ir mailto:hvatandoost1@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 408–415 y rassi et al.: efficiency of two … 409 http://jad.tums.ac.ir published online: december 31, 2020 found in rodent burrows, human and domestic animal shelters in the central plateau of iran (3). phlebotomus papatasi is more prevalent at indoor compared to other species of sand flies. the larval stage develops at animal shelters and rodent burrows (4). in terms of endophilic behavior, ph. papatasi generally rests in human and animal shelters and becomes as a domestic species in many regions, so that no sand fly could ever be so accustomed to adapting to the residential places (5). with development the construction of human dwelling in the plains, abundance of ph. papatasi rapidly increases and when the settlements are near to zcl foci, the transmission condition is more likely to become epidemic (6). it seems that the sandflies, which were caught from residential places, moved from adjacent infected rodent burrows, where ph. papatasi is considered as the prevailing species (7). in terms of biting and blood-feeding behaviors, ph. papatasi can repeatedly feed on its hosts both indoor and outdoor. the presence of multiple lesions on affected persons indicating evidence for recurrent bites of humans by sand flies (8). considering the blood-feeding preference of ph. papatasi, it can be argued that it mainly feeds on any host available nearby with more tendency to humans and rodents. the seasonal activity of this species starts from the beginning of may to the end of october in the northern and central regions of the country and from mid-april to january in southern regions with peaks in july and september (10). the lepomonad infection of ph. papatasi has been reported from isfahan, lotfabad, esferayen, torkaman sahra, ahvaz, dezful, shush, abardej, balochistan, sarakhs, semnan, damghan and shahrud in iran, all of which are known as the foci of zcl (11-13). transmission of zcl by ph. papatasi occurs outside the residential places, especially on the rooftops of houses or outdoor in front of the house in the regions with high endemicity for zcl, whilst in regions with low endemicity, the transmission is more common in agricultural fields and in localities near the rodent burrows (10). therefore, the transmission chain of zcl seems to be interrupted by the use of pyrethroids impregnated bed nets, curtains, or uniforms and personal protection using repellents and occasionally application of residual spraying at indoors (14). the preliminary trials for determining the susceptibility levels of ph. papatasi were reported in the jordan valley in 1967 (15) and the subsequent report revealed the resistance in northern bihar, india (16) and followed in turkey (17). although numerous susceptibility tests have been carried out in the foci of zcl in iran, no evidence has yet been presented for the resistance of ph. papatasi to insecticides (18-22). this study was the first comparative trial to determine the susceptibility of ph. papatasi collected from both indoor and outdoor in zcl foci in lorestan province, west of iran. materials and methods study area this study was conducted in pol-e dokhtar, rumeshgan, and kuhdasht districts where the highest prevalence of zcl was reported. the geographical coordinates of the research sites were 47º27´–48º22´e, 32º37´–33º20´ n, the mean elevation 662 meters above sea level (amsl) for pol-e dokhtar, 47º20´–48º65´e, 33º16´–33º35´n, 1089m amsl for rumeshgan, and 46º51´–47º50´e, 33º09´–33º56´n, and 1191m amsl for kuhdasht. sand fly collections sand flies were caught from indoor using hand catch method with mouth aspirators in the early morning as well as from outdoor using baited traps in the evening till midnight. each baited double net trap had equipped with mini gas lamps as a light attraction as well as a rooster in a cage as an attractant host for blood-feeding of sand flies. the baited traps set up in the evening using four wooden bases, so that the lower edges of the nets lie 10 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 408–415 y rassi et al.: efficiency of two … 410 http://jad.tums.ac.ir published online: december 31, 2020 cm above the ground. the location of traps was close to the rodent burrows and fox nests adjacent to the agricultural lands in the studied villages. the attracted sand flies were collected with the mouth aspirators also released into paper cups with net cover and transferred in a cool-box to the laboratory. a cotton pad soaked in 10% sucrose solution was put on the top of cups for feeding of sand flies. susceptibility tests the insecticide-impregnated papers were purchased from the who’s collaborating center, malaysia. susceptibility tests were carried out on both indoor and outdoor collected sand flies and exposed to ddt 4%, deltamethrin 0.05%, permethrin 0.75%, bendiocarb 0.1%, and cyfluthrin 0.15% impregnated papers using who test kits. at each replicate, 20–25 sand flies were exposed for 60min. if the mortality criterion was in the resistance border, the tests were continued using an increasing trend of logarithmic times until observing 98–100% mortality. both living and dead sand flies treated with each insecticide were preserved in ethanol 70% till mounted in puri’s medium for species identification. data analysis data of susceptibility tests including the number of live, dead, and total sand flies were grouped based on physiological conditions for each of the treated and control groups. the sex ratio is calculated by dividing the number of males by females. mortality was calculated by counting the dead and live mosquito species after 24h exposure to insecticide paper. the mortality was then corrected by applying abbott’s formula when control mortality was observed between 5% and 20%, whereas tests with more than 20% control mortality were discarded and repeated. median and mean of mortality used for plotting box and whisker plot used for showing the efficiency of two capture methods providing live sand flies and the independent-samples t-test used for show ing a significant difference between two capture methods. who criteria were used for interpretation, with 98–100% mortality indicating susceptibility, 90–97% indicating needs further confirmation or verification required, and mortality < 90% indicating resistance. the probit analysis was used in analyzing the timemortality response to estimate lc50 for a fixed concentration of insecticides to kill a defined proportion of sand flies, known as lethal time (lt). result efficacy of two collecting methods a total of 2486 live sandflies were caught using hand-catch and baited trap methods both from indoor and outdoor places with spending 12 and 4 working rounds. from indoor, 1637 sand flies (65.4%) were collected with sex ratio (sr) 0.6vs from outdoors 859 sand flies (34.6%) with sr= 0.1 (table 1). a significant difference was shown between the sr values of sand flies collected in the hand-catch and baited trap methods (t= 7.245, df= 3, p= 0.005) (fig. 1). susceptibility levels of phlebotomus papatasi collected indoor and outdoor the susceptibility of ph. papatasi collected from both indoor and outdoor studied areas were assessd at discriminative doses and 60 min exposure time. the comparative percent mortality were 87.7±1.1 and 92.0±0.4 for ddt, 96.8±0.3 and 97.8±1.4 for deltamethrin, 92.4±0.9 and 97.9±0.0 for permethrin, 93.6± 0.9 and 94.0±0.7 for bendiocarb, and 100±0.0 and 100±0.0 for cyfluthrin while the values in the control group were 1.1±0.1% and 1.3±0.1 respectively (fig. 2). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 408–415 y rassi et al.: efficiency of two … 411 http://jad.tums.ac.ir published online: december 31, 2020 table 1. efficacy of two capture methods used for collectiong live sand flies for susceptibility tests both indoors and outdoors, lorestan province, western iran c a p tu r e m e th o d t y p e o f c o lle c tio n p la c e r e p e titio n o f tr ia l t o ta l liv e c o lle c te d p e r c e n ta g e m a le f e m a le s e x r a tio m e a n o f t e m p e r a tu r e (°c ) r e la tiv e h u m id ity (% ) baited trap outdoor 4 849 34.6 83 766 0.1 30 47 hand-catch indoor 12 1637 65.4 1042 595 0.6 32 38 fig. 1. box plot showing sex ratio of sand flies abundance collected using hand-catch and baited traps methods, lorestan province, iran fig. 2. comparison of susceptibility level of phlebotomus papatasi collected from indoor and outdoor with hand-catch and baited trap methods following the who's criteria (32), lorestan province, iran http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 408–415 y rassi et al.: efficiency of two … 412 http://jad.tums.ac.ir published online: december 31, 2020 discussion due to the widespread outbreaks of zcl in iran and the world, the practical approaches are interrupting the transmission chain using different individual protection methods such as insecticide-impregnated bed nets and curtains, application of repellent when working outdoors, and in limited scale application of indoor residual spraying (14). prior to any control measures during outbreaks of zcl, certain information is required about the bionomy of the vectors in order to take appropriate preventive measures during natural disasters and epidemics. insecticides play a crucial role in controlling the vectors of zcl, and the type of insecticide should be determined using standard tests before use (23). the larval habitats of sand flies are located in rodent burrows and other small mammals nests as well as in domestic animal shelters (24). due to the extensive use of pesticides in animal husbandry and agricultural lands, the larval and adult sand flies are exposed to different chemicals which may lead to tolerant against various insecticides in the long term (25). given that no comparative study has been conducted as a concurrent for determination on susceptibility level between two separate populations living indoors and outdoors using two live capture methods; this study was the first research attempt in this subject. according to the results of this study, the mortality of indoor-collected ph. papatasi was 87%±1.1 when exposed 60min to ddt, and the for deltamethrin, permethrin, and bendiocarb were ranged from 92.4 to 96.8%. with regard to the sufficiency of test replication in this study, it became evident that ph. papatasi was resistant to ddt, under‘verification required’ to bendiocarb, permethrin, and deltamethrin, and susceptible to cyfluthrin. the main cause for the occurrence of resistance to ddt is believed to be the frequent use of this insecticide for controlling malaria vectors during past decades, and the gene responsible for organochlorine resistance was transferred to the next generations (26). it has been reported that the increased use of pyrethroid insecticide the health and agriculture sectors has resulted in cross-resistance to both organochlorine and pyrethroid insecticides (27). the sand flies collected from outdoor using baited traps were tolerant to ddt 4%, deltamethrin 0.05%, permethrin 0.75%, and bendiocarb 0.1% and the mortality rates ranged from 92.0 to 97.8% comparison of susceptibility of ph. papatasi collected from both indoor and outdoor using hand-catch baited traps methods showed the under ’verification required’/susceptibility of sand flies caught from outdoor compared to indoor against ddt, deltamethrin, permethrin, and bendiocarb. investigations on the susceptibility of sand flies to insecticides were initiated by seyedi rashti in mashhad, northeastern iran in 1970. he found that ph. papatasi was susceptible to ddt and dieldrin (18). a study in isfahan showed no resistance in ph. papatasi (12). according to research in mashhad and isfahan (2006–2007), the lt50 of ph. papatasi was increased up to 2.3–3% (22). the first occurrence of under ’verification required’ status to ddt was reported from isfahan in 1992 (29), while another study in the varzaneh county (isfahan province) showed complete susceptibility to ddt in the same period. in the latter study, it was concluded that the discontinuation application of the organochlorine insecticides could result in returning the under ’verification required’ status (12). some other studies conducted in fars, kerman, and isfahan during 1993–2001 reported the susceptibility of ph. papatasi to ddt (18-21). similarly, the susceptibility of ph. papatasi to ddt, propoxur, and deltamethrin were confirmed in the studies conducted in sabzevar, isfahan, baft, and bam during 2002–2013 (20-21). this study was the first report on occurrence of resistance and under ’verification required’ status of ph. papatasi to ddt, deltamethrin, permethrin, and bendiocarb as well as susceptibilhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 408–415 y rassi et al.: efficiency of two … 413 http://jad.tums.ac.ir published online: december 31, 2020 ity to cyfluthrin in zcl foci, west iran. there are several novel investigations of the main vector-borne diseases in the country (28-31) and monitoring the susceptibility/resistance of related vectors is a vital responsibility of corresponding ministries (28-31). conclusion the findings of the present study indicated ph. papatasi resistance to ddt, occurrence of under ’verification required’ status to bendiocarb, permethrin, and deltamethrin, and complete susceptibility to cyfluthrin. acknowledgements the present article was a part of a master thesis entitled “determining the susceptibility of ph. papatasi to common insecticides using hand-catch and baited trap methods in cutaneous leishmaniasis foci in lorestan, 2019” under the code 9611263009, which was financially supported by tehran university of medical sciences and with the cooperation of lorestan university of medical sciences. the authors of this study would like to express their gratitude to the deputy of health of lorestan university of medical sciences as well as the staff of rural health centers in pol-e dokhtar, rumeshgan and 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ferdowsi university of mashhad, mashhad, iran 2department of clinical sciences, faculty of specialized veterinary sciences, science and research branch, islamic azad university, tehran, iran (received 17 nov 2013; accepted 12 april 2014) abstract background: the aims of this study were to determine the seroprevalence of canine ehrlichiosis and risk factors of this disease in companion dogs’ population of mashhad, north east of iran. canine monocytic ehrlichiosis (cme) is a zoonotic disease transmitted by ticks, rhipicephalus sanguineus, and caused by an obligate intracellular bacterium, ehrlichia canis. methods: during september 2009 until november 2010, 250 companion dogs from mashhad, north-east of iran, were examined for serum antibody detection against e. canis by means of immunofluorescence assay test (ifat) and factors associated with a positive antibody response. results: there was a very low prevalence of anti-e. canis antibodies (0.8%, 2/250) among studied dogs. the antibody titers for two seropositive dogs were 1:80 and 1:160, respectively. one (0.4%) of seropositive dogs was infested with, r. sanguineus. in blood smears from one of infested dogs (0.4%), typical morulae of e. canis was observed in lymphocytes. the results confirm that the lowest occurrence of reactive dogs indoors probably related to low tick infestion. conclusion: this is the first report that describes serological evidences of canine monocytic ehrlichiosis in northeast of iran. results suggested that e. canis infection in owned pet dogs from north of khorasan was not endemic from 2009 to 2010. additional molecular studies are necessary to confirm e. canis infection and to identify the local strains of the organism. keywords: ehrlichia canis, indirect immunofluorescence assay, prevalence, dog, ifa introduction canine ehrlichiosis, a tick borne disease, was first recognized by donatien and lestoquar (1935) and has since been reported in dogs geographical widespread (bretischwerdt 1995). at the end of 1960 an epidemic outbreak of the disease with high mortality has been reported in american military dogs and south asia. this severe form was initially given the name canine tropical pancytopenia (william 1981). ehrlichia species are bacteria of the family anaplasmataceae. ehrlichia canis is a gram negative highly pleomorphic, obligate intracellular bacterium which is enveloped with a rippled thin outer membrane (marvomatis et al. 2006). it is considered to be the major causative agent of canine monocytic ehrlichiosis (cme) in dogs (huxsoll et al. 1969). rhipicephalus sanguineus, a brown-dog tick, kennel tick or pan-tropical dog tick belonging to ixodidae family is a ubiquitous tick responsible for transmitting e. canis, (jeremy et al. 2013). it is a one-host tick that feeds on dogs in all three stages of life cycle. ticks acquire e. canis by feeding *corresponding author: dr maneli ansari-mood, e-mail: manelli.ansari@hotmail.com http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 184–194 m ansari-mood et al.: seroprevalence and risk … 185 on infected dogs and transmit infection for at least 155 days afterward to other dogs (groves et al. 1975, breitschwerdt et al. 1995). they can also act as vector of important pathogens of humans such as coxiella burnetii, rickettsia conorii, r. rickettsii and bartonella henselae being of zoonotic concern (wikswo et al. 2007, dantus et al. 2008). this tick species is known to be a vector of e. canis, babesia canis, b. gipsony, hepatozoon canis, and anaplasma platys in dog (gal et al. 2007, anonymous. 2012). three clinical stages have been proposed for cme, acute, subclinical and chronic. the acute phase is characterized by fever, anorexia, lymphadenomegaly, epistaxis and petechia (neer and harrus 2006). during the subclinical phase dogs appear healthy and have the potential to remain persistent carrier (waner et al. 1996). in chronic cases, infected dogs fail to mount an effective immune response. bone marrow involvement leads to pancytopenia (moriera et al. 2005). the disease can be diagnosed by the detection of e. canis morulae in monocyte in blood smears or serologically detection of specific antibodies by the use of ifa test, dot-elisa and western blot immunoassay or by the detection of e. canis in tissue and blood by means of pcr (matthewman et al. 1993, futch and corstvet. 1996, mylonakis et al. 2003). ifa is considered the “gold standard” serological diagnostic technique for e. canis (ristic et al. 1972). the objectives of this study were to determine the seroprevalance of canine ehrlichiosis and risk factors of this disease in companion dogs’ population of mashhad, north khorasan of iran. materials and methods the study was performed on total 250 owned pet dogs (119 females and 131 males) between september 2009 until november 2010 referred to veterinary teaching hospital of ferdowsi university of mashhad for their annual vaccination, as well as with clinical illness. the following details were obtained for each dog: sex, breed, age, body temperature, location of dog’s home, appetite status, examination of lymph node, crt, infestation by tick, epistaxis and reason for referred to the hospital. after physical examination blood samples were taken in edta and non-anticoagulant tube. blood with edta were examined for hematology and complete blood count. sera were separated by centrifuge and stored at -20 ºc until assayed. for each case blood smear was prepared and stained with giemsa and direct microscopic examination was performed to detect morula on white blood cells especially on monocytes and lymphocytes. hematocrite and white blood cell count were recorded for all dogs. antie. canis antibodies were detected by flu ehrlichia immune fluorescence kit (flu ehrlichia canis, megacor, austria) with following method: sera were added to the slides after dilution (1:40) in phosphate-buffered saline (pbs) ph 7.2. positive and negative control sera were also tested. slides were placed in humid chamber and incubated for 30 minute at 37 ºc after that, those were washed twice in pbs. then we added one drop anti-dog fitc (conjugate) to each slides and those were returned to humid chamber and incubated for 30 minute at 37 ºc. incubation was performed in the dark place to protect photosensitive conjugate. after these steps, slides were washed as described before and were air-dried then 2–3 drops of mounting fluid were added to each slides and a cover slip was placed. the slides were analyzed at ×400 magnification with ifa microscopy and were compared each wall with negative and positive control. each serum sample at titer 1:40 or more http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 184–194 m ansari-mood et al.: seroprevalence and risk … 186 was considered positive. a positive reaction appears as bright sharp regularly stained inclusion bodies in cytoplasm of infected cell. the size, appearance and density of the inclusion were compared with positive control. sera were positive at the 1:40 were prepared serial dilution 1:80 1:160 1:320 1:640 1:1360 and tested again with ifa. all data were collected and because of low seropositive cases for e. canis, statistical analysis was not performed. results complete blood count showed 67 anemic (26.8%), 40 thrombocytopenic (16%) dogs. furthermore, 101 dogs in study population were diagnosed with abnormal leukogram findings including 20 leukopenia (8%), 24 leukocytosis (9.6%), 24 neutropenia (9.6%), 33 neutrophilia (13.2%). 7 dogs (2.8%) showed anemia and thrombocytopenia synchronously. 1 dog (0.4%) had morulae (fig. 1). in physical examination, 12 dogs (4.8%) were infested with tick. all ticks were r. sanguineus. 15 dogs (6%) had lymph node enlargement, 6 dogs (2.4%) had fever and 4 dogs (1.6%) had epistaxis (table 1). two (0.8%) of the 250 dogs have been examined were found to be seropositive by the ifa. both of them were adult (above 1 year) and the number of platelets, leukocytes and neutrophils were normal. morula was found in lymphocytes of one the seropositive dog. this dog showed inappetance and depression, had large submandibular lymph node and infested with r. sanguineus on physical examination. fig. 1. a morulae of ehrlichia canis (arrowed) in a blood smear from one of seropositive dogs (morulae in cytoplasm of lymphocyte) table 1. signalment and antibody titer in seropositive dogs variable dog number 171 dog number 235 reference ranges age sex breed hematocrit thrombocyte total wbc count total neutrophil count morulae body temperature appetite status lymph nodes epistaxis ifa titer 13 years old female german shepherd 37 2.5 8000 6500 38.5 normal normal 1:80 9 years old male mixed terrier 42 3.34 7300 5986 in lymphocyte 39 inappetite submandibular l.n enlarged 1:160 37-55 % 1.6-4.3×106/µ l 6000-17000/µ l 3000-11500/µ l http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 184–194 m ansari-mood et al.: seroprevalence and risk … 187 discussion this study is the first investigation of the seroprevalence of e. canis antibodies among dogs in north khorasan of iran. the results revealed low prevalence of e. canis (2 dogs, 0.8%). prevalence of ehrlichiosis was also reported from other regions of iran: jafari et al. (1997) in shiraz (south west of iran) have examined 180 dogs. seventeen dogs (9.44%) were found positive for the presence of e. canis in their white blood cells. akhtardanesh et al. (2009) used ifa and ica to detect antibodies against e. canis in 123 apparently healthy dogs. the overall seroprevalence was 14.63 %. seventeen (13.8%) dogs in ifa test and 13 dogs (10.6%) in ica were seropositive for cme. in blood smears from three infected dogs (16.6%) morulae were observed in monocytes. avize et al. (2010) have reported seroprevalence of cme in 198 companion dogs of different ages by means of ifa and ica 9.6 % in ahvaz (west of iran). morulae of e. canis were observed in monocyte of four infected dogs (2.02%). blood samples from 980 dogs (510 domestic dogs and 475 wild dogs) in west azerbaijan and 820 dogs (520 domestic dogs and 300 wild dogs) in east azerbaijan of iran were obtained by asri and others in 2001 and tested by ifa for diagnosis of canine ehrlichiosis. sixty seven percent of wild dogs and 38 % of domestic dogs in west azerbaijan and for east azerbaijan 58 % and 39 % were serologically positive for ehrlichia. the main variants have been diagnosed were e. canis (75%), e. platys (20%) and e. equi (5%). in our study because of low seroprevalence of e. canis, we could not reach any correlation between age and cme but in many investigations the prevalence has been significantly differed among age groups. in shiraz (jafari et al. 1997) the animals of all ages seemed equally susceptible to disease. in ahvaz (avize et al. 2007) prevalence rate have been significantly higher in adult dogs than juniors. the prevalence rate was 16.8 % in above 3 years old and 11.86 % in 1–3 years old compared with dogs less than 1 year old (1.41%). in kerman (akhtardanesh et al. 2009) high association was observed between age and seropositive dogs. possible explanations for more infection in older group include the immunologic status of the host or more exposure to the vector ticks (rodriguez-vivas ri et al. 2005). german shepherd dog has been reported to be more susceptible to cme (nyindo et al. 1980, harrus et al. 1997). in shiraz (jafari et al. 1997) 21.1 % of infected dogs were german shepherd. some research showed higher prevalence in male dogs (batmaz et al. 2001, costa et al. 2007). in some studies no significant difference was proved between sex and various breeds with presence of e. canis antibodies (waner et al. 2000a, rodriguez-vivas et al. 2005, hernandez et al. 2005, solanogallego et al. 2006, akhtardanesh et al. 2009, avize et al. 2009, roqueplo c et al. 2009). the clinical signs of cme may vary among and within geographic locations (harrus et al. 1997a,b). the probable reasons include e. canis strain pathogenicity, dose of infectious organism, breed of dog, immuno status of the host and co-infection with other tick-borne parasites (rodriguezvivas et al. 2005, neer and harrus 2006). thrombocytopenia is the most common hematological finding in patients with acute cme. this change is found in all stage of disease but is more severe in chronic phase as a result of bone marrow hypoplasia (troy et al. 1980). death may occur as a http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 184–194 m ansari-mood et al.: seroprevalence and risk … 188 consequence of hemorrhages and secondary infections (hendricks and bob 2004). in our study because of low prevalence of e. canis we could not show any relationship between seropositive dogs and hemathologic changes but platelet and leukocyte count in both seropositive dogs were normal. in ahvaz (avize et al. 2009) the prevalence of ehrlichiosis was higher in dogs with thrombocytopenic although the difference was not significant and correlation was not observed between seronegative and seropositive dogs for hemathologic changes. in kerman (akhtardanesh et al. 2009) thrombocytopenia, leukopenia and anemia were just observed in dogs with high ifa titer (>1:320). rodriguez-vivas ri et al. (2005) have found that the presence of thrombocytopenia, platelet-related bleeding and a seropositive response to e. canis in a patient increase the index of possibility for infection. the only known vectors of ehrlichiae are ixodid ticks (rikishia 1991). rhipicephalus sanguineus and possibly the american dog tick, dermacentor variabilis are the vector for e. canis (groves et al. 1975, johnson et al. 1998). rhipicephalus sanguineus is widely distributed in the world but it is mainly in tropical and subtropical regions and also well adapted to the indoor environment where owned dogs are kept (uspensku and ioffe-uspensky 2002, dantas-torres 2010). dogs may acquire ticks in the city areas in parks and housing estates (siuda 1993). infestation by r. sanguineus has significant risk factor for e. canis seropositivity in brazil (trapp et al. 2002). in this study, 4.8 % (12 dogs) were infested by r. sanguineus. one of the seropositive dogs also had this tick on his trunk. r. sanguineus was also the most common species in north-east of iran (razmi et al. 2003). diagnostic method can affect on prevalence results of e. canis. as said above the indirect immunofluorescence antibody (ifa) test is considered the serological “gold standard” for diagnosis of cme (ristic et al. 1972). serological cross-reactivity occurs with other members of ehrlichiae like e. equi (baneth g et al. 1996), e. ristici (ristici et al. 1999), e. ewingii (anderson et al. 1992), e. chaffeensis, neorickettsia helminthoeca (rikisha 1991). in this study, ifa test was used and seropositive titers were 1:80 and 1:160. ifa test is more susceptible than other test but supplementary test such as pcr and western immunoblotting is needed for detection of active infection and distinguished between infections with different type of species. possible explanations for low seroprevalence of e. canis in this study are: 1. selected population: exposures to tick in domestic dogs are lower because of location and observance of health condition by owners. the life conditions of dogs affected the seroprevalence of e. canis (roqueplo et al. 2009). lim et al. (2010) indicate that risk of exposure to vector-borne disease in rural hunting dogs can be quite high in korea. ploneczka et al. (2003) showed that dogs in non-urban areas (9.9%) or they have living in outdoor (12.7%) had a higher prevalence of e. canis. rural dogs had more parasite infestation than urban dogs (dagnone et al. 2002, carvalho et al. 2008). 2. weather conditions: the prevalence of e. canis is largely dependent on the distribution of the vector, r. sanguineus, which occurs mainly in tropical and subtropical regions but it has worldwide distribution (jeremy et al. 2013). jafari et al. (2008) have determined the prevalence of canine ectoparasite infestation in pet dogs from the shiraz area of southern iran. overall, 160 dogs were examined for ectoparasites, and 142 r. sanguineus ticks were found on 13 dogs. a http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 184–194 m ansari-mood et al.: seroprevalence and risk … 189 significant correlation was observed between increases in temperature and decreases in humidity and increased ectoparasite infestation. the number of dogs infested with ectoparasites in summer and spring was significantly higher than in winter (p= 0.007). morales-soto and cruz-vazquez (1998) found r. sanguineus along the year in cuernavaca, mexico but the peaks of tick were found in april, july and november and the lower prevalence were in january. so season of sampling can affect seroprevalance of e. canis. 3. type of serological test: ifa detects antibodies as early as 7 days after initial infection but some dogs may be negative until 28 days after infection or in acute phase of disease and also in chronic phase because of injury to immune system (groves et al. 1975) when e. canis antibody titers results are negative, a follow up examination in 2 to 3 weeks or serotesting for other agents is recommended (neer and harrus 2006). the cme has a worldwide distribution and a significant seroprevalence in dogs from southeast asia, africa, europe, central and south america was reported (cardenas et al. 2007). antibodies against e. canis were detected in neighbors and close countries to iran as 44.4 % in saudi arabia (sacchini et al. 2007), 21 % in turkey (batmaz et al. 2001), and 33 % in egypt (botros et al. 1995). in our study, seroprevalence of e. canis was estimated less than 1 %. so cme is not endemic in mashhad, but in kerman, ahvaz and azerbaijan is considered endemic (asri et al. 2001, akhtardanesh et al. 2009, avizeh et al. 2010). besides, e. canis is a human health hazard and causes clinical signs of disease (perez et al. 2006). human ehrlichiosis is caused by e. chaffeensis, a. phagocytophilum and e. ewingii (dumler et al. 2007). co-infection of e. canis and a. phagocytophilum is possible (amusategui et al. 2007). a. phagocytophilum was reported in ixodes ricinus in north of iran (bashirbod et al. 2004). it is possible that more tick-transmitted pathogens can infect dogs, including e. canis, a. phagocytophilum, b. canis, hepatozoon canis, bartonella spp. (baneth et al. 1998, breitschwerdt et al. 1998, yabsley et al. 2008). so in dogs with clinical signs of thrombocytopenia, leukopenia, fever and epistaxis if they have negative result for e. canis, consider the possibility of infectious with other organisms. there is significant correlation between ehrlichiosis with leptospirosis, leishmaniasis and babesiosis. so in dogs that have one of these diseases, e. canis infectious should be considered (matthewman et al. 1993, suksawat et al. 2001, hernandez et al. 2005, 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vinsonii berkhoffii, and rickettsia spp. in dogs from grenada. vet parasitol. 151: 279–278. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 144 http://jad.tums.ac.ir published online: june 30, 2020 original article purification and partial characterization of agglutinin lectin from heamolymph of german cockroach, blattella germanica zohreh nabavi; mozhgan baniardalani; *hamidreza basseri department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 23 feb 2018; accepted 28 may 2020) abstract background: lectin molecules have crucial biological role in insects’ immune system. the aim of present study was to find the agglutinin activities in haemolymph of german cockroach, belatella germanica with appropriate screening and purification. methods: the heamolymph of cockroach was collected and agglutinin test performed against different animal and human red blood cells (rbc). then sugar inhibition assay was carried out to find carbohydrate specific binding lectin. the proteins of haemolymph was purified using ion-exchange chromatography (hplc) and each fraction was tested for agglutinin activity. finally the molecular weight of the agglutinin protein was determined using sds-page. results: the most agglutinin activity of haemolymph was found against rbc of mouse at titer 1/128ml/l dilution and sugar inhibition assay showed that fucos, n-acetyglucoseamine and galactose reduced titer of agglutinin to ½ml/l. only one fraction of heamolymph at rotation time of 36 minute showed agglutinin activity. the molecular weight of this lectin was measured as 120kds. conclusion: the range of agglutinin activities against different rbc indicates that the isolated lectin is not specific for a particular carbohydrate. in addition, the isolated lectin at low concentration present in heamolymph should be an innate lactin not secreted, because we found it without any trigger immunity of the insect. keywords: german cockroach; heamolymph; lectin introduction insects lack specific acquired nonself recognition systems such as antibody in vertebrates, and lectins molecules have main role of nonself recognition in the innate immune response (1-4). lectins can agglutinate foreign matter and thus become to be it more suitable for understanding. these molecules can also mediate haemocytes invasion to nonself particles, and consequently cause lysis, increase phagocytosis of foreign particles and activate the coagulation system (5, 6). lectins are proteins or glycoproteins that recognize specifically and bind reversibly the carbohydrate-containing molecules of foreign cells (7-10). lectin molecules have a range of immunologic action including antitumor, antifungi and bacteria activities which may find practical applications (11, 12). carbohydrate binding lectins with similar properties are presence on the surfaces of many pathogens. therefore, a wide variety of pathogens can be recognized by specific binding of lectins (13, 14). however, the interaction between lectins and carbohydrates as ligand receptors is common in living organisms (15). it has been shown that two c-type lectins has roles in antibacterial defense as well as in the melanization response to plasmodium berghei in anopheles gambiae (16). in addition, lectins as immune substances have been reported in american cockroach (periplaneta americana) and desert locusts (shistocerca gregaria) (17-19). multiple lectins have been purified from only two species of cockroaches, namely, the american cockroach, p. americana; and the west indian leaf cockroach, *corresponding author: dr hamidreza basseri, e-mail: basserih@tums.ac.ir mailto:basserih@tums.ac.ir j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 145 http://jad.tums.ac.ir published online: june 30, 2020 blaberus discoidalis (20, 21). in addition, fucose-binding lectin in the heamolymph of p. americana showed the maximum phenoloxidase activity against dopamine (6). according to our knowledge and survey of literatures, there are no many references exist about agglutinin activity in the heamolymph of german cockroach, blattella germanica. therefore, in the present study, we tried to identify and purify lectin molecule s from heamolymph of german cockroach. our results provide a new insect lectin from b. germanica. it seems c-type lectin as receptors provide main role for penetration of cockroach allergens (22). this is the first report on characterization of hemagglutinin activity in german cockroach heamolymph. hence, in the present study, we also attempt to isolate and purify lectin from the heamolymph of b. germanica. the finding of current study provides initial information about presence of lectin the heamolymph of german cockroach. these results provide preliminary information for further studies such as allergenic actives cause by german cockroach. materials and methods insect rearing and bleeding german cockroaches, blatella germanica, were maintained in an insectary at 25±2 °c with humidity of 60–65%, and fed on dried bread, sugar cubic with water. adult cockroaches (450 insects) were anaesthetized with co2 and then the ventral surface of sternum of each cockroach was sterilized with 70% ethanol. the coxa membrane and ventral joint of abdomen to thorax were pierced with a sterile needle to pull out haemolymph. the haemolyphm was collected, homogenized and then centrifuged at 1800 rpm for 15 minutes. the supernatant was kept in -80 °c until used. the protein concentration of each sample was measured by bradford assay method (23). haemagglutination assay haemagglutination assays were performed presented by chen et al. (24). the red blood cells (rbcs) of rabbit, rat, sheep, guinea pig, syrian mouse and human (a, b, ab and o groups) were used to exam haemagglutination activities of the haemolymph of b. germanica. all rbcs were washed three times in 10mm phosphate buffered saline (pbs) containing 150mm nacl and 10mm sodium phosphate at ph 7.4 and 380mosm, 1mm cacl2 at 4 ºc and the concentration of the rbcs suspensions to the buffer and adjusted to final concentration of 2%. five microliters of the buffer was dispensed in each microtiter plate well and then two folded serial dilution of the haemolymph extract was prepared in each row of plates to give final dilution ranges of 2 -1 to 2 -10 , prior to the addition of 5μl rbcs to each well. the plates were covered and kept at room temperature for two hours. the end points of agglutination examined under a stereomicroscope and by the flow characteristics of the erythrocyte pellets when the plate was held at an angle. all experiments were replicated three times and the controls always included pbs plus rbcs alone. carbohydrate inhibition assays carbohydrate inhibition assays were followed as mentioned by chen et al. (24). the sugar specificities of cockroach lectins were investigated by competitive binding using the following carbohydrates: d-(+)-glucose, d-(+)-galactose, d-(+)-mannose, l-(-)-fucose, lactose, n-acetyl-d-glucosamine, n-acetyld-galactosamine, fructose and arabinose (all purchased from sigma co.). stock solutions of sugars were prepared in pbs at 0.2m and stored in -4 °c until use. two folded serial dilutions of haemolymph (each of 5μl) were prepared in pbs followed by addition of 5μl of appropriate carbohydrate at the above initial concentration. the plates were incubated at room temperature for 60min and then 5μl j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 146 http://jad.tums.ac.ir published online: june 30, 2020 of the mouse rbcs added to the respective wells. the controls were consisted of carbohydrate plus pbs, and plus rbcs alone. inhibitory effects were recorded as those reducing agglutination in the wells. the end points of agglutination were examined under a stereomicroscope and by the flow characteristics of the erythrocyte pellets when the plate was held at an angle. this experiment was replicated three times. lectin purification ion exchange-high performance liquid chromatography (hplc) (knauer co., germany) was utilized to separate protein from extracted haemolymph. the samples once more were centrifuged for 15 minutes at 1800 rpm and supernatant filtered three times through a 0.45µm filter. the sample was loaded to column resin (asahi chemical co., japan) with carboxymethyl as the functional group as particles, 7.5×100 id, 150mm length, 2000 pore size). the mobile phase was 1m of nacl in 20mm tris-hcl (ph= 7.5–8) which applied at a flow rate of 1ml/min at room temperature. the column was previously equilibrated with 20mm tris-hcl buffer at volume 300µl from 0–20min and a flow rate of 1.0ml/min. the sample at 300µl volume was loaded to an ionexchange column. the proteins were eluted with a 50min linear gradient of 90–10% nacl/tris-hcl at a flow rate of 1.0ml/min at room temperature. the chromatographic run was monitored at 280 nm of absorbance. the fractions were collected manually and then the eluted fractions were concentrated using freeze-dryer device. finally agglutinin activity of each eluted protein was assayed. the molecular weight of agglutinin protein was measured by polyacrylamide gel electrophoresis (sds-page). polyacrylamide gel electrophoresis (sdspage) the molecular weight of protein, purity and molecular mass of fractions were estimated using sds-page. tris base 30.3g, glycine 144g, sds 10g and make to 1l with dh2o. twenty μl of the eluted protein were loaded on the sds-page gels (12%) and run. the gel was stained with coomassie brilliant blue r 250/silver. as well as standard protein marker was loaded on sdspage (mix of seven proteins, 14.4–116kda). biorad apparatus was used, the gels were run at 200v (constant voltage) until the bromophenol blue dye was just off. gels were run at room temperature and 50–60 minutes. ethics approval ethics approval was not required for this study. results haemagglutination assay the results of haemagglutination activities of whole cockroache serum against a range of erythrocytes were represented in fig. 1. the highest activity were found against mouse erythrocytes (titer ≤128) followed by sheep (titer ≤ 64) but these activities were relatively less against different human erythrocytes (fig. 1). the lowest activity was observed with human oerythrocytes (titer ≤8). thus, mouse erythrocytes were candidate for further inhibition assays. carbohydrate inhibition assay the results of haemagglutination inhibition assay are represented in fig. 2. the haemolymph lectin activity was reduced by all tested sugars except fructose. the inhibitory effect of galactose, fucose and n-acetyl-dglucosamine was more than other carbohydrates while the agglutination of hemolymph reduce to titer of ≤2. fructose did not show any specific binding to lectins of haemolymph. glucose, mannose, lactose and n-acetyl-d-galactosamine showed similar inhibitory effect on the lectin activity at titer of ≤8 j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 147 http://jad.tums.ac.ir published online: june 30, 2020 which indicate that non-specific binding exist in the haemolymph lectins for these carbohydrates. arabinose also blocked agglutinin activity at titer of ≤ 4 which it was moderate inhibitor. ion-exchange chromatography following the separation and purification of the protein molecules using ion exchange hplc, five peaks at chromatogram were appeared. all fractions were applied for haemagglutination activities and the main activity was found by fifth peak which appeared at 36min retention time (tr) in chromatogram (fig. 3). the molecular weight of the purified protein was estimated to be about 66 kda by sodium dodecylsulfate polyacrylamide gel electrophoresis (fig. 4). fig. 1. agglutination activities of haemolymph of the german cockroach, blattela germanica, against different red blood cell (rbc) at 2% concentration in phosphate buffered saline. all experiments were replicated three times and the control was phosphate buffered saline (pbs) free haemolymph j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 148 http://jad.tums.ac.ir published online: june 30, 2020 1 2 4 8 16 h a a ct iv it y ( lo g 2 ) fig. 2. haemagglutination inhibition assay of hemolymph lectins of the german cockroach, blattela germanica. the assay was performed with mice red blood cells (rbcs) (2%) in phosphate buffered saline (pbs). the control was haemolymph free carbohydrate fig. 3. ion exchangehigh performance liquid chromatography (ie-hplc) of blattela germanica hemolymph. 300µl of the diluted hemolymph was loaded into the column (7.5×100 id, 150mm length, 2000 a° pore size). the column was equilibrated with 300µl water from 0–20min at a flow rate of 1.0ml/min. proteins were eluted with a 50min linear gradient of 90–10% nacl / tris-hcl at a flow rate of 1.0ml/min at room temperature. the chromatographic run was monitored at 280nm of absorbance. five peaks were observed in chromatogram, only fifth peak was determined presence of lectin in hemagglutination assay j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 149 http://jad.tums.ac.ir published online: june 30, 2020 fig. 4. sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page) of whole heamolymph proteins of belattella germanica and the fractions which showed agglutinin activity. all fractions prepared using reversed phase high performance liquid chromatography (rp-hplc) and applied for agglutinin activity assay. the molecular weight of isolated lectin was approximately 66kda. gel was stained with coomassie brilliant blue r 250 discussion in the current study, we assessed haemagglutinin activity in the heamolymph of german cockroach. the mouse and sheep erythrocytes showed high agglutinin reaction with the heamolymph lectins. similarity, the erythrocytes of mice showed high agglutinin activity against heamolymph of discoid cockroach, blaberus discoidalis. therefore, the authors used the mice erythrocytes for study on two isolated lectins called as bdl1 and bdl2 (24). in the current study, we also used mouse erythrocytes for inhibition assays. german cockroaches have developed effective innate immunity of protecting themselves against pathogenic microorganisms including epidermal immunity (25) or humoral immune defense (24). lectins are one of the pattern-recognition proteins concerned in innate immunity in insects (4). the presence of lectin activities in the heamolymph is more important for immun ity of b. germanica against microbial pathogens. as recently showed, c-type lectin has crucial role to response to pathogen infection by the expression of antimicrobial peptides and the agglutination of bacteria immunity in red flour beetle tribolium castaneum (3). therefore, further work needs to characterize the role of lectins in immunity of the german cockroach. we optimized ion exchange-hplc to purify that protein which showed agglutinin activities. the multiple lectins had been purified from different species of cockroaches e.g. the american cockroach, periplaneta americana (6, 19, 25-27) and the west indian leaf cockroach, bl. discoidalis (21). at least four lectins have been reported in this species, namely, bdl1, bdl2, bdl3, and gsl (24). in addition, based on carbohydrate specificity, we characterized specificity of protein binding carbohydrates to the hemolymph 66 kda j arthropod-borne dis, june 2020, 14(2): 144–152 z nabavi et al.: purification and partial … 150 lectins of the german cockroaches using different carbohydrates. the lectins of german cockroach showed more affinity to glucose, n-acetyl-d-gluctosamine and fucose. based on makela category (28), in the current study, the lectin in b. germanica heamolymph could be categorized in groups ii and iii. these types of lectins are more involve in immunity of insects and have crucial role in phagocytosis of microorganisms (20). in addition, the heamolymph lectin of b. germanica agglutinated different types of animal and human blood cells which indicated this lectin is non-blood type–specific. generally, lectins recognized α-linked on carbohydrate surface of red blood cells, as we could not find rbc specificity, it may indicate the lectins failed to distinguish between α and ß-linked of carbohydrates on the surface of the blood cells. it was also possible that the lectin have independent binding sites, which can be used for attachment to the erythrocyte surface. in the present study, only one fraction of the heamolymph with molecular weight of 66kda (with no subunits) showed agglutinin activities against mice rbc (fig. 1). generally, the molecular weights of all lectins of invertebrates vary from 26kda to 1500kda (29-31). this range of difference could be depended on species or methods used for purification, analysis and protocols. the maximum molecular weight of insect lectin (1500 kda) was reported in p. americana (25) whereas the lowest molecular weight lectin was that of agrotis segetum with 69kda and no subunits (32) while we introduce a lectin from heamolymph of b. germanica with molecular weight of 66kda. according to our results, this agglutinin activities could be calcium dependant-lectin. while the c-type lectin family consists of members that bind their ligands in a calcium-dependent manner, many other c-type lectins show the hemoagglutinin activity in a calcium-independent manner. conclusively, our current results provide a new insect lectin from german cockroach, b. germanica. this finding would be helpful in future studies on lectins concerning. acknowledgements the authors would like to thank dr behrouz vaziri and mrs fatemeh torkashvand in protein 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sexta lipopolysaccharide-specific immulectin-2 protects larvae from bacterial infection. dev comp immunol. 27: 189– 196. 32. zheng x, xia y (2012) beta-1, 3-glucan recognition protein (betagrp) is essential for resistance against fungal pathogen and opportunistic pathogenic gut bacteria in locusta migratoria manilensis. dev comp immunol. 36: 602– 609. j arthropod-borne dis, sep 2022, 16(3): 217–224 s ghodrati et al.: a sero-epidemiological study on … 217 http://jad.tums.ac.ir published online: sep 30, 2022 original article a sero-epidemiological study on visceral leishmaniasis among volunteer children and adults in rural areas of shahroud, iran 2018–2019 sajjad ghodrati1, *behnaz akhoundi1, *mehdi mohebali1,2, mohammad zeinali3, homa hajjaran1, zahra kakooei1 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran 3zoonoses control unit, center of diseases control, ministry of health and medical education (mohme), tehran, iran *corresponding authors: prof mehdi mohebali, e-mail: mohebali@tums.ac.ir, dr behnaz akhoundi, email: behnazakhoundi@yahoo.com (received 06 jul 2020; accepted 30 june 2022) abstract background: visceral leishmaniasis (vl) also known as kala-azar is considered as one of the zoonotic infections in mediterranean countries. the disease reservoir and vectors are dogs and sandflies respectively. due to reported sporadic cases of kala-azar in the past five years in shahroud county, semnan province, iran, this study aimed to investigate the status of this infection in this area and to determine its seroepidemiology to take required measurements for infection control and treatment. methods: this study was conducted on 504 subjects residing in seven villages in shahroud county. blood samples were randomly selected using the cluster sampling method and were collected from subjects aged up to 13 years old (90%) and adults over 13 years old (10%) from september to may 2019. after separating sera from whole blood, samples were subjected to direct agglutination test (dat) to detect anti-leishmania infantum antibodies. a range of 1:10 to 1:800 dilutions were prepared from the samples. results: results of 1:800 titration indicated that no sample was positive for antibodies against l. infantum. after the secondary screening, 10 cases (1.98%) showed the antibody titer of 1:100, while four cases (0.79%) showed the antibody titer of 1:400. of 14 cases with the l. infantum antibodies, all were detected from the children <13 years old. according to clinical findings, no patient was suffering from fever, weight loss, splenomegaly, hepatomegaly, and cachexia and therefore did not show the kala-azar symptoms. conclusion: the results of the current study indicate that kala-azar is not prevalent in shahroud county. keywords: visceral leishmaniasis; seroepidemiology; direct agglutination test; human; iran introduction protozoan parasitic infections are considered major health problems globally. leishmaniasis is one of the most common contagious infections in tropical regions, and it is considered as one of the leading causes of death due to parasitic infections after malaria (1-2). this infection is observed in different cutaneous, mucocutaneous, and visceral forms. mucocutaneous forms are not common in iran; however, visceral leishmaniasis (vl) also known as kala azar is observed sporadically or endemically. kala-azar is a systemic infection, mainly transmitted to humans by sand flies from the genus phlebotomus in the old world (3-5). the incidence of this infection has been reported at 500,000 cases and the fatality subsequent to this disease has been estimated at 75,000 globally; however, vl is mostly found in developing countries with poor hygiene (6). symptoms in humans include prolonged copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:mohebali@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 217–224 s ghodrati et al.: a sero-epidemiological study on … 218 http://jad.tums.ac.ir published online: sep 30, 2022 fever, hepatosplenomegaly, weight loss, pancytopenia, fatigue, and delayed treatment may result in death due to secondary infections or internal bleeding. in most cases, kala-azar may not directly lead to death; nonetheless, patients with the suppressed immune system and hiv patients may contract this infection leading to complications and death (4, 7-8). kalaazar is mostly observed in young children below the age of 12 years old, and it is most common in rural areas. acute forms of infection are mostly present in children aged < 2 years old or immunocompromised patients and the chronic forms are mostly observed in healthy adults. the gold standard for diagnosis of vl is the observation of amastigotes in aspirates of spleen or bone marrow. however, in endemic areas, serological testing is more common (9-10). the reservoir of mediterranean vl, is majorly dogs or beavers, and the etiological agent for this type of infection is mostly leishmania infantum which is present in iran although there is no evidence for its etiology (11-14). in iran, the first case of vl was reported by pouya, in 1949 in rural areas of tonekabon, in the western region of mazandaran province, north of iran. ever since, vl has been endemically reported in some regions in iran such as fars province, eastern azarbaijan, ardabil and boushehr (15). the presence of ph. neglectus, ph. keshishiani, ph. kandelakii, ph. alexandri, ph. major, and ph. tobbi, known vectors of l. infantum, in iran highlights the global distribution and significance of these species in the transmission of leishmaniasis (1621). to our knowledge, no epidemiological studies have been conducted on the prevalence of this infection in shahroud county, semnan. epidemiological studies are highly important as they would provide insight into the status of the infections and thereby, help us take the necessary measures for infection control and treatment. therefore, this study aimed at investigating the epidemiological status of vl and determining the seroprevalence of this infection in shahroud county located in semnan province. materials and methods study area this cross-sectional descriptive study was carried out from september 2018 to may 2019 to determine the seroepidemiology of vl in both children with < 13 years of age and in adults residing in shahroud county. shahroud county in semnan province is located in the east of tehran, at the junction of the road between mashhad and gorgan. regarding the climate of the city, it has a dry desert climate with cold winters and hot summers. rain is not frequently seen as the city ends in the central desert of iran on the southern side. the average precipitation is 167.7mm a year and its average relative humidity is 48% during a year. this county, with a population of 218628 is the most populated county in semnan province. study design in this study, a randomized cluster sampling was carried out corresponding to seven villages, namely dehmolla, abarsij, moghan, abr, bedasht, haq ol khvajeh, and kalateh motahhar from all geographical regions of the county (fig. 1). the sample size of 504 patients was estimated based on cochran’s method. as only 454 children the age of <13 years old volunteered for the test, 50 volunteered persons the age of >13 years old with suspected clinical manifestations were randomly chosen for the sampling. subsequently, multistage sampling was carried out and informed consent was taken from every subject and demographic information of each subject, including age, sex, clinical symptoms, and any disease background was recorded. all subjects were permanent residents of the mentioned regions. for blood collection, 50μl of blood samples were collected from subjects at rural health http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 217–224 s ghodrati et al.: a sero-epidemiological study on … 219 http://jad.tums.ac.ir published online: sep 30, 2022 care centers by disposable sterile lancets and capillary tubes that were non-heparinized. blood samples were then transferred to the department of parasitology, school of public health, tehran university of medical sciences, tehran, iran, in cold chain conditions to prevent the disruption of antibodies and hemolysis. sera were then separated using centrifugation at 3000rpm for 5min. all sera were then stored at -20 c for further tests. for serological tests, sera were tested by direct agglutination test (dat) to detect antibodies against l. infantum according to the methods previously described by harith et al. (22). in this serological study, antigens used in dat are the promastigote forms of l. infantum (23). if the antibodies against l. infantum are present in the sera of infected patients, the antigens and antibodies form a mixed blueish suspension without sedimentation. on the other hand, in case antibodies against l. infantum are absent, a dark blue spot is observed at the bottom of the plate following the accumulation of dat antigen. for the dat test, serial dilutions of sera were prepared with 0.9% saline solution, 0.2g gelatin (1.04070/ merck, germany), 0.78ml 2-me (m-6250/ sigma, usa), and 100ml sterile distilled water at 1:10–1:800 in v-bottom polystyrene microplates. then, 5μl of dat antigen was added to each well. leishmania infantum antibodies were used as positive control and the sera of a l. infantum-free person was used as a negative control to avoid any possible test errors including autoagglutination and so on. finally, after 12–18 h incubation at room temperature, the plates were assessed for antibody agglutination, and the test was repeated one more time for any suspicious agglutination. for initial screening, dilution of 1:800 of samples was used for antibody detection. according to previous studies, antibody titer of ≥ 1:3200 in samples of patients with clinical manifestations is indicative of kala-azar, while antibody titer of 1:800 and 1:1600 are considered negative and suspected of infection, respectively. in cases of samples suspected of vl, sampling should be carried out again 2–3 weeks later to confirm the results. in case a significant increase is observed in antibody titer, vl is confirmed. in case no positive result was observed in the initial screening using 1:800 titration, all sera were screened again using lower titers. finally, results from dat and demographic data were analyzed using spss software v. 22. fisher’s exact and chi-square tests, with a 95% confidence interval, were performed to compare values relative to age, gender, and region. the existence of statistical significance was assumed if p≤ 0.05. results this study was conducted on a total of 504 subjects residing in seven villages in shahroud county. of 504 subjects, 302 (60%) cases were male, and 202 (40%) cases were female. the age range of the participants was 3 months to 59 years old. in fact, 90% of subjects were up to 13 years old and 10% of subjects were 13 or above 13 years old. serum samples were screened at dilution of 1:800. at the initial screening stage, using dat, no antibody titer of ±1:800 was found, however, after the secondary screening, 10 cases (1.98 %) showed an antibody titer of 1:100, while four cases (0.79%) showed the antibody titer of 1:400. demographic data are presented in table 1. http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 217–224 s ghodrati et al.: a sero-epidemiological study on … 220 http://jad.tums.ac.ir published online: sep 30, 2022 table 1. demographic characteristics of the participants and the geographical distribution of subjects showing 1:100 and 1:400 titration with dat antibodies against l. infantum in 2018–2019 in shahroud characteristics no. percent 1:100 titration (no.) 1:400 titration (no.) gender male 302 60 female 202 40 age group ≥13 years old 50 10 2-13 years old 411 82 <2 years old 43 8 resident area dehmolla 75 14.8 1 abarsij 75 14.8 2 1 moghan 100 19.8 2 1 abr 100 19.8 3 2 bedasht 75 14.8 2 haq ol khvajeh 40 7.9 kalateh motahhar 39 7.7 fig. 1. schematic map of shahroud and the geographical locations of sampling villages discussion visceral leishmaniasis (vl) also known as kala-azar is a deadly parasitic disease if left untreated, especially in infants, and a global public health concern. this infection has been reported sporadically or endemically in some regions in iran (15). kala-azar has several types of which mediterranean kala-azar has been reported in iran. since there were unreported sporadic incidences of infection with l. infantum, we aimed at investigating the status of vl in this region. however, the following two studies have shown there was no vl sand fly vector species (ph. kendelaki, ph. tobbi, ph. major, ph. perfiliewi, and ph. alexandri) in the region (shahrood and damghan where is very close to sharood) (24, 25). the clinical manisemnan sharoud tehran http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 217–224 s ghodrati et al.: a sero-epidemiological study on … 221 http://jad.tums.ac.ir published online: sep 30, 2022 festations of kala-azar like fever, weight loss, splenomegaly, hepatomegaly, and cachexia were absent in the individuals, and the subclinical infection might be present in asymptomatic patients and dat can be used for the detection of antibodies against l. infantum. generally, for epidemiological studies, dat is commonly employed in endemic areas in large-scale screening of vl. this technique is simple, valid, and cost-effective with high sensitivity and specificity of 90–100% and 72–100% respectively (26, 27). we could not find any study regarding the prevalence of vl in shahroud county; however, there have been several other studies that have investigated the prevalence of vl using serological testing in other regions in iran. during a four-year period of 2002–2005, a survey of 6558 subjects from different regions of iran known as endemic regions indicated the prevalence of 4.9 and 4.4% of vl in northeastern and northwestern iran, respectively. the prevalence of this infection was lower in the central and southwestern parts of the country with rates of 1.5% and 1.9%, respectively (15). our study showed zero prevalence of vl in shahroud which is somehow in accordance with the rates reported in different parts of iran. in another study by mahami et al. (2003) 1155 children up to 12 years old and adults in ardabil were tested for vl using dat. according to their results, 32 cases (2.8%) showed the antibody titer of 1:800, while 7 cases (0.6%) were serologically positive for vl with the antibody titer of 1:3200 (28). in 2013, the clinical implications, as well as the epidemiology of vl, were studied in different parts of iran using dat. a total of 1698 cases showed the anti-l. infantum titer of 1:3200 and these cases were mostly aged up to 12 years old and almost 75% of cases with antibody titer of 1:3200 were in endemic areas and were having clinical manifestations (15). in 2011, 402 human blood samples were taken from mazandaran province and tested for vl using dat. among these subjects, 8 cases (2%) showed the antibody titer of 1:1600 and were therefore suspected as vl cases. the results showed no definite positive case of vl which is in accordance to the current study (29). on the other hand, in 2015, blood sampling was conducted on 800 subjects residing in lorestan province, and the prevalence of vl was determined using dat. the anti-l. infantum titer was 1:800 and 1:1600 in 21 cases (2.62%) and 5 cases (0.62%) showed the antibody titer of 1:3200 of which only one case had the clinical manifestation of anemia (30). in several kala-azar endemic regions in iran and the globe, most cases of vl infections are in the asymptomatic form. in another study in iraq, 20% of children who were either healthy or asymptomatic showed anti-leishmania antibodies by the dat method. the existence of infected patients who are showing no symptoms of kala-azar in vl endemic regions may affect the transmission cycle of the parasite responsible for this infection. therefore, evaluating the residents in these regions by employing serological testing such as dat can facilitate the on-time identification of vl patients to reduce the rate of transmission (31). similarly, in 2015, in a survey, 1007 blood samples of children under 10 years old residing in rural areas of alborz province were collected and 37 children showed anti-leishmania infantum antibodies with titers of ≥ 1:800 and 2 children showed anti-leishmania infantum antibodies with titers of ≥ 1:3200 with clinical features and then treated with anti-leishmaniasis drugs in pediatric hospital (32). on the other hand, in 2017, blood samples were collected from 617 children up to 12 years old residing in rural areas of fars province with no clinical manifestations and 17 cases showed antibodies against l. infantum, and the highest rate of infection was observed among children aged 5–8 years old, indicating the subclinical vl in children from rural areas in southern iran (33). according to the results of the current study and the presence of sub-clinical cases, it seems that the prevalence of vl in shahroud county http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 217–224 s ghodrati et al.: a sero-epidemiological study on … 222 http://jad.tums.ac.ir published online: sep 30, 2022 is very low. increased availability of healthcare centers and improved policies of the country's health system to control diseases transmitted by insects play a major role in increasing or decreasing the incidence of this infection. according to multiple studies in endemic areas of vl in iran, not only symptomatic dogs, but also asymptomatic dogs can harbor l. infantum infection, thereby affecting the transmission cycle in endemic areas. therefore, it seems necessary to conduct additional epidemiological studies on reservoirs to prevent the spread of the disease in these regions. conclusions the results of the current study indicate that kala-azar is not circulating in shahroud county. additional epidemiological studies are required on vectors and reservoirs of l. infantum to prevent any sporadic incidences in the region. acknowledgments this study was extracted from the first author’s msc. thesis under the ethical supervisory committee code of ir.tums.sph.rec.1397. 129 at the school of public health, tehran university of medical sciences, tehran, iran. the authors of this study would also like to thank mrs farahani, dr elikaee. the authors declare that they have no conflict of interest. ethical considerations to maintain ethics, before sampling, informed consent was obtained from the parents of all children participating in the study. this study had the approval code ir.tums.sph. rec.1397.129 from the research ethical review committee of tehran university of medical sciences, tehran, iran. conflict of interest statement the authors declare that there is no conflict of interest regarding the publication of this paper. this research was conducted in an objective and unbiased manner, and the authors have no financial or personal relationships that may have influenced the results or interpretation of the data presented. references 1. world health organization (2010) who technical report series; no. 949-control of the 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(in persian) 31. gani zh, hassan mk, jassim a (2010) sero-epidemiological study of visceral leishmaniasis in basrah, southern iraq. j pak med assoc. 60(6): 464–469. 32. heidari ae, mohebali m, kabir k, barati h, soultani y, keshavarz h, akhoundi b, hajjaran h, reisi h (2015) visceral leishmaniasis in rural areas of alborz province of iran and implication to health policy. korean j parasitol. 53(4): 379–383. 33. layegh gigloo a, sarkari b, rezaei z, hatam gr, davami mh (2018) asymptomatic leishmania infected children: a seroprevalence and molecular survey in a rural area of fars province, southern iran. j trop med. 2018(8167247): 1–6. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 84 original article morphological and genotypic variations among the species of the subgenus adlerius (diptera: psychodidae, phlebotomus) in iran alireza zahraei-ramazani 1, dinesh kumar 2, hossein mirhendi 3, shyam sundar 4, rajnikan mishra 2, vahideh moin-vaziri 5, hassan soleimani 6, mohammad reza shirzadi 7, reza jafari 8, ahmad ali hanafi-bojd 1, sodabe hamedi shahraky 9, *mohammad reza yaghoobi-ershadi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of zoology, centre of advanced study, banaras hindu university, varanasi 221 005, india 3department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 4department of medicine, institute of medical sciences, banaras hindu university, varanasi 221 005, india 5department of parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 6yazd health research station, national institute of health research, yazd, iran 7communicable disease management centre, ministry of health and medical education, tehran, iran 8esfahan health research station, national institute of health research, esfahan, iran 9school of health, zabol university of medical sciences, zabol, iran. (received 16 mar 2014; accepted 3 may 2014) abstract background: female sand flies of subgenus adlerius are considered as probable vectors of visceral leishmaniasis in iran. the objective of this study was to determine the morphological and genotypic variations in the populations of this subgenus in the country. methods: sand flies collected using sticky traps from 17 provinces during 2008–2010. the morphometric measurements were conducted with an ocular micrometer. data was analyzed by spss. the cytb gene was used to estimate population genetic diversity and identify the female specimens. upgma phenetic tree was used for dna haplotypes of cytb gene. results: six species of subgenus adlerius identified from which one species, p. (adlerius) kabulensis, is new record. the identification key is provided for males. results revealed the molecular systematic in the species of subgenus adlerius and determine the relationship of three females of p. comatus, p. balcanicus and p. halepensis. conclusion: the positions of three females and the males in the upgma tree are correct and the similarities among them confirm our results. the branches of each species are not genetically distinct which justify the overlapping morphological characters among them. molecular sequencing of cytb-mtdna haplotypes can be used for female identification for different species of subgenus adlerius in iran. keywords: phlebotomine sand flies, subgenus adlerius, p. comatus, p. kabulensis, mitochondrial dna cytochrome b gene introduction zoonotic visceral leishmaniasis (zvl) is a potentially fatal disease in human, primarily in children. it is caused by leishmania infantum in which dogs, foxes, and jackals are the main reservoir hosts. four species of the subgenus larrousius and one species of subgenus paraphlebotomus are considered as probable vector species in iran (yaghoobi-ershadi 2012). the disease is endemic in many rural communities of 7 out of 31 prov*corresponding author: prof mohammad reza yaghoobi-ershadi, email: yaghoobi.reza@gmail.com http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 85 inces and also sporadically reported from other regions of the country (yaghoobi-ershadi 2012, zahraei-ramazani et al. 2013). the subgenus adlerius nitzulescu includes about 20 species (seccombe et al. 1993) in which some are suspected or proven vectors of zvl. in the old world these include: phlebotomus longiductus in china, kazakhstan, kyrgyzstan, ukraine, and uzbekistan. p. halepensis ranges in syrian arab republic, azerbaijan and georgia (who 2010). it should be noted that p. (adlerius) arabicus has also reported as the vector of l. tropica in palestinian territories (svobodova et al. 2006a, 2006b). the subgenus adlerius is almost entirely palaearctic (seyedi-rashti and nadim 1992) in various eurasian countries and is thought to have originated from central asia where biodiversity is most abundant (léger and pesson 1987). females from about 20 species of the subgenus adlerius can transmit leishmania major (sadlova et al. 2003, volf and myskova 2007), l. tropica (jacobson et al. 2003, sadlova et al. 2003, svobodova et al. 2006a,volf and myskova 2007), l. donovani (zhang and leng 1997, who 2010) and l. infantum (killick-kendrick 1999, volf and myskova 2007, giorgobiani et al. 2012). there is no published morphological key for species identification that are generally recognized on the basis of male morphology (lewis 1982, seyedi-rashti and nadim 1992). these sand flies are found in diverse natural microhabitats, especially in mountainous areas, such as: cracks, crevices and around the nests and gatherings of the visceral leishmaniasis reservoirs (artemiev 1973). most are also found indoors (theodor and mesghali 1964). because of the short sunny season in the mountains and highland provinces, the collection time of the sand flies is too short. prior to this study, the systematic (morphological and molecular) and molecular studies of some medically significant arthropods such as aedes, anopheles, culex, mites, and sand flies have been studied by molecular methods. the high sensitivity and accuracy of the molecular methods related to dna in terms of morphological identification of species are well established. these are undetectable such as females of the subgenus adlerius is important (munsterman and conn 1997, artemiev 1980, artemiev and neronov 1984). according to previous studies, the cytbmtdna is broadly used for sand flies systematics (esseghir et al. 2000, parvizi and ready 2006). the studies on genetic variation and molecular systematics of the subgenus adlerius sand flies present evidence of ecological differences between the group. access to the genetic variation and molecular systematics of the subgenus adlerius , in addition to assisting the systematic delineation and their classification may offer evidence to interpret and explain the ecological differences among the species. species knowledge, abundance, and distribution of males and females, and changes in their population are useful for all research programs in control of leishmaniasis. the objective of this study was to determine the morphological and genotypic variations among the species of the subgenus adlerius in their distributional areas of iran. materials and methods study area iran is an arid land of 1.6 million km2, extending north to armenia, azerbaijan, turkmenistan, and the caspian sea, east to afghanistan and pakistan , south to the persian gulf and sea of oman, and bordered by turkey and iraq on the west. mountains span the nation in the shape of a large v. between these ranges lies a high plateau where flowing water from the mountains disappears into desert sand. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 86 the caspian sea littoral zone comprises the northern slopes of the alborz mountains and the caspian plain. this is a narrow strip of land, forest covered, with mediterranean climate, with the average temperature ranging between 10 and 35o c and the average relative humidity between 70 and 100%. the central plateau, situated between the alborz and zagros ranges, are very mountainous in the northwest where the ranges originate and becomes a lower desert in the east. the climate is dry with average temperature between 0 and 40 oc, with hot, dry summers and cold, snow-laden winters. several rivers originate on the southern slopes of the alborz mountains. the persian gulf littoral and the khuzestan plain, to the south of the foothills of the zagros mountains, are characterized as a tropical climate (fig. 1). the average temperature ranges between 12 and 50 oc. the average relative humidity ranges between 40% and 80%. the extent and distribution of the subgenus adlerius sand flies collection was conducted from 40o north to 29o south latitude and 44o western to 62o eastern longitude respectively (statistical culture of iran, 1990, personal communication). a detailed study of distribution of the subgenus adlerius species was performed in rural areas of 17 of 31 provinces including [(1) azarbaijan-e-sharqi, (2) azarbaijan-egharbi, (3) ardabil, (4) kordestan, (5) ghom, (6) ilam, (7) lorestan, (8) khuzestan, (9) bushehr, (10) fars, (11) sistan va baluchestan, (12) kerman, (13) kohkiluyeh va boyer ahmad, (14) chahar mahal-o-bakhtiari, (15) esfahan, (16) markazi, and (17) khorasan-e-razavi] (fig. 1). sand flies collection the sand flies were collected using 200 sticky paper traps (consisted of white sheets 15x21 cm, coated with castor oil) placed in different habitats and various biotypes. this included areas outdoors, such as gardens, mountain caves, animal shelters, wall cracks, burrows, tree holes, under stones, and rocks in 3–4 villages suitable for sand fly breeding. these were located in the plains and mountains areas within each province and sampling was conducted twice each year (june and september) from 2008–2010. traps were placed before sunset and collected the next morning before the sunrise. sand flies were preserved in 96 % ethyl alcohol glass containers and were kept at 4 °c in the fridge. the number of the trap sample, date, and location was recorded on the glass containers (moin-vaziri et al. 2007). mounting and identification for species identification, sand flies were mounted in puri’s medium, which was manufactured in our leishmaniasis laboratory in iran and identified after 24 h (yaghoobi-ershadi and javadian 1997) using the keys of theodor and mesghali (1964), artemiev (1973), and lewis (1982). morphological characters used for male identification of the subgenus adlerius included antennal segments, shape of the antennae and ascoid, number of hairs on coxite, length of style on ventral process, length of filament , length of pump, and sub-terminal tubercle of the aedeagus (lewis 1982). morphometric study the eight organs were measured for our morphometrical study included: (1) length of the third segment of antennae, (2) length of style, (3) length of epipharynx, (4) length of coxite, (5) width of style, (6) length of surstyle, (7) length of aedeagus and (8) number of hairs on coxite. the morphometric measurements were conducted with an ocular micrometer and olympus microscope (ch-2) (fig. 2). magnification (40x). statistical analysis statistical analyses were used to determine http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 87 if morphometric characters among the mounted specimens in each species were similar with the species found in different geographical areas. we used spss-version 17.0, and evaluated for differences with the t-test, anova, and kruskal-wallis test with an a priori level of significance set at p< 0.05. if statistical tests were significantly different, the post hoc-bonferroni test was used. when the result of the kruskal-wallis test was significantly different, the mann-whitney test was used for comparing the results. molecular study sections of the abdomen, wings, and legs of the sand flies used for dna extraction were stored in 1.5 ml sterile microtubes containing 96 % ethanol. dna from 120 specimens were extracted, of which 50 were used for mtdna-pcr having 16 sequences (table 1). the washed, dried body of each sand fly was frozen then defrosted with liquid nitrogen and homogenised and ground with a heavy cylindrical metal and shaking vigorously. the content was washed with 300 ml lysis buffer [10 mm tris, 25 mm edta (ph= 8), 1 % sds, 25 mm nacl, 2% triton×-100] and boiled for 10 minutes. this was followed by extraction with a phenolchloroform method. after the ethanol precipitation, dna was dissolved in 15 µ l distilled water and stored at -20 °c. a 25 µ l pcr reaction mixture consisted of 12.5 µ l premix 2x[10x pcr buffer, mgcl2, 10 mm dntps and dna taq polymerase], 1 µ l of cb3-pdr forward primer [5'-ca (y= t/c) attcaacc (w= a/t) gaatgata-3'], 1 µ l of nin-pdr reverse primer [5'-ggta (y= c/t) (w= a/t) ttg cctc ga (w =t/a) ttcg (t/a) tatga3'], 5.5 µ l ddh2o and 5µ l of sand fly genomic dna were used for amplifying of 550 bp. the pcr amplification was carried out with the following thermal profile using a gene amp® pcr system 2700 thermal cycle (ab applied biosystems): 6' min. for initial denaturation at 95 °c, 35 cycles of denaturation at 94 °c for 45 sec., annealing at 55 °c for 1 min., extension at 72 °c for 1 min. sequence alignment and analysis in this study the chromatogram of the dna sequencing samples were edited by using bioedit software. the program clustal w (thompson et al. 1997) was used to study the alignment sequences and similarity scores. our sequences were compared with the sequences available in genbank by using blast available on, www.ncbi.nlm.gov/. the blast 2 sequences were used to find multiple local alignments and detect the best homologous between the female and the male specimens. dna haplotypes of cytb-mtdna gene were used to build an upgma tree. we measured the eight different morphological characters in all specimens of each adlerius species which captured by sticky traps from outdoors of the provinces, and then the similarity scores (%) have been identified by sequence comparing alignment pairwise. therefore, if the more similarity score and less e-value were identified between the pairwise comparison, it showed that they are same species. also, the haplotypes have been confirmed by the polymorphic sites. results composition, frequency and distribution of the adlerius species a total of 9,319 sand flies were collected, of which 3,847 (41.3%) and 5,472 (58.7%) were from the genus phlebotomus and sergentomyia, respectively. from the total number of sand flies of the genus phlebotomus, 167 (1.8%) were from the subgenus adlerius [26 (15.57%) were female and 141(84.43%) were male]. of the males, phlebotomus (adlerius) brevis 8 (5.67%), p. (adl.) halepensis 66 (46.81%), p. (adl.) longiductus 30 (21.28%), p. (adl.) balcanicus 23 (16.31%), p. (adl.) kabulensis http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 88 13 (9.22%) and p. (adl.) comatus 1(0.71%). the p. (adl.) kabulensis artemiev, 1978 is a new record from iran. in case of p. (adl.) comatus which was reported by the authors as a new record by morphological characters from the country (zahraei-ramazani et al. 2013) reconfirmed by the molecular studies (fig. 3). we updated the morphological key for the male subgenus adlerius species of iran as follows: (1) one ascoid on antennal segments 9–15. antenna 8 with two ascoids..................... (2). -one ascoid on antennal segments 9–15. antenna 8 with one ascoid. antenna 6 and 7 with one ascoid or with one long and one short ........................................................ (6). (2) coxite with 14–27 hairs in group, rarely 29 ............................................................ (3). -coxite with 29–115 hairs in group, rarely 27. part of 27–85 hairs of group on distal half of coxite .......................................... (4). (3) whole hair-group on basal half of coxite. tubercle of aedegus 6–16 μm from tip ......... ............ p. turanicus (akhoundi et al. 2011). -part of hair-group on distal half of coxite. tubercle of aedegus 19–28 μm from tip ....... ....... p. brevis (theodor and mesghali 1964). (4) aedegus with rectangular subterminal notch ................ p. halepensis (adler 1946). -aedegus with normal obtuse-angle subterminal notch ....................................................... (5). (5) coxite with 27–50 group-hairs ............... .. p. kabulensis (probably p. cf. kabulensis). -coxite with 50–85 group-hairs. sperm tubes are long (1200–1700 μm) .............................. ........................ p. longiductus (theodor and mesghali 1964) (also p.cf. longiductus). (6) coxite with 90–220 group-hairs. ventral process of style long (about 20 μm ........ (7). -coxite with 30–85 group-hairs. ventral process of style long or short ................................ ......... p. salangensis (akhoundi et al. 2011). (7) coxite very wide, whole group of 125– 200 hairs on its basal half .............................. .................. p. comatus (zahraei et al. 2013). -coxite narrow, part of hair-group on its distal half .................................... p. balcanicus (theodor and mesghali 1964) (also p.cf. balcanicus). the male and female sand flies of subgenus adlerius were collected from 12 provinces and the male were collected from 11 out of 17 provinces. the males were not collected from markazi, bushehr, kerman, kordestan, ghom, and kohkiluyeh va boyer ahmad. the maximum number collection was from ardabil (52 specimens) and the minimum number was from khuzestan and azerbaijan-e-gharbi (1 specimen) provinces. statistical analysis of eight morphological characters of the males of subgenus adlerius sand flies the subgenus adlerius specimens collected from some provinces were very few in number. so for the statistical comparison of the 8 measureable morphological characters of the specimens of p. halepensis, we sorted them according to the four geographical regions (north-west, north-east, south-west and south-east). there was no significant difference between the 8 characters in the specimens. the p. halepensis specimens collected from three provinces (ardabil, azerbaijan-e-sharqi and esfahan) showed significant difference between the length of style and length of aedeagus. the statistical analysis of eight morphological characters of 30 males of p. balcanicus specimens confirmed that there is no significant difference between the characters in the specimens of ardabil and azerbaijane-sharqi. there is a significant difference between the lengths of the coxite of the p. kabulensis specimens in the three provinces (esfahan, lorestan and ilam). the post hoc-bonferroni test shows that the length of coxite of p. kahttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 89 bulensis specimens in lorestan province is significantly more than ilam. the statistical analysis between the 7 specimens of p. brevis in chahar mahal-o-bakhtiari and 1 specimen in azarbaijan-e-gharbi did not confirm any significant difference. the statistical analysis of 30 p. longiductus specimens in four geographical regions (northwest, north-east, south-west and southeast) confirmed that there are significant difference value between the length of surstyle, the length of aedeagus, the number of hairs on coxite, the length of coxite, and the length of styles (p< 0.05) molecular studies on the subgenus adlerius species the pcr experiments amplified a fragment of 550 bp of the mitochondrial genome and it was successfully achieved for 6 species of the subgenus adlerius (table 1, 2). these were registered on jx885982 to jx 885998 accession numbers in the genbank. in the pair wise comparison of the specimens, we focused on the genetic similarities of the specimens among each other and determined the level of such similarities of male and female specimens and identified the females among 6 male species. the pair wise comparison in azv8 female specimens with nucleotides of six male species showed that the maximum similarity, i.e. 98% is found in p. comatus. in total, 528 nucleotides were compared with these 2 sequences which show 8 (1.5%) polymorphic sites and it is confirmed that they are 2 haplotypes (table 3). the pair wise comparison in males of the p. balcanicus species with nucleotides of the three female specimens shows that there are minimum 98% and maximum 99% similarities among the male and the shc11 female specimen. it shows 22 (4.3%) polymorphic sites and it confirms that there are 4 haplotypes (table 3). the nucleotides comparison of the 5 males of the p. halepensis specimens with the ckhe1342 female specimen shows that there are minimum 90.1% and maximum 92.6% similarities between them .the polymorphic sites is 46 (10%) and the sequence comparison revealed that there are 6 haplotypes. also for the two males of p. brevis, it shows 99% similarities with 2 (0.4%) polymorphic sites and 2 haplotypes between these two sequenced specimens (table 3). cytb-mtdna sequences results of the subgenus adlerius species the sequence result of the length 550 bp of cytb gene shows that 5 male species: p. halepensis, p. brevis, p. kabulensis, p. comatus and p. balcanicus are in the areas of our study. also two male specimens: p. cf. balcanicus and p. cf. longiductus which are close to p. balcanicus and p. longiductus are on neighbor-joining tree (fig. 4). the specimens of p. halepensis and p. brevis are in independent and different branches in the tree, but other species are close together in one branch. p. comatus and the female azv8 specimen are in the same branch, which shows that they are the same species. also the branch of female shc11 specimen is near the branch of azv4 and azv5, which shows that females of this species are close to these specimens. the branches of two p. balcanicus specimens i.e. ash10 and azv11 are close together and p. kabulensis is in a different branch. the position in the phenetic tree of the 3 females species and the male species are correct and the similarities among the males and females confirm our above results (fig. 4). http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 90 table 1. number of sand flies used in molecular studies no. species number of specimens that dna has been extracted number of specimens that have been mtdna-pcr number of mtdna-pcr that have been sequenced 1 p. halepensis 55 22 5 2 p. longiductus 23 5 1 3 p. kabulensis 12 5 1 4 p. balcanicus 13 10 3 5 p. brevis 8 3 2 6 p. comatus 1 1 1 7 p. (adlerius) female 8 4 3 total 120 50 16 table 2. profile of sequenced species based on cytb gene in the subgenus adlerius species no. code no. sex collecting area township province genbank accession no. p. halepensis 1 arm754 male niaz village meshkinshahr ardabil jx885982 2 azv2 male ahar-varzaghan road varzaghan azarbaijan-esharqi jx885984 3 ek77 male konjan natanz esfahan jx885991 4 fj103 male mountainous region jahrom fars jx885992 5 msh458 male ghortapeh meshkinshahr ardabil jx885995 p. balcanicus 1 ash10 male sharbian sarsb azarbaijan-esharqi jx885983 2 azv11 male ahar-varzaghan road varzaghan azarbaijan-esharqi jx885989 3 azv5 male ahar-varzaghan road varzaghan azarbaijan-esharqi jx885986 p. longiductus 1 azv4 male mountainous region tabriz azarbaijan-esharqi jx885985 2 fj476 male mountainous region jahrom fars jx885993 p. kabulensis 1 lp7 male mountainous road poldokhtar lorestan not clear 2 lpm5 male mountainous road poldokhtar lorestan jx885994 p. brevis 1 shc7 male chelgerd koohrang chahar mahal-obakhtiari jx885996 2 shc22 male chelgerd koohrang chahar mahal-obakhtiari jx885998 p. adlerius group 1 arm911 famele niaz village meshkinshahr ardabil not clear 2 azv8 famele ahar-varzaghan road varzaghan azarbaijan-esharqi jx885987 3 ckhe1342 famele irandegan khash sistan va baluchestan jx885990 4 shc11 famele chelgerd koohrang chahar mahal-obakhtiari jx885997 p. comatus 1 azv10 male ahar-varzaghan road varzaghan azarbaijan-esharqi jx885988 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 91 fig. 1. provinces where adlerius species were collected. (http://commons.wikimedia.org/wiki/file%3amy-iran-climate-map-simplified.png) fig. 2. organs measured in morphometrical studies of the subgenus adlerius in iran caspian mild mountains desert and semi-desert 1. the length of the third segment of antennae (a3) 5. the length of the style2. the length of epipharynx 3. the length of coxite 8. the numbers of hairs on coxite 7. the length of aedeagus 6. the width of style 4. the length of surstyle http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 92 fig. 3. number of males of subgenus adlerius species collected from 17 provinces fig. 4. neighbor-joining tree for dna haplotypes of cytb-mtdna of the subgenus adlerius sand flies species p. brevis n u m b er p. halepensis p. longiductus p. balcanicus p. kabulensis p. comotus male of adlerius species 8 66 30 23 13 1 70 60 50 40 30 20 10 0 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 93 table3. comparison of cytb gene nucleotide sequences in the subgenus adlerius species discussion phlebotomine sand flies of the subgenus adlerius transmit leishmania parasites in various eurasian countries. but, the species involved have seldom been identified because despite the taxonomic efforts of artemiev (1980) and others. the morphological characteristics do not distinguish between the females of adlerius species that are generally recognized on the basis of male morphology (lewis 1982, sadlova et al. 2003). the detection of p. comatus (zahraeiramazani et al. 2013) and p. kabulensis during the present study has assisted to revise the morphological key. the access to new molecular techniques and genetic information about these probable vectors (artemiev 1973, ardehali et al. 1995) and their systematics, may aid in resolving the problem of identification in morphological similarity, as well. we provide evidence to interpret and explain their morphology and ecology in detail. the systematics of the phlebotomine sand flies due to the nature of their diagnostic characteristics is highly variable. the latest revision of the genus phlebotomus sand flies in old world is included: (1) adlerius, (2) anaphlebotomus, (3) euphlebotomus, (4) kasaulius, (5) transphlebotomus, (6) synphlebotomus, (7) phlebotomus, (8) larroussius, (9) paraphlebotomus (rispail and leger 1998). secombe et al. (1993) believed that the genus phlebotomus had few morphological deficiencies. according to the morphological characteristics they are monophyletic. the upgma is one of the most popular methods in ecology for the classification of sampling units on the basis of their pairwise similarities in relevant descriptor variables (such as species composition) (legendre and legendre 1998). in bioinformatics, upgma is used for the creation of phenetic trees (phenograms). in our molecular analysis, all of the cytb gene sequences confirm the monophyletic of the subgenus adlerius and the morphological identification of these six species are confirmed by the reliable morphological identification keys. the sequence analysis of p. halepensis populations showed 2 lineages in different geographical regions. the haplotypes are 6 in this species. according to our morphological, statistical, and molecular findings, three specimens: azv2, msh458 and arm754 which were collected from the north-west of iran, fall in one branch along with three p. halepensis of genbank. arm754 and msh458 were captured from ardabil and azv2 was captured from azerbaijan-e-sharqi, two different areas. in the statistical analysis, the length of the style in p. halepensis specimens in ardabil province was significantly different from that of esfahan and azerbaijan-e-sharqi. azv2 is in a different branch but is closer to msh458 and arm754 specimens. fj103 and ek77 are in one branch too. in the statistical analysis, the mean of length of style of ek77 is more than that of msh458, arm754 and azv2. so in the species number of sequence identity of sequence (%) number of nucleotides compared number of haplotype polymorphic site (%) p. comatus 2 98 528 2 1.5 p. balcanicus 4 98-99 516 4 4.3 p. halepensis 6 90.1-92.6 458 6 10 p. brevis 2 99 478 2 0.4 p. kabulensis 1 1 p. cf. longiductus 1 1 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 94 upgma tree it shows that they are close to the fj103 specimen in fars province. the fj103 and the three p. halepensis specimens from the genbank found in the north of iran are closer or these specimens in two different areas and are near to their phenetic characters. some geographical event such as desert winds in the central plateau (fig. 1) causes movement and mixing in its population. the pairwise comparison in males of p. halepensis specimens with nucleotides of ckhe1342-female shows that there are minimum 90.1 % and maximum 92.6 % similarity among them. in statistical analysis, the mean of the length of labrum, surstyle, and aedeagus of the p. halepensis specimens in the south-east (ckhe1342) of iran are different from the specimens of other geographical regions. however, ckhe1342-female is in different branch because of the geographical distance between these specimens. in this study all the p. halepensis specimens are on different branch and are in a larger group. it also seems that this species has been separated from another species of adlerius group many years ago than the other adlerius species. according to the statistical analysis and molecular findings, further studies are required to resolve the taxonomic status of this species. the present study on molecular analysis of p. brevis population shows that one lineage with two haplotypes for cytb. the p. brevis (theodor and mesghali 1964) was firstly found in iran. the shc7 and shc22 were captured from chahar mahal-o-bakhtiari province. statistical analysis did not show any difference between them and the specimens from azerbaijan-e-gharbi. along with them, another species i.e 306450786 was submitted to the genbank from iran which is in one independent branch. it seems that p. brevis is not a common ancestor of the other adlerius but diverged early and shares one common ancestor with other species. the second group of the phylogeny tree including lpm5, azv11, ash10, azv5, azv4, azv10 and two females specimens: shc11 and azv8 show that cytb gene does not have sufficient resolution for them and indicates the morphological character overlapping. the length of the branches in this group is short, which indicates that the changes among them are low. this can also be said that the specimens of each species are not far genetically. in statistical analysis by using t-test, we did not find any difference between the specimens of p. balcanicus in ardabil and azerbaijan-e-sharqi provinces. in the molecular study, the pairwise comparison in three males of this species with nucleotides of shc11-female specimen shows that there are minimum 98 % and maximum 99 % similarities among themselves. also, it is confirmed that there are 4 haplotypes among the four sequenced specimens. in the present study, azv4 and azv5 have been identified by using both morphological and molecular methods. however, in our upgma tree, we found that they are differents between p. balcanicus specimens and they are in an independent branch but closer to p. balcanicus branches. the pairwise distance between them is zero. it is clear that the taxonomy of these species should be revised. the results of the molecular investigations indicate that according to the concept of the species they (azv5 and azv4) cannot be considered as a separate species. because the concept of a species is based on the reproductive isolation, while the existence of the intermediate forms and the same mitochondrial gene sequences in this study indicates that there is no reproductive isolation between azv5 morphotypes and azv4 morphotypes. also, we cannot assume these two specimens as subspecies because two subspecies cannot be found as sympatric. so, this indicates that these species e.g p. longiductus and p. balcanicus in iran, including their populations http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 95 have morphological and genetic differences. the similarity between azsh10 and azv11 is 97% and e-value between them is 0.0. further, in the statistical analysis, no difference was found between them. the branches of the four haplotypes of p. balcanicus show that genetically they are not far. the similarity and e-value between the shc11 and azv5 are 99 % and 0.0 respectively. so in the upgma tree they are close together. the pairwise comparison of the nucleotides of cytb-mtdna gene in azv8 female specimen with nucleotides of six male species shows that the maximum similarity i.e 98% is found in azv10. results of the molecular studies reveal that there are 2 haplotypes. these two specimens are in the same branch in the upgma tree. the pairwise distance between them is 0.0. so, these evidences confirm our results as both azv8 and azv10 are p. comatus. in the upgma tree, p. kabulensis is seen as independent branch. the length of the branch in this group compared with other branches is long. this indicates that many changes have occurred during the course of evolution of this species. also this species seems a common ancestor for the other species in this group of the upgma tree. the location and the number of hairs on coxite is the most important morphological characteristic feature for identifying and separating one adlerius species from the other. at times it is difficult for the researchers as the numbers and locations of the hairs of different species are the same and overlap among themselves. in p. kabulensis, the hairs on coxite are 27–50 and in p. longiductus the hairs on coxite are 50–85. if we collect a specimen which has 50 hairs on its coxite, we cannot identify that it belongs to which one of the afore noted two species. also for p. balcanicus and p. longiductus, we encountered the same difficulty. in this case, the researchers use ‘cf’. the ‘cf’ is the abbreviation of the latin word ‘confers’ means “to compare” or “to consult”. it is mainly used in academic writings to indicate a reference to a contrasting finding or viewpoint (it is often used instead of “but see”). it can also appear occasionally in binomial nomenclature by placing before the species name to indicate that the species is not confirmed (strunk and white 1979). to arrive at a result, we use p. cf. longiductus or p. cf. balcanicus. in our study, these species have been confirmed by using both morphological and molecular methods. but in our phenetic tree it is found that they are different from p. balcanicus and p. longiductus. but in an independent group they are close to p. balcanicus branch. obviously, detailed studies for detection of genetic markers to identify the females of six adlerius species are essential. also, further studies on morphology and genetic diversity of the populations of the subgenus adlerius will be of particular importance. conclusions phlebotomus (adlerius) kabulensis is new record for the iran. the positions of three female and males of subgenus adlerius in the upgma tree are correct and the similarities among them confirm our results. the branches of each species are not genetically distinct that justify the overlapping morphological characters among them. molecular sequencing of cytb-mtdna haplotypes can be used for female identification of different species of subgenus adlerius in iran. acknowledgements the authors thank the medical entomology and leishmaniasis staff of esfahan health research station, national institute of health research for their contribution in sand fly collection. the study was funded by the deputy of research, tehran university of medihttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 84–97 ar zahraei-ramazani et al.: morphological and … 96 cal sciences through project number: 514627-01-86. the authors of study declare that they have no competing interests. references ardehali sv, rezaei hr, nadim a (1995) leishmania parasite and leishmaniasis. 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4: 299–306. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 310 http://jad.tums.ac.ir published online: september 30, 2019 original article prone regions of zoonotic cutaneous leishmaniasis in southwest of iran: combination of hierarchical decision model (ahp) and gis elham jahanifard1,2; ahmad ali hanafi-bojd1; hossein nasiri3; hamid reza matinfar4; zabihollah charrahy5; mohammad reza abai1; *mohammad reza yaghoobi-ershadi1; *amir ahmad akhavan1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical entomology and vector control, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran 3faculty of geography, university of tehran, tehran, iran 4department of soil science, collage of agriculture, lorestan university, khoramabad, iran 5open training center, school of geography, tehran university, tehran, iran (received 3 mar 2018; accepted 10 apr 2019) abstract background: cutaneous leishmaniasis due to leishmania major is an important public health problem in the world. khuzestan province is one of the main foci of zoonotic cutaneous leishmaniasis (zcl) in the southwest of iran. we aimed to predict the spatial distribution of the vector and reservoir(s) of zcl using decision-making tool and to prepare risk map of the disease using integrative gis, rs and ahp methods in endemic foci in shush (plain area) and khorramshahr (coastal area) counties of khuzestan province, southern iran from mar 2012 to jan 2013. methods: thirteen criteria including temperature, relative humidity, rainfall, soil texture, soil organic matter, soil ph, soil moisture, altitude, land cover, land use, underground water depth, distance from river, slope and distance from human dwelling with the highest chance of the presence of the main vector and reservoir of the disease were chosen for this study. weights of the criteria classes were determined using the expert choice 11 software. the presence probability maps of the vector and reservoir of the disease were prepared with the combination of ahp method and arc gis 9.3. results: based on the maps derived from the ahp model, in khorramshahr study area, the highest probability of zcl is predicted in gharb karoon rural district. the presence probability of zcl was high in hossein abad and benmoala rural districts in the northeast of shush. conclusion: prediction maps of zcl distribution pattern provide valuable information which can guide policy makers and health authorities to be precise in making appropriate decisions before occurrence of a possible disease outbreak. keywords: decision model; cutaneous leishmaniasis; risk map; iran introduction cutaneous leishmaniasis (cl), a neglected vector-borne disease, is an important public health problem in various parts of iran. two endemic forms of cl have been identified in the country: zoonotic cutaneous leishmaniasis (zcl) due to leishmania major and anthroponotic cutaneous leishmaniasis (acl) due to l. tropica (1, 2). iran is a high-risk country for leishmaniasis with approximately 20,000 re ported cases annually, out of which about 80% are zcl cases (3). khuzestan province is one of the main foci of zcl in the southwest of iran (4). in 2012, annual incidence of cutaneous leishmaniasis was 9.5 to 21.7 per 100000 inhabitants in the province (5). this variation may be due to migration of non-immune persons to endemic areas, living people near rodent colonies, migration of rodent and increas*corresponding authors: prof mohammad reza yaghoobi-ershadi, e-mail: yaghoobia@tums.ac.ir, dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 311 http://jad.tums.ac.ir published online: september 30, 2019 ing synanthropic index for the reservoir and also lack of knowledge and attitude toward zoonotic cutaneous leishmaniasis (6). comprehensive epidemiological and molecular studies in iran have indicated that phlebotomus (phlebotomus) papatasi is the main vector of the disease (2, 7-9). the presence of this sand fly has been reported in different parts of khuzestan province (10-13). a high synanthropic index in this species in their natural habitats in shush and khorramshahr counties is reported (14). natural promastigote infection of ph. papatasi has been reported in khuzestan (12). moreover, l. major has been detected and isolated from this species in roffyeh district of the province (15). based on the distribution of rodent reservoirs of the zoonotic disease, 4 foci of zcl have been identified in the country. leishmania major was detected in tatera indica (rodentia: cricetidae) and nesokia indica (rodentia: muridae) using pcr technique in the west and southwest of khuzestan (16, 17). risk factors such as development of agricultural projects, travelling to the endemic areas, road construction, land cover, groundwater level, sand fly and rodent distribution, soil type, humidity, composting animal manures around homes, presence of secondary reservoirs such as dogs, socio-economic status, sleeping outside, precipitation, altitude, temperature, and the species composition of sand flies have all contributed to the increase in the prevalence of zoonotic cutaneous leishmaniasis (4, 18, 19). cross-sectional studies on the identification and detection of the leishmania parasite in sand flies showed the distribution of the vector and the disease in different geographical areas of the world, however, the incidence of the disease could not be predicted (20). geo-environmental factors, which play important role in the spread of zcl, are often neglected (21). passive and active surveillances of the disease can be useful for finding and monitoring patients in endemic areas, particularly in the in itial stages of an outbreak (22). the spatial distribution of the main vector of zcl is mainly affected by relative humidity and temperature (23). leishmaniasis occurrence is correlated with environmental variables such as the el nino phenomena, which is the effect of climate change on sand fly and reservoir population, parasites and emergence and transmission of leishmaniasis (24). based on the effects of environmental factors on cutaneous leishmaniasis, risk maps of the disease have been prepared in various regions of iran (21, 25). remote sensing (rs) and geographical information systems (gis) are computer based programs used for determining the presence and abundance of vectors and predicting risk map of vector-borne diseases over the last 25 years (26). rs technique provided outstanding results when used to evaluate the risk of arthropod-borne diseases such as malaria, rift valley fever, west nile, lyme, rocky mountain spotted, leishmaniasis and onchocerciasis. satellite images may be used to determine environmental variables such as land use/land cover and temperature where data are otherwise not available (22). gis combines software and hardware systems to analyze, manage and display all spatial distributions in a map framework. gis technique has been used in some studies for the preparation of risk map of diseases and displaying hazardous zones (26-28). the gis was used for health policy purposes due to the potential of estimating hospital requirement and health facilities (29). moreover, gis is able to determine the correlation between some factors like land use, climatic variables, distance to health center, and malaria transmission (30). this technique has also been used for the study of biodiversity, the presence, and abundance of vector and vector-borne diseases with respect to time (26). today, it plays an important role in the health sciences, and it is useful in understanding and visualizing disease distribution and epidemiological data (27). application of gis in spatiotemporal (space and time) ephttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 312 http://jad.tums.ac.ir published online: september 30, 2019 idemiology research is useful in predicting disease distribution in regions at risk (31, 32). multiple linear regressions with gis were used to predict malaria susceptibility zone (33). analytic hierarchy process (ahp) technique has been applied in conjunction with gis and rs to determine at-risk regions in healthcare over the past years (34-36). gisbased multicriteria decision analysis (mcda) is the combination of geographical data and valuable judgments of experts that concentrates on solving spatial decision problems (37). the aims of this study were to determine the spatial distribution of vector(s) and reservoir(s) of zcl using decision-making tools and to prepare risk map of the disease using integrative gis, rs and ahp methods in two foci in khuzestan province (shush and khorramshahr counties). the results of this study can be helpful to health authorities in making precise decisions before disease outbreaks. materials and methods study areas khuzestan province (29° 57ˈ and 33° 00ˈ n, 47° 40ˈ and 50° 33ˈ e) is one of the 31 provinces in iran. it is located in the southwest of the country with 27 counties, 76 cities, and 67 rural districts. this province shares border with iraq and the persian gulf. the weather condition is generally warm, but some parts of the northeast have a temperate climate. three climate zones are identified in the province including mountain, desert, and semidesert zones. shush county (32° 11` 21” n, 48° 15ˈ 28” e) has a land area of 3577km2 and it is situated in the northwest of khuzestan province with hot and arid weather. the average maximum and minimum temperatures are 46.9 °c and 9.5 °c, respectively. the average annual rainfall in this county is about 180mm. khorramshahr county (30° 26ˈ 21̎ n, 48° 10ˈ 45̎ e) is located in the southwest of the province and shares border with iraq and the persian gulf. it is situated at a height of 3 meters above sea level. the weather is hot in summer but mild in winter. the mean maximum and minimum temperatures were 47.5 °c and 9 °c respectively, and mean annual precipitation of 150.6mm in 2013 (fig. 1). however, khorramshahr and shush counties were regarded as plain and coastal areas in these endemic study areas. sampling sites were chosen based on villages had the most cases of cutaneous leishmaniasis in the last five years. data collection and preparation reservoir sampling the sampling of reservoirs was carried out during four seasons from mar 2012 to jan 2013. rodents were captured mostly during the months of may, july, sep, dec, jan and feb. thirty sherman traps with cucumber, nut, date, bread and butter, puff and tomato baits were set in natural, agricultural, semi urban and urban ecotypes. animals were preserved by taxidermy technique and were identified based on morphological characteristics (38). locations of each of the sampling sites were obtained using gps technique (global positioning system). only t. indica and n. indica were included as layers in the arc map. these data were used for determining the accuracy of the proposed reservoir map. vector sampling vector sampling carried out monthly from 2012 to 2013. sand flies were collected by 90 sticky traps (30 in human dwelling, 30 in stables and 30 in rodent burrows or outdoor) from various ecotypes (natural, agricultural, semiurban and urban) in shush and khorramshahr. trapping was conducted before sunset till sunrise in the next morning and location coordinates were determined using gps technique. captured sand flies were preserved in 70% ethanol. permanent microscopy slides of sand flies were prepared using puri’s medium (39). all samples were identified using reliable keys http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 313 http://jad.tums.ac.ir published online: september 30, 2019 (40, 41). only the collection points of the main vector (ph. papatasi) were included in arcmap as a layer to determine the accuracy of the model used for the estimation of presence probability of the vector. human infection and eco-environmental data data on cases of human leishmaniasis due to l. major infection were collected from the health centers at khorramshahr and shush counties from 2004 to 2011. indian remote sensing (irs) satellite images, topographical, land use, river and soil maps, and point shapefile of the rural areas were obtained from the iranian national geographical organization of armed forces. distance from human dwelling and river layers were estimated using the buffer operation in arcgis. climate variables were collected from the meteorological organization of khuzestan province. the annual averages of precipitation, humidity and temperature raster layers were obtained by idw (inverse distance weighted model) surface analysis of the data. these layers were then clipped based on the boundaries of the study area (shush and khorramshahr counties). water information was obtained from the water research center of iran. databank in excel format included water and disease cases used as layers in arcgis 10.2 software. envi (environment for visualizing images) software was employed for analyzing the irs images. normalized difference vegetation index (ndvi) was calculated and used as land cover map in the modeling process. ahp model and processing the analytic hierarchy process (ahp) is a decision-making tool based on mathematics, pioneered by thomas l. saaty in the1970s (42). it has been applied in several decisionmaking scenarios such as choosing the best alternative, prioritizing or determining the relative merit among a set of alternative, resource allocation, bench markings and quality management (43). this multi-criteria decision making (mcdm) technique consists of an ultimate goal, series of alternatives for reaching the goal and a group of criteria that evaluates the alternatives for reaching the goal. it helps decision makers to evaluate factors by pairwise comparisons using standard scales (table 1) (44). the numerical scale (1-9) reflects the importance of one factor relative to others. comparison with the numerical scale also shows which elements are more dominant over the others. analytic hierarchy process can be summarized in three main steps: a. arranging the elements in a hierarchy such that the goal of the decision making occupies the top level, with criteria and alternatives occupying intermediate and lower levels, respectively. b. establishing the matrices of pairwise comparison (judgment matrices) and determining priorities among the elements. c. checking judgment consistency assigned by the consistency ratio (cr). saaty (42) suggested that cr less than 0.1 is acceptable whilst cr more than 0.1 needs judgment revision. thirteen criteria including temperature, relative humidity, rainfall, soil texture, soil organic matter, soil ph, soil moisture, altitude, land cover, land use, distance from human dwellings, underground water depth, and distance from rivers with a higher probability of presence and abundance of the main vector (ph. papatasi) of zcl were chosen, but slope element was used instead of distance from human dwellings for the study conducted on the presence and abundance of the main reservoirs (t. indica and n. indica) in the study areas. pairwise comparison matrix was designed based on thirteen criteria by iranian leishmaniasis experts who have expertise in the ecology and biology of zcl vector and reservoirs; climatic and environmental factors affecting in the spatial distribution of ph. papatasi, t. indica and n. indica. the experts’ answers were based on saaty’s pairwise comparison method http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 314 http://jad.tums.ac.ir published online: september 30, 2019 (table 1). the weights of factors affecting the spatial distribution of vector and reservoirs of zcl were determined by expert choice 11 software. the judgment matrixes were constructed regarding sub-criterion (divisions of criteria) and completed using fundamental scale to pairwise comparison by iranian leishmaniasis experts. then, the matrix tables were analyzed by expert choice 11 software. every layer was reclassified according to sub-criteria matrix analysis. final maps were obtained by multiplying standard weight derived from experts’ ideas by weighted maps of the criteria. hazard map of cutaneous leishmaniasis due to l. major was obtained by overlaying the probability maps of vector and reservoirs presence in the study areas. the presence probability maps of vector and reservoirs e were divided into five classes using the natural breaks method in arcmap (very low, low, moderate, high and very high), and only areas with high and very high risk were mapped as hazard zones. the high and very high strata were considered as the hot spots for zcl transmission. accuracy assessment the accuracy of the presence probability maps of the vector and reservoir were calculated by overlaying the shape files of ph. papatasi, t. indica and n. indica to the proposed maps in arc map. only vector and reservoir sampling sites with high and very high classes, according to the prepared maps, were considered and the results were reported as a percentage. results comparison matrices of the thirteen criteria affecting the presence probability of the vector and reservoirs in shush and khorramshahr counties are presented in tables 2 and 3. the weight of each criterion is showed in table 4. cr was less than 0.1 in all pairwise comparison matrices. the experts’ opinion indicated that the most effective factor affecting the spatial distribution of the zcl vector is temperature followed by humidity and precipitation. on the other hand, the most important factor affecting the presence of the reservoir was soil texture followed by land cover and land use. the weighted maps of layers like temperature, relative humidity, rainfall, soil texture, soil organic matter, soil ph, soil moisture, altitude, land cover, land use, distance from human dwelling, underground water depth, distance from river and slope were prepared for both counties. the presence probability maps of the vector and reservoirs of each county were calculated in arcmap by multiplying weight of the criteria in their weighted maps (figs. 2 and 3). through the combination of the presence probability maps for ph. papatasi, n. indica and t. indica, the zcl risk maps in shush and khorramshahr counties were derived (fig. 4). based on the maps derived from the ahp model, in khorramshahr study area, the highest probability of vector existence is predicted in gharbe karoon and small part of homeh gharbi rural districts and the highest probability of reservoir presence and zcl risk was found in gharbe karoon rural district. there was no risk of disease transmission in homeh-gharbi rural district. the presence probability of the vector was high in sorkheh rural district in the north of shush, but the highest probability of the existence of this species was found in hossein abad and benmoala rural districts in the northeast of shush county. the presence probability maps of the reservoirs showed that the probability of reservoirs presence was high in benmoala. however, the risk of zcl was found to be higher in hossein abad and benmoala rural districts. the accuracy of the models used for the estimation of presence probability of the vector and reservoirs were 90% and 75% respectively in the study areas of shush (fig. 5) and 80% http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 315 http://jad.tums.ac.ir published online: september 30, 2019 and 83.33%, respectively, in khorramshahr county (fig. 6). comparison between the zcl case distribution and spatial distribution of the vector (ph. papatasi) and reservoirs (t. indica and n. indica) in the two counties showed that majority of the zcl cases were reported from the high and very high strata of the presence probability of vector and reservoirs. in other words, the spread of cutaneous leishmaniasis due to l. major was directly related to vector and reservoirs’ distribution probabilities. table 1. fundamental scale to pairwise comparison (44) intensity of importance definition and explanation 1 equal importance (two activities contribute to the objective) 3 moderate importance(experience and judgment slightly favor one activity over another) 5 strong importance (experience and judgment strongly favor one activity over another) 7 very strong importance (an activity is favored very strongly over another) 9 extreme importance (the evidence favoring one activity over another is of the highest possible order of affirmation) 2, 4, 6, 8 intermediate values between the two adjacent judgments (when comparison is needed) fig. 1. map of study areas in khuzestan province, southern iran http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 316 http://jad.tums.ac.ir published online: september 30, 2019 table 2. comparison matrix of the criteria affecting the presence probability of the main vector (phlebotomus papatasi) criteria t e m p e r a tu r e r e la tiv e h u m id ity r a in fa ll s o il te x tu r e s o il o r g a n ic m a tte r s o il p h s o il m o istu r e a ltitu d e l a n d u se l a n d c o v e r d ista n c e fr o m h u m a n d w e llin g u n d e r g r o u n d w a te r d e p th d ista n c e fr o m r iv e r temperature 1 2.605 3.728 5.194 5.194 4.384 2.702 5.674 4.02 2.477 1.431 3.347 3.126 relative humidity 1 2.724 2.569 3.987 4.555 2.569 4.241 3.758 2.239 1.037 3.936 4.02 rainfall 1 3.728 3.987 4.416 1.393 4.241 1.644 1.246 0.591 3.882 3.707 soil texture 1 2.537 3.017 0.903 4.004 1.719 1.673 0.684 3.022 3.898 soil organic matter 1 2.993 1.059 3.005 1.719 1.149 0.591 2.237 3.594 soil ph 1 0.506 1.516 0.422 0.384 0.476 0.974 1.105 soil moisture 1 3.692 2.016 2.477 0.728 3.63 3.845 altitude 1 0.833 1.099 0.459 2.112 2.091 land use 1 1.262 0.59 2.605 2.713 land cover 1 2 2.569 3.594 distance from human dwelling 1 2.085 2.876 underground water depth 1 0.994 distance from river 1 table 3. comparison matrix of the criteria affecting the presence probability of main the reservoirs (tatera indica and nesokia indica) criteria t e m p e r a tu r e r e la tiv e h u m id ity r a in fa ll s o il te x tu r e s o il m o istu r e s o il o r g a n ic m a tte r s o il p h s lo p e a ltitu d e l a n d c o v e r l a n d u se u n d e r g r o u n d w a te r d e p th d ista n c e fr o m r iv e r temperature 1 2.569 3.201 0.762 1.695 2.424 2.268 1.516 3.245 0.718 0.693 2.713 0.944 relative humidity 1 1.585 0.703 0.725 1.461 1.398 1.431 1.741 0.578 0.509 1.719 0.616 rainfall 1 0.520 1.059 0.922 1.563 1.246 1.888 0.328 0.509 1.046 0.631 soil texture 1 3.273 3.277 4.258 5.357 5.123 1.431 2.667 4.324 2.954 soil moisture 1 1.974 1.585 3.438 3.227 0.544 0.474 1.572 0.668 soil organic matter 1 1.657 2.512 2.268 0.564 0.552 1.552 0.725 soil ph 1 0.833 1.217 0.343 0.328 1 0.484 slope 1 1.38 0.333 0.340 1.585 0.525 altitude 1 0.476 0.527 1.32 0.668 land cover 1 2.29 4.891 3.129 land use 1 5.619 3.064 underground water depth 1 0.53 distance from river 1 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 317 http://jad.tums.ac.ir published online: september 30, 2019 table 4. the weight of each criterion affecting the presence of reservoirs and vector derived from ahp technique criteria ahp weight t e m p e r a tu r e r e la tiv e h u m id ity r a in fa ll s o il te x tu r e s o il o r g a n ic m a tte r s o il p h s o il m o istu r e a ltitu d e l a n d u se l a n d c o v e r d ista n c e fr o m h u m a n d w e llin g u n d e r g r o u n d w a te r d e p th d ista n c e fr o m r iv e r s lo p e t o ta l vector 0.207 0.148 0.11 0.077 0.06 0.03 0.08 0.034 0.051 0.055 0.095 0.027 0.026 1 reservoir 0.105 0.063 0.052 0.174 0.058 0.038 0.072 0.036 0.117 0.139 0.034 0.072 0.04 1 fig. 2. presence probability maps of (a) reservoir and (b) vector in shush county, khuzestan province of iran, 2014 fig. 3. presence probability maps of (a) reservoir and (b) vector in khorramshahr county, khuzestan province of iran, 2014 a b a b http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 318 http://jad.tums.ac.ir published online: september 30, 2019 fig. 4. risk map of zoonotic cutaneous leishmaniasis in (a) khorramshahr and (b) shush counties, khuzestan province of iran, 2014 fig. 5. accuracy of distribution probability maps of (a) reservoir and (b) vector based on analytic hierarchy process model in shush county, khuzestan province of iran, 2014 fig. 6. accuracy of distribution probability maps of (a) reservoir and (b) vector based on analytic hierarchy process model in khorramshahr county, khuzestan province of iran, 2014 a b a b a b http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 319 http://jad.tums.ac.ir published online: september 30, 2019 discussion this study provides spatial distribution of the main vector and reservoirs and risk map of zcl using decision-making tools and integrated gis and rs in shush and khorramshahr counties in the southwest of iran. data on human leishmaniasis cases collected from the two counties show that most of the zcl cases occurred in benmoala, hossein abad, shavoor and sayedabbas rural districts of shush and homeh-sharghi rural district of khorramshahr, where there are many agricultural fields. the hazard maps of the disease revealed that most of the cases were recorded in areas where there are agricultural lands. human factors such as population pressure, urbanization and new agricultural projects affect the distribution of leishmaniasis. land use including agricultural lands, rivers, canals and irrigation water are suitable circumstances for the growth of vectors and provide nesting habitats for reservoirs (45, 46). sand flies prefer humid habitats for the laying of eggs, survival, and development of immature stages (47). inland waters and streams affect the abundance and distribution of vectors. increase in sand fly abundance can be facilitated by shortening the reproductive cycle due to an increase in surface moisture in areas such as inland waters and river banks. in the central part of bihar in eastern india, inland water bodies increased the transmission risk of visceral leishmaniasis (vl) by providing suitable breeding places for the vector ph. argentipes (48). in the present research study, disease cases and spatial distribution maps of ph. papatasi derived from a combination of ahp and gis revealed that most of the cases due to l. major occurred in areas with high and very high presence probability of sand flies. a decrease in the depth of underground water provides suitable condition such as moisture, critical factor that increases the population of sand flies. sand fly activities are strongly associated with two environmental parameters; precipi tation and temperature (49). precipitation, mean temperature and slope are the main environmental variables that affect the distribution of the main zcl vector in iran, using the maxent model (50). increased vector abundance is due to the presence of infected rodent and increased soil moisture in the region. the epidemiology of cl is mostly related to the spatiotemporal distribution of the vector and reservoir (49). there is a correlation between the geographical distribution of zoonotic cutaneous leishmaniasis and its main vector, and l. major has been frequently isolated from ph. papatasi in various foci in the country (2). several studies have been conducted on the effects of environmental factors on the distribution and incidence of cl (51, 52). the risk map of visceral leishmaniasis was prepared in two districts in east azarbaijan province (ahar and kalaybar) using group decision-making tools and gis (34). fuzzy ahp model is widely used as a predictor of the prevalence of the disease with an accuracy of more than 80%. in a research study, among eight parameters only dogs, nomads and altitude were the most effective factors affecting the incidence of vl. two studies have been conducted on mapping hazard areas of zcl in esfahan and golestan provinces using the fuzzy ahp method (21, 25). altitude and land cover have a negative influence on disease incidence. temperature and relative humidity are the two main factors that directly correlate with the incidence of zcl (21). environmental factors that affect vector and reservoir population influence the spread and incidence of leishmaniasis. moreover, distribution of cutaneous and visceral leishmaniasis was found to be affected by temperature fluctuations (53). in the present research, variables such as soil texture, land cover, land use and temperature, in order of descending influence, were found to have the highest impact on the presence probability of the reservoirs. the most efhttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 310–323 e jahanifard et al.: prone regions of … 320 http://jad.tums.ac.ir published online: september 30, 2019 fective criteria affecting the presence probability of ph. papatasi, based on experts’ judgment, were temperature, relative humidity and precipitation. there were some differences between our results and that of some previous studies on leishmaniasis. the difference may be due to the different forms of the disease studied and the various criteria selected for the study. in some previous study, criteria such as average altitude, health center distance, population and evaporation were chosen whilst ignoring the two most important factors of zcl cycle like vector and reservoir in the preparation of hazard maps of the disease. environmental elements like slope and seasonal precipitation contribute to the spreading prediction of n. indica and t. indica (zcl reservoirs), respectively, in iran (17). the risk map of kala-azar was prepared using geo-environmental factors such as land cover/land use, condition of vegetation, surface dampness, climate, illiteracy and unemployment rate of the inhabitants in the vaishali district of bihar in india (48). in the present investigation, the hazard map of zcl was prepared using climate and environmental elements in shush and khorramshahr counties. according to the multicriteria decision-making technique, the highest probability of the disease presence is predicted in gharb-ekaroon rural district of khorramshahr whilst sorkheh, hoseinabad and benmoala rural districts where the high-risk areas of zcl in shush county. different multicriteria decision analysis techniques for disease susceptibility mapping were compared (54). ahp model, with high accuracy, was the best among the tools used for the prediction of vl in the northwest of iran and dengue virus disease in ecuador. conclusion using a combination of gis, rs and ahp decision-making techniques, valuable risk maps of cutaneous leishmaniasis due to l. major were prepared while gis and rs are judged only on the base of evidence and environmental factors, and there is no possibility of intervention by experts. our study confirms the usefulness of ahp and gis techniques in preparing the spatial distribution of ph. papatasi, n. indica and, t. indica and the risk map of zcl in areas where similar vector and reservoirs exist. furthermore, it is possible to update the maps by entering new data and satellite images. in the present study, eco-environmental factors were found to have a greater influence on the presence of vector and reservoirs, which can affect the incidence of zcl. we recommend the use of multi-criteria decision making technique for the preparation of risk maps of zcl in other counties of the province. the proposed maps are visual tools that highlight areas with the potential for the disease transmission and/ or outbreak and the population at risk. educating and increasing the knowledge of the people on reservoir, vector, transmission and control of the disease is deemed necessary. in addition, hazard maps are valuable data for health authorities in allocating budget for effective disease control programs and health care for the people living in high-risk zones. acknowledgements the authors are grateful to responsible persons and staffs of health centers in shush and khorramshahr counties. this survey was financially supported by tehran university of medical sciences (tums); project no.9004-27-16311. the authors declare that there is no 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[master thesis]. department of physical geography and ecosystem science, lund university, sweden. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 250 http://jad.tums.ac.ir published online: september 30, 2020 original article investigating on the residue of organophosphate pesticides in the water of the hablehrood river, garmsar, iran mohammad safari1,2; ali ahmadfazeli2; hassan vatandoost3,4; mostafa karimaee5; davood panahi1; mohammadali shokri1; mehdi moradian1; *zahra soleimani1,2 1student research committee, semnan university of medical sciences, semnan, iran 2department of environmental health engineering, school of public health, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 5department of environmental health engineering, semnan university of medical sciences, semnan, iran *corresponding author: dr zahra soleimani, e-mail: ronak_soleimani@yahoo.com (received 27 sep 2018; accepted 17 aug 2020) abstract background: organophosphate pesticides are of concern to the drinking water supply and high risks on human health. methods: an occurrence survey was performed in the spring and summer of 2016 for 6 months to assess the preva lence of organophosphate pesticides in the hablehrood river that is located in semnan province. besides, 10 kinds of organophosphate pesticides were sampled in 5 stations. moreover, were measured by gas chromatography (gc) was supported by an electron capture detector (ecd) and turbochrom software. in all stations, diazinon was detected in the spring and summer, but malathion only in the spring. results: the highest concentration of diazinon was observed at the mahmoud abad station in spring (0.94ppb) and the bonekooh station in the summer (0.93ppb). the highest and lowest concentrations of malathion were detected in mahmoud abad (0.35ppb), and gache station, respectively. conclusion: the concentration of pesticides in bonekooh and mahmood abad was higher than the standard. keywords: organic pollutant; agriculture; water; river introduction in a society, healthy drinking water has the most significant impact on human health (1, 2). pesticides are widely used to control pests in agriculture to increase their productivity (3-6). these compounds are one of the sources of water pollution with serious health risks for humans and animals. furthermore, this makes changes in ecosystems with dangerous consequences for the environment and agriculture, as they cause the emergence and spread of new pests and diseases, thereby increasing the need for them to be used (7). as in previous decades, concerns about water pol lution have increased due to the identification of a large number of pesticides (8). despite global efforts to eliminate or reduce the release of these compounds, there is still evidence of their presence in various matrices of living and non-living organisms (4). the presence of organic pollutants in surface waters may indicate acute exposure of humans and wildlife, and consequently requires continuous monitoring of these pollutants in water resources (1). pesticides used in agriculture enter water resources through direct washing, irrigation, and rainfall in contaminated copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 251 http://jad.tums.ac.ir published online: september 30, 2020 areas (4-7, 9-12). the resistant nature of organochlorine pesticides in the environment leads to problems like chronic toxicity in humans and animals through water, food, and air (3, 5, 10-11). these compounds are less commonly used due to their effect on the nervous system because they quickly pass through cell membranes due to their hydrophobic properties (11). on the other hand, it is estimated that around 200,000 people die annually from pesticide poisoning. the effects of these compounds on humans and the environment depend on the concentration of input toxin, chemical quality, duration of use, exposure time, and toxicity (9). in iran, these compounds are widely used in agriculture and horticulture (13). the first step to manage pesticide residues in water resources is measuring them and compare with existing standards (9). for drinking and environmental waters, the eu has 0.1μg/l; the maximum permitted concentration for a compound’s 0.51μg/l for all pesticides (7-9). because of the high resistance of these compounds in the environment, there is a high tendency to examine their contamination (10). because the river is the most valuable source of drinking water, it is vital to know the presence of these compounds in water before using it (14). studies by the us environmental protection agency (epa) show that conventional water treatment processes (coagulation, sedimentation, filtration, softening, and chlorination) cannot effectively eliminate pesticides. it was demonstrated that, throughout the chlorination process, the sulfur atom double-bonded to the central phosphorus atom in organophosphate pesticides (opps) is replaced by an oxygen atom, which considerably increases the toxicity of these compounds (15). recent studies have revealed that chlorpyrifos and malathion derivatives are at least 100 times, and diazinon derivatives are ten times more toxic than their original forms. this can be posed as a serious problem because the most common method for disinfecting is chlorination (15). due to the serious problem of low water amounts in the country, it is important to manage and monitor the quality and quantity of available water resources. awareness of the residuals of these pesticides in this river is critical for the supply of safe and healthy drinking water so that, if necessary, measures will take to control and prevent any environmental and health crisis. therefore, this study aimed to determine the residual amounts of organophosphate pesticides in the hablehrood river, which is the main source of drinking water in garmsar city. materials and methods study area hablehrood basin is located in the northwest of semnan and northeast of tehran province. the region with an area of about 37570 hectares is placed between the eastern longitudes 52 o14´, 52 o35´, and the northern latitudes 35 °07´, 35 °16´. the average temperature and rainfall are 7.8 °c, 318mm, respectively, which ultimately enter garmsar plain. the basin includes the agricultural land of garmsar and its suburbs that located in the western part of semnan province. about the use of pesticides for agriculture activity, the amount of pesticides used in the southern and northern parts of the hablehrood basin in 2014 was 26667kg and 2251kg, respectively (16). sampling in this study, sampling was done monthly in the spring and summer of 2016 (6 months). hence, five sampling stations select according to the active water basin and agricultures activity including; simin dasht, bonekooh, mahmoud abad, zarrin dasht, and gache were identified along the river (fig. 1). we chose these stations for two reasons: first, there was no easy access to the river from different places along the river; http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 252 http://jad.tums.ac.ir published online: september 30, 2020 second, these stations were downstream of agricultural activities. each month, two samples were taken from each station (60 samples in the whole study). samples collected in 1-liter dark-colored bottles, which completely covered with aluminum foil, were filled in 0.5m depth from the surface of the water. the preparation of bottles comprised washing with detergent without phosphorus compounds and distilled water. then, by adding the preserving agent (0.5g ascorbic acid to the samples with free chlorine), samples were collected according to the standard method and transferred to the laboratory, they were stored in a refrigerator at 2 °c for analysis (17). extraction and analysis of samples chromatography techniques are perceived as the best way to detect pesticides (1, 18). the samples were filtered by fiberglass filter (0.45 microns) to separate suspended solids. all samples transport to the laboratory in cold box (between 1 °c and 4 °c); water samples were extracted using a solid-phase extraction (spe) system according to the established procedures; samples were analyzed over three days (19). the extraction epa methods 8141b was chosen for determination pesticide (19). the gas chromatography used to measure residual pesticides was supported by electron capture detector (ecd) and turbochrom software. this detector can detect contaminants at low concentrations (ppb range). the column capillary silica pe-17 was used for chromatographic separation of pesticides. nitrogen was used as a carrier and make-up gas, and the injection method was in the division state (table 1) (20). statistical analysis spss 21.0 and microsoft excel software was used for data analysis. data on pesticide concentration in different stations and seasons were analyzed by independent t-test and one-test was used to compare two stations or compare each season and compare with level standard concentration pesticide respectively. besides, in various stations and seasons were analyzed by anova, the tukey test was used to compare the two seasons and station in duration sampling. results the results of the analysis of water samples of the hablehrood river during the spring and summer revealed that only the diazinon and malathion were present in water, and the rest were absent in any of the samples. the results of the analysis of samples are summarized in table 2. according to table 2, the only pesticide detected in the spring was diazinon, and in the summer, diazinon and malathion were detected. generally, the results of this part of the research indicate that the average concentration of diazinon in different stations is significantly higher in the summer (p< 0.05). diazinon in the hablehrood river was significantly the highest concentration of related to the mahmoud abad station in spring (0.94ppb) (p< 0.05), and the bonekooh station in the summer (0.93ppb) figures 2 and 3 show the concentrations of diazinon residuals in different stations in spring and summer (p< 0.05). malathion was observed only in summer samples. the highest concentration of malathion in the hablehrood river was detected in mahmoud abad (0.35ppb) and the lowest at gache (0ppb). the concentrations of malathion in different stations in summer are presented in fig. 4. other organophosphorus pesticides that were measured in this study comprise dichlorvos, trifluoralin, methyl-parathion, fenitrothion, profenofos, ethion, and tepp. but did not detect in any of our samples. from sampling stations, diazinon was observed in spring at all stations. the largest and lowest http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 253 http://jad.tums.ac.ir published online: september 30, 2020 amount of its concentration was in mahmoud abad, gache, respectively. in summer, diazinon and malathion have detected at all stations. which the highest concentration of diazinon was in bonekooh and lowest in gache. the highest and lowest amount of malathion was observed in mahmoud abad and ghache respectively. total pesticides in water samples of bonekooh and mahmoud abad in summer were significantly over the permitted of ec (1μg/l total allowable pesticide in surface waters) (p< 0.05) (21). table 1. gas chromatography condition for pesticide measurement amount modes 1microliter injection volume 7 psi inlet pressure 300 °c injector temperature 300 °c detector temperature 40 ml/min gas flow ecd make up 220–240 °c at 2 °c/min 240–260 °c at 1 °c/min 260–280 °c at 5 °c/min oven programming table 2. results from analysis of water samples of hablehrood river during spring and summer detected amount of pesticides (ppb) station compound name season of sampling zarrin dasht ghache simin dasht mahmoud abad bonekooh dichlorvos spring nd nd nd n.d n.d summer nd nd nd nd nd trifluralin spring nd nd nd nd nd summer nd nd nd nd nd diazinon spring 0.74 0.2 0.6 0.94 0.5 summer 0.6 0.5 0.6 0.89 0.93 methyl parathion spring nd nd nd nd nd summer nd nd nd nd nd fenitrothion spring nd nd nd nd nd summer nd nd nd nd nd malathion spring nd nd nd nd nd summer 0.2 nd 0.15 0.35 0.2 profenophos spring nd nd nd nd nd summer nd nd nd nd nd ethion spring nd nd nd nd nd summer nd nd nd nd nd azinphos methyl spring nd nd nd nd nd summer nd nd nd nd nd tepp(is) spring nd nd nd nd nd summer nd nd nd nd nd nd= non detectable http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 254 http://jad.tums.ac.ir published online: september 30, 2020 fig. 1. hablehrood basin and sampling stations http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 255 http://jad.tums.ac.ir published online: september 30, 2020 fig. 2. diazinon concentration (ppb) at different stations in the spring fig. 3. diazinon concentration (ppb) at different stations in the summer http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 256 http://jad.tums.ac.ir published online: september 30, 2020 fig. 4. malathion concentration (ppb) at different stations in the summer discussion most farmers use more organophosphate pesticides than others because of their low cost and a wide range of uses. malathion and diazinon have been used in all over iran (22). as it is discussed, these organophosphate pesticides were detected in all stations of hablehrood river consequently, it is suggested to survey these pesticides in other water supplies. furthermore, due to the lack of education and awareness, farmers do not take caution when using pesticides, which increases pesticide-related diseases in agricultural areas. organophosphate pesticides have adverse effects on the body by inhibiting the activity of acetylcholinesterase enzyme. the effects of carcinogenesis, mutagenesis, and teratogenicity of this group of pesticides have been proven. moreover, these disrupt sexual hormones, reproductive problems, and stop human growth (23). it is well-known that many of pesticides that are used do not remain in their target area and are transferred through penetration into the soil and runoff, and also become new compounds, and many of them enter surface and underground waters, and their metabolites may remain for many years (24-25). the amount of pesticide residues in the water resources depends on different factors, such as the application of each pesticide, proximity of agricultural land to the river, metabolism of pesticides to other compounds, absorption of pesticides into organic matter in water and soil, water temperature and ph (26). for this reason, the residual concentrations of diverse types of organophosphate pesticides are various in different sources. in the present study, dichlorvos, trifluoralin, methyl-parathion, fenitrothion, profenofos, ethion, and tepp were not detected in any of the analyzed samples due to their low application and high hydrolysis rates. the study of dehghani et al. (2012) on kashan water resources, which is consistent with the results of the present study, showed that the rate of removal of chlorpyrifos is higher than diazinon and its amount was much lower than of it (27). in the study by kent et al. (2005), who studied the pesticide residues http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 257 http://jad.tums.ac.ir published online: september 30, 2020 in california's surface waters, it has been observed that 92% of the samples contained at least one pesticide. moreover, the concentration of diazinon in some samples was higher than the standard values (28). the results of this study were also consistent with kent's study, and in summer, the total pesticide concentrations at mahmoud abad station was higher than standard values, which could be due to low knowledge of farmers about the application of pesticides. farmers to prevent the occurrence of diseases for their products and higher productivity, use various fertilizers and pesticides. due to many problems of pesticides on the environment and human health, increasing farmers' awareness about the correct way of cultivation, amount of pesticides, and the natural combating means for agricultural pests can be effective in decreasing the concentration of pesticides in aquatic resources and environment. the consumption of diazinon for agricultural products is high in spring and summer. additionally, the high residue of diazinon in water samples can be due to the high solubility of diazinon in water and also atmospheric falls in the early spring and the entry of this to surface waters (29). besides, diazinon is used more often than other pesticides. among the studies that are consistent with the results of the present study and the level of pesticides exceeds the maximum permissible standard concentration can be found in the study by chowdhury maz et al. (30), lari et al. (31), and székács et al. (32). because of its high solubility rate relative to other pesticides, huge levels of its application, and frequency of use during the crop year, its residual amount was also higher than other pesticides studied in water samples, which is consistent with the results of khazaei et al. (33). malathion has a lower consumption than of diazinon. malathion was not observed in any of the spring samples but was observed in summer samples at different stations except gache station. another reason for the high level of diazinon and malathion at mahmoud abad and bonekooh stations could be due to extensive agricultural and horticultural activities in this area. therefore, as much as we move alongside the river from upstream to downstream, the concentration of pesticides used increases. this can be due to increased agricultural activity and the accumulation of pesticides used in the study area. in other words, a part of a pesticide is degraded and converted to its derivatives, but another part of it goes downstream. the pesticides used in each area are added to it. the residual pesticides in bonekooh station were lower than other stations, because this station had a large distance from the other station, and there is a considerable distance from the agricultural activity area to this station; so part of the pesticides will be decomposed into their derivatives before reaching to this station. conclusion diazinon and malathion were observed in hablehrood river samples. because there is no comprehensive national standard for the maximum residual concentration of pesticides in water resources, the european union (ec) standard was used (34). the concentration of pesticides in bonekooh and mahmood abad in summer was higher than the standard. the mobility of chemicals and their traits, as well as the proximity of agricultural land to hableh rood, affects the possibility of contamination of the river by pesticides. the presence of pesticides in water resources can be due to reasons such as uncontrolled manual spray, poor management in the process of pesticides using, and unnecessary and inappropriate application of pesticides. however, the proper use of pesticides at the right time and preventing the entrance of pesticides into the water can reduce their concentration in water resources. furthermore, continuous monitoring and measuring of pesticides should be a http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 250–260 m safari et al.: investigating on the residue of … 258 http://jad.tums.ac.ir published online: september 30, 2020 priority for drinking water resources to protect the health of the community and the environment. acknowledgements this article is a result of the a-334, a1031 project sponsored by the student research committee of semnan university of medical sciences. semnan, iran. the authors declare that they have no conflict of interest. references 1. maloschik e, ernst a, hegedűs g, darvas b, székács a (2007) monitoring water-polluting pesticides in hungary. microchem j. 85(1): 88–97. 2. miranzadeh m, heidari m, mesdaghinia a, younesian m (2011) survey of microbial quality of drinking water in rural areas of kashan-iran in second half of 2008. pak j biol sci. 14(1): 59–63. 3. 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https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3895686/ j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 369 http://jad.tums.ac.ir published online: december 31, 2019 original article faunistic composition and spatial distribution of scorpions in north khorasan province northeast of iran faranak firoozfar1,2; *abedin saghafipour3; hassan vatandoost4; mulood mohammadi bavani5; masoumeh taherpour2; nahid jesri6; mahmood yazdani1; kourosh arzamani1 1vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 2department of public health, faculty of north khorasan university of medical sciences, bojnurd, iran 3department of public health, faculty of health, qom university of medical sciences, qom, iran 4department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 5department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran 6remote sensing and gis center, shahid beheshti university, tehran, iran (received 16 oct 2018; accepted 18 dec 2019) abstract background: scorpions pose one of the most important public health and medical problems in tropical and subtropical regions of the world, especially in developing countries. this study was conducted to determine the fauna and spatial distribution of scorpions. methods: in this descriptive study, scorpions were captured using ultra-violet (uv) light, pitfall traps and digging methods in north khorasan province, northeastern iran in 2017. after being encoded, the collected scorpions were stored in plastic containers of 70% ethanol and then transferred to the medical entomology lab of tehran university of medical sciences for species identification based on morphological keys. in addition, arc gis 9.3 software was utilized for mapping spatial distribution of scorpions. results: overall, 143 scorpions were captured and identified. all of collected scorpions belonged only to buthidae family. they were also classified into four genera (androctonus, mesobuthus, odontobuthus, orthochirus) and five species: m. eupeus (59.44%), a. crassicauda (16.78%), o. doriae (12.59), m. (olivierus) caucasicus (9.09%), and o. farzanpayi (2.10%). furthermore, spatial distribution of scorpions was performed in this area. conclusion: regarding the diversity, high frequency and wide geographical distribution of scorpions and their longterm seasonal activity in this area, the probability of occurrence of scorpion sting is high. therefore, in order to pre vent the occurrence of this public health problem, health educational programs be implemented by healthcare providers in the area. keywords: scorpions; spatial distribution; gis; iran introduction scorpions (arachnida: scorpions) are one of the oldest terrestrial arthropods, dating back to about 450 million years ago (1). these animals are nocturnal, meaning that they leave their shelter at night for hunting and other biological activities but in daytime, they are hidden in their sanctuaries including seams and gaps of walls, underground holes, undersides of rocks, under the leaves and shells of trees, etc. scorpions have venomous stings that they use to capture insects and defend themselves against natural enemies (2). scorpions feed on insects, spiders, mites and some small vertebrates, while some species of them are cannibals (3, 4). these creatures often live in desert areas, but some of their species are found under rocks in mountainous and forest areas. non-digger and semi-digger scorpions can enter human habitats (2). although the scorpions do not transmit any *corresponding author: dr abedin saghafipour, email: abed.saghafi@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 370 http://jad.tums.ac.ir published online: december 31, 2019 pathogens and parasitic agents to humans, they have always been one of human concerns due to their venomous and deadly stings, as well as scary appearance. some species of scorpions are medically important and cause relatively high mortality in some areas of the world (5). therefore, scorpionism turns out to be one of the most important public health and medical issues in tropical and subtropical regions of the world, especially in developing countries (6). in iran, in addition to the anxiety and concern caused by scorpion sting, its high cost of treatment, and the resulting health-related consequences, deaths from scorpion stings are also reported annually (7). according to the centers for disease control and prevention (cdc) of iranian ministry of health and medical education, approximately 40000–50000 cases of scorpionism occur annually in iran (8, 9). systematically in the world, the scorpions are classified into six super families, 13 families, 18 subfamilies and 10 tribes and more than 2,000 species (10, 11). a review of the previously published articles in this regard showed that there are three families of scorpions in iran, including buthidae, scorpionidae, hemiscorpidae, identified in the form of 19 genera and 59 species (12-14). there is no information about scorpion fauna of this province and there is a big gap in the case of spatial distribution of scorpions in this area, regarding the reports of annual scorpion sting cases from north khorasan health centers (15) and having no information about scorpions, therefore, we for the first conducted a survey to determine scorpion fauna and their spatial distribution in north khorasan province, northeast of iran. materials and methods study area the north khorasan province has an area of 28179km2. it has 8 counties including bojnurd, shirvan, esfarayen, mane, raz jargalan, jajarm, faruj and garme. the population of this province is 811,572. geographically, this province has common borders with turkmenistan in the north, khorasan razavi province in the east and south, semnan province in the southwest, and golestan province in the west (16). scorpion collection and identification in this descriptive study, all of urban and rural areas of north khorasan province were investigated by considering factors such as climatic conditions (such as temperature, humidity), geographical factors (such as elevation, vegetation status, soil type, rainfall) and the different climate areas (desert, semi-desert, foothills and mountainous areas) during jan– dec 2017 (17). in each of the counties, two or three sites were selected as sampling locations. locations were selected in terms of being plain or mountainous. twenty-four locations were determined for collecting scorpions (fig. 1). at each location, residential and non-residential areas were also taken into account. scorpions were captured using three methods: a): ultra-violet (uv) light, in which the researchers made a search for scorpions for two hours in probable scorpions’ habitats including under the stones, under the leaves and shells of trees. at night and daytime before sunset (18). b): pitfall traps in which plastic containers (with 8cm width and 1cm depth) were placed at stations located in the plains on a possible path of scorpion movement at a depth of 11cm of soil, and in order to draw scorpions, moist cotton was placed inside the traps (19). ten traps were used at each location, amounting to a total of 80 traps and the scorpions were collected using iris forceps; and c): digging method by which the researchers identified probable scorpions’ habitats (underground cavities drilled by scorpions) at stations located in plain areas with soft soil and poured some water into their habitats in order to collect digger scorpions. finally, scorpions were collected with the use of an iris http://jad.tums.ac.ir/ https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjbu82n-nbyahuqipokhyxtdzmqfgglmaa&url=https%3a%2f%2fwww.cdc.gov%2f&usg=aovvaw347qtt6pi6cdnkfm8vfdw6 j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 371 http://jad.tums.ac.ir published online: december 31, 2019 forceps (20). for morphological identification, after being encoded, the collected scorpions were stored in plastic containers having 70% ethanol and then transferred to the medical entomology lab of tehran university of medical sciences for species identification based on morphological keys (5). statistical analysis arc gis 9.3 software was used for mapping spatial distribution of scorpions. results overall, 143 scorpions were captured and identified. all collected scorpions belonged only to buthidae family. moreover, they were classified into four genera (androctonus, mesobuthus, odontobuthus, orthochirus) and five species namely, m. eupeus (59.44%), a. crassicauda (16.78%), o. doriae (12.59), m. (olivierus) caucasicus (9.09%), and o. farzanpayi (2.10%) (table 1). the highest frequency of scorpions (72.94%) belonged to desert areas. in addition, the majority of them (58.04%) were captured in areas with clay soil. the frequency of captured scorpion was approximately similar in urban and rural areas (48.95% in urban areas versus 51.05% in rural areas). most of collected samples were gathered from outdoors areas (84.61%). the majority of scorpion’s samples (70 of 143) were captured in summer (august) using ultra-violet (uv) light (75.52%) method (fig. 2). mesobuthus eupeus totally, 85 samples of m. eupeus (59.44% of the gathered specimens) were gathered. according to its spatial distribution, these scorpion species were collected from all 8 counties in the province; that is, from jajarm to raz jargalan (table 1, fig. 3). it was captured in different climate areas. the other ecological characteristics of this species have been presented in table 2. these non-digger scorpion species were collected in urban and rural regions from desert and mountainous areas which had the altitude range of 850–2118m. in this study, 75.29% of the samples were collected from outside human dwellings such as under stones and bark of trees, on the farm, around the houses, in deserts, old homes, and grasslands. the others (24.71%) were caught inside human habitats (storage place, under carpets, under furniture, inside wardrobes). fig. 1. (a) the study area in north khorasan province, northeast of iran, (b) study area based on altitude http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 372 http://jad.tums.ac.ir published online: december 31, 2019 table 1. spatial distribution of scorpions captured in northeast of iran species location longitude (decimal degree) latitude (decimal degree) m. eupeus m. caucasicus o. farzanpay a. crassicauda o. doriae total county altitude (m) n % n % n % n % n % n % garme 2118 56.290284° 36.985493° 15 17.65 0.00 3 100 6 25.00 4 22.22 28 19.58 esfarayen 1244 57.496673° 37.066677° 16 18.82 3 23.08 0.00 5 20.82 3 16.67 27 18.88 mane 850 56.741207° 37.662061° 13 15.29 0.00 0.00 5 20.82 3 16.67 21 14.68 bojnurd 1070 57.314313° 37.470214° 18 21.18 0.00 0.00 1 4.17 1 5.55 20 13.99 raz jargalan 1279 57.109775° 37.932390° 10 11.77 0.00 0.00 4 16.68 5 27.27 19 13.29 faruj 1200 58.098205° 37.085118° 5 5.88 7 53.84 0.00 2 8.34 1 5.56 15 10.49 shirvan 1097 57.927618° 37.409236° 5 5.88 0.00 0.00 1 4.17 1 5.56 7 4.90 jajarm 1000 56.371311° 36.963614° 3 3.53 3 23.08 0.00 0.00 0.00 6 4.19 total 57.101294° 37.471000° 85 59.44 13 9.09 3 2.10 24 16.78 18 12.59 143 100 table 2. some ecological characteristic of scorpions captured in northeast of iran scorpion species ecological characteristic different climate areas type of soil time of capture season of capture deserts mountains semi desert foothills sandy clay night n (%) day n (%) spring summer autumn winter m. eupeus 62 (72.94) 13 (15.30) 3 (3.53) 7 (8.23) 49 (57.65) 36 (42.35) 69 (81.18) 16 (18.82) 8 74 2 1 a. crassicauda 22 (91.66) 2 (8.34) 3 (12.50) 21 (87.50) 17 (70.83) 7 (29.17) 5 18 1 o. doriae 18 (100) 2 (11.11) 16 (88.89) 12 (66.67) 4 (33.33) 2 13 1 m. caucasicus 7 (53.84) 3 (23.08) 3 (23.08) 3 (13.08) 10 (76.92) 7 (53.8) 6 (46.2) 2 11 o. farzanpayi 3 (100) 3 (100) 3 (100) 3 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 373 http://jad.tums.ac.ir published online: december 31, 2019 0 25 50 75 ja n u a ry f e b ru a ry m a rc h a p ri l m a y ju n e ju ly a u g u st s e p te m b e r o c to b e r n o v e m b e r d e c e m b e r s c o r p io n f r e q u e n c y fig. 2. average monthly scorpion collection, northeast of iran, 2017 fig. 3. spatial distribution of captured scorpions, northeast of iran, 2017 androctonus crassicauda overall, 24 samples (16.78% of the collected specimens) from all of counties were cap tured from deserts and foothills areas with the exception of jajarm county at an altitude of http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 374 http://jad.tums.ac.ir published online: december 31, 2019 10m (fig. 3). this species was collected both at indoor and outdoor human dwellings. this species was mostly caught in clay soil at nights and during the summer (table 2). odontobuthus doriae this digger scorpion species wascollected in all north khorasan counties except jajarm county (fig. 3). this species was caught only outside human settlements and in plain and desert areas. the majority of this species was captured in clay soil at nights and during the summer (table 2). mesobuthus caucasicus this species was gathered in jajarm, faruj and esfarayen countries from desert, semi-desert and foothill areas with an altitude range of 1000 to 1244m (fig. 3). the other ecological characteristics of this species have been presented in table 2. orthochirus farzanpayi this scorpion species was captured only in garme county; the highest area of north khorasan province with three specimens (fig. 3). this species was collected in desert area and in sandy soil at nights and during the summer. (table 1, 2). discussion the results of the present investigation indicate that five scorpion species were identified in the studied areas. all of collected species belonged to buthidae family, which is in agreement with the results of research carried out in other regions of iran. according to studies conducted in taibad county of razavi province, darmian county of south khorasan, sistan, and baluchestan (southeastern iran), kerman and shiraz (southern iran), most of captured scorpion species were of buthidae family as well (21-23). factors such as the high adaptability of species in this family due to different climatic conditions have led to their extensive geographical distribution in different regions, as species of this family have been widely dispersed in different areas of the world and iran (21). based on gis findings, the members of this family was captured in all the counties and habitats with different climatic areas, and ecologic conditions in urban and rural areas outside human dwellings such as under stones and bark of trees, on the farm, around the houses, in deserts, old homes and grasslands. in line with previous studies in different areas of iran, in the current research, m. eupeus was the dominant species in the study area (22, 24). additionally, in the present study a. crassicauda were gathered from all of counties in deserts and foothill areas except for jajarm county. this species was collected both at indoor and outdoor human dwellings. furthermore, this species was mostly collected in clay soil during the summer season and at nights. this species has been reported in most provinces of iran such as bushehr, semnan, khuzestan, ilam, west azarbaijan, kurdistan, khorasan razavi, south khorasan, kerman, kermanshah and sistan and baluchistan (25), all of which verify the results of this research. the other scorpion species collected in this present study was m. caucasicus. it in terms of frequency, it ranked second among the collected scorpions in north khorasan province. this is also in line with the results of previous studies carried out in other areas of iran such as west azerbaijan, sistan and baluchestan, isfahan, khorasan, tehran, and semnan provinces (26). the presence of this scorpion species in the current study can be confirmed by previous reports of this species in razavi khorasan and sistan and baluchestan provinces, in all of which the study areas had climatic conditions similar to that of the current study. in the present study, o. farzanpayi was found with very low frequency only in garme county. this species was collected in mountainous areas and in sandy soil at nights and during summer. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 369–377 firoozfar f et al.: faunistic composition and … 375 http://jad.tums.ac.ir published online: december 31, 2019 the size of adult o. farzanpayi is 3 centimeters and often distributed in desert and dry areas. spatial distribution of this scorpion species was reported in plain and flat regions of hormozgan and south khorasan provinces (south and east of iran) and has been previously proven (27). odontobuthus doriae as a digger scorpion species was captured in all north khorasan counties except in jajarm county. this species was caught only outside human settlements and in plain and desert areas. the majority of this species was captured in clay soil at nights and during summer. this digger scorpion is able to dig a cavity up to 40 centimeters deep within the earth. data of the current research indicated that the scorpion activity started in march, and reached its peaked in august when the weather was favorable, leading to a gradual increase in the frequency of scorpion species. this finding was corroborated by others (21, 22). the presence of suitable environmental conditions for the emergence of species has caused the presence of scorpion species to reach its peak in the study area in august. therefore, due to the high incidence of scorpions in the environment in aug, the scorpion sting cases also tend to increase, which call for an urgent need to implement training programs by healthcare providers in an effort to prevent scorpionism in this month. in this investigation, most of scorpions were collected at night. this in agreement with the results of research carried out in other regions of iran such as south khorasan province (22, 28). according to the scientific fact that scorpions are nocturnal creatures, this finding is theoretically expected. therefore, personal protection against scorpion stings at night be undertaken. to the current authors’ knowledge, among the limitations of this study mention can be made of the potential risks of scorpion stings and the problems of collecting scorpions in mountainous areas. conclusion we captured five scorpions’ species including m. eupeus, m. caucasicus, o. doriae, a. crassicauda and o. farzanpayi, among which the first four species are among most medically important 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(buthidae). acta soc zool bohem. 61: 39–52. 28. ramezani avval riabi h, motallebi m, rafinezhad j, amiri m (2010) the ecofaunistics of scorpions in gonabad. horizon med sci. 15(4): 54–61. http://jad.tums.ac.ir/ http://hms.gmu.ac.ir/browse.php?mag_id=27&slc_lang=en&sid=1 j arthropod-borne dis, june 2019, 13(2): 198–205 a barazesh et al.: molecular identification of … 198 http://jad.tums.ac.ir published online: june 24, 2019 original article molecular identification of species caused cutaneous leishmaniasis in southern zone of iran *afshin barazesh1; mohammad hossein motazedian2; moradali fouladvand3; gholamreza hatam2; saeed tajbakhsh3; sepideh ebrahimi2; danial purkamal4 1department of microbiology and parasitology, faculty of medicine, bushehr university of medical sciences, bushehr, iran 2department of parasitology and mycology, school of medicine, shiraz university of medical sciences, shiraz, iran 3the persian gulf marine biotechnology research center, bushehr university of medical sciences, bushehr, iran 4diagnostic lab of leishmaniasis, valfajr health center, shiraz university of medical sciences, shiraz, iran (received 14 apr 2018; accepted 30 apr 2019) abstract background: leishmania major and leishmania tropica are two main species causing cutaneous leishmaniasis (cl) in iran. recently, crithidia spp. has also been reported in the wound of patients with cl. in this study, we determined the species causing cl in the southern of iran and the role of crithidia spp. in creating skin ulcers. methods: in this cross-sectional study from apr to sep 2016, 66 patients with cl referred to diagnostic lab of leishmaniasis, valfajr health center, shiraz, iran, were selected. after dna extraction from the giemsa stained smears, all samples were amplified in two separate steps using specific primers, firstly, to differentiate leishmania species and then to identify crithidia spp. results: two species l. major and l. tropica were responsible for 60 and 6 cases, respectively. moreover, in two patients, mixed infection with crithidia was confirmed. in mix infection cases, the morphology of the cutaneous ulcers was not different from the wounds of other patients. conclusion: leishmania major is responsible for the most common cl in southern iran. in addition, in two patients with l. major and l. tropica, mix infection with crithidia was confirmed. the potential role of crithidia as the main factor for cl and the probability of this parasite to have synergistic effects on leishmania, as a hypothesis, requires more comprehensive researches on the ambiguity of this protozoon. keywords: cutaneous leishmaniasis; leishmania; crithidia; iran introduction cutaneous leishmaniasis (cl) is one of the six important diseases in tropical and subtropical regions which who has recommended and supported researches on its various aspects (1, 2). the factor causing cl is various species of bloody-tissue protozoan parasites called leishmania (3). this disease has distributed to all continents of the world except australia and it can be seen in 88 countries. annually, 1–1.5 million people in the world become infected with leishmania and a population of about 350 million people are at risk for this disease (4, 5). about 90% of cl cases worldwide are reported from seven countries: afghanistan, algeria, brazil, iran, peru, saudi arabia and syria (6) which among these countries, iran and saudi arabia have the highest incidence of the disease (7). about 20,000 cl cases are reported annually from different parts of iran estimated that the real rate is several times higher (8). in *corresponding author: dr afshin barazesh, email: afshin914@gmail.com mailto:afshin914@gmail.com j arthropod-borne dis, june 2019, 13(2): 198–205 a barazesh et al.: molecular identification of … 199 http://jad.tums.ac.ir published online: june 24, 2019 recent years, the prevalence cl in iran, especially in the southern regions, has been growing gradually and alarmingly and is endemic in 17 out of 31 provinces of iran (9), as the number of positive cases has increased from 1560 in 1991 to 3861 in 2001 (10, 11). in an epidemiological survey about cl in iran during 2013–2015, cl is an endemic disease in fars province, and it annually infects numerous people (12). in general, provinces such as fars and isfahan with an incidence of 1.66 cases per 1,000 people, have the highest incidence of the disease in the country (13). leishmania major and l. tropica are two main species causing cl in iran (9), which have a different distribution in different parts of the country (14). in recent studies, besides these two species, some trypanosomatidal protozoa including herpetomonas spp., leptomonas spp. and crithidia spp. have also been reported in the culture of cutaneous ulcers (15-17). crithidia spp. are classified in kinetoplastida order and mastigophora subphylum and are often referred to as insects’ parasites (16). two species (crithidia lucilia and crithidia fasciculata) are also considered to be infectious agents of the leishmania culture medium (18, 19). however, the interesting thing is that the patients who the crithidia spp. have been isolated from their wounds are often drug resistant to various drugs, and their cutaneous ulcers sometimes remain for 2–3yr and become chronic (17), which is probably due to the presence of crithidia alone or simultaneously with leishmania spp. as mix-infection in these groups. in iran, crithidia spp. has been reported frequently from patients suspected of drug-resistant cutaneous leishmaniasis and the genomic sequence of the ribosomal region has proven its belonging to the genus crithidia (17). in another study, crithidia was isolated from the spleen and foot-pad of tetera indica in fars province, iran which considers the presence and role of this protozoon in infecting vertebrate hosts (20). crithidia spp. are able to present simultaneously with some trypanosomatidal parasites such as leishmania spp. and be transferred to different hosts (21). for the first time reported that c. deanei has the ability to infect mouse fibroblast cells (22). in the following in 2010, it concluded the ability of this species to cause infections in balb/c (23). since the establishment of a specific species of leishmania parasite in a geographical region depends on several factors such as the presence of a specific vector and a suitable reservoir (24), the determination of the dominant species in an endemic or hyper-endemic region can be important for adopting an appropriate strategy to control vectors and reservoirs. furthermore, identification of species is essential for more effective treatment of the patients due to the fact that the response to treatment varies in different species (13). therefore, considering the high incidence of cl and its health importance, we decided to determine the species causing cl in the southern region of iran and the role of crithidia spp. in creating skin ulcers by using the molecular methods. materials and methods sample preparation this study was approved by the ethics committee of the bushehr university of medical sciences (ethics committee code: ir.bpums.rec.1395.125) and informed consent was signed by each of the patients. in this cross-sectional study, in a period of 6 months from apr to sep 2016, patients with cutaneous ulcers referred to the diagnostic lab of leishmaniasis, valfajr health center, shiraz, iran were tested. smears of them from exudates of margin of the wounds were prepared and stained with giemsa. finally 66 patients with cl were selected. then a questionnaire containing demographic information and some of the variables such as j arthropod-borne dis, june 2019, 13(2): 198–205 a barazesh et al.: molecular identification of … 200 http://jad.tums.ac.ir published online: june 24, 2019 age, sex, etc. were completed separately for each patient. dna extraction dna was extracted from the giemsa stained smears using the commercial kit (favorgen biotech corp, cat no. fabgk001, taiwan). briefly, the smears on the slides were scratched and collected by lysis buffer into the microtubes and after the addition of proteinase k, incubated initially for 1 h at 60 ºc and then 10min at 70 ºc. by addition of absolute ethanol and transferring the samples to the column, the rest of the process was carried out according to the kit's manufacturer's protocol. pcr and gel electrophoresis all extracted dna samples were pcramplified in two separate steps. first, a pair of primers lin4r and lin17 were used to detect and differentiate of three species l. major, l. infantum and l. tropica, and in the next step, in order to identify crithidia spp., crf and crr primers were used in this purpose. further details about the sequence of primers used and the pcr programs adjusted for the amplification of both genomic pieces, has been presented in table 1. statistical analysis the results and data of the questionnaires were analyzed using spss software version 18 and chi-square test and p-values. at levels < .05, the p-values were considered as statistically significant. results of the 66 patients, l. major and l. tropica were responsible for 60 and 6 cases, respectively (fig. 1). moreover, in two patients with l. major and l. tropica, mix infection with crithidia was confirmed (fig. 2). there was no significant relationship between the disease-causing species and the factors such as the number, appearance, and location of the wounds in the body, the residence as well as the age and gender of the patients (table 2). in two patients with mix infection (leishmania and crithidia), the morphology of the cutaneous ulcers was not different from the wounds of the other patients. table 1. characteristics of primers and temperature patterns used in pcr tests genus primers sequence program processes cycles temp time leishmania lin4r (f) 5‘ggg gtt ggt gta aaa tag gg -3‘ p. denaturation 1 95 ⁰c 5min denaturation 35 94 ⁰c 30sec annealing 52 ⁰c 30sec lin17 (r) 5‘ttt gaa cgg gat ttc tg -3‘ extension 72 ⁰c 45sec final extension 1 72 ⁰c 8min crithidia fcr (f) 5‘tcc atg tgc gag gac aac gtg ct -3‘ p. denaturation 1 94 ⁰c 3min denaturation 30 95 ⁰c 30sec annealing 62 ⁰c 30sec rcr (f) 3‘cgc gtc gtt gat gaa gtc gct -5‘ extension 72 ⁰c 45sec final extension 1 72 ⁰c 5min pcr products were separated on 1.2% agarose gel and tae (tris, acetate, and edta) buffer, and the obtained bands were detected by a uv detector (bio-rad, usa) j arthropod-borne dis, june 2019, 13(2): 198–205 a barazesh et al.: molecular identification of … 201 http://jad.tums.ac.ir published online: june 24, 2019 fig. 1. electrophoresis of pcr products using lin4r and lin17 primers on agarose gel 1.2% to differentiate leishmania species. lane 1: molecular weight marker, lane 2: positive control for l. major, lane 3: positive control for l. tropica, lane 4: negative control, lane 5, 6: positive samples for l. major, lane 7, 8: positive samples for l. tropica fig. 2. electrophoresis of pcr products using crf and crr primers on agarose gel 1.2% to identify crithidia spp. lane 1: molecular weight marker, lane 2: positive control for crithidia spp, lane 3: negative control, lane 4, 5: positive samples for crithidia spp., lane 6: molecular weight marker j arthropod-borne dis, june 2019, 13(2): 198–205 a barazesh et al.: molecular identification of … 202 http://jad.tums.ac.ir published online: june 24, 2019 table 2. demographic features of the patients with cl and the relationship of disease-causing species and some variables variables species p-value l. major n (%) l. tropica n (%) crithidia+leishmania n (%) sex male female 33 (50) 26 (39.4) 2 (3) 3 (4.5) 1 (1.5) 1 (1.5) 0.78 age (yr) < 10 10–20 20–30 30–40 > 40 6 (9.1) 9 (13.6) 9 (13.6) 11 (16.7) 24 (36.4) 1 (1.5) 0 (0) 1 (1.5) 2 (3) 1 (1.5) 0 (0) 0 (0) 1 (1.5) 0 (0) 1 (1.5) 0.73 residence city village 46 (69.7) 13 (19.7) 5 (7.6) 0 (0) 1 (1.5) 1 (1.5) 0.30 city shiraz marvdasht daryun kharameh tiun meimand maharlou sharifabad 41 (62.1) 1 (1.5) 7 (10.6) 1 (1.5) 2 (3) 2 (3) 1 (1.5) 4 (6.1) 5 (7.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.99 number of wounds 1 2–5 > 5 23 (34.8) 22 (33.3) 14 (21.2) 3 (4.5) 2 (3) 0 (0) 1 (1.5) 0 (0) 1 (1.5) 0.53 duration the advent of the wounds 1–4 wk 4–8 wk > 8 wk 22 (33.3) 24 (36.4) 13 (19.7) 1 (1.5) 2 (3) 2 (3) 1 (1.5) 0 (0) 1 (1.5) 0.64 discussion cutaneous leishmaniasis is one of the vector-borne diseases and is caused by various species of leishmania spp. (3, 25). establishment of a specific species of this parasite in any geographical region depends on several factors such as the presence of appropriate reservoir and vector, and determining the dominant species in each area can be very important in controlling the life cycle as well as the more effective treatment of the disease (13). the isolation and mass cultivation of parasite in order to determine the species is not only very time-consuming but also is expensive (26, 27), therefore, in the present study, giemsa stained slides were used for this purpose to reduce the costs and overcome the problem. the success of dna extraction from these smears has already been studied (26). furthermore, the primers used for pcr in various studies were able to identify the genus, so to determine the parasite species, the researchers had to develop advanced molecular methods, such as pcr-rflp or sequencing. in this j arthropod-borne dis, june 2019, 13(2): 198–205 a barazesh et al.: molecular identification of … 203 http://jad.tums.ac.ir published online: june 24, 2019 study, we used the primers previously designed based on the variable part of dna minicircles in kinatoplast (13, 28, 29). these primers have the ability to identify three species so that by electrophoresis of the pcr product, l. major, l. infantum and l. tropica create a specific band in the 650, 720 and 760 bases, respectively. the specificity of 100% has been reported for pcr tests using primers lin4r and lin17 to detect leishmania in atypical cases (13, 30). in some endemic and hyper-endemic regions of iran l. major and in other areas, l. tropica plays a role in causing disease, although, in southern regions such as fars province, considered as one of the most important foci in iran, both species are listed as endemic factors (24, 31). recently, alongside these two dominant parasites, crithidia spp. has been reported from cultivation of cutaneous ulcers of patients (15, 16). in this study, in order to determine the role of crithidia spp. in cutaneous lesions, samples were again amplified with two specific fcr and pcr primers. in two cl cases, there was a mix infection by leishmania and crithidia and, as shown in fig. 2, a band in 850 bases characterizes this parasite (fig. 2). crithidia spp. are classified in flagellate subphylum and are often considered as insect parasites (16). since the sequence of their rrna (ribosomal rna) gene is very close to leishmania, they are usually associated with together (16, 18). crithidia spp. are considered as infectious agents of the leishmania culture medium. crithidia lucilia and c. fasciculata are two important species of this genus (18). over the past 20 years, various methods have been developed to determine the different subspecies of leishmania and crithidia, as well as to study the molecular diversity and the interaction between this parasite and the host (32-34). in recent reports, besides two l. major and l. tropica species, crithidia spp. has also been proven in the wound of patients with cl, and interestingly, even in some of these reports, crithidia has been reported as the only factor isolated from the wound of mentioned patients. in a study to investigate the polymorphism of two species l. tropica and l. major in the central and desert regions of iran, crithidia has been isolated from 6 of 215 and 3 of 125 patients with cutaneous ulcers in isfahan and bam, respectively. molecular analysis of 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iran j parasitol. 5(4): 1–8. 32. baker jr, muller r, rollinson d (1998) advances in parasitology. london, uk: academic press. pp. 215–240. 33. bailey ms, lockwood dnj (2007) cutaneous leishmaniasis. clin dermat. 25 (2): 203–211. 34. banuls al, brisse s, sidibe i, noel s, tibayrenc m (1999) a phylogenetic analysis by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic dna fingerprinting of the leishmania genome project friedlin reference strain. folia parasitol. 46(1): 10– 14. j arthropod-borne dis, june 2020, 14(2): 126–133 r ranjbar et al.: anaplasma infection in … 126 http://jad.tums.ac.ir published online: june 30, 2020 original article anaplasma infection in ticks in southeastern region of iran reza ranjbar 1 ; mehdi anjomruz 2 ; ahmad ali enayati 3 ; mehdi khoobdel 4 ; atiyeh rafinejad 2 ; *javad rafinejad 2 1 molecular biology research center, systems biology and poisonings institute, baqiyatallah university of medical sciences, tehran, iran 2 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3 department of medical entomology, school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 4 health research center, baqiyatallah university of medical sciences, tehran, iran (received 02 jul 2016; accepted 12 nov 2016) abstract background: anaplasmosis and ehrlichiosis are the most important tick-borne diseases. this study was conducted in three cities of kerman province in iran to investigate the circulation of the bacteria in ticks collected from sheep. methods: ticks were collected from animals using srkj forceps and transferred to the entomology lab in cold chain. after specimen’s identification, they kept at -70 ºc. tick dna was extracted using bioneers dna extraction kits followed by nested pcr technique to amplify ribosomal 16s rrna gene to detect anaplasma infection in ticks. results: 472 sheep were examined from which 349 ticks were collected and identified in laboratory using valid keys. tick specimens belonged to two genera and four species; hyalomma marginatum (62.47%) was the most frequent and hylomma asiaticum (5.73%) showed the least abundance. the infestation rate to different tick species was different in three regions of kerman province. observation revealed that 24 specimens (58.3%) were positive for anaplasma. there is a significant difference between male and female infection rate. however, there is no significant difference between these variables in each of these cities. conclusion: this study shows high infection rates to anaplasma in hard ticks. it is essential for health and veterinary authorities and farmers to use appropriate strategies to control ticks to reduce the infestation. keywords: anaplasma; molecular; ticks; iran introduction ticks are mandatory blood-sucking ectoparasites of vertebrate’s especially wild animals and only about 10 percent of its species feed from humans and livestock, particularly sheep and cow. anaplasmosis usually occurs in tropical and subtropical regions and is reported from africa, the middle east, asia, australia, usa, south and central america and southern europe (1, 2, 3). ticks directly affect their host by causing irritation, allergic reactions, anemia, weight loss, paralysis and even death. spotted fever, rocky mountains fever, siberia tick typhus, tularemia, lyme disease, relapsing fever, cri mean-congo hemorrhagic fever, anaplasmosis and ehrlichiosis are among the most important tick-borne diseases transmitted to humans by ticks (4, 5, 6, 7). ticks are also involved in transmitting certain diseases to livestock the most important of which include swine fever in asia, viral hemorrhagic fever, anaplasmosis, theileriosis and babesiosis (8, 9, 10). certain pathogens transmitted by ticks can be spread by contaminated airborne particles, hence, they can be used for military purposes (bioterrorism), many diseases having bioterrorism potential are among diseases transmitted by arthropods including plague, yellow *corresponding author: dr javad rafinejad, email: jrafinejad@tums.ac.ir mailto:jrafinejad@tums.ac.ir j arthropod-borne dis, june 2020, 14(2): 126–133 r ranjbar et al.: anaplasma infection in … 127 http://jad.tums.ac.ir published online: june 30, 2020 fever, tularemia, rift valley fever, q fever, viral encephalitis, etc. the first practical use of insects was by the japanese during world war ii using ticks infected with plague agent (yersinia pestis), musca domestica with cholerae bacteria (vibrio cholerae). during world war ii, the russians used lice infected with typhus agent (rickttesia typhi) against the germans (11, 12, 13, 14, 15). development of genetics and production of arthropods with the ability to transmit life-threatening diseases like human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids), high potency sting, resistance to pesticides and also production of biological agents with more virulence, drug resistance and stability at ambient conditions have greatly contributed to the development of bioterrorism (13) a fact that increases their importance (16). anaplasmosis, a rickettsia zoonosis, is one such disease, among 30 new ones that have emerged since 1973. the rickettsia is a mandatory intracellular parasite of red blood cells. infected red blood cells lose their functionality in circulatory system and are destroyed. this causes diseases in livestock and wild ruminants like cow, sheep, goats, and deer although this disease causes clinical symptoms only in cow and giraffes. the disease is transmitted through red blood cells of infected animals. when an animal becomes ill, despite its recovery, it remains as a source of infection forever. anaplasmosis is transmitted to others hosts by ticks that have bitten infected animals. the pathogen will also multiply in infected ticks. then infected ticks transmit the disease to healthy animals. the pathogen can be transmitted to healthy animals by mosquito bites or contaminated equipment with infected animal blood such as infected syringe or infected equipment of cutting horn as well (17, 18, 19, 20, 21). anaplasmosis begins with a mild fever, but it gradually becomes more serious. treatment is usually effective if it is provided early. tetracycline and amido propionate are two first line drugs used for treatment (18, 19, 22, 23). in iran, the first case of this disease was reported in 2003 from north of the country. anaplasma bacteria are detected in animal blood samples in provinces of khorasan, esfahan, fars, mazandaran and sistan and baluchistan (24, 25, 26, 27, 28). considering the relatively high tick burden of animals and importance of anaplasmosis and other tick-borne diseases in livestock as well as humans, determining the species of infecting bacteria and tick vectors and their infection rate are very important. this could be an effective step in diagnosis of diseases transmitted by ticks and development of strategies to preventing diseases by controlling ticks and patient treatment. materials and methods study area this study is undertaken in three cities of kerman province including kerman, kohbanan and sirjan. kerman is located in the south-east of iran central plateau, between 53º 26' to 59º 29' east longitude and 25º 55' to 32º north latitude. encompassing an area of more than 183/285 square kilometers and approximately eleven percent of the country’s area, kerman is the largest province in iran located in southeast of the country with a history of human settlement since four millennium bc. kohbanan is located at 31º.42' 36" north and 56º.28' 75" east in the far northern parts of kerman province, southeast of iran with a population in 2011 of 21721 people. sirjan is another city of kerman province (29°.45' 55°.67') is located in the west of this province and has a population in 2011 of about 269000 (fig. 1). sample collection ticks were separated from body of sheep, goat and cow (ear, groin, tail, back and neck) using srkj forceps so as not to harm the ticks and animals. they were then put in appropriately labeled tubes and transferred to the entomology lab in cold chain. after specij arthropod-borne dis, june 2020, 14(2): 126–133 r ranjbar et al.: anaplasma infection in … 128 http://jad.tums.ac.ir published online: june 30, 2020 mens identification, selected ticks were kept at -70 ºc in micro tubes for pcr (29, 30). dna extraction bioneers dna extraction kits (intron biotechnology, korea) was used to extract dna according to the instructions of the manufacturer. nested pcr technique with primers designed by rar et al. was used to amplify ribosomal 16s rrna gene in order to detect anaplasma infection in ticks (31). the primers are designed based on conserved and highly changeable region v1 of ribosomal gene 16s (16s rrna) of anaplasma sp. and ehrlichia sp. producing dna fragments of about 500bp (table 1). polymerase chain reaction (pcr) protocol consisted of 35 cycles of a preheating phase at 94 ºc for 5min, denaturation at 94 ºc for 1min, annealing at 57 ºc for 1min, extension at 72 ºc for 1min and a final incubation at 72 ºc for 7min. before finishing the first phase of pcr reaction, master mix of the second phase is prepared. in the second phase of the nested pcr, different primers are used (table 1) and also two micro liter of (pcr) product is used as template and annealing was set at 54 ºc. pcr products were kept in a refrigerator before electrophoresis using 1% agarose gel. results during this study, a total of 472 sheep were examined for tick infestation in three cities of kerman province in spring 2015. a total of 349 ticks were collected from 154 animals resulting in a tick burden of 2.26. collected ticks were identified in laboratory using valid diagnosis keys (32, 33). as present ed in fig. 2, collected ixodidae tick specimens belonged to two genera and four species including hy. marginatum (62.47%), hy. anatolicum (9.47%), hy. asiaticum (5.73%), hyalomma.sp (7.44%), rhipicephalus sanguineus (14.33%) and unidentified specimens (0.58 %) (fig. 2). among identified species, hy. marginatum with 62.47% showed the highest frequency and hy. asiaticum (5.73%) the lowest (fig. 2). totally, from 349 tick specimens, 63 were collected in kerman, 98 in sirjan and 188 in kohbanan. according to the results in kerman, rh. sanguineus with a prevalence of 10.05% showed the highest frequency and hy. anatolicum, hy. marginatum and hy. asiaticum species with 3.43%, 2.58% and 0.29 % were in the next orders respectively. hyalomma marginatum, hy. anatolicum, rh. sanguineus and hy. asiaticum with 20.92, 2.87, 1.72 and 0.85 percent showed the highest frequency in sirjan city respectively. hyalomma marginatum with 39.97 percent had the highest frequency and hy. asiaticum, hy. anatolicum and rh. sanguineus with 4.59, 3.15 and 2.56 percent showed the highest frequency in kohbanan respectively (table 2). fig. 1. geographical location of kerman province, iran table 1. list and details of primers used in this study primer nucleotide sequences forward: ehr1 5′-gaacgaacgctggcggcaagc-3′ reverse: ehr2 reverse: ehr3 reverse: ehr4 5′-agtatccgagaccagatagccgc-3′ 5´-tgcataggaatctacctagtag-3´ 5´-ctaggaattccgctatcctct-3´ j arthropod-borne dis, june 2020, 14(2): 126–133 r ranjbar et al.: anaplasma infection in … 129 http://jad.tums.ac.ir published online: june 30, 2020 table 2. species composition in kerman province (a=kohbanan and b=sirjan and c=kerman) genus/species a b c province (100%) a b c hy. marginatum 136(38.97%) 73(20.92%) 9(2.58%) 218(62.47%) hy. anatolicum 11(3.15%) 10(2.87%) 12(3.43%) 33(9.45%) hy. asiaticum 16(4.59%) 3(0.85%) 1(0.29%) 20(5.73%) hy. sp 16(4.59%) 4(1.14%) 6(1.71%) 26(7.44%) rh. sanguineus 9(2.56%) 6(1.72%) 35(10.05%) 50(14.33%) unidentified sp. 0(0%) 2(0.58%) 0(0%) 2(0.58%) total 188(53.86%) 98(28.08%) 63(18.06%) 349(100%) table 3. infection rates of anaplasmosis in ticks collected from kerman province, southern iran in 2015 city genus/species nymph female)♀( male)♂( (100%) (100%) negative positive negative positive negative positive negative city a hy. marginatum 0(0%) 0(0%) 6(16.22%) 2(5.4%) 13(35.14%) 2(5.41%) 19(51.35%) 21(56.76%) hy. mnatoilicum 0(0%) 0(0%) 2(5.4%) 0(0%) 4(10.82%) 0(0%) 6(16.22%) 6(16.22%) hy. msiaticum 0(0%) 0(0%) 2(5.4%) 0(0%) 4(10.82%) 0(0%) 6(16.22%) 6(16.22%) hy. sp 0(0%) 0(0%) 1(2.7%) 0(0%) 0(0%) 0(%) 1(2.7%) 1(2.7%) rh. manguineus 0(0%) 0(0%) 1(2.7%) 0(0%) 2(5.4%) 0(0%) 3(8.1%) 3(8.1%) total a 0(0%) 0(0%) 12(32.43%) 2(5.4%) 23(62.17%) 2(5.41%) 35(94.59%) 37(100%) city b hy. marginatum 0(0%) 2(5.71%) 5(14.29%) 5(14.29%) 10(28.57%) 7(20%) 15(42.86%) 22(62.86%) hy. anatoilicum 0(0%) 0(0%) 2(5.71%) 0(0%) 4(11.43%) 0(0%) 6(17.14%) 6(17.14%) hy. asiaticum 0(0%) 0(0%) 0(0%) 0(0%) 2(5.71%) 0(0%) 2(5.71%) 2(5.71%) hy. sp 0(0%) 0(0%) 2(5.71%) 0(0%) 0(0%) 0(0%) 2(5.71%) 2(5.71%) rh. ssanguineus 0(0%) 0(0%) 1(2.86%) 0(0%) 2(5.71%) 0(0%) 3(8.58%) 3(8.58%) total b 0(0%) 2(5.71%) 10(28.57%) 5(14.29%) 18(51.42%) 7(20%) 28(80%) 35(100%) city c hy. marginatum 0(0%) 1(4.17%) 0(0%) 1(4.17%) 3(12.5%) 2(8.33%) 3(12.5%) 5(20.83%) hy. mnatoilicum 0(0%) 0(0%) 0(0%) 2(8.33%) 3(12.5%) 2(8.33%) 3(12.5%) 5(20.83%) hy. msiaticum 0(0%) 0(0%) 0(0%) 1(4.17%) 0(0%) 1(4.17%) 0(0%) 1(4.17%) hy. sp 1(4.17%) 0(0%) 0(0%) 0(0%) 0(0%) 0(0%) 1(4.17%) 1(4.17%) rh. manguineus 0(0%) 8(33.33%) 2(8.33%) 1(4.17%) 1(4.17) 9(37.5%) 3(12.5%) 12(50%) total c 1(4.17%) 9(37.5%) 2(8.33%) 5(20.84%) 7(29.16%) 14(58.33%) 10(41.67%) 24(100%) total abc 1(1.05%) 11(11.45%) 24(25%) 12(12.5%) 48(50%) 23(23.95%) 73(76.05%) 96(100%) j arthropod-borne dis, june 2020, 14(2): 126–133 r ranjbar et al.: anaplasma infection in … 130 http://jad.tums.ac.ir published online: june 30, 2020 fig. 2. the infestation rate to different tick species in kerman province, southern iran in 2015 discussion the results revealed that 23 out of 96 analyzed specimens were positives for anaplasma; 11.45% hy. marginatum, 2.08% hy. anatolicum, 1.04% hy. asiaticum, 0% hy. sp, and 9.37% rh. sanguineus were positives. nested-pcr is a sensitive molecular technique to determine infection to anaplasma sp in ticks collected from animals. in 1910, anaplasma marginale was described first by theiler in african cows’ red blood cell (rbc) as a marginal spot (34); afterward, in 1911, he introduced an. centrale, a subspecies of an. marginale which is located in the center of erythrocytes and has less pathogenicity (35). lesto kard, in 1924, demon-trated an. ovis in sheeps’ rbc. donatien and lestoquard observed an. bovis in french cows’ rbc, during an experimental transmission of theileria through hyalomma ticks (36). dumler et al. (2001), after the modification of ehrlichia equi, ehrlichia phgocytophila species plus unknown factor of human ehrlichia granulocytic, category zed them under a species named an. phgocytophilum (37). from the amount of 60 cows, 391 sheep, and 385 goats, through a blood smear test observed that 19.37% of cows were infected to an. marginale, and 80.3% of sheep and 38.92% of goats were infected to an. ovis, during a study between 1999 and 2002 in khorasan province in iran by razmi et al. (2005), there was no a significant difference between age and gender groups regarding the infection rate of anaplasma and the amount of anaplasma bacteria was 0.005–0.5%, 0.01–3%, 0.01–3% in cows, sheep, and goats, respectively (25). spitalska et al. in 2005 investigated 100 samples of sheep blood, and 89 ticks using pcr for 16s and 18s rrna in shiraz city; they observed that 25% of ticks and 29% of blood samples were positive for anaplasma sp. and ehrlichia sp. also, they found that 59.5% of ticks and 76% of blood samples were infected with babesia sp. and theileria sp. (26). a study by n’aman et al. in isfahan city j arthropod-borne dis, june 2020, 14(2): 126–133 r ranjbar et al.: anaplasma infection in … 131 http://jad.tums.ac.ir published online: june 30, 2020 (1386) revealed some anaplasma-like bodies in 75 blood smears out of 150 cow blood samples using microscopic observation. also, they observed an. marginale in 50 blood samples using nested-pcr (27). another research in qa’emshahr city in the province of mazandaran performed on 98 specimens of ix. ricinus using pcr on the 16s rrna gene revealed that 5.1% of the ticks were infected with an. phgocytophilum. these results can be a cause for concern for the appearance of human an. granulocyte (age) disease in that area (28). in another study in qa’emshahr using nested-pcr on 54 rh. sanguineus, 1 rh. bursa, 39 ixodes ricinus, 4 bo. annulatus, 2 haemaphysalis numidiana, and 1 haemaphysalis punctaterevealed that 49.5% of the ticks were positives. at the same time, 65 samples of sheep blood, 90 samples of cow blood, and 4 samples of goat blood were studied showing 43.1%, 22.2%, 25% of sheep, cows, and goats were infected respectively. the researchers reported 25% of human samples (40 blood samples) of at risk individuals from different age groups and genders were infected (24). conclusion results of this study show preliminary data on relatively high infection rates in hard ticks collected from sheep in kerman province. therefore, it is essential that health and veterinary authorities and farmers to adopt appropriate strategies to control ticks to reduce the infestation and infection rates in livestock and humans. it is also necessary that those high risk individuals including herders observe adequate health and safety measures and protect themselves against diseases transmitted by ticks. more studies are recommendded in kerman province as well as in other parts of iran regarding the infection of ticks with anaplasmosis. acknowledgments this study was conducted by molecular 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120. 33. hoogstraal h (1956) african ixodidae. 1. ticks of the sudan with special reference to equatoria province and with preliminary reviews of the genera boophilus margaropus and hyalomma. department of the navy, bureau of medical surgery, washington, dc. 34. theiler a (1910) anaplasma marginale (gen. and spec. nov.): the marginal points in the blood of cattle suffering from a specific disease. pretoria: government printing and stationary office. available at: http://hdl.handle.net/2263/10409 35. theiler a (1911) further investigations into anaplasmosis of south african cattle. first report of the director of veterinary research, union of south africa, pp. 7–46. 36. donatien a, lestoquard f (1936) rickettsia bovis, novelle specie pathogen pour le boeuf. bull soc pathol exot. 29: 1057–1061. 37. dumler js, barbet af, bekker c, dasch ga, palmer gh, ray sc, rikihisa y, rurangirwa fr (2001) reorganization of genera in the families rickettsiaceae and anaplasmataceae in the order rickettsiales: unification of some species of ehrlichia with anaplasma, cowdria with ehrlichia and ehrlichia with neorickettsia, descriptions of six new species combinations and designation of ehrlichia equi and 'hge agent' as subjective synonyms of ehrlichia phagocytophila. int j syst evol micr. 51: 2145–2165. http://hdl.handle.net/2263/10409 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 22 original article susceptibility of culicidae mosquitoes to some insecticides recommended by who in a malaria endemic area of southeastern iran mousa fathian 1, *hassan vatandoost 1, *seyed hassan moosa-kazemi 1, ahmad raeisi 1,2, mohammad reza yaghoobi-ershadi 1, mohammad ali oshaghi 1, mohammad mehdi sedaghat 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2malaria unit, cdc, ministry of health and medical education, iran (received 30 may 2012; accepted 14 may 2014) abstract background: according to the national strategy plan on monitoring of insecticides resistance, this study was carried out to determine the base line susceptibility of the culicidae mosquitoes to the who-recommended insecticides in an endemic focus of malaria in southeastern iran. methods: larval collection was carried out by dipping method and adult collection occurred by suction tube from january to december 2010. the susceptibility test was assessed to ddt 4 %, malathion 5 %, propoxur 0.1 %, deltamethrin 0.05 %, lambda-cyhalothrin 0.05 %, and cyfluthrin 0.15 % at different interval times (discriminative dose) followed by 24 h recovery period . the lt50 and lt90 values were calculated for plotting the regression line using microsoft office excel software ver. 2007. results: anopheles stephensi was quite resistant to ddt and showed susceptible or tolerant to other insecticides. the lt50 and lt90 values to ddt in this species were 29.07, and 98.26 minutes, respectively. anopheles culicifacies and anopheles dthali were found susceptible or tolerant to insecticides. culex pipiens was found resistance to ddt, propoxur, lambda-cyhalothrin and cyfluthrin whereas observed susceptible to malathion and tolerant to deltamethrin. ochlerotatus caspius sl. was resistant to ddt, whereas found susceptible to other insecticides. culisita longiareolata was susceptible to deltamethrin, whereas tolerant to other insecticides. the lt50 and lt90 values of cs. longiareolata to ddt were 17.82, and 51.26 minutes. conclusion: we suggested the same study in different parts of the country for monitoring and evaluation of control measures. keywords: culicidae, malaria, susceptibility test, southeastern iran introduction arthropod borne diseases are very important in the world. malaria occurs often in poor, tropical and subtropical areas and still took an estimated 627 000 lives in 2012, mostly those of children under five years of age in africa. in fact 1300 young lives lost to malaria every day (who 2013). the culicidae mosquitoes are responsible for transmission of the worm parasites of heart dogs such as dirofileria immitis, dirofileria repense, wuchereria bancrofti, some arboviral diseases such as japanese encephalitis, rift valley fever, western equine encephalitis and eastern equine encephalitis, tahyna, sagiyama, trivitatus, lymphocytic choriomanangitis, west nile virus, st. louis encephalitis, california encephalitis. about 70 anopheles species in the world are able to transmit malaria and of these 40 species have been identified as the major vectors (azari-hamidian and harbach 2009). by now, 64 species of culicidae mosquitoes due to 3 sub genera and 7 genera are reported from iran (azari-hamidian and harbach 2009). anopheles stephensi is the main malaria vector in iran. by now, seven *corresponding author: prof hassan vatandoost, email: hvatandoost1@yahoo.com, dr seyed hassan moosa-kazemi, e-mail: moosakazemi@tums.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 23 species of anopheles reported as the malaria vectors in the country including: an. fluviatilis s.l, an. culicifacies s.l, an. sacharovi, an. maculipennis s.l, an. superpictus, an. stephensi and an. dthali (faghih 1969, vatandoost et al. 2004c, vatandoost and borhani 2004a, vatandoost and moinvaziri 2004b, vatandoost et al. 2005b, sedaghat and harback 2005, hanafi-bojad et al. 2006, oshaghi et al. 2006, vatandoost et al. 2006a, 2006b, 2007, 2008, 2009a, 2009b, moosa-kazemi et al. 2009, 2010, omrani et al. 2010). in addition, zaim et al. reported the an. pulcherrimus as secondary vectors of malaria in the south east of iran (zaim et al. 1993). oocyt of plasmodium found at the first time in an. multicolor, while not found in salivary glands (eshghy 1978). avian malaria reported in iran by ghaffari (ghaffari 1955). spraying with residual insecticide (irs) considered an important mosquito control measure. twelve insecticides recommended by who for irs currently, which belong to four chemical groups including one organochlorine, six pyrethroids, three organophosphates and two carbamates (pluess et al. 2010). ddt resistance in the adult of an. stephensi was reported in iran in 1957, subsequently to dieldrin in 1960, and then to malathion in 1976 (eshghy 1978, manouchehri et al. 1974). propoxur was used after reports about to malathion tolerance of an. stephensi in 1978 (eshghy 1978). release of larvivorous fish, using the bacillus thuringiensis, and larviciding by chlorpyrifos-methyl are the main larval control measures and pyrethroid as new insecticides are being used as irs and llins in iran (vatandoost and hanafi bojd 2005a, moosa-kazemi et al. 2007). in spite of more than 50 years malariacontrol programming, 42%–60%of the total malaria cases reported from sistan and baluchistan province.out of 112 malaria cases representing 79 males and 33 females have been reported in chabahar county, southeastern iran in 2010. out of 38 cases were from bangladesh, pakistan and afghanistan and the rest were the local aborigines (moh and me 2010). chabahar is commercial and industrial free zone as well as important due to agricultural and husbandry in sistan and baluchistan province in southeastern iran and also the border line of pakistan. there are scatter data about susceptibility level of malaria vectors in iran, the susceptibility level of culicinae mosquitoes in southeastern country had not been reported. therefore, this study was carried out to determine susceptibility level of mosquitoes to monitoring and evaluation of insecticides resistance. this information could provide an essential clue for judicious use of insecticides and will be very useful to health authorities for future planning of vector control in this endemic malarious area. materials and methods study area the study was carried out in chabahar city, siatan and baluchistan province, southeastern iran (25˚17’gn, 60˚37’ge) (fig. 1). the people engaged to agriculture, horticulture, livestock, fishing sailing, and hand crafts including needlework, making carpet and musical instruments. the absolute maximum and average of temperature was reported 36 ˚c and 16.5 ˚c, respectively (meteorological organization of iran, 2010). average annual rainfall and humidity was 90mm and 79 %, respectively. kajoo and bishmont rivers are the largest rivers located in this area. chabahar county with 24, 729 square kilometers (19.3%) comprised 3 cities, 5 distracts and 11 rural districts. the population of chabahar reported 211,081 including 143,535 rural (68%) and 67,546 urban (32%). naval base near the 22 bahman squar (25° 18’gn,60°37’ge) in chabahar city selected as fix station including, 30 households, and 114 population. baluchi-adam village (25˚ 47’gn, 61˚16’ge) with 71 households, and http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 24 379 populations, and bahoo-kalat village (25˚43’gn, 61˚25’ge), 198 households and 1064 population selected randomly as fixed stations. sampling methods such as larval collection, hand catch was carried out during january to december 2010 (who 1975, who 1975). these studies were conducted once every 15 days and collected mosquitoes were identified by specific systematic keys (shahgudian 1960, zaim and cranston 1986). larval collection and rearing in each fixed and variable station larvae was collected from january to december 2010. larval mosquitoes picked up from the water using a dropper, pipette or fine net and inserted into the bulb. the related data such as water temperature, larval type, number and date sampling was recorded. larvae and pupae in holding container filled with water were transferred to the laboratory for rearing. mosquito larvae feed by dry fish food. adult mosquitoes live quite well on bowl of sucrose 10% in bottom of the cage. the adults were kept in the temperature (22–24 ˚c), relative humidity (60–70%) and 12l12d photoperiod (who 1975a, 1975b). hand collection culicidae mosquitoes were collected from the villages that spraying was not performed before, between 06.30 and 09.30 am. sampling was carried out in each human dwelling, cattle and goat sheds for 15 min using suction tube and torch (who 1975b). the mosquitoes were inserted in the cage as dimensions of 40× 40 cm and sent to the laboratory. total of 200 to 250 mosquitoes were entered in each cage and covered with wet towel. the sucrose 5 % solution was placed inside the cage. the mosquitoes were kept in standard condition (2 5˚c, 75% h). insecticide impregnated papers impregnated papers with ddt 4 %, malathion 5 %, propoxur 0.1 %, deltamethrin 0.05 %, lambda-cyhalothrin 0.05 %, cyfluthrin 0.15 %, and control papers were supplied by world health organization. adult susceptibility test the adult susceptibility test kit including holding and exposure tubes, slides, copper and silver rings and related covers were washed with detergent, and then washed three times with tap water to be free of detergents and insecticides. after drying of the pipe holders, standard paper impregnated with insecticides fixed inside the tubes marked with red dot and copper ring. for holding tube used paper control (no insecticide) tube with a green sign and silver pipe fixed to the wall (who 2006). each time 4– 5 mosquito collected and insert to holding tube overall 20–25 mosquito were kept into holding tube. the susceptibility tests performed on their standard condition (22–26 ˚c, 60% h). the susceptibility of the wild strain of culicidae mosquitoes was assessed to the insecticides impregnated papers. the mosquitoes were exposed to different insecticides by different interval times and 24 hours recovery period. smoking and use of pesticides during the test was strictly prohibited. who criteria for susceptibility test the following criteria have been used for interpretation and classification of results, based on who recommendations (african network on vector resistance 2005). a. susceptibility test carried out with less than 20 mosquitoes per test have not been considered b. at least 80 mosquitoes used per test, three resistance classes as defined. resistant when the mortality was lower than 80 %, susceptible when mortality was 98 % or higher and possible resistant or tolerant when mortality was between 97 % and 80 %. c. three resistance classes as defined when twenty to 79 mosquitoes used per test, sushttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 25 ceptible when mortality was 98 % or higher, resistance suspected, when mortality was between 95 % and 97 %, and resistant when the mortality was lower than 95 %. identification of mosquitoes using morphological characteristics the mosquitoes after the test were mounted and identified by specific systematic keys. the samples were recorded in the special forms by and the appropriate time of deaths associated with history of collection, relative humidity and temperature (shahgudian 1960, zaim and cranston 1986, azari-hamidian and harbach 2009). statistical analysis results were considered reliable if the control mortality was less than 5 % and rejected if more than 20 %. results were corrected by abbott's formula when mortality rates of control group were between 5 to 20 % (abbott 1925, who 2006). data were analyzed by probit analysis (finney 1971, 1978). regression lines of the species were measured through the χ2 test. the lt50 and lt90 were calculated for plotting the regression line using microsoft excel software ver. 2007. results totally, nine species were collected including: an. stephensi, an. dthali, an. culicifacies, an. fluviatilis, cx. pipiens, cx. quinquefasciatus, cx. theileri, cs. longiareolata, oc. caspius s.l (fig. 2). in adult collection an. stephensi was dominant species 34.76 % allocated mosquitoes collected. anopheles dthali and an. culicifacies were followed 15 %, 12.92 %, respectively. culiseta longiareolata had the lowest density with 1.09 %. an. culicifacies, an. stephensi, cx. pipiens, cx. theileri were collected in all months. in larval collection, an. stephensi, with 1495 specimens (28.9%) was predominant followed by cx. pipiens 753 (14.1%), an. culicifacies 12.8 %, cx. quinquefasciatus 6.3 % in the same month. it should be noted that an. fluviatilis larva was collected in may and december. mortality of culicidae mosquitoes exposed to ddt and other insecticides has shown in tables 1 and 2. lt50 and lt90 values of an. stephensi to ddt 4% were 29.70 and 98.26 minutes, respectively. this species was quite resistant to ddt while susceptible to deltamethrin and tolerant to other insecticides (fig. 3). it is concluded that an. culicifacies is tolerant to ddt, malathion, and propoxure whereas susceptible to deltamethrin, cyfluthrin and lambda-cyhalothrin (table 2). the lt50 and lt90 values of this species to ddt 4 % were 18.12 and 46.42 minutes (table 1, fig. 3). anopheles dthali was tolerant to ddt and propoxur whereas susceptible to deltamethrin, lamda-cyhalotrin, cyfluthrin and malathion (table 2, fig. 3). the lt50 and lt90 values to ddt 4 % were 17.86 and 48.42 minutes (table 1). it is concluded that cx. pipiens is quite resistance to ddt, propoxur, lambda-cyhalothrin, and cyfluthrin whereas susceptible to malathion (table 2, fig. 3). in our study oc. caspius found to be resistant to ddt whereas susceptible to other insecticides (table 2, fig. 3). the lt50 and lt90 values to ddt 4 % were 44.68, and 164.01 minutes (table 1). culiseta longiaerolata observed tolerant to ddt, propoxur, cyfuthrin, lambda-cyhalothrin whereas susceptible to deltamethrin (table 2, fig. 3). the lt50 and lt90 values to ddt 4 % were 17.82 and 51.26 minutes (table 1). http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 26 table 1. regression line parameters of various species exposed to ddt 4% in chabahar county, sistan and baluchistan province, southeastern iran, 2010 y=a+ bxp valuex2 (df)****lt90 95%c.i. (minute) ***lt50 95%c.i. (minute) **b±se*aspecies y= -8.0186+ 2.4666 x<0.0598.062(2)98.2629.702.4666±1.655-8.0186an. stephensi y= -9.5283+ 3.1379 x<0.05265.565(2)46.4218.123.1379±3.222-9.5283an. culicifacies y= -8.9653+ 2.9588 x<0.05397.042(2)48.4217.862.9588±3.713-8.9653an. dthali y= -7.3266+ 2.2321 x<0.0559.087(2)119.7231.932.2321±1.042-7.3266cx. pipiens y= -8.2580+ 2.3891 x<0.05409.134(2)164.0144.682.3891±3.687-8.2580oc. caspius y= -8.4636+ 2.7939 x<0.0541.551(2)51.2617.822.7939±1.086-8.4636cs. longeareolata *intercept. **b± se= slope and its standard error. ***lt50, 95 % ci= lethal time causing 50 % mortality and its 95 % confidence interval. ****lt90, 95 % ci= lethal time causing 90 % mortality and its 95 % confidence interval. fig. 1. location of study area in southeastern iran, 2010 fig. 2. the species composition of adult culicidae mosquitoes collected by different sampling methods in chabahar county, sistan and baluchistan province, southeastern iran, 2010 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 27 table 2. susceptibility level of various culicidae mosquitoes exposed to some insecticides in chabahar county, sistan and baluchistan province, southeastern iran, 2010 species insecticide *mr±er **resistance status an. culicifacies ddt 4% 96±2 t malathion 5% 95±2 t propoxur 0.1% 97±2 t deltamethrin 0.05% 100 s cyfluthrin 0.15 % 98±1 s lambda-cyhalothrin 0.05% 99±1 s an. dthali ddt 4% 97±2 t malathion 5% 98±1 s propoxur 0.1% 97±2 t deltamethrin 0.05% 100 s cyfluthrin 0.15 % 98±1 s lambda-cyhalothrin 0.05 % 99±1 s an. stephensi ddt 4% 67±5 r malathion 5% 90±3 t propoxur 0.1% 95±2 t deltamethrin 0.05% 100 s cyfluthrin 0.15 % 96±2 t lambda-cyhalothrin 0.05 % 96±2 t cx. pipiens ddt 4% 54±5 r malathion 5% 100 s propoxur 0.1% 20±4 r deltamethrin 0.05% 93±2 t cyfluthrin 0.15 % 72±4 r lambda-cyhalothrin 0.05 % 72±5 r cs. longiareolata ddt 4% 92±3 t malathion 5% propoxur 0.1% 92±3 t deltamethrin 0.05% 100 s cyfluthrin 0.15 % 88±3 t lambda-cyhalothrin 0.05 % 96±2 t oc. caspius ddt 4% 33±5 r malathion 5% 100 s propoxur 0.1% 100 s deltamethrin 0.05% 100 s cyfluthrin 0.15 % 100 s lambda-cyhalothrin 0.05 % 100 s *mortality rate ±error bar **r resistance, s susceptible, t tolerance http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 28 fig. 3. regression line of adult culicidae mosquitoes exposed to ddt 4 % in chabahar county, sistan and baluchistan province 2010 discussion in our study, 4 genera and 9 species of mosquito larvae and adults were identified based on morphological characters. culicidae species were belongs to the genus of anopheles, culex, culisita and ochleratatus. the species of cx. quinquefasciatus and cs. longiareolata found by larval collection. the most predominant species was an. stephensi with 34.76 % of adult and 29.36 % of larvae collection. vatandoost et al. (2004c) reported three biological forms of this species including type, intermediate and mysorensis in southern iran. type and intermediate forms cited as vector in urban areas whereas, mysorensis form as vector in rural area (vatandoost et al. 2006b). in iran, indoor residual spraying (irs) with ddt was carried out for malaria control during 1950–1968. in this species, resistance to ddt was first recognized in 1958 (mofidi et al. 1958, 1960, manuchehri et al. 1976a, 1976b) and subsequently to dieldrin in 19609 (mofidi and saminmi 1960) and matlathion in 1976 (eshghy 1978). following the emergence of resistance of anopheles stephensi to ddt, other organophosphours, carbamate and pyrethroid insecticides were used. the susceptibility level of an. stephensi to ddt and dieldrin was studied at various parts of iran bordered in persian gulf and oman sea during 1985–88. the results revealed that this species was resistant to ddt and it is quite susceptible to dieldrin (manouchehri et al. 1976). in a similar study in south of iran, the results showed that this species was resistant to ddt (eshghy and janbakhsh 1976). prior of this study, vatandoost et al. (2004) reported three biological forms of an. stephensi was susceptible to bendiocarb, propoxur, malathion, fenitrothion, deltamethrin, permethrin, cyfluthrin and lambda-cyhalothrin, resistance to ddt and tolerant to dieldrin in hormozgan province, south of iran. the same study was carried out on an. stephensi of bandar abbas strain, showed a relatively high adult resistance of http://jad.tums.ac.ir published online: july 16, 2014 an. stephensi an. culicifacies an. dthali cx. pipiens time cs. longeareolata oc. caspius p r o b it j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 29 4.66-fold (lt50= 108min) to 4 % ddt (davari et al. 2007). in our study, the lt50 values of ddt 4.0 % against this species were 29.7 minutes, a manifestation of ddt resistance. some investigations indicated an. stephensi, the main malaria vector, resistant to pyrethroid insecticides in southeastern iran (vatandoost and hanafi-bojd 2012). anopheles culicifacies comprised of five sibling species of a, b, c, d and e in north and south of india reported as a and b, in north east found d associated sympatric with a and b whereas in central area reported all of sibling species (goswami et al. 2006). polytene chromosome examination has been reported only available method that able to differentiate four members of this complex in areas where species e is not prevalent (goswami et al. 2006). species a has been reported in oman and iran (zaim and javaherian 1991). this species is known as vector of malaria in east afghanistan (zahar 1990). in our study, an. culicifacies was tolerant to ddt, malathion and propoxur while susceptible to other insecticides. in contrast, ddt resistance reported for the first time after residual spraying in iran by vatandoost et al. (2011). they reported susceptibility of an. culicifacies to pyrethroid and irritability to fenitrothion, cyfluthrin, and permethrin while high extremely resistance to ddt. there are several reports revealing ddt resistance of an. culicifacies in afghanistan, and also resistance to dieldrin, malathion and ddt in india (goswami et al. 2006). in our study, an. dthali found tolerant to ddt and propoxur while susceptible to deltamethrin, lambda-cyhalothrin, cyfluthrin and malathion. in contrast, there are also some reports emphasizing to susceptibility levels of this species in different countries (who 1992, hanafi-bojd et al. 2007). in spite, the existence of many reports about to susceptibility of an. dthali to insecticides, resistance reported to malathion, chloropyriphos, bromophos, carbamate in egypt and temephos in jordan (who 1992). in our research, cx. pipiens larvae with 1073 (51.14%) collection, was found one of the most predominant species. in adult collection, cx. pipiens catches 12.14 % and cx. quinquefasciatus found as 4.82 % of total collection gjullin and peters (1952) cited decrease of susceptibility level of cx. pipiens complex to ddt and organophosphorus insecticide. first report related to resistance of the cx. quinquefasciatus to organophosphorus insecticide was cited by isaak in 1961 (isaak 1961). increasing in the resistance level of cx. pipiens to organophosphorus insecticides reported by toma et al. (2011). in our study, cx. pipiens was quite resistance to ddt, propoxur, lambda-cyhalothrin, and cyfluthrin, whereas tolerant to deltamethrin and susceptible to malathion. in parallel, there are many reports in relation to some organophosphorus and pyrethroid insecticides resistance in tunisia (ben cheikh et al. 1998), cuba (bisset et al. 1991), burkina faso (chandre et al. 1998), saudi arabia (amin and hemingway 1998) and china (jinfu 1999). another study showed, pyrethroid insecticides had tolerance of cx. pipiens pipiens var molestus in north america (mcabee et al. 2003). in contrast, ddt resistance of cx. pipiens complex reported in southern tehran (nazari and janbakhsh 2000). lt50 value for propoxur and malathion calculated as 51, and 31 minutes respectively (nazari and janbakhsh 2000). the species reported quit susceptible to dieldrin, propoxur and malathion (nazari and janbakhsh 2000). in our study, cx. pipiens was highly resistance to ddt, propoxur, cyfluthrin and lambda-cyhalothrin as evidenced by the discriminating concentrations of times. the results may be related to the present of the species complex, application of irs and distribution of llins in southern iran. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 22–34 m fathian et al.: susceptibility of culicidae … 30 in the present study, oc. caspius found to be quite resistance to ddt whereas susceptible to other insecticide. the lt50 and lt90 values to ddt 4 % were 47.68 and 164.01 minutes. in addition, our study showed cs. longiaerolata quite resistance to ddt and susceptible to other insecticides the lt50 and lt90 values to ddt 4 % were 17.82 and 50.26 minutes. based on the literature, no reports were available on the susceptibility levels of oc. caspius and cs. longiaerolata to who recommended insectidides in the world. conclusion iran is in the malaria elimination stage (who 2011). by now, irs in human dwellings and animal shelters, space-spraying, personal protection through distribution of llins and curtains (icns), repellents measures used to control of malaria vectors in iran. in addition, some biological and chemical agents against larval and adult stages of culicidae mosquitoes had been evaluated in the laboratory. results obtained from susceptibility tests of the malaria vectors on ddt revealed that only an. stephensi was highly resistance to ddt in chabahar area, precautionary measures should be taken in future vector control operations. moreover, the status of resistance in other locations in malaria endemic area should be investigated. since the country relies on deltamethrin for irs operation, tolerant populations of cx. pipiens implies careful consideration and regular monitoring of susceptibility level of mosquitoes in the future. acknowledgement the authors are grateful to dr. nazari head of chabahar health center for coop-oration during the study. the authors thank to dr hanafi-bojd, school of public health, tehran university of medical sciences, for reviewing the manuscript. we express thanks to mr gorgij, mr jadgal, mr amiri, mr lalo field technician of chabahar health center. this study was financially supported by grant of tehran university of medical sciences (id: no: 10513-27-01-89). references abbott ws (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–267. african network on vector resistance (2005) world health organization african region. atlas of insecticide resistance in malaria vectors of the who african region. harare, zimbabwe amin am, hemingway j (1998) preliminary investigation of the mechanisms of ddt and pyrethroid resistance in culex quinquefasciatus say (diptera: culicidae) from saudi arabia. bull entomol res. 79: 361–366. azari-hamidian s, harbach re (2009) keys to the adult females and fourth-instar larvae of the mosquitoes of iran (diptera: culicidae) zootaxa. 2078: 1–33. barr ar, kabtman l (1951) biometrical 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assoc. 7(2): 324–326. zaim m, zahirnia ah, manouchehri av, zaim m, zahirnia ah, manouchehri av (1993) survival rates of anopheles culicifacies s.l and anopheles pulcherrimus in sprayed and unsprayed villages' in ghassreghand district, baluchistan, iran. j am mosq cont assoc. 9: 421–425. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 8 http://jad.tums.ac.ir published online: march 31, 2020 original article molecular identification of leishmania species in phlebotomus alexandri (diptera: psychodidae) in western iran abdollah naghian1,2; mohammad ali oshaghi1; vahideh moein-vaziri3; yavar rassi1; mohammad mehdi sedaghat1; ehsan mostafavi4; arshad veysi5; hassan soleimani6; hossein dehghan7; alireza zahraei-ramazani1; hossein mirhendi8; mohammad hassan amini1; *mohammad reza yaghoobi-ershadi1; *amir ahmad akhavan1 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 department of medical entomology and vector control, school of public health, urmyia university of medical sciences, urmyia, iran 3 department of medical parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 4 department of epidemiology, pasteur institute of iran, tehran, iran 5 zoonoses research center, research institute for health development, kurdistan university of medical sciences, sanandaj, iran 6 yazd health research station, national institute of health research, yazd, iran 7 department of public health, school of public health, jiroft university of medical sciences, jiroft, iran 8 department of medical parasitology and mycology, school of medicine, isfahan university of medical sciences, esfahan, iran (received 06 aug 2018; accepted 05 dec 2018) abstract background: visceral and cutaneous leishmaniasis are common in some areas of iran and consider as health problems. phlebotomus alexandri has been incriminated as a suspected vector for the both form of leishmaniasis. methods: this study was carried out in 4 western provinces of iran. sand flies were collected using sticky traps and light traps from indoor and outdoor resting places. nested pcr was employed to detect leishmania parasites among collected sand flies. results: seven hundred and twenty two p. alexandri females were collected and pooled in 179 batches. results of nested pcr showed, out of 9 samples from east azerbaijan province, only one sample was infected by leishmania infantum. of 34 individual and pooled samples from kermanshah province, only one pooled sample was infected with leishmania major and among 30 individual and pooled samples in fars province, five specimens were infected by l. major, l. infantum, leishmania donovani and leishmania tropica. furthermore, out of 108 individual and pooled samples from khuzestan province, 10 samples showed infection with l. major and l. infantum. conclusion: the results of this study showed that p. alexandri is more active in hot zones than in moderate zones and this species may be considered as a permissive species. keywords: leishmaniasis; phlebotomus alexandri; leishmania; iran introduction leishmaniasis is a parasitic infectious disease that is transmitted by the bite of infected phlebotominae sandflies, and it is grouped in the seventeen neglected tropical diseases (ntd) (1) which has been recorded from 98 countries (2). there are three main forms of disease in the world including visceral leishmaniasis (vl), cutaneous leishmaniasis (cl) and mucocutaneous leishmaniasis (mcl) (3). so far two types of the disease including cl and vl, has been reported in iran (4, 5). cutaneous leishmaniasis is the most common form of the disease in the *corresponding authors: prof mohammad reza yaghoobi-ershadi, email: yaghoobia@gmail.com, dr amir ahmad akhavan, email: aaakhavan@tums.ac.ir http://jad.tums.ac.ir/ mailto:yaghoobia@gmail.com mailto:aaakhavan@tums.ac.ir j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 9 http://jad.tums.ac.ir published online: march 31, 2020 world as well as in iran (5, 6). although the new vl foci have increased remarkably, zoonotic visceral leishmaniasis (zvl) incidence has decreased recently in the country (ministry of health and medical education (mohme)), 2015, unpublished data). there are about 1000 sand fly species and subspecies reported in the world (7), of these only about 93 species transmit 20 leishmania species to humans (2). till now, 29 species and 2 subspecies of phlebotomus and 19 species of sergentomyia sand flies were reported in iran (8). six sand fly species including phlebotomus salehi, phlebotomus mongolensis, phlebotomus alexandri, phlebotomus andrejevi, phlebotomus caucasicus and phlebotomus ansarii known as suspected vectors and phlebotomus papatasi known as the main proven vector of zoonotic cutaneous leishmaniasis (zcl) due to leishmania major in the country (4). moreover, anthroponotic cutaneous leishmaniasis (acl) due to leishmania tropica occurs in 14 foci of 7 provinces in iran. four species of the subgenus larrussious and p. alexandri of the subgenus paraphlebotomus were incriminated as probable vectors of zvl due to leishmania infantum in seven new and old foci of the country (4, 5). phlebotomus alexandri reported as a proven vector for zvl (2) and anthroponotic visceral leishmaniasis (avl) in china (9). furthermore, it is incriminated as the probable vector of avl and zvl in iraq, oman, mongolia, turkey and china (2, 10). leishmania infection rate for p. alexandri reported as 1.74% in khuzestan endemic regions (11). it was also found infected by l. infantum (12) and l. major (13) using molecular methods in fars province in south west and sarakhs in north eastern of iran respectively. furthermore, p. alexandri was found infected in turkemanistan (14) and it is suspected to transmission of leishmania killicki in tunisia (15, 16). considering the studies have been conducted so far in iran and other countries, it seems that more studies are needed to clarify the role of p. alexandri in different leishmania parasite transmission cycles. the current study was designed and conducted in four western endemic and non-endemic provinces of iran. materials and methods study area this study was carried out in 4 western and south western provinces of iran including east azerbaijan, fars, kermanshah and khuzestan from 2011 to 2012. in eastern azerbaijan province (36˚, 45’ to 39˚, 26’ n and 45˚ 5’ to 48˚ 21’ e) in northwest of iran, maximum and minimum temperature were +45 ˚c in julfa and -25 ˚c in bostanabad respectively and the rainfall varied between 196 to 563mm. in kermanshah province (33˚, 41’ to 35˚, 17’ n and 45˚ 24’ to 48˚ 6’ e) in the west of iran, maximum and minimum temperature were +50.4 ˚c in soomar and -17.4 ˚c in sonqur respectively and the rainfall varied between 273 to 573mm. in fars province (27˚, 3’ to 31˚, 40’ n and 50˚ 36’ to 55˚ 35’ e) in south west of iran, maximum and minimum of temperature were +49.8 ˚c in lamard and -14.6 ˚c in safashahr respectively and the rainfall varied between 83 to 1007mm. in khuzestan province (29˚, 53’ to 33˚, 0’ n and 47˚ 40’ to 50˚ 33’e ) in south west of iran, maximum and minimum of recorded temperature in this province were +52.6 ˚c in shushtar and 5.6 ˚c in dehdaz respectively and the rainfall varied between 73 to 702mm (17). sand fly collection and identification sand flies were collected using light traps and sticky traps during active seasons of sand flies in 127 places and 599 places in 2011, respectively (fig. 1). collected sand flies were conserved in 96% alcohol, kept in 4 ˚c refrigerator. the head and the last two segments of sand flies were detached on the slide in sterile condition and mounted in puris medium. the rest of the bodies including abdomen and thorax were transferred into a sterile 1.5ml micro tube and were kept in -20 ˚c until use. the specimens were identified using valid morphological keys (18, 19). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 10 http://jad.tums.ac.ir published online: march 31, 2020 molecular experiments genomic dna was extracted and purified using a geneall kit (exgene™ tissue sv mini) according to the protocol of the kit. pcr conditions polymerase chain reaction amplification has done in an applied biosystems thermocycler. the first step of nested-pcr contained 0.6μm of each forward (aaactcctctct ggtg-cttgc; leish out f) and reverse (aaa caaaggttgtcggggg; leish out r) external primers (20), 12.5μl taq dna polymerase, 2x master mix red (amplicon, denmark) and sterile distilled water to a final volume of 25 μl. first denaturation step at 95 °c for 5min was continued by 30 cycles of denaturation at 94 °c for 30s, annealing at 60 °c for 45s and extension at 72 °c for 1min, with a final extension step of 72 °c for 5min. the second step of nested-pcr was performed in a final volume of 20μl containing 1μl of a 1:10 dilution in distilled water of the first-round pcr product as template, 0.3μm of each forward (aatt caa cttcgcgtt ggcc; leish in f) and reverse (cctctc ttttttctc-tgtgc; leish in r) (20), 10μl of taq dna polymerase and 2x master mix red. the second round pcr was cycled under the following conditions: 95 °c for 2min, 25 cycles of 94 °c for 15s, 62 °c for 30s, 72 °c for 45s followed by 72 °c for 5min. pcr products were electrophoresed on 1.5% (w/v) agarose gel in tbe buffer (0.09 mm tris, 0.09mm boric acid and 20mm edta, ph 8.3), visualized with safe stain (0.5μg/ml) and photographed. reference strains of l. infantum, l. major and l. tropica were used as positive controls and distilled water was used as negative control accordingly. the pcr product of the negative control of the first step of pcr was used as the negative control in the second step and the pcr product of the positive control of the first step pcr was used as positive control in the second step as well. to avoid cross-contamination, necessary precautions were taken. the products of nested pcr were sent to corresponding company for nucleotide sequencing. results sand flies were collected using 207 light traps and 7633 sticky traps during active seasons of sand flies in the studied areas. totally, 722 female p. alexandri were collected in 4 studied provinces including 9 from east azerbaijan, 114 from fars, 90 from kermanshah, and 509 specimens from khuzestan. collected sand flies were pooled in 179 batches according to five parameters including: locality (counties of provinces), places of catch (indoor or outdoor), type of trap (light trap or sticky trap), topography (mountains, foothills or plains) and physiological status (unfed, blood-fed, semi-gravid and gravid) of the sand flies. the locations where p. alexandri sand flies were caught and their leishmania infection are shown in (fig. 1). in kermanshah province, of 34 pooled samples, only one was infected by l. major (fig. 2). this sample was contained 13 unfed female p. alexandri sand flies, which collected from 4 villages (kalashi lulem, kalashi bakhan, mezran and melah rash) of javanrud county (table 1). in east azerbaijan, collected p. alexandri sand flies were pooled in 9 samples. among these samples only one was infected by l. infantum (fig. 1, table 1). the infected sample was contained 1 unfed female sand fly that was collected in a mountainous cave near the village of ghyzil gaya in ahar county. in fars province, collected p. alexandri sand flies were pooled in 31 batches. of these 5 samples were infected by leishmania parasites (fig. 1, table 1). two samples were infected by l. infantum. one of the infected sample by l. infantum was contained 3 blood-fed p. alexandri female which were collected in 2 villages (shorab-chamgole, and pirehsorkh) of mamasani county. one of the infected sample was infected by l. donovani contained an unfed p. alexandri female that was collected in a cave located in nur abad city. the other one showed a mixed http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 11 http://jad.tums.ac.ir published online: march 31, 2020 infection of l. infantum and l. tropica, contained an unfed p. alexandri female which was collected in pireh sorkh village of mamasani. two samples were found to be infected by l. major, one of them was collected in khumeh zar village of mamasani county that was a pooled of 14 unfed female sand flies and another was a mixed infection of l. major and l. tropica contained an unfed female that was collected in shir espari village of mamasani county. in khuzestan province, all collected p. alexandri female sand flies were pooled in 108 samples, of these 10 samples were infected by leishmania parasites and all were unfed. results showed, in seyyed taher village that located in plain area in north of ahvaz county, three samples were infected by l. major, and three samples were infected by l. infantum (fig. 1, table 1). furthermore, another sample was infected with l. infantum in sar dasht (kohe gandom) village in mountainous area in north of dezful county (fig. 1). apart from these, a sample contained one p. alexandri sand fly had infection of l. infantum was collected in dehenow bagher village located in plain area in south of dezful county, (fig. 1, table 1). moreover, a sample contained a sand fly that was infected with l. major collected in magtu vil lage in the south of ahvaz county, (fig. 1, table 1). the geographical and epidemiological parameters of pooled samples are shown in table 1. fig. 1. the map of leishmania infection of phlebotomus alexandri in the studied areas, iran, 2011 and 2012 table 1. the geographical and epidemiological parameters of positive pooled samples of phlebotomus alexandri in the studied regions geographical status province place pooled samples code leishmania species no. sand flies in sample plain khuzestan indoor 108 l. infantum 1 indoor 98.3 l. infantum 23 outdoor 119 l. infantum 2 indoor 97.3 l. major 16 indoor 89.2 l. major 18 indoor 89.1 l. major 4 outdoor 152 l. infantum 1 outdoor 116 l. major 1 foothills fars outdoor 24.2 l. major 14 indoor 3 l. infantum 3 outdoor 19 l. major and l. tropica 1 indoor 2m l. infantum and l. tropica 1 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 12 http://jad.tums.ac.ir published online: march 31, 2020 mountain khuzestan indoor 122 l. infantum 1 fars outdoor 28 l. donovani 1 kermanshah outdoor 41 l. major 13 east azerbaijan outdoor 65.1 l. infantum 1 fig. 2. the 1.5% agarose gel electrophoresis of nested pcr products in khuzestan and kermanshah provinces samples. lanes 1 and 17 are 50bp ladder; lane 2negative control; lane 3leishmania infantum positive control; lane 4 leishmania major positive control; lane 5leishmania tropica positive control; lane 6–15samples of khuzestan province; lane 16 sample from kermanshah province fig. 3. the gel electrophorus image of nested pcr results in fars and eastern azerbaijan provinces’ samples. lene 1 and 12 are 50bp ladder; lane 2 is negative control; lane 3, 4 and 5 are positive control of leishmania infantum, leishmania major and leishmania tropica, respectively; lane 6, 7, 9 and 11 samples from fars, lane 8 a sample from east azerbaijan table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 13 http://jad.tums.ac.ir published online: march 31, 2020 discussion the result of this study showed that, p. alexandri harbored different species of leishmania parasites in the endemic and non-endemic areas. sand flies infection by l. major from javanrud county villages in kermanshah province showed, despite there is no cl cases in these areas, it is postulated that, there is an enzootic foci in this region which can pose a risk to extend zcl to people in the future. this is the first report of p. alexandri sand fly infection due to l. major in javnrud county in kermanshah province. this region is located near qasr-e shirin and sare pole zahab counties, where the previous studies reported zcl cases among endogenous people (21). in east azerbaijan, the study regions are located near meshkin shahr city in the ardabil province where has been known as the first main endemic foci of zvl in iran (4). the infection of p. alexandri due to l. infantum has been reported in fars province as well (12). recently, phlebotomus tobbi was found infected with l. infantum in east azerbaijan (22). in northwest of iran, p. kandelakii (23) and p. perfiliewei (24) were reported infected by l. infantum as well. here, p. alexandri has been introduced as the fourth suspected zvl vector in the north west of iran. since the infected p. alexandri with l. infantum was collected in a fox nest in the border of karamlu and khalifan villages of ahar county, it implies that foxes can play a role in enzootic cycle of parasite in this region. the results of our studies in fars province showed that, the infection of p. alexandri with leishmania parasites is higher comparing to other provinces. this province known as acl and zvl foci in the country (4). according to recent report of the mohme, the occurrence of cl in this province is very high. leishmania major infection in p. alexandri is reported for the first time in mamasani county in fars province, although l. infantum infection in p. alexandri has been reported in this area previously (12). since the occurring mixed infection in p. alexandri, this species might play role as a permissive vector which supports multiple leishmania parasite inside the digestive tract. additionally, it is the first report of p. alexandri infection with l. donovani in nurabad city as well. in the current study p. alexandri found infected with l. infantum in chamgol and pireh sorkh villages of mamasani county for the first time in fars province. in khuzestan province, p. alexanderi was found infected with 2 leishmania parasites including, l. major and l. infantum. on the other hand, in east azerbaijan, p. alexandri was found infected with l. infantum in a fox nest as well. in contrast to the results of previous studies and also current study in other regions, p. alexandri mostly collected in the plain areas in khuzestan province. according to the mohme internal reports in 2015, fars and khuzestan provinces had the first and second incidences of cl in iran respectively, but kermanshah province had the ninth incidence at the same time. also these reports state that fars and east azerbaijan provinces had the first and second incidence of zvl in iran respectively, but khuzestan had just 1 case in the year 2015. microscopic leishmania infection of p. alexandri in shush and ahvaz counties was reported 4 decades ago, but was not detected by molecular techniques and did not show any successful inoculation in sensitive animals (25). in agreement with previous work, our study introduced p. alexandri as a probable vector of zcl and zvl in iran. in the current study the majority of infected p. alexandri sand flies was unfed and it reflects that, they are capable to transmit the parasite to the new host through next blood feeding. furthermore, the finding highlights that p. alexandri mostly occurred in hot regions (khuzestan) rather than cold regions (other studied provinces). this is the first report of l. infantum and l. major infections in p. alexandri in khuzestan province, and is the first report of infection to l. tropica by p. alexandri using molecular technique in the world. in khuzestan province, the occurrence of cl is common ac http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 14 http://jad.tums.ac.ir published online: march 31, 2020 cording to the mohme reports, but only 1 case was reported in this province for zvl in 2015 (26). on the other hand, according to who and some other studies, in eastern provinces of iraq zvl is endemic (27-29). the 90% of zvl cases in iraq occur among less than 5 years old children. the number of reported cases were 3900 in iraq in 1992 and increased to 1050 cases in 2012 (28). conclusion the results of this study showed, p. alexandri could harbor infection of 4 leishmania species (l. major, l. tropica, l. donovani and l. infantum) so that, it supports the hypothesis of permissiveness of this species. furthermore, the reporting of natural infection of p. alexanderi in some presumably free areas of zcl, e.g. in kermanshah, revealed the cycling of parasite among its host and sand flies vector at least in enzootic cycle. acknowledgements this study was financially supported by school of public health, tehran university of medical sciences. our sincere thanks go to mrs kazeruni, mr ghazizadeh, mr kamali, mr alizadeh for their kind collaboration and assistance in the sand flies collection. we are very grateful to prof homa hajjaran, department of parasitology and mycology, school of public health, tehran university of medical sciences (tums), mr motazakker, cellular and molecular biology research center of urmyia university of medical sciences (ums), mrs jalali and mr jafari, the staff of esfahan training and health research center, national institute of health research, tehran university of medical sciences (tums), esfahan, iran for their kind collaboration during this study. the authors declare that there is no conflict of interest. references 1. who (2015) investing to overcome the global impact of neglected tropical diseases: third who report on neglected tropical diseases. world health organization, geneva, p. 211. 2. who (2010) control of the leishmaniasis. who technical report sereis 949. report of the 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leishmaniasis in khuzestan, iran. part i. the leptomonad infection of sandflies. bull soc pathol exot filiales. 67(5): 513–516. 26. iran ministry of health and medical edu http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=killick%e2%80%90kendrick+r+(1990)+phlebotomine+vectors+of+the+leishmaniases https://www.ncbi.nlm.nih.gov/pubmed/?term=phlebotomus+(paraphlebotomus)+alexandri%3a+a+probable+vector+of+leishmania+infantum+in+iran.++annals+of+tropical+medicine+and+parasitology https://www.ncbi.nlm.nih.gov/pubmed/?term=molecular+detection+of+leishmania+infection+in+sand+flies+in+border+line+of+iran%e2%80%93turkmenistan%3a+restricted+and+permissive+vectors http://www.irimo.ir/far/wd/2703 https://www.ncbi.nlm.nih.gov/pubmed/?term=javadian%20e%5bauthor%5d&cauthor=true&cauthor_uid=22808407 https://www.ncbi.nlm.nih.gov/pubmed/?term=molecular+detection+of+leishmania+infantum+in+naturally+infected+phlebotomus+perfiliewi+transcaucasicus+in+bilesavar+district%2c+northwestern+iran https://www.ncbi.nlm.nih.gov/pubmed/?term=molecular+detection+of+leishmania+infantum+in+naturally+infected+phlebotomus+perfiliewi+transcaucasicus+in+bilesavar+district%2c+northwestern+iran https://www.ncbi.nlm.nih.gov/pubmed/?term=studies+on+cutaneous+leishmaniasis+in+khuzestan%2c+iran.+part+i.+the+leptomonad+infection+of+sandflies j arthropod-borne dis, march 2020, 14(1): 8–16 a naghian et al.: molecular identification of … 16 http://jad.tums.ac.ir published online: march 31, 2020 cation (2016) infectious disease management, annual meeting of the zoonosis group in sarein, ardabil, iran. 27. who (2003) communicable disease profile iraq. working group on emergencies, hq division of communicable disease control, emro, who office, baghdad. who office, baghdad. communicable disease toolkit, iraq crisis, p. 39–44. 28. salam n, al-shaqha wm, azzi a (2014) leishmaniasis in the middle east: incidence and epidemiology. plos negl trop dis. 8 (10): e3208. 29. rahi aa, ali ma, valian hk, mohebali m, khamesipour a (2013) seroepidemiological studies of visceral leishmaniasis in iraq. sch j app med sci. 1(6): 985– 989. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=salam+n%2c+al-shaqha+wm%2c+azzi+a+(2014)+leishmaniasis+in+the+middle+east%3a+incidence+and+epidemiology. https://www.ncbi.nlm.nih.gov/pubmed/?term=salam+n%2c+al-shaqha+wm%2c+azzi+a+(2014)+leishmaniasis+in+the+middle+east%3a+incidence+and+epidemiology. j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 56 http://jad.tums.ac.ir published online: march 31, 2020 original article impact of environmental and climate factors on spatial distribution of cutaneous leishmaniasis in northeastern iran: utilizing remote sensing mohammad reza shirzadi1; mohammad javanbakht2; hassan vatandoost3,4; nahid jesri5; *abedin saghafipour6; reza fouladi-fard7; alireza omidi-oskouei6 1 communicable diseases management center, ministry of health and medical education, tehran, iran 2 department of remote sensing and gis, faculty of geography, tehran university, tehran, iran 3 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4 department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 5 remote sensing and gis centre, shahid beheshti university, tehran, iran 6 department of public health, faculty of health, qom university of medical sciences, qom, iran 7 research center for environmental pollutants, qom university of medical sciences, qom, iran (received 24 jun 2018; accepted 10 jun 2019) abstract background: cutaneous leishmaniasis (cl) is a dermal and parasitic disease.. the aim of this study was to determine the effect of environmental and climate factors on spatial distribution of cl in northeastern iran by utilizing remote sensing from 20 march 2016 to 19 march 2017. methods: in this ecological study, the data were divided into two parts: the descriptive data on human cl cases were gathered from communicable diseases center of iran. the remote sensing techniques and satellite imagery data (trmm, modis-aqua, modis-terra and amsr-2 with spatial resolution 0.25°, 0.05°, 5600m and 10km) of environmental and climate factors were used to determine the spatial pattern changes of cutaneous leishmaniasis incidence. results: the incidence of cl in north khorasan, razavi khorasan, and south khorasan was 35.80 per 100,000 people (309/863092), 34.14 per 100,000 people (2197/6,434,501) and 7.67 per 100,000 people (59/768,898), respectively. the incidence of cl had the highest correlation with soil moisture and evapotranspiration. moreover, the incidence of disease was significantly correlated with normalized difference vegetation index (ndvi) and air humidity while it had the lowest correlation with rainfall. furthermore, the cl incidence had an indirect correlation relation with the air temperature meaning that with an increase in the temperature, the incidence of disease decreased. conclusion: as such, the incidence of disease was also higher in the northern regions; most areas of north khorasan and northern regions of razavi khorasan; where the rainfall, vegetation, specific humidity, evapotranspiration, and soil moisture was higher than the southern areas. keywords: cutaneous leishmaniasis; remote sensing; climate; iran introduction cutaneous leishmaniasis (cl) is considered a dermal and protozoan infectious disease which is caused by some species of leishmania parasite (1). the disease is more prevalent in many underdeveloped and developing countries (2). cutaneous leishmaniasis is classified into two forms in terms of clinical symptoms and epidemiological characteristics: zoonotic cutaneous leishmaniasis (zcl) or cutaneous leishmaniasis due to leishmania major (clm) and an throponotic cutaneous leishmaniasis (acl) or cutaneous leishmaniasis due to leishmania tropica (clt). the vectors of cl are some species of female sand flies belonging to the genus phlebotomus such as ph. papatasi and ph. sergenti. besides, reservoirs of disease are some wild rodents, domestic dogs and humans in zcl and acl, respectively (3). the globally recognized foci of the disease are located approximately between 28–42 degrees of north latitude (4). *corresponding author: dr abedin saghafipour, e-mail: abed.saghafi@yahoo.com http://jad.tums.ac.ir/ mailto:abed.saghafi@yahoo.com j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 57 http://jad.tums.ac.ir published online: march 31, 2020 cutaneous leishmaniasis is an endemic disease in tropical areas of the united states, africa, the indian subcontinent, and in the subtropical regions of southwest asia (5). according to the who reports, cl has local transmission in 98 countries and over 350 million people are globally at risk (5, 6). more than 20,000 cases of cl are reported annually in iran, but the actual cases are estimated to be 4 to 5 times more than that figure (7, 8). iran's geographic and climatic conditions are suitable for the growth of sand flies as vectors of zcl, and rodents as reservoirs of disease (3, 9). this zoonotic disease is more prevalent in plain regions in central areas of iran; such as qom province (10) and in the old texture of the towns (11, 12). nowadays, zcl has endemic foci in 17 provinces of 31 provinces in iran, such as the three studied provinces (north khorasan, khorasan razavi and south khorasan) (13-15). investigating the correlation between climatic factors and incidence of the arthropod born diseases as applied epidemiologic studies can help epidemiologists in prevention and disease control, ultimately leading to taking preventive measures in vulnerable areas (16). in this regard, salmon et al. (2012) studied the climate change and the prevalence of leishmaniasis in argentina. the findings of this study indicated that the prevalence of this disease has increased with nonlinear climate changes (17). chamaillé et al. (2010) revealed that the disease prevalence is high in two regions; one region with low winter temperatures, 1042mm average annual rainfall and much forest cover and second area with higher temperatures, lower rainfall and less forest cover. they declared two region groups probably responsible to the environment factors favored by the two sand fly vectors species; ph. ariasi and ph. perniciosus respectively (18). in addition, gonzalez et al. studied the relationship between climate change and risk of leishmaniasis in north america. the results of the study showed that climate changes and increase in temperature provide a natural environment and suitable breeding places for living vectors of this disease (19). in 2012, tommy et al. conducted a standard multiple regression study to investigate the effect of climate factors on zcl incidence in central tanzania and showed that in case of rising rainfall as 1mm and a 1% increase in humidity from july to september, lead to an increase in incidence of the disease (20). ghatee et al. in a study on role of environmental and climatic risk factors on the occurrence of cutaneous leishmaniasis, found that some environmental and climatic factors including land cover, slope, elevation, close proximity to cattle sheds, rainfall, and temperature were the most effective factors (21). in addition, the iranian medical entomologists also have studied on impact of environmental and climate factors on spatial distribution of cl using remote sensing (22, 23). following the launch of the first meteorological satellites in the 1960s, and landsat-1 in 1972, the use of remote sensing in human health studies have increased for more than four decades. the information received from satellites and their various sensors with the ability to separate temporal and spatial distribution of data has created a new field in the scientific researches on human health. several ecological indicators have been used in remote sensing related to vector borne diseases, including normalized difference vegetation index (ndvi), near surface air and sea temperatures, soil moisture and cold cloudy periods. the term ‘remote sensing’ (rs) is a technique for diagnosis of objects from afar (24). so, this study aimed for determining the impact of environmental and climatic factors on spatial distribution of cl in northeastern iran from march 2016 to march 2017 utilizing remote sensing. materials and methods study area one of the main and confirmed foci of cl in iran is the northeastern areas of the country (8). therefore, the three provinces of north khorasan (55°53′–58°20′ e and, 36° 37′-38°17′ n), razavi khorasan (35° 60' n–59° 15' e and, 36° 35' n–60° 25' e) and south khorasan (57° 46´– http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 58 http://jad.tums.ac.ir published online: march 31, 2020 60° 57´ e and 30° 35 -́34° 14´ n) were selected as the study area of this research (fig. 1). according to the general census of population and housing in 2017, the totally residents in the study area was 8,066,491 people (north khorasan, razavi khorasan and south khorasan provinces had a population of 863092, 6434501 and 768898, respectively). these three provinces have an area of 28311, 118854, and 148669km2, respectively (north khorasan, razavi khorasan and south khorasan provinces). study design the data used in this study included two parts: the field data related to the incidence of disease and remote sensing data in a one-year period (from 20 march 2016 to 19 march 2017). estimation of various climatic components in large spatial scales has always been a frequent problem due to lack of availability of appropriate data. in recent years, a global database based on satellite estimates has reduced these problems. in this research, various products of the modus sensor and the global land data assimilation system (gldas) were used. the gldas aims to produce hydrological and meteorological parameters using the combination of ground-level models and satellite imagery with the aim of studying the changes in the energy and water bill on the ground. satellite imagery data (trmm, modis–aqua, modis– terra and amsr-2 with spatial resolution 0.25°, 0.05°, 5600m and 10km) of environmental and climate factors were used to determine the spatial pattern changes of cutaneous leishmaniasis incidence. this system was developed jointly by the national aeronautics and space administration (nasa), the goddard space flight center (gsfc), the national oceanic and atmospheric administration (noaa), and the national centers for environmental prediction (ncep) (25). the parameters extracted from gldas included evapotranspiration, near surface temperature, rainfall, soil moisture and air humidity (table 1). the gldas data has been used and evaluated in many scientific studies (26-28). furthermore, the normalized difference vegetation index (ndvi) and modis data were used in order to study variations in vegetation. in addition, mean of climatic and environmental variables and cutaneous leishmaniasis incidence was evaluated (table 2). field data on cl disease is routinely collected from urban and rural health centers in these three provinces using standard epidemiological forms and reported to the center for disease control and prevention of the ministry of health and medical education of iran. in order to investigate the spatial changes of the disease incidence, as well as the relationship between climatic and environmental factors and the incidence of cl, one-year data of this disease were taken from the cdc. statistical analysis the data related to cl disease were entered into excel software and the disease incidence was calculated. also, pearson correlation coefficient was used to determine the relationship between climatic and environmental factors and cl. the correlation coefficient is a value between -1.0 and 1.0, positive correlation implies the presence of a direct relationship between two variables in which, when one variable increases as the other increases. as well as, negative correlation means there is a reverse change between the two variables. the zero correlation, however, means that the variables are independent of each other. correlation is significant at the 0.05 level. results the incidence of cl in all three provinces was 25.66 per 100,000 people (2565/8,066,491). also, cl incidence in north khorasan, razavi khorasan, and south khorasan was 35.80 per 100,000 people (309/863092), 34.14 per 100,000 people (2197/6,434,501) and 7.67 per 100,000 people (59/768,898), respectively (table 2 and fig. 2). spatial distribution of cl showed that the northern regions had a higher incidence than the southern ones. therefore, the http://jad.tums.ac.ir/ https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjn_8jp49nxahwebvakhe7xaguqfggkmaa&url=https%3a%2f%2fwww.cdc.gov%2f&usg=aovvaw347qtt6pi6cdnkfm8vfdw6 https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjn_8jp49nxahwebvakhe7xaguqfggkmaa&url=https%3a%2f%2fwww.cdc.gov%2f&usg=aovvaw347qtt6pi6cdnkfm8vfdw6 j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 59 http://jad.tums.ac.ir published online: march 31, 2020 disease had a north to south trend which tended to increase from south to the north. in terms of changes in the three provinces, the southern regions of north khorasan and northern regions of razavi khorasan provinces had a high incidence. the southern regions of razavi khorasan province and the whole region of south khorasan had a lower incidence. table 2 shows the mean of six climatic and environmental variables (rainfall, temperature, vegetation, specific humidity, evapotranspiration and soil moisture) in northeastern provinces of iran. the relationship between climatic and environmental factors and incidence of cl was evaluated. rain precipitation rate (mm) the results showed that the mean annual precipitation in these three provinces was 15.08mm. the rate of change in average rainfall across these provinces showed a downward trend at a rate of 21.13 to 15.75 and 8.38mm in north khorasan, razavi khorasan and south khorasan, respectively (table 2). the average annual rainfall in southern regions; all areas of south khorasan, central and southern areas of razavi khorasan was lower than that in northern regions; most areas of north khorasan and northern areas of razavi khorasan. so that north khorasan province had the highest rainfall during the year. the razavi khorasan province also had different conditions; the northern areas of the province had specifically good rainfall, and the southern and western parts had a lower rainfall than the northern parts. furthermore, south khorasan province had a low rate of rainfall in most regions (fig. 3a). there was a significant correlation between the incidence of cl and the precipitation mean (p= 0.046). according to the figures 2 and 3, it was also observed that the maximum precipitation areas were matched with the maximum incidence of the disease. land surface temperature (ºc) the average temperature in all of these provinces was 35.9 °c. it was 42.67, 34.96 and 30.07° for the three provinces of south khorasan, razavi khorasan, north khorasan and respectively. in this study, the trend of temperature decreased from south to north areas. in terms of temperature conditions in the three studied provinces, north khorasan province had a lower average temperature. the northern and northeastern parts of the razavi khorasan province had lower temperatures than those in the southern and eastern parts of the province. south khorasan province residents had also experienced a different temperature in one year, and its southern and western parts had a higher temperature than the eastern and northern regions of the province (fig. 3b). there was a correlation between incidence of cl and air temperature. it was observed that the incidence of disease had decreased in southern regions with high temperatures. normalized difference vegetation index (ndvi) spatial variations in vegetation revealed that the northern regions; most areas of north khorasan and northern regions of razavi khorasan had a better status than the southern regions (fig. 3c). the ndvi was 0.11 in all studied provinces, while it was 0.8, 0.12 and 0.15 in south khorasan, razavi khorasan and north khorasan, respectively. the southern and western regions of razavi khorasan and all regions of south khorasan province had less vegetation. statistical analysis showed that there was a direct correlation between the trend of vegetation changes and the incidence of disease. specific humidity (sh %) spatial variations of specific humidity during this course indicated that the air humidity was low in most regions (table 2). the highest sh was observed in northwest of north khorasan province. the northern parts of razavi khorasan province had higher humidity. in the south khorasan province, the southwest areas were less humid (fig. 3d). in total, the maximum moisture content was consistent with areas having the maximum incidence of disease. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 60 http://jad.tums.ac.ir published online: march 31, 2020 evapotranspiration (kg/ m2 -y) in this study, evapotranspiration had a northwest-southeast trend, showing a decreasing trend from northwest to south east (table 2). the evapotranspiration of the north khorasan province was more than that of the other two provinces. in the province of razavi khorasan, the northern and eastern regions of the province had higher evapotranspiration than the southern and western regions. the spatial variations of evapotranspiration in south khorasan province showed a low level of this index with most of the regions having low evapotranspiration (fig. 3e). the comparison of evapotranspiration and spatial changes in the incidence of the disease showed that evapotranspiration was also an effective factor in the incidence of this disease. soil moisture (%) the spatial variation of soil moisture suggest ed that the northern regions (most areas of north khorasan and northern regions of razavi khorasan) had better conditions than the southern regions (table 2). soil moisture was specifically high in most parts of north khorasan province. in razavi khorasan province, the northern and southern regions had higher rates of soil moisture than in the central regions of the province. moreover, the soil moisture content reached the lowest level in southern khorasan province (fig. 3f). there was also a significantly direct correlation between spatial variation of soil moisture and disease incidence (p= 0.007). pearson correlation coefficient was used to study the relationship between every environmental and climatic factors and the incidence of the disease. table 3 shows the pearson correlation coefficient indices with the incidence of cl. table 1. the characteristics of parameters extracted from gldas in this study index source unit spatial resolution precipitation rate trmm mm 0.25° land surface temperature modis aqua c 0.05° normalized difference vegetation index modis terra ndvi 5600m specific humidity glads model kg kg-1 0.25° evapotranspiration glads model kg m-2 s-1 0.25° soil moisture amsr-2 % 10km fig. 1. location of the study area, northeastern iran http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 61 http://jad.tums.ac.ir published online: march 31, 2020 table 2. mean of climatic and environmental variables and cutaneous leishmaniasis incidence in northeastern provinces of iran from march 2016 to march 2017 index north khorasan razavi khorasan south khorasan total precipitation rate (mm) 21.13 15.75 8.38 15.08 land surface temperature (c º ) 30.07 34.96 42.67 35.9 normalized difference vegetation index (ndvi) 0.15 0.12 0.08 0.11 specific humidity (kg kg-1) x1e-3 4.64 4.12 3.73 4.16 evapotranspiration (kg/ m2 -y) x1e-7 70.0 60.01 43.21 57.74 soil moisture (%) 23.51 14.65 5.01 14.36 cl incidence/100,000 35.80 34.14 7.67 25.66 cl cases 309 2197 59 2565 table 3. pearson correlation coefficient indices with the incidence of cutaneous leishmaniasis in northeastern iran index correlation coefficient pvalue precipitation rate 0.239 0.046 land surface temperature -0.322 0.028 normalized difference vegetation index 0.314 0.032 specific humidity 0.303 0.038 evapotranspiration 0.465 0.001 soil moisture 0.392 0.007 fig. 2. spatial distribution of cutaneous leishmaniasis in northeastern iran http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 62 http://jad.tums.ac.ir published online: march 31, 2020 fig. 3. time averaged map of monthly rate of all environmental and climate factors in northeastern iran discussion the incidence of cl in north khorasan, razavi khorasan, and south khorasan was 35.80, 34.14 and 7.67 per 100,000 people, respectively. the epidemiological studies on cl disease in iran indicated that the majority of cases were reported in eastern, central, and southern provinces in the country (29). in addition, the incidence of cl in iran were evaluated 27.5 per 100, 000 populations in 2011 (30). so, we can conclude that the cl incidence in two provinces out of 3 studied provinces; north khorasan, razavi khorasan was higher than the average incidence of the country. in the present study, the incidence of cl was highly correlated with soil moisture and evapotranspiration. in addition, there was a moderately positive relationship between vegetation and humidity index with the incidence of the disease. besides, the incidence of the disease had the lowest correlation with precipitation. in addition, there was also a significantly positive relationship between cl incidence and land surface temperature, so that the incidence of the disease decreased with the increasing temperature. in this study, there was a weak significant positive correlation between the incidence of cl and the mean of precipitation. it was also observed that the maximum precipitation areas were matched with the maximum incidence of the disease. in contrast, entezari and eskandari have been found that there was an inverse correlation between climatic parameters of rainfall and specific humidity with cl prevalence in fars province, southern iran (31). it has already been proven that as rainfall http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 63 http://jad.tums.ac.ir published online: march 31, 2020 increases at the right level, it can usually adjust air temperature and increases humidity. but, flood-causing rainfalls destroy the breeding places of sand flies in addition to reducing their populations, leading consequently to a decrease in the incidence of the disease (32, 33). for example, the ph. argentipes larvae, when exposed to monsoon floods in northern india, ascends the mud walls, and descend after the deposition of water and the normalization of the conditions, but in other species, these floods lead to the death of the sand flies (34). in a study in french guyana, it was observed that the precipitation had a weak relationship with the cl incidence (35). in another study in brazil, a direct correlation was found between incidence of disease and precipitation (36). in the present study, there was an inverse correlation between cl incidence and land surface temperature. it has been observed that the incidence of disease has decreased in southern regions with high temperatures. this finding is in contrast with the basic concepts about the disease. it had been previously proved that the growth of all species of sand flies occur at temperatures above 18 °c, which is the threshold temperature for their life (37). in general, the findings of various researchers show that the optimum temperature for the development of their life stages before maturation of sand flies is in range of 18– 28 °c (37). in an ecological study on phlebotomine sand flies in the greek aegean islands showed that temperature preferences of them were 21–29 °c (38). it seems in addition to temperature, other environmental and climatic factors are associated to the disease. in addition, adult sand flies activate during sunset and night when the land surface temperature is balanced and humidity rises. in this condition, the infected female phlebotomine sandflies can cause the transmission of leishmania parasites into susceptible hosts such as humans (39). in the present study, spatial distribution of vegetation revealed that the northern regions; most areas of north khorasan and northern regions of razavi khorasan had a better situation than the southern ones. statistical analysis based on the ndvi showed that there is a direct correlation between vegetation changes and the incidence of disease. contrary to the results of the present study, mozafari et al. in a study comparing spatial distribution of disease cases with vegetation status in ardakan, central iran, found that cl is more prevalent in areas with less vegetation (40). such differences in the results of various studies can indicate the impact of different geographic locations on the cl incidence. sometimes the effects of the same climatic parameters will vary from one region to another. vegetation is a function of temperature and humidity. the vegetation cover status and the cl incidence are affected by climatic factors (41). vegetation can be increased under the influence of higher precipitation and lower temperatures. the situation also holds true about cl incidence. in addition, the incidence of cl disease also depends on sand flies’ density (3). therefore, cl is indirectly affected by climatic factors. the incidence of the disease depends on a variety of factors in addition to the population of sand flies. for example, the change in population and the density of the disease reservoirs (rodents) can affect the incidence of disease (42). at the time of drought, the population of wild rodents approaching human settlements may increase. this situation, in turn, can lead to an increase in the population of rodents infected with leishmania parasite. so, the probability of bloodsucking of sand flies from these rodents rises as well as biting on humans and inoculation of the leishmania parasite to humans (43, 44). therefore, a vegetation index at a low level may ultimately lead to an increase in disease as well. the average specific humidity in the study area was 32.22%. the highest humidity was observed in north khorasan and northern regions of razavi khorasan. in general, areas with maximum moisture content were consistent with areas with the highest incidence of disease. the specific humidity is favorable for egg hatching, being between 80–90% (45). hesam-mohammadi et al. also found that a specific humidity of 24.83– http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=hesam-mohammadi%20m%5bauthor%5d&cauthor=true&cauthor_uid=26114129 j arthropod-borne dis, march 2020, 14(1): 56–67 mr shirzadi et al.: impact of environmental … 64 http://jad.tums.ac.ir published online: march 31, 2020 36.33% was desirable for sand flies collected from kashan district, central iran (46). hence, in the present study area, climatic conditions are favorable for the activity of sand flies in terms of humidity, their biting habit on hosts such as humans and the transmission of cl disease. although specific humidity is required in breeding places of sand flies, high moisture can make larvae migrate to other places (37). mozafari et al. analyzed the role of biochemical factors (climatic factors affecting the life of living creatures) and the outbreak of cl in the yazd-ardakan plain, central iran, using data from meteorological data during the statistical period of 1997–2009. their research showed that maximum disease incidence occurs in the second half of the year, especially in autumn, and there is also a weak positive correlation between specific humidity and incidence of the disease (40). in the present study, evapotranspiration has a northwest-southeast trend which decreases from northwest to south east of all study areas. the comparison of evapotranspiration index with spatial distribution of disease confirmed that evapotranspiration is also an important factor in cl incidence. water movement in river beds can have an effective role in increasing evapotranspiration and consequently in increasing specific humidity, ultimately leading to temperature modification. as such, north khorasan with large rivers and streams characterized by sufficient evapotranspiration, has provided suitable breeding places for the activities of sand flies as vectors of the disease. previous studies have also proved the presence of infected female p. papatasi species with leishmania major parasite in endemic areas of north khorasan province (13). spatial changes in the soil moisture indicate that the northern regions have more favorable conditions than the southern ones. in south khorasan province, the rate of soil moisture was also the lowest, which associated to the areas with the lowest incidence of the disease. researchers believe that rotten plants in the soil are the main source of food for sand flies larvae in nature since larvae cannot survive without water and moisture. the findings of the current study support the previous studies evaluating the effect of soil moisture on the trend of the disease. in a study on the mapping of vulnerable areas of kala-azar disease using rs and gis in parts of the bihar province of india, it was found that ponds, streams, irrigation canals, and rivers are directly interrelated and also very effective in maintaining soil moisture and subsoil levels with an average of 65–80% for the growth of larva and adult sand flies (47). conclusion according to the findings, the incidence of disease was also higher in the northern regions; most areas of north khorasan and northern regions of razavi khorasan; where the rainfall, vegetation, specific humidity, evapotranspiration, and soil moisture was higher than the southern studied areas. acknowledgements the authors are grateful to the research deputy of razavi khorasan university of medical science. ethical clearance was earned from the institutional ethics committee of razavi khorasan university of medical sciences (khru. rec.1395.112). the authors declare that there is no conflict of interest. references 1. torres-guerrero e, 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caused by chrysomya bezziana (diptera: calliphoridae) *fariba berenji, bibi razieh hosseini-farash, narges marvi-moghadam deptartment of parasitology and mycology, faculty of medicine, mashhad university of medical sciences, mashhad, iran (received 29 may 2013; accepted 20 apr 2014) abstract myiasis is the invasion of vertebrates' tissue by the larvae of a fly of the order diptera. the objective of this paper is to present a rare case of secondary myiasis of ocular infestation by chrysomya bezziana. a 55-year-old female from sar village of mazandaran province, northern iran), referred to khatam al-anbia hospital of mashhad with extensive destruction of left orbital cavity. existence of larvae was the major complaint in recent months. some live larvae were removed from her destructed left eye. primary diagnosis was myiasis of left upper lid (lul) and suspected recurrent basal cell carcinoma (bcc). the laboratory diagnosis was done in parasitology lab of imam reza hospital of mashhad and collected larvae were identified and confirmed to be larvae of the c. bezziana (diptera: calliphoridae). it is a case report of secondary ophtalmiomyiasis due to c. bezziana of a patient lives in mazandaran province. keywords: myiasis, ophthalmomyiasis, chrysomya bezziana introduction myiasis is the invasion of vertebrates' tissue by the larvae of a fly of the order diptera. “myiasis is broadly divided into obligatory,facultative and accidental myiasis depending on the biology of the fly species causing the myiasis and its likely effect” (janovy et al. 1996, chan et al. 2005). the condition is further categorized on the tissues that are involved. there are two types of myiasis: primary myiasis is caused by the biontophage larvae caused primary myiasis that feed on living tissue and secondary form is caused by necrobiontophage fly larvae that feed on necrotic tissue (raposo et al. 2012). there are more than 160000 diptera species. however few species cause ocular injuries (borror et al. 1989). the larvae of certain flies can invade to orbital tissue [myiasis-orbital], external ocular structures [ophthalmomyiasis externa], internal ocular structures [ophthalmomyiasis interna], and the skin of the eyelid [myiasis-dermal]. causes of myiasis of the eyelid include cuterebra and dermatobia hominis (klintworth 2012). the c. bezziana female lays approximately 150–200 eggs at a time in wounds and mucous membranes of a live mammal and will hatch after 24 hours. the larvae invade to the tissues of the host by sharp mouth-hooks and anchoring intersegmental spines (klintworth 2012). the first stage of larva is similar to a worm with a white color and 1.5 mm length. the measures of second and third stages larvae are 4 to 9 and 18 mm, respectively. the worm like larva is formed from eleven segmented with the anterior spiracle on the second and posterior spiracle located on the last segment. there are also compact spurs surrounding each segment of the body (walker 1994). after being embed*corresponding author: dr fariba berenji, e-mail: fberenji@yahoo.com  http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 125–130 f berenji et al.: a case of secondary … 126 ded in living tissue for 3–4 days the third instar larvae wriggle out of the tissues in search of a suitable substrate for pupation. however, some mature larvae die in the infested tissue. while feeding, only the posterior spiracle are visible (cook and zumla 2009). larvae can invade the conjunctiva and ocular bulb, provoking conjunctivitis, corneal ulcer and destruction of the ocular bulb, eyelids and orbit, since it feeds on the surrounding tissues (tsuda et al. 2007). the pupal stage is temperature dependent with warm weather favoring growth. depending on the temperature, the pupal stage can last anywhere from 1 week to 2 months. the males become sexually mature after 24 hours of leaving their puparium (the hardened shell the pupae mature in), while females take about 6–7 days to become fully sexually mature. if the weather is tropical (29 ˚c or 84.2 ˚f), the entire life cycle will last about 24 days, however, at cooler temperatures (below 22 ˚c or 71.6 ˚f), the life cycle can take 2–3 months to complete (oie committee 2008). case description a fifty five-year-old female from sar village of mazandaran province, northern iran, referred to khatam alanbia hospital with extensive destruction of left orbital cavity. she had eye pains for 4 years and the chief complaint was existence of larvae lasting months. the patient had been visited by ophthalmologist and many larvae were seen in her destructed left eye (fig. 1). primary diagnosis was myiasis of left upper lid (lul) and suspected recurrent bcc (basal cell carcinoma). the ophthalmologist referred her to the laboratory of parasitology of imam reza hospital of mashhad. some live larvae were removed by parasitologist (fig. 2). the collected larvae were evaluated and confirmed to be larvae of the c. bezziana by diagnosis of one pair of anterior spiracle larvae and with 4 to 6 lobes at the ends, located on the first segment, and one pair of posterior spiracles at the bottom of the third instar of the larvae (fig. 3). the maggots placed on a dish of raw meat in a glass jar containing moist sand. the jar closed with cotton and incubated at room temperature. the maggots burrowed into the meat and stayed for some time. at last they leave meat to enter the sand in order to pupate. then the jar examined periodically for the emergent flies (fig. 4). tissues from lul and medial canthus and remaining of larvae were removed by surgery. the eye was patched. after pathological studies, revealed basal cell carcinoma and this disease could provide an appropriate condition for larvae growth of c. bezziana and caused a secondary ophthalmomyiasis. fig. 1. the patient with extensive destruction of left orbital cavity fig. 2. maggots of chrysomya bezziana removed from the patient's eye http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 125–130 f berenji et al.: a case of secondary … 127 fig. 3. larval spiracles of chrysomya bezziana fig. 4. emergent flies from pupae discussion ophthalmomyias is accounts for <5% of human myiasis (wilhelmus 1986) but the status of this disease is not clear in iran. immigration and traveling are two important reasons for increasing the imported cases (langan et al. 2004). some studies have been done on ophthalmomiyasis in our country that most known cases were caused by o. ovis and one by flesh fly (janbakhsh et al. 1977, razmjou et al. 2007, yaghoubi and heydari 2013). dermatobia hominis and o. ovis (sheep botfly) which are endemic to tropical or subtropical areas are two major causes of ophthalmomyiasis in the world (lagacé-wiens et al. 2008). the first reported case of ophthalmomyiasis by o. ovis has been reported in 1977 (janbakhsh et al. 1997). many parts of iran are located in subtropical area and people are in contact with sheep, goats and many other domestic animals in rural regions. according to this information, the number of infected cases are probably more than reported (lagacé-wiens et al. 2008). external ophthalmomyiasis is the most common form of ocular myasis which symptoms are like conjunctivitis (khataminia et al. 2011). in one study on 8 farmers with external ophthalmomyiasis in fars province of iran, all the larvae from the bulbar conjunctiva were removed and the symptoms resolved as soon as removal action. the number of larvae obtained from each person were about 1 to 5 and all of them were identified as o. ovis (diptera: oestridae) larvae (masoodi et al. 2004). ophthalmomyiasis interna occurs when larvae penetrate into the eye (ramonas et al. 2006). two cases of ophthalmomyiasis interna have been reported form iran in a 70 years old woman with bilateral subretinal migration of larvae and 12 years old boy with migration of blowfly larvae (diptera: calliphoridae) form anterior chamber to posterior chamber after 3 days (farahvash et al. 1998, sharifipour et al. 2008). this study described a case of secondry ophthalmomyiasis caused by c. bezziana. chrysomya bezziana belongs to calliphoridae family and suborder brachycera (suborder cyclorrhapha), also named as screw worm of the old world (chemonges-nielsen 2008). human infestation is uncommon, but may occur with outbreaks in dogs, pigs and cattle in rural areas (goddard 2003). human myiasis infestation by c. bezziana has been reported from south asia, africa, saudi arabia, indonesia islands, phillipines, guinea and persian gulf of iran and is well documented in india (lane and crosskey 1993). ophthalmomyiasis caused by c. bezziana is particularly damaging. there are two reported cases for orbit myiasis caused by c. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 125–130 f berenji et al.: a case of secondary … 128 bezziana in iran. the first has described in sina hospital in a 90-year-old woman from andimeshk with ocular rupturing and outpouring of hundreds of c. bezziana larvae and the second was in imam khomeini hospital in ahvaz .the patient was an 87-yearold woman with a severely necrotized left orbit and several live larvae of c. bezziana (radmanesh et al. 2000, khataminia et al. 2011). a pre-existing ocular lesion such as gonococcal conjunctivitis [gonorrhea] and periocular neoplastic ulcers, endophthalmitis may predispose to ophthalmomyiasis by attracting flies. in neglected patients, fly larvae may destroy orbital tissue and cause a marked inflammation and secondary bacterial infection. certain fly larvae can penetrate through the conjunctiva and sclera and cause internal ophthalmomyiasis of the parasitic dipterous larvae. when this occurs, a single larva usually gains access into the eye. most affected individuals are asymptomatic, but severe visual loss may occur (klintworth 2012). in present case predisposing factor was bcc. chrysomya bezziana is an obligate ectoparasite in the animals and afflicts humans especially in some conditions such as poor hygienic lifestyle, working in contaminated areas during the warm seasons (soleimani ahmadi et al. 2009). infestation in humans and livestock are often observed in the wound, normal body orifices such as eye, ear, nose and mouth. the manifestations include pruritus, pain, inflammation, redness, eosinophilia, secondary bacterial infections. it rarely results in death of the patient (wall and shearer 1997). in the present study, the patient lived in rural area of mazandaran with poor sanitary conditions where there is a relationship between people and animals. ophthalmomyiasis caused by c. bezziana is particularly damaging and a rare case of orbital myiasis due to c. bezziana has been documented (klintworth 2012). periorbital tissues that have been compromised by surgery, tumors, infections or ischemia may predispose towards myiasis since the flies feed on exudates, blood, secretions and decomposing tissue (balasubramanya et al. 2003). in this study, the patient had none diagnostic bcc which made a risk factor for internal ophthalmomyiasis. to our knowledge this is the third case of ophthalmomyiasis caused by c. bezziana that leads to a sever destruction. in all of cases the patients were elderly with underlying disease, two with bcc and one with a type of skin cancer (radmanesh et al. 2000, khataminia et al. 2011). conclusion although human ophthalmomyiasis is a rare disease but it may be emerging and increasing. besides the status of this disease is not clear in iran but in this paper we report a rare case with ophthalmomyiasis due to c. bezziana which basal cell carcinoma could provide an appropriate condition for larvae growth of mentioned fly and caused a secondary ophthalmomyiasis. it is considered that many metabolic diseases (diabetes mellitus) and cancers (basal or squamous cell carcinoma), as well as poor hygiene can provide a suitable ground for myiasis because of delaying in wound healing in these diseases. dermatologist and ophthalmologist must be alert about myiasis that can affect the patients with cancerous lesion. acknowledgement the authors greatly acknowledge mr. majid ganjbakhsh for his kind efforts. the authors declare that there is no conflict of interests. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 125–130 f berenji et al.: a case of secondary … 129 references balasubramanya r, pushker n, bajaj ms, rani a (2003) massive orbital and ocular invasion in ophthalmomyiasis. can j ophthal. 38(4): 297–298. borror dj, triplehorn ca, johnson nf (1989) an introduction to the study of insects. 6th ed. saunders college publishing, philadelphia, p. 875. chemonges-nielsen s (2008) chrysomya bezziana in pet dogs in hong kong: a potential threat to australia. aust vet j. 81(4): 202–205. chan jcm, lee jsw, dai dlk, woo j (2005) unusual cases of human myiasis due to old world screwworm fly acquired indoors in hong kong. trans r soc trop med hyg. 99(12): 914–918. cook gc, zumla ai (2009) manson's tropical diseases. saunders, philadelphia. farahvash ms, aalami harandi z (1998) bilateral ophthalmomyiasis interna posterior: report of a case with severe visual loss. arch iran med. 2: 38–41. goddard j (2003) physician's guide to arthropods of medical importance. crc press llc, florida. janovy john, schmidt gerald d, roberts larry s (2012) foundations of parasitology: ninth edition. mcgraw-hill, chicago. janbakhsh b, pirouz ms, tirgari s, agha mohammadi a (1977) a case of ophthalmomyiasis in man by oestrus ovis linneaeus in tehran (insecta: diptera, oestridae). acta med iran. 20(1–2): 19–26. khataminia g, aghajanzadeh r, vazirianzadeh b, rahdar m (2011) orbital myiasis. j ophthalmic vis res. 6(3): 199–203. lagacé-wiens pr, dookeran r, skinner s, leicht r, colwell dd, galloway td (2008) human ophthalmomyiasis interna caused by hypoderma tarandi, northern canada. emerg infect dis. 14: 64– 66. lane rp, crosskey rw (1993) medical insects and arachnids. chapman and hall, london. langan sm, dervan p, o'loughlin s (2004) a moving scalp nodule in a returning traveler. br j dermatol. 151(6): 1270. masoodi m, hosseini k (2004) external ophthalmomyiasis caused by sheep botfly (oestrus ovis) larva: a report of 8 cases. arch iran med. 7: 136–139. radmanesh m, khataminia g, eliasi p, korai mk, ebrahimi a (2000) chrysomyia bezziana-infested basal cell carcinoma destroying the eye. int j dermatol. 39: 455–457. raposo aa, schettini ap, massone c (2012) concurrent primary and secondary myiasis on basal cell carcinoma. an bras dermatol. 87(2): 292–295. razmjou h, mowlavi gh, nateghpour m, solaymani-mohamadi,kia eb (2007) ophthalmomyiasis caused by flesh fly (diptera: sarcophagidae) in a patient with eye malignancy in iran. iran j arthropod-borne dis. 1(2): 53–56. ramonas km, friedman ah (2006) systemic infectious and inflammatory diseases. in: tasman w, jaeger ea (eds). duane’s phthalmology [cd-rom]. vol 5. lippincott williams and wilkins, philadelphia. sharifipour f, feghhi m (2008) anterior ophthalmomyiasis interna: an ophthalmic emergency. arch ophthalmol. 126: 1466–1467. soleimani ahmadi m, nasirian h, nazemi gheshmi am, yaghoobi ershadi mr (2009) human extensive head skin myiasis. iran j publ health. 38(1): 134–138. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 125–130 f berenji et al.: a case of secondary … 130 tsuda s, nagaji j, kurose k, miyasato m, sasai y, yoneda y (2007) furuncular cutaneous myiasis caused by dermatobia hominis larvae following travel to brazil. int j dermatol. 35(2): 121– 123. walker ar (1994) arthropods of humans and domestic animals: a guide to preliminary identification. springer, new york city. wilhelmus kr (1986) myiasis palpebrarum. am j ophthalmol. 101(4): 496. world organisation for animal health (2008) manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees). 6th ed. vol. 2. available at: http://www.oie.int/doc/ ged/d7709.pdf yaghoubi gholamhossein, heydari behrouz (2013) external ophthalmomyiasis presenting to an emergency department: corneal findings as a sign of oestrus ovis. korean j ophthalmol. 27(5): 341– 344. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, december 2020, 14(4): 344–352 e ahmadi and j khajehali: dichlorvos resistance in … 344 http://jad.tums.ac.ir published online: december 31, 2020 original article dichlorvos resistance in the house fly populations, musca domestica, of iranian cattle farms ebrahim ahmadi; *jahangir khajehali department of plant protection, college of agriculture, isfahan university of technology, isfahan, iran *corresponding author: dr jahangir khajehali, e-mail: khajeali@cc.iut.ac.ir (received 12 may 2019; accepted 30 nov 2020) abstract background: insecticide resistance is one of the most important problems associated with the control of musca domestica, due to the potential of the rapid development of resistance to different chemical insecticides. the present study was carried out to evaluate dichlorvos resistance in the house fly populations collected from central regions of iran, isfahan province and chaharmahal and bakhtiari province, during 2017 to 2019. methods: bioassays were carried out using a standard topical application method as well as a fumigation method. the koohrang population (susceptible) with the lowest ld50 values to dichlorvos was chosen to calculate the resistance ratios (rr). altered sensitivity of acetylcholinesterase (ache), a target enzyme for dichlorvos, was investigated. results: according to the results, very high levels of dichlorvos resistance were observed in the mobarake population (rr= 80.25-fold by topical application and 33-fold by fumigation bioassay), and isfahan population (rr= 107.30-fold by topical application and 43-fold by fumigation bioassay) compared to the koohrang population. acetylcholinesterase of the koohrang population was the most sensitive to inhibition by dichlorvos based on the determination of median inhibitory concentration (ic50), but ache of mobarake and isfahan populations were 741.93and 343.94fold less sensitive to inhibition. conclusion: the insensitivity of ache was possibly involved in dichlorvos resistance in the house fly populations. keywords: organophosphorus insecticides; target site resistance; acetylcholinesterase; median inhibitory concentration introduction the house fly, musca domestica linnaeus, is a crucial pest in medical and veterinary. house fly is a vector of different kinds of pathogens in humans and animals. the flies not only act as a source of annoyance but also transfer pathogens mechanically when moving to residential, commercial, livestock, and poultry places (1). the chemical control, often against the adult stage of house flies, is mainly by synthetic insecticides such as pyrethroids, neonicotinoids, organophosphates, and carbamates (2-5). in the dairy and poultry industry, house flies are considered as a major pest, and for its control, pyrethroids and organophosphates are extensively applied in iran. several organophosphorus (ops) compounds including of dichlorvos, diazinon, fenchlorphos, malathion, fenthion, dimethoate, and trichlorfon are used for house fly control (4). organ ophosphorus bind to ache, which leads to an accumulation of acetylcholine (ach) in cholinergic synapses and subsequently disrupt nervous functions, resulting in paralysis and death (6). among ops, dichlorvos (o, odimethyl-o2,2-dichlorovinylphosphate or ddvp) has been recognized as one of the widely used insecticides for the management of house fly and other arthropod pests (7). however, the world health organization has classified dichlorvos as a highly hazardous pesticide (8), and harmful to human and animal health by long-term low-level dietary uptake of food containing dichlorvos residues (9). furthermore, there are several reports on the development of dichlorvos resistance in this species all over the world (10-16). development of resistance may cause increasing the dosage and frequency of copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:khajeali@cc.iut.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 344–352 e ahmadi and j khajehali: dichlorvos resistance in … 345 http://jad.tums.ac.ir published online: december 31, 2020 insecticide applications in the residential, commercial, livestock, and poultry places, which enhances the cost of control, and it also has negative impacts on the environment (17, 18). also, metabolic resistance via the enhanced activity of detoxification enzymes has been associated with resistance to ops, and modified ache has been reported as the main mechanism of resistance (19, 20). biochemical characterization of altered aches has shown that there is a wide range of insensitivity between insect species and between ops compounds (21). insensitivity of ache to ops insecticides in house fly was documented for the first time in 1973 (22). several mutations in the ache gene of the house fly have been proved to be involved in ops resistance (21). despite numerous cases of insecticide resistance in the house fly, there is a broad spectrum in insecticide sensitivity between populations. thus, the assessment of resistance to different insecticides in regional populations of house flies can provide useful information for the fly control and insecticide-resistant management programs (17, 23). in the present study, efforts were made to understand dichlorvos resistance status and mechanisms in the different house fly populations. materials and methods chemicals technical grade of dichlorvos insecticide (98.7%) was provided by golsam sepahan company (iran), acetylthiocholine iodide (atchi), coomassie brilliant blue g-250, bovine serum albumin, 3,3′,5,5′-, fast blue rr salt were purchased from sigma-aldrich (germany). insect rearing the house fly populations were collected from dairies in mobarake (isfahan province 32.3347°n, 51.5571°e) and isfahan (isfahan province -32.6546° n, 51.6680° e). the population of koohrang (chaharmahal and bakhtiari province -32.3297° n, 50.1112° e) was collected from a rural area where insecticides had not been used. adults were reared under laboratory conditions of 25±2 ºc, 16:8 (l: d), and 60±5% relative humidity. the adult diet consisted of sesame meal and wheat bran (1: 3) in a plastic container. also, a mixture of water and sugar (10%) was provided in another plastic container. larvae were transferred to plastic buckets containing 20g a diet, included sesame meal and wheat bran (1: 3), 1.5g milk powder, 1.5g honey mixed with 8ml of water. female house flies were used for bioassays. topical and fumigation bioassays topical bioassays were followed, as described by kasai et al. (24). briefly, technical grade dichlorvos solved in acetone, and then 1μl were topically applied by micropipette (nichiryo model 8100, tokyo, japan) on the notum of co2-anesthetized flies (3–5-day-old females, n= 20 flies per concentration/per replication). in control groups, flies only received topical application of acetone. the treated flies were released in plastic jars (250ml) containing cotton moistened with a 20% sugar solution. mortality data were recorded after 24h exposure to insecticide. for bioassays, 5 to 6 concentrations of insecticide were prepared as serial dilutions in acetone and replicated three times. the fumigant bioassay was conducted, according to rossi et al. (25), with slight modifications. briefly, female house flies (3–5-days adult) were placed in a fitted glass jar (650ml). serial dilutions prepared dichlorvos concentrations in acetone were then placed on a cotton pad inside a petri dish; the dish was sealed to prevent fly contact and was placed inside a glass bottle. the bottle was sealed tightly and kept in a home temperature (25±2 °c) for 30 min. mortality data were recorded after 30min of exposure. each test was replicated three times. acetylcholinesterase activity and inhibition by dichlorvos twenty heads of 3–5-day old females from each population were homogenized in 1ml of http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 344–352 e ahmadi and j khajehali: dichlorvos resistance in … 346 http://jad.tums.ac.ir published online: december 31, 2020 sodium phosphate buffer (0.1m, ph 7.5), containing 0.1% (w/v) triton x-100 in ice-cold conditions. after centrifugation (12,000g at 4 °c for 15min), the supernatant was used for the enzyme assay. the ache activity was assayed based on the method of ellman et al. (26) with slight modifications. the atchi (10 mm) was used as the substrate. to determine median inhibitory concentrations (ic50s) aliquots (10μl) series of dichlorvos concentrations mixed with 20μl enzyme source and 70μl sodium phosphate buffer solution and the mixture was incubated for 5min at room temperature (25±2 °c). the reaction was started by adding 900 μl of substrate reagent solution, containing 1ml atchi, 250μl dtnb reagent (10mm) dissolved in 8.750ml sodium phosphate buffer (0.1m, ph 7.5). the control treatments were prepared by adding 10μl of acetone without insecticide. the acetone concentration of all reactions was 1%. the change in absorbance was measured using a spectrophotometer (unico, model uv-2100, usa) at 412nm for 10min with a read interval of the 30s at room temperature (25±2 °c). the tests were replicated three times. protein content was determined by the bradford method (25), and bovine serum albumin was used as the standard. to convert absorbance into molarity an extinction coefficient of 13.6mm−1 cm−1 was used. the specific activity of ache expressed as nmol of acetylthiocholine iodide hydrolyzed per min per mg protein (nmol min−1 mg of protein−1). the inhibition rate was calculated as a percentage with respect to the control by the following formula (26): % inhibition= 100– [(enzyme activity of treatment÷ enzyme activity of control)× 100] statistical analyses percentage mortality data of the topical application and fumigant assay were corrected by using abbott formula (27), and data were inputted to the polo-plus software for analysis (28). median inhibitory concentration (ic50) values were determined by probit analysis between the inhibition percentages against the insecticide concentrations (29). the ache enzyme activities were subjected to anova, and differences among means were compared by the lsd test (p< 0.05) using sas 9.4 software (30). results topical and fumigation bioassays the ld50 values of dichlorvos in the isfahan, mobarake, and koohrang populations by the topical application were estimated 515.29, 385.22, and 4.80μg/fly, respectively. the koohrang population exhibited the lowest ld50 value and was used as the reference strain to evaluate the resistance ratios (rr). therefore, high rr values were observed in mobarake (80.25-fold) and isfahan (107.30fold) populations (table 1). the fumigation assay of the dichlorvos insecticide on female house flies on different populations was performed, and the ld50 values against dichlorvos in the isfahan, mobarake, and koohrang populations were 1.34, 1.00, and 0.03 (μl/l), respectively (table 2). fumigant bioassay revealed high resistance levels to dichlorvos in mobarake and isfahan populations, 33and 43-fold, respectively. acetylcholinesterase activity and inhibition by dichlorvos the activity of ache was highest in the mobarakh population (155.65±2.83nmol/min/ mg protein) and isfahan population (234.79± 7.07nmol/min/mg protein). it was lowest in the koohrang population (79.25±2.28nmol/min/mg protein) (table 3). the ache activities in the mobarakh and isfahan populations were significantly higher than that of the koohrang population (p< 0.05). sensitivities of three aches to dichlorvos as an inhibitor were also determined. the estimated ic50 values by series of dichlorvos concentrations showed the insensitivity of ache to the insecticide in the resistant populations. acetylcholinesterase of the koohrang population was the most sensitive, and aches of mobarakh and isfahan populations showed insensitivity to dichlorvos (table 3). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 344–352 e ahmadi and j khajehali: dichlorvos resistance in … 347 http://jad.tums.ac.ir published online: december 31, 2020 table 1. contact toxicity of dichlorvos insecticide on the field populations of house flies insecticide populations n ld50 (μg/fly) (95% fl) slope±se chisquare df rr dichlorvos koohrang 360 4.80 (4–5.8) 1.885±0.173 0.72 4 mobarake 322 385.22 (296–502) 1.337+-0.156 0.8 4 80.25 isfahan 300 515.29 (387–710) 1.242±0.161 0.078 4 107.3 n: number of flies used in bioassays fl: fiducial limits df: degrees of freedom rr: ld50 of mobarake or isfahan/ ld50 of koohrang table 2. fumigant toxicity of dichlorvos on the field populations of house flies insecticide populations n1 ld50 (μl/l) (95% fl) slope±se chi-square df rr dichlorvos koohrang 360 0.03 (0.029–0.044) 1.651±0.167 0.845 4 mobarake 360 1.00 (0.87–1.3) 1.565±0.158 0.174 4 33 isfahan 360 1.3 (1–1.6) 1.479±0.154 0.229 4 43 n: number of flies used in bioassays fl: fiducial limits df: degrees of freedom rr: ld50 of mobarake or isfahan/ ld50 of the koohrang population table 3. mean ache activity (nmol/min/mg protein) and its inhibition by dichlorvos populations ache ratioa ic50 (m) ratiob koohrang 79.25±2.28c 3.14×10 -7±1.56 mobarake 155.65±2.83b 1.96 2.32×10 -4±5.78 741.93 isfahan 234.79±7.07a 2.96 1.08×10 -4±2.33 343.94 a: ratio of enzyme activity in the resistant population/ enzyme activity in the susceptible population b: ratio of ic50 in the resistant population/ ic50 in the susceptible population a, b, c significantly different by applying lsd (p< 0.05) discussion dichlorvos is one of the most common insecticides used for house fly control in cattle farms of isfahan province. therefore, monitoring of susceptibility of the fly to dichlorvos is necessary for managing programs of m. do mestica. to our knowledge, no information is available on the resistance status of house flies of iranian cattle farms. in the present study, levels of resistance to dichlorvos were determined through both topical and fumigation applicahttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 344–352 e ahmadi and j khajehali: dichlorvos resistance in … 348 http://jad.tums.ac.ir published online: december 31, 2020 tion methods in different field-collected populations. because of a known reference strain was not available, in this study the most susceptible strain, i.e. the koohrang population, was considered as the reference strain. although this strain possibly was not fully susceptible, field populations showed significantly high resistance ratios, justifying the resistance mechanisms. according to the results, high rr values to dichlorvos were observed in both cattle farm populations of mobarake and isfahan, ranging from 33to 107.30-fold. the rr values in topical bioassay were estimated higher than fumigant bioassay in the resistance populations of the house fly. it could be associated with the cuticular penetration factor involved in the resistance that can decrease insecticide penetration (31, 32). several studies have already documented house fly resistance to dichlorvos and other ops. wang et al. (35) reported a 14 to 28-fold resistance to dichlorvos. moderate levels of resistance have also been reported from the house fly population in argentina (33) and denmark (34). resistance to dichlorvos has also been found in other medically important diptera, such as aedes aegypti (35). also, low rr value to temephos was observed in anopheles stephensi in the chabahar sea of iran (36). furthermore, ops resistance has been investigated in crop pests. high levels of resistance to ops insecticides were reported in spodoptera litura (229-fold) (37) and tetranychus urticae (4164 -fold) (38). the main mechanism of ops resistance is altered ache, which led to enzyme insensitivity to inhibition by insecticides. also, 2to 5fold increases in the activity of ache were associated with ops resistance in drosophila melanogaster and m. domestica, respectively. the higher ache activity in strains with altered ache could be directly contributed to ops resistance or compensate for decrease ache hydrolysis (11, 39). soltani et al. (43) have reported the altered ache in anopheles stephensi in the south of iran, which causes resistance to temephos insecticide. in this study also an increase in ache activity was observed in resistant populations of mobarake (1.96-fold) and isfahan (2.96 fold). walsh et al. (21) have reported a higher than 500-fold insensitively of ache to dichlorvos in house fly resistant populations. moreover, it has reported that resistance to ops in schizaphis graminum contributed to increased ache activity through elevated expression of the ache gene (40) and ache insensitivity (41). based on the biochemical assays on the ache inhibition by dichlorvos and obtained ic50 values, target-site modification is possibly involved in resistance to dichlorvos in house fly populations. the ache of the koohrang population was most sensitive, while ache of mobarake and isfahan populations showed 741.93and 343.94fold insensitivity to dichlorvos. several point mutations in the ache gene can confer enzyme insensitivity to inhibition by ops insecticides (38, 42-44). metabolic detoxification has been demonstrated to be a key ops resistance mechanism in the house fly, which is mediated by cytochrome p450 monooxygenases (p450s), the glutathione s-transferases (gsts) and the carboxylesterases (cares) (45, 46). ahmadi et al. (5) determined detoxification enzyme activities in koohrang, mobarake, and isfahan populations and reported significantly lower activities of p450s, gsts, and cares in the koohrang population than that of mobarake and isfahan populations. p450s have been implicated to play major role in conferring ops resistance in house fly strains. for example, ops resistance in the rutgers strain has been linked to the overexpression of p450s (47). thus, enhanced activity of p450s in mobarake and isfahan populations (more than 2.2 folds) possibly contribute to dichlorvos resistance. in house flies, enhanced production of cares has been implicated as contributing to resistance to ops and other insecticides (48). in the alhf house fly strain, ops resistance have been found due to increased achttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 344–352 e ahmadi and j khajehali: dichlorvos resistance in … 349 http://jad.tums.ac.ir published online: december 31, 2020 tivities of care (49). the care activity in the ops resistant strains of mobarake and isfahan was reported significantly higher than that of koohrang population (3.7and 2.01-fold, respectively). ops resistance in house flies has been also linked to increased activities of gsts (45). previously, 4.9 and 5.2fold higher activities of gsts were found in mobarake and isfahan populations compared to the koohrang population (5). it seems that enhanced detoxification by p450s, gsts, and cares is an important mechanism of dichlorvos resistance in mobarake and isfahan populations. the enhanced detoxification could also be responsible for cross-resistance to other insecticides in house fly populations (50). insecticide resistance can increase costs and doses of insecticides in the house fly control, as well as decrease inefficiency of control (18). it also has environmental pollution problems and human health risks and negatively effects the non-target organisms (51). conclusion the present study revealed high levels of dichlorvos resistance in house fly populations. ache insensitivity and enhanced metabolic detoxification identified as the conferring mechanisms. however, the molecular mechanisms involved in ache insensitivity and enzyme detoxification remain uncharacterized. further studies in iranian cattle farms are needed to confirm these findings and to design management strategies to delay the development of insecticide resistance in house fly populations. acknowledgements the authors are the highly grateful isfahan university of technology and barij essence pharmaceutical company for providing financial assistance to carry out this study. the authors declare that there is no conflict of interests. references 1. nayduch d, burrus rg (2017) flourishing in filth: house fly–microbe interactions across life history. ann entomol soc am. 110(1): 6–18. 2. kaufman pe, nunez sc, mann rs, geden cj, scharf me (2010) nicotinoid and pyrethroid insecticide resistance in houseflies (diptera: muscidae) 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monoterpenes against housefly, musca domestica (diptera: muscidae). ecotoxicol environ saf. 100: 1–6. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 88 http://jad.tums.ac.ir published online: march 31, 2020 original article frequency and antimicrobial resistance patterns of bacterial species isolated from the body surface of the housefly (musca domestica) in akure, ondo state, nigeria *babatunde odetoyin1; babatunde adeola2; olarinde olaniran1 1department of medical microbiology and parasitology, college of health sciences, obafemi awolowo university, ile-ife, osun state, nigeria 2department of microbiology, federal university of technology, akure, ondo state, nigeria (received 05 may 2017; accepted 12 mar 2020) abstract background: the emergence and spread of antibiotic resistant bacteria has become a serious problem worldwide. houseflies are potential carriers of pathogenic and resistant bacteria and could be contributing to the global spread of these strains in the environments.we investigated the prevalence and antimicrobial resistant profiles of bacteria isolated from houseflies in akure. methods: twenty-five houseflies were captured by a sterile nylon net from the slaughterhouse, garbage dump, human house, hospital, and eatery from 9:00am to 1:00pm when the flies were active and transported immediately to the laboratory in sterile containers for processing. bacterial loads were enumerated by serial dilution and plating on nutrient agar and selective media. bacteria species were isolated by conventional isolation technique. antibiotic susceptibility test was determined by the kirby-bauer disc diffusion technique. results: sixty-seven bacterial species were isolated from 25 samples that were collected. the predominant bacterial species was escherichia coli (n= 31, 45%), followed by klebsiella pneumoniae (n= 17, 25%), staphylococcus aureus (n= 11, 16%) and pseudomonas aeruginosa (n= 3, 4.3%). the bacterial load of the samples ranged from 9.7×105cfu/ml to 1.65×106cfu/ml. the results revealed that all isolates of pseudomonas aeruginosa, salmonella spp, and proteus mirabilis were resistant to streptomycin and cotrimoxazole, augmentin and amoxicillin respectively. none of the s. aureus isolates was resistant to cotrimoxazole, chloramphenicol, sparfloxacin, augmentin, and ofloxacin. all isolates were multi-drug resistant. conclusion: house flies that were collected from the slaughterhouse, garbage dump, human house, hospital, and eatery may participate in the dispersal of pathogenic and resistant bacteria in the study environment. keywords: houseflies; antimicrobial resistance; pathogenic bacteria; vector; infection introduction the emergence of antimicrobial resistance in bacteria has become a serious global problem (1). a who report indicates that this problem is contributing to the increase in the cost of diagnosing and treating resistant infections (2). today, the main concern is that antimicrobial production pipelines are drying up and very few available therapeutic options are effective for common infections. antimicrobial resistant bacteria are usually spread from person to person, or from the non-human sources in the environment. available data indicate that the house fly, a known cosmopolitan pest with a worldwide distribution and commonly found in close association with human activities could also contribute to the global spread of pathogenic as well as resistant bacteria in the environments (3-5). houseflies are known to act as mechanical vectors of pathogenic bacteria like vibrio cholerae, escherichia coli, salmonella and shigella (6-8). they pick up the pathogens on their bodies with the aid of their hairy proboscis and feet and disseminate them by regurgitating vomits and depositing faecal droplets during the feeding process. these vectors have also been report*corresponding author: dr babatunde odetoyin, e-mail: odetoyin@yahoo.com http://jad.tums.ac.ir/ mailto:odetoyin@yahoo.com j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 89 http://jad.tums.ac.ir published online: march 31, 2020 ed to be carriers of multi-drug resistant bacteria in hospital environments, and they may participate in the spread of resistant as well as pathogenic pathogens within hospitals (9). adequate control of these vectors would allow a reduction of the transmission of these pathogenic and resistant bacteria. in spite of the awareness of the dangers posed by these flies and their link with poor environmental sanitation, factors/ practices such as indiscriminate refuse dumping and waste disposal, bad drainage systems coupled with improper handling of food still abound in nigeria. since pathogenic microorganisms are widespread in our environment, there is abundant opportunity for flies to become contaminated and, in turn, contaminate the environment. hence, the aim of the study was to isolate and characterize the bacterial pathogens on the external surfaces of houseflies and determine the antibiotic resistance patterns of the isolated organism to commonly used antimicrobials. the data from this study will provide information on the dangers posed by these flies in the environment. effective communication of the information is expected to generate apposite ideas for acceptable and workable interventions to control the spread of resistant pathogens in our environment. materials and methods study location, sample collection and processing the study was carried out in akure, the capital of ondo state which is located in the southwestern part of nigeria. akure lies on latitude 7o15՛north of the equator and on longitude 5o15՛east of the greenwich meridian (10). twenty-five samples of houseflies were captured by sterile nylon nets from five different locations. flies were caught from the selected sites (slaughterhouse, garbage dump, human house, hospital, and eatery) during the period of study with sterile nets from 9:00am to 1:00 pm when the flies were active. the collected flies were placed in sterile vials and transported to the laboratory for identification by an entomologist. all species other than musca domestica were removed. after identification, 2ml of sterile normal saline solution was added to each vial that contained the fly and shaken vigorously for one minute. the fly was removed from the saline and was checked for bacteria dislodged from the external surface of the fly (6). bacterial counts and isolation of microorganisms the diluents used for the samples were the sterile saline solution. one ml was taken from each sample using a sterile syringe and added to nine ml of sterile distilled water in the test tubes. this dilution process was repeated until the 4th dilution was obtained. from the serially diluted samples, 1ml each of the 10-4 dilutions of the housefly samples was taken aseptically with the use of a sterile syringe, and pourplated on the nutrient agar plates, and then incubated at 37 °c for 24 hours. the colonies on the plates were then counted and their morphological features recorded (11). from the nutrient agar plate, sterile inoculating loop previously flamed to red-hot and cooled was used to pick different colonies from all the isolation plates and then streaked on nutrient agar and incubated at 37 °c for 24 hours; the morphological features of the distinct colonies along the line of streak were observed and used to infer the type of organisms present on the isolation plates (12). the biochemical characterization of isolates was carried out as described by cheesbrough (13). antibiotic sensitivity test the antimicrobial susceptibility patterns of the isolates were determined by the kirby-bauer disc diffusion technique on mueller-hinton agar (cm0337) (oxoid ltd., basingstoke, hampshire, england). antibiotics tested were augmentin (30µg), ofloxacin (10µg), chloramphenicol (30µg), gentamicin (10µg), (10µg), sparfloxacin (10µg), amoxicillin (25µg), ciprofloxacin (10µg), streptomycin (30µg), pefloxacin (5µg) and cotrimoxazole (30µg) (remel, usa). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 90 http://jad.tums.ac.ir published online: march 31, 2020 the plates were incubated at 37 °c for 24 hours. the diameters of the zones of inhibition were measured with a ruler and interpreted according to the guidelines of the clinical and laboratory standard institute (clsi) (14). statistical analysis data were presented as frequencies and percentages. independent t-test and analysis of variance test of spss version 20 software package (spss, inc. chicago, illinois) were used to determine the significance of the data. all pvalues were two-sided and a p-value that was less than or equal to 0.05 was considered to be statistically significant. results microbiological analysis the results of the microbiological analysis showed that total bacterial counts ranged from 9.68×105cfu/ml to 1.65×106cfu/ml. the highest load of bacteria was found in samples from hospital (1.65×106cfu/ml), followed by eatery (1.60×106cfu/ml), garbage dump (1.59x106cfu/ml) and human house (1.28×106 cfu/ml). there was no significant difference in the mean load of bacteria from the five sampled sites (f= 2.7836, p= 0.0547). however, the mean load of bacteria from hospital was significantly higher (1.276×106cfu/ml) than the mean load of bacteria from slaughterhouse (9.68 ×105cfu/ml) (t= -2.79503, p= 0.0233) (table 1). organisms isolated from different samples sixty-seven bacterial species were isolated from the external surfaces of 25 identified house flies. these were divided into six genera comprising klebsiella, staphylococcus, escherichia, pseudomonas, proteus, and salmonella (table 2). the commonest bacterial species identified was e. coli (n= 31, 46.3%), followed by klebsiella pneumoniae (n= 17, 25.4%), staphylococcus aureus (n= 11, 16.4%) and pseudomonas aeruginosa (n= 3; 4.5%). of all the different sites sampled, garbage dumps and human houses harboured the highest number of bacteria species (n= 15). escherichia coli was the commonest bacterial species isolated in the slaughterhouse (n= 3), human house (n= 8), eatery (n= 6) and garbage dump (n= 9). however, k. pneumoniae was the commonest bacterial species isolated from the hospital (n= 7). baseline resistance rates of isolated bacteria all isolates (100%) of p. aeruginosa, salmonella spp, and proteus spp were resistant to streptomycin, cotrimoxazole, augmentin, and amoxicillin. no isolate of s. aureus was resistant to chloramphenicol, sparfloxacin, augmentin, and ofloxacin. isolates of e. coli were commonly resistant to augmentin (n= 28, 90.3%) and cotrimoxazole (n= 20, 64.5%). the least resistance rates were exhibited by k. pneumoniae (n= 1, 5.9%), e. coli (n= 1, 3.2%), and s. aureus (n= 1, 9.1%) to ciprofloxacin (table 3). multidrug resistance was defined as the resistance of isolates to at least one antibiotic in three or more classes of antibiotics. as shown in (fig. 1), all isolates were multidrug-resistant, with 66.6% of isolates of p. aeruginosa, 45.4% of s. aureus, 33.3% of salmonella spp., 19.3% of e. coli strains and 5.9% of k. pneumoniae resistant to three or more classes of antibiotics. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 91 http://jad.tums.ac.ir published online: march 31, 2020 table 1. bacterial load of sample (cfu/ml) (104)a colony slaughterhouse human house hospital eatery garbage dump plate 1 86 84 102 184 206 plate 2 102 184 158 146 178 plate 3 56 98 196 96 164 plate 4 84 128 204 206 142 plate 5 156 144 166 168 108 average 96.8b 127.6 165.2b 160 159.6 a= f= 2.7836, p= 0.054752; b= t= -2.79503, p= 0.0233 table 2. number of isolates from all sample locations isolates sample locations slaughter house (n= 12) human house (n= 15) hospital (n= 14) eatery (n= 11) garbage dump (15) total (67) klebsiella pneumoniae 0 (0) 4(26.7) 7(50) 2(18.2) 4(26.7) 17(25.4) escherichia coli 6 (50) 8(53.3) 2(14.3) 6(54.5) 9(60) 31(46.3) staphylococcus aureus 3(25) 3(20) 3(21.4) 0 (0) 2(13.3) 11(16.4) pseudomonas aeruginosa 1(8.3) 0 (0) 2(14.3) 0 (0) 0 (0) 3(4.5) proteus spp 2 (16.7) 0 (0) 0 (0) 0 (0) 0 (0) 2(3) salmonella spp 0 (0) 0 (0) 0 (0) 3(27.3) 0 (0) 3(4.5) table 3. antimicrobial resistant pattern of identified bacteria in all sample locations isolates antimicrobial agents per gen amo cpx s sxt ch sp au ofx staphylococcus aureus (n= 11) 2 (18.2) 3 (27.3) 7 (63.6) 1 (9.1) 3 (27.3) 5 (45.5) 0 (0) 0 (0) 0 (0) 0 (0) salmonella spp (n= 3) 1 (33.3) 0 (0) 1 (33.3) 1 (33.3) 3 (100) 1 (33.3) 1 (33.3) 0 (0) 3 (100) 1 (33.3) proteus spp (n= 2) 1 (50) 1 (50) 2 (100) 1 (50) 2 (100) 2 (100) 1 (50) 1 (50) 0 (0) 2 (100) klebsiella pneumonia (n=17) 4 (23.5) 5 (29.4) 8 (47.1) 1 (5.9) 9 (52.9) 2 (11.8) 1 (5.9) 2 (11.8) 13 (76.4) 11 (64.7) escherichia coli (n= 31) 2 (6.5) 3 (9.7) 18 (58.1) 1 (3.2) 23 (74.2) 20 (64.5) 4 (12.9) 2 (6.5) 28 (90.3) 5 (16.1) pseudomonas aeruginosa (n= 3) 2 (66.7) 1 (33.3) 1 (33.3) 1 (33.3) 3 (100) 3 (100) 1 (33.3) 1 (33.3) 0 (0) 1 (33.3) per= perfloxacin (per 30µg), gen= gentamycin (gen 30µg), amo= amoxicillin (amo 30µg), cpx= ciprofloxacin (cpx), s= streptomycin (s 30µg), sxt= cotrimoxazole (sxt 30µg), ch= chloramphenicol (ch 30µg), sp= sparfloxacin (sp 10µg), au= augmentin (au 30µg), and ofx= ofloxacin (ofx 10µg) http://jad.tums.ac.ir published online: march 31, 2020 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 92 http://jad.tums.ac.ir published online: march 31, 2020 fig. 1. multidrug resistance patterns of isolates discussion in the present study, we investigated the frequency and antimicrobial resistant profiles of bacteria isolated from houseflies in five different locations (slaughterhouse, garbage dump, human house, hospital, and eatery). six bacterial species, comprising salmonella, proteus, s. aureus, p. aeruginosa, e. coli, and k. pneumoniae were isolated from m. domestica. similar findings have also been reported by previous investigators who have highlighted the importance of houseflies as mechanical vectors of various pathogens (15-17). the external organs of m. domestica constitute a large source of bacteria, and their persistent association with humans, animals, food, refuse, and excreta makes them potential mechanical or biological vectors for the dissemination of pathogenic and multidrug-resistant bacteria (18-22). the results of this study indicate that m. domestica plays a great role as a mechanical carrier of bacteria in this environment, most of the bacteria isolated which have also been isolated by previ ous investigators are of medical importance (17, 23, 24). of all the different sites sampled, garbage dump and human house harboured the highest number of bacterial species (n= 15). garbage dumps are sites where waste products are kept, and they serve as media for breeding microbes. also, these dumpsites serve as breeding sites for flies which while feeding could also convey microbes from one place to another thereby affecting the health of the community. apart from garbage dumps, we also observed the highest number of bacteria from human house and hospital which may be due to improper waste disposal and environmental sanitation. our finding is in tandem with the study of nazari et al. (25) that also reported in large numbers the isolation of bacteria from hospital and non-hospital environments which they attributed to a low level of general hygiene. escherichia coli was the commonest bacterial sp isolated in the slaughterhouse (n= 3), http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 93 http://jad.tums.ac.ir published online: march 31, 2020 human house (n= 8), eatery (n= 6) and garbage dump (n= 9). however, k. pneumoniae was the commonest bacteria sp isolated from the hospital (n= 7). klebsiella spp and e. coli are gramnegative organisms that occupy a variety of niches like other members of enterobacteriaceae. their isolation from nearly all sources may not be unconnected with their ubiquity, and with the fact that house flies feed mainly on feaces and other animal waste, which is a rich source of enteric bacteria (26). escherichia coli is known to cause diarrhoea including traveller's diarrhoea, and haemolytic uremic syndrome, which people can contract by eating contaminated food. isolation of this pathogen from the eatery, the slaughterhouse, the human house could lead to outbreaks of e. coli gastroenteritis as these insects may deposit the pathogens when they feed on food that people consume (27). we observed a preponderance of isolates of proteus from the slaughterhouse and isolates of salmonella from the eatery. this observation is in line with the findings of urban and broce (28) that reported proteus spp. as the most common bacteria among gram-negative bacteria isolated from flies that were associated with raw meat, followed by providencia spp., pseudomonas spp., and salmonella spp. (29). proteus species are well known as human opportunistic pathogens and intestinal microorganisms indicating fecal pollution of water or soil. their isolation from flies from the slaughterhouse may be due to the use of such contaminated water for meat processing. in addition, proteus spp. are able to produce volatile components such as putrescine and ammonia, which are important for their swarming ability and, are also able to attract flies to animal carcasses (30). the isolation of salmonella spp from eateries may portend grave danger to public health as this pathogen is associated with gastroenteritis. of all the different bacterial species isolated from flies in this study, s. aureus was the only gram-positive organism. this organism was isolated from nearly all the locations we sampled demonstrating its ubiquity. our finding supports previous reports indicating that fly can carry s. aureus (25, 31). staphylococcus aureus, an opportunist pathogen, is responsible for a number of human diseases ranging from skin lesions, wound infections and food poisoning to more serious conditions, such as osteomyelitis, endocarditis and septicaemia (32). therefore, its isolation from the human house and the slaughterhouse portends serious danger to public health. the results of antibiotic susceptibility tests showed that all isolates were multidrug-resistant. all isolates of p. aeruginosa, salmonella spp, and proteus spp were resistant to streptomycin, cotrimoxazole, augmentin, and amoxicillin. isolates of e. coli were commonly resistant to augmentin and cotrimoxazole. the least resistance rates were exhibited by k. pneumonia, e. coli and s. aureus to ciprofloxacin. even though, there is a paucity of data on the occurrence of antimicrobial resistance in bacteria associated with flies in nigeria, independent studies across the globe have nevertheless emphasized the role of flies in the dissemination of resistant bacteria. in a recent global review of the role of flies in the spread of antimicrobial resistance, onwugambaa et al. (33) revealed that ‘filth flies’ are colonized with clinically relevant antimicrobial resistant bacteria, such as extended-spectrum beta-lactamase, carbapenemase-producing, and colistinresistant bacteria. in iran, davari et al. (5). isolated cephalexin, chloramphenicol, ampicillin, and tetracycline-resistant bacteria from flies, with resistance against the antibiotics above 32.5%. in a similar study conducted in china, multidrug-resistant enterococci, staphylococci, e. coli, k. pneumoniae, p. aeruginosa and aeromonas hydrophila were identified in flies that were collected beside poultry feeding operations (34, 35). likewise, wei et al. showed that antibiotic resistant bacteria can persist in the gut of house and green bottle flies. animal http://jad.tums.ac.ir/ http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=aeromonas+hydrophila j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 94 http://jad.tums.ac.ir published online: march 31, 2020 remains, garbage, hospital waste, and sewage samples are potential sources of resistant bacteria. hence, flies that are exposed to these sources can easily pick up and disseminate resistant bacteria (36). interestingly, some multidrug-resistant bacteria particularly, klebsiella, pseudomonas, and s.aureus were isolated from hospital environments. therefore, hospitals houseflies may also participate more in the spread of antibiotic resistance in the environment. the occurrence of multidrug resistance in clinical isolates is a serious problem due to the waning number of antibiotics used to treat human infections (2, 37). data from independent studies suggest a link between the antibiotic resistance of food of animal origin, the antibiotic resistance of clinical isolates, and community health (38, 39). nevertheless, this link remains a contentious issue because of insufficient information on the ecology of antibiotic resistance and virulence genes in the environment (36, 37). conclusion in conclusion, this present study indicates that house flies (m. domestica) are carriers of pathogenic bacteria that pose a possible health risk to communities. the isolated bacteria were resistant to various commonly used antibiotics. in view of the findings of this study, there is a need for public health education programmes and awareness to be given to the peasants, elites, and patients in the study environment and epidemiological surveillance of food vending joints, major water sources, and hospitals to prevent ingestion of contaminated food and water. suitable steps should be taken to control the flies and monitor the susceptibility pattern of the pathogens they carry. further study is needed to determine the type and diversity of all microorganisms spread by flies as well as the epidemiology of the resistant bacteria they carry. acknowledgements we thank mrs oluwabunmi esther yinka and mr ogunleye festus ajayi for their technical support. the authors declare that there is no conflict of interest. references 1. kumarasamy kk, toleman ma, walsh tr, bagaria j, butt f, balakrihnan r, chaudhary u, doumith m, giske cg, irfan s, krishnan p, kumar av, maharjan s, mushtaq s, noorie t, paterson dl, pearson a, perry c, pike r, rao b, ray u, sarma jb, sharma m, sheridan e, thirunarayan ma, turton j, upadhyay s, warner m, welfare w, livermore dm, woodford n (2010) emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study. lancet infect dis. 10: 597–602. 2. who (2001) who global strategy for containment of antimicrobial resistance. available at: https://www.who.int/drugresistance/wh o_global_strategy.htm/en/ 3. nmorsi opg, agbozele g, ukwandu ncd (2007) some aspects of epidemiology of filth flies: musca domestica, musca domestica vicina, drosophilia melanogaster and associated bacteria pathogens in ekpoma, nigeria. vector-borne zoonotic dis. 7: 107–117. 4. bouamamaa l, sorlozano a, laglaoui a, lebbadi m, aarab a, guterrez j (2010) antibiotic resistance patterns of bacterial strains isolated from periplaneta americana and musca domestica in tangier, morocco. j infect dev 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yeasts from adult house fly (diptera: muscidae) captured from the hospital environments in ahvaz city, southwestern iran. j med entomol. 52: 1351–1356. 9. hemmatinezhad b, ommi d, hafshejani tt, khamesipour f (2015) molecular detection and antimicrobial resistance of pseudomonas aeruginosa from houseflies (musca domestica) in iran. j venom anim toxins incl trop dis. 21: 18. 10. agbelade ad (2013) land use mapping and tree species diversity of federal university of technology, akure. am int j contempt res. 3(2): 104–113. 11. fawole mo, oso ba (2007) laboratory manual of microbiology, revised edition. spectrum books, ibadan, p. 23. 12. olutiola po, sonntag, hs famurewa o (measurement of microbial growth. in: an introduction to general microbiology (a practical approach). p. 111 13. cheesbrough m (2006) district laboratory practice in tropical countries part ii. cambridge university press. 113: 319–329. 14. clinical and laboratory standards institute (2013) m100-s11, performance standards for antimicrobial susceptibility testing. clin microbiol newsl. 23(5): 49. 15.pai hh, chen wc, peng cf (2003) isolation of non-tuberculous mycobacteria from hospital cockroaches (periplaneta americana). j hosp infect. 53: 224–228. 16. barreiro c, albano h, silva j, teixeira p (2013) role of flies as vectors of foodborne pathogens in rural areas. isrn microbiol. 2013: 718780. 17. oyeyemi ot, agbaje mo, okelue ub (2016) food-borne human parasitic pathogens associated with household cockroaches and houseflies in nigeria. par epide con. 1: 10–13. 18. alam mj, zurek l (2004) association of escherichia coli o157:h7 with houseflies on a cattle farm. appl environ microbiol. 70: 7578–7580. 19. rahuma n, ghenghesh ks, ben aissa r, elamaari a (2005) carriage by the housefly (musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in misurata, libya. ann trop med parasitol. 99: 795– 802. 20. macovei l, miles b, zurek l (2008) potential of houseflies to contaminate readyto-eat food with antibiotic-resistant enterococci. j food prot. 71: 435–439 21. chakrabarti s, kambhampati s, zurek l (2010) assessment of house fly dispersal between rural and urban habitats in kansas, usa. j kans entomol soc. 83: 172–188. 22. ahmad a, ghosh a, schal c, zurek l (2011) insects in confined swine operations carries a large antibiotic resistant and potentially virulent enterococcal community. bmc microbiol. 11: 23. 23. sulaiman s, othman mz, aziz ah (2000) isolations of enteric pathogens from synanthropic flies trapped in downtown kuala lumpur. j vector ecol. 25: 90–93. 24. vartivarian se, papadakis ka, anaissie ej (1996) stenotrophomonas (xanthomonas) maltophilia urinary tract infection: a disease that is usually severe and complicated. arch intern med. 156: 433–435. 25. nazari m, mehrabi t, hosseini sm, http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 88–96 b odetoyin et al.: frequency and antimicrobial … 96 http://jad.tums.ac.ir published online: march 31, 2020 alikhani my (2017) bacterial contamination of adult house flies (musca domestica) and sensitivity of these bacteria to various antibiotics, captured from hamadan city, iran. j clin diagn res. 11(4): dc04–dc07. 26. khamesipour f, lankarani kb, honarvar b, kwenti te (2018) a systematic review of human pathogens carried by the housefly (musca domestica l.). bmc pub health. 18: 1049. 27. kobayashi m, agui n, sasaki t, saito n, tamura k, susuki, watanabe h (1999) houseflies: not simple mechanical vectors of enterohemorrhagic escherichia coli o157:h7. am j trop med hyg. 61: 625– 629. 28. urban je, broce a (1998) flies and their bacterial loads in greyhound dog kennels in kansas. curr microbiol. 36: 164–170. 29. drzewiecka d (2016) significance and roles of proteus spp. bacteria in natural environments. microb ecol. 72: 741–758. 30. ma q, fonseca a, liu w, fields at, pimsler ml, spindola af, tarone am, crippen tl, tomberlin jk, wood tk (2012) proteus mirabilis interkingdom swarming signals attract blow flies. isme j. 6: 1356–1366. 31. nayduch d, cho h, joyner c (2013) staphylococcus aureus in the house fly: temporospatial fate of bacteria and expression of the antimicrobial peptide defensin. j med entomol. 50: 171–178. 32. tong syc, davis js, eichenberger e, holland tl, fowler vg (2015) staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. clin microbiol rev. 28: 603–661. 33. onwugamba fc, fitzgerald jr, rochon k, guadabassi l, alabi a (2018) the role of ‘filth flies’ in the spread of antimicrobial resistance. travel med infect dis. 22: 8–17. 34. liu y, yang y, zhao f, fan x, zhong w, qiao d, cao y (2013) multi-drug resistant gram-negative enteric bacteria isolated from flies at chengdu airport, china. southeast asian j trop med public health. 44: 988–996. 35. graham jp, price lb, evans sl, graczyk tk, silbergeld ek (2009) antibiotic resistant enterococci and staphylococci isolated from flies collected near confined poultry feeding operations. sci total environ. 407: 2701–2710. 36. wei tty, miyanaga k (2014) persistence of antibiotic-resistant and -sensitive proteus mirabilis strains in the digestive tract of the housefly (musca domestica) and green bottle flies (calliphoridae). appl microbiol biotechnol 98: 8357–8366. 37. witte w (1999) antibiotic resistance in gram-positive bacteria: epidemiological aspects. j antimicrob chemother. 44 suppl a: 1–9. 38. salyers aa (2002) an overview of the genetic basis of antibiotic resistance in bacteria and its implications for agriculture. anim biotechnol. 13: 1–5. 39. smith dl, harris ad, johnson ja, silbergeld ek, morris jg (2002) animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. proc natl acad sci. 99: 6434–6439. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 78 http://jad.tums.ac.ir published online: march 31, 2020 original article relationship between some environmental and climatic factors on outbreak of whiteflies, the human annoying insects abedin saghafipour1; *alireza zahraei-ramazani2; hassan vatandoost2,3; amin asadollahi2; reza fouladi-fard4; amir hamta5; ali hasanwand2 1 department of public health, faculty of health, qom university of medical sciences, qom, iran 2 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3 department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4 research center for environmental pollutants, qom university of medical sciences, qom, iran 5 clinical research development center (crdu), qom university of medical sciences, qom, iran (received 27 mar 2019; accepted 21 mar 2020) abstract background: the reports of numerous outbreaks of whiteflies from different parts of the world have increased its medical importance. the aim of this study was to determine relationship between environmental changes and climatic factors with the outbreak of the whitefly population in tehran, the capital of iran. methods: this study was carried out in urban areas of tehran, where the increasing population of whiteflies was reported frequently during 2018. in order to entrap the whiteflies, 20 yellow sticky cards smeared with white refined grease were installed on the trunks of the trees at twice per month as trapping time intervals. the captured flies were transferred and conserved in cans containing 70% alcohol and were counted accurately under a stereomicroscope. to determine the relationship between air quality index, precipitation, air temperature and air humidity as environmental and climatic factors with the abundance of whiteflies, change point analysis and generalized estimating equations (gee) was used. results: the most density of white flies per trap was 256.6 and 155.6 in early october and late september respectively. the number moved closer to zero from november to april. the population of whiteflies was inversely correlated with the level of air quality index (p= 0.99) and precipitation (p= 0.95), and it had a direct correlation with the high temperature. also, the population of whiteflies had a direct correlation with the level of air humidity in the first half of the year conclusion: according to these findings, during spring and summer from early may to early october. keywords: change point analysis; climatic factors; environmental change; outbreak; tehran introduction whiteflies (hemiptera: aleyrodidae) feed on a wide range of hosts in a way that for some species more than 900 plant species have been identified (1, 2). cucurbits and ornamental plants, agricultural crops, palms, and weeds are the main hosts of this pest, though there are many weeds which are the secondary hosts (3). the life cycle of whiteflies from egg to adult complete one month depending upon environmental temperature. adult whiteflies may be surviving for one to two months (4). this insect is con sidered a health problem and an important medical pest that can also threaten human safety in some cases. accidental entry of a whitefly into the human respiratory tract can cause inflammation and infection in the upper respiratory tract leading to the emergence of opportunistic fungal and bacterial infections (5). the population of this insect has increased in many parts of the world which has caused many problems for humans, especially in urban areas (6). according to experts, repeated and uncontrolled use of various formulations and concentrations *corresponding author: dr alireza zahraeiramazani, e-mail: azahraei@tums.ac.ir http://jad.tums.ac.ir/ mailto:azahraei@tums.ac.ir j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 79 http://jad.tums.ac.ir published online: march 31, 2020 of pesticides can have many adverse effects, such as the resistance of pests to pesticides, the emergence of new pests, and the eradication of natural enemies (parasitoids and predators). whiteflies are among the pests that have been evolved by continuous use of chemicals and the lack of proper management of pesticides (7). in addition to direct physical and biological harm for human, these insects cause a sharp decline in the production of agricultural products. also, these flies cause the growth and development of saprophytic fungi on their honeydew which reduces the quality and nutritional value, as well as the consistency and shape of the products. moreover, they physically damage non-productive plants. nowadays, whiteflies, due to their increased resistance to various types of pesticides (8), cause more damage to a large number of crops and ornamental plants. male and female winged insects feed on the leaf juice of plants. this leads to yellow spots on the leaves that directly damage the host plant and make it seem short and sick. insect secretions on plant leaves can cause fungal growth (9). so far, about 1556 species of whiteflies have been identified in different parts of the world (10). whiteflies in iran were identified for the first time during the faunistic research and the identification of flies in fars province in 1995 (11). after that 14, 18 and 24 species of whiteflies have been reported in isfahan (9), gilan (12) and golestan provinces respectively (13). subsequently, in 2000, morphological and biological studies were conducted on common species in esfahan, which revealed that whiteflies in esfahan were of the european race (9). such flies are greenhouse pests, but, unfortunately, the lack of proper management of chemicals and pesticides has caused resistance to some of the pesticides and adverse environmental conditions in these insects. as a result, these flies have been able to adapt themselves to greenhouse conditions and easily grow and reproduce. some experts also believe that these flies have been released into the open air through whitefly infestations, and since the ecosystem cycle and the population of predator insects have not been balanced in the environment, this has led to a widespread outbreak of whiteflies in the open air (14). moreover, since these insects are expected to reproduce in places more similar to greenhouse in terms of climate and food resources, it is necessary to identify the ecological factors in the reproduction of the insects, and take measures to control them outdoors (15). over the past few years, the outbreak of whiteflies in iran, especially in the residential areas of tehran, has caused different allergies to humans. therefore, the present ecological study was conducted with the aim of determining the relationship between some environmental and climatic factors on outbreak of whiteflies, the human annoying insects tehran; district 6 in 2018. materials and methods study area the current study was carried out in the urban area of tehran; the capital of iran (district 6) suffers from a severe air quality index, where the abundance of whiteflies has been reported frequently during 2018 (fig. 1). the city of tehran is located between the mountainous region and the plain. tehran's climate is generally described as mild in spring and fall, hot and dry in summer, and cold and wet in winter. based on the 2016 census, tehran has a population of approximately 10 million. vegetation coverage in tehran is including natural and hand-planted forests (16). the air pollution indicators including air quality index (aqi) with monthly activity of whiteflies were received from the iran meteorological organization (17). study design to entrap whiteflies, 20 yellow sticky cards (fly traps) of 30×50cm (18), depending on the diameter of the tree, were installed on the trees each time at twice per month as trapping time intervals from early april to late march 2018. totally 480 yellow sticky cards have been aphttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 80 http://jad.tums.ac.ir published online: march 31, 2020 plied for catching adult whiteflies. in addition, we replaced the old traps with new one in each visit. the chemicals on the traps were odorless and smeared with purified white grease purchased wholesale from reputable stores. the traps were installed on the trees of maple, acacia, european ash, sycamore, and berries for the purpose of catching whiteflies. in this regard, to examine the effect of colorful traps on whiteflies, three different colors of yellow, blue and green for attracting and capturing of them were used in a selected greenhouse. in this study, colored cards of 10×22cm in two heights of 1.3 and 2.3m were installed on tomato plants. after two weeks, the numbers of trapped insects on colored cards were counted. whiteflies were isolated and counted in two ways: traps (sticky cards) were immersed in warm water for 2 minutes (min) until the grease on the surface containing the flies was softened, and the flies were released in hot water. then, the insects were removed from the hot water using a brush and were transferred and conserved in cans containing 70% alcohol. then, at appropriate times, the whiteflies in the canned glass were released into the appropriate plates and carefully counted under a stereomicroscope. in another method, the traps containing whiteflies were peeled off the trees, and given the grid pattern on the surface of traps, the number of flies in every grid was randomly counted and the total number of whiteflies was estimated on the surface of the traps. the yellow and blue colored traps used in this study were supplied by russel ipm company; and the green traps were made of tangle foot adhesive supplied by kerman chemistry company (applied on green cards with brush). statistical analysis change analysis is organized to answer two questions 1) whether there are any change points or not 2) if so in which times change point(s) occurs. we deal with hypothesis test and estimation in first and second question, respectively. generally, suppose x1, x2, …, xn to be sequence of independent random variables with probability distribution function f1, f2, …, fn respectively. change point analysis intended to test following null hypothesis h0: f1= f2= … = fn versus alternative hypothesis h1: f1= … ≠ … = ≠ = ≠ = … = where 1< k1< k2< … < k q< n and q is unknown parameter that shows number of change points and k1, k2, …, xq are the change points and have to be estimated. although in majority of studies which point analysis has been applied probability distribution function supposed to be normal (19), in current study with the respect to response variable, it is let be poisson. so our problem to detect change point (s) refor to x1, x2, …, xc that are consequence of poisson random variable with parameters , i= 1, 2, c respectively. the aim was to test following null hypothesis h0: 1= … c versus alternative hypothesis that shows there are k unknown change points. likelihood function based on null hypothesis is: so likelihood estimation based on that is: and under alternative hypothesis likelihood function is obtained by letting , , the likelihood estimation function of and are given by , . sic 1 under null hypothesis is found as and corresponding alternative hypothesis determined from 1 schwarz information criterion http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 81 http://jad.tums.ac.ir published online: march 31, 2020 so, according to the information criterion principle was rejected if . in order to estimating change point(s) was used (19, 20). after determination of change point (s) in the next stage for considering correlation between data, which were collected over time, marginal longitudinal model was applied. link function because of being count response variable, log poisson fulfilled. coefficients were estimated by generalized estimating equations (gee). this method provides predictive model for the response variable by explanatory variables and it takes into account possible correlation between repeated measures of depend variable of a subject. in this study since data are collected over time it is likely that repeated measure on an individuals are correlated. when gee method is used in order to analyze longitudinal data, correlation structure is formulated and possible correlations between measurements over time is incorporated (21). suppose that is a count and we are interested to investigate changes in expected count to the covariates. since counts are often modeled as poisson random variable, using poisson variance function and log link function, marginal model for would be illustrated as follow: the mean of ( is related to the independent variables by log link function: also the variance of each depends on : in addition to these, an unstructured pairwise correlation pattern is assumed for the within-subject association among the repeated responses vector: the vector of parameters shows the pairwise correlation among responses. the marginal model specified above is a log linear regression model, with an extra-poisson variance assumption (22). so, to determine the relationship between air quality index, precipitation, air temperature and air humidity as environmental and climatic factors with the abundance of whiteflies this model was applied. the goodness of fit of model was evaluated by qic. results the density of whiteflies per trap in different seasons were calculated during the 12-month period of sampling in district no. 6 of tehran in 2018. the most density of white flies per trap was 256.6 and 155.6 in early october and late september respectively due to low temperature and rainfall and high humidity (fig. 2). the density of whiteflies per trap in other times of year was shown in table 1. the number moved closer to zero from november to april. the population of whiteflies was inversely correlated with the level of air quality index (p= 0.99) and precipitation (p= 0.95), and it had a direct correlation with the high temperature. also, the population of whiteflies had a direct correlation with the level of air humidity in the first half of the year, and it was inversely correlated in the final months of the year. in the current study, up to the 13th whiteflies trapping (early october), using the marginal model, the estimates were calculated by the gee method, and considering the poisson link function, the following results were obtained: a) for a unit of increase in air quality index (aqi), the population of whiteflies decreased by 0.98 (decreases). b) for a unit of increase in temperature, the population of whiteflies increased by 1.18 (increases). c) for a unit of increase in humidity, the population of whiteflies increased by 1.04 (increases). d) for a unit of increase in precipitation, the population of whiteflies decreased by 0.99 (dehttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 82 http://jad.tums.ac.ir published online: march 31, 2020 creases) (table 2). in this method, all four variables were significant. in addition, the following results were obtained after the 13th measurement (early october): a) for a unit of increase in air quality index (aqi), the population of whiteflies decreased by 0.98 (decreases). b) for a unit of increase in temperature, the population of whiteflies increased by 2.37 (increases). c) for a unit of increase in humidity, the population of whiteflies decreased by 0.92 (decreases). d) for a unit of increase in precipitation, the population of whiteflies decreased by 0.44 (decreases) (table 3). in this method, none of the four variables were significant. in both gee models, qic showed models were well-fitted. table 1. comparison of air quality index (aqi) with monthly activity of whiteflies, tehran, iran in 2018 variable month density of whitefly per trap air quality index (aqi) early april 00.00 71.52 late april 00.00 71.68 early may 22.55 75.53 late may 24.45 79.56 early june 24.70 82.93 late june 24.75 94.31 early july 24.55 93.67 late july 25.45 87.81 early august 34.00 89.47 late august 50.00 90.75 early september 127.8 102.73 late september 155.6 100.68 early october 256.6 88.00 late october 99.25 84.47 early november 00.00 93.06 late november 00.00 128.12 early december 00.00 136.15 late december 00.00 138.14 early january 00.00 142.28 late january 00.00 145.85 early february 00.00 121.45 late february 00.00 112.15 early march 00.00 110.59 late march 00.00 108.14 table 2. generalized estimating equation (gee) models predicting number of flies before 13th measurement, tehran, iran, 2018 variable coefficient s.e. pvalue air pollution 0.98 0.001 <0.001 temperature 1.18 0.004 <0.001 humidity 1.04 0.003 <0.001 rain fall 0.99 0.000 <0.001 constant 12.98 2.65 <0.001 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 83 http://jad.tums.ac.ir published online: march 31, 2020 table 3. generalized estimating equation (gee) models predicting number of flies after 13th whiteflies trapping, tehran, iran in 2018 variable coefficient s.e. pvalue air pollution 0.98 7.40 0.99 temperature 2.37 3.31 0.53 humidity 0.92 <0.0001 rain fall 0.44 6.35 0.95 constant 1 556.34 1.00 fig. 1. geographic location of the study area in district 6, municipality of tehran, iran fig. 2. average monthly rainfall, temperature and humidity, tehran, iran in 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 84 http://jad.tums.ac.ir published online: march 31, 2020 fig. 3. change point of whiteflies activity, tehran, iran, 2018 (n= number of collected whiteflies; index= times of trapping) discussion the results of this study showed that the population of the whiteflies was inversely correlated with the level of air quality index and precipitation, and it had a direct correlation with high temperature. also, the population of whiteflies had a direct correlation with the level of air humidity in the first half of the year, and it was inversely correlated in the final months of the year. modern transportation and rapid trading of plants, cuttings, branches and other parts of plants, which often contain eggs, larvae and whitefly nymph, have led to the transfer of these pests into new places. however, there may be few whiteflies that enter a new place independently and without encountering the natural enemies with which they are evolving. appropriate level of humidity and temperature is another factor for the developing population of whiteflies. the whiteflies were commonly able to complete their life cycle including egg, larvae, nymph and adult in the temperature of 15–35 °c while survival was usually reduced at temperatures <20 °c or >30 °c (23, 24). the rate of survival of the whiteflies at unfavorable low and high temperatures were also affected by host plants (25). for instance, according to life span of mulberry trees in tehran city, white flies strongly infest these trees (26) and when the leaves fall, the whiteflies population also decreases. importing of seedlings and cuttings of flowers, as well as the types of wood susceptible to the growth of whiteflies are one of the most important factors in the spread of whiteflies in different places. whiteflies unable to fly long distances (27). also, lack of precipitation is another factor in the survival of whiteflies. the high level of pollutants containing co2 in the air of tehran, due to defective fuel consumption of worn out vehicles and other airborne pollution, is another important factor in the survival of whiteflies in urban areas since these flies live in conditions similar to greenhouse. it should be mentioned that, in fact, the high level of pollution and weather conditions are important factors in the reproduction of whiteflies in tehran (28). the abundance of whiteflies in tehran can cause health problems including itching, red and sore eyes, runny nose, allergies, and problems in the respiratory system of individuals, especially asthmatics (29). children and people with poor immune system, the elderly populations and pregnant women are more susceptible to these problems. also, the presence of whiteflies in food and beverages, besides causing fear, is concerning in terms of contamination with pathogenic microorganisms. therefore, this study was conducted in different months of the year to consider the effects of temperature, precipitation, humidity and environmental contaminants. the materials on the wings and bodies of the whiteflies can act as the pollen and cause allergic reactions in individuals (30). rain and low humidity (below 60%) and low or high temperatures disrupt development of the insects. whiteflies stay in one place because they cannot migrate long distances. the longest reported displacement of this insect is 7km (31, 32). whiteflies can also cause problems for the eyes and respiratory tracts (31). the reasons for eradicahttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 78–87 a saghafipour et al.: relationship between … 85 http://jad.tums.ac.ir published online: march 31, 2020 tion of the natural enemies of whiteflies include the destruction of their reproductive habitats, uncontrolled use of authorized and unauthorized pesticides, and the lack of using specialized biological controlling methods. these factors influence the growth of whiteflies in metropolitan cities like tehran. reducing the use of smoky and pollutant vehicles and monitoring the factories that produce vehicles with incomplete combustion (33, 34) is one of the major factors in making the environment unstable for whiteflies. the use of different least-hazardous pesticides to reduce the resistance of whiteflies, as well as the use of systemic toxins for nonproductive trees (35), and reducing the use of insecticides as the only methods of controlling whiteflies, and ultimately the use of growth regulators and integrated pest management (36) will help control these pests in such cities as tehran. conclusion it should be mentioned that, in fact, the high level of pollution in different times of year and weather conditions are important factors in the reproduction of whiteflies in tehran. according to these findings, during spring and summer from early may to early october that temperature and humidity for development of withe flies are supplied in tehran city, personal protection against these pests was recommended by tehran residents. acknowledgements this study was part of a postgraduate thesis in medical entomology and vector control, supported financially by tehran university of medical sciences (grant number: 29826). ethical clearance was earned from the institutional ethics committee of tehran university of medical sciences. authors thankful for the support of the department of medical entomology and vector control sincerely. the authors declare that there is no conflict of interest. references 1. doukas d, payne cc (2007) greenhouse whitefly (homoptera: aleyrodidae) dispersal under different uv-light environments. j econ entomol. 100: 389–397. 2. sadeh d, nitzan n, shachter a, chaimovitsh d, dudai n, ghanim m (2017) whitefly attraction to rosemary (rosmarinus officinialis l.) is associated with volatile composition and quantity. plos one. 12: e0177483. 3. de barro pj, liu ss, boykin lm, dinsdale ab (2011) bemisia tabaci: a statement of species status. annu rev entomol. 56: 1–19. 4. bogran ce, heinz km (2002) whiteflies. texas agricultural extension service. p.7. 5. tracy jm (2011) insect allergy. mt sinai j med. 78: 773–783. 6. polston je, anderson pk 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https://www.ncbi.nlm.nih.gov/pubmed/21624919 possible synergistic effect of combined metarhizium anisopliae and fenitrothion for control of german cockroach (blattella germanica) as a novel approach http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... original article possible synergistic effect of combined metarhizium anisopliae and fenitrothion for control of german cockroach (blattella germanica) as a novel approach mohammad reza khaksar1, majid kababian2, roohollah fateh3,*abedin saghafipour4, hassan vatandoost5, 6, mona sharififard7 1department of occupational health, faculty of health, qom university of medical sciences, qom, iran 2student research committee, qom university of medical sciences, qom, iran 3cellular and molecular research center, qom university of medical sciences. qom, iran 4department of public health, school of health, qom university of medical sciences, qom, iran 5department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 6department of environment chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 7department of medical entomology and vector control, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran *corresponding author: dr abedin saghafipour, e-mail: abed.saghafi@yahoo.com (received 01 sep 2020; accepted 30 aug 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: cockroaches are one of the most common pests in many residential areas. in this study, the simultaneous effects of fungi, metarhizium anisopliae and fenitrothion-coated baits on the mortality rate of the german cockroach nymphs were investigated. methods: to determine the lethal level of fenitrothion insecticide, a bioassay test was performed on the last instar nymphs of the german cockroach reared at insectarium conditions. various toxic concentrations of fenitrothion (0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.5%, and 2%) were used. different concentrations of m. anisopliae (1×104, 1×105, 1×106, 1×107, 1×108 conidia/ ml) were also applied to nymphs. eventually, we combined the effective dose of fenitrothion (0.93%) with the effective concentration of m. anisopliae (6.6 ×106 conidia/ml) to provide the fungus-coated bait to attract insects. mortality was recorded 24–96 hours after exposure to the toxic bait. the resulting data were subjected to probit analysis. results: the results of applying m. anisopliae spores with fenitrothion composition showed that the mortality rate of german cockroach nymphs was significant. therefore, the optimal dose of fenitrothion used in combination with m. anisopliae seems essential to reduce the german cockroach nymphs. conclusion: the results of this study can be considered a suitable method as a mixture with low cost and minimal damage to the environment and other organisms. keywords: blattella germanica; metarhizium anisopliae; fenitrothion; synergism introduction the german cockroach, (blattella germanica), is considered one of the most annoying pests in the world because of its widespread distribution in residential areas (1-2). of four major cockroaches (american, oriental, german, and brown315 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... banded cockroach), the german cockroach (b. germanica) is the most common and most frequently reported cockroach that has developed insecticide resistance (3). thus, their control is very important because they are capable of carrying a variety of pathogenic bacteria, viruses and fungi that can be dangerous to human health (2, 4-5). currently, the german cockroach is resistant to many common insecticides because it has been exposed to pesticides continuously and for a long time; therefore, further exposure to pesticides cannot seriously harm the german cockroach (5-7). thus, regarding some adverse consequences of using insecticides, such as the persistence of toxins in food, development of potential cancers, exposure of vertebrates and nontarget insects, environmental pollution and development of insect resistance, it is essential to applicate more effective pesticides to ensure safety and efficiency (8-9). it seems that in order to prevent insecticide resistance forcing us to use more doses, first, we need to use insecticides that have more effects on pests, and secondly, we can enhance the effects of other factors resulting in a lower dose of insecticides (10). the result is that, in addition to reducing insecticide consumption and reducing costs, we can reduce the adverse effects of insecticides on the ecosystem and human health. in line with other parts of the world, insecticides are being used in iran to control the german cockroach (5). cockroaches collected from homes, dorms, restaurants and hospitals have shown varying degrees of resistance to insecticides from organochlorines, organophosphates, carbamate and pyrethroids insecticides groups. the highest level of environmental pollution is related to organochlorine, organophosphate and carbamate insecticides and the least of it is related to pyrethroids (9, 11). thus, since heavy use of these compounds has harmful impacts on the life of living organisms in the environment as well as humans, it is necessary to look for agents or factors that are less hazardous to the environment in addition to pest disposal. there are methods for the biological control of insects using fungi insecticides which can be very effective in terms of pest control and high reliability (12). to fight against insects, more than 700 species of fungi have been identified as entomopathogenic agents (13). the entomopathogenic fungi (epf) found in nature are readily digestible and nonpathogenic to livestock and plants (14). it has also been observed that the results of exposure to metarhizium anisopliae fungus in reducing the number of the german cockroachs is evident, and these results include m. anisopliae species and their impact on killing the german cockroach (15). there are several reports on the use of m. anisopliae in combination with inorganic insecticides as solid constituents in the prey of the german cockroach. also, there has been no well-documented study of the use of fungal pathogens in beetle prey in iran (16). the assessment of conidiadust formulation of m. anisopliae strain iran 437 has shown that this insecticide is highly effective in reducing the number of german cockroaches which have been recently widely distributed in ahwaz city, khuzestan province, southwestern iran, especially in wards and hospitals (8, 17). in 2007, hartelt and colleagues investigated the effects of entomopathogenic fungi at different nymphal stages of ixodes ricinus ticks and observed that all the tested fungi were effective on i. ricinus. in another study, it was reported that m. anisopliae strain 97 was the most effective type of fungus (18). various studies have been conducted on different pest-producing products reporting that some species of entomopathogenic fungi have desirable insecticidal effects (19). in a study conducted on the relationship between cone germination growth and fungal growth with nine conventional pesticides, it was observed that the efficiency of pesticides was compatible with entomopathogenic fungi because some pesticides had the potential to inhibit the germination of entomopathogenic fungi in vitro. if used at low concentrations with the fungus, their 316 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... effect on the germination of the fungus is negligible, which occurs only in vitro; in the in vivo environment, there is little effect on their virulence against the target insect, so they can be successfully used for various pest-control purposes (20-22). in this study, the synchronous effects of entomopathogenic fungus (m. anisopliae) and fenitrothioncoated baits on the mortality rate of the german cockroach (blattella germanica) were investigated. materials and methods rearing german cockroaches the german cockroach nymphs were reared under insectarium conditions at the department of public health, faculty of health at qom university of medical sciences. in this study, the last instar nymphs of the german cockroach were used for the experiments. plastic boxes were used to hold cockroaches with food as prey, including 10.5 g of rice bran, wheat bran, flour, sugar and rodent food. the environmental condition was 27±2 °c, 60±10% relative humidity and a photoperiod of 12:12h (l: d). nymphs were anesthetized with carbon dioxide gas and sealed in a container with a lace. fungi resource entomopathogenic fungus (m. anisopliae, iranian strain v245) was obtained from the stock of the regional center for the collection of industrial fungi and bacteria of iran with the code number of ptcc 2881. the fungi were cultured on pda (potato dextrose agar) medium and kept in relative humidity of 70±5% and 28 °c± 2 for two weeks. the conidia were scraped of 14-day-old culture with a sterile metal loop and a suspension of conidia was then diluted as the determinate concentration in conidia/ml by using tween 80 solution 0.01% in physiological serum. the resulting mixture was stirred for 10 minutes with a magnetic stirrer. the concentrations of conidia suspension were performed by neobar lam while the direct count by using a hemocytometer (neubauer chamber) was down (23-24). chemicals and pesticides fenitrothion insecticide in the form of technical grade (95%) was used for toxicity testing and was purchased from moshkfam fars chemical co (mfc), iran. preparing the baits the baits were prepared to trap live german cockroaches (25). in order to prepare baits, a mixture of rice bran, wheat flour, rodent food and sugar was blended proportionally and gradually distilled with water to obtain a consistent semi-solid food in various weights up to about 10.5 grams. subsequently, the same baits were exposed to different concentrations of fenitrothion solution according to the doses prescribed in the experiments. baits were inoculated with 1ml of the conidia suspension of m. anisopliae isolate iranian strain v245 with intended doses in conidia/ml (22). then, the last instar nymphs were placed in the plastic container and remained without a food source for 72 h to be kept in a starvation status. thereafter, baits containing different doses of insecticide and conidia suspension were given to the cockroaches for 3 hours. after the exposure time, treated baits were replaced with untreated ones. for each bioassay test, concentrations were applied for at least four times. each test included a control group which fed on untreated bait. mortality rate was recorded daily until all samples died; this procedure was repeated for two weeks. bait bioassay the german cockroach insectary strain, kept at insectarium conditions and clean containers, was used. seven concentrations of insecticides, including 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.5%, and 2%, were tested by control dish with four replicates. the control group was designed as follows: one group consisting of 10 nymphs that received water with the prey dry composition. for each target concentration of insecticide as well as control tests, 10 cockroach nymphs were exposed four times by contact exposure 317 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... test; thus, a total of 320 cockroaches were required for the insecticide resistance test. for entomopathogenic fungi bioassay, five different concentrations of the aqueous suspensions of m. anisopliae, including 1×104, 1×105, 1×106, 1×107 to 1×108 conidia/ml, were regulated. for each concentration, 10 cockroach nymphs were exposed to inoculated bait containing one ml of the fungal suspension for each repetition and then were placed in plastic containers containing filter paper. the control group was designed as follows: one group consisting of 10 nymphs that received water with the prey dry composition. for each target concentration of fungi, 10 cockroach nymphs were exposed four times by contact exposure test; thus, a total of 240 cockroaches were required for fungi resistance test. synergism effect of fungi in this section, the effective dose of fenitrothion and fungal conidia included sublethal doses that had the best and highest lethality effects on the target insects, obtained through statistical analysis which included the effective dose of fenitrothion (0.93%) and m. anisopliae fungi (6.6×106 conidia/ml). cockroach nymphs were applied to test the insecticide and fungi bioassay for 10 cockroaches per container with four replications. the control group at this stage included one group comprising 10 nymphs that received water with the prey dry composition. also, a total of 80 cockroaches were required for the synergistic effect of fungi. finally, 640 german cockroaches were needed to complete all the bioassay tests. statistical analysis after the conversion of mortality data to percentages, the mean and standard error mortality rates for the intended concentrations of fenitrothion insecticide and fungal spores per each experiment were assessed from the first day to the fourth day after the exposure to the poisonous bait. by using the data obtained, the probit regression relationship was plotted as an equation among the death of german cockroach nymphs, insecticide concentrations and fungal spores by spss version 16. the significance level was set at p < 0.05 for all the analyses. results the results of the mortality rate of the german cockroach nymphs resulting from the exposure to fenitrothion insecticide are presented in table 1. it was observed that the mortality rate increased in accordance with the increase in the concentrations of insecticide as the mortality rate of nymphs ranged from 5% to 70% in the minimum and maximum concentrations in the first day of exposure. the highest mortality rate was observed on the fourth day ranging from 22.5% to 92.5% (p ˂ 0.001). therefore, there was a significant relationship between the increase in mortality due to the increase in insecticide concentration and the exposure table 1. the percentage of deaths of the german cockroach nymphs* based on insecticidal concentration the percentage of cockroaches wasted (mean±se) insecticidal concentration (%) first day second day third day fourth day 5.0±5.8 15.0±5.8 20.0±8.2 22.5±9.6 0.1 20.0±8.2 27.5±9.6 32.5±18.9 37.5±23.6 0.3 25.5±10.0 37.5±5.0 52.5±9.6 52.5±9.6 0.5 37.0±23.6 50.0±21.6 55.5±17.3 57.5±17.1 0.7 45.0±19.1 50.0±28.3 60.0±21.6 62.5±18.9 0.9 55.0±30.0 60.0±25.8 67.5±26.3 72.5±26.3 1.5 70.0±25.8 75.0±25.2 87.5±9.6 92.5±9.6 2.0 0.002 0.006 < 0.001 < 0.001 p-value *each treatment contained 10 german cockroach nymphs replicated four times. treatments were significantly effective at p < 0.05. table 1. the percentage of deaths of the german cockroach nymphs* based on insecticidal concentration 318 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... time from the first day to the fourth day. the best time to investigate mortality was 48 to 72 h after the treatment. the values of lc50 and lc90 were 1.29 mg/l and 2.76 mg/l respectively on the first day, decreasing to 0.61 mg/l and 2.08 mg/l with 95% confidence interval on the fourth day (table 2). the lethal dose values showed a significant decrease over time from the day of exposure as the deceasing amount of lc50 had a lower percentage of toxicity after exposure compared to lc90; thus, it can be concluded that this gradual decrease in insecticide concentration dose was very significant. the mortality rate of german cockroach nymphs with increased doses of fungal spores in four consecutive days is shown in table 3. the mortality rate of nymphs exposed to the lowest dose (10%) on the first day increased three-fold (30%) until the fourth day of exposure. moreover, the mortality rate of nymphs exposed to the highest dose of fungal spores 1×108 conidia/ml on the first day was 30%, increasing three-fold on the fourth day. the values of ld50 and ld90 on the first day were 5.4×108 conidia/ml and 3.6×1011 conidia/ml, respectively, decreasing to 2.3×105 conidia/ml and 1.5×109 conidia/ml on the fourth day (table 4). the lethal dose values showed a significant decrease over time from the first day of exposure, and the decreasing amount of ld50 showed a lower percentage of toxicity after the exposure day, table 2. the lc50 and lc90 concentrations of insecticide in the probit model of the death of the german cockroach nymphs on different days post exposure day lc90 (95% ci) lc50 (95% ci) p-value 1 2.76 (2.46, 3.14) 1.29 (1.11, 1.49) < 0.001 2 2.48 (2.21, 2.82) 1.01 (0.83, 1.19) < 0.001 3 2.19 ( 1.94, 2.51) 0.72 (0.54, 0.90) < 0.001 4 2.08 (1.84, 2.39) 0.61 (0.43, 0.79) < 0.001 table 2. the lc50 and lc90 concentrations of insecticide in the probit model of the death of the german cockroach nymphs on different days table 3. the deaths percentage of german cockroach nymphs* based on different doses of fungal spores the percentage of cockroaches wasted (mean ± se) dose of fungal spores (spore/ml) first day second day third day fourth day 10.0±8/2 12.5±9.6 22.5±5.0 30.0±8/2 1×104 12.5±12.6 17.5±15.0 35.0±23.8 37.5±22.2 1×105 22.5±12.6 37.5±17.1 47.5±5.0 60.0±18.3 1×106 25.0±10.0 42.5±9.6 47.5±34.0 67.5±9.6 1×107 30.0±8.2 55.0±5.8 75.5±12.9 92.5±9.6 1×108 0.037 0.001 0.023 < 0.001 p-value *each treatment contained 10 german cockroach nymphs replicated four times. treatments were significantly effective at p < 0.05. table 3. the deaths percentage of german cockroach nymphs* based on different doses of fungal spores table 4. the ld50 and ld90 values of fungal spore dose in the probit model against the german cockroach nymphs on the post-exposure day post exposure day ld90 (95% ci) ld50 (95% ci) p-value 1 (1.9×10 10–5.4×1012) 3.6×1011 (1.1×108 – 4.4×109) 5.4×108 0.001 2 (2.3×10 11 –2.0×1014) 1.9×1012 (7.7×10–1.4×108) 2.9×107 0.001 3 (1.3×10 9 –1.3×1011) 8.7×109 (3.8×105–4.8×106) 1.3×106 0.001 4 (2.6×10 8– 1.7×1010) 1.5×109 (5.8×104 –8.0×105) 2.3×105 0.001 table 4. the ld50 and ld90 values of fungal spore dose in the probit model against the german cockroach nymphs on the post-exposure day 319 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... compared with ld90. however, according to the probit model used in the data analysis, it can be concluded that this decrease was quite significant in terms of fungal spore concentration. the results of applying fungus (m. anisopliae) spores combined with fenitrothion insecticide composition, performed by the bait method, is demonstrated in table 5. in all samples, the mortality trend significantly increased and reached 100% on the fourth day of exposure. nymph mortality of german cockroaches was significantly increased with incremental concentrations of insecticides and fungi separately on the first day of exposure (tables 1 and 3). significant differences on the first to the fourth days indicate that the best time for insect mortality with the selected dose of fungus and the selected concentration of insects initiated from the second day onward. the results of the analysis of the data obtained from the seven-dose fenitrothion-covered baits on mortality rate in terms of percent (%) for four days are depicted in fig. 1. the mortality rate of cockroach nymphs rose with an increase in concentration levels, and the chosen concentration of 0.93% between the second and third days of average chart data was obtained for lc50% concentration determination. the results of the analysis of the data table 5. the results of the mortality rate of german cockroach nymphs* due to exposure to the effective combination of insecticide and fungal spores based on the rising trend of mortality in exposure days days of exposure day1 day2 day3 day4 p-value mortality rate 70% 80% 90% 100% 0.001 70% 80% 100% 100% 0.001 80% 100% 100% 100% 0.001 30% 60% 90% 100% 0.001 *each treatment contained 10 german cockroach nymphs replicated four times. treatments were significantly effective at p < 0.001. table 5. the results of the mortality rate of german cockroach nymphs* due to exposure to the effective combination of insecticide and fungal spores based on the rising trend of mortality in exposure days fig. 1. regression line from the mortality test of the german cockroach nymphs using bait methods on the first day to the fourth day after the application of fenitrothion fig. 1. regression line from the mortality test of the german cockroach nymphs using bait methods on the first day to the fourth day after the application of fenitrothion 320 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... obtained from the five-dose m. anisopliae concentration logarithm in terms of conidia/ ml for four days are shown in fig. 2. as observed, the mortality rate of cockroach nymphs increased as the concentrations moved up, and a chosen concentration of 6.6×106 conidia/m between the second and third days of average chart data were obtained for the ld50% concentration determination. in terms of forms, the lower the slope of the graph, the lower the strain of insects and the greater the impact of insecticide and fungi on insects. moreover, it was observed that the more heterogeneous the population, the harder it is to cope with the homogeneous population. discussion the german cockroach has been known to be the effective transmitter of various infectious agents such as aspergillus fungi, yeast, candida and bacteria such as enterobacter, klebsiella spp. and so on. the majority of these agents have been proven to have resistance to antibiotics (5, 26). a recent study on the resistance of the german cockroach in iran has reported that although adult cockroaches have been abundantly investigated, further studies on the susceptibility of nymphs are needed (27). since german cockroaches are more resistant to controlling agents than other species are, such as brownbanded cockroaches (28), it is logical to fight them with combined chemical and biological controlling methods in minimal doses and a shorter period. cockroaches collected from homes, dorms, restaurants and hospitals have shown varying degrees of resistance to insecticides made from carbamate, phosphorus and pyrethroids. studies on the german cockroach indicate that they are often resistant to insecticides which are usually expected to have a marked and lethal effect. the results included organophosphorus chlorpyrifos, pirimiphos-methyl and malathion toxins, carbamates such as propoxur, bendiocarb, carbaryl and pyrethroid such as permethrin, deltamethrin and cypermethrin (9, 11). the study on the effects of actellic, ficam, diazinon, fenitrothion and coopex on cockroaches in tehran, iran, demonstrated that their sensitivity to pesticides was relatively low; therefore, they suggested fig. 2. the regression line of the mortality test of the german cockroach nymphs using bait methods on the first day to the fourth day after the application of fungal spores fig. 2. the regression line of the mortality test of the german cockroach nymphs using bait methods on the first day to the fourth day after the application of fungal spores 321 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... that other compounds and methods be used to control cockroaches (29). the mechanisms of insecticide resistance may include changes in insecticide target sites, the regulation of degradative enzymes and increased insecticide excretion (29-30). the inappropriate and unscientific application of insecticides by non-specialists may not only result in gradual resistance but also increase economic costs and damage the environment. previous studies on the biological control of mosquito larvae (culex pipiens) have indicated that m. anisopliae fungus has a high potentiality in this regard. studies have also revealed that the combined fungal formulations can have a greater impact on the biological control of brown-banded german cockroach (23, 28). the bulk of insect cuticle contains protein constituents, including hydrophobic and hydrophobic constituents, with long-chain hydrocarbons in the outermost layer or epicuticular layer. these structures are the main target of degradation by the enzymatic function of chitinase and protease of m. anisopliae (31). metarhizium anisopliae as an entomopathogenic fungus is a unique arthropod pathogenic fungus whose infection is caused by direct infiltration into the mite cuticle so that its cuticle is pierced by the fungus. in fact, by infecting the cells with growing spores, the fungal structures of the fungus are destroyed by the production of enzymes such as chitinase, protease and lipase (32). a study conducted on german cockroach nymphs on the ninth and 15th days, after exposure to 6.5×106 conidia inoculated with fungi, reported over 70% mortality rate (15). considering the ld50 and ld90 exposure values of m. anisopliae (iranian strain v245), the mortality rate of cockroach nymphs was 1.3×106 and 8.7×109 within the third and 2.3×105 and 1.5×109 within the fourth day, respectively. compared to the m. anisopliae fungus that had a lethality of four days in a relatively lower range, the beauveria bassiana fungus was shown to have a longer effective range of three to seven days resulting from higher fungal efficacy (33). the results of this study showed a higher degree of effectiveness for the m. anisopliae fungus. the use of insecticides such as chlorfenapyr, pyridaben and fenpyroximate with fungal m. anisopliae sequences was shown to have a significant effect on killing mites in cotton products (21). the synergistic effects between m. anisopliae and boric acid as a mineral insecticide to control important agricultural pests was investigated, and it was demonstrated that the effects occurred when these compounds were used as a liquid prey for the german cockroach (16). the interaction between m. anisopliae and spinosad insecticide against the house fly, musca domestica, was observed to have a synergetic effect increasing the house fly mortality and reducing the lethal time (22). in this study, it was found that the dose of the simultaneous use of fenitrothion with m. anisopliae to control the german cockroach had no suppressive effects on the viability and growth of conidia. however, accelerating the synergistic virulence of the impact on the german cockroach exposed to fenitrothion-coated baits and m. anisopliae was proved to be effective more than other methods in reducing cockroach nymphs. it should be noted that using a combination of m. anisopliae and fenitrothion for household or agricultural purposes may not have any adverse effects on mammals if used in an appropriate dose. the results of this study revealed that using the appropriate combination of insecticides and spore-fungi, with a minimal concentration of these compounds, could result in a good chemical and biological control. on the other hand, the presence of insecticides seems to be necessary as well. the reason may be that the fungus itself needs to be used in a higher dose that can damage the living organisms, which in turn can have harmful and toxic effects. the reason behind using fungi as a synergist is to try to reduce the concentration and amount of insecticides used in society to minimize the number of chemicals while minimizing the use of insecticides. low-risk biological 322 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 314–324 mr khaksar et al.: possible synergistic... fungi (fungi) are used to enhance the killing rate of insecticides. the findings of this study are in agreement with the idea that the simultaneous use of pesticides and fungal pathogens cause more host vulnerability. the results demonstrated that the lethal dose of fungal spore corresponded to ld50 and ld90 values and was suitable for killing cockroaches in the range of 1×106–1×107. after seven days of exposure to the iranian strain 437c (by the amounts ranging from 2×107 up to 4×106/cm2), the mortality rates of brown-banded cockroaches were 97.8% and 93.6%, respectively. the values by which the cockroach nymphs were treated were within the range of ld50 and ld90. in another study, it was demonstrated that the amount of time required to kill brownbanded nymphs when exposed to the same fungal spore dose ranged from 1.4 to 2.7 days (28). similarly, a study on the german cockroach nymphs showed that if the exposure rate to a fungal spore dose was 6.5×106 conidia/cm2, the life cycle of the cockroaches would only last six days (15). nevertheless, depending on the type of exposed cockroaches, the dose and the final time after the exposure would vary. in this study, the mortality rate of cockroach nymphs was monitored up to four days after exposure and remained favorable. the mortality rate of the german cockroach nymphs by the fenitrothion in the caddy had a gradual decrease in the concentration required. thus, the concentration of 0.5 mg/ kg insecticide on the third/fourth days and 0.7 on the first day can be considered as the appropriate dose to exterminate cockroach nymphs. hence, the combination of fungal spore and insecticide doses should be required only in doses that have lethality greater than 50% provided by taking into account the results of lethality studies and post-exposure mortality rates. however, previous results from the concomitant use of pesticides with the fungal pathogen suggest that exposure to both pests can make them more susceptible and result in more pest damage (16, 21). conclusion the results of this study regarding the effects of pesticide and fungal spore on the mortality rate of cockroach nymphs over four days showed that the optimal dose of fenitrothion used in combination with m. anisopliae seems essential for controlling of german cockroach nymphs, and it can be considered a suitable method with low cost and minimal damage to the environment and other organisms. acknowledgements this study received financial support from qom university of medical sciences, qom, iran. in addition, it was approved by the ethical committee of qom university of medical sciences, qom, iran (code of ethics: ir.muq. rec.1397.022). the authors declare no conflict of interest. references 1. wang c, bennett g w(2009) cost and effectiveness of community-wide integrated pest management for german cockroach, cockroach allergen, and insecticide use reduction in low income housing. j econ entomol. 102(4):1614 23. 2. shahraki gh, parhizkar s, nejad ar (2013) cockroach infestation and factors 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(blattaria: blattellidae), with entomopathogenic fungus, metarhizium anisopliae. j arthropod borne dis. 10(3):335-46. 29. salehzade a, mahjub h (2007) comparison of effect of actelic, ficam, diazinon, fenitrothion and coopex on cockroaches from tehran urban areas. j ilam univ med sci. 15: 24–31. 30. whalon me, mota-sanchez d, hollingworth rm (2008) global pesticide resistance in arthropods. oxford university press, oxford, uk. 31. butt t m, coates c j, dubovskiy i m, ratcliffe n a (2016) entomopathogenic fungi: new insights into host-pathogen interactions. adv genet. 94:307-64. 32. ranjbar bahadori sh, pirali kheirabadi kh (2010) biological control of parasites.(1st eds.), islamic azad university of garmsar, garmsar, iran. 33. gutierrez ac, garcía jj, alzogaray ra, urrutia mi, lópez lcc (2014) susceptibility of different life stages of blattella germanica (blattodea: blattellidae) and periplaneta fuliginosa (blattodea: blattidae) to entomopathogenic fungi. int j curr microbiol app sci. 3: 614–621. possible synergistic effect of combined metarhizium anisopliae and fenitrothion for control of germa abstract keywords introduction materials and methods rearing german cockroaches fungi resource chemicals and pesticides preparing the baits bait bioassay synergism effect of fungi statistical analysis results discussion conclusion acknowledgements references j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 165 http://jad.tums.ac.ir published online: june 24, 2019 original article using ecological niche modeling to predict the spatial distribution of anopheles maculipennis s.l. and culex theileri (diptera: culicidae) in central iran najmeh hesami1; *mohammad reza abai1; hassan vatandoost1,2; mostafa alizadeh3; mahboubeh fatemi1; javad ramazanpour3; *ahmad ali hanafi-bojd1,2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of communicable diseases control, deputy for health, isfahan university of medical sciences, isfahan, iran (received 30 may 2018; accepted 24 apr 2019) abstract background: mosquitoes are very important vectors of diseases to human. we aimed to establish the first spatial database on the mosquitoes of isfahan province, central iran, and to predict the geographical distribution of species with medical importance. methods: mosquito larvae were collected from eight counties of isfahan province during 2014. collected data were transferred to a database in arcgis and the distribution maps were created. maxent model and jackknife analysis were used to predict the geographical distribution of two medical important species, and to find the effective variables for each species. results: totally, 1143 larvae were collected including 6 species, anopheles maculipennis s.l., an. superpictus s.l., an. marteri, culex hortensis, cx. theileri and culiseta longiareolata. the area under curve in maxent model was 0.951 and 0.873 rather 1 for an. maculipennis s.l. and cx. theileri, respectively. culex theileri had wider and more appropriate niches across the province, except for the eastern area. the environmental variable with highest gain was mean temperature of the wettest quarter for cx. theileri and temperature seasonality for an. maculipennis. culex theileri, an. maculipennis s.l. and an. superpictus, three important vectors of parasitic agents to humans, were collected in this study. conclusion: the mosquito collected and mapped can be considered for transmission of malaria and filariasis in the region. bearing in mind the results of niche modeling for vector species, more studies on vectorial capacity and resistance status to different insecticides of these species are recommended. keywords: culicidae; spatial distribution; culex theileri; anopheles maculipennis s.l.; ecological niche modeling introduction mosquitoes are one of the most important groups of medical arthropods and transmit malaria, filariasis, different arboviruses and encephalitis as well as annoyance due to their bites (1). a serological study in 1970s showed west nile infection was relatively common in iran with a prevalence of 30%, while infection with sindbis virus was very rare (2). culex theileri is the principal epidemic vector of rift valley fever virus (bunyaviridae: phlebovirus) on the inland plateau of southern africa (3). dirofilariasis due to dirofilaria immitis and d. repens and setariasis have been reported in current studies from some parts of iran, while west nile virus is also detected in current studies from the birds, horse, human and mosquitoes (4–14). this is in accordance with the global trends of these diseases, although both animal and human cases of this disease are under estimated (15, 16). *corresponding authors: dr ahmad ali hanafi-bojd, email: aahanafibojd@tums.ac.ir, mr mohammad reza abai, email: abaimr@tums.ac.ir mailto:aahanafibojd@tums.ac.ir mailto:abaimr@tums.ac.ir j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 166 http://jad.tums.ac.ir published online: june 24, 2019 study on the mosquitoes of iran has a long history and is conducted on different aspects such as fauna, distribution, parasitic infection, resistance to insecticides and modeling distribution (5, 6, 17–20). culex theileri is reported from 27 out of 31 provinces of iran including ardabil, west azarbaijan, east azarbaijan, bushehr, charmahal and bakhtiari, fars, guilan, hamadan, hormozgan, ilam, isfahan, kerman, kermanshah, south khorassan, north khorassan, khorassan-e razavi, khuzestan, kohgiluyeh and boyerahmad, kordestan, lorestan, markazi, mazandaran, qom, sistan and baluchestan, tehran, yazd, and zanjan (21). anopheles maculipennis s.l. is one of the main malaria vectors in its distribution areas including iran (22). it is reported from 20 provinces of iran including ardabil, west azarbaijan, east azarbaijan, charmahal and bakhtiari, guilan, golestan, hamadan, isfahan, kermanshah, khorassan-e razavi, north khorassan, kohgiluyeh and boyerahmad, kordestan, qazvin, lorestan, markazi, mazandaran, semnan, tehran, and zanjan (22). despite activities regarding mosquito control in iran, there is still the problem of painful bites of these insects, especially in central areas like isfahan, arak, semnan, and tehran provinces. due to the vital role of health in development programs, and because the study on ecology and bionomics of mosquitoes is one of the important indices that have a fundamental role in development of ecotourism industry, proper knowledge from behavioral characteristics and bionomics of mosquitoes in different ecological conditions is one of the main factors in planning the strategy to combat the mosquitoes. isfahan province is one of the main poles for industry and tourism in iran. although rare studies have been done in past (23, 24), but still there is no comprehensive survey on vector(s) bioecology in different climates, seasons, temperatures, and so on. with due attention to the extensive climate change in the world and iran as well, accurate study on the ecology and bionomics of mosquitoes are necessary due to their important role in disease transmission, especially arboviruses. data collection on their biodiversity, distribution and ecology will provide guideline for appropriate vector control. geographic information systems (gis) is a rapidly growing technology that combines graphics features with the environmental data obtained from the vectors of disease. this ability helps us to assess the distribution and bioecology of vector species. in last two decades using gis in vector-borne diseases has increased and a new field of investigation has been opened. creating databases, mapping, spatial and statistical analysis of vector-borne diseases are results of this new branch of science (25). this study aimed to establish the first spatial database on the mosquitoes of isfahan province and to predict the geographical distribution of medically important species. materials and methods study area the isfahan province covers an area of approximately 107,027km2 and is situated in the center of iran (fig. 1). the province experiences a moderate and dry climate, on the whole, ranging between 40.6 °c and 10.6 °c on a cold day in the winter season. the average annual temperature has been recorded as 16.7 °c and the annual rainfall on an average has been reported as 116.9mm. more than 5 million peoples are living in 24 counties of this province. isfahan is destination of millions of tourists from different parts of iran and other countries. entomological survey sampling was conducted two times during summer 2015 from 8 counties (khomeinishahr, golpayegan, faridan, khansar, mobarakeh, fereidoonshahr, najafabad, samirom) j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 167 http://jad.tums.ac.ir published online: june 24, 2019 in three main topographic areas of isfahan province. mosquito larvae were collected by the standard dipping method. coordinates of the collections sites were recorded using a gps device. species were transferred to the laboratory, mounted and identified morphologically (26). all the mounted slides were deposited in the medical entomology museum, school of public health, tehran university of medical sciences under code of gc22st11-94. creating database and mapping all collected data obtained from this entomological survey were transferred to a relevant database in arcgis and then the distribution maps were created. modeling maxent model and bioclimatic variables were used to predict and to map the geographical distribution of medically important species which had enough occurrence data points (27). jackknife test in maxent model was used to find the effective variables for each species. the bioclimatic data were downloaded from the worldclim database in 1km2 spatial resolution (version1.4, http://www.worldclim.org/past). they were included bio1 (annual mean temperature (oc), bio 2 (mean diurnal range: mean of monthly (max temp–min temp) (°c)), bio3 (isothermality: (bio2/bio7)× 100), bio4 (temperature seasonality (sd× 100)), bio5 (maximum temperature of warmest month (°c)), bio6 (minimum temperature of coldest month (°c)), bio7 (temperature annual range (bio5−bio6) (°c)), bio8 (mean temperature of wettest quarter (°c)), bio9 (mean temperature of driest quarter (°c)), bio10 (mean temperature of warmest quarter (°c)), bio11 (mean temperature of coldest quarter (°c)), bio12 (annual precipitation (mm)), bio13 (precipitation of wettest month (mm)), bio14 (precipitation of driest month (mm)), bio15 (precipitation seasonality (coefficient of variation)), bio16 (precipitation of wettest quarter (mm)), bio17 (precipitation of driest quarter (mm)), bio18 (precipitation of warmest quarter (mm)) and bio19 (precipitation of coldest quarter (mm)). two environmental variables were also used for modeling i.e. altitude (m) and normalized difference vegetation index (ndvi). the first variable was derived from the digital elevation model (dem) of iran with the same resolution, while ndvi was acquired from aug 2014 image of modis satellite. results species composition overall, 1143 mosquito larvae were collected including 6 species: anopheles maculipennis s.l. (24), an. superpictus s.l. (4), an. marteri (10), culex hortensis (454), cx. theileri (429) and culiseta longiareolata (222). the most and the least density was due to cx. hortensis (39.72%) and an. superpictus s.l (0.35%), respectively (table 1). anopheles maculipennis s.l. was collected from both plain and foothill areas, but we could not find this malaria vectors in mountainous areas of isfahan province. culex theileri and cx. hortensis were the most aboundant species. regardless of the topography of the area, they were caught in all the studied areas. maxent modeling this method was used to find the appropriate niches for cx. theileri and an. maculipennis s.l. to do this, all collection points in this study, as well as earlier studies in isfahan province (1984–2015), were used to have enough occurrence data for modeling (fig. 1). figure 2 shows the collection sites for the studied species. the area under curve was calculated as 0.951 rather 1 for an. maculipennis (fig. 3). more appropriate niches for an. maculipennis were restricted to the western and southwestern areas (fig. 4). based on jackknife test, the environmental variable with the highest gain when used in isolation is temj arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 168 http://jad.tums.ac.ir published online: june 24, 2019 perature seasonality (bio4), which therefore appears to have the most useful information by itself. the environmental variable that decreases the gain the most when it is omitted is ndvi, which therefore appears to have the most information that is not present in the other variables. these numbers show an acceptable validity for the exported maps. for cx. theileri, the area under curve was calculated as 0.873 rather 1 (fig. 3). based on the maps, cx. theileri had wider and more appropriate niches across the province comparing an. maculipennis s.l., except for the eastern area (fig. 4). figure 5 shows the result of the jackknife test of variable importance for cx. theileri. the environmental variable with highest gain, when used in isolation, was mean temperature of the wettest quarter (bio8). this variable appears to have the most useful information for modeling this species. in fig. 5 three jackknife plots for cx. theileri were compared. the auc plot shows that temperature seasonality (bio4) is the most effective single variable for predicting the distribution of this mosquito species used for testing. the relative importance of temperature seasonality also increases in the test gain plot, when compared against the training gain plot. mean temperature of wettest quarter is helping the model to obtain a good fit to the training data, but the temperature seasonality variable gives better results on the set-aside test data. figure 6 shows result of the jackknife test of variable importance for an. maculipennis s.l.. the environmental variable with highest gain, when used in isolation, was temperature seasonality (bio4). this variable appears to have the most useful information for modeling this species. comparing the three jackknife plots can be very informative. the auc plot shows that isothermality (bio3) is the most effective single variable for predicting the distribution of the occurrence data that was set aside for testing when predictive performance is measured using auc, even though it was hardly used by the model built using all variables. the relative importance of isothermality also increases in the test gain plot, when compared against the training gain plot. temperature seasonality is helping the model to obtain a good fit to the training data, but the isothermality variable gives better results on the set-aside test data. table 1. spatial distribution of collected mosquito larvae according to topography and species, isfahan province, 2015 topography total larvae (no.) no. and frequency of species county a n . m a c u lip e n n is s.l a n . su p e rp ic tu s s.l. a n . m a rte ri c x . h o rte n sis c x . th e ile ri c s. lo n g ia re o la ta no. % no. % no. % no. % no. % no. % khomeinishahr plain 129 16 12.4 2 1.56 2 1.56 12 9.3 86 66.66 11 8.52 golpayegan plain 50 4 8.3 2 4.2 0 0 6 12.5 36 75 0 0 mobarakeh plain 65 0 0 0 0 0 0 33 50.77 32 49.23 0 0 najafabad plain 90 0 0 0 0 0 0 57 61.95 35 38.05 0 0 faridan foothill 115 4 3.47 0 0 8 6.96 0 0 34 29.57 69 60 khansar foothill 247 0 0 0 0 0 0 152 61.54 42 17 53 21.46 fereidoonshahr mountain 354 0 0 0 0 0 0 129 36.64 134 38.08 89 25.28 samirom mountain 93 0 0 0 0 0 0 65 69.89 28 303.11 0 0 total 1143 24 2.1 4 0.35 10 0.88 454 39.72 429 37.53 222 19.42 j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 169 http://jad.tums.ac.ir published online: june 24, 2019 fig.1. study sites in isfahan province, iran fig. 2. spatial distribution of collection sites for anopheles maculipennis and culex theileri in isfahan province, central iran, 1984–2015 j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 170 http://jad.tums.ac.ir published online: june 24, 2019 fig. 3. the area under roc curve (auc) for anopheles maculipennis s.l. and culex theileri in isfahan province, central iran, 2015 fig. 4. probability of presence for anopheles maculipennis s.l. and culex theileri in isfahan province, central iran, 2015 j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 171 http://jad.tums.ac.ir published online: june 24, 2019 fig. 5. jackknife test of auc for culex theileri in isfahan province, central iran, 2015 j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 172 http://jad.tums.ac.ir published online: june 24, 2019 fig. 6. jackknife test of auc for anopheles maculipennis s.l. in isfahan province, central iran, 2015 discussion in this study six culicidae mosquito species were collected and identified. based on the previous studies in the province, 24 mos j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 173 http://jad.tums.ac.ir published online: june 24, 2019 quito species are reported as follows: ae. vexans, an. algeriensis, an. claviger, an. dthali, an. maculipennis s.l., an. marteri, an. messeae, an. multicolor, an. sacharovi, an. superpictus, an. turkhudi, cx. modestus, cx. hortensis, cx. mimeticus, cx. perexiguus, cx. pipiens, cx. theileri, cx. territans, culiseta longiareolata, cs. annulata, cs. subochrea, ochlerotatus caspius, oc. pulcritarsus and uranotaenia unguiculata (16, 22, 23). our findings report no new record for the province. the dominant species in our study was cx. hortensis (39.72%) followed by cx. theileri. earlier studies in mobarakeh county found cx. theileri as the dominant species in both larval and adult collections (23). studies during 2003–2005 were also introduced cx. theileri as the dominant species in isfahan province (24). although in our study, the current species had the second density, it can be due to the number of collection times. on the other hand, if field sampling is done at a better time, a greater abundance may be due to this species. generally cx. theileri is a rice field mosquito species (28). it has usually exophilic and exophagic behavior and has a high anthropophilic index, and comprised more than 77% of the mosquitoes catched on human bait (21). other studies were also reported cx. theileri as the main species in some parts of iran (29, 30). culex theileri, an. maculipennis and an. superpictus as three important vectors of parasitic agents to humans were collected in this study. in northwest of iran, cx. theileri infected was reported to d. immitis (7). therefore, in addition to numerous bites on human in isfahan area, it can be considered as probable vector of dirofilariasis. among the collected mosquitoes, two main malaria vectors of iran were identified: an. maculipennis and an. superpictus. in this study, an. maculipennis was only found in khomeinishahr, golpayegan and faridan counties. however, this species has reported from most counties of the province (23, 24). anopheles superpictus was collected from khomeinishahr and golpayegan counties in our survey, but there are reports of this species from 12 counties of the province (23, 24). there is potential for malaria transmission in the study area. since the province has attractions and job opportunities, there are likely infected foreign immigrants from the endemic countries visit the area, at least in some months of the year. three other species in this survey were an. marteri, cx. hortensis and cs. longiareolata. these species have no tendency to feed from human. just cx. hortenis known as birds malaria vector under laboratory condition (31). culiseta longiareolatsa may rarely attack human and domestic animals, although there is little information on this species. maxent model was used to predict the distribution of some anopheles mosquitoes around the world, such as an. albimanus in americas (32), malaria vectors of africa, europe and middle east (33), and an. minimus in southeast of asia (34). a more recent study in iran used this model to find the best ecological niches for three main malaria vectors in south of iran, i.e. an. stephensi, an. culicifacies s.l. and an. fluviatilis s.l. (19). it is also used to predict the potential areas for cx. pipiens, cx. tarsalis and ae. vexans in the united states and cx. tritaeniorhynchus in saudi arabia (35, 36). auc value in this research was 0.951 and 0.873 for an. maculipennis and cx. theileri, respectively. previous studies on maxent model considered predicting auc> 0.75 as good value for suitable niche for species (37). this suggests the maxent prediction in our study is well. a new survey in iran reported auc values of 0.943, 0.974 and 0.956 for an. stephensi, an. culicifacies s.l. and an. fluviatilis s.l., respectively (19). in other countries auc values were reported between 0.77 and 0.99 for different anopheles species (36, 38-40). this difference is due to various ecological needs of these species. j arthropod-borne dis, june 2019, 13(2): 165–176 n hesami et al.: using ecological niche … 174 http://jad.tums.ac.ir published online: june 24, 2019 conclusion in addition to the problems caused by bites on humans, they can be considered for transmission of malaria and filariasis in the region. with due attention to tourist and business attractions of isfahan province, some people from different parts of the world will travel to different counties of the province annually. considering the results of niche modeling for vector species, it is recommended to do additional studies on vectorial capacity of these species, their physiological age, and parasitic infection, and insecticide resistance susceptibility status to different insecticides to predict the risk of establishing the foci of filariasis, west nile fever and malaria in isfahan province. acknowledgements collaboration of deputy for health, isfahan university of medical sciences in providing facilities for this study is highly appreciated. moreover, the authors are grateful to mr h davoodi, technician of museum of medical entomology (tume), school of public health, tehran university of medical sciences for his kind assistance for organizing of 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https://www.ncbi.nlm.nih.gov/pubmed/?term=yang%20d%5bauthor%5d&cauthor=true&cauthor_uid=26868185 https://www.ncbi.nlm.nih.gov/pubmed/?term=feng%20x%5bauthor%5d&cauthor=true&cauthor_uid=26868185 https://www.ncbi.nlm.nih.gov/pubmed/?term=xia%20z%5bauthor%5d&cauthor=true&cauthor_uid=26868185 https://www.ncbi.nlm.nih.gov/pubmed/?term=zhou%20xn%5bauthor%5d&cauthor=true&cauthor_uid=26868185 https://www.ncbi.nlm.nih.gov/pubmed/?term=wang%20j%5bauthor%5d&cauthor=true&cauthor_uid=26868185 j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 202 http://jad.tums.ac.ir published online: june 30, 2020 original article evaluation of different attractive traps for capturing sand flies (diptera: psychodidae) in an endemic area of leishmaniasis, southeast of iran saideh yousefi 1,2 ; *ali reza zahraei-ramazani 1 ; yavar rassi 1 ; hassan vatandoost 1,3 ; mohammad reza yaghoobi-ershadi 1 ; mohammad reza aflatoonian 4 ; amir ahmad akhavan 1 ; abbas aghaei-afshar 5 ; masoumeh amin 1 ; azim paksa 6 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 sirjan school of medical sciences, sirjan, iran 3 department of chemical pollutants and pesticide, institute for environmental research, tehran university of medical sciences, tehran, iran 4 research center for tropical and infectious diseases, kerman university of medical sciences, kerman, iran 5 leishmaniasis research center, kerman university of medical sciences, kerman, iran 6 department of parasitology, faculty of medicine, tabriz university of medical science, tabriz, iran (received 06 sep 2019; accepted 10 dec 2019) abstract background: the attraction of phlebotomine sand flies to plant and animal hosts is due to the produced chemical compounds, affecting the olfactory receptors of the insects. therefore, novel and effective methods, such as attractive toxic sugar baits (atsb) and attractive toxic baits (atb), are based on the effective materials that attract sand flies toward the host. the present study was designed to identify the attractive materials in plants and animals for using in atsb and atb. methods: this cross-sectional study was carried out in july 2018 on endemic areas of leishmaniasis in iran. different baits, including mango, nectarine, grape, banana, melon and watermelon, defibrinated blood of cattle, sheep, goat and chicken, urine of cattle, sheep, goat and ultimately, simple and complex chemicals, such as co2, 1-octanol, lactic acid and human sweat were placed inside the traps, and the rate of the sand flies attraction to these materials was studied. furthermore, data were analyzed using the kruskal-wallis test and mann whitney u test. results: there was a significant difference in the sand flies attraction between the traps containing watermelon, urine of cattle, and sheep, and chemicals such as co2 and human sweat and the control trap (p< 0.05). conclusion: this study showed that watermelon and co2 are the potential candidates for using in atsb and atb, respectively. keywords: control; leishmaniasis; plant component; chemicals; iran introduction some species of phlebotominae sand flies (diptera: psychodidae) transmit a group of metaxenic diseases, such as leishmaniasis, sand fly fever, summer meningitis, vesicular stomatitis, chandipura virus encephalitis, and carrion disease (1). in terms of mortality and morbidity, leishmaniasis is the most important diseases transmitted by some species of sand flies (phlebotomus and lutzomyia genera) and they are also among the 9 most important infectious diseases in the world (2). leishmaniasis mainly occurs in tropic and sub-tropic regions of africa, america, asia, and europe continents (3, 4). almost half of the countries in the eastern mediterranean region (emro) of the world health organization (who) are endemic foci of cutaneous and visceral leishmaniasis. iran, along with afghanistan, iraq, saudi arabia, syria, yemen, and pakistan are *corresponding author: dr ali reza zahraeiramazani, e-mail: alirezazahraei@yahoo.com mailto:alirezazahraei@yahoo.com j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 203 http://jad.tums.ac.ir published online: june 30, 2020 the major foci of anthroponotic cutaneous leishmaniasis (acl), and leishmania trop ica is the causative agent in these areas (5). kerman, tehran, khorasan, fars, yazd, and esfahan provinces are the main active foci of acl in iran. researches have proven that in these foci, phlebotomus sergenti is the main vector, l. tropica is the causative agent, human is the main and dog is the secondary reservoir hosts (6, 7). bam county is the most infectious focus of acl in kerman province (8). after the earthquake in 2003, which resulted in the inhabitants’ homelessness and increased breeding places for sand flies, epidemics of this disease were reported in the city and several rural areas which previously were free of leishmaniasis (9). currently, the main prevention activities for controlling the disease in bam county, are case diagnosis and treatment with glucantim, health education, and occasionally, indoor residual spraying (10, 11). there is no effective vaccine against leishmaniasis. the usual drug (glucantime) is expensive and has several side effects on heart, kidney, and liver (12). indoor residual spraying not only increases the vector resistance to insecticide, but also is expensive and has side effects on the environment and non-target organisms (13). up to now, controlling the vector populations is the most effective way to control metaxenic diseases, including leishmaniasis (14). attractive toxic sugar bait (atsb) is an effective and environmentally friendly method for controlling phlebotomine sand fly populations. in this method, attractive plant substances, such as, fruit juice combined with an oral insecticide, are used and this compound attracts and kills insects. atsb can be sprayed on non-flowering vegetation or it can be used as bait station, and this controlling method has low side effects on non-target organisms (honey bees, beetles, etc.) and environment (13). in the studies carried out in iran, morocco and israel (jordan valley), sand fly populations were reduced using this method (13, 15, 16). attractive toxic bait (atb) uses simple or complex chemicals produced by the host body (human and animal) such as co2 (in animal breath), lactic acid and octanol (in host sweat), urine, sweat, and blood to attract the insects. such compounds have been introduced as effective materials in attracting diptera (17-22). considering the who emphasis on identifying affordable and effective methods for use in integrated vector management (ivm) programs (12), this study examines the rate of phlebotomine sand flies attraction to traps using different baits containing fruits such as mango, nectarine, grape, banana, melon and watermelon, the defibrinated blood of cattle, sheep, goat and chicken, the urine of cattle, sheep, and goat, as reservoir hosts, and simple and complex chemicals such as co2, 1octanol, lactic acid, and human sweat. materials and methods study area this study was conducted in july 2018, in bam county (29° 06′.52" n latitude and 58° 22′.67" e longitude, 1050m above sea level) of the kerman province, iran (fig. 1). the topography of the studied area is lowland plain. this area has hot and dry summers and temperate winters, the mean annual temperature, precipitation and relative humidity is 25 ˚c, 68mm, and 20%, respectively. palm (phoenix dactylifera) is the most common tree in the region (23). study on the rate of the sand flies attraction to different baited traps traps containing different fruits mango (mangifera indica), nectarine (prunus persica), grape (vitis vinifera), banana (musa sapientum), melon (cucumis melo) and watermelon (citrullus lanatus) were used as attractive baits for trapping sand flies based j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 204 http://jad.tums.ac.ir published online: june 30, 2020 on the method used by mong'are et al. (22), with changes in the appearance of the trap. the trap consisted of two main components: a 5-liter plastic bottle and a castrol oil-impregnated paper at a size of 29×20cm, which was glued to the inner wall of the bottle by the castor oil. the upper and one of the lateral parts of each bottle were cut to provide the sand flies with access to the fruits (fig. 2). also, a container was placed on the bottom of the bottle for holding the fruits. the fruits were bought from local stores, 200g of which was daily placed inside the trap. the negative controls included a trap containing a water impregnated sponge, a trap containing sucrose solution 10%-impregnated sponge, and an empty trap. these traps were placed in various biotopes for 10 days in july 2018, from 6:00pm to 6:00am, and to minimize the location error in the sampling, the traps were rotated every day through different locations. the distance between the traps was at least 2m. traps containing the defibrinated blood of animals blood samples of cattle (bos taurus), sheep (ovis aries), goat (capra aegagrus), and chicken (gallus gallus) were freshly collected from slaughterhouses and they were defibrinated using glass balls. the bottles containing defibrinated blood were kept in the refrigerator. a total of 200ml of the animals’ blood was transferred into the container and a sponge with an appropriate size was inserted into the container to absorb all the blood. then, the sponge was placed inside the trap. to prevent the access of wild animals to the blood sponges, the traps were placed inside metal nets designed for this purpose. the traps were placed in domestic, peridomestic, agricultural, and sylvatic biotopes for 10 days in july 2018. moreover, 2 negative controls were used in this stage, including a trap containing water impregnated sponge and an empty trap. traps containing the urine of different animal taxa the urine of cattle, sheep, and goat, prepared from local livestock was used in this study. for this purpose, 200ml of animal’s urine was placed inside the container and a sponge was inserted to absorb all the urine; then the sponge was placed inside the traps. the research was performed in 4 different environments. two controls were used in this stage, including traps containing water-impregnated sponge and empty ones traps containing simple and complex chemicals in each biotope, 200g of dry ice (co2) was used inside the traps. the traps were rotated every day through different locations. furthermore, 16 bright nylon socks, that were worn by children (girls and boys) and adults (men and women) for at least 24 hours, were used in each trap (approximate weight: 200g), in order to investigate the rate of phlebotomine attraction to human sweat. the sweaty socks were placed in 4 different biotopes for 10 days, and new pairs of socks were replaced by the old ones in each sampling. in order to determine the rate of phlebotomine sand flies attraction to 1-octanol and lactic acid, a sponge impregnated with 200ml of pure 1-octanol and lactic acid was placed inside the traps. also, 2 controls, including water-impregnated sponge trap and an empty trap, were considered for the investigations. identification of the captured sand flies the sand flies collected from each trap were stored in ethanol (96%). the identification was based on the morphological characteristics of the male (genitalia and cibarium) and female (spermathecae and pharynges) using the morphological key by seyedirashti et al. (24). statistical analysis the normality of the quantitative variable j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 205 http://jad.tums.ac.ir published online: june 30, 2020 (attraction rate) was investigated by the kolmogorov-smirnov test. non-parametric kruskal-wallis test was used to study the difference between the median attractions among different traps. the mann whitney u test was also used to compare the attraction rate between each trap and control groups, and also to compare the control groups to each other. statistical analysis was done by spss version 24. results the composition of sand flies species a total of 1651 sand flies specimens including, 14.3% female and 85.7% males, belonging to the 2 genera of phlebotomus and sergentomyia, were captured. the distribution of the captured sand flies was 66.87% in peri domestic, 18.83% in sylvatic, 13.45% in agricultural, and 0.85% in domestic sites. the relative abundance of each species was ph. sergenti (73.53%), ph. papatasi (5.87 %), sergentomyia baghdadis (18.71%), se. sintoni (1.39%) and se. dentata (0.5%). the attraction of sand flies to traps containing various fruits the results of the kolmogorov-smirnov test showed that the distribution of the desired variable (attraction rate) was not normal (p< 0.001). according to the kruskal-wallis test, the attraction rate was significant, especially in the female group (p< 0.001) (table 1). the mann whitney u test indicated that the attraction rate was significant in the male group among all of the fruit and control traps. in the female group, there was a significant difference only between watermelon and the control trap (table 1). the attraction of sand flies to traps containing the defibrinated blood of different animals the normality of the attraction rate was investigated by the kolmogorov-smirnov test. the results showed abnormality of the desired variable (p< 0.001). the difference between the median attraction rates were analyzed, using the kruskal-wallis test. the results showed that there was a significant difference in attraction rate only in the males group (p< 0.001) (table 2). therefore, the mann-whitney u test was only used in this group to compare the attraction rate of each blood trap to the control, and also to compare the control groups with each other. the results showed that there was a significant difference in the attraction rate between the traps containing cow, chicken, sheep, and goat blood compared to the control trap (table 2). the rate of sand flies attraction to traps containing the urine of different animals in this study, the normality of the attraction rate variable was investigated by kolmogorov-smirnov test. the kruskal-wallis test was used to examine the difference in the median attraction rate between the different traps. there was a significant difference in attraction rate between male and female groups (p< 0.001). nonparametric mann whitney u test was used to compare the attraction rate of each sample (urine of different animals) to the control trap, and also to compare the control groups to each other. results show that, in the male sand flies group, there was a significant difference in the attraction rate between the traps containing urine from goat, sheep, and cattle in comparison to the control trap (p< 0.05 ) (table 3). in the female group; only the difference in the attraction rate between the traps containing the urine of sheep and cow was significant compared to the control trap (table 3). the rate of sand flies attraction to traps containing various chemicals the results of the kolmogorov-smirnov test showed that the attraction rate variable was abnormal. j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 206 http://jad.tums.ac.ir published online: june 30, 2020 based on the kruskal -wallis nonparametric test, there was a significant difference in attraction rate between both male and female groups (p< 0.001) (table 4). the results of the mann-whitney u test showed that only the differences in attraction rate between traps containing co2 and human sweat were significant compared to the control trap (table 4). table 1. comparison of the sand flies attraction rate between different fruit and control traps in bam county, 2018 sand fly sex fruit type median interquartile range p-value comparison pvalue male mangifera indica 8 11 0.018 mangifera indica vs.* water 0.009 cucumis melo 5.5 7.75 cucumis melo vs. water 0.011 musa sapientum 5.5 9.25 musa sapientum vs. water 0.023 prunus persica 9.5 11.25 prunus persica vs. water 0.015 vitis vinifera 5 6.25 vitis vinifera vs. water 0.023 citrullus lanatus 5.5 6 citrullus lanatus vs. water 0.003 water control trap 2 4.25 sugar solution vs. water 0.393 sugar solution control trap 3 4.25 empty vs. water 0.739 empty control trap 2 4.5 female mangifera indica 1 4.25 < 0.001 mangifera indica vs. water 0.218 cucumis melo 0.5 2.25 cucumis melo vs. water 0.481 musa sapientum 0 0 musa sapientum vs. water 0.068 prunus persica 0 0.25 prunus persica vs. water 0.585 vitis vinifera 0.5 1.25 vitis vinifera vs. water 0.529 citrullus lanatus 3 3.5 citrullus lanatus vs. water 0.004 water control trap 0 1.25 sugar solution vs. water 0.358 sugar solution control trap 0 0 empty vs. water 0.486 empty control trap 0 0 *vs. is the abbreviated form versus table 2. comparison of the sand flies attraction rate between different blood and control traps in bam county, 2018 sand fly sex type of blood median interquartile range p-value comparison p-value male goat blood 4 5.25 < 0.001 goat blood vs.* water 0.004 sheep blood 4.5 4.5 sheep blood vs. water 0.002 cow blood 5 7 cow blood vs. water 0.001 chicken blood 2.5 3.25 chicken blood vs. water 0.001 water control trap 1 1 empty vs. water 0.77 empty control trap 0.5 1 female goat blood 0 0 0.313 goat blood vs. water sheep blood 0 1 sheep blood vs. water cow blood 0 0.25 cow blood vs. water chicken blood 0 2.5 chicken blood vs. water water control trap 0 1 empty vs. water empty control trap 0 0.25 *vs. is the abbreviated form versus j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 207 http://jad.tums.ac.ir published online: june 30, 2020 table 3. comparison of sand flies attraction rate between different urine and control traps in bam county, 2018 sand fly sex type of urine median interquartile range p-value comparison pvalue male goat urine 5.5 16 < 0.001 goat urine vs.* water 0.001 sheep urine 6 14.5 sheep urine vs. water 0.001 cow urine 7 8.25 cow urine vs. water 0.002 water control trap 1 2 empty vs. water 0.910 empty control trap 1 2 female goat urine 1 3 < 0.001 goat urine vs. water 0.28 sheep urine 1 1.5 sheep urine vs. water 0.045 cow urine 3 1.75 cow urine vs. water 0.014 water control trap 0 1 empty vs. water 0.436 empty control trap 0 0.25 *vs. is the abbreviated form versus fig. 1. the study area in bam county, kerman province, iran, 2018 fig. 2. baiting traps for attracting sand flies j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 208 http://jad.tums.ac.ir published online: june 30, 2020 table 4. comparison of the sand flies attraction rate between different chemicals and control traps in bam county, 2018 sand fly sex chemicals median interquartile range p-value comparison p-value male human sweat 6 8.25 < 0.001 human sweat vs.* empty 0.007 co2 15.5 9 co2 vs. empty < 0.001 lactic acid 2 2.75 lactic acid vs. empty 0.247 1-octanol 1 1.5 1-octanol vs. empty 0.529 water control trap 1 2 water vs. empty 0.631 empty control trap 1 1.5 female human sweat 1 2.25 < 0.001 human sweat vs. empty 0.023 co2 2.5 5.25 co2 vs. empty < 0.001 lactic acid 0 1.25 lactic acid vs. empty 0.28 1-octanol 0 0 1-octanol vs. empty 0.99 water control trap 0 0 water vs. empty 0.739 empty control trap 0 0 discussion like the other blood-sucking vectors, sand flies need blood for egg development and, therefore they transmit some sand fly borne diseases to humans and animals (22). identifying methods, for reducing the population of sand flies, decrease the burden of the disease, as well as economic and mental stress caused by these insects. currently, in order to control leishmaniasis, chemical control, and environmental management are also used in addition to case finding and treatment (25). also, using of some chemical control methods, such as indoor residual spraying (irs), space spraying, insecticide-treated curtains, bed nets, and sleeping bags, not only increase vector resistance to insecticides but also have not been effective in some area, probably due to the limitation of contact with insecticides in these methods (26, 27, 15). moreover, successful environmental management methods require sufficient funds and facilities and complete knowledge on the ecological and biological characteristics of sand fly species in each region (28). with the constraints and limitations of conventional sand fly control methods in mind, the world health organization (who) recommends the identification and evaluation of affordable and effective methods for using in integrated vector management (ivm) strategies (12, 29). so far studies have reported the effectiveness of newly applied methods, known as atsb and atb in controlling sand flies and other vector families in different ecological regions around the world (17, 20, 30-32). therefore it is essential to identify the most effective sources attracting vectors in each region in order to use them in atsb and atb. sugars are the only sources of energy for vital activities of male sand flies and one of the most important sources of energy for females. therefore, sugar is an important and major factor in the life of these vectors, and one of the factors limiting the survival and fitness of these insects in dry areas is restricted sugar access (33). fruits, plant tissues, flower nectars, and aphid and coccid honey are the main sources of sugar for sand flies (33-35). although, it is still not fully determined that which compounds produced by or derived from fruits attract the sand flies. however, the abundance of glucose, fructose, and sucrose has been proposed as one of the important sources of energy supply for sand flies various activities (36). j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 209 http://jad.tums.ac.ir published online: june 30, 2020 in order to study the effectiveness of the atsb method in controlling mosquitoes (culicidae), different fruit extracts combined with insecticides have been used and the success rate was reported as 36–100% (30, 37, 38, 39). moreover, in studies conducted on the rate of sand flies attraction to different fruits, the highest rates have been reported for mango, banana, apple, and grape (22). according to a study conducted by schlein (16), using nectarine extract, along with ingested insecticides, could reduce the population of sand flies, and thus this combination is useful in leismaniasis control programs. in studies carried out in iran, it has been confirmed that using brown sugar as an attractant material, combined with boric acid and water for spraying on plants, and the application of fences treated with these materials against colonies of rodents, that are reservoirs of disease, are effective in controlling the population of ph. papatasi as the main vector of zcl in the country (15). despite the importance of sugar in the life cycle of sand flies and the importance of fruits as one of the important sources of sugar supply, no studies have been yet conducted in iran, on the rate of sand flies attraction to different fruits used in atsb. therefore in the present study fruits such as mango, banana, nectarine, grape, melon, and watermelon were used in traps designed for this purpose. based on the results, watermelon (citrullus lanatus) or its combinations are more attractive for male and female of sand flies, compared to other fruits. thus, watermelon due to its ability to attract sand flies, especially males and females of ph. sergenti, seems like a good candidate for using in atsb. in addition to the proper performance of atsb in attracting flies, atb containing materials from the host body has also been proven to be effective in deviating mosquitoes from the human host toward artificial baits containing these materials (40). female blood-sucking sand flies use co2 to find the location of their host for sucking blood and therefore they are attracted to the traps containing this material. aside from the female sand flies, male sand flies are also attracted to the co2 emitted from the host body in order to find females for mating (41, 42). in egypt, italy, and israel co2 has been introduced as an effective attractive material for collecting sand flies (42-44). hesam-mohammadi et al. (45) showed that co2 is not an attractive substance for capturing sand flies in kashan district. in the current study, the highest rate of sand flies attraction, especially male and female ph. sergenti as the main vector of acl, has been reported in traps containing co2, thus, it is clear that co2 is one of the most effective substances for attracting sand flies in the region, and it can be effectively used in atb to capture and control the population of sand flies or for entomological surveillance programs. studies have shown that the human body produces hundreds of fumigation molecules with simple and complex structures. some of these materials can be used to attract medically-important insects (17, 18, 20). various studies have shown that using socks worn by humans in traps can attract a great number of malaria vectors, and due to the persistence of the smell of sweat, closed areas containing worn clothes are good places for attracting anopheles spp. mosquito and sand flies (1921, 46). also, some compounds in human sweat, such as lactic acid, have been successfully used in malaria-vector capture programs due to their extensive attraction for mosquitos. in addition to the attraction of diptera to lactic acid, octanol has also been proposed as an effective material in attracting nyssomyia neivai, which is the vector of cutaneous leishmaniasis in the united state (19, 21). a recent study showed that sand flies are attracted to human sweat; therefore, although the chemicals such as human sweat have been proven to be effective in attracting sand flies to humans and causing disease transmission, j arthropod-borne dis, june 2020, 14(2): 202–213 s yousefi et al.: evaluation of different … 210 http://jad.tums.ac.ir published online: june 30, 2020 using this material in atb can cause distraction of sand flies from humans to traps, and thus help reduce their population and diseases transmission. like many other blood-sucking insects, sand flies require proteins for their egg development, and animal and human blood are the most important supplies of protein (46). in studies conducted by mong'are et al. (22), on the rate of the sand flies attraction to traps containing the blood of cattle, sheep, goat and chicken, the highest rates were reported for goat blood and the lowest was reported for chicken blood. this study demonstrated that, the male sand flies tend to be attracted to the blood of various animals, which seems to contain a material attracting male sand flies to the host for finding females. however, it seems that, the method used to attract sand flies to blood resources is not appropriate for female sand flies, or perhaps the preference of the female sand flies is to other hosts. the animals’ urine containing, substances such as 4-methyl phenol and 3-n-propyl phenol, has been reported to be effective in capturing some of diptera belonging to the culicidae and glossinidae families (47). in studies conducted by keweka et al. (48), cattle urine was proposed to be effective in the attraction of mosquitoes belonging to the culicidae family to breeding places containing this material for laying eggs. few studies have examined the relationship between the host’s urine and its rate of sand fly attraction. according to studies in kenya, the urine of cattle, goat and sheep is effective in attracting ph. martini, se. africana, se. antennata and se. schwetzi (22). the results of the recent study in bam county showed that the urine of animals such as sheep and cattle attracts male and female sand flies, which is probably due to certain components emitted from the host’s urine as a guide for female sand flies to find a host location for laying eggs, and in the case of male sand flies, to find a proper site for mating. therefore, it seems that these materials can be used in atb to attract sand flies. as noted earlier, the largest number of sand flies was captured from peri domestic biotopes, which demonstrate that the animal kairomones are highly attractive for sand flies, and this biotope has been reported to be one of the most dangerous environments due to the sand flies accumulation and subsequently, diseases transmission to humans. conclusion the recent study showed that watermelon and co2 were the potential candidates for using in atsb and atb, respectively. these two substances are more effective and affordable than other examined materials. the data obtained from this study can help to identify and introduce effective baits for using in attractive toxic sugar baits (atsb) and attractive toxic baits (atb), in order to control the sand fly populations and reduce disease cases. acknowledgements this research has been funded by tehran university of medical sciences (project no. 36142). the ethics board of the tehran university of medical sciences was permitted the study procedures with ethical approval number: ir.tums. sph. rec. 1396. 3601. the authors declare that they have no conflicts of 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mwang’onde bj, mahande am, nyindo m, mosha f (2011) the role of cow urine in the oviposition site preference of culicine and anopheles mosquitoes. parasit vectors. 4: 184. j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 94 http://jad.tums.ac.ir published online: april 27, 2019 original article the challenges of the malaria elimination program in the south east of iran, a qualitative study khodamorad soofi1; *narges khanjani2; fatemeh kamiabi3 1department of epidemiology and biostatistics, faculty of health, kerman university of medical sciences, kerman, iran 2research center for environmental health engineering, school of public health, kerman university of medical sciences, kerman, iran 3department of environmental health and medical entomology, faculty of health, kerman university of medical sciences, kerman, iran (received 2 nov 2015; accepted 30 jan 2019) abstract background: despite all the efforts made to control and eliminate malaria in iran, this disease is still considered as a priority health problem in the south east of iran. we aimed to determine the cultural obstacles which have prevented the elimination of malaria in this region. method: this study was carried out through qualitative content analysis. purposeful sampling was done from people who had malaria or were involved with malaria patients in 2015, in sarbaz city, sistan and baluchestan province, eastern iran. data were collected through interviews using open questions and continued until data saturation. results: the most important barriers in malaria prevention was delay in visiting health centers, delay in diagnosis and treatment due to superstitious beliefs, lack of information about the disease, misdiagnosis and fake doctors. other obstacles were lack of trust and cooperation with interventions offered by the health system, lack of proper use of the available facilities to prevent malaria and commuting in the high-risk neighbor countries. conclusion: raising awareness in people, officials and health workers about malaria and preventive health interventions as well as health risks associated with fake doctors, following up and re-examination of peripheral blood smear in suspected cases, establishing malaria control stations in border areas and specific measures to refer immigrants and people crossing the border toward malaria diagnosis stations is suggested. keywords: malaria; elimination; challenge; iran introduction overall, 212 million cases of malaria and 429 thousand deaths due to malaria have been reported around the world (1). currently, about 40% of the world's population who live in lowincome countries are at risk of malaria. although the disease has existed in many parts of the world, today it is mainly found in tropical and subtropical countries and 90% of these cases are in poor african countries (2). iran had 799 microscopic confirmed cases that means the total number of cases were 799 and 166 cases were local or autochthonous cases (1). in 2006, 15896 cases of malaria were reported in iran and the incidence of this disease was 0.2 per thousand people (3) and about 95% of the cases had been reported from three provinces, including sistan and baluchestan, kerman and hormozgan (4). environmental, climatologic, economic, social and cultural conditions and also the province's shared border with afghanistan and pakistan and illegal immigration seem to play an important role in the high number of malaria cases in this province. this province has 1265km shared border with afghanistan and pakistan (5). the south and southeast area of *corresponding author: dr narges khanjani, email: n_khanjani@kmu.ac.ir https://translate.googleusercontent.com/translate_f#_enref_1 https://translate.googleusercontent.com/translate_f#_enref_2 j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 95 http://jad.tums.ac.ir published online: april 27, 2019 iran is still a troubled region in terms of malaria and it is not comparable with any other part of the country based on social, developmental, economic, cultural, and geographical conditions. these areas are socially similar to underdeveloped and deprived areas (3). in recent years, there has been a lot of effort to control and eliminate malaria at global level by interrupting the cycle of disease transmission. the world health forum identified a 15yr framework for all countries that work on malaria control and elimination in 2015 and called for the elimination of malaria in at least 10 countries by 2020, 20 countries by 2025, and 35 countries by 2030. less than half of the 91 malaria-prone countries are on track to achieve the 2020 goals of the who (1). there is a risk of relapse or reemergence of malaria in cleared areas and even epidemics in malarious areas of iran (6). now, local distribution and transmission of the disease in iran have been limited to the provinces of sistan and baluchistan, hormozgan and kerman. the ultimate goal of the malaria elimination program in the horizon of 2025 is to stop local transmission of the disease. sarbaz city with annual parasite incidence (api) equal to 4.5per ten thousand people in 2015 is a candidate for intensified malaria control operations (7). about 10% of local (autochthonous) malaria cases in 2015 were from sarbaz city, which is one of the active foci of malaria in the south-east of iran. therefore, there are a series of cultural and social barriers that despite all efforts to control and eliminate malaria, this disease is still an important health problem in the south east of iran. although some qualitative studies have been conducted about malaria control in world countries, no such study has been conducted in iran, yet. a qualitative study about malaria prevention in gambia suggested control programs should be conducted at the right time, the target population should be properly educated, sensitization meetings should be held continuously and roles should be defined beforehand (8). in peru, malaria was believed to be embedded within their culture and people blamed this for lack of attention to prevention. people thought malaria prevention was their government's responsibility not their own. the other barriers to malaria prevention were people not using bed nets at the right time, not knowing the purpose of space spraying and misconceptions about the cause and prevention of malaria (9). some mothers in gabon preferred to consult a nganga (traditional healer) and not a physician for their children’s malicious fever and thought it was supernatural (10). this qualitative study was carried out to identify the challenges to control and eliminate malaria in the south-east of iran. materials and methods this research was a qualitative content analysis study conducted in 2015. participants were purposefully selected from co-operative malaria patients and medical doctors willing to share their experiences and beliefs with researchers. informed consent was taken from them before the study. malaria patients were selected from the list of diagnosed malaria cases that were under treatment. most of these patients were from sarbaz city. sampling continued until data saturation was achieved. sarbaz city is located in sistan and baluchestan province and because of its high malaria incidence was a suitable place for doing this research. the county has 121km common border with pakistan and the farthest point is about 250km away from the border. there is no permanent river in this city but suitable larval habitats are present. the surrounding mountains make access to health services difficult for rural people who are 92% of the residents. interviews were conducted after participants' consent. the interviews were recorded and then transcribed. interviews were done if necessary in local language (baluchi) and then translated. the average duration of the j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 96 http://jad.tums.ac.ir published online: april 27, 2019 interviews was 45 to 60min. the questions were semi-structured. the interviewer attempted to understand the experience, views, and beliefs of the interviewees. during the interviews, a better understanding of the situation was achieved and some more questions were added to the interviews. interviews were conducted until data saturation was achieved. results seventeen malaria patients and two medical doctors were enrolled. six of the patients were drivers who worked across the iranpakistan border (table 1). the data about the challenges of the malaria elimination program were classified into 4 main groups of concepts. concept 1: lack of on-time referral to health services and delays in diagnosis and treatment can be due to several reasons: a. superstitious beliefs superstitious beliefs are one of the most important factors that prevent patients from visiting health care services and therefore by causing delay in diagnosis and treatment lead to persistence and transmission of the disease. one of the malaria patients confessed that when she was very ill and unconscious instead of taking her to a doctor, her relatives burned espand for her and thought she was stricken by the evil eye. burning espand (a desert plant) is believed to prevent the evil eye in superstitions. “… i was fine, someone cast an evil eye on me, my daughter in law burned espand for me, but i didn't get better …” some patients especially falciparum cases (the researcher knew the parasite type from the lab result), after showing severe malaria symptoms, believed that they were haunted by elfs or nasty spirits and needed to see pray drafters, local witches, or religious figures to exorcize the evil spirits. one patient said: “... i was shivering, i felt bad, my eyes turned white, i felt i was haunted, they took me to the mullah …” b. lack of information about the disease most patients did not have enough information about malaria, its contagiousness, and prevention to avoid being infected or seek treatment on time. one patient said: “... first, when a headache and fever started i felt i have a cold, but later when i took the test they said i got malaria! i don't know how i got malaria?! how should i know that a mosquito bite can cause malaria?! ...” another patient said: “... i went to pakistan and it was very hot there and i was heatstroke … i don’t know how i got malaria when my fever and headache began, i thought it was icter [liver disease] …” many patients felt that there is no serious problem and the disease will get better on its own and ignored it, tried home remedies and did not see a doctor which led to delayed diagnosis and treatment. for example, a mother whose child was sick said: “… first, we gave him a syrup and thought he will get better, after one week still he did not feel better, … later we took him to a doctor …”. another patient said: “… when my fever started i thought it is a usual fever and i took fever pills”. some of the men said they thought they do not need to visit health centers and health centers are for women, and unless they feel really bad or are under pressure from their families they would not visit the health centers. this ignorance makes the disease worse, causes delayed diagnosis and treatment, leads to persistence of infection in the area and may even cause the patient's death. for example, one of the people said: “here, men do not visit the health centers unless they really felt bad… when i had a fever, i didn’t go to the doctor right away, although my family insisted… on the fourth day, i felt really bad and my family urged me j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 97 http://jad.tums.ac.ir published online: april 27, 2019 to go to the clinic …” also by observing the patients living conditions, the researchers realized that the people living in this area and especially their men are very tough and tolerating. they have grown up in harsh conditions and won't take a fever serious unless they are well informed about the dangers of malaria. c. fake doctors another reason people do not visit the health care centers is their trust in the fake doctors known as bengali doctors. fake doctors are fraudulent people who have no academic degree and attempt to cure patients for money. they are an important threat to public health in this region. a third of the patients that we spoke with had initially visited a fake doctor and then after getting a series of medications and not recovering and feeling worse, eventually visited the health centers. “the second day because i felt so much worse i went to the bengali doctor, he gave me two injections, but i didn’t get better, on the fourth day because i got worse my family insisted that i should go to the health center …” the mother of a sick child said: "first thing, i took my baby to a bengali doctor and he gave him medicine. he took the medicine for two days but he did not get better …” two of the doctors working in sarbaz city also believed that the presence of bengali doctors is a threat to public health and complained about the wrong treatments, fake and illicit medications and other harmful treatments. d. false diagnoses sometimes, despite the onset of symptoms, due to general and non-specific symptoms of the disease, low number of parasites in blood samples, lab mistakes, and non-native doctors’ that are not aware of the common diseases in the area, early diagnosis of malaria is delayed. a false diagnosis may also keep a patient confident for a long time that his disease is not serious and therefore lead to more delayed diagnosis and more time for disease transmission in the area. a patient said: “... i went to the clinic because of my weakness and abdominal pain, but the doctor did not even get suspicious that it was malaria and he just gave me a shot and a serum …” or another patient said: “i had fever for 4 days, the first day i went to the clinic and they took a blood sample and said you do not have malaria and on the fourth day because i got worse i went to the clinic again and they took another blood sample and diagnosed that it was … malaria and i was taken to the hospital by an ambulance …” concept 2: lack of trust in interventions done by the health system many patients said they did not believe the interventions done by the health care system work. for example, one of the health interventions done in the malaria-prone areas is indoor residual spraying. in order to prevent malaria, people have to believe that this method works and allow proper spraying to be done. but most patients were not fully aware of its effects and considered it useless and futile and were reluctant to cooperate with the healthcare officers. they thought the pesticides are ineffective and the workers who do the job do not have the passion and commitment. sometimes due to the frustration of repeated pesticide spraying, hot weather, the trouble of unloading home appliances, ruining paint and … people are reluctant to cooperate. comments of some patients were as follows: “we spray to kill mosquitoes, but the insecticides don't work.” “... sometimes we don't feel like spraying, because it is hot and unloading the house is hard …” “... we did spray insecticides but we did not see any benefit, the insecticides don't kill anything since there is dust, spraying becomes ineffective ...” another patient had a strange thought and j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 98 http://jad.tums.ac.ir published online: april 27, 2019 said: “… spraying insecticides are useless, the workers don't spray well … some workers take [steal] the insecticides. they spray water, if they did spray insecticides well, mosquitos would get killed”. concept 3: improper use of the available facilities to prevent malaria one of the ways for preventing malaria in the area covered by sarbaz city health system is the use of insecticide-treated bed nets. however, they are not used and appreciated by the residents for various reasons. people might use this protective tool only when electricity is gone, they have no appropriate cooling system, or when they rest in the yard. for example, one of the patients stated: “... when electricity is available and air conditioning is on we do not use bed nets; we only use them when power is cut off or when we are sleeping in the yard…” the small number of people who use the net, do this in order to get rid of annoying insect bites rather than the prevention of malaria. for example, one of the people said: “... if we don't use bed nets mosquitos do not let us sleep …” it was understood from the statements of the participants that even this limited use was not done correctly either. the common time to use mosquito nets in this area is usually after the evening prayers, after having dinner and bedtime, while the anopheles mosquitoes are active from sunset and there is a possibility to get bitten before the time that they actually use their nets. patients commented that: “... we have bed nets and we use them when we are going to sleep. we cannot use them from the beginning of night …” moreover, in some households, there are not enough bed nets and everyone cannot sleep under the mosquito net. “… we have bed nets but it is not enough for 4 adults, only little kids sleep under a mosquito net ...” “… we have bed nets but we don’t use them. it is not enough, for 6 people we only have one net, if the air conditioning is on, we don’t use it …” but according to the participant's comments, even in households where there are enough bed nets, they might not be used properly: “… we are 7 people and we have 4-bed nets, but only 2 or 3 of us use them …” participants also mentioned other reasons for not using mosquito nets at the beginning of night and one was the disturbance of livestock. “... if we use them at the beginning of the night, the goats will tear them apart …” moreover, sometimes the mosquito nets they use are defective and there is a risk of mosquito bites. “… we have bed nets but they have been torn apart …” these statements can confirm that people have not been well educated about how to properly use and take care of their nets. in addition, in order for spraying to be efficient, it should be done in accordance with correct principles to have a good performance. but people do not allow their houses to be sprayed for different reasons and they rely on spraying their non-residential premises. “… if they come for spraying we will let them spray the outdoors, there is no need to spray the indoors…” concept 4: traveling to high-risk countries people in this area travel to pakistan for various reasons, such as treatment of illnesses, seeing their relatives, occupation (such as truck driver), and trade, and become infected by malaria there. most of the interviewed patients had a history of traveling to pakistan. "... i went to pakistan for 20 days, i am a truck driver and i always travel between iran and pakistan”. “… i am a cross-border driver and i usually travel to pakistan, i think i was infected in pakistan because there was no malaria in our village…” j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 99 http://jad.tums.ac.ir published online: april 27, 2019 "... i traveled to pakistan and stayed for a month and a mild fever began there, then i came back to iran and after 5 nights my fever and chills worsened…” table 1. some characteristics of the malaria patients participating in this study gender education job male female illiterate elementary school high school truck driver housewife farmer unemployed labor worker 12 5 7 5 5 6 4 2 3 2 discussion one way to prevent malaria and reduce the risk of death in patients with this disease is early detection and proper treatment (11-13). one of the expected outcomes of the malaria elimination program for 2025 is diagnosis in less than 24h after the onset of symptoms and starting the treatment within 24h after diagnosis (7). our study showed that social and cultural factors are among the factors that have led to malaria elimination failure in the south-east of iran. lack of adequate information about the disease, superstitious beliefs and prejudices, not taking the disease and its treatment serious, visiting fake doctors and incorrect diagnoses are among the factors that have led to late diagnosis and treatment and eventually longer opportunities for disease transmission and its persistence in the region. the role of social factors such as employment status and location of residence in the persistence of malaria in the south east of iran, has been discussed in previous studies (14). in ghana, the economic and social situation was significantly related to malaria (15). in gambia, social factors such as lowquality housing were related to malaria (16). in minab, jask and roudan in hormozgan province, iran; a significant relationship between social factors such as illiteracy and distance from health facilities and the incidence of malaria was observed (17). cultural problems such as the low rate of literacy and health education have had a role in the incidence of malaria in iran (12). this was also mentioned in the case-control study in chabahar city in which the education level of the control group was higher than the patients with malaria (18). in southern iran, a direct relationship was found between the level of education and people's preparedness to participate in the malaria control programs on a voluntary basis (19). high literacy levels through increased people's participation in malaria control programs, use of protective equipment, child care, disease knowledge and appropriate job and housing safety can be effective in controlling malaria (18). implementation of educational intervention programs can increase knowledge, attitude and the practice of preventing malaria in the society (20). the reduced incidence of malaria had taken place for reasons such as an increase in knowledge, attitude and proper performance of people (21). therefore it seems necessary to plan to increase awareness in the health personnel, public and authorities by local facilities and also take the necessary measures to identify arrest and punish fake doctors who are a threat to public health and take advantage of people nescience to earn money. the low number of parasite in peripheral blood is one of the most important problems and challenges in the malaria elimination program. a negative blood smear does not mean the patient does not have malaria. therefore, whenever a person is suspected of having malaria but the blood test is negative, his test should be repeated in the next few days (13) j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 100 http://jad.tums.ac.ir published online: april 27, 2019 rather than assuring the patient that he does not have malaria. lack of people's trust in the interventions done by the health system is another challenge facing malaria elimination programs. indoor residual spraying and bed net distribution are among the preventive interventions performed by the health network. although many people are acquainted with spraying and bed nets, in practice, the results of our study and other research show that we are still far away from the appropriate use of these preventive interventions (12, 22, 23). in this study, some patients commented that the insecticides were not effective. studies have reported mosquito resistance to insecticides as well (24-26). in order to prevent mosquitoes from developing tolerance, the type of insecticide is changed periodically (23). on the other side, health authorities try to use insecticides that have the least danger for humans. these insecticides might not kill the mosquito immediately, but decrease the mosquito's lifetime and therefore prevent the transmission of disease. however local people expect not to see any insects at all after spraying and therefore might think the insecticides are not effective. the results of this study indicate the lack of willingness and cooperation of people for indoor spraying which is the main obstacle against the success of malaria elimination programs (12). in iranshahr, only 30% of highrisk places were sprayed by insecticides and the reason was residents’ noncooperation despite the fact that most people knew residual spraying was an effective method in reducing malaria transmission. although more than 95% of iranians and 90% of afghan participants knew that spraying, drying swamps and using bed nets prevent malaria but only 24% of participants used mosquito nets and about 90 % of afghans and 62% of iranians did not use nets, 77.7% of the participants slept inside and 64.9% of iranians did not use flyscreens' for windows (22). this is despite the fact that in sistan and baluchestan there are two major malaria vectors including anopheles stephensi and an. culicifacies that have endophilic (rest indoors) and endophagous (feed indoors) tendencies (27). improper use of the existing facilities, despite the high cost and financial burden that it has for the health system, is among the barriers of prevention and eliminating malaria. several studies have considered using bed nets and spraying, effective to control and prevent malaria (12, 18) and the use of treated bed nets as one of the most promising tools for malaria control in most parts of the world (14). however, misuse of for example window fly screens increases the risk of malaria even up to 5 times (17). the proper use of mosquito nets especially the treated ones requires continuous training to ensure the correct use by the majority of people (18). diverting mosquitoes from humans to animals reduces the mosquito-human contact and has a protective role (12) but in case of incomplete and inaccurate spraying, or only spraying outside places and leaving the indoors intact, it will divert the mosquitoes to indoor spaces (13). in order to just satisfy the health authorities, some people allow pesticide spraying in areas such as baths, toilets, animal barns and other places. spraying these places alone can divert mosquitoes into the residential buildings and people's sleeping places and therefore trouble malaria control programs. if the spraying is done properly in place, the abundance of vectors is reduced (28, 29). one of the main features of successful spraying is its comprehensiveness (spraying at-least 70% of all places in the foci), completeness (covering all eligible surfaces) and sufficiency (using the necessary amount of insecticide on surfaces) and repetition at regular intervals (13). another challenge for southeastern iran's malaria elimination program is its shared borders with high-risk countries. this province has 1265km of shared border with pakistan and afghanistan in which legal and illegal movement j arthropod-borne dis, march 2019, 13(1): 94–103 k soofi et al.: the challenges of … 101 http://jad.tums.ac.ir published online: april 27, 2019 of people takes place. among illegal commuters, no supervision or control in regard to their health status is imposed. many studies have been performed on traveling and migrating between malaria-infected centers and its role in malaria transmission. for example, traveling during the last one month before illness was among the factors that increase one's risk of getting malaria and people with a history of travel during the last month had a 21 times higher chance of being infected in comparison to others. chabahar a city near the pakistan border has been introduced as one of the important foci of malaria transmission (18). moreover, in the united arabic emirates, most malaria cases were pakistani immigrants (30). newly arrived immigrants with less than three months of residence in iran had a significantly higher incidence of malaria than the ones with more than 3 months of residence (31). the presence of foreigners in this city in 2008, in addition to increasing the number of malaria cases, has led to changes in malaria foci (32) and people traveling from endemic to nonendemic areas had a great role in the outbreak of malaria (33). the important role of migrants in the transmission and outbreak of malaria has been studied by other researchers (34, 35). in sarbaz county in the former crossing point, a malaria passive post or rdt (rapid diagnosis test) passive post was established to discover malaria cases. however, real success is achieved when all local people and officials understand the importance of malaria and cooperate. in addition, since most commuting occurs in other parts of this county (such as hong, mortan, kastag, kozour, eshkastag, and ashar) which is illegal and uncontrolled, planning to put these areas under surveillance seems necessary. conclusion in order to achieve the goal of eliminating malaria the proper use of existing facilities such as insecticide spraying, mosquito nets, etc., and establishing portable sites for rapid diagnosis of malaria in border areas, is necessary. emphasis on cross-sectoral and inter-sectoral cooperation, to attract more local health officers who are familiar with the customs and common diseases in the region, and planning long-term educational programs, is also suggested. moreover, in order to raise awareness among health professionals, local people and officials; local media, health networking, and the influence of religious leaders should also be incorporated. holding training sessions in accordance with the culture, to increase public awareness and participation; and penalizing fake doctors who sometimes have the support of local people and local authorities is also necessary. acknowledgements the authors appreciate the cooperation of the sarbaz county health staff, as well as the patients who participated in this study. the authors declare that 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https://www.ncbi.nlm.nih.gov/pubmed/?term=azizi%20a%5bauthor%5d&cauthor=true&cauthor_uid=25629068 https://www.ncbi.nlm.nih.gov/pubmed/?term=ali%20aa%5bauthor%5d&cauthor=true&cauthor_uid=25629068 https://www.ncbi.nlm.nih.gov/pubmed/?term=nasser%20a%5bauthor%5d&cauthor=true&cauthor_uid=25629068 https://www.ncbi.nlm.nih.gov/pubmed/?term=hasaballah%20i%5bauthor%5d&cauthor=true&cauthor_uid=25629068 https://www.ncbi.nlm.nih.gov/pubmed/?term=hanafi-bojd%20aa%5bauthor%5d&cauthor=true&cauthor_uid=25629068 http://irje.tums.ac.ir/search.php?sid=1&slc_lang=en&auth=parvin https://www.ncbi.nlm.nih.gov/pubmed/?term=abedi%20f%5bauthor%5d&cauthor=true&cauthor_uid=21570940 https://www.ncbi.nlm.nih.gov/pubmed/?term=yeryan%20m%5bauthor%5d&cauthor=true&cauthor_uid=21570940 https://www.ncbi.nlm.nih.gov/pubmed/?term=pakari%20a%5bauthor%5d&cauthor=true&cauthor_uid=21570940 https://www.ncbi.nlm.nih.gov/pubmed/?term=hanafi-bojd%20aa%5bauthor%5d&cauthor=true&cauthor_uid=28032101 https://www.ncbi.nlm.nih.gov/pubmed/?term=abai%20mr%5bauthor%5d&cauthor=true&cauthor_uid=28032101 https://www.ncbi.nlm.nih.gov/pubmed/?term=salim-abadi%20y%5bauthor%5d&cauthor=true&cauthor_uid=28032101 https://www.ncbi.nlm.nih.gov/pubmed/?term=rafi%20f%5bauthor%5d&cauthor=true&cauthor_uid=28032101 https://www.ncbi.nlm.nih.gov/pubmed/?term=adungo%20ni%5bauthor%5d&cauthor=true&cauthor_uid=7903238 https://www.ncbi.nlm.nih.gov/pubmed/?term=githeko%20ak%5bauthor%5d&cauthor=true&cauthor_uid=7903238 https://www.ncbi.nlm.nih.gov/pubmed/?term=ekisa%20ws%5bauthor%5d&cauthor=true&cauthor_uid=7903238 https://www.ncbi.nlm.nih.gov/pubmed/?term=mahadevan%20s%5bauthor%5d&cauthor=true&cauthor_uid=7903238 https://www.ncbi.nlm.nih.gov/pubmed/?term=ouma%20jh%5bauthor%5d&cauthor=true&cauthor_uid=10344652 https://www.ncbi.nlm.nih.gov/pubmed/?term=ouma%20jh%5bauthor%5d&cauthor=true&cauthor_uid=10344652 https://www.ncbi.nlm.nih.gov/pubmed/?term=githure%20ji%5bauthor%5d&cauthor=true&cauthor_uid=10344652 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https://www.ncbi.nlm.nih.gov/pubmed/?term=quint%c3%b3%20l%5bauthor%5d&cauthor=true&cauthor_uid=18479645 https://www.ncbi.nlm.nih.gov/pubmed/?term=gasc%c3%b3n%20j%5bauthor%5d&cauthor=true&cauthor_uid=18479645 j arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 193 http://jad.tums.ac.ir published online: june 30, 2020 original article epidemiological characteristics of scorpionism in west azerbaijan province, northwest of iran samira firooziyan 1,2 ; ali sadaghianifar 2 ; javad rafinejad 1 ; hassan vatandoost 1,3 ; *mulood mohammadi bavani 4 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 urmia health center, disease control unit, urmia university of medical sciences, urmia, iran 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4 department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran (received 26 oct 2019; accepted 22 may 2020) abstract background: there are four medically important scorpion species (mesobuthus eupeus, mesobuthus caucasicus, androctonus crassicauda and hottentotta saulcyi) in the west azerbaijan province, northwestern iran. scorpionism is considered as a health problem in this region, because there is no information about scorpion envenomation, this study was designed to study epidemiological characteristics of scorpionism to optimize prevention and treatment of scorpion sting in northwest of iran. methods: all the data from epidemiological surveys completed in west azerbaijan hospitals over four years (2014– 2017) for scorpion victims were collected. this information includes the number of victims, sex, age, signs and symp toms, site of sting, body parts of victims, history of previous sting, the condition of the patient in terms of recovery and death, and the time to receive anti venom, all data were analyzed by the excel software. results: a total of 2718 cases of scorpionism were reported from march 2014 to march 2017 in the study area. the most cases occur in both sexes in the age groups of 25 to 34 years old. in urban areas 40.3% of people and in rural areas 59.7% of them have suffered. the poldasht and chaldoran cities, had the most and least incidence respectively. conclusion: in this study, the high risk areas in the case of scorpion stings were identified. results of this study can be used to design preventive programs to educate more people about dangerous areas to prevent scorpion sting and even death. keywords: scorpion; scorpionism; west azerbaijan introduction so far 64 species of scorpions have been reported from iran belonging to buthidae (86%), hemiscorpiidae (9.5%), and scorpionidae families (4.5%) (1). scorpions are venomous animals that can sting people and cause public health problems and sometimes can be fatal (2). the medically important scorpion species in iran belong to two families of buthidae and hemiscorpiidae (2). from the buthidae family the species of mesobuthus eupeus, mesobuthus phillipsii, mesobuthus caucasicus, odontobuthus doriae, odontobuthus bidentatus, orthochirus iranus, buthacus macrocentrus, apistobuthus susanae, compsobuthus matthiesseni, hottentotta saulcyi, hottentotta zagrosensis and hottentotta jayakari are the most medically relevant scorpion in iran (2-8). from hemiscorpiidae the hemiscorpius lepturus and hemiscorpius acanthocercus species are considered as dangerous and deadly scorpion in iran (7, 9). buthidae is the largest family in iran (2). most medically important scorpion species have been reported from south and south western provinces of *corresponding author: dr mulood mohammadibavani, e-mail: mulud.muhammady@yahoo.com j arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 194 http://jad.tums.ac.ir published online: june 30, 2020 iran, among which khuzestan is regarded as high risk area (2, 10). buthidae species have mostly neurotoxic effect, but hemiscorpiidae such as h. lepturus (local name: gadim), causes cell death with hemolytic effect cause more death in the country (11-13). in iran for scorpion sting treatment polyvalent antivenom is used. this antivenom is prepared by razi institute against six medically relevant scorpion species: m. eupeus, a. crassicauda, o. doriae, h. saulcyi, h. zagrosensis and h. lepturus (2, 3). there are four medically important scorpion species: m. eupeus, m. caucasicus, a. crassicauda and h. saulcyi: in west azerbaijan province, northwestern iran, among which m. eupeus is the most prevalence and androctonus crassicauda is the deadliest (3, 14). the people most affected by scorpion stings usually live in poor communities where medical resources are often sparse. scorpion sting is a common health problem all over the world, including iran. nearly 50000 cases of scorpion stings have been reported annually from iran (3, 15). some studies on scorpionism in iran shows: the most cases of scorpion stings occurred in rural area in summer season (1620). the age groups of 10–24 and 25–44 years old are more at risk (17, 20-22). foot and hand are more frequent bitten by scorpions (16, 20, 22-25). scorpion venoms, which are especially lethal in young children, release autonomic nervous system mediators causing myocardial damage, cardiac arrhythmias, pulmonary edema, shock, paralysis, muscle spasms and pancreatitis (26). early administration of anti-venom is highly effective, together with intensive care support in severe cases. however, the rapid tissue distribution of scorpion venom toxins and their ability to cause early death especially in young children, demands early treatment with anti-venom and full cardio-respiratory support (27). the true incidence of scorpion sting envenoming is not known because many cases do not seek medical attention. however, it has been estimated that there are approximately 1 million stings per year in the world. in northern africa, the middle east (notably iran), india and latin america scorpion stings are an emergent health problem, due to the adaptation of some scorpion species to the urban environment (15). since over the past 4 years, deaths from scorpion sting have been reported from west azerbaijan province and scorpion sting is a public health problem in this region of iran. there is no information about epidemiological characteristics of scorpionism in this area. therefore, this study was designed to describe epidemiological characteristics of scorpion envenomation in west azerbaijan province, northwest of iran to optimize prevention and treatment through community awareness. materials and methods study area west azerbaijan province is located in the northwest of iran (fig. 1), bordering turkey, iraq and azerbaijan's nakhchivan autonomous republic, as well as the provinces of east azerbaijan, zanjan and kurdistan. it is separated from armenia by turkey's short border with the azerbaijan republic. this province covers an area of 39,487km², or 43,660 km² including lake urmia, between 37.5528° n and 45.0759° e. in 2012 the province had a population of about 3 million (estimate). the capital and largest city of the province is urmia. the people of this province are active in agriculture and animal husbandry. for this reason, the province has been selected to examine the status of scorpion sting and important medical species. data collection the data from epidemiological surveys completed in west azerbaijan hospitals over four years for scorpion victims were collected. this information includes the number of victims, sex, age, signs and symptoms, body parts of victims, time of sting, history of previous sting, https://en.wikipedia.org/wiki/azerbaijan_(iran) https://en.wikipedia.org/wiki/turkey https://en.wikipedia.org/wiki/iraq https://en.wikipedia.org/wiki/azerbaijan https://en.wikipedia.org/wiki/nakhchivan_autonomous_republic https://en.wikipedia.org/wiki/nakhchivan_autonomous_republic https://en.wikipedia.org/wiki/east_azerbaijan_province https://en.wikipedia.org/wiki/east_azerbaijan_province https://en.wikipedia.org/wiki/zanjan_province https://en.wikipedia.org/wiki/kurdistan_province https://en.wikipedia.org/wiki/armenia https://en.wikipedia.org/wiki/azerbaijan_republic https://en.wikipedia.org/wiki/lake_urmia https://en.wikipedia.org/wiki/urmia j arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 195 http://jad.tums.ac.ir published online: june 30, 2020 the condition of the patient in terms of recovery and death and the time to receive anti venom. the above information was analyzed by the excel software. a spatial distribution map of scorpionism was introduced using gis 4.2. results from march 2014 to march 2017, a total of 2718 cases of scorpion sting from west azerbaijan province were recorded. the largest number (821 cases) was in 2014 and the lowest (548 cases) in 2015 (fig. 2). many scorpion stings occurred during the hot months of may to september with a peak in august (fig. 2). in these four years, 53.6% of cases occurred in men and 46.4% in women. in males, the most cases were in the age group of 25 to 34 years old and the least cases were in the age group of 5 to 9 years old (table 1), while in the females, the highest number of victims was in the age group of 25 to 34 years, and the smallest in the age group of 0–4 years old (table 2). therefore, the most cases of scorpion sting in both sexes were in the age group of 25 to 34 years old. 40.3% of stung people in urban areas and 59.7% in rural areas were reported. among 2718 patients, in 80 cases (2.9%) the head and neck, in 1364 cases (50.2%) the hands, in 1069 cases (39.3%) the leg and in 205 cases the trunk were bitten (table 3). in this study, the most cases of scorpion stings including 1263 cases (46%) occurred: at 00:00 to 6:00am and the lowest cases including 456 cases (16.8%) occurred at 12:00 am to 6:00pm (table 4). 8 percent of the cases (n= 217) had previous history of sting and 2.2% (n= 61) had used previous scorpion anti venom in the past four years. the time of treatment between sting and anti-venom injection in 70.3% of patients were less than 6 hours, in 8% of patients were 6 to 12 hours, in 6.4% of patients lasted more than 12 hours and 15.3% of patients did not receive anti venom (table 5). the percentage of recovered cases were 99.93% and the deaths due to scorpion sting were 0.07% (2 out of 2718 cases), of which two deaths were observed in the last two years (one case in 2016 and another in 2017). the average incidence of scorpionism per 1000 people was calculated and poldasht and chaldoran counties had the most and least incidence (table 6, fig. 3). table 1. stung persons classified by age group (males) total >65 55–64 45–54 35–44 25–34 15–24 10–14 5–9 0–4 years 305 313 262 380 1260 100 19 12 15 20 66 5.2 28 20 13 26 87 6.9 41 42 46 56 185 14.7 57 55 46 66 224 17.8 68 76 61 93 298 23.6 56 61 48 83 248 19.7 17 20 13 16 66 5.2 6 19 8 13 46 3.7 13 8 12 7 40 3.2 2017 2016 2015 2014 total % table 2. stung persons classified by age group (females) total >65 55–64 45–54 35–44 25–34 15–24 10–14 5–9 0–4 years 356 375 286 441 1458 100 18 17 15 22 72 4.9 20 22 10 22 74 5.1 34 41 44 45 164 11.2 66 58 47 90 261 17.9 82 96 77 111 366 25.1 77 82 54 103 316 21.7 20 27 14 18 79 5.4 15 19 12 11 57 4 24 13 13 19 69 4.7 2017 2016 2015 2014 totally % j arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 196 http://jad.tums.ac.ir published online: june 30, 2020 table 3. stung persons according to body part total trunk leg hand head and neck years 661 688 548 821 2718 100 56 43 47 59 205 7.5 258 286 228 297 1069 39.3 330 344 254 436 1364 50.2 17 15 19 29 80 3 2017 2016 2015 2014 total % table 4. time of scorpion stings in stung persons total 18–24 12–18 6–12 0–6 years 661 688 548 821 2718 100 142 152 123 83 500 18.4 163 122 106 65 456 16.8 153 166 115 65 499 18.3 203 248 204 608 1263 46.5 2017 2016 2015 2014 totally % table 5. time of anti-venom injection after sting total >12 6–12 <6 without years 661 688 548 821 2718 100 61 44 34 36 175 6.4 48 65 31 74 218 8.0 480 501 338 591 1910 70.3 72 78 145 120 415 15.3 2017 2016 2015 2014 totally % table 6. the average incidence of scorpionism per 1000 people in different areas years 2017 2016 2015 2014 totally % per1000 naghadeh 16 17 14 44 91 3.35 0.71 miandoab 3 10 12 36 61 2.24 0.22 mahabad 43 47 31 46 167 6.14 0.71 maku 38 63 33 73 207 7.62 2.18 shoot 50 8 0 7 65 2.39 1.17 shahin dej 21 26 29 44 1120 4.42 1.30 salmas 48 103 91 66 308 11.33 1.57 sardasht 64 35 27 54 180 6.62 1.51 khoy 15 28 11 22 76 2.80 0.22 chaldoran 1 0 5 0 6 0.22 0.13 chaipareh 6 1 8 3 18 0.66 0.40 takab 51 48 14 58 171 6.29 2.12 piranshahr 7 18 5 3 33 1.21 0.24 poldasht 74 107 92 46 319 11.74 7.56 bukan 21 19 23 38 101 3.72 0.40 oshnavieh 23 4 4 10 41 1.51 0.55 urmia 180 154 149 271 754 27.74 0.72 total 661 688 548 821 2718 100 0.83 j arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 197 http://jad.tums.ac.ir published online: june 30, 2020 fig. 1. the status of study area, west azerbaijan province in iran 0 50 100 150 200 250 jan feb mar apr may jun jul aug sep oct nov dec 2014 2015 2016 2017 fig. 2. monthly scorpion sting cases, west azerbaijan, northwest of iran, 2014–2017 (x: month, y: scorpion sting cases) j arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 198 http://jad.tums.ac.ir published online: june 30, 2020 fig. 3. spatial distribution of incidence of scorpion sting from west azerbaijan of iran, 2014–2017 discussion scorpion stings are considered as one of the most medically important problem in iran. there are 64 species of scorpions belonging to tree families (buthidae, hemiscorpiidae and scorpionidae) in iran (1). four medically important species of scorpions including: m. eupeus, m. caucasicus, a. crassicauda and h. saulcyi have been reported from west azerbaijan province and cause public health problems in this region. m. eupeus has been captured in the most parts of northwest corner of iran (3). scorpionism is a common health problem all over the world, yet neglected. the true incidence of scorpion sting envenoming is not known because many cases do not seek medical attention. however, it has been estimated that there are approximately 1 million stings per year. in mexico, tunisia, brazil and khuzestan, south-west iran, 250000, 40000, 37000 and 25000 stings were reported in 2005 (15). about 37535 scorpion stings were registered in 2009 and in comparison with cases of 2002, we observed notably increasing on incidence of scorpion sting in iran (6, 18). although scorpion sting is a public health problem in the west azerbaijan province but there is a big gap of information on scorpionism in this region. our results showed that over four last years (2014–2017), a total of 2718 cases of scorpion stings have been recorded from west azerbaijan. in this region, the age group of 25 to 39 is more at risk and the health system in community education should address this age group, while some studies have suggested that most of the victims of scorpionism are younger than 25 years old (18, 19, 28). results of some studies like our results shows that the age of the most of stung persons are more than 25 years old (17, 21, 22). this age group is more active in farming, ranching and gardening. because of their greater activity, they are more likely to be contacted by scorpions. the health system in community education should address this age group. our results showed that 53.6% of cases occurred in men and 46.4% in women that scorpion sting among males sex were 7.2% more than in females. according to reports from the world health organization and some studies, the same result was reported (15, 29, 30). while in some other studies unlike our results scorpion stings in females were more than in males (22, 31, 32). however, in west azerbaijan province, because men are more active than women and most of the farming and gardening work is done by men so they are more at risk in the case of scorpion stings, therefore men should be given more training in order to prevent further scorpion sting. in this study, similar to some other studies, hand, leg, trunk, head and neck, respectively, are more likely to be stung by scorj arthropod-borne dis, june 2020, 14(2): 193–201 s firooziyan et al.: epidemiological characteristics of … 199 http://jad.tums.ac.ir published online: june 30, 2020 pions (18, 29, 33-36). like our results in some studies foot and hand have been more frequent bitten by scorpions (16, 20, 22-25). this may be because most people do not take protective measures like using gloves and boots while working in the fields and gardens. therefore, these organs are easily exposed to scorpion sting. we have to educate people to carefully examine shoes before wearing them and to use gloves and safety shoes when working in an open environment and do not move stones for no reason. in this study, about 15.3% of the victims didn’t receive antivenin. as most cases occur in the village (60%), the villagers should be informed that antivenin is available in hospitals and health centers. because more sting occur per day, they can easily get antivenin from the health center for free. our results showed that many scorpion stings occurred during the hot months of may to september with a peak in august. the results of some studies on scorpion envenomation in iran, in this case are similar to our results that the peak of scorpion stings occurs in hot month of the year (16-20). this can be for two reasons: in the hot months of the year the scorpions are more active and also people's activities such as agriculture, gardening and other activities are increasing. these makes people more likely to encounter scorpions and bitten by them. in our study 99.93% of stung people were recovered and 0.07% of them died. because the species m. eupeus has a wide distribution in this area (3). likely this species caused the most scorpion stings in this region. androctonus crassicauda is regarded as a deadly scorpion in iran (2), and this species has been reported from this area (3). this black species is probably responsible for the death from the scorpion sting in this corner of iran. conclusion in this study, the high risk areas of the province were identified as scorpion sting by using spatial distribution. the results of this study can be used to design preventive programs, to educate more people about the important areas of the province's area at the risk of scorpion sting, and the prevention of scorpion sting and even death. 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venom 2d-lc/ms and data clustering analyses to infer phylogenetic relationships in some scorpions from the buthidae family (scorpiones). toxicon. 47(6): 628–639. 36. e oodt a arc a a om n segre l, dolab ja, funes rf (2003) epidemiological and clinical aspects of scorpionism by tityus trivittatus in argentina. toxicon. 41(8): 971–977. j arthropod-borne dis, december 2018, 12(4): 325–333 r dehghani et al.: a review on … 325 http://jad.tums.ac.ir published online: december 25, 2018 review article a review on scorpionism in iran *rouhullah dehghani 1, esmail charkhloo 2, nedasadat seyyedi-bidgoli 1, elahe chimehi 1, maedeh ghavami-ghameshlo 1 1social determinants research center of health and engineering department of environmental health, medical science university of kashan, kashan, iran 2department of environment health engineering, faculty of health, jiroft university of medical sciences, jiroft, iran (received 17 dec 2017; accepted 15 oct 2018) abstract background: scorpions are one of the most important venomous animals in iran. their sting has more prevalence in the south and southwest areas. the aim of this study was to introduce their sting agent species in the country. methods: data were extracted by a mini review on scorpion stinging articles in iran until early 2018 and then the sting agent species in each area were studied. geographical and provincial distribution of each species also was provided. results: twelve scorpion species are causative agents of sting. according to their deadly rate and clinical symptoms, some of them are considered the most dangerous venomous animals in iran. some death cases have been reported because of the sting of 3 species of hemiscorpius lepturus, h. acanthocercus and androctonus crassicauda. remaining species have not deadly sting but because of their frequency, they encounter the individuals and cause the stinging. conclusion: the highest number of sting agents is in khuzestan, however gilan and mazandaran have the lowest frequency. because of the high prevalence of sting agent species in that province, the necessity of providing control and prevention programs is very important. keywords: province, sting agent, scorpion, iran introduction scorpion stinging has been reported in most of the warm areas of the world. these animals belong to the arthropoda. scorpion’s geographic distribution is all over the world and the latitude between equator north 50 degree and south 52 degree (1) but even in this geographic area, they have different distribution. these animals have been seen in many habitats and are able to live in very tough conditions. they use the least energy (2). they sting in order to defend and feed. they are opportunist in terms of selecting their habitat and using any natural and artificial or human-made spaces and gaps for hiding and habitat (3, 4). some of them are nest makers and diggers. they make nests in the soil with smother soil pattern and proper physical structure (5-7). some species of these animals have adapted their activity inside or around the human residential areas and for this reason their probable encounter with humans has increased. therefore, in these cases, sting threat is more in comparison with active species which are out of and farther from the human residential places (8-11). in iran, the species which sting humans are more opportunist in terms of habitat selection. they use ready spaces and gaps provided in the buildings because of using traditional building materials. this arthropod starts its activities at night and uses its venomous sting to defend or hunt insects to feed. the habitat of most of them is desert and non-residential places (12-14). since these animals are hunters, like tarantulas, this kind of habitats attract the animals which are these hunters’ food. on the other hand, the scorpion hunters are attracted and make a complete food web with different food chains. therefore, this kind of plac*corresponding author: dr rouhullah dehghani, email: dehghani37@yahoo.com j arthropod-borne dis, december 2018, 12(4): 325–333 r dehghani et al.: a review on … 326 http://jad.tums.ac.ir published online: december 25, 2018 es, in addition to providing proper shelter and habitat, make their food available (15-17). scorpions are dangerous for humans because of having toxic and deadly sting and for this reason they are medically important so that according to the available statistics, they have the highest human casualties by venomous arthropod in the world (8, 18). till now, 64 scorpion species have been reported in iran (2) but no report is available if they all sting or not. the population frequency of scorpion species in iran is more than other stinging and biting animals like venomous and non-venomous snakes; therefore, their consequence is more stings (19, 20). in iran, scorpion sting is about 10 times more than snake biting. the highest human fatalities are caused by venomous arthropods in the world (21-23). scorpion sting which threatens many people to death annually is one of the most important health issues in tropical and subtropical areas like iran. the highest statistics of stings and fatalities belong to khuzestan and hormozgan (24-30). since many species live in iran but sting of all of them has not been reported, the purpose of this study is introducing the sting agent species and determining their province distribution during the past 50 years in iran. materials and methods in this review article, keywords like scorpion, sting agents, dangerous species, provincial distribution, iran, identification, studies, family and species were used in the sites related to valid medical and health journals, searching in databases like web of science, ovid, pubmed, systematic review, sid, iran medex, scirus, google scholar and medline to have access to the articles during 1977 until early 2018. the including criteria for entry in this study articles were as follows, the first all iranian articles about animal bites were searched. in the next step, they study focused on venomous animal bites and stings. then the sting agents among of the scorpions were noticed, and then all articles of the scorpion sting agents in the past decades till now have been noticed. overall, 150 sources were found, but only 75 of these considering the purpose of the study; i.e., report of the sting agent and concentration of study on iran, 73 sources were surveyed. in addition to the survey of these studies, their application in iran was done. then the gained results were provided in tables, graph and figure. results up to now, three scorpion families have been reported in iran. the sting agent scorpions in iran include two families of buthidae and hemiscorpiidae. they have 12 species from the 8 genus which 10 species belong to buthidae family and 2 species belong to hemiscorpiidae family. more than 83.5% of the identified sting agent species in iran belong to buthidae family and 16.5% belong to hemiscorpiidae family (29, 2, 31, 32, 33). identification of sting agent species among the scorpions of iran has been done by different researchers especially in the field of medical sciences. still, there are changes in the number of families, genus and species of sting agents in iran so that in the initial reports of researchers, sting agent scorpions in iran were introduced to be 3 to 4 species while they are 12 species now (2, 31). according to the last studies about scorpion sting agent species, there are 2 families in iran: buthidae and hemiscorpiidae. the species of mesobuthus eupeus are in ardabil, kerman, isfahan, markazi, mazandaran, sistan and baluchistan, yazd, kohgiluyeh and boyer-ahmad, semnan, fars, khuzestan, hormozgan, golestan, tehran, kordistan, kermanshah, ilam, west azarbaijan, khorasan razavi and khorasan jonoobi. then, compsobuthus matthiesseni in bushehr, chaharmahal and bakhtiari, fars, hamadan, kerman, kohgiluyeh and boyer-ahmad, kordistan, lorestan, markazi, qom, khuzestan, hormozgan, khorasan, kermanshah, ilam, west azarbaijan and isfahan, hottentotta saulcyi in lorestan, hamadan, chaharmahal and bakhtiari, khuzestan, west j arthropod-borne dis, december 2018, 12(4): 325–333 r dehghani et al.: a review on … 327 http://jad.tums.ac.ir published online: december 25, 2018 azarbaijan, kermanshah, hormozgan, ilam, sistan and balochistan, kordestan, kohgiluyeh and boyer-ahmad, fars, isfahan, kerman and ardabil, odontobuthus doriae in hormozgan, kerman, yazd, isfahan, markazi, ghazvin, tehran, alborz, semnan, west azerbaijan, kermanshah, busher, hamedan, hormozgan, hemiscorpius lepturus in khuzestan, semnan, fars, kordestan, hormozgan, bushehr, ilam, lorestan, kermanshah, isfahan, hamedan, kohgiluyeh and boyer-ahmad and kerman, orthochirus scrobiculosus in khuzestan, hormozgan, tehran, sistan and balochestan, qom, isfahan, median (razavi) khorasan, khorasan jonobi, gilan, semnan, kermanshah, ilam, androctonus crassicauda in bushehr, semnan, khuzestan, ilam, west azarbaijan, kordestan, khorasan razavi, south khorasan, kermanshah, kerman and sistan and balochistan, mesobuthus or olivierus caucasicus in west azarbaijan, sistan and balochestan, isfahan, south khorasan, tehran, markazi and semnan, hottentotta jayakari in qom, hormozgan and fars, hottentotta schach in fars and khuzestan, hemiscorpius acanthocercus in hormozgan and apistobuthus pterygocercus in khuzestan have been reported, respectively (31-53) (table 1, fig. 1). all the provinces of iran have at least one or some species of scorpions of scorpionida order which cause the sting. in iran, the number of sting agents of order scorpionida in fars province is 12 species. overall, 64 species of scorpions have been reported in iran so far (2, 29). mesobuthus eupeus has a wide geographic distribution in iran and at least has been reported in 20 provinces. most of the sting agent species are found in the south of iran specially khuzestan. during the last years, about 80% of all the reported sting cases in iran have been from this area. the sting of h. jayakari has been first reported from iran (8, 18). according to the reports, scorpions’ venoms which their ld50 is measured less than 1.5mg/ kg, in mice are considered to be in the dangerous and or deadly group. among the species in iran, ld50 of 5 species of this arthropod has been measured among which m. eupeus is in the dead border and has been measured as 1.45mg but ld50 of others is less (table 2). ld50 of h. lepturus is much more than buthidae family species but because of the delay mechanism of the venom of this arthropod, it is considered as one of the most deadly species in iran (8, 18, 33, 54, 55). the highest geographical distribution of sting agent scorpions is related to m. eupeus and c. matthiesseni reported in 20 and 18 provinces, respectively. in this study, the minimum geographical distribution belongs to h. acanthocercus reported from hormozgan and a. pterygocercus reported from khuzestan (8, 18, 21). apistobuthus pterygocercus is described as a. susanae based on new samples in khuzestan province. however, in the reports, the name is a. pterygocercus as a stinging agent in khuzestan (8, 18, 56) (fig. 2). discussion the number of scorpion species has been rapidly increasing in the last 3 decades in the world (57-61). the number of described species in the world has reached 2231 classified in 208 genus and 20 families. the family buthidae with a higher frequency than others are scattered all over the world. this family includes the most dangerous species. from this family, deadly species live in iran. in addition, the family hemiscorpiidae from hemiscorpius genus includes dangerous and deadly species in the middle east especially iran and iraq and are classified as the most deadly scorpions of the world (62-71). at present, one of the control methods of health is the use of pesticides, that it may cause resistance to several of urban pests such as scorpions, flies and etc., therefore, the improvement of the environment and the removal of shelters could reduce the risk of scorpion stings (72-75). j arthropod-borne dis, december 2018, 12(4): 325–333 r dehghani et al.: a review on … 328 http://jad.tums.ac.ir published online: december 25, 2018 table 1. scorpion sting agent in iran based on family, genus and species family genus species number of provinces author buthidae mesobuthus mesobuthus eupeus (c. l. koch,1839) 20 2, 30, 29, 28, 24, 23 buthidae compsobuthus compsobuthus matthiesseni (birula, 1905) 18 8, 13, 14, 2, 16 buthidae hottentotta hottentotta saulcyi (simon, 1880) 15 8, 18, 23–28 buthidae odontobuthus odontobuthus doriae (thorell, 1876) 14 8, 23, 25, 31, 32, 33 hemiscorpiidae hemiscorpius hemiscorpius lepturus (peters, 1862) 13 34–51 buthidae orthochirus orthochirus scrobiculosus (birula, 1900) 12 8, 18 buthidae androctonus androctonus crassicauda (olivier, 1807) 12 8, 18, 28, 27, 30, 52 buthidae mesobuthus or olivierus mesobuthus or olivierus caucasicus (nordmann, 1840) 7 8, 18 buthidae hottentotta hottentotta jayakari (pocock, 1895) 3 28 buthidae hottentotta hottentotta schach (birula, 1905) 2 8, 18 hemiscorpiidae hemiscorpius hemiscorpius acanthocercus (monod et lourenço, 2005) 1 53 buthidae apistobuthus apistobuthus pterygocercus (finnegan,1932) 1 8, 18 2 8 12 table 2. ld50 of sting agent scorpions in iran based on the injection method species ld50* method** family androctonus crassicauda 0.08–0.50 sc/iv buthidae odontobuthus doriae 0.19 iv buthidae hottentotta saulcyi 1.01 iv buthidae mesobuthus eupeus 1.45 iv buthidae hottentotta schach 3.36–4.2 iv buthidae hemiscorpius lepturus 5.81 iv hemiscorpidae *the dose is expressed in mg of venom per kg of mouse **method: iv= intravenous injection, ip= intraperitoneal injection, sc= subcutaneous injection fig. 1. provincial abundance of scorpion sting agents in iran j arthropod-borne dis, december 2018, 12(4): 325–333 r dehghani et al.: a review on … 329 http://jad.tums.ac.ir published online: december 25, 2018 fig. 2. species provincial distribution of major scorpion sting agents in iran conclusion at present, the most dangerous species of scorpions are in the south and southwest of iran. however, completing the data about the sting agent scorpions’ species in iran needs more efforts of young researchers. meanwhile, the completion of data in the field of iran’s sting agent scorpion species and different aspects of it needs cooperation between the physicians of the venomous animal's sting therapy units and the entomologist in this field. more accurate studies will be done with the cooperation of specialists of different fields about the sting agent species and the clinical effects of each species. this work necessitates a complete research in the country with a similar method and in the provinces and cities. still, the highest species diversity is seen in the south and southwest provinces but the diversity of scorpions in the northeast 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sedaghat mm, latifi m, alavi-moghaddam s (2018) rodent-borne and rodent-related diseases in iran. comp clin pathol. pp. 1–13. 75. dehghani r, sedaghat mm, bidgoli ms (2012) wound myiasis due to musca domestica (diptera: muscidae) in persian horned viper, pseudocerastes persicus (squamata: viperidae). j arthropod borne dis. 6: 86–89. j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 84 http://jad.tums.ac.ir published online: june 30, 2022 original article molecular survey of mitochondrial genes in different populations of the black fat-tailed scorpion, androctonus crassicauda *mohammad bagher ghavami1, zohreh alibabaei1, fatemeh ghavami2 1department of medical entomology and vector control, school of medicine, zanjan university of medical sciences, zanjan, iran 2department of electronics, faculty of electrical and computer engineering, tarbiat modares university, tehran, iran *corresponding author: dr mohammad bagher ghavami, e-mail: ghavami@zums.ac.ir (received 18 may 2019; accepted 18 dec 2021) abstract background: androctonus crassicauda is the most medically relevant scorpion and understanding its genetic forms is essential for improvement of anti-venom sera, and risk management of scorpionism. present study was designed to identify the variations of mitochondrial genes in different populations of a. crassicauda. methods: adults of a. crassicauda were collected from zanjan province during 2016–2017. genomic dna of samples was extracted and fragments of mitochondrial 16s, coi and nd1 genes were amplified and some of the amplicons were sequenced. haplotype of samples were identified by multiple alignment of sequences, then phylogenetic trees of haplotypes were constructed. results: fragments of 352bp, 618bp and 680bp were amplified from 16s, coi and nd1 genes respectively. nucleotide sequence in coi fragments was conserved, however, five haplotypes with some specific polymorphic sites were detected in 16s and nd1 fragments. haplotype i was dominant and found in all areas. other haplotypes were rare and limited to specific regions. analysis of the phylogenetic trees inferred from 16s and coi genes, confirmed a strong positive correlation between geographic and genetic distance. conclusion: mitochondrial coi, 16s and nd1 genes were detected suitable for identifying the population structure. five genotypes were found using 16s and nd1 genes. to prepare and improve the anti-venoms quality, additional studies are necessary to identify the toxin electrophoretic profile and geographical/ecological niche models of these genotypes in future. keywords: androctonus crassicauda; 16s; nd1; coi; mitochondrial genotypes introduction scorpions are potentially fatal venomous animals whose venom consist of a variety of toxic compounds specifically target ion channels and other cellular receptors (1). scorpion envenomation is a major global health problem and anti-venom is still widely used for its treatment because there is no vaccine or other effective agents (2). among different families of the scorpions, buthidae with the universal distribution is the greatest and the most dangerous scorpions in this family belong to the androctonus (2). androctonus, is a typical saharo-sindian taxon that occurs frequently from the atlantic coast of north africa through western india. this genus includes the largest known buthid scorpions that some of them have extremely toxic venom to humans (2). the taxonomic relationship of androctonus types is not well resolved. this genus was described by vachon in 1952, but since then it has been catalogued first by fet et al. (3) and last by teruel et al. (4). after these revisions, many species were described as new, resurrected from synonymy, or copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 85 http://jad.tums.ac.ir published online: june 30, 2022 raised from subspecies to species level (4–8). nowadays there are correctly seven known species and 40 formerly valid subspecies in this genus (9). the black fat-tailed scorpion, a. crassicauda, is the most dangerous species of androctonus due to causing several human deaths and antivenom production. this species is abundant in various natural habitats ranging from subtropical to temperate regions mostly in the middle east and africa (3). from 33 provinces of iran, the black fat-tailed scorpion has been found in 28 provinces (10) extensive distribution of this species exhibits its adaptation to different ecological conditions and probably possess different types (11). diagnosis of taxonomic groups in this species is traditionally based mainly on morpho-sculpture and coloration characters (12). although few comparative morphological studies have been done on the black fattailed scorpion, no modern precious analyses method exists to differentiate various types and the only existing key is out of date and its types remain largely unresolved. despite the wide distribution of this species in different regions, its ecological requirement brings it into close contact with human settlements regarded as a potential threat then responsible for several deaths (11). given its medical importance, understanding the intraspecific diversity and their spatial distribution are essential for, production of effective anti-venoms, and treatment of envenomation. over the last four decades, mitochondrial dna (mtdna) has been the most popular marker in the study of intra and interspecies variations (13). the genetic information of these markers including gene arrangement patterns, coding usage patterns, nucleotide sequence from protein-coding genes, amino acids and a+t contents (skewness) could be applied as a powerful tool for taxonomy and identification of some species/subspecies. these markers also have been efficiently applied to detect introgression between taxa, answer fundamental questions of the population structure, and resolve their phylogenetic problems (14). moreover, they are assumed to be clock-like and could generate some signals about population history and could reflect its divergence time (13). recently molecular tools have been used to assess the phylogeny of androctonus using nuclear dna its regions (15), barcoding markers for species determination using 16s-rdna (16) and clade identification of species with coi genes (17). in recent years also two genetic groups were found in studied populations of a. crassicauda from turkey according to the sequence analysis of 16s gene (18). in addition, the phylogeographical patterns in six species of androctonus were exhibited in north africa by coi, 16s and nd1 mtdna markers (19). moreover, coi as a molecular tool has been used for phylogeny of different species of scorpions (20– 23). black fat-tailed scorpion consists of the main species of scorpions in zanjan province, northwest of iran (24, 25). this species is the most significant scorpion species in iran and other countries of the middle east and includes many cases of scorpionism in iran (26–28), turkey (29) and egypt (30). this paper is a part of an ongoing study of a. crassicauda biodiversity. we report the first pilot data based on the analysis of coi, 16s and nd1 mtdna genes from different populations of this scorpion in zanjan province. the aims of the current report are identification of intraspecific variations of mitochondrial genes in populations of a. crassicauda and expression of relevant molecular markers in order to select of candidate groups for improving anti-venom quality. materials and methods sampling a total of 84 adult specimens of the black fat-tailed scorpion were collected from twenty localities of zanjan province, northwest of iran, from spring 2016 to autumn 2017 (fig. 1). the study areas have different ecological conditions and include low (below 700 meters above sea level (masl)), semi-high (700–1500masl) and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 86 http://jad.tums.ac.ir published online: june 30, 2022 high (above 1500masl) lands. sample size from each locale were rather low (n< 12) because we found the scorpions in general at extremely low densities. main morphological characters used in identifying this species include carapacial and metasomal carination and coloration, and absence of trichobothria under pedipalp patella (fig. 2). scorpion samples were stored at -20 °c pending molecular analysis. the sex of the scorpions was assessed by the general sexual dimorphism, male having significantly higher number of pectinal teeth than females (fig. 2, d, e). dna extraction total dna was extracted from muscle of dissected tissue of coxa iii–iv after two washes in distilled water. to extract genomic dna (gdna), samples were transferred to 1.5ml eppendorf tubes and suspended in 500ml of tens lysis buffer (10mm tris-hcl (ph 8), 1mm edta, 150mm nacl, 0.5% sds) in the presence of 0.1g of acid-washed glass beads (400–600μm in diameter) and two stainless steel ball bearings (5mm in diameter). the extraction mixture was centrifuged at 10,000g for 5min. the upper phase was extracted and added with 100µl of 5m potassium acetate, and an equal volume of ethanol incubated for 1h at -20 °c. after incubation the extracted suspension was centrifuged at 17,000g for 15min, washed twice with cold 70% ethanol, dried and resuspended in a 50μl nuclease-free te buffer (sinaclon, iran). evaluation of dna quantity and quality dna samples were analyzed by electrophoresis in 1.2% agarose gels containing safe stain, nucleic acid gel stain (sinaclon) in tris-borate-edta buffer and visualized with uv light using a gel doc system (uvitec, cambridge united kingdom). a spectrophotometer (nanodrop; thermo fisher scientific inc.) was also used to measure absorbance at 260, 280 and 230nm and estimate concentration and contamination with protein. amplification of mtdna fragments the 16s, coi and nd1 fragments were amplified in all samples using specific primers (table 1). the amplifications were performed in a final volume of 25µl containing 1u taq dna polymerase (sinaclon), pcr master mix, 10 pm each primer and 1µl purified dna. all amplification conditions were slightly modified for the dna polymerase requirements and consisted of 1min at 94 °c; followed by 40 cycles of 94 °c for 30s; 53 °c for coi and 48 °c for 16s and nd1 genes for 60s and 72 °c for 60s; and a final step of 72 °c for 10min. amplicons from the pcr were separated by electrophoresis as described previously and a 100bp dna ladder (invitrogen) was used to estimate amplicon size. great precautions were taken to minimize the risk of dna contamination of pcr amplification. pcr amplifications, dna extractions and electrophoresis were set up in separate areas and using specific sets of materials including gloves, pipettes, filter tips and laboratory coat. working positions were repeatedly cleaned with 10% naocl to denature potential contaminating nucleic acids. amplified products were analyzed by electrophoresis and single bands of the expected size were sequenced with both forward and reverse primers by seqlab (www.seqlab.dc). analysis of sequences and identification of genotypes chromatograms of nucleotide sequences were checked by eye using chromas pro software (http://www.technelysium.com.au) and multiple sequence alignment was done with clustralw. all sequences were aligned and edited by using bioedit software (https:// bioedit.software.informer.com) and checked indels and single nucleotide polymorphisms within homologous groups. some of the sequences of this study have been deposited in genbank. the resulting alignments were checked by eye and phylogenetical relationships were performed using maximum likelihood (ml), neighbor joining (nj) and minimum evolution (me) and http://jad.tums.ac.ir/ http://www.seqlab.dc/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 87 http://jad.tums.ac.ir published online: june 30, 2022 maximum parsimony (mp) methods with mega x software by using bootstrap with 1000 replications and kimura-2 parameter model (33). the alignments were also applied to construct haplotype networks using the transitive consistency score (tcs) method (34). additionally, the alignments were checked against coi, nd1 and 16s sequences available in genetic data banks and consensus of nucleotide and amino acid sequences were achieved by blast software. androctonus australis was included in phylogenetic analysis as an outgroup. this species was the most appropriate species in the buthid taxa phylogeny and has been repeatedly used in previous studies (14). results the gdna was extracted from 86 samples of adult black fat-tailed scorpions. the quality of purified dna was an a260:a280 ratio of 1.8–2.1 and a260:a230 ratio of 1.3–2.0 for scorpion dna extracts. the concentration of nucleic acids ranged between 50 and 100ng/µl. fragments of 325bp, 618bp, and 680bp were produced by pcr from 16s, coi and nd1 mitochondrial genes, respectively. nucleotide contents a total of 80 high-quality amplicons of the 16s, coi and nd1 fragments were subjected to sequencing. the representative sequences of each group were deposited in genbank with accession numbers mh352581mh352611. from twenty-five subjected sequences of 16s rrna fragments, 16 samples were selected for analysis. these sequences were deposited in genbank with accession numbers of mh352581mh352596. in these samples the pcr product size was 325bp and the a+t content reached a range of 72.84-73.54%. the nucleotide alignment of these samples showed five haplotypes that differed in eight single nucleotide polymorphic sites (snps) (fig. 3). haplotype i, distributed in all regions, was the dominance form and comprised 82% of samples (fig. 4). blast analysis of this haplotype along with sequences available in genbank databases showed 100% identity with the sample of west azerbaijan (sardasht, accession no aj277598) and 93.4% with the sample of south anatolia from turkey (accession no ej217735). like the 16s gene, 25 samples of pcr products of the nd1 gene which showed a sharp band were selected randomly for sequencing. the nucleotide sequences of six samples of these products were submitted in the genbank with accession numbers mh352597 to mh352602. multiple alignments of the sequences showed a length of 680 base pairs (bp) with 17 snps (fig. 5). the a+t content in these sequences were in the range of 70.91–71.18%. in the alignments of the deduced sequences 415 nucleotides decode nd1 and the remaining (265 nucleotides) involve trna genes blast analysis of these sequences showed 89% identity with nd1 gene of a. australis (kj538181) and 87% similarity with buthus occitanus nd1 gene. five haplotypes were obtained from tcs network tree of the nd1 gene sequences (fig. 6). haplotype i was a dominant group which contained 75% of samples. this haplotype is distributed in all areas. from 17 snp sites of nd1 fragments, 2 sites were parsimonious informative including one transversion and one transition substitution. in the 15 remaining sites, 4 mutations were transition substitutions (g↔a, and t↔c) and 11 mutations were transversion substitutions (a↔t, a↔c, g↔c, and g↔t). multiple alignment of decoded amino acids in nd1 peptide showed the study populations clustered in 3 isoforms. nucleotide sequencing of 20 randomly selected coi pcr products showed the products with 618bp. the deduced nucleotides in this fragment decode 206 amino acids. data analysis revealed that all sequences have unique nucleotide arrangement, without any variation. the a+t content in these sequences was the lowest amount (59.51%) in the mtdna fragment. nine representative samples of these sequences were deposited in genbank with achttp://jad.tums.ac.ir/ https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-020-04364-z#ref-cr46 j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 88 http://jad.tums.ac.ir published online: june 30, 2022 cession numbers of mh352603–mh352611. analysis of the blast showed 99.4% identity with the a. crassicauda sample of makoo (accession no mh814933) and 98.1% with sample of sardasht (accession no mk814934) west azerbaijan, northwest of iran. these sequences also shared 99.4% and 98.1% similarity with coi fragments of a. crassicauda from iraq (accession no mt2298940) and egypt (accession no mt636858), respectively (fig. 4). phylogenetic analysis phylogenetic analysis of coi and 16s sequences of a. crassicauda retrieved from the genbank database revealed a close genetic relationship among studied populations and the samples of makoo and sardasht, northwest of iran, turkey, iraq and egypt. constructed trees by ml, nj, me and mp methods strongly support the monophyly of black fat-tailed scorpion populations in the middle east. analysis of the phylogeny indicates that the haplotypes of this scorpion in zanjan and northwestern iran, makoo and sardasht districts, form a clade that its sister group is the populations of iraq, turkey and egypt. this analysis confirms the relationship between geographic distance and genetic diversity; as the geographical distance increases, the genetic diversity also increases (figs. 6, 7). fig. 1. distribution map of sampling sites of androctonus crassicauda in zanjan province http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 89 http://jad.tums.ac.ir published online: june 30, 2022 table 1. details of the primers used in this study gene nucleotide sequence (5’ reference 16s forward cgatttgaactcagatca (29) reverse gtgcaaaggtagcataatca nd1 forward cgacctcgatgtttgaattaa (31) reverse tcgtaagaaattatttgagc coi forward ggtcaacaaatcatcataaagatattg (32) reverse taaacttcagggtgaccaaaaaatca fig. 2. schematic presentation of external body parts in androctonus crassicauda. dorsal view of carapace (a) and metasomal segments (b), pedipalp patella without ventral trichobothria (c), pectinal area of male (d) and female (e), and lateral view of telson (f) http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 90 http://jad.tums.ac.ir published online: june 30, 2022 fig. 3. multiple alignment of 16s fragments in different haplotypes of androctonus crassicauda. the numbers in the parentheses after the name of each haplotype denote the representative accession no of sample that each haplotype belongs to fig. 4. tcs network tree of the 16s (a) and nd1 (b) mitochondrial gene haplotypes of androctonus crassicauda in zanjan province. each dash represents one single nucleotide difference between two neighboring haplotypes. the numbers in the parentheses after the name of each haplotype denote the representative accession no of sample http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 91 http://jad.tums.ac.ir published online: june 30, 2022 fig. 5. nucleotide alignments of nd1 mtdna gene in different haplotypes of androctonus crassicauda in zanjan province. the numbers in the parentheses after the name of each haplotype denote the representative accession number of sample that each haplotype belongs to http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 92 http://jad.tums.ac.ir published online: june 30, 2022 fig. 6. phylogenetic tree of coi haplotypes in different populations of androctonus crassicauda in zanjan province, northwest of iran, based on maximum likelihood, neighbor-joining, minimum evolution (a) and maximum parsimony (b) analyses methods. the numbers in the parentheses denote the accession no. the number on each branch represents percentage of tree containing that branch. the scale bar at the bottom of the trees shows the amount of genetic changes (the number of changes per 100 nucleotide sites) fig. 7. phylogenetic tree of 16s mtdna fragments of androctonus crassicauda in zanjan province, northwest of iran. the constructed tree is inferred by using the maximum likelihood, neighbour-joining and minimum evolution (a), and maximum parsimony (b) analyses methods. the numbers in the parentheses denote the accession no. the value on each branch demonstrates the possibility of branching. the scale bar at the base of trees indicates the genetic diversity (the number of changes per 100 nucleotide sites) discussion the most dangerous scorpion species, a. crassicauda, has been recorded from low to high areas with various climatic conditions in present study. these findings reflect wide range adaptation and tuning of optimal ecological conditions necessary to survive for this scorpion in different areas. the remarkable distribution of this scorpion in these areas calls for the author ities to take the necessary measures to prevent and cure envenomation. the results of the present study showed that the a+t content in coi fragments of fat-tailed scorpion is 59.51%. it is slightly lower compared to those of other studied genes, 16s and nd1, in this species. multiple alignments of coi fragments in study samples indicated that nucleohttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 93 http://jad.tums.ac.ir published online: june 30, 2022 tide sequences in this fragment are conserved. in previous studies phylogenetic analysis of coi gene in mesobotus eupeus showed clear divergence between northern and southern clades in different areas of iran (21). high levels of genetic diversity that represent some geographical coherences also were found in subspecies of scorpio mourus from moroco and turkey (20, 23). in addition, gene diversity of maghrebian hottentottus is accessed by coi gene (22). despite genetic diversity of coi gene in different species of scorpions, findings of present study suggest propriety of this molecular marker for accurate species diagnosis and interspecific taxonomic relationships of a. crassicauda. despite the stability of nucleotide sequences in the coi gene, the genetic diversity of 16s and nd1 genes was observed from different populations of fat-tailed scorpions in the study areas. these findings provide considerable detail on the diversity and valuable information about the population structure of this scorpion. different reports have revealed diversity of 16s gene in this scorpion and other species (35–38); however, to the best of author’s knowledge, this study represents the first evidence of nd1 diversity in the black fat-tailed scorpion. significantly genetic diversity representing 5 haplotypes have been found with 16s and nd1 gene analyses of study populations. more than half of study groups clustered in one clade. according to phylogenetic analyses, all the detected haplotypes in the present study were shared with isolates of worldwide origin, anatolia, west azerbaijan, and northwest of iran. the recorded high genetic variation within 16s sequences suggests the hypothesis that multiple introductions have occurred in populations of this species. therefore, accurate population analysis and determination of the source of genotypes would be necessary to confirm this hypothesis in further studies. phylogenetic analysis results in this study showed that the haplotype i of this scorpion is a dominant group that is represented in all areas, and the other haplotypes which were separated from this haplotype distributed in special localities. finding of this analysis suggests that one monophyletic lineage exists within study populations. further detailed investigations should be carried out to test this hypothesis and many more populations should be tested for other data tests (toxin structure, geographic information system/ecological niche model and nuclear genes) to establish the true genetic structure of populations currently existing in this species. conclusion the black fat-tailed scorpion was recorded from different altitudes in studied areas. this call for authorities to take the necessary preventive and envenomation measures for this scorpion. among molecular markers, coi gene is suitable for determining the interspecific relationship of this scorpion. however, nd1 and 16s genes were suggested to identify the interspecies and population structures in this scorpion. additional complementary works in further studies are needed to elucidate the facts behind the different haplotypes and population groups in this species. acknowledgements this work was financially supported by the deputy for research and technology, zanjan university of medical sciences (zums) with reference number a-12-84-8. the authors are grateful to mr taghiloo and mr torabi for their valuable assistance during surveys and samplings. ethical considerations this study was approved by the ethics committee in biomedical research (zums.rec. 1394.141) of zanjan university of medical sciences, zanjan, iran. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 84–96 mb ghavami et al.: molecular survey of … 94 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http://jad.tums.ac.ir published online: june 30, 2022 ics of two common species, androctonus crassicauda and scorpio maurus kruglovi. bull br arachnol soc. 15(6): 193–200. 37. ojanguren-affilastro aa, mattoni ci, ochoa ja, ramírez mj, ceccarelli fs, prendini l (2016) phylogeny, species delimitation and convergence in the south american bothriurid scorpion genus brachistosternus pocock 1893: integrating morphology, nuclear and mitochondrial dna. mol phylogenet evol. 94(pt a): 159–170. 38. soltan-alinejad p, rafinejad j, dabiri f, onorati p, terenius o, chavshin ar (2021) molecular analysis of the mitochondrial markers coi, 12s rdna and 16s rdna for six species of iranian scorpions. bmc res notes. 14(1): 40. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 51 http://jad.tums.ac.ir published online: march 31, 2022 original article ectoparasites diversity on rodents and shrews at scrub typhus endemic vellore district of tamil nadu, india *philip samuel paulraj1, govindarajan renu1, krishnamoorthi ranganathan2, rajamannar veeramanoharan1, ashwani kumar2 1indian council medical research (icmr), vector control research centre field station, madurai, tamil nadu, india 2indian council medical research (icmr), vector control research centre, puducherry, india *corresponding author: dr philip samuel paulraj, email: philipsamuelpaulraj@gmail.com (received 1 may 2020; accepted 22 jan 2022) abstract background: rodents and shrew living in the vicinity of human beings perform significant role to harbor different species of ectoparasites and thus act as the reservoir host for the spread of diseases to human and animals. methods: this study was undertaken to determine the species composition of the medically important ectoparasites present in the scrub typhus affected places of vellore district using wonder and sherman traps for trapping of live rodent/shrew during september 2017 to august 2018. results: rodent/shrew hosts belonged to two families, three sub families and five genera and five different species. these animals carried 23 species of ectoparasites including 17 trombiculid mites, two non-trombiculid mites, two ticks and two fleas. a total of 940 chigger mites were collected which belonged to three tribes, six genera, two subgenera and 17 species. adult mites collected belonged to two families, three genera and three species. ticks collected were classified under one family, two genera and two species. fleas fall under one family, one subfamily, one tribe, one genus and two species. conclusion: this is the first record of 14 species of chigger mites and three species of adult mites from vellore district, tamil nadu. surveillance of these keystone ectoparasites helped to identify the medically important disease vectors causing acari-borne zoonotic diseases. keywords: diversity; ectoparasites; rodents; scrub typhus; tamil nadu-india introduction mites, ticks, lice, and fleas are important haematophagous arthropod ectoparasites found associated with rodents and play vital role in the transmission of different vector-borne diseases like scrub typhus, plague etc (1–3). rodents and shrews that live nearby human dwellings are considered as the synanthrope species causing major role in the transmission of diseases to human (4). in the rodents and shrews, chigger mites are predominant organism which are the larval stages of mites belonging to trombiculidae family comes under subclass acari of class arachnida, transmitting scrub typhus disease. many acarines act as the vectors of zo onotic diseases and live in close association with different animal host. in india, two major traps are commonly used, which are sherman and wonder traps to capture rodents and shrews alive to harvest different ectoparasites (5). scrub typhus was a major out-break disease during 2017 in tamil nadu. a study on serological evidence of distribution of spotted fevers, scrub typhus and other typhus fevers transmitted through ectoparasites of rodents were already reported in tamil nadu (6). an account of the abundance of ectoparasites on the host at tamil nadu and pondicherry in different seasons was documented already (7). copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 52 http://jad.tums.ac.ir published online: march 31, 2022 based on our earlier study, the prevalence of chigger mites was reported in the scrub typhus affected areas of trivandrum district, kerala state, india (8). even though maximum number of scrub typhus cases was reported from vellore district, tamil nadu, very little study has been undertaken to understand the diversity of the ectoparasites from the different rodents/shrew present in vellore district. a comprehensive survey on the ectoparasites was undertaken in the scrub typhus endemic vellore district of tamil nadu to identify the different species of ectoparsites present here and to bring out the medically important ectoparasites from this area. this study brings out for the first time the presence of a variety of diverse ecto-parasites of zoonotic importance available in vellore district, tamil nadu. materials and methods site selection and trapping vellore district situated in tamil nadu state of india is one of the 37 districts in the tamil nadu state. it is one of the 11 districts that form the north region of tamil nadu. vellore district lies between 12° 15' to 13° 15' north latitudes and 78° 20' to 79° 50' east longitudes in tamil nadu state. the geographical area of this district is 5920.18km2 and a housing population of 3, 936,331 as reported by the 2011– 2022 census (9). the average annual temperature in vellore is 27.9 °c and the annual rain fall is 971mm. based on the earlier history of scrub typhus, as the maximum numbers of scrub typhus cases (80–161 from 2013 to 2016-source dph and pm chennai, tamil nadu) were reported in vellore district it was selected for this study. in vellore district, in consultation with the local health officials seven localities were selected such as ponnai poigai, annaicut, munjurpet and lalapet (rural areas) and nowlok thoppu and vellore town (urban areas) for the collection of ecto-parasites. sample collection the ectoparasites collection was made from, trapping of rodents and shrew was conducted based on scrub typhus affected areas during 2018 (fig. 1). sherman traps and wonder traps were placed in the study villages to capture rodents and shrew, found freely close to human habitats. traps were set in the evening (6.00 pm) and the traps were retrieved the next day morning (7.00am). rodents were anesthetized (7) for the collection of various ectoparasites. the ectoparasites were harvested from the captured rodents/shrews (8). identification all the ectoparasites collected from these animals were preserved in 80% ethanol, transferred to clearing agent, mounted in hoyer’s medium and examined under the microscope for species level identification using standard taxonomical keys (10–15). all collected ectoparasites specimens were preserved in the slide and deposited in mosquito museum entomology laboratory of icmr-vector control research centre field station, madurai, tamil nadu. ethics this study was approved by the institutional animal ethics committee (iaec) of madurai medical college madurai, tamil nadu, india (ref. no. 2339/e1/5/2017 dated 23. 3.2017; s.no. 10). data analysis the data analysis was performed using spss ver.15 (statistics package for social sciences). results this study showed distribution pattern of chiggers and mites in vellore district, tamil nadu. a total of 350 traps were placed, keeping equally 50 numbers of traps at each rodent trapping site. there were only 36 traps found to be positive capturing 56 rodents/ shrew behttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 53 http://jad.tums.ac.ir published online: march 31, 2022 longing to five species rattus rattus, suncus murinus, tatera indica, bandicota bengalensis and mus musculus. the results of the collected rodent/shrew hosts were identified as comprising of two families (muridae, soricidae), three subfamilies (gerbillinae, murinae and soricinae) and five genera (tatera, mus, bandicota, rattus and suncus). rodent trapped per trap was 1.56. these five different species of rodents/shrew collected from vellore district of tamil nadu carried 23 species of ectoparasites included 17 trombiculid mites, two non-trombiculid mites, two ticks and two fleas representing five families namely trombiculidae (17), laelapidae (1), dermanyssidae (1), ixodidae (2), and pulicidae (2). large sampling size helped to gather many species of chigger mites. a total of 940 chigger mites (94.76%) were harvested predominantly from rats/shrew followed by 45 fleas (4.5%), 4 ticks (0.4%) and 3 adult mites (0.3 %). chigger index, flea index, tick index and mite index were 16.79, 0.80, 0.07 and 0.05 respectively (table 1). the collected 940 chigger mites from all the rodent/shrew hosts were identified belonging to one family (trombiculidae), subfamily trombiculinae, which belonged to three tribes (trombiculini, schoengastiini and gahrliepiini), six genera (leptotrombidium, trombicula, neotrombicula, schoengastia, ascoschoengastia and schoengastiella), two subgenera (leptotrombidium and ericotrombidium) and 17 species. only three adult mites were collected belonging to two families (laelaptidae and dermanyssidae) two genera (echinolaelaps and dermanyssus) and two species. similarly, only four ticks were collected in this study classified under one family (ixodidae) two genera (haemophysalis and liponyssoides) and two species. this study showed a total of 45 fleas coming under one family, one subfamily (pulicidae), one tribe (xenopsyllini), one genus (xenopsylla) and two species. species of ectoparasites recovered from field rodents and shrew at vellore district, tamil nadu is given in table 2. maximum 273 chiggers were collected from single rodents rattus rattus. the dominant numbers of chigger per rodent was recovered from the village munjurpet (141) and anaikattu (140). in the urban sites, from the area nowlok thoppu, 464 chiggers were collected. no flea was collected at the urban site. maximum number of fleas per rodent (11) was collected from munjurpet village. adult mites and ticks were recovered at a low number from both urban and rural sites. a taxonomic checklist of available ecto-parasites and rodents/shrew are listed in the appendix: 1 and 2. ectoparasite infestation rate on the rodent rattus rattus was 65%, followed by suncus murinus (60%) and mus musculus 33%. only one bandicota bengalensis was captured and infested with only 18 chiggers. number of ectoparasites collected in a shrew suncus murinus was 58.8, followed by 23.2 in rattus rattus, 18 in bandicota bengalensis and 15 in mus musculus. infestation rate for chiggers in rural area was 87.47% and in the urban locality was 99.23%. fleas reported only from rural areas showed infestation rate 9.89%. ticks were collected both in rural (infestation rate 0.22%) and urban areas (0.46%). adult mites were prevalent in both rural (0.22%) and in urban areas (0.31%). fleas showed a significant difference between its distribution pattern in rural (45) and urban areas (no fleas at urban) (t= -1.927, df= 348, p< 0.05). however, there was no significant difference between the presence of chiggers on rodents between rural and urban sites (t= -0. 173, df= 348, p> 0.05), for ticks (t= -0. 183, df= 348, p> 0.05) and for adult mites (t= -1. 467, df= 348, p> 0.05) (table 1). from the total collection, 67% mites, 25% fleas, 5% ticks and 3% other mites were collected at rural collection sites. likewise, 82% mites, 9% ticks and 9% adult mites were collected at urban trapping sites. more rodents were trapped at rural villages mainly from munjurpet (15) and anaikattu (14) villages. ectoparasites collection was dominant in an urban site http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 54 http://jad.tums.ac.ir published online: march 31, 2022 nowlok thoppu (47%). among the hosts rattus rattus was trapped dominant (71%). chiggers were recovered more at vellore study sites (94.76%). the chigger species leptotrombidium deliense and leptotrombidium indicum were found in all species of rodents and shrews except bandicota bengalensis and mus musculus. next to the chiggers leptotrombidium delicense and leptotrombidium indicum, schoengatiella ligula were collected more in rattus rattus, bandicota bengalensis and tetera indica. the fleas xenopsylla cheopis was found only in rattus rattus and mus musculus at rural areas. the fleas xenopsylla astia was found only in rattus rattus, bandicota bengalensis and tatera indica. there was no significant difference between rural and urban localities in rodent positivity (t= -0.406, df= -54, p> 0.05). this is the first record of 14 different species of chiggers and 3 species of adult mites from vellore district, tamil nadu (table 2). table 1. areawise ectoparasites collected from rodents and shrew at vellore district, tamil nadu, india area rural urban total no of traps fixed 205 100 350 no of traps positive 27 9 36 trap positivity rate 10.8 9.0 10.3 no. of rodents/ shrew collected 45 11 56 no. of rats positive for chiggers 24 9 36 percentage positive for chiggers 53.3 81.8 64.3 no. chiggers collected 398 542 940 chigger index 8.8 58.3 16.79 no. of rats positive for fleas 9 0 9 percentage positive for fleas 20.0 0.0 64.3 no. fleas collected 45 0 45 flea index 1.0 0.0 0.80 no. of rats positive for ticks 2 1 3 percentage positive for ticks 4.4 9.1 64.3 no. ticks collected 3 1 4 tick index 0.04 0.27 0.09 no. of rats positive for mites 1 1 2 percentage positive for adult mites 2.2 9.1 3.6 no. adult mites collected 1 2 3 adult mite index 0.02 0.1 0.05 fig. 1. study areas selected in vellore district, tamil nadu, india http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 55 http://jad.tums.ac.ir published online: march 31, 2022 table 2. species wise ectoparasites recovered from field rodents and shrew trapped at vellore district, tamil nadu, india location rural urban ectoparasites species recorded in present survey villages a1 a2 a3 a4 a5 a6 a7 r (7) r (14) r (13) # ## ectoparasites/hosts sm rr sm rr rr ti bb mm sm rr rr rr sm rr chigger mites leptotrombidium deliense ● ● ● ● ● ● ● ● ● ● ● leptotrombidium rajesthanense ● ● ● ● ● ● ● fr leptotrombidium keukenschrijveri ● ● fr leptotrombidium indicum ● ● ● ● ● ● ● ● ● ● ● fr leptotrombidium delimushi ● ● fr leptotrombidium insigne ● ● ● leptotrombidium kulkarni ● ● ● fr leptotrombidium spilleti ● ● ● fr leptotrombidium dehraduense ● ● fr leptotrombidium jayewickremei ● ● fr schoengastia kanhaensis ● ● fr schoengastiella argalea ● ● fr schoengatiella ligula ● ● ● ● fr schoengastiella.sp ● ● ● ● schoengastia sp. ● ● ● ascoschoengastia indica ● ● fr trombicula hypodermata ● ● ● ● ● ● fr neotrombicula.sp ● ● fr adult mites echinolaelaps echininus ● ● fr dermanyssus gallinae ● ● fr liponyssoides sanguineus ● ● fr ticks haemophysalis sp ● ● ● ● ● rhipicephalus turanicus ● ● ● fleas xenopsylla cheopis ● ● ● ● xenopsylla astia ● ● ● ● ● ● ● a1ponnai, a2poigai, a3annaicut, a4munjurpet, a5lalapet, a6vellore town, a7nowlok thoppu smsuncus murinus, rrrattus rattus, mmmus musculus, titetera indica, bbbandicota bengalensis rreference, #present survey, ##remarks, frfirst record http://jad.tums.ac.ir published online: march 31, 2022 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 56 http://jad.tums.ac.ir published online: march 31, 2022 discussion this present study was proposed to explore the ectoparasites fauna in the vellore district of tamil nadu, india, an area endemic for scrub typhus in tamil nadu. this study was conducted during one full year and the locations were chosen as per the scrub typhus report. we could demonstrate the rodent-associated ectoparasites present in all the study sites. earlier studies have documented more than 3000 species of chiggers all over the world and over 400 mite species were recorded in china (16). a total of 274 species of mites were collected from yunnan province, southwest china (16). fujian province documented 53 species of chiggers, hubei province recorded 41 chigger mite species and beijing recorded 7 species of chigger mites (16). in india so far 204 chigger mite species were recorded. this study recorded more than 10 % of the total collection of ectoparasites from india. in this study, vellore district situated in tamil nadu state is 209 times smaller than yunnan province, southwest china; but showed 23 species of chiggers which clearly showed that this area has a great species diversity of chigger mites. this collection has even outnumbered the collections from other investigations on ectoparasites. total number of the vertebrate host species play very significant factor to influence the species diversity of chigger mites from different places. a total of 11,883 chiggers were collected comprising of 65 species in 14 genera recorded from himalayan region (17) which includes 5 genera leptotrombidium, neotrombidium, ascoschoengastia, schoengastia and schoengastiella collected from the present vellore study. during 1966, leptotrombidium akamushi, leptotrombidium delicense, and schoengastiella ligula were collected from eastern himalayan foothills (18). except for leptotrombidium akamushi, the other 2 species of mites were collected in our study. a survey conducted between 1966 and 1970 in western himalayas, sikkim and hill districts of west bengal showed 66 species of ixodoidea, 15 species of mesostigmatid mites and a large number of trombiculid mites in addition to other haemotophagous arthropods (19). the mite’s collections reported 23 genera and 54 species belonging to 10 families from western himalayas, himalayan districts of west bengal and sikkim between 1966 and 1970 (20). in the worldwide distribution pattern of acarines approximately 12% of 50,000 live as the parasites and less than 100 species are reported to have medical and veterinary importance (21). many acarines act as the vectors of zoonotic diseases and live very close to the animal host. moreover, environmental degradation led to global climate changes in many places. this led to the proliferation of the vectors and in turn transmission of rodent borne parasitic diseases (3). many ectoparasites of rodents play crucial role in the disease transmission to human and animals. thus, this study on ectoparasites is an invaluable data to take up appropriate control programme. medically important chigger mites, leptotrombidium (leptotrombidium) deliense and schoengastiella ligula are the powerful vectors of scrub typhus. abundant collection of these species denotes the potential risk for the transmission of this emerging disease in these areas. echinolaelaps echidninus has medical importance and could induce cross-reactivity with other allergic mites (22). scrub typhus is caused by orientia tsutsugamushi which is transmitted by an arthropod vector of the trombiculidae family chigger mites leptotrombidium deliense. this is the most common re-emerging rickettsial infection in india and many other southeast asian countries. this vector is collected aplenty in this present collection. an outbreak investigation conducted in kurseong, darjeeling established schoengastiella ligula as the vector of scrub typhus due to its abundance (23). in this study, schoengastiella ligula was also collecthttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 57 http://jad.tums.ac.ir published online: march 31, 2022 ed. redbird mite dermanyssus gallinae acting as the vectors of fowl tick fever, endemic typhus, and st. louis encephalitis was reported from this collection (24). similarly, tropical rat mite ornithonyssus bacoti was found infested on the laboratory-reared mice colony from nilgiris, tamil nadu. during 2013, ornithonyssus bacoti was recovered from the domestic and peri-domestic rattus rattus (25). one more rickettsial pox vector species liponyssoides sanguineus was also collected from this study (22). this study reported ticks like rhipicephaus turanicus which plays a potential role as bridge vector carrying tick-borne protozoan sporozoite pathogen hapatozoon canis among various hosts like dogs, foxes, and golden jackals. in this study a hard-bodied tick haemophysalis bispinosa belonging to the genus haemophysalis was found in india. it is obligate ectoparasites of mammals and a potential vector of kyasanur forest disease virus. tropical rat fleas xenopsylla cheopis and xenopsylla astia were also collected in this study. xenopsylla cheopis and xenopsylla astia are responsible for the transmission of yersinia pestis the causative agent for the spread of plaque and generally collected from rattus rattus and rattus norvegicus. the predominance of fleas was reported from angola (26). in indonesia, xenopsylla cheopis was the most common on rattus rattus (27). in iran, fleas catch was related to rattus norvegicus (28). in this present study, xenopsylla cheopis and xenopsylla astia were collected from both rattus rattus and suncus murinus. there was no flea collected from the urban site which may not be a suitable area for the survival of fleas as observed in other areas in malaysia (3). according to rodent host and location, the distribution pattern of the ectoparasites differed (29). in egypt mus muculus harbored less ectoparasites but the other species like rattus rattus frugivorus, rattus norvegicus and meriones shawi were vulnerable to ectoparasites (30). in our present study, suncus murinus and rattus rattus were more vulnerable than the rest of the rodent species. 348 chiggers, two fleas, one tick and two adult mites were collected from six suncus murinus. similarly, 568 chiggers, 34 fleas, three ticks and 1 adult mites were collected from 26 rattus rattus. from the rest of the rodents tatera indica, bandicota bengalensis and mus musculus, very few ectoparasites were collected. a taxonomic checklist of ectoparasites collected from vellore district, tamil nadu, india chigger mites family: trombiculidae ewing, 1929 subfamily: trombiculinae ewing, 1929b tribe: trombiculini vercammen-grandjean, 1960 genus: leptotrombidium nagayo et al. 1916 subgenus: leptotrombidium nagayo et al. 1916 1. leptotrombidium (leptotrombidium) deliense (walch, 1922) 2. leptotrombidium (leptotrombidium) dehradunense stan fernandes and kulkarni, 2003 3. leptotrombidium (leptotrombidium) delimushi vercammen-grandjean and langston, 1976 4. leptotrombidium (leptotrombidium) keukenschrijveri (walch, 1923) 5. leptotrombidium (leptotrombidium) insigne stan fernandes and kulkarni, 2003 6. leptotrombidium (leptotrombidium) kulkarnii vercammen-grandjean and langston, 1976 7. leptotrombidium (leptotrombidium) spilletti mitchell and nadchatram, 1966 8. leptotrombidium (leptotrombidium) jayewickremei (womersley, 1952) subgenus: ericotrombidium vercammengrandjean and andre, 1966 9. leptotrombidium (ericotrombidium) rajasthanense stan fernandes and kulkarni, 2003 10. leptotrombidium (ericotrombidium) indicum stan fernandes and kulkarni, 2003 genus: trombicula berlese, 1905 11. trombicula (trombicula) hypodermata, nadchatram and traub, 1966 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 58 http://jad.tums.ac.ir published online: march 31, 2022 genus: neotrombicula hirst, 1925 12. neotrombicula spp hirst, 1925 tribe: schoengastiini vercammen-grandjean, 1960 genus: schoengastia oudemans, 1910a 13. schoengastia kanhaensis mitchell and nadchatram, 1966 14. schoengastia spp. oudemans, 1910a genus: ascoschoengastia ewing, 1946a 15. ascoschoengastia indica (hirst, 1915) tribe: gahrliepiini nadchatram and dohany, 1974 genus: schoengastiella hirst, 1915 16. schoengastiella argalea (traub and morrow, 1957), 17. schoengastiella ligula radford, 1946b mites ii. family: laelaptidae berlese, 1892 genus: echinolaelaps ewing, 1929 18. echinolaelaps echidninus (berlese, 1887) iii. family: dermanyssidae kolenati, 1859 genus: dermanyssus duges, 1834 19. dermanyssus gallinae (de geer, 1778) genus: liponyssoides hirst, 1913 20. liponyssoides sanguineus (hirst, 1914) ticks iv. family: ixodidae murray, 1877 genus: haemophysalis koch, 1844 21. haemophysalis (kaiseriana) bispinosa newmann, 1897 genus: rhipicephalus koch, 1844 22. rhipicephalus turanicus pomerantzev, matikashvili and lotozki, 1940 fleas v. family: pulicidae billberg, 1820 subfamily: xenopsyllinae tribe: xenopsyllini glinkiewicz, 1907 genus: xenopsylla glinkiewicz, 1907 23. xenopsylla cheopis (rothschild, 1903) 24. xenopsylla astia rothschild, 1903 a taxonomic checklist of rodents/shrew collected from vellore district, tamil nadu, india rodents i. family: muridae illiger, 1811 subfamily: gerbillinae gray, 1825 genus: tatera lataste, 1882 1. tatera indica hardwicke, 1807 subfamily: murinae illiger, 1811 genus: mus clerck1757 2. mus musculus linnaeus, 1758 genus: bandicota gray, 1873 3. bandicota bengalensis gray, 1834 genus: rattus fischer de waldheim, 1803 4. rattus rattus (linnaeus, 1758) shrew ii. family: soricidae fischer, 1814 subfamily: soricinae fischer von waldheim, 1817 genus: suncus ehrenberg, 1832 suncus murinus (linnaeus, 1766) conclusion this is the first record of 14 different species of chiggers and three species of adult mites from vellore district, tamil nadu. the results of the present surveillance provided key scientific data that identifies potential disease vectors which will help the health authorities for the development of appropriate corrective measures to prevent and control the different acari-borne zoonotic diseases in this region. acknowledgements we are extremely thankful to the secretary, department of health research, ministry of health and family welfare and the director general, icmr for financial support. we want to express our deep sense of thanks and gratitude to my director, icmr-vcrc, puducherry and the previous director dr jambulingam, for providing all the necessary facilities, constant encouragement, guidance and useful suggestions for undertaking this study. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 51–60 ps paulraj et al.: ectoparasites diversity on … 59 http://jad.tums.ac.ir published online: march 31, 2022 we want to express our deep sense of gratitude to all our departmental colleagues of icmrvcrc field station, madurai (erstwhile crme, icmr, madurai). we express our sincere thanks to dph and pm, chennai, tamil nadu, especially ddhs, vellore, mr srinivasan, dmo, vellore and other health officials for their guidance and help. ethical considerations this study was approved by the leac madurai medical college madurai, tamil nadu, india (ref no. 2339/e1/5/2017 dated 23.3.2017). conflict of interest statement the authors declare that there is no conflict of interests. references 1. mullen gr, durden la (2019) medical and veterinary 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with notes on species of medical importance. med vet entomol. 5: 1– 7. 28. kia eb, moghddas-sani h, hasssanpoor h, vatandoost h, zahabiun f, akhavan aa, hanafi-bojd aa (2009) ectoparasites of rodents captured in bandar abbas, southern iran. iran j arthropod borne dis. 3: 44–49. 29. deeb ehi, zidan zh, hawashy en, mourad aa (1999) rodent ectoparasites relationships at new reclaimed areas of egypt. ann agr sci. 44: 799–810. 30. kandy ega, shazly eam, mikhail mw, bhat im (2007) ectoparasites of commensal rodents in talkha center, dakahlia governorate, egypt. j egypt soc parasitol. 37: 825–833. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 108 http://jad.tums.ac.ir published online: june 30, 2022 original article bionomics of anopheles culicifacies sensu lato in two malaria endemic districts of central gujarat, india *rajendra k baharia1, jaspreet kaur2, ankita sindhania2, gunjan sharma2, harish c srivastava1, chandra s pant1, manju rahi3, kamaraju raghavendra2, om p singh2 1indian council of medical research (icmr)-national institute of malaria research, field unit, civil hospital nadiad, gujarat, india 2indian council of medical research (icmr)-national institute of malaria research, dwarka secctor-8, new delhi, india 3indian council of medical research (icmr), division of epidemiology and communicable diseases, ansari nagar, new delhi, india *corresponding author: dr rajendra kumar baharia, e-mail: rajendrabaharia@gmail.com (received 20 dec 2020; accepted 26 apr 2022) abstract background: gujarat state has been witnessing large scale urbanization, in last two decades, resulting changes in local environment and microclimate may have also influenced the resting, feeding habits and development of anopheles culicifacies sensu 1ato. therefore, a systematic longitudinal study was undertaken to know the bionomics of an. culicifacies s.l. in present study. methods: the study was conducted in four sentinel villages in kheda and panchmahal districts. the mosquitoes resting indoors and outdoors were collected in early morning hours, using mouth aspirator, pyrethrum space spray and light traps. mosquito landing collections on human volunteers was carried out from dusk to dawn. species composition, abundance, seasonal prevalence, resting behavior (endophily and exophily), sibling species composition, vector potential and insecticide susceptibility status of malaria vectors was studied. results: six anopheles species were collected, an. subpictus s.l. was the predominant species followed by an. culicifacies s.l., a known malaria vector was resting indoor and zoophagic behaviour. anopheles culicifacies, sibling species b (89%) was found. the sporozoite rate (%) and entomological inoculation rate in kheda was 2.33%, 3.09 per bite/ person/annum and they were 1.05% and 0.475 bite/person/annum in panchmahal, respectively. anopheles culicifacies s.l. was found possible resistance to alpha-cypermethrin. conclusion: anopheles culicifacies s.l. showed endophillic, zoophagic behaviour and found possible resistance to alpha-cypermethrin. early biting behaviour of an. culicifacies s.l. in this area is a cause of concern. therefore, there is need for frequent monitoring and evaluation of vector control measures in order to achieve the elimination target of malaria in this area. keywords: endophilic; zoophagic; alpha-cypermethrin; susceptibility; sibling species introduction vector spatial selection is greatly influenced either due to species competition or changes in environmental factors such as temperature and lead to the local adaptation (1). it has been described that climate change will escalate the transmission of vector borne diseases. several authors have claimed decline in certain areas or no change in disease prevalence due to over looked ecological factors (2–3) and development in community (4–5). of the 58 anophelines in india, only six taxa are major malaria vectors with significant regional distribution (6). anopheles culicifacies s.l. has been reported as the major malaria vector in the plains in indian sub-continent. it has been incriminated as malaria vector from rural area of various states copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 109 http://jad.tums.ac.ir published online: june 30, 2022 of india (7–11) including in gujarat state (12). various aspect of bionomics of an. culicifacies s.l. was studied in india (13). the abundance, prevalence, biting and vector potential of an. culicifacies s.l. in gujarat state was studied in 1980s and 1990s (14–21). cytotaxonomic examination of an. culicifacies showed five 5 sibling species named as a, b, c, d and e in various parts of the india (22–25). the species has shown differential sympatricity, resting and feeding behavior, insecticide susceptibility, vectorial capacity (12, 14, 17, 19, 21– 22) and have indicated their differential role in transmission of malaria (10–11, 26–27). anopheles culicifacies s.l. is primarily zoophagic in behavior and its impact on malaria transmission was assessed in gujarat state (12–14). resistance in an. culicifacies against three insecticides, used in public health programme for vector control namely ddt, malathion and diealdrin has been reported from gujarat during 1960s and 1970s (28). gujarat being a progressive state has witnessed rapid urbanization, industrial development, and construction of development projects and associated deforestation during last two decades in the state, resulting in changes in local climate and environment. accordingly, change in microclimate may have influenced some of the bionomic attributes such as resting and feeding habits that may need renewed attention. malaria is unstable in gujarat state, western part of india. during last one-decade malaria has declined to annual parasite incidence (api) of 0.31 in 2019, at the verge of elimination (29). therefore, a systematic longitudinal study was undertaken to know the bionomics of an. culicifacies s.l. in some districts of gujarat state. the results will help in making evidence-based decision for suggesting effective vector control strategy and understand existing lacunae to control and eliminate malaria in similar ecological settings in gujarat. materials and methods study area the study was undertaken in kheda district (22°45' n, 72°45' e) and panchmahal (godhra) district (22°45′ n, 73°36′ e) located in central gujarat (fig.1). based on malaria incidence, potential mosquito breeding sites and baseline entomological survey, four (2 riverine and 2 canal irrigated) villages in each district were included in the study in mayjune 2017. the selected villages angadi, pali (kheda), juni dhari, vinzol (godhara) are riverine whereas the villages such as ravaliya, muliyad (kheda), khazoori and nandisar (godhara) have perennial irrigation canal. the villages were approachable at ease by road with good condition throughout the year. most of the houses are made up of brick with concrete roof; however, a few mud houses with tin or tile roof also exist. major crops in this area are paddy, millet, corn and tobacco. the maximum and minimum external air temperature, relative humidity and rainfall in this area were obtained from anand agriculture university, field unit at thasra, district of kheda. mosquito collections the indoor and outdoor resting, bait and trap collections were carried out at monthly frequency in sentinel villages following standard who methods (30). in each village, 6 human dwellings (hd) were fixed for indoor resting mosquito collection. outdoor resting mosquitoes were collected from underneath of culverts, abandoned temples and bushes. indoor resting mosquito collection from human dwellings adult female anopheles species resting indoors in fixed six human dwellings and two cattle sheds were collected with the help of flashlight and mouth aspirators in early hours of the day (0600 to 0800hrs) for 15 minutes in each of the fixed dwellings. after 15 minutes of hand catch, pyrethrum spray collection (psc) http://jad.tums.ac.ir/ https://tools.wmflabs.org/geohack/geohack.php?pagename=panchmahal_district&params=22_45_n_73_36_e_type:city(2390776)_region:in-gj j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 110 http://jad.tums.ac.ir published online: june 30, 2022 was done from the same room. mosquito collected through hand catch were kept in paper cups and psc kept in petri dishes lined with wet cotton and filter paper at the bottom and transported to the laboratory for processing. the total number of mosquitoes/species resting per structure was calculated as density (per man per hour density for hc) or numbers of mosquitoes/room (psc). outdoor resting mosquito collection mosquitoes resting outdoors were collected with the help of flashlight and mouth aspirators during morning hours (0600 to 0800hrs) from available different outdoor sites in the village. the mosquitoes collected were kept in paper cup with wet cotton and transported to the laboratory for further processing. the number of mosquitoes/species collected was calculated as the density (man hour density). human landing collection (hlc) hourly whole night collection of mosquitoes landing on human bait (volunteer) was carried out from dusk to dawn (1800 to 0600hrs). care was taken to collect mosquitoes as soon as they land on the host to evade mosquito bites on volunteer. a paper cup fastened with netting was used for each hour of collection. the collected mosquitoes were brought to field laboratory identified to species and vector(s). mosquitoes were processed for parity rates and the head and thorax was preserved for enzymelinked immunosorbent assay (elisa) test. light-trap catch (by cdc light trap) two light-traps, one each in indoor and outdoor was used for mosquito collection. the indoor trap was placed 60cm above the ground in the sleeping unit of the household. for outdoor collections, traps were placed just outside the houses where people sit and sleep or in open away from the habitations in the village. mosquitoes were collected in morning from the trap was used for estimating proportion of different species prevalent in the area. these collections expressed as numbers of mosquitoes per trap-night (number of mosquitoes/ trap). processing of mosquito samples anopheline mosquitoes identified to species based on morphological characters using key (31–35), the physiological condition of stomach was determined such as unfed (uf), freshly fed (ff), half gravid (hg) and gravid (g), blood meal of an. culicifacies s.l. were collected on whatman no. 1 filter paper for host source using gel diffusion technique to determine the feeding preference (human blood index), half gravid ovaries of an. culicifacies s.l. was collected and preserved for sibling species identification, the head and thorax of each specimen was preserved for sporozoite elisa test and unfed females used for estimating parity. susceptibility status of vectors to different insecticides anopheles culicifacies s.l. susceptibility status against pyrethroids was estimated in october 2017, january 2019 and march 2019, according to standard who method (36). field collected mixed age population of an. culicifacies s.l. of villages anghadi, pali, revaliya, and vinzol were tested using who prescribed discriminatory concentration impregnated papers of malathion, deltamethrin and alpha-cypermethrin. a minimum of 75 mosquitoes (25 mosquito/replicate) for test and 50 (25/replicate) for control were used. the test was conducted in national institute of malaria research (nimr) laboratory maintained at 27±2 ºc temperature and 60–70% relative humidity during test period. percent mortality was determined post 24 hrs of holding period after one-hour exposure from the total number of alive and dead mosquitoes in the replicates. if control mortality was greater than or equal to 5% and less than or equal to 20%, the value for exposure mortality was corrected by using the abbott’s formula (37). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 111 http://jad.tums.ac.ir published online: june 30, 2022 sibling species identification morphologically identified specimens stored in isopropanol were processed for an. culicifacies sibling species (38). species a/d and species b/c/e were identified by two allele specific primers aca (forward, sequence 5’-gcc gtc ccc ata cac tg-3’) and acb (reverse, sequence 5’ ccg taa tcc cgt gat aac tt-3’). pcr conditions were: one cycle of denaturation at 95 °c for 5min followed by 35 cycles of each of denaturation at 95 °c for 30s, annealing at 55 °c for 30s and extension at 72 °c for 60s, and final extension at 72 °c for 7min. vector incrimination the head and thorax of an. culicifacies s.l. preserved were assayed for presence of sporozoite using elisa (39–41). the specimens were tested for species specific circumsporozoite antigen (csp) of pf, pv210 and pv247 using the protocol as described by akhatar et al. (41). results mosquito abundance and seasonal prevalence a total of 14056 mosquitoes were collected by all methods used of which, 73.94% (10389/ 14056) was anopheles along with 25.46% culex and 0.62% aedes. among anophelines an. subpictus s.l. (69.03%) species followed by an. culicifacies s.l. (17.93%), an. annularis (12.35 %), an. stephensi (1.02%), an. fluviatalis s.l. (0.16%) and an. vagus (0.0096%). anopheles culicifacies s.l. was encountered from all habitats in both districts. from outdoor resting habitats an. subpictus s.l., an. culicifacies s.l. species and culex spp were found. indoor resting collections human dwelling the indoor resting mosquito collections were made for 13 months (july 2017 to july 2018) from fixed human dwellings in sentinel villages of kheda and panchmahal districts. in both the districts, bimodal population increase of an. culicifacies s.l. densities were observed, a minor peak in march and a major peak in august. the indoor resting density in canal irrigated area was higher during most of the study period as compared to in riverine areas in both the districts. in post-monsoon months (december to march), indoor resting density was significantly high in both the areas of panchmahal compared to kheda (fig. 2). during the transmission season, an. culicifacies s.l. density of kheda irrigated versus panchmahal riverine and kheda riverine versus panchmahal irrigated were significant (p= 0.0092, t= 2.77, p< 0.05 and p= 0.0357, t= 2.0, p< 0.05). cattle shed indoor resting sampling in cattle sheds revealed that the density of an. culicifacies s.l. was greater in cattle sheds than human dwellings in kheda and panchmahal. in riverine area of kheda, an. culicifacies s.l. density was higher as compared to irrigated area throughout the study except in post monsoon period, whereas in panchmahal, it was high in riverine area in all the seasons. physiological condition it was observed that in indoor resting samples from riverine villages of kheda district, the proportion of half-gravid + gravid an. culicifacies s.l. was higher compared to full fed and unfed in pre-monsoon, monsoon, and postmonsoon seasons. in canal-irrigated areas of this district, the proportion of fully fed mosquito was higher compared to half-gravid + gravid, and no unfed was encountered during pre-monsoon period. while in monsoon and post-monsoon period, canal-irrigated villages had higher proportion of half-gravid + gravid as compared to fully fed and unfed. the riverine and canal-irrigated villages of panchmahal had higher proportion of half-gravid + gravid compared to fully fed. it indicated that http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 112 http://jad.tums.ac.ir published online: june 30, 2022 large proportion of an. culicifacies s.l. bites in the evening and late evening and rests indoor in both the districts indicating endophillic behavior. parous rate altogether 1223 ovaries (kheda 534 ovaries, panchmahal 689 ovaries) of an. culicifacies s.l. were examined for parity. overall, 30 % (200/689) were found to be parous in panchmahal district throughout the study period while in kheda district it was low in winter 13% (3/22) and summer 7% (1/14). anopheles culicifacies s.l. parity varied in different months, ranging from 7% in may to 60% in july. in the transmission season, parity rate of kheda and panchmahal district were significant (p= 0.0305, t= 3.9, p< 0.05). light trap in kheda, the densities of an. culicifacies s.l. (3.08 per trap) and other mosquitoes (25.58 per trap) were higher in outdoor trap compared to indoor. in panchmahal, the density of other mosquitoes (22.41 per trap) was 10-fold higher than an. culicifacies s.l. in both traps. in outdoor traps, the density was higher in kheda compared to in panchmahal (figs. 3 and 4). anopheles culicifacies s.l. density from the districts of kheda and panchmahal were significant with outdoor other mosquitoes’ density (p= 0.0030, t= 3.4, p< 0.05 and p= 0.0044, t= 3.18, p< 0.05). human landing collection (hlc) the hlc yielded five anopheline species comprising of an. culicifacies s.l., a few an. stephensi, an. annularis, an. fluviatilis s.l., an. vagus, and a few specimens of aedes spp and culex spp. in general, the bite rate of an. culicifacies s.l. was greater in kheda compared to panchmahal in all seasons and found biting throughout the night in both districts. in kheda, the mean biting rate of an. culicifacies (9.0 bite/bait/night) was high during post-monsoon period, september to october. whereas biting activity of other mosquitoes was maximum during march-may, it was 20 times higher than an. culicifacies s.l. in both the districts, most of the landing mosquitoes were caught either in early night or in early morning. thus, two peaks of biting activities were observed in each night, first between 20–21hrs and second at 02–04hrs in both districts (fig.5). human landing catch were significant in between kheda and panchmahal districts (p= 0.0292, t= 2.11, p< 0.05). host blood preference a total of 577 (320, riverine and 257 irrigated area) blood meal samples of an. culicifacies s.l. were collected from sentinel villages in kheda district. from villages in panchmahal district altogether 573 (327 riverine and 246 irrigated villages) blood meal samples were assayed for blood meal sources. the anthropophilic index (ai) of an. culicifacies s.l. in kheda district, in canal irrigated area was 2.33 % (6/257) and in riverine area none was positive for human blood (0/320). in panchmahal district, ai was 1.62% (4/246) in canal irrigated area and 1.52% (5/327) in riverine area. majority of blood meal of an. culicifacies s.l. were found bovine indicating its established zoophagic behavior in both the districts. insecticide susceptibility status the susceptibility status of an. culicifacies s.l. was assessed against malathion, deltamethrin, permethrin and alpha-cypermethrin using who discriminatory dose impregnated papers. it was observed that an. culicifacies s.l. has developed resistance against deltamethrin (24 hrs mortality: 75%) and malathion (24hrs mortality 70%), whereas it was found possible resistance to alpha-cypermethrin with 24hrs mortality 95% in kheda (table 1). in panchmahal district, an. culicifacies s.l. was possible resistance to pyrethroid alpha-cypermethrin with 24hrs mortality 95%, but has developed resistance against malathion (24hrs mortality 75%). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 113 http://jad.tums.ac.ir published online: june 30, 2022 sibling species composition altogether 67 and 223 an. culicifacies s.l. from kheda and panchmahal, respectively were examined for sibling species identification. its sibling species distribution pattern was found similar in both the study areas. the primers were specified for detection of the a/d and b/c/e sibling species complex. in kheda, an. culicifacies species b (82%; 55/67) was predominant species followed by species a/d (12 %; 8/67), in panchmahal b and a was 91% (203/ 223) and 8% (17/223), respectively (table 2). sporozoite and entomological inoculation rates in kheda and panchmahal districts 916 and 900 an. culicifacies s.l. were analyzed for vector incrimination studies. in kheda, 12 (plasmodium falciparum: 7, p. vivax: 5) and in panchmahal district, 04 (p. falciparum: 3, p. vivax:1) specimens were found positive for sporozoite. the sporozoite and biting rate in kheda was 1.33% and 2.33 bite/bait/night and 0.45% and 1.05 bite/bait/night in panchmahal, respectively. entomological inoculation rate (eir) in kheda and panchmahal was estimated to be 3.09 and 0.475 per bite/person/annum, the higher eir in the kheda may be the result of high biting rate of an. culicifacies s.l. (table 3). additional data additional observations were made on cattle shed location, sleeping behaviour of inhabitants (indoors/outdoors) and livestock enumeration to assess any possible impact on the behaviour of vector and its transmission potential. a total of 120 households were visited in the both districts to know the location of cattle shed in study villages. the maximum cattle sheds were adjacent to human dwelling in kheda (60%) and panchmahal (55%). the mix-dwellings (cattle and human habitations under same roof) were more than those of cattle shed away from human dwellings in both districts. in all the sentinel villages, livestock census was undertaken by nimr staff to estimate the cattle human ratio in 2018. the buffalos were found as major livestock followed by cow and other animals (goat and chicken) in each village. the cattle to human ratio were low in both panchmahal (0.19: 1) and kheda (0.20: 1) districts. these indicate the greater risk of human vector contact in both districts and explain the observed epidemiological attributes sporozite rates and eir. the inhabitants of these dwellings were interviewed to know the sleeping behavior (indoor/outdoor) of villagers in different seasons. the behaviour of villagers was similar in all three seasons in both the districts. more than 60% inhabitants sleep indoors during winter and monsoon in both districts and > 50% sleep outdoors in summer. table 1. susceptibility status of anopheles culicifacies s.l. against different insecticides insecticides (%) kheda panchmahal 1h knockdown (%) 24hrs mortality (%) 1h knockdown (%) 24hrs mortality (%) control group 0.0 0.0 0.0 0.0 deltamethrin (0.05%) 70 75 75 85 permethrin (0.75%) 80 85 83 90 alpha cypermethrin (0.05%) 90 95 90 95 malathion (5%) 60 70 65 75 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 114 http://jad.tums.ac.ir published online: june 30, 2022 table 2. sibling species composition of the anopheles culicifacies complex from july 2017 to july 2018 district n genotype (%) a/d b/c/e na (not amplified) uk (unknown) kheda 67 08 (12) 55 (82) 04 00 panchmahal 223 17 (8) 203 (91) 01 02 grand total 290 25 (9) 258 (89) 05 02 fig. 1. location of study districts of kheda and panchmahal in gujarat state fig. 2. mean indoor densities of anopheles culicifacies s.l. in kheda and panchmahal districts from july 2017 to july 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 115 http://jad.tums.ac.ir published online: june 30, 2022 table 3. sporozoite rate, biting rate and entomological inoculation rate (eir) of anopheles culicifacies s.l. in study area from july 2017 to july 2018 districts n pf +ve pv 210 +ve pv 245 +ve total +ve sporozoite rate % biting rate eir kheda 916 7 4 1 12 1.33 2.33 3.09 panchmahal 900 3 1 4 0.45 1.05 0.472 fig. 3. anopheles culicifacies s.l. and density of other mosquitoes per light trap indoors and outdoors in kheda district from august 2017 to july 2018 fig. 4. the density of anopheles culicifacies s.l. and other mosquitoes per light trap indoors and outdoors in the district of panchmahal from august 2017 to july 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 116 http://jad.tums.ac.ir published online: june 30, 2022 fig. 5. mosquito landing rates of anopheles culicifacies s.l. on human baits from dusk to dawn from july 2017 to july 2018 discussion the study was carried out in riverine and canal irrigated villages of kheda and panchmahal in july 2017 to july 2018, to update the information on bionomics of an. culicifacies s.1. in these districts of central gujarat. in villages, the number of brick houses were more compared to mud houses, 50–60% of inhabitants reportedly slept indoors in all seasons. such sleeping behaviour of inhabitants explains observed increased endophagic behaviour of mosquitoes. using various collection methods, six anopheles species were collected namely an. culicifacies s.1., an. stephensi, an. fluviatilis s.1., an. subpictus s.1., an. annularis and an. vagus which include three primary vectors and a secondary vector species (16–22). in earlier studies in kheda, varied prevalence of an. culicifacies s.l, an. stephensi, an. annularis, an. subpictus s.1., an. aconitus, an. barbirostris, an. nigerrimus and an. tessellatus was reported (16–22). among anophelines, an. subpictus s.1. was predominant in human dwellings and cattle sheds. while observations from jabalpur reported an. culicifacies s.1. as most predominant species followed by an. subpictus s.1. (8). from outdoor habitats, an. culicifacies s.1., an. subpictus s.1. and culex spp. could be captured, and similar observations were made in earlier studies conducted at kheda district (14–22). the previous studies have reported large numbers of an. culicifacies resting in outdoor habitats in gujarat (16, 42). on the contrary we found low outdoor resting densities of an. culicifacies s.l. throughout the study, probably due to non-availability of suitable resting habitats outdoors that were hot and humid due to changes in the local environment during last few decades. hence, these observations indicate the need for suitable micro-climate niche for prevalence of mosquitoes either indoors or outdoors. anopheles culicifacies s.1. was prevalent throughout the year in both the districts in varying proportions. in our observations, the density of an. culicifacies s.1. started to build up in the month of february with the increase of temperature suitable for breeding and survival and reached to first peak in march thereafter it declined gradually by july, and with the onset of monsoons increase in density was obhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 117 http://jad.tums.ac.ir published online: june 30, 2022 served with second peak in september and was similar to observations made in earlier reported studies in 1990s (17–18, 21), indicating no differences in seasonal prevalence in major vector, an. culicifacies s.l. in last few decades. light traps collections yielded an. culicifacies s.l., an. stephensi, an. annularis, an. fluviatilis s.1., an. vagus, aedes and culex in varied proportions. during post monsoon period, an. culicifacies s.1. density in trap were higher in both the districts. the density of other mosquito species in traps was nearly ten times more than the density of an. culicifacies s.l. which was like densities in studies in madhya pardesh (8). in study from jabalpur, madhya pradesh, in light trap collection, an. culicifacies s.1., an. fluviatilis, an. subpictus s.1., an. annularis, an. vagus, an. pallidus, an. splendidus, an. barbirostris and an. theobaldi have been reported (8). in bastar, chattishgarh, in light trap catches, an. culicifacies, an. subpict us s.l., an. vagus and an. annularis were recorded (43). in the odisha state, an. fluviatilis s.l. and an. culicifacies s.l., the recognized primary vectors, and an. aconitus, an. annularis, an. jeyporiensis, an. maculatus and an. varuna the reputed secondary vectors of malaria in india (44). however, significantly high number of mosquitoes were trapped in tribal villages of tribal panchmahal district due to availability of large number of suitable mosquito breeding sites. another possible reason could be low infrastructure development in these villages compared to villages in plain areas. high proportion of fed and half-gravid + gravid an. culicifacies s.1. in indoor resting catches in human dwellings in riverine and irrigated villages in both the areas, exhibited endophilic and endophagic behaviour of an. culicifacies s.1. which was similar to observations in earlier reported studies in kheda district in early 1990s (17) indicating no change in resting and feeding behaviour of mosquitoes inspite of regular use of insecticidal interventions and changes in housing. the parous rate of an. culicifacies s.1. was nearly 30% in panchmahal district in all the seasons. while, in kheda district it was low in winter and summer and varied in different months ranging from the lowest in may to the highest in july. the prolonged ovarian cycle or multiple feeding during same cycle may be responsible for low parous rate in winter and summer. another study from kheda district reported overall parous rate of 38.2% during 1991–92 and 36.4% during 2000–2001 (14). an earlier study on seasonal prevalence of an. culicifacies s.1., estimated > 35% parous rate in kheda district (22). the study clearly shows higher survivorship of an. culicifacies s.l. population in all the seasons in panchmahal, owing to suitable climatic condition. in both the districts, an. culicifacies sibling species ‘b’ was predominant but it was not involved in the malaria transmission in this area. our study confirms the results of earlier study in central gujarat (nimr, unpublished report, 2009). the study conducted in 1990s in kheda district also supports our results which reported > 60% sibling species b (14). these observations contrasted with the distribution in other districts. in orissa an eastern part of india predominant species was an. culicifacies c (77.9%) followed by b (21.1%) and a (48%) (45–46). earlier studies in madhya pradesh in central part of india also reported highest prevalence of an. culicifacies c (8, 47). in chhattisgarh state, central india a study reported equal prevalence of sibling species b and c (48). in uttar pradesh, only an. culicifacies sibling species a and b were found with predominance of a (49). the results of the above reported studies are contradictory to our present study as in the eastern and central states of india, malaria is stable and most of the parts of state are hilly forested. anopheles culicifacies a and c are known malaria vector in most part of the india (13, 50). in kheda district, the human biting activity of an. culicifacies s.1. was intense during post-monsoon (september to november) and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 118 http://jad.tums.ac.ir published online: june 30, 2022 in panchmahal (september and november) and low biting in summer in both districts. similar seasonal shift in biting period was also reported in previous studies in early 1990s (17–18, 21). the biting occurred throughout the night with maximum biting in the first quarter and third quarter of night in villages of both districts, our observations agreed with previous reports from gujarat (14, 18). the early night biting of an. culicifacies s.1. is concern in area where long lasting insecticide nets (llins) is main component of vector control strategy. the low anthropophagic index of an. culicifacies s.1. indicated high feeding preference to bovine blood which is like previously reported studies from gujarat (12, 48, 51–53). anthropophagic index may be due to low cattle–human ration in gujarat state (14). during summer, villagers were found sleeping outdoors close to cattle shed without protection against mosquito bites in both the districts. it has been observed by previous authors that indoor resting anopheline comes out at dusk, bite outdoor and enters indoor at dawn for resting (20). the present study shows that villagers having low cattle population are at greater risk of malaria transmission by an. culicifacies s.1 in these districts, despite zoophagic nature. this could be one of the reasons for the increased eir in kheda district. anopheles culicifacies s.1. was found possible resistance to alpha-cypermethrin in the both the districts which is also presently used for indoor residual spraying in this area for malaria vector control under the national vector borne disease control programme. anopheles culicifacies s.1. showed resistance to malathion in both the districts. in kheda district, it has developed resistance to deltamethrin too. another study from kheda district reported resistance in an. culicifacies s.l. against ddt, dieldrin and malathion (14). similarly, an. culicifacies s.1., was reported resistant to ddt, malathion and deltamethrin in surat district of gujarat in 2005–2006 (28). extensive use of insecticides increased the resistance. some of the point mutation in the voltage-gated sodium channel (nav) was responsible for the resistance for the pyrethroids and ddt insecticides (54). in the indian subcontinent, the point mutation leading to leu-to-phe substitution in the voltage gated na+ channel (vgsc) at residue 1014, a most common knockdown resistance (kdr) mutation was reported in an. culicifacies s.l.-a major malaria vector which is responsible for resistance against ddt and pyrethroids (55–57). the susceptibility of an. culicifacies s.1. status to insecticides reported multiple resistance including to pyrethroids and could be a concern for vector control as the programme is still reliant on chemical insecticides mainly pyrethroids. anopheles culicifacies s.1. vector is resistant to multiple insecticides, and this underlines the need for development/use of new insecticides for management of insecticide resistance (58). in present study, a low anthropophilic index (ai) of an. culicifacies s.1. was recorded in both the districts revealing it’s predominant zoophagic behaviour. earlier studies carried out on host preference of an. culicifacies s.1. in india have recorded a wide range of variation in the anthropophily (13). however, there are evidence that feeding preference to human changes in different situations, a high ai in areas with high cattle population (14) or during epidemic period (59). owing to zoophagic nature of an. culicifacies s.1., low sporozoite rate was recorded in both the districts. earlier studies have observed varied degree of sporozoite rates in naturally infected an. culicifacies s.1. in india (12–14). mean annual entomological inoculation rate was estimated to be low in both the districts. similar observations were made from studies in central gujarat have also estimated low eir ranging 0.022 to 0.110 infective bite/person/annum in different periods (12, 14). in plain areas of sundargarh district in odisha where an. culicifacies s.1. plays main role in malaria transmission, eir was estimated to 0.014 infective bites/ person/ night (44). therefore, eir estimates in our study indicate http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 119 http://jad.tums.ac.ir published online: june 30, 2022 active malaria transmission in kheda and panchmahal districts of gujarat. conclusion for preparation of effective vector control strategy updated knowledge on vector biology and behavior is an essential requirement. our present study generated important information on prevalence and behaviour of an. culicifacies s.l. and other attributes related to malaria transmission. the abundance, physiological conditions, and blood meal analysis of indoor resting an. culicifacies s.l. showed endophilic and endophagic behavior in both districts. in both the districts, its highest biting during monsoon and post monsoon coincides with malaria transmission season. we found an. culicifacies s.l. possible resistance to alpha-cypermethrin, currently used insecticide. hence, indoor residual spray (irs) with alpha-cypermethrin may be evaluated in other villages of the districts to control an. culicifacies s.l. in this area. effectiveness of llins depends on site of use (indoor/outdoor) and on feeding time of biting hence early biting behaviour of an. culicifacies s.l. in this area is a cause of concern. although, there have been changes in human housing and local ecology due to developmental activities in villages of both districts during last two decades in gujarat, but our study clearly demonstrated no major change in behaviour of an. culicifacies s.l. therefore, there is need for frequent monitoring and evaluation of vector control measures to achieve the elimination target of malaria in this area. acknowledgements we are thankful to the director general, indian council of medical research for kindly permitting to undertake the study and also for providing fund. we also thank to director, nimr for their kind support in all aspects. we also thank state programme officers of nvbdcp, gujarat, for providing support during the study period. we appreciate sincere efforts of the staff of the national institute of malaria research and health staff of government of gujarat in successful conduct of the study. ethical considerations institutional ethical committee approval was obtained vide letter no. ecr/nimr/ec/2017/ 142 dated 21 june 2017. a meeting with villagers was convened with the help of sarpanch and other opinion leaders to apprise them about the purpose of the study. nimr staff coordinated with the primary health centre staffs, medical officer, multipurpose health worker (mphw) (male and female) and accredited social health activist (asha) to solicit the cooperation of the villagers during study. informed consent of householders of selected sentinel sites for collection and of volunteer to be baits for landing mosquito collections were obtained prior to initiating studies. conflict of interest statement authors declare that there is no conflict of interest. references 1. eleanore d sternberg, matthew b thomas (2014) local adaptation to temperature and implications for vector-borne diseases. trends parasitol. 30(3): 115–122. 2. thomas cj, davie g, dunn ce (2004) mixed picture for 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raghavendra k, velamuri ps, verma v, elamathi n, barik tk, bhatt rm, dash http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 108–123 rk baharia et al.: bionomics of … 123 http://jad.tums.ac.ir published online: june 30, 2022 ap (2017) temporo-spatial distribution of insecticide-resistance in indian malaria vectors in the last quarter-century: need for regular resistance monitoring and management. j vector borne dis. 54(2): 111– 130. 59. pal r (1945) on the bionomics of anopheles culicifacies (giles) part iii. the behaviour adults. j mal inst ind. 6: 217–238. http://jad.tums.ac.ir/ a comprehensive database and geographical distribution model of vectors and vector borne diseases in ardabil province, borderline of iran and azerbaijan republic 2001–2018 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... original article a comprehensive database and geographical distribution model of vectors and vector borne diseases in ardabil province, borderline of iran and azerbaijan republic 2001–2018 davoud adham1; *hassan vatandoost2, 3; *eslam moradi–asl1,4 1department of public health, school of public health, ardabil university of medical sciences, ardabil, iran 2department of environmental chemical pollutants and pesticides, national institute for environmental research, school of public health, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4arthropod borne diseases research center, ardabil university of medical sciences, ardabil, iran *corresponding authors: dr hassan vatandoost, e–mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir, dr eslam moradi–asl, e–mail: moradiasl83@yahoo.com (received 26 nov 2020; accepted 27 july 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: vector borne diseases (vbds) are the infectious diseases reported from all parts of the world and iran. the main vectors of vbds belong to the phylum of arthropod and insects. the aim of this study was providing the database of important vbds and vectors and geographical distribution model in ardabil, northwest of iran. methods: this retrospective cross–sectional study was conducted from 2001–2018. all the earlier published studies, reports and documentations related to vectors and vector–borne diseases searched systematically as well as the data of diseases was collected from the center for disease control (cdc) of ardabil university of medical sciences. arcgis 10.4.2 (http://www.esri.com/ arcgis) were used to spatial analysis mapping, normalized difference vegetation index (ndvi) index and high/low clustering. results: totally 110076 cases of vbds reported in ardabil province during the past 18 years including malaria, visceral leishmaniasis (vl), cutaneous leishmaniasis (cl), crimean congo hemorrhagic fever (cchf), tick–borne relapsing fever (trf), pediculosis, scorpionism, scabies and anthrax. these diseases were transmitted in ardabil province by eight arthropod families, 19 genera and 70 spices of arthropods. most species belonged to culicidae with 24 species followed by psycodidae with 22 and ixodidae with 16 species. the incidence rate of vbds was 63/100000 in 2001 which decreased to 7/100000 in 2010 and then increased to 21 /100000 in 2018. conclusion: the distribution model of the vbds was plotted based on the geographical and ecological of the vectors will help the authorities for decision. keywords: vectors; vector borne diseases; geographical model; ardabil; iran introduction vector borne disease (vbds) are the infectious diseases transmitted by the infected bite of the arthropods such as mosquitoes, sand flies, ticks, fleas, bugs, lice and some species of flies (1, 2). vector borne diseases are responsible for more http://www.esri.com/arcgis http://www.esri.com/arcgis 288 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... than 17 percent of infectious diseases burden and more than 700, 000 deaths annually. more than 3.9 billion people in 128 countries are at risk of transmitting dengue fever which is estimated to be happening 96 million per year. malaria causes more than 400,000 deaths annually worldwide, most of which are children under the age of five. other vbds like chagas disease and leishmaniasis affect hundreds of millions of people around the world (3–5). according to the iranian ministry of health, the malaria morbidity was decreased from 12294 cases in 2000 to 150 cases in 2015. who reported iran to be located in pre–elimination phase (6–7). the incidence of dengue fever has increased 30–fold in the last 50 years and 50–100 million dengue happen every year in the world (8). according to the latest study, the incidence of cl in iran has decreased from 174/100000 to 124/100000 cases while the spatial distribution of cl and vl has increased in new places(9). the important vbds occurring in iran include visceral leishmaniasis (vl),cutaneous leishmanisis (cl), crimean congo hemorrhagic fever (cchf), tick relapsing fever (trf), malaria, pediculosis (10–11). the most important endemic area for vl is the northwest of iran. recently, some vbds such as the pediculosis epidemic were increased in ardabil province (12). the incidence of vl in ardabil province was increased from 2.9 in 2009 to 9.2 /100000 in 2015 and the new sides was increased to 2 time (13). the main vectors’ ecology and biology of vbds as well as its spatial distribution should be identified for prevention and control (14–15). the most important factors affecting the life cycle, growing, developing, reproduction and the survival of generations of arthropods and insects include environment temperature, relative humidity, rainfall, altitude, vegetation, amount of food and suitable shelters (16–17). the aim of this study was to establish a database of vbds and their vectors, and to determine their spatial distribution model in ardabil province. materials and methods study area ardabil province is located in the northwest of iran (37.45–39.42o n, 47.30– 48.55o e), near the border of azerbaijan republic. the province has an area of 17,953 km2 with 1,247,042 populations according to the last census conducted in 2015. the province consists of 10 counties, 21 districts, 26 cities, 71 rural districts and 1477 permanent villages. about 2/3 of the ardabil province is mountainous areas and the remaining is plains. the occupation of most people is farming and animal husbandry (fig. 1) (18). data collection data were collected from the center for disease control (cdc) of ardabil university of medical sciences from 2001–2018 and classified by location, years, months and type of diseases. the vectors database was collected according to the last studies (projects and thesis) in ardabil province from 1950–2018. previous studies, reports and documentations related to vectors and vectors borne diseases in ardabil province were searched in international databases such as cochrane, medline/pubmed, google scholar, science direct, scopus, web of science, veterinary information network, vet med resource, zoological records, biological abstracts, cab abstracts as well iran’s databases (for persian articles) including iran medex, scientific information iran medex, scientific information database (sid) and magiran. meteorological data were obtained from the ardabil meteorological center during the study period. the data included annual precipitation, average annual temperature, altitude, and relative humidity. data and spatial autocorrelation analysis arcgis 10.4 (http://www.esri.com/ arcgis) was used for spatial analysis and mapping. ndvi (normalized difference in vegetation index, and was also used to determine high risk areas of the disease 289 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... across the study area. moran’s i index and general g value were measured, and both the score and p-values (p < 0.05) were calculated and used to evaluate the significance of the index. moran’s i is a commonly used indicator of spatial autocorrelation. in this study, global moran’s i was used as the first measure of spatial autocorrelation. ethics approval and consent to participate this study was approved by the ethical committee of ardabil university of medical sciences, iran (code of ethics: ir. arums. rec.1397.261). results totally 110076 cases of vbds were reported in ardabil province during the past 18 years including nine different types such as malaria, vl, cl, cchf, trf, pediculosis, scorpionism and anthrax. the highest infectivity belongs to pediculosis (96.47%) and the lowest to cchf (0.007 %). the incidence rate of diseases excluding pediculosis was 63/100000 in 2001, which decreased to 7/100000 in 2010, and then it increased to 21/100000 in 2018. the prevalence of pediculosis was increased 110 times during 2001(16/100000) to 2018 (1768/100000). the most pediculosis cases have occurred in schools (table 1 and fig. 2). the results of this study showed that in the past 18 years, abds were transmitted by eight families, 19 genera and 70 species of arthropods in ardabil province. most species belonged to culicidae with 24, psycodidae with 22 and ixodidae with 16 species (table 2). the climate of ardabil province is divided into three parts in north, central and south. the northern part located in the azerbaijan republic borderline with low altitude, minimum precipitation, moderate ndvi, maximum annual temperature, maximum rh, in this part the most important vbd is malaria. in the central part located around sabalan skirt with high attitude (altitude = 4,811 m), moderate precipitation, over average ndvi, moderate average temperature, low rh in the central climate, the main vbd is vl. the southern part is located in the neighborhood of guilan province covered mainly by forests which its climate characterized by high altitude, high precipitation, high ndvi, low annual temperature, moderate rh. the main vbd is rtf in south parts of province. malaria: totally 931 cases were reported which more than 98% of them fig. 1. the study area in north west of iran fig. 1. the study area in north west of iran 290 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... table 1. the spices of vector–borne diseases vectors in ardabil province, northwest of iran (2001–2018) class order family genus spices (adult and larva) references insecta diptera culicidae anopheles an. maculipennis, an. hyrcanus, an. superpictus, an. claviger, an. sacharovi, an. superpictus, an. pesudopictus (19, 20) culex cx. hortensis, cx. modestus, cx. pipiens, cx. theileri, cx. torrentium, cx. tritaeniorhynchus, cx. perexiguus, cx. mimeticus aedes a. caspius s.l., a.vexans, a. flavescens culiseta cs. annulata , cs. longiareolata, cs. subochrea dahliana da. geniculatus coquillettidia coquillettidia richiardii uranotaenia uranotaenia unguiculata insecta diptera psychodidae phlebotomus ph. perfiliewi, ph. papatasi, ph. sergenti, ph. tobbi, ph. major group, ph. caucasicus, ph. wenyoni, ph. elenorae, ph. major, ph. mongolensis, ph. kandelakii, ph. caucasicus group, ph. andrejevi, ph. halepensis, ph. longidnctus, ph. balcanicus, ph. brevis, ph. chinensis group, ph. simici, ph. jacusieli (13, 21–25) sergentomyia se. sintoni, se. dentata arachnida ixodida argasidae ornithodoros ornithodoros lahorensis, o. tholozani (26–29) argas argas persicus, a. reflexus ixodidae dermacentor dermacentor marginatus, d. niveus, hyalomma hy. anatolicum, hy. asiaticum, hy. aegyptium, hy. detritum, hy. dromedarii, hy. marginatum, hy. turanicum, hy. schulzei haemaphysalis h. choldokovskyi, h. erinacei, h. inermis, h. punctate, rhipicephalus r. bursa, r. sanguineus sarcoptiformes sarcoptidae sarcoptes s. scabiei scorpiones scorpionidae scorpio scorpio maurus (30–32) buthidae mesobuthus mesobotus eupeus insecta phthiraptera pediculidae pediculus pediculus capitis (12, 33) table 1. the spices of vector–borne diseases vectors in ardabil province, northwest of iran (2001–2018) fig. 2. the prevalence rate of vector–borne diseases in ardabil province, northwest of iran (2001–2018) fig. 2. the prevalence rate of vector–borne diseases in ardabil province, northwest of iran (2001–2018) 291 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... reported in northern and 2 % in central parts of province. since 2005, there was no report on malaria case in this province (figs. 3 and 7). 55 % of patients were male and 45% female from them 70% located in rural and 30 % in urban area. more than 99% of the malaria cases were proved plasmodium vivax and remaining 1% was not identified. so far, four adult and six larva stage of anopheles have been reported, the most abundant form in adult was an. claviger followed by an. hyrcanus and an. superpictus. totally 17 species of the subfamily culicinae were reported in ardabil province. except for malaria, only one study has been conducted on mosquito– borne diseases in ardabil province. the larvae of setaria labiatopapillosa and dirofilaria immitis were found in an. maculipennis and cx. theileri, respectively. eight spicies of culex, three spices of culiseta, three species of aedes, dahliana geniculatus, coquillettidia richiardii and uranotaenia unguiculata (table 2). the results of malaria spatial autocorrelation showed that the general g index 0.023755 value, z–score of 2.47999448315, there is a less than 5% likelihood that this high– clustered pattern could be the result of random chance (p = 0.0131) (fig. 7a). fig. 3. the frequency of malaria cases in ardabil province, northwest of iran (2001–2018) fig. 3. the frequency of malaria cases in ardabil province, northwest of iran (2001–2018) fig. 4. the frequency of visceral leishmaniasis cases in ardabil province, northwest of iran (2001–2018) fig. 4. the frequency of visceral leishmaniasis cases in ardabil province, northwest of iran (2001–2018) 292 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... visceral leishmaniasis (vl): vl is a zoonotic disease and one of the important endemic diseases in ardabil. during the study, 379 cases of the disease were reported. vl is reported mostly in central and northern part while in the southern part are sporadic (figs. 4, 7). the highest prevalence of vl were reported in meshgin shahr (50%) and germi (20%). 64% of the disease occurs in rural areas and 36% in urban with male to female ratio of 2/1. more than 85% of reported cases were children under 10 years. l. infantum is the causative agent of vl in these areas. the most important reservoirs of this disease are canids which its infection rate increased from 5% in 2000 to 38% in 2018. vl parasites have been isolated and reported from human, dogs, wolfs, fox and jackal in the ardabil province. 22 species of sand flies were identified and reported in this area from them three species (ph. kandelakii, ph. perfilewi and ph. tobbi) has been confirmed by parasitological and molecular methods to l. infantum. p. kandelakii distributed in central and ph. perfilewi and ph. tobbi in northern parts of ardabil province. the results of vl spatial autocorrelation showed that the general g index 0.006569 value, z– score of 0.918861150657, the pattern does not appear to be significantly different than random (p = 0.358) (fig. 7a). cutaneous leishmaniasis (cl): totally 412 cases of cl were reported in this area which the annual incidence rate was 2–5/100000 (figs. 5 and 6). the causative agent of cl not identified in ardabil province. cl were reported from seven counties (bilehsavar, germi, parsabad, ardabil, khalkhal, nir and namin) of the province. 50% of cases had a history of travel to endemic areas of cl. from 22 spices of sand flies, two spices ph. papatasi and ph. sergenti had the highest frequency. phlebotomus papatasi and ph. sergenti were collected in all parts of province but the parasite species was not identified. the cl reservoirs of infection has been reported from the meriones libycus. the results of cl spatial autocorrelation showed that the general g index 0.000037 value, the z–score of 6.80150065796, there is a less than 1% likelihood that this high–clustered pattern could be the result of random chance (p = 0.000). (fig.6a). tick–borne relapsing fever (trf): trf has been reported in the southern regions of ardabil province for a long time. 400 cases of this disease were reported, of which 94% were in rural and 6% urban areas. 52% were female and 48% male and 88% of cases were under the age of 20 year table 2. the frequency of vector–borne diseases in ardabil province, northwest of iran (2001–2018) year malaria visceral leishmaniasis cutaneous leishmaniasis tick relapsing fever lice anthrax scorpionism scabies cchf total prevalence 2001 541 18 32 124 200 5 42 3 1 966 79.5 2002 323 64 23 120 187 6 55 5 0 783 64.3 2003 53 32 32 56 295 2 41 2 1 514 42 2004 10 46 21 44 289 0 48 1 0 459 37.6 2005 0 19 19 22 372 1 43 0 0 476 39 2006 0 16 30 14 481 0 48 1 1 591 48 2007 1 16 20 6 533 0 49 0 0 625 50.8 2008 1 29 27 6 420 8 55 1 0 547 44.3 2009 1 45 31 4 425 0 52 0 1 559 45 2010 0 22 17 2 494 2 37 6 0 580 46.7 2011 0 12 13 1 359 0 87 3 0 475 38 2012 0 10 18 0 715 0 101 1 0 845 67.7 2013 0 3 19 0 1981 1 125 0 0 2129 170 2014 0 7 25 0 15987 0 119 1 1 16140 1286 2015 0 9 16 0 18961 0 155 4 0 19145 1519.5 2016 0 14 21 0 20148 2 182 5 0 20372 1613 2017 1 8 25 0 21879 2 222 8 2 22147 17.47 2018 0 9 23 1 22465 0 214 10 1 22723 1789 total 931 379 412 400 106191 29 1675 51 8 110076 – table 2. the frequency of vector–borne diseases in ardabil province, northwest of iran (2001–2018) 293 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... (figs. 6 and 9). more than 91% of cases have been reported from southern parts and 9% from other areas of province. four species of soft ticks; ornithodoros lahorensis, o. tholozani and argas persicus, a. reflexus have been reported that the o. lahorensis with 46% and the o. tholozani 40% had the high frequencies. the species of borrelia has not been studied and identified in the fig. 4. the frequency of visceral leishmaniasis cases in ardabil province, northwest of iran (2001–2018) fig. 5. the frequency of cutaneous leishmaniasis cases in ardabil province, northwest of iran (2001–2018) fig. 6. distribution of malaria, visceral leishmaniasis, cutaneous leishmaniasis (a), tick relapsing fever, cchf, scabies (b) and pediculosis, anthrax, scorpionism (c) in ardabil province, northwest of iran (2001–2018). fig. 6. distribution of malaria, visceral leishmaniasis, cutaneous leishmaniasis (a), tick relapsing fever, cchf, scabies (b) and pediculosis, anthrax, scorpionism (c) in ardabil province, northwest of iran (2001–2018). 294 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... region. however, in a study conducted in 2004, the infection rate of borrelia sp in the three–tick species as; o. lahorensis, o. tholozani and a. persicus was 14.33% in 2004. the infectious of o. tholozani was estimated to be 17.5% in 2005. the results of trf spatial autocorrelation showed that the general g index 0.000108 value, the z– score of –0.416500980084, the pattern does not appear to be significantly different than random (p = 0.677). (fig. 6b). crimean–congo hemorrhagic fever (cchf): in the province of ardabil, cchf is sporadic as 8 cases have been reported over the past 18 years (figs. 7, 6b). all cases were fig. 7. the frequency of tick–borne relapsing fever cases in ardabil province, northwest of iran (2001–2018) fig. 7. the frequency of tick–borne relapsing fever cases in ardabil province, northwest of iran (2001–2018) table 3. the frequency of vector-borne diseases data on the most important climate factors in ardabil province, northwest of iran (2001–2018) a b d s n o. a b d s in fe ct ed c ou nt y (% ) o f 1 0 in fe ct ed v ill ag e (% ) o f 1 47 7 in fe ct ed c it y (% ) o f 2 6 in fe ct ed d is tr ic ts (% ) o f 2 1 in fe ct ed r ur al d is tr ic ts (% ) o f 7 1 t op og ra ph y of a re a n d v i % a ve ra ge a lt it ud e (m ) a ve ra ge o f a nn ua lly t em pe ra tu re © a ve ra ge o f a nn ua lly r h % p re ci pi ta ti on (m m ) malaria 931 3(30) 18 4 2 12 plain 0.21–0.6 15–500 14–16.5 66–70 290–330 vl 379 6(60) 93 12 14 36 mountain, valley and hill 0.41–0.86 510–2000 10.1–14 55–65 330–400 cl 412 7(70) 35 20 19 56 plain, hill 0.41–0.86 510–2000 10.1–14 55–65 330–400 trf 400 2(20) 15 3 5 18 mountain 0.6–0.86 1600–2500 8.94–12 55–60 370–430 lice 106191 10(100) 1395 26 21 71 mountain, valley, hill, plain 0–0.86 15–2600 8.94–16.5 55–70 290–430 anthrax 29 2(20) 11 2 3 4 mountain 0.6–0.86 1600–2500 8.94–12 55–60 370–410 scorpionism 1675 8(80) 118 21 18 48 plain, mountain 0–0.41 15–2600 8.94–14 55–70 330–430 scabies 51 3(30) 4 2 3 7 mountain 0.6–0.86 1500–2600 8.94–14 55–65 370–430 cchf 8 3(30) 8 4 3 8 mountain, plain 0–0.86 15–2600 8.94–14 55–70 290–400 table 3. the frequency of vector-borne diseases data on the most important climate factors in ardabil province, northwest of iran (2001–2018) 295 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... from rural areas in rancher or farmer. patients had a history of contact with animals, tick bites and no death has been reported. totally 20 spices of soft and hard ticks were reported in ardabil province (table 2). cchf virus infection has been reported from five species by rt–pcr methods. the infection rate were in h. marginatum, h. dromedarii and fig. 8. the frequency of crimean–congo hemorrhagic fever cases in ardabil province, northwest of iran (2001–2018) fig. 8. the frequency of crimean–congo hemorrhagic fever cases in ardabil province, northwest of iran (2001–2018) fig. 9. the frequency of pediculosis in ardabil province, northwest of iran (2001–2018) fig. 9. the frequency of pediculosis in ardabil province, northwest of iran (2001–2018) fig. 10. the frequency of anthrax in ardabil province, northwest of iran (2001–2018) fig. 10. the frequency of anthrax in ardabil province, northwest of iran (2001–2018) 296 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... h. schulzei (0.8%), r. bursa (13.84 %) and o. lahorensis (6.92 %). scabies: 51 cases of scabies were reported in this area. the disease has been integrated into the health care system of the iranian ministry of health since 2016. 65% of the disease was reported in male and 35 % in female. more than 60% of cases were in rural areas and 40% urban (fig. 6b). scorpionism: a total of 1675 cases of scorpion bites occurred in the province during 2001–2018. the incidence of the scorpionism is between 12–20% per 100,000 people. the highest cases (25%) were reported from khalkhal in south part and the lowest from nir county in central part (1.5%). 57% were male and 43% female, and most cases (43%) were in the 10–20 years old and the lowest (18%) over the age of 70 years. 58% scorpion bites were occurred in homes and 42% outside homes. scorpionims have been increased more than two times since 2015. most scorpion’s bites occurred in october (73%) and november (72.5%) (figs. 1, 6c). pediculosis: during the study, 106191 cases of head lice infestation were reported in ardebil province. since 2013, pediculosis has doubled and then is increasing exponentially. 40% of cases have been reported from the central and 28.5% in the northern parts of the province. more than 80% of the cases have been reported in school students. until 2014, most cases were reported from rural areas, but after which 65% of the cases were reported from urban and peri–urban areas. the prevalence of pediculosis was increased from 44/100,000 in 2001 to 1,780/100,000 in 2018 year (figs. 9, 6c). anthrax: the disease was mostly observed in the southern and northern regions. 29 cases were reported from which 90% were in southern parts and 10% of northern parts (figs. 10–7c). discussion according to the who reports, annually more than one billion people are affected by vbds leading to one million deaths. the important distributed vbds fig. 11. general g index for high/low clustering of malaria (a), visceral leishmaniasis (b), cutaneous leishmaniasis (c) and tick–borne relapsing fever (d) in in ardabil province, northwest of iran (2001–2018) fig. 11. general g index for high/low clustering of malaria (a), visceral leishmaniasis (b), cutaneous leishmaniasis (c) and tick–borne relapsing fever (d) in in ardabil province, northwest of iran (2001–2018) 297 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... in world include malaria, dengue fever, schistosomiasis,leishmaniasis, chagas disease, yellow fever, lymphatic filariasis and onchocerciasis (4). malaria was also seemed in the northern parts of ardebil which is one of the oldest endemic areas in borderline of azerbijan republic. the cases of malaria have been zero since 2004 in ardabil province maybe due to plan to eradication of malaria in iran (19). similar to the pattern of global dispersion of p. vivax, the main cause of malaria in northwestern iran is also p. vivax (20). ardabil province is one of the most important endemic foci of visceral leishmaniasis in iran. in recent years, cases of vl have been reduced, but geographical dispersion has increased and new sides were reported. this pattern is also observed in india (21), brazil (22) and previous report from ardabil (13). most of the cases of vl were recorded in the meshgin shahr and germi counties with significant differences from other areas (p > 0.05). in these areas, most of the residents are farming and animal husbandry who keep dogs. the main reservoirs of vl are dogs and canids (23). large numbers of reservoirs may be one of the reasons to high prevalence of the disease in these areas. increasing the prevalence of canine visceral leishmaniasis (11) and distribution of infected dogs in different parts of the region is one of the most important causes of the distribution of vl in new sides in the areas. the high diversity of sand fly spices, high infection of reservoirs and high numbers of livestock has led to a high prevalence of vl in the northwest of iran. cl is a non–endemic disease in northwest of iran. the incidence rate of cl is a 2–5/100,000. the highest reported incidence was 27.5/100000 in iran (24). therefore, the ardebil province is among the regions with a low incidence. the main challenge is that, 47% of cases are children with no history of travel to endemic areas. so far, the causative agent of cl has not been detected in this area. there is a need for further study in this area, because according to the findings of this study, the province of ardebil is likely to be an endemic area with a moderate prevalence. the most important tick–borne diseases (tbd) reported in the ardabil province is cchf. according to the results of this study, tbd are increasing in some parts of iran as the latest reported in the international conference of cchf, the increasing and spread of the disease in the eastern mediterranean have been confirmed (25–26). the most important factors that may effect on tbd prevalence in northwest of iran includes high population density, high livestock rates, mountainous region and high rainfall that causes animal husbandry and number of livestock have increasing. as livestock increases the number of ticks increases and which lead to high incidence of tbd. one of the other causes could be the increase in smuggling of infected animals across the borders of iran. since 2013, the incidence of head lice infestations has increased exponentially in all parts of iran and ardabil province. these epidemics are more common among primary school students. in this area, the prevalence of head lice is between 5–28 /100,000. the reported prevalence was 3–28 in iran and 5–40/100000 in the world (12, 27–28). according to previous studies in ardabil province, pediculosis cases on the outskirts of city was more than villages. it seems family size, students and their parents’ history of infestation, type of bathrooms, and history of use shared hygiene items were probably risk factors associated with head lice infestation among students of primary schools in ardabil province (12, 29). the results of this study showed that malaria is reported only in the north of the province, with a lower elevation and along the aras river bank. the diversity and abundance of anopheles species also have been reported in this region in consistent with previous studies (p < 0.05) (30–32). vl and cl were reported in central part of the province and three of the most important main vectors of vl are reported. the most infection reservoirs of the vl are reported from the central part of the province (11). the matching of three factors proven vectors, 298 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 287–299 d adham et al.: a comprehensive... main reservoirs and fauna of leishmania parasite in this area resulted the ardabil province to be the most important endemic foci of vl in the northwest of iran. tick– and tick–borne diseases most were reported in south and central parts of ardabil. these areas have moderate to high rainfall with denser vegetation and livestock keeping. according to the prevalence and species diversity of ticks in this area, the incidence of tick–borne diseases could be increased (33–36). conclusions suitable climate, sufficient food, appropriate habitats, high abundance of arthropod and reservoirs of abds are the most important factors affecting the prevalence of abds in ardabil province. the climate change and ecological environments have a direct impact on the frequency of arthropods in ardebil province. the fauna, the frequency of the vectors, and main reservoirs be should be identified to prevent abds. the best method to control and prevention of abd disease should be selected based on the geographical conditions. acknowledgements this study was funded ardabil university of medical sciences (arums), project 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ghiasi s m, moradi m, vatandoost h, eshraghian mr, faghihi f, zarei z, haeri a, chinikar s(2010) a survey of crimean–congo haemorrhagic fever in livestock and ticks in ardabil province, iran during 2004– 2005. scand j infect dis. 42(2):137-41. 34. mostafavi e, bagheri amiri f, khakifirouz s, es maeili s, kazemi–lomedasht f (2016) serologic survey of crimean–congo haemorrhagic fever among sheep in ardabil province, northwest iran. j med microbio infec dis.4(1):16–9. 35. vatandoost h, moradi asl e, telmadarreiy z, mohebali m, masoumi asl h, abai mr (2012) field efficacy of flumethrin pour–on against livestock ticks in iran. inter j acarol.38(6):457– 64. 36. eslam moradi asl, hassan vatandoost, zakie telmadarreiy, mehdi mohebali, mohammad reza abai (2018) repellency effect of flumethrin pour–on formulation against vectors of crime an–congo haemorrhagic fever. east mediterr health j. 24(11):1082-1087. a comprehensive database and geographical distribution model of vectors and vector borne diseases in abstract keywords introduction materials and methods study area data collection data and spatial autocorrelation analysis ethics approval and consent to participate results malaria visceral leishmaniasis (vl) cutaneous leishmaniasis (cl) tick-borne relapsing fever (trf) crimean-congo hemorrhagic fever (cchf) scabies scorpionism pediculosis anthrax discussion conclusions acknowledgements references j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 228 http://jad.tums.ac.ir published online: september 30, 2020 original article first report of target site insensitivity in pyrethroid resistant anopheles gambiae from southern guinea savanna, northern-nigeria mustapha ahmed-yusuf1,2; *hassan vatandoost1,3; *mohammad ali oshaghi1; ahmad ali hanafi-bojd1,3; ahmad ali enayati4; rabiu ibrahim jalo5 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical microbiology and parasitology, college of health sciences, bayero university, kano, nigeria 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4department of medical entomology, school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 5department of community medicine, bayero university, kano, nigeria *corresponding authors: dr hassan vatandoost, email: hvatandoost1@yahoo.com, dr mohammad ali oshaghi, email: moshaghi@tums.ac.ir (received 01 dec 2019; accepted 11 jul 2020) abstract background: malaria is a major public health problem and life threatening parasitic vector-borne disease. for the first time, we established and report the molecular mechanism responsible for anopheles gambiae s.l. resistance to pyrethroids and ddt from yamaltu deba, southern guinea savanna, northern-nigeria. methods: the susceptibility profile of an. gambiae s.l. to four insecticides (ddt 4%, bendiocarb 0.1%, malathion 5% and deltamethrin 0.05%) using 2–3 days old females from larvae collected from study area between august and november, 2018 was first established. genomic dna was then extracted from 318 mosquitoes using livak dna extraction protocol for specie identification and kdr genotyping. the mosquitoes were identified to species level and then 96 genotyped for l1014f and l1014s kdr target site mutations. results: the mosquitoes were all resistant to ddt, bendiocarb and deltamethrin but fully susceptible to malathion. an. coluzzii was found to be the dominant sibling species (97.8%) followed by an. arabiensis (1.9%) and an. gambiae s.s (0.3%). the frequency of the l1014f kdr mutation was relatively higher (83.3%) than the l1014s (39%) in the three species studied. the l1014f showed a genotypic frequency of 75% resistance (rr), 17% heterozygous (rs) and 8% susceptible (ss) with an allelic frequency of 87% rr and 13% ss while the l1014s showed a genotypic frequency of rr (16%), rs (38%) and ss (46%) with an allelic frequency of 40% rr and 60% ss, respectively. conclusion: this study reveals that both kdr mutations present simultaneously in northern-nigeria, however contribution of l1014f which is common in west africa was more than twice of l1014s mutation found in east africa. keywords: anopheles gambiae s.l.; insecticide resistance; northern nigeria; voltage-gated sodium channels (vgsc) introduction insecticide resistance is mainly associated with genetic factors that are inherited and can be defined as “the ability in a population to tolerate doses of insecticide which would prove lethal to the majority of individuals in a normal population of the same species, developed as a result of selection pressure to the insecticide” (1, 2). the resistance is mainly acquired through two methods which include the target site insen sitivity and metabolic resistance (3). the target site insensitivity is operated through one of the following methods (insensitive acetylcholinesterase (ache), gaba receptor mutation, or mutations in the voltage-gated sodium channel). the nervous system of mosquitoes is been targeted by specific insecticide through which it acts, though sometimes the sensitivity of the site may reduce as a result of mutations leading to copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 229 http://jad.tums.ac.ir published online: september 30, 2020 resistance (4). the organophosphate and carbamates target the acetylcholinesterase (ache) through carbamoylating the active serine site thereby stopping it from hydrolyzing the acetylcholine (5-7). substitution in the gaba receptor of an alanine to serine has been reported in drosophila melanogaster, d. simulans, aedes aegypti, anopheles stephensi and an. gambiae as the cause of resistance (8). resistance in the pyrethroids and ddt is mainly due to mutations in the gene that encodes the voltage-gated sodium channel called the knockdown resistance (9). in the an. gambiae and an. arabiensis mosquitoes, two different knockdown resistance have been reported (10). the leucine-phenylalanine substitution at position 1014 of the sodium channel gene (l1014f), was the first mutation reported from burkina faso and ivory coast in west africa (11). while a leucine-serine substitution (l1014s) at the same position was reported as the second mutation from western kenya in east africa (12). a study conducted in northern nigeria reported high resistance of an. gambiae to permethrin and ddt with less resistant to bendiocarb (13). the resistance profile and kdr mutation of an. gambiae s.l. populations was also reported from two locations (auyo and bunkure) in northern nigeria (14). high presence of an. coluzzii has been reported from previous studies (13-17). two different studies conducted in northern nigeria both reported lower kdr mutations from both resistant and susceptible mosquitoes (18, 19). similarly, studies conducted in kenya reported lower kdr mutations to an. gambiae (20, 21). the aim of this study was to investigate species composition, the insecticide susceptibility status, and to explore type of kdr mutations conferring pyrethroids and ddt resistance in members of the an. gambiae complex from northern-nigeria. materials and methods study location yamaltu deba (10° 13′ 0″ n, 11° 23′ 0″ e) is one of the eleven local government areas in gombe state, nigeria (fig. 1). it has a population of 255,248, an area of 1,981km² and is located in the north-eastern part of nigeria, stretching through the sudan savannah, northern and southern guinea savannah (22, 23). study sample dipping method was used to collect larvae samples from different breeding places in the study site as described by (13) in order to provide laboratory stock of mosquitoes. the samples were transported to the insectary at bayero university kano with a rearing condition of 28±2 °c temperature, 65±5% relative humidity (rh) and 12:12 hrs d: l. two to three days old female sugar fed mosquitoes were used for susceptibility tests (24). who susceptibility tests adult susceptibility test was conducted according to the recent who bioassay guideline (25). twenty five female mosquitoes of 2–3 days old fed on 10% sugar solution, were exposed to malathion 5%, bendiocarb 0.1%, ddt 4.0% and deltamethrin 0.05% impregnated papers for 60 minutes in the standard who test kit. oil-impregnated papers were used for the control group. there were four replicates for the treated and two replicates for the control group. at the end of the exposure time, both the treated and control mosquito groups were allowed to recover in holding tubes with cotton pads containing 10% sucrose solution on the top for 24 hours and then the number of dead and alive mosquitoes were recorded. a mosquito is considered alive if it is able to fly, regardless of the number of legs remaining. dna extraction genomic dna was extracted from 318 individual mosquitoes using livak dna extraction protocol template preparation kit (26, 27). specie identification the mosquitoes were first identified morphologically using morphological identification http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 230 http://jad.tums.ac.ir published online: september 30, 2020 keys (28, 29). molecular species identification was performed by pcr-sine200 technique as previously described (14). sine pcr reagents were carried out in 15ul master mix containing amplification reaction of 0.51mol of each primer sine 200f and sine 200r, 0.12mm of each dntp, 0.75mm of mgcl2, 1.5u taq dna polymerase, pcr buffer 10x [200mm tris hcl (ph 8.4), 500mm kcl], 1.0µl of template dna extracted from each mosquito. the primer sequence and thermal cycling conditions are shown in (table 1). pcr for kdr west (l1014f) and kdr east (l1014s) the amplification protocol used for the detection of 1014f and 1014s mutations was performed using allele specific pcr in a 12.5µl reaction containing 1µl of template dna, 1x qiagen pcr buffer, 0.5mm mgcl2, 0.5nm of each primer, 0.5µm of dntps, and 1u of taq dna polymerase revised from the protocols previously established by martinez-torres et al. (11) and ranson et al. (30). the primer sequences and thermal cycling conditions are shown in (table 2). the primers agd1, agd2, agd4 and agd5 were used to detect the 1014s mutation whereas primers agd1, agd2, agd3 and agd4 were used to detect the 1014f mutation. data analysis the 24hrs mortality was accessed manually, while the susceptibility was defined as; 98– 100% mortality indicates susceptibility, 90–97 % mortality requires confirmation of resistance and between 0–89% suggests resistance (25). the hardy-weinberg equilibrium equation was used to calculate the genotypic and allelic frequencies. microsoft office excel, version 2003 was used to create charts, calculate the standard deviation, sort and clean the data. abbott’s formula (30) was used to correct for natural mortality, if the control mortality was between 5 and 20%. the results of the tests with >20% mortality in controls, were discarded and the test repeated (25). results female mosquitoes exposed to deltamethrin, ddt and bendiocarb showed 74% (95%, ci: 68–79); 53% (ci: 49–56) and 44% (ci: 38– 49) mortalities after 24 hours, respectively (fig. 2). whereas, malathion was found to be susceptible (fig. 2). molecular specie identification a total of 318 mosquitoes composing of 138 alive (45 exposed to deltamethrin, ddt, bendiocarb and 3 exposed to malathion) and 180 dead (45 exposed to deltamethrin, ddt, bendiocarb and malathion) were identified to specie level. all the dead mosquitoes identified were an. culluzzi, the alive mosquitoes exposed to ddt, malathion and bendiocarb also were an. culluzzi while for deltamethrin exposed, 53.3% were an. culluzzi, 40% an. arabiensis and 6.7% an. gambiae s.s (table 3). genotyping kdr west (l1014f) a total of 96 mosquitoes: 76 alive (69 an. coluzzii, 6 an. arabiensis, 1 an. gambiae s.s) and 20 dead (13 an. coluzzii, 6 an. arabiensis, 1 an. gambiae s.s) were used for kdr genotyping. the following result was recorded: alive mosquitoes, the only an. gambiae s.s 1/1 (100 %) was homozygote resistant (rr); an. arabiensis 4/6 (67%) rr, 2/6 (33%) heterozygote resistant (rs); an. coluzzii 55/69 (80%) rr, 9/69 (13%) rs, 5/69 (7%) homozygote susceptible (ss). dead mosquitoes, the only an. gambiae s.s 1/1 (100%) rr; an. arabiensis 3/6 (50%) rr, 2/6 (33%) rs, 1/6 (17%) ss; an. coluzzii 8/13 (62%) rr, 3/13 (23%) rs, 2/13 (15%) ss (table 4). genotypic and allelic frequencies of l1014f the hardy-weinberg equilibrium equation was used to calculate the genotypic and allelic frequencies. the result was found to be 72 (75%) rr, 16 (17%) rs and 8 (8%) ss; 87% rr and 13% ss (table 5). http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 231 http://jad.tums.ac.ir published online: september 30, 2020 genotyping kdr west (l1014s) a total of 96 mosquitoes were used, l1014s mutation was only found in 37 (39%) mosquitoes distributed as follows: 24 alive (15 an. coluzzii, 8 an. arabiensis, 1 an. gambiae s.s) and 13 dead (8 an. coluzzii, 4 an. arabiensis, 1 an. gambiae s.s) were used for kdr genotyping. the following result was rec orded: alive mosquitoes, the only an. gambiae s.s 1/1 (100 %) rs; an. arabiensis 2/8 (33%) rr, 5/8 (56%) rs, 1/8 (11%) ss; an. coluzzii 4/15 (27%) rr, 3/15 (20%) rs, 8/15 (53%) ss. dead mosquitoes: the only an. gambiae s.s 1/1 (100%) rs; an. arabiensis 3/4 (75%) ss, 1/4 (25%) rs; an. coluzzii 3/8 (38%) rs, 5/8 (62%) ss (table 4). genotypic and allelic frequencies (l1014s) the hardy-weinberg equilibrium equation was used to calculate the genotypic and allelic frequencies. the result was found to be 6 (16 %) rr, 14 (38%) rs and 17 (46%) ss: 40% rr and 60% ss (table 5). table 1. (a) pcr primer sequences and (b) thermal cycling conditions used for specie identification of the anopheles gambiae complex from yamaltu deba (gombe state), northern nigeria, 2018 a primer name sequence (5′ to 3′) identified species size of the pcr product (bp) sine200f sine200r tcg-cct tag acc ttg cgt ta cgc ttc aag aat tcg aga tac an. gambiae s.s. an. coluzzii an. arabiensis 240 470 220 b step temperature °c time cycle initial denaturation 95 5mins 1 denaturation annealing extension 94 54 72 30sec 1min 1min 35 final extension 72 10mins 1 table 2. (a) pcr primer sequences and (b) thermal cycling conditions used for detection knockdown resistance mutations (l1014f and l1014s) in the anopheles gambiae complex from yamaltu deba (gombe state), northern nigeria, 2018 a primer name sequence (5′ to 3′) primer type combination and size of pcr product (bp) agd1 atagattccccgaccatg common forward agd1+agd2=293 agd2 agacaaggatgatgaacc common reverse agd3 aatttgcattacttacgaca specific reverse for l1014f agd1+agd3=195 agd4 ctgtagtgataggaaattta specific forward for susceptible l1014l agd2+agd4=137 agd5 atttgcattacttacgactg specific reverse for l1014s agd1+agd5=195 b step temperature °c time cycle initial denaturation 95 3mins 1 denaturation annealing extension 94 60 72 30sec 1min 1min 35 final extension 72 5mins 1 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 232 http://jad.tums.ac.ir published online: september 30, 2020 table 3. molecular species identification anopheles gambiae s.l. specimens (a: alive, b: dead) following exposure to the insecticides from yamaltu deba (gombe state), in northern nigeria, 2018 a insecticide no. exposed an. coluzzii (%) an. arabiensis (%) an. gambiae s.s (%) deltamethrin 0.05% 45 53.3 40 6.7 ddt 4% 45 100 0 0 malathion 5% 3 100 0 0 bendiocarb 0.1% 45 100 0 0 b insecticide no exposed an. coluzzii (%) an. arabiensis (%) an. gambiae s.s (%) deltamethrin 0.05% 45 100 0 0 ddt 4% 45 100 0 0 malathion 5% 45 100 0 0 bendiocarb 0.1% 45 100 0 0 table 4. genotyping of kdr west (l1014f) and east (l1014s) mutations of anopheles gambiae s.l. from northern nigeria, 2018 kdr mutation (l1014f) alive dead species an. gambiae s.s. an. arabiensis an. coluzzii an. gambiae s.s. an. arabiensis an. coluzzii homozygote resistance 100% 67% 80% 100% 50% 62% heterozygote resistance 0 33% 13% 0 33% 23% homozygote susceptible 0 0 7% 0 17% 15% n=1 n=6 n=69 n=1 n=6 n=13 kdr mutation (l1014s) alive dead species an. gambiae s.s an. arabiensis an. coluzzii an. gambiae s.s an. arabiensis an. coluzzii homozygote resistance 0 33% 27% 0% 0% 0% heterozygote resistance 100% 56% 20% 100% 25% 38% homozygote susceptible 0 11% 53% 0 75% 62% n=1 n=8 n=15 n=1 n=4 n=8 table 5. the allelic and genotypic frequencies of the l1014f and l1014s, mutations of anopheles gambiae s.l. from northern nigeria, 2018 genotypic frequencies (l1014f) homozygote resistance heterozygote resistance homozygote susceptible 75% 17% 8.00% n=96 allelic frequencies (l1014f) homozygote resistance homozygote susceptible 87% 13% genotypic frequencies (l1014s) homozygote resistance heterozygote resistance homozygote susceptible 16% 38% 46% n=96 allelic frequencies (l1014s) homozygote resistance homozygote susceptible 40% 60% http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 233 http://jad.tums.ac.ir published online: september 30, 2020 fig. 1. map (adapted from google earth) showing the geographical location of the study site, gombe state (red circle) in northern nigeria, 2018 fig. 2. susceptibility (24hrs) profile of anopheles gambiae s.l. to four insecticides from yamaltu deba (gombe state), northern nigeria, 2018 discussion susceptibility test bendiocarb showed very high level of resistance. this finding agrees with previous study (32), where they reported a percentage mortality range of 2.3–100%. similarly, a study from kumasi in ghana, reported 38–56% mortality to bendiocarb (33). this study reports moderate level of resistance to deltamethrin from the study site. this finding is in agreement with study conducted in the northern guinea savanna of nigeria (34) where they reported percentage mortality of 83%, and from north western part of nigeria where they reported 78% mortality to http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 234 http://jad.tums.ac.ir published online: september 30, 2020 deltamethrin (14). however, a study conducted around the study location disagrees with our finding where they reported a very high resistance of 38% mortality to deltamethrin (35). ddt was found to be resistant and this finding is in agreement with previous studies from sudan, guinea and sahel savanna of nigeria (13, 14, 19, 30, 36, 37). malathion was found to be susceptible and agrees with studies from different regions within and outside nigeria (14, 19, 33, 34, 37). species identification anopheles coluzzii was found to be the dominant sibling species followed by the an. arabiensis and an. gambiae s.s (table 3). this is in agreement with a study conducted in northern nigeria, where they reported an. coluzzii as the dominant specie 86.8% followed by an. arabiensis 77% (14). also, the high presence of an. coluzzii reported is supported by previous studies (13, 15, 17). however, a study conducted on molecular identification of an. gambiae s.l mosquitoes in kamuli district of uganda, disagrees with our finding where they reported 98% of the mosquitoes to be an. gambiae s.s (38). another study conducted in nigeria by oyewole and colleagues (2011), reported an. gambiae s.s as the dominant species (74.6%) followed by an. arabiensis 26.4% in contrast to our finding (39). knockdown resistance (kdr) west (l1014f) and east (l1014s) the kdr mutations were observed in the study location with high frequency of the l1014f in the an. coluzzii species (table 4). this agrees with study conducted by ibrahim et al. (2014) where they found the kdr mutations in 80.1% of the an. coluzzii and 13.5% in an. arabiensis mosquitoes (12). oyewole et al. (2011), in their study from south-western nigeria reported that 87% of the mosquitoes resistant to deltamethrin carried the kdr mutations and 80% of the ddt resistant mosquitoes as well (36). furthermore, increase l1014f was reported from ghana by lynd et al. (2010) niger, by czeher et al. (2008) and sharp et al. (2007) from equatorial guinea (40-42). a study by awolola et al. (2003) contradicts our finding, where they reported high frequency of l1014f in an. gambiae s.s compared to an. coluzzii (43). derrick et al. (2011) from kenyan, also reported increased presence of kdr in an. gambiae s.s compared to the an. coluzzii. also, increased presence of l1014s was reported by protopopoff et al. (2008), from burundi and verhaeghen et al. (2010), from uganda (44, 45). genotypic and allelic frequencies this study reports high genotypic frequency particularly in the l1014f kdr compared to the l1014s gene from the study location (table 5). this is in agreement with previous study where they reported homozygous resistant of 74.1%, heterozygous resistant of 19.7% and homozygous susceptible of 6.2% for the l1014f in the an. coluzzii. while from the an. arabiensis; 69.2% were homozygous susceptible, 23.1% heterozygous and 7.7% homozygous resistance (14). a study by habibu and colleagues (2017), contradicts our finding, where they reported 65.6% homozygous susceptible, 10% homozygous resistance and 24.4% as heterozygous resistance in the l1014s (36). while the l1014f showed 54.4% as homozygous susceptible, 21.6% as homozygous resistance and 24% as heterozygous resistance. the l1014s was seen both in the an. coluzzii and an. arabiensis (36). our study also reports a very high allelic frequency in the l1014f compared to the l1014s (table 5). this agrees with the results of habibu et al. (2017) where they reported an allelic frequency of 48.9% and 65.9% in an. gambiae s.s and 20% and 61.8% in an. arabiensis in the l1014f (13). while the l1014s mutation recorded an allelic frequency of 40% and 55.3% in an. coluzzii; 20% and 30.8% in an. arabiensis. the l1014f has higher association with an. coluzzii (36). studies by derrick (2011) and stump (2004) from kenyan rehttp://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 228–238 m ahmed-yusuf et al.: first report of … 235 http://jad.tums.ac.ir published online: september 30, 2020 ported lower allelic frequency compared to our findings (20, 21). the high level of insecticide resistance observed may be associated with increased use of pyrethroids treated bed nets and carbamate for indoor residual spraying (irs) in public health and agricultural applications (35, 46). farmers in the study location use a wide range of pesticides and herbicides to protect their crops and these pesticides marketed under different trade names belong to all the chemical classes including organophosphates, organochlorine, pyrethroids and carbamates (36). the high presence of kdr gene seen in this population of mosquitoes could be explained by the increase usage and abuse of insecticides by farmers and the increase coverage of llin distribution. conclusion this study reveals the co-occurrence of l1014f and l1014s mutations with dominance of an. coluzzii and high genotypic and allelic frequencies in the l1014f over l1014s-kdr. very high level of resistance to ddt, deltamethrin and bendiocarb was also observed. acknowledgements the authors wish to thank the sponsors. this research was co-sponsored by international campus, tehran university of medical science (project no. 9513494001) and institute of environmental research (grant. no. 97-02-27-39814), iran with technical support from center for infectious diseases research bayero university, kano, nigeria. the authors declare that they have no competing interests. references 1. mohammed br, abdulsalam ym, deeni yy (2015) insecticide resistance to anopheles spp. mosquitoes (diptera: culicidae) in nigeria. a review. inter j mosq res. 2(3): 56–63. 2. brown g, nossal g (1986) malaria, yesterday, today, and tomorrow. perspect biol med. 30(1): 65–76. 3. william gb, janet c (1997) heme peroxidase activity measured in single mosquitoes identifies individuals expressing an elevated oxidase for insecticide resistance. j am mosq control assoc. 13(3): 233– 237. 4. scott ja (1995) the molecular genetics of resistance: resistance as a response to stress. fla entomol. 78(3): 399–404. 5. weill m, lutfalla g, mogensen k, chandre f, berthomieu a, berticat c, pasteur n, philips a, fort p, raymond m (2003) comparative genomics: insecticide resistance in mosquito vectors. nature. 423 (6936): 136–137. 6. davari b, vatandoost h, oshaghi ma, ladonni h, enayati aa, shaeghi m, basseri hr, rassi y, hanafi-bojd aa (2007) selection of anopheles stephensi with ddt and dieldrin and cross-resistance spectrum to pyrethroids and fipronil. pest biochem physiol. 89(2): 97–103. 7. limoee m, enayati aa, ladonni h, vatandoost h, baseri h, oshaghi ma (2007) various mechanisms responsible for permethrin metabolic resistance in seven field-collected strains of the german cockroach from iran, blattella germanica (l.) 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gorouhi ma, vatandoost h, oshaghi ma, raeisi a, enayati aa, mirhendi h, hanafi-bojd aa, abai mr, salim-abadi y, rafi f (2016) current susceptibility status of anopheles stephensi (diptera: culicidae) to different imagicides in a malarious area, southeastern of iran. j arthropod borne dis. 10(4): 493–500. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 17 http://jad.tums.ac.ir published online: april 27, 2019 original article comparison of cdc bottle bioassay with who standard method for assessment susceptibility level of malaria vector, anopheles stephensi to three imagicides *hassan vatandoost1,2; *mohammad reza abai1,2; morteza akbari1; ahmad raeisi3; hemn yousefi1; soraya sheikhi1; akbar bagheri1 1department of medical entomology and vector control, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of malaria, center for disease control (cdc), ministry of health and medical education, tehran, iran (received 8 jan 2018; accepted 16 apr 2018) abstract background: the detection of insecticide resistance in natural populations of anopheles vectors is absolutely necessary for malaria control. cdc bottle bioassay as a new tools has been employed for detecting the insecticide resistance. for a limit number of mosquito vectors, diagnostic doses and diagnostic times for some insecticides have already been determined using this new assay. for the first time in the area, susceptibility levels of anopheles stephensi was done with ddt, deltamethrin, and bendiocarb using cdc bottle bioassay and compared results with who standard test method. methods: anopheles stephensi were collected in larvae stage from the cisterns of drinking water in chabahar port which considered as old malaria foci, sistan and baluchistan province. the field collected larvae were colonized at the insectary of school of public health (sph), tehran university of medical science. the susceptibility tests were carried out on sugar fed female mosquitoes aged 2–3 days, against ddt 4%, bendiocarb 1% and deltamethrin 0.05% using who and cdc susceptibility methods. the mortality and knockdown rates, as well as the parameters of regression analysis, including lt50 and lt90, was calculated separately for the who and cdc methods. results: the 24h mortality rates of an. stephensi were 28.6% and 25.6% for ddt, 60.8% and 64.6% for bendiocarb and 100% for deltamethrin using both who and cdc assay at 30 and 60min respectively. the 50% lethal times (lt50) were estimated 44.9 and 66.2min, 38.9 and 81.8min and 0.7 and 15.0min respectively using both who and cdc susceptibility tests. conclusion: the similar results of susceptibility levels were shown for ddt, bendiocarb and deltamethrin. the lethal times (lt50) showed significant difference using both who and cdc bioassay methods. keywords: susceptibility; insecticide; who bioassay; cdc bioassay; anopheles stephensi introduction malaria and other mosquito-borne diseases are the major problems worldwide. currently, there are proven and effective tools to combat against vectors (1). more than 80% of malaria cases in the country are reported from three provinces southern and southeastern areas of the country (2). the most routes of malaria cases are immigratio from eastern borders (3) which affected by ex tensive population movement across the border with pakistan, there are several works on different aspects of malaria in the country which is useful for decision making (4). among of anopheles mosquitoes, anopheles stephensi liston (diptera: culicidae) for both plasmodium falciparum and p. vivax with the geographical range from the middle east to india and china and southern slope of areas located in zagros *corresponding authors: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, mr mohammad reza abai, e-mail: abaimr@tums.ac.ir j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 18 http://jad.tums.ac.ir published online: april 27, 2019 mountain chains (5). recent studies in iran have been revealed the presence of 31 anopheles species including genotypes and sibling species, eight of them involved in malaria transmission in iran (6). vector control as the main measures used to reduce malaria transmission at the community level and considered as the most effective measure for eradicating malaria. it has been proven to be the only measure that can reduce malaria transmission from very high to a low level (7). resistance to insecticides among the mosquito vectors is the most important growing concerns in many countries and bioassays allow for the detection and characterization of insecticide resistance in a vector population. world health organization (who) susceptibility test is the main assay for assessing the susceptibility /resistance status among mosquito vectors. cdc bottle bioassay is a new tool for assessing resistance to insecticides in the world (8). the goal of cdc bioassay is to measure the mortality rate of members of a population at a given dose of the insecticide. in order to know the advantages, disadvantages and features, the who test compared with the cdc bottle bioassay using three insecticides against an. stephensi. the resistance parameters of this research allowed comparing data across other countries. to determine the diagnostic dose and the diagnostic time for use in the cdc bottle bioassay, the assay was calibrated. materials and methods mosquito tested the larvae of an. stephensi were collected from the artificial ponds in the urban area of chabahar port, sistan and baluchistan province and colonized at the insectary of school of public health (sph), tehran university of medical science (tums). the rearing condition was 29±1 and 60–65% relative humidity with 12l: 12d photoperiod cycle. larvae were fed with fish flakes and adults were fed a sugar water solution consisting of 10% sucrose. for mosquito mass colonization, female mosquitoes were fed on guinea pin hold in a restrainer. females 3 to 4 days post emergence were used in all experiments. insecticide materials the choice of deltamethrin and bendiocarb were based on conventional use in residual spraying in malaria foci and ddt selected as indicator insecticide for revealing of resistance. the insecticide impregnated papers were purchased with who representative in penang, malaysia. the technical active ingredient of ddt, bendiocarb and deltamethrin were provided from ecotoxicology laboratory, school of public health, tehran university of medical sciences. who’s susceptibility test protocol in each replicate, 20 to 25 sugar-fed female mosquitoes aged 2–5 days gently released into the holding tubes with green dots (4–5 as treated group and 1 as control one) lined inside each tube with untreated papers following the who’s protocol (9). after resting the mosquitoes 15 minutes at the vertical position, they gently blown into the exposure tubes with red dots lined inside the tubes with who’s insecticide impregnated papers including ddt 4%, bendiocarb 1% and deltamethrin 0.05%. the mortality rate of an. stephensi was assessed at logarithmic trend around the diagnostic time. after each exposure time, the mosquitoes were transferred back into the holding tubes marked with green dots and provided the cotton pad moistened with the 10% sucrose solution at top of the nets. the knockdown and mortality rates were recorded after 60 minutes and 24 hours respectively. the environmental condition of test room was 28±1 °c and 60–65% relative humidity. cdc bottle test protocol the cdc bottle test was conducted as a new surveillance tool for detecting the insecticide resistance among malaria vector at the given j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 19 http://jad.tums.ac.ir published online: april 27, 2019 diagnostic doses and the diagnostic exposure times. the stock solutions were prepared by adding 10 and 1.25µl of technical grade of ddt, deltamethrin and bendiocarb per 100ml pure ethanol. the inside surfaces of 250ml glass bottles were coated with 1ml of stock solution and control bottles were coated only with pure alcohol according to the cdc protocol. for each concentration five bottles were coated. the diagnostic doses of ddt, deltamethrin and bendiocarb for anopheles species are respectively standardized as 100, 12.5 and 12.5µg/bottle (8). the coated bottles were placed in incubator equipped to ventilator and set temperature at 35 °c to allow to evaporate the solvent overnight. after the alcohol was fully evaporated, 15–25 mosquitoes aged 3–4 days fed with 10% sucrose solution were released to each bottle using a hand aspirator. the exposure time was set to 30min and for possible estimating of median lethal time (lt50), different exposure times 0.5, 1.0, 1.9, 3.8, 7.5, 15, 30, 45, 60, 75, 90, 105 and 120min were used. the mosquitoes were removed from the bottles and sorted into “alive” and “knocked-down” groups. mosquito groups were kept in separate paper cups with 10% sucrose solution under insectary conditions. after 24h, they were scored as alive or dead in order to determine delayed mortality. at least five replicates were performed for each exposure time. statistical analysis and data interpretation the data interpretation for who susceptibility tests for susceptible/resistance status of anopheles species were adopted according to the latest related criteria (1) as follows: 1. susceptible status: when 98–100% mortality rate resulted at the recommended diagnostic dose and time. 2. tolerance status: when, 90–97% mortality rate resulted at the recommended diagnostic dose and time which suggests the possibility of resistance and needs to be confirmed. 3. resistance status: when less than 90% mortality rate resulted at the recommended diagnostic dose and time. the data interpretation of the cdc bottle test was adopted according to the related protocol. the susceptibility thresholds was considered at the diagnostic time of 30 minutes for all insecticide tested at the recommended diagnostic doses (8): 1. susceptible status: the tested mosquitoes with insecticide-coated bottles are considered susceptible when died before the diagnostic time. 2. resistance status: if tested mosquitoes survive beyond diagnostic time, these survivors represent a proportion of the population with some degree of resistance. the susceptibility tests can be carried out beyond the diagnostic time to evaluate the intensity of resistance. the data from the cdc bottle bioassay using test mosquitoes need to be compared with data from susceptible mosquitoes or from a population that will serve as baseline. resistance thresholds for each insecticide can be determined by calibrating the cdc bottle bioassay. the bioassay data were analyzed using the probit program and the values for the lethal times 50% and 90% mortality (lt50, lt90) and 95% confidence interval were estimated. the abbott’s formula was not applied to the mortality rate in treated and control group were less than 5% in all experiments (10). the regression line of the tested insecticides were plotted using microsoft excel (ver. 2013). results susceptibility to ddt the 24h mortality rate of an. stephensi was 28.6% and 25.6 % exposed respectively 30min and 60min to ddt using who and cdc assays (table 1). the logarithmic exposure times of ddt were ranged from 15 to 120min for cdc bottle test and from 30 to 240 minutes for j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 20 http://jad.tums.ac.ir published online: april 27, 2019 who test. the 50% lethal times (lt50) were estimated 44.9min and 66.2min respectively. the ddt regression equations of the mortality an. stephensi were set as y= -6.4192+3.5250x and y= -4.9652+3.0045x with who and cdc assays (table 2). susceptibility to bendiocarb the 24h mortality rates of an. stephensi were 60.8% and 64.6% exposed 30min and 60 min to bendiocarb using who and cdc assays (table 1). the exposure times of bendiocarb were ranged from 7.5 to 60min for who tests and from 15 to 120 minutes for cdc bottle tests. the 50% lethal times (lt50) were 38.9min and 81.8min respectively. the regression equations of the mortality an. stephensi were set as y= 4.6479+2.9241x and y= -4.4782+2.3413x with who and cdc assays (fig. 2). susceptibility to deltamethrin the 24h mortality rates of an. stephensi were 100% exposed 30min and 60min to deltamethrin using who and cdc assays (table 1). the exposure times of deltamethrin were ranged from 0.5 to 120min for who tests and from 15 to 120 minutes for cdc bottle tests. the 50% lethal times (lt50) were 0.8min and 15.4min respectively. the regression equations of the mortality an. stephensi were set as y= 0.3371+2.7535x and y= -3.4038+2.8646x with who and cdc assays (fig. 3). table 1. susceptibility data recorded according to both who and cdc methods against an. stephensi insecticides susceptibility method range of exposure time (min) total tested range of mortality rate (%) at logarithmic exposure time mortality rate (%) after 24h maintenance resistance status ddt who 30–240 371 18.6–100 28.6 (60min) r* cdc 15–120 1107 13.9–100 25.6 (30min) r bendiocarb who 7.5–60 369 13.3–97.8 60.8 (60min) r cdc 15–120 389 57.3–100 64.6 (30min) r deltamethrin who 0.47–120 380 28.3–100 100 (60min) s** cdc 15–120 1080 100–100 100 (30min) s * resistance ** susceptible table 2. probit regression line parameters of anopheles stephensi exposed to different insecticides with who method insecticide susceptibility assay a b±se lt50, 95% c.i. (min) lt90, 95% c.i. (min) x2 (df) p value ddt 4% who -6.4192 3.5250±0.298 59.3593 66.2271 73.6338 131.8797 152.9699 185.3884 15.265 (2) 0.01 ddt 1% cdc -4.9652 3.0045±0.151 42.0031 44.9348 48.0928 107.3886 119.9894 136.7425 81.874 (2) 0.01 bendiocarb 1% who -4.6479 2.9241±0.262 34.4114 38.8617 43.7645 88.9256 106.6132 135.9259 9.045 (2) 0.05 j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 21 http://jad.tums.ac.ir published online: april 27, 2019 bendiocarb 1% cdc -4.4782 2.3413±0.254 70.1951 81.7899 98.9898 209.6077 288.4610 464.5222 21.696 (2) 0.05 deltamethrin 0.05% who 0.3371 2.7535±0.249 0.651 0.7543 0.8571 1.8701 2.2029 2.7275 4.545 (2) 0.05 deltamethrin 1% cdc 3.4038 2.8646±0.346 12.3959 15.4251 18.0780 36.7971 43.2137 54.0015 46.788 (2) 0.05 fig. 1. mortality rate and regression analysis of bioassays of anopheles stephensi (chabahar strain) exposed to ddt using who and cdc methods fig. 2. mortality rate and regression analysis of bioassays of anopheles stephensi (chabahar strain) exposed to bendiocarb using who and cdc methods, 2015–2016 table 2. continued … j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 22 http://jad.tums.ac.ir published online: april 27, 2019 fig. 3. mortality rate and regression analysis of bioassays of anopheles stephensi (chabahar strain) exposed to deltamethrin using who and cdc methods, 2015–2016 discussion the who standard method is a major and widely used method in the country, but the cdc bottle bioassay method was used for the first time in order to compare the resistance levels. information about the susceptibility of the vectors to insecticide is essential for chemical interventions, so routine determining the susceptibility levels of malaria vector is an integral part of vector control program. in this study, two important methods were used to perform susceptibility levels on malaria vector, an. stephensi that has been colonized at the insectary of sph, tums which the first generation transported from chabahar district have been showed resistance or tolerance to several types of insecticides including organochlorine, pyrethroid and carbamate (11). approximately in all previously studies on susceptibility status of an. stephensi in iran, resistance to ddt have been reported, although tolerance to ddt only reported from southeastern part of the country (12). this is the first study for using cdc bottle test for detecting insecticide resistance in the mosquito vectors in iran. there are some published studies on using cdc bottle test for detecting ddt resistance on disease vectors with 30min exposure time including an. gambiae in madagascar with 99 % mortality rate (13), same species in nigeria with 25% mortality (14, 15), an. gambiae in zambia with 90% (16) and on an. nuneztovari in columbia with 85% mortality (17). our finding also showed highly resistance of an. stephensi to ddt with similar values (25.6% and 28.6%) for mortality rate using both test methods. this is the second evidence of similarity of the results using both cdc and who bioassay method for revealing of susceptibility status compared the first one (22). the cdc bioassay conducted in the macha, zambia, the ddt at 300 μg/bottle, with delayed mortality 13–69% (18). the cdc bioassay method was also applied j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 23 http://jad.tums.ac.ir published online: april 27, 2019 to reveal the resistance status of malaria vectors to bendiocarb, with 30min exposure time including an. gambiae in the benin with 78.9100% mortality depending to different tested localities (19, 20), an. gambiae in northern benin with 100% mortality both in the field (16) and laboratory condition (18). the cdc bottle assay test showed that an. gambiae s.l. was fully susceptible to bendiocarb in nigeria (15) and in almost all of the 18 sampled districts in ethiopia except in omonada, which 25% mortality was recorded (14). regarding an. gambiae from tanguieta site, a mortality of 78.9% was recorded after exposure to cdc bottles treated with bendiocarb showing an indication of resistance of this population to this insecticide (20). in this study, an. stephensi showed 57.3– 100% mortality (table 1). regarding anopheles gambiae s.l. population from the tanguieta, benin, resistant to bendiocarb was reported according to both who and cdc methods with mortality rates of 56.14% and 78.94% respectively (16). with who tube test, the exposure of an. stephensi to the bendiocarb 0.1% showed 13.3–97.8% in this study, and 71.4% on an. stephensi in other study carried out in the nikshahr, southeastern iran (23). due to the extensive use of pyrethroids both for indoor residual spraying and impregnating bed nets and some reports for resistance occurrence among the anopheles vectors (2324), the comparative study using who and cdc were carried out for revealing the resistance level in the world. this study revealed the susceptibility of an. stephensi to the deltamethrin (table 1, fig. 3). gorouhi et al. in 2015 indicated that field strain an. stephensi is the resistant candidate to deltamethrin (25). a bioassays conducted in north and south benin, 100% mortality of an. gambiae was also shown to deltamethrin with cdc assay at 12.5μg/bottle and 30min exposure time (21) as well as with deltamethrin 0.05% impregnated paper (22). the cdc insecticide susceptibility test was used on an. funestus and an. masca rensis in three districts including farafangana, fenerive est and vavatenina in the zambia and revealed the susceptibility of these species to the deltamethrin (13) and the high resistance of an. gambiae was revealed 0–90% mortality in nigeria (15). in other study in dangbo district, west africa, the resistance of an. gambiae to deltamethrin was indicated both with cdc and who bioassay methods respectively with 73.8% and 50.8% mortality (27). the increasing of kdr allelic frequency correlated with the cdc bioassay data on malanville and surelere population an. gambiae (27, 16) as well as the biochemical assays were implicated the mono-oxygenase enzymes as mechanisms of pyrethroid resistance in an. gambiae from misserete, west africa (28, 29). a diagnostic dose of 10μg a.i./bottle was identified as the most sensitive discriminating dose for characterizing resistance in an. darlingi and ae. aegypti and both the bottle assay and the who assay were equally able to differentiate deltamethrin-resistant and susceptible an. albimanus populations (30). there are some published studies on using cdc bottle assay for detecting pyrethroids resistance of malaria vectors with 3min exposure time including an. gambiae in madagascar with 99% mortality rate (13), an. gambiae in nigeria with 25% mortality (14, 15), an. gambiae in zambia with 90% (16) and on an. nuneztovari in columbia with 85% mortality (17). the current study emphasizes that the results of both who and cdc bioassays were similar. another recent study was also emphasizes that both who and cdc bioassays give similar results with regard to mosquito susceptibility to deltamethrin and bendiocarb insecticides (21). the efficacy wheaton coated bottle with deltamethrin could be used at least three times during four consecutive days in laboratory conditions (22). who method requires more mosquitoes j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 24 http://jad.tums.ac.ir published online: april 27, 2019 than cdc method, the comparison between the results of both methods is clear. when the who susceptibility kit is not readily available, bottle bioassays can be used to determine insecticide resistance status of mosquito populations. who bioassays utilize cylinder plastic tubes whereas cdc bottles bioassays use 250 ml wheaton bottles which are made of glass. world health organization (who) papers do not need to be treated by oneself before their utilization because they are ordered in the impregnated form. conversely, cdc bottles need to be coated with insecticide by oneself before each bioassay. in fact, the shelf life and reuse of prepared bottles are still not well documented or studied in laboratory conditions (21). however, in field conditions, the studies of perea et al. showed that bottles treated with 10μg ai deltamethrin per bottle could be stored for at least 14 days and reused on three occasions (27). the major advantages of the bottle assays are that any concentration of a custom insecticide (pure or formulated) may be evaluated and the technique is simple, rapid and economical. one of the disadvantages of who method is the transfer of mosquitoes to the tubes, which can damage the mosquitoes and cause disturbances in the test results so requires care during who susceptibility tests. this problem is partly resolved in the cdc method because cdc bottles bioassays do not need mosquitoes to be transferred from the exposure bottle. in who susceptibility tests mosquitoes must remain in recovery period (stable conditions of temperature and relative humidity) during the 24 hours after exposure to insecticide impregnated paper. the environmental conditions that mosquitoes have in recovery period can affect the test results, which is one of the disadvantages of this method. in cdc bottle bioassays method, this problem has been solved. the cdc bottles need to be clean, dry and coated with insecticide by oneself before each bioassay that takes a long time. if the bottles are contaminated before the coating, there is an error in the test results and this is one of the disadvantages of cdc bottle bioassays method. the assessment of the diagnostic dosage and time for each insecticide used against malaria vectors, in each region and for each of the main vector species is absolutely necessary. according to the cdc method, any concentration of any insecticide (pure or formulated) may be evaluated. who insecticide susceptibility test is the most common method for assessing resistance status in iran. in this study, for the first time in the country, cdc bottle bioassay method has been used to evaluate the level of mosquito susceptibility to the conventional insecticides, and additional supplementary studies are required. conclusion the current study emphasizes that the results of both who and cdc bioassays were similar with the studied insecticides with no significant difference in the related values. the cdc bottle bioassay may be applied as part of a broader insecticide resistance monitoring program especially combined with results of bioassays using synergists and those of biochemical and molecular assays. also the cdc bioassay have been used for determining of biochemical mechanisms that involve the detoxifying enzymes. acknowledgements the authors would like to appreciate the efforts of mrs rafi, staff of the culicidae insectary, sph, tums for their technical assistance in mass colonization of field collected an. stephensi. this study was funded and supported by tehran university of medical sciences (grant no. 9211263018). references 1. world health organization (2016) world j arthropod-borne dis, march 2019, 13(1): 17–26 h vatandoost et al.: comparison of cdc … 25 http://jad.tums.ac.ir published online: april 27, 2019 malaria report 2016. world health organization, geneva, switzerland, p. 136. 2. vatandoost h, mesdaghinia ar, zamani g, madjdzadeh r, holakouie k, sadrizadeh b, atta h, beales pf (2004) development of the regional malaria training centre in bandar abbas, islamic republic of iran. east mediterr health j. 10(1–2): 215–224. 3. soleimani-ahmadi m, vatandoost h, shaeghi m, raeisi a, abedi f, eshraghian mr, madani a, safari r, shahi m, mojahedi a, poorahmad-garbandi f (2012) vector ecology and susceptibility in a malaria endemic focus in southern islamic republic of iran. east mediterr health j. 18: 1034–1041. 4. vatandoost h, mashayekhi m, abai m, aflatoonian m, 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area, southeastern of iran. j arthropod borne dis. 10(4): 493–500. 26. aïzoun n, azondekon r, akogbéto m (2014) does the dynamic of insecticide resistance affect the results of susceptibility tests? int j curr microbiol. app sci. 3(10): 885–892. 27. aïzoun n, aïkpon r, akogbéto m (2014) evidence of increasing l1014f kdr mutation frequency in anopheles gambiae s.l. pyrethroid resistant following a nationwide distribution of llins by the beninese national malaria control programme. asian pac j trop biomed. 4(3): 239–243. 28. aïzoun n, ossè r, azondekon r, alia r, oussou o, gnanguenon v, aikpon r, padonou gg, akogbéto m (2013) comparison of the standard who susceptibility tests and the cdc bottle bioassay for the determination of insecticide susceptibility in malaria vectors and their correlation with biochemical and molecular biology assays in benin, west africa. parasit vectors. 6: 147. 29. aïzoun n, aïkpon r, padonou gg, oussou o, oké-agbo f, gnanguenon v, ossè r, akogbéto m (2013) mixed-function oxidases and esterases associated with permethrin, deltamethrin and bendiocarb resistance in anopheles gambiae s.l. in the south-north transect benin, west africa. parasit vectors. 6: 223. 30. perea ez, león rb, salcedo mp, brogdon wg, devine gj (2009) adaptation and evaluation of the bottle assay for monitoring insecticide resistance in disease vector mosquitoes in the peruvian amazon. malar j. 8: 208. microsoft word 1-dr. sallahodin .doc iranian j arthropod-borne dis, 2007, 1(2): 1-6 s sulaiman et al.: evaluation of pyrethrin … 1 original article evaluation of pyrethrin formulations on dengue/dengue haemorrhagic fever vectors in the laboratory and sublethal effects *s sulaiman, k fadhlina, o hidayatulfathi department of biomedical science, faculty of allied health sciences, university kebangsaan malaysia, kuala lumpur, malaysia (received 3 may 2007; accepted 2 oct 2007) abstract in southeast asia, aedes aegypti (l.) has been incriminated as principal vector of dengue viruses and ae. albopictus as the secondary vector of dengue fever. therefore, the aim of this study was to investigate the effectiveness of three formulations of pyrethrin derived from tanacetum cinerariaefolium against the dengue/dengue haemorrhagic fever vectors aedes aegypti and ae. albopictus in the laboratory. the testings employed 2 methodologies: the who larval bioassay and who adult bioassay. the results showed that all the three pyrethrin formulations had larvicidal and adulticidal activities. the impact of the sublethal doses of pyrethrin formulations on aedes spp. larvae resulted in 4-6% of alive adult emergence compared to 90% of ae. aegypti emerging adults and 96% ae. albopictus alive adult emergence in the control. the impact of sublethal doses of the pyrethrin formulations caused very low fecundity on both aedes spp. compared to the control (p< 0.05). keywords: pyrethrin, dengue vectors, sublethal effects introduction pyrethroids have been widely used for dengue vector control. lambda-cyhalothrin had been shown to be effective against dengue vectors under laboratory and field conditions in malaysia (lim and visalingam 1990, lim and lee 1991, sulaiman et al. 1991, 1993). alphacypermethrin had been shown to be effective against dengue vectors in the field in malaysia, demonstrating both adulticidal and larvicidal effects (sulaiman et al. 1995). pyrethrin derived from pyrethrum daisy tanacetum cinerariaefolium is a highly effective insecticide for controlling insect pests. pyrethrum had shown repellent activity against mansonia mosquitoes (hadis et al. 2003), sampling indoor resting african malaria vectors is done traditionally by hand catches with oral or mechanical aspirators and pyrethrum catches (harbison et al. 2006) and entomological evaluation of malaria vectors at different altitudes (kulkarni et al. 2006). pyrethrum is being used for impregnating of bednets and curtains, made of polypropylene fibre (curtis et al. 1992). the objective of this study was to evaluate the efficacy of various pyrethrin formulations on the larval and adult stages of the dengue vectors, and the effect of sublethal dose on the larval stage of the dengue vectors on adult emergence and fecundity in the laboratory. materials and methods the pyrethrin formulations used were pyrethrin 50%, pyrethrin 0.4g/l+ pbo 1.5g/l, pyrethrin 44g/l+ pbo 160g/l and insecticides abate® and malathion. the pyrethrin formulations were sup*corresponding author: prof dr s sulaiman, tel: +03 40405416, fax: +603 26929032, e-mail: salsul@medic.ukm.my iranian j arthropod-borne dis, 2007, 1(2): 1-6 s sulaiman et al.: evaluation of pyrethrin … 2 plied by botanical resources australia pty ltd, tasmania, australia. larval bioassay the larval bioassay was conducted on aedes aegypti and aedes albopictus, based on who instruction (1981a). the experiment was conducted at 25± 1 ºc and relative humidity 80± 5%. twenty five ae. aegypti and ae. albopictus 4th instar larvae were exposed to 250 ml of prepared pyrethrin concentrations and abate® in 600 ml beakers. four replicates were conducted and mortality was recorded after 24 h. the lc50 and lc90 levels, regression slopes and associated 95% fiducial limits were determined by computer using probit analysis (raymond 1985). adult bioassay the adult bioassay was conducted on ae. aegypti and ae. albopictus using who test kit (1981b). fifteen 2-5 day old female mosquitoes were exposed for 1h to the filter papers in the who test kit, impregnated with various concentrations of pyrethrin formulations and malathion as positive control, then transferred to holding tubes. sugar solution soaked in cotton was provided as food. four replicates were conducted and the number of adult mortality was recorded after 24 h. lc50 and lc90 levels, regression slopes and associated 95% fiducial limits were determined by probit analysis (raymond 1985) studies on sublethal effects were based on loh and yap study (1989) with some modifications. the concentration used was the lc50 of each pyrethrin formulation from the larval bioassay. fifty 4th instar larvae of ae. aegypti and ae. albopictus were used for each sublethal dose of pyrethrin formulation. mortality was recorded after 24 h exposure and the surviving larvae were transferred into beakers containing distilled water. the number of surviving larvae, pupae and emerging adults was recorded. the emerging adults were blood fed and allowed to oviposit and the eggs were counted. the eggs were allowed to hatch into larvae, pupae and adults and recorded. the control includes non insecticide treated mosquitoes. data were analyzed using mann-whitney u test. results table 1 showed that both ae. aegypti and ae. albopictus 4th instar larvae were more susceptible to pyrethrin 44g/l+ pbo 160g/l than pyrethrin 0.4g/l + pbo 1.5g/l or pyrethrin 50%. the pyrethrin 44g/l+ pbo 160g/l lc50 and lc90 for ae. aegypti 4th instar larvae were 0.002 ppm and 0.007 ppm. the lc50 and lc90 for ae. albopictus 4th instar larvae were 0.004 ppm and 0.012 ppm, respectively. the lc50 and lc90 values for pyrethrin 50% were higher than lc50 and lc90 for pyrethrin 0.4g/l+ pbo 1. 5g/l for ae. aegypti 4th instar larvae (0.038 ppm and 0.135 ppm:0.028 ppm and 0.080 ppm). similarly, the lc50 and lc90 for pyrethrin 50% were higher than lc50 and lc90 for pyrethrin 0.4g/l+ pbo 1.5g/l for ae. albopictus 4th instar larvae (0.069 and 0.336: 0.056 and 0.166), respectively. using mann-whitney u test values of lc50 and lc90 for each insecticide tested against 4th instar larvae of both aedes spp. showed significant difference (p< 0.05). however, abate® showed a much lower susceptibility to both aedes spp. 4th instar larvae compared to the three pyrethrin formulations. although the pyrethrin formulations have larvicidal effects against both aedes spp. but abate still remains the insecticide of choice as larvicide. iranian j arthropod-borne dis, 2007, 1(2): 1-6 s sulaiman et al.: evaluation of pyrethrin … 3 table 1. mortality response of pyrethrin formulations to aedes aegypti and aedes albopictus 4th-instar larvae in the laboratory aedes aegypti aedes albopictus insecticides lc50 (95%ci) (ppm) lc90 (95%ci) (ppm) slope±se lc50 (95%ci) (ppm) lc90 (95%ci) (ppm) slope ± se pyrethrin 50% 0.038 (0.029-0.052) 0.135 (0.090-0.280) 2.334±0.371 0.069 (0.039-0.083) 0.336 (0.268-0.487) 2.276±0.674 pyrethrin 0.4g/l + pbo 1.5g/l 0.028 (0.022-0.036) 0.080 (0.058-0.131) 2.842±0.413 0.056 (0.044-0.071) 0.166 (0.120-0.281) 3.226±0.464 pyrethrin 44g/l+pbo 160g/l 0.002 (0.002-0.003) 0.007 (0.005-0.011) 2.973±0.428 0.004 (0.003-0.006) 0.012 (0.008-0.021) 2.974±0.603 abate® (control) 0.00003 (0.00001-0.00007) 0.0016 (0.00045-0.01382) 0.909±0.136 0.00001 (0.0000-0.00002) 0.004 (0.0006-0.1409) 0.594±0.112 table 2 indicates that both ae. aegypti and ae. albopictus adults are more susceptible to pyrethrin 44 g/l + pbo 160 g/l than pyrethrin 0.4 g/l + pbo 1.5 g/l and pyrethrin 50%. the lc50 and lc90 of pyrethrin 44 g/l + pbo 160 g/l for ae. aegypti were 0.209 µg/cm² and 0.469 µg/cm². the lc50 and lc90 for ae. albopictus were 0.197 µg/cm² and 0.492 µg/cm², respectively. the lc50 and lc90 for pyrethrin 50% were higher than that of pyrethrin 0.4g/l + pbo 1.5g/l for ae. albopictus adults. however, the lc50 for pyrethrin 50% was higher than that of pyrethrin 0.4g/l + pbo 1.5g/l for ae. aegypti but the regression slope of pyrethrin 0.4g/l + pbo 1. 5g/l indicated that a slight increase in its concentration would cause a higher mortality of ae. aegypti adults, compared to pyrethrin 50%. using mann whitney u test the value of lc90 for each insecticide formulation tested against adults of both aedes spp. were significantly different (p< 0.05). however, malathion showed a much lower susceptibility values to both ae. aegypti and ae. albopictus adults, compared to the three pyrethrin formulations. table 2. mortality response of pyrethrin formulations to aedes aegypti and aedes albopictus adults in the laboratory aedes aegypti aedes albopictus insecticides lc50 (95%ci) (µg/cm²) lc90 (95%ci) (µg/cm²) slope ± se lc50 (95%ci) (µg/cm²) lc90 (95%ci) (µg/cm² ) slope ± se pyrethrin 50% 1.331 (0.901-2.128) 3.982 (2.080-4.013) 2.745±0.604 1.980 (1.007-2.202) 5.244 (4.896-5.993) 3.036±0.573 pyrethrin 0.4g/l + pbo 1.5g/l 1.701 (0.967-1.996) 3.743 (2.765-4. 010) 3.636±0.699 1.671 (1.010-2.100) 4.721 (3.787-5.220) 3.104±0.564 pyrethrin 44g/l+pbo 160g/l 0.209 (0.160-0.277) 0.469 (0.339-0.875) 3.713±0.753 0.197 (0.091-0.298) 0.492 (0.287-0.992) 3.227±0.622 malathion (control) 0.00006 (0.00003-0.00007) 0.00050 (0.00048-0.00098) 1.303±0.266 0.0008 (0.0006-0.0013) 0.0071 (0.0058-0.0071) 1.364±0.249 iranian j arthropod-borne dis, 2007, 1(2): 1-6 s sulaiman et al.: evaluation of pyrethrin … 4 table 3 shows the impact of aedes spp. larvae which was treated with sublethal doses (ld50) of pyrethrin formulations. all the three pyrethrin formulations resulted in 4-6% of emerging adults of ae. aegypti and ae. albopictus alive, compared to 90% of ae. aegypti adults and 96% of ae. albopictus alive adults in the control, respectively. thus, all the pyrethrin formulations at ld50 when treated on both aedes spp. larvae caused low production of emerging adults. table 3. impact of treatment of mosquito larvae with sublethal doses of pyrethrin formulations pyrethrin 50% pyrethrin 0.4g/l + pbo 1. 5g/l pyrethrin 44g/l + pbo 160g/l control ae. aegypti ae. albopictus ae. aegypti ae. albopictus ae. aegypti ae. albopictus ae. aegypti ae. albopictus no. of larvae tested 50 50 50 50 50 50 50 50 mortality of larvae after 24 hr (%) 44 48 50 52 52 48 2 2 mortality of pupae (%) 2 0 8 4 10 12 0 0 mortality of emerging adults (%) 6 8 16 16 20 14 0 0 emerging adults alive (%) 6 6 4 4 4 4 90 96 table 4 indicated that when aedes spp. larvae were treated with sublethal doses of pyrethrin formulations, the fecundity of subsequent adults produced very low number of eggs for both aedes spp. in the range of 0-4 eggs, compared to 198-227 eggs produced in the control. none of the eggs derived from the treated larvae hatched. however, 99% of the control eggs of both aedes spp. became adults. it was observed that the female mosquitoes derived from different formulations of pyrethrin treated larvae were not feeding well on argus spp. this would affect the number of eggs laid. table 4. impact of mosquito fecundity after the larval exposure to sublethal doses of pyrethrin formulations pyrethrin 50% pyrethrin 0.4 g/l + pbo 1. 5 g/l pyrethrin 44 g/l + pbo 160 g/l control ae. aegypti ae. albopictus ae. aegypti ae. albopictus ae. aegypti ae. albopictus ae. aegypti ae. albopictus no. of eggs produced 2 4 0 2 0 3 1189 2270 % of eggs hatched 0 0 0 0 0 0 99 99 % of larvae becoming pupae 0 0 0 0 0 0 99 99 % of pupae becoming adults 0 0 0 0 0 0 99 99 discussion according to rayman (2006), a 2% pyrethrum solution, a naturally occurring pyrethroids found in chrysanthemum spp. flowers is the recommended agent for aircraft disinsection because they are extremely effective insecticide and pose minimal health risks. according to gerry et al. (2005) the aerial application of ulv pyrethrin insecticide for control of adult mosquitoes did not result in undue exposure to the pilot. jensen et al. (1999) conducted ulv application of pyrethrin, malathion and permethrin on non-target invertebrates, sentinel mosquitoes iranian j arthropod-borne dis, 2007, 1(2): 1-6 s sulaiman et al.: evaluation of pyrethrin … 2 and mosquitofish in california seasonal wetlands, usa. all the insecticides tested including pyrethrin were able to control adult mosquitoes without substantial effects on the aquatic insects or fish in the seasonal wetlands. victor et al. (2002) in their application of temephos and fogging with pyrethrum 2% extract in villages at tamil nadu, india, were effective against immatures and adults aedes aegypti. thus, the pyrethrin formulations can be widely used in controlling dengue vectors. mohapatra et al. (1999) evaluated cyfluthrin and fenfluthrin on their activity against different developmental stages of three vector species viz., anopheles stephensi, ae. aegypti and culex quinquefasciatus. both compounds were more active against the fourth larval instars of all mosquito species tested, cyfluthrin in culicines and fenfluthrin in anophelines brought about maximum inhibition of adult emergence. mohapatra et al. (1999) also found that pyrethroids cyfluthrin and fenfluthrin significantly reduced the fertility rates (p< 0.001) of anopheles stephensi, aedes aegypti, and culex quinquefasciatus. loh and yap (1989) who studied the efficacy and sublethal effects of pyriproxyfen on ae. aegypti, found that the eggs hatchability was reduced by 36.8%. according to xue et al. (2005) application of deet forced egg retention time reduced the number of eggs laid per female aedes albopictus. the rate of egg hatched was considerably reduced after three weeks of retention. the fecundity and fertility of gravid female ae. albopictus were affected by the time duration of forced egg-retention. ali et al. (2006) found that fecundity and fertility based on number of laid eggs per female and percentage of egg hatch in stegomyia albopicta when exposed to 0. 1% boric acid sugar bait were significantly reduced and ovarian development retarded. focks et al. (1991) studied aedes aegypti which were fed on rabbits subcutaneously injected with ivermectin exhibited reduced survival and egg production compared to females fed on the control rabbits. eggs from these rabbits were also less likely to hatch and subsequent larval survival was lower than the controls. similarly, our studies also indicated that pyrethrin formulations affected the produced eggs and then a few produced eggs could not hatch into larvae. to conclude, all the pyrethrin formulations had larvicidal and adulticidal effects on aedes aegypti and aedes albopictus. furthermore, treatment at sublethal doses at the larval stage produced very low emerging adults and fecundity. acknowledgements we wish to thank faculty of allied health sciences, university kebangsaan malaysia for providing research facilities and botanical resources australia pty ltd for providing the pyrethrin formulations. references ali a, xue rd, barnard dr (2006) effects of sublethal exposure to boric acid sugar bait on adult survival, host-seeking bloodfeeding behavior, and reproduction of stegomyia albopicta. j am mosq control assoc. 22: 464-468. curtis cf, myamba j, wilkes tj (1992) various pyrethroids on bednets and curtains. mem inst oswaldo cruz. 87 suppl. 3: 363-370. focks da, mc laughlin re, linda sb (1991) effects of ivermectin (mk-933) on the reproductive rate of aedes aegypti (diptera: culicidae). j med entomol. 28: 501-505. gerry ac, zhang x, leng g, inman ad, krieger ri (2005) low pilot exposure to pyrethrin during ultra-low-volume (ulv) aerial insecticide application for control of adult mosquitoes. j am mosq control assoc. 21: 291-295. hadis m, lulu m, mekonnen y, asfaw t (2003) field trials on the repellent activity of four plant products against mainly mansonia population in western ethiopia. phytother res. 17: 02-205. 5 iranian j arthropod-borne dis, 2007, 1(2): 1-6 s sulaiman et al.: evaluation of pyrethrin … 3 harbison je, mathenge em, misiani go, mukabana wr, day jf (2006) a single method for sampling indoor-resting malaria mosquitoes anopheles gambiae and anopheles funestus (diptera: culicidae) in africa. j med entomol. 43: 473-479. jensen t, lawler sp, dritz da (1999) effects of ultra-low-volume by pyrethrin, malathion, and permethrin on nontarget invertebrates, sentinel mosquitoes, and mosquitofish in seasonally impounded wetlands. j am mosq control assoc. 15: 330-338. kulkarni ma, kweka e, nyale e, lyatuu e, mosha fw, chandramohan d, rau me, drakeley c (2006) entomological evaluation of malaria vectors at different altitudes in hai district, northeastern tanzania. j med entomol. 43: 580-588. lim jl, lee kf (1991) efficacy and relative potency of lambda-cyhalothrin and cypermethrin applied as a group-based ulv aerosol for the control of houseflies and mosquitoes. trop biomed. 8: 157-162. lim jl, visalingam m (1990) relative potency of lambda-cyhalothrin and cypermethrin applied as thermal fogs for the control of houseflies (musca domestica) and mosquitos (aedes aegypti). southeast asian j trop med public hlth. 21: 77-84. loh py, yap hh (1989) laboratory studies on the efficacy and sublethal effects of an insect growth regulator, pyriproxyfen (s-31183) against aedes aegypti (l.). trop biomed. 6: 7-12. mohapatra r, ranjit mr, dash ap (1999) evaluation of cyfluthrin and fenfluthrin for their insecticidal activity against three vector mosquitoes. j comm dis. 31: 91-99. rayman rb (2006) aircraft disinsection. aviat space environ med. 77: 733-736. raymond m (1985) log probit analysis basic programme of microcomputer. cha orstm series: med entomol parasit. 22: 117-121. sulaiman s, omar b, jeffery j, busparani v (1991) evaluation of pyrethroids lambdacyhalothrin, deltamethrin and permethrin against aedes albopictus in the laboratory. j am mosq control assoc. 7: 322-323. sulaiman s, karim ma, omar b, jeffery j, mansor af (1993) the residual effects of the synthetic pyrethroids lambda-cyhalothrin and cyfluthrin against aedes aegypti (l.) in wooden huts in malaysia. mosq -borne dis bull. 10: 128-131. sulaiman s, karim ma, omar b, omar s (1995) field evaluation of alphacypermethrin and lambda-cyhalothrin against aedes aegypti and aedes albopictus in malaysia. j am mosq control assoc. 11: 54-58. victor tj, malathi m, gurusamy d, desai a, ravi v, narayanasamy g, anuradha l, rani c, krishnamurthy p (2002) dengue fever outbreaks in two villages of dharmapuri district in tamil nadu. indian j med res. 116: 133-139. world health organization (1981a) instructions for determining the susceptibility or resistance of mosquito larvae to insecticide. who/vbc/81. 807 world health organization (1981b) instructions for determining the susceptibility or resistance of adult mosquitoes to organochlorine, organophosphate, and carbamate insecticides. establishment of base-line. who/vbc/ 81. 805 xue rd, ali a, barnard dr (2005) effects of forced egg-retention in aedes albopictus on adult survival and reproduction following application of deet as an oviposition deterrent. j vect ecol. 30: 45-48. 6 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 49 original article the fauna and ecology of mosquitoes (diptera: culicidae) in western iran *seyed hassan moosa-kazemi 1, amir hosein zahirnia 2, farid sharifi 1, behroz davari 2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran 2department of medical entomology and vector control, school of medicine, hamadan university of medical sciences, iran (received 30 sep 2013; accepted 21 apr 2014) abstract background: the aim of this study was to obtain new data which would be valuable to develop programs for future planning of mosquito controls in in western iran. methods: larvae and adult collections were carried out from different habitats using standard dipping and animal baited trap methods during may, june and july 2012 in two provinces (kurdistan and kermanshah) in the west of iran. characteristics of breeding places were studied based on the habitat type (river edge, ground pool), water conditions (clear or turbid, stagnant or running), vegetation (with or without vegetation), water temperature, sunlight exposure (full or partial sunlight) and so on. results: overall, 4081 thirdand fourth-instars larvae and 2013 adult were collected. five genera and eleven species of the family culicidae were identified. mosquitoes collected in larval and adult stages including, anopheles maculipennis s.l. (4.1%, 4.42%), an. superpictus (1.35%,1.39%), an. turkhudi (1.75%,1.68%), aedes vexans (2.5%, 8.78%), culex hortensis (1.59%, 1.04%), cx. mimeticus (5%, 2.38%), cx. pipiens (16.5%, 8.15%), cx. theileri (10%, 46.4%), culiseta longiareolata (24%, 4.27%), and cs. subochrea (24%, 4.27%) and ochlerotatus caspius s.l. (9.1%, 21.46%). culiseta longiareolata and cs. subochrea found predominant species in larval collection, whereas culex theileri was dominant in adult collection. anopheles turkhudi is reported for the first time in kermanshah province. conclusion: due to the geographical location of the two provinces, extensively studies with emphasis on mosquito ecology, to having comprehensive and up to date information is essential. keywords: culicidae, larval habitat, fauna, iran introduction the family culicidae (diptera) comprises at least 3531 species representing 112 genera divided into two subfamilies, culicinae and anophelinae (harbach 2013). according to the newest checklist of iranian mosquitoes, 64 species representing seven genera occur in the country (azari-hamidian 2007). oshaghi et al. (2008) recently identified anopheles superpictus grassi as a complex of three genotypes (x, y, and z). naddaf et al. (2010, 2012) reported an. fluviatilis james species u from fars province. besides, mehravaran et al. (2011) recorded the species u in southeastern iran. mosquito-borne diseases such as malaria, west nile, as well as dirofilariasis are a major public health threat in iran (naficy and saidi 1970, azari-hamidian et al. 2007, ahmadnejad et al. 2011). malaria cases in kurdistan province, western iran, increased from 1994 to 1996 (543, 633, and 236, respectively), however, they dramatically decreased after that. there were no cases of malaria in 2009, and just 2 and 3 cases in 2010 and 2011, respectively. all were imported from outside of the province (banafshi et al. 2013). there are only three recent studies on mosquitoes in kurdistan province, including *corresponding author: dr seyed hassan moosakazemi, e-mail: moosakazemi@tums.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 50 the faunistic investigation of anophelines by vahabi et al. (2001) and culicines in sanandaj county by moosa-kazemi et al. (2010). banafshi et al. (2013) studied fauna and ecology of mosquito larvae in the province. by now, 6 genera and 18 species of mosquitoes have been recorded in kurdistan province (macan 1950, lotfi 1973, zaim 1987, vahabi 2001, moosa kazemi et al. 2010, banafshi et al. 2013). seven species of anopheles have been found in the province (macan 1950, vahabi 2001) including an. algeriensis theobald, an. claviger (meigen), an. maculipennis s.l., an. marteri senevet and prunnelle, an. sacharovi favre, an. sergentii (theobald), and an. superpictus grassi. of the maculipennis group, an. maculipennis s.l. and an. sacharovi have been recorded in the province based on morphological characters. zaim (1987) mentioned 6 species of culex linnaeus and 2 species of culiseta genus in the province: culex hortensis ficalbi, cx. mimeticus noe, cx. perexiguus theobald, cx. theileri theobald, cx. pipiens linnaeus, cx. territans walker, culiseta longiareolata (macquart), and cs. subochrea (edwards). moosa-kazemi et al. (2010) recently reported aedes vexans (meigen) and ochlerotatus caspius (pallas) s.l. in kurdistan province for the first time. zaim and cranston (1986) noted that a female specimen from koolan of marivan, kurdistan province, which had been previously identified by lotfi (1973) as cx. impudicus ficalbi, was possibly a new species of coquillettidia dyar. there is little and scattered information about the fauna of culicidae in kermanshah, including at least 4 genera and 22 species (macan 1950, manouchehri et al. 1976, zaim 1987, harbach 1988). eleven species of anopheles have been reported in the province including an. algeriensis, an. claviger, an. dthali patton, an. fluviatilis, an. marteri, an. maculipennis s.l., an. pulcherrimus theobald, an. turkhudi liston, an. superpictus, an. sacharovi, an. stephensi liston (macan 1950, manouchehri et al. 1976, unpublished data from institute of malariology, tehran university of medical sciences). certainly, kermanshah province has not been recognized as an endemic region for malaria at least in 4 past decades, but some sporadic cases were reported in several regions (vejdani 2012). this paper presents the results of recent surveys of culicidae mosquitoes from samples taken from a range of larval breeding sites in which they occur in nature, and adult mosquitoes captured in residential and sparsely populated areas by animal bait traps collection in the two provinces. the aim was to obtain new data which would be valuable to develop programs for future planning of mosquito control in this area of western iran. materials and methods to better understand the culicidae fauna of kurdistan and kermanshah provinces and gather information on their distribution and the kinds of larval breeding sites in which they occur in nature, a descriptive cross-sectional study was performed from may, june and july 2012 in western iran. this study took place in three randomly selected rural villages in kermanshah province (34˚31’ 76’’n 47˚08’69’’e) and three villages in kurdistan province (35˚.31’13’’n 46˚99’ 60’’e). the district is bounded by the iraq in the west, hamadan and lorestan provinces in the east, ilam province in the south, and western azerbaijan and zanjan provinces in the north (fig. 1). in 2011 the maximum and minimum mean monthly temperatures in kurdistan were 30 ˚c and -1 ˚c in july and february, respectively. the total annual rainfall was 800 mm, the minimum of 3.9 mm in april and maximum of 126.2 mm in november. the mean annual relative humidity was 39%. in the same time the maximum and minimum mean monthly temperatures in kermanshah were 28.7 ˚c and 6.9 ˚c in august http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 51 and december, respectively. the total annual rainfall was 406.4 mm, the minimum of 0.1mm in july and maximum of 155.7 mm in november. the mean annual relative humidity was 41%. the main economic activity of the people is agriculture and husbandry of cow and goats. three villages in the different topographical areas, including: hassan-abad-e-sofla, hassan-abad-e-olia, and hossain-abad in kurdistan province and cham-e-seyed sadegh, kaleh ghandi and saleh abad in kermanshah province were selected. to study the mosquito fauna, sampling was carried out using dipping method for collecting larvae and animal baited traps using suction tubes for adult mosquitoes. female mosquitoes were collected primarily through the use of suitable bait (cow) by suction tube, between 20.00 and 03.00 h monthly in fixed animal shelter randomly on each village. before catches, all cows were fixed by tight were closed the shelter. the collected mosquitoes were transferred to cups container with moist cotton. all collected mosquitoes were pinned and then identified using the keys of azari-hamidian and harbach (2009). mosquito name abbreviations are cited based on reinert (2009). larval habitats present within 500 m radius of each village were identified, and sampled for mosquito larvae two per month. three randomly selected breeding places located within a 500 m radius of the study site were also included and formed the sampling unit for mosquito larvae. all larvae were collected from natural habitats, generally feeding with seepage water, such as swamps, seepages, streams, river banks, drying river beds, pools, and grasslands. up to 20 dipper samples were taken at intervals along the edge of each larval breeding place using a standard mosquito dipper (350 ml) depending on the size of the habitat. the larvae for each habitat were placed separately in whirl packs and transported to the laboratory where they were sorted by genera and instars counted and recorded. all third and fourth instars of mosquitoes were preserved in lactophenol solution and later identified morphologically (shahgudian 1960, azari-hamidian and harbach 2009). in order to determine the association of mosquito larvae, samples for each habitat type were pooled together in each site irrespective of date of collection. physical characteristics of the larval habitats including distance to the nearest homestead, habitat stability, water depth, turbidity, presence of other aquatic invertebrates, and presence of floating, emergent, and submerged vegetation were recorded. water depth was measured using a metal ruler. the habitat was recorded as unstable if it did not contain water during the next weekly sampling visit. the culicinae larvae along with the description of their breeding sites were sent to the school of public health, tehran university of medical sciences, tehran, iran. results a total of 2013 adult and 4081 larvae of mosquitoes were collected, in which 5 genera and 11 species were recognized including, aedes vexans, ochlerotatus caspius s.l., anopheles maculipennis s.l. an. superpictus, an. turkhudi, culex hortensis, cx. pipiens, cx. mimeticus, cx. theileri, cs. longiareolata, and cs. subochrea. results of adult and larvae mosquitoes collected presented in table 1 and 2. these species were collected from the plain, slope and mountainous areas of the kurdistan and kermanshah provinces on 6 six selected villages. an. turkhudi is recorded for the first time in kermanshah province. culiseta longiarolata collected only in the adult stage in hossain abad of kurdistan province. the number and location of the mosquito larvae at the ecological station of this area is shown in table 1. in the larval collection, 3913 culicidae larvae were collected, both http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 52 cs. longiareolata (25.9%), and cs. subochrea (25.9%) predominated, followed by cx. pipiens (17.56%), cx. theileri (10.68%), ochlerotatus caspius s.l. (8.35%), cx. mimeticus (5.26%), aedes vexans (3.066%), an. turkhudi (1.84%), an. superpictus (1.43 %), and cx. hortensis (1.66%) (table 1). number and prevalence of mosquitoes catches by animal bait trap through the night, are shown in table 2. overall, culex theileri was found predominant species (46.4 %), followed by ochlerotatus caspius s.l. (21.46%), aedes vexans (8.78%), cx. pipiens (8.14%), an. maculipennis s.l. (4.42%), cs. subochrea (4.27%), cx. mimeticus (2.38%), an. turkhudi (1.68%), an. superpictus (1.39 %), cx. hortensis (1.04%), and cs. longiareolata (0.04%). two species including culex theileri, and oc. caspius s.l., with 46.4% and 21.46% respectively formed about 68% of the total adult collections. the other species were less frequent and in total accounting for 32% of the collections (table 2). culex theileri was the most frequent mosquitoes collected in this area, with total of 934, and 418 specimens, by animal bait trap and larval collection respectively. feeding activity of oc. caspius s.l. was increased in the first third of the night and decrease slowly in the second and the end of the third night whereas blood feeding activity of cs. subochrea was increased in the second and end third of night and then decreased in first third of the night. culex theileri was active thought the night and others mosquitoes species such as cx. pipiens, cx. hortensis, cx. mimeticus, and ae. vexans were active in the second third of the night. blood feeding activity of an. superpictus and an. turkhudi was increased in the second and end third of night and then decreased in first third of the night. two species including culex theileri, and oc. caspius s.l. accounted for 82.02% of the adult collection after the sun set in the animal bait trap collection whereas in the second third of the night these species with cx. pipiense were predominant and accounted for 76.22% of the adult collection. in the end of night culex theileri, oc. caspius s.l., and ae. vexans were predominant and about 85.36% of the total mosquito catches. the larval habitat characteristics and occurrence percentages of different mosquito larvae and their association are shown in table 3 and table 4. anopheles maculipennis s.l. was collected from 45 larval breeding sites. this species was found mostly in transient, stagnant, clear water in natural habitats without vegetation, partial sunlight. natural habitats without vegetation transient, stagnant, clear water in, partial sunlight were found for the species of an. superpictus. anopheles turkhudi were collected from 4 larval breeding sites. this species was found most in transient, stagnant, clear water in natural habitats without vegetation, partial sunlight. natural habitats without vegetation, permanent, slow ranning, turbid water in, full sunlight were found for the species of ae. vexans. culiseta longiareolata was collected from 14 larval breeding sites. this species was found most in transient, stagnant, clear water in natural habitats without vegetation, partial sunlight. natural habitats without vegetation, permanent, slow running, turbid water in, full sunlight were found for the species of cs. subochrea. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 53 table 1. composition and localities of the larvae of mosquitoes collected in the west of iran, may–july 2012 species kurdistan selected villages kermanshah selected villages n % hassanabad-esofla, hassanabad-eolia, hossainabad chame-seyed sadegh, kaleh ghandi saleh abad an. maculipennis s.l. 9 12 14 19 101 13 168 4.1 an. superpictus 10 14 19 13 56 1.35 aَn. turkhudi 18 51 3 72 1.75 ae. vexans 9 98 4 9 120 2.5 cx hortensis 12 14 19 20 65 1.59 cx. mimeticus 8 12 114 19 53 206 5 cx. pipiens 12 100 119 156 229 69 685 16.5 cx. theileri 114 48 124 26 101 5 418 10 cs. longiareolata 221 146 231 58 223 103 982 24 cs. subochrea 265 230 198 289 982 24 oc. caspius s.l. 89 12 98 19 101 8 327 9.1 total 453 626 728 662 1047 565 4081 100 fig. 1. map of iran indicating the location of the study area in kurdistan and kermanshah provinces situated in western iran 1= hossain-abad 2= hassan-abad-e-olia 3=hassan-abad-esofla 4= cham-eseyed sadegh 5=kaleh ghandi 6= saleh abad http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 54 table 2. number and prevalence of adult culicidae mosquitoes catches by animal baited trap in the west of iran, may–july 2012 species kurdiatan selected villages kermanshah selected villages n % hassanabad-esofla, hassanabad-eolia, hossainabad cham-eseyed sadegh, kaleh ghandi saleh abad an. maculipennis s.l. 3 14 18 6 39 9 89 4.42 an. superpictus 2 6 5 8 2 5 28 1.39 aَn. turkhudi 18 13 3 34 1.68 ae. vexans 13 26 98 20 19 176 8.78 cx hortensis 3 5 2 6 5 21 1.04 cx. mimeticus 8 10 9 9 12 48 2.38 cx. pipiens 26 26 19 67 19 7 164 8.147 cx. theileri 201 262 199 133 89 50 934 46.4 cs. longiareolata 1 1 0.04 cs. subochrea 7 29 31 19 86 4.27 oc. caspius s.l. 89 17 98 19 101 108 432 21.46 total 329 358 380 380 329 237 2013 100 table 3. the larval habitat characteristics and occurrence percentages of different mosquito larvae in the west of iran, may–july 2012 o c. c as pi u s s. l. (% ) c s .s u bo ch re a (% ) c s. l on gi ar eo la ta (% ) c x. th ei le ri (% ) c x. pi pi en s (% ) c x. m im et ic u s (% ) c x h or te n si s (% ) a e. v ex an s (% ) aَ n . t u rk h u di (% ) a n . s u pe rp ic tu s (% ) a n . m ac u li pe n n is s. l. (% )larval breeding site: characteristics and habitats a) habitat type 1001009958.674.59954.699.476.489.640.5permanent 00141.425.5145.40.623.610.459.5transient b) water conditions 9995.69892.663.440.599.80.695.295.795.5stagnant water 14.427.436.659.50.299.44.84.34.5slow-running water c) water situation 9887.69795.5989895.50.881.784.299.5clear 212.434.5224.599.218.315.80.5turbid d) vegetation situation 1009910043.667.281.53.50.939.644.839.4with vegetation 01056.422.818.596.799.159.456.251.6without vegetation e) sunlight exposure 0118.664.665.56994.53.96.36.4full sunlight 100999981.433.434.5315.596.193.793.6partial sunlight f) habitat type 100998960.5739871.389659982.4natural 011139.527228.71135117.6artificial http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 55 table 4. the association of different mosquito larvae with other species in west of iran, may–july 2012 o c. c as pi u s s. l. (% ) c s. s u bo ch re a (% ) c s. l on gi ar eo la ta (% ) c x. th ei le ri (% ) c x. pi pi en s (% ) c x. m im et ic u s (% ) c x h or te n si s (% ) a e. v ex an s( % ) a n . t u rk h u di (% ) a n . s u pe rp ic tu s (% ) a n . m ac u li pe n n is s. l. (% ) t ot al o cc as io n s larval breeding site: characteristics and habitats 2128148304445an. maculipennis s.l. (%) 41121221146an. superpictus (%) 121011001413an. turkhudi (%) 21101210007ae. vexans (%) 013221102312cx hortensis (%) 211151212822cx. mimeticus (%) 2124521111432cx. pipiens (%) 001412002823cx. theileri (%) 301213111214cs. longiareolata (%) 10011122116cs. subochrea (%) 130220114218oc. caspius s.l. (%) discussion by now 7 species of anopheles have been recorded in kurdistan province and 11 species in kermanshah province (macan 1950, vahabi 2001, manouchehri et al. 1976, moosa-kazemi et al 2010, banafshi et al 2013). in addition two species including an. stephensi and an. pulcherrimus has been reported in kermanshah province in past decades (manouchehri et al. 1976). three anopheles species were collected in this investigation, including an. maculipennis s.l., an. superpictus, and an. turkhudi. the species of an. algeriensis, an. marteri, an. sacharovi, and an. sergentii were not found. in the previous study, vahabi (2001) found only one adult an. marteri in the province, and an. algeriensis and an. sergentii were not common species. he also did not distinguish the maculipennis group specimens in larval stage and identified the adults of an.maculipennis s.l. and an. sacharovi using morphological characters. an. maculipennis reported as the complex species and molecular study was carried out to determine the species complex in the northern areas of the country (sedaghat et al. 2003). anopheles turkudi is reported in kermanshah province for the first time, this species found in 15 provinces in central plateau, western, southwestern, eastern, southeastern, and southern iran (saghafipour et al. 2012). however, the occurrence of an. turkhudi in the islam abad-e-gharb, kermanshah province was mentioned only in the unpublished documents of the school of public health and the institute of public health research (saebi 1987, unpublished data). in our study only an. maculipennis s.l of the maculipennis group was identified using the mean number of seta 2 branches of the fourthand fifth-abdominal segments of larvae, which is 36.8 in an. sacharovi whereas it is 16.5 in an. maculipennis s.l. (azarihamidian and harbach 2009). in our study, also an. superpictus found 1.35% and 1.39% of larval and adult collection respectively. vahabi (2001) also reported anopheles superpictus (49.8%) and the an. maculipennis http://jad.tums.ac.ir published online: july 15, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 56 group (40.5%) as the most abundant anopheline species in kurdistan province. in parallel, mousakazemi et al. (2000) reported the an. maculipennis s.l. (including 2.08% of the whole samples) and an. superpictus (0.16%) from rice fields in lenjan and mobarakeh areas (isfahan province, central iran). abai et al. (2007) found an. maculipennis s.l. one of the most abundant species in east azerbaijan province, northwestern iran. azarihamidian et al. (2009) found the species of an. maculipennis s.l. (19.0%) in ardebil province, northwestern iran. out of 11 species of culicine mosquitoes, which had been recorded previously in kurdistan province (zaim. 1987, vahabi 2001, moosa-kazemi et al. 2010, banafshi et al. 2013) 8 species were collected in this investigation. culex theileri, oc. caspius s.l., and ae. vexans were the most prevalent species respectively, and collected in almost all of the sampling methods (table 1 and 2). in this study, two species of the tribe aedini including, oc. caspius s.l. and ae. vexans were collected in larvae and adult stages. there is no information about the oc. caspius sibling species (a or b) in the country (azari-hamidian 2007). the species of cs. longiarolata was one of the most frequent culicine mosquitoes collected in this area, with total of 982 specimens, by larval collection. this species was the fewest culicine mosquitoes collected at adult collection. culex pipiens can be separated from cx. torrentium, and cx. vagans by harbach’s key (1988) using seta 1-iii-v, seta 1-m, seta 1-x, seta 1-c, and some other characters. based on this key, in this survey only cx. pipiens was identified. results of this survey were almost as the same of findings by previous study in kurdistan province (zaim 1987, vahabi 2001, moosa-kazemi et al. 2010, banafshi et al. 2013). ochlerotatus caspius s.l. and ae. vexans was reported as the potential vectors of human and domesticated animal pathogens (horsfall 1955, harbach 1988). the present study confirms the occurrence of these species. the species of cx. theileri was one of the culicine mosquitoes collected in the kermanshah, with total of 934, and 418 specimens, by adult and larval collection respectively. this mosquito is the more prevalent species at higher altitudes and in rural areas of isfahan (mousakazemi et al. 2000), east azerbaijan (abai et al. 2007), ardebil (azari-hamidian et al. 2009) and west azerbaijan provinces (khoshdelnezamiha et al. 2014). azari-hamidian et al. (2009) reported cx. theileri as 27.1% in ardebil province, northwestern iran, more than any other culicidae species, our finding also showed the most prevalent species in western iran. in eastern turkey, cx. theileri was the most prevalent culex species (aldemir et al. 2009, alkan and aldemir 2010). in agreement with previous investigations (mousakazemi et al. 2000, ghavami and ladonni 2005, abai et al. 2007, azari-hamidian et al. 2009, khoshdel-nezamiha et al. 2014) this species is most abundant mosquito in the northwestern iran. observations made during the present study agree with those of simsek (2004) who noted that cx. theileri in turkey prefers large natural larval habitats that are generally present in rural areas. conclusion in our research, some potential vectors in medical and veterinary importance identified such as aedes. vexans, an. maculipennis s.l., an. superpictus, cx. pipiens, cx. theileri, and oc. caspius s.l. on the other hand, malaria and west nile have been reported in this area shows the importance of mosquito control programmes (ahmadnejad et al. 2011, vejdani 2012). the ecology of other mosquitoes in iran needs to be studied extensively. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 57 acknowledgements the authors would like to express our appreciation to the people of villages of near sanandaj and kermanshah cities for their kind cooperation through the study. many thanks also decided for efforts of field staff of the school of public health. this investigation received technical and financial support from the tehran university of medical sciences, id no 240/1596. the authors declare that there is no conflict of interests. references abai mr, azari-hamidian s, ladonni h, hakimi m, mashhadi-esmail k, sheikhzadeh k, kousha a, vatandoost h (2007) fauna and checklist of mosquitoes (diptera: culicidae) of east azerbaijan province, northwestern 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(in persian with english abstract). vejdani m (2012) new success for controlling malaria in kermanshah province in west of iran. the first international congress on malaria elimination, 25– 27 january 2012, hormozgan university of medical sciences, kish island, hormozgan, iran, pp.72–73. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 49–59 s h moosa-kazemi et al.: the fauna and … 59 zaim m, cranston ps (1986) checklist and keys to the culicinae of iran (diptera: culicidae). mosq syst. 18: 233–245. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq contr assoc. 3: 568–573. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 224 http://jad.tums.ac.ir published online: june 24, 2019 original article a study on the identification of five arboviruses from hematophagous mosquitoes and midges captured in some parts of northern turkey emre ozan1; harun albayrak2; semra gumusova2; cenk s. bolukbas3; mithat kurt2; gokmen z. pekmezci4; yunus e. beyhan5; hamza kadi1; selma kaya1; ismail aydin1; *zafer yazici2 1veterinary control institut, ministry of food agriculture and livestock, samsun, turkey 2department of virology faculty of veterinary medicine ondokuz mayıs university, samsun, turkey 3department of parasitology, faculty of veterinary medicine ondokuz mayıs university, samsun, turkey 4department of aquatic animal diseases, faculty of veterinary medicine, ondokuz mayis university, samsun, turkey 5department of parasitology, faculty of medicine, yuzuncu yil university, van, turkey (received 18 jan 2018; accepted 11 may 2019) abstract background: whether zoonotic or not, arboviral infections are continuing to be a major threat to human health as well as the livestock industry all around the world. this project presented the results of the identification study on five arboviruses, including west nile virus (wnv), bovine ephemeral fever virus, akabane virus, bluetongue virus, and epizootic hemorrhagic disease virus, in mosquitos and midges from eight provinces of the black sea region. methods: during 2011 and 2012, 3193 mosquitoes were captured around natural streams, rivers, lakes, and ponds using dry-baited miniature light-traps. identification studies were concluded by employing molecular methods. results: according to the morphological identification, blood-sucking mosquitoes and biting-midges belonged to aedes (44.69%), anopheles (28.34%), culex (22.14%) and culicoides (4.83%) species. overall, 146 pools were made up of captured mosquitos and midges. none of the five viruses were directly identified by mosquitoes. conclusion: mosquitoes and midges have got a crucial role in the transmission of arboviruses. the risk of occurrence for the investigated arboviruses will continue depending upon many factors including the presence of these viruses in turkey and its neighboring countries, uncontrolled livestock movements, global warming and climate changes. keywords: mosquito; midge; identification; real-time rt-pcr; arbovirus introduction viruses transmitted by arthropod vectors can either be zoonotic or non-zoonotic. currently, more than 100 species of arbovirus are categorized as zoonoses due to their transmissibility from arthropods to human (1). most arboviruses belong to the virus families flaviviridae, togaviridae, reoviridae as well as both peribunyaviridae and phenuiviridae within the order bunyavirales, formerly known as bunyaviridae (1-3). mosquitoes and ticks are the main vectors for the majority of arboviruses (1, 4). there are approximately 300 species of mosquito and 116 species of ticks known to be vectors of arboviruses (5, 6). the vast majority of mosquitos are aedes and culex species, with 115 and 105 species respectively (6). in addition, 25 midge species, mainly culicoides, and sandflies, lasiohelea blackflies, stink-bugs, lice, mite, might have played a role in transmitting arboviruses (5). even though certain viruses are transmitted by specific vectors, some arboviruses, for example, west nile virus (wnv), can be transmitted additionally by ticks and other arthropods (1). infection with certain arboviruses can result in serious symptoms, including high fever, *corresponding author: dr zafer yazici, e-mail: zyazici@omu.edu.tr j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 225 http://jad.tums.ac.ir published online: june 24, 2019 haemorrhage, meningitis, encephalitis and death (1). moreover, in animals, abortions, stillbirth, congenital anomalies arthrogryposis-hydranencephaly are also seen in as a result of infection by certain arboviruses, for example, bluetongue virus (btv), and akabane virus (akav). equally, some arboviral infections are asymptomatic and the patients recover from infection after a few weeks. wnv, dengue virus (denv) and chikungunya virus (chikv) are the best known and globally distributed human arboviruses (1, 7). btv, akav and bovine ephemeral fever virus (befv) are important, non-zoonotic arboviruses of animals. in particular, large epidemics of btv-serotype 8 in eu countries including holland, czech republic, belgium, sweden, the uk, and switzerland emerged between 2006–2008 and caused major economic losses in all of these countries (8). in turkey, the presence of wnv, btv, akav, befv and epizootic hemorrhagic disease virus (ehdv) has been reported following serological studies (9-12). furthermore, a limited number of studies have been conducted to identify arboviruses from invertebrate vectors (7). the spread of arboviruses is influenced by many factors, such as increasing international travel, globalization of trade and climate change. whether symptomatic or not, arboviral infections may cause a large social and economic burden for societies (1). the detection of arboviruses in vectors is important for public health and the livestock industry and allows a lot of detailed information to be gathered about seasonal arbovirus circulation, helping to create time-dependent preventive measures. as the geographical bridge between europe and asia, turkey has a rich capacity as for natural and ecological conditions that provide a suitable habitat for arthropod populations, thus paving the way for the emergence of new arthropod-borne viruses. at the same time, turkey is also at risk due to the known presence of these viruses in neighboring countries such as iran, iraq, syria, the balkan and blacksea states. the aim of this study was to identify five arboviruses from field trapped mosquitos and midges at different provinces in northern turkey. captured vectors were examined by employing the real-time rt-pcr and rt-pcr. materials and methods samples during summer months of 2011 and 2012, eight provinces of northern turkey (fig. 1) were chosen to collect mosquito and midge samples. in the sampling period, two significant points were taken into account for the installation of traps: (i) areas of sampling where mosquito and midges were particularly abundant, such as cowsheds and sheepfolds, near or close to lakes, ponds, dam-lakes, natural streams and rivers, were chosen in the immediate vicinity of least 3 residential area in each province. (ii) mosquitos and midges were collected on days that were without rain and wind during the night between 6pm and 12pm. mosquitos and midges were caught in glass containers containing water and then were transported immediately to the laboratory under cold chain. mosquitoes and midges were separated under the stereomiscroscope in order to morphologically identify species (13, 14). overall, 146 pools from 3193 mosquitos and midges were made up according to each capture province and capture time, and were stored at -80 °c until use. the vast majority of them were aedes spp. corresponding to 1427 (44.69%) mosquitoes. furthermore, we identified the numbers of anopheles spp., culex spp. and culicoides spp. as 905 (28.34%), 707 (22.14%) and 154 (4.83%), respectively (table 1). homogenization of mosquitoes and midges insect pools were transferred into magnalyser green bead tubes (roche germany). 1ml of j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 226 http://jad.tums.ac.ir published online: june 24, 2019 pbs (gibco) was then added to each tube. pools were homogenized using a 1.4mm magnalyser ceramic bead by shaking magna lyser (roche, germany). following homogenization, the unsoluable particulates were pelleted by centrifugation at 4 °c, 3000rpm for 15min. the supernatant from each pool was then transferred into a new tube and used for nucleic acid purification using the total rna extraction kit (qiagen, spain) according to the manucfacturer’s instructions. rna extractions and rt-pcr tests positive controls of viral rna used for pcr tests were kindly provided by university of texas medical branch (galveston, usa) for wnv, pendik veterinary control institute (istanbul, turkey) for btv, veterinary diagnostic technology inc (usa) for ehdv, veterinary control institute (samsun, turkey) for akav and department of virology, faculty of veterinary medicine, university of selcuk (konya, turkey) for befv. detailed information regarding primers and taqman probes labelled fam-tamra are given in table 2. viral rnas of wnv, btv, ehdv and akav was detected in 96 well plates using a light cycler 2.0 real-time pcr systems (roche, germany). the results were assessed using the lightcycler software 4.05 (roche, germany). arktik thermal cycler (thermo scientific, uk) were used for rt-pcr in order to detect befv rna. the concentration and purity of rna were used by absorbance at 260–280nm wavelenghts using a uv spectrophotemeter (helios gamm, thermo spectronic, uk). to perform a real-time rt-pcr for the detection of wnv-rna in mosquito pools, specific primers and a probe (15) were used. the taqman probe was labeled at the 5’ end with the reporter dye fam and labeled at the 3’ end with quencher dye tamra. briefly, the reaction mixture was made up in a total volume of 50µl containing 2µl sample rna, 75mm tris-hcl (25 ºc, ph 8.8), 20mm (nh4)2 so4, 6mm mgcl2, 0.2mm of dntp mix, 50 pmol of primers 10pmol of prob, 1µl of triton x-100, 5u rnase inhibitor, 100u moloney murine leukemia virus reverse transcriptase and 2,5u taq dna polimerase (thermo scientific, uk) and water. the mixture was cycled as follow: 1 cycle 30min at 42 ºc for reverse transcription, 1 cycle of 2min at 94 ºc, 40 cycles of 30sec at 94 ºc, 30sec at 55 ºc and 20sec at 72 ºc and 1 cycle of 30sec at 40 ºc. to investigate btv-rna in pools, the real-time rt-pcr method (16) was carried out using the qiaamp viral rna mini kit (qiagen, spain) according to the manufacturer's instructions. rna was eluted in 50μl nuclease-free water and then stored at -20 °c. for rna sample denaturation prior to the initial rt step, heat denaturation of rna was evaluated by heating 6μl samples at 98 °c for 5 min followed by rapid cooling on ice. the reaction mixture contained, 20pmol of each primer, 5pmol of prob, 1.25mm mgcl2, 0,4mm dntps, 0.8µl of enzyme mix, 4µl of 5x rtpcr buffer, 3µl denatured rna and water to a final volume of 20µl.the mixture was cycled in using following procedure: after 1 cycle of 30min at 50 ºc for reverse transcription, 1cyle of 15min at 95 ºc for denaturation, 45 cycles of 15sec at 95 ºc and 1min at 60 ºc. test was evaluated at the end of 30s at 40 ºc. to investigate akav-rna in pools, we used touch-down rrt-pcr with some modifications (17). the reaction mixture was made up in a total volume of 50µl containing 3µl sample rna, 75mm tris-hcl (25 ºc, ph 8.8), 20mm (nh4)2so4, 6mm mgcl2, 0.2mm dntp mix, 20pmol of primers 10pmol of prob, 1µl of triton x-100, 5u rnase inhibitor, 100u moloney murine leukemia virus reverse transcriptase and 2.5u taq dna polymerase (thermo scientific, uk). cycling conditions included: rna was reverse transcribed at 42 ºc for 30min and was followed by an inactivation step at 94 ºc for 2min). after reverse-transcription and inactivation steps, 10 cycles of 15sec j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 227 http://jad.tums.ac.ir published online: june 24, 2019 at 95 ºc, 60sec at 60 ºc (decreased by 2 ºc per cycle so that it reached 50 ºc after 10 cycles) and 15min at 72 ºc were carried out. finally, a further 35 cycles of 15sec at 95 ºc, 1min at 50 ºc and 15sec at 72 ºc and 40 ºc for 2min followed. to investigate ehdv-rna in pools, the mixture was prepared in a total volume of 20 µl containing 3µl sample rna, 4µl of 5x master mix, 1.25mm mgcl2, 0.5mm dntps, 20pmol of primers, 5 pmol of probe, 1µl of enzyme mix (qiagen, spain) and water. cycling conditions of the reaction were as follows: 1 cycle of 30min at 50 ºc for reverse transcription step, 15min at 95 ºc for inactivation and denaturation step, and 40 cycles of 10sec at 95 ºc, 1min at 55 ºc, 30 sec at 72 ºc and finally incubation 30sec at 40 ºc. seven primer pairs and 3 probes were used to investigate all of the ehd serotypes. ehdv-1 and 2 were tested using the same primer pair. the different probes were used for ehdv 3 and 4 while the same probes were using for ehdv-1, 2, 5, 6, 7 and 8 (18). befv-rna in pools were tested by using one-step reverse transcription pcr method. specific primers with the qiagen onestep rt-pcr kit were used for ampflying a 809bp dna fragment (19). the rt-pcr mixture was prepared in a total volume of 50µl containing 5µl sample rna, 10µl of 5x mastermix, 0.2 mm of dntp mix, 10pmol of primers, 10u rnase inhibitor and 2µl of enzyme mix and water. amplification was conducted using thermal cycler according to the reaction conditions as follow: following reverse-transcription at 50 ºc for 30min, 1 cycle of 5min at 95 ºc, then 35 cycles of 30sec at 95 ºc, 1min at 56 ºc and 1min at 72 ºc. the amplication process was terminated after a final extension of 10min at 72 ºc. 5µl of pcr products were visualized on a 1.5% agarose gel stained ethidium bromide. results hematophagous mosquitos and biting midges were caught using dry-baited miniature light-traps located near to natural streams, rivers, lakes and ponds. following morphological identification under the stereomicroscope, 146 pools were made, representing 3193 mosquitos and biting midges. at the end of morphological identification, overall proportions of hematophagous mosquitos and biting-midges were found to be 95.17% and 4.83%, respectively. captured mosquitos were found to belong to aedes (44.69%), anopheles (28.34%) and culex (22.14%) species. midges were identified as culicoides (4.83%), the amplication results of four arbovirus including wna, btv, ehdv and akav by a real time rt-pcr test were shown in fig. 2. positive controls for 4 viruses worked very well and gave a good amplification earlier. a detectable fluorescence signals above the threshold for positive controls occurred at 12 cycles for wnv, 28 cycles for btv, 28 cycles for ehdv and 11 cycles for akav. no viral rna for these viruses could be identified by employing real-time rt-pcr in any of the insect pools. similarly, befv-rna was not detected using one-step rt-pcr in any of the mosquito pools. in all of 8 provinces, none of five viruses could be determined during the sampling period. table 1. the numbers of captured insect species according to provinces where the traps were installed captured mosquitos and midges longitude latitute trapping aedes anopheles culex culicoides (°e) (°n) provinces 35.09 42.01 sinop 19 540 158 27 36.20 41.17 samsun 1372 195 168 44 j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 228 http://jad.tums.ac.ir published online: june 24, 2019 36.43 40.19 amasya 3 20 46 20 35.50 40.40 tokat 23 136 181 35 37.53 41.00 ordu 3 3 82 26 38.24 40.55 giresun 4 35 2 39.43 41.00 trabzon 5 4 60 40.31 41.02 rize 2 3 7 total 1427 905 707 154 table 2. primers and probes sequences that employ for this research primer/probes sequences(5’-3’) position wn3 nc-f cagaccacgctacggcg 10,668–10,684 wn3 nc-r ctagggccgcgtggg 10,770–10,756 wn3 nc-p tctgcggagagtgcagtctgcgat 10,691–10,714 btvrsa 291–311f gcgttcgaagtttacatcaat 291–311 btvrsa 387–357r cagtcatctctctagacactctataattacg 387–357 btvuni 291–311f gcttttgaggtgtacgtgaac 291–311 btvuni 381–357r tctcccttgaaactctataattacg 381–357 btv 341–320p cggatcaagttcactccacggt 341–320 btv 346–323p tcctccggatcaagttcactccac 346–323 ehd 1/2f gcgttggatatattggacaaagc 165–187 ehd 1/2r gcatacgaagcataagcaacctt 275–253 ehd 1/2p tcaaatcaaacgggcgcaactatgg 192–216 ehd 3f agccctcgacattctggataag 164–185 ehd 3r cgcgactttctccactttttg 259–239 ehd 3p caaatcaaactggtgccacgatggc 206–230 ehd 4f aagttgccctcgatatcctagataag 160–185 ehd 4r gtaagcgaccttttctaccttttga 266–242 ehd 4p caatgtctaaccaaacaggcgctacaatgg 199–228 ehd 5f gctggatatactcgacaaagcaatg 167–191 ehd 5r acgcgaccttttctactttttga 262–240 ehd 6f gagtcgcgctggatatactc 160–179 ehd 6r gcatatgatgcatacgcgaccttt 276–253 ehd 7f caaaagttgcccttgacattttag 157–180 ehd 7r catatgctactttttctaccttctgc 266–243 ehd 8f ccaaaagtcgcccttgatattt 156–177 ehd 8r catatgccactttttctaccttctgc 268–243 ak151f gctagagtcttcttcctcaaccagaa 151 ak231r aaaagtaagatcgacacttggttgtg 231 ak182p ccaagatggtcttacataag 182 bef346 f tattaccctcctgccggatgcttt 346–369 bef1155 r aggtctgtattcgcaccaagctct 1155-1132 fig. 1. a map showing the provinces targetted in htis study for the capture of mosquitoes and biting-midges during 2011 and 2012. provinces that indicated with numbers are 1: sinop, 2: samsun 3: ordu, 4: giresun, 5: trabzon, 6: rize, 7: amasya, 8: tokat table 1. continued … j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 229 http://jad.tums.ac.ir published online: june 24, 2019 fig. 2. the amplication results of four arbovirus including wna, btv, ehdv and akav by a real time rt-pcr test. curves indicated the real time pcr plots of positive controls. a:wnv, b:btv, c: ehdv, and d:akav. a detectable fluorescence signals above the threshold for positive controls of four viruses occurred at 12 cycles for wnv, 28 cycles for btv, 28 cycles for ehdv and 11 cycles for akav.as it seen in all figures, no positive pools for 4 viruses could be determined discussion whether zoonotic or not, arboviral infections are continuing to be a major threat to human health as well as the livestock industry all around the world (1, 20). in the last decade, many outbreaks in humans and animals caused by arboviruses have been reported. identification of arboviruses in vectors can provide us much information to help develop control strategies against them. historically, there are only a few studies on arboviruses in vectors in turkey, limited to the flaviviridae and togaviridae (7, 21). in one such study, 15.9% of collected mosquitos were reported as wnv positive (7). our study was planned for five animal arboviruses, including wnv, btv, ehdv, akav and befv to identify from their vectors. with the exception of wnv, all of the viruses are non-zootic. this study can be defined as the first compherensive identification study of mosquitoes and midges caught in the middle and east black sea region in turkey. during 2011–2012, 3190 mosquitoes and midges were collected using light traps in 8 provinces of the central and east blacksea region of northern anatolia, turkey. according to the morphological identification results, the vast of majority of the overall vector population was mosquito (95.17%), comprising 44.69% aedes, 28.34% anopheles and 22.14% culex, species known as vector for wnv, befv (22, 23). culicoides midges, known as the important biological vector of btv (8), ehdv (11) and akav (24), represented 4.83% of catches. j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 230 http://jad.tums.ac.ir published online: june 24, 2019 in turkey, serologically the presence of wnv for the first time was reported in different animal species including horses (13.5%), dogs (37.7%), cattle (4%) and sheep (1%) (9). the first identification of wnv from haematophagous mosquitoes in eastern thrace territory of turkey was carried out (7). in the previous years, a few identification studies on wnv had been reported in the black sea region and no wnv could be identified from insects and blood samples of various animal species such as horses and wild birds (25, 26). moreover, the serological diagnosis of wnv could also not be possible in various animal species with the exception of goats that seropositivity rate was reported as 2.85% (27, 28). in our comprehensive study, although 80% of the captured insects were a specific vector for wnv, the identification of wnv was not possible using real-time rt-pcr. the lack of presence of wnv may be due to the absence of certain vector specific determinats of emergence, such as the seasonal activity, climate change, ecological and environmental conditions (7, 9). btv and ehdv both belong to the family reoviriadae, and both viruses are transmitted between ruminant hosts by culicoides biting midges. during the past decade, btv outbreaks have occurred in the western part of turkey (8, 29). an ehdv outbreak was also reported in western anatolia in 2007 (11). however, infections caused by these viruses in northern anatolia had not been seen before. on the other hand, there are serological data regarding both viruses showing the historical presence of these viruses. seropositivity for ehdv had been reported in cattle as 1.33%, with evidence of btv seropositive animals, including 11% for cattle, 3% for sheep and 4% for goat (25, 27–28, 30). in our study, no btv-rna and ehdv-rna could be identified by using real-time rt-pcr. when scrutinized, these results could depend upon two main causes. first, historically neither disease has been clinically evident in this region until recently and, secondly, the proportion of culicoides species was far fewer than other insects captured during sampling periods. furthermore, livestock movements inside turkey may confound some interpretations of serological data. culicoides is also the main vector for akav. despite a serological prevalence of 22% in our study area (27), in neither mosquito nor midge pools could be not detected akav-rna by real-time pcr. this result can be interperented similarly to btv and ehdv. in previous years, numerous befv outbreaks were reported in turkey, primarily in south and sout-east anatolia. the climate of these regions is suitable for befv vectors. in addition, there are common borders with syria and iraq where the virus has been previously reported. the latest outbreak began in the same regions in 2012 and reached a case fatality rate (31, 32). in the central and eastern blacksea region of turkey, antibodies against befv were shown to be 13.5% (27). despite the serological indications, clinical befv has not been reported. in the current study, we attempted to identify befv from captured insects using rtpcr. at the end of the experiments, no befv-rna could be found in all pools. according to our standpoint, there are a few unsuitable climatic conditions and vector ecology may represent important factors in determining whether or not infection occurs. no viral rnas of five arboviruses could be found in pools made up of captured various mosquitos and midges during this project. infections caused by these five viruses were not observed clinically in the central and east blacksea region where the project was carried out. however, in the light of serological data obtained from previous studies, five viruses are nevertheless considered to circulate between animals and prospective vectors. furthermore it is not ruled out that mosquitos and midges cannot be found in some locations of targeting regions or were never infected with five viruses. j arthropod-borne dis, june 2019, 13(2): 224–233 e ozan et al.: a study on the … 231 http://jad.tums.ac.ir published online: june 24, 2019 reservoir, vector, and climate are the most important aspects for the epidemiology of arboviruses. the climatic condition of a region, such as amount of rainfall, wind status and insecticide application are particularly important factors in vector biology. in the black sea region, yearly averages of temperature, humidity and rain are reported as 13.0 ºc (4.2–22.1 ºc), 71%, 842.6mm3, respectively. when compared, these values are lower than the western anatolia region of turkey where the disease is seen frequently, reportedly 16.3 ºc (6.4– 26.8 ºc), 63%, 2, 725.9mm3 respectively. these data are important factors affecting vector abundance. hence the existence of culicoides species in the black sea region was far less than the western and southern part of turkey (14). conclusion mosquitoes and midges have got a crucial role in the transmission of arboviruses. the risk of occurrence for the investigated arboviruses will continue depending upon many factors including (i) the presence of these viruses in turkey and its neighbouring countries (ii) uncontrolled livestock movements (iii) global warming and climate changes. a large scale project, inclusive of both people and animals, should be planned. in addition, effective arboviral disease 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13(1): 9–16 h gevorgyan et al.: evidence of crimean-congo … 9 http://jad.tums.ac.ir published online: april 27, 2019 original article evidence of crimean-congo haemorrhagic fever virus occurrence in ixodidae ticks of armenia *hasmik gevorgyan1,2, gohar g. grigoryan1, hripsime a. atoyan1, martin rukhkyan1, astghik hakobyan2, hovakim zakaryan3, sargis a. aghayan4 1scientific center of zoology and hydroecology, yerevan, armenia 2national institute of health, moh ra, yerevan, armenia 3institute of molecular biology nas ra, yerevan, armenia 4laboratory of zoology, research institute of biology, yerevan state university, yerevan, armenia (received 3 mar 2018; accepted 8 oct 2018) abstract background: crimean-congo hemorrhagic fever (cchf) causes serious health problems in humans. though ticks of the genera hyalomma play a significant role in the cchf virus transmission it was also found in 31 other tick species. methods: totally, 1412 ticks from 8 remote sites in armenia during 2016 were sampled, pooled (3-5 ticks per pool) and tested for the presence of cchfv antigen using elisa test. results: from 359 tick pools, 132 were cchf virus antigen-positive. from 6 tick species, four species (rhipicephalus sanguineus, r. annulatus, r. bursa, hyalomma marginatum) were positive for the virus antigen and r. sanguineus was the most prevalent (37.9%). dermacentor marginatus and ixodes ricinus revealed no positive pools, but both revealed delectable but very low virus antigen titers. the highest infection rate (50%) was observed in r. sanguineus, whereas h. marginatus rate of infection was 1 out of 17 pools. conclusion: for the first time in the last four decades cchf virus antigen was detected in ixodid ticks of armenia. this finding substantiates the role of r. sanguineus in the disease epidemiology; however, the role of h. marginatum in the cchf virus circulation in the country could not be excluded. keywords: cchf; ixodid ticks; armenia; elisa; infection rate introduction ixodid ticks present significant epidemiologic and health concerns in the transmission of dangerous arboviruses to humans (1, 2). in addition, the potential for person to person transmission makes the control of the spread of infection more difficult and unpredictable (3, 4). the crimean-congo hemorrhagic fever (cchf) is one of the zoonotic viral infections that manifests with influenza-like symptoms and can progress to hemorrhagic disease with mortality rate ranging from 5% to 50% (5, 6). there are several routes and sources of human infection with cchf virus, e.g., a direct tick bite, contact with virus-containing killed ticks, direct contact with blood and other tissues of infected animals or humans (3, 7-9). the caus ative agent is cchf virus (cchfv), a member of the bunyaviridae, genus nairovirus. the virus has been isolated from at least 31 species of ticks belonging to ixodidae (hard ticks) and argasidae (soft ticks) families (10). a number of ticks in genera like rhipicephalus and dermacentor are able to care cchf virus, but ticks of the genus hyalomma are the essential vector for the pathogen transmission (11, 12). except direct, through bites, disease transmission there are additional factors that can potentially increase the virus survival in nature. the livestock maintains the stable circulation of the virus in nature. the virus replicates to high titers in some organs of the carrier livestock and usually causes only subclini*corresponding author: dr hasmik gevorgyan, e-mail: hasgevorgian@yandex.ru j arthropod-borne dis, march 2019, 13(1): 9–16 h gevorgyan et al.: evidence of crimean-congo … 10 http://jad.tums.ac.ir published online: april 27, 2019 cal disease in domestic animals, that goes unnoticed and untreated (13). the member of the crow family (corvidae) are the reservoir of the ticks immature stage, whereas, infected animals are the main reservoirs for fully developed adult stage of ticks (14). epidemics and sporadic cchf cases have been reported from eastern europe, russia, africa, the middle east, and central asia (15-17). tick-born arbovirus infections present epidemiologic and health challenge in armenia, as well. being represented by a distinct variety of landscape zones with several climate zones in relatively small territory, the countryside supports potential distribution of the vector tick species (18). although historical review related to arbovirus surveillance in armenia has revealed the circulation of different arthropod-transmitted viruses (19), the first detection of cchfv in ticks and the only laboratory-confirmed severe case of human cchfv disease in armenia is dated back to 1970s (20). to the best of our knowledge there are no published reports about cchfv activity in the country in the last 5 decades. in spite of recent worsening of cchfv situation in neighboring turkey and iran (9, 21–24), there is no consistent monitoring of cchf key vectors distribution and abundance in armenia. moreover, there are no efforts to determine the prevalence and the level of cchfv in ixodid ticks. this study was designed to study the possible presence of cchf virus in ixodid ticks in target provinces of armenia. the initial studies are highly relevant in the context of transformed socio-economic conditions, climatic and environmental changes, and particularly in the context of a significant interest towards tickborn arboviral infections worldwide (25). materials and methods the ticks’ collection was conducted in 2016 and covered 8 locations in five provinces of armenia that included southern, central and northern parts of the country (fig. 1). the selection of these territories was based on preliminary but limited knowledge of high level of hyalomma and rhipicephalus ticks abundance in these areas (26). ticks were collected using two methodologies by flagging in pasture lands of each locality and direct collection of the ticks from livestock. the sampling was linked to the ticks’ seasonal activities and covered period from apr to oct 2016 (27). the field sampling of freeliving questing ticks was performed using flannel strips (1000x100mm). at each pastureland, 5 areas were randomly selected. each selected area was walked in a slow manner dragging the flannel through the grass. at the end of each area caught ticks were collected. for collection of ticks from host animals (cattle and sheep), 10 animals were randomly selected for examination in each herd. ticks were collected with forceps. each individually collected tick was placed in separate vials, labeled and kept alive until species identification. species identification was implemented under the light microscope with magnification of 100x, using the published taxonomic keys and species accounts (28). overall, 1412 ticks were sampled of which only 548 were identified to species level. in 864 cases the identification was difficult due to inadequate life stage or the damage of the sample. however, these “unidentified species” were pooled in groups according to sampling area and tested separately to determine whether or not these ticks may contain any titer of cchfv antigen. prior to serological detection of cchfv antigen, each collected tick was rinsed with 70% ethanol and then placed in physiological solution with antibiotics (mixture of 100u/ml penicillin, 100μg/ml streptomycin). sampled ticks were clustered and labeled according to genera, collection area, season, and placed in separate vials and stored at -18 °c for further processing. stored ticks were pooled into groups of j arthropod-borne dis, march 2019, 13(1): 9–16 h gevorgyan et al.: evidence of crimean-congo … 11 http://jad.tums.ac.ir published online: april 27, 2019 three to five (depending on size) by species and collection area. all pooled ticks were put in the liquid nitrogen for 20min then homogenized using glass pestle. homogenized samples were resuspended in 200μl of dilution buffer in case of flat ticks and 400μl in case of engorged ticks. after the short vortexing tubes were centrifuged at 2000rpm for 5min and supernatant was collected to test for cchf viral antigen using antigen-capture elisa (vectocrimeachf elisa, vector-best, russia) (29). the sensitivity and specificity of the standard panels of positive and negative samples were indicated as 100%. based on obtained optical density (od) values we calculated critical od (odcr) according to the following formula: odcr= odm (k-)+ 0.2 where odm (k-) is the medium value of negative controls’ od. the sample was considered as positive if od sample was higher than odcr. according to the manufacturer protocol if the od sample was higher than 0.8*odcr but less than odcr the reading was considered as inconclusive, i.e., doubtful. the data were analyzed using previously published technique developed for pooled sample studies (30). results of 1412 collected ticks only 548 were identified to species level which belonged to four genera of the family ixodidae: rhipicephalus, hyalomma, ixodes, dermacentor. the dominant tick species were from the genera rhipicephalus (364 ticks, 66.4%). further, 6 tick species were identified, and r. sanguineus was identified as the most common tick species (n= 208, 37.9%) followed by r. annulatus (n= 68, 12.4%), r. bursa (n= 88, 16%), d. marginatus (n= 64, 11.7%), i. ricinus (n= 53, 9.5%) and h. marginatum (n= 67, 12.4%). the analysis of tick species according to geographic location showed that rhipicephalus spp. was the most abundant tick species in kotayk region. i. ricinus and r. annulatus are the most recorded species in gegharkunik province, while sheep in ararat and tavush provinces suffered from d. marginatus ticks. collected ticks were separated into 137 pools of identified tick species and 222 pools of not identified and assayed using elisa test. from 137 pools comprising six tick species, 38 tested positive for the cchfv antigen. these pools included four tick species (r. sanguineus, r. annulatus, r. bursa, h. marginatum) and were registered in all surveyed areas. noteworthy, r. sanguineus showed the highest cchfv infection rate meanwhile only 1 of 17 h. marginatum pools showed positive response. the other six species contained doubtful (non-interpretable) pools (table 1). identified and non-identified tick pools were also clustered to analyze occurrence of cchf virus antigen in carriers in targeted geographic areas. totally, 359 pools were tested. the observation of ticks’ infection rate in studied areas is shown in table 2. the highest tick infection level was found in kotayk and tavush provinces. ticks from other areas demonstrated uneven distribution, with lower prevalence in southern and central part of the country. table 1. serological results of crimean-congo hemorrhagic fever antigen detection in pools according to tick species* tick species no ticks infection rate no pools positive pools doubtful pools rhipicephalus annulatus 65 107.73 17 6 1 dermacentor marginatus 68 0.00 16 0 2 hyalomma marginatum 70 17.85 17 1 3 ixodes ricinus 51 0.00 13 0 1 rhipicephalus bursa 86 71.87 22 5 3 j arthropod-borne dis, march 2019, 13(1): 9–16 h gevorgyan et al.: evidence of crimean-congo … 12 http://jad.tums.ac.ir published online: april 27, 2019 rhipicephalus sanguineus 208 175.30 52 26 4 total 548 137 38 14 *infection rate per 1000 ticks and 95% confidence interval table 2. results of cchfv antigen detection in tick pools according to geographic location* town/village (province) infection rate no pools positive pools doubtful pools zorak (ararat) 0.00 16 0 2 chambarak (gegharqunik) 42.80 39 6 2 tsaghkadzor (kotayk) 195.79 37 20 3 khndzoresk (syunik) 10.87 25 1 2 dilijan (tavush) 156.65 220 100 18 ijevan (tavush) 65.76 8 2 0 artsvaberd (tavush) 73.58 14 3 3 total 359 132 30 *infection rate per 1000 ticks and 95% confidence interval fig. 1. study area discussion changes in climatic, environmental, social and anthropogenic factors have contributed to the spread of cchf infection in new areas and increased incidence in endemic regions. armenia is one of the countries where no studies on circulation of cchf virus among ticks and livestock were conducted during last 5 decades. the first alarming data on the cchf risk in armenia occurred after the detection of agdp antibodies to the virus in cattle sera from 5 areas of the country (31). the next evidence of the virus was the isolation of cchf strains from h. marginatum (5), h. anatolicum (1), r. bursa (1), boophilus (rhipicephalus) annulatus (1), r. rossicus (1) (32). we have embarked on this study based on these 2 references (31, 32). the specific study areas were selected based on preliminary known distribution of hyalomma and rhipicephalus ticks in certain territories, as well as contrasting bio-climatic conditions between targeted provinces. our samples were represented predominately by rhipicephalus, hyalomma, ixodes, dermacentor genera distinguished by their high magnitude of populations and variety of domestic and wild animal hosts (15, 18). ticks were identified, pooled and analyzed using elisa according to species and geographic location. from 1412 collected tick samples by table 1. continued … j arthropod-borne dis, march 2019, 13(1): 9–16 h gevorgyan et al.: evidence of crimean-congo … 13 http://jad.tums.ac.ir published online: april 27, 2019 the time ticks were delivered to the laboratory the condition of 60% of ticks were not appropriate for the species identification. since the aim of this study was to determine the presence of cchf virus in ticks in a specific area identified for the study, we pooled together the unidentified ticks and analyzed these pools only according to geographic area. the rhipicephalus is the most prevalent genus in kotayk region. i. ricinus and r. annulatus are the most prevalent species in gegharkunik province. the most abundant tick species in the current study is r. sanguineus which is, in general, in agreement with published data (34, 35). comparison of results obtained from different regions of the county showed that ticks collected from central regions were more infected than those from southern and northern regions. first, this can be explained by the composition of sampled ticks from this region, where r. sanguineus the most abundant species that were also showing the highest infection rate among all other tick species. second, it could be due to the concentration of livestock and the quality of breeding management including poor hygienic conditions of livestock breeding sites. among all areas studied, we identified the highest ticks’ infection rate in kotayk province with prevalence of 54.05%. the second highest prevalence was identified in dilijan town (tavush province) where along with r. sanguineus two more species (i. ricinus and r. bursa) were obtained. prevalence of infected ticks was decreased in the north – ijevan (25%) and artsvaberd (21.5%) as well as in the east -chambarak (15%). according to our data in artsvaberd and ijevan the most abundant tick species were r. bursa and in chambarak r. annulatus, demonstrating that the infection prevalence in study sites coincides with tick species abundance and infection rates. while in previously published study no virus was observed in h. detritum, h. scupense, i. ricinus, or argas persicus (31), in our study one pool of i. ricinus and 2 pools of d. marginatum demonstrated low-level titers of cchfv antigen. some of ticks could be the virus carriers and these finding may suggest that further studies may be necessary to determine the role of these other tick species in the epidemiology of the cchfv transmission. in our opinion, a significant attention should be devoted to the recognized vector of cchf virus h. marginatum. in our study from 17 h. marginatum ticks only one pool showed positive response and two were rated as doubtful. if compared with rhipicephalus genera figures, there are indications to consider the latter as an acceptable vector for the virus in armenia. at the same time, however, relatively high number of unidentified tick species with significant number of positive pools (132) may avert from an objective assessment of hyalomma impact on cchf cases in armenia. therefore, a geographically wider area and more directed studies should be conducted to be able to answer this question. according to scientific consensus, although, many tick genera are capable of becoming infected with cchf virus, several species of genus hyalomma are the principal vector for cchf virus. to some extent our results and the only serologically proven human cchf case recorded in 1974 (20) support this statement. hyalomma and rhipicephalus ticks are widely distributed and abundant in armenia, but rhipicephalus spp. is likely to have a greater role in the circulation of cchfv in armenia and by serving as a virus reservoir. hence, the occurrence of the pathogen in ticks can represent a significant risk for human population and this risk should not be overlooked. further studies should be focused on animals’ seroprevalence and virus genetic diversity for identification of high-risk areas for human infection. conclusion for the first time in the last five decades, cchf virus antigen was detected in tick samj arthropod-borne dis, march 2019, 13(1): 9–16 h gevorgyan et al.: evidence of crimean-congo … 14 http://jad.tums.ac.ir published online: april 27, 2019 ples in armenia. serological test (elisa) enabled to prove the existence of cchfv antigen mostly in rhipicephalus ticks. our results showed the important role of r. sanguineus tick species in supporting of cchfv circulation in the natural foci, although without officially registered cases of human cchf. moreover, the highest level of infection based on collection area is registered in kotayk region which coincided with abundance of r. sanguineus. our results demonstrated the necessity to conduct pcr-based studies to determine genetic diversity of cchfv in the country. 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(arthropoda) of armenia of medical importance, their biological and ecological peculiarities. med sci armen. 6(1): 81–89 (in russian). 35. dilbaryan kp (2015) blood-sucking ticks (family ixodidae murray, 1887, genus dermacentor koch, 1844) of armenia being the transmitters of human and animal diseases. theory pract parasitol dis. 16: 120–122 (in russian). j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 124 http://jad.tums.ac.ir published online: june 30, 2022 original article pcr positivity of gerbils and their ectoparasites for leishmania spp. in a hyperendemic focus of zoonotic cutaneous leishmaniasis in central iran sahar azarmi1, *alireza zahraei-ramazani1, mehdi mohebali2, yavar rassi1, amir ahmad akhavan1, amrollah azarm1, omid dehghan1, samira elikaee2, rahimeh abdoli1, mohsen mahmoudi2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran *corresponding author: dr alireza zahraei-ramazani, e-mail: azahraei@tums.ac.ir (received 02 sep 2020; accepted 02 may 2022) abstract background: various arthropods, including rhipicephalus sanguineus and ctenocephalides felis felis have been suggested as secondary vectors of leishmania spp. many years ago. this study was conducted to determine zoonotic cutaneous leishmaniasis (zcl) pcr positivity of reservoir hosts and their ectoparasites for leishmania spp. in segzi plain in esfahan province from october 2016 to october 2017. methods: microscopic examination and nested pcr were used to detect and identify leishmania spp. isolated from rodents' ears and ectoparasites, and then, the results were confirmed by two methods, pcr-restriction fragment length polymorphism and sequencing. results: totally, 93 rodents (92 rhombomys opimus and one nesokia indica) and nine different species of ectoparasites (n=527) including fleas, mites, and ticks were collected during different seasons in the study area. fourteen r. opimus were positive for leishmania spp. by microscopic examination while one n. indica and 77 r. opimus were positive by nested pcr. the infection rate of rodents with leishmania major and leishmania turanica was 39.79% (n=37) and 15.05% (n=14), respectively. mixed natural infections with l. major and l. turanica were seen in rodents. moreover, 72.22% of fleas (39/54), 75.0% of mites (5/8), and 100% of tick nymph (1/1) were pcr positive for leishmania parasites. conclusions: the highest rate of infection with l. major and l. turanica in r. opimus populations was observed in summer and spring, respectively. it is suggested that the role of l. turanica and the probable role of ectoparasites in the epidemiology of disease should be investigated. a xenodiagnostic test is recommended for future study. keywords: ectoparasite; leishmania gerbilli; leishmania major; leishmania turanica; rhombomys opimus introduction zoonotic cutaneous leishmaniasis (zcl) caused by leishmania major yakimoff and schokhor, 1914 is a public health problem in several countries affecting a large number of people (1). the disease is endemic in in 18 out of 31 provinces of iran (2). the great gerbils (rhombomys opimus lichtenstein, 1823) is the main reservoir hosts of zcl in iran and this rodent acts as a reservoir host of zcl in esfahan province, central iran (1). the main and biological vector of l. major is phlebotomus pa patasi scopoli, 1786 in iran (3). however, secondary forms of leishmania transmission by other arthropods have been reported in the literature, but their role is still unclear in the epidemiology of leishmaniasis (4–7). some arthropods, such as the brown dog tick (rhipicephalus sanguineus latreille, 1806), have been introduced as probable mechanical vectors for some leishmania parasites (4–8). ticks and fleas may play a role in the transmission of leishmania infantum nicolle, 1908 between copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:azahraei@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 125 http://jad.tums.ac.ir published online: june 30, 2022 dogs (8). two recent studies have suggested that the leishmania parasite could be transmitted by tick and flea bites (6, 7). ticks that have high reproductive rates and long lifespan, are widely distributed and abundant, and can maintain high population densities. they are potential vectors of several vertebrate pathogens (9). according to reports of human parasitism by r. sanguineus (10) and transmission of leishmania parasites by the brown dog tick (5, 6), ticks may have a role in the survival of leishmania parasites between dogs and sporadic transmission of this parasite from dogs to humans (6, 11). like ticks, fleas, which are widely distributed due to their high reproductive rate, can attain a high population density. if a source of blood is available, fleas can live for 200 days. their blood feeding habit and high longevity may turn fleas into a potential vector of leishmaniasis in endemic areas over time (12). a recent study found that the ctenocephalides felis felis bouche, 1835 fleas collected from dogs showed the presence of promastigotes in smears stained by giemsa. moreover, it was also shown that fleas could transmit leishmania chagasi cunha and amp, chagas, 1937 from the infected dogs to hamsters in laboratory conditions. this investigation was confirmed by polymerase chain reaction (pcr) and the indirect fluorescent antibody test (ifat) assays (13). this study was conducted to determine the pcr positivity of zcl reservoir hosts and their ectoparasites for leishmania spp. in a hyperendemic area of esfahan province, iran. materials and methods study area this study was done in segzi plain (32.694866° n, 52.120171° e), 35km east of the city of esfahan, esfahan province over a period of 12 months from october 2016 to october 2017. segzi is located at an average altitude of 1545 meters above sea level and has hot summers and cold winters. most of the vegetation in the desert area of segzi is salsola rigida and haloxylon ammodendron (black saxaul). collection and identification of rodents and their ectoparasites rodents were captured using 30 sherman live traps (18×18×30cm) at intervals of 45 days, 2 times in each season from different residential, agricultural, and desert areas in segzi. the traps were placed in front of the holes of active colonies before sunset and sunrise. in case of trapping before sunset, the traps were collected the next morning. also, the traps placed before sunrise were removed until noon of the same day. the captured rodents were transferred to the laboratory of the esfahan health research station and anesthetized (14). ectoparasites were collected from each rodent by brushing the hairs of the hosts on a water surface and stored in 96% ethyl alcohol separately. the ectoparasites were mounted on microscopic slides and then identified morphologically (15–18). the rodents were identified based on their morphological and morphometrical characteristics using identification keys (19). microscopic examination while the rodents were anesthetized, two different microscopic slides (thin smear) were prepared from each ear lobe and cutaneous lesions for the examination of the presence of leishmania amastigotes. after giemsa staining, the slides were evaluated using a light microscope (×1000). before and after smear preparation, the ears of the rodents were sterilized with 70% alcohol (1). the rodents were transferred to the esfahan health research station animal house and bred for other educational and research purposes. nested pcr assay the prepared smears from ear lobes and cutaneous lesions in the rodents' ears were used for dna extraction and investigation of leishmania infection in rodents. the genomic dna http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 126 http://jad.tums.ac.ir published online: june 30, 2022 was extracted and purified according to the geneall® kit instruction. the extracted dna samples were kept in 40μl of elution buffer at -20 °c. the partial sequence of the internal transcribed spacer (its2) of the ribosomal rna gene (rdna) was used for detection and identification of different species of leishmania parasites. the sequences of the primers were as follows: external forward primer (5'-aaa ctc ctc tct ggt gct tgc-3'), external reverse primer (5'-aaa caa agg ttg tcg ggg g-3'), internal forward primer (5'aat tca act tcg cgt tgg cc-3') and internal reverse primer (5'-cct ctc ttt ttt ctc tgt gc-3') (20). three microliters of template dna, 1.5μl of each external primers, 12.5μl of ampliqon (taq dna polymerase 2x master mix redmgcl2 1.5mm/2mm), and 6.5μl of sterile distilled water were used in the initial pcr. thermal cycle steps of the initial pcr were as follows: at 95 °c for 5 minutes (initial denaturation), 30 cycles at 94 °c for 30 seconds (denaturation), 60 °c for 45 seconds (annealing), and 72 °c for 1 minute (extension) with a final extension step at 72 °c for 5min. the second-round (nested) pcr included 3μl of the diluted product of the first-round pcr (1:10 dilution in distilled water), 1.5μl of each internal primer, 10μl of ampliqon, and 6 μl of sterile distilled water. the thermal cycle steps of the second-round pcr were as follows: 95 °c for 2 minutes (initial denaturation), 30 cycles at 94 °c for 15 seconds (denaturation), 62 °c for 30 seconds (annealing) and 72 °c for 45 seconds (extension) with a final extension step at 72 °c for 5min. the pcr products were analyzed by 1.5% agarose gel electrophoresis. bands created in the gel were visualized using uv ray and gel stain and photographed. the standard strain l. major (mrho/ir/75/er) was used as positive control and distilled water was used as a negative control. the predicted size of its2 products in the second-round pcr was 247bp for l. major (genbank accession numbers: fj753394), 206bp for leishmania gerbilli wang, qu and guan, 1964 (genbank accession numbers: aj300486), and 141bp for leishmania turanica strelkova etc, 1990 (genbank accession numbers: aj272382). also, the ectoparasites collected from pcr positive rodents were tested for detection of leishmania parasites using nested pcr assay as described. pcr-rflp and sequencing the results of the second-round pcr products were confirmed by two methods; sequencing and polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) using restriction digestion with mnli (jena bioscience, germany). pcr-rflp was done by mixing 1μl of the mnli enzyme and 1.5μl of g buffer with 12.5μl of the positive nested pcr product in a final volume of 15μl (21). in this technique, 120, 73, 43 and 11bp band lengths were expected for l. major (genbank accession number: fj753394), 158, 37 and 11bp band lengths for l. gerbilli (genbank accession number: aj300486), 131 and 10bp band lengths for l. turanica (genbank accession number: aj272382). moreover, 30μl of 10 positive nested pcr products were sent to macrogen, south korea for sequencing after purification using the bioneer kit. the fragments were sequenced using internal forward and reverse primers. nucleotide sequences were edited and aligned using the chromas pro v2.1.3 and were compared with the sequences in the genbank using the basic local alignment search tool (blast) in the national center for biotechnology information (ncbi) (http://www.ncbi.nlm.nih.gov/blast). then, the phylogenetic tree was constructed by the mega 7 software and maximum likelihood tree method with a bootstrap value of 1000 replicates. all the obtained sequences in this study were recorded in the genbank. http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/blast j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 127 http://jad.tums.ac.ir published online: june 30, 2022 statistical analysis fisher's exact test was used to evaluate the difference in the infected rodents between different seasons by spss 22.00 software and the p-values less than 0.05 were considered significant. to estimate evolutionary relationships between two nucleotide sequences, the correlation coefficient was calculated using mega 7 software and the pairwise distance matrices method using the p distance model. so, the correlation coefficient between two distance matrices was used to estimate the similarity of two sequences and infer the exact genetic relationship between species of the leishmania. results totally, 92 r. opimus (52 females and 40 male) and one nesoki indica gray and hardwicke, 1832 (one male) were captured. nineteen rodents were trapped in the fall, 11 in the winter, 28 in the spring, and 35 in the summer and 9 different species of ectoparasites (527) were collected from them. the identified ectoparasites included one tick species (one nymph of r. sanguineus), one sucking louse species (polyplax spp. enderlein, 1904), five flea species (399 xenopsylla nuttalli ioff, 1930, 46 echidnophaga oschanini wagner, 1930, 5 nosopsyllus ziarus klein, 1963, 5 coptopsylla mesghalii farhang-azad, 1966 and one nosopsyllus turkmenicus turkmenicus vlasov and ioff, 1937) and two mite species (28 hirstionyssus sp. fonseca, 1948 and 41 dermanyssus sanguineus hirst, 1914 (synonym: liponyssoides sanguineus)) (fig. 1). the amastigote form of leishmania parasites was seen in 14 (15.2%) out of 92 collected r. opimus using microscopic examination. while 77 of r. opimus and one n. indica (83.87%) were positive using nested pcr. table 1 shows the infection of rodents with different species of leishmania in different seasons using nested pcr. all positive smears were positive by nested pcr. the percentage of rodents infected with l. major and l. turanica was 39.79% (37) and 15.05% (14), respectively. mixed natural infections were seen in 15.05% of rodents (14) with l. major and l. turanica, 7.53% of the rodents (7) with l. major and l. gerbilli, and 6.45% of the rodents (6) with the three leishmania species. nesoki indica was infected with l. major. fisher's exact test showed a significant difference in the leishmanial infection rate between different seasons (p= 0.004). the rodents leishmania spp. infection rate was the highest (94.29%) in the summer and the lowest (45.45%) in the winter. mixed leishmanial infection of l. major and l. turanica was observed in all the seasons (table 1). moreover, 39 out of 54 fleas (72.22%), five out of eight mites (75%), and one tick nymph (100%) were found leishmania pcr positive using nested pcr. leishmania dna was detected from 67.5% (27/40) of x. nuttalli, 100% (3/3) of n. ziarus, 100% (3/3) of c. mesghalii, 75% (6/8) of e. oschanini, 80% (4/5) of d. sanguineus, 66.66% (2/3) of hirstionyssus sp., and 100% (1/1) of the nymphs of r. sanguineus. of five pcr positive specimens of x. nuttalli fleas with gravid abdomen status, one (female) was l. major pcr positive for, two (males) were positive for both of l. major and l. turanica, and two (females) for both of l. major and l. gerbilli. moreover, one of two l. major pcr positive hirstionyssus sp. mites was gravid and the rest of the pcr positive ectoparasites were blood-fed. dna of the three species of leishmania parasites and l. turanica was found in two e. oschanini (male) and one n. ziarus (male) fleas, respectively (table 2). out of ten positive its2 nested pcr products, nine were sequenced well. all these sequences were compared with the sequences of its2 in the genbank. the genbank accession numbers for the its2 region of the leishmania parasites included mk372246 (l. turanica; host: r. opimus), mk372247 (l. infantum; host: r. opimus), mk372248 (l. major; host: hirstionyssus sp.), mk372249 (l. major; host: x. nuttalli), mk372250 (l. major; host: r. opimus), mk372251 (l. major; host: n. ziarus), http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 128 http://jad.tums.ac.ir published online: june 30, 2022 mk372252 (l. major; host: r. sanguineus), mk372253 (l. major; host: c. mesghalii) and mk372254 (l. major; host: d. sanguineus). one of the sequenced samples was similar to l. infantum with accession number mg831328.1 (query cover= 98%, e value= 1e-72 and ident= 96%). leishmania tropica wright, 1903 its2 sequence was obtained from genbank and used as an out group for construction of phylogenetic tree. all the l. major (n=7) sequences obtained in this study clustered with l. major from genbank (accession number: aj786164) and formed a separate clade in the tree. also, l. trunica obtained in this study was sister taxon with l. turanica from genbank (accession number: hf545838) and both associated with l. gerbilli (accession number: hf545839) and formed separate clade (fig. 2). the genetic distance coefficient showed no difference between the sequences of l. major in this study and the sequence recorded in the genbank. the evolutionary correlation coefficient between the l. tropica and the sequence of l. major and l. turanica was 0.516 and 0.550, respectively. in addition, the difference of evolutionary correlations between the l. turanica sequence recorded in the genbank and the sequence of l. turanica in this study was 0.008 (table 3). table 1. abundance and rate of seasonal leishmania parasite infection in rodents detected by nested-pcr assay of its2-rdna gene, segzi area, esfahan province, october 2016–october 2017 season a ll c a p tu r e d r o d e n ts infected rodents with leishmania spp. total infected rodents (%) l. major (%) l. turanica (%) l. major + l. turanica (%) l. major + l. gerbilli (%) l. major + l. turanica + l. gerbilli (%) fall 19 11(57.9) 1(5.26) 1(5.26) 3(15.79) 0(0) 16(84.21) winter 11 1(9.09) 0(0) 3(27.28) 0(0) 1(9.09) 5(45.45) spring 28 9(32.14) 7(25) 3(10.72) 2(7.14) 3(10.72) 24(85.71) summer 35 16(45.73) 6(17.14) 7(20) 2(5.71) 2(5.71) 33(94.29) total 93 37(39.79) 14(15.05) 14(15.05) 7(7.53) 6(6.45) 78(83.87) fig. 1. different species of collected ectoparasites collected from rodents in segzi, esfahan province, 2016-2017; a: coptopsylla mesghalii, b: echidnophaga oschanini, nosopsyllus turkmenicus turkmenicus (c), nosopsyllus ziarus (d), xenopsylla nuttalli (e), rhipicephalus sanguineus (f), polyplax spp. (g), hirstionyssus sp. (h) and dermanyssus sanguineus (i) (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 129 http://jad.tums.ac.ir published online: june 30, 2022 table 2. abdominal status and leishmania parasite infection in rodents’ ectoparasites detected by nested-pcr assay of its2-rdna gene, segzi area, esfahan province, october 2016–october 2017 species of ectoparasites n o . o f e x a m in e d s a m p le s pcr-positivity of the ectoparasites with leishmania spp. n o . o f p o si ti v e sa m p le s b lo o d -f e d b lo o d -d ig e st e d l. major l. turanica l. major + l. turanica l. major + l. gerbilli l. major +l. turanica + l. gerbilli xenopsylla nuttalli 40 27 22 5 23 0 2 2 0 echidnophaga oschanini 8 6 6 0 4 0 0 0 2 nosopsyllus ziarus 3 3 3 0 2 1 0 0 0 coptopsylla mesghalii 3 3 3 0 3 0 0 0 0 dermanyssus sanguineus 5 4 4 0 4 0 0 0 0 hirstionyssus sp. 3 2 1 1 2 0 0 0 0 rhipicephalus sanguineus 1 1 1 0 1 0 0 0 0 total 63 46 40 6 39 1 2 2 2 fig. 2. phylogenetic tree of the partial its2 gene sequences of the isolated leishmania parasites from ectoparasites (mk372248-49 and mk372251-54) and smear of rodents' ear lobes (mk372246-50) and the its2 sequences obtained from genbank. bootstrap values that are reported as percentages at the nodes were obtained from 1000 bootstrap replicates. scale bar corresponds to 0.1 change per nucleotide http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 130 http://jad.tums.ac.ir published online: june 30, 2022 table 3. comparison of the its2 sequences of the isolated leishmania parasites from ectoparasites and smears of ear lobes of rodents, segzi area, esfahan province, october 2016–october 2017 leishmania species with genbank accession numbers 1 2 3 4 5 6 7 8 9 10 11 12 m k 3 7 2 2 4 6 h f 5 4 5 8 3 8 h f 5 4 5 8 3 9 m k 3 7 2 2 4 8 m k 3 7 2 2 4 9 m k 3 7 2 2 5 0 m k 3 7 2 2 5 1 m k 3 7 2 2 5 2 m k 3 7 2 2 5 3 m k 3 7 2 2 5 4 a j 7 8 6 1 6 4 k r 7 0 6 3 7 4 1 l. turanica mk372246 0.000 2 l. turanica hf545838 0.008 3 l. gerbilli hf545839 0.042 0.042 4 l. major mk372248 0.203 0.211 0.203 5 l. major mk372249 0.203 0.211 0.203 0.000 6 l. major mk372250 0.203 0.211 0.203 0.000 0.000 7 l. major mk372251 0.203 0.211 0.203 0.000 0.000 0.000 8 l. major mk372252 0.203 0.211 0.203 0.000 0.000 0.000 0.000 9 l. major mk372253 0.203 0.211 0.203 0.000 0.000 0.000 0.000 0.000 10 l. major mk372254 0.203 0.211 0.203 0.000 0.000 0.000 0.000 0.000 0.000 11 l. major aj786164 0.203 0.211 0.203 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 12 l. tropica kr706374 0.550 0.559 0.542 0.516 0.516 0.516 0.516 0.516 0.516 0.516 0.516 0.000 note. these sequences were compared with the available its2 sequences in genbank (aj786164.1, hf545839.1, hf545838.1 and kr706374.1). the correlation coefficient was calculated using mega 7 software and pairwise distance matrices method using p distance model. discussion the current study showed that r. opimus was found in large numbers in segzi. the distribution of r. opimus has a strong correlation with the climatic conditions and topography of the area, such as the seasonal rainfall and average annual temperature (22). in recent years, because of the population growth, making factories and residential houses near the colonies of the reservoir hosts, and expansion of the city, leishmanial infections in rodent populations and humans have increased (23). also, with the emergence of drought in the segzi plain, rodents have invaded the outskirts of the city. so, contact of rodents with humans has increased and subsequently, the leishmanial infection rates have increased. in a study on zcl by akhavan et al. (20) in three rural districts (borkhar, segzi and badrood) of esfahan province, 21 out of 95 r. opimus were positive by microscopic examination and 48 of them by nested pcr. the results of our and other recent studies (20, 21, 24) indicate that l. major, l. gerbilli, and l. turanica, and mixed natural infections exist in r. opimus populations (table 1). in this http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 131 http://jad.tums.ac.ir published online: june 30, 2022 study, the highest infection rates with l. major and l. turanica in r. opimus populations were observed in summer and spring respectively, and the highest percentage of l. major and l. turanica coinfections was seen in winter. the least infection rate of l. major was in the winter (table 1). mixed natural infections with l. major and l. turanica in r. opimus populations are typical in central asia (25). leishmania turanica raises the persistence of l. major infection in r. opimus (26). therefore, mixed natural infections with l. major and l. turanica help to preserve leishmaniasis in rodent populations. according to the results of this study, young r. opimus gerbils matured after the winter which more than 40% of them were infected (table 1) at the emerging of p. papatasi and probable vectors in late may. due to the suitable environmental and climatic conditions and the consequent abundance of food resources, gerbils reproduce twice or more per year (19, 27). in the segzi region, which receives the most rainfall in late winter and early spring, a significant increase in population rates was observed in spring. from early spring to summer, the increasing trend in the numbers of r. opimus declined (27) and the rate of bites per rodent increased. so, it is expected that the chances of parasite ingestion by p. papatasi and ectoparasites from infected rodents are then greater. in our study, phylogenetic analysis results showed no significant difference between the l. major sequences and l. major isolated from rodents and ectoparasites were located in the same clade or monophyletic group. it means that l. major parasite extracted from rodents were not genetically different from those extracted from ectoparasites and were similar (fig. 2 and table 3). zoonotic cutaneous leishmaniosis is increasing esfahan province, central iran (28), while the leishmanial infection rates of the main vector are low in the endemic areas (29). therefore, it could be suggested that other vectors could have a role in disease transmission. in aran and bidgol city in esfahan province, doroodgar et al. (29) reported that 17.8% of r. opimus, 71.4% of human isolates and 1.9% of p. papatasi infected with l. major. the possibility of the transmission of leishmania parasites by ectoparasites has long been discussed (6, 11, 30) and has been already proved in laboratory conditions (5, 13, 31). the present study showed that ectoparasites of the rodents can easily ingest leishmania parasites during blood-feeding. however, just only this evidence cannot prove that these ectoparasites are vectors of zcl. in our study, pcr analysis of ectoparasites specimens to detect the dna of leishmania spp. showed 73.02% (46/63 ectoparasites) positivity and the dna of l. major was detected in 7 of ectoparasite species, including r. sanguineus, x. nuttalli, e. oschanini, n. ziarus, c. mesghalii, hirstionyssus sp. and d. sanguineus by nested pcr (table 2). the high positivity rate in the ectoparasites is related to their life habits and the long duration of blood feeding. around 52.6 % (41/78) of the rodents infected with leishmania spp. had been infested with the ectoparasites. two studies by mckenzie and ferreira et al. showed transmission of leishmania parasites by r. sanguineus and c. felis in laboratory conditions (6, 7). in the mid-1980s, mckenzie (6) demonstrated that the collected r. sanguineus from naturally infected dogs can inject leishmania parasites into the healthy dogs during blood-feeding and infect them. ferreira et al. (7) demonstrated that c. felis fleas collected from infected dogs could transmit leishmania spp. noninfected laboratory hamsters and it was observed that 18.1% of the hamsters were positive by both methods of pcr and enzyme-linked immunosorbent assay (elisa). coutinho et al. (5), found that six r. sanguineus ticks (15.4%) were positive for l. chagasi using the pcr technique. they showed that r. sanguineus could transfer l. chagasi from infected dogs to hamsters in laboratory conditions. in another study, the promastigotes of l. chagasi were observed in 4 stained smears out of 207 (1.9%) c. felis felis specimens collected from dogs, whereas leishmania spp. infection was reported in 43 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 132 http://jad.tums.ac.ir published online: june 30, 2022 out of 144 (29.9%) fleas by pcr (13). in a study in northwest of iran by azarm, in which three species of fleas; c. canis curtis, 1826, c. felis and pulex irritans linnaeus, 1758 were collected from dogs, 75% of c. canis and 66.7% of c. felis collected from infected dogs were pcr-positive for l. infantum. but leishmania dna was not detected from p. irritans (32). in fact, coutinho et al. (5), mckenzie (6), ferreira et al. (7), and coutinho and linardi (13) demonstrated that leishmania parasites can be viable in the blood-sucking arthropods (such as tick and fleas) in laboratory conditions and infected their vertebrate hosts, but, they did not prove that these arthropods could act as vectors of the leishmania parasite in nature. in the current study, five specimens of x. nuttalli fleas and one hirstionyssus sp. mite, which were gravid, were found leishmania dna pcr positive (table 2). in xenopsylla spp. fleas, blood digestion lasts 2–9 days depending on the temperature, relative humidity, and the host species. in fleas, the duration of digestion is shorter at low relative humidity than high relative humidity (33). in a study by colombo, l. infantum was detected in 23% of the fleas and 50% of the ticks collected from the infected dogs by reverse transcription pcr (rt-pcr), real time pcr, and elisa. in addition, rna analysis of the tick specimens collected from infected dogs after seven to ten days showed that the parasites were alive. moreover, the alive parasites were isolated from adult ticks that had molted recently (34). in a study conducted in brazil, the results of immunohistochemistry (ihc) and real time pcr showed leishmania spp. promastigotes in the intestine, ovaries, and salivary glands of the r. sanguineus ticks collected from infected dogs (35). probably leishmania spp. may remain in these ectoparasites such as x. nuttalli and hirstionyssus sp., at least until full digestion of blood meal. however, the detection of leishmania dna is not sufficient evidence of parasite survival in x. nuttalli and hirstionyssus sp., and this re quired more careful study. in another study in turkey, to investigate the presence of l. major, the pools of r. sanguineus ticks on meriones unguiculatus milneedwards, 1867 were examined by rt-pcr. the results showed that none of the pools was infected with l. major (36). however, in this study the main reservoir has not been tested for the potency of ticks in the transmission of l. major. a study by rakhshanpour et al. (37), in iran showed that approximately 67% of the r. sanguineus ticks collected from dogs were infected with l. infantum using the semi-nested pcr. however, none of the parasitological (giemsa staining and cultivation of parasite) and molecular (nested pcr) tests results were positive when the transmission of l. infantum by stained r. sanguineus between dogs was studied in laboratory conditions. in fact, blood-feeding arthropods are susceptible to infection with different types of pathogenic microorganisms, but it does not mean that they can transmit all of them (38). although there is no strong evidence indicating that ectoparasites act as a vector of the leishmania parasites; recent studies suggest that this theory is important. evolutionarily, it is unclear how long it would take for the leishmania parasites to adapt to other blood-feeding arthropods. this is a fact that the leishmania parasites have a long evolutionary history with the main ectoparasites of leishmanial spp. reservoirs, such as l. infantum with r. sanguineus and c. felis (dog ectoparasites) (5, 7) and l. major with x. nuttalli (ectoparasite of r. opimus) (39, 40) and they have been in contact during this time. therefore, it is possible that the ectoparasites will retain leishmania parasites over time and act between reservoirs as potential vectors. conclusion in conclusion, this study found that l. major, l. gerbilli, and l. turanica exist in r. opimus populations in segzi area. the highest infection rates of r. opimus populations with l. mahttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 124–135 s azarmi et al.: pcr positivity of … 133 http://jad.tums.ac.ir published online: june 30, 2022 jor and l. turanica were observed in the summer and spring respectively, and the highest percentage of l. major and l. turanica coinfection was seen in the winter. according to the results of this study, it is possible that l. major remains in the ectoparasites such as x. nuttalli and hirstionyssus sp., at least until the blood fed is digested. therefore, the presence of l. major in the ectoparasites indicates the probable importance of rodents’ ectoparasites in zcl dispersion. thus, to study the probable role of ectoparasites in the transmission of l. major in rural areas between reservoirs and humans, it should be investigated whether the parasites remain viable inside the ectoparasites and performed xenodiagnosis test on the main reservoirs for experimental infections. acknowledgements all authors sincerely thank the head and staff of esfahan health research station for their assistance in the project. ethical considerations animal experiments were admired by the ethics committee of the tehran university of medical sciences, tehran, iran (the ethical code: ir.tums.sph.rec.1396.2804). conflict of interest statement authors declare that there is no conflict of interest. references 1. bakhshi h, oshaghi ma, abai mr, rassi y, akhavan aa, mohebali m, hajaran h, mohtarami f, mirzajani h, maleki-ravasan n (2013) mtdna cytb structure of rhombomys opimus (rodentia: gerbellidae), the main reservoir of cutaneous leishmaniasis in the borderline of iran-turkmenistan. j arthropod borne dis. 7(2): 173–184. 2. norouzi b, hanafi-bojd aa, moin-vaziri v, noorallahi a, azari-hamidian s (2020) an inventory of the sand flies (diptera: psychodidae) of rudbar county, a new focus of leishmaniasis in northern iran, with a taxonomic note on the subgenus larroussius. j arthropod-borne dis. 14 (3): 302–316. 3. ghatee ma, taylor wr, karamian m (2020) the geographical distribution of cutaneous leishmaniasis causative agents in iran and its neighboring countries, a review. front public health. 8: 11. 4. owens sd, oakley da, marryott k, hatchett w, walton r, nolan tj, newton a, steurer f, schantz p, giger u (2001) transmission of visceral leishmaniasis through blood transfusions from infected english foxhounds to anemic dogs. j am vet med assoc. 219(8): 1076–1083. 5. coutinho mtz, bueno ll, sterzik a, fujiwara rt, botelho jr, de maria m, genaro o, linardi pm (2005) participation of rhipicephalus sanguineus (acari: ixodidae) in the epidemiology of canine visceral leishmaniasis. vet parasitol. 128 (1–2): 149–155. 6. mckenzie kk (1984) study of the transmission of canine leishmaniasis by the tick, rhipicephalus sanguineus (latreille) and an ultrastructural comparison of the promastigotes. 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(5.48%). aedes pulcritarsis and cx. perexiguus were found for the first time in guilan province. the active season of adult mosquitoes extended from early may to early oct in the fixed site. there was no significant regression between the abundance of adult mosquitoes and the meteorological data during active season in the fixed site (p> 0.05, r2= 0.31). conclusion: though no significant regression between the abundance of mosquitoes and the meteorological data was observed during active season, temperature and rice fields had a great influence in starting and ending active season in the region. keywords: dirofilaria, flavivirus, flaviviridae, vectors, iran introduction west nile virus (wnv) (flaviviridae: flavivirus) and its subtype kunjin is distributed in eurasia, africa, north and central america and australia. mosquitoes (diptera: culicidae), especially ornithophilic species, are the principal vectors of the virus and some virus isolations have been reported from soft and hard ticks. wild birds, especially wetland species, are the principal vertebrate hosts, the virus has also been isolated from mammals and frogs (1, 2). wnv infection is recorded from horses in at least 26 provinces (out of total 31) in iran (3–5), humans (6–12) and birds (13). guilan province in the caspian sea littoral of northern iran, with vast wetlands, is one of the foci of wnv where infections are found in humans (1.4–10%) (4, 7–8), horses (2.2–25%) (3–4) and birds (especially the common coot, the main reservoir) (62.7%) (13). recently, the virus was found in aedes (ochlerotatus) caspius (pallas) s.l. [ochlerotatus caspius s.l.] in west azerbaijan province, northwestern iran, and *corresponding author: dr shahyad azarihamidian, e-mail: azari@gums.ac.ir j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 399 http://jad.tums.ac.ir published online: december 25, 2018 in cx. pipiens linnaeus in guilan province, northern iran, respectively (14, 15). dirofilariasis is a disease caused by different species of the nematode genus dirofilaria (spirurida: onchocercidae), especially d. immitis (canine or dog heartworm) and d. repens, transmitted by mosquitoes. the disease is cosmopolitan. the reservoirs of the nematodes are many different mammals, especially canids. previously, human dirofilariasis (hd) was considered a rare disease, but at the present time, hd is classified as an emerging disease in some areas because the number of reported cases was dramatically increased (16). dirofilariasis is found in humans, dogs, wolves, jackals, foxes and cats in at least 15 provinces of iran (17–22). guilan province is one of the foci of dirofilariasis, where d. repens infection is found in humans (17, 23) and d. immitis found in 4.4% (24) to 51.4% of local dogs (25, 26). culex theileri theobald is a known vector of d. immitis in northwestern iran (27). the last checklist of iranian mosquitoes comprises 64 species and seven genera (28, 29). subsequently, anopheles superpictus grassi includes two species in iran based on the internal transcribed spacer 2 (its2) sequences of rdna (30), later listed as species a and b (31). a new species of the anopheles hyrcanus group (an. hyrcanus spir) was recognized from southwestern iran, also based on its2 sequences (32). more recently, the occurrence of aedes (stegomyia) albopictus (skuse) [stegomyia albopicta] and ae. (stg.) unilineatus (theobald) [stegomyia unilineata] were reported in southeastern iran and orthopodomyia pulcripalpis (rondani) in northern iran, respectively (33–35). overall, 30 species of mosquitoes representing seven genera were listed in guilan province (36). a large amount of available data on mosquitoes in iran is based on collections and ecology of larvae (27, 36–40 and many other references cited by aforementioned articles). different methods of collecting adult mosquitoes, such as using light traps, aspirators, pit shelters and total catch (pyrethrum space spray), have been used mostly in relation to anopheline vectors of malarial protozoa (41–47). there are a few published documents in the country that deal with adult sampling, especially using light traps, which include culicines (27, 48–50), but there are no studies of seasonal activity. that is why there is very little information about the seasonal activity of culicine adults in iran. this study was carried out by means of light traps to study the seasonal activity of mosquitoes, especially probable and proven vectors of wnv and dirofilaria, in guilan province, northern iran. materials and methods study area guilan province locates in the caspian sea littoral of northern iran, between the caspian sea and the alborz mountain range. it has coastal, plain, foothill, and mountainous areas with an area of approximately 14,700 square kilometers. the province is bordered by mazandaran province in the east, ardabil province in the west and zanjan and qazvin provinces in the south. it is also bordered by the republic of azerbaijan in the north as well as russia across the caspian sea (fig. 1). the province has a temperate climate and relatively warm-humid summer. it is located between 36o33'–38o27' n latitude and 48o32'–50o36' e longitude and formally includes 16 counties. most areas of guilan province with about 1000–2000mm of rainfall annually, have the greatest amount of rainfall in iran, and the main agricultural crop is rice. the province has vast deciduous forests of hyrcania, temperate climate, vast wetlands and rice fields, which provide abundant habitats for mosquitoes. specimen and data collection in seven counties (including different topographical areas of the province) adult collections were carried out at least two times from j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 400 http://jad.tums.ac.ir published online: december 25, 2018 random (variable) sites during aug–dec 2015 and apr–oct 2016 (fig. 1, table 1). sampling was also carried out at a fixed site (pareh village of rudbar county, 36o 50.800' n, 49o 32.650' e, altitude 487m) every two weeks from apr to oct 2016. rudbar county in southern guilan has about 200–500mm annual rainfall and showed mountainous and less humid temperate climate similar mediterranean region. pareh village is in a foothill area and close to natural hyrcanian forest and manmade woodland that includes trees such as olive, walnut, fig, persian ironwood (parrotia persica) and caucasian elm or caucasian zelkova (zelkova carpinifolia). the main livelihood of the people in the village is husbandry and the main domestic animals are cattle and sheep. dogs, horses, donkeys and fowls are also common animals in the region. the meteorological data of pareh village during 2016 is shown in table 2. two cdc light traps were used in each variable and fixed site. the light traps were suspended from the ceiling in animal shelters from sunset to sunrise, i.e. from 1800 to 0600 hrs. the electricity of traps was provided by 6-volt rechargeable batteries. moreover, ad hoc collections were carried out using manual aspirators (hand catch) in the fixed and random sites. the specimens were identified using the morphological-based keys (29). the abbreviations of mosquito genera and subgenera follow reinert (51). the specimens are deposited at the museum of medical and veterinary entomology, school of health, guilan university of medical sciences, rasht, iran. determining species dominance structure the dominance structure of a species is expressed as the percentage of specimens of the species in the whole sample. the following five percentage representation categories (52, 53) were used: eudominat (ed) species (> 30%), dominant (d) (10–30%), subdominant (sd) (5–10%), recedent (r) (1–5%) and subrecedent (sr) (< 1%). mapping collected mosquitoes and statistical analysis arcgis 10.3 was used to create a geo-database of mosquitoes and to map the collection sites and the distributions of the most medically important species. the statistical analysis of mosquito abundance and meteorological data was carried out using the linear regression test of spss software (ver. 16 for windows, spss inc., chicago, il). results mosquito fauna overall, 16327 adult mosquitoes were collected during 29 surveys (aug–dec 2015 and apr–oct 2016): 15959 (97.75%) were captured using light traps and 368 (2.25%) by ad hoc hand catches (table 3). eighteen species representing seven genera were identified morphologically: anopheles (anopheles) claviger (meigen), an. (ano.) hyrcanus (pallas), an. (ano.) maculipennis meigen s.l., an. (ano.) pseudopictus grassi, an. (cellia) superpictus, aedes (aedimorphus) vexans (meigen) [aedimorphus vexans], ae. (dahliana) geniculatus (olivier) [dahliana geniculata], ae. (ochlerotatus) caspius s.l. [ochlerotatus caspius s.l.], ae. (och.) pulcritarsis (rondani) [oc. pulcritarsis], coquillettidia (coquillettidia) richiardii (ficalbi), cx. (culex) mimeticus noè, cx. (cux.) perexiguus theobald, cx. (cux.) pipiens, cx. (cux.) theileri, cx. (cux.) tritaeniorhynchus giles, culiseta (culiseta) annulata (schrank), orthopodomyia pulcripalpis and uranotaenia (pseudoficalbia) unguiculata edwards (table 3). aedes pulcritarsis and cx. perexiguus were found for the first time in guilan province. species dominance structure overall, 2734 anopheline adults (16.71%) and 13,593 culicine adults (83.29%) were collected. the most prevalent species were cx. theileri (23.59%, dominant), cx. tritaeniorhynchus (20.75%, dominant), cx. pipiens (19.37%, dominant), ae. vexans (18.18%, dominant), an. j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 401 http://jad.tums.ac.ir published online: december 25, 2018 pseudopictus (10.92%, dominant) and an. maculipennis s.l. (5.48%, subdominant). these six species included 16,054 specimens (98.1%) of the whole sample (table 3). moreover, they showed the widest distributions in the province (table 4, fig. 2). regarding the dominance structure of subfamily anophelinae, an. pseudopictus and an. maculipennis s.l. with the abundance percentages of 65.2% and 32.8% respectively, were both eudominant. in the case of percentage representation of subfamily culicinae, cx. theileri (28.34%), cx. tritaeniorhynchus (24.93%), cx. pipiens (23.27%) and ae. vexans (21.84%) were dominant, as in the case of all mosquitoes, i.e. the total for both subfamilies. seasonal activity and the fluctuations of rainfall and temperature in general, the active season of adult mos quitoes extended from early may to early oct in the fixed site (pareh village of rudbar county). the peak of activity was late june for cx. theileri, mid-july for an. maculipennis s.l., an. pseudopictus and cx. pipiens, and late july for cx. tritaeniorhynchus. while the peak of activity of most adult mosquitoes was late june to mid-july, and the abundance dramatically decreased after that, the monthly mean temperature increased by aug. also after apr rainfall decreased in the fixed site during jun and jul and the rainy season started in sep (figs. 3–6). there is no significant regression between the abundance of adult mosquitoes and the meteorological data (table 2) during active season in the fixed site (p> 0.05, r2= 0.31). table 1. collection data for adult mosquitoes captures at variable sites in guilan province, iran, august–december 2015 and april–october 2016 locality (city/ village) topography county coordinates altitude (m) rostamabad plain rudbar 36o 52.999' n, 49o 29.385' e 215 joben foothill rudbar 36o 53.072' n, 49o 27.658' e 399 khaskool foothill rudbar 36o 50.789' n, 49o 32.669' e 470 lafandsara foothill rudbar 36o 50.522' n, 49o 32.271' e 620 rudbar foothill rudbar 36o 49.314' n, 49o 25.322' e 270 klayeh foothill rudbar 36o 50.992' n, 49o 32.132' e 438 rudabad plain rudbar 36o 52.397' n, 49o 30.871' e 192 harkian foothill rudbar 36o 59.592' n, 49o 33.491' e 149 siahroodposhteh foothill rudbar 36o 59.862' n, 49o 33.432' e 269 upper harzavil (manjil) foothill rudbar 36o 44.495' n, 49o 26.072' e 506 lower harzavil (manjil) foothill rudbar 36o 44.837' n, 49o 25.735' e 453 halaj (loshan) foothill rudbar 36o 40.306' n, 49o 26.792' e 307 kacha foothill rasht 37o 05.173' n, 49o 36.973' e 124 saghalaksar plain rasht 37o 09.596' n, 49o 31.334' e 53 ghazian coastal anzali 37o 27.347' n, 49o 28.663' e -21 kandbon plain rudsar 37o 03.415' n, 50o 20.987' e 20 kalesara foothill talish 37o 42.251' n, 48o 55.577' e 93 eivazmahaleh foothill astara 38o 23.964' n, 48o 46.715' e 77 sechekeh foothill siahkal 37o 06.755' n, 49o 50.985' e 205 asooyebala (tootaki) mountainous siahkal 37o 03.556' n, 49o 52.542' e 355 j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 402 http://jad.tums.ac.ir published online: december 25, 2018 table 2. the meteorological data of the fixed site (pareh village of rudbar county), guilan province, iran, 2016 meteorological data april may june july aug september october november maximum temperature 20.88 26.71 30.00 29.68 33.53 27.94 20.64 14.91 minimum temperature 10.79 16.56 20.17 21.52 22.60 18.77 12.92 6.25 average temperature 15.84 21.64 25.08 25.60 28.06 23.36 16.78 10.58 relative humidity 69.06 67.04 59.58 65.86 56.53 67.63 72.65 67.39 rainfall 40.34 17.89 10.73 36.95 2.22 56.71 58.87 73.30 fig. 1. map of iran highlighting the location of guilan province including mosquito collection sites surveyed in 2015–2016 j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 403 http://jad.tums.ac.ir published online: december 25, 2018 table 3. the collection method and composition of adult mosquitoes collected in guilan province, iran, august– december 2015 and april–october 2016 species light trap hand catch total dominance structure n % n % n % an. claviger 18 0.11 2 0.54 20 0.12 subrecedent an. hyrcanus 29 0.18 29 0.17 subrecedent an. maculipennis s.l. 752 4.71 144 39.13 896 5.48 subdominant an. pseudopictus 1760 11.03 23 6.25 1783 10.92 dominant an. superpictus 6 0.04 6 0.04 subrecedent ae. caspius s.l. 96 0.60 96 0.60 subrecedent ae. geniculatus 1 0.01 1 0.01 subrecedent ae. pulcritarsis 7 0.04 7 0.04 subrecedent ae. vexans 2930 18.36 39 10.59 2969 18.18 dominant cq. richiardii 94 0.59 1 0.28 95 0.60 subrecedent cx. mimeticus 8 0.05 1 0.28 9 0.06 subrecedent cx. perexiguus 6 0.04 6 0.04 subrecedent cx. pipiens 3030 18.98 134 36.41 3164 19.37 dominant cx. theileri 3844 24.09 9 2.44 3853 23.59 dominant cx. tritaeniorhynchus 3375 21.15 14 3.80 3389 20.75 dominant cs. annulata 1 0.01 1 0.01 subrecedent or. pulcripalpis 1 0.28 1 0.01 subrecedent ur. unguiculata 2 0.01 2 0.01 subrecedent total 15959 100 368 100 16327 100 table 4. the distribution of adult mosquitoes collected in different counties in guilan province, iran, august– december 2015 and april–october 2016 locality species county rudbar rasht anzali rudsar talish astara siahkal an. claviger * an. hyrcanus * * an. maculipennis s.l. * * * * * * an. pseudopictus * * * * * * an. superpictus * ae. caspius s.l. * * ae. geniculatus * ae. pulcritarsis * * ae. vexans * * * * * cq. richiardii * cx. mimeticus * * cx. perexiguus * cx. pipiens * * * * * * * cx. theileri * * * * cx. tritaeniorhynchus * * * * * * * cs. annulata * or. pulcripalpis * ur. unguiculata * j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 404 http://jad.tums.ac.ir published online: december 25, 2018 fig. 2. distribution map of the most prevalent and medically important mosquitoes in the study areas in guilan province, iran, 2015–2016 fig. 3. biweekly abundance of the most prevalent anopheline mosquitoes and monthly mean temperature in the fixed site, pareh village of rudbar county, guilan province, iran, april–october 2016 (total includes anopheles claviger, an. maculipennis s.l., an. pseudopictus, an. superpictus, ae. pulcritarsis, culex mimeticus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus) j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 405 http://jad.tums.ac.ir published online: december 25, 2018 fig. 4. biweekly abundance of the most prevalent anopheline mosquitoes and monthly rainfall in the fixed site, pareh village of rudbar county, guilan province, iran, april–october 2016 (total includes anopheles claviger, an. maculipennis s.l., an. pseudopictus, an. superpictus, ae. pulcritarsis, culex mimeticus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus) fig. 5. biweekly abundance of the most prevalent culicine mosquitoes and monthly mean temperature in the fixed site, pareh village of rudbar county, guilan province, iran, april–october 2016 (total includes anopheles claviger, an. maculipennis s.l., an. pseudopictus, an. superpictus, ae. pulcritarsis, culex mimeticus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus) j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 406 http://jad.tums.ac.ir published online: december 25, 2018 fig. 6. biweekly abundance of the most prevalent culicine mosquitoes and monthly rainfall in the fixed site, pareh village of rudbar county, guilan province, iran, april–october 2016 (total includes anopheles claviger, an. maculipennis s.l., an. pseudopictus, an. superpictus, ae. pulcritarsis, culex mimeticus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus) discussion mosquito fauna during the present investigation, 18 species representing seven genera of mosquitoes were collected in guilan province in which ae. pulcritarsis and cx. perexiguus were new records for the province. thus, the number of species recorded in the province increased from 30 (36) to 33 (35 and the present investigation). seven species, an. (ano.) plumbeus stephens, ae. (dah.) echinus (edwards) [dahliana echinus], cx. (maillotia) hortensis ficalbi, cx. (cux.) torrentium martini, cx. (neoculex) territans walker, cs. (allotheobaldia) longiareolata (macquart) and cs. (culicella) morsitans (theobald) whose larvae were collected during recent years in guilan province (36), were not captured in the present study. the reason is the rarity of some aforementioned species and the tendency of some, such as cx. hortensis, cx. territans and cs. morsitans, to feed on birds, amphibians or reptiles (54), thus they were not attracted to light traps used in animal (cattle and sheep) shelters during the study. also, there are seven species of the maculipennis group in guilan province differentiated by egg patterns or the polymerase chain reaction (pcr) technique (36), which could not be differentiated with the morphologybased keys of females and larvae (29) used in the present study. among the species collected, ae. caspius s.l. is known to vector wnv in west azerbaijan province in northwestern iran (14). shahhosseini et al. (55) referred to the virus later as kunjin-related west nile virus. moreover, wnv was found in cx. pipiens in the sepid-rud valley of guilan province (15). in addition, an. maculipennis s.l., ae. vexans, cq. richiardii, cx. perexiguus, cx. theileri, cx. tritaeniorhynchus and ur. unguiculata is believed to play role as vectors of wnv in different countries of the old world (1, 56). among the aforementioned species, cq. richiardii and cx. pipiens in europe and cx. tritaeniorhynchus in asia are the main vectors of j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 407 http://jad.tums.ac.ir published online: december 25, 2018 the virus (1). culex theileri has been found to be the vector of dirofilaria, the causal agent of dirofilariasis, in ardebil province in northwestern iran (27), and an. claviger, an. hyrcanus, an. maculipennis, an. pseudopictus, an. superpictus, ae. vexans, ae. caspius, ae. geniculatus, cq. richiardii, cx. pipiens, cx. tritaeniorhynchus, cs. annulata and ur. unguiculata are known the vectors of dirofilaria in different countries of the western palaearctic region (54, 57–59). species dominance structure in the present study, the most abundant species were cx. theileri, cx. tritaeniorhynchus, cx. pipiens, ae. vexans, an. pseudopictus and an. maculipennis s.l., respectively. with the exception of an. maculipennis s.l., which is subdominant, they are all dominant according to the classification (52, 53) (table 3). this is concordant with the previous findings in the province based on collections of larvae (36– 38), as well as adults (37, 60). the exception is cx. theileri. the species had been found less often in the larval stage than any other culex in the province (38). one reason is probably due to sampling, the heavy rainfall in the province results in a great number of different natural larval habitats that are favorable for cx. theileri (38), but those habitats are not easily located and sampled. on the other hand, the favorable larval habitats of some species, such as cx. pipiens and cx. tritaeniorhynchus, i.e. artificial containers and rice fields, respectively, are easier to find and sample. another reason is probably the biology of the species, some species such as cx. hortensis and cx. territans, which do not bite humans and mammals but mostly feed on amphibians, reptiles or birds (54), have been collected very often as larvae (38). they were not collected during the present investigation by means of aspirators and light traps from animal shelters which attract cx. theileri (table 3). the most prevalent species of the province, an. maculipennis s.l., an. pseudopictus, ae. vexans, cx. pipiens, cx. theileri and cx. tritaeniorhynchus are known vectors of both wnv and dirofilaria (1, 57, 59). seasonal activity and the fluctuations of rainfall and temperature during the present investigation, ae. vexans was one of most abundant species and most prevalent aedine species (table 3), as noted previously (37, 60). however, most specimens were collected from anzali (table 4) and the species was not collected from the fixed site, so its seasonal activity is not discussed here. anopheles maculipennis s.l. showed the peak of activity in the mid-jul (figs. 3, 4). the peak of monthly activity of anophelines (including an. maculipennis s.l. and an. superpictus) was reported during jul–aug in kalaleh county of golestan province, northern iran (47). the most an. maculipennis s.l. was captured in aug in aras valley, turkey, adjoining iran (61). there are no records for the seasonal activity of culicine adults in iran. the peak of activity of cx. pipiens was found in jul in northern italy (62). that is in concordance with the result of the present study (figs. 5, 6). however, the peak of activity of cx. pipiens was recorded in aug in the belek region and aras valley of turkey (61, 63). most cx. theileri was found in jun in aras valley of turkey (61), which is similar to the present study (figs. 5, 6), however, the peak activity of this species was reported in aug in ankara, turkey (64). moreover, most cx. tritaeniorhynchus was captured in aug in belek region of turkey (63), while the peak of activity was observed in jul in the present study (figs. 5, 6). differences between the results of the present investigation and the findings in other regions may be explained by differences in the topography and climates (especially temperature) which influence the bionomics of mosquitoes. on the other hand, some differences are due to sampling regimes. for example, mosquito abundance was reported based on weekly catches (62), and on monthly catches (61, 63, 64), whereas the seasonal j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 408 http://jad.tums.ac.ir published online: december 25, 2018 activity is based on biweekly captures in the present study and another study (47). besides, dry-ice baited traps was used for sampling by alten et al. (63), bg-traps by roiz et al. (62) and spray sheet collections by sofizadeh et al. (47), whereas light traps were used by simsek (64), alkan and aldemir (61) and in this study. though the mean monthly temperature of about 16 °c is a limiting factor in the activity of adult mosquitoes in the study area (figs. 3, 5), no significant regression was observed between different meteorological data (table 2) and the abundance of adult mosquitoes during active season (p> 0.05, r2= 0.31). during the present study, the peak of activity of most adult mosquitoes was late jun to mid-jul, only the peak of activity of cx. tritaeniorhynchus was in late jul (figs. 3–6), after which the abundance dramatically decreased as temperature increased by aug (figs. 3, 5). high temperature (> 35 °c) is generally a limiting factor for the abundance of adult mosquitoes, especially in localities with warm climate such as southern iran (41) and saudi arabia (65). however, it does not seem that temperature was a key factor in decreasing the abundance of adult mosquitoes in the area of the present study, because the temperature does not exceed 34 °c and the monthly mean temperature is lower than 30 °c in the fixed site in aug (figs. 3, 5). in addition, rainfall decreased in the fixed site during jun and jul while the abundance increased. the rainy season started in sep while the abundance of mosquitoes dramatically decreased (figs. 4, 6). a key factor mentioned here is rice fields, the main larval habitats of the most prevalent species, are dry during aug. temperature decreases significantly during sep and oct, consequently, the prevalence of mosquitoes decreases. in view of integrated vector management, ecological data, especially seasonal activity, is very important for intervention measurements. on the other hand, one of main intervention measurements is using pesticides yet. there is little-published data about the susceptibility status of mosquitoes, especially culicines (66, 67), in northern iran. this subject can be a goal for forthcoming studies in guilan province. conclusion though there was no significant regression between the abundance of adult mosquitoes and the meteorological data in the fixed site during active season, temperature and rice fields had a great influence in starting and ending active season in the region. the seasonal activity of the important species ae. vexans, other species found less abundant in this study, host preference analysis and filarial and arbovirus screening should be the subjects of future investigations in the region. acknowledgements the authors appreciate the administrative support of dr sm rezvani, mr m mirzanejad, mr m moslem and mr a rasaei, department of disease control and prevention, health vice-chancellorship of guilan university of medical sciences, rasht, iran, and the personnel of aforementioned vice-chancellorship for their cooperation in the field. ms r farhadi, and mr omidi, school of health, guilan university of medical sciences, are appreciated for helping in providing the graphs of seasonal activities and data analysis, respectively. the authors are grateful to dr re harbach, department of life sciences, natural history museum, london, uk, for reviewing the manuscript. the research was supported financially by the research vice-chancellorship of guilan university of medical sciences (project number: 93062502). the project was carried out in the school of health, guilan university of medical sciences and the research center of health and environment, guilan university of medical sciences, with the collaboration of health vice-chancellorship of guilan uni j arthropod-borne dis, december 2018, 12(4): 398–413 sh azari-hamidian et al.: seasonal activity of … 409 http://jad.tums.ac.ir published online: december 25, 2018 versity of medical sciences. the authors declare there are no conflicts of interest. references 1. hubálek z, halouzka j (1999) west nile fever-a reemerging mosquito-borne viral disease in europe. emerg infect dis. 5: 643–650. 2. medlock jm, snow kr, leach s 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evaluation of pyrethroid … 370 http://jad.tums.ac.ir published online: december 25, 2018 original article evaluation of pyrethroid susceptibility in culex pipiens of northern izmir province, turkey onur guntay 1, mehmet salih yikilmaz 1, huseyin ozaydin 1, *savas izzetoglu 1, asli suner 2 1section of molecular biology, department of biology, faculty of science, ege university, turkey 2department of biostatistics and medical informatics, faculty of medicine, ege university, turkey (received 6 mar 2018; accepted 21 oct 2018) abstract background: mosquitoes, being a nuisance species, are considered as one of the most important species in public health control programs due to their role as a vector in mosquito-borne diseases observed in humans and animals. we evaluated the susceptibility status of culex pipiens collected from northern izmir, turkey in 2011-16. methods: mosquito larvae, collected from three different locations in northern i̇zmir, were reared in the laboratory. adult susceptibility bioassays were performed using the who insecticide-impregnated papers including deltamethrin 0.05%, permethrin 0.75%, α-cypermethrin 0.05% and cyfluthrin 0.15%. in addition, adult bioassays were performed after the pre-exposure to piperonyl butoxide (pbo) to determine the contribution of p450 detoxification enzymes to the phenotypic resistance. results: in all of the three populations, high levels of resistance were observed (mortalities<63%) to all of the four pyrethroids. different pyrethroids but with the same mode of action can exhibit significantly different phenotypic resistance in a single population. pbo bioassays also showed that p450 detoxification enzymes can have diverse effects on different pyrethroids. conclusion: using just one chemical in a class of insecticide can be misleading for resistance studies. keywords: bioassay, insecticide resistance, pyrethroids, pbo, culex pipiens introduction more than 48 mosquito species have been identified in turkey including culex pipiens, is one of the major nuisances (1). culex species are important vectors for lymphatic filariasis and many other viral diseases such as west nile (2, 3). since the 1950s, the control of pests has gained momentum with the inclusion of chemical compounds in the integrated pest management. pyrethroids, which are in the recommended insecticides list of who for the adult mosquito control (4), are widely used in both indoor and outdoor in turkey. long-term and frequent applications of the same insecticide may select resistant individuals and this selection pressure may eventually lead to resistant populations becoming established (4-6). hence, monitoring of the resistance/susceptibility status of mosquito pop ulations to insecticides is very important for the success of the vector-control programs. the most common form of resistance to pyrethroids is called knock-down resistance (kdr), linked to a single nucleotide substitution in the vssc gene encoding the voltage-sensitive sodium channel protein and involves reduced targetsite sensitivity to pyrethroids (7, 8). the second common mechanism involved in pyrethroid resistance involves detoxification enzymes belonging to the three major enzyme families; namely, cytochrome p450 monooxygenases (cyps), glutathione-s-transferases (gsts) and carboxyl/ cholinesterases (cces). these enzymes cause metabolic detoxification of pyrethroids before they reach their target site (9-11). a few studies have reported the occurrence, distribution and some mechanisms involved in *corresponding author: dr savas izzetoglu, e-mail: savas.izzetoglu@ege.edu.tr j arthropod-borne dis, december 2018, 12(4): 370–377 o guntay et al.: evaluation of pyrethroid … 371 http://jad.tums.ac.ir published online: december 25, 2018 insecticide resistance among certain mosquito species throughout turkey and in neighboring countries such as greece and iran (12-22). yet, we have a very limited knowledge of the insecticide resistance status of mosquito populations in turkey. in this study, our aims were to determine resistance/susceptibility status of three natural populations of cx. pipiens in northern izmir, turkey and to understand to role of p450 detoxification enzymes in different pyrethroids (deltamethrin, permethrin, α-cypermethrin and cyfluthrin). materials and methods mosquito samples mosquito larvae were collected from three different field locations: maltepe village, menemen (n38°37’06”, e26°53’25”) in 2011, sasalı, çiğli (n38°28’23”, e26°56’51”) and ege university botanical garden, bornova (n38°27’33”, e27°14’01”) in 2016. sites were located in the same climatic area with a slight difference (the average winter and summer temperatures are 7.9 °c and 26.3 °c, respectively and the average annual rainfall is 687mm). larvae were transported to the laboratory and reared under conditions at a temperature of 25 °c and 70% (±5%) rh with a 12:12h light: dark photoperiod. preparation for morphological identification species identification based on morphology was performed by selecting 4th stage larvae from the three abovementioned populations. for the preparations, the method (23) has been used with slight modifications. culex pipiens larvae are yellowish-brown in color and have long-medium thickness siphon (24). these characteristics were used for identification. species determinations based on morphological keys (2527) were completed using stereomicroscope with an installed argenit camera (camera m5 cmos). susceptibility bioassays adult bioassays were performed following who guidelines (who/vbc/81.80) using the who susceptibility test kits. all specimens used in the bioassay were at fixed ages (3–5d old) and non-blood fed female adults. mosquitoes were exposed to filter papers impregnated separately with 0.05% deltamethrin (batch no: de 499, expiry date: apr 2018), 0.75% permethrin (batch no: pe 406, expiry date: apr 2018), 0.05% α-cypermethrin (batch no: al 237, expiry date: apr 2018), 0.15% cyfluthrin (batch no: cy 123, expiry date: apr 2018) and control (batch no: py 249, expiry date: mar 2018) for 1h. in order to evaluate the p450 detoxification enzyme activity, 4% pbo-impregnated papers (batch no: pb 019, expiry date: mar 2018) were pre-exposed for 1h before deltamethrin and permethrin applications. this test was not carried out for α-cypermethrin and cyfluthrin applications. we have called this test as synergist bioassay test within this manuscript. oil-impregnated papers were used for controls. all of the impregnated papers were obtained from who vector control research unit at universiti sains malaysia. during the experiments, knock-down (kd) rates were recorded every 15min (28). after completing the exposure time, mosquitoes were gently transferred into plastic cups and provided with 10% sucrose solution for 24h. experimental mortality was recorded after 24h recovery period. for each test session, 4 batches of 20–25 mosquitoes and 2 batches of 20–25 mosquitoes were exposed to insecticide-impregnated and oil-impregnated papers, respectively (28). for each insecticide, three replicates were carried out and the results were pooled to obtain the mean value of each test. temperature and humidity were maintained at 27 °c (±1), 80% (±10) rh throughout the bioassay and recovery period (28). data analysis statistical analyzes of the bioassay results were performed using kruskal-wallis and mannwhitney u tests with the ibm spss ver. 21.0 (chicago, il, usa). the normal distribution of the data was examined using shapiro-wilk j arthropod-borne dis, december 2018, 12(4): 370–377 o guntay et al.: evaluation of pyrethroid … 372 http://jad.tums.ac.ir published online: december 25, 2018 normality test. for all the hypotheses testing, an alpha of 0.05 was used as the cutoff for significance. results morphological identification some characteristics used for genus and species identification are shown in fig. 1. before applying bioassays, we identified that all populations collected were cx. pipiens. bioassays we have first analyzed the kd rates at the end of a 1h-application of each pyrethroid in each population. the pairwise comparisons of kd rates within the populations were tested for significance. out of 18 pairwise comparisons (3 populations, 4 different pyrethroid applications, therefore 6 pairwise comparisons within each population), 7 were found to be significant (*p< 0.05). these significant results were obtained from the comparisons between the kd rates of permethrin and deltamethrin, permethrin and cyfluthrin, and α-cypermethrin and cyfluthrin applications in both çiğli and bornova populations. moreover, one more significant result was also revealed from the pairwise comparison between kd rates of deltamethrin and cyfluthrin applications in menemen population. the knock-down rates at 1h observed in all the populations of the study were summarized in fig. 2. all of the cx. pipiens populations showed different levels of resistance to all tested insecticides with a mortality rate ranging between 0% and 63%. overall, the lowest mortality rates were obtained with permethrin (ranged between 0% and 21%) and the highest mortality rates with cyfluthrin (ranged between 8% and 63%) in all populations. mortality rates resulted from α-cypermethrin and deltamethrin applications ranged between 0% and 25% and between 0% and 38%, respectively. all the results obtained from susceptibility tests carried out in all of the three populations were summarized in table 1. mortalities observed in cyfluthrin applications were significantly different than the mortality rates obtained from the other three pyrethroids used in the study in both menemen and bornova populations (*p< 0.05). for çiğli population, we have found statistically significant difference (*p< 0.05) between mortality rates of cyfluthrin and permethrin applications as well as that of between cyfluthrin and α-cypermethrin applications. however, there was no significant difference (p> 0.05) between mortality rates of cyfluthrin and deltamethrin applications. in addition, there was no significant difference between mortality rates of permethrin and α-cypermethrin applications in çiğli population. furthermore, the pairwise comparison of mortality rates of permethrin, α-cypermethrin and deltamethrin applications in bornova and menemen populations, separately, did not reveal a significant difference. the synergist bioassay test carried out in menemen and bornova populations in order for the assessment of the contribution of p450 detoxification enzymes did not restore susceptibility to permethrin and deltamethrin. however, in both populations, pre-exposure to pbo significantly increased the effect of the pyretroids applied. in menemen population, the mortality rates significantly (*p< 0.05) increased from 1.24% (deltamethrin) to 23.96% (pbo+ deltamethrin) (estimated p= 0.000) and from 0.96% (permethrin) to 4.26% (pbo+ permethrin) (estimated p= 0.014). similarly, in bornova population, mortality rates significantly (*p< 0.05) increased from 16.98% (deltamethrin) to 55.96% (pbo+deltamethrin) (estimated p= 0.000) and from 8.15% (permethrin) to 27.95% (pbo+permethrin) (estimated p= 0.000). this bioassay was not carried out in çiğli population. the comparative results of the synergist bioassays were summarized in table 2. j arthropod-borne dis, december 2018, 12(4): 370–377 o guntay et al.: evaluation of pyrethroid … 373 http://jad.tums.ac.ir published online: december 25, 2018 table 1. mortality rate results (izmir, turkey, 2017) of permethrin (0.75%), α-cypermethrin (0.05%), deltamethrin (0.05%) and cyfluthrin (0.15%) tests obtained at 24h after the application in each of the tested culex pipiens populations were presented below (95% ci) populations % mortality (range) permethrin α-cypermethrin deltamethrin cyfluthrin menemen 0.96 (0–4) 1.83 (0–9) 1.24 (0–7) 17.97 (8–29) çiğli 2.52 (0–7) 2.59 (0–8) 13.12 (3–22) 27.42 (14–46) bornova 8.15 (0–21) 11.39 (0–25) 16.98 (0–38) 46.27 (36–63) table 2. susceptibility test results of culex pipiens with and without pre-exposure to pbo in northern izmir, turkey, 2017 (95% ci) populations insecticide tested % mortality (range) menemen deltamethrin 1.24 (0–7) pbo+deltamethrin 23.96 (8-35) permethrin 0.96 (0–4) pbo+permethrin 4.26 (0–9) bornova deltamethrin 16.98 (0–38) pbo+deltamethrin 55.96 (37–68) permethrin 8.15 (0–21) pbo+permethrin 27.95 (16–50) fig. 1. some characteristics used in morphological identification. a) the last abdominal segment, anal segment and siphon of 4th stage larvae. b) pecten teeth on siphon (shown by) and setae on siphon (all labeled with asterisk,). c) setae on the head; labeled as 4c-7c. d) combs on the last abdominal segment as shown by the arrow j arthropod-borne dis, december 2018, 12(4): 370–377 o guntay et al.: evaluation of pyrethroid … 374 http://jad.tums.ac.ir published online: december 25, 2018 fig. 2. knock-down rates of culex pipiens at 1h of each tested population (a-c) in northern izmir, turkey, 2017 discussion culex pipiens is the most common mosquito in turkey and mosquito control is mainly directed against larval stages besides, many pyrethroids are currently registered and used for adult control (15). many pesticides commonly used in agricultural areas and public health cause mosquito populations to become resistant. the insecticides with same mode of action used in these different areas cause cross-resistance (29, 30). resistance was expected in these populations due to frequent use of agricultural pesticides in the vicinity of the settlement areas where populations were collected. nevertheless, some mortality rates for permethrin, deltamethrin and αcypermethrin were 0%, and therefore the detection of such a high resistance was surprising. resistance to permethrin and deltamethrin was also detected in cx. pipiens populations collected from similar regions in 2012 and 2013, but such low mortality rates have not been determined (18). in our study, the overall mortality rate for deltamethrin was 17%. in contrast, value of this species for deltamethrin in northwestern and southeastern iran reported as 91% and 93% respectively (21, 22). nevertheless, in tehran, mortality rate for deltamethrin in same species was reported as 18% (20). in greece, the lowest mortality rate was reported 64% in 13 different populations of the same species (19). different mortality rates can occur within different pyrethroids with the same mode of action (31). however, in the same population, the difference between cyfluthrin and other pyrethroid mortality means was not expected to be as high as 38% (table 1). this may be related to the chemical structure of the pyrethroid used. the presence of the α-cyano group in the structure of the pyrethroid has an effect on the resistance mechanism(s) (32). this difference can also be explained by the reflection of different kdr mutations in phenotypic resistance in different ways (33). a possible third explanation is the different detoxification enzymes involved in metabolic resistance mechanisms. cyps are major mechanism of insecticide metabolic resistance (34). previous years, different results have been obtained about the role of gsts and cces in pyrethroid resistance (10, 35, 36). under different pyrethroid pressures, metabolic resistance mechanisms can change and phenotypic resistance can reflect differently. under low pyrethroid selection pressure, metabolic resistance is mainly mediated by cyps, but under high pyrethroid selection pressure, high level of metabolic resistance is related to cyps and j arthropod-borne dis, december 2018, 12(4): 370–377 o guntay et al.: evaluation of pyrethroid … 375 http://jad.tums.ac.ir published online: december 25, 2018 cces (11). in this study, we used pbo, the cyps inhibitor (37, 38) to demonstrate the effects of cyps in metabolic resistance of deltamethrin and permethrin. our results showed that cyps indeed play a role in the metabolic resistance of both deltamethrin and permethrin (table 2). in menemen and bornova populations, synergistic value between permethrin and pbo+ permethrin was 3.3% and 19.8% respectively. synergistic results between same pyrethroid and pbo reported in two different population of same species in marin county, california as 55.5% and 12.8% respectively (39). interestingly, in both populations with preexposure pbo, the effect of cyps was higher in deltamethrin than in permethrin. besides the detoxification enzymes, the physical properties and chemical structures of the insecticide used may also play a role together in metabolic resistance mechanisms. conclusion culex pipiens mosquitoes collected from the three different localities in northern izmir have high levels of resistance to permethrin, deltamethrin, α-cypermethrin and cyfluthrin. in additions, p450 detoxification enzymes have an effect on phenotypic resistance. however, the major mechanism is due to the kdr resistance. further studies are needed to explain these mechanisms responsible for pyrethroid resistance in cx. pipiens. the resistance found in this study is highly likely to be caused by pyrethroid spraying in agricultural areas. if some measures are not taken, these resistant populations will increase and lead to serious problems in public health in izmir. acknowledgements we thank dr evren koban from ege university to editing the manuscript and azulia binti zul azman from universiti sains, malaysia and mr atabak naghavi for helping to purchase the test kits from who. the authors declare that there is no conflict of interests. references 1. tuzun n (2010) mosquito species and reproduction areas on the datca peninsula. 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2department of medical entomology, shiraz university of medical sciences, shiraz, iran 3department of pediatrics, college of medicine, ahvaz jundi shapour university of medical sciences, ahvaz, iran 4department of parasitology and mycology, ahvaz jundi shapour university of medical sciences, ahvaz, iran 5department of medical entomology,ahvaz jundishapour university of medical sciences, ahvaz, iran (received 15 mar 2011; accepted 7 dec 2011) abstract background: scorpion sting is a public health problem in khuzestan, south-west iran. the aims of the current study were to monitor the hospitalized children, due to scorpion sting, and releasing more clinical and epi demiologic data related to scorpionism in this province. methods: in this retrospective study, the data of scorpion sting victims, among the hospitalized children in abuzar children hospital of ahvaz jundi shapur university of medical sciences, was analyzed from the points of epidemiological and clinical aspects in 2006. results: the scorpion species of 18 files out of 57 were recognized accurately, using farzanpay's keyof iranian scorpions, as androctonus crassicauda (scorpionida: buthidae) and hemiscorpious lepturus (scorpionida: hemiscorpiidae). conclusion: the most scorpionism emergencies among the children in the khuzestan should be paid to those species. however, h. lepturus sting emergencies are in the top of attention among the children. keywords: scorpion, androctonus crassicauda, hemiscorpius lepturus, scorpionism, children, iran introduction scorpion sting is a public health problem in khuzestan, south west province of iran. a few epidemiologic and clinical studies related to scorpionism have been documented in this area regarding the children. there have been released different data in this case. one of the largest populations of scorpionism victims in khuzestan are the children. vazirianzadeh et al. (2008) have reported that the children, after the housekeepers, are the largest population stung by scorpions. vazirianzadeh et al. (2005) and chitnis et al. (1994) have obtained the similar results to the above study from khuzestan hospitals. pipelzadeh et al. (2006) reported that 66.4% of victims were less than 20 years old and 39.6% under 10 years old. they also reported that h. lepturus hemiscorpius leptururs (peters, 1861) is responsible for 10– 15% of scorpion sting. however vazirianzadeh et al. (2008), chitnis et al. (1994) and pipelzadeh et al. (2006) reported that h. lepturus was responsible for 30% of scorpion stings. *corresponding author: dr babak vazirianzadeh, e-mail: babakvazir@yahoo.co.uk j arthropod-borne dis, 2012, 6(1): 62–69 b vazirianzadeh et al.: an epidemiological and ... 63 in a retrospective study which conducted by mir dehghan et al. (2001) among 1538 scorpion envenomed children in abuzar children's hospital, ahvaz jondishapour university of medical sciences between 1994 and 1999, 17.5% of these children were stung by h. lepturus and the remaining were due to other scorpion species. children potentially are in the greater risk of being damaged and fatal conditions such as developing severe cardiac, respiratory and neurological complications following scorpion sting (mahaba, 1997, bonsak et al, 2009). the information regarding to the hospitalized children of scorpion sting is also not too much in iran. therefore, the aims of the current study were to monitor the hospitalized children, due to scorpion sting, and releasing more clinical and epidemiologic data related to scorpionism in khuzestan. materials and methods the data of scorpion stings, either epidemiologic or clinic, has obtained from files of both health care and children emergency departments from 57 hospitalized scorpions stung children in the abuzar children's hospital, ahvaz jondishapour university of medical sciences during spring 2006. the data belonging to the definite scorpion species were selected using farzanpay's (1987) key of iranian scorpions. then the related data were described epidemiologic and clinically using percentage basis and crude data. the epidemiologic data were geographical locality of the event, spices of scorpion, age distribution of patients, sex of patients and sting sites in hospitalized children. the clinic data were divided into 3 categories: clinic signs and symptoms, lab data and treatments. the signs and symptoms data were divided into local, respiratory, gastroenteritis, neurologic and the other data (fever and death). results totally 57 files (3 of them missed the age parameter) belonging to the children admitted to the emergency department of the hospital were monitored during spring 2006. forty scorpion stung children (70.17%) were from city of ahvaz (center of khuzestan) and its villages and the rest 17 children were from other rural and urban regions of khuzestan (29.81%). thirty-three cases (82.5%) of ahvaz belonged to the urban area; however the rest of 7 cases (17.5%) belonged to the rural area. the species of scorpions were recognized accurately as androctonus crassicauda (scorpionida: buthidae) and hemiscorpious lepturus (scorpionida: hemiscorpiidae) in 13 and 5 cases, respectively. most of the patients were female (53.70%) and 46.29% were male. the related results are summarized in the table 1 and 2. table 2 indicates that the 52.04% of the patients belonged to the 0–6 year old, 40.73% to the 7–12 year old and 3.70% to the 13–18 year old children. the range of age among the stung children by a. crassicauda, was 14 months–12 years old, and among the children, the stung children by h. lepturus, was 2–13 years old. table 3 shows frequency of sting site in the bodies of patients. it explains that 45.61% of stings have been taken place in the head, neck and trunk and the rest in the upper limbs and lower limbs. frequencies of signs and symptoms following stung are shown in the table 4. the most frequent signs and symptoms data were skin disorders (erythema, rash and itching) regarding h. lepturus stung patients and gastroentritis (abdominal pain and diarrhea and vomiting) regarding a. crassicauda stung patients. in the both cases of a. crassicauda and h. lepturus the most frequent of lab data were haemoglobinuria among the child patients following stung with 53.84% and 80% of j arthropod-borne dis, 2012, 6(1): 62–69 b vazirianzadeh et al.: an epidemiological and ... 64 data, respectively. hemoglobinuria were recorded among 35.89% and 43.85% of scorpion stung children regarding unknown species and totally, respectively. however, hematuria was the most frequent lab data among the child patients after hemoglobinuria in the case of h. lepturus stung patients with 60% frequency. hematuria were recorded among 15.38% and 15.78% of scorpion stung children regarding unknown species and totally, respectively. there was not any hematuria recorded regarding a. crassicauda stung children in the current study. duration of hospitalization among the stung children by a. crassicauda, was 1–4 days. this duration for the stung children by h. lepturus, was 2–9 days. this duration was 1–9 days among the stung children by unknown species. all the children were discharged after recovery without death in 18 cases that are described above. however one death case has been recorded in the category of unknown species. the medications that were used as treatments among the stung children are in the table 5. antivenin was used in 73.68% of the cases. however, calcium gluconate, diazepam, dexamethazone and phenobarbital were applied as the least medications. table 1. distribution of scorpion stings according to locality, abuzar children's hospital, ahvaz jondishapour university of medical sciences, spring 2006 locality scorpion stings according to locality a. crassicauda h. lepturus unknown species total number % number % number % number % ahvaz rural 6 10.52 0.00 1 1.57 7 12.28 urban 1 1.57 1 1.57 31 54.38 33 57.89 khuzestan rural 6 10.52 4 7.01 3 5.26 13 22.80 urban 0.00 0.00 4 7.01 4 7.01 table 2. distribution of patients according to age and sex characteristics, abuzar children's hospital, ahvaz jondishapour university of medical sciences, spring 2006 characteristics stung by scorpions age (yr) sex a. crassicauda h. lepturus unknown species total number % number % number % number % 0–6 ♀ 5 38.46 2 40 10 27.77 17 29.82 ♂ 4 30.76 8 22.22 12 22.22 7–12 ♀ 2 15.38 1 20 9 25.00 12 22.22 ♂ 2 15.38 8 22.22 10 18.51 13–18 ♀ ♂ 1 20 1 2.77 2 3.70 total ♀ 7 53.84 3 60 19 52.77 29 53.70 ♂ 6 45.15 2 40 17 47.22 25 46.29 j arthropod-borne dis, 2012, 6(1): 62–69 b vazirianzadeh et al.: an epidemiological and ... 65 table 3. sting sites in hospitalized children in the abuzar children's hospital, ahvaz jondishapour university of medical sciences 2006 part of body frequency (%) head, neck, trunk 45.61 upper limps(hands) 19.29 lower limp(legs) 35.08 total 100 table 4. frequencies of signs/symptoms following stung by scorpions, abuzar children's hospital, ahvaz jondishapour university of medical sciences, spring 2006 clinical signs/ symptoms stung by scorpions a. crassicauda h. lepturus unknown species total number % number % number % number % local : pain 3 23.07 1 20 10 25.64 14 24.56 swelling 2 15.38 8 20.51 10 17.54 edema 5 12.82 5 8.77 erythema , rash and itching 3 60 14 35.89 17 29.82 cellolite 2 5.12 2 3.50 respiratory: respiratory distress 2 15.38 1 20 4 10.25 7 12.28 gastroentritis: abdominal pain and diarrhea 1 7.69 2 5.12 3 5.26 vomiting 6 46.15 1 20 7 17.94 14 24.56 sialorrehoea 1 2.56 1 1.75 neurologic: convulsion 2 5.12 2 3.50 headache 1 20 1 1.75 unconscious 1 2.56 1 1.75 vertigo 1 20 2 5.12 3 5.26 others: fever 2 15.38 11 28.20 13 22.80 death 1 2.56 1 1.75 table 5. medications that were used as treatments among the stung children among hospitalized children in abuzar children's hospital, ahvaz jondishapour university of medical sciences, spring 2006 treatments stung by scorpions a. crassicauda h. lepturus unknown species total number % number % number % number % antivenin 12 92.30 3 60 27 69.23 42 73.68 alkaline diuresis 10 76.92 5 100 17 43.58 30 55.10 calcium gluconate 1 7.69 1 1.75 diazepam 1 7.69 1 2..56 2 3.50 phenobarbital 1 7.69 2 5.12 3 5.26 antibiotic penicillin 2 5.12 2 3.50 antibiotic kefline 4 10.25 4 7.01 dexamethazone 1 2..56 1 1.75 atropine 2 5.12 2 3.50 j arthropod-borne dis, 2012, 6(1): 62–69 b vazirianzadeh et al.: an epidemiological and ... 66 discussion scorpionism is a widespread phenomenon in the tropical and sub-tropical regions. soulaymani et al. (2002) have also expressed that 90% of the fatal scorpionism victims were younger than 15 years old, in the south and central parts of morocco. children younger than 15 years represented 30% of the stung population per year. ozkan and kat (2005) in a study of turkey have also explained that 25% of cases due to scorpion stings belonged to the age group of 0–14 years old. jarrar and al-rowaily (2008) have reported that 21.8% of scorpion sting cases were younger than 16 years old in a study from saudi arabia. yildizdas et al. (2008), ghalim et al. (2000) and goyffon et al. (1982) have discussed that scorpion stings represent an important and serious public health problem worldwide due to their high incidence and potentially severe and often fatal clinical manifestations, especially among children. according to the classification of abroug et al. (1994) the clinical severity was mainly class ii envenomation including 46.15% vomiting (as simpatic or parasipatic response) and 60% hematuria among a. crassicauda or h. leptorus stung children, respectively as the major systemic signs, which were seen in the current study. however, the clinical severity was stronger in the h. lepturus stung cases, because of hematuria. this is confirmed by radmanesh studies (1987, 2002). the results of this study showed that the hematuria was a main sign of systemic toxicity among h. lepturus stung children rather than a. crassicauda stung children whom were suffered of vomiting. reporting hemoglobinuria among the a. crassicuda stung children is not accordance to the radmanesh studies (1987, 2002) which mentioned only hemiscorpioiids caused hemolysis signs among the patients. this could be explained by difference scorpion systematic between two species, which a. crassicauda belonging to buthidae family in contrast to h. lepturus stung belonging to hemiscorpioiidae family. most of the patients were females (53.70%) and 46.29% were males. this rate is accordance with results of vazirianzadeh et al. (2005) in khuzestan. however, it is not consist with the results of dehgani et al. in kashan that they reported that the scorpion stung people were males (53.04%) than females (46.95%) (dehghani et al. 2010). it is due to different methods and geographical locations in the two studies. the results of this study approved that the 64.90% of scorpion stung people have been recorded in urban area which is confirmed that scorpionism in khuzestan is going to be an urban problem. vazirianzadeh et al. reported that the most of patients who referred to ahvaz hospitals regarding scorpion stings had been stung by scorpions in urban area (vazirianzadeh et al. 2008). according to mahaba (1997) the severity of symptoms and signs following scorpion stings are greater among infants than adults, significantly. consequently, the treatments seem to be more important in infants and preschool children than in adults (mahaba 1997). the severity of signs among hospitalized children in this study agrees with the results of mahaba (1997). he explained that the greater number of stings to the head, neck and body among the children because of poor withdrawal reflex when suffering from a sting gives a chance to scorpions injecting more venin. in the current study 45.6% of stings happened in the head, neck and trunk. the severity of hemolysis signs among the hemiscorpioid stung children is greater than the a. crassicauda stung children. in addition, the hematuria has caused longer hospital duration in regarded to the children j arthropod-borne dis, 2012, 6(1): 62–69 b vazirianzadeh et al.: an epidemiological and ... 67 with h. lepturus scorpionism than a. crassicauda stung children in the current study, however, the neurologic effects such as convulsion, laryngospasm and respiratory distress by a. crassicauda are more deadly among the children, totally. pipelzadeh et al. (2007) have reported that clinical signs and symptoms were both local and systemic among the 345 cases of scorpion stings. this confirms the findings in the current study. also cutaneous findings including: erythema, echimosis, ulcer and necrosis among h. lepturus stung children, were recorded in this study. this is accordance to the findings of pipelzadeh et al. (2007) and radmanesh (1987, 2002). totally, 42 cases out of 57 scorpion stung children received antivenin of scorpions. this antivenin is a 5 ml polyvalent ampoule against 6 species including both h. lepturus and a. crassicauda and four other species: mesobuthus eupeus, odonthobothus doriae, hottentotta saulcyi and h. schach. this is made in razi research vaccine and serum institute, iran. except one case that leaded to death despite receiving the antivenin, in the other cases recovery was recorded in the current study. this confirms that using the antivenin is useful to treat the exposed children to scorpion stings. this is almost accordance to the results of gajre and gajre and dammas (1999) that made a pessimist conclusion in the effectiveness of using the earliest species-specific antivenin because it reduces mortality and morbidity of scorpion stings in the cases of definite envenomation with scorpions. the main of afzali and pezeshki (1999) results are similar to the obtained results of the current study. afzali and pezeshki (1999) have reported that the renal failure due to h. lepturus sting is a second phenomenon and the venom this species is not nephrotoxic. they have also explained that hemoglobinuria is the most important sign of h. lepturus sting which can be followed by renal failure. however they have recommended the scorpion sting in the face and body would lead to the renal failure than the scorpion sting in the other part of human body. the average age of patients in their study was 6 years old which agrees to our study that revealed that the age of 52.04% of child scorpion sting victims were between 0-6 years old. the most frequency of applied medicines after using antivenin was alkaline diuresis. this treatment applied to alkali the urine as a medical approach against renal failure due to hemolysis effects of h. lepturus venom. however, studies of farzanpay (1978) and dehghani et al. 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online: march 31, 2020 original article molecular and seroepidemiological survey of visceral leishmaniasis in owned dogs (canis familiaris) in new foci of rural areas of alborz province, central part of iran: a cross-sectional study in 2017 aliehsan heidari1; *mehdi mohebali2,3; mozhgan vahed3; kourosh kabir4; zabihollah zarei2; behnaz akhoundi2; samira elikaee2; hojatallah barati5; monireh sezavar6; hossein keshavarz2,3; zahra kakooei2; homa hajjaran2 1 department of parasitology, school of medicine, alborz university of medical sciences, karaj, iran 2 department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 3 center for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran 4 social determinant of health research center, alborz university of medical sciences, karaj, iran 5 center of against infectious diseases, health depatment, alborz university of medical sciences, karaj, iran 6 department of experimental sciences, faculty of allied medicine, alborz university of medical sciences, karaj, iran (received 24 feb 2019; accepted 12 mar 2020) abstract background: mediterranean form of visceral leishmaniasis (vl) is endemic among some provinces of iran. the present study was designed to determine the prevalence of canine visceral leishmaniasis (cvl) in the owned dogs of the rural areas of alborz province near tehran as the capital of iran. methods: this study conducted on 303 owned dogs that selected using a stratified random sampling method. the direct agglutination test (dat) was used to determine the frequency of vl. the spleen biopsy was taken from the serologypositive dogs for the confirmation of cvl in the suspected dogs. nested pcr and sequencing methods were used to determine the type of leishmania species in the dogs which were parasitological positive. results: overall, the dat results of 9 dogs (2.97%, ci: 1.57–5.55) showed anti leishmania antibodies at titers ≥ 1:320 indicating vl infection. one dog (0.33%, ci 95%: 0.06–1.85) showed clinical signs and symptoms of vl. there was a significant correlation between the positive cases of cvl and rural area (p< 0.001). the leishmania was observed in the impression smears that were prepared from spleen biopsy of five the studied dogs. leishmania infantum were confirmed in all them using nested–pcr assay. the sequence analysis of all five isolates was 95% similar to l. infantum. conclusion: this study shows that domestic cycle of l. infantum has been established in rural areas of alborz province where located near tehran as capital city of iran. it is necessary to increase the awareness and monitoring of the disease periodically. keywords: leishmania infantum; dog; nested polymerase chain reaction (nested-pcr); direct agglutination test; iran introduction mediterranean form of visceral leishmaniasis (vl) is an important zoonotic parasitic disease caused by leishmania infantum (1, 2). this form of the disease has been reported from the mediterranean, three major areas of china, and brazil (1, 3). this epidemiological form of leishmaniasis is endemic among some provinces of iran including ardebil, east azarbaijan, fars and bushehr (4). the disease foci are expanding in other parts of the country, and cases have been reported in other provinces, including the alborz province (5). the most reported cases of vl (98%) were found in the rural area of iran especially in children under 10 years of age (4, *corresponding author: dr mehdi mohebali, email: mohebali@tums.ac.ir http://jad.tums.ac.ir/ mailto:mohebali@tums.ac.ir j arthropod-borne dis, march 2020, 14(1): 38–46 a heidari et al.: molecular and … 39 http://jad.tums.ac.ir published online: march 31, 2020 6). domestic dogs are principal reservoir hosts and source of the infection for humans (7, 8). the causative agent of the disease is transmitted from infected dogs to human by sand fly bite (9). identification of infected reservoirs can play an important role in preventing the transmission of leishmania to humans, especially children. since most infected dogs are clinically asymptomatic, identifying and detecting anti leishmania antibodies in sera is appropriate diagnostic method for screening dogs in epidemiological studies and evaluating control programs (10). there are several serological tests for identifying anti leishmania antibodies, among which the direct agglutination test (dat) is more useful due to its simplicity, no need for special tools, and high reliability and validity (11, 12). determining the type of leishmania in vl is important for controlling the disease. pcrbased methods are applied for the isolation and differentiation of leishmania species in infected dogs, especially in areas where both cutaneous and visceral leishmaniasis existed (13, 14). internal transcribed spacer (its) 2 is one of the best parts of the genome to detect closely related species. according to studies, its2 has sufficient diversity among different species of leishmania parasites and can be used to identify the species (15). determining the prevalence of cvl especially the owned dogs, which are in more contact with humans and sand flies that feed on them might bite humans, is necessary to adopt an appropriate strategy for controlling the mediterranean vl (16, 17). since there are few studies on the epidemiological characteristics of vl in owned dogs in alborz province, and considering the importance of the disease in iran and reported sporadic cases of cvl in recent years from alborz province, and in order to find local foci of the infection in high risk areas of the province, the present study was designed using the dat serological test, parasitology and molecular pcr methods to determine the prevalence of cvl in the owned dogs of the rural areas of alborz province near tehran as the capital of iran. materials and methods alborz province is located in the alborz mountain range slopes. its area is 833.5km2 and its population is 2,712,000 people (18). nazarabad, savojbolagh, taleghan, eshtehard and mahdasht, and karaj-chalus road are the main rural areas of the province (fig. 1). alborz province is located 30 kilometers from tehran, the capital city of iran. the climate of alborz province is to some extent cooler than tehran’s, and it receives 260mm of rain annually. this study was a cross-sectional descriptive study conducted in 2017 on 303 owned dogs in rural areas of alborz province. according to the information of the veterinary organization, there were 6560 dogs with collars in the province. the five main rural regions of alborz province were considered as strata and a stratified random sampling method was used for the selection of the dogs. the samples were selected randomly based on the number of dogs with collars in each stratum. dat was applied to determine the frequency of infection with l. infantum. pcr and sequencing methods were used to confirm the infection and detect the type of leishmania species in the dogs which were parasitological positive. blood samples (3–5cc) were taken from the cephalic or saphenous veins and collected in test tubes. samples were then transferred to the laboratory of alborz university of medical sciences and centrifuged at about 3000rpm for 15 minutes to separate the serum. separated serums were stored in a freezer at -20 °c until performing dat. frequencies and percentages were calculated, chi square and fisher exact tests were used to compare the results. dat test dilution of serum samples of dogs serial dilution of serum samples was prepared by adding a 0.9% saline and 0.78% 2-mercaptoethanol. dat antigen was made from l. infantum [mcan/ir/07/moheb-gh. [(genbank accession no. fj555210)] in rpmi-1640 medium (biosera, south america) plus 10% fetal http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 38–46 a heidari et al.: molecular and … 40 http://jad.tums.ac.ir published online: march 31, 2020 calf serum (biosera, south america), following trypsinization of the parasites, staining with coomassie brilliant blue r-250 (sigma, st. louis, missouri, usa) and fixing with 1.2% formaldehyde (11, 19). the antigens were stored in the refrigerator at 4 °c. the antigens then were added to the diluted serums, and the plates were shaken for one minute. the plates were transferred into a damp room and placed on a horizontal surface in the laboratory temperature (22–25 °c) at least for 12–18 hours. the serum sample with the highest dilution that agglutination was produced in was considered as the maximum positive titer for dat. positive and negative controls were also used in each test. the presence of an antibody in a dilution equal to or above 1:320 was considered as an infection or disease in the dog. parasitology assay the spleen biopsy was taken from the serology-positive dogs. the biopsy samples were quickly transferred in the vicinity of spirit lamp flames to several tubes containing the novy mac neal and nicol and rpmi 1640 culture media. impression smears were also prepared from the spleen, stained with the giemsa technique, and examined for the leishmania under a light microscope with high magnification (1000 x). nested-pcr dna was extracted from smears stained with giemsa using the kit protocol of roche company (high pure template preparation kit). its2 was used to identify the leishmania species. the following primers were used in this study: leish out f (5'aaa ctc ctc tct ggt gct tgc-3'), leish out r (5'-aaa caa a gg ttg tcg ggg g-3'), and leish in f (5' aat tca act tcg cgt tgg cc-3'), leish in r (5'-cct ctc ttt ttt ctc tgt gc-3'). pcr was performed according to the method described in the previous study (15). sequencing positive samples with nested-pcr method were sent to the bioneer company (south ko rea) for sequencing. ethical considerations getting a biopsy of the infected dogs was performed according to coordination made with the health care network, environmental and municipal authorities, and observing the ethical considerations of the least pain and suffering. this study was as joint projects approved by the research ethical review committee of tehran university of medical sciences (project no.95 01-160.31439) as well as alborz university of medical sciences, karaj, iran with (approval number: abzums.rec.1395,8). results direct agglutination test in this study, 303 dogs of the rural areas of alborz province in iran were tested by dat method with positive and negative control serum. one dog (0.33%, ci 95%: 0.06–1.85) showed clinical signs and symptoms of cvl. overall, the dat results of 9 dogs (2.97%, ci: 1.57–5.55) showed anti leishmania antibodies at titer ≥ 1:320 indicating vl infection (fig. 2). in terms of age distribution 194 dogs (64%) belonged to ≤ 3 year-old group and among dogs with anti leishmania antibodies at titers ≥ 1:320, the highest frequency (55.6%) of positive cases was in the 0–3 years age group and the lowest frequency (11.1%) of positive cases was in the > 7 years age group (table 1). out of 303 dogs, 254 (83.8%) were male and 49 (16.2%) were female. in terms of sexual distribution, among dogs with anti leishmania antibodies at titers ≥ 1:320, 1 dog (11.1%) was female and 8 dogs (88.9%) were male. in terms of distribution of dogs with vl in dat in rural areas of alborz province, the highest frequency of positive cases was observed in taleghan (table 2). there was a significant correlation between the positive cases of cvl and rural area (p< 0.001). a spleen biopsy was taken from 5 of the 9 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 38–46 a heidari et al.: molecular and … 41 http://jad.tums.ac.ir published online: march 31, 2020 dogs with anti leishmania antibodies at titers ≥ 1:320 in the dat test. the leishmania was observed in the impression smears that were prepared from spleen biopsy of five the studied dogs under light microscope with high magnification (1000 x). examining the culture media, live parasites (promastigotes) were observed in samples of two dogs. molecular results dna was extracted from the positive slides prepared from the spleen of five dogs. leishmania infantum were confirmed in all the five parasitological positive using nested–pcr assay and 230bp fragment was showed on the agarose gel electrophoresis (fig. 3). the pcr products were sequenced for species identification. the sequence of all five isolates was 100% similar to each other and was similar 95% to l. infantum. the sequence was recorded in the world gene bank under the mk054276 accession number. fig. 1. map of alborz province, iran table 1. frequency of studied dogs in rural areas of alborz province in terms of age and sex in 2017 odds ratio ((95% ci) 95% ci positive dogs tested (n) male/female 1.56 (0.19 to 12.77) 0.36 to 10.69 1 (2.04%) 49 female sex 1.60 to 6.09 8 (3.15%) 254 male ≥ 4 years/0-3 years 2.28 (0.60 to 8.69) 0.80 to 5.18 4 (2.06%) 194 0–3 years age 1.98 to 10.29 5 (4.60%) 109 ≥ 4 years ------- 1.57 to 5.55 9 (2.97%) 303 (100%) total http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 38–46 a heidari et al.: molecular and … 42 http://jad.tums.ac.ir published online: march 31, 2020 table 2. distribution of positive and negative cases of dat test in rural areas of alborz province in 2017 95% ci positive antibodies ≥ 1:320 number (%) dogs tested (n) area 0 (0%) 75 eshtehard ----0 (0%) 76 savojbolagh 0.83 to 10.39 2 (3.03) 64 nazarabad 5.32 to 20.60 7 (10.77) 65 taleghan ----0 (0%) 23 karaj-chalus road 1.57 to 5.55 9 (2.97%) 303 (100%) total fig. 2. distribution of reciprocal titers of anti-leishmania antibodies using dat among owned dogs of rural areas of alborz province in 2017 fig. 3. patterns of its2 nested-pcr products in test isolates and standard leishmania strains. lanes 1–5: dogs biopsy samples, lane 6: l. infantum (mcan/ir/07/moheb-gh), lane 7: l. major (mrho/ir/75/er), lane 8: l. tropica (mhom/ir/01/yaza), lane 9: none template control (negative control), lane 10: marker (100bp) discussion dogs are the main reservoir of mediterranean vl. infected dogs can keep leishmania parasite in their body for a long time and even until death without any symptoms, causing infection of the sand flies (2). from the veterinary and medical point of view, the identification of infected dogs is hence essential for the control of vl in dogs and humans, especially children. in the present study, 3% (9 cases) of owned dogs had anti leishmania antibodies at titers ≥ 1:320 in the dat test indicate vl infection. this was lower than the 17% of vl reported in a study on 384 serum samples of owned dogs with dat test in meshkin-shahr, iran (16), and then the 12% l. infantum infection reported in the dogs of nomads in ardabil province of vl http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 38–46 a heidari et al.: molecular and … 43 http://jad.tums.ac.ir published online: march 31, 2020 endemic area by dat test in 2002 (20). it seems that the climate and geography differences, the level of sand flies’ activities and their contact with the reservoirs, and the economic and social conditions, including less livestock farming in alborz province, are contributing to the difference in infection levels with the northwest of iran. in a study on humans in alborz province, the rate of human infection with vl was lower than endemic areas of northwest and south of iran, which is consistent with the rate of infection of dogs (5). in the present study, according to the statistical findings, there was no significant relationship between the age and infection rate of cvl. this was consistent with the results of a study in brazil, which found no relationship between age and infection rates (21). however, some studies have reported the rate of infection in older dogs due to the possibility of more contact with the sand flies (4, 22). there was no statistically significant difference between sex and leishmaniasis in this study, which was consistent with studies in 1992 in portugal, 1996 in greece, and 2018 in brazil (21, 23, 24). in terms of clinical symptoms in dogs studied in this study, only 1 dog (11.1%) was symptomatic, indicating no significant relationship between positive cases and symptomatic dogs, which was consistent with study conducted in meshkin-shahr (13%), but not with a study in croatia, in which 54% of seropositive dogs from 306 dogs studied with dat test had clinical symptoms (25, 26). it seems that fewer clinical symptoms are observed in dogs infected with vl in iran, especially in alborz province that is consistent with 5 to 10 percent symptomatic infected dogs in endemic regions (4). the absence of vl infection symptoms in dogs is significant epidemiologically because infected dogs can easily transmit leishmania to sand flies (1). in other hand, even treated symptomatic dogs are not cleared from l. infantum and can transmit the infection (1, 12). therefore, regular examination of dogs in cvl high risk areas is necessary. determining leishmania species is involved in the adoption of the strategies for preventing, controlling and treatment cvl. epidemiological evidences and clinical features alone are not sufficient to differentiate the species causing cvl. parasitology examinations and the use of molecular techniques are hence applied for detecting species and even strains of leishmania parasite. based on the results of nested-pcr and sequencing of the pcr products, it was found that the disease agent in all five autopsied dogs was l. infantum, which is consistent with other studies conducted in iran (4, 27). sequencing results provided general view, although limited, of the genetic status of the species causing vl in the studied regions. since slides prepared from a biopsy of bone marrow and lymph nodes in dogs with vl, especially in asymptomatic dogs, have a low sensitivity for diagnosis (2), spleen biopsy was used for microscopic examination. in this study, the findings of the microscopic slide examination were consistent with the results of the molecular and serologic tests. this suggests that in the biopsy of the dog's spleen, the probability of observing the parasite is very high. compared with invasive and high risk methods such as taking biopsy from visceral organs including the spleen, liver, lymph nodes and bone marrow, serological methods can be a proper alternative method for diagnosis of cvl (22). since the disease is prevalent in rural areas with minimum health facilities, it is important to use simple, inexpensive, and reliable diagnostic methods for cvl. therefore, although dat was developed several years ago for the laboratory diagnosis of vl, it is still known as the best, most economical and easiest way to detect the disease in dogs. the geoghraphical distribution pattern of vl infection in dogs was consistent with its distribution pattern among children in alborz province. in a study on children under the age of 10, vl cases were observed in nazarabad and taleghan. the present study found cases of infection of dogs in these two regions, too. based on the available evidence and overlap of http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 38–46 a heidari et al.: molecular and … 44 http://jad.tums.ac.ir published online: march 31, 2020 vl in humans and dogs, and the presence of infected carriers, it seems that taleghan and nazarabad are endemic foci of vl in the alborz province. cvl was previously reported in koohsar, a region near taleghan too (28). the spatial overlap between human and dog visceral leishmaniasis also reprted in spain (8). the information of this study can help health and treatment planners and managers to prevent and control the infection in the main reservoir of the infection. in conclusion this study shows that domestic cycle of l. infantum has been established in rural areas of alborz province where located near tehran as capital city of iran. the high population of dogs in the alborz slopes and the high population of humans in the province that live the near of the dogs, the infection of about 3% of dogs with the l. infantum parasite and the presence of sand flies in the region have provided the condition for transmission the parasite between dogs and humans. therefore, it is necessary to increase the awareness and monitoring of the infection in dogs periodically. health authorities should also plan to prevent and control cvl in the alborz province. acknowledgements this study was financially supported by alborz university of medical sciences, karaj, iran (project no. 2815895) and tehran university of medical sciences, tehran, iran (project no. 31439). the authors are 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province, north of iran. iran j parasitol. 6 (4): 51–59. 28. haddadzade hr, fattahi r, mohebali m, akhoundi b, ebrahimzade e (2013) seroepidemiologcal investigation of visceral leishmaniasis in stray and owned dogs in alborz province, central iran using direct agglutination test. iran j parasitol. 8(1): 152– 157. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 416–419 mh aelami et al.: urinary canthariasis … 416 http://jad.tums.ac.ir published online: december 31, 2019 case report urinary canthariasis due to tenebrio molitor larva in a ten-year-old boy mohammad hassan aelami1; alireza khoei2; hamidreza ghorbani3; farrokh seilaniantoosi4; elham poustchi5; bibi razieh hosseini-farash5,6; *elham moghaddas5 1department of pediatrics and hand hygiene and infection control research center, imam reza hospital, mashhad university of medical sciences, mashhad, iran 2department of pathology, faculty of medicine, mashhad university of medical sciences, mashhad, iran 3kidney transplantation complications research center, mashhad university of medical sciences, mashhad, iran 4department of radiology, faculty of medicine, mashhad university of medical sciences, mashhad, iran 5department of parasitology and mycology, faculty of medicine, mashhad university of medical sciences, mashhad, iran 6cutaneous leishmaniasis research center, mashhad university of medical sciences, mashhad, iran (received 10 aug 2018; accepted 26 oct 2019) abstract canthariasis is a human disease caused by infestation of beetle larvae. we report here an unusual cause of urogenital infection due to tenebrio molitor in a 10-year-old boy suffering from severe and intermittent suprapubic pain from nehbandan city, northeastern iran in 2018. after 9 months, three larvae were excreted. keratinization of bladder wall was observed in histopathology. all laboratory evaluations were normal except for presence of microscopic hematuria. this re port implicated that t. molitor could infest bladder accidentally and cause canthariasis and clinical symptoms that may lead to severe pain and bladder inflammation and hyperemia. keywords: canthariasis; bladder; human; child introduction canthariasis is a parasitic disease caused by beetle larvae, either humans or animals (1). canthariasis in humans and animals due to tenebrio molitor is an uncommon phenomenon. the reports are very rare and pathological effects are poorly known (2). the first and only report on canthariasis due to t. molitor in bladder was reported about 375 years ago in a book entitled “observations medicae” (3). moreover, t. molitor has invaded umbilicus and tonsils (4). however, the majority of reported cases of t. molitor larva in humans have been related to gastrointestinal tract (2, 5). recently accidental ulcer infestation due t. molitor has been reported in a case with hiv/aids and skin ulcers (6). tenebrio molitor is a yellow mealworm commonly found as a stored-product pest. the life cycle of this organism comprises four stages including egg, larva, pupa and adult forms. the entire life cycle lasts approximately one year. adult and the larvae feed on grains (hence the name mealworm), meat or decomposing animals including birds, spiders, rodents, lizards and some other beetles. human is infected by the ingestion of eggs or larvae of t. molitor (7). tenebrio molitor can also promote allergic reactions in exposed individuals (8). epidermolysis bullosa (eb) is the name for a group of rare genetic skin disorders that cause fragility in skin. any trauma or friction to the skin can cause painful blisters (9). in this case report, we describe an unusual case of canthariasis due to t. molitor in a 10-year-old boy with eb. *corresponding author: dr elham moghaddas, e-mail: moghaddase@mums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 416–419 mh aelami et al.: urinary canthariasis … 417 http://jad.tums.ac.ir published online: december 31, 2019 case presentation the patient was a 10-year-old boy (23kg) who had epidermolysis bullosa (eb) disorder referred from a local clinic from nehbandan city in 2018, northeastern of iran. he suffered from periodic painful episodes in urinary system for the past 9 months. symptoms were intermittent, and urine contained brown sediments similar to bladder stones (fig. 1). there were no signs of gross hematuria and fever during the mentioned time. complete medical examinations including urine analysis, urine culture, urine and blood biochemistry, hematology, immunoassays for autoimmune diseases, thyroid function tests and renal ultrasound were performed. ultrasound was performed after observation of the excreted brown sediment from urethra for finding kidney stones. kidney and bladder appeared normal in ultrasound. urinalysis revealed no abnormality except for occult blood. urinary culture was negative. blood cell count and creatinine levels showed the normal ranges. moreover, urine biochemistry was normal. erythrocyte sedimentation rate (esr) was 21 mm/1h. the boy’s mother found three larvae in his urine more than two weeks after symptoms start. morphologically, the larvae had six short legs close together near the head. the head has a pair of short hooks and was creamy white in color. the larvae had 26mm length and 0.5 mm width. they had three pairs of feet on the belly near the head and with each foot had 4 sections and nodes hocks curved claws (fig. 2). based on the characteristics, the larva was diagnosed as t. molitor. debris, hyperemia and inflammation were identified in bladder and in the urethra cystoscopy (fig. 3). however, no larva was seen in bladder and urethra. a single dose of 3mg of ivermectin was orally administrated. the patient recovered after oral ivermectin therapy. following treatment, high volume of insect shells was repulsed (fig. 4). in addition, the abdominal pain completely resolved within a few months following treatment. histopathology of excreted particles from urethral duct showed keratinizing squamous metaplasia after treatment. informed consent was taken from the patient’s parents. this study was reviewed and approved by the ethics committees of mashhad university of medical sciences, iran. fig. 1. brown sedimentations excreted via urethra fig. 2. macroscopic examination of the larva http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 416–419 mh aelami et al.: urinary canthariasis … 418 http://jad.tums.ac.ir published online: december 31, 2019 fig. 3. cystoscopy finding including debris, hyperemia and inflammation fig. 4. disposal shell of insect after treatment by ivermectine discussion this case was initially misdiagnosed as kidney stones because the shedding of stone like substance without excretion of any larva from the urethra. ultrasonography failed to detect the larva probably because the initial request was ordered for checking of kidney or urinary tract stones. unlike most stones that are hard, the observed particles from this patient had soft structure. this beetle has 9–20 instars, the brown sedimentations were probably larvae shells during molting (https://en.wikipedia.org/wiki/mealworm). on the other hand, we could speculate that the class of the scanner for determination of larvae was inadequate. beetle larvae have been recovered from human organs including tonsils (4), nose and bladder (10), umbilical cord (11), the gastrointestinal tract (4, 5), as well as from subcutaneous tissue in wild bird (1). canthariasis is a rare ectoparasitic condition. among the reported cases, most of them were gastrointestinal. no previous cases in hiv/aids patients have been reported, neither associated with skin ulcers. beetles could be found in houses where dried grains are stored, particularly this is case for t. molitor. adult tenebrio laid eggs on the skin of our patient. females lay eggs and larvae develop within few weeks at necrotic tissue. in fact, scars attract the beetle and its larvae are fed on a variety of dried plant or animal matter and are known to scavenge on carcasses of dead animals. moreover, canthariasis has been reported in a case with hiv/aids patients associated with skin ulcer (6). ulcers in hiv patients and other diseases related to skin damages could increase susceptibility to a wide range of infections such as canthariasis. the present case had eb is a genetic disorder that result in easy blistering of the skin and mucous membranes. the patients with eb are susceptible to infection due to damage in skin physical barrier (9). beetles are a common household pest found in stored grain and stored food products. adult of t. molitor laid eggs on the in or around the urogenital opening of our patient when he was resting. then, the eggs hatch and larvae migrate along the urethra with consequent canthariasis. some previous studies suggested this route of transmission for urinary myiasis (12,13) this larva is unable to dwell in subsequently of the bladder wall. because of this, our patient showed no macroscopic hematuria, and the bladder wall thickness was also normal (3mm). however, because of long stimulation time, wall bladder showed pathological changes. there were no lesion(s) in radiographic investigation. tenebrio molitor can be the intermediate hosts of the rat tapeworm, hymenolepis diminuhttp://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/mealworm j arthropod-borne dis, december 2019, 13(4): 416–419 mh aelami et al.: urinary canthariasis … 419 http://jad.tums.ac.ir published online: december 31, 2019 ta (4). on the other hand, human is the accidental host of h. diminuta by ingestion of beetles or meal worms containing metacestode. up to now, seven human patients have been reported from iran to be infected with h. diminuta (14). therefore, unintentional swallowing of t. molitor may be a possible route of transmission. conclusion infection with t. molitor can present as an emergency condition with acute pain episodes in urinary system. understanding of this infection should be considered when there are no evident signs for stones in urinary system in routine management. acknowledgements we appreciate the patient's parents and their child for their support at all stages of the diagnosis of disease. the authors declare that there is no conflict of interests. references 1. park bk, park sj, ryu sy, chae js, park j, choi ks (2016) subcutaneous canthariasis due to tenebrio molitor larva (coleoptera: tenebrionidae) in egretta intermedia. j dair vet anim res. 3: 00086. 2. palmer ed (1946) intestinal canthariasis due to tenebrio molitor. j parasitol. 32: 32–54. 3. finke md (2002) complete nutrient composition of commercially raised invertebrates used as food for insectivores. zoo biol. 21: 269–285. 4. hinman eh, faust ec (1932) the ingestion of the larvae of tenebrio molitor, l. (meal worm) by man. j parasitol. 19: 119–120. 5. senior-white ra (1920) on the occurrence of coleóptera in the human intestine. in dian j med res. 7: 568–569. 6. mullen gr, durden la (2009) medical and veterinary entomology. academic press, new york. 7. freye hb, esch re, litwin cm, sorkin l (1996) anaphylaxis to the ingestion and inhalation of tenebrio molitor (mealworm) and zophobas morio (superworm). allergy asthma pro. 17: 215–219. 8. tulp n (1739) observationes medicae. wishoff g, leiden. 9. bateman t (1811) an account of the larvae of two species of insects discharged from the human body. edinburgh med surg j. 7: 41–48. 10. mowlavi g, mobedi i, mamishi s, rezaeian m, ashtiani mh, kashi m (2008) hymenolepis diminuta (rodolphi, 1819) infection in a child from iran. iran j publ health. 37: 120–122. 11. rodriguez-morales aj, vera-ospina jj, berthel-vergara jm, silvera-arenas la, villamil-gómez we (2018) accidental ulcer infestation due tenebrio molitor in an aids patient: canthariasis. int j infect dis. 73: 253–254. 12. azizkhan rg, mami a (2017) epidermolysis bullosa: epidemiology, diagnosis, complications, and treatment. in: till h, thomson m, foker j, holcomb iii g, khan k (eds) esophageal and gastric disorders in infancy and childhood. springer, berlin, heidelberg 13. salimi m, goodarzi d, karimfar m, edalat h (2010) human urogenital myiasis caused by lucilia sericata (diptera: calliphoridae) and wohlfahrtia magnifica (diptera: sarcophagidae) in markazi province of iran. iran j arthropod borne dis. 4: 72–76. 14. el-dib na, el wahab wma, hamdy da, ali mi (2017) case report of human urinary myiasis caused by clogmia albipunctata (diptera: psychodidae) with morphological description of larva and pupa. j arthropod borne dis. 11: 533–538. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 286–292 m salehi-vaziri et al.: molecular assay on … 286 http://jad.tums.ac.ir published online: september 30, 2020 original article molecular assay on detection of crimean congo hemorrhagic fever (cchf) virus in ixodid ticks collected from livestock in slaughterhouse from south of iran mostafa salehi-vaziri1,2; hassan vatandoost3,4; alireza sanei-dehkordi5,6; mehdi fazlalipour2; mohammad hassan pouriayevali2; tahmineh jalali2; tahereh mohammadi2; mahsa tavakoli2; azim paksa7; *yaser salim abadi8 1research centre for emerging and reemerrging infectious diseases, pasteur institute of iran, tehran, iran 2department of arboviruses and viral hemorrhagic fevers (national ref lab), pasteur institute of iran, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 5department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 6infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran 7department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran 8department of health services and health promotion, school of health, rafsanjan university of medical sciences, rafsanjan, iran *corresponding author: dr yaser salim abadi, e-mail: yasernls@gmail.com (received 08 sep 2020; accepted 29 sep 2020) abstract background: ticks are vectors of a wide variety of pathogens that can be transmitted to humans, and tick-borne diseases are a significant public health issue worldwide. the present study was carried out on the hard tick infestation of livestock transported to rafsanjan slaughter house in the southeast of iran. methods: a cross-sectional survey was carried out biweekly from april to september 2016 to determine tick infesta tion of the meat-producing animals. all the livestock included in our study were thoroughly inspected for the presence of hard ticks on different parts of their bodies. results: a total of 258 hard ticks were collected from the body of livestock hosts. the ticks that were sampled were classified into two genera and five species: hyalomma marginatum, hy. anatolicum, hy. asiaticum, hy. dromedarii, and rhipicephalus sanguineus. hyalomma dromedarii was the most abundant species in the study area. more than 50 percent of the sampled ticks were collected from the body of camels brought to the slaughter house however molecular analysis showed no crimean congo hemorrhagic fever (cchf) virus infection in tick specimens. the sex ratio of the sampled hard ticks shows that female tick infestation was more common among the study livestock. conclusion: due to the crucial role of hard ticks in the transmission of different pathogens to humans, additional investigations are necessary to determine the risk of consumption of infested meat-producing animals in the study area. keywords: ticks; livestock; abattoir; rafsanjan; iran introduction deply (1, 2) introduced hard ticks (ixodidae) and two genera of these arthropods (hyalomma and haemaphysalis) for the first time in iran. filipova et al. (3) reported the collection of 642 ixodid ticks from small-size mammals, mainly rodents, in iran. later, hoogstral copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://www.google.com/search?client=firefox-b-d&q=slaughterhouse&spell=1&sa=x&ved=2ahukewivl6b6ik_rahwobwmbhuz8dpsqkeeckab6baguecq https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 286–292 m salehi-vaziri et al.: molecular assay on … 287 http://jad.tums.ac.ir published online: september 30, 2020 and valdez (4) investigated and explained the medical and veterinary implications of ixodoidea ticks from wild sheep and goats in iran. during the past quarter-century, many studies were conducted on fauna, biology, and geographical distribution of both soft and hard ticks in different parts of iran (5-13). in a study conducted by nabian et al. (7) 1720 tick specimens belonging to fourteen tick species were collected from cattle, sheep, and goats in different localities of caspian sea areas in the north of iran. seven species of hard ticks were similarly sampled from cattle and sheep in the southeast part of iran (5). the distribution of ticks and tick infestation rate of sheep in the west of iran was studied by nasiri et al. (8) who identified 864 hard ticks, which were classified into two genera and five species. in a survey conducted in the eastern part of iran on species diversity and distribution of ticks in sheep, goats, cattle, and camels, 469 adult ticks belonging to 9 species were collected (6). hard tick infestation of livestock and seasonal population dynamics of these ticks were investigated in the central part of iran, and 583 hard ticks belonging to three genera and seven species were identified (12). due to the crucial role of ticks in the transmission of pathogens such as the crimean congo hemorrhagic fever virus (cchfv) to humans, they are considered as a significant problem in public health. crimean congo hemorrhagic fever virus cchfv has been isolated from at least 31 species of ticks (14-16). crimean congo hemorrhagic fever virus cchfv was also isolated from many genera and species of hard ticks and soft ticks in a study conducted on the distribution of ticks. hard ticks of the genus hyalomma were identified as numerically dominant hard tick vectors for the transmission of cchfv in different climatic zones of the country (14, 16). crimean congo hemorrhagic fever is currently present in most areas of iran (17). the aim of the present study was to identify species composition of hard ticks (acari: ixodidae) parasitizing livestock transported to rafsanjan abattoir for the purpose of meat production and to investigate cchfv infection rate in collected ticks. materials and methods sample collection in the present study, a cross-sectional study was carried out biweekly from april to september 2016 in rafsanjan city of kerman province. the cities that livestock brought to rafsanjan’s abattoir have been shown in figure 1 using arc-gis10.2 software (redlands, ca). animals included in our study were livestock considered as hosts of hard ticks, including cattle, sheep, and camels, which were brought to rafsanjan’s abattoir for the purpose of meat production. all the livestock included in our study were surveyed for the presence of hard ticks on their body. tick specimens collected from each host were kept alive in separate labeled holding tubes, and the information related to each tube was recorded. using the key identification guide (18), all collected hard ticks were identified according to their morphological characteristics until the species determination. molecular analysis of crimean congo hemorrhagic fever (cchf) tick specimens were homogenized individually in pbs using the mortar and pestle. total rna extraction was purified by the use of the rneasy mini kit (qiagen) according to the manufacturer’s kit and stored at -70 °c freezer until needed for cchfv detection. we performed rt-pcr for a 536bp fragment in s segment of cchfv as previously described (19). briefly, rt-pcr reaction was carried out in a final 50µl volume with 10µl of 5x qiagen onestep rt-pcr buffer, 2µl of dntp mixture (containing 10mm of each dntp), 2µl of qiagen onestep rt-pcr enzyme mix, 0.2µm of primer f2 (5'-tggacacctt cacaaactc-3), 0.2µm of primer r3 (5'gacaattccctacacc-3'), 1µl of qiagen rnase inhibitor (4 units/µl), and 5µl of extracted rna as a template. the rt-pcr conhttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 286–292 m salehi-vaziri et al.: molecular assay on … 288 http://jad.tums.ac.ir published online: september 30, 2020 ditions began with 30min at 50 °c for reverse transcription reaction step and then 15min at 95 °c for initial denaturation, hot start activation and inactivation of reverse transcriptase enzyme followed by 40 cycles of 95 °c for 30s, 50 °c for 30s, 72 °c for 45s, and a final extension at 72 °c for 10min. polymerase chain reaction (pcr) products were analyzed into 1.5% w/v electrophorese gel agarose. results during the study period, a total of 258 hard ticks were collected from different livestock hosts, including cattle, sheep, and camel. the number and type of hard tick species collected from each livestock have been summarized in table 1. the sampled ticks were classified into two genera, including hyalomma and rhipicephalus. these two genera of ticks were further classified into five species based on their morphological characteristics. the tick species comprised of hyalomma marginatum, hy. anatolicum, hy. asiaticum, hy. dromedarii and rhipicephalus sanguineus (table 1). hyalomma dromedarii was the most abundant species among the collected ticks, followed by hy. marginatum, as seen in table 1. rhipicephalus sanguineus was the least abundant tick species collected from the livestock in the study area. between the different livestock hosts, the occurrence of hard ticks was more common on the body of camels; more than 50 percent (n= 137) of all collected samples were related to this host (table 1). the sex ratio of the collected hard ticks shows that female tick infestation (60%) was more prevalent among the study animals. after a thorough inspection, ticks were collected from different body parts of the livestock. the rump region of sheep, under the tail region of cattle and camels, were the most favorable location for tick attachment (table 2). molecular assay results indicated no cchfv infection in collected ticks. table 1. the frequency of collected ticks in different hosts in slaughterhouse from rafsanjan city, kerman province, iran, 2016 species hosts total cattle camel sheep number percentage hy. marginatum 19 31 38 88 34.11 hy. dromedarii 0 97 0 97 37.60 rh. sanguineus 0 0 10 10 3.88 hy. anatolicum 9 4 24 37 14.34 hy. asiaticum 3 5 18 26 10.07 total 31 137 90 258 100 table 2. distribution of ticks on different body parts of the animals' body in slaughterhouse from rafsanjan city, kerman province iran, 2016 hosts body parts under tail/rump belly thigh neck cattle 27 4 0 0 camel 118 9 8 2 sheep 52 23 15 0 total 197 36 23 2 http://jad.tums.ac.ir/ https://www.google.com/search?client=firefox-b-d&q=slaughterhouse&spell=1&sa=x&ved=2ahukewivl6b6ik_rahwobwmbhuz8dpsqkeeckab6baguecq j arthropod-borne dis, september 2020, 14(3): 286–292 m salehi-vaziri et al.: molecular assay on … 289 http://jad.tums.ac.ir published online: september 30, 2020 fig. 1. map of iran, sites of livestock brought to rafsanjan’s abattoir in, kerman province, 2016 discussion a total of 258 hard ticks from the different livestock hosts were collected and identified. hyalomma dromedarii was the most abundant among the tick species collected from study animals. on the other hand, the presence of ticks on the body of camels was more common compared with the other livestock hosts. the results of the present study also show that the rump region of sheep and under the tail region of cattle and camel are the most favorable regions for tick attachment. in our study, two genera of ixodid ticks were identified and further classified into five species. hyalomma dromedarii and hy. marginatum belonging to the genus hyalomma was the most numerically dominant among sampled ticks. similar results were observed in other studies conducted in different parts of iran (5-8, 11, 12, 2022). the results of the present study also indicate that the adults of hy. dromedarii, which were the most abundant tick species among all sampled hard ticks, prefer camels belonging to the genus camelus dromedarius (mammalia: camelidae) for feeding. this finding is consistent with that of other studies that investigated the tick infestation rate of camels, but in some other studies, other species of hard ticks were collected from camels (6, 12, 22-26). in addition to camels, hy. dromedarii has also been collected from sheep and cattle in some other studies (6, 8, 11, 12). five tick species belonging to two genera (hyalomma and rhipicephalus) were identified in our study, including hy. marginatum, hy. anatolicum, hy. asiaticum, and r. sanguineus. rh. sanguineus, which was the least abundant species among all collected ticks, mainly infested sheep. similar to the results of other studies, it seems that rh. sanguineus is one of the most common tick species that parasitize sheep and goats (5-7, 11, 27, 28). in some other studies that surveyed hard ticks which infested domestic ruminants, some other genera of hard ticks that were not found in our study were reported. for instance, http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 286–292 m salehi-vaziri et al.: molecular assay on … 290 http://jad.tums.ac.ir published online: september 30, 2020 in 2009, a study on tick infestation rate of sheep conducted in abdanan county (a west area in iran) identified heamaphysalis sulcate, which belongs to the family ixodidae, and other species of hard ticks which infested sheep (8). hosseini-vasoukolaei et al. (28) reported that four genera of ixodidae (hard ticks), including ixodes ricinus, boophilus annulatus, h. punctate and h. numidiana infested domestic ruminants in ghaemshahr county (north of iran), but these species were not found in the present study. even so, we did not expect to find i. ricinus in our study area due to its habitat and bio-ecology requirements, based on the results of previous studies which mentioned that the distribution of i. ricinus is just limited to the caspian sea region in the northern part of iran (7, 9, 28, 29). in the present study, we found that ticks prefer to attach to the rump and under tail (perineum) regions, compared with other body parts, and this finding is in agreement with the results of some other studies. the preferred site of attachment of ticks has been associated with infestation season, and it seems that during warm months of the year, hard ticks prefer perineum region for attachment (5, 28). conclusion in conclusion, regardless of no evidence of cchfv infection in this study, due to the crucial role of hard ticks in the transmission of different pathogens to humans, more investigations in the study area are needed for identification of tick pathogens in order to prevent important diseases that can be transmitted to humans by ticks. acknowledgements this study was funded by the deputy for research and technology of rafsanjan university of medical sciences and pasteur institute of iran and ethics code ir.rums.rec. 1395.70. all authors declare that there is no conflict of interest. references 1. delpy l (1936) notes sur les ixodidés du genre hyalomma (koch). ann parasitol hum comp. 14(3): 206–245. 2. delpy l (1938) les especes iranienes du genre haemaphysalis koch 1844. ann parasitol hum comp. 16(1): 1–10. 3. nabian s, rahbari s (2008) occurrence of soft and hard ticks on ruminants in zagros mountainous areas of iran. iran j arthropod borne dis. 2(1): 16–20. 4. hoogstraal h, valdez r (1980) ticks (ixodoidea) from wild sheep and goats in iran and medical and veterinary implications. fieldiana zool. 6: 1–16. 5. dehaghi mm, fathi s, asl en, nezhad ha (2011) prevalence of ixodid ticks on cattle and sheep southeast of iran. trop anim health prod. 43(2): 459–461. 6. ganjali m, dabirzadeh m, sargolzaie m (2014) species diversity and distribution of ticks (acari: ixodidae) in zabol county, eastern iran. j arthropod borne dis. 8(2): 219–223. 7. nabian s, rahbari s, shayan p, haddadzadeh hr (2007) current status of tick fauna in north of iran. iran j parasitol. 2(1): 12–17. 8. nasiri a, telmadarraiy z, vatandoost h, chinikar s, moradi m, oshaghi m (2010) tick infestation rate of sheep and their distribution in abdanan county, ilam province, iran, 2007–2008. iran j arthropod borne dis. 4(2): 56–60. 9. rahbari s, nabian s, shayan p (2007) primary report on distribution of tick fauna in iran. parasitol res. 101 suppl 2: s175–7. 10. rahbari s (1995) studies on some ecological aspects of tick fauna of west 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rafinejad j, pourmand mr, maleki-ravasan n, salehi-vaziri m ( 2019) effect of meteorological factors on hyalomma species composition and their host preference, seasonal prevalence and infection status to crimean-congo haemorrhagic fever in iran. j arthropod borne dis. 13(3): 268–283. 21. salari lak s, vatandoost h, telmadarraiy z, entezar mahdi r, kia e (2008) seasonal activity of ticks and their importance in tick-borne infectious diseases in west azerbaijan, iran. iran j arthropod borne dis. 2(2): 28–34. 22. pasalary m, arbabi m, pashei s, abdigoudarzi m (2017) fauna of ticks (acari: ixodidae) and their seasonal infestation rate on camelus dromedarius (mammalia: camelidae) in masileh region, qom province, iran. persian j acarol. 6(1): 31–37. 23. apanaskevich da, schuster al, horak ig (2008) the genus hyalomma: vii. redescription of all parasitic stages of h. (euhyalomma) dromedarii and h. (e.) schulzei (acari: ixodidae). j med entomol. 45(5): 817–831. 24. gharbi m, moussi n, jedidi m, mhadhbi m, sassi l, darghouth ma (2013) population dynamics of ticks infesting the one-humped camel (camelus dromedarius) in central tunisia. ticks tick borne dis.4(6): 488–491. 25. nazifi s, tamadon a, behzadi ma, haddadi s, raayat-jahromi ar (2011) onehumped camels (camelus dromedaries) hard ticks infestation in qeshm island. vet res forum. 2(2): 135–138. 26. zeleke m, bekele t (2004) species of ticks on camels and their seasonal population dynamics in eastern ethiopia. trop anim health prod. 36(3): 225–231. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 286–292 m salehi-vaziri et al.: molecular assay on … 292 http://jad.tums.ac.ir published online: september 30, 2020 27. ebrahimzade e, pazhoom f, nabian s, shayan p, bakhshani a (2014) ticks fauna of sheep and goats in some suburbs of mazandaran province, iran. iran j vet med. 8(4): 275–279. 28. hosseini-vasoukolaei n, telmadarraiy z, vatandoost h, yaghoobi-ershadi mr, hosseini vasoukolaei m, oshaghi ma (2010) survey of tick species parasiting domestic ruminants in ghaemshahr county, mazandaran province, iran. asian pac j trop med. 3(10): 804–806. 29. razmi gr, glinsharifodini m, sarvi s (2007) prevalence of ixodid ticks on cattle in mazandaran province, iran. korean j parasitol. 45(4): 307–310. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 376 http://jad.tums.ac.ir published online: december 31, 2020 original article employing different traps for collection of mosquitoes and detection of dengue, chikungunya and zika vector, aedes albopictus, in borderline of iran and pakistan jalil nejati1; morteza zaim2; *hassan vatandoost2,3; *seyed hassan moosa-kazemi2; rubén bueno-marí4; shahyad azari-hamidian5; mohammad mehdi sedaghat2; ahmad ali hanafibojd2,3; mohammad reza yaghoobi-ershadi2; hassan okati-aliabad1; francisco collantes6; ary a. hoffmann7 1health promotion research center, zahedan university of medical sciences, zahedan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4departamento de investigación y desarrollo (i+d), laboratorios lokímica, valencia, spain 5department of health education, research center of health and environment, school of health, guilan university of medical sciences, rasht, iran 6department of zoology and physical anthropology, university of murcia, murcia, spain 7bio21 institute, pest and environmental adaptation group, school of biosciences, university of melbourne, victoria, australia *corresponding authors: dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, dr seyed hassan moosakazemi, e-mail: moosakazemi@tums.ac.ir (received 25 may 2020; accepted 06 dec 2020) abstract background: southeastern iran has been established as an area with the potential to harbor asian tiger mosquito populations. in 2013, a few numbers of aedes albopictus were detected in three sampling sites of this region. this field study was aimed to evaluate the efficacy of various traps on monitoring mosquitoes and status of this dengue vector, in five urban and 15 suburban/rural areas. methods: for this purpose, four adult mosquito traps (bg-sentinel 2, bednet, malaise, and resting box trap) were used and their efficacy compared. in addition, large numbers of cdc ovitraps were employed, within 12 months. results: a total of 4878 adult samples including 22 species covering five genera were collected and identified from traps. it was not revealed any collection of ae. albopictus. statistical analysis showed no significant difference in meteorological variables between the two periods, the previous report and the current study. there were significant differences in the total number of mosquitoes collected by various traps in the region across different months. conclusion: the resulting data collected here on the efficiency of the various trap types can be useful for monitoring the densities of mosquito populations, which is an important component of a vector surveillance system. while the presence of ae. albopictus was determined in this potential risk area, there is no evidence for its establishment and further monitoring needs to be carried out. keywords: stegomyia albopicta; ovitrap; sistan and baluchistan introduction like many countries around the globe, the dengue vector, aedes albopictus, has been recently detected in iran, with the first report of this species being in 2016 (1). considering a previous report about the presence and estab lishment of this species in the eastern neighbor of iran, pakistan, this was not unexpected (2-4). despite its short flight length, ae. albopictus is considered an invasive species with a rapid potential expansion. it has been copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com mailto:moosakazemi@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 377 http://jad.tums.ac.ir published online: december 31, 2020 able to spread and establish in various areas from southeast asia to america, europe, africa and australian regions (5-7). due to climatic changes, global warming, and increased international traveling, arboviral diseases and their vectors have been expanded from endemic areas (8). the first iranian dengue fever case with travel background to southeast asia was reported in 2008 (9). the health system expressed its growing concern after denvigg detection in blood donors in southeast of the country, an oriental ecozone situated in the transit route between east asia and other countries (10, 11). this area has also been estimated as potentially harboring ae. albopictus (12). this hypothesis was verified by the detection of ae. albopictus, although only five larvae and seven adults were collected (1). therefore, it is crucial to ascertain the establishment status of this species based on ecological studies and using various sampling methods (13). indeed, the efficiency of different mosquito traps needs to be investigated across a region to establish an effective vector surveillance system (14). using a range of trap types, this study tests whether ae. albopictus is established in southeastern iran and explores the presence of other diurnally active mosquitoes in this potential risk area. materials and methods study area this study was carried out in sistan and baluchestan, the largest province in iran (181, 785km²). it includes 19 cities, 37 towns, and 9716 villages. it has a long shared border with pakistan and afghanistan in the east and the arabian sea in the south. aridity, dust storms, and especially the so-called ‘wind of 120 days’ constitute its general climate. nevertheless, periodic monsoon systems contribute to a diverse climate in the region, especially in southern coastal areas where there are summer rainfalls (11). the maximum average rainfall within 5 years (2011–2015), has been 145.6±89.1mm in the south and 87.9±45.5mm in the northern part of this province. while the region does not possess numerous water bodies (12), cement water tanks (ponds,) as usual water sources especially in rural areas, could be considered mosquitoes breeding places. the humidity in this region is mainly due to the bengal gulf streams, which come to iran only in the summer (15). despite the growing of date palm gardens, small farms, and even paddy fields in this province (11), the normalized difference vegetation index (ndvi) shows a thin vegetation cover (12). based on biogeographical considerations, this province has a unique climate including the palaearctic and indo-malaya realms (11). this part of the country still struggles with malaria and other vector-borne diseases, resulting in the establishment of a substantial vector surveillance system in the area (16, 17) (fig. 1). traps description the co2-baited bednet tarp prepared for this study was 2×2×1.2m in size, with 156 threads per inch mesh size and constructed from polystyrene fabrics. co2 gas was released using a pressure reducing regulator with about 0.5 l/min applied in pulses (20s on/ 40s off) (fig. 2a). the bg-sentinnel 2 trap consisted of a black collapsible fabric container with 40cm high and 36cm in diameter. it has a white gauze lid with a black catch pipe that is opened in case of air suction by an electrical fan. the created airflow, released a bg lure (18) (fig. 2b). figure 2c shows the malaise trap used (height 190–110cm, length 165cm, width 115cm) with a pyramid of white net on a black framework, fabricated from polystyrene. two sachets of the new bg-sweet scent were used as a mosquito attractant in this trap. the sachets were hung on the inner side of the black framework. two types of resting box trap (35×35×35cm) were used, depending on the color of the outer walls (black or blackwhite banded). the internal walls were covered with black cotton fabric. on the front side, there was a 35×15cm entry near the top, while the rest of the front was covered by a http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 378 http://jad.tums.ac.ir published online: december 31, 2020 black wooden\cotton wall. a sugar-fermenting yeast solution was applied to attract mosquitoes. the solution was made up of a mixture of dry yeast (7g), sugar (100g) and water (1l) (19) in 1.5l bottles\jars placed in traps or out of them with a connector plastic tube (fig. 2d). mosquitoes in this trap (as well as the bednet and malaise traps) were collected with an oral tube aspirator. the cdc ovitrap is a standardized device for mosquito egglaying. it involves a black glass jar which is 12.7cm high and 7.6cm in diameter at the top (fig. 2e). as an oviposition substrate, a wooden paddle (2.7cm long and 1.9cm wide) was placed in the jar, which was filled with water (20). in our study, the glass jar was replaced by a black plastic pot (19×14cm) due to its lower weight and breaking probability. field sampling procedure and statistical comparison this descriptive–analytic study was conducted from july 2016 to june 2017. in total, five urban (zahedan, rask, nikshahr, chabahar and konarak) 12 rural, and three suburban areas were explored for mosquitoes. in our study, suburb means peri-urban which includes outer residential areas of a city. the sampling points were chosen based on a previous study which had resulted in the ae. albopictus collection. the larvae of this species had been detected in rask (26.28471 °n, 61.40040 °e; elevation 421 meter above sea level [masl]) and adult samples were collected from paroomi (25.44267°n, 60.90731°e; elevation 44 masl) and vashname-dori (25.45919°n, 60.83179°e; elevation 9 masl) (1). besides, the sampling points were exploited from a modeling study conducted in this area covering various subclimates and topographies. adult sampling was undertaken at 10 points; three were fixed (rask, paroomi, vashname-dori) and seven were varied within an area. the first fixed sampling point was urban, and the other two points were classified as rural areas. sampling started before noon until half an hour after dusk. adult mosquito traps were installed 20– 50m apart. during adult sampling, meteorological variables including temperature, humidity and wind speed were recorded every two hours (fig. 3b). the co2-baited bednet, malaise and resting box tarps were checked every 15 minutes for five minutes. however, it was done once only at the end of the sampling day for the bg-sentinel 2 trap. due to air suction by the electrical fan, the collected mosquitoes could not escape. like other traps, the bg-sentinel 2 trap is installed outdoors. trap placement was conducted according to the instruction manual provided by biogents ag. it was protected from wind, rainfall, and direct sunlight, yet visible to mosquitoes. besides, the trap was positioned close to the mosquito breeding sites. the collected adult specimens were identified based on an iranian mosquito key (21). in addition, 505 ovitraps were installed at 31 points (fig. 3a), including cities, villages, customs areas, and seaports. the paddles were checked every two weeks and suspected cases were moved to the insectary for laboratory assessments. finally, the obtained data, collected specimens, month, average meteorological variables and trap types were analyzed statistically. contingency analysis was used to assess the co-occurrence of a species at a location/time point and computed phi from contingency tables as a measure of the strength of species’ association of species between different trap types. to this end, generalized linear models (glms), were ran, and the data were analyzed as a dichotomous variable in ibm spss statistics 24. results adult sampling results a total of 4878 adults were collected from various traps, in which 22 species of five genera (including three of the genus aedes, nine culex, eight anopheles, one culiseta, and one uranotaenia) were identified. overall, ae. caballus (35.5%) and culex quinquefasciatus (23.82%) were the dominant species, followed http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 379 http://jad.tums.ac.ir published online: december 31, 2020 by cx. sitiens (17.6%), cx. tritaeniorhynchus (10.9%) and ae. caspius (4.2%) (table 1). when pooled across species, a clear mosquito collection peak was detected in february, which coincided with a period of low wind speed, low temperature, and high humidity (fig. 4). of the 11 meteorological stations scattered throughout the study area, three points were selected. based on the previous report, ae. albopictus was collected from three sampling sites. the adults had been trapped in chabahar county in 2013, and the larvae were collected in nikshahr and rask counties in 2009. thus, meteorological data including the mean temperature as well as mean relative humidity along with precipitation related to these three counties were obtained from the data center of the provincial meteorological organization. figure 5 shows the monthly comparison of these variables. in december 2013, the maximum rainfall (38.5mm) was recorded in chabahar station, followed by february of the same year (20.4mm). in 2017, the maximum rainfall occurred in february (68.9mm). the mann-whitney test revealed no significant difference concerning these three variables between two years (12 months); 2013 and 2016–2017 (p> 0.05), in three larvae and adult sampling sites (chabahar, nikshahr, and rask). most mosquitoes were collected by co2-baited bednet traps (65.13%), and with a substantial percentage were caught by malaise traps (34.09%). the resting-box traps and bg traps only collected 0.51% and 0.27% of the mosquitoes respectively. the percentage of species collected by malaise and bednet traps tended to be highly correlated (table 1), whereas it varied in the case of other adult trap types (for instance, bg traps captured a high percentage of cx. sitiens). contingency tables (table 2) indicated that the two trap types placed at the same location tended to collect the same species. this was also reflected in the high phi scores associated with these comparisons. when pooled across trap types, the presence/absence of species was only associated with cx. quinquefasciatus and cx. tritaeniorhynchus (x2= 15.90, df= 1, p< 0.001, phi= 0.375) as well as cx. sitiens and ae. caspius (x2= 36.04, df= 1, p< 0.001, phi= 0.565). the data for these four most common species were statistically ana-lyzed. another common species, ae. caballus, was only de-tected in rural areas in one period and it was not analyzed further. aedes ca-ballus was only collected in february by both malaise and bednet trap types. the other species were collected throughout the year (fig. 6). bednet and malaise trap data were combined to evaluate the effects of the three regions (urban, suburban, and rural) along with other environmental variables on the occurrence of the four common species. for ae. caspius, glm indicated the significant effect of region (wald x2= 9.846, df= 2, p= 0.007), such that this mosquito was more common in urban areas (fig. 7). moreover, wind was found to exert a marginally non-significant effect (wald x2= 3.458, df= 1, p= 0.063), such that lower wind speed was associated with a higher probability of positive traps (b= 1.221, 95% ci 0.066, 2.509). for cx. quinquefasciatus, glm only confirmed the significant impact of wind speed (wald x2= 5.153, df= 1, p= 0.023), such that lower wind speed again was related to positive traps (b= 1.699, 95%, ci 0.232, 3.167). regarding cx. tritaeniorhynchus, the results of glm exhibited the significant effect of location, as reflected by the impact of latitude (wald x2= 6.708, df= 1, p= 0.010) and longitude (wald x2= 6.773, df= 1, p= 0.009). additionally, this species was significantly affected by wind speed (wald x2= 5.402, df= 1, p= 0.020). finally, none of the parameters had a significant role in the occurrence of cx. sitiens. ovitrap results no aedes eggs and larvae were detected on ovitraps’ paddles and waters respectively. two species, cx. tritaeniorhynchus (n= 11) and anopheles stephensi (n= 4), were collected from water of ovitraps at two points located in sarbaz county (26.14118 °n, 61.45337 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 380 http://jad.tums.ac.ir published online: december 31, 2020 °e, elevation 329 masl) and (26.15303°n, 61.44254 °e, elevation 360 masl). table 1. the collected mosquito species by trap types, southeastern iran, sistan and baluchestan province, 2016–2017 species co2-baited bednet malaise bg resting box total n % n % n % n % n % aedes caspius 94 3.0 107 6.4 3 12 1 7.7 205 4.20 ae. caballus 1031 32.5 698 42.0 4 16 1 7.7 1734 35.55 ae. vexans 2 0.1 1 0.1 0 0 0 0 3 0.06 culex quinquefasciatus 798 25.1 354 21.3 5 20 5 38.5 1162 23.82 cx. tritaeniorhynchus 335 10.5 191 11.5 3 12 2 15.4 531 10.89 cx. pipiens 87 2.7 19 1.1 0 0 3 23 109 2.23 cx. sitiens 602 18.8 247 14.8 10 40 0 0 859 17.61 cx. prexigus 38 1.2 10 0.6 0 0 0 0 48 0.98 cx. bitaniorhynchus 5 0.2 0 0.0 0 0 0 0 5 0.10 cx. theileri 5 0.2 1 0.1 0 0 0 0 6 0.12 cx. pseudovishnui 15 0.5 2 0.1 0 0 1 7.7 18 0.37 cx. siniticus 41 1.3 21 1.3 0 0 0 0 62 1.27 anopheles stephensi 61 1.9 9 0.5 0 0 0 0 70 1.44 an. culicifacies s.l. 16 0.5 0 0.0 0 0 0 0 16 0.33 an. superpictus s.l. 10 0.3 0 0.0 0 0 0 0 10 0.21 an. turkhudi 1 0.0 0 0.0 0 0 0 0 1 0.02 an. subpictus s.l. 5 0.2 0 0.0 0 0 0 0 5 0.10 an. fluviatilis s.l. 2 0.1 0 0.0 0 0 0 0 2 0.04 an. dthali 1 0.0 0 0.0 0 0 0 0 1 0.02 an. moghulensis 1 0.0 0 0.0 0 0 0 0 1 0.02 urantaenia unguiculata 2 0.1 0 0.0 0 0 0 0 2 0.04 culisets longiareolata 25 0.8 3 0.2 0 0 0 0 28 0.57 total 3177 100 1663 100 25 100 13 100 4878 100 fig. 1. location of the study area, sistan and baluchestan province, southeastern iran, surveyed during 2016–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 381 http://jad.tums.ac.ir published online: december 31, 2020 table 2. co-occurrence of mosquitoes from the same species in the two trap types, sistan and baluchestan province, 2016–2017 species present in both b and m absent in both b and m only in b only in m x2 (df= 1), p phi aedes caspius 26 83 2 2 92.56, <0.001 0.905 culex quinquefasciatus 58 19 35 1 21.71, <0.001 0.438 cx. tritaeniorhynchus 29 60 21 3 30.92, <0.001 0.587 cx. sitiens 17 91 5 0 82.77, <0.001 0.856 b: co2-baited bednet trap, m: malaise trap fig. 2. traps used for mosquito collection, co2-baited bednet tarp (a), malaise trap (b), 450 resting box (c), bg (d), ovitrap (e), southeastern iran, sistan and baluchestan province, 2016–2017 fig. 3. mosquito sampling sites; adults (a), ovitraps (b), southeastern iran, sistan and baluchestan province, 2016–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 382 http://jad.tums.ac.ir published online: december 31, 2020 fig. 4. the collected mosquitoes and meteorological variables by month, southeastern iran, sistan and baluchestan province, 2016–2017 fig. 5. monthly comparison of the meteorological variables between two years, 2013 and july 2016–june 2017 in three sampling sites: (a) rask, (b) nikshahr, (c) chabahar, southeastern iran, sistan and baluchestan province, 2016–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 383 http://jad.tums.ac.ir published online: december 31, 2020 fig. 6. monthly number of mosquitoes from traps as an estimate of population density for (a) aedes caspius, (b) ae. caballus, (c) culex quinquefasciatus, (d) cx. tritaeniorhinchus and (e) cx. sitiens. note log scale apart from (b), sistan and baluchestan province, 2016–2017 fig. 7. box plots for ln density of five common species by two traps in different areas. (ae. cab) aedes caballus, (ae. cas) ae. caspius, (cx. qui) culex quinquefasciatus, (cx. sit) cx. sitiens, (cx. tri) cx. tritaeniorhynchus are plotted. ln counts from (r) rural, (su) suburb, and (u) urban regions are plotted separately. traps were (b) bednet or (m) malaise. asterisks indicate outliers, sistan and baluchestan province, 2016–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 384 http://jad.tums.ac.ir published online: december 31, 2020 discussion aedes albopictus is a successful container breeder. once introduced into an area, its establishment can be considered as evidence. besides, most control efforts focus on the eradication of this species in case their first detection attempts prove unsuccessful. meanwhile, this is dependent on the species’ environmental adaptability and the quality of control measures applied (22). our study was conducted in a broad area with imported dengue cases and historical vector collection. using different mosquitoes sampling methods within 12 months, did not obtain any collection of ae. albopictus. in a similar investigation performed in southern california, surveillance of this species after about one year from discovery indicated no presence of adults or signs of oviposition. in that research, two types of trap were used: battery-operated cdc/co2-baited light traps and ovitraps. it was carried out after a report supporting the collection and detection of significant numbers of ae. albopictus in the cargo containers of lucky bamboo shipped from china. the rapid control response to the first report of this species and the cooperative efforts aimed at eliminating it were regarded as reasons for this success (22). meanwhile, we do not have any evidence on the eradication efforts carried out by the iranian health system and their aftereffects on the ae. albopictus community consequently. nevertheless, some modeling studies have suggested that northern parts of iran can be susceptible to the establishment of this vector. in a highresolution map, it was found that enhanced vegetation index (evi) annual mean, evi range, annual monthly maximum precipitation, annual monthly minimum precipitation, temperature suitability, and urban as well as peri-urban areas are environmental covariates affecting the occurrence probability of ae. albopictus. specifically, in order of significance, temperature suitability, minimum precipitation, and evi demonstrated the most relative contribu tions to predicting this map (23). studies conducted in southeast of iran have indicated that low vegetation index, high temperature, along with low rainfall associated with this part of the country affect the establishment of ae. albopictus in this region (12). in a previous study in this area, ae. albopictus adults were collected during november/december 2013 after a heavy rainfall (1). although this species was not collected in our study, the highest peak of mosquito activity was preceded by a torrential rainfall. some researchers attribute an important role to rainfall in establishing ae. albopictus, such that at least 500 mm of annual rainfall is needed for this species to develop (24, 25). meanwhile, some studies suggested 290mm of annual rainfall for this to happen (26, 27). evidently, ae. albopictus could be established in areas with 200–500 mm annual rainfall (5). despite the correlation between rainfall and the presence of ae. albopictus, some researchers have highlighted the behavior of ae. albopictus as a container mosquito which could exist in the rainfall independently from its breeding sites (28). in a study in india, this species was collected from arid and semi-arid areas. analogous to the region investigated in the present study, these were plain sandy places with thin vegetation and water stored in containers including a large number of cement and plastic tanks (29). as a result, the risk of ae. albopictus establishment in this area can never be ignored. although ae. albopictus was not collected in the current study, the efficacy of four types of trap on catching six aedes and culex species were evaluated. some species such as ae. vexans, ae. caballus, cx. quinqaefasciatus, cx. tritaeniorhynchus, and cx. sitiens can be regarded as west nile fever and chikungunya vectors (30-34) that occur in iran (8, 35). in fact, cx. quinqaefasciatus and ae. vexans have been previously reported as dominant species in southeast of iran (36, 37). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 385 http://jad.tums.ac.ir published online: december 31, 2020 our results showed that the meteorological factors of wind speed and temperature have a direct significant correlation with the total number of collected mosquitoes. in particular, in southern norway, mosquito abundance displayed a negative association with wind speed, and no mosquitoes were collected at speeds above 7·5m/s (38). our results confirmed that wind speed affects the collection likelihood of several mosquito species.monitoring densities of mosquito populations can be performed by various sampling methods to evaluate the vectors’ behavior, especially the effects of vector control interventions (39). in the current study, co2-baited bednet trap was associated with the majority of collected mosquitoes. we selected this trap based on a previous study in iran where it was proved effective in collecting ae. albopictus (1). also, this trap has been used for sampling in other vector-borne diseases such as dirofilariasis (40) and malaria (41) in iran. however, no report has been made so far concerning the efficacy of this trap type for mosquito sampling. compared to other traps, co2-baited bednet trap covers a larger space, possibly increasing its ability to collect mosquitoes especially in a semi-desert climate with low mosquito density. furthermore, co2 used in this trap might have served as an efficient mosquito attractant (42). most studies on trap efficacy have focused on evaluating bg traps versus other traps. for instance, a study in germany observed that the bg trap was better at collecting a range of mosquito species than three other types of traps: heavy duty encephalitis vector survey trap (evs trap), centers for disease control miniature light trap (cdc trap) and mosquito magnet patriot mosquito trap (mm trap). also contrary to our findings, the widest range of mosquito species was collected by bg traps (43). the field efficacy of the bg trap for collecting of ae. albopictus has been also studied in new jersey, us, where it collected more mosquitoes compared with the cdc trap and the gravid trap (gt). therefore, this trap type has been recommended as an important part of vector monitoring and surveillance programs (44). although most entomological studies with bg traps have been undertaken in europe, north and south america, australia, and southeast asia, covering tropical regions or areas with dense vegetation, the poor performance of bg traps in the current study was surprising (44-46). perhaps this type of trap, especially in the absence of co2, is less effective in a dry climate with a low mosquito density. on the other ahnd, malaise traps (47) and bednet traps performed well in our study. there are few published papers on the usage of malaise traps for mosquitoes in tropical areas of the world (48). in the us, townestype malaise traps (49) were used for about five months to collect mosquitoes, with aedes species being the most widely collected, followed by culex species. vision as well as dark color can play an important role in the attraction of some insects such as tabanid flies and mosquitoes (50). mosquitoes possibly showed a positive response to the malaise trap due to its black color and its bg lure as an attractant. since this trap and the bednet traps tend to catch common mosquitoes in similar proportions at the same locations and times, both trap types could be useful for general mosquito surveys. the so-called resting bbox trap has been applied and modified for sampling malaria vectors, particularly outdoor-resting mosquito species (39). we modified it to monitor ae. albopictus with a strong exophilic (resting) behavior (51). however, in our study, this trap did not perform well compared to others. this trap is marked by a low efficiency in collecting cs. inornata (48). there are few published papers regarding the usage of resting box traps for aedes sampling. in thailand, two types of trap consisting of short boxes (45cm) and tall boxes (90cm) were placed inside houses (52). the short boxes, whose size was close to the one employed in our trap, attracted relatively more females. perhaps this type of trap is an efficient method for sampling ae. aegypti rather than the species collected here. ovitraps http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 376–390 j nejati et al.: employing different traps for … 386 http://jad.tums.ac.ir published online: december 31, 2020 are commonly used for sampling containerbreeding mosquitoes such as ae. albopictus and ae. aegypti (53), even though their efficacy in catching malaria vectors has also been evaluated (54). in the current study, ae. albopictus larvae were not detected in ovitraps, but the two species of cx. tritaeniorhynchus and an. stephensi were occasionally observed. in studies designed for mosquito control, cx. quinquefasciatus, cx. tritaeniorhynchus, and an. stephensi have been reported alone or along with ae. aegypti in ovitraps (53, 55-58). ovitraps could, therefore, be used in surveillance but their low return rate needs to be taken into account, given that > 500 ovitraps were placed outdoors in this study. conclusion data collected here on the efficiency of various trap types can be useful for monitoring densities of mosquito populations, which is an important component of a vector surveillance system. although ae. albopictus had been previously collected from the study area, its establishment is now questionable given that we could not collect it despite substantial effort. further inquiry into ae. albopictus establishment in this potential risk area should be rewarding. eventually, in case of local transmission of dengue, studies on the potential transmission of other aedes species should be considered in any new situation. acknowledgements this study was financially supported by tehran university of medical sciences with the grant number 9121260011 and partially by ministry of health and medical education under code number of nimad 982984. the study was also supported by an nhmrc fellowship to aah. this research could not be accomplished without the assistance of our colleagues in zahedan and iranshahr health deputy. hence, we would like to express our deepest appreciation to all those who provided us with the possibility to do this study. we are also grateful to abdolghafar hasanzehi and abdolmohsen parvin, the health staff in charge of cdc dengue management in zahedan and iranshahr universities of medical sciences. we thank farooq askani, aghil birnoor, rasulbakhsh raeisi, ebrahim badroozeh, salim nabatzehi, shahab dehghani, and eng. noormohammad nabatzehi for their assistance in field sampling. in addition, thanks go to dr asghar talbalaghi, freelance entomologist and consultant in ipm in italy, for his invaluable comments during sampling. last but not least, we thank eng. hamid reza behjati in sistan and baluchestan meteorological organization for providing weather data. the authors declare that there is no conflict of interest. references 1. doosti s, yaghoobi-ershadi mr, schaffner f, moosa-kazemi sh, akbarzadeh k, gooya mm, vatandoost h, shirzadi mr, mosta-favi e (2016) mosquito surveillance and the first record of the invasive mosquito species aedes (stegomyia) albopictus (skuse) (diptera: culicidae) in southern iran. iran j 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responses of anopheles stephensi and aedes aegypti to a fatty acid ester-propyl octadecanoate. acta trop. 124(1): 54–61. 58. hesson jc, ignell r, hill sr, östman ö, lundström jo (2015) trapping biases of culex torrentium and culex pipiens revealed by comparison of captures in cdc traps, ovitraps, and gravid traps. j vector ecol. 40(1): 158–163. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 420–425 ke benallal et al.: first detection of … 420 http://jad.tums.ac.ir published online: december 31, 2019 case report first detection of aedes (stegomyia) albopictus (diptera: culicidae) in algiers, the capital city of algeria *kamel eddine benallal, rafik garni, lazhari bouiba, zoubir harrat laboratoire d’eco-épidémiologie parasitaire et génétique des populations, institut pasteur d’algérie, route petit staouéli, dely ibrahim, alger, algérie (received 13 mar 2017; accepted 13 oct 2019) abstract background: based on the reporting of the presence of stripped mosquitoes by a citizen in the algiers residential neighborhood of bir-khadem, where residents experienced huge daytime mosquito nuisance an entomological investigation was carried out in july 2016. methods: ovitraps and bg sentinel traps baited with lure were used during three consecutive days to collect adult mosquitoes. eighteen residential houses of the bir-khadem neighborhood were also inspected to search larvae breeding sites such as water fountains, baskets and flowerpots. results: a total of 57 aedes albopictus specimens were collected in five villas, consisting of 21 eggs, 20 larvae and 16 adults. conclusion: this is the first record of this invasive species in algiers. keywords: aedes albopictus, mosquitoes, arbovirus, algiers, algeria introduction aedes albopictus is currently considered the most invasive mosquito species (1) as its rapid adaptation to a large variety of environments in both tropical and temperate areas has allowed its spread throughout the world (2). indeed, ae. albopictus larvae can breed in different container forms (3). as early as 1968, ae. albopictus was reported as the most common and widespread mosquito in southeast asia (4). in 1979, this mosquito was reported in albania (5) then in several countries in europe, around the mediterranean basin and in morocco (6, 7). doubtlessly, the worldwide dispersion of ae. albopictus is mainly caused either by trade in used tires or lucky bamboo between the continents and by travelers and cars within land (8, 9). beside its huge nuisance, ae. albopictus capacity to spread could engender a serious threat for the public health since it is competent to transmit several viruses (10). this mosquito has been responsible for the large out break of chikungunya in la réunion island and focal transmission of dengue and chikungunya in europe (11–13). since then because of its ability to adapt to different environments, this mosquito species is under scrutiny by many health services. it has also been shown to be competent for zika virus (10, 14) but its role in the current zika epidemics needs further investigation. aedes albopictus is also known as a vector for west nile virus (15, 16), dirofilaria immitis and d. repens (17, 18). in algeria, it was reported on three occurrences (19-21), but never in algiers, the capital city of algeria. in july 2016, a citizen contacted our laboratory to complain from strange mosquitoes that caused painful bites and huge daytime nuisance. the herein entomological survey led to the identification of ae. albopictus in the district of bir-khadem and assessed its spread in the district. *corresponding author: dr kamel eddine benallal, e-mail: benallalkamel4@yahoo.fr http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 420–425 ke benallal et al.: first detection of … 421 http://jad.tums.ac.ir published online: december 31, 2019 materials and methods bir-khadem is a locality of algiers (altitude 84m, 36.716460° n, 3.042561° e), population number 77. 749 persons located about 6km from the commercial port and 16km off the international airport. in algiers, mediterranean climate occurs with a mean 686mm rainfall per year and a mean temperature of 23 °c. a citizen living in a residential neighborhood brought to our laboratory some mosquito specimens collected in his house. these mosquitoes bite during daytime and made a huge nuisance, which was suggestive of ae. albopictus. following this, sampling procedures for detecting invasive mosquitoes were implemented in that neighborhood. two biogents-sentinel™ trap (bgs) (biogents ag, regensburg, germany) baited with bg-lure (bgl) a mosquito attractive were run outside in the gardens (first at the house of the complainer and the second at the house of the asian persons) for three consecutive days to collect adult mosquitoes. a total of 18 residential houses of the bir-khadem neighborhood were also inspected in order to search possible larval breeding sites such as water fountains, baskets and flowerpots plates. ovitraps consisting of black plastic container filled with 400ml of water and leaves provided with a 20× 20× 5cm of polyester piece as a substrate for aedes spp. mosquito oviposition device were placed under trees and in shaded areas (11). the collected adult and larvae specimens were morphologically identified (22). a survey with local citizens (18 households) about the first appearance of this mosquito in the area was carried out mainly was around: i) the period of biting and activity of this species (daytime or night), ii) what is new in the neighborhood (the installation of new people, a new commercial activity especially that of used tires or the sale of exotic plants), iii) the notion of travel in countries endemic to ae. albopictus. all these questions were asked in order to find an explanation how this mosquito has came to this area since it was reported established only in oran which is distanced about 500km from algiers (21). results the eight mosquitoes (two males and six females) collected by the citizen belonged to aedes genus showed black and white stripes showed black and white stripes on their legs and a medio-dorsal white stripe on the thorax characteristic of ae. albopictus. out of the 18 residential houses prospected 4 were positives with ae. albopictus in association with culex pipiens and culiseta longiareolata around the asian foreign villas (table 1, fig. 1). a total of fifty-seven adults of ae. albopictus (male and female) were identified either collected at adult stages or emerged from larvae and nymphs. the survey carried out with local citizens revealed that this species bites in daytimes and had appeared one month before in june and coincided with the installation of asian persons who work for a building company in algiers. they had not reported new commercial activities (tires or plants) since it was a residential quarter. concerning travelling notion no one had been in an endemic area of ae. albopictus. table 1. location and stage of aedes albopictus collected during the survey house specimen numbers adultes larvae eggs p1 p2 p3 p4 total 13 11 / / 24 15 / / 5 20 7 / 6 / 13 p1: villa where first specimens were collected, p2: foreign’s villa, p3: second positive villa, p4: third positive villa http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 420–425 ke benallal et al.: first detection of … 422 http://jad.tums.ac.ir published online: december 31, 2019 fig. 1. location of aedes albopictus captures at different development stages discussion an entomological survey was implemented in a residential neighborhood of bir-khadem in order to identify the mosquito species implicated in the source of nuisance. as suspected, several specimens of ae. albopictus were found. residential houses with garden, swimming pool, vegetation, trees and animals provide suitable environmental conditions for this species to reproduce. in addition, the stability of good climate conditions during june till august 2016 (mean temperature 25 °c) has allowed ae. albopictus to reproduce and colonize easily this area. the occurrence of this species in this locality with mean annual precipitation of 686 mm is of great interest since over most of its range is associated with very heavy rainfall (23). bg-sentinel traps and ovitraps allow the collection of few specimens only. this suggests that ae. albopictus population is still at low level despite favorable environmental conditions or at the beginning of its introduction. besides, the questionnaire survey carried out with the citizens revealed that this mosquito had appeared since june 2016. one might wonder whether the source of ae. albopictus could be some eggs carried in the plants that they brought from their country and cultivated in their garden for their own consummation since no commercial activity nor tire manufactory are registered in this area. aedes albopictus is well established all around the mediterranean basin and risk-mapping modeling showed that northern algeria is suitable for its installation (24). since the report of dengue and chikungunya in europe (25, 26) and several cases of west nile in algeria (27, 28), the algerian government has established an arbovirus surveillance system according to the international health regulation. unfortunately, the surveillance system setup has not allowed the early detection of ae. albopictus introduction into algeria but thanks to the vigilance of inhabitants who reported these mosquitoes which proves that implication of citizens remains the most important way to report and monitor the establishment of invasive mosquitoes (11). even if insecticide (allethrin) spraying was performed by algiers board of http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 420–425 ke benallal et al.: first detection of … 423 http://jad.tums.ac.ir published online: december 31, 2019 health to eliminate adults before possible establishment, a continuous surveillance with ovitraps and bg-sentinel traps is highly recommended to monitor its extension area since ae. albopictus is known to skip oviposition and to release its eggs in several breeding sites (29). a good communication campaign in radio and television should be also planned as it is a new mosquito species with new behavior in order to explain to citizens how avoiding the development of this species in their garden and how to limit its propagation by simple gestures like covering the water barrels, emptying the flowerpot plates, removing unnecessary utensils from the gardens and the surrounding areas that may contain water and help the pullulation of these mosquitoes. such measures need to be planned before for limiting ae. albopictus propagation to other areas and to avoid the massive using of insecticides for vector control which could favor the appearance of resistance with time. conclusion the fourth record of a. albopictus in algeria suggests that the surveillance system efficiency must be improved by sensitizing and implicating population awareness for this mosquito species in time. furthermore, involvement of ae. albopictus in the transmission of dengue, chikungunya, and zika viruses from viremic persons highlights the importance of monitoring this invasive species to assess the health risk. further studies are recommended to determine the origin of the specimens collected in algiers, and to investigate the ecology and sensitivity to insecticides of this species in case it establishes in algiers for planning adapted and efficient control measures. acknowledgements we would like to thank all the inhabitants of the residential area especially mr karim oudina for their help during entomological investigations, and c bourgouin (gfmi, institut pasteur-paris) for useful comments. the authors declare that there is no conflict of interests. references 1. enserink m (2008) a mosquito goes global. science. 320: 864–866. 2. gould ea, higgs s (2009) impact of climate change and 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and new infect. 27: 9–13. 29. davis tj, kaufman pe, hogsette ja, kline dl (2015) the effects of larval habitat quality on aedes albopictus skip oviposition. j am mosq control assoc. 31 (4): 321–328. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 50 http://jad.tums.ac.ir published online: april 27, 2019 original article widespread report of multiple insecticide resistance in anopheles gambiae s.l. mosquitoes in eight communities in southern gombe, north-eastern nigeria *adedayo olatunbosun-oduola1; ezra abba2; olukayode adelaja1; adeolu taiwo-ande1; kennedy poloma-yoriyo2; taiwo samson-awolola3 1department of zoology, university of ilorin, ilorin, kwara state, nigeria 2department of biological sciences, faculty of science, gombe state university pmb 127, gombe, nigeria 3public health division and epidemiology, nigerian institute of medical research, yaba, lagos, nigeria (received 20 mar 2018; accepted 8 jan 2019) abstract background: timely entomological and insecticide resistance monitoring is a key to generating relevant data for vector management. we investigated the insecticide susceptibility status of anopheles gambiae s.l. in eight rural farming communities in southern gombe, nigeria. methods: overall, 3–5 days-old adult female anopheles mosquitoes reared from field-collected immature stages between september and november, 2014 were exposed to the diagnostic doses of pyrethroids, organophosphate and carbamate insecticides using the center for disease control bottle bioassay. the observatory knockdown time from exposure to each insecticide was recorded up to two hours. the dead mosquitoes were then identified morphologically and by molecular assays. results: mortality results showed resistance in an. gambiae s.l. populations to bendiocarb (2.3–100%), deltamethrin (39–70%), pirimiphos-methyl (65–95%), dichloro-diphenyl-trichloroethane (0–38.1%), permethrin (0–46.3%) and lambda-cyhalothrin (42.5–86.4%). the few cases of full susceptibility were observed from lamdacyhalothrin exposed population of an. gambiae s.l. in banbam and pantami respectively. an. gambiae 177 (45%) was significantly higher (p< 0.05) than an. arabiensis 64 (16.3%), an. coluzzii 34 (8.7%) and an. gambiae/an. coluzzii hybrid 78 (19.8%). conclusion: a strong evidence of widespread resistance in the major malaria vector species in southern gombe to all common classes of insecticides is a justification for the state malaria elimination programme to consciously consider incorporating insecticide resistance management strategies into control programs in order to sustain the future of current control interventions. keywords: multiple; insecticide resistance; anopheles gambiae; gombe south; nigeria introduction malaria is a major public health burden in nigeria with over 90% of her 167 million people at constant risk. nigeria accounts for 29% of the global malaria burden and together with the democratic republic of congo contribute up to 40% of the global burden for malaria (1). anopheles gambiae s.l. have been reported to be the most widespread mosquitoes responsible for malaria transmission in all the ecological zones (1). controlling these vectors pose major challenges as the an. gambiae s.l. mosquito belongs to a complex with each playing different role in transmission and exhibiting varying insecticide susceptibility status across the several regions in nigeria. in nigeria, vector control strategy remains one of the frontline and effective tools for controlling malaria and other insect-borne diseases. the strategy relies heavily on the use of insecticides from just four available classes of insecticides. in the face of increasing reports of insecticide resistance, sustained efficacy of *corresponding author: dr adedayo olatunbosun oduola, e-mail: dayooduola@yahoo.co.uk j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 51 http://jad.tums.ac.ir published online: april 27, 2019 these chemicals is desired. resistance to at least one or more classes of insecticide have assumed a geographical scale in more than 50 countries (2). this is a major threat to pyrethroids which is the only class of insecticide approved for use in long lasting insecticide nets) (llins as compared to indoor residual spray (irs) which utilizes all the four classes (3). the delicate gains made in the global reductions of malaria deaths since 2010 due to increased funding and concerted efforts to attain control is threatened by emerging resistance to insecticides in anopheles mosquitoes (2). insecticide resistance data exist in nigeria; however, most of these data are not consistently monitored for all communities in nigeria. despite an increase in the reporting of insecticide resistance in southern (4-9) and northern nigeria (10-11), only very little information on insecticide resistance exist for gombe state. this suggests a major setback for vector control and weak existing capacity for monitoring insecticide resistance. where these are available, they are not consistently generated to guide implementation of vector control. similarly, there is also limited information on the insecticide susceptibility status of members of the major malaria vector species: an. gambiae s.l. to available classes of insecticides used in malaria vector control in the southern gombe state. this study therefore provides information meeting the demand of the nigerian national malaria strategic plan to generate surveillance data that can be used to inform policy. this study for the first time provides such information on the susceptibility status and identity of the major malaria vectors in southern gombe to different available classes of insecticides used in vector control. materials and methods study area the study was carried out in the four local government areas of southern part of gombe state, 10°17`n, 11°10`e. gombe is a state in north-eastern nigeria, with its capital at gombe. the state has an area of 20,265km2 and a population of around 1.8 million. the state is characterized by two distinct seasons, which are dry season (nov to mar) and wet season (apr to oct). the vegetation of gombe state can be described as sudan savanna with open grassland which dries up during the dry season. gombe state shares boundaries with yobe state to the north, adamawa and taraba states to the south, borno state to the east, and bauchi state to the west. the people of gombe south are mainly farmers. they produce both food and cash crops. among its food crops are yam, cassava, maize, millet, sorghum, cowpea, tomato, groundnut, while cotton are produced for cash. indigenes also keep cattle, goats, sheep, horses, and donkeys and practice the traditional crafts of weaving and dyeing cotton the study was carried out in 8 farming rural communities namely: kalorgu (n09°49′34.9″, e01117′27.1″), ture (n09° 49′09.8″, e011° 22′ 44.2″), bambam (n09° 42′22.7″, e011°32′ 23.5″), pantami (n09° 41′35.4″, e011°28′ 23.4″), pokolin (n09°52′39.8″, e011°12′57.5″), zazzagawa (n09°53′20.1″, e011°12′34.7″), filiya (n09°34′38.5″, e011°06′52.4″), diga (n09°39′41.5″, e11⁰07′10.5″) (fig. 1). collection of anopheles larvae third to fourth instar larvae and pupae of anopheles mosquito larvae were collected from breeding sites in the eight communities from four local government areas (billiri, kaltungo, balanga and shomgom) of gombe south, nigeria. anopheline larvae were collected from their natural breeding sites (rice farms, small pools, puddles, and potholes) using the dipping method during the months of september to nov 2014. coordinates of the study sites were established using the global positioning system (gps). the immature anopheles mosquitoes collected were transported to the gombe state j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 52 http://jad.tums.ac.ir published online: april 27, 2019 malaria control insectary where they were reared to adults. the emerging adult mosquitoes were placed in the adult mosquito cages and fed with 10% sugar solution soaked in cotton wool. separation of female anopheles mosquitoes and morphological identification of the mosquitoes the adult female anopheles used for the test were separated from the males. using morphological characters (12-13) the adult female mosquitoes were identified under a dissecting microscope. the identification focused on dark spots at the upper margins of the wings which is common to all anopheles. the palpis are elongated and segmented into three. speckles on the legs, third pre-apical dark area on vein 1 with a pale interruption and tersi 1–4 with conspicuous pale bands are morphological features for the identification of an. gambiae. cdc bottle bioassay method ten to fifteen unfed female anopheles mosquitoes of 3–5 days old were introduced into four 250ml wheaton bottles coated with technical grade insecticide and one control bottle coated with acetone. these were provided by center for disease control (cdc), atlanta georgia as described by the guideline for evaluating insecticide resistance in vectors using the cdc bottle bioassay). the numbers of dead and live mosquitoes were monitored at different time intervals (0, 15, 30, 35, 40, 45, 60, 75, 90, 105, 120min). this allowed the determination of the total percent mortality against time for all replicates. the tested mosquito samples were stored in eppendorf tubes containing silica gel for further analysis. pcr identification of members of the anopheles gambiae complex mosquitoes identified as an. gambiae complex was subjected to species-specific polymerase chain reaction assays to identify members of the members. the molecular identification method was based on specific dna nucleotide sequences in the intergenic spaces of the ribosomal dna (14). further analysis to identify the an. gambiae (formerly ‘s’ form) and an. coluzzii (formerly ‘m’ form) was determined by incubating amplified material with hhai restriction enzymes at 37 degrees over a period of 3h to detect restriction fragment length polymorphisms (rflps). the enzyme hhai produced patterns of dna bands which differentiated an. gambiae from an. coluzzii (15). data analysis the percentage mortality of the mosquitoes exposed to each of the insecticides was calculated as the proportion of mosquitoes that died at the diagnostic time for each of the insecticides. correction with abbott’s formula was not necessary as control mortalities was less than 5% throughout the test. who recommendations for assessing the significance of detected resistance was used. ccording to the criteria, ≥98% mortality at the recommended diagnostic time indicates susceptibility, ≤97% mortality indicates resistance (16). data were entered into spss ver. 17 software (chicago, il, usa) and species composition in the study communities were analyzed for significant differences using analysis of variance. a general linear model procedure (t-test independent means at p< 0.05 significance) was also used to compare mortality in a gambiae populations exposed to two different insecticides within the same (deltamethrin/ lambdacyhalothrin, deltamethrin/ permethrin, and lamdacyhalothrin/ permethrin) and different classes (pyrethroids and ddt, organophosphate and carbamate) but having the same target site. ethical approval this study received ethical approval from university of ilorin ethical research committee. j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 53 http://jad.tums.ac.ir published online: april 27, 2019 results insecticide susceptibility tests three batches of adult female anopheles mosquitoes each containing 331, 333 and 345 an. gambiae s.l. mosquitoes were exposed to deltamethrin, lambda cyhalothrin and permethrin insecticides (table 1). there was resistance (0–86.4%) in all the study communities to the three different pyrethroids tested except in bambam community where full susceptibility (100%) of the an. gambiae s.l. to lamdacyhalthrin was observed (table 1). in the same manner another batch of 342, 324 and 344 anopheles mosquitoes exposed to bendiocarb, pirimiphos-methyl and ddt showed resistance (0–95%) in all the study communities (table 2). except in bambam and pantanmi where full susceptibility (100%) to bendiocarb was observed (fig. 2). the resistance profile of the an. gambiae population showed that susceptibility (percentage mortality ≥97%) were found only in bambam and pantanmi communities. a glm procedure to compare mortality in an. gambiae s.l. populations exposed to two different insecticides showed no significant difference between mortality of an. gambiae populations exposed to lambdacyhalothrin and deltamethrin (t=-1.58906, p=0.1344). however, mortalities were significantly higher in both lambda cyhalothrin (t= 5.1234, p= 0.0002) and deltamethrin (t= 5.09965, p= 0.0002) exposed populations of an. gambiae s.l. mosquitoes compared to permethrin. lambda cyhalothrin and deltamethrin insecticides produced the highest mortality in the population of an. gambiae in most of the study sites compared to permethrin. in comparing mortalities in an. gambiae populations exposed to two different classes of insecticides (organochlorine/ pyrethroids and bendiocarb/ organophosphate), mortality rate was significantly higher in an. gambiae populations exposed to deltamethrin (t= -0.55542, p=0.587372) and lamdacyhalothrin (t=5.07487, p= 0.000169) compared to ddt. in contrast, there was no significant difference between mortalities observed from an. gambiae s.l. populations exposed to ddt and permethrin (t= -0.55542, p= 0.587372). similarly, a comparison of differences in mortality rates between an. gambiae s.l. populations exposed to bendiocarb (carbamate) and pirimiphos methyl (organophosphate) in all the study sites were also not significant (t= -0.84482, p= 0 .412419). species composition of members of anopheles gambiae s.l. out of the 2019 an. gambiae s.l. mosquitoes subjected to the bioassay, three hundred an. gambiae s.l. mosquitoes selected across the study communities were assayed to detect sibling species. anopheles gambiae (formerly referred to as ‘s’ molecular form) and an. coluzzii (formerly referred to as ‘m’ form), an. arabiensis and hybrid (an. gambiae/ an. coluzzii) were all found across the study communities (table 3). anopheles gambiae (formerly sform) was the significantly dominant species across the study communities constituting 177 (45%) of the total samples analyzed (table 3). anopheles arabiensis accounted for 64 (16.3 %) of the total samples scored in the assay. on the other hand an. coluzzii (m-form) constituted 34 (8.7%). the mosquitoes unaccounted for constituted about 40 (10.2%) and these were samples either misidentified samples or samples with poorly preserved dna (table 3). outside the 40 (10.2%) samples not successfully amplified, analysis of variance to test difference in species composition of members of the an. gambiae s.l. was significant (p= 0.011369). results showed that an. gambiae was significantly higher compared to an. coluzzii (p= 0.006433.) and a. arabiensis (p= 0.024755). there was no significant difference between an. gambiae and the hybrid species (p= 0.103996) (fig. 2). j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 54 http://jad.tums.ac.ir published online: april 27, 2019 table 1. susceptibility status of anopheles gambiae s.l. mosquitoes to pyrethroids insecticides study sites deltamethrin lambda-cyhalothrin permethrin number assayed (331) mortality n (%) status number assayed (333) mortality n (%) status number assayed (345) mortality n (%) status kalorgu 43 21 (48.8) r 41 25 (61) r 44 6 (13.6) r ture 44 25 (56.8) r 43 30 (70) r 41 19 (46.3) r bambam 40 28 (70) r 40 40 (100) s 47 0 (0) r pantami 40 24 (58.5) r 44 38 (86.4) r 44 2 (4.5) r pokolin 41 16 (39) r 41 21 (51.2) r 40 13 (32.5) r zazzagawa 42 26 (61.9) r 40 17 (42.5) r 44 7 (15.5) r filiya 40 21 (52.5) r 40 17 (42.5) r 45 13 (28.9) r diga 41 16 (39) r 44 35 (79.5) r 40 5 (12.5) r n= number of mortality, r=resistant, s=susceptible, numbers in brackets represent percentages no mortality was observed in the control experiment set up for the insecticides in each study sit table 2. susceptibility status of anopheles gambiae s.l. mosquitoes to bendiocarb, pirimiphos-methyl and ddt insecticides study sites bendiocarb ddt pirimiphos-methyl number assayed 342) mortality n (%) status number assayed (344) mortality n (%) status number assayed (324) mortality n (%) status kalorgu 46 40 (87) r 45 16 (35.6) r 40 38 (95) r ture 41 31 (75.6) r 46 13 (28.3) r 40 34 (85) r bambam 41 41 (100) s 42 7 (16.7) r 43 31 (72.1) r pantami 43 43 (100) s 40 10 (25) r 40 27 (67.5) r pokolin 42 12 (28.6) r 41 0 (0) r 40 26 (65) r zazzagawa 44 1 (2.2) r 44 8 (18.2) r 41 27 (65.9) r filiya 44 29 (65.9) r 44 10 (22.7) r 40 30 (75) r diga 40 24 (60) r 42 16 (38.1) r 40 32 (80) r n= number of mortality, r= resistant, s= susceptible, numbers in brackets represent percentages no mortality was observed in the control experiment set up for the insecticides in each study site table 3. distribution and composition of members of anopheles gambiae s.l. in the study areas study sites number assayed (n) species composition a. arabiensis an. gambiae (s-form) an. coluzzii (m-form) hybrid no amplification (na) kalorgu 70 13(18.6) 6(8.6) 2(2.9) 41(58.6) 8(11.4) ture 52 13(18.6) 22(42.3) 2(3.8) 10(19.2) 5(9.6) bambam 21 6(28.6) 10(47.6) 0(0) 3(14.3) 2(9.5) pantami 63 7(11.1) 34(54) 6(9.5) 7(11.1) 9(14.2) pokolin 58 7(12.1) 34(58.6) 7(12.1) 5(8.6) 5(8.6) zazzagawa 74 8(10.8) 49(66.2) 11(14.9) 6(8.1) 0(0) filiya 15 1(6.6) 10(66.7) 3(20) 0(0) 1(6.6) diga 40 9(22.5) 12(30) 3(7.5) 6(15) 10(25) total 393 64(16.3) 177(45) 34(8.7) 78(19.8) 40(10.2) n= total number of an. gambiae s.l., na= no amplification, numbers in brackets represent percentage j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 55 http://jad.tums.ac.ir published online: april 27, 2019 fig. 1. map of gombe south, nigeria showing the study area and sampling localities fig. 2. insecticide susceptibility/resistance profile of anopheles gambiae s.l. mosquitoes in eight selected communities in southern gombe nigeria. error bars represent variability in the data all charts below the red line indicate that population of an. gambiae in the study communities that are resistant to the classes of insecticides j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 56 http://jad.tums.ac.ir published online: april 27, 2019 fig. 3. composition of members of the anopheles gambiae s.l. and hybrid (a gambiae/ an. coluzzii species in southern gombe discussion resistance of an. gambiae s.l. mosquitoes to all the four major classes of insecticides were observed in all the study communities in southern gombe. this widespread observation of resistance in the major vector an. gambiae s.l. and factors responsible for this upsurge in these communities is not known and should be investigated. however, the absence of continuous monitoring data in these communities may have denied vector control managers the opportunity to have detected this trend earlier. resistance of an. gambiae s.l. to several classes of insecticides is not entirely new in nigeria (4, 17). permethrin, bendiocarb and ddt resistance were have been reported in an. coluzzii mosquitoes in kano, northern nigeria (11). summary of insecticide resistance cases and number of countries reporting this have constantly been on the rise (2). however, a consideration of factors (usage of itn, irs, agricultural pesticides) previously reported selecting for insecticide resistance (17, 18) provided a reasonable clue of what could be the driver of insecticide resistance in southern gombe. utilization of insecticide nets by persons in gombe is 34%, which is just below the national average of 37% (1). gombe state ranked 8th out of the 38 states with the highest percentage of households with at least one itn (1). there is a significant usage of insecticide treated nets in gombe state in nigeria hence, these insecticide based tools may have contributed to the insecticide pressure in selecting for resistance in the anopheles gambiae population. on the other hand, the use of irs may not have added significantly to this present scale of insecticide resistance because coverage of irs in all the states in the 6 geopolitical zones of nigeria is just 2.5% (1). as such, there seem to be some other major contributors to the multiple insecticide pressure occasioned in these localities. a literature search for recent update on pesticide utilization in gombe state yielded a positive result on what may have been the source of these insecticide pressures. waap (19) reported the widespread cultivation of horticultural crops on which considerable amount of pesticides of different classes are used in gombe state. hence, by inference, agricultural use of insecticides therefore may have conj arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 57 http://jad.tums.ac.ir published online: april 27, 2019 tributed to the current modification of mosquito susceptibility to the pesticides in southern gombe. the status of pyrethroid resistance status of an. gambiae s.l. population in all the study sites is worrisome considering that the major malaria vector control tools in nigeria rely on this class of insecticides (20). however, the disparities in mortality rates expressed by the anopheles population to each of the pyrethroid insecticides indicate that rate of resistance development vary with different insecticides in each location and population. for example, while higher mortality rates (51.2–100%) to lamdacyhalothrin was observed in the anopheles. populations at all the study sites, lower mortality rates were observed in populations exposed to permethrin (0–46.3%) and deltamethrin (39–70%). this variation is an important indication of the need to introduce insecticide resistance management strategies to slow down the rate at which these mosquitoes develop resistance to the pyrethroids insecticides. this will invariably help in prolonging the shelf life of the few available classes of insecticides on which itns and irs rely upon. recent report from an agricultural setting in jigawa state: another northern state in nigeria showed multiple resistances to a pyrethroid, carbamate and an organochlorine (10). similar evidences of full susceptibility to lambda-cyhalothrin as reported in bambam study community have also been reported in kenya (21), sudan (22), tanzania (23) and nigeria (24). in observing the pattern of resistance in the vectors, full susceptibility to bendiocarb was observed in, bambam and pantami which are both neighbhouring communities. this suggests the focal nature of the insecticide resistance status of anopheles populations in each of these communities. while bendiocarb resistance was observed in anopheles population in 6 out of the 8 communities, resistance to pirimiphos-methyl was observed in the an. gambiae populations from all the communities tested. resistance to pirimiphosmethyl (an organophosphate) in gombe is of great concern to vector control manager because it is not currently used in any of the current vector control tools. however, the widespread resistance in anopheles population in 8 communities to pirimiphos-methyl (organophosphate) and the co-resistance to bendiocarb (a carbamate with same target site with organophosphate) point towards other sources as these have not been used for vector control. in contrast, findings from auyo, a community in jigawa, nigeria, kouande and tanguieta in atacora communities, in republic of benin, the an. gambiae populations were susceptible to pirimiphos-methyl (10, 25). in this study, all the anopheles populations were resistant to deltamethrin exposure in all the study communities. similar findings of deltamethrin resistance have been reported in benin, west africa (26, 27). contrary to the findings of this study, in khartoum city of sudan, high susceptibility to deltamethrin was reported (22). in this study, similar lower mortality of anopheles mosquitoes from all the study communities to permethrin have been reported severally in nigeria: oyo, lagos, niger and bauchi states (4, 24). similar evidence of resistance to permethrin have also been reported in atacora, benin (27). evidence of ddt resistance was high in this present study and this was not surprising considering that both pyrethroids and ddt (organochlorine) share the same target site. high resistance to one class may have led to cross-resistance in the other class. insignificant differences in the mortality rates from permethrin and ddt mortality in the anopheles population also showed similar patterns of resistance. a molecular analysis would have provided additional information on probable existence of similar resistance mechanisms in the populations; however, logistics was a major limitation to this study. several studies in nigeria have reported high levels of permethrinddt resistance in an. gambiae and an. coluzzii in j arthropod-borne dis, march 2019, 13(1): 50–61 a oduola et al.: widespread report of … 58 http://jad.tums.ac.ir published online: april 27, 2019 nigeria (4, 6, 8 and 24). evidence of resistance to pirimiphos-methyl (mortality rate between 65–95%) was observed in the an. gambiae populations in all the study communities. in comparing mortalities in an. gambiae populations exposed to two different classes of insecticides (organochlorine/ pyrethroids and bendiocarb/ organophosphate), showed there is significant differences in mortality rates of populations exposed to deltamethrin and lambda cyhalothrin (class ii pyrethroids). deltamethrin and lambda cyhalothrin are still effective compared with permethrin (a class i pyrethroids). similarly, a comparison of differences in mortality rates between an. gambiae s.l. populations exposed to bendiocarb (carbamate) and pirimiphos-methyl (organophosphate) in all the study sites were also not significant suggesting possible shared resistance mechanisms. this however requires laboratory validation. identification of the anopheles species collected in this study showed the predominance of an. gambiae s.l. as the major malaria vectors in all the study communities. the an. gambiae complex includes sibling species that are the most important vectors of malaria in subsaharan africa (28). of these species an. gambiae (formerly s-form), an. coluzzii (formerly m-form) a. arabiensis and hybrid species (an. gambiae/ an. coluzzii) were found to be sympatric in all the study communities. in this study, an. gambiae (formerly s form) was the predominant species over an. arabiensis, an. coluzzii and the hybrid species identified. the sympatric occurrence of an. gambiae, an. coluzzii and an. arabiensis species have been reported in north–central nigeria (29). while this study reported, a higher proportion (19.8%) of an. gambiae/an. coluzzii hybrids than any previous report in nigeria (6, 12, 30). information on the role of these hybrids and their contribution to malaria transmission is currently unavailable. in this study, an. gambiae s.s (formerly s form) was found to be the most predominant species. in contrast, this species was either found to be entirely absent or in insignificant proportions where the hybrid occurred with predominant an. coluzzii (6, 10, 12, and 30). this raises questions on the fitness status of these hybrids compared to the disappearing an. gambiae when exposed to insecticides. the preponderance of an. gambiae (formerly s form) over an. coluzzii species has always been attributed to ecological preference for drier areas of the savannah regions or areas with temporary breeding habitats. the species composition outcome of this study is in agreement with previous nationwide mapping of the distribution of members of the an. gambiae complex in the sudan savannah where the study communities are located (31, 32). conclusion monitoring insecticide resistance is an integral part of measures against malaria vectors and the resistance management strategies depends on the knowledge of the composition of malaria vectors in an area as well as their resistance profiles. anopheles gambiae, an. coluzzii, an. arabiensis, and the hybrid species identified in all the study communities in southern gombe were resistant to all the four classes of insecticide approved for vector control. there is an urgent need for the implementation of insecticide resistance management strategies in these localities. further studies must also be implemented to assess the operative resistance mechanisms in order to inform the choice of alternative vector control interventions to 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case report gastrointestinal myiasis due to sarcophaga argyrostoma (diptera: sarcophagidae) in mashhad, iran: a case report *mohsen najjari1; bilal dik2; gamze pekbey3 1department of parasitology and mycology, faculty of medicine, mashhad university of medical sciences, mashhad, iran 2department of parasitology, faculty of veterinary medicine, selçuk university, selçuklu, konya, turkey 3department of plant protection, faculty of agriculture, bozok university, yozgat, turkey *corresponding author: dr mohsen najjari, e-mail: najjarimh@mums.ac.ir (received 07 jan 2019; accepted 05 jul 2020) abstract myiasis is infection with a fly larva, usually occurring in tropical and subtropical areas. a 32-years-old immunocompromised woman with fever, gastrointestinal pain, cramps, vomiting; and fatigue was referred to ghaem hospital in mashhad in july 2018. entomological characterisation of cephaloskeletons, posterior spiracles of the excreted larvae and genital dissection of male fly were diagnosed as sarcophaga (liopygia) argyrostoma. accidental intestinal myiasis caused by unplanned intake of dipterous larvae by contaminated food staff or water. patients with immune deficiency may be more at risk for the threat of maggot’s infestation, so in such cases, hospital standard should be more in attention. to best of our knowledge, s. argyrostoma has not been reported before in the indexed literature from iran. keywords: sarcophaga argyrostoma; intestinal myiasis; iran introduction for many years human body cavities have been attacked by insects’ larvae. the word myiasis has been derived from ancient greek language in which “mya” means fly and “isasis” means disease (1). afterward, in year 1840 the term “myiasis” appeared to describe a human ailment that arose from dipterous maggots (2). more recently, it has been more cleared and depicted as invasion of live vertebrate animals and human with fly maggots which in any event for a specific period, feed on the host’s dead or living tissue, liquid body substances, or ingested sustenance (3). in general, it starts when gravid female flies are attracted by the odors of infected wounds, decaying organic matter, feces, urine, and human food, where they deposit larvae or eggs (4) and principally, classified in two groups: entomologically and anatomically. from entomological aspect, it can be classified into two major categories named primary and secondary my iasis. in primary myiasis larvae more found in animals, and rarely found in humans and feeding on living tissue in other side, flies could feed on dead tissues and necrotic lesions in patients known as secondary myiasis. it also classified according to the anatomy and position involved in the host body and had classified as oral, nasopharyngeal ocular, cutaneous, urogenital and intestinal (5). myiasis can be also classified as obligatory, facultative and accidental. this also categorize as entomological point of view (6). myiasis of the intestinal tract (=intestinal myiasis) caused by accidental ingestion of dipterous eggs or larvae usually transited and asymptomatic. the flies that cause gastrointestinal myiasis usually are the members of the families sarcophagidae and muscidae (7). the aim of the study was to report intestinal myiasis that is uncommon in iran though so far there are just three reports of sarcophaga (bercae) africa (as s. haemorrhoidalis), eristalis copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/eristalis_tenax https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 317–324 m najjari et al.: gastrointestinal myiasis due to … 318 http://jad.tums.ac.ir published online: september 30, 2020 tenax and lucilia illustris can be seen in literature (8, 9). case description a 32 years-old woman from golestan province was referred to ghaem hospital in mashhad, northeastern iran with fever, weakness, gastrointestinal pain, cramps, vomiting, fatigue and weight loss in july 2018. her main complication was lasted profound and repeated watery diarrhea started since six months ago. she had a double kidney transplant history seven years ago. other significant clinical results were: hb: 8, wbc: 3400, crp: +++ and esr: 100, cd4 102, level of cyclosporine in her sera was 3.5. she was before roll out for hiv, hcv, htlv1, cryptosporidium spp., entamoeba histolytica, toxoplasma gondii and intestinal tuberculosis due to flask like wound in her the large intestine appeared in colonoscopy. complementary diagnostic process was performed and cytomegalovirus confirmed by real time-pcr. she was treated with ganciclovir (5% (oral), hoffmann la roche) and using a standard protocol. after ten days of her remedy and subside of her gastric pain and diarrhea due to new complaints of abdominal spasms, gastric pain and diarrhea a new stool sample was sent to parasitology laboratory. the result was negative for parasite but in macroscopic examination 15 whitish moved worm like observed that later confirmed for maggots of a fly. to investigate whether the feces were infested by larvae from the canteen in which the patient was defecating a sigmoidoscopy was performed in next day and feces directly obtained from the large intestine. the sample was examined and appeared the presence of five live maggots. due to the threat of maggot tissue penetration, the patient was situated under colonoscopy to examine the presence of bare larvae in the tissue. there were no larvae although in her biopsy milled inflammatory cells were seen and also evidence of wounds healing showed no additional antibiotic and the extra drug prescribed because of any evidence of continued infestation in her next stool samples examination. due to the advent of symptoms of healing, get a relative general improvement and disappearance of diarrhea and gastrointestinal symptoms by complete exertion of larvae, after tree week of hospitalization, she was temporarily discharged from the hospital regarding her request to continue the treatment at home. in the laboratory of parasitology, ghaem hospital at first glance and macroscopic examination of feces, the larvae were miss-diagnosed as pinworms (10) by lab technician while in microscopic examination afterward they were detected as the early stage of unknown fly larvae. obtained wormy like creature was washed using phosphate-buffered saline solution (ph: 7.4). to early diagnosis the larvae one of them cleared by lactophenol and examined again under a light microscope. five live cylindrical maggots measured they were around 9mm in length and 3mm in diameter and transferred on blood agar medium which usually used for microbiology investigation. after three days the larvae grow as instars iii. three of them saved and fixed by formalin 10% for further larval diagnosis and transferred to department of parasitology, veterinary faculty of selçuk university in konya, turkey while the remained two other transferred in a garden soil glass jar (5cm high), they immediately penetrated deep into the soil. five days later, pupas were observable and photographed when they were gently removed from the soil. finally, after ten days, two adult flies appeared (fig. 1). the adult flies transferred in a carton box to the entomology lab in bozok university, yozgat, in turkey for the identification. the other early stage larvae were preserved in ethanol 70% and save in the collection of museum of mashhad university of medical sciences using assigned voucher numbers. the larvae washed by distilled water, transferred to ethanol 70% and 99% for a few hours in each step, dissected and mounted on the slides in canada balsam. they were examined, and captured photos of cephaloskeleton, http://jad.tums.ac.ir/ https://www.google.com/search?q=oral&stick=h4siaaaaaaaaaongvulsz9u3mcppzjxiawcrhvlmdgaaaa&sa=x&ved=2ahukewisntlnqctfahvlabokhxsjcpqqmxmoataxegqichan j arthropod-borne dis, september 2020, 14(3): 317–324 m najjari et al.: gastrointestinal myiasis due to … 319 http://jad.tums.ac.ir published online: september 30, 2020 anterior and posterior spiracles with nikon smz camera mounted stereo zoom microscope with toupcam u3cmos application unit, and leica dm750 camera mounted trinocular microscope with leica dfc295 application unit. the larvae were identified as two and third instars of sarcophaga spp. (diptera: sarcophagidae) (11). the body of larva in follows the general pattern of sarcophagidae, i.e., anterior spiracles are seen (fig. 2). the third instar of s. argyrostoma was easily recognizable from second instar larva; it has three slits in each one of posterior spiracles by the presence of two slits in posterior spiracles (figs. 3, 4). the larva had a pair of strong oral hooks and a cephaloskeleton (figs. 5, 6). in the entomology lab at bozok university, genital dissection of the adult male was performed under leica s8apo stereomicroscope; only the fifth abdominal sternite was softened for 24h in a cold solution of 10% koh to facilitate the further dissection. identification, classification, and terminology followed and, the specimen was identified as s. (liopygia) argyrostoma (fig. 7) (12). digital photos of the genitalia were captured with leica mz 125 camera mounted stereo zoom microscope and leica application suite (ver. 3.8.0). fig. 1. adult fly of sarcophaga argyrostoma appeared in parasitology lab, ghaem hospital, mashhad, july 20, 2018 fig. 2. sarcophaga argyrostoma, third instar larva, anterior stigma, entomology lab selçuk university in konya, august 19, 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 317–324 m najjari et al.: gastrointestinal myiasis due to … 320 http://jad.tums.ac.ir published online: september 30, 2020 fig. 3. sarcophaga argyrostoma, second instar larva, posterior stigma, entomology lab selçuk university, august 19, 2018 fig. 4. sarcophaga argyrostoma, third instar larva, posterior stigma, entomology lab selçuk university, august 19, 2018 fig. 5. sarcophaga argyrostoma, second instar larva -cephaloskeleton and anterior stigma, entomology lab selçuk university, august 19, 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 317–324 m najjari et al.: gastrointestinal myiasis due to … 321 http://jad.tums.ac.ir published online: september 30, 2020 fig. 6. sarcophaga argyrostoma, third instar larva, cephaloskeleton and anterior stigma, entomology lab selçuk university, august 19, 2018 fig. 7. sarcophaga argyrostoma; aaedeagus lateral view bepandrium with cerci and surstylus cfifth abdominal sternite, entomology lab at bozok university, august 29, 2018 discussion it is necessary to consider two distinct subjects in the diagnosis of intestinal myiasis; the principal issue considered is the presence of fly larvae in feces does not affirm the significance of intestinal myiasis. most species of fly eggs or larvae that coincidentally may be intake with http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 317–324 m najjari et al.: gastrointestinal myiasis due to … 322 http://jad.tums.ac.ir published online: september 30, 2020 nourishment cannot continue in the gastrointestinal environment. in such cases, true host infestation is never established, despite the fact, the dead larvae might be distinguished on last stool examinations. such a circumstance is named pseudomyiasis (13). pseudomyiasis can likewise happen when female flies lay eggs on uncovered fecal samples pot before performing laboratory processing (14). the other encounter challenge is the coexistence of concomitant infection as parasites, fungi, and viruses which may produce common related signs as cramp, gastric pain, vomiting and mislead to final intestinal myiasis diagnosis. it is important, therefore, for the symptomatic and asymptomatic patient to find live larvae more than once in stool examination and the best way is to obtain samples through sigmoidoscopy and directly from the large intestine (15). intestinal infestations with fly larvae have been rarely reported, especially from developing countries, however, there are such reports as africa in zimbabwe (harare) (16), north america in laramie, wyoming (17), taiwan, japan, thailand, china, india, iran (9) and turkey (18). clinical presentations have been varied, including abdominal pain, nausea and vomiting, or even anal pruritus, and rectal bloodless; moreover, in some cases, it could be asymptomatic (9). to date myiasis agents associated with accidental intestinal myiasis reported as sarcophaga spp., fannia canicularis, hermetia illucens, eristalis tenax, muscina stabulans, fannia scalaris, musca domestica, lucilia cuprina, stomoxys calcitrans and phormia regina (15). to best of our knowledge, this is the first report of s. argyrostoma reported from the family sarcophagidae in the review of the literatures revealed for intestinal myiasis. in this case, the patient was admitted to a private room. on the other hand, the patient has been under a special diet by a dietitian during the hospitalization period also the consumed food have been served under appropriate catering health conditions. in an interview with the patient, we found that she has a homemade meal [traditional dish] which ingredients were starch, sugar and a kind of herbal extract was prepared out of the hospital. we discovered the remaining in her room refrigerator infested by a few tiny flies’ larvae. this myiasis agent can successfully pass the gastrointestinal tract and overcome the acidic environment of the stomach and enzymatic digestion in the small intestine reported for the first time as human intestinal myiasis, caused by s. argyrostoma in this case. although cited fly has reported previously in wound myiasis in an old woman in italy (19), but has a very low occurrence rate and, does not cause any remarkable disorder for patients. patients with immune deficiency may be more at risk for the threat of maggot’s infestation (20), so in such cases, hospital standards should be more in attention. it is also necessary to monitor such patients to use only hospital meal and minimize the consumption of uncared foodstuff with outside origin. the importance of the current study is hospitalized patients, who encountered to such larvae in their stool samples, are more concerned about; moreover, for laboratory staff, to avoid misdiagnosis with some other kind of worms in the stool; furthermore, it could be attracted for health professionals to aware that this infestation may happen regardless of the socioeconomic conditions. conclusion accidental intestinal myiasis regularly happened due to unplanned intake of dipterous larvae by contaminated food staff or water. patients with immune deficiency may be more at risk for the threat of maggot’s infestation so health care professionals should be aware for different routs of such infestations and hospital standards elevation moreover to reach an early and on time laboratory diagnosis, using entomologist collaboration undoubted led to salient decrease of related morbidities not overlooked. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 317–324 m najjari et al.: gastrointestinal myiasis due to … 323 http://jad.tums.ac.ir published online: september 30, 2020 acknowledgements i take this opportunity to express my gratitude to mr seyed masud marjani for his support and lab assistance. the authors declare that there is no conflict of interests. references 1. khan m, mehboob b, noor ulw, mansoor n (2014) oral myiasis: a case series of 11 patients treated at khyber college of dentistry hospital peshawar. pak oral dent j. 34(1): 56–60. 2. sankari ls, ramakrishnan k (2010) oral myiasis caused by chrysomya bezziana. j oral maxillofac pathol. 14(1): 16–18 3. aguilera a cid, regueiro a, prieto bj, noya jm (1999) intestinal myiasis caused by eristalis tenax. j clin microbiol. 37(9): 3082. 4. hall m (1991) screwworm flies as agents of wound myiasis. world animal review. available at: http://www.fao.org/3/u4220t/u4220t0 7.htm 5. mullen gr, durden la (2009) medical and veterinary 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scientificworldjournal. 2017: 9064531. 20. nagy v (2012) unusual presentation of the urogenital myiasis caused by lucilia sericata (diptera: calliphoridae). ann agric environ med. 19(4): 802–804. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 153 http://jad.tums.ac.ir published online: june 30, 2020 original article environmental factors associated with larval habitats of anopheline mosquitoes (diptera: culicidae) in metema district, northwestern ethiopia *esayas aklilu 1 ; mizan kindu 2 ; araya gebresilassie 3 ; solomon yared 4 ; habte tekie 3 ; meshesha balkew 5 1 department of biology, mada walabu university, bale-robe, ethiopia 2 department of parasitology, school of medicine, microbiology and immunology, madawalabu university, bale-robe, ethiopia 3 department of zoological sciences, addis ababa university, addis ababa, ethiopia 4 department of biology, jigjiga university, jigjiga, ethiopia 5 aklilu lemma institute of pathobiology, addis ababa university, addis ababa, ethiopia (received 09 nov 2018; accepted 26 may 2020) abstract background: malaria is one of the major public health concerns in ethiopia. control options available for the management of malaria, include case detection, personal protection and larval source management. effective control of anopheles larvae largely depends on understanding of the habitats of the vectors. the aims of this study were to identify the breeding habitats of mosquitoes and characterize the larval habitats in gende wuha town in northwestern ethiopia. methods: different aquatic habitats were sampled and characterized for anopheline larvae from november 2012 to june 2013. results: in total, 2784 larvae of anopheles mosquitoes were collected from various breeding habitats. microscopic identification of the iii and iv instars revealed the presence of seven anopheles species. of the anopheles spp, anopheles gambiae s.l. (80%) was the most predominant species in the study area. spearman correlation coefficient results also determined that the density of an. gambiae s.l. increased significantly with habitat temperature (r= 0.346, p< 0.01). significantly higher an. gambiae s.l. larvae were obtained in non-shaded habitats (z= -3.120, p< 0.05) when compared with shaded habitats. conclusions: the current study demonstrated an. gambiae s.l., the principal malaria vector in the country, is the predominant species in the larval sampling habitats. it was also noted the importance of edge of stream as larva breeding habitats for this species during the dry season of the year. therefore, attention should be given for this breeding habitat for control of the vector during dry season. keywords: anpheles gambiae s.l.; larval habitat; metema; ethiopia introduction malaria is a disease caused by parasitic protozoans of the genus plasmodium and transmitted from person to person by the bites of infected female anopheles mosquito. it is endemic in tropical and subtropical regions and prevalent in more than 91 countries. nowadays almost half of the world’s population is at risk of the disease, mostly those living in resource limited countries. despite tremendous efforts have been made to curb malaria, the disease is still a leading cause of morbidity and mortality globally. for instance in 2015, 212 million cas es and 429,000 malaria deaths were reported. ninety percent of all malaria cases and 92 % of deaths occurred in the sub-saharan african countries (1, 2). in ethiopia malaria is one of major public health concerns. in 2017, 2.6 million cases and 5369 deaths were reported in the country (3). about 60% of the country’s populations live in malarious areas, and 68% of the country’s landmass is favorable for transmission, mainly at altitude below 2000m (4). transmission of the disease is mainly described by seasonal *corresponding author: dr esayas aklilu, e-mail: reyeesayas@gmail.com mailto:reyeesayas@gmail.com j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 154 http://jad.tums.ac.ir published online: june 30, 2020 and predominately unstable. the main transmission follows the major rainy season (june– september) and takes place from september– december while minor transmission happens in april–may immediately after the short rainy season (february–march) (5). so far, in ethiopia, 43 species and subspecies of anopheline mosquitoes have been registered (6). of these, four species of anopheles are linked with malaria transmission, namely, an. arabiensis, an. pharoensis, an. funestus and an. nili. the former one is the major vector whereas the rest are secondary vectors (5, 7). larvae of an. arabiensis breeds in small, temporary, sun-lit water collections created during and after the rains albeit it can also breed in a wide variety of other types of water bodies. sometimes it also breeds in seasonal breeding habitats that are created by human activity, for instance brick making pits, sand mining pools, human foot and hoof prints of animals, tyre-rut of tractor and other vehicles in agricultural development, drainage and irrigation canals (7, 8). the larval breeding habitat is important for mosquito population dynamics, due to it is a place where a number of crucial life cycle processes take place such as ovipostion, sub-adults development, and emergence of adult occur (9). better understanding of larval ecology and delineating of larval habitat attributes in terms of environmental characteristics are indispensible for devising novel malaria vector control strategies (10). in ethiopia, a few previous attempts have been undertaken to identify and characterized the breeding habitats of anopheline larva. teklu et al. (11) pointed out the major vector of malaria prefer to breed in slightly turbid and shallow aquatic habitat in the shoreline of the koka reservoir, central ethiopia. in the same general area, kenea et al. (8) revealed other environmental factors, which determine the relative abundance of an. arabiensis. accordingly, the density of larvae of this species was significantly and inversely associated with aquatic vegetation and water current. more recently, animut et al. (12) reported that density of larvae of an. arabiensis had direct and indirect association with water temperature and depth of larval habitat, respectively in butajira, south central ethiopia. however, nothing has been known about species composition of anopheles larvae and their larval breeding habitats in gende wuha town where malaria is one of major public health problems. hence, the aim of this study was to determine the species composition of anopheles larvae, identify and characterize the larval habitats of anopheline mosquitoes in malaria endemic area of gende wuha town, northwest ethiopia. materials and methods study area the study was conducted in gende wuha town in metema district, southwest gondar. it is located 150km and 860km from gondar and addis ababa, respectively. the town is located at latitude of 12 o 46n and longitude of 36 o 24 e and at altitude of 734 meter above sea level (a s l). the rainfall is characterized by a unimodal distribution with annual rainfall that ranges between 500–800mm. the rainy season begins from mid-june and lasts until the end of september. the dry season is much extended and it starts from october and lasts until the end of may. the average day temperature is usually high (25–30 o c) most of the year reaching 40 o c during the months of april–may. larval sampling and processing anopheline larvae were sampled fortnightly for eight consecutive months from november 2012 to june 2013. sampling of anopheline larvae was made from any potential larval breeding habitat that was encountered during the survey. during each survey, a habitat was first visually inspected for the presence of mosquito larvae, and then depending on the size of the breeding habitat ten to twenty samples were taken with a soup ladle (350ml capacity) from each breeding habitat (13). samplings were alj arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 155 http://jad.tums.ac.ir published online: june 30, 2020 ways done by the same individual in the morning (9:00–12:00). the anopheles larvae were separated from the culicine larvae based on the position of the larvae to the surface of water. the former larvae were classified as early instars (i and ii) and late instars (iii and iv). the early instars were counted and discarded whereas all the late instars were killed with warm water and preserved in 70% alcohol for species identification based on morphological characters. in the laboratory, each larva was mounted on a glass slide individually in a drop of hoyer's medium and covered with a cover slip (14), and species identification was carried out using the key of gillies and coetzee (15). characterization of larval habitat at the same time with larva sampling, different environmental characteristics including turbidity, ph, temperature, speed, distance to the nearest house, length, width, depth, trees nearby to the habitat (shade), presence of aquatic vegetation, presence of algal mat, type of substrate, origin of habitat, habitat permanence of each larva habitat were measured or estimated and recorded. turbidity was measured by putting water samples in a glass test tube and holding against a white background and was categorized either clean or turbid. water ph was measured using ph indicator, whereas water temperature was measured using mercury thermometer. the speed of aquatic habitat was delineated visually as fast flowing, slow flowing and still (stagnant). habitat distance to nearest house was estimated visually and classified either greater than or less than 100m. habitat length, width and depth were measured using measuring tape; shade was recorded as absent or present by observing terrestrial vegetation and/or trees and their branches near to the breeding habitat. aquatic vegetation was determined visually and classified into four groups (none, emergent, floating debris and emergent and floating debris). the presence or absence of algal mats was visually determined. type of substrate was categorized as muddy or sandy. origin of habitat type was classified as either natural or man-made; whereas habitat permanence was estimated visually and classified as permanent, semi-permanent and temporary (10, 16). data analysis data analyses were done using spss software (version 20 for windows). the association of the larval density of the anopheline mosquitoes with habitats characteristics such as temperature, ph and depth was analyzed using the spearman correlation coefficient, while association of larval density with some categorical habitat characteristics such as substrate type (stony or muddy), turbidity (low or medium), shade (present or absent) and surface algae (present or absent) were analyzed using the mann-whitney u test. results habitat diversity and species composition of anopheles larvae in the study period, a total of 56 aquatic habitats comprising of edge of stream (n= 33), rain pool (n= 7), puddle (n= 6), spring (n= 5) and streambed (n= 5) were sampled. fifty-two percent of these habitats were positive for anopheles larvae. a total of 2784 anopheles larvae were collected. out of these, 64.8% (n= 1804) larvae were early instars (i and ii) and the rest (35.2%) were late instars (iii and iv). seven species of anopheles were identified (table 1). these are anopheles gambiae s.l., an. pretoriensis, an. rufipes, an. rhodesiensis, an. sergentii macmahoni, an. rivulorum and an. coustani group. of these, an. gambiae s.l. is the most preponderate species, which constituted 80% and, followed by an. pretoriensis (16.4%). anopheles coustani was the least abundant which comprised 0.1%. overall, an. gambiae s.l. and an. pretoriensis jointly accounted for 96.3% of all mosquitoes collected. monthly larval variation during the study period, monthly variations in the population density of an. gambiae s.l. larval were observed. the highest larval denj arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 156 http://jad.tums.ac.ir published online: june 30, 2020 sity of an. gambiae s.l. was observed in may. from february to april the larval density of an. gambiae s.l. was nil as during these time there was no a single breeding habitat of anopheles mosquito (fig. 1). habitat characteristics associated with larval occurrence the spearman correlation coefficient results indicated that the density of an. gambiae s.l. had positive and statistically significant with habitat temperature (r= 0.346, p< 0.01), whereas the density of an. pretoriensis had negatively associated with habitat temperature notwithstanding the association was very weak and not significant (table 2). depth and ph of the habitats had weak positive association with larval density of both species but not significant. the mean comparison analysis revealed the characteristics of larval habitats and mean densities of anopheline larvae (table 3). significantly higher mean densities of an. gambiae s.l. larvae were collected from aquatic habitats that had turbid, natural, free of shade and nearby to human dwellings (< 100m). likewise, significantly higher mean densities of an. pretoriensis larvae were obtained from aquatic habitats that had clear, natural, muddy with mats of algae. analysis using the mann whitney u test indicated that significantly higher an. gambiae s.l. larvae density were obtained in nonshaded habitats (z= -3.120, p< 0.05) when compared with shaded habitats. similar analysis revealed that significantly higher an. pretoriensis larvae were collected in habitats that were clear (z= -1.967, p< 0.05), presence of algae (z= -3.034, p< 0.05) and muddy (z= 2.225, p< 0.05) when compared with turbid, absence of algae and stony substrate, respectively. fig. 1. mean monthly larval density of an. gambiae s.l. in the study area (nov 2012 to jun 2013) j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 157 http://jad.tums.ac.ir published online: june 30, 2020 table 1. anopheles larvae collected from various breeding habitats in gende wuha town (november 2012–june 2013) habitat no. of habitats species total (%) an. gambiae s.l. an. pretoriensis an. rufipes an. rhodesiensis an. sergentii macmahoni an. rivulorum an. coustani edge of stream 33 92 74 4 1 4 10 1 186 (18.9) spring 5 0 60 0 0 0 3 0 63 (6.4) puddle 6 88 26 0 1 1 0 0 116 (12) stream bed 5 0 1 10 0 0 1 0 12 (1.2) rain pool 7 603 0 0 0 0 0 0 603 (61.5) total (%) 56 783 (80) 161 (16.4) 14(1.4) 2 (0.2) 5 (0.5) 14 (1.4) 1(0.1) 980 table 2. anopheline larval density association with different habitat characteristics habitats characteristics mean±se species an. gambiae s.l. an. pretoriensis r p-value r p-value temperature 25.61±0.7 0.346 0.008* -0.61 0.654 ph 6.97±0.1 0.208 0.121 0.168 0 .211 depth (cm) 5.2±0.34 0.058 0.669 0.173 0.055 r-spearman correlation coefficient, *correlation is significant at the 0.01 level j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 158 http://jad.tums.ac.ir published online: june 30, 2020 table 3. anopheline larval density association with some of categorical habitat characteristics categorical habitat characteristics species an. gambiae s.l. an. pretoriensis mean±se x 2 p-value mean±se x 2 p-value turbidity clear 0.61±0.26 241 0.759 0.36±0.15 181.5 0.049* turbid 4.55±3.25 0.00±0.00 origin of habitat natural 1.62±0.79 173.0 0.23 0.35±0.14 157.5 0.08 man made 0.08±0.08 0. 0±0.0 presence of algae present 1.77±1.28 374.5 0.53 0.56±0.24 265.5 0.002* absent 1.01±0.57 0.04±0.03 shade present 0.00±0.00 207.0 0.02* 0.03±0.02 283.0 0.112 absent 2.00±0.97 0.41±0.17 presence of aquatic vegetation noon 1.88±1.1 2.97 0.39 0.41±0.19 1.01 0.798 emergent 0.98±0.54 0.15±0.09 emergent and floating 0.00±00 0.00±0.00 floating 0.08±0.06 0.06±0.05 water current still 1.6±0.87 273.0 0.5 0.34±0.15 294.5 0.87 slow flowing 0.67±0.49 0.15±0.08 type of substrate muddy 1.80±1.04 0.71 0.7 0.02 ± 0.02 7.67 0.022* stone 1.33±1.01 0.48±0.19 habitat permanence permanent 0.22±0.08 3.10 0.21 0.34±0.13 4.8 0.08 semipermanent 2.29±2.26 0.2±0.12 temporary 1.46±0.66 0.32±0.23 distance to the nearest house < 100 2.11±1.05 317.5 0.2 0.15±0.08 307.5 0.11 > 100 0.11±0.56 0.53±0.29 discussion of the forty-three known species and subspecies of anopheline mosquitoes in ethiopia (6), seven of them were found in the present study in larva form. these species were anopheles gambiae s.l., an. pretoriensis, an. rufipes, an. rhodesiensis, an. sergentii macmahoni, an. rivulorum and an. coustani group. the main malaria vector in the country (5, 7) was the most abundant species and followed by an. pretoriensis. five larval breeding habitat types were identified in the present study area, namely edge of stream, spring, puddle, streambed, and rain pool. of these habitats, edge of the stream was the most common breeding habitat. during the dry season, this habitat was the main source for anopheline larvae in the area. the same observations were reported in south-central ethiopia (12) and western kenya (17). the current study indicated that edge of streams play significant role in maintaining larva of anopheles mosquitoes, especially at the time low or no rainfall occur. as a result, attention should be given for these breeding habitats when larval management strategies are developed to reduce density of anopheline mosquitoes. in the present study, larval density of an. gambiae s.l. increased significantly as with water temperature, as reported previously in central rift valley of ethiopia (11) and south-central ethiopia (12). these results indicated that water temperature along with other factors might j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 159 http://jad.tums.ac.ir published online: june 30, 2020 have significant role in larval development and egg hatchability. in contrary to the current observation, mala et al. (18) reported inverse relationship between the habitat temperature and larval density of an. gambiae s.l. in baringo district of kenya. furthermore, dejenie et al. (19) did not show any association between this environmental parameter and the species in central zone of tigray. unlike larvae of an. gambiae s.l., larval density of an. pretoriensis had negative correlation with water temperature but not statistically significant. the average temperature in water bodies where the anopheles larvae were sampled was 25.61±0.7 o c (ranged from 19 o c to 35 o c). such temperature is within the range of conducive water temperature in which anopheline larvae survive and develop into adult stage (20). mann-whitney test analysis indicated that the larvae of an. gamibae s.l. breed significantly on free of shaded habitats as compared to shaded habitats. similar result was also reported by tuno et al. (21) in western kenya. contrary to our observation and earlier studies, mala et al. (18) found that significantly higher larval density of this species in shaded habitats in baringo district of kenya. in the current study, larval density of an. pretoriensis was significantly more abundant in the habitats which had algae than without algae. importantly, previous works showed that the presence of algae is a key factor for the growth of some anopheline species. for instance, manguin et al. (22) underscored the importance of algae for the presence of an. pseudopunctipennis larvae in much of their collection breeding habitats. likewise, gimnig et al. (23) demonstrated the strong association between the larval densities of an. gambiae s.l and algae in western kenya, although larvae of an. gambiae s.l. were rare or absent in the presence of algae in the present study. furthermore, kenea et al. (8) stressed the importance of the algae for the presence of an. pharoensis in central rift valley of ethiopia. conclusion in conclusion, our study demonstrated that an. gambiae s.l. is the predominant species of anopheles mosquitoes in the larval sampling habitats of gende wuha town. breeding of this species in the study area is driven predominately by turbid, natural, free of shade and nearby to human dwelling larval breeding sites. it was also noted the importance of edge of stream as larval breeding habitats for an. gambiae s.l. during the low precipitation period of the year. the edge of streams may be an important breeding habitat for different anopheles spp and for low transmission of malaria during the dry season. therefore, control programs designed to contain the transmission of malaria in this particular area through the management of larvae during dry season should take into account the importance of such breeding habitats. however, as the edge streams may not be the only larval breeding habitats in the area, further detailed longitudinal larval surveillance should be carried on in order to make control of the immature stages of anopheles mosquitoes. moreover, the role of these anopheles species in malaria transmission should be clarified through detailed entomological investigations in the future. acknowledgment the authors are grateful to our field and laboratory assistant mr gashaw mihiret for his helps in carrying out this study. references 1. gilles m (2002) the malaria parasites. in: gilles h, warrell d (eds): essential malariology. vol. 97. oxford press, london, pp. 8–34. 2. world health organization (2016) world malaria report. world health organization, geneva, p. 186. j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 160 http://jad.tums.ac.ir published online: june 30, 2020 3. world health organization (2018) world malaria report. world health organization, geneva, pp. 210. 4. federal minster of health (2014) an epidemiological profile of malaria in ethiopia. federal ministry of health, addis ababa, ethiopia. 5. ghebreyesus a, deressa w, witten k, getachew a, seboxa t (2006) malaria. in: berhane y, haile-marim d, kloos h (eds). epidemiology and ecology of health and disease in ethiopia. shama books, addis ababa, pp. 556–576. 6. walter reed biosystematics unit (2013) systematic catalog of culicidae. walter reed biosystematics unit, smithsonian institution, washington dc, usa. available at: http://www.mosquitocatalog.org. 7. abose t, yebbyo y, olana d, alamirew d, yikuno amlake b, regassa l, mengesha a (1998) the epidemiology and control of malaria with special emphasis on the distribution, behavior and susceptibility of insecticides of anopheline vectors and chloroquine resistance in awash, central ethiopia and other area. world health organization/ mal. 8. kenea o, balkew m, gebre-michael t (2011) environmental factors associated with larval habitats of anopheline mosquitoes (diptera: culicidae) in irrigation and major drainage areas in the middle course of the rift valley, central ethiopia. j vector borne dis. 48: 85–92. 9. overgaard hj, tsuda y, suwonkerd w, takagi m (2002) characteristics of anopheles minimus (diptera: culicidae) larval habitats in northern thailand. environ entomol. 31: 134–141. 10. mwangangi jm, mbogo cm, muturi ej, nzovu jg, githure ji, yan g, minakawa n, novak r, beier jc (2007) spatial distribution and habitat characterization of anopheles larvae along the kenyan coast. j vector borne dis. 44: 44–51. 11. teklu m, tekie h, mccartney m, kibret s (2010) the effect of physical water quality and water level changes on the occurrence and density of anopheles mosquito larvae around the shoreline of the koka reservoir, central ethiopia. hydrol earth syst sci. 14: 2595–2603. 12. animut a, gebre-michael t, balkew m, lindtjørn b (2012) abundance and dynamics of anopheline larvae in a highland malarious area of south-central ethiopia. parasite 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(2011) dry season ecology of anopheles gambiae complex mosquitoes at larval habitats in two traditionally semi-arid villages in baringo, kenya. parasite vector. 25: 4. 19. dejenie t, yohannes m, assmelash m (2011) characterization of mosquito http://www.mosquitocatalog.org./ j arthropod-borne dis, june 2020, 14(2): 153–161 e aklilu et al.: environmental factors … 161 http://jad.tums.ac.ir published online: june 30, 2020 breeding sites in and in the vicinity of tigray microdams. ethiop j health sci. 21: 57–66. 20. bayoh mn, lindsay sw (2003) effect of temperature on the development of aquatic stages of anopheles gambiae sensu stricto (diptera: culicidae). bull entomol res. 93: 375–381. 21. tuno n, okeka w, minakawa n, takagi m, yan g (2005) anopheles gambiae sensu stricto (diptera: culicidae) larvae in western kenya highland forest. j med entomol. 42: 270–277. 22. manguin s, roberts d, peyton de, rejmankova e, pecor j (1996) characterization of anopheles pseudopunctipennis larval habitats. j am mosq control assoc. 12: 619–625. 23. gimnig je, ombok m, otieno s, kaufman mg, vulule jm, walker ed (2001) density-dependent development of anopheles gambiae (diptera: culicidae) larvae in artificial habitats. j med entomol. 39: 162–172. j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 399 http://jad.tums.ac.ir published online: december 31, 2019 original article comparative performance of different traps for collection of phlebotominae sand flies and estimation of biodiversity indices in three endemic leishmaniasis foci in north khorasan province, northeast of iran kourosh arzamani1; *yavar rassi2; *hassan vatandoost2,3; amir ahmad akhavan2; mohammad reza abai2; mohammad alavinia1,4; kamran akbarzadeh2; mehdi mohebali5; sayena rafizadeh6; fateh karimian2; mehdi badakhshan2; azad absavaran2 1vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4toronto rehabilitation centre, university health network, toronto, canada 5department of medical parasitology, school of public health, tehran university of medical sciences, tehran, iran 6ministry of health and medical education, tehran, iran (received 04 aug 2019; accepted 30 dec 2019) abstract background: phlebotominae sand flies (diptera: psychodidae) are the vectors of leishmaniasis. there are different methods for sand fly collection with different performance. the purpose of the current study was to compare the effect of different traps for collection of phlebotominae sand flies in three endemic leishmaniasis foci in north khorasan province, northeast of iran. methods: sand flies were collected using seven different traps from three villages, three times each twenty days during peak periods of seasonal activity in 2016. results: a total of 7253 sand flies were collected. the specimens belonged to19 species. phlebotomus sergenti was the most predominant species in the study area. light trap baited with carbon dioxide (clt) and sticky paper trap (spt) caught 22.6% and 22.3% of sand flies respectively. animal baited trap (abt) and white shannon trap (wst) caught significantly fewer sand flies than the other traps. the sex ratio was different by phlebotominae sand fly species and collection methods. the sex ratio was highest in spt and lowest in black shannon trap (bst). species diversity and species richness in spt were more than other traps. conclusion: our findings confirm that clt and spt are the most efficient sand fly collection methods. clt is higher attractive for females and phlebotomus genus and is an ideal method for monitoring the population of phlebotomus ge nus during surveillance. spt is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities and provide a more realistic estimation of sand flies biodiversity. keywords: collection method; biodiversity; sandflies; leishmania; iran introduction leishmaniasis is transmitted to human in 98 countries and 3 territories on 5 continents in the world. there are three main forms of leishmaniases, visceral leishmaniasis (vl), cutaneous leishmaniasis (cl), and mucocutaneous leishmaniasis (1). in iran, vl and cl are endemic and this country is one of the ten countries with the highest estimated cases of cl. in the north khorasan province vl and cl are endemic. leishmania infantum have been detected in reservoir and vector of the disease. numerous cases of vl have been recorded during the last decades in humans. about 160 confirmed *corresponding authors: dr yavar rassi, e-mail: rassi184@gmail.com, dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com http://jad.tums.ac.ir/ mailto:rassi184@gmail.com mailto:hvatandoost1@yahoo.com j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 400 http://jad.tums.ac.ir published online: december 31, 2019 human cases of vl have been diagnosed and registered especially from the north half of the province during the period 1990–2010. during 2005 to 2008, about 1453 and between the years 2006–2013 at least 2831 patients with cl were reported from different districts of the province (2-7) . phlebotominae sand flies (diptera: psychodidae) are the vectors of leishmaniasis and some human pathogens. there are approximately 1,000 valid described species of sand flies in the world (8). the first step for study of phlebotominae sand flies, their behavior, taxonomy, ecology or determination of infection is collection of them. there are several methods and traps for collection of phlebotominae sand flies (9). a variety of phlebotominae sand flies collection methods have previously been studied in different countries. in a study in turkey among different traps, light trap baited with carbon dioxide (clt) were found to be more stable and productive method than others trap for both estimating the species composition and the population density of sand flies in the study area (10). in an investigation in morocco statistical analysis showed that significantly more number of sand flies were obtained using sticky paper traps (spt) compared with light traps (ltp), therefore spt was more effective than ltp (11). in northeastern of italy three standard methods for collection of sand flies (spt, ltp, and clt) were compared. clt were more attractive for females of p. perniciosus and p. neglectus. ltp showed an intermediate efficiency and were more attractive for p. neglectus, compared to other two traps. results suggest that in northern italy the clt is a suitable sampling method for sand fly monitoring programs (12). in center of iran eight methods for collection of sand flies were compared and 37.3% of all phlebotominae sand flies were collected using disney trap (dst). these proportions were 24.2%, 10.5%, 8.5% and7.3% in spt, black shannon traps (bst), animal baited trap (abt) and white shannon traps (wst) respectively. clt (5.0%), ltp (3.3%) and malaise trap (0.1%) showed a low efficiency (13). in brazil to compare the relative attractiveness of bst and wst for sand flies, several pairs of traps were placed side by side in front of caves in four areas. the result revealed that bst was much more productive than the wst, especially for anthropophilic species (14). according to the previous study tolerance to ddt reported in north khorasan province, therefore collection and monitoring of phlebotominae sand flies is necessary (15). there is no sufficient document for evaluation of different collection methods of phlebotominae sand flies in iran. the purpose of this study was to compare performance of different traps in three endemic leishmaniasis foci in north khorasan province, northeast of iran to assess which of these traps could catch a large number or show the most diversity of sand flies. materials and methods study area this cross-sectional study was conducted during 2016 in north khorasan province, between 36°37′-38°17′ n latitudes and 55°53′58°20′ e longitudes. the province has a desert, mountain and temperate climate with cold winters and receives about 250mm of rainfall annually. the total area was approximately 28,434km2. the province is bordered by turkmenistan in the north and situated in northeast of iran. bojnurd is the capital city of the province (fig. 1). selection of study villages three villages in the province were selected, where human vl and/or cl had been reported in the last 5 years. bacheh-dareh village (a): it is located in raz and jargalan county, 15km from the border of turkmenistan. several human vl and cl cases had been reporthttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 401 http://jad.tums.ac.ir published online: december 31, 2019 ed in the last 5 years. collection site was a valley 3km far from the village and sand flies captures have been restricted to outdoor and wild environments. kohne-jolgeh village (b): it is located in maneh and samalqan county. several human vl and cl had been reported in the last 5 years. collection site was indoors and domestic environments. arg village (c): it is located in jajarm county. only human cl cases had been reported from this village. collection site was indoors and outdoors and domestic environments. sand fly collection sand flies were collected for three times each twenty days during the peak periods of seasonal activity. sample collection began from early jul and continued until late aug in 2016. sand flies were collected using seven different traps including: 1. sticky paper traps (spt) consisting of a white paper sheet with size of 15×21cm that coated with castor oil. we used 10 paper traps for each rotation and totally 60 papers per night. 2. cdc light trap (ltp). the light traps powered by alternating current and were suspended at 1.5 meters above the ground. 3. cdc light trap baited with carbon dioxide (co2) gas (clt) that obtained by a co2 gas tank. 4. white shannon trap (wst) was made of white cloth and consisted of a large central compartment and two smaller lateral ones. the measurements, (width, length and height), of the central and the lateral compartments were, respectively: 1.3×1.3×2 meters and suspended by cords from supports and base of the traps touched the ground. a portable stove was used as co2 and light source inside the traps. 5. black shannon trap (bst). this trap was similar to wst except that were made of black cloth. 6. disney trap (dst). a cage, holding the chicken bait in the middle and surrounded by sticky paper traps. 7. animal baited trap (abt). a net trap (2×3×2 meters) was used to sample sand flies attracted to a chicken as animal bait (fig. 2). all of the traps were placed randomly with a distance of about 20 meters of each other. the traps were set before sunset, were changed every two hours and remained in operation during the night (20:00 to 08:00). as the traps were changed, the new ones were replaced in the same location. traps were rotated clockwise between the trap locations in site “a” and “c” but were fix in site “b”. collected sand flies were stored in 96% ethanol alcohol. the specimens were mounted on glass slides in puri’s medium. species identification was carried out according to morphological characters with several taxonomic keys of sand flies (1617). statistical analyses the statistical analyses were performed in spss program. we evaluated the comparative performance of different traps for each collection site. shannon-wiener index have been calculated to estimate species biodiversity of phlebotomine sand flies in different traps (1819). ethical statement specimen collection was performed in accordance with the procedures approved by the ethical committee of north khorasan university of medical sciences. results a total of 7253 sand flies were collected. frequency, gender and percent of phlebotomine sand flies collected by different traps in three collection sites are shown in table 1. on collection site a we collected the most abundance of sand flies and in collection site c the fewer of specimens collected. the specimens were belonged to19 species, 10 species of the genus phlebotomus and 9 of the genus sergentomyia. in a collection site, we collected 18 species and more sand fly spehttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 402 http://jad.tums.ac.ir published online: december 31, 2019 cies collection appears in this area. significantly fewer sand flies collected in site c with a collection of only four species and in site b eight species were caught. phlebotomus sergenti was the most predominant species being recorded in all localities. abundance of phlebotomine sand fly species, collected by different traps is shown in table 2. more than 89.5% of specimens belong to genus phlebotomus and 62.6% of collected specimens belong to subgenus paraphlebotomus. frequency of sand flies by genus and percent collected by different traps are shown in table 3. clt (22.6%) and spt (22.3%) caught significantly more sand flies and abt (7%) and wst (7.6%) caught significantly fewer sand flies than the other traps. statistical analysis showed significant difference between collection methods and genus of collected sand flies, χ2= 577.8 and p< 0.001. the sex ratios were different in phlebotominae sand fly species. sex ratios in ph. sergenti, ph. alexandri and ph. papatasi were 130, 166 and 75 respectively. in sergentomyia genus sex ratio were 21 and 181 in s.sintoni,and s. sumbarica respectively. species diversity based on shannon-wiener index and species richness has been calculated to estimate species biodiversity of phlebotomine sand flies in different collection methods in study area and is shown in table 5. table 1. frequency, gender and percent of sand flies collected by different traps in three endemic leishmaniasis foci, north khorasan province, 2016 method location a b c female male total % female male total % female male total % abt 59 140 199 6.5 39 68 107 4.5 122 80 202 11.3 bst 183 147 330 10.7 80 85 165 6.9 245 45 290 16.2 clt 316 328 644 21.0 401 289 690 28.8 172 133 305 17.1 dst 330 373 703 22.9 118 220 338 14.1 101 61 162 9.1 ltp 134 137 271 8.8 253 289 542 22.7 71 67 138 7.7 spt 257 581 838 27.3 134 272 406 17.0 226 144 370 20.7 wst 30 58 88 2.9 68 76 144 6.0 204 117 321 18.0 total 1309 1764 3073 100.0 1093 1299 2392 100.0 1141 647 1788 100.0 abt: animal baited trap; bst: black shannon trap; clt: light trap baited with carbon dioxide; dst: disney trap; ltp: cdc light trap; spt: sticky paper traps; wst: white shannon trap. a: bacheh-dareh; b: kohne-jolgeh; c: arg table 2. species abundance of sand flies collected by different traps, north khorasan province, 2016 species collection methods abt bst clt dst ltp spt wst ph.sergenti 179 421 1090 918 642 869 189 ph. papatasi 238 298 386 179 207 226 319 ph. alexandri 20 2 94 3 23 56 15 ph. major 3 15 11 3 6 23 0 ph. halepensis 5 2 5 2 5 10 1 ph. caucasicus 0 0 1 0 3 4 0 ph. mongolensis 3 0 1 2 1 2 0 ph. longiductus 0 0 0 2 3 0 0 ph. turanicus 0 0 1 1 0 1 0 ph. ansarii 0 0 0 0 0 1 0 se. sintoni 17 18 13 41 11 219 12 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 403 http://jad.tums.ac.ir published online: december 31, 2019 se. sumbarica 22 2 22 16 15 53 5 se. pawlowskyi 2 3 0 12 7 48 0 se. theodori 0 0 8 11 5 43 0 se. dreyfussi 8 21 4 7 3 20 11 se. hodgsoni 7 1 3 2 10 17 1 se. dentata 4 0 0 4 8 17 0 se. clydei 0 2 0 0 2 1 0 se. grekovi 0 0 0 0 0 4 0 frequency 508 785 1639 1203 951 1614 553 abt: animal baited trap; bst: black shannon trap; clt: light trap baited with carbon dioxide; dst: disney trap; ltp: cdc light trap; spt: sticky paper traps; wst: white shannon trap table 3. frequency of sand flies by genus collected by different traps, north khorasan province, 2016 cm genus total percentage phlebotomus percentage sergentomyia percentage abt 448 6.9 60 7.9 508 7.0 bst 738 11.4 47 6.2 785 10.8 clt 1589 24.5 50 6.6 1639 22.6 dst 1110 17.1 93 12.2 1203 16.6 ltp 890 13.7 61 8.0 951 13.1 spt 1192 18.4 422 55.4 1614 22.3 wst 524 8.1 29 3.8 553 7.6 total 6491 100.0 762 100.0 7253 100.0 cm: collection methods; abt: animal baited trap; bst: black shannon trap; clt: light trap baited with carbon dioxide; dst: disney trap; ltp: cdc light trap; spt: sticky paper traps; wst: white shannon trap table 4. the sex ratio of phlebotomine sand flies collected by different traps, north khorasan province, 2016 cm phlebotomus sergentomyia female male total sr female male total sr abt 191 257 448 135 29 31 60 107 bst 467 271 738 58 41 6 47 15 clt 856 733 1589 86 33 17 50 52 dst 497 613 1110 123 52 41 93 79 ltp 424 466 890 110 34 27 61 79 spt 342 850 1192 249 275 147 422 53 wst 284 240 524 85 18 11 29 61 total 3061 3430 6491 112 482 280 762 58 cm: collection methods; abt: animal baited trap; bst: black shannon trap; clt: light trap baited with carbon dioxide; dst: disney trap; ltp: cdc light trap; spt: sticky paper traps; wst: white shannon trap table 5. species diversity and species richness of phlebotomine sand flies collected by different traps, north khorasan province, 2016 collection methods abt bst clt dst ltp spt wst species richness 12 11 13 15 16 18 8 species diversity based on shannon-wiener index 1.39 1.05 0.99 0.88 1.09 1.60 1.01 abt: animal baited trap; bst: black shannon trap; clt: light trap baited with carbon dioxide; dst: disney trap; ltp: cdc light trap; spt: sticky paper traps; wst: white shannon trap table 2. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 404 http://jad.tums.ac.ir published online: december 31, 2019 fig. 1. study area of phlebotomine sand flies collection, north khorasan province, iran, 2016. a: bacheh-dareh, b: kohne-jolgeh, c: arg fig. 2. different collection methods used for sampling of phlebotomine sand flies, north khorasan province, iran, 2016. above: white and black shanon and animal baited traps in location c. below: light trap and light trap baited with carbon dioxide gas in location a discussion this is the first research conducted in northeast of iran to evaluate different sand fly collection methods in three endemic foci of cutaneous and visceral leishmaniasis in north khorasan province, iran. a total of 19 species of sand flies collected and identified. some proven or suspected vectors of cl and vl in iran including ph. papatasi, ph. sergenti, ph. caucasicus, ph. alexandri and ph. major were collected in the study area. our findings confirm that these traps differ in performance and demonstrated that clt and spt are the most efficient sand fly collection methods. clt collected 24.5% of all phlebotomus genus, 13 species and its shannon-wiener index was 0.99 while spt collected 18.4% of phlebotomus genus, 55.4% of sergentomyia genus, 18 species of phlebotominae sand flies and its shannon-wiener index was 1.60. the result of the performance of clt and spt is in agreement with some studies about the productivity of these methods (10, 12) but is in contrast with the result of some studies (11, 13). it seems in arid and dry area the performance of spt is more appropriate than other methods, but in moisture and mountainous area this method has low efficiency. spt become ineffective in habitats with high relative humidity because of the viscosity of castorl oil and only dead specimens are collected (20). in our study the performance of spt in arid area was more than mountainous area. species with very low abundance may be difficult to detect using non-attractive traps such as spt. however, we captured some of rare species only with spt, so this method could assess the species composition and is a perfect http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 399–406 k arzamani et al.: comparative performance of … 405 http://jad.tums.ac.ir published online: december 31, 2019 method for determination of biodiversity indices of phlebotominae sand flies. collection of high abundance of sergentomyia genus and male specimens illustrated in this method. according to our study, clt seems to be one of the most effective collection methods and could assess the species composition of phlebotominae sand flies. this trap showed higher performance in capturing females and phlebotomus specimens; therefore clt can be used for monitoring the population of phlebotomus genus during surveillance. it seems in moisture and mountainous areas this method has high efficiency and the result is in agreement with some studies. some researchers obtained co2 for clt using dry ice, which is difficult for regulation of gas emission so we used a co2 gas tank could easily, regulated and increased the efficacy of this method. the performance of clt was more than ltp. co2 is usually a long-range attractant for sand flies, whereas light is probably perceived by sand flies at much closer range (20). conclusion our results suggest that clt and spt are suitable for entomological surveys in the country. clt is higher attractive for females and phlebotomus genus and is an ideal method for monitoring the population of phlebotomus genus during surveillance. however, spt is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities and captured some of rare species and provide a more realistic estimation of sand flies biodiversity. therefore spt is probably the best choice for monitoring of phlebotominae sand flies. acknowledgments this study received financial support from tehran university of medical sciences (project no: 27811) and vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran (project no: 95/60/729). the authors would like to thank the staff of the department of medical entomology and vector control, tehran university of medical sciences and vector-borne diseases research center of north khorasan university of medical sciences for their kind cooperation. the authors declare that there is no conflict of interest. references 1. alvar j, velez id, bern c, herrero m, desjeux p, cano j, jannin j, den boer m; who leishmaniasis control team (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7(5): e35671. 2. yaghoobi-ershadi m (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod borne dis. 6(1): 1–17. 3. alavinia s, arzamani k, reihani m, jafari j (2009) 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culicidae), a dengue virus vector, from multiple geographical locations of samut songkhram, thailand *tanawat chaiphongpachara 1, nattapon juijayen 2, kitthisak khlaeo chansukh 3 1college of allied health sciences, suan sunandha rajabhat university, thailand 2bachelor of public health, college of allied health sciences, suan sunandha rajabhat university, thailand 3department of applied thai traditional medicine, college of allied health sciences, suan sunandha rajabhat university, thailand (received 21 feb 2017; accepted 21 july 2018) abstract background: dengue haemorrhagic fever (dhf) is a mosquito-borne disease and remains a major public health problem, especially in tropical and temperate countries. studying wing morphometric of aedes aegypti as a mosquito vector of dhf can help to better understand biological process of the mosquito adaptation to the environment. we aimed to study the geometric morphometric of ae. aegypti from multiple geographical areas. methods: samples were collected from samut songkhram province in thailand, including coastal, residential and cultivated areas, by ovitrap once per month during oct to nov 2016. results: according to size variation analysis of ae. aegypti, the female mosquito in a cultivated area was significantly different from those in the coastal and residential areas (p< 0.05). whereas male ae. aegypti in a cultivated area were significantly different from those in a residential area (p< 0.05). the shape variation of both female and male ae. aegypti from all areas was statistically different (p< 0.05). conclusion: normally, living organisms, including mosquitoes, are adapted to their environment. the studied geographical locations affect ae. aegypti morphology. keywords: aedes aegypti, mosquito vector, geometric morphometric, dengue hemorrhagic fever introduction dengue haemorrhagic fever (dhf) is a mosquito-borne disease and major public health problem in several countries worldwide (1). aedes aegypti (diptera, culicidae) is the primary mosquito vector of dhf that carries and transmits dengue virus to humans. moreover, ae. aegypti is closely associated with humans (it is active in the daytime and prefers resting in and around human houses) (2). in addition, ae. aegypti can be resistant to chemical insecticides (3). the combination of these factors is associated with outbreaks of dhf. the geographical environment affects the size and shape of mosquitoes (4). the geographical is relating to the landscape of the earth, where it has an environmental and habitat associated with mosquito vector (5). thailand is currently experiencing a disease epidemic. regarding the current dhf situation in thailand, the bureau of vector borne disease at the department of disease control (within the ministry of public health) has compiled retrospective data for recent years. between 2011 and 2013, the dhf incidences rate were 100.789, 116.24, and 234.86 cases per 100000 population, the number of dhf patients is increasing year on year, and the disease remains a major public health problem in thailand. samut songkhram is the smallest province in thailand. samut songkhram is among those provinces with the most dhf cases (671 dhf patients [345.54 per 100000 population] *corresponding author: dr tanawat chaiphongpachara, e-mail: tanawat.ch@ssru.ac.th elham.jahani56@gmail.com j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 352 http://jad.tums.ac.ir published online: december 25, 2018 in 2015). this province consists of a coastal area influenced by tides and having diverse coastal plants, a residential area (high population density), and a cultivated area (low population density). these environments produce morphological variation in ae. aegypti (6). studying the morphological variation of the mosquito helps us have a better understanding of causes, factors, and biological process of the mosquito’s adjustment to the environment (7). recently, transmission of dengue virus by ae. aegypti in any geographic area depends on many factors, including extrinsic features related to the environment and intrinsic factors associated with the virus and vector interaction (6). therefore, to reduce the presence of the vector, it is necessary to study the variability of mosquitoes as a basis for medical entomology. geometric morphometric (gm) is a newly developed morphometric technique for analysis of shapes and sizes of organisms using the principles of geometry (8). many reports have applied gm to medical mosquitoes (9). gm can also be applied to study morphological variations of organisms and analyze the evolution of wing morphometric (10). as with aedes spp., the gm technique was used to identify three species in thailand (ae. aegypti, ae. albopictus and ae. scutellaris), found to be highly capable (11). molecular techniques are popular and powerful tools for the identification of species (12). pcr-based methods are often used for species identification because of their sensitivity, reliability, and specificity for species identification (13). in addition, molecular methods are also commonly used to study genetic variation across different areas (14). although high-efficiency molecular techniques can use for species identification and genetic variation of mosquito, these are expensive and require specialized training (9). gm is an attractive alternative approach to identification and variation because it is cheap, easy, and fast. we aimed to study the gm to investigate the impact of geography on ae. aegypti from multiple geographical areas of samut songkhram province, including coastal, residential and cultivated areas. materials and methods mosquito collection larvae of ae. aegypti were collected in three different geographical locations across samut songkhram province (coastal, residential, and cultivated sites) by ‘ovitrap’ once per month during oct to nov 2016. the geographical locations were selected by spatial data on land utilization information in samut songkhram from the department of provincial administration of thailand. the coastal area was bang cha kreng subdistrict (13° 23'31.57"n 100°1'59.36"e), the residential area was mae klong subdistrict (13°24'32.52" n 100°0'41.40"e), and the cultivated area was jompluak subdistrict (13°28'23.7"n 99°55' 05.1"e) (fig. 1). one village in each of the study areas reporting the highest dengue cases was selected, according to data from the report of samut songkhram provincial bureau of epidemiology in 2015. six ovitraps (one trap per house) were set around houses or spaces under houses in each geographical location. field collected larvae were then reared in the laboratory of college of allied health sciences (suan sunandha rajabhat university, thailand). emerged adults were morphologically identified by illustrated keys to the mosquitoes of thailand (15). mosquito preparation only the right wing of ae. aegypti was analyzed. the right wings were dissected and mounted on microscope slides with coverslips using hoyer solution. all of the sample wings were photographed using a nikon ds-ri1 sight digital camera connected to a nikon az 100m stereo-microscope (nikon corporation, tokyo, japan). the images were analyzed using the morphometric clic program. j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 353 http://jad.tums.ac.ir published online: december 25, 2018 geometric morphometric fourteen landmarks were digitized (fig. 2). for these selected landmarks, there is a selection criterion that must be clearly visible in order to prevent mistakes when plotting. the precision and measurement error of images was estimated by the ‘repeatability’(r) index (7, 8). ten images of mosquito wing in each geographical location were randomly chosen for r testing and repeatability of plotting landmark from random images. after that, both two sets of images were computed as 1-r, with r, which is a model ii one-way anova on repeated measures (7, 8). the landmark-based gm analyzed the size and shape of the mosquito. for wing size estimation, the size was measured by estimating the centroid size (cs) defined as the square root of the sum of the squared distances between the center of the configuration of landmarks and each individual landmark (11). statistical differences between the centroid sizes of the male and female mosquito wings from the different areas were analyzed by non-parametric permutation tests (1000 cycles). for wing shape evaluation, shape variables were measured and analyzed by principal components (pcs) of the “partial wrap” scores calculated after generalized procrustes analysis of raw coordinates. wing shape differences between geographical locations were calculated based on the mahalanobis distance. similar to wing size estimation, statistical differences in wing shape were analyzed by non-parametric permutation tests (1000 cycles). neighbor-joining (nj) trees were generated and calculated based on procrustes distances between ae. aegypti from different locations. validated reclassification accuracies were estimated for testing variation of ae. aegypti in each geography location yielded by gm, each individual was reclassified by comparing the shape based on the mahalanobis distances. software data collection and analyses were performed using the various modules of the clic version 97 (collecting landmarks for identification and characterization), which is freely available at http://mome-clic.com (7). the following modules used coo for landmark collection, tet for the transformation of data to be analyzed, mog for centroid size and shape variables analysis to compute procrustes distances, pad for statistical significance analysis of shape variables and to compute mahalanobis distances, and var for statistical significance analysis of size variables. results by applying the gm technique, we analyzed 220 samples comprised of 103 female and 117 male mosquitoes. according to geographical classification, 68 analyzed samples were from a coastal area, 82 samples from a residential area, and 70 samples from a cultivated area (table 1). for repeatability, comparison of two sets of repeated measurements for the same images of ae. aegypti wing for gm testing showed good scores (0.994). size variation size variation of aedes aegypti wings was analyzed from the centroid size average of the wings from the different areas. classified by sex, female mosquitoes in the residential area had the highest average (2.06vs 2.04 and 1.95mm) in the coastal and cultivated areas respectively. similarly, male mosquitoes in the residential area had the highest average (1.62 vs 1.57 and 1.54mm) in coastal and cultivated areas respectively (table 2). female mosquitoes in the cultivated area had significant smaller wing size than those in the coastal and residential area (p< 0.05). male mosquitoes from the cultivated area had smaller wing size than those from the residential area (p< 0.05) (table 4). shape variation after superimposition of the mean land j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 354 http://jad.tums.ac.ir published online: december 25, 2018 mark configuration of the wings of the males and females from the different areas, polygons as connected mean landmark positions were demonstrated. both polygons as connected mean landmark configurations of female and male mosquito in each different environmental types were clearly different (fig. 3). both factor map of female and male ae. aegypti from landmark-based discriminant analysis by partial wrap showed overlapping of wing shapes in each different geographical type (fig. 4). mahalanobis distances of female ae. aegypti wing shapes in cultivated and coastal areas had the highest value (3.80). similarly, male ae. aegypti wing shapes in the cultivated and coastal areas had the highest value (both 2.63) (table 3). tested by non-parametric permutation tests (1000 cycles), there was a statistical difference (p< 0.05) in both male and female of ae. aegypti mosquitoes in each geographical location (table 4). the neighbor-joining trees, based on the mahalanobis distances between pcs, separated each sex and each location (fig. 5). the validated reclassification accuracy scores confirmed separation from shape differences of ae. aegypti in each geography location, males and females are slightly different. reclassification scores of male mosquitoes were 80% to 89%, while these scores for female mosquitoes were 84% to 90%. the highest percentage of reclassification of male mosquito showed of 89% for the cultivated area, as well as the highest percentages of female mosquito was 90% of cultivated area. the lowest percentages of reclassification were 73% (male mosquito from the residential area) and 75% (female mosquito from the residential area) (table 5). table 1. the number of aedes aegypti used for analysis to classify by sex and geography sex number of aedes aegypti total coastal area residential area cultivated area female 103 32 40 31 male 117 36 42 39 total 220 68 82 70 table 2. means of wing centroid size of aedes aegypti classified by sex and geography sex geography n means±sd (mm.) range (min-max) female coastal area 32 2.04±0.11 1.93-2.15 residential area 40 2.06±0.21 1.85-2.27 cultivated area 31 1.95±0.16 1.79-2.11 male coastal area 36 1.57±0.18 1.39-1.75 residential area 42 1.62±0.15 1.47-1.77 cultivated area 39 1.54±0.14 1.40-1.68 n= number of aedes aegypti table 3. mahalanobis distances between wing shapes of aedes aegypti classified by sex and geography females males geography coastal area residential area cultivated area coastal area residential area cultivated area coastal area 0.00 0.00 residential area 2.53 0.00 2.06 0.00 cultivated area 3.80 3.13 0.00 2.63 1.79 0.00 j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 355 http://jad.tums.ac.ir published online: december 25, 2018 table 4. statistical significance of size and shape differences of aedes aegypti by non-parametric permutation tests (1000 cycles) females males geography coastal area residential area cultivated area coastal area residential area cultivated area size coastal area 0.00 0.00 residential area 0.66 0.00 0.15 0.00 cultivated area 0.01* 0.00* 0.00 0.35 0.02* 0.00 shape coastal area 0.00 0.00 residential area 0.00* 0.00 0.00* 0.00 cultivated area 0.00* 0.04* 0.00 0.02* 0.00* 0.00 *statistically significant (p< 0.05) table 5. validated reclassification accuracies of male and female of ae. aegypti in each geography location geography percentage of reclassification male female coastal area 80% (29/36) 84% (27/32) residential area 73% (31/42) 75% (30/40) cultivated area 89% (35/39) 90% (28/31) fig. 1. map of aedes aegypti collection sites in different geographical locations (1= bang cha kreng subdistrict as coastal area, 2= mae klong subdistrict as residential area, and 3= jompluak subdistrict as cultivated area) j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 356 http://jad.tums.ac.ir published online: december 25, 2018 fig. 2. aedes aegypti wing showing the 14 landmarks used in the morphometrics analysis fig. 3. superimposition of the mean landmark configurations of aedes aegypti in different areas [coastal area (blue), residential area (red), cultivated area (green)]. top: females of ae. aegypti, bottom: males of ae. aegypti fig. 4. factor map from landmark-based discriminant analysis by partial wrap for females (a) and males (b) of aedes aegypti classified by geographical locations j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 357 http://jad.tums.ac.ir published online: december 25, 2018 fig. 5. neighbor-joining trees for shape based on gm analyses of male and female aedes aegypti from different geographical locations discussion here, gm was used to study the wing morphometric of ae. aegypti in samut songkhram province, thailand. two-hundred-twenty ae. aegypti mosquitoes were collected from three different geographical sites. size variation of mosquito females of ae. aegypti have a larger centroid wing size than males, which is in line with other studies (10). moreover, we detected a statistical difference in size variation of the female ae. aegypti. the mosquitoes in the cultivated area were significantly different from those in the coastal and residential areas (p< 0.05). in addition, male ae. aegypti were significantly different from those in the residential area (p< 0.05). the difference between ae. aegypti in the cultivated area and the other areas might be a result of environmental factors, such as temperature, food quality and quantity, and suitable habitat (16). in fact, we did not study the environmental factors that affect ae. aegypti. however, population density differences and the number of households in the cultivated area might have contributed to our findings. other studies have addressed the relationships between population and household number and ae. aegypti numbers (2). urban areas are more suitable for ae. aegypti than rural and sub-rural areas (17). residential areas support the viability and breeding of ae. aegypti, the mosquitoes can easily feed on human blood within houses, and there are many water containers (breeding sites for the dhf vector) (2). high nutrient levels produce larger mosquitoes (18). however, here we did not detect significant differences between the coastal and residential areas for either male or female ae. aegypti. the might be because the coastal area of samut songkhram is a tourist attraction and, therefore, similar to the residential area (tourists plus water containers provide food and breeding sites). mosquitoes from the residential area were largest (female, 2.06±0.21, male, 1.62±0.15). in 2015, the samut songkhram provincial bureau of epidemiology reported that this residential area produced more dhf patients than the other two tested areas. the body size of a female mosquito is correlated with fecundity, larger females lay more eggs during the first gonotrophic cycle (18). moreover, male mosquito size correlates with total sperm numbers within a male and the number transferred to females (19). thus, we would expect a relatively high density of dhf vector in the residential area, which is a factor of disease risk in areas of dengue virus transmission (20). shape variation of mosquito by visualized factor mapping of the landmark-based discriminant analysis (partial wrap), we detected separated and overlapping areas in the wing shape morphospace of male and female ae. aegypti classified geographical location. after being tested with non-parametric permutation tests (1000 cycles), we found that the males and females wing shapes were statistically different in all geographical areas (p< 0.05), likely because of environmental factors j arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 358 http://jad.tums.ac.ir published online: december 25, 2018 (e.g., wind current and weather) (21). in each geographical location of samut songkhram province, there were different environmental factors affecting ae. aegypti shape, for example, storms and wind in the coastal area. the size and shape of ae. aegypti and ae. albopictus were examined from nakhon nayok province of thailand, cucuta municipality of colombia, and florida and hawaii in the united states and detected significant geographic differentiation (10). although we found no statistical difference in size variation in some areas, we detected shape variation across all groups. this might be because the mosquitos are adapted to their noticeably different environments. geography has an impact on aedes mosquito in each geographical location (22). mosquito wing shape is correlated with mosquito population density and food quality. studying these variations helps to better understand how ae. aegypti adapts to its environment. in addition, such morphology data is important for taxonomy studies (11). samut songkhram is a small province. therefore, our ability to detect difference here suggests that gm is a very effective technique for studies addressing mosquito variation. our validated reclassification accuracies scores demonstrate the efficiency of the separation in each geographical location. thus, gm should be considered an alternative method for studying the variation of mosquito vectors. conclusion gm it is a newly-developed morphometric technique used to classify medical insect types and study morphology variants (8). gm is advantageous because it is easy to use, is low-cost, and rapid. gm does not require high entomological skills (7). gm is an effective tool for studying morphology variants of ae. aegypti. aedes aegypti from different locations differ in size and shape, which is important for understanding its local adaptation. such knowledge might help to control mosquitoes in dhf endemic areas. acknowledgements we would like to thank college of allied health science, suan sunandha rajabhat university, thailand for their kind support of our research. this work was supported by suan sunandha rajabhat university, bangkok, thailand. the authors declare that there is no conflict of interests. references 1. kyle jl, harris e (2008) global spread and persistence of dengue. annu rev microbiol. 62: 71–92. 2. rodrigues m de m, marques gram, serpa lln, arduino m de b, voltolini jc, barbosa gl, andrade vr, de lima vlc (2015) density of aedes aegypti and aedes albopictus and its association with number of residents and meteorological variables in the home environment of dengue endemic area, são paulo, brazil. parasit vectors. 8(1): 115. 3. ishak ih, jaal z, ranson h, wondji cs (2015) contrasting patterns of 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five major brazilian ecoregions. inj arthropod-borne dis, december 2018, 12(4): 351–360 t chaiphongpachara et al.: wing geometry analysis … 360 http://jad.tums.ac.ir published online: december 25, 2018 fect genet evol. 12(6): 1246–1252. 22. morales vargas re, ya-umphan p, phumala-morales n, komalamisra n, dujardin jp (2010) climate associated size and shape changes in aedes aegypti (diptera: culicidae) populations from thailand. infect genet evol. 10(4): 580–585. j arthropod-borne dis, december 2020, 14(4): 416–424 f talebzadeh et al.: molecular species identification of … 416 http://jad.tums.ac.ir published online: december 31, 2020 original article molecular species identification of six forensically important iranian flesh flies (diptera) fahimeh talebzadeh1; *mohammad ali oshaghi1; *kamran akbarzadeh1; somayeh panahimoghadam2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of cell and molecular biology, faculty of life sciences and biotechnology, shahid beheshti university, tehran, iran *corresponding authors: dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir, dr kamran akbarzadeh, e-mail: kakbarzadeh@tums.ac.ir (received 21 june 2020; accepted 22 dec 2020) abstract background: flesh flies (diptera: sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. however, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. this study investigated to test utility of mitochondrial cytochrome oxidase subunit i (mt-coi) sequences for differentiation of six forensically important iranian flesh flies namely, sarcophaga crassipalpis, s. flagellifera, s. hirtipes, s. aegyptica, s. africa and s. argyrostoma. methods: male specimens were morphologically identified to species level and then the genomic dna of the flies were extracted and subjected to polymerase chain reaction (pcr) against mt-coi gene. the pcr products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (rflps). results: rate of genetic variation between species was 6–10% which was enough to find restriction enzymes (re) that were able to produce species-specific rflp profiles. combinations of three res: bsrfi, rsai and hinfi, provided diagnostic bands for identification of the six sarcophaga species. conclusion: the results of this study showed that molecular markers such as rflps enhancing the use of evidence from flesh flies in forensic investigation. however, lack proper restriction sites in the coi region inhibited introduction of a single restriction enzyme for easy species identification. it is recommended to apply larger part of dna such as combination of coi and coii genes to provide better rflp markers for species identification of flesh flies. keywords: sarcophagidae; forensic entomology; flesh fly, mitochondrial cytochrome oxidase subunit i (mt-coi) introduction a range of insects present on a corpse can be used as evidence in forensic investigations to estimate the post-mortem interval (pmi). since rate of development in insects is species-specific, commonly, estimation of pmi using insect evidence requires accurate species identification, with subsequent examination of thermos biological profiles to determine insect age (1-4). when larvae or pupae in various stages of development are collected from the site of investigation and the growth rates of samples are known, an approximate time of oviposition or larviposition can be es timated (5). for accuracy, forensic entomologists preferentially use evidence from initial corpse colonizers, such as flesh flies (diptera: sarcophagidae) and blow flies (diptera: calliphoridae) (2). it can be argued that sarcophagids have the potential to provide a more accurate pmi estimate than calliphorids, based on differences in their life cycles (6). sarcophagids lay live larvae (viviparous), providing developed immatures ready to start feeding immediately. however, most calliphorids lay eggs (oviparous), which will only hatch into larvae when the correct environmental condi copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:moshaghi@tums.ac.ir mailto:kakbarzadeh@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 416–424 f talebzadeh et al.: molecular species identification of … 417 http://jad.tums.ac.ir published online: december 31, 2020 tions are met (3). despite the prospective use of sarcophagids in forensic investigations, their use to date has been overshadowed by calliphorids. this is due to the difficulties of morphological species-level identification at any life stage of flesh flies, and a lack of documented thermos-biological profiles of these insects. sarcophagid flies comprise more than 2,500 species in more than 100 genera, of which approximately 800 species belong to the genus sarcophaga meigen (7), widespread, and often among the first insects to colonize a corpse (6). the genus is notorious for having many species with a highly similar morphological appearance, females are difficult to identify, and immature stages are often unknown or lack species-specific diagnostic characters (6, 8). adult sarcophagids can be easily identified at the family level, as most species share the characteristic features of longitudinal stripes on the thorax and a tessellated/chequered abdominal pattern. identification of the species of the genus sarcophaga is traditionally accomplished on males using characters of the phallus (9, 10). however, species-level identification is difficult and requires examination of subtle morphological variation of bristle placement and length, hair coloration, body pigmentation and genitalic structure of adults (7). this is unfortunate because the pmi is sometimes estimated using larvae and pupae. rearing larvae and pupae to the adult level may allow identification but is time consuming, often not possible for all species, and may result in a number of unidentifiable females. considering this, molecular-based approaches for species identifications have been proposed to eliminate issues with identifications based exclusively on taxonomy (11, 12). species identification is essential for determining growth rates, as these rates are species-specific (2). therefore, species identification is a key step in estimating the pmi from entomological evidence. the traditional species identification method is dependent on the morphological features of insects and is not easily applicable to immature samples such as eggs, larvae, and pupae (4-9). moreover, only a few expert taxonomists specialize in forensically important insect species. dna-based approaches have been developed in an effort to improve accessibility to methods of species identification. the mitochondrial encoded cytochrome c oxidase subunit i (coi) gene has been shown to be a major candidate gene to identify forensically important insects (12-14). numerous studies have evaluated the effectiveness of coi gene, with the approach shown to be unreliable for some diptera (15, 16) but also proven successful for many groups of invertebrates, such as mosquitoes, cockroaches, and black flies (17-22). although the coi gene has been used widely for species identification of forensic insects (23-29), however, only a few previously reported studies have used the coi gene for a few forensically important species in iran (30). therefore, there has been little effort to characterize the coi haplotypes of iranian sarcophagidae fly species. this study examined about 711 nucleotide sequences of the 5’ end of coi gene of six sarcophagidae fly species from the genus sarcophaga collected in iran. it is hoped that the results from this study will assist with the implementation of iranian sarcophagidae in forensic investigations. materials and methods specimen collection trapping at decayed meat baits (sheep’s or chicken’s liver and fish carcasses) in plastic bottle fly traps, and hand netting were employed to collect adult sarcophagid specimens in tehran provinces at the laleh park in center of tehran as well as the livestock shopping center and close vicinity of slaughter houses in the east and south of tehran, iran. the captured flies in entomological nets immediately transferred into glass jars. the jars or baits harboring flies were then transported in a polystyrene icebox to the laboratory of medical http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 416–424 f talebzadeh et al.: molecular species identification of … 418 http://jad.tums.ac.ir published online: december 31, 2020 entomology, school of public health, tehran university of medical sciences (sph-tums). live adult were transferred individually into a bottle trap including sand and meat to lay eggs at 28 °c±1, 40%±5 relative humidity and 12h photoperiodicity, protected with an external net curtain to avoid the entry of other insect species. after laying eggs, the dead specimens were identified morphologically by using the taxonomic keys (7, 30, 31). to identify the specimens to species level, the male specimens were dissected and the genitalia of each male specimen were examined. all specimens then were kept in 70% ethanol and stored at -20 °c for further molecular investigation in the laboratory of sph-tums. dna extraction, amplification and sequencing one to three legs from each known adult male sarcophagid specimen were used as tissue for total genomic dna extractions using the qiagen dna extraction kit, following the protocols recommended by the manufacturer (qiagen, hilden, germany). the dna was resuspended in 50–100µl of fresh te solution (1 mm tris–hcl (ph 8), 0.1mm edta) and subsequently stored at 4° c. the coi region of approximately 711bp was amplified and sequenced using the primers of coi-f 5’-gg tcaacaaatcataaagatattgg-3’ and coi-r 5’-taaacttcagggtgaccaaa aaatca-3’ and thermal cycling conditions explained previously (18, 19, 33). the pcr products were visualized on a uv transilluminator following electrophoresis on a 1.2% agarose gel containing ethidium bromide. the pcr products were purified from seized gels according to the supplier's guidelines (amersham pharmacia biotech, usa) and used directly for dna sequencing in an automatic sequencer (seqlab, goä ttingen, germany). the sequences were generated in both directions, and the resultant chromatograms were edited using chromaspro version 1.33 (available online at www.technelysium.com.au/chromaspro.html) and aligned using clustalw2 (34). homology of the sequences with genbank sequences was assessed using fasta search (https://www.ebi.ac.uk/tools/sss/). consensus sequences for the coi region was deposited in the genbank database. multiple alignments of the nucleotide sequences were performed using clustalw2 program (http://www.ebi.ac.uk/tools/msa/clustalw2). following sequencing, polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) assay was used to distinguish the six species. based on sequence variation among the six species, three restriction enzymes were selected by manufacturer’s nebcutter v2.0 program (http://nc2.neb.com/nebcutter2/) and a physical map provided (fig. 1). for restriction fragment assays, the 711bp coi pcr products were digested separately with restriction endonuclease bsrfi, rsai, and hinfi (vivantis) according to the manufacturer’s recommendation. then, the digested sample was resolved by electrophoresis in 2.5% tbe agarose gel, stained with ethidium bromide and viewed under uv-illumination. the restriction patterns were photographed and analyzed. results morphological species identifications in the present study, 273 specimens were collected from tehran district and 86 specimens comprising six species of sarcophagidae were identified using the taxonomic literature. these specimens collectively represent six identifiable taxa: s. argyrostoma robineau-desvoidy 1830, s. africa wiedemann 1824, s. aegyptica/dux salem 1935/thomson 1869, s. crassipalpis macquart 1839, s. flagellifera grunin 1964, and s. hirtipes wiedemann 1830. these identifications were based on male morphological characters, as these are the only specimens that can be reliably identified. difficulties were encountered in accurately identifying some of the 273 specimens using the available taxonomic keys. to further assist with identifications, each ‘unknown’ sequence was comhttp://jad.tums.ac.ir/ http://www.technelysium.com.au/chromaspro.html http://www.technelysium.com.au/chromaspro.html http://www.ebi.ac.uk/tools/msa/clustalw2 j arthropod-borne dis, december 2020, 14(4): 416–424 f talebzadeh et al.: molecular species identification of … 419 http://jad.tums.ac.ir published online: december 31, 2020 pared with the available data in genbank, however no conclusive matches were obtained. we cannot confidently associate the unknown sarcophaga species with a particular subgenus, and it is possible that these could represent new species, given that no broad work on the iranian sarcophaga fauna has been carried out in the past. it is notable that there are 68 reported sarcophagidae species in iran (35). mitochondrial cytochrome oxidase subunit i (coi) analysis the qiagen dneasy blood and tissue kit (qiagen) was used to extract genomic dna from one to three legs of each specimen in each extraction to retain the integrity of the specimens. known specific mitochondrion coi primers were used to minimize the possibility of amplifying nuclear pseudogenes. about 711 bp products of 5’ end of coi gene were able to be amplified using the primer combination of coi-f and coi-r from the dna extracted from the specimen legs. direct pcr-sequencing were used and only strongly amplified products were sequenced. further evidence that the obtained sequences were of mitochondrial origin came from blast search followed by the observation that they did not contain base ambiguities or premature stop codons upon translation. this mtdna region was observed to have a strong at bias (average 67%), which is typical of insect mitochondrial dna (36). the ambiguous parts from the beginning and end of sequences for the six species were removed and finally 621bp reliable data were analyzed. sequence comparison among species showed a maximum of 0.2% intra-specific variation for the species whereas the interspecific variation ranged 6.23–10%. the most divergence rate (10%) was observed between s. argyrostoma and s. flagellifera and the least rate (6.23%) was seen between s. hirtipes and s. aegyptica. based on nucleotide sequence analysis of the mtdna-coi gene, a pcr-rflp assay was developed for the diagnosis and differentiation of the six iranian sarcophaga species. it was accomplished at first stage by digesting the pcr products (711bp) with bsrfi restriction enzyme. this enzyme cuts the pcr–amplified mtdna-coi fragment of s. aegyptica into two bands of 511 and 200bp whereas it does not cut the amplicons of other five species and the amplicons remain intact (fig. 2). at next step, rsai enzyme was used for the five remaining species. this enzyme produces three profiles, 1) a unique profile for s. hirtipes with 485 and 226bp, 2) a specific profile for s. felagellifera by producing 643 and 68bp bands, and 3) a profile with two bands of 580 and 130bp for three species of s. africa, s. crassipalpis, and s. argyrostoma (fig. 2). at third stage, hinfi enzyme was used to separate 1) s. africa with three bands of 514, 172, and 25bp, 2) s. argyrostoma with two bands of 550 and 80bp, and 3) s. crassipalpis with an intact 711bp band (fig. 2). fig. 1. physical map of 711bp of 3’ end of mtdna-coi for six iranian sarcophaga species. b: bsrfi, r: rsai, h: hinfi restriction enzymes http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 416–424 f talebzadeh et al.: molecular species identification of … 420 http://jad.tums.ac.ir published online: december 31, 2020 fig. 2. pcr-rflp assay of 711bp of coi region digested with bsrfi, rsai, and hinfi. lane 1 is a 100bp plus ladder (sinaclon, iran), 2: sarcophaga hirtipes, 3: s. africa, 4: s. crassipalpis, 5: s. flagellifera, 6: s. aegyptica, 7: s. argyrostoma discussion this study was aimed at carefully evaluating the coi gene for species identification of six forensically important iranian sarcophagidae. calculation of the percentage divergence between sequences is used to quantitatively evaluate the success of coi dna. for successful species-level resolution using the coi approach, genetic variation between species (interspecific) exceeds that of within species (intraspecific) in which intraspecific variation should be less than 3% and interspecific variation should be more than 3% (37, 38). this was the case for all of the six iranian sarcophagidae examined in this study, where the percentage divergences lie between 6.23 to 10 percent through the 621bp compared. similar result to this was obtained when different part of coi gene were previously evaluated for identification of sarcophagidae (23, 24, 26, 27). indeed, various fragments of the coi gene (for example: mini-barcode 127bp, 272–278bp, standard barcode 658bp, 700bp, and entire gene 1,535bp) that show low sequence divergence within species but high divergences among species. hence, this gene can be employed as taxon “barcodes” and unknown samples can be placed accurately into species groups simply by calculating their pairwise genetic distances with reference sequences of a “barcode library” (37, 39). as such, an online coi identification system will provide a cost-effective and widely accessible identification tool for flies of forensic interest, even for immature or damaged specimens. at present, such practice are being increased for species of the genus sarcophaga, since coi sequence database has been developed for 12 species from the south of the czech republic (12), for seven species within canada and the usa (6), for 17 malaysian, two indonesian, one japanese species (40), for 16 australian species (26), for 56 species in west europe (25), for several chinese species (23, 24, 28), for six korean species (27), ten iranian species (41) and recently for ten indian species (29). however, a database for other regions in the world is lacking. since the above mentioned studies have shown that intraspecific sequence divergence in sarcophaga is usually well below 1.5%, whereas interspecific divergence is more than 2–2.5%, it can be indicated that the coi gene has a large potential as an identification tool in this forensically interesting taxon. in this study, we introduce pcr-rflp markers using 711bp of 3’ end of coi gene to identify six species of the genus sarcophaga that occur in iran. this is the first mtdna coi pcr-rflp assay for species identification of sarcophagidae in the literature. from the pcr-rflp analysis using combination of three restriction enzymes, each species showed http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 416–424 f talebzadeh et al.: molecular species identification of … 421 http://jad.tums.ac.ir published online: december 31, 2020 a distinct and unique restriction pattern compared to their closely related species. these six species are synanthropic and commonly present in the same collection site, and show high similarities in morphology and behavior. these results suggest that pcr-rflp assay are useful to distinguish these species. pcrrflp technique is a very cheap and rapid method for species identification of many organisms. this molecular assay was used to identify many species of different group of organisms such as rodents (42), pathogens (43-46), anopheles spp (17, 18, 42) and to determine blood type within insect guts (47, 48). this will provide a cost-effective solution that so many specimens can be studied with no need to provide dna sequencing. in this method, using a set of conserved primers, a tiny amount of dna can be amplified and then be identified by restriction enzyme that overcomes lack of ample amount of flesh fly body parts. conclusion the coi sequences of each species were unique and distinguishable from each other, although they showed high homology. species identification from immature dipteran by the dna sequences was simple and time-saving because there was no need to wait for adult emergence or knowledge of morphological keys. nevertheless, given that 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accepted 30 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: culex pipiens has a significant public health importance since it is an important vector of west nile virus and rift valley fever virus. we, therefore, aimed to determine the in secticide resistance level in cx. pipiens populations in the aegean and mediterranean regions of turkey. methods: bioassays have been carried out against dichlorodiphenyltrichloroethane (ddt) (4%), malathion (5%), fenitrothion (1%), propoxur (0.1%), bendiocarb (0.1%), permethrin (0.75%) and deltamethrin (0.05%). biochemical analyses have been performed to detect non-specific esterase, mixed function oxidase, glutathione-s-transferase and acetylcholinesterase levels. a knockdown resistance (kdr) (l1014f) and acetylcholinesterase (ace-1) (g119s) mutations have been detected by using allele-specific primers and a polymerase chain reaction (pcr) amplifica tion of specific alleles (pasa) diagnostic test was performed for detection of f290v mutation. results: bioassay results showed that all cx. pipiens populations were resistant to ddt, mal athion, fenitrothion, bendiocarb, propoxur and some of the populations have started to gain permethrin and deltamethrin resistance. biochemical analyses results revealed that altered glu tathione-s-transferases, p450 monooxygenases, esterase levels might be responsible for ddt, or ganophosphate, carbamate and pyrethroid resistance in cx. pipiens populations. results showed mild to high frequency of l1014f, low frequency of f290v but no ace-1 g119s mutation within the populations. additionally, acetylcholinesterase insensitivity was not significantly high within the most of these populations. conclusion: overall results may help to fulfil the lacking information in the literature regarding insecticide resistance status and underlying mechanism of culex pipiens populations of the mediterranean and aegean region of turkey by using all bioassays, molecular tests and biochemical assays. keywords: kdr; acetylcholinesterase; monooxygenase; glutathione s-transferase introduction mosquitoes can transmit many different pathogenic organisms such as viruses, bacteria, protozoa and nematodes and impact over half of the worlds’ population through the transmission of harmful diseases to both human and animal. they are also responsible for transmitting malaria, lymphatic filariasis and arboviruses such as yellow fever virus, dengue virus and zika virus (1). millions of people are threatened and killed by mosquito-borne diseases every year (2). mosquito control strategies have played a major role in reducing the global burden of mosquito-borne diseases since the introduction of dichlorodiphenyltrichloroe thane (ddt) in the 1940s (3). malaria 406 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... control has been achieved during recent years as a result of insecticide-based strategies including indoor residual spraying (irs), insecticide-treated nets (itns) and long lasting insecticide-treated nets (llitns) (4). current control of mosquitoes is still heavily dependent upon the use of chemical insecticides and four classes of synthetic insecticides called organochlorines (oc), organophosphates (op), carbamates (cb), pyrethroids (py) are recommended by world health organisation (who) for irs (3). however, pyrethroids are recommended for use in itns and llitns (3). despite their environmental pollution concerns and toxicity to non-target organisms and humans, insecticides are still heavily used in current mosquito control strategies. however, this is not sustainable because extensive use of insecticides has also resulted in the development of resistance in mosquito populations around the world. culex pipiens is amongst the most important mosquito species and responsible of maintaining several viruses such as west nile virus (wnv), rift valley fever virus (rvfv) and japanese encephalitis virus (jev) which can be pathogen of human and livestock animals (5). according to the european centre for disease prevention and control (ecdc) report, 1605 wnv infection and 166 deaths due to wnv infection was reported from 11 eu/eu member states in 2018 (6). there have been no reported wnv cases until 2009 in turkey. however, both epidemic and wnv-related central nervous system diseases have been reported in the western part of anatolia since 2009 (7). a total of 47 and 5 wnv were reported from different regions of turkey in 2010 and 2011, respectively (8). recently, one human case of wnv has been reported in 2019 (6). while many integrative techniques can be used for cx. pipiens control, the fastest and cheapest way to control vector densities in endemic regions, control epidemics and reduce annoying mosquito populations is to apply insecticides. however, as it was stated before, rapidly developing insecticide resistance in mosquito populations is the biggest obstacle in vector management and control. therefore, it’s so crucial to understand the status and mechanisms of insecticide resistance for overcoming or delaying resistance to existing compounds and preventing the development of resistance to new pesticides (9). to date, a few studies have been reported regarding the insecticide resistance status of turkish cx. pipiens populations. akıner et al. (10) reported high ddt resistance in cx. pipiens populations in antalya, ankara, çankırı, mersin, hatay, birecik and viranşehir. they also reported high fenitrothion resistance in birecik, mersin, çankırı and antalya; high temephos resistance in birecik, viranşehir, mersin, ankara and antalya. similarly, high ddt, malathion, permethrin and deltamethrin resistance have been reported in cx. pipiens populations of mediterranean region (mersin, adana and antalya). high mixed function oxidase (mfo) and non-specific esterase (nse) activities have been attributed to high ddt, malathion and pyrethroid resistance in these populations (11). taşkın et al. (12) reported high malathion, bendiocarb and dieldrin resistance of cx. pipiens populations collected from the aegean region. kdr mutation (l1014f and l1014c) frequencies were also high in these populations. however, ace-1 (g119s and f290v) and rdl (a302s) mutation frequencies were too low to explain malathion and dieldrin resistance. finally, guz et al. (13) reported high frequencies of diflubenzuron resistance in muğla for the first time. the chitin synthase 1 gene mutations (i1043l and i1043m) were ranging from 15.7% to 52.7% and the kdr l1014f mutation was ranging from 40% to 50%. turkey is a country with high agricultural activities. because of the fact that wide ranges of insecticides are still used for different kinds of pest control in turkey (14). although many insecticides have been used for many years, studies to regularly monitor resistance development in mosquito populations are not sufficient. additionally, this study aims to evaluate both the molecular 407 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... and biochemical mechanisms underlying the current insecticide resistance at the same time in cx. pipiens populations of the mediterranean and aegean region during 2017 and 2018. materials and methods mosquito strains larva and adult cx. pipiens samples were collected from a total of 10 locations in both the mediterranean and the aegean regions of turkey between april 2017 and september 2018 (fig. 1, table 1). larval samples were collected using larval dippers and transferred to the transport container until it was brought to the laboratory. gravid females were collected from stables and houses by using mouth aspirators and brought to the laboratory in a cardboard cup sealed with a thin cloth. following the egg laying of gravid females, f1 generations were obtained. larval samples were reared to adults under the standard laboratory conditions at 26–28 oc, 12:12 h photoperiod and 70–80% relative humidity in an insectarium. morphological identifications were performed using an identification key (15). an unfed 3–5 days old f1 generation females were preferred to use in bioassays, biochemical assays and molecular study. samples were stored in a -80 oc freezer until biochemical assays have been carried out. we have sensitive culex quinquefasciatus fig. 1. sampling localities of culex pipiens populations in turkey (1. hatay-dörtyol, 2. adanakarataş, 3. mersin-tarsus, 4. antalya-manavgat, 5. burdur-gölhisar, 6. afyon-dinar, 7. denizlihonaz, 8. muğla-dalaman, 9. aydın-söke, 10. manisa-hacıhaliller) fig. 1. sampling localities of culex pipiens populations in turkey (1. hatay-dörtyol, 2. adana-karataş, 3. mersintarsus, 4. antalya-manavgat, 5. burdur-gölhisar, 6. afyon-dinar, 7. denizli-honaz, 8. muğla-dalaman, 9. aydınsöke, 10. manisa-hacıhaliller) 408 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... laboratory strain which had not been exposed to insecticides for more than 15 years and raised in aydın adnan menderes university vector biology laboratory. these sensitive cx. quinquefasciatus lab strain was used as a reference strain for comparison in biochemical analyses. bioassays bioassays have been carried out through who’s insecticide susceptibility bioassay tubes (16). susceptibility tests was performed against ddt (4%), malathion (5%), fenitrothion (1%), propoxur (0.1%), bendiocarb (0.1%), permethrin (0.75%) and deltamethrin (0.05%) using who papers supplied by the whopes collaborating centre at university sains malaysia. bioassays were performed in the insectarium, under the same physical conditions in which mosquitoes were raised. a total of 660 adult females were used with controls of each of seven types of insecticides, 60 individuals from each population. for each test, we used 20 adult mosquitoes in assay tubes in triplicate. mosquitoes were transferred into the holding tubes after one-hour exposure to each insecticide-impregnated paper and fed on 10% sugar solution for 24 h. control group were exposed to insecticide free, impregnated only with the excipient without any active ingredient control papers for one hour. mean values of three replicates were used to calculate mortality rates. resistance levels were calculated as susceptible if mortality rates are ≥ 98%, possible resistance if mortality rates are between 90–97% and resistant if mortality rates are lower than 90% (17). biochemical assay a total of 330 (30 samples from each population in addition to the control group) individuals were used for biochemical analyses. an unfed 3–5 days old cx. pipiens f1females were used to perform enzyme assays. homogenization was first carried out using liquid nitrogen and then followed by 250 μl 50 mm sodium phosphate buffer onto the ice to keep the samples from heat denaturation. homogenates were centrifuged at 10.000 g for 10min. at 4 oc. spectrophotometric analyses were carried out in 96 well microtiter plates using biotek elx808 microplate reader (biotek instruments, usa). protein content was measured using a bradford assay which includes bradford dye reagent prepared with coomassie brilliant blue, and the absorbance was measured at 595 nm (18). total protein content was measured using a standard curve of bovine serum albumin (bsa). all biochemical analyses including nse, mfo, glutathione-s-transferase (gst) and acetylcholinesterase (ache) assay were carried out by following the test procedure provided by who (16). all tests were conducted as two replicates. alpha naphthyl acetate, beta-naphthyl acetate and p-nitrophenyl acetate (pnpa) were used as substrates of esterase enzyme for the table 1. sampling localities of culex pipiens populations in turkey, 2017-2018 region province locality abbreviation locality no field property mediterranean region hatay dörtyol hd 1 tourism and agriculture adana karataş ak 2 agriculture mersin tarsus mt 3 agriculture antalya manavgat am 4 tourism and agriculture burdur gölhisar bg 5 agriculture aegean region afyon dinar ad 6 agriculture denizli honaz dh 7 tourism and agriculture muğla dalaman md 8 tourism and agriculture aydın söke as 9 tourism and agriculture manisa hacıhaliller mh 10 agriculture table 1. sampling localities of culex pipiens populations in turkey, 2017-2018 409 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... calculation of non-specific esterase activity. standard curves of alphanapthol, beta naphtol and 4-nitrophenol were created and enzyme activity was calculated as enzyme units (eu, μmol/min) using these standard curves of alpha, beta and p-naphthol acetate. specific enzyme activities were calculated based on enzyme units and stated as eu/ mg protein for each esterase. mfo level was calculated using heme-peroxidase assay based on heme-protein amount (16, 19). heme protein content was calculated using a standard curve of cytochrome c protein to calculate mfo levels. similarly, who (16) were followed for the calculation of gst levels. the extinction coefficient (ε): 4.39 mm−1 was used to calculate specific gst enzyme activities as described by who (16). finally, ache and insensitive ache levels were measured by following the instructions described by who (16). for this assay, the inhibition rate was calculated based on well optic density (od) and the remaining ache rates were calculated by dividing the od of the well with propoxur by that without propoxur for the same mosquito, i.e. rate or end point with propoxur * 100= % remaining activity in propoxur inhibited replicate rate or end point without propoxur. molecular assays a total of 95 and 100 dna was extracted from the mediterranean and aegean region, respectively by using invitrogen purelink genomic dna isolation kit. totally 195 individuals were tested for the presence of kdr (l1014f), ace-1 g119s and ace-1 f290v mutations from the study area. a voltage gated sodium channel (vgsc 1) generegionwasamplifiedusingprimerscgd1: gtggaacttcaccgaactt c, cgd2: g c a a g g c ta a g a a a a g g t ta a g , cgd3:ccaccgtagtgataggaaattta and cgd4: ccaccgtagtgataggaaatttt (20). a polymerase chain reaction (pcr) were performed in a final volume of 25 µl containing 1 µl 2.5 mm dntp, 2.5 µl 10 reaction buffer, 0.3 µl 20 mm each of the primers, 0.3µl (5 u/ml) taq dna polymerase, 1µl template dna and 19.3 µl dh2o. the amplification program consisted of an initial denaturation at 94 °c for 15min, 40 cycles of denaturation at 94 °c for 1min, annealing 50 °c for 1 min, extension 72 °c for 2min and followed by 72 °c for 10 min. subsequently, amplified fragments were loaded on 1% agarose gel and visualized under uv light. a 194 base paired ace-1 gene amplicon was amplified using primer pairs ace1-f1: 5′-ccgggggccaccatgtggaa-3′ and ace1-r2: 5′ gttctcctccgaggccagcgtccg-3′ (21). the pcr was performed in a final volume of 25 μl containing about 50 ng dna, 1.5 u taq dna polymerase, 20 μm each of primers, 2.5 mm dntp, and 10x reaction buffer. pcr reaction conditions were denaturation at 94 oc for 5 min, 30 cycles of denaturation at 94 °c for 30 s, annealing at 54 oc for 30 sn, extension at 72 oc for 1 min, and the final extension step of 72 °c for 5 min. since the g119s mutation creates restriction sites, we used alui enzyme which generates a 120 bp and 74 bp fragment to create fragments and visualize the fragment lengths on agarose gel as a result of the restriction fragment length polymorphism analysis (rflp). the restriction fragment length polymorphism analysis content consisted of 2 μl 10x buffer, 1 μl alu-1 enzyme (2 u), 2μl bsa (0,1 mg/ ml), 15 μl pcr product and 5 μl dh2o. this mixture was left in incubation for 4 hours at 37 °c and then the reaction was stopped at 65 oc for 20 minutes. pcr products then were visualized under uv light on 2% agarose gel. a pcr amplification of specific alleles (pasa) diagnostic test was performed for detection of f290v mutation as previously described by taşkın et al. (12). a 543 bp control band, a 148 bp phenylalanine band and a 435 bp valine band were obtained after a pcr reaction. amplified fragments were then visualized on 1.5% agarose gel electrophoresis under uv light. statistical analyses median enzymatic activities were calculated for cx. pipiens mosquito populations and compared with sensitive cx. 410 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... quinquefasciatus laboratory strain by way of kruskal-wallis test using statistica version 12. results bioassay all of the mediterranean populations were resistant to ddt, malathion, fenitrothion, bendiocarb and propoxur. mortality rates ranged between 71.6–85%; 71.6–81.6%; 70–81.6%; 71.6–83%; 78.3–83%; 91.6– 98.3% and 91.6–100% for ddt, malathion, fenitrothion, bendiocarb, propoxur, permethrin and deltamethrin, respectively. the only permethrin and deltamethrin susceptible population was gölhisar. all of the populations were possible resistant to deltamethrin and permethrin except the gölhisar population. mortality rates are given in fig. 2. all of the aegean region populations were resistant against ddt, malathion, fenitrothion, bendiocarb and propoxur. mortality rates ranged between 7.6–81.6%; 75–81.6%; 76.6–81.6%; 80–88.3%; 80– 83.3%; 96.6–98.3% and 95–98.3% for ddt, malathion, fenitrothion, bendio fig. 2. world health organization insecticide susceptibility test results of culex pipiens populations of the mediterranean region, turkey (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd). calculations are based on the mean results of three replicates. error bars represent standard deviations.) 0 20 40 60 80 100 ddt malathion fenitrothion bendiocarb propoxur permethrin deltamethrin pe rc en ta ge m or ta lit y (% ) insecticides ak am bg mt hd c fig. 2. world health organization insecticide susceptibility test results of culex pipiens populations of the mediterranean region, turkey (population name abbreviations represented as: adana-karataş (ak), antalyamanavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd). calculations are based on the mean results of three replicates. error bars represent standard deviations.) fig. 3. world health organization insecticide susceptibility test results of culex pipiens populations of the aegean region, turkey (population name abbreviations represented as: afyondinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). calculations are based on the mean results of three replicates. error bars represent standard deviations.) 0 20 40 60 80 100 ddt malathion fenitrothion bendiocarb propoxur permethrin deltamethrin pe rc en ta ge m or ta lit y (% ) insecticides ad as mh md dh c fig. 3. world health organization insecticide susceptibility test results of culex pipiens populations of the aegean region, turkey (population name abbreviations represented as: afyon-dinar (ad), aydın-söke (as), manisahacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). calculations are based on the mean results of three replicates. error bars represent standard deviations.) 411 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... carb, propoxur, permethrin and deltamethrin, respectively. all the hacihaliller, honaz and dalaman populations were possible resistant to permethrin and deltamethrin, whereas the dinar and the söke populations were still susceptible to permethrin and deltamethrin. mortality rates are given in fig. 3. biochemical analysis results the median specific alpha, beta and pnpa esterase activities were calculated as 0.242 eu/mg protein, 0.328 eu/ mg protein and 0.265 eu/mg protein in susceptible reference strain, respectively. median specific enzyme activities for alpha esterase, beta esterase and pnpa ranged between 0.325 eu/mg protein and 1.041 eu/mg protein; 0.508 eu/ mg protein and 0.915 eu/mg; 0.754 eu/ mg protein and 1.526 eu/mg protein in the mediterranean region populations of cx. pipiens, respectively. median specific alpha esterase, beta esterase and pnpa activities were significantly increased in all of the mediterranean populations in relation to reference strain (p < 0.05) except the manavgat population in which median alpha and beta esterase activities were not altered significantly and the gölhisar population in which alpha esterase and pnpa activities were not significantly increased comparing to reference strain (p > 0.05). median specific enzyme activities for alpha esterase, beta esterase and pnpa ranged between 0.599 eu/mg protein and 1.590 eu/mg protein; 0.630 eu/mg protein and 1.384 eu/mg; 0.942 eu/mg protein and 1.649 eu/mg protein in the aegean region populations of cx. pipiens, respectively. all of the populations had significantly higher median alpha, beta and pnpa activities in relation to the reference strain (p < 0.05). median alpha and beta esterase and pnpa activities of the populations are given in fig. 4, fig. 5 and fig. 6, respectively. the median specific mfo activity was calculated as 2.605 µg cytochrome-c/mg protein in susceptible reference strain. median mfo activities ranged from 5.310 and 11.373 µg cytochrome-c/mg protein and 9.643 and 21.059 µg cytochrome-c/ mg protein in the mediterranean and aegean populations, respectively. all tested populations displayed increased mfo levels (p < 0.05) except the gölhisar population which did not have significantly increased mfo levels in accordance to reference strain (p > 0.05) (fig. 7). fig. 4. median alpha esterase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisahacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) fig. 4. median alpha esterase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) 412 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... the median specific gst activity was calculated as 0.138 eu/mg protein in susceptible reference strain. median specific gst activities changed between 0.217 eu/ mg protein and 0.743 eu/mg protein in the mediterranean and between 0.261 eu/ mg protein and 0.795 eu/mg protein in the aegean region cx. pipiens populations. all of the mediterranean and the aegean populations had significantly higher median specific gst activities (p < 0.05) except the tarsus, manavgat and gölhisar populations in the mediterranean and the honaz population in the aegean region (p > 0.05) (fig. 8). the median specific remaining ache rate was calculated as 7.2% in susceptible fig. 5. median beta esterase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisahacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh).c: control) fig. 5. median beta esterase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh).c: control) fig. 6. median p-nitrophenyl acetate activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisahacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) fig. 6. median p-nitrophenyl acetate activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hataydörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizlihonaz (dh). c: control) 413 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... reference strain. median remaining ache rates ranged between 36.9% and 17.4% in the mediterranean region while they were ranged between 30.5% and 21.3% in the aegean region. the tarsus and karataş populations had insensitive ache rates which are higher than 30% (p < 0.05) while the others did not have significantly altered ache levels in the mediterranean region (p > 0.05). median remaining ache rates changed between 21.3% and 30.5% in the aegean region. the only populations were the dalaman and söke which had insensitive ache rates expressed as higher than 30% critical level (p < 0.05). all of the remaining aegean populations had sensitive ache rates (p > 0.05). (fig. 9). median levels of detoxifying enzyme activities and p values against control were given in table 2. fig. 7. median mixed function oxidase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisahacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) fig. 7. median mixed function oxidase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hataydörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizlihonaz (dh). c: control) fig. 8. median glutathione-s-transferase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisahacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) fig. 8. median glutathione-s-transferase activities of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hataydörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizlihonaz (dh). c: control) 414 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... molecular test results both 1014l (tta) and 1014f (ttt) alleles were detected in the mediterranean and aegean populations of cx. pipiens. the highest l1014f allele frequency was calculated in the gölhisar population as 0.8, however, the population did not show a statistically significant deviation from hw balance (p > 0.05). the l1014f allele frequencies were 0.56, 0.55, 0.62, 0.45 in the dörtyol, karataş, manavgat and tarsus populations, respectively. all of the populations were in hw balance (p > 0.05) except the manavgat and tarsus populations which had lower heterozygosity rate (p < 0.05). the highest l1014f allele frequency was calculated as 1.00 in the honaz population in which l1014f allele was fixed. the l1014f allele frequency was 0.90 in the fig. 9. median remaining acetylcholinesterase inhibition rates of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydınsöke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) table 2. median level of detoxifying enzyme activities and p values (2 tailed) in culex pipiens populations collected from the mediterranean and aegean region, turkey. r eg io n province locality median level of detoxifying enzyme activities and p values (2-tailed) against control alpha esterase eu/mg protein beta esterase eu/mg protein pnpa eu/mg protein oxidase µg cytochromec/mg protein gst eu/mg protein aschi (%) median p median p median p median p median p median p m ed it er ra ne an hatay dörtyol 0.966 0.000 0.915 0.000 1.299 0.000 10.652 0.000 0.743 0.000 28.125 0.000 adana karataş 0.706 0.000 0.675 0.000 1.393 0.000 11.000 0.000 0.486 0.000 32.211 0.000 mersin tarsus 1.041 0.000 0.908 0.000 1.526 0.000 10.617 0.000 0.384 0.051 36.986 0.000 antalya manavgat 0.325 1.000 0.508 0.930 0.815 0.051 11.373 0.000 0.232 1.000 17.421 0.031 burdur gölhisar 0.497 0.510 0.651 0.024 0.754 0.061 5.310 1.000 0.217 1.000 17.586 0.086 a eg ea n afyon dinar 1.366 0.000 1.384 0.000 1.091 0.000 18.520 0.000 0.795 0.000 27.048 0.000 denizli honaz 1.590 0.000 1.303 0.000 1.649 0.000 21.059 0.000 0.261 0.177 26.635 0.000 muğla dalaman 0.599 0.000 0.667 0.000 0.942 0.001 15.399 0.000 0.559 0.000 30.582 0.000 aydın söke 0.697 0.000 0.630 0.000 1.530 0.000 9.643 0.005 0.626 0.000 30.444 0.000 manisa hacıhaliller 1.434 0.000 1.303 0.000 1.325 0.000 20.585 0.000 0.401 0.000 21.388 0.000 control 0,242 0.328 0.265 2.605 0.138 7.2 table 2. median level of detoxifying enzyme activities and p values (2 tailed) in culex pipiens populations collected from the mediterranean and aegean region, turkey. fig. 9. median remaining acetylcholinesterase inhibition rates of culex pipiens populations (population name abbreviations represented as: adana-karataş (ak), antalya-manavgat (am), burdur-gölhisar (bg), mersin-tarsus (mt), hatay-dörtyol (hd), afyon-dinar (ad), aydın-söke (as), manisa-hacıhaliller (mh), muğla-dalaman (md), denizli-honaz (dh). c: control) 415 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... söke, hacıhaliller and dinar and 0.85 in the dalaman populations. all of the aegean populations were in hw balance (p > 0.05). the kdr genotype, allele frequencies and fisher’s exact test results of cx. pipiens populations along the mediterranean and aegean region are given in table 3. no ace-1 mutation was found in either in the mediterranean or in the aegean cx. pipiens populations based on rflp analyses by using alu-i restriction endonuclease. sequence analysis results confirmed rflp results and did not detect any g119s allele in the study area. f290v mutation frequency was changed between 0–0.075% within the cx. pipiens populations collected from both the mediterranean and aegean region. genotypes and allele frequencies of f290v mutations are given in table 4. discussion the mediterranean and aegean regions are located in the west and south part of turkey and both have favourable climate conditions for mosquito survival. the availability of vast and fertile soils within this geographic area leads high agricultural activities therein. in addition to that, intensive human activity, table 3. genotypes, allele frequencies and exact test results of voltage gated sodium channel 1 gene region of culex pipiens populations in the mediterranean and aegean region, turkey (ss: homozygous susceptible; rr: homozygous resistant; sr: heterozygous resistant; se: standard error; wc: weir and cockerham value; asterisks represent statistical significance (p < 0.05)) region locality no genotypes allele frequency p se wc ss sr rr s r mediterranean adana-karataş 20 3 12 5 0.45 0.55 0.6538 0.0018 -0.1875 antalya-manavgat 20 7 1 12 0.375 0.625 0.000 0.000 0.8984 burdur-gölhisar 20 2 4 14 0.2 0.8 0.1269 0.0018 0.3968 hatay-dörtyol 15 4 5 6 0.43 0.56 0.2935 0.0025 0.3519 mersin-tarsus 20 9 4 7 0.55 0.45 0.0079* 0.0005 0.6122 aegean aydın-söke 20 0 4 16 0.1000 0.9000 1.0000 0.0000 -0.0857 muğla-dalaman 20 0 6 14 0.1500 0.8500 1.0000 0.0000 -0.1515 manisahacıhaliller 20 0 4 16 0.1000 0.9000 1.0000 0.0000 -0.0857 denizli-honaz 20 0 0 20 0.0000 1.0000 afyon-dinar 20 0 4 16 0.1000 0.9000 1.0000 0.0000 -0.0857 table 3. genotypes, allele frequencies and exact test results of voltage gated sodium channel 1 gene region of culex pipiens populations in the mediterranean and aegean region, turkey table 4. genotypes, allele frequencies and exact test results of f290v mutations of culex pipiens populations in the mediterranean and aegean region, turkey table 4. genotypes, allele frequencies and exact test results of f290v mutations of culex pipiens populations in the mediterranean and aegean region, turkey (ss: homozygous susceptible; rr: homozygous resistant; sr: heterozygous resistant; se: standard error; wc: weir and cockerham value; asteriks represent statistical significance (p< 0.05)) region locality no genotypes allele frequency p se wc ss sr rr s r mediterranean adana-karataş 20 20 0 0 1.000 0.000 antalya-manavgat 20 20 0 0 1.000 0.000 burdur-gölhisar 20 18 2 0 0.95 0,050 1.000 0.000 -0.0270 hatay-dörtyol 15 15 0 0 1.000 0.000 mersin-tarsus 20 18 2 0 0.95 0.05 1.000 0.000 -0.0270 aegean aydın-söke 20 17 3 0 0.925 0.075 1.000 0.000 -0.0556 muğla-dalaman 20 20 0 0 1.000 0.000 manisahacıhaliller 20 20 0 0 1.000 0.000 denizli-honaz 20 19 1 0 0.975 0.025 afyon-dinar 20 20 0 0 1.000 0.000 (ss: homozygous susceptible; rr: homozygous resistant; sr: heterozygous resistant; se: standard error; wc: weir and cockerham value; asterisks represent statistical significance (p < 0.05)) (ss: homozygous susceptible; rr: homozygous resistant; sr: heterozygous resistant; se: standard error; wc: weir and cockerham value; asteriks represent statistical significance (p< 0.05)) 416 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... tourism and industrialization induce wide spread of mosquito species and accordingly mosquito-borne diseases. since chemical insecticide-based control management is the primary method for mosquito control efforts in turkey, understanding the insecticide resistance levels of the mosquito populations and underlying mechanisms are crucial for the deployment of appropriate insecticides. in this study, insecticide resistance levels of cx. pipiens populations from the aegean and mediterranean region against various insecticides were investigated. results indicated that all of the populations were resistant to ddt even though ddt use was banned in the 1980s in turkey. ddt resistance has been recorded in many mosquito populations both in turkey and the world since it was used extensively in the fight against malaria until the 1960s (22-25). several researchers have previously reported that gsts that are involved in xenobiotic detoxification, some mfos that are capable of metabolizing ddt (for example cyp6z1) and a kdr mutation causing receptor structure change in voltage-gated ion channels are all responsible for ddt resistance (26-27). all of the mediterranean and aegean cx. pipiens populations showed significantly high gst activity profile except the antalya and gölhisar populations, indicating that ddt resistance is still maintained through increased gst activities in most of the populations. however, the manavgat and gölhisar populations support the hypothesis that more than one mechanism might play a role rather than the fact that gst alone is responsible for ddt resistance. increased mfo levels and l1014f allele frequency in the manavgat population indicates that ddt resistance might be maintained through high mfo levels and kdr mutation even if the gst level is not increased. similarly, increased mortality rate against ddt, permethrin and deltamethrin after treatment with piperonyl butoxide (pbo) in cx. pipiens populations of mersin demonstrates that mfo is responsible for both ddt and pyrethroid resistance of the mersin populations (11). interestingly, high ddt resistance seems to be sustained via l1014f mutation alone in the gölhisar population in which mfo and gst levels are not significantly increased. another interesting conclusion about the gölhisar population is that both kdr mutation and decreased mfo levels, which are thought to be responsible for pyrethroid resistance in addition to ddt resistance, which is still sensitive to permethrin and deltamethrin. in addition to the gölhisar population, when we consider the dinar and söke populations that are sensitive to pyrethroids and have high kdr frequencies because of the ddt resistance, the question of what is the usability of pyrethroids for control purposes comes to mind. allele specific primers indicated that mechanism causing kdr resistance of cx. pipiens populations in both the mediterranean and aegean regions is the increase of l1014f mutation frequencies. however, taşkın et al. (12) reported that the kdr resistance responsible for ddt and pyrethroid resistance is maintained by both l1014f and l1014c mutations in cx. pipiens populations collected from the aegean region. studies on kdr alleles responsible for ddt and pyrethroid resistance have been conducted in cx. pipiens populations obtained from many countries of the world. for example, both l1014f and l101c kdr mutations were detected in cx. pipiens populations in greece (28). additionally, high l1014f allele frequency was reported in cx. pipiens populations obtained from morocco and mohammadiye cities of morocco (29). the kdr mutation is maintained by l1014s allele in cx. pipiens quinquefasciatus populations obtained from some parts of china (30) by l1014f allele in cx. pipiens populations of new jersey (31) and by l1014c in cx. pipiens populations of china (32). following the occurrence of ddt resistance, in the 1970s, cb and op insecticides such as malathion, fenitrothion, and bendiocarb and propoxur began to be applied instead of ddt in the mosquito control studies (33). bioassay results showed that all cx. pipiens populations from the mediterranean and aegean region are 417 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... resistant to both op and cb insecticides. this situation can be explained by the intensive use of op and cb insecticides, especially malathion, in the control of agricultural pests and as a result, creates a high selection pressure in agricultural areas (34). similar to turkish cx. pipiens populations, several researchers from neighbouring countries such as iran, russia and greece reported high insecticide resistance against different insecticides. for instance, kioulos et al. (2014) reported temephos and deltamethrin resistance in some parts of greece (35). fenitrothion, ddt, dieldrin, propoxur, bendiocarb, malathion, deltamethrin and permethrin resistance has been reported in cx. pipiens populations collected from the centre of moscow (36). resistance t oddt, malathion, bendiocarb, propoxur, fenitrothion, deltamethrin, permethrin, lamda-cyhalothrin, etofenprox and cyfluthrin has been reported from iranian cx. pipiens populations (37). the significant increase in esterase enzyme activity in all cx. pipiens populations of the mediterranean region except the manavgat population, indicates that op and cb resistance is maintained with general esterases in populations of this species. regarding op ve cb resistance, molecular and biochemical data results were consistent with each other. ache insensitivity was generally lower along populations except the adana karataş, mersin tarsus, muğla dalaman and aydın söke populations which had slightly high remaining ache activity. consistently, pcr-rflp did not detect any g119s allele in any of the populations indicating that ace-1 g119s mutation is not responsible for the op and cb resistance in cx. pipiens populations. additionally, op and cb resistance could not be explained by ace-1 f290v mutations since the frequency of f290v mutation was also too low. several researchers reported that individuals carrying ace-1 mutation (g119s and f290v) have a fitness cost including longer period time, reduced owerwintering survival, smaller adult size and increased risk of predation (38-41). this might explain why culex pipiens populations in the study area had low ace-1 (g119s and f290v) mutations. it has been reported that fitness cost might be diminished by duplication of the ace-1 gene in some culex pipiens populations (42-43). however, we were lack of duplication data set in that study. similarly, g119s mutation frequency was found to be 0.11 and 0.08 and f290v mutation frequency was found to be 0.05 and 0.06 in cx. pipiens populations obtained from the aegean and marmara regions of our country in 2012 and 2013, respectively (12). the ace-1 (g119s and f290v) mutations leading to insensitivity to organophosphates and carbamates were detected at low frequencies in cx. pipiens populations in greece (28). in addition to that, in cx. pipiens populations obtained from urban and rural populations of morocco, g119s mutation was found to be at a higher frequency in urban areas compared to rural areas due to temephos (op) used to fight cx. pipiens larvae. however, the absence of g119s mutation in some individuals who did not die after being treated with op shows that the only mechanism underlying op resistance is not the g119s mutation (44). similarly, tmimi et al. (2018) found that the g119s mutation frequency was very low in cx. pipiens populations of morocco (29). this study demonstrates that multiple insecticide resistance exists in cx. pipiens populations from the mediterranean and aegean regions of turkey. medium to high kdr (l1014f) mutation frequency and extremely low f290v mutation frequency detected in these populations. however, no g119s mutation was detected within these populations as well as low ache activity levels have been detected. effective implementation of insecticide resistance management strategies is needed in order to delay the fixation of resistance alleles currently occurrs within these populations. the data obtained from this study will be valuable for vector control interventions in turkey. 418 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 405–420 si yavaşoğlu and fm şimşek: insecticide resistance... conclusion understanding the resistance mechanisms and monitoring resistance patterns of the populations regularly are crucial for insecticide resistance management. we used both who’s bioassay tests, biochemical assays and molecular markers at the same time to evaluate the insecticide resistance status and underlying mechanisms for the first time for cx. pipiens populations collected from the mediterranean and aegean region of turkey. the mediterranean and aegean regions are important agricultural regions as well as high tourism activities. as a result of high insecticide use in these regions, we hypothesised the occurrence of high resistance status against different classes of insecticides which have been used until today. we showed the complicated role of detoxification enzymes. however, one of an important limitation of our study was the absence of synergist assays to get more reliable data set regarding the biochemical mechanisms. we also detected mild to high frequency of kdr l1014f allele frequency as a result of ddt and/or pyrethroid use. however, we did not detect any other kdr alleles probably because of the restricted ability of allele-specific primers to detect different kinds of alleles. interestingly, we did not detect ace-1 g119s allele in any populations. furthermore, we detected too low ace-1 f290v mutations and insensitive acetylcholinesterase levels in some of the populations. further studies with higher sample sizes are needed to establish insecticide resistance profiles in order to evaluate more accurately and avoid resistance problems before it is spread to the whole mosquito populations. acknowledgements this research was funded by the scientific research department of aydın adnan menderes university (project number: bap-fef-15005). we are also grateful to professor oğuz türkozan for proofreading for his support in statistical analyses. ethical approval this article does not contain any studies with animals performed by any of the authors. declaration of competing interest the authors declare that they have no conflict of interest. references 1. benelli g , jeffries c l , walker t(2016) biological control of mosquito vectors: past, present, and future. insects. 7(4):52. 2. caraballo h, king k(2014) emergency department management of mosquito-borne illness: malaria, dengue, and west nile virus. emerg med pract. 16(5):1-23. 3. world health organization (2012) global plan for insecticide resistance management in malaria vectors (gpirm). geneva. 4. hemingway j(2014) the role of vector control in stopping the transmission of malaria: threats and opportunities. philos trans r soc lond b biol sci. 369(1645):20130431. 5. brugman va, hernández-triana lm, medlock jm , fooks ar, carpenter s, johnson n(2018) the role of culex pipiens l. 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insecticide resistance and mechanisms of culex pipiens populations in the mediterranean and aegean r abstract keywords introduction materials and methods mosquito strains bioassays biochemical assay molecular assays statistical analyses results bioassay biochemical analysis results molecular test results discussion conclusion acknowledgements ethical approval declaration of competing interest references j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 35 original article aerobic bacterial community of american cockroach periplaneta americana,a step toward finding suitable paratransgenesis candidates sanaz akbari 1, *mohammad ali oshaghi 2, saedeh sadat hashemi-aghdam 3, sara hajikhani 4, ghazaleh oshaghi 5, mohammad hasan shirazi 4 1department of microbiology, islamic azad university, damghan branch, damghan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of animal science, islamic azad university, damghan branch, damghan, iran 4department of pathology, school of public health, tehran university of medical sciences, tehran, iran 5department of nutrition, national nutrition and food technology research institute (nnftri), faculty of nutrition sciences and food technology, shahid beheshti university of medical sciences, tehran, iran (received 29 oct 2013; accepted 16 apr 2014) abstract background: cockroaches mechanically spread pathogenic agents, however, little is known about their gut microbiota. identification of midgut microbial community helps targeting novel biological control strategies such as paratransgenesis. here the bacterial microbiota of periplaneta americana midgut, were identified and evaluated for finding proper paratransgenesis candidate. methods: midgut of specimens were dissected and cultivated in different media. the bacterial isolates were then identified using the phenotypic and 16s-rrna sequencing methods. results: the analytical profile index (api) kit showed presence of 11 bacterial species including: escherichia coli, shigella flexineri, citrobacter freundii, e. vulneris, enterobacter cloacae, yersinia pseudotuberculosis, y. intermedia, leclericia adecarboxylata, klebsiella oxytoca, k. planticola, and rahnella aquatilis in the cockroach midguts. the first three species are potentially symbiotic whereas others are transient. the conventional plating method revealed presence of only four isolates of salmonella, e. coli, and proteus which in three cases mismatched with api and 16s-rrna genotyping. the api correctly identified the four isolates as shigella flexneri, citrobacter freundii, and e. coli (n= 2). 16s-rrna sequence analysis confirmed the api results; however the c. freundii sequence was identical with c. murliniae indicating lack of genetic variation in the gene between these two closely related species. conclusion: a low number of potentially symbiotic bacteria were found in the american cockroach midguts. among them enterobacter cloacae is a potential candidate for paratransgenesis approach whereas other bacteria are pathogens and are not useful for the approach. data analysis showed that identification levels increase from the conventional to api and to genotyping respectively. keywords: periplaneta americana, midgut bacteria, enterobacter cloacae, 16s rrna, analytical profile index (api) kit introduction cockroaches are one of the most important insects in medicine. they inhabit in the dirty environment such as home sewage and could contaminate human foods with pathogenic agents. they can spread germs mechanically as they move freely from areas that may harbor pathogenic organisms: for example, from sewers to food or food preparation surfaces. a number of cockroaches have become pests and live in or around homes where they are omnivorous scavengers. the two most significant pest cockroaches worldwide are the german cockroach blattella germanica and the american *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 36 cockroach periplaneta americana. german cockroach is more common inside homes particularly in kitchens while the later is found around the home close to water pipes and drainage systems (bell et al. 2007, hashemi-aghdam and oshaghi 2014). the presence of the american cockroach, in human dwellings causes damage and distress worldwide. cockroach body has bad smell secretions, which are one of the sources of allergens responsible for asthma (arruda et al. 2001, litonjua et al. 2001, arlian 2002). a numerous pathogens counting 32 species of bacteria (including salmonella and shigella species), 15 species of fungi and moulds, 7 helminths (intestinal parasites), 2 protozoans, and 1 virus which are all harmful to humans being found in or on cockroaches or in the faeces (mille and peters 2004, pai et al. 2005, zarchi and vatani 2009, allotey et al. 2009). intestinal microbial communities of some omnivorous cockroaches such as p. americana, and b. orientalis (blattidae), b. germanica (ectobiidae), and eublaberus posticus (blaberidae) have been examined using cultivation-based studies (burgess et al. 1973, cruden and markovetz 1984, 1987, tachbele et al. 2006, vahabi et al. 2007, akbari et al 2014). however, a comprehensive analysis of the midgut microbiota of the american cockroach is so far lacking. the only cockroach whose gut microbiota has been characterized with cultivation-independent molecular methods is turkistan cockroach shelfordella lateralis (schauer et al. 2012). traditionally, control of cockroaches relies on application of different classes of insecticides which is often associated with environmental toxicity, adverse effects on human health and the emergence of insect resistance (limoee et al. 2006, enayati and motevalli-haghi 2007). a new control strategy named paratransgenic, symbiotic or commensally microbes of host insects are transformed to express gene products that reduce fitness of insect or interfere with pathogen transmission (hurwitz et al. 2011, chavshin et al. 2012, wang and jacobs-lorena 2013). these genetically modified microbes are reintroduced back to the insect where expression of the engineered molecules decreases the host's fitness or ability to transmit the pathogen. recently, jiang et al. (2007) successfully utilized this strategy to develop an engineered densovirus (pfdnv) to control the smoky-brown cockroach (periplaneta fuliginosa) nymphs. identification of a causative pathogen is essential for the choice of treatment as well as evaluation of the presence of virulence factors and antibiotic resistance determinants for most infectious diseases. methods for the identification and discrimination of bacterial isolates can be separated into traditional and molecular groups (nazarowec-white and farber 1999). the traditional microbiological techniques (phenotyping) are based on secondary characteristics of bacteria including staining, cultures, biochemical reactions, antibiograms, serotyping, and bacteriophage typing. phenotypic methodologies still play a significant role in identifying, verifying, and providing antibiotic susceptibility testing for many microbial pathogens. however, the application of molecular techniques to microbiology has led to the development of new and rapid methods for the detection, identification and characterization of many microorganisms including bacteria. these applications are stepwise replacing or complementing phenotypic assays in microbiology laboratories (weile and knabbe 2009). the most common molecular typing methods used in microbiology include chromosomal dna restriction analysis, plasmid typing, ribotyping, pulsed-field gel electrophoresis (pfge) and pcr-based methods such as randomly amplified polymorphic dna (rapd) typing, restriction fragment length polymorphisms (pcr-rflp), and 16s-rrna sequencing (eisenstein 1990, grant and kroll http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 37 1993, maslow et al. 1993, farber 1996, krishna and cunnion 2012, chavshin et al. 2012). other molecular techniques such as whole or partial genome sequencing, real-time pcr, microarrays are also being used (weile and knabbe 2009, krishna and cunnion 2012). the rdna gene sequences are highly conserved within living organisms of the same species, but that they differ between organisms of other species. 16s-rrna is commonly used for taxonomic studies in bacteria because it is present in almost all bacteria, its function over time has not changed, and is large enough for informatics purposes (barney et al. 2001, harmsen and karch 2004, janda and abbott 2007, woo et al. 2008). in this study we analyzed the bacterial flora of american cockroach midgut and evaluated their possible usefulness as proper candidate for paratransgenesis approach. to identify the potential symbiont bacteria two routinely used commercial phenotypic methods namely the traditional methods (including staining, cultures, and biochemical reactions) and analytical profile index (api) biochemical fingerprinting kit compared to genotyping (16s-rrna sequencing) as a reference method. materials and methods isolation and purification of cockroaches’ midgut bacteria adult american cockroach specimens were collected alive from underground of households and confectionary premises in center of tehran, iran. specimens were trapped using small box or food-baited traps and transferred to the laboratory of school of public health, tehran university of medical sciences (sph-tums), iran. the specimens were kept in freezer about 5 min until they became immobilized. before dissection, the specimens were surface sterilized for 2 min in 70% ethanol. bacterial isolation was conducted in a sterile environment on a sterile petri-dish. the legs were then removed, and the alimentary canal was exposed by making a ventral incision extending from the terminal sternum to the prothorax. the intact midgut from each specimen was dissected and transferred to sterile tubes separately. to separate transient bacteria from potential symbiotic bacteria, the midgut specimens were divided randomly into three following groups before homogenization: 1) intact midgut with its contents 2) removing midgut contents without washing, and 3) removing midgut contents with three times washing by pbs and distilled water. phenotypic identification traditional method the midguts were homogenized with glass pestles in 200 μl pbs buffer. homogenized midguts from p. americana adults were poured in a 1.7 ml micro tube containing bhi broth and incubated at 37 °c for 24 h. the media was serially diluted in sterile pbs, and 0.1 to 0.5 ml of each dilution was spread on plates of four different media including brain heart infusion broth (bhi broth), bhi agar, mc conkey agar and blood agar (merck, germany) media. spread plates were placed in incubator until colonies developed (24 h). the colonies with different phenotype were selected for further characterization and were streaked for purification onto fresh plates of the same medium from which they had been picked. colonies grown on the media were purified by several sub-cultures and then were stored at 4 ºc for further analysis. a test tube containing bhi agar open near the dissection area constituted our sterility control during the dissection process. besides, we used a laboratory strain of escherichia coli as reference bacterium in the study. characteristics of the bacteria were evaluated by routine microbiological methods. cell morphology such as size, pigment, smoothness of colony’s surface, having reghttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 38 ular or irregular edges and hemolysis was observed by photonic microscopy after gram staining. identification of bacteria to genus level was performed by using biochemical tests (motility, utilization of citrate, indole formation, lysine degradation, sh2 production and fermentation carbohydrate) according to methods described by sneath (1984). results of biochemical tests were compared with the test results with dichotomous keys. api biochemical fingerprinting kit this phenotypic identification was carried out using the api test kit of ds-difentro-24 (microgene) which is commercially available in iran as described by maleki-ravasan et al. (2013). prior to testing, isolates were fresh cultured overnight at 37 °c on blood agar. suspension was prepared from the colonies with the turbidity equivalent to 2 mcfarland and injected to the wells, incubated for 24 h at 37 °c and reagents was added. positive and negative results using created color changes were determined based on api kit protocol. tests were carried out according to manufacturers’ instructions and results were interpreted using the appropriate laboratory computer software or reference indices recommended by the manufacturer. bacteria can be identified to species level and sometimes to subspecies level. genotypic identification genomic dna from fresh colonies incubated overnight in liquid cultures in nutrient broth was extracted using boiling method. for genotyping, a 1500bp of 16s rrna gene was amplified with specific primers according to weisburg et al. (1991) protocol. amplification was carried out on the isolates with the following pcr cycling conditions: an initial denaturation at 94 °c for 10 min, followed by 30 cycles of denaturation at 94 °c for 30 s, annealing at 56.5 °c for 40 s, and extension at 72 °c for 40 s, and final extension at 72 °c for 10 min (karimian et al. 2011). dna sequencing the pcr products of the isolates were sequenced by seqlab, germany using the amplification primers. consensus sequences obtained from forward and reverse sequences and their homologies with the available sequence data in genbank were tested by using the basic local alignment search tool (blastn) alignment program and the ncbi nucleotide database ncbi (www.ncbi.nlm. nih.gov/blast). results phenotypic identification totally 45 adult american cockroaches were collected from the premises during the may-august 2011. the mid-guts of the specimens were homogenized individually and cultured in different media as described in m and m section. totally 20 purified bacterial colonies in different media cultures were isolated from the midgut of the adult american cockroaches. these colonies were identified as escherichia coli (n= 11), salmonella (n= 7), and proteus (n= 2) using the traditional methods. the classical method also identified the e. coli reference strain correctly. details of biochemical characters used for classical identification of the isolates are shown in table 1. escherichia coli and salmonella were the most prevalent isolates. when the isolates were tested using api kit, the number of genus and species greatly raised and the specimens were categorized in eight genera of escherichia, citrobacter, shigella, yersinia, klebsiella, rahnella, enterobacter, and leclercia (table 2). the api biochemical analysis revealed presence of 11 bacterial species in the midgut of the american cockroaches, however, the bacterial community http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 39 was varied in the number and composition when the midgut tested with or without its contents and or before and after washing with pbs. the number of species was seven, five, and three when the midgut tested with its contents, without its contents and no washing, and without its contents plus washing with pbs respectively (table 2). escherichia coli, shigella flexneri, and citrobacter freundii were present before and after washing the midgut contents. the isolates that were present in the midgut after washing seem to be potentially symbiotic since the transient bacteria normally would wash out and the remaining presumably are symbiotic. genotypic identification and its congruence with phenotypic as previously indicated the classical bacteriological method identified the midgut bacteria as salmonella, proteus and e. coli. the classical method also identified the e. coli reference strain correctly (table 1). we selected a single colony of proteus, salmonella (positive to citrate), salmonella (negative to citrate), and e. coli for further api biochemical fingerprinting and genotyping. the api kit using 24 biochemical tests (table 3) identified the four isolates as s. flexneri, e. coli (n= 2), c. freundii, and the e. coli reference strain. this result was not in accordance with the traditional method in three out of four cases. the two salmonella isolates were identified as s. flexneri and c. freundii, and the proteus isolate identified as e. coli (table 4). then the four isolates plus the reference strain of e. coli were analyzed by genotyping using 16s rrna pcr-direct sequencing. dna extractions from the isolates contained enough bacterial dna for pcr amplification. all samples except negative control produced visible pcr products of about 1500 bp whereas the negative controls were blank. the four isolates that were biochemically (api kit) identified as s. flexneri, c. freundii and e. coli (n= 2) respectively, were identified accordingly as s. flexneri, c. murliniae/ c. freundii, and e. coli (n= 2) based on partial sequences of 16srrna (table 4). their sequences with length of 766, 766, 764, and 768 bp were deposited in genbank with accession numbers of kc 017349, kc017346, kc017348, and kc017 347 respectively. the correspondences of the phenotypic and genotypic methods as well as details of the homologous species and their homology percentage of the species identified in this study with the ones available in genbank are shown in table 4. table 1. details of biochemical characters used for classical identification of bacteria in the american cockroach midgut species biochemical characters sim kia and gas production lysine h2s citrate indole motility salmonella + r/y + + + e. coli + + y/y + + proteus + r/y + salmonella + r/y + + + e. coli reference strain + + y/y + + http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 40 table 2. aip phenotypic identification of bacterial community of american cockroach midgut before and after washing and removing contents intact removing contents without washing removing contents with three times pbs washing bacterial species escherichia coli escherichia coli escherichia coli shigella flexineri eischercia vulneris shigella flexineri citrobacter freundii klebsiella planticola citrobacter freundii yersinia pseudotuberculosis enterobacter cloacae yersinia intermedia rahnella aquatilis klebsiella oxytoca leclericia adecarboxylata table 3. details of reaction tests used in api kit for species identification of four bacteria isolated from midgut of the american cockroaches results of reactiontest reactionno. ++--indole1 +++-vp2 --+-citrate3 ----sh24 ++++urease5 ---+phenylalanine6 ++--laysine7 ++--argentine8 ----ornitine9 ++--malonate10 ----glocuse11 ----adonitole12 ----arabinose13 --++dolicitole14 ----inositole15 ++++lactose16 --++maltose17 ++++mannitol18 ++++ramnose19 ----sucrose20 ++++surbitole21 ++++trehalose22 ++--eskoline23 ++--onpg24 e.colie.colicitrobacter freundiishigella flexneriidentified species table 4. details of congruence between phenotypic and genotypic methods in diagnosis of bacteria isolated from the american cockroach midgut. an: accession number classic api 16s rrna sequencing (a.n in genbank) homolog species (a.n in genbank) homology rate % salmonella shigella flexneri s. flexneri (kc017349) s. flexneri (hq701686) s. flexneri (cp001383) s. flexneri (cp001386) 100 100 100 proteus escherichia coli e. coli (kc017348) e. coli (cp003034) e. coli (jn578644) s. sonnei (nr074894) 99 99 99 e. coli e. coli e. coli (kc017347) e. coli (jq609683) e. coli (cp001925) e. coli (cp002967) e. coli (cp002970) e. coli (cp002291) 100 100 100 100 100 salmonella citrobacter freundii c. murliniae (kc017346) c. murliniae (jn092600) c. freundii (jx860618) 100 100 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 41 discussion the results of this study using api kit revealed that the intact midgut of p. americana harbors fairly a diverse community of gram negative aerobic bacteria of enterobacteriaceae. however, most of these bacteria were transient and acquired from the environment and or the food sources the cockroaches live and feed on as previously described by kane and breznak (1991). therefore the non-transient bacteria comprise small part of the midgut community. the low number of non-transient (possibly symbiotic) microbial community of the american cockroaches found in this study concord to earlier perceptions of the cockroach midgut microbiota, which were based on cultivation-based studies that yielded mostly isolates of the genera enterobacter, klebsiella, and citrobacter (cruden and markovetz 1987). it is worth mentioning that since the bacterial community obtained by culture media is limited by the selectivity of the media employed, the species (number and diversity) estimated in this study are not definitely estimates of the total real community. initial using of nonselective medium (bhi broth) to promote growth of bacteria generally favored the growth of gram negative enterobacteriaceae in the media. however, it is the case for almost all of the studies analyzing the insect gut bacterial communities (hillesland et al. 2008, mukhopadhyay et al. 2012, chavshin et al. 2012). even the studies that have implemented molecular tools used these tools only in the identification and analysis of isolated pure colonies from plate culture, not in the initial isolation of bacteria from the guts (gouveia et al. 2008, hillesland et al. 2008). the molecular tools used in both studies were implemented in the identification of bacterial colonies obtained by culturing, thereby limiting the findings to the small proportion of cultivable microbes. taking into account the limitations of culture dependent techniques makes these findings incomplete. the microbial community of different compartments of intestinal tract of cockroaches including crop, midgut, rectum and colon of p. americana and shelfordella lateralis (turkistan cockroach) previously have been compared using culture media or molecular tools (bignell 1977, bracke et al. 1979, schauer et al. 2012). these studies showed that each compartment harbor diverse community of bacteria, where the anterior colon of the cockroaches contained the highest abundance of microorganisms. the diversity in different compartments of cockroach gut is related to the microbial activities, such as the accumulation of hydrogen and the other microbial products, and the physiochemical characteristics of each part of the gut, such as ph and redox potential (schauer et al. 2012 and references herein). previous studies of cockroaches have reported a decrease in redox potential along the gut, with oxidizing conditions in crop and midgut and reducing conditions in the hindgut. the low redox potential in the hindgut lumen is consistent with the accumulation of hydrogen and the presence of a large and diverse community of clostridiales (bignell 1977, vinokurov et al. 2007, schauer et al. 2012). besides, it is shown that in p. americana, the foregut is a site of considerable lactate production owing to the abundance of lactic acid bacteria (kane and breznak 1991). in this study we focused only on the midgut of the american cockroach because the midgut has an endodermic origin which does not destroy in molting (ecdysis) and hence its microbial community remains intact or is less prone to diminish during multiple molting of cockroach life span. this fact is important for selection of a http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 42 proper candidate bacterium for paratransgenesis approach. in the present study enterobacter cloacae was found among the transient bacteria after removing midgut content but did not remained after washing with pbs. however, this bacterium could be a potential candidate for paratransgesis approach because it is found as the normal gut flora of many humans and is not usually a primary pathogen (keller et al. 1998). enterobacter cloacae have already been used for paratransgenesis and successfully could deliver, express, and spread foreign genes in termite colonies (husseneder and grace 2005) and sand fly phlebotomus papatasi (maleki-ravasan et al. 2014). this species was genetically transformed with defensin (small cysteine-rich cationic proteins found in both vertebrates and invertebrates) to reduce leishmania parasites in vitro conditions (maleki-ravasan et al. 2014). enterobacter cloacae also has been transformed with an ice nucleation gene to reduce the mulberry pyralid moth, glyphodes pyloalis (watanabe et al. 2000). these documents show that the species is amenable for transformation with foreign genes. in this study we also found that e. coli is possibly a symbiotic bacterium in the midgut of american cockroaches. this bacterium was genetically manipulated and tested for paratransgenic approach (riehle et al. 2007, chavshin et al. 2013). however e. coli, and other two potential symbiotic bacteria s. flexneri and c. flexineri are not appropriate candidate for paratransgenesis approach due to their pathogenic effect that can cause diarrhea or meningitis in humans (badger et al. 1999, niyogi 2005). further investigation need to test the utility of e. cloacae or to find another appropriate symbiont for genetic manipulation and delivering effector molecules to control and diminish cockroach pest populations. the results of this study showed the importance of choosing the correct identification method for exact speciation of bacterial species. correct identification impacts directly on treatment outcomes and on the epidemiological analysis of emerging bacterial infections in arthropod borne diseases. the present study revealed that 75% of classical test systems including staining, cultures, and biochemical reactions, yielded wrong speciation results when compared to api kit and genotyping (table 3). for example, classical method identified two isolates as salmonella, however these two isolates were subsequently identified by api and genotyping as s. flexneri and c. murliniae which could mislead the diagnostician and subsequent treatment methods. on the other hand, species classification based on phenotypic features is often time-consuming and is not always easy to carry out (springer et al. 1995, nagy et al. 2006, erme et al. 2009). the classical methods cannot detect the heterogeneity in species, are not reproducible, and challenge with limited database for phenotypic characteristics for common species. common strains are easily identified with charts or keys but when we are facing to rare or intermediate strains, identification is difficult with these tools (barkeley et al. 1984). inability of classical method in recognition of some bacterial species is result of phenotypic variation, phenotypic homogeneity without enough differential characteristics and tendency of traditional method toward established taxa. also some species are complex (phenospecies) and have more than one dna group then classic method cannot separate them phenotypically (janda and abbot 2007). besides, some bacteria particularly anaerobic ones are extremely slow growing or not cultivatable at all. one of the arguments for using classical tests is that they are less costly than api and genotyping, however, the potential penalty of misidentification must be considered. on general there was a great congruence among the results of api and 16s rrna http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 43 genotyping: both methods identified the four selected isolates as s. flexneri, e. coli (n= 2), and c. freundii. however, the 16s rrna sequencing revealed possible mismatching of c. freundii with c. murliniae because both species had identical sequences in the region sequenced. studies of geraghty et al. (2013) on the efficacy of routinely used phenotypic methods compared to genotypic approaches for the identification of staphylococcal species showed that the api staph 32 kit correctly identified all s. aureus isolates (11/11), but 83% (10/12) of the sig species, and 66% (19/29) of the coagulase negative species. they concluded that although the api staph 32 test performed with the highest degree of accuracy for the coagulase positive staphylococci, however they are inadequate for the correct identification of both coagulase negative and coagulase positive staphylococcal species. the api kits are produced for the human diagnostics market and are interpreted against databases with reference strains of human origin. therefore it is suggested that the reproducibility and reliability of these kits are uncertain when applied to none human origins. the api test is based on the evaluation of expression of genetically encoded characteristics and erroneous identification may be due to variable expression of biochemical traits within species (blaiotta et al. 2010). other disadvantages of api kits included long incubation period (18 h), lack of the required turbidity in suspension produced, bubbles in kit’s well, incorrect incubation period which leads to error while reading the results of the color changes. also it is necessary that the databases identification kits updated by their manufactures to include new genus and species (laclaire and facklam 2000). however, the biochemical api kit is less expensive than the molecular method and is more suits for most small clinical laboratories where the implementation of advanced molecular techniques is not feasible owing to the high cost of instrumentation and reagents. nowadays, dna based methods, particularly pcr techniques, for the detection and characterization of microorganisms have revolutionized diagnostic microbiology and are now part of routine specimen processing. these methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterization by genotyping (mellmann et al. 2003, woo et al. 2003, 2008, cloud et al. 2004, speers 2006, sibley et al. 2012). literature show that 16s rdna sequencing is essential and key tool for bacterial identification, particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow growing bacteria, uncultivable bacteria and culture-negative infections (clarridge 2004, woo et al. 2008). there are many conserved primers to amplify a region of a gene, such as the 16s rrna bacterial gene, and the amplified product is usually sequenced and compared to about 4 million 16s rna sequences from different bacteria in internet databases such as genbank (www.ncbi. nlm. nih.gov/genbank), embl data library (www. ebi.ac.uk/embl), and the dna data bank of japan (www.ddbj.nig.ac.jp) with daily data exchange between them, and more specialized high quality databases such as ridom (www.ridom-rdna.de/) for bacterial rdna sequences used for mycobacterial speciation (speers 2006). in spite of above mentioned advantages, experience showed that 16s rrna gene sequences is able to identify most cases (>90%) at genus level but in species level decrease to 65 to 83% and 1 to 14 % of isolates remains unidentified (mignard and flandrois 2006). some other limitations are 1) identifying of new taxa is impossible, 2) limited number of sequences or partial sequences existence in the nucleotide database, 3) similarity of 16s rrna gene sequence in some species, 4) error in nomenclature species http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 35–48 s akbari et al.: aerobic bacterial community … 44 and 5) use of sequences with gap to comparison (hall et al. 2003, bosshard et al. 2006). conclusion this study suggests that midgut of american cockroaches harbor few symbiotic bacterial community at least in case of aerobic gram 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bacterial isolates with ambiguous biochemical profiles. j clin microbiol. 41: 1996–2001. woo pc, lau sk, teng jl, tse h, yuen ky (2008) then and now: use of 16s rdna gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. clin microbiol infect. 14(10): 908–934. zarchi aa, vatani h (2009) a survey on species and prevalence rate of bacterial agents isolated from cockroaches in three hospitals. vector borne zoonotic dis. 9(2): 197–200. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 324 http://jad.tums.ac.ir published online: september 30, 2019 original article characterization of glycoproteins of native 19kda c-terminal merozoite surface protein-1 from native antigen of plasmodium falciparum sahar tajik1; sedigheh sadeghi2; ayda iravani2; mitra khalili1; mohammad arjmand2; nassir-ud din3; farideh vahabi2; hossein feiz-haddad4; behzad lame-rad1; saied reza naddaf5; *zahra zamani2 1department of biochemistry, payame-noor university, tehran, iran 2department of biochemistry, pasteur institute of iran, pasteur avenue, tehran, iran 3institute of bioinformatics, lahore, pakistan 4department of parasitology, ahwaz medical university, ahwaz, iran 5department of parasitology, pasteur institute of iran, tehran, iran (received 25 july 2016; accepted 21 sep 2019) abstract background: plasmodium falciparum is the protozoan parasite which causes malignant malaria of medical concern. prime candidates for recombinant vaccine development are asexual stage antigens of p. falciparum, for example, merozoite surface proteins (msp1 and msp2) not given satisfactory results to date. in this study, the 19kda c-terminal of msp1, a vaccine candidate was purified in its native form in the ring stage, and its glycoproteins studied. methods: the study was carried out at the biochemistry department of pasteur institute of iran in the years 2015–2016. large scale culture of p. falciparum was performed in vitro with 80% ring stage parasitemia. isopycnic ultracentrifugation with 36% sucrose and analytical sds-page on the supernatant and precipitate performed, and the 19kda antigen was obtained by cutting it from strips of preparative sds gels. purified protein was concentrated and analyzed by sdspage and immunoblotting, using antibodies raised to recombinant c-terminal msp1. results: the purified protein gave a single band of 19kda antigen as shown by silver staining of sds-page and a single bond in immunoblotting. bioinformatics also confirmed the likelihood of the presence of glycans on the antigen. conclusion: the presence of n and o-glycoproteins were detected by q proteome kit. this work was done on the ring stage, and earlier workers confirmed the presence of glycoproteins on msp1 in the other stages. this glycosylation is present in all stages, and maybe incomplete protection elicited by recombinant msp1 antigens is due to lack of n and o-glycoproteins. keywords: merozoite surface protein1; c-terminal 19kda; plasmodium falciparum; glycoproteins introduction plasmodium falciparum is one of the causative agents of human malaria, a disease which accounts for almost 900000 deaths per year, the significant portion of whom are children and pregnant women in developing countries according to the malaria fact sheet (1). today, many investigations aim to achieve more efficient medications to conquer drug-resistant malaria parasites or to find new candidate proteins to develop vaccines for disease prevention in endemic areas (2). the biology of p. falciparum has been studied to figure out its interactions with the host. in all stages of the parasite life cycle, carbohydrate-recognizing proteins play a major role in virulence of the disease and its severity (3). current vaccine approaches focus on recombinant proteins or synthetic peptides of the asexual stage. some synthetic peptides have reached the clinical trials stae and seem to be promising, but until today, the lack of success with recombinant vaccines could be due to lack of their glycosylation (4). vaccine research on blood-stage malaria has concentrated on antigens expressed on the surface of merozoites. red blood cells (rbcs) are ruptured, releasing merozoites that quickly in*corresponding author: dr zahra zamani, e-mail: zamani@pasteur.ac.ir, zamanizahra786@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 325 http://jad.tums.ac.ir published online: september 30, 2019 vade other rbcs. the specific antibodies raised against merozoite surface proteins (msps) have only a short time to counteract with the target sites before parasites invasion to red blood cells. the most widely studied merozoite surface protein 1 (msp1) is polymorphic and has a complicated folding pattern. plasmodium falciparum msp1 has a molecular weight of (200kda) and is processed into a complex of polypeptides on the merozoite surface, with an 82kda n-terminal polypeptide and 30kda and 38kda central regions, along with the 42kda c-terminal region (msp1 42). at the time of rbc invasion, msp1-42 is processed, by proteolytic cleavage, into a 33kda fragment (msp1 33) and shed from the parasite with the rest of the msp1 complex, and a c-terminal 19kda fragment (msp1-19). only the c-terminal msp1-19 portion remains on the surface and enters into the infected rbcs, this processing is suggested to be a critical step in the successful attack of parasites to rbcs (5). the presence of glycosylation on the surface proteins of merozoites is still controversial, with studies showing different results, in particular on the msp1 19kda which is a vaccine candidate (6, 7). the extended range of cysteinerich, glycan-recognizing proteins at all stages of the parasite lifecycle play essential roles in cell attachment and invasion (3). carbohydrates (with terminal α-d-galactosyl residues) attached to malaria parasite proteins are suitable antigenic determinants and that they are recognized by the host's immune system (8). it is possible that the recombinant antigens used in the vaccines lacked glycosylation, which could be responsible for their failure in eliciting immunity. the presence of n and o-glycans on the msp1 antigen is still controversial though some workers have shown its presence in the schizont and trophozoite stages. in this study, glycosylation was investigated in the native form of 19kda c-terminal of the msp1 antigen in the ring stage using lectin affinity kits for detection of total and o-glycans along with bioinformatic work to verify the results. materials and methods the study was carried out at the biochemistry department of pasteur institute of iran in the years 2015–1016. plasmodium falciparum 3d7 was cultured by the method of trager and jensen (9) using 7ml rpmi (gibco, in vitrogen ab, sweden) medium containing 5% hematocrit and 10% human ab negative serum with 0.1% gentamycin in a mixture of (92% n2, 5 % co2, 3% o2) in a 25ml flask. the medium was changed every 48h, and the growth of the parasite was monitored by examination of giemsa-stained smears under an oil-immersion objective lens. synchronization of parasites: an equal volume of 5% sorbitol (sigma-aldrich, st louis, missouri, united states) was added to the parasite pellet followed by incubation for 10min at room temperature. the cultures were centrifuged and washed twice with rpmi and diluted to 5% hematocrit (10). all reagents used were sigma-aldrich unless stated otherwise. the collection of ring-stage parasites: free parasites were obtained by adding 40 times the volume of 0.15% saponin at 4 °c for 30min. the cells were centrifuged at 1300rpm at 4 °c for 20 min. the pellet was washed with pbs and centrifuged at 4000rpm 3 times for 1min and stored at -20 °c. large scale production of parasites: the method of radfar et al. (11) was adopted in which about 10% parasitemia was transferred to a 75ml flask with 1% hematocrit with daily changes of medium. when the parasites reached 20%, they were transferred to a 150ml flask with 1% hematocrit followed by synchronization described above. daily changes of the medium were carried out until the parasites reached 60 %. then, the parasites were harvested by centrifugation at 1500rpm for 7min and washed with sterile pbs. the antigen was purified by isopycnic ultracentrifugation using 500µl of the pellets mixed with tkm (50mm tris-hcl containing 25mm http://jad.tums.ac.ir/ https://www.google.com/search?client=firefox-b&biw=1600&bih=789&q=st.+louis&stick=h4siaaaaaaaaaopge-luz9u3slc0sk5u4gaxzcoryrw0spot9pol0hpzmqssszlz81a4vhmpismfpylfjalfxqdmhhgvqwaaaa&sa=x&sqi=2&ved=0ahukewj80qi-jrbnahwposakhyqcahyqmxmiiqeoatat https://www.google.com/search?client=firefox-b&biw=1600&bih=789&q=st.+louis&stick=h4siaaaaaaaaaopge-luz9u3slc0sk5u4gaxzcoryrw0spot9pol0hpzmqssszlz81a4vhmpismfpylfjalfxqdmhhgvqwaaaa&sa=x&sqi=2&ved=0ahukewj80qi-jrbnahwposakhyqcahyqmxmiiqeoatat j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 326 http://jad.tums.ac.ir published online: september 30, 2019 kcl, 5mm mgcl2, 1mm edta containing 5% triton-x, 0.01% 36% sucrose and protease inhibitors) to solubilize the parasite membrane (12) and centrifuged at 110,000xg for 16h after which the supernatant and the precipitate were both dialyzed separately to remove sucrose (13). both fractions were analyzed by sds-page using 12% reducing gel with 125×145×1mm dimension by laemmle's method (1970). the upper fraction contained the c-19 terminal of msp1, and this was named as "parasite extract." further purification was performed using preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) 125× 145×5mm using negative staining. strips were cut at the 19kda and the protein electro-eluted with a dialysis bag in tris-glycine buffer ph 7.5 using 100 volts and 30mamps overnight at 4 °c (14). sds-page staining methods: i) silver staining was carried out using the method of blum et al. (15). ii) coomassie blue r (cbb-r250) staining was carried out using 0.1% dye in 10% acetic acid, 40% methanol and 0.1% cbb-r250. iii) periodic acid schiff's (pas): staining was used to distinguish polysaccharides using method of konat et al. (16). iv) reversible negative staining was carried out on preparative gels using imidazole zinc staining by the method of castellanos et al. (17). immunoblotting: was carried out using (18) with the anti-c-terminal msp1 p. falciparum 3d7 antibody prepared in rabbit, kindly donated by national institute of immunology, new delhi, india glycoprotein analysis: was performed on the purified c-terminal fraction using o-glycan glycoprotein kit and total glycan glycoprotein kit (qiagen, india ltd.) (19). briefly, for o-glycan kit, two affinity lectin columns ail (javelin which is the jacalin lectin from artocarpus integrin folia the jackfruit tree which attaches sialic acid and pna from peanut agglutinin which binds o-galactose units) and then eluted with their respective eluting buffers and the eluted proteins analyzed on sds-page using silver staining. for total glycoproteins, mannose-binding lectins and sialic glycoproteins were bound on concanavalin a and wheat germ agglutinin lectin columns and then eluted with respective eluting buffers. the eluted proteins were then analyzed by sds-page and silver staining. the bioinformatic analysis was performed using c-terminal msp1 genes: different cterminal partial cds were checked for glycosylation sites using expasy software (20). genes of cterminal msp1 found in wild-type iranian p. falciparum isolates registered earlier by the authors in the gene bank: abq52496, abq 52497, abq52498, abq57306, abq52495, abm54034, baa77608, (21) were used to detect predicted n and o-glycosylation (20). results large scale preparation of parasites: a large number of ring-stage parasites were grown (fig. 1). the sds-page analysis of parasite extract exhibited proteins stained by coomassie blue staining in (fig. 2). preparative gels are used for cutting out the pure native msp1-19kda c-terminal antigen (fig. 3) by reversible negative staining. glycoproteins are exhibited in the msp1 antigen using schiff's staining (fig. 4). sds analysis of pure native c-terminal 19kda antigen: shows a single band with silver staining (fig. 5). immuno-blotting showed a single band with monoclonal anti-19kda c-terminal antibody (fig. 6). silver staining for glycoproteins detected o-glycans in sialic acid and o-mannose binding units (fig. 7) by the o-glycoprotein kit. n-glycans comprising of complex bi-antennary and tri-antennary types, including sialic acid, were identified in the c terminal region of msp1 19kda (fig. 8). bioinformatic analysis: the expasy bioinformatic studies show the presence of 62 probable o-glycosylation sites (fig. 9) and five potential n-glycan sites (fig. 10) on the 250 base sequence. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 327 http://jad.tums.ac.ir published online: september 30, 2019 fig. 1. synchronized large scale preparation of ring-stage p. falciparum fig. 2. coomassie blue staining of sds-page of parasite extract. mw is the molecular weight marker, and the right lane is p. falciparum "parasite extract" fig. 3. preparative negative imidazole staining of sds-page of p. falciparum "parasite extract". mw is molecular weight marker fig. 4. glycoprotein schiff's staining of sds page of p. falciparum "parasite extract". mw is molecular weight markers fig. 5. silver-staining of sds-page of p. falciparum of purified c-19 kda antigen. mw is molecular weight markers http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 328 http://jad.tums.ac.ir published online: september 30, 2019 fig. 6. immunoblotting of purified c-terminal 19kda antigen using anti-c-terminal monoclonal antibody raised in rabbits. mw is molecular weight markers fig. 7. silver staining of sds-page of purified antigen from o-proteome kit. mw is molecular weight markers. lane a purified antigen c-terminal 19kda, lane b glycoproteins containing galactose and galactosamine eluted from pna kit, lane c containing n-acetyl neuraminic, galactose and galactosamine eluted from ail column fig. 8. silver staining of sds-page of purified antigen from total q proteome kit, mw is molecular weight markers. lane a purified antigen c-terminal 19kda, lane b glycoproteins containing nacetylglucosamine and sialic acid eluted from wga column; lane c mannose-containing glycoproteins eluted from con a columns; lane d n-acetyl glucosamine and sialic acid glycoproteins eluted from wga column http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 329 http://jad.tums.ac.ir published online: september 30, 2019 fig. 9. expasy file graphs showing 62 probable o-glycosylation sites of p. falciparum c-19kda c-terminal msp1 antigen and 5 potential n-glycan sites on the 250 base sequence fig. 10. expasy file graphs showed five probable n-glycosylation sites of p. falciparum c-19kda c-terminal msp1 antigen discussion numerous researchers on glycobiology of p. falciparum have reported contradictory findings of the occurrence of glycoproteins on the plasmodium surface proteins, especially msp1 and merozoite surface protein 2 (msp2). currently, no reported of glycans on the c-terminal 19kda msp1 antigen. however, our studies have shown the presence of both n and o lectin binding glycans on the 19kda c-terminal of msp1. the n-glycans comprise asparagine binding n-acetylglucosamine including those bound to sialic acids. o-glycans are serine/ threonine binding sialic acids with n-acetylgalactosamine and o-galactose units. neither msp1 nor msp2 shows an affinity for lectin binding or sensitivity to pngase f, suggesting the absence of n-linked glycans (7). however, after the expression of msp1 in mammalian cells (22) and the baculovirus system (23), the proteins are n-glycosylated. glycosylation in the genus plasmodium is mostly active in the biosynthesis of glycol phosphatidylinositol moiety (gpis) attached to the ctermini of the proteins to anchor them to the bilipid bilayers (24). the msp1s as their name denotes is on the surface and are closely associated with the gpis. comparative stoichiometric analysis has shown that two-thirds of http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 330 http://jad.tums.ac.ir published online: september 30, 2019 all gpi-anchored proteins are associated with msp1 and msp2. thirty gpi-anchored proteins are predicted to be expressed, and the existence of some potential n-glycosylation sites are shown in both proteins (25). the availability of the p. falciparum genome has assisted research on the enzymes involved in n-glycosylation and even though the strain lacks most of the enzymes participating in the assembly of n-glycans which participates in a secondary loss of asparagine-linked glycosyltransferases enzymes for asparagine-linked glycans. the presence of two enzymes alg7 and alg14 responsible for the synthesis of dolicholpyro phosphoryl-oligosaccharides have been observed (26-28). the type of n-glycans produced in p. falciparum are shorter than their mammalian counterparts and transferred to glucosaminelabeled proteins and bind the corresponding lectin g. simplicifolia (29). as p. falciparum merozoite maturation takes place within an intraerythrocytic network of modified (parasitophorous vacuolar membrane) and newly made (tubo-vesicular network) membranes (30), it is likely that parasite surface proteins also comprise substrates for carbohydrate-modifying enzymes of the erythrocyte. there are reports of the absence of n-glycosylation machinery in p. falciparum parasites (30) even though nlinked carbohydrates have also been described in alliance with asparagines on msp1 (31). however, our results showed the presence of lectin binding n-glycans on the c19 terminal of native msp1 antigen by the total glycoprotein kit. we have confirmed the earlier studies that n-glycosylation is seen in the native antigen. the expasy bioinformatic studies show the presence of some probable n-glycan binding sites. the partial cds of the c-19kda msp1 genes (ef563844.1, ef563845.1, ef563846.1 (21) of a wild strain isolated from patients revealed the presence of asparagine residues corroborating the results of the expasy website. we have confirmed the presence of o-glycans in native 19kda c-terminal of msp1 by their binding to ail and pna lectin columns which exhibit the presence of sialic acid with n-acetylgalactosamine and o-galactose units. our results confirm that of nasir-ud-din 1991 for msp1 indicating the presence of glucosamine and galactose in the protein (31), and also gilson (30) treated galactose, fucose and glucosamine-radiolabeled proteins with pngasef to remove possible n-glycans, and purified the resulting material by gel filtration and subjected the void volume fraction to alkaline β-elimination. about half of the radioactivity was shifted to the monosaccharide volume, proposing the discharge of o-glycans. they also characterized o-glycosylation reactions using exogenous galactosylases to identify o-glcnac in proteins. other studies (25, 32) reported no o-glycosylation in the blood during the asexual stage of the parasite. however, our work on the asexual stage shows the presence of o-glycosylation which confirms another the work (13). it was used immunoprecipitation to show the c-terminal fragment of msp1 contains o-glycosylation sites. moreover, the whole protein, as well as both n and c-terminal msp-1 fragments, could be exo-galactosylated. recently, the presence of ogt (genes for o-glcnacylation) has been investigated using anti-ogt antibodies and these studies have proposed the presence of oglcnacylation in p. falciparum (33) supporting earlier observations of glcnacβ-ser/ thr in msp2 (6). in our study, the five different 19 kda c-terminal msp1 sequences obtained from various p. falciparum strains showed the presence of both threonine and serine in the 19kda c-terminal sequence, needed for glycosylation. hoessli and co-workers (13) have worked on the schizont and late trophozoite stage, but we have shown the presence of n and o-glycans in the ring stage of the c-19kda antigen. the expasy bioinformatics site showed the probable presence of more than 60 o-glycosylation sites for the seven genes isolated from iranian strains verifying our experimental work. the life cycle of the p. falciparum comprises of the ring stage in the rbc changed to trophozoites and then finally to schizont stage. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 324–333 s tajik al.: characterization of … 331 http://jad.tums.ac.ir published online: september 30, 2019 the schizonts release the merozoites, which once again infect the red blood cells. we have shown n and o-glycans in the ring stage of the native msp1 c-19kda antigen. as earlier workers have demonstrated its presence in the other two stages, it is likely that contrary to all other speculations, the glycosylation of this antigen in its native form is seen in all the stages. the purity of our antigen was quite high as it is was purified by both ultracentrifugation and preparative sds-gel electrophoresis and gave a single band on immunoblotting using the specific monoclonal antibody against c-terminal 19 kda. specific schiff's staining for carbohydrates also shows the presence of glycoproteins in the c-terminal 19kda antigen along with the q proteome kit. bioinformatics analysis too shows the presence of many likely o-glycoprotein sites on the c-terminal antigen and also n-terminal binding ones. conclusion both n and o-glycan antigens are present in the native form of the c-terminal of 19kda antigen in the ring stage. it is possible that the lack of elicitation of protection by recombinant msp1 antigen could be due to their pre-requisition of glycosylation. further research on recombinant antigens should concentrate on their glycosylation if they are to be used as vaccine candidates. the presence of n and o-glycoproteins were detected by q proteome kit on the native 19kda c-terminal msp1 purified from the ring stage of p. falciparum. taking into account the data of earlier scientists that it is likely that msp1 is glycosylated in all the stages of the parasite life cycle and incomplete protection elicited by recombinant msp1 antigens is due to lack of n and o-glycoproteins. acknowledgements we wish to acknowledge east mediterranean regional organizations (emro) for 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proteins of intraerythrocytic stage plasmodium falciparum. j biol chem. 272(10): 6428–6439. 33. perez-cervera y, harichaux g, schmidt j, debierre-grockiego f, dehennaut v, bieker u, meurice e, lefebvre t, schwarz rt (2011) direct evidence of o-glcnacylation in the apicomplexan toxoplasma gondii: a biochemical and bioinformatic study. amino acids. 40(3): 847–856. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2015, 9(1): 131–136 m rajabloo et al.: nymphal linguatulosis in … 131 case report nymphal linguatulosis in indian crested porcupines (histrix indica) in southwest of iran *mohammad rajabloo 1, seyed mostafa razavi 1, hossein shayegh 2, amir mootabi alavi 1 1department of pathobiology, school of veterinary medicine, shiraz university, shiraz, iran 2school of veterinary medicine, shiraz university, shiraz, iran (received 7 oct 2013; accepted 29 mar 2014) abstract linguatula serrata is one of the important zoonotic parasites. carnivores serve as definitive host. the larvae existed in mesenteric lymph nodes (mlns), liver, lungs, etc of intermediate herbivores. the definitive host becomes infected by ingesting viscera containing the infective nymphal stage. humans may be infected with linguatula either by ingestion of nymphs resulting in a condition called nasopharyngeal linguatulosis or halzoun syndrome or by ingestion of infective eggs which develop in internal organs resulting in visceral linguatulosis. indian crested porcupine (hystrix indica) is a common rodent in middle east. based on some tradition, consumption of histrix meat and viscera is common in some parts of iran. the present study reports the occurrence of linguatula serrata nymph in h. indica as a new intermediate host from southwest of iran. keywords: nymphal linguatulosis, linguatula serrate, indian crested porcupine, rodent host introduction linguatula serrata is one of the important zoonotic parasites. adults inhabit in respiratory system of canine (final hosts). infective eggs containing larvae are discharged into the environment by nasopharyngeal secretions or coughed up, swallowed, and passed out of the host in the feces. when suitable intermediate host (herbivorous animal), swallows these eggs, the larvae migrate to mesenteric lymph nodes (mlns), liver, lungs, etc. after six to nine molting, the nymphs are infective for final host. canine become infected by ingesting viscera containing the infective nymphal stage. the parasite is tongue-shaped, lightly convex dorsally and flattened ventrally. males measure 1.8–2 cm, while females measure 8–13 cm in length (soulsby 1982). humans may be infected with linguatula either by ingestion of nymphs resulting in a condition called nasopharyngeal linguatulosis or halzoun syndrome or by ingestion of infective eggs which develop in internal organs resulting in visceral linguatulosis (shekarforoush et al. 2004). halzoun syndrome may arise via consumption of raw or undercooked liver and lymph nodes. the syndrome has been reported in many countries including africa, south-east asia and the middle east (beaver et al. 1984, drabick 1987, el-hassan et al. 1991). some researchers described clinical signs of the syndrome including pharyngitis, salivation, dysphagia and coughing. visceral linguatulosis generally remains asymptomatic (khalil and schacher 1965, yagi et al. 1996). clinical signs associated with infections in intermediate hosts have not yet been described carefully (mehlhorn 2008). there are reports of linguatulosis in other organs such as eye (lazo et al. 1999). the prevalence of l. serrata have been studied in various animals in iran, including dogs (meshgi and asgarian *corresponding author: dr mohammad rajabloo, e-mail: rajabloo.mohammad@gmail.com http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 131–136 m rajabloo et al.: nymphal linguatulosis in … 132 2003, oryan et al. 2008, rezaei et al. 2011), sheep (shekarforoush et al. 2004, nourollahi fard et al. 2011), goats (razavi et al. 2004), camels (shakerian et al. 2008, radfar et al. 2010, rajabloo et al. 2011, oryan et al. 2011, rezaei et al. 2012, bamorovat et al. 2013) and cattle (hami et al. 2009, tajik et al. 2011). there are also some reports of human linguatulosis in iran (sajjadi et al. 1998, maleki 2001, anaraki mohammadi et al. 2008, tabibian et al. 2012). the indian crested porcupine (hystrix indica) has been distributed in iraq, iran, palestine, syria, lebanon, turkey, southern and eastern states of the former soviet union and indian subcontinent (arsalan 2008). hystrix indica is nocturnal and spend daylight hours in natural caves or excavated burrows. porcupines consumed storage organs of at least twelve species of geophytes and hemicryptophytes and ate potatoes, peanuts, peppers, fallen fruit, and other agricultural crops (alkon and saltz 1988). the present study reports the occurrence of l. serrata nymph in h. indica from southwest of iran. case report during a six month period, september 2012 to march 2013, the carcasses of two h. indica were transferred to department of pathobiology, school of veterinary medicine, shiraz university, shiraz, iran. one of them was died in september 2012, when it was crossing the road in bamu national park (29° 40′ 20.52″ n, 52° 46′ 1.21″ e), shiraz, iran, and the other carcass was confiscated from unauthorized hunters. the animals were necropsized systematically. the body cavity was opened by a longitudinal incision from throat to vent. mesenteric and mediastinal lymph nodes and liver were collected from each animal, cut into small pieces and massaged in tepid water and left for 30 minutes to allow nymphs to come out from tissue. the livers were sliced in 4–5 mm thick and observed carefully to find the encapsulated or free nymphs. the samples were examined for the presence of nymphs using a stereomicroscope. recovered nymphs were fixed in ethanol 70%, slightly pressed between two flat glasses. they were stained with carmine and then mounted in canada balsam® as permanent slides measurements of morphological features were made with a light microscope using a calibrated ocular micrometer. samples (liver, mlns, and mediastinal lymphatic nodes) were studied separately. in the case of liver, no nymphal stage was found. the results were also negative for mediastinal lymphatic nodes of both animals but the mlns were infected with 12 and 8 l. serrata nymphs. based on morphologic characteristics, the parasite was identified as the nymphal stage of l. serrata (fig. 1). the parasites were tongue-shaped, segmented and covered with several spines in linear arrangement. total body length was 52±3 mm, the body width was 1.2±1 mm in anterior and 0.25±0.02 mm in posterior ends. the hooklets surround elliptical mouth. fig. 1. nymphs of l. serrata recovered from mesenteric lymphatic nodes of h. indica, stained with carmine (a×4, b×40) discussion canine are definitive hosts and the herbivores such as sheep, goat, cattle and camel act as intermediate host in life cycle of l. serrata. cats (esmaeilzadeh et al. 2008), fox http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 131–136 m rajabloo et al.: nymphal linguatulosis in … 133 (gicik et al. 2009), dingo, canis lupus dingo, chrysocyon brachyurus, procyon cancrivorus cancrivorus and wolf, canis lupus lupus can also be definitive hosts (christoffersen and de assis 2013), l. serrata nymphs were found in european porcupine hystrix (hystrix) cristata, degu, octodon degus, common hare, lepus (eulagos) europaeus europaeus [as lepus europaeus] and common rabbit, oryctolagus cuniculus cuniculus [as lepus cuniculus]. (christoffersen and de assis 2013). it has been found commonly in cottontail rabbits (sylvilagus floridanus) in the usa (moore and moore 1947). linguatula sp. is maintained in australia by a fox-rabbit cycle since foxes, not dogs, have been found infested (pullar 1946). our study revealed that h. indica can be regarded as another intermediate host for this parasite and a sylvatic cycle between porcupine and carnivores may exist in fars province. the prevalence rate of nymphal stages was studied by many researchers in variety of hosts. shekarforoush et al. (2004) reported a prevalence of 11.5% and 3% in the mesenteric lymph nodes and liver of sheep, respectively and razavi et al. (2004) recorded 29% infection in the mesenteric lymph nodes and 6% in the livers of goats in shiraz area, fars province, southern iran. prevalence rates of nymphal linguatulosis in onehumped camels were reported 12.9% in yazd (oryan et al. 2011), 64% in tehran (rajabloo et al. 2011), 20.5% in rafsanjan (bamorovat et al. 2013), 21.12% in isfahan (rezaei et al. 2012), 21.0%, 4.5% in mlns and liver respectively, in najaf-abad (shakerian et al. 2008). it has also been reported from twohumped camel (camelus bactrinus) in tabriz, iran (haddadzadeh et al. 2009). the prevalence rates in sheep were 16.1% in kerman (razavi et al. 2004) and 11.5 % and 3% in mlns and liver, respectively, in shiraz (shekarforoush et al. 2004). in goat, the prevalence rates were reported 29.9% and 6.4% in mlns and liver, respectively in shiraz (razavi et al. 2004) and in cattle, 44% in urmia and 0.25% in tabriz (hami et al. 2009). it seems that the parasite is common among herbivores and canine in iran. halzoun syndrome was reported from shiraz (sajjadi et al. 1998), tehran (maleki 2001, anaraki mohammadi et al. 2008) and isfahan (tabibian 2012). all of these reports showed the high rate of infection in both definitive and intermediate hosts in various parts of iran and it is not unexpected to consider a canine-porcupine sylvatic cycle in this region. the parasite has been reported from human beings in iran (sajjadi et al. 1998, maleki 2001, anaraki mohammadi et al. 2008), austria (koehsler et al. 2011), morocco (le corroller and pierre 1959) and turkey (yılmaz et al. 2011). these reports showed zoonotic importance of infection. hystrix indica is a common rodent in iran. it fed from potatoes, peanuts, peppers, fallen fruit, and other agricultural crops and classified as an herbivore rodent (alkon and saltz 1988, arsalan 2008). infection of mlns suggests a similar migration and life cycle of the parasite in this animal. with our knowledge there is no report about nymphal linguatulosis in indian crested porcupine species but it has been reported from lungs of european porcupine hystrix (hystrix) cristata (linnaeus 1758). one part of the porcupine is always a delicacy the liver, which is easily removed by making a cut just under the neck into which the hand is thrust, and the liver pulled out. it may be fried with bacon, or baked slowly and carefully in the baker-pan with slices of bacon (kephart 2001). many locals relish the porcupine's meat (al nees) as a delicacy and for medicinal purposes. perhaps this is responsible for the decline of h. indica population throughout jordan (amr et al. 2014). consumption of histrix meat and viscera has been observed among native people for medicinal purposes in south and west of iran (personal observation). http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 131–136 m rajabloo et al.: nymphal linguatulosis in … 134 oryan et al. (2008) reported a prevalence of 76.5% in stray dogs in shiraz, southwest of iran. the prevalence of adult stage has also been reported 27.83% in north west (rezaei et al. 2011) and 62.2% in central part of iran (meshgi and asgarian 2003). stray dogs and other wild canines can hunt porcupine and this animal can be a part of their diets. our study revealed that porcupine can act as an intermediate host for sylvatic cycle of the parasite in this area. these facts showed that the sylvatic cycle could support the distribution of l. serrata. further investigations are needed to be carried out to clarify the exact role of canine and other wild rodents in the life cycle of l. serrate. conclusion in conclusion, our results and high prevalence of linguatulosis in both definitive and intermediate host in fars province showed that sylvatic cycle should be considered as a risk factor for human beings and other hosts. acknowledgements the authors declare that there is no conflict of interests. 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september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 302 http://jad.tums.ac.ir published online: september 30, 2020 short communication an inventory of the sand flies (diptera: psychodidae) of rudbar county, a new focus of leishmaniasis in northern iran, with a taxonomic note on the subgenus larroussius behzad norouzi1; ahmad ali hanafi-bojd2; vahideh moin-vaziri3; ayoob noorallahi4; *shahyad azari-hamidian5 1research center of health and environment, guilan university of medical sciences, rasht, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 4department of disease control and prevention, health vice-chancellorship, guilan university of medical sciences, rasht, iran 5department of health education, research center of health and environment, school of health, guilan university of medical sciences, rasht, iran *corresponding author: dr shahyad azari-hamidian, e-mail: azari@gums.ac.ir (received 27 jan 2020; accepted 24 sep 2020) abstract background: different forms of leishmaniasis are significant infectious diseases in iran. while, rudbar county of guilan province has been introduced as a new cutaneous leishmaniasis focus, there are few published data about the phlebotomine sand flies (diptera: psychodidae) of the province. methods: to study the phlebotomine fauna of rudbar county, the sampling was performed in 12 collection sites by light traps, sticky traps and manual aspirators throughout august–december 2015. sand flies were removed from the sticky traps, rinsed in acetone and stored in 80% ethanol along with the collections of light traps and hand catches. results: in total, 2186 sand flies were collected and ten species representing two genera were morphologically identified: phlebotomus (adlerius) halepensis (0.27%), ph. (larroussius) kandeladii (0.10%), ph. (lar.) neglectus (0.91%), ph. (lar.) perfiliewi (53.88%), ph. (lar.) tobbi (43.45%), ph. (paraphlebotomus) sergenti (0.82%), ph. (phlebotomus) papatasi (0.10%), sergentomyia (parrotomyia) baghdadis (0.27%), se. (sintonius) clydei (0.05%) and se. (sin.) tiberiadis (0.10%). the species ph. halepensis, ph. neglectus, ph. perfiliewi, se. baghdadis, se. clydei and se. tiberiadis were reported for the first time in guilan province. this study also verified the presence of ph. neglectus (ph. major krimensis as a synonym and morphotype) in iran. moreover, the taxonomy of the subgenus larroussius of the province was discussed. conclusion: the prevalence of suspected or proven cutaneous and visceral leishmaniasis vectors is noteworthy. the study of ecology of sand flies and detecting the exact vectors of leishmaniasis and phlebotomine fever by molecular specific tests in guilan province are recommended. keywords: phlebotomus; sergentomyia; visceral leishmaniasis; cutaneous leishmaniasis; sand fly fever introduction by now, approximately one thousand phlebotomine species (diptera: psychodidae) have been described worldwide (1). traditionally, sand flies include six genera of the old world chinius, phlebotomus, sergentomyia and the new world brumptomyia, lutzomyia and warileya (2-3), however, more recent classifi cation has been recognized 31 genera in the subfamily (1). among phlebotomine sand flies, at least 98 species of phlebotomus and lutzomyia are proven or suspected vectors of human leishmaniasis, also sand flies play a role in the transmission of viral infections caused by copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 303 http://jad.tums.ac.ir published online: september 30, 2020 phlebovirus (phenuiviridae) and vesiculovirus (rhabdoviridae) and the causal agent of bartonellosis (the bacterium bartonella bacilliformis) (4). the last checklist of iranian sand flies composes 54 species, 31 species of phlebotomus and 23 species of sergentomyia (5). at least 62 species of sand flies have been reported from iran (5-12), however there are some controversial debates about the occurrence of some species and the numbers of iranian species are mentioned from 44 to 50 by different authors (13-16). the old world subgenus larroussius includes at least 25 species (8), in which at least 12 species are proven or suspected vectors of leishmaniasis (17). eleven species of the subgenus have been reported from iran by now which are as follow: ph. ilami [type locality in iran (ilam and mehr)], ph. kandelakii, ph. keshishiani, ph. langeroni, ph. major, ph. neglectus, ph. notus, ph. perfiliewi, ph. smirnovi, ph. tobbi [type locality in iran (rasht) and palestine] and ph. wenyoni [type locality in iran (hamadan)] (5, 8, 9, 18). there are few published documents about the sand flies of guilan province in the caspian sea littoral of northern iran. only four species including ph. kandelakii, ph. papatasi, ph. sergenti and ph. tobbi have been recorded in the province by now (19-20). more than 20000 human cases of cutaneous and visceral leishmaniasis are annually reported from iran (21). adler et al. (19) have noted a few autochthonous cases of human leishmaniasis in rasht, capital of guilan province. golchay et al. (22) have reported three cases of cutaneous leishmaniasis (cl) in the province and introduced cl as an endemic disease in guilan province. nadim et al. (23) have mentioned that rudsar, located in eastern guilan province, could be considered as a less important focus of cl. majidi-shad et al. (24) have introduced rudbar county of the province as a new focus of cl. during the period of 2006–2016, one to 15 autochthonous cases of cl and/or visceral leishmaniasis (vl) (kala-azar) have been recorded in guilan province yearly, especially rudbar county (center of disease control, health vice-chancellorship, guilan university of medical sciences and 21). also, sand fly fever is found in the province (25-27). moreover, lizard leishmaniasis is reported in guilan province (28). there is no recent study on phlebotomines in guilan province. the aim of this study was to determine the phlebotomine fauna of rudbar county during 2015. materials and methods study area guilan province in the caspian sea littoral of northern iran, between caspian sea and alborz mountain range, has coastal, plain, foothill, and mountainous areas with an area of about 14700km2. the province is surrounded by mazandaran province in the east, ardebil province in the west and zanjan and qazvin provinces in the south. it is also bordered by azerbaijan republic in the north as well as russia across caspian sea (fig. 1). guilan province with temperate climate and relatively warm-humid summer is located between 36o33'–38o27' n latitude and 48o32'–50o36' e longitude and formally composes 16 counties. most areas of the province with about 1000– 2000mm annual rainfall have the highest precipitation in iran and the main agricultural crop is rice. rudbar county is located in the southern part of guilan province with about 200–500 mm annual rainfall and showed mountainous and less humid temperate climate similar to the mediterranean region (gilan meteorological organization). the county has an area of 2574km2 and the main agricultural crop is olive. specimen and data collection the specimens of phlebotomines were collected from 12 localities of rudbar county by means of light traps, sticky traps and manual aspirators throughout august–december 2015 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 304 http://jad.tums.ac.ir published online: september 30, 2020 (fig. 1) (table 1). the collection localities were mostly selected based on the previous reported cases of leishmaniasis (department of disease control and prevention, health vice-chancellorship of guilan university of medical sciences and 24). two cdc light traps and 50 sticky traps were used in each locality. the light traps were suspended from ceiling in animal (sheep, cattle and poultry) shelters (near houses) from sunset to sunrise, from 18:00 pm to 06:00 am. the electricity of traps was provided by 6-volt rechargeable batteries. sticky papers consisted of 10x15cm white sheets coated with castor oil. sticky traps were used in animal shelters and yards around animal shelters and houses. also ad hoc hand catch collections were performed by manual aspirators. phlebotomines were removed from the sticky traps and separated from other insects, rinsed in acetone and then preserved in 80% ethanol as well as the collections of light traps and hand catches. the microscope slides of all specimens were prepared using puri's fluid. the specimens were identified using the morphological-based keys of perfil’ev (29), nadim and javadian (30), lewis (31), seyedi rashti and nadim (32), rassi and hanafi-bojd (33) and zahraei-ramazani et al. (12). also, absavaran et al. (18), léger et al. (34) and killick-kendrick et al. (35) were consulted for differentiating the females of the subgenus larroussius. galati et al. (1) was followed for the phlebotomine name genera and subgenera abbreviations. the sex ratio (males: females) was calculated for each species. sampling ratio (samples of sticky traps/ samples of light traps: st/lt) was also calculated. species dominance structure the dominance structure of a species is described as the percent of specimens of the species in the whole specimens. the following five dominance structure categories were used according to tischler (36) and heydemann (37): eudominat (ed) species (> 30 %), dominant (d) (10–30%), subdominant (sd) (5–10%), recedent (r) (1–5%) and subrecedent (sr) (< 1%). gonotrophic cycle stages of female sand flies abdominal appearance of female phlebotomines is used for determining ovary development status and follows the same stages of anopheles (29). world health organization (38) introduced a simplified classification for abdominal appearances follows: empty or unfed (u), freshly fed (f), semi-gravid (sg) and gravid (g). density of sand flies the following formulae were used to calculate the mean density of sand flies collected by sticky traps: density (dn)=number of specimens/number of sticky traps, density (dm2)= number of specimens/m2 of sticky traps (3940). the density was corrected according to the following formula (41): density (ds)= results sand fly inventory in total, 2186 sand flies (896 males, 1290 females) were collected during 8 surveys from 11 localities during august–december 2015 (tables 2 and 3). ten species representing two genera were found. only 0.42% of the specimens belonged to the genus sergentomyia and 99.58% to the genus phlebotomus (table 2). the species ph. halepensis, ph. neglectus, ph. perfiliewi, se. baghdadis, se. clydei and se. tiberiadis were found for the first time in guilan province. the overall abundance of gonotrophic cycle stages of female phlebotomines based on abdominal appearance was presented in table 4. in total, 71.6% of female sand flies were unfed. the percentages of unfed female sand flies were 75 and 71.6 for sergentomyia and phlebotomus, respectively. the percentages were 79.8 and 57.7 for the most abundant species of ph. perfiliewi and ph. tobbi, respectively (table 4). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 305 http://jad.tums.ac.ir published online: september 30, 2020 the overall sex ratio (m: f) of sand flies was 0.69. the ratio was 1.25 and 0.69 for sergentomyia and phlebotomus, respectively (table 2). regarding the trapping method, the sex ratio of sergentomyia specimens was 1.33 and 1 using light traps and sticky traps, respectively. the ratio was 0.55, 10.88 and 0.48 using light traps, sticky traps and hand catches, respectively, for phlebotomus (table 5). general sampling ratio of phlebotomines (st/lt) was 0.11 and the ratio was 0.28 and 0.11 for sergentomyia and phlebotomus, respectively. 22.2% of sergentomyia specimens were collected by sticky traps and 77.8% by light traps. the percentages were 9.8 and 87.5 for phlebotomus, respectively. no sergentomyia specimen was collected by hand catches. regarding the collection methods and sex of sand flies, 64.4% and 67.2% of sand flies were females when light traps and hand catches were used, respectively. however, 91.2% of collected sand flies by sticky traps were male (table 5). the subgenus larroussius with four species and 2150 (98.4%) specimens of the total collection was eudominant. the sex ratio (m: f) was calculated 0.68 (874: 1276) for the subgenus (table 2). the species belong to this subgenus showed the widest distribution and were collected from all localities, except for rostamabad (table 3). regarding the collection methods, most of the larroussius species, 87.5% (1882 specimens), were collected by light traps and 9.8% (210) and 2.7% (58) by sticky traps and manual aspirators, respectively (table 5). the subgenus showed the most average density (134.42) in light traps. the species ph. halepensis, se. clydei and se. tiberiadis were collected only by light traps and ad hoc collections by manual aspirators yielded only ph. perfiliewi and ph. tobbi (tables 5 and 6). phlebotomus perfiliewi and ph. tobbi were the most abundant and eudominant species and showed the widest distribution in the studied areas (tables 2 and 3). also, both species showed the most density in sticky and light traps (table 6). taxonomic note the subgenus larroussius species (ph. kandelakii, ph. neglectus, ph. perfiliewi and ph. tobbi) were distinguished mostly by using the morphology of parameral sheath (aedeagus) and coxite in the males and the base of spermathecal ducts, pharyngeal teeth (pharyngeal armature) and spermatheca segment numbers in the females (figs. 2–5). the ranges, numbers and means of spermatheca segments were as follow: ph. kandelakii (27–33, n=4, mean=28.5, sd=3, se=1.5), ph. neglectus (11, n=1), ph. perfiliewi (12–20, n=20, mean=15.35, sd=1.66, se=0.37) and ph. tobbi (9–15, n=20, mean=12.26, sd= 1.62, se=0.37). this study also verified the presence of ph. neglectus (ph. major krimensis as a synonym and morphotype) in iran. fig. 1. map of iran highlighting guilan province and its 16 counties, including rudbar county, surveyed in 2015 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 306 http://jad.tums.ac.ir published online: september 30, 2020 table 1. the data collection of phlebotomines for localities in rudbar county of guilan province, iran, august– december 2015 locality (city/ village) topography coordinates altitude (m) rostamabad plain 36o 52.999' n, 49o 29.385' e 215 khaskool foothill 36o 50.789' n, 49o 32.669' e 470 lafandsara foothill 36o 50.522' n, 49o 32.271' e 620 rudbar foothill 36o 49.314' n, 49o 25.322' e 270 klayeh foothill 36o 50.992' n, 49o 32.132' e 438 rudabad plain 36o 52.397' n, 49o 30.871' e 192 harkian foothill 36o 59.592' n, 49o 33.491' e 149 siahroodposhteh foothill 36o 59.862' n, 49o 33.432' e 269 upper harzavil (manjil) foothill 36o 44.495' n, 49o 26.072' e 506 lower harzavil (manjil) foothill 36o 44.837' n, 49o 25.735' e 453 halaj (loshan) foothill 36o 40.306' n, 49o 26.792' e 307 pareh foothill 36o 50.800' n, 49o 32.650' e 487 table 2. the abundance and dominance structure of phlebotomines in rudbar county, guilan province, iran, 2015 species males females sex ratio (m:f) total % dominance structure ph. (adl.) halepensis 6 6 6 0.27 subrecedent ph. (adl.) sp. 1 1 0.05 subrecedent ph. (lar.) kandelakii 2 2 0.10 subrecedent ph. (lar.) neglectus 18 2 9 20 0.91 subrecedent ph. (lar.) perfiliewi 360 818 0.44 1178 53.88 eudominat ph. (lar.) tobbi 496 454 1.09 950 43.45 eudominat ph. (par.) sergenti 10 8 1.25 18 0.82 subrecedent ph. (phl.) papatasi 1 1 1 2 0.10 subrecedent se. (par.) baghdadis 4 2 2 6 0.27 subrecedent se. (sin.) clydei 1 1 0.05 subrecedent se. (sin.) tiberiadis 2 2 0.10 subrecedent total 896 (41.0%) 1290 (59.0%) 0.69 2186 100 table 3. the distribution of phlebotomines in rudbar county, guilan province, iran, 2015 species locality r u d b a r p a r e h l a fa n d sa r a k h a sk o o l k la y e h r u d a b a d h a r k ia n s ia h r o o d p o sh te h u p p e r h a r z a v il l o w e r h a r z a v il h a la j (l o sh a n ) ph. (adl.) halepensis * ph. (adl.) sp. * ph. (lar.) kandelakii * * ph. (lar.) neglectus * * * * * * ph. (lar.) perfiliewi * * * * ph. (lar.) tobbi * * * * * * * * * * * ph. (par.) sergenti * * * * * ph. (phl.) papatasi * * se. (par.) baghdadis * se. (sin.) clydei * se. (sin.) tiberiadis * http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 307 http://jad.tums.ac.ir published online: september 30, 2020 table 4. the abundance of gonotrophic cycle stages of female phlebotomines based on abdominal appearance in rudbar county, guilan province, iran, 2015 (*u: unfed, f: fed, sg: sub-gravid, g: gravid) species abdominal appearance (%) total % u* f sg g ph. (adl.) sp. 1 (100) 1 0.07 ph. (lar.) kandelakii 2 (100) 2 0.16 ph. (lar.) neglectus 1 (50.0) 1 (50.0) 2 0.16 ph. (lar.) perfiliewi 653 (79.8) 48 (5.9) 97 (11.9) 20 (2.4) 818 63.41 ph. (lar.) tobbi 262 (57.7) 67 (14.8) 94 (20.7) 31 (6.8) 454 35.19 ph. (par.) sergenti 5 (62.5) 1 (12.5) 2 (25.0) 8 0.62 ph. (phl.) papatasi 1 (100) 1 0.07 se. (par.) baghdadis 2 (100) 2 0.16 se. (sin.) tiberiadis 1 (50.0) 1 (50.0) 2 0.16 total 924 (71.6) 117 (9.1) 193 (15.0) 56 (4.3) 1290 100 table 5. the abundance of phlebotomines based on collection method in rudbar county, guilan province, iran, 2015 species method of collection (%) total (%) light trap sticky trap hand catch male female male female male female ph. (adl.) halepensis 6 6 (0.27) ph. (adl.) sp. 1 1 (0.05) ph. (lar.) kandelakii 1 1 2 (0.10) ph. (lar.) neglectus 9 2 9 20 (0.91) ph. (lar.) perfiliewi 217 773 130 14 13 31 1178 (53.88) ph. (lar.) tobbi 437 443 53 3 6 8 950 (43.45) ph. (par.) sergenti 7 8 3 18 (0.82) ph. (phl.) papatasi 1 1 2 (0.10) se. (par.) baghdadis 3 1 1 1 6 (0.27) se. (sin.) clydei 1 1 (0.05) se. (sin.) tiberiadis 2 2 (0.10) total (%) 680 (35.6) 1232 (64.4) 197 (91.2) 19 (8.8) 19 (32.8) 39 (67.2) 2186 (100) 1912 (87.5) 216 (9.9) 58 (2.6) table 6. the mean density of phlebotomines in rudbar county, guilan province, iran, 2015 [density (dn)=number of specimens/number of sticky traps, density (dm2)=number of specimens/m2 of sticky traps, corrected density (ds)=√(1+(number of sand flies/number of traps)×100)] species sticky trap light trap dn dm2 ds ph. (adl.) halepensis 0.42 ph. (adl.) sp. 0.07 ph. (lar.) kandelakii 0.002 0.09 1.09 0.07 ph. (lar.) neglectus 0.025 0.81 1.87 0.78 ph. (lar.) perfiliewi 0.411 13.06 6.48 70.71 ph. (lar.) tobbi 0.160 5.08 4.12 62.85 ph. (par.) sergenti 0.008 0.27 1.34 1.07 ph. (phl.) papatasi 0.002 0.09 1.09 0.07 se. (par.) baghdadis 0.005 0.18 1.22 0.28 se. (sin.) clydei 0.07 se. (sin.) tiberiadis 0.14 mean total 0.610 19.60 7.78 136.57 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 308 http://jad.tums.ac.ir published online: september 30, 2020 fig. 2. the parameral sheaths of the subgenus larroussius species in rudbar county, guilan province, iran, 2015, a: phlebotomus kandelakii, b: ph. neglectus, c: ph. perfiliewi, d: ph. tobbi (original photos) fig. 3. the bases of spermathecal ducts (showed by arrows) and spermathecae of the subgenus larroussius species in rudbar county, guilan province, iran, 2015, a: phlebotomus kandelakii, b: ph. perfiliewi, c and d: ph. tobbi (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 309 http://jad.tums.ac.ir published online: september 30, 2020 fig. 4. the pharyngeal teeth of the subgenus larroussius species in rudbar county, guilan province, iran, 2015, a: phlebotomus kandelakii, b: ph. neglectus (ph. major krimensis morphotype), c: ph. perfiliewi, d: ph. tobbi (dashes show the pharynx, the wide part of pharynx and pharyngeal teeth) (original photos) fig. 5. the ventrally-directed setae of coxite (showed by arrow) in phlebotomus neglectus (ph. major krimensis morphotype), rudbar county, guilan province, iran, 2015 (original photo) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 310 http://jad.tums.ac.ir published online: september 30, 2020 discussion sand fly inventory during this study 10 species representing two genera were identified in which six species were new records for the sand fly fauna of guilan province. considering the role of sand flies in disease transmission, among collected phlebotomines, two species are known the vectors of cl in iran: ph. sergenti (suspected) (42-43) and ph. papatasi (proven) (42, 44) and four species the suspected vectors of vl: ph. kandelakii (45-46), ph. neglectus (as ph. major s.l.) (4748), ph. perfiliewi (45, 49) and ph. tobbi (50). phlebotomus halepensis is known a suspected or proven vector of vl in other countries of the western palaearctic region (4). in view of the occurrence of four suspected vectors of vl in guilan province and locating one of the most important foci of vl, ardebil province (51) in the west of guilan province, this can be very important for the health system of guilan province. sergentomyia clydei is assumed to play a role in the transmission of lizard leishmaniasis in iran (28, 42, 52). also, ph. papatasi is known a vector of sand fly fever (27). in the present investigation, the sand flies of the genus sergentomyia were collected in a very low prevalence (0.42%) (table 2). other investigations in northwestern iran also showed the low abundance of the genus in comparison to the genus phlebotomus, for example: 4.3% using just sticky traps (49) and 9.2% by means of both light traps and sticky traps (53). however, much more specimens of sergentomyia (29.62%) were collected in an arid area of iran (qom province) using sticky traps (13). many sergentomyia species mainly inhabit natural microhabitats, for example the burrows of animals and caves (29). in this regard, a large amount of sergentomyia species have been found in rodent burrows in iran (52-53) where were not studied in the present investigation. also, sergentomyia species usually suck blood from reptiles and phlebotomus species principally from warm-blooded vertebrates (2). in the present study, the collections were performed mostly in domesticated animal shelters and yards around those shelters and houses that is may be another reason of low abundance of sergentomyia specimens. interestingly, in this investigation, all species of sergentomyia and ph. papatasi were sampled only from loshan city (halaj) and manjil city (lower and upper harzavil) with a more arid climate than other localities (table 3). it seems that ph. papatasi mostly inhabits semi-arid areas (53) and high precipitation is a limiting factor in the distribution of the species (29). despite the vast distribution of ph. papatasi in iran, the species is infrequent in the lowland and humid areas of guilan province with high precipitation (54-55). in contrast, the species of the subgenus larroussius mainly inhabit regions with higher humidity and are intermediate between xerophilous and hygrophilous species (29). that might be the reason why the subgenus composed the majority of the specimens (98.4%) in the present investigation (table 2). another important factor is the collection method. many species of sergentomyia and the species ph. papatsi and ph. sergenti may not be or less attracted by artificial lights in contrast to some species of larroussius (ph. major s.l., ph. kandelakii and ph. perfiliewi) and the subgenus adlerius (29). that may explain the high prevalence of phlebotomines sampled by light traps (87.5%) (tables 5 and 6). in this investigation, the sampling ratio (st/lt) was more in sergentomyia (0.28) than phlebotomus (0.11) which is similar to the previous results in northwestern iran (53). moreover, the majority of females were unfed (table 4). that was also seen in the phlebotomine collection by light traps in northwestern iran (53). in this study, the numbers of sergentomyia specimens and the specimens collected by manual aspirators were a few (table 2), however phlebotomus displayed different sex ratio based on the collection method. the ratio was very lower in the collection http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 311 http://jad.tums.ac.ir published online: september 30, 2020 by light traps (0.55) than sticky traps (10.88). in a study in northwestern iran, the major part of phlebotomines collected by light traps was female and by sticky traps was male (53). they indicated sex ratio 1.3 for phlebotomus and 1.1 for sergentomyia. this higher abundance of males to females may be explained by the higher collection of phlebotomines by sticky traps (2701) than light traps (1281) in that study (53). the females of the subgenus adlerius are not identifiable (10), and the species ph. halepensis was identified based on the male characters in the present investigation (tables 2 and 3). this species is the most prevalent and widespread species of the subgenus in iran (10). three other species of the subgenus: ph. balcanicus, ph. brevis and ph. longiductus which are found in ardebil province (10) adjoining the west of guilan province could also be found in guilan province. in the present study, the subgenus larroussius and especially the species ph. perfiliewi and ph. tobbi were the most abundant and widespread sand flies (tables 2–6). the previous findings displayed that ph. perfiliewi and ph. kandelakii were the most abundant species in northwestern iran (45, 53). in the present investigation, ph. kandelakii was found in a lower prevalence in comparison to ph. perfiliewi and ph. tobbi (tables 2–6). this species had also been introduced as an infrequent species around rasht and anzali of guilan province before (8). taxonomic note the major group of the subgenus larroussius includes ph. major (type locality in india), ph. neglectus (type locality in albania, former yugoslavia, italy, including ph. major krimensis as a synonym, type locality in the former ussr), ph. notus (type locality in afghanistan), ph. syriacus (type locality in palestine and syria), ph. wenyoni (type locality in iran) and probably ph. wui (type locality in china) (8). there are controversial debates about the situation of ph. wui. lewis (31) considered it a subspecies of ph. major as it was originally described. artemiev and neronov (56), léger and pesson (57), xiang and jin (58) raised it to specific rank, but leng et al. (59) synonymized it with ph. smirnovi. on the other hand, killick-kendrick et al. (35) considered it outside the major group because they assumed a bell-shaped spermathecal duct for the species. four species of the major group; ph. major, ph. neglectus, ph. notus, and ph. wenyoni have been reported in iran by now (5, 9, 18). in view of wide distribution of ph. syriacus in the mediterranean and caucasus regions (31), this species may be occurring in iran. the identification of male sand flies of larroussius is usually not difficult, but some female species of the subgenus are not easily differentiated (35). the key of nadim and javadian (30) does not differentiate the female sand flies of ph. perfiliewi and ph. tobbi. the keys of seyedi-rashti et al. (32) and rassi and hanafi-bojd (33) do not distinguish the females of ph. neglectus (as ph. major), ph. perfiliewi and ph. tobbi from each other. morphological characters which are used to identify the female sand flies of the subgenus are: pharyngeal teeth (pharyngeal armature), spermatheca segment numbers, length of spermathecal neck, palpal and ascoid formulae, length of wings, base of spermathecal ducts (18, 29, 31, 34, 35). though, the shape of base of spermathecal ducts is mentioned as a reliable character (34-35) that is not used in the keys to identify the female sand flies of the subgenus in iran (30, 32, 33). in the areas where different species of larroussius sympatrically occur, including the vl foci of northwestern iran, the shape of base of spermathecal ducts has not been used to identify the females of the subgenus in many studies (18, 50). as far as the authors know only akhoundi et al. (53) noted and used that character in their investigation. during the present study, the male sand flies of larroussius were easily differentiated using the parameral sheeth (adeagus) morphology (fig. 2). the females of ph. kandelakii were easily identified based on the number of spermatheca http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 312 http://jad.tums.ac.ir published online: september 30, 2020 segments and the base of spermathecal ducts which is simple (fig. 3a) without a common duct or lateral structure (fig. 3b) or bell-shaped duct opening (fig. 3c, d). the number of spermatheca segments of ph. kandelakii is mentioned about 30 by perfil’ev (29) and 30–35 by artemiev (7) and lewis (31). in the present investigation, the number was 27 in three specimens and 33 in one specimen. the mean number of spermatheca segments is less in ph. tobbi (mean=12.26, sd=1.62, se=0.37) than ph. perfiliewi (mean=15.35, sd=1.66, se=0.37), but because of overlap it cannot be used as an exact character of differentiation. unfortunately, the number of females of ph. neglectus was not enough in this investigation to provide range and mean for this character. léger et al. (34) and killick-kendrick et al. (35) have mentioned that the base of spermathecal ducts is a very reliable character to differentiate aforementioned species (fig. 3), although this character is not easy to see in some specimens, especially when they are not dissected or in gravid or semi-gravid specimens. that is why no photo of the base of spermathecal duct for ph. neglectus is presented here. however, the character could be seen even in the microscope slides provided by puri’s medium using usual microscopes. the main feature of differentiation of females of larroussius species in this investigation was the base of spermathecal duct and then other characters were checked. the pharyngeal teeth (pharyngeal armature) of the present investigation specimens for ph. neglectus (fig. 4b) are exactly similar to the subspecies ph. major krimensis described by perfil’ev (29) (plate iii, b). perfil’ev (29, 60) mentioned that the females of ph. major krimensis differ from other subspecies or species of the major group by the form of pharyngeal teeth which occupy the whole wide part of pharynx which was exactly observed in this investigation (fig. 4b). in males, another specific character of ph. major krimensis is about 20 ventrally-directed setae situated in the middle of inner surface of coxite which forms a sparser group of setae than in other species of the major group (29, 60). this feature is also observed in the collected specimens of this study (fig. 5). lewis (31) considered ph. major krimensis a subspecies of ph. major as it was originally described by perfil’ev (29), but artemiev and neronov (56) synonymized ph. major krimensis with ph. neglectus. on the other hand, there is a suggestion for a possible species rank (ph. krimensis) for it (40). badakhshan et al. (9) reported two morphotypes using the male terminalia characters (the shape of parameral sheath and the ventrally-directed setae of coxite) including the morphotype ph. major krimensis (as a synonym of ph. neglectus) and another morphotype including ph. neglectus or ph. notus. they reported ph. notus for the first time in iran. according to aforementioned differentiating morphological characters which observed in the present study and by badakhshan et al. (9) and absavaran et al. (18), the presence of ph. major krimensis (as a synonym and morphotype of ph. neglectus) verifies in iran. other morphological characters which are used to identify the females of larroussius including the length of spermathecal neck, palpal and ascoid formulae and length of wings (18, 29, 31, 34, 35) were not found to be reliable enough for the identification in the study area. léger et al. (34) and killick-kendrick et al. (35) provided the figures of the base of spermathecal duct for ph. tobbi using the specimens from syria and greece. in the present article, the photos (figs. 2–4) were taken from specimens collected from localities close to the type locality (rasht of guilan province). conclusion this is the first investigation of sand flies in guilan province which includes six new records for the province. this study verifies the presence of ph. neglectus (ph. major krimensis as a synonym and morphotype) in iran. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 302–316 b norouzi et al.: an inventory of the … 313 http://jad.tums.ac.ir published online: september 30, 2020 more investigation on the major group using morphological and molecular data is suggested. all collected phlebotomus species are suspected or proven vectors of cl and/ or vl in iran or adjoining countries. the investigation of bionomics of suspected or proven vectors and detecting the exact vectors of leishmaniasis and three-day fever by means of molecular specific tests in guilan province should be the subject of future studies. acknowledgements the research was supported financially by the research vice-chancellorship of guilan university of medical sciences (project number: 93062502). the authors appreciate the administrative support of dr sm rezvani, mr m mirzanejad, mr m moslem and mr a rasaei, department of disease control and prevention, health vice-chancellorship of guilan university of medical sciences, and the personnel of aforementioned vice-chancellorship for their cooperation in the field. the project was carried out in the research center of health and environment, school of health, guilan university of medical sciences, with the collaboration of health vice-chancellorship of guilan university of medical sciences. references 1. galati eab, galvis-ovallos f, lawyer p, léger n, depaquit j (2017) an illustrated guide for characters and terminology used in descriptions of phlebotominae 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(larroussius) major s.l. (psychodidae-diptera). status of species present in greece. bull soc pathol exot filiales. 80(2): 252–260 (french). 58. xiong gh, jin cf (1987) on the classification of the subgenus larrousius nitzulescu, 1931 and phlebotomus wui stat. nov. (diptera: phlebotomidae). zhongguo ji sheng chong xue yu ji sheng chong bing za zhi. 5(1): 45–48 (chinese). 59. leng yj, lane rp, lewis dj (1987) the identification of two phlebotomine sandflies from xinjian. annual bulletin of the guangdong society of parasitology. 8– 9: 40–43 (chinese). 60. perfil’ev pp (1969) 7. family phlebotomidae. in: bei-bienko gya (ed) keys to the insects of the european part of the ussr. volume v. diptera and siphonaptera part i. nauka publishers, leningrad, pp. 197–204 (sharma br, translator. amerind publishing co., new delhi, 1989) (russian). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 162 http://jad.tums.ac.ir published online: june 30, 2020 original article evaluation of larvicidal efficacy of ricinus communis (castor) plant extract and synthesized green silver nanoparticles against aedes albopictus muhammad waris, *shabab nasir, azhar rasule, iqra yousaf department of zoology, government college university, faisalabad, pakistan (received 14 nov 2018; accepted 19 may 2020) abstract background: aedes mosquitoes are the most important group of vectors having ability of transmitting pathogens including arboviruses that can cause serious diseases like chikungunya fever, dengue fever and zika virus in human. biosynthesis and the use of green silver nanoparticles (agnps) is an important step in the search of reliable and ecofriendly control of these vectors. methods: in this study an aqueous leaves extract of ricinus communis (castor) and silver nanoparticles (agnps) synthesized from this extract were evaluated as larvicidal agent for 2 nd and 3 rd instar larvae of the aedes albopictus. different concentrations (50, 100, 150, 200 and 250ppm) of plant extract and synthesized nanoparticles were prepared and applied on second and third instar larvae. the percent mortality was noted after 6, 12, 18, 24, 30, 36, 42 and 48h of exposure and subjected to probit analysis to calculate lc50 and lc90. results: synthesized ag + nanoparticles were characterized by uv-vis spectroscopy, fourier transform infrared spectroscopy (ft-ir), and energy-dispersive x-ray spectroscopy (xrd). the nanoparticles were more toxic against larvae of ae. albopictus with lc50 value (49.43ppm) and lc90 value (93.65ppm) for 2 nd instar larvae and lc50 (84.98ppm) and lc90 (163.89ppm) for 3 rd instar larvae as compared to the plant extract (149.58ppm, 268.93ppm) and (155.58ppm, 279.93ppm) for 2 nd and 3 rd instar larvae of ae. albopictus respectively after 48h. conclusion: our results suggest the extract of r. communis and synthesized nanoparticles as excellent replacement of chemical pesticides to control the vector mosquitoes. keywords: dengue mosquito; larvicidal; ricinus communis; mosquito larvae; agnps introduction mosquitoes cause a serious threat to public health (1–2). vector-borne diseases such as malaria, dengue, chikungunya, zika virus, japanese encephalitis, filariasis, are being spread by mosquitoes (3). these diseases are found all over the world and cause millions of deaths annually (4). pakistan is at the great risk of vectorborne diseases especially dengue due to it's over crowded cities, insanitation and poor vaccination. in pakistan, dengue cases are reported throughout the year but situation, usually, become worst in the post monsoon period (5). pakistan had the worst dengue epidemic in 2011, during which more than 20,000 cases and 300 deaths were reported officially. in khyber pakhtunkhwa of pakistan, july to end of september 2017 a total of, 52 926 cases of den gue fever including 38 deaths were reported (6) chikungunya virus was detected in 1983 (7) and more than 4000 cases have confirmed through qualitative rt-pcr. zika virus has reached near border areas in neighboring countries like china and india, so outbreak of the disease may occur in pakistan (8). the easy solution to avoid mosquito-borne diseases is the management of mosquito population. this management through chemicals causes health risks to human beings, environmental pollution and insecticidal resistance in mosquitoes (9). this prompted the need of searching for new chemical compounds and alternative strategies, as novel biological tools. so, medicinal plants can be used as an alternate for this purpose because these plants have *corresponding author: dr shabab nasir, e-mail: flourenceshabab@yahoo.com mailto:flourenceshabab@yahoo.com j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 163 http://jad.tums.ac.ir published online: june 30, 2020 many types of target specific, rapidly biodegradable, ecofriendly, and less toxic to human health larvicidal phytochemicals such as saponins, isoflavonoids, tannins, terpenes, steroids, etc. (1011). plants are good source of bioactive insecticidal phytochemicals that can kill mosquito larvae with high mortality rate (12-14) by bringing changes in development, midgut epithelium (15), mutation in dna and production of reactive oxygen species (16-17). they have different mechanisms of action that reduce the chance of resistance development in mosquitoes (18). biologists have begun the use these phytochemicals as larvicides to control the mosquitoes (19). one step ahead, green silver nanoparticles (agnps) synthesized from plant extracts are proved more toxic than phytochemical as larvicides (20). green silver nanoparticles (agnps) have high larvicidal effect because of small size ranging 1–100nm (21) and large surface area. these characteristics of agnps made them a unique larvicide at very low concentrations. these have been tested in a variety of entomological research because these are safe, low cost, easily available and have a simple easy biosynthesis process (21-22). ricinus communis (castor) plant belongs to a big family euphorbiaceae contains nearly about 300 genera and 7500 species. ricinus communis (castor) is a flowering plant, has high medicinal value for healthy human life. this plant is used as laxative, fungicide, anti-oxidant, antiasthmatic, antiulcer, wound healing, insecticidal and larvicidal agent. it has important phytochemicals like glycosides, alkaloids, flavonoids, steroids and saponins. that are helpful in controlling mosquitoes (23-24) (fig. 1). due to these reasons, the present study was designed to evaluate the larvicidal potential of the plant extract and agnps synthesized from this extract of r. communis (castor) against 2 nd and 3 rd larvae of ae. albopictus under laboratory conditions. uv-vis spectroscopy analysis, powdered x-ray diffraction (pxrd) and fourier transform infrared radiation (ftir) spectroscopy were used to confirm the biosynthesis of agnps. materials and methods preparation of leaf extract healthy and fresh leaves of the r. communis (castor) plants were collected (hand plucked) from the old campus of university of agriculture faisalabad, pakistan during the month of may, 2017. leaves were cleaned with cloth and washed with tap water to remove dust. then the leaves were dried in shady place at room temperature and grinded in an electric grinder (anex germany) (25). fifty grams powder of leaves was mixed with 250ml acetone as solvent in the soxhlet apparatus and boiled gently at boiling point range 55.5–56.5 ºc for complete extraction (8h) and stored at 4 ºc (26). preparation of green agnps silver nitrate (agno3) of sigma was purchased and 1mm solution of silver nitrate (agno3) was prepared in 250ml erlenmeyer flask in the darkness to avoid action of light. 10ml acetone plant extracts of r. communis (castor) was put in 250ml conical flask having 90ml of 1mm silver nitrate solution. two to three drops of 1% naoh were added for the adjustment of ph at 8 and mixed continuously by magnetic stirrer. this mixture was kept at 40 °c for one hour under clear sky condition for irradiation. colour change of the solution indicated the formation of agnps. reaction completed on attaining reddish brown colour. solution was cooled and stored in amber bottle at 4 °c, then centrifuged for three times at 5000rpm for 20 minutes to obtain pellets. purified suspension was made by dissolving pellets in double distilled water and was frozen for further use (27). characterization of agnps. the biosynthesized silver nanoparticles were characterized by uv-vis spectroscopy anaj arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 164 http://jad.tums.ac.ir published online: june 30, 2020 lysis, powdered x-ray diffraction (pxrd) and fourier transform infrared radiation (ftir) spectroscopy assistance through high tech central laboratory of government college university faisalabad. uv-vis absorbance spectroscopy. to monitor formation of the green silver nanoparticles, absorption spectra were taken at a scanning speed of 200 to 800nm using a cary 60 double beam uv-vis spectrophotometer (spectramax m3 molecular devices) operating at the resolution of 1nm. uv-vis spectra were recorded after 15 and 30 minutes (28). powdered x-ray diffraction (pxrd) the shape of structure and size of the silver nanoparticles was calculated through diffracted intensities at 40kv voltage and 30ma current with the range of 0°–80° 2θ in cukα radiation (rigaku, ultima iv, and x-ray diffractometer system). fourier transform infrared (ftir) spectroscopy the residue solution of 100ml was centrifuged at 5,000rpm for 10 minutes after reaction to remove impurities. to obtain pure pellet of agnps the supernatant was again centrifuged at 10000rpm for 60 minutes. all measurements were carried out in the range of 400–4000cm -1 at a resolution of 4cm -1 (29). fresh samples having volume of 1–2ml in aqueous form were sent for ftir analysis to hi-tech lab, government college university faisalabad. collection and rearing of mosquitoes larvae and pupae were collected with dippers from a forest near faisalabad, punjab, pakistan (31° 25' 7.3740'' n and 73° 4' 44.7924'' e, 192 meters above the sea level). collected immature stages of mosquito were brought back to the zoology lab, department of zoology, government college university, faisalabad, inside beakers closed with muslin cloth. larvae and pupae of ae. albopictus were identified with the help of identification keys (30–31), reared to adults in 1000ml beakers containing water under lab conditions at 25±5 °c and 80±5% rh (32). adults further reared in separate glass cages. male adults were fed with 10% sugar solution and females with blood on live white rats in separate glass cages for egg laying (33). larvae emerged from the eggs were reared in batches of 300 each, in 1200ml deionized water in stainless steel trays (35x 30x 5cm) for the bioassays (34). fifth generation larvae were used for the bioassay. bioassay group of 20 actively swimming 2 nd and 3 rd instar larvae (identified from the shed cuticle and from the size and colour of the larvae) of ae. albopictus were released in 250ml beaker containing 200ml distilled water separately. five concentrations including 50, 100, 150, 200 and 250ppm of larvicidal solution of r. communis extract and green agnps synthesized from the extract were prepared using distilled water and subjected for mortality assays separately. bioassay was conducted at 27±3 °c, 80±3% relative humidity (rh) and a photoperiod of 16: 8 (l: d) hours (35). the control was set up with dechlorinated tap water and five replications were done for each treatment. mortality rates were calculated using the who (3) bioassay protocol with slight modifications. the percentage mortalities were corrected by using abbott’s formula (36). the average larval mortality data was subjected to probit analysis using minitab -17 statistical software (2017) for calculating lethal concentration 50% (lc50) and 90% (lc90) of larvae and for getting dose and time mortality regression lines. j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 165 http://jad.tums.ac.ir published online: june 30, 2020 results synthesis of silver nanoparticles formation of green agnps through the reduction of the silver metal ions by the extract of r. communis that turned the colour of mixture (plant extract +agno3 solution) into reddish brown in 1h at 40 ºc (fig. 2). uv-vis spectrum of silver nanoparticles the progress of the reaction between ag + and the leaf extract was monitored by uv– visible spectra of silver nanoparticles in aqueous solution with different reaction times that are shown (fig. 3). it was observed from the figure that localized surface plasmon resonance band showed maximum absorbance at 430nm after 30 minutes of reaction time. powdered x-ray diffraction (pxrd) studies result of pxrd (fig. 4) showed intense silver nanoparticle (agnps) diffraction peaks at 38.10, 44.46, 64.45, 77.51, and 81.60, corresponding to facets 113, 202, 221, 310, and 223 of the face-centered cubic crystal structure. fourier transform infrared radiation spectroscopy (ftir) analysis the ftir spectra of silver nanoparticles prepared from the r. communis leaf extract (fig. 5) showed transmittance peaks at 1263.2, 978.6, 849.1, 710.5, 662.8, 502.7, and 435.6/cm. the carbonyl group formed amino acid residues which capped the silver nanoparticles indicated by these peaks. these residues prevent from agglomeration of agnps, and made the medium stable. ftir clearly indicate role of proteins and other compounds of leaf extract in the formation and stabilization of agnps. larvicidal activity of leaf extracts and synthesized silver nanoparticles the results of the larvicidal activity of leaf extract of r. communis (castor) and synthesized agnps with different concentrations (50–250ppm) after different exposure times (6, 12, 18, 24, 30, 36, 42 and 48h) showed a dose and time-dependent toxic effects against 2 nd and 3 rd instar larvae of ae. albopictus. no mortality was observed in the control group. agnps synthesized from r. communis showed 100% mortality for all the exposed larvae after 36h at the concentration of 250ppm (table 1). the synthesized agnps showed least values of lc50 (49.43ppm) and lc90 (93.65ppm) after 48h with regression equation y= -1.208+0.1521x against 2 nd instar larvae of ae. albopictus. similarly for the 3 rd instar larvae, the least values of lc50 and lc90 were 84.98 and 163.89ppm respectively after 48h with regression equation y= 1.072+0.1461x as shown in table 1. the mortality rate of 2 nd and 3 rd instar larvae of ae. albopictus was noted as 98 and 96% respectively after 48h at 250ppm concentration of r. communis leaves extract (table 2). the least values of lc50 and lc90 were 149.57 and 268.92ppm for 2 nd instar larvae and 155.57 and 279.92ppm for 3 rd instar respectively with regression equations y= 1.16+0.129x and y= -1.210+0.113x after 48h. the extract of r. communis exhibited prominent larvicidal activity against the 2 nd instar larvae of ae. albopictus. all the concentrations of plant extracts used in the present study exhibited repellency activity. fig. 1. ricinus communis (castor) plant (original photo) j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 166 http://jad.tums.ac.ir published online: june 30, 2020 table 1. larvicidal activity of agnps synthesized from r.communis against ae. albopictus larvae time larval instars %mortality±sd lethal concentration fl at 95% c.i chisquare p value regression equation 6h 2 nd 10±0.27 lc50 523.02 384.98-1129.65 3.52 0.319 y= -2.30+0.0044x lc90 813.34 564.21-1925.07 3.52 0.319 y= -2.30+0.0044x 3 rd 9±0.27 lc50 565.14 400.52-1494.50 3.72 0.292 y= -2.27+0.0040x lc90 883.53 589.09-2568.35 3.72 0.292 y= -2.27+0.0040x 12h 2 nd 15±0.24 lc50 471.43 363.43-827.90 2.16 0.539 y= -2.19+0.0046x lc90 747.16 543.88-1430.95 2.16 0.539 y= -2.19+0.0046x 3 rd 10±0.21 lc50 513.08 380.19-1034.82 1.34 0.712 y= -.96+0.0038x lc90 847.98 591.32-1873.27 1.34 0.712 y= -1.96+0.0038x 18h 2 nd 20±0.20 lc50 437.37 344.45-706.71 0.54 0.909 y= -1.96+0.0038x lc90 728.90 541.17-1285.97 0.54 0.909 y= -1.96+0.0038x 3 rd 16±0.21 lc50 513.08 380.19-1034.82 0.13 0.718 y= -.96+0.0038x lc90 847.98 591.32-1873.27 0.13 0.718 y= -1.96+0.0038x 24h 2 nd 25±0.19 lc50 384.70 316.42-547.57 0.84 0.809 y= -1.89+0.0049x lc90 645.16 501.53-998.63 0.84 0.809 y= -1.96+0.0038x 3 rd 20±0.19 lc50 455.96 351.97-786.41 0.17 0.918 y= -1.83+0.0040x lc90 774.01 561.84-1463.36 0.17 0.918 y= -1.83+0.0040x 30h 2 nd 50±0.16 lc50 245.24 222.17-280.11 0.34 0.809 y= -1.70+0.0059x lc90 430.12 526.76-372.84 0.34 0.809 y= -1.70+0.0059x 3 rd 35±0.19 lc50 339.95 291.79-435.60 0.17 0.244 y= -1.99+0.0058x lc90 558.36 455.79-770.54 0.17 0.244 y= -1.99+0.0058x 36h 2 nd 70±0.15 lc50 188.19 172.39-207.37 0.77 0.809 y= -1.42+0.0075x lc90 357.11 317.50-418.58 0.77 0.809 y= -1.42+0.0075x 3 rd 50±0.16 lc50 242.61 220.36-275.70 4.17 0.253 y= -1.719+0.0070x lc90 423.45 368.57-514.79 4.17 0.253 y= -1.719+0.0070x 42h 2 nd 100±0.15 lc50 104.99 90.40-117.45 5.77 0.033 y= -1.43+0.1545x lc90 217.85 202.21-238.34 5.77 0.033 y= -1.43+0.1545x 3 rd 95±0.14 lc50 115.02 103.75-125.31 1.67 0.000 y= -1.077+0.0108x lc90 229.82 211.09-225.49 1.67 0.00 y= -1.077+0.0108x 48h 2 nd 100±0.12 lc50 49.43 37.51-59.44 6.77 0.032 y= -1.208+0.1521x lc90 93.65 81.06-110.33 6.77 0.032 y= -1.208+0.1521x 3 rd 100±0.12 lc50 84.98 70.40-101.45 1.27 0.303 y= -1.077+0.1461x lc90 163.89 151.09-175.49 1.27 0.303 y= -1.077+0.1461x lc50: lethal concentration that kills 50% of the exposed larvae; lc90: lethal concentration that kills 90% of the exposed larvae. j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 167 http://jad.tums.ac.ir published online: june 30, 2020 table 2. larvicidal activity of leaf extracts of r. communis against ae.s albopictus larvae time larval instars %mortality±sd lethal concentration fl at 95% c.i chisquare p value regression equation 6h 2 nd 9±0.28 lc50 572.94 403.52-1584.71 5.12 0.163 y= -2.31+0.004x lc90 889.79 589.68-2705.85 5.12 0.163 y= -2.31+0.004x 3 rd 6±0.27 lc50 725.56 448.49-1927.47 4.22 0.238 y= -2.19+0.003x lc90 1148.36 664.68-2035.2 4.22 0.238 y= -2.19+0.003x 12h 2 nd 15±0.22 lc50 537.82 403.69-1002.15 1.35 0.717 y= -2.06+0.041x lc90 778.85 559.72-1546.13 1.35 0.717 y= -2.06+0.041x 3 rd 11±0.27 lc50 725.56 448.49-1927.47 4.22 0.238 y= -2.19+0.003x lc90 1148.31 664.68-2035.20 4.22 0.238 y= -2.19+0.003x 18h 2 nd 25±0.20 lc50 407.93 329.78-608.36 0.92 0.81 y= -2.06+0.041x lc90 679.10 518.69-1101.86 0.92 0.81 y= -2.06+0.041x 3 rd 18±0.19 lc50 507.42 374.13-1045.96 0.06 0.995 y= -1.79+0.003x lc90 869.61 601.13-1974.48 0.06 0.995 y= -1.79+0.003x 24h 2 nd 30±0.16 lc50 313.16 265.99-411.52 2.08 0.554 y= -1.48+0.005x lc90 583.50 465.24-845.33 2.08 0.554 y= -2.06+0.041x 3 rd 22±0.19 lc50 470.34 356.20-869.28 0.49 0.921 y= -1.18+0.004x lc90 820.08 582.04-1670.4 0.49 0.921 y= -1.18+0.004x 30h 2 nd 40±0.16 lc50 313.16 265.99-411.52 2.08 0.554 y= -1.48+0.005x lc90 583.50 465.24-845.33 2.08 0.554 y= -1.48+0.041x 3 rd 30±0.18 lc50 370.21 303.81-530.08 0.054 0.997 y= -1.65+0.004x lc90 656.65 506.69-1031.3 0.054 0.997 y= -1.65+0.004x 36h 2 nd 50±0.16 lc50 258.97 229.21-309.36 0.67 0.880 y= -1.46+0.006x lc90 485.52 406.70-633.31 0.67 0.880 y= -1.46+0.046x 3 rd 40±0.17 lc50 317.15 272.97-403.39 2.05 0.532 y= -1.71+0.005x lc90 554.63 451.92-766.05 2.05 0.532 y= -1.71+0.005x 42h 2 nd 88±0.15 lc50 197.93 176.04-209.75 3.26 0.114 y= -1.51+0.153x lc90 323.98 301.79-336.20 3.26 0.114 y= -1.51+0.153x 3 rd 87±0.15 lc50 276.36 244.35-288.73 0.55 0.832 y= -1.517+0.010x lc90 372.02 350.53-401.52 0.55 0.832 y= -1.517+0.010x 48h 2 nd 98±0.14 lc50 149.57 138.29-159.74 2.06 0.518 y= -1.16+0.129x lc90 268.92 254.26-280.00 2.06 0.518 y= -1.16+0.129x 3 rd 96±0.15 lc50 155.57 139.29-170.74 1.65 0.102 y= -1.210+0.113x lc90 279.92 252.26-295.00 1.65 0.102 y= -1.210+0.113x lc50: lethal concentration that kills 50% of the exposed larvae; lc90: lethal concentration that kills 90% of the exposed larvae j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 168 http://jad.tums.ac.ir published online: june 30, 2020 fig. 2. bioreduction of agno3 into agnps using plant extract (colour change) fig. 3. ultraviolet-visible spectra of silver nanoparticles synthesized by treating r. communis leaf extract with 1 mm agno3 solution fig. 4. powdered x-ray diffraction j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 169 http://jad.tums.ac.ir published online: june 30, 2020 fig. 5. fourier transform infrared radiation spectra of agnps synthesized from leaf extract of r. communis (castor) discussion nanotechnology is an emerging technology in modern era that enables scientists to synthesize particles of different sizes, forms and components. hence synthesized nanoparticles of gold, silver and platinum are being used for insect vectors control and in pharmaceutical industries (17). during current study, change in colour (reddish brown) of the solution indicated the formation of agnps due to the reduction of silver metal ions by the extract of r. communis and was confirmed by the localized surface plasmon resonance band absorbance at 430nm after 30 minutes of reaction time. previous studies also indicated the similar colour changes and maximum absorbance at 430nm (37). our result of pxrd indicated intense silver nanoparticle (agnps) diffraction peaks at 38.10, 44.46, 64.45, 77.51 and 81.60 corresponding to facets 113, 202, 221, 310 and 223 of the face-centered cubic crystal structure. sathyavathi et al. (25) also reported diffraction peaks at 44.50, 52.20, and 76.7, which correspond to the 111, 200, and 222 facets of the face-centered cubic crystal structure. xrd result of silver nanoparticles reported by nirmala et al. (38) is also close to the cited results. the ftir spectra of current study showed transmittance peaks at 1263.2, 978.6, 849.1, 710.5, 662.8, 502.7, and 435.6/cm. the carbonyl group formed amino acid residues which capped the silver nanoparticles indicated by these peaks. these residues prevent from agglomeration of agnps, and made the medium stable. ftir clearly indicate role of proteins and other compounds of leaf extract in the formation and stabilization of agnps (37–40). in our study, the r. communis agnps showed 100% mortality at 250ppm for ae. albopictus after 48h, with lc50 and lc90 values 49.43, 93.65ppm and 84.98, 163.89ppm for 2 nd and 3 rd instar larvae respectively, while lc50 and lc90 values of the leaf extract of r. communis after 48h exposure were 149.57, 268.92ppm and 155.57, 279.92ppm for 2 nd and 3 rd instar larvae respectively. these results clearly indicated that r. communis agnps were more potent than leaf extract of r. communis due to less lc50 and lc90 values. these results are in line with the results of other scientists who also reported more potency of agnps than simple plant extracts (38–39). karthikeyan et al. (39) also reported the toxicity of agnps synthesized from euphorbia hirta leaf extract against an.stephensi 1 st to 4 th instar larvae with lc50 (10.14, 16.82, 21.51, and 27.89ppm, respectively) after 24h. this high potency of agnps was due to high surface area-to-volume ratio, that imparts different biological and catalytic activities in them (30–40). hemant et al. (40) also reported the potency of agnps of euphorbia j arthropod-borne dis, june 2020, 14(2): 162–172 m waris et al.: evaluation of … 170 http://jad.tums.ac.ir published online: june 30, 2020 tirucalli against 2 nd and 4 th instar larvae with least lc50 values (3.50 to 7.01ppm) and (4.44 to 8.74ppm) respectively after 24 hrs. our findings are at par with the previous findings that simple leaf extracts are less potent than agnps. the larvicidal effects of leaves extracts of r. communis showed the lc50 values of 1091.44, 1364.58 and 1445.44ppm against 2 nd , 3 rd and 4 th larval instars of cx. quinquefasciatus (41). basheer (42) also used r. communis extracts through different solvents and found ethyl acetate extract more potent than others against 3 rd instar larvae of ae. albopictus. he also noted that lc50 values decreased with time. these results are similar with our findings. mandal (43) also noted that r. communis seed extract exhibited larvicidal effects with 100% mortality at concentrations 32–64μg/ml, with lc50 value 16.84μg/ml for ae. albopictus larvae. all previous scientists studied either plant extracts or their agnps separately but the present study compared the plant extract with its agnps. cited results are close to our findings but not exactly same due to using different plant, mosquito species, larval stage and solvent for plant extraction. results of our study suggested that the leaf extract of r. communis is toxic to ae. albopictus larvae and toxicity increased when extract combined with agnps. our results clearly proved the excellent larvicidal efficacy of r. communis against ae. albopictus. conclusion it is concluded from our findings that, the leaf extract and synthesized silver nanoparticles of r. communis had excellent potential for killing the of mosquito larvae. the application of this plant extract along with silver nanoparticle on mosquito breeding places surely decrease the population of vector mosquitoes, control many dreadful 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biotherapeutic dis. 3: 1. 41. elimam am, elmalik kh, ali fs (2009) larvicidal, adult emergence inhibition and oviposition deterrent effects of foliage extract from ricinus communis l. against anopheles arabiensis and culex quinquefasciatus in sudan. trop biomed. 26(2): 130–139. 42. basheer agm (2014) ricinus communis (castor) as larvicide on anopheles arabiensis patton. int j adv pha biol chem. 3: 2277–4688. 43. mandal s (2010) exploration of larvicidal and adult emergence inhibition activities of ricinus communis seed extract against three potential mosquito vectors in kolkata, india. asian pac j trop med. 3(8): 605–609. j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 206 http://jad.tums.ac.ir published online: june 24, 2019 original article identification of alleles in the msp1 gene related to complicated malaria in patients infected with plasmodium falciparum in southeast of iran bentol hoda habibi-shorkaei1; afsaneh motevalli-haghi1; *mehdi nateghpour1, 2; leila farivar1; homa hajjaran1; soudabeh etemadi1 1department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran (received 20 jun 2017; accepted 12 jan 2019) abstract background: to overcome human malaria problem several solutions have been employed including extensive studies in the field of plasmodia relevant antigens. the aim of this study was to determine allelic variation in the msp1 gene of plasmodium falciparum among some falciparum malaria-infected patients in southeastern iran. methods: twenty p. falciparum positive cases were enrolled from sistan and baluchistan province, southeastern iran in 2013–15. from each case, 1.5ml of peripheral blood was collected into edta contained tubes. thick and thin blood smears were stained with standard giemsa stain and were checked with conventional microscopical method. dna was extracted from blood samples and amplification of block 2 msp1 was performed using specific primers. gel electrophoresis was done and results showed some amplification fragments corresponding to block 2 regions of pf msp1 gene. finally, four samples from different allelic types were sent for sequencing process. results: fragments were different in size, so classified into six allelic types as kinds of 1–6 based on happening frequencies. digestion of pcr products revealed two sub allelic types (a and b) within allelic types 2 and 3, but not in allelic types 1, 4, 5 and 6. twenty percent of samples were sent for sequencing. sequence alignment showed 78.95% to 91.83% identity between samples. conclusion: identity between samples and phylogenetic tree revealed that there is an extensive diversity range among isolates. fifty percent of the isolates were under the risk of complicated malaria. two of these patients (10%) needed special care and recovery was obtained after getting hospital services. keywords: plasmodium falciparum; sever malaria; iran; pcr-rflp introduction malaria is an ancient problem created many calamities against human for many centuries. parasites of the infection cause illness to 216 million persons and almost 445000 deaths per year. pregnant women and children under 5yr old are two greatest groups damaged from this infection (1–3). the fearful condition of the disease in human is caused by plasmodium falciparum. cerebral malaria, blackwater fever, severe anemia and some other disorders occur in individuals who infected with p. falciparum. such problems can result in death among the infected people if is not being treated with suitable drug. to combat these conditions several malaria vaccines were produced and tested amongst some volunteer in clinical trials, but with poor consequences due to some reasons including gene diversities phenomenon in p. falciparum isolates. however, diversities in msp1 gene demonstrate different alleles which such diversities can reveal different aspects of phenomena in p. falciparum. one of the most important aspects of the phenomena is emergence a number of complications in patients who infected with p. falciparum (4, 5). presence of some msp1 sub allelic types have been seen in those individuals who infect*corresponding author: dr mehdi nateghpour, e-mail: nateghpourm@tums.ac.ir j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 207 http://jad.tums.ac.ir published online: june 24, 2019 ed with cerebral malaria. studies of genetic diversity can provide valuable data in different aspects of p. falciparum malaria such as forecasting complicated cases in the infection. plasmodium falciparum msp1 gene produces a surface protein in merozoites in blood schizogony phase and it is a major candidate for providing a vaccine (6), but its notable diversity in different isolates is an obstacle within the progress. the gene produces a 190kda glycoprotein including seven variable blocks putting at different places among five conserved and five semi-conserved blocks (5). diversity recognition of the gene is important to determine alleles resulting severe malaria. the aim of this study was to determine allelic variation in the msp1 gene of p. falciparum among some malaria patients living in malarious areas of sistan and baluchistan province of iran in view of presence or absence of msp1 alleles that cause cerebral malaria, using pcr-rflp technique. materials and methods study area sistan and baluchistan province is located in southeastern iran. the latitude and longitude of the province are 27º0′ n and 62º0′ e respectively. the weather is warm and low humid in north of the province with average temperature of 31 ºc, and warm and humid in south with average temperature of 30.2 ºc. plasmodium vivax and p. falciparum are prevalent plasmodia species in the studied area. moreover, anopheles culicifacies and an. stephensi are the main vectors in southeastern iran. the province is located on neighboring pakistan and southwestern part of afghanistan with 978 and 300km joint borderline respectively. immigration load of afghani and pakistani population in the province is high with notable imported malaria infection. all the samples for this study were collected from those individuals who were living for long time in the studied areas. blood samples twenty p. falciparum positive cases were enrolled between 2013 and 2015 for this study. from each case, 1.5ml of peripheral blood was collected into edta contained tubes. previous to sampling an informed consent was prepared of the registered individuals. detection of parasites thick and thin blood smears were stained with standard giemsa stain and the slides were checked with conventional microscopical method with two expert microscopists. mix infections were taken out from the study. samples were stored at -20 ºc until dna extraction processes. dna extraction dna of p. falciparum was extracted from blood samples by using a high pure pcr template preparation kit (roch ref: 11796828001) according to the manufacturer instructions. pcr-rflp pcr amplification of the block2 msp1 was performed in 25µl reaction that containing, 10 µl distilled water, 10µl master mix, 3ng dna, 1µl forward primer, 1µl revers primer. designing primers and pcr condition were performed (7). the sets of following primers were used for amplification: forward 5′ctagaagcttta gaagatgcagtattg3′ revers 5′gtacg tctaattcatttgcacg aat3′ pcr amplification was carried out using initial denaturation about 5min in 95 ºc, 30 cycles of 1 min at 94 ºc, 2min in 57 ºc, 2min in 72 ºc and final extension 5min in 72 ºc in a thermal cycler. four µl of pcr product was run on 1.5% agarose gel with simply safe (eurex, cat: e 4600-01). finally the process was visualized with gel duct and resulted in some photographs. rflp (restriction fragment length polymorphism) product amplification of msp1 gene block 2 zones was digested with dra-1 endonuclease with active site of ttt aaa (cat: fd0224). j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 208 http://jad.tums.ac.ir published online: june 24, 2019 the digestion process was carried out in a 30 µl composition of 17µl distilled water, 2µl 10 x fast digest buffer, 10µl pcr product and 1 µl fast digest enzyme. the compound was incubated at 37 ºc for 5min and the incubation was continued at 65 ºc for 5min to inactive the enzyme. overall 18µl of the digested pcr product was analyzed on a 3% agarose gel and then stained with simply safe stain. visualizing process was conducted as mentioned previously. sequencing four pcr products were selected for sequence processes and analyzed with clustalw2 and blast software. results genetic diversity of p. falciparum block2 msp1 gene was considered from clinical isolates collected from sistan and baluchistan province in southeastern iran (table 1). all of the 20 isolates showed gene amplified expression in block 2 regions of msp1 gene. variation of amplified products was identified from 480 to 600bp (fig. 1). the isolates were classified into six allelic types as kinds of 1–6 based on happening frequencies. type 1 possessed the highest frequency (50%) and allelic types 4, 5, 6 the lowest frequency (5%) (table 2). pcr products were digested with dra-1 restriction enzyme. rflp identified the subtypes of allelic types among the isolates. digestion of pcr products revealed two sub allelic (types a and b) within allelic types 2 and 3. while allelic types 1, 4, 5, 6 did not show any subtype (table 3, fig. 2). the phylogenetic tree showed a wide diversity among the studied samples which some of them had considerable homology with 3d7 isolate and some others had more homology with hn2 china isolate. the belem strain of p. vivax was used as outgroup for this analysis (table 4). table 1. demographic data of patients based on nationality, gender and age nationality gender age (yr) total male female < 20 20–40 > 40 iranian 10 4 3 9 2 14 afghan 2 2 1 2 1 4 pakistani 0 2 1 1 0 2 total 12 8 5 12 3 20 table 2. allelic types of msp1 (block2) of plasmodium falciparum isolates based on molecular weight of pcr products (n= 20) size of pcr product allelic type % no of p. falciparum isolate allelic frequency % 500bp 1 10 50 550bp 2 5 25 600bp 3 2 10 480bp 4 1 5 570bp 5 1 5 580bp 6 1 5 j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 209 http://jad.tums.ac.ir published online: june 24, 2019 fig. 1. pcr product of msp1 gene (block 2) of plasmodium falciparum eight isolates were visualized with 2% agarose gel and stained with simply safe. m= dna size marker; lane1–8 isolates, nc= negative control table 3. allelic and sub allelic types of msp1 (block2) of plasmodium falciparum isolates on the basis of pcr-rflp (digested with dra-1 restriction enzyme) (n= 20) allelic type size of pcr product no of isolates sub allelic type size of digested fragment no of isolate frequency % 1 500 10/20=50% --------250,250 10/10 100 2 550 5/20=25% 2a 2b 270,280 250,300 3/5 2/5 60 40 3 600 2/20=10% 3a 3b 280,320 250,350 1/2 1/2 50 50 4 480 1/20=5% -------250,230 1/1 100 5 570 1/20=5% -------280,290 1/1 100 6 580 1/20=5% -------280,300 1/1 100 table 4. percent identity among isolates collected from iran (accession number: ky026097.1, kyo26098.1, ky026095.1, ky026096.1) and plasmodium falciparum 3d7 (accession number xm_001352134.1) and hn2 isolate from china (accession number af062349.1). plasmodium vivax belem isolate used an out group 1 2 3 4 5 6 7 vivax 3 d7 if 77a if 140 if 133 if 95a china 100.00 43.69 43.54 43.74 40.41 39.28 41.36 43.69 100.00 90.26 92.20 78.21 79.71 83.63 43.54 90.26 100.00 91.83 78.95 79.15 78.85 43.74 92.20 91.83 100.00 80.38 79.75 79.27 40.41 78.21 78.95 80.38 100.00 90.37 91.10 39.28 79.71 79.15 79.75 90.37 100.00 91.60 41.36 83.63 78.85 79.27 91.10 91.60 100.00 j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 210 http://jad.tums.ac.ir published online: june 24, 2019 fig. 2. rflp pattern of msp1 gene block 2(digested with dra-1) plasmodium falciparum isolates. digested products were resolved on 4% agarose gel. lane m-dna size marker, lane1 type 4 allele, lane 2 type 3 allele (3a sub allel), lane 3 type 1 allele, lane 4 type 5 allele, lane 5 type 6 allele, nc: negative control. fig. 3. partial nucleotide sequences alignment of p.f. msp1 gene in plasmodium falciparum isolated collected from malarious areas of iran and were compared to the genes of china (hn2), vivax (belem) and 3d7 reference strains. j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 211 http://jad.tums.ac.ir published online: june 24, 2019 fig. 4. phylogenetic tree between isolates collected from southeastern iran (accession number: ky026097.1, kyo26098.1, ky026095.1, ky026096.1) and plasmodium falciparum 3d7 (accession number xm_001352134.1) and china hn2 (accession number af062349.1). plasmodium vivax belem isolate used an out group. discussion investigations about genetic diversities in parasite population can prepare much information in the field of geographical distribution, genomic markers, clinical manifestation, drug resistance and gene polymorphism for relevant researchers (8-12). genetic diversity plays a crucial role in the natural acquisition of immunity in malaria disease and it is a major problem in the pathway of control strategies to prevent the distribution of the infection. combating drug resistance in malaria parasites or producing potential vaccines require comprehension of population structure of the parasite (13). for this purpose, a lot of studies have been conducted by researchers to access genomic markers and polymorphism of the parasite. among the studies about diversity of pfmsp1 gene, a few of them have been focused on relationship between the gene and probable malignancy of p. falciparum (14). there is a significant correlation between diversity in msp1 gene and complicated cases of malaria, and in contrast to them, some others mentioned there is no association among them (15-18, 7, 10). obtaining different results about the mentioned subject are due to the different employed methods to found diversity in this gene. for example nested pcr was used to found frequency of k1, mad20 and ro33 alleles in the parasite population (18-20). however, pcr-rflp was used to report diversity in the gene and reported five allelic types. nine different alleles were reported from of 67 isolates and mentioned msp1 gene in p. falciparum have shown an extended variation in size and pattern (19). any correlation was mentioned between these alleles and malignancy in p. falciparum. there was no significant relationship between genetic diversity of msp1 and severity in malaria, however, mad20 allelic type increased with the severity of malaria. on the other hand, there are a few alleles in pf msp1 gene which are more predominant in cerebral malaria when they used pcr-rflp to investigate diversity in the gene. five allelic types including eight genetic types were identified, they implied that sub allelic types 2a and 3a with 300–200 and 280–200bp were more prevalence in patients got involved with cerebral malaria these sub allelic types were recognized when they used pcr-rflp with dra-1 restriction enzyme. moreover, sub allelic 2b with 250–25bp was seen in both cerebral and noncerebral malaria cases. presence of certain alleles may cause severe disease in patients. in the present study six allelic types were classified by pcr-rflp, also two sub allelic types were identified when pcr products digested with dra-1, results showed that there is a large diversity in the weight of the products fragments when compared with farooq et al results (17). so, diversity of the gene is high in various areas. anyway, 65% of the patients who registered in the present study showed allelic types like reported by farooq and 50% were under the risk of complicated malaria (17). two of these patients (10%) needed special care and j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 212 http://jad.tums.ac.ir published online: june 24, 2019 recovery was obtained after getting hospital services. isolating the genes that involve in producing cerebral malaria provides a valuable opportunity for prevention of deleterious complications of falciparum malaria in endemic areas. moreover, sequence alignment among the samples showed 78.95% to 91.83% identity. there is an extension of diversity among the samples. phylogenetic tree revealed that there is an extensive diversity range between isolates so that some of the isolates achieved in the present study showed a close relationship with isolate hn2 from china and the others have this association p. falciparum 3d7. there is high diversity in the gene in the specific population of the parasite. conclusion msp1 gene of p. falciparum in iranian isolates showed an extensive diversity, moreover, potential characteristics to produce sever malaria particularity cerebral malaria is a burglar alarm for the health authorities to provide accessible facilities for residents in malaria endemic area. acknowledgements the authors would like to convey their gratitude to mr abdolsamad amiri and hamid azariyan for their useful helping in sample collection. we also express our thanks to the staff of department of medical parasitology and mycology, school of public health, tehran university of medical sciences. this work was supported financially by the school of public health, tehran university of medical sciences (tums) under the code no. 3039427-01-95. the authors declare that there is no conflict of interests. references 1. philips rs (2001) current status of malaria and potential for control. j clin microbiol rev. 14: 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plasmodium falciparum isolates by the pcr and potential uses in the epidemiological studies. bull world health organ. 73(1): 85–95. 8. contamin h, fandeur t, bonnefoy s, skouri f, ntoumi f, mercereau-puijalon o (1995) pcr typing of field isolates of plasmodium falciparum. j clin microbiol. 33(4): 944–951. 9. kaneko o, kimura m, kawamoto f, ferreeira mu, tanabe k (1997) plasmodium falciparum: allelic variation in the merozoite surface protein 1gene in wild isolates from southern vietnam. exp parasitol. 86(1): 45–57. j arthropod-borne dis, june 2019, 13(2): 206–213 bh habibi-shorkaei et al.: identification of … 213 http://jad.tums.ac.ir published online: june 24, 2019 10. snounou g, zhu x, siripoon n, jara w, thaithong s, brown kn, viriyakosol s (1999) baised distribution of msp1 and msp-2 allelic variants in plasmodium falciparum populations in thailand. trans r soc trop med hyg. 93(4): 369–374. 11. zwetyenga j, rogier c, tall a, fontenille d, snounou g, trape jf, mercereaupuijalon o 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falciparum: a rapid technique for genotyping the merozoite surface protein -2. exp parasitol. 77: 372–375. 16. felger i, taval l, kabintik s, marshal v, gentonb, alpers m, beekh-p (1994) plasmodium falciparum extensive polymorphism in msp-2 in an area with endemic malaria in papua new guinea. exp parasitol. 79: 106–116. 17. farooq u, dubey ml, malla n, mahajan rc (2006) plasmodium falciparum: polymorphism in the msp-1gene in indian isolates and predominance of certain alleles in cerebral malaria. exp parasitol. 112: 139–143. 18. yohanna s, alfred p (2015) genetic diversity in msp-1gene of plasmodium falciparum and its association with malaria severity, parasite density, and host factors of asymptomatic and symptomatic patients in papua, indonesia. int j med sci public health. 4(11): 1584–1593. 19. heidari a, keshavarz h, rokni mb, jelinek t (2007) genetic diversity in merozoite surface protein (msp)-1 and msp2 genes of plasmodium falciparum in a major endemic region of iran. korean j parasitol. 45(1): 59–63. 20. bouyou-akotet mk, mbondoukwe np, mawili-mboumba dp (2015) genetic polymorphism of merozoite surface protein1 in plasmodium falciparum isolates from patients with mild to severe malaria in libreville, gabon. parasite. 22: 12. https://www.ncbi.nlm.nih.gov/pubmed/?term=keshavarz%20h%5bauthor%5d&cauthor=true&cauthor_uid=17374980 https://www.ncbi.nlm.nih.gov/pubmed/?term=rokni%20mb%5bauthor%5d&cauthor=true&cauthor_uid=17374980 https://www.ncbi.nlm.nih.gov/pubmed/?term=jelinek%20t%5bauthor%5d&cauthor=true&cauthor_uid=17374980 https://www.ncbi.nlm.nih.gov/pubmed/?term=jelinek%20t%5bauthor%5d&cauthor=true&cauthor_uid=17374980 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2526331/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2526331/ https://www.ncbi.nlm.nih.gov/pubmed/?term=bouyou-akotet%20mk%5bauthor%5d&cauthor=true&cauthor_uid=25786326 https://www.ncbi.nlm.nih.gov/pubmed/?term=m%27bondoukw%c3%a9%20np%5bauthor%5d&cauthor=true&cauthor_uid=25786326 https://www.ncbi.nlm.nih.gov/pubmed/?term=mawili-mboumba%20dp%5bauthor%5d&cauthor=true&cauthor_uid=25786326 https://www.ncbi.nlm.nih.gov/pubmed/25786326 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 195 original article bovine ephemeral fever in iran: diagnosis, isolation and molecular characterization *mehran bakhshesh 1, darab abdollahi 2 1animal virology department, research and diagnosis, razi vaccine and serum research institute, karaj, iran 2iranian veterinary organization, tehran, iran (received 16 mar 2014; accepted 28 june 2014) abstract background: bovine ephemeral fever (befv) is an arthropod-borne disease of cattle and water buffaloes. befv occurs seasonally in tropical, subtropical and temperate regions of africa, asia and australia. it has been known for the past decades in iran based on clinical signs but lack of an accurate diagnosis has made the real feature of disease obscured. this is the first scientific report on isolation and identification of the agent in which molecular diagnosis of befv was also set up with high sensitivity and specificity. methods: the viral agent was successfully isolated through serial passages in brain of suckling mice and cell culture. in addition, the circulating virus during the autumn 2012 in iran was molecularly characterized based on partial g gene. results: alignment of 3 virus sequences from different parts of iran revealed that they are identical suggesting that the circulating viruses were most likely the same in this period. phylogenetic analysis of the iranian sequences with 17 sequences in the genbank from the world showed that it is identical to the virus circulated in turkey during the same period suggesting that the virus was circulated in a large geographic region. conclusion: these results offer primary information about befv in iran. to better understanding the epidemiology of the virus, further studies based on seroepidemiology, molecular epidemiology, entomology and meteorology together with finding the model of animal transportation in the region are necessary. keywords: bovine ephemeral fever, iran, diagnosis, isolation, phylogenetic analysis introduction bovine ephemeral fever (befv) is a noncontagious, arthropod-borne disease of cattle and water buffaloes proposed to be transmitted by culicoides biting midges and mosquitoes. befv has been isolated from culicoides brevitarsis, culicoides coarctatus, and anopheles bancroftii, however the arthropod vectors are yet to be defined (walker et al. 2012). the viral agent is classified as the type species of the genus ephemerovirus within the rhabdoviridae family. befv is bullet shaped and its genome consists of five structural protein genes; n, p, m, g and l (walker et al. 2012). the g gene encodes glycoprotein g which is responsible for cell attachment and entry, and also contains typespecific and neutralizing antigenic sites (cybinski et al. 1990, walker et al. 1991). befv occurs seasonally in tropical, subtropical and temperate regions of africa, asia, australia and the middle-east and is a disabling disease with considerable economic impact due to loss of milk production, loss of condition in beef herds, infertility and abortion. the morbidity rate is high (up to 100%), however the mortality rate is generally low (rarely exceeds 1%). typical clinical signs include a sudden onset of fever as high as 41 °c, a sudden and severe drop in milk production, inappetence, lethargy, sali*corresponding author: dr mehran bakhshesh, e-mail: m.bakhshesh@rvsri.ac.ir http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 196 vation, nasal discharge, depression, stiffness, ruminal stasis and dyspnea (walker et al. 2012). treatment is based on remission of clinical signs using non-steroidal anti-inflammatory drugs and administration of calcium borogluconate to hypocalcemic animals. vaccination is considered as the only effective approach to control the disease. liveattenuated, inactivated, subunit and recombinant vaccines have been applied experimentally and commercially while live vaccines generally induce more-prolonged immunity (radostitis et al. 2007). because of the short course of viremia in affected animals and proper shipping requirements of blood samples to the laboratory, detection and isolation of befv is not usually successful. bovine ephemeral fever has been reported for the past decades in iran and some efforts have been taken to isolate the virus (hazrati et al. 1975). according to clinical observations, the economic losses due to the virus seem to be significantly high, however due to the lack of an accurate laboratory diagnosis the real feature of the disease remained unknown. the aim of this study was isolation and adaptation of the viral agent on cell culture, and molecular characterization of the circulating virus in iran during the autumn 2012 while accurate diagnosis of befv was also set up. the results are useful for any epidemiological study and are prerequisites for further research to manufacture a potent vaccine against the virus in iran. materials and methods sampling blood samples with anticoagulant were taken from clinically suspected animals during febrile phase and shipped on ice to the animal virology department, research and diagnosis of razi vaccine and serum research institute. viral rna isolation and pcr amplification viral rna was extracted from whole blood, cell culture and tissues using rntx™ solution (cinagen), according to manufacturer's protocol. the purified rna was subjected to reverse transcription (rt). reverse transcription (rt) was carried out using revertaid™ first strand cdna synthesis kit (fermentas) as follows: 6 μl rna and 1 μl oligo (dt) primer with 5 μl depc-treated water heated at 65 °c for 10 minutes and cooled on ice. then, 4 μl 5x reaction buffer, 1 μl ribolock™ rnase inhibitor (20u/μl), 2 μl of 10 mm dntp mix, 1 μl revertaid™ m-mulv reverse transcriptase (200 u/μl) were added to the solution to a final volume of 20 μl. the resulting cdna was amplified by polymerase chain reaction (pcr) using accupower® pcr premix (bioneer), in which 2 μl of cdna was mixed with 10 pmol of each forward: 5'-atgttcaaggtcctc ataattacc-3' (nt 1–24) and reverse: 5'aatgatcaaagaacctatcatcac-3' (nt 1874–1871) (wang et al. 2001) and dionized water to a final volume of 20 μl to amplify full length of g gene. thermal cycling condition was initial denaturation at 95 °c for 5 min then 35 cycles of 95 °c for 40 s, 52 °c for 40 s, 72 °c for 2 min and 30 s and final extension of 72 °c for 10 min. the pcr product (1μl) was diluted (1/100) and 1 μl of the dilution was subjected for nested-pcr using accupower® pcr premix (bioneer) as described above. nested-pcr amplified an 809 bp pcr product (nt 346–1155) using forward: 5'tattaccctcctgccggat gttt-3' and reverse: 5'aggtctgtatt cgcaccaagctct-3' primers (aziz-boaron et al. 2012). thermal cycling program was carried out as initial denaturation at 95 °c for 4 min then 35 cycles of 95 °c for 30 s, 56 °c for 30 s, 72 °c for 40 s and final extension of 72 °c for 5 min. all pcr products were run on agarose gel and stained with http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 197 ethidium bromide and amplicons were observed in all positive samples. in order to minimize the risk of nucleotide miscorporation for phylogenetic analysis of the 809 bp amplicon, additional pcr reaction was carried out on cdnas using pfu enzyme with the same thermal cycling condition. the pcr reaction mixture (50 μl total volume) comprised 5 μl 10× pfu buffer, 2 μl dntp mix (5mm), 1 μl (10 pmol) of each primer, 5 μl cdna, 3.2 μl mgso4 (25 mm) and 0.8 μl pfu polymerase (2.5 u/μl, fermentas). virus isolation befv was successfully grown and adapted on cell culture by inoculating the virus intracerebrally into suckling mice primarily (van der westhuizen 1967). using this approach, viral isolation was achieved as explained briefly. the blood samples were centrifuged at 2000 ×g for 5 min. buffy coats of blood in anticoagulant were washed with phosphate-buffered saline (pbs). the buffy coats were resuspended in pbs and were frozen and thawed 3 times and 20 μl was inoculated into suckling mice' brains. the suckling mice were examined carefully every day for clinical signs. after 6 days, their brain were extracted and were grinded with pbs, the homogenates were frozen and thawed 3 times, centrifuged at 2000 × g for 5 min and the supernatant was inoculated into suckling mice's brains again until the 3rd passages when clinical signs were observed. the controlled mice were injected with 20 μl pbs repeatedly in all 3 passages. viral rna extraction and rt-pcr were performed on all inoculated and controlled mice. the homogenates from the 3rd passage were used for inoculation of cell culture. confluent green monkey kidney (vero) cell monolayers in 25 cm2 flask were inoculated with 0.5 ml of homogenate from the 3rd passages of virus in brains of suckling mice and incubated at 37 °c for 1 h, then cell maintenance medium containing: dmem (gibco), glucose, calf serum (5%), nahco3, penecillin and streptomycin, was added into the monolayers. the cultures were maintained at 37 °c for 8 days and observed every 24 h intervals. the flasks were frozenthawed 3 times, they were centrifuged and the supernatant were inoculated into new monolayers. a monolayer flask was also considered as control which was inoculated with pbs in serial passages. viral rna extraction and rt-pcr were also carried out on all inoculated and controlled monolayers. sequence and phylogenetic analysis the 809 pcr products were purified by high pure pcr product purification kit (roche) and directly sequenced in both orientations with pcr primers mentioned above. the obtained sequences were assembled by genious (4.8.3) software. the partial g gene sequences from miandoab (36° n, 46° e), shahriar (35° n, 51° e) and mamasani (30° n, 51° e) located in north-west, center and south of iran, respectively, were aligned using clustal w program (thampson et al. 1994). an iranian sequence named sh (shahriar) was deposited in genbank with accession number of kf 14868. this sequence was then aligned with 17 sequences from australia, taiwan, japan, china, egypt, israel and turkey (table 1). the phylogenetic tree was constructed by the maximum likelihood method (tamura et al. 2011) using kimura's two-parameter formula (kimura 1980). the reliability of the branching was evaluated by the bootstrap test with 100 replicates (felsenstein 1985). results polymerase chain reaction (pcr) all pcr reactions were successfully carried out. the first reaction amplified an 1871 bp product spanning the whole g gene and the nested reaction amplified an 809 bp product. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 198 phylogenetic analysis of the partial g gene sequences using clustal w program, the 3 sequences obtained from different parts of iran including shahriar, miandoab and mamasani were absolutely identical as depicted in fig. 1. additionally, alignment of iranian sequence with 17 sequences from the world revealed that the iranian sequence is 99.9 % identical with the turkish isolate tretlik-2-bef which was isolated in 2012. according to the phylogenetic tree, all sequences grouped into three distinct lineages including australian, middleeast and east asian. the iranian 2012/sh with the turkish 2012/ tr-etlik-2-bef and trcp3 were placed with east asian in one cluster (fig. 2). virus isolation from the third passage on suckling mice, clinical signs including paralysis, stiffness in hind legs and death were observed on fourth day after inoculation, while no clinical sign and death were observed in the control mice which were inoculated with pbs (fig. 3). moreover, the monolayer vero cells showed cpe (cytopathic effect) including rounding of cells, chromatin condensation followed by detachment from flasks on fifth day after inoculation from the second passage (fig. 4). the presence of befv genome was confirmed in brains of suckling mice and cells inoculated with the virus by rt-pcr while the control mice and cells were negative. table 1. bovine ephemeral fever virus sequences used for the phylogenetic analysis accession numberyear of isolationcountryisolates af2345331968australiabb7921 af0583231998australiacs1619 af0583221984australiacs1647 ab4620281966japanyhl ab4620442004japanon-04-1 ay9352391984taiwan1984/tw/tn1 ay8181942004taiwantn-2004-124 gq2294522008turkeytrcp77 kc6094262012turkeytr-et/ik-2 kc4703102012turkey2012/tr/cp3 kc3371182012turkey2012/tr/skr gq2277452005egyptegy-2005 jn6460902008israelil-2008 jn8336352012israelisr10/3 jx5666401976chinajb76h jx1746612011chinashandong jx5646392002chinajt02l kf1486852012iransh http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 199 ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 390 400 410 420 430 440 450 460 470 mamasani ttataaga tccgatccca taatgattct ttttaaccat tccccctctt gttgatgttc tccaatctat actgcaatcc tttgaaactc ga miandoab ttataaga tccgatccca taatgattct ttttaaccat tccccctctt gttgatgttc tccaatctat actgcaatcc tttgaaactc ga shahriar ttataaga tccgatccca taatgattct ttttaaccat tccccctctt gttgatgttc tccaatctat actgcaatcc tttgaaactc ga ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 480 490 500 510 520 530 540 550 560 mamasani cctgaatc tggtagggac agacaaagtt tttcttgcca acttgtttgc tcaaataaat atgcataatc tcttcctggt cttgtgggag ct miandoab cctgaatc tggtagggac agacaaagtt tttcttgcca acttgtttgc tcaaataaat atgcataatc tcttcctggt cttgtgggag ct shahriar cctgaatc tggtagggac agacaaagtt tttcttgcca acttgtttgc tcaaataaat atgcataatc tcttcctggt cttgtgggag ct ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 570 580 590 600 610 620 630 640 650 mamasani aagtaaga catatccaag gtatttatat tttctttatt aagtatcttg ctaatggtat ttaaacatct ttcatgttct aattctgcct ta miandoab aagtaaga catatccaag gtatttatat tttctttatt aagtatcttg ctaatggtat ttaaacatct ttcatgttct aattctgcct ta shahriar aagtaaga catatccaag gtatttatat tttctttatt aagtatcttg ctaatggtat ttaaacatct ttcatgttct aattctgcct ta ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 660 670 680 690 700 710 720 730 740 mamasani atatctaa ttcttcaaat tctgttctat ctgtgtgcat cctaaaaccc ggttttttac ccttacatct ttcaatttta aaattttgga aa miandoab atatctaa ttcttcaaat tctgttctat ctgtgtgcat cctaaaaccc ggttttttac ccttacatct ttcaatttta aaattttgga aa shahriar atatctaa ttcttcaaat tctgttctat ctgtgtgcat cctaaaaccc ggttttttac ccttacatct ttcaatttta aaattttgga aa ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 750 760 770 780 790 800 810 820 830 mamasani tcagattc agtttggttt tctatactcc accattctcc atcctcaaaa atgatgccat ttcttccaca aaatgtagac agacatccct ta miandoab tcagattc agtttggttt tctatactcc accattctcc atcctcaaaa atgatgccat ttcttccaca aaatgtagac agacatccct ta shahriar tcagattc agtttggttt tctatactcc accattctcc atcctcaaaa atgatgccat ttcttccaca aaatgtagac agacatccct ta ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 840 850 860 870 880 890 900 910 920 mamasani tttacatg gactaacccg atatctggag cctcccataa tctctctgtc tcatttaatt cactcttaaa ggatttaact gtaatgcatt cc miandoab tttacatg gactaacccg atatctggag cctcccataa tctctctgtc tcatttaatt cactcttaaa ggatttaact gtaatgcatt cc shahriar tttacatg gactaacccg atatctggag cctcccataa tctctctgtc tcatttaatt cactcttaaa ggatttaact gtaatgcatt cc ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 930 940 950 960 970 980 990 1000 1010 mamasani caatgttt gctattacag tgttcactta tttctttgac cctcacatct ggtatccatg tcccagttat atctttaaga ggacatcctt tt miandoab caatgttt gctattacag tgttcactta tttctttgac cctcacatct ggtatccatg tcccagttat atctttaaga ggacatcctt tt shahriar caatgttt gctattacag tgttcactta tttctttgac cctcacatct ggtatccatg tcccagttat atctttaaga ggacatcctt tt ..|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. 1020 1030 1040 1050 1060 1070 1080 1090 1100 mamasani gtcttact gtcattaata gtacaaggag ttagaaaacg agaatcataa atcaaattat cataaggatt caaaaaaggc ttgtgttgta tt miandoab gtcttact gtcattaata gtacaaggag ttagaaaacg agaatcataa atcaaattat cataaggatt caaaaaaggc ttgtgttgta tt shahriar gtcttact gtcattaata gtacaaggag ttagaaaacg agaatcataa atcaaattat cataaggatt caaaaaaggc ttgtgttgta tt ..|....| ....|....| ....|....| ....|....| .. 1110 1120 1130 1140 mamasani aagacata aaattctatt tcttgattca tttcagtatt cc miandoab aagacata aaattctatt tcttgattca tttcagtatt cc shahriar aagacata aaattctatt tcttgattca tttcagtatt cc fig. 1. alignment of partial g gene nucleotides (at residues 383–1142) of three bovine ephemeral fever isolates collected from different parts of iran during autumn 2012 http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 200 fig. 2. phylogenetic tree of partial g gene sequences of the iranian shahriar isolate with 17 isolates from turkey, australia, taiwan, egypt, israel, china and japan during the years 1966-2012. the scale represents 1% sequence divergence fig. 3. suckling mice inoculated with bovine ephemeral fever presented paralysis, stiffness and death while control mouse marked with blue dot on the head (left) showed no clinical signs fig. 4. a) cytopathic effect in vero cell inoculated with bovine ephemeral fever. b) control vero cell line showed no cytopathic effect http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 201 discussion bovine ephemeral fever is one of the most important infectious diseases of livestock in iran. the disease occurs usually in southern regions but sometimes observed as epidemic in other parts. differential diagnosis of bef based on clinical observation is sometimes difficult and complicated. therefore, tentative diagnosis is essential and necessary to implement urgent and proper prophylactic action, evaluation of the extent of the disease and further epidemiological studies. this study represents an accurate and reliable diagnosis using molecular approach for detection of the widely spread viral agent in iran. in future, application of a real time pcr as introduced by stram et al. (2005) will definitely increase the sensitivity of befv diagnosis and will also help to reveal the real extent of the virus. in this research, scientific procedure for the isolation of befv on cell culture was also successfully set up in the country. complete characterization of the isolated viruses will certainly raise our knowledge about the epidemiology of the virus in the middle-east and the world. these viruses can also be applied for manufacturing an appropriate domestic vaccine against the virus and are also efficient for seroepidemiologic studies using viral neutralization (vn) test. as previously reported, the sequences from the world are clustered into three distinct lineages (kato et al. 2009, aziz-boaro et al. 2012, lin and qiu 2012, zheng and qiu 2012, walker et al. 2012). in the present study, australian lineage is also a completely distinct lineage, israeli and some turkish isolates are grouped into a separate lineage and the east asian isolates together with iranian, egyptian and the other turkish sequences construct the third lineage. this geographic distinction explained to be resulted from circulation of the virus in a vast distance by vectors and winds in australia and east asia (murray 1970, ogawa 1992, shirakawa et al. 1994, finlaison et al. 2010). however, the circulation of befv in the middle east has been proposed to occur by both winds and animal transport, this hypothesis was supported by close genetic relationship between some middle east (egyptian-2005) and east asian sequences, and supportive documents indicating animal transport from china to jordan in 2004 (aziz-baron et al. 2012). the iranian 2012/sh was placed with the turkish 2012/tr-etlik isolate in one branch (99.9% nucleotide identity), this branch with chinese 2011/shandong and turkish 2012/ trcp3 constructed a sublineage within the east asian lineage, the high similarity of some turkish and the iranian sequences with that of chinese again provide supportive evidence for the possible two routes of transmission in the middle east. alignment of partial g gene sequence from 3 different regions (north-west, centre and south) of iran during the autumn of 2012 revealed that they are 100 % identical implying that the circulating virus was most likely the same in this period. however, this finding is not consistent with the sequences submitted in the genbank from turkey in the same period which are even separated into different lineages. these divergent sequences may also support the role of animal transport beside the wind and insect in transmission of the virus in the region. turkey faced a severe bef in 2012 with mortality rate of 15–20 % (tonbak et al. 2012), this significant increase in mortality with acute form of bef was also reported in china in 2011 (zheng and qiu 2012), but it is not clear whether the highly similar (96.4% nucleotide identity) turkish (2012/tr-etlik) and chinese (2011/shandong) isolates or environmental factors (walker 2013) are responsible for the change in virulence in these two far regions. it is also remain to be elucihttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 195–203 m bakhshesh and d abdollahi: a checklist of … 202 dated how a number of turkish viruses were co-circulated in a specific period and whether this co-circulation affected the feature of the disease. conclusion the geographic situation of iran which is located between the east asian and middleeast countries places the country at high risk of befv transmission by both winds and animal transport. characterization of more viruses together with analysis of meteorological and entomological data and finding the model of animal transport in years will help in evaluating the risk of the virus transmission in iran and the middle-east. in particular, further research is essentially required to define the vector species and their ecology in iran. due to the high risk of befv transmission and its consequences in the region, implementation of a specific surveillance system and large scale vaccination are recommended. acknowledgements the author gratefully thanks to razi vaccine and serum research institute for financial support of this study. the authors declare that there is no conflict of interests. references aziz-boaron o, klausner z, hasoksuz m, shenkar j, gafni o, gelman b, david d, klement e (2012) circulation of bovine ephemeral fever in the middle east-strong evidence for transmission by winds and animal transport. vet microbiol. 158: 300–307. cybinski dh, walker pj, byrne ka, zakrzewski h (1990) mapping of antigenic sites on the bovine ephemeral fever virus glycoprotein using monoclonal antibodies. j gen virol. 71: 2065–2072. felsenstain j (1985) confidence limits on phylogenesis: an approach using the bootstrap. evolution. 39: 783–791. finlaison ds, read aj, kirkland pd (2010) an epizootic of bovine ephemeral fever in new south wales in 2008 associated with 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j. 9: 268. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, september 2019, 13(3): 259–267 s sayono: effect of d-allethrin … 259 http://jad.tums.ac.ir published online: september 30, 2019 original article effect of d-allethrin aerosol and coil to the mortality of mosquitoes *sayono sayono, puji lestari mudawamah, wulandari meikawati, didik sumanto department of epidemiology and tropical diseases, school of public health, universitas muhammadiyah semarang, semarang, indonesia (received 20 mar 2018; accepted 16 jun 2019) abstract background: commercial insecticides were widely used by communities to control the mosquito population in their houses. d-allethrin is one of insecticide ingredients widely distributed in two different concentrations namely 0.15% of aerosol and 0.3% of coil formulations. we aimed to understand the mortality of indoor mosquitoes after being exposed to d-allethrin 0.15% (aerosol) and 0.3% (coil) formulations. methods: this quasi-experiment study applied the posttest-only comparison group design. the aerosol and coil d-allethrin were used to expose the wild mosquitoes in twelve dormitory bedrooms of smkn jawa tengah, a vocational high school belonging to central java provincial government, on march 2017. the compounds were exposed for 60 min to each bedroom with four-week interval for both of formulations. the knockdown mosquitoes were collected into a plastic cup and delivered to the laboratory for 24h holding, morphologically species identification and mortality recording. history of insecticide use in the dormitory was recorded by an interview with one student in each bedroom. data were statistically analyzed with independent sample t-test and mann-whitney. results: as many as 57 knockdown mosquitoes belonging to three species were obtained namely culex fuscocephala, cx. quinquefasciatus and aedes aegypti with mortality rate of 50.88% after 24h holding. knockdown and mortality of mosquitoes were significantly different based on d-allethrin formulations. d-allethrin concentrations were not effective for controlling culex mosquitoes but effective for ae. aegypti. conclusion: further efficacy of d-allethrin 0.15% aerosol to eradicate ae. aegypti is necessary to be conducted in supporting the dengue vector control. keywords: d-alletrin; insecticide; culex fuscocephala; culex quinquefasciatus; aedes aegypti introduction mosquitoes play an important role as vector of several kinds of diseases. the different species of mosquito could transmit different disease agents, although some species can transmit a number of pathogens (1, 2). aedes mosquitoes have been known as vector of dengue, chikungunya and zika viruses (3) as well as filarial parasites in certain condition (4). culex mosquitoes can transmit japanese encephalitis virus and filarial worms, while anopheles transmits malaria and filarial worms. armigeres spp. and mansonia spp. also transmit filarial worms (1, 2). some mosquito species are found in residential environments with different abundance. eight mosquito species were reported from six habitats in thailand with the order of relative abundance were cx. quinquefasciatus, cx. vishnui, cx. gelidus, ae. aegypti, mansonia spp., anopheles spp., and cx. bitaeniorhynchus, respectively (5). nine mosquito species were found in urban settlement, in nigeria namely cx. quinquefasciatus, cx. annulioris, anopheles gambiae, an. funestus, an. rhodesiensis, an. arabiensis and ae. aegypti. three mosquito species showed the highest relative abundance i.e., cx. quinquefasciatus (50.24%), anopheles spp. (26.5%), and ae. aegypti (0.2%) (6). in mojokerto, east java province, indonesia, five mosquito species were found in settlements namely ae. aegypti, ae. albopictus, ae. laniger, cx. bitaeniorhynchus and cx. quinquefasciatus (7). aedes aegypti is the dominant species in urban settlements (7) and the dengue endemic areas (8), while cx. *corresponding author: dr sayono sayono, e-mail: say.epid@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 259–267 s sayono: effect of d-allethrin … 260 http://jad.tums.ac.ir published online: september 30, 2019 quinquefasciatus is the dominant species in rural area (7). urban people in the dengue endemic areas often used insecticide to repel and against mosquito exposure with several formulations such as repellent, aerosol, mosquito coil and electric mat. the most common of insecticide compounds are d-allethrin, prallethrin, transfluthrin, and diethyl toluamide (deet) (9, 10). commercial insecticides usually applied by community with ignored the written instruction (11). this unstandardized practice can cause a negative impact such as mortality of non-target organisms, environmental pollution, and the emergence of insect vectors resistance (12). the resistance of mosquito vectors to pyrethroid insecticide class has been reported from several countries. aedes aegypti is resistant to several pyrethroid compounds in vietnam (13), martinique (14), and indonesia (15, 16). anopheles barbirostris was resistant to some insecticide compounds namely lambda-cyhalothrin 0.05% and etofenprox 0.5% and tolerant to bendiocarb 0.1% (17). the resistance of cx. quinquefasciatus to pyrethroid was reported from japan and china that indicated by a genetic mutation (18). this species was reported resistant to permethrin, deltamethrin, and bendiocarb in benin, nigeria (19). permethrin resistant of cx. quinquefasciatus also reported from central java, indonesia (20), and mansonia is tolerant to pyrethroid insecticide (21). cx. pipiens is reported resistant to four of insecticide classes namely pyrethroid, organochlorine, carbamate, and organophosphate in iran (22). household insecticides circulating in indonesia mostly contain pyrethroid compounds in either single or combination formulations, including d-allethrin. these insecticides are often used in the dense population settlement with uncontrolled doses. susceptibility of mosquitoes to the compounds is necessary evaluated. we aimed to understand the susceptibility of mosquitoes to d-allethrin 0.15% aerosol and 0.3 % coil compounds in an urban settlement. materials and methods study site this quasi-experimental study was conducted in twelve of sixteen bedrooms at the dormitory of smkn (vocational high school) jawa tengah on march 2017. each of the 4x6 square meter bedrooms contains six beds and represents the densely populated settlement. the two of d-allethrin formulations were applied at the twelve of bedrooms sequentially. application of d-allethrin aerosol and coil insecticide the d-allethrin aerosol insecticide was applied in the morning when the bedrooms are empty. clothes and snacks were moved out from bedrooms. all of ventilation, windows, door, and holes in the rooms were closed. temperature and humidity of each room were measured by hygrometer and recorded. each bedroom was divided into two sections and six quadrant direction for spraying of aerosol insecticide (fig. 1). six sprayings were done in each bedroom for five seconds according to the directions based on the previous study (23) with modifications. all of the bedrooms were closed for 60min after the aerosol insecticide was sprayed. the volume of sprayed liquid is equalized by measuring the weight of the insecticide bottle before and after it is sprayed. the d-allethrin mosquito coil formulation was obtained from the market and applied four weeks following application of the aerosol formulation based on the previous study with modifications (24). a mosquito coil was burned on the tip and placed for 60min in the center of each bedroom. application of those different insecticide formulations was done in the equal condition of bedrooms. laboratory works the knockdown mosquitoes from each bedroom were collected in the plastic cups and labeled by name of the bedroom and delivered to epidemiology laboratory, faculty of public health of universitas muhammadiyah sehttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 259–267 s sayono: effect of d-allethrin … 261 http://jad.tums.ac.ir published online: september 30, 2019 marang for twenty-four-hour holding. each cup was placed in different mosquito cage. temperature and humidity of the recovery room were maintained at the 27±2 celsius degree and 80± 10%. the ten percent of sugar solution was placed in each mosquito cage for feeding the live mosquitoes. mortality of mosquito was calculated based on species. mosquito identification used the arthropod identification key of walter reed biosystematics unit (25). data analysis the relative abundance, exposure frequency and dominance rate of mosquitoes were calculated based on the formulas below (26): dominance rate= relative abundance x frequency the different of knockdown and mortality of mosquitoes were analyzed based on d-allethrin formulation. history of insecticide use in the dormitory was showed in a frequency table. ethical approval protocol of this study was approved by ethics committee of faculty of medicine of universitas diponegoro semarang. informed consent and research permission were obtained from dormitory management. results knockdown and mortality of mosquitoes as many as 57 knockdown mosquitoes were obtained from this study, and mortality of mosquito was 50.88%. majority of the knockdown (94.44%), dead (96.43%) and alive (92.31%) mosquito were obtained from the d-allethrin aerosol exposed-group, and contrast conditions were found in the mosquito coil exposed-group. a number of the knockdown, dead and alive mosquitoes were significantly different based on the d-allethrin formulations (table 1). morphological identification results of morphological identification of mosquito were found three species. two species belong to culex genus and one species belongs to aedes genus, namely cx. fuscocephala, cx. quinquefasciatus and ae. aegypti. mention order of the three mosquito species also represents the order of relative abundance, exposure frequency and dominance rate (tables 2, 3). history of commercial insecticide use results of interview with respondent of each bedroom showed that majority of dormitory inhabitants used insecticide to expel the mosquitoes almost every day, mainly the repellent. the d-allethrin compound was also used among inhabitants (table 4). fogging or residual spraying was not used in this dormitory. table 1. knockdown, dead and alive mosquitoes based on the insecticide (d-allethrin-0.15% compound) formulations insecticide formulation mosquito conditions knockdown dead alive aerosol number 57 29 28 minimum 1 1 0 maximum 13 7 6 mean 4.50 2.33 2.17 standard deviation 3.26 1.92 1.75 mosquito coil number 3 1 2 minimum 0 0 0 maximum 2 1 2 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 259–267 s sayono: effect of d-allethrin … 262 http://jad.tums.ac.ir published online: september 30, 2019 mean 0.25 0.08 0.17 standard deviation 0.62 0.29 0.58 p-value 0.01 0.02 0.01 the higher number of mosquitoes was obtained from the aerosol formulation rather than mosquito coil indicating the faster effect of this formulation table 2. abundance, frequency, and dominance rate of mosquito species spices number of mosquitoes relative abundance (%) frequency dominance rate culex fuscochepala 41 71.93 0.45 32.96 culex quinqueasciatus 15 26.31 0.29 7.67 aedes aegypti 1 1.75 0.4 0.07 three mosquitoes species were found in the dormitory with the high abundance of the culex genera table 3. knockdown, dead, alive and mortality of mosquitoes based on the species and d-allethrin formulations species insecticide formulation knockdown dead alive mortality (%) culex fuscochepala aerosol 41 20 21 48.78 mosquito coil 0 0 0 0.00 culex quinquefasciatus aerosol 12 7 5 58.33 mosquito coil 3 1 2 33.33 aedes aegypti aerosol 1 1 0 100.00 mosquito coil 0 0 0 0.00 total 57 29 28 50.88 mortality of mosquitoes after exposed with d-allethrin 0.15% and 0.30% insecticide compounds were very low. this condition indicated that culex mosquitoes were resistant to these concentrations table 4. information about insecticide use among students at the boarding school variables f % commercial insecticide use 1. yes 10 83,3 2. no 2 16,7 insecticide formulation 1. aerosol 1 10,0 2. electric mat 3 30,0 3. repellent (lotion) 6 60,0 insecticide compounds 1. diethyltoluamid 12.5% 4 40,0 2. diethyltoluamid 13% 1 10,0 3. diethyltoluamid 15% 1 10,0 4. d-aletrin 0.15% and praletrin 0.2% 2 20,0 5. praletrin 13.16g/l 2 20,0 frequency of insecticide use in a week 1. 1–3 times 4 40,0 2. 4–5 times 2 20,0 3. 6–7 times 4 40,0 majority of inhabitants at the dormitory use insecticide, mainly in repellent formulation with deet compound, although the daily use is under 50% of students. forty percent of inhabitants used pyrethroid compounds table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 259–267 s sayono: effect of d-allethrin … 263 http://jad.tums.ac.ir published online: september 30, 2019 fig. 1. direction of insecticide spraying. the bedroom was divided into six spraying directions that indicated by the rows discussion this result showed that d-allethrin aerosol formulation can eradicate and kill more mosquitoes rather than the coil formulation, although this compound indicated effective result for aedes mosquitoes only. this finding required further investigation to obtain sufficient evidence of ae. aegypti susceptibility to d-allethrin. using the aerosol formulation allowed that the droplets of insecticide will effectively contact with the mosquito’s body, and inhale via respiratory system of mosquitoes (27). the mortality rate of mosquitoes in this study indicated the low efficacy of d-allethrin 0.15% to the mosquito population in the dormitory rooms. this result matched with the facts that majority of dormitory residents use commercial insecticide for preventing mosquito attack with high frequency, mainly repellant, electric mat, and aerosol formulation. those insecticide formulations also contain the d-allethrin 0.15% compound (table 4). high frequency of household insecticide use will correlate with insecticide resistance among culex mosquitoes (28). resistance of mosquitoes to d-allethrin compounds is similar to the previous report from malaysia and indonesia that the mosquito coil containing d-allethrin 0.2% compound resulted in the low mortality rate (24, 29, 30), but different from the reports of aerosol insecticide application containing d-allethrin and d-trans allethrin from thailand that showed mortality rate of mosquitoes were 96% (31) and 90% (32), respectively. although distinctly different with the findings in thailand, results of this study correspond to similar finding in central java province that cx. quinquefasciatus mosquitoes in endemic areas of lymphatic filariasis have been resistant to the pyrethroid compound, in particular, permethrin 0.75% (20). in iran cx. pipiens linn. was resistant to some pyrethroid compounds and other insecticide classes (22). permethrin and d-allethrin derived from the same class of insecticide, pyrethroid. this insecticide class has target site the voltage-gated sodium channel gene (33). the different types of pyrethroid compounds will have a similar effect to disrupt the voltage-gated sodium channel gene (34) and cause the genetic mutation of codon 1014 of the gene that indicated by substitution of amino acid leucine to phenylalanine or serine (35). although the dormitory is located in the dengue endemic of urban residential, there is only a few the aedes mosquito found. this phenomenon is caused that the dormitory is surrounded by a two-meter high wall fence, and there was no puddle of clean water as a place for microhabitat of this species. this wall can prevent the entrance of mosquito into the dormitory areas. the female ae. aegypti use the opened clean water of the domestic containers in laying their eggs rather than natural water containers such as leaf midrib, tree hole or bamboo stems (36). culex is the dominant mosquito genus in this settlement. this result is in accordance with the findings in thailand (5) and nigeria (6), but different from the findings in mojokerto, indonesia where the results of larval surveys in various breeding places in the urban settlement obtained the dominant mosquitoes are ae. aegypti (7). aedes aegypti was the dominant mosquito species in the dengue endemic areas (8). in this case, the dormitory is located in an urban environment with dense building and surrounded the sewers with stagnant water. this condition provides a http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 259–267 s sayono: effect of d-allethrin … 264 http://jad.tums.ac.ir published online: september 30, 2019 good habitat for breeding site of culex mosquitoes. theoretically, the favorite habitat of this species is stagnant of water surface such as hoofprints, rain pools, irrigation channels, discarded tubes, ditch, sewer, and ponds (37, 38). other studies on the abundance of mosquitoes in the educational environment were reported from thailand (39), sabah, malaysia (40), makassar, indonesia (41) that the dominant mosquito is the genus culex, similar to the dormitory of this study although cx. quniquefasciatus is not dominant species (42, 43). the further investigation is needed to analyze the wild mosquito susceptibility to d-allethrin compound by using the laboratory strain of mosquito as the control group, and the causation between environmental profiles, kinds of breeding site and insecticide use to the abundance and dominance of mosquito species, and the occurrence of diseases. conclusion there were three mosquito species found in the bedrooms of smkn jawa tengah belonging to two genera namely culex and aedes. exposure of d-allethrin 0.15% in space spray formulation caused higher knockdown and dead mosquito rather than d-allethrin 0.3% in mosquito coil formulation. overall, the mortality rate of mosquito reached 50.88% which indicated a resistant status to this insecticide compound, except ae. aegypti. further investigation is needed to determine ae. aegypti abundance at the dormitory and resistant status of culex mosquito to another insecticide compounds. acknowledgements we would like to thank for smkn jawa tengah (vocational high school of central java government) given the research permission. all authors declare that there is no conflict of interest. references 1. sarwar m (2015) insect-borne disease transmitted by some important vectors of class insecta hurtling public health. int j bioinform biomed enggin. 1(3): 311– 317. 2. singh n, shukla s, gupta v, tandia n, singh p (2015) mosquito borne zoonotics disease. livest sci. 6: 65–72. 3. leta s, beyene tj, clercq emd, amenu k, kraemer mug, revie cw (2018) global risk mapping for major diseases transmitted by aedes aegypti and aedes albopictus. 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17 http://jad.tums.ac.ir published online: march 31, 2020 original article modeling spatial distribution of sergentomyia minuta (diptera: psychodidae) and its potential implication in leishmaniasis transmission in morocco morocco mohamed daoudi1; *samia boussaa1,2; ali boumezzough1 1 laboratory of ecology and environment (l2e), (urac 32), cadi ayyad university, faculty of sciences semlalia, marrakesh, morocco 2 ispits-higher institute of nursing and technical health occupations, ministry of health, marrakesh, morocco (received 07 aug 2018; accepted 07 mar 2020) abstract background: leishmaniases are parasitic diseases caused by leishmania species and transmitted by the bite of sand flies. the genus lutzomyia and phlebotomus of sand flies are known to be the responsible vector for transmitting almost all leishmania species to humans. the detection of leishmania dna in species of the genus sergentomyia, in different regions, suggests their likely role in leishmania transmission. methods: our objective was to determine the potential geographical distribution of sergentomyia minuta, the most dominant sergentomyia species in morocco, using ecological niche modeling. results: the results showed the widespread geographical distribution of s. minuta in morocco, specifically in northern and central morocco where visceral and cutaneous leishmaniasis foci occur. there were six abiotic factors affecting the distribution of s. minuta whose annual precipitation, precipitation seasonality and precipitation of driest month as the most important ecological variables of the model. conclusion: a positive statistical correlation between human leishmaniasis cases and s. minuta abundance was noted suggesting the potential involvement of s. minuta in local leishmania transmission cycles. keywords: sergentomyia minuta; ecological niche modeling; maximum entropy (maxent); morocco introduction phlebotomine sand flies (diptera: psychodidae) are small nocturnal insects of which only the female is a hematophagous. currently, about 1000 species of sand flies are distributed in almost all biogeographic regions of the world (1). these insects play a crucial role in the epidemiology of relevant diseases, some being of great veterinary and medical importance. indeed, certain species of sand flies are vectors of various infectious and parasitic agents such as agents of canine and human leishmaniasis, bartonellosis and several arboviruses. allergic reactions can also be caused by the exposure of sand fly bites, though is not linked to the spread of disease (2). leishmaniasis remains the most related diseases to sand flies. the genera phlebotomus, lutzomyia and sergentomyia contain all hema tophagous species of sand flies (3), with only the genus lutzomyia in the new world, and phlebotomus in the old world responsible for transmitting almost all leishmania species to humans (4, 5). like most countries around the mediterranean, leishmaniases in morocco are a serious public health problem. these affections are widely represented, from the moroccan rif mountains to the palm groves of the moroccan anti-atlas (6). three epidemiological entities are known in morocco: zoonotic cutaneous leishmaniasis due to leishmania major, anthroponotic cutaneous leishmaniasis due to l. tropica and visceral and cutaneous leishmaniasis due to l. infantum (6). regarding disease transmission, only vectorial role of species of the genus phlebotomus (namely, phlebotomus papatasi, p. sergenti, p. ariasi, p. longicuspis and p. perniciosus) was *corresponding author: dr samia boussaa, e-mail: samiaboussaa@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 18 http://jad.tums.ac.ir published online: march 31, 2020 investigated in morocco (7–9); where sand fly fauna has 22 species including, nine species of the genus sergentomyia (10): sergentomyia christophersi, s. clydei, s. africana, s. minuta, s. dreyfussi, s. schwetzi, s. antennata, s. fallax and s. lewisi. considering the epidemiological status of leishmaniasis in morocco, it is critically important to highlight the probable role as a vector for other sand fly species. vector incrimination depends on the accumulation of evidence based mainly on its geographical and temporal abundance. according to many authors, sergentomyia species are spread throughout morocco, especially s. minuta and s. fallax (11, 12). sergentomyia minuta is collected in urban as well as in rural area (13) up to 1,200m (14). it was considered as ubiquitous species in morocco (15) with high density and a long activity period (16, 17). in addition, leishmania dna was detected in species of the genus sergentomyia in different regions suggesting their likely role in leishmania transmission (18). studies conducted in leishmaniasis foci in iran, mali and portugal have reported the detection of l. major dna in sergentomyia sintoni (19), s. darlingi (20) and s. minuta (21). earlier, it has also been isolated from s. garnhami in kenya (22). other reports also detected l. donovani dna in s. babu in india (23) and more recently, l. siamensis detected in s. gemmea in thailand (24). moreover, several viruses have been isolated from sand flies of the genus sergentomyia such as the saboya virus, viruses rna of ard 95737 and ard 111740 (25, 26), which has made it possible to suspect the vector role of sergentomyia species (27). despite the presence and the abundance of sergentomyia species in morocco, no study has invested its potential involvement in leishmaniasis transmission cycles. therefore, we used ecological niche modeling to identify the distribution of the most dominant species of sergentomyia genus in morocco and consequently discuss its potential involvement in local leish mania transmission cycles. materials and methods study area morocco is a northern african country, bordering the north atlantic ocean and the mediterranean sea. the current study covered sand fly sampling in 190 localities with altitude ranges between sea level and up to 2123m above sea (fig. 1). these localities covered mainly northern and central morocco, where human cutaneous and visceral leishmaniases are endemic (28). the climate in morocco is mostly mediterranean; however, seven bioclimatic regimes are occurred due to topographic differences across the country (fig. 1). in 2014, the total human population in morocco is about 33.7 million, with an urbanization rate of 60% (29). occurrence data and environmental variables occurrence records of sand flies were collected from our field survey (published and unpublished data) and literature using the keyword “species name, morocco” as a search word to retrieve all data archived in pubmed database. thus, about 70% of the 190 localities were investigated by the ecology and environment laboratory (l2e) team between 2005 and 2016. the rest of the localities were published observations of the investigations in morocco (20 publications on pubmed). the coordinates of all localities were registered with a global positioning system (gps). for ecological niche modeling, 19 climatic variables (bio1-bio19) were used with elevation precision (alt) for each locality (table 1). altitude and bioclimatic data were obtained from the worldclim project (http://www.worldclim.org), with a spatial resolution of 30 arc seconds (about 1km2). construction of the model the modeling was performed using maximum entropy (maxent) version 3.3.3 (30), which http://jad.tums.ac.ir/ http://www.worldclim.org/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 19 http://jad.tums.ac.ir published online: march 31, 2020 uses an optimization procedure comparing the presence of the species with the characteristics of the environment, based on the principle of maximum entropy. this program allows for inference from incomplete information despite limited occurrence data (31–33). distribution data for the sand fly species and environmental data were imported into maxent v. 3. 3. 3 (31), a total of 15 model replicates were run, with 30% of the points of presence used to test the model and 70% for model construction (34). model evaluation to evaluate the quality of model produced by maxent, we analyzed the operating characteristics curve receiver operating characteristic (roc) which assigns a unique value according to the model performance area under the curve (auc). roc analysis is a measure of sensitivity, which corresponds to the true positive rate (no error of omission), compared to the false positive rate (superfluous forecast error). also, the roc analysis evaluates the ability of the model to correctly predict the occurrence of the species. the closer the auc value is to 1, the closer the correlation of the model (31) (figs. 2, 3). the importance of the variables in explaining the potential geographic distribution of s. minuta was estimated using the jackknife environmental variables importance test (31) (table 2), which assesses the relative contribution (%) of the variables used to generate the distribution model produced by maxent. statistical analysis the chi-squared test (𝜒2) was used to analyze the correlation between variation of s. minuta abundance and the presence/absence of human leishmaniasis cases (cutaneous leishmaniasis by l. tropica and visceral leishmaniasis by l. infantum) in all sampling sites. epidemiological data (human cutaneous and visceral leishmaniasis cases) were obtained from official moroccan ministry of health reports (28). results in this study, we assembled 190 sampling sites for sand fly species. five sergentomyia species were noted: sergentomyia minuta (n= 150), s. fallax (n= 110), s. dreyfussi (n= 57), s. africana (n= 10) and s. christophersi (n= 15). phlebotomus species were also recorded, specifically phlebotomus sergenti (n= 102), p. papatasi (n= 82), p. longicuspis (n= 78), p. perniciosus (n= 76) and p. ariasi (n= 43), while p. alexandri (n= 09), p. kazeruni (n= 03), p. langeroni (n= 02), and p. mariae (n= 02) were noted with very low reporting. sergentomyia minuta was the most predominant species and it was collected in the 150/ 190 sampling sites between 14 and 2123m (table 3). to identify habitat suitability and potential limiting factor of s. minuta, all ecological variables were analyzed. there were 6/19 ecological variables (bio 3, bio 4, bio 12, bio 14, bio 15, bio 19), which were the most important factors affecting the distribution of s. minuta in the study area. figure 4 shows a representation of the maxent model for the s. minuta. warmer colors show areas with better predicted conditions. the white dots indicate the presence locations used for training, while the purple dots show the locations of the tests. since 1995, leishmaniases are notifiable diseases in morocco (ministerial decree n°683-95). all human cases are listed in leishmaniasis epidemiological information collection system and published yearly. we used data recorded of cl and vl between 2005 and 2016 (28) corresponding to 190 localities. statistical analysis of the presence and absence of leishmaniasis cases (cutaneous cl and visceral leishmaniasis vl) and the distribution of s. minuta in morocco showed a positive correlation between vl by l. infantum and s. minuta abundance (r= 0.211) and between cl by l. tropica and s. minuta abundance (r= 0.166). according to chi-squared test (𝜒2) test, s. minuta was more associated to vl distribution http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 20 http://jad.tums.ac.ir published online: march 31, 2020 (𝜒2= 7,166, ddl= 2, p= 0,028) compared to cl (𝜒2= 3,256, ddl= 2, p= 0,196). table 1. description and sources of environmental variables collected for the model environmental variables abbreviation unit source annual mean temperature bio 1 °c worldclim mean diurnal range (mean of monthly (max temperature−min temperature)) bio 2 °c worldclim isothermality (bio2/bio7) (×100) bio 3 _ worldclim temperature seasonality (standard deviation ×100) bio 4 °c worldclim max temperature of warmest month bio 5 °c worldclim min temperature of coldest month bio 6 °c worldclim temperature annual range (bio5–bio6) bio 7 °c worldclim mean temperature of wettest quarter bio 8 °c worldclim mean temperature of driest quarter bio 9 °c worldclim mean temperature of warmest quarter bio 10 °c worldclim mean temperature of coldest quarter bio 11 °c worldclim annual precipitation bio 12 mm worldclim precipitation of wettest month bio 13 mm worldclim precipitation of driest month bio 14 mm worldclim precipitation seasonality (coefficient of variation) bio 15 mm worldclim precipitation of wettest quarter bio 16 mm worldclim precipitation of driest quarter bio 17 mm worldclim precipitation of warmest quarter bio 18 mm worldclim precipitation of coldest quarter bio 19 mm worldclim elevation alt m gtopo30 fig. 1. distribution of the sergentomyia spp in morocco according to bioclimate map http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 21 http://jad.tums.ac.ir published online: march 31, 2020 table 2. analysis of the contribution of the most important ecological variables of the model variable percent contribution (%) auc without the variable auc with only the variable bio 12 41.2 0.917 0.867 bio 15 23.9 0.922 0.892 bio 14 9.4 0.916 0.859 bio 4 5.9 0.914 0.890 bio 19 5.5 0.914 0.906 bio 3 4.8 0.914 0.868 table 3. description of sergentomyia species on sampling sites sergentomyia spp. total of specimens number of stations occupied altitude range (m) s. minuta 6746 150 [14–2123] s. fallax 5582 110 [14–1351] s. dreyfussi 474 57 [282–1309] s. africana 43 10 [402–1340] s. christophersi 99 15 [282–1500] fig. 2. analysis of the omission / commission of sergentomyia minuta http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 22 http://jad.tums.ac.ir published online: march 31, 2020 fig. 3. sensitivity and performance of the model for sergentomyia minuta species fig. 4. estimation of the distribution of sergentomyia minuta species in morocco by maxent discussion morocco is located in the subtropical zone. it benefits from a mediterranean climate, characterized by a dry warm season, nuanced by three essential influences: the progressive remoteness of the atlantic coast, the altitude and the approach of the southern desert (36). the geographical location of morocco is a major factor of its bioclimatic diversity which favors the abundance and the diversity of phlebotomine species; including sergentomyia species that can tolerate the aridity (37-39). thus, a considerable specific richness in morocco for sergentomyia genus counter to the others mediterranean’s regions such as tunisia, where only six species of sergentomyia have been reported (40-42). sergentomyia species were collected in different environments but are rather subservient to the extradomestic environment (43). this is related to their trophic preferences, and their adaptability to dry and open habitats. thus, they find suitable conditions in wild environments. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 23 http://jad.tums.ac.ir published online: march 31, 2020 according to who (43), sand flies of the genus sergentomyia have long been known to feed on reptiles. they are involved in the transmission of sauroleishmania, of reptiles in the old world. they do not present an epidemiological risk for humans (44), although some species can feed on humans (45). recent work on sergentomyia spp shows that these species can be infected by human leishmania spp. and the blood meal analysis shows that it is a mammalian blood, including humans (18). leishmania tropica dna was detected from s. ingrami and s. hamoni collected in ghana (46). all of these results raise questions about the role of sergentomyia spp. as a possible vector of leishmania spp. which has a great impact on human and animal health. among all sergentomyia species, s. minuta is widely distributed around the mediterranean area. it is the most abundant sergentomyia species in the present study (table 3) and early in morocco (12). in greece, the sergentomyia genus represented 84% of the total sand flies fauna with 38% of s. minuta (47). in france, rioux et al. (48) reported a percentage of 73.7% for both genus and species. in tunisia, it was noted that sergentomyia accounted for 70% of sand flies, of which 41.5% belonged to the species s. minuta (49). in algeria, belazzoug et al. (50) found this species with a rate of 39.75% of total specimens. regarding its potential vector role, periera et al. (51) have isolated l. infantum from s. minuta, and blood meal analysis has shown that it is human blood. in italy, recently, latrofa et al. (52) detected l. infantum dna in s. minuta from endemic area of canine leishmaniasis. s. minuta was also naturally infected with l. major in portugal (21) and in tunisia (53). in addition, maia et al. (54) detected leishmania spp. in s. minuta collected in southern portugal; while bravo-barriga et al. (55) identified leishmania dna in s. minuta of spain. in morocco, though the well-known role of phlebotomus papatasi, p. sergenti and larroussius species (p. perniciosus, p. longicuspis and p. ariasi), as competent vector of l. major, l. tropica and l. infantum, respectively (56), that of s. minuta needs further investigation. ecological niche modelling has been widely used in order to identify the probability of sand fly species occurrence (57). to the best of our knowledge, the present study constitutes a first modeling of sergentomyia species. auc values of our model were greater than 0.9 (table 2) indicating that the model performed has a good robustness (31). in the 190 localities used for the model, we noted the coexistence of sergentomyia species and phlebotomus species including the proven vectors of leishmaniasis forms in morocco: p. papatasi, p. sergenti, p. perniciosus, p. ariasi and p. longicuspis. according to the region, p. sergenti and p. papatasi were more abundant in the central morocco, while p. perniciosus was more recorded in northern morocco. the presence and the abundance of s. minuta in the endemic area of leishmaniasis in morocco can be the most important criteria for its possible vectorial incrimination. our results showed the widespread geographical distribution of s. minuta in morocco, specifically in northern and central morocco (fig. 4) where vl and cl foci occur (16, 58). in addition, a positive correlation between visceral leishmaniasis (r= 0.211) and cutaneous leishmaniasis (r= 0.166) with s. minuta abundance was noted and confirm this s. minutaleishmania spatial overlapping. sand fly behaviors can also determine its vector implication. s. minuta presented an anthropophilic behavior in portugal (54). in morocco, s. minuta is a ubiquitous species (13) which shows an adaptation and an important ecological tolerance. it was collected in the different bioclimatic floors with preference to a semiarid climate and altitudes between 800 to 1,000m (12). in addition, the seasonal distribution of s. minuta showed abundance from may to september in sub-humid and semi-arid stages in morocco (15). in arid bioclimate, it http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 24 http://jad.tums.ac.ir published online: march 31, 2020 was active during two periods, october–november and april–may–june (59). this long period of s. minuta activity favors the s. minutaleishmania temporal overlapping in these areas. sand flies of morocco, including sergentomyia species, were found highly dependent on environmental conditions; and their abundance and geographical distribution were affected by several ecological variables (59-62). according to our model, annual precipitation (bio 12) and precipitation seasonality (bio 15) were significantly more important in the distribution of s. minuta in morocco (table 2). precipitation plays an important role in sand flies’ life cycle (63). modeling approach of chargaff et al. (57), found that annual precipitation has a significant role in distribution of leishmania vector species in mediterranean basin (57). s. minuta is recorded in arid region of tunisia with an annual precipitation between 88 and 157mm (53), also in portugal with an annual precipitation equal to 730mm (64). in the present study, it prefers regions with an annual precipitation (bio 12) between: 50–1100mm, and precipitation seasonality (bio 15) between: 30–100mm (supporting information s1). conclusion effective vector control requires accurate vector identification. the spatial and temporal overlapping of s. minuta with leishmania species seemed to present the important criteria necessary to be incriminated as a potential vector of the leishmania species in morocco. hence, the present study opened a debate on the potential role of sergentomyia species, especially s. minuta in the transmission of leishmaniasis in morocco. hypothesis deserves further researches including, but not limited to, protozoan isolation from engorged specimens as well as experimental transmission. acknowledgements the authors would like to thank elouise gaylard for linguistic consultation. the authors declare that there is no conflict of interest. references 1. hazratian t, vatandoost h, oshaghi ma, yaghoobi-ershadi mr, fallah e, rafizadeh s, shirzadi mr, shayeghi m, akbarzadeh k, rassi y (2016) diversity of sand flies (diptera: psychodidae) in endemic focus of visceral leishmaniasis in azar shahr district, east azarbaijan province, north west of iran. j arthropod borne dis. 10(3): 328–334. 2. belkaid y, valenzuela jg, kamhawi s, rowton e, sacks dl, ribeiro jm (2000) delayed-type hypersensitivity to phlebotomus papatasi sand fly bite: an adaptive response induced by the fly?. proc natl acad sci usa. 97(12): 6704–6709. 3. killick-kendrick r, killick-kendrick m (1999) biology of sand fly vectors of mediterranean canine leishmaniasis. in: canine leishmaniasis: an update. the first international canine leishmaniasis forum, 1999 february, barcelona, spain, pp. 28–31. 4. mukherjee s, hassan mq, ghosh a, ghosh kn, bhattacharya a, adhya s (1997) leishmania dna in phlebotomus and sergentomyia species during a kala-azar epidemic. am j trop med hyg. 57: 423– 425. 5. berdjane-brouk z, koné ak, djimde aa, charrel rn, ravel c, delaunay p, del giudice p, diarra az, doumbo s, goita s, thera ma, depaquit j, marty p, doumbo ok, izri a (2012) first detection of leishmania major dna in sergentomyia (spelaeomyia) darlingi from cutaneous leishmaniasis foci in mali. plos one. 7: e28266. 6. kahime k, boussaa s, bounoua l, ouanaimi f, messouli m, boumezzough a (2014) leishmaniasis in morocco: diseases and vectors. asian pac j trop dis. 4: 530–534. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 17–28 m daoudi et al.: modeling spatial distribution of … 25 http://jad.tums.ac.ir published online: march 31, 2020 7. rioux ja, guilvard e, dereure j, lanotte g, denial m, pratlong f, serres e, belmonte a (1986) infestation naturelle de phlebotomus papatasi (scopoli, 1786) par leishmania major mon-25. a propos de 28 souches isolées dans un foyer du sud marocain. in rioux j.a. 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phlebotomus papatasi and p. sergenti (diptera: psychodidae) in cutaneous leishmaniasis endemic foci, morocco. parasit vectors. 9: 60. 63. kasap oe, alten b (2006) comparative demography of the sand fly phlebotomus papatasi (diptera: psychodidae) at constant temperatures. j vector ecol. 31: 378–385. 64. branco s, alves-pires c, maia c, cortes s, cristovão jms, gonçalves l, campino l, afonso mo (2013) entomological and ecological studies in a new potential zoonotic leishmaniasis focus in torres novas municipality, central region, portugal. acta trop. 125(3): 339–348. http://jad.tums.ac.ir/ microsoft word 3-aghighi .doc iranian j arthropod-borne dis, 2007, 1(2): 14-18 z aghighi et al.: distribution of soft ticks and … 14 original article distribution of soft ticks and their natural infection with borrelia in a focus of relapsing fever in iran z aghighi1, m assmar1, n piazak1, e javadian2, ma seyedi rashti2, eb kia2, y rassi2, *h vatandoost2 1department of medical parasitology, pasture institute of iran, tehran, iran 2 department of medical entomology and vector control, school of public health, medical sciences/university of tehran, tehran, iran (received 10 may 2007; accepted 15 oct 2007) abstract tick-borne diseases such as relapsing fever and crimean-congo hemorrhagic fever (cchf) are of public health importance in iran. there are 471 reported cases of relapsing fever in 2003, according to the ministry of health of iran.the number of cases has been increased in recent years. its distribution is more or less prevalent in different parts of iran. the aim of this study was to find out the fauna and natural infection of soft ticks with borrelia in qazvin province, during their seasonal activity. the province covers 15821 km² between 48-45 to 50-50 east of greenwich meridian of longitude and 35-37 to 36-45 north latitude of the equator. for this purpose a field study was carried out in the region. a total of 54 villages from 19 districts were selected randomly and ticks were collected from their habitats according to the standard method. a total of 3197 argasidae ticks were collected from human dwellings, poultry and animal shelters. they belonged to argas and ornithodoros genera which 36.8% were argas persicus, 4% a. reflexus, 6.4% o. canestrini, 45.5% o. lahorensis and 7.3% o. tholozani. it should be noted that 12 ticks of o. erraticus were collected from 12 rodents borrows. we found that 8.82 % of o. tholozani ticks were infected with borrelia persica and half of the o. erraticus were infected with borrelia microti. all the people who are involved with veterinary activities should be aware of disease transmission by the ticks. in the endemic area of the disease tick control is recommended. keywords: ticks, borrelia, relapsing fever, iran introduction tick borne relapsing fever (tbrf) is a zoonotic disease involving many species of rodents in widely distributed areas throughout the world (gray et al. 2002). relapsing fever–like spirochetes infect 3.5% of questing vector ticks in european sites near the rhein vally (richter et al. 2003). tbrf is caused by one of many borrelia designated by the species of ornithodoros ticks responsible for its transmission. tbrf occurs sporadically and in small clusters throughout rural in the northern temperate region of the world. crimean-congo hemorrhagic fever (cchf) tick-borne and relapsing fever (tbrf) are the most common tick borne diseases in country. soft ticks play an important role in transmission of bacteria, rikettsia and viral diseases to human (cunha 2000). in iran soft ticks of the genus ornithodoros spread the tick-borne diseases. the responsible borrelia species are identified closely with its tick vector and they share parallel nomenclature, for example: borrelia persica infected o. tholozani in northern provinces of iran. b. microti is transmitted by o. erraticus which are found in rodent burrows. they are reported from several areas located in central and southern iran. *corresponding author: dr h vatandoost, tel/fax: +98 21 88951393, e-mail: vatando@tums.ac.ir iranian j arthropod-borne dis, 2007, 1(2): 14-18 z aghighi et al.: distribution of soft ticks and … 15 according to the ministry of health of iran (unpublished documents), the total number of 201, 205 and 471 relapsing cases have been reported in 2001, 2002 and 2003, respectively. the disease distribution is different in various parts of iran. the aim of this study was to find out the fauna and infection of soft ticks with borrelia in qazvin province during their seasonal activity. the province is between 48-45 to 50-50 east of greenwich meridian of longitude and 35-37 to 36-45 north latitude of the equator. materials and methods study area qazvin province is located in northern of iran. among 540 villages, the total number of 54 (10%) were randomly selected for investigation. this study is a filed and experimental study. tick collection tick collection was carried out in human dwellings, animal and poultry shelters in 30 min. ticks were collected from cracks, crevices, ceiling, floor and rodents burrows and transferred into the holding tubes. all the specimens were identified on the basic of their morphological characteristics. some of the specimens were selected and prepared for electron microscopy studies. infection of ticks with borrelia o. tholozani was fed on guinea pig but o. erraticus on new borne mice for 30-45 minutes. after 2 weeks blood samples were obtained from infected guinea pig and mice were examined microscopically to determine the presence of borrelia in their blood. o. tholozani were smashed and then injected subcutaneously to sensitive animals. the animals’ blood samples were examined for the presence of borrelia after one week using dark field microscopy stained with wright and geimsa dye. the ethical issue was approved by the institute of pasteur of iran. results a total number of 3197 soft ticks were collected and identified. the percentages of o. lahorensis, o. canestrini, o. tholozani. o. erraticus, a. persicus, and a. reflexus were 45.4%, 6.3%, 7.2%, 0.4%, 36.7% and 4%, respectively (fig. 1). out of 3197 ticks, 243 of o. tholozani (fig. 2) and o. erraticus from qazvin province only ticks in amirabad from central area were infected with b. persica and b. microti, respectively. fig. 1. distribution of ticks in the study area iranian j arthropod-borne dis, 2007, 1(2): 14-18 z aghighi et al.: distribution of soft ticks and … 16 fig. 2. dorsal view of o. tholozani discussion relapsing fever is caused by spiral-shaped bacteria of the genus borrelia. this bacterium lives in rodents and in insects, specifically ticks and body lice. the form of relapsing fever acquired from ticks is slightly different from that acquired from body lice. in tbrf, rodents (rats, mice, chipmunks, and squirrels) which carry borrelia are fed upon by ticks. the ticks then acquire the bacteria, and are able to pass it on to humans. tbrf is most common in sub-saharan africa, parts of the mediterranean, areas in the middle east, india, china, and the south of russia. soft ticks of the genus ornithodoros spread tick borne disease in iran. tbrf is a systemic spirochetal disease and patient spikes a very high fever, with chills, sweating, terrible headache, nausea, vomiting, severe pain in the muscles and joints, and extreme weakness. the patient may become dizzy and confused. the eyes may be bloodshot and very sensitive to light. a cough may develop. the heart rate is greatly increased, and the liver and spleen may be swollen. because the substances responsible for blood clotting may be disturbed during the illness, tiny purple marks may appear on the skin, which are evidence of minor bleeding occurring under the skin. the patient may suffer from a nosebleed, or may cough up bloody sputum. all of these symptoms last for about three days in tbrf lasting 2 to 9 days alternating with a febrile periods of 2 to 4 days. symptoms of infection include high fever, headache, prostration, myalgia, and sometimes gastrointestinal manifestations. the responsible borrelia species are identified closely with its tick vector and they share parallel nomenclature for example: b. persica infected o. tholozani, b. microti infects o. erraticus. b. latychevyi infects o. tartakovsky and b. baltazardy infects unknown vector. the main reservoirs of the borrelia are wild rodents and ornithodoros ticks. it can be concluded that o. lahorensis and o. erraticus encompass the highest and lowest frequency, respectively. among 231 specimens of o. tholozani only 8.82% revealed infection with b. persica and 12 specimens of o. erraticus (50%) were infected with b. microti .the infection of ticks is correlated with disease distribution in the province. in an investigation in iran, o. tholozani was infected with borrelia in kazeroon area, southern iran (janbakhs and ardelan 1997). in the west azerbaijan of iran of the 1421 ticks collected from ardebil, northwestern iran, 45.9%, 40.3% and 13.8% were of the o. lahorensis, o. tholozani and a. persicus species, respectively. the prevalence of ticks was highest in khandabil village. the ticks collected from three villages were found to be infected with borrelia (arshi et al. 2002). a case of meningitis from borreliasis was found in ardebil province (majid-pour 2003). telmadarraiy et al. (2004) described the fauna of ticks in west azerbaijan. they found 15 species of hard and soft ticks. vatandoost et al. (2003) conducted a survey in a borreliasis focus in hamadan province, iran. they found the a. persicus, a. reflexus, o. tholozani, o. canestrini and o. lahorensis are present in the region. o. iranian j arthropod-borne dis, 2007, 1(2): 14-18 z aghighi et al.: distribution of soft ticks and … 17 tholozani was found infected by b. persica robinson and spradling (2006) cited the main important tick borne disease transmitted by different ticks in iran and neighboring countries of caucasus region. they referred tick-borne relapsing fever transmitted by o. tholozani, o. erraticus and o. asperus. it should be noted that in europe the main vector of borrelia is ixodes and there are several species of borrelia reported from this region including: b. burgdorfri, b. afzeli, b. garini, b. valaisi and b. lusitaniae (gern et al. 2007). a relapsing fever–like spirochete, b. miyamotoi has been added to this array of parasitic microbes (fukunaga et al.1995). for control of ticks there are several measures which can be classified as follows: prevention of rodent access to the foundations and attics of homes or vacation cabins reduces the potential for tick exposure in these dwellings. structures infested with soft ticks should be treated professionally with acaricides, rodentproofing measures. interior residual application, animal treatment, personal protection using repellent and impregnated bed nets, removal of ticks from body, impregnation of clothing with permethrin can provide long–term protection against infestation, the development of a biological tick control method has been neglected as compared to the control of plant pests or dipterous insects harmful to men and animal (samish 2007) vaccination, environmental sanitation, active case detection, prompt treatment and health education. reporting of suspected cases of relapsing fever to health authorities is important for initiating prompt epidemiological investigation and control measures. acknowledgements the authors would like to appreciate very much for kind collaboration of staff of department of medical parasitology, pasteur institute of iran as well as department of medical entomology and vector control, school of public health, medical sciences/tehran university. the research was financially supported by the education department of the school of public health. references arshi sh, majidpoor a, sadeghi h, asmar m (2002) relapsing fever in ardabil, a northwestern province of iran. arc iran med. 5(3): [4]-145. cunha ba (2002) tick-borne infectious disease. diagnosis and management. marcel dekker. inc. publication, new york. fukunaga m, takahashi y, tsuruta y, matsushita o, ralph d, mcclelland m (1995) genetic and phenotypic analysis of borrelia miyamotoi sp. nov, isolated from the ixodid tick ixodes persulcatus, the vector for lyme disease in japan. int j syst bacteriol. 45: 804-810. gern l, perret jl, gremion f, guigoz e, rais o, mosman y (2002) five years survey on the prevalence of borrelia burgdorferi senso lato in ixodes ricinus, on tick density and clinical cases of lyme borreliosis in an endemic area of switzerland. int j med microbiol, 291 (supp l33): 21. gray js, kahl o, lane rs, stanek g (2002) lyme borreliosis: biology and control. cabi publishing book. janbakhs b, ardelan a (1977) the nature of sporadic cases of relapsing fever in kazeroon area, southern iran. bull path exot. 70: 587-89. majid-pour a (2003). a case of borrelia meningitis. arch iran med. 6(3): 222-223. richter d, schlee db, matuschka fr (2003) relapsing fever–like spirochetes infecting european vector tick of lyme disease agent. emerg infect dis. 9(6): 697-701. robinson rg, spradling ms (2002) vector – borne infectious disease in iran. washington dc publication. usa. available in: http://www.afpmb.org/pubs/dveps/iran.pdf samish m (2000) biocontrol of ticks. ann new york acad sci. 916: 127-8. iranian j arthropod-borne dis, 2007, 1(2): 14-18 z aghighi et al.: distribution of soft ticks and … 18 telmadarraiy z, bahrami a, vatandoost h (2004) a survey on fauna of ticks in west azerbaijan province, iran. iranian j publ health. 33: 65-69. vatandoost h, ghaderi a, javadian e, zahir nia ah, rassi y, piazak n, kia eb, shaeghi m, telmadarreiy z, aboulghasam m (2003) distribution of soft ticks and their infection with borrelia in hamadan province, iran. iranian j publ health. 32(1): 22-24. j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 378 http://jad.tums.ac.ir published online: december 25, 2018 original article geographical features and seroprevalence of borrelia burgdorferi in erzincan, turkey *aytekin cikman 1, merve aydin 1, 2, baris gulhan 1, faruk karakecili 3, levent demirtas 4, ozan arif kesik 5 1department of medical microbiology, erzincan university school of medicine, erzincan, turkey 2department of medical microbiology, kto karatay university school of medicine, konya, turkey 3department of infectious diseases, erzincan university school of medicine, erzincan, turkey 4department of internal medicine, erzincan university school of medicine, erzincan, turkey 5department of geography, faculty of arts and sciences, erzincan university, erzincan, turkey (received 14 june 2017; accepted 8 oct 2018) abstract background: we aimed to determine the geographical features and seroprevalence of borrelia burgdorferi in erzincan, turkey, which has a high tick population due to its geographical position and climatic conditions. methods: from january to december 2014, 368 people living in erzincan, northeastern turkey were enrolled. b. burgdorferi igg antibodies were investigated in the collected serum samples using the elisa method in 2015. positive and borderline results were confirmed using the western blot (wb) method. results: borrelia burgdorferi igg positivity was found to be 4.1% by elisa and 2.17% by wb. of the seropositive people according to wb, 25% resided in areas within 2000m of rivers, 50% in areas with a slope of 0–5°, and 62.5% in areas with an altitude of lower than 1500 meters. conclusion: the seroprevalence of lyme borreliosis was high in erzincan, particularly among people engaged in animal husbandry in rural areas. in addition, the seroprevalence of borrelia varied according to geographical features, increasing in areas with a lower slope and altitude. keywords: borrelia burgdorferi, seroprevalence, geographical features, altitude, slope degree, turkey introduction lyme borreliosis (lb), also known as lyme disease, is the most common tick-borne infectious disease caused by spirochetes of the borrelia burgdorferi sensu lato (s.l.) complex and is transmitted by ixodes ticks (1, 2). to date, 19 different species have been identified in the b. burgdorferi s.l. complex. lb is mainly caused by three pathogenic genomic species: borrelia burgdorferi sensu stricto (b. burgdorferi), b. garinii, and b. afzelii (3, 4). there are 14 different species of ticks in the ‘ixodes ricinus complex’, in which borrelia continues its life cycle in natüre (5). in addition to the ixodes, which are the vector of spirochetes, other hematophagous insects, rodents and some warm-blooded animals such as birds are also important in the ecological transmission of the disease (6). lb seroprevalence is increased in areas where these ticks live and cases of tick bites are commonly seen. lb is known to have a widespread distribution, particularly in forests and woodlands (7, 8). the risk areas for ticks in cities are green areas and parks. therefore, the highest risk group for lb is forest and agricultural workers, hunters, livestock farmers, and individuals living in areas with a large tick population (9, 10). lb has different clinical episodes. a typical lesion in the early stage of the disease is erythema chronicum migrans. however, the majority of patients also present with fever, flulike symptoms, and regional lymphadeno*corresponding author: dr aytekin cikman, e-mail: draytekin65@hotmail.com j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 379 http://jad.tums.ac.ir published online: december 25, 2018 megaly (9, 11-13). in untreated cases, systemic manifestations such as musculoskeletal involvement, cardiovascular, and neurologic involvement may also develop during the later stages of the disease due to hematogenous dissemination (11, 14). it is difficult to diagnose lb due to the different clinical manifestation of overlapping symptoms and the high antigenic variability of b. burgdorferi. a two-step standard diagnostic protocol is recommended for the laboratory confirmation of lb. in the first step, an elisa or an indirect fluorescent antibody (ifa) method is used for the detection of antibodies. in the second step, western blot (wb) test is recommended to confirm the positive results obtained from the first step (15-17). lb is the most common tick-borne infectious disease in the northern hemisphere, and climatic, economic and social changes in recent years have been reported to increase the incidence of the disease (6, 18, 19). located in the northern hemisphere, turkey has similar geographical and climatic conditions to many european countries, in which lb is frequently reported (20). ixodes are the most common type of ticks observed in turkey (21). however, in turkey, there are only limited studies on the seroprevalence of lb. therefore, we aimed to determine the seroprevalence of lb in erzincan located in the northeast of turkey using the elisa and wb methods and to evaluate some of the risk factors. materials and methods study area the study was carried out from january to december 2014 in erzincan, located between 39° 02'–40° 05' north latitude and 38° 16 '–40° 45' east longitude in northeast turkey. erzincan comprises nine districts, refahiye, kemah, kemaliye, tercan, çayırlı, i̇liç, otlukbeli, üzümlü, and central district. erzincan is located in the kelkit valley which has a large tick fauna. it has many rivers, streams, and wetlands, which facilitate farming and animal husbandry practices. erzincan has the characteristics of a terrestrial climate characterized by relatively cold and rainy winters and hot and dry summers. the annual average temperature is 10.9 °c and the coldest and hottest months are january and july with average temperatures of -6.7 °c and 31.4 °c, respectively. erzincan has an average rainfall of 380.6mm with the maximum being 633.1mm and the minimum being 206.1mm. the annual average humidity is 64.26% (22). collection of blood samples this study was conducted with the approval of erzincan university ethics committee (approval no: 2014/7). the study was planned as cross-sectional epidemiological research. the sample size was determined using a cluster sampling method. overall, 368 healthy volunteers were included in the study. all the participants were informed about the study and signed the informed consent forms. the participants were also asked to complete a short questionnaire to reveal in-formation on their gender, age, occupation, place of residence, engagement in animal husbandry, and tick exposure. a 10ml venous blood sample was taken from all participants. the samples were transferred to the laboratory maintaining the cold chain and then centrifuged at 1610g for 10min to separate the sera and stored at -80 °c until the time of serological tests. detection of antibodies using elisa borrelia burgdorferi immunoglobulin g (igg) antibodies were determined in all serum samples using a serion elisa classic kit (institut virion\serion gmbh, wurzburg, germany) according to the manufacturer’s recommendations. the samples were diluted 1:100 using a dilution buffer. the standards and diluted samples were transferred to microtiter j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 380 http://jad.tums.ac.ir published online: december 25, 2018 wells and incubated at 37 °c for 60min in a moist chamber. the residual serum was removed from the wells by washing four times with a wash buffer. anti-human igg conjugated to alkaline phosphatase was added and incubated at 37 °c for 30min in a moist chamber. the wells were washed four times with the wash buffer; then, substrate p-nitrophenyl phosphate was added and incubated at 37 °c for 30min, followed by the addition of a stop solution. the optic density was determined at 405nm (at the 650nm reference wavelength) using an epoch elisa spectrophotometer (biotek instruments, inc., winooski, vt, usa). each kit was used with a negative control, positive control, and standards in dup-licate. interpretation of the results was performed assisted by serion easy base 4pl software, and the results were expressed in units /milliliter (igg, <3u/ml [negative], 3– 5u/ml [borderline], >5 u/ml [positive]). detection of antibodies using wb the serum samples that had a positive or borderline value in the elisa igg test were further analyzed using a viro-blot kit (viroimmun labor-diagnostika gmbh, germany) to confirm the presence of b. burgdorferi igg antibodies according to manufacturer’s recommendations. briefly, nitrocellulose strips containing electrophoresis-separated b. afzelii/ b. garinii/ sensu stricto proteins were blocked and then incubated with 1020µl of the diluted serum sample (1:51) for 60min. the membrane strips were washed and incubated with alkaline phosphatase (ap)-conjugated anti-human igg antibody for 30min. following a final wash, strips were incubated with a chromogensubstrate solution for 10–15min, washed and air-dried on a rocking platform. the quantitative analysis of bands on each blot was carried out using the blotrix software (viro-immun labor-diagnostika gmbh, germany). the software corrected the background and determined the cutoff values for positivity for the recombinant vlse borrelial protein bands. the igg assay was considered to be positive if five or more of the following ten bands were present: p17/p19 complex, p20/p21 complex, p25 (ospc), p30, p31 (ospa), p35, p45, p59/ p62 complex, p100, and vise. mapping arcgis 10.1 and google earth programs were used to draw the maps and conduct analyses. first, a map of erzincan including its districts and river and stream branches was drawn to be used in the spatial analysis. next, baseline data were created using the arcgis basemap openstreetmap service. then, the locations of the seropositive cases were detected in google earth program and transferred to arcmap software. addition, the relationship between seropositive cases and the altitude and slope degrees of their places of residence was evaluated. to determine the geographic correlations with seropositive samples, we obtained data from the advanced spaceborne thermal emission and reflection radiometer global digital elevation model (aster gdem), national aeronautics and space administration (nasa), and the ministry of economy, trade, and industry (meti). in addition, spatial analysis was performed through buffer analyses (buffer, multiple ring buffer) on the river and streams and their branches. statistical analysis the data were evaluated using the (ibm spss statistics for windows, version 20.0, ibm corp., armonk, ny, usa, released 2011). the values of the variables were expressed in mean±standard deviation and median (max–min) percent and frequency. the categorical data were analyzed by fisher's exact test and the chi-square test. when the expected frequency was less than 20%, the monte carlo simulation method was used to evaluate and determine the frequencies to be included in the analysis. p< 0.05 were accepted as the significance levels for the tests. j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 381 http://jad.tums.ac.ir published online: december 25, 2018 results of the 368 individuals included in the study, 225 (61.1%) were female and the average age was calculated as 51.43±16.91 year. there was no statistically significant difference between the participants in terms of average age and gender (p= 0.578). using the elisa method, b. burgdorferi igg antibodies were found to be positive in 15 individuals (4.1%) and the results of 36 individuals were within borderline value range. these 51 samples with positive or borderline values were further analyzed using the wb technique to determine their b. burgdorferi igg levels. wb confirmed positivity in 8 of the 51 samples. thus, the overall b. burgdorferi igg positivity was calculated as 2.17% (8/368) (table 1). among the 8 individuals with b. burgdorferi positivity confirmed by the wb method, 7 lived in rural areas, 6 were engaged in animal farming and 4 were exposed to ticks. three of these eight cases were men aged 65 to 75yr engaged in farming, lived in rural areas and were exposed to ticks. when all the indi viduals were evaluated, living in rural areas was found to be statistically significant in relation to elisa and wb values (p= 0.010). furthermore, tick exposure and engagement in animal farming had a statistically significant effect on the positivity of the elisa igg values, but not the positivity of the wb values (table 1). the relationship between wb positivity and geographical features namely distance to rivers, altitude, and slope was investigated. twenty-five percent of people with igg positivity lived within 2000m of rivers or their main branches in the study area. the slope of the study area ranged from 0 to 72.44°. the slope degrees of the residential places of the seropositive people were 0–5° for 50%, 5– 10° for 13%, and greater than 10° for 37%. lastly, the study area had an altitude of 817 to 3518m above sea level, and 62.5% of seropositive cases lived in places with an altitude of fewer than 1500 meters (fig. 1, 2). table 1. the seroprevalence of borrelia burgdorferi and associated risk factors elisa igg (n=368) wb (n=51) borderline (n=36) positive (n=15) p borderline (n=17) positive (n=8) p gender female 22 11 0.405 10 5 0.861 male 14 4 7 3 age ≤ 45 10 6 0.391 4 3 0.468 > 45 26 9 13 5 animal farming yes 28 12 0.010* 14 6 0.668 no 8 3 3 2 tick exposure yes 16 3 0.010* 9 4 0.891 no 20 12 8 4 living area rural 30 13 0.010* 15 7 0.010* urban 6 2 2 1 *p< 0.05 j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 382 http://jad.tums.ac.ir published online: december 25, 2018 fig. 1. the relationship between igg positivity and altitude (meter), slope degrees (º) and distance (meter) according to the wb method fig. 2. the geographical features of areas in which seropositive individuals lived according to the results of the wb method j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 383 http://jad.tums.ac.ir published online: december 25, 2018 discussion lb poses serious problems among tickborne diseases. lb is particularly well known in the northern hemisphere and is the most common infectious disease caused by ticks in north america and europe (23, 24). the incidence of lb in europe has been reported to range from 5% to 25% (25). the incidence of lb is high in the south of scandinavia and the north of mediterranean countries increasing from the west to the east. central and eastern europe are the endemic areas in which the disease is most frequently seen (26). although different borrelia species can cause lb, the most common agents in europe are b. afzelii and b. garinii (27). turkey has a similar tick fauna to many european countries (21). in this study, taking into consideration the reported results (21), we used an elisa kit containing b. burgdorferi sensu stricto, b. afzelii and b. garinii antigens (second generation). in the second phase of the study, we employed a highly specific immunoblot method including the vlse surface antigen, reported to have the highest sensitivity and specificity (28, 29). in turkey, although lb is not a notifiable infectious disease and it is relatively less known, studies conducted in recent years have revealed its prevalence. the seroprevalence of b. burgdorferi was evaluated among people engaged in agriculture and animal husbandry and reported it to be 3.3% (30). the b. burgdorferi igg antibody positivity in risk groups was found as 3.8% using the elisa method (20). the wb technique confirmed the positive result in 0.9% of the cases. in the current study, we found a similar percentage of b. burgdorferi igg positivity (4.1%) using elisa compared to previous studies. however, the percentage of wb b. burgdorferi positivity (2.17%) was higher than previous reports. this higher ratio can be attributed to the current study area having a large tick fauna. agricultural workers, hunters, and livestock farmers form the risk group of lb. however, these groups also vary according to the natural habitat of different tick species (31, 32). in the current study, using the elisa method, risk factors were determined to be living in a rural area, exposure to ticks, and engagement in animal husbandry. however, with the wb method, only living in rural areas was found to have a statistically significant effect on the incidence of lb. this can be explained by the lower number of positive cases detected by wb. lb cases in europe are generally reported between the latitudes of 40°n and 60°n. the incidence of lb also varies according to geographical location and species of ticks (9, 33). tick species infected with borrelia were more common in areas with an altitude lower than 1300 meters (9). in norway, lb seroprevalence was lower in inland areas but increased in coastal areas and those close to the south. however, the seroprevalence of lb also differed according to geographical characteristics (34). similarly, in the current study, 62.5 % of the seropositive individuals lived in areas with an altitude below 1500m. furthermore, concerning the slope degrees of the places of residence of seropositive cases, 50% had a 0–5o slope. the results of the current study are similar to those reported for norway in terms of the higher percentage of seropositive individuals living in coastal areas. lb is more frequently seen in areas with a lower slope and lower altitude. as another geographic feature, we also examined the distance of the positively identified people to the rivers in the study area and found that 25% lived within 2000 m from the rivers. contrary to our expectation, this data indicates that lb is more common in areas away from wetlands. the seroprevalence of lb will vary according to the geographical characteristics. this is one of the rare studies, in which the relationship between lb seroprevalence and geographical features (such as distance to river, j arthropod-borne dis, december 2018, 12(4): 378–386 a cikman et al.: geographical features … 384 http://jad.tums.ac.ir published online: december 25, 2018 altitude, and slope degree) was explored. in addition to geographical features, environmental factors such as climate, living conditions and the presence of reservoir animals affect lb seropositivity. the current study is also important in terms of being the first to investigate lb seroprevalence in erzincan, an area with a large tick fauna. conclusion the seroprevalence of lb was found to be high in erzincan, particularly among people engaged in animal husbandry and exposed to ticks. the seroprevalence of borrelia varies according to geographical features and is higher in areas with a lower slope degree and altitude. acknowledgements this project was financially supported by the scientific research and project unit of erzincan university (project no: sag-a300614-0090). the authors declare that there is no conflict of interests. references 1. melaun c, zotzmann s, santaella vg, werblow a, zumkowski-xylander h, kraiczy p, klimpel s (2016) occurence of borrelia burgdorferi s.l. in different genera of mosquitoes (culicidae) in central europe. 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of the borrelia burgdorferi sensu lato species complex in north america. int j syst evol microbiol. 64(1): 128–130. 34. vestrheim df, white ra, aaberge is, aase a (2016) geographical differences in seroprevalence of borrelia burgdorferi antibodies in norway, 2011–2013. ticks tick borne dis. 7(5): 698–702. microsoft word 8dr_telma_rtl.doc iranian j arthropod-borne dis, (2007), 1(1): 58-62 z telmadarraiy et al.: determination of rodent… 58 short communication determination of rodent ectoparasite fauna in sarpole-zahab district, kermanshah province, iran, 2004-2005 *z telmadarraiy, h vatandoost, s mohammadi, aa akhavan, mr abai, j rafinejad, eb kia, f faghih naini, m jedari, m aboulhasani dept. of medical entomology and vector control, school of public health and institute of health research, medical sciences/university of tehran, iran (received 8 oct 2006; accepted 22 apr 2007) abstract ectoparasites of various species of rodents were examined from sarepole zehab, kermanshah province iran during 2005. this city is bordered with iraq. a total of 139 rodents included 6 species of hosts were captured during all different seasons and examined for ectoparasites using live trap. the area of study was mainly plateu (70%) and mountanous. the hosts including: microtus socialis, mus musculus, rattus rattus, nesokia indica, meriones persicus and tatera indica. the 9 species of ectoparasites recovered included 3 fleas (pulex irritans, xenopsylla buxtoni, nosopsyllus medus), one sucking lice (polyplax spinolosa), two ticks (rhipicephalus sp., hyalomma sp.), and 3 mites (lealaps nuttalli, dermanysus sanguineus, ornithonussus bacoti). among all arthropod collected, the lice and flea had the most and least frequency, i.e. 77.7% and 4.4%, respectivelly. the data showed that the ectoparasites on some rodent hosts tend to prefer particular host body sites, and that some ectoparasite species sites may overlap owing to their inaccessibility to the host. keywords: rodents, ectoparasites, iran introduction most medically important rodents belong to the families of muridae and the cricetidae. rodents play a role in many diseases, such as plague, transmitted by the rat flea xenopsylla cheopis and weil's disease, a severe form of leptospirosis transmitted via infected rat urine. rodents play a role in conditions such as echinococcosis (e. multilocularis), trichinellosis, lyme borreliosis, salmonellosis, rat bite fever, tularemia, lymphocytic choriomeningitis, hymenolepis diminuta and rickettsioses such as rmsf, scrub typhus and murine typhus. haemorrhagic fevers that are transmitted by rodents ("rodent-borne") include hantaviruses and arena viruses such as junin, machupo and lassa fever. infection with penicillium marneffei is essentially a disease of rodents, but can occur in aids patients in southeast asia (bell et al. 1988, vatandoost et al. 2003, telmadarraiy et al. 2004). the aim of this study was to find geographical distribution of rodents and their ectoparasites for implementation of any prevention and control measures for zoonotic diseases in the region. materials and methods geographical information sarpole zahab area, 971 km2, longitude 54° 52, attitude 34° 24́, elevation 550 meter, precipitation 500 mm, maximum temperature 44.8 °c, minimum temperature 3.4 °c. *corresponding author: dr z telmadarraiy, tel: +98 21 66465404, fax: +98 21 66462267, e-mail: ztelma@ yahoo.co.in iranian j arthropod-borne dis, (2007), 1(1): 58-62 z telmadarraiy et al.: determination of rodent… 59 rodent collection rodents were live trapped at different localities including markaziand humeh ghalehshahin zahab and posht-tangh beshive and patagh. the collection was carried out on various occasions in years 20042005. live traps were randomly set in different habitats (e.g., woodland transects, woodland, old fields, refuse heaps, beside dump, in and around demolished buildings at different localities). traps were baited with favorable food of rodents according to the season. collecting of ectoparasites captured animals were transported to laboratory and their ectoparasites were picked up. fleas, mites, lice and ticks were collected using brushing against the fur of rodents. in some cases of ticks they were collected by forceps. ectoparasites were stored in 70% ethanol for their preservation and identification. ectoparasite and rodent identification ectoparasites specimens were mounted using different stages of clearing, dehydration and mounting procedures. finally specimens were fixed in between microscope slides and cover glass using canadabalzam. different criteria of morphology of rodents were used for species identification. field operation, site selection, active reconnaissance, trap timing, trapping method, trap baiting, trap collection, biometrics measurement, data recoding, ectoparasites collection laboratory operation, preparation of rodent skulls, mounting of ectoparasite, identification using valid keys, confirmation of some species by expertise institute was carried out according to standard methods provided. some ticks were collected at the immature stage, so that they species name was announced as sp. results in four different epidemiological parts of the county a total of 139 rodents of both sexes were captured and from them 753 sample of ectoparasites were collected. subsequently all of them were identfied in labratoary. rodents belong to suborder myomorpha, family of muridae (subfamily: murinae), critidae (subfamily: gerbillinae and microtinae) (table 1). three species of mus musculus, rattus rattus, and nesokia indica were found in all parts of the study area. there was no significant difference in distribution of two commensal rodent in their distribution (p> 0.05), however wild rodents was found significantly different in their abundance and frequency (p< 0.05). there was significant difference among rodent species in their habitats (p< 0.05). tatera indica was found more infested by different ectoparasites than other species (78.1%). the figure for microtus socialis was very low (0.3%). the ectoparasites were found mainly from different species of flea, mite, lice and ticks (table 2, fig.1). several indicators such as: ectoparasite species richness, mean abundance, ectoparasite catch range, and specific diversity index were determined for each rodent and ectoparasites. these indicators mainly depend on season, rodent species, ectoparasite species, location, method of catch, geographical situation, ecological condition, rodent predators, seasonal activities, and population dynamics. xenopsylla buxtoni was found in all seasons; however this species was more prevalent in summer. in contrast, pulex irritans are active during winter. nosopsyllus medus was found mainly in spring. polyplax spinulosa are found mainly in autumn. hyalomma sp. and laelaps nuttalli in spring, and rhipicephalus sp. in summer were more abundant. iranian j arthropod-borne dis, (2007), 1(1): 58-62 z telmadarraiy et al.: determination of rodent… 60 table 1. species composition of rodents, sarpolezahab district, kermanshah province, 2004-2005 rodents catch at different area markaziand humeh ghalehshahin zahab and posht-tangh beshive and patagh rodent catch rate rodent species catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) chi-square test (p-value) mus musculus 9 25.0 8 22.2 10 27.8 9 25.0 36 25.8 0.794 rattus rattus 6 31.6 4 21.1 3 15.8 6 31.6 19 13.6 0.782 nesokia indica 8 22.9 7 20.0 5 14.3 15 42.9 35 25.2 0.042 microtus socialis 3 7.1 0 0 0 0 2 12.5 5 3.6 0.023 meriones persicus 5 100.0 0 0 0 0 0 0 5 3.6 tatera indica 0 0 0 0 39 100 0 0 39 28.1 0.000 total 31 30.2 19 45.3 57 13.0 32 11.5 139 100.0 0.000 table 2. ectoparasite groups among rodents, sarpolezahab district, kermanshah province, 2004-2005 rodents catch at different area total catch rate fleas mites lice ticks rodent species catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) chi-square test (p-value) mus musculus 4 18.2 18 81.8 0 0 0 0 22 2.9 0.003 rattus rattus 6 50.0 0 0 0 0 6 50.0 12 1.6 1.000 nesokia indica 4 4.2 0 0 34 35.4 58 60.4 96 12.8 0.000 microtus socialis 2 100 0 0 0 0 0 0 2 0.3 meriones persicus 7 21.2 14 42.4 10 30.3 2 6.1 33 4.4 0.026 tatera indica 10 1.7 10 1.7 540 92.0 27 4.6 587 78.1 0.000 total 33 4.4 42 5.6 584 77.7 93 12.4 752 100.0 0.000 fig. 1. different ectoparasites collected from rodents in the study area xxeennooppssyyllllaa bbuuxxttoonnii ppuulleexx sspp.. nnoossooppssyylllluuss mmeedduuss hhyyaalloommmmaa sspp.. rrhhiippiicceepphhaalluuss sspp.. llaaeellaappss nnuuttttaallllii o orrnniitthhoonnyyssssuuss bbaaccoottii ddeerrmmaannyyssssuuss ssaanngguuiinneeuuss ppoollyyppllaaxx ssppiinnuulloossaa iranian j arthropod-borne dis, (2007), 1(1): 58-62 z telmadarraiy et al.: determination of rodent… 61 discussion there are several reports in the world indication variation of those indicators (abulhab 1984, meehan 1984, shoukry et al.1986, durden et al. 2000, shayan 2003, kia et al. 2004). laelaps nuttalli, and polyplax spinulosa was also reported from mongolia (chuluun et al. 2005). the prevalence and general indices of some ectoparasites in this area showed differences related to the locality of their rat hosts. seasonal changes in the general indices of some ectoparasites paralleled seasonal changes in the relative abundance of their rat hosts. the indices of infestation by the mites laelaps nuttalli, the louse polyplax spinulosa and the flea xenopsylla cheopis, on rattus norvegicus norvegicus in brazil were related to seasonal period, sex of the host and area of capture (linardi et al. 1985). mode of transmission of disease from rodents to human are; feces, urine, saliva, blood and milk. factors explaining the emergence of a zoonotic or potentially zoonotic disease are usually complex, involving mechanisms at the molecular level, such as genetic drift and shift, and modification of the immunological status of individuals and populations. social and ecological conditions influencing population growth and movement, food habits, the environment and many other factors may play a more important role than changes at the molecular level. the probability of disease transmission from animals to man is influenced by several factors such as: length of time the animal is infective, length of the incubation period in animals (this is important in some diseases with long incubation periods, because the animals may be studied and euthanatized before they become infective for humans, the stability of the agent. (acha and szyfres 1987). in conclusion, results of our study are able to provide a clue for prevention and control of zoonotic diseases in the region for local authorities and in the emergency situations. acknowledgements the authors would like to thank of the staff of sarpol-e-zahab health center for their excellent assistance in achieving this study. this project received financial support from deputy for education affairs of school of public health, medical sciences/university of tehran. references abulhab j (1984) some ectoparasites of the commensal rodent in baghdad. bull endemic dis. 24(25): 51-62. acha, pn and szyfres b (1987) zoonoses and communicable diseases man and animals. pan american health organization, washington, dc. bell jc, palmer sr, payne jm (1988) the zoonoses: infections transmitted from animals to man. edward arnold press, london, uk. chuluun b, mariana a, ho t, mohd kulaimi b (2005) preliminary survey of ectoparasites of small mammals in kuala selangor nature park. trop biomed. 22(2): 243-247. durden la, hu r, oliver jh, cilek je (2000) rodent ectoparasites form two location in northwestern florida. j vect ecol. 25(2): 222-228. kia e, telmadarraiy z, vatandoost h, homayouni mm, satvat m, jedari m, kasiri f (2004) ectoparasites and arthropodaborne parasites of brown rat, rattus norvegicus in ahvaz, southwest iran. xxii international congress of entomology, queensland, australia. linardi pm, botelho jr, cunha hc (1985) ectoparasites of rodents of the urban region of belo horizonte, mg. ii. variations of the infestation indices in rattus norvegicus norvegicus. mem inst oswaldo cruz. 80(2): 227-232. meehan ap (1984) rat and mice, their biology and control. rentokit ltd [publ]. england. london. iranian j arthropod-borne dis, (2007), 1(1): 58-62 z telmadarraiy et al.: determination of rodent… 62 shayan a (2003) study of rodents and their ec toparasites in khoramabad township [msc thesis]. tarbiat modarres university, iran. shoukry a, morsy ta, abu hashish ta, el kady ga (1986) seasonal activities of two commensal rats and flea index in north sinai governorate, egypt. j egypt soc parasitol. 16 (2): 385-393. telmadarraiy z, bahrami a, vatandoost h (2004) a survey on fauna of ticks in west azer-baijan province, iran. iranian j publ health. 33: 65-69. vatandoost h, ghaderi a, javadian e, zahir nia ah, rassi y, piazak y, kia eb, shaeghi m, telmadarreiy z, aboulghasani m (2003) distribution of soft ticks and their infection with borrelia in hamadan province, iran. iranian j publ health. 32(1): 22-24. j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 134 http://jad.tums.ac.ir published online: june 30, 2020 original article emerging of cutaneous leishmaniais due to leishmania major in a new focus in esfahan province, central iran reza jafari1; hamid abdoli1; mohammad hossein arandian1; nilofar shareghi1; maryam ghanei1; nilofar jalali-zand1; shahram nekoeian2; arshad veysi3, ahmad montazeri4; amirabdollah ghasemi4; javad ramazanpour2; reza fadaei2; *amir ahmad akhavan5 1 esfahan health research station, school of public health, tehran university of medical sciences, esfahan, iran 2 esfahan province health center, isfahan university of medical sciences, esfahan, iran 3 zoonoses research center, research institute for health development, kurdistan university of medical sciences, sanandaj, iran 4 naein health care network, isfahan university of medical sciences, esfahan, iran 5 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 06 dec 2018; accepted 04 feb 2020) abstract background: esfahan province is considered as one of the main focus of zoonotic cutaneous leishmaniasis (zcl) in iran. recently, zcl distribution is expanding through this province leading to report of new cases in non-endemic areas. in the current study epidemiological aspects of zcl has been investigated in naein county in esfahan province. methods: adult sand flies were collected from beginning to the end of their seasonal activity. rodents were caught by sherman live traps once a month for one year. to active case detection, a hundred households in each selected village were visited in november and december 2016. nested-pcr was employed to detect leishmania parasite in the vector, reservoir and human. results: totally 1562 sand flies including phlebotomus sergenti, phlebotomus papatasi, sergentomyia sintoni and sergentomyia mervinae were collected and identified. no leishmania infection was detected in the collected sand flies. all of the 30 collected rodents were identified as rhombomys opimus, and of these 3.3% and 26.7% were infected by leishmania major using microscopic and molecular technique respectively. totally, 914 individuals were investigated and the ulcer and scar rates of zcl calculated to be at 1.1 and 15.3 per 1000 population, respectively. molecular results confirmed l. major infection in human and reservoir samples. conclusion: it is concluded that zcl is established in the area in low endemicity, and it is extrapolated the disease will not be a serious increasing health problem in the near future in this region. keywords: cutaneous leishmaniais; leishmania major; epidemiology; central iran introduction although estimated to cause ninth largest disease burden among infectious diseases, leishmaniasis is mostly neglected among tropical diseases. based on a recent report, it is estimated that, annually 0.2 to 0.4 and 0.7 to 1.2 million visceral leishmaniasis (vl) and cautaneus leishmaniasis (cl) new cases, occur respectively in the world (1). high level of reported cases listed iran in ten countries which together account for 70 to 75% estimated cases of cl globally (1). more than 20000 cases of cl cases are reported in iran annually, although the actual figures are estimated to be 4 to 5 folds (2). cutaneous leishmaniasis manifest in two forms in the country, zoonotic (zcl) and anthroponotic cl (acl), and about 80% of reported cases has been zcl form; especially in the rural *corresponding author: dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir http://jad.tums.ac.ir/ https://www.google.com/search?q=cutaneous+leishmaniasis&spell=1&sa=x&ved=0ahukewi_3-_dklxzahxk66qkhaswboaqkeecccqoaa https://www.google.com/search?q=cutaneous+leishmaniasis&spell=1&sa=x&ved=0ahukewi_3-_dklxzahxk66qkhaswboaqkeecccqoaa mailto:aaakhavan@tums.ac.ir j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 135 http://jad.tums.ac.ir published online: june 30, 2020 areas of the country (3). zoonotic cutaneous leishmaniasis is caused by a protozoan parasite leismania major, and phlebotomus papatasi plays as the main vector in the disease foci inside the country. seventeen out of 31 province deal with the disease (4). rodents belonging to gerbillinae subfamily play as reservoir host of zcl in different part of the country, as well as some other parts of the world. rhombomys opimus (great gerbil) known as the main reservoir host of zcl in central and the northeast of iran (5). esfahan province known as one of the most important foci of zcl, and in 2012 this province was categorized as one of the high reported cases of zcl (6). since the last two decades, iranian scientists along with health authorities have employed and conducted several measures such as spraying of rodent burrows with pesticides (7), baiting the reservoir hosts with rodenticides (8-13) and using deltamethrin-impregnated bed nets and curtains (14, 15) to control zcl in the country. in addition, despite extensive research in the field of vaccine, still there is no licensed vaccine against any form of leishmaniasis (16). iranian researchers have conducted a successful leishmanisis prevention through leishmanization, unfortunately it was banned due to very rare case of un-healing lesions, and since then it was recommended just for military personnel in the high risk areas (17). from 2012 in naein county the number of indigenous cases which were visited in the health centers with signs of cutaneous leishmaniais gradually increased and based on local health authorities report, surprisingly the confirmed recorded cases increased to 26 cases in 2015. considering the importance of this county as a main roads intersection of the country and passengers commuting, it is assumed, without taking serious measures to conduct research and control of the disease in this region, it might become a potential focus to spread zcl to other free areas. regarding this issue as well as lacking any comprehensive study on zcl in this county, conducting an epidemiological research seemed to be necessary. hence, this survey is the first epidemiological study that has ever been conducted in these areas. the current study aimed to study the different epidemiological aspects of the disease in this new emerging focus of zcl. materials and methods study area the present study was carried out in naein county (32°51'49.1"n 53°05'04.5"e), esfahan province, iran from october 2015 to february 2016. naein is located at 170km from north of yazd and 140km from east of esfahan with an area about 22,570km2 and it is considered as the widest county in esfahan province (fig. 1). the selected areas have desert climate, hot in summer and cold in winter. in 2015 the maximum and minimum mean monthly temperature was 40.8 and -10.6 °c in july and december, respectively; with the total rainfall of 40.8mm. entomological survey adult sand flies were collected from the beginning (april) to the end (november) of their active season, once a month using sticky traps in the rural selected areas (bafran, mohamadieh and mazraee emam) of naein. the sticky traps (15 in outdoor resting places and 15 in indoors in each selected point) were installed before sunset and collected next morning before sunrise in three selected villages. the captured sand flies were preserved in ethanol until the time of use. head and the last two abdomen segments of sand flies detached, mounted in puri’s media and the rest of the sand flies’ body was kept in 96% alcohol to examine for leishmania infection using molecular techniques. the species identified using valid keys based on morphological characters (18, 19). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 136 http://jad.tums.ac.ir published online: june 30, 2020 rodent collection and parasitological examination active colonies of gerbils have been found around bafran, mohamadieh and mazraee emam villages of naein. rodents were caught by 40 sherman live traps, baited with cucumber and/or carrot, once a month along a year. during spring to fall, the traps were installed in the vicinity of the gerbil holes in the afternoon and collected at the following morning, while during winter traps were installed at early mornings and collected at the afternoons. the collected gerbils were transferred to the esfahan health research station to further examination. the captured rodents were anaesthetized using ketamine hydrochloride (60mg/kg), and xylazine (5mg/kg) and were identified by morphological characters (20). then impression smears were prepared from the ear lobes of the anaesthetized animals, fixed by absolute methanol and stained by giemsa, and then the slides were microscopically examined at high magnification (1,000x). afterward the ear lobes were cut and transferred into 500μl cold pbs (ph= 7.4), and then disrupted by grinding using a pestle and stored in -20 °c until use. animal ethics was considered in treating with animals during this study. human infection to determine the prevalence and incidence of the disease in the selected village areas (bafran, mohamadieh and mazraee emam) of naein, 100 households in each village were visited in november and december 2016. epidemiologic data including id, presence or absence of scar (s) or active lesion (s), number of the lesion (s) or scar (s), and history of travelling to the other zcl foci were filled out in a questionnaire for all households. individuals who had travelled to other foci of zcl were excluded from the study. new cases of the disease and the number of active lesions were recorded on each visit. samples were prepared from the active lesions; before sampling, lesions were disinfected with alcohol 70% then the margin of ulcer was scratched using vaccinostyle. the prepared serosity was allocated in two parts, one part transferred into alcohol 96% for molecular parasite detection and another part was smeared on a slide, fixed with methanol, stained by giemsa and examined under microscope at high magnification (1,000x). molecular detection of leishmania infection genomic dna from samples of rodents, human and sand flies was extracted by geneall® exgene tm tissue kit (cat no: 101– 109) following the protocol of cultured animal cell or lymphocytes. the its2 region of leishmania parasite was amplified by nested-pcr, using the following primers (21) leish out f (5′-aaa ctc ctc tct ggt gct tgc-3′), leish out r (5′-aaa caa agg ttg tcg ggg g-3′), leish in f (5′aat tca act tcg cgt tgg cc-3′) and leish in r (5′-cct ctcttt ttt ctc tgt gc-3′). the targeted gene of ribosomal dna was amplified by a biosystems thermocycler. the volume of first reaction micro-tube of pcr was 25μl containing 0.6μm of both forward (leish out f) and reverse (leish out r) external primers, 12.5μl taq dna polymerase enzyme, 2x master mix (amplicon, denmark) and sterile distilled water. the first denaturation step was carried out at 95 °c for 5min and was followed by 30 cycles of denaturation at 94 °c for 30s, annealing at 60 °c for 45s and extension at 72 °c for 1min, finally ended with a final extension step of 72 °c for 5min. the second step of nestedpcr was carried out in a volume of 20μl containing 1μl of a 1:25 dilution of distilled water and the first-round pcr product as a template, 0.3μm of each forward and reverse internal primers, 10μl of taq dna polymerase and 2x master mix. the amplification was performed as initial denaturation at 95 °c for 2min, 25 cycles of 94 °c for 15s, 62 °c for 30s, 72 °c for 45s followed by the final exhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 137 http://jad.tums.ac.ir published online: june 30, 2020 tension at 72 °c for 5min. finally, pcr products were loaded on 1.5% (w/v) agarose gel electrophoresis in tbe buffer (0.09mm tris, 0.09mm boric acid, and 20mm edta, ph= 8.3); ethidium bromide (0.5μg/ml) was used to visualize the amplified dna bands on the gel and photographs were taken. leishmania major (mrho/ir/75/er), and distilled water were used as positive and negative controls respectively. to prevent any accidental contamination, primary precautions such as using filter pipette tips, and sterilizing equipment by 10% sodium hypochlorite solution were taken. polymerase chain reaction-restriction fragment length polymorphism (pcrrflp) to distinguish common species of leishmania genus, the products of nested-pcr were subjected to rflp using rapid digest mnl1 (cat. no: rd 1191) restriction enzyme. to this purpose, 10µl pcr products of the nested-pcr, 2µl buffer, 1µl mnl1 restriction enzyme, were added to a micro tube and reached to final volume of 30μl by distilled water. to activate the restriction enzyme, the mixture was kept in 37 °c for 1 hour, and then the products were loaded onto 2.5% (w/v) agarose gel electrophoresis in tbe buffer. finally loaded dna was visualized by ethidiumbromide (0.5µg/ ml), and photographed. results entomological survey from april to november 2016, totally 1562 sand flies (1451 from outdoors, 111 from indoors) were collected and identified based on the valid morphological keys. four species of sand flies including two species of phlebotomus genus and two species of sergentomyia genus were identified as follows: phlebotomus (paraphlebotomus) sergenti: this sand fly was the dominant species in indoors, which 70 (63.1%) and 8 (0.6%) of collected sand flies in indoors and outdoors were belonged to this species respectively. phlebotomus sergenti was caught in all months of sand flies active season in indoor places. the maximum relative frequency of this phlebotomine in indoor places was recorded in june (fig. 2). phlebotomus (phlebotomus) papatasi: the number of caught sand flies belonging to this species were 34 (30.6%) and 48 (3.3%) in indoors and outdoors respectively. phlebotomus papatasi was caught in all months of sand flies active season in either indoors or outdoors. the monthly activity of this species recorded a remarkable pick in june, both in indoor and outdoor places (fig. 3). sergentomyia (sergentomyia) sintoni: the results showed se. sintoni was the most dominant species in outdoor places. of all collected sand flies, 4 (3.6%) from indoor and 1002 (69%) from outdoors places were identified as se. sintoni. similarly, the maximum activity of this species occurred in june (fig. 4). sergentomyia (sergentomyia) mervinae: this species comprised 3 (2.7%) of the sand flies from indoors, and 393 (27.1%) from outdoor resting places. similarly, the maximum number of this species was caught in june (fig. 4). to find leishmania infection among ph. papatasi randomly 30 females were examined by nested-pcr technique. results of molecular experiment showed none of the sand flies were infected by leishmania parasite. sufficient numbers of ph. sergenti from outdoors and se. sintoni and se. mervinae from indoors were not collected to demonstrate monthly activity. reservoir host investigation totally 30 desert rodent were collected by sherman live-tarps during a year, and all identified as r. opimus based on the morphological characteristics. out of 30 examined rodents, only one (3.3%) was infected http://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 138 http://jad.tums.ac.ir published online: june 30, 2020 by amastigote form of leishmania through microscopic examination. in addition, by using molecular examination eight (26.7%) of the captured rodents were found to be infected by l. major (fig. 5). human infection survey to determine scar rate and ulcer rate of zcl among inhabitants, active case finding was carried out at the end of fall in mohamadieh, bafran and mazraee emam villages. taken together, 914 individuals including 449 males (49.1%) and 465 females (50.9%) were investigated and demographic data were recorded as well. among all visited individuals only one woman (0.1%) in ≥25 age-group had one active lesion of zcl on her hand. out of 914 examined persons, 14 individuals (1.5%) had scar which eight (1.8%) were man and six (1.3%) were woman belonging to ≥ 25 age-group. the distribution rate of scar site/s on the body of examined people is shown in fig. 6. the ulcer and scar rates of zcl calculated to be at 1.1 and 15.3per 1000 population, respectively (table 1). molecular experiments on one sample of active lesion confirmed l. major infection. table 1. the prevalence of scar rate of the examined population in the studied villages, naein county, esfahan province, iran, 2011 age groups (yr) male female total no. observed no. of scar/s no. observed no. of scar/s no. observed no. of scar/s no % no % no % 0–4 22 0 0 25 0 0 47 0 0 5–9 29 0 0 24 0 0 53 0 0 10–14 25 0 0 26 0 0 51 0 0 15–19 26 0 0 25 0 0 51 0 0 20–24 49 0 0 42 0 0 91 0 0 ≥ 25 298 8 2.7 323 6 1.9 621 14 2.3 total 449 8 1.8 465 6 1.3 914 14 1.5 fig. 1. map of the studied areas, naein county, esfahan province, iran m a y j u n j u l a u g s e p o c t 0 1 0 2 0 3 0 4 0 5 0 m o n t h n o . p e r 3 0 t r a p s fig. 2. monthly fluctuation of phlebotomus sergenti in indoors of the studied areas, naein county, esfahan province, iran, 2011 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 139 http://jad.tums.ac.ir published online: june 30, 2020 m a y j u n j u l a u g s e p o c t 0 1 0 2 0 3 0 m o n t h s n o p e r 3 0 t r a p s i n d o o r o u t d o o r fig. 3. monthly fluctuation of phlebotomus papatasi in studied areas, naein county, esfahan province, iran, 2011 m a y j u n j u l a u g s e p o c t 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 m o n t h s n o p e r t r a p s e . s i n t o n i s e . m e r v i n a e fig. 4. monthly fluctuation of sergentomyia sintoni and sergentomyia mervinae in outdoors of the studied areas, neain county, esfahan province, iran, 2011 fig. 5. gel electrophoresis of nested-pcr targeted sequences of dna extracted from the smears of the captured rodents. m; 100bp dna ladder (fermentas), 2, 7, 8, 9 and 12 showing the samples were positive for leishmania major. pos; positive l. major reference. neg; negative control 7 7 .0 8 % h a n d 1 8 . 7 5 % l e g 4 . 1 7 % f a c e fig. 6. the percent of scars on the body of visited inhabitant in the studied areas, neain county, esfahan province, iran, 2011 discussion the current study is the first epidemiological survey on zoonotic cutaneous leishmaniasis in naein county, esfahan province. in the current study four species of sand flies including two species of phlebotomus and two species of sergentomiya genus were collected and identified. in indoor places ph. sergenti with the 63.1% density was the most abundant species and ph. papatasi with 30.6 % density ranked in the second place. these two species were caught throughout the sand flies’ active season. generally, sergentomyhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 140 http://jad.tums.ac.ir published online: june 30, 2020 ia species prefer to feed on cold-blooded vertebrates, thus they are known as vectors of reptile leishmania species. it is widely believed, they cannot transmit leishmania to human, although promastigote infection of se. sintoni have shown by direct examination previously (22). however, they are usually occurred sympatrically with phlebotomus species, even though in some cases, same to this study, they have higher density comparing to phlebotomus genus. hence, the role of these sand flies in transmitting of human leishmaniaisis needs more investigation. majority of studies conducted in central iran have shown ph. papatasi as the predominant species in either indoors and outdoors places (23, 24). in agreement with our result, generally it has been shown there were two peaks in the density curve of the most species in the hot and arid areas of central part of the country; one in june or july and the second in august or september (23, 24). of 30 ph. papatasi were passed to nested-pcr experiment none of them were infected by leishmania parasite. natuaral leishmania infection has been reported from ph. papatasi, phlebotomus caucasicus, phlebotomus ansarii and se. sintoni (22-24), and also l. major was detected in ph. papatasi using molecular methods (25-27). in the current study out of 30 captured r. opimus, 8 (26.7%) were infected by l. major using nested-pcr, and reconfirmed by pcrrflp technique. till now, several studies have conducted in the country showed leishmania infection in r. opimus by either direct examination or molecular methods (5, 28-30). as the results showed, the detected leishmania species from r. opimus and human lesion was the same. in the previous studies l. major has been isolated from naturally infected ph. papatasi, ph. caucasicus, r. opimus, meriones libycus and human in endemic area of zcl in the country (5). ulcer and scar rates among surveyed population were 0.1% and 1.5% respectively, these rates were reported as 1.3% and 3.26% in other county of esfahan province (31). the most infected age-group was ≥ 25, while in the previous study was 10–14 age-group (31). in an epidemic situation, the rates of ulcer and scar were reported as 3% and 10.4% respectively, and also the most infected agegroup was 0–4 years old (22). this finding showed high level of endophilic behavior for ph. sergenti. it is worth to mention that high density of ph. sergenti, the main vector of acl, in indoors companioning to a case of acl or dogs infected by l. tropica in the areas, pose a potential threat of acl establishment in the region. on the other hand, despite the high density of ph. sergenti and its refractory to l. major, so it cannot play a role in transmitting of l. major in the areas. considering the results of the current study, this area is not categorized as a high risk area for zcl transmission due to l. major. due to low density and no leishmania infection in potential vector of zcl, low population of reservoir host, sporadic cases and lack of history of the disease below 25 years old group, this area could be considered as a low risk focus. conclusion it is concluded that zcl has established in low endemicity in this area. it is extrapolated that, the disease will not be a serious health problem in the near future in this region. based on the results of this study, presently no vector and/or reservoir host control is recommended. only treatment of rare new cases with active lesion following the national protocol is recommended. considering climate changes and ecological status, consequent probable epidemiological change of zcl is not unexpected, so that continuing of disease surveillance to take urgent action in special condition is recommended. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2020, 14(2): 134–143 r jafari et al.: emerging of cutaneous … 141 http://jad.tums.ac.ir published online: june 30, 2020 acknowledgements authors wish to appreciate the personnel of naein county health centre, esfahan university of medical sciences, for their kind collaboration in the field operation. this research was financially supported by national institute of health research, tehran university of 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leishmaniosis due to leishmania major in ardestan town, central iran. acta trop. 79(2): 115–121. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 60 original article species composition and seasonal activities of malaria vectors in an area at reintroduction prevention stage, khuzestan, south-western iran naimatallah maghsoodi 1, 2, hossin ladonni 2, *hamid reza basseri 2 1health center of izeh district, jondishapour university of medical sciences, ahvaz, iran 2department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran (received 26 oct 2013; accepted 11 may 2014) abstract background: the most part of iran become malaria-free region and fall in prevention of re-introduction stage. these regions however are struggling with imported of malaria cases where malaria vectors exist. therefore, understanding the situation of mosquito vectors is crucial. this study was carried out to find out the present situation of malaria vectors and malaria transmission potential in a malaria-free area. methods: the study was conducted in a malaria free area, izeh county, khuzestan province during 12 months in 2011–2012. five villages, including 2 in highlands and 3 in plain area, were selected randomly. the mosquito sampling methods were conducted using spray sheet and hand catch collection methods from indoor/outdoors, window trap and larvae collections. results: in total, 3352 female anopheles were captured, 1826 mosquito from highland and 1526 from plain areas. five species, an. stephensi, an. fluviatilis s.l., an. dthali, an. superpictus and an. pulcherrimus were identified. the seasonal activities were started from april to march. the abdominal conditions of collected mosquitoes from indoor/outdoor places pointed to exophilic propensity of an. fluviatilis.l. s.l. and endophilic behaviour for rest of the vectors. the results of window trap also confirmed these behaviors. the larval habitats of four species were widely dispersed and included spring, margin of rivers, irrigation channels, stagnant water and rice filed. conclusion: understanding the present situation of malaria vectors in free-malaria area is crucial particularly where is struggling with imported cases. the results of present study can be expanded to other area of northern khuzestan for malaria vector control planning in reintroduction prevention stage. keywords: malaria, anopheles vector, seasonal activity, iran introduction malaria is very geographically specific and remains an important cause of mortality and morbidity in many parts of the world. in 2012, there were 99 countries and territories which highly struggling malaria transmission and 5 countries in the prevention of reintroduction phase (who 2013). although this disease has been eradicated from many regions, there is more possible to reintroduce to an area particularly where anopheles vectors are present circumstances are suitable for parasite transmission (roll back malaria 2013). therefore, to prevent malaria reintroduction is a big challenge (hemami et al. 2013). malaria was native in the most parts of iran but after six decade effort, the disease has been limited to south-eastern part of the country (raeisi et al. 2013). anti-malaria operation was started in iran in 1945 and which cause malaria infection rate considerably decreased during 1948–1956 in the most endemic areas. later malaria eradication programme started and since 1980 almost interrupted malaria transmission in the north and middle parts of the country. then the programme shifted to malaria control programme which has been *corresponding author: dr hamid reza basseri, email: basserih@tums.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 61 continuing up to present time (azizi and bahadori 2013). seven anopheles including an. maculipennis s.l., an. sacharovi, an. superpictus, an. fluviatilis s.l., an. stephensi, an. culicifacies and an. dthali are considered to be main malaria vectors in iran (zahar 1974, hanafi-bojd et al. 2011). in addition, an. pulcherrimus has been reported as suspected potential vector of malaria in the district of ghassreghand, baluchistan, iran (zaim et al. 1993). more recently four anopheles species, an. stephenis, an. dthali, an. pulcherrimus and an. superpictus have been reported from mahshar county located in south of khuzestan province, iran (farhadinejad et al. 2013). at present, iran fall to pre-elimination of malaria but the main challenges to sustaining elimination are addressing the potential reintroduction of cases, either via long border with malaria-endemic countries at east or from migrant populations scatter to whole country (raeisi et al. 2013). however, malaria transmission is highly depend on community and anopheles vectors behavior particularly in southern iran where is threaded by imported cases (shahandeh et al. 2010). khuzestan province recently became free from malaria transmission but the area is threatened by imported malaria cases (akbari et al. 2013). in this circumstance, vector control plays a main role to prevent malaria outbreak. therefore, understanding the vectors' situation in advance is so essential for hampering of malaria occurrence. izeh county is located at north of khuzestan known as a malaria eliminated zone. due to travel of job seeker labors or immigrants into the county, reintroduction of malaria particularly cases that lead to re-establishment of local transmission is the most important issue (personal communication with izeh district health centre). izeh has suitable environments for mosquito development but for more than five years no any malaria transmission has been reported, therefore, vector control programmes have been limited to larviciding operation and environmental management (county health centre of izeh, personal communication). the present study illustrates the current situation of malaria vectors and their activities in the izeh county with a scope of role of the vector in reintroduction of malaria. the results of this study can be used for similar area which has imported malaria cases and potent vectors. materials and methods study area izeh county is located at north of khuzestan province. the county is situated in slope of zagrus mountain chain at north and khuzestan plain in south (fig. 1). it is geographically located on latitude 31°34′8″n, and longitude 49°34′0″e. generally, the area comprises mountainous, hilly regions in the north, with plains regions in the south and west. the region has a mild climate and temperature reaches to 40 °c at maximum in summer and minimum near 10 °c in december and january. the water sources are permanent and temporary rivers, wells springs, ponds and pools. the annual rainfall ranged from 100 to 120 mm. the rainfall occasionally received during winter. izeh has primary health care (phc) since 1988 and all rural and urban areas have phc services. by integrating malaria control activities into health system, the health workers involved in malaria cases finding as well as vector control programme. mosquito collection mosquito collections were standardized as fully described by who (who 2003). during the 12 months, mosquitoes were collected from 3 villages located in mountainous lands and 2 villages in plain lands. collections were carried out from eight indoor and outdoor shelters in each village as follow. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 62 indoor collection the mosquitoes were collected from human dwellings and sheep or cattle sheds by pyrethrum space spray method as fully described by service and who (service 1976, who 2003). briefly, before spray, all the eves, windows, doors, and other exit points in each indoor shelter were closed and then white cloth sheets were spread on the floor. pyrethrum extract (0.2% in kerosene) was sprayed in the entire space of the room and the room was closed for 15 minutes. after 15 minutes, all the knocked-down insects lying on the cloth sheet were collected carefully with the forceps and placed in petri dishes lined with moist filter paper and brought to the laboratory for further studies. the time of application in each village was early in the morning and during sunrise between 0530 and 0730 h. outdoor collection natural shelters inside and around the indicator villages were periodically searched during early morning and mosquitoes were collected using sucking tube and torch light. the mosquitoes were then captured using sucking tube and touch. all mosquitoes were identified based on species taxonomic keys of shahgudian (shahgudian 1960). all details of the shelters including kind of habits, temperature, humidity, and date and time of collection were recorded on forms. in addition, collected female mosquitoes were graded to abdominal conditions in each sampling technique as described by (who 2003). generally, endo/exophilic behavior of each mosquito species was categorized based on abdominal appearance of the collected mosquitoes as follows. the gravid (g) and/or semi-gravid (sg) appearance of the female abdomen demonstrate as resting stages of female mosquitoes, and the females with unfed (u) and freshly fed (f) guts are indicative of the seeking stages (seeking for blood meal or resting places). therefore, ratio of the resting stages classified as tendency to rest in indoor or outdoor places (who 2003). window trap collection both entry and exit window traps (length x width x height= 30× 30× 30 cm) were utilized to find moving behavior of mosquitoes in to the indoor shelters. two entry and two exit traps were fixed on the windows of selected rooms in each villages from sun set until sun rise. all mosquitoes were collected alive with sucking tube and transferred to laboratory for identification. larval sampling and processing larval surveillance was conducted twice a month over a 12 month period in 2011. the larvae collection was carried using standard mosquito dipper from all potent breeding places out around the selected villages with a radius of about one kilometer. all collected larvae were counted and iii-iv instars larvae transferred to lacto-phenol. the larvae were then identified 24 hours after conservation using morphological taxonomic key (shahgudian 1960). results in total, 3352 female anopheles spp. were captured including 1826 mosquitoes from mountainous and 1526 from plain areas. the mosquito species were an. stephensi (ntotal= 837, nindoor= 701, noutdoor= 21, window traps= 115), an. superpictus (ntotal= 1100, nindoor= 871, noutdoor= 48, widow traps= 181), an. fluviatilis s.l. (ntotal= 326, nindoor= 129, noutdoor= 126, window traps= 71), an. dthali (ntotal= 1088, nindoor= 993, noutdoor= 47, window traps= 137) and an. pulcherrimus (nindoor= 1). as illustrated in fig. 1, the seasonal population dynamics of the mosquitoes in indoor places were varied among four species either sampled from mountainous or plain http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 63 areas. totally, an. stephensi, an. superpictus and an. dthali population trend in mountainous followed the same pattern in plain areas (fig. 1a, b, c) but an. fluviatilis s.l. had low indoor abundance in the plain areas comparing with mountainous areas (fig. 1d). the seasonal activities of four species were started in april and the highest population of an. superpictus s.l. and an. dthali was observed in june while the maximum population of an. stephensi and an. fluviatilis s.l. occurred in october. an. superpictus exhibited more tendencies to be in indoor shelters (fig. 1b) followed by an. stephensi and an. dthali populations (fig. 1a). generally, the population density of adults declined to minimum in winter. abundance of female mosquitoes collected from animal and human shelters was varied in either highland or low land areas (table 1). anopheles stephensi showed slightly more tendency to human shelters rather than animals while an. superpictus, an. dthali and an. fluviatilis s.l. were captured more in animal shelters comparing with human places. totally, the pattern of using human/ animal shelters as resting places by each species in highland areas was same as lowlands (table 1). four species also showed propensities for outdoor shelters (fig. 2). the population dynamics of mosquitoes in outdoor shelters displaced similar pattern to indoor places. the mosquitos’ activities started from april to december or january. anopheles fluviatilis s.l. showed more propensities for resting in outdoor shelters either in plain or mountain areas (fig. 2d). in addition, female mosquitoes were graded according to abdominal conditions collected indoors and outdoors. the gravid (g) and/or semi-gravid (sg) appearance of the abdomen demonstrate as resting stages, while the female mosquitoes with unfed guts (u) and/ or freshly fed (f) are indicative of the seeking stages. the ratio of resting stages to seeking stages for an. stephensi showed that this species had a greater tendency to rest inside (g, sg/e, f= 0.75) rather than outdoors (g, sg/e, f= 0.50). anopheles fluviatilis s.l. had a low proportion for endophilic behavior (g, sg/e, f= 0.17) and its preference for resting outside (g, sg/e, f= 0.56) was five times more than an. stephensi and nearly two times more than an. superpictus (table 2). anopheles superpictus showed slightly more exophilic behavior (g, sg/e, f= 0.71) than endophililc (g, sg/e, f= 0.73). though the majority of an. dthali was collected in indoor shelters, resting tendency to indoor (g, sg/e, f= 0.88) was more than outdoors (g, sg/e, f= 0.51). the number of mosquitoes caught by window traps is showed in table 3. generally, indoor tendency exhibited by four anopheles species either in higher in lowland or highland. entry behavior of an. stephensi, an. superpictus, an. dthali and an. fluviatilis s.l. was observed during whole activity seasons (fig. 3). the dynamic of entry movement of four anopheles species was alike to population dynamics of them sampled from indoors. totally, 931 larvae were collected from plain (418 larvae) and mountainous (513 larvae) areas. the collected larvae species in plain area were an. stephensi (117 larvae), an. superpictus (170 larvae), an. dthali (80 larvae) and an. fluviatilis s.l. (51 larvae) and in mountainous area an. stephensi (160 larvae), an. superpictus (105 larvae), an. dthali (222 larvae) and an. fluviatilis s.l. (26 larvae). the larvae were generally found in spring, streams, margin of rivers, irrigation channels, stagnant water such as waste water, drainage, borrow pits as well as rice fields. the ratio of anopheles species in each type of breeding places was highly varied. in addition high different between mountainous and plain areas was observed with respect to using different breeding places (fig. 4, 5). anopheles stephensi larvae were found mostly in stagnant waters or waters with low flow http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 64 rate such as rice farm. comparing to larvae combination in each type of breeding place, an. stephensi larvae ratio in the lowland area (plain) was mostly rice farm (fig. 3c) while in the highland, it found mostly in springs. anopheles dthali larvae collected in various breeding places with relatively high ratio comparing with other anopheles mosquitoes (fig. 4, 5) except in rivers margin of highland region (fig. 4a). in contrary, high abundance of an. superpictus was collected in river margins particularly in highland (fig. a). anopheles fluviatilis s.l. was found at low ratio in stagnant waters and spring of highlands while more ratio of an. fluviatilis.l. s.l. larvae (19%) was collected in stagnant waters of in plain areas (fig. 3b, fig. 4b, c). table 1. number of female mosquito captured from human and animal places in two different areas, izeh county, southwest of iran, 2011–2012 species mountainous areas total plain areas total animal shelters (%) human shelters (%) animal shelters (%) human shelters (%) an. stephensi 230 (48.1) 248 (51.8) 478 103 (46.2) 120 (53.8) 223 an. dthali 302 (63.8) 17 (35.2) 473 299 (57.5) 221 (42.5) 520 an. superpictus 249 (50.1) 244 (48.9) 498 201 (53.8) 172 (46.1) 373 an. fluviatilis s.l. 46 (38.3) 74 (61.6) 120 9 (100) 0 (0.0) 9 an. pulcherrimus 0 0 0 1 (0.002) 0 1 (0.0008) table 2. abdominal condition of female mosquitoes based on collecting sites in izeh county, southwest of iran, 2011–2012 species indoor collections g,sg/f, e (indoors) outdoor collections g,sg/f, e (outdoors) outdoors/ indoorsf, e g,sg f, e g, sg an. stephensi 399 (57.1%) 302 (42.9%) 0.75 14 (72.2%) 7 (27.8%) 0.50 0.66 an. superpictus 504 (57.8%) 367 (42.2%) 0.73 28 (64.7%) 20 (35.3%) 0.71 0.97 an. fluviatilis s.l. 110 (85.3%) 19 (14.7%) 0.17 81 (70.0%) 45 (30.0%) 0.55 3.27 an. dthali 528 (53.21%) 465 (46.8%) 0.88 31 (68.1%) 16 (31.9%) 0.52 0.59 f: fresh fed female mosquito e: empty or unfed female mosquito g: gravid female mosquito sg: semi-gravid female mosquito table 3. frequency of female mosquito captured from exit andentry window trap in two different areas, izeh county, southwest of iran, 2011–2012 species mountainous areas total plain areas total exit (%) entry (%) exit (%) entry (%) an. stephensi 11 (34.3) 21 (65.5) 32 32 (38.5) 51 (61.4) 83 an. dthali 17 (36.1) 30 (63.8) 47 46 (51.1) 44 (48.8) 90 an. superpictus 40 (61.5) 25 (38.4) 65 62 (53.4) 54 (46.5) 116 an. fluviatilis s.l. 23 (62.1) 14 (37.8) 37 26 (76.4) 8 (23.5) 34 http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 65 fig. 1. seasonal prevalence of female mosquitoes: anopheles stephensi (a), an. dthali (b), an. superpictus (c) and an. fluviatilis s.l. (d) collected from indoor places in both mountainous and plain areas of izeh county (2011–2012). the population trends of mosquitoes were similar in both distinguishable areas fig. 2. seasonal density of female mosquitoes: an. stephensi (a), an. dthali (b), an. superpictus (c) and an. fluviatilis s.l. (d) collected from outdoor places in both mountainous and plain areas of izeh county (2011–2012) http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 66 fig. 3. mean number of female mosquitoes, anopheles stephensi (a), an. dthali(b), an. superpictus (c) and an. fluviatilis s.l. (d) collected by exit and entry window traps in izeh county (2011– 2012). the population trends of mosquitoes were similar in both distinguishable areas fig. 4. larvae ratio of four anopheles species, collected from different habitants in plain areas a: river side b: stagnant water c: rice farm d: spring fig. 5. larvae ratio of four anopheles species collected from different habitants in mountainous area. a: river side b: stagnant water c: spring discussion indeed, mosquito collections were carried out on probably occurrence of malaria outbreaks in an eliminated zone such as north of khuzestan province not based on a more comprehensive seasonal or temporal collection protocol may have resulted in a lower mosquito vectors activities overall. however, our results indicate that the activities and behavior http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 67 of four anopheles vectors in izeh county indicates that the area may have potent for malaria transmission depend on mosquito vectors contact with any malaria cases. totally, the population trend of three mosquito species in mountainous area followed the same pattern in plain (fig. 1a, b, c). in contrary, an. fluviatilis s.l. had low indoor abundance in the plain area rather than mountainous. these results may indicate that the environmental condition in both areas is similar for endophilic species. in the present study, an. superpictus and an. dthali were the most abundant and prevalent species showing preference to be indoor places while an. fluviatilis s.l. at low prevalent noticeably to outdoor places. the abundant of an. stephensi was also high in both indoor and outdoor shelters with more intendancy to indoors in izeh areas. anopheles fluviatilis s.l. was collected at very low frequency in indoor places particularly in plain areas indicating that this mosquito is more exophlic and this has been confirmed by the results of collection using window trap methods. similar behavior of an. fluviatilis s.l. was declared by several authors (viswanathan et al. 1945, eshghi et al. 1976, nanda et al. 1996, sahu et al. 2009, sahu et al. 2011). it was illustrated that an. fluviatilis s.l. as a wild mosquito had relatively high tendency to human blood (manouchehri et al. 1976a, basseri et al. 2010). totally, it seems host seeking behavior of four anopheles adults may slightly different when they exhibited varied movement and resting behaviour in izeh county. we also found that larval habitats of an. fluviatilis s.l. may be small, widely dispersed, and transient, and this may be reason we could not find the larvae at high abundance in izeh county. overall, control of an. fluviatilis s.l. is big challenge due to the behavior of this mosquito vector in izeh county. however, this mosquito considered as one of the most important malaria vector for stable malaria in middle east and south of asia countries (eshghi et al. 1976, manouchehri et al. 1976a, gunasekaran 1994, dev et al. 2003). anopheles stephensi was recognized as a domestic mosquito (sharma 1995, kar et al. 1996, chakraborty et al. 1998). it was responsible for malaria epidemic in some part of southern iran (manouchehri et al. 1976b). in addition, this species exhibits a strong preference for human blood in south and south eastern iran (basseri et al. 2005, 2010). in the present study, an. stephensi showed endophilic behavior as well as using natural shelters for rest. therefore, at outbreak situation of malaria, using indoor residual spraying may not be effective method for control of this vector, particularly this anopheles has become resistant to several insecticides in iran (enayati et al. 2003, vatandoost et al. 2006). anopheles dthali was identified as secondary malaria vector in iran (manouchehri et al. 1972). as in previous study stated this mosquito use human dwelling as well as animal shelter in south of iran (manouchehri and rohani 1975), we also found an. dthali in the same places at relatively high abundance. anopheles dthali is still susceptible to all insecticide which used for indoor residual spraying (vatandoost et al. 2007). however, it was stated that an. dthali can be incriminated for transmission malaria at high density where we comparatively found in izeh county. in the present study, anopheles superpictus was found relatively at high density in both mountainous and plain areas of izeh county. this mosquito was also captured by window traps more than other mosquito vectors. anopheles superpictus is one of the most widespread malaria vectors in iran (hanafibojd et al. 2011). overall, the results obtained from window traps collection method (table 3) and density of the female mosquito per room (fig. 1) as well as abdominal condition ratio (table 2) together indicating that this species has more propensity to indoor http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 60–70 n maghsoodi et al.: species composition … 68 places than outdoors. therefore, in any malaria outbreak situation, the behavior of an. superpictus should be considered as a potent vector in north of khuzestan province. this species had been responsible for maintaining malaria transmission due to its refractory behavior in southern slopes of the zagros chain (zahar ar 1990). the study area is very fertile with different water source for breeding of anopheles larvae. the agricultural activities start from beginning of spring and provide many jobs for immigrant labors. furthermore, the environment of izeh provides suitable condition for activity of four malaria vectors. according to report of izeh health centre, the area is struggling with imported malaria cases annually. therefore, occurrence of malaria transmission should be expected. conclusion although malaria has been eliminated from north of khuzestan, but due to population movement form malaria endemic area, understanding the present situation of malaria vector is essential. the results of present study can be expanded to other area of northern khuzestan for malaria vector control planning in reintroduction prevention stage. acknowledgements we thank the personnel of the izeh health centers for kind assistances. the authors would like to thank dr ahmad raeisi, the head of malaria control office, ministry of public health, for his great technical supports. we appreciate the kind cooperation of khuzestan province health centre and special thank to mrs shahla bigdeli for her kind official supports. this work received financial support from tehran university of medical sciences. the authors declare that there is no conflict of interests. references akbari h, majdzadeh r, rahimi foroushani a, raeisi a (2013) timeliness of malaria surveillance system in iran. iran j publ health. 42: 39–47. azizi mh, bahadori m (2013) brief historical perspectives of malaria in iran. arch iran med. 16: 131–135. 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southern iran. iran j arthropodborne dis. 1: 21–27. vatandoost h, oshaghi ma, abaie mr, shahi m, yaaghoobi f, baghaii m, hanafi-bojd aa, zamani g, townson h (2006) bionomics of anopheles stephensi liston in the malarious area of hormozgan province, southern iran, 2002. acta trop. 97: 196–203. viswanathan dk, rao tr, rao ts (1945) the behaviour of anopheles fluviatilis, the behaviour of gravid females. j malar inst india. 6: 243–245. who (2013) malaria. avaiable at: www. who. int/mediacentre/factsheets/fs094/en/. who (2003) malaria entomology and vector control. who/cds/cpe/smt/ 2002.18 rev.1.part i. geneva. zahar ar (1990) vector bionomics in the epidemiology and control of malaria the who african region and the southern who eastern mediterranean region vbc/90.2, mal/90.2. geneva. zahar ar (1974) review of the ecology of malaria vectors in the who eastern mediterranean region. bull world health organ. 50: 427–440. zaim m, subbarao sk, manouchehri av, cochrane ah (1993) role of anopheles culicifacies s.l. and an. pulcherrimus in malaria transmission in ghassreghand (baluchistan), iran. j am mosq control assoc. 9(1): 23–26. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 353 http://jad.tums.ac.ir published online: december 31, 2019 original article scorpions and scorpionism in roudan county, southern iran mehran shahi1; reza habibi-masour2; mehrdad salehi2; mehdi ghasemi-nang3; emadaddin rafizad4; madineh abbasi5; *ahmad ali hanafi-bojd5 1department of medical entomology and vector control, school of public health and infectious and tropical diseases research center, hormozgan heath institute, hormozgan university of medical sciences, bandar abbas, iran 2department of diseases control, hormozgan university of medical sciences, roudan, iran 3department of diseases control, hormozgan university of medical sciences, bandar abbas, iran 4department of diseases control, hormozgan university of medical sciences, bandar abbas, iran 5department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 29 apr 2018; accepted 25 nov 2019) abstract background: every year, thousands of cases and many deaths from scorpion sting are reported in tropical areas of south and southwestern parts of iran. the aim of this study was to identify the fauna and dangerous species of scorpions in roudan county, southern iran. methods: this descriptive study was conducted in 10 stations in hormozgan province. scorpion sampling was done randomly by searching for their shelter and digging out their nests during day, and with the use of uv light during night from february 2013 to october 2014. data of scorpion stings were obtained from health center of hormozgan province during 2014–2016. results: overall, 155 scorpions were collected on a set of eight species belonging to buthidae and hemiscorpiidae families. these species were identified as mesobuthus persicus, mesobuthus phillipsi, hottentotta schach, odontobuthus doriae, compsobuthus persicus, orthochirus farzanpayi, androctonus crassicauda and hemiscorpius acanthocercus. one thousand and twenty-seven cases of scorpion sting were recorded during 2014–2016 with a peak period in summer. most of cases were <44yr old. five out of six medically important scorpions in iran were actively identified in the study area. conclusion: results of this study would greatly help to identify risk factors of scorpion sting in high-risk areas for planning, management and treatment of patients with scorpion sting in these areas. keywords: distribution; scorpion; scorpion sting; scorpionism; iran introduction scorpion sting is one of the most important medical issues in tropical countries of the world, threatening the health of residents in these areas. every year, thousand cases and many deaths from scorpion sting are reported in tropical areas of south and southwestern parts of iran. iranian scorpion fauna consists of 64 species with the highest frequency been related to buthidae family (1). among the known species of scorpions, the most reported deaths due to scorpionism are related to three species androctonus crassicauda, h. lepturus and h. acan thocercus (2). more than 75% of deaths from scorpion sting occurred in the southern provinces of iran including, khuzestan, hormozgan, bushehr and ilam each year (3). in the southern regions of iran, the diversity and density of scorpions are very high. however, information on their fauna, ecology and distribution is very low (4). hormozgan province is one of the most important foci of scorpion sting in southern part of iran. each year, several cases of deaths are reported due to scorpion stings from this area *corresponding author: dr ahmad ali hanafibojd, e-mail: aahanafibojd@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 354 http://jad.tums.ac.ir published online: december 31, 2019 (5, 6). scientific research in the field of scorpions has been very limited in hormozgan province (7-9). by the end of year 2012, about six species of scorpions were identified in the jask county located in eastern part of hormozgan province (10). in an earlier study conducted in hormozgan province, 20 scorpion species were reported from this area (11). a recent study at the provincial capital of hormozgan province reported 22 scorpion species in which 5 species belong to hemiscorpius genus including, h. acanthocercus, h. enischnochela, h. lepturus, h. persicus and h. gaillardia (12). in southern region of iran, h. lepturus is the major cause of death owing to scorpionism (13-18). roudan county is among the high-risk foci of scorpion sting in hormozgan province. annually about 400 cases of scorpion sting are reported from this county. two deaths due to scorpion stings have been reported in 2012 from this county. so far, no study has been done on the fauna of scorpions in roudan county of hormozgan province. due to the reported deaths from scorpion sting in this county, the present study was being conducted to determine the fauna and distribution of dangerous scorpions’ species in this area. therefore, the main objective of this study was to identify and the introduction of dangerous scorpions’ species to provide implement the best treatment method for patients in this highrisk area of southern part of iran. materials and methods study area roudan county is located in the eastern part of hormozgan province with land area of 3,725km2. this county is bordered to the southwestern part by kerman province. in terms of geographical features, this county is composed of two areas thus plains and mountains. roudan county is located at latitude 27o 27' north and longitude 57o 11' east with an altitude of 150 to 700m above sea level. this county has sub-tropical climate with average annual rainfall of 250mm and mean relative humidity of about 45%. the average annual minimum and maximum temperature of this county ranges between 7 °c and 49 °c, respectively. this descriptive cross-sectional study was conducted in 10 stations in hormozgan province (table 1). scorpion samplings scorpions were collected from 10 sites in urban and rural areas of mountainous and plain regions (table 1). scorpion sampling was done randomly using hand catch method by searching under stones and clod, rift racks, under the bark of trees and other shelters, also digging out their nests during the day, and with the use of uv light at night from feb 2013 to oct 2014. the captured samples were placed in 75% alcohol container with an identification label. gps device receiver (garmin international) was used for recording the geographical coordinates. the scorpions were identified by scorpiologist using morphological keys (11, 19) under an nikon xn model stereomicroscope at medical entomology department lab, bandar abbas city. the scorpion samples were kept in entomology lab, school of public health, hormozgan university of medical sciences (hums), bandar abbas, iran. scorpion sting data collection data about cases of scorpion sting in this descriptive cross-sectional study include, age, gender, site of the sting, geographical location of the scorpion sting, monthly cases, sting time, time referred to the hospital and total serum used in the treatment of patients were obtained from health center of hormozgan province during 2014–2016. data analysis was performed using descriptive statistics, and graphs were plotted using excel software. results scorpion fauna overall, 155 scorpions were collected and identified in a set of 8 species, belonging to buthidae and hemiscorpiidae families. of the http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 355 http://jad.tums.ac.ir published online: december 31, 2019 total samples collected, 61 (39.3%) were female and 94 (60.7%) male. the family and habitat of the collected scorpion species were identified at each site of collection. androctonus crassicauda and mesobuthus phillipsi were collected from all stations in the study areas (table 1). hemiscorpius acanthocercus with a frequency collection of 48 was the predominant species. the lowest frequency was from h. schach, c. persicus and o. farzanpayi species (fig. 1). in this study, only one genus of the species hemiscorpius was identified. this species was collected only from roodkhaneh and markazi districts in mountainous area. the distribution map of the scorpion species identified has been shown in fig. 2. epidemiological data the majority of scorpion stings in the study area happened in summer, with most cases (71.1%) been reported in rural areas (fig. 3). according to the recorded evidence on scorpion sting checklist, in the rural and urban areas of roudan the most cases of stinging were related to mesobuthus genus scorpions. more people (36%) were stung in the first half of the night and the frequency of stings was higher among women (50.9%) than men. aged 25 to 44yr (28.5%) were the most frequently affected age groups (fig. 4). most stings sites (41.2%) were in the hands of patients and more than half (54.6%) of patients were referred to treatment centers within one and half hours after the sting. mean annual consumption of anti-scorpion venom was 510 vials. in general comparison, most cases of scorpion related to women occurred in the young and working age group in rural areas (table 2). table 1. species of scorpions collected in roudan county, southern iran, 2014 collection site topography altitude (m) latitude and longitude species faryiab mountain 325 27° 28' 10.96'' n, 57° 4' 16.30'' e a. crassicauda, h. acanthocercus, m. phillipsi, m. persicus, ho. schach, c. persicus rahdar mountain 591 27° 36' 58.97'' n, 57° 6' 3.83'' e a. crassicauda, m. phillipsi, o. doriae, o. farzanpayi abnama plain 218 27° 27' 37.66'' n, 57° 15' 21.14'' e a. crassicauda, m. phillipsi, m. persicus bika plain 180 27° 21' 17.60'' n, 57° 10' 20.95'' e a. crassicauda, m. phillipsi berentin plain 184. 27° 17' 44.99'' n, 57° 14' 59.51'' e a. crassicauda, m. phillipsi jaghin-e shomali plain 234 27° 13' 28.48'' n, 57° 22' 38.44'' e a. crassicauda, m. phillipsi, m. persicus jaghin-e jonoobi plain 212 27° 12' 17.99'' n, 57° 20' 41.33'' e a. crassicauda, m. phillipsi, m. persicus, o. doriae ziyarat ali mountain 456 27° 44' 35.87'' n, 57° 13' 57.55'' e h. acanthocercus, o. doriae, a. crassicauda, m. phillipsi, m. persicus roodkhane bar mountain 498 27° 49' 45.64'' n, 57° 17' 41.69'' e h. acanthocercus, o. doriae, m. phillipsi, m. persicus mosafer abad plain 534 27° 52' 54.94'' n, 57° 11' 51.87'' e a. crassicauda, m. phillipsi http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 356 http://jad.tums.ac.ir published online: december 31, 2019 table 2. cases of scorpion sting in roudan county, southern iran, 2012–2014 year no. of cases no. of antivenom doses age groups area gender urban rural male female <9 10–24 25–44 45–64 >65 2014 110 334 213 231 697 90 120 143 70 21 2015 46 157 106 97 257 62 55 49 21 16 2016 141 239 186 194 575 114 101 99 47 19 total 297 730 505 522 1529 266 276 291 138 56 fig. 1. species composition of scorpions collected in roudan county, southern iran, 2014 fig. 2. distribution map of scorpion species in roudan county, southern iran, 2014 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 357 http://jad.tums.ac.ir published online: december 31, 2019 fig. 3. distribution of cases of scorpion stings by months in roudan county, southern iran, 2014–2016 fig. 4. patients age distribution of cases of scorpion sting in roudan county, southern iran, 2014–2016 discussion in this study, seven species of scorpions belonging to two families (buthidae and hemiscorpiidae) were identified in the study areas. buthidae with seven species showed the highest frequency. results of other studies in southern iran about scorpion fauna were similar to our findings (7-10, 12). most species, distribution and frequency of scorpions in our study were related to m. phillipsi, m. persicus and a. crassicauda. these three species were collected from all sampling sites; our findings in species composition were in accordance with other studies carried out in other counties of hormozgan province (10, 11). results of studies in other parts of the country have also shown that mesobuthus and androctonus have the highest frequency (20-23). distribution of a. crassicauda and m. eupeus has been reported from the northern and southern provinces in iran (24-26). androctonus crassicauda is one of the most danhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 358 http://jad.tums.ac.ir published online: december 31, 2019 gerous scorpions’ species in iran. several cases of death from stings of this species have been reported from hormozgan and khuzestan provinces, in southern part of iran (10, 27). different species of this genus are adapted to different geographical conditions in hormozgan province. the second family detected in the study area was hemiscorpiidae. only one species of this family named h. acanthocercus was identified. in this study, h. acanthocercus has been collected with high density in roodkhaneh district. this district has so far, reported several cases of deaths from scorpion stings. however, bandar abbas county for the first time has reported one case of death from envenomation of h. acanthocercus venom (5). it seems this species is the main cause of this event in roudan county. previously, h. acanthocercus reported from bandar abbas and khamir counties in hormozgan province (11, 12, 19). envenomation by this species cause severe complication or death (5). lack of knowledge about this scorpion species among people and health care staff can be dangerous to health residents in their dispersal areas. hemiscorpius acanthocercus is the main etiologic agent of death in hormozgan province (12). this scorpion genus is the most important cause of death from scorpion sting in khuzestan provinces (3, 16-18). according to the results of previous studies and the findings of the present study, h. acanthocercus has the highest distribution and the dominant species of the genus hemiscorpius in mountainous areas of hormozgan province. the polyvalent anti-venom produced in iran does not contain the venom of this scorpion. in the present study, only one species of digger scorpion, o. doriae was collected. this species was reported from all parts of iran (11, 24). odontobuthus species distribution in most areas of hormozgan province has been reported in earlier studies (8, 10, 11). in this research, in daily scorpion catch, the odontobuthus nests were mainly identified in the plain areas. the lowest range distribution was related to or. farzanpayi and ho. schach these species were collected only from mountainous areas. other researchers have reported similar results (11, 25, 28). hottentotta schach and ho. saulcyi have also been reported from mountainous area of hormozgan province including, bandar abbas, khamir, bastak, parsian and hajabad counties (8, 12). hottentotta schach is one of the six medically important scorpions in iran that has been reported widely distribution in all parts of country (24, 29). hottentotta jayakari and m. eupeus were reported in previous studies from abu-moosa, great tunb, hengam and qeshm islands in the persian gulf (7, 9). this epidemiological study showed that the annual epidemiological factors of scorpion sting have a consistent pattern in roudan county. most cases of scorpion sting were in the summer. the results of other studies about scorpion sting in iran are similar to our study results (30, 31). scorpions have hibernation and because of the favorable temperatures in the warmer seasons, they are more active. this results in increased contact with residents and increased scorpion sting cases. the highest percentage (71.1%) of sting occurred in rural areas and this is consistent with other researchers results in iran (32). similar results have also been reported in turkey and saudi arabia (33-35). sitting and sleeping on the ground, walking barefooted out of house, and putting clothing and bedding on the floor are the effective factors that increase scorpion sting in rural areas. rural areas are ideal habitat for scorpions due to ecological and ecological conditions. in terms of sting time, most stings occurred in the night. scorpions are nocturnal arthropods and come out from their shelters after sunset. the scorpion's nightly search for nourishment enters them into residential areas and increases the risk of scorpion sting. the frequency of scorpions’ sting in women was higher than men. this is in accordance with results of other researchers in iran (36-39). results of studies conducted in saudi arabia did http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 353–361 m shahi et al.: scorpions and scorpionism … 359 http://jad.tums.ac.ir published online: december 31, 2019 not match our finding (40, 41). these differences in results might have occurred due to cultural differences between tribes in different areas. in high-risk areas, some activities such as collecting firewood and working in the agricultural fields raised the risk of a sting. in our study, the highest percentage of scorpion stings was observed among 25–44yr old patients with the next high-risk groups being 10 to 24yr old group. these results were in agreement with some other studies (30, 42, 43). these two age groups have the most social activity in rural areas. the activities of these age groups in agricultural lands, gardens and desert areas put them at greater risk. the high rate of sting in the high-risk group of children also indicates the need to pay attention to the implementation of the scorpion prevention program in these areas. comparing the results of this study showed that most cases of scorpions in rural areas occurred among women and youth age groups. it can be of great help to identify the risk factors of scorpion sting for planning, management and appropriate treatment of scorpion sting among patients in highrisk areas of south part of iran. conclusion due to high frequency of scorpion sting cases and the presence of h. acanthocercus in mountainous areas, hilly villages of roudan county are located in high risk zone of scorpion sting in hormozgan province, southern parts of iran. therefore, planning should be focused on preventive measures including training of residents of these high-risk areas, health personnel and immediate on the treatment of patients with a scorpion sting in 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arthropod-borne dis, march 2022, 16(1): 45–50 a moshfe et al.: anti leishmanial effect of … 45 http://jad.tums.ac.ir published online: march 31, 2022 original article anti leishmanial effect of plantago psyllium (ovate) and white vinegar on leishmania major lesion in balb/c mice abdolali moshfe1,2, keianoush karami1, maryam bahmani1, mohsen naghmachi1, shahrbanoo askarian1, abbas rezaei1, roohallah zare1, *ali jamshidi3 1cellular and molecular research center, yasuj university of medical sciences, yasuj, iran 2department of microbiology, school of medicine, yasuj university of medical sciences, yasuj, iran 3behbahan faculty of medical sciences, behbahan, iran *corresponding author: dr ali jamshidi, email: ajamshidi@behums.ac.ir (received 13 mar 2019; accepted 18 jan 2022) abstract background: leishmania major is the etiologic agent of zoonotic cutaneous leishmaniasis in iran, and glucantime injection is currently used for its treatment. this study aimed to evaluate the anti-leishmanial effect of topical plantago psyllium and white vinegar in l. major infected balb/c mice. methods: thirty infected mice were divided into five groups as follows: group 1: treated with the combination of ovata powder and white vinegar, group 2: treated with glucantime, group 3: treated with white vinegar, group 4: treated with the combination of ovata powder and water, and group 5: without any treatment. all the groups were treated for 18 days. lesion size was measured, and final smears were prepared for microscopic examination. results: the findings indicated that the difference in the mean areas of the ulcers in all the groups before and after treatment was not significant, except for the second (glucantime) and third (vinegar) groups. also, the results showed that in the first, second, third, and fourth group, 6 (60%), 4 (80%), 3 (60%), and 2 (40%) mice were healed, respectively. however, ulcers remained in all the five mice of the control group. conclusion: the combination of ovata powder and white vinegar has been traditionally used to treat leishmanial lesions in iran. it seems the most anti-leishmanial effect is related to vinegar and supported by plantago. the route of treatment with this combination is very simple and painless in comparison with injection. thus, further studies on this issue could help to design more effective and easy-to-use drugs. keywords: leishmaniasis; plantago psyllium; ulcer; mice introduction leishmaniasis is one of the most important zoonotic diseases caused by more than 20 leishmania species. this disease has a worldwide distribution and is transmitted by sandflies between animals and human hosts (1, 2). according to the world health organization (who) reports, leishmaniasis has a high mortality rate and is a health problem in endemic areas, especially in the developing countries (3, 4). cutaneous leishmaniasis (cl) can be observed in the two forms of rural and urban in 97 different countries, and approximately 1.5 million new cases are reported throughout the world annually, with over 80% of the cases occurring in the developing countries (5, 6). brazil, iran, afghanistan, and sudan are the most infected countries (7). leishmania major is the main etiologic agent of zoonotic cutaneous leishmaniasis in some parts of iran, and it has caused economic and health problems in these areas (8). for a long time, pentavalent antimonial drugs have been used as the first drugs of choice in the treatment of leishmaniasis. currently, glucantime is used in many endemic countries because of its production as a generic drug. in copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 45–50 a moshfe et al.: anti leishmanial effect of … 46 http://jad.tums.ac.ir published online: march 31, 2022 some regions, mainly where resistance has developed, miltefosine, paramycin, and liposomal amphotericin b are gradually replacing the antimonials (9, 10). nowadays, due to some factors such as side effects, drug resistance, excessive and economic costs of medical services, lack of easy access to these medications in some areas, researchers are looking for effective alternative drugs to reduce the side effects of cl treatment (11-13). one way to do so is the use of safe plant extracts in the treatment of the disease. the utilization of herbal extracts for the treatment of cutaneous leishmaniasis in many cities of iran has a long history based on avecina traditional medicine. according to the studies conducted in iran (refer to the review studies of moghaddas et al. 2017, oryan et al. 2015, and soosaraei et al. 2017) in this field, more than fifty different local plants and extracts, oil, and powder of them (such as hydroalcoholic, dichloromethane, ethanolic, methanolic, and aqueous extract) have been used to treat cutaneous leishmaniasis, the findings of the mentioned studies showed that some of them like achillea millefolium (persian name: bumadaran), tanacetum parthenium (persian name: babouneh), nigella sativa (persian name: siah daneh), and satureja khuzestanica (persian name: marzeh khuzestani) were exhibited anti-leishmania effects activity (14-16). in the present study, plantago psyllium (ovate) and white vinegar was used in the treatment on leishmania major lesion in balb/c mice, which was not previously reported in the anti-leishmania experimental studies. materials and methods in this experimental study, after disinfecting the leishmanial ulcer surface in mice, which were previously infected by the standard strain of leishmania major (acquired from shiraz school of medicine with mhom/64/ir/er75 code), some of the ulcer margins were collected by a scalpel and dissolved in 6ml of normal saline. then, by an insulin syringe, 0.2ml of the prepared solution (containing l. major amastigote) was injected into the tail of 30 male balb/c mice aged 10 weeks old. this strain of mice was used because it is the most sensitive to l. major; the mice were purchased from razi institute in shiraz in april 2016. after infecting the mice, they were kept on special shelves under the same conditions. after three weeks, an ulcer appeared in the tail of all the mice. then, to confirm the presence of the parasite (amastigote or leishman body) in the lesions, giemsa-stained slides were observed at 100× magnification. afterwards, all of the 30 infected balb/c mice were randomly divided into five groups. the first group was treated with the combination of plantago psyllium powder and white vinegar (10 mice). for this purpose, plantago psyllium seeds were powdered by grinding. then, 6.2g of p. psyllium powder was mixed in 25ml of white vinegar and a dough was prepared. a 4mm thick layer of the dough was used for treating the ulcers. the second group (as positive control), which consisted of five mice, was treated with 0.2ml of glucantime by intraperitoneal injection (according to the treatment method in many articles such as nilforoushzadeh et al. 2008 and taran et al. 2010). for the treatment of the third group (5 mice), a cotton ball soaked in 5ml of vinegar was used and placed on the wound for 2 minutes. also, p. psyllium powder and water combination (4mm thick layer) were used for treatment in the fourth group (5 mice). group five (5 mice) as the negative control did not receive any treatment. before and after treatment (once every 72 hours), the ulcers’ diameters were measured and recorded in all the groups by a caliper (ulcer size was calculated by measuring two diameters perpendicular to each other). all the groups were treated for 18 days, and at the end of the treatment period, smears were prepared for microscopic examination. the data obtained from microscopic http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 45–50 a moshfe et al.: anti leishmanial effect of … 47 http://jad.tums.ac.ir published online: march 31, 2022 observations, diameter changes, and lesions’ healing were analyzed by spss version 20 using paired samples t-test. a p-value of less than 0.05 was considered significant. results the findings of paired samples t-test indicated that the mean areas of the ulcers in all the groups were not significantly different before and after treatment, except for the second (glucantime) and third (vinegar) groups, (table 1). the mean difference of ulcer area in all the groups was evaluated before and after treatment, and each group was compared separately with the second group (glucantime), which revealed a significant difference between the third, fourth, and fifth groups and the glucantime group (p< 0.05, table 2). also, in the first, second, third, and fourth groups, 6 (60 %), 4 (80%), 3 (60%), and 2 (40%) mice were healed, respectively. on the other hand, ulcers remained in all the five mice of the control group. the results showed a significantly (p< 0.05) smaller lesion size in the treated groups, especially in the vinegar and p. psyllium with water groups, compared to the control group. anti-leishmanial effect of vinegar and glucantime were more than p. psyllium on under treatment mice. table 1. comparison of the mean diameter (± sd) of lesions area in balb/c mice due to l. major, before and after treatment in cases and control group group name numbers x±sd before x±sd after sig/ns p. psyllium with vinegar 10 53.7±5.60 42.4±1.99 ns t= 0.36, df= 9, p= 0.72 glucantime 5 69±6.44 1.4±0.62 sig t= 3.39, df= 4, p= 0.028 vinegar 5 23.5±4.13 9.2±5.8 sig t= 3.3, df= 3, p= 0.045 p. psyllium with water 5 38.7±2.47 34.5±9.47 ns t= 0.17, df= 4, p= 0.87 control 5 56.67±5.42 81.6±10.3 ns t= 0.64, df= 4, p= 0.55 sd= standard deviation, x= mean ulcer area, df= degrees of freedom, ns= non-significant, sig= significant, p= p value, and t= the t-value of the paired sample t-test table 2. the mean lesions diameter (± sd) of area in balb/c mice due to l. major, in cases and control group compared with glucantime group, before and after treatment. group name sig/ns mean sd mean sd p. psyllium with vinegar -30.11 44.98 -45.68 15.45 ns t= 1.21, df= 9, p= 0.12 vinegar -60.20 41.12 -45.68 15.45 sig t= -2.3, df= 7, p= 0.038 p. psyllium with water -4.23 23.54 -45.68 15.45 sig t= -2.3 df= 8, p= 0.038 control 96.24 86.08 -45.68 15.45 sig t= -2.14, df= 8, p= 0.032 sd= standard deviation, x= mean ulcer area, df= degrees of freedom, ns= non-significant, sig= significant, p= p value, and t=the t-value of the paired sample t-test http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 45–50 a moshfe et al.: anti leishmanial effect of … 48 http://jad.tums.ac.ir published online: march 31, 2022 discussion cutaneous leishmaniasis is a great health problem in iran. at the present time, no effective drug and vaccine for the inhibition of pathogenic leishmania spp and chemicals for eradication of its vectors is provided. according to the published research in the world, efforts are still ongoing to discover an effective treatment to cure cutaneous leishmaniasis with negligible side effects and low price. the current and standard medicine against cutaneous leishmaniasis is glucantime, but it has many side effects. therefore, traditional treatment of cl is a common habit of natives in many endemic areas in iran. more than fifty different local plants are used to traditional treatment of cutaneous leishmaniasis lesions in iran (14-16). most of these herbal and traditional medicines (same as the findings of present study) showed anti-leishmanial effect, and also, they can play an effective role in healing wounds and finding an effective way to reduce injection pain and treatment costs. on the other hand, production of ointments containing herbal extract or essential oil are noninvasive method for treatment of cutaneous leishmaniasis (17-20). the results indicated that there was a significant difference between the third, fourth, and fifth groups, and no significant difference between the glucantime group and the p. psyllium and vinegar groups. the findings of hejazi et al. showed a significant difference in the means of lesion diameter before and after treatment in the control, yarrow, and thyme groups (p< 0.05). also, paired t-test showed no significant difference in mean lesion diameter after treatment between treatment and glucantime groups (p> 0.05) (21). according to paired ttest, there was no significant difference in lesions’ healing among different groups after treatment. furthermore, the difference in the mean lesion areas before and after treatment in each group was compared with a positive control group (group 2: treated with the glucantime) separately. only the study by wester hof et al. investigated the effect of p. psyllium on the treatment of cutaneous leishmaniasis. that study was aimed at investigating the effect of mucopolysaccharides on wound healing. the results of that study indicated that mucopolysaccharides limited the formation of scars (22). other studies have also evaluated on the effects of herbal extracts on the treatment of cl. for instance, ahmadi et al. examined the effect of garlic extract on cl, and their findings showed that the diameters of lesions were reduced by the hydrous extract of garlic over 30 days of treatment. however, the maximum reduction was achieved when mice were exposed to 10 days of vitamin a ointment, then treated for 45 days with garlic extract (23). doroodgar et al. showed that artemisia sieberi extract had no effect on the treatment of leishmania major ulcers in balb/c mice (24). most studies on the treatment of cl using herbal extracts and natural products with anti-leishmanial activity so far indicate that none of these herbal extracts had a 100% effect on wound healing. although some of these herbal extracts have been somewhat effective in the wound healing process and reduced the period of the disease (25-27). conclusion the combination of plantago p. powder and white vinegar has been traditionally used to treat leishmanial lesions in iran. it seems the most anti-leishmanial effect is related to vinegar and supported by plantago. the route of treatment with this combination is very simple and painless in comparison with injection. thus, further studies on this issue could help to design more effective and easy-to-use drugs. acknowledgements this article was financially supported by yasuj university of medical sciences and aphttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 45–50 a moshfe et al.: anti leishmanial effect of … 49 http://jad.tums.ac.ir published online: march 31, 2022 proved by the ethics committee of yasuj 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products with leishmanicidal activity. nat prod rep. 18(6): 674– 688. 27. da silva bjm, hage aap, silva eo, rodrigues apd (2018) medicinal plants from the brazilian amazonian region and their antileishmanial activity: a review. j integr med. 16(4): 211–222. 28. nilforoushzadeh ma, shirani-bidabadi l, zolfaghari-baghbaderani a, saberi s, siadat ah, mahmoudi m (2008) comparison of thymus vulgaris (thyme), achillea millefolium (yarrow) and propolis hydroalcoholic extracts versus systemic glucantime in the treatment of cutaneous leishmaniasis in balb/c mice. j vector borne dis. 45(4): 301–306. 29. taran m, mohebali m, esmaeli j (2010) in vivo efficacy of gum obtained pistacia atlantica in experimental treatment of cutaneous leishmaniasis. iran j public health. 39(1): 36–41. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 47 http://jad.tums.ac.ir published online: march 31, 2020 original article development of a loop-mediated isothermal amplification (lamp) assay for detection of relapsing fever borreliae faezeh houmansadr1; *mohammad soleimani2,3; *saied reza naddaf4 1 department of cellular and molecular biology, science and research branch, islamic azad university, tehran, iran 2 department of microbiology, faculty of medicine, aja university of medical sciences, tehran, iran 3 tasnim biotechnology research center, faculty of medicine, aja university of medical sciences, tehran, iran 4 department of parasitology, pasteur institute of iran, tehran, iran (received 18 mar 2019; accepted 04 mar 2020) abstract background: this study aimed to develop a loop-mediated isothermal amplification (lamp) assay for the rapid detection of tick-borne relapsing fever in resource-limited areas. methods: a set of six primers were designed based on the conserved regions of the glycerophosphodiester phosphodiesterase (glpq) gene of borrelia species. for sensitivity assay, serial dilutions of a recombinant plasmid containing a 219bp sequence of the glpq were prepared and used as the template dna. the lamp reactions containing the six primers and the reagents required for amplification were incubated at 60–65 °c for 60min in a loopamp real-time turbidimeter. for the specificity test, dna from 14 other bacteria were included in the assays, and double-distilled water was used as the negative control. also, dna from dried blood spots (dbss) of spirochetemic mice, and blood samples from relapsing fever-suspected patients were examined by the lamp along a borrelia-specific nested pcr that targets the rrs-rrl-igs region. results: the lamp detected as low as 90glpq copies in reactions. the primers reacted with dna from dbs of spirochetemic mice showing spirochete concentrations of ≤ one per a 1000x microscopic field. in clinical samples, the lamp assay showed a higher sensitivity compared to nested-pcr. the lamp specificity was 100%, as the primers did not react with other bacteria dna. conclusion: the high sensitivity and specificity of the test, along with the simplicity of the dna extraction procedure, make the lamp a reliable and adaptable tool for the diagnosis of tick-borne relapsing fever in rural endemic areas. keywords: relapsing fever; loop-mediated isothermal amplification (lamp); iran introduction the genus borrelia comprises two distinct groups of spirochetes with the difference in diseases they cause. one group includes the causative agents of lyme disease and the other the relapsing fever borreliae (rfb). currently, there are 22 confirmed rfb, and six other taxa have been proposed (1). except for the louse adapted borrelia recurrentis, the majority of species pathogenic to humans are transmitted by the soft ticks of the genus ornithodoros. a few species, such as borrelia miyamotoi and borrelia lonestari, are vectored by hard ticks and yet share genetic similarities with the rfb (2). soft tick-borne relapsing fever (stbr) is endemic to iran (3-5). until now, despite the improvement of housing and the removal of the disease from mandatory reporting to the ministry of health and medical education (mhme), no year has passed without reports of human infections. in iran, four rfb, including borrelia persica, borrelia microti, borrelia latyschewii and borrelia baltazardi have been described (5). borrelia persica is the primary cause of the disease especially in the *corresponding authors: dr mohammad soleimani, e-mail: soleimanidor@yahoo.com, dr saied reza naddaf, e-mail: saiedrezanaddaf@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 48 http://jad.tums.ac.ir published online: march 31, 2020 west and northwest of the country (5-7), while in the south, epidemiological data and molecular approved human infections indicated b. microti and other b. microti-like borreliae as the other cause of relapsing fever (3, 8, 9). in iran, until recently, confirmation of relapsing fever merely relied on observation of the spirochetes in peripheral blood of febrile patients using darkfield microscopy or giemsa-stained blood smears. the disease is easily diagnosed by microscopy during fever peaks with a massive spirochetemia. however, between the peaks and in milder diseases, the bacteria are scanty and are hard to identify in blood smears. in iran, pcr assays using various molecular markers like flab, glpq and rrs have successfully detected the borrelia spp in relapsing fever patients (3, 8) and animals (10). the pcr assays exhibit high sensitivities but are not commonly affordable in resource-limited laboratories of rural areas, where most of the relapsing fever infections occur. hence, we prompted to develop an alternative dna amplification assay of lower cost for the detection of the disease in these areas. loop-mediated isothermal amplification (lamp) has proved as a robust, cheap, highly sensitive/ specific tool for the detection of various pathogenic agents including viruses, bacteria, fungi, and parasites (11-14). this assay employs a dna polymerase and a set of 4–6 primers that operate in isothermal conditions forming loop structures that ultimately precipitate in the reaction mixture (13). this assay has also shown to be less prone to inhibition from dna preparations and allows adaptability to field conditions (15, 16). this study aimed to develop a (lamp) assay for the rapid detection of relapsing fever borreliae based on the glycerophosphodiester phosphodiesterase (glpq) gene, a sequence conserved among all relapsing fever borreliae, but absent from lyme disease spirochetes (17). materials and methods bacteria species and dna extraction we used b. microti strain ir-1, which was maintained via continual passages in nmri mice for more than 15 years in the parasitology department of pasteur institute of iran. blood samples were obtained from b. microtiinfected mice when spirochetes reached 1.4× 106/ml of blood. dna extraction from 1ml of blood was performed using a genomic dna purification kit (promega, madison, usa) as described by the manufacturer. primer design initially, glpq sequences of 10 relapsing fever borreliae (b. microti, acc. no. jf825473; b. microti, acc. no. eu914144; b. recurrentis, acc. no. kj003842, borrelia sp., acc. no. kx683865; borrelia duttonii, acc. no. dq 346785; borrelia crocidurae, acc. no. cp 004267; borrelia hispanica; acc. no. gu 357573; b. persica, acc. no. eu914143; b. recurrentis, acc. no. af247152; borrelia duttonii, acc. no. gu357577) were obtained from the genbank database and aligned by using clc sequence viewer 7 (clc bio, aarhus, denmark). a set of six primers including two loop primers were designed based on the conserved regions of the glpq sequence, corresponding to the nucleotides 261015–261682 of b. duttonii strain ly (fig. 1), by the online software program, primer explorer v4 (eiken chemical co., tokyo, japan; http://primerexplorer.jp/e/). the theoretical specificity of the designed primers was confirmed by in silico analysis using blast and primer-blast software available in ncbi (http://www. ncbi.nlm.nih.gov/). the primers were synthesized by a commercial company (generay biotechnology, shanghai, china). the main primers (glpq-f3 and glpq-b3) and (glpq-fip and glpq-bip) target fragments of 219bp and 161bp size, respectively, and the loop primer (glpq-lf and glpq-lb) produce http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 49 http://jad.tums.ac.ir published online: march 31, 2020 amplicons of various size with a ladder-like pattern (table 1). glycerophosphodiester phosphodiesterase gene (glpq) cloning a 219bp fragment of the glpq gene was amplified by the primers glpq-f3 and glpqb3 (table 1). the reaction contained 3mm mgso4 (biobasic, toronto, canada), 1.6mm dntps (kawsar biotech co, tehran, iran), 1µl 10x buffer [100mm kcl, 100mm (nh4)2so4, 200mm tris hcl (ph 8.75)], 1% triton x-100, 1mg/ml bsa (biobasic, toronto, canada), 1u taq dna polymerase (biobasic, toronto, canada), 0.4µm of each primer, 1µl template dna, and double-distilled water (ddw) to the 25µl final volume. the amplification was programmed in a thermal cycler (eppendorf, hamburg, germany), for an initial denaturation at 94 oc for 4min followed by 35 cycles of 94 oc for 45sec, 45 oc for 45sec, and 72 oc for 30sec, and a final extension at 72 oc for 10min. the pcr products were run on a 2% agarose gel (min run gel electrophoresis system; bioequip co, shanghai, china), stained with ethidium bromide (cinnagen, alborz, iran), and visualized under uv in gel documentation system (e-box vilber, marne-la-vallée, france). the pcr product was purified using a pcr purification kit (bioneer, daejeon, south korea), cloned into a ta vector (instaclone™ pcr cloning kit, thermo scientific, ma, united states), and transformed in escherichia coli top10f’. the bacteria were incubated at 37 °c for 24h on luria-bertani medium (merck, kgaa, darmstadt, germany) containing 24mg/ml iptg (isopropyl-betad-thiogalactopyranoside) (fermentas, ontario, canada), 20mg/ml x-gal (5-bromo-4-chloro3-indolyl beta d-galactoside) (fermentas, ontario, canada), 10mg/ml tetracycline (razak, alborz, iran) and 50mg/ml ampicillin (cosar, tehran, iran). the recombinant bacteria were identified by blue/white screening, with the while colonies representing recombinant ones. one white colony was added to luria-bertani broth medium containing 10mg/ml tetracycline and 50mg/ml ampicillin followed by incubation at 37 °c for 16h while shaking at 180 rpm. plasmid purification was performed by the accuprep plasmid mini extraction kit (bioneer, daejeon, south korea) and the presence of the glpq gene in the recombinant plasmids was confirmed by pcr amplification with the primers glpq-f3 and glpq-b3. the recombinant plasmid was named ptz57r/t-glpq. sensitivity assay a serial 10-fold dilution of the recombinant plasmid ranging from 9×108 to 9×10-1 copy numbers per microliter, equivalent to ≈32ng to ≈32×104 fg/µl of dna was prepared and used in assays. specificity assay for specificity assays, we used dna of 14 other bacteria including shigella sonnei atcc 9290, klebsiella pneumoniae atcc 7881, bacillus subtilis atcc 6051, staphylococcus aureus atcc 25923, enterococcus faecalis atcc 29212, enteropathogenic escherichia coli (epec) atcc 43887, yersinia enterocolitica atcc 23715, pseudomonas aeruginosa atcc 27853, clinical specimens of escherichia coli, salmonella typhi, acinetobacter baumannii, citrobacter sp, enterobacter sp, and leptospira interrogans in all assays. all the bacteria, including l. interrogans, the closely related bacteria to borrelia species contained glpq sequence. loop mediated isothermal amplification (lamp) assay the lamp reactions contained 40pm of the inner (glpq-fip and glpq-bip) and 10pm of outer (glpq-f3 and glpq-b3) and loop primers (glpq-lf and glpq-lb) (table 1), 11.2 mm dntps (kawsar biotech co, tehran, iran), 0.8m betaine (sigma aldrich, taufkirchen, germany), 20mm tris-hcl, 10mm kcl, 10mm (nh2)so4, 0.05% triton x-100 (ph 8.8) (biolabs, new england, uk), 8mm mgso4 (bihttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 50 http://jad.tums.ac.ir published online: march 31, 2020 obasic, toronto, canada), 0.1% tween 20 (acros organics, vernon, usa), 8u of bst dna polymerase, large fragment (biolabs, new england, uk), 1μl of serial dilutions of the recombinant plasmid, and ddw to the 25 μl final volume. the reactions were incubated at temperatures ranging from 60–65 °c for 60min in a loopamp real-time turbidimeter (la-320c; teramecs, kyoto, japan), followed by heating at 80 °c for 5min for enzyme inactivation. in all assays, dnas from 14 other bacteria were included and ddw was used as a negative control. during the optimization of the assay, some amplifications were performed without the loop primers. also, in some reactions, we added 1μl of fluorescent detection reagent containing calcein (eiken chemical co., tokyo, japan), an indicator of dna amplification. detection of lamp products the amplification in lamp reactions was examined by 1) naked eye observation of white turbidity resulting from the accumulation of magnesium pyrophosphate, a by-product of the reactions, 2) a loopamp real-time turbidimeter that records the optical density of reactions every 6sec at 650nm (the reactions were considered positive when the turbidity reached ≥ 0.1 within 60min), 3) the color change from orange to green, as an indication of dna amplification, and 4) gel electrophoresis of amplicons on 2% agarose gels. a lamp product resulting from the 9×108 dilution was purified using pcr purification kit (bioneer, daejeon, korea) and sequenced in both directions by (abi 3730xl/bioneer 3730xl, daejeon, republic of korea). preparation of dbss from borrelia-infected mice amounts of 200µl blood from b. microtiinfected mice with various degrees of the spirochetemia (225±61.34, 71±36.71, 3.3±1.63, 1.1±1.44, and 0.8±0.78 spirochetes per microscopic field) were dotted on 30 dna banking cards (dbc) (kawsar biotech co, tehran, iran). blood from non-infected mice was used as controls on dbcs. the blood spots were allowed to dry at room temperature, and the dbcs were kept in the same condition until used. loop mediated isothermal amplification (lamp) and pcr with dbs circles of 2mm from dbss were cut and washed three times with dna extraction buffer (provided by the manufacturer), followed by ddw. the circles were allowed to dry at room temperature and then used in lamp and pcr assays as described above except that instead of template dna, dbs circles were included in the reactions. the sensitivity and specificity of the lamp assay were calculated in comparison with microscopy as the gold standard assay using medcalc (2018 medcalc software bvba) software available online (https://www.medcalc.org/calc/diagnostic_tes t.php). loop mediated isothermal amplification (lamp) and nested-pcr amplification of clinical specimens dna was extracted from 39 sera of febrile patients residing in the relapsing fever endemic areas in the south and west of iran using a commercial dna extraction kit as described above. the dna samples were examined by the lamp, and a borrelia-specific nested pcr that amplifies the rrs-rrl-igs region using the outer primers f, 5´-gtatg tttagtgaggggggtg-3´ and r, 5´-gg atcatagctcaggtggttag-3 ́ and inner nested primers f, 5´-aggggggtgaagtc gtaacaag-3 ́and r, 5 -́gtctgataaacc tgaggtcgga-3´ (18). this pcr has exhibited high sensitivity in detecting relapsing fever borreliae (3, 8, 19, 20). in all assays, for specificity test, dnas from other bacteria species were included and ddw was used as a negative control. the lamp reactions were checked for dna amplification as described above, and the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 51 http://jad.tums.ac.ir published online: march 31, 2020 pcr products were resolved on 2% agarose gels, visualized under uv and photographed. results glycerophosphodiester phosphodiesterase gene (glpq ) cloning amplification of the glpq sequence from the recombinant plasmid (ptz57r/t-glpq) with the primers glpq-f3 and glpq-b3yielded the expected 219bp indicating the insertion of this sequence in the plasmid. loop mediated isothermal amplification (lamp) assay with recombinant ptz57r/ t-glpq we observed amplification of glpq sequence in the lamp reactions by naked eye observation of turbidity and the loopamp real-time turbidimeter down to 9×103 copies of recombinant plasmid equivalent to 0.32fg dna (fig. 2a). however, gel electrophoresis revealed amplifications in two lower dilutions of 9×102 and 9×101, equivalent to 3.2fg and 0.32fg dna, respectively (fig. 2b). our lamp assay was 100% specific as the primers reacted with none of the other 14 bacterial dna, including l. introgans. in the reactions containing calcein and the borrelia dna, the color turned from orange to green. the 137bp sequence resulted following the sequencing of the lamp amplicon matched with the glpq sequence of b. microti strain ir-1 (acc. no. jf825473) corresponding to nucleotides 261158–261295 of the whole genome sequence of b. duttonii ly (acc. no. cp000796). the results were the same within the temperature range of 60–65 °c. in the reactions, in abscence of loop primers, the optimum time for isothermal amplification was 60min, whereas, in those with the loop primers, the incubation time reduced to 45min. loop mediated isothermal amplification (lamp) and pcr with dbss of the 30 b. microti-positive dbs, all (100%) showed turbidity with the naked eye indicating amplification, while the glpq-pcr (with the primers glpq-f3 and glpq-b3) yielded the expected 219bp band in 20 positive dbss (66.67 %). the negative pcr reactions belonged to the mice with the lowest level of spirochetemia [1.1±1.44 (n= 4) and 0.8±0.78 (n= 6) spirochete per microscopic field]. neither lamp nor pcr amplification was observed with the negative controls (ddw) or reactions containing other bacteria dna as the template. by considering microscopy as the gold standard, the sensitivity of glpq-lamp and glpqpcr were 100% and 66.67%, respectively. the specificity was 100% for both assays. loop mediated isothermal amplification (lamp) and pcr with clinical samples of 39 clinical samples, 11 became turbid in the lamp assay indicating amplification of glpq gene, whereas pcr amplification of igs sequence only yielded the expected 540bp band in three specimens (fig. 3). table 1. the primers designed and used for amplification of glpq gene by lamp assay primers name sequences (5' to 3') glpq-f3 forward outer primer aatgcacgatcctgaact glpq-b3 backward outer primer tcttcttctagggttggaatt glpq-fip forward inner primer tgctaatgtgaaatcgacggaataacaacaacaaatgttgcaaagc glpq-bip backward inner primer aatcactaagccttagcgaaagattgttgcaggaaaacggtta glpq-lf forward loop primer tctctagctcttcctggaaaca glpq-lb backward loop primer cctgaaacacaacaaccaatatacc http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 52 http://jad.tums.ac.ir published online: march 31, 2020 fig. 1. the regions of the glpq from which the primers designed for lamp assay. forward outer primer, glpq-f3; backward outer primer, glpq-b3; forward inner primer, glpq-fip (f2)+glpq-fip (fic); backward inner primer, glpqbip (b2)+glpq-bip (bic); forward loop primer, glpq-lf; backward loop primer, glpq-lb fig. 2. the sensitivity of the loop mediated isothermal amplification (lamp) assay measured by a 10fold serial dilution of a recombinant plasmid ptz57r/t-glpq plasmid ranging from 9×109 to 9×101/µl. a) the amplification curves generated by the loopamp real-time turbidimeter, colored lines 1–7, serial dilutions 9×109, 9×108, 9×107, 9×106, 9×105, 9×104 and 9×103; line 8, serial dilutions ≤ 9×102. b) the lamp products resolved on agarose gel, lane m, 50bp dna ladder, lane 1, dilution 9×109; lane 2, dilution 9×108; lane 3, dilution 9×107; lane 4, dilution 9×106; lane 5, dilution 9×105; lane 6, dilution 9×104; lane 7, dilution 9×103; lane 8, dilution 9×102; lane 9, dilution 9×101; lane 10, dilution 9×100; lane 11, dilution 9×10-1; lane 12, negative control fig. 3. gel electrophoresis of pcr amplification of the rrs-rrl-igs region. lane m, 5bp dna ladder; lanes 1– 6, human blood samples (a 540bp in lanes 4 and 5 indicates amplification of borrelia dna); lane 7, negative control (ddw) discussion in iran, blood analysis of relapsing fever patients by examination of wet smears using dark-field microscopy or giemsa-stained blood slides has been a common practice for years. this approach was efficient in endemic areas, where the disease commonly appeared in clushttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 47–55 f houmansadr et al.: development of … 53 http://jad.tums.ac.ir published online: march 31, 2020 ters (5), and overlooking the spirochetes in the blood of some febrile patients did not question the identity of the causative agent. however, with the decline of the disease in the endemic regions and reports of sporadic cases from other areas application of more sensitive approaches for the identification rfb became necessary. in iran, over the past decade, pcr assays by based on various molecular markers, such as glpq, rrs, and flab, were developed for the identification of borrelia infection in ornithodoros ticks (21, 22) or characterization of the tick-originated relapsing fever borreliae (9, 23), but rarely clinical samples were included. lately, qpcr and conventional pcrs identified a b. microti-like strain, presumably, an ecotype of african b. duttonii, in relapsing fever patients from southern iran (3, 8). in our previous work, using b. persica-spiked blood samples, we consistently observed bacteria by microscopy in the blood samples with densities ≥ 800–1000 spirochetes/µl (24). herein, with the dbss prepared with the spirochetemic murine blood, the lamp could detect borrelial dna in blood specimens showing less than one spirochetes per 1000x microscopic field. our lamp assay also showed a higher sensitivity in comparison with a nested-pcr amplification of the igs in the diagnosis of clinical samples. the specificity of the lamp assay for the diagnosis of b. microti dna was 100% as the designed primers exhibited no cross-reaction with dna from the other 14 bacteria used in this study. our lamp method requires to be further tested with blood samples from rf patients infected with other borrelia species to ensure the sensitivity and specificity of the assay. lamp method has also shown promise for the detection of borreliae dna and other pathogens in the tick vectors. in china, the lamp could detect b. burgdorferi s. l. in ticks, with a higher sensitivity than a conventional pcr (25). also, in recognizing spotted fever group rickettsia, the lamp appeared ten times more sensitive than an end-point pcr targeting the same gene fragment (26). conclusion such a reliable sensitivity and specificity, as well as the simplicity of the dna extraction procedure, make the lamp a suitable and adaptable assay for field studies in relapsing fever endemic areas. in resource-limited rural health centers, lamp can readily check fieldcollected blood samples, preferably on dbs, for relapsing fever borreliae with the least equipment accessible, i.e., a heat block. having access to a loopamp real-time turbidimeter is preferable. however, lamp is a qualitative assay, and the result can also be monitored by color change with calcein in the fluorescent detection reagent or by the turbidity of magnesium pyrophosphate in the reaction tubes. hence, a heating system that provides a temperature within the range of 60–65 °c would suffice. acknowledgements we thank the tasnim biotechnology of 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g, niu q, liu z, li y, liu j, ma m, ren q, liu a, yin h (2013) development and application of a loopmediated isothermal amplification assay for rapid detection of borrelia burgdorferi s. l. in ticks. transbound emerg dis. 60(3): 238–244. 26. noden bh, martin j, carrillo y, talley jl, ochoa-corona fm (2018) development of a loop-mediated isothermal amplification (lamp) assay for rapid screening of ticks and fleas for spotted fever group rickettsia. plos one. 13(2): e0192331. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=barmaki%20a%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=rafinejad%20j%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=vatandoost%20h%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=vatandoost%20h%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=telmadarraiy%20z%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=mohtarami%20f%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=leghaei%20sh%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=leghaei%20sh%5bauthor%5d&cauthor=true&cauthor_uid=22808396 https://www.ncbi.nlm.nih.gov/pubmed/?term=oshaghi%20m%5bauthor%5d&cauthor=true&cauthor_uid=22808396 j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 341 http://jad.tums.ac.ir published online: december 25, 2018 original article emergence and associated risk factors of vector borne west nile virus infection in ilorin, nigeria *olatunji matthew kolawole, glory adelaiye, jeremiah ikhevha ogah infectious diseases and environmental health research group, department of microbiology, faculty of life sciences, university of ilorin, ilorin, nigeria (received 29 aug 2016; accepted 5 aug 2018) abstract background: west nile virus (wnv) is a mosquito-borne viral pathogen that is the causative agent of west nile fever and encephalitis. diagnostic tools for wnv infection in nigeria are not well established hence the current prevalence rate of wnv infection in nigeria is unknown. we aimed to establish the serological prevalence of wnv infection in febrile patients in ilorin, nigeria in 2016, and to assess the risk factors associated with the acquisition of the virus. methods: this was a cross-sectional study involving the screening of subjects presenting with febrile illnesses. while specific igm elisa was used to determine the seroprevalence, a closed-ended questionnaire was used to ascertain the risk factors associated with this viral infection. results: fifteen (7.5%) of the respondents were positive for wnv infection. subjects living in areas in close proximity to trees and bushes (p= 0.011) and stagnant water (p= 0.001) were at a higher risk of having wnv infection. other risk factors associated with wnv among the respondents include the none use of insecticide (p= 0.001), sitting out at night (p= 0.044), hiv positivity (p= 0.003) and having an organ transplant (p= 0.002). conclusion: this study clearly shows a considerable wnv infection in ilorin, with the presence of factors that can promulgate an outbreak, hence a need for further surveillance in the study area. keywords: mosquito, vector-borne, west nile virus, nigeria, prevalence introduction west nile virus (wnv) is a neurotropic pathogen which belongs to the flaviviridae family, and the causative agent of (wnv) fever and encephalitis (1, 2). the virus was first isolated in 1937 in ugandan and named after the west nile district (3). it was largely limited to africa and parts of asia until it was introduced in new york, usa in 1999 (4). between 1999 and 2010, over 2.5 million people across the world have been infected by this virus, with about 12000 of them developing to encephalitis and 1300 deaths (5). moreover, an outbreak of wnv infection was recorded in greece between 2010 and 2011 which resulted in about 44 deaths. wnv is a single strand, positive-sense rna virus with an open reading frame of about 11kb (6). phylogenetic analysis shows that the virus has two distinct lineages (lineage 1 and 2) although lineages 3, 4 and 5 have also been identified. outbreaks of wnv infection have majorly been associated with lineages 1, 2 and 5 (7, 8). while lineage 1 is transmitted both in africa and other parts of the world, lineage 2 is restricted principal to the african continent (9). however, in 2010–2011 wnv outbreak which occurred in greece lineage 2 was also implicated (3). wnv is a vector-borne virus which infects not just humans but birds and horses. transmission is mainly via vectors like mosquitoes (culex spp.) during the course of blood feeding (2). this is usually achieved by the vector’s injection of at least a vasodilator, platelet inhibitor and coag*corresponding author: dr olatunji matthew kolawole, e-mail: omk@unilorin.edu.ng, tomak7475@gmail.com j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 342 http://jad.tums.ac.ir published online: december 25, 2018 ulation inhibitor in addition to digestive enzymes (2). mosquito saliva in vivo plays a crucial role in the transmission of wnv (10). apart from culex spp, aedes mosquitoes are also known to transmit wnv (11, 12). the human skin acts as a natural barrier to different infections, including wnv although the virus remains in the skin where inoculation occurred for 14 days after infection (13). majority of the infections associated with wnv in humans are asymptomatic, incubation period of the virus last amid 2 to 15 days (14, 15). symptomatic cases of wnv fever occurs in about 20% of infected persons with flu-like symptoms emerging. various risk factors have been associated with the development of wnv infection, they include age, hypertension, compromised immunity, organ transplantation (16–18). factors which generally encourage contact with the vector responsible for the transmission of wnv usually increase the risk of acquisition of the infection. these factors include, proximity to bushes, presence of stagnant water, housing type and weather (19, 20). although very few studies have shown the prevalence of wnv in nigeria (21), none have shown its presence in the study area. moreover, the vectors culex sp. responsible for the spread of the virus have been very common in the study area (22). we aimed to access the prevalence of wnv infection in nigeria and to determine factors influencing the spread of the virus and viral infection. materials and methods study design/study site this research was a hospital-based crosssectional study of febrile malaria patients attending hospitals in ilorin west and south of kwara state. patients that presented symptoms similar to malaria and typhoid fever were recruited for the study for a period of 6 months from jan–jun 5th, 2016. this study was conducted in two local government areas (ilorin west and south) in kwara state. ilorin is the state capital of kwara state in nigeria and located on (8o30’n 5o00’ e). the state is made up of about 1.5 million people and has a land mass of 32500km2. study population/sampling technique the study population consisted of patients that presented with symptoms of malaria and typhoid fever in sobi specialist hospital and civil service clinic, ilorin, kwara state. febrile patients were screened and a well-structured close-ended questionnaire was administered to them after an informed consent form was dully filled and signed. participant’s blood was taken as specimen for the necessary clinical examination. inclusion criteria  febrile patients of all ages.  patients with hypertension, diabetes, chronic renal failure, and hiv.  patients who met the criteria and consented to partake in the study. exclusion criteria  patients vaccinated against or recently infected with related flaviviruses.  patients that did not give their consent. blood sample collection overall, 200 blood samples were collected from febrile patients who met the inclusion criteria. five-millilitre venous blood were collected intravenously using sterile needle syringe from each study participant into a sample bottle marked with a unique number that tallied with the number on their questionnaire. serum separation was done by centrifugation at 1600 revolutions per minute for five minutes. serum samples were then collected and stored at -20 °c. assay the preserved sera were screened for wnv j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 343 http://jad.tums.ac.ir published online: december 25, 2018 igm antibodies using elisa. igm results were expressed in international unit (iu) with calibration performed against reference standards of 5.0 and 10.0iu/ml where samples with index values ≤0.10 are negative, ≥1.0 are positive and samples that fell within the 0.11– 0.99 are equivocal. igm was performed by an indirect elisa assay. analysis and interpretation of results were done according to the manufacturer’s instruction (wkea med supplies corp human (wnv) igm, china: wh-1754). the igm used for this study has a sensitivity of 95% and specificity of 95% for west nile virus. statistical analysis all data generated from the study was checked manually for errors in filling responses. descriptive statistics such as mean, frequency, standard deviation, percentage, and graph were used in the discussion of the results, in order to give a lucid representation of the data analyzed. the interaction between the prevalence of wnv infection and associated risk factors were tested using χ2 (chisquare) test at 5% (p< 0.05) confidence interval. all data generated from the study was checked manually for errors in filling responses. ethical approval the approval for the study was obtained from the ethical review committee of the kwara state ministry of health and the informed consent was obtained from patients. results overall, 200 febrile patients who met the inclusion criteria were enrolled. while majority of respondents (44.0%) were within the age range of 21–30yrs, female subjects accounted for a larger portion (60.0%) of the study population. other socio-demographic characteristics of the study population are also shown in table 1. among the 200 respondents that participated, 15 (7.5%) were positive to (wnv) while 184 (92%) were negative with 1 (0.5%) equivocal result. a positive relationship between wnv and the socio-demographic character of the study population is shown in table 2. establishing the prevalence of (wnv) in relation to presence of trees and bushes around the habitat of respondents revealed of the 83 (41.5%) with trees 7 (3.5%) were wnv positive. of 62 cases (31%) with stagnant water, 12 (6%) were wnv positive. table 3 shows the relationship between wnv prevalence and potential risk factors associated with either the prevalence of the viral infection. table 1. socio-demographic characteristics of the study population socio-demographic characteristics (n=200) study population (percentage) age 11-20 yr 25 (12.5) 21-30 yr 88 (44.0) 31-40 yr 47 (23.5) 41-50 yr 24 (12.0) 61-70 yr 8 (4.0) 71-80 yr 8 (4.0) gender male 60 (30.0) female 140 (70.0) j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 344 http://jad.tums.ac.ir published online: december 25, 2018 tribe yoruba 156 (78.0) hausa 15 (7.5) igbo 22 (11.0) others 7 (3.5) religion christians 60 (30.0) muslims 140 (70.0) marital status married 127 (63.5) single 68 (34.0) divorced 2 (1.0) widowed 3 (1.5) occupation farmers 7 (3.5) civil servants 53 (26.5) traders 61 (30.5) students 50 (25.0) others 29 (14.5) level of education no education 24 (12.0) primary education 25 (12.5) secondary education 62 (31.0) post-secondary education 89 (44.5) table 2. level of education of the respondents in relation to their wnv status socio-demographic characteristics positive (%) negative (%) χ2 (p) level of education 29.63 (0.001) fisher’s exact no education 6 (3.0) 18 (10.5) primary education 7 (3.5) 18 (10.0) secondary education 1 (0.5) 61 (29.0) post-secondary education 1 (0.5) 88 (44) p< 0.05 is statistically significant table 1. continued … j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 345 http://jad.tums.ac.ir published online: december 25, 2018 table 3. characteristics of west nile virus positive individuals risk factor positive (%) diabetes yes 0 (0) no 15 (7.5) hypertension yes 2 (1.0) no 13 (6.5) hiv yes 1 (0.5) no 14 (7.5) organ yes 1 (0.5) no 14 (7.5) vaccination yes 1 (0.5) no 11 (5.5) not sure 3 (1.5) blood transfusion yes 2 (1.0) no 13 (6.5) surgery yes 2 (1.0) no 13 (6.5) presence of trees and bushes yes 7 (3.5) no 8 (4.0) presence stagnant water yes 12 (6.0) no 3 (1.5) use of mosquito net yes 7 (3.5) no 8 (4.0) use of insecticides yes 3 (1.5) no 12 (6.0) traveling frequently yes 0 (0) no 15 (7.5) night sit out yes 14 (7.0) no 1 (0.5) discussion this study provides data on the serological prevalence of (wnv) among febrile subjects in ilorin. it also describes some of the sociodemographic attributes of the participating population while establishing the presence or absence of a relationship between known risk factors of west nile viral infection, the social demographic characteristics of the respondents and the prevalence of west nile virus. young adults and middle-aged people had the highest rate in this study. this is likely due to the fact that majority of the subjects presenting with febrile illnesses at the time of sampling were within this age group. similarly, the female respondents were more than the male ones in a ratio of 2 to 1. this was because women attending antenatal clinics of the hospitals surveyed who also had febrile illness participated in the study. this shifted the population density in favor of the female subjects. this is similar to results that determined the prevalence of wnv among febrile patients in sudan (23). other demographic characteristics such as tribe and religion were largely a reflection of the study area where the study was carried out (24, 25). the most popular housing type among the respondents was single room apartments. this is a reflection of the socio-economic status of the respondents. in nigeria, most low and medium income earners in nigeria lived in single room apartments (26). however, there was no evidence that the housing of the subjects influenced mosquito bite or the prevalence of wnv infection. the educational level of the subjects varied extensively, while less than half of the subjects had tertiary education, 24 (12%) of them had no formal education at all. wnv serological positivity was found among 15 respondents. this represents a 7.5% prevalence rate among the study population. this was lower than the 25% prevalence rate gotten from a similar study in maiduguri, nigeria (21). the difference in these prevalence rates could either be due to the difference in j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 346 http://jad.tums.ac.ir published online: december 25, 2018 geographical location, prevailing climate at the time of sample collection, or differences in the type of test use. while igm elisa was used in this study to ascertain subjects with current or recent wnv infection, baba et al. used plaque reduction neutralization (21). the high level of specificity of the igm elisa used in this study (95%), helps to eliminate largely the risk of cross-reactivity from other flaviviruses especially dengue virus, which is common in serological test of wnv. although, this does not entirely rule out the possibilities of crossreactivity among other flaviviruses, which remains one of the pitfalls of using elisa in testing for wnv. similar wnv prevalence rate of 9.5% and 13.2% was obtained in kenya and sudan (23, 27), respectively. a prevalence rate of 24% in a different part of kenya (28) and an extremely high rate of 66% was reported (29). the differences in the results from this study and other studies could also be largely influenced by environmental conditions (30). the distribution of wnv with respect to age among the respondents was widespread and not limited to a particular age group. age has not been shown to be a potential risk factor in the acquisition of wnv infection. this is similar to another study (23) with a p-value of 0.811, where age had no role in the sero-detection of wnv. this is also in concordance with the reports in maiduguri, nigeria (30). although 11 of the 15 subjects that were positive for wnv were female, this was not statistically significant (p= 0.765) as more females participated in the study than males. in addition, gender is not a known risk factor necessary for the acquisition of wnv. this is also buttressed (27) which showed that gender did not constitute a potential risk factor for wnv acquisition among kenyan adults. however, this is not in agreement with anotehr study (21) which noticed differences between the frequencies of positive diagnoses in males and females for wnv (p= 0.022, by fisher’s exact test). the educational level of the respondents showed that the prevalence of wnv reduced as their educational level increases as 13 of the 15 subjects with wnv infection had either primary education or no formal education at all. increased level of education increases the probability of the respondents having knowledge of methods of preventing wnv infection. a statistical relationship existed between the educational level of the respondents and prevalence of wnv infection. the centre for disease control (31) emphasizes the need for education (formal/ informal) of the populace about wnv in order to reduce the burden of wnv infection. moreover, the housing type of the respondents did not contribute statistically (p= 0.719) to the prevalence of wnv infection. although housing type measured by roofing type could contribute to the prevalence of mosquitoes which are the vectors for wnv. the housing type did not statistically influence the prevalence of arboviruses including wnv among the respondents (27). in chicago (32), living in older houses increased the risk of acquiring wnv infection among the studied population presence of trees and bushes influenced the prevalence of wnv among the respondents. presence of trees and bushes provides habitat for mosquitoes known vectors of wnv. environmental conditions that increase the prevalence of mosquitoes and birds increase the risk of wnv infection (33). similarly, stagnant water is a known factor which encourages the increase in population of mosquitoes which invariably increases the risk of wnv infection (33). wnv case-patients were significantly more likely to reside near slow-moving/stagnant water sources with heavier vegetation (20). results from this study are similar to reports from el paso, texas, where wnv case-patients resided close to yards that were flooded regularly by irrigation canals (19). in romania, the risk for wnv infection was higher among persons with j arthropod-borne dis, december 2018, 12(4): 341–350 om kolawole et al.: emergence and associated … 347 http://jad.tums.ac.ir published online: december 25, 2018 mosquitoes in their homes and with flooded basements (34). although factors that help to reduce incidence of mosquitoes invariably helps to reduce the burden of wnv infection, the use of mosquito nets did not contribute to the prevalence of wnv among the respondents. this might be due to improper use of these mosquito nets. various studies in nigeria have reported various misuse of insecticide-treated nets (35). however, the use of insecticide among the respondents played a major role in the acquisition of wnv. preventing wnv is strongly linked to preventing mosquito bite (33) and this can be achieved through the use of insecticide. adulticiding (36) which involves killing of the adult mosquitoes using insecticides and larviciding (36) which involves the use of insecticide has been shown to be effective in controlling the spread of mosquitoes which are the main vectors of wnv (33). risk for becoming infected can be influenced by time spent outdoors and decisions on whether to adopt personal precautions against mosquito bites (33), this was reflective of the results obtained in this study. in houston, a serosurvey of homeless individuals found that time spent outdoors greatly influenced infectivity, with 12.5% of those who reported spending >12h outdoors being positive for wnv, compared to only 2% of those who reported spending ≤6h outdoors (37). in this study majority of the subjects with (wnv) infection spent considerable time outdoors. although diabetes and hypertension did not constitute factors influencing the acquisition of wnv in this study, factors that reduce the overall immunity of humans will help propagate the development of wnv infection. moreover, hypertension and diabetes have been shown to be independent risk factors in the development of encephalitis from wnv infection (17). in new york city, age of ≥75 yr and diabetes mellitus were both shown to be independent risk factors for death from wnv infection (38). although only one of the subjects had hiv, this subject was also positive for wnv. a statistical association (p= 0.003) exists between the hiv status of the respondents and the prevalence of wnv. hiv infection is known to cause reduced immunity in individuals having the disease, thus such persons are at a higher risk of having wnv infection (39) and are regarded as a major risk factor in the development of wnv infection (9). in this study, only one of the respondents has had an organ transplant, with this same respondent being positive for wnv infection. although a significant value was obtained from this factor, much cannot be deduced due to the very low population of subjects with organ transplant and hiv in this study. organ transplant is a major cause of reduced immunity, which could increase the risk of developing wnv (16). conclusion the risk factors which could promulgate an outbreak of wnv infection in the study area are quite evident. the presence of wnv infection in febrile patients also show a need to establish reliable protocols for the detection of the virus in subjects to avoid wrong diagnosis, as presently, such patients with febrile illnesses are not screened for wnv. environmental and behavioral factors which promote the spread of the virus should also be curbed. acknowledgements no financial support was received for this study. the authors declare that there is no conflict of interests. references 1. sule ms, oluwayelu do (2016) analysis j 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14(4): 353–362 ff ouma: application of high-resolution … 353 http://jad.tums.ac.ir published online: december 31, 2020 original article application of high-resolution melting (hrm) technique towards the detection of asymptomatic malaria in a malaria endemic area of southeastern iran under elimination program francan felix ouma1; *mehdi nateghpour1; afsaneh motevalli haghi1; mehdi mohebali 1,2; leila farivar1; ahmad hosseini-safa1; sayed hussain mosawi3 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran 3cellular and molecular research center, qom university of medical sciences, qom, iran *corresponding author: dr mehdi nateghpour, e-mail: nateghpourm@sina.tums.ac.ir (received 30 sep 2018; accepted 03 dec 2020) abstract background: asymptomatic malaria, which usually exists in low parasitemia, acts as the plasmodium species reservoirs contributing towards malaria transmission. this situation hinders malaria elimination programs in endemic areas, thus necessitating an active case detection with a high sensitive method and treatment of cases. this is why we used a high resolution melting (hrm) assay to monitor the trend of asymptomatic malaria in a malaria endemic area of iran which is under elimination program. methods: the peripheral blood was sampled from 271 clinically approved non-febrile individuals from a malaria endemic zone of southeastern iran for asymptomatic malaria prevalence detection by microscopy, rapid diagnostic tests (rdts) and hrm methods. the hrm assay was done based on the amplification of 18s ssu rrna gene. results: the hrm assay revealed infections from three individuals out of 271 (1.1% asymptomatic malaria prevalence) from the participants, two iranian natives with plasmodium vivax infection and one pakistani immigrant with p. falciparum infection. neither microscopy nor rdts detected plasmodium spp infections from the 271 non-febrile individuals. the nucleotide sequencing analysis of the positive controls used in this study showed a close homology with the reference gene bank sequences of p. falciparum 3d7 (cpo16995.1) and p. vivax sal-1(uo3079.1). conclusion: this study revealed a low frequency of asymptomatic malaria trend within malaria endemic areas of southeastern iran which are under intense elimination program and also the ability of hrm assay in detecting low plasmodium spp parasitemia beyond the limits of microscopy and rdts. keywords: asymptomatic malaria; high resolution melting (hrm); microscopy; rapid diagnostic test (rdts); iran introduction malaria remains an infection of global concern with almost half of the world’s population being at risk (1, 2). the disease burden remains high, in spite of 21% and 29% reported reduction in prevalence and deaths respectively between 2010 and 2015 (3, 4). the estimated global malaria cases and deaths in 2018 stood at 228 million people and 405,000 people respectively. malaria annual global funding estimate stands at us dollars 2.7 billion (4). iran, which is in malaria elimination phase, reports a low malaria prevalence (of 0.87%) with the most endemic areas (including sistan and baluchestan, kerman and hormozgan provinces) being in the south and southeastern part of the country where most cases are imported from malaria endemic neighboring countries of afghanistan and pakistan (5). the continuous exposure of human to malaria parasites in malaria endemic areas may create asymptomatic carriers which act as parasite reservoir harboring gametocyte and contribute towards malaria transmission (6). a severe malaria disease in these areas occurs most copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 354 http://jad.tums.ac.ir published online: december 31, 2020 ly in non-immune individuals such as children or visitors. asymptomatic malaria is the malaria parasitemia of any density without any ordinary symptom in an individual who has not received any recent antimalarial treatment. asymptomatic malaria results from a partial immunity which only arrests but does not eliminate the infection (7). the asymptomatic malaria cases were formerly common in high transmission settings in sub saharan africa and some regions of southeastern asia but currently have also been reported in low transmission settings of south america as well (8). these infections may be associated with chronic and debilitating episodes of symptomatic parasitemia. early detection and treatments of these cases is of paramount importance towards successful malaria elimination in endemic areas. plasmodium spp. parasitemia involved in asymptomatic malaria infection is usually low and would thus require a high sensitive diagnostic tool (7, 6). microscopy and rapid diagnostic tests (rdts) which are the commonly used methods for malaria diagnosis because of their simplicity and inexpensiveness, have a challenge of low sensitivity (9-12). molecular based techniques can overcome this challenge by not only being able to detect plasmodium spp. in lower densities but are also capable of reliably identifying and differentiating between plasmodium spp and mixed infections as well (10, 11, 13, 14). high resolution melting (hrm) assay is a post pcr molecular method of genotyping which is attached to pcr amplification. hrm assay is a high sensitive, simple, rapid and accurate method based on the generation of melt curves and is very instrumental towards diagnosing plasmodium spp. in low parasitemia based on a previously conducted study (10). the aim of this study was to detect plasmodium spp infections among non-febrile individuals living in a malaria endemic area of southeastern iran which is under intense elimination exercise using real time polymerase chain reaction coupled with high resolution melting (real time pcr-hrm) analysis. materials and methods the study settings this cross sectional study was conducted from 2016 to 2017. the study involved sample collection from sistan and baluchestan province in southeastern iran and laboratory experimental implementation in malaria referral laboratory of tehran university of medical sciences. ethical consideration the study was approved by the ethical committee of tehran university of medical sciences (approval ref. no: ir.tums.vcr.rec. 1396.3314). the importance of the study was explained to the participants in their local language. consent was obtained from all of the study participants. the parents and the guardians consented on behalf of the study participants with age below 18 years. study sites this study was conducted in sarbaz and chabahar districts of sistan and baluchestan province in southeast of iran (fig. 1). this province is the largest in iran with an area of 181,785km2 and a population of 2.775 million. the east of the province borders malaria endemic countries of afghanistan and pakistan and oman sea to its south. the province accounts for one of the driest regions of iran with a slight increase in rainfall from east to west, and an obvious rise in humidity in the coastal regions. it has hot and dry weather with about 65mm rainfall annually and accounts for the most malaria cases in iran. about 92% of malaria cases in this region are caused by p. vivax and the balance by p. falciparum. in the recent years, anopheles stephensi and an. culicifacies s.l. are the most important malaria transmission vectors in this region. the area experiences two malaria transmission peaks which are spring and autumn seasons (15, 16). the hospitals in these areas rely majorly on microscopy and rdts for malaria diagnosis. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 355 http://jad.tums.ac.ir published online: december 31, 2020 human blood sampling the sample collection was done from 271 healthy individuals (171 native iranians and 96 immigrants from pakistan and afghanistan) who were residing in the study areas for at least 6 months and had no any signs of febrile illness including fever, chills and rigor. the blood sample collection was done with strict adherence to the universal precautions for handling and disposal of human blood including: cleaning and puncturing the middle finger for preparation of thick and thin giemsa stained blood slides according to the world health organization (who) malaria microscopy standard operating procedure (17). one blood spot from every study participant was put into a dna banking card (kawsar® genomics, iran) for molecular analysis. another blood spot was put in rdt cassette (premier medical, india) and the results read immediately in the field in accordance to the who recommendations (18). the samples were then transferred by air to tehran for molecular analysis. patients who were perceived to have febrile illnesses resembling malaria infection were excluded from this study but were referred to the hospital. microscopy examination microscopic examination was done with strict adherence to world health organization (who) malaria microscopy standard operating procedure (17). the examination was done by two experienced microscopy experts who checked each duplicate of samples from each and every individual using x100 power objective power ocular lens. the slides were labelled negative if there were no any stage of plasmodium spp seen after 100 microscopic fields were checked (17). molecular analysis primer selection a pair of primers sequence used in this work was selected based on a previous study by chua et al. 2016 and was aimed at amplifying the target sequence located at 18s ssu rrna gene of plasmodium spp. the forward primer was 5’-graactsssaacggctcatt-3’ and reverse primer 5’actcgattgatacacacta 3’ (10). dna extraction dna extraction was done based on a previous study by bereczky using tris-edta buffer (19) tris-edta buffer contains 10mm tris which is a mixture of tris base and tris hcl to a ph of 8.0; and distilled water dissolved 0.1mm edta. after the preparation, the tris edta buffer stock solution was kept at a room temperature. punches from a dna banking cards were cut into smaller pieces and put in a specific eppendorf tube. 65µl of te buffer was added to the eppendorf tube containing the sample and vortexed. the samples were then put in a water bath at 50 oc for 15 minutes accompanied by gentle pressing with a fresh micropipette tips for every sample. the water bath temperature was adjusted to 95 oc with 15 minutes heating to elute the dna from the samples. the samples were then centrifuged at 13,000 rpm for 1 minute to condense the liquid on the lid and the wall of the tubes. to confirm the concentration of the dna, spectrophotometry (nanodrop 2000®, wilmington usa) was performed. the dna extracts were then stored at -20 oc until was required to be used for hrm analysis (19). the control templates the positive controls were laboratory samples from p. falciparum and p. vivax infected human patients. these patients tested positive for both the microscopy and rdts. for further scrutiny of the positive control, 30µl of their dna templates was sent for sequencing (macrogen, korea). the nucleotide sequence was then compared with the sequences in the gene bank. the negative control was distilled water. reaction mixture preparation the hrm reaction mixture was made in a final volume of 20µl. the components includhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 356 http://jad.tums.ac.ir published online: december 31, 2020 ed: 9µl of distilled water, 0.5µl of the forward primer (5’-graactsssaacggctcatt3’), 0.5µl of the reverse primer (5’-actcga ttgatacacacta-3’), 5µl of evagreen® dye and 5µl of dna template. the final volume of each sample was put in hrm strips and fixed with optical caps (gunster biotech, taiwan) and loaded into the hrm machine (step one plus tm) for analysis. high resolution melting (hrm) analysis the hrm analysis set conditions included primary denaturation at 95 oc for 5 minutes, followed by 50 cycles each of: denaturation at 95 oc for 25 seconds, annealing at 55 oc for 30 seconds, extension at 72 oc for 30 seconds and final extension at 72 oc for 5 minutes. the hrm section included temperature increase from 65 oc to 90 oc with a temperature shift interval of 0.3 oc. to check for the consistency of the result, the positive clinical samples were repeated 4 times in different times but under the same condition. the hrm curves were generated and analyzed using high resolution melt software v 3.0.1 (step one plus). results characteristics of study participants the study participants were residents of sistan and baluchestan province, southeastern of iran. 64% (n= 175) of the study participants were iranians and the balance (n= 96) were immigrants from afghanistan and pakistan. all of the study participants had no clinical symptoms of malaria or any other febrile disease that seems to resemble malaria paroxysm including fever, chills and rigor. 140 of the study participants were males and the rest (131) were females. about 46.4% of the males (n= 65) were native iranians and the rest (n= 75) were immigrants. about 83.9% of the females (n= 110) were native iranians and the balance (n= 21) were immigrants. none of the participants had either a history of malaria or has been on an antimalarial therapy within the past six months. sequence analysis for the positive control samples a multiple sequence alignment between the nucleotide sequences of experimental positive controls of p. falciparum and p. vivax (fig. 2) and the nucleotide sequences from the gene bank standards of plasmodium falciparum 3d7 (cpo16995.1) and plasmodium vivax sal-1 (uo3079.1) done using clustal w2 confirmed a close homology (99.44% and 90.37% respectively) between them as shown in the percentage identity matrix. the phylogenetic tree also confirmed a close relation (fig. 3). plasmodium spp prevalence detection using the rapid diagnostic tests (rdts), microscopy and high resolution melting analysis microscopy (through checking of 100 fields in thin blood films) and rdts did not detect plasmodium infection from 271 clinical samples (table 1). the nanodrop carried out confirmed a good concentration of dna. the hrm revealed three infections from 271 clinical samples: two p. vivax infections and one p. falciparum infection. high resolution melting analysis the hrm analysis was done using hrm melt software v 3.0.1 (step one plus) (fig. 4) various forms of curves were generated including amplification plots, derivative melt curves, and aligned melt curves for the three clinical positive plasmodium spp. based on the two positive controls. the positive clinical samples were easily identified based on the nature of the curves that aligned with the positive control curves. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 357 http://jad.tums.ac.ir published online: december 31, 2020 fig. 1. study area in sistan and baluchestan province, iran, (http://ircmj.com/168.fulltext) fig. 2. the nucleotide sequence for the experimental positive controls (a) plasmodium falciparum (mg551534) and (b) p. vivax (mg551535) fig. 3. the homology between the plasmodium falciparum and p. vivax experimental positive control samples and the p. falciparum 3d7 (cpo16995.1) and p. vivax sal-1 (uo3079.1) gene bank standards nucleotide sequences. (a) percentage identity matrix and (b) phylogenetic tree. both the identity matrix and the phylogenetic tree shows a close identity between the experimental positive controls and the standards from the gene bank http://jad.tums.ac.ir/ http://ircmj.com/168.fulltext j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 358 http://jad.tums.ac.ir published online: december 31, 2020 fig. 4. melt curves of the positive clinical samples and the experimental positive controls (a) the amplification plot, (b) derivative melt curves and (c) aligned melt curve http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 359 http://jad.tums.ac.ir published online: december 31, 2020 table 1. asymptomatic malaria cases detection results based on rapid diagnostic tests (rdts), microscopy and high resolution melting assays nationality number of study participants number of microscopy positive cases number of rdts positive cases number of hrm positive cases iranian 175 0 0 male 1 female 1 immigrants 96 0 0 male 1 female 0 total 271 0 0 3 discussion the curves for the three clinical plasmodium spp positive samples aligned to the curves of the positive control samples consistently throughout the four trials. 18s ssu rrna was selected for use in this study based on the previously molecular studies. this region is much conserved and highly variable having multiple copies which are scattered throughout the genome of plasmodium spp making it suitable for molecular amplification (10, 20). upon the sequencing of the positive control samples for this study, the nucleotide sequence of the positive controls used (p. falciparum and p. vivax) showed a close homology with the gene bank reference sequences for p. falciparum 3d7 (cpo16995.1) and p. vivax sal-1(uo3079.1). the principals behind hrm analysis towards detections and differentiation of plasmodium spp is based on the nature of melt curves and melting temperature (tm). the hrm assay set up is the same as with the rt-pcr procedure with a difference of incorporation of a fluorescent intercalating dye in hrm that is used to monitor the transition of dna from double stranded (dsdna) to single stranded (ssdna) and assigning the tm in the process. tm is a point in which dsdna is equal to ssdna, this point is very significant towards species differentiation and plasmodium spp mixed infections detection (21, 22). in this study, three individuals out of 271 non-febrile study participants (1.1 %) from the malaria zone of iran have been found to be infected with plasmodium spp. this is in a close conformity with other molecular based asymptomatic malaria surveillance findings from the same malaria endemic areas of iran: amirshakeri et al. and also turki et al. reported 0.33% and 1.5% asymptomatic malaria infections respectively from the surveys they conducted (22, 23). the parasitemia involved in these infections could neither be detected by microscopy nor rdts. however in another microscopy based asymptomatic malaria prevalence survey that has been conducted by nateghpour et al. in iranshahr district of sistan and baluchestan province, neighboring the studied districts, they reported 1.6% and 0.6% plasmodium spp prevalence among afghanistan immigrants and native iranians respectively (24). obviously, according to who guidelines the number of parasites per microliter of blood is crucial for detecting them either with microscope or rdts. the density of parasite is not less than ten and forty for microscopy and rdts respectively. so, discrepancies among results of the mentioned references may be interpreted due to the above mentioned reason. these infections if left undetected and untreated would act as reservoirs and play a crucial role towards the transmission of malaria within a population where they are prevalent and would thus consequently delay or prevent malaria control and elimination exercises in malaria endemic areas. on the other hand, some http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 353–362 ff ouma: application of high-resolution … 360 http://jad.tums.ac.ir published online: december 31, 2020 molecular based studies conducted in the southeastern malarious areas of iran resulted in absence of asymptomatic malaria (25, 26). kipanga and colleagues were able to diagnose and detect plasmodium spp from microscopically negative febrile patients by using pcr coupled with hrm from hospitals in kenya who could not be put on antimalarial drugs (12). chua et al. identified plasmodium spp in 11 out of 14 microscopically negative febrile patients using rt-pcr coupled with hrm (10). iran has achieved tremendous successes towards the elimination of malaria by using methods like: passive and active case detection, vector control methods, and effective treatment using artemisinin combination therapy (act). these strategies have reduced malaria infections in the country from 12,294 to 1,251 between 2000 and 2014 (22-24). asymptomatic malaria may however rekindle malaria infections or slow down the pace of the elimination programs being undertaken. apart from the aforesaid, hrm assay has also been successfully used in other parasitological studies including: identification of mutant strains of p. falciparum that are drug resistant; diagnosing the genetic diversity of dientamoeba fragilis isolates of irritable bowel syndrome patients, analyzing the single nucleotide polymorphism of echinococcus granulosus, detection and identification of old world leishmania, detection and identification of human hookworm infections. these studies have demonstrated a high sensitivity nature of hrm in diagnosis compared to other parasitological methods (27-31). although hrm assay is very sensitive in diagnostics, its initial cost and operational costs are more expensive than microscopy and rdts. conclusion in conclusion hrm assay is a favorable method towards the rapid detection of malaria in low parasitemia below the lod of microscopy and rdts and it is therefore important in areas where malaria elimination exercises are being carried out particularly if the malaria policy makers want to remove most hidden obstacles including asymptomatic malaria from their pathway. this method is also capable of differentiating between plasmodium spp. by just using a single pair of primer sequence making it simpler to use compared to other diagnostic methods. coming up with this kind of an assay which would incorporate dna extraction from the samples prior to their amplifications would be a great development as it would avoid the errors associated with the dna extractions that may affect the results of hrm assay. acknowledgements the authors would like to convey their appreciations to miss s askari for her useful technical collaboration and also to the study participants and their families for their willingness to partake in this study. this study was msc thesis of 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https://www.ncbi.nlm.nih.gov/pubmed/?term=tajaddini%20m%5bauthor%5d&cauthor=true&cauthor_uid=26567833 https://www.ncbi.nlm.nih.gov/pubmed/?term=tajaddini%20m%5bauthor%5d&cauthor=true&cauthor_uid=26567833 https://www.ncbi.nlm.nih.gov/pubmed/?term=rostami-nejad%20m%5bauthor%5d&cauthor=true&cauthor_uid=26567833 https://www.ncbi.nlm.nih.gov/pubmed/?term=mohtashami-pour%20m%5bauthor%5d&cauthor=true&cauthor_uid=26567833 https://www.ncbi.nlm.nih.gov/pubmed/?term=mohtashami-pour%20m%5bauthor%5d&cauthor=true&cauthor_uid=26567833 https://www.ncbi.nlm.nih.gov/pubmed/?term=pestehchian%20n%5bauthor%5d&cauthor=true&cauthor_uid=26567833 j arthropod-borne dis, june 2015, 9(1): 1–6 m nateghpour et al.: endoparasites of wild … 1 original article endoparasites of wild rodents in southeastern iran mehdi nateghpour 1, *afsaneh motevalli-haghi 1,2, kamran akbarzadeh 3, amir ahmad akhavan 3, mehdi mohebali 1, iraj mobedi 1, leila farivar 1 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2national institute of health research, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 9 dec 2012; accepted 27 apr 2014) abstract background: this study was aimed to collect wild rodents for endoparasites determination in some parts of sistan and baluchistan province, southeastern iran nearby pakistan and afghanistan countries. methods: a total of 100 wild rodents were captured alive with cage traps. various samples were collected from blood and feces, also impression smear prepared from different organs. the samples were prepared by formalin-ether or stained with giemsa, after that were examined under microscope. results: all the caught rodents (47 tatera indica, 44 meriones hurriana, 5 gerbilus nanus and 4 meriones libycus) were studied for endoparasites emphasizing to their zoonotic aspects. endoparasites including spirurida, hymenolepis diminuta, hymenolepis nana feraterna, trichuris trichiura, skerjabino taenia, trichostrongylus spp, entamoeba muris, chilomastix mesnili and leishmania spp were parasitologically identified. conclusion: among 9 genera or species of the identified parasites at least 5 of them have zoonotic and public health importance. keywords: wild rodent, endoparasite, iran introduction many infectious diseases classified as zoonotic infections can be caused by parasitic, viral and bacterial agents transmitted to human by different types of animals such as rodents (etemad 1978, weiss et al. 2008). ectoparasites such as fleas, lice and ticks can also transmit some infectious agents from rodents to human (daniels and hutchings 2001). meerburg et al. (2009) showed a large spectrum of rodent borne pathogens. helminth parasites are a large group of metazoan organisms that infect vast numbers of human and livestock (anthony 2007). similarly, some protozoan zoonoses such as toxoplasma and leishmania can also affect hu man and become malignant for those who are positive hiv (alvar et al. 2008, naqi et al. 2010). metazoan and protozoan zoonoses are responsible for a large number of morbidity and mortality of human around the world (chai et al. 2005) and induce significant public health and socioeconomic problems. this study was conducted to collect some informative data about the rodents and relevant parasites in some parts of sistan and baluchistan province in iran with emphasis on finding worms and protozoan parasites which have medical importance to human. *corresponding author: dr afsaneh motevalli-haghi, e-mail: amh.mot@gmail.com  http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 1–6 m nateghpour et al.: endoparasites of wild … 2 materials and methods study areas the study was performed at the iranshahr and nikshahr districts in sistan and baluchistan province, southeastern iran (fig. 1). the weather in the districts is hot and dry in summer with usual maximum temperature of 45 ºc but temperate and low humid in winter with usual minimum temperature of 15 ºc. six sites in each district nearby the inhabitant locations along the rodent's routes were randomly selected for trapping. rodent collection a total of 100 rodents were captured alive with cage traps. the traps were collected and transfered to iranshahr health research station. tail punctured and thin blood smears were made from all of the rodents and then the animals were anesthetized under chloroform inhalation for careful dissection and preparing impression smears of spleen, liver and lungs. one smear also was made from any papilla on the surface of ears. the smears were stained with giemsa stain diluted in natural distilled water up to 3% for 30 min. spots that prepared from dissected brains were also stained as the same method and precisely observed by light microscope with magnification of 1000. some amounts of the gastrointestinal tract content were collected for gastrointestinal parasites examination. ether-chloroform method was applied to identify parasites. both protozoan and helminthes parasites isolated as well as the rodents were distinguished by skilled persons using a number of valid references (khalil and jones 1994, david et al. 2004, jones et al. 2004, rodney et al. 2008). results four species of rodents including tatera indica (47%), meriones hurrianae (44%), gerbilus nanus (5%) and m. libycus (4%) were identified. classification of the animals was performed according to family (cricetidae), subfamily (gerbillinae), genus (tatera, meriones, gerbilus) and species. the maximum and minimum quantities belong to t. indica 47(47%) and m. libycus 4(4%) respectively (table 1). the microscopic examination of gastrointestinal tract contents revealed the presence of spirurida spp. and trichostrongylus eggs in seven and two t. indica respectively. the rest helminthes isolated including hymenolepis diminuta, h. nana feraterna, trichuris trichiura, skerjabino taenia and rictularia were found in adult stages. moreover protozoan parasites, entamoeba muris and chilomastix mesnili were isolated from the feces. in one case of liver tissue leishmania spp., was identified (table 2). table 1. distribution of wild rodents caught from iranshahr and nikshahr districts in southeastern iran genus/species iranshahr nikshahr total n(%) tatera indica 38 9 47 (47) meriones hurrianae 0 44 44 (44) gerbilus nanus 5 0 5 (5) meriones libycus 4 0 4 (4) total 47 53 100 (100) http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 1–6 m nateghpour et al.: endoparasites of wild … 3 table 2. frequency of protozoan and helminthes parasites identify captured in 100 wild nikshahr and iranshahr districts rodents parasites wild rodents total meriones libycus meriones hurrianae tatera indica gerbilus nanus n (%) hymenolepis diminuta 0 0 11 0 11 (23.4) hymenolepis nana feraterna 0 0 8 0 8 (17.0) spirurida spp. 0 0 7 0 7 (14.9) trichuris trichiura 0 0 6 0 6 (12.7) skerjabino taenia spp. 0 0 4 0 4 (8.5) trichostrongylus 0 0 2 0 2 (4.2) rictularia spp. 0 0 1 0 1 (2.1) entamoeba muris 0 2 (%4.2) 2 0 4 (8.5) chilomastix mesnili 0 3 (%6.3) 0 0 3 (6.3) leishmania spp. 0 0 1 0 1 (2.1) total 0 5 (%10.6) 42 0 47 (10) fig. 1. sistan and baluchistan province located at southeastern iran : location of the study areas in the province discussion this study was conducted to consider the endoparasites of wild rodents in some parts of southeastern iran where located nearby western borderline of pakistan and afghanistan countries. control of zoonotic parasites depends on reliable knowledge of their life-cycles, reservoirs, distribution and transmission patterns in each zoogeographical situation. many rodents particularly commensal species enable to take place in transmission cycle of parasitic infections as an important reservoir (ghadirian and arfaa 1972, sadighian et al. 1973, mohebali et al. 1998, 2004, kia et al. 2001, mowlavi et al. 2004). reports released by some authors about rodent borne parasitic infections from some parts of iran made more obvious the role of rodents as reservoir of many protozoan and helminth parasites (edrissian et al. 1975, 1976, yaghoobi-ershadi 1996, mohebali 1997, javadian et al. 1998, sadjjadi and massoud 1999 kia et al. 2001, 2010). during this study four species of rodents, m. libycus, m. hurrianae, t. indica and g. nanus were identified that the most prevalent species was t. indica with 47% (n= 47). in a zoonotic helminth study conducted in khuzestan, a province in southwestern iran, t. indica was found as the most dominant species (sadjjadi and massoud 1999). while in another study performed by kia et al. (2001) rattus norvegicus was the most prevalent rodent in ahvaz, centre of khuzestan province. in the present study the samples were collected from rural areas so our results are comparable with those studies that emhttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 1–6 m nateghpour et al.: endoparasites of wild … 4 phasis gerbils are dominant species in the rural areas, while in the urban areas rattus can be usually found more than gerbils. in kamranrashani et al. study in maraveh tappeh, golestan province located in northeast of iran a heavy burden of infectivity with helminth parasites (81.8%) was found in r. opimus (kamranrashani et al. 2012). in our study m. hurrianae (n= 44, 44%), g. nanus (n= 5, 5%) and m. libycus (n= 4, 4%) stayed at the second to fourth ranks respectively after t. indica. tatera indica bore the maximum parasitic infectivity and none of the parasites was isolated from m. libycus and g. nanus (table 2). in a previous study performed in the leishmaniasis endemic areas of iran t. indica was accounted the main leishmania major reservoir in mehran district of iran where located nearby eastern borderline of iraq (mohebali et al. 2004). some promastigotes of leishmania spp. were isolated from phlebotomous papatasi and ph. salehi sand flies those were collected from t. indica and m. hurrianae burrows in chabahar district located at the south of iranshahr district (kasiri and javadian 2000). in our study leishmania spp. was isolated from t. indica which is consistent their results. in addition, examination of gastrointestinal content of this rodent showed infection with entamoeba muris. although e. muris is assumed to be a common protozoan parasite of the most rodents, results of this study recorded the parasite only for t. indica and m. hurrianae with equal burden of infection. chilomastix mesnili as the third protozoan parasitic infection in this study was found among three m. hurrianae. although c. mesnili has been isolated from a few infected individuals, it is a common parasite among rodents and usually none pathogen for human. a scientific report indicated that 0.6% of adolescent girls from two boarding schools, in southern benin were carrier of c. mesnili at the time of study (alaofe et al. 2008). all the identified helminth parasites were isolated only from t. indica in this study. these results pointed to the more activity and prevalence of t. indica and also capability of the rodent for assuming a wide variety of parasitic infections in comparison with the other captured rodents in the studied areas. among the parasites isolated from rodents in this study, hymenolepis diminuta, the rat tapeworm, was the most prevalent helminth species. in kia et al. study (2010) h. diminuta was the most common parasite that could be found in different species of rodents. some helminthes such as trichuris trichiura, h. feraterna, skerjabino taenia, trichostrongylus spp., spirurida and rictularia spp. are infective to human and deleterious for public health (keney et al. 1975, mowlavi et al. 2006, 2008, ok 2009). infectivity of h. diminuta for human had been distinguished from long time ago in iran (ghadirian and arfaa 1972). among eight t. indica that were infected with order of spirurida one of them was identified as adult rictularia spp. infection but the others remained at the level of order because the infectivities were distinguished only with presence of the eggs. some studies performed in khuzestan province indicated presence of rictularia spp. and gongylonema spp. that were isolated from a number of wild rodents and carnivores (farahnak1998, sadjjadi and massoud 1999, kia et al. 2001). spirurida includes a great number of genera and species which all have invertebrate intermediate hosts. both rictularia and gongylonema have been isolated from human (urch et al. 2005, keney et al. 1975). to our knowledge among nine genera or species of the identified parasites at least five of them have zoonotic and public health importance. conclusion harboring a wide variety of zoonotic parasites by t. indica particularly when the rohttp://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 1–6 m nateghpour et al.: endoparasites of wild … 5 dent lives nearby the native population residences represents a potential risk to the health of the population. although the infection of m. hurrianae was not comparable with t. indica, nevertheless the rodent can be accounted at the second potential risk to the health of human at the studied areas. acknowledgements the authors would like to thank prof m rezaeian, dr m kazemi, dr g mowlavi, mr a shahbakhsh, mr a amiri, mrs e ghodratimoghaddam, mrs f tarighei, mr f damani and mr r sheikh for their useful cooperation. this study was financially supported by national institute of health research, tehran university of medical sciences. the authors declare that there is no 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carnivores and rodents in isfahan, iran. j parasitol. 59 (6): 986. sadjjadi sm, massoud j (1999) helminth parasites of wild rodents in khuzestan province, south west of iran. j vet parasitol. 13(1): 55–56. urch t, albrecht bc, büttner dw, tannich e (2005) human infection with gongylonema pulchrum. dtsch med wochenschr. 130(45): 2566–2568. weiss lm (2008) zoonotic parasitic diseases: emerging issues and problems. int j parasitol. 38(11): 1209–1210. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996) meriones libycus and rhombomys opimus are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans roy soc trop med hyg. 90: 503–504. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 387 http://jad.tums.ac.ir published online: december 25, 2018 original article chemical composition and repellency of origanum vulgare essential oil against cimex lectularius under laboratory conditions mona sharififard 1, 2, ismaeil alizadeh 1, 2, *elham jahanifard 1, 2, changlu wang 3, mohammad ebrahim azemi 4 1infectious and tropical diseases research center, health research institute, ahvaz jundishapur university of medical sciences, ahvaz, iran 2department of medical entomology and vector control, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran 3department of entomology, rutgers university, new brunswick, nj, usa 4department of pharmacognosy, medicinal plant and natural product, research center, school of pharmacy, jundishapur university of medical sciences, ahvaz, iran (received 16 sep 2017; accepted 27 june 2018) abstract background: the common bed bug (cimex lectularius), a nocturnal blood-feeding ectoparasite, is considered an urban pest with public health importance in human environments. we aimed to determine the repellency effect of oregano essential oil, origanum vulgare, against this pest under laboratory conditions. methods: the essential oil was prepared from dried leaves using hydro-distillation method. a gas chromatographymass spectrometer (gc-ms) was used for analysis and identification of oregano essential oil compounds. treated surface method in petri dish was carried out to evaluate repellency potential of the oil using 4th and 5th instar nymphs and adults. the concentration-repellency response of oregano essential oil was calculated and compared with a commercial insect repellent stick containing 33% n, n-diethyl-meta-toluamide (deet). results: the oregano essential oil consisted of 158 compounds with terpineol (22.85%) and α–terpinene (20.60%) being the major components by volume. the ec50 and ec99 of oregano oil (effective concentrations causing 50% and 90% repellency of bed bugs) were 1.61 and 6.57mg/cm2 at 9h after application, respectively. the 40% oregano essential oil exhibited 100% repellency against bed bug at 3, 5, 9 and 24h after application while the repellency index of deet 33% was 100% at 3 and 5h and it decreased to 80% and 27% at 9 and 24 hours. conclusion: oregano oil 40% exhibited more repellency compared to commercial insect repellent stick containing 33% deet. further studies are warranted to confirm the effectiveness of oregano essential oil in personal protecting against bed bug biting. keywords: cimex lectularius, essential oil, origanum vulgare, repellency introduction bed bug (cimex lectularius linnaeus), is a nocturnal blood-feeding ectoparasite considered as urban, common and nuisance pest in the recent past (1, 2). this insect affects everyone in different social status and infests everywhere. the case of infestation has been reported from countries including united kingdom, denmark, norway, italy, spain, sweden, scandinavia, switzerland, australia, brazil, iran, thailand, malaysia, singapore, kuwait, nigeria and the united states (2–11). the bed bug infestation became rare with the new pest control techniques in north america and europe a century ago (12), but the united states has significant resurgence as a perfect storm in 2006 (13). the global resurgence of bed bug infestations was related to increased levels of international transport through tourism and *corresponding author: dr elham jahanifard, e-mail: jahanifard-e@ajums.ac.ir, elham.jahani56@gmail.com mailto:jahanifard-e@ajums.ac.ir j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 388 http://jad.tums.ac.ir published online: december 25, 2018 trade, immigration insecticide resistance and possibly increased temperatures (14). besides pain and itchiness, the problems caused by bed bug infestations include psychological distress with nightmares, insomnia, anxiety, social isolation, and quality of life (15). bed bugs hide in protected places such as crevices on beds and other furniture, inside devices, inside walls, floor cracks. this partially explains why they are difficult to detect and control (16). surface treatment with pyrethroids and other classes of insecticides, physical control, and personal protection are the main recommendations for the control and prevention of bed bugs infestation (1, 17, 18). application of insecticides poses an immediate risk to human health and the environment especially when insecticides are applied on furniture (17). personal protection using repellent compounds on human skin is an effective and in some cases the only practical approach to the control of biting insects. the most common synthetic repellent used on blood-sucking arthropods is n, n-diethyl-meta-toluamide (deet) produced in 1954 (19, 20). due to reports of allergic and toxic effects especially on children and pregnant women from deet, it is placed under investigation (21– 23). therefore, finding a safer repellent compound for use on human body surface is necessary. botanical essential oils are regarded as environmentally friendly products, ecological alternatives and low mammalian toxicity materials (24). however, some plant-based repellents should be used with caution due to their compounds (25). essential oils have been introduced as green pesticides and most of them are non-toxic to humans, animals and are safe and friendly to the environment (26). these products are good alternatives to synthetic insecticides and can delay the development of resistance to insect pests (27). essential oils (eo) are secondary metabolites of plants. they are complex mixtures of volatile organic compounds such as hydrocarbons and oxygenates (28, 29). today, many essential oil products have been developed and are effective as repellents or surface spraying compounds on pest of medical important (17, 30). essential oils have various activities against insect pests, including insecticidal, antifeedant, repellent, oviposition, deterrent and growth regulatory (30, 31). repellent activity of essential oils is an effective way of controlling biting insects including bed bugs and they can be applied on luggage, fabric materials and furniture in order to reduce bed bug infestation by preventing bed bugs into their home (27). oregano oil is considered as an excellent antiseptic and insect repellent. it has some active ingredients such as carvacrol, thymol and α-terpinene reported being highly effective in repelling mosquitoes (32, 33). moreover, it showed significant repel activity at concentration range of 2.5–30% against supella longipalpa (34). the aim of this study was to determine the repellency activity of oregano essential oil (o. vulgare) on bed bugs in comparison with deet against bed bugs (c. lectularius). materials and methods bed bugs the adult and nymph stages of c. lectularius were collected from infested homes in ahvaz city, southern iran and transferred to the laboratory of medical entomology department, school of public health, ahvaz, jundishapur university of medical sciences, ahvaz, iran. the insects were reared in plastic containers (12cm in height and 6cm in diameter) with folded papers as harborage and kept at 26±1 °c, 50±5% relative humidity (rh), photoperiod of 12:12 (l: d) (17, 27). the colony of c. lectularius were fed weekly on rabbits and allowed to suck blood for 10min (2). the bed bugs were starved for 7 d before bioassays. j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 389 http://jad.tums.ac.ir published online: december 25, 2018 chemical compounds insect repellent stick (33% deet) as commercial formulation was purchased from reyhan naghsh jahan pharmaceutical cosmetic and hygienic company. it is used widely in iran on blood-sucking insects such as mosquitoes, sand flies, ticks and bed bugs. essential oil isolation oregano plant (o. vulgare) was collected from its natural habitat from yazd province, central part of iran (latitudes 54.20257 and longitudes 32. 00315). collected oregano specimens were identified by the department of pharmacognosy, ahvaz jundishapur university of medical sciences. firstly, fresh leaves dried with air, and next 200gr of oregano was mixed with 400ml of distilled water and placed in a 1l flask. in the following the essential oil oregano used in the assay was isolated from dried fresh leaves by the hydrodistillation method using a clevenger-type apparatus (model bp, british pharmacopoeia, manufacturer pyrex fan company, iran and mantle model h610 manufacturer fater company, iran) at 90±5 for 5 hours. we extracted 0.9cc pure (about 100%) essential oil of 100gr of dried leaves of o. vulgare. sodium sulfate was used for dehydration. the extracted essential oils were stored at 4 °c in dark glass vials for further experiment (35). gas chromatographic-mass spectral analysis gas-chromatography-mass spectrometer (gc-ms) was used for the analysis and identification of oregano essential oil compounds (hewlett-packard 6890, agilent technology, santa clara, california, usa). it is equipped with hp–5ms column (30m× 0.25mm× 0.25 µm). the initial temperature used was 40 °c for 1min and was later raised to 220 °c at a rate of 3 °c/min and finally raised to 270 °c for 5min at a rate of 20 °c/min. other parameters of the gc-mc machine included carrier gas helium (99/999%), injector temperature (260 °c), detector temperature (fid, 270 °c), split-less mode, ionization potential of 70ev, scan rate of 1 scan/sec, the scan range of m/z 40–48 was used for all analysis. the essential oil constituents were identified by comparing their retention indices, mass spectra fragmentation with those in a stored wiley 7n.1 mass computer library and those of national institute of standards and technology (nist) (36). petri dish repellency assay oregano essential oil at concentrations of 0.625, 1.25, 2.5, 5, 10, 20 and 40% (v/v) were evaluated against the laboratory-reared bed bugs in plastic petri dishes and compared with deet (33%). moreover, the concentrations were calculated as doses of 0.1, 0.21, 0.43, 0.86, 1.72, 3.45, 6.9mg/cm2 for drawing doseresponse curve. plastic petri dishes of 8cm diameter by 1.5cm height were used in this experiment. ethanol was used as solvent for essential oil. white filter papers were divided into two equal halves and inserted into the bottom part of the petri dishes. a piece of folded filter paper was placed in the middle as bed bug harborage (fig. 1). this part (bed bug harborage) was treated with 0.7ml of essential oil solution using a micro-sampler and the other half was left untreated (27). in the control (negative and positive) plastic petri dishes group, one half of the filter paper and the harborage were treated with 0.7ml of 96% ethanol or 33% deet, and the other half was left untreated. ten-bed bugs (4th and 5th instars, nymphs and adults) for each treatment group were released into the center of each petri dish (fig. 1). the number of bed bugs on each side of the petri dish was recorded at 3, 5, 9, and 24h post treatment by visual inspection. the experiments were replicated four times for any concentrations in oregano essential oil, ethanol, and 33% deet. all petri dishes were kept at 26±2 °c, photoperiod of 12:12 (l: d) and 55±5% relative humidity (rh). j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 390 http://jad.tums.ac.ir published online: december 25, 2018 statistical analysis probit analysis was used to calculate the effective concentration (ec50 and ec99). repellency indices were calculated using: repellency =(c–t)/c×100, where c is the mean number of bed bugs located on the treated filter paper in all control plastic petri dishes, and t is the number of bed bugs located in half part of the treated filter paper at unfixed concentration test in the plastic petri dishes (27). repellency indices were compared using analysis of (anova) followed by dunnett test to distinguish between the treatments. all statistical analyses were performed using spss ver. 16 (chicago, il, usa). results yields and chemical constituents of essential oil the content of essential oil was obtained from 0.8 to 1ml per 100gr of dried leaves and the density of the essential oil was calculated as d eo= 0.94g ml -1. oregano essential oil was found to contain 158 compounds using gcms. the most common compounds are shown in table 1 and fig. 2. in addition, chemical analysis of essential oil of o. vulgare is shown in fig. 3. dose-response of essential oil the results of dose-response test are showed with the calculation of repelling effective concentration as mg essential oil per cm2 (ec50 and ec99) in table 2. the dose-response data was calculated from repels effective concentration of essential oil in mg per cm2 of the surface. the data obtained from 9h exposure time was important because this time represents the typical time period when people are in bed and exposure to common bed bug biting. they were 1.61 and 6.57mg/cm2 for ec50 and ec99 at 9h after exposure, respectively with slope (±se) = 0.469±0.073 and chisquare (df)= 17.17 and p< 0.904. calculated dose-response curve for oregano essential oil after 9h is shown in fig. 4. bed bug repellency activity of essential oil repellency indices of oregano essential oil and 33% deet on c. lectularius are shown in table 3. the repellency means are noticeable even in very low concentrations at 3h after exposure. the dunnett test showed, there was no significant difference in repellency between 33% deet and oregano essential oil in the concentrations of 10, 20 and 40% (p= 0.4). after 5h, the repellent activity of oregano oil was similar to 33% deet at the concentrations of 10% to 40% and it varied between 86% and 100%. the repellency of oregano oil at the concentrations of 10% and 20% was still obvious (86–93% repellency). significant difference in repellency was observed between 0.625%, 1.25%, and 2.5% concentrations of oregano essential oil and 33% deet after 3, 5 and 24h (p< 0.001), while the differences in repellency of 5%, 10%, 20%, and 40% oregano essential oil with 33% deet were not significant after 3 and 5h (p= 0.25). the concentration of 40% oregano essential oil had 100% repellency against bed bugs at 3, 5 and 24h post exposure, while the repellency index of 33% deet decreased to 27% after 24h (table 3). the repellency of oregano essential oil at concentrations of 0.625– 20% significantly declined from 3h to 24h, but it was constant at a concentration of 40% and exhibited similar repellency to 33% deet at a concentration of 20%. at 9h after exposure, significant differences were observed between repellency of 0.625%, 1.25%, 2.5%, and 5% concentrations of oregano essential oil and 33% deet (p< 0.001), while the differences between 10%, 20%, and 40% oregano essential oil and 33% deet were not significant (p=0.21) (fig. 5). j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 391 http://jad.tums.ac.ir published online: december 25, 2018 table 1. constituents of oregano essential oil by gc-ms analyses compound ri * major constituents )%( pinene 939 1.15 γ-terpinene 1056 0.14 α – terpinene 1016 20.60 α – terpinolene 1186 2.16 terpineol 1192 22.85 thymol 1290 4.53 carvacrol 1299 4.9 caryophyllene 1419 6.75 other compounds 36.92 *ri, retention index as determined on an hp-5ms column using the homologous series of n-hydrocarbons table 2. effective doses of oregano essential oil against cimex lectularius by treated surface method in petri dishes time ec50 (cl*) mg/cm2 ec99 (cl*) /cm2 slope (±se) chi-square ** (df) p value after 3h 0.27(0.05-0.42) 1.96 (1.52-2.97) 1.377 (±0.279) 21.57 (26) 0.712 after 5h 0.87(0.57-1.16) 4.10 (3.31-5.55) 0.720 (±0.112) 31.70 (26) 0.203 after 9h 1.61(1.18-2.09) 6.57 (5.29-8.92) 0.469 (±0.073) 17.17(26) 0.904 after 24h 4.53 (***) 6.10 (***) 1.19 (±0.877) 12.70 (26) 0.986 ec50 and ec99: effective concentration cause 50% and 99% repellency against bed bug. *cl: confidence limits. **since chi-square goodness of fit test is not significant (p> 0.05), no heterogeneity factor is used in the calculation of confidence limits. ***probit model did not work because <25% repellency occurred. table 3. repellency index of essential oil, ethanol and 33% deet against bed bugs treatment concentration (%) repellency index (mean%±se) 3h 5h 9h 24h 0.625 20±0.88a 14±1.15a 13±1a 3±0.33a 1.25 32±0.57ab 27±1.33ab 27±0.33a 7±0.57a oregano essential oil 2.5 44±0.88bc 39±0.33bc 34±0.88ab 10±0.33a 5 68±0.33cd 61±0.33cd 42±0.57abc 14±0.33ab 10 100±0.0d 86±0.33d 65±0.57bcd 17±0.57b 20 100±0.0 d 93±0.66d 73±0.33c 25±0.57b 40 100±0.0 d 100±0.0d 100±0.0d 100±0.0c 33% deet 33 100±0.0 d 100±0.0d 100±0.0d 27±0.57b ethanol 96 values in columns followed by different letters are significantly different (p< 0.05) j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 392 http://jad.tums.ac.ir published online: december 25, 2018 fig. 1. petri dish repellency assay of oregano essential oil, ethanol and 33% deet on bed bugs fig. 2. dose-response curve for oregano essential oil after 9 hour in laboratory assay fig. 3. chemical structures of major compounds of the essential oil of origanum vulgare j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 393 http://jad.tums.ac.ir published online: december 25, 2018 fig. 4. a typical gc-ms chromatogram showing the chemical analysis of essential oil from origanum vulgare fig. 5. repellency of ethanol, different concentrations of oregano essential oil and 33% deet against bed bugs in petri dish assays. different letters above the bars indicate significant differences at α= 0.05 discussion in this study, terpineol (22.85%) was the most abundant chemical component of oregano essential oil followed by α-terpinene with 20.6% (v/v). the component of α-terpinene in o. vulgare collected from mazandaran was 1.018% (38). the plant phenology and composition was likely affected by ecologic con ditions. the percentage of carvacrol obtained 4.9% in the present investigation. the percentage of carvacrol, collected from various localities in west azarbaijan province of iran were reported in the range of 23.54% to 67.09% (39). the chemical composition of oregano oil changed due to plants’ development, the popuj arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 394 http://jad.tums.ac.ir published online: december 25, 2018 lation or variation, and the cultivation conditions. the content of carvacrol depends on climatic condition and also it is can be variable in budding period and in full bloom (40). in addition, difference in the chemical composition of oregano oil from previously published papers with our study may is regarding the type of oregano subspecies. two percent of α-terpinene, monoterpenes derived from thymus vulgaris were reported with stronger repellency activity than deet against culex pipiens (33). in our findings, oregano essential oil showed more potential as repellent compound than 33% deet, a commercial product, against bed bugs. the high concentration α-terpinene may be responsible for the oil’s repellency. the aim of using repellant compound towards hematophagous pest is to reduce the frequency of biting in endemic areas and prevent bites in areas where allergic reactions to biting insects are common (41). very few studies have been conducted to evaluate insect repellent activities of essential oils against bed bugs (17, 27). more tolerance of bed bugs against insect repellents were compared with other blood-sucking insects (27). according to the petri dish repellency assay, the oregano essential oil of 2.5% showed 10% repellency after 24h. while the same concentration of oil demonstrated 99.1% repellent activity on the brown-banded cockroach (supella longipalpa) after the same time (34). the sub-lethal concentrations (lc10 and lc30) of oregano essential oil had an average repellency of 22.8% and 49.8% against the diamondback moth (plutella xylustella) respectively (42). insect species, method of test, formulation, and concentrations of the oil were responsible for the different results observed in the reported investigations. however the repellency indices were different between selected concentrations of oregano oil and there was no significant difference between efficacy of deet (33%) with 20% oregano oil at 3, 5, 9h post exposure. no repel activity was observed in the control group of our study and all the bed bugs stayed under harborage at 3, 5, and 9h post ethanol exposure. our investigation showed that repellency of 40% oregano oil was 100% against the common bed bugs in petri dish test. it repellency index was similar to 33% deet at 3 and 5h and it was more effective than 33% deet at 9h after application. the 40% concentration of oregano oil was found to be safer for application in human environments and also for personal protection. while ecoraider (1% geraniol, 1% cedar extract, and 2% sodium lauryl sulfate) and bed bug patrol (0.003% clove oil, 1% peppermint oil, and 1.3% sodium lauryl sulfate) as botanical repellents did not exhibit detectable repellency against bed bug in the presence of carbon dioxide source (17). deet, picaridin, tea tree oil, peppermint oil, and citronella oil were tested for repellency activity on triathoma rubida using small restrained rats. no long-range repellency was observed. only citronella oil was able to stop all probing and feeding of t. rubida and appears to be a promising potential repellent to prevent sleeping people from being bitten by kissing bugs (41). the efficacy of any pesticide or repellent compound can vary with the testing method, rate of application, bed bug strain and physiological stage (17). conclusion preferably, the ec99 dose should be estimated to determine the protection time against blood-sucking insects. 6.57mg/cm2 had 99% repellency against c. lectularius in treated surface after 9 hours. further studies are recommended to determine the protection time and repellency of oregano essential oil using ec99 on animal and human hosts. acknowledgements the authors wish to thank dr mohammad j arthropod-borne dis, december 2018, 12(4): 387–397 m sharififard et al.: chemical composition … 395 http://jad.tums.ac.ir published online: december 25, 2018 mahmoodi sourestani for helping in interpretation of gc-ms results. this article is part of ismaeil alizadeh msph thesis and it was financially supported by and conducted in infectious and tropical diseases research center, health research institute, ahvaz jundishapur university of medical sciences (ajums). 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(lepidoptera: pyralidae). j saudi soci agric sci. 16: 184–190. j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 243 http://jad.tums.ac.ir published online: sep 30, 2022 original article molecular identification of leishmania infantum kdna in naturally infected dogs and their fleas in an endemic focus of canine visceral leishmaniasis in iran amrollah azarm1, *abdolhossin dalimi1, mehdi mohebali2, anita mohammadiha1, majid pirestani1, zabihollah zarei2, *alireza zahraei-ramazani3 1department of medical parasitology and entomology, faculty of medical sciences of tarbit modares university, tehran, iran 2department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran *corresponding authors: dr alireza zahraei-ramazani, e-mail: azahraei@tums.ac.ir, dr abdolhossin dalimi, e-mail: dalimi_a@modares.ac.ir (received 24 june 2020; accepted 09 aug 2022) abstract background: fleas (insecta: siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. the existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mecha nism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. the aim of this study was to assess leishmania infantum natural infection of dog fleas in northwest iran in 2018. methods: a total of 20 infested domestic dogs (canis lupus familiaris) were randomly selected from 5 villages. fleas were collected using brushing against dog hairs and fine forceps. then, they were morphologically identified and preserved in ethanol for molecular assay. the kinetoplast dna of the parasite was used for detection of leishmania infantum using a semi-nested polymerase chain reaction (pcr) assay. results: the human flea, pulex irritans, and the cat flea, ctenocephalides felis were identified on 40% and 35% of dogs, respectively. the results of pcr indicated that l. infantum was found in the ctenocephalides canis (75%) and c. felis (66.7%) collected from infected dogs. no leishmanial infection was observed in p. irritans. conclusion: it is concluded that fleas could be infected by leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined. keywords: ctenocephalides canis; ctenocephalides felis; leishmania infantum; pulex irritans introduction fleas (insecta: siphonaptera) are considered as highly specialized bloodsucking on mammals such as humans, livestock, dog, cat, rabbit, squirrels, rats, and mice (1). they transmit agents of some diseases such as bubonic plague, murine typhus, tularemia, and listeriosis (2). one of the most important families of fleas that transmit dangerous diseases to humans is pulicidae. ctenocephalides canis, c. felis, pulex irritans, xenopsylla cheopis, and x. austria, are species of this family (3). several studies have shown that already various species of fleas have been reported from dogs and in most of these studies, three species c. canis, c. felis, and p. irritans have been observed (4). ctenocephalides canis and c. felis are the known vector of many pathogens of dogs that some of them have a zoonotic role (5). due to the biological flexibility of c. canis, c. felis, and the global distribution of dogs and cats, these species are copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:azahraei@tums.ac.ir mailto:dalimi_a@modares.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 244 http://jad.tums.ac.ir published online: sep 30, 2022 the most common flea in the world. in addition, these insects can transfer a great variety of bacterial, viral, and fungal diseases from dogs to humans and other hosts (5). leishmaniasis is endemic in many parts of the world and considered as a major public health problem. there are two forms of this disease in iran: cutaneous and visceral leishmaniasis. visceral leishmaniasis is a life-threatening disease caused by leishmania infantum and is one of the most important diseases transmitted by vectors. leishmania parasites are transmitted by sand flies infected with the parasite. in the old world, phlebotomus (diptera: psychodidae) is the only known biological vector of leishmania spp. parasites (6). in the mediterranean area, canidae has a definite role in the transmission of visceral leishmaniasis (6). previous studies have shown that even if there are no phlebotomus, healthy dogs may receive leishmania parasites from infected dogs. it shows that other species of arthropods such as fleas, ticks, mites, and lice may be involved (7). fleas can receive many types of microorganisms during blood-feeding. this study was focused on the fleas gathered from healthy dogs and those infected with leishmania parasites and assessing natural infection of dogs to leishmania spp. and their fleas in meshkinshahr county in ardabil province. materials and methods study area meshkin-shahr county locates in the ardabil province in the northwest of iran (fig. 1). meshkin-shahr county with geographical coordinates 38° 44’ n and 47° 40’ e and an altitude of 4811 meters above sea level (8). sample population in this study, a population of 20 dogs and their fleas were examined to detect leishmania parasites. the dogs were collected from five different villages, named ahmad abad, kojanagh, parikhan, ur kandi, and sarikhanlou in meshkin-shahr county, ardabil province in 2018. the dogs’ age range was between 1 and 5 years old, which was estimated by the teeth. blood collection and dat examination blood samples from dogs were prepared. a blood sample (8ml) was collected from the saphenous vein of each dog. then, each blood sample was divided into two parts. one part was used for direct agglutination test (dat) and another part was transferred to the laboratory for molecular tests. blood samples were stored at -20 °c until tested. the collected blood samples were centrifuged at 800g for 5–10 min and the sera were separated and stored at -20 °c until examined by dat. afterward, samples were tested according to the protocol described by harith et al. (9). collection and morphological identification of fleas all fleas were collected from the body of dogs using brushing against their hair. in some cases, fleas were collected by fine forceps. then, the collected specimens from each dog were finally conserved in 70% ethanol and identified by the standard keys. at the laboratory, fleas were cleaned by water and immersed in lactophenol for 1 hour. finally, temporary mounts were prepared with lactophenol to identify the collected fleas. after mounting, the slides of fleas were evaluated using a light microscope (×400) and identified by the standard keys (10). molecular study in this study, the method of ish-horowitz was used to extract the dna of l. infantum from fleas (11). also, dna of parasites was extracted from blood samples using the genomic dna extraction kit (bioneer) and stored at -20 ºc for later use. for the detection and identification of species of leishmania parasites, the sequence of kinetoplast dna (kdna) was used. a semi-nested pcr assay was carried out to detect the leishmania infection of http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 245 http://jad.tums.ac.ir published online: sep 30, 2022 blood samples and collected fleas from healthy and infected dogs. the primers introduced by aransay et al. (2000) were applied for the detection of leishmanial infection of blood samples and fleas. the sequences of the primers were linr4 (5'ggg gtt ggt gta aaa tag gg -3') for forward and lin17 (5'ttt gaa cgg gatttctg -3') and lin19 (5' cagaacgcccctacccg -3') for reverse used in a semi-nested pcr technique (12). in the semi-nested pcr assay, the kdna was amplified using lin primers (length 720bp). the pcr test was performed individually for each species of fleas. in the first round of the pcr, 8µl of the master mix, 1µl of each primer (linr4, lin17), 2µl of sterile distilled water, and 3µl of dna were used. the amplification conditions were 94 ºc for 5min, followed by 30 cycles of denaturation at 94 ºc for 30sec; annealing at 52 ºc for 3sec and extension at 72 ºc for 40sec with a final extension step at 72 ºc for 5min. the second round of the pcr was performed with 8µl of the master mix, 1µl of each primer (linr4, lin19), 3µl of sterile distilled water, 2µl of the product of the first round of the pcr, in 33 cycles (94 ºc for 30sec, 58 ºc for 30sec and 72 ºc for 1min) and a final extension at 72 ºc for 10min. pcr products were visualized by uv rays in a 1.5 % agarose gel after electrophoresis with a safe stain solution. results twenty dogs, including seven females (35%) and 13 males (65%) were examined. using dat (8), a high level (titer) of antibody (above 1/ 320) was observed in 12 out of 20 dogs and eight dogs did not have a high level (titers) of antibody. a total of 974 fleas belonging to the genus ctenocephalides and pulex were collected from dogs in different localities. three species c. canis, c. felis, and p. irritans were identified which c. canis was the most abundant (95.48%) followed by p. irritans (3.28 %) and c. felis (1.23%). the dog flea, c. canis was the most common flea on all dogs (100%), and p. irritans and c. felis were found on 8 (40%) and 7(35%) of dogs respectively (fig. 2, table 1). out of 12 types of a blood sample which were positive by dat, 10 samples were positive using semi-nested pcr method (fig. 3). the molecular test was done on 192 c. canis, 12 c. felis, and 18 p. irritans that had been collected from infected dogs to identify leishmania parasite. 160 (83.3%) of c. canis (fig. 4) and 9(75%) of c. felis (fig. 5) were infected with l. infantum and no infection was observed in p. irritans. by semi-nested pcr, it was found that the collected fleas from healthy dogs are not infected with l. infantum (table 1). table 1. the number of collected fleas from healthy and infected dogs and the results of leishmania infantum molecular tests in fleas collected from dogs using the pcr method, meshkin-shahr county, ardabil province in 2018 species the number of collected fleas the number of tested fleas fleas infection with leishmania infantum the infected fleas with healthy host the infected fleas with infected host canis lupus familiaris healthy dog infected dog healthy dog infected dog number % number % ctenocephalides canis 72 858 72 192 0 0 160 83.4 ctenocephalides felis 0 12 0 12 0 0 9 75 pulex irritans 10 22 10 18 0 0 0 0 http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 246 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 1. location of meshkin-shahr county in ardabil province, iran, 2016 fig. 2. a: adult of ctenocephalides felis; 1 shape of the head, 2 length of the first spine of the genal comb, and 3 one short, stout bristle in the interval between the postmedian and long apical bristles of the dorsal margin of the hind tibia, b: adult of ctenocephalides canis; 1 shape of the head, 2 length of the first spine of the genal comb, 3 two short, stout bristles in the interval between the postmedian and long apical bristles of the dorsal margin of the hind tibia, c: adult of pulex irritans; 1 eye hair in this type is under the eye, 2 the borderline of moral rood does not exist in this type, 3 the first part of spermatheca not distinguishing, 4 the picture of the ending part in the male gender http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 247 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 3. agarose gel (1.5%) electrophoresis of semi-nested pcr products (720bp) of dna extracted from blood samples of leishmania-free and infected dogs with l.eishmania infantum. lad: a 100bp ladder (sinclon, iran); lanes 1–6, 8–10, and 12: pcr products of infected blood; lanes 7 and 11: pcr products of leishmania-free blood samples; neg: a negative control free of dna template and lane +: positive control for l. infantum fig. 4. agarose gel (1.5%) electrophoresis of semi-nested pcr products (720bp) of dna extracted from ctenocephalides canis collected from leishmania-free and infected dogs with leishmania infantum. lad: a 100bp ladder (sinclon, iran); lanes 1–4, 6–9 and 11: pcr products of infected fleas; lanes 5, 10, and 12: pcr products of leishmania-free fleas; neg: a negative control free of dna template and lane +: positive control for l. infantum fig. 5. agarose gel (1.5%) electrophoresis of semi-nested pcr products (720bp) of dna extracted from ctenocephalides felis collected from infected dogs with leishmania infantum. lad: a 100bp ladder (sinclon, iran); neg: a negative control free of dna template; lanes 2, 4, 5, and 8: pcr products of infected fleas; lanes 1, 3, and 7: pcr products of leishmania-free fleas and lane +: positive control for l. infantum http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 248 http://jad.tums.ac.ir published online: sep 30, 2022 discussion fleas are one of the most important arthropods in transmitting many diseases. there are many reasons that fleas may have a role in the transmission of infectious pathogens. some of them include a long time of blood-feeding, the mode of obtaining blood, digestion, and contact with the host, and the frequency of host exchange. in the present study, 12 out of 20 (60%) dogs were infected with l. infantum using dat. but pcr assay confirmed leishmanial infection in 10 out of them (fig. 2). three species of fleas collected including c. canis, c. felis, and p. irritans. all the caught dogs (100%) were infested with 974 fleas. the fleas are the most common ectoparasites in domestic dogs of the meshkin-shahr area. in recent studies, the probable role of fleas in the transmission of leishmania parasites has been discussed. in our study, l. infantum was observed in 9/12 (75%) of c. canis (fig. 3) and 4/6 (66.67%) of c. felis (fig. 4) using semi-nested pcr and p. irritans fleas did not infect with the parasite. also, no infection was observed in fleas collected from leishmania-free dogs by pcr assay. lainson and shaw (1985) isolated agent of visceral leishmaniasis from 8 lutzomyia longipalpis and 3 lutzomyia antunesi on the island of maraio (13). there are sporadic cases of visceral leishmaniasis on this island (13). in another study conducted, kdna of l. infantum was isolated from salivary glands of collected ticks (rhipicephalus sanguineus) from infected dogs in southern italy. in this study dantas-torres et al. performed by pcr technique, kdna of l. infantum in the larvae of r. sanguinus tick was isolated after 4 months of experimental infection, which represents the possibility of transovarial transmission of leishmania parasites in the ticks (14). dabaghmanesh et al. (2016) collected a total of 180 leishmania-free ticks collected from fields and bred on lab rodents, were divided into eight groups, and allowed to feed on a dog for fixed periods of time. the infection rate was significantly higher in female than male ticks. the rates were higher among nymphs than adult ticks. the kdna of l. infantum was not detected in ticks 24h post-feeding. it was, however, positive among the second to fourth groups of nymphs and adult ticks. eggs and unfed larvae recovered from the third and fourth adult groups were 100% pcr-positive. the data revealed the passage of l. infantum kdna in nymphs and adults of brown dog tick following fixed time intervals post blood feeding on an infected dog (15). coutinho and linardi in 2007 conducted a study on the possibility of transmission of leishmania chagasi caused by devouring collected fleas (c. felis) from infected dogs in the golden hamsters (mesocricetus auratus. in this study, of the fleas collected, 4/207 (1.9%) showed the presence of promastigotes in smears stained by giemsa, whilst 43/144 (29.9%) exhibited positive pcr assay for dna of leishmania parasites. fourteen of the hamsters tested pcr amplification and 4 of them by indirect fluorescent antibody test (ifat) were positive with leishmania parasites. in addition, out of 16 infected hamsters, 11 had been infected peritoneally and 5 orally (16). in 1932, wenyon reviewed most existing theories about the transmission of leishmania infection and believed that ticks have no role in the transmission of visceral leishmaniasis in the mediterranean region (17). prior to this study, similar cases on the likelihood of transmission of the disease had not been made on dogs. the results are very important because it has been shown in practice that r. sanguinus ticks have failed to transfer l. infantum from infected dogs to healthy dogs. conclusions visceral leishmaniasis has been increasing worldwide, principally due to a substantial rise in human and domestic animal traffic contributing to spreading leishmanial infection in low or non-endemic areas (18). female sand flies of http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 249 http://jad.tums.ac.ir published online: sep 30, 2022 some species of the genus phlebotomus are the main and proven vectors of l. infantum transmission in humans and dog. there has long been speculation about the role of fleas as biologically or mechanically vectors of l. infantum and recent studies have reinforced this hypothesis (15, 18). the human flea, p. irritans, and the cat flea, c. felis were identified on 40% and 35 % of dogs, respectively. the results of pcr indicated that l. infantum was in the collected c. canis (75%) and c. felis (66.7 %) from infected dogs. the result of the present study confirms that fleas can be infected by l. infantum. but it is not yet clear whether these insects as vectors are able to transmit leishmania parasites from an infected host to healthy hosts and the vectorial competence of fleas needs to be determined. given this, it is recommended that the xenodiagnoses test be carried out to determine the probable role of leishmania spp. transmission. acknowledgements the authors would like to thank mrs f baghkhani for her laboratory support and cooperation. ethical considerations the protocols conducted in this study followed the guidelines of the institutional ethical committee (tarbiat modares university). the protocols were approved by tmu ethical committee under registry tmu-3674. conflict of interest statement authors declare that there is no conflict of interest. references 1. darvishi mm, youssefi mr, changizi e, lima rr, rahimi mt (2014) a new flea from iran. asian pac j trop dis. 4: 85– 87. 2. ratovonjato j, rajerison m, rahelinirina s, boyer s (2014) yersinia pestis in pulex irritans fleas during plague outbreak, madagascar. emerg infect dis. 20(8): 1414– 1415. 3. hamzaoui el, zurita b, cutillas a, parola c (2020) fleas and flea-borne diseases of north africa. acta trop. 211: 105627. 4. takahashi k, takahashi m, hamasaka k (2016) finding of the human flea pulex irritans (siphonaptera: pulicidae) in hokkaido, japan with human dermatitis caused by this flea. med entomol zool. 67(4): 233–235. 5. perez-martinez l, venzal jm, gonzalezacuna d, portillo a, blanco jr, oteo ja (2009) bartonella rochalimae and other bartonella spp in fleas, chile. emerg infect dis. 15(7): 1150–1152. 6. mohebali m (2013) visceral leishmaniasis in iran: a review of the epidemiological and clinical features. iran j parasitol. 8 (3): 348–358. 7. ferreira mgpa, fattori kr, souza f, lima vmf (2009) potential role for dog fleas in the cycle of leishmania spp. vet parasitol. 165(1–2): 150–154. 8. kakeh mami a, ghorbani a, kayvan behjoo f, mirzaei mosivand a (2017) comparison of visual and digital interpretation methods of land use/cover mapping in ardabil province. journal of rs and gis for natural resources. 8(3): 121–134 (persion). 9. harith a, kolk a, kager p, leeuwenburg j, muigai r, kiugu s, kiugu s, laarman jj (1986) a simple and economical direct agglutination test for serodiagnosis and seroepidemiological studies of visceral leishmaniasis. trans r soc trop med hyg. 80(4): 583–536. 10. kumsa b, abiy y, abunna f (2019) ectoparasites infesting dogs and cats in bishoftu, central oromia, ethiopia. vet para http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 243–250 a azarm et al.: molecular identification of … 250 http://jad.tums.ac.ir published online: sep 30, 2022 sitol reg stud reports. 15: 100263. 11. ish-horowicz d, burke j (1981) rapid and efficient cosmid cloning. nucleic acids res. 9(13): 2989–2998. 12. aransay a, me scoulica, tselentis y (2000) detection and identiþcation of leishmania dna within naturally infected sand ßies by seminested pcr on minicircle kinetoplastic dna. appl environ microbiol. 66: 1933–1938. 13. lainson r, shaw j, ryan l, ribeiro r, silveira f (1985) leishmaniasis in brazil. xxi. visceral leishmaniasis in the amazon region and further observations on the role of lutzomyia longipalpis (lutz and neiva, 1912) as the vector. trans r soc trop med hyg. 79(2): 223–226. 14. dantas-torres f, lorusso v, testini g, de paiva-cavalcanti m, figueredo la, stanneck d, mencke n, p. brandao-filho sc, alves l, otranto d (2010) detection of leishmania infantum in rhipicephalus sanguineus ticks from brazil and italy. parasitol res. 106(4): 857–860. 15. dabaghmanesh t, asgari q, moemenbellah-fard md, soltani a, azizi k (2016) natural transovarial and transstadial transmission of leishmania infantum by naïve rhipicephalus sanguineus ticks blood feeding on an endemically infected dog in shiraz, south of iran. trans r soc trop med hyg. 110(7): 408–413. 16. coutinho mtz, linardi pm (2007) can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? vet parasitol. 147 (3–4): 320–325. 17. wenyon cm (1932) the transmission of leishmania infections: a review. trans r soc trop med hyg. 25: 319–348. 18. colombo fa, odorizzi rmfn, laurenti md, galati eab, canavez f, pereirachioccola vl (2011) detection of leishmania (leishmania) infantum rna in fleas and ticks collected from naturally infected dogs. parasitol res. 109(2): 267– 274. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2015, 9(2): 267–273 h zendehfili et al.: ectoparasites of … 267 short communication ectoparasites of rodents captured in hamedan, western iran hamid zendehfili 1, *amir hossein zahirnia 1, amir hossein maghsood 2, mohammad khanjani 3, mohammad fallah 2 1department of medical entomology, school of medicine, hamedan university of medical science, hamedan, iran 2department of parasitology, school of medicine, hamedan university of medical science, hamedan, iran 3department of plant protection, faculty of agriculture, bu-ali sina university, hamedan, iran (received 4 jan 2014; accepted 29 mar 2014) abstract background: rodents with a population greater than the entire population of other mammals on earth are the source of economic losses and health conflicts. one of the major health problems with the rodents is their role as reservoir hosts of zoonotic diseases. the aim of this study was to assess the infestation of commensal rodents with ectoparasites in hamedan city, western iran. methods: the samples were collected by live traps during years 2012–2013. after transferring the samples to the entomological laboratory of hamedan university of medical sciences, their ectoparasites were collected and identified. results: a total of 171 slides were prepared from 105 captured commensal rodents: mus musculus, rattus rattus and r. norvegicus comprising three orders namely mesostigmata: hypoaspis (laelaspis) astronomica, dermanyssius sp, pachylaelapidae (male). metastigmata: rhipicephalus sp and anoplura: polyplax spinulosa were recovered in hamedan city. seventy (66.6%) rodents were found infested with at least one species of ectoparasites. conclusion: the results of our study indicate that ectoparasites infestation in commensal rodents of hamedan city is high and more attention by local health authorities is needed to prevent zoonotic diseases. keywords: rodents, ectoparasites, iran introduction over the past decades we have been facing with growth and uncontrolled urban development with problems such as environmental pollutions and increase of harmful organisms (dezfooli et al. 2009). rodents with a population greater than the entire of other mammals on earth have probably caused more human suffering than any other vertebrate pest (meerburg et al. 2009). they are responsible for the spread of many diseases, either directly, by contamination of food with their urine and feces or indirectly through vectors like fleas (azizi et al. 2008). commensal is defined as “sharing one’s table.” commensal rodents, by definition, thrive in close proximity to human activities and include rattus norvegicus (norway rats), r. rattus (roof rats) and mus musculus (common house mice). today, commensal rodents' outbreak in human and industrial centers has become an international problem. ectopara-sites are organisms that temporarily or per-manently live on the host (rodents) that pro-vide their biological needs (oormazdi 1995). many of these ectoparasites are important from medical and veterinary point of view and have a great role in transmission of dis-eases such as crimean-congo hemorrhagic fever (cchf), theileriosis, babesiosis, anaplasmosis, ehrlichiosis, omsk hemorrhagic fever, plague, salmonellosis, murine typhus, hemorrhagic jaundice, leptospirosis and rural cutaneous leishmaniasis (suntsov et al. 1997, inokuma et al. 2001, kia et al. 2009). in addition to transmission of diseases, rodents as important pests can cause the loss of economic *corresponding author: dr amir hossein zahirnia, e -mail: zahirnia@umsha.ac.ir http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 267–273 h zendehfili et al.: ectoparasites of … 268 resources by feeding on crops, grain and food stored and also, with chewing on telephone and electrical wires and damage to electrical devices encounter installations and houses with the major risks. losses to the crops pre-and post-harvest by rodents and especially rats is in a wide range. it is estimated that each year, approximately to 20 percent of the world's food supply are consumed or damaged by rodents. the world health organization estimate that annually about 33 million tons of food is lost by mice, which it is equivalent to 5 % of the total world production of food to feed 130 million hungry people is enough. even more the worst situation is in the third world, for example, rodents destroyed, 40 % of produced in the culture and the storage stage in bangladesh, each year (dehghani 2007, kaboodvandpour et al. 2010). due to the diverse climatic and geographic conditions in different regions of iran, fauna of ectoparasites in different areas should be investigated to be able to draw a map of the distribution of ectoparasites. although the study and identification of ectoparasites in rodents has a long history in the world, the history of research on rodent’s ectoparasites are limited in iran, despite the importance of rodents in terms of health compared with other mammals. this study conducted in hamedan city during 2012–2013 to collecting and identifying commensal rodents and ectoparasites on them to provide basic information for further control measures. materials and methods study area hamedan city as the center of hamedan province located in the west of iran. the neighboring provinces are zanjan and qazvin from the north, kermanshah and kurdistan from the west and markazi from the south and east (fig. 1). the main economic activity in the province is agriculture and animal husbandry and is considered as a center of tourism in the country. maximum and minimum temperature is 40 ˚c in july 2012 and -12 ˚c in january 2013, respectively. the total amount of rainfall in the study area over study period was 454 mm and the mean annual relative humidity is 45 %. rodent capture and identification the samples were collected by using livetraps measuring 29× 22× 50 cm (paramasvaran et al. 2009). these traps were placed in different locations in the city including under the bridges, inside canals, sewage and garbage at sunset and were collected on the next morning. trapped rodents were killed lenity by placing them in a plastic bags containing cotton soaked in chloroform. captured rodents were identified after recording accurate and complete information (etemad 1978). isolation and identification of ectoparasites the bodies of collected rodents were brushed after general anesthesia to remove ectoparasites on a white tray filled with water. also their ears were carefully examined by a forceps. the collected specimens were stored in 70 % alcohol for other process. ectoparasites were cleared by koh solution (10%), then all specimens were mounted with solution hoyer as microscopic slides. all lice, mite and ticks were examined and identified by valid keys (keirans and litwak 1989, faraji et al. 2008, cannings and scudder 2013) under an olympus microscope (bx51) (fig. 2). also all collected specimens are deposited in entomological laboratory, department of medical entomology, school of medicine, university of medical sciences, hamedan, iran. results this survey was carried out on the collection and identification of ectoparasites assohttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 267–273 h zendehfili et al.: ectoparasites of … 269 ciated with commensal rodents in hamedan city during 2012–2013. a total of 105 rodents in both sexes from different parts of the city were captured and from them 170 slides of ectoparasites were collected. the rodents included three species belong to suborder myomorpha, family of muridae (subfamily: murinae): rattus norvegicus (berkenhout 1769), r. rattus (l., 1758) and mus musculus l., 1758. among captured rodents r. norvegicus and m. musculus had the most and least frequency (fig. 3). ectoparasite samples were identified to species level if possible, representing to three orders, namely mesostigmata (mites), metastigmata (hard ticks) and anoplura (sucking lice) (table 1). the 140 specimens were mites belonged to three families, laelapidae [hypoaspis (laelaspis) astronomica (koch, 1839)] (7.6%), dermanyssidae (dermanyssus sp) (72.3%) and pachylaelapidae (male) (2.4%). nine of 170 specimens were lice belonged to a single family, polyplacidae (polyplax spinulosa) (burmeister 1839) and 21 individual hard ticks were from family ixodidae (rhipicephalus sp). the predominant collected ectoparasite groups were mites (82.3%), followed by hard ticks (12.3 %) and lice (5.3%). seventy (66.6%) rats were found infested with at least one species of ectoparasites. interestingly, no fleas were found on rodents sampled. rattus rattus was the predominant host species with the highest record of ectoparasite infestation. the ectoparasite groups and their abundance in each host are shown in table 1. according to this table the catch rate of ectoparasite on r. norvegicus, r. rattus and m. musculus were 87.6, 11.2 and 1.2 respectively. fig. 1. geographic location of collected commensal rodents in hamedan city (google earth © 2011 europe technologies)∗ sampling stations in hamedan city http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 267–273 h zendehfili et al.: ectoparasites of … 270 table 1. ectoparasite arthropods among commensal rodents in hamedan city, hamedan province, 2012–2013 rodent species n ectoparasite total catch mites lice ticks catch n catch rate (%) catch n catch rate (%) catch n catch rate (%) catc h n catch rate (%) r. norvegicus 92 123 82.5 9 6 17 11.4 149 87.6 r. rattus 8 15 78.9 0 0 4 21 19 11.2 m. musculus 5 2 100 0 0 0 0 2 1.2 total 105 140 82.3 9 5.3 21 12.3 170 100 fig. 2. microscopic photos of collected ectoparasites in hamedan city, 2012–2013 a: polyplax spinulosa, b: rhipicephalus sp, c: hypoaspis (laelaspis) astronomica, d: dermanyssius sp fig. 3. total captured rodents in hamedan city, hamedan province, 2012–2013 discussion in this study, despite retaining the basic principles in collecting and preparing specimens of commensal rodent ectoparasite, fleas were not found. it may be due to a low distribution and infestation rate or ecological situation in hamedan city is not suitable for surviving them. according to who plague manuel (1992), the flea (xenopsylla cheopis rothschild 1903) as most important plague vector spends more time in the nest of their host than on the host itself. our results on ectoparasites in small mammals were similar to other studies such as nadchatram et al. (1966), ho and krishnasamy (1991), chulan et al. (2005) and paramasvaran et al. (2009). within all collected specimens, the lice (polyplax spinulosa), were observed frequently in iran already (telmadarraiy et al. 2007, kia et al. 2009, rasouli et al. 2011, nateghpour et al. 2013). this is considered to be of public health importance because this rat louse is known to harbor plague agent and transmit tularemia and bartonellosis to humans and play an adjunctive role in the transmission of murine typhus and plague from rat to rat (zahedi et al. 1984). three species of rodents were infested with at least one species of mesostigmatic mites. these mites (dermanyssius sp) were found frequently on all captured rodents, although this genus was rarely discovered on iranian rats. it has a worldwide distribution and has been incriminated to cause pruritic rattus rattus 5% mus musculus 7% rattus norvegicus 88% http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 267–273 h zendehfili et al.: ectoparasites of … 271 dermatitis in men (telmadarraiy et al. 2007, kia et al. 2009, nateghpour et al. 2013). the host (r. norvegicus) with the majority of the ectoparasites of medical importance was found in areas with human activities (meerburg et al. 2009). in a similar study r. norvegicus reported as prefer host for mesostigmatic mites (easterbrook et al. 2007). family ixodidae have a main role in zoonotic diseases as medically important vectors for far-eastern tick-borne encephalitis virus in many parts of the world (nadchatram et al. 1966, roberts and john 2001, rat zooman 2006). several reports of tick-borne zoonotic diseases were reported from iran for example: crimean-congo hemorrhagic fever (mostafavi et al. 2013, sadeghi et al. 2013). global climate changes may cause rapid environmental modification such as altering the ecology of rodents and creating new foci resulting in the proliferation of vectors and increase of rodent borne parasitic diseases and this situation will increase contact between human and rodents, resulting in a heavier disease burden that would challenge the efficiency of the public health services (paramasvaran et al. 2009). unfortunately updating the current data on ectoparasites of rodents in iran is not sufficient. conclusion ectoparasites infestation rate of commensal rodents in hamedan city is high (66.6%) and among different arthropods species identified, some had zoonotic importance. therefore, the potential health hazard of these species needs to be considered by human and veterinary health authorities to prevent infectivity in humans and domestic animals. acknowledgements this paper is a part of msc thesis of the senior author financially supported by hamedan university of medical sciences, hamedan, iran. the authors declare that there is no conflict of interests. references azizi k, davari b, kalantar m, fekri s (2008) survey of rodents (muridae: gerbillinae) and determine reservoirs of cutaneous leishmaniasis using nestedpcr in the city of jask, hormozgan. journal 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of … 148 http://jad.tums.ac.ir published online: june 30, 2022 original article bionomics of phlebotomine sand flies in different climates of leishmaniasis in fars province, southern iran kourosh azizi1, zahra soltani2, mohsen aliakbarpour2, hassan rezanezhad3, 4, *mohsen kalantari1 1research center for health sciences, institute of health, department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran 2control disease center, shiraz university of medical sciences, shiraz, iran 3department of parasitology, jahrom university of medical sciences, jahrom, iran 4zoonoses research center, jahrom university of medical sciences, jahrom, iran *corresponding author: dr mohsen kalantari, e-mail: kalantari_m@sums.ac.ir (received 30 june 2020; accepted 11 june 2022) abstract background: phlebotomus and sergentomya are distributed in the old-world regions and transmit leishmaniases through mammalian and reptile hosts. cutaneous leishmaniasis (cl) is one of the most important diseases in iran. iranian sand flies belong to three oriental, palearctic, and afrotropical regions. fars province is located in palearctic, but southern parts are affected oriental region situations on phglebotomine population variety. therefore, a comprehensive study was required on the vectors of the disease in this province. methods: regarding the approved role of the environmental factors in creating the fauna and distribution of living creatures, the “de martonne climate method” was used, and the climate was noticed as an important environmental factor for the determination of vector distribution. accordingly, 14 sampling sites were selected from 10 foci in different climates of fars province. 19648 sand flies were collected from the studied areas in this province during 2016. results: phlebotomus papatasi and se. antennata were the most frequent species, which were caught from in/outdoor areas. phlebotomus sergenti and ph. alexandri were caught from both palearctic and oriental zones but were more prevalent in the cold semi-arid climate of the palearctic zone. moreover, they were not caught from the hot desert and summer mediterranean climates of the oriental zone. conclusions: it seems that ph. papatasi as the main vector of cl could be well distributed in different climates in fars. moreover, some species like ph. sergenti preferred especial climates in the palearctic zone. therefore, these data could be helpful to control leishmaniases more efficiently. keywords: cutaneous leishmaniasis; sand flies; phlebotomus papatasi; geographic information system; iran introduction leishmaniases, as one of the most common group of vector-borne diseases, are transmitted to humans through female sand flies’ bites infected with leishmania parasites in the world (1). there are more than 3,000 described psychodid species distributed among six subfamilies of phlebotominae, bruchomyiinae, sycoracinae, trichomyiinae, psychodinae, and horaiellinae in the world (2). the last one, with its sole genus horaiella and its four species, is restricted to the himalayas, china, and thailand (1). in addition, there are about 1000 species of phlebotominae sand flies in the world, of which 98 species are identified as the proven or suspected vectors of human leishmaniases (4). sand flies have different breeding places in tropical and subtropical areas and their variations directly affect the prevalence, incidence, and transmission rates of the disease in different areas (5). copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 149 http://jad.tums.ac.ir published online: june 30, 2022 phlebotomus and sergentomya (diptera: psychodidae) are distributed in the old-world regions and transmit the parasite through humans, different mammalian, and reptile hosts (6). they have a wide range of mammalian hosts, such as canines, rodents, cats, hyrax, and humans. consequently, human leishmaniases have different zoonotic and anthroponotic transmission patterns (7). phlebotomine sand flies usually have different ecological habits and may tend to rest in or outdoors of residential places. some sand fly species prefer to live within active rodents’ burrows across plain or mountainous regions (8). cutaneous leishmaniasis (cl) is endemic in iran and reported from different regions of the country (9). the study of iranian sand flies began in the early years of this century, mainly by researchers such as adler, theodore, and laurie. the first comprehensive study was carried out by mesghali in 1961, which reported 12 species of phlebotomus and 11 species of sergentomyia from iran (10). in continue, javadian and mesghali reported a total of 42 species in the country during 1975. thereafter, 54 species of sand flies were reported from different regions of the country. accordingly, 48 species were finally confirmed from two genera of phlebotomus and sergentomyia based on the studies carried out since 1930 (11). species, such as ph. caucasicus, ph. mongolensis, ph. kazeruni, ph. brevis, ph. adlerius, ph. papatasi, ph. sergenti, ph. alexandri, se. sogdiana, se. hodegson, se. grekovi, se. hodgsoni, se. tiberiadis, se. sintoni, se. clydei, se. dentata, and se. antennata have been usually reported from the northern zones of iran. on the other hand, some species, such as ph. ansarii, ph. longiductus, ph. papatasi, ph. sergenti, ph. alexandri, ph. bergeroti, ph. major, ph. mongolensis, se. sintoni, se. baghdadis, se. theodori, se. antennata, se. clydei, se. squamipleuris, se. dentata, and se. mervynae have been more reported from the southern zones of the country including fars province. in this regard, ph. papatasi and se. sintoni species were reported as the dominant sand flies’ species (9). phlebotomus papatasi and ph. sergenti have been reported as the suspected or proven vectors of cl. moreover, ph. kandelakii, ph. perfiliewi, ph. sergenti, ph. papatasi, ph. ansarii, ph. salehi, and ph. caucasicus have been reported as the suspected or proven vectors of visceral leishmaniasis (vl) in iran (12, 13). additionally, ph. papatasi, ph. keshishiani, ph. alexandri, and ph. major have been reported as the vectors of cl and vl in southern provinces of the country including fars and bushehr (14). the palearctic zone covers eurasia, including europe, asia north of the oriental region, and northern africa, and. the oriental region includes eastern iran, china south of the yangtze river, india and sri lanka, southeast of asia, the philippines, the east indies and indo-malayan archipelago, and the large island regions of taiwan (15). iranian sand flies belong to three oriental, palearctic, and afrotropical regions (16). environmental factors, such as the mean temperature of the wettest quarters, play an important role in sand flies’ distribution (especially ph. papatasi) in iran and can be reported in the regions where biological situations are suitable (17). in fact, such factors as appropriate temperature and relative humidity are very important for their survival in different areas of the country (18). moreover, rainfall, altitude from the sea level, land cover type, annual mean or minimum temperature in the coldest months, and mean temperature of the driest or wettest quarters have been reported to be effective in the distribution of different sand flies’ species (19). changes in the environment cause variations in the transmission pattern of communicable diseases. the consciousness of the relations between the environment changes and the incidence of vectorborne diseases can be helpful in planning effective control strategies (20, 21). due to the high incidence of leishmaniasis in fars province, a comprehensive study was required on the vectors of the disease in this province. the precise control of cutaneous and/ http://jad.tums.ac.ir/ https://www.sciencedirect.com/topics/earth-and-planetary-sciences/archipelago j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 150 http://jad.tums.ac.ir published online: june 30, 2022 or visceral leishmaniasis helps to identify endemic vectors as well as reservoir hosts of the disease. the present study was designed to explore the fauna and the bio-ecology of sand flies in fars province, southern iran, during 2016. materials and methods study area fars province includes 23 counties with an area of 122400km² in south of iran (22). shiraz as the capital city of the province is situated at 29° 59' 18" north, 52° 58' 37" east and about 1200m above the sea level. recently, fars has been the most important foci of cl in southern iran. this province has many different climates, but the most prevalent ones are cold semiarid climate (in shiraz, kharameh, marvdasht, darian, and sarvestan), hot desert climate (in larestan, zarin-dasht, hajiabad, qir, banaruiyeh, and shahr-e pir), hot semi-arid climate (in farashband, zahedshahr, now bandegan, duzeh, sheshdeh, and sahrarud), hot-summer mediterranean climate (in khan-e-zenyan and kazerun), cold desert climate (in mehrdasht), and hot humid continental climate in southern parts. shiraz has been considered to have a local steppe climate. the average annual temperature is 16.8 °c and there is little annual rainfall (the average rainfall is equal to 316mm) in shiraz. recently, shiraz, marvdasht, and kharameh have been the most important foci of cl in fars province, southern iran (fig. 1). selection of villages considering the approved role of the environmental factors in creating the fauna and distribution of living creatures, the “de martonne climate method” was used (23, 24), and climate was noticed as a key factor for determination of vector distribution affected by atmospheric precipitation, elevation, and vegetation. accordingly, 14 sampling sites were selected from 10 foci in plain and mountainous areas regarding the contribution of different climates. among these foci, eight were in endemic areas of zcl and six belonged to non-endemic foci (table 1). sampling phlebotomine sand flies were caught using sticky paper traps and aspirating tube from zafar-abad, mahmood-abad, ahmad-abad, koh-sabz, tole-mahtabi, bahman, deh-dagh, and khsoyeh villages from the palearctic zones and eslam-abad, bid-karz, baba-monir, hossein-abad, deh-mian, and ali-abad from the climate zone of oriental in fars province every month during 2016. sand flies were sampled from indoors, such as bedrooms and bathrooms, and outdoors, such as rocks, rodent burrows, agricultural lands, and wall gaps. in each sampling round, 60 sticky paper traps (30 indoors and 30 outdoors) were fixed in the sunset and collected in the next morning before sunrise. phlebotomine sand flies were collected, kept in ethanol (70%), mounted in the puri’s media, and taxonomically identified according to valid taxonomic criteria studies (25). pearson’s correlation coefficient was used to find the relationship between the disease incidence rate which was reported from the iranian control disease center and the number of collected p. papatasi as the main vector of zcl from different studied villages in 2016. the data were analyzed using the arc explorer software. this application is included three extensions of 3d analyst, spatial analyst, and geostatistical analyst. at first, topology data will be added to the geodatabase part, which is a feature originally available only with arcinfo coverages. then, these three extensions facilitate the ability to access data online, directly from the geography network site or other arcims map services (26). results in this study, a total of 19648 sand flies were collected from different villages of the studied areas in fars province during 2016. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 151 http://jad.tums.ac.ir published online: june 30, 2022 among the collected sand flies, 11778 specimens (59.95%) were male and 7870 (40.05%) were female. in addition, 14030 (71.41%) and 5618 (28.59%) specimens were identified as the various species of phlebotomus and sergentomyia, respectively. phlebotomus species included 10654 ph. papatasi (54.22%), 2052 ph. sergenti (10.44%), 672 ph. alexandri (3.42%), 548 ph. bergeroti (2.79%), 80 ph. major (0.41%), 10 ph. keshishiani (0.05%), 8 ph. tobbi (0.04%), and 6 ph. salehi (0.03%). the most dominant species was ph. papatasi caught from both indoor and outdoor places of all studied foci. on the other hand, among the 5618 collected specimens of sergentomyia, 2250 were se. antennata (11.45%), 1071 were se. sintoni (5.45%), 884 were se. baghdadis (4.5%), 662 were se. clydei (3.37%), 473 were se. theodori (2.41%), 118 were se. squamipleuris (0.60%), 106 were se. mervynae (0.54%), 47 were se. dentata (0.24%), and 7 were se. palestinensis (0.04). besides, se. antennata was the most frequent species caught from in/outdoors (table 2). in the palearctic zone, all above-mentioned phlebotomus species were caught, except for ph. keshishiani that was only collected from bid-karz village in mamasani focus in the oriental zone (fig. 2). besides, among the collected sergentmyia species, se. palestinensis was not caught from the palearctic zone and was only recorded from eslam-abad village of kazerun in the oriental climate region. in the current study, no ph. major, ph. tobbi, and ph. salehi species were caught in the studied villages of the oriental zone (fig. 2). indeed, the first two species were merely recorded from koh-sabz from marvdasht focus and the last one was only collected from tole-mahtabi from neyriz in the palearctic zone (table 2). the results revealed significant variation (p< 0.001) between the incidence rates of cl reported from the iranian control disease center and the number of caught ph. papatasi in different endemic and non-endemic foci of fars province during 2016. but it does not indicate a positive or negative relationship between cl cases and the number of ph. papatasi sand flies caught in different foci. for example, in niriz, where the number of cl cases was higher, the number of ph. papatasi sand flies caught was less than in shiraz with fewer cl cases (table 3). table 1. the geographic coordinates of the studied sites climate zone climate type topographical type foci village x_long altitude y_lat altitude zcl cases palearctic cold semi-arid plain shiraz zafar-abad 653501.1 3254461 864 mountainous shiraz mahmood-abad 659075.2 3249343 plain kharameh ahmad-abad 708846.3 3259576 224 mountainous marvdasht koh-sabz 664812.1 3310268 901 mountainous neyriz tole-mahtabi 823232.7 3237904 95 plain abadeh bahman 641681.6 3450615 29 plain abadeh deh-dagh 662026 3445878 hot desert plain zarin-dasht khosoyeh 831592.2 3161876 135 oriental hot desert mountainous larestan deh-mian 803992.9 3048333 240 plain larestan ali-abad 864440.2 3067707 hot semi-arid plain farashband hossein-abad 612280.1 3186581 69 hot-summer mediterranean plain kazerun eslam-abad 596084.8 3244258 44 mountainous mamasani bid-karz 501180.5 3311488 11 plain mamasani baba-monir 519969 3326872 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 152 http://jad.tums.ac.ir published online: june 30, 2022 table 2. sand fly species collected from different zone and climate regions of fars province during 2016 no. of collected phlebotomine sand flies z o n e s c lim a te s f o c i v illa g e s p h le b o to m u s p a p a ta si p h le b o to m u s se rg e n ti p h le b o to m u s a le x a n d ri p h le b o to m u s b e rg e ro ti p h le b o to m u s m a jo r p h le b o to m u s k e sh ish ia n i p h le b o to m u s to b b i p h le b o to m u s sa le h i s e rg e n to m y ia a n te n a ta s e rg e n to m y ia sin to n i s e rg e n to m y ia c ly d e i s e rg e n to m y ia d e n ta ta s e rg e n to m y ia m e rv y n a e s e rg e n to m y ia th e o d o ri s e rg e n to m y ia b a g h d a d is s e rg e n to m y ia sq u a m ip le u ris s e rg e n to m y ia p a le stin e n sis t o ta l p a le a r c tic c o ld se m i-a rid a a 1473 654 480 0 0 0 0 0 0 88 0 9 9 213 492 0 0 3418 b 652 321 0 0 0 0 0 0 0 53 0 7 8 92 161 0 0 1294 b c 828 99 29 164 0 0 0 0 320 24 25 0 0 21 0 19 0 1529 c d 1839 217 0 112 80 0 8 0 78 0 0 0 60 8 0 0 0 2402 d e 1818 0 0 102 0 0 0 6 492 85 0 0 0 6 0 0 0 2509 e f 19 78 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 97 g 9 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 * h d f h 1038 106 0 24 0 0 0 0 536 364 344 0 7 64 0 0 0 2483 subtotal 13743 t h e o r ie n ta l o r ie n ta l h d g i 428 0 0 51 0 0 0 0 337 369 144 0 0 49 0 0 0 1341 j 763 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 800 * * h s a h k 275 0 57 0 0 0 0 0 60 0 36 31 0 7 91 51 0 608 * * * h s m i l 864 0 0 92 0 0 0 0 95 0 27 0 0 0 140 44 7 1269 j m 2 249 106 3 0 10 0 0 332 76 81 0 22 13 0 4 0 898 n 646 326 0 0 0 0 0 0 0 12 5 0 0 0 0 0 0 989 subtotal 5905 t o ta l % 1 0 6 5 4 2 0 5 2 6 7 2 5 4 8 8 0 1 0 8 6 2 2 5 0 1 0 7 1 6 6 2 4 7 1 0 6 4 7 3 8 8 4 1 1 8 7 1 9 6 4 8 5 4 .2 2 1 0 .4 4 3 .4 2 2 .7 9 0 .4 1 0 .0 5 0 .0 4 0 .0 3 1 1 .4 5 5 .4 5 3 .3 7 0 .2 4 0 .5 4 2 .4 1 4 .5 0 .6 0 .0 4 1 0 0 a, shiraz; b, kharameh; c, marvdasht; d, neyriz; e, abadeh; f, zarin-dasht; g, larestan; h, farashband; i, kazerun; j, mamasani a, zafar-abad; b, mahmood-abad; c, ahmad-abad; d, koh-sabz; e, tole-mahtabi; f, bahman; g, deh-dagh; h, khosoyeh; i, deh-mian; j, ali-abad; k, hossein-abad; l, eslam-abad; m, bid-karz; n, baba-monir. *hot desert; ** hot semi-arid; ***hot summer mediterranean table 3. comparison of the number of collected sand flies and incidence rates of cl in different endemic and nonendemic foci of cl in fars province during 2016 foci cl cases incidence per 100000 no. of caught phlebotomus papatasi p value marvdasht 901 150.1 1839 <0.001 shiraz 864 57.6 2125 kharameh 224 373.3 828 neyriz 95 135.7 1818 zarin-dasht 135 184.4 1038 abadeh 29 49.1 28 mamasani 11 9.4 648 larestan 240 386.8 1191 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 153 http://jad.tums.ac.ir published online: june 30, 2022 kazerun 44 45.5 864 farashband 69 339.6 275 total 2612 94.9 10654 fig. 1. map of iran showing the sample sites in different locations of fars province, southern iran during 2016; abadeh (nos. 1 and 2), marvdasht (no. 3), shiraz (nos. 4 and 5), kharameh (no. 6), neyriz (no. 7), farashband (no. 8), mamasani (nos. 9 and 10), kazerun (nos. 11 and 12), and larestan (nos. 13 and 14) fig. 2. map of the geographic distribution of phlebotomus species in the palearctic and influenced oriental zones of fars province during 2016 table 3. continued ... http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 154 http://jad.tums.ac.ir published online: june 30, 2022 discussion in this study, phlebotomines were caught from various climates in the palearctic and affected zones of oriental in fars province. among the 19648 sand flies caught, 14030 (71.41%) and 5618 (28.59%) specimens were identified as various species of phlebotomus and sergentomyia, respectively. phlebotomus papatasi was the most frequent species and was collected from all in/outdoors of people’s residual places in both palearctic and affected zones of oriental in fars province. indeed, wall gaps of old rooms and agricultural lands were the most frequent breeding places of these species. resting sites of this species is naturally infected with l. major and reported from different parts of iran, including fars province (27). the high biodiversity of the iranian fauna is the result of its area and the influences of four ecozones from palaearctic and old periodical connections with the nearctic by bering strait in north, afrotropical from the arabian peninsula in south, and oriental in southeast includes fars (28, 29). fars province includes a wide variety of sand fly fauna. species such as ph. papatasi, ph. alexandri, ph. keshishiani, and ph. major were formerly reported as the vectors of zcl and vl in this province. but ph. papatasi has been noticed as the main proven vector of zcl in all endemic foci of fars. also, this species was the main frequent species (54.22%) in the current study in both geographical zones. in a similar study conducted in southern foci of this province in palearctic zone, it seems that relative humidity was the main environmental factor affecting phlebotomine activity and spatial distribution. accordingly, their activity increased significantly when the average humidity was induced to more than 10% and reduced rapidly when the average humidity average was over 50% (30). these findings are in contract with those who believe that low humidity which followed by low wind speed and high temperature were the main factors affecting phlebotomine activities (31). however, some other studies indicated that low wind velocity, light intensity, temperature, and low relative humidity (maximum wind speed of 3m/s, humidity of 10%, and minimum temperature of 11 °c) were the main factors affecting their activity and distribution (32). softwares such as geographic information system (gis) and ecological niche models (enms) have been used to generate the distribution map for phlebotomine sand flies to find effective environmental factors on these vectors on the prevalence of leishmaniasis (21). therefore, more research are needed about the seasonal variations and abiotic situations (cloud cover, lunar cycle, wind speed and so forth) in order to improve our knowledge of these epidemiologically important subjects. however, species such as ph. sergenti were formerly recorded as the sole proven vector of leishmania tropica in iran (33). also, ph. sergenti has been found positive with leishmania parasite in shiraz and some other important cities in the country (34). in addition, ph. alexandri was previously recorded as the probable vector of leishmania infantum in southern regions (35-37). in the current study, ph. sergenti and ph. alexandri were caught from both palearctic and affected zones of oriental in the province, but were more prevalent in the cold semiarid climate of the palearctic zone. moreover, they were not caught from the selected sites of larestan and kazerun in hot desert and hot summer mediterranean climates of the zones influenced by oriental zone. phlebotomus major has been reported from northern and southern regions of iran and has been naturally infected with l. infantum in fars (38). however, in the current study, this species was only collected from koh-sabz village from marvdasht in the palearctic zone. the incidence and distribution of leishmaniasis are both influenced by environmental variables affecting the phlebotomine sand flies (as vectors) and reservoirs populations and human behaviors (37). due to the high incidence of leishmaniasis in fars province (39, 40), a comprehensive http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 148–158 k azizi et al.: bionomics of … 155 http://jad.tums.ac.ir published online: june 30, 2022 study was required on the vectors of the disease in this province. the precise control of cutaneous and/or visceral leishmaniasis helps identify the endemic vectors as well as the reservoir hosts of the disease (40). moreover, careful monitoring of environmental variables in relation to biology of vector and leishmaniasis is important for designing and implementation of control plans. conclusion phlebotomus papatasi as the main vector of cl could be well distributed in different climates in fars. moreover, some species like ph. sergenti prefered especial climates in the palearctic zone. it seems that environmental factors play an important role in distribution of phlebotomine sand flies and can be reported in the regions where biological situations are suitable. therefore, the obtained data could be helpful to control leishmaniases more efficiently. acknowledgements this investigation was extracted from an approved msc thesis (proposal no. 92-6823) written by zahra soltani and financially supported by the research vice-chancellor of shiraz university of medical sciences. hereby, the authors would like to thank ms a keivanshekouh at the research improvement center of shiraz university of medical sciences for improving the use of english in the manuscript. ethical considerations ethical approval for this study was obtained from the ethics committee at shiraz university of medical sciences (iran). conflict 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https://link.springer.com/article/10.1007/s12639-021-01360-7#auth-hadi-mirahmadi https://link.springer.com/article/10.1007/s12639-021-01360-7#auth-ali-soleimani j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 136 http://jad.tums.ac.ir published online: june 30, 2022 original article phytochemical composition and bioassay on iranian teucrium polium extracts against anopheles stephensi (diptera: culicidae) saeedeh ghafari1, *azar tahghighi2, khadijeh shamakhte3, hamzeh alipour4, naseh malekiravasan5, mehdi nateghpour6 1traditional medicine and materia medica research center, shahid beheshti university of medical sciences, tehran, iran 2medicinal chemistry laboratory, clinical research department, pasteur institute of iran, tehran, iran 3department of biochemistry, payame noor university, tehran, iran 4research center for health sciences, institute of health, shiraz university of medical sciences, shiraz, iran 5department of parasitology, pasteur institute of iran, tehran, iran 6department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran *corresponding author: dr azar tahghighi, e-mail: atahghighi2009@gmail.com (received 26 nov 2019; accepted 16 may 2022) abstract background: anopheles stephensi is an important malaria vector mosquito in iran and other western asian countries. in many human communities, plant products have been used traditionally instead of synthetic pesticides for mosquito control due to their minimal hazardous effects. teucrium polium, known popularly as felty germander, has been introduced in persian medicine (pm) as an insect repellent from a long time ago. methods: the present study was undertaken to evaluate repellent and larvicidal activity of dichloromethane (dcmetp) and ethanolic extracts (ee-tp) of t. polium against an. stephensi under laboratory conditions. the possible chemical components of the extracts were also investigated through gas chromatography/mass spectrometry (gc-ms) technique. results: based on the results, dcme-tp showed better repellent activity than ee-tp with 56.67 and 28.33 % protection, respectively. larvicidal activity of dcme-tp with 49.41% mortality was also higher than ee-tp (20.24%). the main identified constituents of dcme-tp were long chain alkanes, phenol, aromatic ester, oxaspiro and triterpenoid. while phenolic and aliphatic acid were only the identified components in ee-tp. it is notable that lupeol was detected in dcme of t. polium for the first time. conclusion: dcme-tp can be considered as a new herbal candidate to control an. stephensi mosquitoes. further studies are required on this extract for the fractionation and identification of the active compounds, and the evaluation of their bioactivity in the laboratory and field. keywords: larvicidal; repellent; teucrium polium; anopheles stephensi; phytochemical introduction according to world health organization (who) report, “no significant gains were made in reducing malaria cases in the period 2015 to 2017. the estimated number of malaria deaths in 2021, at 619000, remained virtually unchanged over the previous year” (1). malaria is caused by five different species of plasmodium: p. falciparum, p. vivax, p. ovale, p. malariae, and p. knowlesi, which are transmitted by mosquitoes of the genus anopheles. anopheles mosquitoes are bloodsucking insects and responsible for the transmission of malaria, filariasis and arboviruses. there are more than 30 species currently recognized as anopheles species, out of which seven of them have important roles in malaria transmission in iran (2, 3). anopheles stephensi is the copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 137 http://jad.tums.ac.ir published online: june 30, 2022 primary vector of malaria in the southern parts of iran and other west asian countries (2). while the centralized application of malaria control programs has strongly diminished malaria transmission in malarious regions in the past year, malaria elimination remains an unattainable aim in high-endemic areas (1). there are various methods for malaria control, such as, drug chemotherapy, personal protection, and mosquito control using chemical insect repellents and insecticides (4–5). control of mosquitoes by repellents is an excellent strategy to avoid biting mosquitoes which can decrease the incidence of mosquito-transmitted diseases. another control strategy is killing mosquitoes and their larvae by chemical insecticides such as pyrethroids, organophosphates and carbamates (5). although, control of anopheles mosquitoes with synthetic repellents and insecticides is possible, their environmental effects and the emergence of resistance are the main concerns in the worldwide (5). in this situation, novel control tools can play an important role in the effort to control and eventually eliminate malaria. application of herbal preparations is a suitable tool which can be used as an alternative to synthetic repellents and insecticides. rapid action, minimal side effects on the skin and quick decomposition in the environment are minimum benefits of medicinal plants which encourage the scientists to assess their anti– insect activities (6). several studies have shown that some extracts and essential oils of plants presented repellency, ovicidal, larvicidal and pupicidal activities against mosquitoes (7–11). in mosquito control programs, products with botanical origin and traditional medicine may have the potential to be used successfully (12). persian medicine (pm), as one of the oldest types of complementary medicine, has a long history of using plants for treatment and prevention of diseases (13). pm has introduced many plants as repellents or insecticides which could be the suitable alternatives to mosquito control programs (14). one of these plants is teucrium polium, traditionally named “joadah” (15) or “kalpooreh” (16), which belongs to the family lamiaceae. in this regard, pm manuscripts recommended “joadah” could be spread in the environment or smoke to repel insects (15). in a study, the repellency effect and fumigant toxicity of t. polium essential oil against callosobruchus maculatus f. (coleoptera: bruchidae) and tribolium castaneum (herbst) (coleoptera: tenebrionidae), as the stored-product insects, have been also evaluated (17). in recent years, the recognition of bioactive chemical agents from plants and other natural sources has been noticed by researchers to control mosquitoes. the aims of this study were to evaluate the repellency and larvicidal activities of dichloromethane extract (dcme) and ethanolic extract (ee) of t. polium against an. stephensi mosquito and identifying the chemical components of these extracts using gas chromatographymass spectrophotometry (gc-ms). materials and methods plant materials the aerial parts of t. poilium were collected in march 2008 from sarkouh, one of the parts of bandar lengeh city in hormozgan province, located on the north coast of persian gulf, south of iran. originality of the plant was identified by h moazeni and a pirani, botanists of traditional medicine and materia medica research center (tmrc), shahid beheshti university of medical sciences, iran. the voucher specimen 2095 (tmrc) of the plant has been deposited in the tmrc herbarium. extraction the dried aerial parts of t. polium (200g) were powdered and defatted with hexane, and then macerated in dichloromethane (5:1). dcme-tp was separated by filtration and the residue of the plant was dried at room temperature and then macerated in ethanol solhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 138 http://jad.tums.ac.ir published online: june 30, 2022 vent (5:1). all extraction operations have been performed for 24 hours at room temperature with constant shaking. each extraction process was repeated three times and finally three replicate extracts were added to each other. the dichloromethane) and ethanol extracts of t. polium were concentrated by a vacuum rotary evaporator under reduced pressure at a maximum temperature of 50 °c to yield 4.1 and 19.88g of residue, respectively and stored at 4 °c. sample preparation for repellency test formulation of samples composed of dcme-tp and ee-tp were prepared using oil and aqueous phases (18). the oil phase consisted of stearic acid (1.5g), cetyl alcohol (0.4g) and isopropyl myristate (0.3g) separately. the aqueous phase consisted of dcme-tp and eetp (2.5g) solved in glycerin (1.5g) and water (7.8ml). finally, oil and aqueous phases were added to each other under heating about 65– 70 °c in a water bath and mixed suitably. the base formulation without extracts was used as control. mosquito rearing the laboratory bred an. stephensi strain of chabahar were used for repellency tests which was reared and maintained at 27±3 °c and 70– 80% relative humidity with a photoperiod of 12h light and 12h dark in the insectarium of the school of public health, shiraz university of medical sciences (sum). repellency assay five to seven days old non-blood fed females an. stephensi were used for repellency tests. the repellency study was conducted by a modified 4-celled klun and debboun (k and d) module (19) which is used for quantitative measurement of the efficacy of mosquito repellents on 6cm2 of the forearm skin of healthy male volunteers aged about 45 (fig. 1). the module was built by plexiglas to minimize visual error such as: four cells with larger dimensions to hold 20 mosquitoes in each cell. on the test day, the volunteer had no contact with any cream, lotions, perfumes, or perfumed soaps. before the application of the formulated samples for repellency test, the arm of the volunteer was cleaned by distilled water. after drying, marked areas on the skin (6cm2) were covered with 100mg of sample for three cells and a k and d module bioassay system was put on these areas. only samples foundation served as a control in cell number 4. twenty mosquitos were then release into each cell by an aspirator. observations on the number of bites of an. stephensi mosquitoes were recorded at 30 minutes post treatment. the percentage of repellency was calculated by the following equation: % protection= [nm/nt]× 100 where nm is the mean number of unfed females in the treatment group and nt is the total number of mosquitoes. larvicidal bioassay based on the preliminary tests, fourth and third instar larvae of an. stephensi, chabahar strain were exposed to serially diluted test concentrations of 62.5, 125, 250, 500, 1000, and 2000ppm of ethanolic and dichloromethane extracts of t. polium for 24 hours according to who protocol with minor modifications such as: application a surfactant to distribute monotonous of extracts (20). as the extracts do not dissolve in water completely, ee-tp was dissolved in ethanol and dcme-tp was dissolved in acetone. test solutions were prepared by adding 1ml of appropriate dilution of extracts in ethanol and mixed with 99ml of dechlorinated water containing 0.05% tween 80 (v/v aq). in the control beaker for ee-tp, 1% ethanol was added into water and tween 80 (0.05%). for dcme-tp, control beaker was including 1% acetone and 0.05% tween 80. also, dechlorinated water served as untreated control. a minimum of 25 healthy larvae per each concentration were used for all the experiments. the dead larvae were counted after 24 hours recovery period, and the percentage of mortality was reported as the average of the four replicates. http://jad.tums.ac.ir/ https://www.researchgate.net/publication/328303444_klun_debboun_modules_uses_and_data_analysis j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 139 http://jad.tums.ac.ir published online: june 30, 2022 gas chromatography-mass spectrometry (gc-ms) analysis of the extracts gc–ms analysis of the extracts was performed using an agilent 7000, triple quad, gc 7890a system operating in ei mode (70ev) equipped with a capillary column (hp-5ms, 30m (length), 0.25mm (diameter), film thickness of 0.25µm) as the stationary phase (7). the column oven temperature was initially held at 50 °c for two minutes, and then increased to 290 °c at the ramp rate of 5 °c/min and held for 10 minutes at the same temperature. the total run time was 60 minutes. the temperatures of the injector and detector were set at 250 and 300 °c, respectively. the flow rate of helium as a carrier gas was 1ml/min. compounds were identified by comparison of mass spectral fragmentation patterns and retention indices with national institute of standards and technology (nist) mass spectral library. the relative percentages of the components were obtained according to the peak area in the chromatogram. adverse effect of the formulations on the skin of human volunteers tests in human volunteers performed to detect allergic contact sensitization following application of extracts formulations. the cutaneous reactions were monitored for any abnormalities including erythema, edema, pruritus and urticaria, skin allergy and irritation after 15-minute, 1 hour and 24 hours. results repellency assay the results obtained from repellency test based on the k and d module bioassay with prepared formulations of t. polium extracts on an. stephensi are presented in table 1. all formulations showed good results with the 18% concentration used to achieve protection from mosquito bites. lower blood feeding rate was observed in dichloromethane extract of t. polium with the protection percent 56.67% whereas it was 28.33% for ethanolic extract of t. polium. the base of formulated sample without extract as a control did not exhibit t repellent activity (table 1). larvicidal bioassay the consequence of different concentrations of the dcme and ee of t. polium at 62.5, 125, 250, 500, 1000 and 2000ppm on the larvicidal activity against an. stephensi after 24h exposure is depicted in table 2. the highest and lowest larval mortality of 49.41% and 0% was observed at 2000 and 62.5ppm concentrations, respectively. the control groups didn’t have any mortality. the result indicated the larvicidal activity of dcme-tp was highly dose dependent. whereas ee-tp with weak larvicidal activity was not completely dose dependent. this study showed medium larvicidal activity of dcme of t. polium against fourth and third-instar larvae of an. stephensi. the cutaneous reactions on the skin of human volunteers tests in human volunteers to detect allergic contact sensitization of extracts formulations proved negative. the results did not show any skin irritation, skin sensitization, and skin keratinization. chemical composition of the extracts different alkanes such as tetradecane, hexadecane, octadecane and eicosane, as well as 2,4-di-tert-butylphenol and di-isooctyl phthalate as the aromatic compounds and 7,9-di-tert-butyl-1-oxaspiro (4, 5) deca-6,9-diene-2,8-dione as a di-keton compound were identified in dcme-tp (table 3). lupeol was another chemical component identified by gc-ms in dcmetp. chemical components of 2,4-di-tert-butylphenol and 1-methyl-pyrrolidine-2-carboxylic acid were only identified in ee-tp (table 3). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 140 http://jad.tums.ac.ir published online: june 30, 2022 table 1. repellency assay by the prepared formulation of dichloromethane and ethanolic extracts of teucrium polium (18% concentration) against anopheles stephensi blood feeding/non blood feeding sample repeat 1 repeat 2 repeat 3 control %protection dcme-tp 3/17 4/16 4/16 15/5 56.67 ee-tp 8/12 6/14 8/12 13/7 28.33 abbreviation: dcme; dichloromethane extract, ee; ethanolic extract, tp; teucrium polium table 2. larvicidal activity of dichloromethane and ethanolic extracts of teucrium polium against anopheles stephensi larvicidal activity of dcme-tp concentration (ppm) repeat 1 dead/t* repeat 2 dead/t* repeat 3 dead/t* repeat 4 dead/t* mortality%** 2000 12/21 10/21 11/21 9/22 49.41 1000 5/21 6/23 4/20 5/22 25.26 500 4/22 4/24 3/20 2/26 14.13 250 2/23 2/20 1/22 2/24 7.86 125 0/24 0/20 1/20 1/22 2.5 62.5 0/21 0/25 0/24 0/20 0 larvicidal activity of ee-tp concentration (ppm) repeat 1 dead/t* repeat 2 dead/t* repeat 3 dead/t* repeat 4 dead/t* mortality%*** 2000 4/21 3/21 6/21 4/21 20.24 1000 6/23 4/22 4/25 4/24 19.15 500 4/22 4/23 5/25 4/22 18.48 250 7/21 2/25 3/20 3/25 16.48 125 3/22 2/21 4/22 4/26 14.23 62.5 3/22 2/21 2/21 1/18 9.5 *t: total number of anopheles stephensi larvae. abbreviation: dcme; dichloromethane extract, ee; ethanolic extract, tp; teucrium polium. **the control of dcme-tp containing 1% acetone, 0.05% tween 80, and 99ml dechlorinated water did not have any mortality. ***the control of ee-tp containing 1% ethanol and 0.05% tween 80, and 99ml dechlorinated water did not have any mortality fig. 1. modified 4-celled klun and debboun module used for the repellency assay of dichloromethane (dcme-tp) and ethanolic extracts (ee-tp) of t. polium against anopheles stephensi http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 141 http://jad.tums.ac.ir published online: june 30, 2022 table 3. the identified compounds in dichloromethane and ethanol extracts of teucrium polium by gc-ms analysis the identified compounds in dcme-tp s. no retention time (min) area% name family structure 2 21.864 6.25 tetradecane alkane (ch2)12 ch3ch3 4 24.585 1.49 2,4-di-tert-butylphenol phenol (ch3)3c c(ch3)3 oh 5 26.574 21.96 hexadecane alkane (ch2)14 ch3ch3 11 30.826 25.5 octadecane alkane (ch2)16 ch3ch3 14 33.264 10.02 7,9-di-tert-butyl-1oxaspiro(4,5)deca-6,9 diene-2,8-dione oxaspiro o o o (ch3)3c (ch3)3c 20 34.695 21.08 eicosane (alkane) alkane (ch2)18 ch3ch3 33 43.799 100 di-isooctyl phtalate aromatic ester o o o o (ch2)5ch(ch3)2 (ch2)5ch(ch3)2 49 57.088 2.78 lupeol triterpenoid ho h3c ch3 ch3 ch3 ch3 ch3 ch3 h2c the identified compounds in ee-tp s. no retention time (min) area% name family structure 1 16.048 0.27 2,4-di-tert-butylphenol phenol (ch3)3c c(ch3)3 oh 2 48.525 100 1-methyl-pyrrolidine-2carboxylic acid aliphatic acid n o oh ch3 abbreviation: dcme; dichloromethane extract, ee; ethanolic extract, tp; teucrium polium discussion various species of anopheles mosquitoes showed resistance to synthetic anti-mosquito agents, whilst their environmental risk is worrying due to adverse effects on human, and non-target organisms (7). for these problems, the effectiveness of chemical control agents has been limited in malaria control and eradication programs in recent years. herbal anti-mosquito agents are environmentally friendly alternatives for synthetic inhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 142 http://jad.tums.ac.ir published online: june 30, 2022 secticides with high biodegradability, low residual effect, novelty in mechanism of action, with insignificant effect on the health of humans and pets, and with negligible harm to the non-targeted occupants of the area. given these facts, mosquitoes control agents from natural sources can be considered as safe and effective alternatives. several studies introduced mosquito repellent and larvicidal activity of some extracts and essential oils of plants (7–11, 21-22). according to these studies, the products of traditional medicine have the potential to be used successfully in mosquito control programs. the control of adult malaria vectors with adulticides and repellents is a suitable method to effect on longevity, mosquito densities and other transmission agents. personal protection is a common approach for preventing mosquito bites which helps indirectly in diminishing the mosquito population by depriving the blood meal which is vital for nourishment of the mosquito eggs in the female anopheles mosquito. people are mostly being bitten during peak mosquito biting hours, early in the evening, and sometimes throughout the night. therefore, finding a way to protect people from malaria is essential during these hours which this gap can fill with mosquito repellents including spatial, and topical repellents. the use of repellents protects local people and travelers in endemic areas and consequently reduces the occurrence of mosquito-borne diseases. there are the various repellents, such as n,n-diethyl-mtoluamide (deet), para-methane-3,8-diol (pmd), icaridin, 3-[n-butyl-n-acetyl]-aminopropionic acid, ethyl ester (ir3535) (23). deet is the most effective insect repellent available for human which has broad-spectrum activity on most mosquitoes, ticks and fleas (24). today, application of deet has been restricted due to the adverse effects. icaridin was also classified as slightly hazardous by the who hazard classification category (25). ir3535 and pmd have eye irritation. larvicides, pupicides and ovicides treatments can also help to control transmission parameters (7-11, 21-22). a larvicide is a type of insecticide that effects on the larval life stage of an insect and consequently, reduces the adult mosquito population in breeding areas. there are different formulations of larvicides which can be applied directly on water. therefore, these should not make an unreasonable health risk to humans or other wildlife. methoprene, an insect growth regulator agent prevents the normal maturation of insect larvae, has moderate and high toxicity on different aquatic animals and organisms. temephos, an organophosphate larvicide, affects the central nervous system through inhibition of cholinesterase and results in death before reaching the adult stage. similarly, there are the concerns about its toxicity on non-targeted aquatic species. in this situation, achievement to new alternatives especially, natural products, is one of the most important approaches of research groups. teucrium polium is a traditional plant in iran belonging to the family lamiaceae which is recommended in iranian traditional medicine manuscripts and some papers as an insect repellent (12, 17, 26-27). the present study is the first report of repellent and larvicidal activities of different extracts of t. polium against an. stephensi. dcme of t. polium showed 49.41% larvicidal activity against an. stephensi larvae and 56.67% repellency against mosquitoes. repellent and larvicidal activity of dcme-tp was greater than ee-tp. gc-ms analysis of t. polium extracts identified eight components in dcme containing alkanes (tetradecane, hexadecane, octadecane, and eicosane); 2,4-di-tert-butylphenol; 7,9-ditert-butyl-1-oxaspiro (4, 5) deca-6,9-diene-2,8dione; di-isooctyl phthalate; and lupeol (table 3). 2,4-di-tert-butylphenol and 1-methyl-pyrrolidine-2-carboxylic acid were identified in ee-tp(table 3). the various studies reported larvicidal and repellent activities of plants containing alkanes (28–30). dcme of t. polium was rich in alhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 143 http://jad.tums.ac.ir published online: june 30, 2022 kanes which might be responsible for its antimosquito activity. citrus hystrix and kaempferia galanga essential oils presented larvicidal activity against aedes aegypti (28). both were containing tetradecane. good larvicidal and adulticidal efficacy of acacia nilotica seed essential oil has been reported against larval mosquitoes; an. stephensi, ae. aegypti and culex quinquefasciatus (29). hexadecane was identified in a. nilotica as a major component that probably affects its insecticidal activity. the acetone leaf extract of melia azedarach was tested by ranchitha et al. (30) against larvae and pupae of ae. aegypti. eicosane was determined as one of its components. the various extracts of ocimum canum were evaluated against ae. aegypti and its chloroform extract showed significant larvicidal, pupicidal and adulticidal activity. eicosan was determined as a major component of this extract (31). phthalates are used as plasticizers in the production of plastics which are widely distributed in the environment. fortunately, their rapid photochemical and biological degradation has led to their low level (32). there are little reports of biological active plants containing phthalates. di-isooctyl phthalate was identified as a major component of dcme of t. polium. ramamurthy et al. (33) reported moderate larvicidal and pupicidal activity of ethanolic leaf extract of mukia maderaspatana containing di-isooctyl phthalate against ae. aegypti. based on babu et al. report (34), di-isooctyl phthalate was identified as a major component in the crude extract of pongamia pinnata. antifeedant and larvicidal activity of di-isooctyl phthalate against spodoptera litura showed remarkable results. lupeol was also one of compounds identified in this plant (34). the nanoparticles of acalypha indica including di-isooctyl phthalate showed the mosquito repellent and larvicidal properties against ae. aegypti, an. stephensi and cx. quinquefasciatus (35). 2,4-di-tert butylphenol, another identified component of t. polium has been found in various species of microorganisms, plants, and animals (36). chen and dai (37) reported the ovicidal, larvicidal, and adulticidal activities of 2,4di-tert butylphenol against tetranychus cinnabarinus in a concentration-dependent manner after treatment. lupeol, a pentacyclic triterpene also known as fagarsterol (38), identified in dcme of t. polium. according to the literature review, there are no reports about the identification of lupeol in t. polium. duan et al. (39) have claimed the acaricidal activity of lupeol, derived from inula japonica, and its potential as a botanical pest control agent. díaz et al. (40) isolated lupeol from dodonaea viscosa and reported its anti-insect activity against myzus persicae (green peach aphid) and epilachna paenulata (ladybird beetle). lupeol identified in vernonia brasiliana showed a mild inhibition activity against p. falciparum growth (41), since the lupeol is a lupane type of triterpenoids, it was considered as an interesting template for derivatization and was led to identify more potent antiplasmodial compounds (42). ajaiyeoba et al. (43) also reported the antiplasmodial activity lupeol isolated from ethyl acetate fraction of cassia siamea against multi-resistant strain of p. falciparum (k1). dichloromethane extract of dendranthema grandiflorum containing lupeol presented larvicidal activity against a. aegypti third instar larvae (44). larvicidal activity of chloroform extract of carica papaya latex and silver nanoparticles (cpagnps) of aqueous latex extract confirmed better activity of cpagnps in lower dose against ae. aegypti and cx. quinquefasciatus (45). diisooctyl phthalate, 2,4-di-tert-butylphenol, hexadecane, and 7,9-di-tert-butyl-1-oxaspiro (4, 5) deca-6,9-diene-2,8-dione were some identified compounds in chloroform extract of c. papaya similar to t. polium in this study (45). according to the studies mentioned above, each of the main components of t. polium identified in the present study has displayed proper anti-insect activity. the larvicidal and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 136–147 s ghafari et al.: phytochemical composition and … 144 http://jad.tums.ac.ir published online: june 30, 2022 repellent activity of dcme of t. polium can be probably related to the synergistic effect between the mentioned components. conclusion this study introduces an ideal eco-friendly mosquito repellent from the extract of teucrium polium which has a proper potential as a promising candidate for mosquito control. however, further complementary studies such as fractionation extracts to get the molecule(s), mostly responsible for repellent activity, other formulation of extracts, and field trials should be performed to confirm repellent activity. acknowledgements the authors would like to thank the staff members of the insectarium of the school of public health, shiraz university of medical sciences (sum) to perform bioassay. ethical considerations this study was performed based on ethical considerations and national regulations in animal experiments (no. 1080). conflict of interest statement authors declare that there is no conflict of interest. references 1. who (2022) world malaria report 2021. world health organization. geneva, switzerland. available at: https://www.who.int/teams/globalmalaria-programme/reports/worldmalaria-report-2022. 2. azari-hamidian s, norouzi b, harbach re (2019) a detailed review of the mosquitoes (diptera: culicidae) of iran and their medical and veterinary importance. acta tropica. 194: 106–122. 3. sedaghat mm, sanei-dehkordi ar, abai m, khanavi m, mohtarami f, abadi ys (2011) larvicidal activity of essential oils of apiaceae plants against malaria vector, anopheles stephensi. iran j arthropod borne dis. 5(2): 51–59. 4. hemingway j (2014) the role of vector control in stopping the transmission of malaria: threats and opportunities. philos trans r soc lond b biol sci. 369(1645): 20130431. 5. who (2018) global report on insecticide resistance in malaria vectors: 2010–2016. world health organization. geneva, switzerland. available at: https://apps.who.int/iris/handle/10665/2 72533. 6. bekele d (2018) review on insecticidal and repellent activity of plant products for malaria mosquito control. biomed res rev. 2(2): 1–7. 7. tahghighi a, ravasan nm, djadid nd, alipour h, ahmadvand r, karimian f, yousefinejad s (2019) gc–ms analysis and anti–mosquito activities of juniperus virginiana essential oil against anopheles stephensi (diptera: culicidae). asian pac j trop biomed. 9(4): 168–175. 8. krishnappa k, dhanasekaran s, elumalai k (2012) larvicidal, ovicidal and pupicidal activities of gliricidia sepium (jacq.) 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https://pubmed.ncbi.nlm.nih.gov/?term=ashidi+js&cauthor_id=17705142 https://pubmed.ncbi.nlm.nih.gov/?term=okpako+lc&cauthor_id=17705142 https://pubmed.ncbi.nlm.nih.gov/?term=wright+cw&cauthor_id=17705142 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 104 original article biological activities and composition of ferulago carduchorum essential oil fereshteh golfakhrabadi 1, mahnaz khanavi 1,2, seyed nasser ostad 3, soodabeh saeidnia 4, hassan vatandoost 5, mohammad reza abai 5, mitra hafizi 1, fatemeh yousefbeyk 6, yaghoob razzaghi rad 1, ameneh baghenegadian 1, *mohammad reza shams ardekani 1,2 1department of pharmacognosy, faculty of pharmacy, tehran university of medical sciences, tehran, iran 2department of traditional pharmacy, faculty of traditional medicine and persian medicine and pharmacy research center, tehran university of medical sciences, tehran, iran 3department of toxicology and pharmacology, faculty of pharmacy and pharmaceutical sciences research center, tehran university of medical sciences, tehran, iran 4medicinal plants research center, tehran university of medical sciences, tehran, iran 5department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 6department of pharmacognosy, faculty of pharmacy, guilan university of medical sciences, rasht, iran (received 18 aug 2013; accepted 28 june 2014) abstract background: ferulago carduchorum boiss and hausskn belongs to the apiaceae family. this plant grows in west part of iran that local people added it to dairy and oil ghee to delay expiration date and give them a pleasant taste. the aim of this study was to investigate the antioxidant, antimicrobial, acetyl cholinesterase inhibition, cytotoxic, larvicidal activities and composition of essential oil of f. carduchorum. methods: acetyl cholinesterase (ache) inhibitory, larvicidal activities and chemical composition of essential oil of f. carduchorum were investigated. besides, antioxidant, antimicrobial and cytotoxic activities of essential oil were tested using dpph, microdilution method and mtt assay, respectively. results: the major components of essential oil were (z)-β-ocimene (43.3%), α-pinene (18.23%) and bornyl acetate (3.98%). among 43 identified components, monoterpenes were the most compounds (84.63%). the essential oil had noticeable efficiency against candida albicans (mic= 2340 µ g ml-1) and it was effective against anopheles stephensi with lc50 and lc90 values of 12.78 and 47.43 ppm, respectively. the essential oil could inhibit ache (ic50= 23.6 µ l ml -1). the essential oil showed high cytotoxicity on t47d, hep-g2 and ht-29 cell lines (ic50< 2 μg ml-1). conclusion: the essential oil of f. carduchorum collected from west of iran had anti-candida, larvicidal and cytotoxicity effects and should be further investigated in others in vitro and in vivo experimental models. keywords: ferulago carduchorum, essential oil, antimicrobial, antiacetyl cholinesterase, larvicidal activity introduction ferulago carduchorum boiss and hausskn (apiaceae) known as an endemic plant of iran, grows in west part of iran (mozaffarian 2007). in west of iran, f. carduchorum has been traditionally added to dairy and oil ghee to increase hold time and give them a pleasant taste. in the past, this plant was used as natural preservative to delay expiration date of meat, too. some species of ferulago are benefit for remedy of digestive pains, hemorrhoid (sodeifian et al. 2011), disease of spleen, headache, ulcers and snake bites (demetzos et al. 2000). phytochemical studies on ferulago species have led to identification of different coumarins (ognyanov et al. 1969, andrianova et al. 1975, serkerov et al. 1976, sklyar et al. 1982, de pascual et al. 1979, doganca et al. 1991, *corresponding author: dr mohammad reza shams ardekani, email: shams@tums.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 105 doganca et al. 1992, ruberto et al. 1994, jimenez et al. 2000, khalighi-sigaroodi et al. 2006). there are also some reports about the acetyl cholinesterase inhibitory (dall'acqua et al. 2010), cytotoxic (rosselli et al. 2009), antimicrobial and antioxidant (basile et al. 2009) activities of coumarins of f. campestris furthermore composition of essential oil of 6 different ferulago species (erdurak et al. 2006, kilic et al. 2010) and antimicrobial activity of essential oil of f. bernardii (khalighi-sigaroodi et al. 2005) and f. campestris (cecchini et al. 2010) have been reported later. alzheimer’s disease (ad) is the most common and important degenerative disease of brain among the elderly and is the fourth leading cause of death in western countries (shen et al. 2005, zhou et al. 2008). reports suggest that ad is caused by reduced synthesis of acetylcholine (weinstock et al. 1997), so the use of acetyl cholinesterase enzyme inhibitors (acheis) could help improve ad (grutzendler et al. 2001). traditional ad drugs exhibit side effects and many efforts have been made to achieve a variety of natural acheis with less adverse effects (karimi et al. 2010). furthermore, a lot of medications for cancer treatment are not effective enough so, researchers are trying to find the most effective medicinal plants to treat cancers (rahimifard et al. 2009). mosquitoes are important vectors in transmission of some human diseases such as malaria, dengue fever, yellow fever and filariasis, which they are substantially among the greatest health problems all over the world (james et al. 1992). different species of anopheles transmit malaria, filariasis and certain arboviruses (sedaghat et al. 2005). malaria is one of the most important diseases in southern iran (vatandoost et al. 2011). in previous reports different plants showed toxic effects against public health pests (hadjiakhoondi et al. 2005, hadjiakhoondi et al. 2006, hadjiakhoondi et al. 2008a, hadjiakhoondi et al. 2008b, vatandoost et al. 2008) the in vitro investigation of essential oils is important to estimate their potential to use as an antibiotic or as a drug for ad, cancers, malaria and as a supplement to edible or pharmaceutical products. the aim of this study was to investigate the antioxidant, antimicrobial, acetyl cholinesterase inhibition, cytotoxic, larvicidal activities and composition of essential oil of f. carduchorum. materials and methods plant material the aerial parts of ferulago carduchorum (apiaceae family) were collected from manesht mountain of illam province in june 2011. the longitude and latitude of manesht mountain are 33°40'60" n and 46°28'0" e. this plant is an endemic plant of iran, which grows in west part of iran. the plant was identified and authenticated by mr yousef ajani using flora iranica (rechinger 1978). the voucher specimen is deposited in herbarium institute of medicinal plants (acecr), karaj, iran (herbarium number: 1450). isolation of the volatile oil the essential oil was obtained by hydrodistillation using a clevenger type apparatus (advanced technocracy inc., india) for 4 h according to the european pharmacopoeia (1975) (maisonneune1975). the aerial parts of ferulago carduchorum (apiaceae family) were dried under shade and powdered. the air-dried parts of f. carduchorum (150 g) and 1,000 ml distilled water placed in a round bottom flask (using a fire source from below) connected to a clevenger-type apparatus. hydrodistillation is often used to isolate non-water soluble compounds. hydrodistillation is a method that the plant parts being boiled in water, using a heating source http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 106 from below the vessel. in the presence of boiling water volatile compounds are volatilized at a temperature close to 100 °c, at atmospheric pressure. the volatile materials escape in vapor form through some tubes and then are cool. the essential oil is removed from the top of the hydrosol. 1 ml essential oil obtained duration 1 h hydrodistillation. the oil was dried over anhydrous sodium sulphate and kept at 4 °c in the sealed brown vial until required. 1.3 ml essential oil has been obtained from 100 gram dried plant. the oil yield of the plant was determined as 1.3% v/w. the isolation of the volatile oil has been carried out in laboratory of pharmacognosy, faculty of pharmacy, tehran university of medical sciences, tehran, iran. gas chromatography mass spectroscopy analytical gas chromatography (gc) was carried out using a shimadzu 15a gas chromatograph with capillary column hp-5ms (60m×0.25mm, ft 0.25mm), carrier gas, he, split ratio 1:25 and using a flame ionization detector (fid). the column temperature was programmed at 60 °c for 3 min and then it was heated to 260 °c at a rate of 5 °c min-1 and the temperature was then kept constant at 260 °c for 15 min. gas chromatography mass spectroscopy (gc/ms) was carried out on a hp 68900 with a hp 5973 quadruple detector, on capillary column hp-5ms (5% phenyl methyl siloxane) (60m×0.25mm, ft 0.25mm), carrier gas, he, flow rate, 1 ml min-1. the column was held at 60 °c for 3 min and programmed up to 260 °c at the rate of 5 °c min-1, then kept constant at 260 °c for 15 min. the ms was operated at 70 ev ionization energy. retention indices were calculated using the retention time of n-alkanes that were injected after the oil at the same chromatographic conditions. quantitative data were obtained from the electronic integration of the fid peak areas. the components of the oils were identified by comparison of their mass spectra and retention indices with wiley library and those published in the literature (adams 1995). dpph radical scavenging activity the 1, 1-diphenyl-2-picryl hydrazyl radical (dpph) (merck, germany) has a maximum absorption at 517 nm which was used for the investigation of the free radical-scavenging activity of the essential oil (yokozawa et al. 1998, khanavi et al. 2009). antimicrobial activity antimicrobial activities of essential oil of aerial parts of f. carduchorum was determined against both gram-positive (staphylococcus aureus atcc 6538), gram-negative (escherichia coli atcc 8739, pseudomonas aeruginosa atcc 9027) bacteria and a fungal strain (candida albicans atcc 1023) by microdilution method. negative control was prepared using dimethylsulphoxide (dmso), which was solvent used to dissolve the essential oil. gentamycin was used as positive control against s. aureus, e. coli, p. aeruginosa while, nystatin was used as positive control against c. albicans. their dilutions ranged from 10 to 0.009 μg/ml concentrations in microtitre plates. the antimicrobial activity of essential oil was assessed by the agar well diffusion method. the plates were incubated at 37 oc for 24 hours for bacteria and 20–25 oc for c. albicans. inhibition was detected by measuring clear zones around the wells in millimeters. minimum inhibitory concentration (mic) of the essential oil was assessed by broth microdilution method with visible growth observed by using 96 u-shaped wells plates (nccls 2006). after 24 h of incubation at 35 oc (bacteria) and 20–25 oc (c. albicans), the microdilution plates were tested for the absence or presence of visible growth in comparison with that of the growth in drug-free control well. the endpoint of mic is the lowest concentration of http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 107 the compound at which the test strain does not demonstrate visible growth. acetyl cholinesterase (ache) inhibition the enzymatic activity was measured using by the method described by aazza et al. (2011) with minor modifications. 50 µ l of buffer 0.1 m (ph 8), 25 µl of essential oil dissolved in dimethyl sulphoxide (dmso) with different concentrations and 25 µ l of 0.22 u/ml of ache enzyme were mixed. after 15 min incubation at 37 °c, 25 µl of 15 mm acetyl thiocholine iodide (achi) and 125 µ l of 3 mm 5,5’dithiobis [2-nitrobenzoic acid] (dtnb) were added and the resulting mixture incubated for 30 min at room temperature. absorbance of the mixture was measured at 405 nm by using a microplate reader (elx808, biotek, usa). the inhibitory effect of test compound was calculated by comparing to the negative control: %= [(a0–a1)/ a0]* 100 where a0 was the absorbance of the blank sample and a1 was the absorbance of the sample. the test was repeated three times. the inhibition of enzyme activity was expressed as ic50 (the concentration of the sample (µ l ml 1), required to inhibit 50% of enzyme), calculated by a linear regression analysis. bioassays and larval mortality fourth instar larvae of anopheles stephensi bandar-abbas strain was exposed to test concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 ppm of essential oil (solvent: ethanol) for 24 hours according to standard method described by who (1981). in briefly 1 ml of appropriate dilution of essential oil with 224 ml of water and 25 larvae in 25 ml water mixed and total volume was 250 ml (dharmagadda et al. 2005). for control, only 1 ml of ethanol with 224 ml of water and 25 larvae in 25 ml water mixed and total volume was 250 ml. the experiment was repeated four times on different days. the percentage of mortality was reported from the average for the four replicates after 24 hour exposure period. from the regression line between logarithmic dose and probit mortality, the lc50 was determined. the investigation of larvicidal activity has been carried out in the insectarium of department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. cytotoxicity assay the colon carcinoma (ht-29), breast carcinoma (t47d), hepatocellular carcinoma (hepg2) was obtained from pasture institute of iran, tehran, iran. the colon carcinoma ht-29 and t47d (breast carcinoma) cell lines were mentioned as exponentially growing cultures in rpmi 1640 cell culture medium (paa, germany), supplemented with 10% fetal bovine serum (fbs: gibco, usa), for ht-29 cells and 15% fbs for t47d cells. the hepatocellular carcinoma (hepg2) were cultured in dulbecco’s modified eagle’s medium (dmem, paa, germany) supplemented with 10% fbs. the swiss mouse embryo fibroblast (nih 3t3) cell line was kept in dulbecco’s modified eagle’s medium (dmem, paa, germany) supplemented with 10% fbs. 100 iu/ml penicillin and 100 μg/ml streptomycin (roche, germany) were added to the media. all the cell lines were cultured at 37 °c in air /carbon dioxide (95:5) atmosphere. cytotoxic activity was measured using modified mtt assay (newman dj 2007). 1×104 cells/well were plated in 96-well plates (nunc, denmark) and incubated for 24 h before the addition of drugs. after 48 h of incubation in ht-29, nih/3t3 and mcf-7 cells, 20 μl of mtt (merck, germany) reagent (5 mg/ml) in phosphate buffered saline (pbs) was added to each well. the plates were incubated at 37 °c for 4 h. the medium was discharged and the formazan blue, which had been formed in the cells, were dissolved with 100 μl dimethyl sulphoxide (dmso). after the incubation at 37 °c for http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 108 10 min, absorbance at 570 nm at the dissolved solutions was detected using a micro plate reader (anthos, austria). the cell viability in mtt assay was calculated as the percentage of control value (khanavi et al. 2012b). methotrexate was used as the positive control. cytotoxicity was expressed as the concentration of extract inhibiting cell growth with 50% (ic50±sd). all tests and analysis were run in triplicate. statistical analysis in antioxidant and antiacetyl cholinesterase assays, analyses were carried out in triplicate and the data were expressed as mean ±sd. one-way anova and tukey post-hoc multicomparison tests were used for the analyses after data normality test. in cytotoxicity assay, ic50 (the median growth inhibitory concentration) values were calculated from the ic50 of dose-response curve in the sigma plot 11 software. data representative of three independent experiments with similar results were presented as mean ± sd. for larvicidal activity was used from microprobit software (version 3.0). the percentages of mortality were corrected for the mortality in controls by using abbott's correction. from the regression line between the logarithmic dose and probit mortality, all the parameters including lc50, lc90, confidence interval (ci) and slope values were determined (abbott 1925). results chemical composition of the essential oil the results of essential oil analysis of f. carduchorum were led to identification of 43 compounds (table 1), represented 92.3% of the total oil. gas chromatography-mass diagram of f. carduchorum essential oil showed in figure 1. the oil yield of the plant was determined as 1.3% v/w. major component of essential oil were identified as (z)-βocimene (43.3%), α-pinene (18.23%), bornyl acetate (3.98%) and myrcene (3.15%). antioxidant and antimicrobial activities in this research radical scavenging activity of essential oil of f. carduchorum was determined and ic50 value was calculated as 29.61 µ l. vitamin e also used as a reference and positive control compound. in our antimicrobial investigation, the results of minimum inhibitory concentration (mic) are shown in table 2. the results of gentamycin and nystatin were used as positive control against bacterial and fungal strain, respectively. the results of positive (gentamycin and nystatin) and negative (dmso) controls are shown in table 2. acetyl cholinesterase inhibition activity acetyl cholinesterase inhibition activity of f. carduchorum essential oil was studied for the first time. the results showed that the ic50 of essential oil was 23.6 µ l ml -1. larval mortality the larvicidal activity of our essential oil and methanol against anopheles stephensi larvae under laboratory conditions are presented in table 3. essential oil of f. carduchorum was effective against an. stephensi with lc50 and lc90 values of 12.78 and 47.43 ppm, respectively. cytotoxicity effect the effects of f. carduchorum essential oil on the proliferative response of the ht29, hepg2 and t47d cell lines have been analyzed by treating the cells with different concentrations of the essential oil and significant decrease in cell lines proliferation were observed. the results of cytotoxic tests have been shown in table 4. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 109 table 1. chemical composition of essential oil of ferulago carduchorum no. component rta rib composition (%) 1 α-pinene 10.133 937 18.23 2 camphene 10.403 946 1.63 3 verbenene 10.485 958 trace 4 β-pinene 11.443 971 1.81 5 myrcene 12.306 989 3.15 6 α-phellandrene 12.697 997 0.4 7 p-cymene 13.654 1017 0.21 8 o-cymene 13.500 1020 0.61 9 -3-carene 13 1029 0.25 10 β-phelandrene 13.819 1031 2.07 11 (z)-β-ocimene 14.969 1045 43.3 12 (e)-β-ocimene 15.47 1055 2.71 13 -terpinene 16.807 1062 1.82 14 terpinolene 16.917 1088 0.16 15 linalool 17.236 1100 0.16 16 1,3,8-para-menthatriene 18.155 1115 trace 17 allo-ocimene 18.744 1129 2.29 18 cis-verbenol 18.904 1135 0.26 19 neoallo-ocimene 18.799 1150 trace 20 trans-verbenol 19.162 1156 1.01 21 p-mentha -1,5-dien-8-ol 20.043 1165 0.39 22 4-terpineol 20.516 1170 0.13 23 α –terpineol 21.159 1180 0.06 24 dodecane 22.234 1200 1.02 25 bornyl acetate 25.446 1277 3.98 26 α cubebene 33.892 1366 2.65 27 α -ylangene 31.025 1370 0.16 28 α -copaene 29.649 1378 0.36 29 β–bourbonene 29.952 1382 0.43 30 β -cubebene 30.156 1389 0.14 31 β -elemene 30.271 1399 0.14 32 α -gurjunene 36.654 1421 0.32 33 -elemene 31.944 1428 0.1 34 aromadendrene 31.306 1433 0.31 35 α -humulene 32.659 1446 trace 36 transβ-farnesene 33.017 1452 trace 37 germacrene-d 33.198 1475 trace 38 bicyclogermacrene 34.431 1498 0.68 39 β bisabolene 35.047 1500 0.15 40 -cadinene 35.482 1513 0.5 41 spathulenol 37.424 1568 0.71 42 salvial-4(14)-en-1-one 37.754 1589 trace 43 α-cadinol 39.862 1650 trace total 92.3 monoterpenes hydrocarbons 78.64 monoterpenes oxygenated 5.99 sesquiterpenes hydrocarbons 5.94 sesquiterpenes oxygenated 0.71 nonterpenoids 1.02 notes a retention time, bri retention index on hp-6890 with reference to nalkanes injected after the oil at the same chromatographic conditions. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 110 table 2. minimum inhibitory concentration (mic) of ferulago carduchorum essential oil against selected bacteria and candida albicans mica positive control positive control negative control microorganism essential oil gentamycin nystatin dmso staphylococcus aureus 9000 0.62 escherichia coli 23000 0.009 pseudomonas aeruginosa 18000 2.48 candida albicans 2340 0.02 notea: mic was determined by broth micro dilution method and expressed in µ g ml-1. no effect. table 3. probit regression line of anopheles stephensi exposed to different interval concentrations of essential oil of ferulago carduchorum intercept slope±se* lc50 (ppm) 95% ci lc90 (ppm) 95% ci χ2 χ2table (df) p-value -2.4906 1.9303± 0.323 12.7818 19.5101– 33.4434 47.4356 89.9890– 357.8734 35.78 * 22.458 (6) 0.001 *: no heterogeneity; se: standard error, lc50: lethal concentration to cause 50% mortality in population; lc90: lethal concentration to cause 90% mortality in population, ci: confidence interval, χ2 (df) = heterogeneity about the regression line (degrees of freedom). table 4. cytotoxic activity of essential oil of ferulago carduchorum cell linesa (mtt assay) essential oil methotrexate doxorubicin nih-3t3 0.64±0.02 0.24 ± 0.013 0.21 ± 0.03 ht-29 1.74±0.14 0.23 ± 0.02 t47d 0.17±0.01 0.16 ± 0.09 hep-g2 0.35±0.02 1.04 ± 0.07 a: results are expressed as ic50 values (μg ml -1), key to cell lines employed: ht-29 (colon carcinoma), t47d (breast carcinoma), hep-g2 (hepatocellular carcinoma), nih 3t3 (swiss embryo fibroblast). fig. 1. gas chromatography-mass diagram of ferulago carduchorum essential oil http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 111 discussion among 43 identified components, monoterpenes were the most identified compounds (84.63%) that only 5.99% of them were oxygenated whereas, sesquiterpenes were totally detected about 6.65% with 0.71% oxygenated sesquiterpenes. (z)-β-ocimene and myrcene as acyclic monoterpene hydrocarbons and α-pinene as a cyclic monoterpene hydrocarbon were identified as main compounds. moreover, the major oxygen containing monoterpene and sesquiterpene are bornyl acetate (3.98 %) and spathulenol (0.71 %), respectively. composition of the oil obtained from air-dried aerial parts of f. carduchorum from kerman province, was found to contain (z)-β-ocimene as the major component (samiee et al. 2006), which agrees with our research. the amount of (z)-β-ocimene in our investigation (43.3 %) was more than previous study (21.2 %). more ever f. carduchorum from illam and kerman provinces had shown the constituent of α-pinene 18.23 % and 4.8 % respectively. moreover, other reports showed that in f. humillis, f. trachycarpa and f. angulata (z)-β-ocimene is the main component (baser et al. 2002, khanahmadi et al. 2006). the important compound in three species (f. aucheri and f. mughlae and f. sandrasica) was α-pinene. the major components in essential oil of f. macroseiadia, f. sylvatica and f. bernardii were methyl carvacrol, p-cymene and 2, 4, 5-trimethyl benzaldehyde, respectively (baser et al. 2002, khalighi-sigaroodi et al. 2005). evaluation of mic of the essential oil showed that sample had antimicrobial effect against both gram-positive (staphylococcus aureus), gram-negative (escherichia coli, pseudomonas aeruginosa) bacteria and a fungal strain (candida albicans). the essential oil indicated noticeable efficiency against c. albicans (mic= 2340 µ g ml-1). the antioxidant assessment of fruits and roots of f. campestris were demonstrated that their ic50 were less than ic50 of essential oil of f. carduchorum (cecchini et al. 2010). moreover, antimicrobial effect of f. campestris was better than f. carduchorum, that mic of roots and fruits essential oils were less than mic of f. carduchorum essential oil (cecchini et al. 2010). the results of antimicrobial activity of f. bernardii essential oil were shown mic of essential oil againt gram-positive, gram-negative bacteria and fungal strain were <1000 µ g ml-1 (khalighi-sigaroodi et al. 2005). so, antimicrobial activity of f. bernardii is better than f. carduchorum. anti-candida effect of ferulago capillaris essential oil was investigated and the results were demonstrated that mic is less than f. carduchorum (pinto et al. 2013). there have been reports on the ache inhibitory activity of some bicyclic monoterpenoides including α-pinene and 3carene (miyazawa et al. 2005). hence in our study, ache inhibitory activity of essential oil could be attributed to its α-pinene. in a previous study, larvicidal activity of eucalyptus camaldulensis essential oil against an. stephensi was investigated and the lc50 and lc90 values were 89.85 and 215.26 ppm, respectively (sedaghat et al. 2010). in one study, the larvicidal activities of methanolic extracts of some iranian plants (lawsonia inermis, thymus kotschyanus, cedrus deodara and eight species from stachys) against malaria vector, an. stephensi were investigated (khanavi et al. 2013). these results indicated that essential oil of f. carduchorum was more potent than these plants against an. stephensi. compared to another studies on larvicidal activity of essential oils of heracleum persicum, foeniculum vulgare, coriandrum sativum (sedaghat et al. 2011), cymbopogon olivieri (hadjiakhoondi et al. 2003) and nepeta menthoides (khanavi et al. 2012a) it was found that f. carduchorum was most effective against an. stephensi http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 104–115 f golfakhrabadi et al.: biological activities … 112 (khanavi et al. 2011). therefore, the essential oil of f. carduchorum can be used in insect control as an alternative for chemical compounds on the environment. the essential oil of f. carduchorum showed a high cytotoxicity on t47d, hepg2 and ht-29 cell lines (ic50< 2 μg ml-1). the effect of essential oil on t47d cell line was much stronger than ht-29 and hep-g2. it indicated that the essential oil had potential cytotoxic selectivity on t47d cell line similar to methotrexate (positive control), whereas, the essential oil had lower cytotoxic effect than methotrexate on nih 3t3 (swiss embryo fibroblast). so, the oil could be safer than this drug. another study on vinca minor revealed that its cytotoxicity activity on t47d (ic50= 1.34±0.29) and ht29 (ic50= 3.63±1.24) was less than f. carduchorum (khanavi et al. 2010). in results, this plant has active cytotoxic components. conclusion the essential oil of f. carduchorum collected from west of iran give good biological activity including anti-candida, ache inhibitory, larvicidal and high cytotoxic effects and should be further investigated in others in vitro and in vivo experimental models. the determined biological activities of f. carduchorum essential oil are important information for its future application in treatment. furthermore, the 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mohaghegh ardabili, ardabil, iran 4social determinants of health research center, urmia university of medical sciences, urmia, iran (received 2 mar 2018; accepted 4 dec 2018) abstract background: west nile fever, as an expanding zoonotic disease, has been reported from different creatures involved in the disease from iran. in addition to biological mosquito-associated factors, various elements such as their activities, distribution, behavior and vectorial capacity could be affected by environmental factors. we determined the distribution of west nile virus (wnv) vectors, the environmental factors affecting wnv transmission and the high-risk areas across west azerbaijan province (northwestern iran), regarding the potential of wnv transmission using geographical information system (gis). methods: mosquitoes’ larvae and adults were collected from different habitats of the province in 2015 and identified using standard morphological keys. the data regarding the distribution of mosquitoes across the studied area were organized in arcmap databases. inverse distance weighted (idw) interpolation analysis was conducted on the data of synoptic stations to find climatic variables in the collection sites of different mosquito species. layers of transmission-related environmental factors were categorized and weighed based on their effects on disease transmission. results: overall, 2813 samples of different mosquito species from different regions of the province were collected and identified. according to the gis analysis, areas in the northeastern province, which have lower altitudes and slopes with higher temperatures and more water bodies, were found to have better condition for the activity of mosquitoes (as high-risk areas: hot spots). conclusion: the precision of our results was proven to be in line with previous study results that identified high-risk areas, where wnv-infected vectors were captured from these same areas. keywords: mosquitoes; aedes caspius; culex pipiens; iran introduction due to notable problems caused by mosquitoes and mosquito-borne diseases (mbds), the studies of factors influencing the presence, activities, and distribution of mosquitoes and mbds are important and form absolute parts of the epidemiology of mbds, in which environmental conditions and their changes are components of this process (1). in addition to biological factors, mosquitoes’ activities, distribution, behavior, and even their vecto rial capacity could be affected by environmental conditions (2-5). mosquitoes’ feeding rates vary expressively with temperature. moreover, feeding behavior and host availability are affected by climate. additionally, length of the gonotrophic cycle as an important factor in diseases transmission could be influenced by precipitation patterns (6). finally, a positive correlation between temperature and west nile and west *corresponding author: dr ali reza chavshin, email: chavshin@umsu.ac.ir, chavshin@gmail.com j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 28 http://jad.tums.ac.ir published online: april 27, 2019 nile virus (wnv) incidence in mosquitoes have been reported (7, 8). among the different tools used to identify the effects of environmental factors on mbds and their epidemiology, is geographical information system (gis) (9). in addition, global positioning systems (gps), remote sensing, and spatial statistics could also play important role in mbds research, surveillance, and control programs (10). among the mbds, west nile fever is an emerging zoonosis rapidly spread and caused expansive health threat in different parts of the world (11, 12). the transmission cycle of wnv includes a wide range of migratory birds as reservoirs (13), equines (14) and humans (15) as dead-end hosts, and numerous mosquito species including different species e.g. culex pipiens, cx. restuans, cx. salinarius, cx. tarsalis, aedes vexans, ae. albopictus, and coquillettidia perturbans as biological vectors (16-18). wnv has been isolated and identified in numerous mosquito species including different species of culex, aedes, and anopheles (19–21). due to the wide use of gis in the study of vector-borne diseases (including mbds) (22, 23), several studies have employed gis across the world to predict, risk assessment (18), surveillance (25, 26) and the environmental factors influencing wnv transmission (27, 28). because of the presence of theoretically favorable environments for the establishment of wnv across iran, the presence of wnv has been investigated and showed the seroprevalence rate of (1.3%) in human (29-33), 23.7% in equines (34), overall (15%) infection in birds (35) and recently among the potential vector species, in which among them wnv has been isolated and reported from ae. caspius (36) and culex spp (37). iranian west azerbaijan province in the northwestern part of the country, could be considered as one of the most suspicious areas in iran regarding the possibility of establishment and transmission of wnv, because of its abundant water resources and wetlands for migratory birds from different parts around the world, which serve as reservoirs of wnv (38, 39) and also, the presence of potential vector species of mosquitoes in this region e.g. culex pipiens s.l., ae. caspius, anopheles maculipennis s.l., culiseta longiareolata (40-42). as the first isolation of wnv from its potential vectors was reported from this region (36), and this region borders several countries such as turkey, iraq, armenia, and the republic of azerbaijan, more attention is needed at this area. due to the special circumstances mentioned above about west azerbaijan province, we aimed to determine: 1) the distribution of probable wnv vectors, 2) the environmental factors affecting wnv transmission and 3) the high-risk areas across the province regarding the potential of wnv transmission using gis. materials and methods study area west azerbaijan province is located in the northwest of iran between latitudes 35° 58´– 39° 46´ n and longitudes 44° 3´–47° 23´ e. this province formally includes 17 counties. it is bordered by turkey, iraq, armenia, and the republic of azerbaijan. in addition, it is also bordered by iranian provinces such as east azerbaijan, zanjan and kurdistan (fig. 1). according to information obtained from forests, range and watershed management organization, west azerbaijan province have five types of micro-climates, including highly semi-arid (hsa), moderate semi-arid (msa), slight semi-arid (ssa), semi-wet (sw) and wet (w). distribution of potential west nile vectors in west azerbaijan province mosquitoes were collected during may– nov 2015 from 24 localities (wetlands) across the province (table 1). adults and larvae were j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 29 http://jad.tums.ac.ir published online: april 27, 2019 collected from different habitats using standard methods (43). collected samples were quickly identified using a stereo microscope and a standard morphological key at the collection sites (44). climatic and environmental data as distribution of mosquitoes and their transmitted diseases are notably dependent on environmental and climatic factors, the effective climatic data, including maximum monthly temperature, minimum monthly temperature, mean monthly temperature, mean relative humidity and rainfall data of decade (2004–2014) were obtained from 16 stations of west azerbaijan meteorological organization (table 2). datasets at district level were created in excel sheets for further analysis by arcgis 10.3. the pattern of recent decade of maximum, mean and minimum temperature of different areas in west azerbaijan province was analyzed and mapped in fig. 2 and summarized in table 2. spatial analysis for proper analysis, the data regarding distribution of mosquitoes across west azerbaijan province were acquired from previous studies (30, 33) and added to the findings of this survey in database created in arcmap. inverse distance weighted (idw) interpolation analysis was conducted on the data of synoptic stations to find the climatic variables in the collection sites of different mosquito species. this analysis interpolates a raster surface from points and estimates cell values by averaging the values of nearby sample data points. the closer a point is to the center of the cell which is being estimated, the more weight it is given. the equation for idw analysis is: where vˆ= value to be estimated vi= known value di..., dn= distances from the n data points to the point estimated n layers of environmental factors that are important in transmitting the wnv were categorized and weighted based on their effects on disease transmission, and important impact on vector-borne diseases (3, 45-47) (table 3). the categorized and weighted environmental factors were overlaid with the vectors distribution across the west azerbaijan province for determination of high-risk areas for the establishment of the transmission cycle of wnv based on the mentioned environmental factors. as the five species (ae. caspius, an. maculipennis, cs. longiareolata, cx. pipiens and cx. theileri) reported from study area, also are known proven and suspected vectors of wnv in different parts of the world, they have been included in final analysis (fig. 3). ethics approval and consent to participate prior to the approval of all projects by the urmia university of medical sciences (umsu), they are reviewed and endorsed by the ethics committee of the umsu. sample collection was carried out from private human and animal dwellings. at least one day prior to any sample collection, the owners were informed by the local health system officers. the whole process was coordinated, managed and documented by the “local health system officer” in the study areas. results overall, 2813 mosquito specimens from different regions of the province were collected and identified (table 1). analysis of some climatic variables across the collection sites for different mosquito species is summarized in table 4. the most frequent species in our study was cx. pipiens (22 out of 24 collection j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 30 http://jad.tums.ac.ir published online: april 27, 2019 sites), while ae. caspius was found only in 10 localities. the altitudinal activity of suspected vectors was determined across the areas with an average of 1214m for ae. caspius to 1364m for cs. longiareolata (table 4). considering the altitude of the study area which ranges from 605m to 3600m, the values of the altitude were classified into five categories, by overlying the presence of potential vectors and their distribution map on altitude values of the province, height less than 1615m (605–1500m), are more prepared and favored places for mosquitoes in the study areas. finally using the scale of the most important environmental variables (table 3), determination of high risk areas for wnv and distribution of potential vectors across the studied areas was analyzed and revealed in fig. 3. the map of the mosquitoes including proven and suspected vectors of wnv, ie cx. pipiens, cx. theileri and ae. caspius were collected mostly in hot spots, determined by the model (fig. 3). table 1. collection sites of mosquitoes in west azerbaijan province, northwestern iran, 2015 county collection site longitude latitude altitude a e . c a sp iu s a n . m a c u lip e n n is c x . p ip ie n s c x . th e ile ri c s. lo n g ia re o la ta bazargan bazargan 44.38944 39.38833 1400 0 25 1 13 0 yarim-ghiye 44.43656 39.44604 1409 175 65 0 420 0 khoy hashiyeh rood 45.06315 38.57117 1058 3 0 6 18 0 mahabad mahabad 45.71667 36.75 1351 0 10 89 25 4 khoor-khooreh 45.72341 36.98732 1279 205 2 120 0 30 kani barazan-wetland 45.77716 36.98689 1275 0 0 68 1 0 hajib khosh 45.79718 36.90788 1288 0 0 136 0 0 gapis 45.74795 36.93805 1286 0 0 4 7 0 beytas 45.69427 36.67645 1396 3 0 28 39 0 makoo makoo 44.43333 39.3333 1411 6 7 82 23 3 sangar 44.43429 39.31578 1355 288 132 100 169 90 milan 44.4332 39.34351 1445 37 115 25 22 0 keshmesh tappeh 44.40093 39.33351 1385 0 0 150 94 0 glik gadim 44.66767 39.71264 807 31 0 26 0 0 deim-gheshlagh 45.07119 39.34766 784 0 58 0 0 0 deimgeshlag 44.7987 39.62488 797 190 0 20 0 0 miandoab miandoab 46.06947 36.98592 1291 0 0 98 0 0 naghadeh naghadeh 45.41667 36.95 1338 0 56 0 0 7 yadegarloo 45.52839 37.03822 1284 0 10 0 23 19 plodasht poldasht 45.07111 39.34778 788 0 10 55 89 0 khol-kholeh 44.74524 39.67784 784 0 3 0 40 0 ghooch-ali 44.74391 39.66296 1401 0 25 1 5 0 sardasht sardasht 45.48333 36.15 1556 0 18 0 0 0 urmia urmia 45.05 37.66667 1328 0 128 151 163 246 naz-loo 44.98442 37.65172 1358 0 0 441 67 10 ghahraman-loo 45.17485 37.64896 1000 170 0 24 0 0 koor-abad 44.64239 37.72749 1545 0 68 34 0 6 silvana 44.85142 37.42867 1577 0 120 0 0 0 gojar 44.83373 39.48785 1736 0 7 15 19 7 mavana 44.79643 37.56658 1617 0 13 0 32 24 shaharchay dam 44.98628 37.4952 1433 0 11 0 10 3 talebin 44.83398 37.54025 1608 0 0 0 149 0 j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 31 http://jad.tums.ac.ir published online: april 27, 2019 table 2. average of environmental variables in meteorological stations of west azerbaijan province of iran, 1997– 2016 localities geographical properties environmental variable longitude latitude altitude total rainfall mean relative humidity mean temp min temp max temp bookan 46.21667 36.53333 1386.1 360.5 46 14.5 6.3 19.9 chaldoran 44.38333 39.06667 1788 449.5 58 9.2 2.7 13.9 ghareh ziahadin 45.01667 38.9 1108 357.3 52 13.6 8 18 khoy 44.96667 38.55 1103 289.2 59 12.1 5.5 18.6 mahabad 45.71667 36.75 1351.8 403.8 53 13.0 6.9 19.15 makoo 44.43333 39.3333 1411.3 302.8 57 10.6 5.5 15.6 miandoab 46.05 36.96667 1300 303.8 53 14.4 5.6 20.0 naghadeh 45.41667 36.95 1338 339.0 53 13.9 5.8 19.2 oshnavie 45.13333 37.05 1415.9 437.3 52 13.5 4.5 18.6 piranshahr 45.15 36.7 1443.5 672.7 52 12.0 6.2 17.9 poldasht 45.07119 39.34778 787 198.8 51 14.7 4.8 20.2 salmas 44.85 38.21667 1337 247.5 57 11.5 5.2 17.8 sardasht 45.48333 36.15 1556.8 841.2 49 13.1 9.2 16.8 shahindej 46.73333 36.6667 1395 334.8 47 14.9 6.7 20.4 takab 47.1 36.4 1817.2 338.6 55 9.4 2.5 16.4 urmia 45.05 37.66667 1328 338.9 61 11.6 5.4 17.7 table 3. categorizing the environmental factors based on their effects on disease transmission, and important impact on vector-borne diseases (3, 45–47) environmental factor ratio standard weight category group effect on diseases transmission 55 0.55 26–30 4 very high mean temperature (˚celsius) 20–26 3 high 18–20 2 medium 14–18 1 low 35 0.35 60< 4 very high relative humidity (%) 50–60 3 high 40–50 2 medium <40 1 low 5 0.05 0–800 4 very high altitude (m) 800–1200 3 high 1200–2000 2 medium <2000 1 low 5 0.05 <8 3 very high slope (%) 8–15 2 medium 15< 1 low j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 32 http://jad.tums.ac.ir published online: april 27, 2019 table 4. average of some environmental and climatic variables in the collection sites of mosquitoes, west azerbaijan province, northwestern iran, 2015 species n o . o f c o lle c tio n site s p r e c ip ita tio n (m m ) a v e r a g e o f a ltitu d e (m ) a v e r a g e o f r e la tiv e h u m id ity (% ) a v e r a g e o f m a x im u m t e m p e r a tu r e ( oc ) a v e r a g e o f m in im u m t e m p e r a tu r e ( oc ) a v e r a g e o f m e a n t e m p e r a tu r e ( oc ) ae. caspius 10 326.3 1214.7 56 17.21 5.61 11.74 an. maculipennis 20 347.3 1273.9 54.91 17.75 5.66 12.4 cx. pipiens 22 328.6 1227.5 55.33 17.89 5.6 12.3 cx. theileri 21 330.65 1275.1 55.78 17.65 5.6 12.1 cs. longiareolata 12 345.1 1364.25 56.14 17.95 5.66 12.3 fig. 1. study area in northwestern iran j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 33 http://jad.tums.ac.ir published online: april 27, 2019 fig. 2. environmental and meteorological variables affecting mosquito distribution in west azerbaijan province, northwestern iran, (a): altitude, (b): slope, (c): average of mean temperature, (d): average of minimum temper ature, (e): average of maximum temperature, (f): relative humidity and (g): total precipitation. fig. 3. spatial distribution of mosquitoes and hot spots for vectors of west nile virus in west azerbaijan province regarding environmental factors affecting the transmission of wnv, northwestern iran, 2015 j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 34 http://jad.tums.ac.ir published online: april 27, 2019 discussion the study area has a large number of wetlands that host of migratory birds. although wnv is an enzootic disease among birds (48), however, humans transmission is possible through bites of infected mosquitoes (49). wnv is responsible for disease outbreaks among human in the united states, europe, and the middle east (50). it has been detected in recent studies from birds, horse, human and mosquitoes in iran (31–36). in the present study, environmental factors affecting the transmission of wnv were studied and high-risk areas were determined (fig. 3). earlier study on the impact of climate and environmental variables on wnv in iran, using data from seropositive horses, found four studied factors that correlated with wnv infection in equine, these factors were temperature, distance to wetlands, and local and regional normalized difference vegetation index (ndvi) (27). results from previous studies also indicated the presence of wnv vectors (cx. pipiens, cx. theileri, ae. caspius) in the region (36, 40) and their infection with wnv in iran (36). in addition, the studied areas have the potential for establishment of wnv transmission. the results of current study regarding the determination of the high-risk areas are interesting because they are in accordance with results from previous study that isolated west nile virus from vectors (36). the isolation of the virus in the mentioned study has been reported from the highrisk areas identified in current study and this point shows the acceptable accuracy of the results of the current study which analyzed environmental factors (temperature, relative humidity, altitude and slope). these areas should be considered in planning of wnv epidemic control programs. among the probable reasons increasing the risk of establishing transmission cycle of wnv in these areas is the wide range of wetlands in the area and the presence of migratory birds as the reser voirs of disease and also the appropriateness of environmental conditions for the presence and abundance of various species of mosquitoes, as potential vectors of wnv. also it seems northeastern areas of the province, which have lower altitudes and slopes with warmer temperatures and more water bodies, were found to have better condition for the activity of mosquitoes. diversity of environmental conditions in west azerbaijan province, which provides suitable environment for the establishment of various species of mosquitoes (table 1) have been found in this current and related studies (36, 40, 42). other results have shown the effect of temperature in transmission of wnv by cx. pipiens, and the effect of temperature on the replication of the virus within the mosquito’s body and the incubation period, as well (51). wnv has the ability to replicate in mosquitoes in wider range of temperatures between 14 °c in mosquitoes (52) to 45 °c in birds (53). by increasing the temperature, wnv propagation rate could be increased. the suitable temperature for wnv replication is provided in the northeastern and southern parts of the provinces of iran. therefore, these areas need more investigation on blood feeding pattern of mosquitoes and their infection with wnv. recent study on the feeding patterns of potential wnv vectors in southwest spain showed that cx. modestus, cx. perexiguus and cx. pipiens mainly feed on birds, while cx. theileri and ae. caspius mainly feed on mammals. cx. perexiguus had the highest potential for enzootic virus transmission, followed by cx. modestus and cx. pipiens. according to results of the south-west spanish study, potential transmission risk to humans was low for cx. pipiens, cx. theileri and ae. caspius (54). the frequency of feeding on humans was only affected by season, while the low number of human blood meals was related to their study site. on the other hand, j arthropod-borne dis, march 2019, 13(1): 27–38 m amini et al.: the potential of … 35 http://jad.tums.ac.ir published online: april 27, 2019 the researchers worked in natural areas with very low anthropic presence (54). most of these mosquito species have been reported from our study area as well (36), therefore, they may have some roles in both avian to avian enzootic cycle and avian-to-mammal transmission. certainly, the current study did not cover all important environmental factors affecting the potential of wnv transmission regarding the environmental factors and the effect of other important factors such as wetland, normalized difference vegetation index (ndvi) and land use should be analyzed and determined in future studies. conclusion the present study serves as a preliminary guide that shows the important effects of environmental conditions on one of the important members (mosquitoes) in the transmission cycle of wnv and should be continued with supplementary studies. taking into account the vector bio-ecologic conditions, other environmental factors and the interaction of the virus and the vectors as well as other important rings in the transmission of disease, the data obtained from this current study will be very useful and effective in knowing the exact nature of the disease transmission pathways and help in designing its control strategies. acknowledgements this article is part of the results of the first author’s dissertation for fulfillment of msc degree in medical entomology and vector control from the department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran. this study was financially supported by urmia university of medical sciences, urmia, iran (project no. 1579). the authors would like to thank the health staff of the studied regions for their kind support and contributions. the authors declare that there is no conflict of interests. references 1. khormi hm, kumar l (2011) examples of using spatial information technologies for mapping and modelling mosquitoborne diseases based on environmental, climatic, socioeconomic factors and different spatial statistics, temporal risk indices and spatial analysis: a review. j food agr environ. 9: 41–49. 2. drakeley cj, carneiro i, reyburn h, malima r, lusingu jp, cox j, theander tg, nkya wm, lemnge mm, riley em (2005) altitude-dependent and independent variations in plasmodium 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trop med hyg. 12: 657–667. 52. cornel aj, jupp pg, blackburn nk (1993) environmental temperature on the vector competence of culex univittatus (diptera: culicidae) for west nile virus. j med entomol. 30: 449–456. 53. kinney rm, huang cy-h, whiteman mc, bowen ra, langevin sa, miller br, brault ac (2006) avian virulence and thermostable replication of the north american strain of west nile virus. j gen virol. 87(pt 12): 3611–3622. 54. muñoz j, ruiz s, soriguer r, alcaide m, viana ds, roiz d, vázquez a, figuerola j (2012) feeding patterns of potential west nile virus vectors in south-west spain. plos one. 7: e39549. j arthropod-borne dis, december 2020, 14(4): 391–399 s sayono et al.: larvicidal activity of … 391 http://jad.tums.ac.ir published online: december 31, 2020 original article larvicidal activity of ethyl acetate extract of derris elliptica root against the third-instar larvae of cypermethrin-resistant aedes aegypti offspring *sayono sayono1; risyandi anwar2; didik sumanto3 1faculty of public health, universitas muhammadiyah semarang, semarang, indonesia 2herbal medicine research of dentistry faculty, universitas muhammadiyah semarang, semarang, indonesia 3laboratory of epidemiology and tropical diseases, faculty of public health, universitas muhammadiyah semarang, semarang, indonesia *corresponding author: dr sayono sayono, e-mail: say.epid@gmail.com (received 30 nov 2019; accepted 15 dec 2020) abstract background: derris elliptica extracts have a high larvicidal potential against the laboratory strain of aedes aegypti larvae, but the effect on offspring larvae of pyrethroid-resistant strains of the species is lack understood. this study aimed to determine the larvicidal activity of the ethyl acetate extract of tuba root against the third-instar larvae of the cypermethrin-resistant ae. aegypti offspring. methods: the experimental study occupied four levels of ethyl acetate extract of d. elliptica namely 10, 25, 50, and 100 ppm, and each level was four times replicated. as many as twenty of healthy third-instar larvae, offspring of cypermethrin-resistant ae. aegypti were subjected to each experiment group. larval mortality rate and lethal concentration 50% subject (lc50) were calculated after 24 and 48 hours of exposure time. results: mortality of larvae increased directly proportional to the increase of extract concentration. larval mortality rates after 24 and 48 hours of exposure were 40–67.5% and 62.5–97.5%, and lc50 were 34.945 and 6.461ppm, respectively. conclusion: the ethyl acetate extract of d. elliptica has the high effectiveness larvicidal potential against the thirdinstar larvae, offspring of the cypermethrin-resistant ae. aegypti. isolation of the specific compound is necessarily done to obtain the active ingredient for larvicide formulation. keywords: larvicidal activity; ethyl acetate extract; derris elliptica root; cypermethrin resistant; aedes aegypti introduction the resistance of ae. aegypti to several pyrethroid and organophosphate insecticide compounds such as deltamethrin, lambda cyhalothrin, cypermethrin, malathion, and temephos (1, 2) inhibits the public health action in eradicating the dengue vector, and intrigues researchers to find the other active ingredients as the alternatives. natural chemical compounds (3), including d. elliptica roots (4), are interesting to study for several reasons including but not limited to readily degraded and there is no bioaccumulation in the environment (5). researchers have proven that d. elliptica extracts have high larvicidal potential against the laboratory strain larvae of ae. aegypti (3, 4, 6, 7). however, when methanol extract of d. elliptica was exposed to the filed-caught larvae of ae. aegypti showed a lower larvicidal potential (8). this fact showed that the different extract types of the tubal root have different effects against the different strains of ae. aegypti larvae where the field-caught larvae were more resistant to the phytochemical compound. the results of monitoring of dengue vector susceptibility in central java, indonesia showed a wide spread of resistance to cypermethrin 0.05 % (1), as occurs in various dengue endemic areas in other countries (4, 9, 10). cypermethrin is one of the active ingredients of pyrethroid class insecticide which has caused knockdown resistance (kdr) (11). this resistance mechanism was indicated with the target site insensitivity copyright © 2020 iranian scientific society of biology & control of diseases vectors, and tehran university of medical sciences. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/by-nc/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 391–399 s sayono et al.: larvicidal activity of … 392 http://jad.tums.ac.ir published online: december 31, 2020 in the voltage-gated sodium channel (vgsc) gene (1, 12). the action mechanism of cypermethrin is different from the temephos. this compound is an active ingredient of organophosphate insecticide class which inhibited the acetylcholinesterase enzyme (13). this different mechanism of action is interesting to be studied in understanding the larvicidal activity spectrum of d. elliptica root extracts. the main biochemical compounds of d. elliptica are alkaloids, flavonoids, sterols, tannins, and triterpenoids (14, 15), and rotenone is the most important of a specific compound of flavonoid (16). these compounds have a toxic effect that kills insect larvae through disrupting mechanisms of the endocrine and hormonal systems (14) and reducing the esterase and monooxygenase enzymes (16). initial studies showed that ethyl acetate, methanol, and n-hexane extracts of d. elliptica that have different polarity effectively killed ae. aegypti larvae which were susceptible to temephos 0.02ppm (17), but on the other hand, the ethanol extract type has a lower effect against the temephos-resistant strains (18). the lethal effects of different specific phytochemicals contained in d. elliptica root extracts against the offspring larvae of the cypermethrin-resistant strain ae. aegypti is still lack understood and is interesting to be studied. it is important to evaluate the larvicidal effect of the semi polar extract, ethyl acetate against this strain. this study aimed to determine the larvicidal activity of ethyl acetate extract of d. elliptica root against the thirdinstar larvae of the cypermethrin-resistant ae. aegypti offspring. materials and methods this experiment is the early part of an ongoing study ‘isolation of specific compounds of derris elliptica as the larvicidal ingredients against aedes aegypti mosquito in the dengue control’. ethyl acetate extract is the last step of the sequential extraction process (19, 20). in the summary modification, the extraction process started by maceration of the tuba root powder in methanol solvent for 3 x 24 hours, and then filtered. the clean part of the liquid is evaporated and produced the methanol extract (the crude extract). furthermore, the crude extract was partitioned liquid-liquid with n-hexane solvent to bind the nonpolar lead compounds and resulted in water fraction and nhexane fraction. the water fraction obtained was partitioned with ethyl acetate to bind the semi-polar lead compounds and produced the water fraction and ethyl acetate fraction. all fractions produced were evaporated by using a rotary evaporator to produce four types of extracts, including the ethyl acetate extract which was first completed. the subjects of this study were the offspring filial 2 (f2) larvae of the cypermethrin 0.05% resistant strain of ae. aegypti. the parental mosquitoes were the f1 larvae of the ae. aegypti that is reared from f0 larvae obtaining from a household survey in the dengue endemic areas in the community health center of kedung mundu, tembalang district, semarang city, and subjected to bioassay test using the cypermetrhin-0.05% compound. the result of the bioassay test showed a mortality rate of 85%, which indicated that the mosquito population was resistant to the pyrethroid compound (21). larvae were maintained in the epidemiology and tropical diseases laboratory of public health faculty of universitas muhammadiyah semarang, indonesia. the larvae were placed on a plastic tray containing tap water. conditions of temperature and humidity were maintained in the range of 28±2 °c and 75±10%. the larvae were fed dog food. bioassay tests apply the who standard procedures for larvicide testing (21). there are three important parts at this stage, namely preparation of extract concentration, selection of research subjects, and exposure of research subjects with various d. elliptica extracts. the concentration of the bioassay test used a range of 10, 25, 50, and 100 ppm, the effective concentration in another study using ae. aegypti larvae of laboratory http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 391–399 s sayono et al.: larvicidal activity of … 393 http://jad.tums.ac.ir published online: december 31, 2020 strains (17). the subject of the research was the third instar larvae offspring of the cypermethrin-resistant ae. aegypti, in intact condition, and actively moving. as many as twenty larvae were subjected to each experiment group. two control groups, negative (tap water) and positive (temephos 0.02ppm) control were followed. the effectiveness of the larvicidal activity of the ethyl acetate extract of d. elliptica root was determined by the lc50 that was obtained from probit analysis. this lc50 will be compared with the lc50 of previous experiment results of the same extract type against the laboratory (susceptible) strain of ae. aegypti larvae (17). analysis data was performed descriptively and analytically by using spss statistical software version 15.0. the research protocol obtained ethical approval from the ethics committee of health research of public health faculty of universitas muhammadiyah semarang with registration number 231/kepk-fkm/ unimus/2019. results the ethyl acetic extracts of d. elliptica root showed the larvicidal activity against the ae. aegypti larvae of offspring from the resistant parental to cypermethrin adulticide. there was an increase in the larval mortality rate of ae. aegypti larvae after 24 and 48 hours of exposure to the ethyl acetic extract from 40–67.5% to 62.5–97.5% (table 1), with lc50 of 34.945 and 6.461ppm, respectively (table 2). the larval mortality rate increased directly with the extract concentration. there were no larvae died in the negative control, and 100% of larvae died in the positive control. the trend of the knockdown larvae showed that the slow larvicidal activity of the ethyl acetate extract of d. elliptica root to the third-instar larvae, offspring f2 of cypermethrin-resistant ae. aegypti (fig. 1). statistical analysis showed the differences in larval mortality rate based on the dosage and exposure time (fig. 2). fig. 1. the trend of larval knockdown rate in each extracts concentration based on the exposure time. the colored-line represents the concentrations of the extract http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 391–399 s sayono et al.: larvicidal activity of … 394 http://jad.tums.ac.ir published online: december 31, 2020 table 1. larvicidal activity of the ethyl acetate extract type of derris elliptica against the third-instar larvae of cypermethrin-resistant aedes aegypti offspring concentration (ppm) 24-hours mortality rate (%) 48-hours mortality rate (%) min max mean min max mean 0 (dw) 0 0 0 0 0 0 10 30 40 35 60 65 62.5 25 40 40 40 80 90 85 50 40 70 55 90 100 95 100 65 70 67.5 95 100 97.5 0.02 (tem) 100 100 100 100 100 100 dw= distilled water; tem= temephos table 2. the lc50 of larvicidal activity of the derris elliptica ethyl acetate extract against offspring larvae of the cypermethrin-resistant aedes aegypti exposure time (hours) regression equation lc50 (95% confidence limits) 24 y= -1.331+0.862x 34.945 (18.179–70.780) 48 y= -1.419+1.751x 6.461 (2.206–10.359) 35 40 55 67.5 62.5 85 95 97.5 0 20 40 60 80 100 120 10 25 50 100 m o r ta li ty ( % ) dosage (ppm) 24h 48h fig. 2. mortality rate comparison of aedes aegypti larvae after 24h and 48h exposure of ethyl acetate extract of derris elliptica root. the differences of larval mortality rate are indicated by the letters a, b and c discussion results of the experiment showed that the ethyl acetic extract of d. ellitpica has a high larvicidal activity against the third-instar larvae of the cypermethrin-resistant ae. aegypti f2 offspring, although at the concentration of 100ppm for 24 hours, the larvicidal effect of the ethyl acetate extract type is still lower than the larvicidal activity of temephos 0.02ppm. although the ethyl acetate extract type of tuba root shows lower larvicidal activity than temephos, this extract still indicates high larvicidal potential because its lc50 is lower than 50ppm. a previous study reported that the effective larvicidal activity of plant extracts was categorized into three levels, namely high (lc50< 50ppm), moderate (lc50< 100ppm), and low lc50< 750 ppm) (4). the results of this experiment indicate that the potency of the ethyl acetate extract http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 391–399 s sayono et al.: larvicidal activity of … 395 http://jad.tums.ac.ir published online: december 31, 2020 of d. elliptica root against the third-instar larvae of cypermethrin-resistant ae. aegypti f2 offspring is 84.4% lower than the same extract potency against the susceptible (laboratory) strain (17) indicating by the lc50 bioassay test for 24 hours, respectively 34,945ppm and 21,063 ppm. this extract also had a higher larvicidal potential against f2 offspring larvae of cypermethrin-resistant ae. aegypti rather than the temephos-resistant offspring (18). the larvicidal activity of this extract was also better than the methanolic extract of m. glaziovii peel (22), a. pinata (23), t. patula (24), h. forskaolii (25), o. campechianum and o. quixos (26), and a. occidentale (27) against the third-instar larvae of ae. aegypti, although lower than the specific isolate compound of f. vulgare (28) and p. aduncum (26) essential oils, and p. foetida ethyl acetate extract (29). these preliminary data and information have given new hope that this extract can be an alternative ingredient of larvicide to inhibit the growth of ae. aegypti larvae, even to the strains that are already resistant to cypermethrin adulticide. these results also indicate that temephos is still effective in killing the third-instar larvae offspring f2 of the cypermethrin-resistant ae. aegypti. this condition showed that there is still a way to eradicate the dengue vector, even from strains that have been resistant to other insecticides because each insecticide compound has a different mode of action (30, 31). cypermethrin is a compound of the pyrethroid class, an insecticide group that disrupts the function of sodium channels in insect nerves (32). under normal conditions, the voltage-gated sodium channel (vgsc) gene works ‘open’ and ‘close’ to regulate electrical impulses into the cell. the linkage of the pyrethroid insecticide molecule to the gene disrupts the nerve regulation and impulse of the nerve flowing continuously so that the insects become convulsion and died (33). however, if the point mutations occur in this gene, the linkage of the pyrethroid insecticide molecule does not affect the life of the insect, and this condition caused the kdr (34). the high larvicidal activity of the ethyl acetic extract of d. elliptica root against the third instar larvae, the offspring of the cypermethrinresistant ae. aegypti indicates that this extract has a different mechanism of action than adulticide cypermethrin. the d. elliptica extract contains several lead compounds such as tannins, phlobatannins, terpenoids, cardiac glycosides, and flavonoids (35) mainly rotenone and rotenoids (36). mode of action of rotenone is inhibition the cellular respiration, while pyrethrins the active compound of the pyrethroid insecticide has a mode of action in disruption of the sodium and potassium ions exchange (37). it means that the exploration of specific isolates of d. elliptica extract has the opportunity to be developed into larvicidal bioactive compounds with different modes of action. on the other hand, the effectiveness of temephos larvicide in killing the offspring larvae of the cypermethrin resistant strain of ae. aegypti proved that rotational insecticide with different modes of action and target sites is necessary done. temephos is a compound that plays a role in protein carbonylation so that it causes the general oxidative damage in larval cellular of insects (38). the maximum effect of the ethyl acetate extract of d. elliptica was achieved at 48 hours of exposure time. this condition indicated that the mode of action of this extract is slower than temephos. it can be understood that temephos is a pure chemical compound, while the plant extract still contains many chemical compounds, which may have antagonistic effects (39). extraction with ethyl acetate solvent has selected chemical compounds that are semi-polar, according to the nature of the solvent. however, the extraction results still allow the dissolution of many plant chemical compounds with various modes of action although flavonoid was the dominant compound (40). therefore, the pure compounds from these extracts that have the best larvicidal activity are necessarily understood. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 391–399 s sayono et al.: larvicidal activity of … 396 http://jad.tums.ac.ir published online: december 31, 2020 conclusion the ethyl acetate crude extract of d. elliptica root has a high larvicidal activity against the third-instar larvae, offspring of the cypermethrin-resistant strain of ae. aegypti, although the effect is lower and slower than temephos. isolation of the pure compounds of the extract is needed to find the specific active compounds for larvicide formulation. acknowledgements the authors wish to thank president of universitas muhammadiyah semarang for the research permission for experimenting in laboratory study at the epidemiology and tropical diseases laboratory; dean of mathematical and natural sciences faculty of universitas garut, west java, indonesia; directorate general of research and development strengthening, ministry of research, technology and higher education for the funding of the study. references 1. sayono s, hidayati apn, fahri s, sumanto d, dharmana e, hadisaputro s, asih pbs, syafruddin d (2016) 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insufficientia renalis-case report http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 421–425 a begolli gerqari et al.: scabies crustosa... case report scabies crustosa in a patient with insufficientia renalis-case report *antigona begolli gerqari1, mybera ferizi1, sadije halimi1, ilir begolli2, mirije begolli2, idriz gerqari3 1dermatology clinic, university clinical center of kosovo 2aab university-prishtina 3nuclear medicine department, university clinical center of kosovo *corresponding author: dr antigona begolli gerqari, e-mail: antigonagerqari@gmail.com (received 09 oct 2019; accepted 31 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: sscabies is caused by the parasite sarcoptes scabiei. the disease was first described in ancient greece, while ferdinand von hebra described the stages of development of the arthropodite from the egg to larvae and the adult form of sarcoptosis. the final stage is characterized by the travel of the female parasite and the laying of eggs in stratum corneum and followed by the death of the causer. the main characteristic symptom of scabies is relentless pruritus which is a result of such an infestation inside the skin, as the body reacts to parasites and their excretions, where mites burrow and lay eggs. methods: here we present the 85-year-old patient with scabies crustosa, latent diabetes and renal insufficiency. results: persistent pruritus and scabies rash, where large numbers of mites were detected, was left untreated and further progressed into sores due to scratching. weakened immunity and chronic illness in the patient resulted in a more severe form of the disease which was diagnosed as scabies crustosa. after treatment with a supplemented antiscabiotic regimen, the patient remained symptom free. conclusion: large population movements, one of the main factors of spread and frequent incidence of scabies, contact with the sick, hygiene means, exchange of wardrobes, sharing bed with the sick, schools, preschools, old-age-care institutions and hospitals, enable spread of the disease. in addition, severe types of scabies that form thick crusts on the skin and present with pruritus as a symptom should be considered in immunocompromised and elderly patients with renal insufficiency where pruritus is also prevalent. keywords: scabies crustose; pruritus; sarcoptes scabiei introduction scabies is a parasitic infectious disease caused by the parasitic mite sarcoptes scabiei (acari: sarcoptidae) (1). disease in humans is usually caused by sarcoptes scabiei hominis, the parasitic itch mite that burrows in the epidermis. the female of the parasite has a size of 0.4 x 0.3 mm and the male 0.2 x 0.15 mm. in the skin, the female parasite opens canals where she lays 10 to 25 eggs, and after which the parasite dies. the egg develops into nympha octopode scabies, then into hexapod larvae and after approximately 2–3 weeks develops into an adult parasite by resuming the cycle (1-2). copulation is performed in places where the skin has dents and wrinkles. excretions and decomposing bodies of the parasite provide the characteristic 422 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 421–425 a begolli gerqari et al.: scabies crustosa... appearance of type iv immunological reactions, accompanied with pruritus and scratching symptoms which become even more pronounced in cases of superinfections with other pathogens and skin eczema. scabies crustosa (scabies norvegica) is the most advanced condition of scabies in patients with reduced sensitivity, such as damaged nerves in syphilis, leprosy, neuropathy of various forms, as well as in immunodeficient persons such as aids patients. the clinical picture is abundant with dry and thick crusty skin in different parts with a tendency towards enlargement throughout the body. the number of parasites is large and the conformability is high. treatment consists of the application of antiscabiotics in the form of sulphur in cream, peruvian balm and permethrin cream as well as in resistant cases and scabies crustosa, particularly ivermectin administered orally (3). in practise, topical application of medication (cream) is recommended to all family members who have been in contact with the severely affected patients to prevent recurrence and spread. european guidelines suggest first-line treatment with permethrin 5% cream with two additional treatments with a pause of one week apart in order to destroy mature forms of larvae (3-4). case presentation we present the case of an 85-year-old patient with renal insufficiency, latent diabetes and post-cerebral stroke presenting with intensive pruritus. the patient was admitted to the clinic due to obtuse thick skin layers on the hands and body (figs. 1 and 2), follicular papules and excoriation. itching was obvious. the patient had been treated for three months for symptomatic pruritus based on previous history of latent diabetes and unidentified kidney problems. the obtained laboratory test results in the clinic were as follows: glycemia 7.8, erythrosedimentation rate 50, urea 20.44 (maximal reference value 8.30), creatinine 206.2 (maximum reference value 100), ast 52 (maximal reference value 37) and crp 29 (maximal reference value 6). full blood count results were without pathological deviations, fpsa 0.215 (normal) and ige 2500 (maximum reference value 100). staphylococci were isolated from the swab of the skin, while upon admission microscopy revealed sarcoptes scabiei mites (figs. 3 and 4). we consulted an infectiologist, a nephrologist, a cardiologist and a haematologist. in consultation with the nephrologist and after routine review of creatinine clearance and kidney scintigraphy, the nephrologist detects renal insufficiency, while the infectiologist diagnoses secondary infection with cephalosporin susceptible strain of staphylococcus aureus. to exclude chronic haematological diseases accompanied with chronic intensive pruritus, we performed a peripheral blood smear test which resulted in normal appearance. during the patient’s stay in our clinic, we treated her with antibiotics (cephalosporin ampule) and antihistaminic with antiscabiotic sulphur precipitate 15%, while the inflamed surfaces were treated with fusidic acid antibiotic cream and corticosteroid creams applied on eczemaic areas. after treatment, the patient was discharged and continued treatment by a nephrologist. during the patient’s treatment for scabies, other members of the family were all invited for consultation with the dermatologist and were also treated with antiscabiotic. the patient responded very well to 15% precipitated sulphur that allowed the convenience of not initiating oral ivermectin to begin with. during the patient’s check ups, the patient presented with a few post inflammatory hyperpigmentations, without itching symptoms or re-appearance of the disease. discussion scabies is a frequent parasitic infectious disease with the main symptoms of persistent pruritus due to large numbers of mites in the skin (1, 5). characteristic is the relentless itching during the night. however, it is not unusual for itching to also 423 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 421–425 a begolli gerqari et al.: scabies crustosa... persist during the day, particularly during changes in the weather to warmer. the typical skin changes are follicular papules accompanying excoriations that in cases of long-term persistent pruritus can lead to scabies rush, pimples, blisters and pus. the severe form of the disease develops in immunocompromised individuals and elderly patients. diagnosis of the disease is done through a typical clinical picture and anamnesis of the disease (2, 6). in the native skin preparation in untreated or poorly treated cases, we can observe the presence of the sarcoptes scabiei and/ or its damaged and degraded parts (figs. 3 and 4). the treatment consists in the application of antiscabiotic (3-4), whereas per european guidelines topical permethrin 5% (first-line treatment) is recommended (7). sulphur in neutral creams is also suggested in uncomplicated cases, while in crust and resistant forms of scabies the drug of choice is considered ivermectin (7) taken orally. in pyodermized and eczematous extreme cases in order to prevent glomerulonephritic complications, antibiotics are applied according to the scheme and antibiogram (8). pruritus as fig. 1. scabies crustosa presenting with diffuse cutaneous redness and obtuse thick skin layers fig. 1. scabies crustosa presenting with diffuse cutaneous redness and obtuse thick skin layers fig. 2. scabies crustosa characterised by areas of scaly rash, hyperkeratotic plaques and crusted skin, involving body and arms fig. 2. scabies crustosa characterised by areas of scaly rash, hyperkeratotic plaques and crusted skin, involving body and arms 424 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 421–425 a begolli gerqari et al.: scabies crustosa... a symptom may accompany various other diseases such as melanoma, lymphoma and leukosis, especially diabetes and renal insufficiency (9-10), which can be disorienting when scabies is present. in many situations, the scabies is overlooked, as diagnostics may be outdated, and as a consequence, even more severe forms of development are possible (6, 11). this is more true, for example, in cases of dialysis patients (12) where intensive pruritus is expected (9, 13-14), and therefore may escape the fact that the patient has scabies. in this instance, as a result of non-intentional negligence, the illness may escalate into a micro-epidemic character in the dialysis unit that combined with the poor immune system of the patients allows the possibility of presenting even more severe forms. in conclusion, it is of great importance that in the chronic diseases cases accompanied with pruritus the patient is also tested for scabies. a small commitment in this regard fig. 4. sarcoptes scabiei dead form observed by microscopy (taken from the crust of the same patient) fig. 3. sarcoptes scabiei hominis, the human parasitic itch mite (material taken from the patient) fig. 4. sarcoptes scabiei dead form observed by microscopy (taken from the crust of the same patient) fig. 3. sarcoptes scabiei hominis, the human parasitic itch mite (material taken from the patient) saves the patient from severe suffering and protects others from possible infections. abbreviations aids: acquired immune deficiency syndrome; crp: c reactive protein: free psa: free prostate specific antigen test; ige: immunoglobulin e funding no source of founding to be declared. ethic approval and consent to participate not applicable. competing interests the authors declared that they have no competing interests. 425 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 421–425 a begolli gerqari et al.: scabies crustosa... references 1. hengge ur, currie bj, jäger g, lupi o, schwartz ra(2006) scabies: a ubiquitous neglected skin disease. lancet infect dis. 6(12):769-79. 2. ramos-e-silva m (1998) giovan cosimo bonomo (1663–1696): discoverer of the etiology of scabies. int j dermatol. 37(8):625-30. 3. 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international results from the dialysis outcomes and practice patterns study (dopps). nephrol dial transplant. 21(12):3495505. 10. tseng wc, chu sy, tarng nc (2011) a neglected cause of pruritus in a hemodialysis patient. acta nephrol. 25: 209–210. 11. roberts l j, huffam s e, walton s f, currie b j (2005) crusted scabies: clinical and immunological findings in seventy-eight patients and a review of the literature. j infect. 50(5):37581. 12. lempert k d, baltz p s, welton w a, whittier f c (1985) pseudouremic pruritus: a scabies epidemic in a dialysis unit. am j kidney dis. 5(2):117-9. 13. keithi-reddy s r, patel t v, armstrong a w, singh a k (2007) uremic pruritus. kidney int. 72(3):373-7. 14. patel ts, freedman bi, yosipovitch g(2007) an update on pruritus associated with ckd. am j kidney dis. 50(1):11-20. scabies crustosa in a patient with insufficientia renalis case report abstract keywords introduction case presentation discussion abbreviations funding ethic approval and consent to participate competing interests references j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 159 http://jad.tums.ac.ir published online: june 30, 2022 original article bioecological study on the sand flies (diptera: psychodidae, phlebotominae) in sari county, north of iran *nasibeh hosseini-vasoukolaei1, leila ghavibazou2, amir ahmad akhavan3, ahmad ali enayati1, elham jahanifard4, mahmoud fazeli-dinan1, jamshid yazdani-charati5, seyed hasan nikookar1, zahra saeidi3, atieh shemshadian1 1department of medical entomology and vector control, health science research centre, faculty of health, mazandaran university of medical sciences, sari, iran 2research committee, faculty of health, mazandaran university of medical sciences, sari, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4department of medical entomology and vector control, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran 5department of biostatistics, health sciences research center, addiction institute, mazandaran university of medical sciences, sari, iran *corresponding author: dr nasibeh hosseini-vasoukolaei, email: nasibeh.hoseini@gmail.com (received 25 nov 2020; accepted 11 june 2022) abstract background: phlebotomine sand flies are vectors of leishmania species, the causative agents of leishmaniasis in the world. present study aimed to evaluate the bioecological aspects of sand flies in different ecotopes in sari county, north of iran. methods: sand flies were collected from four villages in mountainous, forest, plain and peri-urban areas monthly using sticky traps in may–october 2016. mounted specimens were identified using valid identification keys under optical microscope. the arc gis 10.5 software was applied for showing the distribution of sand flies. shannon-weiner, simpson and evenness species diversity indices were calculated. results: generally, 334 specimens were captured and identified, namelly phlebotomus kandelakii, ph. papatasi, ph. major, ph. sergenti, ph. longiductus, ph. halepensis, ph. tobbi, sergentomyia dentata, se. theodori, se. sintoni, se. antennata and se. sumbarica. the most common species was ph. kandelakii (n= 128, 38.32 %). the highest simpson index (0.81) and abundance (n= 141) were recorded in the mountaineous area. shannon diversity index was higher in the forest (h'= 1.53) and the highest evenness index was in the plain area (j'= 0.93). the highest richness (s= 9) and shannon indices (h'= 1.57) were observed in june. conclusions: phlebotomus kandelakii, ph. sergenti, ph. tobbi, ph. longiductus, se. theodori, se. antennata and se. sumbarica were recorded for the first time in the study area. since some species are incriminated for leishmaniasis transmission, further studies are required in the northern regions of iran to timely control measures planning. keywords: sand fly; ecology; biodiversity; northern iran introduction sand flies belong to the order diptera, suborder nematocera, family psycodidae and subfamily phlebotominae (1, 2). they are vectors of various types of leishmaniasis. it is a neglected tropical disease caused by obligate protozoan parasites of the genus leishmania (tryp anosomatida: trypanosomatidae) transmitted by the bites of infected female sand flies that host animals such as canids, rodents, marsupials, hyraxes or humans (2, 3). leishmaniasis is endemically extended to extensive areas of the tropics, subtropics and mediterranean basins of about copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:nasibeh.hoseini@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 160 http://jad.tums.ac.ir published online: june 30, 2022 102 countries, with a total of 350 million people at risk and 12 million infected (2, 4, 5). almost 53 species of leishmania have been presented in diferrent parts of the world. of these, 31 species are mammalian parasites, and 20 species are pathogenic to humans. leishmania parasites cause four main clinical types of visceral, cutaneous, diffuse cutaneous and mucocutaneous leishmaniasis (6). cutaneous leishmaniasis is the well-known form of the disease and is estimated to occur 87% of its global incidence in ten countries including afghanistan, algeria, colombia, brazil, iran, syria, libya, tunisia, pakistan and iraq (7). phlebotomine sand flies are belonging to two genera of phlebotomus in the old world and lutzomyia in the new world. according to the latest classification, 53 species of sand flies have been documented from different parts of iran, including 34 phlebotomus species of six subgenera and 19 sergentomyia species of six subgenera (8). phlebotomus sergenti sergenti and ph. sergenti similis were considered as subspecies, and the presence of ph. (adlerius) kabulensis, ph. (adl.) salangensis, ph. (adl.) turanicus, ph. (larroussius) langeroni, ph. (transphlebotomus) mascittii, ph. (lar.) smirnovi, ph. (euphlebotomus) caudatus and se. (grassomyia) indica were doubtful in the country (9). phlebotomus (adl.) comatus is reported as the new species from northwest of the country by zahraei-ramazani et al. (10). sand flies are commonly observed and adapted to natural environments (human and animal places), especialy ph. papatasi and ph. sergenti. this may be advantageous due to the potential breeding and resting sites and easy food source, which can ultimately enhance the risk of transmitting pathogens to humans and domestic animals (11). also, various environmental and physiological conditions of sand flies can influence their capacity to transmit the parasite (12). therefore, understanding the biological and ecological aspects of sand flies is essential. geographic information systems (gis) are software tools regarded to determine the probability of presence, biodiversity, distribution and abundance of vectors and to predict the risk map of vector-borne diseases (13, 14). spatiotemporal epidemiological researches by gis can be applied to predict disease distribution in the regions at risk (15). gis technique is also applied for insect ecology by combining climatic and insect parameters to assess pest risk areas (16). biodiversity is the set of differences between organisms at all levels of the life spectrum, from genes and species to higher levels of taxonomy, including habitat and ecosystem types, and is represented in different types of alpha (α), beta (β) and gamma (ϫ). it is an important ecological issue because changes in the biodiversity of communities can propably lead to the emergence of some dominant species, create new ecological niches for proliferation of them, and enhance the prevalence of sand fly-borne diseases (17). no studies have been conducted on the biodiversity of sand flies in mazandaran province. therefore, the study of biodiversity in the region is momentous and can clarify the transmission of disease to humans and reservoirs. mazandaran province is considered as one of the sporadic foci of leishmaniasis. in previous studies, sporadic cases of cutaneous and visceral leishmaniasis have been documented in some parts of the province (18-20), some of which have a history of travel to endemic areas of leishmaniasis. since mazandaran province borders with the provinces that are endemic foci of the disease, disregarding the characteristics of the vectors can lead to an increase in the incidence of this disease. few studies have been carried out in the area to identify vectors that complete the disease cycle. therefore, this study focuses on collecting sand flies in sari county of mazandaran province and identifying their ecological and biological characteristics. this data set will serve as the basis for other epidemiological studies and the design of disease control strategies. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 161 http://jad.tums.ac.ir published online: june 30, 2022 materials and methods study area this descriptive cross-sectional study was conducted from may to october 2016 in sari county, the capital of mazandaran province, which is located in the central part of the province, on the foothills of alborz mountains in north of iran with a longitude of 53°5 and a latitude of 36°4. the county is divided into two major mountainous and plain areas. sari has a moderate and humid climate in summer and a relatively cold climate in winter. the southern mountainous part of the county has rather long and very cold winters. the average daily temperature and annual rainfall were reported 17.7 °c and 824.4mm, respectively. the maximum and minimum temperatures were in august (36.6 °c) and december and february (2.7 °c), respectively. the maximum and minimum average daily relative humidity were 82% in november and 72% in september, respectively. in this study, sampling areas were selected in such a way that appropriate coverage in the entire study area is ensured by considering the diversity in terms of geography and type of ecosystem. accordingly, four villages were selected from each mountainous (era), forest (shekta), plain (kordkheil) and, peri urban (zoghal chal) areas of sari county (fig. 1). in each area, altitude, longitude and latitude were recorded using a global positioning system (gps) device. in addition, due to the lack of information about the distribution of leishmaniasis vectors in sari, the areas in which the health center had reported the admission of native leishmaniasis patients in the past years, received more attention. sand fly collection (sampling) and identification in this study, sampling was conducted once a month to determine the seasonal and monthly activity of sand flies from the beginning to the end of the sand fly activity season. specimens were collected using sticky paper traps coated with castor oil. during sampling in each selected area, three permanent stations were selected for trapping and 60 sets of sticky traps were installed per station: 15 traps in human indoor places (bathrooms, bedrooms, warehouses), 15 in animal indoor places (stables, poultry houses, animal habitats) and 30 in outdoors (yards) before sunset and picked up the next morning before sunrise. sand fly specimens were taken from sticky paper using a fine needle, washed in acetone, and stored in small vials with 70% ethyl alcohol, then transferred to the department of entomology, faculty of health, mazandaran university of medical sciences for determination of abdominal condition, mounting and identification. the main morphological characters used in this study were pharyngeal armature, cibarial teeth, female spermatheca and male genitalia. to determine the physiological condition of the abdomen, each sample was dissected separately in physiological serum using dissecting needles under a stereomicroscope. to prepare a permanent microscopic slide, the head and the last three segments of the abdomen were seperated. the isolated pieces were then mounted on a clean slide in puri medium. in the case of the genus phlebotomus, the head was mounted upwards and in the case of the genus sergentomyia, the head was mounted downwards on the slide (21). the mounted slides were immediately transferred to an incubator at 37 °c. after drying the slides were identified according to valid morphological keys (22). gps points of the collected sand flies were used as a layer for analysis. arc gis 10.5 software was used to prepare the distribution map of sand flies in the study area. the gis layer containing sand fly data was transferred to arc map which is the main component of arc gis software for geospatial processing. statistical analyzes were performed using spss v.20 (ibm, new york, usa). nonparametric chi-square and binomial statistical tests were used to compare the data. p values less than 0.05 were considered significant. http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/alborz_mountains j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 162 http://jad.tums.ac.ir published online: june 30, 2022 biodiversity of species and statistical analyses the number of taxa (s), number of individuals (n), simpson’s diversity index (d), shannon’s diversity index (h) and pielou’s evenness index (j) were calculated to estimate alpha-biodiversity with the following equations: shannon index: where, pi is the relative abundance of the ith species (n/n), n; the total number of collected individuals. evenness index (pielou’s index formula): where, h' is the shannon-wiener function and s is the total number of species observed. simpson’s index: where, pi=ni/n; ni, the relative abundance of a species calculated as the proportion of individuals of a given species to the total number of individuals in a community, n (23). software pastv.3 (paleontological statistics software package) was applied to perform whole developed calculations (24). results a total of 334 sand flies were collected using sticky traps from may to october 2016 in the study area. two hundred and four (61. 07%) specimens belonged to genus phlebotomus and 128 (38.32%) specimens to genus sergentomyia, including six subgenera of which 146 (43.71%) subgenus larroussius, 123 (36. 82%) sergentomyia, 52 (15.56%) phlebotomus, 2 (0.52%) adlerius, 1 (0.29%) paraphlebotomus, and 1 (0.29%) parrotomyia (nine samples (2.69%) were undetectable) (table 1). in the present study, among 12 species of phlebotominae, the most frequent phlebotomus species was ph. kandelakii (38.32%) followed by ph. papatasi (15.56%) and ph. major (5.08 %). sergentomyia dentate with 22.45% (n= 75) was the most frequent sergentomyia sand flies followed by se. theodori (n= 27, 8.08%) and se. sintoni (n= 15, 4.49%) (table 1). in terms of gender, out of 334 phlebotominae, 194 (58.08%) were identified male and 140 (41.91%) were female. the male to female ratio was 1:0.72. this difference was statistically significant (p= 0.004). from the perspective of abdomen physiological condition, among the 140 female sand flies, 18 (12.85%) were fed, 120 (85.71%) were unfed, and 1 (0.71%) was semi-gravid and the abdomen physiological condition was unclear in one sample because of color change (table 1). this difference was significant (p= 0.004). the results showed that 107 (46.3%) sand flies were collected from human hosts and 124 (53. 7%) from animal hosts. in total, 231 (69%) sand flies were collected from indoors and 103 (31%) were from outdoors (p= 0.004). the most prevalent species were ph. kandelakii and se. dentata with rate of 34.19% and 23.8% in indoors, and 47.57% and 19.41% in outdoors, respectively (table 1). genus phlebotomus was mostly observed in indoors (141/204, 69.1%) (p= 0.004). phlebotomus kandelakii, ph. papatasi, and ph. major were caught more in indoor places, and consequently, they have more access to human and animal hosts (table 1). the activity of sand flies started in earlymay and ended in late-october. the highest abundance of sand flies was observed in june (n= 102, 30.53%) and july (n= 155, 46.40%) (p= 0.004). among the collected species, ph. papatasi was active in all months of the year. the highest prevalence of ph. kandelakii was observed in july (n= 124, 80%) and ph. papatasi and se. dentata in june (fig. 2). analysis of alpha-biodiversity indices showed that the highest richness (s) was observed in shekta area (s= 8) and in june (s= 9), while the lowest was found in kordkheil (s= 3) and in october (s= 2). the highest value of shannon was in shekta (h'= 1.53) and evenness was in kordkheil (j'= 0. 93) and both in may (h'= 1.57, j'= 0.95). the maximun values of shannon were presented in shekta and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 163 http://jad.tums.ac.ir published online: june 30, 2022 may, whereas simpson index indicated that the highest dominance was in era (d= 0.81) and in july (d= 0.67), due to the presence of the dominant species ph. kandelakii. other data on the status of sand fly biodiversity by region and month are shown in table 2 and 3. figure 3 shows that ph. kandelakii, ph. papatasi and se. dentata are the most prevalent species in era, zoghal chal, shekta and kordkheil areas, respectively. phlebotomus longiductus, ph. halepensis, and se. sumbarica were collected only from the mountainous area (era) and the foothills. while ph. kandelakii and ph. major were collected from both mountainous area (era) and forest (shekta). phlebotomus papatasi and se. antennata were collected from both forest and peri urban areas (zoghal chal). sergentomyia theodori, se. dentata, and se. sintoni were collected from three areas: the plain (kordkheil), forest and peri urban. phlebotomus sergenti was collected from forest and ph. tobbi from peri urban area. temporal distribution of ph. kandelakii in the study region is shown in figure 4. this species was not collected from study area in may. table 1. phlebotomine sand flies according to species, sex, and collection places in sari county, north of iran, 2016 species human places animal places outside places total male female male female male female n % phlebotomus (larroussius) kandelakii 15 19 21 24 36 13 128 38.3 sergentomyia (sergentomyia) dentata 7 22 13 13 7 13 75 22.5 phlebotomus (phlebotomus) papatasi 8 3 30 3 5 3 52 15.6 sergentomyia (sergentomyia) theodori 6 4 4 1 10 22 27 8.1 phlebotomus (larroussius) major 5 4 2 3 3 0 17 5.1 sergentomyia (sergentomyia) sintoni 5 3 2 0 4 1 15 4.5 sergentomyia (sergentomyia) antennata 2 1 2 0 1 0 6 1.7 phlebotomus (adlerius) longiductus 0 0 0 0 1 0 1 0.3 phlebotomus (adlerius) halepensis 0 0 1 0 0 0 1 0.3 phlebotomus (larroussius) tobii 0 0 1 0 0 0 1 0.3 phlebotomus (paraphlebotomus) sergenti 0 0 0 0 0 1 1 0.3 sergentomyia (parrotomyia) sumbarica 0 0 0 0 0 1 1 0.3 unknown 0 3 2 2 1 1 9 2.7 total 48 59 78 46 68 35 334 100.0 fig. 1. sampling sites in sari county, north of iran http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 164 http://jad.tums.ac.ir published online: june 30, 2022 fig. 2. monthly frequency and percentage of sand flies, collected in sari county, north of iran, 2016 fig. 3. geographical distribution and frequency of phlebotomine sand flies in sari county, north of iran, 2016 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 165 http://jad.tums.ac.ir published online: june 30, 2022 table 2. biodiversity indices of sand flies, based on spatial distribution in sari county, north of iran, 2016 zoghal chal kordkheil shekta era biodiversity indices 6 3 8 5 number of species (s) 45 10 129 141 abundance (n*) 1.27 1.03 1.53 0.39 shannon (h') 0.35 0.38 0.28 0.81 simpson (1-d) 0.59 0.93 0.59 0.29 evenness (j') *: nine unknown specimens were not included in the biodiversity index analysis fig. 4. temporal distribution of phlebotomus kandelakii in sari county, north of iran, a-f: may-october 2016 [a] [b] [c] [d] [e] [f] http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 166 http://jad.tums.ac.ir published online: june 30, 2022 table 3. biodiversity indices of sand flies based on temporal distribution in sari county, north of iran, 2016 october september august july june may biodiversity indices 2 5 7 7 9 5 number of species (s) 2 17 50 153 97 6 abundance (n*) 0.69 1.1 1.4 0.75 1.57 1.57 shannon (h') 0.5 0.45 0.34 0.67 0.27 0.22 simpson (1-d) 1 0.61 0.45 0.30 0.54 0.95 evenness (j') *: nine unknown specimens were not included in the biodiversity index analysis discussion the study of leishmaniasis vectors is necessary as a prerequisit to many epidemiological studies. the results of this study can inform authorities to make evidence-based decisions regarding appropriate interventions to control vectors and the diseases they transmit. in the present study, seven phlebotomus species and five sergentomyia species were collected. this is the first report after 1998 (25) on different biological and ecological aspects of sand flies in sari county, mazandaran province, north of iran. in the study of rahbarian et al. in mazandaran province, a total number of 194 sand flies were collected which included six species of ph. major, se. sintoni, ph. halepensis, ph. andrejevi, ph. papatasi, and se. dentata (25). compared to previous studies, in the present investigation ph. kandelakii, ph. sergenti, ph. tobbi, ph. longiductus, se. antennata, se. theodori, and se. sumbarica were reported as new records in mazandaran province. other related studies in northern iran were recently conducted in gilan province, neighboring mazandaran province, in which eleven species of sand flies were reported (26, 27). in the present study, twelve species of sand flies were identified, so the species richness is higher than the previous studies, it probably indicates a thorough sampling effort in the present study. regarding the sex of collected sand flies in this study, the sex ratio (male: female) was 1:0.7, which indicates that male sand flies were collected 1.4 times more than female sand flies. in accordance with this study, previous studies reported that the male to female ratio was 1:0.74 in thiland (28), 1:0.92 in western saudi arabia (29) and 1:0.5 in central iran (30). another study in norhtwest of iran reported a higeher sex ratio than the current study, in which male sand flies were captured 2.5 times more than female sand flies (31). the sticky paper trap method used in our study showed that males were more attracted to traps than females. another study in thiland using the method of cdc light trap, suggested that the frequency of collected male sand flies was higher than that offemales, which could be explained by the natural behavior of males that follow females to mate (28). in terms of the abdomen physiological condition, in our study most of the captured female sand flies were unfed, showing that they were more active and flew more than fed ones, probably for sugar or blood feeding. one of the main findings of this study was the abundance of species such as ph. kandelakii, ph. papatasi, and ph. major that are either proven or possible vectors of leishmaniasis in the world (32, 33). additionally, se. sintoni and se. dentata were reported as lizard leishmaniasis (34). according to the current study, ph. kandelakii was the most abundant species collected in indoors and outdoors in the study area. the value of simpson dominance index was high in some areas of study because of the abundance of this species. the higher dominance value in a region may indicate the higher potential of vector for disease transmission. the first report of visceral leishmaniasis in http://jad.tums.ac.ir/ file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_25 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_25 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_26 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_27 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_28 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_29 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_30 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_31 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_28 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_32 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_33 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_34 j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 167 http://jad.tums.ac.ir published online: june 30, 2022 iran was reported from mazandaran province in northern iran in 1949 (35). until recently, visceral leishmaniasis was reported only based on epidemiological evidence and the high prevalence of sand fly vectors of kala azar. in previous studies, leishmania promastigote infection was identified in ph. kandelakii in the endemic focus of meshkin-shahr in northwestern iran (36). in latter studies, l. infantum was detected in ph. kandelakii using molecular methods at the same focus in northwestern (37) and in northeastern iran in north khorasan province (38). thus, according to previous studies, ph. kandelakii, the most abundant species collected in this study area, is of paramount importance considering the fact that it is a vector of zvl in iran. phlebotomus papatasi was another species collected in this study. various studies have shown that ph. papatasi is the main and proven vector of zcl which is distributed all over the country (9, 32). in the past studies, the parasitic infection of ph. papatasi by l. major, l. turanica and l. gerbilli were identified (39). presence of non-pathogenic l. turanica in ph. papatasi and animal reservoirs can imply the role of this parasite in sustaining the disease cycle (40-44). another species collected in the current study was ph. major, which was reported from 17 out of 31 provinces of iran with human cases of zvl and according to the current study, this species was mostly collected from mountainous areas (32, 45). previous morphological and morphometric study in the northwest of the country, showed other morphotypes including ph. major neglectus and ph. major krimensis for this species (46). it is not usually difficult to identify the male laroussius sand flies, but females of some species are not easily distinguishable. further studies using morphological and molecular data are suggested. in the present study, ph. kandelakii, ph. papatasi and ph. major were mainly collected from indoor places, thus they had more access to human and animal hosts. in accordance with the current study, in an investigation on phlebotominae ecology in an endemic cutaneous leishmaniasis focus in isfahan conducted by yaghoobi ershadi in 2001, the most prevalent specie in indoors was ph. papatasi (47). in another study in south of iran, ph. papatasi was collected mostly from indoors (48). in the studies conducted in qom province (49), central iran and in ardabil province (31), northwest of the country, ph. kandelakii was collected more from indoors.this finding could indicate the habituation of these species to human habitats. in this study, ph. kandelakii was collected from the mountains and peaked in june, while ph. papatasi was collected from areas other than mountains. according to previous studies, ph. papatasi usually prefers the plains over the mountains (50, 51). in the present study, the highest temporal distribution was associated to ph. papatasi, which was collected in all months of sand fly activity in the area. in the current study, sand fly activity began in may and continued until october, while the peak of seasonal activity was in july. according to our previous study in the same study area, in november, more disease transmission and higher incidence rate of leishmaniasis occured after the incubation period, from sand fly bites to nodule development (19). during a study conducted in an endemic cutaneous leishmaniasis focus in golestan province, north east of iran, sand flies were collected in 7 months of the year and the activity of sand flies was identified from may to november (52). it has been admitted that phlebotomine sand flies have a more or less definite seasonal distribution pattern in mediterranean countries, become visible in late spring or early summer, and are active until early autumn (53, 54). however, various studies have verified the bimodal pattern of temporal dispersion of phlebotomine sand flies with one peak in spring and another in summer or autumn (11, 55, 56). one of the main findings of this study was the collection of eight species of phlebotomhttp://jad.tums.ac.ir/ file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_36 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_37 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_38 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_9 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_32 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_39 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_40 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_32 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_45 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_46 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_47 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_48 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_49 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_31 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_50 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_51 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_19 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_52 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_53 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_54 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_11 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_55 file:///d:/&nbsp;/jad2/no.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/dr%20hosseini-vasoukolaei.doc%23_enref_56 j arthropod-borne dis, june 2022, 16(2): 159–172 n hosseini-vasoukolaei et al.: bioecological study on … 168 http://jad.tums.ac.ir published online: june 30, 2022 inae from the forest, five species from the mountains, three species from the plains, and six species from the peri-urban areas. the forest region had a richer collection of species composition compared to other regions due to its moderate and humid climate, which created a proper environment for sand fly development. in current study, twelve species were captured. species diversity and richness in this study was high compared to some other species composition studies of sand flies in iran. in the present study, species diversity, richness and evenness in the forest (h'= 1.53, s= 8, j'= 0.59) was higher than the mountainous area. less diversity in mountainous area was because of the presence of the dominant species ph. kandelakii, and the greater value of simpson dominance index (d= 0.81). contrary to our study, shannon and evenness indices in the study of qom (50) province (h'= 1.36, j= 0.62) was higher in mountaineous area, and in north khorasan (51) province, shannon index (h'= 1.033) was higher and evenness was similar (j'= 0.357) in mountainous area. in accordance with this study, in khouzesten province, south of iran, species diversity was higher in plain areas (48). it should be mentined that the values of shannon biodiversity index, affected by richness, eveness and simpson, were high in the studied areas, jointly in the months of may and june. the high diversity indices could be because of humid climate due to low distance to the caspian sea and suitable condition for sand fly breeding. conclusion this study determined some bioecological aspects of sand flies in sari county, mazandaran province, northern iran. the diversity and richness of the species was high, which could be because of the moderate climate due to its geographical proximity to the caspian sea. according to the results of this study, leishmaniasis vectors ph. kandelakii, ph. papatasi and ph. major were present in the study area. considering mazandaran province as one of the sporadic foci of leishmaniasis and geographical proximity of mazandaran province with zcl and zvl foci of golestan province in the northeast and azerbaijan province in the northwest, respectively, and the lack of study of disease vectors in this region, health authorities are recommended to highten the surveillance system and conduct more studies on the ecology of sand flies and leishmanial infections, to prevent the emergence of a new focus of leishmaniasis. acknowledgements this work was supported by research deputy of mazandaran university of medical sciences, project no. 1958. we thank the staff of health centers for their assistance in the project. ethical considerations this project has been approved 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247–255. 55. azizi k, parvinjahromi h, moemenbellah-fard md, sarkari b, fakoorziba mr (2016) faunal distribution and seasonal bio-ecology of naturally infected sand flies in a new endemic zoonotic cutaneous leishmaniasis focus of southern iran. j arthropod borne dis. 10(4): 560–568. 56. hazratian t, rassi y, oshaghi ma, yaghoobi-ershadi mr, fallah e, khanmohamm adi m (2011) study on species composition and seasonal activity of sand flies in a new focus of visceral leishmaniasis, azarshahr district, east azerbaijan province, iran. adv environ biol. 5(10): 3097– 3102. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2019, 13(2): 185–190 f bagheri et al.: detection of diazinon … 185 http://jad.tums.ac.ir published online: june 24, 2019 original article detection of diazinon residue in honey and honey bee (apis mellifera) in bandar-abbas and meshkinshahr, iran fatemeh bagheri1; *hassan vatandoost1,2; mansoureh shayeghi1; yavar rassi1; ahmad ali hanafi-bojd1,2; abbas rahimi-foroushani3; alireza razavi4; fatemeh nikpour-alkaran2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute of environmental research, tehran university of medical silences, tehran, iran 3department of epidemiology and biostatistics school of public health, tehran university of medical sciences, tehran, iran 4department of pathobiology, school of public health, tehran university of medical sciences, tehran, iran (received 24 sep 2018; accepted 15 dec 2018) abstract background: the excessive use of pesticides for crops by farmers, their destructive effects on beneficial organisms, such as bees, have become a big problem today. this study was designed to find out if the honey bee (apis mellifera) and the honey be affected by diazinon. methods: six hives were purchased, 3 hives in bandar-abbas and remaining were considered for meshkinshahr. plants around the hive were sprayed with diazinon at a concentration of 2/1000. sampling took place 15d after spraying, and diazinon residue was measured by the hp tlc. the study was conducted in 2017–2018. results: the amount of diazinon residue in honey and honey bee was measured and compared with existing studies. the amount of diazinon residue in honey bee was found to be 0.017mg/kg in bandar abbas, and 0.005mg/kg in meshkinshahr hives. there was nothing in honey. conclusion: honey is safe for consumers. keywords: honey; apis mellifera; diazinon reside; hp tlc introduction honey is a sweet, sticky and thick liquid produced by bees from nectarine flowers and contains a significant amount of minerals, vitamins, and enzymes (1). honey is composed of main fructose (about 38.5%) and glucose (about 31.0%) (2). it is known as a nutrient and much healthier than sugar (3, 4). honey is widely used for nutritional and medicinal purposes and can be used to treat wound infections and cancers (5). it can be used to treat cough and sore throat, stomach ulcer, earache, measles, and eye diseases (6). honey is widely used worldwide as a food or medicine. feeding babies with honey help to improve the memory and growth of children, as well as reduce anxiety and increase the growth performance during the life of individuals (7). honey is used in cosmetics as well as a natural sweetener in food production. while nutritional value and different aspects of the quality of honey are important, ensuring its chemical safety is also important for consumer acceptance. benefits of health and nutrition honey if it is contaminated with toxic chemicals, such as contamination of honey with residues of pesticides or other environmental pollutants (8). it can be severely exposed to various types of contaminations in the environment. even *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com mailto:hvatandoost1@yahoo.com j arthropod-borne dis, june 2019, 13(2): 185–190 f bagheri et al.: detection of diazinon … 186 http://jad.tums.ac.ir published online: june 24, 2019 small amounts of contaminants in the environment, such as residues of pesticides, can enter the honeybee and enter honey during processing. since pesticides are widely used in agricultural fields and gardens to control pests and plant diseases, and hives are often found alongside the same fields and gardens, the potential for contamination of bees and consequently honey is very high. while the use of synthetic pyrethroids and carbamates as insecticides, herbicides, and fungicides are increasing, but the use of organochlorines (mainly as insecticides in food products) has been severely reduced since their ban in 1999 (9). however, organochlorine pesticide residues are still recognized in the environment and in various types of foods because of their resistance to staying in the environment and illegal use. even the newer generation of pesticides (artificial pyrethroids, organophosphates, and carbamates) is not as stable as the first generation organochloride, but they have very high acute toxicity. organophosphate pesticides have been found in some areas even in fruits and vegetables that enter the market (10). all pesticides are poisonous and many of them have high potential for carcinogenesis and may even cause severe chromosomal aberrations (11). pesticides can also interfere with endocrine disruptions, reproductive organs, fertility and the nervous system (12-15). this study was designed to find out if the honeybee and honey are affected by diazinon. materials and methods this study was conducted in two cities of bandar abbas and meshkinshahr in 2017– 2018. meshkinshahr is located in the province of ardebil in the northwest of iran. this city is near the mountain and with relatively cold weather. both plants that grow naturally in this area, as well as different herbaceous plants, will make beekeepers in the area more prevalent. bandar abbas is located in hormozgan province in southern iran. the climate of this city is warm and humid. the cold winters of the northern regions of iran forced beekeepers to bring their hives to a warm tropical climate, which in winter is pleasant spring weather like bandar abbas. measurement of residues of diazinon in honey for this purpose, 6 hives were purchased. plants around the hive were sprayed up to radius of 200 meters in a hive with diazinon at a concentration of 2/1000, the same conventional concentration that farmers used to control the pests. fifteen days after spraying, honey was harvested. the control beehive was placed at a point where no spraying is done and then honey is picked up. honey harvested from hives should be free of wax. to remove wax from honey, wax honey was placed inside stainless steel and sub filters in a clean container that was washed with conventional detergents and placed under the sun. after 24 h, wax and honey were easily separated. subsequently, samples of harvested honey were transferred to the laboratory. steps to prepare honey samples dissolve 50g of honey in a 50ml tube in 10ml of deionized water and place it in a blender for 1min, then acidify with acetonitrile with acidic (10ml) and 1g sodium acetate and 4g magnesium sulfate. add water and shake for 1min. the samples were centrifuged for 4min at 2,000min. 6ml of high transparent liquid was added to 15ml polyethylene tubes containing 0.4g psa and 0.6g magnesium sulfate without water and centrifuged at 4000 rounds per 2min. in this way, the solution was prepared to be placed on the chromatography device (16). stages of preparation of bee body samples the specimen from worker honey was transferred to disposable polyethylene bags. j arthropod-borne dis, june 2019, 13(2): 185–190 f bagheri et al.: detection of diazinon … 187 http://jad.tums.ac.ir published online: june 24, 2019 honey bees were collected from honey from the honeycombs located in the furthest walls of the hive. these are typically older working honeybees, which are likely to have the most residual pesticide residues, depending on their age. all samples were transported to a laboratory in a cool box and stored at 20 °c in the laboratory until freezing. standard method for diazinon extraction was used (17). subsequently, the extracted solution was measured by chromatography method and the amount of pesticide residual was measured and compared with mrl and adi. detection and measurement of diazinon in honey samples to detect and determine the amount of pesticide, the first step is to apply a stain carried out using 20x20 plates covered with silica gel. a standard insecticide sample of diazinon is required to stain. spotted plates are placed at a depth of 1.5cm inside the chromatography tank. to see the spots of the plate they were placed in a uv cabinet and observed at appropriate wavelengths. regarding standard spots and unknown spots, they can be compared with each other and determine the presence or absence of pesticides in this way qualitatively. hp tlc scanner spot scan is done by the hp tlc. this device is designed to monitor the donometry of chromatographic and electrophoretic purposes. after inserting the plates into the device chamber and adjusting the beam on the first spot, enter the necessary information to start the scan, such as the size of the gap, the speed of the scan, the wavelength, and the type of lamp in the corresponding section if the wavelength and the size of the correct gap are selected. record spectrum the spectrum from the detected peaks is automatically used to determine the identification of the courier or the purity comparison spectrum. quantitative evaluation of results all plates are checked after chromatography and the proper wavelength detector determines this device, and the time required for this operation is very small. using this device, the quantitative evaluation of the material at a higher speed and more precision is performed. results the maximum residue of diazinon was in hive no. 2 in bandar abbas and the minimum s in meshkinshahr no. 2 (table 1). table1. the number of diazinon residues in the honeybees' body in the examined hives bandar-abbas hives diazinon residue sample 1 (control) 0 sample 2 0.025mg/kg sample 3 0.009mg/kg meshkinshahr hives diazinon residue sample 1 (control) 0 sample 2 0.003mg/kg sample 3 0.007mg/kg after analyzing the samples, the residues of diazinon in honey were not observed, and its value was considered zero. so the honey was completely clean and free of insecticide. discussion diazinon residue in bandar abbas hive was 0.025 and 0.009mg/kg respectively in samples. the figures in meshkinshar was 0.003 and 0.007mg/kg. in a study (16), 46 organochlorine, organophosphate, pyrethroid, and organonitrogen pesticides residue were analyzed in honey samples from 18 hives in 9 different locations, followed the gas chromatography. the remaining pesticides tested in 55.6% of the samples collected, and most of them were detected more and more pesticides identified belong to organochlorine and organophosphorus groups. the highest amount of pesticide j arthropod-borne dis, june 2019, 13(2): 185–190 f bagheri et al.: detection of diazinon … 188 http://jad.tums.ac.ir published online: june 24, 2019 residues belonged to diclofen used in the hive, because of fighting the varroa mites, and in 38.9% of honey samples and 81.8% of identified pesticides exceeded the eu-approved honey limit, that is, mrl so the residue of diazinon was set above the limit (16), while in our study, the amount of residual diazinon was less that mrl and honey was free of pesticide (16). in ghana, the amount of residue of a large number of pesticides in honey was measured. pesticides such as deltamethrin, permethrin, fenvalerate, chlorpyrophos, syphlotrin, diazinon and several other pesticides were studied. the residual of all measured pesticides is very low and below the maximum limit set by the european union. so the analysis of residues of pesticides in honey samples shows that there are no health hazards for consumers (10). the results of this study are quite similar to the present study. in our study samples, the amount of residual diazinon was much less than detectable the pollen of the environment can be identified with respect to the residual types of pesticides and heavy metals in honey and honey bee bodies. as one of the goals of our study was to measure the residue of the diazinon pesticide in honey and the honey bee, it is important to ensure the integrity of this essential food product and the impact of spraying the environment on the bee, which is a useful and strategic insect be aware. in our study, although some contamination was detected in the bee body, there was no contamination in honey (18). six villages were considered as research sites and by sampling bees and feces they measured the remaining heavy metals by using the method of atomic absorption spectrometry and showed that the bees changed to chemical in the environment in which they live, especially the increase in the number of heavy metals in soil, air and plants, and the residues of these substances in the body of the bee can represent the presence of these substances in nature. although there may not be honey in the bee product, so these studies indicate that the bees are a reliable indicator of the cleanliness and lack of environment of any chemical (19). the l residue of different pesticides was investigated in local honey samples produced from different places in jordan and from imported honey during 1995–1995. the remaining 50 pesticides were determined in 26 samples of honey. the highest amount of pesticides in the samples was assigned to the chlorine group. lower levels remained in some organophosphates and pyrothyroids. in the 11 samples examined, the remaining αhch, βhch, and lindane residues were detectable. in some of the samples, ddt, heptachlor, heptachlor oxide, dieldrin, and aldrin were also detected. the fluvalinate residue was found only in 4 honey samples. the amitraz residues, tetradifon and bromopropylate were not detectable in honey samples. the measurement of these pesticides in honey was carried out using the gc method. the similarity of this study with our finding is that the diazinon pesticides in honey samples were zero (20). conclusion there was no pesticide residue in honey so it seems honey is safe for consumers. acknowledgments the authors would like to highly appreciate the collaboration of the staff of department of medical entomology and vector control school of public health, tehran university of medical sciences, tehran, iran. this research was financially supported by the vice-chancellor of research affairs, tehran university of medical sciences and center for communicable diseases control, ministry of health ir. iran. project code number, 9121260002. j arthropod-borne dis, june 2019, 13(2): 185–190 f bagheri et al.: detection of diazinon … 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honeybees -bioindicators for environmental quality. bulg j agric sci. 18: 435–442. 20. al-rifai j, akeel n (1997) determination of pesticide residues in imported and locally produced honey in jordan. j apic res. 36: 3–4. http://www.tandfonline.com/author/al-rifai%2c+jawad http://www.tandfonline.com/author/akeel%2c+nageh http://www.tandfonline.com/toc/tjar20/current http://www.tandfonline.com/toc/tjar20/current http://www.tandfonline.com/toc/tjar20/36/3-4 j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 26 http://jad.tums.ac.ir published online: march 31, 2022 original article detection of malaria parasites and other haemosporidia in migratory and native birds in mazandaran and golestan provinces, iran seyedeh fatemeh ehsani-amrei1, *afsaneh motevalli-haghi1, *mehdi nateghpour1, mehdi mohebali1, mohammad reza youssefi2, omid raiesi1,3, seif ali mahdavi4, leila farivar1 1department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2department of veterinary parasitology, babol branch, islamic azad university, babol, iran 3department of parasitology, school of allied medical sciences, ilam university of medical sciences, ilam, iran 4amol faculty of paramedics, mazandaran university of medical sciences, sari, iran *corresponding author: dr afsaneh motevalli-haghi, email: a-motevalli@tums.ac.ir, amh.mot@gmail.com, dr mehdi nateghpour, email: nateghpourm@sina.tums.ac.ir (received 4 may 2020; accepted 6 nov 2021) abstract background: a variety of haemoprotozoa including plasmodium, haemoproteus and leucocytozoon cause infections in birds and are transmitted by some known vectors. these parasites cause anemia, low appetite, weakness and ultimately death in birds. the present study was aimed to determine these parasites, in birds of mazandaran and golestan provinces in iran. methods: the project was performed on 340 live birds in 2016. the samples were collected from february to september 2016, from each bird, two thin and thick blood smears were prepared and the remaining blood about 1ml was kept in edta-containing tubes for molecular studies. the slides were stained with 10% giemsa, then examined microscopically. about ten percent of the negative samples were considered for polymerase chain reaction (pcr) technique, using specific primers to diagnose plasmodium and haemoproteus spp. electrophoresis was done for pcr products and relevant bands to the parasites were identified based on the size. the considered birds belonged to ducks, chickens, roosters, and pigeons. results: from 340 microscopically examined blood samples 32 (9.5%) samples were positive. twenty-five (7.35%) of them were infected with the genus haemoproteus. seven samples (14%) out of 50 microscopically negative samples were found as haemoproteus or plasmodium spp when pcr technique was employed. conclusion: this study revealed the existence of malaria parasites and other haemosporidia in birds in iran. employing molecular methods (pcr examination) could detect more infections. keywords: avian malaria parasites; microscopy; pcr technique; iran introduction apicomplexa is a wide range of protozoa which contains many agents with the ability of to produce important diseases. avian malaria is a parasitic disease of birds caused by species of plasmodium and haemoproteus. these parasites belong to haemosporidia which contains more than 200 species of avian parasites and these have placed into three genera including plasmodium, haemoproteus and leucocytozoon (1). avian malaria is transmitted by some mosquitoes. incidence of the disease depends on some factors such as an abundance of vectors, temperature, weather, quality of water and breeding place (2). species of plasmodium and haemoproteus are pathogens and life-threatening agents for some groups of copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:a-motevalli@tums.ac.ir mailto:amh.mot@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 27 http://jad.tums.ac.ir published online: march 31, 2022 birds particularly those are kept in one place and those are living in warm and humid regions (3). plasmodium and haemoproteus parasites are intercellular agents and growing in red blood cells. these parasites affect birds’ red blood cells (rbcs) in a way that the birds will first suffer anemia and then weakness, lethargy, and distress followed by low appetite, reduced feeding, and consequently death (4). vectors of avian malaria can transmit the parasites to the blood circulation of animals even human but rarely results in infection. moreover, noninfected vectors can pick up the circulating sporozoite from human blood in feeding time (5). parasites of haemosporina are responsible for millions of infections and thousands of deaths in human, wild and domestic animals. if vectors of these parasites share blood feeding between human and animals, the human community is exposed to a wide range of parasites existing in the animal community (3). some authors believe that a specific host of a parasite can be modified by natural selection. that is, parasites can naturally adapt themselves to new hosts such as human. it is important to know which kind of animal parasites may be transmitted to human even if these parasites cannot evolve in human in the current situation (5). indeed, zoonosis may start its primary process based on such transmission form which will adapt in new host gradually. since these parasites are in close contact with human societies identifying them can help individuals to protect themselves against probable infections. over the past five years, few studies have been performed about identifying birds’ parasitic infections in the northern part of iran. the results imply that there is a wide range of parasitic infection among birds. therefore, it was necessary to renew the relevant study about avian malaria in the north of iran regarding the broad spectrum of carriers, vectors, a specific climate of the region, frequent rainfalls, changes in the level of hygiene, especially in rural areas and population growth in this region. on the other hand, besides the health issues portion of the economic life of the native people in the studied areas depends on breeding and selling the birds, so such studies can give some informative hints to the relevant policymakers. materials and methods study areas samples were collected from mazandaran and golestan provinces from february to september 2016. mazandaran province has stretched from the latitude of 35° 47' to 36° 35'n and the longitude of 50° 47'e. the weather in mazandaran province is mild with an average temperature of 25 °c in summer and 8 °c in winter. the average annual rainfall in mazandaran is 700mm. golestan province has extended from the latitude of 36° 30' to 38° 8'n and the longitude of 53° 57' to 56° 22'e with an average temperature of 30 in summer and 7 in winter. the average annual rainfall in golestan is 450mm. golestan province has a variety of climates. sampling and testing a total of 244 and 96 samples were collected from different districts of mazandaran province (including babol, fereidounkenar and sari) and golestan province (including gorgan, gonbad, and kordkuy). domestic birds and wild carnivorous birds were the target groups for this study. the legal guardian was approved from tehran university of medical sciences. the birds were captured randomly using traditionally way including provide plenty of food and water in trap cages, then two milliliters whole blood were collected of their wings and put into edta containing tubes via venipuncture to prepare thin and thick blood smears and designing molecular tests as well. finally the birds were released. thin and thick smears were air-dried and stained with 10% giemsa stain for 20 minutes, http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 28 http://jad.tums.ac.ir published online: march 31, 2022 then the slides were washed up smoothly and after drying assessed under an optical microscope (leitz, germany), with 1000 x magnification. molecular analyses in this study, 50 blood samples including all the suspected samples and 40 of negative samples were examined using the polymerase chain reaction (pcr) test. dna was extracted from the blood samples using a dna extraction kit (bioneer, cat no: k-3032). as rbcs of birds contain a nucleus and thus a large amount of dna, so the blood samples were kept at 95 °c for 15 minutes as an additional step to obtain favorable results. to identify the plasmodium genus, a set of primer (pls and unr primers as the following) were used to amplify the small subunit ribosomal dna gene. development of a 787bp band revealed birds’ infection with plasmodium (6). the (p1) and (p2) primers were used to amplify the cytochrome b gene where the formation of a 500bp band showed the birds’ infection with haemoproteus (7). nucleotide sequences of the primers used in this study are shown in table 1. to identify the genus haemoproteus a total volume of 25µl comprising 10µl of master mix [including dntps, mgcl2, 10x buffer, taq polymerase], 3µl of dna extract (50ng), 1µl of each (p1) and (p2) primers in concentration l0pmol and distilled water (d.w) up to 25µl were prepared. to detect the plasmodium genus the needed materials were as the same to haemoproteus identification except replacing primers with pls and unr primers. after a brief centrifuge, tubes were transferred to a thermocycler (thermofisher, usa). the program used for the pcr test to determine haemoproteus was as follows: initial denaturation at 94 °c for 3 minutes, then 35 cycles including denaturation at 94 °c for 1 minute, annealing at 55 °c for 45 seconds, extension at 72 °c for 45 seconds, and eventually extension at 72 °c for 10 minutes. pcr program to detect plasmodium genus was run as follows: initial denaturation 4 minutes at 94 °c then 35 cycles of: denaturation at 94 °c for 45 seconds, annealing at 50 °c for 90 seconds, extension at 72 °c for 2 minutes and finally, extension at 72 °c in 8 minutes. positive and negative controls were used to compare with samples. a positive and negative control came from samples which was clearly positive and negative in microscopic exams respectively. the products of pcr were put under the electrophoresis process with 1% agarose gel to observe bands corresponding to amplification of specific gene to diagnose parasites genus. gel agarose was prepared using tae buffer, including tris base 242g, acid acetic glacial 57.1 ml, edta 100ml (0.5m, ph: 8) up to 50 times concentration. the utilized products were photographed with a uv illuminator. spss software was used for analyzing the results of microscopic examination and molecular tests. results the captured bird in this study belonged to ducks (anas platyrhynchos), chickens (gallus gallus domesticus), roosters (gallus domesticus), pigeons groups (columba livia) and wild carnivorous birds including egyptian vultures (neophron percnopterus), kite bird (milvus migrans), western marsh harrier (circus aeruginosus), common buzzard (buteo buteo), and eagles (accipitridae). microscopical examination showed that 23 of 244 blood samples (9.42%) that were collected from mazandaran province and two samples (2.08%) of those from golestan province were infected as haemoproteus spp. all these infections were detected from the 23 (35.38%) and 25(30.12%) pigeons in mazandaran and golestan provinces, respectively (fig. 1 and table 2). none of those examined samples showed plasmodium infection using microscopy method. to study molecular investigation a total of fifty samples including microscopically suspicious and ten percent of negative blood samhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 29 http://jad.tums.ac.ir published online: march 31, 2022 ples were assessed by pcr technique. gel electrophoresis of pcr product of haemoproteus cytochrome b gene showed two (4%) haemoproteus spp infected cases in pigeons with a band of 500bp. moreover, gel electrophoresis of pcr products showed five (10%) cases of plasmodium infection in ducks with a band of 787bp (figs. 2, 3 and table 3). employing the spss software confirmed that significant differences were observed between the microscopic method and pcr technique, df= 49 and p< 0.05. table 1. nucleotide sequences of the primers used in this study genus gene length of pcr (bp) oligonucleotides 5’-3’ reference plasmodium small subunit ribosomal dna 787 pls: caatcgagtttagtgtgtatc unr: gatcgtcttcgacggtatct (6) haemoproteus cytochrome b 500 p1: atgcgatatatgcatggtgcttt p2: ataatgatgtgatgtgcattatctgg (7) table 2. number of collected blood samples from mazandaran and golestan provinces including type of birds and parasitic infection using microscopic examination type of bird number plasmodium spp haemoproteus spp. mazandaran province ducks 87 0 0 pigeons 65 0 23(35.38%) chickens and roosters 92 0 0 total 244 0 23 (9.42%) golestan province ducks 41 0 0 pigeons 18 0 2 (11.11%) chickens and roosters 17 0 0 wild (carnivorous) birds 20 0 0 total 96 0 2 (2.08%) fig. 1. sampling sites (section of iran): mazandaran and golestan provinces. stars show collection sites http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 30 http://jad.tums.ac.ir published online: march 31, 2022 table 3. number of plasmodium spp and haemoproteus spp in 50 suspicious and negative blood samples among the studied birds in mazandaran and golestan provinces using pcr tests type of bird number plasmodium spp haemoproteus spp ducks 15 5 (33.33%) 0 pigeons 10 0 2 (20%) chickens and roosters 11 0 0 wild (carnivorous) birds 14 0 0 total 50 5 (10%) 2 (4%) fig. 2. morphology of haemoproteus spp in thin blood smear of pigeon based on microscopic method with 1000x magnification. nrbc: nucleated red blood cell, (original picture) fig. 3. gel electrophoresis of the pcr product of haemoproteus cytochrome b gene, 1 and 2: positive samples of pigeons, no. 3: negative control, no. 4: positive control, sm: size marker. a 500bp band related to haemoproteus infection is shown in pigeon birds in comparison of positive control http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 31 http://jad.tums.ac.ir published online: march 31, 2022 fig. 4. gel electrophoresis of the pcr product of plasmodium small subunit ribosomal dna gene, no. 1, 2, 3, 4 and 5: positive samples of ducks. no. 6: negative control, no. 7: positive control, sm: size marker. a 787bp band corresponding to plasmodium infection is shown in duck birds in comparison of the positive control discussions bird's malaria parasites are transmitted mostly by culicine mosquitoes that some species of them bite a wide range of hosts including human. therefore, a human can be exposed to some transmitted parasites through the biting processes. a large number of birds migrate over the winter from siberia to the province of mazandaran, and if they carry some species of malaria parasites the disease can be spread over the native birds that are in close contact with the human community, moreover avian have a crucial role in economic life of native residents in this area. there are a lot of studies about avian malaria and other haemosporidia in the world. according to these revisions the prevalence rate of haemoproteus and plasmodium species among different birds in new zealand, colombia, bulgaria, and the united states differs between 20% and 32% (8-11) while based on the other reports prevalence rate of the parasites in japan, costa rica, and alaska was less than 10% (12-14). in a cross-sectional study conducted by zhang and colleagues (15), 7.8% of captured birds were infected with some species of plasmodium. a study performed in brazil showed that the level of infection with species of plasmodium was 16.5% (16), where as this rate in the present study was 1.5% in considered birds for both of provinces. moreover, this range can differ even in one country. it is important to note that the parasite prevalence and diversity will change with some geographical features such as temperature, climate, humidity, the prevalence of the vector and so on. in iran, various research showed different results related to study areas. yousefi and colleagues (17-18) could isolate haemoproteus spp from a pigeon in the north of iran. dehghani samani et al. (19) found that haemoproteus infection among the old pigeons was higher than the younger pigeons in the west of iran. shemshadi and colleagues (20) reported that the rate of parasitic infection was 6.1% among the caspian coastal birds in the north of iran. moreover, there are another information about existence of avian malaria in iran. in this published data authors declared that the rate of infected birds was as follow: turkeys (10.2%), hens/roosters (1.7%), pigeons (6.1%) and migratory waterfowl (6.4%). these published data were recorded from fars, isfahan, and mazandaran provinces (21). in the present study, microscopic examination revealed haemoproteus spp infections in 25 samples (7.35%) of the 340 samples, also http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 32 http://jad.tums.ac.ir published online: march 31, 2022 7 samples (14%) of the 50 suspicious and negative samples were positive as haemoproteus and plasmodium using pcr test. regarding these two methods, the microscopic method is easy and cost-effective and thus it is the most common method for diagnosing plasmodium and haemoproteus species. however, it may sometimes show false negative results due to the low level of parasites in birds’ blood, inappropriate staining, or presence of artifacts. in this concern, molecular analyses might be complementary methods for the microscopic of the diagnostic method. so, further studies are required for adjusting and preparing stable conditions of those analyses. the present study revealed the existence of malaria parasites and other haemosporidia in birds in iran and it is a necessity to provide further studies to establish new prevention strategies to block transmission of the parasites in birds’ community. pcr examination showed that there are more infections in the birds when researchers employ molecular methods. as it was implied before, assessment was based on size of the bands in pcr examination but considering that gene sequencing is a reliable way to confirm the presence of parasites, it is suggested to design a new study in this field. conclusion study on avian malaria in addition to knowledge of the condition of the disease among birds, can provide appropriate strategies for preventing further spread of the disease. results of this study prompts scientist to conduct more studies in the field of avian malaria particularly in the studied areas. acknowledgements the authors would like to thank mr farhad pourghase, vahid kheirabadi and mr abdolah mehdipour for their useful collaboration and field sampling. ethical considerations the study was approved by the joint ethical committees of tehran university of medical sciences ethic no. ir.tums.sph.rec. 1397.247. conflict of interest statement authors declare that there is no conflict of interest. references 1. valkiūnas g, iezhova ta (2022) keys to the avian haemoproteus parasites (haemosporida, haemoproteidae). malar j. 21(1): 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identify plasmodium species http://jad.tums.ac.ir/ https://pubmed.ncbi.nlm.nih.gov/?term=bensch%20s%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=graves%20gr%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=jhala%20yv%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=peirce%20ma%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=rahmani%20ar%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=fonseca%20dm%5bauthor%5d https://pubmed.ncbi.nlm.nih.gov/?term=fleischer%20rc%5bauthor%5d https://www.ncbi.nlm.nih.gov/pmc/articles/pmc1639517/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc1639517/ j arthropod-borne dis, march 2022, 16(1): 26–33 sf ehsani-amrei et al.: detection of … 33 http://jad.tums.ac.ir published online: march 31, 2022 in human blood samples: the semi-nested multiplex malaria pcr (snm-pcr). trans r soc trop med hyg. 96(supplement_1): s199–s204. 7. bermúdez‐humarán lg, garcía‐garcía a, leal‐garza ch, riojas‐valdes vm, jaramillo‐rangel g, montes‐de‐oca‐luna r (2002) molecular 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prevalence in blood parasites of adult passerine birds during the breeding season in alaska. j wildl dis. 37(1): 28–35. 15. zhang y, wu y, zhang q, su d, zou f (2014) prevalence patterns of avian plasmodium and haemoproteus parasites and the influence of host relative abundance in southern china. plos one. 9(6): e99501. 16. ribeiro s, sebaio f, branquinho f, marini m, vago a, braga e (2005) avian malaria in brazilian passerine birds: parasitism detected by nested pcr using dna from stained blood smears. parasitology. 130(3): 261–267. 17. youssefi mr, rahimi mt (2011) haemoproteus columbae in columba livia domestica of three areas in iran in 2010. glob vet. 7(6): 593–595. 18. youssefi m, sadeghian a, esfandiari b (2010) prevalence of haemoproteus columbae infection in columba livia in north of iran. j zool. 5(4): 275–277. 19. samani ad, kheirabadi kp, samani ad (2013) prevalence and rate of parasitemia of haemoproteus columbae in columba livia domestica in southwest of iran. iran j parasitol. 8(4): 641. 20. shemshadi b, ranjbar-bahadori s, faghihzadeh-gorgi s (2015) occurrence of parasitic protozoa in wild waterfowl in southern coastal caspian sea lagoons. iran j vet med. 8(4): 261–267. 21. azari-hamidian s, norouzi b, harbach re (2019) a detailed review of the mosquitoes (diptera: culicidae) of iran and their medical and veterinary importance. acta trop. 194: 106–122. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2015, 9(2): 238–245 e a yağmur et al.: determination of the … 238 original article determination of the median lethal dose and electrophoretic pattern of hottentotta saulcyi (scorpiones, buthidae) scorpion venom *ersen aydın yağmur 1, özcan özkan 2, k zafer karaer 3 1alaşehir vocational school, celal bayar university, alaşehir, manisa, turkey 2drug and medical device agency of turkey, turkey 3department of entomology and protozoology, faculty of veterinary medicine, ankara university, turkey (received 27 oct 2013; accepted 2 aug 2014) abstract background: in this study, we investigated the lethal potency, electrophoretic protein pattern and in vivo effects of hottentotta saulcyi scorpion venom in mice. methods: scorpions were collected at night, by using a uv lamp from mardin province, turkey. venom was obtained from mature h. saulcyi scorpions by electrical stimulation of the telson. the lethality of the venom was determined by i.v. injections using swiss mice. in vivo effects of the venom were assessed by using the intraperitoneal route (ip) injections into mice (20±1g) and monitored for 24 h. the protein profiles of the scorpion venom were analyzed by nupage® novex® 4–12 % gradient bis-tris gel followed by coomassie blue staining. results: the lethal assay of the venom was 0.73 mg/kg in mice. we determined the electrophoretic protein pattern of this scorpion venom to be 4, 6, 9, 31, 35, 40, 46 and 69 kda by sds-page. analysis of electrophoresis indicated that h. saulcyi scorpion intoxicated mice exhibited autonomic nervous system symptoms (tachypnea, restlessness, hyperexcitability, convulsions, salivation, lacrimation, weakness). conclusions: hottentotta saulcyi scorpion venom includes short-chain neurotoxins and long-chain neurotoxins according to the electrophoretic protein patterns. the stings of h. saulcyi scorpion must be considered of risk for humans in the southeastern region, turkey. keywords: scorpion, hottentotta saulcyi, venom, in vivo effects introduction in the world, approximately 2091 species and 197 genera of scorpions are described (rein 2013), currently distributed in 15–19 families (soleglad and fet 2003, prendini and wheeler 2005). scorpion envenomation is a major public health problem in developing countries especially in tropical and subtropical regions (özkan et al. 2011). cases of scorpion envenomation into humans are common in turkey due to its geographical location, climate and socioeconomic structure (özkan et al. 2004, özkan and kat 2005). therefore, scorpion envenomation is common in several regions of the country, especially in southeastern anatolia (adıguzel et al. 2007, al et al. 2009, boşnak et al. 2009, özkan et al. 2008, 2011). in the southern region of turkey, scorpion stings and envenomation cases are a major public health problem, especially in southeastern anatolia (altınkaynak et al. 2002, adıguzel et al. 2007, boşnak et al. 2009). children are at greater risk of developing severe cardiac, respiratory and neurological complications (altınkaynak et al. 2002, boşnak et al. 2009). so far, the most medically important and dangerous scorpion species responsible for envenomings described in turkey are: androctonus crassicauda, leiurus quinquestriatus (now leiurus abdullahbayrami), mesobuthus *corresponding author: dr ersen aydın yağmur, e-mail: ersen.yagmur@gmail.com http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 238–245 e a yağmur et al.: determination of the … 239 gibbosus and mesobuthus eupeus, all of which belong to the buthidae (uçar et al. 2005, boşnak et al. 2009, adıguzel 2010, özkan et al. 2011). hottentotta is one of the most widely distributed genera of the family buthidae, with species present throughout africa, the middle east and asia (kovařík 2007, yağmur et al. 2008). the minor numbers of h. saulcyi scorpion sting cases were reported in iran (dehghani and fathi 2012) while the scorpion sting was not reported in turkey. in turkey, h. saulcyi was first recorded in mardin province (crucitti, vignoli 2002) and later observed in batman, şırnak, and hakkâri provinces, by yağmur et al. (2008). adıguzel (2010) reported that there is no data related to venom toxicity and in vivo effects of h. saulcyi scorpion species in turkey. in the present work, we studied the toxicity of h. saulcyi from mardin province of turkey. the lethality and effects of the venom were assayed in mice and the protein profiles (molecular weight) of the venom determined. materials and methods scorpions and venom this study was approved by the ethic committee of refik saydam public health agency, ankara, turkey under process number 33/13.11.2009. scorpions were collected at night, by using a uv lamp from eskikale village, in mardin province of the southeastern region in 05.09.2009, turkey (fig. 1). avoiding scorpion cannibalism, captive scorpions were housed in individual plastic boxes at the department of entomology, faculty of veterinary medicine, ankara university, turkey. the scorpions were fed with crickets or cockroaches and received water daily. venom was obtained from mature h. saulcyi scorpions from mardin province by electrical stimulation of the telson (özkan and filazi 2004). obtained venom from scorpions was collected into an eppendorf tube. the venom was dissolved with sterile double-distilled water and centrifuged at 15,000 rpm for 15 min at 4 ºc (özkan and filazi 2004). the precipitate was discarded and the supernatant was stored at -20 oc until use. protein concentrations were determined using a bca kit (pierce, usa) with bsa as the standard. experimental animals swiss mice of both sexes (20±2g) were employed to determine the median lethal dose (ld50) by intravenous (iv) route of administration. they were bred in the animal facility of the refik saydam public health agency (rspha). the animals were housed under controlled temperature (20±2 °c), with a 12:12 light/dark schedule and were fed commercial rodent pellets and water ad libitum throughout the experiment. determination of the median lethal dose the lethality of the venom was determined by iv injections as described by behrens and karber (1935) sing swiss mice. five mice per each dose group were injected iv with increasing volume doses of venom (11.22, 13.02, 14.80, 16.63, and 18.40 µ g/ 19g mouse), diluted in 0.2 ml physiological saline solution (pss: 0.85% nacl). an equivalent volume of pss was injected into five mice as negative control group. the mice were observed for 24 h after venom injection. deaths occurring during the first 24 h after injection were recorded in order to determine the median lethal dose. the lethality was expressed as the median lethal dose (ld50). experimental protocols for animal experiments were approved by the ethical committee of the rspha. evolution of the experimental envenomation the experimental envenomation was asseshttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 238–245 e a yağmur et al.: determination of the … 240 sed by using the intraperitoneal route (ip) injections into mice (20±1g). one group of mice (n: 6) was injected i.p. with 3 ld50 doses of h. saulcyi venom (2.2 mg/kg), diluted in 0.5 ml pss. the symptoms were monitored for 24 h after venom injection in order to assess the results of experimental envenomation. gel electrophoresis of the venom polyacrylamide gel electrophoresis of venom sample was carried out following the laemmli (1950) method. for separation of proteins, a sample was run on nupage® novex® 4–12 % gradient bis-tris gel (invitrogen corparation, usa) in mes sds running buffer (invitrogen: 50 mm mes, 50 mm tris-hcl, 1 % sds, 1.025mm edta) using xcell surelock mini cell (invitrogen) following standard manufacturer protocol. seeblue® plus2 pre-stained standard (invitrogen, lc5925) was run in parallel in order to calculate the molecular weights of proteins. detection of proteins was carried out initially by coomassie blue staining. the gel was then scanned and molecular weights of the proteins were calculated with molecular imaging software (kodak mi). results scorpions and venom all scorpions were observed as being very aggressive in the course of all keeping milking time. a colorless watery secretion was obtained during capturing and was followed by more viscous milky droplets or ejaculate (mucous accompanied with the venom) during stimulation. after centrifugation of whole venom, the supernatant was of a more viscous form. the protein content of the venom sample was 2.3 mg/ml. lethal potency of the scorpion venom the median lethal dose for h. saulcyi scorpion venom was determined in mice. the ld50 of the scorpion venom was found to be 0.73 mg/kg by iv injection route. assessment of the experimental envenomation after venom injection when intoxicated ip with 2.1 mg/kg (3 x ld50 iv doses) of venom from h. saulcyi, the mice showed the following signs of intoxication: immediately following venom injection, mice showed intense and longterm squeaking (indicating pain), jumping and later restlessness, aggressive behavior, fight, tachypnea, deep dyspnea, weakness, convulsions, paralysis and coma resulting in death. but none of the animals exhibited any hypersalivation or lacrymation symptoms after venom injection by ip route. however, these signs were observed in mice after iv venom injection. determination of protein profiles the protein profiles of h. saulcyi scorpion venom were analyzed by nupage® 4–12 % gradient bis-tris gel followed by coomassie blue staining. proteins of the venom (30 µ g) were determined to be between 3 and 188 kda on electrophoresis on gradient gel as shown in (fig. 2). eight different protein bands with molecular masses of 4, 6, 9, 31, 35, 40, 46 and 69 kda were detected in the venom sample. the proteins in venom secretion were more strongly determined to be 4, 6, 9, 46 kda than other bands after staining by coomassie blue. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 238–245 e a yağmur et al.: determination of the … 241 fig. 1. distribution (red circles) of hottentotta saulcyi in southeastern anatolia region (mardin, batman, şırnak, hakkâri provinces), turkey. the scorpions were captured in mardin fig. 2. the proteins of venom (i) of hottentotta saulcyi (a) from mardin province were separated by using 4–12 % gradient gel electrophoresis (b). molecular weight (ii): 188 kda myosin, 98 kda phosphorylase, 62 kda bsa, 49 kda glutamic dehydrogenase, 38 kda alcohol dehydrogenase, 28 kda carbonic anhydrase, 17 kda myoglobin red, 14 kda lysozyme, 6 kda aprotinin, 3kda insulin, b chain (seeblue® plus2 pre-stain) discussion scorpions do not harbour any agent capable of causing disease. however, scorpions can cause serious health problems by stinging humans, most of the time, they use their venom to protect themselves (adıguzel 2010). scorpion sting is a life-threatening emergency, especially in children and older individuals who suffer from respiratory and/or cardiovascular diseases. the clinical manifestations of scorpion envenomation are predominantly sympathetically and parasympathetically mediated, depending on the scorpion (ismail 1995, boşnak et al. 2009). on the other hand, scorpion species responsible for the stings often are unclear, and this is due to the lack of knowledge of health professionals regarding the scorpion species and the fact that the scorpion is neither seen http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 238–245 e a yağmur et al.: determination of the … 242 nor identified in most scorpion sting cases (latifi et al. 1979, shahbazzadeh et al. 2009). the types of scorpions are categorized as yellow or black scorpion by patients in the southeastern part of turkey. therefore in vivo studies are required to be performed to determine health effects and toxicity of scorpion species’ venom. up to now, no data has been found about toxicity and the effects of h. saulcyi scorpion venom from turkey. in our current study, we have determined the lethal potency and health effects in mice and the protein profiles of h. saulcyi scorpion venom. to our knowledge, this is the first work on protein profiles and the toxicity of the venom of h. saulcyi in turkey. androctonus crassicauda, mesobuthus phillipsii, h. saulcyi, compsobuthus matthiesseni of the buthidae family, scorpio maurus of scorpionidae and calchas birulai of the iuridae family have been recorded in mardin fauna up to now (crucitti, vignoli 2002, yağmur et al. 2008a, 2008b, kovařík et al. 2011). hottentotta saulcyi is distributed in mardin, batman, şırnak, and hakkâri provinces of the southeastern part of turkey (yağmur et al. 2008c) (fig. 1) and neighbouring countries, syria, iran and iraq (kovařík et al. 2007). in southern anatolia region of turkey, as in numerous tropical countries, envenomation by scorpion stings is a major public health problem since most of medically important species in scorpion fauna of turkey are found in this region. in khuzestan province of iran, the prevalence of h. saulcyi sting is 3.35 % (dehghani et al. 2009, dehghani and fathi 2012) while no epidemiologic data have been found on h. saulcyi scorpion sting in turkey (adıguzel 2010). on the other hand, the largest numbers of scorpion sting cases were reported in the southeastern anatolia region (30.4%) of turkey. mardin province (18%) has one of the highest incidences of scorpion stings in the southeastern anatolia region (özkan et al. 2008). in batman, siirt, mardin, şırnak and hakkâri provinces of the southeastern anatolia region of turkey, epidemiological and clinical studies reported that local and systemic symptoms were seen after scorpion sting (soker and haspolat 2000, boşnak et al. 2009). on the other hand, al et al. (2009) stated that 120 patients who were older than 16 years old by scorpion stung did not exhibit cardiac dysfunction, myocardial damage and deaths secondary to major systemics envenoming. during the course of the experimental envenomation, the effects of the venom in mice, its toxicity showed characteristics similar to those described for other medically important scorpion species (a. crassicauda, m. gibbosus, m. eupeus, and l. abdullahbayrami) in turkey. in our study, similar autonomic nervous system symptoms (sympathetic signs [tachypnea, restlessness, hyperexcitability and convulsions] and parasympathetic signs [salivation, lacrimation, and weakness]) were observed in envenomed mice. therefore, h. saulcyi could be considered as a potential scorpion responsible for severe envenomation and probably death in humans. scorpion venom contains simple proteins with short neurotoxin and low-molecularweight polypeptides. additionally, scorpion venom also contains serotonin, which is thought to contribute to the pain associated with scorpion envenomation (adam and weiss 1958). medically important scorpion stings are almost universally characterised by intense local pain. systemic effects occur in a smaller proportion of scorpion sting depending on various factors (luca and meier 1995). in the current work, interestingly, we observed intense and long-term squeaking in envenomed mice according to our experience. to our knowledge, no data have been found on envenomation by scorpions in human. however, squeaking in mice indicated pain, therefore the venom of h. saulcyi can be considered to be rich in terms of serotonin. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 238–245 e a yağmur et al.: determination of the … 243 the geograpical origin of scorpions have an important role with regard to toxicity of venom. latifi and tabatabai (1979) determinated that ld50 of buthotus saulcyi (=h. saulcyi) scorpion venom from iran was 0.95 mg/kg while hassan (1984) stated this to be 1.01 mg/kg in the same country by iv injection route. in our investigation, the median lethal dose of h. saulcyi scorpion venoms was found to be 0.73 mg/kg by iv injection route. scorpion venoms can be classified into two groups according to their molecular sizes, and long-chain and short-chain neurotoxins. among these more-studied groups are the short-chain neurotoxins that present 3,000 to 4,400 da and act on potassium or chloride channels. long-chain neurotoxins that have 6,500 to 8,500 da, act mostly on sodium channels (possani et al. 1999, possani et al. 2000, rodriguez de la vega and possani 2004, 2005). in our study, we have exhibited the electrophoretic protein pattern of h. saulcyi venom to be between 4 and 69 kda by gradient gel. four protein bands with molecular masses of 4, 6, 9 and 46 kda are more strongly detected than other protein bands in the venom sample. analysis of electrophoresis indicated that h. saulcyi scorpion venom possesses both short-chain neurotoxins and long-chain neurotoxins according to the electrophoretic protein patterns. conclusion hottentotta saulcyi scorpion venom has neurotoxin proteins and lethal potency in mice. therefore, the stings of h. saulcyi scorpion must be considered of risk for humans in the southeastern region, turkey. scorpion envenomation in the southeastern anatolia region is an increasing public health problem. even if the scorpion is not captured, each victim should be asked about the sting history. all patients and especially pediatric patients should also be admitted to the hospital. in further studies, the identification of the scorpion may be considered as a useful clinical and epidemiological tool in determining the incidence and risk of scorpion envenomations. therefore, the effects of this species in victims should be described and strengthened with the epidemiological and clinical studies in these regions. in addition, the monovalent a. crassicauda antivenom has commonly been used in turkey up to now. therefore, neutralization experiments should be carried out to test the usefulness of monovalent antivenom on the venom of h. saulcyi scorpion. acknowledgements we wish to thank mr mehmet özkörük for his help during the field trips and collecting the specimens. the authors declare that there is no conflict of interests. references adam kr, weiss c (1958) the occurrence of 5-hydroxytryptamine in scorpion venom. j exp biol. 35(1): 39–42. adıguzel s, özkan ö, i̇nceoğlu b (2007) epidemiological and clinical characteristics of scorpionism in children in şanlıurfa, turkey. toxicon. 49(6): 875–880. adıguzel s (2010) in vivo and in vitro effects of scorpion venoms in turkey: a mini-review. j venom anim toxins incl trop dis. 16(2): 198–111. al b, yılmaz da, sögut o, orak m, üstündağ m, bokurt s (2009) epidemiological, clinical characteristics and outcome of scorpion envenomation in batman, turkey: an analysis of 120 cases. jaem. 8(3): 9–14. 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euscorpius. 76: 1–8. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 215 original article cardiotoxic and arrhythmogenic effects of hemiscorpius lepturus scorpion venom in rats khalil pourkhalili 1, euikyung kim 2, navid reza mashayekhy 3, mostafa kamyab 4, seyed mehdi hoseiny 5, reihane evazy 6, abbas zare mirakabadi 7, *ramin seyedian 8 1department of physiology, school of medicine, bushehr university of medical sciences, bushehr, iran 2college of veterinary medicine, gyeongsang national university, jinju, south korea 3department of cardiology, amir kabir hospital, arak university of medical sciences, arak, iran 4department of biotechnology, persian gulf research institute, persian gulf university, bushehr, iran 5department of hematology and oncology, bushehr university of medical sciences, bushehr, iran 6department of biology, jahrom branch, islamic azad university, jahrom, iran 7department of venomous animals and antisera produstion, razi vaccine and serum research institute, hesarak, iran 8department of pharmacology and toxicology, bushehr university of medical sciences, bushehr, iran (received 1 feb 2014; accepted 22 june 2014) abstract background: envenomation by hemiscorpius lepturus is not painful and the clinical manifestations include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions,cardiac arrhythmia and in minority of cases acute renal failure which may lead to death following disseminated intravascular coagulation in infants. cardiac effects of envenomation by h. lepturus venom including inotropic, chronotropic and arrhythmogenic properties are not studied as now in rat hearts with langendorff apparatus. methods: rat hearts were allowed to equilibrate in its buffer and cardiotropic plus arrhythmogenic effects induced by injection of different doses of h. lepturus venom were detected and recorded by computer acquisition based data in langendorff apparatus. the neutralizing effects of razi institute antivenom and autonomic drugs were assayed in parallel studies. results: hemiscorpius lepturus venom (25 µ g/100 l) treatment caused a negative inotropic (65.4 ± 3.2 versus 110.2 ± 3.4) and chronotropic effects (186.3 ± 4.2 versus 302 ± 6.3) in comparison to normal saline. arrhythmogenic aspects including bradycardia, qrs widening and st depression were induced by venom injection. pre venom treatment (20 min) of razi institute antivenom (10 µ l) neutralized cardiotropic effects but post venom injection (15 min later) had no therapeutic role. pre (10 min before) and post (15 min after) injection of adrenaline (10 µ l) neneutralized cardiotropic effects while pre venom injection (20 min) of propanolol (10 µ l) had aggravating effects. conclusion: our study paves the way for further in vivo investigation of cardiovascular effects of this venom for finding suitable treatments instead of its ordinary antivenom medication in cardiogenic shock induced by the venom. keywords: hemiscorpius lepturus, contractility, arrhythmogenicity, envenomation introduction scorpion envenomation is common in tropical and subtropical areas of the world including southwestern part of iran especially in khuzestan province (prendini 2000, shahbazzadeh et al. 2009). envenomation by h. lepturus belonging to family hemiscorpiidae is not painful and the clinical manifestations in stung patients include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions and in minority of cases acute renal failure which may lead to death following disseminated intravascular coagulation in infants (radmanesh 1990, jalali et al. 2010). this creature is one of the most dangerous scorpions, and responsible for 95 % of the scorpionassociated mortalities in iran, that is probably due to the various pathological enzymes in its venom like hemitoxin, hemicalcin and *corresponding author: dr ramin seyedian, email: raminseyedian@gmail.com http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 216 heminecrolysin (shahbazzadeh et al. 2007, srairi-abid et al. 2008). generally there is only a few case reports on direct cardiotoxic effects of scorpion envenomation in human beings (gueron et al. 1967, bawaskar and bawaskar 1998, shapira et al. 1998, cupo and hering 2002). however previous studies on the envenomation by h. lepturus suggest that there are in vivo arrhythmogenic and negative inotropic effects in rabbits in spite of its small amount of accumulation of radiolabelled venom following subcutaneous injections in animal models during biodistribution study (mirakabadi et al. 2011, seyedian et al. 2012). in this study, the direct effects of h. lepturus venom on rat hearts were investigated with langendorff apparatus. first, inotropic and chronotrpic changes upon time were evaluated and then, neutralization properties of iranian razi institute antivenom and other drugs including epinephrine, propranolol and atropine were employed to analyze direct or indirect cardiotoxic effects of this venom thoroughly promoting further in vivo studies. materials and methods drugs and chemicals the lyophilized venom was acquired by direct electrical stimulation of h. lepturus telsons (15 mv) in iran. the venom was centrifuged, lyophilized and stored at -20 °c until reconstitution by addition of normal saline in our laboratory. the iranian pepsin digested polyvalent antivenom from horse plasma against 6 common endemic scorpions (odontobuthus doriae, mesobuthus eupeus, androctonus crassicauda, buthotus saulcyi, buthotus sach and hemiscorpius lepturus) (latifi and tabatabai 1979, dehghani and fathi 2012) were purchased from razi vaccine and serum research institute. the neutralizing ability of the used batches was 26 ld50/ml. adrenaline, atropine and propranolol were purchased from sigma (st loius, mo). animals female wistar rats (body weight 180–230 g) were obtained from jundishapur medical university breeding center (ahvaz, iran) and placed in groups of three in pvc cages with free access to water and hard food pellets in animal house of bushehr university of medical sciences. they were kept at 20 ± 2 °c and maintained at 12 hours light-dark cycle starting at 7 am. the experimental protocol used in this study conforms to the guidelines of the national institute of health (nih). langendorff perfused heart preparation rats were heparinized (300–400 iu) and anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). after a bilateral thoracotomy, hearts were rapidly excised and after insertion of an aortic cannula perfused retrogradely at a constant perfusion pressure of 90 cm h2o with gassed (5% co2, 95% o2) krebs-henseleit buffer (ph 7.4) containing nacl (118.5 mm), nahco3 (25 mm), kcl (4.7 mm), kh2po4 (1.2 mm), cacl2. 2h2o (1.8 mm), mgso47h2o (1.2 mm), dglucose 11 mm) at 37 °c. an epicardial electrocardiogram (ecg) was continuously recorded using two fine stainless steel electrodes, one attached to the apex of the heart and the other placed on the right atrium and a metal clip attaching to the aortic cannula as the reference electrode. left ventricular pressure was recorded throughout the experiment by a water filled latex balloon placed in the left ventricle and connected to a pressure transducer (mlt 844) through a fluid filled catheter. volume of the balloon was adjusted to obtain end-diastolic pressure of 8–10 mm hg at the end of stabilization period and unchanged for the remainder of the experiment. the electrical and hemodynamic functions of the heart were continuously monitored with a computer-based data acquisition system (powerlab system with chart 5 software, ad instruments, australia). left ventricular developed pressure (lvdp), rate pressure http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 217 product (rpp), max and min dp/dt and heart rate were calculated. coronary flow (cf) was measured by timed collections of the coronary effluent. experimental protocol hearts were allowed to equilibrate for 20 min with its buffer. after the stabilization period, different doses of h. lepturus venom (25, 50 and 100 µ g) dissolved in normal saline (100 µ l) were infused directly acquiring cardiotropic, inotropic and arrhythmogenic changes with langendorff apparatus. razi institute multivalent antivenom (10 µ l) dissolved in normal saline (100 µ l) was injected pre (20 minutes) and post venom (15 min) treatment. in another set of experiments incubated venom (25 µ g) with razi institute antivenom (10 µ l) was titrated to 100 µ l with normal saline for 30 min at room temperature to investigate its neutralizing effect against cardiac aspects of h. lepturus venom. in order to evaluate the neutralizing effects of β adrenoceptors and cholinoceptors drugs, propranolol (10 µ l) in addition to adrenaline (10 µ l) and finally atropine (100 µ l) were used pre and post venom injection. coronary blood flow, left ventricular developed pressure, dp⁄ dtmax and heart rate were recorded and outflow samples were collected every 1 min initially and every 10 min sequentially until the end of our experiments (60 min) to evaluate the coronary flow changes induced by the venom. statistical analysis results are expressed as mean ± sem of three experiments. anova plus post hoc test was carried out in cardiotropic experiments. values of p< 0.05 were considered significant. results inotropic and chronotropic effects of hemiscorpius lepturus venom cardiotropic effects of various doses (25, 50 and 100 µ g ) of h. lepturus venom reconstituted in normal saline (100 µ l) are shown in fig. 1–3 (n=3). representative image of negative chronotropic effects of different doses from h. lepturus venom with the same volume (100 µ l) on isolated rat heart in langendorrf apparatus upon time versus normal saline injection was depicted in fig 1. results present mean ± sem of 3 independent experiments. reducing effects on the contractile potency of the heart was induced shortly (two min later) following h. lepturus injection in langendorrf apparatus.venom caused a statistically significant decrease in heart contractility (negative inotropic) and rate (negative chronotropism) for 40–60 min before ending the examination at 60 minutes. coronary flow was significantly decreased (7.83 ± 0.21 ml to 2.81 ± 0.62 ml) post venom injection (25 µ g) in our experiments. the neutralizing capacity of razi institiute antivenom against cardiogenic effects of hemiscorpius lepturus razi institute antivenom (10 µ l) was used as pre (20 min) and post (15 min) venom (25 µg) injection and its neutralizing effect against the venom were depicted in fig. 4 a, b. razi institute antivenom pretreatment (20 min before venom injection) or incubation with venom for 30 min at room temperature significantly neutralized negative inotropic effects while post venom injection had no effect. in another study incubated venom and antivenom (25 µg in 10 µl) for 30 min at room temperature was titrated with normal saline (100 µ l) and injected to evaluate the neutralizing effects of this remedy on rat hearts (fig. 4c). there was no bradycardia and negative inotropic effects following h. lepturus http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 218 venom (25 µ g) injection pre incubated with the antivenom and the post treatment of antivenom failed to counteract the cardiotoxic effects of venom injection. arrhythmogenic effects of hemiscorpius lepturus venom venom perfusion prominently induced bradycardia, with widened qrs complexes and st depression after passing 50 min in all the envenomed hearts as shown in fig. 5a. normal heart rate and ecg parameter were induced by normal saline (100 µ l) infusion to serve as control (not shown). antivenom administration (10 µ l), 20 min before venom injection had neutralized qrs widening and bradycardia induced by h. lepturus venom (fig. 5b). injection of adrenaline and atropine in our study counteracted the late arrhythmogenic effects induced by h. lepturus venom (data not shown). protection and aggrevating experiments using adrenaline, atropine and propranolol pre and post injection of adrenaline (10 µ l) following using of h. lepturus venom are represented in fig. 6a, b. adrenaline (10 µ l) treatment 10 min before and 15 min after venom injection neutralized negative inotropic effects upon time. according to these diagrams negative inotropic effects were significantly neutralized by this drug since there was no decreasing of lvdp (left ventricular diastolic pressure) following venom injection upon time. in another experiment, pretreatment of propranolol (10 µ l) resulted in additive negative effects on lvdp in isolated rat hearts (fig. 7) and in our final examinations incubation of atropine (100 µl) as an anticholinergic drug with our venom (25 µg) prior to injection neutralized its inotropic effects showing the probability of its indirect mechanism (fig. 8). pretreatment of isolated rat hearts with propranolol, 20 min before venom injection had no neutralizing effect on negative inotropic aspects as represented in this figure. incubation of venom and atropine for 30 min had no negative inotropic effects in isolated rat hearts upon time. fig. 1. heart rate changes by different doses of hemiscorpius lepturus venom in rats in langendorff apparatus # significantly different from normal saline at p< 0.05 heart rate changes induced by different doses of h. lepturus venom b ea ts ( b ea ts /m in u te ) time (minutes) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 219 fig. 2. contractility changes induced by hemiscorpius lepturus venom in isolated rat hearts # significantly different from normal saline at p< 0.05 ##significantly different from normal saline at p< 0.01 fig. 3. inotropic effects induced by injection of hemiscorpius lepturus venom (25µ g) in rat hearts with langendorff apparatus a b time (min) l v d p (l ef t v en tr ic u la r d ev el op ed p re ss u re ) (m m h g) 4 min 4 min 4 min venom (25 µg) venom (25 µg)venom (25 µg) antivenom (10 µg)antivenom (10 µg) l v d p ( m m h g) l v d p ( m m h g) l v d p ( m m h g) inotropic changes induced by doses of hemiscorpius lepturus on isolated rat hearts (n= 3) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 220 fig. 4. inotropic changes following pre and post venom (25 µ g) injection of razi institute antivenom (10 µ l) a b fig. 5. arrhythmogenic affects of hemiscorpius lepturus venom injection (25 µ g) upon time (5a) versus normal saline (5b). bradycardia, qrs widening and st depression were induced with venom injection in langendorff apparatus 15 minutes before venom injection 10 minutes following venom injection 30 minutes following venom injection 50 minutes following venom injection 50 minutes following venom injection 30 minutes following venom injection 10 minutes following venom injection 15 minutes before venom injection 1 sec 1 sec 1 sec 1 sec 1 sec 1 sec 1 sec 1 sec time (minutes) v ol ta g e (m v ) cardiac effects of scorpion venom (25 µg) incubated with antivenom (10 µl) for 30 minutes v ol ta g e (m v ) v ol ta g e (m v ) v ol ta g e (m v ) v ol ta g e (m v ) v ol ta g e (m v ) v ol ta g e (m v ) v ol ta g e (m v ) c http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 221 a b fig. 6. changes in inotropic effects upon the pre and post treatment of epineprine (10 µ l) added before and after venom (25 µ g) injection fig. 7. lvdp changes following pretreatment with propranolol (10 µ l), added 20 min before venom (25 µ g) injection fig. 8. lvdp chnages by the venom (25 µ g) preincubated with atropine (100 µ l) for 30 min discussion in this study, in vitro negative inotropic, chronotropic and late arrhythmogenic effects of h. lepturus envenomation in rats were shown with langendorff apparatus. the most interesting findings in envenomed patients by h. lepturus are hemoglobinuria, hematuria, cutaneous necrotic ulcers and in minority of cases disseminated intravascular coagulation leading to death (radmanesh 1990, shayesteh et al. 2011) as seen in loxosceles envenomation (bay et al. 1997, tavares et al. 2004). envenomation with h. lepturus which is not painful is commonly seen in south west provinces of iran and treated with intramuscular injection of razi institute poly valent antivenom and followed by close monitoring. in previous in vivo studies, bradycardia (decreasing from 227 beats/min to 94 beats /min) and st elevation in lead ii was in4 min 4 min 4 min 4 min atropine (100 µl)+ venom (25 µg) venom (25 μg)propranolol (10 µl) venom (25 µg)epi (10 µl) venom (25 µg) epinephrin (10 µl) l v d p ( m m h g) l v d p ( m m h g) l v d p ( m m h g) l v d p ( m m h g) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 222 duced with intravenous injection of extreme doses (50 µ g/kg) of venom in rabbits with no significant changes of lactate dehydrogenase and creatinine phosphokinase one hour after envenomation showing no myocardial necrosis (mirakabadi et al. 2010). in this paper, a langendorff model was assembled to analyze the direct and indirect chronotropic and inotropic effects of h. lepturus venom on rat hearts. our venom induced a persistant negative dose response inotropic and chronotropic effects in rat hearts until up to 60 min, similar to loxosceles envenomation (shahbazzadeh et al. 2007, chatzaki et al. 2012). this study prudently approved that hemiscorpius lepturus cardiotoxicity was highly dependent on dose and time of its administration. coronary blood flow was significantly decreased (10.4 ± 2.21 to 3.16 ± 2.58 ) in envenomed hearts with no changes in ldh and cpk before and 60 min after envenomation (data not shown) ruling out the myocardial necrosis. according to the nature of poisonous animals, cardiotoxic effects of scorpion venoms could be explained with prevention or releasing of neurotransmitters including acetylcholine and epinephrine from nerve terminals inducing changes in heart rate and contractility (tarasuik et al. 1994, sauviat et al. 1995). our data suggest that h. lepturus venom could be suppressing, at least partially on the adrenoceptors mimicking agent presenting in venom or from varicosities in the heart like other poisonous creatutres (gomes et al. 2010) in addition to direct binding to its myocardial receptors that their shape and origin will be clarified in further studies. in our experiment, pre and post injection of epinephrine (10 µ l) counteracted negative inotropic and chronotropic effects induced with h. lepturus venom. it should be noted that coronary blood flow was not decreased by pre and post venom injection of adrenaline (data not shown) indicating h. lepturus indirect effect via supression of β receptors as one of the most autonomic determinants of cardiac contractility and coronary perfusion (gordon et al. 1998). in a parallel study, preinjection of propranolol (10 µ l ) as a sympathetic antagonist had an additive role on cardiotropic effects of h. lepturus venom supporting our hypothesis (fig. 7). based on our study pre venom injection of atropine or incubated mixture of venom with this drug, had significant neutralizing effect in contractility following envenomation clearly showing that negative inotropic effect of this venom possibly was also dependent on cholinergic system by direct stimulation or activation of muscarinic receptors in atria with releasing of acetylcholine from nerve terminals like tityus cerrulatus scorpion venom (teixeira et al. 2001). post venom injection of atropine had no neutralizing effect on cardiotropic aspects possibly due to binding of the venom to its ion channel receptors strongly. there is a great debate about the efficacy of scorpion antivenom in neutralization of the detrimental effects induced by envenomation (gueron et al. 1967, abroug et al. 1999, ismail 2003, chatzaki et al. 2012) since clinical efficacy of treatment for envenomed patients is affected deeply with the time between scorpion sting and administration of antivenom. generally h. lepturus venom like other poisonous animals have low molecular weight diffusing rapidly to target organs including kidney and heart while the heavy chain molecules of antivenom have not this ability making it unsuitable for treating envenomed patients (ismail et al. 1983, seyedian et al. 2010, seyedian et al. 2012). in our experiment, prevenom injection of antivenom in addition to incubation of venom and antivenom completely antagonized all deterimental effects including negative inotropic, chronotropy, decreasing coronary blood flow and even late arrhythmogenic effects (bradycardia, qrs prolongation and st depression after 60 min) in isolated rat http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 223 hearts while post venom injection had no effect. it seems that antivenom could bind firmly to venom and prevents its direct and indirect effects while injected before envenomation but due to its high molecular weight structure and different pharmacokinetic and biodistribution profile versus h. lepturus venom had no treating effects in envenomed hearts even when injected 5 min after envenomation (data not shown). scorpion venoms generally act mainly on na+ and to lesser degrees on calcium and potassium channels provoking electrocardiographic changes (gordon et al. 1998). arrhythmogenic changes in our envenomed hearts could be induced by late ischemia or prolongation of repolarization phase with some purified toxins from h. lepturus as hemicalcins and heminecrolysin (shahbazzadeh et al. 2007, borchani et al. 2011) acting on ion channels. those effects were neutralized by prevenom injection of razi institute multivalent antivenom. arrythmogenic changes with venom including qrs prolongation and st depression were not observed during treatments with other drugs (atropine, propranolol and adrenaline) and antivenom as control. conclusion it seems that cardiotropic changes including negative inotropism and chronotropism of h. lepturus venom in addition to late arrhythmogenic manifestations has a detrimental effect in heart failure induced by envenomation in human beings. we believe that indirect and direct mechanisms are involved in this phenomenon since injection of adrenaline and atropine according to their high and low presence in the rat heart changed our results. pre venom injection of razi institute antivenom as the customary treatment could neutralize its cardiotropic effects but unfortunately it had no effect even 5 min after envenomations making its beneficial role on detrimental cardiac consequences questionable. acknowledgements the authors would like to thank the authorities of bushehr university of medical sciences for providing facilities. references abroug f, elatrous s, nouria s, haguiga h, touzi n, bouchoucha s (1999) serotherapy in scorpion envenomation: a randomized controlled trial. lancet. 354: 906–909. bawaskar h, bawaskar p (1998) indian red scorpion envenoming. ind j pediatr. 65: 383–391. bey ta, walter fg, lober w, schmidt j, spark r, schlievert pm (1997) loxosceles arizonica bite associated with shock. ann emerg med. 30: 701–703. borchani l, sassi a, shahbazzadeh d, strub jm, tounsi-guetteti h, boubaker ms, akbari a, van dorsselaer a, el ayeb m (2011) heminecrolysin, the first hemolytic dermonecrotic toxin purified from scorpion venom. toxicon. 58: 130– 139. chatzaki m, horta c, almeida m, pereira n, mendes t, dias-lopes c, guimarães g, moro l, chávez-olórtegui c, horta m (2012) cutaneous loxoscelism caused by loxosceles similis venom and neutralization capacity of its specific antivenom. toxicon. 60: 21–30. cupo p, hering se (2002) cardiac troponin i release after severe scorpion envenoming by tityus serrulatus. toxicon. 40: 823–830. dehghani r, fathi b (2012) scorpion sting in iran: a review. toxicon. 60: 919– 933. gomes, hl, andrich f, mauad h, sampaio kn, de lima me, figueiredo sg, moyses mr (2010) cardiovascular effects of scorpionfish (scorpaena plumieri) venom. toxicon. 55: 580–589. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 224 gordon d, savarin p, gurevitz m, 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disseminated intravascular coagulation like syndrome after a sting of hemiscorpius lepturus: a case series. trop biomed. 28: 518–523. srairi-abid, n, shahbazzadeh d, chatti i, http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 215–225 k pourkhalili et al.: cardiotoxic and … 225 mlayah-bellalouna s, mejdoub h, borchani l, benkhalifa r, akbari a, el ayeb m (2008) hemitoxin, the first potassium channel toxin from the venom of the iranian scorpion hemiscorpius lepturus. febs j. 275: 4641– 4650. tarasiuk a, sofer s, huberfeld si, scharf sm (1994) hemodynamic effects following injection of venom from the scorpion leiurus quinquestriatus. j crit care. 9: 134–140. tavares f l, sousa-e-silva mcc, santoro m l, barbaro kc, rebecchi imm, sanomartins is (2004) changes in hematological, hemostatic and biochemical parameters induced experimentally in rabbits by loxosceles gaucho spider venom. hum exp toxicol. 23: 477–486. teixeira a, fontoura b, freire-maia l, machado c, camargos e, teixeira m (2001) evidence for a direct action of tityus serrulatus scorpion venom on the cardiac muscle. toxicon. 39: 703–709. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2018, 12(4): 414–420 x cai et al.: insecticidal and … 414 http://jad.tums.ac.ir published online: december 25, 2018 original article insecticidal and acetylcholinesterase inhibition activity of veratrum nigrum alkaloidal extract against the german cockroach (blattella germanica) xianghai cai 1, qingfeng li 2, lei xiao 3, hailiang lu 3, jian tang 3, jianbo huang 2, *jianzhong yuan 2 1state key laboratory of phytochemistry and plant resources in west china, kunming institute of botany, the chinese academy of sciences, china 2institute of plant physiology and ecology, shanghai institutes for biological sciences, the chinese academy of sciences. shanghai, china 3infinitus (china) company ltd, guangzhou, china (received 30 mar 2018; accepted 13 nov 2018) abstract background: veratrum nigrum (liliaceae) is perennial medicinal plant widely used to treat various conditions. to determine its insecticidal properties against the german cockroach (blattella germanica), several laboratory tests were carried out. methods: a 4kg dry sample of v. nigrum root was purchased from the medicinal material market in yunnan province in 2015, china. in contact toxicity tests, v. nigrum alkaloidal extract was topically applied to the abdomen of cockroaches using a micro-applicator. in vitro acetylcholinesterase (ache) activity tests were performed using a modified ellman method. results: veratrum nigrum alkaloidal extract was toxic to male adults and 4th nymphs cockroaches, with median lethal dose (ld50) values of 14.90µg/insect, 14.21µg/insect for adults and 41.45µg/insect, 39.01µg/insect for 4 th nymphs after 24h and 48h exposure, respectively. there was a significant difference between adults and nymphs in terms of tolerance to v. nigrum alkaloidal extract. there was no significant difference in mortalities at 24h and 48h, the lethal effect of v. nigrum alkaloidal extract on german cockroach was quick. ache activity tests showed that v. nigrum alkaloidal extract had an excellent inhibitory effect on ache: inhibition in the 4th nymphs and male adults had 50% inhibiting concentration (ic50) values of 3.56mg/ml and 5.78mg/ml respectively. the inhibitory effect of ache activity was positively correlated with inhibitory time (0–20min), at a concentration of 1mg/ml, inhibition of nymph and adult ache activity had 50% inhibiting time (it50) values of 8.34min and 16.75min, respectively. conclusion: v. nigrum may be explored as a potential natural insecticide for control of the german cockroach. keywords: insecticidal activity, acetylcholinesterase inhibition, veratrum nigrum, medicinal plant, german cockroach introduction the german cockroach, blattella germanica linnaeus is both a serious pest and a mechanical vector for bacteria and other pathogens (1–3). to date, the control of cockroaches in china mainly has relied on two types of insecticides administered as sprays or toxic baits based on synthetic insecticides. toxic baits may be more effective than spray formulations against german cockroaches (4). synthetic insecticides are an effective tool to manage pests but are associated with negative effects such as insecticide resistance, environmental pollution and human health problems. over 500 insect species had developed resistance (5). the resistance of the german cockroach to different insecticides is also very serious (6, 7). therefore, it is essential to develop a new type of environmentally-friendly insecticide. plant extracts are undoubtedly an ideal candidate due to their low mammalian toxicity, readily biodegradable and low risk to the environment (8). many medicinal plant extracts have been reported to show insecticidal properties against public health pests, such as mosquitoes (9, 10), *corresponding author: dr jianzhong yuan, e-mail: yuangang188@126.com mailto:yuangang188@126.com j arthropod-borne dis, december 2018, 12(4): 414–420 x cai et al.: insecticidal and … 415 http://jad.tums.ac.ir published online: december 25, 2018 houseflies (11, 12) and german cockroaches (b. germanica) (13, 14). one such example is veratrum nigrum linnaeus which belongs to the liliaceae family. it is a perennial traditional chinese medical plant and is very prevalent in the wild in china. it has been extensively used for the treatment of hypertension, stroke, excessive phlegm and epilepsy (15, 16). in recent years, it had other properties, such as antifungal (15) and insecticidal activity (17, 18). however, there are few studies on the insecticidal activity of v. nigrum against public health pests such as the b. germanica. therefore, we investigated the insecticidal activity of v. nigrum against b. germanica. materials and methods preparation of v. nigrum alkaloidal extract a 4kg dry sample of v. nigrum root was purchased from the medicinal material market in yunnan province in 2015, china. the sample was crushed and extracted three times with 15l methanol at room temperature. the extraction liquid was made up to 1000ml with water after removal in vacuo. the extract was partitioned between 1% hydrochloric acid solution and ethyl acetate. components of the acidic layer, adjusted to ph 9–10 with 10% ammonia solution, were extracted with ethyl acetate to give an alkaloidal extract (69g). compounds acetylthiocholine iodide, 5, 5’-dithiobis (2nitrobenzoic acid) (dtnb), triton x-100, and eserine were purchased from sigma chemical company. positive control, tetramethrin (95%) was purchased from shanghai forever chemical co, ltd. insect rearing blattella germanica strain maintained at 26±1 °c and 60% relative humidity under 16:8h (l:d) cycle in the laboratory for 40yr without exposure to any insecticides. contact toxicity test a group of 20 male adult or 4th nymph cockroaches were anesthetized using ether. a droplet (1µl) containing five different concentrations of v. nigrum alkaloidal extract (0.375–12%) or tetramethrin (0.375–6%) was topically applied to the abdomen of cockroaches using a microapplicator (burkard, uk). a 1µl droplet of acetone was applied to the control group. after treatment, cockroaches were transferred to a 200ml glass bottle maintained at a temperature of 261 °c and a humidity of 7510% rh. mortality was assessed 24h and 48h after treatment. all experiments were repeated in triplicate. in vitro acetylcholinesterase (ache) activity test in vitro acetylcholinesterase (ache) activity tests were performed as follows: 10 male adults and 4th nymph cockroaches were homogenized using a glass tissue grinder in ice in 20mm phosphate buffer (ph 7.0) containing 0.5% triton x-100. the homogenate was then centrifuged at 12000gr for 15min at 4 °c. the supernatant was used as an enzyme source for measuring ache activity via a modified ellman method (19). to determine the 50% inhibiting concentration (ic50) of the alkaloidal extract, 50µl of the enzyme was added to a test tube containing 50µl of six different concentrations (0.375–12 mg/ml) of the alkaloidal extract and pre-incubated at 25 °c for 5min. to determine the 50% inhibiting time (it50), the above procedure was followed and pre-incubated at 25 °c for six different time periods (0.5min, 1min, 5min, 10min, 15min, 20min). after incubation, 1300µl of 20 mm ellman solution containing 1mm acetylthiocholine iodide, 0.23mm 5', 5'-dithiobis (2-nitrobenzoic acid) and 0.45mm nahco3 were added to the test tube which was then incubated at 25 °c for 20min. the reaction was stopped by 100µl 10mm eserine and the absorbance at 412nm was measured. each assay was replicated five times. j arthropod-borne dis, december 2018, 12(4): 414–420 x cai et al.: insecticidal and … 416 http://jad.tums.ac.ir published online: december 25, 2018 statistical analysis mortality corrected using abbott's formula. all analyses were performed using spss 13.0 software (chicago, il, usa). ld50, ic50 and it50 were considered to be significantly different on the basis of non -overlapping of 95% confidence intervals (cls). results contact toxicity test contact toxicities of v. nigrum alkaloidal extract against male adults and 4th nymphs cockroaches are shown in table 1. veratrum nigrum alkaloidal extract has significant insecticidal activity against the b. germanica. there was no significant difference in mortality at 24h and 48h (overlapping of 95% cl) at ld50 values of 14.90µg/insect, 14.21µg/insect for adults and 41.45µg/insect, 39.01µg/insect for 4th nymphs after 24h and 48h exposure, respectively. the lethal effect of v. nigrum alkaloidal extract on b. germanica is quick. in vitro acetylcholinesterase (ache) activity test the results of in vitro acetylcholinesterase (ache) activity tests are shown in table 2 and fig. 1. veratrum nigrum alkaloidal extract was found to have an excellent inhibitory effect on ache, showing ic50 values were 3.56mg/ml for nymphs and 5.78mg/ml for adults, respectively (table 2). inhibition of ache activity was positively correlated with inhibition time. when the concentration of v. nigrum alkaloidal extract was 1mg/ml, inhibition of ache activity in both nymphs and adults was at it50 values of 8.34 min and 16.75min, respectively (fig. 1). table 1. toxicity of veratrum nigrum alkaloidal extract against male adults and 4th nymphs of blattella germanica after exposure for 24 and 48h compounds (µg/insect) age exposure time ld50sd 95%fiducial limits slopesd chi square (2) alkaloidal extract adult 24 14.901.35 13.40–16.68 3.330.42 1.99 4th nymph 24 41.454.45 36.02–47.68 2.270.33 3.73 tetramethrin adult 24 7.890.88 6.89–9.93 3.390.40 2.89 4th nymph 24 13.981.26 10.82–18.22 2.790.38 2.43 alkaloidal extract adult 48 14.211.40 13.12–15.78 3.230.38 2.25 4th nymph 48 39.014.03 35.55–46.89 2.650.37 3.68 tetramethrin adult 48 7.720.78 6.52–8.90 3.210.37 2.76 4th nymph 48 13.301.28 10.32–18.00 2.810.33 2.41 fig. 1. residual ache activity in male adults and 4th nymphs of blattella germanica at 1 mg/ml veratrum nigrum extract j arthropod-borne dis, december 2018, 12(4): 414–420 x cai et al.: insecticidal and … 417 http://jad.tums.ac.ir published online: december 25, 2018 table 2. ic50 values of veratrum nigrum alkaloidal extract against male adults and 4th nymphs of blattella germanica ache activity age ic50 (mg/ml) (sd) 95% confidence limit slope (sd) chi square (2) adult 5.780.47 4.78–6.98 1.240.13 1.54 4th nymph 3.560.49 2.36–4.72 1.350.11 1.35 discussion the chinese pharmacopoeia includes traditional chinese medicine plants, shown to have anti-oxidant, free radical scavenging, anti-inflammatory and anticancer properties (20). in previous studies, v. nigrum extracts had insecticidal effects against mythimna separata, aphis craccivora, cx. pipiens pallens, tetranychus cinnabarinus, and plutella xylostella (17, 18). certain chinese medicinal plant extracts have been reported to be bioactive against b. germanica, such as pogostemon cablin at a ld50 of 23.45 µg/insect (14), and chenopodium ambrosioides at a ld50 of 64.47µg/insect (13). our results showed that v. nigrum alkaloidal extract has significant insecticidal activity against b. germanica. compared with the control (a chemical insecticide: tetrarmethrin, ld50 of 7.89 µg/insect, 7.72µg/insect for adults and 13.98µg/ insect, 13.30µg/insect for 4th nymphs after 24h and 48h exposure, respectively), v. nigrum alkaloidal extract showed a little weaker or equivalent toxic to the b. germanica. contact toxicity of v. nigrum alkaloidal extract against male adults was stronger than 4th nymphs b. germanica. moreover, the result was similar to insecticidal activities of tetramethrin against above both. the tolerance level of the nymphs was significantly higher than that of adults (non-overlapping of 95% cl), as also reported by (21-23). the weight difference between males and females may be one reason for the different susceptibility to insecticide (24, 25). in our research, the mean nymph weight (30.18 mg) was lighter than the adult male (54.33mg) but did not appear to be a factor for the increase intolerance. therefore, there may be other modes of action responsible for this difference, but the mecha nisms of plant extract toxicity against b. germanica are poorly reported. the insecticidal target site of the phytochemicals against cockroaches has been previously researched, such as the insect gaba receptor was target site for carvacrol, pulegone, and thymol (26). octopamine receptor was target site for carvacrol, -terpineol and pulegone (27, 28). in addition, the ache inhibition activity of phytochemicals on b. germanica has been previously studied in vitro (24, 29, 30). in the present study, v. nigrum had an excellent inhibitory effect on ache, showing ic50 and it50 values were 3.56mg/ml，8.34min for nymphs and 5.78mg/ml, 16.75min for adults, respectively. ache in 4th nymph b. germanica was more susceptible to insecticide than in male adult cockroaches, similar to results found by (24, 29). the mechanisms of insecticide resistance in b. germanica have many factors, including target site insensitivity, metabolic detoxification and decreased skin permeability (31, 32). therefore it is difficult to explain the higher tolerance of nymphs against v. nigrum alkaloidal extract through ache inhibition alone. in addition, plant extracts have many active ingredients and biological activity is, therefore, the result of a combination of various components (13, 33). it is necessary to determine the active components of v. nigrum which are currently poorly understood for insecticidal efficacy. in recent years, 35 active ingredients were identified in v. nigrum alkaloidal extract and the main components were veratramine, followed by veratrosine and jervine (34). this would provide a strong foundation for screening the insecticidal constituents of v. nigrum. further work focuses j arthropod-borne dis, december 2018, 12(4): 414–420 x cai et al.: insecticidal and … 418 http://jad.tums.ac.ir published online: december 25, 2018 on insecticidal activities of v. nigrum ingredients against b. germanica. conclusion insecticidal activities of phytochemicals derived from chinese medicinal plants have been previously investigated against b. germanica. v. nigrum (the family of liliaceae) alkaloidal extract have excellent insecticidal activity compared to tetramethrin in vivo and inhibitory effect on ache in vitro. it showed potential for further development as possible natural insecticide against b. germanica. however, further research is also necessary, such as the activities of ingredients and the modes of action of the individual ingredient. acknowledgments we thank prof lm tao (school of pharmacy, east china university of science and technology) for his editorial assistance. this 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mil med univ. 33(5): 536–539 (in chinese). j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 13 http://jad.tums.ac.ir published online: march 31, 2022 original article morphometric indices and venom protein profile in different populations of androctonus crassicauda *mohammad bagher ghavami1, zohreh alibabaei1, fatemeh ghavami2 1department of medical entomology and vector control, school of medicine, zanjan university of medical sciences, zanjan, iran 2department of electronics, faculty of electrical and computer engineering, tarbiat modares university, tehran, iran *corresponding author: dr mohammad bagher ghavami, email: ghavami@zums.ac.ir (received 28 jan 2019; accepted 27 oct 2021) abstract background: androctonus crassicauda is the most medically relevant animal and understanding its morphological characteristics is essential in the production of antiscorpion sera. methods: adults of a. crassicauda were collected from different areas of zanjan province and the morphometric parameters and the cuticular fluorescence patterns of samples were studied. the crude venom of samples was extracted by electric stimulation, and their biochemical properties were analyzed by the sds-page method. results: values of the morphometric parameters depended on sex and altitude of the area. except for values of the pectinal organ, these parameters in females were higher than in males. no significant difference was in the number, shape, and intensity of cuticular fluorescence patterns. the body length of males in high and lowlands was 72.53±1.53 and 77.33±2.70mm, respectively. females' body lengths in that area were 81.66±2.19 and 86.55±2.33mm, respectively. analysis of toxin proteins showed two isotypes that the 12, 13, 15, 16, 18, and 19kda proteins were in all areas. however, the 41 and 74kda proteins, and 46 and 63kda proteins were detected in low and highlands, respectively. conclusion: black fat-tailed scorpion has a considerable dominancy and developing preventive programs and providing treatment facilities in studied areas are necessary. values of the morphological parameters and venom electrophoresis patterns depended on the geographical location. therefore, pool crude toxin is suggested for the production of effective antivenoms. moreover, additional field complementary works in the geographic information system based niche modeling and mass fingerprinting of scorpion venoms are suggested for screening effective isotypes. keywords: androctonus crassicauda; black fat-tailed scorpion; morphometric parameters; scorpion venom sds-page; scorpion venom extraction introduction scorpions are potentially fatal venomous animals, and their envenomation is a major public health problem in the world. buthidae is the largest family of scorpions with worldwide distribution and contains the most dangerous species (1). the black fat-tailed scorpion, androctonus, is a large and ancient group of buthidae scorpions that has successfully adapted to various ecological conditions and occurs in different habitats. the overall body plan of this group has changed slightly and its systematics at the species level remains poorly defined. at present, eight species and 17 subspecies are reported within the androctonus genus and among them, a. crassicauda is the most dangerous, causing many human deaths (2-5). populations of this scorpion have a widespread distribution in the middle east and africa. the wide distribution of this species mostly in subtropical and temperate regions, between 23o–38o latitudes, indicates it's adapting to various ecological conditions and has different types (6). despite intensive research performed on external morphology in differ copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:ghavami@zums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 14 http://jad.tums.ac.ir published online: march 31, 2022 ent species of scorpions, few investigations have been conducted on a. crassicauda, and the characterization of some parameters of this species such as reproductive apparatus, is still unknown (7, 8). in the past decades, scorpion biology has been revolutionized using ultraviolet (uv) lamps (black lights) for their detection in the field and morphological surveys (9, 10). these animals are strongly fluorescent when illuminated with long uv light. molecules associated with the cuticular fluorescence of scorpions have been identified as the beta-carbon and 4-methyl, 7-hydroxy coumarin. the fluorescence exhibited by the tegument and the intensity of the emitted light is depended on the amount of these compounds in the cuticle. however, the biological function of fluorescence has not been definitively demonstrated (6, 11, 12). no difference in fluorescence spectra between genders of species is detected. however, statistical significance between the two sympatric species and subspecies of some scorpions was observed previously (13). black scorpions have a protected body form with black coloration and current morphological methods, using white light, cannot be accurate enough to identify the minor morphological differences in these scorpions. therefore, studying fluorescence patterns could provide complementary information to the morphological study of a. crassicauda. scorpion venom is the other most important item in the study of scorpions. it is a secretion compound of water, salt, and low molecular weight peptides with 13–76 amino acid residues. it resembles typical short and long-chain toxins specific for ion channels and receptor target cells. biochemical analyses of scorpion toxins show about 44 different eluting fragments, from which 30 fragments are completely separated. these fractions are unique defense, feeding weapons, and effective semiochemicals that modulate their behavior. scorpions apply these compounds in several sophisticated ways, for subduing prey, and deterring predators, probably during mating and frequently for deferring against humans (6). molecular and biochemical characterization of a. crassicauda venom in anatolia, southeast of turkey, represented at least 44 different fractions with toxicity to mice and insects. the analysis of toxin in this scorpion identified 80 distinct molecular mass compounds varying from 267–44551 da peptides that could modify k and na channels. to date, only eight peptides have been identified from a. crassicauda venom in that region, three of which have been fully sequenced (acra1, acra3, acra4), and one (acra2) is partially sequenced, and the other four are putative (acra5-8) (14, 15, 16). however, due to the increment of new strategies of proteome analysis and gene cloning from transcriptomes, the venom variation in different areas, and the number of identified components may increase significantly (17). the black fat-tailed scorpion is the most significant scorpion species in iran and the middle east countries. it is the main scorpion species in zanjan province, northwest of iran, causing many cases of scorpionism in this area (18, 19). due to the multifunctional role of venom in scorpions, its analysis could exhibit the variation of population groups in a. crassicauda. moreover, detection of venom components in a. crassicauda is extremely important to produce effective antivenom and understanding of clinical symptoms of patients. although a. crassicauda causes many human fatalities, the knowledge of this species is restricted and limited to past decades. in addition, despite the long history of venom research in the world, the venom of a few iranian scorpions has investigated. therefore, it is important to study the polypeptide electrophoretic patterns, and morphometric indices of a. crassicauda in distributed areas, as these characteristics, may have been applied in therapeutic management. therefore, the aims of this study were to characterize the morphometric indices, the fluorescence pattern of exhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 15 http://jad.tums.ac.ir published online: march 31, 2022 ternal anatomy, and the polypeptide electrophoresis pattern of venom in different populations of a. crassicauda in zanjan province. materials and methods study area zanjan province is located in northwest iran, from 35°35' to 37°15'n and 47°15' to 49°25'e (fig. 1). the altitude in this region varies from 270m to 3400m above sea level (asl). the average maximum temperature in this area is around 27 °c, whereas the average minimum temperature stands at -19 °c. meanwhile, the temperature rises to 32 °c on hot days; it drops to -27 °c on icy days. the average annual rainfall in the first month of spring stands at 72 millimeters, while in the second month of summer, it slips to a meager 3.6mm. the rate of humidity in the morning stands on average at 74% and at noon at 43% (https://en.climatedata.org/asia/iran/zanjan/zanjan-764536/). sample collection adults of a. crassicauda specimens were collected in different periods between jun and september 2015–2016 from twenty-three localities of zanjan province (table 1, fig. 1). the collection sites are grouped in two lowlands (under 700m asl) and highlands (above 1200m asl). the geographical characteristics of sampling stations were determined by the global positioning system (gps). scorpions were caught at night by using ultraviolet light, a portable flashlight equipped with 3w 375– 380nm ultraviolet led, detection method, and a few were captured in the daytime by rock rolling in the field. avoiding scorpion cannibalism, captive specimens were housed in individual plastic boxes. they were kept alive in laboratory conditions (40% relative humidity, 12:12 l: d, and 24±2 °c) and fed with ground meat and living crickets and received daily water ad labitum. food was given a weak after venom extraction in order to allow time for the animals to recover from stress. dead specimens were transferred into 75–96% ethyl alcohol and kept at -20 °c for further studies. they were deposited in the scorpion collection at the department of medical entomology, zanjan university of medical sciences, iran. morphological survey in the laboratory, sexing and taxonomic study of the samples were conducted with the help of stereomicroscope (olympus, szx9) and the keys suggested by farzanpey (20). examination of cuticular sculpture and morphology were facilitated using white light and ultraviolet fluorescence photomicrography. preserved specimens were attached to a dark, non-fluorescent plastic surface. images were acquired when the light was excited by the 3w indium gallium nitride (ingan) light emitting diodes (leds) emit light within a narrow band in the near uv wavelength range (395–410nm) and 455–470nm in white light posited at the distance of 0.25m from the specimen. imagines were produced using a digital camera (nikon ds camera ds-fi1) fitted on a nikon smz 1500 stereomicroscope. biometric measurements were taken with a >0.001mm accuracy by using digimizer software. (https://www. digimizer.com). measurements of 27 morphometric characters of adult specimens were adapted from mirshamsi et al. (21) for subsequent statistical analysis. morphometric variables abbreviations of morphometric characters include bl: body length, ca_l: carapace length, ca_aw: anterior width of carapace; ca_pw: posterior width of carapace, x: distance between anterior margin of the carapace and anterior margin of median eyes; y: distance between anterior margin of median eyes and posterior margin of carapace; mt_l (i–v): length of metasomal segments i–v; mt_w (i–v): width of metasomal segments i–v; mt_h (i– http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 16 http://jad.tums.ac.ir published online: march 31, 2022 v): height of metasomal segments i–v; tl_l: telson length; tl_w: telson width; tl_h: telson height; ch_l: chela length; m_l: manus length; mf_l: movable finger length; pl-l: pectinal left lamella length, and pr-l: pectinal right lamella length. venom extraction and preparation the crude venom was obtained by electrical stimulation of the telson of scorpion using an electro-pulse stimulator. in summary, one person held the pre-abdomen and telson of a scorpion individual with tweezers to keep it stationary and another person collected the venom from the aculeus of the scorpion. for electrical stimulation of the membrane anterior to the telson, we used the transcutaneous electrical nerve stimulation tensmed 400 (arman poya co, iran) connected with modified tweezers to apply power. to improve stimulation and enhance electrical contact, the tweezers were lubricated with glycerin saline gel. the intensity of the stimulation current was adjusted in pulse width from 200–250µs, a pulse rate of 60–80hz, and amplitude of 12– 16v. the extracted venom was collected with a microcapillary glass tube (inner diameter 1.5mm) that was attached to a flexible silicon tube and disposable insulin syringe and transferred into a 1.5ml centrifuge tube. crude venom was suspended in deionized water, centrifuged (12,000×g, 4 °c, 10min), and the supernatant that contained soluble venom proteins were transferred to a clean 1.5ml tube. finally, the taken supernatants were immediately lyophilized and stored at -20 ºc until use according to their geographical origins. after milking venom (venom collection), the animals were housed in individual boxes for another milking process. measurement of protein concentration protein concentrations was determined by absorbance measurements at 280nm and expressed as mg protein/ml. for each venom type, we prepared a solution of venom with a final concentration of 5mg/ml. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of venoms electrophoretic analysis of venoms was performed on 18% polyacrylamide gel in the presence of sds. all samples were dissolved in a sample loading buffer (50mm tris–hcl, ph 6.8, 1% β-mercaptoethanol, 6% glycerol, 2% sds, and 0.01% bromophenol blue). for the separation of proteins, samples were run on running buffer (25mm tris, 192mm glycine, 0.1% sds, ph 8.3). the molecular mass standard (sinaclon, pr911654) was run parallel to calculate the molecular weights of proteins. a constant electric current of 80ma was applied for 4–5 hours for the migration of proteins. after migration, the gel was stained with 0.1% coomassie blue r-250. the gel was then scanned, and the molecular weights of the proteins were calculated. results a total of 98 samples of adult scorpions comprising a. crassicauda, mesobuthus eupeus odontobuthus doriae, and scorpio maurus townsendi were collected from the studied areas. the black fat-tailed scorpion was the most frequent species and included about 70 % (68 scorpions) of the samples. the frequency of the rest scorpions, m. eupeous, od. doriae, and scorpio maurus, was 19%, 4%, and 3%, respectively. samples of a. crassicauda were collected mainly in the summer months (table 1). morphological survey the descriptive statistics (minimum and maximum values) of morphometric characters for each sex of a. crassicauda in study groups are presented in table 2. the schematic view of representative males and females in low and highlands are illustrated in fig. 2. analysis of morphometric parameters showed a statistical difference between sexes for the length http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 17 http://jad.tums.ac.ir published online: march 31, 2022 of pedipalps, carapace, telson, and pectinal organs. a univariate comparison of morphological parameters showed that these parameters also vary with the local altitude. female scorpions were presented as larger than males. the values of some characters were generally large in females except for the pectinal features. small spectrum variation for long, wide, and high metasomal 1-v characters is considered to have minimal scalar overlapping with their homologous character. the body length values of male scorpions in high and lowlands were 72.53±1.53 and 77.33±2.70mm, respectively. this parameter also varied in females in different areas and average body length reached 81.66±2.19 and 86.55±2.33mm in low and highlands, respectively. a comparison of these values showed that the body length of female scorpions in highlands overlapped with the size of males in lowlands. the values of the pectinal organ as other morphometric parameters depended on scorpion sex and local altitude. male scorpions showed pectinal lengths of 9.25±0.25 and 9.43±0.32mm in high and lowlands, respectively. despite the body length, the length of the pectinal organs in female scorpions did not vary and its value was 8.04±0.18mm in both study areas. a survey of the genital operculum of scorpions showed that it is more oval in males and slightly triangular in females. in addition, the genital operculum is fused in females and split among males. carination the emitted florescent light from different parts of scorpions and cuticular florescent patterns are illustrated in fig. 3. no significant difference was seen in the number, shape, and intensity of emitted light from carina in the carapace, prosoma metasomal segments, telson, and pedipalps of specimens. scorpion venom all scorpions were observed as being very aggressive during all keeping milking time. a total of 75µl crude venom was extracted from each scorpion sample. a colorless watery secretion was obtained during capturing followed by more viscous milky droplets or ejaculate (mucous accompanied with the venom) during stimulation and did not turn blue after milking. after centrifugation of the whole venom, the supernatant was of a more viscous form. sds-page analysis of the venom the protein profiles of a. crassicauda venoms were analyzed by sds-page followed by coomassie blue staining. in these analyses, 2 different isotypes, each with a different molecular mass, were detected. the number of protein bands for the investigated scorpions in two groups was 9. out of all protein bands, the bands of 12, 13, 15, 16, 18, 19 and 57kda consistently appeared in all venom samples (fig. 3). in addition to the above share proteins, two protein bands of 47kda in lowlands and 63kda in highlands were noticed unique in each group, and two bands of 41kda in lowlands and 46kda in highlands were not clearly different between two groups (fig. 4). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 18 http://jad.tums.ac.ir published online: march 31, 2022 table 1. frequency of collected androctonus crassicauda from different areas of zanjan province, northwest of iran studied areas coordinates latitude and longitude altitude (meters asl) time of collection frequency district locality zanjan subtotal zanjan 36.698138 n 48.514163 e 1600–2000 sept, 2016 4 4 4 3 4 3 3 24 gavazang 36.719335 n 48.521419 e aug, 2016 valarood 36.716381 n 48.372671 e aug, 2016 sarimsaghlu 36.759271 n 48.371128 e sept, 2016 do asb 36.706016 n 48.566930 e sept, 2015 sayan 36.647160 n 48.541718 e sept, 2015 khodabandeh subtotal khodabandeh 35.940912 n 48.144053 e 1600–1800 july, 2016 1 1 1 1 2 2 8 tatardeh 35.956463 n 48.102211 e sept, 2015 khalife 35.994119 n 47.993913 e july, 2016 sohrevard 36.074354 n 48.432798 e july, 2016 qeshlaq vakil 36.144121 n 48.046618 e july, 2016 garmaab 35.850285 n 48.198390 e july, 2016 mahneshan subtotal mahneshan 36.768985 n 47.668715 e 1200–1500 jun, 2016 5 4 3 2 14 sari aghol 36.823192 n 47.629671 e jun, 2016 sahand e sofla 36.773948 n 47.541359 e jun, 2016 sahand e olia 36.777957 n 47.519815 e sept, 2015 tarom subtotal chavarzagh 36.994055 n 48.777488 e 400–700 july, 2016 2 5 4 3 2 4 2 22 daraam 37.024636 n 48.778250 e aug, 2016 haronabad 36.834549 n 49.025299 e aug, 2016 abbar 36.921868 n 48.960540 e aug, 2016 dastjerdeh 36.849681 n 48.943995 e sept, 2016 sansooz 36.835585 n 48.943389 e sept, 2016 tashvir 36.789542 n 49.002457 e sept, 2016 total 68 fig. 1. map of study areas in zanjan province. locality of collection sites ( ), district center ( ) lowlands ( ) and highlands ( ) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 19 http://jad.tums.ac.ir published online: march 31, 2022 table 2. minimum (min) and maximum (max) values of the morphometric indices (with 95% confidence interval) in specimens of androctonus crassicauda collected from low and highlands of zanjan province parameter lowlands (400-700m asl) highlands (1200-2000m asl) ♂(n=27) ♀(n=19) ♂(n=12) ♀(n=10) min max min max min max min max bl 72.03 82.63 81.98 91.12 69.53 75.53 77.36 85.96 cal 8.50 9.32 9.29 10.93 8.38 9.01 9.29 10.49 ca-aw 4.23 5.08 4.87 5.62 4.44 4.93 5.04 5.55 ca-pw 8.60 9.69 9.99 11.96 8.55 9.13 9.98 11.21 mt(i)-l 5.51 6.22 5.85 7.06 5.56 6.24 5.70 6.47 mt(i)-w 3.58 4.27 3.78 4.80 3.70 4.37 4.05 4.41 mt(i)-h 4.20 5.10 4.11 5.27 4.16 4.74 4.40 4.94 mt(ii)-l 6.47 7.14 6.86 7.79 6.34 6.97 6.75 7.45 mt(ii)-w 4.17 5.17 4.48 5.67 4.47 5.12 4.69 5.21 mt(ii)-h 4.31 5.13 4.29 5.66 4.28 4.86 4.42 4.99 mt(iii)-l 6.66 7.48 7.14 8.01 6.49 7.06 6.94 7.62 mt(iii)-w 5.11 6.13 5.14 6.29 5.13 5.68 5.33 6.07 mt(iii)-h 4.59 5.34 4.73 5.94 4.56 5.20 4.90 5.50 l mt(iv) 7.52 8.59 7.88 9.22 7.62 8.17 7.89 8.60 mt(iv)-w 5.66 6.67 5.46 6.85 5.54 6.16 5.64 6.43 mt(iv)-h 4.77 5.42 4.73 5.74 4.56 5.19 5.14 5.48 mt(v)-l 7.33 8.48 8.37 9.64 7.58 8.21 7.65 8.68 mt(v)-w 5.26 6.20 5.19 6.57 5.28 5.85 5.42 6.13 mt(v)-h 2.97 3.46 3.05 3.69 2.92 3.40 3.29 3.72 tl-l 8.01 9.32 9.23 9.81 7.71 8.72 8.83 9.98 tl-w 2.94 3.51 3.38 4.22 3.02 3.39 3.63 3.91 tl-h 2.82 3.33 3.04 3.64 2.64 3.06 3.19 3.58 x 3.44 3.82 3.86 4.49 3.47 3.73 3.94 4.41 y 5.12 5.73 5.47 6.67 5.01 5.44 5.49 6.18 chl 14.69 15.64 16.41 18.60 14.64 15.93 15.83 17.79 m-l 6.36 7.01 6.91 8.16 6.49 7.24 6.81 7.71 mf-l 10.43 11.37 11.99 13.02 10.19 11.13 11.56 12.59 pl-l 8.64 9.89 7.11 8.83 8.94 9.92 7.35 8.36 prl 8.52 9.77 7.25 8.90 8.94 9.88 7.69 8.39 fig. 2. schematic view of the representative male and female of androctonus crassicauda in study areas. male (a, c) and female (b, d) samples in lowland (a, b) and (c, d) highland http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 20 http://jad.tums.ac.ir published online: march 31, 2022 fig. 3. schematic presentation of external body parts in androctonus crassicauda under uv fluorescence. carapace (a); tergite (b); ventral aspects of prosoma in male (c, e, g) and female (d, f, h); legs and pedipalp (i, j, k, l); metasomal segments in ventral (m, n), dorsal (o, p), and lateral (q, r) aspects; metasomal segment v and telson in lateral (s) and ventral (t) aspects http://jad.tums.ac.ir/ https://www.researchgate.net/figure/schematic-representation-of-external-and-internal-body-parts-of-octopus_fig14_305329627 j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 21 http://jad.tums.ac.ir published online: march 31, 2022 a b fig. 4. electrophoretic profile of androctonus crassicauda venoms on poly acryl amide gel in the presence of sds. part a: venom obtained from captured scorpions in highlands and part b: venom obtained from collected scorpions in lowlands. lanes 6 in highlands and 7 in lowland are molecular weight (kda) markers, and the others are venom samples discussion the results of the present study showed that a. crassicauda is a dominant scorpion in studied areas and occurs in different conditions. the appearance of this species in study areas reflects the tuning of optimal ecological conditions necessary for its survival in these areas. the presence of this scorpion in these areas calls for the authorities to take the necessary measures to prevent and cure envenomation. the findings of this study showed that the body size of the specimens is depended on gender and local conditions (altitude). local dependence on morphometric indices causes both sexes to have a wide range of body lengths. although body size can distinguish the sex of the same population, geographical distance, and the overlap of the sizes of both sexes can challenge gender discrimination. therefore, it is suggested that the values of the morphological parameters of each region must be used to analyze the samples of that region. our data allowed us to evaluate the size variation of a. crassicauda with respect to local conditions. season length is an important ecological factor that may influence the variation of deduced parameters. in high altitudes the average ambient temperature is lower, and the duration of the activity period is shorter than the lowlands. because the black fat-tailed scorpion is a slowly developing animal, therefore, its encounter in short activity season reduces the growth period, resulting in reduced body size in highlands. moreover, the patterns in body size variation we found in a. crassicauda might be adaptive under the given ecological conditions and food availability in these two lands. in the current study sexual dimorphism among scorpions was performed by morphometric parameters; length of body, carapace, pedipalp, and structure of the pectinal organ. these findings are similar to the results of studies conducted in turkey and fars province, southern iran (7, 8). many studies have been carried out on the use of uv light to the finding of scorpions (9, 10), but the present study is the first study, in collecting of scorpions and determining the pattern of carina in different parts of the scorpion. therefore, the protocol of this study is recommended for the study of scorpions, especially those that have dark tegument. the findings of this study revealed that a. crassicauda in the study areas represented a http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 22 http://jad.tums.ac.ir published online: march 31, 2022 homogenous species and all the samples had one fluorescent pattern. this process may be related to the presence of selective pressure or specific ecological behavior (sex finding and mating) of specimens. further studies could solve this problem in different situations. in the current study, the electrophoretic protein patterns of the black fat-tailed scorpion venom were presented in the range from 12 to 74kda. among these proteins, six bands 12, 13, 15, 16, 18 and 19kda appeared in all samples. analysis of electrophoretic patterns also indicated that this scorpion possesses other proteins whose molecular weight was more than 40kda and varied in different strains of a scorpion. previews studies indicated that venom proteins of a. crassicauda, collected from sanliurfa and mardin provinces (southeastern anatolia) of turkey, were in six bands with 12, 15, 29, 35, 53, and 58kda. they showed that 35, 53, and 58kda proteins played an important immunogenic role in the production of antivenom against this scorpion (22, 23). scorpion venoms in the present study were documented into two groups according to their molecular sizes. these groups include the short and long chain neurotoxins with molecular size below 20kda and the target aging peptides with up 40kda weight. among these groups more studies are conducted about neurotoxins, however, there is scant information on the other study groups, and the studies have been limited to their toxicity and immunogenic. the low molecular weight biogenic amines (histamine, dopamine, nor-adrenalin, etc.) found in samples are involved in local reactions and their release from a single sting can lead to systemic reactions. they can act on blood vessels and nerve endings inducing swelling, redness, pain, and itching. the major toxic effects of venom are attributed to the presence of large peptides (4, 6). these peptides can cause damage to the cell membrane, leading to the release of enzymes from lysozymes and mast cell granules, resulting in cytolysis. additionally, they can act as neurotoxic provoking hyper-excitability. thus, the individual variability in venoms is extremely important for evaluating the venom yield and the resulting toxicity after a scorpion sting. further studies are needed to evaluate the toxicity of venoms and the median effective dose (ed50) of scorpion anti-venom against envenomation (24) in various populations of this scorpion in the future. analysis of electrophoresis peptide patterns in the present study indicated that a. crassicauda venom possesses different peptides both with neurotoxins and target aging according to the electrophoretic protein patterns. polymorphisms of protein contents also have been observed in the venom of individual a. australis hector and a. mauretanicus (25, 26). in addition, differences in venom content have been described in other species of scorpions; m. tamulus (27), and leiurus quinquestriatus (28). abdel-rahman et al. (29) suspected that a combination of local environmental conditions, geographical separation, and genetic separation may play a major role in the intra-specific variation of venom of scorpio maurus palmatus. moreover, ozkan and ciftci (22) indicated that variation of protein bands detected in the venom of captive male m. gibbosus from the same biotope in turkey might result from the physiological condition of scorpions. the peptide variation in different populations of deduced scorpions might result from adaptive radiation to environmental conditions. nevertheless, this might represent only the tip of the iceberg and the number of novel toxins will still be expanding in the future. moreover, differences in the band pattern of separated protein in venom samples clearly suggest the existence of genetic variation among the scorpion strains of different regions in the study areas. results of this study showed that the molecular pattern of the venom proteins varies in geographical areas. it should be noted that this variation may have a significant role in the venom toxicity of geographical or ecologhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 13–25 mb ghavami et al.: morphometric indices and … 23 http://jad.tums.ac.ir published online: march 31, 2022 ical groups of scorpions. due to the effectiveness and toxin neutralizing capacity dependence on intraspecific variations of this scorpion, it is recommended that pooling of the numerous venoms from population groups is used in the production of antivenoms. obviously, before mixing the crud venoms, it is necessary to identify the biochemical fractions and toxic and immunogenic proteins of each venom in different populations. moreover, further studies are recommended for the mass fingerprinting of scorpion venoms for barcoding, chemotaxonomy, and screening of effective isotypes in the future. conclusion different populations of black fat-tailed scorpions have the same fluorescence patterns. however, morphometric parameters and venom electrophoretic patterns of this scorpion vary in different situations. based on these findings the morphometric parameters of each region are suggested to analyze the samples of that area. moreover, additional complementary field works in the gis ecological niche model together with the mass fingerprint of scorpion venoms are recommended for a screening of effective isotypes in future studies. acknowledgment this work was supported by research project no. a-12-84-8 offered by vice-chancellor for research and technology of zanjan university of medical sciences. the authors are grateful to mr taghiloo and mr mohammadi for their valuable assistance during the surveys and samplings. ethical consideration ethical approval for this study was obtained from the ethics committee at zanjan university of medical sciences (iran). conflict of interest statement authors declare that there is no conflict of interest. references 1. mullen gr, stockwell sa (2019) scorpions. in: mullen gr, durden la (eds): medical and veterinary entomology, 3rd edition. elsevier, london, pp. 489–501. 2. radmanesh m (1990) androctonus crassicauda sting and its clinical study in iran. am j trop m hyg. 93(5): 323–326. 3. ismail m, abd-elsalam ma, al-ahaidib ms (1994) androctonus crassicauda (olivier), a dangerous and unduly neglected scorpion-i. pharmacological and clinical studies. toxicon. 32(12): 1599–1618. 4. rodriguez de la vega rc, possani ld (2005) overview of scorpion toxins specific for na+ channels and related peptides: biodiversity, structure-function relationships and evolution. toxicon. 46(8): 831–844. 5. dehghani r, fathi b (2012) scorpion sting in iran: a review. toxicon. 60(5): 919–933. 6. stockmann r (2015) introduction to scorpion biology and ecology. in: gopalakrishnakone p, possani ld, schwartz ef, rodríguez de la vega rc (eds): scorpion venoms. springer, new york, pp. 25–59. 7. ozkan o, adiguzel s, kar s (2006) parametric values of androctonus 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waghmare a (2006) intraspecific variation in protein of red scorpion (mesobuthus tamulus, coconsis, pocock) venoms from western and southern india. j venom anim toxins incl trop dis. 12(4): 612–619. 28. omran ma, mcvean a (2000) intraspecific variation in scorpion leiurus quinquestriatus venom collected from egypt (sinai and aswan deserts). j toxicol toxin rev. 19 (3–4): 247–264. 29. abdel-rahman ma, omran maa, abdelnabi im, veda v, mcvean a (2009) intraspecific variation in the egyptian scorpion scorpio maurus palmatus venom collected from different biotopes. toxicon. 53: 349–359. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 234 http://jad.tums.ac.ir published online: september 30, 2019 review article conducting international diploma course on leishmaniasis and its control in the islamic republic of iran *mohammad reza yaghoobi-ershadi1; amir ahmad akhavan1,3; mohammad reza shirzadi2; yavar rassi1; ali khamesipour4; ahmad ali hanafi-bojd1; *hassan vatandoost1,3 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2zoonosis control department, ministry of health and medical education, tehran, iran 3department of chemical pollutants and pesticides, institute for environmental research tehran university of medical sciences, tehran, iran 4center for research and training in skin diseases and leprosy (crtsdl), tehran university of medical sciences, tehran, iran (received 26 may 2019; accepted 21 sep 2019) abstract background: leishmaniasis represents the important public health problem in the world. one of the main objectives of world health organization is capacity building of managers and authorities who are involved with diseases control activities. methods: the course was conducted in esfahan health research and training center (e.h.r.t.c) in summer 2005 and 2009. the course carried out jointly by the ministry of health and medical education (moh) of iran, world health organization-eastern mediterranean regional office (who-emro) and school of public health, tehran university of medical sciences (sph-tums) and designed for medical officers, senior technicians and managers involved in leishmaniasis control. prior to initiate the course, pre-test evaluations including different subjects on leishmaniasis and its control were carried out. the examinations include multiple choice questions. the duration of the course was 3 weeks. a total of 206 contact hours were taught. the main subjects were basic epidemiology, leishmaniasis parasitology, leishmaniasis entomology, control of vectors and reservoirs, principles of integrated pest management, field work and planning. different methods of teaching including lecture, laboratory, workshop, team work, field exercise and presentation were used. requirement for achievement of the course was to have at least 60% of the total mark for awarding the diploma certificate. results: a total of 45 participants from iraq, afghanistan and iran graduated from this course. conclusion: the course is providing the skill for managers, how to combat against disease in their country and is parallel to the policy of the leishmaniasis control for capacity building in endemic areas of their countries. keywords: leishmaniasis; diploma course; disease control; iran introduction human leishmaniasis with a wide clinical spectrum is the neglected form of neglected tropical diseases with a wide variety of parasite species, reservoirs and vectors which are involved in transmission. the causative agent is more than 20 species of the protozoa leishmania and is transmitted to animals and humans through the bite of certain species of female sand flies (1). out of about 1000 species of sand flies, 93 of them spread leishmaniasis. they are usually 2 millimeter large and can tear the skin in feeding of blood (2). the disease is extended in 100 countries in the world with a global prevalence of 12 million people and 2 million new cases are reported each year (3, 1). cases mostly occur in developing countries around the subtropical regions and its incidence is rising significantly. the presence of the disease is linked directly to poverty, but the factors such as social, poor housing, en*corresponding authors: prof mohammad reza yaghoobi-ershadi, email: yaghoobia@tums.ac.ir, prof hassan vatandoost, email: vatando@tums.ac.ir http://jad.tums.ac.ir/ mailto:yaghoobia@tums.ac.ir mailto:vatando@tums.ac.ir j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 235 http://jad.tums.ac.ir published online: september 30, 2019 vironmental and climate change influence the epidemiology of the disease. more than half of the world's population lives in endemic areas and is at risk of infection (1). available tools for prevention and control are limited in the world, which means that exposed individuals should take steps to reduce contact with the vector. furthermore, the health authorities should implement surveillance actions and carry out public health interventions when necessary. early diagnosis and proper treatment are essential for halting this disease (4). cutaneous and visceral leishmaniasis are endemic in iran and continue to be a growing health threat to community development and in the environment. cutaneous leishmaniasis (cl) occurs in two forms, anthroponothic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl) (5). the incidence of cl has been reported between 36–49 cases per 100 000 population during 1983–2017 but in recent years it has had a decreasing trend (fig. 1). the number of reported cl cases in 2017 was 12491, with minimum of 443 in july and maximum of 1661 in november (fig. 2). fig. 3 shows the frequency of cutaneous leishmaniasis cases by province in the country in 2017. the most affected province was ilam with incidence rate of 135/1000 and the lowest incidence rate (0-11) has been reported from the northwest and southwest areas. a total of 60 cases of vl have been reported from iran in 2017, the most affected area was meshkin-shahr district in the northwest (fig. 4). the trend of visceral leishmaniasis cases shows a sharp reduction during 1998–2017 (fig. 5). the main elements of cutaneous and visceral leishmaniasis control programs in the islamic republic of iran have focused on the establishment of a national committee for leishmaniasis control, are as follows: strengthening the leishmaniasis surveillance system and establishing a laboratory network from the national to peripheral level, establishment of standard diagnosis and treatment centers, provision of educational programs for different social levels and revision of the national guideline, standardization of case definition and treatment results, early diagnosis and rapid treatment of patients accompanied by dressing wounds to prevent sand fly bites, reservoir control programmes, collecting the rubble and rubbish tips and vector control programs, applying direct agglutination test in periphery levels for visceral leishmaniasis diagnosis, identification and collection of infected owned and stray dogs in visceral leishmaniasis endemic foci, intersectional coordination with related organizations and institutions (6). leishmaniasis and its control has been a matter of interest by different researchers at school of public health, tehran university of medical sciences (tums), in coincidence with the beginning of epidemiological studies on cl in 1965 and have been continued by scientists and young researchers so far. some of their several publications on different aspects of leishmaniasis is worthy of mention in recent years (7-21). course contributors the course was organized jointly by the ministry of health and medical education, islamic republic of iran, the school of public health and national institute of health research, tehran university of medical sciences, world health organization, eastern mediterranean region (emro). it provided the participants with the knowledge and skills in different aspects of leishmaniasis and its control, through small group work, field exercises, exchange of experiences and discussions with qualified specialists. contents of the course epidemiological trends of leishmaniasis description, history and geographical distribution of leishmaniasis in the world and eastern mediterranean regional office (emro), epidemiology of leishmaniasis in iran and neighboring countries, effects of climate change and disaster on leishmaniasis situation. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 236 http://jad.tums.ac.ir published online: september 30, 2019 treatment clinical manifestation and treatment of old world cutaneous and visceral leishmaniasis: physical systematic, interleisonal, oral drugs, management of visceral leishmaniasis immunology of leishmaniasis natural immunity and acquired immunity, experience of vaccination in leishmaniasis, serological surveillance of asymptomatic visceral leishmaniasis and case detection, leishmaniasis surveillance in iran. human disease methods of studies on human disease: rates, ratios, proportions, prevalence and incidence of leishmaniasis, data presentation: common statistical tests, tables, graphs, charts. leishmaniasis entomology an overview of vectors and reservoirs of leishmaniasis, biology and ecology of sand flies, taxonomy of sand flies, identification criteria, explaining of identification keys. identification of sand fly genera: different sub-genera of phlebotomus genus. sampling methods of sand flies: sucking tube, cdc light traps, funnel traps, sticky oil traps and other techniques, preservation, mounting and identification of sand flies, collection of sand flies on human and animal baits, dissection of sand flies for detection and isolation of leishmania parasites, molecular entomology of leishmaniasis vectors. reservoir hosts rodent leishmaniasis, biosystematics of rodents, identification of main reservoir hosts, biology, ecology, distribution and behavior of rodents, criteria for incriminating rodents as reservoirs, methods of field and laboratory studies of reservoirs of cutaneous and visceral leishmaniasis, canine visceral leishmaniasis, molecular epizootiology of rodent leishmaniasis. control of leishmaniasis different methods of prevention and control of cutaneous and visceral leishmaniasis, methods for evaluation of leishmaniasis control, different methods for elimination of stray dogs, leishmanization and its effectiveness in the control of cl in iran, vaccine for visceral leishmaniasis, principals of integrated pest management (ipm planning), rodenticides and environmental management and sanitation, destroying of chenopods, different methods of rodent control, insecticide susceptibility tests on sand flies, calculation of lt50 and lt90 using probit analysis, insecticides recommended by who against leishmaniasis vectors, formulation of insecticides for residual spraying, measurement and calculation of insecticides for impregnation of bed nets and curtains, safe use and judicious use of insecticides and precaution measures, insecticide storage (condition of safe storage), space spraying, ultra low volume (ulv), thermal fog. fieldwork and practical demonstrations collection of sand flies by different methods from outdoors and indoors in a rural district (field work) around esfahan, carrying of traps from the field to laboratory, preservation and mounting of collected sand flies, identification of sand fly genera: different sub-genera of phlebotomus genus (laboratory practice), collection of sand flies by different methods (from rodent burrows, on human and animal bait, field work), carrying of traps from the field to laboratory, dissection of collected sand flies for detection and isolation of leishmania parasites, mounting, identification of collected sand flies, collection of rodents by sherman traps in the field and carrying of them to the laboratory, identification of collected rodent reservoirs, preparations of smears from the ear of each rodent, fixation of slides, staining with giemsa microscopic examination of slides, isolation of parasites from infected rodents, measurement of rodenticides for application in the field circumstances, preparing of poisoned bait, education of workers, domestic dog examination and conhttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 237 http://jad.tums.ac.ir published online: september 30, 2019 trol, field application of rodenticides, its monitoring and evaluation impact, visiting households in 3 villages for case finding , examination of people for the presence of ulcers or scars, recording the necessary information on the related forms, preparing smears from ulcers, fixation of slides, staining with giemsa, microscopically examination of slides, visiting health centers for examination and treatment of patients of cl in esfahan, different methods of prevention and control of cutaneous and visceral leishmaniasis, methods for evaluation of leishmaniasis control, principals of integrated pest management, integrated pets management (ipm) planning, different methods of rodent control, insecticide susceptibility tests on sand flies, calculation of lt50 and lt90 using probit analysis, insecticides recommended by who against leishmaniasis vectors, formulation of insecticides for residual spraying, measurement and calculation of insecticides for preparation of bed nets and curtains, safe use and judicious use of in insecticides and precaution measures, space spraying, ulv, thermal fog, different parts and function of hudson pump and thermal fog, measurement and calculation of insecticides for impregnation of bed nets and curtains (practical work), practical work for indoor residual spraying at the station, collection of sand flies by aspirator from the villages, carrying out insecticide susceptibility tests by who standard method on collected sand flies, results of susceptibility tests on sand flies: recording and analysis, suggestion of a control program for an anthroponetic cutaneous leishmaniasis (acl) focus, suggestion of a control program for a zoonotic cutaneous leishmaniasis (zcl) focus, suggestion of a control program for a visceral leishmaniasis (vl) focus. 19 83 19 84 19 85 19 86 19 87 19 88 19 89 19 90 19 91 19 92 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 01 20 02 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14 20 15 20 16 20 17 inc. 36 48 32 26 42 35 28 33 25 21 23 35 27 36 39 30 30 20 22 21 32 40 32 35 32 32 31 28 26 27 22 29 25 19 15 case 15 21 15 12 21 18 15 19 14 11 13 20 16 22 23 18 18 12 14 13 21 27 21 24 22 23 22 21 19 20 16 22 19 14 12 0 5000 10000 15000 20000 25000 30000 0 10 20 30 40 50 60 inc/100,000 fig. 1. trend of cutaneous leishmaniasis in iran, 1983–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 238 http://jad.tums.ac.ir published online: september 30, 2019 fig. 2. number of cutaneous leishmaniasis cases by month, iran, 2017 fig. 3. frequency of cutaneous leishmaniasis cases by province, iran, 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 239 http://jad.tums.ac.ir published online: september 30, 2019 fig. 4. trend of visceral leishmaniasis in iran 1998–2017 fig. 5. visceral leishmaniosis cases by province, iran, 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 240 http://jad.tums.ac.ir published online: september 30, 2019 evaluation of the course and participants in evaluating the training course, almost all the training units in the module were covered during the period. there were adequate facilities for the delivery of lectures. these include a well-furnished spacious room for lectures with adequate audio-visual equipment. these include a white board, a slide projector and screen and a dedicated computer for the training. each participant was provided with a copy of the relevant books and handouts. the participants were given practical field experience and laboratory demonstrations. the laboratory was well equipped with dissecting and compound microscopes which made it possible for each participant to individually and independently do the laboratory work. there was enough reference collection of sandflies available for practical work. all the facilitators tried to make the course as participatory as possible and there was very good relationship between the facilitators and the participants. almost all the participants through verbal communication rated the entire course as very good, delivery of lectures as excellent and practical demonstrations and fieldwork as good. the onsite accommodation for participants enabled all the activities to be organized on time. conclusions in general, based on the assessments and interviews, also considering the fact that most of the participants were programme managers and medical health professionals who have had very little knowledge on entomology, vector and reservoir control, the course achieved its purpose of providing them with basic information needed for decision making in disease control activities in their respective countries. the participants and facilitators for the first time shared experiences with regard to vector control activities between different countries. we will recommend that all the participants should be monitored and given the necessary support to enable them contribute to diseases control and elimination activities in their respective countries. acknowledgements the authors would like to appreciate very much for kind support of school of public health (tums), cdc of iran (from moh), world health organization, eastern mediterranean region, and also prof nadim, prof javadian, prof gouya, prof asilian, dr zahraeiramazani, eng abdoli, eng jafari, prof mohebali, mr arandian, ms shareghi, ms ghanei, for their contribution as course tutors and field works. references 1. jhingran a, chatterjee m, madhubala r (2008) leishmaniasis: epidemiological trends and diagnosis in: leishmania after the genome by: myler pj and fasel n, caister academic press, norfolk, uk, pp. 1–14. 2. maroli m, feliciangeli md, bichaud l, charrel rn, gradoni l (2012) phlebotomine sand flies and the spreading of leishmaniases and other diseases of public health concern. med vet entomol. 27(2): 123– 147. 3. choi cm, lerner ea (2001) leishmaniasis as an emerging infection. j invest dermatol symp proc. 6: 175–182. 4. yaghoobi-ershadi mr, akhavan aa, mohebali m (2001) 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phosphide for the control of zoonotic cutaneous leishmaniasis in a hyperendemic area, central iran. j vector borne dis. 51 (4): 307–312. http://jad.tums.ac.ir/ https://www.researchgate.net/profile/zahra_saeidi?_sg=0mpahmcqorlcyzerym_f_vj73qtc73p7xgqpp_7oyicsw9scszyr7vpj4r3nfnac3kmzpa8.sovlccuauq5pbjjq2umxljkzudwrma9myazd9pvcztfctzezeh_kdb-to_xelql0rqaoxlw2bzv7vwzfdojfhg https://www.researchgate.net/profile/alireza_sanei-dehkordi?_sg=0mpahmcqorlcyzerym_f_vj73qtc73p7xgqpp_7oyicsw9scszyr7vpj4r3nfnac3kmzpa8.sovlccuauq5pbjjq2umxljkzudwrma9myazd9pvcztfctzezeh_kdb-to_xelql0rqaoxlw2bzv7vwzfdojfhg https://www.researchgate.net/profile/alireza_sanei-dehkordi?_sg=0mpahmcqorlcyzerym_f_vj73qtc73p7xgqpp_7oyicsw9scszyr7vpj4r3nfnac3kmzpa8.sovlccuauq5pbjjq2umxljkzudwrma9myazd9pvcztfctzezeh_kdb-to_xelql0rqaoxlw2bzv7vwzfdojfhg https://www.ncbi.nlm.nih.gov/pubmed/?term=akhavan%20aa%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=veysi%20a%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=arandian%20mh%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=vatandoost%20h%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=yaghoobi-ershadi%20mr%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=hosseini%20m%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=abdoli%20h%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=heidari%20k%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=sadjadi%20a%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=sadjadi%20a%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=fadaei%20r%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=ramazanpour%20j%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=aminian%20k%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=aminian%20k%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=shirzadi%20mr%5bauthor%5d&cauthor=true&cauthor_uid=25540963 https://www.ncbi.nlm.nih.gov/pubmed/?term=jafari%20r%5bauthor%5d&cauthor=true&cauthor_uid=25540963 j arthropod-borne dis, september 2019, 13(3): 234–242 mr yaghoobi-ershadi et al.: conducting international … 242 http://jad.tums.ac.ir published online: september 30, 2019 18. yaghoobi-ershadi mr, akhavan aa, jahanifard e, vatandoost h, amin gh, moosavi l, zahraei-ramazani ar, abdoli h, arandian mh (2006) repellency effect of myrtle essential oil and deet against phlebotomus papatasi under laboratory conditions. iran j pub health. 35(3): 7–13. 19. yaghoobi-ershadi mr (2016) control of phlebotomine sand flies in iran, a review article. j arthropod borne dis. 10(4): 429–444. 20. veisi a, vatandoost h, yaghoobi-ershadi mr, arandian mh, jafari r, hosseini m, abdoli h, rassi y, heidari k, sadjadi a, fadaei r, ramazanpour j, aminian k, shirzadi mr, akhavan aa (2012) comparative study on the effectiveness of coumavec and zinc phosphide in controlling zoonotic cutaneous leishmaniasis in a hyperendemic focus in central iran. j arthropod borne dis. 6(1): 18–27. 21. yaghoobi-ershadi mr, akhavan aa, zahraei-ramazani ar (2003) bionomics of phlebotomus papatasi (diptera: psychodidae) in an endemic focus of zoonotic cutaneous leishmaniasis in central iran. j vect ecol. 30(1): 115–118. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 126 http://jad.tums.ac.ir published online: june 24, 2019 original article clinical profiles of dengue fever patients, during an outbreak *shafia saba1,2; ata ur rehman khan2; unsar naeem-ullah1; syed haroon masood bokhari1 1department of entomology, muhammad nawaz shareef university of agriculture multan, pakistan 2epidemic prevention and control program, district health authority punjab, pakistan (received 18 apr 2018; accepted 14 apr 2019) abstract background: dengue fever (df) has become a major public health concern globally. it is an infection caused by a virus of the family flaviviridae, with five serotypes (denv 1–5). recent years have seen an increase in the prevalence of the disease in pakistan. the current study was carried out to evaluate the clinical features, laboratory find ings and demographic information of the patients reported during the dengue outbreak in multan of pakistan in 2015. methods: the hospital documentation-based data of confirmed df cases were collected for the 6 months period from a tertiary care hospital in multan, pakistan. the patients were labeled as confirmed on the basis of ns1 and igm positivity by elisa. the data collected were analyzed using spss. results: overall, 361 patients were investigated (78.67% males and 21.33% females), with maximum infection rate in the age group of 18–35yr (50.41%). mean hospital stay was 2.64d (sd 1.2), while mean fever duration was 5.27 (sd 1.57). outbreak occurred during the months from jul–dec, while maximum patients were reported in oct (287). no mortality was reported, and all patients recovered. conclusion: better management practices and timely reporting can reduce the risk factors associated with the disease. keywords: dengue fever; outbreak; public health, vector-borne diseases introduction our planet is going through a critical phase due to vector-borne diseases (vbds) of humans. besides others, arthropods are the major group to transmit diseases in human. among the vbds spread by insects, dengue fever (df) is the most prevailing disease in human communities after malaria, with vast geographic distribution (1). primary vector responsible for transmission of the disease is mosquito species aedes aegypti, followed by ae. albopictus (2), and the rate of infection is higher in rainy months/days because of increasing populations and breeding sites of vector mosquitoes (3). dengue fever is a febrile disease also known as “break-bone fever” (4) caused by a virus of the family flaviviridae (genus fla vivirus) (5) with five serotypes viz. den-1, den-2, den-3, den-4 and den-5 (6). den1 and den-3 have been stated to cause more serious primary infections while remaining are extra problematic and serve as a source of secondary infection (7). once, the patients have any of the said serotypes they cannot get infected with the same again, but the situation goes worst as the patient lose self-immunity against the other serotypes. however, the condition becomes more critical when individuals get second infection (after first infection), thus resulting in extreme illness (8). the disease can occur in more than one forms ranging from dengue fever (df), dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) (9). the symptoms *corresponding author: dr shafia saba, e-mail: sabashafia@yahoo.com mailto:sabashafia@yahoo.com j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 127 http://jad.tums.ac.ir published online: june 24, 2019 shown by patients of df are fever, musculoskeletal pain, retrobulbar pain, headache, and morbilliform rashes. dhf patients have highgrade fever and hemorrhagic attacks. dss is characterized by negative change in mental situation and normally low blood pressure (10). classical dengue is reported mainly in children, youngsters, and adults (11). the worldwide spread of the disease has increased radically in past few years with half of the world population under the peril of infection and it is claimed that 3.9 billion people inhabiting 128 countries are under the threat of df (12). about 50–100 million cases are reported every year and the death rate in various countries is not less than twelve thousand per year (1). the disease has spread to more areas over time; for example, before 1970 the dhf was endemic only in nine countries. whereas during the year 1995, the number increased more than four times and in 1998 about 2500 million people were at verge of the disease (5). high numbers of cases were from tropical countries with poor economies (13). initially, dengue cases were only recorded from urban areas but in recent past cases have been reported from rural areas as well (14). travelers are responsible to disseminate the disease from across the countries. a large proportion of people who traveled through tropical and sub-tropical countries acquired the disease. this condition alone increased the dengue cases up to 16% by 2005. now dengue is reported more than malaria in travelers from countries of south east asia (15). since last many years, pakistan is facing regular epidemics in one or another area in all of its provinces during and soon after rainy seasons (16). the disease was reported in the country for the first time in 1985, in youth of 16yr with a strange fever (17). in hub, baluchistan in 1995, 75 dengue cases were reported out of which 57 doomed to death (18). in 2011, pakistan faced the worst strike of dengue in which more than 300 deaths and 20000 cases were reported (19). among the affected cities, lahore was severely hit followed by faisalabad, multan, bahawalpur and sargodha (20). in 2015, the multan district of punjab witnessed a devastating outbreak of dengue fever in all towns of the district. the objective of present study was to analyze vulnerability of local people for the disease in various demographic traits like gender and age, geographical prevalence of the patients in the district, monthly trend of infection and clinical findings of the patients reported in a tertiary care hospital. materials and methods description of the study area the historic city of saints “multan” is situated on the bank of “chenab river” and lies at 30°11′52″n latitude and 71°28′11″e longitude in punjab province of pakistan. multan is the 7th most populous city of pakistan and is the cultural and economic center of south punjab. its total area is 781km2 with highest elevation of 423ft. the district multan comprises 6 autonomous towns including shah rukn-e-alam (sra), musa pak shaheed (mps), sher shah (ss), bosan, shujabad (sb) and jalal pur pirwala (jpp) town. population of the district consists of 1.871 million inhabitants. the climate of the district is hot and dry. the summer season starts from may and continues till september. may, june, and july are the hottest months. rainfall occurs during monsoon i.e., from july to sep, while july is the wettest month. during winter season there is very little rain (21). the current study was carried out with the collaboration of epidemic prevention and control program, district health authority, multan by collecting data of patients reported in nishtar hospital multan from jul to dec 2015. collection of clinical data a hospital documentation-based study was conducted from 1st july 2015 to 31st dec 2015 in nishtar hospital multan, punjab, pakistan. the data regarding patients reported with fever https://tools.wmflabs.org/geohack/geohack.php?pagename=multan&params=30_11_52_n_71_28_11_e_region:pk_type:city(3117000) j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 128 http://jad.tums.ac.ir published online: june 24, 2019 and other symptoms related to dengue from all above-mentioned towns of multan was included in the study. the deag (dengue expert advisory group) case definitions were used as a guideline to label the patients as suspect, probable and confirmed dengue fever patients (22). however, only the confirmed patients were enrolled. the patients were labeled as confirmed df on the basis of serological tests (23) i.e., rapid diagnostic test ns1 antigen (accudiag™ dengue ns1 antigen elisa) and igm antibody detection (calbiotech dengue virus igm elisa) as per instructions of the manufacturer. medical records and demographic data of all 361 confirmed df patients were obtained using a predesigned performa and analyzed by spss (version-16) (chicago, il, usa). results were expressed in terms of means, standard deviation (sd) and proportions. results epidemiological findings overall, 2477 patients with suspicion of df were reported at nishtar hospital multan in the year 2015, out of which 1794, 292 and 361 were marked as suspected, probable and confirmed respectively based on serological positivity of igm and ns1 tests. the current study is based on confirmed endures, comprising 284 (78.67%) males and 77 (21.33%) females. patients with <18yr of age were 7.2%, 16.1% were >50yr while 26.3% were in the age group of 3–50. the majority of patients were 50.4% and belonged to the age group between 18–35yr (fig. 1). all the patients had fever from early phase of the illness (mean 5.27d, sd 1.57) while mean hospital stay was 2.64d (sd 1.2). all the patients were recovered and discharged, and no mortality was reported. geographical distribution among the 4 urban and two rural towns of multan district, the highest number of df patients was recorded from sher shah town (58.72%), followed by bosan town (19.96%), shah rukn-e-alam town (11.36%) and musa pak shaheed town (9.42%), while only 0.54% of the patients were reported from the rural towns (fig. 2). from the confirmed patients, 99% (355) were residents of multan district while only 1% (7) was reported from other districts, whereas 8% (32) had travel history from other districts before onset of fever. clinical findings all the patients were reported with fever and other symptoms like vomiting, myalgia, headache, arthralgia, epistaxis, loose motion, and abdominal pain. among all the 361 patients 64.26 (190 men and 42 women) were positive for ns1 antigen, and 35.73% (94 men and 35 women) cases were igm positive (fig. 3). the platelet count in 63 (17.45%) patients was less than 50x103/microliter, in 254 (70.36%) it was between 50x103–100x 103/ microliter while in 44 (12.18%) patients the count was above 100x103/microliter. white blood cells (wbc) count was also recorded on the date of admission in hospital which was ≤ 4x103/microliter in 70.08% (253) and > 4x 103/microliter in 29.91% (108) patients. during the study period the number of patients was highest in the month of october with 287 (79.51%) cases, and a fair reduction was observed in sep and nov with 30 (8.31%) and 40 (11.09%) patients, respectively. while only 4 (1.09%) patients were admitted in the months of jul, aug and dec collectively (table 1). table 1. monthly infection rate of dengue fever patients studied during 1st july to 31st dec 2015 in nishtar hospital multan, punjab, pakistan month july august september october november december patients 2 1 30 287 40 1 https://www.google.com/search?client=firefox-b-ab&biw=1366&bih=659&q=epistaxis&spell=1&sa=x&ved=0ahukewipv4ick6nwahvdmi8khtjrbuyqbqgkkaa j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 129 http://jad.tums.ac.ir published online: june 24, 2019 fig. 1. age and gender ratio of dengue fever patients studied during 1st july to 31st dec 2015 in nishtar hospital multan, punjab, pakistan fig. 2. prevalence of dengue fever patients in different towns of multan, pakistan studied during 1st july to 31st dec 2015 fig. 3. serological representation of dengue fever patients studied during 1st july to 31st dec 2015 in nishtar hospital multan, punjab, pakistan discussion the present study was conducted during the df outbreak in the year 2015 in multan district. infection rate was high in men (78.67%) as compared to women (21.33%). the similar trends were observed by various workers (22-25), the df ratio between men and women was 55.3:4.7, 58:42 and 59.3:40.7 (in percentage), respectively. similar results were j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 130 http://jad.tums.ac.ir published online: june 24, 2019 observed in kolkata, india where highest infection rate was reported in males than in females (26). the same tendency was noted in a recent study in sri lanka in which 66.2% were males and 33.8% females (27). males get more infections as compared to females. the main reason for this trend may be that men are more exposed to outdoor activities and environmental factors as compared to females (28). another reason may be that females wear long sleeves and full trousers in pakistan which give them protection from mosquito bites (23). it is, therefore, suggested to adopt personal protective measures from mosquito bites. in the current study, among the age groups, the individuals of 18–35yr of age got maximum infections i.e., 50.41% followed by 26.31% in age group of 36–50, 16% >50yr and 7.2% in <18yr of age. in a similar study, maximum infection rate was observed in the age group of 21–40yr (29), while in another study highest number of patients were reported between 11– 30yr (25). the maximum infection rate in age group of 15–45yr was also reported (30). however, contrary to our findings a study showed the highest number of df patients in the age group of 0–10yr (31). in puerto rico, in 1994– 1999, elders (> 65yr) and infants (1 year) were more infected as compared to the youth (2 to 18yr) and adults (19 to 64yr). infants and elderly are more delicate and easily carry and develop the infection (32). in the present study, assumption can be made that people between 18–50yr have more outdoor activities and chances of vector contact than the other age groups and thus magnitude of infection is higher in this age group. in the study under discussion, mean duration of fever was 5.27d (sd 1.57) which was in accordance with the findings of another study i.e., 5.5d, sd 2.7 (33). the mean hospital stay was 2.64d (sd 1.2), which was 4.76±1.53 in another study (28). in a study carried out in sri lanka, the same was reported to be 2.7d (sd 0.6) (25). on the contrary, in another work mean hospital stay was reported to be 7–12d (30). taking into account the history of infection in the outbreak under discussion, 91% of the patients were local residents with no travel history and only 1% were from other districts, whereas 8% had a travel history to other districts. the maximum infection was acquired locally, which means that the local vector was infected by some means. the majority of cases were reported in the month of oct i.e., 79.51%, with 8.31% in sep and 11.09% in nov, while only 1.09% of positive df cases were reported in the rest of the 3 months (july, aug and dec). in district swat, kpk of pakistan the same pattern was observed. the rate of infection was low in the month of july and with gradual increase. the highest number was reported in oct i.e., 36.11 %. the increasing number of patients was associated with the pattern of rain (29). dengue outbreaks are clearly linked with rainy season (34). patient count was zero from jan to mar, with only 8 reported from apr to aug. the patient count started to rise, and the maximum number of confirmed df cases were reported from sept to nov and again declined in dec. in the same description, maximum numbers of patients were observed in the months from aug to oct 2006 which was a period of heavy rainfall (20). all these findings are in accordance with the current study and the disease incidence is clearly related to the monsoon and pre-monsoon period. the high rainfall provides increased number of potential breeding sites for vector mosquitoes, consequently increase in number of df cases. the number of patients was also linked to increased temperature and rainfall, and during high-temperature mosquito got the chances to feed more on humans as compared to low-temperature season (23). mosquito population is linked with raised temperatures (35) and increase in temperature by 1 °c resulted in increase the risk of df transmission by 1.95 times (36). among the four urban and two rural towns j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 131 http://jad.tums.ac.ir published online: june 24, 2019 of multan district, the highest number of patients (212) was reported from sher shah town. the reason for this might be the maximum number of tyre shops present in the town. tyres serve as breeding place for mosquitoes and the same can serve as a mean of shipping the dengue vectors in and outside an area (23). moreover, multan is situated almost in the center of the country and serves as main perching place on major roads and rail track. the city is connected by rail with all parts of the country and lies on the main track between karachi, peshawar, lahore and quetta. for the whole year, people travel through the city from all around the state. in year 2015, karachi, lahore and rawalpindi were also under dengue outbreak. the presence of railway station in sher shah town can be a potential reason for more dengue burden in the locality under discussion. the evidence supporting the statement is that a large number of df patients were the residents of railway colony multan located around the railway station of sher shah town. the movement of infected persons is one of the main causes of the swift spread of this disease in pakistan (20). taking into account the clinical aspects, all the patients were reported with high-grade fever which is the clear sign of the disease. these observations are in accordance with the studies carried out by different workers (2529). in the outbreak under consideration, not a single patient was diagnosed with dhf or dss, all endure were fully recovered and discharged from the hospital. the reason for this may be easy and in time access of the infected patients to the medical facilities and adaptation of proper remedial measures. all this was assured by the epidemic prevention and control program in the district. the blood samples of the patients were confirmed through igm and ns1 positivity. ns1 antigen test is used for early detection of infection. this antigen is detected from 1st to 5th day from onset of fever. whereas igm is an antibody produced in mammals in response to any infection and it takes at least one week to formulate. it can be detected from 5th to 9th day from onset of fever (37). in the present study among the confirmed patients, 64.26% reflected the positivity for ns1 while the rest were positive for igm antibodies. in a study conducted in 2013 with 62 patients, 38 cases were positive for ns1 and 24 were negative. on the other hand, out of 24 ns1 negative tests, 6 patients revealed the positivity of igm and the rest 18 were found to be negative for igm antibodies (26). similarly, it was scrutinized in an experiment that out of 6000 confirmed df cases 4121 were positive for ns1 (28). in a recent study carried out in india, 23% were ns1 positive (38). platelet count was also taken into consideration in the present study. in 17.45% cases platelet count was < 50x 103/microliter, while it was between 50x 103–100x 103/microliter in maximum patients i.e., 70.36%, while in rest of the patients it was > 100x 103/microliter. the same was found to be < 50x 103/microliter in 15.16%, between 50x 103–100x 103/microliter in 28.65% and > 100x 103/microliter in 56.18 % (29). while others reported the platelet count <100x 103/microliter in 55%, 82% and 89% patients respectively (29, 39, 40). conclusion epidemiology and the clinical manifestations of the df patients were revealed and observed that the disease is most prevalent in adult males. the reason behind this is more outdoor activities of adult males and hence more exposure to the disease vector. therefore, vector control measures should be especially adopted at workplaces. on the clinical side, all the cases were of df, with no report of dhf or dss which is a clear indication of timely reporting and better management practices. same pattern is suggested to be followed to avoid mortalities and reduce the risk factors of the disease. https://en.wikipedia.org/wiki/peshawar https://en.wikipedia.org/wiki/quetta j arthropod-borne dis, june 2019, 13(2): 126–134 s saba et al.: clinical profiles of … 132 http://jad.tums.ac.ir published online: june 24, 2019 there is no multivariate vaccine available for control of the disease and the only way to avoid the infection is preventive measures. in this context, community participation and awareness regarding better preventive and control measures of dengue vector is necessary. there is also need for large scale entomological and epidemiological surveys. otherwise, df is likely to become a much serious health issue in pakistan. acknowledgements the authors express their heartfelt gratitude and sincere thanks to the officials of the epidemic prevention and control program, district health authority multan, for their active support and cooperation throughout the study period. gratitude is also expressed to prof dr shafqat saeed, chairman department of entomology, muhammad nawaz shareef university of agriculture multan for his valuable suggestions and guidance. appreciation and gratefulness are also articulated to dr azhar ali khan, deputy registrar, muhammad nawaz shareef university of agriculture multan for reviewing the manuscript and suggesting necessary corrections regarding write up. no funding was taken from any organization to carry out the study. the authors declare that there is no conflict of interests. references 1. world health organization (2000) scientific working group on dengue. in meeting report, geneva, switzerland. 3–5. available at: http://www.who.int/tdr/publications/do cuments/dengue-swg.pdf 2. nathan mb, dayal-drager r, guzman m (2009) epidemiology, burden of disease and transmission. dengue guidelines for diagnosis, 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mehta r (2018) clinical and hematological profile of patients with dengue fever at a tertiary care hospital–an observational study. mediterr j hematol infect dis. 10(1): e2018021. 39. ratageri vh, shepur ta, wari pk, chavan sc, mujahid ib, yergolkar pn (2005) clinical profile and outcome of dengue fever cases. indian j pediatr. 72(8): 705– 706. 40. karoli r, fatima j, siddiqi z, kazmi ki, sultania ar (2011) clinical profile of dengue infection at a teaching hospital in north india. j infect dev ctries. 6 (7): 551–554. j arthropod-borne dis, september 2015, 9(2): 274–280 mb ghavami and a djalilvand: first record of … 274 case report first record of urogenital myiasis induced by megaselia scalaris (diptera: phoridae) from iran *mohammad bagher ghavami 1, ahmad djalilvand 2 1department of medical entomology and vector control, faculty of medicine, zanjan university of medical sciences, zanjan, iran 2department of pathology, faculty of medicine, zanjan university of medical sciences, zanjan, iran (received 14 sep 2013; accepted 2 july 2014) abstract myiasis is the infestation of organs and tissues of human and animals with fly larvae. this article reports an 18 year-old man with urogenital myiasis, the passing of live megaselia scalaris larvae in the urine, from zanjan city, northwest of iran. we discourse the importance of diagnosis and management of urogenital myiasis in medicine. keywords: urogenital myiasis, megaselia scalar, scuttle fly, phorid larva introduction myiasis, the infestation of organs and tissues of human and animals with fly larvae, is a common phenomenon especially in people of tropical and subtropical areas. in spite of occasional reports of obligatory myiasis, the majority of reported cases are facultative myiasis and more than a dozen families of flies can cause this form (gullan and durden 2009). phoridae, commonly known as humpback or scuttle flies, is the most important family of dipteran that causes myiasis and various problems for human. in this family genus megaselia, perhaps the largest genera of living organisms, has a wide variety of life styles and polyphagus diet (disney 2008). among identified species of this genus, m. scalaris is a cosmopolite and synanthropic one. it has been unwittingly carried around the world by human (disney 2008) and previously has been reported from alborz province in iran (zamani 2009). adults of scuttle flies which are very small (2–4 mm in length), can explore a large variety of environmental and ecological habitats. females of these flies are highly attracted to strong of foul odors and lay their eggs on different decomposing materials such as fruits, stole, meat, excrement and carrions (disney 2008). the scuttle flies which are important agent of human facultative myiasis, exhibit a greater diversity of larval habitat than other insects. these flies can explore a large variety of environmental and ecological niches (disney 2008). in spite of predation and parasitation of some arthropods by this species (costa et al. 2007), previous studies showed them as a cause of human myiasis (francesconi lupi 2012). moreover, they have already been a polyphagus species and may be harmful to health as they become vector of pathogens (disney 2008). several clinical forms of myiasis such as cutaneous (biery et al. 1978, carpenter and chastain 1992, hira et al. 2004) and intestinal (kaneko et al. 1975, sing et al. 1989, mazayad et al. 2005) myiasis have been induced by m. scalaris. however, the urogenital form is rarely seen and all of reported cases are restricted to females. so the study of men clinical manifestation as well as morphology of third instars larvae of these *corresponding author: dr mohammad bagher ghavami, e-mail: ghavami@zums.ac.ir http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, september 2015, 9(2): 274–280 mb ghavami and a djalilvand: first record of … 275 flies seems to be important. the aim of this paper is to report a case of man urogenital myiasis involving and to discourse the importance of its diagnosis and management in clinical science. case presentation an 18 year-old man living in zanjan city, northwest of iran, presented to ayatollah mosavi hospital at zanjan university of medical sciences with history of difficulty in urination and maggot discharging. he was uncircumcised. for his history of nocturnal enuresis and depression, he was medicated by venlafazine, alpirazolam and nortriptrin. physical examination and sonography of cortex of bladder and kidneys were normal. however, small echogenic floating particles were seen in bladder (fig. 1). he received mebendazole (2 mg tablet) orally and washed his urogenital area with solution of xylocain (2%) and iodine (1%). after an hour the urine sample was collected in clean container. the urine was yellow in color and laboratory examination showed microhematuria, proteinuria and leukocyturia. moreover, 2 larvae were found upon collected urine. the larvae were forwarded to the department of medical entomology and vector control for further studies. all symptoms disappeared on the next day of specimen collection. microscopic examination showed that all larvae were alive, whitish in color and pale intestinal content in the third terminal part (fig. 2). one larva was preserved in 70 % ethylic alcohol and the other cultured in nutrient rich medium to complete its life cycle but it died after 1 day. the length and width of larvae ranged from 6.8 to 6.9 and 0.68 to 0.69 mm respectively (fig. 2). precision microscopic examination of larvae revealed the presence of short spinous (peglike) processes on the integument. the larval head was noncapsulate, being reduced to mouth hooks and supporting cephalopharyngeal skeleton which was developed and supported large areas of the head (fig. 3). the anterior spiracles were located on each latero-posterior edge of the prothorax each had about 8–10 rays. a pair of posterior spiracles protruded dorsally on the 12th segment each appears with large and slender plate. the spiracle hairs appear centrally, at the area of construction. dorsal and lateral trunks were present and well developed. in addition to their anterior and posterior union the dorsal and lateral trunks were joined by transverse connectives (fig. 4). based on characters and previous descriptions of researchers (sukantason et al. 1994, harison and cooper 2003, boonchu et al. 2004, disney 2008) the larvae were definitely identified as megaselia scalaris, facultative myiasis parasite belonging to the phoridae family. the voucher specimens were retained in the department of entomological systematics at university of tarbiat modarres. fig. 1. sonography of cortex of bladder with, small echogenous floating particles fig. 2. third instars larva of megaselia scalaris http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, september 2015, 9(2): 274–280 mb ghavami and a djalilvand: first record of … 276 fig. 3. anterior section of third instars larva of megaselia scalaris fig. 4. posterior section of third instars larva of megaselia scalaris discussion urogenital myiasis, the infestation of genitourinary organs by maggots, based on the anatomical location could be classified as external or internal myiasis. external urogenital myiasis is clinically, epidemiologically, and entomologically similar to wound myiasis but it affects women more commonly. internal urogenital myiasis is a rare event and occurs when the maggot reaches an internal genitourinary organ. all described cases are considered as accidental cases and uncommon myiasis agents are associated with m. scalaris, psychoda albipenis and muscoid flies (francesconi and lupi 2012). several previous researchers reported cases of wound (disney 2008), intestinal (mazyardand rifaat 2005) and nasocominal myiasis (hira et al. 2004), due to m. scalaris. however, the published cases of genital myiasis are very few in number and these reports refer to patients of various ages. the first report of human urogenital myiasis due to this species was reported in 1978 by disney and kurahashi and then other cases were reported (ramalingam et al. 1980, cilla et al. 1992, saleh and elsibae 1993, yildiz et al. 1997, delir et al. 1999, perez-eid c, mouffok 1999, rodriguez and, rashid 2001, passos et al. 2002). this is the first report of urogenital myiasis due to m. scalaris in iran. there is a paucity of such information from this country, probably because specimens tend to be discarded without study, due to lack of experience in identifying fly larvae and in the absence of reference center. we hope that our report will stimulate others to present similar clinical findings and to contribute to the pool of knowledge on the subject in iran. to the best of our knowledge, only one paper documented the distribution of m. scalaris in iran. so we hope that in the coming future special aspects of this species will http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, september 2015, 9(2): 274–280 mb ghavami and a djalilvand: first record of … 277 be studied in iran. the identification of larvae of megaselia sp posed difficulties here because none could be reared to adult for conformation based on adult morphology. however, the available characteristics were enough to distinguish m. scalaris larvae. the passing of live m. scalaris larvae in the urine indicated infestation of the urogenital system. the reported infestations (pryianond et al. 1973, biery et al. 1978, sing et al. 1989) were normally, a secondary consequence of some obstructions to the normal flow of the urine, such as stone formation in the bladder. our microscopic examination showed that the larvae were probably mature; at least second to third instars transition, and may have been seeking a place to pupate. it is worth mentioning that identification of the immature larvae of parasite causing myiasis posed difficulties particularly the parasite with little available knowledge. however, the available morphological characters of the larvae especially the cephalopharyngeal skeleton and the structure of spiracles can be used in differentiation of specimens. phorid flies basically find in tropical regions. however the global warming and the globalization of commerce have established conditions suitable for expand their wider distribution. the flies infesting the patients probably originated from a natural population near home in the residential area. the present case is probably accidental infestation related to lack of personal hygiene. it's likely that female of megaselia deposited her eggs on the underwear clothing or directly on the urogenital area after being attracted by urine odor. the rational treatment of genitourinary myiasis is to remove the offending larvae. in many cases when the diagnosis is done the maggot has already been expelled. several substances such as antiseptic and anesthetic solutions have been used in the form of lavage (francesconi and lupi 2012). it seems, in our case, oral administration of 2 mg mebendazole and washing the urogenital area with solution of xylocain (2%) and iodine (1%), cured the myiasis. since the urogenitoury tract has inaccessible area, removed of maggots is difficult by conventional instruments, to overcome this problem we used urogenital lavage with xylocaine and iodine solution in our patient. xylocaine causes spastic paralysis in parasites straight muscles and this way the paralyzed larva was easily removed. medical personnel should take care of susceptible patients especially those with nocturnal enuresis, need to bear in mind the possibility of infestation with scuttle flies larvae, be able to make a prompt diagnosis of myiasis and implement relevant intervention to prevent tissue infestation. prevention of this condition is important and involves use of insect repellents and insecticides for control of fly population, adequate protective clothing, good skin and wound hygiene to keep flies from reaching the skin, covering open wounds, change dressing daily, hang clothes to dry in bright sunlight and/or iron them and improve hygiene and sanitation. because the size of gravid flies is very small, window screening and mosquito nets cannot effect on personal protection. besides, so much attention is paid to large muscoid flies that may cause myiasis, it is likely that the small phorids are unnoticed or ignored, which may lead to an increase in the occurrence of phorid myiasis. we need to emphasize that this myiasis rarely occurs in healthy individuals and neglected personal hygiene is the single most important factor in human infestation. conclusion nowadays the global warming and commerce globalization change the geographical map of scuttle flies and observation of the http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, september 2015, 9(2): 274–280 mb ghavami and a djalilvand: first record of … 278 scarce case of urogenital myiasis in iran, represents their wide distribution. since m. scalaris can possess considerable larval habitat, its creation of various clinical forms of myiasis is imminent. therefore, we hope that our reported case will stimulate researches to present similar findings and we expect several aspects of this species will be studied in future. in addition medical personell should take special attention to susceptible cases and be able to diagnose myiasis as well as implicate relevant interventions to prevent new infections. acknowledgements we would like to express our sincere thanks to dr talebi, department of entomology, tarbiat modarres university for confirmation of larval identification, to dr barati for skillfully produced sonography, to dr fazaeli, department of parasitology, zanjan university of medical sciences for invaluable advice and encouragement, to mr m asadi for urine analyzing and to mr j mohammadi for the 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[msc thesis]. tarbiat modares university, tehran. zwart p, disney rhl, de batist p, mutschmann f (2005) the phoridscuttlefly (megaselia scalaris) a threat to zoological collections and especially to amphibians. zool med bvzs. 5: 27–30. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 277 http://jad.tums.ac.ir published online: september 30, 2020 original article insecticide susceptibility status of wild population of phlebotomus kandelakii and phlebotomus perfiliewi transcaucasicus collected from visceral leishmaniasis endemic foci in northwestern iran yavar rassi1; *eslam moradi-asl2,3; *hassan vatandoost1,4; malek abazari2, abedin saghafipour5 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of public health, school of public health, ardabil university of medical sciences, ardabil, iran 3arthropod borne diseases research center, ardabil university of medical sciences, ardabil, iran 4department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 5department of public health, school of public health, qom university of medical sciences, qom, iran *corresponding authors: dr eslam moradi-asl, e-mail: moradiasl83@yahoo.com, prof hassan vatandoost, email: hvatandoost1@yahoo.com (received 08 apr 2020; accepted 28 sep 2020) abstract background: phlebotomus kandelakii and phlebotomus perfiliewi transcaucasicus sand flies are the vectors of visceral leishmaniasis in iran. the aim of this study was to evaluate the susceptibility of ph. kandelakii and ph. perfiliewi transcaucasicus, collected from an endemic focus of visceral leishmaniasis (vl) in the northwest of iran, to different selected insecticides. methods: sand flies were collected from the villages of meshkinshahr and germi counties using light traps and aspirators from may to october 2019. the sand flies were identified as ph. kandelakii and ph. perfiliewi transcaucasicus using standard identification keys. susceptibility test was carried out against ddt (4%), malathion (5%), propoxur (0.1%) and lambda-cyhalothrin (0.05%) according to the who standard method. medcalc statistical software was employed to calculate lt50 and lt90 and to compare the chances of sand flies surviving the exposure to the studied insecticides. results: a total of 1,278 female specimens were used for the susceptibility tests. out of which 1,063 samples were used for the test and 215 for the control group. the estimated lt50 values for ddt (4%), malathion (5%), propoxur (0.1%), and lambda-cyhalothrin (0.05%) for ph. kandelakii were 15.1, 13.4, 15.4 and 5.8 minutes respectively, and for ph. perfiliewi transcaucasicus were 11.9, 15.6, 15.9 and 5.8 minutes respectively. conclusion: this susceptibility studies revealed different lt50 values for different insecticides efficient against ph. kandelakii and ph. perfiliewi transcaucasicus. the regular monitoring for the resistance of ph. kandelakii and ph. perfiliewi transcaucasicus sand flies seems necessary in diseases control programs in this area. keywords: phlebotomus kandelakii; phlebotomus perfiliewi transcaucasicus; insecticide resistance; insecticide susceptibility; lambda-cyhalothrin introduction leishmaniasis is transmitted to human in 98 countries and 3 territories on 5 continents in the world and approximately 0.2 to 0.4 cases of visceral leishmaniasis (vl) and 0.7 to 1.2 million cases of cutaneous leishmaniasis (cl) occur each year. leishmaniasis is a parasitic disease found in tropical and subtropical areas around the world. it has been categorized as a neglected tropical disease (ntd). leishmaniasis is triggered by infection with leishmania parasites, which spread through the bite of phlebotomine sand flies of phlebotomus and lutzomyia. there are several distinct types of leishmaniasis in humans. the most prevalent copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:moradiasl83@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 278 http://jad.tums.ac.ir published online: september 30, 2020 types are cl (causing skin sores) and vl (causing impacts on several internal organs) (1). vl, also known as kala-azar, is a severe health problem in endemic areas. the disease is of great significance to the health care system around the globe and can be fatal without adequate therapy (2). the primary causative agent of vl is leishmania infantum in mediterranean areas such as iran (3). in the mediterranean region, there are two major forms of severe and fatal visceral leishmaniasis and cutaneous leishmaniasis. the worldwide mortality rate for leishmaniasis is between 26,000 and 65,000 cases per year (4). vl is revealed to be endemic in 7 areas in iran and the most significant endemic foci are ardabil province in northwestern iran (5). untreated vl has a 100% mortality rate (6). of 44 confirmed species of sand flies in iran, three of them have been identified as the main or probable vectors of vl in ardabil province (7-10). to date, 22 phlebotomus species have been recognized in ardabil province, and two major vector species, ph. kandelakii and ph. perfiliewi transcaucasicus, have been recorded from various regions with a large population (11). leishmania infection of the two listed species with l. infantum and leishmania donovani from the north and center of the province has been reported using parasitological and molecular techniques (12). at present, the chemical control of sand flies, including insecticide residual spraying (irs) or the use of insecticide-impregnated bed nets (itns), are effective means to control visceral leishmaniasis (13). in order to use insecticides against leishmaniasis, the who recommends the use of a safety insecticide previously tested for vectors’ susceptibility (14-20). accordingly, this is the first study to test the sensitivity of two major vectors of visceral leishmaniasis in an endemic foci in northwestern iran to four types of insecticides based on the who guidelines. materials and methods study sites this research was performed in ardabil province located in northwestern iran from may to october 2019. the geographic coordinates of the province are 38.4853 °n, 47.8911 °e. the province is 17,953km2 in area (about 1.09 percent of the country's total region), 1,338 meters above sea level and has a population of 1,249,000 people. sandflies collection a total of 1,278 sand flies were collected for testing and control purposes. samples were gathered from the villages of meshkinshahr and germi districts using light traps and aspirators from human homes, dog nests and livestock stables. sand flies were immediately transferred to the arthropod-borne research center in ardabil university of medical sciences. the specimens were kept in a laboratory in a maintenance cage under 25±2 °c and 60±10% humidity. the specimens were fed with sugar-soaked cotton during storage. they were recognized by using valid keys (21-23). procurement of insecticide impregnated papers and their concentration impregnated papers with ddt (4%) (batch no. dd186, exp. date: july 2016); malathion (5%) (batch no. ma 177, exp. date: april 2018); propoxur (0.1%) (batch no. pr 104, exp. date: april 2018), and lambda-cyhalothrin (0.05%) (batch no. la 262, exp. date: july 2018), as well as papers for control were supplied by the collaborating center of the world health organization in penang, malaysia. susceptibility tests female sandflies were subjected to susceptibility tests according to the guidelines of who. sandflies were exposed to impregnated papers (ddt (4%) (organochlorine), malathion (5%) (organophosphate), propoxur (0.1%) (carbamate) and lambda-cyhalothrin (0.05%) (pyrethroid) at different logarithmic times. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 279 http://jad.tums.ac.ir published online: september 30, 2020 the sample exposure period was 3.75, 7.5, 15, 30 and 60 minutes. samples were recovered at a suitable temperature for 24 hours under laboratory circumstances and supplied with sugar water. the mortality rate was recorded after 24 hours. dead and live samples were gathered and stored in 70% alcohol and then mounted in a drop of puri's medium and recognized using valid morphological keys. four replicates were performed for each experiment. data analysis data collected in microsoft excel 2016 were used to plot slopes and graphs. the medcalc statistical software was used to calculate lt50 and lt90 and to compare the chances of sand flies surviving the exposure to insecticides in the study areas. results during the study period, 1,278 sand fly specimens were used for sensitivity test on two main proven vectors species of vl in the ardabil province of which 1,063 were for the test and 215 for the control group. seven hundred and forty-nine specimens of ph. kandelakii and 529 specimens of ph. perfiliewi transcaucasicus were evaluated respectively. all the samples of the above-mentioned species were found fully susceptible to lambdacyhalothrin (0.05%) insecticides with 100% mortality. the estimated lt50 values for ddt (4%), malathion (5%), propoxur (0.1%), and lambda-cyhalothrin (0.05%) for ph. kandelakii were 15.1, 13.4, 15.4 and 5.8 minutes (table 1), respectively, and for ph. perfiliewi transcaucasicus were 11.9, 15.6, 15.9 and 5.8 minutes respectively (table 2). the mortality rates for ph. kandelakii after 60 minutes of exposure to ddt (4%), malathion (5%), propoxur (0.1%) and lambdacyhalothrin (0.05%) were 91.17%, 93.18%, 97.61% and 100%, respectively. the mortality rates for ph. perfiliewi transcaucasicus were 95.83%, 90.90%, 91.30% and 100%, respectively (table 3). the slope diagrams and the trend of the mortality rates are shown in fig. 2. fig. 1. the study area and sand fly collection sites in the northwest of iran http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 280 http://jad.tums.ac.ir published online: september 30, 2020 table 1. lethal time, lt50 and lt90 values for phlebotomus kandelakii to ddt (4%), malathion (5%), lambdacyhalothrin (0.05%) and propoxur (0.1%), in ardabil province, iran, 2019 insecticide a b±se lt50±95% ci (minutes) lt90±95% ci (minutes) wald statistic p-value ddt (4%) -3.063 2.596±0.367 11.884 34.783 49.855 <0.0001 15.123 47.113 18.943 76.134 malathion (5%) -3.144 2.789±0.338 10.911 29.906 68.032 <0.0001 13.397 38.582 16.235 56.021 lambda-cyhalothrin (0.05%) -2.501 3.278±0551 4.084 10.012 35.296 <0.0001 5.794 14.253 8.221 20.292 propoxur (0.1%) -3.060 2.579±0326 12.375 36.518 62.416 <0.0001 15.351 48.183 18.811 72.833 fig. 2. comparison of regression lines, equations and lt50 of phlebotomus kandelakii and ph. perfiliewi transcaucasicus as exposed to ddt (4%), malathion (5%), propoxur (0.1%), and lambda-cyhalothrin (0.05%) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 281 http://jad.tums.ac.ir published online: september 30, 2020 table 2. lethal time , lt50 and lt90 values for phlebotomus perfiliewi transcaucasicus to ddt (4%), malathion (5%), lambda-cyhalothrin (0.05%) and propoxur (0.1%), in ardabil province, iran, 2019 insecticide a b±se lt50±95% ci (minutes) lt90±95% ci (minutes) wald statistic p-value ddt (4%) -2.621 2.436±0.402 8.703 27.897 36.682 <0.0001 11.901 39.949 15.725 73.761 malathion (5%) -3.207 2.690±0.431 11.757 32.870 38.864 <0.0001 15.565 46.619 20.495 84.098 lambda-cyhalothrin (0.05%) -3.430 4.506±0.908 4.467 8.821 24.606 <0.0001 5.772 11.111 7.059 17.158 propoxur (0.1%) -3.089 2.568±0.404 12.221 35.565 40.336 <0.0001 15.944 50.291 20.679 90.236 table 3. susceptibility tests using the who bioassays for phlebotomus kandelakii and ph. perfiliewi transcaucasicus in endemic foci of visceral leishmaniasis in the northwest of iran, 2019 insecticide exposer time (min) ph. kandelakii ph. perfiliewi transcaucasicus no. of sandflies exposed no. of sandflies dead mortality rate (%) no. of sandflies exposed no. of sandflies dead mortality rate (%) ddt (4%) 3.75 20 1 5 18 3 16.67 7.5 26 5 19.23 21 5 23.80 15 31 16 51.61 19 11 57.89 30 29 24 82.75 22 19 86.36 60 34 31 91.17 24 23 95.83 malathion (5%) 3.75 29 2 6.89 21 1 4.76 7.5 32 4 12.5 17 3 17.64 15 38 26 68.42 19 9 47.36 30 35 30 85.71 20 17 85 60 44 41 93.18 22 20 90.90 lambda cyhalothrin (0.05%) 3.75 24 3 12.5 18 2 11.11 7.5 26 21 80.76 21 18 85.71 15 31 30 96.77 25 23 92 30 28 26 92.85 19 19 100 60 38 38 100 26 26 100 propoxur (0.1%) 3.75 26 2 7.69 18 1 5.56 7.5 34 8 23.52 24 4 16.67 15 32 14 43.75 21 11 52.38 30 40 29 72.50 26 20 76.92 60 42 41 97.61 23 21 91.30 control 110 2 1.81 105 3 2.85 discussion since ardabil province, along with east azerbaijan, is known as the primary focal point of vl in iran (24) and 100–300 new cases of the disease are recorded annually (5), most of which are from the northwest (25), care must be taken against any sudden epidemics. one http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 282 http://jad.tums.ac.ir published online: september 30, 2020 way to control the epidemic is to use suitable and safe insecticides to kill sand flies (22). several trials have been performed in separate areas of iran on vectors of cutaneous leishmaniasis, including ph. papatasi and ph. sergenti, and it has been verified that ph. papatasi is susceptible to the assessed insecticides (2629), except tolerance to ddt in isfahan province, central iran (30). furthermore for the first time, resistance against ddt was observed in wild strain of p. sergenti population in north khorasan province, iran (14, 27). according to the results of current study, lambda-cyhalothrin 0.05% with 100% lethality had the greatest impact on ph. kandelakii and ph. perfiliewi transcaucasicus in ardabil region. this revealed that these vectors are extremely susceptible to this insecticide and can be used to regulate vectors at the period of the epidemic. the results of the lambda-cyhalothrin (0.05%) insecticide in brazil against lutzomyia longipalpis, the main vector of vl in the americas, showed that this vector was also highly sensitive to insecticides (31). the ph. kandelakii and ph. perfiliewi transcaucasicus vectors’ resistance to insecticides has not been recorded to date in iran. in 1994, susceptibility tests were conducted on ph. kandelakii and p. perfiliewi transcaucasicus, in ardabil province (northwest), iran. the results revealed that the both species were susceptible to the insecticides and mortality rate with 60min exposure to 4% ddt was 100%. in india, in numerous areas, ph. argentipes showed resistance to ddt (32-34), but was susceptible to malathion and deltamethrin with 98% and 100% mortality, respectively (20). the resistance of the main vectors of vl against ddt (4%) and deltamethrin (0.05%) was reported in borderline of nepal (17). the reports on insecticide susceptibility status of ph. kandelakii and ph. perfiliewi transcaucasicus are limited and there are no records of insecticide resistance till now. according to results of current study and the who guidelines for malaria vectors at the diagnostic dose ph. kandelakii and ph. perfiliewi transcaucasicus, proven vectors of visceral leishmaniasis in northwestern iran were possible resistance to propoxur (0.1%), malathion (5%) and ddt (4%) insecticides. so the results of earlier studies were contradictory to this study. phlebotomus kandelakii distributed in some important vl foci of iran and leishmania infection reported previously so further studies should be conducted in other regions (8). the strength of this study was that we conducted insecticide susceptibility status of two main vectors of vl in an endemic area of iran with four different kinds of insecticides and in five different logarithmic times but because of limitation, the enough amount of sand flies collection was not possible. also there are specific guidelines approved by who for diagnostic dose of mosquitoes, and there are no such guidelines for phlebotominae sandflies; however, it is of great value to establish a baseline data and to assess the insecticide susceptibility to different insecticides in sand fly vectors. there are several novel investigations of main vector borne diseases in the country (3538). evaluation of diseases monitoring is a vital responsibility of corresponding ministries. conclusion the findings of this study showed that according to the criteria for resistant level of ph. kandelakii and ph. perfiliewi transcaucasicus, the proven vectors of vl in northwestern iran were susceptible to lambda-cyhalothrin (0.05 %), however these species are possible resistance to propoxur (0.1%), malathion (5%) and ddt (4%) insecticides. regular monitoring for the resistance of ph. kandelakii and ph. perfiliewi transcaucasicus sand fly populations are necessary for vl control programs in this area. furthermore, these insecticides should also be assessed for ph. papatasi and ph. sergenti, as the main vectors of cl in endemic foci in iran. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 277–285 y rassi et al.: insecticide susceptibility status of … 283 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hosseini-vasoukolaei n, oshaghi ma, shayan p, vatandoost h, babamahmoudi f, yaghoobi-ershadi mr, telmadarraiy z, mohtarami f (2014) ana-plasma infection in ticks, livestock and human in ghaemshahr, mazandaran province, iran. j arthropod borne dis. 8(2): 204–211. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=shirani-bidabadi%20l%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/?term=zahraei-ramazani%20a%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/?term=yaghoobi-ershadi%20mr%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/?term=rassi%20y%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/?term=akhavan%20aa%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/?term=akhavan%20aa%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/?term=oshaghi%20ma%5bauthor%5d&cauthor=true&cauthor_uid=28870534 https://www.ncbi.nlm.nih.gov/pubmed/28870534 https://www.ncbi.nlm.nih.gov/pubmed/28870534 https://www.ncbi.nlm.nih.gov/pubmed/22397726 https://www.ncbi.nlm.nih.gov/pubmed/15239308 https://www.ncbi.nlm.nih.gov/pubmed/16295685 https://www.ncbi.nlm.nih.gov/pubmed/26354219 j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 68 http://jad.tums.ac.ir published online: march 31, 2020 original article effects of resident education and self-implementation of integrated pest management strategy for eliminating bed bug infestation in ahvaz city, southwestern iran ismaeil alizadeh1,2; elham jahanifard1,3; *mona sharififard1,3; mohammad ebrahim azemi4 1 infectious and tropical diseases research center, health research institute, ahvaz jundishapur university of medical sciences, ahvaz, iran 2 department of biology and vector control, school of public health, kerman university of medical sciences, kerman, iran 3 department of medical entomology and vector control, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran 4 department of pharmacognosy, medicinal plant and natural product, research center, school of pharmacy, jundishapur university of medical sciences, ahvaz, iran (received 01 aug 2018; accepted 12 mar 2020) abstract background: bed bugs as blood-sucking insects have become a public health problem in urban communities throughout the world. the objective of this study was to determine the effects of resident education and self-implementation of integrated pest management (ipm) strategy for eliminating bed bug infestation in infected apartments in ahvaz city, iran. methods: this interventional study was conducted in seventy apartments infested with bed bug (cimex lectularius) in ahvaz city, southwest iran, during a 6-month period. the bed bug infestations reported to health centers were inspected visually and confirmed by medical entomology experts. then, the heads of the households were received techniques/ tools of the ipm program and trained by medical entomology experts before self-implementation of control methods. finally, the infected apartments were inspected by the experts at 1, 3, and 6 months after intervention and data were recorded in a checklist. results: from the seventy infected apartments, 57%, 28% and 15% were considered as low, moderate, and high level infestation respectively. the bed bug infestation was eliminated from 53 apartments (76%) after one month and it reached to 62 apartments (88%) by the end of third month. finally, after six months of applying ipm program, bed bugs infestation was eliminated from all infected apartments (100%). residents expressed their 100% satisfaction with applying the bed bug ipm strategy. conclusion: training residents to implement the ipm program can reduce pest control costs, the volume of pesticides consumed, and human exposure to chemical pesticides, resulting in increased human and environmental health and safety. keywords: bed bug; urban pest; integrated pest management; strategy introduction the bed bugs (cimex lectularius and c. hemipterus) as blood-sucking insects have become a serious pest and public health problem in urban communities throughout the world. they affected human health through blood feeding that can lead to pain, itching, secondary infections, loss of sleep, psychological distress and other allergic responses to humans (1-8). these urban pests have adapted to live in close to indoor human environments including houses, hotels, dormitories, sports, workplace environments, and transport systems all over the world during the past decade (1, 2, 5, 9). generally, increased international travels, insecticide resistance, climate changes, lack of awareness, lack of effective monitoring and management tools and the decreased use of broad-spectrum insecticides in human dwellings can be outlined as *corresponding author: dr mona sharififard, email: sharififard-m@ajums.ac.ir http://jad.tums.ac.ir/ mailto:sharififard-m@ajums.ac.ir j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 69 http://jad.tums.ac.ir published online: march 31, 2020 leading reasons involve with the bed bug emerging (2, 5, 9–11). also social interactions between residents, home visits, or exchange of infested furniture are revealed to cause bed bug distribution in recent studies (5, 12, 13). elimination of bed bug infestation is costly and time-consuming because of activity and nocturnal blood feeding, habit of hiding, spreading around the house (5, 14, 15). they are providing to be one of the most challenging urban pests. most people prefer to use chemical insecticides for removing the bed bug infestation due to more convenient application of chemical method compared with other control tools. even if relatively proper insecticides are used, the challenges with pesticide resistance can occur. also, using chemical insecticides directly on furnishings in human dwellings increases the risk of human exposing to pesticides (1, 5). integrated pest management (ipm) approach is considered as a promising, comprehensive and safe strategy for humans and environment recently. it exploits cooperation between residents and professional pest experts to use both chemical and non-chemical tools and technics simultaneously to eliminate of pest infestation (13). non-chemical techniques include heat treatment, vacuuming, mattress encasements, laundering, freezing and heat steam (3, 5, 14, 16, 17). in order to effectively eliminate bed bug infestations, recognition, management, and eradication are the components of successful control program (13). bed bug infestation is increasing in ahvaz city, south west of iran based on health services reports, as well as resident’s reports to department of medical entomology. the average cost of bed bug control is about $ 0.78 per m2 for chemical control based on pest control services reports in ahvaz city in 2019. while the bed bug infestation usually occurs in low-income communities, control by those companies is not cost-effective and the people prefer to apply some conventional control methods by themselves. little studies have been documented the usefulness of ipm strategy for control bed bugs (5, 13, 18, 19). but there are no studies on resident education and self-implementation of ipm strategy for control of bed bug infestation. the objective of this study was to determine the effects of resident training to apply ipm strategy for eliminating bed bug infestation in infected apartments. materials and methods design of interventional study this interventional study was one arm prepost study conducted in 70 apartments with bed bug infestation in ahvaz city, southwest iran during a 6-month period from may 2015 to october 2016. ahvaz city is one of the metropolitan cities in the southwestern of iran, with a population of 1.3 million. it is located in a dry area of iran with subtropical hot desert climate. the infected apartments were located in different parts of the city both in west and east of the karun river (fig. 1). all the apartments were including bedroom, living room, kitchen, and bathroom. at first, a list of the apartments with reported bed bug infestation was prepared from the health centers and department of medical entomology of ahvaz jundishapur university of medical sciences in ahvaz city. inspection of the infected site is the first step of each ipm program (fig. 2). the houses were inspected visually by medical entomology experts to confirm the bed bug infestation (fig. 3). from the reported infected houses, 70 houses were selected randomly for the study. residents of infected apartments were informed about the objectives and procedures of the investigation. all the heads of households signed written consent prior to implementation of the program. a self-administered checklist was used to evaluate the bed bugs infestations level. the bed bug infestation was assessed through the visual observation of live and dead bed bugs, fecal spots, cast skins, and eggs and categorized to three levels of low, medium and high infestation (low: 1–10 live or dead bed bugs, few fecal http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 70 http://jad.tums.ac.ir published online: march 31, 2020 spots and few cast skins; medium: 11–50 live and dead bed bugs, fecal spots and cast skins, high: > 50 live or dead bed bugs, lots of fecal spots and cast skins). bed bugs were collected and preserved in 75% ethanol and sent to the laboratory of medical entomology and vector control for identification. training of residents after inspection, identification of the infestation, signing the consent form, the head of the households were trained by medical entomology experts to control the bed bug infestation by self-applying of ipm strategy. they also provided guidelines to improve the prevention and control programs based on ipm approach. eight techniques/tools and six steps were considered in the bed bug management program are explained in (figs. 4 and 5). totally two hours was spent to train the head of each house in this survey. the steps of t1, t3, t7 and t8 were performed only once by residents in the first week but the use of other steps includes laundering, vacuuming, steam cleaner and wipes were continued several times until bed bug elimination (fig. 4). entomological experts were on-call with the residents until complete bed bug elimination. data collection the infected apartments were inspected by the entomological experts at 1, 3, and 6 months after intervention. the bed bug infestations in each home were recorded in self-administered checklist based on visual inspection. also, after one, three and six months, the residents were interviewed for three questions: (1) do you still have bed bug infestation in your apartment? (2) how effective do you think this program is? (3) how worried you are about bed bug infestation? a total of 241 residents from seventy infected apartments were interviewed. data analysis the data were collected to determine the overall effectiveness of ipm strategy in infected apartments. excel version 2013 software was used to analysis the frequency and descriptive variables. results evaluation of the self-implementation integrated pest management program totally, 70 infected apartments were inspected during this survey. the levels of infestation were recognized as low, moderate and high in 57%, 29% and 15% of infected houses based on initial inspection. the collected specimens were identified as common bed bug (c. lectularius). during the home inspection phase, it was found that the infestations rate of bedrooms and living rooms were 73% and 27%, respectively. also in 38%, 32%, 18% and 14% of the infected houses, the bed bug harborages were crack and cervices, bedding, wooden furniture and wallpaper and belka, respectively (fig. 8). residents of all apartments attended to the integrated bed bug control program in this study (fig. 5). the bed bugs infestations from 53 apartments (76%) were eliminated after one month. in this time 100%, 90% and 30% of apartments with low, moderate and high level of infestations were cleaned from bed bug infestations, respectively. the bed bug infestation was eliminated from apartments with low infestations without using pesticide. then, the bed bug elimination from 62 apartments (88%) was occurred after three months of self-implementation of ipm strategy. in this time 100% and 70% of apartments with moderate and high levels of infestation were cleaned, respectively. finally, after six months of applying ipm strategy by residents, the bed bug infestation was eliminated from all infected apartments (100%) (fig. 6). all residents have high cooperation in this study. in total, all residents expressed their 100% satisfaction with applying the ipm strategy. as shown in (fig. 6), elimination of bed bugs from apartments with low and moderate level of infestation occurred faster than those with high bed bugs infestations. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 71 http://jad.tums.ac.ir published online: march 31, 2020 resident’s interview during this survey, 286 people were lived in the infested apartments. from them, 241 people with the age of > 16 years were interviewed. all of them were satisfied with ipm strategy as an effective approach for bed bug elimination. bed bug bite signs were observed in 23% of the residents. among the interviewed resident, 82% were very concerned, experienced sleep loss, and psychological distress, 10% of them had a somewhat concern and 8% showed no concern. fig. 1. study areas in ahvaz city, southwestern iran fig. 2. different steps of running an integrated pest management program http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 72 http://jad.tums.ac.ir published online: march 31, 2020 fig. 3. (a) and (b) infested living room, (c) common bed bug colony over the bed, (d) bed bug infestation under wallpapers (f) visual inspection by medical entomology expert fig. 4. techniques/tools of the integrated bed bug control program http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 73 http://jad.tums.ac.ir published online: march 31, 2020 fig. 5. flowchart of applying the ipm program in bed bug infested houses in ahvaz city, southwest iran fig. 6. (a) vacuuming, (b) infested belka in living room, (c) vacuum airless bag, (d) sealing wall cracks and crevices, (e) discarded wooden furniture, photo by ismaeil alizadeh http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 74 http://jad.tums.ac.ir published online: march 31, 2020 fig. 7. bed bugs infestations rate in different sources of the infected apartments fig. 8. effect of integrated pest managaemnt (ipm) on bed bug eliminating rate with low, moderate and high level of infestation discussion the role of education in raising knowledge of the public and consequently reducing the prevalence rate of vectors and vector-borne diseases is completely clear. a study by alizadeh et al. (2018) in ahvaz city reported residents' knowledge about transmission of bed bug, medical importance, habitat, infestation control, prevention of bed bug were 36.7%, 13.3%, 35%, 75% and 66.7% respectively (20). most people get their necessary information by searching the internet, but they are not practically familiar with pest control techniques. our study is the first documented practically application of selfimplementation ipm strategy in order to eliminating bed bug infestation in iran. the purpose of this study was to examine the overall effectiveness of ipm approach to removing bed bug infestation, not to evaluate specific techniques/ tools. the ipm program was attributed to five practices including: a) inspection of infected apartments, b) identification of insect species, (c) monitoring the infestation level, d) action http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 75 http://jad.tums.ac.ir published online: march 31, 2020 including training residents to apply the ipm techniques/tools according to protocol, (e) evaluation the effectiveness of control program. education of residents regarding biology, behavior, medical importance of bed bug, and what actions should be to take for infestation removing is an important component of any ipm effort in multifamily housing (13). bed bug infestation was eliminated completely (100%) using non-chemical techniques/tools in houses with low infestation in our study. this finding is confirmed by wang et al. 2016, cooper et al. 2.16. they also reported that residents training, non-chemical techniques/tools, and periodic monitoring of bed bug can effectively remove light bed bug infestations (5, 13). although the ipm strategy is time consuming, reducing the use of pesticides and exposing them to humans are two of the main goals of an ipm program that can offset the time delay. generally, integrated pest management strategy which uses various non-chemical techniques/ tools before chemicals becomes more prevalence and promising in last decades. furthermore, ipm considered as practical strategy in urban environment where bed bug control is challenging in low-income communities and multiunit housing building (18). due to the high cost of bed bug elimination (about $ 0.78 per m2) by pest control services, residents in low-income communities prefer to control the infestation themselves. therefore, the role of education of residents and their involvement in the implementation of the pest control program can reduce their costs (1). a few studies have documented the effectiveness of ipm strategy for reducing or eliminating of bed bug infestation around the world (1, 5, 9, 13, 19). ongoing education and commitment of the housing residents will play an important role in program success and elimination of bed bug infestation (13). we continued our inspection and training weekly until complete clean-up of the houses. these actions resulted in complete removing of bed bug infestation after one, three and six months in 76%, 88% and 100% of the houses, respectively. in the study of cooper et al. (2015), bed bug infestation was eliminated from 52 apartments out of 62 treated apartments using ipm strategy and the mean bed bug counts was reduced to 96% after 6 month of inspection treated apartments. moreover, the infestation rate was decreased from 15 to 2.2% after 12 months (13). the effectiveness of ipm strategy for deleting bed bug infestation was reported by all the residents. the results of our study are also consistent with wang et al. (2019) which reported ipm program as a much more effective approach for building-wide control of bed bugs than conventional pest control service(19). generally, the failure of ipm program and the prolonged infestation are probably related to not completely follow-up the control instruction, lack of resident cooperation (5, 10, 13, 21) or bed bug re-infestation of the houses. additionally, physical disabilities, financial problems, no concern about bed bug infestation and resident`s distrust to non-chemical methods can be the reasons of lack cooperation of residents (9). continuous monitoring and evaluation of control operations are two key factors in assessing the effectiveness of every ipm program (19). close supervision of medical entomology experts until the full bed bug elimination have been the strengths of this study in addition to high level of resident’s cooperation. residents' home attendance during weekly monitoring was one of the drawbacks of the program, making it difficult to accurately estimate the infestation level and quality of the program execution. also, the reduction in bed bug infestation during the implementation of the program was not quantitatively estimated. conclusion the present study provides first evidence for using ipm strategy in control bed bugs infestation in iran. this study was focused on training the residents of infected apartments to eliminate http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 76 http://jad.tums.ac.ir published online: march 31, 2020 the bed bug infestation using ipm strategy. in addition, this present research provided a good model that is effective for self-implementation in infected apartments suffering from bed bug infestations in ahvaz city. in conclusion, to get rid of the bed bugs infestations, residents must have high motivation and cooperation in this model. the use of sticky trap and dusts (silica gel or diatomaceous earth± pyrethrum and pbo) (17) in combination with the ipm strategy is suggested in future studies. we also recommend that residents who involve in controlling this urban pest can be trained by mobile health application (22) to improve knowledge and awareness of ipm strategy and arrive to successful bed bug control. acknowledgement this article is a part of ismaeil alizadeh msph thesis and it was financially supported by ahvaz jundishapur university of medical sciences (ajums). (project no. og-95102) and ethical number: ir.ajums.rec.1394.709. the authors are grateful to dr changlu wang, department of entomology, rutgers university, new brunswick, nj, usa, for his helpful comments on an earlier draft and reviewing the manuscript. we would like to thank all the residents who participated in our study and help us to complete the study. references 1. wang c, cooper r, dhang p (2011) environmentally sound bed bug management solutions. in book: urban pest management: an environmental perspective chapter: four publisher: cab international editors: partho dhang. 18: 44–63 2. bennett gw, gondhalekar ad, wang c, buczkowski g, gibb tj (2016) using research and education to implement practical bed bug control programs in multifamily housing. pest manag sci. 72 (1): 8–14. 3. stedfast ml, miller dm (2014) development and evaluation of a proactive bed bug (hemiptera: cimicidae) suppression program for low-income multi-unit housing facilities. j integr pest manag. 5(3): e1–e7. 4. wang c, singh n, cooper r (2015) field study of the comparative efficacy of three pyrethroid/ neonicotinoid mixture products for the control of the common bed bug, cimex lectularius. insects. 6(1): 197–205. 5. wang c, singh n, zha c, cooper r (2016) bed bugs: prevalence in low-income communities, resident’s reactions, and implementation of a low-cost inspection protocol. j med entomol. 53(3): 639–46. 6. sharififard m, alizadeh i, jahanifard e, wang c, azemi me (2018) chemical composition and repellency of origanum vulgare essential oil against cimex lectularius under laboratory conditions. j arthropod borne dis. 12(4): 387–397. 7. alizadeh i, sharififard m, jahanifard e, rajaei f, mousavian g, mehraghaei m (2018) identification, knowledge, and awareness of people regarding public health nuisance insect (cimex lectularius) in southwest of iran. jundishapur j health sci. 10(3): 1–7. 8. alizadeh i, sharififard m, jahanifard e (2017) allergic reactions and dermatitis to common bed bug bites: a case report from ahvaz, southwest of iran. iran j dermatol. 20: 65–67. 9. wang c, saltzmann k, bennett g, gibb t (2012) comparison of three bed bug management strategies in a low-income apartment building. insects. 3(2): 402–409. 10. romero a, potter mf, potter da, haynes kf (2007) insecticide resistance in the bed bug: a factor in the pest’s sudden resurgence? j med entomol. 44 (2): 175– 178. 11. zhu f, wigginton j, romero a, moore a, ferguson k, palli r, potter mf, haynes http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 68–77 i alizadeh et al.: effects of resident … 77 http://jad.tums.ac.ir published online: march 31, 2020 kf, palli sr (2010) widespread distribution of knockdown resistance mutations in the bed bug, cimex lectularius (hemiptera: cimicidae), populations in the united states. arch insect biochem physiol. 73(4): 245–257. 12. wang c, saltzmann k, chin e, bennett gw, gibb t (2010) characteristics of cimex lectularius (hemiptera: cimicidae), infestation and dispersal in a high-rise apartment building. j econ entomol. 103(1): 172–177. 13. cooper ra, wang c, singh n (2016) evaluation of a model community‐wide bed bug management program in affordable housing. pest manag sci. 72(1): 45–56. 14. wang c, gibb t, bennett gw (2009) evaluation of two least toxic integrated pest management programs for managing bed bugs (heteroptera: cimicidae) with discussion of a bed bug intercepting device. j med entomol. 46(3): 566–571. 15. potter mf, haynes kf (2014) bed bug nation: is the united states making any progress. proceedings of the 8th international conference on urban pests, 20– 23 july 2014, zurich, switzerland, pp. 51–58. 16. doggett sl, orton cj, lilly dg, russell rc (2011) bed bugs: the australian response. insects. 2(2): 96–111. 17. davies tg, field lm, williamson ms (2012) the re-emergence of the bed bug as a nuisance pest: implications of resistance to the pyrethroid insecticides. med vet entomol. 26(3): 241–254. 18. romero a, sutherland a, gouge d, spafford h, nair s, lewis v, choe dh, li s, young d (2017) pest management strategies for bed bugs (hemiptera: cimicidae) in multiunit housing: a literature review on field studies. j integr pest manag. 8(1): 1–10. 19. wang c, eiden a, cooper r, zha c, wang d (2019) effectiveness of building-wide integrated pest management programs for german cockroach and bed bug in a high-rise apartment building. j integr pest manag. 10(1): 33–38. 20. alizadeh i, sharififard m, jahanifard e, rajaei f, mousavian g, mehraghaei m (2018) identification, knowledge, and awareness of people regarding public health nuisance insect (cimex lectularius) in southwest of iran. jundishapur j health sci. 10(3): 1–7. 21. wang c, singh n, cooper r, liu c, buczkowski g (2013) evaluation of an insecticide dust band treatment method for controlling bed bugs. j econ entomol. 106(1): 347–352. 22. alizadeh i, gorouhi ma, aghaei afshar a, hayati r, mirr i (2019) satisfaction of mobile users with mobile application" identification, prevention, and control of bed bugs": designing and developing mobile health application. j health biomed info. 6(1): 24–31. http://jad.tums.ac.ir/ javascript:; javascript:; javascript:; j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 261 http://jad.tums.ac.ir published online: september 30, 2020 original article establishment and validation of theileria annulata sporozoite ak-93 infection in laboratory-reared hyalomma anatolicum tick using in vivo and in vitro assays *asghar afshari1; gholamreza habibi1; mohammad abdigoudarzi2; fereshteh yazdani3 1department of parasitic vaccines research and production, razi vaccine and serum research institute, agricultural research education and extension organization (areeo), karaj, iran 2department of parasitology, razi vaccine and serum research institute, agricultural research education and extension organization (areeo), karaj, iran 3department of brucellosis, razi vaccine and serum research institute, agricultural research, education and organization extension (areeo), karaj, iran *corresponding author: dr asghar afshari, e-mail: a.afshari@rvsri.ac.ir (received 15 jun 2019; accepted 27 sep 2020) abstract background: tropical theileriosis caused by theileria annulata is a tick-borne disease which transmitted by the ixodid tick members of the genus hyalomma. studies on different aspects of disease require to access infective sporozoite of parasite which produced by tick vector. this study was carried out to establish of t. annulata life cycle to achieve t. annulata infected ticks. methods: laboratory rabbit and calf were used for rearing of hyalomma anatolicum different instars. unfed nymphs were fed on t. annulata infected calf. clinical signs, giemsa stained smears and polymerase chain reaction (pcr) methods were used for detection of infection in blood and tick specimens. susceptible calf was used for confirmation of sporozoites maturation and infectivity in bioassay test. results: hyalomma anatolicum two and three-host strategies of life cycle was lasted 90 and 116 days respectively. the pcr confirmed t. annulata infection in blood and tick samples. maturation of t. annulata sporozoites was confirmed in bioassy test. first clinical symptom of disease was seen earlier in the case of transmission of disease through feeding of live ticks in comparison with blood injection method. conclusion: complete life cycle of t. annulata was done and confirmed by clinical signs, microscopic examination, molecular methods and bioassay test. according to published reports to date, this is the first report of establishment of h. anatolicum tick infection with t. annulata using susceptible calf under controlled conditions in iran. keywords: theileria annulata; hyalomma anatolicum; tick rearing; polymerase chain reaction (pcr); bioassay introduction bovine tropical theileriosis caused by theileria annulata is one of the most important tick borne disease in cattle. the disease is transmitted trans-stadially by species of ixodid ticks of the genus hyalomma and it is distributed in tropical and subtropical areas from northern africa, southern europe, the middle east (including iran) to far east (1, 2). complex life cycle of t. annulata involve schizogony and merogony in vertebrate host and sporogony in invertebrate hosts. sporozoites of t. annulata enter the blood stream of host by feeding of inf ected tick on cattle and multiply as schizonts in lymphocytes and macrophages and from these stages merozoites developed in red blood cells that called piroplasm or erythrocytic forms. some of these differentiate to gametocytes. by uptake of blood with ticks, the gamogony and sporogony stages occurred in tick midgut lumen and salivary glands of tick respectively (3). lymph nodes enlargment, fever, anemia and icterus are main clinical signs of disease (4). tropical theileriosis can be controlled by methods such as application of acaricides for copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 262 http://jad.tums.ac.ir published online: september 30, 2020 tick vector control, administration of live attenuated vaccine or by combination of both acaricides and vaccination (1). in case of infected animals the specific treatment against t. annulata is recommended by using parvaquone and buparvaquone (5). hyalomma anatolicum as main vector of t. annulata is predominant species of hyalomma ticks in domestic ruminants in iran (6). succesfull laboratory rearing of h. anatolicum tick using white rabbit has been reported (7). artificial tick feeding has been used in some studies (8, 9). in this study laboratory animals were used as host for nymph and larva of ticks and preservation of tansstadial transmission of t. annulata. sprozoite stage of theileria annulata that present in salivary glands of tick vector is important in parasite life cycle (10). the maturation is necessary for infectivity of sporozites and occurred in tick salivary glands by both incubation at 36–37 °c or feeding on rabbit for 4–5 days (11, 12). infective sporozoites can be used in many studies on different features of disease specially in vaccine efficay test (13). this study was carried out to establish of sexual stage of t. annulata life cycle using laboratory white rabbit and t. annulata infected calf for rearing of different tick instars to achieve h. anatolicum ticks infected by sporozoites of t. annulata ak-93 local strain. although the bovine theileriosis vaccine immunity assay has performed by inoculating cryopreserved t. annulata infected blood stabilates, but here for the first time we tried to install the process of preparation and confirmation of t. annulata infected tick by in vitro molecular methods and in vivo assay by inoculation to susceptible calf. materials and methods parasite a highly virulent t. annulata infected blood ak-93 strain has been previously cryopreserved in cell bank unit of the department of parasite vaccines research and production, razi vaccine and serum research institute, (rvsri). the virulent t. annulata ak-93 strain has been isolated from an infected calf with severe theileriosis from takestan county in qazvin province of iran in 2014. the infectivity and virulence of the ak-93 strain was earlier confirmed by in vivo assay through subcutaneously injection to a healthy intact calf (14). tick rearing the adult engorged female hy. anatolicum tick obtained from department of parasitology, razi vaccine and serum research institute, incubated for oviposition. all incubation conditions in tick rearing experiments were carried out in constant temperature 28 oc and relative humidity 85% unless specifically noted. to avoid fungal contamination the tick was rinsed by distilled water, dried in room temperature and kept in glass tube before incubation. the glass tube outlet closed by cotton for good ventilation. egg hatching after complete egg laying, the dead tick was removed from tube and eggs were incubated in the same condition as mentioned in the case of oviposition. larvae feeding three month old white laboratory rabbit was provided from department of laboratory animal production of razi vaccine and serum research institute 1.5 to 2kg of weight was used for feeding of tick larvae. the ear’s hair of rabbit and also calves completely shaved and cleaned before releasing of larvae on animal’s ears. aproximately 1000 larvae released on rabbit ears using cotton earbags. moulting of larvae the free detached engorged larvae have been collected from rabbit's earbag and kept in glass tube and incubated for moulting. one of six-month-old calves was injected with ak-93 virulent strain of t. annulata for infection induction. another calf was remained safe for future challenge of the infective tick. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 263 http://jad.tums.ac.ir published online: september 30, 2020 experimental infection of calf experimental infection with theileria annulata in calf was done by subcutaneous injection of 20ml of virulent t. annulata infected blood (ak-93 strain) around prescapular area. the clinical features of bovine theileriosis in inoculated calf such as fever and lymph node enlargement were monitored daily. giemsa stained smears prepared from aspirate of swelling lymph node were used for early detection of disease. feeding of nymphs unfed nymphs were divided in two groups, one for feeding on intact calf and another for feeding on t. annulata experimentally infected calf using earbags as described before in the case of larvae feeding. moulting of nymphs both groups of engorged nymphs collected from intact and theileria infected calves were incubated under conditions similar to moulting of larvae. feeding of adults and maturation of sporozoites adult male and female unfed hyalomma anatolicum ticks that obtained from moulted nymphs were used for rearing of next generation of tick by feeding on intact calf, as well as for t. annulata sporozoites maturation in tick’s salivary glands by incubation at 37 oc, relative humidity 85% for 3 days in the case of ticks that their nymphal stage had been fed on theileria infected calf. bioassay test unfed adult t. annulata infected ticks were fed on intact six-month-old calf for confirmation of sporozoite maturation as bioassay test and the remainder ticks were used for stabilate preparation that could be applied in other future studies. animal working all the procedures were accomplished in accordance with animal care and ethics committee (acec) of razi vaccine and serum research institute instructions. there was no deviation of planned protocols and working methods and no animal wasting in research was occurred. in the last part of the tests, all rabbits and calves were dismissed according to the standard protocols. molecular test primer design two sets of primers were used: (i) tams (theileria annulata merozoite surface antigen) for confirmation of theileria annulata infection in calf blood and nymph specimens and (ii) haec (hyalomma anatolicum excavatum calreticulin gene) for pcr and reverse transcription polymerase chain reaction (rtpcr) internal control of hyalomma ticks. the forward and reverse haec primers designed from two exons of mentioned gene (in order to produce two different pcr product lengths) on genomic dna and cdna in pcr/rt-pcr reactions (table 1). dna extraction phenol-chloroform method were used for genomic dna extraction from blood and ticks samples (15). each tick was grounded by mortar and pestle in 2ml of pbs (phosphate-buffered saline) and supernatant was used for dna extraction. rna extraction and cdna synthesis total rna was extracted from the tick stabilate using the one step rna reagent (ytzol pure rna, yekta tajhiz azma, iran) in accordance with the manufacturer’s instruction. dnase treatment was done to assure the rna response in rt-pcr assay (dnase i, jena bioscience, germany). the cdna synthesis and further pcr were performed using the mmulv reverse transcriptase and pcr mix (yekta tajhiz azma, iran) according to the manufacturer’s instructions. briefly, 2μg of total dnase treated rna and 160pm of oligo (dt) 15 primer were incubated at 70 oc for 5 minutes http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 264 http://jad.tums.ac.ir published online: september 30, 2020 and then 4ul rt buffer 5x, 20u rnase inhibitor, 200 units of m-mulv reverse transcriptase, 500μm each of dntp were added in a total volume of 20μl. the reaction was incubated at 42 oc for 1 hour, followed by 10min at 70 oc to inactivate the enzyme. pcr and rt-pcr dna and cdna obtained from nymphal stages of h. anatolicum ticks before and after feeding on experimentally infected calf were used in pcr and rt-pcr reactions. each reaction preformed in total volume of 20μl containing 10μl of master mix (yekta tajhiz azma, iran), 7μl of depc water, 1μl of each forward and reverse of mentioned primers and 1μl of dna and synthesized cdna in pcr and rt-pcr reactions, respectively. both pcr andrt-pcr cycling started an initial denaturation at 94 oc for 2min followed by 38 cycles of denaturation at 94 oc for 30sec, annealing at 60 oc for 1min and extension at 72 oc for 1min. final extension preformed at 72 oc for 7 minutes. expected lenght of pcr and rtpcr products were detected by running on 1.5% agarose gel for 60 minutes. results tick rearing the adult h. anatolicum preoviposition and complete ovipostion periods lasted 7 and 14 days respectively. the maximum egg laying occurred in days 4–7 of oviposition period. the egg hatching was initiated after incubation for 7 days and lasted for 12 days. the resulted larvae were kept for 8 days for pre-feeding period. during larvae feeding on rabbit's ear, two and three-host behaviors were seen. due to needs for unfed nymphs according to transstadial transmission nature of theileria parasites, to get unfed infected adult ticks for bioassay test, some engorged larvae detatched manually from rabbit's ear in day 6 after releasing and remainder larvae moulted to nymphs on rabbit and engorged nymphs dropped and collected in day 13 of feeding period. after incubation, the resulted unfed nymphs and adults were fed on experimentally infected and intact calves respectively for 8 days. the obtained engorged nymphs and adults were incubated for moulting and oviposition. complete two and three-host life cycle of h. anatolicum tick lasted 90 and 116 days. some stages of tick rearing are shown and results of tick rearing in two and three-host manner were summarized in table 2. experimental infection of calf absence of previous exposure to t. annulata in calf was confirmed with pcr using species specific tams primers (table 1). prescapular lymph node enlargement were observed in 15th day after injection of infected blood. the schizont stage of t. annulata was detected using giemsa stained biopsy smear (fig. 2 left). parasitemia (fig. 2 right) and fever above 40 °c (up to 42) were observed at day 20 post infection. polymerase chain reaction and reverse transcription polymerase chain reaction the expected pcr/rt-pcr products resulted from tams (597bp) and haec (480/ 330bp) primers on dna/cdna of nymphs samples before and after infection with t. annulata were seen in agarose gel (fig. 3). bioassay test theileria annulata infected nymphs moulted to unfed adults after incubation. additionally, to maturation of sporozoites of t. annulata in tick salivary glands, the adult ticks incubated at 37 oc and rh 85% and then three males and four females ticks were used for confirmation of sporozoite maturation by in vivo bioassay test using intact calf (fig. 4) and remaindor tick will used in stabilate preparation according oie's instruction for vaccine efficacy test. the absence of theileria infection in experimental calves was confirmed by specific pcr assay. clinical signs such as lymph node enlargement and fever occurred http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 265 http://jad.tums.ac.ir published online: september 30, 2020 around in days 10 and 11 after releasing of ticks, however, the t. annulata infection was verified by pcr and giemsa blood and lymph node smear staining. the clinical signs of theileriosis in experimental calves using two employed methods were summarized in table 3. table 1. specifications of primers used in this study primer sequence 5'-3' expected size of product genbank reference accession no. pcr rt-pcr tamsf gag aca agg aat att ctg agt cc 597bp 597bp z48739.1 tamsr tta agt ggc ata taa tga ctt aag c haecf cat ttt gct tcc cct ggt cgg 488bp 330bp ay395259.1 haecr ctg cag tcg aac agc ttg agg table 2. duration of rearing of tick's different developmental stages developmental stage duration (day) two-host manner three-host manner adult female preoviposition 7 7 oviposition 14 14 pre-hatching 7 7 hatching 12 12 larvae pre-feeding 8 8 larvae feeding 13 6 larvae pre-moulting 5 larvae moulting 7 nymph pre-feeding 10 nymph feeding 8 nymph pre-moulting 7 9 nymph moulting 7 9 adult pre-feeding 7 7 adult feeding 8 7 total life cycle 90 116 fig. 1. different stages of hyalomma anatolicum tick rearing: a (eggs contain larvae), b (feeding of larvae on rabbit), c (engorged larvae), d (nymph and adult feeding on calf), e (engorged nymphs before molting) and f (adult tick feeding on calf's ear) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 266 http://jad.tums.ac.ir published online: september 30, 2020 table 3. comparative clinical signs of theileriosis using two infecting methods method of infection day of observation onset of clinical signs lymph node enlargement fever prasitemia subcutaneously injection of blood 15 19 20 feeding of live infected ticks 10 11 13 fig. 2. theileria annulata in giemsa stained lymph node (left) and blood smear (right). macroschizont in infected leukocytes and merozoites in rbcs are shown in lymph node biopsy and blood smears respectively fig. 3. agarose gel electrophores of tams (1, 3, 6, 8) and haec (2, 4, 7) products in pcr (1–4) and rt-pcr (6-9) reactions on hyalomma anatolicum nymphs before (1, 2) and after (3, 4, 6, 7), theileria annulata vaccine strain as positive control (8), no cdna as negative control (9) and 100 bp dna ladder (m) fig. 4. adult unfed infected hyalomma anatolicum male and female ticks in bioassay test discussion here in this study, t. annulata sporozoite ak-93 infection was established and validated in laboratory-reared hy. anatolicum tick vector by in vivo and in vitro techniques. since, there is no appropriate laboratory animal model for bovine theileriiosis, the susceptible taurine calves breeds are used for in vitro establishment of parasite’s life cycle. many efforts were performed to feed tick larvae on calf’s ear in this study, but were not successful. however, the laboratory rabbits were used for feeding of larval stage and calves for nymph and adult stages of h. anatolicum tick. the results of laboratory tick rearing studies rehttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 267 http://jad.tums.ac.ir published online: september 30, 2020 vealed that h. anatolicum tick has two-host behavior (7). a similar behavior was observed here in this study. depending on the geographical distributions of hyalomma in iran, the minimum duration of complete life cycle of h. anatolicum ticks have been reported from 76–100 days (16). in the present study, according to the stable conditions for temperature and relative humidity and regardless of times elapsed for prefeeding periods, the complete life cycle for h. anatolicum continued 75 and 91 days in two and three-host behaviors respectively. in addition, as we have to provide unfed nymphs to further feeding on experimentally t. annulata infected calves, the engorged larvae were detached and collected manually from rabbit’s ear and this physical intervention resulted in prolongation of complete life cylce in three-host manner. molecular detection of t. annulata infection using specific primers designed for various genes such as tams (17), internal transcribed spacers (its1-its2) (18) and sporozoite surface antigen (spag) (19). in the present study, the species specific tams primers were used for detection of t. annulata infection in calves and tick specimens. additionally, rt-pcr reactions using tams and haec genes were carried out in order to confirm the presence of active parasite infection (sporozoites formation) in prepared samples. it should be noted that the calreticulin is a multifunctional gene that expressed in all eukaryotic cells and has a 158bp intron spanned between two exons in h. anatolicum (20). this specific character is useful for rt-pcr assay. moreover, giemsa-stained blood and lymph node needle biopsy smears were also examined for early detection of t. annulata infection in calves. in acute tropical theileriosis, death may be occurred between 5 and 7 days after onset of clinical signs in calves (21). thus, early detection of infection is important for well-timed releasing of nymphs on infected calf according to the required time for nymphs feeding. sporozoite maturation could be achieved in tick's salivary glands by two methods; feeding of infected ticks on vertebrate hosts for several days or incubation of infected ticks at 36– 37 oc in laboratory condition. however, it has been reported that the sporozoite maturation could be better formed by feeding on live animals than in vitro incubation (12). since, the infected ticks supposed to be used in bioassay test, thus the ticks were incubated for 3 days to induce sporozoites maturation. this period has been reported for six days in h. a. excavatum as a very close species to h. anatolicum (11). comparison of clinical symptoms in two methods of disease transmission including injection of t. annulata infected blood and through live infected ticks indicates the severity of disease was more by t. annulata sporozoites transfer via live infected ticks. tropical theileriosis in iran was described for the first time in 1935. control of disease has been performed using live attenuated theileriosis vaccine manufactured by razi vaccine and serum research institute initiated since 1973 and continues to date (1). according to oie’s recommendation, vaccine efficacy test has to be done by challenge test using live infected ticks or t. annulata sporozoite tick stabilate. despite the absence of international agreed standards for the challenge dose size, 5 to 10 females and the same number of infected unfed male hyalomma ticks have to be used for infection of cattle (13). here in this study, we used three male and four female unfed infected adult hyalomma ticks for sporozoite maturation by subjecting to an intact naïve calf in bioassay test. therefore, the presence of clinical signs of theileriosis after feeding of ticks has verified the sporozoite maturation and successful complete formation of t. annulata life cycle in calf and tick under in vitro controlled condition. similar results have been reported in transmission of t. annulata by h. dromedary (22) and theileria hirci by h. anatolicum previously (23). in recent years, due to ethical, economical and practical problems associated with tick rearing using anihttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 268 http://jad.tums.ac.ir published online: september 30, 2020 mals led researchers to use artificial membranes for tick feeding as a new approach (24). hyalomma anatolicum tick adult stage was successfully fed artificially through silicone membrane, although this method did not worked for nymphs, however, replacement of silicone membrane by mouse skin sheet showed satisfactory results (9). although artificial tick feeding was used for adults and a little for nymph stages, but rabbit utilization for rearing of larval stage is unavoidable. according to oie recommendation (13) the efficacy test of theileriosis vaccine must be conducted using live or stabilte of infected tick thus the obtained theileria annulata infected tick from this study will used in vaccine efficacy test in future studies. based on the available published reports to date, this is the first report of establishment of h. anatolicum tick infection with t. annulata under controlled condition in iran. ethics we hereby declare all ethical standards have been respected in preparation of the submitted article and all experiments on laboratory animals were performed according to regulations set by ethical committee of razi institute. acknowledgements the authors thank dr ghader khezrian for providing blood sample. this study was supported by the parasitic vaccine research and production department, razi vaccine and serum research institute. this study was supported by the razi vaccine and serum research institute, iran with the research project code of 2-18-18-94123. the authors declare that they have no conflict of interest. references 1. hashemi-fesharki r (1988) control of theileria annulata in iran. parasitol today. 4(2): 36–40. 2. purnell re (1978) theileria annulata as a hazard to cattle in countries on the northern mediterranean littoral. vet sci commun. 2(1): 3–10. 3. jalovecka m, hajdusek o, sojka d, kopacek p, malandrin l (2018) the complexity of piroplasms life cycles. front cell infect microbiol. 8: 248. 4. shkap v, de vos aj, zweygarth e, jongejan f (2007) attenuated vaccines for tropical theileriosis, babesiosis and heartwater: the continuing necessity. trends parasitol. 23(9): 420–426. 5. mchardy n, wekesa ls, hudson at, randall aw (1985) antitheilerial activity of bw720c (buparvaquone): a comparison with parvaquone. res vet sci. 39(1): 29– 33. 6. nabian s, rahbari s, changizi a, shayan p (2009) the distribution of hyalomma spp. ticks from domestic ruminants in iran. med vet entomol. 23(3): 281–283. 7. ghosh s, azhahianambi p (2007) laboratory rearing of theileria annulata-free hyalomma anatolicum anatolicum ticks. exp appl acarol. 43(2): 137–146. 8. kröber t, guerin pm (2007) an in vitro feeding assay to test acaricides for control of hard ticks. pest manag sci. 63 (1): 17–22. 9. tajeri s, razmi g, haghparast a (2016) establishment of an artificial tick feeding system to study theileria lestoquardi infection. plos one. 11(12): e0169053. 10. mehlhorn h, schein e (1984) the piroplasms: life cycle and sexual stages. in advances in parasitology. adv parasitol. 23: 37–103. 11. samish m (1977) infective theileria annulata in the tick without a blood meal stimulus. nature. 270(5632): 51–52. 12. walker ar, mckellar sb (1983) the maturation of theileria annulata in hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites. vet parasitol. 13(1): 13–21. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 261–269 a afshari et al.: establishment and validation of … 269 http://jad.tums.ac.ir published online: september 30, 2020 13. oie terrestrial manual (2018) chapter 3.4.14. theileriosis. pp: 1185–1209 https://www.oie.int/fileadmin/home/en g/health_standards/tahm/3.04.14_thei lieriosis.pdf 14. modirrousta h, habibi g, shayan p, afshari a, mirjalili a, abdigoudarzi m (2019) determination of the infectivity of cryopreserved theileria annulata sporozoites in tick derived stabilates iran ak-93 strain, by in vivo and in vitro methods. iran j parasitol. 14(4): 572–583. 15. sambrook j, fritsch ef, maniatis t (1989) molecular cloning: a laboratory manual. proc natl acad sci. usa, 74: 5463–5467. 16. abdigoudarzi m, rivaz s (2011) tick rearing studies of hyalomma anatolicum anatolicum (acari: ixodidac) and preparing of its life cycle data at laboratory conditions. vet j. 24(1): 1–12. 17. habibi gr, esmaeil-nia k, bozorgi s, najjar e, hashemi-fesharki r, bordbar n (2007) pcr-based detection of theileria infection and molecular characterization of tams1 t. annulata vaccine strain. arch razi inst. 62(2): 83–89. 18. aktas m, bendele kg, altay k, dumanli n, tsuji m, holman pj (2007) sequence polymorphism in the ribosomal dna internal transcribed spacers differs among theileria species. vet parasitol. 147(3–4): 221–230. 19. schnittger l, katzer f, biermann r, shayan p, boguslawski k, mckellar s, doreen beyer d, shiels b, ahmed j (2002) characterization of a polymorphic theileria annulata surface protein (tasp) closely related to pim of theileria parva: implications for use in diagnostic tests and subunit vaccines. mol biochem parasitol. 120(2): 247–256. 20. xu g, fang qq, sun y, keirans je, durden la (2005) hard tick calreticulin (crt) gene coding regions have only one intron with conserved positions and variable sizes. j parasitol. 91(6): 1326– 1332. 21. gill bs, bhattacharyulu y, kaur d (1977) symptoms and pathology of experimental bovine tropical theileriosis (theileria annulata infection). ann parasitol hum comp. 52: 597–608. 22. mazlum z (1969) transmission of theileria annulata by the crushed infected unfed hyalomma dromedarii. parasitology. 59(3): 597–600. 23. hooshmand-rad p, hawa nj (1973) transmission of theileria hirci in sheep by hyalomma anatolicum anatolicum. trop anim health prod. 5(2): 103–109. 24. tajeri s, razmi gr (2011) hyalomma anatolicum anatolicum and hyalomma dromedarii (acari: ixodidae) imbibe bovine blood in vitro by utilizing an artificial feeding system. vet parasitol. 180(3–4): 332–335. http://jad.tums.ac.ir/ https://www.oie.int/fileadmin/home/eng/health_standards/tahm/3.04.14_theilieriosis.pdf https://www.oie.int/fileadmin/home/eng/health_standards/tahm/3.04.14_theilieriosis.pdf https://www.oie.int/fileadmin/home/eng/health_standards/tahm/3.04.14_theilieriosis.pdf j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 362 http://jad.tums.ac.ir published online: december 31, 2019 original article toxicity of ocimum basilicum and ocimum gratissimum extracts against main malaria vector, anopheles gambiae (diptera: culicidae) in nigeria kayode david ileke1; *jacobs mobolade adesina2,3 1department of biology, school of science, federal university of technology, akure, ondo state, nigeria 2department of crop, soil and pest management technology, rufus giwa polytechnic, owo, ondo state, nigeria 3insect chemical ecology laboratory, institute of bioresources and sustainable development, department of biotechnology, govt. of india, takyelpat, manipur, india (received 20 june 2018; accepted 13 oct 2019) abstract background: anopheles gambiae (diptera: culicidae) transmit malaria parasite that causes malaria fever in humans, causing millions of deaths every year among infants in tropical countries. this study was undertaken to assess the toxicity of ocimum basilicum and ocimum gratissimum against pre-adult stages and adult malaria vector, an. gambiae and non-targeted aquatic organism, fingerlings of clarias garipienus. methods: ethalonic extracts of o. basilicum and o. gratissimum were prepared according to the method described by who. the larvae and pupae of an. gambiae were exposed to plant extracts for 24h and their mortality was recorded. toxicity of ocimum species on non-targeted organism, fingerlings of c. garipienus was also investigated. results: ocimum basilicum showed remarkably potency against pre-adult stages and adults an. gambiae causing 100% mortality at 0.4% concentration within 24h of treatment. the lc50 and lc90 of o. basilicum were lower than o. grattisimum in all stages of an. gambiae studied. ocimum basilicum and o. gratissimum extracts significantly reduced the number of bites by the vector given a range of 72.25% to 81.75% protection. ocimum species at the tested concentrations did not significantly reduce the number of fingerlings introduced. conclusion: ocimum species at the tested concentrations did not significantly reduce the numbers of non-targeted organisms, fingerlings introduced. therefore, o. basilicum and o. gratissimum could be used to reduce malaria prevalence in the endemic areas of nigeria as it poses no threat to aquatic organisms. keywords: ocimum basilicum; ocimum gratissimum; insecticide; anopheles gambiae; clarias garipienus introduction arthropod insect vectors are blamable for spreading serious human diseases like malaria, encephalitis, yellow fever, dengue and filariasis (1). in africa, malaria is a vector-borne infectious disease that causes the death of infants and this had led to public health concern throughout the tropical and subtropical regions (2). alteration in the natural environment have also contributed to the widespread of the disease and change in behavior of the vector toward chemical insecticides. in the beginning, synthetic chemical insecticide proves to be the only effective means of combating mosquito until its adverse effects on the environment and user health become pronounce (3). malaria vectors have developed a resistant mechanism against chemical insecticides because of continuous application of synthetic products in our environment for their management (1, 4, 5). the problems of these environmental and health hazards limit their success in vector control, which has led to the development of eco-friendly, biodegradable and readily available plant-based mosquitocides with low-cost implication (3, 6, 7). botanicals are considered as one of the harmless sources for controlling insect vectors and stored products pests (8, 9). botanical based insecticides have been reported by many entomologists and parasitologists to control larvae, pupae, and adult mosquitoes (8, 10, 11). ocimum species belong to the family lami *corresponding author: dr jacobs mobolade adesina, e-mail: mobolade72@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 363 http://jad.tums.ac.ir published online: december 31, 2019 aceae. recent research has investigated the health benefits associated with ocimum essential oils. studies revealed the anti-viral, anti-microbial, antioxidant, and anti-cancer properties of the plants (12). hence, in view of an increasing interest in developing plant-based insecticides as an alternative to chemical mosquitocides, this study was undertaken to assess the toxicity of o. basilicum and o. gratissimum against pre-adult stages and adult malaria vector, anopheles gambiae and non-targeted aquatic organism, fingerlings of clarias garipienus. materials and methods mosquito rearing anopheles gambiae mosquitoes were breed in the hatchery laboratory, department of animal and environmental biology, adekunle ajasin university akungba akoko, ondo state, nigeria (7° 28' n, 5° 44' e) and maintained at ambient temperature of 28±2 °c with 12:12 light and dark photoperiod in 75±5% relative humidity. plant materials and extractions ocimum gratissimum and o. basilicum leaves were collected fresh from supare akoko, ondo state (7° 26' 0" north, 5° 43' e). plant leaves were authenticated by plant taxonomist in the plant science and biotechnology department, adekunle ajasin university, akungba akoko, ondo state (7° 28' n, 5° 44' e). the leaves were rinsed in distilled water to remove any form of impurities, air-dried in laboratory and ground into powder. about 150g of o. gratissimum and o. basilicum leaves powders were soaked separately in an extraction bottle containing absolute ethanol. stirred occasionally with a glass rod and extraction terminated after 3 days. the resulting mixture was filtered and the solvent was evaporated using a rotary evaporator. extracts were kept in a vial and preserved in the refrigerator until further use. larval, pupal and adult mortality bioassay bioassay tests were carried out on ocimum species extracts using five different concentrations (0.1%, 0.2%, 0.3%, 0.4% and 0.5%) prepared according to the standard methods recommended by who (13-15). twenty larvae and pupae of an. gambiae were separately introduced into the treated water, solvent treated and untreated water was set as control. each treatment was replicated four times. mortality was observed over 24h of treatment. twenty an. gambiae adults were introduced into a test-tube that contain suspended filter papers soaked with 0.1%, 0.2%, 0.3%, 0.4% and 0.5% ocimum species extracts separately in four replicates for adult bioassay according (2, 3). mortality of adult insect was accessed after 2h of post-exposure. percentage larvae, pupae and adult mortality were corrected (16). mosquito coil toxicity bioassay chemical mosquito coils were mimic to form plant-based coils derived from o. basilicum and o. gratissimum separately. this is done by thoroughly mixed 10ml of 50% concentrated plant materials with 5g coconut shell, charcoal powder and distilled water to form semi-solid material that solidify with time under shade (3). coil toxicity bioassay was evaluated using a glass chamber size 100× 70× 30cm. two control coils were also set up, one made without extracts and the second control made using synthetic chemical as positive control. one hundred 2–3d old adult an. gambiae, fed with 10% sucrose solution was released into the chamber for 60min. the procedure was repeated four times on separate days. mortality of adult insect was accessed after 60min of post-exposure. percentage adult mortality was corrected using the methods described by abbott (16). non-targeted aquatic organism bioassay fingerlings of c. garipienus of not more than 4–5wk old were collected from the hatchery laboratory, department of animal and environmental biology, adekunle ajasin university akungba akoko, ondo state, nigeria (7° http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 364 http://jad.tums.ac.ir published online: december 31, 2019 28' n, 5° 44' e). the fingerlings were acclimatized for 14d in a dechlorinated borehole water. fingerlings were fed twice daily until 24h prior to their exposure to ocimum species in accordance with the recommendation of adekunle ajasin university, akungba akoko ondo state, nigeria ethical committee. twenty fingerlings were exposed to each concentration tested, fed with fish pellets (1mm) and daily monitored for 5d. mortality was recorded daily for four days. they were considered dead when no visible movement was observed when agitated and probe with a sharp object. analysis of data the percentage of mortality of larvae, pupae and adults were calculated and corrected relative to the associated controls using abbott’s formula. lethal concentrations (lc50 and lc90) and their 95% confidence limits were determined using probit analysis (17). result larvicidal, pupicidal and adulticidal activity of ocimum grattisimum and o. basilicum extracts ocimum grattisimum and o. basilicum significantly affects the mortality of an. gambiae larvae at all the concentrations tested (table 1). the toxicity of o. grattisimum and o. basilicum leaves extracts were significantly (p< 0.05) different from the solvent treated and control experiment. ocimum basilicum extract was the most toxic causing 100% mortality at 0.4% concentration within 24h of post-exposure and its effect was not significantly (p< 0.05) different from o. grattisimum extract. ocimum grattisimum and o. basilicum extracts were able to cause 100% mortality at 0.5% concentration within 24h of post exposure and its effect was significantly (p< 0.05) different from solvent treated and control. similarly, o. basilicum caused 100% pupae mortality within 24h of treatment at 0.5% concentration and its effect was significantly not different from o. grattisimum extract who caused 90% pupae mortality (table 2). the results of fumigant toxicity of o. basilicum leaf extract were able to achieved 100% insect mortality at 0.5% concentration while o. grattisimum caused 95% adult mortality after 60 min of exposure period (table 3). ocimum grattisimum and o. basilicum leaves extracts effects were significantly (p< 0.05) different from solvent treated and the control at all tested concentrations. the lc50 and lc90 of o. basilicum were lower than o. grattisimum in all stages of an. gambiae studied (table 4). the lethal concentration of o. grattisimum and o. basilicum to achieve 50% mortality was lower in larvae stage (0.104% and 0.093% respectively) compared to pupae (0.145% and 0.121% respectively) and adult (0.118% and 0.088% respectively) of an. gambiae. similarly, the lethal concentration of o. grattisimum and o. basilicum to achieve 9 % mortality was lower in larvae stage (0.363% and 0.246% respectively) compared to pupae (0.728 % and 0.485% respectively) and adult (0.519 % and 0.305% respectively) of an. gambiae. mosquito coil toxicity smoke toxicity of o. grattisimum and o. basilicum extracts on an. gambiae adults were presented in table 5. ocimum grattisimum and o. basilicum extracts and the positive control (baygon insecticide) were not able to give 100% protection against adult a. gambiae from sucking blood. positive control (baygon insecticide) had the lowest number of fed mosquito (10.5). this is followed by o. basilicum extract which had 6.75 and 11.5 fed mosquitoes in o. grattisimum extract which was not significantly different from other treatments apart from untreated control that recorded 47.5 fed an. gambiae. ocimum grattisimum, o. basilicum extracts and positive control were able to achieve 72.25%, 81.75% and 84.5% protection which was not significantly from each other. toxicity of ocimum basilicum and o. gratissimum extracts on aquatic habitat organism there was no mortality of fingerlings rec http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 365 http://jad.tums.ac.ir published online: december 31, 2019 orded after 2d of water treatment (table 6). however, 10.25% mortality were recorded on fingerlings water treated with 0.5% o. grattisimum and o. basilicum extracts after 3d of treatment. similarly, 10% and mortality of fingerlings were recorded in water treated with 0.4 % o. grattisimum and o. basilicum extracts after 4d of exposure period. at 0.5% concentration, 20% mortality were recorded in fingerlings water treated with o. grattisimum and o. basilicum extracts after 5 d of treatment. table 1. toxicity of ocimum grattisimum and o. basilicum on larvae of anopheles gambiae plant extracts concentration (%) 0.1 0.2 0.3 0.4 0.5 ocimum grattisimum 55.00+3.75b 65.00+3.75b 85.00+3.75b 90.00+4.25b 100.00+0.00b ocimum basilicum 60.00+4.25b 75.00+3.75b 92.50+4.20b 100.00+0.00b 100.00+0.00b solvent treated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a each value is a mean ± standard error of four replicates. means followed by the same letter along the column are not significantly different using duncan’s new multiple range test. table 2. toxicity of ocimum grattisimum and o. basilicum on pupae of anopheles gambiae plant extracts concentration (%) 0.1 0.2 0.3 0.4 0.5 ocimum grattisimum 45.00+3.75b 50.00+4.25b 70.00+4.25b 77.50+4.10b 90.00+4..25b ocimum basilicum 50.00+4.25b 60.00+4.25b 75.00+3.75b 82.50+4.20b 100.00+0.00b solvent treated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a each value is a mean ± standard error of four replicates. means followed by the same letter along the column are not significantly different using duncan’s new multiple range test. table 3. fumigant toxicity of ocimum grattisimum and o. basilicum on anopheles gambiae adults plant extracts concentration (%) 0.1 0.2 0.3 0.4 0.5 ocimum grattisimum 50.00+4.25b 60.00+4.25b 75.00+3.75b 85.00+4.25b 95.00+3.75c ocimum basilicum 60.00+4.25b 75.00+3.75b 85.00+4.20b 95.00+0.00b 100.00+0.00c solvent treated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a each value is a mean + standard error of four replicates. means followed by the same letter along the column are not significantly different using new duncan’s multiple range test table 4. lc50 and lc90 of ocimum grattisimum and o. basilicum extracts an. gambiae plants lc50 (lcl–ucl)% lc90 (lcl–ucl)% larvae o. grattisimum 0.104 (0.007–0.168) 0.363 (0.232–0.494) o. basilicum 0.093 (0.013–0.143) 0.246 (0.165–0.770) pupae o. grattisimum 0.145 (0.025–0.223) 0.728 (0.409–1.047) o. basilicum 0.121 (0.032–0.210) 0.485 (0.273–0.697) http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 366 http://jad.tums.ac.ir published online: december 31, 2019 adults o. grattisimum 0.118 (0.026–0.180) 0.519 (0.330–0.708) o. basilicum 0.088 (0.013–0.140) 0.305 (0.208–0.904) keys: lc lethal concentration, lcl–lower concentration limit, ucl–upper concentration limit table 5. smoke toxicity of ocimum grattisimum and o. basilicum extracts on adults an. gambiae extracts total number of adult mosquitoes fed mosquitoes unfed mosquitoes % protection ocimum grattisimum 50 11.50±1.15a 38.50±2.24b 72.25±3.24b ocimum basilicum 50 6.75±0.08a 43.25±2.44b 81.75±3.18b control i (synthetic insecticide) 50 5.25±0.35a 44.75±2.37b 84.50±3.39b control ii (untreated) 50 47.50+2.20b 2.50+0.04a 0.00 ± 0.00a each value is a mean + standard error of four replicates. means followed by the same letter along the column are not significantly different using new duncan’s multiple range test table 6. toxicity of fingerlings treated with ocimum grattisimum and o. basilicum extracts extracts of plants conc. mortality%±s. e after 1day 2 days 3 days 4 days 5 days ocimum grattisimum 0.1 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.2 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.3 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b 0.4 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.04b 10.00+0.04b 0.5 0.00+0.00a 0.00+0.00a 10.00+0.04b 10.00+0.04b 20.00+1.14b ocimum basilicum 0.1 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.2 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.3 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b 0.4 0.00+0.00a 0.00+0.00a 0.00+0.00a 10.00+0.25b 10.00+0.25b 0.5 0.00+0.00a 0.00+0.00a 10.25+0.04b 10.00+0.02b 20.00+1.14b untreated 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a 0.00+0.00a each value is a mean ± standard error of four replicates. means followed by the same letter along the column are not significantly different using duncan’s new multiple range test discussion the use of plant-derived products as mosquitocides have proved to be an alternative approach to the control of insect vectors since the use of synthetic insecticides have been discouraged due to their food and environmental health concerns, toxicity to untargeted organisms and insect vector resurgence rates have made their exploitation undesirable (18). according to the results of our study, the extracts of the ocimum species caused high mortality of an. gambiae larvae, pupae and adults. the high lethal effects on pre-adults and adults an. gambiae may be ascribed to the active compounds presents in these plants such as alkaloids (8). the lc50 and lc90 of o. basilicum were lower than o. grattisimum in all stages of an. gambiae studied. the lethal concentration of o. grattisimum and o. basilicum to achieve 50% mortality was lower in larvae stage (0.104% and 0.093% respectively) comtable 4. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 367 http://jad.tums.ac.ir published online: december 31, 2019 pared to pupae (0.145% and 0.121% respectively) and adult (0.118% and 0.088% respectively) of an. gambiae. similarly, the lethal concentration of o. grattisimum and o. basilicum to achieve 90% mortality was lower in larvae stage (0.363% and 0.246% respectively) compared to pupae (0.728% and 0.485% respectively) and adult (0.519% and 0.305% respectively) of an. gambiae. larvicidal, pupicidal and adulticidal activity of the ocimum extracts has been collaborated ted by the findings of other studies (1, 8, 19). the authors reported the effectiveness of o. sellio and o. basilicum essential oils on mosquitoes (19). larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent activities towards three mosquito species were evaluated (1). recently, afolabi et al. (8) reported the adulticidal and repellent activity of different extracts of o. caninum and o. gratissimum against adult an. gambiae. our present study, o. basilicum and o. gratissimum extracts significantly reduced the number of bites by the vector given a ranged of 72.25% to 81.75% protection. preliminary study on non-targeted organism, fingerlings of c. garipienus showed that ocimum species at the tested concentrations did not cause lethal effects on the numbers of fingerlings introduced. no previous study on the effects of o. basilicum and o. gratissimum extracts on non-targeted organism, fingerlings of c. garipienus was found in literature. however, the addition of o. americanum to diet of sciaenops ocellapus did not significantly affect the reproductive parameter and whole body composition after 7wk (20). conclusion extracts of o. grattisimum and o. basilicum are promising in disease-vector mosquito’s management. these findings have demonstrated to be one of the alternative approach to manage mosquito vectors than the use of synthetic chemical insecticides that causes adverse effect on humans, environment and on non-target aquatic organisms. further studies should be conducted to describe toxicological and histological effects of ocimum species on non-target organisms, fingerlings of c. garipienus. acknowledgements the authors thank dr obembe of the department of plant science and technology, adekunle ajasin university akungba akoko, nigeria for the identification of botanicals used and mr eniade of hatchery unit, department of animal and environmental biology, adekunle ajasin university akungba akoko, nigeria for the provision of fingerlings of clarias garipienus. the authors declare that there is no conflict of interest. references 1. veena p, tripathi ak, aggarwal kk, khanuja sps (2005) insecticidal, repellent and oviposition-deterrent activity of selected essential oils against anopheles stephensi, aedes aegypti and culex quinquefasciatus. biores technol. 96: 1749– 1757. 2. akinkurolere ro, adedire co, odeyemi oo, raji o, owoeye ja (2011) bioefficacy of extracts of some indigenous nigerian plants on the developmental stages of mosquito (anopheles gambiae). jord j biol sci. 4(4): 237–242. 3. ileke kd, ogungbite oc (2015) alstonia boonei de wild oil extract in the management of mosquito (anopheles gambiae), a vector of malaria disease. j co lif med. 3(7): 557–563. 4. kuntal b, goutam c (2014) phagodeterrence, larvicidal and oviposition deterrence activity of tragia involucrata l. (euphorbiaceae) root extracts against vector of lymphatic filariasis culex quinquefasciatus (diptera: culicidae). asian pacif j trop dis. 4(1): 226–232. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 362–368 kd ileke and jm adesina: toxicity of … 368 http://jad.tums.ac.ir published online: december 31, 2019 5. ajaegbu ee, danga ysp, chijoke iu, okoye fbc (2016) mosquito adulticidal activity of the leaf extracts of spondias mombin l. against aedes aegypti l. and isolation of active principles. j vect bor dis. 53: 17–22. 6. dwivedi sc, kavitha kc (2001) ricinus communis: a potential larvicide for mosquitoes. pestol. 25(5): 48–50. 7. rambabu b, aruna dm, durga pb (2014) larvicidal activity of an indigenous plant, euphorbia nivulia. de pharmacol sini. 1(1): 7–9. 8. afolabi oj, simon-oke i, adepeju eo, oniya mo (2018) adulticidal and repellent activities of some botanical oils against malaria mosquito anopheles gambiae (diptera: culicidae) beni-suef university. j bas appl sci. 7: 134–138. 9. ileke kd, adesina jm, obajulaye eo (2016) synergetic effects of two botanicals entomocides as pest-protectants in maize grains. j biolog res. 89(2): 33–39. 10. hossain e, rawani a, chandra g, mandal sc, gupta jk (2011) larvicidal activity of dregea volubilis and bombax malabaricum leaf extracts against the filarial vector culex quinquefasciatus. asian pac j trop biomed. 4: 436–441. 11. ileke kd, oyeniyi ea, ogungbite co, adesina jm (2015) nicotiana tabacum: a prospective mosquitocide in the management of anopheles gambiae (giles). int j mosquitoes res. 2(4): 19–23. 12. akinyemi ko, oladapo o, okwara ce, ibe cc, fasure ka (2005) screening of crude extracts of six medicinal plants used in south-west nigerian unorthodox medicine for anti-methicillin resistant staphylococcus aureus activity. bmc complement. alternative med. 5: 6–14. 13. who (2006) guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mosquito nets. who bull. 3: 27–39. 14. who (2009) guidelines for efficacy testing of mosquito repellents for human skin. who bull. (4): 1–28. 15. who (2013) larval source management: a supplementary measure for malaria vector control: an operational manual. world health organization, geneva. 16. abbott ws (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–267. 17. finney dj (1971) probit analysis. 3rd ed. cambridge university press, cambridge. 18. ghosh a, chowdhury n, chandra g (2012) plant extracts as potential mosquito larvicides. indian j med res. 135(5): 581–598. 19. padilha de paula j, gomes-carneiro mr, paumgartten frj (2003) chemical composition, toxicity and mosquitoe repellency of ocimum selloi oil. j enthnopharma. 88: 253–260. 20. subili fj, velasquez a, pinheiro cg, heinzmann bm, gatlin iii dm, baldisserotto b (2016) evaluation of ocimum americanum essential oil as an additive in red drum (sciaenops ocellapus) diets. fish shellfish immunol. 56: 155–161. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/27417228 j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 97 http://jad.tums.ac.ir published online: march 31, 2020 original article epidemiology of visceral leishmaniasis with emphasis on the dynamic activity of sand flies in an important endemic focus of disease in northwestern iran ehssan mozaffari1; *hassan vatandoost1,2; *yavar rassi1; mehdi mohebali3; amir ahmad akhavan1,2; eslam moradi-asl4; zabihola zarei3; alieza zahrai-ramazani1; esmail ghorbani4 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3 department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 4 school of public health, ardabil university of medical sciences, ardabil, iran (received 28 dec 2019; accepted 06 mar 2020) abstract background: leishmaniasis diseases are known to be one of the most important public health problems in world and iran. visceral leishmaniasis is considered to be the most serious form and transmitted by sand flies species. the aim of this study was to investigate the dynamic activities of sandflies in northwestern iran. methods: this crass-sectional study was conducted from april to december 2018 in meshkinshahr county, ardabil province. sticky traps have been used to collect sand flies. they are stored in 70% alcohol and finally identified using valid keys. the aspects of the synoptic information were inquired from the meshkin shahr weather department and results analyzed for spss24. results: totally 259 sandflies were collected during study period. from collected samples 78.7% were male and 21.3% female. there were 8 different species. phlebotomus kandelakii was the most prevalent one (30.8%). the average temperature, relative humidity, and average wind speed was 13.5 °c, 84%, and 2 meters per hour on the onset of sandflies’ activity, respectively. these values were 18.3 °c, 85% and 1.5 meters per hour at the peak of their activity and 16 °c, 62% and 5 meters per hour at the final stage of their activity. sand flies had one peak in july which is strongly influenced by temperature and humidity conditions. two species of ph. kandelakii and phlebotomus perfilewi transcaucasicus, had the highest activity in this endemic area. conclusion: the results of current study will provide a guideline for control of diseases in the country. keywords: phlebotomus kandelakii; phlebotomus perfilewi transcaucasicus; visceral leishmaniasis; iran introduction arthropod-borne diseases are known to be one of the most important public health problems. today, more than one third of infections are the direct result of arthropod-borne diseases (1). leishmaniasis is a complex disease which is transmitted by vectors that carrying more than 20 different types of leishmania parasites belonging to the kinetoplastida class (2, 3). leishmaniasis has been reported in more than 101 countries worldwide (4), with more than 350 million people living in risky areas around the world (2, 5). the most important vectors of leishmaniasis were phlebotomus sandflies in the past and are currently lutzomyia sandflies (6). in terms of clinical symptoms, leishmaniasis infection can be classified as cutaneous leishmaniasis (cl), visceral leishmaniasis (vl) and mucocutaneous leishmaniasis (mcl) in which visceral leishmaniasis (also called kala-azar) is considered to be the most serious form (7). visceral leishmaniasis affects between 0.2 and 0.4 million people and causes approximately 40,000 *corresponding authors: dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, dr yavar rassi, email: rassiy@tums.ac.ir http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com mailto:rassiy@tums.ac.ir j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 98 http://jad.tums.ac.ir published online: march 31, 2020 deaths each year (6). visceral leishmaniasis in iran belongs to the mediterranean type with the leishmania infantum agent. dogs and canines have been identified as the main reservoirs of the disease and approximately 100-300 human cases are registered every year in iran (8, 9). to date, a total of 44 sandflies species have been identified, and 10 more species are still under investigation (10). the most recent study in the endemic focus of the province of ardabil has described 22 species of sandflies dispersed throughout the region (11). definitive vectors of visceral leishmaniasis in the province of ardabil belong to the larrossious subgenus, including the species phlebotomus kandelakii, phlebotomus perfilewi transcaucasicus and phlebotomus tobbi (12–14). parasitic infection has also been confirmed in three areas of the province of ardabil: meshgin shahr, germi and bileh savar (15). phlebotomus kandelakii, the main vector of visceral leishmaniasis in northwestern iran, is a mountainous sandfly that lives in special conditions. the study of the ecological behavior and dynamic activities of such vectors may help to prevent and control leishmaniasis diseases (16). the aim of this study is therefore to investigate the dynamic activities of sandflies with an important endemic focus in northwestern iran. materials and methods study area the province of ardabil is located in the north-west of iran. this study was conducted in the district of meshgin shahr, ahmad abad village located in the center of ardabil phlebotomus, from april to december 2018 (fig. 1). more than 50 percent of visceral leishmaniasis cases have been identified in the selected area all through the province. data collection information on cases of disease was obtained from a health center in meshkin shahr. the relevant information regarding temperature, humidity, and wind speed were measured and recorded by maximum/minimum thermometer and silver barometer on a daily basis in 5 different, fixed locations. other aspects of the synoptic information were inquired from the meshkin shahr weather department. a maximum of 60 sticky traps (30 indoors and 30 outdoors) have been used to capture sand flies. sticky traps were installed once every two weeks before the sunset and were collected before the sunrise. trapped sandflies were removed from sticky traps by the insulin syringe and stored in 70% alcohol. they were then transferred to the laboratory, mounted with 1–2 drops of puri's solution and finally identified using valid keys (16, 17). results in this endemic focus, from 2000 to 2018, 30 disease cases were reported of which 60% were male and 30% were female. of these 56% were less than 2 years old, 24% from 2 to 5 years old, and 20% above 5 years of age. using the direct agglutination test (dat), the highest number of cases (32%) was found to be positive with antibody titer 1:3200 and the lowest number (4%) with antibody titer to 1:51200. the highest number of visceral leishmaniasis cases (68%) were confirmed in areas at an altitude of 1310–1380 meters and areas with average vegetation, average tree height, scarp topography, 60% rural population, farming, livestock as the main occupation, and 268 domestic dogs and flocks. on the other hand, the smallest number of visceral leishmaniasis cases have been reported from areas located along the valley topography at an altitude between 1250 and 1300 meters above sea level with dense vegetation and tall trees, 40% urban population, shop keeping as main occupation, and 98 domestic dogs and flocks (fig. 2). a total of 259 sandflies were trapped by sticky traps during the 30 weeks of the study period (april to december 2018) of which 78.7 % were male and 21.3% were female. there were 8 different species identified with ph. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 99 http://jad.tums.ac.ir published online: march 31, 2020 kandelakii as the most frequent one (30.8%), followed by phlebotomus papatasi (28.5%) and ph. perfiliewi transcaucasicus (3%). of those sandflies, 32.8% were trapped in the house yards and 3.4% in the bathrooms. the onset of the activity and appearance of vectors in this region started from the 5th week of study (may 5), the peak of activity started from the 13th and 14th week of study (late july), and the ending point of activity started from 26th and 27th weeks of the study (october). the largest numbers of sandflies were trapped in barns (33%), and the lowest number in the restrooms (3.4%). phlebotomus kandelakii was trapped mostly in bedrooms and barns and ph. papatasi in aviculture and barns. the average temperature, relative humidity, and average wind speed was 13.5 °c, 84%, and 2 meters per hour on the onset of sandflies’ activity, respectively (figs. 3–5). these values were 18.3 °c, 85% and 1.5 meters per hour at the peak of their activity and 16 °c, 62% and 5 meters per hour at the final stage of their activity (table 1). fifty-nine per cent of sand flies are caught in high-risk disease areas and 40.2% from low-risk areas. phlebotomus kandelakii was mostly (80%) trapped in highrisk areas where infection with leishmaniasis has been reported in both humans and animals. during the 12th to 16th weeks of study, 42.5% of the sand flies were caught when the wind speed was 3–4 meters per hour with an average speed of 1–2 meters per hour. the highest recorded temperature in this period was 25–30 °c and the lowest temperature was 12–15 °c; the temperature for suitable activity was 17–18.5 °c. in the peak of activity, the highest humidity was 85–96%, the lowest was 61–84%, and the average of sufficient humidity was 76–86% (table 2). fig. 1. field of research and areas where sandflies are caught in northwestern iran http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 100 http://jad.tums.ac.ir published online: march 31, 2020 table 1. frequency of captured sand flies based on location in the endemic focus of northwestern iran spices male female room bathroom toilet barn birdman depot yard total ph. kandelakii 56 24 16 2 3 35 4 5 15 80 ph. perfiliewi transcaucasicus 6 2 2 1 1 2 0 0 2 8 ph. longidactus 14 0 0 0 4 5 1 3 1 14 ph. balcanicus 20 0 0 0 8 9 0 2 1 20 ph. papatasi 68 6 3 6 5 26 21 8 5 74 ph. sergenti 15 5 0 0 0 0 12 2 6 20 ph. mongolensis 25 0 0 0 3 7 2 3 10 25 ph. caucasicus group 0 18 0 0 4 1 2 2 9 18 total 204 55 21 9 28 85 42 25 49 259 fig. 2. topographic characteristics of the endemic area of visceral leishmaniasis in northwestern iran fig. 3. weekly activity of sand flies based on the temperature of the region in the endemic focus of northwestern iran http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 101 http://jad.tums.ac.ir published online: march 31, 2020 table 2. dynamic activity of sandflies on the basis of weather variables in the endemic area of northwestern iran w e e k ly a c ti v it y m a x w in d s p e e d m in w in d s p e e d m a x t e m p e r a tu r e m in t e m p e r a tu r e m e a n t e m p e r a tu r e a v e r a g e d a il y c lo u d y m a x r e la ti v e h u m id it y m in r e la ti v e h u m id it y m e a n r e la ti v e h u m id it y t o ta l d a il y r a d ia ti o n a v e r a g e d e w p o in t te m p e r a tu r e a v e r a g e w e tb u lb t e m p e r a tu r e a v e r a g e r e la ti v e h u m id it y a t 0 3 o 'c lo c k a v e r a g e r e la ti v e h u m id it y a t 1 5 o 'c lo c k n o . s a n d f ly a c ti v it y 1 6 2.375 15.6 4.4 9.9 2 62 28 44.6 2801 -2.3 4.8 53 41 0 2 6 1.375 18.7 7.1 13.1 2 66 38 54.8 2756 3.9 8.5 63 44 0 3 7 2.125 20.4 9.6 14.4 4.4 75 48 63.1 2147 7.3 10.6 68 54 0 4 6 1.5 17.6 10.8 13.2 4.4 100 64 84 1386 10.3 11.6 81 88 0 s ta r t 5 6 2 17.4 11.6 13.5 4.4 98 72 84.2 1243 10.7 11.9 89 87 1 6 10 1.625 20.4 11 14.5 4.4 84 52 70.6 1822 9.1 11.5 84 62 2 7 14 3.375 22.6 13.4 18.2 4.4 60 35 44.1 2516 5.6 11.5 47 38 6 8 16 4.25 21.7 14.9 17.2 4.4 81 26 46.5 1234 4.8 10.8 46 26 9 9 15 4.125 19.6 11.2 15.6 4.4 55 34 45.2 1292 3.6 9.5 55 48 10 10 14 3.5 19.6 9.4 14.5 4.4 64 29 46.3 2017 2.7 8.7 64 41 14 11 11 1.375 19.2 11 16 4.4 94 56 67.6 1801 6.7 9.6 62 63 16 12 8 1.5 23 12 17 4.4 91 84 85 677 10.2 10.3 100 100 22 13 3 1 28 14 18 4.4 96 82 84 1806 9.4 9.6 100 84 35 p e a k 14 3 1.8 29 14 18.5 4.4 94 72 86 1905 8.6 9.4 98 98 31 15 4 2.4 29.8 15 17.6 4.4 85 61 76.2 1720 8.1 10 83 61 24 16 4 2.6 26 13 16.2 4.4 64 30 49.2 1452 -1.4 4.9 64 30 20 17 3 2.6 23 12 15.5 4.4 52 35 41.6 2160 2.3 9 52 35 14 18 4 2.6 21.9 11 16.8 4.4 45 20 33.6 2058 -4.2 5.2 38 30 19 19 2 2 22.6 12 18 4.4 51 31 40.2 2016 3.9 10.8 51 31 10 20 5 2.6 24.8 12.4 18.4 4.4 100 32 53.8 1906 3.6 9.1 50 100 8 21 3 2 20.8 9.2 17.4 4.4 74 50 65.2 1845 10.5 13.4 71 74 6 22 2 1.2 20.2 12 16.3 4.4 86 65 74 1789 11.58 13.5 68 86 3 23 3 1.4 20.4 11 16.3 4.4 80 58 66.2 2047 9.82 12.6 80 59 4 24 2 1.2 20 12 15.3 4.4 74 44 59.6 2149 7.2 11 59 74 2 25 5 1.4 22 11.4 18.4 4.4 70 49 56.4 2015 9.3 13.2 70 51 2 26 4 2.4 25 13 19.9 4.4 66 33 46.2 2049 7.4 13 66 36 0 e n d 27 7 1.57143 21.6 11.4 16.4 4.4 78 45 62 1980 8.9 12.2 61 56 1 28 4 0.875 24.4 10 17.3 4.4 88 33 60.7 1895 8.8 12.5 86 33 0 29 8 2 18.8 13.8 14.3 4.4 91 66 77.6 1987 10.3 12 77 85 0 30 6 1.25 16 7.4 10.8 4.4 95 71 85.5 2105 8.4 9.5 95 71 0 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 102 http://jad.tums.ac.ir published online: march 31, 2020 fig. 4. weekly activity of sand flies based on the relative humidity of the region in the endemic focus of northwestern iran fig. 5. weekly activity of sand flies based on the wind speed of the region in the endemic focus of northwestern iran discussion visceral leishmaniasis infection cases in the endemic focus of northwestern iran, including ardabil province, have started to rise since 2013 and were up to four times higher in 2018. in addition, canine visceral leishmaniasis has also risen from 4% to 38% in recent years (9, 18). previous studies in this region have already shown that 50% of certain vectors of visceral leishmaniasis in iran have been registered from ardabil province (12–14) with ph. kandelakii as the most common species trapped in the city of meshgin shahr and in the villages of ahmad abad, parikhan, oor-kandi and khalaf (19, 20). the identification of sandflies’ activity areas, determination of monthly frequency, activity season, the appearance and disappearance time of main vectors, and identification of the dynamic activity of vectors in endemic foci are among the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 97–105 e mozaffari et al.: epidemiology of … 103 http://jad.tums.ac.ir published online: march 31, 2020 most important factors that can be taken into account for vector and diseases control in this area (21-24). the appearance of sand flies in this region started from the first half of may, the peak time of their activity was at the end of july, and the final activity period is in the second half of october. however, climate change may affect these periods from 7 to 14 days. the activity period of sand flies lasts between 150–170 days on average per year. nonetheless, it should be noted that these indexes will vary depending on the specific topographical and climatic conditions, as in the northwesterly regions, sand flies have one generation and one peak time, whereas they have 2–3 generations and 2 peak times in the southern regions (16, 25). in most areas of the who emro countries, sand flies transmitting visceral leishmaniasis have one or two peaks of activity and start to act from early april to june (22). most of the sand flies in the current study were caught in the outdoors and previous studies in this region and other areas showed the same findings, indicating that the vectors of visceral leishmaniasis prefer to take blood and rest outside the housing areas (11, 19, 26, 27). the sand flies activity period in the study area was between 13.5 °c and 20 °c, with a relative humidity of 80–86 percent and a wind speed of between 1.5 and 2.5 meters per hour. temperature and humidity conditions that may have a direct impact on the life cycle of the vectors were among the most important factors influencing the development of sand flies and their life cycle (16). in this research, the peak activity of sand flies was found to be exactly 18–18.5 °c and the average humidity was 80–86 percent. two studies for india and brazil have found similar results with respect to the direct effects of temperature and humidity on leishmaniasis vectors development (28-30). conclusion sand flies were active from april to october (5–6 months on average), with on peak in july which is strongly influenced by temperature and humidity conditions. the vectors of visceral leishmaniasis in this study belonged to the larrossius subgenus 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(2012) risk zone modelling and early warning system for visceral leishmaniasis kala-azar disease in bihar, india using remote sensing and gis. isprs int arch photogramm remote sens spat inf sci... xxxix-b8, pp. 71–75. 30. sudhakar s, srinivas t, palit a, kar s, battacharya s (2006) mapping of risk prone areas of kala-azar (visceral leishmaniasis) in parts of bihar state, india: an rs and gis approach. j vector borne dis. 43(3): 115– 122. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=rahola%20n%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=thierry%20m%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=thierry%20m%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=sereno%20d%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=bongiorno%20g%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=bianchi%20r%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=bianchi%20r%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=khoury%20c%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=tsirigotakis%20n%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=dokianakis%20e%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=dokianakis%20e%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=antoniou%20m%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=christodoulou%20v%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=christodoulou%20v%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=mazeris%20a%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=karakus%20m%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=ozbel%20y%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=arserim%20sk%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=arserim%20sk%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=erisoz%20kasap%20o%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=gunay%20f%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=oguz%20g%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=kaynas%20s%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=tsertsvadze%20n%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=tskhvaradze%20l%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=tskhvaradze%20l%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=giorgobiani%20e%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=gramiccia%20m%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=gramiccia%20m%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=volf%20p%5bauthor%5d&cauthor=true&cauthor_uid=26900688 https://www.ncbi.nlm.nih.gov/pubmed/?term=gradoni%20l%5bauthor%5d&cauthor=true&cauthor_uid=26900688 j arthropod-borne dis, december 2019, 13(4): 407–415 t hazratian et al.: baseline susceptibility of … 407 http://jad.tums.ac.ir published online: december 31, 2019 original article baseline susceptibility of culiseta longiareolata (diptera: culicidae) to different imagicides, in eastern azerbaijan, iran teimour hazratian1; azim paksa1; mohammad mahdi sedaghat2; hassan vatandoost2,3; seyed hassan moosa-kazemi2; alireza sanei-dehkordi4,5; yaser salim-abadi6; masoumeh pirmohammadi2; saideh yousefi2; masoumeh amin2; *mohammad ali oshaghi2 1departmemt of parasitology, faculty of medicine, tabriz university of medical sciences, tabriz, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 5infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran 6department of health services and health promotion, school of health, rafsanjan university of medical sciences, rafsanjan, iran (received 25 oct 2019; accepted 30 dec 2019) abstract background: culiseta longiareolata is an important vector for many human diseases such as brucellosis, avian influenza and west nile encephalitis. it is likely an intermediate host of avian plasmodium that can transmit malta fever. the aim of this study was to determine the susceptibility level of cs. longiareolata to different classes of imagicides which are recommended by world health organization . methods: larval stages of the cs. longiareolata were collected from their natural habitats in marand county at east azerbaijan province, northwestern of iran in 2017. adult susceptibility test were carried out with using impregnated papers to insecticides including ddt 4%, cyfluthrin 0.15%, deltamethrin 0.05%, propoxur 0.1% and fenitrothion 1% by standard test kits. results: results showed that cs. longiareolata adult is more susceptible to pyrethroid and carbamate insecticides. among tested insecticides, cyfluthrin was the most toxic against cs. longiareolata with lt50 value of 11.53 minutes and fenitrothion had the least toxic effect (lt50: 63.39 min). conclusions: this study provided a guideline for monitoring and evaluation of insecticide susceptibility tests against cs. longiareolata mosquitoes for further decision making. keywords: baseline susceptibility; culiseta longiareolata; insecticides introduction mosquitoes transmit many important human diseases such as malaria, filariasis, several types of encephalitis, many arboviral diseases and also cause serious nuisance and irritation (1-5). west nile virus has been detected in 62 mosquito species, including genera of aedes, anopheles, culiseta and culex in the united state of america (6-9). about 3500 species of mosquitoes reported worldwide, and approximately 64 of those can be found in iran (10-17). culiseta longiareolata is a vector for brucellosis, avian influenza and west nile encephalitis. these mosquitoes are likely an intermediate host of avian plasmodium and can transmit malta fever (18, 19). the mosquito cs. longiareolata is a common and abundant species in many countries of europe, africa and also asia, such as iran, albania, azores, botswana, bulgaria, canary islands, croatia, cyprus, djibouti, egypt, southern england, ethiopia, france, greece, *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 407–415 t hazratian et al.: baseline susceptibility of … 408 http://jad.tums.ac.ir published online: december 31, 2019 hungary, india, iraq, italy, jordan, lebanon, lesotho, madeira, mauritania, morocco, namibia, pakistan, portugal, romania, russia, slovakia, somalia, south africa, spain, sudan, switzerland, syria, tajikistan, tunisia, turkey, ukraine and yemen (1). eggs and larvae of cs. longiareolata is found mostly in tires, so it can be spread across the world through tire trading (20). culiseta longiareolata is found common in human habitations. the larvae are rarely found in natural waters that are found mostly in temporary pools, rock pools, artificial containers, wooden and metal barrels and tanks built of concrete, which are rich in decaying organic materials (18). early growth stages larvae of cs. longiareolata are more found in shallow areas of pools, whereas late growth stages are found deeper areas of the pools (21). chemical insecticides such as organophosphates, organochlorine, carbamate, and pyrethroid are principal weapon against both adult and larval stages of mosquitoes vectors (2224). increasing and inappropriate use of synthetic insecticides in mosquito control in parallel to pest control agriculture is one of the main causes of increased tolerance and resistance in different species of mosquitoes across the world (25-27). according to reports in recent years, the level of tolerance and resistance of some mosquitoes and other arthropods has increased in some parts of the world which is a major barrier to the success of vector control programs (28-34). in our knowledge, there was no comprehensive study on monitoring the susceptibility level of cs. longiareolata to various insecticides in the world. the aim of this study was to evaluate the susceptibility of cs. longiareolata to five common insecticides recommended by world health organization. materials and methods study area this study was carried out in marand county in east azerbaijan province, northwestern of iran. the county located at latitude 38°42'n, longitude 45°76'e and altitude 1342 meter (fig. 1). bioassay procedure in this experimental study larval stage of cs. longiareolata were collected from larval habitats, then all specimens were transferred to insectary of department of medical entomology and vector control with 27±1 °c temperature, 12:12 light and dark period and 60±5% of relative humidity. adult susceptibility test of mosquitoes were carried out using standard impregnated papers insecticides such as ddt 4%, cyfluthrin 0.15%, deltamethrin 0.05%, propoxur 0.1% and fenitrothion 1%. according to the standard procedures recommended by the world health organization (who). in brief, twenty-five unfed female mosquitoes were exposed to insecticide-impregnated papers at different exposure interval times, moreover for each different exposure time 4 replicates of mosquitos were used and 2 replicates of 25 adult mosquitoes were considered as controls with untreated papers. probit analysis was conducted on mortality data collected after 24 hours exposure to different times of insecticides using finney’s statistical method to determine the lethal time causing for 50% and 90% mortality (lt50 and lt90) values and their 95% confidence limit of upper and lower confidence levels (35-37). the percentage mortality was calculated and corrections for mortality when necessary were done by abbot’s formula (38). according to the who criteria, the susceptibility level of the mosqueitoes was considered in three classes as susceptible, tolerant and resistant. the mortality between 98–100% was considered as susceptible, less than 90% demonstrated resistance and between 90–97% was determined as resistance candidate (36, 39, 40). results table 1 and 2 show the probit regression line parameters for females of cs. longiareolata to http://jad.tums.ac.ir/ https://www.google.com/search?q=organochlorine&spell=1&sa=x&ved=0ahukewjvi7gamrnaahvs3qqkhfwid4aqbqghkaa https://www.google.com/search?q=carbamate&spell=1&sa=x&ved=0ahukewibwpm9mrnaahxscuwkhztoceyqbqghkaa https://www.google.com/search?q=pyrethroid&spell=1&sa=x&ved=0ahukewif7pfpmrnaahuqmewkhvcdbimqbqghkaa https://www.google.com/search?q=pyrethroid&spell=1&sa=x&ved=0ahukewif7pfpmrnaahuqmewkhvcdbimqbqghkaa j arthropod-borne dis, december 2019, 13(4): 407–415 t hazratian et al.: baseline susceptibility of … 409 http://jad.tums.ac.ir published online: december 31, 2019 different insecticides. in addition, probit regression lines of insecticides against adult of cs. longiareolata were drown which showed a linear relationship between mortality and time (fig. 2). the lt50 values were 52.38, 28.79, 11.53, 63.39 and 28.05min after treatment with ddt 4%, deltamethrin 0.05%, cyfluthrin 0.15%, fenitrothion 1% and propoxur 0.1%, respectively (fig. 3). the highest toxicity against cs. longiareolata was found on cyfluthrin (lt50: 11.53 and lt90: 43.37min) while the lowest toxicity was observed for fenitrothion 1% (lt50: 63.39 and lt90: 183.26min) (table 1). table 1. the parameters of probit regression line of five insecticides on culisita longiareolata in marand county at east azerbaijan province, northwestern of iran, 2017 insecticide name a b±se lt50 , (lcl-ucl) 95% c.i. lt90, (lcl-ucl) 95% c.i. x2 (df) p value cyfluthrin -2.37 2.23±0.27 9.18 32.05 4.36 (3) > 0.05 11.53 43.37 14.27 68.03 ddt -4.41 2.57±0.32 43.35 125.78 5.8 (3) > 0.05 52.38 165.47 63.02 250.91 deltamethrin -3.67 2.53±0.30 23.72 70.17 6.46 (3) > 0.05 28.79 92.24 34.8 138.22 fenitrothion -5.01 2.78±0.34 53.26 140.51 2.39 (3) > 0.05 63.39 183.26 75.85 274.31 propoxur -5.85 4.04±0.46 24.19 48.64 2.81 (3) > 0.05 28.05 58.2 32.36 74.68 a= y-intercept, b= the slope of the line, se= standard error lt50, 95% ci= lethal time causing 50% mortality and its 95% confidence interval lt90, 95% ci= lethal time causing 90% mortality and its 95% confidence interval lcl: lower confidence limit, ucl: upper confidence limit x2= heterogeneity about the regression line df= degree of freedom, p= represent heterogeneity in the population of tested table 2. susceptibility level of culisita longiareolata exposed to different groups of insecticides at diagnostic dose in marand county at east azerbaijan province, northwestern of iran, 2017 insecticides mr±eb* resistance status** cyfluthrin 95±0.25 rc ddt 42.5±0.25 r deltamethrin 70±0.41 r fenitrothion 37.5±0.48 r propoxur 87.5±0.48 r *mortality rate±error bar **rc: resistance candidate ***r: resistance http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 407–415 t hazratian et al.: baseline susceptibility of … 410 http://jad.tums.ac.ir published online: december 31, 2019 fig. 1. map showing iran, highlighting the location of east azerbaijan province and marand county fig. 2. regression lines of culiseta longiareolata exposed to different group of insecticides in marand county at east azerbaijan province, northwestern of iran, 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 407–415 t hazratian et al.: baseline susceptibility of … 411 http://jad.tums.ac.ir published online: december 31, 2019 fig. 3. lethal time causing 50% mortality of culiseta longiareolata exposed to different group of insecticides in marand county at east azerbaijan province, northwestern of iran, 2017 discussion the excessive use of synthetic pesticides in agriculture plays an important role in the development of insecticide resistance in arthropods (41, 42). resistance in medically important arthropods is developing and this is a major problem in their control (43). considering the current who criteria for insecticide resistance evaluation, cs. longiareolata is resistant to fenitrothion, ddt, deltamethrin, propoxur and candidate of resistance to cyfluthrin. some studies showed that cs. longiaerolata is resistance to ddt, propoxur, lambda-cyhalotrin and tolerant to malathion and deltamethrin more over lt50 value found as 131.94, 5.21, 17.60, 5.19 and 29.12min for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur respectively (43). lt90 value of cs. longiaerolata for ddt, deltamethrin, lambda cyhalothrin, malathion and propoxur calculated as 588.13, 29.24, 229.26, 26.69 and 371.76 minutes respectively(41). our results based on probit regression line showed that adult of cs. longiareolata is more suseceptible to pyrethroid and carbamate insecticides. lt50 value of this species for ddt, cyfluthrin, deltamethrin, fenitrothion and propoxur calculated as 52.28, 11.53, 28.79, 63.39 and 28.05 minutes respectively. lt90 value found as 165.47, 43.37, 92.27, 183.26 and 58.2 minutes for ddt, cyfluthrin, deltamethrin, fenitrothion and propoxur respectively. previous studies reported that cs. longiareolata larvae was susceptible to bacillus sphaericus and b. thuringiensis (44). some reports showed that the lc50 and lc90 values of novaluron (insect growth regular) against cs. longiareolata were reported as 0.51–0.91μg/l and 2.32–4.30μg/l, respectively (45). in many regions of iran, results of susceptibility test on cx. pipiens, cx. quinquefasciatus, anopheles stephensi, and cs. longiareolata showed that high resistant to different classes of insecticides, such as ddt, deltamethrin, lambda-cyhalo-thrin, propoxur and cyfluthrin and this finding is similar to our results for cs. longiareolata (29-31, 41, 46, 47). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 407–415 t hazratian et al.: baseline susceptibility of … 412 http://jad.tums.ac.ir published online: december 31, 2019 the lack of data on mosquito susceptibility to insecticides is a limiting factor for the success of control programs. therefore, this finding can be useful in future vector control programs and investigations in order to prevent the development of resistance to insecticides. due to the emergence of resistance in mosquitoes to different classes of insecticides, the use of biological agents can be an effective method to control mosquitoes (42). however, the use of botanical insecticide, which have no adverse effects on the environment and humans, can be appropriate and an alternative control method for insecticide in vector control programs (48-55). conclusion this study confirms the resistance of the cs. longiareolata to fenitrothion, ddt, deltamethrin, propoxur and candidate of 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48. sanei-dehkordi a, soleimani-ahmadi m, akbarzadeh k, salim abadi y, paksa a, gorouhi ma (2016) chemical composition and mosquito larvicidal properties of essential oil from leaves of an iranian indigenous plant zhumeria majdae. j essent oil bear pl. 19(6): 1454–1461. 49. soleimani-ahmadi m, abtahi sm, madani a, paksa a, abadi ys, gorouhi ma (2017) phytochemical profile and mosquito larvicidal activity of the essential oil from aerial parts of satureja bachtiarica bunge against malaria and lymphatic filariasis vectors. j essent oil bear pl. 20 (2): 328–336. 50. soleimani-ahmadi m, gorouhi ma, azani s, abadi y, paksa a, rashid g (2017) larvicidial effects of essential oil and methanol extract of achillea wilhelmsii c. koch (asteraceae) against anopheles stephensi liston (diptera: culicidae), a malaria vector. j kerman univ med sci. 24(1): 58–67. 51. soleimani-ahmadi m, sanei-dehkordi a, turki h, madani a, abadi ys, paksa a (2017) phytochemical properties and insecticidal potential of volatile oils from tanacetum persicum and achillea kellalensis against two medically important mosquitoes. j essent oil bear pl. 20(5): 1254–1265. 52. vatandoost h, sanei-dehkordi a, sadeghi s, davari b, karimian f, abai m (2012) identification of chemical constituents and larvicidal activity of kelussia odoratissima mozaffarian essential oil against two mosquito vectors anopheles stephensi and culex pipiens (diptera: culicidae). exp parasitol. 132(4): 470–474. 53. sanei-dehkordi a, soleimani-ahmadi m, salim-abadi y, paksa a (2019) wild chive oil is an extremely effective larvicide against malaria mosquito vector anopheles stephensi. asian pac j trop med. 12(4): 170–174. 54. sedaghat mm, sanei-dehkordi ar, khanavi m, abai mr, mohtarami f, vatandoost h (2011) chemical composition and larvicidal activity of essential oil of cupressus arizonica e.l. greene against malaria vector anopheles stephensi liston (diptera: culicidae). pharmacogn res. 3(2): 135–139. 55. davari b, vatandoost h, oshaghi ma, ladonni h, enayati aa, shateghi m, basseri hr, rassi y, hanafi-bojd aa (2007) selection of anopheles stephensi with ddt and dieldrin and cross-resistance spectrum to pyrethroids and fipronil. pestic biochem phys. 89(2): 97–103. http://jad.tums.ac.ir/ https://www.sciencedirect.com/science/article/abs/pii/s0048357507000557#! https://www.sciencedirect.com/science/article/abs/pii/s0048357507000557#! https://www.sciencedirect.com/science/article/abs/pii/s0048357507000557#! https://www.sciencedirect.com/science/article/abs/pii/s0048357507000557#! j arthropod-borne dis, december 2018, 12(4): 361–369 m osanloo et al.: larvicidal activity of … 361 http://jad.tums.ac.ir published online: december 25, 2018 original article larvicidal activity of essential oil of syzygium aromaticum (clove) in comparison with its major constituent, eugenol, against anopheles stephensi mahmoud osanloo 1, 2, mohammad mehdi sedaghat 3, fariba esmaeili 1, *amir amani 1, 4 1department of medical nanotechnology, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran 2students’ scientific research center, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4natural products and medicinal plants research center, north khorasan university of medical sciences, bojnurd, iran (received 22 may 2017; accepted 31 june 2018) abstract background: in this study, larvicidal activity of clove essential oil (eo), as a green and relatively potent larvicide, was compared with its main constituent, eugenol, against anopheles stephensi. methods: high-performance liquid chromatography (hplc) was used to determine the amount of eugenol, major constituent of clove eo. in addition, larvicidal activity of clove eo and eugenol was evaluated against an. stephensi. results: the amount of eugenol in clove eo was determined as 67% using hplc analysis. lc50 and lc90 of clove eo (57.49 and 93.14ppm, respectively) were significantly lower than those of eugenol (86.96 and 128.18 ppm, respectively). conclusion: eo showed more effective than its major component. considering the lower cost of the essential oil and lower risk in occurrence of resistance in larvae, use of clove eo is preferred as larvicide in comparison with eugenol, against an. stephensi. keywords: larvicidal activity, hplc, syzigium aromaticum, eugenol, essential oil, anopheles stephensi introduction more than 17% of all infectious diseases around the world are vector-borne diseases, such as dengue fever, yellow fever, and malaria (1). the number of death for such diseases is more than 1 million annually e.g. malaria caused 429000 death just in 2015 (1, 2). in order to control malaria, who recommends control of larva which now used in 55 countries (2). unfortunately, due to frequent use of synthetic larvicides (such as temephos), not only environmental pollution have appeared, but also many cases of resistance has occurred in mosquitoes around the world (3-6). essential oils (eos) have been suggested as alternative sources for insect control as repellents, insecticides or larvicides and they offer ad vantages such as biodegradability and negligible effects on non-target specious and environment (7, 8). recently, comparisons of larvicidal activity of eos with their major components have been reported (9, 10). however, there is no conclusion so fare, eos are better or their major constituent(s) in terms of larvicidal activity. syzygium aromaticum (clove) belongs to the myrtaceae family which considers as an important medicinal plant with wide range of biological activities such as anti-bacterial or anti-oxidant activities (11, 12). clove eo has also shown larvicidal activity against field collected larva of ae. aegypti with lc50 of 92.56 and 62.3ppm in two different reports (13, 14). *corresponding author: dr amir amani, e-mail: aamani@tums.ac.ir mailto:aamani@tums.ac.ir j arthropod-borne dis, december 2018, 12(4): 361–369 m osanloo et al.: larvicidal activity of … 362 http://jad.tums.ac.ir published online: december 25, 2018 eugenol, as the major constituent of clove eo, has also indicated larvicidal activity against laboratory reared and field collected of ae. aegypti (lc50: 33 and 93.3ppm, respectively) (14, 15). its larvicidal activity against other population of mosquito has been documented, with lc50 of 25.4, 28.14 and 30.8ppm against an. subpictus, ae. albopictus and cx. tritaeniorhynchus (16). anopheles stephensi is an important malaria vector with wide distribution in the arabian peninsula, the indian subcontinent, afghanistan and iran (17-19). in this research, for the first time, larvicidal activity of clove eo and eugenol against an. stephensi was evaluated and compared. materials and methods materials high-performance liquid chromatography (hplc) grade methanol, pure eugenol (99%) and ethanol were supplied by merck (germany). clove eo was purchased from green plants of life co (iran). determining of eugenol contents in clove eo oil by hplc hplc analysis was used to determine the amount of eugenol in clove eo. the apparatus consisted of a 30cm× 3.9mm reverse phase c18 column (waters, milford, usa), a pressure less injection pump (model l-6200, hitachi, japan) to drive solvent and loading of samples, a uv visible detector (model l4000, at 280nm, hitachi, japan) for detection, a chromato-integrator (model d-2500, hitachi, japan) for analyses. a mixture of methanol and distilled water was used as mobile phase with flow-rate of 1ml/min. analytical procedure was started with dissolving 50µl of eugenol or 0.5ml of clove eo in 10ml of methanol, then, 20µl of this solution was injected into system at a flow-rate of 0.7ml/ min. the optimum mobile phase with a methanol: water ratio of 80:20 was used for elution. by comparing peak areas of eugenol with solution of clove eo, its amount in clove eo was determined. evaluation of larvicidal activity third and fourth instar larvae of an. stephensi were used, they obtained from insectarium of tehran university of medical sciences. they reared in special condition: 28± 2 °c, 12:12 dark and light periods and relative humidity of 65±5%. larvicidal bioassay was performed in line with who recommendation test in lab, with some modifications (20). standard solutions were prepared by dissolving in ethanol at appropriate concentrations (i.e. eugenol 60 µl/ml and clove eo 30µl/ ml). by adding 1ml from each sample (0.5%v/v) to cups containing 199ml of no chlorine water, desired concentrations of samples were prepared. using separated nets, 25 larvae of an. stephensi were added slowly to all containers. dead and moribund larvae (unable to respond to stimulating agent) were counted after 24h of exposing in all cups. larvicidal bioassays were performed in 16 repetitions at 4 different replicates at concentrations (ppm) of 12.5, 25, 50, 75, 100, 150 for clove eo and 12.5, 25, 50, 100, 150, 200 and 300 for eugenol. in each replicate, two control groups were considered having ethanol (0.5%v/v) with similar treatments. lethal concentrations of each sample (i.e. lc50 and lc90), were determined using a probit regression model in spss ver. 19 (chicago, il, usa). comparison of larvicidal activity of clove eo with eugenol evaluation of overlaps between confidence intervals (ci) of two groups is an easy and common approach to compare various lc values. if no overlap is observed, the difference is considered as significant (21). lc50 and lc90 of clove eo and eugenol were calculated and compared using independent sample test by spss. j arthropod-borne dis, december 2018, 12(4): 361–369 m osanloo et al.: larvicidal activity of … 363 http://jad.tums.ac.ir published online: december 25, 2018 results determining content of eugenol in clove eo results of hplc analysis of eugenol and clove eo samples are shown in fig. 1. comparing the graphs, the peak related to eugenol is observed at retention time of 6.43 and 6.41min, for eugenol and clove eo, respectively. by comparing peak areas of eugenol and clove eo, percentage of eugenol in clove eo was calculated as 67%, considered relatively high. evaluation of larvicidal activity of clove eo and eugenol results of larvicidal activities of clove eo and eugenol against an. stephensi are shown in fig. 2. calculated lc50 and lc90 values were 57.49 and 86.96ppm for clove eo and 93.14 and 158.2ppm for eugenol, respectively (table 1). larvicidal activity in both samples (i.e. clove eo and eugenol) appeared at 25ppm and enhanced with increasing the concentration of those. nevertheless, perfect larvicidal activities were achieved at 100 and 200ppm for clove eo and eugenol, respectively. furthermore, no overlap in ci of lc50 and lc90 for clove eo and eugenol are observed, thus, larvicidal activity of clove eo is significantly better than eugenol (table 1). moreover, probit regression line of the both clove eo and eugenol are illustrated in fig. 3. table 1. calculated parameters by probit analysis specimen a b±se lc50 (ppm) ci: (lclucl) lc90 (ppm) ci: (lcl–ucl) χ2 (df) sig clove eo -2.50 0.04±0.002 57.49 86.96 47.96 (3) 0.15 > sig* (43.28–74.24) (71.19–128.18) eugenol -1.83 0.02±0.001 93.14 158.2 39.13 (4) 0.15 > sig* (75.60–113.33) (133.85–201.20) a= intercept; b±se= slope and standard error of the line; ci= confidence interval (0.05), ucl= upper confidence limit, lcl= lower confidence limit, χ2 (df)= chi 2 and degree of freedom. *since the significance level is less than 0.15, a heterogeneity factor is used in the calculation of confidence limits. fig. 1. hplc profile of solution of eugenol and clove eo, related peak for eugenol appeared at retention time of 6.43 and 6.41min, respectively j arthropod-borne dis, december 2018, 12(4): 361–369 m osanloo et al.: larvicidal activity of … 364 http://jad.tums.ac.ir published online: december 25, 2018 fig. 2. larvicidal activity of clove eo and eugenol against anopheles stephensi fig. 3. probit regression line of clove eo and eugenol discussion determining content of eugenol in clove eo in this study, amount of eugenol in clove eo was found as 67%, which is comparable with other reports. reviewing other reports, content of eugenol has been reported in values more or less similar to this value: e.g. 58.29% (22), 59.29% (23), 76.8% (24), 86.61% (25) and 88.58% (26). evaluation of larvicidal activity of clove eo and eugenol obtained lc50 of clove eo and eugenol against an. stephensi were 57 and 93ppm, respectively. lc90 values were 86 and 158ppm, respectively. there are many reports about larvicidal activities of other eos against an. stephensi. for example, larvicidal activities (lc50) of some essential oils such as artemisia dracunculus (11.36ppm), anethum graveolens (38.80ppm) and kelussia odoratissima (4.77ppm) were evaluated (27-29). determined lc50 of clove eo in this research is lower than many reports against an. stephensi for instance, lawsonia inermis (69.40ppm) (30), cionura erecta (77.30ppm) (31) and cupressus arizonica (79.30ppm) (32), zhumeria majdae (61.34ppm) (33). however, calculated lc50 in some other reports is lower than our reported lc50, for instance, bunium persicum (27.72ppm) (34), tanacetum persicum and achillea kellalensis (48.64 and 35.42ppm respectively) (35), satureja bachtiarica (24.27ppm) (36) and citrus aurantium (31.20ppm) (37). larvicidal activity of either of clove eo and eugenol, against other species of mosquito, has already been reported. ae. aegypti is shown to be more susceptible than cx. quinquefasciatus when using clove eo (i.e. lc50: 92.56 vs. 124.42ppm) (13). larvicidal activity of synthetic derivatives of eugenol against ae. aegypti has been shown (lc50~ 62.3ppm or higher) (14). recently, many reports have been released j arthropod-borne dis, december 2018, 12(4): 361–369 m osanloo et al.: larvicidal activity of … 365 http://jad.tums.ac.ir published online: december 25, 2018 about comparison of larvicidal activity of eos with their major components. however, there is no conclusion so far whether eos outperforms or their major constituent in terms of larvicidal efficacy. lc50 in allium tuberosum eo against aedes albopictus was found to be 17.9ppm, lower than that of its two major components, allyl methyl trisulfide and dimethyl trisulfide, with lc50 of 27.5 and 36.4ppm, respectively (38). lc50 of ruta chalepensis eo has been evaluated against anopheles quadrimaculatus (14.9 ppm) and aedes aegypti (22.2ppm). while, 2-undecanone, its major component, showed similar lc50 to that of total eo against an. quadrimaculatus (14.2ppm), significantly lower values against ae. aegypti (14.37ppm) were obtained (21). lc50 of eo of allium macrostemon (72.86 ppm) was better than a major constituent, methyl propyl disulfide (86.16ppm), while its efficacy was lower than the other major component, dimethyl trisulfide (36.36ppm), against ae. albopictus (40). synergistic effects of constituents of some eos are nowadays well-known when they are used as anti-fungal or anti-bacterial agents (39, 40). our findings in this research and our previous study also show that an. stephensi is more susceptible to the clove eo or eo of kelussia odoratissima (with lc50 of 57.49 and 4.77ppm, respectively), compared with their major constituents, eugenol (93.14ppm) and zligustilide (8.73ppm), respectively (28). a type of synergism may have occurred in larvicidal activity of the eos too. eos are mixtures of many constituents such as flavonoids, alkaloids, and monoterpenes (41, 42). modes of action of mentioned constituents are different e.g. main site action of flavonoids is acetylcholinesterase (43), while alkaloids and monoterpenes target na-k-atpase or na+ and k+ channels (44-46). this could be the main reason for occurring synergism in larvicidal activity in our study. having mentioned that resistance against larvicides is mostly observed when a single active agent is used compared with those having multi components (47-49). conclusion use of clove eo as a green larvicide against an. stephensi is preferred compared with its major constituent (eugenol). considering the fact that the eo is a lot cheaper than eugenol and is composed of several components, thus, has lesser chance of occurring resistance, the whole eo may be suggested as a proper larvicide. acknowledgements this research has been supported by students’ scientific research center, tehran university of medical sciences, grant no. 9501-61-31420, and also had ethical approved by its ethical committee center, ir.tums. rec.1395.2480. the authors declare that there is no conflict of interests. references 1. world health organization (2017) vectorborne diseases fact sheet. available at: http://www.who.int/mediacentre/factsh eets/fs387/en/ 2. world health organization (2016) world malaria report. available at: 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and citrus sinensis (l.) osbeck essential oils and their antagonistic effects with temephos in resistant populations of aedes aegypti. mem inst oswaldo cruz. 111(7): 443–449. 48. intirach j, junkum a, tuetun b, choochote w, chaithong u, jitpakdi a, riyong d, champakaew d, pitasawat b (2012) chemical constituents and combined larvicidal effects of selected essential oils against anopheles cracens (diptera: culicidae). psyche j entomol. 2012: id 591616. 49. okumu fo, knols bg, fillinger u (2007) larvicidal effects of a neem (azadirachta indica) oil formulation on the malaria vector anopheles gambiae. malar j. 6 (1): 63. j arthropod-borne dis, march 2020, 14(1): 1–7 m abbasi et al.: study on the fauna of … 1 http://jad.tums.ac.ir 2031, 20 marchpublished online: original article study on the fauna of aquatic insects in northwestern iran madineh abbasi1,2; sara doosti3; *hassan vatandoost1,4; nasibeh hosseini-vasoukolaei5 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of communicable diseases, vice health, tabriz university of medical sciences, tabriz, iran 3department of medical entomology and vector control, medical school, zanjan university of medical sciences, zanjan, iran 4department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 5department of medical entomology and vector control, health science research center, faculty of health, mazandaran university of medical sciences, sari, iran (received 07 nov 2019; accepted 12 mar 2020) abstract background: aquatic insects include groups of arthropods which at least one step of their life happens in water. some of these insects play an important role in the transmission of various diseases to human and animals. because there is a little information about the fauna of aquatic insects in iran, this study was aimed to collect and identify aquatic insects in northwestern iran. methods: a descriptive cross sectional study was performed in july 2017 in rivers of three villages of osku county of east azerbaijan province, northwestern iran. the specimens were collected using different methods including d frame net-collector, standard mosquito dipper, sweep netting and plastic pipette. the collected specimens were identified based on the systematic keys of aquatic insects. results: totally 285 samples were collected. four orders including diptera, hemiptera, ephemeroptera and coleoptera were identified. collected samples belonged to seven families of tipulidae, chironomidae, corixidae, culicidae, hydrophilidae, baetidae and dytiscidae. the most predominant family was culicidae belonging to order diptera. culiseta longiareolata was the most frequent species collected in the study area. conclusion: aquatic insects usually play an important role in the food chain of animals and some species of them act as predators and play an important role in the biological control of vectors. therefore, more studies are required to carry out in the field of aquatic insects. keywords: aquatic insects; fauna; azerbaijan; iran introduction aquatic insects include groups of arthropods which at least one steps of their life happens in water. about more than 30000 species of aquatic insects were identified which can live in the freshwater and some species are living in the brackish water (1). according to the fossil records, aquatic insects appeared in the triassic (2) more than 150 million years after the appearance of insects. the most important places for aquatic insects are various and include shallow holes with stagnant water, pools and floodgates, large and small rivers, streams, beaches, lakes, mineral water and drinking water pools (3). several of them live near the water and their life cycle can be completed in or depended to water. therefore, these insects called semi aquatic insects (4). many of these insects spend their primary stages in the water while the adults are completely terrestrial, for example the order of ephemeroptera (mayflies), odonata (dragonflies and damselflies), plecopter (stoneflies), diptera (flies), trichopter (caddisflies). the adults lay their eggs singly or patches in or around the water (5). some of these insects play *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ mailto:vatando@tums.ac.ir j arthropod-borne dis, march 2020, 14(1): 1–7 m abbasi et al.: study on the fauna of … 2 http://jad.tums.ac.ir 2031, 20 marchpublished online: an important role in the transmission of various diseases to human and animals such as dengue virus, zika virus, west nile virus, encephalitis, malaria, filariasis and other arboviral diseases (6, 7). some of diseases are transmitted via biological or mechanical pathway to human by tabanidae and simuliidae (8). some of them like dragonflies and damselflies can be the host of termatodes (9). a few of them cause the mental annoyance and dermal damage on the human and animal hosts by their painful bite (10). some aquatic insects play the role of contamination indicators of water (for example ephemeroptera, plecoptera and trichoptera) and are mentioned as “biological indicators” of water quality (11, 12). many of them are the main food supply for fishes and amphibians (13–16). because there is little information about the fauna of aquatic insects in iran, this study was aimed to collect and identify the aquatic insects in the northwestern iran in order to open a new window to the vast aquatic insects of the world. materials and methods study area a descriptive cross sectional study was performed in july 2017 in the rivers of three villages of osku county (amghan, ansrood and kandowan). this county is one of 16 counties of east azerbaijan province of iran located in the vicinity of tabriz. the county with the geographical coordinates of 37° 51′ 29.54″ n, 45° 56′ 24.18″ e is located on the northwest of sahand mountain range at an altitude of 1579 meters. based on the results of the general census of population and housing in 2011, the population of the county of osku is about 98,988 people (2.7% of the province's population) and the population of the center of this county is estimated as 16983 people. like other counties in azerbaijan area, it has a short, mild summer and cold and long winters. its precipitation is mostly in the cold seasons and its summers are dry and sometimes rainy (17) (fig. 1) sampling methods the specimens were collected using different methods including: d frame net-collector, standard mosquito dipper (350ml), sweep netting and plastic pipette. sampling carried out in different part of breading places in several occasions (fig. 2). the samples were collected, transferred to individual jars containing some water obtained from their habitat. subsequently they were put in glass vials contained 90% ethylic alcohol. the date and location of sampling were written on the label and stick on the vials. all samples were sent to school of public health, tehran university of medical sciences, where the author identified the specimens using the keys of aquatic insects based on guide to aquatic invertebrate families of mongolia 2012 and other relevant systematic keys (1824). in this study, we used stereo-typed microscope and microscope for identification of samples. the results were recorded on a data sheet based on the order and family and number of its. all of the photos are original. results totally, 285 samples were collected belonging to four orders: diptera, hemiptera, ephemeroptera, and coleoptera (table 1). seven families were identified as tipulidae, chironomidae, corixidae, culicidae, hydrophilidae and dytiscidae. the most predominant family was culicidae (71.2%) belonging to the order diptera. in the family culicidae, the species belong to culiseta longiareolata (200 cases) and culex hortensis (3 cases). http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/counties_of_iran j arthropod-borne dis, march 2020, 14(1): 1–7 m abbasi et al.: study on the fauna of … 3 http://jad.tums.ac.ir 2031, 20 marchpublished online: table 1. abundance of some aquatic insects’ larvae collected in the study areas percent sample no family order 8.7 25 baetidae ephemeroptera 3.2 9 tipulidae diptera 71.2 203 culicidae 7.1 20 chironomidae 1.1 2 dytiscidae coleoptera 1.6 4 hydrophilidae 7.1 20 corixidae hemiptera 100 285 total fig. 1. geographical status of sampling sites in the study area in osku county of east azerbaijan province, iran fig. 2. (a, b) two sampling sites, aghbolagh river, east azerbaijan, iran http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 1–7 m abbasi et al.: study on the fauna of … 4 http://jad.tums.ac.ir 2031, 20 marchpublished online: discussion during study, the specimens were collected via different methods such as d-frame netcollector, standard mosquito dipper (350 ml), sweep netting and plastic pipette. a total 285 samples have been grouped in four orders: diptera, hemiptera, ephemeroptera, coleoptera and the most abundant of them belong to the order diptera. this order is one of the richest groups of insect and has worldwide distribution with more than 152000 described species based on biosystematics database of world diptera (25). however, in aquatic insect ecosystems, diptera play an important role and more than half of them belong to this group. the colonies of diptera are often found in clean and fresh water and in many cases they are the most abundant taxa (26). in our study, the main family and species of diptera belong to culicidae and cs. longiareolata (71.2%). knight and stone in 1977 reported that the genus of culiseta consists of 37 species in 7 subgenus which most of them found in the palearctic and nearctic regions (27). in iran, two subspecies allotheobaldia and culiseta reported up now (28). the subgenus allotheobaldia has only one species (cs. longiareolata) in the world and reported from the palearctic and ethiopian regions, pakistan and indian (29). this species reported for the first time by gusevich in 1943 from north of iran (30). then dow in 1953 reported it from gorgabad, sharafkhane port and maraghe in northwest of iran (31). culiseta longiareolata larvae usually could be able grow in transient water, without vegetation and mud bottom. also it collected from rain water pools with shaded and partial sunlight larval habitats. this species has not been reported from artificial habitat until now (32). azari-hamidian in 2003 explains this species from guilan (33). in the same study which carried out by shayeghi et al. in isfahan province, most of the specimens which collected belong to diptera order (cx. theileri) and their result was very closely to our result (34). abai et al. (2007) presented the same results in their investigation (35). in study of shayeghi et al. 2017 in northern of iran different species of trichoptera, ephemeroptera, plecoptera, hemiptera and odonata have been collected and diptera order was the most prevalent samples (24.5%) (36). in other study was carried out in markazi provinces, 24 species of aquatic beetles in five families identified (37) and hydrophilidae is one of the species that we also reported it in our study. shayeghi et al. in 2015 reported three orders of plecoptera, trichoptera and ephemeroptera from karaj river (38). the main family in current study belongs to perlidae (49.7%) (order: plecoptera). in shayeghi study in jajroud river, five families (blephariceridae, simulidae, hydropsychidae, baetidae, and dytiscidae) were identified and the main order belongs to diptera that is the same as our results (36). ostovan et al. (2004) studied on biodiversity and fauna of aquatic insects and beetles in ardabil and fars provinces (39). also, atamehr in 2002 and 2004 reported 51 species in 40 genera and 14 families from east azerbaijan province (40, 41). eight orders of aquatic insects includes ephemeroptera, odonata, plecoptera, hemiptera, megaloptera, coleoptera, trichoptera and diptera have been reported from reese voshell study in usa (42) and we also collected ephemeroptera, hemiptera and diptera in our studies, which is similar to the above results. the second order, which had a high abundance in this study, belonged to mayfly or ephemeroptera, (family: baetidae). 40 families of mayflies found in worldwide, consisting of about 3330 species (43). the families of mayflies are divided into tree sub-orders, setisura, pisciforma and rechtracheata (44). mayflies have distributed throughout the world and live in freshwater and sometimes brackish waters on all continents except antarctica (42). there are different organisms that live in or on mayfly's body, including a variety of bacteria, protozoa, nematodes (round worms), cestodes (tape worms) and trematodes parasite. as well as http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 1–7 m abbasi et al.: study on the fauna of … 5 http://jad.tums.ac.ir 2031, 20 marchpublished online: they act as final hosts for parasites and intermediate hosts, notably for fish parasites (43). in shayeghi et al. studies in 2017, mayflies including tree families: baetidae (91.5%), heptagenidae (5.5%) and caenidae (3%) have been collected from rice-field, slow moving river, temporary pond and shallow stream (36). the results of their studies was very similar to our results. baetidae act as a major component of invertebrate drift in running water (45). conclusion according to the results, it can be concluded that various species of aquatic insects were collected and identified during study. the main order in study was diptera. aquatic insects usually play an important role in the food chain of animal. there are need to do more studies about this important subject. there are some current studies on aquatic insects in different parts of iran (46-52). some species of aquatic insects act as predators and play an important role in biological control of vectors as well as bioindicator for aquatic conditions. the authors declare that there is no conflict of interests. references 1. merritt r, cummins k (1996) an introduction to the aquatic insects of north america. p. 861. 2. belayeva nv, rasnitsyn ap, quicke dlj (2002) history of insects. first edition. kluwer academic publishers. p. 503. 3. kleber dc, rhainer g (2019) aquatic insects, behavior and ecology. springer international publishing, springer nature switzerland, p. 428. 4. voshell r (2002) a guide to common freshwater invertebrates of north america. mcdonald and woodward publishing company, granville, ohio, p. 456. 5. subramanian ka, sivaramkrishnan kg (2015) aquatic insects of indiaa field guide. ashoka trust for ecology and envi ronment (atree), bangalore, india, p. 62. 6. american mosquito control association (2019) mosquito-borne diseases. available at: https://www.mosquito.org/page/diseases 7. fatalmoudou t, ogobara d, alpha seydou y, sékou ft, philippe p, vincent r (2018) mosquitoes (diptera: culicidae) and mosquito-borne diseases in mali, west africa. parasite vectors. 11: 467. 8. foil ld (1989) tabanids as vectors of disease agents. parasitol today. 5(3): 88–96. 9. chae sj, pusterla n, johnson e, derock e, lawler sp, madigan je (2000) infection of aquatic insects whit trematode metacercariae carry ehrlichiaristicii, the case of potomac house fever. j med entomol. 37(4): 619–625. 10. villiers de (1987) simulium dermatitis in man-clinical and biological features in south africa. a case report. s afr med j. 71(8): 523–525. 11. suhaila ah, che salmah mr (2017) application of aquatic insects (ephemeroptera, plecoptera and trichoptera) in water quality assessment of malaysian headwater. trop life sci res. 28(2): 143–162. 12. suhaila ah, che salmah mr (2017) ephemeroptera, plecoptera and trichoptera (insecta) abundance, diversity and role in leaf litter breakdown in tropical headwater river. trop life sci res. 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k, marvi-moghadam n, ahmad yusof m (2018) the species diversity of aquatic insects in karaj river, central iran. j marine sci res dev. 8(5): 258. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 226 original article laboratory evaluations of the fractions efficacy of annona senegalensis (annonaceae) leaf extract on immature stage development of malarial and filarial mosquito vectors *younoussa lame 1, elias nchiwan nukenine 1, danga yinyang simon pierre 1, ajaegbu eze elijah 2, charles okechukwu esimone 3 1department of biological sciences, university of ngaoundere, cameroon 2department of pharmaceutical and medicinal chemistry, nnamdi azikiwe university, nigeria 3department of pharmaceutical microbiology and biotechnology, nnamdi azikiwe university, nigeria (received 12 jan 2014; accepted 9 july 2014) abstract background: within the framework to control mosquitoes, ovicidal, larvicidal and pupicidal activity of annona senegalensis leaf extract and its 4 fractions against anopheles gambiae and culex quinquefasciatus were evaluated in the laboratory conditions. methods: ovicidal test was performed by submitting at least 100 eggs of mosquitoes to 125, 250, 500, 1000 and 2000 ppm concentrations, while larvicidal and pupicidal effects were assessed by submitting 25 larvae or pupae to the concentrations of 2500, 1250, 625 and 312.5 ppm of plant extract or fractions of a. senegalensis. results: the eggs of an. gambiae were most affected by n-hexane (0.00% hatchability) and chloroform (03.67% hatchability) fractions compared to cx. quinquefasciatus where at least 25 % hatchability were recorded at 2000 ppm. for larvicidal test, n-hexane (lc50= 298.8 ppm) and chloroform (lc50= 418.3 ppm) fractions were more effective than other fractions on an. gambiae larvae while, a moderate effectiveness was also observed with nhexane (lc50= 2087.6 ppm), chloroform (lc50= 9010.1 ppm) fractions on cx. quinquefasciatus larvae. the highest mortality percent of the pupae were also recorded with n-hexane and chloroform fractions on an. gambiae at 2500 ppm. as for cx. quinquefasciatus only 50 % and 36 % mortality were recorded with n-hexane and chloroform fractions respectively. conclusion: the extract of a. senegalensis was toxic on immature stage of mosquito species tested. by splitting methanolic crude extract, only n-hexane and chloroform fractions were revealed to possess a mosquitocidal effects and could be considered and utilized for future immature mosquito vectors control. keywords: fractions, pupicidal, annona senegalensis, anopheles gambiae, culex quinquefasciatus introduction apart nuisances they inflict to human beings, mosquitoes are responsible of dreadful diseases such as malaria, filariasis, dengue haemorrhagic fever, etc widespread in the world (murugan et al. 2007, kamaraj et al. 2009). most of sub-saharan africa countries have stable endemic malaria because climatic conditions, which are ideal for the transmission, coincide with the range of an. gambiae, an. arabiensis and an. funestus, the most efficient vector mosquitoes in the world (foko et al. 2011). in cameroon an. gambiae is the principal vector of malaria in rural and urban areas (foko et al. 2011). filariasis, a disease affecting the arms, legs and genitals, is much prevalent in the world. filariasis caused by wuchereria bancrofti is transmitted by cx. quinquefasciatus, mosquitoes widespread in the countries now and lymphatic filariasis infects 80 million people annually of which 30 million cases exist in chronic infection (samidurai et al. 2009). synthetic pesticides have been extensively used for vector control by either killing, pre*corresponding author: dr younoussa lame, e -mail: youlasobi@yahoo.fr http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 227 venting adult mosquitoes to bite human beings or by killing mosquito immature stages at the breeding sites of the vectors (joshep et al. 2004). development of insect resistance to synthetic pesticides such as malathion, ddt, deltamethrin and even biopesticides such as bacillus thuringiensis (tabashnik 1994), added to high operational cost, environmental pollution and deleterious effects on non-target organisms are the problems people are facing to control vector borne diseases (mittal et al. 2003). these problems have highlighted and need for the development of new strategies for selective mosquito control. with these problems in focus, it becomes increasingly necessary to search for an alternative in the development of environmentally safe, biodegradable, low cost, target specific insecticides for mosquito control and which can be used with minimum care by individuals and communities in specific situations (dua et al. 2010). plants may be a source of alternative agent to replace the synthetic insecticides for mosquito control. toxicity of phytochemicals in mosquitoes was first reported by campbell et al. (1933). review papers from all over the world have documented the toxic effect of plant extracts on mosquito eggs, larvae and pupae (tare et al. 2004, de lima et al. 2006, promsiri et al. 2006, govindarajan et al. 2012, dhivya and manimegalai 2013). earlier, the plant extracts belonging to the family of annonaceae such as annona muricita, a. cherimolia, a. squamosa, cananga odorata, ferula hormonis, etc have shown larvicidal effect against anopheles sp and culex quinquefasciatus (saxena et al. 1993, bodadillaalvarez et al. 2002, moore and lenglet 2004, isman 2006). das et al. (2007) reported that the ethanol leaf extract of annona squamosa was found to have the most promising larvicidal activity against cx. quinquefasciatus larvae. ovicidal, larvicidal and pupicidal effects of hyptis suaveolens, calotropis gigantea and delonix elata against an. gambiae and cx. quinquefasciatus were also reported (ivoke et al. 2009, govindarajan et al. 2012, dhivya and manimegalai 2013). gueye et al. (2011) reported insecticidal activity of extract a. senegalensis extract on eggs and adults groundnut weevils caryedon serratus. in northern part of cameroon, the leaves of this plant are used locally to protect maize, millet and sorghum against weevils’ attacks (ngamo et al. 2007). in ethno-medicine, the leaves of a. senegalensis are used as antidrepanocitory and antitrypanosomic (ogbadoyi 2007), antidiarrheic (suleiman 2008), cures snake bite, generalized eodemes, aches and constipation (akoegninou et al. 2006). with a large scale of activity as insecticide and medicinal plant, it should be advisable to extent biological properties of this plant on mosquitoes. this study was aimed to evaluate ovicidal, larvicidal and pupacidal activity of a. senegalensis methanolic crude extract and its fractions (n-hexane, chloroform, ethyl-acetate and methanol fractions) on an. gambiae and cx. quinquefasciatus mosquito species. materials and methods collection and processing the plant material the fresh leaves of a. senegalensis were collected at dang, a village of ngaoundere in the adamaoua region (latitude 7°24.949’n, longitude 13°32.870’e and altitude 1093 masl), cameroon in december 2011. the plant was identified by the herbalist, pr. mapongmetsem pierre-marie, department of biological science, university of ngaoundere, cameroon and then confirmed at the national herbarium in yaoundé, where voucher sample was deposed. the leaves are then dried at room temperature, then grounded with an electric grinder and then stored at 4 oc in the refrigerator until use. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 228 extraction and fractionation the method of gueye et al. (2011) was followed for the purpose. indeed, five hundred (500) grams of powder were macerated in 2500 ml of methanol for 72 h at room temperature and then the maceration was filtrated using filter paper whatman no.1. the filtrate was summited to rotary evaporator apparatus to obtain a residue called crude extract. part of this crude extract was separated successively by the method of differential solubility in four solvents of different polarity: n-hexane, chloroform, ethyl-acetate and methanol. the crude extract was mixed with silica gel (70–260 mesh size) and macerated in n-hexane, then filtered with whatman no. 1 filter paper after phase separation. n-hexane fraction and marceration (1) are recovered. marceration (1) is dried in the open air and then soaked in chloroform, phase chloroform fraction filtrated and marceration (2) are also recovered. marceration (2) after dried in open air is soaked in ethyl-acetate; phase ethyl-acetate fraction filtered and marceration (3) are also recovered. marceration (3) is finally taken up in methanol to recover the polar compounds in the methanol fraction after filtration. each fraction has been concentrated using rotary evaporator and the solid fractions gotten were stored at -4 oc until bioassays. mosquito breeding the larvae of cx. quinquefasciatus were collected from the laboratory of national arbovirus research center (navrc), enugu, nigeria in february 2013 while an. gambiae larvae were collected from stagnant water in the gutter at awka market, anambra state, nigeria (06°12'23"n, 07°03'23"e) and identified also in navrc in january 2013. larvae were kept in plastic trays containing tap water. larvae were fed a diet containing crayfish and biscuit in a ratio of 3: 1, respectively. pupae were transferred from the trays to a cup containing tap water and were maintained in our insectary cage (45×45×40 cm), where adults emerged. adults were maintained in cages and were continuously provided with 10 % sucrose solution in a jar with a cotton wick. on day five, blood meal was given to the female adults for eggs maturation. beaker with 100 ml of tap water lined with filter paper was kept inside the cage for oviposition (kamaraj et al. 2008). all the experiments were carried out, at 27±2 °c and 75–85 % relative humidity under 12: 12 light and dark cycles. ovicidal activity for ovicidal activity, the method of kumar et al. (2012) was followed for the purpose. the freshly laid eggs were collected by providing ovitraps in mosquito cages kept 2 days after the female mosquitoes were given a blood meal. indeed, 100 gravids female mosquito were placed in a screen cage where ten oviposition cups were introduced for oviposition, 30 min before the start of the dusk period. the eggs were laid on filter paper no. 1 provided in the ovitrap. out of these ten cups, nine were filled with test solution of 125, 250, 500, 1000 and 2000 ppm concentration of plant extract/ fractions of the leaves of a. senegalensis while one was filled with 99 ml of distilled water mixed with 1 ml of tween-80 used as a control. a minimum of 100 eggs was used for each treatment, and the experiment was replicated four times. after treatment, the eggs were sieved through muslin cloth, thoroughly rinsed with tap water, and left in plastic cups filled with tap water for hatching assessment after counting the eggs under microscope (su and mulla 1998). the percent of egg mortality was calculated on the basis of nonhatchability of eggs with unopened opercula (chenniappan and kadarkarai 2008). larvicidal test the larvicidal activity of extract/fractions http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 229 of a. senegalensis was evaluated against an. gambiae and cx. quinquefasciatus according to the method described by who (1996). the extract/fractions were dissolved in 0.5 ml of tween-80. the concentrations of 2500, 1250, 625 and 312.5 ppm of extract/fractions were prepared in the volume of 100 ml with tap water in the 250 ml beakers. twenty five fourth instar larvae were transferred into the used and four replicates were maintained for each concentration. mortality was recorded after 24 h of exposure, during which no food was given to the larvae. larvae were considered dead if appendages did not move when probed with needle in the siphon or cervical region. larvae incapable of rising to the surface or not showing the characteristic diving reaction when water was disturbed, were considered moribund and added to the dead larvae for calculating percentage of mortality. data were adjusted for control mortality using abbott’s formula (abbott 1925), if mortality in the control sets exceeded 5 %. pupicidal effect pupicidal effect was assessed according to the method applied by ashfaq and ashfaq (2012). twenty five freshly emerged pupae of each mosquito species were transferred into beakers of 250 ml volume, containing 75 ml of tap water. the extract/fractions of the plant used were dissolved in tween-80 and then added with tap water to make up to 100 ml corresponding to the concentrations of 2500, 1250, 625 and 312.5 ppm of extract/ fractions of a. senegalensis were made. each treatment was replicated four times and the number of emerged adults for each replication was recorded after 48 h. phytochemical screening the qualitative phytochemical analyses of the components responsible of toxicity on insects were carried out according to the methods of harborne (1973) and trease and evans (1989). these methods are founded on detection the presence of secondary metabolites such as alkaloids, flavonoids, saponins, tannins, phenolic compounds, steroids, terpenoids, oil and fats which possess insecticidal properties in the extract and fractions of a. senegalensis. statistical analysis the values recorded from toxicity essays were transformed in percentage of mortality, hatchability using microsoft excel 2010 and were corrected using abbot’s formula whenever required. the percentage of mortality, hatchability data were subjected to the anova procedure using the statistical package for the social science (spss 16.0). duncan test at p= 0.05 was applied for mean separation. probit analysis (finney 1971, spss 16.0) was applied to determine lethal concentrations causing 50 % (lc50) and 90 % (lc90) mortality of larvae and pupae 24h after treatment application. results the results of ovicidal activity of a. senegalensis extracts against against an. gambiae and cx. quinquefasciatus are presented in table1. a significant (p< 0.001) variation of percentage of eggs hatchability is observed in mosquito species assessed. the rate of hatchability has significantly (p< 0.001) reduced with the increasing of concentration. in comparison with the methanolic crude extract, the fractionation process pointed out the effectiveness of n-hexane followed by chloroform fractions on mosquito eggs. at the highest concentration (2000 ppm), less than 5 % rate of hatchability of an. gambiae were recorded with n-hexane (0.00%), methanolic crude extract (04.67%), chloroform fraction (03.67%) contrary to ethyl-acetate and methanol fractions where, the high values of 22.67 % and 60.33% were recorded respectively. the eggs of cx. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 230 quiquefasciatus were less affected by the extracts used. the percentage of hatchability was 100 % with methanol fraction with all concentrations tested and at least, 25 % of eggs hatched were observed in others extract or fractions of a. senegalensis. the toxicities of the fractions of a. senegalensis extract on early four instar an. gambiae and cx. quinquefasciatus larvae were noted, and the lc50, lc90, 95 % confidence limits of lcl and ucl and chisquare were also calculated (table 2). at 2500 ppm and 1250 ppm, 100 % mortality of the larvae of an. gambiae was recorded with n-hexane and chloroform fractions. among the fractions, n-hexane (lc50= 298.8 ppm, lc90= 572.9 ppm) and chloroform (lc50= 418.3 ppm, lc90= 822.0 ppm) fractions were found to be more effective than other fractions against an. gambiae larvae. as for cx. quinquefasciatus, a moderate effectiveness was also observed with the mortality percent of 48% for n-hexane fraction, 29.33 % for methanolic crude extract and 13.33 % for chloroform fraction recorded at 2500 ppm. in comparison of fractions, n-hexane fraction (lc50= 2087.6 ppm) was most toxic followed by methanolic crude extract (lc50= 5884.1 ppm) and chloroform fraction (lc50= 9010.1 ppm) while, no mortality was recorded with ethyl-acetate and methanol fractions. apart ethyl-acetate and methanol fractions of a. senegalensis, others fractions especially nhexane and chloroform fractions possessed a significant (p< 0.001) efficacy against pupae of mosquito species assessed. at the highest concentration (2500 ppm), 80 %, 70 % and 62 % mortality of an. gambiae pupae were recorded with nhexane, chloroform fractions and methanolic crude extract respectively (fig. 1). a moderate mortality of cx. quinquefasciatus pupae were noted with n-hexane fraction (50%) chloroform fraction (36%) and methanolic crude extract (34%) at the highest concentration (2500 ppm) (fig. 2). at all concentrations were tested and no mortality of the pupae of an. gambiae and cx. quinquefasciatus was recorded with ethyl-acetate and methanol fractions of a. senegalensis. the fractions of a. senegalensis leaves extract were screened for the presence of major phytochemical groups responsible of insecticidal activity. the preliminary phytochemical screening of the crude extract revealed the presence of alkaloids, flavonoids, saponins, tannins, phenolic compounds, steroids, terpenoids, oil and fats except steroids (table 3). by splitting the crude extract, the same phytochemicals were found in n-hexane fraction excepted saponins, steroids and terpenoids. alkaloids, flavonoids tannins and phenolic compounds were also found in chloroform fraction. table 1. hatchability percent of anopheles gambiae and culex quinquefasciatus eggs treated with extract/fractions of annona senegalensis mosquito species conc (ppm) percentage of egg hatch ability fractions mce nhf chf eaf mtf f anopheles gambiae 0 100±0.00fa 100±0.00fa 100±0.00fa 100±0.00ea 100±0.00ea 0.00ns 125 69.67±1.53eb 62.67±2.08ea 79.00±2.00ec 96.33±1.53ed 98.67±0.58ed 285.59*** 250 52.00±2.00db 39.67±2.08da 58.33±2.08dc 81.33±2.08dd 95.67±1.15de 421.70*** 500 34.00±1.00cb 19.67±3.21ca 29.67±1.53cb 65.0±3.61cc 88.67±1.53cd 423.60*** 1000 17.67±1.53bc 04.00±2.00ba 10.00±2.00bb 41.33±2.08bd 77.00±2.00be 716.29*** 2000 04.67±1.53aa 00.00±0.00aa 03.67±1.15aa 22.67±3.06ab 60.33±2.08ac 545.37*** f 1850*** 1153*** 1689*** 516.18*** 348.42*** culex quinquefasciatus 0 100±0.00ea 100±0.00fa 100±0.00fa 100±0.00ea 100±0.00aa 0.00ns 125 98.00±1.00eb 88.00±2.00ea 96.67±1.53eb 100.0±0.00ec 100.0±0.00ac 50.63*** 250 92.67±1.53dc 74.00±2.00da 84.67±1.53db 98.00±1.00dd 100.0±0.00ad 176.69*** http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 231 500 81.67±3.51cc 58.33±1.53ca 69.67±1.53cb 95.67±0.58cd 100.0±0.00ad 263.67*** 1000 66.67±2.08bc 42.67±3.06ba 55.67±0.58bb 90.33±0.58bd 100.0±0.00ae 594.27*** 2000 57.00±3.00ac 25.33±2.08aa 40.67±2.52ab 84.67±1.53ad 100.0±0.00ae 644.53*** f 192.67*** 592.68*** 729.05*** 168.93*** 0.00ns mce: methanolic crude extract, nhf: n-hexane fraction, chf: chloroform fraction, eaf: ethyl-acetate fraction, mtf: methanol fraction means ± se in the same column for the same category of concentration, followed by the same small letter and in the same row for the same category of extract, followed by the same capital letter do not differ significantly at p= 0.05 (duncan‘s test). each datum represents the mean of three replicates of 100 eggs each. ns= p> 0.05, ***= p< 0.001 table 2. lc50 and lc90values [ppm (95% fiducial limits)] at 24 h of fractions of annona senegalensis extract against fourth instar larvae of anopheles gambiae and culex quinquefasciatus mce= methanolic crude extract, nhf= n-hexane fraction, chf= chloroform fraction, eaf= ethyl-acetate fraction, mtf: methanol fraction, fl= fiducial limit, lc= lethal concentration, ns= no significant. *= p< 0.01, variances and covariances have been multiplied by the heterogeneity factor in computing fiducial limits of lethal concentrations because the probability of χ2 value p< 0.05, = undetermined values because of low or no mortality table 3. qualitative phytochemical screening of some components of extract/fractions of annona senegalensis photochemical components annona senegalensis mce nhf chf eaf mtf alkaloids + + + _ _ flavonoids + + + + _ saponins + _ _ + + tannins + + + + + phenolic compounds + + + _ _ steroids _ _ _ _ _ fats and oils + + _ _ + terpenoids + _ _ + + mce= methanolic crude extract, nhf= n-hexane fraction, chf= chloroform fraction, eaf= ethyl-acetate fraction, mtf= methanol fraction, +=present, = absent mosquito species extracts slope±se r2 lc50 (95% fl) lc90 (95% fl) χ 2 an. gambiae mce 2.69±0.14 0.98 973.3 (840.9-1132.7) 2914.8 (2290.3-4130.2) 33.44* nhf 4.54±0.38 0.77 298.8 (274.6-320.1) 572.9 (530.6-629.0) 8.63ns chf 4.37±0.27 0.85 418.3 (394.3-441.9) 822.0 (760.6-902.6) 16.19ns eaf 2.38±0.18 0.97 2789.3 (2464.3-3256.2) 9662.1 (7427.4-13697.9) 17.31ns mtf 2.45±0.46 0.70 8511.4 (5608.7-20304.7) 28360.3 (13685.4-133304.7) 15.37ns cx. quinquefasciatus mce 1.64±0.17 0.94 5884.1 (3888.6-12610.7) 35659.9 (15583.6-175387.5) 19.36* nhf 1.68±0.14 0.98 2807.6 (2382.6-3467.3) 16240.9 (11105.9-27263.4) 14.01ns chf 1.95±0.28 0.88 9010.1 (6024.9-18200.9) 40915.2 (19789.1-149218.8) 16.05ns eaf mtf table 1. continued… http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 232 fig. 1. pupicidal activity of extract/fractions of annona senegalensis against anopheles gambiae fig. 2. pupicidal activity of extract/fractions of annona senegalensis against culex quinquefasciatus discussion overall, ovicidal, larvicidal and pupicidal activity of various extract or fractions of the leaves of a. senegalensis were demonstrated on an. gambiae and cx. quinquefasciatus mosquito species. thus, toxicity of fractions varied with solvent used. n-hexane and chloroform fractions showed promising mosquitocidal activity. indeed, plants are extensively reported to possess toxic effect on the mosquitoes and can be utilized as a potent source of mosquito control (kumar et al. 2012). the secondary metabolites are known to be effective against a wide range of insect pests as well as mosquito vectors (sriwattanarungsee et al. 2008). these compounds may jointly or independently prove its efficacy against the mosquito targets by its ovicidal, larvicidal, pupicidal, adulticidal and by inhibition of growth activity (borah et al. 2010). earlier, some plants such as annona squamosa l., gloriosa superba l., millingtonia hortensis, abuta grandifolia, minthostachys setose, azadirachta indica, ocimum gratissimum and hyptis suaveolens, etc have been reported to control mosquito population (ciccia et al. 2000, kaushik and saini 2008, bagavan et al. 2009, okigbo et al. 2010). the ovicidal effects were generally dose dependent. rajkumar and jebanesan (2004) recorded similar observations in their study of ovicidal activity of moschosma polystachyum leaf extract against cx. quinquefasciatus. compared to the present study, a complete inhibition of egg hatching of an. gambiae with methanolic extract of hyptis suaveolens was reported by ivoke et al. (2009). the ethanolic flower extract calotropis gigantea has exhibited toxic effect on egg rafts of cx. quinquefasciatus with 100 percent mortality at 200 ppm (dhivya and manimegalai 2013). govindarajan et al. (2011) recorded similar findings in which the methanol leaf extract of coccinia indica exerted zero hatchability (100% mortality) at 150 ppm for cx. quinquefasciatus. no hatchability eggs of ae. aegypti were also recorded with methanol, benzene and acetone extracts of cassia fistula at 160 mg/l (govindarajan 2009). on the contrary of this study, ovicidal effect of a. senegalensis was obtained with ethyl-acetate and methanol fractions on the eggs of caryedon serratus (gueye et al. 2011). this difference can be explained by the difference on insect species used and the phytochemical constituents of mce nhf chf eaf mtf extract/ fractions mce nhf chf eaf mtf extract/ fractions % m o rt al it y % m o rt al it y culex quinquefasciatus anopheles gambiae 100.00 80.00 60.00 40.00 20.00 0.00 100.00 80.00 60.00 40.00 20.00 0.00 http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 233 plant extracts and extraction solvent used (shalaby et al. 1998). in comparison to this study, the chloroform soluble fractions of tagetes erecta showed the highest toxicity than the other samples and consequently, the lowest lc50 values (75.48 mg/l) in fourth instar larvae of cx. quinquefasciatus (farjana et al. 2011). the hexane extracts of cleistanthus collinus and murraya koeingii plants showed 100 percent mortality at 24 h bioassay against the third instar larvae of cx. quinquefasciatus at 1000 ppm concentration (tennyson et al. 2012). a significant mortality of four instar larvae of cx. quinquefasciatus was also recorded with methanol extract of cliona celata with lc50= 95.63 ppm and lc90= 242.16 ppm (appadurai et al. 2013). the results are also comparable with an earlier report by fred-jaiyesimi and anthony (2011) reported that the effects of the methanol extract, petroleum ether and chloroform fractions of paullinia pinnata leaf have been investigated against the third and fourth instar larvae of an. gambiae. govindarajan et al. (2008) reported also that methanolic leaf extract of acalypha indica was more lethal to the egg and larvae of cx. quinquefasciatus and an. stephensi. kamaraj et al. 2011 reported values at 1000 ppm of 78, 73, 75 and 100 % mortality larvae of an. subpictus with n-hexane, chloroform, ethyl-acetate and methanol bark extracts of annona squamosa respectively. anupam et al. (2012) reported that n-hexane is the most non polar (polarity index of 0.1) that mainly extracts essential oil, chloroform or ethyl acetate are moderately polar (polarity index of 4.1) that mainly extracts steroids, alkaloids, etc. the presence of these phytochemicals distributed in each fraction confers to the fraction, its larvicidal property. prabhu and murugan (2011) observed a significant pupicidal effect of plant extracts of moringa oleifera against an. stephensi and pupal mortality of greater than 70 % was encountered, similar to the results of the present study with an. gambiae. the pupae of ae. aegypti were found greatly susceptible to higher dose (230 ppm) of plant extract catharanthus roseus which caused mortality of 79 % (remia and logaswamy 2010) confirms the findings of the present study with n-hexane fraction on an. gambiae. the effectiveness of the extract or fractions of a. senegalensis could be explained by the presence of alkaloids and others compounds which are toxics for eggs, larvae and pupae of mosquito species. earlier, jolad et al. (1984) reported the presence of alkaloids, carbohydrate, lipids, amino acids, polyphenols, essential oils terpenoids the genus annona sp plant. conclusion annona senegalensis contained active ovicidal, larvicidal and pupicidal compound in its leaves. however, these phytochemical compounds found in n-hexane and chloroform fractions could be the key candidates in insecticide values of this plant. this makes it a more suitable candidate for the development of new potential eco-friendly insecticides. further investigation is needed to identify the active compounds of n-hexane and chloroform fractions responsible for its activity. acknowledgements the authors are grateful to the help of the botanist, pr mapongmetsem pm of the department of biological sciences, university of ngaoundere, cameroon, for identification of the plant. the staff of faculty of pharmaceutical sciences, nnamdi azikiwe university, anambra state, nigeria where the laboratory experiments were carried out is highly acknowledged for their kind collaborations. we are also grateful to the laboratory of national arbovirus research center http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 234 (navrc), enugu, nigeria for the mosquito species supplied and identification. no fund was received for conducting this study. the authors declare that there is no conflict of interests. references abbott ws (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–267. akoègninou a, van der burg wj, van der maesen ljg (2006) flore analytique du bénin, éd. backhuys plublishers. anupam g, nandita c, goutam c (2012) plant extracts as potential mosquito larvicide. indian j med res. 135: 581–598. appadurai dr, arokia vk, michael gp, savarimuthu i (2013) larvicidal, ovicidal, and repellent activities of marine sponge cliona celata (grant) extracts against culex quinquefasciatus say and aedes aegypti l. 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(annonaceae). j ethnopharm. 116: 125–130. tare v, deshpande s, sharma rn (2004) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 226–237 y lame et al.: laboratory evaluations of … 237 susceptibility of two different strains of aedes aegypti (diptera: culicidae) to plant oils. j econ entomol. 97: 1734–1736. tennyson s, ravindran kj, arivoli s (2012) screening of twenty five plant extracts for larvicidal activity against culex quinquefasciatus say (diptera: culicidae). asian pac j trop biomed. s1130–s1134. trease ge, evans mc (1989) text-book of pharmacognosy, 13th ed, bailere tindall. who (1996) report of the world health organization informal consultation on the evaluation and testing of insecticides. ctd/ whopes/ic/96, world health organization, geneva, switzerland. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 253 original article fauna and larval habitat characteristics of mosquitoes in neka county, northern iran seyed hassan nikookar 1, *seyed hassan moosa-kazemi 2, mohammad reza yaghoobiershadi 2, hassan vatandoost 2,3, mohammad ali oshaghi 2, abolfazl ataei 2, mehdi anjamrooz 2 1health sciences research center, department of medical entomology and vector control, faculty of health, mazandaran university of medical sciences, sari, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3institue for environmental research, tehran university of medical sciences, tehran, iran (received 29 may 2012; accepted 12 oct 2013) abstract background: ecological studies on mosquitoes are very important in vector control programs. there are a few studies about the ecology of mosquitoes in northern iran. this study was carried out to detect fauna and larval habitat characteristics of mosquitoes. methods: this study aimed to determine fauna and the ecology of mosquitoes in neka county, mazandaran province, northern iran from april to december, 2009. the larval collection was conducted using standard dipper, and the characteristics of larval habitat were investigated based on degree of transparency of water, type of water (stagnant or running), plant vegetation, sunny or shady, temperature and altitude of the natural or artificial breeding places. results: the mosquito larvae were collected from 72 habitats and identified using systematic keys. nine species of mosquitoes were identified: anopheles claviger (0.31%), an. maculipennis (0.54%), an. plumbeus (10.28%), an. superpictus (0.01%), culiseta annulata (1.07%), cs. longiareolata (8.91%), culex mimeticus (0.03%), cx. pipiens (63.99%), and ochlerotatus geniculatus (14.85%). the range of temperature in the larval habitats was 19.6–22.5 °c. significant difference was observed in the rate of temperature among the species in the larval habitats (p< 0.05). a checklist of mosquitoes including seven genera and 32 species has been provided for mazandaran province. conclusion: the most dominant species were cx. pipiens. they were collected from the larval habitats like border rivers, ponds, rain water pools, discarded tires and tree holes. culiseta annulata was included to the checklist of mosquitoes in mazandaran province. keywords: larval habitat, culicidae, ecology, iran introduction according to the latest classification of culicidae, the family includes two subfamilies, 11 tribes, 112 genera and 3537 species. the subfamily anophelinae has three genera and culicinae has 109 genera (harbach 2007). many of the world's major diseases such as malaria, west nile, sindbis, dirofilariasis, dengue fever, yellow fever, rift valley fever are transmitted by mosquitoes. west nile and sindbis viruses have been reported in northern parts of iran (naficy and saidi 1970, saidi et al. 1976). the mosquito borne filarial worms, dirofilaria immitis and d. repens, setaria (s. labiatopapillosa, s. digitata and s. equina) and diptalonema evansi (camel filariasis) have been reported in iran (siavashi and massoud 1995, eslami 1997, azari-hamidian et al. 2007, oryan et al. 2008). anopheles maculipennis and culex theileri were reported as vectors of setaria labiatopapillosa and *corresponding author: dr seyed hassan moosakazemi, e-mail: moosakazemi@tums.ac.ir http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 254 dirofilaria immitis respectively, in ardebil province (azari-hamidian et al. 2009). dengue fever cases were reported in tehran, imported from malaysia, india and thailand. in addition, six df cases were reported from sistan and baluchistan province in southeast iran (chinikar et al. 2010, 2012). the systematic key of anopheles species in iran was reported by shahgudian (1960). many studies have been carried out about the genus of culex and its medical importance (ghaffary 1956, lotfi 1970, lotfi 1973, lotfi 1976, zaim et al. 1984a, 1984b, 1985, 1986). the results of these studies have led to the publishing of the iranian culicinae checklist (zaim and cranston 1986). the role of mosquitoes in malaria transmission and its geographical distribution have been studied by saebi (1987). the fauna and ecological characteristics of culicidae with emphasis on their medical importance were conducted in isfahan and hormozgan provinces as well as chabahar county (mousakazemi et al. 2000, moosa-kazemi et al. 2005, 2009) and guilan province (azarihamidian et al. 2002a). moreover, the study on the genus of aedes is very scattered in the northern parts of the iran. azari-hamidian et al. (2002a) stressed the occurrence of oc. caspius, oc. echinus, ae. vexans and oc. geniculatus in guilan province, northern iran. more than 30 culicidae species reported from guilan province (azari-hamidian et al. 2003a). distribution of larval habitat of cx. pipiens was reported by dehghan et al. (2011) in central iran. among the 467 species of anopheles identified in the world (harbach 2007), about 70 anopheles species are malaria vectors in which about 40 species are important vectors (service 1993). anopheles maculipennis complex, one of the most important vectors of malaria, was reported in northern iran (eslami 1997, azari-hamidian 2007a). anopheles maculipennis, and cx. pipiens were reported as dominant species in isfahan province, central iran (mousakazemi et al. 2000a) and in east azarbaijan and ardebil provinces (abai et al. 2007, azarihamidian et al. 2009). by now, seven species of malaria vectors have been reported in iran (vatandoost et al. 2009a). they include: an. culicifacies, an. dthali, an. fluviatilis, an. maculipennis, an. sacharovi, an. stephensi, and an. superpictus (edrissian 2006, vatandoost et al. 2008, vatandoost et al. 2009a, moosa-kazemi et al. 2010). anopheles pulcherrimus has been identified as potential vector in iran using serological method (zaim et al. 1993). although oocyte of plasmodium was found in the stomach of an. multicolor but sporozoite never found in its salivary gland and not considered as the malaria vector (eshghy 1977). anopheles hyrchanus was found as a malaria vector using the pcr method in northern iran (dinparast djadid et al. 2009). at present, 64 species, three subspecies and seven genera of culicidae have been reported from iran (azari-hamidian 2007a). the first study about the mosquitoes in mazandaran province was carried out by gutzevich (1943) and zolotarev (1945). the occurrence of the species of an. maculipennis, an. melanoon (an. subalpinus), an. superpictus, an. sacharovi and an. messeae have been reported by zolotarev (1945). however, gutsevich (1943) reported the species of an. pulcherimus, ae. vexans, oc. geniculatus and cq. richiardii in mazandaran province. subsequently, garrett jones (1951) reported the species of an. claviger, an. maculipennis, an. plumbeus and an. hyrcanus in klardasht. institute of parasitology and malariology (1953) reported the species of an. marteri in ramsar. dow (1953) reported the species of an. maculipennis, an. melanoon, an. subalpinus (synonym of melanoon), an. hyrcanus, an. pseudopictus, an. superpictus, an. sacharovi, cx. pipiens, cx. mimeticus and cx. tritaeniorhynchus. however, faghih (1969) stressed the occurrence of the species http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 255 of an. melanoon, and an. messeae. minar (1974) reported the species of an. maculipennis, an. pseudopictus, oc. pulcritarsis. however, zaim et al. (1984a) cited the species of ae. vexans, oc. echinus, oc. geniculatus and oc. pulcritarsis. zaim (1987) cited the species of ae. vexans, oc. echinus, oc. geniculatus, oc. pulcritarsis, cx. perexiguss, cx. theileri, cx. mimeticus, cx. tritaeniorhynchus, cx. hortensis, cx. pipiens, cs. longiareolata and cs. subochrea. saebi (1987) stated the species of an. plumbeus, an. hyrchanus, an. psudopictus, an. maculipennis and an. claviger. momeni (1991) reported the species of an. messeae, an. melanoon, and an. maculipennis. glick (1992) reported the species of an. psudopictus. karami (2000) cited the presence of cx. bitaeniorhynchus, ur. unguiculata, oc. caspius. sedaghat et al. (2003) reported the species of an. maculipennis, an. sacharovi, an. persiensis in mazandaran province. sedaghat and harbach (2005) stated the occurrence of the species of an. melanoon, an. persiensis, an. pseudopictus. moreover, dinparast et al. (2007) cited the species of an. atroparvous and an. persiensis. dinparast et al. (2009) stressed the presence of the species of an. hyrchanus. nikookar et al. (2010) reported the species of an. plumbeus, oc. echinus, oc. geniculatus, oc. pulcritarsis, cx. pipiens and cs. annulata. iran is in the malaria pre elimination stage (who 2008), thus the bionomic study of mosquitoes in this country is very important. there are scattered studies on the fauna and ecological properties of mosquitoes in mazandaran province. many travelers may travel to mazandaran province and neka county during visiting iran. this part of iran holds specific location due to access to the caspian sea and to other aspects such as trade, commerce and navigation. this study aimed to determine fauna and the ecology of mosquitoes in neka county, mazandaran province, northern iran from april to december, 2009. the data obtain in this study will be valuable to develop programs for future planning of mosquito control in this area. materials and methods the study area mazandaran province is located in the northern iran between the caspian sea and the alborz mountains (36°39 n, 53°17 e) (fig. 1). this area located in the north with caspian sea, in the south with tehran and semnan provinces, in the east with golestan province and in the west guilan province, including 19 counties (geography of mazandaran province 2013). maximum and minimum temperatures were 27.6 and 9.1 °c in july and december 2009, respectively. the maximum annual rainfall has been reported 204.6 mm in october, and the least was 0.1 mm in july 2009. the most important crop of the region is rice, and hyrcanian forest area and mild and rainy weather caused this province to be a suitable mosquito habitat (mazandaran local meteorological office 2009). three fixed villages, namely darvishan, chalmardi, and khairabad, were randomly selected for field survey. moreover, six variable villages (golbestan, elmiyeh, baghearab, nimchah, chalehpol, and khorshid) were selected with regard to existing facilities in neka county. in each fixed village, six fixed and two variable locations were selected in plain, slope and mountain conditions. larvae sampling was collected from the natural larval habitats of fixed villages including river margins, ponds and water craters, craters rain water, marsh, tree holes, rice fields, worn tires, irrigation canals, wells, and sewage, using the standard dipper rate of 350 mm capacity every 15 days (who 1963). in addition, larval collection carried out in variable villages randomly. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 256 we attempted to collect the data about the biological and physical characteristics of the larval habitats, including running water, residents (permanent or temporary), the type of larval habitats natural or artificial, presence or absence of the plant, type of substrate (sand, clay, or rocky), to be sunny (sunny or shady), water status (clear or opaque) and water temperature. the larval stages iii and iv were preserved in 100 % lactophenol solution and later identified morphologically using the keys of shahgudian (1960), zaim and cranston (1986a), harbach (1985, 1988), and azarihamidian and harbach (2009). the results were analyzed by spss 11.5 software. the abbreviations of the genus names were regulated based on reinert (2009). results a total of 32530 larvae were collected: 3625 larvae of anophelinae and 28905 culicinae. table 1 shows the results of anophelinae and culicinae species in the fixed and variable breeding places and tree holes. four species of anopheles were collected: an. claviger (0.31%), an. maculipennis (0.54%), an. plumbeus (10.28%), and an. superpictus (0.01%). anopheles claviger collected 0.31 %, 2.78 % of total culicidae and anopheles collection, respectively (table 1). the characteristics of the habitats under different temperature and altitude conditions are shown in table 2. the minimum and maximum temperature of water of the larval habitats was calculated as 19 °c and 22 °c, respectively (average 20.5 °c). on the other hand, the maximum and minimum altitude of the habitats was 300 and 200 meters, respectively (average 250 meters). anopheles maculipennis was collected 0.54 %, 4.82 % of the total and anopheles collection, respectively. maximum and minimum water temperature was reported as 26 °c and 17 °c, respectively (average 22.5 °c). maximum and minimum altitude was 300 and 180 meters respectively, (average 240 meters). anopheles plumbeus was collected 10.28 %, 92.27 % of total and anopheles collection, respectively. moreover, this species was collected in tree holes and the artificial habitats such as pond water. maximum and minimum temperature was found as16 °c and 14 °c, respectively (average 15 °c). the minimum and maximum sea level of larval habitat was calculated at least 180 and 250 meters, respectivelly and the average were 215 meters. anopheles superpictus was collected 0.01 %, 0.13 % of the total and anopheles collection, respectively. minimum and maximum temperature of the larval habitat was reported as 17 °c and 19 °c, respectively and average was 180 °c. the minimum altitude was 280 meters, and the maximum was 300 (average 290 meters).the optimum of temperature and favorable altitudes of other species are presented in tables 1 and 2. the majority of an. plumbeus was collected in natural, permanent, and stagnant water with vegetation, turbid and transparent water and semi-shade tree holes are shown in table 3. an. superpictus larvae were collected from natural, permanent, stagnant water with vegetation, clear and transparent breeding places. on the other hand, an. claviger larvae were collected mostly in permanent, and stagnant, transparent water with semi-shade and vegetation. culex mimeticus was found in the natural habitats such as river and ponds from permanent river, and stagnant pools with vegetation. the larvae of cx. pipiens, the most abundant species, were found in artificial habitats and sewage breeding places in agricultural lands and low abundance along the river. moreover, oc. geniculatus, the most dominant species, was found in permanent water with vegetation in slow running with clay substrate water, semihttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 257 shade in natural breeding places. the larvae of cs. longiareolata were found in most the larval habitats. the larval habitat characteristics of the other species are shown in table 3. checklist of culicidae in mazandaran province family culicidae subfamily anophelinae subgenus anopheles meigen 1an. (ano.) algeriensis 2an. (ano.) claviger 3an. (ano.) marteri 4an. (ano.) maculipennis 5an. (ano.) atroparvus 6an. (ano.) melanoon 7an. (ano.) messeae 8an. (ano.) persiensis 9an. (ano.) sacharovi 10an. (ano.) plumbeus 11an. (ano.) hyrcanus 12an. (ano.) pseudopictus subfamily culicinae subgenus cellia theobald 13an. (cel.) pulcherrimus 14an. (cel.) superpictus subgenus aedimorphus theobald 15ae. (adm.) vexans subgenus finlaya 16oc. (fin.) echinus 17oc. (fin.) geniculatus subgenus ochlerotatus 18oc. (och.) caspius 19oc. (och.) pulcritarsis subgenus culex 20cx. (cux.) pipiens 21cx. (cux.) perexiguss 22cx. (cux.) territans 23cx. (cux.) theileri 24cx. (cux.) mimeticus 25cx. (cux.) tritaeniorhynchus 26cx. (cux.) hortensis subgenus allotheobaldia 27cs. (all.) longiareolata subgenus culiseta 28cs. (cus.) annulata 29cs. (cus.) subochrea subgenus coquillettidia 30cq. (coq.) richiardii subgenus pseudoficalbia 31ur. (pfc.) unguiculata fig. 1. map of iran and neka county, northern iran http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 258 table 1. abundance of larval mosquitoes (density per 10 dip) in neka county, mazandaran province, northern iran, 2009 %n villages species variablekhairabadchalmardidarvishan 0.311000262an. claviger 0.5417503.5410an. maculipennis 10.283345033301.5an. plumbeus 0.015000.50an. superpictus 1.0735008252cs. annulata 8.9129001620221.550.5cs. longiareolata 0.031000.50.50cx. mimeticus 63.992081569991494527.5cx. pipiens 14.854830048300oc. geniculatus table 2. species composition of mosquito larvae collected from different temperatures and altitudes in neka county, northern iran, 2009 altitudetemperature c◦ meanmaximumminimummeanmaximumminimumspecies 25030020020.52219an. claviger 24030018022.52617an. maculipennis 215250180151614an. plumbeus 290300280181917an. superpictus 240300180172212cs. annulata 24030018018.52512cs. longiareolata 25030020012.51312cx. mimeticus 24030018018.52512cx. pipiens 24025023012.51411oc.geniculatus table 3. larval habitat characteristics in neka county, mazandaran province, northern iran, 2009 a n . claviger a n . m acu lipen n is a n .plu m beu s a n . su perpictu s c s.an n u lata c s. lon giareolata c x. m im eticu s c x.pipien s o c.gen icu latu s habitat status permanent 91 95.3 91.3 66.2 82.5 75.1 100 71.6 99 temporary 9 4.7 8.7 33.8 17.5 24.9 0 28.4 1 water slow running 10 16 20 29.5 10 2 3 30 0 stagnant 90 84 80 70.5 90 98 87 70 100 vegetation without vegetation 2 2 7.6 20 5 29 19.6 13 0 with vegetation 98 98 92.4 80 95 71 80.4 87 100 type of bed clay 47 1 84.5 50 98 88 66 56 94 sand 3 17 15.5 20 2 5 30 33 6 stone or cement 50 82 0 30 0 5 4 11 0 http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 259 water status opaque 1 40 50 9.1 50 40 40 45 50 transparent 99 60 50 90.9 50 60 60 55 50 status light sunny 1 33.2 0 14.7 0 2.26 26.7 37.4 0 shaded 34 28.4 5 26.1 5 26.9 19.4 18.9 5 semi-shade 65 38.4 95 59.2 95 70.5 54.9 43.7 95 habitat type natural 99 55 98 89 98 97 70 41 100 artificial 1 45 2 11 2 3 30 59 0 discussion in the present study, an. plumbeus larvae were collected from tree holes and artificial habitats such as permanent and stagnant water. however, the maximum and minimum of temperature of the larval habitat was as 16 °c and 14 °c, respectively. in parallel, azarihamidian (2011a) found this species in tree holes and discarded tire. the most favorable temperature is 15–18 °c; however, the species has tolerated to freeze (up to -6° c to -8 °c) in the laboratory (horsfall 1955). however, garrett jones reported the occurrence of this species in kelardasht of mazandaran province (garrett jones 1951). in our investigation, cs. annulata was found in natural, permanent, stagnant, semishade habitats with vegetation. this species has been reported in guilan province, alborz and zagros mountains, east bushehr and robat tork of isfahan province (azarihamidian et al. 2003b). zaim and cranston (1986) included cs. subochrea in their systematic keys and cs. annulata in their checklist. the taxonomy and distribution of cs. annulata, cs. subochrea and cs. alaskaensis need to be more investigated in iran (azarihamidian 2005). record of cs. annulata in our research was based on adults, whereas cs. subochrea were not found in our collection, and neither were the adults of this species (table1). culiseta subochrea and cs. annulata were reported in ardebil province (azari-hamidian et al. 2009). the larval habitats of cs. annulata have also been reported from various places except tree holes (azari-hamidian et al. 2003b). however, an. plumbeus and oc. geniculatus species have been reported in tree holes (nikookar et al. 2010). in our study, the larvae of cs. longiareolata were collected from all larval habitats except tree holes, which comprised 8.91 %, 10.03 % of the total and culicinae collection, respectively. this species was reported first time in north of the country by gutzevich (1943). the occurrence of this species was also reported by the previous study in mazandaran province (zaim 1987). the larval habitat of this species has been reported from many parts of iran (zaim 1987). at the same study, the larval habitat has been reported as contaminated with organic materials, in old pit located in outdoor places and rice fields (kitron and pener 1986, mousakazemi et al. 2000, azari-hamidian 2005). it has also been reported from the north khorasan and qom provinces (azarihamidian et al. 2011, saghafipour et al. 2012). there is scatter information about the distribution and taxonomy of the three species of cs. alaskaensis, cs. annulata, and cs. subochrea in iran (zaim et al. 1986, azari-hamidian et al. 2003b, azari-hamidian 2005). moosa kazemi et al. (2010) found the species of cs. subochrea and cs. longiareolata http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 260 in sanandaj county of kurdistan province, northwestern iran. in our research, an. claviger was found in permanent, stagnant water with vegetation habitat, as well as in the clay and stone substrate of water. in parallel, macan (1950) has reported the existence of this species in the sunny/shadowy springs, and slow running pools water temperature of 14–16 °c in iraq and western iran. dow (1953) reported the larval habitat of this species in shallow and small pools with little vegetation. it was also found within the larval habitats with temperature of 12–15 °c (horsfall 1955). the occurrence of this species in mazandaran was reported previously by garrett jones (garrett jones 1951). zaim (1987) collected an. claviger larvae from kashan, central iran. in our study, an. maculipennis was found in permanent, transparent, semi-shady natural breeding places with vegetation habitat, as well as in the cement or stone substrate of water. in parallel, azari-hamidian (2011) reported the presence of this species in the stagnant, gravel substrate, sunny springs, and pools with stagnant water. anopheles maculipennis complex formally comprises 12 palearctic members, including an. atroparvus, an. beklemishevi, an. labranchiae, an. maculipennis, an. martinius, an. melanoon, an. messeae, an. sacharovi, an. persiensis, an. daciae, an. lewisi and an. artemievi (dinparast djadid et al. 2007). anopheles maculipennis species associated by an. hyrcanus, an. claviger from mazandaran province previously (nikookar et al. 2010). this species has been identified in guilan (azari-hamidian et al. 2004), mazandaran and golestan provinces (zaim et al. 1986). anophelesmesseae and anopheles melanon reported from guilan province, and an. sacharovi and an. hyrcanus from golestan province (saebi 1987). anopheles sacharovi has been reported from mazandaran province (sedaghat et al. 2003). dow reported the species of an. subalpinus in sari, babolsar, chalus and astaneh cities in mazandaran province. he also reported an. melanoon in astaneh city, guilan province (dow 1983). anopheles subalpinus is a synonyms an. melanoon. initial studies mainly focused on identification of the mosquitoes and their role in disease transmission (gutsevich 1943, macan 1950, dow 1953, minar 1974). in our investigation, an. superpictus was found as 0.01 %, 0.13 % of the total and anopheles collection, respectively. this species was found in permanent, stagnant, with clay substrate, transparent water, semi-shade, natural with vegetation habitat. studies in different parts of mazandaran province confirmed the occurrence of this species (zolotarev 1945, dow 1953). moreover, azari-hamidian (2011) stated its presence in stagnant, transient, mud substrate, fully sunlight places with vegetation in natural habitats. further support for our results comes from a study by mousakazemi et al. (2000) in zarrin-shahr and mobarakeh areas of isfahan province. some information on the larval stage of an. superpictus was reported by yaghoobi-ershadi et al. (2001) in ardebil province, northwestern iran. some ecological aspects of this species have been in rasht county of guilan province (azarihamidian et al. 2002b). ghanbari et al. (2005) reported the larval breeding places of mosquitoes including anophelines in iranshahr, southeastern iran. however, the edges of stony streams with sunlight and semishallow have been mentioned as the main breeding places of this species in western iran (macan 1950). three genotypes named x, y, and z within an. superpictus have been reported in iran by oshaghi et al. (2008). there are no reports about the genotypes of this species in mazandaran province, however, it needs to be studied by the future investigations. in our study, culex pipiens was found as 63.99 % of the total and 72.01 % of culicinae larvae collection in natural and http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 261 artificial larval habitats such as tree holes. in addition, hollow pipes, hot water, holes hungarian sewage, stagnant water, and toilet field were reported as the breeding places of this species (jupp 1970, kulanin 1980, zaim 1987, vinogradova 2000, mousakazemi et al. 2000). this species were reported in natural habitat (rain pool, river edge) with standing water azari-hamidian (2007b). the species of cx. pipiens and cx. quinquefasciatus are very similar. there are no reports in regarding the existence of cx. quiquefasciatus in mazandaran province. the shape of siphon of larvae in the tip, middle and the base has been mentioned to identification of two recent species (zaim and cranston 1986). however, azari-hamidian and harbach (2009) mentiond the existence of two branches of seta 1 on abdominal segment iii-vi for cx. pipiens and one branch for cx. quinquefasciatus . the common characteristics among the species of cx. vagans, cx. torrentium, and cx. pipiens, were reported as the presence of two pairs of long seta on the base of siphon (zaim and cranston 1986). the existence of seta 1 with 4–5 branches and seta 1-x with two branches is attributed to cx. torrentium and two branches of seta 1 on abdominal segment iii-v mentioned to cx. vagans (harbach 1985). in our study, ochlerotatus geniculatus was collected as comprising 14.85 %, 16.7 % of the total and culicinae larvae collections, respectively. in addition, the most favorable temperature was 11–14 °c. maximum and minimum altitude of the larval habitat was 250 meters and 230 meters, respectively and the average was 240 meters. further support of this result comes from some previous studies (gutsevich 1943, zaim et al. 1984a, zaim 1987, azari-hamidian et al. 2001, nikookar et al. 2010). this species is very similar to oc. echinus as reported in a recent study (nikookar et al. 2010). ochlerotatus echinus is distributed in palaearctic, europe, north africa, and south east asia, and in iran, it has been reported from guilan province in the north (knight and stone 1977, azarai-hamidian et al. 2002). in addition, this species were collected by janbakhsh in sari county and reported by zaim (1984a). studies from some parts of mazandaran province showed the occurrence of this species (zaim et al. 1984a, zaim 1987, nikookar et al. 2010). moreover, the distribution of this species has been reported from both guilan, mazandaran provinces (azari-hamidian et al. 2002a), as well as from gulestan (gutsevich 1943), and ardebil (azari-hamidian et al. 2009). in our study, culex mimeticus was found as comprising 0.03 % of the total larvae collection in permanent, stagnant, transparent water, clay substrate, semi-shade natural breeding places. in parallel, zaim (1987) stressed the permanent, slow running water, full sunlight, mud substrate, transient and fresh water with vegetation in natural stream bed pools and rice fields as the breeding places of this species. studies from different parts of mazandaran confirmed the occurrence of this species (dow 1953, zaim 1987). conclusion our results indicate that the presence of the dominant species of cx. pipiens in larval habitat natural and artificial oc. geniculatus and an. plumbeus in natural such as tree hole. these species dramatically increased and reach to peak in warm season. the recent species could be creating nuisance and health risks in this area. acknowledgements the authors are grateful to dr azarihamidian for giving the useful comments, dr davood nikzad, mr hamid ravesh, mr mojtaba asghari, mr mirramazan nikookar, http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 253–266 s h nikookar et al.: fauna and larval … 262 mr mehrdad badangiz, and mr mohammad badangiz (environmental health director of neka) for supporting the investigation. we also would like to express our appreciation to the people of darvishan, khairabad and chelmardi villages in neka county for their kind cooperation during the study. this study was financially supported by tehran university of medical sciences (no: 1051327-01-89). the authors declare that there is no conflict of interests. references abai mr, azari-hamidian s, ladonni h, hakimi m, mashhadi-esmail k, sheikhzadeh k, kousha a, vatandoost h (2007) fauna and checklist of mosquitoes (diptera: culicidae) of east azerbaijan, northwestern iran. iran j arthropod-borne dis.1: 27–33. azari-hamidian s, yaghoobi-ershadi mr, javadian e (2001) a faunistic study of mosquito (diptera: culicidae) fauna in rasht county (iran). modarres j 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potent lethal/repellent agents against rhipicephalus bursa, probable carrier for zoonosis samin madreseh-ghahfarokhi1; *amir dehghani-samani2,3; yaser pirali4; azam dehghani-samani5 1department of clinical sciences, faculty of veterinary medicine, ferdowsi university of mashhad, mashhad, iran 2faculty of medicine, birjand university of medical sciences, birjand, iran 3department of clinical sciences, faculty of veterinary medicine, shahrekord university, shahrekord, iran 4department of pathobiology, faculty of veterinary medicine, shahrekord university, shahrekord, iran 5faculty of veterinary medicine, shahrekord university, shahrekord, iran (received 20 jun 2017; accepted 17 june 2019) abstract background: rhipicephalus bursa is a hard tick with importance in transmission of tick-borne diseases and zoonosis. natural products are excellent alternative to pesticides. in this study for the first time, lethal and repellent activity of zingiber officinalis and eucalyptus globulus against rh. bursa were studied. methods: in july till september of 2017, essential oils were extracted in shahrekord university, iran from fresh plant materials and engorged mature ticks were collected from infested sheep herd located in saman, iran. ticks were challenged by different concentrations of essential oils including 300, 500 and 800µl/ml and 1 (pure) singly and/or in combination. percentages of killed and repellent ticks as efficacy of acaricidal and repellent activity of essential oils against rh. bursa were calculated and analyzed statistically. results: efficacy of eucalyptus essential oils was more in whole concentrations and its highest performance was observed in concentration 1 (pure). efficacies of ginger and combined essential oils were different for each concentration but their highest efficacies were observed in concentration 1 (pure), too. conclusion: this study showed considerable values of acaricidal and repellent activity against rh. bursa for both essential oils singly and in combination, so they can be considered as potent lethal/repellent agents for control of ticks, but more studies need for this purpose, study on lethal/repellent activity of these essential oils and/or other plants against other important arthropods can be considered as subjects for next experiments. keywords: acaricidal activity; essential oil; eucalyptus; ginger; repellent activity introduction ticks can transfer some of important diseases that named tick-borne diseases (1). rhipicephalus bursa is a hard tick from the genus rhipicephalus which is the fourth largest in the family ixodidae and has an important role in transferring of different diseases (2). sheep, goats, cattle, horses are the preferred hosts of rh. bursa, it is a two-host species, usually has a mono-tropic type of behavior, with immature stages feeding on the same host species as the adult tick stages. however, the immaturestages can also feed on rodents and rabbits (3). rhipicephalus bursa is an important member of common tick fauna in different part of iran (4–6) and several important microorganisms including: crimean congo hemorrhagic fever (cchf) virus (7), ehrlichiosis bacteria (8), anaplasma phagocytophilum (9), rickettsia aeschlimannii (10) and other bacteria (11) were isolated repeatedly from rh. bursa. use of chemical pesticide agents is increasing every day in order to control ticks populations. about 2.5 million tons of pesticides are used on crops each year and the *corresponding author: dr amir dehghani-samani, e-mail: amirds2008@gmail.com mailto:amirds2008@gmail.com j arthropod-borne dis, june 2019, 13(2): 214–223 s madreseh-ghahfarokhi et al.: zingiber officinalis and … 215 http://jad.tums.ac.ir published online: june 24, 2019 worldwide damage caused by pesticides reaches $100 billion annually. the reasons for this are: (a) the high toxicity and non-biodegradable properties of pesticides and (b) the residues in soil, water resources and crops that affect public health (12). addition to environmental concerns, there are several reports about insecticide-resistant against chemical pesticides like pyrethroid and propetamphos in ticks including rh. bursa (13, 14). thus, searching for replace of chemical pesticides is necessary in order to firstly solve the problem of long term toxicity to mammals and secondly to find environmental friendly pesticides and develop techniques used to reduce pesticide use while maintaining crop yields. natural products are an excellent alternative to chemical insecticides. several plants contain compounds that they use in preventing attack from arthropods and/ or insects. these chemicals fall into several categories, including repellents, feeding deterrents, toxins, and growth regulators (15). ginger (rhizome of zingiber officinale roscoe) is one of the most widely used herbal medications in oriental medicine against pain, inflammation, stomach problems, nausea, vomiting, epilepsy, sore throat, cough, common cold, bruises, wounds, liver complaints, rheumatism, muscular pains, atherosclerosis, migraine headaches, high cholesterol, ulcers, and etc. (16). ginger essential oil can produce from fresh rhizomes and it has many efficient effects like antibacterial, antiviral, antifungal and other properties (17, 18). moreover, the genus eucalyptus that knows by over 700 species distributed throughout the world (19), provides variety of components extracted from its essential oil known as insecticide and repellent agent (20). antimicrobial and antioxidant activity of eucalyptus essential oil were reported (21, 22), also effect of eucalyptus essential oil on respiratory bacteria and viruses, traditional use of eucalyptus in treatment of rhino-sinusitis and anti-diabetic effect of eucalyptus were identified (23–25). there is no study on lethal and repellent activity of z. officinalis and e. globulus essential oils against rh. bursa, and this is the first survey in this way. in this study acaricidal and repellent activity of essential oils of ginger and eucalyptus against rh. bursa as a widedistributed species of ticks in different parts of iran with an important role in transmission of zoonosis were studied singly and in combination with together. materials and methods extraction of essential oils current study were done from july till september of 2017, fresh rhizomes of z. officinalis and leafs of e. globulus were used to essential oil extraction. fresh rhizomes of z. officinalis were prepared from the shops and fresh leafs of e. globulus were collected in summer (july 2017) from grown trees located in najafabad city, isfahan province in center of iran (32°50′43″n 51°36′00″e). essential oils extractions were done separately for each sample via conventional hydrodistillation method. hydrodistillation in a clevenger-type apparatus consists of immersing the ground plant material directly in a flask filled with water that is then brought to the boil. vapors carry volatile compounds and the condensate drops on the pentane/ether trap in the inner tube of the apparatus where the volatiles are retained. in current study, 200gr of each sample were crushed and added to 800ml distilled water in a round bottom flask. the flask was heated and the clevenger apparatus was attached. the mixture was boiled at 100 °c and then the temperature was reduced to 60 °c and kept for 3 h, the recovered mixture was allowed to settle and finally essential oil was withdrawn for each sample separately (26). collection of ticks mature engorged male and/or female ticks j arthropod-borne dis, june 2019, 13(2): 214–223 s madreseh-ghahfarokhi et al.: zingiber officinalis and … 216 http://jad.tums.ac.ir published online: june 24, 2019 (disregard of their sex) were collected from a highly infested sheep herd located in the saman city, chaharmahal and bakhtiari province, southwest of iran (32°27′06″n 50°54′ 38″e). infestation was so high in the herd and ticks were mostly presented around or inside of outer ear, perineal area and around the vent and under the groin area, ticks dis-attaching was done via sterile forceps, without any palpation and with the low pressure of forceps and their rotation. guard glass, disposable dress, and multi-layer disposable gloves were used during the work and whole the health concerns were considered. in order to species-specific collecting, morphological aspects of rh. bursa species like color were considered during the collecting procedure. collected ticks were immediately transferred to specific glass boxes, kept in cool place away from any chemical agents and sunlight, transferred to laboratory for experiment. note that dis-attaching of ticks was a difficult procedure and some ticks were injured during the collecting or transferring, that they were removed for experiment. totally, about 700 ticks were collected. confirmed species identification was done under the laboratory optic loop via identification keys for tick's species (3), also health of ticks was controlled again under the loop and probable injured ticks were removed. differentially, 93 % of collected ticks were identified as rh. bursa and about 7% were identified as other species of hard ticks. finally, 640 engorged health ticks were prepared for experiment, in order to deletion of biases in experiment, collected ticks were immediately examined after collection and till the experiment, ticks were incubated in 25 °c and 80% humidity, in common glass box with air circulation. treatments preparation concentrations of 300, 500 and 800µl/ml and 1 (pure) were prepared from essential oils of e. globulus and z. officinalis separately via combination by different amount of normal saline, also mixed concentrations of 150µl/ ml eucalyptus plus 150µl/ml ginger, 250µl/ ml eucalyptus plus 250µl/ml ginger, 400µl /ml eucalyptus plus 400µl/ml ginger and 500 µl/ml eucalyptus plus 500µl/ml ginger were prepared with the same method. in order to sure of tests validity, a positive control group with concentration of 100mg/ml was prepared via dilution of 25ml of ripcord ® (cypermethrin 40%, spiagri company, tehran, iran) in 75ml normal saline, also a negative control group was prepared from pure normal saline without any additive components. evaluation of acaricidal activity of essential oils three replication tests were done for every treatment (including positive and negative control groups) and for each replication 13 health ticks were transferred to plastic nets and dipped in different concentrations of essential oils, and/or positive and negative control solution, with same temperature (25 °c) for 30sec, then they were transferred to specific glass dishes with same condition including humidity, light and air circulation for all of treatments and then were incubated in 25 °c and 80% humidity for two hours. note that all the methods were same for every groups and replications. after the two hours, percentage of died ticks as acaricidal efficacy of each treatment were counted and mean of efficacy for each treatment were calculated and analyzed statistically. for sure whole of died ticks were carefully observed under the laboratory optic loop. evaluation of the repellent activity of essential oils repellent activity of each essential oils against rh. bursa in concentration of 1 (pure) singly and 500µl/ml eucalyptus plus 500µl/ ml ginger in combination were studied by ytube olfactometer bioassay. y-tube olfactometer consists of a glass y-tube with the main arm (the stem) and 2 arms containing one rej arthropod-borne dis, june 2019, 13(2): 214–223 s madreseh-ghahfarokhi et al.: zingiber officinalis and … 217 http://jad.tums.ac.ir published online: june 24, 2019 pellent and control in another one, where a low rate air movement is created by sucking the air in the two arms of the y-tube with a pump connected to the stem. the essential oil sample and control are applied on a paper attached to the arms of the tube. arthropods are introduced into the tube by a hole located at the center (the joint point of the three tubes). after introduction, the hole is closed with a rubber stopper and the pump is operated. after specific time of exposition, the number of arthropods on each of the 2 tubes (treated and control) are scored to assess the percentage of repellency (27, 28). in this study, triplicate tests were done and in each replication 13 ticks were put into every arm and 10 ml of each mentioned treatments was added into treated tube and nothing into control tube. hard ticks are slow, so more time considered and five hours after pumping, the number of repellent ticks was reported as repellent percentage of treatment and control groups. note that after each application of y-tube olfactometer, it was cleaned, washed and dried for next examination. statistical analysis the analyzed data were expressed as the mean±standard error of the mean (sem) using sigma stat (ver. 3.1) software. groups were compared using one-way anova for repeated measurements. a value of (p≤ 0.05) was considered significant. results acaricidal activity of essential oils results of current study showed different amounts of efficacy (lethal effect) for different concentration of essential oils, singly and in combination. highest acaricidal activities against rh. bursa were observed for concentration 1 (pure) of eucalyptus and gingers essential oils singly. the most potent treatment (about 54%) was eucalyptus essential oil in concentration 1 (pure) and the lowest activity (about 8.5%) was observed for combined essential oils in concentration 150+150 µl/ml of essential oils. significant differences (p≤ 0.05) between different concentrations of eucalyptus essential oil were also observed and the highest increase in its acaricidal activity was observed between concentration 800µl/ ml and 1 (pure) of this essential oil. ginger essential oil also had different values o acaricidal activity in different concentrations, its highest activity was observed in concentration 1 (pure) too, but its activity had equal increase between different concentrations and the highest increase in its acaricidal activity of combined essential oils was observed between concentrations mixed 150+150µl/ml and mixed 250+250µl/ml of essential oils. table 1 shows different values of acaricidal activity in different concentrations of essential oils singly and in combination. comparison between efficacies of different essential oil treatments in each concentration shows that eucalyptus essential oils were the most potent agents than others in whole concentrations. efficacies of ginger essential oils in different concentration were between the efficacies of eucalyptus and combined essential oils except for concentration 500µl/ml that efficacy of combined essential oils was intermediate. performances of different treatments in each concentration are compared in fig. 1. different values of repellent activity were observed for different essential oils group versus control group. eucalyptus essential oil had the highest repellent activity significantly (p≤ 0.05) against rh. bursa and other groups had different efficacies. comparison between repellent activities of examined groups is shown in fig. 2. j arthropod-borne dis, june 2019, 13(2): 214–223 s madreseh-ghahfarokhi et al.: zingiber officinalis and … 218 http://jad.tums.ac.ir published online: june 24, 2019 table 1. acaricidal activity (mean±sem)% of different concentrations for each treatment (essential oils) versus negative and positive control groups groups z. officinalis combined e. globulus negative control 0*a 0a 0a 300µl/ml singly or mixed 150µl/ml 11.47±0.29b 8.53±0.86b 15.21±1.09b 500µl/ml singly or mixed 250µl/ml 16.73±1.54c 21.38±2.92c 24.38±0.42c 800µl/ml singly or mixed 400µl/ml 28.36±2.09d 25.62±1.37c 37.65±2.64d 1 (pure) singly or mixed 500 µl/ml 39.08±1.29e 33.75±2.83d 53.83±3.14e positive control 100f 100e 100f *presence of different superscript lowercase letters (a-f) shows the significant differences (p≤0.05) between different concentrations (rows) of each essential oil treatment (column). fig. 1. comparison between acaricidal activities of different treatments in each concentration. presence of different lowercase letters (a–f) in each concentration shows the significant differences (p≤ 0.05) between different essential oils (lines). fig. 2. repellent activities of different essential oils versus control group. presence of different uppercase letters (a– d) shows the significant differences (p≤ 0.05) between groups. j arthropod-borne dis, june 2019, 13(2): 214–223 s madreseh-ghahfarokhi et al.: zingiber officinalis and … 219 http://jad.tums.ac.ir published online: june 24, 2019 discussion role of rh. bursa in transmission of many important zoonosis microorganisms (7-11), failure of chemical pesticides and presence of pesticides-resistant ticks (13, 14) and lack of studies on acaricidal and repellent natural products against rh. bursa were the reasons for design of current study. natural products, such as essential oils produced by the secondary metabolism of herbs and are used in human consumption as functional food, food additives, medicines, nutritional supplements and the manufacture of cosmetics due to their properties (29) are good candidates as replace for chemical products. there is no similar study about acaricidal and/or repellent activity of essential oil of natural plants like z. officinalis and e. globulus against rh. bursa. in this study for the first time acaricidal and repellent activity of essential oils of z. officinalis and e. globulus against rh. bursa as a wide-distributed species of ticks in different parts of iran with an important role in transmission of zoonosis were studied singly and in combination with together. acaricidal and repellent activities of eucalyptus essential oil were studied against the poultry red mite, dermanyssus gallinae (acari: mesostigmata) and its efficacies were reported about 90% and 94% for acaricidal and repellent activity respectively (20), in current study acaricidal and repellent activities of eucalyptus essential oil against rh. bursa (acari, ixodidae) were determined about 54% and 76% respectively, that maybe its lowest performances occurred due to different resistance between these ticks species. insecticidal and repellent activity of essential oil of z. officinalis and e. globulus against culex theileri theobald were reported in another study, the highest value of insecticidal activity of eucalyptus essential oil was 66% and its highest repellent activity against culex theileri theobald was 74% and for ginger essential oil insecticidal and repellent activities was 39% and 62%, respectively (30). results of current study agreed with them. in current study eucalyptus, essential oil had the highest acaricidal (54%) and repellent (76%) activates against rh. bursa. ginger essential oil had intermediate effects between treatments and its acaricidal and repellent activities was 39% and 62% respectively and values of 34% and 42% were observed as the lowest results for acaricidal and repellent activities of combined essential oils respectively. acaricidal effect of pelargonium roseum and eucalyptus essential oils against adult stage of rhipicephalus annulatus were also studied and dose-dependent effects were reported, highest acaricidal effects were observed for ticks treated with 5% dilutions of pelargonium roseum (79.2%) and eucalyptus (16.7%) after one day incubation (31), that higher value of acaricidal activity of eucalyptus essential oil in current study versus their results maybe was observed due to higher concentration of this essential oil in current study. pesticide and/or repellent activity of eucalyptus essential oil against acanthoscelides obtectus (say) (32), culex quinquefasciatus (33), housefly, musca domestica (34), pediculus humanus capitis (anoplura:  pediculidae) (35) and lutzomyia longipalpis (36) were reported. result of current study agreed with them and shows acaricidal/repellent activity for eucalyptus essential oil due to presence of components such as 1, 8-cineole, citronellal, citronellol, citronellyl acetate, p-cymene, eucamalol, limonene, linalool, a-pinene, gterpinene, a-terpineol, alloocimene, andaromadendrene in eucalyptus essential oil (37). there is a little data about insecticidal and/ or repellent activity of ginger essential oil. insecticidal, repellent and oviposition-deterrent activity of ginger essential oil against anopheles stephensi and aedes aegypti (38) and larj arthropod-borne dis, june 2019, 13(2): 214–223 s madreseh-ghahfarokhi et al.: zingiber officinalis and … 220 http://jad.tums.ac.ir published online: june 24, 2019 vae of spodoptera littoralis (39) were reported. result of current study was agrees with them and showed acaricidal/repellent activity of essential oil of ginger due to presence of presence of bioactive compounds such as gingerols, shogaols, diarylheptanoids, phenylbutenoids, flavanoids, diterpenoids and sesquiterpenoids in it (40). moreover, briefly, insecticidal and/or acaricidal effect of micromeria fruticosa, nepeta racemosa and origanum vulgare (lamiaceae) essential oils against tetranychus urticae and bemisia tabaci (41) were reported in another study, also essential oils of cuminum cyminum, pimenta dioica and ocimum basilicum had different values of acaricidal effects against the cattle tick rhipicephalus (boophilus) microplus (42). in another study, acaricidal activity of essential oils of lippia graveolens, rosmarinus officinalis, and allium sativum against rhipicephalus microplus were identified (43). acaricidal properties of artemisia absinthium and tanacetum vulgare essential oils against tetranychus urticae were also studied (44). conclusion briefly, eucalyptus essential oil had the highest acaricidal (54%) and repellent (76%) activates against rh. bursa. ginger essential oil had intermediate effects between treatments and its acaricidal and repellent activities were 39% and 62% respectively and values of 34% and 42% were observed as the lowest results for acaricidal and repellent activities of combined essential oils respectively. these agents can be considered as potent lethal/repellent agents for control of ticks, but more studies need for this purpose, study on lethal/repellent activity of these essential oils and/or other plants essential oils against other/this important arthropods can be considered as subjects for next experiments. acknowledgements authors are grateful to prof yaser pirali (designer and adviser during the study), dr azam dehghani samani: chief technology officer (cto) of tolou tejarat khavar company, mr ayat dehghani samani (owner of farm and animals) and all of the persons that helped them to do this research. there was no financial support received from any real/legal person, organization, institute or etc. authors certify that there are no financial and personal relationships with 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obtained by three methods of extraction. j econ entomol. 94(1): 167–171. j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 173 http://jad.tums.ac.ir published online: june 30, 2020 original article seroprevalence study on west nile virus (wnv) infection, a hidden viral disease in fars province, southern iran masoumeh amin 1,2 ; morteza zaim 1 ; *hamideh edalat 1 ; *hamid reza basseri 1 ; mohammad reza yaghoobi-ershadi 1 ; farhad rezaei 3 ; kourosh azizi 2 ; mostafa salehivaziri 4 ; mohsen ghane 5 ; saideh yousefi 1 ; sorna dabaghmanesh 2 ; sedigheh kheirandish 6 ; mohammad esmaeil najafi 7 ; jalal mohammadi 2 1 department of medical entomology and vector control, school of health, tehran university of medical sciences, tehran, iran 2 department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran 3 department of medical virology, school of health, tehran university of medical sciences, tehran, iran 4 department of arboviruses and viral hemorrhagic fevers (national ref lab), pasteur institute of iran, tehran, iran 5 department of clinical sciences, school of veterinary science, shiraz university, shiraz, iran 6 department of oral and maxillofacial pathology, bushehr university of medical sciences, bushehr, iran 7 environmental health unit, faculty of health, shiraz university of medical sciences, shiraz, iran (received 12 sep 2019; accepted 10 dec 2019) abstract background: west nile virus, a mosquito-borne flavivirus, causes a variety of symptoms in human, from asymptomatic infection to neuroinvasive disease. several studies have been conducted on the seroprevalence of wnv infection in different areas from iran. this study was performed to find the presence of antiviral antibodies in human serum among some high risk population and awareness of health care staff about symptom of the wnv infection. methods: study performed in five geographical districts based on high population of immigrant and domestic birds and prevalence of the antiviral antibodies in horses which was reported previously. totally 150 human blood samples were collected during 2018. the samples collected from patients referred to the clinics. the elisa method used to detect igg and igm antibody against wnv. logistic regression models used to analyze the effect of sex, age, keeping birds and urban/rural residence on the risk of infection. the awareness of health care staff about symptom of infection surveyed. results: from all blood donors, 41 samples (27.33%) showed positive to igg antibody. from which 56.10% were males and remaining females. none of the mentioned factors had a significant relationship. health care staff had less attention to the infection. conclusions: although the prevalence of antibodies was relatively high, due to the similarity to other viral diseases, health care staff had less attention to the disease. the study showed that people in these areas have been exposed to the virus. further research activities are recommended for control of this arbovirus. keywords: west nile virus; seroprevalence; iran introduction west nile virus is a mosquito-borne virus that infects various mammal species especially horses and humans; however, the most commonly west nile virus infected animals are birds that serve as the reservoir host (1, 2). the birds show different signs of wnv infection as some of them become ill with high symptom of the disease and also die, while some others show no symptom. it is stated that house sparrows and crows are so susceptible to wnv infection but they are poor reservoir for transmission of virus via *corresponding authors: dr hamideh edalat, e-mail: edalat@tums.ac.ir, dr hamid reza basseri, e-mail: basserih@tums.ac.ir http://shirazu.ac.ir/en/node/1510#veterinary mailto:edalat@tums.ac.ir mailto:basserih@tums.ac.ir j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 174 http://jad.tums.ac.ir published online: june 30, 2020 seems to be more responsible for conservation and transmission of the virus in united states (5). furthermore, human and equines are incidental as well as dead-end hosts (6, 7). the virus was first found from the blood of a woman in northern uganda in a region close to the nile river in 1937. wnv is widespread in africa, europe, asia, america and caribbean islands. in nature, birds are the reservoir of virus and mosquitoes get the virus from birds (8-10). the mosquitoes of the culex species play an important role in the wnv transmission cycle (11-13). humans become infected via mosquito bites. it can also get the virus through organ transplantation, breast feeding and blood transfusion. human infections are mild or asymptomatic. severe disease is stated in older patients. most human infections have no signs (80%); nearly 20% of those infected develop a mild infection known as west nile fever. symptoms of the disease include vomiting, diarrhea, headache, backache, muscle aches, skin rash and enlarged lymph nodes. in severe cases, nervous system problems like inflammation of brain and spinal cord with hemorrhage, perivascular cuffing and neuronal degeneration have been detected. wnv has been measured a mild pathogen causing self-limiting outbreaks, but some newer isolates of virus seem to be lethal; therefore an increased incidence of neurological disease and a higher case fatality rate have been associated (4, 15, 16). several studies have been carried out on the presence of antiviral antibodies in human serum in the world (17, 18, 19). these antiviral antibodies are immunoglobulin m (igm) and immunoglobulin g (igg). igm is the first antibody to be immediately developed when any foreign particle is introduced. wnv-specific igm antibodies are usually detectable 3 to 8 days after onset of illness and persist for 30 to 90 days, but longer persistence has been documented. therefore, positive igm antibodies occasionally may reflect a past infection. if serum is collected within 8 days of illness onset, the absence of detectable virus-specific igm does not rule out the diagnosis of wnv infection, and the test may need to be repeated on a later sample. igg is a long term response for any disease and thus protect our body from viral and bacterial attacks. wnv igg antibodies generally are detected shortly after igm antibodies and persist for many years following a symptomatic or asymptomatic infection. therefore, the presence of igg antibodies alone is only evidence of previous infection and clinically compatible cases with the presence of igg, but not igm, should be evaluated for other etiologic agents. the effect of igm is temporary and they disappear after 2–3 weeks of their production. igg is produced at the later stage of infection, but their effect is for a long time and helps in complete eradication of infections or diseases. given that we only found igg antibodies in the blood samples of people living in this study area, this indicates their contact with the virus in the past and possibly their wnv infection. igm provides immediate response while igg responds later with the permanent eradication of antigen and its effect is lasting. the amount of igm produced at the time of exposure to antigen is six times greater than igg (20-24). a study from 2008 to 2009 showed widespread circulation of wnv in iran, mainly in southwestern provinces where the virus probably circulates every year (25). previous seroepidemiological studies in iran revealed the presence of wnv antibody in different regions of iran (26-29). apart from the study in chabahar, south of iran, no further study has been conducted in south of iran (30). although a study on the serum of horses in fars province has reported a high incidence of antibody against the virus (25), no coherent study has been carried out on the human infection in fars province, southern iran. therefore, the aim of this study was to determine the presence of antiviral antibodies and previous exposure in human serum as well as of awareness of health staff j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 175 http://jad.tums.ac.ir published online: june 30, 2020 and clinicians about symptom of the wnv. the results of current study demonstrate the primary information about situation of wnv in the intended area. materials and methods geographical area and date of research this study was carried out in five selected geographic counties of fars province, southern iran during 2018 (fig. 1). fars province is located in the south of iran between 29°37′ n and 52°32′ e and covers an area of 122,400km 2 . the study areas include shiraz, zarqan, marvdasht and sepidan counties as well maharloo wetland area. generally, shiraz district has urban texture and the habitants do not keep domestic animals at home. therefore, the habitants are more common host for mosquito bites. in contrary, marvdasht, zarqan, sepidan and maharloo counties have rural texture with many livestock and thus the mosquito vectors have more access to animal blood source rather than human. in addition, the criteria for chosen areas were presence of high population of immigrant and domestic birds as suspected reservoirs of wnv and high prevalence of antiviral antibodies in horses (according to previous study). furthermore, the occurrence of suspected mosquito vectors in the area was considered (according to primary pilot in the current study). awareness of sign and symptom about wnv a cross sectional study was conducted which assessed awareness of sign and symptom of wnv disease among health care staff and practitioners of selected health facilities by using an open-ended questionnaire. the data were collected from 12 health centers. according to data obtained from a student thesis at shiraz veterinary school (31), antibody titers were higher in horses in shiraz, zarqan, marvdasht and sepidan district than other areas of fars province. the maharloo wetland area, which hosts the largest number of migratory birds in the province, was also selected as one of the study areas. three centers from marvdasht, three centers from sepidan, one center from maharloo, two centers from zarqan and three centers from shiraz were selected. at these centers, all staff were asked about their knowledge of the symptoms of wnv infection. serum sample collection the serum samples were collected from patients who referred to the clinics of five selected areas during 2018. the sample size was 150 individuals. the desired parameters of each participant including age, gender, keeping birds and urban/rural residency were considered in a questionnaire. five milliliter of venous blood was taken and the sera were stored at -20 °c until tested. all serum samples were referred to the viral laboratory, department of medical virology, school of public health, tehran university of medical sciences and were examined for antibody detection (igg and igm) against wnv. enzyme linked immuno sorbent assay (elisa) this test was carried out by euroimmune kit (ei_2662-9601g/m) manufactured in germany to detect anti wnv antibodies (immunoglobulin g and immunoglobulin m) in the serum sample. the test was performed following the kit manufacturer’s protocol. the chosen kit was so specific without any cross activity to other flaviviruses. the antigen source was a recombinant, detergent-extracted glycoprotein e of wnv from the membrane fraction of human cells (32). igm capture immunoassays: testing was performed according to the manufacturers’ instructions. serum samples, along with the controls and cutoff calibrator, were diluted 1:100 in sample diluent and then added to microwells coated with anti-human igm antibodies. folj arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 176 http://jad.tums.ac.ir published online: june 30, 2020 lowing a 1h incubation, the wells were washed and reconstituted recombinant wnv antigen was added. an incubation of 2h occurred, followed by a second wash and the addition of horseradish peroxidase (hrp)-conjugated mouse anti-flavivirus conjugate. after 30 min, a third wash step was employed, and a substrate consisting of tetramethylbenzidine (tmb) and hydrogen peroxide was added to each well. after 10min, 1molar sulfuric acid was added to each well to stop the reaction, and the absorbance of each well was determined spectrophotometrically at 450nm (32). wnv igg enzyme immunoassays: samples were diluted 1:100 in sample diluent and added to micro wells coated with recombinant wnv antigen and incubated for 60min. after the wells were washed, hrp-conjugated fc fragment-specific anti-human igg was added and the wells were incubated for 30min. a second wash was performed, and tmb substrate was incubated in the wells for 10min. to stop the reaction, 1molar sulfuric acid was added to each well, and the absorbance of each well was determined spectrophotometrically at 450nm (32). statistical analysis logistic regression method (spss statistical package version 19.0; ibm spss institute, usa) was used to evaluate significant differences in the seroprevalence positive rates by different demographic factors and a significance level of 0.05 was considered statistically. ethical statement the study suggestion was accepted by the research ethics committee of the tehran university of medical sciences, tehran, iran (no.ir.tums.sph.rec.1396.4204) and informed written consent was obtained from the subjects for blood sampling. results the demographic characteristics of par ticipants are presented in table 1. overall, 150 volunteers participated in the study included 63 (42.00%) from urban and 87 (58.00%) from rural area. the majority of participants was from shiraz county (40.00%) and the minimum of them was from sepidan (13.33 %) followed by zarqan (14.67%), maharloo (16.00%) and marvdasht district (16.00%). about one third of the participants (32.67%) stated that they have birds at home. the majority of participants belonged to the middle age group (30–50 years). overall the prevalence of positive igg antibody was detected among 27.33% participants (41 out of 150), which included 23 males (56.10%) and 18 females (43.90%). among them, 27 (18.00 %) cases were from shiraz county, 3 (2.00 %) were from zarqan county, 4 (2.67%) cases were from maharloo wetland area, 2 (1.33%) cases were from sepidan county and 5 (3.33 %) cases were from marvdasht county (table 2). the prevalence of antibodies against wnv was not statistically significant by gender (p= 0.493). the positive cases based on the age group in study area are shown in table 3. the infection rate to wnv among different age groups was so varied in different counties. none of the positive cases was found among 20–30 years old group in all counties, while the majority of positive case from shiraz county occurred at age group of 31–40 followed by 41–50 year olds. considering the age groups, the prevalence of antibody against wnv was not significantly different (p= 0.832). the positive cases based on keeping birds (as potent reservoirs) at home in five counties are shown in table 4. the positive wnv igg antibody was categorized based on keep birds as suspected reservoirs. the residents who live in rural district had more infection to wnv virus comparatively. about one third of participants (32.66%) stated that they keep birds j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 177 http://jad.tums.ac.ir published online: june 30, 2020 at home and among them 14 (28.56%) was positive to wnv igg antibody. the participants from maharloo who kept birds at home comparatively had more positive cases (12.24 %) and the minimum positive cases was occur among inhabitants of shiraz county (2.04%). however, the prevalence of positive antibodies against wnv was not significantly different among participants who kept birds (p= 0.632).the positive cases according to living in urban/rural areas have been shown in table 5. among all participant 42 % live in urban and 58% in rural areas whereas the positive cases were comparatively higher in the urban areas. between all positive cases, 32.92% were urban and 21.84% in rural areas. the majority of positive cases were found in urban area of shiraz county. the prevalence of positive cases was not statistically significantly different between rural and urban cases (p= 0.291), but it was significantly different between rural and urban cases of shiraz county. although the prevalence of wnv infection was relatively high, due to the similarity of symptoms to other viral diseases, health care staff had less attention to the disease. table 1. demographic characteristics of participants from five districts, fars province, southern iran, 2018 district no. percent from total (%) gender male percent from total (%) female percent from total (%) shiraz 60 40.00 31 20.66 29 19.33 zarqan 22 14.67 12 8.00 10 6.67 maharloo 24 16.00 12 8.00 12 8.00 sepidan 20 13.33 10 6.67 10 6.67 marvdasht 24 16.00 13 8.67 11 7.33 total 150 100.00 78 52.00 72 48.00 age (year) 20–30 22 14.66 17 11.33 5 3.33 31–40 45 30.00 23 15.33 22 14.67 41–50 43 28.67 21 14.00 22 14.67 51–60 28 18.67 14 9.33 14 9.33 ≥60 12 8.00 3 2.00 9 6.00 total 150 100.00 78 52.00 72 48.00 keeping birds 49 32.67 28 18.67 21 14.00 residence place urban 63 42.00 34 22.67 29 19.33 rural 87 58.00 53 35.33 34 22.67 table 2. frequency of positive west nile virus igg antibody according to the sex, fars province, southern iran, 2018 region number of samples percent from total (%) positive cases gender of positive cases p-value no. percent from total (%) male percent from total (%) female percent from total (%) shiraz 60 40.00 27 18.00 15 36.58 12 29.26 zarqan 22 14.67 3 2.00 2 4.88 1 2.44 maharloo 24 16.00 4 2.67 1 2.44 3 7.32 sepidan 20 13.33 2 1.33 1 2.44 1 2.44 marvdasht 24 16.00 5 3.33 4 9.76 1 2.44 total 150 100.00 41 27.33 23 56.10 18 43.90 0.493 j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 178 http://jad.tums.ac.ir published online: june 30, 2020 table 3. frequency of positive west nile virus igg antibody according to the age groups, fars province, southern iran, 2018 region age (year) no. positive cases gender of positive cases p-value no. percent from total (%) male percent from total (%) female percent from total (%) shiraz 20–30 12 0 0.00 0 0.00 0 0.00 31–40 23 18 12.00 10 24.39 8 19.51 41–50 13 9 6.00 5 12.19 4 9.76 51–60 8 0 0.00 0 0.00 0 0.00 ≥60 4 0 0.00 0 0.00 0 0.00 zarqan 20–30 5 0 0.00 0 0.00 0 0.00 31–40 4 0 0.00 0 0.00 0 0.00 41–50 7 2 1.33 2 4.88 0 0.00 51–60 3 1 0.67 0 0.00 1 2.44 ≥60 3 0 0.00 0 0.00 0 0.00 maharloo 20–30 5 0 0.00 0 0.00 0 0.00 31–40 6 2 1.33 0 0.00 2 4.87 41–50 3 0 0.00 0 0.00 0 0.00 51–60 9 2 1.33 1 2.44 1 2.44 ≥60 1 0 0.00 0 0.00 0 0.00 sepidan 20–30 4 0 0.00 0 0.00 0 0.00 31–40 3 0 0.00 0 0.00 0 0.00 41–50 7 2 1.33 1 2.44 1 2.44 51–60 4 0 0.00 0 0.00 0 0.00 ≥60 2 0 0.00 0 0.00 0 0.00 marvdasht 20–30 2 0 0.00 0 0.00 0 0.00 31–40 8 2 1.33 2 4.88 0 0.00 41–50 11 3 2.00 2 4.88 1 2.44 51–60 2 0 0.00 0 0.00 0 0.00 ≥60 1 0 0.00 0 0.00 0 0.00 total 150 41 27.32 23 56.10 18 43.90 0.832 fig. 1. location of study area and sampling sites in fars province, southern iran, 2018 j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 179 http://jad.tums.ac.ir published online: june 30, 2020 table 4. frequency of positive west nile virus igg antibody based on the history of keeping birds at home, fars province, southern iran, 2018 region number of samples keeping birds percent from total (%) positive cases p-value total percent from total (%) male percent from total (%) female percent from total (%) shiraz 60 6 4.00 1 2.04 1 7.14 0 0.00 zarqan 22 8 5.33 2 4.08 1 7.14 1 7.14 maharloo 24 17 11.33 6 12.24 2 14.29 4 28.57 sepidan 20 10 6.67 2 4.08 1 7.14 1 7.14 marvdasht 24 8 5.33 3 6.12 2 14.29 1 7.14 total 150 49 32.66 14 28.56 7 50.00 7 50.00 0.632 table 5. frequency of positive west nile virus wnv igg antibody according to living in urban/rural areas, fars province, southern iran, 2018 region number of samples urban rural positive cases total percent from total (%) p-value no. percent from total (%) no. percent from total (%) urban percent from total (%) rural percent from total (%) shiraz 60 37 24.67 23 15.33 18 28.57 9 10.34 27 18.00 zarqan 22 8 5.33 14 9.33 1 1.59 2 2.30 3 2.00 maharloo 24 0 0.00 24 16.00 0 0.00 4 4.60 4 2.67 sepidan 20 7 4.67 13 8.67 1 1.59 1 1.15 2 1.33 marvdasht 24 11 7.33 13 8.67 2 3.17 3 3.45 5 3.33 total 150 63 42.00 87 58.00 22 34.92 19 21.84 41 27.33 0.291 j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 180 http://jad.tums.ac.ir published online: june 30, 2020 discussion this is the first record on wnv infection on human in fars province, southern iran. the study was conducted in five different districts and we found that the infection rate to wnv was varied based on sex and age of participants. it seems, males at middle age were more exposing to the mosquito vector bites (table 2, 3). the statistical analysis showed that none of the demographic factors had a significant relationship with the incidence of antibodies (p> 0.05). we found some evidence indicated that the risk of wnv infection can be age-dependent in the study areas. similarity, a study in ohio showed that titer of antibody against wnv in the children was 2.5 times higher than adult (33). while another study conducted on immunocompromised and immunocompromised patients, age was not a significant seroconversion factor. their findings suggest that immune factors affect seroconversion (34). however, the age dependency of wnv infection is controversial. in addition, no significant association was observed between sex and seropositivity of wnv igg antibody among the voluntaries. previously, equal infection of wnv was reported between both sexes in in united states (35). however, our results may indicate that both sexes had same exposure to the infected bites. as birds are potent reservoirs of wnv, we tried to find out any correlation between keeping birds in the inhabitant houses and wnv infection rate. noticeably, 28.56% of residents which kept birds were positive to wnv igg antibody. of course, these positives may be related to exposure to the virus in previous years. generally, birds are attractive blood source for culex spp (36, 37), therefore, keeping birds at home can cause distraction of mosquitoes from human to birds and thus help reduce disease transmission to human. we also found higher infection rate in urban areas comparing to rural. cycle of wnv in nature always occur between birds and mosquito vectors. therefore, density of birds around inhabitants rise transmission of the disease among communities′ members in endemic area, while only 28.56 % of participants who kept birds at home were positive and we could not find any association between wnv igg positive rate and those who kept birds at home. it likely, presence of birds around human can be good alternative blood source for the mosquito vectors. therefore, the relationship between rate of wnv infection and keeping birds is so controversial and it is depended to situation of virus transmission in area. west nile virus transmission may be varied based on rural and urban areas (38-40). it is stated that the rate of wnv infection was higher in rural areas comparing with urban areas in bucharest and culex pipiens was dominant mosquito species (41). while we also found higher positive antibody among urban residents where cx. pipiens was relatively dominant (unpublished). in addition, poor drainage, catch basins, sewage, and manmade containers around houses of urban residents provide good larval development sites for cx. pipiens. moreover, poor of alternative blood source for vectors may convey the mosquito to human inhabitants in urban area. as there is no complete cure or effective vaccine for the virus, therefore monitoring and surveillance studies regarding wnv prevalence is an important tool for health care specialists to have a future regarding this lifethreatening arbovirus in order to choose the best protective plans. as well, climate variation due to worldwide warming in recent years may increase mosquito activity period that can lead to escalating virus prevalence. because of climate differences in iran, the dispersal and richness of mosquito differs in different provinces of j arthropod-borne dis, june 2020, 14(2): 173–184 m amin et al.: seroprevalence study on … 181 http://jad.tums.ac.ir published online: june 30, 2020 iran. therefore, in provinces where the weather is warmer, the life span of the mosquitoes is also increased, resulting in a higher chance of transmission. seroepidemiological studies are necessary to define the prevalence of wnv in other parts of iran for better decision making in the future. conclusions these results suggest circulation and exposure of the human population to wnv in south of iran. the knowledge generated from this study will further contribute to our understanding of the ecology and epidemiology of wnv fever in iran and will assist in the provision of public health measures to reduce the risk of exposure to the virus. it is notable that the high volume of tourist traffic in fars province and other economic significance of the province, awareness of the cycle of this disease is necessary and the information from this project will be used to plan the control of transmission and outbreak in fars province and other neighboring provinces. previous studies have shown that horses have antibodies in the study area. the study also found that people living in the area were also in contact with the virus. of course, the authors of this article have also researched on the probable vectors of wnv in this study area which will be published in the future. certainly, future studies on the birds and their infection with the west nile virus can help to clarify the disease situation in this area. acknowledgements the authors would like to thank school of public health, tehran university of medical sciences, tehran, iran, for their assistance in carrying out this research (project no.37365). this study was supported by national institute for medical research development (grant no.940947). the authors de clare that there is no conflict of interests. references 1. meshkat z, chinikar s, shakeri m, manavifar l, moradi m, mirshahabi h, jalali t, khakifirouz s, shahhosseini n (2015) prevalence of west nile virus in mashhad, iran: a populationbased study. asian pac j trop med. 8 (3): 203–205. 2. cao l, fu s, lu z, tang c, gao x, li x, lei w, he y, li m, cao y, wang h, liang g (2019) detection of west nile virus infection in viral encephalitis cases, china. vector borne 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in culex pipiens mosquitoes, associated with disease outbreak in greece, 2017. acta trop. 182: 64–68. https://www.ncbi.nlm.nih.gov/pubmed/?term=mavridis%20k%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=fotakis%20ea%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=kioulos%20i%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=mpellou%20s%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=mpellou%20s%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=konstantas%20s%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=varela%20e%5bauthor%5d&cauthor=true&cauthor_uid=29474832 https://www.ncbi.nlm.nih.gov/pubmed/?term=gewehr%20s%5bauthor%5d&cauthor=true&cauthor_uid=29474832 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3faculty of medicine, yuzuncu yil university, van, turkey 4department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran *corresponding author: dr teimour hazratian, email: hazratiant@tbzmed.ac.ir (received 28 apr 2018; accepted 30 nov 2020) abstract background: myiasis is a disease caused by infections of tissues and organs of human and vertebrates body by the larvae of real flies of diptera which feeding on living or dead tissues of host for a period of time. this report aims to present a case of urogenital myiasis caused by the larvae of psychoda albipennis (diptera: psychodidae) for the first time in iran. methods: in this case report, we present a case of a 9-year-old girl with urogenital myiasis caused by p. albipennis. she presented to sina hospital with dysuria and claimed that he had observed several black-grayish colored mobile particles in his urine at different times. the patient lived in miandoab, west azerbaijan province, iran. results: in the hospital her urine sample, containing 3 larvae was referred to entomology lab of the medical faculty for identification and characterization. according to morphological factors, the larvae were identified to approximate size of 8–10mm long, white to gray color, thorns and pale scales and a siphon at the posterior end of the body. by comparing the larvae with the reported ones from turkey, diagnosis was confirmed. conclusion: according to our survey, this is the first observation of urogenital myiasis in east azerbaijan province, iran. our case illustrates urogenital myiasis caused by p. albipennis in iran. urogenital myiasis has not been previously reported from iran as a human disease. keywords: myiasis; urogenital myiasis; psychoda albipennis; iran introduction diptera is the most important order of insects in the medical field. this order includes tow suborder: nematocera and brachycera (1). the term “myiasis” was derived from a greek word “myia” which mean fly (2). "myiasis" was first used by fw hope in 1840. nowadays, myiasis is a medical term for a disease caused by fly larvae in human and vertebrates tissues and organs. in other words, myiasis is a disease caused by infections of tissues and organs of human and vertebrates body by the larvae of the suborder brachycera or real flies of diptera which feeding on living or dead tissues of host for a period of time (3, 4). myiasis could be classified by both entomological and clinical procedures. in entomological method, the classification of myiasis is based on parasitic characteristics of fly larvae in which it is divided into obligatory, facultative and accidental types. in clinical method, myiasis is classified according to the infected area of the body. myiasis can manifest in various body parts, such as the eye, nose, ear, oral cavity, throat, skin, gastrointestinal system and copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:hazratiant@tbzmed.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 425–429 t hazratian et al.: first record of … 426 http://jad.tums.ac.ir published online: december 31, 2020 urogenital system of hosts. depending on the location of the dipterous larvae in the eye, ear, intestine, oral cavity, skin of hosts, the parasite can cause a broad range of medical conditions (5). limited number of urogenital myiasis are reported from iran and the etiological agents are chrysomyia bezziana and wohlfahrtia magnifica (2). actually, this disease is one of the causes of urogenital disorders in human (6). studies have shown that urogenital myiasis is associated with urinary tract obstruction, poor hygiene and or the presence of ulcers in the genitourinary system (7). sometimes urogenital myiasis by infection of the larvae of flies such as p. albipennis is dangerous and should be removed as soon as possible with surgical procedures (8). clinical manifestations of urogenital myiasis could include abdominal pain, diarrhea and dysuria (9). in recent time a peculiar case of urogenital myiasis was reported from egypt, china, japan, argentina, chile, saudi arabia, england, spain, india and particularly from turkey in which the causative species were identified from the subfamily pychodinae called p. albipennis (10-19). in our case report, in the first stage we observed larvae in the urine and then bladder bag, which was identified as p. albipennis, therefore this has been the first reported case in iran. case description in this case report, we present a case of a 9-year-old girl with urogenital myiasis caused by p. albipennis. she presented to the sina hospital with dysuria and claimed that he had observed several black-grayishcolored mobile particles in her urine at different times.the patient lived in miandoab, west azerbaijan province, iran. after observing larvae in urine in her sample, was referred to the health center of that city and due to diagnosis of filariasis, mebendazole was prescribed. despite taking the medication, the patient was still suffering from stomach ache and larvae still present in the urine. she was finally referred to tabriz sina hospital. in the hospital, her urine sample containing 3 larvae was submitted to the entomology lab of tabriz medical faculty for characterization.valid larval identification keys were used to identify larvae collected based on morphological characterizations such as size, color, setae and scales of the abdominal segments and siphon (20-22), and the larvae were identified to be p. albipennis (figs. 1–3). comparing the reported causative agents of myiasis from turkey (12), our present was proven to be p. albipennis. the morphological characteristics of the larvae were completely consistent with the reported cases in turkey. the larvae were grayish-white, with approximate 8–10mm size. they have quite cylindrical body with pale hairs and scales on their surfaces. also, there were teeth-like serrations on the periphery of the body. the body has 8 segments with the last segment containing a quite distinct respiratory siphon. two larvae of p. albipennis were mounted in puri's medium and one of them was preserved in alcohol in the laboratory. fig. 1. the larva of psychoda albipennis (original, hazratian 2018) fig. 2. the head of psychoda albipennis (original, hazratian 2018) http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 425–429 t hazratian et al.: first record of … 427 http://jad.tums.ac.ir published online: december 31, 2020 fig. 3. the siphon of psychoda albipennis (original, hazratian 2018) discussion the family psychodidae consists of six subfamilies, of which only two are medically importance. phlobotomines (sand flies) are blood feeding larvae which live in places rich in organic materials, such as animal burrows, termite hills and tree holes (23-25). these flies are known as leishmaniasis vectors. the subfamily psychodinae (moth flies), includes small mosquitoes (2–5mm) as phlobotomines with stretched legs and hairy body. but mouth segments are shorter and are not compromised for blood feeding. these insects reproduce in moist conditions and they are mostly found around sewage pipes. thus, they are called "sewage flies" or "fitter flies" and lay their eggs within a jelly-like mass. sometimes their population grows so high that they enter the eyes, ears and noses and bother the hosts. in spite of living in dirty environment, they are not considered disease vectors. but some bacteria are spread from them. in addition, their hair and scales are allergic (26). adult p. albipennis is approximately 2mm long, covered with intense hairs, and primarily reside in houses, particularly in moist areas such as, toilets and bathrooms. the adult female flies lay their eggs in groups of 30–40 in moist, damp places, on hard objects and stones. the fly larvae are found in dirty, moist places, spoiled vegetables and fruits, sewage, irrigated plants and rubbish dumps (27). in the fourth stage of growth, the larvae are commonly grayishwhite. typically, their bodies consist of 7 or 8 segments and have a siphon at the end of the last ring. they possess short pale hairs and scales on the surface. the edges of larvae are bordered with tooth-like projections (28). by the end of 2017, about 80 cases of human myiasis were reported in iran where most of them (52%) were oral myiasis. about 65% of the reported cases were caused by oestrus ovis larvae. in terms of age, two categories were considered, one infected individuals from 21–40 years old and the other from individual over 65 years old (4). a questionnaire-based study revealed that more than 88% of cases occurred among individuals involve in sheep and boat rearing (29). reported cases of myiasis from different areas in iran, often involve submission of sample to the entomological lab of medical faculties for characterization. thus, it is suggested that consultation should be made with medical entomologist to detect the various types of myiasis diagnosed in iran. particularly, in this recent case report involving urogenital myiasis caused by p. albipennis. it is worthy to consider this fly larva as the causal agent of urogenital myiasis, because of high population of this fly in the breeding places, the occurrence of ear, ocular and nasal myiasis are possible. conclusion to the best of our knowledge, this is the first report ever urogenital myiasis caused by p. albipennis in iran. this type of myiasis has not been previously reported from iran as a human disease. the authors declare that there is no conflict of interest. acknowledgements we would like to appreciate the staff of sina hospital med laboratory for the sample provision and special thanks to the contributive effort of dr kamran akbarzadeh, the dehttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 425–429 t hazratian et al.: first record of … 428 http://jad.tums.ac.ir published online: december 31, 2020 partment of medical entomology and vector control, health school, tehran university of medical sciences. the authors declare that there is no conflict of interest. references 1. hackman w, vaisanen r (1982) different classification systems in the diptera. ann zool fennici. 19: 209–219. 2. hazratian t, tagizadeh a, chaichi m, abbasi m (2017) pharyngeal myiasis caused by sheep botfly, oestrus ovis (diptera: oestridae) larva, tabriz, east azarbaijan province, iran: a case report. j arthropod borne dis. 11(1): 166–170. 3. erzincllioĝlu y (1983) the application of entomology to forensic medicine. med sci law. 23(1): 57–63. 4. howard lo (1930) a history of 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gk (2015) urogenital myiasis caused by psychoda albipennis: a case report. j urol surg. 4: 195–196. 29. akbarzadeh k, rafinejad j, nozari j, rassi y, sedaghat mm, hosseini m (2012) a modified trap for adult sampling of medically important flies (insecta: diptera). j arthropod borne dis. 6(2): 119– 128. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 127 http://jad.tums.ac.ir published online: january 05, 2016 original article genetic diversity of crimean congo hemorrhagic fever virus strains from iran *sadegh chinikar 1, saeid bouzari 2, mohammad ali shokrgozar 3, ehsan mostafavi 4, tahmineh jalali 1, sahar khakifirouz 1, norbert nowotny 5, anthony r. fooks 6, nariman shah-hosseini 1 1arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory), pasteur institute of iran, tehran, iran 2department of molecular biology, pasteur institute of iran, tehran, iran 3national cell bank of iran, pasteur institute of iran, tehran, iran 4department of epidemiology, pasteur institute of iran, tehran, iran 5institute of virology, department of pathobiology, university of veterinary medicine, vienna, austria 6animal health and veterinary laboratories agency, wildlife zoonoses and vector-borne diseases research group, weybridge, new haw, addlestone, surrey, united kingdom (received 10 may 2013; accepted 29 june 2014) abstract background: crimean congo hemorrhagic fever virus (cchfv) is a member of the bunyaviridae family and nairovirus genus. it has a negative-sense, single stranded rna genome approximately 19.2 kb, containing the small, medium, and large segments. cchfvs are relatively divergent in their genome sequence and grouped in seven distinct clades based on s-segment sequence analysis and six clades based on m-segment sequences. our aim was to obtain new insights into the molecular epidemiology of cchfv in iran. methods: we analyzed partial and complete nucleotide sequences of the s and m segments derived from 50 iranian patients. the extracted rna was amplified using one-step rt-pcr and then sequenced. the sequences were analyzed using mega5 software. results: phylogenetic analysis of partial s segment sequences demonstrated that clade iv-(asia 1), clade iv-(asia 2) and clade v-(europe) accounted for 80 %, 4 % and 14 % of the circulating genomic variants of cchfv in iran respectively. however, one of the iranian strains (iran-kerman/22) was associated with none of other sequences and formed a new clade (vii). the phylogenetic analysis of complete s-segment nucleotide sequences from selected iranian cchfv strains complemented with representative strains from genbank revealed similar topology as partial sequences with eight major clusters. a partial m segment phylogeny positioned the iranian strains in either association with clade iii (asia-africa) or clade v (europe). conclusion: the phylogenetic analysis revealed subtle links between distant geographic locations, which we propose might originate either from international livestock trade or from long-distance carriage of cchfv by infected ticks via bird migration. keywords: cchfv, molecular epidemiology, rt-pcr, phylogeny, reassortant virus, iran introduction endemic regions of crimean congo hemorrhagic fever (cchf) have been reported in africa, the middle east, eastern europe and western asia (elevli et al. 2010). in the past 10 years, turkey, bosnia, and iran have been reported the most frequent outbreaks of cchf worldwide (mohammadi and razmi 2014). the virus is transmitted to humans through the bite of infected ixodid ticks (mostly of the hyalomma genus) (mehravaran et al. 2013, champour et al. 2014), or direct contact with *corresponding author: dr sadegh chinikar, email: sadeghchinikar@yahoo.com j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 128 http://jad.tums.ac.ir published online: january 05, 2016 infected blood, by meat from infected animals or by nosocomial transmission (chinikar et al. 2012c). cchfv is a single stranded rna virus with segmented negative sense genome consisting of a small (s), a medium (m) and a large (l) segment. the s rna segment encodes the nucleocapsid (n) protein, and the m rna segment encodes the glycoprotein precursor, resulting in the two envelope glycoproteins gn and gc, while the l segment encodes the putative rna-dependent "rna" polymerase (papa et al. 2002). it has been proved that segmented rna viruses have the potential of segment reassortment and form new distinct genomic variants if the host cells are subject to dual infection by more than one genetically distinct virus (hewson et al. 2004b, lukashev 2005). cchfvs are relatively divergent in their genome sequence and grouped geographically into seven distinct clades based on the s-segment sequence analysis (han and rayner, 2011). west-africa in clade i, central africa in clade ii, south-africa and west-africa in clade iii, middle-east and asia in clade iv, europe in clade v and greece in clade vi (deyde et al. 2006, hewson et al. 2004a). the clade iv may be devided into two distinct clades, asia-1 and asia-2 (hewson et al. 2004a). beside the genome analysis on s segment, cchfvs are divided into six clades based on m-rna sequences; clade iv (asia or middle east) comprising strains in china, pakistan, oman, and south africa, clade iii (s africa or w africa 2) comprising those in uzbekistan, tajikistan, china, pakistan, iran, iraq, south africa and nigeria, clade i (w africa1) comprising those in congo, senegal, china, clade v (europe) comprising those in russia, kosovo and turkey, clade vi (greece) including isolated strain from greece, and clade vii (mauritania) including isolated strain from mauritania (papa et al. 2005, morikawa et al. 2002, morikawa et al. 2007). in iran, the first phylogenetic study on ssegment of cchfv in 2004 showed that the cchfv strains were clustered within clade asia 1 with the highest similarity to matin strain from pakistan (chinikar et al. 2004), previously a pioneering genetic characterization study had showed that the iranian strain (arteh 193-3) obtained from ticks in the north-eastern region of khorasan province in 1978 clustered within clade africa i and had similarity with cchfv strains from west africa, mainly from senegal and mauritania (sureau and klein 1980). in 2008, further phylogenetic analysis in tick populations in isfahan province in central iran, demonstrated that a variant strain clustered within clade iv (asia 1) with the highest similarity to an iraqi strain (chinikar et al. 2012a). genetic analyses in 2012 of one strain from northern iran illustrated that the russian cchfv genome is circulating in this area, and accordingly the existence of european clade v in iran was confirmed (chinikar et al. 2013). according to the previous phylogenetic studies, there are seven clades of cchfv and iranian strains of cchfv distributed within three clade i (africa i), clade iv (asia 1) and clade v (europe) (chinikar et al. 2013). the main objective of this study was to obtain new insights into the molecular epidemiology of cchfv in iran by analyzing the partial and complete nucleotide sequences of the s and m segments of cchfv genome obtained from iranian patients and several sequences available from genbank. materials and methods investigation areas and sampling according to the distribution of cchfv in iran, cchfv have been reported from 27 of 31 provinces until now. nineteen provinces j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 129 http://jad.tums.ac.ir published online: january 05, 2016 encompassed all geographical areas and were randomly selected and investigated in a span of 11 years between 2002 and 2013. a total of 50 human sera samples were selected among previously rt-pcr positive samples which were stored in the arboviruses and viral hemorrhagic fevers laboratory (national ref. lab) sample bank; pasteur institute of iran and also transferred samples to the this laboratory during the study. geographical distribution and the year of sampling related to each strain are shown in table 1. serological and molecular identification to investigate each human serum sample for the presence of cchfv-specific antibodies, an elisa was initially used to detect igm (garcia et al. 2006). molecular analysis was subsequently applied. for this purpose, viral rna was extracted from 140 μl of serum using a qiaamp rna mini kit, according to of the manufacturer's instructions (qi agen gmbh, hilden, germany) (yashina et al. 2003). specific primers for amplification of partial s, whole s and m segments were designed by clc main workbench software version 5.0, based on available cchfv sequences in genbank. to amplify the partial of the s-segment, a primer pair (psf5’-gaatgtgcatgggtt agctc-3’) and (psr 5’-gacatcacaa tttcaccagg-3’) was designed and used to amplify a 536 bp section. for reverse transcription, 50 °c (30 min) used. an initial enzyme activation step at 95 °c for 5 min was succeeded by 40 reaction cycles carried out with 30 sec at 94 °c, 30 sec at 50 °c, and 45 sec at 72 °c followed by a final incubation at 72 °c for 10 min. for amplification of the full-length of the s-segment, a touch down rt-pcr used with the following primers: wsf: 5’-tctcaaa gaaacacgtgccgc-3’ and wsr: 5’tctcaaagatatcgttgccgc-3’ to amplify a 1680 bp section of the s-segment. thermal cycle condition designed as 45 °c (30 min) for reverse transcription and 95 °c (15 min) as an initial enzyme activation, and then followed by 40 reaction cycles at 94 °c (10 sec), 66-52 °c (30 sec), 68 °c (100 sec), eventually, 68 °c (10 min) as final extension. for amplification of the partial of the msegment, the primers of pmf 5’-tgcac ttgagcatctgcaa-3’ and pmr 5’-ag ctgattcctgtcctttc-3’ was designed and used to amplify a 557 bp section of the m-segment. for reverse transcription, 50 °c (30 min) used an initial enzyme activation step at 95 °c for 15 min was succeeded by 40 reaction cycles carried out with 30 sec at 94 °c, 30 sec at 45 °c, and 1 min at 72 °c followed by a final incubation at 72 °c for 10 min (rodriguez et al. 1997). pcr products were amplified using one-step rt-pcr, according to rodriguez et al. the amplified products were visualized by ethidium bromide agarose gel staining (yadav et al. 2012). sequencing the pcr products were then sequenced using big dye terminator v3.1 cycle sequencing kit with modified sanger sequencing method by abi genetic analyzer 3130. for full-length s-segment sequencing, internal primers were designed as follow: forward: 3'aatgcaaacacggcagcttt 5' and reverse: 3'ggaactgtgagacag tcggg5'. the obtained sequences were optimized (gaps and noises were deleted) (chinikar et al. 2010). nucleotide sequence accession numbers whole s segment isolates iran-gilan 69, iran-isfahan 78, iran-kerman 43, iran-kh razavi 72, iran-tehran 65 and iran-zahedan 19 submitted to genbank and assigned accession no. kj027521, kj027522, kj196326, kj485700, kj566219 and kj676542 respectively. sequence alignments and phylogeny analysis in addition to the 50 cchfv sequences ob j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 130 http://jad.tums.ac.ir published online: january 05, 2016 tained, several sequences available from genbank at www.ncbi.nih.gov were incorporated into the alignments for phylogenetic analyses (table 2). the sequence alignment was performed by clustalw and a scaled phylogenetic tree generated by the maximum likelihood (ml) with kimura 2-parameter distance using mega5 software. bootstrap confidence limits were based on 1000 replicates. this method evaluates the topologies of different trees and chooses the best tree based on a specified model. this model is based on the evolutionary process that can account for the conversion of one sequence into another (tonbak et al. 2006, aradaib et al. 2011). results viral rna was extracted from 50 selected sera of infected patients originated from 19 provinces of iran including khorasane-razavi, khorasane-jonobi, sistanvabalochestan, kerman, tehran, gilan, mazandaran, isfahan, yazd, qom, azarbaijane-sharghi, azarbaijanegharbi, zanjan, kermanshah, hormozgan, fars, boshehr, khuzestan and lorestan collected between 2002 and 2013. samples included in this study were correlated to the prevalence of the cchfv from each region. accordingly, 11 sera were selected from the southeast of iran, 10 sera from central iran, 9 sera from northeast iran, 8 sera from southern iran, 4 sera from west, 3 sera from northwest, 3 serum samples from north, 1 serum sample from east and 1 serum sample from southwest. partial sequence phylogenetic analysis of s segment the phylogenetic analysis conducted by the neighbor joining (nj) and maximumlikelihood (ml). only ml are presented here, however phylogenetic tree that were created using the nj algorithm had the same topology and strongly supported the same phylogenetic groups as the ml tree. phylogenetic analysis using the partial ssegment (~500 bp) demonstrated, of 50 sequenced cchfv obtained from iranian patients, 40 sequences (80%), mainly in the southeast and east of iran, were located within clade iv (asia 1). two iranian sequences (4%), both in the southeast of iran, formed a distinct cluster in the clade iv (asia 2). these two iranian sequences fell in the subgroup consisting of the india. notably, two sequences had strong similarity to each other with 100 % bootstrap support. seven sequences (14%) of iranian strains, mainly in the northwest of iran, were located within clade v (europe). one sequence (iran-kerman/22) showed itself as out-group and had the highest differences with other clades. no iranian sequences were positioned within clade iii (s africa and/ or w africa 2), clade ii (central africa), clade i (w africa 1), and clade vi (greece ap92). as expected, all sequences from an outbreak in mashhad city, khorasane-razavi province, in 2012 grouped in a cluster with the highest similarity to each other (irankhrazavi 2, 3, 4, 5, 16 and 17) (fig. 1). complete genome sequence phylogenetic analysis of s segment the topology of complete genome sequence analysis of s-segment (~1500 bp) was in accordance with partial genome sequence analysis of the s-segment. the phylogeny of the complete s segment nucleotide sequences from strains obtained in genebank indicated that eight major groups could be recognized. the iranian s segment from strain iran-kerman/22 was least similar to all other s segments and stands as an outgroup and considered as clade vii (iran) (fig. 2). j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 131 http://jad.tums.ac.ir published online: january 05, 2016 partial sequence phylogenetic analysis of m segment based on phylogenetic analysis on the partial sequence of m segment (~520 bp), six clades were distinguishable. the tree showed that seven iranian strains clustered in the clade iii (s africa and/ or w africa) and v (europe) with other strains of middle east and europe. the iranian cchfv strains iran-kerman/ 43, iran-tehran/65 and iran-khrazavi/72 in accordance with the iranian strains (dq 446216 and dq446215) formed a separate cluster within the clades iii and showed maximum closeness with the pakistan (aj 538199). in addition, iran-gilan/69 and iranisfahan/78 strains fell in clade v (europe) and showed maximum association with the isolates from russia, kosovo and turkey. interestingly, iran-kerman/22 showed the highest similarity to clade v in the m segment analysis (fig. 3). table 1. details of the iranian cchfv strains isolated from human serum used in this study. the sequences used for analysis of partial s, complete s and partial m segments are indicated in table strain location date partial s complete s partial m iran-khrazavi/1 northeast 2013 * iran-khrazavi/2 northeast 2012 * iran-khrazavi/3 northeast 2012 * iran-khrazavi/4 northeast 2012 * iran-khrazavi/5 northeast 2012 * iran-khrazavi/16 northeast 2012 * iran-khrazavi/17 northeast 2012 * iran-khrazavi/80 northeast 2012 * iran-khrazavi/72 northeast 2012 * * * iran-kerman/22 southeast 2012 * * * iran-kerman/27 southeast 2012 * iran-kerman/24 southeast 2006 * iran-zahedan/25 southeast 2012 * iran-zahedan29 southeast 2012 * iran-kerman/43 southeast 2013 * * * iran-zahedan/74 southeast 2012 * iran-kerman/77 southeast 2013 * iransistanbalochestan/ 85 southeast 2011 * iran-zahedan/19 southeast 2012 * * iran-zahedan/20 southeast 2012 * iran-shiraz/39 south 2012 * iran-minab/49 south 2007 * iranbandarabas/50 south 2011 * iranhormozgan/84 south 2011 * iran-booshehr/52 south 2010 * iranhormozgan/87 south 2011 * iran-fars/89 south 2011 * iran-booshehr/97 south 2002 * iran-khjonobi/73 east 2012 * iran-zanjan/23 west 2012 * iran-zanjan/41 west 2006 * j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 132 http://jad.tums.ac.ir published online: january 05, 2016 iran-kermanshah/55 west 2008 * irankhorramabad/56 west 2010 * iran-ahvaz/54 southwest 2010 * iran-yazd/57 central 2009 * iran-qom/58 central 2011 * iran-tehran/65 central 2011 * * * iran-isfahan/78 central 2013 * * * iran-isfahan/81 central 2011 * iran-kashan/15 central 2005 * iran-qom/71 central 2008 * iran-yazd/86 central 2010 * iran-tehran/90 central 2012 * iran-tehran/91 central 2010 * iran-tabriz/99 northwest 2003 * iran-oroomieh/100 northwest 2003 * iran-tabriz/102 northwest 2004 * iran-babol/14 north 2012 * iran-amlash/21 north 2012 * iran-gilan/69 north 2012 * * * table 2. details of cchf virus strains originated from iran and other countries retrieved from genbank and used for sequence analysis virus strains location date clade/ name (s segment) genbank accession no s segment clade/name (m segment) genbank accession no m segment niv 112143 india 2011 iv/asi-1 jn572089 sct ex afghanistan afghanistan 2012 iv/asi-1 jx908640 afg09-2990 afghanistan 2009 iv/asi-1 hm452305 artec193-3 iran 1978 i/wafr-1 u15022 766/02 iran 2004 iv/asi-1 ay366373 787/02 iran 2004 iv/asi-1 ay366379 iran 52 iran 2002 iii/s afr,wafr-2 dq446215 iran53 iran 2002 iii/s afr,wafr-2 dq446216 baghadad12 iraq 1979 iv/asi-1 aj538196 iii/s afr,wafr-2 aj538197 china79121 china 1979 iv/asi-2 af358784 i/w afr-1 ab069673 chinac68031 china 1968 iv/asi dq211629 kosovo 1917 kosovo 2009 v/eur jn173797 kososvo9553 kosovo 2001 v/eur ay675511 kosovohoti kosovo 2001 v/eur eu037902 armg951 madagaskar 1994 iv/asi-1 u15024 ibar10200 nigeria 1966 iii/s afr,w afr-2 u88410 iii/s afr,w afr-2 af467768 matin pakistan 1976 iv/asi-1 af527810 4/asi af467769 pakistansr3 pakistan 2000 iv/asi-1 aj538198 iii/s afr,w afr-2 aj538199 drosdov russia 1967 v/eur dq211643 v/eur dq211630 kashmanov russia 1967 v/eur dq211644 v/eur dq211631 spu 128817 south africa 1981 iii/s afr,w afr-2 dq076415 iii/s afr,w afr-2 dq157174 spu9785 south africa 1985 iii/s afr,w afr-2 dq211646 iv/asi dq211633 ard39554 mauritania 1984 iii/s afr,w afr-2 dq211641 vii/mauritania dq211628 ab1-2009 sudan 2009 iii/s afr,w afr-2 hq378187 ard8194 senegal 1969 i/w afr-1 dq211639 i/w afr-1 dq211626 ard15786 senegal 1972 i/w afr-1 dq211640 table 1. countinued… j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 133 http://jad.tums.ac.ir published online: january 05, 2016 semunya uganda 1958 ii/central afr dq076413 ug3010 congo 1956 ii/central afr dq211650 i/w afr-1 dq211637 hodzha uzbekistan 1967 iv/asi-2 ay223475 iii/s afr,w afr-2 ay223476 hu8966 tajikistan 1990 iii/s afr,wafr-2 ay179962 ap92 greece 1975 vi/greece dq211638 vi/greece dq211625 oman oman 1997 iv/asi-1 dq211645 iv/asi dq211632 dubai 616 dubai 1979 iv/asi-2 jn108025 kelkit06 turkey 2006 v/eur gq337053 200310849 turkey 2003 v/eur dq211649 v/eur dq211636 kt281/75 dugbe virus af434165 jc280 pakistan 1992 hazara virus m86624 fig. 1. phylogenetic tree of cchfvs based on the 520-nucleotide s rna sequences. the tree was constructed by using the maximum likelihood method with mega 5. the sequences obtained from this study are shown by asterisk. the numbers above the branches indicate the bootstrap values in percentages (of 1000 replicates) table 2. countinued… j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 134 http://jad.tums.ac.ir published online: january 05, 2016 fig. 2. phylogenetic tree of cchfvs full-length s rna sequences (1500 bp). the tree was constructed by using the maximum likelihood method with mega 5. the sequences obtained from this study are shown by asterisk. the numbers above the branches indicate the bootstrap values in percentages (of 1000 replicates) j arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 135 http://jad.tums.ac.ir published online: january 05, 2016 fig. 3. phylogenetic tree of cchfvs partial m rna sequences (500 nucleotides). the tree was constructed by using the maximum likelihood method with mega 5. the sequences obtained from this study are shown by asterisk symbol. the numbers above the branches indicate the bootstrap values in percentages (of 1000 replicates) discussion in 1992, the first complete nucleotide sequence of s-rna segment of chinese strain c68031 of cchfv was determined (marriott and nuttall 1992). afterwards, during an outbreak of cchf in the united arab emirates between 1994 and 1995, nested reverse transcriptase polymerase chain reaction (rtpcr) amplifying of the partial s-rna segj arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 136 http://jad.tums.ac.ir published online: january 05, 2016 ment of cchf virus was developed and used for analysis of cchfvs (schwarz et al. 1996). phylogenetic studies relied on the sequence data of the s-rna segment have demonstrated genetic diversity for many cchfv strains from different regions of the world (iashina et al. 2002). despite the fact that recombination is relatively scarce in cchfv genome (chare et al. 2003) and a partial sequence of s-rna segment can be used for phylogenetic analyses, but it is recommended to use the full sequence data of the srna segment for obtaining more precise phylogenetic analysis, as the possibility of recombination is still expected (lukashev 2005). there have been several genetic analyses of cchfvs obtained from iran; however, all the reported iranian strains were obtained mostly from the south-east of iran and just four obtained from central and recently one strain from the northern regions of iran (chinikar et al. 2012a, chinikar et al. 2004, chinikar et al. 2010, chinikar et al. 2013). what makes this study unique is in that a large number of cchfv strains have been characterized genetically by partial and complete nucleotide sequencing of virus small (s) and medium (m) segments with a wide geographical distribution. fifty partial sequences of s segment and six partial sequences of m segment and seven complete sequences of s segments from various locations have been studied. in this study, we have used a bioinformatics approach to analyze an alignment by estimating the phylogenetic relationship between the obtained sequences from iranian patients and genbank available data. phylogenetic analysis based on large collections of partial and complete sequences of the s segment has indicated the existence of eight distinct clades for cchfv (drosten et al. 2002). accordingly, the most surprising finding in this study is discovery of a novel genomic variant of cchfv. in other words, this study suggests the emergence of viiiran clade for cchfv in iran as a novel clade based on s segment analysis. in general, this phylogenetic analysis based on sequences of s-rna of cchfvs reveals that the majority of cchfv sequences from iran belongs to clade iv (asia 1), and clade v (europe), which is in consistent with previous reports from iran (chinikar et al. 2004, chinikar et al. 2012b). in addition, for the first time, two sequences (iran-zahedan/19 and iran-sistanbalochestan/ 85, which were obtained from the southeast iran, were seen within clade iv (asia 2) with the highest proximity to strains from india (jn572089). the data of this study are in conformity with previous studies which showed similarity between iranian s segment of cchfv strains with pakistan, afghanistan (chinikar et al. 2004) and iraq (chinikar et al. 2012a), interestingly findings regarding all phylogeny studies during these years is that cchfv strain iran/artec 193-3 is likely fade away from iran and it has been never reported since 35 years ago when it was reported as the first cchfv strain from iran by sureau (sureau and klein 1980). our data based on the s segment shows that cchfvs are grouped in eight different clades and have correlation with their geographical location (morikawa et al. 2007). previous phylogenetic studies based on lrna segment sequences have showed that the l tree topology is similar to the s tree topology (hewson et al. 2004b). however, the phylogenetic topology based on m-rna segment sequences of cchfvs is different from that of s-rna segments (seregin et al. 2004, ahmed et al. 2005). these analyses show that cchfvs are likely to be grouped in six different phylogenetic clades based on m-rna sequences (deyde et al. 2006, carroll et al. 2010). to obtain a phylogenetic tree based on m segment, a partial sequence data can be used (morikawa et al. 2007), indicating that rej arthropod-borne dis, june 2016, 10(2): 127–140 s chinikar et al.: genetic diversity of … 137 http://jad.tums.ac.ir published online: january 05, 2016 combination within the m segments is not common during evolution of the cchfvs. however, genetic re-assortment occurs frequently in cchfvs, when host ticks co-infected with different types of cchfvs (deyde et al. 2006). the reason why m-rna segment re-assortment is more frequently observed is not clear, however, strong interrelation between n protein encoded in the srna segment and rna polymerase encoded in the l-rna segment may be required to produce viable virus (chamberlain et al. 2005). different examples of rna segment reassortment can be seen in the m segment phylogenetic tree. tree based on m segment nucleotide and deduced amino acid sequence differences were very similar to each other. like mauritanian strain ard39554 that belongs to group iii in s and stands as an outgroup vii in m segment trees, surprisingly in our obtained strains, the iranian strain iran-kerman/22 forms a unique group and considered as clade vii in s tree (fig. 2), whereas it grouped within group v (europe) with 100 % bootstrap support for this topology in m tree (fig. 3), suggesting that it representing m segment re-assortment. other potential m segment re-assortment events include iranian strains iran-kerman/43 and iran-khrazavi/72 that were in group iv (asia 1) in s tree, while cluster within group iii in the m segment tree. conclusion the cchfvs in iran have multiple origins with vivid geographical relationships between virus strains. moreover, phylogenetic results reveal subtle links between distant geographical locations, which may originate either from livestock trade or from long-distance carriage of virus by infected ticks during bird migration. according to all previous phylogeny studies, four genetic lineages of cchf viruses, clade iv (asian 1 and ii), clade v (europe) and new clade vii (iran) exist in iran. based on mentioned above data, having a high genomic variation of cchfv, have been made iran as a miniature model of the world for genetic analysis of cchfv with various strains for cchfv. acknowledgements this manuscript is based on the results of corresponding author's phd thesis. we thank all members of the arboviruses and viral hemorrhagic fevers laboratory (national reference lab), pasteur institute of iran, and keyhan azadmanesh, and ms niknam and mohsen 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127–140 s chinikar et al.: genetic diversity of … 140 http://jad.tums.ac.ir published online: january 05, 2016 mourya dt (2012) genetic characterization and molecular clock analyses of the crimean-congo hemorrhagic fever virus from human and ticks in india, 2010–2011. infect genet evol. 14: 223–231 yashina l, vyshemirskii o, seregin s, petrova i, samokhvalov e, lvov d, gutorov v, kuzina i, tyunnikov g, tang y w. (2003). genetic analysis of crimeancongo hemorrhagic fever virus in russia. j clin microbiol. 41: 860–862. j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 214 http://jad.tums.ac.ir published online: june 30, 2020 original article evaluation of susceptibility of aedes caspius (diptera: culicidae) to insecticides in a potent arboviral-prone area, southern iran sahar hassandoust 1 ; *seyed hassan moosa-kazemi 1 ; *hassan vatandoost 1,2 ; mohammad mehdi sedaghat 1 ; kamran akbarzadeh 1 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 department of chemical pollutants and pesticide, institute for environmental research, tehran university of medical sciences, tehran, iran (received 29 jul 2019; accepted 20 jun 2020) abstract background: southern part of the country is a high risk for mosquito transmitted arboviruses. this study was carried out to determine the base line susceptibility of the aedini mosquitoes to the who-recommended insecticide. methods: larval collection was carried out by dipping method and adult collection occurred by suction tube from january to december 2017. the adult susceptibility test was assessed to bendiocarb 0.1%, ddt 4%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, malathion 5% and, permethrin 0.75% at different interval times as well as at discriminative dose recommended by who. the larval susceptibility test was occurred using temephos and bacillus thuringiensis serotype h-14, at different concentrations. the lt50, lt90 and lc50, lc90 values were calculated for plotting the regression line using microsoft office excel software ver. 2007. results: aedes caspius was quite resistant to ddt, malathion, bendiocarb and showed susceptible or tolerant to other insecticides.the lt50 and lt90 values to ddt in this species were 157.896, and 301.006 minutes, respectively. the lc50 and lc90 values of ae. caspius to temephos were 0.000068, and 0.000130ppm, the figures for b. thuringiensis was 111.62 and 210.2ppm, respectively. conclusion: a routine and continuous study for monitoring and evaluation of different species of aedes to insectides is recommend at different parts of country for decision making. keywords: aedes caspius; susceptibility; iran introduction arthropod borne diseases are very important in the world. the tribe aedini (family culicidae) contains approximately one-quarter of the known species of mosquitoes, including vectors of deadly or debilitating disease agents. this tribe contains the genus aedes, which is one of the three most familiar genera of mosquitoes (1). the aedini mosquitoes are responsible for transmission of the barmah forest, batai, babanki, bouboui, bunyamwera, chikungunya, cache, valley, dengue, eastern equine encephalitis, edge hill, everglades, getah, gangan, highlandj, ilheus, james canyon, kedougou, la crosse, lebombo, murray valley river, nyando, ngari, oriboca, orungo, pongola, ross river, rift valley fever, semiliki forest, sindbis, st louis, encephalitis; spondweri, tahyna, tensaw, trivittatus, uganda s, venezuelan equine encephalitis, west nile, wslv, wesselbron, wyeomyia, yellow fever, and zika (1). the number of dengue cases reported annually by who ranged from 0.4 to 1.3 million in the decade 1996–2005 (2). as an infectious disease, the number of death cases varies substantially from year to year (3). at the present, culicidae includes; 2 sub families, 11 tribes, 113 genera and 3526 species *corresponding authors: dr seyed hassan moosakazemi, e-mail: moosakazemi@tums.ac.ir, dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com mailto:moosakazemi@tums.ac.ir mailto:hvatandoost1@yahoo.com j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 215 http://jad.tums.ac.ir published online: june 30, 2020 (4). the iranian mosquitoes includes 69 species, that 7 or 11 genera depending on the classification used for aedines (5-6). recent epidemics of mosquito-borne viral infections in countries neighboring iran i.e. dengue, chikungunya and west nile infections in pakistan, dengue and rift valley fever in saudi arabia, and west nile infection in iraq have placed this country at a serious risk for mosquito-borne diseases (7-9). aedes caspius (pallas) is the vector of tahina and west nile viruses (7-8, 10). at the present seven anopheles species reported as the malaria vectors in the country including: an. fluviatiliss. l, an. culicifaciess. l, an. sacharovi, an. maculipenniss. l, an. superpictus, an. stephensi and an. dthali (11). in addition, zaim et al. reported the an. pulcherrimusas secondary vectors of malaria in the south east of iran (12). oocyte of plasmodium found at the first time in an. multicolor, while not found in salivary glands (13). avian malaria reported in iran by ghaffari (14). spraying with residual insecticide (irs) considered an important mosquito control measure. twelve insecticides recommended by who for irs currently, which belong to four chemical groups including one organochlorine, six pyrethroids, three organophosphates and two carbamates (15-16). ddt resistance in the adult of aedes aegypti, ae. albopictus and susceptibility to temephos, bacillus thuringiensis and metabolic resistance of the current species to deltamethrin and ddt have been reported in africa (17). resistance of ae. aegypti larvae to temephos has been reported in asia (18-19). in addition, larval resistance of aedes albopictus to temephos have been reported in malaysia (20), thailand (21).adult susceptibility test on ae. aegypti against some payrethroids has been reported in various research study (21-24). in spite of some reports due to resistance of an. stephensi against ddt, dieldrin and malathion in iran (13, 25-31). mechanism of resistance of an. stephensi against temephos has been reported by (3233). by now there are no evidence of resistance of ae. vexans and ae. caspius in iran. release of larvivorous fish and microbial agent using the bacillus thuringiensis, and larviciding by chlorpyrifos-methyl are the main larval control measures and pyrethroid as new insecticides are being used as irs and llins in iran (34-35). in spite of more than 50 years’ malaria control programming more than 60% of the total malaria cases reported from southern iran. malaria is one of the most important communicable diseases transmitted by anopheline mosquitoes (diptera: culicidae) to humans. in 2013, there are 97 countries and territories with ongoing malaria transmission, and 7 countries in the prevention of reintroduction phase, making a total of 104 countries and territories in which malaria is presently considered endemic. based on who estimate, 207 million cases of malaria occurred globally in 2012 resulted to 627 000 deaths (2). malaria is one of the important infectious diseases in iran with an average of about 15000 annual cases in the last decade, while total recorded cases has dropped to less than 500 locally transmitted cases in 2013. more than 80% of malaria cases in iran are reported from three provinces of sistan and baluchistan, hormozgan and kerman in southern and southeastern areas of the country. the most routes of malaria cases are immigration from afghanistan and pakistan to southern and southeastern areas of the country (36). over the last 20 years there has been a dramatic reduction of the malaria burden in iran. while in 1991, nearly 100,000 cases were reported, less than 100 locally transmitted cases in 2017 (ministry of health, annual reports unpublished data). all observations indicate that the data reflect the real situation and that the overwhelming majority of cases, which occur, are included in the national system, although there is room for improvement in the surveillance system. the spectacular progress can be ascribed to effective implementation of approj arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 216 http://jad.tums.ac.ir published online: june 30, 2020 priate curative and preventive control interventions through a strong health care infrastructure. social and economic development allowing better housing, use of air-conditioning etc. has also played a role. locally transmitted cases are now concentrated in the southeastern part of the country, which are affected by extensive population movement across the border with pakistan, where malaria control faces serious difficulties. in 2009, iran set time-bound elimination objectives for its malaria program. there has been excellent progress since, but the continued risk of importation of malaria cases from pakistan poses a huge challenge, politically, socially, operationally and technically, to malaria elimination in iran. the situation in the next decade will be absolute elimination or one where a few small short-lived foci emerge from time to time as a result of importation. the latest number of autochthonous cases in the whole country is 42 including 23 local malaria patients, 7 relapsed cases, 12 imported from the other districts by end of july 2016 (ministry of health, annual reports unpublished data). aedes albopictus and ae. aegypti has been recently reported in algeria, lebanon, palestine, syria, and turkey (37). aedes albopictus has been identified along the south eastern iran (6) and mediterranean coast of europe for decades along with local transmission of denv and chikungunya since 2007 (38). near the pakistan border, serologic evidence suggests possible denv transmission in iran (39-41), in afghanistan (42), though local transmission has not been confirmed to our knowledge (41). the presence of aedes or denv transmission in these areas should not be ruled out (41). qeshm and kish are commercial and industrial free zones in hormozghan province. this area also is important due to agricultural and husbandry in southern iran in the border line of persian gulf and oman sea. the study area is endemic to malaria, however in recent years, the nuisance's aedini species have been increased. there are no data about susceptibility level of aedini vectors in iran, so, the susceptibility level of aedeini mosquitoes has been studied during this research. the results could provide an essential clue for judicious use of insecticides and will be very useful to health authorities for future planning of vector control. material and methods study area the study was carried out in hormozgan (27°11′18″n 56°16′36″e/27.1884°n 56.276 8°e), province, southern iran. the people engaged to agriculture, horticulture, livestock, fishing sailing, and hand crafts including needlework, making carpet and musical instruments. the absolute maximum and average of temperature was reported 52 °c and 26.5 °c in hormozghan province, respectively. average annual rainfall and humidity was 140.28mm and 79%, respectively. the absolute maximum and average of temperature in isfahan was reported 40.6 °c and 17.1 °c. in this area average annual rainfall and humidity was 63.5 mm and 22%, respectively (43) (fig. 1). hormozghan province with 70,697km 2 (27,296 sq. mi) square kilometers comprised of 21 counties (or districts), 69 municipalities, 13 major cities and 2,046 villages. in 2011 a little more than 1.5 million people resided in hormozghan province. daregaz village (27˚ 49’gn, 56˚17’ge) with 268 households, and 926 populations, and kovae village (27˚44 ’gn, 56˚22’ge), 38 households and 112 populations, talsooro village (27˚46’gn, 56˚23 ’ge), 92 households and 309 populations, as fixed stations and zakin village (27˚49’gn, 56˚16’ge), 158 households 571 population selected randomly as variable stations. sampling methods sampling methods such as larval collection, hand catch was carried out during january to december 2017 (44). these studies https://tools.wmflabs.org/geohack/geohack.php?pagename=hormozgan_province&params=27.1884_n_56.2768_e_type:city(1578183)_region:ir https://tools.wmflabs.org/geohack/geohack.php?pagename=hormozgan_province&params=27.1884_n_56.2768_e_type:city(1578183)_region:ir j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 217 http://jad.tums.ac.ir published online: june 30, 2020 were conducted once every 30 days and collected mosquitoes were identified by specific systematic keys (5, 45). larval collection and rearing in each fixed and variable station larvae was collected from january to december 2017. mosquitoes larvae were picked up from the water using a dropper, pipette or fine net and inserted into the bulb. the related data such as water temperature, larval type, number and date sampling was recorded. larvae and pupae in holding container filled with water were transferred to the laboratory for rearing. mosquito larvae feed by dry fish food. adult mosquitoes live quite well on bowl of sucrose 5% in bottom of the cage. the adults were kept at 28 °c, relative humidity (80%) and 14l10d photoperiod (46). hand collection aedini mosquitoes were collected from the villages between 06.30 and 09.30am. sampling was carried out in each human dwelling, cattle and goat sheds for 15min using suction tube and torch (44). the mosquitoes were transferred in the cage as dimensions of 40×40cm and then sent to the laboratory. total of 200 to 250 mosquitoes were entered in each cage and covered with wet towel. the sucrose 5% solution was placed inside the cage. the mosquitoes were kept in standard condition (25 ˚c, 75% rh). in hormozghan province, totally, ten species were collected including: an. stephensi, an. dthali, an. culicifacies, an. fluviatile, cx. pipiens, cx. quinquefasciatus, cx. theileri, culiseta longiareolata, ae. caspiuss. l and ae. vexans. in adult collection an. stephensi was dominant species 34.76% allocated mosquitoes collected. an. dthali and an. culicifacies were followed 15%, 12.92%, respectively. culiseta longiareolata had the lowest density with 1.09%. an. culicifacies, an. stephensi, cx. pipiens, cx. theileri were collected in all months. in larval collection, an. stephensi, with 1495 specimens (28.9%) was predominant followed by cx. pipiens 753 (14.1%), an. culicifacies12.8%, cx. quinquefasciatus 6.3% in the same month. it should be noted that aedes caspius larvae was collected in may and december. insecticide impregnated papers impregnated papers with ddt 4%, malathion0.08%, bendiocarb 0.1%, deltamethrin 0.05%, lambda-cyhalothrin 0.03%, permethrin 0.25%, and control papers were supplied by world health organization. larvicides solutions. five concentration of temephos as (0.000015, 0.000031, 0.000062, 0.000125, 0.000250ppm) and four concentrations of bti as (4, 36, 296, 2368ppm) were immersed in 249ml of tap water separately and larval test was applied based of who criteria guideline 2016 (2). adult susceptibility test the adult susceptibility test was carried out according who guideline (2). each time 4–5 mosquito collected and insert to holding tube overall 20–25 mosquito were kept into holding tube. the susceptibility tests performed on their standard condition (22–26 °c, 60% h). the susceptibility of the wild strain of aedini mosquitoes was assessed to the insecticides impregnated papers. the mosquitoes were exposed to different insecticides by different interval times and 24 hours’ recovery period. who criteria for susceptibility test based on who recommendations (2), the following criteria have been used for interpretation and classification; mortality in the range 98–100% indicates susceptibility. a mortality of less than 98% is suggestive of the existence of resistance and further investigation is needed. the observed mortality (corrected if necessary) is between 90% and 97 %, the presence of resistant genes in the vecj arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 218 http://jad.tums.ac.ir published online: june 30, 2020 tor population must be confirmed. the confirmation of resistance may be obtained by performing additional bioassay tests with the same insecticide on the same population or on the progeny of any surviving mosquitoes (reared under insectary conditions) and/ or by conducting molecular assays for known resistance mechanisms. if at least two additional tests consistently show mortality below 98%, then resistance is confirmed. if mortality is less than 90%, confirmation of the existence of resistant genes in the test population with additional bioassays may not be necessary, as long as a minimum of 100 mosquitoes of each species was tested. however, further investigation of the mechanisms and distribution of resistance should be undertaken. when resistance is confirmed, pre-emptive action must be taken to manage insecticide resistance and to ensure that the effectiveness of insecticides used for malaria vector control (2). identification of mosquitoes using morphological characteristics the mosquitoes after the test were mounted and identified by specific systematic keys. the samples were recorded in the special forms by and the appropriate time of deaths associated with history of collection, relative humidity and temperature (5, 45). statistical analysis results were considered reliable if the control mortality was less than 5% and rejected if more than 20%. results were corrected by abbott's formula when mortality rates of control group were between 5 to 20% (47-48). data were analyzed by probit analysis (49). regression lines of the species were measured through the χ2 test. the lt50 and lt90values were calculated for plotting the regression line using microsoft excel software ver. 2013. results adult bioassay adult bioassays using various insecticides showed that lt50 and lt90 values for ddt 4% against ae. caspius were ranged from 157.896–301.006 minutes for the band strain. bioassay test for other insecticides against is shown in table. 1, fig. 2. larval bioassay larval bioassays using temephos showed that lc50 and lc90 for ae. caspius ranged from 0.000068–0.000130mg/l for the band strain (susceptible reference strain) to 111.62–210.2 mg/l for the b. t (table. 2, figs. 3, 4). mortality of aedes caspius exposed to ddt and other insecticides has shown in tables 1 and 2. lt50 and lt90 values of this species to ddt 4% were 157.89 and 301.006 minutes, respectively. this species was quite resistant to ddt and other insecticides except deltamethrin (fig. 2). it is concluded that an. caspius is resistant to ddt, malathion, and bendiocarb, permethrin, lambdacyhalothrin whereas susceptible to deltamethrin, (table 1). the lt50 and lt90 values of this species to ddt 4% were 157.896 and 301.006 minutes (table 1, fig. 2). mortality of aedes caspius larvae exposed to temephos and bti has shown in (table 2 and figs. 3, 4). lc50 and lc90 values of this species to temephos were 0.000068 and 0.000130ppm, respectively. lc50 and lc90 values of this species to bti were 111.62 and 210.2ppm, respectively. https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-4-79#tab1 j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 219 http://jad.tums.ac.ir published online: june 30, 2020 table 1. regression line parameters of aedes caspius adult stage exposed to some insecticides recommended by who in a arboviral-prone area southern iran, 2017 insecticide a b±se lt50 95% c.i (minute) lt90 95% c.i (minute) (df) p-value book y= bx+a ddt4% 1.1511 0.0167±0.196 157.896 301.006 2.925 (2) p>0.05 5.99 y= 0.0167x+1.1511 malathion 5% 1.2944 0.0081±0.190 160.229 304.435 0.289 (2) p>0.05 5.99 y= 0.0081x+1.2944 bendiocarb 0.1% 1.6845 0.0135±0.087 42.124 80.0356 0.357 (2) p>0.05 5.99 y= 0.0135x+1.6845 deltamethrin 0.1% 1.7745 0.0141±0.077 48.735 92.5965 0.08 (2) p>0.05 5.99 y= 0.0141x+1.7745 lambdacyhalothrin 0.05% 1.8494 0.0132±0.166 46.129 87.6451 11.307 (2) p>0.05 y= 0.0132x+1.8494 permethrin 0.75% 1.5955 0.0156±0.196 29.652 56.3388 10.890 (2) p<0.05 5.99 y= 0.0156x+1.5955 table 2. regression line parameters of aedes caspius larval stage exposed to some larvicides recommended by who in arboviral-prone area southern iran, 2017 larvicide a b±se lc50 95% c.i lc90 95% c.i p-value book y= bx+a temephos 6.8275 3322.2±0.385 0.000068 0.000130 0.872 (3) p>0.05 7.81 y= 3322.2x+ 6.8275 b.thuringiensis 1.7839 0.0004±0.256 111.62 210.2 173.914 (2) p<0.05 7.81 y= 0.0004x+1.7839 fig. 1. map of study area, hormozghan province, iran j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 220 http://jad.tums.ac.ir published online: june 30, 2020 fig. 2. regression line of aedes caspius adult stage exposed to some insecticides recommended by who in arboviral-prone area southern iran, 2015 fig. 3. regression line of aedes caspius larval stage exposed to temephos larvicide recommended by who in a potent dengue endemic area of central and southern iran, 2015 fig. 4. regression line of aedes caspius larval stage exposed to b. thuringiensis larvicides recommended by who in arboviral-prone area southern iran, 2015 j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 221 http://jad.tums.ac.ir published online: june 30, 2020 discussion in our study, 4 genera and 10 species of mosquito larvae and adults were identified based on morphological characters. culicidae species were belongs to the genus of anopheles, culex, culisitaand aedes. the species of ae. caspius and ae. vexans found by larval collection. the most predominant species was an. stephensi with 34.76% of adult and 29.36 % of larvae collection. vatandoost et al. (2004b) (50), reported three biological forms of this species including type, intermediate and mysorensis in southern iran. type and intermediate forms cited as vector in urban areas whereas, mysorensis form as vector in rural area (51). in iran, indoor residual spraying (irs) with ddt was carried out for malaria control during 1950–1968. in this species, resistance to ddt was first recognized in 1958 malathion in 1976 (13). following the emergence of resistance of an. stephensi to ddt, other organophosphorus, carbamate and pyrethroid insecticides were used. the susceptibility level of an. stephensi to ddt and dieldrin was studied at various parts of iran bordered in persian gulf and oman sea during 1985–2016. the situation of dengue fever and dengue hemorrhagic fever has been changed in imported to indigenous cases in iran and probable aedes albopictus is responsible for these endemic diseases due to unplanned urbanization (6). in southern iran, the climatic conditions are suitable for mosquito’s life cycle. the changes in temperature, humidity and wide range of water grades may have a significant effect on the population growth and also vector control programmers (52). potent dengue vector in iran has exophilic behavior, so, the efficacy of larvicing materials is very important to vector control programs. temephos and bti were evaluated in lab scale against ae. caspius larvae in the current study. in this research work, different concentrations of bti were prepared as done by previous workers (53-54). bacillus turingiensis is safe and ef fective biocontrol agent used widely to control of mosquitoes for the recent years (5558). the experiment was conducted in tape water. abdalmagid et al. (2012) (53) checked the efficacy of bti dunks in field water and studied the physio-chemical properties of water. they concluded that these properties have no impact on the efficacy of bti (p> 0.05). mulla (1990) (59) studied that it was difficult to handle 1 st instar larvae because of high mortality rate during handling. due to this reason we used 3 rd and 4 th instar larvae for our experiments. in the present study we found low mortality rate in case of bti. in agreement with this study, rodrigues et al. (1999) (60) reported the low mortality of ae. agypti post treatment by bti and 24h. recovery periods. ramathilaga et al. (2012) (61) studied the impact of bti against 3rd instar larvae of ae. aegypti as was recorded in the present study against 3 rd and 4 th instar larvae of ae. caspius. in the present study, 40% and 78% mortality was recorded for 592 and 1184ppm of bti respectively after 24h in tape water while ramathilaga et al. (2012) (61) recorded (16%) mortality at the 1mg concentration of bti for 24h treatment in tap water. haung et al. (1993) (62) recorded 52.1, 69.5 and 78.2% mortality after 12, 24 and 48h respectively in 0.10ppm against ae. aegypti larvae while 97.1, 97.1 and 97.1% mortality after 12, 24 and 48h in 0.20ppm. gbehou et al. (2010) (63) compared the efficacy of bti on aedes, culex and anopheles species and observed 40, 80 and 100% mortality after 2, 4 and 6h against aedes species. many other factors such as species, genera susceptibility, feeding behavior of larvae, instar susceptibility to biocides, suspended organic matter, water temperature, larval density, and water depth influence the efficacy of bti against mosquitoes (boisvert 2005) (64). some of these factors like organic, inorganic, muddy, food and floating particles decreased the efficacy of bti due to j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 222 http://jad.tums.ac.ir published online: june 30, 2020 adsorption of bti onto suspended particles followed by a slow sedimentation (65-66). in the present study, we found higher concentration of bti is enquired for 100% mortality rate. in parallel, ohana et al. 1987 (67), mulla 1990 (59) reported more concentration need to control of ae. agypti larvae due to bti a few toxic suspended crystals particles ingested by larvae. in this research study, different concentrations of temephos were prepared as done by previous workers (68). this larvicides is safe and effective agent used widely to control of mosquitoes for the recent years. kemabonta and nwankwo 2013 (68) checked the efficacy of temephos in field water with comparison to spinozad. they concluded that these properties have good impact on the 3 rd and 4 th ae. agypti larvae (p> 0.05). the lc50 values for wild aedes caspius larvae were 0.000068mg/l and 0.000130mg/l, while the lc50 values for the laboratory bred and wild aedes aegypti larvae were 7.418g/l and 8.150 g/l respectively (68). in the present study, 100% mortality was recorded at 0.000250mg/l of temephos respectively after 24h in tape water while kemabonta and nwankwo (2013) (68) recorded (100%) mortality at the 30g/l concentration of temephos for 24h treatment in tap water. many other factors such as species, genera susceptibility, feeding behavior of larvae, instar susceptibility to biocides, suspended organic matter, water temperature, larval density, and water depth influence the efficacy of bti against mosquitoes (64). some of these many other factors like organic, inorganic, muddy, food and floating particles decreased the efficacy of temephos. in addition, many factors effects of efficacy of bti due to adsorption of bti onto suspended particles followed by a slow sedimentation (6566). in the present study, we found higher concentration of bti will be needed for 100% mortality rate. in parallel, ohana et al. 1987 (67), mulla 1990 (59) were reported more concentration need to control of ae. agypti larvae due to bti a few toxic suspended crystals particles ingested by larvae. the interruption in the efficacy of bti was found to be caused by bacterial adsorption to soil particles, but the inactivation could be inverted by washing the mud away (44). due to these reasons, the mean value of lc50 was higher against ae. caspius larvae in comparison to temephos. the mean lc50 values of bti and temephos were 111.62ppm and 0.000068ppm after 24h for tape water respectively. the results of the present study revealed the higher mortality post treatment by temephos in tape water because temephos is considered as contact larviciding in comparison to bti as digestive effects and it is free of any particles due to suspended particles. based on the literature, no reports were available on the susceptibility levels of ae. caspius. conclusion iran is near the dengue endemic area, aedes albopictus was reported for the first time in southeastern iran in 2014. by now, irs in human dwelling sand animal shelters, space-spraying, personal protection through distribution of llins and curtains (icns), repellents measures used to control of vectors in iran. in addition, some biological and chemical agents against larval and adult stages of mosquitoes had been evaluated in the laboratory. results obtained from susceptibility tests of ae. caspius on some who recommended insecticides revealed that highly resistance to them in southern iran. precautionary measures should be taken in future vector control operations. moreover, the status of resistance in other locations in this area should be investigated. since the country relies on deltamethrin for irs operation, tolerant populations of aedini species implies careful consideration and regular monitoring of susceptibility level of mosquitoes in the future. j arthropod-borne dis, june 2020, 14(2): 214–227 s hassandoust et al.: evaluation of susceptibility of … 223 http://jad.tums.ac.ir published online: june 30, 2020 acknowledgements the authors are grateful to dr majdzadeh head of national institute of health research, isfahan institute of health research for cooperation during the study. the authors thank to dr holakoei-naeini, head of bandar-abbas health training and research center, for help us during this 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( 2009) dengue guidelines for diagnosis, treatment, prevention and control: new edition. available at: world health organization. https://apps.who.int/iris/handle/10665/ 44188 . p. 147. https://onlinelibrary.wiley.com/journal/14390418a https://onlinelibrary.wiley.com/journal/14390418a https://doi.org/10.1111/j.1439-0418.1984.tb03785.x https://doi.org/10.1111/j.1439-0418.1984.tb03785.x https://apps.who.int/iris/handle/10665/44188 https://apps.who.int/iris/handle/10665/44188 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 204 original article pfcrt gene in plasmodium falciparum field isolates from muzaffargarh, pakistan *sumrin sahar 1, akhtar tanveer 1, akbar ali 2, hazrat bilal 3, rana muhammad saleem 3 1lab of parasitology, zoology department, university of the punjab, lahore, pakistan 2department of basic health sciences, faculty of pharmacy, northern border university, rafha, saudi arabia 3medical entomology and disease vector control, health services academy, lahore, pakistan (received 21 dec 2014; accepted 22 june 2014) abstract background: the aim of the study was to identify the prevalence of different species of plasmodium and haplotypes of pfcrt in plasmodium falciparum from the selected area. methods: overall, 10,372 blood films of suspected malarial patients were examined microscopically from rural health center sinawan, district muzaffargarh, pakistan from november 2008 to november 2010. p. falciparum positive samples (both whole blood and fta blood spotted cards) were used for dna extraction. nested pcr was used to amplify the pfcrt (codon 72–76) gene fragment. sequencing was carried out to find the haplotypes in the amplified fragment of pfcrt gene. result: over all slide positivity rate (spr), p. vivax and p. falciparum positivity rate was 21.40 %, 19.37 % and 2.03% respectively. fta blood spotted cards were equally efficient in the blood storage for pcr and sequencing. analysis of sequencing results of pfcrt showed only one type of haplotype sagtvmnt (agtgtaatgaataca) from codon 72–76 in all samples. conclusion: the results show high prevalence of cq resistance and aq resistant genes. aq is not recommended to be used as a partner drug in act in this locality, so as to ward off future catastrophes. keywords: malaria, pfcrt, resistance, pakistan introduction globally 106 countries from tropical and subtropical are endemic for malaria (who 2010). these are in tropical or subtropical zones with are suitable breading places far anopheline mosquito. the regions included in this area are asia, africa, islands of south, west and central pacific ocean, latin america, turkey and certain caribbean islands (lamar et al. 2007). in the areas of malaria transmission, epidemics of malaria have been leading cause of high morbidity and mortality. complex epidemics of malaria have been observed in kenya (may 1999–august 1999, burundi (september 2000–may 2001), south sudan (june 2003–november 2003), in two areas of ethiopia (july 2003–february 2004) and india (january 2005–march 2005) (checchi et al. 2006, kumari 2009). in 3rd world countries including pakistan, malaria has always been major public health problem and it accounts for more than 95% of regional burden (huda and zamani 2009). the main reason of malariogenic potential in pakistan is extensive agricultural practices, vast irrigation network, monsoon rains and poverty (who 2005). annually, about 500000 episodes of malaria infection occur (yasinzai and kakharsulemankhel 2008) in rural areas, were associated with more people live below poverty line (38.65%) than urban areas (22.39%). the main-stay in the control of malaria is *corresponding author: dr sumrin sahar, e-mail: sumrinsahar@yahoo.com http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 205 the increasing frequency of drug resistant parasites especially p. falciparum (gama et al. 2010). first reports of resistance against cq came from south america (moore and lanier 1961), south east asia (harinausta et al. 1965). later, it reached to africa. across the world, cqr strain of p. falciparum originated from at least six locations independently: in java, philippines, papua new guinea, south east asia and two locations in south america (wootton et al. 2002, chen et al. 2003). in pakistan, the resistance of p. falciparum to cq was first detected in 1981 from sheikhupura and national institute of malaria research and training (nimrt) revealed that r1 level of cq resistance was widespread in pakistan (frequency ranging from 30–84%) (shah et al. 1997). chloroquine was used as a first line treatment in pakistan from 1950– 2007 (asif 2008). in the district muzaffargarh, response of p. falciparum to chloroquine was found out during the time period of 2003– 2005. ri was 29.36 %, and rii was 29.41 % (rana 2009). in 1990s, chloroquine resistant p. falciparum spread widely in pakistan (rab et al. 2001). factors involved in the spread of drug resistance are drug use practices, drug half life, transmission intensity, clone multiplicity, parasite density, host immunity and genetic basis of drug resistance (hasting and watking 2006, pongtavornpinyo et al. 2008). resistance against many antimalarials has arisen in the same geographic areas. these areas have low transmission rate, low immunity level and higher parasites biomass (white and pongtavornpinyo 2003). use of substandard medicines can also be involved in the emergence of drug resistance (leslie et al. 2009). with the advancement in molecular biology and isolation of cq resistance clone (dd2) helped in identification of molecular markers in p. falciparum associated with drug resistance (jiang et al. 2008). in 1990, thomas wellem along with his team performed a cross between hb3, a chloroquine sensitive (cqs) and dd2, a chloroquine resistant (cqr) strain (wellem et al. 1990). it was found that the main determinant of cq resistance is a locus on chromosome 7 of p. falciparum (wellem et al. 1991). it was further noted that it is a 38 kb fragment (10 genes) (su et al. 1997). fidock et al. (2000) reported that a highly polymorphic 3.1 kb gene (composed of 13 exons) is involved in resistance. martin and kirk (2004) reported that it is a 48.6 kda protein of 424 amino acids long. it forms 10 transmembrane domains. the protein is located on digestive vacuole and it has transporter-like properties. it was called p. falciparum chloroquine resistant transport gene (pfcrt) and considered as a member of drug metabolite transporter super family (tran and saier 2004). in the past, svmnt (ser-val-meth-asn-thr) allele carrying parasites had been reported twice, in 1996–1997 from africa and in 2004 from tanzania (alifrangis et al. 2006). three common haplotypes of pfcrt are cvmnk (cqs, present in all geographic regions), cviet (cqr found in africa and south east asia), svmnt (cqr, sagtvmnt is present in some countries of asia and stct vmnt is found in south america) (wootton et al. 2002, ursing et al. 2006). the high level of resistance to aq metabolite desethylamodiaquine (deaq) is found to be associated with the presence of pfcrt codon 72–76 haplotype svmnt. it replaces cvmnk found in cqs strain (wootton et al. 2002, warhurst et al. 2003, sa et al. 2009, beshir et al. 2010). in oocyte system, competition of drugs with cq was checked by mutated pfcrt. drug quinine and aq at normal concentration and mefloquine and primaquine at higher concentration were able to compete with cq for transport by mutated pfcrt while piperaquine and artemisinin did not interact with pfcrt (martin et al. 2009). keeping in view the situation of malaria, http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 206 this study was designed to identify the chloroquine resistant genes of p. falciparum which convert a chloroquine sensitive parasite (cqs) to chloroquine resistant (cqr) type parasite. materials and methods selection of area for sampling muzaffargarh is located at latitude 30.06˚ longitudes 71.02˚, between the two rivers, chenab in the east and indus in the west. for present study, the area of muzaffargarh was selected on the basis of high endemicity. data obtained by the curtsy of directorate general health punjab, pakistan (through personal communication) reported “1388 laboratory confirmed cases” in punjab were with 15 % in district muzaffargarh in 2006. in 2007, total number of positive malarial cases was 1903, again highest positive cases (7.5%) were observed in muzaffargarh. sample collection blood samples were collected from individuals presented with fever, persistent headache and malaria like symptoms. patient’s consent was obtained prior to sampling, finger prick blood was used for slide preparation. thick and thin blood smears were prepared and stained by giemsa stain (3% solution in 7.2 ph phosphate buffer) (who 1991). two ml blood of p. falciparum positive patients was collected in edta vacutainers and spotted on flinders technical associates (fta) classic cards (whatman) by micropipette according to manufacturer’s instruction. vacutainers were stored at 4 ˚c and cards were stored at room temperature till use for dna extraction. sampling was done at rural health centers muzaffargarh from november, 2008 to november, 2010 (25 months) as shown in fig. 1. dna extraction for amplification of pfcrt gene, dna was eluted both from fta cards and whole blood. p. falciparum positive samples (by microscopic review and rdts method). fta cards contain chemicals that lyses (removes) blood cells, denature proteins and protect nucleic acids from nucleases, oxidation and uv damage. captured nucleic acid is ready for downstream applications in less than 30 min and is stable for years at room temperature. dna was eluted according to manufacturer’s instructions (whatman). while from whole blood, 200 μl of sample was used for dna extraction with the qia amp midi kit, as described by the manufacturer (qiagen). extracted dna was analyzed by using 1 % agarose gel. amplification of pfcrt gene fragment two-step nested pcr was done to amplify pfcrt gene fragment of 145 base pairs. primers were designed, using dna sequence of p. falciparum chloroquine resistant strain dd2/indochina (by pub med accession number af030694). the sequences of primers used in round 1 and round 2 are given in table1. preparation of pcr reaction mix for round 1 (pfcrt) the pcr mixture contained 1x pcr buffer (promega), 2.0 mm mgcl2 (promega), 0.2 mm dntps, 0.25 µm each primer, 1.25 units, 0.5 units taq polymerase (promega) and 5 µ l template dna. the total reaction volume was 50 µ l. cycling conditions for pcr were: one cycle of 95 ˚c for 5 minutes followed by 30 cycles of 95 ˚c for 10 seconds, 57 ˚c for 30 seconds and 72 ˚c for 30 seconds, followed by a final extension at 72 ˚c for 10 minutes. pcr reaction mix preparation for round 2 (pfcrt) the pcr mixture contained 1x pcr buffer (promega), 2.0mm mgcl2 (promega), 0.2 mm dntps, 0.25 µ m each primer, 0.5 units taq polymerase (promega) and 5 µltemplate dna. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 207 the total reaction volume was 50 µ l. cycling conditions for pcr were: one cycle of 95 ˚c for 5 minutes followed by 35 cycles of 95 ˚c for 30 seconds, 48 ˚c for 30 seconds and 65 ˚c for 30 seconds, followed by a final extension at 65 ˚c for 3 minutes. amplified pcr products of each gene fragment were electrophoresed using 3 % agarose gel and visualized on uv. pcr products were cleaned using exonuclease i (biolabs inc) and sequenced in both directions using big dye® terminator system. statistical analysis results of sequencing were analyzed by the programme of applied bio-system (sequence scanner v, 0.1). results sampling was carried out in muzaffargarh, pakistan from november 2008 to november 2010 (25 months), in order to find out the malaria frequency. during this period, 10372 blood films of suspected malarial patients were prepared and examined. their positivity rates for the infection of plasmodium (both falciparum and vivax), p. vivax and p. falciparum were 2220, 2009 and 211 respectively. in this duration, slide positivity rate (spr), p. vivax positivity rate % (vpr) and p. falciparum positivity rate (fpr) were 21.40 %, 19.37 % and 2.03 % respectively. the difference in p. vivax (90.49%) and p. falciparum (9.51%) infection was found highly significant (χ2= 1456, p< 0.001). extracted dna samples positive for p. falciparum (211) were used for dna extraction. genomic dna is shown in fig. 2, 3. amplification of pfcrt (codon 72–76) all the 211 p. falciparum positive samples were amplified using polymerase chain reaction. amplification was carried out of the extracted dna from whole blood and from fta blood spotted cards. pfcrt (codon 72–76) amplification (by using disc of blood spotted fta cards) amplification by using the disc (2mm) was also conducted by using punched disc from fta blood spotted cards. product of (145bp) was visible as show in fig. 4. similar pcr results were obtained using dna extracted from whole blood and fta blood spotted cards which showed that fta cards were found equally efficient for storage of blood for pcr reaction. sequencing result sequencing results were analyzed by using applied-biosystem vi software. it showed only one type of haplotype sequence sagt vmnt (agtgtaatgaataca) from codon 72–76 in all samples. no wild type cvmnt or other drug resistant (mutant) haplotype was found. fig. 5 is depicting sequencing results of pfcrt (codon 72–76) with forward primer. table 1. primers used for amplification of pfcrt gene fragment of 145 base pairs pcr round primer primer sequence (5/–3/) tm 1st round crtda (forward primer) 5/ggtggaggttcttgtcttggta -3/ 57.5 ˚c crtdb (reverse primer) 5/gacctatgaaggccaaaatgactg-3/ 56.7 ˚c 2nd round crtdc (forward primer) 5/tgtgctcatgtgtttaaactt -3/ 50.06 °c crtd –d (reverse primer) 5/caaaactatagttaccaatttt -3/ 46.10 °c http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 208 fig. 1. map of district muzaffargarh showing rhcs fig. 2. gel electrophoresis picture showing extracted genomic dna of falciparum positive malarial patients (lane l shows 100bp ladder, lane 1–7 show genomic dna) fig. 3. gel electrophoresis picture showing amplified pfcrt gene fragment (145bp) (by using dna extracted from the whole blood). lane 1 and 2 show negative and positive controls respectively, lanes 3 to 12 show patient samples, lane 13 shows 100bp dna marker fig. 4. picture of gel electrophoresis showing amplified pfcrt gene fragment (145bp) (by using disc of blood spotted card). lane 1 and 2 show negative and positive controls respectively, lanes 3 to 12 show patient samples, lane 13 shows 100bp dna marker fig. 5. sequencing results of pfcrt (codon 72–76) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 209 discussion present findings showed that overall spr observed in muzaffargarh, pakistan was 21.40 %. according to the report of ministry of health, (2006) malaria was the second most prevalent disease in pakistan (murtaza et al. 2009). akin figures of spr (19.7% and 19.40%) were reported from punjab and khyber pakhtunkhwa (idris et al. 2007, mohammad et al. 2010), which were respectively 1.64 % and 2.00 % less than present study. spr reported from india has shown the percentages of 25.2 % and 24.54 % (dhiman et al. 2010, panigahi et al. 2011), which were 3.80% and 3.14% higher than in current study. although malaria is rife worldwide but the maximum numbers of devastations caused by it are in sub-saharan africa (who 2010). in africa 30.20 %, 27.07 % and 22.60 % spr was reported from uganda, ethiopia and tanzania (jensen et al. 2009, yohannes and haile 2010, mubi et al. 2011) and these figures indicated an enhancement of 8.80 %, 5.67 % and 1.20 % respectively than the figures of the current study. these differences may be due to geographical, climatic, economic and cultural variations. in the current study, p. vivax, was the predominant specie of this region. the overall infection caused by p. vivax and p. falciparum was 90.49 % and 9.51 % respectively, and the difference between the infection of p. vivax and p. falciparum was highly significant (χ2= 1456, p< 0.001). similar figures were collected from muzaffarabad, azad kashmir and khyber pakhtunkhwa, occurrence of p. vivax infection (90.40% 92.20%, 92.20%) was far more than that caused by p. falciparum infection (9.6%, 7.8% and 7.8%) (jan and kiani 2001, jamal et al. 2005, jalalul-din and ally 2006). similarly higher incidence of p. vivax infection (72.4%) than p. falciparum infection (24.1%) was reported from baluchistan province (idris et al. 2007). some other figures from the same province showed higher incidence of p. vivax infection (58.9%, 51.8%, 88.6%, 72.3%) than p. falciparum infection (41%, 48.1%, 11.3%, 27.6%) (yasinzai and kakarsulemankhel 2008). infections caused by p. vivax were also reported more than those of p. falciparum from some areas of sind (mahmood et al. 2005, atif et al. 2009). p. vivax infection (11.70%, 75%) was less than p. falciparum infection (88.29%, 85%) in some areas or indian (dhiman et al. 2010, panigrahi et al. 2011). similarly, p. falciparum (90%, 70%, 90.7% 68.53%) was predominant specie in some african regions (pinto et al. 2000, yohannes and haile 2010, lemma et al. 2011). currently, parasites strains are exhibiting resistance to all major classes of drug present in the field and some of them have been withdrawn from use because of their ineffectiveness. unfortunately we are unaware that where the next generation of antimalarial will come from (grimberg and mehlotra 2011). core objective of the study was the scrutiny of mutant and wild types of pfcrt (codon 72–76). pfcrt gene fragment of 145bp was amplified in all positive p. falciparum (211) samples. results showed 100 % sagt vmnt (ser-val-meth-asn-thr). no stctvmnt haplotype or wild type (cvmnk) was observed. origin of sagtvmnt had been hypothesized from mato grasso, brazil (vieira et al. 2004) and was reported from guyana, peru, suriname, venezuela (contreras et al. 2002, cortese et al. 2002, peek et al. 2005, mehlotra et al. 2008, griffing et al. 2010). clinical resistance to chloroquine (cq) and amodiaquine (aq) was found associated with mutations in pfcrt gene (picot et al. 2009, beshir et al. 2010). sa et al. (2009) correlated high level resistance to aq metabolite desethylamodiaquine (deaq) in tests (in vitro) with the presence of svmnt haplotype of pfcrt. mehlotra et al. (2008), zakeri et al. (2008), and beshir et al. (2010) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 204–214 s sahar et al.: pfcrt gene in plasmodium … 210 detected high prevalence of this haplotype in the parasitic population of brazil, papua new guinea, laos, afghanistan and iran. esmaeili et al. (2008) also reported very high prevalence of k76t mutation in an endemic district of iran. these results were also supported by the study carried out (in vitro) on natural p. falciparum isolate, laboratory reference strain, parasites transfected with allelic replacement and progeny of genetic crosses (sidu et al. 2002, sa et al. 2009). in the past, svmnt (ser-val-meth-asn-thr) allele carrying parasites had been reported twice, in 1996–1997 from africa and in 2004 from tanzania (alifrangis et al. 2006). three common haplotypes of pfcrt are cvmnk (cqs, present in all geographic regions), cviet (cqr found in africa and south east asia), svmnt (cqr, sagt vmnt is present in some countries of asia and stct vmnt is found in south america) (wootton et al. 2002, ursing et al. 2006). in the area of muzaffargarh p. vivax was pre-dominant specie so cq exposure was higher. it may be the main reason of high prevalence of spreading cqr parasite (having pfcrt “svmnt”) in this area. similar findings were reported from india, mutant pfcrt “svmnt” haplotype was endemic in those areas, where p. vivax was dominant specie (mallick et al. 2012). conclusion prior to everything else, appropriate diagnosis of plasmodium and its species is highly necessary. a treatment initiated without proper diagnosis not only increases the probabilities of drug resistance, but also invites complications. malaria controlling policies should be decentralized: made and implemented at province and district levels. proper understanding and careful evaluation of local epidemiological pattern and the other aspects of malaria can produce base-line data in planning task for devising control strategies. the drug resistance issue is likely to pose long term challenges for us. hybrid of conventional and neo drugs can help to conserve the efficiency of valuable anti malarial medicines, but choosing the right combination partners will continue to require the study of presently known and emerging mechanism. the results show high prevalence of cq resistance and aq resistant genes. excessive presence of cq and aq resistant genes in plasmodium population isolated from the people of muzaffargarh suggest establishing the base line for monitoring of mutations. this is evident now that in this area, cq or aq will not be effective for the victims of p. falciparum infection. aq is not recommended to be used as a partner drug in act in this locality, so as to ward off future catastrophes. acknowledgements financial assistance was provided by university of the punjab research committee 2010–2011 and higher education commission pakistan (pin# 074-0551-(bm-4-033) is gratefully acknowledged. thanks to dr hanan el mohammady ismail head, immunology section clinical trials research programme us naval medical research unit #3 (namru-3) cairo, egypt for valuable suggestions to improve the manuscript. this paper is produced from phd project conducted by the principle author. the authors declare that there is no conflict 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*mousa tavassoli; bijan esmaeilnejad department of pathobiology, faculty of veterinary medicine, urmia university, urmia, iran (received 29 nov 2016; accepted 1 jul 2019) abstract background: this study was carried out to identify theileria spp. infections in goats and ticksin kermanshah province, western iran from may–sep 2015. methods: for differentiation of different theileria spp. both blood and tick samples were examined by nested pcrrflp. results: light microscopy of blood smears revealed theileria spp. infection in 22 (5.5%), while 68 (17%) of blood samples were positive using nested pcr. out of 68 positive samples, 85.3% (58/68) and 11.7% (8/68) were respectively positive for theileria ovis and t. lestoquardi. mixed infection was detected in 3% (2/68) cases. overall, 420 ixodid ticks belong to seven different hard ticks species were collected from goats. rhipicephalus turanicus 112 (26.7%), r. sanguineus 95 (22.6%), r. bursa, 91(21.7%), hyalomma anatolicum, 55(13.1%), h. excavatum 27(6.4%), h. marginatum, 22(5.3%) and dermacentor marginatus, 18(4.2%) were the main tick species infesting goats. the pcr products obtained from ticks were subjected to the differentiation of theileria species. respectively, 2 and 8 pools of h. marginatum and r. turanicus salivary glands were infected with t. ovis and t. lestoquardi. in addition, t. annulata and t. lestoquardi infection weredetected in three pools of h. anatolicum. conclusion: this is the first report of goats and collected ticks to theileria spp infection in iran. the results suggest that t. ovis has a higher prevalence than t. lestoquardi. it is also postulated h. marginatum, r. turanicus and h. anatolicum might play an important role in the field as a vector of theileria spp in this area. keywords: theileria; tick; goat; nested pcr-rflp; iran introduction theileriosis is a hemoparasitic disease of domestic and wild ruminants caused by species of the genus theileria that transmitted by species of ixodid ticks. it is one of fatal diseases of sheep and goats in tropical and subtropical regions, where it causes significant economic losses as well as reduced production (1). the disease occurs due to at least six species of theileria spp. including t. ovis, t. separata, t. recondita, t. lestoquardi (t. hirci), and theileria sp. (china 1) and theileria sp. (china 2) that recently was reported from north of china (1). among theileria parasites, t. lestoquardi, theileria sp. (china 1) and theileria sp. (china 2) are considered highly pathogenic (2). two species, t. lestoquardi and t. ovis, cause ovine/caprine theileriosis in iran (3). the presence of these two species was also confirmed by sequencing analysis and nested pcr-rlfp in iran (4). theileria annulata, which is a causal agent of tropical malignant theileriosis in cattle, can also infect sheep (3). hyalomma species have been implicated in the transmission of t. lestoquardi and t. annulata, namely h. anatolicum, h. savignyi, h. aegyptium, and h. impeltatum (5). rhipicephalus bursa, r. evretsi, and r. sanguineus are considered as the main field vectors of t. ovis (6). gold standard for diagnosis of theileriosis is usually based on microscopic examination of giemsa-stained blood smears, clinical signs and the species of *corresponding author: dr mousa tavassoli, e-mail: mtavassoli2000@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 298 http://jad.tums.ac.ir published online: september 30, 2019 theileria specified purely based on the animal host, which the sample was taken. on the other hand, since in most endemic areas, t. lestoquardi and t. annulata infections can occur naturally in both cross host and vectors (4, 7). the exact identification and differentiation between theileria spp. parasites are important for understanding their epidemiology (8). achieving to identify theileria species and vector ticks in goat is not feasible by using conventional theileria diagnostic methods based on morphological features, especially during mixed infections and low parasitemias (4). serological assays e.g. indirect immunofluorescence and elisa have been developed for the laboratory diagnosis of theileriosis. however, the antibodies cannot always be detected in long-term as well as cross-reactivity of the antibodies against other theileria species has limited the specificity of serological tests (7). the use of alternative techniques, such as nested pcr-rflp molecular tool based on 18s rrna has become necessary to provide complementary diagnostic information for low-costly and effectively identification of theileria or babesia species in the common host (4, 9-11). to date, earlier investigations about molecular epidemiology of ovine theileriosis in iran restricted to eastern geographical areas and none so far has addressed the question of the theileria infection in goats and vector ticks in iran (4, 12-15). considering the importance of goat farming in iran and paucity of data about species of theileria, which can infect goats and fieldcollected ixodid ticks, we aimed to accurate, identify theileria species in goats and ticks by using microscopic examination and nested pcr-restriction fragment length polymorphism (rflp) screening assay. materials and methods field study area the study was carried out during tick active season, from early may through late sep 2015, in kermanshah province (an area of more than 24640km2), located in an important livestock production region and tropical area in western iran between 33°37'–35°17' n latitudes and 45°20'–48°1' e longitudes and 1420 above sea level. ecologically, this area is classified as a semi-arid zone. goat rising is a very important economically occupations in this province. animals, blood sampling, collection and examination of blood smears and ticks forty goat flocks were randomly selected by the local veterinary service of kangavar, songhor, kermanshah, gilan-e gharb and qasr-e shirin areas and 400 peripheral blood samples were collected and then immediately thin blood smears prepared from ear capillaries were fixed in methanol for 5min and stained in 10% giemsa solution in phosphate buffer solution (pbs), ph 7.2, for 20min and examined under a magnification of 1000x for the presence of intracellular forms of the parasite with morphology similar to theileria spp. the percentage of infected red cell per 100 red blood cells was calculated. for estimating parasitemia, 100 microscopic fields containing approximate 1000 red blood cells per field were reviewed and the number of parasites per 100,000 red blood cells was enumerated. even the presence of a single piroplasm was considered as positive. from each flock, at least 8 animals were randomly chosen. during the sampling, data on the variable parameters of the flock and animals were recorded. based on size, the flocks divided into flocks with 15– 50 goats and flocks with more than 50 goats. age was the individual-level factor. in each flock, goats were categorized into two age classes (<1yr old versus ≥1yr old). flocks were divided into two categorized: flock with tick infestation and no tick infestation. at the same location where blood samples were collected, ticks were collected by inspecting the whole body of each goat for the presence of ticks, mainly on their ears, along their nape of neck, http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 299 http://jad.tums.ac.ir published online: september 30, 2019 perineum, and udder/orchid, inner thighs, shoulder region and tail base. ixodid ticks were manually collected from the body surface of examined animals by rubbing alcohol pads surrounding the skin to remove embedded living ticks (16). the hard ticks were placed into labeled glass vials with 70% ethanol (merck, germany) and transferred to the parasitology laboratory. tick species were identified using taxonomic keys (17, 18). whole tick were washed once in roccal (1% benzalkonium chloride) and three times in 70% ethanol and then dried. the ticks’ salivary glands were dissected out and placed into 200µl lysis buffer containing 20µl proteinase k, incubated for 10min at 55 °c. after adding of 360µl binding buffer and incubation for 10min at 70 °c, 270µl ethanol (100%) was added to solution, and after vortexing, complete volume was transferred to the mbst column. the mbst column was first centrifuged and then washed twice with 500µl washing buffer. finally, dna was eluted from the carrier with 50µl elution buffer and stored at -70 °c until use. they were divided into 55 pools comprising 4–10 tick specimens according to the source species and bioclimatic zones. dna isolation and nested pcr total dna was extracted from blood and tick samples using molecular biological system transfer kit (mbst iran). briefly, 200µl blood and tick samples were first lysed in 180μl lysis buffer and the proteins were degraded with 20μl proteinase k for 10min at 55 °c. after addition of 360μl bindings’ buffer and incubation for 10min at 70 °c, 270μl ethanol (100 %) was added to the solution and after vortexing, the complete volume was transferred to the mbst column. the mbst column was first centrifuged and then washed twice with 500μl washing buffer. finally, dna was eluted from the carrier with elution buffer. purity of dna was tested spectrophotometrically at wavelength of 260 and 280nm. extracted dna of theileria was diluted to give a final con centration of 50ng. a nested pcr was used to detect theileria spp. dnas. the homologous and variable regions of 18s rrna gene were amplified by employing two-pair primers. outer primers for the primary pcr were forward strand primer thei f1 5'-aac ctg gtt gat cct gcc ag-3' and reverse strand primer thei r1 5'-aaa cct tgt tac gac ttc tc-3'. the amplicon size of the primary pcr was 1700bp. the nested inner primers were forward strand primer thei f2 5'-tga tgt tcg ttt yta cat gg-3’, and reverse strand primer thei r2 5'-cta ggc att cct cgt tca cg-3'. after the second pcr, the size of nested pcr products for different species ranged from 1417 to 1426bp. the primer's specificity and sensitivity was assessed (11). primary pcr was performed in a 30μl total reaction volume containing 3μl dna(45–150 ng), outer primer (20pg), dntp (250μm of each deoxynucleotide triphosphates), 10x pcr buffer (100mm tris-hcl (ph 9), 500mm kcl, 1% triton x-100), taq polymerase (1.25u, promega madison, wi, usa), and mgcl2 (1.5 mm) in an automated thermocycler (corbet research, australia) under following program: initial denaturation stage (5min at 94 °c), 25 cycles (denaturation step, 30 sec at 94 °c, annealing step, 30sec at 51 °c, extension step, 30sec at 72 °c) and final extension of 5min at 72 °c. fifty ng of pcr products were used as template in nested pcr. in this stage, the amplification mixture was the same as that used in primary pcr, except that the inner primers were used. nested pcr conditions were at 94 °c for 2min followed by 30 cycles of 94 °c for 30sec, 52 °c for 30sec 72 °c for 30sec, and final extension, 5min at 72 °c. then, 10μl aliquots of the pcr products were stained with cyber green solution and electrophoresed through a 1.5% gel. after electrophoresis, amplified samples were visualized by uv transilluminator (bts-20m, japan). expected pcr products for the different theileria species are shown in table 1. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 300 http://jad.tums.ac.ir published online: september 30, 2019 the extracted dna from salivary glands of ticks were amplified according to the protocol was previously described for blood samples. the positive dna from theileria spp. schizont was kindly provided by razi vaccine and serum research institute (tehran branch, iran) (accession numbers: kf429799, mg 208059, kp019206). sterile water was served as negative control. restriction enzyme analysis the pcr product was purified using the mbst pcr product purification kit. 10–15µl of purified pcr product were then digested with 1ml (10u) of hpaii and haeii restriction enzymes in 3µl 10× buffer and 5µl h2o at 37 °c for 2h according to supplier recommendations (jena bioscience, jena, germany), and analyzed using 2% agarose gel(jena bioscience, jena, germany), and analyzed using 2% agarose gel. the restriction analysis patterns for theileria spp. are listed in table 2. statistical analysis the fisher’s exact test and mantel-haenszel test were used to show association between the presence (positive and negative blood samples) of theileria and the various parameters i.e. flock size, gender and age of animal, tick infestation of goats and presence of ticks in the flock. mcnemar’s chi-square test was used to compare the data of blood smears with blood pcr method. results were displayed as p-values as well as relative risk values (with 95% confidence intervals). p< 0.05 was accepted to be statistically significant. results microscopic examination of thin blood smears showed parasitemia in infected animals ranging from 0.011% to 0.012% piroplasms. low numbers of highly polymorphous parasites inside the red blood cells were detected in most of the blood smears. samples with round, oval, ring and anaplasmoid forms were tentatively classified as theileria spp. microscopic examination of 400 blood smears obtained from five different areas of kermanshah province revealed that 22 (5.5%) goats were positive for piroplasms. the percentage of positive smears for theileriosis was determined between 1.25% and 10% in this region. the highest cases of theileria piroplasmosis was seen in qasr shirin (10%), followed by gilan gharb (8.75%), kermanshah (5%), kangavar (2.5%) and songhor (1.25%) (table 3). the results of nested pcr assay showed that 17% (68/400) of the goats were infected with theileria spp. (table 3, fig. 1). the highest rate of infection was observed in qasr-e shirin, 26.25% (21/68), that was followed by gilan gharb, kermanshah, kangavar, and songor, 22.5% (18/68), 17.5% (14/68), 12.5% (10/ 68), and 6.25% (5/68), respectively. rflp analysis proved presence of t. ovis in majority (85.3%) of theileria positive samples, while t. lestoquardi and mixed infection was detected in 11.7% (8/68) and 3% (2/68) of the samples, respectively. moreover, enzymatic digestion of pcr products of mixed infections revealed that 1 sample was infected with t. lestoquardi and t. ovis, and one sample with t. lestoquardi and t. annulata. all microscopically positive samples were confirmed by nested pcr. no theileria piroplasms were seen on blood smears of samples that were negative in nested pcr. however, there were 46 pcr positive samples, which were negative in microscopic examination (table 4). the prevalence of theileria spp. infection in age groups and different gender were not significantly different (table 5). in this study, 420 ticks of seven different species, r. turanicus 112 (26.7%), r. sanguineus 95 (22.6%), r. bursa 91 (21.7%), h. anatolicum 55(13.1%), h. excavatum 27 (6.4 %), h. marginatum 22 (5.3%) and d. marginatus, 18(4.2%) (table 6) were isolated and then analyzed using nested pcr in terms of the presence of theileria spp. infection. there are 45 tick pools, of which, 13 pools http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 301 http://jad.tums.ac.ir published online: september 30, 2019 that belong to the salivary glands of h. marginatum, r. turanicusand h. anatolicumwere positive for theileria spp. infection (table 7) (fig. 1). the frequency of theileria spp. infection was higher in flocks with tick infestation than no tick infestation (p< 0.05). frequency of theileria spp. infection was significantly (p< 0.05) higher in female ticks. molecular identification results by nested pcr-rflp screening technique demonstrated that at least three genetically distinct theileria spp. are found in both blood and ticks samples in surveyed-area. digestion of pcr product sobtained from goats and ticks, which were positive for theileria spp. using hpaii and haeii, was shown in table 2. out of 68 positive samples, 85.3% (59) and 11.7% (8) were positive for t. ovis and t. lestoquardi, respectively. mixed infection was detected in 3% (2) cases. digestion of pcr products obtained from animals revealed that one sample with t. ovis and t. lestoquardi, as well as one sample with t. lestoquardi and t. annulata mixed infections were detected (table 3, fig. 2). respectively, 2 and 8 pools of h. marginatumand r. turanicussalivary glands were infected with t. ovisand t. lestoquardi (table 7). table 1. expected pcr products for different theileia spp (11) species the size of nested-pcr product t. lestoquardi 1417bp t. annulata 1420bp t. ovis 1426bp table 2. the pattern of rflp of pcr products of different theileria spp by using of hpaii and haeii (13) species hpaii haeii t. lestoquardi 900-, 278-, 106-, 94and 39bp no digestion t. annulata 1178-, 106-, 94and 39bp n digestion t. ovis 856-, 326-, 204-and 39bp 295 and 1131bp table 3. results of microscopic examination and nested pcr and rflp for theileria spp in different areas of the kermanshah province area mea nested pcr t. ovis t. lestoquardi t. annulata mixed nsb pc % parasitemia (%) p % p % p % p % p % kangavar 80 2 2.5 0.011 10 12.5 10 100 0 0 0 0 0 0 songar 80 1 1.25 0.011 5 6.25 5 100 0 0 0 0 0 0 kermanshah 80 4 5 0.011 14 17.5 14 100 0 0 0 0 0 0 gilan-e-gharb 80 7 8.75 0.011 18 22.5 14 77.8 3 16.7 0 0 1 5.5 qasr-e-shirin 80 8 10 0.012 21 26.25 15 71.4 5 23.9 0 0 1 4.7 total 400 22 5.5 0.011 68 17 59 85.3 8 11.7 0 0 2 3 amicroscopic examination, bnumber of sample, cpositive sample http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 302 http://jad.tums.ac.ir published online: september 30, 2019 table 4. comparison of microscopic examination and nested pcr analysis results in theileria infection diagnosis in goats result test thin blood smear nested pcr positive 22 68 negative 378 332 total 400 400 table 5. association between the presence (pcr-positive and negative blood samples) of theileria spp infection in goats and the evaluated parameters (flock size, age, gender and tick burden of animal) flock size age of animal gender og animal tick burden of animal 15–50 animals >50 animals <1 year >1 year male female no tick tick infestation number 210 190 100 300 85 315 305 95 negative 17.7 (84.2%) 155 (81.5%) 84 (84%) 248 (82.4%) 70 (82.4%) 262 (83.2%) 274 (89.8%) 58 (61%) positive 33 (15.8%) 35 (18.5%) 16 (16%) 52 (17.4%) 15 (17.6%) 53 (16.8%) 31 (10.2%) 37 (39%) p(f)* p(f)= 0.59 (ns) p(f)= 0.88 (ns) p(f)= 0.40 (ns) p(f)= 0.007 *p (f): fisher's exact test, p value, ns: not significant table 6. frequency of tick species collected on the body of goats tick species tick number (%) tick pool of salivary glands (no. of male) (no. of female) r. teranicus 112 (26.7%) 8 (84) 3 (28) r. sanguineus 95 (22.6%) 6 (60) 5 (48) r. bursa 91 (21.7%) 5 (54) 4 (41) h. anatoliaum 55 (13.1%) 4 (39) 2 (19) h. excavetum 27 (96.4%) 2 (17) 1 (10) h. marginatum 22 (5.3%) 2 (18) 1 (4) dermacentormargintus 18 (4.2%) 1 (12) 1 (6) total 420 28 (284) 17 (156) table 7. results of molecular methods for detection theileria spp in salivary glands of ixodid ticks tick species t. ovis t. lestoquardi t. annulata total (%) no. of male (%) no. of female (%) no. of male (%) no. of female (%) no. of male (%) no. of female (%) hyalomma marginatum 1 (1) 1 (2) 2 (3) rhipicephalus turanicus 1 (1) 3 (21) 1 (3) 3 (20) 8 (45) hyalomma anatolicum 1 (2) 1 (1) 1 (14) 3 (17) total 2 (2) 4 (23) 1 (3) 4 (22) 1 (1) 1 (14) 13 (65) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 303 http://jad.tums.ac.ir published online: september 30, 2019 fig. 1. product of theileria spp using primary pcr and nested inner primers. lane m: molecular marker (100bp plus ladder); lane p.ct1and pct2: positive controls; lane n.c: negative control, lanes 1–2 and s1–s3: pcr products from infected goats; lane 3and t1–t3: pcr products from infected adult ticks. fig. 2. rflp patterns of theileria spp amplification products. (a) lane m: molecular marker (100bp plus ladder), lane pc, undigested pcr product, lane 1: t. lestoquardi hpaii digest, lane 2: t. ovis hpaii digest, lane 3: t. annulata hpaii digest, lane 4: mixed t. lestoquardi, t. ovis and t. annulata with hpaii. (b) lane m: molecular marker (100bp plus ladder), lane 1: undigested pcr product, lane 2: t. ovis haeii digest isolated from infected goats; lane 3: t. ovis haeii digest from infected tick. discussion the highest infection rate of theileria in qasr shirin and gilan gharb may probably be associated to that these regions bordered with iraq where has been known as a foci of this disease. this finding is the closest to other results (19, 20) that have reported 28.8% and 20.8% of theileria spp. infections in iraq, respectively. moreover, several studies performed to detect theileria infection by the microscopic method in goats in different geographical regions of turkey, western neighbor of iran (2123). 3.8% of sheep and goats blood samples http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 304 http://jad.tums.ac.ir published online: september 30, 2019 were theileria positive in pakistan by microscopy observation (24). in italy, the prevalence of theileria infection was 1.6% (25). the results concerning the lower incidence of theileria infection in the goat are compatible with the results in this paper. the reason that the goat had a low prevalence of theileria infection may probably be ascribed to the ability of the goat to graze in unreachable and inclined areas. in this situation, goat is less exposed to the bite of infected-ticks (25). however, in contrast with the results collected in this paper regarding the low prevalence of theileria infection in goat, the high-frequency rates of theileria spp. infection in goats were reported from various northern zones of egypt (85.33%) and iran (66.7%) (26).this difference is likely due to the differences between surveyed-areas and sampling design as the present work sampled-goats were selected randomly. in the present study, the parasitemia was not exceeded by 10.012%. in similar studies, (22, 27, 28) goat infected with theileria spp. commonly had low parasitemia. it could be attributed to first, in caprine benign theileriosis, this normally gives very low parasitaemia and second, t. lestoquardi is considered as a pathogenic parasite that causes the death of animals before the appearance of the parasite in lymphoid cells and erythrocytes (29). in iraq, serological tests employing indirect fluorescent antibody test (ifat) were used and the seropositivity rate of t. lestoquardiin goats varied from 7.3% to 3.8% in different regions of the country (30, 31). moreover, using ifat, 8.9% infection of goats by theileria were reported in turkey (22). in the present study, covering five different regions of kermanshah province, the molecular prevalence ranged from 6.25% to 26.25%. although the results of the present and previous study cannot be compared due to the different methods were employed, the results clearly indicated that theileriosis was not broadly dispersed in this region. in the present study, the age and sex of the animals did not show any significant association with theileria spp. infection. in the endemic area, due to the relatively high number of infected ticks found and the fact that young and old goats are being continuously exposed to infected ticks, a stage of enzootic stability may have been developed (32). these results are in agreement with the finding of other researchers (33, 34). on the contrary, age and sex influenced the prevalence of piroplasmosis. these two contradictory findings may be attributed to the difference in the number of examined animals, natural immunity, pregnancy, drug administration for controlling of hemoprotozoan and recent use of acaricides, which had an effect on tick distribution (15, 35). as expected, the prevalence of theileria spp. infection in goats detected by nested pcr was significantly (p< 0.05) higher than that obtained in microscopic examination of thin blood smears. therefore, dna amplification methods had higher efficiency than microscopic examination for detection of theileria. the results were in agreement with a previous report about ovine theileriosis (11, 36). this study also revealed that recovered animals frequently sustain subclinical infections, which are microscopically undetectable. due to no report on caprine theileriosis in iran to compare with the present study, therefore the data was comparable to the previous studies on the prevalence of theileria spp. infection in goats in other countries. in previous similar studies done in other countries in middle east e.g. turkey (10, 21, 37) and syria (38), t. ovis is reported as the most prevalent species. the same result was obtained from tunisia as well (39). in disagreement with these findings, in iraq, the most abundant theileria species identified was t. lestoquardi (19, 30). the dissimilarity amongst results of the two studies may be related to the different diagnostic methods that they used for microscopic examination to assess the frequency of http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 305 http://jad.tums.ac.ir published online: september 30, 2019 theileria spp. infection in goats; obviously molecular methods are most reliable. the low prevalence of t. lestoquardi found in this study is in concern with those reported earlier (10, 21). the lower prevalence of t. lestoquardi infection found in goats might be because of h. anatolicum, which is the natural vector of this theileria species (23). although this species is widely distributed all over iran, low infestation 13.1% (55/420) was recorded during this survey, probably because goats may not be a preferable host for h. anatolicum anatolicum (40). most of the malignant small ruminant theileriosis foci in iran are situated in places with a mean annual temperature (mat) between 20–25 °c that often are under latitude (l) of 30° in the south part of iran (41). in this situation, parasite can infect the goats but failed to reach infective numbers, subsequently, less infection occurs (27). the high prevalence of t. lestoquardi was reported from qasr shirin as 23.9%. this result may probably be attributed to that qasr shirin county situate in nearly of most famous endemic foci of malignant small ruminant theileriosis in the south of iran such as ahvaz and ilam with same geographical and climatic features (4). theileria annulata is transmitted by h. anatolicum in kermanshah province, iran (42), and in most of these areas both cattle and goat are raised close to each other, our findings regarding infections of t. annulata in goat are reasonable. the presence of antibodies against t. annulatawas demonstrated earlier in infected goats (43). in the present study, lower rates (4.7%) of t. annulata infection in goat indicates that the mild to moderate susceptibility of goats to infection with t. annulata (43) and the possibility of goat acting as a reservoir of t. annulata under field conditions. regarding ticks infesting goats, rhipicephalus spp. (namely r. turanicus, r. sanguineu, and r. bursa respectively were found to be the most dominating in this study, as has been also recorded in turkey (22), iran (44), and iraq (45). apart from rhipicephulus spp., the second most common species of tick infesting goats werefound to be hyalomma spp. similarly, the most common tick species of goat in iraq is rhipicephalus spp. followed by the hyalomma spp. (45). in opposition to our results, hyalomma spp. and dermacentor spp. existed only in the mountainous parts of the countries among goats (46). in consistentwith the findings of other studies (44), d. marginatus exhibited the lowest (4.2%) distribution in this work. this is because of a different geographical and metrological structure of the mountainous area versus semi-arid climate. in this work, nested pcr incriminated h. marginatum, r. turanicus and h. anatolicum as suspected vectors for transmission of theileria spp. based on rflp results, h. anatolicum was found to be infected by t. annulata and t. lestoquardi. earlier studies, (5, 25, 42, 47) were corroborated our data. transmission potential and infectivity of r. turanicus for t. ovis infection is in accordance with the other findings (13, 48). however, in contrast to our results, infection of r. bursa, r. sanguineus and h. turanicum were reported by t. ovis (6, 13). a geographical disparity between two regions may have resulted in a better adaptation of one tick’s species to the local conditions, thus replacing with other ones. as shown in the current study, r. turanicus was infected by t. lestoquardi. the obtained results are in conformity with the finding of previous investigations, which reported that rhipicephalus spp. maybe has a transmission role in small ruminant theileriosis (12). there were no molecular evidence about the role of the r. turanicus as a vector in the transmission of t. lestoquardi infection in goat. in iran, in a similar study (47), t. lestoquardi dna was isolated from r. turanicus, obtained from unknown infested host. moreover, r. turanicus could be the main vector of b. ovis (16, 49). however, further detailed experimental studies are needed to demonstrate precisely that whether r. turanicus can transmit t. lestoquardi http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 297–309 m rahmani-varmale et al.: molecular detection and … 306 http://jad.tums.ac.ir published online: september 30, 2019 to goats. in harmony with the findings in the present study, prevalence of theileriosis was higher in herds with tick infestation indicates the presence of a positive correlation between the prevalence of the disease and the presence of ticks (24, 25). in addition, from the results of this work, the difference between the prevalence of theileria spp. infection in female ticks was statistically significant (p< 0.05). in agreementto the findings in this paper, the prevalence of theileria infection has been reported to be higher in female ticks. this may be due to female ticks had many more type iii acini than male ticks and theileria parasites were only detected in type iii acini (8, 50). these statements show why female ticks have greater theileria infection prevalence than males. conclusion theileria ovis is the dominant causative agent in this region but the evidence of t. lestoquardi and t. annulata infection of goats in few cases were noteworthy, as well. theileria annulata was successfully transmitted from cattle to goats and vice versa by h. a. anatolicum. taking into account the possibility of occurrence of t. annulata infection in goats andsuccessful feeding of h. anatolicum on goat even a small portion, goats may be risk factors for theileriosis in cattle in areas where cattle and goats raised together as well as common competent vector coexist in the same area. finally, h. marginatum, r. turanicus and h. anatolicum may play an important role in transmission of different 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tehran university of medical sciences, tehran, iran (received 15 apr 2013; accepted 31 may 2014) abstract background: cockroaches are of vital importance medically and hygienically. they are able to contaminate foods and act as vectors of pathogenic agents such as bacteria, protozoa, and parasites to human environment either mechanically or through their digestive system. cockroaches belong to the phylum arthropoda, class insecta, and order blattodea or blattaria. to date, over 4,500 cockroach species have been reported from different parts of the world. we overviewed the documents involved iranian cockroaches to up-to-date checklist of cockroach species distributed in various provinces of iran. methods: an extensive literature review was performed in 2013 on iranian handbooks, reports and published data available since 1986 to obtain a comprehensive list of iranian cockroaches. furthermore, in an entomological survey in tehran, cockroach specimens were collected and identified based on morphological and the dna sequences of the mitochondrial cytochrome oxidase subunit ii (coii) gene (mt-dna coii) characteristics. results: morphological characterization revealed presence of an un-described species very similar to polyphaga aegyptiaca, p. indica and somehow to pycnoscelus surinamensis, however, supplementary molecular analysis revealed the species was associated with polyphaga of corydiidae (polyphagidae). with regards to the report of the un-described species, the cockroach fauna of iran includes three families, 14 genera, and 26 species. conclusion: some species has not been collected or reported recently and also many geographical regions of the country have not been studied yet, hence a systematic research is required to reveal the real cockroach list of the country. geographical distributions, nomination changes, and synonyms of cockroach species are presented. keywords: cockroach, blattodea, polyphaga sp, mtdna-coii, molecular systematics introduction cockroaches are one of the oldest and the most successful insect lives have been present on the earth from pennsylvanian epoch (the upper carboniferous period). the fossil records indicate that cockroaches have undergone rapid changes correlated with developmental cycles to obtain the ideal body form and survived as resistant taxa (moore et al. 1952). to date, over 4,500 cockroach species have been reported from different parts of the world of which about 30 species are associated with human habitats (valles et al. 1999). cockroaches live all over the world but most of the species are found in tropical and equatorial regions (schal and hamilton 1990). cockroaches hygienically are one of the important pests widely distributed throughout the world. frightening and pesky appearance of cockroaches is another significant factor to call them pest. the sanitary and medical significance of cockroaches is owing to their presence in homes, groceries, food warehouses, restaurants, hotels, hospitals on the one hand and in sewer systems and rubbish *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 162 bins on the other hand. they can act as vectors of pathogenic agents such as bacteria, protozoa (single-celled eukaryotes), and parasite eggs and can transmit diseases to humans either mechanically or through their digestive system. thus they are considered a very important vector of diseases including cholera and typhoid fever to humans. moreover, cockroach infestations, including saliva, feces and cast skins, are a source of allergens and can irritate allergies and asthma in people, especially children (farhoudi et al. 2003, ahmed et al. 2010, sookrung and chaicumpa 2010, shahraki 2011). almost 40%–60% of patients with asthma in urban and inner-city areas possess ige antibodies to cockroach allergens (sohn and kim 2012). cockroaches belong to the order blattodea or blattaria (sometimes considered a suborder, blattaria, of order dictyoptera), and are regarded as the most primitive winged insects. blatta is of latin origin and means shunning light (hanafi-bojd and sadaghiani 2009). many adult cockroaches have fully developed wings, whereas other cockroaches have short wings or lack wings altogether. young, immature cockroaches resemble adults but are smaller and wingless. cockroaches are identified by the following taxonomic features: color, number and the shape of spines on the femora, tarsus and wings, reproductive organs, and head frontal. moreover, at the posterior end of the male abdomen, there are cercus, stylus and hypandriun which are important factors in species identification. historically, according to the classification of mckittrick (1964) and roth (1970), cockroaches have been regarded as a separate order divided into two superfamilies of blattoidea and blaberoidea which totally include five families of polyphagidae, blaberidae, blattellidae, blattidae, and cryptocercidae. however, in more recent classification, number of superfamilies tops out at three including corydioidea (synonym: polyphagoidea), blattoidea (synonym: termitoidea), and blaberoidea. and number of family tops out at nine including blaberidae, ectobiidae (synonym: blattellidae), blattidae, lamproblattidae, tryonicidae, cryptocercidae, termitidae, corydiidae (synonym: polyphagidae), nocticolidae (inward et al. 2007, lo et al. 2007). however, lo et al. (2007) criticized the classification of termites as a family of blattaria and suggested to retain the order of isoptera (termites with seven families including mastotermitidae, termopsidae, hodotermitidae, kalotermitidae, serritermitidae, rhinotermitidae, and termitidae) as an unranked name within blattaria (i.e blattaria: isoptera), until cockroach phylogeny is better resolved and an appropriate ranking can be applied. currently, beccaloni and eggleton (2013) classified the termites as an epifamily (ie termitoidae including 9 families of mastotermitidae, archotermopsidae, hodotermitidae, stolotermitidae, kalotermitidae, stylotermitidae, rhinotermitidae, serritermitidae, and termitidae) and created two other epifamilies to contain the cockroach species also placed in the superfamily blattoidea with the termites. unfortunately, in terms of faunistic studies on the iranian cockroaches, there are no enough records available. the only evidence is a paper written by mirzayans (1986) in which four families, eleven genera and 24 species of cockroaches were reported in iran. however, considering the report of parcoblatta sp by fathpour et al. (2003) and on the base of recent classification, iranian cockroaches could be classified in three superfamilies, three families, 14 genera, and 25 species. different species of family blattidae can be found all over the world. most of the species that are recognized as house pests belong to this family. among family blattidae, five species have been reported from different parts of iran. species of family corydiidae (polyphagidae) live in arid regions particularly in desert and semi desert areas. in iran twelve species of this family have already been recognized. in terms of the number of species, http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 163 family ectobiidae (blattellidae) is the largest order of cockroaches in the world and eight species of this family have been reported from the country. the german roach, blattella germanica, the major pest of residential dwellings, belongs to family blattellidae as well. in an entomological survey, we have found two male cockroach specimens originated from north of tehran which showed morphological characters of corydiidae (polyphagidae) and by some means to pycnoscelus surinamensis. however, further taxanomic investigation revealed some morphological characters distinct the two specimens from polyphaga aegyptiaca, p. indica, and py. surinamensis. this encouraged us to use molecular technique to determine their identity. currently, different mitochondrial and nuclear dna markers have been used for species identification and phylogenetic analysis of different groups of cockroaches (maekawa and matsumoto 2000 and 2003, maekawa et al. 2001 and 2007, lo et al. 2006 and 2000, park et al. 2004, sulaiman et al. 2012). among the loci, mitochondrial cytochrome oxidase subunit ii (mtdnacoii) gene has been commonly used for cockroaches (e.g maekawa et al. 2007) and other insects (liu and beckenbach 1992, chen et al. 2002, oshaghi et al. 2006 and 2007, preativatanyou et al. 2010, chen et al. 2012). in this study we tested the mtdna-coii sequences of the two specimens. meanwhile, this paper overviewed the documents involved iranian cockroaches to up-to-date checklist of cockroach species distributed in various provinces of the country. materials and methods to obtain a complete list of iranian cockroaches, a broad literature evaluation including persian and english documents has been carried out on iranian handbooks, reports and published data which were published since 1986 onwards. besides, in an entomological survey in north tehran, cockroach specimens were collected actively using boxes from indoor of buildings in summer 2011. the collected specimens were transferred to the insect molecular laboratory of school of public health, tehran university of medical sciences (sphtums) and then were morphologically studied using the available pictorial and morphological keys of pratt and stojanovich (1966), mirzayans (1986), cochran (1999), and hanafibojd and sadaghiani (2009). further molecular characters were provided to test sequence homology and phylogenetic relationship with the available entries in genbank. dna extraction, mtdna-coii pcr, sequencing, and phylogeny the male specimens were subjected to dna extraction using two legs from each individual. total genomic dna was extracted using qiaamp® mini kit (qiagen, germany) according to the manufacturer’s protocol. the coii gene of the cockroach mitochondrial genome was amplified using polymerase chain reaction (pcr) and then subjected to dna sequencing. pcr was carried out in 25 μl reactions, with the final concentrations of 1 out 100 μl template dna, 1 unit (0.2 μl) of taq polymerase, 1× pcr reaction buffer, 2mm mgcl2, 200μm of each dntps (fermentas, sinaclon) and 10 μm (1.2 μl) of each forward and reverse primer (sinaclon, iran). the primers used in this study were a-tleu 5’atggcagatt agtgcaatgg-3’ and reverse primer of b-tlys 5’-gtttaagagaccagtactt g-3’ as previously described (liu and beckenbach 1992). pcr thermal program included an initial denaturation step of 94 ˚c of 5 minutes, followed by 5 cycles of 94 ˚c for 1 minute, 45 ˚c for 40 seconds, 72 ˚c for 1 minute, and then followed by 35 cycles of 94 ˚c for 40 seconds, 50 ˚c for 40 seconds, 72 ˚c for 1 minute, and a final elongation step for 72 ˚c for 5 minutes. pcr products were visuhttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 164 alized on 1% agarose gel containing ethidium bromide and using an uv transilluminator. the amplified mtdna-coii amplicons were bidirection sequenced via the same amplification primers by bioneer company (s. korea) and seqlab (germany). the dna sequence reads were edited manually using the chromas software to eliminate discrepancies between forward and reverse sequences. the consensus sequences were analyzed using the blast search available in nucleotide collection of ncbi. to determine association of the undescribed specimens with species of polyphagidae, most of the mtdnacoii entries of corydiidae (polyphagidae) including p. aegyptiaca, ergaula capucina, therea sp, and eremoblatta subdiaphana, and a subset of entries represented species of other families of the order blattodea were retrieved from genbank and used for data analysis. representatives of other families were blattella germanica, symploce pallens, and blattella lituricollis of ectobiidae (blattellidae), shelfordella lateralis, blatta orientalis, periplaneta fuliginosa, and p. americana of blattidae, py. surinamensis of blaberidae, and locusta migratoria from orthoptera as outgroup. the sequences were aligned using the multiple alignment program clustalw2 online program and manually adjusted. the two specimen sequences were identified to genus level by sequence comparison with the genbank entries and by the phylogenetic analysis using the neighbor-joining method embedded in the clustalw2. phylogenetic analysis was performed on a combination of the data obtained from this study plus representatives of the cockroach species mentioned above. results entomological survey the undescribed species (polyphaga sp) were found, out of houses close to mountain area of assad-abad zone, velenjak area in northern part of tehran. morphological investigation showed that they look like two other known iranian species of polyphaga indica and p. aegyptiaca (egyptian) and by some means to py. surinamensis but in some characters they stand as a distinct species. in addition to variation in the size and color with the mentioned species, they had a distinct pronotum with straight edge posteriorly and laterally (fig. 1c, 1d). pronotum was angularly in py. surinamensis and rounded laterally in p. aegyptiaca. they also had a separation line between inferior part of forehead and main part of face which is specific character of p. aegyptiaca but lacked distinctive ocelli (simple eyes) which is specific character of py. surinamensis. it is noticeable that p. aegyptiaca and p. indica belong to corydiidae (polyphagidae) whereas py. surinamensis belongs to blaberidae. the most important morphological traits of this species which shared with the mentioned species include lack of strong spines along the internal margin of the middle and hind femora; pronotum dark brown, except a narrow yellowish band along anterior and lateral margins (fig. 1). morphological characters of the specimens somehow represented a new record for the country. our study using molecular mtdna coii marker revealed that the specimens belong to polyphaga of corydiidae (polyphagidae) however their coii sequences were only 85% homologue to p. aegyptiaca and far more different from other species of corydiidae (polyphagidae) indicating presumably a distinct and new species in iran. the mtdna-coii sequences of the two specimens were submitted to genbank with accession numbers of kj769153-kj769154. the sequences include 683 bp un-ambiguous data and there were no polymorphic sites between the samples. interestingly, alignment of the sequence data showed a fixed six dna base pairs deletion equals to two amino acids (aa) deletion in species of http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 165 corydiidae (polyphagidae) including the specimens found in this study in comparison with other cockroaches (fig. 2) indicating the undescribed specimens definitely belongs to corydiidae (polyphagidae) and presumably to polyphaga genus. further phylogenetic analysis confirmed the close association of the undescribed specimens to the ployphaga genus. phylogenetic analysis was performed using 16 sequences of the mtdna-coii of various cockroach species and of locusta migratoria (orthoptera) as outgroup. the cladogram inferred using the neighbor-joining method strongly supported association of the specimens (we call them as polyphaga sp) with other corydiidae (polyphagidae) species (fig. 3). the tree resolved very well the four families of the cockroaches each one formed a major group. two families of blaberidae and ectobiidae (blattellidae) were shown to be sister groups. checklist of iranian cockroaches with regards to the report of the undescribed species (polyphaga sp), the cockroach fauna of iran includes three families, 14 genera, and 26 species (table 1). superfamily blattoidea family blattidae periplaneta americana (linnaeus 1758) synonymy: blatta aurelianensis fourcroy 1785, periplaneta americana colorata rehn 1902, blatta domicola risso 1826, blatta ferrugineofusca gronovius 1764, blatta heros eschscholtz 1822, blatta kakkerlac de geer 1773, blatta americana linnaeus 1758, blatta orientalis sulzer 1776, blatta siccifolia stoll 1813, periplaneta stolida walker 1868. iranian records: almost all parts of iran (mirzayans 1986), southwest iran (shahraki 2010), hamedan-hamedan (salehzadeh 1995, mahjoob et al. 2010, nejati et al. 2011), isfahan (fathpour et al. 2003), isfahan-kashan (doroodgar et al. 2006), khuzestan-khorramshahr (kassiri and kazemi 2012), kohkiluyeh va boyer-ahmad-yasuj (shahraki et al. 2011), kurdestan-sanandaj (vahabi et al. 2007), mazandaran-sari (motavali haghi et al. 1997), tehran (karimi zarchi and vatani 2009), zanjan-zanjan (mohammadi 1996), qomqom (fahiminia et al. 2010). periplaneta australasiae (fabricius 1775) synonymy: blatta aurantiaca stoll 1813, blatta australasiae fabricius 1775, blatta domingensis palisot de beauvois 1805, periplaneta emittens walker 1871, periplaneta inclusa walker 1868, periplaneta repanda walker 1868, periplaneta subcincta walker 1868, polyzosteria subornata walker 1871, periplaneta zonata haan 1842. iranian records: most southern states and warm (persian gulf coast) (mirzayans 1986), hamedan-hamedan (mahjoob et al. 2010, nejati et al. 2011), khorassan-e-jonubibirjand, yazd (hanafi-bojd and sadaghiani 2009). blatta orientalis (linnaeus 1758) synonymy: blatta badia saussure 1863, blatta castanea blanchard 1851, blatta culinaris de geer 1773, blatta europaea bartsch 1846, blatta ferruginea thunberg 1810, stylopyga orientalis gracilis adelung 1903, blatta hemialata gistel 1856, pulex imperator westwood 1858, blatta lucifuga poda 1761, kakerlac pallipes philippi 1863, kakerlac platystetho philippi 1863, blatta secunda schaeffer 1769, stylopyga orientalis spontanea semenov-tian-shansky 1909, blatta tertia schaeffer 1769. iranian records: almost all parts of iran (mirzayans 1986), southwest iran (shahraki et al. 2010), hamedan-hamedan (salehzadeh 1995, mahjoob et al. 2010, nejati et al. 2011), tehran (karimi zarchi and vatani 2009), zanjan-zanjan (mohammadi 1996). shelfordella lateralis (walker 1868) synonymy: blatta lateralis walker 1868, shelfordella ahngeri adelung 1910, http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 166 paraloboptera sillemi hanitsch 1935, periplaneta tartara saussure 1874, periplaneta lateralis walker 1868, shelfordella tartara zarudnyi adelung 1910. iranian records: sistan va balouchestan (sarbaz, saravan, jalgh, pishin, zabol), ardebil-mogan, kermanshah-kangavar, isfahankashan, tehran (tehran, varamin), fars (firuzabad, kakan), golestan (gonbad-ekavus), kerman-kerman, khuzestan (masjedsoleiman, ahvaz), hormozgan (bandarlengeh, issin), (mirzayans 1986), southwest iran (shahraki et al. 2010), isfahan (fathpour et al. 2003), hamedan-hamedan (salehzadeh 1995), kohkiluyeh va boyer-ahmad-yasuj (shahraki 2011), yazd (hanafi-bojd and sadaghiani 2009), gilan province, assalem district (authors of this article). shelfordella monochroma (walker 1871) synonymy: blatta monochroma walker 1871, shelfordella gussakovskii bei-bienko 1950, shelfordella tertia bei-bienko 1938, periplaneta monochroma walker 1871. iranian record: hormozgan-bandar-abbas (mirzayans 1986). superfamily corydioidea (polyphagoidea) family corydiidae (polyphagidae) polyphaga aegyptiaca (linnaeus 1758) synonymy: blatta lata gistel 1856, blatta omega costa 1839, heterogamia punctata fischer 1846, blatta sphaerica gistel 1856, blatta spinifer kirby 1910, heterogamia spinipes fischer 1846, steleopyga trichoprocta fischer 1833, blatta aegyptiaca linnaeus 1758. iranian records: khuzestan (ahvaz, dezful), fars (shiraz, firuzabad, dehram), lorestan (aligudarz-ghalikuh 2360 m. alt.), tehran (damavand 2070 m. alt.), mazandaran (babol, gaemshahr), golestan-kalaleh, kermanshah (gilan-e-gharb, chuar, sumar, ghasre-shirin), elam-mehran, (mirzayans 1986), isfahan (fathpour et al. 2003), khuzestan-ahvaz (vazirianzadeh et al. 2009). polyphaga indica (walker 1868) synonymy: there is no synonym. iranian records: khuzestan-ahvaz, fars (mount dena 2210 m. alt. western slope, kazerun: gavkoshak, dashte-arjan 1900 m. alt.), azarbayjan-e-sharghi (tabria, moghan), khorassan-e-razavisafi abad, sistan va balouchestan, tehran (varamin, tehran, rudehen, damavand), isfahan-golpayegan, semnan-garmsar, salt desert, kerman-rudbar, mazandaran (mirzayans 1986). polyphaga saussurei (dohrn 1888) synonymy: heterogamia saussurei dohrn 1888, polyphaga camelorum kirby 1903, heterogamia pellucida redtenbacher 1889. iranian records: khorassan-e-razavi (dareh-gaz), hormozgan-minab, fars (midast, firuzabad, farrashband, kazerun, sarmashhad), sistan va balouchestan (zabol, pishin, bampur, iranshahr, khash, sarbaz-dez) (mirzayans 1986), yazd (hanafi-bojd and sadaghiani 2009). note: in the study of salehzadeh (1995), one unknown species of this genus has been reported from hamedan. polyphaga sp (sp nov?) in this study for the first time two specimens of a new but undescribed species (polyphaga sp) (sp nov?) were found in a summer time (agust 2011), in a house (third floor) where was built in close affinity with touchal mountain, asad-abad zone, shemiranat, northern tehran. the vocher specimen (fig. 1) is deposited in entomology meuseum of the school of public health, tehran university of medical sciences. arenivaga livida (brunner von wattenwyl 1865) synonymy: there is no synonym. iranian records: azarbayjan-e-sharghi (jolfa, aras) (bey-bienko 1950), azarbayjane-sharghi (tabriz-khalat pushan) (mirzayans 1986). http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 167 hemelytroblatta subhyalina (chopard 1921) synonymy: arenivaga subhyalina chopard 1921, polyphaga subhyalina chopard 1921, iranian records: gazvin (chopard 1929, beybienko 1950), north of iran (bey-bienko 1950), fars (shiraz, dashte-arjan 1900 m. alt.) (mirzayans 1986). hemelytroblatta roseni (brancsik 1898) synonymy: arenivaga roseni brancsik 1898, homoeogamia roseni brancsik, 1898. iranian records: sistan va balouchestanbampur, isfehan-murchekhort, khuzestan (chopard 1929, bey-bienko 1950), isfahan (kashan, kabutarabad, barzok), semnangarmsar, sistan va balouchestan (zabol, khash), tehran-varamin (mirzayans 1986), isfahan (fathpour et al. 2003). hemelytroblatta longipes (chopard 1929) synonymy: heterogamodes longipes chopard 1929, arenivaga longipes chopard 1929. iranian records: north of iran (chopard 1929, bey-bienko 1950), fars (firuzabad, shiraz, dehram), semnan-garmsar, tehranvaramin (mirzayans 1986). hemelytroblatta marismortui (janson 1891) synonymy: arenivaga marismortui janson 1891, heterogamia maris-mortui janson 1891. iranian records: fars (kazerun-chah chenar), probably in provinces lorestan, azarbayjan-e-gharbhi, kermanshah, khuzestan (mirzayans 1986). hemelytroblatta latifrons (chopard 1929) synonymy: heterogamodes latifrons chopard 1929, arenivage latifrons chopard 1929. iranian records: boushehr (chopard 1929), chaharmahal va bakhtiari (bakhtiari mountains) (bey-bienko 1950), khuzestan (ahvasmolla sani) (mirzayans 1986). heterogamisca persica (chopard 1921) synonymy: polyphaga persica chopard 1921. iranian records: markazi-arak, sistan va balouchestan (khash-mount taftan), khorassan-e-jonubi-nehbandan (chopard 1929), kerman-esfandagheh, isfahan (kabutarabad), sistan va balouchestan (chahbahar, tiss, iranshahr), gazvin, yazd, khuzestan (ahvaz), fars-yazd khast (mirzayans 1986). leiopteroblatta monodi (chopard 1969) synonymy: does not exist. iranian record: lut-desert (mirzayans 1986). tivia inconspicua (bei-bienko 1950) synonymy: there is no synonym. iranian records: sistan va balouchestan (bahukelat, ghasr-ghand, dashtiari, tang sarheh) (bey-bienko 1950), hormozgan (isin: mount geno 750 m. alt.) (mirzayans 1986). superfamily blaberoidea family ectobiidae blattella germanica (linnaeus 1767) synonymy: blatta asiatica pallas 1773, blatta bivittata serville 1838, eublattella cuneivittata hanitsch, 1925, phyllodromia cuneivittata hanitsch 1925, blatta daurica laxmann 1769, phyllodromia magna tepper 1895, phyllodromia niitakana shiraki 1931, blatta obliquata daldorf 1794, ischnoptera paralella tepper 1893, blatta germanica linnaeus 1767, ischnoptera parallela princis 1969, blattella germanica shuguroffi karny 1908, blatta transfuga brünnich 1763. iranian records: almost all parts of iran (mirzayans 1986), southwest iran (shahraki et al. 2010), fars (fallahzadeh et al. 2008), hamedan-hamedan (salehzadeh 1995, mahjoob et al. 2010, nejati et al. 2011), hormozgan-bandar-abbas (shahi et al. 2008), isfahan (fathpour et al. 2003), isfahan-kashan http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 168 (doroodgar et al. 2006), kerman-kerman (kamyabi et al. 2006), kermanshah (limoee et al. 2011), kordestan-sanandaj (vahabi et al. 2007), kohkiluyeh va boyer-ahmadyasuj (shahraki et al. 2011), mazandaransari (motavali haghi et al. 1997, abedi and dayer 2006, enayati and haghai 2007), khorassan-e-razavi-mashhad (naderinasab and moravvej 2012), tehran (ladonni 1997, mousavi et al. 2004, baniardalani et al. 2006, limoee et al. 2006, nasirian 2007, karimi zarchi and vatani 2009, fakoorziba et al. 2010), zanjan-zanjan (mohammadi 1996, ghavami et al. 2005), qom-qom (fahiminia et al. 2010). blattella biligata (walker 1868) synonymy: blatta cercalis walker 1868, ischnoptera evansi chopard 1921, blatta fasciceps walker 1871, ischnoptera fulvastra chopard 1924, blatta mellea krauss 1902, ischnoptera modestiformis karny 1915, symploce mossambica princis 1965, symploce persica bei-bienko 1935, ischnoptera biligata walker 1868, phyllodromia unicolor brunner von wattenwyl 1893, blatta cercalis walker 1868. iranian records: sistan va balouchestan (bey-bienko 1950), hormozgan (bandarabbas, shamil, minab, tonbe bozorg), sistan va balouchestan (sarbaz, saravan, iranshahr, chabahar, tiss, baftan) (mirzayans 1986). note: in the studies of salehzadehs (1995), two unknown species of symploce have been reported from hamedan. supella (supella) longipalpa (fabricius 1798) synonymy: blatta cubensis saussure 1862, blatta extenuata walker 1868, blatta incisa walker 1868, blatta phalerata saussure 1863, ischnoptera quadriplaga walker 1868, blatta subfasciata walker 1871, blatta (phyllodromia) supellectilium serville 1838, blatta transversalis walker 1871, ischnoptera vacillans walker 1868, blatta longipalpa fabricius 1798. iranian records: tehran (bey-bienko 1950), isfahan (fathpour et al. 2003), southwest iran (shahraki et al. 2010), hamedan-hamedan (salehzadeh 1995). symploce zarudniana (bei-bienko 1963) synonymy: there is no synonym. iranian records: sistan va balouchestan (bey-bienko 1950), hormozgan (bandarabbas, shamil), sistan va balouchestan (pishin, iranshahr, baftan), fars (mirzayans 1986). balta ramifera (walker 1871) synonym: blatta ramifera walker 1871. iranian record: sistan va balouchestanbampur (bey-bienko 1950), sistan va balouchestanbaftan (mirzayans 1986). phyllodromica (phyllodromica) polita (krauss 1888) synonym: aphlebia polita krauss 1888. iranian record: gilan (assalem, parehsar 750m. alt) (mirzayans 1986). phyllodromica (lobolampra) persa (beibienko 1935) synonymy: does not exist. iranian record: golestan (gorgan-shahkuh) (bey-bienko 1950). parcoblatta sp hebard 1917 synonym: ischnoptera fox 1917, platamodes scudder 1862. iranian records: isfahan (fathpour et al. 2003). this species was reported in a study that was focused on cockroaches as reservoirs and vectors of drug resistant salmonella spp since this species was new for iran, further investigation will confirm its presence in the country. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 169 fig. 2. partial comparison of the mtdna-coii gene of different cockroach specie. the dashes (-) in the red square in the last three lines show the six dna base pairs deletion in species of polyphaga genus including the undescribed specimens (polyphaga sp) and other two polypahaga aegyptica from japan (j) and london (l). the species from top to down respectively are: ergaula capucina, blattella lituricollis, blattella germanica, symploce pallens, pycnoscelus surinamensis, blatta orientalis, periplaneta americana, periplaneta fuliginosa, shelfordella lateralis, eremoblatta subdiaphana, and polypahaga aegyptica. fig. 3. the neighbor-joining tree for mtdna-coii sequences of some representative species of four known cockroach families. except for polyphaga sp, sequence data of other species were retrieved from genbank. the line underneath shows the genetic distance scale http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 170 table 1. classification of the 26 iranian cockroach species. ns: not stated superfamily family genus species general name blattoidea blattidae periplaneta p. americana american periplaneta p. australasiae australian blatta b. orientalis oriental shelfordella s. lateralis turkistan shelfordella s. monochroma ns corydioidea corydiidae (polyphagidae) tivia t. inconspicua ns polyphaga polyphaga p. aegyptiaca p. indica egyptian ns polyphaga p. saussurei ns polyphaga polyphaga sp unknown hemelytroblatta h. livida ns hemelytroblatta h. subhyalina ns hemelytroblatta h. longipes ns hemelytroblatta h. roseni ns hemelytroblatta h. marismortui ns hemelytroblatta h. latifrons ns heterogamisca h. persica ns leiopteroblatta l. monodi ns blaberoidea ectobiidae (blattellidae) supella s. longipalpa brown-banded symploce s. zarudniana ns blattella b. biligata ns blattella b. germanica german balta b. ramifera ns phyllodromica p. polita ns phyllodromica p. persa ns parcoblatta p. sp wood fig. 1. photos of an adult male specimen of polyphaga sp found in assad-abad, northern tehran, iran, 2011. general top (a and b) and ventral (c) views, pronotum with a narrow yellowish band along anterior and lateral margins and straight posteriorly (d), and end of abdomen with a pair of anal styles (e). (photos: original) discussion result of this study revealed presence of an undescribed species of polyphaga in iran. in this research we tried to use the cockroach morphological characteristics for identification of the unknown specimens but presence of some similar characteristics among three species of p. indica, p. aegyptiaca, and py. surinamensis made identification problematic. this situation highlights usefulness of molecular data in resolving systematic of closely related species. the molecular analysis including phylogenetic analysis using mtdna coii sequences showed that the specimens belong to corydiidae (polyphagidae) and suggested that they are most closely related to polyphaga genus. mtdna coii is the most frequently gene used for molecular systemhttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 161–175 ss hashemi-aghdam and ma oshaghi: a checklist … 171 atic of cockroaches (liu and beckenbach 1992, park et al 2004, lo et al 2000 and 2006, bergamaschi et al. 2007, maekawa et al. 2007), however, due to lack of available data on different polyphaga species in genbank database, identification of the specimens at the species level postpones until other polyphaga species molecular data becomes available. also more specimens especially female specimens of the species should be collected and subjected to further morphological, biological and ecological investigations. with regards to the report of the undescribed species, the cockroach fauna of iran includes three families, 14 genera, and 26 species. however, it should be mentioned that since some species has not been collected or reported recently and many geographical regions of the country have not been studied yet, hence a systematic research is required to reveal the real cockroach list of the country. conclusion the usefulness of molecular markers for species identification. recently regions of mtdna have been used as dna barcodes to identify and classify living organisms. application of dna barcode could put light on many un-identified or questionable taxa. due to lack systematic study on cockroach fauna of iran, available data is not representing real fauna of the iranian cockroaches. it is highly recommended to conduct a comprehensive systematic research using a combination of morphological, ecological, and molecular techniques in various geographical regions to reveal the composition of iranian cockroach species. acknowledgements this work was financially supported by tehran university of medical sciences. the authors declare that they have no conflicts of interest. the authors declare that there is no conflict of interests. references abedi a, dayer ms (2006) evaluation of the effect of the fungus metarhizium anisopliae, as a biological control agent, on german cockroaches blattella germanica. j med sci pathobiol. 8(1): 31–36 [in persian]. ahmed a, minhas k, sahar ne, aftab o, sher-khan f (2010) in silico identification of 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pestiferous blattodea. comp infibous biochem physiol c pharmacol toxicol endocrinol. 124(3): 227–232. vazirianzadeh b, mehdinejad m, dehghani r (2011) identification of bacteria which possible transmitted by polyphaga aegyptica (blattodea: blattidae) in the region of ahvaz, sw iran. jundishapur j microbiol. 2(1): 36–40. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 97 http://jad.tums.ac.ir published online: march 31, 2021 original article molecular identification and genotyping of babesia canis in dogs from meshkin shahr county, northwestern iran *majid khanmohammadi1; reza zolfaghari-emameh2; mehdi arshadi3; elham razmjou4; poorya karimi4 1department of laboratory sciences, marand branch, islamic azad university, marand, iran 2department of energy and environmental biotechnology, division of industrial and environmental biotechnology, national institute of genetic engineering and biotechnology (nigeb), tehran, iran 3al-zahra hospital, tabriz university of medical sciences, tabriz, iran 4department of medical parasitology and mycology, iran university of medical sciences, tehran, iran *corresponding author: dr majid khanmohammadi, e-mail: majid593@gmail.com (received 12 oct 2019; accepted 13 mar 2021) abstract background: canine babesiosis is one of the mainly worldwide-distributed tick-borne haemoprotozoan parasitic diseases in dogs. methods: a total of 43 blood samples were randomly collected from naturally infected dogs in seven villages from different geographical areas of meshkin shahr, ardabil province, iran. the presence of babesia species detected with standard methods including parasitological and gene sequencing techniques targeting the 18s rrna gene. results: our results revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with babesia. all four babesia-infected dogs were confirmed b. canis by the molecular-based method. sequence alignments comparison of the b. canis genotypes a and b, it was revealed that all b. canis isolates belonged to genotype b. conclusion: this study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in iran. keywords: babesia canis; babesiosis; dogs; genotyping; rna, ribosomal, 18s; iran introduction canine babesiosis is a tick-borne parasitic disease with worldwide importance and caused by intra-erythrocytic babesia species. the identification of each babesia species routinely is based on the host specificity and the morphological characteristics of piroplasmids (1). the differences in geographical distribution, vector specificity, antigenic properties, genetic characteristics and severity of the clinical manifestations sub divided the former species into the three subspecies, namely b. canis canis (3–5μm) is transmitted by dermacentor reticulatus in europe, b. canis vogeli transmitted by rhipicephalus sanguineus sensu lato in tropical and subtropical regions, and b. canis rossi transmitted by haemaphysalis leachi in south africa (1, 2). babesia gibsoni (1.5–2.5μm) is present in asia, north america, africa, australia and europe (3-6). the geographical distributions of both species of d. marginatus and d. reticulatus in europe range from portugal to ukraine (continue to the east of kazakhstan), turkey and probably to the northern parts of iran (710). the first report of dermacentor ticks in iran was documented in 1971 by mazlum (11). this study performed among the 30 provinces and the results defined that, dermacentor ticks were found only in six provinces (semnan, khorasan, kurdistan, ardabil, east azerbaijan, and zanjan) with the highest rate of distribution in ardabil in which ticks was found to be restricted to four species; d. niveus, d. margina copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 98 http://jad.tums.ac.ir published online: march 31, 2021 tus, d. raskemensis and d. daghestanicus (1217). in a study 200 adult dermacentor ticks (139 d. niveus and 61 d. marginatus) were collected from a sheep babesiosis infected in the ardabil region of iran, b. ovis was detected in ticks by semi-nested pcr. based on the results obtained d. niveus and d. marginatus, which are distributed in ardabil region of iran, might play a crucial role in the transmission of babesia infection to domestic animals (12). to date, there are no reports of d. reticulatus in iran. dermacentor ticks usually occurred in the mountainous area with cold climatic conditions and high altitude (15). a cross sectional study in southeastern iran, among tick-infested dogs, three dogs were infected with b. gibsoni. in this study, ticks were identified and belonged to r. sanguineus sensu lato and no babesia dna was detected. this study first record of b. gibsoni in dogs in iran (18). it seems that b. canis and b. gibsoni were the major species infecting dogs and causing various clinical symptoms in iran (19-21). based on the morphological features of the infection, it has documented that tick species including r. bursa, r. sanguineus, r. turanicus and d. marginatus have distributed in different regions of iran. r. bursa and r. sanguineus may play the major roles as the vector of the parasite respectively in the case of animal babesiosis in iran (13). the babesiosis infection was detected with molecular and serological methods in dogs and other wild canine (22-27). the molecular-based techniques enable differentiation of morphologically undistinguishable babesia species (28). the molecular diagnostic methods (for example pcr) are costeffective with high sensitivity and specificity and the most reliable techniques for babesia dna detection in blood (29-31). easy application and accessibility of databases and growing amount of annotated genomic sequences in databanks caused an improvement in the phylogenetic studies on b. canis (31). limited information on the canine babesiosis has been documented, while a high number of suspicious clinical cases reported in dogs from iran. this study provides essential and valuable data to insight into the prevalence and distribution of canine babesiosis in iran. thereby, regarding to raise of knowledge on this parasite, the detection and characterization of the babesia species and subspecies from canine babesiosis in meshkin shahr has a great importance through application of pcr and sequencing of 18s rrna gene sequences. materials and methods study area, blood and spleen samples from july 2017 to february 2018, a total of 43 blood samples were randomly collected from shepherd dogs (canis familiaris) (32 males and 11 females, 9 months to 7 years old) in meshkin shahr, ardabil province, northwest of iran. blood samples were collected into 0.001m edta-containing tubes, and transported in iceboxes to the laboratory of protozoology, faculty of medicine, iran university of medical sciences. blood samples aliquoted, smears were prepared from edta-sampled, blood air-dried, and stained with giemsa. genomic dna was extracted from each blood samples using a dna extraction kit (qiagen dna blood minikit, germany). all samples were identified and followed the detection process using pcr. molecular analysis and characterization of the isolated babesia species dna was extracted from whole blood samples using the dna extraction kit from blood (qiagen, hilden, germany) through following manufacturer instructions as previously described (22, 31). to detect babesia species, the gene fragment (~550bp) from 18s rrna was amplified and sequenced using the primers bab gf2 (5′-gyyttgtaattggaatga tgg3′) and bab gr2 (5′ccaaagac tttga tttctctc-3′). all stages were performed using the previously described pcr protocol (23). generally, reactions were performed in a total of 25μl, including 2.5μl of 10x pcr buffer, 2.0μl of dntp (2.5mm each), 1.25u of taq http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 99 http://jad.tums.ac.ir published online: march 31, 2021 dna polymerase (sinacolon co. iran) 1.0μl of template dna, 1.0μl of each primer (10pmol), and 16.25μl of double distilled water (sina colon co. iran). the pcr reaction was 95 °c (3min), [95 °c (30s), 55 °c (30s), 72 °c (90s)]× 35 cycles, 72 °c (5min). in the second round of pcr 418bp of dna fragments were generated using another pair of primers, piro-nest f (5′-ggataaccgtgst aattstagggc3′) and piro-nest r (5′-gtgtgtacaaaggg cagggacg-3′) (4). the amplified pcr products were maintained at -20 °c until analyzed. the products were run on electrophoresis in a 1.5% agarose gel containing 0.2μg of safe stain/ ml in trisacetate-edta buffer at 120v for 30 min and consequently transilluminated under uv light. ethical approve this study was approved by admission with the ethics procedures and guidelines of the respective national the animal ethics use committees of research issued by the council of the iran university of medical sciences (ir. iums.rec. 27899.). sequences analysis sequences subjected to online blast algorithm and were compared with previously registered sequences in the genbank database. to confirm the classification of the parasite, large fragments of the 18s rrna gene were amplified from each sample that was positive for babesia. the18s rrna genes sequences were analyzed by standard technique using a sequencer and bioedit software (perkin-elmer, usa) (32). analysis of dna sequences and phylogenetic relationships for b. canis isolates and the group of isolates from dogs were aligned using clustal w software (33). a phylogenetic tree was created using alignments performed with neighbor joining (nj) phylogenetic tree using kimura-2-parameter algorithm with bootstrap as the tree construction method (34). furthermore, phylogenetic analysis of gene sequences were performed with maximum likelihood method with mega 7.0 software. the representative sequence was annotated in the genbank database with accession number mn173220, mn173221, mn173222 and mn 173223 (table 1). to assess b. canis genotypes (4, 35), the obtained sequences were compared with the members from genotype a (ay 703072) and genotype b (ay649326). results in the direct microscopic diagnostic investigation of blood smear and molecular study of blood samples revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with b. canis (fig 1). in the clinical examinations, all four dogs had major symptoms of babesiosis and most of the infected dogs had fever and splenomegaly. in addition, blood parameters including hemoglobin concentration, haematocrit, rbc count, and direct bilirubin had increased (table 1). out of the total samples subjected to pcr, four dogs was babesia-positive including one female and three males. dna was purified from all blood samples of the collected dogs and used as the pcr template, which a 550bp band was observed in the analysis. the results of sequence analysis were the same as with the other previously annotated sequences. the nucleotide sequences from canine samples were identical to each other and had shown a 99.6–100% identity with b. canis derived from dogs in genbank reference sequences originated from different countries such as turkey (ky247106 and kf499115), china (mk256974), slovakia (dq869307), estonia (kt008057), romania (hq662634), and croatia (ay072926). the comparison of b. canis nucleotide sequences obtained in this study with genotypes a and b revealed that all our isolates were classified as genotype b and the main difference was observed in positions 490 and 491. the difference of the two genotypes in the row of adenine and guanine nucleotides are in genotype b as ag and ga in genotype a (fig. 2). the results of phylogenetic analysis revealed that the 18s http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 100 http://jad.tums.ac.ir published online: march 31, 2021 rrna gene sequences obtained in this study matched with b. canis and alignments showed that all b. canis isolates belonged in the category of genotype b (fig. 3). in present study, the common ancestor of genotype a and b was obtained with confidence level 99%. genetic confidence intervals can help to better understand genealogical relationships to dna matches. fig. 1. direct microscopic detection of babesia canis in the blood of naturally infected dogs. field-giemsa stained thin smears showing various forms of b. canis in erythrocytes. a: closed angle pyri form bodies of b. canis and b: wide angles b. canis near the margin of the infected rbcs fig. 2. multiple sequence alignment of the partial 18s rrna gene and the sequences of genotype a and b. in this position nucleotide changes can be seen http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 101 http://jad.tums.ac.ir published online: march 31, 2021 fig. 3. neighbor-joining analysis of canine babesia sequences obtained from samples submitted. a 550bp fragment of the 18s rrna was aligned with representative sequences derived from genbank. bootstrap values (1000 replications) are shown in the phylogenetic tree. comparison of the b. canis sequences obtained in this study with genotypes a and b. samples sequenced in the present study are marked with red cycle (mn173220mn173223). the tree was inferred using the neighbor joining method of mega7, bootstrap values are shown at each branch point http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 102 http://jad.tums.ac.ir published online: march 31, 2021 table 1. principle information on the animals sampled and the babesia species isolated samples pathogen clinical symptoms accession number (s) blood analysis* diagnostic investigation location and coordinates breed id of animal age sex mongrel mesh 1 1 year female b. canis fever, splenomegaly mn173220 rbc: 4.3 hgb: 99 hct: 27.6% direct bilirubin: 1.08 pcr: positive blood smear: positive tissue: positive ag bolagh 38°20′57″n 47°39′57″e kurd mastiff (pshdar) mesh 2 3 years male b. canis fever, vomitin, lethargy mn173221 rbc: 4.8 hgb: 90 hct: 26.1% direct bilirubin: 1.01 pcr: positive blood smear: positive tissue: positive parikhan 38°24′51″n 47°38′38″e anato-lian karabas mesh 3 4 years male b. canis fever, icter, splenomegaly mn173222 rbc: 4.3 hgb: 91 hct: 29.1% direct bilirubin: 1.15 pcr: positive blood smear: not tested tissue: positive qurt tappeh 38°25′43″n 47°37′29″e mongrel mesh 4 14 months male b. canis cough, splenomegaly mn173223 rbc: 4.1 hgb: 97 hct: 26.6% direct bilirubin: 1.12 pcr: positive blood smear: positive tissue: positive qara darvish 38°56′43″n 47°28′48″e *normal ranges: red blood cell (rbc) count, 4.6–10×109/l; hemoglobin concentration (hgb), 93–153g/l; haematocrit (hct), 28–49%; direct bilirubin, 0.15±0.01 http://jad.tums.ac.ir published online: march 31, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 103 http://jad.tums.ac.ir published online: march 31, 2021 discussion in this study due to the most availability of 18s rdna sequences from b. canis in the genbank, the 18s rdna was used to search for the intraspecific variability and the most available abundant b. canis sequence. out of the 43 samples subjected to pcr, 9.3% (4/43) were found to be positive for babesia infection. this is the first study of a molecular detection and identification of b. canis infection in dogs from iran and our results revealed that b. canis was prevalent in meshkin shahr, iran. on the basis of 18s rrna gene sequence analysis, genetic heterogeneity of b. canis has been reported in poland, croatia, estonia, lithuania, hungary and china (4, 22, 35). two genotypes of b. canis, includes a and b, have been documented so far, and have shown to have variable virulences (4, 36). the results provided principle information toward a better understanding of the epidemiology of canine babesiosis in iran and prepared the situation for implementation of an effective control planning on babesiosis. a variable interspecies pathogenicity of the b. canis genotypes stated by previous studies (1, 4, 25, 28). the clinical manifestations of b. canis infection are mild to acute, and the severity of disease has a significant relationship with the species of babesia causing infection (4). there are few reports and studies on babesia spp. in iran, while a widespread distribution of the parasite vector and suitable weather condition were observed in some areas of iran (18, 37). therefore, there is the probability canine babesiosis and establishment of an infection chain in some geographic areas of iran. niak et al. (1973) studied the blood parasites of 155 dogs and one fox (vulpes vulpes) in the north of iran, b. canis was just found in one splenectomized dog and b. gibsoni was found from fox (21). jalali et al. (2013) applied a pcr method in the study and documented that the prevalence of canine babesiosis was 0.36% (20). in another study, akhtardanesh et al. (2016) detected 60 tick-infested anemic dogs, among which three dogs (5%) were posi tive through using a genus-specific pcr and all infected with b. gibsoni. none of the collected ticks was positive at the babesia specific pcr (18). in the collected blood samples of dogs from seven regions of shiraz in south of iran, only one positive sample was infected with b. canis (19). the results provided useful data on the distribution of b. canis genotypes in dogs from iran, and showed the necessity to use a molecular-based analysis for an accurate diagnosis of canine babesiosis. the pcr-based analysis demonstrated that the molecular techniques can a highly sensitive easy to use and cost-effective tools for the simultaneous detection and differentiation of b. canis genotypes. however, since a limited number of target gene sequences are currently available for molecular detection of this parasite, any consideration on the population genetics of babesia in the study areas would be highly scrupulously (38). genetic intraspecific variability is a vital mechanism for piroplasm parasite survival in hosts (39). it is proven that b. canis transmitted by d. reticulatus, and the distribution area of the parasite is directly related to the presence of this tick species. although b. canis has been observed in dogs in iran (19-21, 37), but so far we have no reports of d. reticulatus ticks in iran. these results demonstrate that probably b. canis and d. reticulatus have infested a dog’s population, at least in the northern part of iran. dermacentor reticulatus, not occurring in the mediterranean climatic zone, is a tick of some cool regions generally in wooded areas. this ticks has a wide spread geographical overlap with d. marginatus. preferred habitats are forests and swamps zones where it can survive for long periods (8). the main clinic pathological sings in babesia infections were a moderate to acute disease haemoglobinuria and a mild to very severe normochromic normocytic haemolytic anemia but, the symptoms of the disease are classified based on clinical sings and severity of the infection (40). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 104 http://jad.tums.ac.ir published online: march 31, 2021 in the present study, the main clinical signs in babesia infected dogs were fever, splenomegaly, vomiting, cough. haemoglobinuria and haemolytic anemia was not seen, which may be due to host immune system status, age and stages of infection. based on clinical signs and mortality rates, genotype b is more virulent than genotype a (4). considering that, all of the positive cases of babesiosis in this study were of genotype b and all of them had typical clinical symptoms of the disease, the results of this study are consistent with those of other studies (4, 36, 40). the babesiosis infection is detected with molecular and serological methods in domestic dogs and other wild canine in the world. the basic method of diagnosis is the observing intracellular parasite, however, this method has limitations such as false positive, co infections and non-identification of the species (41). serological analysis is a very useful diagnostic method, but has some limitations such as cross-reactivity between different babesia species and it cannot differentiate between acute infection and prior exposure with the parasites (40, 41). the molecular-based techniques enable differentiation of morphologically undistinguishable babesia species and the most reliable techniques for babesial dna detection in blood and tissue (36, 39, 42). ardabil province and especially meshkin shahr region have cold and mountainous climate with forest and swamp conditions. the presence probability of this tick, because of the proof of b. canis not far-fetched. it has proven that global warming and climate change will lead to a further spread of the vectors and transmitted pathogens (42). indeed, the climate change is a global challenge, which may explain not only the increase of density and scattering of tick vectors, but also the pattern distribution their hosts, changes in periods of activity, and variations in geographical distribution (42). the studies suggest a possible role of dermacentor spp. as vectors of tick-borne pathogens that affect human and animal health (12, 19). fast diagnostic technique is necessary for the accurate determination of babesia in canine that could be carried and possibly transmitted by dermacentor or other related spp. more studies are needed to increase the knowledge in the epizootiology, ecology and epidemiology of canine babesiosis in ardabil area. epidemio-molecular studies are necessary to provide important data for the development of new vaccines and effective therapies against canine babesiosis (43). through a novel diagnostic strategy, our study could characterize b. canis infection in dogs in meshkin shahr, iran. due to the increasing numbers of piroplasm species, infected dogs may state a drastic health position threat to dog’s population and prevalence of animal infectious disease in iran. conclusion our study identified the presence of b. canis in dogs in meshkin shahr, iran, but further studies are needed on the prevalence of babesia spp. in large sample dog populations from extended areas in iran to understand better about the epidemiology of canine babesiosis and to promote an effective control program to determine the tick species diversity in dogs in different areas of iran. the finding of this study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in iran. acknowledgements the authors are pleased to thank prof mehdi mohebali and dr zabihollah zareei (department of medical parasitology and mycology, tehran university of medical sciences, iran) and prof lameh akhlaghi (department of medical parasitology and mycology, iran university of medical sciences, iran), for their technical advises and valuable assistances. the authors declare that they have no competing interests. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 97–107 m khanmohammadi et al.: molecular identification and … 105 http://jad.tums.ac.ir published online: march 31, 2021 references 1. solano-gallego l, sainz a, roura x, estrada-pena a, miro g (2016) a review of canine babesiosis: the european perspective. parasit vectors. 9(1): 336. 2. uilenberg g (2006) babesia a historical overview. vet parasitol. 138(1–2): 3–10. 3. rene-martellet m, moro cv, chene j, bourdoiseau g, chabanne l, mavingui p (2015) update on epidemiology of canine babesiosis in southern france. bmc vet res. 11: 223. 4. wang j, liu j, yang j, liu z, wang x, li y, luo j, guan g, yin h (2019) molecular detection and genetic diversity of babesia canis canis in pet dogs in henan province, china. parasitol int. 71: 37–40. 5. zygner w, gorski p, 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pact of climate change in the epidemiology of vector-borne diseases in domestic carnivores. comp immunol microbiol infect dis. 36(6): 559–566. 43. baneth g (2018) antiprotozoal treatment of canine babesiosis. vet parasitol. 254: 58–63. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 148 original article dynamic relations between incidence of zoonotic cutaneous leishmaniasis and climatic factors in golestan province, iran mohammad reza shirzadi 1, *abolfazl mollalo 2, mohammad reza yaghoobi-ershadi 3 1communicable diseases management center, ministry of health and medical education, tehran, iran 2department of geo-spatial information system (gis), center of excellence in gis, k. n. toosi university of technology, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 14 june 2014; accepted 1 oct 2014) abstract background: zoonotic cutaneous leishmaniasis (zcl), an important public health problem in iran, is sensitive to climate conditions. this study aimed to examine dynamic relations between the climate factors and incidence of zcl in golestan province, northern iran during 2010–2012. methods: data of monthly climatic factors, including temperature variables, relative humidity variables, evaporation, total rainfall, and number of freezing and rainy days together with monthly zcl incidence were used. spearman rank correlation was carried out to explain associations between the monthly zcl incidence rate and climate factors at 0, 1, 2, 3 and 4 months lagged periods. pearson’s correlation analysis was conducted to examine the type and strength of relationships between the spatially averaged climate factors and zcl incidence rate in district level. stepwise multiple regression was used to find the best combination of independent climatic variables, which predict the zcl incidence. results: spearman correlation analysis indicated that the highest correlations between climate factors and monthly zcl incidence were established when the climate time-series lagged the zcl incidence series, especially two month prior to disease incidence. based on the results of the both spearman rank correlation and pearson correlation analyses, zcl incidences in golestan province tend to be more prevalent in areas with higher temperature, lower relative humidity, lower total rainfall, higher evaporation and lower number of rainy days. the results of stepwise regression analysis indicated that minimum temperature, mean humidity, and rainfall had considerable effect on zcl incidence. conclusion: climate factors are major determinants of zcl incidence rate in golestan province and such climate conditions provide favourable conditions for propagation and transmission of zcl in this endemic area. keywords: zoonotic cutaneous leishmaniasis (zcl), climate factors, correlation analysis, gis, iran introduction leishmaniasis is an environmental dependent disease affected by a variety of factors, amongst which climate factors are considered to play major role in frequency of the disease (patz et al. 2005). it represents significant socio-economic burden to society and psychological disfiguring effect on patients with permanent scars, predominantly in developing countries including iran (yaghoobi-ershadi et al. 2013). the disease is one of the most important health problems in iran and its prevention and surveillance is one of the who priorities (wha 2007). leishmaniasis in iran has mainly three clinical forms including cutaneous leishmaniasis (cl), visceral leishmaniasis (vl) and mucocutaneous leishmaniasis (mcl), amongst which cl is the most common form of leishmaniasis. although, cl can be seen in zoonotic (zcl) and anthroponotic (acl) forms, about 80 % of cases reported in the country are of zcl form (yaghoobi-ershadi 2012). zoonotic cu*corresponding author: mr abolfazl mollalo, email: a_mollalo@yahoo.com http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 149 taneous leishmaniasis due to leishmania major is still a great and increasing public health problem in many rural areas of 17 out of 31 provinces of iran (akhavan et al. 2010). according to the published statistics by the who, number of cl cases in this country during the period of 1998 to 2009 generally increased from 18,560 to 24,586 patients (who 2012). fig. 1 shows frequency of cl occurrence in iran, during the period of 1998–2009. although zcl yearly afflicts considerable numbers of people from different parts of iran, inadequate attention has been paid to monitoring and surveillance of the disease. golestan province, an endemic province of zcl in iran, with several significant spatial and spatio-temporal hotspots of zcl especially in northern and northeastern parts, is constantly at risk of infection (mollalo et al. 2015). previous entomological studies carried out in this province proved that zcl is caused by the leishmania major (yakimoff and schokhor) (kinetoplastida: trypanosomatidae), the main vector is phlebotomus papatasi scopoli (diptera: psychodidae) also the main reservoir host is rhombomys opimus (great gerbil) (rassi et al. 2008, sharbatkhori et al. 2014). climate is one of the most significant factors that might affect the spatial distribution of many infectious diseases including leishmaniasis. it affects through control on host or vector physiology and behaviour directly (e.g. effect of rainfall on parasite development and vector competence) or indirectly (e.g. effect of temperature on the range and abundance of the sand fly species that act as vectors or through socio-economic changes that affect the amount of human contacts with the transmission cycle) (ready 2008). climate would be expected to modify the spatial and temporal distribution of the leishmaniasis (kelly-hope and thomson 2008). new methodological advances, such as geographic information system (gis) over last 30 years has provided an ability to better understand the etiology of the diseases in shorter time and less costs. gis is a worthwhile tool in studying infectious diseases (moore and carpenter 1999). in spite of broad studies on the association between climate variables and incidences of different kinds of infectious diseases throughout the world, very little researches in regards of leishmaniasis has been reported from iran. several studies in various parts of the world had linked different forms of leishmaniasis to environmental factors. in the study region, mollalo et al. (2014), linked the normalized difference vegetation index (ndvi), as a general proxy indicator of climate changes (including temperature, humidity and rainfall), with incidence of cl and demonstrated that most of cases were occurred in nonvegetative or low-density vegetation areas. during 1991–2001 in costa rica, chaves and pascual (2006) studied monthly cl incidence by using mathematical models. they showed that cl has cycles of about three years related to temperature and indices of the el niño southern oscillation. using such a model, they could predict the incidence of cl up to 12 months ahead with an accuracy of between 72 % and 77 % depending on prediction time. in central tunisia, toumi et al. (2012), investigated temporal dynamics and impacts of climate factors (including rainfall, temperature and humidity) on incidence of zcl. their results showed seasonality during the same epidemiologic year so that zcl incidence raised by 1.8 % when there was 1 mm increase in the rainfall lagged by 12 to 14 months and by 5.0 % when there was a 1 % increase in humidity from july to september in the same epidemiologic year. to the best of our knowledge, this is the first attempt in terms of assessing the relationship between climate factors and zcl incidence in quantitative manner in surveyed area. development of low-cost and efficient management tools for effective control of the http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 150 zcl is an important objective, which requires comprehensive efforts and studies including the ecology of the disease and role of the climate change. for this purpose, this study was designed to investigate the relations between the climate factors and incidence of zcl to gain deeper insight into the possible interactions between climate conditions and zcl incidence in golestan province of iran. materials and methods study area golestan province consists of 14 counties with 60 districts located in north-east of iran (36° 30’ to 38° 8’ n and 53° 57’ to 56° 22’ e) bounded on the north by turkmenistan country, on the west by mazandaran province and the caspian sea, on the south by semnan province, and on the east by northern-khorasan province. the geographical position of this province provides a unique area with very diverse climate. as northern regions are located in the arid and semi-arid climate, southern regions represent a mountainous climate, and central and southern west regions are located in a moderate mediterranean climate. based on the climatic conditions of the region during the study period, the monthly maximum and minimum temperature were 30.7 °c and 5.09 °c in july 2011 and february 2012, respectively. the total annual rainfall was a minimum of 0 mm in july 2010 and its maximum of 124.18 mm in october 2011. the minimum monthly relative humidity was 51.78 % (june 2010) and the maximum was 79.64 % (march 2010) (golestan province meteorological center, unpublished data). data collection the iranian primary health care (phc) system was well founded especially in rural areas. more than 16,000 health houses over the country cover almost 95 % of the rural areas. health workers are responsible to deliver primary healthcare and to keep health records of people to the center for disease control and prevention (cdc) of their study area. monthly zcl incidence records over the period of january 2010 to december 2012 (36 months) were considered for analysis of their temporal correlations and lagged effects. during the study period a total of 2,893 zcl cases, diagnosed by direct smear examination, were officially reported by cdc of golestan province. the data were checked in a meticulous manner to prevent any possible mistakes and were mapped at district level using arcgis desktop software version 9.3 (esri inc, redlands, ca). fig. 3 shows the annual zcl incidence rate at district level. climate data data of climate factors were obtained from golestan province meteorological center. the data were collected from synoptic stations in golestan province and synoptic stations in adjacent provinces including northern-khorasan and semnan during september 2009 to december 2012 (40 months) for more accurate interpolation. climatic factors, including the minimum, maximum and mean temperature (°c), minimum, maximum and mean relative humidity (%), mean evaporation (mm), total rainfall (mm), and number of freezing and rainy days were calculated for each district based on the observation of synoptic stations in mentioned provinces. after collection of climate data, they were entered into gis environment for further analyses. this has been done through the following procedure: 1) the monthly average of measurements of the above factors for each synoptic station was calculated. 2) a point layer was created for observation stations and the monthly averages of factors associated with their related points. 3) for any of the factors, using inverse distance weighting (idw) method, a raster was created by interpolating the monthly average http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 151 values of those factors between stations. 4) for each factors, its raster was overlaid with the polygon layer of the district boundaries. for each district, the average of the raster cells inside district polygon was assumed as the value of the factor in that district and analyzed to recognize possible relationships with zcl incidence. fig. 2 shows maps of climatic factors at district level in golestan using idw method. geographic information system was used in conjunction with statistical analytical methods to analyze the relations between zcl incidence and climate factors in the study area. spatial statistics analyses to assess the correlation between climate factors and zcl incidence rate, the monthly zcl incidence was regarded as the dependent variable, while climate variables were considered as independent variables. a fourstage approach was adopted to describe and analyze the possible relations of the climatic variables on the incidence of zcl. firstly, the monthly and annual zcl incidences of each district were calculated and mapped. secondly, spearman rank correlation was used to examine the association between the climate factors and zcl incidence rate. in this regard, cross correlation was performed to detect the lagged effect of climate factors on zcl incidence rates at global (province) level at 0, 1, 2, 3 and 4 months lagged periods. thirdly, pearson’s correlation analysis at local (district) level was conducted to examine the type and strength of relations between the spatially averaged of climate variables and annual zcl incidence rates in districts. finally, multivariate stepwise regression was used to establish the models to determine the contribution rate of all the climatic factors. results the monthly trend of climate factors and zcl incidence rate is shown in fig. 4. with regard to the lagged effects, it can be visually seen positive associations between average temperature and evaporation with increase of zcl incidence rate, and negative associations between relative humidity, and rainfall with increase of zcl incidence rate. however, statistical analyses needs to formally test whether the results are statistically significant or not. associations between monthly zcl incidence rate and climate factors were observed at global (province) level at 0, 1, 2, 3 and 4 months lagged periods. besides, local correlation at district level between zcl incidence rate and climate factors was observed using the pearson correlation analysis. based on the results of the spearman rank correlation at the province level, positive associations were observed between the monthly zcl incidence rates with all temperature variables and evaporation. in addition, negative correlations were seen between the monthly zcl incidence rates with all humidity variables, rainfall, number of rainy days and number of freezing days. the time lag(s) of climatic factors preceding zcl incidence at which the series showed that the strongest correlation were obtained by crosscorrelation analysis of monthly zcl incidence series and monthly climatic data timeseries. among ten climate factors used in this study, except minimum humidity and rainfall, highest correlations were found with 2months lagged period. moreover, lowest correlations were found 0-month (for all temperature variables, evaporation, rainfall and number of freezing days) and 4-month (for all humidity variables and number of rainy days). among the climate factors, temperature variables showed the highest correlation with the monthly zcl incidence rate whereas rainfall showed the least correlations with the monthly zcl incidence rates. detailed information of the results of spearman rank correlation between the http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 152 monthly zcl incidence rates and climate factors at 0, 1, 2, 3 and 4 months lagged periods has presented in table 1. pearson’s correlation analyses revealed that positive associations were observed between annual zcl incidence rates in all 60 districts of province with all temperature variables, evaporation, and number of freezing days. while negative associations were observed with all relative humidity variables, rainfall and number of rainy days. these results indicate that zcl incidences in golestan province tend to be more prevalent in districts with the lowest average relative humidity values as an indicator of drought. however, all the variables except number of freezing days showed similar signs in both analyses. among the climate factors, relative humidity showed the highest correlation, while minimum temperature showed the least correlations with zcl incidence rates. table 2 represents results of the pearson correlation between the monthly zcl incidence rates and climate factors. results of the multivariate stepwise regression showed the best model among others, with highest r, r2, and lowest standard error, was the regression model with equation y= 4.015+0.739* (minimum temperature)-0.841* (mean humidity)-0.7631* (rainfall) (table 3). the multiple regression showed 0.536 changes of monthly zcl incidence contributed to the average monthly minimum temperature, mean humidity, and total rainfall. table 1. spearman rank correlation between the monthly zcl incidence rates and climate factors at 0, 1, 2, 3 and 4 months lagged periods, between september 2009 and december 2012 monthly zcl incidence temperature min. temperature max. temperature relative humidity min. relative humidity 0-month .403* .399* .368* -.505** -.492** 1-month .735** .695** .723** -.764** -.712** 2month .895** .858** .854** -.776** -.649** 3-month .800** .780** .749** -.625** -.445** 4-month .472** .502** .398* -0.278 -0.088 monthly zcl incidence max. relative humidity evaporation rainfall no. rainy days no. freezing days 0-month -.514** 0.314 -0.166 -.493** -0.306 1-month -.736** .695** -.383* -.604** -.540** 2month -.759** .893** -.497** -.611** -.670** 3-month -.636** .853** -.551** -.469** -.646** 4-month -.411* .574** -.336* -0.138 -.451** **.correlation is significant at the 0.01 level *.correlation is significant at the 0.05 level table 2. results of pearson correlation analysis between climate factors and zcl incidences in 60 districts of golestan province, iran, between september 2009 and december 2012 monthly zcl incidence temperature min. temperature max. temperature relative humidity min. relative humidity yearly zcl incidence .150 .035 .106 -.326** -.272* monthly zcl incidence max. relative humidity evaporation rainfall no. rainy days no. freezing days yearly zcl incidence -.320** .249 -.210 -.128 .122 **.correlation is significant at the 0.01 level *.correlation is significant at the 0.05 level http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 153 fig. 1. frequency of cl cases in iran, between 1998 and 2009 fig. 2. climatic maps of golestan province during 2010–2012, generated by idw method http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 154 fig. 3. annual zcl incidence rate at the district level per 100,000 individuals in golestan province, iran, 2010–2012 http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 155 http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 156 fig. 4. temporal trend of zcl cases incidence (per 100, 000) concerning mean of climate factors, golestan province, iran, 2010–2012 table 3. the results of stepwise regression analysis in which 3 out of 10 climate factors were selected model r r-square adjusted r2 p-value 6 0.732 0.536 0.534 <0.01 final regression model: y=4.015+0.739*(minimum temperature)-0.841*(mean humidity)-0.7631*(rainfall) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 157 discussion the results of this study both support and extend the findings of previous works where temporal series of climate variables had associated with other vector-borne infectious diseases such as malaria (nath and mwchahary 2012, zhao et al. 2014) and dengue fever (goto et al. 2013). examining of correlations showed statistically significant association between climate factors behind zcl incidence time series by spearman rank correlation. according to results of the spearman rank correlation, monthly climate factors dynamically correlated with monthly zcl incidence rate during three years period of study. however, climate factors generally did not show strong correlation with zcl incidence rates in the same months. the best correlations between the climate factors and monthly zcl incidence rates were observed when climate factors time-series were lagged the zcl incidence rates. this indicates that climate change prior to the disease active period is an important influencing factor and at least one-month time is needed for climate factors to effect on occurrence of zcl in humans. it should be noted that the above temporal relationship might vary by different geographic areas due to different seasonal patterns in different ecological zones. previous studies have debated the association between leishmaniasis epidemic and climatic factors in some areas of iran. comparison the results of correlation analysis between incidence of cutaneous leishmaniasis and climate factors with studies of yazdanpanah and rostamianpur (2013) in ilam province, west of iran, and mozafari and bakhshizadeh-kolooche (2011) in yazd-ardakan plain in central part of iran, indicates that although positive correlation between the average temperature and cl incidence was observed in ilam, this relation was negative in yazd. in contrast, both of the studies reported that the association between cl epidemic and relative humidity was not significant, while in our analyses in both local (district) and global (province) levels, relative humidity appeared to be the most significant factor. therefore, results of this study are not valid for other study areas of iran. moreover, results of our study were consistent with findings of other researchers around the world including roger et al. (2013) in french guiana located in south america, and sing (1999) in rajasthan, india. both of the studies showed that incidence of disease increased with rise of temperature and decreased with decline of rainfall, and relative humidity, respectively. the results of this study are also consistent with the previous findings of mollalo et al. (2014), at the same study area, who observed significant association between vegetation cover and cl incidence in golestan province. because low vegetation covers almost accompanies by higher temperature, evaporation, lower rainfall, and relative humidity. moreover, it is clear from fig. 3 that districts with high incidence rate of zcl were almost located at northern parts of the province with arid and semi-arid climate conditions indicating that such climate conditions provide favorable circumstances for zcl transmission in this province. the main limitations of this study are related to current surveillance system in iran, which yearly loses considerable numbers of the cases. official reports are probably underestimated due to many reasons such as the not reported, not diagnosed or misdiagnosed cases. therefore, it is possible that incidence of the disease is underestimated in this study. however, there are few published empirical evaluations of reported and underestimated cl cases, the degree of underreporting cl cases in iran was found to be 2.8 to 4.6 fold (alvar et al. 2012). since the registration system is uniform throughout the http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 158 iran, these errors are evenly distributed. in addition, short period of this study (three years) may not lead to robust and reliable results. besides, it should be noted that this study examined only climate factors on zcl epidemics, without taking socio-economic conditions, and other elements influencing the number of new cases, such as herd immunity or individual factors influencing the number of asymptomatic carriers into account. therefore, it is necessary to perform further studies to other environmental factors (such as construction of roads, building dams, etc.) and even culture and life style of people in the study area, which might influence on disease pattern. since the geographical and seasonal distributions of zcl is closely linked to the climate conditions, using climate factors together with the other influencing factors as predictive indicators can be used to establish early warning systems (ewss) to forecast zcl incidence in managing the next epidemic. conclusion climate factors have been greatly caused or affected on the spatial distribution of zcl in golestan province, so that areas with higher temperature and evaporation, and in contrast lower humidity, rainfall, and number of rainy days were more susceptible to disease occurrence. these findings can provide essential guidelines for public health policy makers to monitor and predict the disease based on the climate factors for future control measures. this means that the budget, personnel, and resources can be allocated more efficiently by concentrating on major determinants of zcl epidemic in golestan province. acknowledgments we would like to express our sincere thanks and appreciations both to the authorities of the golestan center for disease control and prevention (cdc) for providing zcl data, and golestan, semnan and northern-khorasan provinces meteorological centers for supplying the climate data used in this study. this project has been financially supported by cdc of ministry of health of iran with the grant number of 1294503 /30318. the authors declare that there is no conflict of interests. references akhavan aa, yaghoobi-ershadi mr, mirhendi h, alimohammadian mh, rassi y, shareghi n, jafari r, arandian mh, abdoli h, ghanei m (2010) molecular epizootiology of rodent leishmaniasis in a hyperendemic area of iran. iran j public health. 39(1): 1–7. alvar j, velez id, bern c, herrero m, desjeux p, cano j, jannin j, boer m, who leishmaniasis control team (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7(5): e35671. chaves lf, pascual m (2006) climate cycles and forecasts of cutaneous leishmaniasis, a nonstationary vector-borne 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tunisia. plos negl trop dis. 6(5): e1633. yaghoobi-ershadi mr, shahbazi f, darvishi m, akhavan aa, jafari r, khajeian m, rassi y, soleimani h, shirzadi mr, hanafi-bojd aa, darabi h, arandian mh, sanei-dehkordi a, heidari m (2013) molecular epidemiological study of cutaneous leishmaniasis in the focus of bushehr city, southwestern iran. j arthropod-borne dis. 7(2): 113–121. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod-borne dis. 6(1): 1–17. yazdanpanah ha, rostamianpur m (2013) analysis of spatial distribution of leishmaniasis and its relationship with climatic parameters (case study: ilam province). bull env pharmacol life sci. 2(12): 80–86. zhao x, chen f, feng z, li x, zhou xh (2014) the temporal lagged association between meteorological factors and malaria in 30 counties in south-west china: a multilevel distributed lag nonlinear analysis. malar j. 13(1): 57–69. world health assembly (2007) the world http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 148–160 mr shirzadi et al.: dynamic relations … 160 health assembly resolution (wha 60.13) on the “control of leishmaniasis”. geneva, switzerland. available at: http://www.who.int/neglected_ diseasesmediacentre/wha_60.13_eng .pdf. who (2012) available at: http://gis.emro. who. int/leishmanya/atlas.html. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 159 http://jad.tums.ac.ir published online: january 05, 2016 original article the effect of hemiscorpius lepturus (scorpionida: hemiscorpiidae) venom on leukocytes and the leukocyte subgroups in peripheral blood of rat mehri ghafourian 1, neda ganjalikhanhakemi 2, ali asghar hemmati 3, rouhullah dehghani 4, *wesam kooti 5 1health research institute, research center of thalassemia and hemoglobinopathy, department of immunology, ahvaz jundishapur university of medical sciences, ahvaz, iran 2department of pharmacology, school of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran 3department of pharmacology and toxicology, school of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran 4department of environmental health, school of health and social determinants of health (sdh), research center, kashan university of medical sciences, kashan, iran 5student research committee, kurdistan university of medical sciences, sanandaj, iran (received 2 mar 2014; accepted 3 dec 2014) abstract background: the aim of this study was to investigate the effect of hemiscorpius lepturus venom on leukocytes and the leukocyte subgroups in peripheral blood of rat. methods: in this experimental study, sixty n-mari rats were divided into three groups of 20 rats. then the rats in each group were divided into four subgroups based on the blood sampling time that was 2, 6, 24 and 48 hours after the venom injection, respectively. the control group did not receive anything, however, the first and the second experimental groups received 0.1 and 0.01mg/kg of venom, subcutaneously. in accordance with a designated four sampling times, the blood sampling was carried out in three groups. after rbc lysis, the leukocytes and leukocyte subpopulations were determined and counted using appropriate hematological standard methods. results: the leukocyte and the neutrophil count at two (p<0.05), six (p<0.01) and 24 (p<0.05) hours after the venom injection showed a significant decline compared with the control group, this decrease was significant at the dose of 0.1 mg/kg until 48 hours after the venom injection (p<0.05). the lymphocyte count showed a significant decline throughout the all hours of the experiment, compared with the control group (p<0.05). conclusion: leukocytes are probably affected by the cytotoxicity effect of the h. lepturus venom in a dosedependent manner. this could be a wakeup call for the medical staff to perform quick and accurate treatment in the least time possible. keywords: hemiscorpius lepturus, venom, leucocyte, rat introduction scorpion envenomation is one of the main problems in the public health system in many countries in the world. this involves 2.3 billion inhabitants in the areas with the scorpion sting threat (chippaux et al. 2008). in 2008, the annual incidence of scorpion stings was 1.200.000 leading to 3250 deaths (chippaux et al. 2008, khoobdel et al. 2013). this number demonstrated a relative growth in a report released in 2012 and exceeded than annually 1,500,000 scorpion stings, however, the mortality rate due to the scorpion stings showed a significant decrease and fell into 2600 deaths per year (jean-philippe 2012). the highest number of scorpion sting in the world has been allocated to iran after mexico (osnaya-romero et al. 2001, dehghani *corresponding authors: mr wesam kooti, e-mail: kweasm@yahoo.com j arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 160 http://jad.tums.ac.ir published online: january 05, 2016 and fathi 2012). the annually 42,500 scorpion stings and 20 following deaths have been reported from 2001 to 2009, in iran (celis et al. 2007). in the middle east, among the 52 known species of scorpions, the most dangerous scorpions are reported in iran (celis et al. 2007). the scorpion sting has been reported from all of the provinces of iran, however, the most common incidence rates have been detected in khuzestan, with incidences of 541 per 100000 individuals (dehghani et al. 2009, rafizadeh et al. 2014). hemiscorpius lepturus belongs to the hemiscorpiidae family, and is the most medically important and a dangerous scorpion in khuzestan, iran and the world (shahbazzadeh et al. 2007). hemiscorpius lepturus has been responsible for 15 % of the scorpion sting bite cases, however, it is the leading cause of 89 % of deaths followed by the scorpion sting (pipelzadeh et al. 2007). the lethality arising from this scorpion is approximately 60 times higher than the average for the remaining venomous scorpion stings in the region (pipelzadeh et al. 2007). the venom of h. lepturus leads to acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia, known as the nephrotoxic, hepatotoxic and hemolytic complications of the scorpion venom (valavi et al. 2008). envenomation by h. lepturus is characterized by various local and systemic signs. the local signs vary from erythema to necrosis, while the patient feels no pain. on the other hand, the nephrotoxicity is the most important systemic complication that if left untreated could result in severe renal, cardiac and pulmonary failure (pipelzadeh et al. 2007). consequently, it may damage the intestinal lamina propria (mojgan heidarpour et al. 2011). the venom may induce severe pathological damages in target organs such as skin, blood cells, central nervous system (cns), and cardiovascular system (seyedian et al. 2010). it leads to an increase in liver enzymes aspartate aminotransferase (ast), alanine aminotransferase (alt) and alkaline phosphatase (alp), indicating the severe hepatic damage (pipelzadeh et al. 2006). khodadadi et al. showed that the h. lepturus causes more rbc lysis and ldh increase, comparing with the complications of envenomation resulting from the stings of two other species of scorpions, mesobuthus eupeus and androctonus crassicauda (khodadadi et al. 2012). during a fiveyear study performed in ahvaz, it was described that 90 % of patients whom were admitted in hospitals due to the general condition worsening, were stung by h. lepturus (mir dehghan et al. 2001). the immune system cells are distributed throughout the body, from the outer most points to the deepest organs and tissues, such as blood, bone marrow, thymus and spleen. despite this diversity, the major group of immune cells is the peripheral blood leukocytes. these cells apply a comprehen-sive supervision on different organs and tissues through the regular circulation from blood to lymph, from inside of the vessels to the outside and from the interior of the organs to the blood, resulting in protection of the body against the pathological factors (vodjgani 2012). the results of an experimental study aiming the investigation of the effect of envenomation by h. lepturus on the hematological indices three days after injection of the venom suggested that the leukocyte number has been increased over the normal range, however, there was not any significant difference with the control group (dehghani et al. 2012). an increase in the peripheral leukocyte count had been demonstrated during the investigation of the blood among h. lepturus scorpion sting victims (chitnis et al. 1993). while comparative studies to investigate the effects of h. lepturus venom on hematologic parameters and vital organs of the body have been done, but so far, any experimental research on the effect of the scorpion venom on the immune sysj arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 161 http://jad.tums.ac.ir published online: january 05, 2016 tem cells and its various sub-groups in the early hours after the sting, and further-more determination and investigation of the period and the intensity of the envenomation have not carried out yet. therefore, the aim of the present study was to investigate the effect of h. lepturus envenomation on blood leukocytes and their subgroups in the early hours after the sting. the results of this study can help physicians, health officials and the medical staff to fast and accurate treatment of the victims of h. lepturuss sting and prevention of complications of the scorpion venom on important factors of the immune system. materials and methods animals sixty male rats from n-mari species (weight range of 300–350 grams) purchased from pasteur institute of iran (tehran), were used during the study. the animals were kept in standard cages in animal house at the school of pharmacy of ahvaz jundishapur university of medical sciences. rats were housed in temperature-controlled rooms (22– 25 °c) with constant relative humidity (40– 70%) and 12h/12h light/ dark cycle before doing experimental protocols. the study was performed in accordance with the principles of laboratory care established by the ethics committee of ahvaz jundishapur university of medical sciences, ahvaz, iran. venom preparation the scorpion electroshocked venom was provided as a lyophylized powder from pasteur institute of iran, (venom and therapeutic biomolecule lab, biotechnology res. center, tehran, iran). the concentration of crude venom protein was determined by using bradford method (bradford, 1976). the 0.1 and 0.01 concentrations of the venom prepared in distilled water. the injection vol ume was 0.1 ml. grouping of animals and sampling the animals were randomly divided into three groups of 20 rats in each. the control group did not receive any thing; however, the first and the second groups received h. lepturus venom at the concentrations of 0.1 and 0.01 mg/kg/bw subcutaneously. thereafter, the animals of each group (n= 20) were divided into four subgroups (n= 5), with respect of the four blood sampling time, that were two, six, 24 and 48 hours after the venom injection, the animals were then kept in separate cages. experimental studies the animals of each subgroup were anesthetized with ketamine and xylazine (alfasan, holland). the blood samples were obtained from the animal’s heart amounted to 0.5–2 ml by syringe. soon after the sampling, the blood was maintained in glasses containing the anticoagulant, edta (ethylene-diaminetetra-acetic acid), and the leukocytes were counted using the diluent solution, marcanu (rbc lysis buffer) and the neobar slide (hemocytometer) by using the light microscope (olympus, 3h-z-japan), in order to count and determine the leukocyte subgroups (including neutrophils, lymphocytes, monocytes and eosinophils) the appropriate peripheral blood smears were prepared on the microscope slides and then fixed by means of the water-free methanol. then giemsa staining (merckgermany) was carried out by the diluted stain with the rate of 1/10. and finally, the differential counting was performed using a 100x lens microscope (mahbod 2008, mansouri et al. 2015). statistical analysis data were analyzed using the spss ver. 13 (version 13, spss inc, chicago, il) and the statistical tests of anova and lsd. data were considered significant statistically j arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 162 http://jad.tums.ac.ir published online: january 05, 2016 when p< 0.05 as presented in fig. 1–5. results the effect of h. lepturus venom on leukocyte count the scorpion venom has led to a significant reduction of leukocytes in the venomreceiving group with the dose of 0.01 mg/kg at two and 24 hours after the injection, compared with the control group (p< 0.05). this decrease has been maximum of six hours after the injection (p< 0.01). after 24 hours from the injection, the effect of the venom has subsided; and after 48 h there was no any significant difference with the control group (p> 0.05). the toxicity effect of the venom is intensified with the increase of the injected venom concentration, so that in spite of a decrease in venom effect in the group receiving 0.01 mg/kg after 48 hours, the decrease in toxicity effect is still significant in the group receiving 0.1 mg/kg compared with the control group (p< 0.01) (fig. 1). the effect of the h. lepturus venom on the peripheral blood neutrophil count the neutrophil mean count showed a significant decline in the venom receiving group with the dose of 0.01 mg/kg at two, 24 and 48 hours (p< 0.05), and six hours after the injection, compared with the control group (fig. 2). the effect of h. lepturus venom on the peripheral blood lymphocyte count the mean of the peripheral blood lymphocyte count in the venom receiving group of 0.01 mg/kg at two, six and 24 hours (p< 0.05), and 48 hours (p< 0.01) after the injection demonstrated a significant decline compared with the control group, respectively. the effect of h. lepturus venom on the peripheral blood eosinophil and monocytes count the comparison of the mean and standard deviation of the eosinophil and monocytes of the peripheral blood in the case and control groups showed no significant differences (p> 0.05) (figs. 4, 5). fig. 1. the comparison of the mean±sd of white blood cells (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg and the control group at different times * significant difference (p< 0.05) between the experimental and control groups ** significant difference (p< 0.01) between the experimental and control groups fig. 2. the comparison of the mean±sd of neutrophils (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg and the control group at different times j arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 163 http://jad.tums.ac.ir published online: january 05, 2016 * significant difference (p< 0.05) between the experimental and control groups ** significant difference (p< 0.01) between the experimental and control groups fig. 3. the comparison of the mean±sd of lymphocytes (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg, and the control group at different times * significant difference (p< 0.05) between the experimental and control groups ** significant difference (p< 0.01) between the experimental and control groups fig. 4. the comparison of the mean±sd of monocytes (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg and the control group at different times fig. 5. the comparison of the mean±sd of eosinophil (× 10 per ml3) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg/kg and the control group at different times discussion the present study was aimed to investigate the effect of h. lepturus venom on leukocytes and their subgroups in peripheral blood of rats. leukocytes are considered as the major group of immune cells.these cells apply a comprehensive supervision on different organs and tissues, through the regular circulation of blood to lymph, from inside of the vessels to the outside and from the interior of the tissues to the blood, resulting in a protection of the body against the pathological factors. in general, leukocytes consist of various groups of cells including lymphocytes, monocytes and granulocytes (vodjgani 2012). the clinical syndrome induced by h. lepturus sting is different from the stings by the other scorpions existing in iran and the world, and exhibits more severe manifestations. lack of local pain or being a mild pain after the sting, cutaneous manifestations such as erythema, swelling and necrosis at the sting site, the red blood cell lysis, and nephrotoxicity, including hemoglobinuria, proj arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 164 http://jad.tums.ac.ir published online: january 05, 2016 teinuria and hematuria are some manifestations among scorpion sting victims (radmansh 1990, radmanesh 1998, pipelzadeh et al. 2006). the results of the present study demonstrated those leukocytes are affected by the venom after two hours, exhibiting sensitivity, decrease in leukocytes continues for six hours after the venom injection. if leukocytes were evaluated after 12 hours, it was possible to notice a decreasing trend, due to prolongation of the presence of the venom into the body. probably these variations in leukocytes are the results of cytotoxic effects of h. lepturus venom, which leads to white blood cell lysis and destruction (shayesteh et al. 2012). there is no any considerable experimental or clinical study in this field, and the decrease in leukocytes in the early hours is one of the new findings of this research. lack of the related reports might be due to the difference in evaluation time of the hematological indices in various studies (dehghani et al 2005, dehghani et al. 2012), it has been the result of the use of the antivenom in human researches, as well (chitnis et al. 1993, ghafourianboroujerdnia and mohebbi 2008). in the present study, the leukocyte count trend increased after 24 hours of venom injection, and there was not any significant difference in the venom-receiving group of 0.01 mg/kg with the control group. the bone marrow has been probably capable to reconstruct white blood cells. various studies have reported leukocytosis as well as the major clinical signs of the envenomation by h. lepturus, after other symptom such as hemoglobinuria, hematuria and proteinuria (chitnis et al. 1993, ghafourian boroujerdnia and mohebbi 2008). therefore, the reconstruction of leukocytes observed in this study is in agreement with the previous findings (dehghani et al. 2005, dehghani et al. 2012). however, the more venom concentration, the more decline in leukocyte count, the slower wbc reconstruction, so that the leukocyte count has not reached to the normal range after 48 hours of envenomation with 0.1 mg/kg. according to the present study, after two hours, the percent of the blood neutrophils has been changed from the normal range of 61 % to 35.2 %. this reduction has been more severe after six hours and has fallen down to 25.5 %. neutrophils make up the most abundant population of white blood cells, and mediate the primary stages of inflammatory response. they are the most effective phagocytes in peripheral blood and have a major role in defense against the extracellular factors (abbas et al. 2011). these mature cells migrate to the inflammation site just four hours after the antigen entrance, and have the capability to invade the antigen. phagocytosis of the particles and waste products in neutrophils is associated with a series of biochemical events and morphological changes in the cell (vodjgani 2012). the majority of the neutrophils at the inflammation site will be wiped out by other cells, such as macrophages, after the phagocytosis of the invader. therefore, this can explain the rapid decrease in neutrophils. jalali et al. (2011) aiming to investigate the effect of h. lepturus on the serum levels of cytokines il-1β, il-6, il-8 and tnf-α, demonstrated a direct relation between the worsening of the patient’s general condition and the above-mentioned cytokines. il-8 is a chemotactic protein that is known as nap-1 or the attractive and activator of neutrophils (vodjgani 2012). the increase of this cytokine leads to fever and hyperthermia, which is probably due to neutrophil aggregation, pathogen killing and the death of the two kinds of cells (taraz 2008). tnf-α enhances the production of some particular serum proteins such as amyloid a, through affecting hepatocytes. this cytokine suppresses stem cell division, which may lead to neutropenia (vodjgani 2012). therefore, the severe dej arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 165 http://jad.tums.ac.ir published online: january 05, 2016 crease in neutrophils in the group receiving high doses of h. lepturusvenom will be reasonable. in the following 24 hours, the neutrophil count especially in the venomreceiving group of 0.01 mg/kg has approached the normal range. regarding the neutrophilia, the neutrophil compensation could be explained. through the neutrophilia the conversion of marginal neutrophils into circulating neutrophils could be noticed. these findings are consistent with the results of the research carried out by ghafourian and mohebby on patients with scorpion referred to the hospital bite (ghafourian and mohebbi 2008). in the present study, lymphocytes have decreased during the first hours after the venom injection, and this reduction was significant even after 48 hours. one of the fractions extracted from the venom of h. lepturus may reduce the lymphocyte count (bigdeli et al. 2006). lymphocytes make up around 20 to 40 % of the leukocytes, and 99 % of the cells in the lymph, respectively. lymphocytes are considered as the most important specific immune cells. the process of recognition and processing of antigen by lymphocytes as well as the clonal expansion requires the time, and the precise determination of the mechanism of the effect of the venom of h. lepturus on lymphocytes cannot be explained during the first 48 hours, perhaps due to immunological reasons. in the present study, the blood lymphocyte decrease was dose-dependent, which could probably be the result of the direct effect of the venom on the lymphocytes, which leads to disruption and lysis of these cells. in fact, it could be concluded that the venom of h. lepturus has a lymphotoxic effect (ghafourian and mohebbi 2008). in the present research, the peripheral blood eosinophil count in the control group showed no significant difference with the experimental groups. eosinophil consists of 2 % of leukocytes found in the normal situation in tissues, especially the epithelium of the respiratory tract, gastrointestinal tract and genitourinary tract. these cells have a weak phagocytic ability, and increase mainly in type-1 hypersensitivity responses and parasitic infections, as well. therefore, the lack of their considerable change could be reasonable. conclusion the venom of the scorpion h. lepturus effect on the leukocytes in the early hours, as well as increasing the concentration of the toxin, its destructive power is increased. in other word, the venom effect is dose dependent. the scorpion venom may induce the release of bradykinin, prostaglandin and corticosteroids. however, the essential role of these factors is to intensify the inflammation, but recently the natural corticosteroid hormones or similar synthetic substances are used to alleviate the inflammatory reactions against the allograft transplantation and immune system suppression. therefore, the extraction of the useful fractions of h. lepturus venom for natural induction of corticosteroids and decreasing leukocytes may be useful in the treatment of some types of leukemia and the graft surgeries, as well. it is recommended to investigate this issue in the future studies. however, the severe and dose-dependent reduction of the immune cells in the first hours after the injection of h. lepturus venom could be an alarm for health officials and medical staff to perform quick and accurate treatment in the least possible time, and to prevent of complications of the scorpion venom is the body's vital organs. acknowledgements the results provided are from md thesis of neda ganjalikhanhakemi approved with number\ 469 and implemented in the student j arthropod-borne dis, june 2016, 10(2): 159–167 m ghafourian et al.: the effect of … 166 http://jad.tums.ac.ir published online: january 05, 2016 research committee, and funded by the research deputy vice-chancellor for research affairs of ajums. the authors appreciate and thank this deputy vice-chancellor for financial support, particularly the research consultation center for technical support and dr delavar shahbazzadeh and miss sara ali-akbari due to their unsparing 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[md dissertation]. school of pharmacy, ahwaz jondishapur university of medical sciences, iran. valavi e, alemzadeh ansari mj (2008) hemolytic uremic syndrome following hemiscorpius lepturus (scorpion) sting. indian j nephrol. 18(4): 166–168. vodjgani m (2012) immunology. 8th ed. jahad daneshgahi publication, tehran. j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 288 http://jad.tums.ac.ir published online: dec 31, 2022 original article the evaluation of androctonus crassicauda antivenom against the effects of aegaeobuthus nigrocinctus scorpion venom on autophagy, apoptosis and necroptosis mehmet eray alcigir1, *ozcan ozkan2 1department of pathology, kirikkale university, faculty of veterinary medicine, kirikkale, turkey 2department of biology, çankırı karatekin university, faculty of science, çankırı, turkey *corresponding author: dr ozcan ozkan; email: ozcanozkan@karatekin.edu.tr (received 27 may 2021; accepted 13 aug 2022) abstract background: in this study aimed to show the role of autophagy acting as a seesaw between apoptosis and necroptosis in certain vital organs under the effects of the aegaeobuthus nigricinctus venom and different dosages of the androctonus crassicauda antivenom administration in mice. methods: in the venom group (vg), mice (n= 6) were inoculated with 2ld50 a. nigrocinctus venom. in the antivenom administered groups (avg), the effects of the potency of the a. crassicauda antivenom were evaluated to have a neutralization effect against 20ld50 of the a. nigrocinctus venom. after histopathological examination, expressions of mammalian target of rapamycin (mtor) as an autophagy activator, receptor-interacting serine/threonine-protein kinase 3 (ripk3) as a necroptosis activator, and caspase-3, caspase-9 as the markers of apoptotic cell death signals were evaluated by the immunoperoxidase method in addition to dna in-situ fragmentations by the terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) method. results: only in vg, caspases and tunel expressions were found to be higher after the envenomation process in contrast to the elevated ripk3 expressions. mtor expressions remained almost stable in the organs. in ag, mtor expressions were further increased in the 30ld50 and 40ld50 groups. conclusion: there were an increased mtor expression and stabilized caspases and tunel expression in these subgroups, the ripk3 expressions were found to be low when compared with all of the antivenom administration groups. increasing doses of the antivenom drifts more the cells to autophagy while cell fate in organs under envenomation getting rid of apoptosis and necroptosis pathways. keywords: scorpion; aegaeobuthus nigrocinctus venom; androctonus crassicauda antivenom; cell death mechanisms introduction venom secretions from scorpions are comprised of a complex mixture of salts, mucoproteins, histamine, serotonin, biogenic amines, low molecular weight peptides and high molecular weight proteins. the venom of each scorpion species has a different component profile. low molecular weight peptides which are neurotoxins is the most important components in scorpion venoms, and it is also the component that is believed to be responsible for envenomation. these peptides stimulate the ion channels of cells such as sodium (na+), potassium (k+), calcium (ca2+) and chloride (cl−) (1). scorpion ven oms can cause oxidative stress on cells and mitochondrial instability depending on overproduction of reactive oxygen species (ros) (1– 5). against this situation, autophagy machinery is activated (2, 3). mammalian target of rapamycin (mtor), a serine/threonine kinase, is likely to be the chief of this orchestra. it has a pivotal role in cellular metabolism, such as cell growth and proliferation (6). however, mtor creates an inhibition in the autophagy induction (7). another reaction in response to cellular damage like mitochondrial distress is necroptosis to be known as a regulated type of necro copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 289 http://jad.tums.ac.ir published online: dec 31, 2022 sis. it includes the spilling of the cellular contents and therefore, the triggering of the chemoattractant factors to independently develop, arising from the absence of caspase activations. the mechanism is continued mainly by the serine-threonine kinase receptor-interacting protein (rip) although the absolute activation is unknown (8–12). however, in programmed cell death or apoptosis, cellular contents cannot always spill out in response to several damages. the cells sometimes prefer silent deaths, triggered by intrinsic and/or extrinsic pathways under the effect of chemoattractant or immunemediated signals (12, 13). amongst these, the cysteine-dependent aspartate driven proteases (caspases) are known to have a well-defined role in the apoptosis complex for a prolonged time. caspase-9 as initiator of apoptosome and caspase-3 as effector have pivotal roles after the mitochondrial cytochrome c. caspases, furthermore, lead to cleaving in cellular proteins to be like in receptor-interacting serine/ threonine-protein kinase 1–3 (ripk1-ripk3) complex in necrosome of dying cells (14, 15). therefore, caspases can take a role in a number of non-apoptotic molecular interaction independently of apoptotic cell death (16). up to now, scorpionism has been one of the lasting global health problems in tropical and subtropical countries (17). therefore, it is extremely important to know about the medical importance of scorpion venom (17, 18). in turkey, the aegaeobuthus nigrocinctus scorpion was reported to exist in the adıyaman, erzincan, gaziantep, hatay, kahramanmaras, kilis and malatya provinces of the south-eastern mediterranean and eastern anatolian regions. in these regions, therefore, this scorpion species may be responsible for most of the cases of scorpion stings (19, 20). nowadays, although there are no vaccines or other effective agents against animal venoms, hence the serotherapy stands as the only and unique treatment option available as reported before (21). the androctonus crassicauda monovalent antidote has been used in the treatment of all cases of scorpion stings in turkey (22). as far as we know, currently there are a few studies about the a. nigrocinctus scorpion venom. moreover, cell death pathways, unfortunately, has have not been observed on different organs, although there have been numerous reports on cellular damage stemming from a few scorpion species. therefore, the species at hand is a neglected scorpion species regarding studies all over the world and in turkey (20). in this respect, there is a requirement to fill the gap over the cell death cascade. both different cell death types have been comparatively evaluated in different organs and the supremacy of the antivenom over the neglected a. nigrocinctus scorpion has been shown by this study. so, the presented study is one of the first studies conducted on the a. nigrocinctus scorpion venom. the aim of this study was to measure the neutralization efficiency of the a. crassicauda antivenom against the a. nigrocinctus venom; (a) the effect of autophagy on the seesaw role between apoptosis and necroptosis (b) and to measure the response in different cell death reactions in the various vital organs. materials and methods the usage of animals and their care in this study which was approved by the local ethics committee (2017/04), 30 healthy cd-1 mice of 20±2g were used in total. until the end of the experiment, mice (n= 30) were housed in a polycarbonate mouse cage (eu type 2) and they were maintained at 22±2 °c on a 12h light / dark cycle with free access to food and water. venom and antivenom handling in all the experimental procedures, the a. nigrocinctus venom was collected from the nemrut mountain national park, which is in the adıyaman province in the southeastern anatolia region of turkey, and its ld50 is 0.38 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 290 http://jad.tums.ac.ir published online: dec 31, 2022 mg/kg on mice (20). the monovalent a. crassicauda antivenom produced in horses by the ministry of health was used by the general directorate of public health. the potency capacity (ed50) of 1ml of antivenom neutralizes 50ld50 of the venom. experimental procedure the mice modelling system is performed simply according to two main caption: antivenom administered envenomation group (avg), only venom administered group (v), and the control (c) group which was not administered any agent. the effect of the monovalent antivenom and the a. nigrocinctus venom in mice: pathological examination in antivenom group, mice were classified into three sub-groups with six mice in each group (n= 18). for each antivenom group, individually, 1ml of the antivenom was mixed with an equivalent volume of doses of 20ld50 (gi), 30ld50 (gii), and 40ld50 (giii) of the a. nigrocinctus venom and incubated for 45 min at 37 °c. then, mice in each ag were subcutaneously (s.c.) injected with 200µl of each of the mixtures. after injection, mice were monitored for abnormal reactions and signs of envenomation for 12h. the mice in the ag were euthanized with overdose of the mixtures of ketamine and xylazine at the end of the observation. the animals (n= 6) in the venom group (vg) as positive control were injected with 2 ld50 of a. nigrocinctus venom in 200 µl physiological saline solution (pss), while the negative control group (cg) were injected with 200 µl of the pss venom. macroscopical and histopathological examinations autopsy procedures were performed immediately on dead mice in vg and the animals (n= 6) in cg and avg groups for the examination of macroscopic changes and histochemical analysis after the animals were euthanized. briefly, the peritoneal cavities of the mice were opened, and tissue samples were collected from the livers, kidneys, lungs, hearts, and brains of the mice in each group and were immediately placed in 10% v/v formalin solution. after embedding in paraffin, the sections at 4µm-thicknesses were taken. they were placed in slides and were stained with hematoxylin and eosin (h and e) for microscopic examination. immunohistochemical analysis detection of mtor, ripk3, caspase-3 and caspase-9 expressions the strep avidin-biotin complex peroxidase (strep abc-p) method was applied following the manual instructions described in the kit (peroxidase detection system, re7110-k, leica, novocastra). the sections at 4µm-thickness were passed through xylol and alcohol series (5min for each), and then de-paraffinized and rehydrated. the sections were boiled in citrate buffer (ph 6.0) at 160 ºc for 15min to reveal the antigenic determinants (bioptica, italy). to eliminate endogenous peroxidase activity, the tissues were kept in 3% hydrogen-peroxide (h2o2)-methanol solution for 15min. nonspecific protein activity was prevented with the use of blocking serum (novocastra, leica). incubation with primary antibodies (mtor-1:250 dilution gene tex, gtx48628, ripk3-1:200 antibodiesonline.com, abin2792102, caspase-3 ls-b22845, lsbio, usa, 1:100 dilution, anti-caspase-9 adi-aap109, enzolife sciences, usa, 1:150) was left overnight at +4 °c. biotin-linked antibody and streptavidin-linked antibody were dripped onto tissue sections and incubated at 37 ºc for 15min. thereafter, they were rinsed twice for 5min, using pbs at the end of each phase, except in the protein blocking phase. for the control sections, pbs was used instead of primary antibody as the negative control. diaminobenzidine (dab) was used as chromogen, while gill’s hematoxylin was used as ground staining. the slices were fixated using entellan® which is a non-aqueous mounting medium. detection of dna in-situ fragmentation the terminal deoxynucleotidyl transferase http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 291 http://jad.tums.ac.ir published online: dec 31, 2022 mediated nick end labeling (tunel) staining assay method was applied according to the kit procedure (in situ cell detection kit, roche, usa, cat no: 11684795910). for control sections, a labeling solution without terminal transferase was dripped onto the slices. evaluation of results all histopathological and immunoexpression results were illustrated in an olympus bx51 and photographed with an olympus df 25 camera attachment. mean scores were performed semiquantitatively counting 10 high power field at 400 magnifications for each collected organs in all groups. histopathological scoring was found to be as follows; negative (-): 0–10%, mild (+): 10–30%, moderate (++): 30–70%, strong (+++): 70–100%. statistical analysis immunoexpressions were evaluated using a two-way anova test to compare the variability in reactions among the groups. the posthoc bonferroni test was used in multivariate comparisons. the data was analyzed using the graphpad (8.0 version) software. a value of p< 0.05 was accepted as statistically significant. results histopathological findings the venom group and control groups regarding the results of the negative control group; the organs aforementioned were not affected by any degeneration or necrotic changes. there was also no inflammatory reaction at all. the only hyperemic changes were present in the liver and kidney vessels at some of the cases (fig. 1). findings of the positive control as vg in liver suggests that there was hyperemia in central and portal vessels. acute cell swelling to vacuolar degeneration were ended in karyolysis and cytoplasmic shrinkage in hepatocytes. in the kidney: hyperemic capillaries and glomerulus were present. acute cell swellings as well as vacuolar degeneration were evaded in particularly cortical tubules. in the spleen, follicular hyperplasia in lymphoid follicle as well as haemorrhage was observed. in the lungs, hyperemic capillary vessels and neutrophil extravasation were considered. in the heart, hyperemic capillaries, parenchyma degeneration as well as inflammatory cell infiltration were again found (fig. 1). antivenom group (avg) in the liver, the degeneration associated with acute cell swelling and vacuolar degeneration was not found in hepatocytes at every field as being in vg. in the kidney, acute cell swelling and vacuolar degeneration in cortical tubules were present although there were not any findings in the medullary region of the kidneys. in the spleen, follicular hyperplasia in lymphoid follicles, intrafollicular hemorrhage as well as the presence of megakaryocytes was observed in some of the cases. in the heart, individual parenchyma degeneration with shrinkage pink cytoplasm in some cardiomyocytes were observed. the findings were not observed in all the cases (fig. 1). immunohistochemical findings mtor expressions expressions were localized in the membrane and cytoplasm of cells. in vg, the expressions were scattered diffusively from the central to peri-central region of the lobules of the liver, on the periphery of lymphoid follicles of the spleen, on the cortical tubule epitheliums of the kidney and cardiomyocytes in the heart. any statistical differences were not found between these groups (p> 0.05). in the control group, there were no expressions (p< 0.05 between this and other groups). in all the antivenom subgroups (avg) including from gi to giii, the expressions were increased when compared to previous subgroups in envenomed mice organs. the expressions were localized in the periphery of lobules of the liver, in the periphery of the http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 292 http://jad.tums.ac.ir published online: dec 31, 2022 lymphoid follicles of the spleen, on the cortical tubule epitheliums of the kidney, and cardiomyocytes in the heart. any statistical differences were not found between these groups (p> 0.05). however, there was a meaningful statistical difference in comparison between the envenomed and antivenom administration groups (p< 0.05). in the control group, no expressions were found (p> 0.05 between this and other groups). caspase-3 and caspase-9 expressions these expressions were localized in the membrane and cytoplasms of the cells. in vg, both expressions were found at high degrees regarding the aforementioned cellular localization which stated in mtor sections. however, when compared between caspase-3 and caspase-9 expressions, there was not any statistical difference between subgroups (p> 0.05). in comparison of two caspases, both had the same distribution with no statistical difference (p< 0.05). in the control group, there were no expressions (p< 0.05 between this and other groups). in gi to giii, both expressions had similar characteristics in terms of localization, the expressions were found at lower degree of positivities (p< 0.05). a meaningful statistical difference was found between envenomed and antivenom administration groups (p< 0.05). in the control group, there were no expressions (p< 0.05 between this and other groups). tunel reactions expressions were the same with the previous ones. in vg, the expressions were at high levels. they were found at the same localization in all of the organs as mentioned in previous markers. in the control group, there were no expressions (p< 0.05 between this and other groups). the expressions were found at the same localization in all of the organs as mentioned in previous markers. however, the positivity degrees were lower in gii and giii of avg when compared to that of gi (p< 0.05). when these expressions were compared to the envenomed groups, such positivities were more elevated and the distribution of positivities were more evaded in tissues in antivenom groups (p< 0.05). in the control group, there were no expressions (p< 0.05 between this and other groups). ripk3 expressions in vg, expressions were seen in the cytoplasms of cells. the expressions had the same localizations as being previous markers. the distribution of positivities were stronger and more prevalent in the tissues when compared to that of gi in avg. among groups, there was a statistical difference (p< 0.05). in the control group, there were no expressions (p< 0.05) between the previous two groups. the expressions were decreased in all of the antivenom subgroups when compared to that of all envenomed groups (p< 0.05). the distribution of positivities were the same as the previous ones. nevertheless, the number of positive cells decreased particularly in the gii and giii of avg. there was a statistical difference with the comparison of gi (p< 0.05). in the control group, there were no expressions (p< 0.05 between this and other groups). all the expressions according to vital organs were shown in figure 2, 3 and fig. 4. statistical evaluations of tabular data were presented in table 1. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 293 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 1. description of the degeneration in the liver, kidney, and heart in the envenomed groups (vg), and antivenom groups (ag1 to 3), no findings to report in the control group (cg), x400, hematoxylin-eosin (h and e) staining fig. 2. immunoexpressions of mtor, caspases, dna in situ fragmentation and ripk3 in the experimental group (a) and the antivenom groups (b) http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 294 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 3. immunoexporessions in the organs of the envenomed animals, diaminobenzidine (dab) chromogen, abc-p, x200 fig. 4. immunoexporessions in organs of the antivenom administered animals, diaminobenzidine (dab) chromogen, abc-p, x200 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 295 http://jad.tums.ac.ir published online: dec 31, 2022 discussion cells may be exposed to numerous dangerous factors during their lifetime. in this case, cells, due to continuous damage may be drifted to death by using self-activation of specific molecular pathways (23). these possible molecular mechanisms and some triggering linkages are a controversial matter for a long time whether if this is a real selfsuicide or only a biological result or reaction against lethal factors. therefore, several terminologies have been produced meeting those mechanisms (24, 25). cell death mechanisms can be diversified within different categories including apoptosis, necroptosis, autophagy, piroptosis etc. in particular, first three mechanism are related to formerly known necrosis. serine-threonine kinase receptor-interacting protein (rip) and kinase (ripk), activate signaling pathways under cellular distress by excessive free radical accumulation. on the other hand mtor which is taken as a role regarding the pi3k/akt/mtor signaling pathway, in terms of creating a downstream in autophagic activation. caspases, cysteine-dependent aspartate driven proteases, are taken into role within necroptosis. caspases, have a role of cleaving in cellular proteins in necrosome. herein, the key role of caspase-9 is associated to be an activator for apoptosis and a deactivator for autophagy. downregulation of caspases, reactivated mtor change autophagic mechanism in order to provide autolysosomal activity. by negative feedback, increased autosomal vesicles can reverse autophagy. if free radical (i.e ros activity) related-damages are excessive and being out of controling mechanism, rip activation, namely necroptosis, is generated because mitochondrial distress is triggered. as seen herein, each mechanism is closely related to each other. these mechanisms can be easily developed under cellular distress to be like in scorpion envenomation (thanks to toxin contents) (8–12). in the light of the current knowledge, autophagy is known to provide cellular homeo stasis by inhibiting catabolic products and generating nutritional substance and some molecular precursors for cells; namely, a cell survival mechanism (25). however, more recently, the subject of autophagy machinery or selfeating has been given emphasis (24). against the cellular damage resourced from toxication, some damages happen in cells due to intoxication. many toxins and metabolites, cause cytotoxicity by their effects on vital organs such as the heart, kidney, spleen, brain, and skin. in addition to the cellular alterations, hemorrhage and disseminated intravascular coagulation can develop as a result of cytotoxicities (5, 26–29). the toxic effects primarily start in the mitochondria by increasing oxidative stress and over time, it begins to affect all organs. as a result of cellular alterations, cells can be drifted by many mechanisms into death. several factors can determine the fate of the cell. these mechanisms include autophagy, programmed cell death or apoptosis and necroptosis which results in necrosis (5, 29, 30). to begin with programmed cell death, it is reported that the execution of cell death plays an orchestrate role between the mentioned processes. apoptosis triggers cellular membrane receptor facilitating to emit death signals to cytosol. in some events, cells under oxidative stress can release cytochrome c from the mitochondria (4, 5, 31). in such situations, the apoptosis cascade begins to develop autonomously by the activation of cysteinyl-aspartases or caspase family of proteases (32). among the caspases family, caspase-9 is known to have an essential role for the mitochondrial signaling pathways. apoptotic cascades continue by the activation of caspase-3 (33). another idea on cell death is related to autophagy. it takes critical responsibilities on cell death as well as many roles including tissue development, differentiation and homoeostasis and taking under control of health and aging in a healthy organism. however, the role of housekeeping http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 296 http://jad.tums.ac.ir published online: dec 31, 2022 and checking the vital functions of cells whether they behave normally is among the evitable responsibilities (34, 35). in our study, we observed that the caspase-3 and caspase-9 expressions in vg, were high. in avg, gi to giii, both expressions had similarly and lower caspases expression of vg. there is statistical significance between vg and avg. but there is no meaningful difference between both caspases’ level. the autophagy mechanism provides this by regulating some proteins to send signals. mtor or rapamycin, phosphoinositide 3-kinase (pi3k), gtpases, calcium and elements of protein synthesis machinery are included among them (36). some regulatory factors control mtor activity in cells according to whether the cell posing a threat or not. the decreased pi3k activations generally show parallel situation and function mtor signaling via aktmediated phosphorylation. therefore, pi3k/akt/ mtor pathway signaling cascade leads to a decreasing activity in the autophagy mechanism (37). on the contrary, the induction of autophagy may lead to cell survival even the cell under stress conditions by oxidative stress. in addition to, it has been stated that autophagy might prevent cells from undergoing apoptosis (38). therefore, for the autophagy mechanism, it has a pro-survival effect to antagonize apoptosis. in this respect, the current study results are consistent with these data. on the other hand, programmed necrotic cell death or necroptosis is triggered by serine/ threonine kinases receptor-interacting protein 3 or ripk3 activation, binding to ripk1 after the regulation by caspases and ubiquitination. thus, these enzymes facilitate to loss of the cellular carbohydrate deposits and to increase glutamine metabolism (39). in our study, we observed that mtor in vg were expressed in the liver, spleen, kidney and heart although there was no statistical difference between remained groups. on the other side, the present study deter mined that venom exposure triggers increased ripk3 activity. however, we observed that of all the dosages of antivenom administration, mtor expression continues to increase under toxin stress. therefore, we concluded that mtor and ripk3 have adverse effects when each dose of antivenom was administered. in our study, the ripk3 expressions of vg were much stronger and more prevalent in the tissues when compared to that of gi in avg. ripk3 expressions at both remained groups and control groups were similar and did not give meaningful statistical results because the expressions were decreased in particularly gii and giii of avg. we believe that the sole envenomation proven increase in ripk3, i.e., necroptosis. so, this situation shows that the hypothesis makes the current study right on envenomation-necroptosis interaction. on the other hand, mtor expressions, i.e., autophagy, were decreased in some organs of vg in spite of increasing in avg. decreasing mtor show that autophagy can be increased in some organs. but other organs were not affected from triggering autophagy as being like in control group. so, these results prove partly the hypothesis regarding the vice-versa effect between ripk and mtor activities. cells’ fate in the way of surviving and drifting to death can be triggered at the same way in some vital organs against venom-associated cellular distress. accordingly, we found that cellular death pathways were triggered by high apoptosis and necroptosis as well as low autophagic activity which resulted in cellular dna breaks. tunel reactions have proven such kind of dna breaks. in vg, the tunel reactions were at high levels. however, these reactions in avg were lower than that of the vg. when compared within the avg, the reactions were found lower in gii and giii than gi. these results showed that antivenom co-administration in the envenomed group inhibit cellular death mechanism. so, dna breaks can be stopped thanks to diminished cellular death and possibly ros-related cellular stress. another important point in our study is the antivenom or immunotherapeutic usage, how http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 297 http://jad.tums.ac.ir published online: dec 31, 2022 it can affect cellular damage or can reverse the adverse effect in such envenomation. it has been reported that the improvement of the immunotherapeutic treatment in such envenomation events require a better knowledge of the pharmacological actions of the scorpion venom and of the mechanism of its in vivo neutralization by the antivenom. selection of the proper antivenom dose has a vital effect on immediate and durable intervention with regards to sting events by complete neutralization of the toxins. as such, the cellular and vascular or other damages can be better prevented by the effective diffusion of antivenom to all the organs (40). in the current study, decreasing mtor and ripk3 expressions as well as relatively increasing caspases and dna in-situ fragments in all the antivenom subgroups show us that necroptosis decreases, and cell death increases as a result of the autophagy mechanism controlling cell survival in the way of preventing apoptosis. by evaluating the results of the current study (a) there is a close relationship between autophagy-apoptosis and necroptosis. (b) in mice, the a. nigricinctus venom and the monovalent antivenom administration can be a useful model for coming to a better understanding of the potential harmful effects over cells in various vital organs such as the liver, kidneys, spleen, heart, and lungs. (c) the monovalent antivenom against high ld value of the venom may reverse the potential necroptotic effects on cells due to envenomation. (d) caspase signals apart from apoptotic cell death can also aid in decreasing mtor expressions in envenomed animals. thus, combined expressions may trigger the activation of autophagy. (e) ripk3 may solely change the fate of cells in the course of necroptosis. conclusion envenomation by scorpion toxin cause a cellular damage in several organs. excessive free radicals disturb de facto cellular membran, cytoplasmic organels and nuclear structure. in this situation is known that cells are drifted to degeneration or necrosis. however, by this experimental study, we show there are definitive mechanisms which is related to each other. according to exposure degree of toxin and serum support, cell fate can be easily changed under free radical distress. these mechanisms can be turned appear as apoptosis, necroptosis and autophagy. we obtained sustainable information from this experimental that autophagic mechanism shows parallel to decreased of venom capacity and increased serum support. at the same time, we understood that this condition gets the irreversible necroptosis capacity decreased. by this, we have also seen that apoptotic mechanism is more effective in initial phase of envenomation. but we have concluded that apoptotic signals get less effective in presence of higher serum support. in this situation, cell makes a decision living or death after such mechanisms are run in cell at the same time. the obtained results can facilitate in order to understand the relations amongst different cell death mechanisms as well as reversal of the monovalent antivenom effectivity on organ damage. at the same time, we inferred from result of this study that serum support the more earlier time is early getting started and suitable dose is selected, the less organ damages are developed. however, the results should be confirmed by correlating with other markers taking place in apoptotic, necroptotic and autophagic cascades. acknowledgements the authors declare that they do not have any conflict of interest with any researchers. the authors express their sincere thanks to the ministry of health in ankara, turkey for kindly providing the a. crassicauda spesific anti-venom. the study has not been funded by any cooperation. the researchers performed the study by own facilities. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 288–300 me alcigir and o ozkan: the evaluation of … 298 http://jad.tums.ac.ir published online: dec 31, 2022 ethical considerations the animal care and all of the experimental protocols were performed in accordance with the guidelines defined by the local ethical committee in experimental animal research comitee, health ministry (2017-e330341). conflict of interest statement the authors declare no conflict of interest with any researchers. references 1. zhang x, zhang x (2016) scorpion venoms in gastric cancer. oncol lett. 12: 3683– 3686. 2. komatsu m, waguri s, koike m, sou y, ueno t, hara t, mizushima n, iwata j, ezaki j, murata s, hamazaki j, nishito y, iemura s, natsume t, yanagawa t, uwayama j, warabi e, yoshida h, ishii t, kobayashi a, yamamoto m, yue z, uchiyama y, kominami e, tanaka k (2007) 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zaru r, micheau o, thome m, attinger a, valitutti s, bodmer jl, schneider p, seed b, tschopp j (2000). fas triggers an alternative, caspase-8independent cell death pathway using the kinase rip as effector molecule. nat immunol. 1(6): 489–495. 40. krifi mn, savin s, debray m, bon c, el ayeb m, choumet v (2005) pharmacokinetic studies of scorpion venom before and after antivenom immunotherapy. toxicon. 45: 187–198. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 315 http://jad.tums.ac.ir published online: dec 31, 2022 original article first report of natural infection of phlebotomus mongolensis to leishmania major and leishmania turanica in the endemic foci of zoonotic cutaneous leishmaniasis in iran azad absavaran1,2, mehdi mohebali3, vahideh moin-vaziri4, alireza zahraei-ramazani1, amir ahmad akhavan1, sayena rafizadeh5, amirhossin rassi6, alireza barmaki1, *yavar rassi1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical entomology and vector control, zanjan university of medical sciences, zanjan, iran 3department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 4department of medical parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 5ministry of health and medical education, tehran, iran 6faulty of medicine, tehran university of medical sciences, tehran, iran *corresponding author: prof yavar rassi , e-mail: rassiy@tums.ac.ir (received 06 july 2021; accepted 03 sep 2022) abstract background: the primary aim of this study is to determine infection to leishmania parasites in the wild population of phlebotomus caucasicus and phlebotomus mongolensis using molecular methods in some important zoonotic cutaneous leishmaniasis foci in iran. methods: sand flies were collected from active colonies of rodent burrows from 16 trapping sites using sticky trap paper. in order to detect and identify of leishmania parasites in females ph. caucasicus and ph. mongolensis, the nested– pcr amplification of its2-rdna region was performed to generate amplicon with 245bp for leishmania major, 206bp for l. gerbilli and 141bp for l. turanica. results: in the current study we found dna of different gerbil parasites such as l. major and l. turanica, and mixed infection of l. major/l. turanica in ph. caucasicus and ph. mongolensis. it should be noted that, in iran, natural infection with leishmania parasites is recorded for the first time in this study in ph. mongolensis. conclusion: both species of ph. caucasicus and ph. mongolensis not only may participate in the zcl transmission cycle between reservoir hosts, but also results of this study support the role of these species as secondary vectors in the transmission of leishmaniasis to humans. keywords: leishmaniasis; phlebotomus caucasicus; phlebotomus mongolensis; leishmania major; leishmania turanica introduction leishmaniasis is a group of vector-borne diseases caused by a protozoan parasite belonging more than 20 leishmania species. the disease spreads to tropics, subtropics, and the mediterranean basin, as well as to 98 tropical countries in asia (middle east), europe (southern europe and the mediterranean), africa (tropics, north, west, and east africa), and the united states (mexico, central and south america). more than 1 billion people are at risk for leishmaniasis in endemic areas. the prevalence of this disease is 12 million cases worldwide and it is estimated that 700 000 to one million new cases occur annually (1, 2). copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:rassiy@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 316 http://jad.tums.ac.ir published online: dec 31, 2022 there are three main forms of leishmaniases, including cutaneous leishmaniasis (cl), visceral leishmaniasis (vl), also known as kala azar, and mucocutaneous leishmaniasis (mcl) (2). cutaneous leishmaniasis is the most common form of the disease and in 2021 over 85 % of new cl cases occurred in 10 countries: afghanistan, algeria, brazil, colombia, syria, libya, tunisia, pakistan, iraq, and iran (2, 3). there are two epidemiological types of cutaneous leishmaniasis in iran: anthroponotic cutaneous leishmaniasis (acl) or urban/ dry form and zoonotic cutaneous leishmaniasis (zcl) or rural/wet form. zoonotic cutaneous leishmaniasis is a major public health problem in iran which is endemic in many rural regions in 19 out of 31 provinces and about 85% of confirmed leishmaniasis cases in the country are of zcl type. the causative agent of zcl in iran is leishmania major and the main animal reservoirs of disease are rodents of the subfamily gerbillinae (4). of more than 1000 species of identified phlebotomine sand flies, 31 species of phlebotomus (old world) and 47 species of lutzomyia (new world) are proven vectors of human leishmaniasis (5–7). according to recent studies, to date 48 confirmed phlebotomine sand flies have been reported from iran, including 30 species of the genus phlebotomus and 18 species of the genus sergentomyia (8–12). phlebotomus (phlebotomus) papatasi is the proven and main vector of l. major to human in endemic foci of zcl in iran. phlebotomus caucasicus group belongs to the subgenus paraphlebotomus, which has been considered as species group including ph. caucasicus, ph. mongolensis and ph. andrejevi, playing a main role in maintenance of enzootic cycle of l. major among rodent reservoir hosts (8). phlebotomus caucasicus and ph. mongolensis not only participate in the transmission cycle of zcl among reservoir hosts but also play an important role as secondary vectors in the transmission of leishmaniasis to humans (8). the females of these species have similar taxonomic characteristics and are isomorphic but based on recent study, morphometric analysis and morphological characters have been used for discrimination of these closely related species (13). natural promastigote infection was isolated from ph. caucasicus collected from gerbil and jird burrows in the focus of esfahan province in iran and typed by isoenzymes assays as l. major zymodeme mon-26 (14). the traditional or classical methods such as sand fly dissection and culture of parasite have been used for leishmania detection, but these techniques are time consuming and requires many sand fly specimens and also are less sensitive than molecular techniques and are not able to differentiate leishmania parasite species (15). the present study has used a nested-pcr method, which able to differentiate leishmania parasite species and is a specific alternative method to classical techniques (16). in recent years, molecular techniques are frequently used in epidemiological studies specifically on phlebotomine sand flies as vectors of zcl in endemic foci of iran for detection and identification of leishmania infection in phlebotomine sand flies (17–27). the objective of present study was to use molecular methods for the first time to detect and identify of leishmania infection within wild caught ph. caucasicus and ph. mongolensis in some important zoonotic cutaneous leishmaniasis foci in iran. materials and methods sand flies’ collection and species identification sand flies were collected from the different allopatric locations in the provinces of esfahan and fars (central and southern iran, respectively) and sympatric locations in golestan province (northeastern iran). from june through october 2016, sand flies from the active rodent burrow colonies were collected using sticky trap papers (castor oil coated white papers, 21×30cm) from 16 collecting sites. colhttp://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 317 http://jad.tums.ac.ir published online: dec 31, 2022 lected sand flies were stored in 96% ethanol and kept in -20 °c for morphological and molecular assays. at first, for removing castor oil on specimen’s body surface, collected specimens were washed twice in 1% detergent and sterile distilled water and then was dissected in a drop of sterile normal saline by sterilized forceps. the head and the last two abdominal segments were cut off and slide mounted in puris' medium for species identification and identified after 24–72 hours using valid identification keys (13, 28–30). the remaining body (abdomen, wings, and legs) were preserved in 1.5ml sterile micro-tubes containing 96% ethanol for dna extraction and detection of leishmania parasite. molecular detection and identification of leishmania species geneall® exgenetm tissue kit (geneall biotechnology company, south korea) was used to extract genomic dna. to detect and to identify leishmania parasites we used the nested-pcr assay developed by akhavan et al. (16). nested-pcr method has been used to amplify the leishmania spp. regions of its2, primers as follows: leish out f (5′-aaa ctc ctc tctggt gct tgc-3′), leish out r (5′-aaa caa agg ttg tcg ggg g-3′), leish in f (5′-aat tca act tcg cgt tgg cc-3′) and leish in r (5′-cct ctcttt ttt ctc tgt gc-3′). pcr products were separated by 1.5% (w/v) agarose gel electrophoresis in tbe buffer (0.09mm tris, 0.09mm boric acid and 20mm edta, ph 8.3), visualized under ultraviolet light after staining with safe stein (0.5μg/ml) and photographed. reference strains of l. major (mrho/ir/75/er), l. gerbilli (mrho/cn/60/gerbilli) and l. turanica (mrho/su/1983/marz-051) were used as positive controls. also, double distilled water was included in each run as a negative control (16). in order to sequencing, the pcr products of the second-round (nested) pcr for all positive samples were purified using the gel purification kit (expintm pcr sv, geneall biotechnology company, south korea). both forward and reverse strands of amplified dna were sequenced with the pcr primers. nucleotide homologies of the sequenced products were evaluated with leishmania spp. sequences available in genbank and then checked by using basic local alignment search tool (blast) analysis software (http://www.ncbi.nlm.nih.gov/blast) results a total of 176 female sand flies were selected in this study (64 specimens of ph. caucasicus and 112 specimens of ph. mongolensis), and extraction of genomic dna was conducted to identify leishmania parasites in these phlebotomine sand flies. in 17 female sand fly specimens including 6 specimens of ph. caucasicus and 11 specimens of ph. mongolensis, leishmania parasites were detected. out of these 17 specimens of leishmania-infected sand flies, seven, eight, and two specimens were infected to l. major, l. turanica, and mixed infection of both l. major and l. turanica respectively. the positive specimens produced species-specific band/s corresponding to l. major (245 and 233bp), and l. turanica (141bp) (fig. 1). it is important to note that l. major and l. turanica parasites were detected in esfahan and golestan provinces in both ph. caucasicus and ph. mongolensis, but in fars province (sadegh abad) only l. major parasite from ph. mongolensis was detected in one case (table 1). in this study, the infection rate for leishmania parasites were estimated to be 9.3 % for ph. caucasicus and 9.8% for ph. mongolensis. details of leishmania parasites detected in six specimens of ph. caucasicus and 11 specimens of ph. mongolensis in different collection sites with their abdominal status mentioned in tables 1 and 2. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 318 http://jad.tums.ac.ir published online: dec 31, 2022 table 1. leishmania parasite positive pcr detected in specimens of phlebotomus caucasicus and ph. mongolensis based on collection sites in esfahan, golestan and fars provinces, iran, 2016 positive samples no of examined specimens collection sites species (leishmania positive samples) mix of l. major + l. turanica l. turanica l. major total 2 2 4 32 habib abad (esfahan) ph. caucasicus (6 specimens) 14 ali abad (esfahan) 12 nik abad (esfahan) 1 1 1 ezhiyeh (esfahan) 1 1 5 agh tagheh (golestan) 1 1 8 sian (esfahan) ph. mongolensis (11 specimens) 1 1 3 abbas abad (esfahan) 2 nik abad (esfahan) 4 heydar abad (esfahan) 3 ezhiyeh (esfahan) 16 raja abad (fars) 1 1 7 sadegh abad (fars) 3 kouh sabz (fars) 2 band amir (fars) 8 ghareh gol (golestan) 1 1 12 ouch quee (golestan) 1 3 4 26 agh tagheh (golestan) 1 2 3 18 narlidagh (golestan) 2 8 7 17 176 total fig. 1. agarose (1.5%) gel electrophoresis of nested-pcr products for leishmania parasite infection in phlebotomus caucasicus and ph. mongolensis in esfahan, golestan and fars provinces. lanes m, 50 bp ladder (excelbandtm, smobio technology); lane 1, l. major (245 bp, detected in ph. caucasicus); lane 2, l. turanica (detected in ph. caucasicus); lanes 3-4-8-9-11, l. turanica (detected in ph. mongolensis); lane 5, l. major (245bp, detected in ph. mongolensis); lane 6, l. major (233 bp, detected in ph. mongolensis); lane 7, mix infection with l. major (233 bp) and l. turanica (detected in ph. mongolensis); lane 10, mix infection with l. major (245 bp) and l. turanica (detected in ph. mongolensis); lane 12, l. major (positive control); lane 13, l. turanica (positive control); lane 14, negative control (distilled water) http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 319 http://jad.tums.ac.ir published online: dec 31, 2022 table 2. leishmania parasite positive pcr detected in phlebotomus caucasicus and ph. mongolensis specimens based on abdominal status in esfahan, golestan and fars provinces, iran, 2016 abdominal status of specimens leishmania positive samples specimens species g sg ff uf l. major + l. turanica l. turanica l. major total 1 1 4 3 3 6 64 ph. caucasicus 2 9 2 5 4 11 112 ph. mongolensis 2 1 1 13 2 8 7 17 176 total uf= unfed, ff= fresh fed, sg= semi gravid, g= gravid discussion identification of phlebotomine sand flies as vectors of leishmaniasis is very important and crucial for leishmaniasis control programs. in this study, molecular detection, and identification of leishmania parasites based on nested-pcr method allowed us to detect more leishmania infections than previously in iranian sand flies. numerous natural sand fly promastigote infections in zcl foci in iran have been reported based on parasitological methods and direct examinations (31–35), identification of leishmania parasite using isoenzyme electrophoresis (14, 36), and molecular methods based on polymerase chain reactions (17– 27). in the present study two species of leishmania parasites including l. major and l. turanica as well as l. major + l. turanica mixed infection in ph. caucasicus and ph. mongolensis were detected using nested-pcr of its2rdna region. these parasites are in concordance with identified gerbils’ parasites and ph. caucasicus and ph. mongolensis belongs to kazakhstan, uzbekistan, turkmenistan and china (37, 38) and iran (23, 39). phlebotomus caucasicus is an asiatic species, first recorded from the transcaucasia region and is distributed from the geographical area of iran to china. it is a common species in sandy deserts and hills, and usually lives in rodent and bird nests, bites rarely humans (40). various transmissions of gerbil parasites have occurred in iran's northern neighbors in central asia, including l. major, l. turanica, and l. gerbilli, within or near the nest of the great gerbil of rhombomys opimus (41). rodent colonies provide habitat for many species of sand flies, increasing the risk of the leishmania parasite being spread to mammals (42). phlebotomus caucasicus was first introduced by adler and theodor in 1957 and was identified among central asian rodents as a suspected vector of l. major and l. gerbilli parasites (43). it is also considered an l. turanica vector in turkmenistan (37, 44), and l. donovani vector in central asia and kazakhstan (45). in this research, three samples of l. major parasite (esfahan and golestan provinces) and three samples of l. turanica parasite (esfahan province) were identified from six ph. caucasicus specimens. typing of parasites isolated from this sand fly species by isoenzyme method led to the precise diagnosis of parasite as l. major mon-26 and proved to be the same type of human parasite, also ph. papatasi as vector and rh. opimus as reservoir (14). phlebotomus caucasicus is also confirmed to be 12.5 and 7.5% natural leptomonad infection in the nikabad and borkhar regions of esfahan province respectively (14). in 2008, parvizi and ready identified two species of l. major and l. gerbilli from ph. caucasicus in http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 320 http://jad.tums.ac.ir published online: dec 31, 2022 esfahan province and l. gerbilli in golestan province based on nested-pcr method by sequencing of its1-5.8s rdna region (17). using the rapd-pcr method, l. major parasites were reported from four ph. caucasicus group specimens (4.2%) caught from rodent burrows in shahroud county (semnan province) in iran (39). once again, using the nested-pcr method of kdna genome and using the rflp method of its1-rdna region, the l. major parasite was reported from ph. caucasicus group of sand flies collected in damghan city (semnan province) in iran (46). according to these reports, along with the 20% anthropohilic index for ph. caucasicus (14, 47), strong evidence shows that ph. caucasicus is a natural vector of l. major, l. turanica and l. gerbilli parasites among reservoir rodents as well as a secondary or suspected vector of l. major for humans in zcl foci in iran (8). in the current study, four samples of l. major (golestan and fars provinces), five samples of l. turanica (esfahan and golestan provinces) and two mixed infection samples of both l. major and l. turanica (golestan province) were found from 11 collected ph. mongolensis specimens. it should be noted that, in iran, natural infection with leishmania parasites is recorded for the first time in this study in ph. mongolensis. phlebotomus mongolensis is also an asiatic species and is a dominant species in the sandy deserts and hills and usually lives in rodents burrow (41). it is also known to be l. turanica and l. gerbilli vector in turkmenistan (37, 44), l. turanica in china (38), l. donovani and l. major in central asia and kazakhstan (45). based on the findings of this study, it is possible that both ph. caucasicus and ph. mongolensis are potential vectors of the gerbil leishmania parasites. however, the detection of genomic dna of leishmania in sand flies does not confirm that they have been vector and pcr-based methods can not differentiate leishmania promastigotes in both infectious and non-infectious forms. the preference of sand flies to human blood, and the growth and development of parasites in the external cycle and experimental bite transmission have been the most important criteria for disease parasite transmission (48–50). considering that another criterion for the definition of a sand fly as a vector is the presence of the infective form of metacyclic promastigotes in the thoracic areas of midgut, foregut and mouth sections of sand flies, the position of the sand flies’ abdomen is also very critical in determining the vector. in this study, among the 17 ph. caucasicus and ph. mongolensis sand flies’ specimens with leishmania infection, 13 specimens with empty (unfed) abdominal status, indicating that the parasite of leishmania was successfully developed in midgut areas. the transmission dynamics of leishmania parasites depend not only on the leishmania species diversity but also on the parasite's intra-species strain diversity. in this research, according to the results of molecular identification of leishmania parasites, it can be concluded that ph. caucasicus and ph. mongolensis were considered as potential vectors of l. major and l. turanica parasites and allowing them to circulate of leishmania parasites among rodents and probably humans. acknowledgements this study was financially supported by the deputy of research, tehran university of medical sciences, grant no. 27252. ethical considerations this experiment was carried out under the guidance of the ethics committee of the tehran university of medical sciences (ir.tums. rec.1394.144). http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 315–324 a absavaran et al.: first report of … 321 http://jad.tums.ac.ir published online: dec 31, 2022 conflict of interest statement authors declare that there is no conflict of interest. references 1. alvar j, vélez id, bern c, herrero m, desjeux p, cano j, jannin j, denboer m (2012) who leishmaniasis control team. 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abdolamir behbahani, email: behbahani-a@ajums.ac.ir (received 21 oct 2017; accepted 14 apr 2021) abstract background: any mosquito control methods requires precise information about population dynamics, variety, biology and mosquito habitat. this research assessed culicid mosquitoes' attraction to a human host and a calf to better understand their behavior. methods: adult mosquitoes were sampled in 22 weeks in southwestern iran's nur ali village from may to october 2015. the mosquitoes were drawn to the person and calf as bait, while the unbaited trap was also used. a substantial statistical difference between attracted mosquitoes to the hosts was determined in the t-test. results: within 22 weeks, 29821 mosquitoes were captured. only 9% were collected from the human baited net trap, 89.1% from the calf baited net trap, and 1.9% from the unbaited net trap. the number of collected female mosquitoes was statistically significantly higher using the calf baited net trap of the total mosquitoes, 916 were randomly identified at the species level by local identification keys. of these, 63 were anopheles stephensi (human: 16%, calf: 75% and unbaited: 9%), 83 an. pulcherrimus (human: 27%, calf: 60% and unbaited: 13%), 118 aedes caspius (human: 24%, calf: 69% and unbaited: 7%), 493 culex tritaeniorhynchus (human: 52%, calf: 37% and unbaited: 11%), 153 cx. quinquefasciatus (human: 44%, calf: 47% and unbaited: 9%), and 6 cx. theileri (human: 33%, calf: 50% and unbaited: 17%). conclusion: the obtained results here provide useful insights into the mosquito population and the possibility of using this information as an essential part of integrated vector management regarding the reemergence of malaria or other mosquito-borne. keywords: mosquitoes; diversity; iran introduction blood feeding of female mosquitoes (diptera: culicidae) plays an essential role in transmitting diseases by mosquitoes, for instance, rift valley fever, yellow fever, west nile, dengue and malaria. the above plus other transmitted diseases by vectors cause about one billion people infected and more than one million deaths worldwide every year (1). parasitic and arboviral mosquito-borne diseases have been reported from iran for a long time. despite considerable progress recently in eliminating ma laria in iran, 799 reported malaria (632 cases were imported) were registered in 2015 (2). the records of other mosquito-borne diseases in iran, including dengue, west nile fever, dirofilariasis and sinbis, were reported in the literature (3-9). based on a retrospective cross-sectional study, 541 cases of malaria were reported in khuzestan province in the southwest of iran between 2001 and 2014. the most prevalent malaria species was plasmodium vivax with 478 (88.35%) cases compared with p. falciparum copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 162–170 p faraji-fard et al.: species variety of … 163 http://jad.tums.ac.ir published online: june 30, 2021 with 63 (11.65%) cases (10). the culicidae has 41 or 113 genera (depending on the classification of the tribe aedini), which are classified in the number of 3583 mosquito species. these 41 (113) genera are divided into the following two subfamilies: anophelinae and culicinae (11). based on the most recent iranian mosquito studies, 70 species were reported in 8 to 12 genera in iran (12-15). several species of culicid mosquitoes are recorded in khuzestan province, including an. stephensi, an. fluviatilis s.l., an. dthali, an. superpictus, an. pulcherrimus, an. culicifacies, an. hyrcanus, an. sacharovi, culex pipiens, cx. perexiguus, cx. sinaiticus, cx. modestus, cx. tritaeniorhynchus, cx. theileri, cx. pusillus, cx. quinquefasciatus, aedes caspius, uranotaenia unguiculata and a culiseta species (14-21). insecticide-treated nets and indoor residual spraying were used as the primary control activities of malaria vectors in the country (2). the explanation behind the progress made against malaria by iran and other countries was that governments were using insecticide-treated nets and indoor residual spraying to avoid malaria in significant transmission areas. it will aid little in managing malaria and zoonotic diseases with zoophilic vectors (22, 23). due to indoor residual spraying and treated mosquito nets by many societies, mosquitoes have adapted to remain alive by being more likely to feed on animal blood than ever. hence, appropriate management steps will be required to eliminate malaria or other vector-borne diseases in some regions of the world where it is caused by zoophilic disease vectors that are difficult to control (22, 24-26). the natural tendency of mosquitoes to feed from domestic animals, especially cattle reduces bites to nearby humans. therefore, the possibility of diversion of mosquitoes to animals was proposed as a malaria control method (27-29). the above concept application has been presented as a part of the integrated vector management, such as applying insecticides on cattle in the areas under specific conditions (30). after insecticide resistance and the harmful effects of certain insecticides in the environment were noted, this approach seemed like the best option. it is inexpensive, effective, and easy to encourage people to participate and contribute to disease vectors' control (31, 32). this research attempts to determine and compare the number of collected female mosquitoes in human baited, calf baited, and unbaited net traps to explain the obtained mosquito population's assortment traps toward providing information about the available potential control methods. besides, it provided some basic knowledge into the vector population to help the health authorities make the right decisions in case of any mosquito-borne disease in the area. materials and methods study area this research was carried out in nour ali village (31o 57' 17" n, 48o 55' 42"e, and 41m above sea level) within the central district in shoushtar county, khuzestan province in the southwest of iran (fig. 1). with 16 families, the village has 79 and an average of 4.9 persons per household. the village, situated along with one of the karoun river branches called gargar, is plain and flat. for several aquaculture farms in and around the village, this river supplies vital water, people farming crops, herds cows, sheep, goats, and ostrich. adult sampling the sampling of adult mosquitoes was conducted for 22 weeks period from may to october 2015. mosquitoes were collected using three types of net traps: first, a big standing rectangle is 2m by 1.5m by 1.5m as a human baited (a volunteer female in her fifties, who weighs 65kg) net trap. a steel frame surrounded an open area with a mosquito net all around. second, a calf baited (four months with an average of 60kg calf) net trap (3m by 3m by http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 162–170 p faraji-fard et al.: species variety of … 164 http://jad.tums.ac.ir published online: june 30, 2021 3m). the calf was held in the center of the net trap inside a steel frame. the third was the unbaited net trap (2 by one by 1.6m). our experiment's unbaited net trap was used to describe and relate the findings to only a human and a calf to attract the mosquitoes. the netting sides were raised approximately 30 centimeters above the ground to enable host-seeking mosquitoes to enter the net trap inside (33). traps were placed at the end of the day and stayed in position until dawn. the next morning, before entering the net trap to catch mosquitoes trapped overnight, the net trap sides were lowered to the floor. mosquitoes were captured by an aspirator from the internal walls and roof of the net trap and placed into labeled paper cups for later identification (33). field identification of mosquitoes all female mosquitoes were sorted. for every night trapping, if sufficient samples were available, 20 specimens were randomly chosen and identified from each baited net trap and five specimens from unbaited net trap to species level according to the local keys available (34, 35). statistics the collected data during 22 weeks from all types of net traps were considered for analysis. the collected data were entered in msexcel 2010 version. the calculations were done using the spss version 25.00. the means of collected mosquitoes in the net traps were compared using a t-test. results of the total collected female mosquitoes, 9 percent were trapped by human baited, 89.1 percent by calf baited and 1.9 percent by unbaited net trap (fig. 2). this research showed that statistically, lesser culicid mosquitoes (25.73±6.57) were attracted by unbaited net trap relative to human baited net trap (122.36±39.44), t (22.16)= 2.42, p< 0.05. this analysis showed that the calf baited net trap was statistically stronger at attracting mosquitoes (1207.41±264.46) than the unbaited baited net (25.73±6.57) t(21.03)= 4.47, p< 0.05. this analysis showed that human baited net trap, relative to calf baited net trap (1207.41±264.46), t(21.93)= 4.05, p< 0.05, attracted fewer culicid mosquitoes (122.36± 39.44) statistically (fig. 2). of the total 916 identified female mosquitoes, they were 42.3% (n= 388) from human baited net trap, 47.3% (n= 433) from calf baited net trap and 10.4% (n= 95) from unbaited net trap. the results of the species identification showed out of 916 identified female mosquitoes were 6.9% (n= 63) anopheles stephensi, 9.1% (n= 83) an. pulcherrimus, 12.9% (n= 118) aedes caspius, 53.8% (n= 493) culex tritaeniorhynchus, 16.7% (n= 153) cx. quinquefasciatus, and 0.6% (n= 6) cx. theileri. table 1. the mosquito's comparison in the trap with human bait against the trap containing a calf baited in iran's southwest in 2015 species mean±std. deviation calf baited mean ±std. deviation human baited f p ae. caspius 81(3.68 ± 4.05) 28(1.27 ± 1.42) 6.95 0.01 cx. tritaeniorhynchus 180(8.18 ± 3.96) 258(11.73 ± 5.17) 8.03 0.00 cx. quinquefasciatus 72(3.27 ± 2.51) 68(3.09 ± 2.38) 0.06 0.80 cx. theileri 3(0.14 ± 0.46) 2(0.09 ± 0.29) 0.16 0.68 an. stephensi 47(2.14 ± 2.94) 10(0.45 ± 0.80) 7.47 0.00 an. pulcherrimus 50(2.27 ± 2.51) 22(1.00 ± 1.41) 5.17 0.02 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 162–170 p faraji-fard et al.: species variety of … 165 http://jad.tums.ac.ir published online: june 30, 2021 fig. 1. the area of this research is shoushtar county (khuzestan province) in iran's southwest 2015 fig. 2. the comparison of human baited, calf baited and an unbaited net trap to collect mosquitoes in 22 weeks from may to october 2015 in southwestern iran discussion the factors that control the distribution of diseases transmitted by vectors are still poorly known in many areas. the arrival of humans into new areas as part of their migration pattern increases exposure to vector and pathogen vectors. determining the significance of mosquitoes as disease vectors requires knowledge of their biology and ecology (36). this research's observations have shown that collected mosquitoes were an. stephensi, an. pulcherrimus, ae. caspius, cx. tritaeniorhynchus, cx. quinquefasciatus and cx. theileri. anopheles stephensi was believed to be the primary malaria vector in the south (37, 38), in similar ways, it was found that blood-feeding females stayed substantially in animal shelhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 162–170 p faraji-fard et al.: species variety of … 166 http://jad.tums.ac.ir published online: june 30, 2021 ters following blood-feeding (17). anopheles pulcherrimus was recorded as a potential malaria vector in southeastern iran (14). the primary rift valley fever vector in saudi arabia has been identified as cx. tritaeniorhynchus, despite positive reports in iran, potential vectors were not established (15, 39-40). nevertheless, the ability to transmit the vector-borne diseases regarding these species' blood-feeding is considered nuisance and annoyance pests for inhabitants in the area. the findings of this analysis revealed that cx. tritaeniorhynchus from culicinae and an. pulcherrimus from anophelinae had the most significant number of specimens among the collected samples (table 1). while cx. theileri was scarce in the traps. due to the abundance of aquaculture farm-related breeding sites in the region, variations between the densities of collected species will occur due to some species' favor to these locations than others. also, the difference in the attractiveness of the hosts for different species may cause different collection densities. the propensity of mosquitoes to have a particular host relies on genetic variations between their various populations. however, regardless of genetic traits, it depends on the environmental conditions, the hosts' presence, density and the interaction between the vector and the disease microorganisms (41). a strong positive relationship (p˂ 0.05) was statistically demonstrated between the collected mosquitoes' densities in the net traps. the differences between the hosts' attractiveness in the current study may refer to the number of odors of different hosts and the olfaction, making the mosquitoes search for their hosts (42). the collected mosquitoes' significant results (1207.41±264.46) were observed in the calf baited net trap against the human baited and unbaited net trap. the higher mean of mosquito density showed significantly in the human baited net trap (122.36±39.44) compared to the unbaited net trap (25.73±6.57). the baited traps' host attraction has been verified by the lowest number of trapped mosquitoes without bait in the trap (p< 0.05). factors affecting the host preference for hematophagy include accessibility of hosts, their protective actions, and the suitability composites of their blood (43). our data indicate that the calf body's relatively large size compared to the exposed human body parts can lead to a calf releasing higher carbon dioxide, attracting more hungry mosquitoes (44–46). as shown in table 1, our results indicate that an. pulcherrimus and an. stephensi were particularly capable of feeding on the calf. this result was close to previous studies (25, 44). however, only the small number of specimens described may be biased as related. the culex spp reported were cx. quinquefasciatus, cx. theileri and cx. tritaeniorhynchus, which are considered effective vectors ((15). many culex mosquitoes have been affiliated with human populations that prepare an optimal environment for mosquito larvae, such as standing water (47). our studies found no statistically important change in the attraction of cx. quinquefasciatus and cx. theileri to the calf or human. these results agree that previous studies that reported cx. quinquefasciatus have different preferences, from 100 percent on humans to a high birdlikeness degree (41, 48). the vast range of hosts that cx. quinquefasciatus can feed blood under many distinct conditions seems to have chosen the most prevalent host type (41). our observation concerning cx. theileri did not follow previous studies that considered much of animals' blood meals than humans (49, 50). in contrast (table 1), cx. tritaeniorhynchus was markedly attracted to humans than the calf. previous experiments have proven this species to be zoophilic, although only a limited number of species reported here could be biased (51). aedes caspius, as a potential vector of some vector-borne diseases (15), was drawn significantly to the calf. more mosquito studies are recommended to better understand and evaluate mosquito tendencies in the field, as a subset of collected mosquitoes has been identified. as figure 2 shows, keeping a calf has been shown to attract ten times as many moshttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 162–170 p faraji-fard et al.: species variety of … 167 http://jad.tums.ac.ir published online: june 30, 2021 quitoes as we would expect the man to have. this protection can improve when implemented as an integrated control measure. nevertheless, some reports have demonstrated that, when animals are kept near humans, it could lead to the person being attacked by mosquitoes (32, 52). therefore, the number of zoophilic mosquitoes that can transmit mosquito vector-borne diseases to humans is increased by keeping an animal within a human settlement in rural areas (53). however, maintaining the different sheds outside the human residences helps eliminate the area's associated vector-borne disease (31, 53). consequently, local studies are critical 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485–490. 54. daluwaththa hs, karunaratne sh, de silva wp (2019) species composition of mosquitoes associated with a livestock field station. ceylon j sci. 48(1): 77–84. http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 225–232 z telmadarraiy et al.: study on hard and … 225 http://jad.tums.ac.ir published online: sep 30, 2022 original article study on hard and soft ticks of domestic and wild animals in western iran zakkyeh telmadarraiy1,2, habibollah kooshki1, hamideh edalat1, hassan vatandoost1, hasan bakhshi1, faezeh faghihi3, asadollah hosseini-chegeni4, *mohammad ali oshaghi1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2rahyan novin danesh (rnd) private university, sari, mazandaran, iran 3cellular and molecular research center, iran university of medical sciences, tehran, iran 4department of plant protection, faculty of agriculture, lorestan university, khorramabad, iran *corresponding author: dr mohammad ali oshaghi; e-mail: moshaghi@tums.ac.ir (received 08 may 2021; accepted 19 july 2022) abstract background: ticks are blood-sucking ectoparasites of many vertebrates and act as vectors of a wide range of vectorborne diseases. alongside pathogens transmission, ticks also cause economic losses in animal industry such as production loss, physical damage, anemia, and poisoning. this study aimed to determine the fauna, geographical distribution and seasonal activity of ticks collected from animals in lorestan province, west of iran. methods: ticks were collected from domestic animals including cattle, sheep, goats, chickens, turkeys, pigeons, as well as wild animals such as jackals in 2017–2018. then, they were identified based on morphological characteristics using valid identification keys. results: out of a total of 706 ticks, 433 (61.33%), 104 (14.73%), 33 (4.67%) and 136 (19.26%) ticks were collected in spring, summer, autumn and winter, respectively. in terms of hard ticks, 4 genera and 6 species were identified: hyalomma asiaticum (22.80%), hyalomma anatolicum (3.68%), hyalomma marginatum (2.40%), rhipicephalus sanguineus (0.84%), dermacentor marginatus (1.13%), and haemaphysalis sulcata (0.64%). additionally, two genera and four species fell into soft ticks: argas persicus (60.48%), argas reflexus (6.65%), ornithodoros canstrini (0.70%) and ornithodoros erraticus (0.42%). there was significant variation in the seasonal activity and abundance of ticks in different seasons but in the tick abundancy among different regions. conclusions: the present study provides a perspective of the distribution status of ticks in lorestan province, their seasonal activity and the likelihood of emergence of related diseases. keywords: ectoparasites; argasidae; ixodidae; lorestan; tick fauna introduction ticks are obligatory blood-sucking ectoparasites of different animals of mammals, reptiles, and birds which act as vectors of a wide range of viral, bacterial, and protozoan diseases. the most important diseases transmitted by ticks are rocky mountain spotted fever, crimean-congo hemorrhagic fever, lyme disease, typhus, ehrlichiosis, tularemia, babesiosis, colorado tick fever, and relapsing fever (1–6). alongside pathogens transmission, ticks cause important economic losses in animal industry. they also cause production losses, phys ical damages, anemia and poisoning, as well as negative impacts on human public health including annoyance, dermatitis, fatigue, malnutrition, and even death (7,8). almost 900 recognized species of ticks fall into three major families: ixodidae, argasidae and nuttalliellidae (a monotypic family representing the most primitive living lineage of ticks) (9). the growing number of ticks has a connection with rising accessibility of new environments and an increase in the number of host species specially in wild animals (10). besides, climate chang copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:moshaghi@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 225–232 z telmadarraiy et al.: study on hard and … 226 http://jad.tums.ac.ir published online: sep 30, 2022 es, especially temperature rise, have expanded the range of many tick species as warm conditions make the environment more suitable for these vectors (11, 12). therefore, the effects of infestation with ticks impose more effects on the lives of humans and animals. also, the need for studies on tick fauna and their environmental ecology in different geographical areas is felt more than before. the first study of ticks in iran was conducted by delpy on the prevalence of hard ticks (ixodidae); furthermore, abbasian-lintzen (13) and mazloum (14) reported a list of ticks, collected from different livestock. over time, many studies were conducted in different parts of iran regarding of ticks infesting livestock (15–20). some studies examined the fauna of ticks in western iran. davari and colleagues investigated the distribution and fauna of ticks in aleshtar county in iran (21). kayedi et al. (22) also identified hard and soft ticks collected from livestock in aleshtar and aligudarz counties, lorestan province, iran. ramezani et al. (23) also studied the ticks of livestock and their seasonal activities in northwest of iran. however, an up-to-date and comprehensive study that examines most of the province counties in all seasons seems necessary. the aim of this study was to determine the fauna, geographical distribution, and seasonal activity of ticks collected from animals in lorestan province, west of iran. materials and methods study area lorestan province (33.4871°n; 48.3538°e) covers an area of 28,294km2 in western iran. it shares borders with hamadan province in the north, markazi province in the northeast, isfahan province in the east, khuzestan province in the south, ilam province in the west, and kermanshah province in the northwest. the highest point of the province is the oshtorankuh mountain (more than 4,000m above sea level, asl) while the low-lying areas are in the southern parts of the province (500m asl). climatically, the province can be divided into two types of the mountainous experiencing cold winters and moderate summers, and warm weather experiencing hot summers and relatively moderate winters. 70% of lorestan villages are located in mountainous areas and 30% in plains. according to iran’s statistics organization in 2016, there were 246,900 cows, 1864,300 sheep and 814,900 goats in lorestan province. collection of ticks and their identification in this investigation, 24 villages from 7 counties of lorestan province including dorud, dowreh, delfan, selseleh, kouhdasht, pol-edokhtar, and khorramabad were selected for sampling. ticks were collected from both plains and mountains during winter 2017 until the fall of 2018 using forceps and were placed into separate labelled vials. a multistage random sampling method was used in which four livestock holding units were randomly selected from each village for tick collection. ticks were collected from domestic animals including cattle, sheep, goats, chickens, turkeys, and pigeons, as well as wild animals such as jackals. samples were transferred to the tick laboratory, department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran, along with the cold chain for species identification. genera and species were diagnosed under stereomicroscope according to valid morphological keys (24). ethical approval for this study was obtained from tehran university of medical sciences board (approval number/id: ir.tums.sph. rec.1399.244). results of 706 ticks collected, 224 hard ticks (31. 73%) and 482 soft ticks (68.27%) were identified during sampling period .in terms of hard ticks, four genera and six species were identified including: hyalomma asiaticum schulze http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 225–232 z telmadarraiy et al.: study on hard and … 227 http://jad.tums.ac.ir published online: sep 30, 2022 and schlottke, 1930 (22.80%), hy. anatolicum koch, 1844 (3.68%), hy. marginatum koch, 1844 (2.40%), rhipicephalus sanguineus latreille, 1806 (0.84%), dermacentor marginatus sulzer, 1776 (1.13%), and haemaphysalis sulcata canestrini and fanzago, 1878 (0.64%). aidditonally, two genera and four species fell into soft tick family including: argas persicus oken, 1818 (60.48%), a. reflexus fabricius, 1794 (6.65%), ornithodoros canstrini bir 1895 (0.70%) and o. erraticus lucas, 1849 (0.42%). chi-square test showed a significant difference between the sampling area and tick species (p= 0.001) (table 1, fig. 1). out of a total of 706 ticks, 433 (61.33%), 104 (14.73%), 33 (4.67%) and 136 (19.26%) ticks were collected in spring, summer, autumn and winter, respectively (fig 2). chi-square analysis revealed a significant difference between the tick distribution and different seasons (p= 0.000). fig. 1. distribution map of tick species collected from different counties of lorestan province, west of iran, 2017–2018 fig. 2. seasonal distribution of hard and soft ticks collected in lorestan province, west iran, 2017–2018 http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 225–232 z telmadarraiy et al.: study on hard and … 228 http://jad.tums.ac.ir published online: sep 30, 2022 table 1. geographical abundance, percentage, and distribution of ticks in lorestan province, 2017–2018 tick species location total n (%) dorud n (%) dowreh n (%) delfan n (%) selseleh n (%) kouh dasht n (%) pol-e-dokhtar n (%) khorramabad n (%) hyalomma asiaticum 11 (6.8) 33 (20.5) 20 (12.4) 18 (11.2) 32 (19.9) 28 (17.4) 19 (11.8) 161 (22.8) hyalomma anatolicum 1 (3.8) 4 (15.4) 0 (0) 0 (0) 7 (26.9) 11 (42.3) 3 (11.5) 26 (3.7) hyalomma marginatum 1 (5.9) 0 (0) 6 (35.3) 7 (41.2) 1 (5.9) 0 (0) 2 (11.8) 17 (2.4) rhipicephalus sanguineus 1 (16.7) 3 (50.0) 1 (16.7) 0 (0) 0 (0) 0 (0) 1 (16.7) 6 (0.85) dermacentor marginatus 0 (0) 2 (25.0) 1 (12.5) 1 (12.5) 0 (0) 0 (0) 4 (50.0) 8 (1.1) haemaphysalis sulcata 0 (0) 1 (16.7) 0 (0) 0 (0) 1 (16.7) 1 (16.7) 3 (50.0) 6 (0.85) argas persicus 14 (3.3) 54 (12.6) 87 (20.4) 42 (9.8) 91 (21.3) 59 (13.8) 80 (18.7) 427 (60.5) argas reflexus 1 (2.1) 14 (29.8) 7 (14.9) 12 (25.5) 0 (0) 3 (6.4) 10 (21.3) 47 (6.7) ornithodoros canstrini 2 (40.0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (60.0) 5 (0.7) ornithodoros erraticus 0 (0) 3 (100.0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (0.4) total 31 (4.4) 114 (16.15) 122 (17.3) 80 (11.3) 132 (18.7) 102 (14.45) 125 (17.7) 706 (100) discussion the results of the present study showed that of 706 collected ticks, 224 belonged to hard ticks and 482 were soft ticks. further to soft ticks, the dominant genus was argas and the dominant species was a. persicus. among hard ticks, the most common genera and species were hyalomma and hy. asiaticum, respectively. variation of hyalomma genus was more demonstrated. statistical analyzes showed that there is a significant relationship between seasons and tick distribution. as a result, the prevalence of ticks is higher in spring and summer than in autumn and winter. we suggest that temperatures increase in spring season and provides favorable weather conditions that facilitates the growth and survival of ticks during the spring season in the study area. also, it is shown that abiotic factors such relative humidity, temperature, and rainfall and biotic (availability of animal richness impact tick densities and activity (25–27). this is in concordance with the results of other researchers indicating variation in seasonal activity of ticks (12). the chi-square test showed a significant difference between the sampling area and tick species. the frequency of hyalomma, dermacentor, haemaphysalis and ornithodoros genera were high in khorramabad which is a mountainous region while argas and repicephalus were the most abundant species in plain region such as delfan and dowreh counties, respectively. in concordance with our study, davari et al. showed that a. persicus was the most frequent species of soft ticks in lorestan province (21). however, they also concluded that rh. sanguineus was the dominant species in the region which is different from our results. this difference could be justified due to limited sampling area in davari’s research. additionally, in both researches the frequency of ticks was higher in spring, which is reasonable due to the pick of tick activity in warmer seasons. furthermore, kayedi and colleagues reported that a. persicus was the dominant soft tick species in the area (22). however, they also concluded that the dominant species in aleshtar and alihttp://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 225–232 z telmadarraiy et al.: study on hard and … 229 http://jad.tums.ac.ir published online: sep 30, 2022 godarz counties was rh. sanguineus. it should also be noted that they only surveyed two counties which were lower than our sampling areas. kayedi et al. (22) also concluded that tick frequency deceased from spring to winter during the year, the same as our result. hyalomma asiaticum is the second most prevalent species in the present study. a survey on fauna of ticks in west azerbaijan province showed high prevalence of rh. sanguineus and hy. asiaticum (24). another study revealed presence of nine species of hard ticks in northwest of iran (23). a study of tick fauna in tandoureh national park, khorasan razavi, east of iran, revealed that six species were present: d. niveus (47.2%), rh. turanicus (32.9%), d. raskemensis (8.6%), hy. turanicum (5.6%), ha. sulcata (3%) and hy. aegyptium (2.6%) (28). in that study, d. niveus was the most predominant species. this species is distributed in semi-deserted areas. this could be the reason behind the absence of this genus in our study area. in another study conducted on hard ticks of domestic ruminants and their seasonal dynamics in yazd province, east of iran, ticks were classified into three genera and seven species including hy. dromedarii (55.92%), hy. marginatum (13.20%), hy. anatolicum (9.78 %), hy. detritum (4.98%), hy. asiaticum (3.94 %), rh. sanguineus (11.84%), and d. marginatus (0.34%). the highest seasonal activities occurred in summer (29). in a study on the border of iran-pakistan, southeast corner of iran, the collected ticks were classified into three genera: hyalomma (90.7%), rhipicephalus (6.1 %), and dermacentor (3.2%) where hy. anatolicum was the most common species in the study area (30). in north of the country, 15 species of ticks were identified based on their morphological characteristics including rh. sanguineus, r. bursa, i. ricinus, ha. punctata, h. sulcata, h. erinacei, h. inermis, hy. marginatum, hy. asiaticum, hy. excavatum, hy. anatolicum, hy. dromedarii, hy. detritum, b. anulatus and a. persicus. dominant species were rh. sanguineus and hy. asiaticum respectively (31). haemaphysalis asiaticum also ranked second prevalent species in our study. however, fewer species were found in the present study. in a study in south khorasan province, east of iran, two genera and seven species of ticks were identified including: rh. sanguineus (21.9%), hy. detritium (25.0%), hy. marginatum (2.4%), hy. anatolicum (0.8%), hy. asiaticum (1.6%), hy. dromedarii (43.0%) and hyalomma sp. (4.7%) (32). other studies in eastern parts of iran also revealed that rhipicephalus and hyalomma are more prevalent (33, 34). in terms of soft ticks’ dominance in iran, argas genus is the only or the dominant species. in general, the differences between different studies of tick fauna can be justified due to variations in sample size, geographic area, hosts, date of collection and vegetation. argas persicus (fowl tick or poultry tick), is found predominantly on chickens, ducks, and geese. lorenz oken reported this species in mianeh for the first time. they are carriers of borrelia anserina, causative agent of avian spirochetosis, one of the most serious infections of the poultry. hyalomma genus is the most important tick species associated with crimeancongo haemorrhagic fever virus (cchfv). the most abundant species found in iran are as follows: h. anatolicum, h. asiaticum, h. detritum, h. dromedarii, h. excavatum, h. marginatum, h. rufipes and h. schulzei (1, 12, 35). repicephalus sanguineus and d. marginatus are also involved in cchfv transmission. due to the predominant tick fauna of the region and the possibility of and borrelia epidemics, further studies in this field seem necessary to prevent the risk of potential occurrence. ornithodoros erraticus which was identified in our study, plays an important role in the transmission of the qalyub virus, african swine fever viruses, borrelia procedure, and b. hispanica (36, 37). clarification of the status of these diseases in the ticks of the region requires molecular approaches to provide a clear view, although the occurrence of these diseases in iran seems rare. http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 225–232 z telmadarraiy et al.: study on hard and … 230 http://jad.tums.ac.ir published online: sep 30, 2022 conclusions the present study provides a perspective of the distribution status of ticks in lorestan province, their seasonal activity and the likelihood of emergence of related diseases. awareness of the potential risk of diseases will assist with animal and public health. acknowledgements this study was supported by tehran university of medical sciences, tehran, iran; (grant no. 43192). ethical considerations ethical approval for this study was obtained from tehran university of medical sciences ethics board (approval number: ir.tums.sph. rec.1399.244). conflict of interest statement authors declare that there is no conflict of interest. references 1. telmadarraiy z, chinikar s, vatandoost h, faghihi f, hosseini-chegeni a (2015) vectors of crimean congo hemorrhagic fever virus in iran. j arthropod borne dis. 9(2): 137–147. 2. oshaghi ma, rafinejad j, choubdar n, piazak n, vatandoost h, telmadarraiy z, mohtarami f, ravasan nm (2011) discrimination of relapsing fever borrelia persica and borrelia microtti by diagnostic species-specific primers and polymerase chain reaction-restriction fragment length polymorphism. vector borne zoonotic dis. 11(3): 201–207. 3. barmaki a, rafinejad j, vatandoost h, telmadarraiy z, mohtarami f, leghaei sh, oshaghi m. 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treatment of head lice (pediculus humanus capitis) in areas covered by health centers of islamshahr city, tehran, iran in 2019 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... original article evaluation of efficiency of ivermectin lotion in comparison with permethrin shampoo and dimethicone lotion for treatment of head lice (pediculus humanus capitis) in areas covered by health centers of islamshahr city, tehran, iran in 2019 leila hamedanian1, *mohammad reza salmani nadoshan2, *hassan vatandoost1,3, mojgan baniardalani1, *javad rafinejad1 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 department of health, south tehran health center, tehran university of medical sciences, tehran, iran 3 department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran *corresponding author: dr. javad rafinejad, e-mail: jrafinejad@tums.ac.ir dr. hassan vatandoost, e-mail: hvatandoost1@yahoo.com (received 31 aug 2021; accepted 30 sep 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: head lice infestation is known as a serious health problem in developing and developed countries. the prevalence of pediculosis in children and females is higher than others. the infestation may cause the absence of student from school and make their parents upset. the aim of current study was to evaluate the who recommended insecticides for control of head lice in islamshahr health center. methods: in the current study infested individuals were older than 6 years old and have been diagnosed with at least 3 head lice adult and 10 live nits from less than 0.7 cm scalp. the pediculicides was applied randomly among groups. permethrin shampoo as a golden standard was used. dimethicone lotion 4% and ivermectin lotion was compared with it. this study was conducted on 179 infested people. results of infestation were evaluated after one month of intervention. different indicators such as: present of head lice adult or live nit, redness and irritation in head skin were invested. results: all the participants were female and 72.6% of them were in the age group of 6-11. the result revealed that aging not only decreases the infection rate but also raised the recovery percentage. the results showed that 79.5% permethrin receiver, 83% of people who treated with dimethicone lotion and 90.6% of ivermectin receiver had no head lice. there was no significant difference among these three pediculicides. conclusion: there was no significant difference among tested pediculicides. an appropriate pediculicide can be selected by training people. keywords: head lice; treatment; permethrin; pediculicide introduction head lice infestation is a global health problem that is not limited to one community or continent. the disease is present in varying proportions in most developed and developing countries (1-7). despite preventive activities in countries, head lice infestations still persist at different ages and exist in densely population areas. economic, social and cultural status are also very important in lice infestation. children 326 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... are infested more than adults. the highest infestation was reported in 5-11 yearold children. it is more common in girls than in boys and in whites than in blacks. however, the prevalence of infestation is higher among primary school students and their families, especially in poor areas receiving substandard health care (8-10). in addition to direct contact and head-tohead contact with an infestation person, lice infestation is also the most common method of transmission through personal stuff such as combs, hats, scarves, underwear and towels (11). head lice have no role in transmitting any diseases, but it may cause discomfort and embarrassment to people in the family and school, especially in the sensitive ages of childhood and adolescence. so far, various methods have been used to treat head lice infestation, the most available pediculicides used to kill adults. there are numerous reports of increased cases of pediculosis and recurrences after treatment and in most cases, after treatment, between 20 and 80 nits remain on the head, which are the main cause of re-infestation. treatment with 1% permethrin shampoo and 1% lindane shampoo has been available in iran since previous years, and dimethicone lotion has been used in the treatment of pediculosis since 2014. while ivermectine lotion has not yet officially entered the drug distribution network system despite receiving a license from the food and drug administration. in this study, the efficiency of ivermectine lotion in comparison with permethrin shampoo as the standard pediculicide and dimethicone lotion has been evaluated so the results can be used in the implementation of pediculosis control policies. material and methods the study was a clinical trial based on direct observation of at least three live adult lice or more than 10 active nits at a special distance (less than 0.7 cm from the scalp) on patients six years and older referred to health centers. also, the infested individuals that were identified by trained personnel of islamshahr city’s school by tehran performer. at the beginning of the study, a workshop was held by the organizers about the necessary training on how to examine, research ethics, how to diagnose infestation and refer infested people, as well as treatment methods for each pediculicide gave to school health’s experts and health network diseases. eligible individuals were divided into three groups, 6 to 11 years, 11 to 15 years and 15 years and elder, the pediculicide were randomly distributed among the groups where as 1% permethrin shampoo is used as a common pesticide in the treatment of head lice, it was considered as a standard and the two groups of 4% dimethicone lotion and 0.05% ivermectine lotion were studied in comparison with the permethrin shampoo. after the intervention for one month, based on the presence of symptoms such as the presence of adult lice or active nits on the head or the presence of itching and redness on the scalp of patients were evaluated. the results were recorded in the relevant forms and finally the results were compared. exclusion criteria included pregnant and lactating women, people with chronic diseases of the scalp and body and using any type of treatment to get rid of lice in a recent month. in order to determine the sample size of the intervention statistical population by considering the 95% confidence interval and 80% test power and also considering the relative accuracy of 20% by using ivermectine lotion compared to permethrin (6, 12, 13, and 14), the sample size was 179; it included 73 permethrin, 53 dimethicone and 53 ivermectine receiver. in order to blinding, the pediculicides were poured at the rate of usage of one person in unlabeled containers with only codes a, b and c written on them, and wilcoxon signed-rank test was used to evaluate the effectiveness of each pediculicide. in order to make the best use of 1% permethrin shampoo, infested people or their companions were instructed to wash their hair with regular shampoo at first, and if the hair was dehydrated eventually, then hair and the scalp of the 327 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... infested person should be thoroughly soaked in permethrin shampoo for 10 minutes, then the shampoo would soak the hair. in order to use dimethicone 4% and ivermectine lotion 0.5% for infested people optimally, considering that the hair shouldn’t be wet or damp, the participants or his/her companion should be instructed to apply dimethicone lotion from the scalp to the ends of dry hair at home. the whole scalp should be completely covered by the lotion. in the case of dimethicone lotion after 8 hours, for ivermectine lotion after 10 minutes, washing the hair with regular shampoo under the pressure of water with the help of a fine-toothed comb and separate the dead lice and nits from the hair. all subjects were accurately evaluated for adult lice or active nits for up to one month and the evaluation results were recorded in the relevant form. the primary outcome in all three groups was the percentage of patients who did not have any adult head lice or nits from less than 0.7 cm of scalp. the 14th and 30th day need to be checked out, and the secondary consequence was lack of local allergic reaction on the scalp, body skin or around the eyes. also, no re-infestation was observed at the end of the month. also, the level of satisfaction of the subjects with the quality of treatment was very important. due to the interventional nature of this study, it was first obtained from the university ethics committee under the ir.tums.sph.rec.1398.305 agreement. then, based on the informed consent of the patient or his guardian, while providing the telephone number of the project manager for any questions and counseling, all the necessary training was carefully given to the infested person or his companion. since this study, like other studies, has limitations such as finding infested cases in a timely manner, follow-up after the intervention, proper training in pediculicide use, providing the required pediculicide, especially ivermectine lotion, outbreak of corona virus at the end of the study, traffic problems were encountered and the necessary arrangements were made for each case. results this study was performed based on the calculation of statistical volume on 179 infused individuals, all were female, and the age was 6 years old to the top. the age distribution of these individuals is shown in table 1. according to the results presented in table 1, the most infested people (72.6%) belonged to the age group of 6-11 years and the lowest (10.6%) belonged to the age group of over 15 years. the infested individuals were randomly intervened with three pediculicides, the most common use of permethrin was accorded in the age group of 11-15 years and to the top, and the highest use of dimethicone and ivermectine lotion occurred in the age group table 1. frequency distribution of people participating in the treatment of head lice infestation by age groups in islamshahr city in 2019 cumulative percentage frequency percentage frequency age groups 72.6 72.6 130 6-11 89.4 16.8 30 11-15 100 10.6 19 above 15 100 179 total table 1. frequency distribution of people participating in the treatment of head lice infestation by age groups in islamshahr city in 2019 table 2. distribution of efficiency of three used pediculicide in treatment of head lice in some age groups in islamshahr in 2019 intervention age group ) (year permethrin total ivermectin total dimethicone total total number percentage number percentage number percentage number percentage number percentage number percentage number percentage 6-11 cured 18 58.1 31 23.8 42 91.3 46 35.4 44 83 53 40.8 104 80 not cured 13 49.1 4 8.6 9 17 26 30 11-15 cured 24 92.3 26 86.6 4 100 4 13.3 0 0 0 0 28 93.3 not cured 3 7.7 0 0 0 0 3 6.6 above15 cured 16 100 16 84.3 2 66.7 3 15.8 0 0 0 0 18 94.7 not cured 0 0 1 33.3 0 0 1 5.3 total 73 41 53 29.5 53 29.5 179 100 table 2. distribution of efficiency of three used pediculicide in treatment of head lice in some age groups in islamshahr in 2019 328 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... of 6-11 years (table 2). the results of the relationship between treatment success and age groups in the study population showed a significant relationship in the permethrin group (r = -.455, p <0.001) and with increasing age, the incidence decreased (increased recovery) so that the chance of infection in the group of 6-11 years, that was 8.8 times higher than the group of 11 years and above, while this relationship was not significant in the group of ivermectine and dimethicone. the results showed that out of 130 infested people in the age group of 6-11 years (80%), out of 30 infested people in the age group of 1115 years (93.3%) and out of 19 infested people in the age group of 15 years and above (94.7%) had been recovered. the results showed that the best efficiency of permethrin shampoo was in the age group 15 years and above, which was 100% of the infested people who had been recovered, and the lowest efficiency was in the age group of 6-11 years, in which only 58.1% of the infested people had been treated. the results also showed that dimethicone lotion was used only in the age group of 6-11 years and 83% of the intervened people were treated with this lotion. ivermectine lotion efficiency in the age group of 11-15 years was 100% and in the age group of 6-11 years was 91.3%. this lotion was not used in the age group over 15 years. in general, the efficiency of the three pediculicides used in the treatment of head lice in islamshahr in 2019 showed that 83.5% of the people recovered and only 16.5% of them did not respond to treatment methods. in order to compare the success ratio of treatment of these three pediculicides with each other in the study population, chi-square test (test statistic value = 2.83, degree of freedom =: 2 and probability value = 0.243) was used and the results showed that the success ratio independent on type of treatment. also, the separate results of the three intervened pediculicides showed that the highest efficiency was related to ivermectine and the lowest was related to permethrin (table 4). as table 4 shows, 79.5% of the subjects who received permethrin, 83% of those who received dimethicone, and 90.6% of those who received ivermectine were recovered. wilcoxon signedrank test was also used for the efficiency of each pediculicide (table 5). wilcoxon signed rank test showed that the treatment used in each group was effective (p <0.001). in order to compare the success ratio of treatment in the treatment groups, the ratio test was used (table 6). according to the probability value, it is concluded that the success ratio of treatment in all three treatment groups is similar, and there is no significant difference between the three pediculicides. table 3. general distribution of efficiency of the three pediculicide used in treatment of the head lice infestation in islamshahr in 2019 percentage frequency result after intervention 83.5 150 recovered 16.5 29 not recovered 100 179 total table 3. general distribution of efficiency of the three pediculicide used in treatment of the head lice infestation in islamshahr in 2019 table 4. intervention results of the pediculicides used intervention results not treated treated percent number percent number 20.5 15 79.5 58 permethrin 17 9 83 44 dimethicone 9.4 5 90.6 48 ivermectin table 4. intervention results of the pediculicides used 329 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... therefore, in case of using any of the treatments methods with good results (though ivermectine was more appropriate), due to almost similar and no significant difference between them will be obtained desirable results. discussion the results of this study, like the results of other researchers, showed that the highest ratio of infestation is in females and in the age group of 6-11 years, therefore sex and age are the two main epidemiological factors in the studies and interventions related to pediculosis (8, 14-25). numerous studies have been performed on the efficiency of pediculicides used to control head lice infestation and comparing pediculicides with each other. the results of this study showed that the success ratio of treatment with the three pediculicides, despite a slight difference, had a relatively similar effect. also, the results of the efficiency of each pediculicide showed that aging had a positive role on efficiency, which may due to increasing awareness and mastery of how to use at this ages, while the lotions studied had more effective results at young ages, this study also had a good result because lotions have much less side effects than permethrin shampoo and this can be used even in children under two years. the results of this study is similar to bashiri study (26) that the efficiency of permethrin shampoo was 97.3% and rafinejad et al. (15) that the efficiency of permethrin in girls was 75%, also dinapli et al. (27) showed that permethrin 1% was the most effective substance with high safety to treat head lice infestation, it has been announced that this pediculicide can be made available to the public without a doctor’s prescription. zahirnia et al. (28) declared the efficiency of permethrin shampoo was 88% was consistent. burgess et al. (29,30) reported the efficiency of 4% dimethicone lotion compared with 5% phenothrin in a randomized clinical trial and obtained almost similar results between 70% and 75%, which was consistent with the present study. feldmeier (2012) study showed that dimethicone lotion and silicone oils worked perfectly physically with low surface tension and complete surface coverage, so this group of compounds is very effective and safe, and there is no risk to use head lice resistant (31). ferrara et al (2013) study in rom, italy, determined the efficiency and safety of dimethicone 4% in the treatment of head lice infestation and they received prevention for all participants, with a cure rate of 82.4% (32). monisha et al. (2018) showed in a cohort study on the efficiency of 0.1% permethrin shampoo in comparison with 0.5% ivermectine lotion in head lice infestation after treatment with topical permethrin and 0.1% ivermectine lotion 0.5%, the presence of active nits were significantly reduced in the ivermectine group (33). saghafipour et al. (2017) (9) studied the effect of 1% permethrin shampoo on head lice in qom province. in this trial, table 5. wilcoxon signedrank test to evaluate the efficiency of each pediculicide probability value z test criterion type of treatment <0.001 -7.616 permethrin <0.001 -6.663 dimethicone <0.001 -6.928 ivermectin table 5. wilcoxon signedrank test to evaluate the efficiency of each pediculicide table 6. comparison test of success rates in treatment groups probability value test criteria degree of freedom treatment success ratio type of treatment 0.243 2.828 2 0.794 permethrin 0.830 dimethicone 0.906 ivermectin table 6. comparison test of success rates in treatment groups 330 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... 378 people infested with head lice were treated with 1% permethrin shampoo and one week after the end of the treatment period, they were examined and the infested people received another course of treatment and this process continued for up to 4 treatment periods. the results showed that 58.2% recovered after one course of treatment, 35.4% in two courses, 2.4% in three courses and 1.6% after four courses of treatment. in 2.4% of cases, the infestation was not eliminated. in total, 93.6% of infested people were free of infestation during one to two treatment periods. there was a relationship between the density of infestation in the family and the level of literacy of the mother in the family with increasing the length of treatment. people over the age of 20 (compared to those under the age of 10) had a better chance of increasing their treatment (9). conclusion the results of this study showed that taking one pediculicide is not particularly preferable to other pediculicides except due to some factors such as economic status, pesticide price, availability, underlying diseases, pesticide sensitivity, how to use and age of people. correctly, the necessary effectiveness and the recurrence of infestation and the problems and challenges are related to wrong training and incorrect practice that can be compensated with proper training and proper use of compounds. also, screening before the beginning of the school year to identify and treat infested cases, use the capacity of medical entomology and vector control graduates in screening and treatment of infested people at the beginning of the school year. holding workshops and training classes for school health educators and experts or employing trained entomologists as school health educators, uploading reliable and simple scientific information for public use on the ministry of health’s system, universities and health centers, using the capacity of cyberspace to teach the correct way to diagnose and treat infestation, introduce common and approved pediculicide of the ministry of health along with the standard treatment guidelines, review, modify and create a national standard 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shampooes in treatment of head lice (pediculus capitis) in primery school-children in hamadan province, iran 2000-2001. 15(49):1624. 29. burgess if, brown cm, lee pn (2005) treatment of head louse infestation with 4% dimeticone lotion: randomized controlled equivalence trial. biomed j. 330(7505): 1423. doi: 10.1136/ bmj.38497.506481.8f. 30. burgess if, lee pn, matlock g (2007) randomized, controlled, assessor blind trial comparing 4% dimethicone lotion with 0.5% malathion liquid for head louse infestation. plos one. 2(11), p.e1127. 31. feldmeier h (2013) pediculosis capitis: new 332 http://jad.tums.ac.ir published online: sep 31, 2021 j arthropod-borne dis, sep 2021, 15(3): 325–332 l hamedanian et al.: evaluation of efficiency... insights into epidemiology, diagnosis and treatment eur j clin microbiol infect dis. 31(9):2105-2110. 32. ferrara p, del bufalo f, romanov, tiberi e, bottaro g, romani l, malamisura m, ianniello f, ceni l, mottini g, gatto, a (2013) efficacy and safety of dimethicone in the treatment of lice infestation through prophylaxis of classmates. iran j public health. 42(7): 700-706. 33. monisha bm, madhumitha m, thilagavathi s (2018) comparison of efficiency of 1% permethrin lotion vs. 0.5% ivermectin shampoo in the treatment of pediculosis capitis. interj res. 4(3): 357-362. evaluation of efficiency of ivermectin lotion in comparison with permethrin shampoo and dimethicone abstract keywords introduction material and methods results discussion conclusion conflict of interest acknowledgments references j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 179 http://jad.tums.ac.ir published online: june 30, 2021 original article diversity of hard tick populations and their geographical variations in northwestern iran rohollah moradi1; eslam moradi-asl2; zakkyeh telmadarraiy1; seyedeh zahra parkhideh1; *yavar rassi1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of public health, school of public health, ardabil university of medical sciences, ardabil, iran *corresponding author: prof yavar rassi, e-mail: rassiy@tums.ac.ir (received 19 jan 2021; accepted 28 jun 2021) abstract background: ticks are forced vertebrate ectoparasites, including humans, and are vectors of serious diseases such as crimean congo hemorrhagic fever, relapsing fever, and various forms of encephalitis. spatial assessment of the prevalence of ticks and detection of high risk areas for tick-borne disease transmission and evaluation of ecological measures are key aims of this research. methods: ticks were collected using standard methods from 27 villages in the region of sarab county in north-eastern iran during the four seasons of 2018–2019 and identified using valid keys. the calculations of indices for biodiversity were based on the margalef index, shannon-weiner index and simpson index. r2.15 statistical software was used for statistical analysis of indices of biodiversity, and arcmap10.4.1 software, idw and generalg methods were used. analysis were used to investigate spatial distribution and to determine important tick hotspots. results: a total of 2500 animals surveyed, 35% of them were infected. in total, 1416 ticks were caught, 74.6 %, 23.9% and 1.4 % were adult, nymph and larvae respectively. 94% of the ticks were hard ticks including 6 genera. according to the margalef diversity index, the highest species biodiversity was related to summer (1.4234), and the lowest was related to winter (0.7379), conclusion: large hotspot area was found in the central part of the study area. the area of study was very prone to tick-borne disease transmission in terms of tick diversity and tick species richness. tick-borne disease control is an important measure. keywords: ticks; species biodiversity; gis; iran introduction ticks are the most important medical and veterinary ectoparasites and are one of the most important arthropod groups (1). ticks belong to the order of metastigmata which feeds on animals and humans and play an important role in the transmission of the disease. this order is composed of two common argasidae and ixodidae families (2). there are about 899 hard tick species, and 185 soft tick species (3). in iran 14, 5, 8, 11, 1 and 3 hard tick species of hyalomma, rhipicephalus, ixodes, haemaphysalis, bophilus and dermacentor has been rec orded respectively. 10% of ticks feed on domestic animals, particularly cattle, sheep and goats. these are of major health significance due to the conditions that cause human and animal diseases (4-6). tick-borne diseases are among the most common emerging and re-emerging diseases in recent years that have spread to different geographic areas (7). several diseases including tick-borne fever, rocky mountain spotted fever, q fever, lyme disease, and crimean-congo hemorrhagic fever (cchf) are among the known tick-borne human diseases copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:rassiy@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 180 http://jad.tums.ac.ir published online: june 30, 2021 (8-10). regardless of the importance of ticks in spreading different pathogens, its population structure, relative abundance and frequency, hosts type, habitats, ecology and diversity of species must be regulated. the study of tick biodiversity using various scientific indices seems to be very helpful (11-12). the aims of this study was to determine the status of species diversity and richness of hard ticks and their geographical distribution in the livestock of sarab region in northwestern iran using simpson, shannon weiner, margalef indices and gis software indicators. materials and methods study area the county of saraba is located in the province of east azerbaijan, iran. the capital of the county is sarab. the county population at the 2006 census was 132,094, out of 31,977 families. the county is subdivided into two districts: the central district and mehraban district. the county has four cities: sarab, mehraban, sharabian, and duzduzan. at the 2006 census, it had a population of 42,057 in 11,045 families. livestock and animal husbandry has traditionally been common in most rural households (fig. 1). tick collection a total of 27 villages were selected from 9 districts. ten locations were selected in each village (human, animal and livestock storage areas, traditional and modern grain deposits, farmland attached to the village and preferably attached to active livestock sables, estimated at 300 places). during the study, 441000 cattle (331000 sheep and goats) and (95000 cows and oxen) (8000 horses) and (7000 donkeys) were in the sarab county. therefore, ticks from a total of 2500 livestock were collected in this study, of which 75% of the sample size were sheep and goats, 23% were cows and oxen, and 2% were horses and donkeys. valid keys were used to identify species of the ticks (13). this experiment was carried out under the guidance of the ethics committee of the tehran university of medical sciences (ir.tums.sph.rec. 1398.058). biodiversity indices different indices of biodiversity were used in this study including species richness (margalef's richness index), species diversity (simpson's and shannon weiner's index). calculation of tick dispersion arcmap 10.4.1 software and interpolation with inverse distance weighted (idw) were used to analyze tick dispersion and to assess the distribution of disease vectors hot spots. the general g formula was used to determine the pattern of distribution of all cchf disease species and main vectors including hyalomma marginatum and h.anatolicum in the study region. results for a total of 2500 animals examined, ticks infested 763 animals (30.5 %). in total, 300 stalls and stables were inspected in 27 villages, 75 of which were infested with ticks (25%). a total of 1,416 ticks have been collected, of which 30% were male, 44.6% were female, 23% were nymphs and 1.4% were also tick larvae. of the 94% of the ticks collected, 11 species have been classified as hard ticks, belonging to 4 genera: hyalomma (65%), haemaphysalis (11%), rhipicephalus (6%), and dermacentor (12%). three species of soft ticks from two genera of ornithodoros and argas were identified (table1). sheep and goats with 58.68%, cows and ox with 13.23%, horses and donkeys with 2.14% respectively were the most infested animals with ticks. in terms of age, ticks were observed more in animals under 3 years of age (49 %) and the lowest infection (10 %) was observed in anihttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 181 http://jad.tums.ac.ir published online: june 30, 2021 mals over 7 years of age, and the most infested section of the animals were sub-tail (29.89 %) and animal ears (25.69 %). biodiversity and species richness tick diversity and species richness were calculated on the basis of two factors: seasonal activity and the host. the margalf species richness index showed that the highest richness was related to summer (1.4234), and the lowest richness was related to winter (0.7379). the species diversity of ticks collected at different seasons in the research region were also significantly different (p< 0.05). the highest species diversity was related to summer (2.1709), and the lowest species diversity was related to winter (0.781), according to the shannon-wiener index measurements. the index of evenness of ticks collected during different seasons in the research region was significantly different (p< 0.05). the evenness index analysis found that the highest uniformity was in relation to the summer season (0.9428) and the lowest uniformity was in relation to the autumn season (0.4428). the simpson species diversity index also showed that the highest diversity was correlated with summer (0.8622), and the lowest diversity was associated with autumn (0.3549). the results of this study found that the species richness of ticks collected in the sarab region during the first and second six months of the year had a significant difference (p< 0.05), and the first six months of the year had higher and more diversity and richness of species than the second six months of the year (table 2). according to the simpson species diversity and the shannon-wiener indices, the findings of species diversity and richness based on different hosts showed that the highest species diversity among the studied hosts were correlated to sheep and goats (0.881and 2.333), and the lowest species diversity was found in horses and donkeys (0.715 and 1.399) respectively. there was a significant difference (table 3) in the evenness index for livestock hosts that ranging from the highest for sheep and goats (0.9096) to the lowest for cows and oxen (0.7945). spatial distribution the most ticks were collected in the villages of dozduzan (6.43%), sharabian (6.28%), mehraban (6.12%), kelian (5.6%), ardha (5.54 %) and mahin bijand (5.3%) and the lowest in dichan village (2.45%). in mehraban district the highest spatial dispersion as well as the highest density of hard and soft tick species was found. the findings of the interpolation study (idw) revealed that there was a large hotspot area in the central part and a small hotspot in the northwestern part of the county of sarab in terms of tick abundance, both of which were high-risk areas in the mehraban region with 4596 and 1150 households in population (fig. 2). the results of the analysis of spatial distribution patterns for all species and for two species of h. marginatum and h. anatolicum using the general g index showed that all species were distributed randomly in all areas of study and did not have a specific pattern such as cluster or regular (fig. 3 and table 4). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 182 http://jad.tums.ac.ir published online: june 30, 2021 table 1. the total number of ticks collected from hosts and residential places in sarab county, 2018–2019 t o ta l l a r v a n y m p h adult ticks hosts species genus family ixodidae f e m a le m a le s h e lt e r s d o n k e y a n d h o r se s h e e p a n d g o a t c o w a n d o x 46 0 0 33 13 0 0 17 11 h.anatolicum hyalomma 319 0 2 111 206 0 12 69 52 h.marginatum hyalomma 60 0 0 25 35 0 0 22 12 h.scupense hyalomma 143 0 0 141 2 0 2 67 39 h.sp hyalomma 55 0 0 17 38 0 2 18 8 h.asiaticum hyalomma 18 18 0 0 0 0 0 2 0 larva hyalomma 279 0 279 0 0 0 7 70 32 nymph hyalomma 104 0 0 87 17 0 0 65 4 he.sulcata haemaphysalis 31 0 0 18 13 0 0 14 4 he.numidiana (erinacei) haemaphysalis 23 0 0 23 0 0 0 19 0 he.concinna haemaphysalis 76 0 0 54 22 3 4 21 8 r.sanguineus rhipicephalus 20 0 0 10 10 0 0 6 8 r.bursa rhipicephalus 169 0 0 105 64 0 2 76 42 marginatus dermasentor 520 0 434 65 21 63 0 42 0 o.lahorensis ornithodoros argasidae 7 0 0 3 4 2 0 2 4 a.reflexus argas 42 0 29 13 0 7 0 0 0 a.persicus argas 1912 18 744 705 445 75 29 510 224 table 2. biodiversity and species richness of ticks captured in the studied areas of sarab county, based on seasonal activity, 2018–2019 spring summer fall winter shannon-wiener index 2.1267 2.1709 0.7934 0.781 simpson's index)d( 0.1449 0.1378 0.6451 0.6385 simpson's index(d-1) 0.8551 0.8622 0.3549 0.3615 evenness index 0.9236161 0.942812 0.4428048 0.485265 margalef diversity index 1.3702635 1.423474 0.8287641 0.7379352 table 3. diversity and species richness of ticks caught separately by the host in the study areas in sarab county, 2018–2019 cow and oxen goat and sheep donkey and horse stables shannon-wiener index 1.905 2.333 1.399 0.447 simpson's index)d( 0.198 0.119 0.285 0.787 simpson's index(d-1) 0.802 0.881 0.715 0.213 evennes index 0.7945 0.9096 0.8693 0.3224 margalef diversity index 1.807 1.708 1.077 0.483 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 183 http://jad.tums.ac.ir published online: june 30, 2021 table 4. the results of hot-spot and autocorrelation analysis of tick species in sarab county, east azerbaijan, northwest of iran, 2018–2019 general g summary global moran's i summary species observed general g expected general g variance z-score p all species 0.000015 0.000016 0.000000 -0.357036 0.721065 hyalomma marginatum 0.000014 0.000016 0.000000 -1.464457 0.143069 hyalomma antalicum 0.000013 0.000016 0.000000 -0.911540 0.362011 fig. 1. map of studay area, sarab county, east azerbaijan province, iran fig. 2. general distribution of ticks caught in the study areas of sarab county, 2018–2019 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 184 http://jad.tums.ac.ir published online: june 30, 2021 fig. 3. results of scattering pattern analysis of ticks caught in sarab county, east azerbaijan, northwest of iran, 2018– 2019 discussion in this study, 2500 different livestock were studied and 30.5% of livestock and 25% of stalls and stables were infested with different ticks. this is one of the first studies to include more samples and tick populations in iran. in related surveys, the infestation rate of ticks was 11%, 9.37%, 43% and 24% in northwest, west and north iran (14-16). the highest frequency of captured ticks was related to spring (37.24%) and the lowest frequency was related to winter (11.83%) and this indicates the seasonal behavior of ticks in the mountainous and northwestern regions of iran, mainly in spring, which can be attributed to the nasiri study in abdanan county, ilam province (17). nonetheless, more seasonal tick activity was recorded in summer and autumn in areas such as golestan province northern iran, which have ecologically different climates and have lower altitudes and higher temperatures than the mountainous areas (18). however, more seasonal tick activity was recorded in summer and autumn in areas such as golestan province northern iran, which have ecologically different climates and have lower altitudes and higher temperatures than the mountainous areas (18). in the study area, the tick biodiversity, according to the shannon-wiener index, was found to be moderate to high (2.432) and the biodiversity index of simpson (d-1) was also determined to be 0.856. the dominant species in the area therefore had a great diversity. we found the highest richness of ticks captured from cows and oxen and the lowest from horses and donkeys, according to the margalef species richness index. the findings of this research did not confirm previous studies in the province of golestan (18) that found more sheep species http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 179–186 r moradi et al.: diversity of … 185 http://jad.tums.ac.ir published online: june 30, 2021 compared to cows. the diversity of the species was highest in autumn, and lowest in winter. the findings of this research were consistent with study for seasonal ticks activities in northwestern iran (19). six genera and 14 species of hard and soft ticks were collected in this research, of which the dominant genus was hyalomma (48.12%). the findings of this research revealed that the region was prone to diseases like cchf. according to the study in 2004 (unpublished data), in the eastern province of azarbaijan, northwestern iran, the hyalomma genus was confirmed to have the highest abundance (52.81%) which was consistent with recent study (unpublished data). interestingly, in a survey conducted in west azerbaijan province, 7 tick species with more abundant in the hyalomma genus (unpublished data) were reported to have the highest abundance and distribution, as our recent research did. based on the findings of this study and the above research, it can be inferred that the provinces located in northwestern iran including east azerbaijan, west azerbaijan and ardabil had a high abundance of hyalomma species and were ecologically suitable habitat for this tick. identifying high-risk and low-risk disease and vector areas allows to make appropriate decisions on the management of the vectors and diseases transmitted by them, and to arrange for the control and preventive management of the region. throughout this research, the spreading of ticks as the vectors was found randomly around the region, but internal analysis revealed that the high-risk area of ticks was very high in the west of the study area as well as being a large hotspot. the high population of livestock and livestock occupation which have been the key hosts of ticks were some of the factors involved in this region. therefore, in terms of tick-borne diseases, this area is indeed one of the high-risk areas and control measures should be taken to prevent bites of ticks and diseases that they transmit. conclusion the main method of application of pesticides against ticks are dipping method, pour-on and sometime oral administration by systemic acaricides in iran as well as in sarab county, eastern azerbaijan province. the findings of the recent study indicate that the number of tick species caught in this region was varied due to the large abundance of livestock in sarab county and the wealth of animal and livestock husbandry practices and the production of dairy products. because of the small scale of the county it reveals the richness of different species of ticks in this region. in the study of tick diversity in the research region among different hosts, it was observed that all the indices observed on sheep and goats have a comparatively higher value that the ticks were more associated with long hair hosts, so periodically picking the wool and hair of these hosts may be a way to reduce tick activity. throughout this research, the spreading of ticks as the vectors was found randomly around the region, but internal analysis revealed that the high-risk area of ticks was very high in the west of the study area as well as being a large hotspot. acknowledgements this study was financially supported by the national institute for medical research development (nimad) grant no.983031 and tehran university of medical sciences, grant no. 40919. the authors declare that there is no conflict of interest. references 1. lane rp, crosskey rw (1993) medical insects and arachnids. chapman and hal, uk, p. 723. 2. goodman jl, dennis dt, sonenshine de (2005) tick-borne diseases of humans: asm press, dc. isbn: 1-55581-23-4. washington, p. 440. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 179–186 r 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[in persian] 17. nasiri a, telmadarraiy z, vatandoost h, chinikar s, moradi m, oshaghi m (2010) tick infestation rate of sheep and their distribution in abdanan county, ilam province, iran, 2007–2008. iran j arthropod borne dis. 4(2): 56–60. 18. sarani m, telmadarraiy z, moghaddam as, azam k, sedaghat mm (2014) distribution of ticks (acari: ixodidae) infesting domestic ruminants in mountainous areas of golestan province, iran. asian pac j trop biomed. 4(suppl 1): s246–251. 19. vatandoost h, moradi-asl e, telmadarreiy z, mohebali m, masoumi-asl h, abai mr, zarei z (2012) field efficacy of flumethrin pour-on against livestock ticks in iran. int j acarol. 38(6): 457–464. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 268 http://jad.tums.ac.ir published online: september 30, 2019 original article effect of meteorological factors on hyalomma species composition and their host preference, seasonal prevalence and infection status to crimean-congo haemorrhagic fever in iran nayyereh choubdar1; *mohammad ali oshaghi1; javad rafinejad1; mohammad reza pourmand2; naseh maleki-ravasan3; mostafa salehi-vaziri4; zakkyeh telmadarraiy1; fateh karimian1; mona koosha1; abbas rahimi-foroushani5; safdar masoomi5; kourosh arzamani6; jalil nejati7; mohsen karami8; ehsan mozaffari1; yaser salim-abadi9; eslam moradi-asl10; behrooz taghilou11; manouchehr shirani12 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of pathobiology, school of public health, tehran university of medical sciences, tehran, iran 3department of parasitology, pasteur institute of iran, tehran, iran 4department of arboviruses and viral hemorrhagic fevers, pasteur institute of iran, tehran, iran 5department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran 6vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 7department of public health, school of public health, zahedan unversity of medical sciences, zahedan, iran 8department of parasitology, faculty of medicine, babol university of medical sciences, babol, iran 9department of health services and health promotion, school of public health, rafsanjan university of medical sciences, rafsanjan, iran 10department of public health, school of public health, ardabil university of medical sciences, ardabil, iran 11zanjan health center, zanjan university of medical sciences, zanjan, iran 12mamasani health center, shiraz university of medical sciences, shiraz, iran (received 26 jan 2019; accepted 30 apr 2019) abstract background: the impact of environmental factors and host on hyalomma spp. community structure and abundance in the main crimean-congo haemorrhagic fever (cchf) foci of iran is largely unknown. methods: biotic and abiotic factors, including host, temperature, humidity, altitude, köppen-geiger climate types, season, and precipitation on hyalomma spp. community structure and abundances in 11 provinces of iran were investigated. additionally, the possible infection of ticks with cchf virus was evaluated using reverse transcription pcr technique. results: species analyses demonstrated the presence of hyalomma anatolicum, h. marginatum, h. dromedarii, h. asiaticum, h. detritum and h. schulzei in the study area. hyalomma anatolicum was the dominant species in the southern and northern parts, whereas h. dromedarii was distributed mostly in central parts of the country. the highest tick infestation was recognized in hot season. spatial variation in tick relative density was observed between habitat types where more ticks were collected in deserts, semi-deserts, and mediterranean habitats. except for h. dromedarii, which was more prevalent on camel (p= 0.044), there were no significant variations in the frequencies of other hyalomma species on different hosts. hyalomma anatolicum, h. dromedarii frequencies had significant positive and negative association with temperature and precipitation respectively. also humidity has positive impact on h. asiaticum frequency. conclusion: data presented here will help improve ecological models to forecast the distribution of hyalomma spp. ticks, to evaluate the risk of cchf and other tick-borne diseases, and to design proper vector control measures to suppress hyalomma populations in iran. keywords: hyalomma; ticks; climate; host; environment introduction ticks are among the major vectors of pathogens for animals and humans in the world. they can play a crucial role in the transmission of a wider range of pathogens including *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=choubdar%20n%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=oshaghi%20ma%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=maleki-ravasan%20n%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=karimian%20f%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=koosha%20m%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=arzamani%20k%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=moradi-asl%20e%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://www.ncbi.nlm.nih.gov/pubmed/?term=shirani%20m%5bauthor%5d&cauthor=true&cauthor_uid=29745300 https://scholar.google.com/citations?view_op=view_org&hl=en&org=16806557757143553874 j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 269 http://jad.tums.ac.ir published online: september 30, 2019 protozoans, viruses and bacteria than other arthropod vectors (1). the main public health problem and the largest geographical distribution related to ticks is crimean-congo haemorrhagic fever (cchf), a viral hemorrhagic fever, for which ticks serve as reservoirs of the virus. humans are infected either through tick bites or due to direct contact with infected blood and tissues of a mammalian host (2). infected domestic animals are known to act as amplifying hosts in the absence of any clinical symptoms (3). the main mode of cchfv transmission to humans in iran is exposure to the blood or viscera of infected livestock. moreover, direct exposure to ticks (tick bites) accounts for noteworthy transmission in humans (4-7). cchf's case mortality rate ranging from 5–80% (8, 9). cchf virus is endemic to africa, the balkans and ukraine, the middle east and central asia (10). it is prevalent in most of iran’s neighboring countries, including pakistan and afghanistan (11). iran is known as one of the main foci of cchf in western asia where the first outbreak of the disease was reported in 1999 and has since become the main public health concern in the country (5, 12-14). cchf virus exists in an enzootic cycle between ticks and mammals, and geographic distribution of the virus mirrors the distribution of the primary tick vector species (15). in addition, other factors such as environment, climate, geographical features, socioeconomic parameters, grazing system, and livestock’s age can affect the distribution and transmission of the disease (16-18). seasonal pattern, temperature, relative humidity, and lower altitude positively affect the occurrence of the disease (15). moreover, the main foci of this disease in humans were in the eastern, northeastern, and central regions of iran (5, 12-14). currently, cchf is prevalent in 23 provinces (ps) of iran, particularly the ones having a long border with three high-risk countries, turkey, afghanistan and pakistan (9, 11, 18, 19). since 2000, the most cchf cases have been reported from sistan and baluchistan province in southeast corner of iran where hyalomma spp. ticks are present and cchfv is endemic (4, 15, 20). the cchfv has been isolated from 30 species of hard ticks; however, the main group of vectors appears to be ticks of the genus hyalomma in most parts of the world (21). hyalomma marginatum koch, 1844, and h. asiaticum schulze and schlottke, 1930, are the main cchfv vector in europe and asia respectively (9, 21). in iran, the virus has been isolated from several species of hard ticks (ixodidae) includes; hyalomma, rhipicephalus, haemaphysalis, and dermacentor genera. the most frequent species of this genus reported in iran include; h. marginatum, h. asiaticum, h. anatolicum koch, 1844, h. detritum schulze, 1919, h. dromedarii koch, 1844, h. rufipes koch, 1844 and h. schulzei olenev, 1931 (22-24). climate may play an essential role in the distribution and seasonal abundance of ticks (25, 26). iran is composed of nine climate types out of 31 possible köppen-geiger climate types (27). most parts of central, eastern and southern iran are characterized by köppen bwh (hot desert climate) and bwk (cold desert climate) climate types. the coastal areas of the caspian sea and most parts of mountainous areas of zagros and alborz in the west and north of iran have moderate climate type csa (temperate with hot and dry summer=hot summer climate). however, the eastern slope of zagros and southern slope of alborz connected to the central arid and semi-arid climate of central iran are distinguished with bsk climate (cold semi-arid climate= cold steppe). the southern parts of zagros region are mostly dominated by bsh climate (hot semi-arid). dsa (hot-summer mediterranean) and dsb (warm-summer mediterranean) climate types are found in some parts of mountainous areas of zagros and alborz, while csb (warm-summer mediterranean) and cfa (humid subtropical) are the localized climate types found in coastal areas of the caspian sea. nevertheless, very few studies on hyalomma spp. community have incorporated biotic and abiotic environmental factors such http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 270 http://jad.tums.ac.ir published online: september 30, 2019 as host, season, altitude, latitude, temperature, humidity, precipitation, and climate features in iran. in this study, we have tried to determine hyalomma species community structure on livestock in different regions of iran to assess their spatial and temporal distribution, and to test overall impact of the environmental factors on hyalomma species density and diversity, and possible association with cchf distribution in the country. we also tested their infection to cchfv to evaluate the abundance of potential cchfv vectors in the region. this would provide data on the relative prevalence of hyalomma spp., which might facilitate the control of ticks and tick-borne diseases in the study areas. materials and methods study area iran lies between latitudes 24° and 40° n and longitudes 44° and 64° e and locates in eurasia and western asia. it is bordered to the northwest by armenia and the republic of azerbaijan; to the north by the caspian sea; to the northeast by turkmenistan; to the east by afghanistan and pakistan; to the south by the persian gulf and oman sea, and to the west by turkey and iraq (fig. 1). iran is considered as an area with wide ranges of altitude (below sea-level in shores of the caspian sea to 5,770m of the damavand mountain), climate (humid and nearly jungle-like forests in the north to arid places in dasht-e lut with less than 100 mm annual rainfall) and temperature (from -35 °c in the northwest to 70 °c in the deserts of dasht-e lut). tick specimens were collected from eleven ps in iran as follows: north khorasan, east azerbaijan, ardabil (pars abad), kerman and fars with bsk climate, yazd, sistan and baluchistan with bwh climate, isfahan of bwk climate, illam, zanjan, and golestan of csa climate and meshkin shahr of ardabil of csb climate (fig. 1, table 1). tick collection samplings were carried out in both hot and cold seasons in 2016 and 2017. since infected livestock is the main mode of cchfv transmission to humans in iran, ticks were collected from livestock during three to five visits in per season from eight farms/stables in each district. thirteen districts were tested for the presence of ticks (table 1). the cattle were randomly selected from farms/stables (nomadic grazing system) and whole body of 20 animals (cow, sheep, goat and or camel, based on availability) were carefully examined, just after whipping, the ticks were manually collected. ticks were carefully removed from animals by forceps. details of sample collection including location, type of animal, and date were recorded. ticks were stored in tubes and covered on top with small piece of muslin cloth and carefully labeled at the collection site. all of the collected specimens were identified to species level by using a stereomicroscope (olympus sz51-japanese) and a taxonomic key (28). environmental factors environmental data were obtained from various data providers (table 1). elevation (altitude) and latitude/longitude data were supplied by www.gps-coordinates.org. mean annual precipitation, relative humidity, temperature, and climate data were obtained from http://www.irimo.ir. the human cchf cases were provided by ministry of health and medical education of iran. rna extraction and rt-pcr alive tick specimens were separately washed with phosphate buffered saline (pbs) and crushed with pestle in 200–300µl of pbs. total rna was extracted from each sample homogenate using the qiaamp rna mini kit (qiagen, germany) according to the manufacturer's instructions. the extracted rna was dissolved in 50µl of rnase-free water and stored at -70 ºc until analysis. reverse transcription http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/eurasia http://www.gps-coordinates.org/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 271 http://jad.tums.ac.ir published online: september 30, 2019 of rna was performed individually using onestep rt-pcr kit and specific primers f2 (5ʹ tggacaccttcacaaactc -3ʹ) and r3 (5ʹ gacaaattccctgcacca -3ʹ) (29), which amplify a 536bp fragment of the s segment of the cchf viral genome. the total cchfv genome, extracted from a previously confirmed rt-pcr-positive serum was used as the positive control, and no template control (ntc) was used as the negative control in the rt-pcr tests. for gel-based analysis, 5µl of the pcr products were mixed with 1µl loading buffer and electrophoresis was performed on 2% agarose gel and visualized on a uv transilluminator. statistical analysis the numbers of ticks per season in each district were counted. data for each individual hyalomma tick were used to determine its species, and its association with factors such as season, host, and location. associations between each specific tick species and categorical parameters were analyzed using χ2 test. because the data were not normally distributed, mann-whitney and kruskal-wallis tests were used to assess the statistical significance of the variables. spearman’s correlation coefficient and univariate and multivariate regression analysis were used to find possible associations between tick density and environmental factors using spss software (ver. 24, chicago, il, usa). for statistical data analysis, we used poisson regression analysis to test if there is an association between climate factors such as humidity, precipitation, altitude, and temperature as predictors and count of ticks and dependent variable. differences in tick species proportions were considered as statistically significant if p< 0.05. all graphs and statistics were performed using graphpad prism ver. 5.00 for windows (graphpad software). results tick composition overall, 3929 hard ticks were collected from the study areas including; 76 nymphs, 451 nonhyalomma, and 3402 hyalomma ticks. the nonhyalomma ticks comprised specimens of rhipicephalus (n= 274, 54.76%), dermacentor (n= 168, 37.26%), haemaphysalis (n= 11, 2.44%), boophilus (n= 17, 3.77%), and ixodes (n=8, 1.77 %) genera. two season investigations resulted in collection of hard ticks of the genus hyalomma from different infested cattle in the study area. details of the collected hyalomma spp. tick specimens are shown in table 2. from mar 2016 to dec 2017, a total of 1536 cattle from 128 farms from 13 districts were inspected and 3402 hyalomma spp. ticks were collected. more than half (53.8%) of specimens were females. the cattle infestation level ranged from zero to 20 hyalomma spp. ticks per animal. the number of hyalomma spp. ticks in hot season were higher than cold season (58.3 vs. 41.7 %), however, the distribution of hyalomma spp. was the same for two seasons (mann˗whitney u= 57.5, p= 0.1091). hyalomma spp tick infestation was observed in all study areas of iran. six species of genus hyalomma includes; h. anatolicum, h. marginatum, h. dromedarii, h. asiaticum, h. detritum, and h. schulzei were found to infest the animals in the study area. hyalomma anatolicum and h. detritum with 31.9% and 1.59% were the most and the least prevalence species in the study area. the distribution of the six tick species among the provinces were not similar (kruskal˗wallis test: p= 0.006, kruskal˗wallis static: 16.33). sistan and baluchistan with six and yazd and fars with one hyalomma species showed the most and the least diversity among eleven p’s (table 1). except for h. detritum found only in hot season, all the other hyalomma species were collected throughout the year (table 1). when the distributions of six species were compared in pairs, results showed that distribution of h. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 272 http://jad.tums.ac.ir published online: september 30, 2019 detritum and h. schulzei in the collection sites were significantly different (mann˗whitney, u= 18.5 and 28, p= 0.0042 and 0.0247 respectively). tick species found in different host species data were analyzed for the suitability and the specificity of the tick species to the hosts from which they were collected. statistical analysis showed that except for h. dromedarii, which was significantly more prevalent on camel (p= 0.044), there were no significant variations in the frequencies of other hyalomma spp on different hosts (fig. 2). hyalomma anatolicum the major tick species were found on camel, cow, sheep and goat but the number of ticks were not significantly different between the hosts (χ2= 4.623, df= 3, p= 0.202). hyalomma dromedarii being the second most prevalent species on the hosts was significantly more abundant on camel (χ2= 8.104, df= 3, p= 0.044). the frequencies of h. marginatum and h. asiaticum ticks were not significantly varied (h. marginatum: χ2 = 2.008, df= 3, p= 0.544, h. asiaticum: χ2= 3.209, df= 3, p= 0.361) between the hosts, although both species were more established on cow. hyalomma schulzei and h. detritum were rarely found and were almost exclusive on cow and cow/camel respectively. impact of latitude/longitude on hyalomma distribution to assess the impact of geographical location on hyalomma species occurrence and community, the country was divided into three divisions of south (5 districts), center (2 districts), and north (6 districts). this analysis showed however, the differences were not significant (p> 0.05) among different parts of the country (fig. 3). impact of season on hyalomma species abundance and community seasonal analysis of tick abundances showed that seasonality positively affects hyalomma spp tick abundances in the study area. tick abundances in hot season were 1.4 times of cold season. when hyalomma species were analyzed individually, tick abundances’ for all species were higher in hot season than cold season, however, differences were significant only for three species of h. dromedarii, h. marginatum and h. detritum (p< 0.05) (fig. 4). except for one location, hyalomma species communities were higher in hot season than cold season (table 1). impact of temperature on hyalomma species abundance and community analysis of data showed a positive association between the annual mean temperature and total tick abundances (fig. 5). when hyalomma species were analyzed individually, tick abundances’ were significantly higher for two species of h. dromedarii (p= 0.03) and h. anatolicum (p= 0.018) (table 3). impact of altitude on hyalomma species abundance and community analysis of the data revealed a weak negative association between the altitude (meter above sea level) and tick abundances (fig. 5). chah˗bahar district in southeastern part of the country with the lowest altitude comprised the highest hyalomma spp tick abundance and species community (table 1). when hyalomma species were analyzed individually, tick abundances’ differences were not significant (p= 0.647–0.947) for the species studied (table 3). impact of relative humidity on hyalomma species distribution and community analysis of data showed there is a general positive association between the annual mean relative humidity (rh) and either the tick abundances (fig. 5) or the tick species community. however, when each hyalomma species were analyzed, tick abundances’ differences were not significant (p= 0.143–0.968) for the species studied (table 3) suggesting no or, at best, a very weakly significant association between rh and hyalomma spp present. impact of precipitation on hyalomma species abundance and community on general, analysis of data showed there is http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 273 http://jad.tums.ac.ir published online: september 30, 2019 a negative association between the annual mean precipitation and either the tick abundances (fig. 5) or the tick species community. reduction in precipitation is favored to hyalomma spp tick abundance and community. however, when individual hyalomma species were analyzed, tick abundances’ differences were significant only for h. dromedarii (p< 0.001) and h. anatolicum (p= 0.05) (table 3). impact of climate type on hyalomma species abundance and community analysis of data revealed that 46% of hyalomma spp. tick specimens were collected from desert climate (bwh) in yazd and sistan and baluchistan. total tick abundance rates were significantly much lower in other climate types of the country including warm-summer mediterranean climate (csb) in meshkin shahr of ardabil, hot-summer mediterranean climate (csa) in illam, zanjan, and golestan, cold desert climate (bwk) in isfahan, cold semi-arid climate (bsk) in north khorasan, east azerbaijan, ardabil (pars abad), kerman and fars (fig. 6). except for csb climate with two hyalomma species, other climate norms had similar number (5) of hyalomma species. statistical analysis showed that abundances’ frequency of four more prevalent hyalomma species was not significantly different in various climate types (p= 0.392). cchfv infection rt-pcr was used to detect the cchf viral infection in less than 10% (390 out of 3929 ticks) of the hyalomma spp ticks collected in the study areas. among this sample (390), including all of the six hyalomma species, no cchfv positive sample was found. table 1. details of meteorological variables in the study areas province district climate norm coordinates aasl (m) map (mm) mat (ºc) marh (%) ardabil meshgin shahr csb 38°24 ʹ 50.8ʹʹ n 47°38ʹ 27.3ʹʹ e 1338 356 9.7 63 pars-abad bsk 39°26 ʹ 44.2 ʹʹn 47°25ʹ 07.1ʹʹ e 32 382 12.1 63 east azerbaijan khodaafarin bsk 39°08’26.5”n 46°57’27.5”e 1144 403 13.6 82 fars marvdasht bsk 30°01’19.7”n 52°38’22.9”e 1620 263 16.7 47 ilam pardeh csa 33°46’48.2”n 46°04’26.9”e 1378 442 16.2 35 golestan gonbad-e kavus csa 37°15’50.7”n 55°12’18.5”e 52 363 18.6 93 isfahan nain bwk 32°50’38.1”n 53°04’29.2”e 1571 76 16.6 50 kerman rayen bsk 29°35’00.8”n 57°28’01.2”e 1756 176 14.2 60 north khorasan raz and jargaland bsk 37°56’32.4”n 57°06’16.3”e 1086 262 11.5 42 sistan and baluchestan chabahar bwh 26°14’27.5”n 61°24’10.0”e 7 106 25.8 70 sarbaz bwh 26°37’35.5”n 61°15’42.9”e 915 134 23.5 70 yazd mehriz bwh 31°35’33.7”n 54°30’25.3”e 1230 55 18.9 32 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 274 http://jad.tums.ac.ir published online: september 30, 2019 zanjan ijrud csa 36°06’00.2”n 48°35’54.4”e 2050 356 10.2 53 aasl: altitude above sea level, map: mean annual precipitation, mat: mean annual temperature, marh: mean annual relative humidity, bwh: hot desert climate, bwk: cold desert climate, csa: hot summer mediterranean climate, csb: warm-summer mediterranean climate, bsk: cold semi-arid climate table 2. details of the collected ticks from 11 provinces of iran cold season hot se hot season district, province (climate) species f m total host species f m total host chabahar, sistan and baluchestan (bwh) h. anatolicum h. asiaticum h. marginatum 182 23 31 11 19 46 312 goat cow h. asiaticum h. dromedarii h. detritum h. marginatum h. anatolicum 45 0 0 7 11 115 150 10 70 76 484 goat cow camel sarbaz, sistan and baluchestan (bwh) h. anatolicum h. marginatum h. dromedarii h. schulzei 32 18 46 10 16 13 52 7 194 goat cow h. dromedarii h. detritum h. marginatum h. asiaticum h. anatolicum 76 0 34 16 132 80 4 14 8 43 407 goat cow camel rayen, kerman (bsk) h. dromedarii h. asiaticum 32 0 9 31 72 camel h. marginatum h. anatolicum h. dromedarii 44 13 0 0 0 12 69 camel marvdasht, fars (bsk) h. marginatum 8 1 9 goat sheep h. marginatum 51 8 59 goat pardeh, illam (csa) h. anatolicum h. dromedarii h. asiaticum h. schulzei 39 8 8 0 0 4 0 6 65 cow h. anatolicum h. dromedarii h. asiaticum h. schulzei 54 13 11 0 0 6 0 7 91 goat sheep mehriz, yazd (bwh) h. dromedarii 47 12 0 69 goat camel h. dromedarii 103 0 103 camel nain, isfahan (bwk) h. dromedarii 48 8 56 camel h. dromedarii h. detritum h. marginatum h. asiaticum h. anatolicum 62 0 0 14 31 53 13 11 40 47 271 camel cow ijrud, zanjan (csa) h. asiaticum h. anatolicum h. dromedarii 102 0 3 53 7 0 165 cow sheep h. anatolicum h. detritum h. marginatum 69 0 22 31 27 51 200 cow khoda-afarin, east azerbaijan (bsk) h. anatolicum h. asiaticum h. dromedarii 24 19 2 7 7 3 62 cow goat h. anatolicum h. asiaticum h. dromedarii 98 74 7 25 27 10 241 cow meshgin-shahr, ardabil (csb) h. marginatum h. asiaticum 15 0 42 9 66 cow h. marginatum h. asiaticum 24 0 65 7 96 cow sheep pars abad, ardabil(bsk) h. marginatum h. anatolicum 24 0 17 19 60 cow goat h. marginatum h. anatolicum 15 0 11 13 39 cow gonbad-e kavus, golestan(csa) h. dromedarii h. marginatum 0 0 7 5 12 camel h. anatolicum h. dromedarii h. marginatum 9 0 0 6 7 4 26 sheep raz and jargaland, north khorasan (bsk) h. anatolicum h. dromedarii 46 0 23 9 78 sheep cow h. schulzei h. anatolicum h. dromedarii h. marginatum 28 13 5 0 7 9 0 26 96 sheep goat table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 275 http://jad.tums.ac.ir published online: september 30, 2019 h. asiaticum 0 8 total 911 509 1220 959 1023 2128 f: female, m: male, bwh: hot desert climate, bwk: cold desert climate, csa: hot summer mediterranean climate, csb: warm-summer mediterranean climate, bsk: cold semi-arid climate table 3. univariate regression analysis between environmental parameters and abundance of each tick species in iran. bold-red represents significant correlations species climate parameter r square sig parameter constant estimates b1 h. anatolicum humidity 0.082 0.222 11.125 0.772 temperature 0.274 0.018 -20.157 4.671 precipitation 0.197 0.050 100.784 -0.157 altitude 0.011 0.667 63.813 -0.007 h. asiaticum humidity 0.122 0.143 -16.804 0.845 temperature 0.015 0.619 21.216 0.727 precipitation 0.000 0.999 33.496 -9.155e-5 altitude 0.013 0.647 40.882 -0.006 h. dromedarii humidity 0.012 0.641 57.447 -0.259 temperature 0.225 0.030 -33.177 4.264 precipitation 0.549 < 0.001 102.277 -0.253 altitude 0.000 0.942 40.866 0.001 h. marginatum humidity 0.000 0.968 33.180 -0.015 temperature 0.003 0.801 36.294 -0.240 precipitation 0.010 0.667 27.467 0.020 altitude 0.001 0.916 31.504 0.001 fig. 1. map of iran, tick collection sites, and climate norms of the study area. 1–2: chabahar and sarbaz, of sistan and baluchestan, 3: raven of kerman, 4: marvdasht of fars, 5: pardeh of ilam, 6: mehriz of yazd, 7: nain of isfahan, 8: ijrud of zanjan, 9: khoda-afarin of east azerbaijan, 10–11: meshgin shahr and pars-abad of ardabil, 12: gonbad-ekavus of golestan, and 13: raz and jargaland of north khorasan table 2. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 276 http://jad.tums.ac.ir published online: september 30, 2019 fig. 2. tick species and their hosts from which they were collected. h. dromedarii was significantly more prevalent on camel (p= 0.044), but there were no significant variations in the frequencies of other hyalomma spp on different hosts fig. 3. hyalomma abundance (a) and species numbers (b) among northern (n= 6), central (n= 2), and southern (n= 5) districts of iran, 2016–2017. bars indicate mean ±sem http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 277 http://jad.tums.ac.ir published online: september 30, 2019 fig. 4. hyalomma species abundances in hot and cold seasons in 13 districts of 11 provinces of iran, 2016–2017. bars indicate mean ±sem, (p< 0.05). tick abundances were significantly different in hot season for three species of h. dromedarii, h. marginatum and h. detritum (p< 0.05) fig. 5. association between environmental factors and hyalomma abundances in iran. red squares refer to significant relationship between environmental factors and the tick species. mat: mean annual temperature, marh: mean annual relative humidity, map: mean annual precipitation, alt: altitude above sea level http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 278 http://jad.tums.ac.ir published online: september 30, 2019 fig. 6. relative abundance of hyalomma ticks in five different climate norms of iran. bsk: north khorasan, east azerbaijan, ardabil (pars abad region), kerman and fars, bwh: yazd and sistan and baluchistan, bwk: isfahan, csa: illam, zanjan, and golestan, and csb: ardabil p (meshkin shahr region) discussion this study is one of the first that documents the spatial-temporal as well as impact of environmental factors on abundance and community of hyalomma spp. ticks in eleven provinces of iran that include hot spots of cchfv infection. the present study revealed the presence of six species of genus hyalomma in the study area. h. anatolicum was the dominant species in the southern and northern parts while h. dromedarii distributed mostly in the central parts of the country. the genus of hyalomma has known the most important tick species associated with livestock in iran (22). due to the importance of ticks as vector, several studies on the distribution and fauna of ticks have been performed in the country. findings of most previous studies (30-36) are in accordance with the tick fauna and distribution reported in the present study. in the present study we have found no specimen of h. rufipes, h. aegyptium, h. excavatum, h. impeltatum which may be reflections of the sample collection method used, time and place of sampling, and ecological changes occurred in the study areas during decades. our results pointed out that hyalomma spp. abundance in hot season was higher than cold season. this observation may be due to other or a combination of factors: greater tick activity under higher temperatures in summer, since adult hyalomma spp. ticks are highly motile in seeking out vertebrate hosts (questing); human hosts are more likely to spend time outdoors during summer (longer daylight hours); increased agriculture and natural grazing for livestock during summer months leading to increased opportunity for bringing humans into contact with ticks. these factors may explain the higher incidence of the disease in the summertime in the country as stated by other researchers (12). these authors analyzed monthly cchf passive data for 15 years (from 2000 to 2014) at the county level and showed that almost 70 % cases occurred in hot seasons (may–sep) peaked in june with more than 180 patients out http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 279 http://jad.tums.ac.ir published online: september 30, 2019 of 1027 cases (17.5%). our results also pointed out that temperature influences generally on hyalomma spp. tick abundance where this impact was significant for h. dromedarii and h. anatolicum, the two most prevalent species in the study areas. increasing tick vector populations directly increase the risk of disease transmission to human and hence tick abundance followed by rising temperature can be considered as an important predictor for cchf incidence. this finding is in agreement with the epidemiological evidence indicating a significant positive correlation with cchf disease and temperature in iran (12, 13, 37) and other countries such as turkey, bulgaria (38, 39). in the present study, we found a positive but not or weakly significant association between tick abundance and the annual mean relative humidity. this weather variable had significant positive impact on the incidence of cchf in iran (13, 40). our results also indicated that average annual precipitations affect negatively tick abundances of the two most prevalent species h. dromedarii and h. anatolicum in the study areas. these variables had reverse impact on the incidence of cchf in iran (14, 37, 40) and senegal (41) higher rainfall provide unfavorable conditions for hyalomma spp. tick activity and development (42). results of this study also pointed out that climate norm may have impact on hyalomma genus tick abundance in the study areas of iran. although frequencies of each species in various climate types were not significantly different, the most relative prevalence (47%) of the hyalomma spp. ticks were observed in hot desert climate (bwh) comprising yazd and sistan and baluchistan in southeastern and south-center of the country. the combination of economic and ecological situation, host availability, and some climate variables including duration of hot season and higher annual average temperature may support hyalomma genus tick abundance and species diversity in these regions. in sistan and baluchistan, livestock is moved across borders and ticks could originate from distant regions. as a matter of fact, thousands of livestock (sheep, goat, and camels) are annually imported from afghanistan and pakistan to this province, where adult ticks carried on imported livestock from the neighboring countries is the main introductory route of cchfv into the province (37, 43). in addition to imported livestock, migratory birds can also play an important role in hyalomma spp. tick abundance and species diversity in these regions. we should consider spread of cchfv by ticks attached to migratory birds flying thousands of kilometers during their annual migration from northern to southern areas to the province. the ecosystem of oman sea in chabahar and konarak coast in south and hamoun˗e puzak and helmand river in northeast of sistan and baluchistan, are habitats of unique species of waterfowl and shore birds as well as migratory birds (44, 45). the infected nymphs of the genus hyalomma carried on migratory birds will molt on arrival into adults and then potentially infect their mammal hosts with cchfv (46-49). however, the role of migratory birds carrying infected ticks has not been studied as a cause for increased cchf in iran particularly in southeast corner of iran. it is suggested to investigate the role of the migratory birds in spreading cchfv through attached ticks. the socioeconomic, environmental, and ecological features of sistan and baluchistan caused the province is being the most cchf endemic region of the country since 2000 (4, 12, 20). we have not found cchf virus infection in the tick specimens collected in this study. one possible explanation for lack of cchf infection is that although the tick specimens were collected from the animals bred on nomadic grazing system, they are usually bred intensively, and grazing is mostly limited with the field around the stables. therefore, there are limited interactions with wildlife and their ticks, which lead to a lower tick density and diversity. therefore, it is necessary to test effect of real nomadic grazing on the tick density and diversity in the counhttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 268–283 n choubdaret al.: effect of meteorological … 280 http://jad.tums.ac.ir published online: september 30, 2019 try. rate of animal infection to the virus was much lower (4.5%) in the animals feeding in the stable grazing system than the ones feeding in the nomadic system (30%) (17). this situation may alter abundance and community of tick population as well. other possible explanation could be the lower age of animals we tested, where it was shown that rate of infection increased in older animals from 4.5% to 23.7% (17). however, the epidemiology of cchf virus in iran reviewed and rhipicephalus sanguineus, h. marginatum, h. anatolicum, h. asiaticum and h. dromedarii were the most frequent species which were positive for cchf virus (50). however, the infection rate was highly variable and ranged 0–16.7% for the tested tick species (48, 50, 51). in this study we had focused on hyalomma species and did not test the virus infection in other tick species such as rhipicephalus and haemaphysalis which also play a role in the cycle of transmission of the disease in nature in the wild cycle and in connection with humans in the domestic cycle and should be considered in cchf epidemiological studies (48, 50, 51). further studies should consider other factors influencing tick life cycle and abundance over long time, including microhabitats, nomadic (natural) system, and host density, three potentially influent factors that were not assessed in the present study. our study was performed in one year, but these climate factors should be assessed for several years. conclusion hyalomma spp. ticks are responsive to changes in their environment and may be particularly sensitive to host, season, temperature, relative humidity, and precipitation, considered as important factors of tick population dynamics. further studies assessing the impact of microclimates and host abundance in hyalomma spp. abundance and species would support our understanding of the environmental factors influencing tick prevalence and diversity. acknowledgements this work has supported by tehran university of medical sciences, iran, grant number 29005. all authors have seen and approved the manuscript and declare that they have no competing interest. references 1. de la fuente j, estrada-pena a, venzal jm, kocan km, sonenshine de (2008) overview: ticks as vectors of pathogens that cause disease in humans and animals. front biosci. 13: 6938–6946. 2. gale p, estrada‐peña a, martinez m, ulrich rg, wilson a, capelli g, phipps p, de la torre a, muñoz mj, dottori m, mioulet v (2010) the feasibility of developing a risk assessment for the impact of climate change on the emergence of crimean‐ congo haemorrhagic fever in livestock in europe: a 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20(8): 1331–1334. 50. telmadarraiy z, chinikar s, vatandoost h, faghihi f, hosseini-chegeni a (2015) vectors of crimean congo hemorrhagic fever virus in iran. j arthropod borne dis. 9(2): 137–147. 51. mohammadian m, chinikar s, telmadarraiy z, vatandoost h, oshaghi ma, hanafibojd aa, sedaghat mm, noroozi m, faghihi f, jalali t, khakifirouz s, shahhosseini n, farhadpour f (2016) molecular assay on crimean congo hemorrhagic fever virus in ticks (ixodidae) collected from kermanshah province, western iran. j arthropod borne dis. 10(3): 381–391. http://jad.tums.ac.ir/ http://wwwsciencemagorg/news/2018/02/can-iran-and-afghanistan-cooperate-bring-oasis-back-dead http://wwwsciencemagorg/news/2018/02/can-iran-and-afghanistan-cooperate-bring-oasis-back-dead http://wwwsciencemagorg/news/2018/02/can-iran-and-afghanistan-cooperate-bring-oasis-back-dead detection of deltamethrin resistance in cattle tick, rhipicephalus microplus collected in western haryana state of india http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... original article detection of deltamethrin resistance in cattle tick, rhipicephalus microplus collected in western haryana state of india snehil gupta1, surbhi2, *sachin kumar3 1department of veterinary parasitology, lala lajpat rai university of veterinary and animal sciences, hisar, india 2department of veterinary physiology and biochemistry, lala lajpat rai university of veterinary and animal sciences, hisar, india 3division of parsitology, icar-indian veterinary research institute, izatnagar, bareilly, india *corresponding author: dr sachin kumar, e-mail: drkumarens@gmail.com (received 12 apr 2020; accepted 15 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: out of 931 species of ticks, rhipicephalus microplus is the most widely studied tick species due to its pivotal role in transmission of babesiosis and anaplasmosis, resulting in huge economic loss in cattle and buffalo’s industry. chemical control using deltamethrin forms the mainstay of tick control strategy because of high potency and low toxicity. however, inadvertent use of deltamethrin has led to the development of deltamethrin resistance in field ticks methods: the engorged female ticks were collected in a plastic container covered with a cotton plug from 10 places (jarwa, barwa, khajakhera, banisi, nakipur, dhangar, badopal, shivalya dharamshala, siwani and meham) of five districts of western haryana. the larval packet test (lpt) was conducted for the characterization of resistance in field tick. results: in the present study, ticks were collected from 10 places from 5 districts of western haryana and evaluated against deltamethrin using larval packed test. the lc50 values (confidence interval) of ticks larvae against del tamethrin collected from nakipur, dhangar, barwa, badopal, shivalya dharamshala, siwani, jarwa, khajakhera, meham and banisi are 73.6 (67.2–81.9), 61.2 (61.6–98.5), 52.7 (14.4–101.0), 140.0 (86.7–448.6), 65.8 (37.1–95.2), 232.1 (201.0–304.7), 3.72 (0.20–9.87), 21.3 (12.0–31.6), 107.6 (96.8–127.6), 54.2 (43.4–58.4) ppm, respectively. the resistance factor ranges from 0.31 to 11.86, indicating variable resistance among field isolates. conclusion: data generated on deltamethrin resistance status in r. microplus from haryana, india can be used as an indicator for the management of the species in the state. keywords: acaricide resistance; rhipicephalus microplus; deltamethrin; haryana introduction ticks (acari: ixodida) are becoming increasingly relevant as a sanitary problem for human, domestic, and wild animals worldwide. ticks belong to the phylum arthropoda, class arachnida, subclass acari, superorder parasitiformes, order ixodida and superfamily ixodoidea. further superfamily ixodoidea contains the 3 families, such as ixodidae, argasidae, and nuttalliellidae. out of 931 species of ticks, there are 722 species of ixodidae which are known and classified till now (1). among the ixodidae, rhipicephalus microplus has been reported as the most economically most important and widely distributed ecto-parasites (2, 3). rhipicephalus microplus is a voracious blood feeder and plays a pivotal role in transmission of babesiosis and anaplasmosis, resulting in huge economic loss in cattle and buffalo’s industry (4, 5). to control ticks, the use of 390 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... chemical acaricides like synthetic pyrethroids, is the predominant approach implemented in the field (6). amongst synthetic pyrethroids, deltamethrin is most widely used acaricide in india because of its high potency and ease of availability under field condition. rhipicephalus microplus being a one host tick species is more prone to selection against deltamethrin due to frequent exposure or under-dosing of drugs. it leads to the emergence of deltamethrin resistance in field ticks (7, 8, 9). inadvertent use of deltamethrin and heavy tick infestation was reported from several organised cattle farms of western haryana. acaricide resistance in the ticks can be noticed and diagnosed at the farmer level based upon the persistence of ticks on animals beside the application of acaricides. however, to facilitate global monitoring of status of acaricide resistance and providing a firm basis for comparison of test results, standardized diagnostic methods are adopted. therefore, fao has recommended the use of the larval packet test (lpt) for field investigations of acaricide resistance. in the present study, status of deltamethrin resistance in five districts of western haryana was evaluated using larval packet test. materials and methods collection of ticks the present study was conducted during the period from april to august, 2019 to determine the status of deltamethrin resistance in r. microplus ticks of western haryana. adult fully engorged dropped down female ticks were collected in a clean plastic container covered with a cotton plug from ten places (jarwa, barwa, khajakhera, banisi, nakipur, dhangar, badopal, shivalya dharamshala, siwani and meham) of five districts of western haryana. ticks collected were immediately transported to the laboratory (25±2 ºc and 75% relative humidity) at the department of veterinary parasitology, college of veterinary science, lala lajpat rai university of veterinary and animal sciences for larval packet test (lpt). identification of ticks was carried out using available standard literature (10, 11). for larvae, two adult female ticks of each isolate were kept in each well cleaned and labelled glass vials, covered with muslin cloth and then, placed in desiccators at 28±1 °c and 85±5% relative humidity for 1–2 months. ticks started laying eggs by 3–4 days of incubation and continued to lay eggs for the next 8–10 days. then the adult ticks were removed and eggs were left for hatching in the vials. after that, the larvae were allowed to mature for 8–10 days. around 10–14 days old larvae were used for larval packet test. the sample collection places was represented in fig. 1. acaricides for the lpt, technical grade pure deltamethrin (sigma-aldrich, usa) was fig. 1. illustration of the sampling sites in haryana, india, 2019 fig. 1. illustration of the sampling sites in haryana, india, 2019 391 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... used to prepare 5,000 ppm stock solution by diluting in acetone. then, the different working concentrations of deltamethrin were prepared in distilled water from the stock solution and tested against larvae of r. microplus ticks. distilled water was used to treat the control packets. larval packet test (lpt) the lpt was conducted according to fao (12) guidelines. briefly, 0.6ml of the working solutions of different concentrations of deltamethrin (12.5, 25, 50, 100, 200 ppm) were used to impregnate 3.75 cm by 8.5 cm filter paper rectangles (541 whatman filter paper) and were kept for 30 minutes in the bod incubator to dry the filter paper. then the rectangles were folded in half and sealed on the sides with adhesive tapes, forming an open-ended packet for placing the ticks’ larvae. control packets were also prepared by impregnating with the distilled water only. approximately 150 larvae (10 to 14 days old) were put inside the packets using a fine brush and sealed with adhesive tape. these packets were then kept in a desiccator separately to avoid contamination, which were then placed in a bod incubator maintained at 28±1 °c and 85±5% rh. the test was performed in triplicate for each concentration of each acaricide. after 24 h of incubation, larval mortality was calculated. larvae capable of walking were considered alive, whereas those which moved their appendages but did not walk were counted as dead. statistical analysis regression analysis of lpt was done by probit method (13) using graphpad prism-5 statistical software. for calculating resistance factor (rf) of ticks, lc50 of indian veterinary research institute (ivri-i) r. microplus) against deltamethrin (11.8ppm) is used as country specific reference susceptible tick line (7). resistant factor (rf) for ticks was calculated as the coefficient of lc50 of field ticks and lc50 of reference susceptible ivri line i tick (14). resistant factor (rf) = lc50 of field ticks / lc50 of reference susceptible ivri line i ticks. on the basis of rf, the resistance status in the field ticks population were classified as susceptible (rf< 1.4), level i resistance (1.5< rf< 10.0), level ii resistance (10.1<rf<25.0), level iii resistance (25.1<rf<40), level iv resistance (rf>41) (15). results on the basis of the dose response data, the mortality slopes, lc50, lc95, 95% confidence limit, resistance factor (rf) and resistance level (rl) of field ticks were determined (table 1 and fig. 2). data generated showed table 1. mortality slope, lc50, lc95 value with 95% ci and rf values of larvae of rhipicephalus microplus against deltamethrin collected from different places of haryana, india, 2019 places district slope±se r2 lc50 (ppm) (95% ci) lc95 (ppm) (95% ci) rf # rl nakipur bhiwani 2.262±0.1285 0.993 73.6 (67.2–81.9) 383.1 (321.6–504.0) 6.23 ii barwa 3.794±0.8104 0.915 52.7 (14.4–101.0) 141.2 (103.5–475.5) 4.46 i siwani 1.436±0.1234 0.985 232.1 (201.0–304.7) 3130.0 (1610.2–7862.7) 19.66 ii dhangar fatehabad 2.115±0.806 0.801 61.2 (61.6–98.5) 284.6 (249.5–319.4) 5.18 ii badopal 2.625±0.392 0.957 140.0 (86.7–448.6) 580.7 (277.5–1088.2) 11.86 ii shivalya dharamshala 2.005±0.525 0.879 65.8 (37.1–95.2) 424.5 (286.8–476.7) 5.57 ii jarwa mahendragarh 0.970±0.0349 0.997 3.72 (0.20–9.87) 176.2 (125.0–378.2) 0.31 s khajakhera sirsa 1.809±0.1446 0.987 21.3 (12.0–31.6) 168.6 (141.0–291.0) 1.80 i meham rohtak 1.286±0.0355 0.991 107.6 (96.8–127.6) 1971.1 (1131.5–3515.8) 9.11 ii bainsi 1.823±0.2202 0.971 54.2 (43.4–58.4) 421.8 (313.7–597.2) 4.59 i #rf resistance factor; rl resistance level; s susceptible; susceptible = rf<1.4; level i= 1.5< rf< 5; level ii= 5.1< rf< 25; level iii= 26< rf< 40; level iv= rf>40.0 table 1. mortality slope, lc50, lc95 value with 95% ci and rf values of larvae of rhipicephalus microplus against deltamethrin collected from different places of haryana, india, 2019 392 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... y= 2.262x+0.726 r2= 0.993 1.0 1.5 2.0 2.5 3.0 3 4 5 6 7 log con p ro bi t m or ta lit y y= 3.794x-1.611 r2= 0.915 1.0 1.5 2.0 2.5 3.0 0 5 10 15 log con p ro bi t m or ta lit y (a) (b) y= i.436x+1.565 r2= 0.985 1.0 1.5 2.0 2.5 3.0 0 2 4 6 log con p ro bi t m or ta lit y y= 2.115x+0.610 r2= 0.801 1.0 1.5 2.0 2.5 3.0 0 2 4 6 8 10 log con p ro bi t m or ta lit y (c) (d) y= 2.625x-0.697 r2 = 0.957 1.5 2.0 2.5 3.0 -50 0 50 100 150 log con p ro bi t m or ta lit y y= 2.005x+1.315 r2= 0.879 1.0 1.5 2.0 2.5 3.0 0 2 4 6 8 log con p ro bi t m or ta lit y (e) (f) fig. 2. individual regression curve showing probit mortality in lpt against log concentration of deltamethrin in rhipicephalus microplus ticks collected from different locations of haryana, india, 2019 (a-j) (a=nakipur, b=barwa, c=siwani, d=dhangar, e=badopal, f=shivalya dharamshala, g=jarwa, h=khajakhera i=meham, j=bainsi) that the lc50 values (confidence interval) of ticks larvae against deltamethrin collected from nakipur, dhangar, barwa, badopal, shivalya dharamshala, siwani, jarwa, khajakhera, meham and banisi are 73.6 (67.2–81.9), 61.2 (61.6–98.5), 52.7 (14.4– 101.0), 140.0 (86.7–448.6), 65.8 (37.1– 95.2), 232.1 (201.0–304.7), 3.72 (0.20– 393 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... 9.87), 21.3 (12.0–31.6), 107.6 (96.8–127.6), 54.2 (43.4–58.4) ppm, respectively (table 1). however, resistance factor ranges from 0.31 to 19.66, indicating variable resistance status among the field isolates. only jarwa (mahendragarh) isolate was found susceptible, three other field isolates (barwa, khajakhera and banisi) showed low levels (level i) of resistance, whereas, six isolates (nakipur, dhangar, badopal, shivalya dharamshala, siwani and meham) showed moderate resistance (level ii) to deltamethrin. slope value ranges from 0.970±0.0349 to 3.794±0.8104 which indicates heterogeneity of field tick population characterized by a flatter curve, therefore leading too y= 0.970x+ 4.44 r2= 0.997 1.0 1.5 2.0 2.5 3.0 5.5 6.0 6.5 7.0 7.5 log con p ro bi t m or ta lit y y= 1.809x+2.572 r2=0.987 1.0 1.5 2.0 2.5 3.0 4 5 6 7 8 log con p ro bi t m or ta lit y (g) (h) y= 1.286x+2.360 r2= 0.991 1.0 1.5 2.0 2.5 3.0 4.0 4.5 5.0 5.5 6.0 log con p ro bi t m or ta lit y y= 1.823x+1.805 r2= 0.971 1.0 1.5 2.0 2.5 3.0 3 4 5 6 7 8 log con p ro bi t m or ta lit y (i) (j) fig. 2. individual regression curve showing probit mortality in lpt against log concentration of deltamethrin in rhipicephalus microplus ticks collected from different locations of haryana, india, 2019 (a-j) (a=nakipur, b=barwa, c=siwani, d=dhangar, e=badopal, f=shivalya dharamshala, g=jarwa, h=khajakhera i=meham, j=bainsi) continued fig. 2. individual regression curve showing probit mortality in lpt against log concentration of deltamethrin in rhipicephalus microplus ticks collected from different locations of haryana, india, 2019 (a-j) (a=nakipur, b=barwa, c=siwani, d=dhangar, e=badopal, f=shivalya dharamshala, g=jarwa, h=khajakhera i=meham, j=bainsi) fig. 3. the larval packet test (lpt) bioassay of deltamethrin against filed collected rhipicephalus microplus, haryana, india, january, 2019 fig. 3. the larval packet test (lpt) bioassay of deltamethrin against filed collected rhipicephalus microplus, haryana, india, january, 2019 394 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... much greater value of lc95 in comparison to lc50 values. the goodness of fit (r 2) value of the isolate ranges from 0.801 to 0.997 indicating a good fit of observed data in statistical model. in 9 out of 10 isolates, lc50 estimate were 1.80–19.66 times higher than the reference susceptible ivri-i tick line. the lpt bioassay was showed in fig. 3. discussion synthetic pyrethroids and organophos phates are most frequently used acaricides in india followed by formamidines and macrocyclic lactones (16). in most of the northern indian states, acaricidal resistance is a rampant problem due to widespread and indiscriminate usages of chemical acaricides (16, 17, 18, 7, 19). reports of deltamethrin resistance in r. microplus ticks are also available from various parts of north india (7, 17, 24-27). in a study at central india (jabalpur, madhya pradesh), deltamethrin treated animals showed re-infestation with ticks by the 14th day of treatment, which provides indication of the reduced efficacy of deltamethrin in tick control in animals and animal sheds (23). later, high resistance ratio (rf= 22.35 to 34.64) of field tick against deltamethrin was also recorded from mhow districts in madhya pradesh (28). several tick isolates from western india (gujarat) were also found resistant (rf= 2.52-22.59) against deltamethrin (29). there are scarce reports on the emergence of deltamethrin resistance from the southern part of india (30, 31). in a recent study from south india, researchers have recorded a positive correlation (r=0.66) between the tick burden on household cattle and resistance factor and suggested that the deltamethrin resistance was one of the contributing factors for persisting tick load on cattle (31). recently, deltamethrin resistance (rf=3.3-21.8) has also been recorded from the assam state of eastern india (32). earlier from different parts of punjab, low to severe (level i to iv) deltamethrin resistance has reported (20) while in the present study, using lpt only a low level (level i-ii) of resistance (rf=1.8– 19.66) was observed in ticks collected from selected organized cattle farms of western haryana. in a similar lpt-based study (21) from haryana, the level ii resistance was recorded from all isolates of fatehabad which corroborate with findings of the present manuscript. some resistance workers screened r. microplus of haryana and reported the emergence of level i (rf=3.5) deltamethrin resistance from hisar district of haryana (22). previously, there were few adult immersion tests with discriminating doses (ait-dd) based on deltamethrin resistance reports from haryana (8). in this study, larval packet test is preferred as it is considered as a more sensitive tool for detection and monitoring of acaricide resistance in ticks (15). in the present report, several organized farms of western haryana are screened for development of deltamethrin resistance in ticks using larval packet test which could be conducted with fewer numbers of ticks (12). characterization of the deltamethrin resistance in field ticks of western haryana indicated that out of 10 isolates, resistance factor of 9 isolates was higher than the reference susceptible ivri-i tick line. on comparing the resistance factor of different isolates, it could be concluded that deltamethrin resistance may not yet prevail in the mahendragarh district of haryana and still the compound is effective for the control of field ticks. other isolates have shown low to moderate (level i-ii) resistance which showed that the situation is still not grave and could be controlled by stringent alternative tick control strategies with emphasis on integrated tick management approaches. conclusion resistance monitoring is a continuous process to provide an accurate pattern of resistance in different time intervals. the present study indicated the clue of resistance pattern to synthetic acaricides that may be helpful to formulate a suitable strategy for tick management in haryana state of india. 395 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 389–396 s gupta et al.: detection of... acknowledgements the authors would like to thank lala lajpat rai university of veterinary and animal sciences university, hisar, haryana for providing 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https://pubmed.ncbi.nlm.nih.gov/?term=srivastava%2bs&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=boruah%2brr&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=nagar%2bg&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=shakya%2bm&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=nath%2bt&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=nandi%2bsp&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=s%2bk&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=s%2bk&cauthor_id=31841946 https://pubmed.ncbi.nlm.nih.gov/?term=ghosh%2bs&cauthor_id=31841946 detection of deltamethrin resistance in cattle tick, rhipicephalus microplus collected in western ha abstract keywords introduction materials and methods collection of ticks acaricides larval packet test (lpt) statistical analysis results discussion conclusion acknowledgements references j arthropod-borne dis, june 2019, 13(2): 191–197 e moradi-asl et al.: investigation on the … 191 http://jad.tums.ac.ir published online: june 24, 2019 original article investigation on the occurrence of aedes species in borderline of iran and azerbaijan for control of arboviral diseases eslam moradi-asl1; *hassan vatandoost2,3; davod adham1; daryosh emdadi4; hassan moosa-kazemi2 1department of public health, school of public health, ardabil university of medical sciences, ardabil, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4center for disease control of ardabil university and medical sciences, ardabil, iran (received 18 jul 2018; accepted 11 may 2019) abstract background: to investigate the diversity of the genus aedes present in the natural areas of ardabil province, northwest of iran. methods: this cross-sectional study was carried out from apr to oct 2016 in north-western of iran. thirty-three areas of 10 cities which are border areas were selected randomly. the larvae were collected 2 times in each month during the seasonal activities of mosquitoes and the larvae were identified morphologically according to the appropriate identification keys. results: overall, 694 larvae were collected from four counties, from which only 7.2% were aedes larvae. three species of aedes were identified which include ae. caspius, ae. vexans and ae. flavescens. aedes flavescens is reported from ardabil province for the first time. conclusion: aedes species were a high density in borderline of iran and azerbaijan. therefore, the north parts of ardabil province are a suitable habitat for aedes species mosquitoes. care should be taken for vector control in the case of occurrence of any arboviruses transmitted by aedes mosquitoes. keywords: larval habitat, aedes, borderline, iran introduction mosquito-borne arboviruses are health threat in the worldwide. for instance, more than 2.5 billion people live in high-risk areas of dengue fever (df) in the world and over 100 million people are infected with this disease (1, 2). currently, there is no vaccine and no specific treatment for df (3). recently, outbreaks of df have happened in malaysia, taiwan, and india (4). endemic df always occurred in southeast asia (5). however, some outbreaks recently have been occurred in parts of the middle east, south asian countries including pakistan (6, 7). some factors affecting the distribution of df in different parts of the world are as follows: increasing urban population density, increased travel, and unsystematically urbanization (8-10). df has been reported in 120 countries (11). the agent of df is a flaviviridae family and the main vectors are aedes aegypti and ae. albopictus (12). thus mosquitoes are an invasive species in world widespread in tropical and temperate regions of the world. the ability to lay eggs and grow in dishes cultural artifacts. in the last two decades and facilitate the movement in the world are impacts factors in distribution of this mosquitoes (13). *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com j arthropod-borne dis, june 2019, 13(2): 191–197 e moradi-asl et al.: investigation on the … 192 http://jad.tums.ac.ir published online: june 24, 2019 aedes aegypti and ae. albopictus are vectors of important diseases such as df, yellow fever and chikungunya (14-16). aedes aegypti is a mainly urban vector and is feeding exclusively from human (17). aedes albopictus are mostly found in suburban and rural environments and are feeding of the different species of mammals, including humans, as well as the different species of birds (18). currently, vector control is the best method to control of the df (19). in iran, studies regarding the biodiversity and distribution of aedes are limited. however, there is no information on aedes mosquito's diversity components in north-western of iran. the aim of this study was to investigate the diversity of aedes genus present in the natural areas, as well as the differences on the faunestic composition of aedes species in function of the climatic and ecological features of ardabil province borderline. various aedes mosquito species are considered as potential vectors of zika virus including ae. africanus, ae. albopictus, ae. polynesiensis, ae. unilineatus, ae. vitiates, ae. apicoargenteus, ae. leuteocephalus, ae. aegypti, ae. vitattus, ae. furcifer, and ae. hensilli. materials and methods study area this cross-sectional study was carried out from apr to oct 2016 in north-western of iran. ardabil province is located in northwestern iran 37.45° to 39.42° n and 47.30° to 48.55° e. the province has an area of 17 953km2. this province is bordered to the north with the republic of azerbaijan and along the border is 282.5km (fig. 1). in 159km from the border, flowing aras and balha rivers. during the border, iran linked to the republic of azerbaijan for two areas bilehsavar and aslanduz. ardabil province in the longitudinal axis of the expansion (1°35`) and high extent to the north-south latitudes (2°31`) have a large variety of climates. about 2/3 textured mountainous with large variation in height and the rest are flat areas and posts. north province (mugan plain) with low altitude has relatively warm weather and central and southern regions have a cool mountain climate (20-22). sample collection overall, 33 areas of 10 cities of priority border areas were selected randomly. during the seasonal activity, the larvae were collected in each month 2 times. using a ladle handling, and the standard ladle of 350ml was collected from natural and artificial larval habitats. in each habitat, sampling was collected from different parts and the ladle was made 10 times on each side. in the case of well water used from the bucket and the limited larval habitats such as cavity trees were used from droppers. the larval stages iii and were stored in lactophenol solution and after about a week and transparent larvae, using berlese's fluid were prepared microscopic slides and identified morphologically using appropriate identification keys. results overall, 2000 mosquito larvae were collected, from which only aedes larvae were selected and identified. from 33 areas, six (18%) were positive for the presence of aedes larvae. in four counties, (40%) aedes larvae were collected. totally, 694 larvae were collected from four counties that 7.2% were aedes larvae. three species of aedes genus were identified which included ae. caspius, ae vexans, and ae. flavescens. these species were reported from thee ardabil province for the first time. all three species were collected from 78–2114 meter above sea level altitude. aedes larvae were collected from two different climate zones. the first zone: the northern part of the province where the climate is hot and humid and low altitude (60–78km) j arthropod-borne dis, june 2019, 13(2): 191–197 e moradi-asl et al.: investigation on the … 193 http://jad.tums.ac.ir published online: june 24, 2019 that includes pars abad and bilehsavar and the second zone: southern part of the province with mountainous climate and high altitude (2114-2110m), which includes the khalkhal and sareyn (table 1). in the first zone the larvae were collected in jun and sep months but in the second zone in jul and aug. all three species aedes more in border areas the maximum aedes larvae from three species were collected in borderline of iran and azerbaijan but in other areas in this study, only two species for low abundance were collected (table 2). the survey of larvae habitat characteristics showed that most of larvae were collected (66%) in areas without trees and shrubs and sunny. totally 80% of larvae habitats were temporary that including holes and marshes around rivers and craters were caused by the overflow waters and 86% were natural habitat. about 50 % of the larvae collected in the afternoon and at cooler temperatures and 33% at mid-day and 17% at am. the water of larval habitat more was mainly stagnant and 50% transparent and type of bed habitat was 80% clay and 20% sand. table 1. total larvae collected from ardabil province, north-western of iran, 2016 location village total larvae aedes larvae genus species elevation y x sareyn alvars 40 1 aedes caspius 2110m 38.14985 47.96122 bilehsavar jafarabad 68 10 aedes caspius 176m 39.50238 48.04068 2 aedes flavescens khalkhal khangahe 213 1 aedes caspius 2114m 37.53637 48.5755 1 aedes flavescens parsabad oltan 251 3 aedes caspius 74m 39.60545 47.76123 mahmoudabad 70 10 aedes caspius 87m 39.54975 47.97872 3 aedes flavescens 3 aedes vexans normohamadkandi 52 10 aedes caspius 165m 39.4721 47.49537 5 aedes flavescens 1 aedes vexans total 694 50 table 2. larval abundance of aedes in ardabil province, north-western of iran, 2016 species aedes caspius aedes flavescens aedes vexans total % kowsar 0 0 0 0 0 bilehsavar 10 2 0 12 24 parsabad 23 8 4 35 70 germi 0 0 0 0 0 khalkhal 1 1 0 2 4 nir 0 0 0 0 0 namin 0 0 0 0 0 meshkinshahr 0 0 0 0 0 sareyn 1 0 0 1 2 ardabil 0 0 0 0 0 j arthropod-borne dis, june 2019, 13(2): 191–197 e moradi-asl et al.: investigation on the … 194 http://jad.tums.ac.ir published online: june 24, 2019 fig. 1. map of study area, ardabil province, iran discussion in this study three spices of aedes larvae (ae. caspius, ae. vexans and ae. flavescens) identified from ardabil province, north-western of iran that one species (ae. flavescens) was reported for first time. aedes larvae were dispersed in all regions of ardabil province but the frequency of aedes larvae were maximum in north regions of this province that borderline of iran and azerbaijan. only two adult species was reported (ae. caspius and ae. vexans) (22). aedes caspius is distributed in palearctic areas (23) and in iran reported from more than 16 provinces such as gilan, west azerbaijan, khorasan, zanjan, lorestan, isfahan, yazd, kerman, hormozgan, bushire and khozestan (24). this species very more frequently collected from six regions (parsabad, aslanduz, bilehsavar, khalkhal and sareyn) in this study that two areas (aslanduz and bilehsavar) were located in borderline of iran and azerbaijan and both are customhouse. this species is also collected with extensive distribution from gilan and ardabil province (25-26). moreover, ae. caspius reported from bushire (27), eastern of iran (28), chaharmahal and bakhtiari (29), east azerbaijan (30) and qom province (31). in 2016, ae. caspius along with ae. albopictus and aedes unilineatus reported from sistan and baluchestan (32). aedes caspius more feeding on mammals and humans (9, 33) and can be transmitted rift valley fever, dirofilariasis and tularemia (34). aedes vexans is distributed in holoarctic and oriental areas (35). this species and ae. eagypti and culex quinquefasciatus have the most distribution in world (33). in iran also reported from gillan, west azerbaijan, mazandaran, bushire and hormozgan (23). in this study ae. vexans larvae were collected 2 times in jun and sep from parsabad and aslanduz. the number of this species were lower in comparison to other aedes species but in gillan province the most larvae collected were ae. vexans (25). the results of this study showed j arthropod-borne dis, june 2019, 13(2): 191–197 e moradi-asl et al.: investigation on the … 195 http://jad.tums.ac.ir published online: june 24, 2019 that the larvae of ae. vexans were collected of less than 200m altitude that this result matched to another study (22) and the adults of this species collected from east azerbaijan in high altitude (30). the feeding preference of this species is on the mammals then birds and reptiles (32). aedes vexans can transmitted various diseases such as eastern and western equine encephalitis, japanese encephalitis and california encephalitis. aedes flavescens was reported first time in ardabil province and 11 larvae of ae. flavescens collected from three parts of this area (parsabad, bilehsavar and khalkhal). totally 100% of larvae habitats were temporary and most whit vegetation. total of these larvae were collected at afternoon. zaim et al. for first time reported ae. flavescens from west azerbaijan in 1987 and one time from large pool whit vegetation (24). these results showed that ae. flavescens were dispersed in northwest of iran. conclusion aedes species were a high density in borderline of iran and azerbaijan. 40% of ardabil province was found aedes mosquitoes. so the north parts of ardabil province are a suitable habitat for aedes species mosquitoes. therefore, more studies need to be done in these areas. acknowledgements the authors are grateful to dr zaim, mr esmail gorbani, mr ramazan gasemian, mr mehdi rafii, and mr shokri. this study was financially supported by ardabil university of medical sciences (no: 9502). the authors declare that there is no 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54–61. 32. clements an (1999) sensory, reception and behavior. biol mosq. 2: 740. 33. lane rp, crosskey rw (2012) medical insects and arachnids. springer science and business media. springer publication, uk. 34. gad am, farid ha, ramzy rr, riad mb, presley sm, cope se, hassan mm, hassan an (1999) host feeding of mosquitoes (diptera: culicidae) associated with the recurrence of rift valley fever in egypt. j med entomol. 36(6): 709–714. 35. knight kl, stone a (1977) a catalogue of the mosquitoes of the world (diptera: culicidae). 2nd edition. entornological society of america. the thomas say foundation, usa. j arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 238 http://jad.tums.ac.ir published online: january 05, 2016 original article specific antivenom ability in neutralizing hepatic and renal changes 24 hours after latrodectus dahli envenomation elham valikhanfard-zanjani 1, *abbas zare-mirakabadi 2, shahrbanoo oryan 1, hamid reza goodarzi 2, mahdise rajabi 1 1department of biology, faculty of sciences, kharazmi university, tehran, iran 2department of venomous animals and antivenom production, karaj, iran (received 18 june 2014; accepted 24 sep 2014) abstract background: latrodectism, a syndrome caused by latrodectus genus, is one of the clinical problems that occur predominantly in north east of iran. nowadays antivenom therapy has become the most useful treatment for animal bites; however there is still a controversy about route and time of antivenom administration in spider bite. the aim of the present study was to determine the efficacy of specific antivenom in neutralizing hepatic and renal symptoms 24 h after latrodectus dahli envenomation. methods: we selected a group of male new zealand white rabbits, weighing 2±0.3 kg. the l. dahli venom (0.5 mg/kg) was injected subcutaneously. specific antivenom (2.5 ml, i.v) was injected 24 h following venom injection. blood sampling was performed before and 24 h after venom injection, as well within 24, 48 and 72 h after antivenom administration. serum levels of (aspartate amino transferase (ast) alanine amino transferase (alt), alkaline phosphatase (alp), urea, bilirubin, creatinine and albumin were determined in all the sam. results: latrodectus dahli venom caused significant increase (p< 0.05) in all foresaid serum parameters. antivenom reversed the ast, alp, creatinine, urea and bilirubin to normal levels, but failed about alt level, also nonsignificant decrease was observed in albumin levels. conclusion: antivenom administration 24 h after venom injection can greatly reverse symptoms caused by venom. future studies in human beings should be conducted to assess the protection against the specific-latrodectus antivenom. keywords: l. dahli, liver, kidney, venom, antivenom introduction arachnid bites are one of the most important clinical problems in tropical and subtropical countries (almeida et al. 2009, lima et al. 2009). although the most spiders are venomous, but just a few are really dangerous for people (vassilevski et al. 2009). latrodectism is a syndrome caused by one of the most dangerous spiders in the world named latrodectus. black widow spider is a common name for this genus due to intrinsic behavior of females that kill their mates after mating (bettini and maroli 1978). among 30 species that distributed worldwide, only 4 species are reported as inhabitant of iran. the clinical problems of latrodectism are common in north east part of iran (afshari et al. 2009, shahi et al. 2011). black widow spider venom contains a neurotoxic compound, αlatrotoxin, that affect motor end plates in neuromuscular junction and presynaptic neurons in synapse region. αlatrotoxin cause great secretion of neurotransmitters at central nervous system and neuromuscular junction. the toxin by interaction with specific receptors that called neurexin, a dependent calcium receptor, caused formation of cation nonspecific channels in the presynaptic membrane, as well latro*corresponding author: dr abbas zare-mirakabadi, e-mail: zareabbas83@gmail.com j arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 239 http://jad.tums.ac.ir published online: january 05, 2016 philin, αlatrotoxin independent calcium receptor, by activation of plc and dag caused release internal calcium from endoplasmic reticulum. in fact, both receptors increase exocytosis level in the presynaptic membrane (henkel and sankaranarayanan 1999, ushkaryov et al. 2008, silva and ushkaryov 2010). previous reports (maretic and stani 1954, levine et al. 2010) indicate that venom of spiders belonging to latrodectus genus can cause cardiovascular changes including blood vessels spasm, arterial hypertension, tachycardia, bradycardia and change in the s-t segment with a prolongation of the q-t interval. same reports also showed urinary changes including oliguria and a high specific gravity of urine, with albuminuria, which are typical of patients with latrodectism. other reports (prior and park 2004, lima et al. 2009) also indicate priapism, hazy vision, diarrhoea, dyspnea, tachypnea and pressure in the chest of patients bitten by this spider. the treatment of latrodectism by various drugs and compounds like calcium gluconate, methocarbamol, benzodiazepines and narcotics have been reported, but in fact, many of them provides only temporary relief. nowadays use of antibodies and antibody fragments are considered as the most effective treatment in animal bites (allen and norris 1991, guti´errez et al. 2003). however there is a substantial controversy about the efficacy, safety and antivenom administration route. previously antivenoms were injected intramuscularly, but according to the data published in recent decades (isbister et al. 2007a, isbister 2007b) intramuscular route is not efficient, so intravenous route was proposed. however further researches are required to obtain doses, time and route of antivenom administration. on the other hand determination of antivenom ability to reverse complications of black widow needs more investigations. in this study we attempted to assess the effects of intravenous antivenom injection in neutralization of renal and hepatic symptoms causing by l. dahli venom. materials and methods venom crude spider venom was provided by department of venomous animals and antivenom production, razi vaccine and serum research institute, karaj, iran. latrodectus dahli were dissected out, and a pair of glands was collected into ice cold phosphate buffered saline (pbs). the glands were washed in pbs in order to remove possible contaminants, and venom was harvested in pbs by gentle compressing of the glands. the suspension was clarified by centrifugation at 8000 rpm, and the venom was stored at -20 °c until use. experimental animals six new zealand male white rabbits weighing 2±0.3 kg were selected. all rabbits were maintained in quarantine for at least 3 days before the experiment. the temperature was controlled at 18–22 ˚c with food and water. rabbits were anaesthetized with intramuscular injection of 0.5 ml ketamine and 0.5 ml xylazine in ratio 1:1 respectively. latrodectus dahli venom (0.5 mg/kg) was injected subcutaneously. blood sampling was carried out for all animals before and 24 h after venom injection as well 24 h, 48 h and 72 h after antivenom injection. blood collected with edta (1 mg/ml of blood), and processed for in clinical signs and symptoms of all the animals were recorded during the experiment. biochemical kits for determination of the changes in aspartate amino transferase (ast) alanine amino transferase (alt) and alkaline phosphatase (alp) enzymes, as well bilirubin, urea, creaj arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 240 http://jad.tums.ac.ir published online: january 05, 2016 tinine and albumin, analytical kits were purchased from pars azmoon company (iran). other chemicals and reagents used in this study were analytical grade and purchased from merck (germany). methods in this study l. dahli’s venom (0.5 mg/ kg) was injected by subcutaneously route to the fore legs of rabbits. blood sampling was performed before and 24 h after venom administration and later antivenom (2.5 ml with neutralization capacity of 500 ld 50/ml) was injected by i.v route. the blood collection was carried out again at 24, 48 and 72 h following antivenom injection. experiment ended by scarifying rabbits for extraction of heart, liver and kidney tissues throughout the surgery. blood collected in the tubes containing edta, were processed for biochemical studies. for this purpose blood samples were centrifuged at 2500×g for 10 min at 4 °c, then the serums were processed for biochemical studies. this article was a part of project work in razi vaccine and serum research institute which was approved by ethics committee of this institute. statistical analysis data were analyzed with statistical software spss 20.0. the comparison was between the data obtained before and after the venom injection as well as before and after antivenom injection. all the results were statistically analyzed using the student "t" test. the results were considered to be statistically significant if (p< 0.05). results the signs and symptoms of envenomation appeared within first few hours by redness, mild swelling as well as muscle cramps at bite site. later the rabbits showed tremors and finally paralysis in legs. difficulty in respiration was observed in most of the animals within 24 hours following venom injection. table 1 shows the changes in all parameters levels, before and after the venom injection. following venom injection (0.5 mg/kg, s.c), increasing in alanine aminotransferase (alt), alkaline phosphatase (alp), creatinine and bilirubin levels were seen within 24 h, which were statistically highly significant (p< 0.01). aspartate aminotransferase (ast) level within 24 h was also increased, that was extremely significant (p< 0.001). although the rise in urea and albumin levels, 24 h after venom injection, were not more than 21.80 % and 16.21 %, respectively, but were significant (p< 0.05) too (table 1). antivenom injection 24 h after venom, compared with pre-injection of the antivenom, leads to changes in the parameters levels within 24, 48 and 72 h. in comparison with before and 24 h after antivenom injection, decrease in alp and alt were statistically significant (p< 0.05), also a highly significant (p< 0.01) decrease was observed in ast level when compared with the level before antivenom injection. the level of creatinine at 24 h after antivenom injection, showed unexpectedly increase, which was highly significant (p< 0.01), however the bilirubin, urea and albumin levels showed nonsignificant decrease at 24 h (table 2). following antivenom injection within 48 h, the levels of all the parameters continued decrease in their levels which were statistically highly significant (p< 0.01) but unexpectedly the levels of alt and creatinine did not decrease but rather increased; the rise were respectively significant (p< 0.05) and nonsignificant (table 3). within 72 h after antivenom injection, compared with pre-injection of the antivenom, ast and alp levels were decreased, which statistical analysis showed that this decline were highly significant (p< 0.01), also extremely significant decrease (p< 0.001) were observed in bilirubin j arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 241 http://jad.tums.ac.ir published online: january 05, 2016 and urea levels. however decrease in albumin and creatinine levels, within 72 h compared with pre-injection of the antivenom, were non-significant. alt level within 72 h did not decrease, rather increased up to 27 %, which was statistically non-significant (table 4). table 1. effects of latrodectus dahli venom at 24 hours on rabbit’s serum alt, ast, alp, bilirubin, creatinine, urea, albumin (mean±sem) parameters before venom after venom (500µg/kg; s.c) % change p value alt (u/l) 48.61 ± 4.6 92.58± 10.49 + 90.7 p< 0.01 ast (u/l) 54.35 ± 6.01 169.81± 15.31 + 213.81 p<0.001 alp (u/l) 49.01± 4.62 199± 23.28 +306.04 p< 0.01 bilirubin (mmol/l) 0.67 ± 0.087 0.93± 0.1 + 38.80 p< 0.01 creatinine (mg/dl) 0.81 5± 0.08 1.12± 0.23 + 37.42 p< 0.01 urea (mg/dl) 27.09 ± 1.92 33± 4.08 + 21.80 p<0.05 albumin (gr/dl) 25.95 ± 3.1 30.1± 4.1 + 16.21 p<0.05 % changes: changes between before and 24 hours after venom administration. table 2. effects of latrodectus dahli antivenom at 24 hours on rabbits serum alt, ast, alp, bilirubin, creatinine, urea, albumin (mean±sem) parameters after venom 24 h antivenom (2.5ml, iv.) % change p value alt (u/l) 92.58±10.49 78.26±8.63 14 p< 0.05 ast (u/l) 169.81±15.31 114±10.77 32.86 p< 0.01 alp 199± 23.28 88.5±22.75 -55.52 p<0.05 bilirubin (mmol/l) 0.93±0.1 0.87± 0.087 6.45 n.s. creatinine (mg/dl) 1.12±0.23 1.4±0.67 + 25 p< 0.01 urea (mg/dl) 33±4.08 32±3.56 3.03 n.s. albumin (gr/dl) 30.1±4.1 29.15±4.75 3.15 n.s. n.s: not significant. % changes: changes between before and 24 hours after antivenom administration. table 3. effects of latrodectus dahli antivenom at 48 hours on rabbits serum alt, ast, alp, bilirubin, creatinine, urea, albumin (mean±sem) parameters after venom 48 h antivenom (2.5ml, i.v.) % change p value alt (u/l) 92.58± 10.49 145±15.20 + 58 p<0.05 ast (u/l) 169.81± 15.31 89±11.30 47.58 p<0.01 alp 199±23.28 67.88±8.78 65.88 p<0.01 bilirubin (mmol/l) 0.93± 0.1 0.74±0.06 20.43 p<0.01 creatinine (mg/dl) 1.12± 0.23 1.23±0.188 + 9.82 n.s urea (mg/dl) 33± 4.08 27.56±3.2 16.48 p<0.01 albumin (gr/dl) 30.1± 4.1 25.5±3.98 15.28 n.s n.s: not significant. % changes: changes between before and 48 hours after antivenom administration. j arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 242 http://jad.tums.ac.ir published online: january 05, 2016 table 4. effects of latrodectus dahli antivenom at 72 hours on rabbits serum alt, ast, alp, bilirubin, creatinine, urea, albumin (mean ± sem) parameters after venom 72 h antivenom (2.5 ml, i.v.) % change p value alt (u/l) 92.58±10.49 116.83±27.98 +27 n.s. ast (u/l) 169.81±15.31 88±9.72 48.17 p<0.01 alp 199±23.28 58±8.71 -70.85 p<0.01 bilirubin (mmol/l) 0.93±0.1 0.65±0.05 -30.10 p<0.001 creatinine (mg/dl) 1.12±0.23 0.9±0.18 -19.64 n.s. urea (mg/dl) 33±4.08 24±2.87 -27.27 p< 0.001 albumin (gr/dl) 30.1±4.1 25.2±3.2 -16 n.s n.s: not significant. % changes: changes between before and 72 hours after antivenom administration. discussion in the present study the effects of l. dahli venom within 24 h on various serum biochemical parameters were determined which showed a significant rise in liver and kidney function tests. so it seems that l. dahli venom in less than 24 h, exerts its destructive effects on foresaid organs. clinical manifestations of black widow spider envenomation indicated that the nervous system is the primary target of alpha-latrotoxin, as by activation of sympathetic nervous system cause intense release of neurotransmitters (vetter and isbister 2007. alt is considered as the principal liver marker enzymes that frequently used as an indicator of hepatocyte damage (barraviera et al. 1995a, frança et al. 2009). aspartate aminotransferase (ast) has a wide distribution, so that can be found in many organs such as heart, kidneys, liver and skeletal muscles (huang et al. 2006). significant increase in alt and ast levels observed 24 hours after venom injection, indicating liver damage caused by the venom of l. dahli. however the results showed that antivenom administration can reverse ast levels but it was failed to return alt level to initial state (before venom injection) within 72 hours. in clinical trials, increasing in alt level represents severe hepatic injury (huang et al. 2006). it seems that black widow spider venom exerts its destructive effects, in less than 24 hours, in liver tissue. marzan and maretic investigated the pathological effects of black widow spider venom on liver, they confirm the results of this study. according to their observations, after 30 minutes to 6 hours of venom injection, hepatic cells swollen and gradually massive hyperaemia appears, pericapillary edema after 10 hours, necrosis and lobular necrosis were seen respectively after 12 and 24 hours of venom injection (maretic 1953, marzan 1955). the ast to alt ratio appears to be a useful index for distinguishing liver disease and according to this report, if this ratio is less than 1 (>1) represents mild liver injury, but if this ratio is more than 1(<1) indicates severe liver damage (siddiqi et al. 2007). in our study, considering the ratio of ast/alt, 24 hours after venom injection, severe damages appeared in the liver, which lasted 24 hours after antidote injection; but within 48 and 72 hours after antivenom injection, the ratio dropped, that indicates the antivenom is able to reduce the severity of the injury. since 1995 several authors (barraviera et al. 1995b, voronov et al. 1999) have described in envenomations the systemic inflammatory response syndrome. j arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 243 http://jad.tums.ac.ir published online: january 05, 2016 this causes transient liver abnormalities with increased enzymes and decreased production of albumin and fibrinogen. this acute phase reaction could have also contributed to the changes observed in this study. other indicators of liver health are measurements of alkaline phosphatase and bilirubin levels in blood. alkaline phosphatase increased significantly, following venom injection, however, it seems that antivenom was able to restore the amount of enzyme to the primary level (before venom injection). high levels of bilirubin can be caused by liver dysfunction, which happens when it is damaged (marzan 1955). results of present study showed that, 24 hours after venom injection, bilirubin level was increased dramatically, this fact was also present by other researchers (maretic 1953, marzan 1955). although antivenom has little effect after 24 hours, but within 48 and 72 hours could improve bilirubin levels significantly. the kidney, because of its extensive blood supply network, is one of the most vulnerable organs to toxin injury by either hemodynamic alterations which lead to renal ischemia or by direct kidney injury (sitprija and sitprija 2012). determination amount of creatinine, urea and albumin in blood serum were basic parameters of kidney health. twenty four hours after venom injection, creatinine, albumin and urea levels were increased significantly, demonstrating venom effects on kidney tissues. this fact also has been approved by other researchers. marzan and maretic in a histological studies have shown that, after 10–20 hours of black widow spider venom injection, degeneration of tubular epithelium with necrosis (within 24 hours) were seen in kidney tissue (maretic 1953, marzan 1955). increase in albumin level, can be due of dehydration that occurs through intense sweating and increased saliva (walker et al. 1990). body dehydration by profuse sweating and increasing saliva are quite compatible to the results of maretic and prior, but increasing albumin level, is contrary to their findings (maretic and stani 1954, prior and park 2004). increasing urea level, 24 hours after venom injection, is reported by maretic and prior and it seems, that is due to dehydration or impaired renal functions (maretic and stani 1954, prior and park 2004). antivenom injection could decrease significantly urea and creatinine levels, within 72 hours and was able in reverse albumin levels to initial state. the α-ltx after entering the circulation, through activating l-type calcium channels, which are abundant on vascular smooth muscle cells, results in calcium influx which subsequently leads to vasoconstriction and hypertension (sitprija and sitprija 2012). as well as, increasing catecholamine levels followed by venom injection, causes vasoconstriction through interaction with alpha adrenoceptors in proximal and distal tubules, and causes renin secretion through interaction with beta adrenoceptors of juxtaglomerular cells of kidney. increasing renin secretion from kidneys will result in enhancement of angiotensin ii production. followed by these events, it seems renal blood flow decreases that can lead to a significant reduction in glomerular filtration rate and induces tissue ischemia (schrier 1974). on the other hand, according to immunological studies of herberth in 2005, some latrophilin receptor (αlatrotoxin independent calcium receptor) genes are expressed in various mammalian tissues, so that highest expression of latrophilin ii can be found in placenta, lungs, liver and mammary glands tissues, respectively (herberth et al. 2005). thus it seems that black widow spider venom can induce its effects by either directly and indirectly mechanisms. the present study provides evidence that latrodectus venom with impressing sympathetic nervous system, increasing blood catecholamines, hypertension and maybe direct interaction with its recepj arthropod-borne dis, june 2016, 10(2): 238–245 e valikhanfard-zanjani et al.: specific antivenom … 244 http://jad.tums.ac.ir published online: january 05, 2016 tors on tissues can induce hepatotoxicity and nephrotoxicity effects in rabbits. conclusion the l. dahli venom can cause kidney and liver function changes within 24 h which are reversed to normal using specific antivenom even 24 h after envenomation. however more investigations are needed to validating route and time of black widow spider specific antivenom administration. future studies in human beings should be conducted to assess the protection against the specificlatrodectus antivenom. acknowledgements we wish to thank the excellent technical assistance 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laboratory examinations. 3rd ed. butterworths, boston. j arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 126 http://jad.tums.ac.ir published online: march 31, 2021 short communication the first study of west nile virus in feral pigeons (columba livia domestica) using conventional reverse transcriptase pcr in semnan and khorasanerazavi provinces, northeast of iran *hamid staji 1 ; morteza keyvanlou 2 ; zeinab geraili 3 ; hedyeh shahsavari 3 ; elnaz jafari 3 1department of pathobiology, faculty of veterinary medicine, semnan university, semnan, iran 2department of clinical sciences, faculty of veterinary medicine, semnan university, semnan, iran 3faculty of veterinary medicine, semnan university, semnan, iran *corresponding author: dr hamid staji, e-mail: hstaji@semnan.ac.ir (received 16 nov 2019; accepted 13 mar 2021) abstract background: west nile virus (wnv) is an arboviral infection continuing to be as major threat to human health as well as the livestock industry all around the world. birds including pigeons are one of the potential reservoirs for wnv. this study aimed to detect the presence of wnv genome in feral pigeons circulating in semnan and khorasane-razavi provinces (iran) including 10 urban and 10 suburban areas. methods: totally, 150 samples (brain and kidney) were collected equally from feral pigeons and the presence of wnv genome was evaluated in these samples after rna extraction. results: all the samples were negative for the presence of wnv-rna in this investigation. conclusion: although obtained result indicated no evidence of wnv genome in feral pigeons but complementary studies regarding serologic detection of wnv in vertebrate hosts as well as pigeons and identification of arthropod vectors seems necessary for comprehensive determination about infection status in these areas. keywords: feral pigeons; west nile virus; rt-pcr; iran introduction west nile virus (wnv) is a mosquito-borne virus related to genus flavivirus (flaviviridae) with nearly worldwide distribution and spread by mosquitoes belonging to the genera aedes, anopheles, culex and ochlerotatus (13). historically, wnv infection was first identified from human cases with encephalitis in africa (1937) and its significance as a zoonotic virus was highlighted (1). west nile virus infection shows wide distribution characteristics throughout southwestern asia, russia, the middle east, europe, australia, and africa. the worldwide distribution of wnv has changed during the last decades by the synergism of predisposing factors such as modern transportation, global warming, and demographic chang es (4). previous studies have demonstrated that the wnv has been detected in at least 27 provinces of iran in different hosts including horses, humans and birds (5). in nature, wnv circulates in birds and mosquitoes feeding from birds as biologic vectors and these hosts are characterized as its natural hosts at which the virus amplifies in their body. for wnv, humans and horses are expressed as accidental dead-end hosts notifying its zoonotic potential more (6). west nile virus infection has been reported from different continents as well asia. the iranian semnan and khorasan-razavi provinces are situated in the central and northeastern geographic parts of iran and these two provinces are well-known for their copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. mailto:hstaji@semnan.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 127 http://jad.tums.ac.ir published online: march 31, 2021 suitable conditions for migratory birds from other countries in cold seasons. migratory birds are one of the major reservoirs of wnv for other avian (pigeons) species and for arthropod hosts and virus distribution occurs via different roots of transmission such as feeding conditions (7). previous studies confirm the existance of wnv in pigeons and their possible role in spreading of wnv to mammalian hosts (8-10). cities and livestock farms are considered as two suitable places for pigeons to make colonies because of the existence of food and for nesting purposes. so, these birds are now well adapted to farms and cities. recently, there has been some concern about zoonotic pathogens that can be transmitted via pigeons to humans and domesticated animals as well horses. specially, when such bird species co-exist with humans and livestock, they are considered to be a high risk factor for transmitting avianderived pathogens such as wnv because of the strong chance of contact with mammalian hosts (8). in iran, some investigations have confirmed the seroprevalence of wnv infection in humans, equines and wild water birds (birds circulating in water like ducks) and genome (wnv rna) detection in mosquitoes but wnv rna-detection found in these studies are very limited (11, 12). however, despite the presence of wnv genome in mosquitoes and human cases with encephalitis from iran (11, 13), there is no information about the existance of wnv in free-living pigeons in iran. therefore, a cross-sectional (5 years) study was carried out to investigate the molecular prevalence of wnv in feral pigeons from semnan and khorasane-razavi provinces (iran) to identify the probable role of these birds as reservoirs of wnv in this two regions. materials and methods sample collection in this cross-sectional study, the sample size was calculated using the following equation: n= 4 pq/l2, where n represents the minimum sample size needed for the prevalence estimation, p: prevalence (assumed prevalence of wnv in pigeons of the screened areas was considered 50% because of unavailability of previous data regarding its prevalence), q: 100p, and l: allowable error or precision (considered 0.1 in the present study). then, the minimum required sample size became 100 samples from birds. brain and kidney samples were taken based on sterile procedure from deeply injured, euthanized with ketamine (30mg/kg) or freshly dead wild pigeons which had been admitted to different vet clinics located in semnan and khorasene-razavi provinces during a 5-year period. the study was performed on 150 pigeons from different regions of (including 70 samples from 10 urban and 80 samples from 10 suburb areas) semnan and khorasane-razavi provinces (fig. 1) in north-eastern iran during for autumn 2014 to winter 2019. in the present study, the 70 samples from urban regions were collected from six cities located in semnan province including: semnan (35.5537810°n, 53.3791795°e), damghan (36.1747867°n, 54. 3542161°e), shahroud (36.3917606°n, 54. 9941698°e), biyarjomand (36.0971499°n, 55. 8126512°e), mayamey (36.4227077°n, 55. 6451097°e), forumad (36.5198894°n, 56. 7492357°e) and four cities located in khorasanerazavi province including: sabzevar (36. 2301945°n, 57.6391283°e), davarzan (36. 3431047°n, 56.8838182°e), sheshtemad (35. 94831686°n, 57.7599779°e) and rudab (36. 0283218°n, 57.3040453°e). also, the 80 samples from suburbs were collected from five regions located in semnan province including: ebrahim abad (36.4160772°n, 55.7220140°e), abkhori (35.8064424°n, 53.8563982°e), ala (35.5431662°n, 53.4924760°e), delazian (35. 4978981°n, 53.4066454°e), armian (36. 3630742°n, 55.4031629°e) and five regions located in khorasane-razavi province including: kahak (36.3584244°n, 56.7727503°e), mazinan (36.3156334°n, 56.8212518°e), sadkharve (36.3119113°n, 57.0695328°e), j arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 128 http://jad.tums.ac.ir published online: march 31, 2021 chesham (36.1717463°n, 57.0416171°e) and karrab (36.3553765°n, 57.5003219°e). these birds were not vaccinated against any viral agents, previously. generally, sampling was accidental and kidney and brain samples were collected from mentioned birds and stored at −70 °c in sterile falcon tubes until performing rna extraction. viral rna extraction and rt-pcr amplification the experiment was carried out using conventional reverse transcriptase pcr (rt-pcr) technique. total rna was extracted from brain and kidney samples using rnxplus solution (sinaclon, iran) as described by the manufacturer’s instructions. then, cdna was synthesized from 500ng of total rna using cyclescript reverse transcriptase kit (bioneer, korea). detection of wnv was carried out by amplification of the 3-untranslated region (3-utr) using previously described primer pair (14). for all rt-pcr reaction sets, a plasmid containing the 3-utr region of wnv was used as positive control and distilled h2o as negative control. each 25μl pcr reaction consisted of 2× concentration mastermix containing polymerase, dntps, mgcl2, reaction buffer, and stabilizers (jena bioscience, germany) by adding 0.8μl of each primer (10μmol/l; forward primer, cagaccacgcta cggcg; reverse primer, ctagggccgcgt ggg) and 50ng of template cdna quantified by nanodrop® (smart nano, canada). reaction mixes were overlaid with mineral oil and subjected to one step of 94 °c for 3min and then 35 cycles of amplification in a thermocycler (bioer xp cycler, china). the cycling condition was as follows: denaturation at 94 °c for 3min, annealing at a temperature specific (achieved by gradient temperature conditions) to the primer pairs 55 °c for 40s, and extension at 72 °c for 60s. then, a final extension step was followed at 72 °c for 5min for completion of the last pcr cycle. polymerase chain reaction amplicons (8–10μl) were electrophoresed in 3% agarose (sigma-aldrich) gel, stained with ethidium bromide (sigma-aldrich), visualized and photographed under a uv illuminator (nanolytik™, england). midrange dna ladder (100 bp, jena bioscience) was used for fragment size determination. results the molecular experiment amplified the expected fragment of dna (536bp) in the positive control but all the 150 kidney and brain samples tested for the presence of west nile virus rna by using conventional rtpcr were negative as shown in fig. 2. fig. 1. location of semnan and khorasane-razavi provinces in iran (a). the magnified area of both provinces at which the sampling regions are distributed within the area (b) j arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 129 http://jad.tums.ac.ir published online: march 31, 2021 fig. 2. gel electrophoresis results of detected gene fragment regarding the west nile virus in the brain and kidney samples of screened pigeons. m: marker (100bp), c+: positive control (3-utr region of wnv weighting 536bp), b: blank (negative control), 1-2: samples showing no amplified fragments (negative samples) discussion wnv genome was detected in none of the 150 screened pigeons in the present study. previous publications have documented the role of migratory birds in the epidemiology of wnv. also, field and experimental studies have shown that the pigeons have high wnv seroprevalence rates in endemic areas and can reproduce significant levels of viraemia (15-17). some investigations have confirmed the presence of wnv genome (rna) in pigeons in asian countries such as korea (8). however, to the best of our knowledge, no detailed study aimed at evaluation of the role of pigeons as reservoir for currently circulating iranian wnv strains has been performed. according to the results regarding the absence of wnv genome in the pigeons of monitored areas in the present study, it is highly recommended to perform a serologic survey on these bird species to find out their probable previous exposure to wnv and their seropositivity. the absence of wnv in these areas and in vertebrate hosts as well pigeons, the very low virus loads in the samples and region’s weather conditions in sampling seasons may provide unsuitable circumstances for the presence of wnv in screened areas. west nile virus is an emerging infectious pathogen for a variety of hosts including mammals, birds and even reptiles with a worldwide geographical distribution as well as asian countries (18, 19). migratory birds species play an important role in spreading wnv and specific species of birds are considered as critical reservoirs for this viral agent because the ornithophilic arthropods especially culex spp. feeding on birds play important role in the natural cycle of wnv infections (23, 25-26). then, birds circulating around humans and farm animals can introduce wnv to these vertebrate hosts via infecting the culex mosquitoes and their bites, subsequently. previous investigations have confirmed the presence of wnv infection in iran and neighj arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 130 http://jad.tums.ac.ir published online: march 31, 2021 boring countries via serologic and virological assays (11). the first report regarding the presence of wnv in iran is documented by naficy and saidi (22) by serological assays in human cases and higher rates of seroprevalence have been observed in central and southwestern iran. because of the presence of different climatic conditions and theoretically suitable environments for the establishment of wnv foci across iran, different investigations have focused on the existence of wnv in arthropods species (11), humans and equine hosts (22-26), and migratory and water birds (19) representing a heterogeneous geographic distribution of wnv in different regions. however, despite the extensive distribution of feral pigeons as potential vector species, there is no information about the existence of wnv in this bird populations and monitoring of feral pigeons in iran seems to be necessary as a factor involved in the cycle of this disease because previous studies have introduced pigeons as reservoirs of wnv (8). in the present study, wnv genome was not detected in screened birds in semnan and khorasane-razavi regions of iran. however, the reasons of these negative results can depend on various impacting factors. conclusion in conclusion, although our result indicated no evidence of wnv infection in feral pigeons but serological surveillance of these birds as potential vectors for wnv is highly recommended to fully understand the actual statues of this infection. for this reason, our investigations will continue for the detection of antibodies against wnv in vertebrate reservoirs including pigeons and determination of mosquitos’ fauna regarding to this viral pathogen existing in these regions. acknowledgements authors are grateful to dr alireza chavshin (department of medical entomology and vector control, school of public health, urmia university of medical sciences) for providing the plasmid as positive control for rt-pcr protocol carried out in this study. no competing financial interests exist. references 1. trevejo rt, eidson m (2008) west nile virus. j am vet med assoc. 232(9): 1302–1309. 2. romi r, pontuale g, ciufolini mg, fiorentini g, marchi a, nicoletti l, cocchi m, tamburro a (2004) potential vectors of west nile virus following an equine disease outbreak in italy. med vet entomol. 18(1): 14–19. 3. turell mj, sardelis mr, dohm dj, o'guinn ml (2001) potential north american vectors of west nile virus. annal new york acad sci. 951(1): 317–324. 4. özer n, ergünay k, simsek f, kaynas s, alten b, caglar ss, ustacelebi s (2007) west nile virus studies in the sanliurfa province of turkey. j vec ecol. 32(2): 202–206. 5. moin-vaziri v, charrel rn, badakhshan m, de lamballerie x, rahbarian n, mohammadi bavani m, azari-hamidian s (2019) a molecular screening of mosquitoes (diptera: culicidae) for flaviviruses in a focus of west nile virus infection in northern iran. j arthropod borne dis. 13(4): 391–398. 6. estrada-franco jg, navarro-lopez r, beasley dw, coffey l, carrara as, da rosa at, clements t, wang e, ludwig gv, cortes ac, ramirez pp (2002) west nile virus in mexico: evidence of widespread circulation since july 2002. emerg infect dis. 9(12): 1604–1611. 7. figuerola j, soriguer r, rojo g, tejedor cg, jimenez-clavero ma (2007) seroconversion in wild birds and local circulation of west nile virus, spain. emerg infect j arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 131 http://jad.tums.ac.ir published online: march 31, 2021 dis. 13(12): 1915–1922. 8. kim c, oh h, song j, hur m, suh jh, jheong wh, kim jt, oh hs, park jh (2016) first detection of west nile virus in domestic pigeon in korea. j vet sci. 17(4): 587– 589. 9. komar n, panella na, burns je, dusza sw, mascarenhas tm, talbot to (2001) serologic evidence for west nile virus infection in birds in the new york city vicinity during an outbreak in 1999. emerg infect dis. 7(4): 621–629. 10. panella na, young g, komar n (2013) experimental infection of eurasian collared‐ dove (streptopelia decaocto) with west nile virus. j vector ecol. 38(2): 210–214. 11. bagheri m, terenius o, oshaghi ma, motazakker m, asgari s, dabiri f, vatandoost h, mohammadi bavani m, chavshin ar (2015) west nile virus in mosquitoes of iranian wetlands. vector borne zoonotic dis. 15(12): 750–754. 12. fereidouni sr, ziegler u, linke s, niedrig m, modirrousta h, hoffmann b, groschup mh (2011) west nile virus monitoring in migrating and resident water birds in iran: are common coots the main reservoirs of the virus in wetlands? vector borne zoonotic dis. 11(10): 1377– 1381. 13. shah-hosseini n, chinikar s, ataei b, fooks ar, groschup mh (2014) phylogenetic analysis of west nile virus genome, iran. emerg infect dis. 20(8): 1419–1421. 14. lanciotti rs, kerst aj, nasci rs, godsey ms, mitchell cj, savage hm, komar n, panella na, allen bc, volpe ke, davis bs (2000) rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a taqman reverse transcriptasepcr assay. j clin microbiol. 38(11): 4066–4071. 15. spedicato m, carmine i, bellacicco al, marruchella g, marini v, pisciella m, di francesco g, lorusso a, monaco f, savini g (2016) experimental infection of rock pigeons (columba livia) with three west nile virus lineage 1 strains isolated in italy between 2009 and 2012. epidemiol infect. 144(6): 1301–1311. 16. michel f, sieg m, fischer d, keller m, eiden m, reuschel m, schmidt v, schwehn r, rinder m, urbaniak s, müller k (2019) evidence for west nile virus and usutu virus infections in wild and resident birds in germany, 2017 and 2018. viruses. 11(7): 674–681. 17. waziri ij, kabir j, kwaga jk, nguku p (2018) serosurvey of west nile virus in household-reared pigeons in bauchi metropolis, nigeria. trans r soc trop med hyg. 112(4): 169–174. 18. yazici z, albayrak h, ozan e, gumusova s (2012) the first investigation of west nile virus in horses using real time rtpcr in middle black sea region in turkey. j arthropod borne dis. 6(2): 151– 157. 19. castillo-olivares j, wood j (2004) west nile virus infection of horses. vet res. 35(4): 467–483. 20. apperson cs, hassan hk, harrison ba, savage hm, aspen se, farajollahi ar, crans w, daniels tj, falco rc, benedict m, anderson m (2004) host feeding patterns of established and potential mosquito vectors of west nile virus in the eastern united states. vector borne zoonotic dis. 4(1): 71–82. 21. rappole jh, derrickson sr, hubálek z (2000) migratory birds and spread of west nile virus in the western hemisphere. emerg infect dis. (4): 319–326. 22. naficy k, saidi s (1970) serological survey on viral antibodies in iran. trop geog med. 22(2): 183–88. 23. meshkat z, chinikar s, shakeri m, manavifar l, moradi m, mirshahabi h, jalali t, khakifirouz s, shahhosseini n (2015) prevalence of west nile virus in mashj arthropod-borne dis, march 2021, 15(1): 126–132 h staji et al.: the first study of … 132 http://jad.tums.ac.ir published online: march 31, 2021 had, iran: a population-based study. asian pac j trop med. 8(3): 203–205. 24. sharifi z, mahmoudian sm, talebian a (2010) a study of west nile virus infection in iranian blood donors. arch iran med. 13: 1–4. 25. ahmadnejad f, otarod v, fallah mh, lowenski s, sedighi-moghaddam r, zavareh a, durand b, lecollinet s, sabatier p (2011) spread of west nile virus in iran: a cross-sectional serosurvey in equines, 2008–2009. epidemiol infect. 139(10): 1587–1593. 26. chinikar s, javadi a, ataei b, shakeri h, moradi m, mostafavi e, ghiasi sm (2012) detection of west nile virus genome and specific antibodies in iranian encephalitis patients. epidemiol infect. 140(8): 1525– 1529. j arthropod-borne dis, december 2015, 9(2): 176–183 sa v zoelen et al.: antigenic cross-reactivity … 176 original article antigenic cross-reactivity anti-birtoxin antibody against androctonus crassicauda venom *suhandan adigüzel van zoelen 1, ozcan ozkan 2, bora inceoglu 3 1netherlands organisation for applied scientific research-innovation for life-hollanda, turkey 2drug and medical device agency of turkey, ankara, turkey 3department of entomology, university of california, davis, usa (received 2 nov 2013; accepted 7 may 2014) abstract background: antivenom is still widely used in the treatment of envenomation as there are no vaccines or other effective agents available against animal venoms. recently, neurotoxins named birtoxin family have been described from parabuthus transvaalicus and androctonus crassicauda. the aim of the present study was to test the antibirtoxin antibodies for their ability to neutralize the lethal effects of a. crassicauda scorpion venom. methods: sds-page and western blotting used the presence of components from a. crassicauda and p. transvaalicus scorpion venoms and to determine the degree of cross-reactivity. the minimum lethal dose (mld) of venom was assessed by subcutaneously (sc) injections in mice. results: the mld of the a. crassicauda venom was 35 µ g/ 20g mouse by sc injection route. western blotting showed the presence of components from a. crassicauda and p. transvaalicus scorpion venoms strongly cross react with the a. crassicauda antivenom. however, western blotting of the a. crassicauda scorpion venom using the refik saydam public health agency (rspha) generated antibody showed that not all the venom components cross reacted with the anti-birtoxin antibody. the antibodies only cross reacted with components falling under the 19 kda protein size of a. crassicauda venom. conclusion: the bioassays and western blotting of a. crassicauda venom with the anti-birtoxin antibodies produced against a synthetic peptide showed that these antibodies cross reacted but did not neutralize the venom of a. crassicauda. keywords: androctonus crassicauda, venom, anti-birtoxin antibody, cross-reactivity introduction most of the medically important scorpion species belong to buthus, parabuthus, mesobuthus, tityus, leiurus, androctonus and centruroides genera of the buthidae family (balozet 1971, bücherl 1971, efrati 1978). scorpion venoms can be classified into two groups according to their molecular sizes, long-chain and short-chain neurotoxins. the short-chain neurotoxins are 3,000 to 4,400 da and act on potassium or chloride channels. long-chain neurotoxins are 6,500 to 7,800 da and act mostly on sodium channels (possani et al. 1999, 2000, inceoglu et al. 2006, ozkan et al. 2008). it has been estimated that 100.000 distinct peptides exist in scorpion venom but only limited number of these peptides have been described (possani et al. 1999, 2000, martin-eauclaire et al. 2005, inceoglu et al. 2006). the unique specific treatment of scorpion envenomations is immunotherapy with antibodies from immunized horses (ghalim et al. 2000). however, the venom is a complex mixture of antigens wherein not all components are equally important for the production of neutralizing antibodies. thus, the identification of immunogenic protein(s) and/or their neutralizing epitopes may lead to the *corresponding author: dr suhandan adigüzel van zoelen, e-mail: suhendantr@gmail.com  http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 176–183 sa v zoelen et al.: antigenic cross-reactivity … 177 use of more clearly defined substances as immunogens to develop efficient antivenoms or to their use as antigens. the venom of p. transvaalicus consists of recently described closely related neurotoxins named birtoxin family (inceoglu et al. 2001, 2005). an antibody developed using a synthethic peptide composed of the first 18 amino acid residues of birtoxin displayed strong reactivity with the whole venom of p. transvaalicus, p. leisoma and pure birtoxin (inceoglu et al. 2006). these antibodies also neutralized the venom of p. transvaalicus in mice. recently, calışkan et al. (2006) also reported the presence of peptides in a. crassicauda venom that belong to the birtoxin-like peptide family. in this study, we tested the anti-birtoxin antibodies for their ability to neutralize the lethal effects of a. crassicauda scorpion venom. materials and methods venoms venom was obtained from mature a. crassicauda scorpions (from sanliurfa) by electrical stimulation of the telson. the venom was mixed with sterile double-distilled water and centrifuged at 15,000 rpm for 15 min at 4 ºc. the supernatant was immediately lyophilized at refik saydam public health agency (rspha) and stored at -80 oc until use. venom of commercially obtained p. transvaalicus scorpions were collected as described (inceoglu et al. 2001, 2006) at university of california, davis, ca. antivenom (rshc anti-ac) antivenom of a. crassicauda was obtained as described (ozkan et al. 2006a). briefly, increasing venom doses, mixed half-and-half with adjuvants, were injected subcutaneously into horses on the 1st, 14th, 21st, 28th, 35th and 42nd days. on the 45th, 48th and 51st, days, blood samples were collected three times from the jugular vein of each animal and stored in containers with 10 % sodium citrate. after plasma separation, antivenom was obtained, from combined plasma, by the digestive method and kept in the dark at 4 ºc. one dose of rsha anti-ac was normalized to neutralize 2 mld of a. crassicauda venom in rats when tested subcutaneously. anti-birtoxin antibody the 18 residues n-terminal portion of birtoxin-like peptides ‘nh2-advpgnypld kdgntykc’ was commercially synthesized by sigma and polyclonal antibodies against this peptide were raised by sigma-genosys (inceoglu et al. 2006). briefly, the synthetic peptide was cross-linked to keyhole limpet hemocyanin and rabbits were immunized. the bleedings were done after the 4th, 5th and 6th booster doses and pooled. igg molecules were purified using a protein a antibody purification kit from sigma following the manufacturer’s instructions. protein concentrations were determined using a bca kit (pierce, usa) with ovalbumin as the standard. determination of the minimum lethal dose (mld) in mice all the experiments were performed according to the guidelines by the ethical committee of the faculty of veterinary medicine in ankara university. the minimum lethal dose (mld) of venom was assessed by subcutaneously (sc) injections in mice (20±2 g). the animals were kept in the experiment room under standard conditions throughout the experiment. five mice per each dosegroup were injected sc with doses of venom, diluted in 0.5 ml saline solution. an equivalent volume of 0.5 ml saline was injected into five mice as negative control group. the animals were observed for 48 h after venom injection in order to determine mld. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 176–183 sa v zoelen et al.: antigenic cross-reactivity … 178 serum-neutralization assays in mice a solution of a. crassicauda venom (3 mld for each mouse) diluted in physiologic saline solution (pss) to a 2.5 ml volume, were prepared. the anti-birtoxin antibody was prepared at doses ranging from 0.5 ml to 1.5 ml. on the other hand a. crassicauda venom (1 mld) also prepared for each mouse and mixed with 1.5 ml anti-birtoxin antibody. the final volume all dilutions were made up to 5 ml with pss. the solutions were incubated for 60 min at room temperature. then, 0.5 ml of each solution was subcutaneously injected into groups of eight mice previously injected with a. crassicauda venom. the control groups were only injected with 1 mld of the venom diluted in pss. the numbers of surviving mice were recorded up to 48 h. after administration, animals were monitored for 48 hours and the number of living animals was recorded. the anti-birtoxin doses that prevented 100 % deaths in the groups were considered the minimum effective doses (med). a solution of a. crassicauda venom (3 mld for each mouse) diluted in physiologic saline solution (pss) to a final volume of 2.5 ml. the anti-birtoxin antibody was prepared at doses ranging from 0.5 ml to 1.5 ml. separately, a. crassicauda venom (1 mld, 62 µ l venom for each mouse) was prepared for each mouse and mixed with 1.5 ml antibirtoxin antibody. all solutions were then diluted to a final volume of 5 ml using pss. these solutions were incubated for 60 min at room temperature. then, 0.5 ml of each solution was subcutaneously injected into groups of eight mice previously injected with a. crassicauda venom. the control groups were only injected with 1 mld of the venom diluted in pss using the same volumes. following administrations the animals were monitored up to 48h and survival was noted. the anti-birtoxin doses that prevented 100 % deaths in the groups were considered the minimum effective doses (med). gel electrophoresis of the venoms and western blotting venoms were analyzed by sodiumdodecylsulfate polyacrylamide gel electrophoretic (sds-page) analysis according to laemmli (1970). venom of a. crassicauda and p. transvaalicus scorpions were separated on precast nupage 12 % bis-tris gel are electrophoretically transferred to the nitrosellulose membrane (ncm) and divided to two sections. the membranes were incubated in blocking buffer (3% bsa in tbst [0.1 tween 20, 150 mm nacl, 10 mm triscl, ph 7.5]) for one hour. the membranes were then washed three times with tbst (tris-buffered saline tween-20, [0.1% tween 20, 150 mm nacl, 10 mm tris-cl, ph 7.4]) and strips of the membrane were exposed to pre-immune serum for each antivenom for 30 min followed by three washes and incubated with antivenom of a. crassicauda (1: 4000) and the anti-birtoxin ab (1: 1000). membranes were again washed 3 times with tbst, and then incubated with horseradish peroxidase-conjugated anti-horse antibody and hrpconjugated anti-rabbit (1: 5000) for 60 min. the membranes were washed with tbst for 10 minutes and antigens were visualized using the immun-star hpr chemiluminescent subtrate (biorad). membranes were exposed to x-ray film in a dark room and developed. results the mld of the a. crassicauda venom was found to be 35 µ g/20 g mouse (1.75 mg/kg) by sc injection route (table 1). the potency of a. crassicauda antivenom (500 μl) has previously been determined to be neutralizing 2 mld in 150g rats according to manufacturer’s instructions. here this was confirmed to be the case. to assess the potency of the anti-birtoxin antibody, increasing doses of the antibody were used while the amount of a. crassicauda http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 176–183 sa v zoelen et al.: antigenic cross-reactivity … 179 venom was kept constant (1 and 3 mld). the ac antivenom (0.8 ml) potently neutralized 3 mld of the venom while all control mice died. however, 1.5 ml of antibirtoxin antibody was not able to neutralize even 1 mld of venom of a. crassicauda scorpion (table 2). despite the lack of the ability of anti-birtoxin antibodies to neutralize the venom of ac western blots indicate that there is a certain level of cross reactivity between this ab and the ac venom (fig. 1). although the peptide toxins fall below 10 kda molecular weight range often times on western blots a smear in the range of 5–15 kda corresponds to these peptides and their heteromers due to the running conditions. as shown by western blotting, a. crassicauda antivenom strongly reacted with the components of both p. transvaalicus and a. crassicauda venoms (fig. 1). similarly, the anti-birtoxin ab strongly reacted with both p. transvaalicus and a. crassicauda scorpion venoms as well. fig. 1 shows that proteins that were detected using the antibirtoxin ab all fall under 19 kda molecular mass. fig. 1. the venoms from androctonus crassicauda (a) and parabuthus transvaalicus (b) were separated and transferred to membranes. proteins were detected using the rshc anti-ac (1: 4,000) on the panel i and anti-birtoxin ab (1: 1,000) on the panel ii. molecular weight (m) markers on the panel ii are seeblue® plus2 (invitrogen corporation, usa) table 1. minimum lethal dose of androctonus crassicauda venom androctonus crassicauda scorpion negative control venom (μg/mouse) mice (death/total) pss (μl/mouse) mice (death/total) 10 15 20 25 35* 0/5 1/5 2/5 4/5 5/5* 500 0/5 mld: 35 μg/ 20g mouse table 2. neutralization capacity of the anti-birtoxin and antivenom was assayed for androctonus crassicauda venom in mice med of androctonus crassicauda antivenom med of anti-birtoxin antibody venom (μg/mouse) antivenom (μl) mice (surviving /total) venom (μg/mouse) antibody (μl) mice (surviving /total) 105 105 105 35 (control) 400 800 1000 5/8 8/8 8/8 0/8 105 105 105 35 500 1000 1500 1500 0/8 0/8 0/8 0/8* med: 800 μl med: no determined http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 176–183 sa v zoelen et al.: antigenic cross-reactivity … 180 discussion venom effect on the autonomic nervous system the antivenom therapy plays an important role in the treatment of scorpionism cases. as several studies stated that upon poisoning caused by scorpion stings, it is often recommended that the patients treated with species-specific antivenom and this must be administered early upon envenomation (alexander 1984, el-amin 1992, ismail 1993, sofer et al. 1994). the potentially dangerous and medically important scorpion species venom effect on the autonomic nervous system (bawaskar 2005). androctonus crassicauda is considered as the most significant species of scorpions in turkey and neighbouring countries iran, iraq and syria causing a large number of envenomations every year (radmanesh 1990, ozkan et al. 2006b, chippaux and goyffon 2008, dehghani and khamechian 2008, antopolsky et al. 2009, bosnak et al. 2009, dehghani et al. 2009, shahbazzadeh et al. 2009, dehghani and fathi 2012). parabuthus species are medically the most important scorpions in south africa thus bergman (1997) reports on the clinical manifestations of human envenomation by p. transvaalicus and the incidence rate of envenomation in zimbabwe. krifi et al. (1998) reported the difficulties in standardization the venom quality and ld50 determination which are partly related to geographical origin, the age of the venomous species, the season and venom extraction procedures, the number of specimens milked, the breeding conditions, and also the species’ strain, of the test animal body weight and administration route. these parameters must be accepted as important factors for standardization of the venom toxicity and the antivenom efficacy. thus, potency of antivenom is estimated by national or regional control authority and is described as a toxin neutralization unit according their standards (theakston et al. 2003, ozkan et al, 2007). therefore in the present study, mld and med were determined instead of ld50 and ed50. in our study, the med of the antivenom against 3 mld a. crassicauda venom was 0.8ml while 1.5ml of anti-birtoxin antibody was not able to neutralize even 1 mld of the venom of a. crassicauda scorpion. therefore the med of the anti-birtoxin was not determined. inceoglu et al. (2001) determined that native birtoxin from p. transvaalicus also has the average molecular mass of 6543.6 da. besides, martin-eauclaire et al. (2005) described new members of birtoxin-like peptides familiy from the venom of a. austrailis. moreover they notified that this new family might probably exist in other “old-world” buthidae venoms (martineauclaire et al. 2005). recently, this species from turkey has been studied and five toxins described by calıskan et al. (2006) two of which (acra 1, 6496.8 da and acra 2 7848.6 da), were lethal to mice. in this study, western blotting showed the presence of components from a. crassicauda and p. transvaalicus scorpion venoms strongly cross react with the a. crassicauda antivenom. however, western blotting of the a. crassicauda scorpion venom using the rhsc generated antibody showed that not all the venom components cross reacted with the anti-birtoxin antibody. the antibodies only cross reacted with components falling under the 19 kda protein size of a. crassicauda venom. this is not unexpected since most of the neurotoxic peptide components fall under this range. however these findings indicate that in contrast to the p. transvaalicus venom, in a. crassicauda venom the birtoxin like peptides contribute minimally to the neurotoxicity. this reiterates the fact that species differhttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 176–183 sa v zoelen et al.: antigenic cross-reactivity … 181 ences and geographical variations which result in a diverse number of neurotoxic peptides in the venom needs to be considered as an important factor in developing antivenoms that are effective. the finding that p. transvaalicus venom can be neutralized with a polyclonal antibody raised against the first 18 amino acid residues of birtoxin seems to be an exception (inceoglu et al. 2006) though it remains to be seen if horse derived antivenom against a. crassicauda will neutralize the venom of p. transvaalicus. similarly, it remains to be seen if the anti-birtoxin antibodies will neutralize the venom of closely related species including p. granulatus. conclusion the bioassays and western blotting of a. crassicauda venom with the anti-birtoxin antibodies produced against a synthetic peptide showed that these antibodies cross reacted but did not neutralize the venom of a. crassicauda. acknowledgements this project was supported by refik saydam hygiene center of ministry of health, ankara, turkey. we are grateful to the directory of refik saydam public health agency. we also thank to dr bruce d hammock and all hammock laboratory (department of entomology, university of california, usa) members for their support and help. the authors declare that there is no conflict of interests. references alexander jod (1984) scorpion stings. in: alexander jod (ed) arthropods and human skin. springer-verlag, berlin, p. 199. antopolsky m, salameh s, stalnikowicz r (2009) need for emergency department observation after scorpion sting: prospective study and review of the literature in the middle east. eur j emerg med. 16: 206–208. balozet l (1971) scorpionism in the old world. in: bücherl w, buckley e (eds) venomous animals and their venoms. volume 3. venomous intervertebrates. academic press, new york, pp. 349– 371. bawaskar hs (2005) management of severe scorpion sting at rural settings: what is the role of scorpion antivenom? j venom anim toxins incl trop 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warrell da, griffiths e (2003) report of a who workshop on the standardization and control of antivenoms. toxicon. 41: 541–557. http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 69 http://jad.tums.ac.ir published online: march 31, 2021 original article fauna and larval habitat characteristics of mosquitoes (diptera: culicidae) in kashan county, central iran, 2019 tahereh sadat asgarian 1 ; *seyed hassan moosa-kazemi 1 ; *mohammad mehdi sedaghat 1 ; rouhullah dehghani 2 ; mohammad reza yaghoobi-ershadi 1 1 department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran 2social determinants of health research center, department of environment health, school of public health, kashan university of medical sciences, kashan, iran *corresponding authors: dr seyed hassan moosa-kazemi, e-mail: moosakazemi@tums.ac.ir, dr mohammad mehdi sedaghat, e-mail: sedaghmm@tums.ac.ir (received 08 feb 2020; accepted 24 jan 2021) abstract background: mosquitoes are responsible for spreading devastating parasites and pathogens causing some important infectious diseases. the present study was done to better understand and update the fauna of culicidae and to find out the distribution and the type of their larval habitats in kashan county. methods: this study was done in four districts of kashan county (central, qamasr, niasar and barzok). mosquito larvae were collected from 23 active larval habitats using a standard 350ml capacity mosquito dipper from april to late december 2019. the collected larvae were transferred to containers containing lactophenol, and after two weeks individually mounted in berlese's fluid on a microscope slide and identified to species by morphological characters and valid keys. results: in this study, a total of 9789 larvae were collected from urban and rural areas in kashan county. the identified genera were anopheles, culiseta and culex. in this study larvae of an. turkhudi, cx. perexiguus, cx. mimeticus, cx. deserticola and cs. subochrea were collected for the first time from kashan county. conclusion: the results of this study indicate the presence and activity of different mosquito species in kashan county that some of them are vectors of arbovirus and other vector-borne diseases. keywords: mosquito; larvae; surveillance introduction arthropod-borne diseases have always been a major health problem in different parts of iran. transmission of malaria, filariasis, enceph-alitis and other arboviral diseases by mosquitoes have made mosquitoes the most important arthropods in medicine and health (1-3). the mosquitoes are from the order diptera, suborder nematocera and the family culicidae (4). seven arboviral diseases, two bacterial diseases, four filarial and two protozoal diseases which are assumed to be transmitted by mosquitoes are found in iran (1, 5). one genus and species (mansonia uniformis) was added to iranian mosquito checklist, thus there are 70 species and 8 (or 12) genera depending the classifica tion of the tribe aedini (6). this family has two subfamilies, anophelinae and culicinae in iran. azari-hamidian et al. (1) reviewed mosquitoes of iran and their medical and veterinary importance. there are diseases caused by mosquito-borne pathogens in isfahan province including avian pox (chickens, pigeons and turkeys), west nile fever (horses and humans), anthrax (sheep, cattle and goats), mosquitoborne filariases (camels) and bird malaria (turkeys, hens/roosters, pigeons and migratory waterfowl). because of their high adaptation to different habitats, mosquitoes are successful organisms and are found all over the world except copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 70 http://jad.tums.ac.ir published online: march 31, 2021 antarctica. mosquito larvae are found in a range of habitats, including natural and artificial habitats, with temporary or permanent, stagnant or flowing waters, contaminated or clean waters, with or without vegetation, and even in the smallest places where water accumulates in it, such as pots, used tires and animal footprints (2, 3). based on literature, in isfahan province, ten species of anopheles: an. algeriensis, an. claviger, an. dthali, an. maculipennis, an. marteri, an. messeae, an. multicolor, an. sacharovi, an. superpictus, and an. turkhudi have been reported (7-15). an. messeae was recorded based on egg chorion pattern (9). zaim (10), saebi (11) and mousa-kazemi et al. (12) reported the anopheles sacharovi and an. maculipennis of the maculipennis group, based on morphological characters. the recent group species was also being verified later by pcr technique (13). ladonni et al. (14) reported three species of an. dthali, an. marteri, and an. turkhudi to the fauna of the isfahan province. seven species of culex have been reported in isfahan province including: cx. hortensis, cx. mimeticus, cx. modestus, cx. perexiguus, cx. pipiens, cx. territans, and cx. theileri (10, 12, 16, 17). culiseta longiareolata, cs. annulata and cs. subochrea are cited in the province (10, 12, 17). three species of ochlerotatus caspius s.l., oc. pulcritarsis, and aedes vexans have been reported in the province (10, 12, 18). zaim (18) reported uranotaenia unguiculata. based on ladonni et al. (14) there are at least 24 species and six genera in the province. little information is available about mosquitoes in kashan county. zaim (10) mentioned some aspects of mosquito larvae and adult ecology in kashan county and relieved that the occurrences of 14 species in this area. doroudgar et al. (15) studied the epidemiology of malaria in kashan. this limited information are not sufficient as the basis to prevent possible epidemics of mosquito borne diseases. because of the importance of mosquitoes in medicine and health and disturbances caused by bites, the special location of kashan county, climate changes in the county, as well as the construction of garden of birds in qamsar that have led to the entry of birds from 17 different countries into the area, therefore, fauna and ecological study and obtain data on larval habitats, species composition, active season, and activity peak of mosquito larvae in kashan county are very important in integrated vector management. an entomology check program was felt after more than three decades in the county and a specialized study was done to better understand and update the fauna of culicidae in kashan county and to find out the distribution and the type of their larval habitats in the region, which can lead to preparation of basic information in the country and region and if necessary, be effective in adopting the right strategy of integrated vector management. materials and methods kashan county is located in the north of isfahan province, between karkas mountains and the central desert of iran. this county is bounded on the north by the central desert and the city of qom, on the east by the desert plain, on the south by the towns of borkhar and meymeh and natanz and on the west by the city of mahallat. kashan county has four districts (central, qamsar, niasar and barzok) and seven cities (kashan, meshkat, qamsar, niasar, jowshaqan, kamoo and barzok). this is a faunistic and descriptive-sectional study that was done in four districts of kashan county including central district (kashan and meshkat cities), qamasr district (qamasr, kamoo and jowshaqan cities), niasar district (niasser city) and barzok district (barzok city). the climate of the county varies depending on ups and downs. the uplands are cold, foots are temperate and plains, and especially on the margins of the desert are tropical. this county has a special position due to located on a large chain of roads in the country, located on the north-south transit route and located on the main communication route between eastern, western, northern and southern cities and provinces (19). in this study, larval habitats were visited in 77 urban and rural areas of the county and 23 j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 71 http://jad.tums.ac.ir published online: march 31, 2021 active larval habitats were selected (figs. 1, 2). permanent or temporary, natural or artificial larvae habitats, with or without vegetation, slow flowing or still water and full or partial sunlight in urban or rural areas, visited and sampled monthly. larvae and pupae of the mosquitoes were collected using a standard 350ml capacity mosquito dipper from april to late december 2019. twenty dips were taken in each larval habitat. sampling were always done by the same individual in the morning (08:00– 12:00h) or afternoon (15:00–18:00h). larvae from small water bodies collected by eye dropper. the collected larvae were transferred to petri dish containing lactophenol and the date, collection site and habitat type of the larvae were recorded with special code on the containers and in the relevant forms and transferred to medical entomology laboratory of tehran university of medical sciences (tums). after that the larvae in lactophenol were transparent, each larva was individually mounted in berlese's fluid on a microscope slide and identified to species by morphological characters and valid keys (20-22). some mosquitoe specimens were deposited in the museum of medical entomology, tums. results in this study, a total of 9789 larvae were collected from urban and rural areas of four districts of central, qamsar, niasar and barzok in kashan county and were identified based on valid diagnostic keys at species level. the identified genera was anopheles 772 (7.89%), culiseta 1706 (17.42%) and culex 7311 (74.69%) which included 13 species. culex pipiens and cx. theileri had the highest abundance with 3658 (37.36%) and 2555 (26.10%), respectively (table 1). ten larval habitats were artificial and 13 were natural. 45.8% of larval habitats were in urban areas and 54.2% in rural areas. among the larval habitats in urban areas, kashan had the highest and meshkat (central district) the least abundance of larvae. in the rural areas, the larvae habitats of khoncheh (from central district) and qazaan (from qamsar district) had the highest and lowest abundance of mosquito larvae, respectively. in terms of mosquito fauna diversity, the central district (kashan and meshkat cities) with 3 species (cx. pipiens, cx. theileri and cs. longiareolata) had the least species diversity and districts of barzok and niasar with 11 species had the highest mosquito larvae fauna diversity. in terms of time distribution, the highest number of larvae was in august and the lowest number of larvae was in december. in this study, cx. pipiens, cx. theileri, cs. longiareolata and an. superpictus s.l. showed the highest time dispersion. all anopheles species in this study were collected from natural habitats. among the culicinae species, cx. pipiens, cx. theileri and cs. longiareolata were collected from natural and artificial habitats and the rest of the species collected only from natural larval habitats. larvae of an. superpictus s.l. and an. maculipennis s.l. were collected from permanent and temporary habitats and larvae an. claviger and an. turkhudi were found from a temporary habitat. the larval habitat characteristics of the species has been listed in table 2. in view of the simultaneous presence of different larval species in larval habitats, cx. theileri was collected from 23 habitats with all others larvae. this species had the highest distribution in kashan county. culiseta longiareolata was collected from 20 larval habitats. anopheles superpictus s.l. was collected from 10 larval sites in four districts of the county and had the highest dispersal among anopheline species. species of an. claviger, an. turkhudi, cs. annulata and cs. subochrea each with a larval habitat had the least distribution in kashan county (table 3). table 4 shows the species caught and coordinates of the collection sites of mosquito larvae in kashan county. j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 72 http://jad.tums.ac.ir published online: march 31, 2021 table 1. abundance of larvae of the mosquito species collected in kashan county, central iran, 2019 species districts of kashan county central qamsar niasar barzok total no. % no. % no. % no. % no. % an. superpictus s.l. 159 3.21 453 14.94 10 1.08 57 6.44 679 6.93 an. maculipennis s.l. 0 0 54 1.78 15 1.63 22 2.49 91 0.93 an. claviger 0 0 0 0 1 0.10 0 0 1 0.01 an. turkhudi 0 0 0 0 0 0 1 0.11 1 0.01 cx. pipiens 2651 53.56 733 24.17 37 4 237 26.78 3658 37.36 cx. theileri 1028 20.8 1233 40.67 176 19.07 118 13.33 2555 26.10 cx. deserticola 175 3.53 105 3.46 68 7.37 92 10.40 440 4.5 cx. perexiguus 205 4.14 29 0.96 71 7.70 31 3.50 336 3.43 cx. hortensis 150 3.03 31 1.02 40 4.33 33 3.73 254 2.60 cx. mimeticus 15 0.30 39 1.29 0 0 14 1.58 68 0.70 cs. longiareolata 566 11.43 355 11.7 460 49.84 280 31.64 1661 16.96 cs. annulata 0 0 0 0 43 4.66 0 0 43 0.44 cs. subochrea 0 0 0 0 2 0.22 0 0 2 0.02 total 4949 100 3032 100 923 100 885 100 9789 100 table 2. characteristics of larval habitats and percentage of mosquito larvae in kashan county, central iran, 2019 percentage of culicidae larvae (%) characteristic variables a n . su p e rp ic tu s s. l . a n . m a c u li p e n n is s . l. a n . c la v ig e r a n . tu rk h u d c x . p ip ie n s c x .d e se rt ic o la c x .h o rt e n si s c x . p e re x ig u u s c x . m im e ti c u s c x . th e il e ri c s. l o n g ia re o la ta c s. a n n u la ta c s. s u b o c h re a permanence permanent 90 33.3 0 0 17.6 75 60 62.5 66.7 39.1 40 0 0 temporary 10 66.7 100 100 82.4 25 40 37.5 33.3 60.1 60 100 100 water current slow flowing 70 33.3 0 0 0 75 60 62.5 66.7 34.8 35 0 0 still 30 66.7 100 100 100 25 40 37.5 33.3 65.2 65 100 100 intensity of light full sunlight 80 33.3 0 0 23.5 75 40 75 66.7 73.9 85 0 0 partial sunlight 20 66.7 100 100 76.5 25 60 25 33.3 26.1 15 100 100 turbidity clear 90 66.7 100 0 52.9 87.5 80 75 66.7 87 85 100 100 turbid 10 33.3 0 100 47.1 12.5 20 25 33.3 13 15 0 0 vegetation with vegetation 90 100 100 100 58.8 100 100 87.5 100 56.52 10 100 100 without vegetation 10 0 0 0 41.2 0 0 12.5 0 43.48 90 0 0 substrate type mud 20 66.7 100 100 76.5 25 40 37.5 33.3 17.4 60 100 100 sand 80 33.3 0 0 23.5 75 60 62.5 66.7 82.6 40 0 0 habitat type natural 100 100 100 100 11.8 100 100 100 100 56.6 50 100 100 artificial 0 0 0 0 88.2 0 0 0 0 43.4 50 0 0 j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 73 http://jad.tums.ac.ir published online: march 31, 2021 table 3. number of simultaneous presence of mosquito larvae in active larval habitats in kashan county, central iran, 2019 species n o . o f l a r v a l h a b it a t a n . su p e rp ic tu s s. l . a n . m a c u li p e n n is s . l. a n . c la v ig e r a n . tu rk h u d c x . p ip ie n s c x .d e se rt ic o la c x .h o rt e n si s c x . p e re x ig u u s c x . m im e ti c u s c x . th e il e ri c s. l o n g ia re o la ta c s. a n n u la ta c s. s u b o c re a an. superpictus s. l. 10 * 2 1 7 6 4 5 3 10 10 an. maculipennis s. l. 3 2 * 1 1 3 3 3 3 2 3 3 1 1 an. claviger 1 1 * 1 1 1 1 1 1 1 1 an. turkhudi 1 1 1 * 1 1 1 1 1 1 1 cx. pipiens 17 7 3 1 1 * 7 5 7 3 17 17 1 1 cx. deserticola 8 6 3 1 1 7 * 5 6 3 8 8 1 1 cx. hortensis 5 4 3 1 1 5 5 * 4 3 5 5 1 1 cx. perexiguus 8 5 3 1 1 7 6 4 * 3 8 8 1 1 cx. mimeticus 3 3 2 1 3 3 3 3 * 3 3 cx. theileri 23 10 3 1 1 17 8 5 8 3 * 20 1 1 cs. longiareolata 20 10 3 1 1 17 8 5 8 3 20 * 1 1 cs. annulata 1 1 1 1 1 1 1 1 1 * 1 cs. subochrea 1 1 1 1 1 1 1 1 1 1 * a b c d e f fig. 1. some of active larval habitats in the study areas of kashan county, central iran from april to late december 2019. a. khoncheh (permanent, slow flowing), b. nabar (natural and permanent), c. niasar (temporary, still), d. barzok (natural and still), e. kashan (natural, slow flowing) and f. kashan (artificial and still) j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 74 http://jad.tums.ac.ir published online: march 31, 2021 table 4. coordinates of the collection sites and mosquito larvae identified in kashan county, central iran, 2019 district site of sampling longitude latitude mosquito larvae identified central meshkat-hamzer 51.25705 34.17636 cx. theileri central meshkat 51.27324 34.18104 cs. longiareolata, cx. pipiens, cx. theileri central taherabad 51.35628 34.0421 cs. longiareolata, cx. pipiens, cx. theileri central ravand (fish farming) 51.37237 34.02696 cs. longiareolata, cx. pipiens, cx. theileri central kashan university of medical science 51.41 34.01705 cs. longiareolata, cx. pipiens, cx. theileri central wastewater treatment 51.39998 34.04713 cs. longiareolata, cx. pipiens, cx. theileri central khoncheh 51.21894 33.98134 an. superpictus s. l., cs. longiareolata, cx. theileri, cx. deserticola, cx. hortensis, cx. mimeticus, cx. perexiguus, cx. pipiens central jazeh 51.377 7 33.84543 cs. longiareolata, cx. pipiens, cx. theileri qamsar moslemabad 51.50039 33.78592 an. superpictus s. l., cs. longiareolata, cx. theileri qamsar road of javinan 51.48367 33.74182 an. superpictus s. l., cs. longiareolata, cx. deserticola, cx. hortensis, cx. pipiens, cx. theileri qamsar farfahan wastewater 51.42875 33.77983 cs. longiareolata, cx. pipiens, cx. theileri qamsar farfahan-river 51.43248 33.75867 an. maculipennis s. l., an. superpictus s. l., cs. longiareolata, cx. pipiens, cx. theileri, cx. deserticola, cx. hortensis, cx. mimeticus, cx. perexiguus qamsar mazgan 51.40234 33.73779 cx. theileri qamsar bonrood 51.42037 33.72226 an. superpictus s. l., cs. longiareolata, cx. deserticola, cx. pipiens, cx. theileri qamsar qazaan 51.39733 33.7058 cs. longiareolata, cx. deserticola, cx. perexiguus, cx. theileri qamsar qohroud 51.42307 33.67773 an. superpictus s. l., cs. longiareolata, cx. deserticola, cx. pipiens, cx. theileri, cx. perexiguus niasar kamal-al-molk spring 51.09115 34.0962 an. superpictus s. l., cs. longiareolata, cx. pipiens, cx. theileri, cx. perexiguus niasar kamal-al-molk 51.11026 34.08385 cs. longiareolata, cx. pipiens, cx. theileri niasar niasar 51.15015 33.97601 an. maculipennis s. l., an. claviger, cs. annulata, cs. subochrea, cs. longiareolata, cx. deserticola, cx. hortensis, cx. pipiens, cx. theileri, cx. perexiguus niasar nashalj 51.07791 33.97987 cs. longiareolata, cx. perexiguus, cx. pipiens, cx. theileri barzok nabar 51.20775 33.88567 an. superpictus s. l., cs. longiareolata, cx. theileri barzok barzok-spring 51.22939 33.78756 an. maculipennis s. l., an. superpictus s. l., an. turkhudi, cs. longiareolata, cx. deserticola, cx. hortensis, cx. pipiens, cx. mimeticus, cx. theileri, cx. perexiguus barzok barzok-khavid 51.22525 33.79302 an. superpictus s. l., cs. longiareolata, cx. theileri j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 75 http://jad.tums.ac.ir published online: march 31, 2021 fig. 2. the location of larva sampling places in kashan county, central iran, 2019 discussion in this study, 13 species of mosquito larvae were collected and identified from three genera of culicidae. larva of an. turkhudi was col lected for the first time from kashan county, the city of barzok. also among the species of culex, larvae, cx. mimeticus and cx. desertij arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 76 http://jad.tums.ac.ir published online: march 31, 2021 cola were found from kashan county for the first time. of the three species of culiseta identified in this study, cs. subochrea was identified for the first time in the county and found from a temporary natural habitat associated with cs. annulata larvae in the city of niasar. zaim (10) reported cx. modestus from mahmoudabad of kouhpayeh. we did not catch this species, because in recent years land leveling, destruction of lowlands and highlands, improvement of streams and cementation of streams have reduced larval habitats in different areas of mahmoudabad (now is named meshkat city). zaim (10) also reported an. multicolor from shurab area. in the new divisions of the country, this area has been separated from kashan county and is an area in aran and bidgol county, therefore, it was not one of our study areas. anopheles superpictus s.l. this anopheles is distributed throughout the iranian plateau and the slopes of the alborz mountains and southern zagros, as well as the coastal plains of the caspian sea and the persian gulf (23) and is one of the seven species of malaria vectors in iran (20). this species was the most abundant and distributed species among anopheles in kashan county with 87.95% of the anopheles collected. its larvae were collected from qamasr, khoncheh, barzok, qohroud, javinan, ka-malal-molk, moslem abad and nabar from june to november. previous studies have also reported larvae of this species from different regions of kashan county (10, 15). barghamadi et al. (24) reported the best larval habitat for this species in kohkiluyeh and boyer-ahmad province, natural habitats with permanent and clear water, still, non-vege-tated with mud core. moosa-kazemi et al. (12) reported its larval habitat primarily on rocky at shallow depths and secondarily on sandy and sunny rivers also shallow creeks with muddy bed. in a study in ardabil, larvae of this species were reported from permanent and temporary habitats with submerged vegetation and from riverbeds (25). in the study done in pol dokhtar county from lorestan province, the larvae of this species was mostly found in natural habitats of edge and bed of rivers with permanent, slow flowing and clear water with external or submerged vegetation (26). soleimani-ahmadi et al. (27) collected this species from larval breeding habitats including permanent habitats in riverbeds mostly without vegetation and algae also still and clear water with full sunlight and sandy substrates. in kashan county, the best larval breeding habitats for this species was the natural habitat of river edge with permanent and slow flowing water and external or submerged vegetation. anopheles maculipennis s.l. this species was included 11.79% of total anophelinae species collected from kashan county. it was found from temporary natural habitats in niasar (july), in barzok (september and october) and a permanent natural habitat in qamasr (july and august), and 100% of its larval habitats in kashan county had low to high vegetation. zaim (10) reported this species only once in july 1984 from qamsar. larvae of this species have been reported in swamps, marshes, natural meadows, and water leaks in rivers and springs and in sandy beds of rivers and stagnant waters. this species is one of the malaria vectors in iran and is the main vector in the caspian coast in northern iran (28). anopheles claviger it occupies a variety of larval habitats but usually prefers non-polluted, permanent or semipermanent waters and it has been reported in almost all provinces of the country and is widely distributed in the north and central parts of iran (29). waters that are warmed by sunlight, on the margins of rivers and canals in stagnant water or with slow flowing in areas with shades of plants and trees, are suitable for larval period (30). it is blood sucking outdoor and transfers tularemia disease (29). in kashan county, this species was found from niasar in a temporary larval habitat with stagnant, semisunny, clear water and high vegetation in august. in 1984, zaim (10) collected these mosquito larvae from a larval breeding place with running water next to a water stream in mahmoudabad area in december. j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 77 http://jad.tums.ac.ir published online: march 31, 2021 anopheles turkhudi this species has been reported from the south and some parts of central iran (29, 31). in this study, the larvae of this mosquito were found from kashan county for the first time from a temporary natural larval breeding place with vegetation also still and turbid water with partial sunlight and muddy substrates in barzok in september. ladonni et al. (14) reported this species from fereydunshahr for the first time in isfahan province. culex pipiens its larval breeding place include both natural and artificial habitats. among the larvae collected in kashan county, this species was most abundant and was collected from april to late december. among the different districts of kashan county, the most larvae of this species belonged to the central district and the city of kashan so that, out of 3,645 larvae cx. pipiens, 2020 were collected from urban and rural kashan, such as kashan wastewater treatment plant, stabilization pond of kashan university of medical sciences and waste water. in iran, this species has been often reported from natural habitats (8). zaim (10) found it from the natural habitats. moosa-kazemi et al. (30) and azeri-hamidian (32) collected it from man-made habitats, artificial habitats and rice fields. in kashan county, the best larval habitat for this species was temporary larval breeding place with still and clear water, vegetation and partial sunlight. many arbovirus diseases are transmitted to humans by cx. pipiens, such as the rift valley fever, japanese encephalitis, and the west nile. this species is primarily an ornithophilic species that feeds on both man and animals (33). culex theileri this mosquito is found in some parts of the world and has been reported from all provinces of iran (34-38). in kashan county, this species had the highest spatial distribution and was collected from all larval habitats in cities and villages all of the time study from april to december. its larval habitats are diverse in kashan county. the best habitat for its larvae was the natural, permanent and temporary habitat, with still and clear water, and fullest sunlight with or without vegetation. zaim (18) reported it from natural habitats, moosa-kazemi et al. (37) and yaghoobi-ershadi et al. (38) reported it from natural and artificial habitats. cx. theileri is a major vector of the rift valley fever virus in south africa (39) and it is involved in the transmission of dirofilaria immitis nematode to humans. in iran, third instar larvae of cx. theileri were naturally infected with d. immitis and may be the main vector of this parasite (35). culex theileri also transmits west nile and sindbis viruses in vitro (40). culex hortensis this species is widely distributed in the mountainous regions of iran and has been reported from at least 23 provinces (10, 18, 32 and 41) and in kashan county was collected from permanent natural larval breeding places in central district, khoncheh village (august), natural and permanent larval breeding places in qamsar (august), natural and temporary larval habitat in niasar (july and august) and natural and temporary larval habitat of barzok (the months of september and october). the best habitat for larvae of this species in kashan county was permanent habitats with slow flowing and clear water, full sunlight with vegetation. zaim (10) reported this species from dehnar, qamsar, qohroud, barzok and niasar in 1984. culex hortensis has been introduced as a vector of avian malaria in vitro (42). culex deserticola larvae of this species were found in larval habitats with cx. hortensis and is reported for the first time from kashan county. larvae were collected in central district from the natural and permanent larval habitats of khoncheh village in september and october, in qamsar district from natural and permanent habitats of qamsar city and villages of qohroud and qazaan in july and september, in niasar district from a natural and temporary habitat in city of niasar in june and july and in barzok district from a temporary natural habitat from june to october. its best larval habitats for this species in kashan county was permanent habj arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 78 http://jad.tums.ac.ir published online: march 31, 2021 itats with slow flowing and clear water with vegetation and full or partial sunlight. culex perexiguus it is distributed in most provinces in iran (18). in kashan county, this species was found from permanent or temporary natural larval habitats in qamasr, niasar, barzok and the villages of qohroud, kamalal-molk and khoncheh from june to october. the best habitat for larvae of this species in kashan was permanent habitats with slow flowing water, full sunlight and with vegetation. zaim (10) found cx. perexiguus (as cx. univittatus) in mahmoudabad of kouhpayeh. culex mimeticus it is widely distributed in iran and its larvae are found along the rocks and on the riverside of mountainous areas with abundant vegetation (10). the best habitat for larvae of this species in kashan county was permanent habitats with clear slow flowing water, full sunlight, and with vegetation, and was found from larval habitats in khoncheh (july), qamasr (september) and barzok (august, september and october). in this study, cx. mimeticus larvae were reported from kashan county for the first time. zaim (10) found only adult of this species from qohroud. culiseta longiareolata this species is widely distributed in iran. this mosquito has a variety of larval habitats, and is found in clear to polluted waters and in fresh and saline waters (10, 43). in kashan county, this species was an active mosquito species and its larvae were collected from different habitats throughout the study from april to late december. 93.2% of larval habitats of this species in kashan county had no vegetation and 100% of them had still water. zahirnia et al. (26) also collected 70% of the larvae of this species in pol dokhtar county from without vegetation habitats. it often is blood sucking from birds and rarely attacks humans. although it feeds only on birds in north africa, it feeds on large domestic animals in europe, including france. this species has medical importance. adults are vectors of brucellosis, avian influenza virus, west nile (44) as well as avian malaria (42). its larvae are known as mosquito larvae predators (45). culiseta annulata in iran, larvae have been found from mountainous areas and the alborz and zagros mountains. it has also been reported from bushehr (46). azari-hamidian (43) studied the larval habitat characteristics and associated species in guilan province. in kashan county, this mosquito larvae was found from a temporary natural habitat, partial sunlight with high vegetation, still and clear water in niasar in june and july. this larval habitat was dried in midjuly. larvae of this species were not found in the other studied areas. zaim (10) reported larvae of this species in june from temporary larval habitats with still fresh water in qohroud and from permanent larval habitats with saline flowing water from mahmoudabad in october and november. azari-hamidian (43) reported it from both natural and artificial habitats. culiseta subochrea culiseta subochrea is found in a number of provinces in iran (47-49). in kashan county, this species was found in a temporary larval habitat with still and clear water, partial sunlight and tall vegetation with cs. annulata in niasar. paksa et al. (48) collected this species from stagnant and turbid water habitats with muddy substrate and shallow depth. conclusion the results of this study indicate the presence and activity of different mosquito species in kashan county. considering the dispersion of an. superpictus s.l. in different districts of kashan county, its abundance and since it's one of the seven malaria vectors in the country, a study is needed to identify its complex species. this study could be a basis for more comprehensive research about the importance of mosquitoes in medicine and health. we declare that we have no conflict of interest. j arthropod-borne dis, march 2021, 15(1): 69–81 ts asgarian et al.: fauna and … 79 http://jad.tums.ac.ir published online: march 31, 2021 acknowledgements authors are grateful to dr sm asgarian for helping with field collections. the authors declare the ethical approval code as no.: ir. tums.vcr.rec.1397.1001. references 1. azari-hamidian s, norouzi b, harbach re (2019) a detailed review of the mosquitoes (diptera: culicidae) of iran and their medical and veterinary importance. acta trop. 194: 106–122 2. zaim m (1987) malaria control in iran. j am mosq control assoc. 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6(1): 54–61. preparation of a nanoemulsion of essential oil of acroptilon repens plant and evaluation of its larvicidal activity agianst malaria vector, anopheles stephensi http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... original article preparation of a nanoemulsion of essential oil of acroptilon repens plant and evaluation of its larvicidal activity agianst malaria vector, anopheles stephensi samira firooziyan1,2, mahmoud osanloo3, seyed hassan moosa-kazemi1, hamid reza basseri1, habib mohammadzadeh hajipirloo4, ali sadaghianifar2, *amir amani 5,6, *mohammad mehdi sedaghat1 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 urmia health center, disease control unit, urmia university of medical sciences, urmia, iran 3 department of medical nanotechnology, school of advanced technologies in medicine, fasa university of medical sciences, fasa, iran 4 medical parasitology department, school of medicine, urmia university of medical sciences, urmia, iran 5 natural products and medicinal plants research center, north khorasan university of medical sciences, bojnurd, iran 6 medical biomaterials research center (mbrc), tehran university of medical sciences, tehran, iran *corresponding author: dr. mohammad mehdi sedaghat, e-mail: sedaghmm@tums.ac.ir; dr. amir amani, e-mail: amani76@gmail.com (received 07 june 2021; accepted 20 sept 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: extensive use of chemical larvicides to control larvae, has led to resistance in vectors. more efforts have been conducted the use of natural products such as plant essential oils and their new formulations against disease vectors. nanoformulation techniques are expected to reduce volatility and increase larvicidal efficacy of essential oils. in this study for the first time, a larvicide nanoemulsion from the essential oil of acroptilon repens was developed and evaluated against anopheles stephensi larvae under laboratory conditions. methods: fresh samples of a. repens plant were collected from urmia, west azarbaijan province, iran. a clevenger type apparatus was used for extracting oil. components of a. repens essential oil (aeo) were identified by gas chromatography–mass spectrometry (gc–ms). all larvicidal bioassay tests were performed according to the method recommended by the world health organization under laboratory condition. particle size and the morphologies of all prepared nanoformulations determined by dls and tem analysis. results: a total of 111 compounds were identified in plant. the lc50 and lc90 values of aeo calculated as 7 ppm and 35 ppm respectively. aeo was able to kill 100% of the larvae in 4 days. conclusion: the nanoemulsion of aeo showed a weak effect on the larvar mortality. it may therefore be suggested that this kind of nanoemulsion is not appropriate for the formulation as a larvicide. it is important to screen native plant natural products, search for new materials and prepare new formulations to develop alternative interventions with a long-lasting impact. key words: acroptilon repens; nanoemulsion; larvicidal effect; vector control; anopheles stephensi 334 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... introduction vector borne diseases are infections which are transmitted by the bite of infected arthropod species and account for 17% of all infectious diseases. every year more than one billion people are infected and more than one million people die from vectorborne diseases including malaria, dengue, schistosomiasis, leishmaniasis, chagas disease, yellow fever, lymphatic filariasis and onchocerciasis (1). the most important diseases transmitted by mosquitoes are malaria, dengue fever, lymphatic filariasis, yellow fever, chikungunya, zika virus, as well as viral encephalitis (2). anopheles mosquitoes are widely distributed and found in the tropics temperate regions (3). the most important disease transmitted by anopheles mosquitoes is human malaria, which is the most important parasitic disease in the world (4). the disease is still a major health problem worldwide, including iran. most malaria cases were reported from the south and southeastern of iran. plasmodium vivax was the dominant species (5). anopheles stephensi is an important malaria vector in the middle east and south asia (6, 7). to control the disease, larval control is currently being performed in 55 countries (8, 9). the use of natural products is an interesting approach in this regards. today, there are loads of studies and recommendations on plant extracts and essential oils as larvicides, insecticides and repellents (10, 11). extracts and essential oils (eos) are biocompatible and have minimum toxic effects on non-target organisms. along with many novel formulations, nanoemulsions of pesticides have been considered recently due to their greater efficiency, lesser adverse effects on non-target organisms (12, 13). however, extracts and eos have volatile components that restrict their use in natural environments (14-16). this can be overcome by formulating them in the form of nanoemulsions. there has been a lot of research recently on eos as natural larvicides, but there are a few available articles on nanoemulsions as larvicides. in a study, the larvicidal activity of eucalyptus essential oil and its nanoemulsion against culex quinquefasciatus was investigated. the result showed that the bioactivity of the nanoemulsion was improved than the bulk eo (17). in a study, nanoemulsion of artemisia dracunculus essential oil showed better larvicidal efficiency on an. stephensi larvae in comparison with its essential oil (18). likewise, encapsulation of a. dracunculus essential oil in chitosan nanoparticles presented very good larvicidal activity with 9 days residual effect (19). volpato et al (2016) investigated the effect of essential oil and its nanoemulsion against alphitobius diaperinus. the nanoemulsion showed a three-fold better effect as compared to the essential oil (20). balasubramani et al (2017) in their experiments obtained similar results with the nanoformulation of vitex negundo essential oil compared to its essential oil against aedes aegypti (21). in a recent study, larvicidal activity of cinnamomum zeylanicum essential oil was compared with its nanoemulsion. the formulated nanoemulsion showed 32% better larvicidal effect as compared to the essential oil, the residual effect of the formulation was 3 days. these results indicated an increase in larvicidal activity and residual effects of an essential oil nanoemulsion compared to bulk essential oil (11). as the extract of a. repens had very good larvicidal activity against anopheles stephensi, culex pipiens and culex quinquefaciatus in the previous work (22), we decided to extract its essential oil and provide the nanoformulation in order to investigate their larvicidal effect against an. stephensi larvae. materials and methods collection, identification and extraction of acroptilon repens fresh samples of a. repens were collected in junjul 2018 from urmia, west azarbaijan province, iran (45.08º e, 37.55º n, elevation ~ 1332 m above sea level) (fig.1). 335 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... collection and identification of plant acroptilon repens plants were collected (fig. 2), rapidly transferred to the laboratory and then was identified by experts in department of plant sciences, tehran university. extraction of essential oil all collected plants were washed with water, then shad dried. dried samples were hydrodistilled, using clevenger type apparatus for five hours. the extracted oil dried over anhydrous sodium sulfate. in total, 65 ml extracted oil obtained from 650 kg of dried plant. to prevent degradation and oxidation, the essential oil was stored in dark glass containers, completely away from sun light at 4-8 °c. analysis of essential oil by gas chromatography–mass spectrometry (gc-ms) gc-ms analysis used to identify compounds of the essential oil. the essential oil diluted using hexane with the specifications given in table 1. the compounds of the essential oil were analyzed using gc-ms and compared with standard mass spectra available in the device library. mosquito rearing anopheles stephensi larvae were reared in the insectary at 29 ± 1˚c with relative humidity of 70 ± 5% under 12 h light/12 h dark conditions. the cages for keeping mosquitoes were wooden cubes with dimensions of 30 cm × 30 cm × 30 cm, covered with fine mesh. the stock culture of adult an. stephensi fed twice a week on sheep blood (artificial feeding). the egg rafts laid transferred to enamel larval trays. the larvae were fed with fish food. preparation of nanoemulsion in this study, surfactant (tween 80) and co-surfactant (span 20) were stirred for 6 minutes at 600 rpm. the essential oil was then added at 90% lethal concentration of the bulk essential oil and stirred for 10 minutes at 600 rpm. water was then added dropwise and stirring was continued at 600 rpm for 38 minutes. ten different nanoemulsion preparations having a constant amount of essential oil (1.4 %) and different amounts of surfactant (2 to 9.2 %) and co-surfactant (0.8 %). the nanoemulsion stored in a dark place at room temperature for 24 hours, then checked visually for signs of phase fig. 1. collection site of plant acroptilon repens in urmia, west azarbaijan province, iran fig. 1. collection site of plant acroptilon repens in urmia, west azarbaijan province, iran 336 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... separation, precipitation or creaming. dynamic light scattering (dls, k-one. ltd, korea) used to determine the particle size (ps) of the prepared nanoformulations. transmission electron microscopy (tem) used to confirm the ps and to investigate the morphology of the particles. determining the larvicidal efficacy larvicidal bioassays performed according to the who guideline. logarithmic dilutions prepared from bulk essential oil by dissolving in ethanol. all the nanoemulsion/ bulk samples diluted 200 times before performing the larvicidal tests (23). third and early 4th instars larvae were used. one ml of the essential oil was added to 249 ml of chlorine-free (ph=7) water and stirred and 25 healthy larvae added to the containers. containers were covered and after 24 hours, the number of living and dead larvae counted. fig. 2. acroptilon repens fig. 2. acroptilon repens table 1. analysis conditions and specifications of gc-ms device instrument specifications manufacturer company agilent technologies 1. gc system 7890a 2. mass selective detector 5975c vl msd with triple-axis detector 3. ion source electron impact (ei) 70ev 4. analyzer quadrupole 5. column rtx 5 ms -length 30m -i.d. 0.250 mm -film thickness 25 μm conditions 1. injection port temperature 230˚c 2. ion source temperature 230˚c 3. carrier gas he 99.999% 4. sample volume 0.2 μl temperature program initial temperature (ºc) 40 initial time (min) 1 program rate (ºc/min) 3 final temperature (ºc) 270 final time (min) 10 split ratio (ml/min) 100 septum purge (ml/min) -- flow rate (ml/min) 1 table 1. analysis conditions and specifications of gc-ms device 337 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... determining the duration of action of bulk and nanoemulsions of a. repens essential oil to determine the duration of larvicidal activity, according to the instructions of the who, the formulation prepared for the larvicidal test diluted 100 times (23). one ml of specified concentrations of bulk or nanoemulsion samples added to 249 ml of chlorine-free (ph=7) water, then, 25 live larvae were added to the solution. after 24 hours, the number of dead/ live larvae was counted, without changing the solution; the larvae (dead and live) were removed from the containers, followed by the addition of 25 new live larvae in the containers. the larvae were replaced for 8 days. all the larvicidal bioassays repeated 16 times in four different replicates. in each replicate, two control groups containing ethanol were considered. statistical analysis the lethal concentrations of 50% and 90% (lc50 and lc90) were calculated using probit analysis (24). the regression line plotted using excel 2007 software. if mortality of the control group was less than 5%, the data from the bioassay tests were considered correct. when the control mortality was between 520%, it was corrected using the abbott formula (24). if the larvae became pupae or the larvae mortality were more than 20% in the control group, the test was repeated. results determination of chemical composition of a. repens essential oil components of aeo identified by gc– ms analysis. one hundred and eleven components were determined, with five major components including caryophyllene oxide (12.055%), α-cubebene (12.054%), 1-heptadecene (5.181%), delta-cadinene (3.771%) and β-cubebene (3.771%) as listed in supplementary data (table 2). characterization of aeo nanoemulsion from preliminary studies to find on the optimum nanoemulsion (i.e., highest stability and lowest particle size), a nanoemulsion preparation with 6.8% tween 80, 0.8% span 20, 1.4% aeo and water was prepared. figure 3 shows dls results of the nanoemulsion with d50= 106 nm. the morphology of nanoemulsion particles was determined using transition electron microscopy (tem) (fig. 4). the results show that the nanoemulsion was well-formed and the particles were almost spherical. the aeo nanoemulsion did not show any sign of phase separation after 4-month storage (4 °c and room temperature) and centrifugation (25000 rpm, 30 min). larvicidal bioassay of a. repens essential oil the results of larvicide activity of six different concentrations of aeo are shown in figure 5. mortality rate at 3.125 ppm was 0% and increased to 100% at 50 ppm. there was no mortality in the control groups. in regression line, a positive correlation was observed between essential oil concentrations and the probit mortality (fig. 6). the lc50 and lc90 values of aeo against an. stephensi larvae calculated as 7 and 34 ppm, respectively. figure 7 shows comparison of the residual larvicidal properties. aeo killed 100% of the larvae in the first four days of the experiment. after the 4th day, larval mortality decreased and reached 76%. aeo nanoemulsion showed weaker activity. it had 84% mortality on the first day and the mortality rate decreased to 0% on the 7th day. to compare larvicidal activity of aeo with aeo nanoemulsion against an. stephensi larvae, equal concentrations of aeo used in the short time test (24 hours). the larvicidal effects of aeo were 88%, while the nanoemulsion properties of aeo reduced to 44% (fig. 8). figure 9 shows the particle size of the nanomeulsion after 200 times dilution, showing instability for the preparation after dilution. 338 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... table 2. chemical composition of the essential oil of acroptilon repens no. rt (min) compound peak area % quality mol weight (amu) 1 37.993 caryophyllene oxide 503332611 12.055 96 220.183 2 29.563 α-cubebene 503308397 12.054 99 204.188 3 41.823 1-heptadecene 216337204 5.181 99 238.266 4 35.537 delta-cadinene 188186597 4.507 98 204.188 5 30.091 β-cubebene 157435883 3.771 97 204.188 6 31.287 caryophyllene 155313108 3.720 99 204.188 7 28.316 α-cubebene 148603368 3.559 98 204.188 8 38.858 3-undecyne 128983727 3.089 55 152.157 9 47.078 2-pentadecanone 108810045 2.606 96 268.277 10 33.756 tricyclo[2210(2,6)]heptane-3,5-diol 96224253 2.305 91 204.188 11 40.881 1,3-cyclooctadiene 93566390 2.241 74 108.094 12 34.201 1-pentadecene 91513407 2.192 99 210.235 13 38.285 dihydrotanshinone i 87244445 2.090 91 220.183 14 37.172 [1,5]naphthyridine-4-carbaldehyde 77809615 1.864 58 220.183 15 38.673 2(1h)-naphthalenone 64550157 1.546 86 220.183 16 41.231 azulene 63504092 1.521 97 198.141 17 38.063 β-copaen 62409995 1.495 70 220.183 18 30.689 4,4-dimethyl-3 56695349 1.358 47 202.172 19 33.406 delta-elemene 53675197 1.286 58 204.188 20 41.422 methyl α-oxo-7-azaindole-3-acetate 50884172 1.219 59 218.167 21 32.655 5,9-undecadien-2-one 46366238 1.110 90 194.167 22 40.652 bicyclo 45358016 1.086 89 204.188 23 29.881 β-damascenone 45288877 1.085 98 190.136 24 36.249 calacoene 44746403 1.072 78 172.125 25 25.752 1-tridecene 41473191 0.993 98 182.203 26 36.612 caryophyllene oxide 39040647 0.935 60 220.183 27 33.972 β-selinene 37808861 0.906 99 204.188 28 39.526 naphthalene 36960428 0.885 90 204.188 29 30.212 3,5-octadiene 33754464 0.808 64 194.203 30 39.869 10,10-dimethyl-2,6-dimethylenebicyclo 33082573 0.792 99 220.183 31 33.546 β-selinene 32759075 0.785 96 204.188 32 40.048 bicyclo 30063246 0.720 84 204.188 33 34.354 delta-cadinene 29461311 0.706 80 204.188 34 32.922 bicyclo[221]heptane 27543459 0.660 98 204.188 35 60.641 tricosane (cas) 25909857 0.621 98 324.376 36 35.212 delta-cadinene 25336335 0.607 62 204.188 37 36.803 bicyclo[310]hexane 24755522 0.593 42 136.125 38 55.424 phytol 23404833 0.561 90 296.308 39 37.579 cyclohexane 22932610 0.549 84 192.188 40 75.287 nonacosane 21848026 0.523 99 408.47 41 51.238 hexadecanoic acid 20517153 0.491 99 256.24 42 34.557 naphthalene 19954822 0.478 99 204.188 43 32.77 trimethylcyclohex 19947825 0.478 80 278.134 44 70.75 heptacosane 18873599 0.452 95 380.438 45 67.226 benzenedicarboxylic acid 18814084 0.451 91 211.012 46 30.925 methanoazulene 18182957 0.435 99 204.188 47 40.474 isoaromadendrene epoxide 18089104 0.433 43 220.183 48 31.904 bicyclo[311]hept 17605938 0.422 98 204.188 table 2. chemical composition of the essential oil of acroptilon repens 339 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... continued table 2. chemical composition of the essential oil of acroptilon repens no. rt (min) compound peak area % quality mol weight (amu) 49 30.409 7-methanoazulene 15474913 0.371 99 204.188 50 35.823 naphthalene 14843167 0.356 99 204.188 51 32.515 khusimene 14686454 0.352 91 204.188 52 21.929 decanal 13680181 0.328 91 156.151 53 33.851 tricyclo 13035063 0.312 91 204.188 54 39.717 dimethyl-2 12491034 0.299 83 220.183 55 40.347 4-methanoazulene 12398829 0.297 55 204.188 56 34.977 tridecanal 12181378 0.292 94 198.198 57 34.474 pentadecane 11697698 0.280 93 212.25 58 49.457 13-pentadecatrien-2-one 11566338 0.277 86 262.23 59 25.161 vitispirane 11436343 0.274 98 192.151 60 28.914 cycloisosativene 11154885 0.267 97 204.188 61 40.226 gamma-selinene 11037619 0.264 64 204.188 62 41.097 quinoline, 2,6-dimethyl 10239966 0.245 35 157.089 63 65.877 heneicosane 9939147 0.238 91 296.344 64 41.988 heptadecane 9896521 0.237 97 240.282 65 36.491 2-methyl-6-nitrophenol 9850609 0.236 53 153.043 66 44.463 alloaromadendrene oxide-(2) 8884878 0.213 83 220.183 67 32.146 4-dimethylaminopyridin-2-amine 8576066 0.205 52 137.095 68 39.316 6-methoxy-1-acetonaphthone 8242200 0.197 78 200.084 69 48.751 nonadecane (cas) 8087481 0.194 98 268.313 70 26.408 1h-indene 7844665 0.188 92 174.141 71 30.835 1h-cycloprop[e]azulene 7747134 0.186 99 204.188 72 54.941 heneicosane 7588497 0.182 99 296.344 73 37.045 naphthalene 7339014 0.176 80 172.125 74 42.192 vulgarol b 7325012 0.175 55 220.183 75 44.164 ledene oxide-(ii) 7233022 0.173 60 220.183 76 39.138 trimethyl-2'-methylidene-9'-oxabicyclo 7090758 0.170 41 220.146 77 43.025 2-dodecen-1-yl(-)succinic anhydride 7006122 0.168 30 266.188 78 45.735 eicosane 6974224 0.167 38 282.329 79 28.443 naphthalene 12 dihydro 1 1 6 trimethyl 6527008 0.156 97 172.125 80 39.221 tricyclo 6429488 0.154 38 220.183 81 45.43 octadecane 6266398 0.150 98 254.297 82 31.618 germacrene-d 5570492 0.133 98 204.188 83 43.407 valerenol 5558781 0.133 70 220.183 84 43.559 isopropylidene 5353049 0.128 42 218.167 85 49.673 hexadecanoic acid 5318288 0.127 98 270.256 86 22.323 pentylthiophene 5122791 0.123 83 154.082 87 54.547 1-heptadecanol 5095930 0.122 95 256.277 88 46.066 bicyclo[1310]hexadecan-2-one 4816216 0.115 55 236.214 89 13.595 dl-limonene 4755721 0.114 99 136.125 90 36.129 α-calacorene 4672444 0.112 38 200.157 91 51.906 eicosane 4631346 0.111 96 282.329 92 28.997 cycloisosativene 4487495 0.107 99 204.188 93 42.554 tetradecanal 4420395 0.106 91 212.214 94 48.178 cyclotetradecane 4361575 0.104 90 196.219 95 16.483 benzene 4265136 0.102 96 132.094 96 22.075 naphthalene, 1,2,3,4-tetrahydro 4132000 0.099 97 174.141 97 44.666 7,8-dihydropyran 4116843 0.099 50 173.084 98 27.68 benzene, 1,2,3,4-tetramethyl4066183 0.097 46 134.11 99 11.991 furan, 2-pentyl 3750856 0.090 91 138.104 340 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... continued table 2. chemical composition of the essential oil of acroptilon repens no. rt (min) compound peak area % quality mol weight (amu) 100 32.019 aromadendrene 3697814 0.089 99 204.188 101 33.189 widdrene 3626118 0.087 83 204.188 102 13.423 benzene, 1-methyl-4-(1-methylethyl) 3546573 0.085 97 134.11 103 57.836 docosane 3527160 0.084 94 310.36 104 17.214 nonanal 3429151 0.082 91 142.136 105 56.277 4,4,6-trimethyl 3326248 0.080 43 140.12 106 42.923 4,4-dimethyl-3 3080628 0.074 89 202.172 107 63.294 tetracosane 2968794 0.071 97 338.391 108 32.324 bicyclo[311]heptane 2911317 0.070 60 204.188 109 43.311 4-tetradecene 2856951 0.068 84 196.219 110 23.316 cis-3-hexenyl isovalerate 2576062 0.062 72 184.146 111 43.19 2-cyclopenten-1-one 2205731 0.053 90 164.12 fig. 3. dls results of aeo nanoemulsion fig. 3. dls results of aeo nanoemulsion fig. 4. transition electron microscopy (tem) image of aeo nanoemulsion fig. 4. transition electron microscopy (tem) image of aeo nanoemulsion 341 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... fig. 5. larvicidal activity of aeo against anopheles stephensi 0 10 20 30 40 50 60 70 80 90 100 3.125 6.25 12.5 25 50 0 48 80 84 100 m or ta lit y% concentration(ppm) fig. 5. larvicidal activity of aeo against anopheles stephensi fig. 6. probit regression line of aeo against anopheles stephensi larvae y = 34.048ln(x) 23.595 0 20 40 60 80 100 120 1 10 100 l ar va l m or ta lit y (p ro bi t) concentration (log) 50 nm fig. 6. probit regression line of aeo against anopheles stephensi larvae larvicidal effects of aeo and aeo nanoemulsion fig. 7. comparison of residual larvicidal effect of aeo vs. aeo nanoemulsion (after diluting 100 times) during an 8-day study 0 20 40 60 80 100 1 2 3 4 5 6 7 8 100 100 100 100 92 84 82 76 84 76 68 56 40 16 0 0 m or ta lit y % days aeo aeo nanoemulsion fig. 7. comparison of residual larvicidal effect of aeo vs. aeo nanoemulsion (after diluting 100 times) during an 8-day study 342 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... fig. 8. comparison of larvicidal activity of aeo vs. aeo nanoemulsion (after diluting 200 times) during an 24 88 44 0 10 20 30 40 50 60 70 80 90 100 aeo aeo nanoemulsion m or ta lit y % fig. 8. comparison of larvicidal activity of aeo vs. aeo nanoemulsion (after diluting 200 times) during an 24 fig. 9. dls results of aeo nanoemulsion after dilution fig. 9. dls results of aeo nanoemulsion after dilution discussion today, associated with extensive use of various chemical pesticides, serious damages have been observed in the environment and non-target organisms which is being carefully considered by international organizations such as united states environmental protection agency (usepa), world health organization (who) and food and agricultue organization (fao) (25). in addition, frequent use of insecticides has led to their resistance for vectors (26). to reduce environmental damages and increase the effectiveness of insecticides on target organisms, the use of novel preparations such as nano-formulations has been suggested (10). in this study the most components of aeo were identified in comparison with the similar studies. total number of components in aeo in earlier studies varied from 11 to 77 compounds (27-32) while we were able to identify 111 components in the essential oil due to timely gc analysis. our research showed lc50 and lc90 of aeo as 7 and 34 ppm against an. stephensi, respectively. reviewing other reports have shown different values for other essential oils against an. stephensi. depending on the obtained results, lc50 values are summarized as follows: lc50 < 10 ppm: 1 eo (kelussia odoratissima), 10 ppm < lc50 < 50 ppm: 22 eos (c. zeylanicum (11), ar. dracunculus, platycladus orientalis, tagetes patula, ferulago carduchorum, chloroxylon 343 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... swietenia, ipomoea cairica, feronia limonia, chloroxylon swietenia, foeniculum vulgare, satureja bachtiarica, bunium persicum, plectranthus amboinicus, citrus aurantium, plectranthus mollis, achillea kellalensis, citrus paradisi, anethum graveolens, achillea wilhelmsii, zingiber nimmonii, zingiber cernuum and blumea eriantha) 50 ppm < lc50 < 100 ppm: 14 eos (murraya exotica, syzigium aromaticum, zanthoxylum armatum, zhumeria majdae, origanum scabrum, boswellia ovalifoliolata, lavandula gibsoni, origanum vulgare, lawsonia inermis, cionura erecta, cupressus arizonica, trachyspermum ammi, eucalyptus camaldulensis, coccinia indica) and lc50 > 100 ppm: 9 eos (kadsura heteroclita, stachys byzantina, heracleum persicum, coriandrum sativum, stachys setifera, thymus vulgaris, stachys inflata, ajuga chamaecistus tomentella and cedrus deodara) (10). according to proposed categories of larvicidal activity of plant essential oils against mosquito larvae, aeo lies in the third category as an active plant (33). in previous work, lc90 and lc50 of a. repens extract against an. stephensi were 0.37 ppm and 3.39 ppm, against culex pipiens were 3.5 ppm and 60 ppm and against cx. quinquefaciatus were 4 ppm and 39.7 ppm, respectively (22). with the help of nano-techniques, the stability of essential oils in nature increases. additionally, nanoproducts cause faster absorption in the target insect (25, 34). in a report, nanoemulsions of azadirachta indica essential oil with different particle sizes (31, 93 and 251 nm) were prepared and tested against cx. quinquefasciatus. the nanoemulsion with smallest particle size was found to be the most effective larvicidal agent (35). in another study, nanoemulsion of ar. dracunculus essential oil was investigated against an. stephensi. the size of the prepared nanoemulsions was 12 to 291 nm. similar to the above, larvicidal properties of the nanoemulsion increased significantly with decreasing droplet size (18). previous studies had shown good results of new nanoformulations as larvicides (11), although all prepared nanoformulations had no similar effects. it is possible that different effects can be observed among different plant natural products and their formulations. in this experiment, the comparison has been made between the larvicidal activity of bulk essential oil and its nanoemulsion against one of the main vectors spreading malaria, an. stephensi. in this study, aeo showed complete mortality of larvae for up to 4 days, while its corresponding nanoemulsion failed to indicate 100% mortality even on the first day after diluting 100 times. besides, the bulk preparation showed more larvicidal effect compared with the nanoemulsion after diluting 200 times (i.e. 88% vs. 44%). in a similar study, nanoemulsion of artemisia dracunculus essential was broken or at least showed substantial changes in its nanostructures; it was not able to show a change in larvicidal activity of the essential oil (18). in another study, after dilution, by breaking nanostructure of anethum graveolens essential oil, practically, no difference may be determined between nanoemulsion and bulk essential oil (36). in total, our nanoemulsion preparation failed to show good efficacy compared with the bulk essential oil. to investigate the possible reason, we measured the particle size after 200 dilutions and found that the nanoemulsion breaks up after dilution. in other studies, nanoemulsions of essential oils have been tested against larvae. the results appear to be promising. for instance, nanoemulsion of copaifera duckei (37), rosmarinus officinalis (32) and ocimum basilicum (38) have shown potential against ae. aegypti however, considering the reports, the nanoemulsions have not been diluted 100 or 200 times (as recommended by who). additionally, in these studies, the results of nanoemulsions have not been compared with the bulk essential oils. it is arguable that by performing the studies similar to ours, the nanoemulsions would not indicate positive results. based on the result of the current study, difficulty in obtaining aeo and the negative larviciding results, we therefore do 344 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 333–346 s firooziyan et al.: preparation of... not recommend considering aeo as a good candidate for the next studies. however, it is suggested that for performing the larvicidal studies, nanoemulsions which are stable after 200 dilutions, should be considered. different extractions of the following iranian native plants were evaluated against main malaria vector, an. stephensi, such as mentha spicata, cymbopogon olivieri, azadirachta indica, melia azedarach, tagetes minuta, calotropis procera, eucalyptus camaldulensis, cupressus arizonica, thymus vulgaris, lawsonia inermis, cedrus deodara, cionura erecta, bunium persicum, carum carvi, artemisia dracunculus, rosmarinus officinalis. (39-44). world health organization recommended several biological and chemical insecticides for mosquito larval control including: bacillus thuringiensis h-14, b. spahericus, chlorpyrifos, chlorpyrifos-methyl, deltamethrin, diflubenzuron, etofenprox, fenitrothion, fenthion, fuel oil, malathion, methoprene, permethrin, phoxim, pirimiphos-methyl, pyriproxyfen, temephos, and triflumuron (45). monitoring and mapping of insecticide resistance is appr-opriate measure for vector control. conclusion the larvicidal effects of aeo compared to its nanoformulation against an. stephensi larvae reported. according to the lc50 and lc90 of aeo, it is considered an active natural product. however, the prepared nanoemulsion did not show even equal efficacy in comparing with aeo, probably due to instability after 200 times dilution. use of nanoemulsions with better stability profiles or other types of nanoparticles such as polymeric ones may be suggested. furthermore because of the increasing importance of these alternative larvicides for vector control, the study and screening of native plant natural products should not be neglected. acknowledgment this research has been supported by tehran university of medical sciences & health services grant no. ir. tums. vcr. rec. 1397. 584. conflict of interest the authors declare that there is no conflict of interest. references 1. who. global plan for insecticide resistance management in malaria vectors: world health organization; 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(2006). pesticides and their application for the control of vectors and pests of public health importance, pp125. preparation of a nanoemulsion of essential oil of acroptilon repens plant and evaluation of its larv abstract key words introduction materials and methods collection, identification and extraction of acroptilon repens collection and identification of plant extraction of essential oil analysis of essential oil by gas chromatography-mass spectrometry (gc-ms) mosquito rearing preparation of nanoemulsion determining the larvicidal efficacy determining the duration of action of bulk and nanoemulsions of a. repens essential oil statistical analysis results determination of chemical composition of a. repens essential oil characterization of aeo nanoemulsion larvicidal bioassay of a. repens essential oil discussion conclusion acknowledgment conflict of interest references j arthropod-borne dis, 2012, 6(1): 86–89 r dehghani et al.: wound myiasis due to … case report wound myiasis due to musca domestica (diptera: muscidae) in persian horned viper, pseudocerastes persicus (squamata: viperidae) r dehghani 1, *mm sedaghat 2, m sabahi bidgoli 1 1dept of environment health, kashan university of medical sciences, iran 2dept of medical entomology, school of public health, teheran university of medical sciences, iran (received 5 feb 2011; accepted 12 oct 2012) abstract a case of myiasis due to musca domestica describes in pseudocerastes persicus for the first time. the snake was found in bari karafs, kashan, iran, with a lesion on its body. fourteen live larvae of m. domestica removed from its wound. this is the first report of a new larval habitat of m. domestica. keywords: myiasis, pseudocerastes persicus, musca domestica, muscidae introduction housefly, musca domestica linnaeus, is a synanthropic species and lives in close relations to human. it is usually considered as a mechanical vector of diseases and is capable to transfer hundreds of pathogenic organisms to human. musca domestica is equally attracted to human food sources or animal wastes (nmorsi et al. 2007, butler et al. 2010). housefly as an opportunistic species can deposit its eggs in various types of moist, decaying organic materials such as compost, garbage, feces, fresh and decaying fruit, most human food, and even carrion. in some cases females lay their eggs on soft tissue wound(s) of human or other animals, called myiasis. myiasis is an important parasitic infestation affecting man and domestic or wild animals in many parts of the world. it can define as "the infestation of live vertebrate animals with dipterous larvae, which, at least for a certain period, feed on the host's dead or living tissue, liquid body substances, or ingested food" (zumpt 1965). it may occur in any part of the world and according to the species of fly may involve obligate, facultative or in some cases accidental parasitic infestations. although m. domestica is found everywhere, but the case of facultative myiasis of this species is rare. there are just few case reports of facultative myiasis in human caused by m. domestica (dogra and mahajan 2010). however, intestinal myiasis, which is accidental myiasis or pseudomyiasis, has been reported in human (sehgal et al. 2002). human cases of pseudomyiasis occur when the larvae contaminates food or drink and passing through the alimentary canal. most cases of myiasis due to m. domestica are facultative myiasis, which may occasionally contaminate living tissue. dermal and sub-dermal myiasis is an important myiasis in animals, which are the invasion of diptera larvae to skin tissues. *corresponding author: dr mohammad mehdi sedaghat, e-mail: sedaghmm@tums.ac.ir 86 j arthropod-borne dis, 2012, 6(1): 86–89 r dehghani et al.: wound myiasis due to … dermal myiasis has been reported in sheep caused by m. domestica in the middle east (amin et al. 1997, schnur et al. 2009). in the present communication, a rare case of snake myiasis due to m. domestica describes. case a 70 cm of persian horned viper was presented to medical entomology lab, department of environment health, kashan university of medical sciences in summer 2010. the snake was captured in bari karafs (34º 02′ n, 51º 03′ e) near mashhad ardehal in west of kashan, isfahan province, central of iran. the area has a warm and dry weather and marginalized from the north and east to the desert. the snake was identified pseudocerastes persicus using the key of mc diarmid et al. 1999. it had a broad head with the short and rounded snout. the supraorbital hornlike structures was obvious above of each its eye. the color of the viper was gray. the snake had a lesion of 10 mm diameter on the dorsal surface of the posterior mid-body (fig. 1a, b). the wound started to be infected shortly thereafter. a close up view revealed its infestation with larvae of dipterous fly. after removing all larvae, they kept to complete their metamorphosis. ten live larvae and 4 pupae of dipterous fly were subject to the further study (fig. 1c). the collected larvae were processed for micro-slide preparation of their spiracles. the pupae were kept in a dry container in laboratory condition to emerge the adult fly. the adult flies emerged after 3 days. the identification key of zumpt (1965) was used to identify the larvae and adults. the larvae were creamy-white and cylindrical with broad and flattened posterior end and tapering anteriorly toward the head. each larva had one pair of dark hooks, with 12 apparent segments. the openings of posterior spiracles had sinuous slits and the peritreme of each spiracle was "d" shape with a button. the four adults were gray, with four longitudinal dark lines on the back. the morphological study of the spiracles of fourth instar larvae and the emerged adults revealed that all were musca domestica. the snake had poor appetite while it was wounded. the wound washed and cleaned with 10% povidone-iodine and then treated with tetracycline topical ointment 3%. the snake had been weakened when it was first found and was not fed during the first week in the laboratory. in the second week, live rock geckoes were accessible to the snake in its container. the persian horned viper started to eat its live prey soon after that. after three weeks treatment and taking care of the snake, it had been recovered but a scar remained on the skin (fig. 1d). the persian horned viper is still alive and is kept at department of environment health, kashan university of medical sciences for further study. fig. 1. a lesion with 10 mm diameter in the dorsal surface of persian horned viper (a and b), larvae and pupae of the dipterous fly after culturing, out of the lesion (c), picture of the same area three weeks after treatment (d) (original photos) 87 j arthropod-borne dis, 2012, 6(1): 86–89 r dehghani et al.: wound myiasis due to … discussion snake myiasis has been reported rarely. although dipterous larvae in dead body of animals and snake carrions are usual, but few myiasis among live snakes has been reported. an earlier report described a case of dermal myiasis due to cuterebra spp. (cutebridae) in crotalus viridis abyssus (garrigues 1964). a case of snake myiasis caused by megaselia scalaris (phoridae) was described in brazil (da silva et al. 1999). in this study, we described a rare snake dermal myiasis due to m. domestica in central iran. musca domestica has been incriminated as a myiasis agent in many parts of the world. it has been found in human or animals tissues. although the adult flies are not parasitic but they can lay their eggs on necrotic tissues or wounds. such kind of myiasis is unusual in good sanitation and clean environment; however, some cases occurred on domestic or wild animals. persian horned viper is distributed throughout the middle east and usually lives far from human in the desserts. pseudocerastes persicus is a native species in iran and its myiasis has not seen before. in this case, m. domestica has been attracted to the infected wound of the snake. the female housefly deposited her eggs in the moist and decaying snake wound and then the larvae migrated into the deep part of the wound. injuries in man and animals are the most important sources to attract gravid flies. in wild animal injuries due to fighting, wounds and the site of the ticks or other arthropods bite are potential sources for invasive of dipterous flies. moreover, rat bite injury may provide substrate for the developing larvae in snake. myiasis is a widespread and completely preventable veterinary problem in livestock or pet animals. besides, the infestations of wild animals with dipterous larvae can affect health of livestock or pet animals as well as human. although, fly larvae have been found in snake carcass (moretti et al. 2009), this species commonly have no myiasis. we suppose this myiasis occurred because the snake previously had been wounded and the moist around the wound attracted the female fly to laying her eggs. this report reveals the new larval habitat of m. domestica that previously has not reported. in this case, pseudocerastes persicus saved successfully with washing the wound with a disinfectant and treatment with topical ointment antibiotic. however, in all cases of dermal myiasis early treatment is a key factor in treatment and prevention of the problem. acknowledgements the authors gratefully acknowledge mr keresi for providing, his assisting in treatment and taking the pictures of the snake, mr varasteh, mr abdolahi, mr hossienkhah, mr davari and mrs sabbaghian the staffs of medical entomology lab, department of environment health, kashan university of medical sciences. the authors also are grateful to dr roya sadeghi, school of public health, teheran university of medical sciences, iran, for critically reviewing the manuscript. the authors declare that there is no conflict of interests. references amin ar, shoukry a, morsy aa, mazyad sa (1997) studies of wound myiasis among sheep and goats in north sinai governorate, egypt. j egypt soc parasitol. 27: 719–737. butler jf, garcia-maruniak a, meek f, maruniak j e (2010) wild florida house flies (musca domestica) as 88 j arthropod-borne dis, 2012, 6(1): 86–89 r dehghani et al.: wound myiasis due to … carriers of pathogenic bacteria. florida entomologist. 93: 218–223. da silva rj, do prado ap, roderigues rr, de magalha˜ eslopes ca, conde godoy wa (1999) megaselia scalaris (diptera: phoridae) causing myiasis in crotalus durissus terrificus (serpentes: viperidae) in brazil. j med entomol. 36: 630. dogra ss, mahajan vk (2010) oral myiasis caused by musca domestica larvae in a child. int j pediatr otorhinolaryngol extra. 5: 105–107. garrigues rm (1964) a cuterebra (diptera: cuterebridae) infestation in the grand canyon rattlesnake, crotalus viridis abyssus, with a list of those recorded from other hosts. trans kan acad sci. 67: 689–692. mc diarmid r, campbell ja, touré t (1999) snake species of the world: a taxonomic and geographic reference, vol. 1. herpetologists' league, washington dc. nmorsi opg, agbozele g, ukwandu ncd (2007) some aspects of epidemiology of filth flies: musca domestica, musca domestica vicina, drosophilia melanogaster and associated bacteria pathogens in ekpoma, nigeria. vectorborne zoonotic dis. 7: 107–117. moretti tc, allegretti silmara m, mellopatiu cátia a, tognolo alexandre m, ribeiro odairb, solis daniel r (2009) occurrence of microcerella halli (engel) (diptera, sarcophagidae) in snake carrion in southeastern brazil. rev bras entomol. 53: 318–320. schnur hj, zivotofsky d, wilamowski a (2009) myiasis in domestic animals in israel. vet parasitol. 161: 352–355. sehgal r, bhatti hp, bhasin dk, sood ak, r, malla n, singh k (2002) intestinal myiasis due to musca domestica: a report of two cases. jpn j infect dis. 55: 191–193. zumpt f (1965) myiasis in man and animals in the old world. butterworths, london. 89 j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 196 http://jad.tums.ac.ir published online: june 30, 2021 original article current status of insecticide susceptibility in the principal malaria vector, anopheles gambiae in three northern states of nigeria mustapha ahmed yusuf 1,2; mohammad ali oshaghi1; *hassan vatandoost1,3; *ahmad ali hanafi-bojd1,4; ahmadali enayati5; rabiu ibrahim jalo6; akande oyebanji azeez aliyu aminu2; isa muhammad daneji2 1department of medical entomology and vector control, school of public health, international campus, tehran university of medical sciences, tehran, iran 2department of medical microbiology and parasitology, college of health sciences, bayero university, kano, nigeria 3department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4zoonoses research center, tehran university of medical sciences, tehran, iran 5department of medical entomology, school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 6department of community medicine, college of health sciences, bayero university, kano, nigeria *corresponding authors: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, dr ahmad ali hanafi-bojd, e-mail: aahanafibojd@tums.ac.ir (received 14 jul 2020; accepted 01 jun 2021) abstract background: malaria is a major public health problem in nigeria with 97% of its population with high morbidity and mortality. mosquitoes play an important role in the transmission of malaria parasites. this study was conducted to evaluate the current resistance status of anopheles gambiae to insecticides. methods: larvae of an. gambiae was collected from three zones; a, b and c differentiated on the basis of variation in agricultural ecosystems between august and november, 2018 in the northeast and northwestern parts of nigeria. they were carefully reared to adult stage and insecticidal susceptibility tests were conducted. results: the mosquitoes tested showed high levels of resistance to all the insecticides used with the exception of malathion. study zone a, recorded 74% mortality after 24h to deltamethrin compared to 81% from zone b and 82% from zone c, respectively. mosquitoes from zone b exposed to ddt had the highest level of resistance at 37% compared to 40% and 53% from zones a and c, respectively. resistant to bendiocarb was also observed, with zone a having the lowest mortality of 44% compared to 48% from zone c and 55% from zone b, respectively. according to the results of knockdown tests, mosquitoes from zone a exposed to deltamethrin recorded the lowest knockdown across the study locations while zone b recorded the lowest knockdown for ddt. conclusion: the results of the study provide an insight into the current status of an. gambiae to four major insecticides in northern nigeria as guideline for mosquitocontrol. keywords: malaria; anopheles gambiae; insecticide resistance; susceptibility; northern nigeria introduction malaria is a life-threatening parasitic vector-borne disease troubling many countries in the tropical and subtropical regions of the world (1). with over 200 million new cases between 2010 and 2017, africa still carries the highest burden (92%) worldwide compared to southeast asia (5%) and the eastern mediterranean regions (2%), respectively (2-3). nigeria, democratic republic of congo, mozambique, india and uganda account for nearly half of all copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com mailto:aahanafibojd@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 197 http://jad.tums.ac.ir published online: june 30, 2021 malaria cases worldwide (3). in 2017 alone, there were reported cases of increased malaria transmission (more than half a million cases) from countries with highest burden in africa (nigeria, democratic republic of congo and madagascar) compared with the year 2016 (3). malaria is a serious problem in nigeria with approximately 100 million cases and over 200,000 deaths annually (45). it also accounts for nearly 60% of outpatients visits, 30% of under-five hospitalization and contributes to 11% maternal mortality, making nigeria the country with the highest burden in the world (4-5). the malaria parasite is mainly transmitted in africa by the anopheles gambiae s.l. and the major vectors are an. gambiae and an. funestus species complexes (6-9). anopheles gambiae forms a species complex comprising of eight morphologically indistinguishable, i.e., identical sibling species in the series pyretophorus in the anopheles subgenus cellia across sub-saharan africa (6, 10). the individual species of the complex exhibit similar traits and are closely related, making it very hard to be identified morphologically except for some few larvae and adult females (11). the an. funestus complex is comprised of nine sibling species of which an. funestus sensu stricto (s.s.) is the principal vector with very high density and found across different geographical regions (12). however, the an. gambiae s.l. complex is the most widely distributed anopheles mosquitoes in nigeria (65.2%) followed by the an. funestus (17.3%), respectively (13). the main control measures adopted in nigeria for malaria vectors are insecticide-treated nets (itns)/long-lasting insecticide-treated nets (llins) (14). these methods when properly used against insecticide susceptible mosquito populations contribute significantly in the reduction of malaria cases (15-16). other vector control measures used for malaria prevention in nigeria include personal protective measures such as the use of repellents and house screening (14). there is a serious threat to these control measures as a result of progression of insecticide resistance to major malaria vectors across malaria endemic countries including nigeria (14, 17). insecticide resistance is defined as the ability of some organism to tolerate a specific dose of a toxic substance that will be deadly to other organisms of the same species and from the same environment (18). over the years, the problem of insecticide resistance is progressing and involving more classes of insecticide, and this can significantly affect the strength of vector control programs leading to failure, thus resistance management is designed to delay or prevent insecticide resistance (19). an important part in the resistance management strategies is identifying the resistance and mechanisms involved by obtaining the baseline information about the vector susceptibility, detection of resistance in the early stage and monitoring its frequency levels over time (20-21). an integrated approach where two or more methods are employed in the vector control programs could help in delaying the sustain progression of the resistance (20). a study conducted in 2013 at bichi (northern nigeria) reported a high resistance of an. gambiae with significant elevation of detoxification enzymes in deltamethrin and bendiocarb resistance strains compared to susceptible species from agricultural and residential areas (22). a similar study from bichi was conducted in 2015 to assess the level of resistance against bendiocarb, permethrin and ddt. very high resistance to permethrin and ddt was reported with less resistant to bendiocarb (14). resistance to permethrin and ddt exposed an. gambiae s.s across all the geographical zones of nigeria with the highest level of the resistance in the forest savannah, mosaic and guinea savanna has been reported (18). the resistance profile and kdr mutation of an. gambiae s.l. populations was also reported from two locations (auyo and bunkure) in northern nigeria (13). other studies have also reported resistance to commonly used insecticides from http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 198 http://jad.tums.ac.ir published online: june 30, 2021 nigeria (6-7). also insecticide resistance was reported in this species from the neighboring country ghana (23-24). periodic monitoring of susceptibility status of an. gambiae to insecticides used in public health practice is vital and will guide stakeholders towards the procurement and strategy used in vector control programs. the aim of this study was to examine the current situation of insecticide susceptibility in the principal malaria vector, an. gambiae s.l. in northern nigeria. materials and methods study area the study was conducted across three different locations within three states (gombe, jigawa and kano) in northeast and northwest nigeria from august to november, 2018 (fig. 1). the locations were designated as study zones a, b and c differentiated by the type of vegetative zone, i.e., a: lies within the sudan, northern and southern guinea savanna; b: is found within the sudan, sahel and northern guinea savanna; and c, lies within the sudan and northern guinea savanna (6, 13). zone a, yamaltu deba (10° 13′ 0″ n, 11° 23′ 0″ e) is one of the 11 local government areas in gombe state, nigeria. it has a population of 255,248 and an area of 1,981 km². gombe state (10° 15′ 0″ n, 11° 10′ 0″ e) is situated in the north-eastern part of nigeria (25, 26). the state shares common borders with borno, yobe, taraba, adamawa and bauchi states. the state has two distinct climates, the dry season (november–march) and the wet season (april–october) with average annual rainfall of 850mm (27). zone b, auyo (12°21′n, 9°59′e) is a locality in jigawa state, north-western nigeria within the sudan and guinea savannah with pockets of sahel savannah. the town is known for its irrigation activities in which rice and vegetables are produced. it has a total population of 132,001 and estimated land mass area of 512km (13, 26). zone c, kumbotso (11°53′17′′n, 8°30′10′′e) is situated in kano state, north-western nigeria in the sudan and guinea savanna with a population of 409,500 and an area of 158km2 (18, 26). the temperature is generally warm, and the annual rainfall is about 1,300mm between april and september (28). study sample sampling was conducted from different breeding places in the study areas using dipping method as described by habibu, 2017 (14) in order to provide laboratory stock of mosquitoes. the samples were transferred to the insectary at bayero university, kano with a rearing condition of 28±2 °c temperature, 65±5% relative humidity (rh) and 12:12h d: l. two to three days old female sugar-fed mosquitoes were used for susceptibility tests (29). the inclusion criteria used for the selection of the sampling areas include: history of pesticide and herbicides use on the land from agriculture activities, vector control and irrigation activity, availability and high density of target species, high intensity of malaria transmission, and paucity of data on susceptibility profile of the malaria vector an. gambiae s.l. susceptibility test papers the impregnated test papers recommended by who used in this study were purchased from the vector control research unit, school of biological sciences, universiti sains malaysia 1800 minden, penang, malaysia. who susceptibility test the current who susceptibility bioassay guideline (21) was followed. at least 120–150 female mosquitoes were aspirated from the mosquito cage into six holding tubes giving six replicate samples of 20–25 mosquitoes per tube. with the mosquitoes in the tubes, the slide unit was immediately closed and the holding tubes set in a vertical position for one hour. damaged mosquitoes were removed at the end of the one hour exposure time. the exposure tubes http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/borno_state https://en.wikipedia.org/wiki/yobe_state https://en.wikipedia.org/wiki/taraba_state https://en.wikipedia.org/wiki/adamawa_state https://en.wikipedia.org/wiki/bauchi_state j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 199 http://jad.tums.ac.ir published online: june 30, 2021 were lined with a sheet of insecticide-impregnated paper, while the plain control tubes were lined with oil-impregnated papers, provided for each group of insecticides by who, fastened into position with a copper spring-wire. the plain holding tubes containing the mosquitoes were attached to the vacant position on the slides and with the slide unit opened, the mosquitoes were blown gently into the exposure tubes containing the following insecticide treated papers with discriminating dosages: ddt 4%, bendiocarb 0.1%, malathion 5% and deltamethrin 0.05 %. the exposure tubes containing all the mosquitoes were set aside in a vertical position for one hour after which they were transferred back to the holding tubes by reversing the procedure outlined. knockdown was recorded on deltamethrin and ddt from all the study zones. a pad of cotton-wool soaked in sugar solution 5% was placed on the mesh-screen end of the holding tubes and the mosquitoes were kept in the recovery period for 24 hours. during this time, the holding tubes were kept in the insectary. after the recovery period, the mortality was scored and recorded. a mosquito is considered alive if it is able to fly, regardless of the number of legs remaining (21). data analysis the 24h mortality was accessed manually, while the susceptibility was defined as: 98–100 % mortality indicates susceptibility, 90–97% mortality requires confirmation of resistance and between 0–89% suggests resistance (21). microsoft office excel, version 2013 was used to create charts, sort and clean the data. while statistical package for the social sciences (spss) version 16 was used to calculate the 95 % confidence interval and the means of the variable using the student’s t test. abbott’s formula (30) was used to correct for natural mortality if the control mortality was between 5 and 20%. the results of the tests with >20% mortality in controls were discarded and the test repeated (31). results a total of 1800 f0 2–3 days old adult female an. gambiae s.l. mosquitoes were used for the bioassays. anopheles mosquitoes from all the three zones were only susceptible to malathion and resistant to ddt, bendiocarb and deltamethrin (table 1, fig. 2). a mortality of 74% (95%, ci: 68–79) was recorded in zone a to deltamethrin compared to 81% (ci: 72– 89) from zone b and 82% (ci: 76–87) from zone c (fig. 2). this study area had a comparatively lower mortality to deltamethrin, indicating a relatively higher resistance to this insecticide. resistance by an. gambiae to bendiocarb in nigeria is usually moderate but was found to be very high in all the three locations with zone a having the lowest mortality of 44% (ci: 38–49) compared to 48% (ci: 44–51) from zone c and 55% (ci: 53–56) from zone b, respectively (table 1). the organophosphate, malathion was the only insecticide mosquitoes from all the study locations were susceptible to, with 100% mortality both in zones b and c, while zone a recorded 99% (fig. 2). according to the results of knockdown tests, mosquitoes from zone a exposed to deltamethrin recorded the lowest knockdown at 30 minutes 32% (standard error, se= 0.333), compared to 37% (se= 0.344) and 48% (se= 0.355) from zones b and c, respectively (fig. 3). similarly, at 60 minutes zone a mosquitoes still recorded the lowest knockdown 44% (se= 0.354) compared to 51% (se= 0.357) and 56% (se= 0.355) from zones b and c, respectively (fig. 3). comparatively, from the deltamethrin knockdown result observed, zone a recorded the lowest knockdown rate at various time intervals shown above, which is in line with the low percentage mortality observed to deltamethrin after 24h 74% (ci: 68–79) from this study area (figs. 2, 3). the highest percentage knockdown to deltamethrin was observed with mosquitoes from zone c, also in keeping with the high percentage mortality to this insecticide http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 200 http://jad.tums.ac.ir published online: june 30, 2021 after 24h 82% (ci: 76–87) recorded from this locality (figs. 2, 3). the highest level of ddt resistance was 37% (ci: 34–39) and seen with mosquitoes from zone b compared to 40% (ci: 37–42) and 53 % (ci: 49–56) from zone a and zone b, respectively (fig. 4). comparatively, mosquitoes from zone b recorded the lowest mortality to ddt, signifying that they are highly resistant to ddt. similarly, in confirming the aforementioned statement, these mosquitoes recorded the lowest knockdown of 2% (se= 0.1) at 30 minutes and 9% (se= 0.204) at 60 minutes compared to 3% (se= 0.122) and 17% (se= 0.268) from zone c and 8% (se= 0.2) and 8% (se= 0.274) from zone a, respectively (fig. 4). the high knockdown observed to ddt with mosquitoes from zone a was in keeping with the high percentage mortality observed with mosquitoes from this study area to the organochlorines (figs. 2, 4) and was statistically significant (p< 0.05). fig. 1. map showing the geographical locations of the study sites in nigeria, 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 201 http://jad.tums.ac.ir published online: june 30, 2021 fig. 2. percentage mortality of anopheles gambiae s.l. collected from some localities in nigeria after 24h exposure to insecticides, 2018 fig. 3. knockdown profile of anopheles gambiae s.l. mosquitoes collected from northern nigeria exposed to deltamethrin 0.05% after 60 minutes, 2018 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 202 http://jad.tums.ac.ir published online: june 30, 2021 table 1. susceptibility status of anopheles gambiae s.l. at 95% confidence interval with the mean and standard deviation collected from the study zones in nigeria after 24h exposure to different insecticides, 2018 mortality (%) locality insecticide exposed mean ± sd control 95% confidence interval resistant status zone a deltamethrin 0.05% 100 74.00±3.13 4.00±0.65 68–79 r ddt 4% 100 53.00± 3.56 0.00±0.00 49–56 r malathion 5% 100 99.00± 0.71 0.00±0.00 97–100 s bendiocarb 0.1% 100 44.00± 3.21 0.00±0.00 38–49 r zone b deltamethrin 0.05% 100 81.00±2.80 4.00±0.65 72–89 r ddt 4% 100 37.00±3.44 0.00±0.00 34–39 r malathion 5% 100 100.00±0.71 0.00±0.00 100 s bendiocarb 0.1% 100 55.00±3.55 0.00±0.00 53–56 r zone c deltamethrin 0.05% 100 82.00±2.74 2.00±0.48 76–87 r ddt 4% 100 40.00±3.49 0.00±0.00 37–42 r malathion 5% 100 100.00±0.71 0.00±0.00 100 s bendiocarb 0.1% 100 48.00±3.56 0.00±0.00 44–51 r r: resistant; s: susceptible fig. 4. knockdown profile of anopheles gambiae s.l. mosquitoes collected from northern nigeria exposed to ddt 4% after 60 minutes, 2018 discussion in the assessment of the resistance level to four insecticides viz; deltamethrin, ddt, mal athion and bendiocarb by an. gambiae mosquito during the present study, bendiocarb showed http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 203 http://jad.tums.ac.ir published online: june 30, 2021 resistance across all the study area, with zone a having the lowest percentage mortality followed by zone c and b respectively. this finding agrees with studies from the north-eastern state of gombe in the sudan savanna of nigeria (7), where they reported a percentage mortality range of 2.3–100%. similarly, a study from the forest zone vegetation of kumasi in ghana reported 38–56% mortality to bendiocarb (24). another study from the coastal savanna vegetation of ghana reported a mortality rate of 12.3 % to bendiocarb (23). most of the studies conducted in the sudan, guinea, sahel savanna and humid forest reported moderate resistance to full susceptibility to bendiocarb (13-14). the major challenge arising from this finding is that the initial thought of using carbamate as an alternative to the rapid spread of pyrethroids resistance may no longer hold (23-24, 32). this study also reports resistance to the pyrethroids (deltamethrin) from the study sites with zone a having the lowest percentage mortality followed by zone b and c, respectively. this finding is in agreement with studies conducted in the northern guinea savanna of nigeria (33) where they reported percentage mortality of 83%, and from the sudan through guinea and some parts of sahel savanna (13) where 78% mortality to deltamethrin was reported. however, a study conducted in the sudan savanna disagrees with our finding where a very high resistance of 38% mortality to deltamethrin was published (22). also, another study from the forest zone vegetation of kumasi in ghana reported very high resistance in the range of 15– 46% (24). recently, high resistance to deltamethrin in the range of 1–70% from the sudan and sahel savanna was reported in nigeria (67). a rise in pyrethroids resistance by mosquitoes is becoming a big threat to the vector control strategy in nigeria, thus necessary and urgent steps need to be taken to avoid complete failure of the measures (18). the resistance profile of the mosquitoes from all the three study locations to ddt shows a high level of resistance, with zone b having the highest level followed by c and a. this finding is in agreement with previous studies from sudan, guinea and sahel savanna (6-7, 13-14, 16, 33). similarly, very high resistance in the range of 12–46% was reported from the forest zone and coastal vegetation of ghana (23-24). in this study, malathion was the only insecticide found to be very active against the mosquito vector tested across all the study locations and agrees with previous studies from within and outside nigeria (6, 13, 24, 32, 34). a slow knockdown to deltamethrin, that increased with increasing time of exposure was observed from all the zones with mosquitoes from zone a having the lowest knockdown followed by zone b and then c, respectively. this finding agrees with previous studies reported (35). ddt also recorded very low knockdown from all the study locations. this finding is similar to previous reports (13, 35). however, a previous study conducted at bichi in kano-nigeria reported high knockdown to ddt (14). the high level of insecticide resistance by an. gambiae observed may be associated with increased use of pyrethroids treated bed nets (22). this is because farmers in these study locations use a wide range of pesticides and herbicides to protect their crops, and these pesticides marketed under different trade names belong to all the chemical classes including organophosphates, organochlorine, pyrethroids and carbamates (16). the insecticide resistance observed in this study along with previous reports of resistance from these locations involving three major classes (carbamate, organochlorine, pyrethroids) of insecticides used in public health practice is disturbing and may have a negative effect on the malaria control programme which ultimately can lead to failure of the vector control programme (6-7, 14, 33). resistance management consisting of all available measures designed to delay or prevent resistance should be implemented in all the malaria endemic zones as soon as possible (18, 24). some methods that can also limit the progression of insecticides resistance to mosquito vectors of malaria inhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 196–206 m ahmed yusuf et al.: current status of … 204 http://jad.tums.ac.ir published online: june 30, 2021 clude: altering the dose and frequency of pesticide application in areas with high seasonal transmission; applying different formulations; avoidance of slow release formulations and identify new pesticides with an alternate mechanism of action (18). conclusion the results of the study provide an insight into the current status of an. gambiae to four major insecticides in northern nigeria. this may form a new baseline data for further studies on other classes of insecticides that can be adopted to guide the control of mosquito vectors of malaria in nigeria. acknowledgements we are very grateful to dr sulaiman s ibrahim, eng mr abai for all the support and advice given during this work. the authors would also like to acknowledge contributions made by mr muhammad m mukhtar and nura abubakar for the help rendered in field sample collections and the performance of susceptibility tests. this research is financially supported by international campus, tehran university of medical sciences under code number 30105. the authors declare that they have no competing interests. references 1. world health organization (2020) world malaria report. p. 125. 2. world health organization (2017) achieving and maintaining universal coverage with long-lasting insecticidal nets for malaria control. p. 156. 3. world health organization (2018) world malaria report. p. 125. 4. nigeria malaria indicator survey (2015) fact sheet. p. 12. 5. sheet nmf (2011) united states embassy in nigeria publication. retrieved from http//photos. state. gov//libraries/nigeria/231771/public 6. ibrahim ss, mukhtar mm, datti ja, irving h, kusimo mo, tchapga w, lawal n, sambo f, wondji cs (2019) temporal escalation of pyrethroid resistance in the major malaria vector anopheles coluzzii from sahelo-sudanian region of northern nigeria. sci rep. 9(1): 7395. 7. olatunbosun-oduola a, abba e, adelaja o, taiwo-ande a, poloma-yoriyo k, 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gambiae) breed in auyo residential and irrigation sites northwest nigeria. europe sci j. 11(9): 315– 326. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 63 http://jad.tums.ac.ir published online: march 31, 2023 original article bacterial contamination of collected cockroaches and determination their antibiotic susceptibility in khorramabad city, iran behroz davari1, ali ezat hassanvand1, aref salehzadeh1, mohammad yousef alikhani2, *seyed mostafa hosseini2,3 1department of medical entomology, hamadan university of medical sciences, hamadan, iran 2infectious disease research center, hamadan university of medical science, hamadan, iran 3department of microbiology, hamadan university of medical sciences, hamadan, iran *corresponding author: dr seyed mostafa hosseini, e-mail: smhoseiny88@yahoo.com (received 18 dec 2021; accepted 05 feb 2023) abstract background: cockroaches are one of the most important carriers of pathogenic microorganisms. therefore, the presence of cockroaches in public places, especially in hospitals, homes, and restaurants, is dangerous, and threatens the health of society, people, and the environment. the aim of this study was evaluation of bacterial contamination of cockroaches and the sensitivity of these bacteria to various antibiotics, captured from khorramabad city, iran methods: this descriptive cross-sectional study was performed on 150 cockroaches collected from hospital environments, homes, and restaurants in khorramabad. the outer surface of the cockroaches was washed with physiological saline. the suspension was centrifuged for 5 minutes at 2000rpm. isolation and identification of bacteria was performed using phenotypic methods. antibiotic susceptibility testing was performed by disk diffusion method according to clinical and laboratory standard institute (clsi) guideline. results: a total of 100 american cockroaches (66.66%), 28 b. germanica (18.66%) and 22 blatta orientalis (14.66%) were identified. in total, 97.33% of the collected cockroaches were infected with bacteria. the most bacterial infection of the cockroaches was escherichia coli, coagulase-negative staphylococci and bacillus respectively. the overall results of the antibiogram test showed that the identified bacteria were resistant to cephalothin, ampicillin, cefotaxime, and kanamycin antibiotics, semi-sensitive to ciprofloxacin and sensitive to tetracycline, gentamicin, nitrofurantoin, trimethoprim/sulfamethoxazole, and chloramphenicol. conclusion: infection of cockroaches with pathogenic bacterial agents in hospital, residential, and restaurant environments, as well as the observation of bacterial resistance to some common antibiotics is worrying. keywords: cockroaches; bacteria; antibiotic introduction insects are one of the most populous classes of arthropods, which are important in terms of economic, health, and medical (1). cockroaches belong to the order blattaria that so far about 4,000 species of cockroaches have been identified, less than 1% of which are considered pests. cockroaches live in warehouses, bakeries, baths, hospitals, and even ships and harbors, hiding in dark, narrow crevices and crevices during the day and leaving their shelters at night to feed (2). today, perhaps few places can be found that are safe from the presence of cockroaches, and therefore increases their potential as a harmful health factor (3). this insect is important in medicine due to its habit of vomit (puking) a portion of the food eaten, defecating in the living environment, having weak bites, especially between the fingers, and producing a foul odor, particularly in the american cockroach, which is potentially dangerous to human health. also, secretions and scabs from changing their skin contain aller copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:smhoseiny88@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 64 http://jad.tums.ac.ir published online: march 31, 2023 gens that dermatitis, itching, and many acute respiratory diseases are their complications (4, 5). laboratory studies by using serological methods on cockroach secretions and fecal particles have isolated and identified several allergens from them. cockroaches are the second important cause of asthma after dust allergies (6, 7). the ability of cockroaches to quickly run and move increase their ability to escape the control and transmission methods of pathogens (8). cockroach feces contains compounds such as xanthurenic acid and quinolinic acid, all of which are derivatives of tryptophan and have mutagenic and carcinogenic properties (9). the importance of cockroaches in the spread of nosocomial bacteria and the incidence of nosocomial infections in patients is greater than previously thought. cockroaches are naturally infected with the pathogenic bacteria that cause leprosy, plague, bloody diarrhea, urinary tract infections, pimples, abscesses, and food poisoning. cockroaches are infected with approximately 150 species of bacteria, 60 species of yeast, 90 species of protozoa, 45 species of ringworm, 12 species of pathogen fungi, and several hookworms and whipworms. the most important and prominent carriers of these bacteria are blattella germanica, periplaneta americana, and blatta orientalis (2). in iran twelve species of this family have already been recognized. in terms of the number of species, the family ectobiidae (blattellidae) is the largest order of cockroaches in the world and eight species of this family have been reported from the country. the german roach, b. germanica, the major pest of residential dwellings, belongs to family blattellidae as well (10). cockroaches are omnivores and due to feeding on a variety of foods and even human waste, they can spread diseases such as typhoid, leprosy, dysentery, etc. by transmitting large numbers of harmful microorganisms. these microorganisms live for a long time in the intestines of cockroaches and are eventually transferred to another organism (11-13). one of the most important challenges that the world is facing in the fight against bacteria is their increasing antibiotic resistance. the resistance of microorganisms to antibiotics is coded by different genes that are also capable of horizontal transfer between bacteria. cockroaches can mechanically transfer antibiotic-resistant bacteria from hospital environments to non-hospital areas and vice versa, causing serious problems for public health (14). due to the abundance of cockroaches and their role in the mechanical transmission of various pathogens, and insufficient knowledge of the level of bacterial contamination on their external surface and prevention of transmission of various infections, and raising the level of public health, this research aimed to evaluate of bacterial contamination of cockroaches and the sensitivity of these bacteria to various antibiotics, captured from khorramabad city, iran. materials and methods sampling this is an analytical descriptive cross-sectional study. the study was conducted from march 2019 to march 2020 in khoramabad, iran. the study sites included two hospitals: shohadaye ashayer and shahid madani; and public places (including homes, and restaurants). in this study, 150 cockroaches were captured by hand catch method. after observing the cockroaches, they were transported to the entomology laboratory of hamadan university of medical sciences using sterile gloves. for preventing (cockroaches) mixing contamination with each other, only one cockroach was used in each container. then each sample was placed at 4 °c for 5 minutes for anesthesia. species of cockroaches were determined according to the identification key (14, 15). bacteria isolation firstly, in the laboratory, the outer surface of the cockroaches was washed with 5ml sterhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 65 http://jad.tums.ac.ir published online: march 31, 2023 ile normal saline. then the solution was centrifuged for 5 minutes at 2000g. the sediments that remained at the bottom of the centrifuge tubes were cultured on the various mediums such as eosin methylene blue (emb) and blood agar (merck, germany) (16, 17). cultivated mediums were incubated in 37 oc for 24 hours. in the next step, for each grown colony, all phenotypic differential tests were performed to isolate the bacteria into the genus or species level. for identification of gram-negative bacteria gram stain, fermentation of sugars, motility test, triple sugar iron (tsi) (merck), sulfide indole motility (sim) (merck, germany), and simmon citrate (merck) tests were applied. for the identification of gram-positive bacteria, gram stain, catalase test, oxidase test, fermentation of sugars (mannitol), and sensitivity to antibiotics were employed (18). antibiogram the resistance of bacterial strains isolated from the cockroaches was investigated using the kirby-bauer disk diffusion method (16, 19). a control strain of pseudomonas aeruginosa atcc 27853 was used for quality control of susceptibility testing. from the tested organisms, a suspension with turbidity equal to 0.5 mcfarland standards was provided for each bacterial strain and transferred to muller-hinton agar medium (merck), and then antibiotic discs including: ampicillin (30µg), tetracycline (30µg), kanamycin (30µg), chloramphenicol (30µg), trimethoprim-sulfamethoxazole (23.75–1.25µg), gentamicin (30µg), ciprofloxacin (5µg), nitrofurantoin (300µg), cephalothin (30µg) and cefotaxime (30µg) that all antibiotic discs were purchased from mast group ltd., (england) and incubated for 24 hours at 37 °c. after 24 hours, the diameter of the growth inhibition zone was measured. the diameter of the growth inhibition zone of the microorganisms for each antibiotic was divided into sensitive, semi-sensitive, or resistant according to the clinical and laboratory standards institute (clsi) guidelines (20). finally, data were analyzed using spss version 20 software package developed by ibm cooperation, using chi-square tests. p< 0.05 was regarded as significant. the amount of bacterial contamination as well as the pattern of their resistance to antibiotics is checked with the type and location of collection of cockroaches. the relationship between the place of isolation of cockroaches (home, hospital, and restaurant) with the frequency of cockroaches and also the dominant species was determined. results the captured cockroaches and extracted bacterial strains 100, 28, and 22 cockroaches were caught from hospitals, homes, and restaurants, respectively. a total of 146 bacterial colonies or strains were isolated from the 150 caught cockroaches. table 1 shows the frequency of bacteria in each location. out of 150 cockroaches, 100 (66.6%) were p. americana, 28 (18.6%) b. germanica, and 22 (14.66%) blatta orientalis. in total, 97.33% of the collected cockroaches were infected with bacteria. the highest bacterial infection of cockroaches was e. coli, and then coagulase-negative staphylococci and bacillus species were the most common bacterial infections. statistical analysis showed that there is a statistically significant relationship between different places of cockroach collection and the rate of bacterial infection (p< 0.05). the results of investigating the relationship between bacterial isolates and cockroaches are shown in figure 1. out of 33 e. coli, 25 strains were isolated from the p. americana, and 15 strains out of 19 enterococcus spp. were isolated from the p. americana, which was statistically significant (p <0.05). the distribution of other bacteria in cockroaches was uniform. sensitivity of bacteria to antibiotics according to the results, it was found that the bacteria isolated from the hospital had a http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 66 http://jad.tums.ac.ir published online: march 31, 2023 higher resistance to antibiotics than the bacteria isolated from homes and restaurants (table 2), which is a statistically significant difference (p< 0.05). table 1. frequency of bacteria isolated from three cockroach species (p. americana, b. germaniaca, b. orientalis) in different parts of khorramabad, 2019-2020 bacteria collection place total hospital no (%) * home no (%) restaurant no (%) escherichia coli 21 (63.6) 9 (27.3) 3 (9.1) 33 (100) bacillus spp. 8 (34.8) 4 (17.4) 11 (47.8) 23 (100) klebsiella spp. 8 (80) 1 (10) 1 (10) 10 (100) enterococcus spp. 16 (84.2) 2 (10.5) 1 (5.2) 19 (100) acinetobacter spp. 1 (33.3) 1 (33.3) 1 (33.3) 3 (100) coagulase negative staphylococci 21 (67.7) 7 (22.6) 3 (9.7) 31 (100) proteus spp. 7 (87.5) 1 (12.5) 0 (0.0) 8 (100) enterobacter aerogenes 9 (100) 0 (0.0) 0 (0.0) 9 (100) pseudomonas aeruginosa 7 (70.0) 2 (20.0) 1 (10.0) 10 (100) total 98 (67.1) 27 (18.5) 21 (14.4) 146 (100) *(p< 0.05) e sc h er ic h ia c ol i b ac il lu s sp p k le bs ie ll a sp p e n te ro co cc u s sp p a ci n et ob ac te r sp p c o n s p ro te u s sp p e n te ro ba ct er a er og en es p se u do m on as a er u gi n os a 0 5 10 15 20 25 30 n u m b e r o f b a c te r ia periplaneta americana blattella germanica blattella orientalis * * fig. 1. relationship between bacterial isolates and cockroach species, *(p< 0.05) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 67 http://jad.tums.ac.ir published online: march 31, 2023 table 2. antibiotic resistance pattern of bacteria isolated from cockroaches; the number of sensitive, resistant, and intermediate resistant bacteria to different antibiotics are listed as numbers for each bacterium antibiotics response bacteria total e . c o li b a c il lu s k le b si e ll a e n te ro b a c te r sp p a c in e to b a c te r sp p c o n s1 p ro te u s sp p s . a u re u s p se u d o m o n a s sp p gentamicin s i r 25 7 1 16 6 1 7 2 1 15 3 1 2 1 0 25 3 2 6 2 0 7 2 0 9 1 0 112* 27 6 kanamycin s i r 7 16 11 3 13 7 5 3 2 2 13 4 0 2 1 6 14 10 4 2 2 5 1 3 3 5 2 35 69 42 cefotaxime s i r 2 2 29 3 2 18 0 1 9 1 1 17 0 2 1 3 2 25 1 0 7 0 0 9 1 1 8 11 11 123* cephalothin s i r 3 1 29 2 2 19 2 4 4 0 2 17 3 0 0 4 3 23 2 0 6 3 0 6 1 1 8 20 13 112* ampicillin s i r 2 6 25 2 3 18 1 2 7 2 4 13 2 1 0 6 4 20 2 2 4 2 1 6 2 1 7 21 24 100* tetracycline s i r 27 4 2 14 7 2 9 0 1 13 3 3 1 0 2 27 3 0 5 3 0 8 1 0 8 1 1 112* 22 11 chloramphenicol s i r 20 11 2 7 9 5 3 3 4 9 3 7 1 1 1 19 6 5 3 3 2 2 4 3 6 2 2 70 42 31 ciprofloxacin s i r 7 14 12 9 7 5 8 2 0 9 5 5 1 1 1 8 14 8 4 2 2 3 2 4 0 9 1 49 56 38 nitrofurantoin s i r 10 10 13 6 10 7 9 0 1 7 6 6 0 1 2 6 16 8 4 1 3 5 1 3 2 2 6 49 47 49 cotrimoxazole s i r 17 4 12 10 2 11 4 2 4 8 3 8 1 1 1 15 2 13 5 3 0 1 2 6 7 2 1 68 21 56 1. coagulase-negative staphylococci s: susceptible, i: intermediate, r: resistant, *(p< 0.05) discussion in the present study, we identified three species of p. americana, b. germanica and b. ori entalis that the most common of them were american cockroaches, which included 100 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 68 http://jad.tums.ac.ir published online: march 31, 2023 cockroaches (67%), followed by german cockroaches 28 (19.4%) and b. orientalis 22(14/8%). in a study conducted by zarchi et al. in tehran hospitals, they showed that 65.6% of cockroaches were american, 12.1% german, and 22.3% oriental, which is statistically consistent with the number of american cockroaches in the present study (21). also in other similar researches in the provinces of iran (zanjan, mazandaran, and esfahan), p. americana and german cockroaches were identified as two dominant species (22, 23). the results of pai et al. studies showed that in clinical settings (environment) most cockroaches are german type and in non-clinical places, most cockroaches are american type but this difference was not statistically significant (24). the most common in hospital samples were coagulase-negative staphylococci and e. coli. the most bacteria found in the house samples were e. coli and coagulase-negative staphylococci. bacillus spp. was the most common bacterium found in restaurant samples. this study showed that the prevalence of bacterial infection in cockroaches caught in hospitals, houses, and restaurants in khorramabad were 97.33%. as in some reports, the role and importance of cockroaches as carriers of bacteria, fungi, and pathogens in humans has been confirmed (25). this study also showed that cockroaches can transmit a variety of bacteria. in the study of chitsazi et al. in mashhad during the years 2010‒2011, the most isolated bacteria were related to enterococcus from cockroaches in mashhad hospital, and the most frequent of cockroaches caught from student dormitories were related to enterobacter aerogenes and most abundant of isolated cockroaches from houses are related to klebsiella oxytoca (26). in bangkok, several species of bacteria were also isolated from cockroaches in residential and hospital areas. escherichia coli and klebsiella pneumoniae were most common in the home, and e. coli and enterobacter cloacae were most common in the hospital, which was consistent with our study of the prevalence of e. coli (27). in a study by lin et al, the most isolated bacteria from cockroaches were e. coli, pseudomonas aeruginosa, salmonella spp., and staphylococcus aurous (28). in another study by chaichanawongsaroj et al. e. coli and klebsiella spp. were commonly isolated from nosocomial cockroaches and showed that the insect, as a carrier of this bacterium, was involved in the transmission of these bacteria (27). the study by fakoorziba et al. in 2010 on p. americana and b. germanica showed the high potential of these organisms to carry pathogenic bacteria, which isolated 25 different species of important medical bacteria that 22 species were gram-negative bacteria. in this study, bacteria from the family enterobacteriaceae of the genera proteus, citrobacter, aerobacter, klebsiella and bacillus were isolated from the outer surface of cockroaches (29). akbari and colleagues conducted a study to determine aerobic bacterial community of american cockroach guts and found 11 bacterial species including: e. coli, shigella flexineri, citrobacter freundii, e. vulneris, enterobacter cloacae, yersinia pseudotuberculosis, y. intermedia, leclericia adecarboxylata, klebsiella oxytoca, k. planticola, and rahnella aquatilis. some of the isolated bacteria of that study were consistent with the present study (13). also in the present study, staphylococcus was the second bacterium that was abundantly identified in the studied cockroaches, this bacterium causes a wide range of infections from simple skin infections (such as decoctions, boils, scabs, eyelashes, and abscesses) to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, and septicemia. staphylococcus aureus is one of the five most common causes of nosocomial infections, especially post-surgical wound infections. each year, 500,000 people in hospitals in the united states become infected with s. aureus (30‒32). the results of the antibiogram test showed that the identified bacteria were resistant to the antibiotics cephalothin, ampicillin, cefotaxime, http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 63–71 b davari et al.: bacterial contamination of … 69 http://jad.tums.ac.ir published online: march 31, 2023 and kanamycin that this bacterial resistance was worrying, and they were also relatively resistant to the antibiotics nitrofurantoin and co-trimoxazole. the cultured bacteria were also semi-sensitive to the antibiotics kanamycin and ciprofloxacin and were sensitive to the tetracycline, gentamycin, and chloramphenicol antibiotics. in a study by stypułkowska-misiurewicz et al. and colleagues, they examined cockroaches in nine warsaw hospitals and found that the bacteria carried by their bodies were the same ones (species) responsible for nosocomial infections. therefore, they stated that there was a microbiological risk of cockroaches in the hospital environment and it was proved that some bacteria in the body of cockroaches are responsible for nosocomial infections, in which there is also antibiotic resistance (5). davari et al. conducted a study with the aim of investigating the frequency of resistant and sensitive bacteria isolated from houseflies, their findings showed, k. pneumonia had the highest bacterial count with 43% and p. aeruginosa 37%, proteus mirabilis 29.1%, and citrobacter freundii were reported 4.28%. among all hospital samples, resistance to cephalexin, chloramphenicol, ampicillin, and tetracycline was high at 32.5%. the result was that gentamicin showed the highest sensitivity among all hospital specimens (33). in our study, the highest sensitivity to tetracycline, gentamicin, and chloramphenicol antibiotics was reported. considering that many diagnostic tests were needed to determine the bacterial strain, and this required a lot of financial resources, so we decided to stop the differentiation tests at the species level in some isolates. conclusions according to the results of this study, it was found that cockroaches can be very effective in transmitting pathogenic bacteria and they can also mechanically transfer bacteria from hospital environments to the outside of the hospital and vice versa, which is very important. in addition, according to the findings of this research, it can be assumed that cockroaches play a major role in the spread of antibiotic resistant bacteria. the next point is that cockroaches in hospital environments were more contaminated with bacteria, one of the reasons for this is that people who visit hospital centers are usually sick and carry various pathogens. therefore, raising the level of health and combating cockroaches seems very necessary. acknowledgments the study was funded by vice-chancellor for research and technology, hamadan university of medical sciences (no: 9404302417). ethical considerations since human samples and animal studies were not used in this study, there were no ethical restrictions. conflict of interest statement the authors declare there is no conflict of interests. references 1. donkor es (2019) nosocomial pathogens: an in-depth analysis of the 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j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 27 original article fauna and checklist of mosquitoes (diptera: culicidae) of east azerbaijan province, northwestern iran mr abai1, *s azari-hamidian2, h ladonni1, m hakimi1, k mashhadi-esmail1, k sheikhzadeh3, a kousha3, h vatandoost1 1 department of medical entomology and vector control, school of public health, medical sciences/ university of tehran, iran 2 school of public health, guilan university of medical sciences, rasht, iran 3 tabriz health center, tabriz university of medical sciences, tabriz, iran (received 14 may 2007; accepted 29 oct 2007) abstract in order to study the mosquito (diptera: culicidae) fauna of east azerbaijan province, some samplings were carried out by dipping method for the larvae and hand catch, night biting catch, total catch, and shelter pit collection as well as using window trap for the adults during june, july, and august 2004 plus july and august 2005. in total, 1305 adult mosquitoes and 603 larvae were collected. seven genera and 15 species were identified in the province including; anopheles claviger, an. hyrcanus, an. maculipennis s.l., an. pseudopictus, an. sacharovi, an. superpictus, aedes vexans, coquillettidia richiardii, cx. pipiens, cx. theileri, cx. tritaeniorhynchus, culiseta longiareolata, cs. subochrea, ochlerotatus caspius s.l., and uranotaenia unguiculata. an. maculipennis complex, cx. pipiens, and cx. theileri were the most prevalent and widely distributed species. an. pseudopictus, ae. vexans, and cq. richiardii are reported for the first time in east azerbaijan province and a checklist for the mosquitoes of the province is also presented. among the mosquitoes of the province, there are many potential vectors of human and domesticated animal pathogens that their ecology needs to be studied extensively. keywords: fauna, iran, mosquito, species checklist introduction the mosquito fauna of iran includes seven genera, 64 species, and 3 subspecies. the records of 12 other species need to be verified (azari-hamidian 2007). by now, five genera and 19 species of mosquitoes have been recorded in east azerbaijan province by different investigators (beklemishev and gontaeva 1943, zolotarev 1945, kalandadze and kaviladze 1947, dow 1953, minar 1974, danilov 1975, saebi 1987, zaim 1987, harbach 1988, vatandoost et al. 2005). six species of anopheles meigen have been recorded in the province (beklemishev and gontaeva 1943, zolotarev 1945, kalandadze and kaviladze 1947, dow 1953, saebi 1987) including; an. claviger (meigen), an. hyrcanus (pallas), an. maculipennis meigen, an. pseudopictus grassi, an. sacharovi favre, an. superpictus grassi. of the maculipennis group, an. sacharovi has been recorded in the province by beklemishev and gontaeva (1943) along with dow (1953) and an. maculipennis by zolotarev (1945) plus dow (1953) based on egg pattern as well as by djadid et al. (2007) using polymerase chain reaction (pcr) technique. there is no published reference about the occurrence of an. pseudopictus in this province (azari-hamidian et al. 2006). kalandadze and kaviladze (1947) reported ochlerotatus caspius (pallas) s.l. and oc. dorsalis (meigen) in the province. oc. dorsalis has not been found in iran recently. zaim and cranston (1986) did not mention this spe*corresponding author: s azari-hamidian, tel/fax: +98 131 3229599, e-mail: azari@gums.ac.ir iranian j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 28 cies in their checklist of iranian culicinae. the occurrence of this species in iran needs to be verified. dow (1953) reported uranotaenia unguiculata edwards in this province. zaim (1987) mentioned seven species of culex l. and three species of culiseta felt in the province as follows: culex modestus ficalbi, cx. mimeticus noe, cx. perexiguus theobald, cx. theileri theobald, cx. tritaeniorhynchus giles, cx. hortensis ficalbi, cx. pipiens l., culiseta longiareolata (macquart), cs. alaskaensis ludlow, and cs. subochrea (edwards). there is only one recent study on the mosquitoes in the province in relation to malaria and anophelines (vatandoost et al. 2005). as mentioned above, the data on the mosquitoes of east azerbaijan province are mostly old and scattered. in order to study the mosquito fauna and providing a primary checklist, an investigation was carried out in the province. materials and methods study area east azerbaijan province is located in northwestern iran between 39º 26´36º 45´ n latitudes and 45º 5´48º 22´ e longitudes and has mostly foothill and mountainous areas with an area of approximately 45490 square kilometers. the province is bounded by the republic of azerbaijan and armenia in the north, west azerbaijan province in the west and the south, zanjan province in the south, and ardebil province in the east. this province formally includes 19 counties. the center of the province, tabriz city, is in almost 1360 m above sea level. the average annual rainfall is about 300 mm. the average relative humidity changes are from 44% (12:30 pm) to 67% (6:30 pm). the averages of the maximum and minimum temperatures are 17.7 °c and 6.8 °c, respectively, and the average temperature is 12.3 °c (these are 30 year data from tabriz synoptic station). the province includes arid and semiarid climates. the main occupations in rural areas are agriculture and husbandry and in urban, service, business, and industry. specimen and data collection to study the mosquito fauna, sampling was carried out in east azerbaijan province by dipping method for collecting larvae and hand catch, night biting catch on human, cow, and donkey, total catch, and shelter pit collection and using window trap for the adults during june, july, as well as august 2004 and july plus august 2005. the specimens were collected from six counties in the different topographical and climatic areas of the province including: ahar, ajabshir, jolfa, kaleibar, maragheh, and tabriz counties. in addition, some adult mosquitoes were reared from the pupae. the larvae were preserved in lactophenol and the microscopic slides of the preserved larvae were prepared using berlese medium. the adult mosquitoes were pinned. the adult specimens and the third and fourth stage larvae were identified using the keys of shahgudian (1960), zaim and cranston (1986), harbach (1988), and darsie and samanidou-voyadjoglou (1997). mosquito name abbreviations are cited based on reinert (2001). results totally 1305 adult mosquitoes and 603 larvae were collected and seven genera along with 15 species were identified, including; an. claviger, an. hyrcanus, an. maculipennis s.l., an. pseudopictus, an. sacharovi, an. superpictus, aedes vexans (meigen), coquillettidia richiardii (ficalbi), cx. pipiens, cx. theileri, cx. tritaeniorhynchus, cs. longiareolata, cs. subochrea, oc. caspius s.l., and ur. unguiculata. these species were collected from the mountainous areas of the province on 12 occasions in june-august 2004 and on 11 occasions in july and august 2005. aedes vexans and cq. richiardii are recorded for the first time in east azerbaijan province and this is the first formal report of an. pseudopictus in the province. uranotaenia unguiculata iranian j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 29 was collected only in the larval stage and ae. vexans, cq. richiardii, cx. tritaeniorhynchus, and cs. subochrea only in the adult stage. anopheles maculipennis complex, cx. pipiens, and cx. theileri were the most prevalent species and collected in almost all of the counties, but an. claviger, an. hyrcanus, an. pseudopictus, ae. vexans, cq. richiardii, cx. tritaeniorhynchus, cs. subochrea, and oc. caspius s.l. were collected only in kaleibar county (table 1 and 2). anopheles maculipennis s.l., an. sacharovi, cx. pipiens, cx. theileri, and oc. caspius s.l. were collected by different methods, but an. claviger, an. pseudopictus, and ae. vexans by hand catch plus night biting catch; an. hyrcanus, an. superpictus, cx. tritaeniorhynchus, and cs. longiareolata only by hand catch; and cq. richiardii along with cs. subochrea only by night biting catch. aedes vexans, cq. richiardii, and cs. subochrea were collected from only outdoors and an. hyrcanus, an. superpictus, cx. tritaeniorhynchus, and cs. longiareolata from only indoors and other species from both indoors and outdoors (table 3). composition and localities of the adult mosquitoes collected in this survey are shown in table 1. composition and localities of the larvae of mosquitoes collected in this survey are shown in table 2. the adult mosquitoes col lected by different methods are shown in table 3. the checklist of mosquitoes of east azerbaijan province is given below. ochlerotatus dorsalis is not mentioned here. as it was explained before the occurrence of this species in iran needs to be verified (the first and new formal records are shown by*): 1anopheles claviger (meigen, 1804) 2an. hyrcanus (pallas, 1771) 3an. maculipennis meigen, 1818 4an. pseudopictus grassi, 1899 * 5an. sacharovi favre, 1903 6an. superpictus grassi, 1899 7aedes vexans (meigen, 1830) * 8coquillettidia richiardii (ficalbi, 1889) * 9culex hortensis ficalbi, 1889 10cx. mimeticus noe, 1899 11cx. modestus ficalbi, 1889 12cx. perexiguus theobald, 1903 13cx. pipiens linnaeus, 1758 14cx. theileri theobald, 1903 15cx. tritaeniorhynchus giles, 1901 16culiseta alaskaensis ludlow, 1906 17cs. longiareolata (macquart, 1838) 18cs. subochrea (edwards, 1921) 19ochlerotatus caspius (pallas, 1771) s.l. 20uranotaenia unguiculata edwards, 1913 table 1. composition and localities of the adult mosquitoes collected in east azerbaijan province during june, july, and august 2004 and july and august 2005 county species ahar ajabshir kaleibar maragheh tabriz no. % an. claviger 3 3 0.2 an. hyrcanus 1 1 0.1 an. maculipennis s.l. 20 266 4 290 22.2 an. pseudopictus 94 94 7.2 an. sacharovi 427 427 32.7 an. superpictus 70 20 90 6.9 ae. vexans 16 16 1.2 cq. richiardii 1 1 0.1 cx. pipiens 23 31 16 12 82 6.3 cx. theileri 4 48 126 10 5 193 14.8 cx. tritaeniorhynchus 1 1 0.1 cs. longiareolata 2 47 49 3.7 cs. subochrea 1 1 0.1 oc. caspius s.l. 57 57 4.4 total 24 71 1094 28 88 1305 100 iranian j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 30 table 2. composition and localities of the larvae of mosquitoes collected in east azerbaijan province during june, july, and august 2004 and july and august 2005 table 3. the adult mosquitoes collected by different methods in east azerbaijan province during june, july, and august 2004 and july and august 2005 county species ahar ajabshir jolfa kaleibar maragheh tabriz no. % an. claviger 57 57 9.4 an. hyrcanus 1 1 0.2 an. maculipennis complex 17 1 49 20 87 14.4 an. pseudopictus 9 9 1.5 an. superpictus 28 28 4.7 cx. pipiens 28 1 20 15 64 10.6 cx. theileri 51 19 2 2 31 40 145 24.0 cs. longiareolata 16 17 33 5.5 oc. caspius s.l. 175 175 29.0 ur. unguiculata 3 1 4 0.7 total 115 20 3 294 71 100 603 100 collecting method night biting catch on species hand catch total catch shelter pit collection window trap human cow donkey rearing pupa an. claviger 2 1 an. hyrcanus 1 an. maculipennis s.l. 265 7 16 2 an. pseudopictus 8 84 2 an. sacharovi 403 2 12 6 4 an. superpictus 70 20 ae. vexans 4 6 6 cq. richiardii 1 cx. pipiens 12 6 16 1 6 1 40 cx. theileri 5 7 1 105 10 2 63 cx. tritaeniorhynchus 1 cs. longiareolata 1 48 cs. subochrea 1 oc. caspius s.l. 7 4 18 28 total 779 15 55 2 227 54 2 171 iranian j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 31 discussion seven genera and 15 species of mosquitoes were identified from east azerbaijan province, including the first record of ae. vexans, and cq. richiardii and the first formal report of an. pseudopictus in the province. all six species of anopheles, which had already been recorded in east azerbaijan province, were also collected in this investigation. however, the occurrence of an. pseudopictus in the province was mentioned only in the unpublished documents of the school of public health (sph) and the institute of public health research (iphr) as a variety or subspecies of an. hyrcanus (azari-hamidian et al. 2006). some recent investigations mentioned only an. hyrcanus in northwestern iran (yaghoobi-ershadi et al. 2001, vatandoost et al. 2005), though many references counted an. pseudopictus as the more common species of the hyrcanus group in northern iran (dow 1953, minar 1974, azari-hamidian et al. 2006). the present study confirms these observations. it seems that those investigations, mentioned an. hyrcanus, referred to the hyrcanus group indeed and did not differentiate an. hyrcanus from an. pseudopictus. these species can easily be separated using hindtarsomere 4 in the adult stage (azari-hamidian et al. 2006). in this study, these species were differentiated from each other in the larval stage based on seta 2-c using darsie and samanidou-voyadjoglou’s key (1997). this seta is simple in an. pseudopictus, but it has some short apical branches in an. hyrcanus. recently, an. superpictus was mentioned as a complex in iran (oshaghi et al. 2004). the biosystematics of this species needs to be studied more in the country. two species of the tribe aedini including; ochlerotatus caspius s.l. and oc. dorsalis (as a subspecies of oc. caspius) were recorded in east azerbaijan province (kalandadze and kaviladze 1947). there is no information about the oc. caspius sibling species (a or b) in the country (azari-hamidian 2007). in this study, ae. vexans, for the first time, and oc. caspius s.l. were found in the province. the genus coquillettidia dyar, with its unique species cq. richiardii in iran, has been found in mazandaran, guilan, ardebil, and probably kurdistan provinces (azari-hamidian 2007 and azari-hamidian, unpublished data). this is the first record of this species in east azerbaijan province. three species of culex were found in this study (out of seven previously recorded species in the province). based on zaim and cranston’s (1986) key, the larval stages of cx. pipiens, cx. torrentium, and cx. vagans are not distinguishable from each other, but they can be separated by harbach’s (1988) key using seta 1-iii-v, seta 1-m, seta 1-x, seta 1-c, and some other characters. among the available specimens from east azerbaijan province only cx. pipiens was identified. two species of culiseta, cs. longiareolata and cs. subochrea, were found in this investigation. kalandadze and kaviladze (1947) found both cs. annulata and cs. subochrea (as a subspecies of cs. annulata) in west azerbaijan province and cs. subochrea in east azerbaijan province. they mentioned that the record of cs. annulata in iran was the first one. zaim and cranston (1986) included cs. annulata in their checklist and cs. subochrea in their keys. the taxonomy and distribution of these two species need to be investigated more in iran (azarihamidian 2007). mosquitoes, especially anopheles, have been studied in iran and east azerbaijan province mostly in relation to malaria. there are many potential vectors of human and domesticated animal pathogens, such as ae. vexans, cx. pipiens, cx. theileri, cx. tritaeniorhynchus, and oc. caspius s.l. (horsfall 1955, harbach 1988), among the mosquitoes of the province that their ecology need to be studied extensively. in this study several methods of collection were used in different areas of east azerbaijan province to show the diversity of the collected mosquito species. however, more mosquito surveillance iranian j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 32 is needed in the province to interpret different aspects of their ecology such as composition, prevalence, active season, host preference, and larval habitat. all specimens are deposited in the medical arthropod museum at the school of public health, tehran university of medical sciences, iran. acknowledgements the authors are grateful to m haghi, tabriz health center, tabriz university of medical sciences, for his cooperation. the authors also wish to thank dr sm omrani, deparement of medical entomology and vector control, school of public health, tehran university of medical sciences for reviewing the article. this study was financially supported by the institute of public health research, academic pivot for education and research, tehran university of medical sciences: project no.: 241.83.77. references azari-hamidian s, abai mr, ladonni h, vatandoost h, akbarzadeh k (2006) anopheles peditaeniatus (leicester) new to the iranian mosquito fauna with notes on anopheles hyrcanus group in iran. j am mosq contr assoc. 22: 144-146. azari-hamidian s (2007) checklist of iranian mosquitoes (diptera: culicidae). j vect ecol. 32(2) [in press]. beklemishev vn, gontaeva aa (1943) anophelogenous landscapes of northwest iran. med parasitol parasitic dis. 12: 17-32 [in russian]. danilov vn (1975) mosquito fauna of northwestern iran. med parasitol parasitic dis. 44: 732 [in russian with english abstract]. darsie re, samanidou-voyadjoglou a (1997) keys for the identification of the mosquitoes of greece. j am mosq contr assoc. 13: 247-254. djadid nd, gholizadeh s, tafsiri e, romi r, gordeev m, zakeri s (2007) molecular identification of palearctic members of anopheles maculipennis in northern iran. malaria j. 6: 6-15. dow rp (1953) notes on iranian mosquitoes. am j trop med hyg. 2: 683-695. harbach re (1988) the mosquitoes of the subgenus culex in southwestern asia and egypt (diptera: culicidae). contrib am ent inst. 24: 1-240. horsfall wr (1955) mosquitoes. their bionomics and relation to disease. hafner publishing, new york. kalandadze lp, kaviladze op (1947) on the blood-sucking mosquitoes of the western part of the iran azerbaijan. med parasitol parasitic dis. 16: 57-65 [in russian, english abstract in trop dis bull, 1948, 45: 296-297]. minar j (1974) result of the czechoslovak-iranian entomological expedition to iran 1970 no 6: diptera: culicidae. acta ent mus nat pragae. 6(suppl.): 87-89. oshaghi ma, yaghoobi-ershadi mr, shemshad k, abaei mr, akbarzadeh k (2004) mitochondrial and ribosomal dna sequence variation between anopheles superpictus populations in iran. ix european multicolloquium of parasitology, valencia, spain, pp. 103-104. reinert jf (2001) revised list of abbreviation for genera and subgenera of culicidae (diptera) and the notes on generic and subgeneric changes. j am mosq contr assoc. 17: 51-55. saebi me (1987) morphological study on anopheline larvae and their distribution in iran [phd dissertation]. school of public health, tehran university of medical sciences, tehran, iran [in persian]. shahgudian er (1960) a key to anophelines of iran. acta med iran. 3: 38-48. vatandoost h, abdoljabari boonab r, abai mr, oshaghi ma, rassi y, gholizadeh s, mashhadi-esmail k, kousah a, haghi m, gorghani m, aliakbarie-sharabiani b, seif iranian j arthropod-borne dis, 2007, 1(2): 27-33 mr abai et al.: fauna and checklist of … 33 farshid m, piazak n (2005) entomological survey in kalibar, a resurgent malaria focus in east-azerbaijan, iran. pak j biol sci. 8: 1466-1471. yaghoobi-ershadi mr, namazi j, piazak n (2001) bionomics of anopheles sacharovi in ardebil province, northwestern iran during a larval control program. acta trop. 78: 207215. zaim m, cranston ps (1986) checklist and keys to the culicinae of iran (diptera:culicidae). mosq syst. 18: 233-245. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq contr assoc. 3: 568-573. zolotarev er (1945) anopheles maculipennis of northern iran. med parasitol parasitic dis. 14: 50-57 [in russian]. j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 135 http://jad.tums.ac.ir published online: june 24, 2019 original article molecular characterization of anopheles sacharovi based on sequences of its2-rdna region and coi gene in north of iran sahereh gholami1; hasan bakhshi1,2; seyyed hassan moosa-kazemi1; alireza zahraeiramazani1; alireza chavshin3; *mohammad mehdi sedaghat1 1department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran 2malaria and vector research group, biotechnology research center, pasteur institute of iran, tehran, iran 3department of medical entomology, school of public health, urmia university of medical sciences, urmia, iran (received 31 aug 2018; accepted 10 apr 2019) abstract background: malaria is an important mosquito-borne disease considered as one of the public health concerns across many countries. anopheles mosquitoes are the main vectors of plasmodium parasites, which cause malaria. some of these vectors such as anopheles maculipennis s.l. and anopheles sacharovi are considered as complex of sibling species distributed in north of iran. methods: this study was conducted in north and northwest of iran including east azerbaijan, west azerbaijan, ardabil, golestan and north khorasan provinces with emphasis on the northern borders of the country during 2015– 2016. adult specimens were collected and subjected to morphological identification as well as molecular analysis. results: overall, 10405 mosquitoes were collected comprising 21 species. culex pipiens and cx. theileri were found as the most frequent species in whole study area. morphological identification showed that out of 1455 female anopheles specimens, 77% belonged to an. maculipennis group. out of the identified species, its2 region and coi gene sequences of 8 an. maculipennis s.s. and 31 an. sacharovi representing all provinces were obtained and submitted to genbank. the coi sequences for an. sacharovi revealed the presence of 9 haplotypes with similarity of 98.17–100%. conclusion: some investigations have reported an. martinius as a member of sibling species of an. sacharovi among iranian anopheles genus; while based on our study, there was no evidence of the presence of this species in north and northwest of iran. keywords: anopheles sacharovi; anopheles martinius; coi; its2-rdna, iran introduction anopheles mosquitoes are responsible for transmission of malaria parasites in humans. there are 30 definitive reported species, 3–4 biological forms and geographical races of anopheles in iran. there are seven primary malaria vectors recognized in iran including an. stephensi, an. culicifacies s.l., an. fluviatilis s.l., an. superpictus s.l., an. dthali, an. maculipennis s.l. and an. sacharovi (1). some of the most important species are in the maculipennis group comprised as a primary or secondary vector of malaria parasites in the palaearctic region (2, 3). there is a report on the bionomics of an. maculipennis and an. sacharovi from iran and iraq and the distribution of the two species in central and northern areas of iran (4). anopheles maculipennis s.l. was reported in central and northern areas of the country (5). twenty-two species of anopheles were listed in iran based on literature records. the list included an. martinius among iranian anopheles species (6); although, there is no evidence of an. martinius occurrence in iran so far. as a result of recent molecular genetic studies, dna sequence data are available for *corresponding author: dr mohammad mehdi sedaghat, e-mail: sedaghmm@tums.ac.ir j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 136 http://jad.tums.ac.ir published online: june 24, 2019 identification of the members of the complex. anopheles persiensis was described as a new species of the an. maculipennis complex in north of iran (2). two members of an. maculipennis complex including an. maculipennis and an. sacharovi have been reported as vectors of malaria parasites in central and northern parts of the country. they are also considered as vectors of plasmodium parasites from armenia, azerbaijan and turkey (7). members of the an. maculipennis complex are distributed mostly in northern and central areas of the country (8). the an. maculipennis complex comprises several sibling species including major vectors of malaria parasites of historic europe (9). currently, there are 10 members of the an. maculipennis complex in the palearctic region including an. melanon, an. messeae, an. persiensis, an. sacharovi, an. martinius, an. atroparvus, an. daciae, an. labranchiae and an. artemievi (10). out of the complex, six species including an. maculipennis s.s., an. maculipennis, an. persiensis, an. messeae, an. atroparvus, an. labranchiae and an. sacharovi have been identified in iran based on molecular methods (3). anopheles melanoon and an. messeae are listed as iranian species based on the egg morphology studies as well as molecular studies (8). this study was carried out based on the molecular and morphological characters of an. maculipennis s.l. collected from north and northwest of iran with emphasis on border lines including east azerbaijan, west azerbaijan, ardabil, golestan and north khorasan provinces where are considered as important biogeographic regions, being the corridor between europe and asia. these provinces have share borders with five countries including turkmenistan, armenia, azerbaijan, iraq and turkey where the members of the an. maculipennis complex play an important role in malaria transmission. the potential occurrence of an. martinius, a close species of an. sacharovi, was also considered in this study. materials and methods adult mosquitoes and larvae were collected from five provinces located in north of iran during 2015–2016 using standard methods (fig. 1, table 1). animal bite traps and shelter pit methods were used for collection of adults. the collection of larvae was carried out by dipping method. all samples were identified to the species level by using morphological keys (11). these identifications were used to target specimens for molecular identification using ribosomal internal transcribed spacer ii (its2-rdna) region and cytochrome oxydase i (coi) gene to differentiate cryptic species within the an. maculipennis complex. genomic dna of the mosquitoes was extracted using (g-spine ™) dna extraction kit, according to the manufacturer's instructions. reactions were carried out in a total volume of 20µl using the pcr kit. the desired its2 fragments were amplified by using universal 5.8s (5´-tgtgaactgc aggacacatgaa-3´) as the forward and 28s (5´-atgcttaaattagggggtagtc 3´) as the reverse primers (12). the pcr conditions were as follows: 94 °c for 2min, followed by 25 cycles of 94 °c for 20sec, 50 °c for 15sec, and 70 °c for 25sec and terminating with a 72 °c for 5min. the desired coi fragments were amplified using lco1490 (5´ggtcaacaaatc ataaagatattgg-3´) as the forward and hco2198 (5´-taaactt cagggtgacc aaaaaatca-3´) as the reverse primers (13). the pcr conditions were as follows: 94 °c for 2min, followed by 5 cycles of 94 °c for 30sec, 45 °c for 40sec, and 72 °c for 1min, followed by 35 cycles of 94 °c for 30sec, 55 °c for 30sec, and 72 °c for 1min, respectively, terminating with a 72 ºc for 5min. accuracy and quality of the amplicon were examined using a 1% agarose gel and visualized by gel doc after staining with sinacolon® (tehran, iran) safe stain. dna chromatograms were inspected using chromas software (version 2.23) and the sequences were submitted to genbank. similarity with other sequencj arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 137 http://jad.tums.ac.ir published online: june 24, 2019 es in genbank was assessed using blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) online tool. phylogenetic tree was constructed by mega7 (ver. 7.0.21) software (molecular evolutionary genetic analysis) using maximum likelihood method with 1000 replicates of bootstrapping. phylogenetic tree of fifteen coi sequences obtained from this study (an. sacharovi: 11 and an. maculipennis: 4) and four coi genbank sequences as outgroup (ku950429: an. martinius, km224658: an. melanoon, km258220: an. messeae and ku 380466: an. gambiae) were created (fig. 2). results morphological investigations out of 10405 specimens, 6556 adult samples and 3849 larvae were collected. morphological identifications revealed that the specimens represented five genera and 21 species including an. maculipennis s.l., an. sacharovi, an. claviger, an. hyrcanus, an. superpictus s. l., an. psudopictus, culex hortensis, cx. pipiens, cx. theileri, cx. modestus, cx. mimeticus, cx. perexiguus, cx. tritaeniorhynchus, aedes caspius, ae. geniculatus, ae. vexans, ae. flavescens, ae. echinus, culiseta longiareolata, cs. subochrea and uranotaenia unguiculata. culex pipiens (30.2%), culiseta loniareolata (23.8%) and cx. theileri (22.3%) were the dominant species, and accounted for 76.3% of the collected samples (table 2). culex pipiens and cx. theileri were found as frequent species in whole study area. uranotaenia unguiculata (n=1, 0.009%), cs. subochrea (n=4, 0.003 %) and ae. vexans (n=4, 0.003%) were considered as infrequent species. uranotaenia unguiculata was only collected in west azerbaijan province. among aedes mosquitoes, ae. caspius (1.2%) was found in whole study area and ae. flavescens in sympatry with ae. vexans in ardabil province. among anopheles mosquitoes, an. claviger and an. hyrcanus were widespread across the whole study area. anopheles maculipennis s.l. (5.8%) was found in five provinces and an. sacharovi (0.3%) was found in sympatry with an. maculipennis s.l. only in three provinces located in northwest. anopheles superpictus s.l. was also found with frequency of 3.9%. the last three species are the main malaria vectors in iran. molecular investigations anopheles maculipennis s.l. and an. sacharovi were subjected to molecular study using its2 and coi sequences. eight sequences of an. maculipennis s.s. for the its2 region (n=4) and coi gene (n=4) were obtained and the sequences submitted to genbank under accession numbers ky225560, ky225561, ky225562, ky225563 and ky196448, ky196449, ky 196450, ky196462 for its2 and coi regions respectively. all specimens were identified as an. maculipennis s.s. by an identity of 100 %. thirty one sequences for an. sacharovi for the its2 region (n=19) and coi gene (n=12) were obtained and the sequences were submitted to genbank under accession numbers ky225557-ky225559, ky263795-ky263806 and ky 196451-ky196461 for the its2 and coi regions respectively. all its2 sequences for an. sacharovi were identical with 100% similarity. the coi sequences for an. sacharovi showed that there were 9 haplotypes with similarity of 98.17–100%. the base composition of the coi fragments showed an at bias with all sequences being between 66.4 and 67.8% at rich (mean= 66.8%). seventeen single nucleotide polymorphisms among the haplotypes were observed (table 3). based on the coi sequences of an. sacharovi and an. maculipennis s.s. (about 601bp), phylogenetic tree was constructed. a tree for 15 coi sequences including 11 an. sacharovi, 4 an. maculipennis as well as 3 sequences of an. maculipennis complex deposited in genbank (an: ku950429 (an. martinius), km 224658 (an. melanoon), km258220 (an. messeae) and ku380466 (an. gambiae) was constructed (fig. 2). the phylogenetic tree revealed the inter-population differences of the studied https://blast.ncbi.nlm.nih.gov/blast.cgi j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 138 http://jad.tums.ac.ir published online: june 24, 2019 species. the smallest genetic distance was shown between the an. sacharovi populations from three provinces including east azerbaijan, west azerbaijan and ardabil provinces rather than golestan and north khorasan provinces. the three mentioned provinces are closely located in northwest of the country (fig. 1). moreover, an. melanoon, an. messeae and an. martinius were recognized in separate clades from all an. sacharovi and an. maculipennis s.s. populations. fig. 1. the study area: 1. north khorasan, 2. golestan, 3. ardabil, 4. east azerbaijan, 5. west azerbaijan table 1. geographical coordinates of the study areas no. province county latitude longitude 1 west-azerbaijan poldasht 39° 20' 49.69'' n 45° 3' 59.61'' e shahindej 36° 40' 26.74'' n 46° 34' 12.48'' e oshnavieh 37° 2' 11.17'' n 45° 5' 49.69'' e makoo 39° 17' 44.24'' n 44° 30' 51.07'' e 2 ardabil parsabad-moghan 39° 37' 14.80'' n 47° 54' 18.22'' e aslan-duz 39° 26' 29.57'' n 47° 24' 40.25'' e meshgin shahr 38° 23' 41.38'' n 47° 39' 53.46'' e 3 east-azerbaijan kaleybar 38° 51' 51.27'' n 47° 2' 25.94'' e azarshahr 37° 44' 39.54'' n 45° 59' 13.95'' e 4 north-khorasan bojnord 37° 28' 12.74'' n 57° 18' 51.61'' e shirvan 37° 24' 33.25'' n 57° 55' 39.42'' e 5 golestan gorgan 36° 50' 44.31'' n 54° 26' 21.61'' e j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 139 http://jad.tums.ac.ir published online: june 24, 2019 fig. 2. phylogenetic tree constructed by 15 coi sequences (about 601bp) obtained from anopheles sacharovi=11 and anopheles maculipennis=4. the sequences ku950429: anopheles martinius, km224658: anopheles melanoon, km258220: anopheles messeae and ku380466: anopheles gambiae (as the out-group) were derived from genbank j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 140 http://jad.tums.ac.ir published online: june 24, 2019 table 2. species composition of mosquitoes collected from east-azarbaijan: ea, west-azarbaijan: wa, ardabil: a, golestan: g, north-khorasan: nkh no. species adults species species larvae adults (%) larvae (%) total (adults and larvae) (%) ea wa a g nkh ea wa a g nkh 1 an. maculipennis 88 97 33 19 24 155 118 72 261 (3.98) 345 (8.96) 606 (5.8) 2 an. sacharovi 26 3 5 34 (0.51) 34 (32) 3 an. claviger 64 43 99 14 2 99 50 104 222 (3.38) 253 (6.57) 475 (4.56) 4 an. hyrcanus 12 23 20 9 64 (0.97) 64 (0.61) 5 an. superpictus 3 13 116 15 266 132 (2.01) 281 (7.3) 413 (3.96) 6 an. psudopictus 15 15 (0.22) 15 (0.14) 7 cx. hortensis 120 9 20 3 180 106 28 152 (2.31) 314 (8.15) 466 (4.48) 8 cx. pipiens 1326 9 63 109 46 336 1254 8 1553 (17.58) 1598 (41.51) 3151 (30.28) 9 cx. theileri 1703 14 39 13 281 243 35 1769 (26.98) 559 (14.525) 2328 (22.38) 10 cx. modestus 3 47 50 3 (0.04) 97 (2.52) 100 (0.96) 11 cx. mimeticus 17 7 6 13 17 (0.25) 26 (0.67) 43 (0.41) 12 cx. perexiggus 3 17 3 18 3 23 (0.35) 21 (0.54) 44 (0.42) 13 cx. tritarincus 15 15 (0.38) 15 (0.14) 14 ae. caspius 1 6 32 31 8 33 15 78 (1.18) 48 (1.24) 126 (1.21) 15 ae. geniculatus 4 6 10 (0.15) 10 (0.096) 16 ae. vexans 4 4 (0.1) 4 (0.038) 17 ae. flavecence 11 11 (0.28) 11 (0.1) 18 ae. echinus 15 15 (0.22) 15 (0.14) 19 cs. loniareolata 2080 42 71 11 215 55 6 2204 (33.61) 276 (7.17) 2480 (23.83) 20 cs. subochrea 4 4 (0.06) 4 (0.038) 21 ur. ungiuiculata 1 1 (0.02) 1 (0.009) total (%) 5420 (52.09) 234 (2.24) 258 (2.48) 393 (3.78) 252 (2.42) 1336 (12.83) 1963 (18.86) 550 (5.3) 6556 (63) 3849 (37) 10405 (100) 140 http://jad.tums.ac.ir published online: june 24, 2019 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 141 http://jad.tums.ac.ir published online: june 24, 2019 table 3. dna sequence comparison of about 601bp of coi gene of anopheles sacharovi distributed in the study area. totally 9 haplotypes were identified within the sequenced samples, *: non-synonymous base change, dots show identical sequences to the top sequence position 38 116 155 170 203 209 278 323 326 341 347 368 401 487 509 557 563 poldasht t g a t a t c a t c a g a c g a a azarshahr . . . . . . . . . . . . . . . . . parsabadmoghan . . . . . . . . . . . . . . t . . shahindej . . . . . . . . . . . . . . t . . makoo . . . . . . . . . . . . . . t . . kaleybar . . . . . . . g . . . . . . t . . azarshahr . . g . . . . . . . g . . . . . g poldasht c a . c g c t . . . g a . . t g . kaleybar . . . . . . . . . . . . g . t . . parsabadmoghan . . g . . . . . c . g a . g* . . . oshnavieh . . . . . . . . . . g . . . t . . aslan-duz . . . . . . . . . t . . . . t . . discussion the maculipennis subgroup currently comprises 10 members including an. artemievi, an. atroparvus, an. daciae, an. labranchiae, an. maculipennis, an. martinius, an. melanoon, an. messeae, an. persiensis and an. sacharovi. six members of an. maculipennis complex have been identified as primary or secondary vectors of malaria parasites in the palearctic region (2). these members are very close related species which are difficult to be identified by morphological characteristics. although it is possible to distinguish an. sacharovi from other members by morphological characteristics, an. martinius is remained as a sibling species of an. sacharovi which makes it impossible to identify them by their morphological characteristics; these two species can be detected by cytological studies (14). although the occurrence of an. martinius in iran had been mentioned, there is no evidence for distribution of this species in the country so far. the most ambiguity is the distribution of an. martinius in east of caspian sea and east of iran. although it was reported in 1941 (15), but there is no new evidence of occurrence of this species in north of tajikistan as well (16). this is in concordance with another study (17). cytogenetic study showed that only an. maculipennis s.s. was present in this region. however, an. artemievi described as a homosequential species with an. maculipennis, could be erroneously identified as an. martinius (18). anopheles artemievi is a new and predominate species in kyrgyzstan, where it was identified as an. martinius (18). on the other hand, there are cytological or molecular evidences for occurrence of an. martinius in northeast of turkmenistan, the turkmen-khorasan mountain range, karakalpakstan and the khorezm areas of uzbekistan (19, 20). our molecular studies on the an. maculipennis complex were conducted to elucidate j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 142 http://jad.tums.ac.ir published online: june 24, 2019 the possible occurrence of an. martinius. members of the an. maculipennis complex were identified by sequence analysis of the its2rdna and coi gene. morphological character-based identification showed that out of 1455 female anopheles specimens, 1121 (77%) belonged to an. maculipennis complex. molecular analysis of the complex indicated the presence of an. sacharovi and an. maculipennis s.s. in northwest and north of iran. this result is in agreement with other studies (2, 3). three genetically distinct species of the an. maculipennis complex were reported in iran (2): an. maculipennis s.s., an. sacharovi and an. persiensis. however, the last species was not found in this study as it was found as a dominant species of the complex in the southern caspian sea littoral provinces of guilan and mazandaran (2). six members of the group were reported based on molecular approach, while there was no an. martinius in the study area in northern iran (21). no species of this complex was found but an. maculipennis s.s. based on molecular study in zanjan province located in the northwest of iran (22). anopheles maculipennis s.s. and an. sacharovi were found in nine provinces from northwest to central regions of iran, it was no molecular evidence for presence of an. martinius either (23). molecular and phylogenetic analysis of the present investigation indicates more species diversity of an. sacharovi than has been recognized up to now. divergence among the members of mosquito complexes varies but can be fundamental. twenty-two species of anopheles were reported in iran including an. martinius (6). apparently, there is no molecular evidence of an. martinius presence across the whole study area. the results correspond with other findings (24). the base composition of the coi fragments showed an at bias with all sequences being between 66.4% and 67.8% at rich (mean=66.8%). these levels fall within the range of at bias in mitochondrial genomes of other members of an. maculipennis complex including an. maculipennis (25), an. messeae (26), an. sacharovi (2, 3) and an. martinius (an: ku950429). the coi sequences of an. sacharovi showed that there were 9 unique mtdna haplotypes with similarity of 98.17–100%. the sequences were translated into amino acids to obtain the mitochondrial code. translation of the sequences into amino acids showed all but one of the twelve specimens shared the same amino acid haplotype. only one specimen (ky196454) from parsabad-moghan in ardabil province showed two nucleotide transversion (g c, c g) at the second and third codon position bases of 478 and 479bp (table 3). these nonsynonymous bases change altered the codon from the consensus ccg (arginine) to cgc (proline), thus resulting in a unique amino acid haplotype. conclusion we collected three important malaria vectors in north of iran. the permanent presence of historical vectors of pathogens results in potential epidemiological threats. some malaria foci have been spotted in the northwest of iran. significant increases in commercial activities and travel from the neighbouring countries has led to increase concern about malaria and other vector-borne diseases in the northern provinces. this has increased the concern of malaria cases occurrence in the area. a better understanding of the accurate identification of sympatric sibling species and their distributions remain important as the malaria control programs depend on the accurate identification of the vectors. here, there is no evidence for occurrence of an. martinius in north of the country. acknowledgements the authors are grateful to prof mohebali and eng. zarei for cooperation during the study. this study was financially supported by tehran university of medical sciences (grant no. 94-02-27-28914). javascript:popref('f3') j arthropod-borne dis, june 2019, 13(2): 135–144 s gholami et al.: molecular characterization of … 143 http://jad.tums.ac.ir published online: june 24, 2019 the authors declare that there is no conflict of interests. references 1. salahimoghadam a, khoshdel a, barati m, sedaghat m (2015) an overview and mapping of malaria and its vectors in iran. hormozgan med j. 18(5): 473–485. 2. sedaghat m, linton ym, oshaghi m, vatandoost h, harbach r (2003) the anopheles maculipennis complex (diptera: culicidae) in iran: molecular characterization and recognition of a new species. bull entomol res. 93(6): 527– 535. 3. sedaghat mm, linton ym, nicolescu g, smith l, koliopoulos g, zounos ak, oshaghi ma, vatandoost h, harbach re (2003) morphological and molecular characterization of anopheles (anopheles) sacharovi favre, a primary vector of malaria in the middle east. syst entomol. 28(2): 241–256. 4. etherington d, 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(blattodea: blattellidae) atefeh khan-ahmadi1, hassan vatandoost1,2, amir ahmad akhavan1, mozhgan baniardalani1, kourosh khalifeh-soltani3, amrollah azarm4, *alireza zahraei-ramazani1 1department of biology and vector control of diseases, school of public health, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of mechatronic, school of electrical engineering, islamic azad university, majlesi branch, isfahan, iran 4department of medical parasitology and entomology, faculty of medical sciences of tarbit modares university, tehran, iran *corresponding author: dr alireza zahraei-ramazani, e-mail: azahraei@tums.ac.ir (received 01 oct 2022; accepted 21 feb 2023) abstract background: the german cockroach, blattella germanica (insecta: blattodea, blattellidae), which occurs widely in human buildings, is a small cockroach species. cockroach control chemical pesticides are toxic to the environment, and it is sometimes impossible to prevent them. controlling blattella germanica through ultrasonic waves can be efficient and less dangerous for the environment. methods: in this study, the repellency and lethal effect of ultrasonic waves on male and female german cockroaches was tested in a twin glass cubic chamber at laboratory condition. the wave frequencies tested ranged from 20 to 100khz with 5khz steps. a signal generator generated these frequencies, and the piezoelectric transmitter of these ultrasonic waves was positioned in the chamber's center on the upper side. results: fisher's test showed that there was the greatest repellency effect in both male and female at frequencies of 35 and 40khz. according to the results of the regression test, the most lethal effect is at the frequencies of 40 and 75khz. conclusion: the operating ultrasonic frequencies investigated in this study can be used to repel and kill german cockroaches as pests endangering human health and environment. keywords: repellency; lethal effect; ultrasonic waves; blattella germanica introduction blattella germanica is one of the most critical and widespread domestic pests worldwide (1). cockroaches have chewing mouthparts and feed on a variety of materials (omnivorous) and aiding in the mechanical transmission of various pathogenic viruses, bacteria, fungi, and protozoans to humans (2–4). there are two main methods for controlling cockroaches, including chemical and non-chemical methods. a third but not very common method of control involves the use of parasitoid wasps belonging to the family evaniidae, which naturally parasitize and destroy embryos inside egg capsules of cockroaches (3). the chemical control method includes residual spraying, dusting spray, mist spray, and poisoned baits (5, 6). the use of chemical insecticides to control cockroaches is the most popular method (3). although the use of chemical insecticides in pest control, including cockroaches, has increased, these pesticides can be dangerous for environment and harmful to non-target organisms (1, copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 84 http://jad.tums.ac.ir published online: march 31, 2023 7–9). one of the unwanted consequences of the widespread use of pesticides is that the insect will become resistant to pesticides, reducing insecticides' impact (7–9). non-chemical control methods for cockroaches include ultrasonic waves, vacuuming, trapping, and the use of heat and cold, non-toxic gases such as nitrogen and carbon dioxide (10). using the nonchemical method with ultrasonic pest repelling seems beneficial for both humans and their environments. cerci receptors of cockroaches receive air vibrations and can act as sound and vibration receptors. cerci tactile hairs respond to sudden vibrations or loud noises by sending spike potentials through the cerci nerve to points of contact with long axons at the synapses of the sixth abdominal nerve node. the long axons enter uninterruptedly into the thoracic nodes, where synaptic haas connect with the leg muscles' motor neurons. this system causes a fast transfer of stimuli and evasion maneuvers (11). ultrasonic pest repellent generates an ultrasonic frequency range that is not audible to humans but is loud for pests. to this end, some studies have examined the performance of some commercial ultrasonic devices that have been used to control german cockroaches (12– 16). the results of these studies showed that these devices did not have an acceptable detrimental effect on pests. the frequency used in these studies was 2–5khz, and the acoustic pressure in one centimeter of the sound source was 68 to 74db. the degree of repellency depends on the ultrasound's intensity and frequency. two main problems with previous ultrasonic devices can be considered two primary mismatches. first, the power of actual sound generated by devices was almost less than what the manufacturers claimed. second, the sound frequency did not match the german cockroach's operational frequencies, which are 20–50khz (17). a study confirms that to date, research on german cockroaches has not yielded an acceptable solution, and controlling this insect is still an outstanding problem. according to the above contents, in this study we examined the lethality and repellent effects of a span range of ultrasonic wave frequencies (20–100) khz on the specimens of german cockroaches. materials and methods in this study, ten male and female german cockroaches (5 females with capsules), were taken from the insectarium of the tehran university of medical sciences (tums) and bred in the esfahan health research station. german cockroaches were raised by keeping them in pyrex glass 2,000ml, temperature 25±2 (°c), humidity 60±5 (%), and, light period 12:12 (hours). they were given bread as food and water with a wet cotton wick. vaseline oil was rubbed on the edges of pyrex glass so that the german cockroaches could not escape from the container. then the container lids were be closed with nets. the test site was two glass square cubic chambers with dimensions 1× 1× 1m3, connected by a glass rectangular cubic chamber with dimensions 1× 0.4× 0.5m3. in one of these two square cubic chambers, a sliding glass door was used to enter and exit the cockroaches (fig. 1). the piezoelectric device, was located in the center of the upper page in first chamber (50 cm from the floor of the first chamber ( (fig. 1). the connection between the two chambers was closed by solid cardboard, and only 10cm was kept open. in fact, the shape of this chamber was taken from article (9), which was changed in this study. the time for each test was every morning, and the duration of each test was one hour. in each experiment, a bit of fresh bread was placed in the center of the bottom page in the first chamber as an attractor for german cockroaches. temperature and humidity were equal in both glass chambers. the experiments were performed to determine the frequency range of annoying german cockroaches. in this study, to generate and to display ultrasonic wave shapes, the signal genhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 85 http://jad.tums.ac.ir published online: march 31, 2023 erator model jsg106h2 and the oscilloscope model mos620ch were used, respectively. experiments were performed at frequencies (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100) khz, and at these frequencies, the sound intensity ranged was 166 to 173db. for each frequency, six german cockroaches (3 males + 3 females) were entered the chamber through the gate and, after one minute, the signal generator and oscilloscope that were set up earlier were turned on and the movements and behavior of german cockroaches were observed. several german cockroaches entered the second chamber without the sound transmitter and moved away from the sound source. for each frequency, three repetitions and one control were considered. in the control test, all conditions (temperature and humidity) were like the ultrasonic wave test, with the only difference being that the generator and oscilloscope were off. a total of 68 experiments were performed. after each test, german cockroaches were collected with an electric aspirator. also the live german cockroaches were transferred into glass containers which was contained with a piece of bread and wet cotton, and then closed the glass with piece of net. statistical analysis in this study two main statistical observations, were tested. first, the fisher test was applied to investigate the relationship between the repellency and lethal effects of ultrasonic waves tested on male and female german cockroaches. second, the logistic regression test was applied to investigate which frequency has the most repellency and lethal effect on german cockroaches examined. in all tests, a p-value of 0.05 was considered. results the sample size of german cockroaches tested was 408 (306 treatment plus 102 control), equal for males and females. several german cockroaches entered the second chamber, which is without a sound transmitter and moved away from the sound source. at the time of testing, some of the far-flung german cockroaches turned around and died after a short paddle. others in the main chamber with the audio transmitter turned around and died. according to the fisher test results (table1), the effect of ultrasonic wave repellency at 35 and 40khz was significant in both males and females with p< 0.05. the p-value is insignificant in other 15 frequencies. at all frequencies tested, the repellency impact is more significant in males than females except at frequencies of 70 and 85khz that females are more than males (fig. 2). at frequencies of 20, 90, and 100khz, no female german cockroach moved away. at 85khz, the male ones did not move away (fig. 2). when 95khz was used, repellency effects were similar for male and female german cockroaches (fig. 2). table 2 shows the results of the fisher test for mortality. it is indicated that the lethal effects of ultrasonic waves at 25 and 80khz are significantly different between males and females with p< 0.05. as for the rest of the tested frequencies, there is no significant difference. the mortality rate for male cockroaches is more than or equal to the female ones except for 80 khz. in fact, the most lethal effect of the female german cockroach is at the frequency of 80khz (fig. 3). moreover, the results specify that no female cockroaches were killed at 20 khz; however, dead male cockroaches were observed at all frequencies (fig. 3). the logistic regression test results can be seen in tables 3 and table 4. table 3 displays that the maximum effect of ultrasonic wave repellency is at 40khz frequency with 61.1% of repellency and with p< 0.05. table 4 determines the highest mortality impact at 40 and 75 khz, which was 72.2% and 66.7%, respectively, and in these two frequencies with p< 0.05. at frequencies 20 and 90khz, p< 0.05, but p-value is not significant because, in these two frequencies, the number of german cockroaches that have not moved away is more than the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 86 http://jad.tums.ac.ir published online: march 31, 2023 number of german cockroaches that have moved away. at frequencies 20, 85 and 90khz, is p< 0.05, but p-value is not significant. because the number of german cockroaches not killed is more than the number of german cockroaches killed. table 1. study of the repellency effect of ultrasonic waves based on the gender of german cockroaches (fisher test) row frequency (khz) df. exacting sig. 1 20 1 0.235 2 25 1 *0.008 3 30 1 0.712 4 35 1 0.681 5 40 1 0.500 6 45 1 0.167 7 50 1 0.681 8 55 1 0.500 9 60 1 0.500 10 65 1 0.319 11 70 1 0.681 12 75 1 0.066 13 80 1 *0.028 14 85 1 0.765 15 90 1 0.765 16 95 1 0.147 17 100 1 0.147 df.= degree of freedom, sig.= significant, *= p< 0.05 fig. 1. two glass square cube chambers are connected to each other by a rectangular cube, and one of the square cube chambers has a door for the entrance and exit of the safe cockroach for testing http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 87 http://jad.tums.ac.ir published online: march 31, 2023 table 2. study of the lethal effect of ultrasonic waves based on the gender of german cockroaches (fisher test) row frequency (khz) df. exacting sig. 1 20 1 0.235 2 25 1 *0.008 3 30 1 0.712 4 35 1 0.681 5 40 1 0.500 6 45 1 0.167 7 50 1 0.681 8 55 1 0.500 9 60 1 0.500 10 65 1 0.319 11 70 1 0.681 12 75 1 0.066 13 80 1 *0.028 14 85 1 0.765 15 90 1 0.765 16 95 1 0.147 17 100 1 0.147 df.= degree of freedom, sig.= significant, *= p< 0.05 table 3. determining the most effective frequency repelling german cockroaches using logistic regression test row frequency (khz) score df. exacting sig. 1 20 41.306 16 0.010 2 25 1.693 1 0.193 3 30 1.693 1 0.193 4 5 341 1 0.068 5 40 8.300 1 *0.004 6 45 0.078 1 0.780 7 50 1.774 1 0.183 8 55 0.61 1 0.804 9 60 3.341 1 0.068 10 65 0.078 1 0.780 11 70 1.693 1 0.193 12 75 0.601 1 0.804 13 80 0.600 1 0.439 14 85 0.649 1 0.421 15 90 5.690 1 0.017 16 95 3.455 1 0.063 17 100 5.690 1 0.017 df.= degree of freedom, sig.= significant, *= p< 0.05 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 88 http://jad.tums.ac.ir published online: march 31, 2023 fig. 2. the repellency rate of ultrasonic wave frequencies (20–100) khz on blattella germanica fig . 3. the mortality rate of ultrasonic wave frequencies (20–100) khz on blattella germanica frequency (khz) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 89 http://jad.tums.ac.ir published online: march 31, 2023 fig. 4. graphical abstract of ; two glass cubic chambers with dimensions 1× 1× 1 m3 , piezoelectric device in the center of the upper page, signal generator model jsg106h2 and the oscilloscope model mos620ch, schematic of ultrasonic waves; shape of a live german cockroach, and the german cockroaches which died from ultrasonic waves http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 90 http://jad.tums.ac.ir published online: march 31, 2023 table 4. determining the most effective frequency killing german cockroaches using logistic regression test row frequency (khz) score df. sig 1 20 459.957 16 0.001 2 25 0.102 1 0.749 3 30 2.746 1 0.097 4 35 1.712 1 0.191 5 40 7.790 1 *0.005 6 45 0.030 1 0.862 7 50 0.102 1 0.749 8 55 0.030 1 0.862 9 60 3.249 1 0.071 10 65 1.712 1 0.191 11 70 1.712 1 0.191 12 75 5.276 1 *0.022 13 80 0.663 1 0.416 14 85 7.000 1 0.008 15 90 7.000 1 0.008 16 95 1.352 1 0.245 17 100 1.352 1 0.245 df.= degree of freedom, sig.= significant, *= p< 0.05 discussion cockroaches, which are abundant in most human places, are known vectors of about 150 species of bacteria, 60 species of fungi, six species of yeast, 90 species of protozoa, 45 species of pathogenic ringworms, and several hookworms and whipworms, as well as they are significant sources of allergens (17). nowadays, all people know the dangers of using various types of chemical insecticides that are used to control cockroaches and especially the german cockroach inside and outside human places. the use of simple, low-cost, and risk-free physical control methods is a choice that should be seriously considered. one of those methods is the use of ultrasonic waves. ultrasound refers to mechanical waves with a frequency higher than human hearing (more than 20khz or 20,000 cycles per second). ultrasonic waves are generally longitudinal and originate from the vibrations of an elastic body. they always require a medium to diffuse and use pressure changes to diffuse. these waves are rapidly absorbed by the air. as a result, they are not transmitted over very long distances (18, 19). the frequency range of ultrasounds is very wide and depends on their use (20, 21). humans are exposed to ultrasonic waves in different ways, through air as well as through direct contact with a vibrating solid or liquid coupling medium. air exposure to ultrasound is used in many areas of life, such as dog whistles, bird, rodent and insect repellants. air ultrasound mainly affects the external organs of the body such as ears and eyes (in case of longterm exposure). adverse effects reported in people exposed to airborne ultrasound include temporary threshold changes in sound perception and hearing loss, changes in blood sugar levels, electrolyte imbalances, fatigue, headache, dizziness, nausea, tinnitus, and irritation. also, in the second method, ultrasound works in the liquid environment of the body based on tissue heating (thermal effect) or cavitation phenomenon. liquid exposure occurs mainly in medicine, and in diagnosis, treatment, and surgery. when used at higher frequencies, the effect of ultrasound on tissue is based on heating. the thermal effect usually becomes dominant as the frequency increases from kilohertz (khz) to megahertz (mhz) levels (22–24). according to their frequency, there are three sources of ultrasound production, low frequency (10–100khz), with many applications from an industrial perspective, medium frequency (100 khz-1mhz), for use in therapeutic applications, and high frequency (1–10mhz), mainly used for medical purposes and non-destructive control devices (20, 24). the potential of ultrasound to cause adverse effects in laboratory animals, plants, and cell culture has been well established, but whether similar effects occur in humans and in sensitive human tissues needs further investigation (16, 24–26). also, despite many animal studies, no human research has been recorded to date http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 91 http://jad.tums.ac.ir published online: march 31, 2023 that shows the major physiological consequences of ultrasound exposure to imaging (13). it is a proven fact that the human body absorbs ultrasonic waves, and the effects of this absorption are unknown depending on the form of application and the dose and frequency of ultrasonic waves, either khz or mhz, in the medium to long term, and can be it varies from positive to very negative (27). to find out the proper instructions for using ultrasound emitting equipment to protect the health of workers, health professionals, patients, and all people and in general for better occupational safety, indoor environment quality and environmental health; it is necessary to formulate regulations by government departments (28). therefore, it is recommended to conduct more studies on the effect of ultrasonic waves on the people who receive these waves so that the negative consequences of these incomprehensible sounds on human health can be prevented. it must be accepted that mankind is increasingly exposed to these sound waves, but its consequences are ignored by most people (13). previous studies have shown that ultrasonic waves cannot have a repellent effect on german cockroaches. in this study we investigated the effects of ultrasonic waves on male and female german cockroaches. the 20–100khz waves were tested in 5 to 5 sine waves to reach this objective. all experiments were performed in two glass chambers with no extra noise in the environment during the test. with regular feeding and standard laboratory conditions, all the control cockroach specimens survived for more than 5 days after testing. furthermore, the ultrasonic wave exposure cockroach specimens (treated cockroaches) started to die after one hour of rest, and all of them died within three days after testing. the results of this study show that the effects of these ultrasound frequencies are more effective on male than female of german cockroaches, and can repel, seriously injure, or kill them (fig. 4). in this study, where the use of ultrasonic sound waves was performed on the german cockroach, its laboratory effectiveness was confirmed. therefore, it is recommended that the country's health and environmental officials pay special attention to this german cockroach method controlling which is nature friendly too. in addition, in cases where there are people sensitive to chemical insecticides or there are insects’ resistant to chemical insecticides, it is recommended to use this alternative control method. of course, our recommendation until formulate regulations by government departments, use this control method outside of human places. it should be known that ultrasonic waves should be periodically tested by researchers on the cockroaches of that country, and if they are effective, they can be used again. in future studies, if these german cockroaches are exposed to higher frequencies with waveforms such as square, triangular, noise, or broom, the effects them may be significantly more intensive and deadly. identifying these appropriate frequencies, will enable electronic devices to control different insects in future more efficiently. conclusion it can be concluded that these ultrasound frequencies can repel, injure, or kill german cockroaches. this study showed that ultrasonic waves could have both repellent and lethal effects on german cockroaches. these effects were more effective on males than females. we found that there is a repellent effect of 30.7% (10.7% female + 19.9% male) in general. its lethal effects were 40.8% overall (17% female + 23.8% male). the highest repellency rate and death rate were observed at 40khz and both 40khz and 75khz, respectively. identifying these appropriate frequencies will enable future electronic devices to control these insects more efficiently. if these insects are exposed to higher frequencies with waveforms such as square, http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 83–93 a khan-ahmadi et al.: evaluation of … 92 http://jad.tums.ac.ir published online: march 31, 2023 triangular, noise, or broom, insects' effects may be significantly better. however, further studies are required to reveal safety of ultrasonic waves on the people who receive these waves before we could recommend this method. acknowledgements the authors would like to express their gratitude to eng. reza jafari, internal manager esfahan health research station. also, the authors thank all colleagues and researchers who participated in this study and supported us. this study was financially supported by the deputy of research, tehran university of medical sciences; grant no. 42088. ethical considerations this experiment was carried out under the guidance of the ethics committee of the tehran university of medical sciences (ir.tums. sph.hcr ec.1397.294). conflict of interest statement the authors declare there is no conflict of interests. references 1. pan xy, zhang f (2020) advances in biological control of the german cockroach, blattella germanica (l.). biol control j. 142: 104104. 2. wannigama dl, dwivedi r, zahraei-ramazani a (2014) prevalence and antibiotic resistance of gram-negative pathogenic bacteria species isolated from periplaneta americana and blattella germanica in varanasi, india. j arthropod borne dis. 8 (1): 10–20. 3. zahraei-ramazani ar, saghafipour a, vatandoost h (2018) control of american 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f, mortimer a, carstensen e, kremkau f, miller d, miller m (1988) bioeffects considerations for the safety of diagnostic ultrasound. j ultrasound med. 7: 1–38. 26. barnett sb, ter haar gr, ziskin mc, rott hd, duck fa, maeda k (2000) international recommendations and guidelines for the safe use of diagnostic ultrasound in medicine. ultrasound med biol. 26(3): 355–366. 27. houston le, odibo ao, macones ga (2009) the safety of obstetrical ultrasound: a review. j ultrasound med. 29 (13): 1204–1212. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 400 http://jad.tums.ac.ir published online: december 31, 2020 original article molecular epidemiology and phylogeny of crimean-congo haemorrhagic fever (cchf) virus of ixodid ticks in khorasan razavi province of iran hossein maghsood1,2; *sedigheh nabian1,2; parviz shayan1,2; tahmineh jalali3,4; meysam saboor darbandi5; mohammad mehdi ranjbar6 1department of parasitology, faculty of veterinary medicine, university of tehran, tehran, iran 2iranian research center for tick and tick-borne diseases, university of tehran, tehran, iran 3department of arboviruses and viral hemorrhagic fevers (national reference laboratory), pasture institute of iran, tehran, iran 4research center for emerging and reemerging infectious disease, pasteur institute of iran, tehran, iran 5veterinary organization of kalat, khorasan razavi, iran 6department of foot and mouth disease vaccine production, razi vaccine and serum research institute, agricultural research education and extension organization (areeo), karaj, iran *corresponding author: dr sedigheh nabian, e-mail: nabian@ut.ac.ir (received 14 june 2017; accepted 21 dec 2020) abstract background: crimean–congo hemorrhagic fever (cchf) is a fatal disease caused by nairovirus classified within the bunyaviridae family. the virus is transmitted to humans through the bites of infected ticks or direct contact with viremic animals or humans. the current study aimed to detect the virus genome in ticks from khorasan razavi province. methods: one hundred hard ticks were collected randomly from 100 sheep in four different areas of the province. collected ticks were kept alive and identified. all the ticks were analyzed for the presence of cchf virus genome using reverse transcriptase polymerase chain reactions (rt-pcr). results: the identified ticks were belonging to hyalomma marginatum (16% female and 6% male), rhipicephalus turanicus (52% female and 25% male), and dermacentor raskemensis (1%). the cchf virus genome was found in hyalomma marginatum (5% male from taibad and sabzevar region and 1% female from taibad). genetic analysis of the virus genome isolated from two regions (sabzevar and taibad) showed 100% identity. conclusion: this study indicated that cchf should be regarded as a risk-borne infection in this province. therefore, special health management is needed to control this disease. keywords: crimean-congo haemorrhagic fever; ixodid ticks; khorasan razavi province introduction crimean-congo hemorrhagic fever (cchf) is a serious zoonotic viral disease which can be fatal. crimean-congo haemorrhagic fever has been reported in asian, african and eastern european countries (1) and can be transmitted to humans via a number of routes including ticks bites, direct contact with infected animal blood or meat, direct contact with infected individuals, and through nosocomial and community outbreaks (2). the virus causing the disease has a single stranded rna genome and is known as nairovirus in the family of bunyaviridae (3-5). although the disease is usually asymptomatic in infected animals, it causes a severe haemorrhagic syndrome in humans, with a fatality rate up to 50% (6). crimean-congo hemorrhagic fever has the highest rate of distribution amongst those areas of the world with global distribution of ticks, especially hyalomma (7). diagnosis of viral infection is achieved using three methods, elisa, isolation of the cchf virus and molecular methods to detect copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 401 http://jad.tums.ac.ir published online: december 31, 2020 the viral genome (8). jorjani, first reported the disease in 1100 ad in his book and pointed out that ticks were associated with the haemorrhagic syndrome (9). in 1975, it was discovered that the virus existed around the caspian sea and the east azerbaijan province of iran. three years later, in iran, the virus was reported from infected ticks (10, 11). chinikar et al. analyzed the cchf virus genetically in iran and deposited the sequence of partial fragments in genbank (ay366373 and ay366979). furthermore, their discovery was recognized globally as they had reported two genetic branches for of the virus in iran (12). so far, cchf virus has been reported in more than 31 different types of ticks including ixodidae and argasidae ticks in the world (13, 14). there had been numerous reports on cchf in khorasan razavi province (8, 12, 15), but not on its vectors. therefore, the purpose of this study was to determine molecular evidence of cchf viruses in ixodid ticks from khorasan razavi, iran materials and methods study area khorasan razavi province (fig. 1) is located in north-east of iran (33◦52′ and 37◦ 42′) (fig. 1). regarding its massive area, this province consists of various climates and natural environments but in terms of the temperature averages is 13.5 °c/56.2 °f and also rainfall categorized as nearly an average receiver of rain (the annual rainfall is 251mm/9.9 inch) (https://www.worlddata.info/asia/iran/climate -razavi-khorasan.php). tick collection and molecular procedure the ticks were collected from 17 regions of 4 cities namely mashhad, sabzevar, kalat and taibad (fig. 1). one hundred ticks were collected from entire body of each 100 sheep and kept in proper humid conditions in the caped tubes and transferred to the department of parasitology, tehran faculty of veterinary medicine. the ticks were identified using identification keys (16). after identification of the ticks, they were analyzed using rt-pcr in the arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory) at the pasteur institute of iran. ticks were individually washed twice with pbs (pbs1×, ph= 7.4) and crushed with mortar and pestle in 200–300μl of pbs 1×. then, viral rna was extracted from each tick by using the qiaamp viral rna kit according to the manufacturer’s instructions (qiagen gmbh, hilden, germany). the extracted viral rna was subsequently analyzed by rt-pcr with the one-step rt-pcr kit (qiagen gmbh, hilden,germany) using specific published primers: f2 5ʹtggacaccttcacaaa ctc3ʹ and r35ʹgacaattccctacacc3ʹ (17). these primers were selected to amplify a 536bp fragment of a highly conserved region inside the s-segment of the cchfv genome. the pcr was carried out in a total volume of 50μl for 30min at 50 ˚c, 15min at 95 ˚c, and 40 cycles including 30s at 95 ˚c, 30s at 50 ˚c, 45s at 72 ˚c, and, finally 10min at 72 ˚c as a final extension. positive and negative controls were used in each pcr reaction (18). then, pcr products were analyzed by performing 1.5% agarose gel electrophoresis in tbe buffer with a 100bp dna ladder. dna bands were visualized with ethidium bromide staining under ultraviolet transilluminator (genius co., usa). sequence analysis for verification of positive samples, amplified fragments were sent for sequencing to takapouzist co. then, finch tv software was used for quality control of sequenced data. subsequently, the sequenced data were compared with the corresponding sequence data registered in data bank by blast program (http://blast.ncbi.nlm.nih.gov/blast.cgi) after verification of similarity, closest hits were selected and all similarity blast results retrieved. for constructing the phylogenetic tree, clustalw and bioedit software version 7.7.9 were used. after alignment of iranian s gene http://jad.tums.ac.ir/ https://www.worlddata.info/asia/iran/climate-razavi-khorasan.php https://www.worlddata.info/asia/iran/climate-razavi-khorasan.php j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 402 http://jad.tums.ac.ir published online: december 31, 2020 sequences of cchf with other sequences, redundant sequences or very short sequences and areas with ambiguous alignment or containing poly-n stretches were excluded from the analyses. for probable similarity and cluster analysis, the mega 6 software package was used for construction of phylogenetic trees using the neighbor joining method with bootstrap 1000 (19). results the ticks collected from the four different regions (mashhad, taibad, sabzevar and kalat) and were classified in 3 different species as: female hyalomma marginatum (fig. 2) (16% female and 6% male), rhipicephalus turanicus (fig. 3) (52% female and 25% male) and dermacentor raskemensis (fig. 4) (1%) (table 1). cchf virus infection was found in hyalomma marginatum (5% male ticks (from taibad and sabzevar and 1% female ticks from taibad) (table 2). genetic analysis of the virus genome isolated from the two different regions (sabzevar and mashhad) showed 100% identity with some registered isolates in genbank. the obtained nucleotide sequences of cchf virus, showed 100% similarity with the cchf virus from several registered sequences from the hamedan province of iran (ay366378.1, ay 366379.1, gu456725.1). moreover, blast data of the ss rna segment of cchf virus with sequences from genbank showed 100% similarity with sequences from pakistan and afghanistan (ay 905662.1, aj538198.1, hm 452305.1) and 99% similarity with three other sequences from iran (gu456728, dq446212.1 and dq446213.1). furthermore, the obtained sequences showed similarity between 85–95% with the registered sequences from various other states as follows: iraq (aj 538196.1), china (dq211642.1), south africa (ay 905664.1, dq211648.1), sudan (gq862372.1), bulgaria (jf807428.1), turkey (hq664913.1, fj601847.1) and with some sequences from iran (u15022, ay 905653.1). in order to construct phylogenetic trees based on the partial nucleotide sequence of the s segment, sequences belonging to neighboring countries to iran and some very similar sequences from a blast search (africa and china) were used. the phylogenetic tree in fig. 5 showed the close relationships between taibad and sabzevar sequences with previous reports of iranian cchf sequences (s segment) and also with isolates from afghanistan and pakistan. they are all located in group 1. sequences from oman (dq211645) and iraq (aj538196) and afghanistan (jx908640) had lower similarity to iranian sequences. also, the nearest relatives to iranian sequences are tajikistan, china and india sequences that are clustered together in group 2. furthermore, isolates from different provinces of turkey grouped together in a separate clade (group 3). interestingly one of the iranian isolates from rasht was located in this group. table 1. the variety of identified ticks in four cities of khorasan razavi province, iran ticks location identified ticks (%) number of collected ticks hyalomma marginatum hyalomma marginatum rhipicephalus turanicus rhipicephalus turanicus dermacentor raskemensis female male female male female sabzevar 30 43/3 10 30 16/6 0 taibad 38 7/8 7/8 57/8 26/3 0 mashhad 14 0 0 35/7 64/2 0 kalat 18 0 0 88/8 5/5 5/5 total 100 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 403 http://jad.tums.ac.ir published online: december 31, 2020 fig. 1. map of iran shows the location of khorasan razavi province (the black area) fig. 2. hyalomma marginatum (male, above and female, below) fig. 3. rhipicephalus turanicus (male, dorsal and ventral surfaces) fig. 4. dermacentor raskemensis (female) table 2. the results of tick infection by crimean-congo haemorrhagic fever virus using reverse transcription polymerase chain reaction dermacentor raskemensis ♀ rhipicephalus turanicus ♂ rhipicephalus turanicus ♀ hyalomma marginatum ♂ hyalomma marginatum ♀ ticks location 0 0 0 0 0 kalat 0 0 0 0 0 mashhad 0 0 0 +3 +1 taibad 0 0 0 +2 0 sabzevar 0 0 0 5 1 total http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 404 http://jad.tums.ac.ir published online: december 31, 2020 fig. 5. the tree of iranian partial s segment nucleotide sequences constructed by mega 6. the tree was constructed by using the neighbor-joining (nj) algorithm based on differences in s sequences of different isolates. units at the bottom of the tree indicate the number of substitution events. the length of each pair of branches represents the distance between sequence pairs. the dataset was re-sampled 10,000 times using the bootstrap method. the sequence information at the tips of the branches includes the accession number of the sequence, name of the isolate and strain discussion crimean-congo hemorrhagic fever (cchf) is a severe and often fatal viral disease in human caused by a bunyavirus and is transmitted by infected body fluids, secretions or persons with ticks (18, 20, 21). it is considered as one of the zoonosis diseases. this disease is reported from asian, african and eastern and central european states. there are some studies about diagnosis of its agents and distribution of this tick borne diseases (12, 18, 20-23). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 405 http://jad.tums.ac.ir published online: december 31, 2020 the global warming and climatic changes in iran, cause changes in tick fauna and tick borne diseases including cchf throughout the different regions. therefore, it seems that conducting more periodic studies on virus distribution and its genetic variation in order to prevent a wider distribution of the virus is needed (12, 18). amongst the different regions in iran, the khorasan province has crucial importance because of neighboring with afghanistan and pakistan where the ticks and cchf exist. reports of cchf in iran were related to sporadic cases between 1970–1978; while there was no evidence of cchf investigations between 1978–1999 (10, 11, 24, 25). virus reported from different provinces (esfehan, sistan and baluchestan, fars, tehran, khorasan and khuzestan) which resulted some death cases in years between 2000–2009 (15). another study which was carried out in yazd, proved that hyalomma species is the best transmitter of the virus. none of the ticks genus rhipicephalus and dermacentor harbored the virus genome (27). telmadarraiy et al detected igg against cchf in examined sheep (27.8%) of hamedan province and showed 16.4% of the ticks were certain transmitters of cchf especially those belonging to the hyalomma, rhipicephalus and haemaphysalis species (23). chinikar et al. also analyzed the cchf virus genetically to show that nucleotide sequences of the s and m segments of viruses from 9 iranian patients had 98% similarity. furthermore, their physiological analysis proved that they were similar to the branches of the same virus found in madagascar and pakistan. they could also establish that there have been at least 2 genetic lineages in circulation in iran (12). analysis of s segment nucleotide sequences of cchf between taibad and sabzevar showed 100% identity. also, comparison of these results with another sequence in genebank showed 100% identity with pakistan (aj 538198.1, ay905662.1) and afghanistan viruses (hm425305.1) related sequences. these similarities can be attributed to the transportation of livestock between neighbor provinces in iran or transmitting the ticks by migrating birds between neighbor countries. the present study points out that h. marginatum ticks in the regions of taibad and sabzevar are certain transmitters of cchf. the above claim can be proven by the help of a comparison between the results obtained by ghorbani who reported numerous similarities in the nucleotide sequences extracted from iranian patients in different regions as well as pakistan’s (28). tahmasebi et al. showed genetic diversity in isolated virus from ticks of different parts of hamedan province, which attributed to virus replication and recombination phenomena in vector ticks (21). conclusion regarding to significance of ticks in cchf transmission and similarity of obtained cchf virus sequences in this study with afghanistan and pakistan sequences, suitable strategy is that infected ticks to virus and illegal transactions of infected livestock in these borders might be considered to control cchf disease in khorasan razavi province. acknowledgements the authors would like to thank the faculty of veterinary medicine, university of tehran for funding this project and staff of the arbovirus laboratory, pasteur institute of iran (national laboratory of iran) for their technical support and veterinary office of sabzewar and dr joghataei. the authors declare that there is no conflict of interests. references 1. swanepoel r, struthers j, shepherd a, mcgillivray g, nel m, jupp p (1983) http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 400–407 h maghsood et al.: molecular epidemiology and … 406 http://jad.tums.ac.ir published online: december 31, 2020 crimean-congo hemorrhagic fever in south africa. am j trop med hyg. 32: 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http://jad.tums.ac.ir published online: june 30, 2022 original article identification of intestinal fungal microflora and bacterial pathogens in the collected adult ixodes ricinus from the northern provinces of iran manijeh yousefi-behzadi1,2, neda moazzezy3, mahdi rohani1,4, saied reza naddaf5, ehsan mostafavi1,2, ali mohamadi1, masoomeh shams-ghahfarokhi6, nasrin pashootan7, *mehdi razzaghi-abyaneh7 1department of epidemiology and biostatistics, research centre for emerging and reemerging infectious diseases, pasteur institute of iran, tehran, iran 2national reference laboratory of plague, tularemia and q fever, research centre for emerging and reemerging infectious diseases, pasteur institute of iran, akanlu, kabudar-ahang, hamadan, iran 3molecular biology department, pasteur institute of iran, tehran, iran 4department of microbiology, pasteur institute of iran, tehran, iran 5department of parasitology, pasteur institute of iran, tehran, iran 6department of mycology, faculty of medical sciences, tarbiat modares university, tehran, iran 7department of mycology, pasteur institute of iran, tehran, iran *corresponding author: dr mehdi razzaghi-abyaneh, e-mail: mrab442@pasteur.ac.ir (received 03 oct 2020; accepted 20 apr 2022) abstract background: ticks are vectors of many pathogens that involve various important diseases in humans and animals, they have several diverse hosts consequently can retain a diverse group of indigenous microbes, from bacteria to fungi. little is known about the prevalence and diversity of tick microflora colonizing the midgut and their effects on ticks and their interaction. this information is important for development of vector control strategies. methods: this study was carried out in northern iran during autumn 2019. ticks, ixodes ricinus caught alive on the bodies of domestic animals in the fall. the tick homogenate was prepared. the identification of fungal isolates was carried out according to a combination of macro and microscopic morphology and molecular sequencing. pathogenic bacteria of the family borreliaceae, francisella tularensis, borrelia burgdorferi and coxiella burnetii were tested by real-time pcr. results: a total of 133 mature i. ricinus ticks were collected from domestic animals, including 71.5% cattle and 28.5% sheep. the tick frequency rates were 87.21% for mazandaran, 8.28% for golestan and 4.51% for gilan provinces. total prevalence of fungal tick contamination was 53.4% (75/133) of which trichoderma harzianum (57%) was the most prevalent species followed by aspergillus spp. (42%), mortierella alpine (19%) and penicillium polonicum (14%). all tick samples were negative for three pathogenic bacteria including francisella tularensis, coxiella burnetii, and borrelia burgdorferi by real-time pcr analysis. conclusion: these results show a first picture of the microbial diversity of ticks and highlight the importance of microbiota and their role in host-pathogen interaction. keywords: microflora; ixodes ricinus; fungal species; mycoflora; microbiome introduction arthropods are vectors of many pathogens that involve various important diseases in humans and animals (1). these organisms are hematophagous which feed on blood and most of these arthropods are blood eaters and during engorgement transmit or acquire microorganisms (2) on the other hand, arthropods have several diverse hosts consequently can retain a diverse group of microbes indigenous, from bacteria to fungi (3, 4). a single tick can carry different pathogens, co-infections are common and can make diagnosis and treatment difficult (4). ixodidae ticks are important in the transmission of a variety of zoonotic microorganisms (viruses, copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://mail.yahoo.com/neo/b/compose?to=mrab442@pasteur.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 98 http://jad.tums.ac.ir published online: june 30, 2022 bacteria and protozoan) (5). ticks are not only carriers of pathogens, but also a diverse group of commensal and symbiotic microorganisms as bacteria, viruses and fungi are also present in ticks whose biology and their effects on ticks and their interaction remain largely unexplored or very often neglected (6, 7). tick-borne diseases seem to be a real challenge threatening public and economic health. some of them can cause physical and cognitive damage which can be very painful (8). lyme disease is the most common tickborne pathogen in the temperate woodlands of north america, europe and asia and is caused by some members of borrelia burgdorferi. the past 20 years the number of reported cases has tripled in the united states and has also increased in parts of europe (9). the first endemic case of lyme borreliosis was reported in 1997 in iran proving the existence of the spirochete b. burgdorferi which had not previously been found in ticks in this region (10). in 2001 a 9-yearold boy was admitted to children's medical center with a final diagnosis of lyme disease (https://acta.tums.ac.ir/index.php/acta/article/ view/3187). in 2020, naddaf et al. reported for the first time the existence of the infection of i. ricinus ticks in the littoral of the caspian sea by the spirochetes lyme borreliosis (11). the tularemia caused by francisella tularensis, zoonosis, is characterized by high morbidity and mortality rates in more than 190 different mammal species, including humans (12). in iran, positive serological tests were first reported in 1973, in wildlife and domestic livestock in the northwestern and southeastern parts of the country. the first human case was reported in 1980 in the southwest of iran, and recent studies conducted among at-risk populations in the western, southeastern, and southwestern parts of iran. the presence of f. tularensis in ticks was confirmed in the province of kurdistan (western iran) and the possible role of ticks in the transmission of the pathogen to livestock and humans by bites has been demonstrated in this region (13). q fever is a zoonosis with a worldwide dis tribution is caused by coxiella burnetii, many species of mammals, birds, and ticks are reservoirs of c. burnetii in nature. coxiella burnetii infection is most often latent in animals (14). according to a study published in 2019 in the case of infective endocarditis (ie) patients hospitalized in rajai cardiovascular medical and research center from august 2015 to september 2017, a high prevalence of q fever was revealed (15). coxiella burnetii dna or its antibodies have frequently been detected in ruminants. since these animals can transmit the infection to humans, q fever can be a potential health problem in iran (16). anaplasmosis is a zoonotic disease, described in various domestic animals and humans and the bacterium in question is anaplasma phagcytophilum transmitted by ticks with a worldwide distribution (17). in iran, in 2014 in the province of mazandaran, we have the first investigation of tick-borne anaplasma infections in domestic animals and humans (18). fungal microbiota comprises an important part of total microbiota of vertebrates and non-vertebrates (19). the determination of the tick fungal microflora (microbiota) and the interactions between its symbiotic microorganisms in the context of pathogen transmission will likely reveal new perspectives and spawn new paradigms for tick-borne diseases (20). the present study for the first time aimed to determine the diversity of the fungal microflora that colonize in the middle intestine of mature i. ricinus ticks in the three provinces of northern iran. materials and methods study area and population this study was conducted in northern iran consists of the southern border of the caspian sea and the alborz mountains with an area of 58,167km2 in three provinces of gilan (37.280 n, 49.59o e) mazandaran (36.2262° n, 52.5319° e) and golestan (37.2898° n, 55.1376° e). the forest cover of northern iran has a total of http://jad.tums.ac.ir/ https://acta.tums.ac.ir/index.php/acta/article/view/3187 https://acta.tums.ac.ir/index.php/acta/article/view/3187 j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 99 http://jad.tums.ac.ir published online: june 30, 2022 3,400,000 hectares of forest on the northern slopes of the alborz mountains and the coastal provinces of the caspian sea (fig. 1). general characteristics of the climate are too much rain in all seasons, especially in autumn and winter, relatively high humidity in all seasons and low temperature difference during the day due to the presence of moisture. domestic animals such as cattle, sheep, and dog due to the large number of forests, can easily move between the forest and the herd. sampling sampling of ticks on domestic animals (cattle, sheep) was carried out in autumn 2019 (from 6 to 11 november) during the period of multiplication of ticks and was carried out at various sites in the provinces of northern iran including mazandaran, gilan and golestan (table 1), (fig. 1). the sample size was estimated at 100 ticks but given the number of ticks collected at this time of the year to increase the accuracy of the study, all 133 ticks collected were entered in this study and collected according to their animal host in 14 sampling sites (table 1). ticks caught alive on the bodies of domestic animals living in barn using forceps and sterile disposable material, were then collected according to their host. the ectoparasites were identified directly under a stereomicroscope without mounting. all ectoparasites have been identified at the species level using available taxonomic morphological key of ticks (21). ticks identified as mature i. ricinus were kept for this study. a number of 133 i. ricinus ticks were isolated from two domestic animals, cattle and sheep from a total of 14 sampling sites (table 1). preparation of ticks’ homogenate the whole ticks were placed in sterile tubes containing 70% ethyl alcohol for 3min then rinsed with sterile water three times to avoid transportation of microorganisms from the outside environment and from the surface of the ticks. homogenization of the samples (ticks) is done with a qiagen tissue lyser ii homogenization device, the samples were placed in sterile microtubes containing metal willow and 300μl of rtl rna buffer extraction kits (qiagen) were added to the microbial tubes. according to the manufacturer's instructions, the qiagen tissue lyser ii microtubes were homogenized at 30 hertz (hz) for 3min to obtain a uniform suspension. the topical solution was removed and transferred to a new sterile microtube to continue the extraction process. one part was used fresh for fungus culture and the rest for dna extraction and the molecular process. mycological identification culture based identification ticks’ homogenate was diluted with phosphate buffer saline (pbs) to give a final volume of 100µl and diluted solutions these were spread on to sabouraud dextrose agar (peptone 1%, glucose 2%, agar-agar 1.5%; merck, germany) and potato dextrose agar (potato infusion 20%, dextrose 2%, agar 2%) plates and incubated for 2 weeks at 25 °c. the plates were periodically checked for fungal colonies. identification of fungal isolates was performed according to a combination of macro and microscopic morphology. molecular identification dna extraction dna was extracted and purified from fungal colonies using the following method, briefly, 10–20mm3 of the fresh colonies grown on sabouraud glucose agar (difco, detroit, mi, usa) were added to 1.5ml tubes that contained 300µl of lysis buffer (200m tris-hcl, ph 7.5; 25mm ethylenediaminetetraacetic acid (edta); 0.5% w/v sodium dodecyl sulfate; and 250mm nacl) and crushed with a conical grinder (micro multi mixer; ieda co. ltd., tokyo, japan) for 1min. the samples were incubated in a boiling water bath for 10min, mixed with 150µl of 3.0m sodium acetate, kept at −20 °c http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 100 http://jad.tums.ac.ir published online: june 30, 2022 for 10min, and centrifuged at 12,000rpm for 10min. the supernatant was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and once more with chloroform. the dna in supernatant was precipitated with 250µl isopropanol, washed with 300µl of 70% ethanol, air dried, and rehydrated in 50µl ultrapure water, and stored at −20 °c until use. polymerase chain reaction (pcr) and sequencing for the polymerase chain reaction with the specific its region primers, its1 and its4 (table 2). each mixture contained 2.5µl of 10× reaction buffer, 0.5µm of universal fungal primers forward its1 and reverse its4, 400µm of deoxynucleoside triphosphate, 1.25u of taq dna polymerase (takara, japan), 1µl of dna extracted and enough ultrapure water to reach a final reaction volume of 25µl. the pcrs were programmed for preheating at 96 °c for 6min followed by 35 cycles at 94 °c for 1min, 56 °c for 1min, and 72 °c for 45s, and a final extension step at 72 °c for 10min. five microliters of the pcr products were electrophoresed onto1.5% agarose gel in tris-acetate-edta (tae) buffer (tris 40mm, acetic acid 20mm, edta 1mm), stained with 0.5µg/ml of ethidium bromide, and observed and photographed under ultraviolet irradiation. the pcr products of the its region were purified using a qi quick purification kit (qiagen, valencia, ca, usa), sequenced in both directions using the same primers. molecular detection of bacterial pathogens the whole genomic dna extracted from ticks were screened for members of the family borreliaceae, f. tularensis and c. burnetii by targeting 16srrna, isftu2 and is1111 genes respectively, using primers and probes listed in table 2. the final 20μl reactions contained 10μl master mix 2× (amplicon, denmark), 500 nm of each primer, 200nm probe, and 4μl (50 nm) of the template dna. amplifications were performed in a rotor-gene 6000 instrument (corbett life science, sydney, australia) for an initial denaturation at 95 °c for 10min, followed by 45 cycles of denaturation at 95 °c for 15sec, and annealing at 60 °c for 60sec. dna of b. borgdorferi senso stricto (amplirun® borrelia dna control), the dna of f. tularensis subsp holarctica nctc 10857 and plasmid contain is1111 from c. burnetii were included as positive controls in all the assays. pcr with no sample dna was considered as negative control. results ticks collected a total of 133 mature i. ricinus ticks were collected from domestic animals including 71.5 % cattle and 28.5% sheep. the tick frequency was 87.21% for mazandaran, 8.28% for golestan and 4.51% for gilan (table 1), (fig. 2). detection of fungal species the isolated extract of tick midgut was cultured on sabouraud dextrose agar (e-merck, germany) medium to isolate the possible fungal species. the morphological and then molecular identification have proven the fungal species include 57% trichoderma harzianum (mt804339, mt803548, mt809136), 42% aspergillus spp., 14% penicillium polonicum (mt809131) and 19% mortierella alpine (mt803487). among the 14 sampling sites, ticks from seven sites were contaminated with fungal species from four distinct genera (fig. 3). detection of bacterial pathogens real-time pcr for three bacterial pathogens including f. tularensis, c. burnetii, b. burgdorferi was negative for all 133 ticks obtained from 14 sampling sites (table 1). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 101 http://jad.tums.ac.ir published online: june 30, 2022 table 1. details of location and host of fungal species (trichoderma harzianum, mortierella alpine, aspergillus sp., penicillium polonicum) were found in ixodes ricinus from the northern provinces of iran, 2019 province city distract n e host no. of sampled ticks (contaminated ticks) fungal spp. mazandaran mahmoud-abad boondeh 36°34'16.0" 52°14'16.0" cattle 7 (5) t. harzianum mazandaran chamestan joorband 36°26'42.2" 52°07'37.9" cattle 6 nf mazandaran chamestan joorband 36°26'45.2" 52°07'26.4" sheep 2 nf mazandaran chamestan joorband 36°26'39.2" 52°07'32.1" cattle 6 nf mazandaran amol razageh 36°19'50.0" 52°21'42.0" sheep 6 nf mazandaran amol esku mahalleh 36°24'19.7" 52°18'30.8" cattle 7 nf mazandaran chamestan joorband 36°26'15.5" 52°07'15.3" cattle 14 (6) (14) t. harzianum m. alpine mazandaran amol komdarreh 36°23'55.2" 52°25'51.7" cattle 20 nf mazandaran savadkuh chay baq 36°20'30.4" 52°52'05.9" sheep 11 (9) (5) t. harzianum, aspergillus sp. mazandaran tonekabon goli jan 36°49'04.1" 50°48'45.8" cattle 16 (10) (5) aspergillus sp., p. polonicum mazandaran nowshahr musa abad 36°37'40.2" 51°30'39.0" sheep 11 (10) (8) t. harzianum, aspergillus sp. mazandaran fereydunkenar boneh kenar 36°39'24.0" 52°30'22.3" cattle 10 (9) (8) t. harzianum, aspergillus sp. golestan kordkuy valaghuz 36°45'59.2" 54°07'10.2" cattle 11 nf gilan rudsar ahmad abad 37°10'13.5" 50°16'57.3" cattle 6 (4) (5) t. harzianum p. polonicum nf: not found fig. 1. map of tick sampling areas in three northern provinces of iran http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 102 http://jad.tums.ac.ir published online: june 30, 2022 table 2. primers and probes used for molecular detection of three pathogens francisella tularensis, coxiella burnetii, borrelia burgdorferi and fungal species target (gene) primer/probe sequence (5’ 3’) product (bp) isftu2 (f. tularensis) isftu2fw acaagaagtcatgcttgattcaac 144 isftu2rv ggattacctaaagcatcagtcatagc probe fam-atagcaagagcacatgcttgtgctacggtamra 16s rrna (borrelia) 6bor16sfw ggtcaagactgacgctgagtca 135 6bor16srv ggcggccacttaacacgttag probe fam-tctacgctgtaaacgatgcacacttggtg –bhq-1 is1111 (c. burnetii) tmq-koorts4-fw aaaacggataaaaagagtctgtggtt 70 tmq-koorts4-rv ccacacaagcgcgattcat tmq-koorts4-probe fam-aaagcactcattgagcgccgcg-tamra fungal its its1 tccgtaggtgaactgcgg 600-750 its4 tcctccgcttattgatatggc fig. 2. the frequency (left panel) and prevalence (right panel) of fungal contamination of the ixodes ricinus ticks collected in three northern provinces in iran, 2019 fig. 3. distribution of fungal genera and species in contaminated ixodes ricinus ticks in northern provinces of iran, 2019 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 103 http://jad.tums.ac.ir published online: june 30, 2022 discussion to our knowledge this project was the first attempt to determine the fungal community that inhabits the middle intestine of ticks in the three provinces of northern iran and contamination with three bacterial pathogens that transmitted to human by ticks, using culture and molecular methods. ixodes ricinus ticks cover a wide geographic region in the eu (22), i. ricinus is an indigenous hard tick species having a wide geographical distribution (23), portugal to russia and from north africa to scandinavia. this wide geographical distribution entails that this tick species could have a role in transmission of tick-borne disease in wide geographic areas (24). a survey of ticks was carried out in four different geographic areas of iran, where the majority of domestic ruminants in iran exist (18, 19) showed ixodidae ticks were found throughout the year. the highest number of adult ixodidae ticks was generally found from april to august, and ixodes ticks were present in the caspian region and in the south and west of country (24-27). the number of ticks obtained is very important in mazandaran which agrees with the previous studies on the abundance of this tick according to favorable climatic conditions in the northern provinces of iran (28, 29). ixodes ricinus is a three-host tick: larvae, nymphs and adults feed on different hosts where larvae and nymphs prefer small to medium-sized animals and adults tend to feed on large animals (30). in this study we have chosen adult ticks because of their size, it is especially the adult females fed or in the process of being engorged with blood which are the most detectable, because they are much larger than during the other stages of development and the amount of blood they eat on large animals such as cattle and sheep. we were expecting to have a much more varied number of fungi species. as this study is the first done by this approach, we have not the element of comparison but in a study carried out on the midgut of sand flies they found six fungi species of which two fungi species are common with our study (penicillium and aspergillus) (31). another most observed species in this study was actinomycetes that is a shared bacterial species with fungi and among 14 sample sites in 83.4%, this species of bacteria was present. the fungal species isolated in this study are among the saprophytes which can be pathogenic in insects, plants and humans (32). studies have shown that fungi can have an entomopathogenic effect under different environmental conditions (33). the predominant pathogenic genera isolated from soil in the united states for winter tick larvae (dermacentor albipictus) being aspergillus spp, beauveria bassiana, mortierella spp, mucor spp, paecilomyces spp, penicillium spp. and trichoderma spp. (34). on i. ricinus ticks, the most important tick species in europe, susceptibility to entomopathogenic fungi shows particularly high potential efficacy and the predominant species of isolated entomopathogenic fungi were hyphomycetes, paecilomyces farinosus and verticillium lecanii, beauveria bassiana, b. brongniartii, p. fumosoroseus and v. aranearum (35). the secondary metabolites of fungi may be involved in their entomopathogenic effect, aspergillus flavus is effective on insect heliozella stenella by secreting aflatoxin, caused histological changes in even at very low doses. metarhizium anisopliae secretes distruxine (a) distruxine (b) when injected into wax and silk moth. beauvaria bassiana (white muscurdine fungus) secret beauvaricine is the peptide depsi (36). the susceptibility to entomopathogenic fungi against two of the most important tick species in europe: i. ricinus and dermacentor reticulatus shows the potential efficacy particularly greater in i. ricinus (37). the definition of the tick's microbiome and interactions between the tick and its symbiotic bactehttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 104 http://jad.tums.ac.ir published online: june 30, 2022 ria in the context of pathogen transmission likely reveal new knowledge for controlling tickborne diseases (38). in this study we have tried to answer this question even though it is very early because in the studies already show microbiome of i. ricinus ticks evaluated over the last 10 years in austria reveals a number of bacteria were affiliated with the genera rickettsia, bartonella and borrelia (39) which are known to be pathogenic and transmitted by ticks indicating that the tick microbiome is mainly composed of gram-negative bacteria of the phylum proteobacteria and intracellular bacterial endosymbionts which regulate the reproductive capacity and vectoral competence of ticks, also maintain the integrity of the epithelial barrier of the tick gut (6). although i. ricinus is of great importance to public health, its microbial communities remain largely unexplored to this day. a pool of adults of i. ricinus collected from two distinct geographic regions of northern italy showed a total of 108 genera belonging to all bacterial phyla and pathogenic bacteria, such as borrelia, rickettsia and candidatus neoehrlichia (40). the microbiome currently focuses mainly on its eubacterial members, but the microbiome is also made up of viruses and eukaryotic microbes such as protozoa, nematodes, and fungi, and their interactions within and between realms could further modulate the human health (41). on the other hand, ecological analysis revealed that the composition of bacterial communities depending on the geographic regions and the life stages of the ticks. this finding suggests that the environmental context (abiotic and biotic factors) and host selection behaviors affect their microbiome (40). entomopathogenic fungi are known to infect different tick species and their effectiveness is very strain specific. numerous studies show the important role of pathogenic fungi in the control of insects, including ixodes (35, 37, 42–44). although entomopathogenic fungi have been widely used for agricultural and forestry pest control, little effort has been made to assess the applicability of the biological control potentials of entomopathogenic fungi against ticks, vectors of human and animal diseases. it can also be said that the susceptibility of ticks to a particular fungus depends largely on the genera and species of ticks; species of fungus, strain, concentration of conidia;, temperature, humidity and geographical area always trying to understand the biology of the fungus, which is necessary to better understand its proper use in field conditions (40). in the case of fusarium stem rot caused by fusarium graminearum strongly affecting the productivity of corn crops by modifying the plant microbiome by promoting the colonization of roots by trichoderma harzianum in the corn rhizosphere, first, we increase plant growth and further protect the environment from harmful agrochemical effects by replacing it with a biological control agent (45). conclusion results of the present study revealed that different human and animal pathogenic fungal genera are among microbial flora of i. ricinus ticks in northern provinces of iran. these results represent only a first image of the microbial diversity of the tick. further studies are needed to determine the roles these genera play in ticks and their effects on human health. these studies could bring us closer to the discovery of the causative agent and interaction between them to know the system of transmission of these agents even save them by ticks. acknowledgments authors wish to thank the personal of mycology department of the pasteur institute of iran for their kind help in culturing tick samples. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 97–107 m yousefi-behzadi et al.: identification of … 105 http://jad.tums.ac.ir published online: june 30, 2022 conflict of interest authors declare that there is no conflict of interest. ethical 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kermaniran: preliminary zoonotic risk assessment shahrzad motaghipisheh 1, *baharak akhtardanesh 2, reza ghanbarpour 3, mohammad reza aflatoonian 4, mohammad khalili 3, saeed reza nourollahifard 3, saghar mokhtari 5 1research center for modeling in health, institute of futures studies in health, kerman university of medical sciences, kerman, iran 2department of clinical sciences, faculty of veterinary medicine, shahid bahonar university of kerman, zoonosis research committee of kerman university of medical sciences, kerman, iran 3department of pathobiology, faculty of veterinary medicine, shahid bahonar university of kerman, zoonosis research committee of kerman university of medical sciences, kerman, iran 4tropical and infectious diseases research center, kerman university of medical sciences, kerman, iran 5department of clinical sciences, faculty of veterinary medicine, shahid bahonar university of kerman, kerman, iran (received 16 mar 2014; accepted 12 jan 2015) abstract background: ehrlichiosis is an emerging tick-borne zoonotic disease caused by the family of anaplasmatacea. recently, outbreak of human monocytic ehrlichiosis was reported in northern part of iran. besides, serological evidence of canine monocytic ehrlichiosis caused by ehrlichia canis was reported from southeastern of iran but the epidemiology of this disease is almost undetermined in iran. the present study was designed to use pcr for detection of ehrlichia spp. in tick infested household dogs and determination of risks of disease transmission to dog’s owners. method: blood samples were prepared from 100 tick infested household dogs after complete clinical examination. complete cell blood count was done for each sample. dna extraction was done and pcr was carried out by a commercial kit afterwards. regarding to pcr results, blood samples were collected from owners and family members who were exposed to infected and non-infected dogs. a similar method was utilized for dna extraction and pcr in human samples. result: ehrlichial dna was detected by pcr in six percent of rhipicephalus sanguineus tick pools and 9% of the examined dogs. no positive sample was detected among the 67 examined human bloods. conclusion: ehrlichiosis could be considered as an emerging canine disease but owning a dog should not be considered a major risk factor for ehrlichiosis in humans. further serological and molecular studies in different parts of iran are required to clarify the epidemiology of ehrlichiosis in canine, ticks, and human population. keywords: ehrlichiosis, dog, ownership risk, tick, iran introduction humans and dogs are both susceptible to tick-borne diseases. whenever dogs have tick infestation, they could be considered as reservoirs for human pathogens, as definitive feeding hosts for vector ticks or as mechanical transporters. borreliosis, ehrlichiosis and anaplasmosis, are the reported emerging zoonotic diseases, which create ownership risks for tick, infested pet dogs (fritz 2009). in the order rickettsiales anaplasma phagocytophilum, e. canis, e. chaffeensis, e. ewingii, rickettsia rickettsii and r. conorii are zoonotic tick-borne pathogens, which expose dogs and their owners (nicholson et al. 2010). ehrlichiosis is a life-threatening emerging human tick-borne zoonosis, caused by obligate intracellular gram-negative bacteria *corresponding author: dr baharak akhtardanesh, e-mail: akhtardanesh@mail.uk.ac.ir j arthropod-borne dis, june 2016, 10(2): 246–252 s motaghipisheh et al.: ehrlichiosis in household … 247 http://jad.tums.ac.ir published online: january 05, 2016 named ehrlichiae. the dog brown tick (r. sanguineus) is the main vector of disease in infested dogs (beugnet et al. 2009). rhipicephalus sanguineus is the most prevalent tick in dogs, which has a worldwide distribution. this tick is a common vector of many dogs and human pathogens. this tick is well distributed in both urban and rural areas in tropical, subtropical and some temperate regions. rhipicephalus sanguineus not only infests dogs but also is highly adapted to live within human dwellings (dantas-torres 2010). recent studies have demonstrated that ticks exposed to high temperatures attach and feed on humans more rapidly. this observation suggests that the risk of human parasitism by r. sanguineus could increase in areas experiencing warmer and/or longer summers, consequently increasing the risk of transmission of zoonotic agents (guglielmone et al. 2006, shoorijeh et al. 2008). the human parasitism by r. sanguineus is relatively common in europe, particularly during the summer. in contrast, the human parasitism is much less common or maybe much less reported in south america and there is no report about asian countries (guglielmone et al. 2006). kerman is located in southeast of iran with warm springs and hot summers which makes it a perfect condition for r. sanguineus activity. on the other hand, most of pet dogs in this area are kept outdoor as guard dogs predisposing them to ectoparasite infestation in warm seasons. since the major route of human infection with ehrlichia spp. is transmission by a vector tick, the presence of infected ticks near humans could be the most important risk factor for human infection (unver et al. 2001, dantas-torres et al. 2006). dogs play an important role in transporting infected ticks into their surroundings and their owners (shoorijeh et al. 2008). in iran, outbreak of human monocytic ehrlichiosis has been reported from northern part (babamahmoodi 2004). on the other hand, serological evidences of canine monocytic ehrlichiosis were reported in kerman and khuzestan provinces respectively (akhtardanesh et al. 2010, avizeh et al. 2010). serological studies usually indicates exposure rather than active infection, and might mislead due to serological cross reactions with other closely related organisms, so the present study was designed to use the polymerase chain reaction assay to confirm presence of ehrlichia spp. in tick infested client-owned dogs and their attached ticks and determine the risks of disease transmission to the dogs’ owners in kerman city. materials and methods blood samples were randomly prepared from 100 owned tick infested dogs regardless of their age, sex and clinical status between april to october 2011. the animals were referred to the teaching veterinary hospital of shahid bahonar university of kerman. each animal was fully clinically examined and attached semior fully engorged ticks were collected from dogs using a forceps and transferred into the labeled holding tubes containing absolute ethanol individually. then detailed questionnaire was filled for each animal and owner (or other family members) who were in close contact with pet dogs. five milliliter of blood were collected from the cephalic vein of dogs which their owner provided verbal consent for attending in the study and divided in two tubes containing edta anticoagulant. two-milliliter aliquot of blood was used for hematological evaluation and remaining three milliliter refrigerated at –18 oc for dna extraction. the same method was used for the owner and family members who accepted to attend in a pathobiology laboratory for blood collection. complete blood counts were perj arthropod-borne dis, june 2016, 10(2): 246–252 s motaghipisheh et al.: ehrlichiosis in household … 248 http://jad.tums.ac.ir published online: january 05, 2016 formed by cell counter (sysmex kx-21n™, usa) for all samples. the presence of hematological disorders such as anemia, leukopenia and thrombocytopenia was recorded in comparison with reference ranges (tefferi et al. 2005, willard et al. 2012). in parasitology laboratory, collected ticks were taken from the absolute ethanol and their identification was carried out by observation with a binocular microscope (40 x magnifications). ticks were classified into family, genus and species using the taxonomic and morphometric keys (walker et al. 2003). at least one to maximum five adult ticks collected from each dog and each species were pooled in sterile eppendorf tubes and minced using a sterile scalpel blade. specimens were incubated overnight after adding of 20-microliter proteinase k. genomic dna was extracted from specimens by using the qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer instructions. dna extraction from dogs and human blood samples was carried out by viral gene-spin kits (vetek™, south korea). finally, pcr was done for all samples by vetek™ ehr detection kit (vetek™, south korea) according to company instructions. ethics the project underwent ethical review and was given approval by an institutional animal care and done by appropriately qualified scientific colleagues. results a total of 408 ticks were collected from 100 selected dogs which all were identified as rhipicephalus sanguineus regarding to specific characteristics including red-brown color, elongated body shape, and hexagonal basis capituli. six of tick pools (6%) and 9% of examined dogs were positive for ehrlichia spp. by pcr (fig. 1). three dogs were ehrlichia spp. positive whereas their tick pools were negative. in dog population, infection rate was not significantly related to age (p value=0.627) and sex (p value=0.682) (table1). no significant changes were seen in owner’s complete blood count tests but some hematological alterations were seen in infected dogs. however, there was no significant difference between the infected and non-infected dogs in pcv level (p value= 0.242), wbc (p value=0.345), neutrophil (p value=0.643), lymphocyte (p value=0.408), eosinophil (p value=0.27) and monocyte count (p value=0.45) (table 2). blood samples from 36 persons (owner and family members) that were in close contact with infected dogs (group 1) and 31 owners of non-infected dogs (group 2) were pcr negative. demographic data for the participants are shown in table 3. all owners were aware about their dog tick infestation but none of the participants reported a tick bite history. table 1. ehrlichia spp. infection status among studied dogs regarding to age and sex groups parameters studied dogs population no. (%) pcr-positive dogs no. (%) age >12 month 25 (25) 2(8) 1-3 years 33(33) 3(9.1) >3 years 42(42) 4(9.5) sex male 58(58) 5(8.6) female 42(42) 4(9.5) j arthropod-borne dis, june 2016, 10(2): 246–252 s motaghipisheh et al.: ehrlichiosis in household … 249 http://jad.tums.ac.ir published online: january 05, 2016 table 2. hematological alteration in studied dogs regarding to ehrlichia spp. infection status parameter unit ehrlichia spp. infection pcr-positive* pcr-negative* pcv % 35.4 ± 3.12 38.5 ± 6.4 wbc ×103/ml 11.2 ± 3.4 12.4 ± 4.3 neutrophil ×103/ml 7.3 ± 2.3 7.6 ± 2.8 lymphocyte ×103/ml 4.1 ± 1.1 4.9 ± 3.1 basophil ×103/ml 0 0 eosinophil ×103/ml 0 0.05 ± 0.01 monocyte ×103/ml 0.04 ± 0.01 0.01 ± 0.01 *data are mean±sem table 3. demographic data of owners variable owners of infected dogs (n=36) owners of non-infected dogs (n=31) age(mean ± sem)* 35.4 ± 2.9 42.0 ± 3.6 sex no (%) male 23 (63.8) 19 (62) female 13 (36.1) 12 (38) place of residence no (%) urban 18 (50) 28 (90.3) rural 18 (50) 3 (9.7) * years fig. 1. agarose gel electrophoresis for identification of ehrlichia spp. dna in ticks and dogs blood samples l: 100 bp dna ladder, cr+: positive control (336 bp), ntc: negative control, lanes 1, 2 positive dogs blood samples, lane 3, 4 positive tick samples. discussion the genus ehrlichia consists of five recognized species, including e. canis, e. chaffeensis, e. ewingii, e. muris, and e. ruminantium. nowadays ehrlichia species are discovered in new areas and new tick species, which emphasize on wider distribution of these agents. host population, migration, changes in climate and control failure is environmental factors, which have been known to exacerbate the spread of ehrlichia species (esemu et al. 2011). dogs can be infected with different ehrlichia species of which e. canis, e. ewingi, and e. chaffeensis are considered zoonotic. ehrlichia canis, which is the most prevalent species in dogs, has been shown to infect humans in venezuela whereas r. sanguineus was a common ectoparasite on household dogs in both urban and rural areas (perez et al. 1996, perez et al. 2006). domestic dogs and ring-tailed lemurs are naturally exposed to e. chaffeensis and human granulocytic ehrlichiosis which caused j arthropod-borne dis, june 2016, 10(2): 246–252 s motaghipisheh et al.: ehrlichiosis in household … 250 http://jad.tums.ac.ir published online: january 05, 2016 by this organism have been reported in north america, asia and europe (spolidorio et al. 2010). ehrlichia canis, e. chaffeensis and e. ewingii have been recently detected synchronously in dogs and their ticks. ndip et al. suggest that r. sanguineus ticks which are primarily infected with e. canis, may get infected with other ehrlichial agents and transmit them to humans (ndip et al. 2007). ehrlichia ewingii and a. phagocytophilum which has been identified as pathogens of both dogs and humans are chiefly granulocytotropic (buller et al. 1999, ganguly et al. 2008). serosurvey showed that dogs be routinely guarded for assessing risk for human granulocytic ehrlichiosis in humans in europe and north america (cizman et al. 2000, day 2011). a. phagocytophilum reported to be prevalent in ixodex ticks, which infest dogs, so it can easily, transmitted to humans (nicholson et al. 2010). on the other hand, a. phagocytophilum was isolated from ixodex ticks in northern parts of iran, which creates risk of human infection in our country (bashiribod 2004). the presented data showed that dogs and their ticks can be part of the epidemiological cycle of ehrlichiosis all around the world and surveillance, diagnosis, treatment, and prevention of tick-borne diseases in humans and dogs can yield mutually beneficial information for public and veterinary health. in this study, three dogs were ehrlichia spp. positive whereas their tick pools were negative. it is not determined that after tick’s blood sucking, how long does it take that transmission of ehrlichiosis from infected dogs to naïve ticks occurs. on the other hand, the rickettsemia levels to which ticks are exposed during feeding may also impacts on the proportion of infected ticks, so noninfected ticks may not have enough time to achieve the infection from their infected hosts (johnson et al. 1998). prevalence of e. canis in different tick species collected from dogs from ardebil in north west of iran was reported (16.66%) by khazeni et al. (2013) while nested pcr detected ehrlichial dna in 63.82% of r. sanguineus ticks and these results warrant studying on vector competence of ticks for the ehrlichiosis agents. all collected ticks in our study were r. sanguineus but results of present study suggest that owning an ehrlichia infected dog should not be considered a major risk factor for human ehrlichiosis in kerman. this finding must be interpreted cautiously due to the widespread distribution of r. sanguinus in hot seasons on owned dogs and increased risk of human parasitism by r. sanguineus in tropical areas like as kerman. population at risk for ehrlichiosis are the elderly, immunosuppressed and infants patients and as all of the examined human populations in this study were healthy adults, the absence of association between dogs and human infections must be interpreted conservatively. low incidence of disease in dogs’ population and small population of dog’s owners who attend in our study are other confinement factors in this study. in conclusion, result of present study confirm the presence of ehrlichiosis as an emerging infectious disease in canine population and their collected ticks in iran, but further research is needed to reveal the importance of pet dogs and their ticks in the cycle of human ehrlichiosis in our country. conclusion based on the result of the present study, ehrlichiosis could be considered as an emerging canine disease but owning a dog should not be considered a major risk factor for ehrlichiosis in humans. although our data sugj arthropod-borne dis, june 2016, 10(2): 246–252 s motaghipisheh et al.: ehrlichiosis in household … 251 http://jad.tums.ac.ir published online: january 05, 2016 gest that ownership of dogs, is not associated with increased risk of ehrlichiosis in human medicine but this finding must be interpreted cautiously due to the widespread distribution of r. sanguinus in hot seasons on owned dogs. further serological and molecular studies in different parts of iran are required to clarify the epidemiology of ehrlichiosis in canine, ticks, and human population. acknowledgements the authors are grateful to zoonosis research committee of kerman university of medical sciences for the financial support with the grant no.168/90 and research council of shahid bahonar university for technical and laboratory support. the authors declare that there is no conflict of interests. references akhtardanesh b, ghanbarpour r, blourizadeh h (2010) serological evidence of canine monocytic ehrlichiosis in iran. comp clin path. 19(5): 469–474. avizeh r, mosallanejad b, razi jalali m, alborzi a (2010) seroprevalence of ehrlichia canis in dogs referred to veterinary hospital of shahid chamran university of ahvaz, iran. arc razi inst. 65(1): 21–26. babamahmoodi f (2004) first outbreak of human ehrlichiosis in mazandaran province. 12th iranian congress of tropical infectious disease, 17–21 january 2004,tehran, iran. bashiribod h (2004) first molecular detection of 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http://jad.tums.ac.ir published online: april 27, 2019 original article the prevalence of antibodies against sandfly fever viruses and west nile virus in cyprus gaetan billioud; christina tryfonos; *jan richter department of molecular virology, cyprus institute of neurology and genetics, nicosia, cyprus (received 14 oct 2017; accepted 2 jan 2019) abstract background: sandfly fever is an incapacitating disease caused by sandfly-borne phleboviruses that can lead to meningitis, encephalitis or meningoencephalitis. west nile virus (wnv), a mosquito-borne flavivirus, can induce neuroinvasive disease manifested by meningitis, encephalitis or acute flaccid paralysis. both vectors are endemic in cyprus and very active during summer. the aims of this study were to determine first the prevalence of sandfly fever viruses (sfv) and wnv infections in cyprus and second, to investigate their role in central nervous system (cns) infections. methods: for the prevalence study, 327 sera collected in 2013 and 2014 were tested for anti-sfv and anti-wnv igg using indirect immunofluorescence assay and elisa, respectively. in order to investigate a possible role of sfv and wnv in cns infections, 127 sera of patients presenting symptoms of sfv or wnv infections were screened for igm specific to sfv and wnv. results: the overall anti-sfv igg seroprevalence was 28% and was increasing with age (p< 0.01). the seroprevalence rate for anti-wnv igg in cyprus was 5%. concerning the role of sfvs in cns infections, anti-sfv igm was detected in 8 out of 127 sera from selected patients presenting relevant symptoms of infections during vector’s active period. in addition, anti-wnv igm were detected in 17 out of the 127 patients with compatible symptoms. conclusion: the findings confirm the presence of sandfly fever and wnv in cyprus and should, therefore, be considered in the differential diagnosis of patients with febrile illness/meningitis. keywords: phleboviruses; west-nile virus; cyprus; seroprevalence introduction sandfly (diptera: psychodidae)-transmitted phleboviruses may cause a transient and moderate febrile illness (named pappatasi fever, 3-day fever or sandfly fever) but can also affect the central nervous system (cns) leading to serious infections. the most common vectors are phlebotomus papatasi, p. perfiliewi or p. perniciosus. the genus phlebovirus has been recently assigned to the newly created family phenuiviridae and the order bunyavirales (1). sandfly-borne phleboviruses are enveloped negative-sense and single-stranded tripartite rna viruses classified around two viral species (sandfly fever naples (sfnv) and salehabad) and two tentative species (sicilian and corfu). infections with sfnv and sandfly fever sicilian (sfsv) viruses are clinically similar and characterized by high fever, headache, retro-orbital pain, malaise, diarrhoea, myalgia, photophobia and anorexia along with thrombocytopenia, leukopenia and elevated liver enzymes (2, 3). toscana virus, a member of the naples serogroup, generally induces a mild febrile illness without cns involvement. however, when the cns is affected, high fever, headache, nausea, vomiting, kernig signs, neck rigidity, myalgia and sometimes unconsciousness, tremors, paresis and nystagmus as well as encephalitis, severe meningoencephalitis and long-lasting sequelae have been reported (4, 5). the mediterranean *corresponding author: dr jan richter, e-mail: richter@cing.ac.cy j arthropod-borne dis, march 2019, 13(1): 116–125 g billioud et al.: the prevalence of … 117 http://jad.tums.ac.ir published online: april 27, 2019 basin is endemic for sandfly-borne phleboviruses infections (4, 6-10) and particularly cyprus where phlebotomines (11, 12) and outbreaks have been reported (13-16). regarding west nile fever, the majority of clinical cases are mild and present with flu-like symptoms characterized by fever, malaise, and myalgia frequently accompanied by rash. severe cases with signs of encephalitis, meningoencephalitis or meningitis are rare (<1%), most often observed among elderly and associated with a 10% fatality rate (17). the disease results from an infection by west nile virus (wnv) (genus flavivirus, family flaviviridae, order unassigned) transmitted through a mosquito (diptera: culicidae) bite, mostly from the genus culex and usually from the culex pipiens complex (18), abundant in cyprus (19, 20). human infection is solely incidental of the enzootic transmission cycle between avian natural hosts and mosquito vectors (21). the virus is considered to be one of the most important emerging arboviruses in recent years because of the multiple outbreaks reported in europe and particularly in the mediterranean area (greece, turkey, spain) (22, 23). to date, no epidemiological data for wnv have been reported for cyprus but the first neuroinvasive case was reported in 2016 (24, 25) and a second confirmed case in 2018 (ecdc update), confirming the presence of the wnv in the island. because of outdated or absent epidemiological data concerning cyprus, a cross-sectional serologic survey was conducted to estimate sfv and wnv igg respective seroprevalences in order to assess the exposure of the cypriot population to these pathogens. in addition, the possible role of these pathogens in cns infections in cyprus was investigated by testing for markers of recent infection (igm) and viral rna in samples of patients with febrile illness and/or cns infection. materials and methods sample collection serum and cerebrospinal fluid (csf) samples from patients were received at the cyprus institute of neurology and genetics, stored appropriately at -20 °c and used subsequently. for igg seroprevalence, 327 samples between jan 2013 and dec 2014 were retrospectively selected following a leftover sampling methodology matching the 2013 census cypriot demography according to patient’s sexes (male or female), age and origin of the sample (famagusta, larnaca, limassol, nicosia or paphos district) (fig. 1). informed consent was taken from the patients before participation. the patients with known disease, medical procedure or treatments affected the antibodies detection were excluded (i.e. autoimmune disease, immunomodulatory treatments, transplanted, neoplasm, etc.). determination of specific igm was performed on samples received during 2013 and 2014 vector active season (warm months, from may to nov in cyprus) from 127 selected patients with infectious pathologic features relating to sandfly or west nile fevers. based on similar selection criteria, 72 csf samples from patients collected between 2015 and 2016 during vector active season were tested for circulating viral rna. sfv igg and igm detection overall, 327 and 127 sera were analyzed for the presence of anti-sfv igg and igm, respectively, by indirect immunofluorescence test (iift) biochip mosaic™: sandfly fever virus igg or igm (euroimmun, lübeck, germany). this test is the only commercially available test that offers the possibility to assess the presence of antibody reacting against two species members of the sandfly sicilian serogroup (sfsv and sandfly fever cyprus viruses, sfcv) and two members of the sandfly naples serogroup (sfnv and toscana virus, tosv), j arthropod-borne dis, march 2019, 13(1): 116–125 g billioud et al.: the prevalence of … 118 http://jad.tums.ac.ir published online: april 27, 2019 without excluding the possibilities of crossreactivity. sera were diluted 100 and 50 times in sample buffer for igg and igm, respectively, and tested according to the manufacturer’s instructions. before determining specific anti-sfv igm, igg antibodies and rheumatoid factors of class m were removed by immune-absorption using the siemens rf-absorbent (marburg, germany) [ref: oucg194] for 15min at room temperature. samples were considered positive if in some cells, but not all, cytoplasmic finely granular structures and inclusion bodies fluoresced with the same pattern obtained as for the positive control serum. wnv igg and igm detection overall, 327 sera were collected for specific anti-wnv igg detection and 127 sera for igm detection, respectively, using an fda-approved enzyme-linked immunosorbent assay (elisa) (euroimmun, lübeck, germany). sera were diluted 100 times for both igg and igm in sample buffer (containing igg/rf-absorbent for igm detection) and tested according to the manufacturer’s instructions. the samples were considered positive when the ratio between the extinction values of the samples over the calibrator (20 ru/ml for igg) was ≥ 1.1. viral rna extraction and rt–pcr amplification viral rna was extracted from 72 csf samples using 400μl of clinical specimen by the iprep™ purelink® virus kit according to manufacturer’s instructions and resuspended in 100μl depc water. ten microliters of the extracted viral rna was used as a template using specific primers with the superscript® iii platinium one-step qrt-pcr kit (invitrogen, usa), according to the manufacturer’s instructions. for phlebovirus detection and quantification, the specific primers used amplified the 3’ end of the small segment n gene and were based on published protocols (26, 27). for flavivirus detection and quantification, the specific primers targeted the ns5 gene and were based on a previously published protocol (28). statistics results were statistically analyzed with the prism™ version 5.01 software from graphpad (la jolla, ca, usa). along with descriptive statistics, univariate analysis was conducted on the results obtained from the seroprevalence study by fisher’s exact tests. comparison of groups’ average age was analyzed by nonparametric two-tailed mann-whitney tests. the logistic regression model was generated from the gnu package r environment. p< 0.05 indicated statistical significance. results prevalence study of anti-sandfly fever viruses and west nile virus igg antibodies to determine the exposure of cypriots to sandfly fever viruses, 327 human serum samples from 2013–2014 were screened for antisfvs and anti-wnv igg. the sampling was distributed by sex and age proportional to cyprus’ population (table 1). by sex, 166 were females (~51%) and 161 males (~49%). by age groups, 81 were <20yr old (~25%), 184 aged 20–60yr (~56%), and 62 were aged >60yr (~19%) with an overall mean age of 37yr old. by districts, 9 specimens were from famagusta (~3%), 32 from larnaca (~10%), 107 from limassol (~33%), 170 from nicosia (~52%) and 9 were from paphos district (~3%) (fig. 1). the overall seroprevalence of anti-sfvs igg was 28% (ci95%: 23.5–33.4%) and increased with age (table 1). difference of mean age in anti-sfv seropositive group (47yr old) compared to the seronegative patient's group (33yr old) was statistically significant (p< 0.0001). logistic regression model confirmed the trend (p< 0.001) and computed an increased risk of ~3% per year (se 0.6%). differences observed in seroprevalence for anti-sfv igg between females (~25%) and males (~31%) j arthropod-borne dis, march 2019, 13(1): 116–125 g billioud et al.: the prevalence of … 119 http://jad.tums.ac.ir published online: april 27, 2019 or between districts (famagusta: ~0%, larnaca: ~19%, limassol: ~31%, nicosia: ~30% and paphos: ~33%) were not statistically significant (p> 0.05) (table 1). difference in prevalence between sera reacting only against sfsv and/or sfcv (~12%) and sera reacting only against sfnv and/or tosv (~11%) was not statistically significant (p> 0.05) (data not shown). the same 327 human sera from 2013–2014 were also screened for anti-wnvs igg. the overall seroprevalence of anti-wnv igg was ~5% (ci95%: 2.5–7.2%) (table 1). the difference of mean age in anti-wnv seropositive group (33yr old) compared to the seronegative patients group (37yr old) was not statistically significant. differences in frequencies observed between each age group, sex and district were also not statistically significant. five patients (~1.5%) were found to have igg antibodies reacting against both sfvs and wnv (data not shown), giving seroprevalence rates of ~31% (5/16) and ~5% (5/93) of antisfvs and anti-wnv igg in anti-wnv and anti-sfvs igg positive groups, respectively. the differences of rates in positive groups compared to the general population (~28% and ~5% for anti-sfv and anti-wnv igg, respectively) were not statistically significant. role of sfv and wnv infections in cns infections in cyprus to assess the morbidity of sfv infections in cyprus, human sera from patients displaying infectious pathologic features in 2013 and 2014 during sandflies’ active period were tested for markers of recent sfv infection, igm. eight sera (~6%) were found positive for antisfv igm out of the 127 selected patients (table 2). difference of sfv igm seroprevalence rates between each age group, sex and district were not statistically significant. however, a higher frequency of samples reacting only against sfv serotypes belonging to the naples serogroup (sfnv and tosv, 7/127) than to the sicilian serogroup (sfsv and sfcv, 0/ 127) (data not shown) was statistically significant (p< 0.05). three of the 8 anti-sfv igm positive patients (~38%) showed neuroinvasive features (meningitis or meningoencephalitis) but no statistical significance was found compared to the anti-sfv igm negative group (34/ 127, ~26%, p> 0.05) (data not shown). of the 8 seropositive patients for anti-sfv igm, four patient’s csf were available and three of them were also found positive for anti-sfv igm (data not shown). all csf positive for sfv igm reacted only against the naples serocomplex, further confirming their neuroinvasion capabilities. detection of phlebovirus nucleic acids by specific quantitative rt-pcr in 72 human cerebrospinal fluid samples from patients displaying infectious pathologic features in 2015 and 2016 during sandflies’ active period was performed, but no specimen was found positive (data not shown). seventeen (~13%) out of the 127 tested human sera from patients displaying infectious pathologic features in 2013 and 2014 during mosquito’s active period were found positive for anti-wnv igm (table 2). the difference of wnv igm seroprevalence rates between sexes, age groups or districts were not found statistically significant. two of the 17 antiwnv igm positive patients (~12%) showed neuroinvasive features (meningitis or meningoencephalitis) but no statistical significance was found compared to the anti-wnv igm negative group (29/110, ~26%) (data not shown). with regard to nucleic acid testing, no wnv rna was detected in the 72 human csf samples from selected patients displaying infectious pathologic features in 2015 and 2016 during mosquitos’ active period (data not shown). j arthropod-borne dis, march 2019, 13(1): 116–125 g billioud et al.: the prevalence of … 120 http://jad.tums.ac.ir published online: april 27, 2019 table 1. sandfly fever virus and west nile igg seropositivity sfv igg wnv igg tested positive % positive % total 327 93 ~28 16 ~5 males 161 52 ~32 6 ~4 females 166 41 ~25 10 ~6 < 20 y 81 6 (*) ~7 4 ~5 20–60 y 184 60 (*) ~33 11 ~6 > 60c y 62 27 (*) ~44 1 ~2 famagusta 9 0 0 0 0 larnaca 32 6 ~19 1 ~3 limassol 107 33 ~31 5 ~5 nicosia 170 51 ~30 10 ~6 paphos 9 3 ~33 0 0 sfv, sandfly fever viruses; wnv; (*), statistically significant (p< 0.05) table 2. sandfly fever virus and west nile igm seropositivity sfv igm wnv igm tested positive % positive % total 127 8 ~6 17 ~13 males 58 6 ~10 5 ~9 females 69 2 ~3 12 ~17 < 20 y 52 2 ~4 8 ~15 20–60 y 54 4 ~7 7 ~13 > 60c y 21 2 ~10 2 ~10 famagusta 5 0 0 2 ~40 larnaca 10 0 0 0 0 limassol 41 6 ~15 9 ~22 nicosia 66 2 ~3 6 ~9 paphos 5 0 0 0 0 sfv, sandfly fever viruses; wnv fig. 1. illustrative map of cyprus’ districts. white, area studied; grey, non-government controlled area; dark grey, un buffer zone; light grey, uk sovereign bases j arthropod-borne dis, march 2019, 13(1): 116–125 g billioud et al.: the prevalence of … 121 http://jad.tums.ac.ir published online: april 27, 2019 discussion because the presence of arboviruses in cyprus had been reported previously (15, 24, 2932), we evaluated the residents’ exposure to sfv and wnv following cyprus’ demography in terms of age, sex and location. the overall seroprevalence rate for antisfvs igg estimated in this study was ~28%. higher seroprevalence of antibodies against sfsv virus (~62%) and neutralizing antibodies against sfnv (~57%), sfsv (~32%) and tosv (~20%) viruses were reported in previous studies on cypriot residents (33, 34). a recent seroprevalence study of phleboviruses in cypriot dogs indicated also high neutralizing rates for sfsv (~60%) and tosv (8.4%) (35). lower rates found here may reflect the different specificity and sensitivity of the assay used (iift or plaque neutralization reduction assay (prnt) (36), sampling method skewed towards specific geographical areas (famagusta, larnaca or paphos districts), test subjects (healthy donors or symptomatic patients), species tested (humans or dogs) or simply a lower exposition of the modern cypriot population, possibly due to arthropods abatement programs to eradicate malaria in the island (37). direct comparison with studies depicting sfv seroprevalences from neighbouring countries is limited, mostly because of the wide use of prnt and the focus on tosv. however, our study reports comparable anti-sfv igg seroprevalence rates described in turkish central/northern anatolia (~33%) (38) but lower compared to the turkish mersin province (~67%) (39), both tested by iif. the anti-sfv igg seroprevalence rate increasing with age is in agreement with the conclusions of previous studies (40, 41) and demonstrates that the cypriot population is exposed to sfvs throughout life. unfortunately, other risk factors previously identified such as living in rural areas or high levels of outdoor activity could not be assessed because of the lack of patient’s information. concerning wnv, the overall igg seroprevalence found in cyprus was 5% and the difference of rates between sexes, age groups or districts were not statistically significant. it is the first time that anti-wnv igg seroprevalence has been estimated in cyprus. wnv-specific igg rates were lower in neighbouring countries such as greece (2.1%) (42), northern italy (0.3–2.1%) (43) or south-eastern france (1.4%) and higher in libya (13.1%) or tunisia (12.5%) (44) but more similar to turkey (2.4– 12.1%) (20, 45), algeria (6.7%) or serbia (~4%) (46). in addition, the sfv igg seropositivity rate in the wnv igg seropositive group (~31%) was comparable to the rate found in the entire population (29.4%). similarly, the wnv igg seropositivity proportion in the sfv igg seropositive group (~5%) was analogous to the sample population tested in this study (~5%). this supports the hypothesis of random chance to contract each viral infection. regarding the role of sfv in cns infections in cyprus, we identified samples (8/ 127) with markers of recent sfv infection (igm) in patients presenting symptoms akin to sandfly fever, supporting a role of sfvs in the aetiology of febrile illnesses/meningitis in cyprus. distinction between antigenically related viruses amongst the sandfly fever naples species (i.e. sfnv and tosv) and virus closely related to sandfly fever sicilian virus (i.e. sfsv and sfcv) based on iift can be difficult due to cross-reactivity. however, there was a significantly higher rate of sera from symptomatic patients tested for anti-sfv igm reacting only against viruses from the naples serogroup than sera reacting only against viruses belonging to the sicilian serogroup. because of similar rates observed for igg between serogroups and that patients were selected based on relevant symptoms, it confirms that j arthropod-borne dis, march 2019, 13(1): 116–125 g billioud et al.: the prevalence of … 122 http://jad.tums.ac.ir published online: april 27, 2019 infections from viruses belonging to the naples serogroup are more symptomatic, particularly tosv (47). anti-sfv igm specific to members of the naples serogroup are at higher concentrations or last longer than the ones specific to sicilian-like viruses (34). however, the detection of anti-sfv igm specific to the naples serocomplex in the csf of affected patients strongly supports their neuroinvasive pathogenesis. possible cross-reactivity with anti-hantavirus and anti-crimean-congo hemorrhagic fever virus (cchfv) immunoglobulins cannot be ruled out based on the iift assay used. however, no hantavirus or cchfv infection nor hemorrhagic fever have ever been reported in cyprus so far. even though their vectors are present on the island (48), it is unlikely that the anti-sfv antibodies seroprevalence rates depicted in this report are markedly influenced due to such cross-reactivity. markers of recent wnv infection (igm) were also detected in patients displaying relevant symptoms, highlighting its circulation and morbidity in the cypriot population. few seropositive patients had neurological symptoms such as meningitis or meningoencephalitis. together with the first cypriot symptomatic case of a 75yr-old man with confirmed wnv infection during summer 2016 (24), these results demonstrate the circulation of wnv in cyprus. all the anti-wnv igm seropositive cases were from 2013, following the trend of marked decreased west nile fever autochthonous cases reported in europe during these yr (49). attempts to detect viral genetic material in more recent (2015 and 2016) patients’ csf by rt-qpcr following published protocols (2628) were unsuccessful. the most likely causes for this shortcoming are probably low sfv and wnv viremia detectable only in a small period of time, usually before patients present to the healthcare personnel, the limited number of samples tested and the use of direct rtqpcr. conclusion this report provides evidence of significant sfv and wnv circulation in cyprus as well as evidence of their pathogenicity among cypriot patients. for 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control. available at: http://ecdc.europa.eu/en/healthtopics/w est_nile_fever/west-nile-fevermaps/pages/historical-data.aspx. http://ecdc.europa.eu/en/healthtopics/west_nile_fever/west-nile-fever-maps/pages/historical-data.aspx http://ecdc.europa.eu/en/healthtopics/west_nile_fever/west-nile-fever-maps/pages/historical-data.aspx http://ecdc.europa.eu/en/healthtopics/west_nile_fever/west-nile-fever-maps/pages/historical-data.aspx j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 278 http://jad.tums.ac.ir published online: dec 31, 2022 original article a comparative study on the biodiversity and species richness of phlebotomine sand flies (diptera: psychodidae) in kermanshah and khuzestan provinces of iran negin yahaghi1, mahboubeh fatemi1, ahmad ali hanafi-bojd1, abdollah naghian2, mohammad reza yaghoobi-ershadi1, alireza zahraei-ramezani1, yavar rassi1, hassan soleimani3, hossein dehghan4, arshad veysi5, *kamran akbarzadeh1, *amir ahmad akhavan1 1department of vector biology and control of diseases, school of public health, tehran university of medical sciences, tehran, iran 2department of medical entomology and vector control, school of health, urmia university of medical sciences, urmia, iran 3yazd health research station, tehran university of medical sciences, yazd, iran 4department of public health, school of public health, jiroft university of medical sciences, jiroft, iran 5zoonoses research center, research institute for health development, kurdistan university of medical sciences, sanandaj, iran *corresponding authors: prof amir ahmad akhavan; aaakhavan@tums.ac.ir, prof kamran akbarzadeh; akbarzadehk@yahoo.com (received 23 oct 2021; accepted 07 dec 2021) abstract background: leishmaniasis is one of the most important vector-borne and neglected tropical diseases in many parts of the world. the main objective of this study was to determine the biodiversity of phlebotomine sand flies (diptera: psychodidae, phlebotominae) in khuzestan and kermanshah provinces of iran. methods: sampling was conducted in khuzestan and kermanshah provinces using sticky paper traps and cdc light traps. the samples were then stored in 96% alcohol-containing vials, mounted, and identified. afterward, the alpha diversity (using simpson’s, shannon-weiner’s diversity, evenness, maghalef’s, menhinick’s, hill and indices) and beta diversity indices (using sorensen’s and jaccard’s coefficients) were calculated. results: a total of 4302 sand flies were caught and identified which comprised mostly of phlebotomus papatasi, ph. alexandri and ph. sergenti. it was found that among the four counties of ahvaz, shush, shushtar and dezful in khuzestan province, shush had the lowest, and shushtar, the highest amount of species diversity and evenness. in the four studied counties of kermanshah province, species diversity in kermanshah county had the lowest amount and sarpol-e-zahab, the highest. the species richness in kermanshah county was the lowest, while qasr-e-shirin county had the highest amount. conclusion: an overview of the biodiversity of phlebotomine sand flies in kermanshah county in kermanshah province, and shush county in khuzestan province, showed less stability in community structure of these vectors, which can be counted as an alarm for emerging dominant vectors among the studied counties and have the potential to increase the prevalence of leishmaniasis. keywords: leishmaniasis; phlebotominae; species richness; biodiversity; iran introduction leishmaniasis is a vector-borne and neglected tropical disease and a tropical infection of poverty as it is associated with malnutrition, population displacement, poor housing, weak immune system, and lack of financial resources. among all parasitic diseases, mortality from leishmaniasis is second only to malaria (1). leishmaniasis affects 102 countries, areas, or copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:aaakhavan@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 279 http://jad.tums.ac.ir published online: dec 31, 2022 territories worldwide (2). the disease causes considerable human morbidity and socio-economic burden to endemic societies. two forms of the disease, visceral leishmaniasis (vl) and cutaneous leishmaniasis (cl), are endemic in iran, and zoonotic cutaneous leishmaniasis (zcl) is more common (about 80%) in the rural areas of 19 out of 31 provinces of the country (3). phlebotomine sand flies (diptera: psychodidae, phlebotominae) are the well-known vectors of leishmaniasis (4). iran's most geographic and climatic conditions are suitable for the growth and proliferation of rodents and sand flies as reservoirs and vectors of this disease (5). up until now, 53 species of sand flies have been identified in iran, of which 34 species belonging to the genus phlebotomus and 19 to the genus sergentomyia (6). two provinces of khuzestan and kermanshah were chosen according to the purposes of previous research done in 2011–2012 (7). khuzestan province in the southwest of iran is considered to be an important endemic focus of zcl due to the environmental impact of the warfare (8). in 2012, the annual incidence of cl was 9.5 to 21.7 per 100000 inhabitants in the province (4). this variation may be due to migration of non-immune people to endemic areas, people living near rodent colonies, migration of rodents and increasing synanthropic index for the reservoir, and lack of knowledge and attitude toward zcl (9). on the other hand, kermanshah province was chosen to study as a non-endemic focus for cl. alpha (or within habitats) diversity is the most common form of inventory diversity, which reflects species packing within a community. species diversity is a central theme in ecology and has two separate components named species richness (s) and evenness (e). two indices of shannon–wiener (shannon index) and simpson’s index are commonly used for species biodiversity which differ in their emphasis on species richness (shannon-wiener) or abundance (simpson’s) (10). beta (or between habitat) diversity is the cat egory of differentiation diversity that measures the variation in taxonomic composition between areas of alpha diversity. some ecological studies such as species richness, species evenness, degree of presence or occurrence, synanthropic index and jaccard’s and sorensen’s similarity indices are important to study the composition of sand fly species in different biotopes, spatial separation among the different populations and the degree of attraction or avoidance of phlebotominae sand flies to human places (11, 12). the main purpose of this study is to determine species richness and biodiversity of the sand flies, and the applied purpose would be determining the areas with high potential in leishmaniasis transmission to implement prevention and control programs. materials and methods study area khuzestan province (29° 57’–33° 00’n, 47° 40’–50° 33’ e) is in the southwest of the country and divided into 27 counties. topographic elevations in the province vary between 0 and 3740m (13). this province consists mostly of lowlands and is generally warm, however, northeastern parts are more elevated and have a more moderate climate. kermanshah province (33° 41’–35° 17’n, 45° 24’–48° 06’e) is situated in the west part of the country and contains 14 counties. mean topographic elevation in the province is 1342 m (14). the province is mostly mountainous, and overall, the weather condition is cool and to some extent rainy during colder seasons. sand fly collection during the activity seasons of sand flies in 2011 and 2012, sand flies were collected using 4239 sticky paper traps and 124 cdc light traps from indoors (human dwelling and stables) and outdoors (mountainous, foothill, and plain areas) in khuzestan and kermanshah provinces, shown in fig. 1 and 2 (7). in this study, samples were http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 280 http://jad.tums.ac.ir published online: dec 31, 2022 mounted using puri’s medium. they were then identified using the sand fly identification key of the eastern mediterranean region (15). data process and analysis to determine alpha and beta diversity, four counties from each province were selected and their data were processed in microsoft office excel 2016 software using related indices and coefficients. the average and standard deviation of all the results were calculated and, their p-value was indicated by performing the analysis of variance (anova) test in ibm spss statistics 26 software. alpha diversity indices to determine the species diversity, shannon–weiner index (h´): h´= -∑( ×ln ) was used where‘ ’ can be calculated by dividing , the number of species, by n, the total number of collected individuals (16). also, indices hill n₁ ( ) to determine the species diversity exponentially, and hill ( ) was used where e is the euler's number and is the symbol for hill (17). species richness was estimated by calculating margalef’s index ( ) which can be calculated through this equation: ( )= (s-1)/ln n (17) and menhinick’s index which is more reliable (18). evenness index (e) was calculated by dividing h´ index by natural logarithm (ln) of the number of collected species (s) (17). to determine the dominant vector’s diversity, simpson’s index (d) was calculated using the formula: . the result is a number between 0 and 1, and 0 indicates unlimited diversity, and on the contrary, 1 represents no diversity at all (19). beta diversity indices beta diversity was also estimated intraprovincially by determining the similarity between different communities using jaccard’s (sj) and sorensen’s indices (ss). sj: a/(a+ b+ c) and ss: 2a/(2a+ b+ c) are the equations for calculating jaccard’s and sorensen’s indices, respectively. in both equations, a represents the number of species (richness) found in both biotopes and b is the number of species found in the first biotope and c biotope is the number of species in the second (20). results a total of 4302 sand flies were collected from both provinces which included 17 and 12 species belonging to the genus phlebotomus and sergentomyia, respectively. species composition and relative abundance of collected sand flies from the studied areas are shown in table 1. 39.3% of the collected sand flies were identified as male and 60.7% as female. in khuzestan province, ph. papatasi and ph. alexandri were the most prevalent species, which respectively consisted of 57.3% and 29.5% of the total sand flies. in kermanshah province, se. dentata was the most prevalent species (29%), but it is neither a proven nor a probable vector, ph. papatasi, ph. alexandri, and ph. sergenti were consecutively the dominant vector species, with 24.3%, 9.1% and 9.02% of the total samples, respectively. alpha diversity results in table 2 show that among the four studied counties in khuzestan province, shushtar county had the highest probability of dominant vector presence, species diversity, and species evenness, albeit not significantly (p= 0.361), also ahvaz county showed the highest amount of species richness (p= 0.759). shush county on the other hand, had the lowest amount of all the mentioned indices. in kermanshah province, kermanshah county also showed the lowest amount of these indices, whereas javanroud county significantly had the highest amount of dominant vector presence (p= 0.029), sar-e-pol zahab county had the highest amount of species diversity (p= 0.007) (significantly), and evenness (p= 0.132), http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 281 http://jad.tums.ac.ir published online: dec 31, 2022 and ghasr-eshirin county was significantly considered to be the richest county, species-wise (p= 0.05). beta diversity results proved that among the four studied counties in khuzestan province, ahvaz and shush county had the least amount of similarity in sand fly species, and equally, dezful with shushtar and ahvaz showed the most (table 3 and 4). as shown in table 5 and 6, in kermanshah province, javanroud county had the least similarity with kermanshah county and the most with ghasr-eshirin county. table 1. species composition, abundance, and relative abundance of phlebotomine sand flies in khuzestan and kermanshah provinces, iran, 2011–2012 species collection sites and percentages khuzestan province % kermanshah province % ph. papatasi 1425 57.344 443 24.380 ph. alexandri 735 29.577 166 9.135 ph. jacusieli 3 0.120 3 0.165 ph. sergenti s.l. 13 0.523 164 9.025 ph. mongolensis 14 0.563 1 0.055 ph. kazeruni 1 0.0402 0 0 ph. caucasicus group 4 0.160 1 0.055 ph. mesghali 1 0.040 1 0.055 ph. major 0 0 72 3.962 ph. brevis 0 0 2 0.110 ph. perfiliewi 0 0 13 0.715 ph. chinensis group 0 0 53 2.916 ph. halepensis 0 0 2 0.110 ph. tobbi 0 0 1 0.055 ph. keshishiani 0 0 8 0.440 ph. wenyoni 0 0 1 0.055 ph. longiductus 0 0 1 0.055 se. clydei 40 1.609 16 0.880 se. tiberiadis 56 2.253 19 1.045 se. iranica 17 0.684 1 0.055 se. squamipleuris 7 0.281 2 0.110 se. dentata 75 3.018 521 28.673 se. sintoni 76 3.058 160 8.805 se. theodori 12 0.482 63 3.467 se. antennata 5 0.201 88 4.843 se. palestinensis 1 0.040 1 0.055 se. mervynae 0 0 2 0.110 se. africana 0 0 1 0.055 se. pawlowskyi 0 0 8 0.440 total 2485 100.0 1817 100.0 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 282 http://jad.tums.ac.ir published online: dec 31, 2022 table 2. the alpha diversity indices for sand fly species in the studied counties of khuzestan and kermanshah provinces, iran, 2011–2012 province county shannonweiner diversity shannonweiner evenness simpson marghalef menhinick hill n₁ hill n₂ = khuzestan shush 0.5933 0.2966 0.6599 0.8079 0.7776 2.0136 1.6661 shushtar 1.2820 0.5980 0.1581 1.5383 1.1180 3.9679 3.3570 ahvaz 0.9725 0.4682 0.3775 1.5083 1.1431 2.7669 2.4675 dezful 1.2423 0.4813 0.3739 1.3159 0.8977 3.7509 3.1337 kermanshah javanroud 1.5706 0.5028 0.2527 1.9201 1.0817 4.8853 3.8484 kermanshah 0.5703 0.3111 0.6748 0.7056 0.4768 1.8051 1.5617 sar-e-pol zahab 1.5768 0.5435 0.2697 1.5537 0.8979 4.9137 3.8275 ghasr-e shirin 1.5021 0.5018 0.3150 1.7301 1.7499 4.8173 3.6834 fig. 1. study areas in kermanshah province, iran, 2011–2012 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 283 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 2. study areas in khuzestan province, iran, 2011–2012 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 284 http://jad.tums.ac.ir published online: dec 31, 2022 table 3. the jaccard’s and sorensen’s coefficient (beta diversity) indices for four counties of khuzestan province, iran, 2011–2012 shush -shushtar shush -ahvaz shush -dezful shushtar ahvaz shushtar -dezful ahvaz -dezful cj 0.54545 0.41667 0.54545 0.46667 0.69231 0.69231 cs 0.70588 0.58824 0.70588 0.63636 0.81818 0.81818 table 4. the jaccard's and sorensen's coefficient (beta diversity) indices for four counties of kermanshah province, iran, 2011–2012 javanrud kermanshah javanrud ghasr-e-shirin javanrud -sare-pol zahab kermanshah ghasr-eshirin kermanshah sar-e-pol zahab sar-e-pol zahab ghasr-e-shirin cj 0.444444 0.578947 0.526316 0.533333 0.466667 0.5625 cs 0.615385 0.733333 0.689655 0.695652 0.636364 0.72 discussion this study is considered as one of the first researches on biodiversity of sand flies in kermanshah province, and a complementary one in khuzestan province, as a main focus of cl in the southwest of iran. our results indicated that after more than four decades (21), ph. papatasi and ph. alexandri are still active in all the studied counties, with ph. papatasi being the dominant species. phlebotomus papatasi normally prefers to live in plain areas rather than in mountains. lower abundance of this species in mountainous areas such as the studied counties in kermanshah province could be explained by its preference to semi-arid and plain areas such as the studied counties in khuzestan province (12, 22). phlebotomus alexandri has been reported as a probable vector of zoonotic visceral leishmaniasis (zvl) in iran (7, 23). this species prefers regions with a high percentage of relative humidity and warmer niches (22), which may be the reason behind its higher abundance in the studied areas of khuzestan province, rather than kermanshah province which is less humid and warm. from all the studied area in this study, 27 species of sand flies were collected and identified. this showed that species richness of sand flies in these areas was higher than some other regions in the country (12, 24, 25). jahanifard et al. (12) found that the urban area of shush county has the highest biodiversity and evenness, and the lowest species richness, which shows that the lowest species number in this area became more adapted to environmental conditions. the highest species richness was in the semi-urban area of this county which accounts for the high species number in peripheral areas of the county. it can be concluded that the natural habitat is under the stress of continuous drought in shush county. however, in the present study, this county itself showed the lowest species richness and diversity among the four studied counties, which could lead to a rise of activities of dominant vectors and a probable increase in leishmaniasis case reports. a study based in kuhpayeh district in isfahan province (26) revealed the importance of elevation in sand fly diversity, where the species diversity in mountainous areas was higher than in plain areas and lowlands. the same conclusions were derived from more research throughout the recent years (25, 27), yet some indicated otherwise such as the study done by sofizadeh et al. (28). therefore, considering the result of the present study that shows higher diversity in plain areas rather than elevated ones, http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 278–287 n yahaghi et al.: a comparative study on … 285 http://jad.tums.ac.ir published online: dec 31, 2022 the degree of species diversity cannot be simply based on the factor of elevation and, we must consider different aspects. due to the restrictions of covid-19, we weren’t able to collect more samples from other counties as well for much more comprehensive research. we recommend the authorities take preventive measures in shush county in khuzestan province and kermanshah county in kermanshah province since they may contain a more suitable habitat for vectors. further research on the biodiversity of the remaining counties in both provinces and other areas of the country is highly recommended. moreover, determining the reasons behind species similarity between different counties in each province needs further research on various ecological, geographical, anthropological variables. conclusions based on the results, it can be concluded that kermanshah county in kermanshah province, and shush county in khuzestan province are ecologically unstable and more likely to welcome vector species, which is critical in planning control programs. acknowledgements this study was conducted as a part of a msc thesis of negin yahagi, by financial supports of school of public health (sph), tehran university of medical sciences (tums), code of 99-2-99-49686. the authors are grateful to all colleagues at the university of medical sciences and declare that there is no conflict of interest. ethical considerations this study has been reviewed and approved by the school of public health (sph), tehran university of medical sciences (tums) ethics committee and has been registered with the code ir.tums.sph.rec.1399.206. conflict of interest authors declare that they have no competing interest. 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mr, yaghoobi-ershadi mr, aghaei-afshar a, akhavan aa, amin m, paksa a (2020) diversity of phlebotomine sand flies (diptera: psychodidae) in mountainous and plain areas of an endemic focus of anthroponotic cutaneous leishmaniasis in iran. asian pac j trop biomed. 10(5): 201–207. 28. sofizadeh a, akbarzadeh k, allah kalteh e, karimi f (2020) relationship between the distribution and biodiversity of sand flies (diptera: psychodidae) with the incidence of zoonotic cutaneous leishmaniasis in endemic foci of golestan province, iran. j med entomol. 57(6): 1768–1774. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 305 http://jad.tums.ac.ir published online: january 06, 2016 original article screening of methanolic plant extracts against larvae of aedes aegypti and anopheles stephensi in mysore *thirumalapura krishnaiah mohankumar, kumuda sathigal shivanna, vijayan valiakottukal achuttan department of studies in zoology, university of mysore, mansagangothri, mysore, karnataka, india (received 25 feb 2014; accepted 6 dec 2014) abstract background: mosquitoes transmit serious human diseases, causing millions of death every year. vector control is facing a threat due to the emergence of resistance to synthetic insecticides. insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. nine different locally available medicinally important plants suspected to posse larvicidal property were screened against fourth instar larvae of aedes aegypti and anopheles stephensi to a series of concentrations of the methanolic extracts. methods: susceptibility tests on ae. aegypti and an. stephensi were conducted using standard who methods. the larvae of two mosquito species were exposed to methanolic extracts and mortality counts were made after 24 hours of exposure as per who method. larvae of ae. aegypti were more susceptible than that of an. stephensi. results: among the nine plant species tested, annona reticulata leaf extract was more effective against ae. aegypti larvae with lc50 and lc90 values of 95.24 and 262.64 ppm respectively and against an. stephensi larvae 262.71 and 636.94 ppm respectively. the least efficacy was in cosmos bipinnatus with lc50 and lc90 values of 442.6 and 1225.93 ppm against ae. aegypti and lc50 and lc90 values of 840.69 and 1334.01 ppm of thespesia populnea against an. stephensi. conclusion: the crude methanolic extract of the an. reticulata with good larvicidal efficacy could be considered for further characterization to control mosquito vectors instead of chemical insecticides. high efficacy found in an. reticulata extract will be considered for further studies to isolate the bioactive compound. keywords: ae. aegypti, an. stephensi, annona reticulata, larvicide, thespesia populnea introduction many new and re-emerging diseases are transmitted by arthropod vectors (brogdon and mc alister 1998). “vector borne diseases account for around 17% of the estimated global burden of all infectious diseases” (who 2006). over 350 species of mosquitoes are vectors of pathogens that cause diseases in humans and domesticated animals. more than fourteen mosquito genera are known to harbour arboviruses (mattingly 1973). “these diseases contribute significantly to disease burden, death, poverty and social debility in tropical countries” (yang et al. 2004). the proliferation of the diseases is not only due to the higher number of breeding places in urban agglomeration, but also due to increasing resistance of mosquitoes to current commercial insecticides such as organo-chlorides, organophosphates, carbamates, pyrethroids and also to biological insecticides (goettel et al. 1992, das and amalraj 1997, yadav et al. 1997). thus, mosquitoes are responsible for the transmission of more diseases than any other group of arthropods and play an important role as etiologic agents of devastating malaria, filariasis, dengue, yellow fever, japanese encephalitis, chikungunya and other viral diseases. in addition, they also cause irritation *corresponding authors: thirumalapura krishnaiah mohankumar, e-mail: mohanvbrl2012@gmail.com j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 306 http://jad.tums.ac.ir published online: january 06, 2016 to human by causing allergic responses that include local skin reactions as well as systemic reactions such as angioedema and urticaria (peng et al. 1999). aedes aegypti, a vector of yellow fever, dengue and chikungunya is widely distributed in the tropical and subtropical zones. about two-third of the world’s population, live in areas infested with dengue vectors, mainly ae. aegypti (hahn et al. 2001). anopheles stephensi is the primary vector of malaria in india and other west asian countries. every year, an estimated 300–500 million new infections and 600,000 cases based on world malaria report 2013 (who 2013) deaths result from malaria worldwide. in the past decade, chikungunya a virus transmitted by ae. spp. mosquitoes has reemerged in africa, southern and southeastern asia, and the indian ocean islands as the cause of large outbreaks of human disease (burt et al. 2012). malaria is a protozoan infection of erythrocytes caused in human beings by five species of the genus plasmodium (p. falciparum, p. vivax, p. ovale, p. malariae, and p. knowlesi). in most cases, malaria is transmitted via the bite of an infected female anopheline mosquito, but congenital malaria and acquisition through infected blood transfusion are well described (falade et al. 2007). “more than 40 per cent of the world’s population-approximately 3 billion people are exposed to malaria in 108 endemic countries “(who 2009). about one million cases of malaria are reported in india every year. many approaches have been developed to control mosquito menace. one such approach to prevent mosquito borne disease is by employing larvicide. the current mosquito control approach is based on synthetic insecticides. even though their effectiveness, they created many problems like insecticide resistance (liu et al. 2005), pollution and toxic side effects on human beings (lixin et al. 2006). this has necessitated the need for research and development of environmentally safe, biodegradable, indigenous method for vector control. botanicals offer great promise as source of phyto-chemicals with proven potential as insecticides which can play an important role in the control of mosquitoes and in the interruption of disease transmission at individual as well at community level (mittal and subbarao 2003). six plant families with several representative species, asteracae, cladophoraceae, labiatae, meliaceae, oocystacae and rutaceae appear to have the greatest potential for providing future mosquito control agents. insecticides of plant origin do not cause toxicity to human and domestic animals and are easily biodegradable. in the present study leaf extract of the plant passifloraceae, annonaceae, asteraceae, lauraceae, malvaceae, lamiaceae was studied for its insecticidal activity against malaria and dengue vectors. the application of botanical derivatives against mosquito has been reviewed in detailed by sukumar et al. (1991). in this regard, of late the researchers have shown more interest on plant derivatives as botanicals offer great promise as sources of molecules for the control of both agricultural pests and medically important insect species. now a days, the increased use of phytochemicals for the control of these insects may be attributed to the fact that population throughout the world are aware of the danger inherent in conventional insecticides, particularly the detrimental effect on the environment (pitasawat et al. 1998). it is in this regard, plant derived products have received increased attention from scientists and more than 2000 plant species are already known to have insecticidal properties (balandrin et al. 1985, sukumar et al. 1991). natural insecticides such as pyrethrum, rotenone and nicotine among others have been extensively used until recently for insect control (balandrin et al. 1985). to search derivatives with insecticidal property from local plants, the present inj arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 307 http://jad.tums.ac.ir published online: january 06, 2016 vestigation has been made in the vector biology research lab, department of studies in zoology at university of mysore. materials and methods aedes aegypti and an. stephensi larvae available at the mosquito colony maintained in vector biology research lab, department of studies in zoology, university of mysore by following standard rearing techniques. the larvae were reared in large enamel or plastic trays (30x24x5 cm) containing dechlorinated water and fed using finely powdered dog biscuits and dry yeast in the ratio of 2: 1. plant material and extraction nine plant species as listed in table 1 were collected from in and around mysore, karnataka, from march 2012 to jan 2013 and the leaves were shade dried, powdered manually and subjected to methanol solvent extraction in a soxhlet apparatus until exhaustion, to obtain non-polar bioactive constituents. the pooled extract was evaporated in a rotory vacuum evaporator at 40 ºc to dryness and stored at 4 ºc in an air tight bottle for further analysis. this was later dissolved in acetone and employed to prepare different concentrations. larval bioassay bioassays on mosquito larvae were performed on late third or early fourth instars, according to standard guidelines of who (2005). the required quantity of plant extract of different concentrations was prepared in acetone as solvent. one ml of each of the concentration was mixed thoroughly with 249 ml of dechlorinated water in 500 ml glass beakers. larvae were exposed in an ascending series of five concentrations according to log dose (table 2). parallel control tests were also maintained by adding 1ml of the solvent to 249 ml of dechlorinated water. twenty five early fourth instar larvae were transferred to each of the beakers. a minimum of three replicates were kept for each concentration along with the control. observation for the dead or moribund larvae was carried out after 24 h duration at 25±2 ºc and 75±5 % of relative humidity (rh). data analysis larval mortality counts were adjusted for the mortality in control, if any, by employing abbott’s formula (abbott 1975) to give an estimate of the plant extract attributable mortality. the corrected mortality data were subjected to regression analysis of probit mortality on log dosage (finney 1971). the significant difference in lc50 is based on the non-overlapping of 95 % fiducial limits. results table 2 provides the mortality rate of mosquito larvae against different concentrations of methanol extracts. table 3 and 4 provide the efficacy of methanol extracts tested against the dengue vector ae. aegypti and malarial vector an. stephensi larvae. fig. 1 and 2 show the log doseprobit mortality responses of all plant extracts. though the plants exhibited larvicidal activity against the two mosquito species, out of the nine plants screened using methanol as solvent, the annona reticulata (annonaceae) was found to possess better larvicidal activity against ae. aegypti followed by an. stephensi. the high percentage of mortality with low concentration was recorded of in the species against ae. aegypti has been with lc50 and lc90 values being 95.24 and 262.64 ppm respectively (table 3). likewise lc50 and lc90 values against an. stephensi are 262.71 and 636.94 ppm respectively (table 4). the 95% fiducial limits (fl) of an. reticulata for lc50 is 46.83–139.69 and 95% fl for lc90 is 172.23–931.30 and the slope is 2.90±0.55 j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 308 http://jad.tums.ac.ir published online: january 06, 2016 against ae. aegypti. similarly, the 95% fl for lc50 is 197.21–137.54 and for lc90 it is 457.41-1434.98 with the slope being 3.33±0.49 against an. stephensi. among these two mosquito species, susceptibility of ae. aegypti was found to be significantly more than that of an. stephensi. further, the larvicidal efficacy of an. reticulata was found to be significant by more compared to other plants (p< 0.05). the figure depicts the log dose-probit mortality responses and slope regression lines of tested plants. 800.00600.00400.00200.000.00 concentrtaion 100.00 80.00 60.00 40.00 20.00 0.00 m or ta lit y thesp passifl laurus hyptis gossip cosmos an.ret an.glab abutilon thesp passifl laurus hyptis gossip cosmos an.ret an.glab abutilon plants r sq linear = 0.957 r sq linear = 0.963 r sq linear = 0.982 r sq linear = 0.98 r sq linear = 0.999 r sq linear = 0.976r sq linear = 0.93r sq linear = 0.999r sq linear = 1 fig. 1. regression graph showing mortality of methanolic extracts of different plant species on aedes aegypti j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 309 http://jad.tums.ac.ir published online: january 06, 2016 table 1. list of plant screened against aedes aegypti and anopheles stephensi no plants name family medicinal/ toxic property 1 passiflora foetida passifloraceae pitta, inflammation,insomnia, depression and anxiety disorders 2 annona glabra annonaceae anticancer effects, substantial antimicrobial, antifungal and moderate insecticidal, sporicidal and cytotoxic 3 cosmos bipinnatus asteraceae jaundice, intermittent fever and splenomegaly 4 laurus nobilis lauraceae rheumatism and dermatitis 5 abutilon indicum malvaceae febrifuge, anthelmintic, antiemetic, anti-inflammatory, and in urinary and uterine discharges, piles, and lumbago 6 gossypium herbaceum annonaceae febrifuge, anthelmintic, antiemetic, anti-inflammatory. 7 annona reticulata annonaceae febrifuge, anthelmintic, antiemetic, anti-inflammatory, and in urinary and uterine discharges, piles, and lumbago 8 hyptis suoveolens lamiaceae gastrointestinal infections, cramps, and pain, skin infections 9 thespesia populnea malvaceae antifertility, antibacterial, antiinflammatory, antioxidant, purgative and hepatoprotective activity table 2. concentration and mortality of nine plant species against aedes aegypti and anopheles stephensi no plant name aedes aegypti anopheles stephensi concentration(ppm) mortality (%) concentration(ppm) mortality (%) 1 passiflora foetida 100 150 200 250 300 14 36 56 78 96 200 350 500 650 800 12 30 54 74 90 2 annona glabra 100 150 250 400 600 16 34 60 74 90 50 200 400 550 700 10 28 48 70 92 3 cosmos bipinnatus 150 300 450 600 750 10 28 52 64 76 50 200 350 500 750 12 30 52 78 90 4 laurus nobilis 100 250 350 500 700 12 28 58 78 88 100 250 400 750 900 14 36 52 74 92 j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 310 http://jad.tums.ac.ir published online: january 06, 2016 5 abutilon indicum 100 300 450 550 750 12 28 62 78 92 150 300 450 700 750 16 36 50 76 90 6 gossypium herbaceum 150 200 250 300 350 10 34 62 76 90 100 250 400 550 800 08 30 48 74 88 7 annona reticulata 30 100 150 200 250 12 40 68 84 96 100 200 300 400 500 12 30 52 70 90 8 hyptis suoveolens 150 250 350 400 550 12 32 48 66 82 150 300 450 600 750 08 32 50 76 94 9 thespesia populnea 100 250 400 550 700 10 28 48 68 90 500 700 900 1100 1300 10 30 52 78 92 table 3. efficacy of methanolic extracts of nine plant species against aedes aegypti no plant name lc50 (ppm) 95% fl lc90 (ppm) 95% fl slope±se heterogeneity (df) regression equation 1 annona reticulata* 95.24 46.83-139.69 262.64 172.23-931.30 2.90±0.55 5.20(3) y=2.90x±0.75 2 annona glabra 114.47 54.99-191.18 320.82 191.84-3738.15 2.86±0.64 6.98(3) y=2.86x±0.89 3 passiflora foetida 172.31 143.20-200.36 300.99 96.24-242.06 5.29±0.70 2.73(3) y=5.29x±6.83 4 gossypium herbaceum 216.23 194.57-239.21 620 524.18-778.30 2.79±0.24 1(3) y=2.79x±1.53 5 laurus nobilis 302.66 207.85-409.02 813.94 555.81-2193.14 2.98±0.47 3.35(3) y=2.98x±2.39 6 hyptis suaveolens 327.18 303.55-352.76 720.37 624.61-880.33 3.73±0.35 1(3) y=3.73x±4.40 7 abutilon indicum 332.98 123.63-546.59 903.76 549.46-692.12 2.95±0.71 7.40(3) y=2.95x±2.45 8 thespesia populnea 353.07 236.81-498.69 969.48 635.18-3376.91 2.92±0.50 3.79(3) y=2.92x±2.44 9 cosmos bipinnatus 442.60 402.11-488.40 1225.93 1013.27-2710 2.89±0.28 1(3) y=2.89x±2.66 note: lc50 median lethal concentration, fl fiducial limits; lc90 lethal concentration, df degree of freedom. *difference in lc50 from the extracts of other eight plants is significant based on non-overlapping 95% fiducial limits (p< 0.05). table 2. continued… j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 311 http://jad.tums.ac.ir published online: january 06, 2016 table 4. efficacy of methanolic extracts of nine plant species against anopheles stephensi no plant name lc50 (ppm) 95% fl lc90 (ppm) 95% fl slope±se heterogeneity (df) regression equation 1 annona reticulata* 262.71 197.21-337.54 636.94 457.41-1434.98 3.33±0.49 2.89(3) y=3.33x±3.06 2 cosmos bipinnatus 258.39 105.36-471.41 1048.00 544.56-1326.18 2.10±0.45 5.74(3) y=2.10x±0.68 3 gossypium herbaceum 298.80 62.13-768.79 1221.72 564.60-1130.12 2.09±0.55 8.40(3) y=2.09x±0.18 4 laurus nobilis 336.57 222.28-475.98 1197.75 757.60-3516.32 2.32±0.33 2.82(3) y=2.32x±0.87 5 abutilon indicum 372.81 231.41-532.21 1000.95 655.62-4054.35 2.98±0.56 4.51(3) y=2.98x±2.68 6 hyptis suaveolens 391.66 361.46-422.54 843.60 748.86-986.10 3.84±0.31 1(3) y=3.84x±4.97 7 passiflora foetida 441.01 409.18-473.62 922.27 820.34-1077.59 3.99±0.33 1(3) y=3.99x±5.57 8 annona glabra 448.78 338.06-570.24 900.18 675.62-1951.54 4.23±0.69 3.94(3) y=4.23x±6.24 9 thespesia populnea 840.69 801.16-880.57 1344.01 1249.14-1478.27 6.28±0.50 1(3) y=6.28x±13.39 note: lc50 median lethal concentration, fl, fiducial limits, lc90 lethal concentration, df, degree of freedom. *difference in lc50 from the extracts of other eight plants is significant based on non-overlapping 95 % fiducial limits (p< 0.05). 1250.001000.00750.00500.00250.000.00 concentration 100.00 80.00 60.00 40.00 20.00 0.00 m or ta lit y thesp passiflo laurus hyptis gossip cosmos annona an.glabr abutilon thesp passiflo laurus hyptis gossip cosmos annona an.glabr abutilon plants r sq linear = 0.984 r sq linear = 0.984 r sq linear = 0.999 r sq linear = 0.946 r sq linear = 0.994 r sq linear = 0.995r sq linear = 0.977r sq linear = 0.996r sq linear = 0.997 fig. 2. regression graph showing mortality of methanolic extracts of different plant species on anopheles stephensi j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 312 http://jad.tums.ac.ir published online: january 06, 2016 discussion the results of the larvicidal bioassay employing different plant extracts against two different mosquito species (table 3, 4) indicate significant larvicidal activity (p< 0.05) with methanol extract. the biological activity of these plant extract may be due to various compounds such as, phenolics, terpenoids, flavonoids and alkaloids (gohil et al. 2010). such compounds may jointly or independently contribute to produce toxic activity against the mosquito species. environmental safety of an insecticide is of paramount importance while employing against pests and vectors. an insecticide need not cause high mortality on target organisms in order to be acceptable (kabaru and gichia 2001). resistance to insecticides dates back to the beginning of application of chemicals, since ddt was initially introduced for mosquito control in 1946 and just in one year, the first case of ddt resistance occurred in ae. tritaeniorhynchus and ae. sollicitans (hemingway and ranson 2000). more than 500 species of arthropods are reported to be resistant to various insecticides (shelton et al. 2007). in this regard, phytochemicals may serve as suitable alternatives to synthetic insecticides in future, as they are relatively safe, inexpensive and are readily available throughout the world. according to bowers et al. (1995), the screening of locally available medicinal plants for mosquito control will be cost effective, reduce dependence on expensive imported products and stimulate local efforts to enhance public health. it is in this regard, the present study adds to our knowledge on the efficacy of the locally available medicinal plants. an earlier report points that, ethanolic extract of annona squamosa leaf has the most promising larvicidal activity against cx. quinquefasciatus larvae (das et al. 2007). the larvicidal and growth regulating activities of annona squamosa and syzygium cumini two related species have been reported against an. stephensi and other mosquitoes (saxena et al. 1993 and kaushik and saini 2008). the significant activity demonstrated by extracts of a. squamosa and a. senegalensis suggest that the two plants may have strong killing effects against insects particularly mosquitoes, hence giving a promising source of larvicidal agents (magadula et al. 2009). previously, a collection of a. squamosa plant materials from brazil indicated larvicidal effect against ae. albopictus and culex quinquefasciatus (das et al. 2007) and against an. stephensi (saxena et al. 1993). however, no reports on the larvicidal efficacy are available on an. reticulata. by comparing our results with the earlier studies, it is evident that the methanolic extracts of an. reticulata leaf have promising larvicidal activity against two mosquito species. it shows the lc50 value of 95.24 against ae. aegypti and 262.71 against an. stephensi (tables 3, 4). karunamoorthi and ilango (2010) have reported that thelc50 and lc90 values of methanol leaf extracts of croton macrostachyus (c. macrostachyus) were 89.25 and 224.98 ppm, respectively against late third instar larvae of malaria vector, an. arabiensis (an. arabiensis). the crude leaf extract of azadirachta indica with different solvents, viz. benzene, chloroform, ethyl acetate and methanol were tested for larvicidal activity against an. stephensi. the lc50 values were 19.25, 27.26, 23.26 and 15.03, respectively. kamaraj et al. (2009) have reported that the peel methanol extract c. sinensis, leaf and flower ethyl acetate extracts of ocimum canum against larvae of an. stephensi (lc50 =95.74, 101.53, 28.96, lc90=303.20, 492.43 and 168.05 ppm) respectively. the highest larval mortality was found in methanol extract of o. canum against the larvae of ae. aegypti (lc50=99.42, 94.43 and 81.56 ppm) j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 313 http://jad.tums.ac.ir published online: january 06, 2016 and against cx. quinquefasciatus (lc50= 44.54, 73.40 and 38.30 ppm), respectively (kamaraj et al. 2008). an. stephensi and ae. aegypti. chowdhury et al. (2009) have reported that the chloroform and methanol extracts of mature leaves of solanum villosum showed the lc50 value for all instars between 24.20 and 33.73 mg/l after 24 h and between 23.47 and 30.63 mg/l after 48 h of exposure period against an. subpictus. govindarajan (2010) evaluated larvicidal activity of crude extract of sida acuta against three important mosquitoes with lc50 values ranging between 38 and 48 mg/l. the crude extract had strong repellent action against the three species of mosquitoes as it provided 100 per cent protection against an. stephensi for 180 min followed by ae. aegypti (150 min) and cx. quinquefasciatus (120 min). dua et al. (2009) showed that the lc50 values of the neem oil were 1.6 and 1.7 ppm while lc90 values were 3.4 and 3.7 ppm against an. stephensi and ae. aegypti respectively. shivakumar and kataria (2011) have reported that an. showed a high susceptibility to very low concentrations of azadirachta indica (1–5 ppm), whereas in the case of ae. aegypti, the diagnostic concentration is slightly higher at 10ppm. the larvicidal action on an. stephensi, at a concentration of 3ppm resulted in 100% mortality within 72 h and a concentration of 15 ppm produces 90% mortality within 24 h of the treatment. in the present investigation out of the nine plants screened using methanol as solvent, annona reticulata (annonaceae) was found to possess better larvicidal activity against early fourth instar larvae of ae. aegypti followed by an. stephensi with lc50 and lc90 values being 95.24 and 262.64 ppm respectively (table 3). likewise lc50 and lc90 values against an. stephensi are 262.71 and 636.94 ppm respectively (table 4). annona muricata was an active larvicide with a 48-hour lc50 value of 67.4 µ g/ml. (jacobson 1958). he has further reported similar results for the seed extracts of another annona species, namely a. cherimola, a. glabra and a. squamosa, which had lethal effects on larvae of ae. sp. its potential as a larvicidal plant was further supported by the results of a recent study by satoto (1993), who has found that a. squamosa seed was one of the most effective larvicides against both culex tritaeniorhynchus and ae. aegypti. earlier studies further indicated that crude extracts of leaves of annona reticulata when evaluated for in vitro anthelmintic activity on indian adult earth worms eisinia fetida, a dose dependent inhibition of spontaneous motility (paralysis) of the worms was noticed (sonal et al. 2011). there are no reports available on the toxicity of annona reticulata against mosquito larvae. the present studies add that, apart from all medicinal property viz, antimicrobial, antitumoric, anthelmintic, antibacterial, cytotoxic etc, mosquito larvicidal property also present in this plant. therefore, the methanolic extracts of this plant inhibiting the development of larval growth, indicates hopes for further characterization of the active compound in our lab. conclusion the results from this study indicate that phyto-products isolated from the plants are more advisable to use for control of mosquito borne diseases. these plants are remarkably economical and ecofriendly with more larvicidal properties. among the plants screened an. reticulata showed high larvicidal efficacy against two mosquito species. hence, an. reticulata will be selected for further chemical isolation of the active ingredient in future studies. it could be considered as a potent resource for controlling mosquito larvae. such practice would not j arthropod-borne dis, september 2016, 10(3): 305–316 tk mohankumar et al.: screening of … 314 http://jad.tums.ac.ir published online: january 06, 2016 only reduce the disadvantages of insecticides on the environment but also promote 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accepted 29 june 2022) abstract background: culex theileri (diptera: culicidae) is a known vector of pathogens that cause dirofilariasis, sindbis fever and west nile fever in iran. the species is one of the country’s most abundant and widely distributed species. methods: in order to conduct a faunistic and bionomic study of mosquitoes, larval collection was carried out in chahar mahal and bakhtiari province of western iran using dipping method. results: in total, some 2096 larvae of cx. theileri were identified. among the larvae, 1024 (48.9%) displayed aberrations and anomalies in the development of cranial seta 1-c (preclypeal seta). morphological aberrations and anomalies of seta 1-c in the thirdand fourth-instar larvae and abdominal terga of the adults of cx. theileri collected in iran are discussed and illustrated. conclusion: more studies are needed throughout the distribution of cx. theileri to analyze the variation of the species, especially using both morphological and molecular data. keywords: anomalous; abnormal; character; morphology; variation introduction the genus culex linnaeus (diptera: culicidae) currently includes 779 species comprising 26 subgenera (1). the genus consists of at least 34 species of seven subgenera in southwestern asia (2) and 19 species representing five subgenera in iran (3). the genus comprises several important vectors of pathogens that cause infections in humans and domesticated animals (3). culex theileri theobald belongs to the theileri subgroup of the pipiens group of culex (culex) (2). the species is found in the afrotropical, palearctic and oriental regions; however, it is mainly a southern palearctic species (2). culex theileri is a known vector of avian plasmodium in portugal (4), turkey (5) and spain (6); avian haemoproteus in spain (6); dirofilaria immitis (canine heart worm) in portugal (7), iran (8) and canary islands of spain (9); west nile virus in south africa (10) and iran (11); sindbis virus in south africa (10, 12) and iran (13); japanese encephalitis virus in china (14); and rift valley fever virus in south africa (15, 16). additionally, some insect-specific flaviviruses (isfs) were isolated from this species in myanmar (17), portugal (18, 19), spain (18) and turkey (20) that are designated culex theileri flavivirus (cxthfv or ctfv). also, the wolbachia pipientis endosymbiont has been found in cx. theileri in portugal (21) and iran (22). lewis (23) noted some variation in the num copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 207 http://jad.tums.ac.ir published online: sep 30, 2022 ber of the branches of setae 5,6-c (inner and median frontal setae) in cx. theileri larvae in sudan. harbach (2) recorded the morphological variation of the larval and pupal chaetotaxy and gynandromorphism has been reported by bedford (24) and sanchez-murillo et al. (25). azari-hamidian et al. (8) showed molecular variation of the barcode region of the cytochrome c oxidase i (coi) gene in the species for the first time in iran. demirci et al. (26, 27) studied genetic and morphometric variation in cx. theileri in turkey. azari-hamidian and harbach (28) observed one abnormal seta 1-c (preclypeal seta) in a larva of cx. theileri collected in kurdistan province of iran. culex theileri is widely distributed in iran, being found in 28 of the 31 provinces of the country (8, 29–32). the species is one of the most abundant species in the country (33, 34). the larvae occur in a wide range of aquatic habitats (29, 33). during a faunal study of mosquitoes in chahar mahal and bakhtiari province, cx. theileri was the most widely distributed and abundant species collected (33, 35). among the larvae collected, 1024 specimens displayed various aberrations and anomalies in the cranial chaetotaxy. also, variation in the abdominal terga of adults was observed in different areas of iran. the present paper illustrates these anomalies, aberrations and variations. materials and methods the larval collection was carried out in chahar mahal and bakhtiari province using dipping method. larvae were temporarily preserved in lactophenol solution and later mounted on microscope slides using berlese’s medium. larvae and adults were identified using the keys of azari-hamidian and harbach (36). the morphological terminology of harbach and knight (37, 38), revised and updated in the mosquito taxonomic inventory (1), is used. photographs were taken by the authors. voucher specimens were deposited in the museum of medical and veterinary entomology, school of health, guilan university of medical sciences and the museum of medical entomology, school of medicine, shahrekord university of medical sciences, shahrekord. azari-hamidian and harbach (28) could be consulted for the definitions of aberration, abnormally, anomaly and variation. a brief explanation of the terms, which were used for anomalies and aberrations, is listed in table 1. results a total of 2096 larvae of cx. theileri (25.1 % of all mosquito larvae collected) were identified in collections made in chahar mahal and bakhtiari province. among the larvae, 1024 (48.9%) displayed aberrations and anomalies in the development of cranial seta 1-c whereas 1072 (51.1%) had normally spiniform seta 1-c (fig. 1). six larvae (0.28%) had one anomalous additional seta 1-c (fig. 2) and 1018 (48.57 %) exhibited aberrations in the development of seta 1-c that are listed in table 2. the most prevalent aberrant feature was bilateral (26.71 %) and unilateral (17.56%) spiculation, receptively (fig. 3). interesting and rare features observed only once in a single larva (0.05%) included a forked seta 1-c on one side and its mate on the other side with 3 branches (fig. 6) and a normal spiniform seta 1-c on one side and a filiform seta 1-c on the other side (fig. 7). additionally, aberrant scaling of the abdominal terga was observed in adults. the abdominal terga of cx. theileri normally have basal pale bands that are produced medially into somewhat triangular patches and apical dark scaling is not interrupted by pale scaling (fig. 9) but in a few specimens, the posterior dark scaling included two patches of pale scales (fig. 10). http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 208 http://jad.tums.ac.ir published online: sep 30, 2022 table 1. brief description of the anomalies and aberrations of seta 1-c in culex theileri larvae encountered in the present investigation, chahar mahal and bakhtiari province, iran normal normal spiniform, normally developed 1-c without any abnormalities including aberrations (spiculation and branching) and anomalies (extra 1-c) (fig. 1) abnormal anomalous (unilaterally double) extra 1-c on one side (fig. 2) aberrant unilateral or bilateral spiculation one or both sides of 1-c with spiculation at mid-length (fig. 3) unilateral or bilateral branching one or both sides of 1-c with a branch at mid-length (fig. 4) unilaterally or bilaterally forked one or both sides of 1-c forked apically (fig. 5) unilaterally forked on one side and other side with 3 branches 1-c on one side forked; its mate on the other side with 3 branches (fig. 6) one side spiniform and the other side filiform 1-c on one side spiniform; its mate on the other side filiform (fig. 7) abnormal shape developed differently than the above conditions (fig. 8) table 2. characteristics of seta 1-c observed in culex theileri larvae collected in chahar mahal and bakhtiari province, iran characteristics n normal 1072 abnormal anomalous (unilaterally double) 6 aberrant unilateral branching 7 unilaterally forked 23 unilateral spiculation 368 unilateral spiculation and unilateral branching 6 unilateral spiculation, unilateral branching and unilaterally forked 1 unilateral spiculation and unilaterally forked 8 bilateral branching 1 bilateral spiculation 560 bilateral spiculation and bilateral abnormal shape 1 bilateral spiculation and bilateral branching 2 bilateral spiculation and bilaterally forked 2 bilateral spiculation and unilateral branching 22 bilateral spiculation, unilateral branching and unilaterally forked 1 bilateral spiculation, unilateral branching and bilaterally forked 1 bilateral spiculation and unilaterally forked 13 one of pair spiniform the other filiform 1 one of pair unilaterally forked the other with 3 branches 1 subtotal 1024 total 2096 http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 209 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 1. culex theileri, normal spiniform seta 1-c, ardal, chahar mahal and bakhtiari province, iran (original photo) fig. 2. culex theileri, anomalous (extra) seta 1-c on one side (unilaterally double), esaabad, chahar mahal and bakhtiari province, iran (original photo) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 210 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 3. culex theileri, aberrant seta 1-c with spiculation at mid-length, chamnar, chahar mahal and bakhtiari province, iran (original photo) fig. 4. culex theileri, aberrant seta 1-c with bilateral branching, sarkhon, chahar mahal and bakhtiari province, iran (original photo) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 211 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 5. culex theileri, one the pair of seta 1-c forked, saman, chahar mahal and bakhtiari province, iran (original photo) fig. 6. culex theileri, one seta 1-c forked and the other with 3 branches, sarkhoon, chahar mahal and bakhtiari province, iran (original photo) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 212 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 7. culex theileri, seta 1-c spiniform (normal) on one side and filiform on the opposite side, razgah, chahar mahal and bakhtiari province, iran (original photo) fig. 8. culex theileri, seta 1-c with abnormal shape, alooni, chahar mahal and bakhtiari province, iran (original photo) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 213 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 9. culex theileri, abdominal terga with normal basal pale bands, kojanagh, ardebil province, iran (original photo) fig. 10. culex theileri, abdominal terga with a pair of postmedian patches of pale scales, astara, guilan province, iran (original photo) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 206–216 s azari-hamidian et al.: morphological aberrations of … 214 http://jad.tums.ac.ir published online: sep 30, 2022 discussion regarding the variability of cx. theileri, harbach (2) stated: "the larvae and adults, excluding the male genitalia, are extremely variable and exhibit the same degree of variation within local populations that is observed throughout the entire range. therefore, it appears that only a single widespread and variable species is involved, yet it is possible that the taxon actually consists of an aggregate of biologically distinct, isomorphic species". therefore, it seems that after more than 30 years the situation is the same. for the first time, azari-hamidian et al. (8) found a substitution at position 518 (g/a) in the barcode region of the coi gene of cx. theileri collected in a northwestern province of iran (ardebil) which caused one amino acid change (v to m). later, demirci et al. (26) studied genetic and morphometric variation in cx. theileri in northeastern turkey and found that although populations did not display genetic differentiation, there was a positive correlation between wing (body) size/shape and altitude. also, demirci et al. 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(for 1981). 13(2): 201–217. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 344 http://jad.tums.ac.ir published online: december 31, 2019 review article efficacy of extractions of iranian native plants against main malaria vector, anopheles stephensi in iran for making appropriate formulation for disease control *hassan vatandoost1,2; fatemeh nikpour1,2; ahmad ali hanafi-bojd1,2; mohammadd reza abai1,2; mahnaz khanavi3; abbas hajiiakhondi3; ahmad raesi2; jalil nejati1 1department of medical entomology and vector control school of public health, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of pharmacognosy, faculty of pharmacy, tehran university of medical sciences, tehran, iran (received 01 oct 2019; accepted 20 dec 2019) abstract background: malaria is the main vector–borne disease worldwide. there are several reports of insecticide resistant in malaria vectors worldwide due to using different insecticides. the aim of this study was to evaluate different native plant extortions against main malaria vector, anopheles stephensi in iran for choosing the appropriate plant for formulation and use for vector control. methods: the larvae of an. stephensi were reared in insectary, extraction of plants were carried out at department of pharmacology. the standard who method for biological tests was used for calculation of lc50 and lc90. probit regration lines were plotted for calculation of lc50 and lc90. results: in this study several plants including: mentha spicata, cymbopogon olivieri, azadirachta indica, melia azedarach, lagetes minuta, calotropis procera, eucalyptus camaldulensis, cupressus arizonica, thymus vulgaris, lawsonia inermis, cedrus deodara, cionura erecta, bunium persicum, carum carvi, artemisia dracunculus, rosmarinus officinalis were used. results showed that mentha spicata and eucalyptus camaldulensis, had the lowest and highest lc50 respectively. conclusion: results indicated that mentha spicata and eucalyptus camaldulensis, had the lowest and highest lc50 respectively. several other plant extract also showed significant mortality. the formulation of these plants should be prepared and evaluate at the field condition against malaria vectors. keywords: plants; malaria vector; pesticide; iran introduction malaria is the most important mosquitoborne disease so that an estimated 212 million cases worldwide in 2015 out of them 3,800,000 cases estimated to be happen in eastern mediterranean region (emro). it was estimated that 429,000 deaths from malaria occurred globally including 7300 cases in emro. the disease in the region had 291 million people at risk, and mostly reported from 5 countries: sudan (36%), pakistan (27%), somalia (18%), afghanistan (11%) and yemen (8%) (1). there were an estimated 219 million cases and 435 000 related deaths in 2017 (2). insecticide resistance is becoming a problem of global importance as it threatens the significant achievements in malaria control. dramatic increase of insecticides use in malaria vector control projects has resulted to growing trend of insecticide resistance among mosquito vectors. currently increased attention to pyrethroids as effective and low-risk insecticides has developed the risk of resistance to this group. nowadays there are two main interventions in malaria control programs: indoor residual spray*corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 345 http://jad.tums.ac.ir published online: december 31, 2019 ing (irs) and long-lasting insecticidal nets (llins). for both methods, pyrethroids are used and this will increase the pressure of selection for resistance. as a result, there are reports indicating resistance to pyrethroids in malaria vectors of the emro region in recent years (3). data are still limited and difficult to consolidate as many countries have not yet carried out adequate routine susceptibility tests, and malaria-free countries don’t usually do the tests. during the last decade, three out of four insecticide classes were applied in malaria control activities in emro countries (4). organophosphates including malathion, dichlorvos, temephos and fenitrothion with a total of 381 tones active ingredient had the highest use, followed by pyrethroids (cypermethrin, alpha-cypermethrin, deltamethrin, lambda-cyhalothrin, permethrin) and carbamates (bendiocarb, propoxur) with 157 and 30.6 tones active ingredient, respectively. in contrast of organophosphates, during these years using of pyrethriods and carbamates seems to be increased. insecticide resistance is the selection of a heritable trait in an insect population that results in an insect-control product no longer performing as intended. establishing the baseline of all plant evaluated against vector and conducting a comprehensive situation analysis is the starting point for overcome against resistance. this will require collecting available background data and, if necessary. interpretation of the data must take into account. countries should design a monitoring plan that includes data on vector distribution and relevant vector attributes for transmission and control, on susceptibility/ resistance to currently used insecticides, and on the quality of vector control interventions. malaria in iran malaria is one of the important infectious diseases in iran with an average of about 15000 annual cases in the last decade. the most routes of malaria cases are immigration from afghanistan and pakistan to southern and southeastern areas of the country (ministry of health, annual reports). during the 2002–2017, 134,273 malaria cases were reported. the malaria incidence decreased from 0.24/1000 cases in 2002 to 0.01/1000 in 2017. from 2009 onward, the number of imported cases increased in comparison with the autochthonous and indigenous cases. most cases were seen in males and people over 15 years of age. moreover, the dominant registered reports were from rural areas. most malaria cases were reported from the south and southeastern of iran. plasmodium vivax was the dominant species (5). there are several activities on different aspects of malaria in the country: including insecticide resistance monitoring (6-17), using bednets and long lasting impregnated nets (18-24). recently resistance of an. stephensi to different insecticides in malarious areas of iran has been reported (3). the last checklist of iranian mosquitoes shows 31 anopheles species including sibling, biological forms and genotypes, 17 out of them are reported to be included in malaria transmission. these vectors are considered as sibling, genotype and type forms. anopheles stephensi, an. culicifacies, an. fluviatilis, an. dthali are the main vector species of south-eastern foci, while an. sacharovi and an. maculipennis are included in malaria transmission in northwest focus, and an. superpictus has wide distribution in all malaria foci of the country (fig. 1). seasonal activity of anopheline mosquitoes varies in different area due to environmental condition. it shows one peak in northwest especially in summer, however, there are two peaks of activity in coastal warm and humid region in the southern part of iran with oriental epidemiological characteristics. the chemical control of vectors now is restricted to endemic malarious areas of south-eastern part of the country with deltamethrin and residual spraying and long lasting permethrin impregnated nets (olyset) for personal protection, while bihttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 346 http://jad.tums.ac.ir published online: december 31, 2019 ological control is conducting by bacillus thuringiensis as larvicide. knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes in local and global scales. the main criteria for susceptibility status, which are recommended by who, were considered. the results showed that there is resistance to ddt and dieldrin, indication of tolerance to some tested insecticides. agriculture in iran remains highly sensitive to climate developments; the country's most important crops are wheat, rice and other grains, sugar beet, fruits, nuts, cotton, and tobacco, which require the use of insecticides. so far different groups of insecticides are using for crops protection in the country. the main governmental use of insecticide in the health sector is their application for adult mosquito control. the campaign against malaria vectors started with organochlorines (ddt, dieldrin and bhc) during the 1960’s, followed by organophosphates (malathion and pirimiphos-methyl) for 2 decades from 1966 and continued with the carbamate, propoxur during 1977–1990, and then with pyrethroids including lambdacyhalothrin and deltamethrin. temephos, reldan and pirimiphos-methyl was used for larviciding (ministry of heath of iran) materials and methods rearing of mosquito larvae rearing and maintaining mosquito larvae was carried out in the temperature of 29±2 ºc and relative humidity of 70±10% and light dark cycle of 16h light and 8h was performed in culicidae insectarium of the school of public health tehran university of medical sciences. the larvae of the late 3rd stage or early 4th stage of an. stephensi were used for larvicidal tests. anopheles stephensi larvae used in this study were obtained from the laboratory of the “school of public health and institute of health research” tehran university of medical sciences, tehran, iran. they were reared under insectary conditions at 25±1, 12/12h (light/dark) photoperiod and 50–70% relative humidity and were fed with 10% sucrose solution. the late 3rd and early 4th instar larvae were used for the tests. the sucrose solution was withdrawn from the cage, 14h prior to the tests. biological tests (larvicidal) the standard who method for biological tests was used. the overall temperature of the lab (28 ºc), test period (24h) and the number of larvae (25 in each 400cc beaker) has to be constant. the best age range of the larvae for the tests are the larvae of the late 3rd stage or early 4th stage range and preferably dechlorinated water should be used in the tests. at least 5 logarithmic concentrations should be made of the eo. in order to find the suitable concentration first the concentrations should be chosen in a larger domain and based on the results the concentration domain becomes narrower. usually the concentration in which has the 50% relative mortality and two concentrations lower than it and two concentrations upper than it are used to draw that regression line diagram. in each test 5 concentrations of pesticide and for each concentration 4 repetitions and in general 2 witnesses are considered. statistical analysis the test results after 24h were read as the following way: the number of alive larvae, the number of dead larvae, the number of moribund larvae, number of larvae and the total number and the results were used to draw the mortality tables. the mortal quantities of 50% and 90 % of eos (lc50 and lc90) and the level of confidence of 95%, the equation of the regression line will be estimated using a regression probit analysis as described by finney (25). when the mortality of the witness group is less than 5% then the resulted data of biometric tests have been correct but if the mortality of the witness group is between 5% to 20% they have to be corrected line. the percentage mortality was calculated using abbot’s formula (26). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 347 http://jad.tums.ac.ir published online: december 31, 2019 extraction solvent fractionation dried whole samples (300g) were extracted with 80% methanol (meoh, 6×1.5l) in a percolator at room temperature for 2 weeks. the combined extract was concentrated to dryness under reduced pressure at 40 °c. the meoh extract was successively dissolved in 100ml meoh: h2o (7: 3) and extracted mosquito rearing and evaluation with petroleum ether (4×200ml), chloroform (chcl3, 4×200ml), h2o-saturated ethyl acetate (etoac, 4×200ml) and h2o-saturated n-butanol (n-buoh, 4×200ml) in separatory funnel. each fraction together with the remaining meoh part after the solvent fractionation, were then evaporated to dryness under reduced pressure at 40 °c for the purpose of test fraction. all solvents were purchased from merck (merck, darmstadt, germany). list and identification of plants in this study the different extractions of the following iranian native plants were evaluated against main malaria vector, an. stephensi, mentha spicata, cymbopogon olivieri, azadirachta indica, melia azedarach, lagetes minuta, calotropis procera, eucalyptus camaldulensis, cupressus arizonica, thymus vulgaris, lawsonia inermis, cedrus deodara, cionura erecta, bunium persicum, carum carvi, artemisia dracunculus, rosmarinus officinalis. results results of efficacy of different iranian native plants against malaria vector an. stephensi at the lc50 and lc90 levels are presented in table 1 and fig. 2. from these results it can be concluded that mentha spicata and eucalyptus camaldulensis, had the lowest and highest lc50 respectively. table 1. efficacy of different plants extract against anopheles stephesni at the lc50 and lc90 level component name lc50 (mg/l) lc90 (mg/l) reference mentha spicata 0.009 hajiakjoondi a et al. (2000) (27) cymbopogon olivieri 321.90 983.6 hadjiakhoondi a, et al. (2003) (28) azadirachta indica 0.35 1.81 vatandoost h, et al. (200 4) (29) melia azedarach 5.51 34.90 hadjiakhoondi a, et al. (2006) (30) tagetes minuta l (dried plant) 1.30 5.07 hajiakhondi a, et al. (2008) (31) tagetes minuta l (fresh plant) 1.05 3.83 hajiakhondi a, et al. (2008) (31) calotropis procera (alcoholic extract) 109.71 234.61 shahi m, et al. (2010) (32) calotropis procera (fresh latex) 13.06 23.53 shahi m, et al. (2010) (32) eucalyptus camaldulensis (methanol extract) 397.75 3085.18 sedaghat m, et al. (2010) (33) eucalyptus camaldulensis (essential oil) 89.85 215.26 sedaghat m, et al. (2010) (33) cupressus arizona e.l. (leaf essential oil) 79.30 238.89 sedaghatm, et al. (2011) (34) centaurea bruguierana ssp. belangerana 15.70 48.34 khanavi m, et al. (2011) (35) sargassum swartzii 11.75 53.47 khanavi m, et al. (2011) (35) chondria dasyphylla 10.62 56.39 khanavi m, et al. (2011) (35) nepeta menthoides (methanol extract) 69.54 175.55 khanavi m, et al. (2012) (36) nepeta menthoides (essential oil ) 234.35 419.86 khanavi m, et al. (2012) (36) kelussia odoratissima mozaffarian (essential oil) 4.88 9.60 vatandoost h, et al. (2012) (37) thymus vulgaris (methanol extract) 191.33 503.98 khanavi m et al. (2013) (38) lawsonia inermis (methanol extract) 69.40 158.75 khanavi m, et al. ( 2013) (38) cedrus deodara (methanol extract) 128.04 292.87 khanavi m, et al. (2013) (38) stachys trinervis (methanol extract) 210.42 604.04 khanavi m, et al. (2013) (38) stachys inflate (methanol extract) 195.84 392.81 khanavi m, et al. (2013) (38) stachys setifera (methanol extract) 181.62 352.35 khanavi m, et al. (2013) (38) stachys laxa (methanol extract) 269.64 602.6 khanavi m, et al. (2013) (38) http://jad.tums.ac.ir/ http://www.openj-gate.org/search/searchresults.aspx?searchterm=%22h.%20vatandoost%22&field=au&res=10&type=2&sub=all&update=none&from=-1&to=2011&pr=2 j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 348 http://jad.tums.ac.ir published online: december 31, 2019 stachys persica (methanol extract) 282.80 515.94 khanavi m, et al. (2013) (38) stachys subaphylla (methanol extract) 252.60 592.37 khanavi m, et al. (2013) (38) stachys byzantine (methanol extract) 103.29 276.99 khanavi m, et al. (2013) (38) stachys turcamanica (methanol extract) 253.45 549.05 khanavi m, et al. (2013) (38) cionura erecta (essential oil) 77.30 199.58 mozaffari e, et al. ( 2014) (39) cionura erecta (methanol extract) 250.38 490.00 mozaffari e, et al. ( 2014) (39) ferulago carduchorum (essential oil) 12.78 47.43 golfakhrabadi f, et al. ( 2015) (40) bunium persicum (essential oil) 27.72 91.35 sanei-dehkordi a, et al (2016) (41) carum carvi (seeds) 21.6 72.44 torabi pour h, et al. (2016) (42) artemisia dracunculus (branches and leaves) 1.33 4.12 torabi pour h, et al. (2016) (42) rosmarinus officinalis (branches and leaves) 93.22 229.29 torabi pour h, et al. (2016) (42) fig.1. map of distribution of malaria vectors in iran fig.2. efficacy of different plants extract against anopheles stephesni at the lc50 and lc90 level table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 349 http://jad.tums.ac.ir published online: december 31, 2019 discussion most botanical components are rapid acting and breakdown quickly in the environment. the extract of whole leaf and essential oil of some certain plants have been investigated against some public health pests. the use of botanical pesticide may help in reducing the environmental side effects by the synthetic insecticides. the results obtained suggest that the extracts of different iranian native plants may be a promising as larvicide against an. stephensi. there are many researches in the field. in other investigation, nathan et al. (2007) (43) reported that the larvicidal activity of essential oil from eucalyptus tereticornis sm. with lc50 and lc90 values were 23.8 and 63.9ppm respectively against an. stephensi larvae. there are some reports about the resistance to these chemicals in mosquitoes. therefore we need to identify alternative insecticide substances from natural products. many scientists reported insecticidal activities of plants belong to different families in different parts of the world. there are several native reports about crude solvent extracts of different parts of plants, essential oils or their chromatographic fractions. they showed various levels of bioactivity against different developmental stages of malaria vectors (44). some plants have phytochemicals constituents for the control of mosquitoes. one of the earliest reports of the use of plant extracts against mosquito larvae is extraction of plants’ alkaloids like nicotine, anabasine, methyl anabasine and lupinine from the russian weed in 1933 (45). some plant families such as asteraceae, cladophoraceae, labiatae, meliaceae, oocystaceae and rutaceae have the maximum potential for development of novel mosquito control agents (46). the genus lawsonia has one species, lawsonia inermis (47-48). henna`s leaves, flowers, seeds, stem barks and roots had been used in iran to treat some diseases such as rheumatoid arthritis, headache, ulcers, diarrhea, leprosy, fever, leucorrhoea, diabetes, cardiac disease. it had hepatoprotective effect and been used as colouring agent too (49). conclusion due to larvicidal effect of some iranian native plants against malaria vector, production of specific formulation is required for evaluation under filed condition. acknowledgements the authors would like to appreciate very much for kind collaboration of department of medical entomology and vector control, school of public health, tehran university of medical sciences (tums) for providing the test mosquito, an. stephensi. all the extraction was carried out at department of pharmacology, tehran university of medical sciences (tums). this study was supported by the ministry of health and medical education of iran with nimad project number 971065. all authors declare that there is no conflict of interest. references 1. world health organization (who) (2016) world malaria report. p. 186. 2. world health organization (who) (2018) world malaria repot. p. 210. 3. abbasi m, hanafi-bojd aa, yaghoobi-ershadi mr, vatandoost h, oshaghi ma, hazratian t, mohammad mehdi sedaghat, sajjad fekri, reza safari, abdol mojahedi, yousef salari (2019) resistance status of main malaria vector, anopheles stephensi liston (diptera: culicidae) to insecticides in a malaria endemic area, southern iran. asian pac j trop med. 12: 43–48. 4. world health organization (who) (2017) regional malaria action plan 2016–2020 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 344–352 h vatandoost et al.: efficacy of extractions … 350 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275–277. 48. chetty km (2008) flowering plants of chittoor. 1st ed. andhra pradesh, india. 49. chopra rn, nayar sl, chopra ic (1956) glossary of indian medicinal plants. new delhi: c sir. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=hafizi%20m%5bauthor%5d&cauthor=true&cauthor_uid=26114148 https://www.ncbi.nlm.nih.gov/pubmed/?term=yousefbeyk%20f%5bauthor%5d&cauthor=true&cauthor_uid=26114148 https://www.ncbi.nlm.nih.gov/pubmed/?term=rad%20yr%5bauthor%5d&cauthor=true&cauthor_uid=26114148 https://www.ncbi.nlm.nih.gov/pubmed/?term=baghenegadian%20a%5bauthor%5d&cauthor=true&cauthor_uid=26114148 https://www.ncbi.nlm.nih.gov/pubmed/?term=ardekani%20mr%5bauthor%5d&cauthor=true&cauthor_uid=26114148 https://www.ncbi.nlm.nih.gov/pubmed/?term=ardekani%20mr%5bauthor%5d&cauthor=true&cauthor_uid=26114148 microsoft word 2-dr vazirian zadeh .doc iranian j arthropod-borne dis, 2007, 1(2): 7-13 b vazirianzadeh et al.: the effects of oral application … 7 *corresponding author: dr b vazirianzadeh, tel: +98 611 3782956, fax: +98 611 3332036, e-mail: babakvazir@yahoo.co.uk original article the effects of oral application of cyromazine and triflumuron on house-fly larvae *b vazirianzadeh1, ma jervis2, nac kidd2 1department of medical parasitology and mycology, school of medicine, ahwaz jundishapoor medical sciences university, ahwaz, iran 2department of ecology and biodiversity, school of biosciences, cardiff university of wales, uk (received 26 nov 2006; accepted 13 oct 2007) abstract accumulations of large quantities of wastes (manure, used litter, dead birds) which are excellent medium for fly-larvae over poultry houses provide breeding places for different groups of fly pests, with house-flies being the dominant species. this project is a comparative lab study. in this research project the larvicidal effects of cyromazine and triflumuron were studied as two insect growth regulators (igrs) to reduce the fly population using oral application. both igrs had a significant effect on larval mortality compared with their controls among the concentrations (p< 0.01, fisher’s lsd with bonfferoni correction) including a dose-dependent relationship. comparisons among lc50 and lc90 values, using fiducial limits, showed that cyromazine was significantly more toxic to the larvae of the two strains than triflumuron. it is concluded that cyromazine should be used in a larvicidal programme to control house-fly rather than triflumuron. keywords: housefly, cyromazine, triflumuron, oral application introduction commercial poultry houses are rapidly expanding worldwide to meet the needs of the increasing human population (axtell 1999). in modern production systems, poultries are housed in high densities with, consequently, accumulations of large quantities of wastes (manure, used litter, dead birds) which are excellent medium for fly-larvae, consequently adult fly production (axtell 1986, 1999). this phenomenon provides breeding places for different groups of fly pests, with house-flies being the dominant species. igrs are a diverse group of insecticides, with a range of effects on insect specific phenomena, disrupting the growth and development of insects and other arthropods. they mainly affect the development of immature stages, and disrupt metamorphosis and reproduction (graf 1993, retnkaran et al. 1985) and are becoming more important in the management of insect pests (grenier and grenier 1993). igrs include various chemical categories including: juvenile hormones, chitin synthesis inhibitors, and triazine derivatives (retnakaran et al. 1985) with different modes of action. cyromazine (cga 72662, n-cyclopropyl-1, 3, 5-triazine-2, 4, 6-triamine) represents a new class of igrs derived from aziodotriazine herbicides (shen and plapp 1990). it was discovered by ciba-giegy ltd. in the mid 1970s and originally developed under the trade name of ‘vetrazine’, a blow-fly control agent. cyromazine is now also applied topically to control housefly larvae in manure (‘neporex’), as a feed– through in poultry (‘larvadex’), as well as in crop protection (‘trigard’) (moreno-mari et al. 1996, graf 1993). ultrastructural studies on larvae of l. cuprina have suggested that the pesticide works at the cuticular level (jimenez-pyedro 1995). however, binnington (1985) and friedel et al. (1988) iranian j arthropod-borne dis, 2007, 1(2): 7-13 b vazirianzadeh et al.: the effects of oral application … 8 suggested that cyromazine did not inhibit chitin formation but might be acting by disruption of the endocrine system. triflumuron is the common name for the chemical 2-chloro-n-[[[4-(trifluoromethoxy) phenyl] amino] carbonyl] benzamide. it was first introduced by bayer in 1979 at the international plant protection congress in washington (senior 1998). triflumuron has been shown to be effective against a variety of insects of medical and agricultural significance, under laboratory and field conditions, belonging to the orders of diptera, orthoptera, siphonaptera, coleoptera and lepidoptera. it has been used to control house-flies, mosquitoes, fleas and cockroaches in public and animal health (retnakaran and wright 1987, weaver et al. 1984, main and mulla 1982, miura and takahashi, 1979). ‘alystin’ and ‘starycide’ are two of its trade names. triflumuron is a benzoylphenyl urea that acts primarily as a stomach poison, but may also have a contact action, depending on the insect species, the developmental stage of the insects, the dose applied, and the method of application (senior 1998). its effects are typical of benzoyl phenyl ureas, it acts to inhibit the deposition of chitin in the cuticle of arthropods. it has both ovicidal (smith and grigarik 1989, broadbent and pree 1984) and larvicidal (smith and grigarik 1989, hejazi and granett 1986, asher and nemny 1984) properties, depending on the species studied. in addition, it induces sterility in adult female house-flies (howard and wall 1995, weaver and begley 1982). there have been several previous investigations using cyromazine, of triazine derivatives, as a larvicide against house-fly larvae (keiding et al. 1991, keiding et al. 1990, kelly et al. 1987, iseki and georghiou 1986) but no comparisons have ever been made with triflumuron. the benzoylphenyl ureas, to which triflumuron belongs, are mainly used as larvicides (reynolds 1987), but very little work has been done on the larvicidal effects of triflumuron on house-fly larvae (weaver and begley 1982). in this study the toxicity effects of cyromazine and triflumuron were evaluated and compared using the oral application. materials and methods igrs both igrs were of technical grade: cyromazine (99.9% a.i., novartis) and triflumuron (99.6% a.i., bayer). insects house-fly larvae were of two strains: rentokil, a non-resistant laboratory strain, and chicken house, a wild strain. the house-flies were reared at insect investigations ltd. (cardiff university of wales) in constant environmental chambers (25 °c, 55%-60% rh and 16hl: 8hd). the rearing method was that of cetin et al. (2006) and kristensen and jespersen (2003) with some modifications. the larvae were reared in a medium made by mixing the following ingredients: 170g dog food pellets, 20g-yeast extract (sigma) and 30g milk powder. the pellets were placed into a plastic container, and sufficient water added to soften them. the pellets were then soaked in the water for at least one hour. the pellets, milk powder and yeast extract were together placed into a food mixer, and blended thoroughly. the resultant mixture was left for another hour, after which its time consistency was checked, and more water added if necessary. the mixture should be not much watery or thick. oral application (oa) to examine the effects of oa, second-instar larvae were used, because of their higher food consumption rate. the larvae were provided with: 0.125, 0.25, 0.5 and 1 mg/kg larval medium (kelly et al. 1987). distilled water was used as the solvent for cyromazine and acetone was used for triflumuron. twenty-five of the second-instar larvae were placed in a 250cm glass-jar containing 70gr of igr-contaminated larval medium. the latter was treated with the highest volume of acetone, 1000µl/kg diet, as this was the volume used in iranian j arthropod-borne dis, 2007, 1(2): 7-13 b vazirianzadeh et al.: the effects of oral application … 9 the treated control (see above). a non-treated control did not receive any igr or acetone. in all cases three replicates were used for both strains. the jars containing larvae were kept in a constant environmental chamber and monitored for two weeks. emergence of the adults usually took< 10 days. the numbers of emerged houseflies at the end of experiment was counted to record the rate of survival which was then converted to the rate of mortality. all the experiments were conducted in school of biosciences, cardiff university of wales, uk. data analysis all percentage mortality data were arcsine square root-transformed. they were checked for the homogeneity of their variances using bartlett or levene tests and for the normality of their residuals using the ryan-joyner test, which is similar to the shapiro-wilks test. oneway anovas were then performed on the data followed by the least significant difference (fisher’s lsd and bonfferoni correction) method for distinguishing between the means to find any significant differences in mortality between the different concentrations and control, also amongst the concentrations. if the anova table indicates a significant difference between the means, then pairs of means were compared, using lsd method to determine if they are significantly different from each other. then concentration-mortality regression was performed by probit analysis (chi package, 2000) to determine the lc 50 and lc 90 for each treatment. the chi package is based on finney’s probit analysis (finney, 1978) and takes abbott’s formula (abott 1925) as a common correction. differences in toxicity at lc50 and lc90 were considered to be significant when fiducial limits did not overlap (sheppard et al. 1992, stark et al. 1991). results overall one way-anovas revealed significant differences in mortality among the treatments and controls (p< 0.001). moreover both igrs had a significant effect on mortality of larval mortality compared with their controls among the concentrations (p< 0.01, fisher’s lsd with bonfferoni correction). the results are summarised in table 1 and 2. there was a dosedependent relationship in the rate of mortality of larvae with both igrs. this was confirmed using probit analysis. comparisons among lc50 and lc90 values, using fiducial limits, showed that cyromazine was significantly more toxic to the larvae of the two strains than triflumuron. besides, there was no significant difference in the susceptibility of the two strains to either cyromazine or triflumuron. there was no difference in mortality between treated and un-treated controls. acetone had no side effects on the larval mortality. table1. probit analysis, using oral application of 2nd instar, for both igrs against both strains of house-fly larvae, mortality of larvae to adults treatment lc50 mg/kg diet fl* of lc50 lc90 mg/kg diet fl* of lc90 chi-squared values df sd of slope cy/re 0.207 0.176-0.238 0.566 0.473-0.727 0.362 2 0.342 cy/ch 0.216 0.190-0.241 0.566 0.473-0.727 0.249 2 0.349 tr/re 0.531 0.475-0.602 3.455 2.567-5.132 0.085 2 0.267 tr/ch 0.594 0.541-0.659 3.905 3.024-5.390 0.005 2 0.272 cy/re= cyromazine # rentokil, cy/ch= cyromazine # chicken, tr/re= triflumuron # rentokil, tr/ch= triflumuron # chicken, fl*= fiducial limits iranian j arthropod-borne dis, 2007, 1(2): 7-13 b vazirianzadeh et al.: the effects of oral application … 10 table 2. toxicity of two igrs against house-fly larvae, using fiducial limits (mortality of larvae to adults), topical application of 2nd instar comparisons ratio of toxicity (lc50) significant ratio of toxicity (lc90) significant trch=trre 1.12 ns 1.10 ns cych=cyre 1.04 ns 1.00 ns trre<cyre 2.57 sig 6.10 sig trch<cych 2.75 sig 6.90 sig trch= triflumuron # chicken, trre= triflumuron # rentokil, cych= cyromazine # chicken, cyre= cyromazine # rentokil discussion the mortality effects of oa of cyromazine agree with the results of kelly et al. (1987) and those of iseki and georghiou (1986) but do not accord with the results of scotte et al. (2000). the reason for these mismatches in the latter case is unclear. the obtained results of cyromazine in this study have been confirmed by using of 50% cyromazine to larval foci followed by cultural and biological control methods as a field trial in poultry houses (crespo et al. 2002). they obtained satisfactory reduction on the house flies population to overcome on insecticide resistance population. kocisova et al. (2004) obtained a 74-81% reduction in the population of house flies over a 5 weeks field trial, using neporex (a.i. 2% cyromazine) against second and third larval instars which confirmed the larvicidal effects of cyromazine. novartis technical brochure (2006) explains a 100% efficacy in the mortality of house fly larvae in the different instar larvae by using an oral application. this result is similar to the results of current study. a dose-dependent relationship in the rate of mortality of larvae with cyromazine in the current study agrees with the results of tomberlin et al. (2002). they obtained similar results using cyromazine in an oral application against black soldier fly, hermetia illucens (l.). the obtained rates of mortality with oa of triflumuron confirm the results of weaver and begley (1982), as they reported general dosedependent of mortality. however they used percentages of puparium formation to calculate the rate of mortality, whereas in this study, successful emergence of adults was used. in addition, the results of howard (1995) based on topical application of adults show a dosedependent for mortality of house-fly larvae, using triflumuron. the results of the present study are confirmed by study of tong and hui (2004), using a similar application method against housefly larvae too, the significant larvicidal properties of triflumuron is confirmed by studies of batra et al. (2005), sulaiman et al. (2004) and ladonni and farashiani (2000) against aedes aegypti and anopheles stephensi larvae in field trials of india, malaysia and iran, respectively. these agree with larvicidal effects of triflumuron in the current study. the obtained results using novularon, an igr of benzoylurea, in a laboratory feeding larvicidal application against house fly larvae, by cetin et al. (2006) agree the results of present study using triflumoron as an igr of benzoylurea. results of the present study reveal that, both igrs, can affect significantly on mortality of house-fly larvae (table 1 and 2), and that cyromazine was more toxic than triflumuron in all cases. cyromazine was 2.57 and 2.75-fold more effective than triflumuron at lc50 and 6.10, 6.90-fold more effective at lc90 against rentokil and chicken house strains, respectively, than triflumuron. furthermore, using oa both strains showed the same degree of susceptibility to each igr (table 1 and 2). therefore it is concluded that iranian j arthropod-borne dis, 2007, 1(2): 7-13 b vazirianzadeh et al.: the effects of oral application … 11 cyromazine should be used in a larvicidal programme to control house-fly. also, several researchers have found a lack of cross-resistance between cyromazine and other igrs: lufenuron (wilson 1997), diflubenzuron (farkas and plapp 1991), methoprene (bloomcamp et al. 1987). furthermore, no cross-resistance was reported between cyromazine and conventional insecticides, organophosphorous, pyrethroids and organochlorines (keiding et al. 1991, shen and plapp 1990) and cyromazine as a larvicide. keiding (1991) reported no increase in lc50 and lc90 values of cyromazine as a larvicide, after five or six selections. in conclusion according to the above mentioned results and facts using cyromazine in a larvicidal program is preferred to triflumuron. acknowledgements we are very thankful to the ministry of health, iran, and department of ecology and biodiversity, school of biosciences, cardiff university of wales, uk, for finance supporting and providing the facilities to carry on this research project, respectively. references abbott ws (1925) a method for computing the effectiveness of an insecticide. j econ entomol. 18: 265-267. asher kr, nemny ne (1984) the effect of cme 134 on spodoptera littoralis eggs and larvae. phytoparasitica. 12(1): 13-27. axtell rc (1986) fly management in poultry production: cultural, biological, and chemical. poult sci. 65: 657-667. axtell rc (1999) poultry integrated pest management: status and future. integrated pest manage rev. 4: 53-73. batra cp, mittal pk, adak t, ansari ma (2005) efficacy of igr compound starycide 480 sc (triflumuron) against mosquito larvae in clear and polluted water. j vector borne dis. 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http://jad.tums.ac.ir published online: january 06, 2016 review article a review of impact of bam earthquake on cutaneous leishmaniasis and status: epidemic of old foci, emergence of new foci and changes in features of the disease mohammad reza aflatoonian 1, *iraj sharifi 2, bahnaz aflatoonian 1, mohammad reza shirzadi 3, mohammad mahdi gouya 4, alireza kermanizadeh 5 1research center of tropical and infectious diseases, kerman university of medical sciences, kerman, iran 2leishmaniasis research center, kerman university of medical sciences, kerman, iran 3zoonoses department, center for disease control, ministry of health and medical education, tehran, iran 4center for disease control, ministry of health and medical education, tehran, iran 5cutaneous leishmaniasis control center, bam university of medical sciences, kerman, iran (received 4 jan 2014; accepted 3 feb 2015) abstract background: global findings indicate that incidence rate of cutaneous leishmaniasis (cl) has significantly increased during the past decade, as documented in many countries. this review was aimed to evaluate the trend of cl cases in terms of demographic and clinical characteristics during a decade after the earthquake (2003–2012) compared to the corresponding period before the earthquake in bam (1993–2003). methods: direct smear preparations along with different intrinsic methods were used for detection and identification of the causative agents. results: overall, 20999 cases of cl have occurred during the last 20 years (1993–2012), 6731 cases before and 14268 cases after the earthquake (p< 0.001). conclusions: following a major earthquake, several risk factors could activate epidemics of cutaneous leishmaniasis in old foci and induce emerging foci in new areas. keywords: cutaneous leishmaniasis, disaster, earthquake, iran introduction leishmaniasis is estimated to cause the ninth largest disease burden in terms of morbidity and mortality among infectious diseases and is largely neglected in tropical and sub-tropical countries (who 2009). cutaneous leishmaniasis (cl) represents an important public health concern of considerable magnitude in many parts of the world, especially in africa, indian subcontinent, eastern and southern mediterranean regions and countries of the middle east including iran (who 2010). both types of cl are present in different parts of iran. leishmania major, the causative agent of zoonotic cl (zcl), and l. tropica, the cause of anthroponotic cl (acl), are endemic in various districts of iran (shirzadi and gouya 2012). phlebotomus papatasi and p. sergenti are the principal sand fly vectors, respectively, for the aforementioned cl diseases (nadim and aflatoonian 1995, rassi et al. 2008). they impose an enormous medical and socio-economic impact here, although they are a cause of global concern. cl has the potential to spread within the cities and across borders. acl is prevalent in kerman province, par*corresponding author: dr iraj sharifi, e -mail: iraj.sharifi@yahoo.com j arthropod-borne dis, september 2016, 10(3): 272–281 mr aflatoonian et al.: a review of impact of … 273 http://jad.tums.ac.ir published online: january 06, 2016 ticularly in the city of bam (sharifi et al. 2011a). global findings have shown that incidence rate of cl cases has significantly increased during the past decade (alvar et al. 2012), as documented in iran (shirzadi and gouya 2012) and across the world (reyburn et al. 2003, kolaczinski et al. 2004, who 2010, ready 2011, alvar et al. 2012, ocampo et al. 2012). several contributing factors play an important role in increasing cl disease which include inadequate vector and reservoir control, urbanization, ecological changes, natural disasters, population movement, poor sanitation and garbage disposal system, human behavioral risk factors and resistance to standard drugs (daszk et al. 2001, desjeux 2001, macpherson 2005, croft et al. 2006). all of these changes can lead to favor epidemic, emergence and spread of cl. natural disasters including earthquakes, tsunamis, floods and cyclones can create suitable conditions for increased incidence of infectious diseases and cause epidemics (dye and wolpert 1988, desjeux 2001, bayramgurler et al. 2002, smith 2005, shultz et al. 2005, sharifi et al. 2011b, kouadio et al. 2012, zhang et al. 2013). iran is prone to natural disasters such as earthquake. following natural disasters, particularly after earthquakes, various risk factors in favor of diseases will be created, which in turn provide a suitable condition for breeding the vector and thus transmission of the causative agent. other confounding factors have contributed to the occurrence of new outbreaks and emergence of cl after disasters in several countries (ashford 2000, daszak et al. 2001, patz et al. 2000, fazaeli et al. 2009, fakoorziba et al. 2010). during the period after the earthquake, the population of bam has significantly increased mainly due to various occupational opportunities related to the large-scale construction works and new project development, which caused massive movement of contractors and labor forces (sharifi et al. 2011c). according to the official report, about 60,000 new comers, mostly males and from non-cl endemic regions (non-immune) arrived to the area and consequently resulted in increased incidence rate. the aim of the present review was to evaluate trend of cl cases in terms of demographic and clinical characteristics during 10 years after the earthquake (2003–2012) compared to the corresponding period before the earthquake in bam (1993–2003). furthermore, we wanted to analyze the impact of control measures applied post disaster in bam, which would serve as a model for planning future control strategies and public health measures not only in our country but also in other foci of endemic cl as well as epidemics. materials and methods scope of this study the review of literature was disappointing as there was no comprehensive study reported. we therefore looked at the operational aspects of acl case-management, prevention and control measures that could be undertaken in disaster situation in an endemic focus of acl. in addition, their possible impact on reduction of the cl disease was assessed. therefore, in this review, epidemic of old foci and emergence of new foci in bam district following the earthquake were reviewed. since field trials of single and multiple doses of killed l. major plus bcg vaccine candidate against acl (sharifi et al. 1998a) had been conducted prior to the earthquake (1993–1998), we had very good information about the epidemiology of acl, prior to the earthquake of 2003 which gave us good background information to compare the post-earthquake situation. the trained and knowledgeable team of personnel created for the vaccine studies, were the basis j arthropod-borne dis, september 2016, 10(3): 272–281 mr aflatoonian et al.: a review of impact of … 274 http://jad.tums.ac.ir published online: january 06, 2016 on which a cl clinic (clc) for cutaneous leishmaniasis control was established which is still functioning. therefore, in addition to using existing published studies, registered data on passive and active case-detections, were extracted, analyzed and presented. study area this study was carried out in district of bam, 180 km south to the city of kerman, center of kerman province, southeast of iran (fig. 1). bam is located 1000 m above the sea level along the zagros mountain range. a massive earthquake struck the city of bam and the neighboring areas on 26th december 2003. the affected population was 250,000, 100,000 in urban and 150,000 in rural areas. the magnitude of this earthquake was 6.6 on the richter scale, leaving 30,000 death, 30,000 injured, 60,000 homeless and damaging over 90% of the city's infrastructures including medical and health facilities, causing l0 million tons of mud and rubbles (abolghasemi et al. 2006). special attention was paid to ensure rapid recovery and rehabilitation of public health services. the city was divided to 12 zones by national and provincial health systems to facilitate accessibility of the population to essential health services and strengthen the medical supply chain. simultaneously, a disease surveillance system was promptly established in the affected areas for delivering primary health care (phc), even though the magnitude of destruction was enormous. however, it took two years to reconstruct the health and medical system following the earthquake. approximately 30,000 people from the province of kerman and additional 30,000 individuals from other provinces of the country arrived at the city for various occupational purposes. cutaneous leishmaniasis clinic in bam, cl is a notified disease and its surveillance has long been integrated into the health system. the clinic was officially established in 2005 (two years after the earthquake) and used as a headquarters to coordinate and control various activities involved in the postearthquake era including training personnel, health education, diagnosing and identifying cases, active and passive casefinding, implementing vector control measures, eliminating stray dogs, treating patients and referring nonhealing cases to the provincial health clinic in afzalipour university hospital in kerman, center of kerman province. a national control program was developed by national center for disease control office, zoonoses department, and several medical universities of ministry of health (moh). case definition and identification skin scraping tissues were obtained from the periphery of lesions using a scalpel and sterile blade. the clinical materials were used for direct smear preparation and microscopic examination, culture and further identification by immunological methods, pcr or characterization by isoenzyme (sharifi et al. 1997, sharifi et al. 1998b, sharifi et al. 2011a). personal protection measures dressing of lesions and stick insect repellents such as diethyl toluamide (deet) to skin or clothing to reduce man-vector contact was applied. face-to-face health education program of the population at risk and retraining of health personnel were in high priority. a large-scale deltamethrin-impregnated screens and curtains were assessed in high-risk areas in the city of bam (aflatoonian and sharifi 2011, noazin et al. 2013). environmental management the activities included improving housing conditions, sanitation of temporary camps, land clearance around dwellings and insecticide spraying, particularly in high-risk areas, to eliminate potential sand fly breeding sites. about 10 million tons of mud and rubbles were removed. j arthropod-borne dis, september 2016, 10(3): 272–281 mr aflatoonian et al.: a review of impact of … 275 http://jad.tums.ac.ir published online: january 06, 2016 vector incrimination so far, four main studies have been carried out on distribution of vectors and their implication in transmission of acl in bam before (nadim et al. 1995) and after the earthquake (aghasi and sharifi 2003, aghaei afshar et al. 2011, aghaei afshar et al. 2013). treatment of patients pantavalent antimonials are the first choice medicines for treating all forms of leishmaniasis in global terms. in bam, meglumine antimoniate (glucantime) was given intramuscularly (im), intralesionally or sometimes intravenously (iv). combination therapy between meglumine antimoniate and cryotherapy (liquid nitrogen) or along with the second line drugs (allopurinol) was used mainly for treating acl resistant forms (esfandiarpour and dabiri 2007, sharifi et al. 2010). results epidemic of anthroponotic cutaneous leishmaniasis in the city of bam all the cases (20540 patients) were identified by direct smear preparation method and overall 150 random cases by intrinsic techniques including 14 cases by immunological and 9 patients by biochemical methods before the earthquake and 253 cases by molecular methods (conventional or nestedpcr) after the earthquake. various methods revealed that all species (100%) were l. tropica after the earthquake, except for the cases identified before the earthquake which were predominantly l. tropica (90%) and was at low frequency (≤10%) l. major. acl due to l. tropica has been endemic in city of bam since ancient times (nadim and aflatoonian 1995). according to the data gathered in the last 20 years (1993–2012), 20999 cases of acl have occurred, 6731 cases before and 14268 cases after the earthquake (p< 0.001) during a similar time (fig. 2). over the same time, acl incidence has steadily risen from 1.2% in 1993 to 2.5 % in the peak epidemic in 2006. there was a significant excess of incidence rate (p< 0.001) among the cases during the two times before and after the earthquake (fig. 2). most of the cases occurred in females (53.4%) and then in males (46.6%) 10 years before the earthquake. in contrast, proportion of females (43.7%) to males (56.3%) was reversing (fig. 2) 10 years after the earthquake (p< 0.001). the majority of cl cases was recorded in individuals of <20 years old 10 years before the earthquake (mean age, 17 years), whereas this proportion changed in the corresponding period after the earthquake (fig. 3) and there was a gradual shift of infection toward older individuals (mean age of 25 years). anatomical distributions of lesions were considerably different during these two time periods, face (60%) was the most site of involvement in the 10 years before the earthquake while location of involvement significantly changed to hands (59%) in the years after the earthquake (p< 0.001). severity of diseases in terms of the number of lesions significantly changed before and after the earthquake (p< 0.05) (sharifi et al. 2014). duration of the cl disease sharply decreased from 10.8 months to 6.1 months (p< 0.001) in the years after the earthquake. majority of the cases originally came from four old zones before the earthquake, whereas in the period after the earthquake, the cases were distributed throughout the 12 zones within the city of bam (fig. 1). epidemic of anthroponotic cutaneous leishmaniasis in adjacent districts a newly established focus of acl with low endemicity occurred following the earthquake in the rural areas of mohammadabad county, jiroft district, adjacent to dehbakri county, bordering bam district (poursmaelian et al. 2010). originally, seven cases sporadically emerged between 1997 and 2002 prior j arthropod-borne dis, september 2016, 10(3): 272–281 mr aflatoonian et al.: a review of impact of … 276 http://jad.tums.ac.ir published online: january 06, 2016 to the earthquake. the cases sharply increased to 181 patients after 2003 to peak in 2006 and become endemic, thereafter. the overall prevalence rate was 5.3%, 6.2% in females and 4.5% in males, resulting in a significant gender difference (p< 0.05). most of the lesions were single and on the face (47%) followed by hands and legs (39%). nested-pcr showed l. tropica, the only causative parasite. emergence of anthroponotic cutaneous leishmaniasis following the earthquake southern villages of bam a newly emerging focus of acl was identified in southern villages (nazamshahr county) of bam district (aflatoonian et al. 2013). overall, 1.2% of the inhabitants were infected (0.5% active and 0.7% scars) and females were more significantly infected (1.7%) than males (0.8%) (p= 0.003). all the age groups were equally affected. most of the lesions were on the face and majority of them had single lesion. most of the cases appeared from 2006 to 2008 during the cl epidemic in the city of bam. pcr indicated l. tropica as the causative agent. northwestern villages of bam another epidemic of acl due to l. tropica occurred in rural areas of dehbakri county, north west of bam district (sharifi et al. 2011c). again, all the age groups were affected although patients ≤10 years of age showed the highest rate of infection (p= 0.0001). the overall prevalence rate was 5.3%, 6.3% in females and 4.3% in males. out of 204 cases, 1.8% had active sores and 3.5% showed scars with a significant difference between the genders (p= 0.005). of the lesions, 47% were on the face and 77.9% had one lesion. the incidence gradually increased 2004– 2005 but grew exponentially 2006–2008. vector susceptibility test baseline susceptibility tests were carried out on field-collected strains of p. papatasi and p. sergenti and tested using who impregnated papers with ddt 4% and deltamethrin 0.05% in the endemic focus of disease in dehbakri county during 2010. lt50 values of ddt4% and deltamethrin 0.05% against p. papatasi were 20.6 and 13.6 min, respectively. the same data for p. sergenti ranged between 21.8 and 17.7 min (aghaei afshar et al. 2011). the impact and outcome indicators following residual spraying with deltamethrin were assessed in a new focus of cl in dahbakri (bam). incidence of the disease was significantly decreased (3.7 vs. 8.5). there was a significant difference between the treatment and control areas on blood fed and gravity of sand flies. the results clearly indicated that indoor residual spraying caused a significant decline of density, blood fed and gravity of vectors, resulting in a sharp reduction of disease incidence (aghaei afshar et al. 2013). cost of treatment and prevention in a descriptive cross-sectional survey, data about treatment and prevention costs of 5320 cl patients during the two years of epidemic (2006 and 2007) were analyzed (aflatoonian et al. 2009). the per capita treatment cost was estimated to be us$ 48, which showed a significant difference in terms of cost effectiveness as compared with the patients in other cl endemic countries. it seems that the implementation of preventive measures had a negligible effect on the acl control strategy (aflatoonian et al. 2010). the authors concluded that establishment of clc in bam reduced the disease costs and provided an efficient, free and easy accessible service for the patients after the earthquake. this experience could be a suitable model to be used in a similar disaster situation in other parts of the world. j arthropod-borne dis, september 2016, 10(3): 272–281 mr aflatoonian et al.: a review of impact of … 277 http://jad.tums.ac.ir published online: january 06, 2016 fig. 1. distribution of cutaneous leishmaniasis before (a) and after (b) the earthquake in bam district fig. 2. distribution and incidence of anthroponotic cutaneous leishmaniosis cases in bam before and after the earthquake by year (1993–2012) fig. 3. mean age distribution of anthroponotic cutaneous leishmaniosis cases in bam before and after the earthquake by year (1993–2012) discussion global data indicate that cl is spreading and extending its range to new foci, especially after major natural and anthropogenic environmental changes (daszak et al. 2001, alvar et al. 2012). such changes can activate various risk factors and lead to alterations in the density and range of vector and reservoir population, thereby enhancing accessibility of humans to infected sand fly bites (patz et al. 2000, desjeux 2001). the review of data indicate that majority of the cl cases occurred in male individuals of higher ages in the period after the earthquake. the reason was mainly due to arrival of sensitive male labor forces to the city of bam for various positions, which was created after the earthquake (sharifi et al. 2011b). similar risk factors have contributed major roles to the emerging epidemics of cl or other infectious diseases in iran and abroad (dye and wolpert 1988, bayramgurler et al. 2002, shultz et al. 2005, smith 2005, fazaeli 2009, fakoorziba et al. 2010, kouadio et al. 2012, zhang et al. 2013). despite implementation of comprehensive control measures acl occurred in several localities within the district of bam. however, j arthropod-borne dis, september 2016, 10(3): 272–281 mr aflatoonian et al.: a review of impact of … 278 http://jad.tums.ac.ir published online: january 06, 2016 such control approaches have not adequately been evaluated. although, large impregnated curtains and screens were assessed in several places within the city of bam. significant effectiveness of these control measures was observed. in addition, the impact and outcome indicators following application of residual spraying with deltamethrin were evaluated in a new emerging focus of acl in dahbakri county, north-west of bam district. the incidence rate was significantly decreased (aghaei afshar et al. 2013). most of the cases occurred in nonindigenous individuals who arrived to the affected areas from neighboring districts and various provinces of the country, after the earthquake. majority of whom were nonimmune labors and contractors who traveled from noncl endemic areas to the city of bam where cl infection was endemic (fig. 1). the incidence of cl was in order of 3fold in nonlocal population during the peak epidemic (2006) compared to the indigenous residents (aflatoonian and sharifi 2010). while the incidence of disease was relatively stable in local residents, it rose gradually from 2005 to 2007 in the city of bam (sharifi et al. 2011b). conclusions the findings presented herein strongly suggested that, following the earthquake, major risk factors were created which were associated with epidemics and emergences of cl in bam district and adjacent areas. such precipitating determinants included suitable environmental conditions, poor hygienic conditions, individual and behavioral changes and arrival of non-immune population. these factors could activate epidemics of old foci and induce new emerging foci. epidemiological and clinical features of the disease in terms of gender, age, location, severity and duration of the lesions significantly changed after the earthquake. proper surveillance, followed by prompt diagnosis and subsequent treatment, should be extended to all areas with high risk in order to prevent and control epidemics and/ or emergences of acl, particularly in endemic foci after a major natural disaster. acknowledgements we thank all the partners and authors who contributed information and added to the scientific merits of this work. we would also like to thank the personnel in bam health system, cutaneous leishmaniasis clinic and kerman leishmaniasis research center for their extensive work during the last two decades. the authors declare that there is no conflict of interests. references abolghasemi h, radfar mh, khatami m, saghafi nia m, amid a, briggs s (2006) international medical response to a natural disaster: lessons learned from the bam earthquake experience. prehosp disaster med. 21: 141–147. aflatoonian mr, sharifi i, fekri ar (2009) evaluation of the cost-effectiveness of cutaneous leishmaniasis treatment after the earthquake. j kerman univ med sci. 4: 365–373. aflatoonian mr, sharifi i, abbasi r, ranjbar l (2010) to evaluate the cost of prevention on 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(doi 10.1007/s12639-014-0435-5). shirzadi mr, gouya mm (2012) national guidelines for cutaneous leishmaniasis surveillance in iran. ministry of health and medical education, zoonoses control department, tehran iran. shultz jm, russell j, espinel z (2005) epidemiology of tropical cyclones: the dynamics of disaster, disease, and development. epidemiol rev. 27: 21–35. smith aw (2005) tsunami in south asia: what is the risk of postdisaster infectious disease outbreaks. ann acad med singapore. 34: 625–631. world health organization (2009) leishmaniasis: background information. a brief history of the disease. available at: www.who.int /leishmaniasis/en. world health organization (2010) control of the leishmaniasis. report of a meeting of the who export committee on the control of leishmaniasis, who technical report series 949, geneva. zhang s, lu z, liu h, xindong xiao, zongguo zhao, genshu bao, jian han, tao jing, gen chen (2013) incidence of japanese encephalitis visceral leishmaniasis and malaria before and after the wechuan earthquake, in china. acta trop. 1: 85–89. j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 106 http://jad.tums.ac.ir published online: march 31, 2020 original article determination of species composition of mosquitoes in lahore, pakistan *farkhanda manzoor1; robeela shabbir2; madiha sana2; sumbal nazir2; muhammad aslam khan3 1lahore college for women university, lahore college for women university, jail road, lahore, pakistan 2department of zoology, lahore college for women university, lahore, pakistan 3university of health sciences lahore, lahore, pakistan (received 05 apr 2017; accepted 25 mar 2020) abstract background: present study was conducted to determine species composition of mosquitoes (larvae, pupae and adults) collected from ten different towns of lahore from september 2014 to august 2015. methods: mosquito larvae, pupae and adults (male and female) were collected by using dippers and aspirators from september 2014 to august 2015 in different sites of lahore comprising of ten towns i.e. iqbal, aziz bhatti, data ganj baksh, gulberg, nishtar, ravi, samanabad, shalimar, wagah, and lahore cantonment. mosquito larvae and adults were identified by standard entomological keys. diversity, richness and rarity of mosquito fauna were analyzed by the shannon, simpson and margalef indices respectively. results: in this study, a total of 8656 mosquitoes belonging to four genera namely anopheles, culex, aedes and mansonia were identified. among fifteen species collected, cx. quinquefasciatus was the most abundant species in the city having 25.8% relative abundance. however an. culicifacies s.l. (sensu lato) was reported as the least abundant species with 0.22% relative abundance. the highest diversity of mosquitoes was shown in the month of august (h= 2.25) while the lowest diversity was recorded june (h= 1.43). extensive sewage water supported the maximum abundance of cx. quinquifasciatus in urban areas of this city. conclusions: this study has significantly elaborated the monthly varying species composition of mosquito fauna of this city. hence this research will help us to find out the control strategies of mosquito borne diseases in this region. keywords: mosquito fauna; lahore; shannon index; simpson index; climatic factors introduction mosquito serves as a vector of various deadly diseases including malaria, dengue fever, zika fever and chikungunya. the vector causes deaths of more than one million people annually (1). worldwide there are about 3541 species of mosquitoes reported belonging to 42 different genera (2). however there are 134 species of mosquitoes found in pakistan belonging to anophelinae and culicinae subfamilies (3). mosquitoes play an important role in food chains of various ecosystems as well (4). pakistan is one of those subtropical countries that have been under threat of vector borne diseases. like other asian countries, pakistan is also withstanding substantial climatic changes that are sufficient to harbor the outbreaks of mosquito borne diseases (5). lahore, an urbanized city of pakistan, was reported to have 11,283 cases of dengue fever in the epidemics of 2012 (6). recent dengue fever outbreaks in khyber pakhtunkhwa resulted in 2179 positive cases (7). the shift in the climate of south asian countries tends to escalate the incidence of mosquito borne diseases (8). moreover, the abiotic factors like temperature, humidity and rainfall are positively supporting the population of mosquitoes with increased sustainability of their breeding sites and long survivorship (9, 10). likewise, certain studies have been conducted in various regions of pakistan reporting the mosquito fauna that have related the relative abundance of different species with the gradual shift in climatic trend (11–15). the relationship between the biodiversity of mosquitoes in la*corresponding author: prof farkhanda manzoor, e-mail: drfarkhanda786@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 107 http://jad.tums.ac.ir published online: march 31, 2020 hore with its climatic conditions was studied in 2013. the reported species were aedes aegypti, culex quinquefasciatus, anopheles stephensi and anopheles subpictus s.l.. the population dynamics of these mosquito species was strongly correlated with seasonal temperature variations of the region (15). the rapid urbanization of this city with extensive road construction projects and elimination of open fields is a reason to provide the flourishing breeding sites for disease transmitting mosquito species. hence, it is so crucial to monitor the abundance of various mosquito species for maintaining vector control programs in this region. the objective of current study was to describe the abundance, diversity and rarity of different species of mosquitoes in different habitat types. morever this study would also find a link between environmental variables and the relative abundance of different species of mosquitoes collected. materials and methods lahore having a semi-arid climate and typical monsoon season was selected as the area of study. the geological coordinates of lahore are 31˚ 32̍ north and 74˚ 20̍ east respectively and is about 223 meter high from sea level. the selected towns of this city were iqbal town, aziz bhatti town, data ganj baksh town, gulberg town, nishtar town, ravi town, samanabad town, shalimar town, wagah town and lahore cantonment as shown in the lahore administrative towns boundary (fig. 1). sampling of larvae, pupae and adult mosquitoes mosquito samples were collected from fixed spots of ten towns four times in a month from september 2014 to august 2015. mosquito larvae and pupae were collected by using strainers and dippers (16) from fresh as well as polluted water bodies of all towns (17). simultaneously, indoor and outdoor collection was done for adult mosquitoes by using sweeper and aspirators (14). open drains, freshwater reservoirs, discarded containers, flower vases, sewage water, tyres and tree-holes were keenly observed during sampling of larvae, pupae and adult mosquitoes. the collected mosquitoes were transferred to plastic jars covered with net for further experimentation. preservation and identification of the mosquitoes the adult mosquitoes were sucked through aspirator from different plastic jars and were then kept in airtight conical jars containing chloroform dipped cotton swab that helped in killing the adult mosquitoes in two to four minutes. these killed mosquitoes were shifted in test tubes with a cork containing small amount of silica gel for preservation of the mosquitoes. the collected larvae and pupae were identified under the dissecting microscope. the mosquito larvae, pupae and killed adults were later identified using standard taxonomic keys given in “the fauna of british india including ceylon and burma, diptera family culicidae, tribe anopheline, vol. iv and tribe megarhinini and culicini, vol. v” (18, 19). data analysis relative abundance was determined by using following formula. where ‘n’ is the number of mosquito, and it is divided by the total number of mosquitoes collected and then it is multiplied by 100 (20). the mosquito species were termed as dominant species with more than 10% relative abundance and subdominant species with 3 to 10% relative abundance while mosquitoes species having lesser than 3% relative abundance were tagged satellite species (21). the distribution status of collected mosquito species in different collection sites was calculated by the formula as given (21), distribution (c)= n x100/ n where n is number of sites where species was http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 108 http://jad.tums.ac.ir published online: march 31, 2020 found, and n is the total number of sites. the distribution status of different species was categorized as constant species (80–100%), frequent species (60–80%), moderate species (40–60%) and infrequent species (20–40%). mosquito species richness was calculated by using margalef’s index of richness (dmg) dmg= (s-1)/ in n. where, s is total number of species and n is total number of individuals. shannon-weaver index was also applied to find out the diversity of mosquitoes with respect to monthly variation (22). h= -∑pilnpi where h= shannon diversity index and pi= ni/n where ni= importance value index of the species and n= importance value index of all species. simpson index (d) was also applied to find out the rarity of mosquito species (23) collected. it was calculated by using formula, d= ∑n(n-1)\n(n-1) whereas, n= no. of mosquitoes of individual species, and n= no. of total mosquitoes species collected. climate analysis annual reports of climate data from september 2014 to august 2015 were retrieved from punjab metrological department for the analysis of temperature, humidity and rainfall. the collected data were further processed by plotting different graphs on ms excel. moreover, poisson regression analysis was also performed on spss 16.0 to develop a correlation between the frequency of mosquitoes collected and changing weather variables. results mosquito species were recovered from the different types of particular habitat such as open drains, fresh water, discarded containers, flower vases, sewage water, tyres and tree holes (table 1). anopheles species and mansonia species was specifically found to be fresh water. culex species were collected from their breeding sites of fresh water, discarded containers and sewage water. aedes species were collected from different sites except sewage water, all of its breeding sites were predominately fresh water in nature. relative abundance of fifteen different species of mosquitoes throughout lahore with their abundance status and distribution class was shown in table 2. culex quinquefasciatus was the most abundant species with 25.80% relative abundance. in contrast an. culicifacies was appeared to be the least in number with 0.22% relative abundance. culex sitiens and cx. tritaeniorhynchus were reported as dominant species during collection. moreover, an. pulcherrimus, cx. vagans, cx. theileri, cx. vishnui, ae. albopictus and ae. aegypti were found sub dominant species. however, an. annularis, an. nigerrimus, an. subpictus s.l., an. stephensi, and an. culicifacies were recovered as satellite species having least abundance and sporadic distribution. mansonia uniformis was found to be infrequent species in its distribution. in this study, total of 8656 mosquitoes were collected. taxonomic identification revealed fifteen species of mosquitoes such as an. annularis, an. pulcherrimus, an. stephensi, an. nigerrimus, an. subpictus s.l., an. culicifacies, cx. vagans, cx. quinquefasciatus, cx. sitiens, cx. tritaeniorhynchus, cx. vishnui, cx. theileri, ae. aegypti, ae. albopictus and ma. uniformis. an overview of month wise abundance of all mosquito species collected in one year of study was shown in table 3. the highest mosquito sample size was shown in the month of september (dmg= 4.17) while the lowest sample size was shown in june (dmg= 1.63). on the basis of shannon index, the highest diversity was shown in august (h= 2.24) while the lowest diversity was shown in june (h= 1.43). due to the highest diversity of mosquitoes in august, less number of rare species of mosquitoes (d= 0.11) was present. the lowest diversity in june showed the highest http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 109 http://jad.tums.ac.ir published online: march 31, 2020 number of rare species (d= 0.25) in this month (table 4). the relationship of seasonal conditions recorded in every month with the abundance of mosquitoes collected was shown in (figs. 2–4). table 5 enlisted the results of poisson regression analysis showing the correlation between various climatic variables and expected number of mosquitoes to be collected. table 1. different breeding habitats of mosquitoes in ten different towns of lahore, pakistan, september 2014 to august 2015 mosquito open drains fresh water discarded containers flower vases sewage water tyres tree holes an. annularis + an. pulcherrimus + an. subpictus s.l. + an. nigerrimus + an. stephensi + an. culicifacies s.l. + cx. quinquefasciatus + + + cx. tritaeniorhynchus + + + cx. vagans + + + cx. vishnui + + cx. theileri + + + cx. sitiens + + + ae. aegypti + + + + + + ae. albopictus + + + + + + ma. uniformis + table 2. relative abundance, status and distribution class of mosquitoes collected from all the ten towns of lahore, pakistan, september 2014 to august 2015 species abundance relative abundance relative abundance status distribution class an. annularis 217 2.51 satellite sporadic an. pulcherrimus 308 3.56 satellite sporadic an. subpictus s.l. 166 1.92 satellite sporadic an. nigerrimus 150 1.73 satellite sporadic an. stephensi 51 0.59 satellite sporadic an. culicifacies s.l. 19 0.22 satellite sporadic cx. sitiens 1029 11.89 dominant sporadic cx. quinquefasciatus 2233 25.80 most dominant moderate cx. tritaeniorhynchus 868 10.03 dominant moderate cx. vagans 795 9.18 subdominant moderate cx. vishnui 141 1.63 satellite moderate cx. theileri 810 9.36 subdominant moderate ae. albopictus 962 11.11 dominant frequent ae. aegypti 730 8.43 subdominant frequent ma. uniformis 177 2.04 satellite infrequent http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 110 http://jad.tums.ac.ir published online: march 31, 2020 table 3. richness and abundance of mosquito population in lahore, pakistan, september 2014 to august 2015 month a n . a n n u la ri s a n . p u lc h e rr im u s a n . su b p ic tu s s. l. a n . n ig e rr im u s a n . st e p h e n si a n . c u li c if a c ie s s. l. c x . s it ie n s c x . q u in q u e fa sc ia tu s c x . tr it a e -n io rh y n c h u s c x . v a g a n s c x . v is h n u i c x . th e il e ri a e . a lb o p ic tu s a e . a e g y p ti m a . u n if o rm is t o ta l sep-2014 90 130 75 68 23 10 209 593 197 168 33 179 255 194 52 2276 oct-2014 37 44 24 21 3 0 64 147 43 33 0 0 4 0 0 420 nov-2014 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 dec-2014 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 jan-2015 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 feb-2015 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 mar-2015 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 apr-2015 0 0 0 0 0 0 167 314 141 152 28 162 138 108 34 1244 may-2015 0 0 0 0 0 0 160 341 123 131 23 165 146 122 25 1236 jun-2015 0 0 0 0 0 0 40 100 85 26 0 35 0 0 0 286 jul-2015 0 0 0 0 0 0 154 239 104 107 18 110 180 130 30 1072 aug-2015 90 134 67 61 25 9 235 499 175 178 39 159 239 176 36 2122 total 217 308 166 150 51 19 1029 2233 868 795 141 810 962 730 177 8656 table 4. monthly analysis of species composition of mosquitoes collected from different towns of lahore, pakistan, september 2014 to august 2015 months abundance margalef’s index (dmg) shannon index (h) simpson index (d) september 2276 4.17 2.15 0.12 october 420 3.43 1.82 0.18 november 0 0 0 0 december 0 0 0 0 january 0 0 0 0 february 0 0 0 0 march 0 0 0 0 april 1244 2.58 1.93 0.15 may 1236 2.59 1.91 0.16 june 286 1.63 1.43 0.25 july 1072 2.64 1.92 0.14 august 2122 2.40 2.24 0.11 table 5. parameter estimates of poisson regression analysis showing correlation between weather variables and expected mosquito frequency from different towns of lahore, pakistan, september 2014 to august 2015 estimate standard errors exp (b) pvalue intercept 2.038 .0068 7.677 .000 temperature -0.012 .0020 0.989 .000 humidity 0.010 .0021 1 .000 rainfall 0.021 .0009 1.021 .000 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 111 http://jad.tums.ac.ir published online: march 31, 2020 fig. 1. location of study sites in towns of urban city lahore, pakistan, september 2014 to august 2015 fig. 2. relationship between temperature and the total number of mosquitoes collected monthly in lahore, pakistan, september 2014 to august 2015 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 112 http://jad.tums.ac.ir published online: march 31, 2020 fig. 3. relationship between the average humidity and the total number of mosquitoes collected monthly in lahore, pakistan, september 2014 to august 2015 fig. 4. relationship between rainfall and the total number of mosquitoes collected monthly in lahore, pakistan, september 2014 to august 2015 discussion fifteen species of mosquitoes belonging to four genera anopheles, aedes, culex and mansonia were recovered from the urban city lahore during the period of september 2014 to august 2015. firstly there were twenty nine species of mosquitoes ever reported in lahore, pakistan (24). in 1971, the mosquito fauna of this city was highly enriched with the identification of 31 different species belonging to seven genera n o . o f c o ll e c te d m o sq u it o e s . a v e r a g e r a in fa ll ( m .m ) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 113 http://jad.tums.ac.ir published online: march 31, 2020 (7). however, current study exhibited reduction in the number of collected mosquito species. this decline in reported mosquito species of this city during last forty years is due to drastic change in environmental conditions, expanding sewage system and due to diminishing freshwater habitats. mosquito collection performed in this study by dippers and aspirator mainly focused on indoor and outdoor breeding sites. however by employing other methods such as, ovitrap, cdc light traps, bg traps, human and animal baits, the species composition of the region can modulate accordingly. culex quinquefasciatus was the most abundant species found in all the towns with the highest diversity. same species was also reported as the most dominant species of pakistan in a study analyzing the mosquito diversity based on barcoding (5). this species breeds in polluted water therefore it is found in sewage canal which is an air conditioned breeding place for this mosquito. mukhtar et al. (2003) investigated the breeding of mosquitoes in waste water irrigation where he found three genera aedes, culex and anopheles. the present study discovered nearly all the species such as an. pulcherrimus, an. culicifacies s.l., an. subpictus s.l., an. stephensi, cx. quinquefasciatus and cx. tritaeniorhynchus which were previously collected from wastewater (25). ali and rashid (26) studied polluted water of palosai stream near peshawar. they identified cx. quinquefasciatus and an. stephensi while aedes was missing. this shows that aedes species are the inhabitants of temporary habitats only. by using different traps and trapping techniques twelve different mosquito species were collected in lahore in 2013 (15). in our study an. subpictus s.l., an. culicifacies s.l., and ma. uniformis were additionally reported. anopheles culicifacies s.l. and an. stephensi are fresh water breeders. they have almost disappeared from main city and are recovered from suburb areas getting more susceptible to diseases like malaria. anopheles annularis and an. nigerrimus are usually found in clean and still water. the species were recovered from all the ten towns of lahore. both of the species are considered as vector of malaria in india, nepal and srilanka (27). the risk of transmission of malaria by these species is quite low, because in pakistan there is no evidence of their vectorial capacity. however further studies can be conducted to investigate these species as suspected vectors of malaria in this region. the disappearance of fresh water in this city has lowered the relative abundance of fresh water dwelling species like an. pulcherrimus and an. subpictus s.l. thus enlisting them as sporadic in distribution. aedes species were largely taken from temporary habitats over the entire city in suitable breeding sites of fresh water like water tanks, broken pots, pans and old tyres that explain the vulnerability of dengue epidemics in lahore. in our study, the recorded optimum temperature, 25 ˚c, optimum humidity, 80% and 15mm rainfall in the months having maximum number of species built the positive correlation between the climatic conditions and abundance of mosquitoes collected. the poisson regression analysis in table 5 proposed that for every unit increase in temperature the expected mosquito frequency increased by e-0.012= 0.988. likewise, for every unit increase in rainfall and humidity, collected mosquito number would be increased by e0.21= 1.233 and e0.01= 1.01 respectively. the climatic analysis of this study showed the most suitable environmental conditions like temperature, humidity and rainfall in the month of august and september that is referred as the typical monsoon period in pakistan. along with suitable climatic variables for survival, this season also provides extensive breeding sites for different species of mosquitoes. the results of this study also exhibited fresh water sources, discarded containers and open drains as more productive breeding sites for dwelling mosquito species and all of these sites are more functioning in the months of monsoon. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 114 http://jad.tums.ac.ir published online: march 31, 2020 the highest mosquito diversity (h= 2.24) was reported in august due to the availability of high rainfall and suitable humidity. this ultimately enhances the dissemination of vector borne diseases. due to the highest diversity of mosquitoes in august, the least number of rare species were recorded. likewise, the highest species richness was found in september (dmg= 4.17). in these months, high temperature in water also boosts larval development making the survival of mosquitoes more feasible (28). conclusions from the findings of this study and previous studies it can be concluded that a worldwide climatic shift along extensive urbanization is influencing the distribution as well as the diversity of mosquito fauna of this cosmopolitan city. hence, there is always a potential risk of the outbreak of the mosquito borne diseases. the results of this research provide significant help to plan effective control strategies against mosquito borne diseases before time. moreover, it is further suggested to plan similar studies by employing other methods of mosquito collection to assess current species composition in lahore, pakistan. acknowledements we thank the staff of department of zoology, lahore college for women university for providing facilities and support to conduct field work. moreover, we pay our gratitutde to mr saeed ahmad for his help during the field trips and collecting the specimens. the authors declare that there is no conflict of interests. references 1. world health organization (2009) dengue: guidelines for diagnosis, treatment, prevention and control. available at: https://www.who.int/neglected_diseases /resources/9789241547871/en/ 2. harrison ba (2014) field identification of adult or larval mosquitoes. available at: http://www.gamosquito.org/resources/fg uideid.pdf 3. aslamkhan m (1971) the mosquitoes of pakistan i. mosq syst newsletter. 3 (14): 147–159. 4. rueda lm (2008) global diversity of mosquitoes (insecta: diptera: culicidae) in fresh water. hydrobiologia. 595: 477– 487. 5. ashfaq m, hebert pdn, mirza jh, khan am, zafar y, mirza ms (2014) analyzing mosquito (diptera: culicidae) diversity in pakistan by dna barcoding. plos one. 9(5): e97268. 6. ali z, zahra g, ali h, khan bn, bibi f, khan ma (2015) prevalence of dengue fever during 2011–2012. j anim plant sci. 25(3 supp. 2): 348–354. 7. world health organization (2017) weekly epidemiological monitor. available at: http://www.who.int. 8. ramasay r, sunderan sn (2012) global climate change and its potential impact on disease transmission by salinity-tolerant mosquito vectors in coastal zones. front physiol. 3: 198–210. 9. sutherst rw (2004) global change and human vulnerability to vector borne diseases. clin micrbiol rev. 17: 136–173. 10. reiter p (2001) climate change and mosquito-borne disease. environ health perspect. 109(1): 141–161. 11. gadahi j, bhutto ib, arijo ag, akhtar n, laghari z, memon mr (2012) population diversity of mosquito fauna in and around tandojam pakistan. int j agro vet med sci. 6(4): 290–295. 12. qasim m, naeem m, bodlah i (2014) mosquito (diptera: culicidae) of murree hills, punjab, pakistan. pak j zool. 46(2): 523– 529. http://jad.tums.ac.ir/ https://www.who.int/neglected_diseases/resources/9789241547871/en/ https://www.who.int/neglected_diseases/resources/9789241547871/en/ http://www.gamosquito.org/resources/fguideid.pdf http://www.gamosquito.org/resources/fguideid.pdf http://www.scopemed.org/?jid=25&iid=2012-6-4.000 j arthropod-borne dis, march 2020, 14(1): 106–115 f manzoor et al.: determination of … 115 http://jad.tums.ac.ir published online: march 31, 2020 13. naeem-ullah u, akram w, suhail a, rana sa (2010) grouping of different mosquito species on the bases of larval habitat, pakistan. j agri sci. 47(2): 124–131. 14. ali n, rasheed sb (2009) determination of species composition of mosquitoes found in palosai stream, peshawar. pak entomol. 31(1): 47–51. 15. manzoor f, nasir a, fazal a (2013) population dynamics of different mosquito species at lahore college for women university, lahore, pakistan. j mosq res. 3: 12–20. 16. aziz at, dieng h, ahmad ah, mahyoub ja, turkistani am, mesed h (2012) household survey of container-breeding mosquitoes and climatic factors influencing the prevalence of aedes aegypti (diptera: culicidae) in makkah city saudi arabia. asian pac j trop biomed. 2(11): 849–857. 17. yee da, allgood d, kneitel jm, kuehn ka (2012) constitutive differences between natural and artificial container mosquito habitats vector communities, resources, microorganisms, and habitat parameters. j med entomol. 49(3): 482– 491. 18. christophers sr (1933) the fauna of british india including ceylon and burma, diptera family culicidae, tribe anopheline, vol. 4. taylor and francis, london, p. 384. 19. barraud pj (1934) the fauna of british india including ceylon and burma, diptera family culcidae, tribe megarhinini and culicini, vol. 5. taylor and francis, london, p. 384. 20. macarthur r (1960) on the relative abundance of species. am nat. 94: 25–36. 21. sengil az, akkaya h gonenc d (2011) species composition and monthly distribution of mosquito (culcidae) larvae in the metropolitan area turkey. int j biol med res. 2(1): 415–424. 22. shannon ce, weaver w (1963) the mathematical theory of communication. university of illinois press, urbana. p. 131. 23. simpson eh (1949) measurement of diversity. nature. 163: 688–689. 24. khan ma, salman c (1969) the bionomics of the mosquitoes of the changa manga national forest, west pakistan. pakistan j zool. 1: 183–205. 25. mukhtar m, herrel n, amerasinghe f, ensink j, hoek w, konradsen f (2003) role of waste water irrigation in mosquito breeding in south punjab, pakistan. southeast asian j trop med public health. 34(1): 72–80. 26. ali n, rasheed sb (2009) determination of species composition of mosquitoes found in palosai stream, peshawar. pak entomol. 31(1): 47–51. 27. service wm, towson h (2002) the anopheles vector, in: arnold (eds): essential malariology, vol 2. london, uk, pp. 59–84. 28. world health organization (2013) world malaria report, geneva. available at: https://www.who.int/malaria/publicatio ns/world_malaria_report_2013/en/ http://jad.tums.ac.ir/ mailto:doc_farkhanda@yahoo.com https://www.who.int/malaria/publications/world_malaria_report_2013/en/ https://www.who.int/malaria/publications/world_malaria_report_2013/en/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 251 http://jad.tums.ac.ir published online: sep 30, 2022 original article epidemiological characterizations, new localities, and a checklist of the known scorpions in the kurdistan region, northern iraq fenik hussen1, *hamid kachel2, gzng hama3, edi kachal2, masoud slo2, ihsan hiwil2, ayoub ahmed4 1department of biology, college of science, salahaddin university, erbil, iraq 2department of general science, college of basic education, university of zakho, zakho, iraq 3department of biology, faculty of science, university of sulaymaniyah, sulaymaniyah, iraq 4department of plant protection, khabat technical institute, erbil polytechnic university, erbil, iraq *corresponding author: dr hamid s kachel, e-mail: hamid.kachel@uoz.edu.krd (received 15 nov 2021; accepted 13 aug 2022) abstract background: annually, thousands of scorpion stings and rare mortalities are recorded in the kurdistan region of iraq. in the present study, we prepared a checklist for the geographical distribution of scorpion fauna composition, new localities data, and scorpion sting profile for the years 2019–2020 in the region. methods: random field surveys for scorpion collection were carried out in 22 localities in the kurdistan region from june 2020 to october 2021. clinical data related to scorpion stings in 2019 and 2020 were obtained from the medical records of public health offices in the region. results: in this survey, 166 specimens were collected. they were composed of seven scorpion species belonging to three families including: buthidae, hemiscorpiidae, and scorpionidae. the collected species were androctonus crassicauda, compsobuthus matthiesseni, hottentotta saulcyi, mesobuthus mesopotamicus, orthochirus fomichevi, hemiscorpius lepturus, and scorpio kruglovi. moreover, three scorpion species comprising buthacus tadmorensis, hottentotta mesopotamicus, and calchas anlasi were recorded in the previous study conducted in the kurdistan region. in 2019–2020, 3726 scorpion stings were reported, with a peak in the summer season. most of the patients were 15–49 years old. in the current study, medically significant scorpion species have been identified. conclusion: the outcome of the present work will increase the awareness of the kurdish community regarding the distribution of dangerous scorpion species and consequently reduce their stings. keywords: scorpion; scorpion sting; distribution; scorpionism; iraq introduction the kurdistan region is located in northern iraq and has a population of around six million people. the total area of the region is approximately 40,643km². the region covers erbil, duhok, and sulaymaniyah provinces. it is located at 36°04′59″n; 44°37′47″e. the region has borders with three countries that have higher scorpion diversity than iraq (19 species) (1). iran, in the west, has the highest scorpion species diversity (78 species, 2 subspecies) as compared to the other neighbouring countries (2), followed by turkey in the north with 41 species and 3 subspecies (3); then saudi arabia in the east with 26 species (4) and syria with 21 species (5). scorpion stings are unpleasant but rarely endanger life. children and older adults are at the highest risk for severe complications. among the 2710 species of known scorpions (6), only about 50 of them develop venom toxic enough to be lethal to humans (7). each year, more than one million scorpion stings occur. deaths from these stings are a major public health concern in tropical countries where access to medical care is limited (8). according to recent publications, 4162 scor copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:hamid.kachel@uoz.edu.krd https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 252 http://jad.tums.ac.ir published online: sep 30, 2022 pion stings were recorded in primary health care centres in the kurdistan region in 2017 and 2018 (9). based on the envenomation epidemiological data of the neighbour countries, androctonus crassicauda (olivier, 1807), androctonus bicolor (ehrenberg, 1828) and hemiscorpius lepturus (peters, 1861) are considered the most dangerous scorpions among the known species in iraq and they are responsible for the most death cases related to scorpion stings (10–11). knowledge of scorpion stings and diversity in iraq generally, and the kurdistan region in particular is extremely poor. according to recent studies on the scorpion fauna of the kurdistan region of iraq, compsobuthus matthiesseni (birula, 1905), h. lepturus, hemiscorpius saulcyi (simon, 1880), orthochirus fomichevi (kovařík, yağmur, fet and hussen, 2019), and scorpio kruglovi (birula, 1910) were listed in sulaymaniyha province. in duhok province, a. crassicauda, c. matthiesseni, hottentotta mesopotamicus (lourenço and qi, 2007), h. saulcyi, o. fomichevi and s. kruglovi were listed. while in erbil province, a. crassicauda, buthacus tadmorensis (simon, 1892), c. matthiesseni, calchas anlasi (yağmur, soleglad, fet and kovařík, 2013), h. saulcyi, h. lepturus, m. mesopotamicus, o. fomichevi and s. kruglovi were listed (1, 11, 12). the aim of this paper is to investigate scorpion stings in 2019–2020 and explain the current geographical distribution of different scorpion species from the kurdistan region. the outcomes of the present work will increase the awareness level of the kurdish community regarding the scorpion’s type and consequently reduce their stings. materials and methods epidemiological data the clinical data relating to the scorpion stings present in this study were obtained from the medical records of primary public health offices of the kurdistan region. statistical data of scorpion envenomation of 2019 and 2020 were checked. the relevant information for each scorpion sting case such as gender, age, and the geographical region of scorpion sting were recorded. excel software is used to analysis data statically and organize it in detailed tables and graphs. study area this cross-sectional study for scorpion species collection was conducted in the kurdistan region administrative of iraq. sulaymaniyah is located at latitude 35°33′26″n, 45°26′08″e at elevation of 882m on the board of iran. duhok is located at latitude 36°52′n, 43°0′e at elevation of 565m on the board of turkey and syria. erbil is located at latitude 36° 11'28.0068''n, 44°0'33.0012''e at elevation of 390m on the board of iran and turkey. the geographical features of three provinces are roughly identical and consist of two areas: mountains and plains. they have semi-arid climate weather with very warm dry summers and cool wet winters. the kurdistan area provinces are separated into 32 districts to better understand the geographical distribution of each scorpion species. twenty-two sites were randomly selected for this study survey (fig. 3). scorpion survey a random field survey for scorpion collection was carried out in twenty-two stations in rural and urban areas of the three provinces of kurdistan during the period of june 2020 to october 2021. sampling at night was done by using a black ultraviolet light (uv) to detect scorpions above ground, while during sunny days the ground was examined by searching under stones, rocks, and tree barks. all the collected scorpion specimens were placed in 80% ethyl alcohol and kept at the zoology laboratory at the university of zakho. all species were identified based on the descriptions and taxonomic keys published by levy and amitai (13) and kovařík (14). http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 253 http://jad.tums.ac.ir published online: sep 30, 2022 review of the scorpion fauna of kurdistan in this section, we reviewed all the data in previous studies related to the identified localities of scorpions in kurdistan. then we build a district-based checklist for the geographical distribution of each scorpion species recorded in kurdistan. results epidemiological data a total of 3726 scorpion sting cases were dealt with by the public hospitals in three provinces of the kurdistan region, iraq during the 2-year period from january 2019 until december 2020. according to the recorded evidence on scorpion stings, the highest numbers of cases were reported in 2019 and the lowest in 2020 (table 1). the scorpion sting victims were mostly between the ages of 15 and 49, accounting for 42.8% of all cases, followed by 30.1% of those between the ages of 4 and 14 (table 1). those over 50 and less than 4 accounted for 13.4% and 13.7% of the cases, respectively. summer was the most common season for scorpion stings (51.9%), with autumn being the next most common season (32.2%). in contrast, scorpion stings were reported at a very low rate of 12.2% and 3.7% in the spring and winter, respectively (fig. 1). the number of male and female scorpion sting victims was 2079 (55.8%) and 1647 (44.2%), respectively. table 2 summarizes the monthly occurrence of scorpion sting cases by gender. scorpion fauna composition out of all the 166 specimens collected in different sites of this survey, the buthidae family involves five species: a. crassicauda, c. matthiesseni, h. saulcyi, m. mesopotamicus and o. fomichevi, while scorpionidae and hemiscorpiidae each have a single species: s. kruglovi and h. lepturus, respectively (table 3, fig. 2). among all the collected samples, 102 (61.4%) were male and 64 (38.6%) females. the most abundant species were h. saulcyi 93 (56.0%), s. kruglovi 46 (27.7%) and a. crassicauda 15 (9.0 %), which were collected from various sites in this survey. the lowest frequency was from c. matthiesseni 4 (2.4%), m. mesopotamicus 3 (1.8%), h. lepturus 3 (1.8%) and o. fomichevi 2 (1.2%) (fig. 2). orthochirus fomichevi was collected only from zakho district in duhok province, while c. matthiesseni, m. phillipsii were collected only from erbil province. fig. 1. seasonal average rate of scorpion stings in three cities north of iraq during 2019–2020 http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 254 http://jad.tums.ac.ir published online: sep 30, 2022 table 1. age distribution for scorpion stings in erbil, duhok and sulaymaniyah provinces during 2019–2020 year city age (years) 1–4 5–14 15–49 50 over total 2019 erbil 98 201 150 98 1990 duhok 129 399 593 164 sulaimaniyah 9 23 97 29 average 236 623 840 291 2020 erbil 102 128 124 87 1736 duhok 155 316 412 105 sulaimaniyah 16 57 219 15 average 273 501 755 207 total (%) 509 (13.6) 1124 (30.2) 1595 (42.8) 498 (13.4) 3726 (100) table 2. monthly gender-based scorpion sting cases recorded in the kurdistan region provinces from 2019 to 2020 month 2019 2020 male female total % january 9 13 11 11 22 0.6 february 12 4 7 9 16 0.4 march 10 25 17 18 35 0.9 april 42 68 70 40 110 3 may 146 166 158 154 312 8.4 june 320 349 403 266 669 18 july 316 196 314 198 512 13.7 august 388 319 341 366 707 19 september 221 312 286 247 533 14.3 october 283 170 257 196 453 12.1 november 181 78 155 104 259 7 december 62 36 60 38 98 2.6 total (%) 1990 (53.4) 1736 (46.6) 2079 (55.8) 1647 (44.2) 3726 100 fig. 2. scorpion species composition collected in this survey in kurdistan region, 2020–2021 http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 255 http://jad.tums.ac.ir published online: sep 30, 2022 table 3. species of scorpions collected in different survey sites in kurdistan region provinces 2020–2021 collection site (district) topography altitude (m) latitude and longitude scorpion species (n.) total collected sulaymaniyah province qaladze (pishdar) plain 882 36° 10' 51.2" n 45° 7' 26.9" e h. saulcyi (10) s. kruglovi (15) a. crassicauda (3) 28 kele border (pishdar) mountain 849 36.18 66 n 45.3243 e s. kruglovi (12) a. crassicauda (1) 13 bawze (pishdar) mountain 1138 36.0878 n 45.3133 e h. saulcyi (16) s. kruglovi (1) a. crassicauda (1) 18 swne (pishdar) mountain 890 36° 14' 53.2" n 45° 7' 34.6" e h. saulcyi (3) 3 makok mountain (ranya) mountain 882 36° 17’ 29.2” n 44° 47’ 29.3” e h. saulcyi (3) 3 chaqizh lower (sulaymaniyah) plain 912 35.626307ºn 45.199277ºe h. lepturus (3) a. crassicauda (1) c. matthiesseni (1) h. saulcyi (3) o. fomichevi (1) 9 duhok province telkabar (zakho) plain 487 37°16′28″n 42°71′78″e h. saulcyi (25) a. crassicauda (1) c. matthiesseni (1) 27 sheransh (zakho) mountain 1439 37°13′48″n 42°50′40″e h. saulcyi (5) 5 betas (zakho) mountain 678 37° 3' 39" n 42° 44' 7" e h. saulcyi (4) o. fomichevi (1) 5 demka (zakho) mountain 873 37° 13' 10" n 43° 4' 36" e h. saulcyi (7) 7 bani nan (bardarash) plain 298 36° 30′ 12.2″ n 43° 35′ 14″ e a. crassicauda (2) 2 erbil province efraz (khabat) plain 291 36°21'51.37"n 43°48'24.84" e a. crassicauda (2) 2 rawanduz (rawanduz) mountain 658 36°35'28.79"n 44°23'32.99" e s. kruglovi (10) 10 choman (choman) mountain 2407 36°38'14.67"n 44°53'21.52"e h. saulcyi (3) 3 pirdawd (erbil) plain 360 36.024650 n 43.927116 e h. saulcyi (2) m. mesopotamicus (3) 5 ankawa (erbil) plain 413 36° 13' 42.1" n 43° 59' 43.2" e h. saulcyi (2) 2 rezan (mergasur) mountain 882 44.1294069" n 36.8551834" e h. saulcyi (2) 2 harir (shaqlawa) mountain 699 44.3467706 " n 36.552188 " e h. saulcyi (3) a. crassicauda (2) s. kruglovi (8) 13 qushtapa (erbil) plain 390 36° 00' 2.0639" n 44° 01' 57.077" e h. saulcyi (4) a. crassicauda (2) 6 bestana (erbil) plain 646 36° 2' 45.6" n 44° 11' 57.2" e c. matthiesseni (1) 1 koysinjaq (koysinjaq) plain 594 36°4'30.33"n 44°37'11.77"e c. matthiesseni (1) 1 dashty hawler (dashti hawler) plain 419 36° 9' 11.5" n 44° 6' 0.3" e h. saulcyi (1) 1 total 166 http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 256 http://jad.tums.ac.ir published online: sep 30, 2022 table 4. comparison of scorpion species distribution in previous and current study in kurdistan region provinces (duhok, erbil and sulaymaniyah) in iraq divided into 32 districts: duhok (1zakho, 3duhok, 4amedi, 5shikhan, 6akre, 7bardarash), erbil (8mergasur, 9soran 10choman, 11rawanduz, 12shaqlawa, 13khabat, 14erbil, 15dashti hawler, 16koysnjaq, 17makhmur), sulaymaniyah (18ranya, 19pishdar, 25sulaymaniyah, 26said sadiq, 29halabja,). green indicates report only in this study, blue indicates report in only previous studies and red indicates report in both current and previous stud studies district per province in kurdistan region of iraq duhok erbil sulaymaniyah scorpion family-species 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 25 26 29 buthidae a. crassicauda b. tadmorensis c. matthiesseni h. mesopotamicus h. salucyi m. mesopotamicus o. fomichevi hemiscorpiidae h. lepturus iuridae c. anlasi scorpionidae s. kruglovi species number per district 6 1 1 2 4 1 6 1 1 1 6 2 5 4 8 2 1 3 6 3 2 fig. 3. kurdistan region provinces divided into 32 districts duhok (1zakho, 2sumel, 3duhok, 4amedi, 5 shikhan, 6akre, 7bardarash), erbil (8mergasur, 9soran 10choman, 11rowanduz, 12shaqlawa, 13khabat, 14erbil, 15dashti hawler, 16koysnjaq, 17makhmur), sulaymaniyah (18ranya, 19pishdar, 20dukan, 21 mawat 22sharbazher, 23penjwin, 24chamchamal, 25sulaymaniyah, 26said sadiq, 27qaradagh, 28sharazur, 29halabja, 30kalar, 31darbandikhan, 32khanaqin). the study sites surveyed represented by black dots http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 257 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 4. general overview of scorpion species collected in this study in the kurdistan region of iraq a. androctonus crassicauda; b. hottentotta saulcyi; c. compsobuthus matthiesseni; d. mesobuthus mesopotamicus; e. orthochirus fomichevi; f. scorpio maurus; g. hemiscorpus lepturus (photos by authors) fig. 5. the distribution map of the known scorpion species in 22 out of 32 districts of the kurdistan region of iraq: duhok (1zakho, 2sumel, 3duhok, 4amedi, 5shikhan, 6akre, 7bardarash), erbil (8mergasur, 9soran 10choman, 11rawanduz, 12shaqlawa, 13khabat, 14erbil, 15dashti hawler, 16koysnjaq, 17makhmur), sulaymaniyah (18ranya, 19pishdar, 20dukan, 21mawat 22sharbazher, 23penjwin, 24chamchamal, 25sulaymaniyah, 26said sadiq, 27qaradagh, 28sharazur, 29halabja, 30kalar, 31darbandikhan, 32khanaqin) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 258 http://jad.tums.ac.ir published online: sep 30, 2022 discussion the current checklist study showed that the kurdistan region had 10 scorpion species belonging to nine genera and four families: buthidae, hemiscorpiidae, iuridae and scorpionidae. this diversity in the region's scorpion fauna indicates scorpions' preferred living habits and climate. seven species (fig. 4) of the 10 known in kurdistan were collected in the current survey and three scorpion species were previously recorded in kurdistan but not in this study: b. tadmorensis, h. mesopotamicus, and c. anlasi (12, 15–16). the buthidae family had the highest frequency of seven species, with a single species for each of the other families. the highest number of scorpion species in this checklist was reported from erbil province, with 9 species, followed by 6 different species in each of duhok and sulaymaniyah provinces. when compared to a recently published checklist of iraq scorpions, the species richness and diversity rose by four scorpion species in each of erbil and sulaymaniyah, and one scorpion species in duhok (1, 12). so, following baghdad province, which had 10 species, erbil province has the second greatest number of scorpion species variety in iraq, followed by diyala province with 7 species, while the scorpion numbers recorded in the other fifteen provinces range from 0 to 6 species (1). the geographical distribution checklist of scorpion species identified in the kurdistan region is shown in table 4 and figure 5 based on the 32 districts of the kurdistan region. the most abundant species, in terms of both species and distribution, was h. saulcy, which was found in sixteen distinct districts across kurdistan's three provinces. it was collected for the first time in four districts (two in erbil and two in sulaymaniyah), and it had previously been reported in 12 districts (11–12). in the iraq scorpion checklist, kachel and his colleagues (1) reported h. saulcy in seven provinces of iraq from three different geographical regions of the country (mr= mountainous and highland region (17), ur= undulated and hilly region, dr= desert region). androctonus crassicauda, one of iraq's most dangerous scorpion species, was the second most widely distributed species in kurdistan and the first in iraq. the distribution of a. crassicauda in kurdistan has been documented in thirteen districts, two of which are new. androctonus species can adapt to a wide range of environmental conditions (18). according to the most recent iraq scorpion checklist (1), a. crassicauda has been found in 14 provinces across the country, in all four geographical conditions (mr, ur, dr, ar= alluvial region). several incidences of mortality from this species' stings have been documented in iran's southern provinces of hormozgan and khuzestan (19–20). orthochirus fomichevi and c. matthiesseni have been found in 8 and 7 districts, respectively, in kurdistan's three provinces. compsobuthus matthiesseni has been found in three different geographical conditions in iraq (mr, ur, ar regions), while o. fomichevi has only been found in the mr region (1). other buthidae species' distribution were restricted to specific provinces. m. mesopotamicus and b. tadmorensis were only found in erbil province districts (12), while h. mesopotamicus was only recorded previously in duhok province's zakho district (15). in iraq, the distribution of razianus zarudnyi was also limited to the province of baghdad (21). the hemiscorpiidae family was the second to be detected in the kurdistan region. the only species of this family found in kurdistan was h. lepturus, which was found in three districts: one in erbil and two in sulaymaniyah province. hemiscorpus lepturus is the most dangerous scorpion species, its envenomation causing severe complications and the main cause of death in khuzestan province in iran (22–24). the key feature for identifying http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 259 http://jad.tums.ac.ir published online: sep 30, 2022 this species was its small, thin stings. people's and health-care workers' lack of knowledge about this scorpion species can be hazardous to residents in their area (18). other non-medically important scorpion families found in kurdistan are the iuridae and scorpionidae, each of which has one species, c. anlasi and s. kruglovi, respectively. because both species' venoms are weak, they must rely on their powerful chelicerae to capture prey. calchas anlasi's distribution is limited to turkey, with a solitary occurrence in the kurdistan district of soran, it requires further verification in iraq (16). while s. kruglovi has a greater geographic range, it has been reported in all of iraq's neighbouring countries except turkey (25). in iraq, it has been reported from six provinces from all four geographical conditions (1, 26). in the current study, s. kruglovi was shown to be the second most common and third most distributed in 13 districts across kurdistan's three provinces. the current study's epidemiological data in the kurdistan region revealed that the number of scorpion stings was lower in the year 2020 than in 2019. the current study's findings over a two-year period are slightly lower than earlier studies conducted in kurdistan (9), which might be due to covid-19's restriction laws or people's fear of visiting hospitals to receive sting treatment. according to the data, men (55.8%) are more likely than women to be stung (44.2%). this conclusion is consistent with findings from india (27) and argentina (28). in contrast, prior research in iran (18, 29) found that the number of female patients presenting with scorpion stings was higher than the number of male patients. overall, scorpion sting epidemiology is related to the victim's gender, study year, and study area. however, cultural factors such as gender disparities in behaviour may be considered. our findings revealed that in kurdistan, annual epidemiological determinants of scorpion stings follow a similar trend. in the summer, the highest rate of scorpion sting cases was reported. the current study's findings are consistent with those of prior research conducted in iraq's kurdistan (7, 10) and iran (30, 31). according to this data, scorpion stings are most common during the hotter months of the year (may to september). as the temperature rises, scorpion activity rises, and their contact with humans increases the number of stings (32). according to our findings, scorpion stings were most common in people aged 15 to 49 years old, followed by the age group of 4 to 14 years. this finding is consistent with findings from a previous study in the kurdistan region (9, 12). this age group has been identified as being at high risk in related studies, which could be due to outdoor work and activity outside the home. according to an iranian study, scorpion stings occur most frequently among young people aged 15 to 24 years old (33). likewise, scorpion stings are more common in people aged 20 to 29 in in the northeast region of brazil (34). conclusion according to the latest research, scorpionism is a severe health problem in iraq's kurdistan region, impacting people of all ages and genders, as well as many stung patients. however, because kurdish society and public health agencies are unaware of the problem and potentially deadly scorpion species, more information on scorpion diversity and sting prevention techniques should be provided. acknowledgements the authors appreciate the collaboration and assistance of the staff of the provincial health centres in erbil, duhok, and sulaymaniyah. conflict of interest statement the authors declare not to have conflicts of interest. http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 251–261 f hussen et al.: epidemiological characterizations … 260 http://jad.tums.ac.ir published online: sep 30, 2022 ethical considerations not applicable. references 1. kachel h, al-khazali a, hussen f, yağmur e (2021) checklist and review of the scorpion fauna of iraq (arachnida: scorpiones). arachnol mitt. 61 (1): 1–10. 2. bavani m, jesri n, sarvi m, saeedi s, saghafipour a, shirani-bidabadi l (2022) new data on medically important scorpion 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1275–1282. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 7 original article assessing the susceptibility status of mosquitoes (diptera: culicidae) in a dirofilariasis focus, northwestern iran abolfazl ataie, *seyed hassan moosa-kazemi, hassan vatandoost, mohammad reza yaghoobi-ershadi, hasan bakhshi, mehdi anjomruz department of medical entomology and vector control, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran (received 13 dec 2011; accepted 3 may 2014) abstract background: mosquitoes are considered as the vectors of dirofilariasis and some vector borne disease in iran. the objective of this study was to determine the susceptibility level of the vectors to various insecticides recommended by who for any control measures in an endemic area in northwestern iran. methods: mosquito larval and adult collections were carried out using different methods provided by who including dipping and hand catch techniques. the susceptibility level was assessed to ddt 4%, malathion 5%, propoxur 0.1%, deltamethrin 0.05% and lambda-cyhalothrin 0.05%. results: totally, 749 adults and 5060 larvae of culicidae mosquitoes were collected comprising seven species of adult and larvae, including: anopheles claviger, an. maculipennis, an. sacharovi, culex hortensis, cx. pipiens, cx. theileri and culiseta longiaerolata. frequency of larvae and adults of an. maculipennis was very low, so susceptibility tests on this species did not performed. results showed that cx. theileri, cs. longiaerolata and cx. pipiens were resistant to ddt 4%, lambda-cyhalothrin 0.05%, and propoxur 0.1% whereas found tolerant to deltamethrin 0.05% and malathion 5%. the lt50 and lt90 values for five insecticides were calculated. conclusion: we suggest the same study in different parts of the world to obtain the data due to bionomic and susceptibility status of dirofilariasis vectors. this information will help the health authorities for monitoring and evaluation of control measures. keywords: culicidae, vector, dirofilaria immitis, susceptibility test, iran introduction mosquitoes (diptera: culicidae) are the most important groups of arthropods in medical and veterinary. they act as vectors of several diseases such as malaria, yellow fever, dengue, filariasis, setariasis and encephalitis, causing serious health problems to humans (service 2003, almeida et al. 2008). the culicidae mosquitoes are vector of dogs heartworm parasites, dirofilaria immitis, di. repens, wuchereria bancrofti, some arboviral diseases such as japanese encephalitis, rift valley fever, western equine encephalitis and eastern equine encephalitis, tahyna, sagiyama, trivitatus, lymphocytic choriomanangitis, west nile virus, st louis encephalitis, california encephalitis (muller 2002, mullen and durden 2009). dirofilariasis is a metazoonotic disease that is transmitted by certain species of mosquitoes. dirofilaria immitis could be found in carnivores especially dogs and reported from various countries including turkey (atas et al. 1997), japan (hatsushika et al. 1997), brazil (reifur et al. 2004), canada (slocombe and villeneure 1993) and other countries (chen and liang 1993, rosa et al. 2002). dirofilaria immitis is commonly causes canine heartworm disease in many countries *corresponding author: dr seyed hassan moosakazemi, e-mail: moosakazemi@tums.ac.ir http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 8 (genchi et al. 2005). human dirofilariasis has been shown as pulmonary or subcutaneous infection, but in korea one case of hepatic dirofilariasis has been reported by di. immitis agent (choi et al. 2002). dirofilariasis produced by di. immitis parasites have been reported from all tropical, subtropical and moderate areas and the highest prevalence is in north america, south america, australia, italy and japan (venco et al. 2004). dirofilariasis is a worldwide distributed disease, but the most endemic areas are those with moderate, tropical and subtropical climates, where mosquito populations are high and stable. other regions with cold climates or with hot summers and with rivers, lakes and widely irrigated lands are also suitable for the development of the disease. endemic occurrence of di. immitis has been reported in the usa, canada, south america, africa, australia, asia and europe (yildirim et al. 2006). dirofilaria immitis was first reported from a dog in iran in 1969 (sadighian 1969). then infection to heartworm was reported from different areas of iran including ardebil (bokai et al. 1998), shiraz (jafari et al. 1996), tehran (meshgi and eslami 2001), tabriz (meshgi et al. 2002) tonekabon (ranjbar-bahadori et al. 2005), and mashhad (razmi 1999). iran reported one of the disease foci in the world (who/fao/oie 1984). zoonotic dirofilariasis has been reported in 11 provinces of iran (azari-hamidian et al. 2007). by now, di. immitis has been isolated from peripheral blood in a cat in tabriz (ashrafi et al. 2001). azari-hamidian et al. (2007) reported di. immitis in dogs, jackals, foxes, wolves and cats, and di. repens in dogs and jackals in different areas of the country. the natural infection has been reported about 26.7% on stray dogs in tonekabon (former shahsavar), mazandaran province (sadighian 1969). in addition, contamination of 0.95 and 36.8 % has been reported in the east and west azerbaijan, ardebil and tehran, fars and khuzestan, mazandaran, golestan and east azerbaijan provinces (sadighian 1969, abai 1990, zarriffard 1993, jafari et al. 1996, jamali and hashemzade farhang 1996, bokaie et al. 1998, farahnak et al. 1998, razm arayi et al. 2000, ariamanesh 2000, mashgi and eslami 2001, sadjjadi et al. 2004, fallah et al. 2005, ranjbar-bahadori and eslami 2005). infection of subcutaneous dirofilariasis has been reported in the anzali port and lahijan, karaj and ahvaz county (siavashi and massoud 1995, athari 2003, maraghi et al. 2006, radmanesh et al. 2006, (khanmohammadi et al. 2011). by now, human and animal dirofilariasis cases have been recorded in 11 of the 31 provinces of iran. azari–hamidian et al. (2009) reported the fourteen human cases of infection, including eight subcutaneous and three ocular cases of di. repens, a testicular hydrocele case of di. immitis and two pulmonary cases probably attributable to di. immitis. two species of dirofilaria include di. immitis (canine heartworm) and di. repens have been reported in iran. microfilarial worms of di. immitis as 258±7µ m, and for infected larvae ranged 0.75–1.3mm (anderson 2000). the length of adult calculated as 250–300×1 mm (muller 2002). female mosquitoes serve as an intermediate host by sucking blood from dog which circulating di. immitis microfilaria (montoya et al. 1998). the diagnosis of canine heartworm infection is based upon the detection of di. immitis circulating in blood or upon the detection of serum antibodies by serologic methods (peribanez et al. 2001). wolbachia (rickettsiales: rickettsiaceae) found in filarial nematodes such as dirofilaria spp., have an important role in the filarial infection pathogenesis (bazzocchi et al. 2000). the immune response of dog against wolbachiain, considered as a diagnostic method for canine heartworm diseases (tiawsirisup et al. 2010). some of mosquito species are able to transmit infected of third-stage larvae of http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 9 dirofilaria parasite (aranda et al. 1998). many of di. immitis vectors reported in the world (grieve et al. 1983). the different species of the genera of aedes, ochlerotatus, psorophora, anopheles, and culex found susceptible to di. immitis in usa (mullen et al. 2009). anopheles maculipennis and cx. theileri reported naturally infected with infective larvae of di. immitis and setaria labiatopapillosa in iran (azari-hamidian et al. 2009). in iran, the insecticides residual spraying against malaria vectors was carried out during 1950–1968. following the resistance of the vectors to ddt (1957), dieldrin (1960) and malathion (1976), carbamate and pyrethroid insecticides were used (zaim 1987, moosakazemi et al. 2007). some enzymes such as monooxygenases, esterases and glutathione s-transferases have been considered as a reason for resistance to permethrin insecticides. the efficacy of these insecticides will be increased by using some synergists like piperonyl butoxide (pbo) that inhibits monooxygenases ortribufos (def) that inhibits esterases (enayati et al. 2003). prior of this study, there are no reports on the susceptibility of mosquitoes to insecticides in ahar county, east azarbaijan province. the aim of this study was to determine the susceptibility level of dirofialriasis vectors to insecticides in the area. materials and methods study area east azarbaijan province is located in the northwest of the country, bordering armenia and the republic of azarbaijan. the investigation was carried out during the summer of 2011 in the rural district of ahar county (38˚45´n, 47˚11΄e). the population of ahar county has been reported 147.781 and 34.067 families. agriculture, horticulture, livestock and making hand crafts making carpet and needlework are introduced as the main jobs. cold and moderate weather provides the suitable condition for agricultural and husbandry activities. three fixed afil, (38˚37´n, 47˚31΄ e), noghdose (38̊ 39´n, 47˚36΄e) , bohol (38˚36´n, 47˚35΄e) and six variable villages selected randomly (fig. 1). mosquito collection hand collection: in each fixed and variable places, mosquitoes were collected from indoor places by the using suction tubes. adult mosquitoes were collected from the villages, 05.00 to 08.00 am by using suction tube. then the adult mosquitoes released in the cage and covered with wet towel and mosquitoes feed by sucrose 5 % solution (silver 2008). larval collection: in each fixed station the mosquitoes were collected from june to august 2011. the related data such as water temperature, larval type, larval number and the date of sampling were recorded. the larvae were collected from the villages, 08.00 to 11.00 am by using standard dipper (350 ml) and eye dropper. the larvae were transferred into a closed container, sent to the laboratory and placed within a few cups into cages to obtain f1 generation. the samples were reared (silver 2008). adult susceptibility test the mosquitoes were transferred into the exposure tubes at different logarithmic exposure time, and mortality was then calculated after a 24 h recovery period (25 ˚c, 75%rh). the mortalities between 5.0 to 20.0 % were corrected by abbott’s correction formula. all the tests were accepted when the mortality was less than 5% and ignored when the mortality was more than 20% in the control group (abbott 1925, silver 2008, who 1998). the susceptibility level of the species was considered in three classes as susceptible, tolerant and resistant based on who criteria (who 2002). the mortality more than http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 10 98 % was considered as susceptible, less than 80 % noted as resistant and between 97 to 98 % defined as tolerant. insecticides impregnated papers impregnated papers, ddt 4 %, malathion 5 %, deltamethrin 0.05 %, propoxur 0.1 % and lamda-cyhalothrin 0.05 % were supplied by who. statistical analysis the lt50 was considered as lethal time causing 50 % mortality and its 95 % confidence interval and lt90 mentioned as lethal time causing 90 % mortality and its 9 5% confidence interval. the lt50 and lt90 values of cx. theileri, cx. pipiens and cs. longiaerolata and probit regression line parameters were calculated to finney’s test (finney 1971). plotting the regression line was calculated through the χ2 test using microsoft excel ver. 2007. identification of mosquito using morphological characteristics the samples were mounted and identified by systematic keys (shahgudian 1960, zaim and cranston 1986, azari-hamidian and harbach 2009). results totally, 749 adults and 5060 larvae of culicidae mosquitoes were collected including seven species, an. maculipennis sl, an. sacharovi, an. claviger, cx. pipiens, cx. hortensis, cx. theileri and cs. longiaerolata. culex theileri was found to be dominant species as 42.8 %, 33.6 % allocated in adult and larval stages, respectively. cx. pipiens was followed by 29.9 %, 25.3 % in adult and larvae respectively. an. maculipennis sl was found 6.9 % in larvae collection. an. claviger was collected as low density (4.7%), an. sacharovi, cx. hortensis and cs. longiaerolata were also collected in larval habitats. the maximum and minimum temperature in larval habitats was 29 ˚c and 22 ˚c, respectively. an. maculipennis sl was collected very low, so susceptibility tests on this vector did not perform. the lt50 and lt90values for five insecticides are shown in table 1, figs. 2–6. the mortality rates of field collected cs. longiareolata, cx. theileri and cx. pipiens to ddt 4 % at diagnostic exposure time were 18 %, 15 %, 23 %, respectively. considering the criteria of resistance of the species described by who (table 2), it should be mentioned that the species of cx. pipiens, cx. theileri and cs. longiaerolata were resistant to ddt 4 %, lambda-cyhalothrin 0.05 %, and propoxur 0.1 %. these mosquitoes were found tolerant to deltamethrin 0.05 % and malathion 5 %. our results based on probit regression line showed that cs. longiaerolata was more susceptible than cx. theileri and cx. pipiens when exposed to deltamethrin 0.05 % and malathion 5 %. in addition, cs. longiaerolata was found more susceptible to cx. pipiens and cx. theileri after exposed to lambda-cyhalotherin 0.05 %, propoxur 0.1 % and ddt 4 %. fig. 1. map of the study area, east azerbaijan province, northwestern iran http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 11 fig. 2. probit regression line of culicinae mosquitoes to ddt 4 % in ahar county, east azarbaijan province, 2011 fig. 3. probit regression line of culicinae mosquitoes to deltamethrin 0.05 % in ahar county, east azarbaijan province, 2011 fig. 4. probit regression line of culicinae mosquitoes to lambda-cyhalothrin 0.05 % in ahar county, east azarbaijan province, 2011 fig. 5. probit regression line of culicinae mosquitoes to malathion 5 % in ahar county, east azarbaijan province, 2011 http://jad.tums.ac.ir published online: july 16, 2014 time/second p r o b it cs.longiaerolata cx.theileri cx.pipiens p r o b it p r o b it p r o b it time/second time/second time/second cs.longiaerolata cs.longiaerolata cs.longiaerolata cx.theileri cx.theileri cx.theileri cx.pipiens cx.pipiens cx.pipiens j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 12 fig. 6. probit regression line of culicinae mosquitoes to propoxur 0.1 % in ahar county, east azarbaijan province, 2011 table 1. probit regression line parameters of insecticides against mosquitoes in ahar county, east azarbaijan province, 2011 pvalue y= a+bxx2 (df)****lt90 95%c.i (minute) ***lt50 95%c.i (minute) **b±se*aspeciesinsecticides <0.05 <0.05 <0.05 y= -7.6975+1.9745 x4.119(2)588.13131.941.9745±0.381-7.6975cs. longiaerolata cx. theileri cx. pipiens ddt 4% y= -6.7965+1.6702 x2.624(2)1144.01195.481.6702±0.363-6.7965 y= -6.6372+1.6985x1.778(2)765.75134.751.6985±0.305-6.6372 <0.05 <0.05 <0.05 y = -4.2737+1.7124 x9.996(2)29.245.211.7124±0.148-4.2737cs. longiaerolata cx. theileri cx. pipiens deltamethrin 0.05% y = -4.6191+1.6581 x7.516(2)60.35010.1791.6581±0.141-4.691 y = -4.9528+1.7710 x5.921(2)55.21110.4321.7710±0.152-4.9528 <0.05 <0.05 <0.05 y= -3.4762+1.1496 x3.329(2)229.2617.601.1496±0.114-3.4762cs. longiaerolata cx. theileri cx. pipiens lambdacyhalothrin 0.05% y= -3.7667+1.1630 x2.621(2)365.228.871.1630±0.123-3.7667 y= -3.7260+1.1772 x4.048(2)298.7724.371.1772±0.121-3.7260 <0.05 <0.05 <0.05 y= -4.4964+1.8031 x9.939(2)26.695.191.8031±0.157-4.4964cs. longiaerolata cx. theileri cx. pipiens malathion 5% y= -4.5115+1.6842 x4.676(2)45.877.951.6842±0.142-4.5115 y= -4.6712+1.7413 x5.028(2)43.708.0251.7413±0.148-4.6712 <0.05 <0.05 <0.05 y= -3.7575+1.1588 x8.809(2)371.7629.121.1588±0.123-3.7575cs. longiaerolata cx. theileri cx. pipiens propoxur 0.1% y= -4.1764+1.2443 x9.727(2)405.6437.861.2443±0.137-4.1764 y= -4.2249+1.2666 x5.825(2)371.0736.1051.2666±0.137-4.2249 *a= intercept. **b±se= slope and its standard error. ***lt50, 95 % ci= lethal time causing 50 % mortality and its 95 % confidence interval. ****lt90, 95 % ci= lethal time causing 90 % mortality and its 95 % confidence interval. http://jad.tums.ac.ir published online: july 16, 2014 p r o b it time/second cs.longiaerolata cx.theileri cx.pipiens j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 13 table 2. susceptibility status of culicinae species to insecticides in ahar county, east azarbaijan province, 2011 species insecticide mr±eb* resistance status** cs. longiaerolata ddt 4 % 18±4 r deltamethrine 0.05 % 87±4 t lambda-cyhalothrine 0.05 73±5 r malathion 5 % 90±3 t propoxur 0.1 % 55±5 r cx. theileri ddt 4 % 15±4 r deltamethrine 0.05 % 88±4 t lambda-cyhalothrine 0.05 65±5 r malathion 5 % 93±3 t propoxur 0.1 % 50±6 r cx. pipiens ddt 4 % 23±5 r deltamethrine 0.05 % 91±3 t lambda-cyhalothrine 0.05 63±5 r malathion 5 % 97.5±2 t propoxur 0.1 % 53±6 r *mortality rate±error bar **r: resistance, t tolerance discussion in this study, a total of 3 genera and 7 species of mosquito larvae and adults were collected in ahar county. culicidae species belonged to the genus of anopheles, culex and culiseta. the most predominant species was cx. theileri as 42.8% of adult and 33.6% larvae collection. this species is reported as one of the most predominant species in ardebil (azari-hamidian et al. 2009), isfahan (moosakazemi et al. 2000) and guilan province (lotfi 1970, azari-hamidian et al. 2000, azarihamidian et al. 2001, azari-hamidian 2007). this species cited as predominant in zanjan (ghavami and ladoni 2005), east azarbaijan (abai et al. 2007), and southeastern iran (moosa-kazemi et al. 2009). simsek (2004) noted the dominant species in ankara, turkey (simsek 2004). zoophilic index on goat, dog, cattle, and human reported as 58.3%, 50%, 44.4% and 13.6%, respectively. culex theileri found 1.49% of the total collection in neka county (nikookar et al. 2010). the seasonal activity of larvae started at the end of may reach to peak in the end of june in isfahan province. seasonal activity of adults began in the end of may reach to peak in mid of july (mousakazemi 1995). the maximum blood feeding activity reported as 22.30 pm–00.30 am, 23% of feeding observed in the second third of the night while 19% found in the first third of the night (moosa kazemi e al. 2010). for the first time di. imimitis was isolated from cx. theileri with 10% infection in meshkin shahr (azari-hamidian et al. 2009). nazari and janbakhsh (2000) noted the resistance of this species to ddt, tolerant to propoxur and dieldrin in southern tehran. in our study, lt50 value of cx. theileri for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur was calculated as 195.48, 10.179, 28.87, 7.95 and 37.86 minutes respectively. lt90 value found as 1144.01, 60.35, 365.2, 45.87 and 405.64 minutes for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur respectively. in contrast, lt50 value of this species for propoxur and http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 14 malathion reported as 31, and 22 minutes respectively (nazari and janbakhsh 2000). at present, seems to, this species was resistance to ddt, lambda-cyhalothrin and propoxur whereas tolerant to deltamethrin and malathion. in our study, cx. pipiens was dominant species comprised as 29.9% of adult and 25.3% of larvae. minimum and maximum temperature in the larval habitat found as 22 ˚c, 29 ˚c respectively. the larvae collected in the larval habitat as a range of altitude 834–1382 meters. based on morphological characteristic cx. pipiens with cx. vagans and cx. torentium has mentioned in one group by zaim and cranston (1986). lotfi (1970) reported the cx. torentium from fars province based on the siphon index and size of siphonal setae (lotfi 1970). culex pipiens complex have a great abundance in north and north west of the country (lotfi 1970, azari-hamidian et al. 2000, azari-hamidian et al. 2001) east azarbaijan (abai et al. 2007), zanjan (ghavami and ladoni 2005). this species reported the dominant species in isfahan province. larvae were collected from rice fields (mousakazemi 1995). the larval activity in isfahan began in late may reached peak in late june and then declined. the seasonal activity of adult started in the end of june and reached peak in mid-july and declined in end of august. the attractiveness of this species to light trap had been reported more than to human bait collection (mousakazemi 1995). azari-hamidian et al. (2001) reported the blood feeding of this species on cattle in north of iran. in contrast mousakazemi (1995) reported the tendency of blood feeding on human in central iran (mousakazemi 1995). it seems that the limitation of animal host in the urban area of lenjan county, leads to attractive of this species to light trap. this species was reported one of the most predominant species in mazandaran province in north of iran (nikookar et al. 2010). the adaptation of cx. pipiens to variety of breeding places with different degrees of pollution near the human residence reported by ghavami and ladoni (2005) and dehghan et al. (2010). culex pipiens and cx. quinquefaciatus larvae were detectable based on siphon/ saddle index and seta 1 on abdominal segment iii-iv (harbach 1985, azari-hamidian and harbach 2009, dehghan et al. 2010). seta 1 on abdominal segment iii-iv in cx. pipiens is usually single while this character in cx. quinquefaciatus is usually double. culex pipiens form molestus can be separated from cx. pipiens pipiens mainly by ecological and physiological characters. culex pipiens form molestus this found in open spaces and the accumulated water. the adults are able to laying the first batch of eggs without a blood-sucking (autogenes). they are mating in small spaces and they hibernate in the winter (stenogamus). the blood feeding of cx. pipiens form molestus reported from mammals more than other animals. the main different characteristic between the adult of cx. quinquefasciatus and cx. pipiens pipiens was reported as the ratio of dv/d in male genitalia (sundaramans 1949). the distance of tip of ventral arm to dorsal arm of phalosoma considered as dv and d mentioned the distance of tip of dorsal arms of phalosoma (barr and kabtman 1951, mattingly et al. 1951). in addition some characters are important including: the number of setae on the side in the ninth tergit hairs on maxillary pulp of the adult male, and dv/d (vinogradova 2000). the most reliable character for identifying larval stages of cx. torentium and cx. pipiens reported as siphon/saddle index and seta 1 on abdominal segment iii-iv. in cx. torentium reported with four branches (harbach 1985, azarihamidian and harbach 2009, dehghan et al. 2010). culex pipiens includs pipiens and molestus forms. culex pipiens pipiens is considered as http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 15 unatogenous laying eggs on open breeding places, and pass the winter with diapose while cx. pipiens form molestus is considered as autogenous, stenogamus, and pass the winter without diapose. culex pipiens pipiens blood feeds on birds than human whereas cx. pipiens form molestus feed on human. in addition blood feeding of cx. pipiens form molestus is reported from birds, rodents and guinea pigs. culex pipiens pipiens, cx. quinquefasciatus and cx. pipiens form molestus as the most common and most widespread in the world. culex pipiens form molestus habitat found in temperate climates, whereas cx. quinquefasciatus has been distributed in most parts of the world and considered as cosmotropical (vinogradova 2000). gjullin and peters (1952) reported the tolerant of cx. pipiens complex to ddt and organophosphoros insecticidae. the first reports related to resistance of the cx. quinquefasciatus to organophosphoros insecticidae was reported by isaak (isaak 1961). in addition, the increase of the resistance to organophosphoros insecticides reported by toma et al. (2011). many studies indicated the tolerance to organophosphoros from tunisia (ben cheikh et al. 1998), cuba (bisset et al. 1991, rodriguez et al. 1993), burkina faso (chandre et al. 1998), saudi arabia (amin and hemingway 1998), china (jinfu 1999) and north america (mcabee et al. 2003). in iran, indoor residual spraying was carried out during 1950–1968 against malaria vector. following the resistance of an. stephensi, the main malaria vector to ddt, other insecticides such as dieldrin, malathion, propoxur, lambda-cyhalothrin and recently deltamethrin used. the resistance of cx. pipiens complex to ddt reported in southern tehran. lt50 for propoxur and malathion calculated as 51, and 31 minutes respectively. the species was quit susceptible to propoxur and malathion (nazari and janbakhsh 2000). khyami-horani et al. (1995) reported two standard strains, bacillus thuringiensis, and ba. sphaericus had killing effects as 0.0006 to 0.006 ppm and 0.003 to 0.06 ppm respectively against cx. pipiens molestus larvae. in our study, lt50 value of cx. pipiens for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur calculated as 134.75, 10.43, 24.37, 8.025 and 36.105 minutes respectively. lt90 value found as 765.75, 55.211, 298.77, 43.70 and 371.07 minutes for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur respectively. lt50 value of this species in our study was lower than the previous study cited by nazari and janbakhsh (2000). in our study, cs. longiaerolata observed resistance to ddt, propoxur, lambdacyhalotrin and tolerant to malathion and deltamethrin. lt50 value found as 131.94, 5.21, 17.60, 5.19 and 29.12 minutes for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur respectively. lt90 value of this species for ddt, deltamethrin, lambda-cyhalothrin, malathion and propoxur calculated as 588.13, 29.24, 229.26, 26.69 and 371.76 minutes respectively. some reports emphasizing to susceptibility of cs. longiareolata larvae to ba. sphaericus and ba. thuringiensis (katbeh bader et al. 1999). the range of mortality found as a 0.003–0.004 ppm. the mortality of larvae in temperature of 28±1 ˚c found more than 20±1 ˚c (katbeh bader et al. 1999). bouaziz et al. (2011) reported the lc50 values of novaluron (igr) against cs. longiareolata as 0.51 and 0.91 μg/l respectively. the lc90 values were reported as 2.32–4.30 μg/l (species was collected to 41.1 % in larval collection (bouaziz et al. 2011). conclusion cross-resistance of cs. longiareolata, cx. pipiens and cx. theileri found after exposed to ddt and lambda-cyhalothrin. also, we recommend the same procedure in field trial http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, june 2015, 9(1): 7–21 a ataie et al.: assessing the susceptibility … 16 in different parts of iran such as west azerbaijan, ardebil, tehran, fars, khuzestan, mazandaran and golestan provinces to obtain the unique conclusion about criteria for susceptibility status. the results could be useful for controlling of vector-borne diseases and dirofolaria immitis in the future. acknowledgements the authors are grateful to m mohebali and eng zare for cooperation during the study. we express our appreciation to people of the villages that helped us during the study. the authors declare that there is no conflict of interests. references abai mr (1990) fauna and seasonal activity of the sand flies and their animal reservoirs of visceral leishmaniasis in meshgin shahr county. 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[phd dissertation]. school of public health, tehran university of medical sciences, iran. http://jad.tums.ac.ir published online: july 16, 2014 j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 233 http://jad.tums.ac.ir published online: sep 30, 2022 original article molecular detection of leishmania infection in phlebotomine sand flies from an endemic focus of zoonotic cutaneous leishmaniasis in iran sadegh mohammadi-azni1, mohsen kalantari2, *behrad pourmohammadi3,4 1damghan health center, semnan university of medical sciences, semnan, iran 2research center for health sciences, institute of health, department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran 3department of parasitology, school of medicine, semnan university of medical sciences, semnan, iran 4research center for health sciences and technologies, school of health, semnan university of medical sciences, semnan, iran *corresponding author: dr behrad pourmohammadi, e-mail: behrad89@gmail.com (received 08 oct 2020; accepted 25 july 2022) abstract background: due to the outbreak of zoonotic cutaneous leishmaniasis (zcl), a disease caused by leishmania major and mainly transmitted by phlebotomus papatasi, in damghan city, semnan province, the probable vectors of the disease were investigated in the city from 20 march 2016 to 20 january 2018. methods: sand flies were collected from indoors and outdoors biweekly by sticky traps in different parts of the city. the trapped sand flies were stored in 70% ethanol. they were identified and checked for leishmania infections using nested-pcr method and specific primers; csb1xr, csb2xf, lir, and 13z. results: overall, 1862 phlebotomine sand flies of ph. papatasi (48.8%), ph. andrejevi (8.3%), ph. caucasicus (7.7), ph. mongolensis (2%), ph. sergenti (1.2%), ph. alexandri (0.7%), sergentomyia murgabiensis sintoni (29.3%), and se. sumbarica (2%) were collected indoors (31.1%) and outdoors (68.9%). the highest and lowest numbers of collected sand flies were belonging to ph. papatasi (48.8%) and ph. alexandri (0.7%) respectively. 2.2% of the examined sand flies were shown to be infected with l. major and all were belonging to ph. papatasi. conclusion: this study confirms the report of ph. papatasi infection with l. major and also the existence of ph. sergenti and ph. alexandri, the potential vectors of l. tropica and l. infantum respectively, in damghan city. according to the findings, it is necessary for health officials to plan and take action to prevent the occurrence of zcl epidemic in the city as well as the occurrence of other forms of leishmaniasis. keywords: molecular survey; leishmania major; sand fly; nested pcr; damghan introduction leishmaniasis, a protozoan parasitic infectious disease transmissible by the bite of the subfamily phlebotomine (diptera: psychodidae) sand flies and classified in the seventeenth neglected tropical diseases (ntd), occurs in tropical and subtropical areas of some 98 countries of the world (1). more than 350 million people are living in areas at risk of leishmaniasis and an estimated 2 million new cases of the disease occur annually. distribution and incidence of leishmaniasis is not the same in endemic areas. seven countries; afghanistan, algeria, brazil, iran, peru, saudi arabia and syria have about 90% of cutaneous leishmaniasis (cl) cases (2). two clinical types of cutaneous leishmaniasis including anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl) with causative agents of leishmania tropica and l. major respectively, have been reported from several parts of iran (3, 4). cutaneous leishmaniasis is the main vectorborne disease in iran with an annual average of more than 22,000 cases that about 80% of copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ file:///g:/jad/no.%2039/1330-1361%20%20%20pourmohammadi/behrad89@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 234 http://jad.tums.ac.ir published online: sep 30, 2022 them are zoonotic form. the endemic foci of this type of the disease are in rural areas of 18 out of 31 provinces (5, 6). incidence of cl in damghan district had been reported around 111 per 100,000 in 2009 (7). there are more than 800 phlebotomine sand fly species, but 166 species are proven or suspected vectors of human leishmaniasis in the world. furthermore, about 50 species of them have been found to be naturally infected with leishmania species and have been introduced as the vectors of leishmaniasis (8-10). gerbil rodents (muridae: gerbillinae) are the main reservoir hosts of zcl in the most areas of iran (5, 11, 12) including damghan district (13, 14). iranian sand flies includes 48 species in two genera of phlebotomus and sergentomyia. each genus has 6 subgenera in the country (15). the subgenus sergentomyia have no role in the transmission of leishmaniasis to human. they are vectors of lizard leishmaniasis. phlebotomus papatasi is the main vector of zcl, while ph. salehi is also found to be infected with l. major in some foci of the disease. phlebotomus salehi is mainly reported in the southern part of the country. phlebotomus ansari, ph. salehi and ph. caucasicus group are known to have a role in zoonotic cycle of cl between rodents (5, 16). phlebotomus alexandri have been reported in the most studies on sand flies in iran and it is found to be infected with l. infantum (17). damghan district in in semnan province, northern iran. it is situated 342km east of tehran on the high-road to mashad, at an elevation of 1,250m above sea level. at the 2016 census, the county's population was 94,190 in 30,296 households. this county is an important focus of zcl, with annually more than 200 new cases of the disease in the last years (18). an outbreak of the disease, with about 1500 cases, has occurred in the rural areas of damghan district in 1999 (18). rodents such as rhombomys opimus, nesokia indica and meriones libycus have been reported as the main reservoir hosts (14, 19) and ph. papatasi has been introduced as the main vector of the disease in rural areas of the district (20). at the beginning of the disease outbreak, leishmaniasis was only limited to the rural areas, but in recent years, zcl agents were detected in the patients who had previously traveled to the endemic rural areas of damghan (7, 18). this finding reinforces the hypothesis that the disease is spreading to the city and that there is a need to consider the necessary measures to prevent and control the disease. for vector control strategies, it was necessary to detect the leishmania parasites in the phlebotomine sand flies as the vectors of zcl in damghan focus. therefore, this study was designed and conducted to identify the vectors (sand fly species) and their infection with leishmania parasites in the city. materials and methods study area this descriptive cross-sectional study was conducted during 20 march 2016 to 20 january 2018 in different parts of damghan city, in semnan province. damghan is located in 1170m above sea level and 350km east of the capital, tehran, in northern part of iran (13). the city is situated between shahroud and semnan cities and is bounded on the mountainous areas in the north (alborz mountains) with 9.8 ℃ and the plain areas in the south (kaveer desert) with 23.5 ℃ average temperatures. the annual rainfall and average temperature of the city are about 120mm and 16.3 ℃ respectively (21). the city has generally a warm weather in summer and cold in winter. sand fly collection sand flies were collected from indoors (bedroom, guestroom, toilet and stable) and outdoors (e.g. rodent burrows and wall cracks) biweekly by sticky traps in different parts of the city. in each sampling, 300 sticky paper traps (30 indoors and 30 outdoors) were fixed in the sunset and collected in the next morning before sunrise. phlebotomine sand flies were collecthttp://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/iran https://en.wikipedia.org/wiki/tehran https://en.wikipedia.org/wiki/mashad j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 235 http://jad.tums.ac.ir published online: sep 30, 2022 ed and kept in ethanol (70%). the head and last abdominal segments of female sand flies were removed and mounted on a microscope slide in a drop of puri’s medium, and taxonomically identified according to valid taxonomic criteria (22). each sample identified at species level, according to the standard identification keys (23, 24). the remaining portion of the female sand flies′ bodies were subjected to dna extraction and molecular detection of leishmania infection. molecular identification totally 179 samples of the female sand flies were selected for molecular detection of leishmania infection. each female sample was separately checked for leishmania infection by nested-pcr assay using specific primers (25). dna extraction each sample were homogenized in a mixture of 200μl lysis buffer (50μl tris-hcl [ph 7.6], 1% tween 20 and 1μl edta), 12μl of proteinase k solution (19μl of the enzyme/ ml), in a 1.5ml sterile microcentrifuge tube. the homogenate incubated at 37 °c overnight, and then 300μl phenol: chloroform: isoamyl alcohol mixture (25:24:1, by vol.) were added. after shaking vigorously, the mixture was centrifuged (10,000 rpm for 10min). the extracted dna in the supernatant was precipitated with 400μl of cold and pure ethanol, resuspended in 50μl of double-distilled water, and stored at -20 °c until using for detection of leishmania parasites′ kdna. amplification of kinetoplast minicircle dna nested pcr assay was carried out in two rounds using the csb1xr (att ttt cgc gat ttt cgc aga acg) and csb2xf (cga gta gca gaa act ccc gtt ca) primers for the first round and lir (tcg cag aac gcc cct) and 13z (act ggg ggt tgg tgt aaa atag) for the second round. first, a total reaction mixture (25μl) was prepared which contained 5μl of template dna, 12μl master mix (containing deoxynucleoside triphosphate (sinaclon, tehran, iran), taq polymerase, mgcl2, tris-hcl (ph 7.6)), 1μl of csb1xr and 1μl of csb2xf, and 6μl of doubled distilled water (ddw). pcr reaction protocol was set in a thermocycler (eppendorf ag; humbug, germany). the temperature program was set at 94 °c for 5min for the first extension, followed by 30 cycle (which was repeated at 94 °c for 30s, 55 °c for 1min, and 72 °c for 1.5min), and then a final extension at 72 ° c for 10min. for the second round, 1μl of the first-round products’ dilution (1:9, by vol.) was used as the templates. the reaction for the second round was the same as the first round with an exception in the volume of the reaction mixture changed to 30μl (as the ddw volume was changed from 6μl to 11μl for the second round), and the use of 13z and lir primers. finally, 5μl of the final products were run on 1.5% (v/v) agarose gel marked with ethidium bromide and visualized by ultraviolet trans-illumination. the size of bands was estimated by comparison with the size of the reference strains (12, 25). reference strains of l. major (mhom/ir/ 54/lv 39), l. infantum (mcan/ir/96/lon 46) and l. tropica (mhom/ir/89/ard 2) were used as standards from department of medical parasitology, shiraz university of medical sciences. also, double-distilled water was included in each run as a negative control. a band of 560bp, 680bp, and 750bp indicated the presence of l. major, l. infantum and l. tropica respectively. results totally, 1862 sandflies (822 females and 1040 males) including 8 species (six phlebotomus spp. and two sergentomyia spp.) were trapped and identified in a period of 22 months. they were belonging to; ph. papatasi (48.8%), ph. mongolensis (2%), ph. caucasicus (7.7%), ph. sergenti (1.2%), ph. alexandri (0.7%), ph. andrejevi (8.3%), se. murgabiensis sintoni (29.3 %) and se. sumbarica (2%) (table 1). http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 236 http://jad.tums.ac.ir published online: sep 30, 2022 the sand flies′ sex ratio (ratio of male to female) was 126.5/100. totally, 580 (31.1%) and 1282 (68.9%) specimens were collected from the indoor and the outdoor places respectively. 43.2% of the sandflies collected from the outdoors and 61% from the indoors were belonging to p. papatasi. 52.3 percent of the abdominal status of sand flies were empty, and the others were blood fed (10.3%), gravid (16%), and semi gravid (21.4 %) (table 1). four (2.2%) of the 179 molecularly examined sand flies were shown to be infected with l. major parasites with band size about 560bp (figure 1). of which, one was from indoor, 2 from rodent barrows and another one was from outdoor. 75% (3/4) of infected sandflies were collected in august and september, but 25% (1/4) were trapped in june. table 1. details of sand flies collected in damghan city, 2016–2018 outdoor (%) indoor (%) abdominal status no. (%) female no. (%) male no. (%) total no. (%) species gravid semi-gravid empty blood fed 554 (61) 354(39) 96 (26) 75 (20.4) 147 (40) 50 (13.6) 368 (40.5) 540 (59.5) 908 (48.8) phlebotomus papatasi 35 (94.6) 2(5.4) 1 (12.5) 2 (25) 3 (37.5) 2 (25) 8 (21.6) 29 (78.4) 37 (2) phlebotomus mongolensis 122 (85.3) 21(14.7) 3 (11.1) 7 (26) 11 (40.7) 6 (22.2) 27 (18.9) 116 (81.1) 143 (7.7) phlebotomus caucasicus 12 (100) 2 (100) 2 (16.7) 10 (83.3) 12 (0.7) phlebotomus alexandri 15 (68.2) 7(31.8) 4 (100) 4 (18.2) 18 (81.8) 22 (1.2) phlebotomus sergenti 142 (91) 14(9) 5 (16.7) 8 (26.6) 12 (40) 5 (16.7) 30 (19.2) 126 (80.8) 156 (8.3) phlebotomus andrejevi 364 (66.7) 182(33.3) 26 (7.2) 82 (22.7) 235 (65.1) 18 (5) 361 (66.1) 185 (33.9) 546 (29.3) sergentomyia murgabiensis sintoni 38 (100) 1 (4.5) 2 (9) 16 (72.8) 3 (13.7) 22 (57.9) 16 (42.1) 38 (2) sergentomyia sumbarica 1282 (68.9) 580 (31.1) 132 (16) 176 (21.4) 430 (52.3) 84 (10.3) 822 (44.1) 1040 (55.9) 1862 total fig. 1. gel electrophoresis of nested-pcr products of leishmania major kdna in phlebotomus papatasi caught from urban areas of damghan, 2016–2018. the wells correspond to l. major in ph. papatasi (1-4), reference strains of l. major (5), negative control (dw) (6), and molecular weight marker (7) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 237 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 2. map of the distribution of leishmania major infected sand flies in damghan city, semnan province, iran discussion the identification of eight sandflies species in this study showed that these insects have a relatively high diversity in the city. so far, 44 species of both genera phlebotomus and sergentomyia of sand flies have been identified and reported in iran (5, 20). only four of the iranian sand fly species were shown to be infected with leishmania parasites. promastigotes of leishmania parasites have been found and reported in 13 various species of these significant biologic vectors from endemic foci of cutaneous and visceral leishmaniasis (5). in the present study ph. papatasi was dominant species and had the highest abundance in indoor and outdoor places and in rodent burrows. this species of sand flies, in most other studies, has also shown the highest frequency and they have been collected and reported from various parts of the country with an eight to 1756m altitude range (5). the study area, damghan city, with an altitude of 1170m, arid and semiarid climate is a suitable place for this species to survive. besides climatic situations, some other factors such as the construction of new buildings and accumulation http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 238 http://jad.tums.ac.ir published online: sep 30, 2022 of their wastes in different parts of the city, the presence of reservoir rodents in those places may affect the spread of the disease. phlebotomus papatasi is spread in the arid and semi-arid zones of mediterranean, europe, north africa, middle east, and the indian subcontinent (11, 26-28) and it has been reported as a dominant species in other cutaneous leishmaniasis foci in iran, notably in rural areas of the country and has been introduced as proven vector of zcl (11, 29-33). among the molecularly examined sandflies, only the species of ph. papatasi were shown to be infected with l. major parasites (4 out of 147; 2.7%). similar infection rates have been reported by other researchers from various parts of the country (5, 20, 32, 34). other species of the examined sand flies were not observed to be infected with l. major parasites. furthermore, natural infection of ph. papatasi and nesokia indica with l. major has previously been reported from rural areas of damghan district (13, 20). based on the detection of l. major in ph. papatasi collected from the studied area and the presence of zcl human cases in this region, it may confirm the zcl cycle in damghan county. another species of the identified sand flies in this study, ph. sergenti, were not shown to be infected with leishmania parasites. phlebotomus sergenti is the main vector of acl in iran and many other countries (35-39). based on the presence of ph. sergenti as the potential vector of acl in damghan city, further epidemiological study is recommended on human and animal reservoirs such as dogs. identification of ph. alexandri was another important finding of this survey. natural infection of ph. alexandri with l. infantum as the causative agent of visceral leishmaniasis (vl) has already been reported from iran (17, 40), and it has been introduced as vector of the disease in some other parts of the world (41-45). moreover, the authors have recently reported the natural infection of nesokia indica with l. infantum in the studied area (11). therefore, beside of animal-sand fly-animal cycle of zoonotic visceral leishmaniais, animal-sand fly-human cycle of the disease can be expected in the studied region. phlebotomus caucasicus accounted for 6.2% of the surveyed sandflies. none of the species was found to be infected with l. major in this study. in a study, 3.3–20% of examined ph. caucasicus collected from rodent barrows have been reported to be infected with l. major promastigotes in central part of iran (4, 20, 40). most of the studied female sand flies (52.3 %) with digestive tract were checked for pcr. of those, the largest number was belonging to ph. papatasi and the largest number of infected sand flies (75%) was observed in the same specimens. in these sand flies, the parasites have passed blood digestion stages behind, and more importantly, they have been able to pass the peritrophic membrane (46). in the current study, 75% of infected sand flies were trapped from rodent's burrows and outside places. therefore, it is recommended that local people wear adequate dress and apply insect repellent to avoid bites outsides. in homes also the use of screens and bed nets impregnated with insecticide to prevent disease is recommended. conclusion this study confirms the report of ph. papatasi infection with l. major from damghan city by other researchers (20). this finding could justify the reason for zcl of people dwelling in the city with any history of travelling to the zcl foci. moreover, the existence of the other species of sand flies, including ph. sergenti and ph. alexandri, the vectors of l. tropica and l. infantum respectively, raises the possibility of future outbreaks of anthroponotic cutaneous leishmaniasis and visceral leishmaniasis in the study area. therefore, further studies accompanied by designing and performing the vector control programs in the city are seriously recommended. http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 233–242 s mohammadi-azni et al.: molecular detection of … 239 http://jad.tums.ac.ir published online: sep 30, 2022 acknowledgements this study was financially supported by semnan university of medical sciences (project no.731). we would like to extend our appreciation to the research deputy of semnan university of medical sciences. ethical considerations ethical approval for this study was obtained from the ethics committee at semnan university of medical sciences, iran (ir.semums. rec.1397.136). conflict of interest statement authors declare that there is no conflict of interest. references 1. naghian a, oshaghi ma, moein-vaziri v, rassi y, sedaghat mm, mostafavi e, veysi a, soleimani h, dehghan h, zahraei ramazani ar, mirhendi h, amini mh, yaghoobi-ershadi mr, akhavan aa (2020) molecular identification of leishmania species in phlebotomus alexandri (diptera: psychodidae) in western iran. j arthropod borne dis. 14(1): 8–16. 2. azari-hamidian s, norouzi b, maleki h (2023) the checklist and distribution of sand flies (diptera: psychodidae) of guilan province and their medical importance with a taxonomic note on the name sergentomyia murgabiensis sintoni. caspian 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bull world health organ. 64(1): 107–112. 46. baharshahi a, behravan m, keikhosravi a (2018) morphological identification of sand flies and their species’ diversity. arch adv biosci. 9(4): 35–41. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 116 http://jad.tums.ac.ir published online: march 31, 2020 original article monitoring of laboratory reared of phlebotomus papatasi (diptera: psychodidae), main vector of zoonotic cutaneous leishmaniasis to different imagicides in hyper endemic areas, isfahan province, iran leila shirani-bidabadi1; ali reza zahraei-ramazani2; mohammad reza yaghoobi-ershadi2; amir ahmad akhavan2; mohammad ali oshaghi2; ahmad ali enayati3; yavar rassi2; fatemeh gholampour2; niloufar shareghi4; elham madreseh5; *hassan vatandoost2,6 1 department of vector biology and control, school of health, kerman university of medical sciences, kerman, iran 2 department of medical entomology and vector control,school of public health, tehran university of medical sciences, tehran, iran 3 department of medical entomology and vector control, school of public health, mazandaran university of medical sciences, mazandaran, iran 4 isfahan health research station, national institute of health research ,tehran university of medical sciences, tehran, iran 5 department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran 6 department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran (received 06 mar 2020; accepted 30 mar 2020) abstract background: in domestic and per domestic area,insecticides such as ddt, malathion, fenitrothion, propoxur and, more recently, synthetic pyrethroids such as deltamethrin and lambda-cyhalothrin, have been successfully used to control sand flies in many countries. the present study reports the results of time-mortality bioassay to ddt 4%, lambdacyhalothrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and deltamethrin 0.05% in recently colonized phlebotomus papatasi populations in iran. methods: the insecticide susceptibility status of p. papatasi laboratory population was assessed during 2016–2017, following the standard who technique for mosquito (who, 2013) based on diagnostic dose. sand flies collected from rural area of badrood (matin abad), natanz county, esfahan province, using aspirator. results: susceptibility test to ddt and pyrethroids was assessed on 3534 laboratory-reared p. papatasi (1746 females and 1788 males). the lt50 and lt90 values were measured using probit analysis and regression lines. the test results against males of p. papatasi revealed that lt50 values to ddt 4%, permethrin 0.75%, deltamethrin 0.05%, cyfluthrin 0.15% and lambdacyhalothrin 0.05% were 439.28, 108.90, 97.75, 5.00 and 57.84 seconds. the figures for females were 641.62, 136.15, 146.44, 8.71 and 72.69 seconds , respectively. conclusion: according to presented results, the reared population of sand flies collected from a hyper-endemic region of esfahan province is still susceptible to prethroids and resistance candidate to ddt 4%. keywords: phlebotomus papatasi; bioassay; insecticide resistance; rearing introduction phlebotomine sand flies (diptera, psychodidae) are hematophagous insects involved in the transmission of viruses (bunyaviridae, reoviridae and rhabdoviridae), bacteria (bartonella bacilliformis) and protozoa (leishmania spp.) to animals and humans (1-3). among protozoa, leishmania spp. are recognized as pathogenic to humans, causing different clinical forms: visceral (vl), cutaneous (cl), mucocutaneous, post-kala-azar dermal and mucosal leishmani*corresponding author: dr hassan vatandoost, e mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 117 http://jad.tums.ac.ir published online: march 31, 2020 asis (4). leishmaniases are neglected diseases worldwide distributed, occurring mainly in tropical and subtropical zones (5). cutaneous leishmaniasis (cl) is caused by leishmania major, l. tropica or l. infantum, while vl is caused by l. infantum (6). there are 56 species (32 phlebotomus and 24 sergentomyia) of phlebotomine sandflies in iran but phlebotomus papatasi is the main vector of zoonotic cutaneous leishmaniasis (zcl) (7-10). few studies have been performed on the level of susceptibility of sandflies reared in the laboratory to common insecticides used in agriculture and health in world. hassan et al. (2012) in sudan studied insecticide susceptibility status of first progeny (f1) of p. papatasi to ddt, permethrin, malathion, and propoxur. this study results showed sand flies were resistance to malathion and propoxur (11). in iran sand flies p. papatasi which were collected from a hyper endemic focus of the disease in central iran breed under laboratory condition and determine sand flies susceptibility level to commonly used insecticides (12). denlinger et al. in 2016 showed that p. papatasi and lutzomyia longipalpis sand flies are highly susceptible to the carbamates as their diagnostic doses are under 7.0μg/ml. both species are also highly susceptible to ddt during the exposure assay as their diagnostic doses are 7.5μg/ml, yet their diagnostic doses for the 24h recovery period are 650.0μg/ml for lu. longipalpis and 470.0 μg/ml for p. papatasi (13). italian populations of p. perniciosus and p. papatasi from campania region and from rome, respectively, were susceptible to the insecticides ddt 2%, lambda-cyhalothrin 0.06% and permethrin 0.2% as compared with the reference strain used (14). the present study reports the results of timemortality bioassay to ddt 4%, lambda-cyhalothrin 0.05%, permethrin 0.75%, cyfluthrin 0.15 % and deltamethrin 0.05% in recently colonized p. papatasi populations in iran. materials and methods study area this study was performed during the summer of 2016, 2017 and sand flies were caught from the rural district (matin abad) of badrood (33˚ 44́ n, 52˚ 2 ́ e), natanz county, esfahan province, central of iran. the area is located in the desert with hot and dry weather in summer and quite cold in winter. this zone is a zoonotic cutaneous leishmaniasis (zcl) focus in iran. in this area were exsistence many farm lands. crops such as wheat, sunflower, alfalfa, clover and other are planted in the area. due to intensive use of insecticides for agriculture pests, the leishmania vectors are exposed to insecticide selection pressures. collected sand flies from field sand flies were collected outdoor near their breeding places using hand aspirator device from sunset to midnight during the period of june to august 2016, 2017. collected alive sand flies were released into a clean cage with a hanging piece of wet cloth for supplying suitable humidity and feeding on a 20% sucrose solution soaked cotton. sand flies cage were placed in a plastic bag to remain wet and to keep stable temperature situation. the cages were transported to sand fly insectary in esfahan health research station, tehran university of medical sciences. appropriate condition for rearing and inbreeding of sand flies in laboratory were 25±2 ˚c and 72±9.6% relative humidity in insectary and 90±7% rh in rearing box (15). temperatures were maintained by automated electric heaters and photoperiod of 14/10 d/l was maintained in the insectary. sand fly rearing method after resting sand flies in insectary, fed and gravid wild-caught female adults were separated by aspirator and were released into individual pots according to volf and volfa method (16) and were fed with honey solution (50 %) and saturated sucrose. engorged female were fed on blab/c that anesthetized with 0.2 cc kethttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 118 http://jad.tums.ac.ir published online: march 31, 2020 amine and xylazine for 30 minutes per mice. after feeding, blood-engorged sand flies were individually put in oviposition vials lined at the bottom with plaster of paris and covered with mesh. the vials containing females were then maintained at 26–28 ˚c and 72±10% rh. after oviposition females died, then females were removed from oviposition vials and were preserved individually in 70% alchohol for mounting and identification up to the species level by using proper identification entomology keys (17). females and males at least one week after mounting, were identified using valid key and if there were any other species, all of them were excluded from the tests. males and females of p. papatasi females were separated from other species for rearing. the pots were checked daily for hatching the eggs. the larvae (l1) were fed with larval food, complex of rabbit food (palette) and rabbit feces with liver powder (18). for mass rearing of sand flies used larger pots that linned with plaster of paris, then we transferred 20–30 blood feeding females with 5–10 males. emerged adults were released in a new cage with wet cloth and sucrose solution (20%). all of them were placed in a plastic bag to remain wet and to maintain stable temperature situation. then the 3–10 days old adults were tested in a standard who susceptibility test method as described for mosquitoes (19). problems with sand fly breeding we have many problems in mass rearing of sand flies in insectarium including: collection of sand flies from field because of wind in night and decrease temperature in midnight, decrease of density of sand flies due to interventions control of esfahan deputy of health and provincial health center in study areas in recent year, preparation of proper humidity and temperature in insectary for sand fly rearing, fungal contamination in all of stage, mites, cannibalism (especially in first larval stage) diapause of fourth larval stage. in caused occasional contamination of food with fungi belonging to genus aspergillus, mucor and cladosporium, mites (super cohort: monogynaspida), insects from the order psocoptera and ants were observed during any stage of rearing process (18). our serious problems have been fungi such as mucor sp., cladosporium sp. and aspergillus sp., bacteria such as pseudomonas sp., salmonella sp., diphteroide sp. and mites (order mesostigmata). susceptibility bioassays of reared sand flies susceptibility test against specimens reared in laboratory condition, were carried out in summer of 2016, 2017. the susceptibility tests were carried out on 3534 laboratory-reared p. papatasi (1746 females and 1788 males). who testkit tubes and impregnated papers were procured from collaborating center of who in malaysia. all the susceptibility tests were done according to standard who testing protocols on unfed female sand flies using at least 8–15 specimens in per test. emerged sand flies transferred to cages, after resting and feeding by sucrose solution (20%), sand flies were tested according to the standard method of who (19). the sand flies were transferred into the exposure tubes by aspirator and were gently transferred to the holding tube at different time intervals and then the mortality was counted after 24h recovery period. after exposure period sand flies fed with 20% sugar solution placed in the top of the holding tube. control test tubes carrying control papers were also held parallel to each set of tests. all the tests were ignored if the mortality was higher than 20% in the control group. the test was done in four to six replicates for each insecticide. during these bioassays tests, laboratory condition was stabilized at (25±2 ˚c and 73±10% rh) in insectary. the resistance status of sand fly specimens was determined according to the latest who criteria (19) as follows, (a) mortality rates between 98– 100% indicate full susceptibility, (b) mortality rates between 90–97% require further investigation, (c) mortality rates < 90 %, the population is considered resistant to the tested insecticides (19). after each test, all the dead and alive sand flies were transferred to 70% alcohol separatehttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 119 http://jad.tums.ac.ir published online: march 31, 2020 ly for subsequent study. the exposure time interval was between 7, 14, 28, 56, 113, 225, 450, 900, 1800 and 3600 seconds. at least 8– 10 interval times were used to gain the mortality between 5 and 95%. in each exposure time at least 4–6 replicates were used comprising 50– 100 sand flies depending on the availability and the same age of the adults. abbott’s formula was not used to correct experimental mortalities if the control group mortality was <5%. if control group mortalities exceeded 20%, the entire testing replicate was not used (20). insecticides papers used in bioassay test impregnated papers with different diagnostic dosages of ddt 4% (batch number: dd 214), permethrin 0.75% (batch number: pe 340), deltamethrin 0.05% (batch number: de 432), lambda-cyhalothrin 0.05% (batch number: la215) and cyfluthrin 0.15% (batch number: cy098) were taken from collaborating center of who in malaysia. obtaining data and analyses the exposure time versus probit mortality were used according to finney 1971. the excel 2013 was used for data entering. data analysis was done with statistic software spss version 22. excel software version 2013 was used for drawing the regression line. results identification of sand flies all females and males specimens (2942 number) used for the establishment of the colonies of sand flies from the badrood population were identified by avalaible key morphologically as p. papatasi. bioassay test of laboratory sand flies in this study 2942 sand fly specimens collected from study area were reared in insectary. the results of susceptibility test against laboratory -reared p. papatasi female revealed lt50 values to ddt (4%), permethrin (0.75%), deltamethrin (0.05%), cyfluthrin (0.15%) and lambda-cyhalothrin (0.05%); 641.62, 136.15, 146.44, 8.71, 72.69 seconds, respectively (table 1). this data for males to ddt (4%), permethrin (0.75%), deltamethrin (0.05%), cyfluthrin (0.15%) and lambda-cyhalothrin (0.05%) were 439.28, 108.90, 97.75, 5.00, 57.84 seconds respectively (table 2). figures 1, 2 show the probit regression lines. the results showed that males were more susceptible than females to all the insecticides tested at lt50 level (fig. 3). the results showed that males were more susceptible than females to all the insecticides tested at lt50 level (fig. 3). table 1. parameters of probit regression lines of different insecticides against female phlebotomus papatasi, laboratory strain, esfahan, iran, 2016–2017 insecticide a a b±se b lt50 c 95%cl (seconds) lt90 d 95%cl (seconds) χ 2 (df) heterogeneity p-value y=a+bx ddt 4% -4.3272 1.5414 ±0.514 350.23 641.62 1283.51 1822.01 4352.28 196819.15 3.281 (2) p> 0.05 y= -4.3272+ 1.5414 x permethrin 0.75% -2.8509 1.9533 ±0.499 87.63 136.15 189.47 778.65 1239.85 2667.51 25.164 (4) p< 0.05 y= -2.8509+ 1.3359 x deltamethrin 0.1% -2.1764 1.0049 ±0.259 13.82 146.44 604.09 651.23 2760.47 2254569.23 13.600 (4) p< 0.05 y= -2.1764+ 1.0049 x http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 120 http://jad.tums.ac.ir published online: march 31, 2020 cyfluthrin 0.15% -0.5400 0.5743 ±0.114 2.50 8.71 18.69 434.62 1485.06 19486.52 9.183 (5) p> 0.05 y= -0.5400+ 0.5743 x lambdacyhalothrin 0.05% -4.0834 2.6391 ±0.410 52.59 72.69 101.65 176.75 279.09 675.84 0.091 (2) p> 0.05 y= -4.0834+ 2.1936 x a a= intercept b b±se= slope and its standard error c lt50, 95% c.l.= leathal time cause 50% mortality and its 95% confidence limits d lt90, 95% c.l.= leathal time cause 90% mortality and its 95% confidence limits 1 2 3 4 5 6 7 8 1 10 100 1000 10000 time(second) p r o b it ddt lambdac yhalothrin cyfluthrin deltamethrin permethrin fig. 1. probit regression lines of different insecticides against female of phlebotomus papatasi laboratory population, esfahan, iran, 2016–2017 1 2 3 4 5 6 7 8 1 10 100 1000 10000 time(second) p r o b it ddt permethrin deltamethrin lambdac yhalothrin cyfluthrin fig. 2. probit regression lines of different insecticides against male of phlebotomus papatasi laboratory population, esfahan, iran, 2016–2017 table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 121 http://jad.tums.ac.ir published online: march 31, 2020 table 2. parameters of probit regression lines of different insecticides against male phlebotomus papatasi laboratory population, esfahan, iran, 2016–2017 insecticide a a b±se b lt50 c 95% c.l (seconds) lt90 d 95% c.l (seconds) x 2 (df) p-value y= a+bx ddt 4% -3.2516 1.2304±0.412 4.700 (3) p< 0.05 y= -3.2516+ 1.2304x 439.28 4834.64 permethrin 0.75% -2.5612 1.2573±0.182 76.00 667.98 5.524 (4) p> 0.05 y= -2.5612+ 1.2573x 108.90 1138.58 148.41 2787.51 deltamethrin 0.1% -1.7559 0.8823±0.297 0.02 444.34 18.26 (4) p< 0.05 y= -1.7559 +0.8823x 97.75 2771.37 1022.94 cyfluthrin 0.15% -0.3278 0.4686±0.105 0.50 608.75 1.73 (5) p> 0.05 y= -0.3278+ 0.4686 x 5.00 2720.51 16.08 80988.38 lambdacyhalothrin 0.05% -1.7522 0.9943±0.198 30.78 422.65 0.213 (2) p> 0.05 y= -1.7522+ 0.9943 x 57.84 1125.10 109.68 8755.23 a a= intercept b b±se= slope and its standard error c lt50, 95% c.l= leathal time cause 50% mortality and its 95% confidence limits d lt90, 95% c.i= leathal time cause 90% mortality and its 95% confidence limits fig. 3. lt50 values of different insecticides against male and female of phlebotomus papatasi laboratory populations, esfahan, iran, 2016–2017 discussion due to the lack of a suitable guide line for sand flies, we had to use protocols of who for mosquito (19) in this study, the who expo sure kit bioassay is widely accepted because it can measure insecticide susceptibility in many species of insect vectors worldwide (21). the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 122 http://jad.tums.ac.ir published online: march 31, 2020 assays can be run with live insects collected in the field or with their progeny reared in the laboratory. to control sand flies, populations around the world have been exposed to the four main classes of insecticides; 1) organochlorines, 2) organophosphates, 3) carbamates, and 4) pyrethroids,via residual spraying, ultra-low volume spraying, insecticide-treated clothing, and insecticide-treated nets. these exposures are either intentional in directed vector control efforts or are inadvertent as part of vector control efforts targeted against other insect vectors (22). some sand fly populations have been found to be tolerant or resistant to the insecticides used in the middle east, southern asia, and south america. in montes claros, brazil, 29 of 80 (36.3%) lu. longipalpis (lutz and nieva) survived a 0.05% deltamethrin exposure (23). hassan et al. (2012) in the surogia village of khartoum state, sudan, reared sand flies in laboratory and they tested with malathion and propoxur. results showed that 51 p. papatasi (79.7%) had insensitive acetylcholinesterase, which is associated with malathion and propoxur resistance. both of these insecticides have been extensively used in this region as part of the anti-malaria mosquito control program (11). denlinger et al. (2015) results showed that both laboratory sand flies l. longipalpis and p. papatasi are susceptible to ddt (24). similar results with our study have been found in insecticide-susceptible italian sand flies reared in laboratory (p. perniciosus and p. papatasi), where the lt50 and lt90 for ddt were longer compared with permethrin and lambdacyhalothrin (14). also, saeidi et al. (2012, 2013) found both insecticide-susceptible male and female p. papatasi field and laboratory population to have much longer lt50 and lt90 to ddt than to permethrin, deltamethrin, cyfluthrin, and lambda-cyhalothrin (6, 12). denlinger results suggested that laboratory colonies of insecticidesusceptible sand flies were not very susceptible to ddt (24). shirani-bidabadi et al. (2017) found that male and female p. papatasi field population to exposure to insecticides had much longer lt50 and lt90 to ddt than to permethrin, deltamethrin, cyfluthrin, and lambda-cyhalothrin (20). our results of the tests at different durations of exposure indicated that laboratory sand fly populations from esfahan province were fully susceptible to pyrethroid insecticides used, whereas the early tolerance (resistant candidate) was detected to ddt 4% after 3600s (1h) contact in the population of esfahan province. for both males and females, the susceptibility levels to ddt4% were greater than to pyrethroids. the high lt50 level of this vector to ddt was attributed to the long term use of insecticides for malaria vector control in the region that was transmitted genetically to their progeny. according to the report of the branch of the ministry of jahad in esfahan province, several herbicides, fungicides and inesticides have been used for agriculture and veterinary pest control in the region, including methalaxile, carbaryl, permethrin, cypermethrin, deltamethrin, metasystox-r and mancozeb. the susceptibility test of a laboratory strain of p. papatasi to ddt4% and pyrethroids in badrood shows that males were more susceptible than females to all the insecticides tested at lt50 level. in comparing our results and the study by saeidi et al. (2013), lt50 values of female and male to ddt 4% was greater than lt50 in this study, males were more susceptible to pyrethroids and the sand flies were more tolerant compared to our results. lt50 values in males and females in our study to ddt 4%, permethrin 0.75%, and cyfluthrin 0.15% were also smaller compared to their results, but lt50 values in males and females to deltamethrin 0.1% and lambda-cyhalothrin 0.05% in our study were greater than theirs (12). this difference was due to the presence of deltamethrin and lambda-cyhalothrin in agriculture fields in recent years in matin-abad. conclusion we observed a clear difference between the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2020, 14(1): 116–125 l shirani-bidabadi et al.: monitoring of … 123 http://jad.tums.ac.ir published online: march 31, 2020 insecticide susceptibilities of the p. papatasi laboratory population that had been exposed to insecticides in varying times intervals, this difference in susceptibility varied with sex. the result presented in this study can serve as starting points for determining the susceptibility of laboratory-reared p. papatasi, for determining diagnostic times for other sand fly species of public health concern. knowing if a population of sand fly is resistant or resistance candidate to an insecticide or insecticide class is critical because it allows control strategies to be effectively implemented while not exacerbating the prevalence of insecticide resistance or resistance candidate in distribution area of sand flies. according to presented results,the reared population of sand flies collected from a hyper-endemic region of esfahan province is still susceptible to pyrethroids and resistance candidateto ddt 4%. acknowledgements we are grateful to all staff of esfahan health research station, national institute of health research, tehran university of medical sciences. this research was financially supported by school of public health tehran university of medical sciences (tums), iran. under code number 95-01-27-30104 and this study protocol has been approved by local institutional review board including the approval no. ir. tums.rec.1394.11.15. the authors declare that there is no conflict of interest. references 1. maroli m, feliciangelli md, bichaud l, charrel rn, gradoni l (2013) phlebotomine sand flies and the spreading of leishmaniases and other diseases of public health concern. med vet entomol. 27(2): 123– 147. 2. gaglio g, brianti e, napoli e, falsone l, dantas-torres f, tarallo vd, otranto d, giannetto s (2014) effect of night timeintervals, height of traps and lunar phases on sand fly collection in a highly endemic area for canine leishmaniasis. acta trop. 133: 73–77. 3. halada p, hlavackova k, dvorak v, volf p (2018) identification of immature stages of phlebotomine sand flies using maldi-tof ms and mapping of mass spectra during sand fly life cycle. insect biochem mol biol. 93: 47–56. 4. akhoundi m, kuhls k, cannet a, voty 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longipalpis and phlebotomus papatasi sand flies (diptera: psychodidae: phlebotominae). j med entomol. 52(5): 1003–1012. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=gontijo%20nf%5bauthor%5d&cauthor=true&cauthor_uid=19772549 https://www.ncbi.nlm.nih.gov/pubmed/?term=gontijo%20nf%5bauthor%5d&cauthor=true&cauthor_uid=19772549 https://www.ncbi.nlm.nih.gov/pubmed/?term=reithinger%20r%5bauthor%5d&cauthor=true&cauthor_uid=19772549 j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 363 http://jad.tums.ac.ir published online: december 31, 2020 original article treatment failure in cutaneous leishmaniasis patients referred to the school of public health, tehran university of medical sciences during 2008–2017 zahra kakooei1; homa hajjaran1; *behnaz akhoundi1; sorour charehdar1; samira elikaee1; zahra shafeghat1; hamid hassanpour1,3; mohammad taghi satvat1; elham kazemi-rad1; *mehdi mohebali1,2 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran 3department of medical parasitology, school of medicine, ilam university of medical sciences, ilam, iran *corresponding authors: dr behnaz akhoundi, e-mail: behnazakhoundi@yahoo.com, dr mehdi mohebali, email: mohebali@tums.ac.ir (received 05 nov 2019; accepted 03 dec 2020) abstract background: cutaneous leishmaniasis (cl) is a vector borne disease predominantly found in tropical and subtropical countries, including iran. for more than 6 decades, pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis, but over the past few years, clinical resistance to these medications has increased. in this study, we evaluated cl patients who did not show any desirable responses to the anti-leishmanial treatment within a 10year period (2008 to 2017). methods: all patients from different parts of iran suspected of having cutaneous leishmaniasis, who were referred to the laboratory of leishmaniosis in tehran university of medical sciences from 2008–2017 were parasitological examined. results: during this period, a total of 1480 suspected cl patients were referred to the laboratory of leishmaniosis. samples from 655 patients (70.8%) suspected of having cl were positive microscopically. the failure rate in patients treated with anti-leishmaniasis medications for a minimum of three complete treatment periods was 1.83% (12 cases). there was no association between the number and size of skin lesions and patient characteristics. also, the route of drug administration had no significant effect on the number and size of lesions. conclusion: in the present study, treatment failure was found in some confirmed cl patients treated with meglumine antimoniate. over the past few years, it seems that had been increased in resistance to these medications. so, a review of the correct implementation of the treatment protocol and/or a combination therapy may be helpful in preventing an increase in the rate of treatment failure. keywords: cutaneous leishmaniasis; anti-leishmania drug; treatment failure; iran introduction protozoan parasites of the genus leishmania cause a wide spectrum of clinical manifestations known as leishmaniases. cutaneous leishmaniasis (cl) is the most common forms of this disease in the world, with more than 350 million people at risk. there is an estimated incidence of 0.9–1.7 million new cases each year (1-2). in iran, two forms of cl have been reported: zoonotic cutaneous leishmaniasis (zcl) and anthroponotic cutaneous leish maniasis (acl). the main pathogen of zcl in iran is leishmania major (l. major), whereas acl is mainly due to leishmania tropica (l. tropica) infections (3). for more than 6 decades, pentavalent antimonial (sbv) compounds, such as meglumine antimoniate (glucantime®) and sodium stiboglucunate (pentostam®), have been successfully used as the first-line treatment for all forms of leishmaniasis (4). in iran, the national treatment protocol for cutaneous leishmaniasis recommends intramuscularly administered 20mg sbv5/kg body weight per copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:behnazakhoundi@yahoo.com mailto:mohebali@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 364 http://jad.tums.ac.ir published online: december 31, 2020 day. the recommended treatment duration is 14 days for zcl and 21 days for acl (5). however, over the past few years, there has been increasing concern about the resistance of parasites to glucantime® (6). anti-leishmania resistance and treatment failure is a major challenge in new and old-world countries (7-9). resistance of l. tropica to glucantime® was first reported in iran in 2006 (10). previously identified risk factors for the failure of treatment with glucantime® are body weight above 68kg, previous anti-leishmania treatment, having ≥ 3 skin lesions, and failure to complete a course of treatment (11). in this study, we evaluated cases of treatment failure in cl patients who were referred to the leishmaniosis laboratory of tehran university of medical sciences (tums) between 2008–2017 (10 years) (12, 13). materials and methods patients all patients suspected of having cutaneous leishmaniasis, who were referred to the leishmaniosis laboratory of tums between 2008– 2017 were considered eligible for inclusion in this study. parasitological confirmation was performed after gaining accurate information about the place of living of patients. after sterilizing the skin around the lesions/nodules with ethanol 70%, a small incision was made in the margin of the lesion with a disposable lancet, and some tissue and exudates were removed by scraping. the scrapings from the margins of the lesions were air dried, fixed in absolute methanol, and stained with giemsa 10%. the specimens were then examined for amastigotes demonstration by light microscopy with high magnification (14). design the following data were obtained and recorded for each patient: age, sex, history of travel to endemic areas of leishmaniasis, number of lesions, size of the largest lesion, duration of infection, and adverse events. in this study, we focused more on cl patients who failed to respond to treatment, and who remained positive for smear prepared from the lesions after receiving at least two complete treatment courses. the necessary criteria for investigating cases of relapse, treatment failure, and clinical resistance were as follows: relapse: patients who received a topical or systemic treatment courses, whose outcome was improvement but the symptoms (any active lesion) reappeared in the original site of the lesion (15). treatment failure: cases in which the lesion remained active after four weeks of complete topical or systemic treatment course (15). clinical resistance: cases of relapse and treatment failure, in which active lesions persisted for weeks after at least two complete courses of systemic treatment (15). according to the criteria above, 40 patients overall did not show any desirable therapeutic response to the following regimen of anti-leishmania drugs: glucantime®: systemic injection (20mg sbv5/ kg for two weeks): (no= 8) glucantime®: local injection 1–2ml of glucantime® intralesionally injection around each skin lesion weekly for 4–6 weeks): (no= 0) glucantime®: systemic and local injection (20mg per kg for two weeks): (no= 3) miltefosine®: oral (2.5mg per kg daily for 28 days) and glucantime®: (no= 4) antibacterial compounds(antibiotics): (no= 25) data analysis the data were analyzed using spss software version 24. chi-square and fisher's exact test were used, and p≤ 0.05 was considered significant for differences between groups. results during the 10-year study period, 1480 individuals suspected of having cl were referred to the leishmaniosis laboratory. of http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 365 http://jad.tums.ac.ir published online: december 31, 2020 these, 655 cases were confirmed positive for leishmaniasis by microscopic examination of samples (amastigotes were seen in microscopic examination). the maximum number of positive cases was recorded in 2018 (116 patients, 17.7 %), whereas the minimum number of positive cases was recorded in 2013 and 2016 (45 cases in each year, 6.9%) (fig. 1). out of the 655 positive cases, 464 (70.8%) were males and 191 (29.2%) were females. the youngest patients with confirmed infection was 2 months old and highest age among the positive cases was 80 years old (fig. 2). about the nationality of the patients, 572 patients (87.3%) were iranians and 83 (12.7%) were from afghanistan. overall, the majority of the patients, 397 individuals (60.6%), lived in tehran but had a history of trip to endemic regions. karaj (the capitol of alborz province) had the second highest number of cases (48 patients, 7.3%). on the other hands, the highest number of lesions were one to four (fig. 3) and also the most common site of the lesions was on the hands with 42/3 % (304) (fig. 4). among the patients who were referred to the leishmaniosis laboratory, 139 cases (21.2%) had relapse and were referred to the laboratory for re-examination. in the next step, for followup and understanding the phase of the disease, the patients were followed up. in 2008 and 2009, 20 patients with positive microscopic test were lost to follow-up due lack of contact information. during 2010–2017, 40 cases were initially identified as treatment failure, but only 34 patients could be followed up due to lack of contact information. out of the 34 patients who were followed up, 22 patients improved by modifying the treatment regimen, such as reinjection of glucantime®, use of supplementary drugs such as miltefosine® capsule, use of ointments, and other traditional drugs. among the remaining 12 cases, even with modification of diet and increase in drug dosage, 7 cases did not show any desirable response to treatment (58.3%) and five cases had treatment failure (41.7%) (table 1, fig. 5). among these 12 individuals with treatment failure, the youngest and oldest were 16 and 62 years old, respectively. among the patients with treatment failure, the shortest duration of disease was six and the longest was 312 months, respectively. 66 65 55 45 90 50 45 64 116 60 0 20 40 60 80 100 120 140 n u m b e r year 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 fig. 1. distribution of confirmed cl cases referring to leishmaniasis laboratory tehran university of medical sciences during 2008–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 366 http://jad.tums.ac.ir published online: december 31, 2020 table 1. the characteristics of patients with no responses to anti-leishmania drugs referring to leishmanisis laboratory tehran university of medical sciences during 2008–2017 no age (year) living place travel place during two past years duration of persistence of lesion site of lesion type of administered drug and the type of injection 1 62 tehran firoozkuh 3 years leg 21 systemic glucantime® 2 22 iranshahr unknown 6 years leg, ankle, toes 200 systemic glucantime® 60 local glucantime® 56 oral miltefosine® 3 47 hesarak karaj qom 4 years hand 76 systemic glucantime® 4 30 qom kashan 3 years hand 100 systemic glucantime® 5 27 neyshabour unknown 13 years face, nose 300 systemic glucantime® 100 local glucantime® 70 oral miltefosine® 6 36 tabriz isfahan 3 years ear, eyes 25 systemic glucantime® 25 local glucantime® oral miltefosine® 7 30 dehloran unknown 13 years back 23 systemic glucantime® 8 20 karaj afghanistan 6 years face 200 systemic glucantime® 100 local glucantime® 9 16 mashhad unknown 9 years in nasal mucosa 84 systemic glucantime® 90 oral miltefosine® 10 25 tehran afghanistan 26 years the entire body 21 systemic glucantime® 11 16 tehran afghanistan 1 year face 21 systemic glucantime® 12 43 unknown pakistan 6 months face 42 systemic glucantime® 0.6% 4.9% 5.9% 3.2% 85.3% 0 10 20 30 40 50 60 70 80 90 ˂1y 1-5y 5-10y 10-15y >15y fig. 2. frequency of confirmed cl patients referring to leishmaniasis laboratory tehran university of medical sciences by age groups during 2008–2017 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 367 http://jad.tums.ac.ir published online: december 31, 2020 42.3% 25.9% 22.3% 9.6% 0 5 10 15 20 25 30 35 40 45 hand head foots body fig. 3. distribution of skin lesions on the bodies of the confirmed cl cases referring to leishmaniasis laboratory tehran university of medical sciences during 2008–2017 304 115 62 33 141 46.4 17.6 9.5 5 21.7 0 50 100 150 200 250 300 350 1 2 3 4 >10 number percent fig. 4. distribution of the number of lesions in confirmed cl patients referring to leishmaniasis laboratory tehran university of medical sciences during 2008–2017 fig. 5. skin lesions with no responses to anti-leishmania drugs in two cl patients referred to leishmaniasis laboratory tehran university of medical sciences http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 368 http://jad.tums.ac.ir published online: december 31, 2020 discussion in this study, 40 cases were initially confirmed as treatment failure, but only 34 could be followed up. out of this, 22 improved by changing therapeutic regimen such as reinjection of glucantime®, use of supplementary drugs such as miltefosine® capsule, use of ointments, and other unknown drugs (12, 13, 15). however, in the remaining 12 cases, even by changing the therapeutic regimen and raising the drug dose, they did not show any desirable response and treatment failure was observed (table 1). furthermore, in the present study, among treatment resistant patients, the longest course of the disease was 312 months (26 years), highlighting the importance of factors such as immune system of the patient, drug kinetics, and existence of resistant strains (11). there have been various reports on the incidence of drug resistance in different foci of leishmaniasis in iran. specifically, treatment failure rate in anthroponotic cutaneous leishmaniasis due to l. tropica infection has been reported to be 10.8 % in mashhad and 11.1% in bam. in isfahan, one of the main foci of zcl, drug resistance was reported as 11.6% in 2005 and in 2013, 3.7% failure rate for topical injection, 4.7% for systemic injection, and 3.4% for concurrent use of both treatment methods were reported (14, 17). one of the mechanisms leading to the reduced response clinical forms of leishmaniases to the antimony pentavalent compounds is the development of acquired resistance to the drug. although the antimony pentavalent compounds have been used for several decades as the firstline treatment for all clinical forms of leishmaniasis, unfortunately the therapeutic effect of these compounds has been jeopardized with the emergence of resistance strains in most endemic regions (16). specifically, in india, sudan, latin america, europe, and middle east, drug resistance is an important threat to effective treatment of the clinical forms of leishman iasis. for example, in india, over 60% of cases of visceral leishmaniasis caused by leishmania donovani do not respond to treatment, which can be due to different reasons such as the development of drug resistance by the parasite, immunologic changes of the patient, ineffective treatment regimen mainly due to lack of patient compliance (23, 24). in this regard, different studies have corroborated the idea that the resistance to the antibiotics could be acquired. to investigate the existence of acquired drug resistance in bihar region in india, lira et al. isolated l. donovani from drug responsive patients and those who were confirmed as treatment failure. using the presence of intra-macrophage amastigote (invitro) in isolates as criteria for resistance, they found that the isolates from patients who had responded to treatment were three times more sensitive to sodium stibogluconate drug in comparison to drug-resistant isolates. these results confirm the existence of acquired resistance in india (25). in other studies conducted in france, acquired resistance in clinically resistant leishmania infantum isolates was confirmed in drug resistance tests under in-vitro conditions (26). also, studies in latin american countries such as columbia using in-vitro tests indicated that some cases of treatment failure in new-world cutaneous and mucocutaneous leishmaniasis have been due to development of resistant strains (24-28). similarly, in iran, studies in 2004 and 2006 on 185 patients with acl in mashhad reported treatment failure in 20 cases (10.8%). in these studies, drug resistance of isolates was confirmed under in vitro conditions through the culture of macrophages. the isolates which were clinically resistant to glucantime® were also resistant in drug sensitivity tests, and required higher doses for their elimination. in the anthroponotic form of the disease, the probability of spread of drug resistant l. tropica strains http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 369 http://jad.tums.ac.ir published online: december 31, 2020 across human populations is very high, and it’s of significant public health importance (17). also, acquired resistance of l. infantum and l. major with zoonotic cycles have been reported in iran, and have been confirmed under in-vitro conditions. the progression of antimonial resistance in anthroponotic forms such as in india resulting from l. donovani and in iran due to l. tropica infections suggests that in the future, resistance to other anti-leishmaniasis drugs such as miltefosine® and amphotericin b may also develop in case of extensive usage (11). considering the fact that drug resistance is one of the major challenge in the successful treatment of the disease, identification of the mechanisms involved in drug resistance can be useful in improving therapeutic strategies. furthermore, identification of suitable markers for monitoring and detecting drug resistant cases and predicting the course of development of resistance in endemic regions could be helpful in this regard. among the most important reasons of resistance are genetic, protein, and enzyme factors, as well as intracellular factors such as signaling pathways and apoptosis. the initial mechanisms in the development of drug resistance include reduction of drug concentration in the parasite through a decreased uptake or increase in drug excretion through cell pumps, deactivation of drug, and inhibition of drug activation in the cell. a recent research indicated that other than these typical mechanisms, other factors such as apoptosis and signaling pathways are also involved in the development of natural drug resistance. accordingly, today various methods such as real-time rt-pcr, microarray, and proteomic methods are used to detect the factors and genes affecting clinical resistance phenomenon (27, 28). in iran, a study conducted by kazemi rad et al. (2013) on l. tropica to detect genes whose expression is different between sensitive and resistant l. tropica using cdna-aflp technique confirmed that 13 genes play a major role, the most important of which were as follows: aqua glycero porine (aqp1), affects drug uptake; multi drug resistance protein a (mrpa), involved in entrapping the drug; phospho glycerate kinase (pgk), involved in carbohydrate metabolism; ubiquitin, involved in degrading oxidized proteins; amino acid permease (aap3), involved in uptake of arginine amino acid; protein kinase (pk), involved in signaling pathways; mitogen activated protein kinase (mapk); and protein tyrosine phosphatase (ptp), involved in phosphorylation pathway. also, in their study, using quantitative real time pcr (qrt pcr) method, it was found that in resistant isolates, there was an increased expression of aap3, ubiquitin, pgk, ptp, and mrpa, whereas aqp1 and mapk had diminished expression (29, 30). in another study conducted by zaeran et al. (2015) on l. major resistance and sensitive to glucantime® using two-dimensional electrophoresis method performed for determining and comparing expression of proteins, it was found that out of 2967 protein points, 89 points in resistant l. major had altered expression compared with sensitive l. major; 60 proteins had increased and 29 proteins had diminished protein expression. also, they found that 11 protein points which did not exist in the sensitive l. major were expressed by resistance l. major. these changes of expression may be one of the major causes of resistance in l. major (31). conclusion considering the increased rate of drug resistance cases and numerous reports in this regard in different endemic regions of iran, complete or hybrid treatment should always be taken into consideration by the treatment team in both private and governmental healthcare centers in order to tackle the acquired resistance of the parasite and relapse of disease. on the other hand, researchers should seek to develop new drugs with high efficacy in order to combat problems such as incomplete treatment http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 370 http://jad.tums.ac.ir published online: december 31, 2020 and the presence of resistant strains in the indigent population. there are several reports on different aspects of leishmaniasis in the country. these reports will provide a guideline for disease control (32-66). acknowledgements the authors highly appreciate the support and cooperation of all colleagues, students, and patients referred to the medical parasitology and mycology department, school of public health, tehran university of medical sciences. the authors declare that there is no conflict of interests. references 1. world health organization (2014) manual for case management of cutaneous leishmaniasis in the who eastern mediterranean region. who regional publications, eastern mediterranean series (35). available at: https://apps.who.int/iris/handle/10665/12 0002 2. world health organization (2002) an increasing risk factor for leishmaniasis (weekly epidemiological record. who press. 77(44): 365–370. 3. nadim a, javadian e, mohebali m, zamen moemeni a (2008) leishmania and leishmaniasis in iran, second edition. academic publication center, tehran (in persian). 4. ferreira fm, castro ra, batista ma, rossi fmo, silveria-lemos d, frezard f, a.l.moura s, rezende sa (2014) association of water extract of green propolis and liposomal meglumine antimoniate in the treatment of experimental visceral leishmaniasis. parasitol res. 113 (2): 533–543. 5. treatment and medical education of iran (2002–2003) diseases management center, ministry of health, treatment and medical education. annual communicable diseases report. ministry of health, treatment and medical education press, iran. 6. firdous r, yasunzai m, ranja k (2009) efficacy of glucantime® in the treatment of old world cutaneous leishmaniasis. int j dermatol. 48(7): 758–762. 7. soleimanifard s, arjmand r, saberi s, salehi m, hejazi sh (2017) treatment outcome of the drug-resistant zoonotic cutaneous leishmaniasis by glucantime®. adv biomed res. 6: 17. 8. croft sl (2001) monitoring drug resistance in leishmaniasis. trop med int health. 6 (11): 899–905. 9. croft sl, sundar s, fairlamb ah (2006) drug resistance in leishmaniasis. clin microbiol rev. 19(1): 111–126. 10. hadighi r, mohebali m, boucher p, hajjaran h, khamesipour a, ouellette m (2006) unresponsiveness to glucantime® treatment in iranian cutaneous leishmaniasis due to drug resistant leishmania tropica parasites. plos med. 3(5): e162. 11. rodrigues am, hueb m, santos ta, fernandes cj (2006) factors associated with treatment failure of cutaneous leishmaniasis with meglumine antimoniate. rev soc bras med trop. 39(2): 139–145. 12. samady ja, janniger ck, schwartz ra (1996) cutaneous and mucocutaneous leishmaniasis. cutis. 57: 13–20. 13. mohammadzadeh m, behnaz f, golshan z (2013) efficacy of glucantime® for treatment of cutaneous leishmaniasis in central iran. j infect public health. 6(2): 120–124. 14. hajjaran h, mohebali h, teimouri a, oshaghi ma, mirjalali h, kazemi-rad e, shiee mr, naddaf sr (2014) identification and phylogenetic relationship of iranian strains of various and visceral cases of leishmaniasis based on n-acetylglucosamin-1-phosphate transferase gene. http://jad.tums.ac.ir/ https://apps.who.int/iris/handle/10665/120002 https://apps.who.int/iris/handle/10665/120002 j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 371 http://jad.tums.ac.ir published online: december 31, 2020 infect genet evol. 26: 203–212. 15. shirzadi mr (2012) handbook of cutaneous leishmaniasis care in iran. razi nahan publication. 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vatandoost h (2020) monitoring of laboratory reared of phlebotomus papatasi (diptera: psychodidae), main vector of zoonotic cutaneous leishmaniasis to different imagicides in hyper endemhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 363–375 z kakooei et al.: treatment failure in … 375 http://jad.tums.ac.ir published online: december 31, 2020 ic areas, esfahan province, iran. j arthropod borne dis. 14(1): 116–125. 66. mozaffari e, vatandoost h, rassi y, mohebali m, akhavan aa, moradi-asl e, zarei z, zahrai-ramazani a, ghorbani e (2020) epidemiology of visceral leishmaniasis with emphasis on the dynamic activity of sand flies in an important endemic focus of disease in northwestern iran. j arthropod borne dis. 14(1): 97–105. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 137 review article vectors of crimean congo hemorrhagic fever virus in iran *zakkyeh telmadarraiy 1, sadegh chinikar 2, hassan vatandoost 1,3, faezeh faghihi 4, asadollah hosseini-chegeni 5 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory), pasteur institute of iran, tehran, iran 3institue for environmental research, tehran university of medical sciences, tehran, iran 4cellular and molecular research center, iran university of medical sciences, tehran, iran 5department of plant protection, faculty of agriculture, university of guilan, guilan, iran (received 17 feb 2014; accepted 6 dec 2014) abstract background: ticks are important vectors and reservoirs of crimean congo hemorrhagic fever (cchf) virus. human beings may be infected whenever the normal life cycle of the infected ticks on nonhuman vertebrate hosts is interrupted by the undesirable presence of humans in the cycle. a total of 26 species of argasid and ixodid ticks have been recorded in iran; including nine hyalomma, two rhipicephalus, two dermacentor, five haemaphysalis, two boophilus, one ixodes and two argas as well as three ornithodoros species as blood sucking ectoparasites of livestock and poultries. the present paper reviews tick vectors of cchf virus in iran, focusing on the role of ticks in different provinces of iran using reverse transcription polymerase chain reaction (rt-pcr) assay. methods: during ten years study, 1054 tick specimens; including two species of argasidae and 17 species of ixodidae were examined for their infection to cchf virus genome. the output of all studies as well as related publications were discussed in the current paper. results: the results show that rhipicephalus sanguineus, hyalomma marginatum, h. anatolicum, h. asiaticum and h. dromedarii were known as the most frequent species which were positive for cchf virus. conclusion: the status of ticks which were positive for cchf virus revealed that unlike the most common idea that hyalomma species are the most important vectors of cchf virus, other ticks including rhipicephalus, haemaphysalis and dermacentor can be reservoir of this virus; thus, considering geographical distribution, type of host and environmental conditions, different tick control measurements should be carried out in areas with high incidence of cchf disease. keywords: vector tick, domestic animals, cchf, iran, rt-pcr a historical perspective of cchf disease in iran the name cchf derives from two regions in asia and africa where an often fatal human hemorrhagic fever disease occurred for the first time in the 1940s and 1950s (nuttall 2001). interestingly, a hemorrhagic disease similar to cchf was reported by dzhurzhoni, an iranian physician, in 1110 a.d. (hoogstraal 1979). the cchfv is the type species of the genus nairovirus, family bunyaviridae. the disease is known with the local name “karamikh typhoid fever” in northwest of iran (chinikar 2007). following publication of some reports about the occurrence of a hemorrhagic disease in human and domestic animals and confirming the presence of viral agent in collected ticks from iran (achrafi and noriyan 1966, chumakov et al. 1970, asefi 1974, saidi et al. 1975, sureau et al. 1980, ardoin and karimi 1982), the disease are considered as a public health problem by iranian health system *corresponding author: dr zakkyeh telmadarraiy, e-mail: ztelma@yahoo.co.in http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 138 in 1999 (labbaf-ghasemi 2006, chinikar 2007). the virus have been detected frequently in livestock (such as sheep, cow, goat and camel), human and tick vectors in many parts of iran (saidi et al. 1975, moradi et al. 2008, telmadarraiy et al. 2008a, telmadarraiy et al. 2008b, telmadarraiy et al. 2009, chinikar et al. 2012, mehravaran et al. 2013, champour et al. 2014). so far, the disease has been reported from 23 out of 31 provinces of iran that mostly centralized in southeast, due to a long border with two high risk countries, afghanistan and pakistan (chinikar et al. 2010). since 2000, the most cchf positive cases have been reported from sistan-o baluchistan province of iran (chinikar et al. 2010, keshtkar-jahromi et al. 2013). tick vectors ticks (acari: ixodidae) are the important vectors/reservoirs of crimean congo hemorrhagic fever virus (cchfv). they play an important role in the survival of the virus in nature (watts et al. 1988). the parasitic behavior of ticks during their all life stages, excessive blood feeding on hosts, wide range of vertebrate hosts, great reproductive potential, long term survival and adaptability to harsh and variable ecological conditions are among considerable characteristics of ticks as potential vectors of a large number of microbial agents (linthicum and bailey 1994). humans may be infected via a tick bite whenever a normal cycle of virus-tick-nonhuman vertebrates is interrupted by the undesirable presence of human (such as herdsmen, tourists, veterinarian researchers) in the nature cycle. domestic animals are known to be susceptible to cchf virus; however, there is no evidence that any symptomatic disease develop (eldridge et al. 2004). the disease occur in palearctic, oriental and some parts of sub-saharan africa wherever one or several hyalomma to be distributed as widespread tick species (linthicum and bailey 1994). a range of disease from mild to severe clinical illness occurs in human. in severe cases, petechial rash on the trunk and limbs, besides the appearance of hemorrhage from the body cavities are among the common clinical symptoms of the disease (nuttall 2001). the approximate mortality rate of 22 % has been reported in cchf cases infected by a tick bite, though the rate is variable in different areas (hoogstraal 1979). the virus has been reported in two groups of ticks. the first group includes aggressive species, which search for human actively. for instance, species such as hyalomma marginatum and h. anatolicum are the main vectors in this group when epidemics is occurring (hoogstraal 1979). second group are less aggressive species seeking humans normally. for instance the genera rhipicephalus, boophilus, haemaphysalis, amblyomma, dermacentor and even other hyalomma are among species that chiefly maintain enzootic foci of cchf virus among tick vectors and wild/domestic mammals (hoogstraal 1979). approximately, 26 soft (argasidae) and hard (ixodidae) tick species have been recorded from iran so far. they include nine hyalomma, two rhipicephalus, two dermacentor, five haemaphysalis, two boophilus, one ixodes, two argas and three ornithodoros species as ectoparasites of livestock and poultries (maghami 1968, mazlum 1971, rahbari et al. 2007). studies on tick vectors of cchfv in iran have begun since 1980s, when sureau et al. (1980) detected the virus in nymphs of ornithodoros lahorensis collected from goat, however, the antibody of cchfv (cchf virus) was detected in the serum of the sheep sent from iran to russia, for the first time (chumakov et al. 1970). few years later, saidi et al. (1975) reported the presence of cchfv antibody in the sera of man, sheep, goats, cattle and some small mammals in iran. iran has different climatic conditions and appropriate landscapes suitable for the occurrence of many species of bloodsucking ticks http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 139 (up to the present 50 species) as external parasites on mammals, birds and other animals (kamali et al. 2001). the circulation of cchf virus in ticks and livestock plays an important role in the maintenance of virus in iran (chinikar et al. 2010). considering to the importance of ticks in the survival of the virus, a series of studies have been conducted by our group on ticks suspected to transmit the cchfv between domestic animals and human. the present paper reviews the tick vectors of cchfv in iran and summarizes the results of ten years study conducted in 14 provinces of iran. similar studies associated with tick vectors of cchfv have also been discussed in the present study. argasidae the argasid or soft ticks are the most common ectoparasites found on the poultries and domestic animals in iran (rafyi and maghami 1965). to date, five human associated soft ticks are known in iran, all of them play important role in transmission of arthropod borne diseases (djanbakhsh 1956, rafyi and rak 1985, barmaki et al. 2010, oshaghi et al. 2011, rafinejad et al. 2011). argas two argas species are widely found in iran namely a. persicus, a parasite of poultries, and a. reflexus, a parasite of pigeon and doves. recently, a third species is also found on pipistrellus pipistrellus which can parasitize bats (hosseini-chegeni and tavakoli 2013). ornithodoros five ornithodoros species have been recorded in iran, including o. erraticus and o. tartakovskyi (parasites of rodents), o. lahorensis and o. canestrinii (parasites of livestock) and o. tholozani (parasites of mammals, birds and even human) (djanbakhsh 1956, rafyi and maghami 1965, rafyi and rak 1985). ixodidae totally, 21 ixodid or hard tick species have been reported in iran. all of the species parasitize domestic animals, including sheep, goat, cattle, camel, horse, and donkey, moreover, humans might be infested accidentally (chinikar 2007). dermacentor the genus dermacentor is a most attractive tick having an ornate scutal pattern. the polymorphic d. marginatus and d. niveus (= d. daghestanicus) are two well-known species in iran (rahbari et al. 2007) as well as a less known species d. raskemensis; however, their species status need to be revised. haemaphysalis six species of haemaphysalis includes h. concinna, h. erinacei, h. inermis, h. parva, h. punctata and h. sulcata were found in iran. the latter species (h. sulcata) is the most widespread species in iran (rahbari et al. 2007, hosseini-chegeni et al. 2014). hyalomma the genus of hyalomma is the most important tick species associates with livestock. the most frequent species of this genus found in iran are as follows: h. anatolicum, h. asiaticum, h. detritum, h. dromedarii, h. excavatum, h. marginatum, h. rufipes and h. schulzei (hosseini-chegeni et al. 2013). ixodes the species i. ricinus belonging to ixodes genus has been reported on livestock in iran. the prevalence of this species is confined to hyrcanian zone in north part of iran. this multi-host species parasitize over three hundred different hosts; including domestic and wild animals (sonenshine et al. 2002). two other species of ixodes genus have been reported on wild animals (filippova et al. 1976). rhipicephalus r. sanguineus is the most common species http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 140 of the genus rhipicephalus throughout iran. as well as a rare species, r. bursa, it may also be found on domestic and wild animals in iran (rahbari et al. 2007). vectors of cchfv in iran, past to present since 2004, we examined different tick species of two families argasidae (two genera, two species) and ixodidae (five genera, 17 species) for detection of cchf virus genome. the infection rate of tick species that were positive for cchf virus has been shown in the table 1. the collection sites of tick in different geographical zones of iran have also been shown in fig. 1. our results showed that rhipicephalus sanguineus, hyalomma marginatum, h. anatolicum, h. asiaticum and h. dromedarii were the most frequent tick species which were positive for cchfv whereas other species were less infected (fig. 2, fig. 3). based on the results obtained from reverse transcription polymerase chain reaction (rt-pcr), argasid ticks, including argas persicus, ixodid ticks, boophilus annulatus, haemaphysalis sulcata, ha. concina, and ha. erinacei as well as rhipicephalus turanicus were negative for cchf virus. after dengue virus, cchfv is the second most widespread arboviruse type disease throughout the world (appannanavar and mishra 2011). the biological role of ticks is also important, not only as virus vectors, but also as reservoirs of the virus in nature (whitehouse 2004). the long term survival of the virus occurs in live dormant ticks at unfavorable climatic conditions (chumakov 1965). hoogstraal (1979) reviewed the epidemiology of tick borne crimean congo hemorrhagic fever virus. he introduced the trans-stadial and trans-ovarial (vertical) transmission of the virus in ticks (hoogstraal 1979). another route that cchf virus could be transmitted among tick vector is an interesting mechanism called co-feeding behavior in which virus transferred between feeding ticks on the host (gordon et al. 1993). except tick bite, the other route for transmissions of virus to human being is the direct contact with blood or tissue of the infected livestock and nosocomial infection. the biting of the infected tick or contact of human to the infected tick hemolymph through naked hands or other parts of the body and also other tick borne transmission routes are considered non-significant in the epidemiology of cchf virus in iran (shirzadi 2003). based on chinikar et al. (2007) the direct infection of humans by tick bite is rare in iran and has been observed only in few cases (chinikar 2007). cchfv propagates in tick vectors and within the natural host vertebrate including domestic animals. from biological point of view, the virus is transmitted from tick to the host via tick saliva during blood feeding. the virus is transmitted from tick to tick through co-feeding phenomenon (nuttall 2001). thus, in iran, the essential role of ticks in the epidemiology of cchf virus may be debatable and never considered merely according to patient’s statements and health worker reports (izadi et al. 2003). ornithodoros lahorensis was reported as the most frequent soft tick infected to cchfv in northwest (ardebil and azerbaijan-e sharqi) northeast and central parts of iran (sureau et al. 1980, shirani et al. 2004). shirani et al. could detect the virus in the body of living ornithodoros lahorensis maintained in the laboratory conditions for three months. the authors also concluded that argasid ticks might be considered as a potential reservoir of cchfv (shirani et al. 2004). however, the few other studies showed that argasid ticks cannot be infected in the laboratory condition (linthicum and bailey 1994, shirani et al. 2004). three argas ticks reported from iran including a. persicus, a. vespertilionis and a. refelexus, all have been considered as the main vectors of animal diseases (hosseinihttp://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 141 chegeni and tavakoli 2013). the infection of the latter species to cchfv was confimed in hamedan province, iran (tahmasebi et al. 2010). an expected reason of the minor implication of soft ticks in the transmission of cchfv is their quick feeding behavior (excluding some exceptions e g, larvae of o. lahorensis, o. canestrinii and argas persicus) (hoogstraal 1985). in many literatures hyalomma ticks were introduced as the main vectors of cchfv thus the correct identification of species is an essential work since it may be a complicated and controversial issue in iran (hosseini et al. 2011, hosseini and tavakoli 2012, tavakoli et al. 2012). in spite of the only report on low infection rate of hyalomma detritum (salim-abadi et al. 2011), tahmasebi et al. (2010) reported 48 positive ticks out of 294 of h. detritum tested for cchfv which is somewhat doubtful (tahmasebi et al. 2010). the other less frequent and patchy distributed tick is rhipicephalus bursa that is the most frequent tick infected to cchfv in fereydoonshahr of isfahan province (chinikar et al. 2012) and northwest of iran (telmadarraiy et al. 2009). many literatures, refer hyalomma tick as main vector of cchfv in nature; however, in our study wherever hyalomma was absent or not considered as the predominant vector (in ghaemshahr county, mazandaran province), cchfv was detected from other ticks such as rhipicephalus and haemaphysalis. it seems that these species are key species in the survival of the virus in the absence of hyalomma. on the other hand, sometimes the infection rate of hyalomma to cchfv was in low level in comparison with rhipicephalus (i.e, totally 5 positive out of 8 rhipicephalus versus only a few positive hyalomma out of 97 specimens in ghaen county, khorasan-e joonobi province). there are little information on genetic structure and molecular phylogenetic analysis on cchf viruses isolated from different tick vectors in iran. however, the phylogeny studies on the iranian cchfv isolates in comparison with a representative set of cchfv sequences from other regions of the world based on s-segment sequences revealed that the iranian isolates are mostly related to asian 1 group particularly to the pakistani strains (chinikar et al. 2012) although a few isolates were associated with iraq (chinikar et al. 2012) or europe 1 strains (morovvati et al. 2014). further molecular epidemiology studies are necessary to find out the origin and the phylogenetic relationship of iranian cchfv to improve the surveillance and control measurements of the disease in the country. fig. 1. the seven geographical zones of iran; numbers 1–14 represented different localities of tick collection, 1. ardabil, 2. azarbayjan-e sharqi, 3. golestan, 4. hamadan, 5. ilam (abdanan township), 6. khorasan-e joonobi, 7. khorasan-e razavi, 8. khorasan-e shomali, 9. mazandaran (pushtekooh district), 10. mazandaran (ghaemshahr county), 11. qom, 12. sistan-obaluchistan, 13. tehran, 14. yazd http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 142 table 1. infection rates (%) of iranian tick species with cchf virus in different localities of iran tick species r . s an gu in eu s r . b u rs a o . l ah or en si s h a. p u n ct at a h a. i n er m is h .s p h . s ch u lz ei h . m ar gi n at u m h . d ro m ed ar ii h . d et ri tu m h . a si at ic u m h . a n at ol ic u m d . m ar gi n at u st/pprovince● 13.86.94.60.80.8----130/35ardebil* -11-5.43.7-756/15azarbayjan-e sharqi** 0.7-0.71.6--2.3130/7golestan 2.31.2-1.23.42.3-1.288/10hamadan*** 1.35-1.351.3574/3ilam 0.90.9--0.9--1.81.8-115/7khorasan-e joonobi 3-4.5-67/5khorasan-e shomali 1.758/1mazandarani 2.42.42.42.4--42/4mazandaranii -7.1--14/1qom -8.3---140/6sistan-o baluchistan**** 2----2,e-50/2tehran -0.72.11.40.70.7-140/8yazd***** 3 8 1 8 .3 1 7 .9 3 .6 1 0 .7 1 7 .1 0 .8 1 4 .9 5 2 3 .9 1 .4 9 .5 5 1 1 .8 5 71 1 1 2 /1 0 8 total references related to other studies including papers authored/co-authored by senior author (zt) including; *: telmadarraiy et al. 2009 **: telmadarraiy et al. 2008a ***: moradi et al. 2008; telmadarraiy et al. 2008a ****: mehravaran et al. 2013 *****: salim-abadi et al. 2011 d: dermacentor, ha. haemaphysalis, h: hyalomma, r: rhipicephalus, o: ornithodoros ●: numbers arranged according to collection sites in the map (fig. 2) t/p: total tested/positive tick specimen, i. pushtekooh district, ii. ghaemshahr county, e: egg mass, blank places: not done, -: negative fig. 2. the male of most frequent iranian ixodid ticks infected to cchfv, i) rhipicephalus sanguineus, ii) hyalomma marginatum, iii) hyalomma anatolicum, iv) hyalomma asiaticum, v–vi) hyalomma dromedarii dorsal and ventral view respectively. (black and white arrows indicated the most taxonomic characters of certain species) fig. 3. the male of less frequent iranian ixodid and argasid ticks infected to cchfv, i) hyalomma schulzei, ii) hyalomma detritum, iii) dermacentor marginatus, iv) rhipicephalus bursa, v–vi) haemaphysalis sulcata ventral and dorsal view respectively, vii–viii) ornithodoros lahorensis dorsal and ventral view. (black and white arrows indicated the most taxonomic characters of certain species) http://jad.tums.ac.ir published online: march 11, 2015 j arthropod-borne dis, december 2015, 9(2): 137–147 z telmadarraiy et al.: vectors of crimean … 143 control of ticks based on cchf vector, different control measurements should be performed in high risk areas where disease presents (hoogstraal 1979). for instance, in ghaemshahr county of mazandaran province, and azarbayjan-e gharbi province, haemaphysalis punctata and ha. inermis are the most prevalent species. in azerbaijan-e sharqi province, d. marginatus is considered as target of control whereas in hamadan, yazd and kurdistan provinces, hyalomma ticks (e g, h. marginatum) are the most important vectors, tick control measures may be focused on domestic mammals and animal housings especially with reference to behaviorally unusual ridiculous ticks, h. detritum and h. anatolicum (telmadarraiy et al. 2004, salari-lak et al. 2008, nasiri et al. 2010). hyalomma and other ticks can parasitize small wild rodents (rattus norvegicus, rhombomys opimus, tatera indica, meriones libycus, meriones hurrianae and the lesser mouse eared bat, myotis blythii) (kia et al. 2009, tajedin et al. 2009, vatandoost et al. 2010, nateghpour et al. 2013). the control measures of livestock ticks recommended by the iranian veterinary organization including the application of acaricides, at animal housing, as anti tick bathroom and pure on or spot on domestic animals (rezaii 2014). infusing ivermectin is currently being used as a relative chief anti tick method in the iranian ruminants, but concerning its harmful residues on animals products (milk and meat), it is not advised by the iranian veterinary organization. flumethrin is also reported as a pesticide suitable for the control of hard and soft ticks especially hyalomma tick parasitizing livestock whenever use as pouron host (vatandoost et al. 2012). conclusion the high frequency of tick species which are infected to cchf virus reveals that unlike a most common idea which believes that hyalomma species are the main vectors of cchf virus, other ticks including rhipicephalus, haemaphysalis and dermacentor can be reservoir of this virus; thus, considering geographical distribution, type of host and environmental conditions, different tick control measurements should be acquired in areas with high incidence of cchf disease. acknowledgements we would like to appreciate staffs of the laboratory of arboviruses and viral hemorrhagic fevers, institute of pasteur of iran as national reference laboratory, for their technical support. we would also like to thank students of department of medical entomology and vector control of school of public health, tehran university of medical sciences for their efforts in collecting and identifying tick specimens. our sincere thanks go to engs. tigi and zarei, dr hosseini, dr shakeri, and ms shirzaiiyan-zaroni for their kind helps in this research. this study 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original article evaluation of the time course on the effectiveness of who standard pyrethroid and carbamate impregnated test papers against anopheles stephensi, the main malaria vector in iran awat dehghan1, seyed hassan moosa-kazemi1, mohammad mehdi sedaghat1, *hassan vatandoost1,2, *ahmad ali hanafi-bojd1,3 1department of vector biology and control of diseases, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3zoonoses research center, tehran university of medical sciences, tehran, iran *corresponding authors: dr ahmad ali hanafi-bojd, e-mail: aahanafibojd@tums.ac.ir; dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com (received 27 apr 2022; accepted 19 mar 2023) abstract background: anopheles stephensi is a major vector of malaria in some parts of the world. a standard method for determining resistance in adult mosquito populations is the bioassay test recommended by the world health organization (who). the papers used in this method have an expiry date. this study aimed to determine the effectiveness of outdated susceptibility test papers for use in insecticide resistance monitoring programs. methods: beech and bandar abbas strains of an. stephensi were reared in the insectary. permethrin 0.75%, deltamethrin 0.05%, and bendiocarb 0.1% impregnated test papers prepared by universiti sains malaysia were used. probit analysis was used to analyze the results and prepare time-mortality regression lines of lt50 and lt90. results: there was a difference in the mortality of both tested strains of an. stephensi was exposed to all tested insecticides. both expired and not expired permethrin and deltamethrin papers induced 100% mortality at the diagnostic time (60min), but their insecticidal properties were reduced gradually in serial times. the highest efficacy of test papers was in the first trimester after the expiry date and decreased over time. conclusion: at the diagnostic time of 60 minutes, the mortality rate of both dated and expired papers was 100% in the pyrethroid insecticides, even three years after expiry dates, if stored in the package provided by the producer, in a refrigerator. this value was reduced to less than 100% in the expired papers of bendiocarb comparing the dated papers that induced 100% mortality. keywords: susceptibility test; anopheles stephensi; insecticide impregnated papers; expiration date introduction malaria is an infectious disease and one of the major health problems worldwide. in 2021, estimated 247 million cases of malaria occurred worldwide, resulting in 619,000 deaths (1). the disease is caused by plasmodium parasites and is transmitted to humans by the bites of anopheles mosquitoes (2). one of the methods to prevent and fight this disease is to control vectors in different ways. due to the importance of transmitting this disease in iran, especially in the southern and southeastern regions, vector control operations are carried out regularly in these areas. in the global malaria strategy, the use of insecticides has maintained its special place in control operations. following the increasing use of various pesticides and the emergence of the phenomenon of physiological and behavioral resistance of vectors, major problems copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:aahanafibojd@tums.ac.ir mailto:hvatandoost1@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 73 http://jad.tums.ac.ir published online: march 31, 2023 have been created in malaria control programs. one of the most important vectors of malaria in iran is anopheles stephensi, whose distribution is limited to the provinces of sistan and baluchestan, hormozgan, bushehr, kerman, fars, khuzestan, ilam, lorestan, kohgiluyeh and boyer-ahmad, and kermanshah (3, 4). extensive use of residual spraying to control malaria vectors has led to resistance of an. stephensi to ddt, dieldrin, malathion and other organophosphates, bendiocarb, and some pyrethroids in the middle east (5). the first report of resistance in an. stephensi was to ddt in 1957 in the south of the country (6). so, dieldrin replaced ddt in the indoor residual spraying (irs) program, but unfortunately, three years after the application of dieldrin, in 1960, cases of resistance to this insecticide were observed in the areas under the coverage of irs (7). malathion, propoxur, lambda-cyhalothrin, deltamethrin, and bendiocarb, are other insecticides used for irs in iran, respectively. unfortunately, recent studies in the last decade show an. stephensi is resistant to all groups of insecticides in some parts of iran (8–13). increasing resistance to insecticides is a complex and dynamic process and depends on many factors. increasing the dose of insecticide is not recommended to maintain effectiveness because it creates environmental problems and adversely affects human life. the resistance gene in the vector population may also increase dramatically. often, when the number of resistant insects in a vector population increases, the effectiveness of a particular insecticide decreases to the point that it must be replaced by another insecticide. therefore, insecticide resistance management is a very important issue that should be considered in malaria control programs. detection of insecticide resistance in natural vector populations is essential for malaria control. a standard method for determining resistance in adult mosquito populations is the bioassay test recommended by the world health organization (14). in the who standard method, mosquito specimens are exposed to a series of different insecticides using insecticide-impregnated papers at differential or diagnostic doses. this method has been widely used in this field and gives acceptable results in detecting insecticide resistance for monitoring purposes. the papers used in this method have an expiration date, so due to the customs consideration in some countries, it is a timeconsuming process from the date they are produced until they reach the malaria vector control authorities; and may expire by the appropriate time for starting the susceptibility tests. in 1957, during studies on the expiration date of insecticide-impregnated papers in susceptibility tests against aedes aegypti using the who standard method, it was found no significant difference between the two groups of papers impregnated with ddt and dieldrin with different ages after production (15). in reviewing previous research around the world, we found that no other study has been conducted in this field since that date. during these years, new insecticides have been introduced and used around the world to control malaria vectors. insecticide-impregnated test papers have also changed a lot. this study aimed to answer the question of whether the expiration date listed on the susceptibility test papers can be a deterrent to the use of these papers or influence their lethality. materials and methods mosquito strains we used a field strain of this mosquito collected from hormoodar village (56.32 oe, 27.31 on), bandar abbas county, southern iran, as well as the beech susceptible laboratory strain. phenotypic resistance profile of the wild population (bandar abbas) of an. stephensi has been identified in a previous study (9). the collected samples from the field were transferred to the insectary and reared to establish the laboratory colony. the rearing conditions were at 30±2 °c, 65±5% relative humidity, and a light to dark period of 12 to 12 hours (16). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 74 http://jad.tums.ac.ir published online: march 31, 2023 an artificial blood-feeding device with human whole blood was used to feed the adult female mosquitoes and this operation was repeated every three days (17). the colony was fed on a 10% sucrose solution. insecticides three insecticides that are used by the iran national malaria control program, including permethrin (in the long-lasting insecticide-treated nets (llins), deltamethrin, and bendiocarb (in irs), were considered in this study. so, for this experiment, we used deltamethrin 0.05%, permethrin 0.75%, and bendiocarb 0.1% impregnated papers prepared by the who collaboration center, universiti sains malaysia (usm) (table 1). two series of each insecticide were used in this study with different impregnation and expiry dates. these papers were provided to us by the center for disease management of the ministry of health and kept in the refrigerator under standard conditions. susceptibility tests tests started on 9 dec 2020 and finished on 27 august 2021. three to five-day-old nulliparous female mosquitoes fed with sucrose 10% were used in insecticide susceptibility tests, according to the who standard method and using standard impregnated papers (15). each experiment for each tested time consisted of four replications and two controls, and in each replication, 25 adult mosquitoes were tested. the tests were performed at serial times of 7.5, 15, 30, and 60 minutes with the diagnostic dose of each insecticide. shorter times were used as needed. with this method, we tested the efficacy of the test papers in serial time under 60 minutes to be able to calculate the lt50 and to compare the lt50s between the different groups of test papers. according to the who instructions, each paper was used only 6 times (15). then, the tested mosquitoes were kept in the insectary condition for 24 hours. the number of dead specimens was recorded at the end of the above time. if the mosquitoes in the control group had no mortality or less than 5%, the test was considered correct. if the control mosquitoes had a mortality of 5–20%, the results were corrected with the abbott formula. also, if the control mortality was more than 20%, the test was unacceptable and repeated (15). data analysis probit software (18) was used to analyze the results and to prepare a lethal time regression line. the results were displayed using descriptive tables and graphs. by comparing the mortality rate of the papers impregnated with a specific insecticide and its standard deviation with different expiration dates, the effect of time course on the lethality of these papers was investigated. results bendiocarb 0.1% beech laboratory and bandar abbas strains of an. stephensi were tested with the papers produced in aug. 2017, expired in aug. 2020, and tested in feb. 2021. these tests were performed in five serial times (225, 450, 900, 1800, and 3600 sec). the regression line equations obtained from probit analysis were calculated as y= 0.0007x+3.5474 and y= 0.0007x+3.405 respectively (fig. 1). also, the two strains were tested with the papers produced in aug. 2019, expired in aug. 2022, and tested in may 2021. these tests were performed with seven serial times (56, 112, 225, 450, 900, 1800, 3600 sec). the regression line equations obtained from probit analysis were calculated as y= 0.0023x+ 4.1386 and y= 0.002x+3.645 (fig. 1). the lt50 and lt90 values for the two bendiocarb papers are presented in fig. 2. permethrin 0.75% beech laboratory and bandar abbas strains of an. stephensi were tested with the papers produced in aug. 2017, expired in aug. 2018, and tested in march 2021. these tests were http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 75 http://jad.tums.ac.ir published online: march 31, 2023 performed in eight serial times (28, 56, 112, 225, 450, 900, 1800, and 3600 sec). the regression line equations obtained from probit analysis were calculated as y= 0.0035x+ 4.5092 (fig. 3) and y= 0.0019x+4.6029 (fig. 5), respectively. also, the two strains were tested with the papers produced in aug. 2019 and expired in aug. 2020. these tests were performed with 8 serial times (28, 56, 112, 225, 450, 900, 1800, 3600 sec), three, six, and nine months after expiration. for the beech strain, the regression line equation obtained from probit analysis was calculated as y= 0.0074x+4.4337, y= 0.0036x+4.7234, and y= 0.0032x+4.7962 for the 1st, 2nd, and 3rd trimesters, respectively (fig. 3). the lt50 and lt90 values are presented in fig. 4. the lt50 and lt90 values are presented in fig. 4. for bandar abbas strain, the regression line equations for the first, second, and third trimesters were y= 0.0088x+ 4.1856, y= 0.0051x+4.4706, and y= 0.0039x+4.6843, respectively (fig. 5). deltamethrin 0.05% beech laboratory and bandar abbas strains of an. stephensi were tested with the papers produced in aug. 2017, expired in aug. 2018, and tested in march 2021. these tests were performed in eight serial times (28, 56, 112, 225, 450, 900, 1800, and 3600 sec). the regression line equations obtained from probit analysis were calculated as y= 0.0015x+4.7598 (fig. 6) and y= 0.0038x+4.0065 (fig. 7), respectively. also, the two strains were tested with the papers produced in aug. 2019 and expired in aug. 2020. these tests were performed with 8 serial times (28, 56, 112, 225, 450, 900, 1800, 3600 sec), three, six, and nine months after expiration. for the beech strain, the regression line equation obtained from probit analysis was calculated as y= 0.0031x+4.6574, y= 0.0015x+5.1529, and y= 0.0016x+5.0414 for the 1st, 2nd, and 3rd trimesters, respectively (fig. 6). the lt50 and lt90 values are presented in fig. 7. for bandar abbas strain, the regression line equations for the first, second, and third trimesters were y= 0.0033x+4.3534, y= 0.0017x+4.6184, and y= 0.0008x+4.9212, respectively (fig. 7). the lt50 and lt90 values are presented in fig. 8. fig. 1. comparison of time-mortality regression lines due to exposure of anopheles stephensi with bendiocarb 0.1% with two different producing and expiration dates. be= beech strain, ba= bandar abbas strain http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 76 http://jad.tums.ac.ir published online: march 31, 2023 table 1. specifications of insecticides used in this study insecticide impregnation date expiry date batch number control number deltamethrin 0.05% august 2017 august 2018 de 527 527 permethrin 0.75% august 2017 august 2018 pe 428 428 deltamethrin 0.05% august 2019 august 2020 de 717 717 permethrin 0.75% august 2019 august 2020 pe 594 594 bendiocarb 0.1% august 2017 august 2020 be 200 200 bendiocarb 0.1% august 2019 august 2022 be 272 272 fig. 2. comparison of lt50 and lt90 values of two strains of anopheles stephensi after exposure with bendiocarb 0.1% with two different expiration dates. be= beech strain, ba= bandar abbas strain. error bars show standard deviation (sd) 2.00 4.00 6.00 8.00 10.00 1 10 100 1000 p r o b it m o r ta li ty exposure time (second) probit2017 probit2019 three month after expired probit2019 six month after expired probit2019 nine month after expired log. (probit2017) log. (probit2019 three month after expired) log. (probit2019 six month after expired) log. (probit2019 nine month after expired) fig. 3. comparison of the regression lines of time-mortality due to exposure of anopheles stephensi (beech strain) to permethrin 0.75% with two different production and expiration dates http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 77 http://jad.tums.ac.ir published online: march 31, 2023 i fig. 4. comparison of lt50 and lt90 values of two strains of anopheles stephensi after exposure with permethrin 0.75% with two different expiration dates. be= beech strain, ba= bandar abbas strain. error bars show standard deviation (sd) 2.00 3.00 4.00 5.00 6.00 7.00 8.00 1 10 100 1000 p r o b it m o r ta li ty exposure time (second) probit2017 probit2019 three month after expired probit2019 six month after expired probit2019 nine month after expired log. (probit2017) log. (probit2019 three month after expired) log. (probit2019 six month after expired) log. (probit2019 nine month after expired) fig. 5. comparison of the regression lines of time-mortality due to exposure of anopheles stephensi (bandar abbas strain) to permethrin 0.75% with two different production and expiration dates http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 78 http://jad.tums.ac.ir published online: march 31, 2023 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 10 100 1000 p r o b it m o r ta li ty exposure time (s econd) probit2017 probit2019 three month after expired probit2019 six month after expired probit2019 nine month after expired log. (probit2017) log. (probit2019 three month after expired) log. (probit2019 six month after expired) log. (probit2019 nine month after expired) fig. 6. comparison of the regression lines of time-mortality due to exposure of anopheles stephensi (beech strain) to deltamethrin 0.05% with two different production and expiration dates 2.00 3.00 4.00 5.00 6.00 7.00 8.00 1 10 100 1000 10000 p r o b it m o r ta li ty exposure time (s econd) probit2017 probit2019 three month after expired probit 2019 six month after expired probit 2019 nine month after expired log. (probit2017) log. (probit2019 three month after expired) log. (probit 2019 six month after expired) log. (probit 2019 nine month after expired) fig. 7. comparison of the regression lines of time-mortality due to exposure of anopheles stephensi (bandar abbas strain) to deltamethrin 0.05% with two different production and expiration dates http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 79 http://jad.tums.ac.ir published online: march 31, 2023 fig. 8. comparison of lt50 and lt90 values of two strains of anopheles stephensi after exposure with deltamethrin 0.05% with two different expiration dates. be= beech strain, ba= bandar abbas strain. error bars show standard deviation (sd) discussion given that our study is the first in the world on susceptibility test papers produced by the who collaboration center in malaysia (usm) and there are no similar studies for it, in this section, we discuss the insecticide used and the reasons for the decrease in the effectiveness of susceptibility test papers at different times after production. although in a study conducted in the 1960s using ddt test papers against ae. aegypti, each set of papers killed very similar proportions of the tested mosquitos (15). in this study, we found in all tests performed using deltamethrin 0.05% and permethrin 0.75% according to the standard method of the who in the diagnostic time of 60 minutes, in both dated and outdated papers, the mortality rate was found to be 100% in beech and bandar abbas strains of an. stephensi. comparing the regression lines of time mortality obtained from probit analysis of bendiocarb insecticide against beech strain of an. stephensi with different expiration dates, it is clear that the insecticidal properties decreased one year after the expiration date of bendio carb 0.1% impregnated papers. also, the values of lt50 and lt90 in expired papers have increased 5.4 and 4.4 times, respectively (fig. 2). also, in the tests performed with 2017– 2020 bendiocarb impregnated papers, the lt90 value is 3 times lt50, while for 2019–2022 bendiocarb papers, the lt90 value is 3.7 times lt50. comparing the regression lines of time mortality obtained from probit analysis of bendiocarb insecticide against bandar abbas strain of an. stephensi with different expiration dates, it is clear that the insecticidal properties decreased one year after the expiration date of bendiocarb 0.1% impregnated papers. also, the values of lt50 and lt90 in expired papers have increased by 3.2 and 2.2 times, respectively (fig. 2). also, in the tests performed with 2017–2020 bendiocarb impregnated papers, the lt90 value is 2.5 times lt50, while for 2019–2022 bendiocarb papers, the lt90 value is 3.5 times lt50 (fig. 2). comparing the regression lines of time mortality obtained from probit analysis of permethrin insecticide against beech strain of an. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 80 http://jad.tums.ac.ir published online: march 31, 2023 stephensi with different expiry dates, the insecticidal properties decreased over time. also, the lt50 and lt90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (1.5 times for lt50 and 2.3 times for lt90). the results showed that in the third trimester (9 months after the expiration of 2019–2020 papers), the values of lt50 and lt90 have increased by 1.04 times for lt50 and 1.7 times for lt90 compared to the first trimester (fig. 4). comparing the regression lines of time mortality obtained from probit analysis of permethrin insecticide against bandar abbas strain of an. stephensi with different expiry dates, the insecticidal properties decreased over time. also, the lt50 and lt90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (2.1 times for lt50 and 6.3 times for lt90). the results showed that in the third trimester (9 months after the expiry date of 2019–2020 papers), the values of lt50 and lt90 increased 1.7 times for lt50 and 3.8 times for lt90 compared to the first trimester (fig. 4). comparing the regression lines of time mortality obtained from probit analysis of deltamethrin insecticide against beech strain of an. stephensi with different expiration dates, the insecticidal properties decreased over time. also, the lt50 and lt90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (1.7 times for lt50 and 2.3 times for lt90). the results showed that in the third trimester (9 months after the expiration of 2019–2020 papers), the values of lt50 and lt90 increased by 1.4 times for lt50 and 1.4 times for lt90 compared to the first trimester (fig. 8). compared the regression lines of time mortality obtained from probit analysis of deltamethrin insecticide against bandar abbas strain of an. stephensi with different expiry dates, the insecticidal properties decreased over time. also, the lt50 and lt90 in the 2017–2018 papers have increased compared to the papers of 2019–2020 (1.4 times for lt50 and 2 times for lt90). the results showed that in the third trimester (9 months after the expiry date of 2019–2020 papers), the values of lt50 and lt90 increased 1.1 times for lt50 and 1.7 times for lt90 compared to the first trimester (fig. 8). the decrease in mortality and the increase in lt50 and lt90 are due to the decrease in the insecticidal properties of the insecticides used, which can be due to several reasons, including breaking and decomposing part of the insecticide or evaporating it later. this issue should be further investigated and the volume of insecticide residues on susceptibility test papers should be measured with special facilities and methods (19, 20). also, another reason for the decrease in the quality of these papers over time can be related to the storage conditions and how these papers are stored. in the interval between tests, the papers should be stored in their original plastic box, to be sealed with adhesive tapes, and stored in the refrigerator at 4 °c or, if this is not possible, in a dark cupboard at room temperature. papers stored at 4 °c should be exposed to room temperature before use. insecticide-impregnated papers should never be exposed to direct sunlight (14). in general, by comparing the mortality rates of two strains of bandar abbas and beech at different serial times in the presence of bendiocarb 0.1%, permethrin 0.75%, and deltamethrin 0.05%, we conclude that the susceptible laboratory strain in exposure to insecticide-impregnated papers is more sensitive because it had a higher mortality rate. the results of the most recent study in bandar abbas showed this species was resistant to bendiocarb 0.1%, permethrin 0.75%, and deltamethrin 0.05% (9). the lower mortality rate in the bandar abbas strain could be due to the presence of resistance genes or metabolic mechanisms in the natural population of this strain because this strain was collected from the field and used after breeding in the insectary. synergist bioassays on the ddt and permethrin-resistant laboratory strains of an. stephensi from iran indicated the metabolic resistance in this species (21). another study in iran confirmed that metabolic mechanisms play a critical role in the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 72–82 a dehghan et al.: evaluation of the … 81 http://jad.tums.ac.ir published online: march 31, 2023 resistance of an. stephensi to cyfluthrin, a pyrethroid insecticide (12). it is recommended that additional studies be performed on insecticide residues and their metabolites on the who susceptibility test papers at different times after production. due to the special conditions of the covid-19 pandemic, access to papers with different production dates was not possible for this study. this is the limitation of this research. conclusions at the diagnostic time of 60 minutes, the mortality rate of dated and expired papers was equal in the pyrethroid insecticides, even three years after expiry dates, if stored in their standard package in the refrigerator. about the bendiocarb, the mortality rate of the tested mosquitoes was less than 100% in the expired test papers. due to the high cost of test papers for the countries which are involved in malaria diseases, especially in the african region, the results of this experiment are interesting. by the way, to chemically analyze the residual amount of insecticides or their metabolites on the papers using hplc or gc is suggested to have an idea about the active ingredient remaining on the papers on different dates. acknowledgments we thank the staff of the mosquito insectary, department of vector biology and control of diseases, school of public health, tehran university of medical sciences, for their cooperation in breeding mosquitoes. special thanks to mr a badzohre for his help in doing tests and dr f nikpour and the center for disease management of the ministry of health for providing the susceptibility test papers. this project has been financially supported by the deputy of research, tehran university of medical sciences, and grant number: 47832. ethical considerations this research has been registered with the ethics code ir.tums.sph.rec.1399.175 in the ethics committee of the school of public health, tehran university of medical sciences. conflict of interest statement the authors declare there is no conflict of interests. references 1. who (2022) world malaria report 2022. world health organization, geneva. 2. hviid l, jensen at (2015) pfemp1–a parasite protein family of key importance in plasmodium falciparum malaria immunity and pathogenesis. adv parasitol. 88: 51–84. 3. shahi m, hanafi-bojd aa, vatandoost h, soleimani ahmadi m (2012) susceptibility status of anopheles stephensi liston the main malaria vector, to deltamethrin and bacillus thuringiensis in the endemic malarious area of hormozgan province, southern iran. j kerman univ med sci. 20: 87–95. 4. oshaghi ma, yaghoobi f, vatandoost h, abai mr, akbarzadeh k (2006) 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[(accessed on 20 march 2023)]; available at: https://www.iaea.org/sites/default/files/2 1/06/nafa-ipc-manual-guidelines-forstandardised-mass-rearing-of-anophelesmosquitoes-v1.0.pdf. 17. nasirian h, ladonni h (2006) artificial blood feeding of anopheles stephensi on a membrane apparatus with human whole blood. j am mosq control assoc. 22 (1): 54–56. 18. finney dn (1971) probit analysis, 3rd edition. cambridge university press, london. 19. wilkinson cf (2013) insecticide biochemistry and physiology. 2nd ed. springer us, new york. 20. shahinfar e, heidari a, alizadeh bh (2015) to investigate the degradation status of expired pesticides based on the formulation and quality control parameters. environ sci. 13(2): 57–66. 21. davari b, vatandoost h, oshaghi ma, ladonni h, enayati a, shaeghi m, basseri hr, hanafi-bojd aa (2007) selection of anopheles stephensi with ddt and dieldrin and cross resistance spectrum to pyrethroids and fipronil. pest biochem phys. 89: 97–103. http://jad.tums.ac.ir/ https://www.iaea.org/sites/default/files/21/06/nafa-ipc-manual-guidelines-for-standardised-mass-rearing-of-anopheles-mosquitoes-v1.0.pdf https://www.iaea.org/sites/default/files/21/06/nafa-ipc-manual-guidelines-for-standardised-mass-rearing-of-anopheles-mosquitoes-v1.0.pdf https://www.iaea.org/sites/default/files/21/06/nafa-ipc-manual-guidelines-for-standardised-mass-rearing-of-anopheles-mosquitoes-v1.0.pdf https://www.iaea.org/sites/default/files/21/06/nafa-ipc-manual-guidelines-for-standardised-mass-rearing-of-anopheles-mosquitoes-v1.0.pdf j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 61 http://jad.tums.ac.ir published online: march 31, 2022 original article pediculicidal activity of foeniculum vulgare essential oil in treatment of pediculus capitis as a public health problem elham jahanifard1,2, hoda ghofleh-maramazi1, *mona sharififard1,2, mohammad mahmoodi sourestani3, amal saki-malehi4, elham maraghi4, sima rasaei5 1department of medical entomology and vector control, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran 2social determinants of health research center, ahvaz jundishapur university of medical sciences, ahvaz, iran 3department of horticultural science, faculty of agriculture, shahid chamran university of ahvaz, ahvaz, iran 4department of biostatistics and epidemiology, school of public health, ahvaz jundishapur university of medical sciences, ahvaz, iran 5department of dermatology, school of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran *corresponding author: dr mona sharififard, email: sharififard-m@ajums.ac.ir (received 2 july 2020; accepted 11 feb 2022) abstract background: pediculosis, caused by pediculus spp is an important public health problem in urban and rural areas around the world. natural compounds such as plant essential oils (eos) have been suggested as a potential alternative for insect pest control recently. the purpose of this study was to investigate the toxicity of foeniculum vulgare essential oil against the head louse, pediculus capitis under laboratory conditions. methods: fennel essential oil components were analyzed using gc-mass apparatus. immersion and contact filter paper bioassays were used to evaluate fennel essential oil toxicity at the two-fold concentrations of 2.5, 5, 10, 20, and 40% against nit and nymph/adult stages of the head louse. results: trans-anethole, α-thujone, and limonene, which consisted of 76.08%, 10.37%, and 5.34% were the most components of fennel oil respectively. the lc50 values for the adult /nymphs were 11.5, 6.4, 3.9, 3.1 and 2.5% and lc99 values were 29.5, 15.2, 12.8, 10.8, and 7.4% at 10, 20, 30, 45 and 60 minutes after exposure respectively. the lethal times (lt50) for adults/nymphs were 5.2, 8.1, 9.5, 20.5, and 45.8 minutes and lt99 were 138.6, 91.3, 23.8, 21.7, and 13.9 minutes in the concentrations of 2.5, 5, 10, 20 and 40%, respectively. lc50 and lc99 values were 2.32% and 7.36% after 5 days for the eggs. conclusion: fennel essential oil at the concentration of 15% after 20min is suggested to develop as an appropriate formulation to evaluate in clinical trials. keywords: head lice; pediculus capitis; pediculusis; foeniculum vulgare introduction pediculus capitis, a blood-sucking insect, belonging to the order anoplura, the family pediculidae, completes its life cycle on the human head as obligatory ectoparasite (1, 2). the insect feeds several times during the day every two-three hour. today, head louse is one of the most important health problems around the world from a village to urban areas (1–3). the main mode of transmission of this infestation is close personal contact and sharing of personal stuff (1–3). head louse infestation is a global health problem that often infests children in school ages (4 to 13 years), teachers and family members, and other people who copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 62 http://jad.tums.ac.ir published online: march 31, 2022 will be in contact with the infected children (1–5). it is higher in girls and women (2, 3). according to who reports approximately 6–12 million people are annually infected by head louse in different areas of the world. this public health problem is prevalent in many developing countries where the primary healthcare program of who is inefficient and haphazard. the prevalence of this infestation worldwide is variable from 0.7–50% and between less than 5% to over 40% among school children, respectively (1–3). the highest prevalence rate of this infestation was seen in central and south america (33%), followed by africa (31%), australia (19%), asia (18%), north america (8%), and europe (5%) (6). epidemiological studies in schools in various countries have shown the different frequencies of pediculosis; 13.6% in mexico, 26.6% in jordan, 15.3% in south africa, 23.32% in thailand, 26.4% in nigeria, and 28.3% in england (1–3). head louse infestation in different regions of iran has been reported as less than 6% to 30% (4). varied prevalence rates have been observed in different provinces of iran such as 4% in urmia, 13.5% in hamedan, 1.8% in kerman, 4.7% in sanandaj, 7.6% in qom province, 27% in sistan-balochistan province, and 0.47% in isfahan (1–4). pediculus capitis infestation is increasing in some areas in iran along with other communicable diseases (5). growth of population, people's immigration from villages to cities, marginalization, and the establishment of satellite settlements with minimal health facilities and welfare services can be the reasons for this increase (5). although head louse does not transmit any disease to humans, it causes problems such as itching, skin lesions, lymph nodes, and secondary fungal and bacterial infections, including yellow ulcers in severe infestation (6–8). it can be caused anemia, particularly in children, headaches, insomnia, bad morals, restlessness, and decentralization, especially in children, social embarrassment, isolation, and mental stress (1–8). physical methods such as combing, separating hair lice from the hair, and scrubbing hair have been used to remove head louse infestation in the past (9, 10). accurate differentiation of nits and hair casts plays important role in treating p. capitis (11). nowadays, anti-pediculosis compounds are recommended to treat the infestation (9, 10). chemical control, as the main treatment for head lice, involves the use of a wide range of synthetic neurotoxic insecticides such as permethrine %1, malathion 0.5%, lindan 1%, permethrin 5%, crotamiton 10%, ivermectin 0.5% , spinodad 0.9%, pyrethrins plus piperonal butoxide and benzyl alcohol 5% (9, 10). most of these compounds may have harmful effects on the patients (10). the high prices of anti-lice products and lice resistance concerns in several countries, including iran (5, 7). therefore, today, the development of new anti-lice combinations with higher safety and performance is considered a serious necessity. alternative control compounds with novel mode of action, low mammalian toxicity and harmless environmental impact are needed to be developed in order to prevent and control vectorborne diseases. it seems the plant based on products and their main components, such as meutropoinids are good alternatives to chemical insecticides because of easily their extracted, biodegradability and little toxicity against mammals (12). also, they are effective against a wide range of insect pests, including head lice, and unlike chemical pesticides, the problem of resistance development is occurring slowly (12, 13). many of these compounds are found in the markets in unusual products offered as pediculicides without proper evaluation (14). the anti-lice properties of the plant compounds may be enhanced by their lipophilic performance and cause better penetration and greater bioavailability in the insect body (15, 16). several factors which include the lipophilicity of the prodhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 63 http://jad.tums.ac.ir published online: march 31, 2022 ucts, the rate of diffusion through the cuticle and some physicochemical variables such as the density and the molecular structure of the eo components may affect the penetration rate and finally, contact toxicity of the eos (18-19). so far, numerous plant essential oils have been studied to determine their pediculicidal properties against head lice around the world (8, 13–26). fennel (foeniculum vulgare) is an aromatic herb of the family apiaceae. this plant is known as a native plant in the mediterranean and southern europe, and warm weather is favorable for its growth. it is distributed in different parts of iran, including gorgan, northern manjil, baluchistan and azerbaijan (27). fennel is one of the medicinal plants that have been introduced in iran's herbal pharmacopoeia (27), which is known for its medicinal properties, including anti-nausea, digestibility, and diuretic properties. different parts of the fennel plant are considered for essential oil extraction. there are more than 30 components in fennel essential oils, the most important of which are trans-anethole, funchon, limonene, α-pinene and estragole (28–30). the main fennel oils have acaricidal, anti-fungal, antibacterial properties, and have recently been shown to have a repellent effect against insects (28–30). its efficiency has been proven against insect-borne diseases such as the mosquitoes culex pipiens and aedes aegypti (29, 30). the aim of this study was to determine the toxicity of f. vulgare essential oil against head lice under laboratory conditions. materials and methods essential oil extraction the fennel plant was harvested from razan area of hamedan province during the harvesting season in late summer and early autumn. the dried seed was used for essential oil extraction (28–30). the seed was crushed and powdered with an electric mill and was extracted by the clevenger apparatus by water distillation. for this purpose, every 100 to 150g of crushed fennel seed was extracted for 4 hours using a clevenger apparatus. extracted essential oils were stored in the refrigerator at 4 °c and in the dark glass until the test was performed. determination of essential oil components gas-chromatography-mass spectrometer (gc-ms) was used for the analysis and identification of fennel essential oil components (gc agilent 7890, ms agilent 5975). it was equipped with hp–5ms column (30m× 0.25 mm× 0.25μm). for this purpose, the essential oil samples were first rehydrated with sodium sulfate (na so4) and diluted with dichloromethane, which was especially spectrophotometric. then 0.2μl of diluted oil was taken by the micro sampler and injected into the gcms apparatus. the essential oil constituents were identified by comparing their retention indices, and mass spectra fragmentation with those in a stored wiley 7n.1 mass computer library and those of the national institute of standards and technology (nist). bioassay tests adults, nymphs, and eggs of p. capitis were collected from the head of children 6–13 years old, who attended primary schools in karoon county, khuzestan province from three schools over a 2-month period. the children were not previously treated with anti-lice products for at least 1 month and the head lice were collected using a finetoothed anti-louse metal comb and transported to the medical entomology laboratory of ahwaz jundishapur university of medical sciences in glass jars with screw caps. adults and nymphs test fennel oil was dissolved in ethanol as solvent to obtain the following two-fold doses: 2.5, 5, 10, 20 and 40% (= 0.39, 0.77, 1.5, 3.1, 6.2mg/cm2). for evaluating pediculicidal activity, the contact bioassay method was used. petri dishes lined with wathman no.1 filter pahttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 64 http://jad.tums.ac.ir published online: march 31, 2022 pers (9cm in diameter) were treated with 1ml of different concentrations of eos and control filter paper received 1ml of ethanol. after drying of filter papers, batches of 10 adults and 4–5th instar nymphs of head lice were placed on each petri dish, containing a few strands of human hair, and the dishes were covered with lids. treated and control groups were left in petri dishes for 15min at 65±5% humidity in the dark chamber and incubated at 35±2 °c and then placed in petri dishes with untreated filter papers and incubated under mentioned conditions (18, 31). lice, exposed to the essential oil within 2h after collection. a control test was performed with lice placed on solvent ethanol-impregnated filter paper dried for 5 min. the plates with adults and nymphs of lice were observed by stereomicroscope at 10, 20, 30, 40, and 60min after exposure. head louse death was defined as the absence of movement of limbs and gut, with or without stimulation using forceps. experiments were repeated at least three times (18). ovicidal test hair-containing eggs were cut from the student's heads with scissors and placed in a container and transported to the laboratory of medical entomology for bioassay testing. ten viable louse eggs were dipped in 1ml of eos solutions of 2.5, 5, 10, 20 and 40 % (= 6.2, 3.1, 1.5, 0.77 and 0.39mg/cm2) for 1 minute and then distributed in petri dishes lined with damped wathman no.1 filter paper. the control group was treated with ethanol as a solvent. treated and control groups were incubated at 35±2 °c and 65±5% humidity chamber in darkness. the louse egg hatching was monitored daily under microscopic inspection until 7 days after hatching of the control group. louse eggs with closed operculum and nymphs inside were the criterion for embryo mortality. experiments were repeated, at least, three times (14–16, 18). data analysis the probit regression model was used for determining lethal doses (ld50 and ld99) and lethal times (lt50 and lt99). the p-value and χ 2tests were used to assess the significance and goodness of fit to the probit regression models, respectively. the mortality means with fennel oil were corrected with abbott`s formula using natural mortality data. abbot`s formula (abbot): statistical analysis was performed using statistical package for social sciences (spss) software (version 16). a p-value of less than 0.05 was considered statistically significant. results essential oil extracted an average of 1.8ml of essential oil was extracted from 150 grams of fennel seed. the density of 1ml of fennel essential oil was calculated to be 0.97g/ml. approximately 19 main compounds were identified in the fennel essential oil, the highest amount was related to trans-anethole (76.08%), α-thujoneα (10.37%), d1-limonene (5.34%) respectively and then the combination of methyl chavicol (3.55%) (estragol) (table 1). bioassay test results the contact toxicity of fennel essential oil against head lice resulted in significant differences in mortality means over the times of 10, 20, 30, 45 and 60 minutes in a constant dose (f= 154.528, df= 4, p< 0.001). in other words, the effect of time in the means’ mortality was significant, so the mortality increases with time increasing. significant differences were observed between fennel essential oil doses, i.e., the effect of dose was significantly on the head lice mortality means (f= 265.658, df= 5, p< 0.001) and the mortality increased with essential oil increasing dose (table 2). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 65 http://jad.tums.ac.ir published online: march 31, 2022 probit analysis of head lice mortality rates (adult/nymph) at different times is presented in table 3. as shown, the p values of the model were < 0.05 at different times which indicates that the data fit the probit model. also, heterogeneity factors are < 1 which confirms the data accuracy (table 3). when the heterogeneity factor (which equals the chi-square divided by degrees of freedom) is > 1, a plot of the data should be examined because the data do not fit the model (32). the concentrations need to eliminate 50% and 99% of adult/nymph stages at different times are presented in table 3. with increasing time from 10min to 60min, lc50 values decreased from 16.05 to 7.38% and lc99 changed from 39.5 to 2.46%. a comparison of the lethal doses of 50% and 99% of head lice mortality at different times showed no overlap between the confidence limits (cl) of lc50 values at 10min with the other times (20, 30, 45 and 60min). so, it can be concluded that the effect of lethal doses on head lice mortality was significantly different. however, there was an overlap between the confidence limits of lc50 at 20, 30, 45 and 60 minutes, indicating no significant differences between the 50% lethal doses at these times. comparison of lc99 confidence limits after different time periods showed cl overlaps at 20, 30 and 45 minutes and so there were no significant differences in lc99 values at these time periods, but the highest overlap can be seen in the confidence limits of lc99 between 20 and 30min and also between 30 and 45min. lc99 values at 10 minutes were significantly different from these values at other times due to a lack of overlap in confidence limits. the lethal doses of 50% and 99% of fennel essential oil against head lice nit were 2.3% and 7.4% after 5 days, respectively. considering the model's p values and the heterogeneity factor calculation which is < 1 also show that the observed data fit the probit model appropriately. head lice lethal time the times required killing 50% and 99% of head lice in adult and nymph stages exposed to different concentrations of fennel essential oil are shown in table 4. it would take 13.9, 21.7, 23.8, 91.3 and 138 minutes to kill 99% of head lice at concentrations of 40, 20, 10, 5 and 2.5%, respectively, which indicate an inverse relationship between lethal times and essential oil concentrations. also, the negative slope values in table 4 indicate a decrease in lethal times with the concentration increasing of fennel essential oil. a comparison of the confidence limits of lt99 at different concentrations also indicates a significantly different between the time of lethality at concentrations of 2.5% and 5% with other lethal times. the 99 % lethal time at a concentration of 10%, 20% and 40% had no significant difference due to their overlap of confidence limit. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 66 http://jad.tums.ac.ir published online: march 31, 2022 table 1. storage time and percentage of essential oil components of fennel using gas chromatography )%( retention time fennel essential oil components 0.97 0.13 0.28 0.06 0.32 0.11 0.16 5.34 0.35 0.11 10.37 0.21 3.55 0.17 0.05 0.68 76.08 0.01 7.896 8.314 9.041 9.121 9.562 9.939 10.546 10.672 10.735 11.564 12.463 14.088 15.655 16.657 16.937 17.218 18.247 24.187 α-pinene camphene sabinene β-pinene β-myrcene 1-phellandrene cymene d1-limonene)limonene( 1,8-cineole terpinene α-thujone camphor methyl chavicol ) estragol( α-fenchyl acetate carvone anisaldehyde trans-anethole delta-cadinene table 2. mean mortality percentages of head louse at adult/nymph stages exposure to different concentrations of fennel essential oil by contact bioassay essential oil concentration (%) time after exposure (min) mortality mean (%) ± se 40 10 20 30 45 85±4.0 90±0.0 90±0.0 90±0.0 60 90±0.0 10 67.5±4.8 20 77.5±4.8 20 30 90±0.0 45 90±0.0 60 90±0.0 10 42.5±4.8 20 75±2.9 10 30 80±5.8 45 85±2.9 60 87.5±4.8 10 25±4.1 20 45±2.9 5 30 57.5±6.3 45 65±2.9 60 80±4.1 10 6±2.4 20 20±3.2 2.5 30 34±4 45 42±3.7 60 48±3.7 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 67 http://jad.tums.ac.ir published online: march 31, 2022 table 3. lethal concentrations (%) of fennel essential oil against adult and nymphs and egg of head lice using filter paper contact p slope (±se) lc99 (95%cl) lc50 (95%cl) time (min) head lice <0.001 21.4 (22) 0.07 (±0.01) 29.5 (23.2-36.9) 11.1 (9.5-21.6) 10 adult/ nymph <0.001 7.9 (22) 0.22 (±0.04) 15.2 (13.6-20.3) 6.4 (2.8-7.2) 20 <0.001 18.9 (22) 0.26 (±0.02) 12.6 (10.6-16.4) 3.9 (1.8-5.6) 30 <0.001 14.4 (22) 0.30 (±0.05) 10.8 (8.9-13.9) 3.1 (1.2-4.6) 45 <0.001 3.9 (22) 0.90 (±0.18) 7.4 (5.9-11.7) 2.5 (2.1-3.7) 60 <0.001 14 (21) 0.50 (±0.09) 7.4 (6.1-11.9) 2.3 (1.5-4.3) 5 days egg 95% cl: 95% confidence limits of lethal concentrations of 50% and 99% head lice, : chisquare (degree of freedom), p: significance level of probit model table 4. lethal times (lt50 and lt99) of head louse (adult / nymph) at different concentrations of fennel essential oil in vitro (min) 95% cl: 95% confidence limits of lethal times of 50% and 99% head lice, a: chi-square (degree of freedom), p: significance level of probit model discussion in this study, the highest percentages of fennel eo components were belonged to transanethole, α -thujone, and limonene which constituted of 76.08%, 10.37%, and 5.34%, respectively. this result is consistent with the other findings. trans-anethole (32% and 30%, respectively), limonene (28% and 18%, respectively) and fenchone (10% in both cases) were the main compounds identified in the fennel eos from cape verde and portugal, respectively (30). trans-anethole constituted 72% of the fennel oil composition in the study by zoubiri et al. 2014 (29). the results of lee (2004) on the properties of fennel essential oil against two dermatophagoides dust mites and the essential oil constituents showed that the highest percentage of essential oil components were belonged to trans-anethole, fenchone and estragole with 53.2, 14.2, and 12.7%, and fenchone had high lethal activity against dust mites (28). fennel essential oil showed appropriate potential for treating the head louse infestation in nit and nymph/adult stages in our study. at the concentration of 10%, it kills 50% and 99% of the adult/ nymph after 9.5 and 24 minutes, respectively. the lethal times of 50% and 99% mortality decreased to 8 and 21.7 minutes at the concentration of 20%. the lc50 and lc99 values decreased with an increase in exposure times. after 20 minutes, these values were calculated as 6.4 and 15.4%. fennel eos from cape verde and portugal resulted in 99% mortality of ae. aegypti larva at 37.1 and 52.4µl l-l, respectively (30). p a slope ( se) lt99 (95%cl) lt50 (95%cl) concentration (%) 0.001 10.1 (18) -1.5±0.21 138 (107.3-214.4) 45.8 (38.9-58.1) 2.5 0.001 8.2 (18) -0.68±0.2 91.3 (74.6-110.3) 20.5 (12.1-26.2) 5 0.001 10.9 (18) -0.52±0.25 23.8 (18.8-30.2) 9.5 (8.8-14.6) 10 0.001 6.2 (18) -0.3±0.19 21.7 (15.5-35) 8.1 (6.5-17.8) 20 <0.001 12.2 (18) -0.37±0.27 13.9 (9.8-32.5) 5.2 (3.6-8.4) 40 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 68 http://jad.tums.ac.ir published online: march 31, 2022 application of 40 and 60mg.l-1 fennel essential oil eliminated 50% and 90% of the second instar larval population of culex pipiens after 2 hours and 4 hours, respectively (29). repellency of antitol and estragole components of fennel essential oil has been reported against stored grain pests, rhyzopertha dominica, sitophilus zeamais and tribolium confusum. the estragole type of fennel essential oil showed more repellency than the anethole type against the three studied pests (26). the mentioned studies indicate that the fennel essential oil has toxicity or repel activity against insect pests. the results of these studies are consistent with our study in terms of the major constituent of fennel essential oil which is trans-anethole and the potential of this essential oil for repelling or killing of insect pests. differences in the obtained results can be attributed to the pest species, plant phenology affected by the geographical condition and climate as well as the bioassay test. adulticidal and ovicidal activity against p. capitis has been reported for some essential oils. many essential oils that are recommended for the treatment of head lice including eucalyptus, rosemary, geranium, tea tree, lemon, and their components were studied for possible adulticide and repellent effects on head and body lice (13–27). tea tree (melaleuca alternifolia) and nerolidol essential oils alone and in combination showed that tea oil with 1% concentration caused mortality of 100% of adult/nymph in 30min and had a better effect than nerolidol oil. the toxicity of nerolidol against head louse egg was better than tea essential oil (50% egg lethality at 1% concentration for 5 days). combining these two substances together killed the entire lice population within 30 minutes (18). the lethal toxicity of wild bergamot, clove, lavender, tea tree, and verbena essential oils was evaluated against adult head lice using impregnated filter paper bioassay method. clove oil, diluted either in coconut oil or sunflower oil, demonstrated the best adulticidal activity of > 90% mortality within 2h in lice exposure to 30min contact toxicity (14). the lt50 values were calculated as 2.5, 8.1, 9.5, 20.5, and 45.8 minutes in the concentration of 40, 20, 10, 5 and 2.5% (equal to 6.2, 3.1, 1.5, 0.77 and 0.39mg/cm2) respectively in our study. thymus vulgaris, aloysia polystachya and aoysia citriodora eos showed that thyme essential oil has significant toxicity for adult and egg stages as well as knock down against the eggs and adults of p. capitis by fumigant and contact toxicity bioassays. the calculated kt50 values for adults at doses of 0.84, 0.63, 0.42 and 0.21mg/cm2 were 3.93, 6.30, 6.49 and 9.90 minutes in contact bioassay, respectively (22). three origanum species essential oils have been shown to decrease the rate of limb, bowel, and abdomen movements of head louse significantly at the concentration of 1%, producing more than 90% mortality after 12h using the adult immersion test for 5min (27). nonconformity in the results can be related to differences in the components of the plant essential oil, the head lice susceptibility, and the bioassay method. variation in insect responses to different essential oils has been studied previously. the qualitative and/or quantitative chemical composition among plant species may be different and it definitely affects the obtained results (25). conclusion fennel oil is a potent and useful compound for human head louse treatment. concentrations of 12.6% to 15.2% of this essential oil killed 99% of adult/nymph at 30 and 20 minutes. it also kills 99% head louse nit in 5 days. but further research is necessary to evaluate the safety of this plant eo on human health and to develop appropriate formulation for improving the pediculicidal activity in clinical trials. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 69 http://jad.tums.ac.ir published online: march 31, 2022 acknowledgements this article is part of hoda ghofleh maremazi’s msph thesis. it was financially supported by ahvaz jundishapur university of medical sciences (ajums). 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volatile oil of foeniculum vulgare miller. in their tissue culture and comparison with the whole plant. j med plants. 3(15): 73–80. 28. lee hs (2004) acaricidal activity of constituents identified in foeniculum vulgare fruit oil against dermatophagoides spp. (acari: pyroglyphidae). j agric food chem. 52(10): 2887–2889. 29. zoubiri s, baaliouamer a, seba n, chamouni n (2014) chemical composition and larvicidal activity of algerian foeniculum vulgare seed essential oil. arab j chem. 7(4): 480–485. 30. rocha dk, matos o, novo mt, figueiredo ac, delgado m, moiteiro c (2015) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 61–71 e jahanifard et al.: pediculicidal activity of … 71 http://jad.tums.ac.ir published online: march 31, 2022 larvicidal activity against aedes aegypti of foeniculum vulgare essential oils from portugal and cape verde. nat prod commun. 10(4): 677–682. 31. who (1981) instructions for determining the susceptibility or resistance of body lice and head lice to insecticides. who/ vbc/81.808, geneva. 32. robertson jl, russell rm, presler hk, savin ne (2017) bioassays with arthropods. crc press, taylor and francis group. boca raton, london and new york. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 34 http://jad.tums.ac.ir published online: march 31, 2022 original article spatial distribution, seasonal abundance and physio-chemical assessment of mosquito larval breeding sites in mardan district, khyber pakhtunkhwa, pakistan sara gul1, *khurshaid khan1, muhammad sajjad1, muhsin jamal2, mujeeb ullah3, gauhar rehman1, abid ali1 1department of zoology, abdul wali khan university mardan, khyber pakhtunkhwa, pakistan 2department of microbiology, abdul wali khan university mardan, khyber pakhtunkhwa, pakistan 3department of zoology, islamia college university peshawar, khyber pakhtunkhwa, pakistan *corresponding author: dr khurshaid khan, email: khurshaidkhan@awkum.edu.pk (received 18 dec 2020; accepted 16 dec 2021) abstract background: mosquitoes (diptera: culicidae) are haemotophagus insects and are vectors of many arthropod-borne diseases. present study aimed to explore species composition, seasonal abundance, spatial distribution and physiochemical properties of larval breeding sites of mosquitoes in district mardan, khyber pakhtunkhwa, pakistan. methods: both adults and larvae of mosquitoes were collected through light traps, insecticide spray, mouth aspirator and larval standard dipping method in district mardan from may to november 2017. water samples from larval sites were physio-chemically analysed. results: 5078 (3704 adults and 1374 larvae) mosquito specimens were collected in mardan, katlang and takhtbhai tehsils. six species in four genera were reported. culex pipiens (89.80%) and armigeres subalbatus (9.20%) were the most abundant species. diversity was high in takhtbhai (0.29) followed by katlang (0.28) and mardan (0.25). greater number of specimens were recorded in peridomestic sites (93.97%) as compared to domestic habitats (6.03%). culex pipiens larval abundance had negative correlation with ph whereas it correlated positively with electric conductivity, salinity, and tds (total dissolved sulphur). mosquito abundance peaked in august and july while the lowest was in may. their monthly abundance had positive correlation with rainfall (r= 0.5069), relative humidity (r= 0.4439) and mean minimum temperature (r= 0.2866). number of mosquitoes was highest at low elevation < 347m asl (above sea level) in agriculture land and near to water bodies (streams). conclusion: culex pipiens being the most abundant species, was susceptible to high ph. mosquitoes preferred habitats were at low elevation in agriculture land. keywords: diptera; culicidae; takhtbhai; temperature; salinity introduction mosquitoes (diptera: culicidae) are hematophagous insects distributed throughout the tropical and temperate regions of the globe. to date, a total of 3,500 species and subspecies of mosquitoes have been recorded and are placed in 4 genera, among which important ones are culex, anopheles and aedes. some species of mosquitoes are the biological vectors of many human and livestock diseases including malaria, dengue, yellow fever and filariasis. malaria is considered a serious mosquitoes born infection that has greatly impacted human health over a century. in addition to their medical importance as vectors of human pathogens, female mosquitoes can attack humans and other animals causing nuisance of a magnitude that negatively affects individuals and society (1). mosquitoes lay their eggs (approximately 100–200 per adult life) in a variety of water sources ranging from small containers to marsh copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:khurshaidkhan@awkum.edu.pk https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 35 http://jad.tums.ac.ir published online: march 31, 2022 land ecosystems. among them anopheles species prefer clean and unpolluted water while culex species breed in water with high organic content. majority of them breed in drainage ditches, ponds, tin cans, old tires, and tree holes. some floodwater mosquitos eggs withstand desiccation for year/s and hatch when flooded again (2). in larval stages they feed on micro-fauna in aquatic habitats and after emergence their survival depends upon plant nectars with addition females take blood from a variety of animals. understanding mosquito species composition, distribution, abundance and seasonality is one of the essential prerequisites for surveillance and epidemiology of the infectious diseases (3, 4). their pattern of community composition are useful aspects for medical entomologists to gain broader insight and projections regarding disease dynamics, spread and maintenance of globally spreading pathogens (5). in mosquitoes ecological studies, geographic information system (gis) and remote sensing (rs) tools are used to identify environmental conditions associated with vector-borne diseases (6). these techniques have been used to identify conditions favourable for larval mosquitos development and factors particularly vegetation, elevation as well as landscape for mosquitos distribution (7). pakistan have a rich fauna of mosquitoes and so far, 134 species of mosquitoes were identified while two species (anopheles culicifacies s.l and an. stephensi) have been reported as malaria vectors (2, 811). inadequate research exists on mosquitoes in khyber pakhtunkhwa province of pakistan (2, 12). the previous published literature of the country mainly addressed aspects of mosquito identification, classification, vector potential and breeding sites of some species. limited researchers documented the general ecology, composition, seasonal variations, and breeding habitats of mosquitoes which is useful for both medical entomologists as well as epidemiologists including health workers. therefore, the present study aimed to explore species composition, habitats preferences, seasonal abundance, and spatial distribution of mosquitoes in district mardan, khyber pakhtunkhwa, pakistan. materials and methods study area the present study was carried out in mardan district (34.20 n and 72.05 e) with total surface area of 1,632km2, khyber pakhtunkhwa, pakistan (fig. 1). the district is bordered in east with buner and swabi districts, in north with malakand district, in south with nowshera district and in west with charsadda district. the region has distinct summer and winter seasons with average temperature 22.2 °c as well annual rainfall 559mm. topographically the area has plain in the south and west while mountainous terrains in the north-east. sugarcane, wheat, maize, tobacco and vegetables are the yielding crops where kalpani (entered from north) is the main stream. various industries, factories and mills are sporadically established in the district. study design, data collection and analysis the entomological study was conducted in 59 localities of three tehsils (administrative subunit of the district) of mardan including mardan, katlang and takhtbhai from may to november 2017. adult mosquito specimens were collected using insecticide flit method, light traps, mouth aspirators and hand net. larvae were captured through standard dipping method (13). light traps were used at nighttimes (8pm to 6am) while flit method was used for indoor sites collection in early morning. larval collection was made from water bodies (runny, stagnant, ponds, water tanks, tyres with 300ml water from the spot to ensure food supply in plastic jars). net cloth was used for closing jar openings and were later shifted to entomology research laboratory where the larvae in jars were reared at room temperature until they emerge into adults. during sampling, coordinates of each collection site was recorded http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 36 http://jad.tums.ac.ir published online: march 31, 2022 through gps (global positioning system) device (garmin etrex, usa). during the field study adults and larvae were collected once per month in 59 fixed sites in mardan from may to november 2017. mosquitoes were identified up to species level through taxonomic keys as described by christopher (14) and barraud (14, 15). water samples of 500ml of each larval site were collected in triplicate and analysed for total dissolved sulphur, salinity, electric conductivity, and ph following ma et al. (15). all data related to mosquitoes were arranged in microsoft excel for further analysis. annual rainfall, minimum, maximum temperature, and relative humidity data for the respective year (2017) were obtained from meteorological department, regional office, peshawar (kp), pakistan. gps co-ordinates data were entered into arcgis version 10.5.0 for spatial distribution where digital elevation model (dem) was extracted from advance spaceborne thermal emission and reflection radiometer (raster). landuse map of the district was obtained from national centre of excellence in geology university of peshawar, kp, pakistan. results a total of 5078 mosquito specimens (adults and larvae) representing four genera (culex, anopheles, armigers and aedes) containing six species were collected in 59 sites in mardan (35), katlang (14) and takhtbhai (10) tehsils. among the collected species cx. pipiens (n= 4560, 89.80%) was the most abundant species followed by ar. subalbatus (467, 9.20%) while ae. albopictus, cx. vishnui, cx. tritaeniorhynchus and an. walkeri showed least abundance <1%. highest mosquito abundance was recorded in mardan (3481, 68.55%) followed by takhtbhai (801, 15.77%) and katlang (796, 15.68%). a total of 1684 males and 3394 females were recorded and male to female ratio was 1:2 (table 1). variations in diversity of mosquitoes among the studied tehsils was observed whereas shannon diversity index h= 0.29 was highest for takhtbhai while species richness was in peak in katlang 0.141 and takhtbhai 0.106 (table 1). greater number of culex pipiens was collected in outdoor habitats (n=3009) as compared to indoor sites (n=105) in mardan tehsil. similarly, number of larvae (n=1089) was highest in mardan as compared to takhtbhai (n=204) and katlang (n=81). culex pipiens larvae were most abundant in mardan (n=1013) followed by takhtbhai (n=186) and katlang (n=77) (table 2). during the present study mosquitoes were collected from may to november 2017 and their peak abundance was observed in august and september while least abundance in may and november (fig. 2) positive correlation was observed between climatic variables and mosquito abundance in all studied tehsils (table 3). culex pipiens larval abundance had negative correlation with ph whereas it correlated positively with electric conductivity, salinity, and tds. majority of mosquito larvae were collected in ph of greater than 7.0 (table 4 and 5). overall mosquito abundance was high at elevation < 347m above sea level (asl) while their abundance was observed at elevation ranging from 347 to 1159m asl in the district. in addition, cx. pipiens abundance was also high at < 347m elevation in the region. culex. pipiens abundance was clustered on agriculture land of the district (fig. 3). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 37 http://jad.tums.ac.ir published online: march 31, 2022 table 1. species composition, diversity and distribution of mosquitoes (adults and larvae) in mardan district, 2017 species tehsils grand total mardan katlang takhtbhai gender ♂/♀ ♂:♀ ♂/♀ ♂:♀ ♂/♀ ♂:♀ cx. pipiens 1041/2073 0.5 182/523 0.34 236/505 0.46 4560 ar. subalbatus 152/185 0.82 36/49 0.73 19/26 0.73 467 ae. albopictus 8/18 0.45 1/1 0 0/0 0 28 cx. vishnui 0/0 0 2/2 1 5/10 0.5 19 cx. tritaeniorhynchus 1/1 1 0/0 0 0/0 0 2 an. walkeri 1/1 1 0/0 0 0/0 0 2 sub total 1203/2278 221/575 260/541 5078 species per site 5 4 3 species richness 0.084 0.141 0.106 diversity index (h) 0.25 0.28 0.29 fig. 1. study area location map http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 38 http://jad.tums.ac.ir published online: march 31, 2022 table 2. mosquito adult/larvae and indoor/outdoor abundance in mardan district, 2017 tehsils species cx. pipiens ar. subalbatus ae. albopictus cx. vishnui cx. tritaeniorhynchus an. walkeri sub total mardan (n/%) indoor 105(53.29) 73(37.05) 15(7.61) 0 2(1.01) 2(1.01) 197 outdoor 3009(91.62) 264(8.03) 11(0.33) 0 0 0 3284 katlang (n/%) indoor 27(24.77) 78(71.55) 2(1.83) 2(1.83) 0 0 109 outdoor 678(98.68) 7(1.01) 0 2(0.29) 0 0 687 takhtbhai (n/%) indoor 0 0 0 0 0 0 0 outdoor 741(92.5) 45(5.617) 0 15(1.87) 0 0 801 mardan (n/%) adult 2101(85.30) 334(13.56) 24(0.97) 0 2(0.08) 2(0.08) 2463 larvae 1013(93.08) 74(6.73) 2(0.18) 0 0 0 1089 katlang (n/%) adult 628(97.52) 12(1.86) 2(0.31) 2(0.31) 0 0 644 larvae 77(95.06) 2(2.65) 0 2(2.65) 0 0 81 takhtbhai (n/%) adult 555(92.96) 42(7.04) 0 0 0 0 597 larvae 186(91.18) 3(1.47) 0 15(7.35) 0 0 204 grand total 4560 467 28 19 2 2 table 3. tehsil wise correlation between abundance and climatic parameters in mardan, 2017 tehsils monthly mean min. temp monthly mean max. temp monthly total rain fall (mm) relative humidity (average) mardan 0.2704 0.0033 0.5666 0.5127 katlang 0.2591 0.12 0.1723 0.2182 tkhtbhai 0.3538 0.1266 0.5054 0.2759 total abundance 0.2866 0.0396 0.5069 0.4439 http://jad.tums.ac.ir published online: march 31, 2022 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 39 http://jad.tums.ac.ir published online: march 31, 2022 fig. 2. tehsil wise seasonal abundance of mosquitoes in mardan district, 2017 fig. 3. mosquito abundance projected on elevation and land cover maps, 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 40 http://jad.tums.ac.ir published online: march 31, 2022 table 4. larvae collection and physiochemical properties of water parameters of the studied sites in mardan, 2017 tehsil collection sites average ph average ec (μs/cm) average tds average salinity (g/l) mosquitoes larvae (number) mardan stagnant water near houses 7.33 1488.33 997.2 1.00 cx. pipiens (234), ar. subalbatus (15) stagnant water under shrubs/ herbs 7.51 1684 1128.3 1.13 cx. pipiens (200), ar. sublbatus (31) stagnant field water 7.39 1946.1 1303.9 1.3 cx. pipiens (224), ar. subalbatus (10), ae. albopictus (2) sewerages 7.3 2213 1482.7 1.48 cx. pipiens (220), ar. subalbatus (2) stagnant water bodies at dung places/ stockyard and garbage’s 7.58 1495.85 1002.23 1 cx. pipiens (135), ar. subalbatus (16) katlang clean water ditches in crop field 7.48 1569 1051.25 1.05 cx. pipiens (77), ar. subalbatus (2), cx. vishuni (2) takhtbhai drainages 7.4 1886 1263.6 1.26 cx. pipiens (186), ar. sulbalbatus (3), an. vishnui (15) mean 7.450 1697.916 1137.614 1.136 average sdv 0.107 412.523 276.392 0.276 tds: total dissolved sulphur, ec; electric conductivity, sd: standard deviation table 5. species wise correlation between larval abundance and water parameters in mardan, 2017 total larvae cx. pipiens ar. subalbatus ae. albopictus average ph -0.42 -0.4064 0.4642 -0.1628 average ec 0.36 0.3395 -0.4352 0.3113 average tds 0.36 0.3396 -0.4351 0.3114 average salinity 0.37 0.3474 -0.4296 0.3077 tds: total dissolved sulphur, ec: electric conductivity http://jad.tums.ac.ir published online: march 31, 2022 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 41 http://jad.tums.ac.ir published online: march 31, 2022 discussion mosquito biodiversity and ecological preferences are poorly known despite the fact that there is a need to explore the diverse mosquitos fauna in south asia including pakistan (16). recent dengue epidemics revealed the need for more detailed understanding of the diversity, habitat preferences and distribution of these arthropods in the region (17, 18). studies on species composition and abundance of local mosquito populations have helped to develop better management strategies for mosquito-borne diseases (19-21). therefore, this study was aimed to provide information related to species composition, seasonal variation, larval breeding sites and spatial distribution of mosquitoes in district mardan, khyber pakhtunkhwa, pakistan. during the study period total of 5078 mosquito’s specimens (adults and larvae) belonged to four genera and six species including cx. pipiens, ar. subalbatus, ae. albopictus, cx. vishnui, cx. tritaeniorhynchus as well as an. walker were recorded in the mardan. among the collected mosquitoes, medical importance of cx. pipens is not yet investigated in pakistan. although this species is source of nuisance due to its biting pattern. the high abundance of this species was reported in different countries globally (22) and locally in pakistan (23). this species could transmit filarial parasites to humans and in immunocompromised individuals. in the present study ar. subalbatus were anthropophilic mosquitoes and are known to feed on birds blood showing an opportunistic feeding behaviour (24). however, their medical importance is not adequately studied in pakistan. although, this mosquito has the vectorial capability of brugian filariasis (25), japanese encephalitis virus (26) and dog heart worm (dirofilaria immitis) (27). in the current study we reported one species of aedes genus, ae. albopictus primarily because the collection sites were rural, and the species has been reported to be preferentially rural. it’s been known to transmit at least 20 arboviruses, filarial worms and a main vector of dengue in asia (28). in pakistan previous studies established that, ae. albopictus is the vector of dengue and is chiefly responsible for the maintaining the disease transmission in rural areas (12). culex tritaenorhynchus, cx. vishnui and an. walkeri were captured in least number in the study area. among them cx. tritaenorhynchus usually prefer human settlement (24). it is responsible for the transmission of wnv (west nile virus) in pakistan (29). other two species, cx. vishnui and an. walkeri are suspected to be the vectors of je (30) and wnv to humans respectively (31). mosquitoes were collected in three tehsils of mardan district whereas, highest number of specimens were recorded in tehsil mardan as compared to katlang and takhtbai. this is probably because the collection had cx. pipiens in highest number, which has been known to prefer polluted and urban landscape for existing and breeding (32). tehsil mardan has congested human settlements, polluted environment and is a main business hub while other two tehsils have freshwater habitats. other reason for variations in species composition and distribution is the variation in various biotic factors (food) and abiotic factors (habitat diversity, water bodies, weather conditions and elevation) (33). investigating seasonal variation has a key importance in the control of vector-borne diseases. in our study we found that mosquitoes had a single peak from july to september in the region. in pakistan, heavy rainfall occurs in these months that provides suitable breeding sites and climate for their existence. during the collection period maximum number of specimens were recorded from stagnant water bodies which are reported to be the breeding sites for culex, aedes and anopheles in the province (12, 34). all ground water breeding mosquito larvae require optimal water temperature, tds and http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 34–44 s gul et al.: spatial distribution … 42 http://jad.tums.ac.ir published online: march 31, 2022 ph for their survival. we observed that mosquito larvae favoured ph of 7.4± (6.8-7.4± preferable for breeding) as recorded by others (35, 36). there was a positive correlation of larval abundance with tds. its known that tds in its higher concentration reduces water transparency and increases oxygen deficiency (37). in our spatial analysis we observed mosquito abundance at low elevation < 347m asl, in agriculture land and near water bodies (streams). utilization of vector distribution maps help to predict the risk for transmission of various diseases (38, 39). the current study highlights the mosquito diversity of mardan region which is poorly explored in the past. the importance of these findings may help medical entomologist and health departments for implementing effective control and surveillance of mosquitoes borne diseases. conclusion a total of sex (06) mosquitoes were collected whereas, culex pipiens being the most abundant species, was susceptible to high ph. mosquitos species preferred habitats at low elevation in agriculture land in the area. this study will help the medical entomologists and health experts for allocation of the budget. acknowledgements we would like to thank dr basit rasheed, department of zoology, university of peshawar in and dr muhammad naeem department of chemistry, abdul wali khan university mardan for assisting in mosquito identification and in water samples analysis. ethical considerations this study was approved by the board of studies, abdul wali khan university mardan, khyber pakhtunkhwa, pakistan. conflict of interest statement authors declare that there is no conflict of 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psychodidae) vectors of leishmaniasis in pakistan. j med entomol. 55(3): 626–633. 39. kitron u (1998) landscape ecology and epidemiology of vector-borne diseases: tools for spatial analysis. j med entomol. 35(4): 435–445. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 301 http://jad.tums.ac.ir published online: dec 31, 2022 original article accurate identification of leishmania parasites in sand flies by polymorphism analysis of cytochrome oxidase subunit 2 gene using polymerase chain reaction and quantitative pcr-high resolution melting techniques in iranian border with iraq seyedeh maryam ghafari1,2, reza fotouhiardakani1,2, *parviz parvizi1 1molecular systematics laboratory, parasitology department, pasteur institute of iran, tehran, iran 2cellular and molecular research center, qom university of medical sciences, qom, iran *corresponding author: dr parviz parvizi, e-mail: parp@pasteur.ac.ir (received 08 nov 2020; accepted 03 sep 2022) abstract background: firmly identification of leishmania in phlebotomus papatasi and understanding of natural transmission cycles of parasites in sand flies are important for treatment and local control. methods: modified and developed method of high resolution melting (hrm) as a preferable technique was employed to accurate identification of leishmania in sand flies from iranian border with iraq, by targeting cytochrome oxidase ii (coii) gene and designing suitable primers. pcr products cloned into ptg19-t vector, then purified plasmid concentration was measured at 260 and 280nm wavelength. the melting curve plots were generated and dna sequences were analyzed using sequencher 3.1.1, clc main workbench 5.5, mega 6, dnasp5.10.01 and medcalc® version 13.3.3 soft wares. results: among about 3000 collected sand flies, 89 female ph. papatasi were identified and two with l. major. in amplified fragment of coii gene among 611bp, 452bp had no genetic variations with low polymorphic sites (p= 0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). leishmania major was discriminated in ph. papatasi with 0.84 °c melting temperature (tm) and unique curve based on thermodynamic differences was an important criterion using hrm technique. conclusion: subsequent war in iraq made a high risk habitat for parasites transmission. it is important to discover accurate diagnostic procedures for leishmaniasis control. keywords: cloning; cytochrome oxidase ii; qpcr-hrm introduction phlebotomine sand flies (diptera: psychodidae, phlebotominae) are the only proven natural vectors of leishmania species, causative agents of a neglected tropical disease, leishmaniasis (1-3). leishmania is a digenetic parasite with the extracellular stage within an invertebrate vector which called promastigote and the intracellular stage within a vertebrate hosts and reservoirs which called amastigote (4). incrimination and identification of leishmania parasite, has been developed and performed using different molecular methodologies and analyses for more than 30 years (2, 5-7). genetic analysis of mitochondrial and nuclear genes has commonly been employed for leishmania species identification considering their sensitivity, simplicity, reliability and specificity in epidemiological studies (2, 8). high resolution melting (hrm) method is an efficient and cost-effective method that eliminates the risk of laboratory contaminations. in recent years, quantitative pcr using sybr green or taqman chemistries have been developed and evaluated for detection, quantification, identification and leishmania species differentiation in human samples, a few in res copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:parp@pasteur.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 302 http://jad.tums.ac.ir published online: dec 31, 2022 ervoir hosts and few in sand flies (9-13). suitable methodology helps to distinguish among leishmania species especially in tiny insect of female sand flies as natural vectors of leishmaniasis. this is crucial and essential in epidemiological studies to determine appropriate procedures (14). in this research for typing at intra-species level of leishmania species, new discriminative molecular markers have been applied (2, 8). qpcr-hrm, may answer the epidemiological questions of zcl in the region of border between iran and iraq such as transmission cycles of animal reservoirs and transmitting of the vectors, infection’s origin, modeling of spreading in this border and disease distinction of imported cases. the objectives of this investigation were to determine leishmania species and genotyping of leishmania parasites in sand flies of ilam province. materials and methods locations, sand flies trapping and morphological identification sand flies were captured from gerbil burrows and nearby domestic animal shelters in two locations of ilam province, border with iraq where ph. papatasi is proven vector of zoonotic cutaneous leishmaniasis (zcl). accordingly, mehran and dehloran were considered (31°, 58’ to 34°, 15’ n and 45°, 24’ to 48°, 10’ e) in west of iran, with altitude of 2740 meters above sea level (masl) (fig. 1 and table 1). the sticky papers, cdc miniature light traps (with the white light bulb 1–2m above ground level) which were set overnight to sample sand flies in domestic animal shelters and inside houses in the morning and manual aspirators were applied for sand flies sampling by expert field collectors. besides, funnel traps were used to sample from rodent borrows. the collected specimens were put in microtubes without ethanol and then were frozen. all female sand flies were identified by morphological characters of the head and terminal genitalia, following a dissection of fed females with sterilized forceps and microneedles and mounted in berlese fluid (15)( fig. 2). dna extraction, pcr and sequencing dna from thorax and abdomen of sand flies were extracted using ish-horovize method with minor modifications and also using genet bio kits (genet bio, daejeon, korea) (5, 15). total dna was extracted from the dissected thorax and attached anterior abdomen of individual females of ph. papatasi. each sample contained the midgut, the location of most l. major promastigotes, and was homogenized in a 1.5ml microfuge tube using a disposable plastic tip of a micropipette. following ethanol precipitation, the dna was dissolved in 15µl 1×te (10mm tris-hcl, 1mm edta ph 8.0), to give a concentration of 5– 10ng/µl, and stored at −20 °c (5, 15). cytochrome oxidase subunit ii (coii) as an enzymatic gene was amplified to detect any leishmania infection among sand flies (2, 8). new sensitive and specific primers were designed and employed to amplify coii gene to detect leishmania parasite in sand flies. pcr was performed on every female ph. papatasi using coii specific new designed forward primer, coii f (5′-atggcttttata ttatcattttg-3′) and reverse primer, coii r (5′ggcataaatccatgtaagac-3′). the amplification reaction was carried out in a total of 20µl containing 1× taq polymerase buffer (promega), 1.5mm mgcl2, 60µm of each dntp, 1µm both forward and reverse primers, 1µm primer unit taq polymerase (promega) and 1.5µl of dna (5–10ng/µl) extracted from individual wild caught sand flies. the mixture was incubated in a pe geneamp ®pcr thermocycler 9700 (0.2ml block) at 94 °c for 3min followed by 37 cycles, each consisting of 30s at 94 °c, 30s at 58 °c and 90s at 72 °c. after the last cycle, the extension was continued for a further 10min then held at 4 °c (16). pcr products were subjected to electropho http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 303 http://jad.tums.ac.ir published online: dec 31, 2022 resis on 1.5% agarose gel and leishmania positive pcr products were used for sequencing by abi prismtm 310 automated sequencer (applied biosystems, thermo fisher scientific, foster city, usa) in order to accurate identification of species and haplotypes (2, 8). sequences of coii gene was compared in different leishmania species to determine conserved areas in sand flies of ilam. the amplified sequences with those from genbank, were compared and analyzed in aspect of their phylogenetic and polymorphism. cloning in this study, in order to prepare standards for qpcr-hrm and besides for re-sequencing of ambiguous sites, pcr products were cloned directly into ptg19-t vector using sinaclon pcr ta cloning kit (sinaclon bioscience, tehran, iran). to confirm cloning results, colony pcr with vector specific primers was performed (17). plasmid was extracted from transformed bacterias, using sinaclon plasmid isolation kit (sinaclon bioscience, tehran, iran). purified plasmid concentration was measured at 260 and 280nm wavelength using nanodrop® nd-1000 spectrophotometer (thermo fisher scientific, waltham, usa). in accordance with the following formula dna copy number was calculated. then, seven serial dilutions of plasmids were prepared in the ratio of one to ten (fig. 3). dna (copy number)= [6.02× 1023 (copy/ mol)× dna amount (g)]/ dna length (dp)× 660 (g/mol/dp) quantitative pcr-high resolution melting high resolution melting (hrm) analysis were carried out by corbett rotor-gene 6000 hrm real time pcr instrument (corbett life science, sydney, australia). a part of coii gene was amplified using designed co.a. 470.2 forward and reverse primers in the presence of evagreen dye (fig. 4a). in this method, the observed differences in the melting temperature were noticed and the use of hrm method was considered for separation of different species. hrm procedure was designed and performed in 20µl containing 2µl of dna or plasmid (10 ng/reaction), 0.7µl of each primer (10pmol/µl or 10µmol/l) and 4µl of 5x hot firepol® evagreen® qpcr mix plus (solis bio byne, tartu, estonia). then, 12.6µl of pcrgrade h2o was added to the volume. the initial denaturation for 1 cycle was 15 minutes at 95 °c then it was followed by 45 cycles, 15 seconds in each cycle for denaturation; 30 seconds at 55 °c for annealing, 30 seconds at 72 °c for extension and final extension was 5 minutes at 72 °c (1 cycle) and then stored for 10 minutes at 4 °c. the qpcr-hrm products were followed by a conventional melting step: melting curve was performed from 65 to 92 °c, with an increasing slope of 0.1 °c each step, with 2 seconds rest at each step afterwards. finally, to determine the average tm for each leishmania spp., the melting curve plots were generated and analyzed using hrm software (corbett life science, sydney, australia). statistical analysis dna sequences were edited and aligned using sequencher 3.1.1 and clc main workbench 5.5 softwares. polymorphism and phylogenetic analyses of our sequences with standard sequences from genbank were carried out using mega 6 and dnasp5.10.01 softwares (18). in order to compare the median and tm, the medcalc® version 13.3.3 software and mann-whitney u test were used. the calculation of diversity between sequences and neutrality were performed by dnasp 5.10.01 software. the ratio of non-synonymous substitution (dn) to synonymous substitution (ds) was assessed using dnasp 5.10.01 software (19). results sand flies sampling and preparation out of more than 3000 collected sand flies, 234 sand flies were dissected, mounted and five http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 304 http://jad.tums.ac.ir published online: dec 31, 2022 species were identified (table 1). more than collected samples were male sand flies and more of the sand flies were not ph. papatasi so 234 were female ph. papatasi. eighty nine out of 234 were fed which were screened in order to detect leishmania infection. leishmania major was found at least in two ph. papatasi out of 89 female sand flies from two locations (mehran and dehloran) (table 1). polymorphism analysis from 89 female ph. papatasi only two were infected with leishmania which identified and sequenced. the monomorphic and polymorphic positions of coii were compared using chi-square test. of 611bp nucleotides, 452bp had no genetic variations. also, the number of polymorphic sites were significantly lower than that of monomorphic sites sequences (p= 0.001). for informative and non-informative sequences in the evaluated gene, genetic variation occurred in 46 nucleotide positions. bioinformatics analyses were performed by dnasp5.10.01 software and number of synonymous sites was significantly greater than non-synonymous sites (p= 0.00). in the expression gene (coii), among 26% segregating sites in nucleic acid variation areas, 20.2% sites were non-synonymous mutations also, more mutations might have occurred in the negative selection side. based on the findings by dnasp5.10.01 software, ds/dn ratio for coii had the low amino acid changes (ds: synonymous mutations in silent sites, dn: non-synonymous mutations in replacement sites). according to tajima's d index, dn/ds ratio was calculated to be 0.14 (in the positive direction) so coii gene has produced through evolutionary process (table 2) (20). a natural evolution was reported, based on the comparison of mean nucleotide diversity (π= 0.075) and expected number of mutations (θ= 41.51) in each sequence of gene. the study of different coii sites of leishmania species showed no significant difference in nucleotide diversity (π) or expected number of mutations (θ) (p= 0.86). the comparison of synonymous mutations in silent sites (ds) and non-synonymous mutations in replacement sites (dn) in coii expression gene showed a significant difference between two leishmania species (p= 0.001). the number of ds (79.8%) was greater than dn (20.2%). the greatest dissimilarities in ds site were detected in the comparison of l. major and l. tropica populations (p= 0.019). among 12 sequences of l. major, 19 nucleotide positions (13 ds and 6 dn) were different in terms of gene expression. also, among four sequences of l. tropica species, 27 nucleotide positions (7 ds and 20 dn) varied in terms of gene expression. haplotype diversity was analyzed for l. major and l. tropica and was observed 81.35% and 28.38%, in l. tropica and l. major populations respectively. the average, standard deviation (sd), and coefficient variation (cv) showed low to high haplotype diversity (p= 0.000). to determine the extent of natural selection in leishmania species, the average number of nucleotide differences (k) and the number of expected genetic differences in the whole sequence (θ) were compared, using tajima’s d index. results showed that evolution was positive in coii gene (table 2). genetic evaluation and phylogenetic analysis 30 leishmania sequences were employed. these species are included two l. major (two haplotype were found in two ph. papatasi in ilam) and 28 leishmania species reference strains: 10 l. major, four l. tropica, three l. donovani, and three l. infantum from old world and five from new world: two l. mexicana and two l. tarentolae and one l. amazonensis. they were employed to evaluate the sensitivity and specificity of coii gene for accurate identification of leishmania species (fig. 5 and table 3). to determine the extent of natural selection in leishmania species, the average number of nucleotide differences (k) and the number of expected genetic differences in the whole http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 305 http://jad.tums.ac.ir published online: dec 31, 2022 sequence (θ) were compared, using tajima’s d index between l. major and l. tropica species. based on the findings, coii gene belongs to l. tropica species has been produced through evolutionary processes (tajima’s d= 2.85, p< 0.01). this indicates that evolution was positive in coii gene. molecular phylogenetic analysis of coii gene is presented in figure 5 based on the maximum liklihood (ml) method. it presents the concatenated tree of coii gene. figure 5a was drawn, based on the nucleotide sequences. kimura’s two-parameter method was used for 30 nucleotide sequences. the topology of the tree revealed that this mitochondrial gene caused the separation of leishmania species, whereas this was unable to separate subspecies or determine haplotype diversity. by applying the ml method, 1326 sites were analyzed. also, under similar conditions, the phylogenetic tree of coii gene based on the mitochondrial genetic amino acid codes, showed a similar topology by ml model (fig. 5b). quantitative pcr-high resolution melting different dilutions of dna template were prepared for validation, accuracy and sensitivity of hrm assay (r2= 0.94, efficiency= 4.49). high sensitivity of hrm could pull out minimum amount of dna to draw appropriate melting curves. serial dilutions of specimens were experienced on three consecutive days with similar reactions. identical dna concentrations were repeated with coii gene. melting curves for serial dilutions were compared. identical dna were analyzed and melt patterns were appropriated for both species on different days. according to results, two common leishmania species in ilam (l. major and l. tropica) were discriminated with 0.84 °c difference in tm, using hrm technique (mean tm were 74.8 °c and 75.64 °c for l. major and l. tropica respectively (fig. 6, 7 and table 4). fig. 1. geographical location of ilam province, and sampling sites table 1. sampled sand flies collected in june to august 2016, from ilam province has shown base on gender, species and locations http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 306 http://jad.tums.ac.ir published online: dec 31, 2022 species sergentomia clydei sergentomia dentata sergentomia sintoni phlebotomus mongolensis phlebotomus papatasi gender f m f m f m f m f m location in rb ash in rb ash in rb ash in rb ash in rb ash mehran dehlouran nirogah bargh 0 0 1 0 0 0 1 0 0 0 1 3 0 0 0 0 0 9* 2 38 imamzadeh hasan 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 2 11 0 4 56 dasht akbar 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 2 0 0 15 7 chehmeh gheer 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 10 janbazan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 12 roosta ali 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 6 0 33* 2 1 1 1 0 6 4 0 7 89 125 total 234 (97f+137m) in: indoor, ash: animal shelter, rb: rodent burrow, f: female, m: male, *leishmania positive fig. 2. phlebotomus papatasi female, a: unfed, b: fed, c: gravid, d: semi gravid fig. 3. a: two leishmania infection were detected by pcr. the sequences were identified as l. major based on sequencing. -ve: negative control, +ve: positive control, m: markers (100bp left). b: coii gene was cloned in the ptg19-t vector. plasmids were extracted and serial dilution of them were prepared. m: markers (1000bp right, 100bp left). 1: pcr products with coii primers for plasmid including coii in l. tropica with 611bp, 2: pcr products with coii primers for plasmid including coii in l. major with 607bp, 3: pcr products with m13 primers for plasmid including coii in l. major with 800bp, 4: plasmid extraction products contains coii gene in l. major with 3500bp, 5: plasmid extraction products contains coii gene in l. tropica with 3500bp, 6: plasmid without ptg19-t vector with 2880bp table 2. polymorphism analysis of coii sequences from leishmania species by dnasp5.10.01 software http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 307 http://jad.tums.ac.ir published online: dec 31, 2022 a gene no. seq. no. nucleotide (bp) v (%) singleton variable (%) parsimony variable (%) synonymous/replacement change 2 v a r ia n ts 3 v a r ia n ts 4 v a r ia n ts t o ta l 2 v a r ia n ts 3 v a r ia n ts 4 v a r ia n ts t o ta l c .c d s (% ) d n (% ) d n /d s r a tio coii 29 611 24 0 0 24 114 21 1 136 15 147 (87) 21 (12) 0.14 b gene no. seq. no. nucleotide (bp) s eta k π ө per site ө per seq. tajima’s d h hd coii 29 611 149 183 45.97 0.075 0.067 41.51 0.42 27 1 a: v: variable (polymorphic) nucleotide site, c·c: total number of sites in other codons (complex codons), which were not analyzed because of their highly variable regions, ds: synonymous mutations in silent sites, dn: non-synonymous mutations in replacement sites b: s: segregation site, eta: total number of mutations, k: average number of nucleotide differences between pairs of sequences, π: mean nucleotide diversity, θ per site: expected number of mutations per a site, θ per seq: expected number of mutations per a sequence, tajimaʼs d: the statistical test proposed by tajima (1996), h: no. of haplotypes, hd: haplotype diversity fig. 4. a: based on obtained sequences and polymorphism analysis results, forward and reverse primers were designed for qpcr of coii gene. co.a.r470.2 (f: tggagaaacaacaatatttagtaa, r: cctaaacttgaaattgcaaatg) b: schematic illustration of coii gene, amplified with specific primers (small black arrows) for leishmania species using clc bioinformatics software (8) http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 308 http://jad.tums.ac.ir published online: dec 31, 2022 table 3. details of coii sequences of leishmania parasites from ilam and genbank sequences for constructing phylogenetic tree the name of species accession no. origin source reference l. major **ku680818 **ku680819 ku680820 af287688 eu140338 ef633106 kf815208 kf815210 kf815211 mh443402 mh443403 mh443404 soviet union iran italy sudan japan not found sudan “ “ “ “ “ “ not found not found not found ulcers of patients ulcers of patients ulcers of patients not found ulcers of patients not found clinical samples:hb,hbm,hln* “ “ “ “ “ “ not found not found not found this study this study 8 21 22 21 23 23 23 direct submission (aghai maybodi et al. 2018), unpublished direct submission (eslami et al. 2019), unpublished direct submission (aghai maybodi et al. 2018), unpublished l. donovani fj416603 kf815198 ay660023 us sudan “ “ “ cultivated parasites ulcers of patients clinical samples:hb,hbm,hln nebohacova et al. 2009 23 23 l. infantum kf815207 kf815206 kf302727 “ “ “ “ “ “ not found “ “ “ “ “ “ not found 23 23 direct submission (soares, 2013), unpublished l. tarentolae ku680825 l07544 germany usa ulcers of patients cultured parasites 8 25 l. amasonensis hq586836 china “ “ “ 26 l. mexicana hq586845 ku680824 “ “ “ brazil “ “ “ cultured parasites 26 8 l. tropica kf302720 ku680821 hq586846 hq586847 brazil iran shandong, china soviet union not found ulcers of patients cultured parasites cultured parasites direct submission (soares, 2013), unpublished 8 26 26 26 l. sp. hq586841 hq586843 china jiangsu, china cultivated parasites “ “ “ 26 26 hb: blood, hbm: bone marrow, hln: lymph node, **: identified l. major in this study, “ “ “: same as above http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 309 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 5. phylogenetic analyses discriminated leishmania species well. a: phylogenetic tree based on the mitochondrial nucleotides (coii gene) drawn by maximum likelihood method. b: phylogenetic tree based on the mitochondrial genetic amino acid codes, drown by maximum likelihood method fig. 6. specifications performance of leishmania detection by qpcr validation. amplification plots derived from serial dilutions of cultured parasites (l. major and l. tropica standards with r and f co.a.470.2 primers), ranging from 20–2× 107 copies / reaction plasmids by qpcr http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 310 http://jad.tums.ac.ir published online: dec 31, 2022 table 4. quantitative pcr-high resolution melting analysis of coii gene for leishmania major and l. tropica in different plasmid copy number fig. 7. melting temperatures and normalized melting profiles obtained with the hrm assays for l. major and l. tropica. a: derivative melt curves, b: aligned and normalized melt curves, c: differences melt curves discussion for this investigation, blood fed female sand flies were screened for detecting leishmania in fections from two locations in ilam province (mehran and dehlouran) in iranian border with leishmania species plasmid copy number qpcr hrm tm mean efficiency r2 tm l. major 104 4.49 4.49 0.94 0.94 74.60 74.8 104 74.83 105 74.65 105 74.58 106 74.55 106 74.63 107 74.63 107 74.85 l. tropica 104 75.08 75.64 104 75.22 105 75.00 105 74.98 106 75.50 106 75.42 107 74.25 107 74.22 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 311 http://jad.tums.ac.ir published online: dec 31, 2022 iraq where the endemic foci of zcl places in these two countries. in these locations, ph. papatasi is proven and main vector of l. major (27). we were not surprised to find only two ph. papatasi which carried l. major out of 89 sampled in our preliminary screen. the infection rates of ph. papatasi can be low even in well studied in different zcl foci in the world while infection rates can be high in human and reservoir hosts (5, 28-29). in about 20 past years, the sensitivity and specificity of molecular techniques was compared targeting different mitochondrial and nuclear genes for identifying l. major (2, 8). now, we have tried to take an effective step with a new modified molecular method and genetic analysis for detection and identification of infection in native ph. papatasi samples, in ilam province. rapid, sensitive and accurate diagnostic procedures are crucial for detecting and characterizing of leishmania parasites in sand flies, in order to provide accurate treatment, precise prognosis and appropriate control measurements. for this research a new technique of hrm was employed for fast detecting of leishmania species and mix infections targeting coii gene in sand flies. coii gene is an expression gene. the silent site (ds) and replacement site (dn) of l. major and l. tropica were significantly different (p= 0.001). nucleotides were 611 positions in the final dataset and 452 out of 611bp had no genetic variations. evolutionary relationships of taxa for coii maxicircle mitochondrial were analyzed using molecular phylogenetic of maximum likelihood method. the nucleotide's substitution in coii gene in l. major had 86% similarity with sequences available in the genbank. coii is a mitochondrial gene and all mitochondrial genes have more mutations to compare with nuclear genes. when coii gene compared with other polymorphic genes in different leishmania species has less diversity. although boite and colleagues (2012) mentioned that coii was less capable for discriminating and distinguishing different leishmania species but this research and other investigations showed that the phylogenetic analyses and trees of this gene is able to identify leishmania strains at the species level (8, 30). coii gene is conserve enough to discriminate leishmania parasites and has polymorphism sites to discriminate some species, more over it has 20–50 copy numbers (8, 26). according to previous findings, coii is the most sensitive as compared with itsrdna, hsp70, nagt and cyt b genes that were tested (2, 8). in this research new primers were designed to detect leishmania species and strains. the prediction’s results were analyzed using clc software and followed by hrm method. the altered regions could make a desirable temperature difference in species of leishmania parasites (31). according to polymorphism and phylogenetic analyses of coii gene, l. major was discriminated in ph. papatasi from ilam province with 0.84 °c tm using hrm technique. determining unique curves based on thermodynamic differences is the important criterion of hrm method. hrm can detect single nucleotide polymorphisms based on small differences in the nucleotide composition for any suitable mitochondrial and nuclear genes (6, 32). hrm to compare with conventional pcr is more sensitive, specific, simpler, less expensive and faster. dna can be extracted directly from samples, blood and other tissues. hrm results can be obtained without additional post-pcr processing in less than 2.5 hours (33). so far, many effective and efficient studies have been conducted to isolate leishmania parasite from sand flies (9, 33-35). because of eight years-imposed war between iraq and iran subsequently kuwait then long time iraq occupation by usa following isis, much transmission of l. major occur in locations in both sides of iranian border with iraq. these situations have provided refuges for many sand fly species as well as reservoir http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 301–314 sm ghafari et al.: accurate identification of … 312 http://jad.tums.ac.ir published online: dec 31, 2022 hosts to make a high risk habitat for transmission of parasites (5, 36). conclusions the transmission cycles of l. major might depend on many criteria including diversity of leishmania species and intraspecific strains, sand fly species, reservoir hosts, locations and migration of two countries people. different parasite species interact to maintain l. major infections in reservoir hosts and people. understanding the roles of ph. papatasi as a proven vector which transmits l. major to the reservoir hosts and people, is important; also it is equally important to discover the complementary roles of rapid, sensitive and accurate diagnostic procedures for leishmaniasis in order to disease control and treatment in iranian border with iraq. acknowledgements the authors wish to thank the healthcare authorities of ilam province as well as the staff of molecular systematics laboratory of pasteur institute for their assistance. there are no disputes over the ownership of the data presented in the paper. this work was supported by pasteur institute of iran (grant number 735) as well as the national institute for medical research development (nimad) (grant number 973166) awarded to prof parvizi. ethical considerations this work was supported by pasteur institute of iran (grant number 735) as well as the national institute for medical research development (nimad) (grant number 973166). conflict of interest statement the authors declare there is no conflict 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northeastern caspian littoral, iran aioub sofizadeh1; *mohammad reza abai2,3; hassan vatandoost2,3; ahmad raeisi4; mohammad sistanizadeh-aghdam2 1infectious diseases research center, golestan university of medical sciences, gorgan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4malaria control unit, center for communicable diseases control, ministry of health and medical education, tehran, iran *corresponding author: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir (received 22 mar 2020; accepted 23 may 2021) abstract background: malaria resurgence has occurred in the northern half parts of iran. the resurgence of malaria in the prone area could arise from various factors, e.g. wide use of pesticides in the agriculture sector and factors such as habitual patterns of movement of local people from problematic southeastern foci in iran toward the caspian littoral. there are no new data on the resistance status of main malaria vectors in the caspian littoral, and this study was aimed at renewal data on conventional insecticides. methods: the field strain of adult anopheles superpictus and anopheles maculipennis were collected using the hand catch method and transferred to the laboratory. the susceptibility tests were carried out against ddt 4%, malathion 5%, permethrin 0.75%, deltamethrin 0.05%, and lambda-cyhalothrin 0.05%, followed by the who’s procedure. results: the primary malaria vector in caspian littoral is an. maculipennis, revealed to be still resistant to ddt and mortality rate, lt50 and lt90 of female mosquitoes were 75.0%, 54.2, minutes and 111.3 minutes. the under ’verification required’ status of an. maculipennis was also revealed to lambda-cyhalothrin based on recent who’s criteria. the malaria vector an. superpictus is also considered the second malaria vectors in the west parts of the studied area, which showed to be susceptible to all insecticides tested. conclusion: ddt resistance is persisted in an. maculipennis despite stopping residual spraying with ddt since 1978 in the caspian littoral, but the occurrence of pyrethroid under ’verification required’ status is a progressive threat to the possible development of cross-resistance in the future. keywords: anopheles maculipennis; anopheles superpictus; insecticide resistance; malaria; caspian littoral introduction malaria is one of the most important vector-borne diseases globally, especially in developing countries, and iran is located in the eastern mediterranean region with lower malaria endemicity. the country's southeastern parts, including the provinces of sistanbaluchistan, hormozgan, and southern kerman are characterized by "refractory malaria". later on up to the year 1944, malaria epidemiology was studied by some iranian and overseas investigators, and it was found the hypo-endemic situation at some littoral parts of the caspian sea in north of iran (1). in the past years, studies were carried out in the golestan province from 1949 to 1957 and, the spleen index was measured in 21 villages. the classical malariometric measure causing splenic enlargement rate was estimated at 52.1% in the bandar-e copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:abaimr@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 171–178 a sofizadeh et al.: resistance status of … 172 http://jad.tums.ac.ir published online: june 30, 2021 gaz and 32.5% in the gorgan area during 1949–1959. however, the annual parasite index was reported as 5.7% in the gorgan area, northern iran. the prevalence of malaria was stated as 100 per 10,000 populations in 1949 to 8 per 10000 populations in 1959, and the mortality decreased from 40% to 2%. malaria cases were recorded 164 in the gorgan, 50 in the bandar-e-shah, 57 in the komish-tapeh, 52 in the gonbad-e-kavous, 44 in the hajilar, and 103 in the gaz among the age group 2–12 years old in 1935. the percentage of malaria parasites was 50.0% plasmodium malariae, 46.1% plasmodium vivax, and 3.9% plasmodium falciparum (2). seven anopheline mosquitoes, including anopheles stephensi, anopheles culicifacies, anopheles fluviatilis, an. superpictus, anopheles sacharovi, an. maculipennis complex and anopheles dthali are involved in the transmission of malaria in iran (3-5). a total of three species of malaria vectors was reported in north of iran, and an. maculipennis was introduced as the primary vector (3) and an. superpictus as a secondary vector (6-7) and anopheles sacharovi are also considered a malaria vector in the northwestern parts of iran (8). malaria was considered a significant health problem from 1941 to 1948, so that no other disease has caused such irreparable financial and human losses in the country. the disease has become more common in the populated areas of the caspian territory, due to the presence of a favorite climate for the development of anopheles mosquitoes. control strategies were using the residual spraying of indoor places with ddt, larval control with oil derivation at different habitats, and treating the patients with quinine. malaria eradication program (mep) was started in 1957 in iran and from 1957–1971 caused interruption of transmission in the north of iran (9). due to prone condition of study area, and favorite climate for malaria vectors, routine movement of local people from the southeastern parts to northeastern of the caspian area as well as the wide application of pesticides in the agricultural sector, this study aimed to determine the susceptibility level of two anopheles species to the conventional insecticides in the kalaleh district, northeastern of the caspian territory. materials and methods study area the study was conducted in the kalaleh district (37° 22' n, 55° 29' e), golestan province, from april to october 2016. this province was split off from mazandaran province in 1998. the province is bounded by the caspian sea and the mazandaran province in the west, the semnan province in the south, the north khorasan province in the east, and a borderline with turkmenistan in the north (fig. 1). this study was carried out in three fixed villages and five randomly selected one in kalaleh district. most parts of the golestan province are plain, and more than 2/3 of the plains have arid and semiarid climates, and 1/3 of the others have a temperate climate. the district area is 1985km2 with 117660 population located in the northeast parts of the golestan province. the main agricultural products are alfalfa, rice, watermelon, and cotton. maximum and minimum temperatures were recorded as 40.8 and -0.2 °c, respectively, and the mean annual relative humidity was recorded as 74.0 %. the total annual rainfall was 772mm, the minimum precipitation in august and maximum in february. the sampling of mosquitoes carried out in 3 villages of kalaleh district, including aziz-abad (37°32'45''n, 61°41'52''e), gharanki-jangal (37°34'31''n, 61°46'43''e) and gorgandoz (37°31'24''n, 61°43'10''e) with the mean elevation of 65 meters above sea level. mosquito collection the sampling plan for collecting of adult mosquitoes was carried out from april to february 2016. the fresh-fed mosquitoes were dominant compared to unfed and gravid physiologic conditions, so only the fresh-fed mosquitoes were used in order to the homogeneity of test http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 171–178 a sofizadeh et al.: resistance status of … 173 http://jad.tums.ac.ir published online: june 30, 2021 data. the indoor-resting mosquitoes were collected by mouth aspirator before sunrise, transferred into the wooden cages, and transported in a cool condition to the laboratory in the health center of kalaleh district, golestan province, northeast of iran. susceptibility test susceptibility levels of field-collected mosquitoes to insecticides were determined by exposing freshly fed females to the diagnostic doses of insecticide-impregnated papers supplied by who, i.e. ddt 4%, malathion 5%, permethrin 0.75%, deltamethrin 0.05%, and lambda-cyhalothrin 0.05%. the exposure time for all the insecticides tested was 60min, followed by a 24h recovery period. to calculate the lt50 for ddt, the logarithmic exposure times ranged 15, 30, 60, and 120 minutes, followed by 24h holding period were carried on. each logarithmic exposure time was replicated four times using 25 female field-caught mosquitoes. the susceptibility exposure tubes were held in the vertical position during testings with pyrethroids, organochlorine and organophosphate insecticides (12). the recovery period of exposed mosquitoes was kept in a room with a temperature of 25±2 °c. simultaneously, the control group also was exposed 60 minutes to untreated papers. after exposure, the mosquitoes spent the recovery period at 25±2 °c and 70–80% relative humidity with access to soaked cotton pads in 10% sucrose solution for 24h until scoring the mortality. if control mortality was within 5–20%, test mortality was corrected by abbott's formula. the mortality rate was ranked as the susceptible, under ’verification required’ status, and resistant, based on who’s criteria e.g., 98–100%, 90–97%, and below 90%, respectively (10-11). results the dominant species were, an. superpictus and an. maculipennis that tested for resistance/susceptibility level to ddt, malathion, deltamethrin, permethrin, and lambda-cyhalothrin (table 1). the response of 100 mosquitoes of an. maculipennis to ddt 4.0% for 1h, followed by a 24h recovery period resulted in the survival of 25 mosquitoes, and the mortality was 75.0%. the regression parameters of ddt time-response, including intercept (a), slope ± standard error (b±se), heterogeneity of mortality data with the degree of freedom (χ2(df)), lt50 ±95% confidence interval (ci), and lt90 ± 95% ci were calculated (table 2). the lt50 and lt90 values for an. maculipennis were 54.2 and 111.3 minutes, respectively. the regression line and the equation was shown in fig. 2. the susceptibility level of an. superpictus to the tested insecticides is summarized in table 1, which showed complete susceptible to all tested insecticides. table 1. susceptibility levels of dominant species of anopheles at the diagnostic doses to different insecticides using who-recommended method (10), northeastern parts of the caspian littoral, iran insecticide anopheles superpictus anopheles maculipennis total mosquito tested no. dead mortality rate (%) resistance status* total mosquito tested no. dead mortality rate (%) resistance status* ddt 4% 100 99 99.0 s 100 75 75.0 r malathion 5% 100 100 100.0 s 100 100 100.0 s deltamethrin 0.05% 100 98 98.0 s 100 98 98.0 s permethrin 0.75% 100 100 100.0 s 100 100 100.0 s lambda-cyhalothrin 0.05% 100 100 100.0 s 100 96 95.5 v control 100 0 0.0 100 0 0.0 *s=susceptible; v=under ’verification required’ status; r=resistant http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 171–178 a sofizadeh et al.: resistance status of … 174 http://jad.tums.ac.ir published online: june 30, 2021 table 2. regression analysis of bioassay data of anopheles maculipennis exposed to ddt 4.0% using whorecommended method, northeastern parts of the caspian littoral, iran a b±se lt50 (min)±95%cl lt90 (min)±95%cl χ2 (heterogeneity) χ2 table (df) p -7.1132 4.1014±0.337 49.4392 54.2415 59.6185 97.2503 111.3806 132.7855 24.685 * 5.991 (2) 0.0 5 fig. 1. map of study area showing kalaleh district, golestan province, northeast of iran where two main species of anopheline were collected fig. 2. regression parameters estimating the lethal time of anopheles maculipennis exposed to ddt 4.0%, northeastern parts of the caspian littoral, iran http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 171–178 a sofizadeh et al.: resistance status of … 175 http://jad.tums.ac.ir published online: june 30, 2021 discussion currently, malaria is regarded as an infectious disease causes financial losses and workforce health. it is still concerned with health authorizes at the sistan and baluchistan, hormozgan, and southern kerman provinces (12). with attention to development achieved during five decades of vector control programs and the reduction of prevalence, malaria elimination is in the joint approach of the iranian ministry of health and the world health organization (12). due to the risk of malaria reemergence in northeastern parts of the caspian littoral, which is caused by numerous climatic, environmental, and social factors, determining of susceptibility level of anopheles vectors was noticed. the maculipennis complex comprised 12 palearctic members that distributed in different provinces including west azarbaijan, east azarbaijan, ardabil, guilan, mazandaran, golestan, isfahan, fars, kohgiluyeh and boyer-ahmad, kermanshah, kurdistan, zanjan, tehran and khuzistan but the exact distribution of each member of maculipennis complex as well as its bioecology is not clear (3). the resistance ratio (rr) which calculated by dividing the lc50 of the resistant population by the lc50 of the susceptible strain had been calculated for an. maculipennis in different localities of iran during 1970–1977, e.g., central parts (isfahan province, rr= 64.2 min), the northwestern parts adjusting to borderlines of republic azarbaijan and armenia (ardabil province, rr= 57.5min, the east azarbaijan province, rr= 58.4min), caspian littoral (guilan province, rr= 77.1min; mazandaran province, rr= 58.1min; golestan province, rr= 63.7min) and northeastern parts (razavi khorasan, rr= 74.6min) (13-14). during the malaria resurgence at the caspian littoral in 2008, it was shown that an. maculipennis (strain astara, guilan province, caspian littoral) exhibited low resistance (84.0%) to ddt, whereas susceptible to malathion, lambda-cyhalothrin, and deltamethrin during 1998– 1999 (15). a similar study conducted at different villages of the mazandaran province, in the caspian plateau during 1988–1989 and the susceptibility level of an. maculipennis was determined against ddt 4% after 60min of exposure time using who’s method. the results showed the resistance of an. maculipennis to ddt ranged 72.5–94.4%, which followed 93.9% mortality after 120min exposure. the latter species was susceptible to dieldrin 4% and malathion 5.0% but surprisingly showed under ’verification required’ status to deltamethrin 0.025 % with a mean of mortality of 96.5% (16). also, the susceptibility level of an. maculipennis to ddt 4.0% was also determined in the guilan province, in the west of the caspian littoral during 1987 with the mortality rate of 87.5– 91.7%, 90.5–94.3%, and 96.1–97.1% after 120, 150, and 180min exposure time which indicated a high resistance level of an. maculipennis to ddt 4% in guilan province (16). another study conducted on the susceptibility level of anopheles messeae against ddt 4% using the who’s method at 60min in sari, amol, and tonekabon districts, mazandaran province, caspian littoral during 1989–1990. the results also revealed a high resistance level of an. messeae to ddt ranged 8.9–61.2%, with a mean of 40.1%. the latter species was reported as susceptible to dieldrin 4% and malathion 5.0% (17). during a recent trial in the northwestern part of iran, it was indicated that an. maculipennis (strain west azarbaijan, the borderline of turkey) displayed high resistance (50.0%) against malathion and under ’verification required’ status to permethrin and deltamethrin (18). in neighboring countries of iran, susceptibility tests on an. maculipennis were carried out since 1974 in turkey, revealing resistance to organophosphate insecticides (19). the resistance of anopheles artemievi, one member of the maculipennis complex, was established to ddt (26.7%) at different parts of uzbekistan. the variations in susceptibility level of http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 171–178 a sofizadeh et al.: resistance status of … 176 http://jad.tums.ac.ir published online: june 30, 2021 an. maculipennis were shown related to seasonal change and mosquito collection months (20-21). the resistance of five strains of an. maculipennis was confirmed to ddt, malathion, permethrin, and deltamethrin in turkey (20, 22). during this study, it was revealed that an. superpictus still remained susceptible to all tested insecticides from different groups. a similar situation of the susceptibility of an. superpictus was shown in different parts of iran during 1971–74 (23) and then in ilam province, west of iran during 2000 (24), whereas a record of resistance (56.0%) of an. superpictus was recently recorded in the sistan and baluchistan province, southeastern iran (25). in the piedmont and mountainous districts of uzbekistan, an. superpictus was also highly sensitive to the insecticides, while the diapausing female an. superpictus mosquitoes in the population were found to be resistant to ddt (82.8%) and highly resistant to malathion (43.8%) (26). more than a half-century has passed since the newer investigations revealed the an. superpictus still remained susceptible to ddt, malathion, and pyrethroids (27). in tajikistan, an. superpictus was proved to be exophile and completely susceptible to the malathion, but with a low ddt resistance (28). a different pattern of susceptibility was shown a low resistance (85.0%) to the deltamethrin, but susceptible to the ddt, malathion, and permethrin between the field population of an. superpictus collected from the badakhshan province, afghanistan (29). the adult an. superpictus that collected from the jordan in the middle east showed a transit susceptibility (96.0%) to the deltamethrin, whereas completely susceptible to the lambda-cyhalothrin (30). the study's results and comparison of the past and present data in different countries indicated a serious alert status for pesticide management both in health and agriculture arthropod control. conclusion susceptibility level of an. maculipennis to ddt remained with the least change in the eastern part of caspian littoral despite withdrawal of indoor spraying with ddt since 1971, but under ’verification required’ status to pyrethroids could be considered a threat to the possible development of resistance in the future. the results of the tests on malathion, deltamethrin, permethrin revealed susceptibility to both an. maculipennis and an. superpictus to these insecticides. acknowledgments this study was supported by the research deputy of golestan university of medical sciences (gums) under research code 35/678. the authors would like to appreciate the kind collaboration of gums deputy and the staff of the health center of kalaleh district. the authors declare that there is no conflict of interest. references 1. edrissian gh (2006) malaria in iran: past and present situation. iranian j parasitol. 1: 1–14. 2. jalali m (1956) history of malaria and it's control in iran to 1955. institute malariology and parasitology, tehran university and ministry of health. iran. 225: 60–74. 3. djadid n, gholizadeh s, tafsiri e, romi r, gordeev m, zakeri s (2007) molecular identification of palearctic members of anopheles maculipennis in northern iran. malar j. 17: 1–10. 4. manouchehri av, zaim m, emadi am (1992) a review of malaria in iran, 1957–1990. j am mosq control assoc. 8: 381–385. 5. salari-lak sh, vatandoost h, entezarmahdi mr, ashraf h, abai mr and nazari m (2002) monitoring of 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mingaleva gn (1992) a comparison of the level of resistance and irritability in anopheles hyrcanus and an. superpictus to insecticides. med parazitol (mosk). 1: 15–17. 29. ahmad m, buhler c, pignatelli p, ranson h, nahzat sm, naseem m, sabawoon mf, siddiqi am, vink m (2016) status of insecticide resistance in high-risk malaria provinces in afghanistan. malar j. 5: 98. 30. khalil a, kanani ka, katbeh-bader a, al-abdallat m, shadfan b (2015) susceptibility tests on insecticides used to control mosquitoes in jordan. jordan j biol sci. 8: 180–183. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 239 http://jad.tums.ac.ir published online: september 30, 2020 original article the phylogenetic analysis of cimex hemipterus (hemiptera: cimicidae) isolated from different regions of iran using cytochrome oxidase subunit i gene awat samiei1; *mousa tavassoli1; karim mardani2 1department of pathobiology, faculty of veterinary medicine, urmia university, urmia, west azerbaijan, iran 2department of food hygiene and quality control, faculty of veterinary medicine, urmia university, urmia, west azerbaijan, iran *corresponding author: dr mousa tavassoli, e-mail: m.tavassoli@urmia.ac.ir (received 05 sep 2018; accepted 24 aug 2020) abstract background: bedbugs are blood feeding ectoparasites of humans and several domesticated animals. there are scarcity of information about the bed bugs population throughout iran and only very limited and local studies are availa ble. the aim of this study is to assess the phylogenetic relationships and nucleotide diversity using partial sequences of cytochrome oxidase i gene (coi) among the populations of tropical bed bugs inhabiting iran. methods: the bedbugs were collected from cities located in different geographical regions of iran. after dna extraction pcr was performed for coi gene using specific primers. then dna sequencing was performed on pcr products for the all 15 examined samples. results: dna sequencing analysis showed that the all c. hemipterus samples were similar, despite the minor nucleotide variations (within the range of 576 to 697bp) on average between 5 and 10 single nucleotide polymorphisms (snps). subsequently, the results were compared with the database in gene bank which revealed close similarity and sequence homology with other c. hemipterus from other parts of the world. conclusion: in conclusion, this study has demonstrated the ability of the coi gene to differentiate between the c. hemipterus populations from a few different locations in iran. the current research is the first report of phylogenetic and genetic species diversity analysis conducted on c. hemipterus in iran. these results provided basic information for further studies of molecular epidemiology, public health and pest control operators in iran. keywords: bed bug; cimex hemipterus; coi; phylogenetic analysis; iran introduction “bed bugs” is a term often applied to the approximately 90 species within the cimicidae; of these, only cimex lectularius, the common bed bug, and c. hemipterus, the tropical bed bug, show a strong host preference for humans (1). cimex hemipterus, is considered as the common tropical bedbug whereas c. lectularius is common in temperate climates (2). it has been well documented that bed bugs harbor at least 40 human pathogens, but there is no tangible evidence regarding the ability of routine mechanical transmission any of them (1, 3-6). all the daytime, bed bugs disappear and hide in inaccessible places such as: cracks and crevices in beds, wooden furniture, floors, and walls and reappear at night to feed from their preferred host, humans (7). apart from the discomfort caused by the bite, bedbugs have been known to cause secondary infections and psychological disorders (4). chronic infestation can cause nervousness, lethargy, pallor, diarrhea, and even iron deficiency (8-10). bedbugs infest any kind of temporary accommodations, especially hotels, serviced apartments, and cause severe problems. a resurgence of bed bugs has been reported copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 240 http://jad.tums.ac.ir published online: september 30, 2020 from the united states, canada, australia, europe, and some asian countries during the past 15 years (11-16). the reasons for the explosion have not yet been clarified; however, several factors such as increased rate of international travel, reduced use of residual insecticides indoors, and insecticide resistance may play a role (17). few studies have been undertaken on these bugs in iran. dehghani et al. examined an outbreak of these bugs in 1998 in villages west of kashan. out of 495 houses in 10 villages there was 6.7% contamination1 (18). in that year, the national association of managing world pests announced the contamination in new york at 6.7%. shahraki et al. in 2000, examined an outbreak of bugs in university dormitories among 180 boys and 145 girls in yasuj, western iran. they reported 28.9% contamination which approximated the current study after taking into consideration that the numbers of individuals were not close to our study (19). haghi et al. reported that in bahman amir, mazandaran, iran most bugs were found in bedrooms (56.54%), living rooms (31.25%), and kitchens (8.59%) (17). also, from the 182 examined containers in polour, mazandaran province, 164 (approximately 90.1%) had evidence of contamination by bed bugs (20). genetic analysis of medically important insect species is absolutely required, because it provides useful information about vector transmission, disease epidemiology and disease control (21). there is scarcity of information about the bed bug population throughout iran and only very limited and local studies are available (17, 20). taxonomic and morphologic identification of bed bugs require highly experienced person and appropriate samples. nowadays, molecular techniques such as nucleotide sequence analysis and phylogenetic tree have been developed for taxonomic identification (22). in recent years, the application of highresolution molecular markers has provided important new insight into the population genetic structure and infestation dynamics of many insect pest species of public health concern (23-25). new molecular tools now make it feasible to not only accurately identify the number of populations actively infesting a building (24, 26) but also to elucidate dynamics and characteristics essential for understanding infestation patterns and history, e.g., levels of genetic diversity (a measure often associated with population health (27), temporal stability of populations after pest control efforts (28), and the presence or absence of genetic mutations associated with insecticide resistance (29). population genetics studies on bed bugs have been completed using nuclear rrna, mt dna genes, and microsatellite loci as markers (25, 30-34). the aim of the first of these studies (szalanski et al. 2008) shed light on the dispersal patterns of bed bugs during their recent global resurgence. all of the studies on bed bugs thus far have found genetic diversity within human associated population to be low, resulting from a great deal of inbreeding (25, 30-32). despite the importance of bed bugs in iran, genetic evaluation has not yet been studied. therefore, we aimed at this shortcoming and analyzed dna sequences of c. hemipterus gathered from different parts of iran, using partial sequences of cytochrome oxidase i gene (coi) and evaluated the genetic relationship between them. materials and methods sample collection all procedures in this study were carried out in accordance with the guidelines of the animal ethics committee of faculty of veterinary medicine, urmia university (aecvu) and supervised by authority of sample collection urmia university research council (uurc). http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 241 http://jad.tums.ac.ir published online: september 30, 2020 geographically, there are 4 different zones in iran, known as region 1: caspian sea (temperature: 8–26 °c, annual rainfall: 400–1,500 mm), region 2: mountainous area (temperature: -5–29 °c, annual rainfall: 200–500mm), region 3: persian gulf (temperature: 12.6–35 °c, annual rainfall: 200–300mm), and region 4: the central desert (temperature: -4–44 °c, rainfall: less than 100mm). the pattern of bed bug species distribution for the 4 different areas was determined according to the method of skerman and hillard (35). considering 10% prevalence, 95% confidence level and 5% error rate, 138 bed bugs collected and were used in this study. that way, adult bed bugs were collected from various locations such as infested hotels, residential houses and industrial buildings during may 2016 to august 2017, with the help of pest control companies. the following cities were selected from each region: region 1: sari and rasht, region 2: tehran, isfahan, urmia, tabriz, shiraz, saghez, sanandaj, kermanshah and hamadan, region 3: ahvaz and bandar abbas, and region 4: semnan. these locations were mapped by collecting the locality data via google earth (fig. 1). individual samples were collected by using forceps then stored in a sample collection bottle and preserved in 95% alcohol and stored at -20 °c until analysis onset. the insects were transferred to the laboratory of parasitology division, faculty of veterinary medicine, urmia university and identified under a stereo microscope (olympus sz61, olympus corporation, tokyo, japan), using morphological keys described by usinger (1) and walpole (36). the pronotum which is the most distinguishing feature used to identify the two bedbugs species. the pronotum of c. lectularius is wider than that of c. hemipterus because of an upturned lateral flange on the margin of the pronotum on the thorax of c. lectularius which is absent in c. hemipterus (1). the dorsal and ventral sides of the bedbug pronotum were observed to get a distinct image of the pronotum because the projecting edge of some were not very clear dorsally. dna extraction genomic dna from individual tropical bed bug of each collection site was extracted using dna isolation kit, mbst (molecular and biological system transfer, tehran, iran) following the manufacturer’s instructions. for this, samples were grinded by pestle and placed into 1.5ml micro centrifuge tube and total dna was eluted in 100µl of elution buffer. dna quality and concentration from each specimen was determined spectrophotometrically using the nanodrop (thermo scientific 2000c, united states) and stored in 20 °c for further procedures. we control the contaminations by check the 260/230 ratio because a poor ratio may be the result of a contaminant absorbing at 230nm or less. also we check the wavelength of the trough in the spectra; this should be at 230nm. amplification of the mitochondrial coi and sequencing for pcr amplification of the coi, the 658bp amplicon, the forward and the reverse primers lep-f (5′-att caa cca atc ata aag ata tng g3′) and lep-r (5′taw act tcw ggr tgtccr aar aat ca-3′) were used (32). the pcr was conducted in 25µl total volume, each containing 2.5µl 10x pcr buffer, 2μl 50mm mgcl2, 0.5µl dntps, 3µl dna template, 10 picomole forward and reverse primers (0.5µl for each), 0.5µl taq dna polymerase (sinaclon, iran) and 15.5µl ddh2o. the pcr cycling conditions set in the program were as follows: initial denaturation at 94 °c for 5min followed by 35 cycles of 94 °c for 30sec (denaturation), 42 °c for 30sec (annealing), 72 °c for 45sec (extension) and a final extension step of 72 °c for 2min. pcr products were analyzed by electrophoresis in 1.5% agarose gel to confirm that the samples conhttp://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 242 http://jad.tums.ac.ir published online: september 30, 2020 tained a single band. then, stained with safe dna stain gel and visualized with uv-transilluminator (bts-20m, japan). finally, purified pcr products were sent to sinaclon company (tehran, iran) for sequencing. nucleotide diversity and phylogenetic tree construction the nucleotide sequences of each species from various regions were aligned for variation positions. sequences were uploaded on ncbi to search for the most similar reference sequences, and positions of the coi were determined with the help of blast, available at ncbi. a total of eleven coi sequences belong to c. hemipterus available in the gene bank were used to phylogenetic analysis, including 3, 3, 2, 1, 1 and 1 sequence related to malaysia, bangladesh, thailand, czech republic, usa and iran, respectively. the triatoma dimidiata (hemiptera: reduviidae), accession number jq575031, was used as an out group. the alignment was manually edited to remove any alignment errors using the aligning tool clustal w (37) and exported as mega and fasta format files. all the obtained nucleotide sequences were deposited in the genbank with the assigned accession numbers (table 1). subsequently, phylogenetic relationship was examined and constructed by maximum-likelihood method (ml) using the molecular evolutionary genetics analysis (mega), version 6.0. the reliability of an inferred tree was tested by 1000 bootstrap. the dna sequence polymorphism analyses for determining nucleotide diversity were estimated using bioedit version 7, 0, 1 and blastn software (38). results the average size of coi fragment obtained from the amplified c. hemipterus was found to be 655bp which was at the expected pcr product size (approximate length 658 bp) for the all 15 examined samples, within the range of 576 to 697bp. the accepted coi sequences found in ncbi genbank database showed that the percentage identity ranged from 97 to 100. nucleotide sequences of coi obtained from this study were submitted to ncbi genbank and then accession numbers of mg770888 to mg77089, mg739319 to mg 739326, mg737714 and mg696803 were assigned to them (table 1). after some processing, for example, deleting and aligning sequences using mega 6 molecular software, 362bp of partial coi from 15 sequences of c. hemipterus were obtained successfully. construction of phylogenetic tree is done based on mitochondrial coi sequencing by maximum likelihood (ml) method. the sequences obtained from the present study were compared with sequences of c. hemipterus and c. lectularius from different parts of the world. as the tree shows, the samples were classified in three major clusters which confirm the genetic variation among different species of cimex. all our isolates and those of other parts of the world were placed in one clustered together (g1) showing no significant difference between various regions despite the minor nucleotide variations. consistently, they were far from c. lectularis and t. dimidiata clusters (g2 and g3, respectively). our isolates were further clustered into three subgroups (fig. 2). the first one (sg1) contained 9 isolates which are shown in the phylogenetic tree in fig. 2. sequences from this subgroup clustered together with the c. hemipterus reference sequences containing three nucleotide sequences from bangladesh (mg552132, mg572242, mg 587917), three nucleotide sequences from malaysia (kt851503 to kt851505), two nucleotide sequences from thailand (jx 826469, jx826470), two nucleotide sequences from czech republic (kf018754, gu 985538), one nucleotide sequences from iran (ky560443) and one nucleotide sequences http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 243 http://jad.tums.ac.ir published online: september 30, 2020 from usa (jq782821). the second one (sg2) contained two isolates collected from kermanshah (mg770888) and esfahan (mg 739326). these sequences showed a significant nucleotide similarity with each other and were distinct from the both subgroups sg1 and sg3, and there is no reference sequence in genbank that corresponds to this subgroup. our third subgroup (sg3) contained four isolates collected from hamedan (mg739319), bandar abbas (mg739324), tehran north (mg739321) and semnan (mg739325). analysis of these sequences showed a significant nucleotide similarity between hamedan and bandar abbas and also between tehran north and semnan. this phylogenetic tree is also supported by a mean pair-wise distance that is calculated at 0.005, suggesting that all of the c. hemipterus populations studied are clustered together, showing no significant variance between different regions despite minor nucleotide variations, on average between 5 and 10 single nucleotide polymorphisms (snps). fig. 1. map of iran showing study locations of cimex hemipterus collected for the present study. abbreviations are listed in table 1 table 1. collection site, isolated code, collection date and accession number of each bed bug in this study isolated code isolation source state collection date length /bp longitude and latitude genbank accession number hm hamadan hamedan 20 june 2016 585bp 34.7989° n, 48.5150° e mg739319 az ahvaz khozestan 25 june 2016 577bp 31.3183° n, 48.6706° e mg739320 tn tehran north tehran 15 may 2016 608bp 35.6892° n, 51.3890° e mg739321 ra rasht gilan 28 june 2017 669bp 37.2682° n, 49.5891° e mg739322 sr sari mazandaran 10 june 2017 706bp 36.5659° n, 53.0586° e mg739323 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 244 http://jad.tums.ac.ir published online: september 30, 2020 ba bandar abbas hormozgan 23 july 2017 683bp 27.1832° n, 56.2666° e mg739324 sm semnan semnan 20 july 2016 683bp 35.2256° n, 54.4342° e mg739325 is isfahan isfahan 15 july 2017 734bp 32.6546° n, 51.6680° e mg739326 kr kermanshah kermanshah 14 august 2016 669bp 34.3277° n, 47.0778° e mg770888 sn sanandaj kordistan 10 august 2017 721bp 35.3219° n, 46.9862° e mg770889 shz shiraz fars 23 may 2016 717bp 29.5918° n, 52.5837° e mg770890 ur urmia west azerbaijan 22 may 2016 684bp 37.5498° n, 45.0786° e mg770891 tb tabriz east azerbaijan 19 august 2017 722bp 38.0962° n, 46.2738° e mg770892 ts tehran south tehran 17 may 2017 587bp 35.6892° n, 51.3890° e mg737714 sz saghez kordistan 16 may 2017 705bp 36.2389° n, 46.2780° e mg696803 fig. 2. maximum likelihood (ml) tree inferred from sequences of the mitochondrial coi gene for 15 cimex hemipterus populations collected in iran (*sign) and one outgroup (triatoma dimidiata accession no. jq575031.), numbers at nodes indicate bootstrap values (%) of ml replicates, obtained by 1,000 replications table 1: continued … http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 245 http://jad.tums.ac.ir published online: september 30, 2020 discussion there is a paucity of data and analysis regarding phylogenetic relationships below infraorder cimicomorpha (39, 40). the proposed evolutionary relationships of the taxa within superfamily cimicoidea and the family cimicidae are based on morphological characters, chromosome numbers, and host associations (1, 32). previous studies have associated the cimicidae with other families within the superfamily cimicoidea using both morphological and molecular characters (39, 41). a total evidence analysis using 16s, 18s, 28s and coi dna sequence data and 73 morphological characters (39) has determined 13 infraorder cimicomorpha and superfamily cimicoidea are both monophyletic. the same study reported that the families cimicidae, polyctenidae and curaliidae form a monophyletic clade (39). in this work, we studied the phylogeny of c. hemipterus populations come from different regions of iran, using the mitochondrial coi gene sequences. the coi gene is a part of the mitochondrial dna genes and has been used as a potent marker for molecular phylogenetic studies, because it is species specific and appropriate for analyses of intra specific variations. the rate of evolvement in the coi gene is also relatively rapid which allow distinction at the species level and the identification of obscure species (42, 43). despite the vast outbreak of bedbug in various rural and urban regions of iran, there are very limited and unsatisfactory reports about the prevalence (17, 20). probably due to resurgence and propagation of bed bugs in other countries, the population also has increased in iran (44, 45). limited public awareness, increase in internal and international travels, increase in utilization of second-hand furniture and resistance to pesticides may contribute to this resurgence (46, 47). therefore, the resurgence and subsequent problems inspired us to perform phylogenetic analysis, as well as study genetic diversity and population dynamics. the perceived near extirpation of bed bugs from many areas around the world suggests a genetic bottleneck would have occurred, which would be reflected in low genetic diversity across current bed bug populations (25, 30-34). however, all of the studies completed thus far have found a relatively high genetic diversity between populations in different locations. such high diversity across populations is atypical for species that have undergone a recent and single founder event (30). szalanski et al. (30) focused on genetic variation among various bed bug populations in usa, canada and australia. they examined a partial sequence of the mitochondrial (mt) 16s rrna gene and nuclear rrna its1 region of 136 adult bed bugs sampled from 22 populations and found a relatively high genetic diversity in the 16s gene, and low diversity in the its-1 gene. the current research is the first report of phylogenetic and genetic species diversity analysis conducted on c. hemipterus in iran. our analysis showed one main cluster in phylogenetic tree. therefore, from the present study it can be concluded that c. hemipterus in iran is an arthropod with low genetic diversity with a potentially high capability for raise the levels of inbreeding. the previous study, done by booth et al. (25) regarding molecular markers of bed bug infestation dynamics within apartment buildings supported the same pattern of genetic diversity in c. lectularius as they reveal restricted genetic diversity. similar researches also have conducted in other countries. seri-masran and majid (45) studied genetic diversity of bed bugs in malaysia. they considered 22 selected infested structures and consistent to our findings, they observed one main monophyletic clade. another study was performed in thailand and one main cluster of c. hehttp://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2020, 14(3): 239–249 a samiei et al.: the phylogenetic analysis of … 246 http://jad.tums.ac.ir published online: september 30, 2020 mipterus was obtained when it was compared with c. lectularis isolates. both of the aforementioned studies support our results. given that the current phylogenetic and taxonomic relationships within the family cimicidae are based on host relationships and morphology, it is possible that the inclusion of molecular data could cause a restructuring of the systematic relationships (1, 32). in addition to coi genes, the dna sequences of the entire mitochondrial genome of c. hemipterus may be useful for population genetics studies. there are several reports in the country using molecular methods for species identification of insects (48-58). conclusion these results provided basic information for further studies of molecular epidemiology and control of c. hemipterus infestation to the public, medical association, entomologists and pest control operators in iran. acknowledgements the authors gratefully thank iranian pest control companies from all states in collaboration in the bed bugs sample collection. we also grateful to dr a nazarizadeh and dr m soltani eini for their fruitful assistance in manuscript writing and helpful advice. references 1. usinger rl (1966) monograph of cimicidae (hemiptera, heteroptera). entomological society of 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abai mr, ebrahimzadeh a, moazeni roodib a, gorouhi a (2011) first report on anopheles fluviatilis u in southeastern iran. acta trop. 117(2): 76–81. 58. oshaghi ma, shemshad kh, yaghobiershadi mr, pedram m, vatandoost h, abai mr, akbarzadeh k, mohtarami f (2007) genetic structure of the malaria vector anopheles superpictus in iran using mitochondrial cytochrome oxidase (coi and coii) and morphologic markers: a new species complex? acta trop. 101(3): 241–248. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ javascript:void(0) javascript:void(0) javascript:void(0) javascript:void(0) javascript:void(0) https://www.ncbi.nlm.nih.gov/pubmed/?term=vatandoost%20h%5bauthor%5d&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=oshaghi%20ma%5bauthor%5d&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=azari-hamidian%20s%5bauthor%5d&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=mianroodi%20ra%5bauthor%5d&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=mianroodi%20ra%5bauthor%5d&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=dabiri%20f%5bauthor%5d&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=oshaghi%20ma%5bauthor%5d&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=vatandoost%20h%5bauthor%5d&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=hanafi-bojd%20aa%5bauthor%5d&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=raeisi%20a%5bauthor%5d&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=nikpoor%20f%5bauthor%5d&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3819495/ molecular evaluation of the novel coronavirus infection of cockroaches and flies collected from kamkar-arabnia hospital in qom city, central iran: with innovated internal control http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... original article molecular evaluation of the novel coronavirus infection of cockroaches and flies collected from kamkar-arabnia hospital in qom city, central iran: with innovated internal control reza fotouhi-ardakani1, majid kababian2, *abedin saghafipour2, melika alirezaei1, hassan vatandoost3, 4 1cellular and molecular research center, qom university of medical sciences, qom, iran 2department of public health, faculty of health, qom university of medical sciences, qom, iran 3department of medical entomology and vector control, school of health, tehran university of medical sciences, tehran, iran 4department of environment chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran *corresponding author: dr. abedin saghafipour, e-mail: abed.saghafi@yahoo.com (received 10 aug 2021; accepted 03 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: due to the confirmation of the presence of the novel coronavirus in the feces and municipal sewerage system, and the feeding of domestic insects from fecal matter, as well as the ability of these insects to mechanically transmit microbes from the sewerage system. this study was aimed at molecular evaluation of the novel coronavirus infection isolated on cockroaches and flies collected from kamkar-arabnia hospital in qom city, iran. methods: totally, 18 samples; (12 samples cockroaches and 6 flies) from the external surface of cockroaches and houseflies as well as their digestive system were prepared. after designed and synthetized exogenous heterologous internal control, the rna was extracted to investigate the contamination of these samples with the novel coronavirus. to detect the virus, the e and rdrp genes were identified. results: investigation of coronavirus e gene using the multiplex one-step qpcr technique on the collected samples showed an amplification plot in ct= 35.70 related to the internal surfaces of cockroaches collected from the treatment and sick room of the hospital. also, the design of internal control to ensure the accuracy of the extraction process was successful. conclusion: according to the findings of the present study regarding detecting the presence of the coronavirus infection in the digestive system of domestic insects such as american cockroaches and considering their ability to mechanically transmit viruses, it is recommended to control the domestic insects that are in close contact with humans in crowded places such as hospitals and health centers during the covid-19 pandemic. keywords: cockroaches; flies; hospital; novel coronavirus infection; one-step qpcr; iran introduction the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a causative agent of a zoonotic disease that emerged in the late dec 2019 in china and caused of the coronavirus disease19 (covid-19) pandemic (1). this novel virus is classified as an rna virus belonging to the coronaviridae family (2). this family of viruses is the 359 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... causative agent of a wide range of viral diseases, from the common cold to more severe respiratory infections such as middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus 2 (sars-cov). the coronaviruses have so far attracted a great deal of attention from international viral pathogenesis and pathophysiology (3). in fact, nowadays, the novel coronavirus, which causes covid-19, is more lethal than the other coronaviruses and is a serious warning to all countries in the world (4). as of october 26, 2020, 08:43 gmt, more than 43 million people (43,376,552) worldwide have been infected with the virus, and more than 1.1 million deaths (1,159,534) have occurred, according to the world health organization (who) (5). the disease has adverse effects on public health and has affected other social and economic aspects of the population (6). the covid-19 is a highly contagious disease, and each infected person can infect an average of at least 3 others (7). human-to-human transmission (hht) of the virus through respiratory droplets and contaminated objects and surfaces have been confirmed by observation of infection with the virus in family members as well as health and medical staff (8, 9). according to recent research, the main route of transmission of the virus is through inhalation of infected respiratory droplets with the infected person or contact with the patient’s secretions. in air borne transmission, infected airborne droplets are spread through the sneezing or coughing of the infected person into the environment and surfaces in the mouth or nose of people close to the patient and then transported into the lungs (10). there is also the possibility of a person becoming infected with the novel coronavirus through contaminated objects or surfaces. after contact with the infected surface and then touching the mouth, nose and eyes with infected hands, the virus enters the body through the mucosa (11). in addition, the presence of the novel coronavirus in the feces and municipal sewage system has been proven (12). already, transmission of the virus by the domestic insects living in and around humans is a scientific hypothesis. domestic insects including houseflies and american cockroaches often live in the municipal sewage system and feed on fecal matter. these insects are able to transmit microbes mechanically from the sewage system to human’s dwellings (13). considering that iran is one of the major foci of sars-cov-2 infection (14) and for the first time in iran on 19 february 2020, two patients infected by the novel coronavirus were reported from qom in kamkar-arabnia hospital as a center for infectious diseases and patients with covid-19; this study was aimed to molecular evaluation of the novel coronavirus infection isolated on external and internal surfaces of cockroaches and flies collected from kamkar-arabnia hospital in qom city, iran. materials and methods study area the study was done in kamkar-arabnia hospital (within the latitudes and the longitudes 34.646765°n, 50.886532°e) in qom city, iran. qom city is located in central iran within the latitudes and the longitudes of 34.15°-35.15° n and 50.30° 51.30° e, respectively. at the same time, as in other countries, the covid-19 infection occurred in iran and qom city was one of the areas of the country where the first cases of this disease were reported (14). patients with the coronavirus infection were referred to kamkar-arabnia hospital as a center for infectious diseases and patients with covid-19 (fig. 1). data collection in the cross-sectional study, the sampling process on american cockroaches and 360 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... houseflies was done at treatment and sick rooms, kitchens, toilets, and bathrooms in august 2020. the hand catch was done using sterile entomological forceps and sticky traps for american cockroaches’ specimens. in the sticky trap method, a tile or plastic sheet with approximate dimensions of 20* 20 cm was used. a small amount of absorbent material, such as bread soaked in beer or a small piece of cake, was placed on it as bait, and a circle of non-dry glue known as mouse glue was placed around it. cockroaches were trapped in these traps. in the hand catch method, after observing american cockroaches, they were taken and placed in separate sterile tubes using sterile gloves, and then transferred to the medical entomology laboratory of qom university of medical sciences. in addition, the hand net was used for collecting house flies. in order to detect probable infection to the novel coronavirus in insects, sampling was performed from the external surfaces of them. then, the external surface of each sample was thoroughly washed with 5 ml of sterile distilled water and rinsed with 70% ethyl alcohol for 2 minutes to remove contaminants from the external surface of their bodies. to eliminate the effects of alcohol on the samples, the samples were then placed in sterile physiological saline for 3 minutes. then the internal surface of captured cockroaches and houseflies was labeled in separate tubes. the samples were stored at 4-80c in the lab of a knowledge base company for routine molecular analysis on coronavirus (dspr, qom, iran). data analysis bioinformatics analysis and primer selec tion the optimization and setting up of multiplex one-step qpcr technique for the detection of different gene regions of sarscov2 depends on the selection or design of multiplex primers and probe in order to screening and definitive diagnosis. for this purpose, the complete genomes of this virus were obtained from gene bank. after aligned the sequences by clc genomics workbench 12 (clc, bio-qiagen, aarhus, denmark), the e gene and rdrp gene were considered for amplifying. the evaluated primers were selected for the multiplex one-step-qpcr method [15]. (table1). non-competitive internal control design to achieve the goal of building internal control, an innovative gene structure was designed and synthesized. this gene structure contains 76 bases; 23 bases at the end of 3 placed as a specific reverse primer for the coii gene. the remaining 56 bases were designed as a stem-loop which denaturant at 600c and is amplified with a primer and probe on the hex channel (fig.2). exogenous heterologous internal control could be added as amplification control in the extraction step and investigation of this process. rna extraction coronaviruses rna was extracted using viral dna/rna extraction kit (padtan gostar isar, tehran, iran) according to a modified manufacture protocol. before the extraction process, the samples must reach room temperature. at the first 500 μl lysis buffer added to a 1.5 ml nuclease-free tube. then 4 μl exogenous heterologous internal control along with 200 μl of the prepared sample was added to the tube and done pulse vortexing for 15 seconds. after the addition of 200 μl of precipitation buffer, the lysate was transferred to the binding columns for rna isolation. finally, the columns were washed and genomic rna eluted by 50 ml prewarmed (60ºc) dnase-rnase free water. an approved positive and negative control was extracted simultaneously with other samples. the quantity and quality of rna extracted was determined by nanodrop one 361 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... spectrophotometer (thermo scientific, wilmington, de, usa) and the gene related to internal control, respectively. multiplex one-step taq-man qpcr opti mization multiplex one-step qpcr amplification was carried out in rotor-gene 6000 instrument (corbett research, sydney, australia) to the detection of the novel coronavirus in three channels of fam, rox, and hex for isolation of rdrp gene, e gene, and internal control receptivity, using the following protocol: 4 µl 5x qpcr probe mix (solis bio byne, estonia), 0.5µl reverse transcriptase enzyme (solis bio byne, estonia), 7 µl of 10 pmol each three pair primers and probes, and 8.5 µl of rna in 20 µl final reaction volume. the amplification condition was optimized by incubating the reaction mixtures at 50˚c for 25 min (revers transcription), 95˚c for 10 min (initial activation), followed by 45 cycles at 94˚c for 15 s and 58˚c for 60 s. all the samples were investigated in duplicate, and novel coronavirus positive samples were detected by analyzing the amplification curve on all three channels simultaneously. results a total of 18 specimens were collected in this study, of which 12 specimens were related to the american cockroaches and 6 specimens to the houseflies. the results of the evaluation of the insect samples using the multiplex one-step qpcr technique in terms of infection with the novel coronavirus were analyzed according to table 2. the internal control gene amplification plot, fig. 1. the study area; kamkar-arabnia hospital in qom city, central iran fig. 1. the study area; kamkar-arabnia hospital in qom city, central iran 362 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... fig. 2. amplification plot of rnase p gene on hex channel of rotor-gene 6000 instrument on insect specimens using multiplex one-step qpcr along with positive and negative control fig. 2. amplification plot of rnase p gene on hex channel of rotor-gene 6000 instrument on insect specimens using multiplex one-step qpcr along with positive and negative control fig. 3. amplification plot of e gene on rox channel of rotor-gene 6000 instrument on insect specimens using multiplex one-step qpcr along with positive and negative control fig. 3. amplification plot of e gene on rox channel of rotor-gene 6000 instrument on insect specimens using multiplex one-step qpcr along with positive and negative control fig. 4. amplification plot of rdrp gene on fam channel of rotor-gene 6000 instrument on insect specimens using multiplex one-step qpcr along with positive and negative control fig. 4. amplification plot of rdrp gene on fam channel of rotor-gene 6000 instrument on insect specimens using multiplex one-step qpcr along with positive and negative control 363 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... which was evaluated of the extraction and qpcr process in this study, for all samples had a ct (cycle of threshold) between 22.21-26.20 (fig.3). investigation of coronavirus e gene using the multiplex one-step qpcr technique on the collected samples showed an amplification plot in ct= 35.70 related to (cockroach internal sample 3) ci3. this sample belonged to the internal surfaces (digestive system) of american cockroaches collected from the treatment and sick room of kamkar-arabnia hospital, qom, iran. in the rdrp gene analysis, no amplification plot was observed in any sample except for positive control (fig.4). discussion respiratory diseases are usually transm itted through respiratory droplets (16). in the case of the covid-19 infection in china and other countries, first most patients were infected with respiratory droplets and contact routes (17). recently, in addition, other ways of transmitting the coronavirus have been mentioned, including airborne transmission (16). it should declare that droplet transmission takes place when an individual is in close contact with a person who has respiratory infection symptoms related to covid-19 infection. also, airborne transmission of the novel coronavirus related to the presence of the virus within droplet nuclei, which are commonly determined to be particles <5μm in diameter, may remain in the air circulation for long times and be transmitted to other people over distances more than 1 meter (18). furthermore, there is some document that covid-19 virus can lead to intestinal infection in humans and be present in feces. so, to date some studies have detected the novel coronavirus from sewerage system and feces (12). besides that, it has been proven that domestic insects such as american cockroaches (periplaneta americana) and houseflies (musca domestica) live in the sewage system and they are the mechanical vectors of microbes and parasites such as viruses, table 1. primers and probes for detection novel coronavirus primer name reference e_sarbeco_p1 rox-acactagccatccttactgcgcttcg-bbq e_sarbeco_f1 acaggtacgttaatagttaatagcgt e_sarbeco_r2 atattgcagcagtacgcacaca rdrp_sarsr-p2 fam-caggtggaacctcatcaggagatgc-bbq rdrp_sarsr-f2 gtgaratggtcatgtgtggcgg rdrp_sarsr-r1 caratgttaaasacactattagcata rp-probe hex-cgcagagccttcaggtcagaacccgc-bhq rp-f tggcggtgtttgcagatttgg rf-r tgagcggctgtctccacaag table 1. primers and probes for detection novel coronavirus table 2. analyzing the results of examining all three genes e, rdrp, and rnase p using the multiplex one-step qpcr technique row positive control negative control hex (ic) fam (rdrp) rox (e) result 1 <35 >40 <45 >40 <39 sarbecov positive 2 <35 >40 <45 <39 <39 2019-ncov positive 3 <35 >40 <30 >40 >40 detectable not 4 <35 >40 <45 <39 >40 repeat test *** 5 <35 <35 <35 +/+/contamination repeat 6 >35 >35 >45 +/+/pcr failure repeat table 2. analyzing the results of examining all three genes e, rdrp, and rnase p using the multiplex one-step qpcr technique 364 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 358–365 r fotouhi-ardakani et al.: molecular evaluation... bacteria, and parasites such as amoebae, shigella infection (shigellosis), helminthes eggs to humans (19-21). in the present study, the theory of mechanical transmission of the novel coronavirus by insects from the sewage system was proposed and a specimen of the digestive system of american cockroaches was positive for the coronavirus in the e gene. findings of other study confirm that the clinical samples with e only amplification should not be dismissed as non-specific results. colton, hayley, et al found that 85% of e only samples were confirmed by a second assay targeting the n gene or an alternative rdrp only assay. furthermore, we observed a mean difference of three ct when comparing the e gene to rdrp values, which may suggest the possibility of higher copy numbers of the e gene (22). the unique transcription strategy of the coronaviruses, in which genes towards the 3’ end of the genome would be present in higher copy numbers during active viral replication, could explain these claims (23). given this hypothesis and the fact that the virus load in the ci3 sample was low, the e gene in ct=35.7 showed a curve. the lack of curve in the rdrp gene is justified. in addition, amplification of innovative internal control designed in this study to prevent the false-negative due outcome from inhibitor interference could sure use the proper performance of the nucleic acid extraction method. these domestic insects are usually found indoors in the living environment and workplace and human services, including hospitals in various wards (24). due to the feeding of these insects from the sewage environment, microbial contamination such as coronavirus is carried out by the external organs of the insect body such as mouth appendages, body surface hairs, legs and wings. these microbes can be transmitted to people by re-feeding from the hospital environment and patients and their companions or are hospitalized in the wards (20). furthermore, american cockroaches and houseflies are omnivorous, and in addition to transmitting the coronavirus through the feces and sewage system, they may become infected with the coronavirus by sitting on infected material in hospitals and transmitting it to others. conclusion in the case of the covid-19 pandemic, due to the close relationship of these insects with humans and their large populations, and their high reproducibility and rapid movement from one place to another, they can be potentially dangerous in relation to the transmission of the coronavirus. it seems necessary to control the population of these insects in central covid-19 infection hospitals in order to prevent the transmission of the corona virus by insects. in addition to health and treatment measures for patients with covid-19 infection, it is recommended taking special measures to control the insect population. acknowledgements the study was funded by the qom university of medial science through project number: 1662 and it was approved by the ethics committee of qom university of medical sciences (ir.muq.rec.1400.006). we thank workers in kamkar-arabnia hospital in qom city for helping with the field working. conflict of interest statement authors declare that there is no conflict of interest. references 1. rodriguez-morales aj, 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https://pubmed.ncbi.nlm.nih.gov/?term=corman%2bvm&cauthor_id=31992387 https://pubmed.ncbi.nlm.nih.gov/?term=landt%2bo&cauthor_id=31992387 https://pubmed.ncbi.nlm.nih.gov/?term=kaiser%2bm&cauthor_id=31992387 https://pubmed.ncbi.nlm.nih.gov/?term=molenkamp%2br&cauthor_id=31992387 https://pubmed.ncbi.nlm.nih.gov/?term=meijer%2ba&cauthor_id=31992387 https://pubmed.ncbi.nlm.nih.gov/?term=chu%2bdk&cauthor_id=31992387 https://dx.doi.org/10.1016%2fj.envres.2020.109819 https://dx.doi.org/10.1007%2fs12098-020-03263-6 https://dx.doi.org/10.1007%2fs12098-020-03263-6 https://apps.who.int/iris/bitstream/handle/10665/112656/9789241507134_eng.pdf%3bjsessionid%3d41aa684fb64571ce8d8a453c4f2b2096?sequence=1 https://apps.who.int/iris/bitstream/handle/10665/112656/9789241507134_eng.pdf%3bjsessionid%3d41aa684fb64571ce8d8a453c4f2b2096?sequence=1 https://apps.who.int/iris/bitstream/handle/10665/112656/9789241507134_eng.pdf%3bjsessionid%3d41aa684fb64571ce8d8a453c4f2b2096?sequence=1 https://apps.who.int/iris/bitstream/handle/10665/112656/9789241507134_eng.pdf%3bjsessionid%3d41aa684fb64571ce8d8a453c4f2b2096?sequence=1 https://apps.who.int/iris/bitstream/handle/10665/112656/9789241507134_eng.pdf%3bjsessionid%3d41aa684fb64571ce8d8a453c4f2b2096?sequence=1 https://apps.who.int/iris/bitstream/handle/10665/112656/9789241507134_eng.pdf%3bjsessionid%3d41aa684fb64571ce8d8a453c4f2b2096?sequence=1 https://doi.org/10.1186/s12889-018-5934-3 https://doi.org/10.1186/s12889-018-5934-3 molecular evaluation of the novel coronavirus infection of cockroaches and flies collected from kamk abstract keywords introduction materials and methods study area data collection data analysis bioinformatics analysis and primer selection non-competitive internal control design rna extraction multiplex one-step taq-man qpcr optimization results discussion conclusion acknowledgements conflict of interest statement references j arthropod-borne dis, 2012, 6(2): 112–118 m limoee et al.: toxicity of pyrethroid and … 112 original article toxicity of pyrethroid and organophosphorous insecticides against two field collected strains of the german cockroach blattella germanica (blattaria: blattellidae) from hospitals in hamadan, iran mojtaba limoee 1, *behroz davari 2,4, seyed hassan moosa-kazemi 3 1depatment of public health, nosocomial infections research center, kermanshah university of medical sciences, kermanshah, iran 2department of entomology, school of medicine, hamadan university of medical sciences, hamadan, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4 kurdistan university of medical sciences and environment health research center, kurdistan, iran (received 29 feb 2012; accepted 26 aug 2012) abstract background: the german cockroach, blattella germanica is a major hygienic pest and mechanical vector for pathogenic agents in hospitals and residential areas. the development of insecticide resistance is a serious problem in controlling of this pest. toxicity of four commonly used insecticides (permethrin, cypermethrin, malathion and chlorpyrifos) against two hospitalcollected strains of the german cockroach was investigated. methods: topical bioassay methods were carried out for detecting insecticide susceptibility of adult male cockroaches. for each insecticide, four to six concentrations resulting in >0% and <100% mortality were used. three to six replicates of 10 cockroaches per concentration were conducted. the differences between ld50 (µ g/g) values were considered statistically significant only when the 95% confidence intervals did not overlap. results: two hospitalcollected strains of the german cockroach showed low to moderate levels of resistance to chlorpyrifos, permethrin, malathion and cypermethrin based on resistance ratios compared with susceptible strain. conclusion: the low level chlorpyrifos resistance suggesting this insecticide may still provide adequate control of these strains. while the obsereved moderate levels of resistance to cypermethrin could imply developing resistance to this compound. keywords: insecticide resistance, pyrethroid, organophosphate, german cockroach introduction the german cockroach, blattella germanica (l) is a major hygienic pest in households, hospitals and residential areas. these insects have been recognized as mechanical vectors and reservoir for pathogenic agents (pai et al. 2003, 2005, kinfu and erko 2008). in addition to mechanical transmission of pathogens, large indoor cockroach populations are also one of the causes inducing asthma (roberts 1996, miller and koehler 2003, kinfu and erko 2008). insecticide resistance is now a serious problem, challenging the control of the german insecticide resistance is now a serious problem, challenging the control of the german cockroach. in addition, selection with some insecticides could confer resistance to the new insecticides through cross-resistance (wei et al. 2006). the development of resistance to different classes of insecticides in field-collected strains of the german cockroach, blattella germanica has been reported frequently (cochran 1995, lee et al. 1996, pai et al. 2005). pai et al. (2005) determined the resistance of the german cockroach from hospitals and *corresponding author: dr behroz davari, e-mail: davaribehroz@yahoo.com j arthropod-borne dis, 2012, 6(2): 112–118 m limoee et al.: toxicity of pyrethroid and … 113 households to propoxur, chlorpyriphos and cypermethrin in taiwan. they concluded that the resistance patterns of propoxur> chlorpyriphos> cypermethrin in hospital strains and propoxur> cypermethrin> chlorpyriphos in household strains, might be due to the frequency of application of the insecticides. chai and lee (2010) determined the resistance of 22 fieldcollected strains of the german cockroach from various localities of singapore to 6 classes of insecticides. among the different classes of insecticides, the effectiveness and low mammalian toxicity of pyrethroids have resulted in these compounds being extensively used for cockroach control. nevertheless, due to frequent use of these compounds, control failure in some field populations have been reported resulting from the development of pyrethroid resistance (walles and yu, 1996, dong 1998, valles 1998, valles et al. 2000, wei et al. 2006). the insecticides from different chemical groups including organophosphorous, carbamates and particularly pyrethroids have been used extensively to control the german cockroach in iran and as a result, insecticide resistance appears to have become a prevalent among its populations (ladonni 1993, 1997, ladonni and sadegheyani 1998, limoee et al. 2006, 2011). earlier, we found pyrethroid resistance (including permethrin, cypermethrin and cyfluthrin) and cross resistance to ddt in seven field-collected strains from tehran, iran (limoee et al. 2006). synergistic studies and biochemical assays revealed that the metabolic mechanisms were involved in resistance to permethrin in those strains mentioned above (limoee et al. 2007). nasirian (2010) summarized the susceptibility of different strains of the german cockroach to different classes of insecticides in iran (nasirian 2010). in a recent article, resistance to organophosphorous carbamate and pyrethroid insecticides, were reported in three hospitalcollected strains from kermanshah, iran. permethrin resistance was suppressible by synergist pbo, suggesting the oxidases involvement in resistance (limoee et al. 2011). although attempts were made to determine the insecticide resistance of the german cockroach populations from different provinces of iran, such as tehran and kermanshah (ladonni 1993, 1997, ladonni and sadegheyani 1998, limoee et al. 2006, 2011, nasirian 2006), there is no information on insecticide resistance status in hospital strains of the german cockroach in hamadan, a western province of the country. therefore, this study was undertaken to detect possible insecticide resistance in two hospital-collected strains of the german cockroach using four commonly used insecticides from different classes including permethrin, cypermethrin, malathion and chlorpyrifos. materials and methods cockroach strains three german cockroach strains were examined in this study: sus is the standard susceptible strain maintained since 1975 in the insectary at the school of public health, tehran university of medical sciences without exposure to insecticides; two strains ech and fh were collected in 2010 from two hospitals in hamadan. spraying with different insecticides including pyrethroids and organophosphates were not very effective in controlling of the german cockroach in those hospitals (personal communication). all cockroaches were maintained in an insectary at 27±2 ºc, 60±10% rh, with a photoperiod of 12:12 h (l: d). each strain was reared in the same size labeled glass jar. cockroaches were provided with unlimited cat food and water. tests were conducted on adult males of f2–f4 generations. chemicals chemicals used were permethrin, 93.7% (technical grade) cis:trans 60:40, cypermethrin, 97.5% (technical grade), (zeneca, haslemere, j arthropod-borne dis, 2012, 6(2): 112–118 m limoee et al.: toxicity of pyrethroid and … 114 uk), chlorpyripfos, 97% (technical grade) and malathion, 92% (technical grade), (cyanamid agro, india). co2 was used as anesthetic and acetone as solvent. bioassays methods bioassay tests were performed by topical application of 1µl of a known concentration of insecticide solution to the first abdominal segment of the insects, using a hand microapplicator (burkard, scientific ltd, uk) equipped with a 1.0 ml hamilton glass syringe. adult male cockroaches were anesthetized with carbon dioxide for 20–30 seconds before insecticide treatment (valles 1994). for each insecticide, four to six concentrations resulting in >0% and <100% mortality were used. three to six replications of 10 cockroaches per concentration were conducted. control groups received acetone alone. treated cockroaches were kept in pyrex glass jars provided with food and water before scoring the mortality. cockroaches were considered dead when they were unable to turn themselves to normal posture within one minute after being turned onto their dorsum. data analysis bioassay data were pooled and subjected to probit analysis (finny 1972), using a personal computer. the differences between ld50 values were considered statistically significant only when the 95% confidence intervals did not overlap. all ld50 values were converted from µg/cockroach to µg/g of cockroach body weight to avoid possible effect of weight differences on insecticide susceptibility. the resistance ratios (rrs) were calculated by dividing the ld50 of the resistant strain by the ld50 of the susceptible strain. results toxicity of permethrin both hospital-collected strains of the german cockroach showed the similar levels of resistance to permethrin based on resistance ratios (rrs) compared with sus strain (rr values for fh and ich strains were 3.36 and 3.15, respectively), (table 1). comparisons made between the 95% confidence intervals of the ld50 values of field strains with susceptible strain (sus) indicated that both strains had significantly different rrs (p< 0.05) (table 1). toxicity of cyprmethrin different levels of resistance to cypermethrin were observed in two hospital-collected strains of german cockroach based on rrs compared with susceptible strain (sus) (table 1) showing resistance ratios, 3.23 and 6.18 at ld50 levels, for ich and fh strains, respectively. comparisons made between the 95% confidence intervals of the ld50 values of field strains with susceptible strain (sus) indicated that both strains had significantly different rrs (p< 0.05) (table 1). toxicity of malathion two hospital-collected strains of german cockroach showed the similar levels of resistance to malathion with significant rrs based on overlap of 95% confidence intervals of the ld50 values, compared with susceptible strain (sus) (p< 0.05). resistance ratio values for fh and ich strains were 5.2 and 6.23, respectively (table 2). toxicity of chlorpyriphos two hospital-collected strains of german cockroach had similar resistance levels to chlorpyriphos with significant rrs based on overlap of 95% confidence intervals of the ld50 values, compared with susceptible strain (sus) (p< 0.05). resistance ratio values for fh and ich strains were 2.2 and 2.4, respectively (table 2). j arthropod-borne dis, 2012, 6(2): 112–118 m limoee et al.: toxicity of pyrethroid and … 115 table 1. lethal dose values for permethrin and cypermethrin for insecticide susceptible and two hospital collected strains of german cockroach strain n yintercept s lope(se) x2 (df) ld50 (95% cl) µg/ga rrb susc permethrin 201 0.99 3.89 (0.49) 3.621 (4) 10.70 (9.44–12.1) 1 cypermethrin 169 3.11 3.3 (0.42) 3.57 (2) 3.74 (3.18–4.44) 1 fh permethrin 169 0.24 3.06 (0.37) 1.03 (3) 35.97 (29.56–43.79) 3.36sig cypermethrin 149 0.87 3.03 (0.40) 0.77 (3) 23.13 (18.96–28.46) 6.18sig ich permethrin cypermethrin 149 190 0.81 2.83 2.74 (0.37) 2.01 (0.30) 1.30 (3) 2.63 (3) 33.70 (27.17–42.00) 12.07 (9.06–15.19) 3.15sig 3.23sig a micrograms of insecticide/g of cockroach body weight bresistance ratio csusceptible strain sigsignificant table 2. lethal dose values for malathion and chlorpyriphos for insecticide susceptible and two hospital collected strains of german cockroach strain n yintercept s lope(se) x2 (df) ld50 (95% cl) µg/ga rrb susc malathion 157 1.39 2.71 (0.38) 1.39 (2) 21.47(17.48–26.11) 1 chlorpyriphos 183 1.31 4.91 (0.64) 6.96 (3) 5.66(5.11–6.33) 1 fh malathion 120 2.02 3.43 (0.52) 5.68 (2) 111.66(92.23–134.29) 5.20sig chlorpyriphos 160 0.05 4.61 (0.61) 5.42 (2) 12.46(10.91–14.00) 2.20sig ich malathion 120 2.32 3.44 (0.56) 0.05 (2) 133.75(111.38–163.17) 6.23sig chlorpyriphos 160 0.28 4.68 (0.66) 0.71 (2) 13.47(11.91–15.20) 2.40sig amicrograms of insecticide/g of cockroach body weight bresistance ratio csusceptible strain sigsignificant discussion in contrast to our previous study (limoee et al. 2006, 2011), the present study showed the low to moderate resistance levels of collected strains to pyrethroid, organophosphorous and carbamate insecticides. hence, it could be suggested that the resistance to those group of insecticides are developing. several researchers previously concluded that the lethal dose (ld) method especially by topical application might be appropriate for determining susceptibility levels of the german cockroach strains to different insecticides because the amount applied could be precisely measured (milio 1987, scott and cochran 1990, choo et al. 2000, ladonni 2001). thus, this method was used throughout this study. according to reierson et al. (1998) 10 fold resistance measured by topical application is the critical point above which operational control failures are likely to occur while, resistance ratio at 5x and below may still achieve a good control of the german cockroach population. although we observed the high level of resistance to permethrin and cypermethrin in different field collected strains of the german cockroach in our previous studies (limoee et al. 2006, 2011), being consistent with another study on pyrethroid resistance in some populations of this insect (ladonni and j arthropod-borne dis, 2012, 6(2): 112–118 m limoee et al.: toxicity of pyrethroid and … 116 sadegheyani 1998), in present study, the low to moderate resistance levels of collected strains from hospitals of hamadan to pyrethroid insecticides compared with the susceptible strain were detected. our findings probably suggested that these strains had not been selected especially by permethrin for several generations. nevertheless, cypermethrin resistance seemed to be higher (rr: 6.18) than resistance to permethrin. continued use of pyrethroids may lead to a relatively high selection pressure. as reported by zhai and robinson (1991), the use of cypermethrin against the german cockroach about 4 years resulted in high level of resistance and control failure (zhai and robinson 1991), while, the pyrethroid insecticides have been recently replaced with some carbamates or organophosphates such as bendiocarb and chlorpyriphos in controlling of german cockroach populations of hospitals in hamadan, iran (personal communication). we can conclude that according to reirson et al. (1998), the resistance ratio of these two strains to chlorpyriphos compared with the susceptible strain probably reflect a relatively low levels of resistance to this compound because these strains have not been selected by chlorpyrifos for several generations. hence chlorpyrifos may still provide adequate control of these strains. these results are consistent with our previous studies on chlorpyrifos resistance in some populations of the german cockroach from kermanshah, iran which resistance ratios ranged from 1.20 to 2.18 for chlorpyrifos (limoee et al. 2011). lee et al. (1996), reported low levels of resistance to propoxure due to low frequency of insecticide applications (bi yearly). similarly, the two strains in this study were subjected to a low frequency of malathion application and then replaced by chlorpyrifos (personal communication). these strains exhibited moderate levels of resistance to malathion (>5x) probably because of low frequency of application and short term usage of malathion. indeed, based on our findings in this study, it can be suggested that chlor-pyrifos could still be a potent toxic organo-phosphate in controlling of german cock-roach populations in the hospitals of hamadan. on the other hand, a mixture of carbamate and cypermethrin has recently been used against hospital strains of german cockroach in kermanshah (limoee et al. 2011). the majority of toxicological and biochemical studies suggest that insecticide resistance in the german cockroach can be mediated by a number of mechanisms, the most common being enhanced enzymatic metabolism (walles and yu 1996, valles et al. 2000, wei et al. 2001). the synergistic studies supported by biochemical assays implicated that p450 monooxygenases hydrolases were involved in permethrin resistance in some strains of this insect in iran (limoee et al. 2007). finally, we concluded that the low level chlorpyrifos resistance suggesting this insecticide may still provide adequate control of these strains. while the observed moderate levels of resistance to cypermethrin could imply developing resistance to this compound. thus, we propose further studies on changes insecticide resistance levels by using the different synergists following insecticide selection in these strains for providing in vivo evidences about the possible mechanisms of insecticide resistance. acknowledgements the authors would like to express their sincere gratitude to authorities of the deputy for research and school of public health of kermanshah university of medical sciences for providing the research grant and administrative assistance. we also thank dr sedegeh tavassoli and the entire staff of the fatemieh and ecbatan hospitals in hamadan who helped us for collecting of cockroaches. the authors declare that there is no conflict of interest. j arthropod-borne dis, 2012, 6(2): 112–118 m limoee et al.: toxicity of pyrethroid and … 117 references chai ry, lee cy (2010) insecticide resistance profiles and synergism in field population of the german cockroach (dictyoptera: blattellidae) from singapore. j econ entomol. 103: 460–471. choo lewc, tang cs, pang fy, ho sh (2000) comparison of two bioassays methods for determining deltamethrin resistance in german cockroach (dictyoptera: blattellidae). j econ entomol. 93(3): 905–910. cochran dg (1995) misuse of the tarsal contact method for detecting insecticide resistance in the german cockroach (dictyoptera: blattellidae). j econ entomol. 90: 1441–1444. dong k, valles sm, scharf me, ziechner b, bennett gw (1998) the knockdown resistance (kdr) mutation in pyrethroidresistant german cockroach. pest biochem physiol. 60: 195–204. finny dj (1972) probit analysis. third ed, cambridge university, london. kinfu a, erko b (2008) cockroaches as carriers of human intestinal parasites in two localities in ethiopica. trans r soc trop med hyg. 102: 1143–1147. ladonni h (2001) evaluation of three methods for detecting permethrin resistance in adult and nymphal blattella germanica (dictyoptera:blattellidae). j econ entomol. 94(3): 694–697. ladonni h (1993) susceptibility of blattella germanica to different insecticides in different hospitals in tehran, iran. j entomol soc iran. 12 and 13: 23–28. ladonni h (1997) susceptibility of different field strains of blattella germanica to four pyrethroids (orthoptera: blattellidae). iranian j publ health. 26: 35–40. ladonni h, sadegheyani s (1998) permethrin toxicity and synergistic effect of piperonyl butoxide in the first nymphal stage of blattella germanica (dictyoptera: blattellidae). iranian j publ health. 27: 44–50. lee cy, yap hh, chong nl, lee rst (1996) insecticide resistance and synergism in field collected german cockroach (dictyoptera: blattelllidae) in peninsular malaysia. bull entomol res. 86: 675–682. limoee m, enayati aa, khassi k, salimi m, ladonni h (2011) insecticide resistance and synergism of three fieldcollected strains of the german cockroach blattella germanica (l.) 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report of biochemical mechanisms of insecticide resistance in the field population of culex pipiens (diptera: culicidae) from sari, mazandaran, north of iran seyed hassan nikookar1; mahmoud fazeli-dinan2; seyyed payman ziapour3; fatemeh ghorbani4; yaser salim-abadi5; hassan vatandoost4,6; ahmad ali hanafi-bojd4; *ahmadali enayati1 1department of medical entomology and vector control, school of public health and health sciences research center, addiction institute, mazandaran university of medical sciences, sari, iran 2department of medical entomology and vector control, school of public health and health sciences research center, mazandaran university of medical science, sari, iran 3department of parasitology, zoonosis research center, pasteur institute of iran, amol, iran 4department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 5department of health services and health promotion, school of health, rafsanjan university of medical sciences, rafsanjan, iran 6department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran (received 03 june 2019; accepted 29 dec 2019) abstract background: culex pipiens play an important role in transmission of infectious diseases. vector control by chemical pesticides, leads inevitably to resistance development. understanding the underlying resistance mechanisms can help improve the control programmes and insecticide resistance management. methods: the total contents of cytochrome p450s and the activities of glutathione s-transferases, alphaand betaesterases and inhibition rates of acetylcholine esterase (by propoxur) were measured in the field population of cx. pipiens collected from sari county, north of iran, in 2016 and the results were compared with those of the laboratory susceptible strain according to the biochemical assay methods of who for adult mosquitoes. independent sample t-test was used to compare the mean values of enzyme activities/contents between filed and laboratory susceptible populations. results: the enzyme ratio of cytochrome p450s, alphaand beta-esterases in the field population was 2.07, 3.72 and 1.36 respectively when compared with the results of the laboratory population. although not statistically significant, the mean gsts activities in the field population was marginally less than the laboratory population (er=0.92). acetylcholinesterase was insensitive to propoxur in 62.82% of the individuals of the tested field population. there was a significant difference (p< 0.05) between all values of the activities/contents of the enzyme in the field population except for gsts compared with the laboratory susceptible strain. the highest enzyme activity was related to alpha esterase. conclusion: the present study showed a range of metabolic mechanisms, comprising p450s and esterases combined with target site insensitivity of ache, contributing to organophosphate, carbamate and pyrethroid resistance in the field population of cx. pipiens. keywords: culex pipiens; insecticide resistance; enzyme; iran introduction mosquitoes are the most important arthropods of medical importance transmitting various diseases to humans and animals. among mosquitoes, the genus culex, especially, cx. pipiens complex are known vectors of several human pathogens including west nile virus, *corresponding author: dr ahmad ali enayati, e-mail: ahmadali_enayati@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 379 http://jad.tums.ac.ir published online: december 31, 2019 st. louis encephalitis virus, rift valley fever virus, japanese encephalitis virus, wuchereria bancrofti (1-3) as well as zoonotic pathogens including dog heartworm (4) and avian malaria (5). mosquito-borne diseases immensely affect public health and cause problems in terms of economy and development all over the world (6). culex pipiens is widely distributed in almost all continents throughout the world (7, 8). lately, cx. pipiens has been arrived on newfoundland’s avalon peninsula, canada and southern sweden (9, 10). it shows a vast geographical distribution in most parts of iran (1113). the distribution, dominance and high abundance of cx. pipiens have been reported from northern iran (14-18). the use of chemical insecticide is one of the most widely practiced strategies for mosquito-borne diseases control (19). given the important role of pesticides in mosquito control programms, the development of resistance to insecticides is of great concern for diseases control (20, 21), recently led to many outbreaks of mosquito-borne diseases (22). resistance to insecticides in cx. pipiens has been reported from many parts of the world (23, 24). in iran, approximately 14,000 tons of active ingredient agricultural pesticides were imported or produced (25). population and environmental studies have alarmingly revealed high levels of pesticide residues in the environment (25), it could possibly be a cause of concern for development of resistance to pesticide in mosquito populations that have a water-related life cycle. culex pipiens was resistant to ddt, propoxur, lambda-cyhalothrin, cyfluthrin and deltamethrin (26–29), tolerant to deltamethrin (27, 28) and susceptible to malathion (28). in a recent study, however, revealed relatively high resistance to propoxur, malathion, fenitrothion and dieldrin in the field populations of cx. pipiens from sari county, north of iran (30). the two basic mechanisms of insecticide resistance in mosquitoes are metabolic resistance and target site insensitivity. metabolic resistance is mainly caused by elevated levels of enzyme activities that lead to detoxification or sequestration of insecticides before they reach to their target site (31). glutathione s-transferases (gsts), cytochrome p450s and esterase’s (ests) are three main enzyme groups responsible for metabolic resistance to the main classes of insecticide applied against insects of public health importance (32, 33). elevated level of activeties of these enzymes was shown to be responsible for resistance to pesticides in a variety of mosquitoes including anopheles stephensi (34, 35), aedes aegypti (36, 37) and an. culicifacies (38). biochemical studies showed the involvement of cytochrome p450s in the metabolic resistance to four major insecticide classes in an. funestus, an. stephensi and an. darlingi (39–41). increased levels of esterases have been associated with resistance to organophosphates, carbamates and pyrethroids in cx. quinquefasciatus and helicoverpa armigera (42). involvement of glutathione s-transferases has been observed in resistance to organophosphates, organochlorines and pyrethroid insecticides in an. subpictus, cx. pipiens and rhipicephalus bursa, musca domestica, drosophila melanogaster and nilaparvata lugens (43–45). glutathione strasferases are principally responsible for resistance to organochlorine insecticides and their involvement in resistance to other groups of insecticides is secondary (44). there is also evidence for involvement of the insensitive acetylcholinesterase (ache) in resistance to organophosphates and carbamates in cx. pipiens, ae. aegypti and ae. albopictus (46, 47). esterases showed more important role in the detoxification of organophosphate insecticides used against cx. pipiens in egypt (48). ddt, organophosphate and pyrethroid resistance was characterized in cx. pipiens from turkey and cytochrome p450s and esterases were involved in resistance (21). overproduced esterases and insensitive ache were involved in enzymatic resistance of the field populations of cx. pipiens from grand tunis area, northeast tunisia (49). other resistance mechanisms, including targetsite resistance mutations, can be involved in insecticide resistance in cx. pipiens populations http://jad.tums.ac.ir/ https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&uact=8&ved=0ahukewjhuose3sbuahwgcrqkhtzncd0qfgg2mam&url=http%3a%2f%2fwww.mosquito.org%2fmosquito-borne-diseases&usg=afqjcnhpknitxjpowevywxt4ivrjhs8jxa j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 380 http://jad.tums.ac.ir published online: december 31, 2019 from the mediterranean region (50, 51). the kdr mutation (l1014f) has been reported in association with resistance to lambda-cyhalothrin from cx. pipiens in morocco region (51). the l1014s mutation has been observed in cx. pipiens pallens from japan and china. the l1014c and v1016g mutations have only been reported in cx. pipiens form molestus from china and saudi arabia, respectively (23). the kdr mutation along with elevated levels of p450s and gsts, are associated with pyrethroid resistance in the populations of cx. pipiens from china (52). different amplified esterases (encoded by the ester super locus) and two substitutions on the ache1 (encoded by the ace1gene) including the g119s and the f290v substitutions have been reported to confer resistance to a great variety of organophosphate and carbamate insecticides in cx. pipiens (50). the g119s mutation is common in cx. pipiens populations in europe, africa and china but f290v mutation does not have a large dispersal in cx. pipiens populations outside the mediterranean region (53). culex pipiens is a dominant species in iran (11), especially mazandaran province (14, 15, 18). it is an important vector of the west nile virus in the world (1). evidence of the west nile virus circulating in the north of the country (54) and the presence of wetlands for migrating birds (reservoir hosts), is a cause for concern for the spread of the virus in the province. in a recent study in mazandaran province, resistance to several insecticides has been documented in cx. pipiens (30), however, there is no report on the mechanisms of insecticide resistance in this species from iran. therefore, determination of the underlying insecticide resistance mechanisms of cx. pipiens are important for better insecticide resistance management and implementation of effective control programs if need be. materials and methods study areas and sample collection the present study was undertaken in sari township, the capital of mazandaran province, northern iran, in 2016 with geographical coordinates of 36◦33′48.70˝ n and 53◦03′36.35 e. it is situated between the caspian sea in the north and the alborz mountain ranges in the south, miandorud township in the east and qaemshahr township in the west. the study area has a population of approximately 296,417 according to the 2011 census with an average annual temperature of 15 °c and precipitation of 789.2mm. it included two distinct geographical areas of mountainous and plain/littoral. larvae collection was carried out using the dipping sampling method from artificial habitats (drainage channels) in the village of qajar kheil located in a plain area of sari township, in summer 2015. ecologically, the sampling site had features including stagnant water, with depth of 50cm, expanse of 5m, muddy floor, without plant and shadow–sun conditions. water temperature and average ph were 20 °c and 7.12, respectively. the main occupations of the residents of the sampling area are agriculture, horticulture, animal husbandry, fishing and handcrafts diazinon, chlorpyrifos, imidacloprid, cypermethrin and glyphosate were the most commonly used pesticides by farmers in agriculture and animal husbandry in plain areas of sari township especially ghajar kheil. rearing and preparation of samples larvae were reared in standard insectary conditions 28±2 °c and 70±5% relative humidity in a 12: 12 dark: light photoperiod. live unfed 2–3d old adult females were put in the -80 °c freezer for conducting biochemical assays. a susceptible laboratory strain of cx. pipiens provided by the department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran was used for comparison. biochemical assays activity levels of glutathione s-transferases (gsts), alphaand betaesterases, and inhibition rates of acetylcholine esterase (by propoxur), contents of cytochrome p450s and total prohttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 381 http://jad.tums.ac.ir published online: december 31, 2019 tein for adult mosquito specimens were measured according to (45, 55, 56) with minor modifications. buffer solutions including 0.625m potassium phosphate ph 7.2, 0.025m sodium acetate ph 5, 1% triton sodium phosphate ph 7.8, 0.1m sodium phosphate ph 7, 0.02m sodium phosphate ph 7.2, 5% sodium dodecyl sulphate (sds) diluted in 0.1m sodium phosphate ph 7, 0.1m sodium phosphate 6.5 and 50mm sodium phosphate ph 7.4 were prepared fresh and used for a maximum of two weeks. the rest of the solutions and reagents were made fresh during the tests being done. forty-five deep-frozen female adult mosquitoes (45 specimens of the field and 45 susceptible) were individually put in wells of a 96-well microtiter plate (maxwell®, china), initially, 100μl of cold dwater was added to each well and the specimens were homogenized by a steel pestle on ice. then another 250μl of cold dwater was added and the plate was centrifuged at 1100g (3000rpm) in a refrigerated centrifuge (beckman coulter®, inc., california, usa) at 4 °c for 20min. the supernatants were used as enzyme source for biochemical assays. absorbance levels were measured spectrophotometrically with a microplate reader (elx808 ultra microplate reader biotek® inc., california, usa) activated by kc-junior software, at specific wavelengths for each enzyme. the mean absorbance was calculated based on data for the two replicate wells per mosquito. three blank replicates were considered in all enzyme assays that include all reagents and working solutions related to activities of each enzyme except the enzyme source (instead of which distilled water was used). cytochrome p450s assay in a fresh microtiter plate, reaction mixture in each well consisting of 20 microliters of the homogenate in duplicate, 80 microliters of 0.625m potassium phosphate buffer ph 7.2, 200 microliters of 3,3′,5,5′ tetramethylbenzidine (tmbz) solution (0.01g tmbz dissolved in 5ml methanol plus 15ml of 0.25m sodium acetate buffer ph 5.0) and 25 microliters of 3% hydrogen peroxide were mixed. then, plates were kept at room temperature for 2h and absorbance was read at 450nm as an endpoint. the enzyme contents were described as equivalent units of cytochrome (euc) p450s /mg protein amended for the known heme content of cytochrome c and p450s using a standard curve of purified cytochrome c. acetylcholinesterase (ache) assay twenty-five microliters of homogenates were put in each well in duplicates followed by adding 145l of triton phosphate buffer (1% triton x-100 in 0.1m phosphate buffer ph 7.8) to each replicate to solubilize ache. ten l of dtnb solution (0.01m dithiobis-2nitrobenzoic acid in 0.1m phosphate buffer ph 7.0) and 25l of the substrate aschi (0.01m acetylthiocholine iodide) were added to one replicate to initiate the reaction. the latter solution was replaced by 25l of the substrate aschi containing 0.2% of the inhibitor propoxur (0.1m) for the second test replicates. the kinetics of the enzyme reaction was monitored continuously at 405nm for 5 min. the percentages of inhibition of ache activity by propoxur in the test were calculated relative to the uninhibited wells. the assay conditions were predetermined so that individuals without an insensitive ache-based resistance mechanism had >60% inhibition of the ache activity. general esterase assay the alphaand beta-naphthyl acetate substrates were used to measure the general esterases activity. reaction mixtures included 20 microliters of the homogenate in duplicate (for each of the alphaand beta esterases) in adjacent microtiter plate wells (assigned alpha and beta) and 200 microliters of alphaand beta-naphthyl acetate solution (120l of 30mm alphaor beta-naphthyl acetate dissolved in 12ml 0.02m phosphate buffer ph 7.2) respechttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 382 http://jad.tums.ac.ir published online: december 31, 2019 tively. after 30min incubation period at room temperature, 50 microliters of fast blue solution (0.023g fast blue dissolved in 2.25ml distilled water and 5.25ml of 5% sds 0.1m sodium phosphate buffer ph 7) was added to each well. after another incubation period at room temperature for 5min, the absorbance was measured at 570nm as an endpoint. the resulting optical densities (od) were converted to product concentration using standard curves of ods for known concentrations of the products alphaand beta-naphthol, respectively. the enzyme activity was stated as µm of product formed/min/mg protein. gsts assay the reaction mixture contains 10µl of the homogenate in duplicate plus 200µl of working solution (10mm reduced glutathione dissolved in 0.1m phosphate buffer ph 6.5 and 3mm cdnb originally dissolved in methanol). the absorbance was kinetically read at 340nm for 5min. the enzyme activity was reported as mm of conjugate produced/min/mg protein using the extinction coefficient of cdnb corrected for the path length of the solution in the microtiter plate well. protein assay protein content of mosquito homogenates was measured using bradford method by adding 300 microliters of bio-rad reagent (prepared as 1: 4 dilution in ddh2o) to 10 microliters of supernatant in duplicates. the absorbance was measured at 570nm after the mixture was incubated for 5min at room temperature. absorbance was converted into protein concentration using a bovine serum albumin standard curve obtained with the same method and reagents. analysis of biochemical assays data raw data of readings from the plate reader were transformed into enzyme activities/ contents using the microsoft excel version 10 and the equations for each enzyme group. independent-sample t-test using spss version 19 software (ibm, usa) was employed to compare the mean values of enzyme activities or contents between filed and laboratory susceptible populations. enzyme ratios (er) were computed by dividing the mean activities or content of different enzyme groups of the field strain with those of the laboratory susceptible strain. the p<0.05 was considered as statistically significant. the percentage inhibition of ache by propoxur compared with non-inhibited reaction was computed and the threshold of >60% was considered as ache insensitive to propoxur. results our previous study on cx. pipiens revealed that this species is highly resistant to all four major classes of insecticides including organochlorine, organophosphate, carbamate and pyrethroids, but the resistance level was lower to pyrethroids compared to other insecticides. the highest and the least mortality rates were produced by etofenprox and propoxur with a rate of 76.47% and 4.25%, respectively. the mean and standard deviation of cytochrome p450s contents in the field and laboratory susceptible populations were 49×10-6± 299×10-7 and 282×10-7±235×10-7euc cytochrome p450s/mg protein, respectively. the ratio of cytochrome p450s in the field population was 2.07 when compared with that of the susceptible laboratory population (table 1). the differences between the contents of p450 in the field and laboratory susceptible populations were statistically significant (p= 0.04). the mean gst activity in the field population was marginally less than that in the laboratory susceptible population (0.119vs 0.129 mm/min/mg protein) with a ratio of 0.92 (table 1). however, the difference of gsts activity in the field and laboratory susceptible populations was statistically not significant (p> 0.05) at 5% level. the mean activity of alphaand beta-naphthyl acetate was 878×10-6 and 1×10-3μm/min/ http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 383 http://jad.tums.ac.ir published online: december 31, 2019 mg protein in the field population and 236× 10-6 and 7×10-4μm/min/mg protein in the laboratory susceptible population, respectively. the enzyme ratios in the field population were 3.72 and 1.36 for alphaand beta-esterases when compared with those of the laboratory susceptible population (table 1). there was a significant difference between the field and laboratory susceptible populations regarding the activity of alpha(p< 0.001) and beta-esterases (p= 0.001). acetylcholinesterase biochemical assay showed that 62.82 % of the tested field population had insensitive ache to propoxur as the inhibition of ache was less than the threshold of 60% (table 2). scatter diagram shows variations in enzyme levels in each individual in field and laboratory populations (fig. 1). fig. 1. scatter diagram of distribution of enzyme activities/contents levels in each individual in the field and laboratory susceptible populations of cx. pipiens from sari, iran in 2016 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 384 http://jad.tums.ac.ir published online: december 31, 2019 table 1. mean enzyme activity/content and enzyme ratio (er) of mfo, gst, alpha and beta esterases in the field and susceptible laboratory populations of cx. pipiens, summer 2016 enzymes population mean er mfo field population 49 × 10-6 2.07 laboratory susceptible population 282 × 10-7 1 gst field population 0.119 0.92 laboratory susceptible population 0.129 1 alpha field population 878 × 10-6 3.72 laboratory susceptible population 236 × 10-6 1 beta field population 1 × 10-3 1.36 laboratory susceptible population 7 × 10-4 1 table 2. inhibition of ache activity by propoxur in the filed population of cx. pipiens collected in summer 2016 no of tested cx. pipiens inhibition rates of ache no of tested cx. pipiens inhibition rates of ache no of tested cx. pipiens inhibition rates of ache no of tested cx. pipiens inhibition rates of ache 1 74.92 21 65.8 41 36.5* 61 45.8* 2 52.3* 22 25.4* 42 86.7 62 60.4* 3 71.3 23 63.1 43 52.8* 63 53.1* 4 59.4* 24 41* 44 54* 64 54.9* 5 54.8* 25 13.4* 45 60.5* 65 71.2 6 45.7* 26 68.8 46 17.3* 66 57* 7 52.8* 27 36.6* 47 67 67 56.8* 8 38.1* 28 57.7* 48 59.9* 68 56.2* 9 73.1 29 58.5* 49 86.5 69 60.3* 10 76.76 30 38.5* 50 60.5* 70 82.5 11 68.4 31 78.1 51 65.3 71 65.6 12 73.4 32 49.6* 52 24.4* 72 51.9* 13 52.5* 33 37.2* 53 43.7* 73 65.2 14 63.5 34 76 54 50.3* 74 43.1* 15 32.3* 35 46.8* 55 21.7* 75 21.4* 16 76.3 36 64.7 56 59.2* 76 84.2 17 68.7 37 67.4 57 33.4* 77 16.8* 18 48.6* 38 59.5* 58 35.2* 78 64 19 84.3 39 75.9 59 10.8* 20 66.4 40 2.6* 60 45.7* *the inhabitation rate is lower than the threshold of 60% http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 385 http://jad.tums.ac.ir published online: december 31, 2019 discussion culex pipiens is considered to be a competent vector of pathogens in the world (1). this species is one of the most common mosquito species in iran (11) and has a widespread distribution in the north of the country in the caspian sea region (14, 15). in a recent study in the area cx. pipiens showed resistance to organochlorine, organophosphorus, carbamates and pyrethroid insecticides (30). development of insecticide resistance can be caused by direct selection pressure from insecticides used in vector control programmes and or indirectly by the use of agrochemicals (57). in this follow up study, elevated enzyme activities of alphaand betaesterases, cytochrome p450s and insensitive ache in the filed population of cx. pipiens confirmed the occurrence of resistance and also elucidated its underlying resistance mechanisms. the results of this study indicated that different enzyme groups may play a role in insecticide resistance in the field population of cx. pipiens. alphaand beta-esterases and cytochrome p450s levels were significantly higher in the filed population of cx. pipiens compared to the laboratory susceptible population. involvement of esterases and cytochrome p450s in ddt, malathion and pyrethroid resistance was described in populations of cx. pipiens from turkey (21). zayed et al. showed an association between elevated levels of esterases activity in cx. pipiens larvae with organophosphorus resistance (43) which is in accordance with the findings of our research. enhancements of cytochrome p450s in pyrethroid resistance were reported in cx. pipiens quinquefasciatus (58), cx. pipiens pallens (59), an. stephensi (32), ae. aegypti and ae. albopictus (60, 61). general esterases were mostly reported to be involved in resistance to organophosphates and carbamates, elevated levels of these enzymes have also been associated with resistance to organophosphates in cx. pipiens quinquefasciatus (58), anopheles stephensi (35) and cross resistance to pyrethroids (34). in the present study, significant increase in alpha-esterases activity was discovered in the field population compared with other enzyme groups relative to the laboratory susceptible population. this higher level of enzyme activity can possibly suggest the significance of development of organophosphorus resistance in the field populations of cx. pipiens. the development of resistance to insecticide in the study area could possibly be due to the heavy use of pesticides. mazandaran is the leading province in terms of pesticide use based on its toxic load in the iran (62). in recent years farmers have converted fields not previously cultivated into horticulture, and this is probably one reason for the high use of pesticides for pest control in the area pesticides applied in agriculture can pollute streams running off farms and this could be the indirect path of how these insecticides may end up in larval habitats of mosquitoes. consequently high usage of insecticides in horticulture and agriculture practices leads to emergence of resistance to insecticides in mosquito populations in many areas (63). similar observations have also been made in populations of cx. quinquefasciatus from malaysia and india (58, 64) and ae. aegypti and ae. albopictus from thailand (47) associated with elevated alpha-esterases levels. in this study the difference in the activities of glutathione s-transferases in susceptible laboratory and field populations were not statistically significant from each other. this is observed in similar studies in iran (29). gsts are mainly involved in organochlorine insecticides resistance (44). whereas p450s and esterases are contributing to resistance to most groups of insecticides. as the population tested in the current study was highly resistant to many different organophosphorous (op) and carbamates insecticides, this is quite natural, according to the economics of resistance, that the insects overproduce those enzymes with higher role in de http://jad.tums.ac.ir/ j arthropod-borne dis, december 2019, 13(4): 378–390 sh nikookar et al.: first report of … 386 http://jad.tums.ac.ir published online: december 31, 2019 toxifying more insecticide groups. acetylcholinesterase (ache) is a key enzyme in the nervous system, hydrolyzing acetylcholine neurotransmitter and terminating neural impulses, and are the target for both organophosphates and carbamate insecticides, two main classes of pesticides applied for pest and vector control in agriculture and public health (22). in the present study, inhibition rates of ache by propoxur showed the frequency of insensitive ache gene in the filed population of cx. pipiens to be higher than the threshold of <60% which confirmed its involvement in the resistance to organophosphates and carbamates. high insecticide resistance caused by insensitivity or reduced sensitivity in response to ops and carbamate insecticides was observed in an. gambiae, an. albimanus, cx. vishnui, cx. pipiens, and cx. quinquefasciatus (22). high insensitivity of acetylcholinesterase can be mainly due to mutations in the ace-1gene (22). thus, molecular studies are recommended to verify the presence of these mutations in ace-1 gene in the field populations of cx. pipiens in mazandaran province. conclusion the findings of this research provide the first evidence on the involvement of metabolic mechanisms including alphaand beta-esterases, mixed-function oxidases and acetylcholinesterase in insecticide resistance in the field population of cx. pipiens from sari, mazandaran province, iran. there is evidence of detection of wnv in the mosquito populations and records of human infection with the virus in north and north west of iran. the findings of the present study provide valuable information on the underlying resistance mechanisms of this 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http://jad.tums.ac.ir/ insecticide resistance status of malaria vectors in a malarious area, southeast of iran http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... original article insecticide resistance status of malaria vectors in a malarious area, southeast of iran jalil nejati 1,2; seyed hassan moosa-kazemi2; mohammad ali oshaghi2; abdollah badzohre2; masoumeh pirmohammadi2; zahra saeidi2; nazanin naserikarimi2; seyedeh zahra parkhideh2; *hassan vatandoost2,3 1 health promotion research center, zahedan university of medical sciences, zahedan, iran 2 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3 department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir (received 23 jun 2020; accepted 30 mar 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: malaria continues to be the main vector-borne disease in iran. the endemic foci of malaria are in sistan and baluchistan province, the borderline of iran and pakistan. by the year 2020 the program of the country is malaria elimination. the main vector control is using insecticide as indoor residual spraying. the aim of the study was to evaluate the susceptibility of main malaria vectors to different insecticides recommended by who. methods: all the insecticides papers supported by who and evaluation of insecticide resistance of anopheles stephensi, anopheles culicifacies, anopheles superpictus to different chemical groups of imagicides including ddt 4%, malathion 5%, propoxur 01.%, lambdacyhalothrin 0.05%, deltamethrin 0.025% and permethrin 0.75% were followed by the who guideline. results: results of the susceptibility test against different insecticides revealed that an. stephensi and an. culicifacies are resistant to ddt and susceptible to other insecticides. an. superpictus is susceptible to all groups of pesticides. conclusion: knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes in local and global scales. keywords: anopheles stephensi; anopheles culicifacies; anopheles superpictus; resistant; pesticides introduction malaria is the main vector borne diseases worldwide. according to the recent record of the world health organization, a total of 228 million cases have been reported in 2018 mainly in the african region (1). according to the report of the ministry of health of iran, less than 89 locallytransmitted cases have been report ed in 2017. the aim of country is to eliminate the disease by 2025 (2). the campaign against malaria vectors started with organochlorines (ddt, dieldrin and bhc) during the 1960’s, followed by organophosphates (malathion and pirimiphos-methyl) for 2 decades from 1966 and continued with the carbamate, propoxur during 1977–1990, and then with pyrethroids including lambdacyhalothrin and deltame thrin. temephos, reldan and pirimiphos-methyl was used for larviciding. the last checklist of iranian mosquitoes 279 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... shows 31 anopheles species including sibling, biological forms and genotypes, 17 out of them are re ported as malaria vector transmission. these vectors are considered as sibling, genotype and type forms. anopheles stephensi, an. cu licifacies, an. fluviatilis, an. dthali are the main vector species of south-eastern foci, while an. sacharovi and an. maculipennis are included in malaria transmission in northwest focus. anopheles superpictus has wide distri bution in all malaria foci of the country (fig. 1). anopheles stephensi is reported from the indi an subcontinent it is also distributed across the middle east and south asia region, exist ing in countries such as: afghanistan, bah rain, bangladesh, china, egypt, india, iran, iraq, oman, pakistan, saudi arabia, and thai land (3, 4). it is also reported from djibouti and ethiopia (5, 6). anopheles culicifacies reported from afghanistan, bahrain, cambo dia, china, eritrea, ethiopia, india, iran, iraq, laos, myanmar (burma), nepal, oman, paki stan, sri lanka, thailand, vietnam, yemen. it has five sibling species as a, b, c, d, and e. anopheles superpictus is a main malaria vec tor in palearctic region, middle eastern coun tries, northern africa, india, afghanistan, pa kistan, central and southern europe, and russia (7). insecticide resistance is the selection of a heritable trait in an insect population that re sults in an insect-control product no longer performing as intended. establishing the base line of insecticide resistance and conducting a comprehensive situation analysis is the starting point for tracking resistance. this will re quire collecting available background data and, if necessary, conducting additional tests on vector susceptibility and on resistance mechanisms. interpretation of the data must take into account the resistance situation in neighbouring countries as well as previous experience elsewhere with the same type of resistance mechanisms. countries should design a monitoring plan that includes data on vector distribution and relevant vector attributes for transmission and control. investigation on susceptibility/resistance to currently used insecticides, and on the quality of vector control interventions. experience suggests that if nothing is done, resistance will stabilize in the vector population and reversal will be difficult or even impossible, so that, some of the most effective insecticides will no longer be usable. materials and methods study area the study was carried out in sarbaz city, sistan and baluchistan province, borderline of iran and pakistan (fig. 2). mosquito collection mosquitoes were collected in the breeding places by a dipper equipment. the larvea were ternsferred to the insectary to become adults. mosquito identification all the adutls females were identified using valuable key identification (8). insecticide papers resource all the insecticides papers were supported by who. insecticide susceptibility tests aduls suceptibiity tests were carried out accordig to the who guideline. susceptible when mortality is 98% or higher, possible re sistant when mortality is between 97 and 90%, and resistant when the mortality is lower than 90%. an excel sheet was created for in secticide resistance based on the applied in secticide at diagnostic dosage recommended by who. statistical analysis the mortality quantities of 50% and 90% of imagicides (lt50 and lt90) and the level of confidence of 95%, the equation of the regres sion line were estimated using a regression probit analysis as described by finney (1971) (9). when the mortality of the control group is less than 5%, then the data of biometric tests have not been corrected, but if the mortality of the control group is between 5% and 20%, they have to https://en.wikipedia.org/wiki/afghanistan https://en.wikipedia.org/wiki/bahrain https://en.wikipedia.org/wiki/bangladesh https://en.wikipedia.org/wiki/china https://en.wikipedia.org/wiki/egypt https://en.wikipedia.org/wiki/india https://en.wikipedia.org/wiki/iran https://en.wikipedia.org/wiki/iraq https://en.wikipedia.org/wiki/oman https://en.wikipedia.org/wiki/pakistan https://en.wikipedia.org/wiki/saudi_arabia https://en.wikipedia.org/wiki/thailand https://en.wikipedia.org/wiki/djibouti 280 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... be corrected. the percentage mortality was calculated using abbot’s for mula (1925) (10). results results of exposure of an. stephensi to different logarimic time of pesticides is shown in table 1. probit regression line is shown in fig. 3. the results of the susceptibility test at diagnosctic dose are shown in fig. 4. the order of lt50 value is ddt> propoxur>malathion >bendiocarb>deltamethrin. mortality at dignostic dose considering mortality less than 90% revealed that an. stephensi is resistant to ddt and suscpetible to other insecticides. results of exposure of an. table 1. parameters of probit regression lines of anopheles stephensi exposed to different insecticides y=a+bx p χ2 (df) lt90 lt50 b±se a insecticide y=-10.2229+2.9472x >0.05 5.747 (2) 8008.1993 2942.2500 2.94 ±0.309 -10.22 ddt y=-9.5860+3.1479x >0.05 1.174 (2) 2833.6861 1109.7414 3.1 ±0.287 -9.58 malathion y=-11.3137+3.6761x <0.05 2.307 (2) 2668.2999 1195.6603 3.67 ±0.324 -11.31 propoxur y=-10.8544+3.6683x <0.05 0.634 (2) 2033.9448 909.8464 3.66 ±0.313 -10.85 deltamethrin y=-13.8762+4.6409x <0.05 0.401 (2) 1845.5122 977.1575 4.6 ±0.410 -13.87 bendiocarb a= (interceptor), b±se (slope± standard error), lt50 (lethal time cause 50% mortality according to seconds), lt90 (lethal time cause 90% mortality according to seconds) table 1. parameters of probit regression lines of anopheles stephensi exposed to different insecticides table 2. parameters of probit regression lines of anopheles culicifacies exposed to different insecticides y=a+bx p χ2 (df) lt90 lt50 b±se a insecticide y=-13.4202+4.2917x >0.05 0.902 (2) 2664.5821 1339.7000 4.3 ±0.362 -13.42 ddt y=-11.0527+3.6346x >0.05 0.002 (2) 2475.2129 1099.0019 3.6 ±0.325 -11.05 malathion y=-12.1599+3.8937x >0.05 1.419 (2) 2832.0720 1327.2653 3.9 ±0.336 -12.15 propoxur y=-11.8132+3.9346x >0.05 1.912 (2) 2128.8572 1005.5845 3.9 ±0.333 -11.81 deltamethrin y=-12.1914+4.0442x >0.05 1.635 (2) 2145.1592 1034.0933 4.0 ±0.358 -12.19 bendiocarb table 2. parameters of probit regression lines of anopheles culicifacies exposed to different insecticides fig. 1. spatial distribution of malaria vectors in iran fig. 1. spatial distribution of malaria vectors in iran 281 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... culicifacies to different logarimic time of pesticides are shown in table 2. probit regression line is shown in fig. 5. the results of susceptibility test at diagnosctic dose are shown in fig. 6. the order of lt50 value is ddt> propoxur>malathion >bendiocarb>deltamethrin. mortality at dignostic dose considering mortality less than 90% revealed that an. culicifacies is suscpetible to all insecticides. results of table 3. parameters of probit regression lines of anopheles superpictus exposed to different insecticides y=a+bx p χ2 (df) lt90 lt50 b±se a insecticide y=-11.8709+3.3259 x <0.05 7.837 (2) 9007.5451 3709.0662 3.3±0.874 -11.87 ddt y=-10.5072+3.4735x >0.05 3.980 (2) 2476.6908 1059.0387 3.4±0.444 -10.50 malathion y=-13.3080+4.2832x >0.05 3.360 (2) 2548.2910 1279.4752 4.2±0.489 -13.30 propoxur y=-11.8821+4.0435x >0.05 2.307 (2) 1801.2125 868.1769 4.0±0.371 -11.88 deltamethrin y=-11.5871+3.9542x >0.05 4.225 (2) 1796.5675 851.7902 3.9±0.380 -11.58 lambdacyhalothrin y=-12.4960+3.9622x <0.05 8.819 (2) 3000.9704 1424.9688 3.9±0.726 -12.49 permethrin table 3. parameters of probit regression lines of anopheles superpictus exposed to different insecticides fig. 2. map of study area, sistan and baluchistan province, iran fig. 2. map of study area, sistan and baluchistan province, iran fig. 3. probit regression line of anopheles stephensi exposed to different insecticides fig. 3. probit regression line of anopheles stephensi exposed to different insecticides 282 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... exposure of an. superpictus to different logarimic time of pesticides are shown in table 3. the results of susceptibility test at diagnosctic dose are shown in fig. 7. the order of lt50 value is ddt> propoxur >malathion> permetrhin> deltamethrin> lamabdacyhalothrin. mortality at dignostic dose considering mortality less than 90% revealed that an. superpictus is resistsnt to ddt and suscpetible to all insecticides. fig. 4. resistance of anopheles stephensi to different insecticides at diagnostic doses fig. 4. resistance of anopheles stephensi to different insecticides at diagnostic doses fig. 5. probit regression line of anopheles culicifacies exposed to different insecticides fig. 5. probit regression line of anopheles culicifacies exposed to different insecticides 283 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... discussion results of susceptibility tests against different who recommended insecticides, in cluding ddt, malathion, propoxur, bendio carb, del tamethrin, lambdacyhalothrin and per methrin against an. stephensi, an. culicifa cies and an. superpictus revealed that only an. superpictus is susceptible to all insecticides. however, an. stephensi and an. culicifacies showed re sistant to ddt. there are several reports on resistant status of malaria vectors including an. stephensi (1117). anopheles ste phensi showed resistance to lambdacyhalo thrin, del tamethrin, permethrin, and bendio carb in bandar abbas county, southern iran (17). anopheles stephensi samples were re sistant bendiocarb, fig. 6. resistance of anopheles culicifacies to different insecticides at diagnostic doses fig. 6. resistance of anopheles culicifacies to different insecticides at diagnostic doses fig. 7. resistance of anopheles superpictus to different insecticides at diagnostic dose fig. 7. resistance of anopheles superpictus to different insecticides at diagnostic dose 284 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... propoxur, deltamethrin, per me thrin, ddt, malathion and pirimiphos-me thyl in somali region (18). resistant to ddt, mal athion, bendiocarb, permethrin and del tame thrin was reported in an. stephensi and an. culicifacies from afghanistan. resistant to only deltamethrin and bendiocarb was observed in an. superpictus (19). there are also report of resistant to pyrethroids in afghanistan (20). anopheles superpictus populations were con firmed resistant to ddt, malathion and propoxur and susceptible to pyrethroid insec ticides in different parts of turkey (21). anopheles cu licifacies was resistant to orga nochlorine in secticides and tolerant to carba mates insecti cides and susceptible to other insecticides (22). insecticide resistance in an. culicifacies was reported (23-24). in order to suggest pyre thrpids for malaria vector control addition studies is necessary to find the mechanisms of resistant to ddt and cross-resistant to pyre throids. according to the re sults of gorouhi et al. (2018) (25), metabolic mechanisms play a crucial role in the development of ddt and cyfluthrin resistance in an. stephensi. global results showed a wide variety of susceptibility/resistance status of malaria vectors to these chemicals according to the location, historical context of pesticide used, ge netic background of vectors, age and abdominal conditions of adults, use of pesticides for agricultural pest control may play important role in the sus ceptibility status of these species to different insecticides (2632). conclusion the results of this study are providing a guideline for the country to manage their vector control activities against insecticide resistance of malaria vectors and provide novel ap proaches. acknowledgments this research is financially supported by the ministry of health and medical education national institute for medical research devel opment. the au thors declare that they have no competing in terests. references 1. world health organization (2019) the 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https://pubmed.ncbi.nlm.nih.gov/28745553/?from_term=oshaghi%2banopheles&from_pos=9 https://pubmed.ncbi.nlm.nih.gov/28745553/?from_term=oshaghi%2banopheles&from_pos=9 https://pubmed.ncbi.nlm.nih.gov/28745553/?from_term=oshaghi%2banopheles&from_pos=9 https://pubmed.ncbi.nlm.nih.gov/28745553/?from_term=oshaghi%2banopheles&from_pos=9 286 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 278–286 j nejati et al.: insecticide resistance... cides. pathog glob health. 111(6):289-296. 32. soltani a, vatandoost h, oshaghi ma, enayati aa, raeisi a, eshraghian mr, soltandallal mm, hanafi-bojd aa, abai mr, rafi f (2013) baseline susceptibility of different geographical strains of anopheles stephensi (diptera: culicidae) to temephos in malarious areas of iran. j arthropod borne dis. 7(1):56-65. https://pubmed.ncbi.nlm.nih.gov/28745553/?from_term=oshaghi%2banopheles&from_pos=9 https://pubmed.ncbi.nlm.nih.gov/?term=soltani%2ba&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=vatandoost%2bh&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=enayati%2baa&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=enayati%2baa&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=raeisi%2ba&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=eshraghian%2bmr&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=soltan-dallal%2bmm&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=soltan-dallal%2bmm&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=hanafi-bojd%2baa&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=abai%2bmr&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/?term=rafi%2bf&cauthor_id=23785695 https://pubmed.ncbi.nlm.nih.gov/23785695/?from_term=oshaghi%2banopheles&from_page=3&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/23785695/?from_term=oshaghi%2banopheles&from_page=3&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/23785695/?from_term=oshaghi%2banopheles&from_page=3&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/23785695/?from_term=oshaghi%2banopheles&from_page=3&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/23785695/?from_term=oshaghi%2banopheles&from_page=3&from_pos=4 https://pubmed.ncbi.nlm.nih.gov/23785695/?from_term=oshaghi%2banopheles&from_page=3&from_pos=4 insecticide resistance status of malaria vectors in a malarious area, southeast of iran abstract keywords introduction materials and methods study area mosquito identifications insecticide papers resource insecticide susceptibility tests statistical analysis discussion conclusion acknowledgments references j arthropod-borne dis, december 2014, 8(2): 228–231 a zamani and j rafinejad: first record of … 228 case report first record of the mediterranean recluse spider loxosceles rufescens (araneae: sicariidae) from iran *alireza zamani 1, javad rafinejad 2 1department of animal biology, school of biology, college of science, university of tehran, tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 3 feb 2013; accepted 2 nov 2013) abstract loxosceles rufescens, commonly known as the mediterranean recluse or mediterranean fiddle-back spider is recorded from iran for the first time. the genus loxosceles contains 103 accepted species, two of them (including l. rufescens) are cosmopolitan. all loxosceles species tested so far possess necrotic venoms, which is a unique characteristic among the order araneae. considering this characteristic, it is of medical importance to determine a specific geographical distribution of these spiders. collecting methods include visual inspection and direct hand collecting. all of the specimens were collected in tehran. six specimens of both sexes were collected from parks, houses and apartments. the characteristics of the genus, which are the unique arrangement of six eyes in three diads, the violin shaped mark on the cephalothorax, and the short tibia of adult male palpi with narrow base and it’s embolus which is about as long as the width of the globular bulb were observed and recorded. having the cytotoxicity of the venom and its urban distribution in mind, l. rufescens is probably among the more important spider species of iran. since there are no reports of loxoscelism from iran, we assume that the bites are either infrequent or misdiagnosed. therefore further studies are needed to clarify the medical importance degree of this species. keywords: cytotoxicity, arachnidism, loxoscelism, distribution, faunistics introduction among all animal orders, with more than 40,000 described species, araneae (spiders) ranks seventh in the global diversity (mirshamsi kakhki 2005). with more than 244 recorded species (ghavami 2006), iran's araneofauna is very poorly known. in the latest published checklist of spiders of iran (ghavami 2006), it is declared that an unidentified loxosceles species was reported from tehran by goodarzi (1994). the genus loxosceles consists of 103 species, several of which have been the subject of medical and biological research due to their necrotic venom, which may produce a set of symptoms known as loxoscelism (appel et al. 2005). loxoscelism is the only proven cause of arachnogenic necrosis in humans (swanson and vetter 2006). once these spiders were placed in their own family, loxoscelidae (gertsch 1949, gertsch and ennik 1983) and in scytodidae (gertsch 1967), but now, along with sicarius spiders, they are placed in the family sicariidae (simon 1893). out of 103 described species of loxosceles spiders, two are reported to have been transported to numerous parts of the world (harvey 1996). one of them is the chilean recluse spider l. laeta (nicolet, 1849), which is originally from south america, but studies have shown their presence in california and finland as well (gertsch and ennik 1983), and the other one, is the more widely distributed l. rufescens (dufour, 1820). the  *corresponding author: mr alireza zamani, e-mail: a.zamani@ut.ac.ir http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 228–231 a zamani and j rafinejad: first record of … 229 latter originated from mediterranean countries, but now can be found in regions including usa, east asia, australia and many others (bonnet 1957, gertsch and ennik 1983, platnick 1989, platnick 1993) and it is now considered as a cosmopolitan species (platnick 2012). the aim of this study was to declare the presence of l. rufescens in iran for the first time. materials and methods study area this study was based on the specimens that were collected from different parts of tehran province, the capital of iran. tehran is geographically located at 35.7117°n 51.4070°e in the northern parts of the central plateau of iran, below the southern slopes of alborz mountains. spiders since these spiders are ground dwelling and usually live inside apartments, under rocks and fallen tree logs, discovery of all l. rufescens specimens was a result of visual inspections and direct hand collecting between years 2010–2013. indoor specimens were found on the walls or were captured while they were foraging on the ground. outdoor specimens were found under large rocks and in the leaf litter. six specimens were collected (first one: 35°45'n, 51°24'e second one: 35°43'n, 51°25'e third one: 35°43'n, 51°25'e fourth one: 35°21'n, 51°22'e fifth one: 35°34'n, 51°02'e and sixth one: 35°46'n, 51°20'e) (fig. 1), preserved in 75 % ethanol and deposited in senior author’s personal collection. half of the specimens were found in apartments/ houses and the rest of them were found in parks. various stereo-microscopic photos of specimens and their structures were captured using a light stereo-microscope, plus a sony dsc-tx10 camera. publication of gertsch and ennik (1983) was used as an identification key. results laboratory examinations clarified that two adult males and four subadult females were collected. description medium-sized, haplogyne, six-eyed spiders. males have simple palpal organs, females without sclerotized epigyne. total body length 7–7.5 mm in both sexes (fig. 2, 3). as in most spiders, the most definite key for identifying to the species level is the form of the adult male palpi and female genitalia (gertsch and ennik 1983). in l. rufescens, the tibia of male’s palpus is short, thick, and its base is narrow and not very prolonged. the embolus is about as long as the width of the globular bulb (greene et al. 2009) (fig. 4). in female l. rufescens, the spermathecae is white and dimpled, and its parts are near each other at the midline and are identified by a single, large rounded lobe (greene et al. 2009). fig. 1. tehran, iran. the collecting site for l. rufescens http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 228–231 a zamani and j rafinejad: first record of … 230 fig. 2. male's cephalothorax, dorsal view. the characteristics of the genus, arrangement of six eyes in three pairs and also the violin shaped mark on the cephalothorax are visible fig. 3. female's cephalothorax, dorsal view. the characteristics of the genus, arrangement of six eyes in three pairs and also the violin shaped mark on the cephalothorax are visible fig. 4. right male palpus, lateral view. note that the tibia is short, thick, and its base is narrow and the embolus is about as long as the width of the globular bulb discussion loxosceles spiders are infamous for their ability to cause severe skin necrosis, and in some cases, highly damaging systematic effects (greene et al. 2009). there are several reports of loxoscelism caused by l. rufescens in numerous parts of the world; such as in western asia, it was blamed for an outbreak of skin lesions in orchard workers (borkan et al. 1995), in turkey, a case of loxoscelism which after 20 days caused severe necrosis has been reported (yigit et al. 2008) and other reports from greece (stefanidou et al. 2006), jordan (amr 1988) and palestine (wong et al. 1987). considering several other medically important spiders of iran which all possess neurotoxic venoms (including latrodectus tredecimguttatus (rossi, 1790), l. dahli levi, 1959, l. hasseltii thorell, 1870 and etc.), having the cytotoxicity of the venom and its urban distribution in mind, l. rufescens is probably among the more important species of iran and should be added to the list of iranian spiders of medical importance. since there are no confirmed reports of loxoscelism from iran, we assume that the bites are either infrequent or misdiagnosed, which probably the latter would be the case. therefore further studies are needed to clarify the medical importance degree of this species. acknowledgements many thanks to dr rick vetter and biology scientific board members of university of tehran for their assistance and reviewing the article, mr barbod safaei mahroo and mr parham beyhaghi for their help in photographing and mrs zohre mirzaee, mrs zahra taghavi and mrs afsaneh ghaffari for their collecting aids. the authors declare that there is no conflict of interest. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 228–231 a zamani and j rafinejad: first record of … 231 references amr zs (1988) arthropods of medical importance in jordan. jordan med j. 22 (2): 125–137. appel mh, bertoni da silveira r, gremksi w, veiga ss (2005) insights into brown spider and loxoscelism. invertebrate surviv j. 2(2): 152–158. bonnet p (1957) bibliographia araneorum. vol. 2(3). les frères douladoure, toulouse. borkan j, gross e, lubin y, oryan i (1995) an outbreak of venomous spider bites in a citrus grove. am j trop med hyg. 52: 228–230. gertsch wj (1949) american spiders. d van nostrand, new york. gertsch wj (1967) the spider genus loxosceles in south america (araneae, scytodidae). bull am mus nat hist. 175: 264–360. gertsch wj, ennik f (1983) the spider genus loxosceles in north america, central america and the west indies (araneae, loxoscelidae). bull am mus nat hist. 175: 264–360. ghavami s (2006) renew checklist of spiders (aranei) of iran. pak j biol sci. 9(10): 1839–1851. goodarzi hr (1994) an introduction to the identification and classification of iranian spiders. [msc thesis]. plant pest and disease research institute, tehran, iran. greene a, breisch nl, boardman t, pegac jr bb, kunickis e, howes rk, brown pv (2009) the mediterranean recluse spider, loxosceles rufescens (dufour), an abundant but cryptic inhabitant of deep infrastructure in the washington, dc area (arachnida: araneae: sicariidae). am entomol. 55(3): 158–169. harvey m (1996) the first record of the fiddle-back spider loxosceles rufescens (araneae: sicariidae) from western australia. rec aus mus. 18: 223–224. mirshamsi kakhki o (2005) faunistic study of spiders in khorasan province, iran (arachnida: araneae). ijab. 1(1): 55–66. platnick ni (1989) advances in spider taxonomy 1981–1987. manchester university press, manchester. platnick ni (1993) advances in spider taxonomy 1999–1991 with synonymies and transfers 1940–1980. new york entomological society, new york. platnick ni (2012) the world spider catalog, version 13. american museum of natural history, new york, usa. available at: http://research.amnh.org/iz/spiders/catalog. simon e (1983) histoire naturelles des araignees. vol. 2(1). libraire encyclopedique de roret, paris. stefanidou mp, chatzaki m, lasitihiotakis kg, ioannidou dj, tosca ad (2006) necrotic arachnidism from loxosceles rufescens harbored in crete, greece. j eur acad dermatol. 20(4): 486–487. swanson dl, vetter rs (2006) loxoscelism. clin dermatol. 24(3): 213–221. wong rc, hughes se, voorhees jj (1987) spider bites. arch dermatol. 123(1): 98–110. yigit n, bayram a, ulasoglu d, danisman t, corakocal l, sancak z (2008) loxosceles spider bite in turkey (loxosceles rufescens, sicariidae, araneae). j venom anim toxins. 14(1): 178–187. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 133 http://jad.tums.ac.ir published online: march 31, 2021 original article immune responses in cutaneous leishmaniasis: in vitro thelper1/thelper2 cytokine profiles using live versus killed leishmania major akram miramin-mohammadi 1 ; amir javadi 2 ; seyyed ebrahim eskandari 1 ; mahmood nateghi-rostami 3 ; *ali khamesipour 1 1center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran 2department of social medicines, qazvin university of medical sciences, qazvin, iran 3department of parasitology, pasteur institute of iran, tehran, iran *corresponding author: dr ali khamesipour, e-mail: ali.khamesipour@gmail.com (received 29 may 2019; accepted 14 mar 2021) abstract background: recovery from cutaneous leishmaniasis (cl) leads to protection against further lesion development. in contrast, vaccination using killed parasites does not induce enough protection; the reason(s) is not currently known but might be related to different immune response induced against live versus killed parasites. in this study, th1/th2 cytokine profiles of cl patients were evaluated against live versus killed leishmania major. methods: in this study peripheral blood mononuclear cells (pbmc) of the volunteers with active cl lesion (cl), history of cl (hcl) and healthy volunteers were cultured and stimulated with live or killed leishmania major, the supernatants were collected and levels of ifn-γ, il-5 and il-10 were titrated using elisa method. results: the results showed that ifn-γ levels in cl patients (p< 0.001) and hcl volunteers (p< 0.005) are significantly higher when stimulated with live than stimulated with killed l. major. ifn-γ production in pbmc volunteers with cl and hcl stimulated with live or heat-killed l. major was significantly (p< 0.001) higher than in unstimulated ones. the level of il-5 in cl patients (p< 0.005) and hcl volunteers (p< 0.001) are significantly lower when stimulated with live than killed l. major. there was no significant difference between the levels of il-10 in pbmc stimulated with either live or killed l. major. conclusion: it is concluded that using live leishmania induces a stronger th1 type of immune response which justify using leishmanization as a control measure against cl. keywords: cutaneous leishmaniasis; live leishmania; heat-killed leishmania; in vitro immune response introduction leishmaniasis is a neglected tropical disease (1) caused by different leishmania species and transmitted by the bite of various phlebotomus species. the disease is endemic in 102 countries, it is estimated that 10th of the world population is at risk to contract one form of the disease, worldwide at least 12 million are affected and 1.5–2 million is the annual incidence. over 90% of vl occurs in six countries of bangladesh, ethiopia, brazil, india, sudan, and south sudan and 70–75% of cl cases occur in 10 countries including afghanistan, algeria, brazil, iran, peru, ethiopia, north sudan, costa rica, colombia and syria. the burden of the disease (dailys) is reported to be 3.3 million (2, 3). leishmaniasis is a major public health problem in some endemic foci, infection with leishmania parasites depends upon the leishmania species causes the disease and the host genetic background which governs the immune response. the outcome of exposure to leishmania might be asymptomatic or might induce a range of clinical manifestations from a simple self-healing skin lesion to non-healing form of cl, diffuse cl, mucocutaneous leishmaniasis, a fatal systemic disease and post copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 134 http://jad.tums.ac.ir published online: march 31, 2021 kala-azar dermal leishmaniasis (pkdl). current available measures are not practically effective to control leishmaniasis; there is ample evidence to prove that in principle development of a vaccine against leishmaniasis is possible. numerous leishmania antigens have been introduced as potential vaccine candidate and a few of first-generation vaccines have been tested in human clinical trials, but so far no vaccine is available against any form of human leishmaniasis. control measures are not effective and chemotherapy accompanies drawbacks and is not always effective. in experimental murine models of leishmaniasis, control of infection and induction of protection require generation of th1 type of immune response represented by production of a high level of ifn-γ in the absence of th2 response, in most of the mouse strains like c57bl/6 mice, infected with leishmania major induce a selfhealing lesion and upon healing the animals are protected against further lesion development, so protection accompanies development of th1 response, whereas in susceptible balb/c mice infected with l. major induces progressive lesions which eventually generalized and kills all the mice, the progressive disease occurs with development of a th2 type of immune response with high levels of il-4, il-5 and il-10 and no ifn-γ production (4, 5). in human leishmaniasis recovery from cl induces by natural infection or leishmanization (lz) usually accompanies with protection against subsequent lesion development. leishmanization is an inoculation of live virulence leishmania intradermally into susceptible individuals to induce artificial cl lesion, lz showed to be an effective control measure against cl. the surrogate marker(s) of cure and protection in human leishmaniasis is not well known, but in some studies, a correlation between the generation of th1 type of immune response and protection is shown (6-13). in this study, volunteers with active of cl lesion, volunteers with a history of cl and healthy volunteers were recruited. blood samples were collected and stimulated with either live or killed l. major and th1/th2 cytokine profile was checked. materials and methods ethical considerations and study population the study was carried out in leishmaniasis clinic of center for research and training in skin diseases and leprosy, tehran university of medical sciences (crtsdl, tums) during march 2017 to march 2019. the study proposal was approved by the ethical committee of tehran university of medical sciences. the study groups are as follow: (i) 16 parasitologically proven patients with active cl lesion(s), (ii) 25 volunteers with history of cl, (iii) 15 healthy volunteers from non-endemic areas with no history of leishmaniasis. the lesion of every cl patient and volunteers with history of cl were parasitologically confirmed by direct smear, and/or culture, pcr was used to identify the leishmania species. isolation of peripheral blood mononuclear cells peripheral blood mononuclear cells (pbmc) were isolated from a heparinized blood sample which was collected from each volunteer by using ficollhypaque density centrifugation. parasites and antigens leishmania major (mrho/ir/75/er) was isolated from the spleen of an infected balb/c mouse, the parasites were cultured in nnn media and sub-cultured in complete rpmi media supplemented with 10% fcs and penicillin/ streptavidin. the promastigotes were harvested at stationary phase, washed 3 times, and resuspended in pbs and then divided into two parts; one part was used as it was to stimulate pbmcs (5×105 live promastigotes/well), and the second part was killed by boiling for 5 minutes and checked microscopically to assure that the parasites are killed, the killed leishmania was used to stimulate pbmcs (the same number as live promastigote, 5×105/well). j arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 135 http://jad.tums.ac.ir published online: march 31, 2021 blood sampling collection heparinized blood samples (10ml) were collected from every volunteer, blood samples were diluted 1:1 using rpmi. the diluted blood samples were overlaid gradually with ficollhypaque (30–40% of blood volume) in 50ml disposable centrifuge tubes. isolated pbmc was washed 3 times using pbs and resuspended in rpmi containing 10% heatinactivated fetal calf serum, 100u/ml penicillin and 100μ/ml/ ml streptomycin. peripheral blood mononuclear cell culture the cell number was adjusted to a final concentration of 1×106pbmc/ml, the cells were cultured in triplicates in u-bottom 96 well culture plates (2×105 cells in 200μl volume/well) for 72h at 37 °c in 5% co2, the pbmcs were stimulated with either pha(5μg/ml), live parasites (5×105/well) or heat-killed l. major (5× 105/well), or culture media alone with no stimulation as a negative control. after 72 hours of incubation, 150μl of the supernatants were carefully collected from each well and the triplicates were pooled and kept at -80 °c until used. cytokine assay the level of il-5, il-10, and ifn-γ were titrated in culture supernatants using elisa method (biosciences, usa) according to the manufacture’s recommendation. briefly, the plates were coated with anti-ifn-γ/il-5/il-10 mab in pbs, ph 7.4, and incubated at 4 °c overnight. after blocking the wells using buffer containing pbs plus 0.05% (v/v) tween 20, supernatants were added to each well. biotinlabeled mab in incubation buffer was added to each well and as enzyme streptavidin-hrp was used. the reaction was developed using 3, 3′, 5, 5′-tetramethylbenzidine (tmb) substrate and stopped with 0.5 m h2so4 solution. the plates were washed after each step of incubation using pbs +0.05% (v/v) tween20. the plates were read at 450nm using a reader (biotek, usa). the mean optical densities (ods) of triplicate cultures were compared with the standard curves prepared using recombinant il-5, il-10, and ifn-γ. the cytokine levels represent the differences between the ods of test and background wells. the results are expressed in pg/ml (mean +sd) of triplicates experiments (11, 12). statistical analysis shapiro-wilk test was used to evaluate the normality of numerical variables. the quantitative variables were expressed as mean ±sd (standard deviation), and qualitative variables as a percentage. chi-square test and fisher’s exact test was used to examine the relation between qualitative variables. pair t-test /wilcoxon signed-rank test was used to the intergroup comparison. one-way anova with a followed post-hoc test (bonferroni) was used to multiple comparisons between the groups. p-value less than 0.05 was considered statistically significant. the spss version 16 (spss inc., chicago, il, usa) software was used for all statistical analyses. results the background information of the volunteers including gender, age and, cutaneous leishmaniasis status are presented in table 1. in healthy volunteers, the mean +sd of ifn-γ production in pbmc stimulated with live or heat-killed parasites versus unstimulated were (408.5+198.2), (369.5+199.07) and (179.7+ 88.1), respectively. there was a significant difference in ifn-γ production in pbmc of healthy volunteers stimulated with live l. major (p< 0.001), heat-killed l. major (p< 0.005) vs. unstimulated, there was no significant difference in ifn-γ production in pbmc of healthy volunteers stimulated with heat-killed l. major or live l. major (fig. 1a), in active lesion of cl volunteers the mean +sd of ifn-γ production in stimulated with live or heat-killed l. major vs. unstimulated were (1,953.2+ 1,032.8), (1,157.0+678.3) and (220+182), respectively. ifn-γ production in pbmc volunteers with active lesion stimulated with live or j arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 136 http://jad.tums.ac.ir published online: march 31, 2021 heat-killed l. major was significantly (p< 0.001) higher than with non-stimulated (fig. 1a), ifn-γ production in pbmc of volunteers with active lesion stimulated with live l. major was significantly (p< 0.001) higher than stimulated with heat-killed l. major (fig. 1a). in volunteers with history of cl, the mean +sd of ifn-γ production in pbmc stimulated with live l. major, heat-killed l. major and unstimulated were (2337.4+879), (1729.4+597.1), and (209+107), respectively. ifn-γ production in pbmc stimulated with live or heat-killed l. major was significantly (p< 0.001) higher than unstimulated pbmc, there was a significantly higher ifn-γ (p< 0.005) production in pbmc of history of cl volunteers stimulated with live l. major in comparison with pbmc stimulated with heat-killed l. major (fig. 1a). in healthy volunteers, the mean +sd of il-5 production in stimulated with live or killed l. major and unstimulated were (157.3+53.9), (191.2+ 86.6) and (160.3+36.8), respectively. there was no significant difference in il-5 production in pbmc of healthy volunteers stimulated with live or killed l. major and unstimulated pbmc, there was no significant difference in il-5 production in pbmc of healthy volunteers stimulated with live l. major or heat-killed l. major (fig. 1b). in pbmc collected from volunteers with active cl lesion, the mean +sd of il-5 production in pbmc stimulated with live, heat-killed l. major or unstimulated were (187.8+69.1), (272.6+113.5) and (198.7+97.5), respectively. there was no significant difference in il-5 production in pbmc of active lesion cl volunteers stimulated with live l. major compared with unstimulated pbmc, but there was a significant difference (p< 0.05) in il-5 production in pbmc of volunteers with active cl lesion stimulated with killed l. major compared with unstimulated pbmc. il-5 production in pbmc of volunteers with active lesion stimulated with live l. major was significantly (p< 0.005) lower than pbmc stimulated with heat-killed l. major (fig. 1b). in volunteers with a history of cl, the mean +sd of il-5 production in pbmc stimulated with live, heat-killed l. major and unstimulated were (133.4+50.4), (196.7+68.8) and (148.6+ 675), respectively. there was no significant difference in il-5 production in pbmc of volunteers with a history of cl stimulated with live l. major in comparison with unstimulated but there was a significant difference (p= 0.005) in il 5 production in pbmc of volunteers with history of cl stimulated with heat-killed l. major in comparison with unstimulated (fig. 1b). il-5 production in pbmc of volunteers with history of cl stimulated with live l. major was significantly (p< 0.001) lower than that of pbmc stimulated with heat-killed l. major (fig. 1b). in healthy volunteers, the mean+sd of il-10 production in pbmc of volunteers stimulated with live l. major, heat-killed l. major and unstimulated were (272.2+180.2), (263.4+ 236.4) and (212.3+160.2), respectively. there was no significant difference in il-10 production in pbmc of healthy volunteers stimulated with live l. major, heat-killed l. major and unstimulated pbmc, there was no significant difference in il-10 production of pbmc of healthy volunteers stimulated by heat-killed l. major and pbmc stimulated with live l. major (fig. 1c). in volunteers with active cl lesion, the mean +sd of il-10 production in pbmc stimulated with live l. major, heatkilled l. major and unstimulated were (451.1+ 291.1), (464.4+264.6) and (286.6+28.1), respectively. there was no significant difference in il-10 production in pbmc of volunteers with active cl lesion stimulated with live l. major, and unstimulated but there was a significant difference (p< 0.05) in il-10 production in pbmc of volunteers with active cl lesion stimulated with heat-killed l. major or unstimulated pbmc, there was no significant difference in il-10 production in pbmc of volunteers collected from active cl lesion stimulated with live l. major or killed l. major (fig. 1c). in volunteers with history of cl, the (mean +sd) of il-10 production in pbmc stimulated with live l. major, heat-killed l. major and unj arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 137 http://jad.tums.ac.ir published online: march 31, 2021 stimulated pbmc were (359.2+120.4), (365.3+ 198.6) and (241+98), respectively. there was no significant difference in il-10 production in pbmc of volunteers history of cl stimulated with heat-killed l. major or live l. major with unstimulated, there was no significant difference in il-10 production in pbmc of volunteers with a history of cl stimulated with live l. major or killed l. major (fig. 1c). there was a significant difference (p< 0.001) between the ifn-γ levels in supernatants of pbmc collected from volunteers with active cl lesion and pbmc of healthy volunteers stimulated with heat-killed or live l. major (fig. 1a). there was a significant (p< 0.001) difference between the ifn-γ levels in supernatants of pbmc of volunteers with history of cl and pbmc of healthy volunteers stimulated with heat-killed or live l. major (fig. 1a). there was a significant difference between the ifn-γ levels in supernatant of pbmc of volunteers with history of cl and volunteers with active cl lesion stimulated with heat-killed l. major (p< 0.005) and but there was no significant difference between the ifn-γ levels in supernatant of pbmc of volunteers with history of cl and volunteers with active cl lesion stimulated with live l. major (fig. 1a). there was a significant difference between the il-5 levels in supernatants of pbmc of volunteers with active lesion and pbmc of healthy volunteers stimulated with heat-killed l. major (p< 0.005) but there was no significant difference between the il-5 levels in supernatants of pbmc of volunteers with active lesion and that of healthy volunteers stimulated with live l. major (fig. 1b). there was a significant (p< 0.005) difference between the il-10 levels in supernatants of pbmc of volunteers with active lesion and that of healthy volunteers stimulated with live or heat-killed l. major. but there was no significant difference between the il-10 levels in supernatants of pbmc of volunteers with a history of cl lesion and that of volunteers with active lesion stimulated with live or heat-killed l. major. there was no significant difference between the il-10 levels in supernatants of pbmc of volunteers with active lesion and that of volunteers with history of cl stimulated with live l. major or heat-killed l. major (fig. 1c). table 1. characteristic of the volunteers hcl cases active lesion healthy number of volunteers 25 16 15 mean of age (year) 32 33.6 45 gender (m/f) 16.9 13.3 13.2 number of lesion 30 51 0 position of the lesion upper limb 11 12 lower limb 2 2 trunk 1 1 face 4 2 mean duration of the lesion (months) 5.4 2 j arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 138 http://jad.tums.ac.ir published online: march 31, 2021 fig. 1. cytokine levels (pg/ml) in culture supernatants of peripheral blood mononuclear cell (pbmc) of different volunteers (healthy, active cutaneous leishmaniasis( cl) lesion, history of cl) stimulated with live l. major or killed l. major, (a) ifn-γ level in culture, (b) il-5 level in culture, (c) il-10 level in culture a c b j arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 139 http://jad.tums.ac.ir published online: march 31, 2021 discussion recovery from leishmaniasis induced by natural infection or leishmanization leads to the development of lifelong protection against further disease development. leishmanization is the most effective control measure against cl (14). experiences with vaccination using killed leishmania vaccines showed limited protection. it seems that the quantity/quality of immune response which generated in vaccines receiving killed leishmania vaccine apparently is not similar to the immune response generated against live leishmania. although the reason of the different immune response against live vs. killed leishmania is not well known but one reason might be that likely live leishmania is presented by both endogenous and exogenous antigen presentation pathways but killed leishmania is only presented by the exogenous pathway. studies on murine model of leishmaniasis indicated that parasite persistence plays a crucial role in the induction of a strong and long-term immunity, another factor such as differences in the nature of the early inflammatory and/or immune responses to live vs. killed parasites might be important, animals inoculated with live leishmania induced more ifn-γ and less il-4 than those which were injected with killed leishmania (it was shown that infection with live parasites, (virulent or avirulent) leads to extensive activation and ifn-γ production by both cd4+ and cd8+ t cells while injection with killed parasites only lead to activation of cd4+ t cells. this activation of cd8+ t cells may play a role in the regulation of the early immune response to live vs. killed parasites. live leishmania promastigotes showed to directly activate primary nk cells to produce ifn-γ in the absence of il-12 and professional antigen presenting cells while killed parasites unable to do so, this means that live leishmania activates nk cells by direct mechanism but killed leishmania do not activate nk cells. in another study, c57bl/6 mice which were clinically cured from l. major lesion, skin-resident cd4+ t cells were found in the skin far from the site of the primary infection and were able to enhance protection against a later challenge by producing ifn-γ and recruiting circulating t cells to the skin. it was shown that live and dead parasites can differ in their ability to induce cellular responses in healthy donors as defined by ifn-γ production and cell proliferation (15-25). historically, leishmanization showed to be highly efficacious control measure against cl, in contrary, the results of several clinical trials using different leishmania species killed by various ways showed that killed leishmania does not induce immune response strong enough to protect against natural infection. in this study, the th1/th2 profile of volunteers with a history of cl and volunteers with active cutaneous leishmaniasis lesions were evaluated against live l. major in comparison with killed l. major. ifnγ production in pbmc of volunteers with active lesion (p< 0.001) and a history of cl (p< 0.005) stimulated with live l. major was significantly higher than stimulated with heat-killed l. major. il-5 production in pbmc of volunteers with active lesion (p< 0.005) and volunteers with history of cl (p< 0.001) stimulated with live l. major was significantly lower than pbmc stimulated with heat-killed l. major. there was no significant difference in il-10 production in pbmc of volunteers with active cl lesion stimulated with live l. major and killed l. major. il-5 and il-10 in the supernatant of pbmc collected from volunteers with active lesion were significantly (p< 0.005) higher than healthy volunteers stimulated by live or killed l. major. the level of ifn-γ in volunteers with a history of cl was higher than active lesion but not significantly, and the level of ifn-γ in volunteers with active lesions was significantly higher (p< 0.001) than healthy volunteers, this means that th1 response which is necessary for protection against leishmaniasis is induced upon recovery. il-5 level in volunteers with active lesion(s) was more than volj arthropod-borne dis, march 2021, 15(1): 133–142 a miramin-mohammadi et al.: immune responses in … 140 http://jad.tums.ac.ir published online: march 31, 2021 unteers with a history of cl. the results of this study showed that the intensity of th1 type of immune response is significantly higher in pbmc stimulated with live leishmania than stimulated with killed leishmania, the current results showed why natural infection/l eishmanization induces protection but vaccination with killed leishmania does not induce enough protection. this study is in agreement with previous studies carried out in which it was shown that responses to live and dead promastigotes are different in the induction of cytokines and response to live in which response to live parasites was parasites produced significantly more ifn-γ than the group of animals injected with killed stronger than dead ones (20). in another study, it was shown that the group of mice inoculated with live leishmania parasites produced significantly more ifn-γ than the group of animals injected with killed parasites (25). conclusion it is concluded that using live leishmania parasites in vitro induces a significantly stronger th1 type of immune response than killed leishmania which justifies using live leishmania (leishmanization) as a control measure to protect against cutaneous leishmaniasis. acknowledgements the study was financially supported 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236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 236 http://jad.tums.ac.ir published online: june 30, 2021 original article synergistic anti-leishmanial activities of morphine and imiquimod on leishmania infantum (mcan/es/98/lim-877) fatemeh ghaffarifar1; masoud foroutan2; *soheila molaei3,4; *eslam moradi-asl5 1department of parasitology, faculty of medical sciences, tarbiat modares university, tehran, iran 2department of parasitology, abadan faculty of medical sciences, abadan, iran 3zoonoses research center, ardabil university of medical sciences, ardabil, iran 4deputy of research and technology, ardabil university of medical sciences, ardabil, iran 5department of public health, school of public health, ardabil university of medical sciences, ardabil, iran *corresponding authors: dr soheila molaie, e-mail: s.molaei@arums.ac.ir, dr eslam moradi-asl, e-mail: moradiasl83@yahoo.com (received 7 jan 2020; accepted 2 may 2021) abstract background: this study was performed to evaluate in vitro and in vivo leishmanicidal potential of morphine (mph), imiquimod (iq), and their combination. methods: leishmania infantum promastigote and amastigote assays were performed at the presence of 0.015–150µm mph, 0.04–416µm iq, and their combination. the inhibition effects of these drugs on promastigotes were evaluated after 24, 48, and 72h. the cytotoxic effects of the drugs were evaluated by mtt as well as flow cytometry after 72h. we explored the therapeutic effects of mph and iq in balb/c mice at the end of the treatment using parasite load determination and cytokine assay. one group of mice received mph for three weeks before infection. results: the results of promastigote and amastigote assays showed the cytotoxic effects of the drugs at low concentrations. the cytotoxic effects were higher on promastigotes than amastigotes (p< 0.05). there was a negative correlation between drug concentration and amastigote/promastigote viability. imiquimod alone or combined with mph showed remarkable cytotoxic effects at all concentrations (p< 0.05). flow cytometry results revealed apoptosis in the parasite following exposure to the drug combinations. accordingly, the reduction of parasite loads in the spleen and liver was observed (p< 0.05) with simultaneous increases in ifn-γ and il-4. we believe that the in vivo leishmanicidal effect was mediated by mph through il-4 and by iq through both il-4 and ifn-γ. conclusion: results pointed out the promising effects of mph and iq at low concentrations, especially when combined. keywords: morphine; imiquimod; synergism effect; leishmania infantum; iran introduction leishmaniasis is one of the neglected tropical diseases, that caused by an obligatory intracellular parasite, the genus leishmania of the family trypanosomatidae (1). leishmania parasites are transmitted by phlebotomus sand flies and their infection leads to cutaneous, mucocutaneous, and visceral leishmaniasis depending on parasite species and host immunity (2). iran is an important focus of cutaneous and visceral leishmaniasis in the middle east, in which the visceral form is caused by the l. in fantum. an epidemiologic study by mohebali, 2013 showed that the seroprevalence of vl in humans and canines in iran are 4.7% and 12.2 % respectively (3, 4). the symptomatic form of the disease in humans, is characterized by irregular fever, anemia, hepatosplenomegaly, severe weight loss, globulinemia, and hyperglycemia. the mortality rate is 90–95% in undiagnosed and untreated cases (5). promastigotes and immotile amastigotes are the two forms of the parasite. amastigotes formed in host cells af copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ http://www.arums.ac.ir/file/download/page/1495617715-affiliation.pdf http://www.arums.ac.ir/file/download/page/1495617715-affiliation.pdf mailto:s.molaei@arums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 237 http://jad.tums.ac.ir published online: june 30, 2021 ter the bite of ingestion by the female sandfly containing promastigotes (6). therefore, the parasite avoids a rapid and direct attack by the host immune system and manipulates hostparasite interactions. these results in inhibiting the host cell apoptotic machinery and ensure the parasite’s extended survival in infected cells (7). different kinds of leishmania spp have been reported to inhibit apoptosis in host cells (8). for example, l. infantum can inhibit drug-induced apoptosis in human macrophages (9). therapeutics employed to treat leishmaniasis are limited and unsatisfactory. they include pentavalent antimony (sbv), amphotericin b (amb) and miltefosine (mf) (10). despite the inadequate understanding of the mechanism of these drugs, their targets are believed to differ substantially. antimony, with a rather complex mode of action, has multiple cellular targets. mf disrupts the biosynthesis of glycolipids and glycoproteins as well as the metabolism of alkyl phospholipids. amb exerts its toxic effects since it has a high affinity for the ergosterol of the leishmania plasma membrane (11). however, these agents have some limitations including high cost, long treatment duration, route of administration (intradermal and intramuscular injection), toxic effects on liver, heart, and kidneys as well as the lack of response to treatment in 10–15% of cases (12). failure response to antimonial compounds has been reported from endemic areas such as india and also iran (13). therapeutics that specifically kill infected macrophages may be beneficial for the treatment of leishmaniasis since the parasite resides in host macrophage or monocytes (7). stimulation of opioid receptors in infiltrating cells may be involved in local immune response control or be a signal to produce specific cytokines or antibodies analogous to other opioid receptors. morphine is the main alkaloid in opium and an active metabolite of heroin (bimonte) (14). morphine acts through opioid receptors (d, l, and j), which is related to mph-induced macrophage apoptosis through reducing the number of murine peritoneal and rabbit alveolar macrophages (15). the mph-induced apoptosis may be mediated by the up-regulation of bax and p53 proteins, increased p38 mapk phosphorylation, or tgf-β production by macrophages (16). in vivo and in vitro studies show that mph inhibits macrophage migration, which is secondary to the apoptotic effects of mph (17). imiquimod, a potent tolllike receptor-7 (tlr7) agonist, exerts its effects by the modification of immune responses and stimulation of apoptosis (18). imiquimod modulates immune responses by activating dendritic cells, macrophages, or other cell types via tlr7, pro-inflammatory cytokines including ifn-a, ifn-c, tumor necrosis factor (tnf) α/β, il-1a, and il-12. treatment with iq has an impact on the expression of various genes involved in apoptotic pathways (19). furthermore, iq decreases growth and/or increases apoptosis in several human cells (20). also, iq has been reported to induce gene expression and protein production of opioid growth factor receptors (21). currently, extensive research is being conducted worldwide to improve the treatment strategies of leishmaniasis. the present study was designed to investigate putative promastigote and amastigote inhibition as well as apoptotic features of mph, iq, or their combination on l. infantum and visceral leishmaniasis in balb/c mice (22-23). materials and methods in this study, all applicable international, national and institutional guidelines for the care and use of animals were followed and approved by the medical ethics committee of faculty of medical science, tarbiat modares university and approved under process no. 52d/3593/2015. in vitro experiments promastigote and macrophage culture promastigotes of l. infantum reference strain jpcm5 (mcan/es/98/lim-877) provided from http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 238 http://jad.tums.ac.ir published online: june 30, 2021 department of parasitology, kerman university of medical sciences (24). the promastigotes were grown in rpmi-1640 medium (gibco, dus) supplemented with 20% heat-inactivated fbs (gibco, us), 100iu/ml penicillin, and 100μg/ml streptomycin in a humidified atmosphere at 24 °c. then, 100µl of medium containing 1×106 promastigote cells/ml in logarithmic phase were subcultured and seeded in 96-well microplates. mouse macrophage cells, j774 a1 (cgbr80052901, kindly offered by professor marcel hommel), were cultured in rpmi-1640 medium supplemented with 10% fbs and 100 iu/ml penicillin/streptomycin at 37 °c and 5% co2 in a humidified atmosphere. then, they were seeded in 12-well microplates with a density of 1×105 cells/well and used for the next experiments (25-26). drug preparation morphine sulfate (temad company, iran) and iq (invivogen, toulouse, france) was purchased. morphine powder was dissolved in 5ml distilled water to obtain a stock solution. one mg/ml stock solution of iq was prepared by dissolving the powder in a commercially available specific solvent. then, the stock solutions of both drugs were diluted in rpmi to obtain the concentrations of 0.015–150µm. glucantime (273µm) was used as control and purchased as a liquid solution (85mg/ml) from sanofi-aventis, france (27-28). study groups and treatment of promastigotes, macrophages, and amastigoteinfected macrophages cell treatment with drugs was performed in three groups including l. infantum promastigotes, j774 macrophages, and l. infantum promastigote-infected macrophages. in each group, treatments were performed with different concentrations of mph, iq, or mph +iq. parallel exposures to 273µm of glucantime or culture medium were applied as controls. the treatment groups included (i) mph (0.015–150 µm), (ii) iq (0.041–416µm), (iii) glucantime (273µm), (iv) 0.015–150µm mph + 0.041– 416µm iq. promastigote and amastigote assay promastigote assay the promastigotes were treated with different concentrations of mph, iq, and their combinations for 24, 48, and 72h. then, the numbers of promastigotes were counted. the percentage of live promastigotes was evaluated by mtt assay after 72h. promastigotes (1× 106cells/ml) were exposed to studied concentrations of mph, iq, and their combination for up to 72h. then, the supernatant was removed, and the cells were treated with 5mg/ml mtt ((3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, sigma-aldrich) solution and incubated for 4h. afterward, dimethylsulfoxide (dmso) was added, and the absorbance was read at 570nm. the results were expressed as the mean percent reduction of parasite number compared to the untreated controls (28). uninfected macrophage cells viability we evaluated the effects of the drugs on uninfected macrophages, and the percentage of live macrophages was calculated. 1×105cells/ ml of macrophage were cultured and treated with different concentrations of mph, iq, and their combinations. the viability of the cells was determined by mtt assay after 72h (2829). amastigote assay after reaching confluency, j774 a1 macrophages were seeded on 12-well microplates (nunc) with a density of 1×105cells/well for 24h. adherent macrophages were infected with the stationary phase of l. infantum promastigotes at a ratio of 1:10 and were allowed to infect the macrophages for 6h. the cells were washed with fresh rpmi to remove nonphagocytosed promastigotes. infected macrophages were further incubated up to 72h in the presence of the drugs. the effects of drugs were http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 239 http://jad.tums.ac.ir published online: june 30, 2021 calculated from the percentage of infected cells and the number of amastigotes per infected macrophage in treated and untreated cultures in methanol-fixed and giemsa-stained preparations. values are means of triplicate exams (29). evaluation of apoptosis by flow cytometry promastigotes, uninfected macrophages, and infected macrophages were treated with different concentrations of mph, iq, and their combinations. then, flow cytometry was performed to detect apoptotic and necrotic cells using annexin-v fluos staining kit (biovision, usa). 1×106 parasites or 1×105 macrophages, and infected macrophage cells were treated with the drugs and incubated at 24 °c. after 24h, they were washed with cold pbs and centrifuged at 1500g for 5min. 5μl annexin-v fitc, 500μl binding buffer and 5μl pi (propidium iodide) were added and incubated for 15 minutes at room temperature. the test was performed using cyflow® space flow cytometry (sysmex-partec, usa) and data were analyzed by flomax software (partec, version 2.3) (30). all in vitro experiments were performed in triplicates. in vivo experiments animals and study groups this experimental study was performed at the laboratories of the parasitology department, faculty of medical sciences, tarbiat modares university (tmu), tehran, iran from october 2016 to july 2017. thirty female balb/c mice, with an average weight of 18– 21g and age of 5–8 weeks, were purchased from pasteur institute of iran. the mice were kept in our animal facility and used for in vivo experiments. the mice were randomly divided into 5 groups with 5 mice in each group (table 1). in all the cases, 100μl of promastigotes (2×107cells/ml) at the stationary phase were injected intraperitoneally into the mice. the infected mice were kept for about 18 days to allow parasite growth. then, the treatments were performed in each group for 3 weeks. 100µl of 1500μm mph once a week and 100µl of 624μm/mouse imq three times a week were injected intraperitoneally. all relevant ethical considerations in animal experiments were used before starting the study (medical ethics committee of tarbiat modares university, no: 52d/3593/tums). before treatment, two mice were sacrificed and evaluated by parasite culture to confirm the parasite growth. in the 6th group, the mice received mph for 3 weeks and were then infected with promastigotes. in all groups, after one month, the all of mice were sacrificed. then, their spleen and liver tissues were evaluated for parasite load by dilution method. briefly, 30mg of spleen or liver tissues were homogenized and transferred to rpmi-1640 medium. five dilutions of the suspensions were prepared, incubated at 26±2 °c, and followed up for two weeks. finally, the least count of the parasite was considered as the final titer, and parasite load was calculated (26). extraction of spleen lymphocytes and cytokine assay to measure ifn-γ and il-4 levels, the mice were sacrificed at the end of the treatments, and lymphocytes were isolated from spleen tissue. the staining of lymphocytes was performed using trypan blue to obtain the percentage of live cells. then, 1×106/ml lymphocytes were cultured in 12-well plates in rpmi1640 medium. then, the lymphocytes were stimulated by 20μg/ml of sla and incubated in the presence of 5% co2 at 37 °c for 72h. finally, inf-γ and il-4 levels in culture supernatants were measured by elisa using u-cytech kits (bioscience, netherlands) according to the manufacturer’s instructions. to isolate soluble leishmania antigens (sla), 108 promastigotes/ml at stationary phase was suspended in pbs and lyzed by five freezethaw cycles. the lysates were then centrifuged at 3000g at 4 °c for 15 minutes. the protein concentration of the supernatant was measured using bradford assay (26). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 240 http://jad.tums.ac.ir published online: june 30, 2021 statistical analysis data are shown as mean ±sd from at least two independent assays. in in vitro experiments, the differences between the groups were evaluated by one-way anova. in in vivo, the differences between treated and control groups were determined by an unpaired students t-test. p< 0.05 was considered significant by spss version 23. results the results of in vitro experiment cultured promastigotes were exposed to 0.015–150µm concentrations of mph, 0.041– 416µm iq, and their combination. the promastigote inhibition was assayed after 24, 48, and 72h exposure to the drugs as shown in fig. 1. the results showed that glucantime, as a positive control, and mph and iq in high concentrations had no effect on promastigotes count (p> 0.05). in mph and iq group, the concentrations of a lesser extent had inhibitory effects on promastigotes in all time intervals. this effect was more obvious after 72h (p< 0.05). in the group treated with mph +iq, similar to mph and iq only, the concentrations showed the least effects on promastigotes after 72h. the observed promastigote inhibition effect can be attributed to the presence of iq due to mostly the lack of response in the mph group. the viability of promastigotes and uninfected macrophages was evaluated after treatment with similar concentrations of mph, iq, and their combination for 72h (fig. 2). promastigote viability results showed a significant effect on the viability for mph in any doses (p< 0.05) except for 150 and 15µm mph, 416 and 41.6µm iq and their combination. the ic50 values of mph, iq, and combination formula on promastigotes after 72h were 0.102± 0.03, 0.235±0.01, and 0.173±0.02µm, respectively. also, the ic90 values of mph, iq, and their combination on promastigotes after 72h were 0.191±0.02, 0.398±0.01, and 0.245±0.02 µm, respectively. the results of the toxic effects of the drugs on uninfected macrophages were similar. glucantime had the most significant effect with no effect of mph even at high concentrations compared to the control group. in contrast, iq and the combination of the drugs showed toxicity on macrophages at higher doses (p< 0.05). the effect of imiquimod on promastigotes in higher doses, whether alone or in combination with mph, was not significant (p> 0.05). the ic50 values for mph, iq, and combination of these drugs were 0.8±0.01, 0.2±0.01, and 0.6± 0.02µm, respectively, after 72h on uninfected macrophages. fig. 3. shows the amastigotes count after exposure to all of the studied concentrations of mph, iq, and their combination for 72h. in contrast to promastigote assay, glucantime showed a significant effect on amastigotes compared to the negative control (p< 0.05). also, drugs in all doses except 150µm showed significant toxic effects both on infected macrophages or intracellular amastigotes compared to the control group (p< 0.05). the same results were observed for iq alone or its combination with mph especially at low doses (p< 0.05). analysis of differences in contaminated macrophages and intracellular amastigotes showed that there were significant differences between the treatment and control groups (p= 0.01) except for high concentrations (mph 150µm, iq 416µm, and their same combinations). the most significant toxicities were related to 0.04µm iq and then 0.015µm mph compared to other groups (p= 0.04). the ic50 values of mph, iq, and mph+iq on amastigotes after 72h were 18.2±0.3, 0.79±0.1, and 8.72± 0.1µm, respectively. also, the ic90 values were calculated 29.7±0.1, 1.1±0.1, and 11.8±0.3µm for mph, iq, and mph + iq respectively. fig. 4. demonstrates the results of flow cytometry analysis. as seen, early apoptosis in promastigote cells was low in morphine treatment, 3.04% and 6.08% at 1.5 and 0.015µm, respectively. the necrosis value was 2.55% http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 241 http://jad.tums.ac.ir published online: june 30, 2021 and 5.96% at 1.5 and 0.015µm mph, respectively. in contrast, in iq treatment, early apoptosis occurred more than necrosis, i.e. 4.11% and 9.18% apoptosis and 3.63%, 2.93% at 4.16µm and 0.04µm of iq, respectively. when these two drugs were combined, the early apoptosis increased and was 13.72%. the live uninfected macrophage cells were 90.75%, 91.59%, 87.56% at 0.015µm mph, 0.04µm iq and 0.015µm mph+0.04µm iq, respectively. apoptosis in infected macrophages was more than in other cells. significant differences were seen in the values of apoptosis and necrosis in all groups compared with the control (p< 0.05). in vivo experiments fig. 5. shows that parasite load in mice was significantly reduced in all drug groups com pared with infected mice treated with no drugs. the highest reduction in parasite load was observed in the groups that received 1500mph treatment before the infection. also, there was a significant difference between these groups in terms of reduction in parasite load (p< 0.05). as shown in fig. 6, the levels of both ifn-γ and il-4 had increased in all treated groups compared to untreated control. in mice treated with mph, there was a significant reduction in ifn-γ. mph seems to inhibit the parasites with a mechanism other than ifn-γ production. as seen, in the pre-mph group, the mice were treated before inoculation of parasites, ifn-γ higher than il-4 level, but there are no noticeable differences between il-4 levels compared to the control group (p> 0.05). table 1. the studied mice groups in the present study (5 mice in each group) abbreviation route of administration prescription amounts treatment mice groups ctrl(-) uninfected-untreated control groups ctrl(+) intraperitoneal 273μm infected-untreated (parasiteinfected treated with glucantime mph intraperitoneal 1500μm parasite-infected treated with morphine treatment groups iq ointment 624μm parasite-infected treated with imiquimod mph+ iq intraperitoneal/ ointment 1500+624μm parasite-infected treated with morphine+ imiquimod treatment pre-mph intraperitoneal 1500μm parasite-infected after pretreatment with mph for 3 weeks http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 242 http://jad.tums.ac.ir published online: june 30, 2021 fig. 1. promastigotes were exposed to 0.015–150µm concentrations of mph, (0.041–416µm) iq, and their combination for 24, 48, and 72h. in the negative control group, no treatment was applied. glucantime was used as a positive control. data are mean± standard deviation of one experiment in triplicate, *p< 0.05, tarbiat modares university, tehran, 2017 fig. 2. viability percentage of promastigotes and macrophages after 72h exposure to 0.015–150µm concentrations of mph, (0.041–416µm) iq, and their combination for 72h. after treatment, the viability was evaluated using mtt. in the control group, no treatment was applied. glucantime was used as a positive control. data are mean± standard deviations of one experiment in triplicate, *p< 0.05, tarbiat modares university, tehran, 2017 fig. 3. amastigotes were exposed to 0.015–150µm concentrations of mph, (0.041–41µm) iq, and their combination for 72h. in the control group, no treatment was applied. glucantime was used as a positive control. data are mean± sd, *p< 0.05, tarbiat modares university, tehran, 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 243 http://jad.tums.ac.ir published online: june 30, 2021 fig. 4. the results of flow cytometry analysis of promastigotes, un-infected, and infected macrophages. early and late apoptosis as well as necrosis has been shown after 72h, tarbiat modares university, tehran, 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 244 http://jad.tums.ac.ir published online: june 30, 2021 fig. 5. the mice were infected by leishmania infantum and then treated with studied drugs. at the end of treatment (one week later), the mice were sacrificed, and the parasite loads were evaluated in their liver and spleen. data were presented as mean±sd of experiments in 6 mice groups: ctrl (-): infected with no treatment, ctrl (+): parasiteinfected with 273μm glucantime treatment, mph: parasite-infected with 1500μm mph treatment, iq: parasite-infected with 624μm mouse iq treatment, mph + iq: parasite-infected with 1500μm mph + 273μm iq treatment, pre-mph: parasite-infected after pre-treatment with 1500μm mph for 3 weeks, tarbiat modares university, tehran, 2017 fig. 6. the mice were exposed to leishmania infantum and then treated with studied drugs. afterward, the mice were sacrificed, and the cytokine levels were evaluated in their spleen using elisa. data are presented as mean±sd deviation of experiments in 6 mice groups: ctrl (-): infected with no treatment, ctrl (+): parasite-infected with 273μm glucantime treatment, mph: parasite-infected with 1500μm mph treatment, iq: parasite-infected with 624μm mouse iq treatment, mph + iq: parasite-infected with 1500μm mph +273μm iq treatment, pre-mph: parasite-infected after pre-treatment with 1500 μm mph for 3 weeks, *p< 0.05, tarbiat modares university, tehran, 2017 discussion finding new treatments for leishmaniasis has been the target of ongoing efforts for dec ades. current medications for the treatment of visceral leishmaniasis are pentavalent antimohttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 245 http://jad.tums.ac.ir published online: june 30, 2021 ny, pentamidine, miltefosine, and amphotericin b. however, problems such as the emergence of resistance and severe toxic effects are limiting these drugs’ usefulness (31). thus, several new drug candidates have been proposed by many researchers (32). in this study, we used mph and iq to evaluate their potential toxicity on l. infantum promastigotes as well as uninfected and infected macrophages. the ic50 values of mph, iq, and combination formula on promastigotes after 72h were 0.102± 0.03, 0.235±0.01, and 0.173±0.02µm, respectively. also, the ic90 values of mph, iq, and their combination on promastigotes after 72h were 0.191±0.02, 0.398±0.01, and 0.245±0.02 µm, respectively. these values for uninfected macrophages were 0.8±0.01, 0.2±0.01 and 0.6± 0.02µm, respectively. these data showed that mph, iq, and their combination could be considered as new antileishmanial drug candidates at a minimum concentration of drugs. jabari et al. showed noticeable results of these drugs on leishmania. major (l. major) in in vitro conditions (28). in our present study, the drugs showed anti-leishmanial effects in the lowest concentration compared to the control group (p< 0.05). on the other hand, simultaneous usage of mph and iq showed promising results against promastigotes compared with the control (p< 0.05). glucantime, as the positive control, showed a poor effect on promastigotes. many studies have pointed that glucantime has low anti-leishmanial activities on promastigotes of leishmania spp (34). the ic50 of mph, iq and their combination was found to be 18.2± 0.3, 0.79±0.1, and 8.72±0.1µm, respectively, on l. infantum amastigotes. also, the ic90 values were calculated 29.7±0.1, 1.1±0.1, and 11.8±0.3µm for mph, iq, and mph +iq, respectively on amastigotes. the low toxicity of these drugs on j774 macrophages indicated the high effect of these drugs on intracellular leishmania parasites. these results were consistent with results obtained by others on l. major amastigotes (27, 33). in the present study, glucantime was more toxic to uninfected and in fected macrophages than mph and iq (p< 0. 05). flow cytometry results indicated a lower value of early and late apoptosis in mph or iq alone than their combinations. the results of early and late apoptosis were consistent with other studies that were performed on l. major (28). the difference between our study and others is perhaps related to drug doses and incubation time. the in vivo experiments on balb/ c mice confirmed the in vitro results. the parasite burden of spleen and liver tissues in test groups decreased significantly compared to the control group (p< 0.05). the positive synergistic effect of mph +iq showed more efficacies in controlling the parasite multiplication rate compared to mph or iq alone. on the other hand, when mph was administered before inoculation of parasites, the growth rate of parasites in balb/c mice was inhibited, and the parasite rate was at a minimum compared to the control and other test groups. the parasite count decreased to 48%, 63%, 67%, and 80% in mph, iq, mph +iq, and premph groups, respectively. the results of cytokine assay showed that il-4 was produced more than ifn-γ in mph group. in other groups, ifn-γ was produced more than il-4. the role of mph as an immunomodulator in protection against leishmaniasis by producing cytokines of cd4 + helper t-cells has been shown in many studies (14). other similar studies on l. major have shown contradictory results (34). in another study, the augmented effect of mph at low doses was approved in murine visceral leishmaniasis (35). many studies have shown that these drugs affect the parasite by involving immune system receptors. this is true especially for mph, which may induce the activation of opioid receptors as an immunomodulator (36). due to the selective pressure of the host defense system, pathogens have evolved different mechanisms helping them to antagonize apoptotic death of the invaded host cells such as macrophages. this not only gives more time for parasite replication (37) but also accelerates the ingestion of apoptotic infected macrophages http://jad.tums.ac.ir/ https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cd4 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/t-helper-cell j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 246 http://jad.tums.ac.ir published online: june 30, 2021 by uninfected ones. this event provides a way for amastigote spreading and subsequent infection (38). promastigotes of l. infantum and their surface lipophosphoglycan have been shown to prevent the apoptosis of macrophages, and this allows the survival of intracellular parasites. to unravel the mechanisms behind the resistance of l. infantum to apoptosis, some experiments have been performed. these experiments indicate that l. infantum inhibits apoptosis via the regulation of the iap (apoptosis inhibitor) family of proteins including ciap1 and ciap2. iap modulation will result in the apoptosis resistance of human macrophages after l. infantum infection. in addition, infection with l. infantum changes the expression of apoptosis-related proteins including bcl-2, bax, caspase-3, caspase-8, and caspase-9 (7). this shows that promastigotes employ multiple signals to confer more anti-apoptotic effects human macrophages. although, pi3k is not directly involved in bcl-2 regulation, a considerable level of pi3k phosphorylation occurs in l. infantum-infected cells. in this regard, mapks and pi3k are probably engaged during leishmania infections and take part in the apoptosis or survival of host cells (39). opioids, including mph, have immunoregulatory effects and do this through interactions with their receptor on immune cells. they are thus classified as cytokine families. the molecular basis underlying this effect is the modulation of cytokines and altered expression patterns of some cytokine receptors (40). several researchers have demonstrated the protective role of mph against leishmaniasis. in a study, low doses of mph were injected subcutaneously to l. donovani-infected balb/c mice or hamsters. the results showed significant suppression or even sterile clearing of the infection. in contrast, high doses exacerbated the infection (41). in another study, poonawala et al. showed that mph improved the healing of ischemic wounds through stimulating nitric oxide (no) via opioid receptors (42). the exact mechanisms of the protection role of mph against leishmaniasis are not completely clear. macrophages also have tlr7, which one of its potent ligands is imidazoquinoline compound iq (18). it has some roles in the modulation of the immune system through the activation of macrophages or other cells via tlr7 (19). in the present study, mph and to a lesser extent iq as well as their combination showed significant toxicity on promastigotes both in vitro and in vivo. in vitro experiments showed that drugs, at high doses, had no effect on promastigotes. the results of mph were similar to in vivo experiments. mph was not able to induce apoptosis in parasites, but prophylaxis with mph before infection made the mice resistant to the parasite, an effect that was far more effective than iq in in vivo. iq was able to reduce parasite load in vivo, but its effect became stronger when it was synergistically used with mph. researchers have shown that iq treatment stimulated genes and protein expression of the opioid growth factor receptor (21). the synergistic effect observed in this study may be attributed to this phenomenon. according to the results of many other studies, iq is effective on leishmania (43), viruses, and tumor cells (44). imiquimod is used as a drug in warts, basal cell carcinoma, and kaposi carcinoma, chronic hepatitis type c, intraepithelial carcinoma, melanoma, lung sarcoma, and breast cancer (45). a clinical trial study conducted by firooz et al. on efficacy and safety of iq combined with glucantime for cutaneous leishmaniasis of l. tropica expressing no beneficial effect of combining treatment with 5% iq cream with meglumine antimoniate in patients (46). another study by mohebali et al. (47), showed safety, but low efficacy (40.40%) of alum-alm mixed with bcg and imiquimod on canine visceral leishmaniasis. several studies in iran and other countries showed contradictory results of imiquimod treatment on cl lesions. the genus of leishmania, treatment duration, type application of iq (topical or subcutaneous injection), and clinical manifestation of lesions were reported responsible for cure rate in http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 247 http://jad.tums.ac.ir published online: june 30, 2021 iq treatment (46-47). the mechanism of action of iq is unknown, but it is an agonist of tlr7 and affects immune responses, induce tnfa, il-1a, and il-12 production, and stimulates apoptosis pathways (48). various cytokines are produced during leishmaniasis such as tumor necrosis factor (tnf) and interferon (ifn), which enhance macrophage activation and other inflammatory responses (49). macrophages are the main cells against leishmania and have prominent roles in immune response such as phagocytosis. therefore, to better understand the mechanisms involved in killing leishmania, the spleen lymphocytes were exposed to the parasite. the results showed that in mph group, increased the level of il-4 strongly. the results for iq or iq +mph -treated mice were contrasted, showing the equal increase of il-4 and ifn-γ. this may imply that the apoptosis mechanism is different in the two treatments. studies have demonstrated that iq enhances ifn-γ production both in human and murine cutaneous leishmaniasis. ifn-γ kills the parasite and causes protective immunity. also, il-4 enhances programmed apoptosis in stimulated human monocytes (50). susceptibility to infection is associated with the activation of th2 or th1 cells and secretion of il-4, il-5, il-6, il-10, il12, ifn-γ, and lymphotoxins. this could help animals to kill parasites and control infections such as leishmaniasis (51). ifn-γ is important to the immune defense against intracellular pathogens. in leishmaniasis, ifn-γ promotes th1 differentiation and macrophage activity. ifn-γ signaling in macrophages results in the activation of host defense mechanisms. additionally, ifnγ induces genes such as nitric oxide synthase, the most important molecule responsible for killing leishmania parasites by macrophages (52). macrophages that are activated by cytokines can produce large amounts of nitric oxide, one of its functions is a defense against intracellular pathogens particularly leishmania. nitric oxide has been demonstrated to kill leishmania parasites by inducing amastigotes apoptosis (53). ifn-γ is important since it has immune-stimulatory, immune regulatory, and immune-modulatory effects. in a study on the effects of artemisinin on vl, the authors demonstrated the ability of lymphocytes of infected mice to produce inf-γ during treatment with artemisinin (54). our study revealed a considerable ifn-γ response in the cultured splenocyte of test groups. there are several reports on different aspects of leishmaniasis in the country including the resistant status of vectors to different who recommended insecticides, reservoirs, reservoir control, vector control, ecology, novel approaches, training, and epidemiology (55-90). these reports will provide a guideline for disease control. conclusion according to the results of the present study, mph and iq alone or in combination with mph at low concentrations could inhibit the multiplication of the l. infantum promastigote and amastigote. the drugs eliminated the parasite growth and the development of murine visceral leishmaniasis. based on current and future in vitro and in vivo studies, mph and iq alone or in combination may be considered as a new therapeutic agent for the treatment of visceral leishmaniasis. the authors declare that they have no competing of interests. acknowledgments the authors wish to gratefully acknowledge parisa ebrahimi-sadr for her technical assistance. this work was supported by the department of parasitology, faculty of medical sciences, tarbiat modares university of iran (approval code: no: 52d/ 1575, date: 15 march 2015). the authors declare that they have no competing interests. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 236–254 f ghaffarifar et al.: synergistic anti-leishmanial activities of … 248 http://jad.tums.ac.ir published online: june 30, 2021 references 1. goto h, lindoso jal (2010) current diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis. expert rev anti infect ther. 8(4): 419–433. 2. dantas-torres f (2010) review of" humananimal medicine-clinical approaches to zoonoses and other shared health risks" by peter m. rabinowitz and lisa a. conti (eds.). parasite vectors. 3(1): 20. 3. mohebali m (2013) visceral leishmaniasis in iran: review of the epidemiological and clinical features. iran j parasitol. 8 (3): 348–358. 4. 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canine visceral leishmaniasis in dogs in ardabil province, northwestern iran. iran j public health. 49(6): 1033– 1044. 88. shirani-bidabadi l, zahraei-ramazani ar, yaghoobi-ershadi mr, akhavan aa, oshaghi ma, enayati aa, rassi y, gholampour f, shareghi n, madreseh e, vatandoost h (2020) monitoring of laboratory reared of phlebotomus papatasi (diptera: psychodidae), main vector of zoonotic cutaneous leishmaniasis to different imagicides in hyper endemic areas, esfahan province, iran. j arthropod borne dis. 14(1): 116–125. 89. mozaffari e, vatandoost h, rassi y, mohebali m, akhavan aa, moradi-asl e, zarei z, zahrai-ramazani a, ghorbani e (2020) epidemiology of visceral leishmaniasis with emphasis on the dynamic activity of sand flies in an important endemic focus of disease in northwestern iran. j arthropod borne dis. 14(1): 97–105. 90. rassi y, asadollahi h, abai mr, kayedi mh, vatandoost h (2020) efficiency of two capture methods providing live sand flies and assessment the susceptibility status of phlebotomus papatasi (diptera: psychodidae) in the foci of cutaneous leishmaniasis, lorestan province, western iran. j arthropod borne dis. 14(4): 408–415. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 187 http://jad.tums.ac.ir published online: june 30, 2021 original article biodiversity indices and medically importance of ticks in north khorasan province, northeast of iran kourosh arzamani1; *abedin saghafipour2; seyed ahmad hashemi1; hassan vatandoost3; mohammad alavinia1,4; saber raeghi5; zakkyeh telmadarraiy3 1vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 2department of public health, faculty of health, qom university of medical sciences, qom, iran 3department of medical entomology and vector control school of public health, tehran university of medical sciences, tehran, iran 4toronto rehabilitation centre, university health network, toronto, canada 5department of laboratory sciences, maragheh university of medical sciences, maragheh, iran *corresponding author: dr abedin saghafipour, e-mail: abed.saghafi@yahoo.com (received 31 dec 2019; accepted 26 apr 2021) abstract background: ticks are considered as the main vectors for the transmission of various pathogens such as relapsing fever and cchf to humans. this study was investigated the biodiversity indices and medically importance of ticks in north khorasan province, northeast of iran during 2015–2019. methods: specimens were captured from infested ruminants including cows, sheep, and goats. additionally, tick collections also were performed on non-domesticated creatures such as turtles, rodents, and hedgehogs. specimens were identified using valid identification keys. species diversity, species richness and evenness indices have been calculated to estimate species biodiversity of ticks. results: a total of 1478 adult ticks were collected. the specimens were from two families: ixodidae (90.05%) and argasidae (9.95%), 6 genera and 17 species including: rhipicephalus sanguineus (55.9%), rhipicephalus bursa (13.4%), hyalomma marginatum (9.5%), hyalomma anatolicum (9.5%), hyalomma asiaticum (0.2%), hyalomma aegyptium (0.5%), hyalomma scupense (1.3%), hyalomma sp (1.2%), haemaphysalis sulcata (0.7%), haemaphysalis erinacea (0.1%), haemaphysalis inermis (0.1%), haemaphysalis punctata (0.2%), haemaphysalis concinna (0.1% boophilus annulatus (1.2), and dermacentor marginatus (6.1%) among hard ticks as well as argas persicus (91.8%) and argas reflexus (8.2%) amongst soft ticks. rhipicephalus sanguineus, rh. bursa, hy. marginatum and hy. anatolicum were known as the most frequent species of hard ticks. tick’s species richness, shannon diversity index and simpson index in this area were s= 17, h’= 1.69, d= 0.294 respectively. conclusion: based on tick distribution veterinary authority, public health organizations and other officials should act for implementation of disease prevention. keywords: tick infestations; ruminant; geographic information system; ixodidae; argasidae introduction there are two major tick (arthropoda: arachnida) families: argasidae and ixodidae. in these families, some genera and species are able to transmit some important pathogens to humans and livestock. the adult ticks live for some years. in the absence of host, they can survive and may endure hunger for several years. both male and female ticks are blood sucking and paly as vector of diseases. some of tick species are able to transmit these agents to the next generation (1). ticks are blood-sucking ectoparasites of vertebrates, especially wild animals. after a tick bite, people may develop any of flu-like symptoms including fever, weakness, pain and swelling (2). the ticks transmit several bacterial, protozoal, rickaettsial, spirochaetal, and viral diseases of humans such as q fever (coxiella burnetii), tick-borne relapsing fever copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:abed.saghafi@yahoo.com https://www.cdc.gov/relapsing-fever/index.html https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 188 http://jad.tums.ac.ir published online: june 30, 2021 (borrelia), rocky mountain spotted fever (rickettsia), tularemia (pasteurella), crimean-congo hemorrhagic fever (cchf) and so on (3). in addition, ticks are able to transmit veterinary pathogens related to babesiosis, theileriosis, ehrlichiosis and anaplasmosis in ruminants (4). although, ticks were not known and familiar to humans for a very long times but their importance in terms of problems in livestock were began from the mid-nineteenth century (5). currently, there are 896 species of ticks all over the world (6). according to many studies have been performed on fauna of medical important ticks, they are distributed vastly in all regions of the world (7-10). a study on ectoparasites of sheep and goats in the eastern part of the amhara region, northeast ethiopia revealed that there are 5 species of ticks including boophilus decoloratus, amblyomma variegatum, amblyomma gemma, rh. pulchellus, rh. evertsi on body of sheep and goats. also, four genera and six species of ticks’ species and treatment of cows, sheep and goats in the sivas-zara region, in turkey were reported (1112). previously, many studies on the distribution of tick fauna in iran have been done (1319). asadollahi et al. (2014) have reported the presence of eight tick species in four different genera and they were identified as hyalomma anatolicum (39%), rhipicephalus sanguineus (25.4%), hy. excavatum (14.3%), hy. asiaticum (6.8%), haemaphysalis sulcata (3.7%), hy. scupense (3.4%), hyalomma spp. (3.4%), rh. anuulatus (2%) and hy. dromedarii (2%) in khouzestan province, south-west of iran (20). ramezani et al. (2014) carried out a study in meshkinshahr county, ardabil province, and reported nine species of ticks including dermacentor marginatus, d. niveus, haem. erinacei, haem. punctata, hy. anatolicum, hy. asiaticum, hy. marginatum, rh. bursa and rh. sanguineus. also, ganjali et al. (2014) reported different species of ixodidae (hard ticks) from sheep, goats, cattle and camels in zabol county, the eastern area of iran that they were included hy. dromedarii (17.3%), hy. schulzei (1.8%), hy. marginatum (0.5%), hy. excavatum (12.60%), hy. anatolicum (11.2%), hy. asiaticum (11.0%), rh. sanguineus (21.2%), rh. bursa (10.2%) and rh. turacunis (13.911%) (21-22). the iranian center for disease control and prevention (cdc) reported some cases of cchf infestation in ticks that collected from endemic areas of disease in iran such as the north khorasan province (23). annually, most cases of cchf in sistan and baluchestan, isfahan, fars, tehran, khorasan, and khuzestan are reported in iran (24). the most recent data indicate a total of 870 confirmed cases of cchf in iran, with 126 deaths from 2000 to 2012 (25). north khorasan province is one of the transit sites of domestic animals from the eastern borders (afghanistan and pakistan) into the country. furthermore, ticks are able to transmit different infectious agents to humans and animals, therefore identification of ticks and geographical distribution of them are essential to evaluate epidemiology and risks of human and animal tick-borne diseases. this study was carried out to investigate the biodiversity indices and medically importance of ticks in north khorasan province, northeast of iran during 2015–2019. materials and methods study area this cross-sectional study was conducted in north khorasan province, northeast of iran, between 36°37′-38°17′ n latitudes and 55°53′58°20′ e longitudes with an area of more than 28,434km2. the province is bordered by turkmenistan in the north (fig. 1). according to the 2011 census in iran, it includes eight cities with a population of more than 867,000 inhabitants. the province has desert and mountainous areas and receives about 250mm of rainfall annually. ticks collection in this cross-sectional descriptive study, the ticks were collected from infested ruminant inhttp://jad.tums.ac.ir/ https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjbpkkkqmjqahwijsakhctqbiwqfggamaa&url=http%3a%2f%2fwww.cdc.gov%2f&usg=afqjcnfzicntgz3jgwl6v2dgv1nb_9dniw&bvm=bv.139782543,d.bgs j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 189 http://jad.tums.ac.ir published online: june 30, 2021 cluding cows, sheep, and goats during the period from 2015 to 2019. additionally, some specimens collected from turtle, rodent, and hedgehog. the tick samples were collected from 53 different areas in the province. specimens were performed by examining the whole body including, ears, abdomen, tail, perianal regions, milk gland area, and back of the animals. collected ticks were placed in separate dry and labeled vials and transferred to the laboratory of vector-borne diseases research center, north khorasan university of medical sciences. determination of the species diversity all specimens were identified based on morphological characteristics and the keys given by estrada-pena (2004) in the laboratory of medical entomology, tehran university of medical sciences (26). all statistical analyses were done by excel software ver. 2010. and alpha diversity indices were calculated using an online biodiversity calculator (27). results in this study, 515 (55.2%), out of the total 933 inspected sheep, goat and cow were infested with ticks. a total of 1478 ticks (770 males and 708 females) were collected (table 1). the identified tick specimens belonged to two families: ixodidae (90.05%) and argasidae (9.95%), 6 genera and 17 species including concerning ixodidae families, rh. sanguineus (55.9 %), rh. bursa (13.4%), hy. marginatum (9.5%), hy. anatolicum (9.5%), hy. asiaticum (0.2%), hy. aegyptium (0.5%), hy. scupense (1.3%), hy. sp (1.2%), haem. sulcata (0.7%), haem. erinacea (0.1%), haem. inermis (0.1%), haem. punctata (0.2%), haem. concinna (0.1), b. annulatus (1.2), and d. marginatus (6.1%) (table 1). rhipicephalus (69.3%) followed by hyalomma (22.2%) were the predominant tick genus of hard ticks. with regards to the seasonal variation, the highest and lowest tick infestation was recognized in spring and winter, respectively. the results show that out of soft ticks only ar. persicus (91.8) and ar. reflexus (8.2) were collected in this region (table 2). all species of hard ticks were collected from sheep except hy. aegyptium and haem. concinna. collected species base on the hosts are shown in table3. some of the most important alpha diversity indices including, species richness, shannonwiener index, simpson index, shannon evenness index (equitability index), buzas and gibson's evenness index, berger-parker dominance index, menhinick richness index, and margalef richness index were calculated (table 4). table 1. the frequency of hard ticks by gender in north khorasan province, northeast of iran during 2015–2019 species male female total % rh. sanguineus 404 340 744 55.9 rh. bursa 87 91 178 13.4 hy. marginatum 80 47 127 9.5 hy. anatolicum 70 56 126 9.5 d. marginatus 41 40 81 6.1 hy. scupense 8 10 18 1.3 b. annulatus 0 16 16 1.2 hy. sp 0 16 16 1.2 haem. sulcata 4 6 10 0.7 hy. aegyptium 3 3 6 0.5 hy. asiaticum 3 0 3 0.2 haem. punctata 2 1 3 0.2 haem. erinacea 0 1 1 0.1 haem. inermis 0 1 1 0.1 haem. concinna 1 0 1 0.1 total of all specimens 703 628 1331 100 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 190 http://jad.tums.ac.ir published online: june 30, 2021 table 2. the prevalence of soft ticks in north khorasan province, northeast of iran during 2015–2019 h o st v is it r h . sa n g u in e u s r h . b u rs a h y . m a rg in a tu m h y . a n a to li c u m d . m a rg in a tu s h y . sc u p e n se b . a n n u la tu s h y . sp h a e m . su lc a ta h y . a e g y p ti u m h y . a si a ti c u m h a e m . p u n c ta ta h a e m . e ri n a c e a h a e m . in e rm is h a e m . c o n c in n a t o ta l o f a ll s p e c im e n s sheep 812 473 107 107 91 81 12 16 10 8 0 3 3 1 1 0 913 goat 81 267 67 2 2 0 0 0 0 2 0 0 0 0 0 0 340 cow 40 0 2 18 33 0 6 0 0 0 0 0 0 0 0 0 59 turtle 2 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 6 rumbomis 1 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 4 meriones 1 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 2 hedgehog 5 4 2 0 0 0 0 0 0 0 0 0 0 0 0 1 7 942 744 178 127 126 81 18 16 16 10 6 3 3 1 1 1 1331 table 3. species and numbers of hard ticks on different host collected in north khorasan province, northeast of iran from 2015 to 2019 species male female total % ar. persicus 59 76 135 91.8 ar. reflexus 8 4 12 8.2 total of all specimens 67 80 147 100 table 4. biodiversity and species richness indices of hard ticks in north khorasan province, north east of iran from 2015 to 2019 index computation amount specimens total number of specimens 1478 species richness (s) the number of species 17 shannon-wiener index (h’ ) 1.69 simpson index 0.294 shannon evenness index (equitability index) h/ln (s) 0.595 buzas and gibson's evenness index 0.317 berger-parker dominance index 0.503 menhinick richness index 0.442 margalef richness index 2.19 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 191 http://jad.tums.ac.ir published online: june 30, 2021 fig. 1. geographical locations of collected ticks of north khorasan province, northeast of iran discussion understanding the fauna species and geographical distribution of ticks will provide appropriate strategies to control and prevention of the tick-borne diseases (28). north khorasan province is bordered with turkmenistan. this province is a suitable area for agricultural production and animal husbandry (29). a considerable large number of sheep, goats, cows and so on graze on pastures of the province and therefore are at a greater risk of tick infestation and their borne diseases (30). in this study, most of ticks were collected on the body of sheep, goats and cows, but some of other animals such as turtles, some rodents of rhombomys and meriones spp. and hedgehog were infested with ticks. most of ticks such as ixodes, dermacentor, rhipicephalus, haemaphysalis have at least three hosts. telmadarraiy et al. have showed that rh. sanguineus, hy. marginatum, hy. anatolicum, hy. asiaticum and hy. dromedarii were known as the most frequent species which were positive for cchf virus in endemic areas of iran (23). most of these proven vectors of cchf in iran were collected and identified in north khorasan in this study. in this present study, 17 species of ticks were identified including rh. sanguineus, rh. bursa, hy. marginatum, hy. anatolicum, hy. asiaticum, hy. aegyptium, hy. scupense, hyalomma sp, boophilus annulatus, dermacentor marginatus, argas persicus, ar. reflexus. it has been shown that the tick infestation on cattle included haem. parva (33.8%), dermacentor marginatus (2.8%), boophilus annulatus (21.1%), haem. concinna (15.5%), hy. marginatum (19.7%) and rh. bursa (7%). besides that, many studies on http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 192 http://jad.tums.ac.ir published online: june 30, 2021 tick fauna have been conducted in different areas of iran. for instance, in west azarbaijan, 7 genera and 15 species were identified including rhipicephalus, hyalomma and orinthodoros and o. lahorensis, h. marginatum, h. asiaticum, rh. bursa had the highest frequency (31). nabian et al. reported the presence of fourteen tick species were identified as hy. anatolicum (5.23%), hy. marginatum (20.34%), h. detritum (3.48%), haemaphysalis punctate (12.79%), haem. parva (0.58%), haem. concinna (0.58%), haem. choldokovsky (6.97%), ixodes ricinus (2.32%), rh. sanguineus (19.76%), rh. bursa (4.65%), boophilus annulatus (9.88 %), dermacentor niveus (6.39%), d. marginatus (1.74 %) and o. lahorensis (5.23%) in ardabil, north west of iran (18). in this research, the genera rhipicephalus (69.3%) and hyalomma (22.2%) were the most predominant ticks. out of them rh. sanguineus, rh. bursa, hy. marginatum and hy. anatolicum had highest frequencies among hard ticks. a similar study was also reported from meshkinshahr (a northwest area in iran) in 2009 by hosseini vasoukolaei et al. in which most of the ticks belonged to rhipicephalus and hyalomma genus (19). based on the results of the mazlum (1971) carry out on ticks rh. bursa, rh. sanguineus, hy. anatolicum were the most common ticks in iran that are widespread in various areas of the country (15). nabian et al. showed that rh. sanguineus was the dominant tick species on body’s seep in mazandaran province, north of iran (18). rhipicephalus bursa and rh. sanguineus are very important in terms of health and medicine in iran because they have basic role in the transmission of pathogens in sheep. the pathogens that are transmitted by these tick species are babesia canis, ba. equi, ba. caballi, theileria ovis, anaplasma marginale and rickettsia oina (32). the hyalomma ticks are most common ticks in iran has been isolated from different domestic animals. mazloum studies indicated that hy. anatolicum is one of the prevalent ticks in iran. hyalomma anatolicum can transmit many infectious agents of humans such as theileria annulata, theileria parva, ba. caballi, q fever and cchf. previous epidemiological studies in khorasan provinces have demonstrated that cchf disease is one of endemic disease in this region (23, 25). it also has an important role in transmission of theileria herci to sheep in iran with regards to the seasonal variation, the highest and lowest tick infestation was recognized in spring and winter, respectively (33). other studies have been indicated the highest tick infestation distribution was spring and summer (16, 34-35). in principle, ticks are most active in hot seasons and humidity. in these areas due to livestock grazing season (spring and summer when the heat is suitable for complete life cycle of ticks) and putting them in the pasture, the tick distribution will be mainly influenced by rainfall (36). six haemaphysalis species have been recorded by different authors in iran and three species of them have been reported previously from north khorasan province (37). in the current study we report five species of this genus, so all species of this genus which reported from iran collected from north khorasan province. there are only limited studies in iran in which the authors calculated the biodiversity indices of ticks. furthermore, to the best of our knowledge, there has not been any report on biodiversity of ticks in north khorasan province and this is the first work that provides a numerical index on biodiversity of ticks in this region. the result showed tick’s species richness, shannon diversity index and simpson index in this area were higher than some other regions in the country (38-41). conclusion the present study showed a high degree of diversity of ticks in north khorasan province, northeast of iran. some of the most medically and veterinary important spices of ticks were collected from this region. it is important to awareness of fauna and the distribution of ticks in order to apply effective tick control prohttp://jad.tums.ac.ir/ https://www.google.com/search?biw=1608&bih=827&q=theileria+ovis&spell=1&sa=x&ved=0ahukewidlokmqnlqahvhd8akhbuwcbqqvwuifyga&dpr=0.9 https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjao6cgqnlqahvgbsakhrjubbmqfggamaa&url=https%3a%2f%2fmicrobewiki.kenyon.edu%2findex.php%2fanaplasma_marginale&usg=afqjcnhghilzlwfmcvjdatolnkxtwfzoeq https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewjao6cgqnlqahvgbsakhrjubbmqfggamaa&url=https%3a%2f%2fmicrobewiki.kenyon.edu%2findex.php%2fanaplasma_marginale&usg=afqjcnhghilzlwfmcvjdatolnkxtwfzoeq j arthropod-borne dis, june 2021, 15(2): 187–195 k arzamani et al.: biodiversity indices and … 193 http://jad.tums.ac.ir published online: june 30, 2021 grams and prevention of the disease in the region. we declare that we have no conflict of interest. acknowledgements the authors are grateful to the research deputy of north khorasan university of medical science. this study received financial support from vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran (project no: code of ethics: 91/390). the authors declare that there is no conflict of interest. references 1. service mw (1980) a guide to medical entomology london and basingstoke: macmillan press. p. 226. 2. kazimirova m, stibraniova i (2013) tick salivary compounds: their role in modulation of host defenses and pathogen transmission. front cell infect. microbiol. 3: 43. 3. jongejan f, uilenberg g (2004) the global importance of ticks. parasitology. 129: s3–14. 4. demessie y, derso s (2015) tick borne hemoparasitic diseases of ruminants: a review. adv biol res. 9: 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7-nasirian .doc iranian j arthropod-borne dis, 2007, 1(2): 40-47 h nasirian: duration of fipronil and … 40corresponding author: h nasirian, tel/fax: +98 21 88951393, e-mail: hanasirian@yahoo.com original article duration of fipronil and imidacloprid gel baits toxicity against blattella germanica strains of iran h nasirian department of medical entomology and vector control, school of public health, medical sciences/ university of tehran, tehran, iran (received 2 dec 2006; accepted 13 oct 2007) abstract the current study was conducted to investigate the duration of fipronil and imidacloprid gel baits toxicity against german cockroach strains in iran during 2003-2004. in order to conduct this study, nine german cockroach strains were used. newly emerged adult male german cockroaches starved for one scotophase (12 h), and ingested fipronil and imidacloprid gel baits for 2 h. after the given time was over, the bait was removed and replaced with mouse pellet. mortality was recorded at 12 intervals for 144 h (6 days). mortality data of the replicates were pooled and was tested using probit analysis. both gel baits were toxic to adult male german cockroaches. in the ingested bait method, the susceptible strain showed lt50 of 47.1 and 11.3 h for fipronil and imidacloprid gel baits, respectively, and the average lt90 was 74.2 and 19.3 h, respectively. lt50 of the feral german cockroach strains varied 14.9 h from 30.5 to 45.4 h and 4.4 h from 12.4 to 16.8 h for fipronil and imidacloprid gel baits, respectively. all german cockroach strains showed a similar susceptibility to fipronil and imidacloprid gel baits, compared with the susceptible laboratory strain. the steep slopes of ingested bait mortality curves indicated that the feral german cockroach strains were homogenous to fipronil and imidacloprid ingested gel baits. these results suggest that fipronil and imidacloprid gel baits appear to have considerable potential as a bait for insecticideresistant strains of german cockroach. keywords: german cockroach, gel bait, fipronil, imidacloprid, iran introduction the german cockroach, blattella germanica (l.), is a common indoor pest in low income housing. cockroaches not only spoil food but also transfer pathogens and cause allergic reactions and psychological distress (brenner 1995). management of cockroach populations in urban environments has recently shifted from the predominant use of insecticide sprays to the inclusion of baits in management programs (reierson 1995). this change has been facilitated by the general perception that baits are safer and therefore they appeal to the public’s concern about risks associated with pesticides in the domestic environment. moreover, baits fit well into the integrated pest management (ipm) objectives of reducing pesticide usage while maintaining effective suppression of cockroach populations (schal and hamilton 1990, rust et al. 1995). application of insecticidal baits is one of the most common and effective strategies for controlling the b. germanica (reierson 1995, bennett et al. 1997). toxic baits are commonly used in urban pest and management programs and provide several advantages over other insecticide application methods. baits greatly reduce problems encountered with run off and drift from liquid and dust insecticide formulations (jech et al. 1993). baits are useful when control programs are conducted near water, or in areas where threatened and endangered species occur, and where preservation of beneficial iranian j arthropod-borne dis, 2007, 1(2): 40-47 h nasirian: duration of fipronil and … 41 species of arthropods is important. baits offer the advantage of low odor, stability, and ease of application compared with aerosol and spray formulations (appel 2004). unlike liquid or dust forms, baits have little dispersion. in addition, using baits substantially reduces the overall amount of active ingredient needed and therefore reduces the amount of insecticide exposed to the environment. baits are often specific to the target organism, or at least provide greater selectivity than liquids and dusts. fipronil (c12h4cl2f6n4os), a phenylpyrazole insecticide, was discovered by rhonepoulenc agro in 1987, introduced in 1993, and registered as a pesticide in the united states in 1996. fipronil (5-amino-1-[2, 6-dichloro-4-(trifluromethyl) phenyl]-4-[(trifluromethyl) sulfinyl] 1h-pyrazole), is a relatively new insecticide that is at the beginning of a widespread use against an array of arthropod pests of agricultural, medical, and veterinary importance (colliot et al. 1992). application rates vary between 0.6 and 200 g a.i./ha, depending on the target pest and formulation. fipronil slowly degrades on vegetation and relatively slowly in soil and water, with a half-life ranging between 36 hour and 7.3 month. this very different time span depends on substrate and conditions. it is relatively immobile in soil and has low potential to leach into underground water. it is moderately toxic to laboratory mammals by oral exposure (ld50= 97 mg/kg for rats, ld50= 91 mg/kg for mice). technical fipronil is in toxicity categories ii and iii, depending on the route of administration, and is classed as a non-sensitizer (tingle et al. 2003). baits containing fipronil have already, in the 5 year use since their introduction, become popular among consumers and professionals alike for control of domestic cockroaches and ants. the great appeal of fipronil can be attributed, in large part, to its considerable lethality (kaakeh et al. 1997), but equally attractive is its distinctly greater toxicity to insects than mammals (gant et al. 1998, hainzl et al. 1998). fipronil kills insects by interacting agonistically with gamma-aminobutyric acid (gaba)-gated chloride channels (gant et al. 1998), a mode of action that colliot et al. (1992) called unique. recently, a toxic gel bait containing fipronil became available commercially (colliot et al. 1992). neonicotinoid insecticides exhibit high selective toxicity to insects over vertebrates. it has been shown that the selective toxicity of neonicotinoids is, at least in part, due to their selectivity to insect nicotinic acetylcholine receptors (nachrs) (shimomura 2005). imidacloprid (c9h10cln5o2), 1-(6-chloro-3-pyridylmethyl)-n-nitro-imidazolidin-2-ylideneamine, is a member of the class of neonicotinic insecticides. produced commercially by bayer cropscience, it is active both topically and by ingestion (lagadic et al. 1993) and is currently registered for crop, ornamental, and turf plant protection as well as seed treatments, companion animal parasite control, and as a soil termiticide. the oral ld50 of imidacloprid is > 4000 mg/kg body weight in rats and 131 mg/kg in mice, the 24 h dermal ld50 in rats is greater than> 2000 mg/kg. it is not irritating to eyes or skin in rabbits and guinea pigs. imidacloprid is rated as "moderately toxic" acutely by the who and the epa (class ii or iii). imidacloprid has low vapor pressure. the chemical breaks down to inorganic molecules by both photolysis and microbial action, in the air and with a half-life of 30 days in water and 27 days in soil anaerobically. because of its activity against a wide range of insects and its relatively low mammalian toxicity, imidacloprid is an excellent candidate for use against cockroaches (appel and tanley 2000). toxic gel baits are used more and more frequently to control urban cockroach populations (appel 1990, koehler et al. 1995). gel baits have been the main method for german cockroach control in the united states for at least 58 year (harbison et al. 2003). gel baits are proven to be convenient to use and highly effective (appel 1992, ross 1993, appel and benson 1995, kaakeh et al. 1997, appel and tanley, 2000). they are also safer and more iranian j arthropod-borne dis, 2007, 1(2): 40-47 h nasirian: duration of fipronil and … 42 environmentally friendly than insecticide sprays due to their targeted application. as a result, gel baits have become a very popular cockroach management tool for pest management professionals. when the active ingredient is incorporated into palatable bait, cockroaches readily consume a lethal dose from a single meal. therefore, baits were considered less likely to select for high-level cockroach resistance than insecticide sprays and other formulations (wang et al. 2004). the current study was designed to investigate duration of fipronil and imidacloprid gel baits toxicity against german cockroach strains in iran. materials and methods cockroach strains nine strains of b. germanica were evaluated for duration of fipronil and imidacloprid gel baits toxicity at the school of public health, medical sciences/university of tehran, iran during 2003 and 2004. a standard susceptible strain (ss) which has been maintained since 1975 without exposure to any insecticide in the insectary (ladonni 2001), was used as reference strain. eight strains of german cockroach (d1 to d7 and m) were collected from different locations in tehran (51° 22′-51° 24′ e, 35° 42′-35° 44′ n) including one infested habitable convened, and the seven different infested student dormitories of different universities (medical sciences/university of tehran, tehran, sanatee sharif and tarbiat modares), after insecticide spraying control failure with pyrethroid insecticides (nasirian 2004). cockroach collecting and rearing cockroaches were collected with a piece of a radiology film (10 × 10 cm) and transferred to an apparatus by hand catch in the last hours of the night. the apparatus was manufactured from two parts, the upper inside surface of the upper part (5 cm) was lightly greased with petroleum jelly to prevent cockroaches from escaping, after collecting cockroaches and in the insectary the lower part separated from the upper part and cockroaches transferred to glass rearing jars to prevent cockroaches from greasing (nasirian 2004). all cockroach collected strains were maintained and colonized at 27± 2 ˚c, 60± 10% rh, and a photoperiod of 12:12 (l: d) h in the insectary at the aforementioned address. each strain was kept in separate labeled glass rearing jars of the same size (500 ml). the upper inside surface of the jars was lightly greased with petroleum jelly to prevent escape. cockroaches were provided with mouse pellet, water ad libitum and a cardboard as a shelter. insecticides chemicals used were imidacloprid gel bait 2.15%, bayer ag leverkusen, germany, fipronil gel bait 0.05%, commercialized as goliath, rhone-poulenc rhodic, lyon, france, and co2 as an anesthetizer. toxic bait method only adult males (1-3 wk old) were used in bait ingested because their weight and physiology were more uniform than those of adult female cockroaches were (appel et al. 1983). moreover, female cockroaches were needed for further reproduction. adult male german cockroaches starved for one scotophase (12 h) with 5-6 bait ingested, and each bait ingested was replicated 3-6 times (10 cockroaches for each replicate), and then ingested fipronil and imidacloprid gel baits (fipronil gel 0.05%, commercialized as goliath, and imidacloprid gel bait 2.15%, for 2 h. after the given time was over, the gel baits were removed and replaced with mouse pellet. control groups (10 cockroaches for each replicate) ingested mouse pellet alone. a 1-6 day monitored giving> 0% and< 100% mortality at 144 h after ingested fipronil and imidacloprid gel baits were used for cockroaches. ingested fipronil gel bait males were placed in 150 by 25-mm plastic petri dishes, provided with mouse pellet food and water, and monitored for mortality at 12 intervals (12 h) for 144 h under the same temperature and photoperiod as the colony. in addition, it should be mentioned that mortality after ingested fipronil and imidacloprid gel baits was recorded for 12 days but mortality became stable after 6 days. if insects on their backs were unable to right themselves when prodded, they were considered dead. iranian j arthropod-borne dis, 2007, 1(2): 40-47 h nasirian: duration of fipronil and … 43 statistical analysis mortality data from the replicates were pooled and the time exposure mortality was assessed by probit analysis (finney 1972), with a spss package. resistance ratios were calculated as the 50% response value (lt50) of rr strain divided by the 50% response value of the ss strain. significant differences (α= 0.05) between lt50 and lt90 of each strain to fipronil and imidacloprid gel baits were verified by one-way anova. distribution of variables was analyzed using onesample kolmogorov-smirnov test. results both gel baits were consumed and toxic to adult male german cockroaches. there was negligible (< 1%) control groups (ingested mouse pellet alone) mortality. after probit analysis (finney 1972) in the ingested bait method, the susceptible strain showed lt50 of 47.1 and 11.3 h for fipronil and imidacloprid gel baits (fig. 1), respectively, and the average lt90 was 74.2 and 19.3 h, respectively (table 1 and 2). lt50 of the feral german cockroach strains varied 14.9 h from 30.5 to 45.4 h and 4.4 h from 12.4 to 16.8 h for fipronil and imidacloprid gel baits, respectively (table 1 and 2). after calculation of the 50% response value (lt50) of rr strain divided by the 50% response value of the ss strain, all german cockroach strains showed a similar susceptibility to fipronil and imidacloprid gel baits compared with the susceptible laboratory strain and the steep slopes of ingested baitmortality curves indicated that the feral german cockroach strains were homogenous to fipronil and imidacloprid ingested gel baits. table 1 shows that the field collected strains are more susceptible to fipronil gel bait than the susceptible strain. it is probably due to the field collected strains which were more attractant to fipronil gel bait than the susceptible strain. no significant differences (p< 0.05) were observed between lt50 and lt90 of each strain to fipronil and imidacloprid gel baits using oneway anova. one-sample kolmogorov-smirnov test was showed that the distribution of variables were normal (fig 1.). table 1. toxicity of fipronil toxic gel bait to a susceptible and feral-reared german cockroach strains strains n slope±se χ2 lt50 a (ci) lt90 a (ci) rr b s 160 0.05±0.01 2.13 47.1 (42.0-53.2) 74.2 (65.7-88.3) d1 160 0.08±0.01 10.13 36.7 (27.2-48.1) 53.5 (43.8-66.3) 0.8 d2 196 0.07±0.01 8.9 32.9 (23.1-43.0) 51.8 (42.0-81.5) 0.7 d3 248 0.1±0.02 3.4 30.5 (27.5-33.6) 53.0 (39.1-69.3) 0.7 d4 160 0.05±0.01 6.1 30.5 (25.2-35.3) 56.5 (49.6-68.0) 0.6 d5 240 0.17±0.03 0.4 32.9 (29.2-35.1) 50.6 (37.9-65.6) 0.7 d6 284 0.04±0.01 3.1 45.4 (39.3-52.2) 76.2 (66.2-85.1) 1.0 d7 160 0.03±0.01 1.4 39.1 (25.3-47.4) 77.3 (65.2-88.5) 0.8 m 220 0.05±0.01 2.5 37.5 (25.9-43.7) 63.1 (55.6-81.2) 0.8 a lt50 and lt90 values in hours (95% ci) b resistance ratio: lt50 of resistant strain/ lt50 of susceptible strain iranian j arthropod-borne dis, 2007, 1(2): 40-47 h nasirian: duration of fipronil and … 44 table 2. toxicity of imidacloprid toxic gel bait to a susceptible and feral-reared german cockroach strains strains n slope±se χ2 lt50 a (ci) lt90 a (ci) rr b s 160 0.16±0.03 1.3 11.3 (9.29-13.05) 19.3 (17.07-23.19) d1 160 0.17±0.03 1.4 12.4 (10.6-14.09) 19.9 (17.72-23.47) 1.1 d2 186 0.12±0.03 0.8 12.4 (7.6-14.09) 22.9 (20.12-30.0) 1.1 d3 228 0.09±0.02 4.8 14.4 (9.72-17.4) 28.5 (24.86-35.39) 1.3 d4 160 0.14±0.02 6.2 16.8 (8.52-24.33) 27.1 (21.73-38.75) 1.4 d5 240 0.02±0.01 0.3 13.8 (7.54-31.22) 23.2 (15.42-33.23) 1.2 d6 264 0.03±0.01 6.7 15.29 (6.0-26.52) 27.0 (14.12-39.61) 1.4 d7 160 0.18±0.02 8.8 15.6 (9.44-20.64) 22.8 (18.60-33.59) 1.4 m 180 0.16±0.02 3.2 13.4 (11.62-15.11) 21.2 (18.88-24.93) 1.2 a lt50 and lt90 values in hours (95% ci). b resistance ratio: lt50 of resistant strain/ lt50 of susceptible strain. 0 10 20 30 40 50 60 70 80 s d1 d2 d3 d4 d5 d6 d7 m strains h ou rs fipronil lt50 fipronil lt90 imidacloprid lt50 imidacloprid lt90 fig. 1. comparison of fipronil and imidacloprid gel baits toxicity to a susceptible and feral-reared german cockroach strains discussion these results indicated that the fipronil and imidacloprid gel baits relatively killed the german cockroaches in ingested bait method, with time until 144 h and becoming stable thereafter. although both fipronil and imidacloprid gel baits tested had similar high efficacy levels after direct ingestion, important differences in death rates were observed under laboratory conditions. cockroaches died more rapidly after ingestion imidacloprid gel bait than after ingestion iranian j arthropod-borne dis, 2007, 1(2): 40-47 h nasirian: duration of fipronil and … 45 of fipronil gel bait. in comparison the fipronil gel bait lt50 (or lt90) of susceptible strain and the average lt50 (or lt90) of the feral german cockroach strains was more than to imidacloprid gel bait. the fipronil gel bait was more slowly affect the german cockroach than the imidaloprid gel bait. the observed differences between the two baits might be inherent to their different modes of actions. appel and tanley (2000) reported that the imidacloprid gel bait was toxic to all stages of the german cockroach (appel and tanley, 2000). baits affected cockroaches differently in relation to their developmental stage, small nymphs died more rapidly than large nymphs and adults. this may be related to differences in size, action on metabolism, or physiology (cornwell 1976, rust et al. 1995). fipronil is active at very low concentration so that cockroaches are killed after eating only a very small amount of bait (kaakeh et al. 1997, valles et al. 1997, durier and rivault 2000). furthermore, the fact that the presence of fipronil in gel did not influence its level of attractiveness indicates that fipronil is not repellent to cockroaches (durier and rivault 2000). extensive use of insecticides has led to the development of resistance in german cockroach to a wide range of insecticides including organochlorines, organophosphates, carbamates and pyrethroids (lee et al. 1996, cochran 1997, ladonni 2001, nasirian et al. 2006) and consequent control failures in some field populations have been reported (cochran 1989, scott et al. 1990, atkinson et al. 1991, valles and yu 1996, dong et al. 1998, valles 1999, valles et al. 2000, wei et al. 2001). fipronil and imidacloprid are relatively new and acting at new target sites, which are currently not affected by resistance than other previous insecticides that will be used for pest control especially against german cockroach in iran. however, nasirian et al. (2006) investigated fipronil toxicity against these strains of german cockroach. the rrs of these strains to fipronil ranged from 1.5to 2.6-fold (nasirian et al. 2006). since fipronil has not been used previously in the area, this relatively low resistance could be due to german cockroaches collected from the field exhibited higher susceptibility level to fipronil than the susceptible strain (kaakeh et al. 1997). therefore, based on the data from nasirian et al. (2006), fipronil could be recommended as a valuable tool in resistance management of german cockroach in this study area. it is concluded that fipronil and imidacloprid toxic gel baits appear to have considerable potential as bait insecticide for insecticide resistant german cockroach strains in iran. acknowledgments the author would like to thank dean of school of public health, and head of medical entomology and vector control department of medical sciences/university of tehran for their support to carry out this research. this investigation received financial support from the school of public health (medical sciences/university of tehran), via msph thesis. references appel ag (1990) laboratory and field performance of consumer bait products for german cockroach (dictyoptera: blattellidae) control. j econ entomol. 83: 153–159. appel ag (1992) performance of gel and paste bait products for german cockroach (dictyoptera: blattellidae) control: laboratory and field studies. j econ 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accepted 5 nov 2007) abstract following annual report of new cases of cutaneous leishmaniasis from different areas of kuhpayeh district, esfahan county, an investigation was carried out on some ecological aspects of phlebotomine sand flies during 2000-2002. sand flies were collected biweekly from outdoor and indoor resting places with the aid of 30 sticky traps from the beginning to the end of the active season. female sand flies from rodent burrows were dissected and examined for the presence of promastigote infection. blood meals of engorged sand flies were identified by elisa method. totally, 4993 sand flies were collected and identified. the following species were found indoors: p. papatasi, p. sergenti, p. caucasicus, p. mongolensis, p. alexandri, p. ansarii, p. major, p. kandelakii, s. sintoni, and the subsequent species were found outdoors: p. papatasi, p sergenti, p. caucasicus, p. mongolensis, p. ansarii, p. major, s. sintoni, s. dentata, s. pawlowskyi. the sand flies active season began from may till october in this region. natural promastigote infections observed in 1.06% of p. papatasi and also in one out of four of p. caucasicus. the human and rodent blood indices in p. papatasi were 61.9% and 20.69%, respectively. it seems that p. papatasi is the probable vector among rodents and also transmit leishmania major, the causative agent of zoonotic cutaneous leishmaniasis to man, and p. caucasicus transmit the agent of the disease among rodents. keywords: ecology, leishmaniasis, phlebotmine sand flies, iran introduction phlebotomine sand flies are the vectors of leishmaniasis and sand fly fever in iran (nadim and seyedi-rashti 1971, tesh et al. 1976, yaghoobi-ershadi et al. 2005). cutaneous leishmaniasis due to l. major (clm) is a great health problem in the country and prevails in rural districts of 15 out of 30 provinces in iran (akhavan et al. 2007). kuhpayeh district is one of the endemic foci of cutaneous leishmaniasis (cl), 30 km east of esfahan city, central iran. the incidence of the disease was reported 4.1/1000 in 1996 (esfahan health center, unpublished data). the infected district is located close to the esfahan city, one of the most important historical and industrial cities of iran and lots of people from various parts of iran and also other countries visit it during the active seasons of sand flies. the main vector to human in iran is phlebotomus (phlebotomus) papatasi scopoli *corresponding author: aa akhavan, tel/fax: +98 21 88951393, e-mail: aaakhavan@sina.tums.ac.ir iranian j arthropod-borne dis, 2007, 1(2): 34-39 h abdoli et al.: some ecological aspects of … 35 (diptera: psychodidae), and the other vectors among rodents in the rural areas are p. (paraphlebotomus) caucasicus marzinowsky, p. (pa.) mongolensis sinton, p. (pa.) andrejevi shakirzyanova, p. (pa.) alexandri sinton, and p. (synphlebotomus) ansarii lewis (nadim et al. 1968, 1994, javadian et al. 1977, yaghoobiershadi et al. 1994, 1995b, rassi et al. 2007). l. major has been isolated from p. papatasi and p. caucasicus in some clm foci of iran (yaghoobi-ershadi et al. 1994, 1995 b, 2001 a, 2001 b, 2005, rassi et al. 2007). the ecological aspects of the cl vectors have not been examined in kuhpayeh district yet. therefore some ecological aspects of sand flies were studied for the first time in this district for implementation of future control measures. the objectives of the present study were to determine the sand flies fauna, species composition, population density, number of generation and also leishmanial infection rate of sand flies in the area. materials and methods the investigation was conducted during 2000-2002 in 4 areas of kuhpayeh district, esfahan province, central iran. the city of kuhpayeh is the center of the district (32º 42′n, 52º 26′e) east of esfahan city. the district has different plain and mountainous climate at various altitudes, about 1550 to 1780 m. the maximum and minimum mean monthly temperatures were 37.9 ºc and –5 ºc in july and february, respectively. the total annual rainfall was 70 mm with a minimum of 0.4 mm in april and maximum of 29.9 mm in december during rainy months. the minimum mean monthly relative humidity was %26 (july) and the maximum was %57.5 (december). sand flies were collected biweekly from indoor (bedrooms, hall, toilets and stables) and outdoor (rodent burrows, mountain and rock rifts) fixed places in 9 parts of the areas for detecting monthly variation and also 11 points for detecting the fauna of sand flies, using 30 sticky traps (castor oil coated white paper 20×32 cm) from the beginning (april) through the end (october) of the active season. for species identification, sand flies were mounted in puri,s medium (smart et al. 1965) and identified after 24 h using the keys of theodor and mesghali (theodor and mesghali 1964). then they were counted and segregated by sex. in order to determine natural promastigote infections of sand flies, some unfed, blood fed, semi gravid and gravid female sand flies of rodent burrows were collected by sticky traps and examined in a fresh drop of sterile saline (9/ 1000) for the presence of promastigotes in alimentary canal. to determine the host preference of p. papatasi, the sand flies collected by aspirating tube and sticky paper traps from human dwelling, rodent burrows and stables. the sand flies were dissected freshly and blood meals smeared onto circles of whatman no. 1 filter paper. they were interleaved with non-absorbent onionskin paper and sent with necessary information to the department of parasitology in the pasteur institute of iran for enzyme linked immunosorbent assay (elisa) testing (edrissian et al. 1985). because of small quantity of blood meals, each one was tested against only one antiserum. results a total of 4993 adult sand flies (4294 from outdoors and 699 from indoors) were collected during may-october 2000 and 11 species of sand flies (8 species of the genus phlebotomus and 3 species of the genus sergentomyia) were identified. the following species were found in indoors: p. papatasi (27.43%), p. sergenti (52.04%), p. caucasicus and p. mongolensis (4.39%), p. alexandri (0.31%), p. ansarii (0.78), p. major (2.04%), p. kandelakii (1.72%), s. sintoni (11.29%) and the subsequent species were found in outdoors: p. papatasi (35.04%), p. sergenti (2.03%), p. caucasicus and p. moniranian j arthropod-borne dis, 2007, 1(2): 34-39 h abdoli et al.: some ecological aspects of … 36 golensis (3.65%), p. ansarii (1.15%), p. major (0.05%), s. sintoni (57.73%), s. dentata (0.28%), s. pawlowskyi (0.07%). six species of phlebotomine sand flies were collected from plain areas and 11 species were found in mountain areas (table 1). the most common sand flies in plain areas were p. papatasi (37.39%) and s. sintoni (58.23%) and the most abundant species in mountain areas was p. sergenti (71.3%). these species were collected in all of the months during the active season. the monthly activity of p. papatasi and p. sergenti began in late april and ended in late september with two peaks of activ ity (fig. 1 and 2). no sand fly was found in the area during november to march due to cold weather. the sex ratio i.e. number of males/100 females of p. papatasi, p. sergenti and s. sintoni was 110.53, 357.89 and 18.64 in outdoors and also 182.26, 1085.71 and 41.18 in indoors, respectively (table 2). natural promastigote infections observed in 1.06% of p. papatasi and also in one out of four of p. caucasicus but attempt to isolate the parasite failed (table 3). elisa testing of 52 p. papatasi showed that the human and rodent blood indexes in p. papatasi were 61.9% and 20.69%, respectively. table 1. the diversity of sand flies in plain and mountain areas,kuhpayeh district, esfahan province, central iran, 2000. table 2. the sex ratio of sand flies collected by sticky paper method from indoor and outdoor resting places, kuhpayeh district, esfahan province, central iran, 2000 table 3. natural leptomonad infection rate of sand flies from rodent burrows, kuhpayeh district, esfahan province, central iran 2000-2002 age group no. sand flies with leptomonads species no. dissected n p ? t g e h infected% p. papatasi 94 13 70 11 1 1 1.06 p. caucasicus 4 3 1 1 1 25 p. ansari 1 n: nulliparous, p: parous,?: age not known, t: total, g: gut, h: head, e: esophagus species area p . papatasi p . sergenti p. m ongolensis p . caucasicus p . alexandri p . ansarii p . kandelakii p . m ajor s. sintoni s. dentata p . paw low sky plain areas + + + + + + mountain areas + + + + + + + + + + + indoors outdoors female male female male resting places species no percent no percent total sex ratio no percent no percent total sex ratio p. papatasi 62 35.43 113 64.57 175 182.26 712 47.5 787 52.5 1499 110.53 p. sergenti 28 8.43 304 91.57 332 1085.71 19 21.84 68 78.16 87 357.89 s. sintoni 51 70.83 21 29.17 72 41.18 2082 84.29 388 15.71 2470 18.64 iranian j arthropod-borne dis, 2007, 1(2): 34-39 h abdoli et al.: some ecological aspects of … 37 fig. 1. the monthly variation of p. sergenti from indoor and outdoor resting places, jebel rural area, kuhpayeh district, esfahan province, central iran, 2000 fig. 2. the monthly variation of p. papatasi from indoor and outdoor resting places, sistan rural area, kuhpayeh district, esfahan province, central iran, 2000 discussion this is the first report of some ecological aspects of sand flies in kuhpayeh rural district, esfahan province, central iran. six species of phlebotomines were identified in plain areas and 11 species in mountain areas. the diversity of sand flies in mountain areas was more than plains. p. papatasi and p. sergenti are the dominant sand flies of the genus phlebotomus with two generations in the plain areas. p. papatasi and p. sergenti are peridomestic species. blood meal analysis shows that the anthropophilic index of p. papatasi (61.9%) is high as has been found by some other investigators (natima et al. 1991).the anthropophilic index of this species has been reported to be 29.6-44.6% and 31.2% in iranian j arthropod-borne dis, 2007, 1(2): 34-39 h abdoli et al.: some ecological aspects of … 38 borkhar and badrood districts of esfahan province, respectively (yaghoobi-ershadi et al. 1995a, 2005). natural promastigote infection rate of p. papatasi from rodent burrows was 1.06%. natural infection of this species was also recorded from badrood (6.7-22%), nikabad (4.76%), sabzevar (4.5%), lotfabad (5.8%) and esfarayen (2.2%) (mesghali et al. 1967, javadian et al. 1976, yaghoobi-ershadi et al. 2001a, 2001b, 2003, 2005). l. major has been isolated from p.papatasi and p. caucasicus in some clm foci of iran (yaghoobiershadi et al. 1994, 1995b, 2001a, 2001b, 2005, rassi et al. 2007). naturally promastigote infection of p.caucasicus has been reported from a focus of zoonotic visceral leishmaniasis (zvl) in iran (rassi et al. 2004). l. major has been isolated from gerbils and also human in some clm foci of iran (yaghoobi-ershadi et al. 1996, akhavan et al. 1998, rassi et al. 2007). the close contact between vectors and reservoir hosts have created a very efficient cycle for transmitting the disease among rodents and also transmission the disease to people living in the villages located near rodent burrows. regarding the results of the present study and based on the isolation and characterization of l. major from p.papatasi and p. caucasicus in neighboring focus of the disease in esfahan province (yaghoobi-ershadi et al. 1994, 1995b) it seems that p. papatasi has the main role to transmit the parasite among rodents and also to man and p. caucasicus transmit the agent of the disease among rodents. to confirm the focus of clm in this district, isolation and characterization of l.major from p. papatasi, great gerbil and also human is recommended. acknowledgements we 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phlebotomus (paraphlebotomus) caucasicus in isfahan province, islamic republic of iran. trans r soc trop med hyg. 88: 518-519. j arthropod-borne dis, march 2023, 17(1): 28–35 v moin-vaziri and m badakhshan: the impact of … 28 http://jad.tums.ac.ir published online: march 31, 2023 review article the impact of covid-19 pandemic on arthropod-related diseases *vahideh moin-vaziri1, mehdi badakhshan2 1department of medical parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 2department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran *corresponding author: dr vahideh moin-vaziri, e-mail: vmvaziri@gmail.com, v.vaziri@sbmu.ac.ir (received 11 july 2021; accepted 12 mar 2023) abstract background: the covid-19 pandemic that caused by the infection with the novel coronavirus sars-cov-2 has revealed individual and global vulnerabilities all over the world. many countries that had been struggled with arthropodborne diseases (vbds) are now embroiled in another challenge caused by covid-19 pandemic. the situation that poses major obstacles 1) by misdiagnosis 2) delay in early and appropriate treatment of vbds 3) difficulties in applying regular strategy for vector control and prevention methods and finally 4) irregularity in financing supports. given the possible scenario of syndemics, it is important to plan integrated and combined measurement with the maximum participation of the people and health authorities. here, the impact of covid-19 pandemic on some major arthropod-related diseases will be discussed. methods: to access the collective data all related databases such as science direct, pubmed, elsevier, google scholar, as well who web page were searched with key words “arthropoda-related diseases, covid-19 with the name of each individual disease”. results: the results showed that the management, control, and treatment of most important arthropod-related diseases could be delayed due to covid-19 pandemic. conclusion: dealing with covid-19, it is crucial to consider the other main killers such as malaria, dengue fever, etc. more especially in vulnerable populations by greater political, financial and global commitment. continued surveillance will be essential to monitor for any possible changes. keywords: arthropoda-related diseases; covid-19; syndemics introduction arthropods are large phylum in the kingdom animalia and include most known animal species mainly belong to insects, spiders, mites, ticks, and scorpions. they may effect on human health by direct effects, such as bites/stings, allergic reactions, entomophobia, myiasis or indirectly by the transmission of some important diseases like malaria, leishmaniasis, dengue fever, yellow fever, and so on (1-2). they caused a lot of morbidity, mortality and economic losses, globally (2). to make a brief reference to the importance of vector borne diseases (vbds), it is suffice to look at the global statistics; malaria global cases in 2020 was reported as 241 million cases, and the estimated number of malaria deaths stood at 627000 (3). annual new cases of leishmaniasis estimated as 700000 to 1 million, out of them 50000 to 90000 cases belong to the visceral leishmaniasis, fatal form of disease (4). the number of dengue cases increased dramatically about eight fold over the last two decades and has been listed as a potential threat for 2020 and recent outbreaks in many countries confirmed this claim, with 100– 400 million infections annually (5). although copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:vmvaziri@gmail.comv mailto:v.vaziri@sbmu.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 28–35 v moin-vaziri and m badakhshan: the impact of … 29 http://jad.tums.ac.ir published online: march 31, 2023 these data help us to have a glimpse on the importance of vbds, but this is not the whole story, just a handful of examples. several factors could affect on vbds such as climate change, trade and travel (esp. global air travel and seaborne trade), human migration, socio-economic and ecological drivers (land use, farming practices, natural fauna, public health situation, human life style…), and as well political agenda. any other factor that changed these elements will affect the prevalence and incidence of vbds. recently, the covid-19 pandemic has led to significant loss of life and impact on other aspects of human life such as public health, food systems and the world of work, resulting in extreme poverty and poor public health (6-7). the purpose of this article is to emphasize on the impact of covid-19 on the most important arthropod problems based on available literatures. covid-19 and malaria malaria caused by different species of plasmodium and transmitted by anopheline mosquitoes, it is one of the highest contributors to morbidity and mortality in the world, with 627000 death in 2021 (3). the declining malaria burden over the past 20 years reflects major advances in disease control due to multidisciplinary interventions in the vector control, early diagnosis and anti-malarial therapies (8). this trend creates a promising outlook to eliminate malaria from asia pacific by 2030 and africa by 2050, a success which seems fragile and probably remains behind due to covid-19. as a prediction, the world will return to the level of mortality in 2000 (7, 9-10). also, it was stated that deaths due to malaria could increase over 5 years up to 36%, compared with no covid-19 pandemic (11). one of the risk factors for malaria expansion is travelling to/or from endemic areas (12) at first glance, travel restrictions due to the covid-19 quarantines could be regarded as a positive hint, but will be faded by some adverse impact of covid-19 on; 1) malaria vector control (irregularities in insecticide-treated nets distribution as well, larval and adult mosquito control measurements), 2) detection and treatment (interruption the health facilities due to the fear of exposure to covid-19, difficulty in transportation, need of additional resources to protect health care workers from covid-19, 3) economic collapse and health system failure, 4) population movement due to the economic crisis (11–. malaria control can be affected by other potential factors, such as the death of health care workers, as well by misdiagnosis of malaria cases in returning travelers in time of covid-19 pandemic (11, 13– 14), 16-17). practical recommendations for decision maker are available to keep the staff safe and to maintain main services at community level (18). continued and more concentrated epidemiological surveillance will be essential to better withstand challenges in malaria endemic area (19). covid-19 and leishmaniasis leishmania is an obligate intracellular pathogen that invades phagocytic host cells, and it was transmitted to vertebrate hosts by approximately 30 species of phlebotomine sand flies. leishmaniasis is among the neglected tropical disease (ntds), with three main clinical forms, visceral (the most serious form), cutaneous (the most common), and mucocutaneous. globally, about 90 countries are considered endemic for leishmaniasis (20) so it is very likely that leishmaniasis and covid-19 have a spatial and temporal overlaps. leishmaniasis is associated with poverty, lack of basic sanitation, malnutrition, population displacement, poor housing, weak immune system and lack of financial resources (4). as covid-19 triggered the individual and global vulnerabilities, it is well clear that it could have an enhancing effect on leishmaniasis burden as reported from brazil recently (21). in the fear of corona virus patients who were suspected to cutaneous form of disease may not refer to the physician in appropriate time that not only exacerbate the lesions but also effect on the transmission http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 28–35 v moin-vaziri and m badakhshan: the impact of … 30 http://jad.tums.ac.ir published online: march 31, 2023 cycle of disease, especially in the anthroponotic form which humans act as main reservoirs. on notice, in visceral form, as the fatal form of leishmaniasis, the situation is more complicated. although it does not share clinical features with covid-19, but because of distinct immunopathogenesis between diseases, vl patients showed vulnerability in their immune system against antiviral responses (22-23) so, endemic neglected diseases like vl should be more considered during covid-19 pandemic. covid-19 and arthropod-borne viruses the term “arboviruses” refers to the viruses that biologically transmitted to vertebrates by different species of arthropods mainly culicine mosquitoes and ixodid ticks (24). it is important to raise the global awareness on the possible impact of covid-19 on arboviral diseases. the most important arboviruses will be discussed following. covid-19 and dengue fever dengue is a major public health problem especially in tropical and sub-tropical regions; it is caused by four serotypes of dengue virus (denv) and transmitted by mosquitos, mainly by aedes aegypti. the global incidence of dengue has grown dramatically recently and about half of the world's population is now at risk. the number of dengue cases increased 8 fold over the last two decades, from 505,430 cases in 2000, to over 4.2 million in 2019. reported death cases between the year 2000 and 2015 increased from 960 to 4032 (5). in syndemic areas of dengue fever and covid-19 the problem increased for the health system(2526). similar clinical manifestations shared by covid-19 and dengue fever have raised concerns in two ways; at first by confusing the physicians in the correct diagnosis of disease, considering this fact that dengue is the leading cause of febrile illnesses in travelers who returns from south-east asia (25-27), secondly by potential false-positive results in the patients with pre-existing denv antibodies that could make a cross-reactivity between sarscov-2 and denv. although, the results of a preliminary study in italy on a small number of serum samples (32 samples) can downsize this concern as they stated that “the concern about false-positive dengue serology in covid-19 patients could be downsized, at least when an elisa is used” (28). by the way, in endemic areas of dengue, covid-19 act as a double punch for patients and for public health by further spread of the virus and the risk of health-workers collapsing (26, 29-35). on the other hand, in the lack of vaccine and specific treatment for dengue fever, the only intervention method that seems to work is vector control, (5) which accordingly might have decreased in the community and dengue prevention programs during covid-19 pandemic (36-37). since simultaneous infections will worsen the epidemiological situation, special attention should be done on the patients who live or had the history of travel to dengue endemic areas. it should be mentioned that the restriction on movement of people to curb covid-19 could decrease the cases in a period of time as reported in sri lanka (38). covid-19 and yellow fever yellow fever which mostly occurs in south america and sub-saharan africa, is an acute viral hemorrhagic disease caused by a mosquito-borne flaviviruses. the "yellow" in the name refers to the jaundice that affects some patients. yellow fever control started by early application of quarantine in 15th century and shifted to more coordinated approach through time, and today includes vaccine requirement at the entrance of the countries (39). there is currently no specific antiviral drug for yellow fever, but the eliminate yellow fever epidemics strategy (eyes) that launched in 2017, aimed to protected more than 1 billion people by 2026 (40). large-scale vaccination is the main arm of this struggle, and unfortunately easily could affected by covid-19 pandemhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 28–35 v moin-vaziri and m badakhshan: the impact of … 31 http://jad.tums.ac.ir published online: march 31, 2023 ic, especially in syndemic areas (41-42). although some restriction rules for covid-19, such as home quarantine, closing the borders, travel restriction and prohibition of social and religious events could mitigate yellow fever dispersion too, but the adverse effects of the current pandemic cannot be ignored. without a doubt, the main obstacle is the economic crisis of the countries that affect the health system and the routine diplomacy. regularly, risk of yellow fever in urban areas could be reduced by eliminating the potential mosquito breeding sites by applying larvicides in water storage containers and anywhere that standing water collect (40), which under the shadow of covid-19 will certainly be affected (43). covid-19 and zika virus zika virus is a flavivirus that firstly identified in 1947 in monkeys, the initial report of humans infection was in uganda and the united republic of tanzania in 1952 which continued by several epidemics in different countries in 2007, 2013 and 2015 (large outbreak in brazil). the main vector is day-biting mosquito aedes aegypti (44). so far, no vaccines or antiviral drugs are licensed for zika, so the only way left is 1) personal protection against mosquito bites during the day and early evening 2) using physical barriers 3) applying insect repellent to skin or clothing and finally 4) elimination mosquito breeding sites. the latter one requires community initiatives to support local government and public health programs to reduce mosquito breeding sites (44), which will certainly be affected by the problems caused by the covid-19 (45). covid-19 and pediclusis human pediclusis is defined as infestation to body lice (pediculus humanus) and head lice (pediculus capitis). pediclusis due to head lice still remains a public health problem, it is extremely contagious, it could be found in any age, but it is more common among young children aged 3 to 12 years (46). transmission had occurred commonly by close human-tohuman contact, through social and family relationships(46). two main strategies against pediculosis are individual control and prevention of its transmission (46). when who declared the covid-19 as a global pandemic in march 2020, the governments of different countries imposed the mandatory quarantine, social distance, and public lock down. these restriction rules forced the students, as the most vulnerable ones for pediculosis, to stay at home and have virtual education; as a result the close contact was interrupted and it can be concluded that it will have a reducing effect on the prevalence of p. capitis, the fact that was proved by a survey in argentina (46). covid-19 and scabies scabies is one of the earliest human diseases caused by sarcoptes scabiei and transmitted by direct skin‐to‐skin contact (47). regardless of socioeconomic status, it is a public health issue in all countries, but its prevalence would be enhanced with poverty, malnutrition, overcrowded conditions, and immune-compromised status (48-49). delay in refereeing to the physician can lead to delay in diagnosis and inadequate treatment which in turn can lead to further dispersion; a condition that occurs more frequently during covid-19 quarantine in comparison to the life before (50-51). conclusions dealing with covid-19, it is crucial to not ignore the other main killers such as malaria, dengue fever and so forth. more especially in vulnerable populations by greater political, financial, and global 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int j dermatol. 59(11): 1391–1392. 51. kutlu ö, güneş r, coerdt k, metin a, khachemoune a (2020) the effect of the “stay-athome” policy on requests for dermatology outpatient clinic visits after the covid-19 outbreak. dermatol ther. 33(4): e13581. http://jad.tums.ac.ir/ j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … original article the seasonal activity of ixodes ricinus tick in amol, mazandaran province, northern iran *nasrollah vahedi-noori ¹, sadegh rahbari ², saeed bokaei ³ ¹phd student, department of parasitology, faculty of specialized veterinary sciences, science and research branch, islamic azad university (iau), tehran, iran ²department of parasitology, faculty of veterinary science and research branch, islamic azad university, tehran, iran ³department of food hygiene, faculty of veterinary medicine, university of tehran, tehraniran (received 2 nov 2010; accepted 29 may 2011) abstract background: the present study aimed to demonstrate the seasonal activities of ixodes ricinus at the pasture level and on the host. methods: a vast pasture in amol countryside (mazandaran province, iran) which had the potential for a considerable number of cattle and sheep to graze was chosen. tick sampling from the skin of 130 cattle and 130 sheep were collected every month interval. simultaneously, the activity of the different stages of i. ricinus on the pasture was considered by dragging method. the collected ticks were placed in jars containing 70% alcohol and sent to the parasitological laboratory for identification. results: the rate of the infestation with adult i. ricinus in cattle and sheep increases gradually with the beginning of fall and reaches its peak in january, february and march while it starts to decline with the beginning of spring as the infestation rate reach to zero in summer months. accordingly, the highest number of adult i. ricinus existed on the cattle during january, february, and march. in addition, the results of dragging have been revealed that the active tick population in the pasture exists during november, december, january, and march. conclusion: ixodes ricinus is regarded a common tick species in amol (mazandaran). due to the biological properties of i. ricinus which is active in the cold and humid months of the year, the prevalence of ruminant infestations with i. ricinus in this area increases from november to march but reaches to zero again with the beginning of summer. keywords: ixodes ricinus, seasonal activity, iran introduction ixodid tick is one of bloodsucker arthropods belonging to acarina, which cause damage to the host skin and anemia in addition; ticks are also the most important vectors of diseases. in this regard, ixodes ricinus is the one of the important vectors of highly pathogenic agents affecting both humans and domestic animals worldwide (kettle 1995). recently, there has been an increase in the amount of attention paid to ticks as they are vectors for pathogenic agent such as: the spirochete borrelia burgdorferi etc. conse quently, numerous researchers in world-wide are engaged in studies on dispersal and seasonal activity of i. ricinus and on the extent of their infestation with b. burgdorferi (grey et al. 1992, talleklint et al. 1993, 1996, hubalek et al. 1998, nilison 1998, o'connell et al. 1998). there are many studies carried out so far related to the tick fauna in iran, a number of studies have been conducted in northern part of iran to investigate the tick diversity. razavi et al. (2006) showed that only 86% *corresponding author: dr nasrollah vahedi noori, e-mail: nsvahedi@yahoo.com 129 j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … of the 696 ticks infesting cows in amol area were i. ricinus, while in kelardasht area i. ricinus comprised 26.8% of the 798 collected hard ticks (yossefi et al. 2008). nabian et al. (2007) showed 2.32% of the 1720 collected ticks were i. ricinus. razmi et al. (2007) showed that 6.8% of the population of the collected ticks from mazandaran province included i. ricinus. the important point related to the studies so far conducted in northern part of iran is that the seasonal activities of tick fauna has not been perspicuous, although some works has pointed out the tick seasonal activity in western azerbaijan (rahbari et al. 1995, davudi et al. 2008, salari et al. 2008) but, it is still a gap in tick ecology. the present study aimed to demonstrate the seasonal activities of i. ricinus on the host and at the pasture level. materials and methods a vast pasture in amol countryside (mazandaran province, northern iran) which had the potential for a considerable number of cattle and sheep to graze was chosen. tick sampling from the skin of 130 cattle and 130 sheep were collected in every month interval from october 2009 to september 2010. simultaneously, an area of 250 m2 of the pasture randomly selected for dragging and the activity of the different stages of i. ricinus on the pasture was considered. the collected ticks were placed in jars containing 70% alcohol and were then sent to the laboratory. tick identification was done according to the morphological structures mentioned by estradapena et al. (2004). climatological data were also collected from central station of amol. results in this study, six species of hard ticks (ixodid ticks) have been identified including i. ricinus, boophilus annulatus, rhipicephalus bursa, rh. turanicus, haemaphysalis concinana, hyalloma deteritium and i. ricinus were allocated 42% of total ticks. monthly inspection of target animals for i. ricinus tick revealed that the rate of infestation in cattle with adult i. ricinus increased gradually with the beginning of fall and reached its peak in january, february, and march while it started to decline with the beginning of spring as the infestation rate reached to zero in summer. (fig. 1). accordingly, the highest number of adult i. ricinus existed on the cattle during winter (table 1). the observations showed that the occurrence of infestation with adult i. ricinus in sheep starts in november and continues to june. in this process, the percentage rate of infestation with adult i. ricinus in sheep increases gradually in fall and reaches its peak in january, february, and march. however, it declines with the beginning of spring in a way that no tick was picked up from sheep in the target area in july, august, and september (fig. 2), while the greatest average number of adult i. ricinus tick on each animal was found during january, february, and march (table 1). in addition, the tick intensity on cow and sheep was 13.81 and 6.45 respectively (table 1). simultaneous with tick collection from the body of the animals in the target area, the activity of the different stages of i. ricinus on the pasture was carried out by dragging method. the tick population dynamic on the pasture was present during november, december, january, and march while the active population of larvae, nymph, and adult i. ricinus ticks in the area begins in november with a rising trend and reaches its population peak in february. the population balance of the ticks in the pasture was accompanied by increase in nymph, larvae, and adult stages of ticks respectively. however, the population of i. ricinus ticks on the pasture had a different pattern of larva, nymph, and adult intensity in december (fig. 3). 130 j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … table 1. the tick intensity of ixodes ricinus on host and on 10 m2 of the pasture in different seasons season and annual tick intensity on pasture tick intensity on hosts larva nymph adult cattle sheep fall 4 2.3 1.7 6.08 3.4 winter 6 3.3 1.3 34.4 13.85 spring 0 0 0 14.76 8.57 summer 0 0 0 0 0 annual 2.5 1.4 0.75 13.81 6.45 fig. 1. the seasonal activity of adult ixodes ricinus on cattle (october 2009 to september 2010) fig. 2. the seasonal activity of adult ixodes ricinus on sheep (october 2009 to september 2010) 131 j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … fig. 3. tick intensity of ixodes ricinus on 10 m2 of the pastrue (october 2009 to september 2010) discussion the studies which dealing with hard ticks in iran so far, indicate that i. ricinus tick is only found in the northern provinces of iran (razavi et al. 2006, rahbari et al. 2007, razmi et al. 2007, yossefi et al. 2008). i. ricinus is hence regarded a common tick species in amol. this tick is present as the most common tick in all zoogeographical regions of the world, which have a humid climate, and especially in cold and humid weather (kott et al. 2004). there is a wide range of hosts for this tick, which mostly feeds on cattle, sheep, horse, deer, wild rabbit, other small mammals, and birds. since this tick is very sensitive to aridity, its behavior on plants in influenced by its need to maintain and absorb humidity from the environment (kettle 1995). although rahbari (1995) showed that the largest population of active hard ticks is found on the body of ruminants in western azerbaijan province in spring and summer, it has the lowest frequency in fall. salim abadi et al. (2008) identified that the largest population of ixodid tick exist in yazd province in summer, the results of this research indicate that the largest number of adult ixodid ticks on each cow or sheep is found in january, february and march and no adult tick was separated from the body of the ruminants in july, august and september (fig. 1 and 2). therefore, we can infer from these results that the presence of adult ticks is more dependent on climatic conditions and its biological properties. these results are the same as those, which was introduced by tuncer et al. (2004). they determined the seasonal activity of i. ricinus in turkey, started in december and last until the may of the next year. also in another work in america, mackay and foil (2005) found that the activity of the adult i. scapularis started in november and last until next may. on the other hand the researches conducted by miroslawa (2004) in poland, foldvari et al. (2007) in hungary and hrklova et al. (2008) in slovak, demonstrated two peaks of activity for the adult of i. ricinus, in spring and in summer. according to these authors, the 132 j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … timing of peaks depends on many factors, such as temperature and humidity. the results of the dragging in the amol area conformed to the results of tick intensity of animal to some extent and showed that the activity of adult i. tick started in november and continues until february (fig. 3). the relative humidity was slow down from may to september, which was less that 80%. hence, adult tick was observed neither in pasture nor on the body of the animals especially after july (fig. 1 and 2). the rate of infestation with adult i. ricinus in cattle and sheep has been increased gradually during fall and reached its peak (75% in cattle and 80% in sheep) in january, february, and may. however, it started to decline with the beginning of spring and reached zero in summer times. in western azerbaijan, the rate of the infestation with hard ticks was 55%, 57% and 62% in sheep, goat and cattle respectively rahbari (1995). the infestation rate with i. ricinus in target animals increased with the decrease in temperature and rise in humidity but decreased with the rise in temperature and decrease in humidity hence, it can be concluded that the activity of i. ricinus was highly dependent to the cold and humid months of the year. the results of dragging on the pasture showed that the activity of the nymph stage of i. ricinus tick started in november and last until february, which showed a rising trend. this activity stopped in spring and summer (fig. 3). in denmark through dragging method, it was found that the nymphal stage of i. ricinus has a dramatic decline in its activity from may to september but slightly increased in september (kalsbeek and frandsen 1996). in ireland, the nymphal stage of i. ricinus activity started in march and reached to zero after a slight increase. then, it indicated a limited rise in activity from august to november (gray 1980). their activities showed bimodal but the comparison between these results with those achieved in amol, revealed that the main reason for this difference could be the different geographical and climatic conditions of the two regions. the results of dragging on the pasture show that the active population of i. ricinus in different stages in the target area has a maximum activity curve during the year (fig. 3). in other words, it has a unimodel activity. the larval stage of i. ricinus has been activated during a period from november until february, this activity stopped in spring and summer, which seems to be due to the biological properties of i. ricinus whose activity decreases and even stopped in the hot and dry months of the year. gray (1985) determined the activity of the larval stage of i. ricinus in ireland showed bimodal activity. the comparison of our result and those achieved in ireland showed a difference in the monthly activity period. it can be concluded that the activity of i. ricinus in mazandaran is limited to the cold seasons of the year, this condition caused enough coldness, and humidity (over 80%) for tick host finding in fall and winter, therefore, the wide and extensive occurrence of i. ricinus could be observed on animals. acknowledgements the authors would like to appreciate to the network amol veterinary experts and specialized veterinary faculty researchshahriar unit. the authors declare that there is no conflict of interests. references davudi j, hoghooghi rad n, golzar abdi sh (2008) ixodid tick species infesting cow and their seasonality in west azerbaijan res. j parasitol. 3: 98–103. estradapena a, bouattour a, camicas jl, walker ar (2004) tick of domestic animals in mediterranean region. a 133 j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … guide to identification of species. 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vatandoost h, chinikar s, oshaghi ma, moradi m, mirabzadeh ardakan e, hekmat s, nasiri a (2010) hard tick on domestic ruminants and their seasonal population dynamics in yazd province, iran. iran j arthropod-borne dis. 4(1): 66–71. 134 j arthropod-borne dis, 2012, 6(2): 129–135 n vahedi noori et al.: the seasonal activity of … talleklint l, jaenson tgt, mather t (1993) seasonal variation in the capacity of the bank vole to infected larvae ticks with the lyme disease spirochete, borrelia burgdorferi. j med entomol. 30: 812– 815. talleklint l, jaenson tgt (1966) relationships between i. ricinus density and the prevalence of infection with borrelia like spirochetes and density of infected ticks. j med entomol. 33: 805–811. tuncer d, mutlu g, karaer z, sayin f (2004) seasonal occurrence of ticks on and borrelia burgdorferi influence in i. ricinus in antalya region. acta parazittologica turcica. 28(3): 158–160. yossefi mr, keighobadi m, asnaashari my (2008) ixodid tick species infestating sheep and cattle in kelardasht part (chaloos) iran. j entomol. 5(1): 56–58. 135 j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 60 http://jad.tums.ac.ir published online: march 31, 2021 original article the effect of different doses of mesobuthus eupeus (scorpionida: buthidae) scorpion venom on the production of liver necrosis in nmri mice sara mojdegani-fard 1 ; *sohrab imani 1,2 ; mahmoud shojaei 1 1 department of entomology, science and research branch, islamic azad university, tehran, iran 2 department of parasitology and toxicology, school of medicine, semnan university of medical science, semnan, iran *corresponding authors: dr sohrab imani, e-mail: imanisohrab@gmail.com (received 27 nov 2015; accepted 22 jan 2017) abstract background: scorpion venom has a variety of different components considerably. some of these compounds are proteins such as phospholipase a2 which is one of the most important. use of scorpion venom for the treatment of any disease requires an initial study to determine the therapeutic dose or safe dose. therefore, due to the necessity of studying scorpion venom, it is of special importance to study the effects of its dose response in animal tissues. methods: to determine the inflammatory effects of scorpion’s venom (mesobuthus eupeus), 50 nmri mice with an average weight of 24±7g were selected for investigation in two experiments. in first-round 25 of them were divided into 5 groups and were exposed to different doses of venom injection paralleling the control group. single-injection of various doses on 25 mice was performed and results were compared. results: there was a significant differences between the test and control groups (in most groups). liver necrosis was one of the important symptoms in this study, the severity of which was measured and statistically analyzed. conclusion: it was determined that 0.05ppm is a safe dose and sub-lethal doses can use for the investigation of therapeutic effects of venom on cancer, diabetes, dermatitis, and so on. keywords: dose-response; liver necrosis; nmri mice; scorpion’s venom introduction animal products’ (such as poison, enzyme and so on) usage for treating some diseases have a good background in history, and in recent years these components have been drow more attention, and several studies have been done worldwide. in various studies, some researches have been investigated on the effects of bees and scorpions' venom on insulin secretion and blood glucose in wistar male rats. melittin polypeptidethe main component in bee venomhas been stimulated insulin secretion from langerhans islands in nmri mice in laboratory conditions. melittin is recommended as a valuable factor for further studies on β-cell’s plasma membrane which has a role in insulin secretion regulation (1-3). in insulin-resistant mice, direct activation of phospholipase a2 by melittin caused insulin output from langerhans islands. phospholipase a2 activation has a role in the compensation for insulin resistance in langerhans islands (4). additionally, treatment with honey bee phospholipase a2 causes the release of insulin from cells and insulin secretion to blood (5). anticancer effects of the compounds of mesobuthus marthensis scorpion’s venom were evaluated in iran. it has been shown that venom can inhibit cancer cell growth significantly and induces cell death. it also prevented glioma cell growth by inhibition of their ion canals (6). these animal’s venom substances can act as potential anti-tumor agents. scorpions with a 300 million years history are one of the oldest animals on the earth and the oldest fossils copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. mailto:imanisohrab@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 61 http://jad.tums.ac.ir published online: march 31, 2021 belong to them as arachnids and arthropods. it is proven by the existence of some basic anatomical features such as single nerve chain nodes. in some cultures and human civilization, there are some documents for using natural toxin for some diseases treatment with the special rank for scorpion’s venom (7). mesobuthus eupeus from the family buthidae is known as a spotted yellow scorpion. water-soluble compounds of scorpion’s venom with ph from neutral to alkaline generally made of mucus, oligopeptides, nucleotides, amino acids, and other organic compounds also from some enzymes such as phospholipase, hyaluronidase and some molecules with relatively low molecular weight like serotonin, histamine, protease inhibitors and histamine releasers (8). high molecular weight compounds such as hyaluronidase and phospholipase a2 can stimulate immune responses. in this study, the effects of different sublethal doses of m. eupeus venom was used to check for necrotic responses and other effects on nmri hepatic cells. materials and methods scorpion collection was conducted using black light at night time from july to october 2015 in the suburbs of omidiyeh and aghajari cities in khuzestan province, iran. out of about 1500 collected scorpions, 362 m. eupeus scorpions have been identified. this scorpion with a yellow body and a size of about 6cm with 3–5 rows of dark longitudinal spots in the dorsal region of the pre-abdomen is called the spotted yellow scorpion. venom extracting was performed by an electroshock device by electrodes attaching to the scorpion’s body and venom gland. a weak electrical current has passed through the scor-pion’s body in a short time causing shock. as a result, it leads to the excretion of venom, which was collected by holding a micro-tube at the end of the sting. usually, the voltages used were 6 to 7.5 volts, depending on the size of the scorpion. after extracting, the venom was dried using a freeze-dryer and stored in the refrigerator at 2–4 °c (9). in this study, a total of 50 non-infected adult male nmri mice weighing 24±7g were randomly divided into 10 experimental groups including treatment and control groups under different doses of venom. they were kept in animal house of the islamic azad university of tehran, science and research unit under the following conditions: 12/12h daylight/ darkness, temperature around 22±2 °c, adequate humidity and freely used the usual dry diet with water (9, 10). the research was conducted in two stages of experimentation as follows: the first stage: studying the effects of m. eupeus scorpion venom: during this stage, injections were performed 6 times in the groin and each time at intervals of one day, and the mice were anesthetized with diethyl ether and dissected one day after the last injection, and the liver and some other organs were studied. 1) control group: used the usual dry diet with water and healthy mice received sterile distilled water by injection equivalent to the volume of injected venom. sterile distilled water was also used to dissolve the venom. the treatment groups were received different doses as follows: 2) the first group received a dose of 0.5ppm 3) the second group received a dose of 0.2ppm 4) the third group received a dose of 0.1ppm 5) the fourth group received a dose of 0.05ppm the second stage: investigation of the effects of m. eupeus scorpion venom: at this stage, the mice were injected at a set dose at one stage, and the next day they were dissected and their livers were removed. at this stage, the same number of mice with the same grouping was considered. 1) control group: operated by the method of the previous control group. 2) the first group received a dose of 1ppm 3) the second group received a dose of 0.4ppm 4) the third group received a dose of 0.2ppm 5) the fourth group received a dose of 0.1ppm j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 62 http://jad.tums.ac.ir published online: march 31, 2021 after removal of the organs, fixing, and tissue preparation, staining was performed by eosin-harris hematoxylin staining method for liver tissue. the slides prepared in this way were examined by light microscope with 40x magnification and necrosis spots were identified in them. the area and size of the spots and their frequency in 100 fields of view were measured by an image analyzer and leica qwin software. results at the first round of experiment by four sublethal doses, we observed that three of them had visible symptoms associated with liver necrosis except for the 0.05ppm (table 1). at the highest dose, in addition to being more frequent, it showed a completely different pattern in terms of color intensity with other samples. table 2 shows the frequency of stains in terms of necrosis severity. in this case, the color intensity of the stains is considered and according to the quality of the stains, which were sometimes very dark and sometimes very light, they are divided into three categories: mild, moderate, and severe. table 2 shows the number of color intense spot and severe necrosis observed in each microscopic field by 40x magnitude. as the concentration increased, the surface area of the spots initially increased, but was not an absolute trend, so that at concentrations of 0.5 to 1ppm, the surface area of the spots did not increase, but their intensity increased in terms of turbidity (fig. 2). also the tables and diagrams show that at the highest concentration, severe spots were not seen, but the frequency of spots in this concentration was much higher than other concentrations and statistically, the difference was significant. there is a 99% probability with other concentrations (figs. 3–6). in the second period of experiments, as shown in table 3, signs of liver necrosis were observed in all doses. as observed in the experiments of the first stage, an increase in the size of the spots was seen with an increase in dose, but this was not an absolute trend. also, in figure 3, the frequency of data is given so that at higher doses the size of the spots is smaller but their number and amount of staining is increased, as shown in table 4, at the dose of 1ppm the highest number of severe spots were seen and figure 4 is comparing the number of color intensity of necrotic stains in sub-lethal dose of mesobuthus eupeus venom on nmri liver tissue in the first round of test as shown in figure 1, no signs of necrosis were observed in the liver tissue sample of the control group, but in other images in figure 2, in the liver tissue of mice receiving sub-lethal doses (0.1, 0.2, 0.4, 0.5 and 1ppm), necrosis is observed with a change in the size of the spots along with a change in the intensity of their staining. in images a and b, the spot level was larger, and in images c and d, the area was smaller and the color intensity was different. fig. 1. liver image of healthy mice receiving sterile distilled water equivalent to the volume of the injected toxin j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 63 http://jad.tums.ac.ir published online: march 31, 2021 table 1. average area of necrotic stains in sub-lethal doses of mesobuthus eupeus venom on nmri hepatic tissue in the first round the intensity of the color of stains dose (ppm)/ number of necrotic stain control 0.5 0.2 0.1 0.05 0 118.924 29.41 26.185 0 table 2. number of color intense spot and severe necrosis in sub-lethal doses of mesobuthus eupeus venom on nmri hepatic tissue in the first round the intensity of the color of stains dose (ppm)/ number of necrotic stain control 0.5 0.2 0.1 0.05 mild 0 7 2 2 0 moderate 0 23 2 0 0 severe 0 0 0 0 0 table 3. average area of necrotic stains in sub-lethal doses of mesobuthus eupeus venom on nmri hepatic tissue in the second period the intensity of the color of stains dose (ppm)/ number of necrotic stain control 1 0.4 0.2 0.1 0 75.518 93.781 60.738 31.176 fig. 2. symptoms of liver necrosis due to effect of sub-lethal dose of scorpion venom of mesobuthus eupeus changes in the images of a–d represent the different intensity of staining j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 64 http://jad.tums.ac.ir published online: march 31, 2021 table 4. number of color intense spot and severe necrosis in sub-lethal doses of mesobuthus eupeus venom on nmri hepatic tissue in the second period the intensity of the color of stains dose (ppm)/ number of necrotic stain control 1 0.4 0.2 0.1 mild 0 1 0 1 0 moderate 0 4 2 3 3 severe 0 26 4 1 0 fig. 3. comparing the average area (μm2) of necrotic stains in sub-lethal doses (ppm) of mesobuthus eupeus venom on nmri liver tissue in the first round fig. 4. comparing the number of color intensity of necrotic stains in sub-lethal dose (ppm) of mesobuthus eupeus venom on nmri liver tissue in the first round of test j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 65 http://jad.tums.ac.ir published online: march 31, 2021 fig. 5. comparing the average area (μm2) of necrotic spots in sub-lethal doses (ppm) of mesobuthus eupeus venom on nmri liver tissue in the second round fig. 6. comparing the number of color intensity of necrotic stains in sub-lethal doses (ppm) of mesobuthus eupeus venom on nmri liver tissue in the second round of test j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 66 http://jad.tums.ac.ir published online: march 31, 2021 discussion phospholipase a2 is an important cause of inflammation in tissues (5). during 2005, some researcher used sub lethal doses of cobra venom and examined its effect on normal tissues as well as cancerous tissues such as skin, liver, kidney and heart tissue in animal models (rabbit), the results showed an increase in the synthesis of nucleic acids and a concentration of 25μg/ml was introduced as a therapeutic dose that prevents the increase in the production of nucleic acids and can be used in the treatment of cancerous tissues (10). in some studies, the inhibitory effect of fraction 3 anticancer peptide of the scorpion venom of botus martensi crush on liver cancer cells and its mechanism of action at different concentrations and at different times were investigated. they found that fraction 3 of the anti-cancer peptide of scorpion venom induces apoptosis endothelial in a dose-dependent manner. after 12 hours of treatment with different concentrations of 5, 10, 50, 100 and 200mg/l of scorpion venom, the rates of apoptosis were reported 6.1±3.0%, 15.3±4.9%, 48.5±5.2%, 66.7±6.5% and 91.2±6.9%, respectively. the results showed that fraction 3 of the anti-cancer peptide of scorpion venom was able to inhibit human liver cancer cells by inducing g2 apoptosis (11) in addition, other studies investigated the effect of the sv-1 fraction of scorpion venom on the growth of skov3 ovarian cancer cells. the results showed that sv-1 could significantly inhibit the growth of skov3 ovarian cancer cells at concentrations of 200, 400 and 800mg/l. inhibition rates were reported at 29.87%, 48.11% and 67.77%, respectively. therefore, they suggested that sv-1 could inhibit the proliferation and growth of skov3 ovarian cancer cells and that changes in the cell cycle and apoptosis may be important mechanisms of sv-1 inhibitory effects (12). necrosis, apoptosis, nuclear caryolysis, shrinkage of the cytoplasm, increase in cytoplasmic vacuoles and mitochondrial destruc tion are the results of the effects of animal venom that may be seen in various tissues (13). in cuba, the native scorpion species rhopalurus junceus has been used in traditional medicine to treat cancer. the researchers found a range of scorpion venom concentrations (0.1, 0.25, 0.5, 0.75 and 1mg/ml) in a panel of human tumor epithelial cell lines (hela, siha, hep2, nci h292, a549, mda-mb231, mdamb-468, ht-29) used hematopoietic cells and normal cells and compared the results. only epithelial cancer cells showed a significant reduction in viability (ic50) of 0.6–1mg/ml and showed no effect on normal cells and hematopoietic cells. hela cells also showed little effect on apoptosis. among tumor cell types, a549 was the most sensitive. necrosis caused by scorpion venom with acridine orange/ ethidium bromide fluorescent dye showed decreased expression of apoptosis-related genes. they concluded that the scorpion's venom has selective toxicity against epithelial cancer cells. in general, we should know that scorpion venom fractions act selectively, and those compounds that inhibit the progression and proliferation of cancer cells by inducing apoptosis have no effect on normal cells. therefore, it does not pose a risk to the person because it only targets cancer cells (15). necrosis studies agree the data of this study according to some others study (10, 12, 14). in this study, the number of spots did not show a significant increase with increasing dose from 0 to 0.4ppm, while increasing the number of necrosis spots increased from 0.5ppm to 1. the area of the spots increased with changes in dose from 0 to 0.5ppm (figs. 3, 4) and did not show a significant change in the dose range of 0.5 to 1ppm (figs. 5, 6). the intensity of necrosis spots in the dose range of 0 to 0.4ppm did not show a significant increase, while with a dose increase of 0.5 to 1ppm, a significant increase was observed. j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 67 http://jad.tums.ac.ir published online: march 31, 2021 conclusion in the current study, the 0.05ppm of scorpion’s venom of the doses used is a threshold dose for the appearance of symptoms. signs began from the above dose and no other symptoms were noticed seen in the lower doses. in the field of scorpion’s venom dose-response and its impacts on nucleic acid’s synthesis further studies are strongly recommended for more explanation. acknowledgements the authors would like to highly appreciate dr h sameni, faculty of medical sciences for cooperation to the observation of microscopic slides and provide useful comments. this study was a part of phd project of sara mojdegani-fard at department of entomology, science and research branch, islamic azad university, tehran, iran. the authors declare that there is no conflict of interests. references 1. xie w, xing d, zhao y, su h, meng z, chen y, du l (2005) a new tactic to treat postprandial hyper-lipidemia in di-abetic rats with gastro paresis by improving gastrointestinal transit. eur j pharmacol. 7: 113– 120. 2. moosavi sm, imani s, haghighi s, mousavi se, karimi a (2012) effect of ira-nian honey bee (apis mellifera) venom on blood glucose and insulin in diabetic rats. j arthropod borne dis. 6(2): 136–143. 3. roodbari l (2012) effect of anderoctonus crassicauda venom in the treatment of type 1 diabetes in rats. 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[msc dissertation]. tehran islamic azad university, science and research, tehran, iran (in persian). 9. kadkhodayi alyadorani m, amouzgari z, hanifi h (2007) phospholipase and hyaloronidase activity in mesobuthus eupeus venom. feyz. 4: 24–30. 10. roghani m, bagheri a, asyabi m, hodavandkhani a (2004) effect of the aqueous extract of coriander leaves in diabetic mice by stz. iran j endocrinol metab. 4: 259–255. 11. shaikh dm, jokhio p (2005) effect of snake venom on nucleic acid and total proteins in various normal and cancerous animal tissues. pak j physiol. 1: 1–2. 12. jian-wei l, jing h, gui-rong z, zheng-ren w (2006) effects of anti-cancer peptide fraction 3 from buthus martensii karsch on apoptosis of human liver cancer cells. j jilin univ med ed. 32: 625–628. 13. shi-bo f, ying y, feng-qin y, shu-chun l, peng-wu l, gui-zhi j (2007) inhibitory effects of fraction from scorpion venom on growth of skov3 ovarian carcinoma cells. j jilin univ med ed. 33: 41–43. 14. shahramyar z, zaremirakabad a, morovat j arthropod-borne dis, march 2021, 15(1): 60–68 s mojdegani-fard et al.: the effect of … 68 http://jad.tums.ac.ir published online: march 31, 2021 h, lotfi m, nouri aq (2011) induction of apoptosis in human leukemia cell line (hl60) by animal´s venom derived peptides (icd-85). the first regional congress on venomous animals and toxin, 2011 december 1–3, razi vaccine and serum research institute, karaj, iran, pp. 174–175. 15. garcía ad, díaz lm, herrera yf, sánchez hr, lorenzo yc, llanes dm, garlobo yr, castro jf (2013) in vitro anticancer effect of venom from cuban scorpion rhopalurus junceus against a panel of human cancer cell lines. j venom res. 4: 5– 12. insecticide resistance in the west nile encephalitis, japanese encephalitis, avian malaria and lymphatic elephantiasis vectors, culex pipiens complex (diptera: culicidae) in iran http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... review article insecticide resistance in the west nile encephalitis, japanese encephalitis, avian malaria and lymphatic elephantiasis vector, culex pipiens complex (diptera: culicidae) in iran amrollah azarm1, mohammad nasrabadi1, fatemeh shahidi1, awat dehghan1, fateme nikpoor1, alireza zahraie-ramazani1, seyede maryam molaeezadeh1, faramarz bozorgomid1, ghazal tashakori1, *hassan vatandoost1,2 (received 23 june 2021; accepted 22 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: culex pipiens complex is considered as a vector of some important diseases such as west nile fever, equine encephalitis, rift valley fever, st. louis encephalitis, elephanthiasis and avian malaria in the world. the main measure for vector control is using insecticides. high use of insecticides caused resistance in the populations. the aim of this study was to review the status of insecticide resistance in the vector. methods: insecticide resistance in this species was found by the available papers and map of the data for carbamates, organochlorine, organophosphates, pyrethroids, microbial and insect growth regulator insecticides were done. an intensive search of scientific literature was done in “pubmed”, “web of knowledge”, “scopus”, “google scholar”, “sid”, and related resources. results: results showed that a wide variety of resistance to different insecticides in the country. due to importance of this species in transmission of diseases. discussion: resistance management strategies should be further considered to prevent from in secticide resistance and replacement of novel approach for vector control. keywords: insecticide; resistance; vector; culex pipiens complex introduction mosquitoes are the most important vectors of pathogens in the world (1). the culex pipiens complex from the culicinae subfamily includes the subspecies cx. pipiens pipiens, cx. p. pipiens form molestus, cx. p. quinquefasciatus, cx. p. ausralicus, cx. p. globocoxitus and cx. p. pallens (2). in recent studies, the species cx. pipiens and cx. quinquefasciatus have been identified as two separate species by molecular methods (3,4). the global distribution of this species is shown in fig.1. cx. pipiens in iran has two biological forms of cx. pipiens pipiens and cx. pipiens molestus. two subspecies are almost semi-domesticated and are found in most subtropical regions (4). the biological form of cx. pipiens form pipiens is mainly found in rural areas, is very interested in the blood of the host bird, preferably lays eggs in stagnant water and outside human habitation, has winter diapause and is non-autogenous. the biological form of cx. pipiens form *corresponding author: dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 350 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... molestus has autogenic behavior and lacks winter diapause (3). in addition to causing allergies and disturbances, the cx. pipiens complex transmit the highest number of pathogens compared to other vectors (4). cx. pipiens transmit several diseases such as west nile fever (wnf), equine encephalitis (ee) , rift valley fever (rfv), st. louis encephalitis (slev) , some worm pathogens such as dirofilaria immitis and wuchereria bancrofti, as well as some avian malaria protozoa such as plasmodium relictum and plasmodium gallinaceum (5) (fig.2). in recent years, cx. pipiens has been accused of transmitting the zika virus in some african countries and the united states (6,7). the preference for taking blood from birds, which are mainly hosts of disease agents and sometimes arbovirus, has led to a widespread global distribution of zoonotic diseases. the distribution and distribution of cx. pipiens is also very effective on the process of economic development resulting from the tourism industry. the increase in abundance and morphological and physiological characteristics in cx. pipiens has caused that after malaria vectors, more attention should be paid to the control of this species (7,8). the best and most standard method of the who to control vector-borne diseases is to use vector control methods (9-12). vector control is performed by one of the biological, physical, and chemical methods. the fast effect in a short time and easy access to chemical compounds have made most of the methods of control of mosquitoes depend on these compounds. chemical control may be carried out at the larval stage as a larvicide or in the adult stage by various methods such as residual spraying, spatial spraying, and insecticide-impregnated mosquito nets. one of the oldest methods of chemical control of culex mosquitoes in iran is the use of oily compounds on the surface of the water, which causes their death by covering the breathing siphon in the larva stage. after that, the use of mineral compounds became common for controlling measures. among the chemical compounds, four groups of organochlorine, organophosphates, carba mates, and pyrethroids are in the first place. organochlorine insecticides were first introduced to the world in 1939 by paul muller. organochlorine insecticides with disruption of the vital enzyme ca mg atpase and imbalance in the axonal transmission system cause repeated and sequential irritation, convulsive movements, paralysis, and eventually insect death. organophosphate insecticides are esters of phosphoric acid. the toxic properties of these compounds were first discovered in 1937 by schrader. organophosphate insecticides are the most diverse and widely used toxins fig. 1. global distribution of the culex pipiens complex mosquitoes (google scholar) (6). fig. 1. global distribution of the culex pipiens complex mosquitoes (google scholar) (6). 351 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... in agriculture and public health. the rapid effect, absence of hazardous residues, and lack of cumulative property of common point are important. organophosphate insec ticides disrupt the nerve function of insects and kill them by inactivating the enzyme acetylcholinesterase and accumulating acet ylcholine at the synapse site. pyrethroid insecticides are of plant origin and their insecticidal properties were first discovered in iran in 1763 from the flowers of the pyrethrum plant. insecticidal properties of pyrethroids are derived from the keto alcoholic esters of chrysanthemum fig. 2. global distribution of diseases transmitted by cx.pipiens complex(google scholar) chemical control of culex pipiens fig. 2. global distribution of diseases transmitted by cx.pipiens complex(google scholar) 352 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... and pyrethroid acids. the core of this group of insecticides is cyclopropane carboxylate. rapid decomposition, lack of hazardous residues, and lack of cumulative properties are characteristic of pyrethroid compounds pyrethroid insecticides affect the sodium channels and lead to the paralysis of the insect. in many parts of the world, pyrethroid insecticides are used in the form of insecticide-impregnated mosquito nets and indoor spraying to control adult mosquitoes. carbamate insecticides were first identified in 1940 by grysin. the main insecticide carbamate is physostigmine, which is extracted from the extract of physostigma venenosum. carbamate insecticides are organic compounds of carbamic acid derivatives (h2ncooh) whose functional group is carbamate esters. carbamate insecticides kill insects by reversibly inactiv ating the acetylcholinesterase enzyme. the most widely used carbamate insecticide used in health programs is carbaryl. the use of chemical pesticides was very effective and useful at first, but gradually and with increasing consumption, it caused environmental hazards, harmful effects on human health, and, most importantly, resistance in culex mosquitoes and other vectors. this phenomenon creates staggering costs in the countries involved. major countries fighting the culex mosquito are also infected with malaria, and various species of anopheles mosquitoes are found in these countries. close ecological and biological characteristics such as larval habitat have made culex mosquitoes resistant to most of the insecticides used against malaria vectors. the phenomenon of resistance is the most important problem in the fight against mosquitoes and other vectors and makes it more difficult every day to fight the pathogens transmitted by vectors. according to the world health organization, the phenomenon of insecticide resistance is an inherited or acquired trait that allows an insect to tolerate different doses of one insecticide or a combination of several insecticides. according to the standard of the who, when the insect mortality rate is less than 90%, the so-called resistant species, if the mortality rate is between 90% and 97%, the species is tolerant, and if the mortality rate is 98% and more be, the species is sensitive (1326). materials and methods data collection in this study, the data were collected from internet sources, contacts with organizations and institutes and reports of the world health organization (who). in online sources, we searched published articles on cx. pipiens resistance and iran in pubmed, scopus, isi, literature retrieval system (afpmb), iranmedex, google scholar, and related resources. using the keywords table 1. the resistance of culex pipiens against organochlorine insecticides in different regions of iran resistance status kt50 (min) lt50 (min) pesticides province year reference resistant ddt 4% tehran city 2015 salim-abadi et al. resistant resistant resistant ddt 4% dieldrin 4% dieldrin 0.4% mazandaran (sari county) 2018 ghorbani et al. resistant 139 ddt 4% kerman (rafsanjan county ) 2017 salim-abadi et al. 1567 240 ddt 4% tehran (southern) 2019 rahimi et al. resistant ddt 4% tehran(varamin county) 2004 vatandoost et al. resistant ddt 4% west azerbaijan 2015 naseri et al. resistant 31.93 ddt 4% siatan & baluchistan (chabahar) 2014 fathian et al. resistant 78.39 ddt 4% tehran(qarchak) 2017 zeidabadi et al. resistant 134.75 ddt 4% east azarbaijan 2015 ataie et al. table 1. the resistance of culex pipiens against organochlorine insecticides in different regions of iran 353 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... resistance, organophosphate, pyrethroid, organochlorine, carbamate, igr, and p450, kdr, monooxygenase, glutathione, or esterase. main data were collected and classified then analyzed using the software. results and discussion in iran, various insecticides have been used against cx. pipiens. according to standard studies, the cx. pipiens has are resistant to some of organochlorine insecticides such as ddt 4% in the cities of varamin, tehran, qarchak, islamshahr, chabahar, tabriz, urmia, sari, and rafsanjan and against the insecticide dieldrin 4% in the city of sari has shown resistance (table 1) (figure 2). among organophosphate insecticides cx. pipiens was susceptible to malathion 5% in chabahar sari and rafsanjan and tolerated in azerbaijan and varamin. cx. pipiens is also resistant to fenitrothion 1% in sari and temephos in tehran and sari (table 2, fig. 3). fig. 3. the resistance of culex pipiens against organochlorine insecticides in different regions of iran fig. 3. the resistance of culex pipiens against organochlorine insecticides in different regions of iran table 2. the resistance of culex pipiens against organophosphate insecticides in different regions of iran resistance status kt50 (min) lt50 (min) pesticides province year reference resistant resistant resistant resistant malathion 5% fenitrothion 1%, malathion (1ppm) temephos (0.02) mazandaran (sari) 2018 ghorbani et al. resistant 33 malathion 5% kerman (rafsanjan) 2017 salim-abadi et al. 74.5 29.7 malathion 5% tehran (southern) 2019 rahimi et al. tolerant malathion 5% tehran (varamin) 2004 vatandoost et al. susceptible malathion 5% siatan &baluchistan 2014 fathian et al. resistant lc50: 0.18 temephos tehran 2016 abai et al. tolerant 8.02 malathion 5% east azarbaijan 2015 ataie et al. table 2. the resistance of culex pipiens against organophosphate insecticides in different regions of iran 354 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... figure 4. the resistance of culex pipiens against organophosphate insecticides in different regions of iran fig. 4. the resistance of culex pipiens against organophosphate insecticides in different regions of iran table3. the resistance of culex pipiens against carbamate insecticides in different regions of iran resistance status kt50 (min) lt50 (min) pesticides province year reference resistant resistant bendiocarb 0.1% propoxur 0.1% mazandaran (sari) 2018 ghorbani et al. resistant 27 bendiocarb 0.1% kerman (rafsanjan ) 2017 salim-abadi et al. 139 140.1 73 60.3 bendiocarb 0.1% propoxur 0.1% tehran (southern) 2019 rahimi et al. resistant bendiocarb 0.1% tehran (varamin) 2004 vatandoost et al. resistant propoxur 0.1% siatan & baluchistan 2014 fathian et al. resistant 36.10 propoxur 0.1% east azarbaijan 2015 ataie et al. table3. the resistance of culex pipiens against carbamate insecticides in different regions of iran figure 5. the resistance of culex pipiens against carbamate insecticides in different regions of iran fig. 5. the resistance of culex pipiens against carbamate insecticides in different regions of iran 355 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... table 1. the resistance of culex pipiens against organochlorine insecticides in different regions of iran resistance status kt50 (min) lt50 (min) pesticides province year reference resistant ddt 4% tehran city 2015 salim-abadi et al. resistant resistant resistant ddt 4% dieldrin 4% dieldrin 0.4% mazandaran (sari county) 2018 ghorbani et al. resistant 139 ddt 4% kerman (rafsanjan county ) 2017 salim-abadi et al. 1567 240 ddt 4% tehran (southern) 2019 rahimi et al. resistant ddt 4% tehran(varamin county) 2004 vatandoost et al. resistant ddt 4% west azerbaijan 2015 naseri et al. resistant 31.93 ddt 4% siatan & baluchistan (chabahar) 2014 fathian et al. resistant 78.39 ddt 4% tehran(qarchak) 2017 zeidabadi et al. resistant 134.75 ddt 4% east azarbaijan 2015 ataie et al. table 4. the resistance of culex pipiens against pyrethroid insecticides in different regions of iran figure 6. the resistance of culex pipiens against pyrethroid insecticides in different regions of iran fig. 6. the resistance of culex pipiens against pyrethroid insecticides in different regions of iran among carbamate insecticides, this species has shown resistance to bendiocarb 0.1% in rafsanjan, sari and varamin counties and resistance to propoxur 0.1% in chabahar, sari, and tabriz counties (table 3, fig. 4). the highest diversity of insecticide used against cx. pipiens in iran is related to a pyrethroid insecticide. cx. pipiens tolerates permethrin 0.75% in varamin city. it is resistance in sari city tehran, west azerbaijan. it is tolerant to deltamethrin 0.05% in chabahar, tabriz and varamin counties. cx. pipiens is tolerant to lambdacyhalothrin 0.05% in varamin city and has shown resistance in tehran, chabahar, tabriz, rafsanjan, uromia and sari cities. cx. pipiens is susceptible to cyfluthrin 0.15% in varamin city and resistant to chabahar, sari and tehran cities. cx. pipiens also tolerant against etofenprox 0.5% in varamin city and resistant in sari city (table 4, fig. 5). there are also several reports of insecticide resistance in the world (26-36). analysis of data from various researches in iran shows that cx. pipiens complex 356 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 349–357 a azarm et al.: insecticide resistance... is resistant to ddt 4%. mechanism of similar effect between ddt and pyrethroid insecticide. examination of the maps revealed that the areas where the cx. pipiens species that have shown resistance to the insecticide ddt is similar to the areas where the species has shown resistance to the pyrethroid insecticide. therefore, in these areas, the use of these two groups of insecticides in control programs should be avoided. analysis of article data also showed that in tehran and mazandaran provinces, cx. pipiens species have shown resistance against most of the tested insecticides, so in these two provinces, insecticides with different mechanisms of action should be used. conclusion according to the who guideline there are several insecticide are being used for control of mosquitoes as indoor residual spraying, fogging, larviciding and insecticide treated net (37). monitoring and mapping of insecticide resistance is a vital guideline for authorities to provide appropriate measure for vector control. conflict of interest the authors declare that there is no conflict of interest. acknowledgments this research is financially supported by by ministry of health and medical education under code number of nimad 995633. references 1. kenney jl, romo h, duggal nk, tzeng w-p, burkhalter kl, brault ac (2017) transmission incompetence of culex quinquefasciatus and culex pipiens pipiens from north america for zika virus. am j trop med hyg. 96(5):1235-40. 2. yoshimizu mh, padgett k, kramer v (2020) surveillance of a kdr resistance mutation in culex pipiens (diptera: culicidae) and culex quinquefasciatus in california. j med entomol. 57(2):645-8. 3. yu d, madras n, zhu h (2018) temperature-driven population abundance model for culex pipiens and culex restuans (diptera: culicidae). j theor biol. 443:28-38. 4. shahhosseini n, kayedi 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(1):22-30. 34. naseri-karimi n, vatandoost h, bagheri m, chavshin ar (2015) susceptibility status of culex pipiens against deltamethrin and ddt, urmia county, west azerbaijan province, northwestern iran. asian pacific j trop dis. 5: s77-s9. 35. salim-abadi y, asadpour m, sharifi i, sanei dehkordi a, gorouhi ma, paksa a (2017) baseline susceptibility of filarial vector culex quinquefasciatus (diptera: culicidae) to five insecticides with different modes of action in southeast of iran. j arthropod-borne dis. 11(4):453. 36. abai mr, hanafi-bojd aa, vatandoost h (2016) laboratory evaluation of temephos against anopheles stephensi and culex pipiens larvae in iran. j. arthropod-borne dis. 10(4):510-515. 37. world health organization (who). pesticides and their application for the control of vectors and pests of public health importance. pp.125. insecticide resistance in the west nile encephalitis, japanese encephalitis, avian malaria and lymph abstract keywords introduction materials and methods data collection results and discussion conclusion conflict of interest acknowledgments references evaluation of nanoemulsion of eucalyptus globulus oil as potent botanical larvicide against malaria vector, anopheles stephensi and west nile vector, culex pipiens under laboratory and semi-field conditions http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... original article evaluation of nanoemulsion of eucalyptus globulus oil as potent botanical larvicide against malaria vector, anopheles stephensi and west nile vector, culex pipiens under laboratory and semi-field conditions sara sabzalizade1, amir amani2,3, hassan vatandoost1,4, fatemeh hosseini1, roya najafi-taher3, *hamid reza basseri1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical nanotechnology, school of advanced technologies in medicine, tehran university of medical sciences, tehran, iran 3medical biomaterials research center (mbrc), tehran university of medical sciences, tehran, iran 4department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran *corresponding author: prof. hamid reza basseri, email: basserih@tums.ac.ir (received 31 aug 2021; accepted 08 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: due to undesired environmental impact of insecticides as well as resistant of vectors to them, the development of organic and natural insecticides has been more considered. in the current study, we developed nanoemulsion of eucalyptus and investigated lavicidal activity of it against malaria vector, anopheles stephensi and culex pipiens under laboratory as well as semi-field conditions. methods: an optimized nanoemulsion was prepared by mixing eucalyptus oil, tween 80 and ethanol at ratio of 1:2:1.5 in distilled water, then, stirred for 20 minutes at room temperature. the product was then used for bioassay tests against 3-4th instar larvae of anopheles stephensi as well as culex pipiens. furthermore, a semi-field trial was carried out to evaluate larvicidal activity of nanoemulsion of eucalyptus. results: nanoemulsion of eucalyptus showed significantly high lavicidal activity comparing with bulk eucalyptus essential oil. the lc50 and lc90 value of nanoemulsion against an. stephensi were 111.0 and 180.8 ppm respectively and 29.5 and 73.7 ppm for cx. pipiens, respectively. in the semi field condition, the nanoemulsion of eucalyptus decreased 1-2nd instar larval density of culicines and anophelines to 90.1% and 85.2%, respectively. conclusion: the nano formulation of eucalyptus oil showed high larvicidal activity. therefore, nanoemulsion of eucalyptus oil can be used as an eco-friendly larvicide against mosquitoes. keywords: eucalyptus; essential oil; nanoemulsion; larvicide; anopheles stephensi; culex pipiens introduction mosquitoes are a serious threat to public health as they act as vectors that help in transmission of diseases that can be lethal (1,2). owing to the lack of proper medication and vaccines for treating mosquito-borne diseases, an alternative and effective approach used to control the vector population at the larvicidal stage is necessary because, during this stage, 381 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... the mosquitoes are in a stationary phase (3 5). the resistance to synthetic pesticides and harmful effects of their accumulation in the environment has created the need for natural and non-persistent insecticides. the essential oils extracted from plants are suitable as they are economically reasonable and have high activity in certain cases and are also bio degradable (4,6,7). eucalyptus is a diverse genus of flowering trees and shrubs in the myrtle family, myrtaceae. the oil extracted from eucalyptus leaves possesses allelopathic property and prevents insects from attacking it, thereby, acting as a natural pesticide (8,9). the fumigation activity (10) and repellency (11) and insecticidal activity of eucalyptus oil has been demonstrated (12). nanoemulsion of natural oils could enhance pesticide activity of the component (13). nanoemulsions are emulsions whose droplet size is uniform and extremely small with the size ranging from 20 to 100 nm. nanoemulsions are metastable and their stability is determined by the method of preparation (14). nanoemulsions can be formulated by two kinds of methods such as high-energy and low-energy emulsification methods. the high energy emulsification method comprises high pressure homogenization and ultrasonication (15). ultrasonication is the most widely used method owing to its ease of use and it is an economical method. the low-energy emulsification technique comprises methods that exploit the chemical properties of a system to convert a microemulsion into a nanoemulsion (16,17). the use of nanopesticides would be a contemporary measure for the control of pests and reducing the toxic effect of synthetic bulk pesticides on the environment (18). recently, controlling for vector borne diseases using neem oil nanoemulsion will be of good alternative against culex quinquefasciatus compared to the synthetic pesticides (13). the present study was carried out to develop a nano formulation of eucalyptus oil as an ecofriendly larvicide and evaluate its larvicidal activity against the larvae of malaria vector, anopheles stephensi and vectors of west nile virus (wnv), culex pipiens in laboratory and semi-field conditions. material and methods preparation of nanoemulsion eucalyptus oil (eucalyptus globulus) was purchased from barich co., iran and stored at room temperature under laboratory conditions; tween 80 (polyoxyethylene 20 monooleate) was supplied from sigma. all other chemicals used were of analytical reagent grade. the oil-in-water nanoemulsion was formulated using eucalyptus oil, nonionic surfactant (tween 80) and water. the concentration of eucalyptus oil (6%, v/v) was fixed for all the formulations. initially, coarse emulsion was prepared by adding water to organic phase containing oil, surfactant and cosurfactant in ratios 1:1: 1.5 (v/v) using a magnetic stirrer, which was then subjected to ultrasonic emulsification using a 20 khz sonicator (ultrasonics, usa) with a power output of 750 w. energy input was given through sonotrode containing a piezoelectric crystal with a probe diameter of 13 mm. sonicator probe generates disruptive forces that reduce the droplet diameter converting coarse emulsion to nanoemulsion. then the characterization of nanoemulsions was carried out and the emulsion stability was investigated. droplet size distribution and polydisper sity index the droplet size distribution (analysis by volume) and poly dispersity index (pdi) of eucalyptus oil nanoemulsion formulation (1:2) was determined using a 90-plus particle size analyzer. the pdi is a measure of the homogeneity and stability of the droplet size in the nanoemulsion system. pdi values below 0.2 indicate a narrow size distribution and thus provide long-term stability to the formulated nanoemulsion. prior to experiment, formulated emulsion was diluted with milli-q (millipore corporation) double-distilled water to trim down multiple scattering effects. morphology of emulsion droplets to visualize the shape and morphology of 382 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... the formulated nanoemulsions, atomic force microscopy (afm) was carried out. one drop of emulsion was negatively stained with phosphotungstic acid and positioned on a copper grid. larvicidal bioassay mosquito rearing: mosquitoes consisting of an. stephensi and cx. pipiens were reared under a uniform condition including larvae and adult nutrition, temperature (28±2 °c), humidity (70±10 %) and on a 12-h light dark cycle. larvae were grown in bowls at a density of 200 larvae / 500 ml of distilled water with 0.01% table salt, and fed on fish food. the pupae were transferred to cages made of muslin cloth before eclosion to the adult stage. the female mosquitoes were fed on 10% fructose and the females were fed on guinea pig blood. larvicidal activity: the larvicidal activity of eucalyptus nanoemulsion against third instar larvae of an. stephensi and culex pipiens were treated with different concentrations of nano and bulk eucalyptus oil emulsions. the bioassays were carried according to the guide line of world health organization for laboratory and field testing of mosquito larvicides (19). initially, serial dilutions of nanoemulsion (5, 50, 100, 160, and 240 ppm) were prepared in sterile glass beakers (250 ml) containing 200 ml of water. then 20 larvae (stage 3-4) of the mosquito species were placed into each beaker. the same procedure was conducted for the bulk of eucalyptus oil. the mortality rate of larvae was recorded after 24 and 48 hours. each test was performed in four replicates. the percentages of larval mortality and standard deviation were calculated for each concentration of nanoemulsion and bulk emulsion. semi-field larval bioassays twelve artificial breeding places each 1×1 meters were provided in a semi-field condition in the kazeroun area, southern iran and allow the wild mosquitoes to lay their eggs on the surface of breeding places. the density of larvae 10 per dipper were measured in each breeding place before any intervention. the spray of three times of obtained lethal concentration (lc90) of bulk-eucalyptus oil (≈114 ppm) and nanoeucalyptus oil (≈90 ppm) were sprayed randomly on the surface of each breeding place. each treatment was replicated in four breeding places and four breeding places of free oils as the control. statistical analysis the mortality quantities of 50% and 90 % of imagicides (lc50 and lc90) and the level of confidence of 95%, the equation of the regression line were estimated using a regression probit analysis as described by finney (20). mosquito identifications all the adults females were identified using morphological identification key identification (21). results characterization of the selected nano emulsion based on the thermodynamic stability study, 1:2 ratio nanoemulsions was selected, and their average diameter is 18 nm and their size distribution as presented in fig. 1. the polydispersity index (pi) of the nanoemulsions is 0.060 which shows that it is uniform. the particles are presented in atomic force microscopy (afm) from 22 to 40 nm, and the droplets are spherical in nature (fig. 2). larvicidal activity of eucalyptus oil nano emulsion the larvicidal activity of both nanoemulsion eucalyptus oil and bulk eucalyptus oil against larvae of an. stephensi and cx. pipiens was varied. high mortality rate was observed among larvae of an. stephensi exposed to 160 ppm of nanoemulsion of eucalyptus oil within 24 hours, while the mortality rate for eucalyptus oil was 74% at the concentration of 160 ppm after 24 hours (table 1). moreover, 383 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... 100% mortality occurred for the larvae exposed to both nanoemulsion eucalyptus oil and eucalyptus oil at a concentration of 240 ppm. overall, the larvicidal activity of nanoemulsion oil against larvae of an. stephensi within 24 hours was significantly higher than eucalyptus at 160 ppm, but absolute mortality was observed among the larvae of an. stephensi after 48 hours in both nanoemulsion formulation and bulk oil of eucalyptus (table 1). similarity, the larvicidal activity of nanoemulsion oil against larvae of cx. pipiens was higher than eucalyptus oil at 160 ppm within 24 hours while 100% mortality occurred by 240 ppm for both formulations of eucalyptus oils (table 2). lethal concentration (lc50and lc90) for nanoemulsion of eucalyptus oil and eucalyptus oil were significantly different within 24 and 48 hours after treatment (table 3). moreover, lc50 and lc90 of nanoemulsion of eucalyptus oil against larvae of an. stephensi significantly lower than eucalyptus oil within both 24 and 48 hours treatment (table 3). similarly, lower lc50 and lc90 was observed for larvae of cx. pipense exposed to nanoemulsion of eucalyptus oil which was significantly different with lc50 and lc90 for the larvae exposed to eucalyptus oil (table4). semi field larval bioassays both nano-eucalyptus and bulk eucalyptus oils decreased the density of mosquito larvae in the treated breeding places one day after fig. 1. size distribution of eucalyptus nanoemulsion oil measured by dynamic light scattering fig. 1. size distribution of eucalyptus nanoemulsion oil measured by dynamic light scattering fig. 2. atomic force microscopy image and size of eucalyptus nanoemulsion oil fig. 2. atomic force microscopy image and size of eucalyptus nanoemulsion oil 384 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... spraying. the high larvae population at stage 1 and 2 in the control breeding sites indicated that the wild mosquitoes laid eggs frequently during the bioassay period (fig. 3). therefore, the density of mosquito larvae was relatively high in the control breeding site while rapidly declining in the treated sites. however, nano eucalyptus emulsion kept the larvae density low for 6 days post spraying while the number of larvae at stage 1 and 2 increased two days after eucalyptus oil spraying (fig. 3). the results indicating that nano-eucalyptus oil fig. 3. residual larvicidal activity of nanoemulsion of eucalyptus oil and bulk eucalyptus oil against different stages of mosquito larvae at semi-field condition. twelve artificial breeding places (1×1 meters) were prepared and then allowed the wild mosquitoes laid eggs. l1, 2 = larvae stage 1 & 2, l 3,4= larvae stage 3 & 4 fig. 3. residual larvicidal activity of nanoemulsion of eucalyptus oil and bulk eucalyptus oil against different stages of mosquito larvae at semi-field condition. twelve artificial breeding places (1×1 meters) were prepared and then allowed the wild mosquitoes laid eggs. l1, 2 = larvae stage 1 & 2, l 3,4= larvae stage 3 & 4 table 1. probit analysis of larvicidal activity of essential and nanoemlusion of eucalyptus globulus oil on 3rd and 4th instar larvae of anopheles stephensi after 24 hours concentration (ppm) essential oil nanoemlusion oil mortality (%) observed probit mortality expected probit mortality mortality (%) observed probit mortality expected probit mortality 60 9 3.659 3.246 8 3.595 3.384 160 74 5.643 5.658 91 5.025 5.204 240 100 7.526 6.654 100 7.576 7.024 360 100 7.526 7.651 100 7.576 8.089 control 0 0 table 1. probit analysis of larvicidal activity of essential and nanoemlusion of eucalyptus globulus oil on 3rd and 4th instar larvae of anopheles stephensi after 24 hours table 2. probit analysis of larvicidal activity of essential and nanoemlusion of eucalyptus globulus oil on 3rd and 4th instar larvae of culex pipiens after 24 hours concentration (ppm) essential oil nanoemlusion oil mortality (%) observed probit mortality expected probit mortality mortality (%) observed probit mortality expected probit mortality 60 8 3.595 3.384 11 3.773 3.520 160 51 5.025 5.204 86 4.900 5.161 240 100 7.576 7.024 100 7.576 6.802 360 100 7.576 8.089 100 7.576 8.443 control 0 0 table 2. probit analysis of larvicidal activity of essential and nanoemlusion of eucalyptus globulus oil on 3rd and 4th instar larvae of culex pipiens after 24 hours 385 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... has slightly longer residual larvicidal activity rather than bulk eucalyptus. discussion due to numerous limitations to control of adult mosquitoes, the ideal method to control them is targeting the larval stage. control of mosquito larvae is based largely on the use of synthetic chemicals. generally, synthetic pesticides have some disadvantages such as health problems, harmful to the environment, pests may develop tolerance to certain chemicals over time and contamination of soil and water resources (22-24). however, the indiscriminate and injudicious use of pesticides has led to the widespread development of resistance among pests as well as insect vectors (25). thus, alternative components to be needed for controlling mosquito vectors. botanical pesticides are considered as safe, easily table 3. lethal concentration (lc50 and lc90) and in the 24 and 48 hours bioassay tests of eucalyptus oil and nanoeucalyptus against 3rd and 4th instar larvae of anopheles stephensi b ± se lc90 (ppm) ± 95%c.l. lc50 (ppm) .± 95%c.l χ2 (heterogeneity) χ2 table (df) p-value exposure time (hours) 5.6608 ± 0.764 169.1937 206.2336 295.0632 103.7845 122.8343 145.0833 15.611* 15.086 (5) 0.01 24 e uc al yp tu s oi l 2.399 ± 1.023 142.402 155.973 175.732 93.761 101.663 145.0833 14.623* 15.035 (5) 0.00 48 6.0457 ± 0.539 102.8420 111.0171 119.7206 63.7924 80.8730 105.7719 5.1802* 9.210 (2) 0.01 24 n an oe uc al yp tu s oi l 7.0457 ± 0.652 46.235 58.595 65.548 68.235 76.869 95.582 4.258* 9.145 (2) 0.00 48 table 3. lethal concentration (lc50 and lc90) and in the 24 and 48 hours bioassay tests of eucalyptus oil and nano-eucalyptus against 3rd and 4th instar larvae of anopheles stephensi table 4. lethal concentration (lc50 and lc90) and in the 24 and 48 hours bioassay tests of eucalyptus oil and nanoeucalyptus against 3rd and 4th instar larvae of culex pipiens b ± se lc90 (ppm) ± 95%c.l. lc50 (ppm) ± 95%c.l χ2 (heterogeneity) χ2 table (df) p-value exposure time (hours) 6.5324 ± 1.034 20.3120 39.4112 78.6321 47.3526 68.6321 76.1356 10.546* 8.235 (2) 0.01 24 e uc al yp tu s oi l .4508 ± 1.127 14.7673 37.3711 86.4146 42.4037 64.2180 74.0175 10.678* 9.210 (2) 0.01 48 3.2200 ± 0.313 25.7675 29.4644 33.0781 43.3775 53.6733 70.0869 15.611* 9.210 (2) 0.01 24 n an oe uc al yp tu s oi l 5.1234 ± 0.4123 19.9645 23.2523 26.2879 31.6885 34.5681 39.0263 15.646* 9.451 (2) 0.01 48 table 4. lethal concentration (lc50 and lc90) and in the 24 and 48 hours bioassay tests of eucalyptus oil and nano-eucalyptus against 3rd and 4th instar larvae of culex pipiens 386 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... biodegradable, environmentally friendly and with low toxicity (26,27). however, usage of them is often limited due to instability and rapid degradation, application frequency as well as requiring higher application rates. hence, the use of botanical insecticides associated with nanotechnology offers considerable potential for increasing efficacy of plant based insecticide (27-29). eucalyptus oil is the oil distilled from the leaves of eucalyptus, a genus of the plant family myrtaceae. the repellent activity of eucalyptus oil was demonstrated against cx. quinquefasciatus (30,31). the seed and leaf extract of eucalyptus oil contain compounds that are toxic to mosquito larvae (32,33). since they do not cause any adverse health effects; they are used as insect repellents and a safe and eco-friendly alternative to synthetic pesticides (34). therefore, in this study, we used eucalyptus oil as a safe and non-toxic larvicide. generally, botanic pesticides have low effective durability; therefore, we utilized nanotechnology to overcome this problem. we synthesized nano-eucalyptus oil to increase larvicidal durability and effectiveness. the method of preparation determines the stability of the formulated nanoemulsion. based on atomic force microscopy analysis, the size of our product was from 22 to 40 nm (fig. 2), and the larvae mortality we achieved in laboratory and semi field bioassays (tables 1 , 2 and fig. 3) was as the results of these nano-size. nevertheless, the larvicidal effects of nano-eucalyptus oil are likely to change as the nano-particle size changes. the stabilization of the nanoemulsions is also dependent on the steric effect of the non-ionic surfactant (35). therefore, the effectiveness of nano-eucalyptus oil may be influenced by chemical component of water in the breeding sites. in semi-field assay, we did not observe 100% mortality of mosquito larvae a day after treatment of nano-eucalyptus oil and eucalyptus. this result may be due to components of water in the breeding sites. in addition, only a single concentration of nano-eucalyptus oil (≈90 ppm) was tested against wild mosquito larvae. according to who guide line, the larvicide dosage for the field trial should be three times of ld90 concentration in a laboratory scale. however, we did not observe convenient results for larvicidal effect of nano-eucalyptus oil in the field condition, thus, more serial concentrations of the nano emulsion should be assays to overcome the highest larvae mortality in the field condition. in this study, eucalyptus oil (6%, v/v) is mixed with a non-ionic surfactant, tween 80 along with water, which is used as an aqueous phase. this emulsion is then subjected to ultrasonication that breaks down the bulk emulsion into an emulsion comprising droplets having the size in the nanometer range. this increases the surface area of the droplets, thereby, increasing the reactivity; thus, making nanoemulsions more effective than its bulk counterpart. however, the insecticidal and antimicrobial activity of eucalyptus oil is linked to its chemical composition such as 8-cineole (eucalyptol), which its concentration ratio varies in eucalyptus trees from different locations (36). conclusion the nano formulation of eucalyptus oil showed high larvicidal activity against mosquito larvae when compared to its bulk counterpart. therefore, nanoemulsion of eucalyptus oil can be used as an eco-friendly larvicide against mosquitoes. from this study, it can be concluded that eucalyptus oil nanoemulsion is a safe and effective alternative larvicide to control mosquito borne diseases. acknowledgements the authors would like to thank the school of public health, tehran university of medical sciences, for their support in carrying out this research. this study was supported by the deputy of research, tehran university of medical sciences, grant no. 29565. the ethical approval was obtained from the ethics committee of tehran 387 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 380–388 s sabzalizade et al.: evaluation of nanoemulsion... university of medical sciences. competing interests the authors declare that they have no competing interests. references 1. lajeunesse mj, avello da, behrmann ms, buschbacher tj, carey k, carroll j, chafin tj, elkott f, faust am, fauver h (2020) infected mosquitoes have altered behavior to repellents: a systematic review and meta-analysis. j med entomol. 57:542-550. 2. nadim ss, ghosh i, martcheva m, chattopadhyay j (2020) impact of venereal transmission on the 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malaria abstract keywords introduction material and methods preparation of nanoemulsion droplet size distribution and polydispersity index morphology of emulsion droplets larvicidal bioassay semi-field larval bioassays statistical analysis mosquito identifications results characterization of the selected nanoemulsion larvicidal activity of eucalyptus oil nanoemulsion semi field larval bioassays discussion conclusion acknowledgements competing interests references j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 156 case report infantile nosocomial myiasis in iran naseh maleki ravasan 1, *mansoureh shayeghi 1, babak najibi 2, *mohammad ali oshaghi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2shaheed rajaei cardiovascular, medical and research center, tehran university of medical sciences, tehran, iran (received 20 nov 2011; accepted 15 oct 2012) abstract myiasis, the invasion of live human tissue by larva of diptera, is reported in the nasal cavity of a 5.5year-old iranian girl. she was referred from golestan province to the shaheed rajaei heart center in tehran. in the 41th day after admission, a live parasite was found in her nasal secretions suction identified presumably as a second instar larvae of a facultative myiasis, woholfartia nuba (diptera: sarcophagidae), on the basis of mtdna-coi and morphological characteristics. since presence of the larva was recorded after hospitalization, by definition, this infestation is considered a nosocomial myiasis. keywords: nosocomial, myiasis, woholfartia nuba, iran introduction among arthropod diseases affecting animals, larval infections-myiasisof domestic and wild animals have been considered important since ancient times. myiasis occurs worldwide, but more often in hot and humid climates. species causing myiasis are categorized into obligatory, facultative, and accidental (hall and wall 1995). common infection sites are skin wounds (safdar et al. 2003) eyes (razmjou et al. 2007), nose (kim et al. 2009), and throat (pasternak et al. 2007) and urogenital tract are less common (salimi et al. 2010). nosocomial infection is defined as the infection that is caused during or after the hospitalization which was neither present nor incubating at the time of admission (wenzel 1997). nosocomial myiasis, although rare, is sometimes reported in debilitated, diabetics, cardiovascular, and encephalopathics patients as well as people having motor vehicle accidents. some of the contributing factors included disturbed unconsciousness or hypoesthesia that prevents the patient’s sensation of fly contact, and paralysis or immobility that prevent from fending off the fly detected (lettau 1991, daniel et al. 1994, amitay et al. 1998). recent findings from the whole parts of the world indicate that nosocomial myiasis is probably under-reported. hospital-acquired myiasis usually occurs during summer months (june to september) when fly populations are in peak activity and the most identified larvae were lucilia sericata. although l. illustris, cochliomyia macellaria, c. hominivorax, sarcophaga crassipalpis, s. cruentata, parasarcophaga argyrostoma, musca domestica, cuterebra buccata and megaselia scalaris belonging to the families of calliphoridae, sarcophagidae, muscidae, cuterberidae, and phoridae were reported as well (table 1). there are no or a few, however, which have become obligatory parasites *corresponding authors: dr mansoureh shayeghi, email: mansorehshayeghi@yahoo.com, dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 157 of warm-blooded vertebrates, like wohlfahrtia magnifica (schiner 1862) in the old world, and w. vigil (walker) and w. opaca (coquillet) in the new world. these species are not easy to separate, and the identification of files reared from maggots must be left to the specialist (hall and wall 1995). the genus wohlfahrtia, which comprises a fair number of species, is distributed over the holarctic and the ethiopian regions. this genus phylogenetically is related to sarcophaga and some of the species also develop in decomposing organic matter. like species of sarcophaga, the flies of this genus are viviparous and give birth to very mobile larvae of the first stage. each female carries approximately 120– 170 larvae. they are deposited near skin lesions, even minute ones like tick bites, which are used for penetrating the tissue. the mucous membranes of nose, eyes and the female genital organs are apparently attacked without the utilization of pre-existing wounds. the larvae grow rapidly and cause widespread destruction of the healthy tissue, and after 5–7 days leave the wound for pupation. the adults are diurnal, favoring the hot hours of the day, and are not normally found on the wing in the morning, the evening or in gloomy weather. the flesh fly, wohlfahrtia nuba (wiedemann) is largely distributed in africa and asia. according to geographical distribution given by spradbery (2002), w. nuba is distributed southerly than w. magnifica including the whole parts of iran (except northwest), pakistan, saudi arabia, countries neighborhood of west sides of the red sea, egypt, libya, tunisia, mauritania, senegal and guinea (spradbery 2002). case report a 5.5-year-old iranian girl was referred from gonbad county, golestan province, northern iran to the shaheed rajaei heart center in tehran. she was operated on her heart ten days later and then was delivered to intensive care unit (icu) of the hospital. in this part of the hospital a live parasite was found in her nasal secretions suction and consignment to the department of medical entomology in the school of public health, tehran university of medical sciences, iran. putative patient died 45 days after admission, unfortunately. since this infestation manifested more than 40 days after the patient hospitalization, by definition, this may be considered nosocomial. current morphological characters as well as sequence of a part of cytochrome oxidase subunit one of mitochondorial dna (mtdna-coi) was used for identification of the mentioned dipterous larva. special dates the most important dates for this myiasis report were: patient hospitalization, 25 august 2010, open heart operation, 4 september 2010, parasite separation, 5 october 2010, and expiration, 9 october 2010. morphological examinations the key morphological characters including size of the larva, spinous bands, position of posterior spiracles and posterior cavity as mentioned by zumpt (zumpt 1965) examined under microscope (olympus szx12). the cephalo-pharyngeal skeleton and both anterior and posterior spiracle dissected and mounted on glass slide by pouri solution. after desiccation the larva was keyed using valid keys (zumpt 1965, ferrar 1987, cdc 1994, hall and wall 1995, spradbery 2002). testing the morphological characteristics revealed that the size of the larva was as long as 5 mm, spinous bands with sclerotized spines, the posterior spiracles located in deep posterior cavity, prothoracic spiracles with 9 openings branches, peritreme present, with 2 distinct slits, spiracular slites pointing toward opening in peritreme, posterior spiracles with the incomplete peritreme, and dorsal arm of cephalo-skeleton longer than ventral arm (fig. 1–4). on the basis j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 158 of above characters the larvae presumably identical to the second instar larva of w. nuba (diptera: sarcophagidae). molecular examinations except the cephalo-pharyngeal skeleton and spiracle the rest of the larva body was used for dna extraction using the qiagen-dneasy blood and tissue kit (germany). the mtdnacoi gene extending 690 bp of 5' fragment as applied by lunt (lunt et al. 1996) was amplified using primers including c1-j-2090/ c1n-2735. this region of gene was am-plified in 20μl reaction mixtures containing 1μl each of the forward and reverse primers, 1μl the genomic dna, 1u of taq dna polymerase, 1.5 mm mgcl2 and 250 μm dntps. the pcr amplification was performed in two thermal circulations. after initial denaturation at 94 °c for 2 min, the first one performed for 5 cycles of 94 °c for 40 s, 45 °c for 40 s, and 72 °c for 1 min, and the last one repeated for 35 cycles of 94 °c for 40 s, 51 °c for 40 s, and 72 °c for 1 min. final extension step was continued at 72 °c for 5 min. pcr products were visualized on a 1% agarose gel containing ethi-dium bromide and using an uv transilluminator. the pcr products were sequenced by seqlab, a german sequencing company. sequence of this fragment was deposited in the genbank database with accession number jf277565. the obtained sequence in this study was compared with the relevant sequences available in public databases using the blast program (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) and then were aligned using clustalw2 (http: //www.ebi.ac.uk/tools/msa/clustalw2/). phylogenetic relationships between the specimen of this study and some representatives of other related flies causing myiasis including eu418542, ab112857, ab112861, aj426041, eu418536, dq345078, af083657, ab1128 51, ab112854, eu418545, aj417708, af295 559, af104625, eu627697, and eu627705 were inferred with the algorithms implemented in clustalw2. sequence of coi region of two member of muscidae (musca ventrosa: eu 627697; neomyia timorensis: eu627705) were used as out groups. sequence comparison of the specimen with the available data in genbank using blast revealed maximum 93% identity with chrysomya albiceps coi region. other entries of the coi sequences with similarity less than 93% belonged to other dipteran flies of calliphoridae, muscidae, scathophagidae and drosophilidae families. the phylogenic trees inferred from these data showed that the specimen placed in a completely isolated branch far from other dipteran species particularly from the closely related species of calliphoridae (fig. 5). however, among different branches on the tree, protocalliphora sialia (calliphoridae) was the most associated fly to w. nuba. table 1. summary data for reported cases of nosocomial myiasis during 1980–2010 family no of reports country common reported species calliphoridae 21 usa (6) korea (3) canada (2) honduras (2) czech (2) jamaica (1) occupied palestine (1) india (1) kuwait (1) french guiana (1) turkey (1) lucilia sericata sarcophagidae 4 usa (1) england (1) turkey (1) italy (1) sarcophaga sp muscidae 2 usa (1) england (1) musca domestica cuterebridae 1 usa (1) cuterebra buccata phoridae 1 kuwait (1) megaselia scalaris j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 159 fig. 1. the second instar larva isolated from nasal secretions suction of a 5.5-year-old girl, 2010, iran. spinous band indicated just around the right down corner (original) fig. 3. posterior spiracles of the second instar larva isolated from nasal secretions suction of a 5.5-yearold girl, 2010, iran (original) fig. 2. the cephalo-pharyngeal skeleton of second instar larva isolated from nasal secretions suction of a 5.5-year-old girl, 2010, iran (original) fig. 4. anterior spiracle of the second instar larva with 9 lobes isolated from nasal secretions suction of a 5.5-year-old girl, 2010, iran (original) j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 160 fig. 5. the phylogenetic relationship of wohlfahrtia nuba (jf277565) (sarcophagidae) found in this study with other dipteran flies inferred from 645 bp of the coi gene. sequence data of other flies are retrieved from genbank, calliphoridae: (chrysomya putoria: eu418542, c. inclinata: ab112857, c. megacephala: ab112861, aj426041, eu418536, c. albiceps: af083657, ab112851, ab112854, c. varipes: eu418545, phormia regina: dq345078, lucilia cuprina: aj417708, protocalliphora sialia: af295559), scathophagidae: (scathophaga stercoraria: af104625), muscidae: (musca ventrosa: eu627697, neomyia timorensis: eu627705) discussion this is the first report of nosocomial case caused by w. nuba in iran. morphological characters of the larva showed that it was approximately at the second to third instar transition (due to presence of developing pre-third slit), therefore, no more than five or six days old (60–72 hours, on average 66 h). this came from the fact that the insect rate of development is temperature dependent (nietschke 2007, oshaghi et al. 2009, shiravi et al. 2011). this can be calculated using the available information on thermal requirement of w. nuba (amudi 1993). total developmental times of flesh flies w. nuba from first larval stage to eclosion was reported 34.7, 23.5, 18.7, 13.4 and 13.6 days when reared at 21, 25, 29, 33 and 37° c respectively. estimated the lower developmental (tl) threshold temperatures were 12.8, 14 and 13.6 °c and the thermal constant (k) were 116.8, 152.2 and 268.2 degree days (dd) for larvae, pupae and total developmental time (egg to adult) respectively (amudi 1993). on the other hand, the temperature of human nostril is approximately 30 °c (greenberg 1984). therefore based on the base threshold temperature of larvae (12.8 °c), the larvae had received 17.3 dd (30–12.8= 17.3) every day. by dividing thermal constant (k) of larval stage to this value (116.8/17.3= 6.75 day), only about 7 days was enough for the larvae to complete their development and convert to pupae. because there are three instars at larval stage, each instar needs roughly 2.25 days (6.75/3= 2.25) to develop. therefore, since the larva was approximately at the second to third inj arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 161 star transition, its age can be about 4 to 4.5 days. we also found, as expected, that this case like most of other cases (greenberg 1984, daniel et al. 1994, joo and kim 2001, yazar et al. 2005, sesterhenn et al. 2009) occurred during the summer when the fly population is in its greatest density. wohlfahrtia nuba is a facultative flesh fly and infests wounds of livestock in north africa and the middle east, but it probably feeds only on dead or diseased tissues rather than on living tissues (amudi 1993). it also was found in head wounds or head cavities but not in dermal sores. wohlfahrtia nuba appears to be an important myiasis causing agent in saudi arabia, because almost one third of the cases reported from different geographical localities of the country were due to this species (el-azazy and el-metenawy 2004). the larvae of this species are also used in maggot therapy (el-azazy and elmetenawy 2004). however; it was observed that w. nuba would feed on healthy tissues at the edges of a wound if all necrotic tissue was exhausted (grantham-hill 1933). the parasite we identified morphologically was a second instar larva. it is worth mentioning that identification of the immature larva of parasite causing myiasis posed difficulties particularly the parasites with little available knowledge. however, the available morphological characteristics of the larva, especially the number of lobes of its anterior spiracles (fig. 4), plus lack of similarity of its coi sequence to any other morphologically related species such as c. albiceps, c. megacephala, c. varipes, c. putoria, c. inclinata, lucilia cuprina, l. illastris, protocalliphora sialia, and phormia regina, together with knowledge of the distribution of w. nuba in the region (spradbery 2002), support correct identification of the larva as w. nuba. generally we have severely limitations with sarcophagid flies because it is difficult or impossible to determine the species of a sarcophagid larvae and in many instances an adult specimen, based on anatomy. up to now there are no suitable key for the identification of the immature stages of sarcophagidae so we used combination of several valid keys (zumpt 1965, ferrar 1987, cdc 1994, spradbery 2002). in addition, for most larval stages of flesh flies identification based on morphological characteristics is still impossible (byrd and castner 2001), so we used molecular toll for identification purposes. of the various types of myiasis, only the secondary (accidental, facultative) myiasis is potentially nosocomial. and in this paper we report a facultative myiasis caused by w. nuba. flesh flies prefer sunlight rather than shaded conditions but hospital condition must be attracting for these flies (smith 1986). based on greenberg (1984) opinion, some of the factors that frequently produce myiasis include (i) helpless and debilitated individuals (ii) blood or odors of decomposition, (iii) neglect of nursing or custodial personnel, and (iv) summer season. in case of our report, facilitator conditions were included the patient’s inability to fend off flies, the initial presence of mucus in the nose and location of the hospital where was in the center of tehran metropolitan. reviewing 35 reported nosocomial cases from various countries showed those myiases were caused by maggots of both facultative and obligatory parasitic species of flies. in practice, there is no indication of ‘true’ myiasis in both community-acquired cases as well as nosocomial infestations, because hospital-acquired myiasis is probably under-reported for a number of reasons and some of the cases are squelched by hospital administrators, risk managers, and public relations departments for obvious medico legal and political reasons (smith and clevenger 1986, lukin 1989, lettau 1991, josephson and krajden 1993, daniel et al. 1994, chigusa et al. 1996). the prevention measures of nosocomial j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 162 myiasis are directly related to the flies. sanitary and efficient waste disposals supplemented by some insecticide sprays should minimize fly density. screens or sealed windows will provide physical barriers. wound care and dressing, as well as attention to the hygiene of the patients will also decrease the attractions of flies to the patients (lettau 1991, josephson and krajden 1993). acknowledgements we would like to thank dr m hall from the department of entomology, natural history museum, london, uk, dr t pape from natural history museum of denmark, and dr s tirgari from tums for giving some impressive comments and eng. k akbarzadeh for providing identification keys. this research was supported by the tehran university of medical sciences. the authors declare that there is no conflict of interest. references amitay m, efrat m, mcgarry jw, shinwell es (1998) nosocomial myiasis in an extremely premature infant caused by the sheep blowfly lucilia sericata. pediatr infect dis j. 17: 1056–1057. amudi ma (1993) effect of temperature on the developmental stages of wohlfahrtia nuba (dip: sarcophagidae). j egypt soc parasitol. 23: 697–705. byrd jh, castner jl (2001) insects of forensic importance. in: byrd jh, castner jl (eds) forensic entomology. the utility of arthropods in legal investigations. crc press, boca raton, pp. 287–302. cdc (1994) pictorial keys to arthropods, reptiles, birds and mammals of public health importance. centers for disease control and prevention (cdc) atlanta. chigusa y, kirinoki m, yokoi h, matsuda h, okada k, yanadori a, yamazaki s (1996) two cases of wound myiasis due to lucilia sericata and l. illustris (diptera: calliphoridae) med entomol zool. 47: 73–76. daniel m, sramova h, zalabska e (1994) lucilia sericata (diptera: calliphoridae) causing hospital-acquired myiasis of a traumatic wound. j hosp infect. 28: 149–152. el-azazy ome, el-metenawy tm (2004) cutaneous myiasis in saudi arabia. vet rec. 154: 305–306. ferrar p (1987) a guide to the breeding habits and immature stages of diptera cyclorrhapha. parts 1 and 2. scandinavian science press. grantham-hill c (1933) preliminary note on the treatment of infected wounds with the larva of wohlfahrtia nuba. trans r soc trop med hyg. 27: 93–98. greenberg b (1984) two cases of human myiasis caused by phaenicia sericata (diptera: calliphoridae) in chicago area hospitals. j med entomol. 21: 615. hall m, wall r (1995) myiasis of humans and domestic animals. adv parasitol. 35: 257–334. joo cy, kim jb (2001) nosocomial submandibular infections with dipterous fly larvae. korean j parasitol. 39: 255–260. josephson rl, krajden s (1993) an unusual nosocomial infection: nasotracheal myiasis. j otolaryngol. 22: 46–47. kim js, seo pw, kim jw, go jh, jang sc, lee hj, seo m (2009) a nasal myiasis in a 76-year-old female in korea. korean j parasitol. 47(4): 405–407. lettau la (1991) nosocomial transmission and infection control aspects of parasitic and ectoparasitic diseases. infect control hosp epidemiol. 12: 179–185. lukin lg (1989) human cutaneous myiasis in brisbane: a prospective study. med j arthropod-borne dis, 2012, 6(2): 156–163 n maleki ravasan et al.: infantile nosocomial … 163 j aust. 150: 237–240. lunt dh, zhang dx, szymura jm, hewitt gm (1996) the insect cytochrome oxidase i gene: evolutionary patterns and conserved primers for phyloge-netic studies. insect mol biol. 5: 153–165. nietschke bs, magarey rd, borchert dm, calvin dd, jones e (2007) a developmental database to support insect phenology models. crop prot. 26: 1444– 1448. oshaghi ma, maleki-ravasan n, javadian e, rassi y, sadraei j, enayati aa, vatandoost h, zare z, emami sn (2009) application of predictive degree day model for field development of sand fly vectors of visceral leishmaniasis in northwest of iran. j vec borne dis. 46: 1–8. pasternak j, joo sh, ganc aj, junior msd, morsh rd, pinto th (2007) a case of throat cochliomyia hominovorax infestation. einstein. 5(2): 170–172. razmjou h, mowlavi gh, nateghpour m, solaymani-mohamadi s, kia eb (2007) ophthalmomyiasis caused by flesh fly (diptera: sarcophagidae) in a patient with eye malignancy in iran. iran j arthropod-borne dis. 1(2): 53–56. safdar n, young dk, andes d (2003) autochthonous furuncular myiasis in the united states: case report and literature review. clin infect dis. 36: 73– 80. salimi m, edalat h, jourabchi a, oshaghi ma (2010) first report of human nasal myiasis caused by eristalisin tenax in iran (diptera: syrphidae). iran j arthropod-borne dis. 4: 77–80. sesterhenn am, pfützner w, braulke dm, wiegand s, wernerja, taubert a (2009) cutaneous manifestation of myiasis in malignant wounds of the head and neck. eur j dermatol. 19: 64–68. shiravi ah, mostafavi r, akbarzadeh k, oshaghi ma (2011) temperature requirements of some common iranian forensically important blow and flesh flies (diptera) under laboratory conditions. iran j arthropod-borne dis. 5: 54–62. smith dr, clevenger rr (1986) nosocomial nasal myiasis. arch pathol lab med. 110: 439–440. smith kgv (1986) a manual of forensic entomology. the trustees of british museum of natural history, london and comstock publishing associates, new york. spradbery jp (2002) a manual for the diagnosis of screw-worm fly. agriculture, fisheries and forestry, australia. wenzel rp (1997) prevention and control of nosocomial infections. 3rd ed. williams and wilkins co, blatimore. yazar s, dik b, yalcin s, demirtas f, yaman o, ozturk m, sahin i (2005) nosocomial oral myiasis by sarcophaga sp. in turkey. yonsei med j. 46: 431–434. zumpt f (1965) myasis in man and animals in the old world. butterworths, london. http://jad.tums.ac.ir published online: april 10, 2013 j arthropod-borne dis, june 2013, 7(1): 99 s wiwanitkit, v wiwanitkit: filter paper for… letter to the editor filter paper for preserving insects, bacteria, and host reservoir dna *somsri wiwanitkit, viroj wiwanitkit wiwanitkit house, bangkhae, bangkok, thailand sir, the recent report on “utility of filter paper for preserving insects, bacteria, and host reservoir dna for molecular testing” is very interesting (karimian et al. 2011). karimian et al. (2011) reported that “the filter paper method is a simple and economical way to store, to preserve, and to distribute dna samples for pcr analysis.” many facts should be discussed. first, this work is only a simple retrospective observational study. there is no economic analysis. no data on the cost, hence, it might not be possible to conclude that the technique is an economical way. second, karimian et al. (2011) concluded on preservation. this might be an extrapolating summary. there must be the proposed mechanism how the simple filter paper can preserve the sample. what the active gradient in the filter paper that helps preserve must be clarified. it seems that the filter paper is only a good alternative sample collection method. references karimian f, sedaghat mm, oshaghi ma, mohtarami f, dehkordi as, koosha m, akbari s, hashemi-aghdam ss (2011) utility of filter paper for preserving insects, bacteria, and host reservoir dna for molecular testing. iran j arthropod-borne dis. 5(2): 42–50. *corresponding author: prof dr somsri wiwanitkit, email: somsriwiwan@hotmail.com 99 j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 143 http://jad.tums.ac.ir published online: june 30, 2021 review article human cutaneous leishmaniosis in iran, up to date-2019 mohamad reza razavi1; *mohammad reza shirzadi2,3; mehdi mohebali4,5; mohammad reza yaghoobi-ershadi6; hassan vatandoost6; mehrshad shirzadi7; mohammad mehdi gouya3; faranak gharachorloo3; shahnam arshi3; behzad amiri3 1department of parasitology, pasteur institute, tehran, iran 2reserch center for zoonoses, ardabil university of medical sciences, ardabil, iran 3center for communicable diseases management, ministry of health and medical education, tehran, iran 4center for research endemic parasites of iran, tehran university of medical sciences, tehran, iran 5department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 6department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 7student of law, islamic azad university, east branch, tehran, iran *corresponding author: dr mohammad reza shirzadi; email: shirzadim@gmail.com (received 18 apr 2021; accepted 31 may 2021) abstract cutaneous leishmaniasis is a neglected and parasitic vector borne diseases that is endemic in tropical and subtropical countries, including iran. the aim of this study was to explain the present status of cl in iran. this report is based on data that recorded by cutaneous leishmaniasis surveillance system in 2019, and evaluated in center for communicable diseases management in ministry of health in iran. iran has been considered an endemic area for cutaneous leishmaniasis in the world. dependent to activities for cutaneous leishmaniasis control the number of cases decreased from 23202 in 2008 (incidence rate 32 per 100000) to 13124 in 2019 (incidence rate 15.8 per 100000), more cases reported from september to december, in 2019, 46% of cases had one lesion and 21% had 2 lesions, 85% of cases diagnosed when the diameter of lesions had 3 centimeters and bellows. although the leishmania control program began in 1977, the incidence of the disease has dropped dramatically since 2008 when the new cutaneous leishmaniasis control program have been implemented. although in some areas the incidence of the disease increased, but the implementation of the new program has reduced the number of cases, in order to continue reducing the disease, permanent support for the control programs is needed. keywords: human cutaneous leishmaniasis; surveillance; iran introduction leishmaniasis are worldwide parasitic infectious diseases caused by protozoa of leishmania genus which among them, approximately 20 species are pathogenic for humans (1-2). natural transmission to mammalian hosts is usually by the bite of female phlebotomus or lutzomya sandflies depending on geography (1). there are three main clinical manifestations with different degrees of severity on the species of parasite involved and elicited host immune responses. these features including cu taneous, mucocutaneous and visceral (1, 3-4). currently, world health organization (who) lists leishmaniasis amongst the nine most important tropical and subtropical diseases which has been reported in all continents except oceania (1, 3). in 2018, 92 countries or territories were considered endemic for, or had previously reported cases of cl. today more than 1 billion people live in areas endemic for leishmaniasis and are at risk of infection. cutaneous leishmaniasis (cl) is the most com copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 144 http://jad.tums.ac.ir published online: june 30, 2021 mon form of leishmaniasis and causes skin lesions, mainly ulcers, on exposed parts of the body, leaving life-long scars and serious disability or stigma. about 95% of cl cases occur in the americas, the mediterranean basin, the middle east and central asia. in 2018 over 85% of new cl cases occurred in 10 countries: afghanistan, algeria, bolivia, brazil, colombia, iran (islamic republic of), iraq, pakistan, the syrian arab republic and tunisia. it is estimated that between 600000 to 1 million new cases occur worldwide annually (5-6). cutaneous leishmaniasis (cl) is a vector borne disease predominantly found in tropical and subtropical countries. it is an important infectious disease in the eastern mediterranean countries, including iran. in the middle east, cutaneous form is more common than visceral leishmaniasis (vl), and roughly 70–75% of cl incidence is reported from six countries: afghanistan, algeria, brazil, colombia, iran (islamic republic), and the syrian arab republic (7-11). some vector borne diseases are endemic in iran and neighboring countries, but the most i important of them is leishmaniasis (12). researchers in iran have conducted several epidemiological studies on leishmaniases since 1941 (5). cl is neglected tropical diseases and cause by various types of intracellular protozoan species of the genus leishmania. iran is endemic for old world cl and cl is one of the differential diagnosis for chronic skin diseases (5-6). two type of cl is endemic in iran as zoonotic cutaneous leishmaniasis (zcl) and antheroponotic cutaneous leishmaniasis (acl). zcl is causes by leishmania major and acl is cause by leishmania tropica. main type the disease (more than 85%) is zcl in iran (5). phlebotomus papatasi has been identified as main vector for zcl in different parts of iran (8, 13, 14) and also the rodents of rhombomys opimus, meriones libycus, tatera indica, nesokia indica, and meriones hurrianae gerbils are repeatedly reported to be infected with l. major in different endemic foci of zcl and act as reservoirs (8, 13, 15-16). in some different places of 18 endemic provinces transmitting the zcl especially in rural areas. phlebotomus sergenti as well as humans and dogs serve as the vectors and reservoirs of acl (8, 13). three main district that where transmitting the acl are mashhad (in khorasan province), kerman and bam (in kerman province) that almost all of cases in this districts are acl, except traveled cases. although in some cities a small number of acl are transmited as neyshaboor, sabzevar, taybad, ghom, shiraz, isfahan, yazd, kashan, zahedan and abadan districts. dependent to national guideline, diagnosis the cases dependent to epidemiological criteria, clinical criteria and laboratory test including confirmation of amastigote forms of leishmania parasites using direct examination with light microscope with high magnification (1000x), and conduct the standard treatment for confirmed cases, these activities needs for surveillance and cl control (17-19). based on description of the legal activities of the ministry of health and medical education in iran for control the diseases (20) as cl, stablished the diagnostic and treatment centers in endemic districts from 2008, in this centers available facilities for giemsa stain and medicine for diagnosis suspected cases and standard treatment the cases free of charge dependent to cl national guide line. and also the effective reservoir and vector control and training population about personal protection and intersectoral cooperation has been explained in national guideline (19, 21). despite the implementation of an effective program for controlling cl in iran the burden of the diseases are still high in some areas of country (22). due to strengthen activities for cl control in iran, who supported the implementation of the training courses for health staff in eastern mediterranean region countries (23). many studies have been conducted on the causes of the spread, increase and outbreak of cl in iran, which have shown that vegetation cover, climatic factors, disasters and population movement play an important roles in this regard (24-29). in addihttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 145 http://jad.tums.ac.ir published online: june 30, 2021 tion, to control the disease while taking special measures in native areas, it is very important to identify high-risk areas to prevent the spread of the disease (30-32). materials and methods study area the cl surveillance system in iran conducting in all provinces and coverage all population in endemic and non-endemic areas. the population of iran is 83,992,949 (2019) and the area of iran is 1,873,959 square kilometers. 18 out of 31 provinces are endemic for cl and around 75% of population in iran are leaving in cl endemic areas. cl is one of the reporting diseases from 1977 and the surveillance system covered this disease in iran. there are surveillance system for case finding, recording and reporting and treatment the cases, the strategic plan for cl control, as inter sectoral cooperation, community education, reservoir control, and vector control. cl is endemic in 18 provinces in iran and some cases diagnosed from non-endemic provinces that had travel to endemic provinces. study design a retrospective, cross-sectional study was carried out using data from the national leishmaniasis surveillance system. the data used in this study include data that collected in centre for communicable diseases control (cdc) in ministry of health and medical education in iran. the portal online computerized recording system stablished from 2000 for record the information of cl cases in district level, and the information of recorded cases in 2019, evaluate in 2020. the iranian cdc in ministry of health and medical education is responsible for surveillance and collection of data on communicable diseases from various districts of the country. leishmaniasis surveillance and control, the information about cl have been collected over 35 years, from 1983 to 2000 data were recorded and reported in paper format and from 2000 to 2006 based on epi-info, version 6 software. online reporting was launched in 2006 for limited districts and after one years the data including microscopical exam as potential tool for the confirmation of all reported cases were gathered online in all districts. the data collected for this survey obtained from surveillance system and include; seasonal trend, individual clinical manifestation, geographical information, laboratory data and treatment methods for all cases of cl. case definitions were based on the national cl control guideline (19). the zoonosis control department was subset of the center for communicable diseases control and information for cl were accessed and analyzed by this department. ethical approval for the study was obtained from the national leishmaniasis expert committee. statistical analysis the data were analyzed using microsoft excel 2013 at the zoonosis control management in centre for communicable diseases control (cdc). results iran has been reported an endemic area for cl in the world. in this report, we describe some cl indicators in iran. after the earthquake in bam district in 2003 number of cases increase from average 400 cases per year before earthquake to 3125 cases in 2006, in this district in 2006 with support the national committee revised the national plan for surveillance and control the cl in iran, and stablished in bam district. after 1 year evaluated the results of activities and the national plan conducted in all provinces. the main activities in national plane are: education health personals, intersectoral cooperation, established leishmaniasis diagnosis and treatment center in endemic districts, community education, environment sanitation, vector control and reservoir control. in cl diagnosis and treatment centers, the facilities for diagnosis and medicine for treatment the cases stablished. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 146 http://jad.tums.ac.ir published online: june 30, 2021 table 1. prevalence of cl in different provinces of iran no endemic provinces geographical zones cl number cl incidence rate/100000 1 semnan center, east 719 95.9 2 ilam west, south 470 78.7 3 golestan north, east 1188 60.9 4 isfahan north, center 3127 59.1 5 fars all 2002 40.0 6 khorasan-shomali south 214 24.0 7 khorasan-razavi all 1581 23.4 8 yazd all 260 21.4 9 ghom center 293 21.3 10 khoozestan all 1013 20.7 11 kerman center, south 565 17.1 12 systan va bloochestan east, south, west 357 12 13 hormozgan west, south, west 160 8.4 14 kermanshah west 117 5.9 15 khorasan-jonoobi east 39 4.8 16 lorestan south-west 69 3.8 17 tehran south 434 3.1 18 booshehr all 28 2.3 fig. 1. trend of cutaneous leishmaniasis iran 1983–2019 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 147 http://jad.tums.ac.ir published online: june 30, 2021 fig. 2. number of c.l cases by month 2019 fig. 3. location of c. leishmaniasis lesions iran 2019 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 148 http://jad.tums.ac.ir published online: june 30, 2021 fig. 4. number of c. leishmaniasis lesions iran 2019 fig. 5. size of c. leishmaniasis lesions iran 2019 trend and distribution in 2019, 13124 cl cases were reported in iran (incidence rate 15.8 cases per 100000 population). in 2008, 23202 cases reported (incidence rate 32 cases per 100000 population) and in this year stablished new surveillance system. and dramatically decreased the cases during 12 years. during this year’s we had two pike in 2014 with 22099 cases and in 2018 with 15714 cases (fig. 1). in 2019, 12636 (96%) of cases reported from 18 endemic provinces and 488 (4%) of cases reported from non-endemic provinces that have traveled to endemic provinces. more cases reported from isfahan province in center in iran with 3127 cases and 51.2 incidence rate per 100000. but highest incidence rate was in semnan province with 95.9 per 100000 population and 719 cases. cl is endemic in 18 out http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 143–151 mr razavi et al.: human cutaneous … 149 http://jad.tums.ac.ir published online: june 30, 2021 of 31 provinces and in tab 1 show the number and incidence rate per 100000 of cl in endemic provinces. seasonal aspects because more than 85% of cases are zoonotic in iran more cases reported from september to december (fig. 2). clinical aspects the cutaneous lesions were mostly on the hand (62%) and foot (26%) and face (21%). in 1177 cases (9%) two or more organ were infected (fig. 3). in 46% of cases had one lesions, and 54% of cases had two or more lesions, 18 cases had more than 21 cases. and maximum number of lesions was 30 lesions (fig. 4). diagnosis the cases in early stage is very important because response to treatment is more effective. and the scar gets smaller. the fig. 5 show the maximum size of the lesions in cl cases and 85% have the 3cm or bellows the diameter. conclusions our study has provided available information on cl in 2019 in iran. the review shows cl is endemic in various parts of 18 provinces in iran and also explain the main clinical manifestation. using national cl control guideline was effective to decreasing dramatically number of cases in recent years but population how are leaving in endemic areas are at risk and also population in other areas is also at risk to take infection if travel to endemic areas. activities for control the cl in national guideline needs to fallows but it dependent to budget, and unfortunately it is not possible to cover all endemic areas. also in non-endemic areas with appropriate climate factors and plants conditions and environment sanitation, that increase population of rodent and sand-flies are high risk areas that cl present in this non endemic places. therefore, strong support is needed for the implementation of the national cl guideline to prevent and control cl spreading and outbreaks. in the other hand the physician have to more attention to cl in differential diagnosis in skin lesions. acknowledgments the author wish to acknowledge all health staff in the provincial and district levelthat their activities do the control of cl in iran. the author declare that they have no competing interests. references 1. torres-guerrero e, quintanilla-cedillo mr, ruiz-esmenjaud j, arenas r (2017) leishmaniasis: a review. f1000 research. 6 (f1000 faculty rev). 750: 1–15. 2. oryan a, akbari m (2016) worldwide risk factors in leishmaniasis. asian pac j trop med. 9(10): 925–932. 3. salam n, al-shaqha wm, azizi a (2014) leishmaniasis in the middle east: incidence and epidemiology. plos negl trop dis. 8(10): 1–8. 4. postigo jar (2010) leishmaniasis in the world health organization eastern mediterranean region. int j antimicrob agents. 36(s suppl1): s62–s65. 5. nadim 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entomology and vector control, school of medicine, hamedan university of medical sciences, hamedan, iran (received 18 dec 2013; accepted 19 feb 2014) abstract background: this study aimed to identify and determine the larval and adult mosquitoes (culicidae) fauna in hamedan county, western iran. methods: it was a cross-sectional study which took place in four area of the hamedan county. sampling methods for larvae, pupae and adults were dipping, hand catch, night catch and total catch. larvae and adult mosquitoes collected and were sent to laboratory of medical entomology, hamedan university of medical sciences, hamedan, iran for further identification to species level to determination of fauna. data analysis was performed using spss software version19. results: three genera and eight species of family culicidae were collected and identified in hamedan county, hamedan province, west iran, during may to october 2013. these species included: culex theileri, cx. pipiens, cx. antennatus, culiseta subochrea, cs. langiareolata, anopheles superpictus, an. maculipennis and an. stephensi. the species cx. antennatus and an. stephensi were reported for the first time in hamedan county. conclusion: an. stephensi and cx. antennatus caught had not been previously recorded in hamedan province. due to vast agricultural activities in the province which provides suitable environment for the establishment of various species of mosquitoes and since many of them are potential vectors of human and domesticated animal pathogens, their ecology needs to be studied extensively. keywords: culicidae, fauna, iran introduction family culicidae, belong to the order diptera, the two-winged flies. the family is a large and abundant group which occurs throughout temperate and tropical regions of the world, and well beyond the arctic circle. the family comprises 3,536 species classified in two subfamilies and 112 genera (harbach 2013). there are many studies on mosquito fauna in iran (zaim 1987, vatandoost et al. 2004, ghavami and ladonni 2005, vatandoost et al. 2006, abai et al. 2007, azari-hamidian 2007, moosa-kazemi et al. 2009, azarihamidian et al. 2010, azari-hamidian 2011, oshaghi et al. 2011, hanafi-bojd et al. 2012, khoobdel et al. 2012, saghafipour et al. 2012, banafshi et al. 2013, soleimani-ahmadi et al. 2013, khoshdel-nezamiha et al. 2014). according to the latest checklist of mosquitoes in iran, seven genera, 16 subgenera and 64 species have been reported (azari-hamidian 2007). in hamedan province, three genera and eleven species of the family culicidae were reported as follows: anopheles superpictus, an. maculipennis sl, an. claviger, an. dthali, an. turkhudi, culex pipiens, cx. theileri, cx. hortensis, cx. perexiguus, culiseta longiareolata and, cs. subochrea (manochehri et al. 1970, saebi 1987, dehghan et al. 2011). among the family culicidae there are vectors of important diseases such as malaria, mosquito-borne viruses, dirofilaria immitis (dog heart worm) and d. repens (dirofilariasis). some of these diseases are particular importance in iran *corresponding author: mr hamid zendehfili, email: hzendehfili@yahoo.com http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 212–218 ah zahirnia and h zendehfili: mosquito fauna … 213 (naficy and saidi 1970, saidi et al. 1976, azari-hamidian 2007, chinikar et al. 2010). these vectors also have an important role in transmission of pathogens to livestock and wild birds and so make them as considerable family for veterinary medicine (marm kilpatrick et al. 2006, azari-hamidian 2011, moosa-kazemi et al. 2012). as a lot of aquatic habitats can be seen around hamedan county which is good places for breeding of culicidae, we decided to conduct a study to identify the species of mosquitoes in the county. materials and methods this was a faunistic and descriptive study from the methodologies point of view, and took place in four geographically direction including villages and rural districts in hamedan county (fig. 1) (saleh abad in the west, dehpyaz in the north, varkaneh in the east and darre morad beyk in the south). twelve fixed and variable stations were selected from may to october in 2013 and larvae, pupae and adult mosquitoes collected by different sampling methods (who 1992). the neighboring provinces are zanjan and qazvin from the north, kermanshah and kurdistan from the west, markazi from the south and east. the main economic activity is agriculture and animal husbandry in the province and maximum and minimum temperature of hamedan city were 35 °c in july and 0 °c in august respectively. the total amount of rainfall in the area is 222 mm, the lowest 2.1 mm in september and the highest was 22 mm in may. the mean annual relative humidity was 35%. sampling was carried out using dipping method for collecting larvae and night catch using suction tubes from animal and human baited traps and total catch with pyrethrum (0.2% in kerosene) spray using a pressurized hand sprayer for adult mosquitoes. after spraying, the room was kept closed for 15 min and the knocked-down mosquitoes were then collected from the floor sheet with forceps and placed in petri-dishes lined with moist cotton. habitat characteristics of adult mosquitoes and environmental data such as temperature and humidity were recorded on special forms. mosquito larvae are founded in many diverse habitats such as ponds, swamps and irrigation canals. the third and fourth collected larvae were preserved in lactophenol medium with recorded information and were sent to the medical entomology laboratory of hamedan university of medical sciences, after a week the specimens were mounted with berlese fluid on microscope slides. the pupae were reared to adult in emergent cages and identified to species by morphological characters. also all collected adult mosquitoes were pinned and then identified using valid keys (zaim and cranston 1986, azari-hamidian and harbach 2009). results a total of 697 adult mosquitoes and 1422 larvae were collected including three genera and eight species which are as follows: culex theileri, cx. pipiens, cx. antennatus, culiseta subochrea, cs. langiareolata, anopheles superpictus, an. maculipennis and an. stephensi (table 1). culex theileri was most abundant species in hamedan county in both adult and larvae stages. two species cx. antennatus and an. stephensi were observed as the first record for the province. number of adults and larvae collected and sampling locations shown in tables 2, 3. the species an. stephensi was collected only in adult stage from saleh abad village in the west of hamedan city. the most and the lowest mosquito collected, were recorded in saleh abad in the west and dehpyaz in the north, respectively. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 212–218 ah zahirnia and h zendehfili: mosquito fauna … 214 table 1. frequency of culicidae adults and larvae collected in the hamedan county, may–october 2013 larvae: n (%)adult: n (%)species 16 (1.1)21 (3.0)an. superpictus 13 (0.9)12 (1.8)an. maculipennis 0 (0.0)2 (0.3)an. stephensi 746 (52.5)342 (49.0)cx. theileri 413 (29.0)191 (27.4)cx. pipiens 53 (3.8)42 (6.0)cx. antennatus 63 (4.4)50 (7.2)cs. subochrea 118 (8.3)37 (5.3)cs. langiareolata 1422 (100)697 (100)total table 2. number and location of larval and adult mosquito collected in four rural regions in hamedan county, may–october 2013 species village saleh abad dehpiaz varcane dare morad beik a (%) l (%) a (%) l (%) a (%) l (%) a (%) l (%) an. superpictus 13(1.8) 6(0.4) 0(0.0) 0(0.0) 8(1.1) 10(0.7) 0(0.0) 0(0.0) an. maculipennis 5(0.7) 9(0.4) 0(0.0) 0(0.0) 7(1) 4(0.2) 0(0.0) 0(0.0) an. stephensi 2(0.2) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) cx. theileri 161(23.0) 324(22.7) 17(2.4) 36(2.5) 76(10.9) 292(20.5) 65(9.3) 41(2.8) cx. pipiens 43(6.1) 118(8.2) 31(4.4) 11(0.7) 48(6.8) 104(7.3) 34(4.8) 115(8) cx. antennatus 42(6) 53(3.7) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) cs. subochrea 11(1.5) 17(1.1) 0(0.0) 0(0.0) 28(4) 39(2.7) 5(0.7) 0(0.0) cs. langiareolata 17(2.4) 23(1.6) 2(0.2) 0(0.0) 15(2.1) 81(5.6) 0(0.0) 0(0.0) total 294(42.1) 550(38.1) 50(7) 47(3.2) 182(16.9) 530(37) 104(14.8) 156(10.8) a: adult l: larva table 3. number and location of larval and adult mosquito family culicidae collected in four urban regions in hamedan county, may–october 2013 species area area1 area2 area3 area4 a (%) l (%) a (%) l (%) a (%) l (%) a (%) l (%) cx. theileri 9(1.2) 26(1.8) 3(0.4) 14(1) 10(1.4) 10(0.7) 1(0.1) 3(0.2) cx. pipiens 11(1.5) 31(2.1) 2(0.2) 6(0.4) 16(2.2) 23(1.6) 6(0.8) 5(0.3) cs. subochrea 3(0.4) 7(0.5) 0(0.0) 0(0.0) 3(0.4) 0(0.0) 0(0.0) 0(0.0) cs. langiareolata 1(0.1) 2(0.1) 0(0.0) 0(0.0) 2(0.2) 10(0.7) 0(0.0) 2(0.1) total 24(3.2) 66(4.5) 5(0.6) 20(1.4) 31(4.2) 43(3) 7(0.9) 10(0.6) a: adult l: larva http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 212–218 ah zahirnia and h zendehfili: mosquito fauna … 215 fig. 1. location of sampling stations in the study area villages: 1. saleh abad, 2. darre morad beyk, 3. varkaneh, 4. dehpyaz sampling stations in hamedan city discussion eight species belonging to three genera were collected and identified from rural and urban locations in four geographical directions in hamedan county. species cx. antennatus and an. stephensi are reported from this region for the first time. anopheles stephensi has a wide distribution in iran including southern slopes and valleys of the zagros chain in ilam, khuzistan, kohgiluye and buyerahmad, lorestan, sistan and baluchistan, bushehr, fars, hormozgan, kerman and also in southern kermanshah provinces (manochehri et al. 1976, zaim and cranston 1986, hanafi-bojd et al. 2012). there are a few published documents about cx. antennatus distribution in the country, but presence of this species has been reported in different parts of iran including homozgan and fars provinces (lotfi 1970). six from eleven species which identified already in the province are reported in this study and two species were observed as new record for the region (manochehri et al. 1970, saebi 1987, dehghan et al. 2011). species an. claviger, an. dthali, an. turkhudi, cx. hortensis and cx. perexiguus which previously reported from the province were not collected in this survey. culex pipiens can be separated from cx. torrentium and cx. vegans by harbach’s key using seta 1-iii-v, seta 1-m, seta 1-x, seta 1-c, and some other characters and our identification was supported with this key (harbach 1988). our results are similar to previous reports that have been made on the mosquito’s larvae in the province (dehghan et al. 2011). some species such as cx. theileri and cx. pipiens are potential vectors for transmission of diseases to humans and animals (lane and crosskey 1993, moosa-kazemi et al. 2012). culex theileri was the most dominant species in this region which is similar to the results of previous studies in sanandaj (moosakazemi et al. 2010), zanjan(ghavami et al. 2005), ardebil (azari-hamidian et al. 2009), isfahan (moosa-kazemi et al. 2000), east azerbaijan (abai et al. 2007) and west azerbaijan (khoshdel-nezamiha et al 2014). this http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 212–218 ah zahirnia and h zendehfili: mosquito fauna … 216 species was collected as the same time in the night which is consistent with the results in sanandaj (moosa-kazemi et al. 2010). most collected mosquitoes were from rural areas, and the results were consistent with similar studies in turkey (simsek 2004) and in iran (ghavami and ladonni 2005). two species cs. langiareolata and cs. subochrea were collected in our study, these findings are consistent with previous results (dehghan et al. 2011). collecting of two adults an. stephensi in saleh abad region (area 1) for the first time in west of the county could be related to climate change and global warming but further studies are needed to confirm this. however climate change will have enormous implication for human health, especially for the burden of vector borne infections diseases (emily and shuman 2010). abundance of suitable larval breeding places such as ponds, wetlands and water ponds, rivers and streams and drainage water in and around the county which many of them are related to agricultural activities contribute to the high frequency of mosquitoes in this area. given the importance of mosquitoes in the transmission of pathogens, and potential risk in emerging and re-emerging diseases, research and monitoring in these places to detect and report the status of the family mosquitoes seems to be essential. conclusion from the results of this study can be concluded that the some species of mosquitoes have a very large distribution in many parts of the province. the results of this and previous studies indicate the presence of 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accepted 02 may 2022) abstract background: the painful bite of pulex irritans; causes wound on the host body and is a vector for bartonella bacteria species, which can cause trench fever, rickettsia species, rocky mountain spotted fever and mediterranean spotted fever. we conducted a study to find out the hosts, abundance, as well as the molecular characteristics of this flea species in meshkin-shahr county, ardabil province, northwest of iran. methods: after collecting fleas from different reservoir hosts and transferring them to the laboratory, identification was done using different morphological characters as well as the internal transcribed spacer1 (its1) molecular marker. results: this morphological study indicated that from the 1053 fleas, which were collected from 162 different animals, including cats and dogs, 74 specimens belonged to human flea, pulex irritans. in addition, in molecular analysis showed a high sequence similarity (99.5%) with the p. irritans counterparts from spain country and zanjan of iran available in genbank. conclusion: pulex irritans species is an obligatory hematophagous ectoparasite of human and animals. therefore, considering the relatively high frequency of this species on the body of cats and dogs, it is suggested to conduct more studies on its distribution and the possibility of being a vector of pathogens among these animals and human. the results of these studies will be used to compile and review the control programs of this vector. keywords: pulex irritans; dog; cat; its1; iran introduction fleas, the common name for the order siphonaptera, have unique properties like the lack of wings, sucking mouthparts, with a specific digestive system. their digestive system is different from other blood-feeding insects structurally and functionally. the flea body pressed laterally. it is different from other insects that have stretched bodies from each side. in addition, a flea has a great leaping ability, which is the result of strong feet and the evolution of the curves of sides. it feeds from a wide range of hosts including birds and mammals. these features make this order of insect different from others (1). the results from the molecular stud ies on dna and morphological studies and ovary features showed that the closest relative of fleas is boreidae from mecoptera species (2). today in the world, over 2500 species of fleas have been identified whose hosts are mostly birds and mammals (3). the desire for blood feeding from warm-blooded animals has led to some of these species being vectors of some infectious diseases, and besides being the main host for some tapeworms like dipylidium caninum (4). the family pulicidae of fleas has a great desire to feed blood from humans or other mammals. the existence of similar morphological characters among different species of copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:dalimi_a@modares.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 197 http://jad.tums.ac.ir published online: sep 30, 2022 pulex, as caused most of the species of this type, to be hard to differentiate (5). one of the most famous species of this genus is pulex irritans. despite its name, this species does not have a definite host and can suck blood from a wide range of mammals, and this factor makes it a carrier of various diseases such as dipylidium caninum and trench fever. rickettsia species, which can cause rocky mountain spotted fever and mediterranean spotted fever (6). the existence of a wide range of hosts and compatibility with different climatic areas results in finding this flea in many different areas of the world (7). most of the studies done on this species in some areas of iran have related to morphological characters and there is little data about its molecular characters. in this study, the internal transcribed spacer1 of ribosomal dna (its1-rdna) gene, was determined to provide a molecular marker for identification of the local iranian p. irritans population. materials and methods study area ardabil province is one of the thirty-one provinces of iran. meshkin-shahr county is one of the counties located in the province in the northwest part of iran (fig. 1). meshkinshahr county with geographical coordinates 38° 44' n and 47° 40' e is at an altitude of 1400 meters above sea level. morphological studies the present study was conducted during march and july 2018 in meshkinshahr county, ardabil province. the sampling was done from random captured cats (felis catus) and dogs (canis lupus) specimens, with two standard methods of combing and separating with forceps (9). after collecting and recording the reservoir's host data; their ectoparasites were transferred to the glass tubes with lids containing 70% ethanol for further identification (fig. 2). first, the flea specimens from each host were cleared with 10% koh individually and the permanent slides in canada balsam were prepared. for the morphological identification of flea specimens, we used the standard key of harimalala et al. (10). after morphological identification and final confirmation, specimens were taken out of slides, washed, and transferred to tubes containing 90% ethanol. molecular studies the identified flea specimens were used for dna extraction and molecular study. after morphological studies, a sample of each flea was placed under a binocular on a slide and its abdominal contents were completely emptied and its information was recorded and transferred to a new microtube. the flea was then crushed into a microtube with a pistol. the microtube was transferred to a nitrogen tank for 5 minutes and then the dna of fleas was extracted using dneasy kit (qiagen, hilden, germany) and stored at -20 ºc for later use. the its1 primers used of p. irritans were nc5 (5′-gta ggt gaa cct gcg gaa gga tca tt-3′) and its1rev (5′-gct gcg ttc ttc atc gac cc-3′) as forward and reverse respectively (11). in each 15µl reaction of pcr, the 0.50–1µl of each extracted dna, 2µl primer (10pmol of each), 7.5µl 2x master mix (taq dna polymerase master mix red, 2mm mgcl2, amplicon) were used. the pcr reaction was conducted with a thermal cycler device along 33 cycles (94 degrees for 30 seconds, 58 degrees for 30 seconds, and 72 degrees for 1 minute) and for the final extension, 10 minutes with 72 degrees was used. the pcr products were visualized in a 1.5% agarose gel containing a dna-safe stain (cat no., ep5082, sinaclon). pcr product of each sample sequenced by the abi3730xl sequence analyzer (macrogen, s. korea). the sequences were edited and aligned using clustal omega (https://www.ebi.ac.uk/tools/msa/clustalo/) and compared with reference sequences from genbank. for its1 sequences, the phylogenetic tree was built with the maximum likelihood (ml) algorithm using molecular evolutionary genetics analysis (mega) software, inhttp://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 198 http://jad.tums.ac.ir published online: sep 30, 2022 cluding sequences representative of three families (pulicidae, leptopsyllidae, ceratophylidae) of order siphonaptera. results the results of the morphological study indicated that among the 1053 flea specimens, which were isolated from two animal hosts, 47 dogs and 115 cats, 74 (7.03%) specimens belonged to p. irritans. it was specified that from 74 specimens of p. irritans fleas, 35 (47.29%) were males and 39 (52.70%) were females (table 1). out of the 74 p. irritans fleas, 49 (66.21 %) were collected from dogs; 32 (43.24%) were collected from female dogs, and 17 from male dogs, and the other 25 samples were taken from male cats. in this study, p. irritans flea were not found on female cats (table 2). the morphological characteristic of p. irritans is shown in figure 3. the specific primers used in this study amplified about 900bp of its1 region of rdna gene. (fig. 4). the analysis of the its1 sequence of the identified isolates approved the morphological identification. the sequence of the isolated from the present study is accessible under genbank accession no. mg745174. after trimming the sequence data, 802bp of sequences were blasted. the sequence obtained in this study displayed 99.5% sequence homology with two p. irritans specimens from spain (genbank id: lt797452) and zanjan (genbank id: kx822017) in west of iran (fig. 5). in addition to these two sequences, there were some other p. irritans its1 sequences in genbank originated from spain, different locations of west of iran (including mahneshan zanjan, khoramabad, kamyaran, hamedan, bahar, gilangharb, kerrmanshah, mahabad, sanandaj, and urmia), china, and india which displayed 95.69-99.38% sequence homology with the sequence obtained in this study. to assess phylogenetic relationship among p. irritans isolates, multiple alignments were generated for a subset of p. irritans specimens plus the sequence obtained in this study, and the alignment was used to construct a phylogenetic tree by neighbor joining (nj) method. phylogenetic relationships of the p. irritants using the its1 region of the rrna gene produced the highest log likelihood as shown in figure 6. the sequence obtained in this study was strongly associated with the specimen from spain (genbank id: lt797452). most of the nodes in the tree had 98-99% bootstrap values indicating very strong support for the nodes created in the tree. table 1. geographical features of the study area, prevalence and distribution of fleas and their animal hosts in meshkinshahr county, ardabil province, iran, 2016-2018. collected fleas cats dogs location sampling area % n % n % n east north 14.43 152 16.52 19 10.64 5 473852 382452 parikhan 9.59 101 21.79 25 14.89 7 474054 382246 meshkinshahr 2.66 28 15.65 18 6.37 3 473110 383105 sarikhanloo 33.33 351 13.04 15 12.76 6 473804 382115 urkandi 17.76 187 6.96 8 6.37 3 473336 382441 ahmad bigloo 0.57 6 5.22 6 12.76 6 474023 382102 aghbalagh 19.37 204 16.52 19 17.02 8 473053 382918 koojangh 2.28 24 4.35 5 19.14 9 473534 382200 ahmad abad 99.99 1053 100 115 99.95 47 …… …….. total http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 199 http://jad.tums.ac.ir published online: sep 30, 2022 table 2. prevalence of host’s flea infestation in meshkinshahr county, ardabil province, iran, 2016-2018. m: male flea, f: female flea pulex no. (%) fleas on cats fleas on dogs total fleas sampling area % f % m % total %/74 pulex % no %/74 pulex % no 12.1 9 8.1 6 18.9 14 4.0 3 15.9 18 14.8 11 14.2 134 152 parikhan 2.7 2 0.0 0 2.7 2 1.3 1 19.4 22 1.3 1 8.5 79 101 meshkinshahr 6.7 5 0.0 0 6.7 5 0.0 0 0.0 0 6.7 5 2.9 28 28 sarikhanloo 33.7 25 16.2 12 50.0 37 8.1 6 33.6 38 41.8 31 33.3 313 351 urkandi 2.7 2 0.0 0 2.7 2 0.0 0 4.4 5 2.7 2 19.3 182 187 ahmad bigloo 0.0 0 0. 0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.6 6 6 aghbalagh 5.4 4 9.4 7 14.8 11 2.7 2 15.0 17 12.1 9 19.8 187 204 koojangh 1.3 1 1.3 1 2.7 2 2.70 2 11.5 13 0.0 0 1.1 11 24 ahmad abad 64.8 48 35.1 26 100 74 18.9 14 100 113 81.0 60 100 94 1053 total fig. 1. map of area study meshkinshahr county, ardabil province, iran. fig. 2. isolation and collection of fleas from a dog in meshkinshahr, northwest of iran using the standard method of combing and transferring them to tubes containing 70% ethanol (original) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 200 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 3. morphological characters of pulex irritans. a: eye hair is under the eye; b: the borderline of moral rood which does not exist; c: the first part of spermatheca which is not distinguishing; d and e: end part of the female and male genus (original) fig. 4. the its1-rdna pcr products (about 900bp) on agar gel 1.5%. ladder: 100bp dna ladder (sinclon, iran), 1: negative control (ddh2o); 2: pulex irritans isolated from stray cats, 3 and 4: pulex irritans isolated from stray dogs http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 201 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 5. the multiple alignments of 802bp of its1-rdna sequence of pulex irritans from this study (meshkinshahr, genbank id: mg745174) and two reference sequences from znajan, iran (genbank id: kx822017) and spain (genbank id: lt797452). there was 99.5% similarity between meshkinshahr specimen with the zanjan and spain specimens. * indicates similar nucleotide, and gaps indicates substitution in the position http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 202 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 6. phylogenetic relationships inferred from neighbor joining analyses constructed by mega10 based on 802bp sequences of its1-rdna gene of pulex irritans obtained in this study and a subset of pulex irritans sequences available in genbank. the genbank id of the sequences are shown in front of branches. xenopsylla cheopis with accession number dq 295061.1 was used as out-group. scale bar indicates genetic distance. our sample is marked with an arrow discussion all human fleas collected specimens are characterized by laterally compressed bodies and large hind legs. pulex irritans lack the combs that lend the appearance of genal and pronotal combs. it has a rounded forehead, which allows differentiation from the anteriorly flattened head of the stick-tight flea. behind the antennae, the head has only a single pair of hairs. it has no pleural rod, a feature that differentiates it from xenopsylla cheopis (12). in terms of medical importance, p. irritans can play the role of the intermediate host of the dog tapeworm (for example d. caninum). in addition, p. irritans may be a vector for erysipeloid (13). moreover, the p. irritans is implicated in the spread of diseases historically associated with x. cheopis (14). the human female flea can lay more than 400 eggs in her lifetime. the ability of pupae and “cocoons” adults to lie dormant for a year or more can confound efforts at flea control. it does not kill adult fleas, but it does prevent reproduction. adverse reactions flea bites appear as tense, pruritic, urticarial papules on exposed areas including the hands, forearms, and lower legs. excoriation with secondary impetiginization is common. vesicular and bullous flea bite reactions occur and can be quite dramatic. histologic examination reveals a wedge-shaped dermal infiltrate that surrounds both venules and arterioles. eosinophils are common. epidermal necrosis, spongiosis, and intraepidermal or subepidermal bullae may be seen. the human flea is distributed throughout the world and has been recorded in a wide range of mammal hosts including humans, domestic dogs, cats, pigs, badgers (meles meles) and foxes (vulpes vulpes) (15). this species is also medically important as a possible vector of flea-borne diseases such as tularemia, murine typhus, and tapeworm (16). in different parts of the world, for isolating and identifying p. irritans, different characters have been used. out of the genus pulex, the species p. irritans is the most abundant in the world and in iran (17). the results of the morphological studies indicated that out of the http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 196–205 a azarm et al.: pulex irritans on … 203 http://jad.tums.ac.ir published online: sep 30, 2022 1053 fleas, 74 specimens belonged to p. irritans. in a similar study for investigating ectoparasite of dogs and cats in kurdistan province west of iran and bordered with the northeast of iraq country, this species was isolated with ctenocephalides felis and c. canis but with a lower percentage of frequency (18). moreover, in a review by maleki-ravassan et al. (19) on ectoparasite of dogs in different parts of iran, p. irritans species and two species of c. felis and c. canis were isolated from hosts. in addition, another study, which was conducted on dogs in shiraz, had similar results (20). in a study in 2015 for identifying ectoparasite on sheep in kurdistan province bordered with the east of iraq country, 1323 specimens of p. irritans were isolated from hosts (21). in addition, sharifdini et al. 2021 were collected p. irritans from seven raccoons from guilan province, northern iran (22). in comprehensive research by barutzki and schaper (23) in 2000 for identifying ectoparasite of dogs and cats in germany, out of 1508 cats and 2653 dogs, c. felis, c. canis, and p. irritans were isolated. already, there are about 2500 species and subspecies of fleas in about 220 genera, but only relatively, few are important human pests. in this study, the result of the morphological analysis showed that the identified flea was p. irritants. the its1 gene sequence obtained in this study plus the phylogenetic tree, and high sequence homology with other available data in genbank, confirm and support the result of our morphological study. conclusion pulex irritans is one of the most important ectoparasites of humans, which transmits various pathogens (including bartonellosis and rickettsioses) and causes skin sensitivity and irritation by biting humans and animals. this flea is transmitted from the cats and dogs body to humans and can bite humans. to achieve control of parasitic skin diseases of p. irritans in developing countries, local epidemiology has to be better understood. studies in iran on epidemiology and the medical significance of p. irritans in different areas of animals are limited. molecular approaches, although in their infancy, are now providing a better understanding of the biology of ectoparasites and will become cornerstones for prevention and control in the future. therefore, this study was conducted in order to accurately identify this vector based on morphological and molecular characteristics. morphological studies show that there is no moral root border in this flea and the first part of the spermatheca is not differentiated. in this study, p. irritans was not caught in the body of female cats, which is not important from the point of view of disease epidemiology because both male and female fleas are able to feed on blood and transmit the disease to cats, dogs, and humans. the specific primers used in this study amplified the its1 regions of about 900bp. it is suggested that to find out the distribution of p. irritans in male and female cats, as well as the possibility of p. irritans being a vector among cats, dogs and human, more studies should be conducted at the country level. acknowledgements the authors would like to thank all personals who work in department of medical entomology tarbiat modares university, tehran, iran for their helping to do this research. ethical considerations 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1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2oral and maxillofacial pathology, school of dentistry, bushehr university of medical sciences, bushehr, iran *corresponding author: dr hamideh edalat, e-mail: edalat@tums.ac.ir (received 08 may 2018; accepted 12 nov 2020) abstract background: malaria has long been regarded as one of the most important public health issues in iran. although the country is now in the elimination phase, some endemic foci of malaria are still present in the southeastern areas of the country. in some endemic foci, there are no data on the malaria vectors. to fill this gap, the present study was designed to provide basic entomological data on malaria vectors in the southeastern areas of iran. methods: adult and larval stages of anopheles mosquitoes were collected by using different catch methods. resistance of the main malaria vector in the study area to selected insecticides was evaluated using diagnostic doses advised by the world health organization in 2013–2014. results: a total of 3288 larvae and 1055 adult anopheles mosquitoes were collected, and identified as: anopheles stephensi (32.1%), anopheles culicifacies s.l. (23.4%), anopheles dthali (23.2%), anopheles superpictus s.l. (12.7%), and anopheles fluviatilis s.l. (8.6%). anopheles stephensi was the most predominant mosquito species collected indoors at the study area, with two peaks of activity in may and november. this species was found to be resistant to ddt 4%, tolerant to malathion 5% and susceptible to other tested insecticides. conclusion: all the five malaria vectors endemic to the south of iran were collected and identified in the study area. our findings on the ecology and resting/feeding habitats of these malaria vectors provide information useful for planning vector control program in this malarious area. keywords: malaria; anopheles; malaria vectors; bio-ecology; iran introduction malaria has long been regarded as one of the most important public health issues in iran. the disease caused irreparable financial and fatality losses in the country, which made initiation of elimination necessary. the most important endemic foci in the country are sistan and baluchistan, hormozgan and kerman provinces, which in total, account for 96% of all cases. at present, iran is in the process of eliminating malaria, and under this condition, even low number of reported cases is very important (1). adequate understanding of the association between the behavioral characteristics of the disease vectors and their ecology is important in the planning and determination of strategies to fight against the disease. anopheles species has been considered an important part in malaria transmission cycle after its role in the transmission of the disease was discovered. the ability of anopheles mosquitoes to transmit plasmodium infections is attributed to the physiology and biochemistry of their bodies, which are different according to species characteristics. other factors such as frequency of blood feeding, longevity, ecological and environmental conditions are important in this respect (2). according to the last checklist of mosquitoes of iran, there are 30 anopheles species (3). kerman province has a long history of ma copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:edalat@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 326 http://jad.tums.ac.ir published online: december 31, 2020 laria, and even though the disease has been well controlled in other endemic areas of the country, malaria is still considered a major health problem in the province. previous studies have identified nine anopheles species in the province, and among them, anopheles fluviatilis s.l., anopheles dthali, anopheles culicifacies s.l., anopheles stephensi and anopheles superpictus s.l. have been reported as vectors of malaria (4-6). residual spraying and use of insecticidetreated bed nets have recently been implemented in qaleh-ganj county in kerman province. due to the higher potential of malaria transmission by anopheles vectors, it was necessary to carry out studies on the fauna and ecology of anopheles species in the area and their susceptibility to some of the conventional pesticides. the aim of this study was to collect information about the fauna and bioecology of anopheles mosquitoes in the area, and to determine their susceptibility to some selected insecticides. our study intended to provide data that can be useful for future vector control programs in the area. materials and methods study area kerman province is located in the southeast of the central plateau of iran (fig. 1). the province has a mean annual rainfall of 152.9mm, and according to the current national counties distribution, the province is composed of 16 counties. qaleh-ganj is a county located in the south of the province (27.5277° n, 57.8651° e), with a population of about 70,000 people, 17% of the people live in urban and 83% in rural areas. according to the koppen-geiger climate classification, qale-ganj county is classified under hot desert climate (bwh); however, in recent years due to reduced rainfall, drought has gripped the area. the maximum and minimum recorded temperatures in the district are 52 and 2 °c, respectively. monthly maximum and minimum relative humidity in the district is 70% and 35%, respectively, and rainfall in the area is between 0–125mm (statistical yearbook of kerman province, 2015). entomological survey in this study, sample collection was carried out in three villages including: shah-kahan (27.519126°n, 57868245°e), marz (27.537 180°n, 57.852005°e), and rameshk (27.521 183°n, 57.857334°e), during 2015. study sites were chosen according to the who standard techniques (7). mosquitoes were collected using the simple sampling method, before the activity season of the vectors. specimen collection was carried out over a period of 12 months using total catch, pit shelter, light trap, window trap and dipping methods (7) and by using night catch method during the month of may in 2015. total catch method was used to determine species richness of each location and monthly changes in species population and diversity, and to determine the physiological status of endophagic and endophilic species. artificial pit shelters were created for the collection of outdoor adult mosquitoes. a pit was drilled in each study location (village). cavities with a dimension of 30×30cm were dug in the walls of the pit shelters about 0.5 meters from the floor of the shelter. these attractive cavities served as resting-sites for mosquitoes entering the pit. mosquitoes were aspirated from the pit shelters using an aspirator, before sunrise (6– 9am) in each day of collection. another method used for mosquito sampling in our study was the light trap method. during the study, a cdc light trap was used, before sunset and until sunrise in the next day, to collect mosquitoes in ramsehk village. to study the feeding and resting behaviors of adult mosquitoes, window exit traps were installed on the outside of window frames in selected locations. collected females were identified at species level and their abdominal condition was recorded. night collection method using human and animal (cow) baits was conducted to identify the host preference of anophhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 327 http://jad.tums.ac.ir published online: december 31, 2020 eles mosquitoes in the study area. in this method, mosquitoes were collected as soon as they landed on the host using torch and aspirator, from sunset to sunrise. to determine the peak of host seeking and blood feeding activity, samples collected in each hour were kept in individual cups covered by a fine net and labeled based on the place and time of capture. after adult specimen collection, the mosquito species were mounted on entomological pins and were identified with a morphological key (8). female mosquitoes collected by the different methods were classified according to their abdominal condition as: gravid (g), semigravid (sg), unfed (u) and/or freshly blood-fed (f) (7). in the present study, anopheline mosquito larvae were collected using the dipping method (7). physical characteristics of the larval habitats were recorded during larval collection. collected larval specimens were preserved in lacto-phenol for least 24h prior to specimen preparation for microscopic identification. the specimens were mounted on glass slides using chloral-gum mounting media and were covered with coverslips. the glass slides were then placed in an incubator at 37 °c to dry before observing under a microscope. identification key was used to identify the mounted samples based on morphological characteristics (8). susceptibility test studies susceptibility tests were performed using insecticide-impregnated papers on 2 to 3 day old dominant anopheles species fed with 5% sugar. mosquitoes were exposed to insecticide-impregnated papers at diagnostic doses, as described by the world health organization pesticide scheme guidelines, for one hour, and mortality after 24-hour recovery period was recorded. the tests were carried out at a temperature between 22–26 °c and relative humidity of 60%. in the present study, ddt 4%, malathion 5%, propoxur 0.1% and deltamethrin 0.05% were used for susceptibility testing. mortality rate of 98–100% was considered as susceptible, 90–97.99% as tolerant, and < 90% as resistant (9). for each insecticide, four replicates of 25 2 to 3day old sugarfed female mosquitoes were tested for susceptibility, whilst two replicates were used as controls. in this study: if control mortality was less than five percent, the results of tests were considered acceptable; if control mortality was between 5–20%, the test results were corrected using abbotts' formula; and if control mortality was > 20%, the tests were repeated (9). results mosquitoes collection a total of 3288 larvae and 1055 adult anopheles mosquitoes were collected and identified as an. stephensi (32.1%), an. culicifacies s.l. (23.4%), an. dthali (23.2%), an. superpictus s.l. (12.7%) and an. fluviatilis s.l. (8.6%). results are described, according to the method of collection, as follows: total catch a total of 541 anopheles species were collected indoors using this method and an. stephensi (47.31%) was the most predominant species. the abundance of other species collected by this method is as follows: 114 (21.07%) belonged to an. culicifacies s.l., 123 (22.73%) belonged to an. dthali, 7 (1.32%) belonged to an. fluviatilis s.l., and 40 (7.57%) belonged to an. superpictus s.l. (fig. 2). anopheles stephensi was collected throughout the sampling period except in january and february. pit shelter a total of 137 anopheles species were collected from shelter pits, and an. fluviatilis s.l. was the most predominant species (35.76%). anopheles superpictus s.l. was also identified by this method (fig. 3). light trap three anopheles species were collected by this method, and an. superpictus s.l. was the most numerically dominant. other species colhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 328 http://jad.tums.ac.ir published online: december 31, 2020 lected by this method include an. culicifacies s.l. and an. stephensi (table 1). window trap a total of 57 female anopheles mosquitoes representing four species were trapped by window traps. anopheles dthali (33.3%), an. culicifacies s.l. (31.6%), an. stephensi (26.3%), and an. superpictus s.l. (8.8%) were collected and identified using this method. night catch using human/animal baits table 2 shows the abundance of adult mosquitoes collected by night catch method using human and animal baits during may 2015. using human baits, the highest collection occurred in the first third of the night, and the species were identified as an. superpictus s.l. and an. fluviatilis s.l. an. culicifacies s.l. and an. fluviatilis s.l. were also collected using animal baits, with the highest collection also occurring at the first third of the night. blood feeding peak of an. fluviatilis s.l. and an. culicifacies s.l. were 8–9pm and 10–11pm, respectively. abdominal status of collected female mosquitoes abdominal condition of the female anopheles mosquitoes collected by three different methods was examined. we classified the females as unfed (u), blood fed (f), and gravid/ semigravid (g/sg), according to their abdominal condition (tables 3–5). total catch the number of gravid and semi gravid an. stephensi collected by this method was higher than the number of unfed and blood-fed an. stephensi. unlike an. stephensi, the number of gravid and semi gravid an. culicifacies, an. fluviatilis s.l., an. superpictus s.l. and an. dthali was lower than the number of unfed and blooded ones (table 3). pit shelter the number of gravid and semi gravid an. culicifacies s.l. species was higher than the number of unfed and blood-fed an. culicifacies s.l. in contrast, the number of gravid and semi gravid an. stephensi, an. fluviatilis s.l., an. superpictus s.l. and an. dthali was lower than that of unfed and blood fed ones (table 4). window trap the number of gravid and semi gravid an. culicifacies s.l. and an. stephensi was higher than the number of unfed and blood-fed ones; in contrast, the number of gravid and semi gravid an. fluviatilis s.l., an. superpictus s.l. and an. dthali was lower than the number of unfed and blood-fed species (table 5). larval collection anopheles stephensi (31.59%) and an. fluviatilis s.l. (7.29%) were respectively the most abundant and least abundant larval species collected in the study area. the results of species abundance per 10 dips have been summarized in fig. 4. characteristics of larval habitats in the study area have been detailed in table 6. susceptibility tests mortality rate of an. stephensi against ddt 4%, malathion, propoxur, and deltamethrin was 25%, 97%, 99% and 98%, respectively. mortality in the control against all insecticides tested were zero except for malathion and propoxur. anopheles stephensi was quite resistant to ddt, but tolerant to malathion and sensitive to the remaining insecticides. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 329 http://jad.tums.ac.ir published online: december 31, 2020 table 1. the number of adult mosquitoes collected by light trap in rameshk cllage, qaleh-ganj county, kerman province of iran, 2015 species j a n f e b m a r a p r m a y j u n j u l a u g s e p o c t n o v d e c t o ta l % an. stephensi 0 2 0 0 0 0 0 0 3 1 0 0 6 27.27 an. culicifacies 0 0 0 0 0 0 0 0 0 2 2 0 4 18.18 an. superpictus 0 2 3 1 0 0 0 2 0 1 3 0 12 54.55 table 2. the abundance of mosquitoes collected on human/animal baits by night catch method, qaleh-ganj county, kerman province of iran, may 2013 bait species catch time (night) total 1st third 2nd third 3rd third human an. stephensi 7 2 1 10 an. culicifacies 5 2 2 9 an. superpictus 9 5 4 18 an. dthali 6 3 1 10 an. fluviatilis 11 6 2 19 total 38 18 10 66 animal an. stephensi 4 3 2 9 an. culicifacies. 15 3 4 22 an. superpictus 9 5 4 18 an. dthali 12 2 1 15 an. fluviatilis 19 6 4 29 total 59 19 15 93 table 3. abdominal status of collected female mosquitoes by total catch, in the study area, qaleh-ganj county, kerman province of iran, 2015 t im e species/abdominal status an. stephensi an. culicifacies an. superpictus. an. dthali an. fluviatilis u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l mar 6 15 28 49 3 9 7 19 1 3 2 6 6 14 12 32 0 0 0 0 apr 7 17 44 68 2 14 25 41 2 5 7 14 0 21 15 36 0 3 2 5 may 5 22 14 41 1 8 6 15 0 2 5 7 3 6 2 11 0 2 0 2 jun 2 6 3 11 0 2 2 4 0 3 0 3 0 2 1 3 0 0 0 0 jul 0 2 3 5 0 2 0 2 0 1 0 1 1 2 0 3 0 0 0 0 aug 0 0 2 2 0 0 1 1 0 0 0 0 0 0 1 1 0 0 0 0 sep 2 2 5 9 1 1 2 4 0 1 2 3 1 4 4 9 0 0 0 0 oct 9 15 24 48 2 5 8 15 0 0 0 0 3 5 4 12 0 0 0 0 nov 2 7 5 14 2 6 2 10 0 3 1 4 2 7 3 12 0 0 0 0 dec 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 jan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 feb 3 3 3 9 0 2 1 3 0 2 1 3 0 3 1 4 0 0 0 0 total 36 89 131 256 11 49 54 114 3 20 18 41 16 64 43 123 0 5 2 7 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 330 http://jad.tums.ac.ir published online: december 31, 2020 table 4. abdominal states of collected female mosquitoes by shelter pit, in the study area, qaleh-ganj county, kerman province of iran, 2015 time species/abdominal status an. stephensi an. culicifacies. an. superpictus an. dthali an. fluviatilis u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l mar 0 3 1 4 0 1 1 2 1 1 3 5 0 0 0 0 1 4 2 7 apr 1 2 2 5 0 1 2 3 1 4 4 9 0 2 1 3 2 7 3 12 may 0 1 1 2 0 1 2 3 1 2 1 4 1 2 0 3 0 3 3 6 jun 0 1 2 3 0 1 0 1 1 1 0 2 1 0 1 2 1 1 1 3 jul 0 1 0 1 0 2 1 3 0 0 2 2 0 1 0 1 0 0 0 0 aug 1 1 0 2 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 sep 1 1 1 3 0 0 1 1 0 2 1 3 0 2 1 3 0 2 1 3 oct 0 3 2 5 0 0 0 0 0 0 1 1 0 0 1 1 1 2 3 6 nov 2 0 1 3 0 1 1 2 0 0 0 0 0 0 0 0 2 2 2 6 dec 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 jan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 feb 0 2 0 2 0 1 0 1 0 0 0 0 0 1 1 2 0 2 2 4 total 5 15 10 30 0 8 9 17 4 10 12 26 2 8 5 15 7 24 18 49 table 5. abdominal status of collected female mosquitoes by outdoor window trap, in the study area, qaleh-ganj county, kerman province of iran, 2015 time species/abdominal status an. stephensi an. culicifacies an. superpictus an. dthali u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l u f g + s g t o ta l mar 0 0 2 2 0 0 1 1 0 0 0 0 0 1 1 2 apr 0 0 1 1 0 1 2 3 0 0 1 1 0 0 2 2 may 0 0 1 1 0 0 1 1 0 0 0 0 0 0 1 1 jun 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 jul 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 aug 0 1 1 2 0 0 0 0 0 0 0 0 0 0 0 0 sep 0 0 0 0 0 1 1 2 0 0 0 0 0 0 0 0 oct 0 1 1 2 0 1 3 4 1 1 0 2 0 2 1 3 nov 1 1 2 4 0 1 1 2 0 0 0 0 0 3 1 4 dec 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 jan 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 3 feb 0 1 2 3 1 1 2 4 0 1 1 2 0 3 1 4 total 1 4 10 15 1 5 12 18 1 2 2 5 0 11 8 19 table 6. characteristics of larval habitats in the study area, qaleh-ganj county, kerman province of iran, 2015 categories an. stephensi an. culicifacies an. superpictus an. dthali an. fluviatilis habitat situation constant 64 75 67 64 72 temporary 36 25 33 36 28 running water 100 100 100 100 100 resident water 0 0 0 0 0 vegetation situation without vegetation 64 75 67 64 72 with vegetation 36 25 33 36 28 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 331 http://jad.tums.ac.ir published online: december 31, 2020 type muddy 36.4 25 33 36 28 sand 27.6 38 23 26 30 stone or cement 36 37 46 38 42 water quality turbid 64 75 67 64 72 fresh 36 25 33 36 28 sunlight situation sunny 27.5 38 23 26 30 semi-shade 72.5 62 77 74 70 shade 0 0 0 0 0 habitat natural 64 75 67 64 72 artificial 36 25 33 36 28 temperature (oc) mean 29.5 28 26.5 31.5 22 ph mean 7.6 7.6 7.6 7.6 7.6 fig. 1. study area in kerman province of iran fig. 2. the abundance of mosquitoes collected from indoors by total catch, in the study area, qaleh-ganj county, kerman province of iran, 2015 http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 332 http://jad.tums.ac.ir published online: december 31, 2020 fig. 3. the abundance of mosquitoes collected by shelter pit method in the study area, qaleh-ganj county, kerman province of iran, 2015 fig. 4. larval abundance per 10 dips in the study area, qaleh-ganj county, kerman province of iran, 2015 discussion anopheles stephensi among the 541 anopheles species collected indoors by the total catch method, an. stephensi was the most predominant species. it was highly abundant in may. in contrast to our study, an. stephensi was sampled throughout the year in another study in southeastern iran, with the peak of activity occurring in february and september–october (10). in another study, the peak of an. stephensi activity was found in may and november in bandar abbas in southern iran (11). however, a recent study conducted in jask county, south of our study area, reported two peaks of activity for an. stephensi in march–april and october (12). the peak of activity of an. stephensi is directly affected by weather variables especially temhttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 333 http://jad.tums.ac.ir published online: december 31, 2020 perature, which varies between different study areas. the density of anopheles stephensi collected in shelter pits was low. a similar result was reported in the southern iran (10). with the night catch method, the highest number of an. stephensi mosquitoes was collected in the first third of the night between 9–10pm. in a study conducted at khesht area in fars province in iran, similar results were obtained (13). it should be mentioned that this method was only performed during the month of may. we recommend this method to be used at least in all months of the malaria transmission season in future studies in the study area. in this study, the number of gravid and semi gravid an. stephensi collected by shelter pit method was lower than the number of unfed and blood-fed ones, but in total catch method, the gravid and semi gravid ratio was more than 1. this shows that an. stephensi has a higher endophilic tendency. in a similar study conducted in sistan and baluchistan and hormozgan provinces of iran, an. stephensi was found to be the most numerically dominant among the sampled mosquito species. moreover, the number of gravid and semi gravid an. stephensi collected in the study, both in shelter pits and indoors, was higher than the number of unfed and bloodfed species. the investigators indicated that an. stephensi is more endophilic compared with the other species sampled in the study areas; with g+sg/f+uf ratio lower than one both outdoors and indoors (18) anopheles culicifacies s.l. in our study, the number of gravid and semi gravid an. culicifacies s.l. collected by total catch method was lower than unfed and bloodfed ones, but in the pit shelters and outdoor window trap methods, the gravid and semi gravid ratio was more than 1. moreover, the high number of unfed mosquitoes compared with gravid and blood-fed mosquitoes collected by indoor window trap method shows a high endophagic tendency of this species. in contrast to our findings, in a study conducted in sistan and baluchistan, the number of gravid and semi gravid an. culicifacies s.l. collected indoors was lower than the number of unfed and bloodfed mosquitoes. the researchers also reported the same abdominal state findings for an. culicifacies s.l. mosquitoes collected from pit shelters (14). another study conducted in sistan and baluchistan indicated that this anopheles species is more endophilic (15, 16). consistent with our study, based on ventral aspect ratio (g+sg/u+f), another study stated that an. culicifacies s.l. prefers both indoors and outdoors as their resting places (10). similar results have also been reported in different regions of india (17). although this species had two peaks of activity during march–april and october–november, a study conducted in sistan and baluchistan in iran, larvae of an. culicifacies s.l. were more abundant in april to december in rice fields with palm trees (18). in terms of breeding sites, this species was mostly collected from sites which have turbid water, semi-shade and have no vegetation. the mean temperature and ph of the study area were 28 oc and 7.6, respectively. an earlier study conducted in the south of our study area reported clear, no vegetation, sunny, and natural breeding sites with average temperature and ph of 25–30 oc and 7.14–8.90, respectively as climate preferences of this species (19). anopheles dthali this species was most abundant in may, june and october during the study period. a previous study conducted in a relatively warmer area in iran reported that the peak of activity of this species occurs in april and september–october, which is one month earlier than observed in our study (20). it seems that weather conditions play crucial role in the period of activity of this species, making it necessary to take into consideration the weather condition of the area before planning any vector control measures. the highest abundance of this spehttp://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 334 http://jad.tums.ac.ir published online: december 31, 2020 cies, using night catch method with human and animal baits, occurred in the first third of the night between 8–9pm. we thus recommend that people be encouraged to use bed nets or avoid outdoors at these times. larvae of an. dthali were mostly found in stagnant, turbid waters, natural breeding sites in river banks which have no vegetation, and semi-shade. in agreement with our results, this anopheles species was sampled from breeding sites without vegetation (19), but unlike our findings, it was mostly collected from clear and sunny sites. although a previous study reported that breeding places with temperature ranging between 13 ºc and 28 ºc and ph between 6.9 and 8.0 are preferred by this species (21), the mean temperature and ph of our study sites were 31.5% and 7.6, respectively. another study also reported that about 50% of an. dthali larvae were collected from breeding sites with temperature ranging between 25.1–30 oc and ph between 7.14 and 8.20 (19). anopheles fluviatilis s.l. this anopheles species is also considered as a secondary vector of malaria in most of its' distribution areas in iran (22). in some studies, it was captured in outdoor habitats and on animal baits (23). we found an. fluviatilis s.l. was the most predominant species (35.76%) caught by pit shelter method, confirms its exophilic habit. it was also collected by night catch method using human and animal baits in may and november, mostly in the first third of the night between 9–10pm. the number of unfed and blood-fed an. fluviatilis s.l. was higher than the number of gravid and semi gravid an. fluviatilis s.l. captured by total catch method, which shows high exophilic tendency of this species. breeding sites for this species in our study area were natural water bodies, stagnant, turbid waters, and semi-arid areas without vegetation. mean temperature and ph of the study sites were 22 oc and 7.6, respectively. this species usually breeds in fresh, slow flowing or even stagnant waters (22). anopheles superpictus s.l. the highest collection of this species occurred in the first third of the night between 8 to 9pm. in our study, the number of unfed and blood-fed an. superpictus s.l. was lower than the number of gravid and semi gravid an. superpictus s.l. captured by total catch method, which shows high exophilic tendency of this species. contrary to our results, a study conducted in south west of iran, reported endophilic habit of this species in agreement with earlier studies (24). studies on the larval habitats of an. superpictus s.l. in iran revealed that this species is frequently abundant in river banks, both in natural breeding sites and artificial habitats created by human activities like mining pools (22). we collected this species from natural breeding sites, stagnant, turbid waters and semi-arid areas with no vegetation. mean temperature and ph of the collection sites of this species were 26.5 oc and 7.6, respectively. an earlier study reported clear, sandy bed, full sunlight and natural water bodies as the main breeding sites for an. superpictus s.l. (19). insecticide resistance our results show that anopheles stephensi is quite resistant to ddt, tolerant to malathion and susceptible to deltamethrin and propoxur. this result is in accordance with that of a study conducted in chabahar (25), in which an. stephensi was found to be resistant to ddt but tolerant to malathion. resistance of this species to ddt in iran has been reported in the last two decades (11, 13, 26-30). fortunately, in our study, this species was found to be susceptible to both deltamethrin and propoxur insecticides. thus, these insecticides can be used in indoor residual spraying for vector control against this main endophilic malaria vector. furthermore, it is recommended that more susceptibility tests be conducted on other species with high endophilic and endophagic tendencies, especially an. culicifacies s.l. and an. dthali, in the area. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2020, 14(4): 325–343 h edalat et al.: ecology of … 335 http://jad.tums.ac.ir published online: december 31, 2020 conclusion in conclusion, five malaria vectors are active in the study area. regular entomological studies are recommended to monitor their monthly activity and susceptibility status to insecticides. the peak of host seeking activity of the indoor mosquitoes occur in the first 3rd of the night, therefore, community-based training programs should be designed and implemented to enforce proper use of mosquito nets and personal protective measures against mosquitoes. country has a long history of work on malaria and publication of several papers on different aspects of malaria including insecticide resistance monitoring, sibling species, molecular study, new record, novel methods for vector control, faunestic study, use of plants for larval control, using bednets and long lasting impregnated nets, morphological studies, malaria epidemiology, ecology of malaria vectors, biodiversity, community participation, vector control, repellent 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current susceptibility status of anopheles stephensi (diptera: culicidae) to different imagicides in a malarious area, southeastern iran. j arthropod borne dis. 10(4): 493–500. 117. abai mr, hanafi-bojd aa, vatandoost h (2016) laboratory evaluation of temephos against anopheles stephensi and culex pipiens larvae in iran. j arthropod borne dis. 10(4): 510–518. 118. sanei-dehkordi a, vatandoost h, abai mr, davari b, sedaghat mm (2016) chemical composition and larvicidal activity of bunium persicum essential oil against two important mosquitoes vectors. j essent oil bear pl. 19(2): 349– 357. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 197 original article biodiversity of aquatic insects of zayandeh roud river and its branches, isfahan province, iran mansoreh shayeghi 1, *hassan vatandoost 1, abdollah gorouhi 1, ali reza sanei-dehkordi 1, yaser salim-abadi 1, mohsen karami 1, mohammad reza jalil-navaz 1, amir ahmad akhavan 1, zahra shiekh 1, sajad vatandoost 3, mohammad hossein arandian 2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2isfahan health research station, national institute of health research, tehran university of medical sciences, tehran, iran 3department of zoology, university of tehran, tehran, iran (received 15 apr 2013; accepted 5 feb 2014) abstract background: aquatic insects are the major groups of arthropods that spend some parts of their life cycle in the water. these insects play an important role for transmission of some human and animal diseases. there is few information about the aquatic insects fauna of iran. methods: to study the aquatic insects fauna, adult, nymphal and larval collections were carried out from different habitats using the standard technique in zayandeh roud river, isfahan province,central iran, during summer 2011. results: in total, 741 speimens of aquatic insects were collected and morphologically identified. they include 7 families and 12 genera representing 2 orders. the order of diptera (92.31%) and coleoptera (7.69%). the families culicidae, syrphidae and chironomidae from diptera order, gyrinidae, dytiscidae, haliplidae, hydrophilidae from coleoptera order were identified. conclusion: some aquatic insects play an important role for transmission of human and animal diseases. these insects also are important for biological control. therefore ecological study on aquatic insects can provide information about ecology of insects in an area for any decision making. keywords: aquatic insects, zayandeh roud river, iran introduction aquatic insects are a major group of arthropods which at least one stage of their life cycle occurs in water. most of them live in water in primary stages that fallowed by terrestrial adult (eg, ephemeroptera, odonata, plecoptera, trichoptera, megaloptera). semi aquatic insect are only associated with aquatic and semi aquatic vegetation, the water’s surface, or the margins of water habitats (merritt and cummins 1996). some species of aquatic insect are medically important vectors that transmit diseases such as malaria, dengue, filariasis, yellow fever, and some other main arboviruses (foil 1989). furthermore few numberof them have a painful bite that cause dermatological effect on human and animal host (villiers 1987). some of them act as a host of termatods such as dragonflyand damselfly (chae et al. 2000). in some countries dragonfly are considered as a threat to the poultry industry because they transmit a parasitc flatworm of prosthogonimus spp (angel 1973). some aquatic insects are used as an indicator of water contamination. water quality is evaluated by comparing the number of tolerant species (some midge larva) to the number of intolerant species (ephemeroptera, plecoptera, *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 198 and trichoptera orders) (voshell 2002). furthermore some of these insects are used in toxicological researches in primary stages (merritt and cummins 1996). aquatic insect are found in a wide variety of aquatic habitats from pond, spring, stream to rivers which are different in salinity, ph and other characteristics. apart from medically importance of aquatic insects, they play an important role in the ecosystem. for instance they serve as food for fish, amphibians, and water birds. they are also involved in the breakdown of organic matter and nutrients. some of the aquatic insects are responsible for breaking down the dead leaves and plant parts that fall on the water surface. this material provides the base of the food chain in some aquatic environments. some of them filter suspended particles in water and cause light reach to bottom of streams where algae grow. another kind of aquatic insects mix soft sediment of bottom while searching for food and this makes bottom appropriate for organisms and this phenomen is due to oxygen enreachment of the bottom. additionally, predator quatic insects reduce the numbers of other invertebrates and help keep to have a balance among different organism and food reservoir (voshell 2002). this paper attempts to investigate aquatic insect fauna in isfahan province. apart from researches that have been conducted on culicidae family members, there is a few studies on aquatic insects. materials and methods study area present study conducted in the zayandeh roud river of isfahan province during summer 2011. the isfahan province located in the center of iran and situated 340 kilometers far away of iran’s capital. isfahan region has generally arid climate with hot summer with maximum temperature around 36 °c and cool winter. it has an average annual rainfall of 150 millimeter zayandeh roud river in this region provides a suitable habitat for aquatic insect. isfahan province is surrounded by qoum, semnan and markazi provinces to the north, fars and kohkiloiye province to the south, yazd province to the east, and lorestan, khuzestan and chaharmahal to the west. with a total area of around 105, 937 square kilometers (6.57 % total area of iran). it lies at an altitude of 1575 meters above sea level at a latitude of 30° 42′ n to 34° 30′ n and a longitude 49° 36′ e to 55° 32′ e. the climate is temperate. it has the warm and semi-humid climate in north and east parts and cold climate in south. the studied areas were selected by clustered random sampling consist of several localities in the study area. we started our survey from zarinshahr city, ca. 1684 m (32º 22’ n, 51º 22’ e) to baghbahadoran city, ca. 1751 m (32º 23’ n, 51º 10’ e), (fig. 1). data sampling aquatic insects collected in different habitats. the sampling was carried out from rifles, under stones, aquatic vegetation, over hanging terrestrial vegetation, within burrows, leaf packs and fine sediment. the specimens collected by d frame net-collector, plastic pipette and forceps. after collection, all specimens were preserved in 70% alcohol, date and time of sampling and place of collection were recorded on each container. the samples were transferred to the laboratory of medical entomology department, tehran university of medical sciences. then the samples were identified using stereo-typed microscope, and valid identification keys (clifford 1991, borror and white 1998, epler 2001, bouchard 2004, sangradub and boonsoong 2006, subramanian and sivaramakrishnan 2007, azarihamidian and harbach 2009, mullen and durden 2009). http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 199 results during several times sampling in the study area a total of 741 aquatic insects were collected that including: 2 orders, 7 families and 12 genera witch summarized in table 1. in the diptera order there are 3 families: culicidae (n=384, 51.82%), syrphidae (n=4, 0.54%) and chironomidae (n=296, 39.95%) and coleoptera order was including 4 families: gyrinidae (n= 6, 0.81%), dytiscidae (n=24, 3.23%), haliplidae (n=7, 0.94%), hydrophilidae (n=20, 2.7%) and culex theileri belong to culicidae family was the most frequent (51.82%) moreover peltodytes in the haliplidae family with 0.40% of all collected samples was the least frequent (table 1 and fig. 2, 3). fig. 1. map of study area in isfahan province, iran table 1. the prevalence of some aquatic insects in the study area order family genus no. percent (%) diptera culicidae culex (culex theileri) 384 51.82 syrphidae eristalis 4 0.54 chironomidae chironomus 296 39.95 coleoptera gyrinidae gyrinus 6 0.81 dytiscidae agabus 5 0.67 dytiscus 13 1.75 hydroporus 6 0.81 haliplidae haliplus 4 0.54 peltodytes 3 0.40 hydrophilidae laccobius 10 1.35 enochrus 4 0.54 hydrobius 6 0.81 total 7 families 12 genus 741 100 http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 200 fig. 2. aquatic insect genus and family composition in study area fig. 3. families of collected aquatic insects, a: hydrophilidae b: haliplidae c: gyrinidae d: dytiscidae e: culicidae f: chironomidae g: syrphidae discussion total number of 741 samples belongs to 2 orders and 7 families and 12 genera were identified using stereo-typed microscope. the results are summarized in table 1. culex theileri with 51.82% of collected sample was the most abundance frequent (table 1 and fig. 2). in the same study that conducted in the our study area by shayeghi et al. in 2011, their result were compatible with our result, for the study on the aquatic insects of isfahan province and also their probable use of biological control, coleoptera order was one of the abundance collected aquatic insect and this result was likely to our result (shayeghi et al. 2012) cx. theileri was one of the collected samples in the study area. research conducted by mousa-kazemi et al. in 2002, cx. theileri was the most abundance frequency and their results were agree with our results (mousa-kazemi et al. 2000). this species is the more prevalent species at higher altitudes in rural areas of zanjan province (ghavami and ladonni 2005) and east azerbaijan province (abai et al. 2007). dehghani et al. (2007) conducted a study for studying fauna of aquatic insects in sewage maturation ponds of kashan. the families of chironomidae and hydrophilidae were prevalent. in the other study that conducted by vafaei et al. 2007 for surveying of the aquatic beetles (coleoptera: polyphaga) of markazi province (central iran) after investigation in freshwater habitats of study area, 24 species (coleoptera: hydrophilidae, helophoridae, hydraenidae, elmidae, and dryopidae) belonging to 13 genera and five families were identified and in this study hydrophilidae family was one of the collected samples like present study (vafaei et al. 2007). some aquatic insect are an important for biological control of larvae and adults of mosquitoes in the breeding places also some of these insects play an important role in transmission of some human and animal diseases, for example in iran several species belong to anophelinae sub family including anopheles culicifacies s.l., an. stephensi, an. dthali, an. fluviatilis s.l., an. superpictus, are known to be the malaria vectors (naddaf et al. 2003, doosti et al. 2006, oshaghi et al. 2006, vatandoost et al. 2006, hanafi-bojd et al. 2011, mehravaran et al. 2011, http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 201 oshaghi et al. 2011, vatandoost et al. 2011, hanafi-bojd et al. 2012a, 2012b, 2012c, vatandoost and abai 2012a, soleimani-ahmadi et al. 2012a, 2012b, vatandoost and hanafibojd 2012). therefore the ecological specifications of these insects could provide a clue for further arthropod-borne disease control. conclusion according to the results it could be concluded that there are several species of insects in the study area. they are involved in the food chain of aquatic insects. they also could be considered as biological control agent for vectors as well as bio indicators. acknowledgements the authors wish to appreciate the staff of isfahan health research centre, national institute of health research, tehran university of medical sciences (tums) for their collaboration along the study. some partially expenses are founded by tehran university of medical sciences grant number 11840. references abai mr, azari-hamidian s, ladonni h, hakimi m, mashhadi-esmail k, kousha a, vatandoost h (2007) fauna and checklist of mosquitoes (diptera: culicidae) of east azerbaijan province, northwestern iran. iran j arthropodborne dis. 1: 27–33. angel lm (1973) the family prosthogonimidae (trematoda) in australia. int j parasitol. 3(6): 853–862. azari-hamidian s, harbach re (2009) keys to the adult females and fourth-instar larvae of the mosquitoes of iran (diptera: culicidae). zootaxa. 2078: 1–33. borror dj, white re (1998) a field guide to insects: america north of mexico. vol. 19. p. 888. bouchard rw (2004) guide to aquatic invertebrates of the upper midwest: identification manual for students, citizen monitors, and aquatic resource professionals. university of minnesota. p. 208. chae sj, pusterla n, johnson e, derock e, lawler sp, madigan je (2000) infection of aquatic insects whit trematode metacercariae carry ehrlichiaristicii, the case of potomac house fever. j med entomol. 37: 619–625. clifford hf (1991) aquatic invertebrates of alberta: an illustrated guide. vol. 1. university of alberta press, alberta, p. 538. de villiers pc (1987) simulium dermatitis in man-clinical and biological features in south africa. a case report. s afr med j. 71: 523–525. dehghani r, miranzadeh mb, yosefzadeh m, zamani s (2007) fauna aquatic insects in sewage maturation ponds of kashan university of medical science 2005. pak j biol sci . 15: 928–931. doosti s, azari-hamidian s, vatandoost h, oshaghi ma, hosseini m (2006) taxonomic differentiation of anopheles sacharovi and an. maculipennis s.l. (diptera: culicidae) by seta 2 (antepalmate hair) of larvae in iran with a historical review and an annotated bibliography of iranian an. maculipennsi complex. acta med iran. 44(1): 21–27. epler jh (2001) identification manual for the larval chironomidae (diptera) of north and south carolina. north carolina department of environmental. natural resources. raleigh, north carolina. vol. 1. special publication sj2001-sp13. p. 526. foil ld (1989) tabanids as vectors of disease agents. parasitol today. 5: 88–96. ghavami mb, ladonni h (2005) the fauna and frequency of different mosquito http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 202 species (diptera: culicidae) in zanjan province. j zanjan uni med sci. 13: 46–54. hanafi-bojd aa, azari-hamidian s, vatandoost h, charrahy z (2011) spatiotemporal distribution of malaria vectors (diptera: culicidae) across different climatic zones iran. asian pac j trop med. 6: 498–504. hanafi-bojd aa, vatandoost h, oshaghi ma, haghdoost aa, shahi m, sedaghat mm, abedi f, yeryan m, pakari a (2012a) entomological and epidemiological attributes for malaria transmission and implementation of vector control in southern iran. acta trop. 121(2): 85–92. hanafi-bojd aa, vatandoost h, oshaghi ma, charrahy z, haghdoost aa, zamani g, abedi f, sedaghat mm, soltani m, shahi m, raeisi a (2012b) spatial analysis and mapping of malaria risk in an endemic area, south of iran: a gis approach for planning of control. acta trop. 122(1): 132–137. hanafi-bojd aa, vatandoost h, oshaghi ma, charrahy z, haghdoost aa, sedaghat mm, abedi f, soltani m, raeisi a (2012c) larval habitats and biodiversity of anopheline mosquitoes (diptera: culicidae) in a malarious area, southern iran. j vector borne dis. 49 (2): 91–100. mehravaran a, oshaghi ma, vatandoost h, abai mr, ebrahimzadeh a, roodi am, grouhi a (2011) first report on anopheles fluviatilis u in southeastern iran. acta trop. 117(2): 76–81. merritt rw, cummins kw (1996) an introduction to the aquatic insects of north america. vol. 3. kendall/hunt publishing company, p. 862. mousa-kazemi sh, zaim m, zahraii a (2000) fauna and ecology of culicidae of the zarrin-shahr and mobarakeh area in isfahan province. armaghan danesh. journal of yasuj university of medical sciences. 5: 46–54. mullen gr, durden la (2009) medical and veterinary entomology. vol. 2. academic press. p. 595. naddaf sr, oshaghi ma, vatandoost h, asmar m (2003) molecular characterization of the anopheles fluviatilis species complex in iran. east mediterr health j. 9: 257–265. oshaghi ma, vatandoost h, goruhi a, abai mr, madjidpour a, arshai s, sadeghi h, nazari m, mehravaran a (2011) anopheline species composition in borderline of iran-azerbaijan. acta trop. 119(1): 44–49. oshaghi ma, yaghoobi f, vatandoost h, abai mr, akbarzadeh k (2006) anopheles stephensi biological forms, geographical distribution, and malaria transmission in malarious regions in iran. pak j biol sci. 9: 294–298. sangpradub n, boonsoong b (2006) identification of freshwater invertebrates of the mekong river and tributaries. mekong river commission, vientiane, p. 274. soleimani-ahmadi m, vatandoost h, shayeghi m, raeisi a, abedi f, eshraghian mr, madani a, safari r, oshaghi ma, abtahi m, hajjaran h (2012a) field evaluation of permethrin long-lasting insecticide treated nets (olyset®) for malaria control in an endemic area, southeast of iran. acta trop. 123(3): 146–153. soleimanai-ahmadi m, vatandoost h, shayeghi m, raeisi a, abedi f, eshraghian mr, safari r, shahi m, mojahedi a, poorahmad-garbani f (2012b) vector ecology and susceptibility in a malaria endemic focus for implementation of long-lasting insecticidal net. eastern mediterr health j. 18(10): 1034–1041. subramanian ka, sivaramakrishnan kg (2007) aquatic insects for biomonihttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 197–203 m shayeghi et al.: biodiversity of aquatic … 203 toring freshwater ecosystems-a methodology manual. ashoka trust for ecology and environment (atree), bangalore, india. shayeghi m, doosti s, bazrafkan s, hosseinivasoukolaei n, vatandoost h, akhavan aa, vatandoost s, arandian mh (2014) prevalence of aquatic entomofauna, the predators of mosquitoes, in the zayandeh roud river of central iran. asian pac j trop med. (in press). vafaei r, ostovan h, incekara ü, pešić v (2007) faunistic study of the aquatic beetles (coleoptera: polyphaga) of markazi province (central iran) with new records. arch biol sci. 59(3): 239–242. vatandoost h, hanafi-bojd aa (2012) the indication of pyrethroid resistance in the main malaria vector,anopheles stephensi from iran. asian pac j trop med. 5(9): 722–726. vatandoost h, abai mr (2012a) irritability of anopheles sacharovi to different insecticides in a malaria-prone area, iran. asian pac j trop med. 5(2): 113–116. vatandoost h, emami sn, oshaghi ma, abai mr, raeisi a, piazzak n, mahmoodi m, akbarzadeh k, sartipi m (2011) ecology of malaria vector anopheles culicifacies in a malarious area of sistan va baluchestan province, south-east islamic republic of iran. eastern mediterr health j. 17(5): 439-445. vatandoost h, oshaghi ma, abaie mr, shahi m, yaghoobi f, baghaii m, hanafi-bojd a a, zamani g, townson h (2006) bionomics of anopheles stephensi liston in the malarious area of hormozgan province, southern iran. acta trop. 97: 196–205. voshell jr (2002) a guide to common freshwater invertebrates of north america. blacksburg, virginia: mcdonald and woodward publishing company. pp. 1–456. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, 2012, 6(2): 98–103 kk swami and m srivastava: blood meal preference of … 98 original article blood meal preference of some anopheline mosquitoes in command and non-command areas of rajasthan, india kailash kumar swami, meera srivastava laboratory of entomology, pg department of zoology, governmental dungar college, rajasthan, india (received 8 aug 2010; accepted 1 nov 2011) abstract background: the present study was undertaken to compare the entomological situation by analyzing the blood meal of mosquitoes of canal irrigated and non-irrigated areas of bikaner in order to explore scientific information on the vector biology and malaria burden profile and to plan proper strategies for malaria control and eradication. methods: adult mosquitoes were collected and the abdomen of the blood fed females were crushed on a filter paper for blood meal analysis and subjected to precipitin test. results: the blood meal analysis showed that anopheles subpictus had a preference towards cattle blood, an. culicifacies and an. stephensi preferred human blood, while, an. annularis was noted to feed only on bovine blood. conclusion: although an. annularis, has been recently reported from the area, was found to feed exclusively on bovine blood, earlier reports suggest that this species is a vector of malaria and therefore preventive measures should be taken well in advance before this species gets established in the area. keywords: malaria, anopheline mosquitoes, canal irrigated (command area), non-command area, thar desert introduction malaria imposes great socio–economic burden on humanity. according to who estimates, 300–500 million malaria cases are reported annually (who 1998). in addition, the estimated annual mortality attributed to malaria ranges from 7 lakhs to 2.7 million globally (kumar et al. 2007). in the south– eastern asian region, 1.2 billion are exposed to the risk of this infectious disease, most of who live in india (kondrachine 1992). of the 2.5 million reported cases in south–east asia, india alone contributes about 70% and currently 80.5% of the 109 million population of the country live in malaria risk areas. of this, 4.2%, 32.5% and 43.8% live in areas of high, moderate and low risk to malaria respectively (http://www.searo.who.int/). until recently, the states of orissa, jharkhand, west bengal, north-eastern states, chattisgarh and madhya pradesh contributed to bulk of malaria (dash et al. 2008), with the state of rajasthan having no case in the list. of late, malaria has become the most dangerous and debilitating vector borne disease of the thar desert, and has been witnessing a spate of epidemics since 1990 every alternate year, while, in 1994 and 1995 it assumed a continuous trend and the total number of malaria positive cases in the later year were the third largest ever reported (tyagi 1997). bikaner, situated in the north-western part of rajasthan is characterized by extremes of temperature, both in summer as well as in winter and low and erratic rains. loonkaransar tehsil lying in bikaner district is a canal irrigated area whose ecology has changed drastically due to the introduction of indira gandhi canal. there is an increase *corresponding author: dr meera srivastava, e-mail: meerayk@rediffmail.com j arthropod-borne dis, 2012, 6(2): 98–103 kk swami and m srivastava: blood meal preference of … 99 in vegetation leading to enhanced ambient relative humidity and availability of water logging at number of places resulting from the canal seepage. after the initiation of irrigation activities in the region, there has been a tremendous increase in the number of malaria cases. kolayat tehsil, lying about 51 km from bikaner city has large water body in the form of lakes. the present study was undertaken to study the blood meal preference of different anopheline species. materials and methods study area district of bikaner bikaner district is a part of the great indian thar desert. it lies in northwestern part of rajasthan located between 27°11' and 29°03' north latitudes and 71°54' and 74°22' east longitudes (www.indianetzone.com>rajasthan> districts of rajasthan). the total area of the district is 30289 sq km with a general elevation lying between 154 to 429 m above the mean sea level, sloping towards north-west. sand dunes range in height from 6 to 30 m. the district has a dry climate with large variation in temperature and has scanty rainfall. the summer months are extremely hot with the day temperature sometime going up to 49 °c, june being the hottest month. during winter the minimum temperature sometimes drops up to 2 °c below freezing point, january being the coldest month. the average rainfall in the district is 259.6 mm and average relative humidity is of the order of 15 to 20 percent. the present study was carried out in three tehsils viz, bikaner, kolayat and loonkaransar. bikaner tehsil bikaner tehsil occupies an area of 3192.52 sq km and lies between 27°15'–28°22' north latitude and 72°06'–73°46' east longitudes above mean sea level of 238 m (www. mapsofindia.com). the total population is 7, 25,810 and density of population per sq km is 235. in this tehsil, the survey was carried out only in bikaner city. five sampling sites were selected in the city for study purpose. kolayat tehsil the main tehsil headquarter of kolayat lies approximately 51 km away from bikaner city. the total area of the tehsil is 7957. 62 sq km lying between 28°28' and 60″ n latitudes and 73°45'0″ e longitudes, 191 m above mean sea level (www.fallingrain. com). the total population of the tehsil is 2, 07,749 with the density of population per sq km being 26. five villages of this tehsil were surveyed. loonkaransar tehsil the main tehsil head quarter of loonkaransar lies 70 km away from bikaner. the tehsil occupies an area of 5036.58 sq km and lies between 28°33'–29°03' north latitudes and 73°05'–74°12' east longitudes, and 191 m above sea level (www.collinsmaps. com). the total population of the tehsil is 1, 74,293 and the density of population per sq km is 35. five villages lying near the canal distributaries were selected for the study under irrigated area. canal irrigated and non–irrigated study area the area under study was demarcated into two major heads viz., canal irrigated (command) and non-irrigated (non–command areas), based on distribution of water from the main canal (ignp) and its distributaries. indira gandhi nahar pariyojna, formerly known as rajasthan nahar pariyojna, measures 649 km long canal with a distributary system of 8776 km which draws water from the sutlej and beas rivers and courses to northwestern arid land of rajasthan. the command area under this system is 0.49 lac hectare at 110irrigation intensity. therefore, the vilj arthropod-borne dis, 2012, 6(2): 98–103 kk swami and m srivastava: blood meal preference of … 100 lages studied under loonkaransar tehsil were a part of irrigated area, while those surveyed in kolayat tehsil and the city of bikaner were placed under non-irrigated area. period of survey and periodicity of collection the survey was carried out over a period of two years from october 2006 to september 2008. samples were collected every week from the selected locations. the anopheline species were collected from vicinities of different water bodies/breeding sites. the adult mosquitoes were collected during dawn and dusk from the field areas. through surveys different breeding and resting sites of anopheline species were identified. the collection sites were puddles, ponds, lake, cattle water points, pipe leakages, construction sites, households, culverts/causeways, and fodder rooms. method of collection adult mosquitoes were collected with the help of aspirator tubes and torch light. these were then transferred into collection tubes plugged with cotton and brought live to the laboratory. the abdomen of the blood fed female were crushed on whatman's filter paper no. 1 for blood meal analysis. it was then subjected to precipitin test using gel-diffusion method for identifying the blood meal as given by collins et al. (1983). in this method the blood obtained by crushing the abdomen of the vector is tested against anti-sera for blood of man and other host animals, the reaction against a particular antiserum is presumptive evidence that the blood belongs to that host. reagents solution 1 this solution was prepared by dissolving 1.03 g sodium barbiturate and 0.81 g of barbituric acid in 100 ml of distilled water. solution 2 this solution was prepared by dissolving 0.45 g of agarose in 50 ml of distilled water. procedure first the dry blood obtained from the vector stomach was dissolved in normal saline (0.7 g/100 ml dw), 4.5 h before the experiment. 50 ml of solution 1 was added to 50 ml of solution 2. microslides (glass) were prepared by spreading 3 ml of mixed solution on each slide, which on drying formed a gel. these slides were then kept in refrigerator for 12 to 15 h. various test antisera and mosquito blood dissolved in normal saline were released in micro quantities onto the wells in the gel slides. when the serum of blood ingested by a mosquito comes in contact with a specific antiserum, a precipitation occurs at the place of contact through gel. the presence of turbidity through transparent gel layer between test sample and antisera is indicative of the blood source (host). results during the study, three species including anopheles subpictus, an. stephensi and an. culicifacies were documented from bikaner, two species namely an. subpictus and an. stephensi were noted at kolayat tehsil, while three species were observed in loonkaransar tehsil which included an. subpictus, an. stephensi and an. annularis. results of blood meal analysis have been presented in table 1. the results of the blood meal analysis showed that an. subpictus had a preference towards cattle blood as compared to human, while an. culicifacies and an. stephensi preferred human blood and an. annularis gut content revealed the presence of only bovine blood. the results therefore indicate that an. annularis seems to be specifically zoophagic while rest of the three species are zoophagic as well as anthropophagic in nature. j arthropod-borne dis, 2012, 6(2): 98–103 kk swami and m srivastava: blood meal preference of … 101 table 1. blood meal preference of anopheline mosquito species (%) on different hosts species bovine mixed human anopheles subpictus 57.44 25.53 17.02 anopheles stephensi 15.60 30.56 53.84 anopheles culicifacies – 28.00 72.00 anopheles annularis 100 – – discussion the findings revealed that an. subpictus had a preference towards cattle blood as compared to human, while an. culicifacies and an. stephensi preferred human blood and an. annularis gut content showed the presence of only bovine blood. according to bruce-chwatt and goeckel (1960) the information on the host preference of an. culicifacies which extends from south eastern arabia and pakistan to indo-china and south across india to ceylon was very confusing. in those parts of india where this species was considered the main local vector, the result of precipitin tests positive to man did not exceed to 2% of all, while afridi and puri (1940) showed that the ratio between the number of cattle and man influenced the feeding behavior while senior white and rao (1943) found that it varied in relation to the type of shelter in which the mosquitoes were collected. the overall anthropophilic index of an. culicifacies was reported to be 11.9% by bhatt et al. (2008). according to joshi et al. (1988), the blood meal results of an. culicifacies indicated the mosquito species to be predominantly zoophagic in nature. roy et al. (1991) observed the percentage of mixed feeds i.e., mosquito feeding both on human and cattle hosts was only 3 to 4%. further, they also found that mosquitoes collected from human dwellings did not show a higher percentage of human feeding than cattle feeding. anopheles dirus was reported to be highly anthropophilic by dutta et al. (1996). thapar et al. (1998) reported a high proportion of an. culicifacies between 76–100% to feed on bovine hosts followed by 0.32–24.2 on human. according to them an. culicifacies and an. stephensi were zoophagic in nature. grieco et al. (2002) documented an. vestitipennis to feed mostly on human blood but it also fed on cattle blood, while an. albimanus and an. punctimacula fed mostly on cattle blood and rarely on humans. parida et al. (2006) reported the overall anthropophilic index of an. culicifacies to be 1.6 and of an. annularis to be 3.2%. further, an. culicifacies was also noted to show seasonality in its anthropophilic index by them. the final choice of host by a hungry mosquito may depend on extrinsic or intrinsic factors. extrinsic factors such as availability of the host, its range of flight and resting habits, etc may influence the selection, but the mosquito is still able to exert a free choice when extrinsic factors lead it to a number of possible hosts (eg, in huts where humans and animals live together). extrinsic factors such as the sleeping habits of the hosts, the odors given out by them, the large exposed area of warm skin, the accessibility of the capillary blood vessels, the palatability of the blood, or possibly simple random selection lead to the final choice. however, intrinsic factor, a genetic requirement of blood from a certain host or a particular capacity to respond to some impulses only-may override these tendencies. in addition to this, certain characteristics of behavior may be due to interplay of both intrinsic and extrinsic factors. this is the case of the difference between the indoorand outdoor-biting behaviors. anopheles which enters houses (endophilic species) will have better opportunities for feeding on man than those which remain out of doors (exophilic species). obviously, knowledge of the behavior of mosj arthropod-borne dis, 2012, 6(2): 98–103 kk swami and m srivastava: blood meal preference of … 102 quitoes (dispersion, gono-trophic cycle, nightly turnover, resting-habits) is most useful for the study of feeding preferences in malaria vectors, and therefore of their epidemiological importance. it might be worth recalling here that females normally seek a blood meal within 24 hours of emergence. the frequency of subsequent feeds is determined by the duration of the gono-trophic cycle and by the rate of digestion, which is more rapid in hot, humid weather. the amount of blood that has been ingested by fully fed female anopheles varies according to the size of the species, but there is also some variation between individual females (bruce-chwatt and goeckel 1960). it could therefore be concluded that although during the present study an. annularis, which has been recently reported from the area, was found to feed exclusively on bovine blood, earlier reports suggest by guresekaran et al. (1989) and amerasinghe et al. (1991) that this species is a vector of malaria and therefore preventive measures should be taken well in advance before this species gets established in the area. acknowledgements authors are thankful to the head, department of zoology and to the principal, dungar college, bikaner for providing necessary facilities. the authors declare that there is no conflict of interests. references afridi mk, puri im (1940) studies on the behavior of adult anopheles culicifacies. j malar inst india. 3: 1. amerasingh fp, amerasinghe ph, peiris js, wirtz ra (1991) anopheline ecology and malaria infection during the irrigation development of an area of the mahaweli project, sri lanka. am j trop med hyg. 45(2): 226–235. bhatt rm, srivastava hc, rajnikant, yadav rs (2008) dynamics of anopheles culicifacies transmitted malaria in the absence of effective zooprophylaxis in a riverine settlement in gujrat, india. curr sci. 95(1): 82–87. bruce–chwatt lj, gockel cw (1960) a study of the blood–feeding patterns of anopheles mosquito through precipitin tests. bull who. 22: 685–720. collins rt, dash bk, agarwal rs, dal kb (1986) an adapation of the gel diffusion technique for identifying the source of mosquito blood meal. indian j malariol. 23: 81–89. dash ap, valecha n, anvikar ar, kumar a (2008) malaria in india: challenges and oppurtunities. j biosci. 33(4): 583–592. dutta p, bhattacharyya dr, khan sa, sharma ck, mahanta j (1996) feeding pattern of anopheles dirus, the major vector of forest malaria in north east india. southeast asian j trop med public health. 27(2): 378–381. grieco jp, achee nl, andre rg, roberts dr (2002) most feeding preference of anopheles species collected by manual aspiration, mechanical aspiration, and from a vehiclemounted trap in the toledo district, belize, central america. j am mosq control assoc. 18(4): 307–315. gunasekaran k, sahu ss, parida sk, sadanandane c, jambulingam p, das pk (1989) anopheline fauna of koraput district, orissa state, with particular reference to transmission of malaria. ind j med res. 89: 340–343. joshi h, vasantha k, subbarao sk, sharma vp (1988) host feeding patterns of anopheles culicifacies species a and b. j am mosq control assoc. 4(3): 248–251. kondrachine av (1992) malaria in who southeast asia region. indian j malariol. 29: 129–160. parida sk, hazra rk, marai n, tripathy hk, mahapatra n (2006) host feeding j arthropod-borne dis, 2012, 6(2): 98–103 kk swami and m srivastava: blood meal preference of … 103 patterns of malaria vectors of orissa, india. j am mosq control assoc. 22(4): 629–634. roy a, ansari ma, sharma vp (1991) feeding behavior patterns of anophelines from uttar pradesh and gujarat states of india. j am mosq control assoc. 7(1): 11–15. thapar br, sharma sn, dasgupta rk, kaul sm, bali a, chhabra k, lal s (1998) blood meal identification by using microdot elisa in vector mosquitoes. j commun dis. 30(4): 283–287. tyagi bk (1997) emerging and re–emerging vector-borne disease in the thar desert, north-western rajasthan, india. reg hlth forum. 2(1): 9–16. senior-white r, venkat vr (1943) on malaria transmission around vishakhapatnam. malaria inst india. 5: 187–205. j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 108 http://jad.tums.ac.ir published online: march 31, 2021 original article spatial modelling of malaria in south of iran in line with the implementation of the malaria elimination program: a bayesian poisson-gamma random field model amin ghanbarnejad1; habibollah turki2; mehdi yaseri1; ahmad raeisi3,4; *abbas rahimiforoushani1 1department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran 2infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran 3departments of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 4center for communicable diseases control, ministry of health and medical education, tehran, iran *corresponding author: dr abbas rahimi-foroushani, e-mail: rahimifo@tums.ac.ir (received 13 aug 2020; accepted 30 mar 2021) abstract background: malaria is the third most important infectious disease in the world. who propose programs for controlling and elimination of the disease. malaria elimination program has begun in first phase in iran from 2010. climate factors play an important role in transmission and occurrence of malaria infection. the main goal is to investigate the spatial distribution of incidence of malaria during april 2011 to march 2018 in hormozgan province and its association with climate covariates. methods: the data included 882 confirmed cases gathered from cdc in hormozgan university of medical sciences. a poisson-gamma random field model with bayesian approach was used for modeling the data and produces the smoothed standardized incidence rate (sir). results: the sir for malaria ranged from 0 (abu musa and haji abad districts) to 280.57 (bandar–e-jask). based on model, temperature (rr= 2.29; 95% credible interval: (1.92–2.78)) and humidity (rr= 1.04; 95% credible interval: (1.03–1.06)) had positive effect on malaria incidence, but rainfall (rr= 0.92; 95% credible interval: (0.90–0.95)) had negative impact. also, smoothed map represent hot spots in the east of the province and in qeshm island. conclusion: based on the analysis of the study results, it was found that the ecological conditions of the region (temperature, humidity and rainfall) and population displacement play an important role in the incidence of malaria. therefore, the malaria surveillance system should continue to be active in the region, focusing on high-risk areas of malaria. keywords: bayesian; spatial; poisson-gamma; hormozgan; malaria elimination introduction malaria is the third most important infectious disease after tuberculosis and aids and one of the tenth diseases under investigation by the who as part of a program, called tropical disease research. malaria threatens more than 40% of the world's population. it is estimated that about 2.5 billion people worldwide are at risk of malaria (1). according to the report by who in 2019, about 80 countries are facing with malaria transmission which had led to 228 million infected cases and 405,000 death (1). the disease is present in most tropical, subtropical and even temperate regions of the world. due to the nature of the disease and the complexity of malaria epidemiology, it is not possible to solve the disease problem with a single strategy. malaria is not only an infectious disease of the tropical areas, but also a copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 109 http://jad.tums.ac.ir published online: march 31, 2021 health problem related to climatic, social, economic and developmental conditions in the world. iran is located in an endemic area of malaria according to the malaria's global distribution map (2). although ongoing efforts to combat malaria in the last five decades have led to a significant reduction in malaria in iran, there are still positive cases of malaria, especially in the south and south-east of the country. the incidence of malaria in iran has decreased from 15378 cases in 2002 to 960 cases in 2017 (3). the three provinces including kerman, sistan and baluchestan, and hormozgan are the most malaria-prone areas of iran (4, 5). this disease has been prevalent as hyper-endemic in many parts of hormozgan province for many years. 12% of malaria cases reported in the south-eastern region and 10% of cases reported in iran belong to hormozgan province (6). who had an elimination program for some countries and first phase of implementation in iran was during 2010 to 2015 and second phase was begun in 2016 and run successfully in the region (7-10). appropriate weather and humidity conditions throughout the year for carrier activity, adjacent to sistan and baluchistan province as the main malarious area of iran, as well as relocation and presence of immigrants infected with malaria, especially pakistani and afghani, are the most important factors affecting the malaria situation in this province (6). many studies around the world have linked the incidence of malaria to weather conditions (11-19). therefore, the use of climate variables will help to improve the prediction of malaria cases. malaria modeling in the context of spatial models and mapping is one of the most useful approaches to study malaria in relation to climate factors. for the mapping of diseases, including malaria, the data applied are mainly the number of occurrences in each area of the country, namely province or county. one set of statistical models that is widely used in disease mapping is bayesian hierarchical spatial models. the most commonly used models in this domain are markov random field models proposed for the first time by besag et al. in 1991 (20) and the ecological regression model developed by clayton and bernardinelli in 1992 (21) . in the aforementioned models, spatial quality and resolution of the risk surface function is similar to the resolution at which the data is measured. to overcome this limitation, wolpert and ickstadt (1998) (22), presented an extension of the random field to the hierarchical model introduced by clayton and kaldor (1987) (23), in which the spatial resolution of risk surface function could be independent of where the data measured. this model was applied in epidemiology by best et al. (2005) (24) and covariates were used to improve the risk surface function in the model. in this study, the relationship between malaria incidence and climate factors will be assessed based on extended poisson-gamma random field model and produce the malaria risk map in hormozgan province at county level. the main goal of this study was to design and develop a model to investigate and predict the conditions of malaria transmission in the endemic region of hormozgan in accordance with successful implementation of the malaria elimination program. materials and methods study area iran has diverse climate and is in the endemic area of the global malaria spread map (2). most of the positive cases are in the south and south-east of the country, where malaria elimination program is in progress. the three provinces of kerman, sistan and baluchestan and hormozgan are malaria-prone areas of iran. hormozgan province is one of the malaria-prone areas of the country and has been hyper-endemic for many years (25). hormozgan province is located in south of iran, has about 1,000km of coastline. its population was 1,578,183 based http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 110 http://jad.tums.ac.ir published online: march 31, 2021 on 2016 census data. its area is 70,697km2 and its provincial capital is bandar abbas. data collection data on malaria incidence during april 2011 to march 2018 in hormozgan province was used for analysis. these data are collected from communicable disease center in health deputy of hormozgan university of medical sciences. the data were grouped based on location of occurrence and categorized at county level. the data on climate variables gathered from hormozgan meteorological organization (hmo). the variables temperature, humidity and rainfall were available as monthly record. for analysis, we calculated mean of the variables in each year, then averaging on 6 surveyed years and considered it as mentioned covariate for each district in the model. statistical model for response variable, we analyzed observed numbers of malaria cases (i: county (1, …, 14)). the model used in this paper was a poisson-gamma random field model with rainfall, temperature and humidity as covariates, the following bayesian hierarchical structure was considered for modeling framework: level 1: points: intensity: level 2: latent sources: level 3: parameter: in the above context, the number of observed malaria cases in each region is modelled by a poisson process on y with mean , where is the population reference measure. as mean of the poisson process depends on a set of covariates which their effect can be considered as excess risk (ja) or relative risk (jm). the spatial effect is introduced with a latent covariate which is modelled as a gaussian kernel mixture of a random measure on space s. we choose s as bounding box of area of hormozgan province. in this paper, a gamma random field with shape measure α(ds) and inverse scale function was used. included latent sources s, , located at with size . bivariate gaussian kernel with correlated longitude and latitude is as follow: we consider four latent sources at fixed location of each kernel for modelling. the sir (standardized incidence ratio) was calculated as below: is the expected number of malaria cases in region i which calculated as, , is the total rate of malaria in the study region. results descriptive statistics the study included 882 registered cases in period from april 2011 to march 2018. based on epidemiological classifications, 716 cases (81.2%) were imported. demographic characteristics of the cases are presented in table 1. about half of the registered cases were located in rural areas. majority of cases were noniranian (81.5%). mean age of the patients was 24.3±13.68 and ranged from infants to 96 years old person. men are more infected than women (84.96 versus 13.44 per 100,000 populations). the data was aggregated and grouped in administrative district, county for model fitting. the incidence rate of malaria per 100,000 persons was calculated as number of case in each region divided by population multiplied by 100,000 and showed in fig. 1. the incidence rate ranged from 0 (abu musa and haji abad districts) to 280.57 (bandar–e-jask). calculated sirs are presented in fig. 2. the distribution of cases according to year and moth is shown in fig. 3. as seen in this http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 111 http://jad.tums.ac.ir published online: march 31, 2021 figure, the trend of registered malaria cases overally declined from 2011 to 2018. also, there is a peak in summer every year. the descriptive statistics of meteorological covariates are presented in table 2. as mentioned in the table, the variation of temperature is small because hormozgan province is located in a warm area, but the humidity has wide range variability from approximately 30% to 71%, this is because of the diversity of land cover of area. in the study period, annual rainfall ranged from 5mm to 16.62mm. model fitting the bayesian poisson-gamma random field model was fitted to the data through mcmc and gibbs sampling method with 200000 iterations after burning of first 50000 iterations. the regression coefficients and risk ratio of covariates influence on malaria incidence with the 95% bayesian credible intervals are reported in table 1. as mentioned in table 2, the effects of temperature and humidity on malaria incidence are positive although rainfall has negative impact on malaria incidence. the smoothed sir for malaria incidence are estimated from the model and shown in fig. 3. table 1. demographic characteristics of the patients with malaria in hormozgan province during 2011–2018 variable category no. (%) sex male 763 (86.5 %) female 119 (13.5 %) age under 5 years 60 (6.8 %) 5–15 years 116 (13.2 %) 16–29 years 481 (54.5 %) 30–64 years 211 (23.9 %) 65 years and older 14 (1.6 %) residency urban 453 (51.4 %) rural 429 (48.6 %) nationality iranian 163 (18.5 %) afghan 270 (30.6 %) pakistani 425 (48.2 %) other nations 24 (2.7 %) job worker 479 (54.3 %) farmer 42 (4.8 %) housewife 40 (4.5 %) school students 18 (2 %) children 99 (11.2 %) other 204 (23.1 %) type of malaria parasites plasmodium vivax 801 (90.8 %) plasmodium falciparum 79 (9 %) mixed 2 (0.2 %) table 2. descriptive statistics of meteorological covariates in hormozgan province during 2011–2018 variables mean standard deviation range temperature (°c) 28.04 0.86 26.09–29.76 humidity (%) 52.70 14.41 29.50–70.71 rainfall (mm/year) 10.14 4.17 5.07–16.62 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 112 http://jad.tums.ac.ir published online: march 31, 2021 fig. 1. incidence rate of malaria in hormozgan province fig. 2. calculated standardized incidence ratio (sir) for malaria in hormozgan province http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 113 http://jad.tums.ac.ir published online: march 31, 2021 table 3. summary statistics of coefficients of bayesian poisson-gamma model variable rc* 95% ci** for rc rr*** 95% ci** for rr temperature (⁰c) 0.8287 (0.65, 1.02) 2.29 1.92–2.78 rainfall (mm/year) -0.079 (-0.107, 0.05) 0.92 0.90–0.95 humidity (%) 0.43 (0.029, 0.057) 1.04 1.03–1.06 latent source 0.0059 (0.0003, 0.02576) *regression coefficients **credible interval ***risk ratio fig. 3. trend of malaria cases in hormozgan province during 2011-apr to 2018-mar fig. 4. smoothed standardized incidence ratio (sir) based on bayesian modelling http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 114 http://jad.tums.ac.ir published online: march 31, 2021 discussion the key point of the present study was mapping malaria incidence in a region which previously known as endemic area for malaria infection and elimination program is implemented in the region. the estimated malaria risk map helps health policy makers to have better realization of the infection risk in the region and effect of climate. as mentioned in results, there is a decline of detected malaria cases from 2011 to 2018 because of good implementation of malaria elimination program and successful malaria surveillance system. in this paper we developed a model for analyze spatial pattern of malaria incidence in hormozgan province, south of iran in presence of some climate covariates. visualizing standardized incidence rate (sir) on the region map is one of the best representation of disease burden in epidemiological studies. in the malaria disease, there are studies which produce smoothed maps via statistical modelling around the world such as works done by taddese et al. in northwest of ethiopia (26), saita et al. in tak province of thailand (27), bui et al. in brazil (28), gwitira et al. in zimbabwe (29), sasane et al. in india (30), nur et al. in indonesia (31), hast et al. in zambia (32). the bayesian framework was used to modeling the malaria incidence and create smoothed map after adjusting for climate variables: temperature, rainfall and humidity. this is the first research that uses a bayesian modeling framework to study the relationship between climatological covariates and malaria incidence in hormozgan province. the last published paper for mapping malaria in hormozgan was conducted by hanafi-bojd in 2012 (33), they analyzed data for only bashagard district and produced malaria risk maps and identified hot spots. also, there is another published work in minab district of hormozgan province that assessed the association between meteorological factors with malaria incidence during 2003 to 2009 us ing time series analysis but not considering the spatial pattern (34). effect of temperature based on bayesian modelling, temperature was important environmental covariate on malaria incidence. the impact of malaria was hazardous due to the approximately 2.3 risk ratio. this result is consistent with the study by mohammadkhani et al. in kerman, southeast of iran (35). because of neighboring of hormozgan and kerman provinces this result is feasible. also, umer et al. in pakistan evaluate the relationship between climate factors and malaria incidence and their findings were in line with our study. in the study by ikeda et al. in south africa (36), high temperature was associated with high incidence which was similar to the present study. kang et al. in madagascar showed that temperature is an affecting factor on malaria (37). laneri et al. assess the impact of climate drivers on malaria incidence in a region of argentina and found that the temperature had positive effect (38) which is consistent with the present study. sempiira et al. in uganda analyzed mis data for children under 5 years and the effect of land surface temperature was positive and significant (39). herekar et al. in karachi, pakistan found the positive correlation between temperature and malaria cases (40). liu et al. in tengchong county of yunnan province in china investigated the effect of climate on malaria incidence and had found that high temperature is an important factor on malaria (41). in some studies, there are negative impact on malaria incidence, m’bra et al. found a negative effect of temperature on malaria incidence in cote d’ivoire (42). the effect of temperature on malaria incidence was negative in a research by santos-vega et al. in northwest of india (43). effect of humidity another climate factor which has positive http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 108–125 a ghanbarnejad et al.: spatial modelling of … 115 http://jad.tums.ac.ir published online: march 31, 2021 and significant effect on malaria incidence in our study was humidity. this result is consistent with other studies around the world. herekar et al. in pakistan concluded the same result for humidity (40). laneri et al. had found a complex relationship between humidity and malaria, in their study the maximum humidity above 85% had negative effect and minimum temperature had positive effect which is consistent with this study in some aspects. simple et al. found a positive impact of humidity on malaria in uganda (44) which is in line with our study. effect of rainfall the impact of rainfall on malaria incidence was estimated to be negative in this study which is consistent with the study conducted by adigun et al. in nigeria (45). millar et al. found that the rainy season was the strongest climate predictor for malaria (46). matthew showed that there is a strong and direct correlation between rainfall and malaria occurrence in ile-ife region in south-western of nigeria (47). but in our study the rainfall had negative impact on malaria incidence. the direct effect of rainfall has seen in the african countries which have tropical and wet climate (48) which is different from the climate of hormozgan. the studied region has a warm and dry summer and in the winter, some of the area experienced rainy days. the spatial distribution in hormozgan showed a hot spots pattern in the areas which have foreign workers from other provinces and other countries. there is noticeable effect of temperature on malaria incidence which is concluded that the people should be aware in hot season and policy makers must have good planning to control and eliminate the malaria in the district. conclusion based on the analysis of the study results, it was found that the ecological conditions of the region (temperature, humidity and rainfall) and population displacement play an important role in the incidence of malaria. therefore, the malaria surveillance system should continue to be active in the region, focusing on high-risk areas of malaria. country has a long history of work on malaria and publication of several papers on different aspects of malaria including insecticide resistance monitoring, sibling species, molecular study, new record, novel methods for vector control, faunestic study, use of plants for larval control, using bednets and long lasting impregnated nets, morphological studies, malaria epidemiology, ecology of malaria vectors, biodiversity, community participation, vector control, repellent evaluation, anthropophilic index of malaria vectors, training is designated as malaria training center by who. there are several reports on different aspects of malaria vectors recently (49-143). acknowledgements the authors would like to express gratitude to the staff of the communicable disease center of hormozgan university of medical sciences specially mr sajjad fekri in gathering the data. we also glad to thanks the staff of meteorological 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493–500. 141. abai mr, hanafi-bojd aa, vatandoost h (2015) laboratory evaluation of temephos against anopheles stephensi and culex pipiens larvae in iran. j arthropod borne dis. 10(4): 510–518. 142. sanei-dehkordi a, vatandoost h, abai mr, davari b, sedaghat mm (2016) chemical composition and larvicidal activity of bunium persicum essential oil against two important mosquitoes vectors. j essent oil bear pl. 19(2): 349–357. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 82 http://jad.tums.ac.ir published online: march 31, 2021 original article comparative testing of susceptibility levels of phlebotomus sergenti, the main vector of anthroponotic cutaneous leishmaniasis, to conventional insecticides using two capture methods in kerman city, southeastern iran yavar rassi 1 ; shahla ebrahimi 1 ; *mohammad reza abai 1,2 ; *hassan vatandoost 1,2 ; amir ahmad akhavan 1,2 ; abass aghaie afshar 3 1 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3 leishmaniasis research center, kerman university of medical sciences, kerman, iran *corresponding authors: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir, abaiemr@gmail.com, prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir (received 01 aug 2020; accepted 13 mar 2021) abstract background: collecting live sand flies from indoor sites is a major challenge for researchers in large cities due to the reluctance of families to survey their homes. this study was conducted to assess the efficacy of two methods for collecting sand flies for use in susceptibility tests in the urban area of kerman, southeastern iran. methods: sandflies were mainly collected using both baited traps and hand catch methods from outdoor and indoor sites. susceptibility tests were separately done according to the standard world health organization testing protocol on phlebotomus sergenti, including 60-minute exposure to ddt 4.0%, propoxur 0.1%, deltamethrin 0.05%, and malathion 5.0%. results: during this research, the natural habitats and suitable indoor sites were selected to predict the density of live sand fly with perfect accuracy. the number of live ph. sergenti caught by hand catch and baited traps methods was 42 and 361 in indoor and outdoor sites, respectively. the mortality rate of ph. sergenti exposed to ddt 4%, deltamethrin 0.05, malathion 5%, and propoxur was 100%. conclusion: the baited traps showed a significant efficiency compared to hand catch for collecting live ph. sergenti for use in susceptibility tests in urban areas. the ph. sergenti collected from both indoor and outdoor sites were susceptible to all insecticides. keywords: phlebotomus sergenti; susceptibility; insecticides; baited traps; hand catch introduction anthroponotic cutaneous leishmaniasis (acl) caused by leishmania tropica is transmitted by phlebotomus sergenti (1). in the acl foci, the humans could act as a l. tropica reservoir for sand fly infection during the year. in this regard, patients with acute ulcers (2), chronic diffuse ulcers, lupoid or tuberculous ulcers (3), non-healing ulcers (4), recurrent ulcers (5) and ulcers resistant to pentavalent antimonial drugs could provide enough parasitic reservoirs for establishing an acl transmission cycle by sandflies in the area (6). dogs are considered as an accidental host (7) in which the lesions usually appear on the snout, interdigit, and sometimes on the corner of the eyelids (1, 8-9). the majority of acl cases in the world occur in morocco (10-11), algeria (12), libya (13), tunisia (14-15), afghanistan (16-21), iran (22), pakistan (23), saudi arabia (24), syria (25), jordan (26-27), iraq (27), israel (28), and turkey (29). anthroponotic cutaneous leishmaniasis is a well-known disease in densely popu copyright © 2020 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. mailto:abaiemr@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 83 http://jad.tums.ac.ir published online: march 31, 2021 lated cities in iran and is predominantly endemic in urban and peri-urban areas including tehran (30), mashhad (31), neishabour (32), shiraz (33), kerman (34-35), bam (37-40), isfahan (41), and jiroft (42). phlebotomus sergenti is a proven vector of acl (43). dissection of many specimens of this species in tehran failed to show the leishmania (1), while there are several reports of the leptomonad of l. tropica in dissected ph. sergenti in mashhad (44). moreover, leishmania parasites have been found in kerman (45-46) and shiraz using restriction fragment length polymorphism (rflp) (47-49). leptomonad infections of ph. sergenti have also been reported from afghanistan, saudi arabia (50) and tunisia (51). in some urban foci, ph. caucasicus is suspected as a secondary vector (1). in the acl foci located in the plains, the behavior of ph. sergenti is completely domestic with more density found at indoor sites versus the mountainous acl foci. this species is found in moderate density in caves and crevices of mountains and foothills where the birds act as a host for blood feeding. the presence of light or other avian hosts attracts the sandflies to the houses located adjacent to the foothills or mountains (43). in southern acl foci, the activity of ph. sergenti begins in late june and ends in mid-september (52). in the central acl foci, the activity starts in early april and ends in mid-november with two peaks at the end of april and in mid-september (53). some ecological aspects and surveillance of ph. sergenti have been studied in endemic foci of acl (54-57). the acl occurrence and emergence are characterized by several factors such as environmental changes, poor housing conditions, unprotected people from sand fly bites, resettlement of non-immune people, deterioration of the infrastructure and migration (5862), natural disasters like the earthquake (6365), displacement of people in borderlines (66), civil wars (67-69), and refugee camps (70-76). at present, spraying operations are limited to the residual foci of malaria in the country (79) and this method is not applied for controlling the acl vectors. based on the endophilic habits of ph. sergenti, some attempts have been made to reduce the density of sandflies (78-81) using other insecticide-based vector control measures such as insecticide impregnation of the bed nets (itns) (82-86), curtains (itcs) (87-88), tents (itts) (89). moreover, integrated vector control methods (89-90) have also been applied to control ph. sergenti population in epidemic conditions. in order to analyze the susceptibility tests on sand flies, testing of insecticides at discriminative doses has been used for ranking the susceptibility level and as a criterion for interpreting the results. it is difficult to collect live sandflies from indoor places in large cities. therefore, the present study was conducted to determine the most convenient capture method for collecting live sandflies from indoor and outdoor sites and to use the collected sand flies for determining their susceptibility to conventional insecticides in the acl foci in kerman, southeastern iran, where there is scarce data in this regard. materials and methods study area the study area was located in kerman, southeastern iran. the city borders khorasan and yazd to the north, the lut desert to the east, bam to the south, sirjan to the west, and rafsanjan to the northwest. kerman is elevated 1793 meters above the sea level and the main coordinates of the study locations were between 30°06'32"n and 57°06'27.6"e. the study area was a wide plain surrounded by stony mountains with a poor vegetation. the studied localities for collecting the adult sand flies were sarasiyab, masjed-e-sahebzaman, shahzadehmohammad, safa cave, allahabad, shahraksanaati, sarbaz mountains and shahrak-ealmahdi (fig. 1). the entomological operations were carried out from july to september 2019. sand fly collection live sand flies were collected using an as j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 84 http://jad.tums.ac.ir published online: march 31, 2021 pirator from the homes located in the periphery of the mountains in the early morning. the collected sand flies were released into paper cups and transferred to the laboratory for testing. the baited traps were equipped with mini gas lamps, and the netted tents were installed close to the caves or crevices near the mountains around kerman city (fig. 2), where the presence of adult sand flies was already proven during pretest trials using sticky paper traps. the collected live sand flies were also released into paper cups and transferred in a box under a wet towel. sucrose solution was provided on the top of cups as a cotton pad soaked in sucrose solution 10%. tested insecticides the insecticide-impregnated papers were purchased from the who representative in penang, malaysia. the papers were impregnated with ddt 4.0% (batch number dd 265, expire date: july 2022), deltamethrin 0.05% (batch number de 527, expire date: august 2019), malathion 5.0% (batch number ma 234, expire date: july 2020), and propoxur 0.1%, (batch number pr, 123 expire date: august 2020) and tested using the who’s testing kit. susceptibility tests susceptibility tests were carried out on sand flies collected from both indoor and outdoor sites. the collected sandflies were tested separately according to the capture method using the world health organization (who) protocols (91-93). the mortality rates were determined at discriminative doses for 60 minutes using the who test tubes for the adult sand flies. at each replicate, 20–25 sand flies were tested. a 10% sucrose solution was provided during the recovery period. the mortality was recorded after 24 hours of the recovery period. both dead and live sandflies were preserved in separate tubes containing 70% ethanol according to tested insecticides and collecting methods until microscopic slides were prepared using the puri’s medium at the appropriate time. the mounted sandflies were identified using valid identification keys (94-96). data analysis the susceptibility data including the number of live, dead, and total sandflies were ranked according to sex (male and female) and physiological condition (blood-fed, unfed and gravid) separately in two treatment groups compared to the control group. the charts of mortality rate were drawn with standard errors to show any significant differences using the microsoft excel 2010 software. the independent t-test was applied to compare the abundance and sex ratio of ph. sergenti between hand-catch and baited trap methods using the ibm spss software version (25). results efficiency of collection methods the efficiency the baited traps for collecting live ph. sergenti from outdoor was 370 compared to 45 sand flies collected using handcatch from indoor sites (table 1) indicating an significant difference (t= 7.214, df= 9, p= 0.001) (fig. 3). the sex ratio of ph. sergenti was 1.0 at indoor compared to 1.2 at outdoor places (table 1) showing no significance difference (t= 2.574, df= 8, p= 0.07) (fig. 4). susceptibility of phlebotomus sergenti collected from indoors the mortality rate of ph. sergenti collected from indoor places using hand-catch method was 100% after exposure to ddt and malathion. the mortality rate was 0.0% in the control group (fig. 5). susceptibility of phlebotomus sergenti collected from outdoors the mortality rate of ph. sergenti collected from outdoor sites using baited traps was 100 %. after exposed to ddt, propoxur, deltamethrin, and malathion. the mortality rate was 0.0% in the control group (fig. 6). j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 85 http://jad.tums.ac.ir published online: march 31, 2021 fig. 1. map showing kerman city and the locations studied the collecting methods of live sand flies used for susceptibility tests, kerman province, 2019 fig. 2. using the baited traps for collection of live phlebotomus sergenti in the mountainous areas around kerman city, southeastern iran, kerman province, 2019 j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 86 http://jad.tums.ac.ir published online: march 31, 2021 table 1. statistical comparison of efficiency of two collection methods used for live collection of phlebotomus sergenti, kerman city, southeastern iran, july–sep 2019 capture methods capture place sessions of collection total collected capture rate (%) no. male no. female sex ratio physiologic conditions environmental condition no. fed no. unfed no. gravid temp °c relative humidity (%) baited traps outdoor 9 370 89.2* 198 172 1.2† 29 (16.8%) 112 (65.1%) 31 (18.0%) 28–30 <40 hand catch indoor 5 45 10.8* 22 23 1.0† 3 (17.4%) 17 (73.9%) 2 (8.7%) 26–28 <40 t=-7.214, df= 9, p= 0.001 † t=-2.574, df= 8, p= 0.07 fig. 3. statistical comparison of abundance of phlebotomus sergenti collected using hand-catch and baited traps from indoor and outdoor places, kerman city, southeastern iran, 2019 fig. 4. statistical comparison of sex ratio of phlebotomus sergenti collected using hand-catch and baited traps from indoor and outdoor places, kerman city, southeastern iran, 2019 j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 87 http://jad.tums.ac.ir published online: march 31, 2021 fig. 5. susceptibility of phlebotomus sergenti to ddt and malathion, collected using hand-catch from indoor places, kerman city, southeastern iran, 2019 fig. 6. susceptibility of phlebotomus sergenti to ddt, deltamethrin, malathion and propoxur collected using baited traps from outdoor places, kerman city, southeastern iran, 2019 j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 88 http://jad.tums.ac.ir published online: march 31, 2021 discussion despite the implementation of the national leishmaniasis surveillance program and the efforts for adopting different control methods during past decades, acl is still a major health challenge with increasing trends in many parts of iran, particularly kerman province. this study was conducted to investigate the susceptibility level of ph. sergenti to conventional insecticides according to the who instruction for the phlebotomine susceptibility test. furthermore, there is an operational gap for employing a capture method for adequate yielding of live ph. sergenti, which is necessary for the standard sample sizes used in susceptibility tests. in this study, efficiency of baited traps showed the more yield of live ph. sergenti compared to handcatch method from indoor places (t= 7.214, df= 9, p= 0.001) with no significant difference for the sex ratio. the most important obstacle for collecting sand flies from indoor places using the hand-catch method is the lack of cooperation and willingness of the households to check their houses for the presence of sand flies. in addition, in urban areas, due to the application of aerosol sprays or using cooling systems in the houses, the abundance of sand flies is usually lower than the suburbs. in the other study, different collecting methods were assessed for live sand flies with a total of 122 live ph. sergenti collected at outdoor sites in the mountainous parts of kashan district. the comparison of different traps showed the most efficiency (59.8%) for the black shannon traps during the summer (97). in other attempts made in the northeastern parts of iran as well as in cukurova plain, southern anatolia, turkey, the co2 cdc light traps showed the most efficiency with 43.2% and 75.0 percent for collecting live ph. sergenti with a total abundance of 2521 and 4 from outdoor sites (98-99). in another testing, different commercial traps were assessed for collecting live ph. sergenti in the north of aswan, southern egypt which the bgs traps showed more efficacy (35.5%) (100). the results of susceptibility tests on ph. sergenti collected from outdoor sites showed 100% mortality exposed to ddt 4.0%, deltamethrin 0.05%, malathion 5.0%, and propoxur 0.1%, indicating the complete susceptibility. the ph. sergenti caught in the houses located adjacent to the mountains using hand-catch method revealed 100% mortality when exposed to ddt 4.0% and malathion 5.0%. due to the obvious differences in the biology and behavior of ph. sergenti with other species of sand flies, the finding of the present study was compared with other studies if the tests were only carried out on ph. sergenti. the number of reports on the susceptibility level of ph. sergenti to insecticides is limited in the world. during the years 1971, 1976, and 1998, three trials were carried out in the acl foci in the large cities located in the northeast including mashhad (31) and neishabur (32) as well as kerman (101) located in the southeast of iran. the findings revealed the complete susceptibility of this species to the ddt. moreover, there are three other published records for assessing the susceptibility status of ph. sergenti in mashhad, northeast of iran (102) and dehbakri county, southeast of iran (103) in 2007 and 2011, which indicated the complete susceptibility of ph. sergenti to ddt 4.0% and deltamethrin 0.05%. in a more recent study carried out in maneh and samalqan county, northeastern iran, all sand flies were collected using cdc light traps from outdoor and tested with papers impregnated with ddt 4.0%, bendiocarb 0.1%, and permethrin 0.75%. the results showed mortality rates of 89.8±1.4, 93.6±1.4, and 95.6±1.7% respectively, indicating the resistance of ph. sergenti to ddt (104). a study carried out in north africa and the middle east in 2007 found the high susceptibility of ph. sergenti to malathion, ddt, cyfluthrin, bendiocarb, permethrin, and resmethrin (105). a susceptibility test conducted in morocco showed the full susceptibility of wild populations of ph. sergenti to ddt, lambdacyhalothrin, and malathion (106). the finding of the present research showed the susceptibility of ph. sergenti j arthropod-borne dis, march 2021, 15(1): 82–96 y rassi et al.: comparative testing of … 89 http://jad.tums.ac.ir published online: march 31, 2021 to ddt, malathion, deltamethrin, and propoxur in indoor and outdoor populations collected in the acl foci of kerman. it is expected that application of the insecticide-based control strategies for sand flies, for example irs, itns, itc and itt, could be an effective method for controlling ph. sergenti in indoor and outdoor places if the measures are performed correctly. conclusion anthroponotic cutaneous leishmaniasis is still a major health problem in kerman, southeast of iran. this research showed the high susceptible status of ph. sergenti in both the indoor and outdoor populations against all tested insecticides. the research-based evidence indicates the success of future insecticide-based vector control methods for controlling sand flies in the acl foci. acknowledgements the authors are grateful to mrs m mozafari due to her sincere assistance in providing the data of the acl cases in kerman. this study was part of a msc dissertation funded and supported by tehran university of medical sciences (tums) [grant no. 98-01-27-41998, research ethics approval id: ir.tums.sph.rec. 1398.079]. the authors 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2021 ulation of phlebotomus sergenti (diptera: psychodidae) to different imagicides in a endemic focus of cutaneous leishmaniasis in northeast of iran. j vector borne dis. 54: 282–286. 105. tetreault ge, zayed aebb, hanafi-bojd aa, beavers gm, zeichner bc (2001) susceptibility of sandflies to selected insecticides in north africa and the middle east. j am mosq control assoc. 17 (1): 23–27. 106. faraj c, ouahabi s, adlaoui eb, el elkohli m, lakraa l, el rhazi m, ameur b (2012) insecticide susceptibility status of phlebotomus (paraphlebotomus) sergenti and phlebotomus (phlebotomus) papatasi in endemic foci of cutaneous leishmaniasis in morocco. parasite vectors. 5: 51. j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 1 http://jad.tums.ac.ir published online: march 31, 2022 original article cellular immunity in mice vaccinated with recombinant phospholipase d toxoid of hemiscorpius lepturus scorpion narges safari-foroushani1, mohammad hossein modarressi2, kamran pooshang bagheri3, mahdi behdani3, *delavar shahbazzadeh3 1islamic azad university, science and research branch, tehran, iran 2department of medical genetics, school of medicine, tehran university of medical sciences, tehran, iran 3venom and bio therapeutics molecules lab, medical biotechnology department, biotechnology research center, pasteur institute of iran, tehran, iran *corresponding author: dr delavar shahbazzadeh, email: shahbazzadeh@yahoo.com (received 15 june 2021; accepted 22 sep 2021) abstract background: hemiscorpius lepturus is one of the most dangerous scorpions in iran and the world. numerous studies have been conducted on phospholipases, especially phospholipase d, in this scorpion’s venom, and the results have shown this protein to be the main cause of death. therefore, one of the most effective ways of preventing fatalities is to produce a toxoid vaccine from the deadly toxin of the venom. the present study was conducted to assess the nontoxicity of this toxoid and the safety of the vaccine candidate in balb/c mice. methods: the production of interferon-gamma and interleukin-4 cytokines in the spleen cells of the mice was measured using elispot assay 28 days following immunization with rpld toxoid. results: the unpaired t-test results showed a significant increase in the concentration of ifn-γ cytokine in the vaccinated mice (p= 0.001), indicating that the immune system is directed toward the th1 pattern, while no significant differ ence was observed in the levels of il-4 (p= 0.16) despite an increase in this cytokine. the in-vivo tests showed that the mice immunized with interval doses of 80µg of toxoid were completely protected against 10 × the ld100 of the venom. moreover, the toxoid had no dermonecrotic effects and caused no necrotic and inflammatory complications in the rabbit skin. conclusion: as a vaccine, the toxoid has the potential to increase the th1 cytokine response and, subsequently, increase acquired cellular immunity. thus, this toxoid appears to be able to provide an effective vaccine against the venom of hemiscorpius lepturus. keywords: hemiscorpius lepturus; toxoid; interferon-gamma (ifn-γ); interleukin 4 (il-4); cellular immunity introduction scorpion sting is one of the main public health problems in the world (1). the six dangerous scorpion species in iran include hemiscorpius lepturus (scorpionidae), androctonus crassicauda, hottentotta saulcyi, hottentotta schach, odontobuthus doriae, and mesobuthus eupeus (buthidae) and among them, h. lepturus is considered the most dangerous (2). hemiscorpius lepturus, or gadim scorpion (its local name), is blamed for 50% of all scorpion stings nationwide, while it is responsible for 89% of the reported deaths (3). in addition to adults, this scorpion’s sting is highly deadly in sensitive groups, particularly children, and causes 60 times more deaths than other scorpion species in iran (3, 4). stinging by this scorpion causes clinical signs and symptoms not previously described for other scorpion species (5). unlike other scorpions, whose venom causes neurotoxic signs and symptoms, this scorpion’s venom produces cytotoxic signs and symptoms with systemic effects such as dermonecrosis, inflammation, arterial leak, and renal failure (6, 7). these signs and symptoms are attributed to the venom’s enzymatic compounds (5, 8). recently, a 32-kda copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:shahbazzadeh@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 2 http://jad.tums.ac.ir published online: march 31, 2022 protein, called phospholipase d, has been identified in h. lepturus scorpion’s venom (9). it is a dermonecrotic toxin that causes signs and symptoms fully similar to those of the complete venom of h. lepturus and is the most important molecule in the scorpion venom (10, 11). these signs and symptoms are fully similar to the main clinical signs and symptoms of the brown loxosceles intermedia sting, which is responsible for many signs and symptoms and deaths in humans (9, 12). the treatment currently used for gadim venom in iran is the equine polyvalent antiserum against the six venomous scorpions in the country but there is still no standard protocol for the management of people afflicted with the venom of this scorpion (2). moreover, there are reports of anaphylactic shock and death in the people stung by this scorpion, especially young children (7). the use of antiserum is particularly effective against pathogens that can activate the immune system in the unstimulated state (13). based on a previous study, we assumed that toxoid (the first non-toxic form of phospholipase d toxin produced using formalin) would be able to induce active immunity in balb/c mice and, consequently, could be used as a vaccine (10). this hypothesis has previously been confirmed by using toxoids as vaccines for the prevention of diphtheria (14, 15, 16), tetanus (15, 17), and botulism (18, 19). accordingly, the present study was conducted to test the non-toxicity of the toxoid and assess the efficiency of the immunogenicity of the produced toxoid for its use as a vaccine. this study assesses the vaccine by investigating inflammatory and anti-inflammatory cytokines in the immunized mice. the authors believe that if it is demonstrated that pro inflammatory cytokines are significantly increased in the present study, the results were confirmed by repeated tests, it can then be concluded that the present vaccine increases cellular immunity in the mice and can also cause acquired immunity of the cellular type. materials and methods expression and purification of recombinant phospholipase d the phospholipase d (pld) gene of the iranian venomous scorpion h. lepturus was expressed and the product purified based on a previous design (10, 11). briefly, pet-22b plasmid was used in gene expression. this gene has the gene bank accession number: ky287766. e. coli bl21 bacterium was transformed using the pld gene. the transformed bacterium was cultured in lb medium containing ampicillin at 37 °c until optical absorption at 600nm wavelength reached 0.6. the induction was carried out using 0.1mm isopropyl beta-d-1thiogalactosidase (iptg) (thermo fisher scientific co. waltham, ma, usa). the cells were kept at 30 °c for 3.5 hours, then centrifuged at 10000×g for 10 minutes at 4 °c and suspended in the lysis buffer. to purify the recombinant protein, bacterial cells were first dissolved in 1ml of the lysis buffer per 0.1g of the precipitate and placed on ice for 30 minutes. then, the host cell membrane was disintegrated by sonication (hielscher co. teltow, germany) over 15 minutes at 30% amplitude with a cycle frequency of 30. next, the lysed cells were centrifuged, and the supernatant containing the soluble protein was purified by affinity chromatography with ni-nta agarose column (qiagen co. hilden, germany). the recombinant protein was dialyzed using pbs and analyzed by sds-page (20). toxoid production from recombinant phospholipase d to produce the toxoid, detoxification, recombinant pld was first diluted in sterile pbs and then added to a 10kd dialysis sac (10). the protein was exposed to 0.25 methanol formalin for 10 days at room temperature. the samples were gently dialyzed with pbs buffer at 4 °c, over 24 hours (the buffer was changed twice during this procedure) and filtered through a 0.22μm membrane to remove excess residue. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 3 http://jad.tums.ac.ir published online: march 31, 2022 the toxoid concentration was determined using the bca kit following the manufacturer’s instructions (i ntron biotechnology co. seoul, korea). the residual formalin test was performed to ensure the absence of formalin in the toxoid samples (20). animals the present study had a code of ethics from pasteur institute of iran for using animals in research (no: pii.rec.1394.38). two 3±1 months-old new zealand white rabbits weighing 2kg each, and one hundredtwenty 6±1 months-old female balb/c mice weighing 20– 25g each were procured from the pasteur institute of iran. before the experiments, the animals were kept in standard laboratory conditions at a temperature of 22–25 °c with 12 hourly alternating light/dark cycles and relative humidity of 40–60% with free access to food and water for one week, after which time, the mice were randomly divided into 2 groups (60 mice per group). toxoid toxicity test the toxoid toxicity was assessed using dermonecrotic activity so that 3μg of the toxoid was brought to a volume of 100μl of sterile pbs and injected subcutaneously into a rabbit with the hair on its back shaven. the presence of inflammation and necrosis was assessed after 4, 8, 16, 24, 48, 72, and 96 hours, and the injection site diameter was measured with a caliper. pbs and rpld1 toxin were used as negative and positive controls. toxoid immunogenicity in mice one hundred-twenty female balb/c mice were assigned into two sixty mice groups. the first group was chosen for vaccinating with toxoidfreund’s adjuvant and the second group for toxoidalum adjuvant as described in detail below. immunization by toxoid-alum adjuvant in this series of experiments each of sixty other mice received 80μg of toxoid. this amount of toxoid was mixed with 100μl of sterile pbs and then mixed with 100μl of alum adjuvant (thermo fisher scientific co. waltham, ma, usa.) followed by stirring until a homogenous solution was obtained. 200μl of this solution was subcutaneously injected to each mouse on days 1, 14, 28, 42, 56 and 70. here also, 6 mice that only received sterile pbs-alum under the same conditions were considered as negative controls. immunization by toxoid-freund’s adjuvant based on previous studies (safari et al. 2018), mice were immunized in 10 groups with two-weeks intervals on days 15, 29, 43, 57 and 71. the first injection was administered using complete freund’s adjuvant and the following injections using incomplete freund’s adjuvant. 80μg of toxoid was homogenized with 100μl of freund’s adjuvant at 4 °c for 4 hours, so that the antigen could completely mix with the adjuvant, diluted in pbs to 200μl, and injected subcutaneously. the control group was injected with 100μl of pbs and 100μl of freund’s adjuvant. the animals were examined in terms of health and weight in all stages of the experiment. here also, 6 mice that only received sterile pbs-freund under the same conditions were considered as negative controls. challenging tests in immunized mice the mice immunized by toxoid were challenged one week after the last injection. in the case of the mice which have been immunized by toxoidfreund’s adjuvant, the first group of them was divided into five groups of six mice. each mouse in group 1 received 10 (37µg), group 2; 20 (74µg), group 3; 50 (185µg), group 4; 100 (370µg) and group 5; 200 (740µg) ld 100 of the rpld. also, the other 30 mice from these mice were assigned to five groups of six mice and received different doses of crude venom, as group 1 received 1 (100µg), group 2= 2 (200µg), group 3= 5 (500µg), group 4= 7 (700 µg) and the group 5= 10 (1000µg) ld 100 of the crude venom. the other 60 mice that were immunized by toxoid-alum adjuvant received http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 4 http://jad.tums.ac.ir published online: march 31, 2022 the same doses of rpld and crude venom respectively as mentioned above. all the injections were done intraperitoneal, and mice were monitored for 2 weeks. those that survived were regarded as the survivors and non-immunized mice were regarded as the negative controls. isolation of splenocytes from mouse spleen the vaccinated mice were put down by spinalization four weeks after the last injection. then, they were submerged in 70% alcohol for a few seconds. the spleen was removed from each mouse by opening the peritoneum in fully sterile conditions under a microbiological hood and was completely cleaned of excess matter and adipose tissue. the spleen was quickly transferred to a small sterile petri dish with 5 ml of rpmi culture medium containing 0.5% fbs and penicillin and streptomycin antibiotics. the spleen cells were suspended using several injections of rpmi culture medium. the resulting suspension was centrifuged at 600×g and 4 °c for 10 minutes and tris-nh4cl was then added to it. the cell mixture was centrifuged after inverting it several times. the cell deposit was suspended in the rpmi culture medium containing antibiotics. cells were counted using a hemocytometer slide, and the percentage of the surviving cells was determined. the resulting cell supernatant was kept at -20 °c until analysis. counting was carried out using the following formula: number of cells counted in 4 squares in the corners of the neobar slide × 2 (dilution coefficient) × 104 (volume coefficient) measurement of il-4 and ifn-γ cytokines to place the mouse anti-il-4 and antiifn-γ monoclonal antibodies, first, 100μl of the antibody in question at a concentration of 5μg/ml in sterile pbs buffer was added to each well in a 96-well plate and kept at 4 °c overnight for 16 hours. the wells were rinsed three times with pbs and kept with 200μl of complete rpmi culture medium at laboratory temperature for 2 hours. the spleen cells isolated from each mouse were diluted, and 2× 105 cells in a volume of 100μl were added to wells in triplicates. a volume of 50μl of the toxoid at a concentration of 10μg/ml was added to each well to stimulate cells. only sterile pbs was added to the control wells. plates were kept at 37 °c for 48 hours. the culture medium was drawn from each well, and wells were rinsed once with cold sterile distilled water and three times with pbs buffer containing 0.05% tween 20; then, 100μl of the mouse biotin-avidin detection mba in pbs buffer with 1% bsa at a concentration of 2μl was added to the wells. plates were kept at laboratory temperature for 2 hours, and then unbound antibodies were removed and each well was rinsed with pbs buffer containing 0.05% tween 20. diluted streptavidin-hrp was added to each well at a 1:1000 ratio in pbs containing 1% bsa and then kept at 37 ºc for 1 hour. the wells were rinsed three to five times with pbs buffer, and 100μl of the final substrate solution bcib-bcip/nbt blue one component ap membrane substrate (surmodics ivd, inc., usa) was added. one hour after incubation at 37 °c, the wells were rinsed with sterile distilled water, and plates were air-dried. the spots that were developed were counted automatically through enzyme-linked immunospot (elispot) assay. il-4 and ifn-γ cytokines were measured using elispot ifn-γ (ab 64029) and il-4 (ab 64033) kits purchased from abcam company, usa. each test was repeated three times, and the results were assessed as mean ± sd. statistical analysis the statistical analysis was performed using ibm spss 19.0 (new york, usa). the results were expressed as the mean ± sd and were compared using student’s t-test. differences were considered statistically significant at values of p≤ 0.05. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 5 http://jad.tums.ac.ir published online: march 31, 2022 results detection of dermonecrotic activity of phospholipase d toxoid given that the main indicator of the effect of h. lepturus scorpion venom is phospholipase d with dermonecrotic properties, this test was used to assess the toxic properties of the produced toxoid, and the results were assessed after conducting the dermonecrotic test. the results showed that in 96 hours, the toxoid injection had no dermonecrotic effect in rabbits and no wounding or inflammation signs were observed on rabbit skin (fig. 1a). therefore, detoxification of phospholipase d to produce toxoid was successful, while 72 hours after phospholipase d injection (positive control), the necrosis area reached 0.72±0.3cm3 (fig. 1b), and pbs was considered as the negative control (fig. 1c). immune response of cytokines il-4 and ifnγ to toxoid vaccine following immunization of the mice with toxoid -freund/alum adjuvants, the supernatant obtained from their spleen was used to evaluate the anti-inflammatory il-4 cytokine and the inflammatory ifn-γ cytokine. no fatalities occurred in the course of the study. the mean immunity indices of ifn-γ and il4 cytokines were measured in the study and control groups. the mean concentrations of ifn-γ cytokine in the vaccinated group with toxoid -freund/alum adjuvants increased compared to the control. as a result, ifn-γ cytokine in the vaccinated group with toxoid alum adjuvant, extremely close results were observed with toxoidfreund adjuvant. the mean concentration of il-4 cytokine in the vaccinated group with toxoid -freund/alum adjuvants slightly increased compared to the control. there was a significant difference between all vaccinated groups in terms of the concentration of the inflammatory ifn-γ cytokine (p≤ 0.05) but no significant difference was observed in the concentration of anti-inflammatory il-4 cytokine between these groups (p≥ 0.05) (table 1). the concentration of inflammatory and anti-inflammatory cytokines was determined in vaccinated and control groups using the elispot technique, and the results were compared. the results showed an increase of 400–500 units or 400–500% in the inflammatory ifn-γ level in the study group compared to the control, and this increase was significant (p= 0.001). the level of the anti-inflammatory il-4 cytokine increased by 1.1–1.33 units or 1.1–1.3% in the study groups compared to the control group. despite this slight increase, the mean level of il-4 showed no significant difference between the study and control groups (p= 0.16). the comparison of the levels of inflammatory ifn-γ and anti-inflammatory il-4 cytokine with each other in the study groups showed an increase of 400– 432 units or 400–432% in the ifn-γ level compared to the il-4 level, and the difference between them was significant (p= 0.001) (fig. 2). thus, an increase in ifn-γ activity leads to a decrease in il-4 activity, and they have a regulatory effect on each other. comparisons were based on the unpaired t-test. p≤ 0.05 indicates a significant difference in all cases. only ifnγ concentration from freund/alum -immunized mice showed statistically significant results (p= 0.001) (fig. 2). thus, an increase in ifn-γ activity leads to a decrease in il-4 activity, and they have a regulatory effect on each other. vaccination trials in the mice the results indicated that the mice vaccinated with toxoid-freund/alum adjuvants challenged either by rpld or crude venom of h. lepturus, survived throughout the study. the results obviously indicated that all mice vaccinated with the toxoid were completely protected against a wide range of rpld or crude venom concentrations, using either of these two adjuvants (table 2). non-immunized negative control mice in all groups died after injecting one ld100 of rpld or one ld100 of crude venom. from these results, it can be deduced that the amount of vaccine (toxoid= 80 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 6 http://jad.tums.ac.ir published online: march 31, 2022 μg/mouse) which promoted the efficacy of the immune response was certainly adequate to protect the mice against rpld or crude venom of h. lepturus. as a result, toxoid vaccine developed an effective cellular immunity against the scorpion venom of h. lepturus. table 1. hemiscorpius lepturus toxoid concentration of inflammatory ifn-γ and anti-inflammatory il-4 in the mice vaccinated with toxoid -freund /alum adjuvants and control groups variable case group control group p* mean sd mean sd ifn-γ (a) ifn-γ (b) 263.33 231.51 6.77 4.13 42.66 40.24 2.51 3.14 0.001 il-4 (a) il-4 (b) 53.55 50.21 5.26 3.42 38.17 35.82 2.31 4.14 0.16 (a): ifn-γ/ il-4 in the mice vaccinated with toxoidfreund adjuvant. the mice that only received freund-adjuvant + pbs (control group) (b): ifn-γ/ il-4 in the mice vaccinated with toxoidalum adjuvant. the mice that only received alum-adjuvant + pbs (control group). *comparison based on unpaired t-test. p≤ 0.05 indicates a significant difference in all cases. the ifn-γ variable is statistically significant table 2. in vivo vaccination trials in immunized mice number of mice rpld challenge (ld100s)a survival rate (%) number of mice crude venom challenge (ld100s)(b) survival rate 6 10 100 6 1 100 6 20 100 6 2 100 6 50 100 6 5 100 6 100 100 6 7 100 6 200 100 6 10 100 neg. cont.(c) neg. cont.(c) 6 1 0 6 1 0 (a): one ld100 of rpld is equal to 3.7μg. (b): one ld100 of hemiscorpius lepturus scorpion crude venom is equal to 100µg (c): the mice that only received adjuvant + pbs (negative controls) fig. 1. hemiscorpius lepturus toxoid intracutaneous injection of 3µg of pld toxoid into rabbit’s shaved skin. no dermonecrotic activity was observed in 96 hours after injection (a). injection of 3µg of pld as the positive control showed a specific dermonecrotic activity, and the necrosis site reached 0.72cm3 after 72 hours (b). pbs was used as the negative control (c). each experiment was conducted with two repeats a b c http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 7 http://jad.tums.ac.ir published online: march 31, 2022 0 50 100 150 200 250 300 350 s f c ifn-γ control infγ il-4 control il-4 * * a b fig. 2. hemiscorpius lepturus concentration of inflammatory ifn-γ from mice vaccinated by toxoid -freund/alum adjuvants and anti-inflammatory il-4 from mice vaccinated by toxoid -freund/alum adjuvants and control groups by elispot method. (a) assay was performed on freund-immunized mice. the mice that only received freund-adjuvant + pbs (negative control). (b) assay was performed on alum-immunized mice. the mice that only received alumadjuvant + pbs (negative control). the mean ± s.e.m is shown in each column discussion given the high percentage of the annual mortality due to h. lepturus sting in the warm regions of iran, especially in the southern areas (khuzestan province) (3), the main solution is prevention instead of treatment. prevention occurs with vaccine production, and currently, the only effective treatment against h. lepturus sting is the use of the polyvalent antiserum obtained from horses immunized against the whole venoms of the six venomous scorpions of iran (2). the main disadvantages of the common antisera on the market include their lack of specific activity against particular scorpion venoms and the complications they cause, especially in susceptible groups of the population (21). various methods are used in the preparation of vaccines, and among them, formalin use is the most common (22) and basic (23) method. toxoid vaccines obtained by using formalin on the venom of cobra (24) and habu (25) snakes are currently used in southern asian countries, especially in india. when toxoid is used as a vaccinating agent, an augmented im mune response ensues that forms an immunological memory against the toxoid immunogenic epitopes (26). based on previous reports, we used formalin for the first time to detoxify the most venomous component of h. lepturus venom (phospholipase d protein) to produce a toxoid vaccine (10). detoxification of this protein and its conversion into toxoid can provide a suitable candidate for vaccinating individuals against h. lepturus venom if the three-dimensional structure of the protein is preserved. the use of formalin modifies histidine residues and produces uncommon imidazole derivatives in the protein (27). the resulting toxoid can neutralize the deadly effect of the phospholipase d toxin and the complete h. lepturus venom (10). after developing this new molecule, in the present study, we assessed the toxicity of this toxoid and the active immunity type of the vaccine. dermonecrotic activity assessment is the most important confirmatory test for toxoids developed from toxins. it can be inferred from the results that the produced toxoid does http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 8 http://jad.tums.ac.ir published online: march 31, 2022 not have any enzymatic activity even to the slightest degree, while all toxins in the whole venom, such as phospholipase d toxin, and the whole venom contain this activity (9, 28). in agreement with our results, it has previously been reported that recombinant pld antisera inhibit the necrotic activity of the whole venom of gadim scorpion (11). other findings confirm that some antibodies against dermonecrotic toxins can fully neutralize the dermonecrotic activity of the scorpion’s whole venom. for instance, in the study conducted in brazil, anti-dermonecrotic antibodies were produced against the brown loxosceles intermedia spider venom, which were fully capable of neutralizing the spider’s venom (29). thus, the antisera obtained by using the toxoid produced in the previous study can be regarded as an effective agent in preventing necrotic wounds caused by h. lepturus venom (10). studies have shown that inflammatory effects are observed following the scorpion sting (30). the toxicity of the toxoid and the effectiveness of the vaccine were assessed through dermonecrotic test and t-helper (th) cell immune response, respectively. to transmit a message and affect other cells, the immune system cells must secrete the products that mediate the transmission of this message (26). in response to microbes and other antigens, cells of the innate and specific immunity system secrete protein molecules, generally known as cytokines, that produce different effects (26, 31). two subgroups of th immune cells, th1 and th2, secrete different types of cytokines (26). among the cytokines secreted by th2 cells, the main interest is in interleukin-4 (il-4), which has the key role in the synthesis of ige (32). the il-4 producing cells stimulate and differentiate lymphocytes and switch isotypes from igm to ige production in b lymphocytes and thus have a major role in inducing and maintaining allergies, inflammation, and necrosis (33). il-4 stimulation can affect mast cell responses through ige signaling (34). studies have shown that the increase in serum il-4 levels is directly associated with allergic symptoms and inflammation (33, 34). il-4 has a central role in type 1 allergic reactions and can have a role as a mediator in the t-cells differentiation (35). on the other hand, as the most important cytokine synthesized by th1 cells, ifn-γ has a role in cell immunity and inhibits th2-related immune response (36, 37). studies have shown that ifn-γ has a highly important role in th2 lymphocyte dependent allergic reactions (37, 38). ifn-γ can cause the release of pro inflammatory cytokines during allergic reactions (39). th1 and th2 cytokines’ role in the immune response and their mutual balance have decisive effects on disease incidence and complications (26, 37). differentiation of t-helper lymphocytes can be induced through the effect of various cytokines and antigens (40). ifn-γ cytokine can cause differentiation of th0 toward th1 (39). activation of the th1 immune response has pro inflammatory properties and activates b cells and macrophages and induces igg toward igg2β, igg2α in mice, resulting in the activation of the cellular acquired immune system (37, 39-40). il-4 cytokine causes differentiation of th0 toward th2 (39). activation of the th2 immune response, which has anti-inflammatory properties, results in the growth and differentiation of lymphocytes, production of immunoglobulin e, basophils, mastocytes, inactivation of macrophages, recruitment of eosinophils, and induction of igg toward igg1 in mice and causes inhibition of the immune system (26, 37). in the present study, to assess the inflammatory effects of the vaccine and type of immunity conferred by it, concentrations of inflammatory ifn-γ and anti-inflammatory il-4 cytokines in the supernatant of the spleen of the mice immunized with toxoid was determined and their relationship with cellular immunity was investigated. given the sensitivity of this experiment, the immune response was measured by elispot assay to evaluate the toxoid as a vaccine candidate. this test is 20 to 200 times more sensitive than the elisa test and http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 9 http://jad.tums.ac.ir published online: march 31, 2022 its results are almost 100% accurate (41). in addition to the increase in humoral immunity that was assessed using the elisa test in our previous article (10), the present study provides evidence for an increase of 400–500 % in the concentration of the inflammatory cytokine in the immunized mice compared to control groups. this increase is significant and has noticeably increased the cellular immunity. however, the anti-inflammatory cytokine increased by 1.1– 1.33% compared to the control group, and this increase was not significant. to evaluate the potency of this toxoid protein as a vaccine candidate and its effectiveness in neutralizing the lethality of crude venom of h. lepturus scorpion, the following experiments were performed. the groups of mice vaccinated by toxoid using freund or alum adjuvants, were challenged by whole venom of h. lepturus. their survival rate was documented for two weeks’ post-injection and over longer time periods. this survival strongly affirms that all mice were completely protected against wide range of different lethal dosages of crude venom (10 ld100, equal to 50mg/kg), thus illustrating a successful and 100% immunization in the mice. the results obtained confirm that the developed vaccine, in addition to inducing humoral immunity, can increase the th1 cytokine response within lymphocytes and consequently increase cellular immunity. in agreement with the present study results, a study conducted by borchani et al. showed that a type of pld in h. lepturus called hemonecrolysin can increase the secretion of pro-inflammatory factors tnf-α and il-6 on human monocytes by 3.5 times compared to the control. this pld can also double the level of the anti-inflammatory factor il-10 compared to the control (42). furthermore, in agreement with the present study results, it has been reported that the recombinant phospholipase d obtained from the brown loxosceles recluse is also able to increase pro-inflammatory cytokines tnf-α, il-1β, il-8 on human fibroblasts (43). these results show that the vaccine produced from toxoid has the same effect on t-cell as the whole venom, recombinant phospholipase d from the scorpion venom, or recombinant phospholipase d of the brown spider and can activate inflammatory processes and that it can be used as a booster of the immunological memory against scorpion/spider stings. in a study conducted by homayoon et al. (44), it was shown that pasteurella multocida vaccine, inactivated by formalin, can significantly increase pro-inflammatory cytokines, and significantly increase cellular immunity in the immunized balb/c mice compared to the control group. in agreement with the results obtained, the vaccines produced from leishmania major also significantly increased the ability to produce ifn-γ and resulted in the th1 response in mice, while the production of il-4 and the th2 cytokine response decreased significantly (45). overall, lack of clinical assessments was among the limitations of the present study and is proposed to be addressed in future studies. moreover, according to the present study results, this vaccine, as a protective agent in people exposed to h. lepturus scorpion sting, can increase th1 cytokines and thus increase the acquired immunity of the cellular kind. conclusion the present study results showed for the first time that toxoid, as a vaccine, can increase the production of certain components of th1 and th2 cytokine response in the serum of mice immunized with rpld toxoid, and this increase is significant in terms of the level of ifn-γ cytokine. furthermore, it was shown that the supportive immunity produced in the mice was associated with the activity of th1 cytokines. acknowledgements this article is a part the ph.d. thesis of narges safari-foroshani approved by the pasteur institute of iran, tehran, iran. this rehttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 1–12 n safari-foroushani et al.: cellular immunity in … 10 http://jad.tums.ac.ir published online: march 31, 2022 search did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ethical considerations male balb/c mice (18–20g) were purchased from pasteur institute of iran. the animals were allowed to adapt for a week in standard conditions with a dark/light cycle of 12h. the room temperature was 22±1 °c and the relative humidity adjusted at 50±5%. the animals were received a standard pellet diet and fresh tap water. all experiments were approved by ethical committee of the pasteur institute of iran (code number ir.pii.rec.1394. 38). conflict of interest statement authors declare that there is no conflict of interest. references 1. jalali a, rahimi f (2014) epidemiological review of scorpion envenomation in iran. j iran pharm res. 13(3): 743–756. 2. latifi m, tabatabai m (1979) immunological studies on iranian scorpion venom and antiserum. j toxicon. 17(6): 617–621. 3. shahbazzadeh d, srairi‐abid n, feng w, ram n, borchani l, ronjat m, akbari a, pessah in, de waard m, el ayeb m (2007) hemicalcin, a new toxin from the iranian scorpion hemiscorpius lepturus which is active on ryanodine‐sensitive ca2+ channels. j biochem. 404(1): 89–96. 4. seyedian r, jalali a, babaee mh, pipelzadeh mh, rezaee sa (2012) a biodistribution study of hemiscorpius lepturus scorpion venom and available polyclonal antivenom in 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with bacterial dnas as adjuant. arch clin microbiol. 11(1): 1105– 1109. 45. roberts mt, b-stober c, mckezie a, blackwell jm (2005) interleukin-4 (il-4) and il-10 collude in vaccine failure for novel exacerbatory antigens in murine leshmania major infection. infect immun. 73(11): 7620–7628. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2013, 7(2): 188–193 a veysi et al.: laboratory evaluation of … http://jad.tums.ac.ir published online: august 31, 2013 short communication laboratory evaluation of a rodenticide-insecticide, coumavec®, against rhombomys opimus, the main reservoir host of zoonotic cutaneouse leishmaniasis in iran arshad veysi 1, hassan vatandoost 1, mohammad hossein arandian 2, reza jafari 2, mohammad reza yaghoobi-ershadi 1, yavar rassi 1, *amir ahmad akhavan 3,1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2esfahan health research station, national institute of health research, tehran university of medical sciences, esfahan, iran 3institute for environmental research (ier), tehran university of medical sciences, tehran, iran (received 11 feb 2013; accepted 22 apr 2013) abstract background: zoonotic cutaneous leishmaniasis is a growing health problem in many rural areas of iran. rhombomys opimus, the great gerbil, is the main animal reservoir of zcl in the northeast and central part of iran. the aim of the current study was to evaluate the rodenticidal effect of coumavec® (a mixture of coumatetralyl 0.5% and etofenprox 0.5%) on r. opimus under laboratory condition. methods: great gerbils were collected from sejzi rural district, esfahan province, iran. four groups of 19 great gerbils were treated with the poisoned baits of different concentrations and one group was considered as control. the bating procedure was conducted in three stages: first, second (a week after first) and third (a month after first stage), in each stage baits were offered in 1 day, based on national protocol for rodent control operation in purpose of zcl control. results: the mortality rate for 0.03, 0.0625, 0.125 and 0.25% concentrations in the first stage of baiting were obtained 36.8%, 31.5%, 52.6% and 36.8%, in the second stage 47.3%, 52.6%, 68.4% and 52.6%, and in the third stage 52.6%, 63.1%, 68.4% and 57.8% respectively. the maximum and minimum mortality has occurred in 5-6 days and 31-40 days intervals consequently. conclusion: the results of this study showed that, coumavec® has some rodenticidal effects on r. opimus in laboratory condition. for the appropriate rodenticide-insecticide contamination of the rodent body and also considering to the economic issues, we suggest the use of 0.125% concentration for rodent control operation in the field condition. keywords: rhombomys opimus, rodenticide, coumatetralyl, zoonotic cutaneous leishmaniasis, iran introduction zoonotic cutaneous leishmaniasis (zcl) is a growing health problem in many rural areas of iran, which involves 17 out of 31 provinces of the country (yaghoobi-ershadi 2012). rodents, especially those belonging to gerbillinae subfamily have significance role as reservoir hosts of zoonotic cutaneous leishmaniasis (yaghoobi-ershadi 2008, yaghoobiershadi and javadian 1996). in addition, different kinds of pathogens like bacteria, rickettsia, viruses, protozoa and helminthes can be transmited by rodents to human and animals (bell et al. 1988). phlebotomus papatasi is the main vector of zcl and leishmania major is the causative agent of the disease in the most parts of the country (yaghoobi-ershadi 2012). rhombomys opimus, the great gerbil, is the main animal reservoir *corresponding author: dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir 188 j arthropod-borne dis, december 2013, 7(2): 188–193 a veysi et al.: laboratory evaluation of … http://jad.tums.ac.ir published online: august 31, 2013 of zcl in the northeast and central part of iran (yaghoobi-ershadi and javadian 1996). this gerbil is relatively large and has diurnal activity whereas the most other species have nocturnal activity (dubrovskiy 1979). a karyotype study revealed that diploid chromosome number for r. opimus was 2n=40 (akhavan et al. 2004). rhombomys opimus is an herbivores rodent and feeds on green dried leaves as well as stems of plants. this species has social life and lives with other members of the colony in their burrows. this species has 1 to 2 and sometimes 3 generations in spring and 1 more generation in summer or fall. they have 5–14 offsprings in each generation. the longevity of this species reaches to 3 years. this long longevity provides appropriate condition for the maintenance of leishmania parasites in the rodent population while sand flies are inactive. many ecological characters of great gerbils and presence of sand flies, directly or indirectly, effect the leishmaniasis cycle in the nature (bell et al. 1988). attempts to control of leishmaniasis in the field condition by rodent control operation have been conducted in some countries including iran. in central asia, a largescale operation against great gerbils through poisoned baits successfully eliminated the rodents (dergacheva and zherikhina 1980). along april to january 1997, a field trial carried out to control zcl by destruction rodent burrows and using zinc phosphide 2.5% baits in radius of 500 meters from houses in central iran. the results showed that the control program reduced the incidence of zcl 12folds at the end of the first year of the operation and 5-folds at the end of the second year (yaghoobi-ershadi et al. 2000, yaghoobiershadi et al. 2005). recently some behavioural resistance and/or bait shyness against the conventional rodenticide, zinc phosphide, among the population of great gerbils has been reported from some endemic areas of the disease (unpublished data, esfahan health research station, iran). it is necessary to introduce some new, safer and more effective alternative rodenticides to control the reservoir hosts and subsequently the disease in endemic areas of zcl in iran. furthermore, coumavec® has insecticidal effect on the ectoparasites and other blood feeding insects of the gerbils but the aim of the current study was the evaluation of rodenticidal effect of coumavec® (a mixture of coumatetralyl 0.5% and etofenprox 0.5%) on r. opimus in laboratory condition. materials and methods rodent collection active colonies of gerbils were identified in sejzi rural district (32˚39˙54.84”n/ 52˚08˙07.38”e), esfahan province, iran. sherman live traps were placed near the rodent holes from january to february 2010. around 80 to 100 live traps baited with cucumber and sometimes carrot were used in each day. they were set up daily in the early morning to evening in winter. the collected gerbils were transferred to the animal house of esfahan health research station. different morphologic criteria of rodents were used for r. opimus identification. captured rodents were identified by valid identification keys (etemad 1978). just r. opimus species were included in the study. all the animals, which were used in this study, had no experience of exposing to the rodenticides. laboratory tests all experiments were conducted with animals maintained individually in metal cages. five groups of 19 great gerbils per group were sexed and selected, four groups were treated with the poisoned baits of each concentration and one group was as control. the commercial formulation of coumavec®, a mixture of coumatetralyl 0.5% and etofenprox 0.5% (levant overseas development ltd, argenteul, france) which was evaluated in 189 j arthropod-borne dis, december 2013, 7(2): 188–193 a veysi et al.: laboratory evaluation of … http://jad.tums.ac.ir published online: august 31, 2013 these tests, recommended for direct application as a rodenticide dust or for dilution in bait. poisoned baits were prepared using a mixture of grain and four concentration of coumavec® (0.03, 0.0625, 0.125 and 0.25%). sexually mature animals were acclimatized to the laboratory condition at least 7 days. the bating procedure was conducted in three stages: first, second (a week after first) and third (a month after first stage), in each stage baits were offered in 1 day, based on national protocol for rodent control operation in purpose of zcl control. along the keeping animals in the animal house, before the tests, they were fed on the standard animal diet (pellet). for providing required water, they were offered carrot. in each stage, the diets were withdrawn and a fresh container, holding 15 gr of poised bait for each rodent, was placed in the cages. the ordinary diet was not resumed until complete consumption of poison bait. the gerbils were then fed on ordinary laboratory diet until the next stage of baiting. dead animals were examined for internal bleeding and other symptoms of anticoagulant poisoning. gerbils, which survived during the test period, were maintained on laboratory diet, and those still alive 30 days after third offered poisoned bait, were considered as survived individuals. animal ethics consideration animal experiments were approved by the ethical committee of tehran university of medical sciences, tehran, iran. data analysing the data were analyzed using spss 11.5 and graphs were prepared using excel. the cumulative mortality rate of gerbils among the treated and control groups was compared using chi-squared test. results totally 95 healthy-mature rodents were used in this investigation. all dead animals were examined for internal bleeding and other symptoms of anticoagulant poisoning (fig. 1). the results of toxicity test based on concentration in each stage using coumavec® is shown in fig. 2. the mortality rate for 0.03, 0.0625, 0.125 and 0.25% concentrations in the first stage of baiting were obtained 36.8%, 31.5%, 52.6% and 36.8%, in the second stage 47.3%, 52.6%, 68.4 and 52.6%, and in the third stage 52.6, 63.1, 68.4 and 57.8% respectively. the maximum and minimum mortality has occurred in 5–6 days and 31–40 days intervals consequently. the maximum rate of mortality for female and male was observed in 0.0625 and 0.125% concentrations consequently (fig. 3). in spite of more mortality rate in 0.125% concentration, statistical differences between the concentrations have not seen. the number of dead rodents in the intervals between 1–5, 6–10, 11–15, 16–30 and 31–40 were summarised in table 1. as the table shows, the maximum and minimum mortality has occurred in 5–6 and 31–40 interval consequently. the statistical analyses showed that there were no significant differences between four selected concentrations of coumavec® on the rodent’s mortality rate. fig. 1. internal bleeding of a rhombomys opimus treated by coumavec® 190 http://jad.tums.ac.ir published online: august 31, 2013 j arthropod-borne dis, december 2013, 7(2): 188–193 a veysi et al.: laboratory evaluation of … http://jad.tums.ac.ir published online: august 31, 2013 table 1. mortality time of the tested rodents with coumavec® under laboratory condition in each stage of the baiting concentration total 1–5 6–10 11–15 16–30 31–40 0.03 19 8 (41.1%) 0 (0%) 1 (5.2%) 1 (5.2%) 0 (0%) 0.625 19 6 (31.5%) 1 (5.2%) 2 (10.5%) 3 (15.78%) 0 (0%) 0.125 19 6 (31.5%) 4 (21%) 2 (10.5%) 0 (0%) 1 (5.2%) 0.25 19 6 (31.5%) 3 (15.7%) 0 (0%) 1 (5.2%) 1 (5.2%) control 19 0 0 0 0 0 fig. 2. mortality rate of the tested rodents with coumavec® in laboratory condition in each stage of the baiting fig. 3. mortality rate of the tested rodents with coumavec® in laboratory condition based on sex discussion the results showed that maximum mortality occurred along 1–5 days after first baiting, slightly more than 40 percent for 0.25, 0.125, and 0.0625% and over 40 percent for 0.03% concentrations. it is suggested that the later baiting stages did not have dramatic effects on the mortality. the complete mortality was not obtained for all concentration as well. the maximum mortality was slightly more than 68% and the minimum was almost 52% occurred in 0.125 and 0.03% concentration, consequently. in addition gill and redfern (gill and redfern 1983) showed that complete mortality with coumatetralyl (15 days feeding, in 0.0375% concentration) against meriones shawi were not obtained. 191 j arthropod-borne dis, december 2013, 7(2): 188–193 a veysi et al.: laboratory evaluation of … http://jad.tums.ac.ir published online: august 31, 2013 all concentrations have an increasing mortality trend, as prepared baits offered in each stage, the mortality increased consequently. in dissected dead animals, the expected organs were checked. bleeding mostly occurred in stomach and after that extent to the intestine, spleen and liver. therefore, all concentrations can be used in the field operations but in view of the fact that this pesticide is an anticoagulant-insecticide mixture, for the appropriate rodenticide-insecticide contamination of the rodents’ body and also considering to the economic issues, this article suggests 0.125% concentration for rodent control operation in the field condition. acknowledgements authors would like to express their appreciation to the staff of esfahan health station, national institute of health research, tehran university of medical sciences (tums) for their collaboration along the study. in addition, the authors are grateful to levant overseas development (lod), ltd, france for providing and sending coumavec® to conduct this project. this research was financially supported by school of public health, tums, institute for environmental research (ier), tums and department of zoonosis, cdc, ministry of health and medical education, islamic republic of iran. the authors declare that they have no conflicts of interest. references akhavan aa (2011) immune response of great gerbil against phlebotomus papatasi saliva. lap lambert academic publishing, saarbrücken, germany. akhavan aa, yaghoobi-ershadi mr, shiranibidabadi l, safari n, abdoli h, derakhshandeh p, zahraei ramazani ar (2004) karyosystemic and morphometric characterization of rhombomys opimus and meriones libycus, the main reservoir hosts of zoonotic cutaneous leishmaniasis in the endemic foci of isfahan provinces, iran. 12th iranian congress on infectious diseases and tropical medicine. 2004 january 17–21, tehran, iran. p 22. bell jc, plamer sr, payne jm (1988) the zoonosis: infection transmitted from animal to man. edward arnold press, london, uk. dergacheva i, zherikhina ii (1980) changes in the population density and species composition of phlebotomidae as a result of eradication of great gerbils in a focus of zoonotic cutaneous leishmaniasis in the karshi steppe. meditsinskaya parazitolojiya i parazitarnye bolezni. 49: 50–55. dubrovskiy ya (1979) biology of great gerbil the principal carrier of zoonotic cutaneous leishmaniasis. who traveling seminar on leishmaniasis control. exussr ministry of health.. etemad e (1978) mammals of iran, vol. 1. rodents and their identification keys. tehran, national society of guardianship of natural resources and human environment (persian). gill j.e. and redfern r (1983) laboratory tests of seven rodenticides for the control of meriones shawi. j hyg camb. 91: 351–357. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod-borne dis. 6(1): 1–17. yaghoobi-ershadi mr (2008) reservoir host of cutaneous leishmaniasis in iran. in: nadim a, javadian e, mohebali m, zamen-momeni a (eds) leishmania parasite and leishmaniasis. academic press center. tehran, iran. pp. 177– 190. 192 j arthropod-borne dis, december 2013, 7(2): 188–193 a veysi et al.: laboratory evaluation of … http://jad.tums.ac.ir published online: august 31, 2013 yaghoobi-ershadi mr, zahraei-ramazani ar, akhavan aa, jalali-zand ar, abdoli h, nadim a (2005) rodent control operations against zoonotic cutaneous leishmaniasis in rural iran. ann saudi med. 25(4): 309–312. yaghoobi-ershadi mr, akhavan aa, zahraeiramazani ar, javadian e, motavalliemami m (2000) field trial for the control of zoonotic cutaneous leishmaniosis in badrood, iran. ann saudi med. 20 (5–6): 386–389. yaghoobi-ershadi mr, javadian e (1996) epidemiological study of reservoir hosts in an endemic area of zoonotic cutaneous leishmaniasis in iran. bull who. 74(6): 587–590. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996) meriones libycus and rhombomys opimus (rodentia: gerbillidae) arethe main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans r soc trop med hyg. 90: 503–504. 193 j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 282 http://jad.tums.ac.ir published online: january 06, 2016 original article immunization of cattle with tick salivary gland extracts *ali nikpay 1, sedigheh nabian 2 1department of pathobiology, amol university of special modern technologies, amol, iran 2department of parasitology, faculty of veterinary medicine, tehran, iran (received 15 oct 2014; accepted 25 feb 2015) abstract background: rhipicephalus (boophilus) annulatus tick is one of the most important ectoparasite of cattle. recently, several laboratories in the world have been concentrated on immunizing cattle against tick using various types of tissue extracts of ticks. the aim of this study was to evaluate the effect of immunization of cattle with tick salivary gland extract on biological parameters of ticks and humoral immune responses of cattle. methods: fourteen more dominant protein bands identified as immunogenic by western-blot analysis were eluted from polyacrylamide gel. test and control groups were injected three times with eluted proteins and sterile pbs (ph= 7.2) respectively with equivalent amount of adjuvant. after four weeks a tick challenge was performed. finally, biological parameters of collected engorged female ticks were recorded and humoral immune responses to immunization measured by elisa. results: the results indicated immunization of cattle resulted in reduction in mean tick counts, attachment, engorgement weights, feeding index, egg mass weight, hatchability and fertility index (respectively 63.1%, 62.6%, 30.2%, 36.4%, 40%, 78.7% and 13.3%) and increased duration of feeding, pre-oviposition and incubation period of eggs (respectively 8.6%, 45 and 31.34%). all changes were statistically significant (p< 0.05). results showed an increase in antibody production of test group from the first week after immunization. the antibody level was boosted following tick infestation. conclusion: this investigation indicates that immunization of cattle with these antigens could induce a protective immune response against rh. (b.) annulatus tick that would be expected to provide a safe non-chemical means of tick control. keywords: rhipicephalus (boophilus) annulatus, immunization, salivary gland, humoral immune responses, tick introduction rhipicephalus (boophilus) annulatus tick is one of the most important ectoparasites of cattle that can occasionally complete its life cycle successfully on sheep, goats and wild ungulates such as antelopes. surviving in asia, africa, america and europe, this species of tick causes economic losses due to direct effects on the preferred hosts and by the pathogens they transmit (murrell and barker 2003, estrada-peña et al. 2004, rajput et al. 2006, wikel 1999a, b). therefore, tick control is a continuing global priority. traditional control methods such as using chemicals have had partially successful results, however, acaricide compounds are expensive and have adverse effects such as a high incidence of resistance among tick populations, as well as harmful effects on vertebrate hosts, human beings and the environment. these increasing problems have stimulated researches on alternative methods of tick control in recent years (mattewson et al. 1975, walker 1994, nari and hansen 1999, fao 2004, rajput et al. 2006). these methods include biological control methods such as pasture spelling and artificial selection for tickresistant cattle. such steps can reduce tick burdens, but enhancement of host resistance through artificial immunization with a vaccine against ticks would constitute a *corresponding author: dr ali nikpay, e-mail: ali.nikpay@gmail.com j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 283 http://jad.tums.ac.ir published online: january 06, 2016 major advance in control process. several attempts have been done to immunize actively cattle against ticks and it has been reported that protective host immune responses can damage female tick internal organs and interfere with tick's feeding and fertility behavior (wikel 1999a, b, willadsen and jongejan 1999). performing various functions in the life cycle of ixodid ticks especially in their feeding procedure, the salivary glands are the major route for pathogen transmission. to facilitate attachment and engorgement, ticks produce biologically active components with anti-haemostatic, anti-inflammatory and immunomodulatory effects. “the initial secretion of the tick saliva contains a cement-like material, which helps the tick to establish a firm attachment with the host tissue” (hager and burgess 1980). exposure to proteins secreted by ticks can provoke antigen-specific humoral and cellular responses in the mammalian hosts. containing the natural antigens injected by the tick during infestation, salivary gland extracts might be an obvious choice to induce immunity against ticks (willadsen 1980, brossard and fivaz 1982, wikel 1996). the purpose of this research was to determine the protein pattern of rh. (b.) annulatus salivary glands by sds-page and to investigate the effects of immunizing cattle with immunogenic fractions of salivary gland extract of this tick against tick infestation. the quality of immune responses measured by elisa and the efficacy of immunization was evaluated by analyzing feeding and fecundity parameters of ticks harvested from treatment group compared to the control groups. materials and methods ticks engorged female rh. (b.) annulatus ticks were collected from healthy cattle (free of any transmissible pathogens), washed with 70% ethanol and then washed three times with sterile phosphate buffer saline (pbs) ph= 7.2, after drying they were kept under a constant temperature of 28 ºc, a relative humidity of 85% and a 12:12 (l: d) photo period. experimental animal eleven healthy holstein calves (3–5 months old) were purchased from a tick-free area (tehran veterinary medicine faculty research institute) and kept in tick-proof pens. antigen preparation semi engorged adult female ticks fed for about 7–10 days on tick-bite free cattle, was collected to rear and maintain clean ticks. after washing with 70% ethanol and sterile pbs, ticks were opened along their dorsal surface as previously described (brown et al. 1984). then, salivary glands were removed, dissected free of other tissues, placed into pbs containing 1 mm phenyl methyl sulphonyl fluoride (pmsf) at 4 ºc. salivary glands were suspended in 200 μl pbs with pmsf, grinded by glass beads, sonicated (dr hielscher gmbh, germany) for 30 minutes with 0.4 s cycles (40w) on ice and completely homogenized by freeze-defreeze alternatively. the mixture then were centrifuged at 12000×g for 15 minutes at 4 ºc , supernatant were collected and stored at -20 ºc until use. the protein concentration of salivary gland extract determined according to the method of warburg (hudson and hay 1984, tietz and andresen 1986). protein electrophoresis protein analysis by sds-page was carried out using a discontinuous gel system (laemmli 1970) with an acrylamide concentration of 5% in stacking gel and 12% in the separation gel. a high molecular weight protein ladder (fermentas, smo661) was used as molecular weight markers. j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 284 http://jad.tums.ac.ir published online: january 06, 2016 western blot analysis the protein bands from polyacrylamide gel were electro blotted to a nitrocellulose paper (ncp) using a mini blotter (biorad) according to wang's method (wang and nuttall 1994). nonspecific reactive sites on the ncp blocked for 1 h in tris-buffered saline with 0.5% tween 20 (tbs-t ph 8), containing 3% bovine serum albumin (habtech) on a shaking plate at room temperature. the ncp was incubated for 1 h at 37 ºc with bovine sera diluted 1:50 in pbs (ph= 7.2), the ncp was incubated for a further 1 h at 37 ºc with sheep antibovine igg (serotech, conjugated with hrpo) diluted 1:2000 in pbs-tween, washed and reacted with di amino benzidine (dab) (sigma) as a substrate. the ncp was washed 3 times between each step of the assay with pbs-tween. finally, the immunogenic protein fractions were appeared on the ncp. protein extraction from polyacrylamide gel after staining the polyacrylamide gel with coomassie blue, the desired bands identified by western blotting were cut out with a scalpel blade, sliced to a minimum and put into a microcentrifuge tube. gel slices were washed twice with distilled water and soaked in 1 mm 2-mercaptoethanol for 15 min. then, gel slices were incubated in elution buffer for 12 hours. after centrifuge, the supernatant was decanted and replaced by elution buffer and incubated for 4 hours. the two solutions were then pooled and added 5 volumes of -20 ºc methanol: acetone solution to remove sds and stain as well as precipitation of the protein. after 24 hours acetone solution were discarded and pellet were air dried on ice. to renaturation of protein with guanidine hcl, the pellet was suspended in the minimum amount of buffer a, containing 100 mm kcl, 1mm dtt, 12.5 mm mgcl2, 6 m guanidine, 10% (v/v) glycerol, 25 mm hepes-koh ph 7.6 and 0.1 mm edta. after 20 minutes incubation at room temperature, several volumes of buffer b, composed of 1mm dtt, 0.1 mm kcl, 0.01% (v/v) igepal ca-630, 12.5 mm mgcl2, 10% (v/v) glycerol, 25 mm hepes-koh ph 7.6 and 0.1 mm edta, were added and dialyzed at room temperature for 2–3 hours against buffer b. protein concentration was measured by warburg method. the protein solution was sterilized with a 0.45 μm filter (schleicher and schuell, germany) and stored at -20 ºc (hager and burgess 1980, hunkapiller et al. 1983, kadonaga 2007). immunization cattle were divided into two groups (treatment and control). the treatment group were intradermally injected with a mixture of 100 μg eluted protein and equivalent amount of freund's adjuvant (biogen) every two weeks for a total three immunizations (first injection with complete freund's adjuvant and two another with incomplete adjuvant). in a similar procedure the control group were injected with sterile pbs (ph= 7.2) and equivalent amount of complete and incomplete adjuvant as followed above. tick challenge four weeks after the last injection, each cattle were infested with about 500 rh. (b.) annulatus tick larvae as described previously (nikpay et al. 2012) on their right flank. tick parameters infested animals were checked daily and recovered ticks were collected, counted, weighed and isolated. engorged ticks were collected twice a day, at 8:00 am and 4:00 pm. each five engorged ticks were placed in a tube and were maintained in incubator with a constant condition as described earlier. the degree of protection was determined by measuring the usual parameters in ticks fed on immunized animals and comparing the results with those fed on control animals. j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 285 http://jad.tums.ac.ir published online: january 06, 2016 these parameters were including: mean of recovered tick counts, attachment rate, engorgement weight, length of feeding period, feeding index (engorgement weight(mg)÷ feeding length (day)), preoviposition period, egg mass weight, incubation period, hatchability rate and fertility index (egg mass weight(mg)÷engorgement weight (mg)). statistical analysis the effect of treatment on level of tick fecundity and tick feeding parameters in two groups were analyzed by statistical methods. t-test and chi square test was used to assess significant differences between immunized group means for each of variables (p< 0.05). all analyses were carried out using the spss program (version 16.0). humoral response the levels of igg antibodies produced against protein fractions of tick salivary gland extract injected to immunized cattle were monitored by elisa according to the protocol described by harlow (harlow and lane, 1988). briefly, elisa plates (greiner) were coated with 5 µ g per well of antigen in 20 mm carbonate buffer (ph= 9.6) by incubation overnight at 4 ºc. they were washed three times and incubated for 1 h at 37 ºc with 1% bovine serum albumin (hab-tech) in pbs. then, test sera diluted 1:200 in pbs, were incubated for 1 h at 37 ºc. after this time, the plates were washed three times and 100 µ l of 1:2000 sheep anti –bovine igg conjugated with peroxidase were added to the individual wells. after incubation at 37 ºc for 1 h the plates were washed. the color developed with 2, 2`, azino-bis 3-ethyl benzthiazoline-6-sulfonic acid (abts) as substrate. the optical density (od) was determined at 405 nm (awarenessstat fax 2100). sera from immunized and control cattle were collected from week 0 (before the first injection) until week 18 and results were compared in two groups. results determination of protein pattern by sdspage coomassie blue staining of polyacrylamide gel visualized a broad protein profile in rh. (b.) annulatus tick salivary gland extract, comprised of 32 fractions with molecular weight of >200, 160, 107, 103, 92, 82, 80, 78, 72, 66, 63, 56, 55, 50, 46, 40, 38, 37, 36, 33, 30, 29, 27, 25, 22, 19, 18, 17, 16, 15 kda and two bands lesser than 15 kda (fig. 1). analysis of salivary gland antigens using bovine serum positive serum from cattle infested with about 10000 rh. (b.) annulatus tick larvae, recognized approximately 22 proteins from female tick salivary gland extract including two bands more than 170 kda, 160, 103, 92, 78, 72, 66, 63, 56, 55, 50, 40, 38, 36, 33, 29, 27, 26, 25, 19 kda and a band less than 17 kda . sera from uninfested animals did not react with any of these antigens by this assay (fig. 2). from these immunogenic fractions, 14 bands, which were more dominant than others were (92, 78, 72, 66, 56, 55, 50, 40, 38, 36, 33, 27, 25 and 19 kda), were dissected from polyacrylamide gel and used for immunization procedure. effects of immunization against tick challenge tick parameters we used two groups of parameters to assess the effects of immunization on ticks feeding parameters and fecundity parameters. feeding parameters effects of immunization on feeding parameters are summarized in table 1. among immunized animals, the number, weight and feeding index of engorged female ticks were significantly reduced compared to the control group. the length of feeding period in j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 286 http://jad.tums.ac.ir published online: january 06, 2016 immunized group was longer than control group (p< 0.05). fecundity parameters female ticks recovered from immunized cattle passed longer period to lay egg after engorgement in comparison with ticks harvested from control cattle. the eggs were laid by ticks engorged on immunized cattle had longer incubation period than eggs produced by ticks fed on control cattle. immunization with eluted antigens significantly reduced egg mass weight, hatchability rate of eggs and fertility index (p< 0.05) (table 1). humoral responses cattle immunized with eluted fractions of salivary gland extract developed antibodies to these antigens. humoral immune responses to immunization with tick salivary glands eluted proteins measured by elisa. results showed a steady increase in antibody production level of treatment group from the first week after immunization until the last injection. after fourth week, it reached a plateau during next four weeks. when a tick challenge was performed at week eighth, it rose sharply and peaked at week 12 and then this trend declined and gradually stabilized at week 18. in control group, there was a period of stability during the first eight weeks. after tick infestation, it surged dramatically and reached a peak at week 12. then, it fell slightly to a constant level. although both groups have had a significant increase in their antibody production level after 18 weeks, this level was boosted following tick infestation in treatment group (fig. 3). table 1. different parameters measured in female engorged ticks recovered from vaccinated and control groups and their change percentage variable group mean percent of changes number of collected ticks vaccinated 161 -63.1 1 control 436 attachment rate vaccinated 32.2 -55 control 87.2 engorgement weight (g) vaccinated 0.206 -30.2 control 0.295 length of feeding period (day) vaccinated 25.250 8.6 control 23.511 preoviposition period (day) vaccinated 2.906 45 control 2.011 egg mass weight (g) vaccinated 0.403 -40 control 0.665 incubation period (day) vaccinated 25.406 31.34 control 19.345 feeding index vaccinated 0.008 -36.4 control 0.013 fertility index vaccinated 1.95 -13.3 control 2.25 hatchability rate vaccinated 747 -78.7 control 3504 1 negative values indicates reduction in that value j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 287 http://jad.tums.ac.ir published online: january 06, 2016 fig. 1. sds-page analysis of rhipicephalus (boophilus) annulatus salivary gland extract. sample: sds-page gel stained with coomassie blue. marker: protein ladder. mw: molecular weight fig. 2. western blot analysis of rhipicephalus (boophilus) annulatus salivary gland extract. neg sera: serum of uninfested cattle. pos sera: serum of infested animals with tick larvae. prestain: prestained protein ladder. mw: molecular weight fig. 3. changes in antibody production to selected antigens in vaccinated animals in comparison with control animals during 18 weeks indicates when tick challenge was carried out. vac: vaccinated animals. control: control group j arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 288 http://jad.tums.ac.ir published online: january 06, 2016 discussion it is generally accepted that tick saliva plays an important role in pathogen transmission, suppression of host immune reactions, and successful engorgement. therefore, biochemical and immunological characterization of tick saliva components introduced to the mammalian host during the feeding procedure is essential (willadsen 1980, brossard and fivaz 1982, wikel 1996, 1999a, b, 2006, willadsen and jongejan 1999). some mammalian hosts acquired resistance to tick bites after repeated experimental infestation with ticks (brossard and fivaz 1982, dipeolu et al. 1992, nikpay et al. 2008, nikpay et al. 2012). according to these researches, although feeding behavior of ticks was affected after several experimental tick infestations, some components of tick saliva acting as host immune response modulator, suppressed host immune system. therefore, this method could not reject ticks from host properly (dipeolu et al. 1992, cobon et al. 1996, wikel 1999a, b, willadsen and jongejan 1999). on the other hand, some researchers used various tissue extracts of ticks as a source of antigens for immunization of hosts. since the salivary gland extracts contain the natural antigens injected by the arthropod during infestation, it appears to be an obvious choice to induce immunity against blood-sucking arthropods. it has been shown immunization with salivary gland extracts of rh. (b.) annulatus tick resulted in a clear reduction in the severity of clinical babesiosis. in addition, fewer babesia bigemina positive ticks were collected from these cattle (jittapalapong et al. 2004a, jittapalapong et al. 2004b). it is obvious that the importance of inhibiting tick feeding to prevent direct damage and pathogen transmission and reliability of using natural antigens to avoid repeated tick infestation justifies further investigations on natural salivary gland antigens of ticks. our results demonstrated that immunization of calves with eluted fractions of salivary gland extracts of rh. (b.) annulatus ticks induced remarkable immunity expressed as an inhibition of feeding and fertility of the ticks with less direct trauma and damage and without the risk of disease transmission. as mentioned in results, immunization of cattle resulted in reduction in mean tick counts, attachment, engorgement weights, feeding index, egg mass weight, hatchability and fertility index (respectively 63.1%, 62.6%, 30.2%, 36.4%, 40%, 78.7% and 13.3%) and increased duration of feeding, preoviposition and incubation period of eggs (respectively 8.6%, 45 and 31.34%). clearly, the host immunity affects the physiology of the tick in some permanent manner persisting even after the parasite detached. the shortening of the feeding time by the vaccine suggests that although the degree of immunity produced in this assay was relatively acceptable, the stronger immunization can probably inhibit feeding totally, which is highly desirable because it will prevent the transmission of tickborne pathogens during feeding. the humoral immune responses, measured by elisa, showed cattle injected with tick salivary gland eluted proteins have expressed a constant increase in antibody production level from the first week of injection which were boosted after tick challenge. we had demonstrated tick infestation elevates the antibody level in infested cattle (nikpay et al. 2008). the results reported in this study confirmed that immunization against rh. (b.) annulatus could be achieved under experimental conditions. therefore, the immunogen fractions of the salivary gland extract would be an appropriate target to design an effective vaccine against blood-sucking parasites especially ticks. in addition, our prej arthropod-borne dis, september 2016, 10(3): 282–291 a nikpay and s nabian: immunization of cattle … 289 http://jad.tums.ac.ir published online: january 06, 2016 vious studies indicated there are protein bands with the same molecular weight in different tissues of rh. (b.) annulatus tick resulted in similar changes in antibody production level in cattle (nikpay et al. 2008, nikpay et al. 2012). comparison of the results of sdspage and western blot analysis in this study and some previous studies reveals that there are several protein fractions having similar molecular weight and similar immunogenicity in different species of ixodid ticks studied (nabian et al. 2005, razmi et al. 2005, manzano-román et al. 2007, norouzi et al. 2007, nikpay et al. 2008, nikpay et al. 2012). interestingly, some of these fractions were reported from the different tissues of ticks. thus finding common crucial antigens in various tissues of different species of ticks would be our final goal in other studies. conclusion results of this investigation indicates that immunizing of cattle with these antigens could induce a protective immune response against rh. (b.) annulatus tick infestation that would be expected to provide a safe non-chemical means of tick control. we would like to follow this study by characterization of each immunogenic fraction and finding common crucial antigens in other tick species to design a feasible and reliable vaccine against tick infestation acknowledgements authors wish to acknowledge the financial support of the research council of tehran university and veterinary medicine faculty of tehran university. the authors declare that they have no conflict of interests. references brossard m, fivaz v (1982) ixodes ricinus l.: mast cells, basophils and eosinophils in the sequence of cellular events in the skin of the infested or reinfested rabbits. parasitol. 85: 583–592. brown sj, shapiro sz, askenase pw (1984) characterization of tick antigens inducing host immune resistance. i. immunization of guinea pigs with amblyomma americanum-derived salivary gland extracts and identification of an important salivary gland 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arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 132 original article seroepidemiologic survey of canine visceral leishmaniasis in tehran and alborz provinces of iran *abdolali malmasi 1, saeedeh janitabar 1, mehdi mohebali 2, behnaz akhoundi 2, nadi maazi 1, mehdi aramoon 1, narges khorrami 1, hesam a seifi 3 1department of clinical sciences, faculty of veterinary medicine, university of tehran, tehran, iran 2department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 3department of clinical sciences, faculty of veterinary medicine, ferdowsi university of mashhad, mashhad, iran (received 25 june 2012; accepted 2 oct 2013) abstract background: a two year seroepidemiological study was designed to find out the seroprevalence and risk factors of canine visceral leishmaniasis (cvl) among stray and owned dogs in tehran and alborz provinces of iran. methods: blood samples of 602 dogs living in 11 counties of tehran and alborz provinces were taken by venipuncture in 2008–2010. after separation of blood sera, anti-leishmanial antibodies were detected by direct agglutination test (dat). results: overall, of the 408 and 194 serum samples collected randomly from dogs in 11 localities in tehran and alborz provinces, 18/408 (4.41%) and 12/194 (6.18%) respectively were found positive. among the localities, shemiran in tehran province and karaj in alborz province had the highest prevalence rates. no statistically significant differences were found between sex and living place but there was significant difference between living status (owned or stray) and cvl infection of dogs in alborz province (p= 0.018). the highest seroprevalence (7.5%) was found in dogs aged 3 to 5 years old. only 20% of the seropositive dogs were symptomatic. conclusion: concerning possible human infections in tehran and alborz provinces, both symptomatic and asymptomatic seropositive dogs should be considered as a risk. keywords: visceral leishmaniasis, direct agglutination test, dog, iran introduction visceral leishmaniasis is one of the most important parasitic diseases affecting humans, domestic and wild canines worldwide. the infection is transmitted by sandflies. reservoir hosts vary within different geographic areas and can include domestic or wild canines (greene 2006). domestic dogs (canis familiaris) are principal reservoir hosts for mediterranean type of visceral leishmaniasis caused by leishmania infantum (mohebali et al. 2005). the clinical manifestation of disease vary from asymptomatic, self-limiting infections to fatal visceral leishmaniasis. seroepidemiologic studies of canine leishmaniasis have shown a large number of asymptomatic seropositive animals in meshkinshahr district of ardabil province (moshfe et al. 2009). canine leishmaniasis is not only a veterinary problem but also a serious public health concern. as the high proportion of infected dogs are asymptomatic, detection of specific antibodies remains the method of choice for mass screening of dogs in epidemiological surveys (moshfe et al. 2008). several diagnostic tests are available to detect anti-leishmania antibodies in canine sera. in the present study, *corresponding author: dr abdolali malmasi, eamalmasi@ut.ac.irmail: http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 133 direct agglutination test (dat) was used as it is a simple as well as a valid test and does not require specialized equipments (moshfe et al. 2008). up to now, ardabil, east azerbaijan, fars, bushehr and qom provinces have been recognized as endemic foci of canine visceral leishmaniasis in iran (mohebali et al. 2001, mohebali et al. 2005, fakhar et al. 2005, habibzadeh et al. 2007, farzam et al. 2007, moshfe et al. 2008, khanmohammadi et al. 2010a, khanmohammadi et al. 2010b). the objective of this first ever study in tehran and alborz provinces of iran was to determine the seroprevalence and distribution of canine visceral leishmaniasis among dogs living in the urban and suburban areas of these highly populated regions with their new developing cities. materials and methods study area this survey was conducted over a period of 2 years (from july 2008 to october 2010) in 11 counties from two provinces of tehran and alborz located across the north of the central plateau of iran at 35° 40' 19" n, 51° 25' 28" e and 34° 43' 42" n, 50° 58' 19" e, respectively (fig. 1). samples were collected from 8 counties (damavand, eslam shahr, rey, robat karim, shahriar, shemiran, tehran, varamin) of tehran and 3 counties (hashtgerd, karaj, nazar abad) of alborz provinces.the study population was selected by convenience sampling. the geographical locations, where this study was carried out are shown in fig. 1. sampling totally, 602 dogs living in tehran (n= 408) and alborz (n= 194) provinces were selected for this study. ages of dogs were determined either by observing their teeth or asking from the owners, all dogs based on their ages put into three classes (0–3, 3–5 and >5 years). after the clinical examination of dogs, blood samples (2.5ml) were taken by venipuncture and put into 10ml polypropylene tubes and centrifuged at 800 x g for 5–10 minutes. sera were separated and stored at -20 oc. all serum samples were tested by direct agglutination test (dat) in leishmaniasis laboratory of the school of public health, tehran university of medical sciences. direct agglutination test (dat) the direct agglutination test antigens were prepared from indigenous l. infantum [mcan/ir/07/moheb-gh. (genbank accession number fj555210)] with the methods described by harith et al. (1989) and mohebali et al. (2005). specific antileishmania antibodies at a titer of ≥1: 320 in sera of indigenous dogs were considered as positive based on previous studies (mohebali et al. 2011). data analysis chi-square (x2) analysis was used to compare seroprevalence values relative to gender, age, geographical zone, clinical signs and living status of dogs. data regarding gender and age of some dogs from total population (30/602 and 36/602 respectively) were missing ,thus we did not consider those data in the analysis. analyses were conducted using spss software version 17 (spss inc, chicago, illinois, usa), with a probability (p) value of <0.05 as statistically significant. results eighteen out of 408 (4.4%) of the canine blood samples collected from tehran province, and twelve out of 194 (6.1%) from alborz province, were seropositive (≥1: 320) by dat. in general, no statistical significant differences were observed between seroprevalence values found in tehran and alborz provinces http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 134 (p= 0.35). sero-prevalence (titer ≥1: 320) of canine leishmania infection by gender in tehran and alborz provinces are shown in table 1. there was no significant difference among two sexes in both tehran and alborz provinces (p= 0.933 and p= 0.349 respectively). referring to three age groups, the highest seroprevalence (7.5%) was found in dogs aged 3 to 5 years old compared with other groups (p= 0.023), however there was no statistically significant difference among three age classes in each province (p= 0.134 and p= 0.082 in alborz and tehran provinces respectively). table 2 shows the seroprevalence (titer ≥1: 320) of canine leishmania infection regarding age classes in tehran and alborz provinces. only 6/30 (20%) of the seropositive dogs showed clinical signs of disease including lethargy, cachexia, skin lesions, alopecia, epistaxis and myopathy and the remaining 80% were asymptomatic. as shown in table 3, 11/226 (4.6%) and 1/79 (1.2%) of stray dogs and 7/182 (5.7%) and 11/115 (8.7%) of owned dogs from tehran and alborz provinces were seropositive respectively. there was statistically significant difference between living status and canine vl infection of dogs in alborz province (p= 0.018 ). as shown in table 4, shemiran and karaj counties in tehran and alborz provinces showed the highest seroprevalence (14.2% and 10%, respectively). fig. 1. map of iran shows 2 provinces and 11 localities (damavand, eslam shahr, rey, robat karim, shahriar, shemiran, tehran, varamin, hashtgerd, karaj, nazar abad) where this survey was conducted http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 135 table 1. sero-prevalence (titer ≥1: 320) of canine leishmani a infection by gender in tehran and alborz provinces. percentages were calculated in each group (male or female) totally and in each region femalemalesex p valuesno. of infected dogs (titer ≥1: 320) (%) no. of sampled dogs (%) no. of infected dogs (%) (titer ≥1: 320) no. of sampled dogs (%) 0.9337 (4.6)146 (95.4)10 (4)232 (96)tehran 0.3494 (4)90 (96)8 (7)104 (93)alborz 0.85211 (4.5)236 (95.5)18 (5.7)336 (94.3)total table 2. sero-prevalence (titer ≥1: 320) of canine leishmania infection regarding age groups in tehran and alborz provinces of iran. percentages were calculated in each age group totally and in each region >53–50–3age (yr) p valuesno.of infected dogs (%) (titer ≥1: 320) no. of sampled dogs (%) no. of infected dogs (%) (titer ≥1: 320) no. of sampled dogs (%) no.of infected dogs (%) (titer ≥1: 320) no. of sampled dogs (%) 0.0824 (6.4)59 (93.6)10 (6.8)138 (93.2)3 (1.6)179 (98.4)tehran 0.1342 (9.5)19 (90.5)7 (7.4)88 (92.3)2 (0.4)83 (99.6)alborz 0.0136 (7)78 (93)17 (7)226 (93)5 (2)262 (98)total table 3. sero-prevalence (titer ≥1: 320) of canine leishmania infection regarding living status in tehran and alborz provinces of iran. percentages were calculated in each group (stray or owned) totally and in each region ownedstrayliving status p valuesno. of infected dogs (%) (titer ≥1: 320) no. of sampled dogs (%) no. of infected dogs (%) (titer ≥1: 320) no. of sampled dogs (%) 0.6187 (5.7)182 (96.3)11 (4.6)226 (95.4)tehran 0.01811 (8.7)115 (91.3)1 (1.2)79 (98.8)alborz 0.8218 (5.7)297 (94.3)12 (3.8)305 (96.2)total table 4. distribution and seroprevalence of l.infantum infection (titer ≥1: 320) among 602 dogs from 11 counties in tehran and alborz provinces of iran province /locality no. of sampled dogs no. / % of infected dogs(titer ≥1: 320) tehran province 408 18 (4. 4) damavand 62 4(6.4) eslam shahr 9 0(0) rey 12 0(0) robat karim 23 1(4.3) shahriar 198 6(3) shemiran 14 2(14.2) tehran 79 5(6.3) varamin 11 0(0) alborz province 194 12 (6.18) hashtgerd 115 6(5.2) karaj 60 6(10) nazar abad 19 0(0) total 602 30(4.98) http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 136 discussion dogs can remain infected by l. infantum without displaying apparent clinical signs of vl for years even for their entire life (moreno and alvar 2002, moshfe et al. 2009). determination of the prevalence of canine leishmania infection is necessary to define control measures for zoonotic visceral leishmaniasis (mohebali et al. 2005). reports from neighboring turkey have shown the seroprevalences of 2.58% and 3.6% (ozensoy et al. 1998, aslantas et al. 2005), jaffe showed that the disease was highly prevalent in central israel and encroaching on urban areas (jaffe et al. 2004), seroprevalence of 18% and 26.6% with dat and elisa, respectively have been reported from pakistan (rab et al. 1995). based on our results, no statistically difference was found between genders. this finding is in consistent with other studies (mohebali et al. 2001, francasilva et al. 2003, mohebali et al. 2005, farzam et al. 2007, moshfe et al. 2008, khanmohammadi et al. 2010a) and in conflict with some others (dantas-torres et al. 2006, khanmohammadi et al. 2010b, zaffaroni et al. 1999). in the present study, the infection was more prevalent in dogs aged 3 to 5 years old. simillarly, bokai et al. (1998) showed the highest infection in middle-aged dogs. in the study of mohebali et al. (2005) dogs aged 8 years or more, showed the highest seroprevalence. the results of the study of dantas-torres et al. (2006) showed that the seroprevalence rate was higher among juvenile (≤1 year) dogs than adults (>1 years). in our study, only 20% of 30 seropositive dogs (titer ≥1: 320) showed the clinical features of disease and the remaining 80% were asymptomatic. fourteen (46.6%) and ten (33.3 %) seropositive dogs in tehran and alborz provinces respectively were found asymptomatic which is in agreement with other studies reporting similar results (mohebali et al. 2005, moshfe et al. 2008, dantas-torres et al. 2006). there was statistically significant difference between living status and cvl infection of dogs in alborz province (p= 0.018). it seems that the owned dogs in this province, were much more likely to be exposed to leishmania infection than stray dogs. owned dogs in tehran province also showed higher prevalence than stray dogs for cvl, but their difference was not significant (table 3). despite lower prevalnce for cvl in owned dogs population of tehran province, difference between two provinces in the same category was not significant (table 3). seroprevalence values of 6.1% and 4.4% for leishmania infection among dogs in alborz and tehran provinces, respectively, reflect the significant risk of exposure for both healthy dogs and human population in these regions. if lower cut-off value was considered (<1: 320) the prevalence should have been more than the current findings. only 20% of the seropositive dogs were symptomatic. since asymptomatic leishmania-infected dogs as well as symptomatic ones, have a potential role in the maintenance of l. infantum infection (moshfe et al. 2009), finding and reporting such number of infected asymptomatic dogs in these provinces of iran for the first time should be considered as a risk from both veterinary and public health aspects. among the localities, shemiran (14.2 %) in tehran province and karaj (10%) in alborz province had the highest prevalence rates. conclusion based on our survey these provinces especially those localities and suburbs with higher seroprevalence should be regarded as new endemic foci for cvl in iran. the huge population living inside these areas particularly in new developing cities around both provinces, should be considered as at risk population. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 137 acknowledgements the authors would like to express their gratitude to all those who helped them running this survey including a aliari for his technical assistance in small animal hospital, faculty of veterinary medicine, university of tehran, p nobakht the local private veterinarian in hashtgerd county, s tehranian for helping in statistical review of the manuscript and s charehdar for her technical assistance in parasitology laboratory, school of public health, tehran university of medical sciences. the authors declare that there is no conflict of interest. references bokai s, mobedi i, edrissian ghh, nadim a (1998) seroepidemiological study of canine visceral leishmaniasis in meshkinshahr, northwest of iran. arch razi inst. 41–46, 48–49. clarisa b, palatnik-de-sousa (2008) vaccines for leishmaniasis in the fore coming 25 years. vaccine. 26: 1709–1724. dantas-torres f, edileuza felinto de brito m, pinto brandao-filho s (2006) seroepidemiological survey on canine leishmaniasis among dogs from an urban area of brazil, vet parasitol. 140: 54–60. fakhar m, motazedian mh, asgari gh, mohebali m, mehrbani d (2006) introduction of a new endemic foci of kalaazar in south of iran. armaghan danesh j. 11(2): 103–113. farzam m, changizi e, mohebali m, akhoundi b, salimi bajestani mr (2007) seroepidemilogical survey of visceral leishmaniasis in shepherd dogs of jahrom county, fars province. vet j iran. 4(3): 58–67. frança-silva j, t da costa r, siqueira a, ll machado-coelho g, a da costa c, mayrink w, p vieira e, s costa j, genaro o, nascimento e (2003) epidemiology of canine visceral leishmaniosis in the endemic area of montes claros municipality, minas gerais state, brazil. vet parasitol. 111: 161–173. greene craig e (2006) infectious diseases of the dog and cat, third edition, wb saunders co, chapter 73, pp: 685–698. habibzadeh s, arshi s, sadeghi-bazargani h, mohebali m (2007) rk39 dipstick test as a practical solution for canine leishmaniasis diagnosis among nomadic populations. j anim vet adv. 6(2): 175–178. harith a, salappendel rj, reiter i, knapen f, korte p, huigen e, kolk rhg (1989) application of a direct agglutination test for detection of specific anti-leishmania antibodies in the canine reservoir. j clin microbiol. 27: 2252–2257. jaffe chl, baneth g, abdeen za, schlein y, warburg a (2004) leishmaniasis in israel and the palestinian authority. trends parasitol. 20(7): 328–332. khanmohammadi m, fallah e, rahbari s, sohrabi i, farshchian m, hamzavi f, mohammadpour asl a (2010a) seroepidemiological survey of canine visceral leishemanisis in sarab district, east azerbaijan province, northwest of iran. afr j microbiol res. 4(19): 2022–2028. khanmohammadi m, fallah e, rahbari s, sohrabi i, farshchian m, hamzavi f, mohammadpour asl a (2010b) study on seroprevalence of canine visceral leishmaniasis (cvl) in ownership dogs of sarab, east azerbaijan province, northwest of iran with indirect immuno fluorescence antibody test (ifat) and its health importance. j anim vet adv. 9(1): 139–143. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 132–138 a malmasi et al.: seroepidemiologic survey of … 138 mohebali m, hamzavi y, fallah e, zarei z (2001) study of canine visceral leishmaniasis in some parts of iran and its health importance. j vet res. 56: 55–59. mohebali m, khamesipour a, mobedi i, zarei z, hashemifesharki r (2004) double blind randomized efficacy trial of alum precipitated autoclaved leishmania major vaccine mixed with bcg against canine visceral leishmaniasis in meshkin-shahr district i.r. of iran. vaccine. 22: 4097–4100. mohebali m, hajjaran h, hazavi y, mobedi i, arshi sh, zarei z, akhoundi b, manouchehri naeini k, avizeh r, fakhar m (2005) epidemiological aspects of canine visceral leishmaniosis in the islamic republic of iran. vet parasitol. 129: 243–251. mohebali m, edrissian ghh, shirzadi mr, akhoundi b, hajjaran h, zarei z, molaei s, sharifi i, mamishi s, mahmoud-vand h, torabi v, moshfe a, malmasi a, motazedian mh, fakhar m (2011) an observational study on the current distribution of visceral leishmaniasis in different geographical zones of iran and implication to health policy. travel med infect dis. 9(2): 67–74. moreno j, alvar j (2002) canine leishmaniasis: epidemiological risk and the experimental model. trends parasitol. 18: 399–405. moshfe a, mohebali m, edrissian gh, zarei z, akhoundi b, kazemi b, jamshidi sh, mahmoodi m (2008) seroepidemiological study on canine visceral leishmaniasis in meshkin-shahr district, ardabil province, northwest of iran. iran j parasitol. 3(3): 1–10. moshfe a, mohebali m, edrissian gh, zarei z, akhoundi b, kazemi b, jamshidi sh, mahmoodi m (2009) canine visceral leishmaniasis: asymptomatic infected dogs as a source of l. infantum infection. acta trop. 112: 101–105. ozensoy s, ozbel y, turgay n, alkan mz, gul k, gilman-sachs a, chang kp, reed sg, ali ozcel m (1998) serodiagnosis and epidemiology of visceral leishmaniasis in turkey. am j trop med hyg. 59(3): 363–369. rab ma, frame ia, evans da (1995) the role of dogs in the epidemiology of human visceral leishmaniasis in northern pakistan. t roy soc trop med h. 89: 612–615. zaffaroni e, rubaudo l, lanfranchi p, mignone w (1999) epidemiological patterns of canine leishmaniosis in western liguria (italy). vet parasitol. 81: 11–19. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, june 2013, 7(1): 90–93 h albayrak , e ozan: seroepidemiological study of... http://jad.tums.ac.ir published online: april 10, 2013 short communication seroepidemiological study of west nile virus and rift valley fever virus in some of mammalian species (herbivores) in northern turkey *harun albayrak1, emre ozan 2 1department of virology, faculty of veterinary medicine, ondokuz mayis university, samsun, turkey 2virology laboratory, veterinary control institute, samsun, turkey (received 23 nov 2011; accepted 24 oct 2012) abstract background: west nile virus (wnv) and rift valley fever virus (rvfv) are mosquito-borne viral diseases. the objective of this study was to investigate the rvfv and wnv infections as serologically in different mammalian species (cattle, horse, goat, sheep and water buffalo) in the northern turkey. methods: blood samples randomly collected from 70 each cattle, horse, sheep, goat and water buffalo were analyzed for the presence of antibodies to rvfv and wnv using an competitive enzyme-linked immunosorbent assay (c-elisa) in northern turkey. results: none of the animals were positive for antibodies to rvfv. in contrast, wnv antibodies were found in two of 350 samples (0.57%). conclusion: this may suggest that the rvfv disease is not present in northern turkey.this is the first serological study on rvfv in turkey. keywords: elisa, herbivore, rvfv, turkey, wnv introduction west nile virus (wnv) is a member of the japanese encephalitis virus complex of the family flaviviridae, genus flavivirus, which also includes japanese encephalitis virus, st. louis encephalitis virus (slev), murray valley encephalitis virus (mvev), and others. these viruses are mosquito borne, primarily transmitted by culex spp, and have wide, overlapping distributions throughout the world (mackenzie et al. 2002). the diagnosis of wnv infection is commonly achieved using serological assays (castillo-olivares and wood 2004). while plaque reduction neutralization tests are still considered the gold standard for specific diagnosis, elisa is now routinely used (dauphin and zientara 2007), as it is less laborious and more suited to highthroughput screening. rift valley fever virus (rvfv), family bunyaviridae is an emerging epidemic disease of humans and livestock, as well as an important endemic problem in sub-saharan africa. the virus is transmitted to livestock and humans by the bite of infected mosquitoes or exposure to tissues or blood of infected animals. massive epizootics are typically observed in livestock during times of unusually high and sustained rainfall because of the presence of breeding sites and overabundance of adult competent mosquito vectors. infections caused by rvfv are characterized by severe disease and abortion in livestock, particularly sheep and cattle (linthicum et al. 1999). the recent rvf outbreaks in the arabian peninsula (shoemaker et al. 2002), the first outbreaks outside af*corresponding author: dr harun albayrak, email: harunalbayrak55@msn.com 90 j arthropod-borne dis, june 2013, 7(1): 90–93 h albayrak , e ozan: seroepidemiological study of... http://jad.tums.ac.ir published online: april 10, 2013 rica, have the implication that it is likely that rvfv will now spread further into non-endemic rvf areas since it is capable of utilizing a wide range of mosquito vectors (turrel et al. 1998). the objective of this study was to investigate the rvfv and wnv infections as serologically in different mammalian species (cattle, horse, goat, sheep and water buffalo) in the northern turkey. materials and methods blood samples were collected from seventy each cattle, horse, sheep, goat and water buffalo, without clinical signs of the diseases in the northern part of turkey between may and october 2010 (fig. 1). the age of the animals varied from 2 to 17 years. blood samples were taken from the jugular veins of the animals. blood tubes were centrifuged at 3, 000×g for 10 min, and the samples were transferred to sterile tubes and stored in -20 °c until used. the commercial c-elisa kits were obtained from id.vet, montpellier, france, and the test was performed according to the producer’s description. plates were read with an elisa reader at 450 nm and results were calculated. suspected samples were retested by c-elisa. results a total of 350 animals (70 each cattle, horses, sheep, goats and water buffaloes) were tested by c-elisa. all animals were negative for antibodies against rvfv. although no wnv antibodies were detected from cattle, horse, sheep and water buffalo samples, out of 70 goats, 2 (2.85%) were found to be seropositive for wnv (table 1). table 1. seroepidemiology of west nile fever and rift valley fever in north of turkey animals number of samples positivity (%) for wnv positivity (%) for rvfv buffalo 70 0 (-) 0 (-) cattle 70 0 (-) 0 (-) goat 70 2 (2.85) 0 (-) horse 70 0 (-) 0 (-) sheep 70 0 (-) 0 (-) total 350 2 (0.57) 0 (-) fig. 1. areas of sample collection 91 j arthropod-borne dis, june 2013, 7(1): 90–93 h albayrak , e ozan: seroepidemiological study of... http://jad.tums.ac.ir published online: april 10, 2013 discussion west nile virus has a wide geographical range that includes portions of europe, asia, africa, australia and america (fauquet et al. 2005). many serological test methods were used in the diagnosis of wnv such as plaque reduction neutralization test (prnt) and elisa. while prnt is still considered the gold standard for specific diagnosis, elisa is now routinely used (dauphin and zientara 2007). the c-elisa has a higher specificity (99.4%) and sensitivity (84.9%) for wnv infection (padilla et al. 2009). west nile virus antibodies have been detected in humans and animals in turkey (ozkul et al. 2006, ergunay et al. 2007a, 2007b) and antibodies and viruses have been detected among mammals and vectors in the neighbouring countries of balkan peninsula (hubalek and halouzka 1999). in addition, mosquito species know to transmit mosquito-borne diseases have been observed in tur-key (dik et al. 2006). however, except three human west nile cases in 2010, there has been no report of acutely infected humans and animals in turkey. all human cases were detected in aegean region of western border of turkey. this region is also border bet-ween turkey and greece where west nile hu-man cases were observed in 2010 and eigh-teen people died in greece. only one sero-logic study has been performed in the cent-ral, southern and western parts of turkey for wnv. ozkul et al. (2005) were carried out a serosurvey in mammalian species. positivity rates for the animals varied and were as fol-lows: ass-mules 2.5%, cattle 4%, dogs 37.7%, horses 13.5%; sheep 1% and humans 20.4%. there is no study on the seroprevalence of wnv infection in buffaloes and goats in tur-key. the determined positivity in goats in this study (2.85%) was found to be very low comparing to the reported value in dogs, hu-mans and horses. albayrak and ozan (2010) performed a molecular study about presence of wnv in wild bird samples in the same re-gion, but they did not detect any wnv nuc-leic acid from these samples. reservoir-vec-tor-climate trio was very im-portant at the ep-idemiology for the all mosquito-borne virus-es. given are average annual values of heat, humidity, and rainfall of aegean region (west-ern) [16.3 °c (6.4–26.8 °c), 63.2%, 725.9 mm3] and black sea region (northern) [13.0 (4.2–22.1), 71%, 842.6 mm3]; additionally, an-nual heat changes are more dramatic in black sea region (tsms 2010). higher vector acti-vity cause to increase in vectordependent di-seases. climate conditions of western, cent-ral and southern parts of turkey were more suitable for mosquitoes than northern part of turkey. it is commonplace knowledge that the result of the seroprevalance studies are influ-enced by many factors such as the number of sampled animals, the age of the animals, the time of sampling, the conditions of care and feeding, individual differences and so on. there has been no report of presence of rvfv in turkey. no antibody response was detected against rvfv in northern turkey. although mosquito species known to transmit rvfv have been observed (dik et al. 2006), there has been no report of acutely infected humans and animals in turkey. this may suggest that the disease is not present in northern turkey. in addition, the vectors in this area may not carry rvfv. the present study indicated that rvfv might not become a risk potential for animals in northern turkey. to beter understand rvfv transmission in turkey, additional studies focusing on major vectors (eg mosquitoes) are needed. the existent data in turkey is not enough to determine regional based profile of the wnv and rvfv infections. beside, further studies are necessary to understanding of vector dynamics, interactions among different sensitive species and risk factors of exposure. 92 j arthropod-borne dis, june 2013, 7(1): 90–93 h albayrak , e ozan: seroepidemiological study of... http://jad.tums.ac.ir published online: april 10, 2013 acknowledgements the authors declare that there is no conflict of interest. references albayrak h, ozan e (2010) molecular detection of avian influenza virus but not west nile virus in wild birds in northern turkey. zoonoses public health. 57: 71–75. castillo-olivares j, wood j (2004) west nile virus infection of horses. vet res. 35: 467–483. dauphin g, zientara s (2007) west nile vi-rus: recent trends in diagnosis and vac-cine development vaccine. 25: 5563–5576. dik b, yagcı s, linton ym (2006) a review of species diversity and distribution of culicoides latreille, 1809 (diptera: ceratopogonidae) in turkey. j nat hist. 40: 1947–1967. ergunay k, saygan mb, aydogan s, menemenlioglu d, turan hm, ozkul a, us d (2007a) west nile virus seroprevalance in blood donors from central anatolia, turkey. vector borne zoonot dis. 7: 157–161. ergunay k, ozer n, us d, ozkul a, simsek f, kaynas s, ustacelebi s (2007b) seroprevalance of west nile virus and tick-borne encephalitis virus in southeastern turkey: first evidence for tickborne encephalitis virus infections. vector borne zoonot dis. 10: 771–775. fauquet cm, mayo ma, maniloff j, desselberger u, ball la (2005) family flaviviridae. in: fauquet cm, mayo ma, maniloff j, desselberger u, ball la (eds) virus taxonomy-classification and nomenclature of viruses. eighth report of the international committee on the taxonomy of viruses. elsevier academic press, san diego. hubalek z, halouzka j (1999) west nile fevera reemerging mosquito-borne viral disease in europe. emerg infect dis. 5: 643–650. linthicum kj, anyamba a, tucker cj, kelley pw, myers mf, peters cj (1999) climate and satellite indicators to forecast rift valley fever epidemics in kenya. science. 285: 397–400. mackenzie js, barrett adt, deubel v (2002) the japanese encephalitis serological group of flaviviruses: a brief introduction to the group. curr top microbiol. 267: 1–10. ozkul a, yıldırım y, pinar d, akcali a, yilmaz v, colak d (2006) serological evidence of west nile virus (wnv) in mammalian species in turkey. epidemiol infect. 134: 826–829. padilla ja, rubio el, romero ee, cordoba l, cuevas s, mejia f, calderon r, milian f, rosa atd, weaver sc, franco jge, saiz jc (2009) the continous spread of west nile virus (wnv): seroprevalance in asymptomatic horses. epidemiol infect. 137: 1163–1168. shoemaker t, boulianne c, vincent mj, pezzanite l, al-qahtani mm, al-mazrou y, khan as, rollin pe, swanepoel r, ksiazek tg, nichol st (2002) genetic analysis of viruses associated with emergence of rift valley fever in saudi arabia and yemen, 2000–2001. emerg infect dis. 8: 1415–1420. turkish state meteorological service (tsms) (2010) update on average annual values of heat, humidity, and rainfall. available at: http//www.dmi.gov.tr/ tahmin/il-veilceler.aspx. turrel mj, bailey cl, beaman jr (1998) vector competence of a houston, tex-as strain of aedes albopictus for rift valley fever virus. j am mosq cont assoc. 4: 94–98. 93 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 original article detection of naturally infected vector ticks (acari: ixodidae) by different species of babesia and theileria agents from three different enzootic parts of iran mohammad abdigoudarzi reference laboratory for ticks and tick-borne diseases, department of parasitology, razi vaccine and serum research institute, karaj, iran (received 16 jan 2012; accepted 14 may 2013) abstract background: diagnostic study of vector ticks for different pathogens transmitted specifically have been done by iranian old scientists working on the basis of biological transmission of pathogens. in this study we decided to confirm natural infection of different collected ticks from three different provinces of iran. methods: ticks were collected from livestock (sheep, goats and cattle) during favorable seasons (april to september 2007 and 2008). slide preparations were stained by giemsa and feulgen and were studied searching for any trace of infection. positive dna from infected blood or tissue samples was provided and was used as positive control. first, pcr optimization for positive dna was done, and then tick samples were subjected to specific pcr. results: eleven pairs of primers were designed for detection of theileria, babesia and anaplasma spp. totally 21 tick samples were detected to be infected with protozoa. hyalomma anatolicum anatolicum and rhipicephalus turanicus from fars province were infected with t. lestoquardi at two different places. hyalomma detritum was infected with t. lestoquardi in lorestan province and rh. turanicus was infected to ba. ovis from fars province. conclusion: totally 21 tick samples were detected to be infected with protozoa. every sample is regarded with hostenvironment related factors. since there are complex relations of vectors and their relevant protozoa, different procedures are presented for future studies. keywords: tick, vector, ixodidae, molecular detection introduction vector capacity for ticks (acari: ixodida, ixodidae and argasidae) transmitting different protozoan pathogens is very specific and has got the attention of old scientists. the first detected vector in the world was boophilus microplus, when smith, and kilborne discovered the etiology of cattle fever, babesia bigemina, and that it was transmitted by tick vectors, in 1893. their important work helped set the stage for the discovery of the mosquito vector transmission of yellow fever by walter reed and colleagues (kaplan et al. 2009). old iranian scientists were working on tick and tick borne diseases also tried to confirm vector ticks using tick rearing on experimentally infected host then inducing the infection to the prone host. review of annual reports of razi institute revealed that from 1961 to 1966, mazlum has recorded 21 species of ixodidae family collected from different livestock. he denoted hyalomma anatolicum anatolicum as a vector of bovine theileriosis on the basis of non-iranian scientists studying their home-land vectors. mazlum also denoted cleverly that on the basis of studies in countries other than iran, rhipicephalus bursa is a vector of theileria hirci (= t. lestoquardi) but, regarding the absence of this species in sistan and khouzestan provinces on that time *corresponding author: dr mohammad abdigoudarzi, e-mail: m.abdi@rvsri.ir 164 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 and regarding the presence of theileriosis due to th. hirci in sheep in those areas, so another vector should be presented. mazlum also could transmit th. annulata by injection of grinded adult infected ticks (hy. dromedarii) to the host. there is not any documented or recorded background study confirming infected ticks by salivary gland staining methods. liebisch et al. (1978) could confirm hy. anatolicum anatolicum as a vector of th. annulata by collecting infective ticks and transmitting ticks on a suitable host for blood feeding. walker and mckellar (1983) could successfully transmit the infectivity from nymphal stage to adult stage of hy. anatolicum anatolicum. all infected adult ticks were mixed and grinded. slide preparations of the mixture could reveal sporozoite of th. auunlata after staining by giemsa. tick and tick borne protozoa have been reviewed in a very inclusive article regarding molecular studies on ticks and the protozoan pathogens they carry. there it implies that a specific vector tick may transmit a specific protozoan or some vectors may transmit different pathogens. when rate of infection in the vector ticks is very low, molecular methods and amplification of a target gene can help for confirming a specific pathogen (sparagano et al. 1999). figueroa et al. (1992, 1993) could confirm dna of ba. bigemina in collected hemolymphs of female bo. microplus ticks. alvarez et al. (1996) confirmed infection rates of up to 50% ba. bovis infection in extracted dna from female bo. microplus blood fed on experimentally infected cattle to ba. bovis. dekok et al. (1993) could confirm an 18s gene sequence of th. annulata in extracted dna from hy. dromedarii and hy. marginatum mar. using primers designed by allsopp (1993) for theileria spp. kirvar et al. (1998), could amplify a specific 785 bp segment for th. lestoquardi after pcr amplification of extracted dna from hy. anatolicum anatolicum. there was no cross reaction of infected hy. anatolicum anatolicum to th. annulata or specific dna for cattle. habibi et al. (2004) in an experimental molecular study could confirm ba. ovis infection in collected blood samples from sheep by pcr method. primers were designed regarding 18s ribosomal rna gene and atp binding protein gene. the purpose of this study was to detect the potential role of ixodidae spp. and natural infection of collected ticks from livestock in three different enzootic geographical regions of iran. two different methods (staining of salivary glands and molecular method) were involved and type of infection was detected and presented. materials and methods three geographical regions were selected according to different climatic information. eastern azerbaijan located in north-western iran, fars province, central-southern iran and lorestan province, western iran. tick infested herds were selected, and ticks were collected from the surface of infested host during favorable seasons (april 2007 to september 2008) (table 1). the infestation rate was regarded and whole ticks were collected when there were less than 10 ticks over the host and in more infested host as much as 10 ticks were collected. ticks were put in a clean glass tube capped tightly with cotton and labeled for each infested host, and then samples were transferred in humid chamber to the laboratory. ticks were identified using different characteristic identification keys (delpy 1946, pomerantzev 1950, delpy 1954, hoogstraal 1956, hashemi-fesharki et al. 2002, estradapena 2004). then they were incubated in humid chamber at 28 °c and 85% relative humidity. confirmation of protozoan infection in ticks 1-staining methodsalivary glands were pulled out of the body of live ticks and a wet 165 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 mount has been prepared for each sample, staining was done by giemsa (giemsa solution from merck company) and feulgen staining method (using basic fuchsin according to a laboratory manual from cellular biology section at shiraz university) (fig. 1 and 2). comparison of slide preparation with normal salivary gland tissue can reveal the infection caused by some of protozoan agents. 2molecular studydna extraction was done for suspected ticks, quantity and quality control was done. each dna was subjected to a specific primer for ticks (its2) (abdigoudarzi et al. 2011) confirming the specificity. then confirmed dna samples were subjected to specific designed primers for theileria, babesia and anaplasma. primer design ten pairs of primers for detection of five protozoa, including th. annulata, ba. bovis, ba. ovis, babesia sp. and anaplasma marginal were designed. the primers were designed according to information at table 1 and ordering from sinagen company. an additional pair of primers specific for amplifying a 785bp segment of gene for a main surface antigen of merozooite, tams1 (30kda) for th. annulata (kirvar et al. 2000) was ordered and have been used in this study too. tams1 f: (5'-atg-ctg-caa-atg-aggat-3' ) and tspms1 r: (5'-gga-ctg-atgaga-aga-cga-tga-g-3'). pcr protocol for tams1 (th. annulata) denaturation, 94 °c for 3 min, then 40 cycles of (94 °c= 1 min, 60 °c =1 min, 72 °c= 1 min) and final extension 72 °c for 10 minutes. the pcr products run on 1% agarose gel and electrophoresis was done. pcr protocol for slag1 (th. lestoquardi) denaturation, 94 °c for 2 min, then loop1 (11 cycles) (94 °c= 20 sec, 57.5 °c= 45 sec, 72 °c= 30 sec) loop2 (12 cycles) (94 °c=15 sec, 57.5 °c= 30sec, 72 °c= 30 sec) loop3 (17 cycles) (94 °c= 10 sec, 57.5 °c= 15 sec, 72 °c= 30 sec) and final extension 72 °c for 5 minutes. results totally extracted dna were divided to 13 groups (171 samples) related to 171 individuals of tick. these are 10% of total samples, 1710 tick samples were collected. experiments are summarized in five stages as follows. stage 1–100 dna samples were subjected to a specific primer for tick identification (its2) and 70% were confirmed (fig. 3). this stage confirms the quantity and quality of extracted dna from ticks and also the specificity of dna from ticks (abdigoudarzi et al. 2011). stage 2positive controlfor the detection of ba. ovis and th. lestoquardi was prepared using positive dna from infected blood from infected animals (fig. 4). stage 3extracted dna from ticks (groups no. 4, 5, 7, 8, 9, 10, 11) (86 dna samples from 86 individuals of ticks) were subjected to a specific primer for th. annulata (tams1-f, tspms1-r). each group was subjected to pcr when a positive control was included. there was no positive band after gel electrophoresis for tick samples. there was a positive band (≈ 800bp) just for positive control. stage 4–33 dna samples extracted from collected ticks of shiraz (groups 4 and 5) subjected to a pair of specific primers (tams1, tams4) and followed by (tams3, tams2) (nested pcr) one sample was detected infected to th. annulata (fig. 5) dna ladder marker is 100bp and size of the amplified segment is 500bp (fig. 5). stage 5eight out of 14 samples (group 5) were subjected to specific primers for th. lestoquardi and protocols of slag1, two samples were positive for infection. agarose gel 166 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 electrophoresis of pcr products related to dna group no. 4 (19 specimens) and specific primers for ba. ovis (bab3, 4). no. 5 (500 bp), 16, 18, 19 and 20 regarded positive and related individual ticks were recorded infected to ba. ovis (fig. 6). stage 6two type of staining methods were applied on salivary glands of suspected ticks. regarding the specific biology of salivary glands in blood fed ticks and protozoan life cycle there was no confirmed particles in salivary glands that could be regarded as evidence for the presence of infection. here in this stage scientific problem are regarded and will be discussed in the following. fig. 1 and fig. 2. wet mount preparation of salivary glands of suspected ticks, staining was done by giemsa and feulgen, clusters are acini of salivary gland. fig. 3. sample photo of agarose gel electrophoresis of pcr products related to extracted dna from ticks (14 specimens) (fars province) and specific primer for its2. (no. 5, 9, 11, 12, 13) are very specific good amplified bands confirming the quality and quantity of dna fig. 4. agarose gel electrophoresis of pcr products related to dna positive for t. lestoquardi (line 5) and babesia ovis (line 7) and specific primers for slag and babesia ovis. these (pcr products)( from known positive dna) have been used as positive control 167 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 fig . 5. agarose gel electrophoresis of a 500 bp positive band (s) from a suspected tick (group 5 of shiraz), primers are (tams1, tams4) followed by (tams3, tams2) (a nested pcr was applied) and the infection of tick with t. annulata was confirmed. (marker is 100 ladder marker) (s= sample) fig. 6. agarose gel electrophoresis of pcr products related to dna group no. 4 (19 specimens) and specific primers for babesia ovis (bab3, 4). no. 5 (500 bp), 16, 18, 19 and 20 regarded positive and related individual ticks were recorded infected to babesia ovis. (m= 100bp marker) table 1. detected ticks and type of protozoan infection by molecular method group number of specimens protozoan type confirmed case of infection 5 8 out of 14 t. lestoquardi 4-hyalomma anatolicum ana. f 9-hyalomma anatolicum ana. f 7 lorestan-aligoudarz-ab-barik (30/1/87) (18th april 2008) t. lestoquardi 1h. detritum f 5 fars-ghir va karzin-baghe no karzin (14/6/86) (4th sep. 2007) (14) t. lestoquardi 12rhipicephalus turanicus f 13rhipicephalus t. n 14hyalomma anatolicum ana. f 1 fars-arjan-benroud-sheepandgoats date: 2/3/86 (22th may 2007) -560mm babesia ovis 4rhipicephalus turanicus f, m 5rhipicephalus turanicus m 7rhipicephalus turanicus m 8rhipicephalus turanicus m 10rhipicephalus turanicus m 13rhipicephalus turanicus m 14rhipicephalus turanicus f, m 15rhipicephalus turanicus f 4 fars-ghir va karzinashayere dashte bilaki (collection date (14/6/86) (4th sep. 2007) temp.40◦c 250mm (sheepand goats) (19) babesia ovis 5hyalomma anatolicum ana. m 16-hyalomma anatolicum ana. f 18-hyalomma marginatum m 19-hyalomma sp. f 20-hyalomma anatolicum ana. m 168 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 discussion regarding sheep and goats, theileriosis due to th. hirci and babesiosis due to ba. ovis and ba. motasi are the most pathogenic protozoa. ba. crassa, anaplasma ovis and eperythrozoon ovis are usually non-pathogenic and do not cause any apparent problem (hashemi-fesharki 1997). in a study in china, the importance of ticks and tick-borne diseases of small ruminants was discussed. of the 109 species of ticks identified to date of study in china, 45 species infest small ruminants. five species have been proved to be involved, or possibly involved, in the transmission of tick-borne diseases. anaplasma ovis, ba. motasi, ba. ovis and two unidentified species of theileria, have been recorded in small ruminants in china (yin and luo 2007). in a study by shayan et al. (2007), using pcr, dna was isolated from 269 salivary glands of rhipicephalus spp. (108 r. bursa, 87 rh. turanicus, 74 rh. sanguineus) collected from sheep with suspected to babesiosis. nested pcr and restriction fragment length polymorphism was performed. as positive control for the dna extraction procedure, the dna was analyzed with the common primers designed from the 18s rrna of the ticks. ba. ovis was detected in salivary gland of 18.5% r. bursa, 9.1% rh. turanicus, and 8.1% rh. sanguineus, respectively (shayan et al. 2007). in the present study the size of the amplified band for ba. ovis in rh. turanicus was 400 bp and it is compatible to anticipated 391 bp from table 1. it is also similar to the size of amplified band (389 bp) for ba. ovis in rh. turanicus in study of the same gene by shayan et al. 2007. there is no information about the geographical region of study by shayan et al. 2007, but in our study ba. ovis infection in rh. turanicus collected from two different places (farsarjanbenroudsheep and goats) from fars province in iran (table 1) have been confirmed. in addition, ba. ovis infection has been confirmed in hy. anatolicum anatolicum and hy. marginatum too (farsghir and karzin 2007) (table 1). there is no documented information of these hyalomma spp. infections to babesia up to now. in a similar study in iran, about 323 ticks were collected from 102 animals in ghaemshahr city in northern parts of iran (88 sheep, 12 goats and 2 cattle). the prevalence of ticks infesting animals was rh. sanguineus (82.35%), rh. bursa (0.3%), ixodes ricinus (i. ricinus) (15.2%), boophilus annulatus (bo. annulatus) (1.2%), haemaphysalis punctata (ha. punctata) (0.3%) and ha. numidiana (0.6%). eleven (55%) tick specimens were pcr positive against genome of th. ovis (t. ovis). sequence analysis of the pcr products confirmed presence of th. ovis in one rh. sanguinus (telmadarraiy 2012). in this study, collected rh. sanguineus ticks have been tested and other collected ticks have been neglected, this is not explained why the main role was directed for rh. sanguineus. in studies by shayan et al. 2007 and telmadarraiy 2012 confirmed tick vectors should be addressed to the specific host and region of study to be able to get epizootical information. there is a well planned study (razmi et al. 2003) and clinically confirmed ill hosts have been regarded, then ticks on their body have been collected and their salivary glands were tested by staining method. during a two year period 188 suspected cases of ovine theileriosis from 28 herds were clinically examined and investigated for the presence of th. lestoquardi in appropriate blood smears and any tick species on body of sheep. in this study, 36.17% of sheep were infected to th. lestoquardi with a parasitemia of 0.01–15%. there was no significant difference between the rate of parasitemia in sheep and the frequency of infected ticks. it has been found that 61.1% of the animals harboured hy. anatolicum anatolicum, 33.42% 169 j arthropod-borne dis, december 2013, 7(2): 164–172 m abdigoudarzi: detection of naturally … http://jad.tums.ac.ir published online: august 31, 2013 rh. sanguineus and 0.05% hy. marginatum m. the examination of 345 tick salivary glands showed that 15% of salivary glands of hy. anatolicum an. and (4%) of rh. sanguineus contained feulgen positive bodies (razmi et al. 2003). regarding the present study, routine monitoring of infected ticks in endemic foci of protozoan infection could be an essential need in addition to monitoring of collected sera (blood) of infected animals and the epizootical information will be raised, then better understanding of tick and tick borne diseases and better planning for control measures for ticks could be achieved. acknowledgements the present study was the result of a confirmed finalized official research project at razi institute and the author wishes to thank all cooperatives including dr gr habibi, dr k esmaeil-nia, dr gr karimi, 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accepted 25 jun 2012) abstract background: although there are about 520 species of birds in iran, but only some of them have been checked for ectoparasites so far.the aim of this study was to check some more available species of the birds of iran for lice. methods: this study was performed between 2008–2010 in northern iran. for this purpose we tried to check some of the wild bird species available and mostly not checked before to identify the lice of them.the birds were found in some of the houses of hunters keeping as trap for catching more birds, some of the bird keepers and a few dead birds from taxidermists. in this way we could check 79 birds of 6 species. results: we identified 11 lice species on the birds and overall 15.2 % of the examined birds were infested by the lice. nine lice species including aquanirmus podicipis, pseudomenopon dolium, ardeicola sp, ciconiphilus decimfasciatus; menacanthus sp, austromenopon transversum, pectinopygus gyricornis, colpocephalum turbinatum and hohorstiella lata were recorded for the first time on the birds in iran. one specimen of menacanthus sp was found on the purple heron (ardea purpurea) that is a new host report for this lice. conclusion: although the infection rate was not very high in the birds, but 11 species of lice in 6 studied birds species in this study, shows there are still other lice species that exist in the birds and should be identified and added to iran lice fauna list. keywords: lice, birds, iran introduction chewing lice (ischnocera, amblycera) are permanent obligate ectoparasites mostly parasitic on bird species and they feed on feathers and skin scales. although they have mouthparts designed for chewing, some of the species of ishnocera can cause skin irritations and suck blood. chewing lice have harmful effects that lead to decrease in productivity in host. there are about 4000 lice species on birds in the world (price et al. 2003). beside the existence of about 520 species of birds in iran (mansoori 2008), there are a few studies relevant to lice infestations and fauna in iran (rafyi et al. 1968, ardalan 1971, 1975, rak et al. 1975). rafyi et al. (1968) detected 15 lice species (goniodes dissimilis, goniodes gigas (= goniocotes gigas), goniocotes gallinae, campanulates compar (= goniocotes bidentatus), lipeurus caponis, cuclotogaster heterographus (= lipeurus heterographus), chelopistes meleagridis, columbicola columbae, anaticola anseris menopon gallinae, menopon pallidulum, menacanthus stramineus and trinoton anserinum) on poultry, pigeons, ducks, geese and turkeys in iran. ardalan (1971) added to these six lice species; columbicola tschulyschman from domestic pigeon (columba *corresponding author: dr ali halajian, e-mail: ali_hal572002@yahoo.com 83 j arthropod-borne dis, june 2013, 7(1): 83–89 b dik, a halajian: chewing lice (phthiraptera) of… http://jad.tums.ac.ir published online: april 10, 2013 livia), philopterus corvi from raven (corvus corax), cuculoecus latifrons (= philopterus latifrons) and philopterus ocellatus from crow (in text: corvus corone), cummingsiella ovalis and saemundssonia humeralis from curlew (numenius arquata). same author (ardalan 1975) reported quadraceps legatus from common tern (sterna hirundo), quadraceps conformis from curlew, laemobothrion vulturis from egyptian vulture (neophron percnopterus) and myrsidea anaspila from raven, for the first time in iran. eslami et al. (2009) detected five lice species (lipeurus caponis, menopon gallinae, menacanthus stramineus, goniodes ocilis and cuclotogaster heterographus) from chicken in golestan province, northern iran. according to the results of the studies performed on the lice species on the birds, approximately 39 lice species have been detected in iran, until now. this study was carried out to identify the chewing lice of some of the bird species of iran. materials and methods this study was performed between 2008– 2010 in two northern provinces of mazandaran and guilan, northern iran. for this aim, seventy nine birds belonging to the six bird species (great crested grebe: podiceps cristatus n= 31, little egret: egretta garzetta n= 11, purple heron: ardea purpurea n= 6, cormorant: phalacrocorax carbo n= 5, blackheaded gull: larus ridibundus n= 21, rock pigeon: columba livia n= 5) were examined for louse. the birds were found in some of the houses of hunters keeping as trap for catching more birds, and some of the bird keepers. grebes were taken for checking from fishermen that catch the birds accidentally in the fishing nets. for collecting the lice, with considering the restricts for handling the wild birds and little time for checking each bird and from among the reliable methods (clayton and drown 2001), visual examination as the main method and based on the situation, post-mortem-ruffling were used. in order to collect the lice, the body of each bird was carefully examined. all lice were collected and placed in tubes with 70% alcohol. the louse specimens were cleared about 24 hours in 10% koh, and then put in distilled water for one day. following dehydration in a graded alcohol series (70%, 80%, 90% and 99%, in consecutive days) the specimens were mounted on slides in canada balsam. after being dried in incubator, lice specimens were identified under a light microscope. the identification of the lice was carried out according to relevant literatures (price and beer 1963, 1965, timmermann 1964, clay 1973, price 1973, zlotorzycka 1976, price et al. 2003). results eleven lice species were identified on the birds in this study. aquanirmus podicipis (denny, 1842): 4 ♀, 1 nymph (n), december 23rd, 2010, host: great crested grebe: podiceps cristatus, locality: nashtarood city, mazandaran province. intensity: 5 in one bird. this species is reported for the first time in iran (fig. 1). ardeicola sp.: 1 ♀, june 30 th, 2010, host: little egret: egretta garzetta, locality: talesh, guilan province. the species was not identified because there was only one female sample that was crashed during the mounting on slide. ardeicola sp is reported for the first time in iran. remarks: ardeicola expallidus blagovestchensky, 1940 lives on e. garzetta. our sample was close to this species, although because of lack of samples it wasnt identified to species level. austromenopon transversum (denny, 1842): 3 ♀ 1 n, december 1st, 2010, host: blackheaded gull: larus ridibundus, locality: cheshmeh kileh river, tonekabon city, mazan84 j arthropod-borne dis, june 2013, 7(1): 83–89 b dik, a halajian: chewing lice (phthiraptera) of… http://jad.tums.ac.ir published online: april 10, 2013 daran province. intensity: 4 in one bird. this species is reported for the first time in iran (fig. 2). campanulates compar (burmeister, 1838): 3 ♀ 1 ♂, 2009, host: rock pigeon: columba livia, locality: nashtarood city, mazandaran province. intensity: 4 in one bird (fig. 3). ciconiphilus decimfasciatus (boisduval and lacordaire, 1835): 3 ♂ 1 ♀, june 30th, 2010; host: little egret: egretta garzetta, locality: talesh, guilan province. intensity: 2 in each bird. this species is reported for the first time in iran (fig. 4, 5). colpocephalum turbinatum denny, 1842: 1 ♀, 2009, host: rock pigeon: columba livia, locality: nashtarood city, mazandaran province. this species is reported for the first time in iran (fig. 6). columbicola columbae (linnaeus, 1758): 3 ♀ 2 ♂, 2009, host: rock pigeon: columba livia, locality: nashtarood city, mazandaran province. intensity: 4 in one and 1 in one bird (fig. 7). hohorstiella lata (piaget, 1880): 3 ♀, 2009, host: rock pigeon: columba livia, locality: nashtarood city, mazandaran province. intensity: 3 in one bird. this species is reported for the first time in iran (fig. 8). menacanthus sp: 1 ♀, december 1st, 2010, host: purple heron: ardea purpurea, nashtarood city, mazandaran province (fig. 9, 10). remarks: menacanthus species are not found on the birds belonging to the family ardeidae. however, one specimen of menacanthus sp. was detected on the heron. the identification of the host species was correct, but in this case maybe the purple heron was an accidental host for this specimen. pectinopygus gyricornis (denny, 1842): 3 ♂ 3 n, april 18th, 2010, host: great cormorant: phalacrocorax carbo, locality: ramsar city, mazandaran province. intensity: 5 in one bird and 1 in another bird. this species is reported for the first time in iran (fig. 11). pseudomenopon dolium (rudow, 1869): 1 ♂, december 23rd, 2010, host: great crested grebe: podiceps cristatus, locality: nashtarood city, mazandaran province. this species is reported for the first time in iran (fig. 12, 13). fig. 1. aquanirmus podicipis, adult female, host: podiceps cristatus (great crested grebe) (original) fig. 2. austromenopon transversum, adult female, host: larus ridibundus (black-headed gull) (original) fig. 3. campanulates compar, adult female, host: columba livia (rock pigeon) (original) 85 j arthropod-borne dis, june 2013, 7(1): 83–89 b dik, a halajian: chewing lice (phthiraptera) of… http://jad.tums.ac.ir published online: april 10, 2013 fig. 4. ciconiphilus decimfasciatus, adult female, host: egretta garzetta (little egret), (original) fig. 6. colpocephalum turbinatum, adult female,host: columba livia (rock pigeon) (original) fig. 8. hohorstiella lata, adult female, host: columba livia (rock pigeon) (original) fig. 5. ciconiphilus decimfasciatus, adult male, host: egretta garzetta (little egret), (original) fig. 7. columbicola columbae, adult male (left), adult female (right), host: columba livia (rock pigeon) (original) fig. 9. menacanthus sp, adult female, host: ardea purpurea (purple heron) (original) 86 j arthropod-borne dis, june 2013, 7(1): 83–89 b dik, a halajian: chewing lice (phthiraptera) of… http://jad.tums.ac.ir published online: april 10, 2013 fig. 10. menacanthus sp, adult female, head, host: ardea purpurea (purple heron) (original) fig. 11. pectinopygus gyricornis, adult male, host: phalacrocorax carbo (cormorant) (original) fig. 12. pseudomenopon dolium, adult female, host: fig. 13. pseudomenopon dolium, adult male, host: podiceps cristatus (great crested grebe) (original) podiceps cristatus (great crested grebe) (original) discussion approximately 4000 valid lice species have been reported on the birds worldwide (price et al. 2003). however, there is little previous information about the louse fauna of the birds in iran and there are not many studies done about the chewing lice on the wild birds in this country. in the studies done relevant to the chewing lice found on wild birds, approximately 39 lice species have been detected in the birds in iran, until today. a few number of wild birds species were examined for the louse species in those studies. although only six species of wild birds including 79 birds overall were examined for the louse in this study, but eleven lice species with nine new species for the fauna of iran are reported and the overall infection rate was 15.2%. out of these birds, only rock pigeon was examined by the previous researchers of iran (rafyi et al. 1968) and the other 5 species of birds are checked for lice for the first time in this study. in this study, eleven lice species, aquanirmus podicipis, ardeicola sp, austromenopon transversum, campanulates compar, ciconiphilus decimfasciatus, colpocephalum turbinatum, col87 j arthropod-borne dis, b dik, a halajian: chewing lice (phthiraptera) of … http://jad.tums.ac.ir published online: april 10, 2013 umbicola columbae, hohorstiella lata, menaca-nthus sp, pectinopygus gyricornis and pseudomenopon dolium were found on the birds. campanulates compar (= goniocotes bidentatus) and c. columbae were the only species reported previously in iran, campanulates compar from pigeon as goniocotes bidentatus by rafyi et al. (1968) and ardalan (1972) and columbicola columbae from rock pigeon (columba livia) by rafyi et al. (1968) and rak (1974). except these two species, the others are recorded for the first time in iran. one specimen of ardeicola sp. collected from little egret was not identified because the specimen was crashed during mounting on the slide. rak et al. (1975) stated that they detected some lice species found on the hosts which were not their normal host. similarly, we found one specimen of menacanthus sp collected from purple heron which is not its normal host and this specimen could not be identified and this issue needs to be studied more with more samples. in conclusion a. podicipis, p. dolium, ardeicola sp, c. decimfasciatus, menacanthus sp, a. transversum, p. gyricornis, c. turbinatum and hohorstiella lata were recorded for the first time on the birds in iran. normally, the species belonging to the genus menacanthus are not found on the birds belonging to the family ardeidae. however, one specimen of menacanthus sp was found on the purple heron for the first time in this study. acknowledgements we wish to thank dr jamshid mansoori for his great help in confirming the identification of birds and to dr sareh tavakol (islamic azad university, tonekabon branch) for providing one of the samples.with special thanks to aynaz. the authors declare that they have no conflicts of interest. references ardalan a (1971) mallophaga of iran: new records. bull soc pathol exot filiales. 64(2): 236–237. ardalan a (1972) notes on mallophaga of iran. the 4th national congress of plant medicine, 2–7 september 1972, evin, tehran, iran. pp. 1–6. ardalan a (1975) mallophaga of iran. ii. 5 new records of mallophaga from iran. bull soc pathol exot filiales. 68(1): 93–94. clay t (1959) key to the species of austromenopon bedford (mallophaga) parasitic on the charadriiformes. proc r ent soc lond (b). 28: 157–168. clay t (1973) the species groups of pectinopygus (phthiraptera: philopteridae). bull brit mus (nat hist) entomology. 29: 201–223. clayton dh, drown dm (2001) critical evaluation of five methods for quantifying chewing lice (insecta: phthiraptera). j parasitol. 87(6): 1291–1300. edwards rl (1965) revision of the genus aquanirmus (mallophaga: philopteridae), parasitic on grebes (podicipidae). canad entomol. 97: 920–935. eslami a, ghaemi p, rahbari s (2009) parasitic infections of free range chickens from golestan province, iran. iranian j parasitol. 4(3): 10–14. mansoori j (2008) a guide to the birds of iran (2nd ed). farzan book publishing, tehran. 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(mallophaga), iii. ann mag nat hist (series 11). 13: 767–780. timmermann g (1954) vorläufige übersicht über das amblycerengenus austromenopon bedord, 1939 (mallophaga). bonn zool beitr. 5: 195–206. timmermann g (1964) gruppen-revisionen bei mallophagen. vii. die pectinopygusarten der grosskormorane (gen. phalacrocorax brisson, 1760 s.str.). mitt hamburg zool mus inst. 61: 271–284. zlotorzycka j (1976) wszolymallophaga. nadrodzina menoponoidea. polskie towarzystwo entomologiczne [klucze do oznaczania owadów polski]. 15(2): 1–189. 89 j arthropod-borne dis, june 2022, 16(2): 173–179 h abbas et al.: concurrent trypanosomosis, … 173 http://jad.tums.ac.ir published online: june 30, 2022 case report concurrent trypanosomosis, babesiosis and toxocarosis in a dog: a case report *haider abbas1, hafiz muhammad rizwan1, muhammad younus2, muhammad sohail sajid3, shahid hussain farooqi4, zeeshan iqbal5, muhammad imran6, usman amanat7, nida wazir7, muhammad ahsan naeem8 1department of pathobiology (parasitology section), kbcma college of veterinary and animal sciences narowal, sub-campus of university of veterinary and animal sciences (uvas) lahore, pakistan 2department of pathobiology (pathology section), kbcma college of veterinary and animal sciences narowal, sub-campus of uvas lahore, pakistan 3department of parasitology, univesity of agriculture faisalabad, pakistan 4department of clinical sciences (medicine section), kbcma college of veterinary and animal sciences narowal, sub-campus of uvas lahore, pakistan 5department animal sciences (livestock management section), kbcma college of veterinary and animal sciences narowal, sub-campus of uvas lahore, pakistan 6department of animal sciences (animal nutrition section), kbcma college of veterinary and animal sciences narowal, sub-campus of uvas lahore, pakistan 7kbcma college of veterinary and animal sciences narowal, sub-campus of uvas lahore, pakistan 8department of basic sciences, kbcma college of veterinary and animal sciences narowal, sub-campus of uvas lahore, pakistan *corresponding author: dr haider abbas, e-mail: haider.abbas@uvas.edu.pk (received 05 mar 2022; accepted 25 jun 2022) abstract background: among the blood-borne parasitic infections of dogs, trypanosomosis and babesiosis are highly prevalent in the indian subcontinent and highly pathogenic. methods: a six-month-old dog was presented with complaint of lethargy, anorexia and loss of body weight over the previous 15 days on october 11, 2021. a full clinical and targeted haematologic and parasitological examination was undertaken. results: wet smears were positive for the motile trypanosomes while stained thin blood smears showed large number of extracellular trypanosomes and intra-erythrocytic ring-like inclusion bodies of babesia spp. a high burden of toxocara canis was identified on coprological examination. conclusion: this report describes the clinical manifestation of concurrent infection of haemoparasites and gastrointestinal nematodes in pakistani dogs, and highlights the importance of prompt veterinary intervention. keywords: trypanosoma; babesia; toxocara canis; dog; pakistan introduction the haemoprotozoan parasites like babesia (b.) canis, b. gibsoni and trypanosoma (t.) evansi are common causes of life-threatening diseases in wide range of animals including dogs throughout the indian subcontinent and in other parts of world (1-5). salivarian trypanosomes are flagellate haemoprotozoa which belong to family trypanosomatidae and the genus tryp anosoma (6), and are biologically transmitted by biting flies (7). trypanosoma evansi is widely recognized as a cause of neuropathy, immune suppression and anemia in camels and horses, referred to as surra (8, 9); but adult dogs are also widely infected in pakistan (10, 11). the copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:haider.abbas@uvas.edu.pk https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2022, 16(2): 173–179 h abbas et al.: concurrent trypanosomosis, … 174 http://jad.tums.ac.ir published online: june 30, 2022 clinical signs of trypanosomosis in dogs include fever, anemia, superficial lymph node enlargement, loss of appetite, lethargy and progressive loss of bodily condition, leading to fatal outcomes if untreated (12). babesia spp. are intraerythrocytic parasites, biologically transmitted by haemaphysalis or rhipicephalus spp. ticks and infecting a wide variety of hosts including humans as well as domestic and wild animals (13). many species of babesia can infect dogs, causing anemia and hemoglobinuria due to haemolysis as the parasite replicates asexually in the red blood cells (14). the parasites complete their life history through sexual reproduction in the tick host mid gut and the formation of sporozoites in the salivary gland (15). canine gastrointestinal nematode (gin) infections, especially toxocara (t.) canis and ancylostoma spp., are important causes of retarded growth and general malaise (16). dogs and humans have a close association (17), and their gins are of great concern due to their serious zoonotic potential (16). toxocara canis is distributed worldwide, causing diarrhea, poor growth and death if present in large number (18). environmental contamination with faeces containing vast number of eggs which shed during patent infections presents a serious public health risk (19), resulting in human disease referred to as visceral larval migrans. this case report presents the co-infection of trypanosoma spp., and babesia spp. haemoparasites along with the gin, toxocara canis, and describes the therapeutic response to treatment in a dog. case presentation a six-month-old male pet dog (pointer crossbred) was brought to the outdoor clinic at the khan bahadhar chaudhry mushtaq ahmad (kbcma) college of veterinary and animal sciences, narowal, sub-campus of university of veterinary and animal sciences lahore, pakistan on october 11, 2021. the owner reported that the dog had been anorexic, dull and depressed for the past 15 days, and that the onset of clinical signs coincided with the dog having consumed a putrified dead lamb carcass. on examination, the dog was thin, weak and did not respond well to its surroundings (fig. 1). the rectal temperature was normal (38.8 oc), oral mucous membranes were pale and the conjunctivae were pale and jaundiced (fig. 2). there were no swollen lymph nodes, but signs of tachypnoea (38 breaths per minute), coughing and a frothy nasal discharge were observed. with the consent of the owner, a 5ml blood sample was collected from the cephalic vein into an edta coated vacutainer to investigate hematologic parameters and detect any haemoparasites. sample was collected for coprological examination and processed qualitatively by the faecal floatation technique and quantitatively by a mcmaster egg counting method using three-chambered slide having threshold of 8 eggs per gram (epg) (chalex, usa). wet mounts were prepared for motile trypanosomes and the thin blood smears were stained with field’s stain and examined at 1,000x under oil immersion to detect haemoparasites. the haematologic parameters (table 1) showed significantly low packed cell volume (pcv) and haemoglobulin (hb) values indicating severe anemia, and marked eosinophilia, lymphocytosis and neutropenia. the wet mount of blood sample showed the presence of large number of motile extracellular trypanosomes. the field-stained thin smear confirmed their identity (fig. 3) and showed the presence of multiple intracellular babesia spp. with ringlike forms in the red blood cells (fig. 4). toxocara canis eggs were identified on coprological examination, being about 75μm x 90 μm in size and having a dark brown colour, a thick pitted shell and sub-globular shape (fig. 5). an adult worm of t. canis was also found in the faeces measuring approximately 18cm in length. the t. canis faecal egg count was 2900 epg, indicating a moderate to high level of infection. the disease history and clinical signs were consistent with chronic haemoprotozoal infechttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 173–179 h abbas et al.: concurrent trypanosomosis, … 175 http://jad.tums.ac.ir published online: june 30, 2022 tion and gin infection (12); hence the dog was treated with intramuscular injection of diminazene aceturate (pronil® injection, selmore) at a dose rate of 3.5mg/kg body weight on the first day and same dose was repeated after 72 hours. doxycycline (vibramycin®, pfizer) was given orally at a dose rate of 5mg/kg of body weight, along with a liver tonic (1teaspoon per day, hepa-merz® syrup, brookes) and multivitamins (1 teaspoon per day, vidaylin-l® syrup, abbott) for 5 days. a single treatment of anthelmintic 1 tablet per 10kg body weight (drontal® plus, bayer) was given. the dog did not respond to supportive therapy and drug treatments, and died 20 days after clinical presentation. fig. 1. dog showing emaciation and persistent recumbency fig. 2. dog showing anaemic conjunctiva and an absence of corneal opacity fig. 3. trypanosoma spp. (arrows) on a field-stained thin blood smear (1000 x magnification) of an infected dog. the genus identity was confirmed by the morphology of extracellular protozoa with a flagellum, kinetoplast and undulating membrane fig. 4. erythrocytes showing ring-like forms of babesia spp. (arrows) on a field-stained thin blood smear (1000 x magnification) of an infected dog fig. 5. toxocara canis eggs at 40 x identified on coprological examination of an infected dog http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 173–179 h abbas et al.: concurrent trypanosomosis, … 176 http://jad.tums.ac.ir published online: june 30, 2022 table 1. summary of haematologic parameters of the infected study dog hematologic parameters observed values reference values erythrocytes (1012/l) 1.69 5.5-8.5 hemoglobin (g/dl) 3.70 12-18 haematocrit (%) 14.0 37-55 leucocytes (109/l) 17.0 6.0-17 neutrophils (109 /l) 1.50 2.9-12 eosinophils (109 /l) 2.0 0.0-1.3 granulocytes (%) 41.1 60–70 lymphocytes (%) 49.7 8.0–21 platelets (109 /l) 33.0 200–500 discussion disruption of the erythrocytes by the release of babesia spp. piroplasms and mechanical injury to erythrocytes by circulating trypanosomes would have accounted for the clinical signs that were observed (20, 21). eosinophilia, and lymphocytosis are typical in case of trypanosomosis (22, 23). trypanosomal infections in dogs have been reported in different countries in asia (10, 21, 24), southern europe (25), africa, north america (26) and south america (27). salivarian trypanosomiasis in various animal hosts caused by t. evansi, t. brucei, t. vivax, t. congolense or t. theileri, is transmitted by biological vectors such as tsetse fly, and tabanids or stomoxys spp. biting flies, depending upon the trypanosoma spp. and geographical location (28). in pakistan, the predominant species is t. evansi, which is considered to be the result of the bites of the flies of family tabanidae and stomoxidae, and the molecular studies should be performed to confirm the presence of species of this parasite in animals (29, 30). babesia canis and b. gibsoni are important causes of disease in dogs with a similar global distribution to trypanosoma spp. but dependent on the distribution of dermacentor spp., haemaphysalis spp. or rhipicephalus tick vectors. the ring-like morphology of the piroplasms identified in this case are consistent with b. gibsoni, which is particularly pathogenic and prevalent in the indian subcontinent, causing mainly chronic disease. the treatment response of these cases to antiprotozoal drugs is generally poor (31). male dogs are considered more susceptible to babesia infection due to more exposure to tick infestation because of their roaming behaviour in search of mates (32), but the dog in the present case was a pet dog, and the development of infection of trypanosoma and babesia might be due to its being less trypanotolerant and having had less exposure to ticks at a young age (due to provision of more care and hygienic environment as compared to roaming dogs), resulting in less protective immunity. babesiosis may also be predisposed to immunosuppression caused by trypanosoma spp. (33, 34). toxocara canis infection is common in dogs worldwide, and particularly common in the indian subcontinent (35, 36). patent infections are usually seen in young puppies, following prenatal placental infection, lactogenic infection, ingestion of eggs or ingestion of a paratenic host. from about 3 months-old, second stage larvae ingested with eggs usually migrate to various tissues, where they become dormant. the high level of infection in a six month-old dog was unusual, and may have been predisposed to by immunosuppression due to trypanosomosis. conclusion the poor response to diminazene aceturate and doxycycline was not unexpected, due to the advanced stage of disease in the dog, and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2022, 16(2): 173–179 h abbas et al.: concurrent trypanosomosis, … 177 http://jad.tums.ac.ir published online: june 30, 2022 highlights the importance of seeking veterinary advice and treatment immediately when signs of ill health are observed. acknowledgements professor neil sargison, personal chair of farm animal practice at the royal (dick) school of veterinary studies, university of edinburgh, easter bush campus, midlothian, eh25 9rg, united kingdom is highly appreciated for reviewing the manuscript both scientifically and grammatically. ethical considerations with the consent of the 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hanafi-bojd 2, h vatandoost 2 1 school of public health, hormozgan university of medical sciences, bandar abbas, iran 2 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 15 apr 2007; accepted 27 jul 2008) abstract background: the german cockroach, blattella germanica l., is a serious household and public health pest worldwide. control of this species has been very difficult to achieve. toxicity of cypermethrin ec10%, deltamethrin ec5%, diazinon ec0.5%, lambda-cyhalothrin ec5% and negon® (permethrin+propoxur oil liquid1%) commercial formulations were investigated against adult males of german cockroaches collected from four hospitals of bandar abbas city, southern iran, during 2006. these insecticides have been used for cockroach control in this city. methods: the tests were carried out only on males by the glass jar contact method recommended by the who. results: maximum mortality rates of 20, 35, 90, and 100% were obtained after one hour contact to label-recommended doses of cypermethrin, deltamethrin, lambad-cyhalothrin, diazinon and permethrin+propoxur insecticides, respectively. kt50 results were different from 5.68 min for permethrin+propoxur mixture to 240.37 min for cypermethrin. german cockroach showed < 80 per cent mortality using three pyrethroid insecticides. conclusion: it seems that the label-recommended concentrations of these insecticides were wrong and lower than who advised for cockroach control. for monitoring of resistance it is recommended to do more tests using the pure active ingredient of these insecticides. keywords: german cockroach, commercial insecticides, resistance, hospital, iran introduction according to world health organization report, cockroaches have been in existence for about 360 million years (cochran 1982). these insects are important pests because they spread filth and ruin food, fabrics and book-bindings. they disgorge portions of their partially digested food at intervals and drop feces. they also discharge a nauseous secretion both from their mouths and from glands openings on the body which gives a longlasting, offensive cockroach smell to areas or food visited by them. cockroaches are not usually the most important cause of a disease, but they may play a supplementary role in some allergic diseases as well as spread of some diseases like: diarrheal diseases, amebic dysentery, cholera, leprosy, plague, typhoid fever and viral diseases such as poliomyelitis. therefore they might be important in transmission of nosocomial infections (pai et al. 2004). the german cockroach, blattella germanica (l.), is a serious household and public health pest worldwide. control of this species has been very difficult to achieve. in order to keep it under control, different organochlorine and organophosphate insecticides have been used, as well as carbamate and pyrethroid compounds. still this species has been developing resistance to these compounds. at pre*corresponding author: a.a. hanafi-bojd, tel: +98 21 88951393, e-mail: aahanafibojd@tums.ac.ir iranian j arthropod-borne dis, (2008), 2(1): 21-27 m shahi et al: evaluation of five local formulated … 22 sent, resistance has become a substantial problem that sometimes causes failure of control operations in many countries (atkinson et al. 1991). heavy infestations of cockroaches can be effectively managed by chemical control measures, followed by environmental management to deprive them of food and shelter. the german cockroach is resistant to different insecticides (cochran 1995, hanafi-bojd and sadaghiani 2001, vatandoost and mousavi 2001). the present study was conducted to determine the susceptibility levels of this species to insecticides currently being used in its control program in four hospitals of bandar abbas city, southern iran. materials and methods study area the investigation was carried out over a period of 10 months of 2006 at bandar abbas city, hormozgan province (25º 24' 28º 57' n. and 52º 41'59º 15' e.), bordered by the persian gulf. this city is the main economic seaport of iran. cockroach strains this research was carried out in four training hospitals of hormozgan university of medical sciences in bandar abbas city, called: shahid mohammadi, shariati, koodakan and ebne-sina. b. germanica specimens were collected from different parts of the hospitals. the collections were made by trapping and hand catch methods. traps were made by glass bottles containing bread and butter. the upper internal edge of the bottles, were coated with a fine layer of butter to prevent the escape of cockroaches. these traps were externally covered with dark paper. the samples were collected weekly and sent to the bandar abbas training and public health research center. cockroach colonies were reared in an insectary, maintained in big glass containers at 27±2° c and a relative humidity of 77±3%. they were fed with rabbit food, bread, soybean and water. chemicals to determine the levels of resistance, five commercial formulations of insecticides were used: cypermethrin ec10% (parto-nar, co.), deltamethrin ec5% (parto-nar co.), diazinon ec0.5% (giah co.), lambda-cyhalothrin ec5% (parto-nar, co.) and negon® (permethrin+propoxur) oil liquid 1% (iran chemical production co.). these insecticides formulated in iran and are using for insect pests (such as cockroaches). contact glass jar test tests were conducted on adult males by who standard method (who 1970). the duration of contact was 1 h and then cockroaches were transferred to holding cups. results of mortality were recorded after 24 h. concentrations were prepared based on label-recommended doses of each commercial insecticide. the label recommended concentrations for pyrethriod insecticides were as follows: deltamethrin 2.5% ec, 50 ml in 5 lit water; lambda-cyhalothrin 5% ec, 50-70 ml in 8 lit water; cypermethrin 10% ec, 50-70 ml in 5 lit water. diazinon was ec 60% (that we prepared its 0.5% solution) and the negon® oil liquid 1% was labeled “ready to use”. therefore, we diluted the insecticides based on their label and then pipetted 1 ml of each solution into labeled glass jars (surface area= 180 cm2). the upper inside lip of the jars were smeared with a thin layer of butter to prevent the cockroach escape. the insecticides were deposited evenly in the inner jar surface by rolling the jar on a flat surface until all liquid solvent had evaporated. ten adult males were introduced into the jar and knockdown of the cockroaches was recorded at 5 min intervals. four replicates and one control were carried out for each strain using every insecticide. data analysis iranian j arthropod-borne dis, (2008), 2(1): 21-27 m shahi et al: evaluation of five local formulated … 23 results from all replicates for each insecticide were pooled and subjected to probit analysis (finney 1971). results results of mortality after 1 h contact to label-recommended dosages of cypermethrin, deltamethrin, lambda-cyhalothrin, diazinon and negon® (permethrin+propoxur) are showed in table 1. although in the case of three pyrethroid insecticides the mortality rates of cockroaches were lower than 91%, but two other insecticides (diazinon and negon®) caused 100% mortality. the highest mortality rates for deltamethrin, lambdacyhalothrin and cypermethrin was 35, 90 and 20%, respectively. kt50 values of tested cockroaches in contact to the insecticides were varied from a minimum of 5.68 min for negon® in the ebne-sina strain to a maximum of 240.37 min for cypermethrin in koodakan strain, respectively (table 2). kt50 levels of three pyrethroid insecticides: cypermethrin, lambdacyhalothrin and deltamethrin were distinctly lower in shahid mohammadi strain. table 1. mortality results of one hour contact of male german cockroaches (blattella germanica l.) to five commercial insecticides, bandar abbas, southern iran, 2006 cypermethrin lambda-cyhalothrin deltamethrin diazinon propoxur+permethrininsecticide hospital no. % no. % no. % no. % no. % shahid mohammadi 2 5 36 90 14 35 40 100 40 100 shariati 5 12.5 33 82.5 6 15 40 100 40 100 ebne-sina 8 20 22 55 7 17.5 40 100 40 100 koodakan 3 7.5 16 40 9 22.5 40 100 40 100 control 0 0 1 2.5 0 0 0 0 0 0 table 2. kt50 (min) values of blattella germanica from four hospitals of bandar abbas city, southern iran, in contact to five commercial formulation of insecticides, 2006 insecticide hospital cypermethrin lmabdacyhalothrin deltamethrin diazinion propoxur+permethrin lower 47.72 18.41 31.66 44.05 7.75 kt50 53.78 20.99 35.49 46.79 9.89 shahid mohammadi upper 63.70 23.57 40.22 50.23 11.89 lower 81.64 32.61 59.68 33.89 5.36 kt50 118.67 35.29 71.49 35.27 7.22 shariati upper 441.97 38.17 109.87 36.65 8.76 lower 31.23 67.67 38.22 3.61 kt50 35.69 85.77 40.26 5.68 ebne-sina upper 41.45 135.29 42.17 7.44 lower 113.66 43.39 53.86 32.68 11.95 kt50 240.37 55.52 69.26 34.94 14.02 koodakan upper 9292.93 90.62 103.61 37.27 15.91 iranian j arthropod-borne dis, (2008), 2(1): 21-27 m shahi et al: evaluation of five local formulated … 24 table 3. who label recommended concentrations against cockroaches for the studied insecticides recommended concentration (%) insecticide who label cypermethrin 0.05-0.2 0.0014 deltamethrin 0.03-0.05 0.025 lambdacyhalothrin 0.015-0.03 0.004 diazinon 0.5 0.5 permethrin 0.25 1 propoxur 1 1 discussion cockroaches are difficult to control with insecticides for several reasons, e.g. they may become resistant to commonly used compounds. moreover, many insecticides are repellent to them and are therefore avoided (wooster and ross 1989). chemical control gives only temporary relief and, wherever possible, it should be accompanied by environmental sanitation and house improvement (schal 1988). in this study, german cockroaches showed less than 80% mortality using cypermethrin, deltamethrin and lambdacyhalothrin (except for two strains) (table1). an investigation on efficacy of synthetic pyrethroids (0.02% deltamethrin+0.13% allethrin) and propoxur aerosol in the control of german cockroach in india showed that 89.36-87.8 and 77.9559.74% reductions were achieved with propoxur and synthetic pyrethroid treatment respectively by first week post-treatment (agrawal et al. 2005). results of another study on german cockroaches using commercial insecticides in north of iran showed 100% mortality after 1h contact with propoxur 0.32%, permethrin 0.32%, bendiocarb 0.24% and pirimiphos methyl 0.16% (haghi et al. 1997). the resistance levels of 12 strains of german cockroach in peninsular malaysia were low to high (2.8 to 92x) for carbamates (propoxur and bendiocarb), low (2.0 to 7.6x) for organophosphate (chlorpyrifos) and low to moderate (1.0 to 23x) for pyrethroids (cypermethrin and permethrin) when compared to a susceptible strain (lee et al. 1996). such levels were observed in our study and we found diazinon and permethrin+propoxur based insecticides very effective, rather than the other tested insecticides. who introduced the insecticides and their concentrations for control of cockroaches (chavasse and yap 1997, rozendaal 1997). table 3 shows the who recommended concentrations of insecticides that are used in this study in comparing with the label recommended dose of them for cockroaches control. it can be observed that some commercial insecticides advised very lower concentrations of active ingredient than who recommendation that may be the cause of low mortality in our tests. so for resistance monitoring of german cockroaches in the studied hospitals of bandar abbas, it is recommended to test the active ingredient of tested insecticides based on who concentrations. study of susceptibility level of german cockroach to lambda-cyhalothrin in tehran, the capital city of iran, showed 4 out of 5 strains of this species are tolerant and only one is susceptible (ladonni 1997). the irregular use of insecticides, especially pyrethroids, in the insect pests control programs, is resulted to increase their tolerance or resistance levels. for a successful control program against german cockroach in these hospitals, it is recommended to use correct concentrations of effective insecticides from different groups. application method and accuracy in spraying with due attention to cockroach shelters, are another factors for success. in the study of permethrin resistance ratio (rr) by tarsal contact tests on first-instar nymphs of b. germanica, kt50 of 8.41 min and lt50 of 12.82 with permethrin 15 mg/m2 were obtained and all wild strains showed significant levels of resistance to permethrin, with rr iranian j arthropod-borne dis, (2008), 2(1): 21-27 m shahi et al: evaluation of five local formulated … 25 of 17-27 fold at the kt50 level (ladonni 2000). our minimum of kt50 that observed in mixed insecticide that contains propoxur+ permethrin 1% was ranged from 5.68 to 14.02 min (table 2). the susceptibility level of eleven strains of germen cockroach was evaluated against permethrin and fipronil by topical application. in all strains, cockroaches showed different degrees of resistance to permethrin (nassirian et al. 2006). we observed 100% mortality using negon® that contains permethrin, but it is a mixture of pyrethroid and carbamate insecticides. this mixture destroys all resistance mechanisms and if there was a compound of only permethrin, we could make a better inference about the resistance status of b. germenica strains of bandar abbas. first nymphal stages of different strains of b. germanica were tested to diazinon 2% using insecticide impregnated papers in tehran city, iran (ladonni et al. 1997). comparison of lt50 of field strain with the susceptible insectary strain showed 3 of them had tolerance (1.5< rr< 2.5). our study showed 100% susceptibility to the who recommended dose of diazinon. insecticide resistance studies on b. germanica from cuba showed evidence of resistance to cypermethrin, deltamethrin and lambda-cyhalothrin (diaz pantoja et al. 2000). the resistance to six insecticides was investigated on field strains of german cockroaches caught in hospitals from various parts of poland. the tested field strains showed high or moderate resistance to permethrin, deltamethrin, cypermethrin and bendiocarb, moderate resistance or tolerance to etofenproks, and tolerance or susceptibility to propoxur (gliniewicz et al. 1996). although we found different rates of mortality for these pyrethroids ranging from 7.5 to 90% (table 1), it may be partly due to incorrect concentrations advised by their manufacturing company. to determine the resistances in cohorts of b. germanica from hospitals and households of thailand to organophosphates, carbamates and pyrethroids, bioassays by topical application were carried out. resistance ratio (rr) was determined by comparing the ld50 of the collected cohorts with that of a susceptible cohort. rr ranges of chlorpyrifos, propoxur and cypermethrin in the hospitals were 2.0428.80, 2.86-30.86 and 1.95-14.05 and those in the household were 1.92-17.72, 6.93-62.50 and 2.80-27.35, respectively. significant correlations among the rrs of the three insecticides were separately found in the hospitals and households, indicating the existence of cross resistance among the three groups of insecticides (pai et al. 2005). the history of cockroach control spraying in our studied hospitals showed that carbamate and pyrethroids were mainly used such as: propoxur, cyfluthrin, deltamethrin and lambd-cyhalothrin. inappropriate use of these insecticides can end to tolerance/resistance in cockroaches. therefore, we suggest the label recommended concentration of cypermethrin, deltamethrin and lambdacyhalothrin commercially insecticides that we used in this study have to change based on the who suggested concentrations to obtain best results in cockroach control program of bandar abbas hospitals. acknowledgements the authors are grateful to staff of bandar abbas training and public health research center, especially mr h javdan, for their close collaboration. this project is financially supported by hormozgan university of medical sciences, bandar abbas, iran. references agrawal vk, talik r, dutta gupta kk (2005) efficacy of synthetic pyrethroid and propoxur aerosol in the control of german cockroaches (dictyoptera: blatellidae) in cookhouses. j vect borne dis. 42: 117-121. iranian j arthropod-borne dis, (2008), 2(1): 21-27 m shahi et al: evaluation of five local formulated … 26 atkinson th, wadlergh rh, koehler pg, patteson r (1991) pyrethroid resistance and synergism in a field strain of the german cockroach (dictyoptera: blattellidae). j econ entomol. 84(4): 1247– 1250. chavasse dc, yap hh (1997) chemical methods for the control of vectors and pests of public health importance, who/ ctd/ whopes/97.2. 129 pp. cochran dg (1982) cockroaches. their biology, distribution and control. document who/cds/cpc/world health organization technical report. 82. 856. geneva: who, pp 1-53. cochran dg (1995) insecticide resistance, in understanding and controlling the german cockroach, ed. by rust mk, owens jw and reierson da. oxford university press, new york, ny. pp.171-192. diaz pantoja c, perez mg, calvo e, rodriguez mm, bisset ja (2000) insecticide resistance studies on blattella germanica (dictyoptera: blattellidæ) from cuba. annals of new york academy of sciences. 916 (1): 628-634. finney dj (1971) probit analysis. third edition. london, cambridge university press. gliniewicz a, krzeminska a, sawicka b (1996) susceptibility of cockroaches blattella germanica l. collected from hospitals to selected pyrethroid and carbamate insecticides. rocz panstw zakl hig. 47(3): 333-41. haghi f, gholami sh, sedaghat mm (1997) determining of susceptibility level of hospital cockroaches of sari to insecticides in 1996. iranian journal of mazandaran university of medical sciences. 16: 1-6. hanafi-bojd aa, sadaghiani sh (2001) cockroaches of iran. tehran university of medical sciences press, tehran, iran. ladonni h (1997) susceptibility of different field strains of blattella germanica (orthoptera: blattellidae) to four pyrethroids. iranian j publ health. 26(3-4): 35-40. ladonni h (2000) permethrin resistance ratios compared by two methods of testing nymphs of the german cockroach, blattella germanica. med vet entomol. 14: 213-216. ladonni h, abolhassani m, shayeghi m (1997) susceptibility of first nymphal stage of different strains of blattella germanica l. (dictyoptera: blattellidae) to diazinon and propoxur, using insecticide impregnated paper. j entomol soc iran. 17: 31-39. lee cy, yap hh, chong nl (1996) insecticide resistance and synergism in field collected german cockroaches (dictyoptera: blattellidae) in peninsular malaysia. bulletin of entomological research. 86: 675-682. nassirian h, ladonni h, shayeghi m, vatandoost h, yaghoobi-ershadi mr, rassi y, abolhassani m, abaei mr (2006) comparison of permethrin and fipronil toxicity against german cockroach (dictyoptera: blattellidae) strains. iranian j publ health. 35(1): 63-67. pai hh, chen wc, peng cf (2004) cockroaches as potential vectors of nosocomial infections. infect control hosp epidemiol. 25(11): 979-84. pai hh, wu sc, hsu el (2005) insecticide resistance in german cockroaches (blattella germanica) from hospitals and households in taiwan. int j environ health res. 15(1): 33-40. rozendaal ja (1997) vector control. world health organization, geneva. pp. 425. schal g (1988) relation among efficacy of insecticides, resistance levels, and sanitation in the control of the german cockroach (dictyoptera: blattellidae). j econ entomol. 81: 536-544. vatandoost h, mousavi b (2001) cockroaches, their biology, distribution and iranian j arthropod-borne dis, (2008), 2(1): 21-27 m shahi et al: evaluation of five local formulated … 27 control. (ed by: cochran dg). tehran university press, tehran, iran. who (1970) tentative instructions for determining the susceptibility or resistance of cockroaches to insecticides. technical report series no. 443, world health organization, geneva. annex 12. pp. 130-133. wooster mt, ross mh (1989) sublethal responses of the german cockroach to vapors of commercial pesticides formulations. entomol exper appl. 52: 49-55. j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 108 short communication hard tick species of livestock and their bioecology in golestan province, north of iran aioub sofizadeh 1, *zakkyeh telmadarraiy 2, abbas rahnama 1, ahmad gorganli-davaji 1, asadollah hosseini-chegeni 3 1infectious diseases research center, golestan university of medical sciences, gorgan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of plant protection, faculty of agriculture, guilan university, rasht, iran (received 13 nov 2011; accepted 3 sep 2013) abstract background: a survey on tick species composition was carried out in golestan province iran during year 2010– 2011.the aim was to determine tick species parasitizing domestic ruminants and their seasonal population dynamics. methods: a total of 124 sheep, 92 goats, 84 cattle, 74 camels and 12 horses in several villages were inspected for tick infestation. the collected ticks preserved in 70% alcohol and then were identified. results: the overall 1059 ticks (453 female, 606 male) were collected. the ticks occur on sheep, goats, cattle, camels and horses as 72.1%, 77.3%, 75.8%, 69.3%, and 50% respectively. the frequency of ticks in spring was more than other seasons and the least was observed in winter. in the spring and summer, infestation rate in domestic ruminants were calculated as 100%. six genus and fourteen hard and soft tick species were identified including rhipicephalus sanguineus, r. bursa, ixodes ricinus, haemaphysalis punctata, h. sulcata, h. erinacei, h. inermis, hyalomma marginatum, hy. asiaticum, hy. dromedarii, hy. excavatum, hy. anatolicum, hy. detritum, boophilus annulatus and argas persicus. rhipicephalus sanguineus was the most abundant species in the study area. the largest number of ticks was collected from animal ears and tails. haemaphysalis, hyalomma, rhipicephalus and boophilus occurred in mountainous, forest and plateau areas of golestan province but ixodes occurred only in mountainous and forest areas, whereas rhipicephalus and hyalomma were present in coastal areas of golestan province. conclusion: the result of this study is a survey on tick species from domestic animals in iran and implication of possible prevention measures for diseases transmitted by ticks. keywords: ticks, domestic ruminants, bioecology, iran introduction medical and veterinary implications of ticks (acari: ixodidae) on human and animals life is very obvious question and proved from many years ago (hoogstraal 1982). annoyance and paralysis of livestock also transmission large number of pathogen agents is only some cosmopolitan associated tick problems (sonenshine 1991, service 2001). approximately only 10% of the currently known 867 tick species are also responsible for damage directly due to their feeding behavior (oliver 1989) and unavoidability act as vectors of a broad range of pathogens of those hosts. ruminants are also affected by direct tick damage including tick bite abscesses, anemia, tick induced dermatophilosis. the pioneer work on tick fauna was delpy l in iran (delpy 1936). later continue by gs pervomaisky, h nemenz (pervomaisky 1948, nemenz 1953). subsequently r abbasianlintzen, z mazlum, g maghami and a rafyi et al. conducted some studies generally on do*corresponding author: dr zakkyeh telmadarraiy, e-mail: ztelma@yahoo.co.in http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 109 mestic animals ticks in iran (abbasian-lintzen 1960, maghami 1968, mazlum 1971). however certain studies also has been carried out on the ticks of wild animals by na filippova et al former presented data for 642 ixodid ticks taken from small-sized mammals mainly rodents in different zoogeographical zones of iran and the latter studied ixodid ticks parasitizing wild sheep and goats in iran focusing on maintaining natural foci of many hazardous diseases for human (filippova et al. 1976). recently nabian et al and rahbari et al published a list of adult ticks collected from domestic animals in north and whole iran (nabian et al. 2007, rahbari et al. 2007a). telmadarraiy et al. published a list of tick species and their prevalence in the northwest and the western part of iran (telmadarraiy et al. 2004, telmadarraiy et al. 2008a, telmadarraiy et al. 2010). furthermore there are several reports on epidemiology, distribution, medically importance and acaricide susceptibility of different tick species through in this country (telmadarraiy et al. 2007b, vatandoost et al. 2010a). since thus far only few studies were accomplished on tick fauna in different areas of iran, it seems to be a gap in our knowledge about distribution of tick species in the iran. in addition there is lack of finding about the frequency of ixodid tick species from domestic ruminants in golestan province. therefore, this study is aimed to figure out the frequency of ticks on domestic ruminants likewise their seasonal and topological abundance and their host in the aforementioned part of iran. materials and methods study area the present study carried out in different zone of golestan province which is located in northeastern of iran and geographically located at 36°25´n and 53°51´e on the southeastern coast of caspian sea, a region suitable for agriculture and animal husbandry. this province geographically divided into three parts: covered forest mountainous parts and plateau areas as well as the coastal plains. ticks tick sampling was carried out on all three part of the study area at different time intervals. a total of 124 sheep, 92 goats, 84 cattle, 74 camels and 12 horses in several villages in golestan province were examined for tick infestation on the whole body of each animal. the sites of tick attachment were recorded during collection, collected ticks were preserved in 70% alcohol, counted and tick identification done by using the suitable and comprehensive keys including pomerantzev (1950) and hoogstraal (1956) (pomerantzev 1950, hoogstraal 1956). results during the study period among several villages the total number of 1009 ticks were collected and identified, the occurrence of ticks on sheep, goats, cattle, camels and horses were 72.1%, 77.3%, 75.8%, 69.3%, 50% respectively. table 1 show the number of hard tick species in whole study area. the frequency of ticks in spring was more than other seasons and the least was observed in winter. in the spring and summer infestation rate of domestic ruminants was higher than 90% and in autumn and winter was lower than 25%. the mean number of ticks on each animal was high in spring and summer (40–50 ticks per animal) and was low in autumn and winter (3–5 ticks per animal), so the largest number of ticks were collected from animal ears and tails. fifteen species of ticks were identified based on the morphological characteristics which comprise rhipicephalus sanguineus, r. bursa, ixodes ricinus, haemaphysalis punctata, h. sulcata, h. erinacei, h. inermis, hyalomma marginatum, hy. asiaticum, hy. excavatum, http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 110 hy. anatolicum, hy. dromedarii, hy. detritum, b. anulatus and argas persicus was collected in nests of hen and domestic ruminants. out of 1059 collected ticks from animal, a significant number of 593 r. sanguineus were identified. this species stands out as being the most prevalent tick species comprising 56% of the all ticks collected from domestic ruminants in golestan province. the tick species prevalence is shown in table 1. latter species was the most abundant tick in both mountainous areas and the coastal plains, as well as hy. anatolicum in the plateau areas. whereas i. ricinus, r. bursa, b. annulatus and h. inermis occurred in mountainous areas and hy. dromedarii, hy. asiaticum, hy. excavatum and hy. detritum occurred in plateau areas only. the species diversity of hard ticks in plateau areas was more than other areas (fig. 1). the tick species diversity on sheep is highest from the other hosts (table 2). table 1. seasonal abundance of hard ticks in golestan province species number (percent) season spring summer autumn winter total ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ hy. anatolicum 148(14/7) 0 35 33 24 0 56 0 0 33 115 hy. asiaticum 2(0/2) 0 2 0 0 0 0 0 0 0 2 hy. detritum 2(0/2) 0 0 0 2 0 0 0 0 0 2 hy. dromedarii 64(6/3) 26 24 0 11 0 1 1 1 27 37 hy. excavatum 4(0/4) 0 2 0 0 0 2 0 0 0 4 hy. marginatum 64(6/3) 8 21 2 27 0 6 0 0 10 54 h. erinacei 4(0/4) 0 0 0 0 3 0 1 0 4 0 h. inermis 24(2/4) 0 0 0 0 22 2 0 0 22 2 h. punctata 66(6/5) 0 0 0 0 52 11 0 3 52 14 h. sulcata 3(0/3) 0 0 0 0 1 0 1 1 2 1 i. ricinus 13(1/3) 6 0 1 0 6 0 0 0 13 0 r. (b.) annulatus 4(0/4) 0 0 0 0 4 0 0 0 4 0 r. bursa 18(1/8) 0 0 10 8 0 0 0 0 10 8 r. sanguineus 593(58/8) 163 250 88 92 0 0 0 0 251 342 total 1009(100) 203 334 134 164 88 78 3 5 428 581 fig. 1. topological preference of hard ticks collected in golestan province (the number of specimens presented between two parenthesis) http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 111 table 2. the host diversity of hard tick species in golestan province species number (percent) host cattle sheep goat camel horse hy. anatolicum 108 0 0 25 15 hy. asiaticum 1 0 0 1 0 hy. detritum 0 0 0 2 0 hy. dromedarii 0 0 0 64 0 hy. excavatum 4 0 0 0 0 hy. marginatum 40 0 0 13 11 h. erinacei 3 1 0 0 0 h. inermis 24 0 0 0 0 h. punctata 63 3 0 0 0 h. sulcata 1 2 0 0 0 i. ricinus 7 0 6 0 0 r. (b.) annulatus 4 0 0 0 0 r. bursa 10 5 0 0 3 r. sanguineus 15 359 202 4 13 total 280 370 208 109 42 discussion in the present study 206 (53.3%) out of 386 livestock was infested with tick. the number of ticks was high in spring and summer (40–50 tick per animal) but low in autumn and winter (3–5 tick per animal, average 21.5–25). hosseini vasoukolaei et al. (2010) in ghaemshahr recorded that tick infestation rate of animal and the number of ticks per animal was 24% and 3–5 respectively (hosseini vasoukolaei et al. 2010). in the study conducted by rahbari et al. (2007) in four different zoogeographical areas in iran where the majority of the domestic ruminants in iran exist the number of ticks per animal (10–20) was low (rahbari et al. 2007a). in respect of this result to result of the present investigation tick infestation rate of animals was high in our studied area. through viewpoint of tick infestation of livestock the percentage of tick per sheep, goat, cattle, camel and horse were equal 45.1%, 47.8%, 64.2%, 62.1%, and 50.0% respectively. tick infestation of cattle, sheep and goat in the study of rahbari et al. (2007) was equal 62%, 55% and 57% respectively (rahbari et al. 2007a). in the other study carry out by davoudi et al. (2008) in northwestern parts of iran 10.16% of cattle and 1.07% of buffaloes were infested by ticks. thus tick infestation of animals in our studied areas than ghaemshahr and northwestern parts of iran was higher but coincided with the result of rahbari et al. (2007). these results reveal significant differences concerning tick abundance on different hosts in different geographical areas. in a survey conducted from april 2001 to january 2003 in tamil nadu of india from 600 sheep and goat was selected randomly 64.6% sheep and 97.66% goat were infested by ticks (vathsala et al. 2008) and therefore tick infestation rate of animals in indian study is higher from tick infestation rate of animals in our country. we observed that infestation rate may be maximized in the spring and summer seasons. this result is in agreement with study of davoudi et al. (2008) he also sees the highest rate of infestation occur in spring (davoudi et al. 2008). however telmadarraiy et al. (2004) and salari lak et al. (2008) in the northwestern iran most of tick collected in the summer and spring respectively (telmadarraiy et al. 2004, salari http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 112 lak et al. 2008). it may be as a result of that climate condition and flora of northwestern iran in summer is similar to climate condition of our studied area in spring. in the studies conducted in iran and also in our study, the lowest number of ticks were collected during winter, whereas interestingly in tamil nadu of india seasonal tick infestation rate of sheep and goat in winter (january to march) was 61% and 97.66% respectively (vathsala et al. 2008). this could be due to indian climate conditions in winter agree with climate condition of northwestern iran in same season. in the present study, the ticks were obtained in more number from animal ears and tails. the possible factors effect question include temperature and humidity of skin also delicateness of it that cause tick attached and engorged on this sites. whereas in winter which is colder than other seasons ticks attached the trunk between wool’s host which is warmer than ears and tail thus become their preference site in the winter season. in our study, five genera of ixodid ticks including rhipicephalus (60.5%), hyalomma (27.1%), haemaphysalis (10.6%), ixodes (1.3%), and boophilus (0.4%) were collected and rhipicephalus ticks were dominant in the investigated area. in a survey on ticks collected from sheep, in bahar township of hamadan province in the western part of iran, the ixodid ticks found were hyalomma, rhipicephalus and haemaphysalis and hyalomma ticks were predominant ticks (telmadarraiy et al. 2008a). the frequency of hyalomma and rhipicephalus species in a study conducted in ardabil province located in the northwest of iran, comprising 43.6% and 41.7% of all collected ticks respectively (telmadarraiy et al. 2010). in the studies performed in west azerbaijan province hyalomma ticks were dominant as 63.2% and 61.3% respectively (davoudi et al. 2008, salari lak et al. 2008). in the other study in west azerbijan rhipicephalus (42%), hyalomma (41%), boophilus (7%), dermacentor (7%), and haemaphysalis (3%) were found where rhipicephalus as well as hyalomma were the most prevalent tick species (telmadarraiy et al. 2004). thus could not be understand that in the most aforesaid studies hyalomma ticks were the most abundant ticks, but the largest number of ticks that in northwest of iran were found belonging rhipicephalus genus and the most abundant species in the study area was rhipicephalus sanguineus which is in agreement with study of hosseini vasoukolaei et al. (2010). along with our findings, nabian et al. (2007) found that r. sanguineus is the major species in mazandaran province and did not find r. bursa (nabian et al. 2007), but result of our study show eight male and ten female r. bursa in contrast with only one female r. bursa (hosseini vasoukolaei et al. 2010). in the study conducted by rahbari et al. (2007) r. sanguineus was the main species in the north of iran (rahbari et al. 2007a). from the studies accomplished by telmadarriy et al. (2004, 2008) and davoudi et al. (2008) two species r sanguineus and r bursa were collected from different animals including sheep, goats, cattle, buffaloes and camels (telmadarraiy et al. 2004, telmadarraiy et al. 2008a), showing rhipicephalus are concerned to blood of different hosts. according to these studies, it can be concluded that r. sanguineus is the tick species with great significance for domestic ruminants in the north of iran. razmi et al. (2007) found that boophilus comprising the majority (51.3%) of ticks collected in a study conducted in mazandaran province following hyalomma (18.5 %), rhipicephalus (16.8%), haemaphysalis (6.3%), ixodes (6.3%) and dermacentor (0.1%) were also present in the study area (razmi et al. 2007), but in our study boophilus collected with low frequency and never we collected dermacentor species. the ticks of the genus ixodes are the important vectors of lyme borreliosis in europe and america commonly collected in iran with species i. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 113 ricinus only in the caspian sea littoral area (nabian et al. 2007 rahbari et al. 2007a, razmi et al. 2007). in our study ixodes tick were found with low frequency (1.3%). the most common ixodid tick vector of human granulocytic ehrlichiosis (hge) in europe is i. ricinus (rymaszewska and grenda 2008). in a study by bashiribod et al. (2004) in ghaemshahr, 5.1% of all i ricinus ticks examined by molecular methods was shown to be infected with anaplasma phagocotophilum (bashiribod et al. 2004). in a study by hosseini vasoukolaei et al. (2010) 49 out of 323 collected ticks were identified as i ricinus, which is an important vector of tickborne disease aforesaid and may be considered in the study area, need for further investigation (hosseini vasoukolaei et al. 2010). in the study undertaken by nabian et al. (2007) genus ixodes was found with frequency 2.32% (nabian et al. 2007). several haemaphysalis species were found in iran. in the present study, h. erinacei, h. sulcata, h. punctata and h. inermis were collected from farm animals, in spite of only h. erinacei was reported in study of hosseini vasoukolaei et al. (2010) from north of iran (hosseini vasoukolaei et al. 2010). rahbari et al. (2007) in a study completed in three geographical zone of iran reported 6 species h. punctata (3.4%), h. parva (0.5%), h. sulcata (0.6%), h. choldokovskyi (1.7%) (now synonymized as h. sulcata), h. concinna (0.06%) and haemaphysalis sp. (0.6%) but never reported h. erinacei and h. inermis (rahbari et al. 2007b). interestingly the latter species recorded by telmadarraiy (2008) in south of sari in mazandaran province (telmadarraiy et al. 2008b). in respect of much haemaphysalis are three host ticks thus they may be serve as competent vectors of different domestic ruminant thereupon human diseases. in the present work several hyalomma including hy. dromedarii, hy. marginatum, hy. asiaticum, hy. excavatum, hy. anatolicum, hy. detritum were collected at least in all studied geographical areas. whereas in a series studies conducted by one of authors (zt) from year 2004 to year 2010 in yazd, ilam, east and west azerbaijan, ardabil, hamadan, khorasan jonoobi provinces as well as south of sari in mazandaran province (telmadarraiy et al. 2004, telmadarraiy et al. 2006, telmadarraiy et al. 2008a, telmadarraiy et al. 2008b, nasiri et al. 2010, salim abadi et al. 2010, telmadarraiy et al. 2010) various hyalomma species were recognized like present study. also author with cooperation other (2007, 2009, 2010) studied ectoparasite of rodents including ticks as well as lesser mouse eared bat, myotis blyti ( telmadarraiy et al. 2007a, kia et al. 2009, vatandoost et al. 2010b). it is necessary indicate which hyalomma species has critical role for transmission of crimean congo hemorrhagic fever. with respect to which is three geographic continents including mountainous, littoral and plateau possess golestan province species diversity and the number of species found in mountainous, littoral and plateau area comprising 6, 2 and 10 species respectively. otherwise following ticks genera and species collected respect to three aforesaid situation constitute r. sanguineus (69%) in mountainous and (93.5%) in littoral area which is more incident from other species. beside hy. anatolicum (47.9%) and hy. dromedarii (29%) in plateau area were dominant species. whereas in a study conducted in plateau area of yazd province hy. dromedarii was more prevalent species (55.92 %) while d. marginatus, hy. marginatum and hy. anatolicum ticks were collected with very slightly frequency (salim abadi et al. 2010). in another study directed in esfahan province r. sanguinus in mountainous and hy. anatolicum in plateau areas were the most abundant ticks respectively (noaman et al. 2008). it may be concluded that genera of rhipicephalus and hyalomma are as principal genera in mountainous and plateau area respectively. in the present study most of http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 114 tick species were collected from cattle, only hy. dromedarii and hy. detritum were found on camel. in addition hy. anatolicum was predominant hyalomma species obtained from cattle and the other tick genera and species including h. sulcata, h. erinacei (haemaphysalis), hy. asiaticum, hy. excavatum (hyalomma) and boophilus (rhipicephalus) annulatus only with low frequency collected from cattle host. likewise the most number of species collected from two hosts camel and horse including hy. dromedarii and hy. anatolicum respectively. acknowledgements this study was supported by the infectious diseases research center of golestan university of medical sciences. references abbasian-lintzen r (1960) a preliminary list of ticks (acarina: ixodidae) occurring in iran and their distributional data. acarologia 2: 43–61. bashiribod h, kazemi b, eslami g, bigdeli s, bandehpour m, rahbarian n, ramezani z (2004) first molecular detection of anaplasma phagocytophilum in ixodes ricinus ticks in iran. j med sci. 4: 282–286. davoudi j, hoghooghi rad n, golzar adabi s (2008) ixodid tick species infesting cows and buffaloes and their seasonality in west azerbaijan. res j parasitol. 3: 98–103. delpy lp (1936) notes sur les ixodidés du genre hyalomma (koch). ann parasitol hum comp. 14: 206–245. filippova na, neronov vm, 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cypermethrin in ornithodoros lahorensis neuman and argas persicus oken (acari: argasidae) field populations. pak j biol sci. 10: 4315–4318. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 108–116 a sofizadeh et al.: hard tick species of … 116 telmadarraiy z, chinikar s, shakeri ma, vatandoost h, faghihi f, faghihi f, moradi n, ranjbar e, motiei m, hosseine vasoukolaei n, aboulhasani m (2008b) crimean-congo hemorrhagic fever: a seroepidemiological and molecular survey in poshete-kooh of sari, mazandaran province iran. tropical medicine and malaria. international convention center jeju, jeju island, korea. 29th september -03 october 2009 vatandoost h, telmadarraiy z, sharifi m, moradi a, kamali m, taran m (2010a) ectoparasites of lessr mouse bat, myotis blyti from kermanshah, iran. asian pac j trop med. 3: 371–373. vatandoost h, telmadarraiy z, sharifi m, moradi a, kamali m, taran m (2010b) ectoparasites of lessr mouse bat, myotis blyti from kermanshah, iran. asian pac j trop med. 3: 371–373. vathsala m, mohan p, sacikumar, ramessh s (2008) survey of tick species distribution in sheep and goat in tamil nadu, india. small rumin res. 74: 238–242. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, december 2013, 7(2): 132–138 m tavassoli et al.: pcr-based detection of … http://jad.tums.ac.ir published online: august 31, 2013 original article pcr-based detection of babesia spp. infection in collected ticks from cattle in west and north-west of iran *mousa tavassoli 1, mohammad tabatabaei 2, mosleh mohammadi 1, bijan esmaeilnejad 1, hemn mohamadpour 3 1department of pathobiology, faculty of veterinary medicine, urmia university, urmia, iran 2department of pathobiology, faculty of veterinary medicine, shiraz university, shiraz, iran 3department of immunology, faculty of medical science, tarbiat modares university, tehran, iran (received 7 feb 2011; accepted 07 apr 2013) abstract background: babesiosis is a haemoparasitic disease of domestic and wild animals caused by species of the genus babesia. babesia bigemina, b. bovis and b. divergens are known to be pathogenic in cattle. the disease is transmitted during blood feeding by infected ticks and is the most economically important tick-borne disease in tropical and subtropical areas. ixodid ticks are vectors in the transmission of babesiosis. the classic presentation is a febrile syndrome with apparent anemia and hemoglobinuria. this study was carried out to determine species of bovine babesia spp. vector ticks collected from naturally occurring bovine babesiosis in west and north-west of iran. methods: two hundred and eleven ticks were collected from 113 cattle and ticks' species were identified using the standard taxonomic keys. after dna extraction from salivary glands of each tick, the presence of babesia spp. infection in ticks was examined by pcr method using primers derived from the gene encoding rhoptry protein. results: rhipicephalus sanguineus and r. bursa ticks were infected with bovine babesia spp. conclusion: rhipicephalus spp. may play a major role in the transmission of infection of bovine babesia spp. in west and north-west of iran. keywords: babesia, rhipicephalus, bovine, pcr, iran introduction babesiosis is a serious disease of cattle caused by protozoan parasites of the genus babesia. it is an important emerging tickborne disease, which causes major economic losses, and affects many domestic animals, mainly cattle and sheep, in tropical and subtropical regions (bock et al. 2004). three babesia species, namely b. bigemina, b. bovis and b. divergens are mainly the agents of bovine babesiosis (uilenberg 1995). rhipicephalus sanguineus, r. decolaratus, r. geiyi, r. annulatus, r. evertsi, r. bursa, ixodes ricinus and i. persulcatus have been implicated in the transmission of babesia spp (estrada-pena et al. 2004). the first demonstrated case of human babesiosis in the world was reported in a yugoslavia farmer, in 1957 (skrabalo et al. 1957). babesia divergens, a parasite of cattle, has been implicated as the most common agent of human babesiosis in europe, causing severe disease in splenectomized individuals. in the us, b. microti, a babesial parasite of small mammals, has been the cause of over 300 cases of human babesiosis since 1969, resulting in mild to severe disease, even in non-splenectomized patients (kjemtrup and conrad 2000). hard-bodies ticks, in particular i. dammini (i. scapularis) and i. ricinus, are the vectors of the parasite. human babesiosis is characterized by fever, chills, sweating, headache, and muscle *corresponding author: prof mousa tavassoli, email: mtavassoli2000@yahoo.com 132 j arthropod-borne dis, december 2013, 7(2): 132–138 m tavassoli et al.: pcr-based detection of … http://jad.tums.ac.ir published online: august 31, 2013 ache. additional symptoms include joint pain, nausea, vomiting, and prostration. repeating episodes of clinical disease can persist for months eventually leading to anemia, jaundice, and blood in urine (michael, 2001). a relatively recently described babesial parasite, the washington (wa1) type, has been shown to be the causative agent in seven human cases in the western us. this parasite is closely related to babesial parasites isolated from large wild ungulates in california (ca-1). like b. microti, wa1-type parasites cause mild to severe disease and the immunopathogenesis of these parasites is distinctly different from each other in experimental infections of hamsters and mice. a b. divergens-like parasite was also identified as the cause of a fatal human babesiosis case in missouri (mo-1). isolated cases of human babesiosis have been described in africa and mexico, but the causative parasites were not well-characterized (michael et al. 2001). most cases were infected by ticks carrying the rodent parasite b. microti, but other emerging. babesia spp. (currently known as wa1, ca1, and mo1) are increasingly involved. a few other cases of human babesial infection have been described in china, egypt, mexico, south africa and taiwan (michael et al. 2001). several cases were the result of blood transfusion (gorenflot et al. 1998). diagnosis of babesiosis can be traditionally achieved by microscopic examination of giemsa-stained blood smears, clinical signs and serological methods (d’oliveira et al. 1997). other diagnostic techniques, based on the detection of dna from the infective agent, such as pcr are able to simultaneously detect and differentiate the infecting organisms in a given animal (schnittger et al. 2004). this study aimed to determine the presence of the bovine babesia spp. in salivary glands of ticks' species collected from naturally infested cattle with ixodid ticks in west and north-west of iran. materials and methods field study area and animals the study was conducted in two semi-arid provinces (kurdistan and west-azerbaijan) located in western and north-western iran where bovine babesiosis is endemic. ticks were collected from 113 cattle suspected of bovine babesiosis during a period of 4 month spanning from june to september 2008. in these areas, the cattle are traditionally grazing on extensively natural pasture. collection of ticks a total of 211 ticks were collected, which were manually removed from cattle clinically suspected to bovine babesiosis. the adults and nymph ticks were collected from cattle and kept in dry plastic tubes containing few fresh grass leaves covered by a lid containing several minute holes. tubes were labeled and conditioned under room temperature for a few days, and then were dispatched to the laboratory. the purpose of this procedure was to maintain ticks alive inside the tubes until the laboratory taxonomic identification. the ticks were identified by morphologic characteristics according to the standard taxonomic keys (estradapena et al. 2004) and then transferred to 70% ethanol until further use. dna isolation from tick ticks were processed individually as described by d’oliveira et al. (1997) with some modifications. briefly, each tick was taken from the 70% ethanol, air dried on a filter paper and the scutum was removed with a microscalpel by cutting across the dorsal shield before removing the salivary glands. for each tick, a new blade and heatsterilized forceps were used. the salivary glands was placed in a 1.5ml micro-centrifuge tube, 200µl phosphate-buffer saline (pbs) was added and the sample boiled for 10min on a hot plate. sds 1% was added to 150µl of the 133 j arthropod-borne dis, december 2013, 7(2): 132–138 m tavassoli et al.: pcr-based detection of … http://jad.tums.ac.ir published online: august 31, 2013 boiled sample, which was then extracted once with phenol, ph 7.8, phenol: chloroform (1: 1) and chloroform: isoamyl alcohol (24: 1), respectively. subsequently, dna was ethanol precipitated and resuspended in 25µl 10 mm tris hcl, ph 7.5. two micro liters was used as template dna in each pcr reaction. pcr reaction for pcr amplification of the rhoptry protein spanning the 239bp amplicon, the forward and the reverse primers 5'caggatt gctttcgcaacaag3' and 5'ccttgac ataaccggcgagg3' were used (shayan et al. 2007). reaction mixture contained 12.5μl of ready to use pcr master kit (containing dntps, taq dna polymerase and mgcl2, cinagen, iran), 2μl of each primers (final concentration: 0.5 μm), 2μl of template dna extract (10 ng) and distilled water to a final volume of 25μl. the pcr amplification reactions were carried out using thermal cycler (corbett research, cp2-003, australia). the reactions were incubated at 94 °c for 5 min followed by 35 cycles of 94 °c for 45 sec, 56 °c for 45 sec and 72 °c for 45 sec. the pcr reactions were ended by a final extension at 72 °c for 10 min. the amplified pcr products were separated by electrophoresis on 1.5% agarose gel in 0.5x tbe buffer and subsequently stained with ethidium bromide and visualized under uv light using a transilluminator (bts-20m, japan). the 100bp dna ladder (fermentas, hannover, germany) was used as a size marker in all gels. the positive control for babesia was obtained from cattle with clinical babesiosis (diagnosis was done based on clinical signs and light microscopic examination giemsastained thin blood smear). venous blood sample, taken from healthy calf without contact with ticks, served as negative control in the study. results a total of 113 cattle suspected of suffering from babesiosis were investigated for the presence of tick species on their bodies. totally, 211 ticks were collected from 113 cattle. the following ticks were isolated, hyalomma anatolicum anatolicum 27.9%, h. asiaticum asiaticum 20.3%, h. anatolicum excavatum 11.8%, h. detritum 12.7%, r. sanguineus 7.5%, r. annulatus 2.8%, r. bursa 13.7%, dermacentor marginatus 3.3% and haemaphysalis punctata 0.9% (table 1). detection of t. annulata in ticks by pcr primer set p1/p2 was used in the pcr performed on tick dna samples taken from suspected cattle (shayan et al. 2007). a 239 bp fragment was generated in all samples (fig. 1). the examination of 211 ticks revealed that 8 out 16 r. sanguineus and 1 out of 29 of r. bursa ticks were infected with babesia spp. (table 1). moreover, out of 11 male and 5 female ticks of r. sanguineus 7 and 1 ticks were infected to babesia spp., respectively. the results also showed the infection in one male r. bursa tick (table 1). fig. 1. agarose gel electrophoresis of amplified dna from different ticks infected with babesia spp. lane nc: negative control, lane pc: positive control, lanes 1–3: positive samples, m: 100 bp molecular size markers (fermentas, germany) 134 j arthropod-borne dis, december 2013, 7(2): 132–138 m tavassoli et al.: pcr-based detection of … http://jad.tums.ac.ir published online: august 31, 2013 table 1. frequency of tick species on the infected cattle and percentage of infection with babesia spp. by pcr thick species tick (n-%) male (n) female (n) total infected tick (%) infected male (%) infected female (%) w* nw** h.a. anatolicum 19(32.2%) 40(67.8%) 35 24 h.a. asiaticum 24(60%) 16(40%) 19 21 h.a. excavatum 10(40%) 15(60%) 12 13 h. detritum 15(55.5%) 12(44.5%) 15 12 r. sanguineus 6(37.5%) 10(62.5%) 11 5 8(50) 7(43.7) 1(6.3) r. annulatus 0 6 0 6 r. bursa 19(65.5%) 10(34.5%) 19 10 1(3.8) 1(3.8) d. marginatus 0 7(100%) 3 4 h. punctata 0 2(100%) 0 2 total 93(44.1%) 118(55.9%) 114(54%) 97(46%) 9(4.3%) 8(3.8%) 1(0.5%) 211 *: west **: north west discussion babesiosis is one of the most important tick-borne zoonoses. human babesiosis is a malaria-like disease caused by a protozoan parasite that develops inside red blood cells (rbcs) of humans and small rodents, including voles, and shrews (senanayake et al. 2012). babesia bigemina and b. bovis are known to be pathogenic in cattle (uilenberg 2006). it is a species that causes human and cattle babesiosis (kjemtrup and conrad, 2000). in 1968, b. divergens and b. microti were identified as the cause of human babesiosis and small mammalian hosts in europe and us, respectively (senanayake et al. 2012). ixodes spp. and rhipicephalus spp. have been implicated in the transmission of human and bovine babesia spp., respectively (friedhoff 1988, uilenberg 2006). diagnosis of babesiosis can be achieved by microscopic examination of giemsa-stained blood smears and clinical signs in acute phase of the disease, but after acute infections, recovered animals frequently sustain subclinical infections, which are microscopically undetectable. they can be served as a source of infection for the potential biological vectors causing natural transmission of the disease. serological methods are not specific for any babesia spp. due to cross-reactivity with other babesia spp. (d’oliveira et al. 1997). furthermore, false positive and negative results are commonly observed in these tests. a problem discussed in protozoan infections is the determination and characterization of transmitter agent. because many analyses were previously performed with the salivary gland smears such as methyl-greenpyronin staining or feulgen staining methods, in some cases, the transfer vector remains unanswered (guglielmone et al. 1997). staining of the ticks' salivary glands can definitely confirm the babesia spp. infection of the ticks, but the main drawbacks for this method are the low sensitivity, time-consuming and the difficulty of differentiating the species involved (oliviera_sequeira et al. 2005). the use of alternative techniques, such as pcr, has become necessary to detect and identify babesia infections effectively. molecular techniques are more sensitive and specific than other traditional diagnostic methods (sparagano 1999, almeria et al. 2001). recently, dna amplification methods have 135 j arthropod-borne dis, december 2013, 7(2): 132–138 m tavassoli et al.: pcr-based detection of … http://jad.tums.ac.ir published online: august 31, 2013 been developed and used for the detection of babesia spp. (schnittger et al. 2004). information on the prevalence of tick-borne pathogens in potential vector ticks of the region is essential for the identification of tick-borne diseases. altay et al. (2008) found that r. bursa was main vector tick for cattle babesia spp. in eastern turkey (where, it is contiguous with present surveyed-areas). several pervious studies that carried out in mediterranean region showed that b. bigemina and b. bovis are transmitted by r. bursa (bouattour and darghouth 1996, ravindran et al. 2006, ghirbi et al. 2010). in addition, authors reported that bovine babesia spp. was transmitted by r. sanguineus (mahoney and mirre 1971, 1977). the results are in agreement with our finding. we found rhipicephalus spp. as major vectors for bovine babesiosis in west and north-west of iran. according to reports by morisod et al. (1972), oliviera et al. (2005), oliviera-sequeria et al. (2005), boophilus microplus and i. ricinus are major vectors of cattle babesia spp. however, we did not determine b. microplus and i. ricinus as vectors. this may be due to geographical disparity between two surveyedregions. babesia microplus has not been found during tick sampling in present study and rhipicephalus spp. was better adapted to climate of current investigated-areas. the present study showed that rhipicephalus spp. might play a major role in the transmission of bovine babesia infection. there are records that hyalomma anatolicum anatolicum, h. a. asiaticum, h. a. excavatum, h. detritum, dermacentor marginatus and haemaphysalis punctata can be the agent for crimean-congo hemorrhagic fever virus (cchf) and tickborne encephalitis virus (tbe) (estrada-pena and jongejan 1999). therefore, we suggest molecular-based diagnostic method can be employed to determine the rate of cchf and tbe virus infection in humans and intermediate host tick collected from naturally infested 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et al.: efficacy of … 156 original article efficacy of different sampling methods of sand flies (diptera: psychodidae) in endemic focus of cutaneous leishmaniasis in kashan district, isfahan province, iran marzieh hesam-mohammadi 1, *yavar rassi 1, *mohammad reza abai 1, amir ahmad akhavan 1, fatemeh karimi 1, sina rafizadeh 2, alireza sanei-dehkordi 1, maryam sharafkhah 3 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2ministry of health and medical education, tehran, iran 3department of epidemiology and biostatistics, tehran university of medical sciences, tehran, iran (received 4 mar 2013; accepted 2 oct 2013) abstract background: the aim of the study was to evaluate and compare the efficiency and practicality of seven trapping methods for adult phlebotominae sand flies. the results of this investigation provide information to determine the species composition and nocturnal activity pattern of different sand fly species. methods: the study was carried out in both plain region (about 5km far from northeast) and mountainous region (about 40km far from southwest of kashan city). seven traps were selected as sampling methods and sand flies were collected during 5 interval times starting july to september 2011 and from 8:00pm to 6:00am in outdoors habitats. the traps include: sticky traps (4 papers for 2 hours), disney trap, malaise, cdc and co2 light traps, shannon traps (black and white nets) and animal-baited trap. results: a total of 1445 sand flies belonging to 15 species of phlebotomus spp. and five of sergentomyia spp. were collected. females and males comprised 44.91% and 55.09% of catches, respectively. of the collected specimens, se. sintoni was found to be the most prevalent (37.86%) species, while ph. papatasi, accounted for 31.76% of the sand flies. conclusion: disney trap and sticky traps exhibited the most productivity than other traps. in addition, in terms of the efficiency of sampling method, these two trapping methods appeared to be the most productive for both estimating the number of sand flies and the species composition in the study area. keywords: psychodidae, sand flies, nocturnal activity, trapping, sampling methods, iran introduction phlebotomine sandflies (diptera: psychodidae) are widespread in the tropics and subtropics, and they transmit leishmania, protozoan parasites which cause visceral leishmaniasis (vl) and various forms of cutaneous leishmaniasis (cl) infecting more than 350 million people in more than 80 countries. they have a wide distribution, though mainly in the tropics and subtropics (lane 1993). approximately 700 species of phlebotomine sand flies distributed among 6 genera but only two of them, phlebotomus in the old world and lutzomyia in the new world are introduced medically importance (lewis 1982, lane 1987, lane 1993, sharma and singh 2008), and only 10% of them act as the disease vector. further, about 30 species of these are important from public health standpoint (who 1990, desjeux 2000, sharma and singh 2008). the blood-feeding females of phlebotomine sand flies (diptera: psychodidae, phlebotomine) include natural vectors of protozoa of the genus leishmania (kinetoplastida: trypanosomatidae), which are the parasitic causative agents of mammalian leishmaniasis (killick-kendrick 1990). *corresponding author: prof yavar rassi, e-mail: rassiy@sina.tums.ac.ir, mr mohammad reza abai, e-mail: abaimr@sina.tums.ac.ir http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 156–162 m hesam-mohammadi et al.: efficacy of … 157 cutaneous leishmaniasis are reported from more than 50% of the 31 provinces in iran (yaghoobi-ershadi et al. 1999, rassi et al. 2004, rassi et al. 2006, rassi et al. 2007, rassi et al 2007, rassi et al. 2011). in the middle east, phlebotomine sand fly populations occasionally occur in great numbers, resulting in biting pressure of 1,000 bites per person per night (coleman et al. 2006). an extraordinarily high number compared with cdc light traps. although the impact on adult sand fly biting activity was not determined (blow et al. 2007), these preliminary results suggested that commercial mosquito traps might be good candidates for inclusion into integrated sand fly control or suppression programs in desert settings (hoel et al. 2000). in this study, sampling of sand flies were carried with disney trap, malaise, sticky traps, cdc light trap, co2 light trap, shannon traps and animal-baited trap. the purpose of the current study was to assess which of these traps could catch a larger number or show the most diversity of sand flies in the interval times. some of them are currently used in routine sand fly surveillance programs (alexander 2000). previous studies aiming at determining the species composition and distribution patterns of sand flies in kashan revealed eleven phlebotomus spp. and six sergentomyia spp. some ideas for precise sampling in order to determine species composition, nocturnal activity and biodiversity of different sand fly was species in cl focus at different topographic condition e.g. mountain/plain. furthermore the effects of some environmental factors such as temperature, relative humidity and height above sea level were studied in relation to fluctuation considered in pattern of nocturnal activity as well as sand fly biodiversity. materials and methods study area the study was carried out in both plain region about 5km far from northeast and mountainous region about 40 km far from southwest of kashan district, isfahan province, in central iran (fig. 1). the geographical coordinates is 51º 29' 54.6" e and 33º 58' 52.9" n for plain region and 51º 13' 54.6" e and 33º 58' 52.9" n mountainous region with at an altitude of 951m and 1823m respectively. the climate is hot and dry in summer and cold during the winter. the maximum and minimum temperatures were 38 °c and 19 °c in plain region and were 45 °c and 15 °c in mountainous region during the study period. sand fly sampling seven traps were employed for sampling of sand flies. the sampling was repeated three times during the peak activity of sand flies in both plain and mountainous regions. turning of sand flies sampling were 5 times per night starting 8:00pm and ending 6:00 am at outdoors using sticky traps (4 papers for 2 hours, totally 20 papers per day), disney trap, malaise, cdc light trap, co2 light trap (0.5kg dry ice), shannon traps (both black and white nets) and animal-baited trap (fig. 2). the study was extended from july to september 2011. the traps were set up at least 15m from each other. sticky traps were changed every two hours and as the traps were changed, the new ones were replaced in the same location. other collection methods were also performed over the same 10 hours period using mouth aspirator to collect the sand flies. all sand flies at these traps were captured during 10–15min in every two hours. collected sand flies for sticky traps (20×30cm papers coated with castor oil) and disney trap were removed from sticky papers using entomological needles or fine brushes, preserved in 70% ethanol, and kept in microtubes before identification. at other traps, sand flies were also collected at interval of 2 hours by aspirator starting 8:00 pm ending 6:00 am. the sand http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 156–162 m hesam-mohammadi et al.: efficacy of … 158 flies were anesthetized with chloroform and transferred to microtube containing 70% ethanol. then specimens transported to laboratory research center for mounting and identification. mounting and morphological identification after recording the sampling data and locations, some sand fly specimens which collected with sticky traps were washed in acetone and rest of them transferred to 70% ethanol. at appropriate time, the sand flies were mounted in puri’s medium. all the identifications were carried out based on their genus and then species. the identification keys of theodor and mesghali 1964, lewis 1982, rassi et al. 2006 and absavaran et al. 2009 were used. the permanent mounted, labelled and identified microscope slides were deposited in medical entomology and zoology museums, school of public health, tums under code no. gc22st5-92. recording climatic conditions to determine the relationship between temperature and relative humidity (rh) and the abundance or activity of sand flies in both study regions (plain and mountain) associated with traps some meteorological data were recorded. the temperature and relative humidity were recorded every two hours during the night. statistical analysis data were analyzed using spss 20. in order to ensure normality and homogeneity of variances before subjecting to statistical analysis, data on the number of flies collected were square-root transformed. the significance difference of effect of the traps and time was analyzed in relation to sand flies species caught in two regions using univariate analysis of variance (anova) and chi-square tests. total sand fly species caught constituted the dependent variable, while sex and traps and time were chosen as the fixed factor. basic correlation matrices were used to determine the existence of a correlation between the abundance of the sand flies, average hourly temperature and relative humidity in the study area. some of graphs were prepared using microsoft excel 2010. results a total of 1445 sand flies belonging to 15 species of the genus phlebotomus and 5 of the genus sergentomyia were collected. females and males comprised 44.91% and 55.09% of catches during the study period respectively. of the collected total, s. sintoni was found to be the most prevalent (37.86 %) species while ph. papatasi the proven vector of l. major, l. turanica and l. gerbili in isfahan accounted for 31.76% of the sand flies that were identified. other species were s.(ser.) dentata (0.3%), s.(ser.) antennata (0.2%), s.(ran.) pawlowskyi (0.3%), s.(par.) palestinensis (0.1%), ph.(syn.) ansarii (0.1%), ph.(para.) kazeruni (1.7%), ph.(par.) caucasicus group (0.4%), ph.(par.) alexandri (1.1%), ph.(lar.) wenyoni (0.6%), ph.(lar.) tobbi (1%), ph.(lar.) major (3.5%), ph.(lar.) keshishiani (0.2%), ph.(lar.) kandelaki (0.1%), ph.(adl.) longiductus (0.1%), ph.(adl.) halepensis (0.1%), ph.(adl.) chinensis group (0.1%) and ph.(adl.) brevis (0.3%). during the study period, the population of sand flies was found to be the lowest in cdc light trap and co2 light trap. population size was raised with two highest peaks in disney trap and sticky trap at 22:00–24:00 pm. the average monthly temperature and relative humidity values ranged between 21.83– 28.33 °c and 24.83–36.33% respectively. during the night, when the maximum number of sand flies (29.25%) was collected at 22:00– 24:00 pm, the average temperature and rh were found to be 26.92 °c and 26.5%, respectively, while the minimum number of sand flies was sampled at 02:00–04:00 am (11.62 %) with an average temperature of 23.08 °c and 33.5% relative humidity (fig. 3). http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 156–162 m hesam-mohammadi et al.: efficacy of … 159 the number of sand flies collected using different types of traps during the study period is presented in table 1. almost all sand fly species were commonly caught with disney trap and sticky traps. approximately 37.3% of the total collection was made up using disney trap. 24.2% and 10.5% of sand flies were collected using sticky traps and shannon trap (black net), respectively. collections using animal baited trap and shannon trap (white net) constituted 8.5% and 7.3% of the total collection. neither co2 light trap (5.0%) nor cdc light trap (3.3%) and malaise trap (0.1%) showed a high efficiency during the study. a significant interaction was revealed between the collection methods and the number of individuals collected using of univariate analysis of variance (anova) (p< 0.05), and there was significant interaction between the trapping methods and the proportion of males and females collected (p< 0.05). when significant effects of traps were established (p< 0.05), differences among traps were exposed that co2 and standard cdc light traps, shannon trap (black and white net) and animal baited trap displayed some similar efficiency, whereas sticky papers and disney trap differed from the others (p< 0.05). results with respect to the nocturnal activity indicated that even though the number sand flies declined rapidly between 02:00 and 04:00h. there was no significant difference between hourly pattern either in the species prevalence or in the activity of the species in different traps (fig. 4). fig. 1. kashan district, isfahan province, central iran fig. 2. different methods used for sand fly sampling fig. 3. correlation of two environmental conditions with nocturnal activity of sand flies fig. 4. correlation between collection methods and density of abundance sand flies species in the study area in kashan city http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 156–162 m hesam-mohammadi et al.: efficacy of … 160 table 1. prevalent sand flies collected by different traps, kashan, isfahan province, 2011 species traps total baited trap cdc co2 light trap disney trap black shannon trap white shannon trap sticky trap ph.(lar.) major 19 3 0 12 2 4 9 49 ph.(phl.) papatasi 14 18 56 208 38 22 86 442 ph.(par.) sergenti 21 4 2 114 73 22 36 272 s.(ser.) sintoni 45 17 14 179 31 46 207 539 total 99 42 72 513 144 94 338 1302 discussion this study is the first detailed research in terms of species composition, density and nocturnal activity of sand flies using different methods of capturing in an endemic focus of cutaneous leishmaniasis in kashan district, isfahan province, iran. trapping over the summer season during the peak of sand flies activity revealed that s. sintoni (37.9 %), p. papatasi (31.8%) and p. sergenti (20.1%) are the most abundant and prevalent species in these regions respectively (doroudgar et al. 1999). both the previous and recent findings showed that p. papatasi the main vector of l. major, l. turanica and l. gerbili in isfahan province, p. sergenti the proven vector of l. tropica and l. gerbili in this region, also s. sintoni were represented with high populations in the study areas (seyedi rashti and nadim 1992, doroudgar et al. 1999, yaghoobi-ershadi 2012). although sand fly abundance was not strongly and significantly influenced by the variations in average monthly temperature and relative humidity during the six months of survey in cukurova plain, south anatolia, turkey, the maximum number of sand flies was recorded in the hottest and driest seasons between june and september when the average temperature was comparatively high, the relative humidity was low and the rainy days ranged between 1.5 to 3.5 days. (kasap et al. 2009). the role of climatic factors on the seasonal distribution of sand flies in the arid areas of india exhibited that the majority of species preferred comparatively higher temperatures and low rh%, a prerequisite for survival in arid and semi-arid conditions (singh 1990). our study results are concordance with the other survey in relation to effects of temperature and relative humidity on abundance of sand flies. it seems that special range of both high temperature and low relative humidity caused the high abundance of sand fly species in our study regions. the results showed that before midnight (between 22:00–24:00 pm) sand flies were very active. although cdc and co2 light traps are used extensively in the field studies of sand flies (alexander 2000), in cukurova plain in turkey sticky traps have no known attractiveness and have generally been used for determining species composition of an area as they randomly sample the species where they are set (kasap et al. 2009), whereas our findings showed that disney trap and sticky traps have been attractiveness more than other traps. our findings in terms of the efficiency of the sampling methods, showed these two trapping methods appeared to be the most productive for both estimating the number of sand flies and the species composition in the study area, in agreement with some previous studies. it seems that the sticky traps provide more realistic results than cdc light traps that could attract additional phototropic sand flies. the effective range of http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 156–162 m hesam-mohammadi et al.: efficacy of … 161 cdc light traps was less than 5m (wheeler et al. 1996). study in northern italy showed that when compared to sticky traps, co2 traps were more effective in collecting sand flies and addition of a light source improved the catches (veronesi et al. 2007). the nocturnal activity patterns of sand flies have been reported for old world species (roberts 1994, guernaoui et al. 2006, coleman et al. 2007). according to our results, sand flies activity was not significantly different over time even though total counts decreased between 04.00–06.00h during late summer. no significant difference was found in the species prevalence throughout the nights. it seems to be the most important factor affecting sand fly nocturnal activity in similar studies was low humidity, followed by low wind velocity and high temperature (roberts 1994, guernaoui et al. 2006, kasap et al. 2009). conclusion disney trap and sticky traps exhibited the most productivity than other traps. therefore, more detailed studies with respect to the seasonal variations and the effects of abiotic conditions, other than temperature and relative humidity, such as cloud cover, wind velocity and lunar cycle on the nocturnal activity of sand flies may improve knowledge of the behavior of sand flies for important epidemiologically surveys. acknowledgements this study was funded and supported by tehran university of medical sciences (tums), grant no. 13967. the authors declare that they have no conflicts of interest. references alexander b (2000) sampling methods for phlebotomine sandflies. med vet entomol 14: 109–122. absavaran a, rassi y, parvizi p, oshaghi ma, abaie mr, rafizadeh s, mohebali m, zarea z, javadian e (2009) identification of sand flies of the subgenus larroussius based on molecular and morphological characters in north western iran. iran j arthropod-borne dis. 3(2): 22–35. coleman re, burkett da, sherwood v, caci j, spradling s, jennings bt, rowton e, gilmore w, blount k, white ce, putnam jl (2007) impact of phlebotomine sandflies on us military operations at tallil air base, iraq: 2. temporal and geographic distributions of sandfies. j med entomol. 44: 29–41. desjeux p (2000) leishmania/hiv coinfection, south-western europe, 1990–1998 geneva, switzerland: who, pp.1–12. doroudgar a, seyedi-rashti 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sticky-traps and cdc light-traps for sampling phlebotomine sandflies entering houses in venezuela. med vet entomol. 10: 295–229. who (2010) control of the leishmaniasis: report of a meeting of the who expert committee on the control of leishmaniases control. technical report series, no. 949. world health organization, geneva, switzerland, pp. 32–138. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod-borne dis. 6(1): 1–17. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 90 original article evaluation of essential oil and its three main active ingredients of chinese chenopodium ambrosioides (family: chenopodiaceae) against blattella germanica wei xiang zhu, kun zhao, sha sha chu, *zhi long liu department of entomology, china agricultural university, beijing, china (received 9 oct 2010; accepted 18 sep 2011) abstract background: the efficacy of essential oil of chenopodium ambrosioides flowering aerial parts and its three main active ingredients was evaluated against blattella germanica male adults. methods: composition of essential oil was determined by gc-ms. topical application bioassay was used to evaluate contact toxicity of essential oil and three main components. fumigant toxicity of essential oil and its main components was measured using a sealed space method. results: twenty-two components were identified in the essential oil and the main components were (z)-ascaridole (29.7%), isoascaridole (13.0%), ρ-cymene (12.7%) and piperitone (5.0%). the essential oil and (z)-ascaridole, isoascaridole and -cymene possessed fumigant toxicity against male german cockroaches with lc50 values of 4.13, 0.55, 2.07 and 6.92 mg/l air, respectively. topical application bioassay showed that all the three compounds were toxic to male german cockroaches and (z)-ascaridole was the strongest with a ld50 value of 22.02 g/adult while the crude oil with a ld50 value of 67.46 g/adult. conclusion: the essential oil from chinese c. ambrosioides and its three main active ingredients may be explored as natural potential insecticides in the control of cockroaches. keywords: blattella germanica, chenopodium ambrosioides, essential oil, fumigant, contact toxicity introduction german cockroach, blattella germanica (l) is an important pest of homes, restaurants, and commercial food processing facilities worldwide. they are a major public health concern in hospitals, kitchens, and food manufacturing plants because they are able to carry a variety of bacteria and other pathogenic organisms. they are the mechanical vectors to a few pathogens that can cause disease such as food poisoning, typhoid, pneumonia and asthma (brenner 1995). body parts, cast skins, and feces of cockroaches are human allergens, 2nd in importance only to the house dust mites. currently, control of cockroach pop ulations primarily depends on continued applications of residual insecticides, such as propoxur, acephate, dimethyl 2, 2-dichlorovinyl phosphate (dichlorvos, ddvp), and pyrethroids and stomach poisons, such as hydramethylnon and sulfluramid. however, the repeated application of these insecticides may possess undesirable side effects, such as the disruption of natural biological control systems, and the development of resistance (chang and ahn 2001). there are also serious concerns about human health. these problems have highlighted the need for the development of new types of selective cockroach-control alternatives. there has been growing interest in the *corresponding author: dr zhi long liu, e-mail: zhilongliu@cau.edu.cn j arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 91 use of plant oils for protection of agricultural products and control of public health insects because they are often of low mammalian toxicity, readily biodegradable and pose low danger to the environment if used in small amounts (rajendran and srianjini, 2008). during our screening program for new agrochemicals from chinese medicinal herbs, chenopodium ambrosioides l (family: chenopodiaceae) was found to possess strong insecticidal activity against german cockroaches (fig. 1). chenopodium ambrosioides is an aromatic herb that grows in central and south america and now distributed throughout the tropical parts of the world (duke et al. 2002). it has also been employed by empirical herbalists and healers against intestinal parasites (especially small tapeworms and round worms) throughout latin america, as well as in the west indies (quinlan et al. 2002). the plant is also distributed in the southern provinces of china. the aerial parts of this plant have been used as condiment, traditional purgative for intestinal worms and acesodyne and in the chinese traditional medicine. this herb can expel wind, treat rheumatism (jiangsu new medical college 1977). essential oil of c. ambrosioides has been shown to possess insecticidal and repellent activities against several stored product insects (su 1991, tapondjou et al. 2002) and medical important insect pests (toloza et al. 2006, gillij et al. 2008). in the present study, insecticidal activity c. ambrosioides essential oil and three main active ingredients against the german cockroaches were investigated. materials and methods test insects male adults (5–10 days old) were collected from a synchronously reared laboratory colony of insecticide-susceptible german cockroaches. cockroaches were supplied ad libitum with purina no 5012 rat chow (laboratory animal centre, chinese academy of medicinal sciences, beijing 100021), and water was provided in glass tubes with cotton stoppers. all colonies were kept in plastic tanks at room temperature. plants fresh aerial parts (15 kg of leaves, stems and flowers) of c. ambrosioides were harvested in august 2008 from fuzhou (26.08 north latitude and 119.28 east longitude), fujian province (fuzhou 350013), pr china. the aerial parts were air-dried for one week and ground to a powder. the species was identified and the voucher specimens (cmh-tujingjie-fujian-2008-08) were deposited at the department of entomology, china agricultural university, beijing 100094. extraction of essential oil the ground powder of c. ambrosioides aerial parts was subjected to hydrodistillation using a modified clevenger-type apparatus (xwd-c-1000, shanghai xinwangde laboratory equipment co, china) for 6 h and extracted with n-hexane. anhydrous sodium sulphate was used to remove water after extraction. essential oil was stored in an airtight container in a refrigerator at 4 c. gas chromatography and mass spectrometry gas chromatographic analysis was performed on the agilent 6890n while the essential oil was identified on a mass spectrometer agilent technologies 5973n. they were equipped with a flame ionization detector and capillary column with hp-5ms (30m× 0.25mm× 0.25μm). the gc settings were as follows: the initial oven temperature was held at 60 c for 1 min and ramped at 10 c min−1 to 180 c for 1 min, and then ramped at 20 c min−1 to 280 c for 15 min. the injector temperature was maintained at 270 c. the samples (1 μl) were injected neat, with a split ratio of 1: 10. the carrier j arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 92 gas was helium at flow rate of 1.0 ml min−1. spectra were scanned from 20 to 550 m/z at 2 scans s-1. most constituents were identified by gas chromatography by comparison of their retention indices with those of the literature or with those of authentic compounds available in our laboratories. the retention indices were determined in relation to a homologous series of n-alkanes (c8–c24) under the same operating conditions. further identification was made by comparison of their mass spectra on both columns with those stored in nist 05 and wiley 275 libraries or with mass spectra from literature (adams 2001). component relative percentages were calculated based on gc peak areas without using correction factors. (z)-ascaridole and isoascaridole were isolated by using bioassay-directed fractionation on repeated silica column from the essential oil of c. ambrosioides and confirmed by ms, 1h-nmr and 13c-hnm data. -cymene (98%) was purchased from aladdin reagent (china) co., ltd, shanghai 201206, prchina. positive control, pyrethrum extract (25% pyrethrine i and pyrethrine ii) was purchased from fluka chemie. fumigant toxicity a serial dilution of c. ambrosioides essential oil (20–1.3%, 5 concentrations) and pure compounds (2.5–0.3% for (z)-ascaridole, 20–0.6% for another two compounds, 5 concentrations) was prepared in n-hexane. a whatman filter paper (diameter 6.0 cm) were each impregnated with 50 μl dilution, and then placed on the underside of the screw cap of a glass vial (diameter 7.2 cm, height 19.0 cm, volume 750 ml). the solvent was allowed to evaporate for 30 s before the cap was placed tightly on the glass vial, each of which contained 10 male cockroaches inside to form a sealed chamber. fluon (ici america inc) was used inside glass vial to prevent insects from contacting the treated filter paper. preliminary experiments demonstrated that 30 s were sufficient for the evaporation of solvents. nhexane was used as controls. five replicates were carried out for all treatments and controls, and they were incubated at 24–26 c, 75% rh, 12:12 ld photoperiod for 24 h and then mortality was recorded. mortality was defined as inability to move when placed on the dorsal side and inability to respond to prodding. results from all replicates were subjected to probit analysis using the priprobit program v1.6.3 to determine lc50 values (sakuma 1998). topical application bioassay groups of ten adult male cockroaches were anaesthetized with carbon dioxide for 15 seconds before treatment. a serial dilution of the essential oil (7.0–1.3%, 5 concentrations) and pure compounds (10–0.6%, 6 concentrations) was prepared in acetone. aliquots of 2 µ l of the solution were dispensed from an arnold automatic micro-applicator (burkard, ricksmanworth, england) and applied to the dorsal thorax of individual insects. controls were determined using acetone. both treated and control cockroaches were then transferred to glass vials (10 insects/ vial) and kept in incubators (24–26 c, 75% rh, 12:12 ld photoperiod). mortality of cockroaches was observed at 24 h post-treatment. five replicates were carried out for all treatments and controls. results from all replicates were subjected to probit analysis using the priprobit program v1.6.3 to determine ld50 values (sakuma 1998). results the results of gc-ms of c. ambrosioides essential oil are presented in table 1. a total of 22 active ingredients were identified in the essential oil, accounting for 88.6% of the total oil (table 1). the main components were (z)-ascaridole (29.7%), isoascaridole (13.0%), and ρ-cymene (12.7%) followed by piperitone (5.0%), isothymol (4.9%), and 3, 4-epoxy-menthan-2-one (4.1%). j arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 93 (z)-ascaridole, isoascaridole and -cymene possessed fumigant toxicity against male german cockroaches with lc50 values of 0.55, 2.07 and 6.92 mg/l air, respectively while the crude essential oil with a lc50 value of 4.13 mg/l air (table 2). (z)-ascaridole also showed the strongest contact toxicity to german cockroaches with a ld50 value of 22.02 g/adult (based on the ld50 values, with no overlap in 95% fiducial limits) by using topical application bioassay while the crude essential oil with a ld50 value of 64.47 g/adult (table 3). isoascaridole and -cymene also had weak contact toxicity against german cockroaches with ld50 values of 96.28 and 119.90 g/adult, respectively. table 1. chemical constituents of essential oil derived from chenopodium ambrosioides compound ri* chemical formula relative area (%) -pinene 931 c10 h16 1.3 -pinene 981 c10 h16 0.3 δ-4-carene 1002 c10 h16 1.9 -terpinene 1017 c10 h16 1.1 -cymene 1024 c10 h14 12.7 ,α-dimenthylstyrene 1118 c10 h12 0.7 trans--mentha-2,8-dienol 1126 c11 h18 o2 0.6 trans--2,8-menthadien-1-ol 1139 c10 h16 o 0.6 2-ethylcyclohexanone 1158 c8 h14 o 0.9 ,-4-trimethylbenzyl alcohol 1182 c10 h14 o 2.8 cis-piperitol 1196 c10 h16 0.5 (z)-ascaridole 1245 c10 h16 o2 29.7 piperitone 1250 c10 h16 5.0 3,4-epoxy--menthan-2-one 1276 c10 h16 o2 4.1 thymol 1292 c10 h14 o 1.1 carvacrol 1298 c10 h14 o 4.9 isoascaridole 1295 c10 h16 o2 13.0 precocene ii 1368 c13 h16 o3 1.5 caryophyllene oxide 1584 c15h24o 2.2 geranyl tiglate 1700 c15 h24 0.8 hexahydrofarnesyl acetone 1842 c18 h36 o 1.7 phytol 2119 c20 h40 o 1.2 total 88.6 *ri, retention index as determined on a hp-5ms column using the homologous series of n-hydrocarbons; table 2. fumigant toxicity of essential oil and components from chenopodium ambrosioides against male cockroach adults compounds lc50 (mg/l air) 95% fiducial limits slope±se chi square (χ 2 ) ascaridole 0.55 0.47–0.63 7.25±0.69 10.15 -cymene 6.92 6.11–7.85 7.06±0.45 8.23 isoascaridole 2.07 1.78–2.43 6.17±0.49 9.46 crude oil 4.13 3.62–4.74 5.06±0.40 11.21 ddvp 0.01* *data from jang et al. (2005) j arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 94 table 3. contact toxicity of essential oil and components from chenopodium ambrosioides against male cockroach adults compounds ld50 (g/adult) 95% fiducial limits slope±se chi square (χ 2 ) ascaridole 22.02 19.92–24.43 6.12±0.73 9.48 -cymene 119.90 102.13–143.51 5.23±0.47 13.40 isoascaridole 96.28 78.04–117.93 3.44±0.35 14.67 crude oil 64.47 59.21–70.46 4.09±0.43 7.40 pyrethrum extract 1.70 1.16–3.78 4.23±0.47 6.80 fig. 1. flowering chenopodium ambrosioides (orginal) rt: 0.00 28.05 sm: 7g 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 tim e (m in) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 r el at iv e a bu nd an ce 7.85 8.764.63 15.26 17.69 13.44 26.7024.2810.42 18.93 nl: 5.77e7 tic ms 3-03 fig. 2. gc-mc graph of chenopodium ambrosioides essential oil profiles j arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 95 discussion among the three isolated compounds, (z)-ascaridole was proved to be the most active (fumigant) compound because it showed 7 times more toxic to the german cockroaches compared with the crude essential oil (table 2). isoascaridole also demonstrated stronger fumigant toxicity against german cockroaches than the crude essential oil (based on the lc50 values, with no overlap in 95% fiducial limits). however, ρ-cymene had less toxic to german cockroaches than the crude essential oil. in the previous report (jang et al. 2005), 41 naturally occurring monoterpenoids was evaluated for fumigant toxicity against german cockroaches and verbenone was most toxic to the german cockroaches with a ld50 value of 11.5 mg/l air. in the present study, (z)ascaridole was 20 times more toxic to the german cockroaches compared to verbenone. however, fumigant toxicity bioassay demonstrated that all the three isolated compounds and the crude essential oil were less toxic to the cockroaches compared to the commercial insecticide, ddvp because of ddvp with a lc50 value of 0.007 mg/l air. the three isolated compounds exhibited contact toxicity against the german cockroaches (table 3). however, compared with the control (pyrethrum extracts, ld50= 1.70 μg/adult), all the three compounds and the essential oil showed less toxic to the german cockroaches in the topical application bioassay. the above findings suggested that the c. ambrosioides essential oil and the three components especially (z)-ascaridole may possess potential to be developed as novel natural insecticides, especially fumigants in the control of cockroaches. in the previous studies, several essential oils have been evaluated and demonstrated to possess insecticidal, antiffedant, and repellent activities against cockroaches, such as mint oil (appel et al. 2001), catnip essential oil (peterson et al. 2002), majoram oil (jang et al. 2005), garlic and thyme oil (tunaz et al. 2009), citrus oils (yoon et al. 2009), and essential oils derived from star anise illicium verum (chang and ahn 2001), american peppertree schinus molle (ferrero et al. 2007), and nutmeg myristic1a fragrans (jung et al. 2007). moreover, naturally occurring monoterpenoids, components of essential oils were also evaluated for insecticidal and repellent activities against cockroaches (ngoh et al. 1998, jang et al. 2005) and cineole, l-fenchone, limonene, linalool, menthone, pulegone, and thujone at 50 g/ml air (14 h exposure) caused 100% mortality of male adult german cockroaches (lee et al. 2003). the aerial parts of this plant have been used as traditional purgative for intestinal worms in chinese medicine (jiangsu new medical college 1977). however, the essential oil of c. ambrosioides is an irritant to the mucous membrane of the gastrointestinal tract, kidney and liver (gadano et al. 2006). overdoses of this oil have caused death in men and rats (monzote et al. 2006). intake of 10 mg/kg of the oil has been known to cause cardiac disturbances, convulsions, respiratory disturbances, sleepiness, vomiting and weakness and even death. moreover, ascaridole is toxic and has a pungent, not very pleasant flavor; in pure form, it is an explosive sensitive to shock (potawale et al. 2008). for the practical use of ascaridole and the crude essential oil as novel natural fumigants/ insecticides, further studies are necessary on the safety of these materials to human, and on the development of formulations to improve efficacy and stability, and to cut cost as well. the main constituents of the essential oil were (z)-ascaridole, isoascaridole, ρ-cymene and piperitone (table1). however, there were great variations in chemical composition of the essential oils of c. ambrosioides. for example, α-terpinyl acetate (73.9%) and ρ-cymene are major constituents of c. amj arthropod-borne dis, 2012, 6(2): 90–97 wx zhu et al.: evaluation of essential oil … 96 brosioides essential oil from mexico and content of ascaridole is only 2% (pino et al. 2003). in another report, limonene (32.5%), transpinocarveol (26.7%) and geranial (5.0%) were main components of c. ambrosioides essential oil from mexico (sagrero-nieves and bartley, 1995). the main components of c. ambrosioides essential oil derived from brazil were (z)-ascaridole (61.4%) and (e)-ascaridole (18.6%) (jardim et al. 2008). however, the c. ambrosioides essential oil from nigeria contained α-terpinene (56.0%), α-terpinyl acetate (15.7%) and ρ-cymene (15.5%) and no ascaridole was detected in the oil (muhayimana et al. 1998). the essential oil from india contained α-terpinene (47.4%), ρ-cymene (25.8%) and ascaridole (14.8%) (singh et al. 2008). however, (z)-ascaridole (29.7%), isoascaridole (13.0%), and ρ-cymene (12.7%) are the three main components of the essential oil from the chinese c. ambrosioides (table 1). for the practical use of the crude essential oil of chinese c. ambrosioides as a new natural insecticide, standardization of the essential oil is needed. acknowledgments 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(anoplura: pediculidae) from argentina. j med entomol. 43: 889–895. tunaz h, er mk, isikber aa (2009) fumigant toxicity of plant essential oils and selected monoterpenoid components against the adult german cockroach, blattella germanica (l.) (dictyoptera: blattellidae). turk j agric for. 33: 211–217. yoon c, kang sh, yang jo, noh dj, indiragandhi p, kim gh (2009) repellent activity of citrus oils against the cockroaches blattella germanica, periplaneta americana and p. fuliginosa. j pestic sci. 34: 77–88. j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 163 original article fauna and larval habitats of mosquitoes (diptera: culicidae) of west azerbaijan province, northwestern iran farahnaz khoshdel-nezamiha 1,2, hassan vatandoost 3 , shahyad azari-hamidian 4, mulood mohammadi bavani 3, farrokh dabiri 2, rasool entezar-mahdi 5, *ali reza chavshin 1,2 1research center of social determinants of health, urmia university of medical sciences, urmia, iran 2department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4school of health, guilan university of medical sciences, rasht, iran 5director of health deputy of west azerbaijan, urmia university of medical sciences, urmia, iran (received 23 june 2013; accepted 26 oct 2013) abstract background: several important diseases are transmitted by mosquitoes. despite of the potential of the occurrence of some mosquito-borne diseases such as west nile, dirofilariasis and malaria in the region, there is no recent study of mosquitoes in west azerbaijan province. the aim of this investigation was to study the fauna, composition and distribution of mosquitoes and the characteristics of their larval habitats in this province. methods: larvae and adult collections were carried out from different habitats using the standard methods in twenty five localities of seven counties across west azerbaijan province. results: overall, 1569 mosquitoes including 1336 larvae and 233 adults were collected from 25 localities. the details of geographical properties were recorded. five genera along with 12 species were collected and identified including: anopheles claviger, an. maculipennis s.l., an. superpictus, culex pipiens, cx. theileri, cx. modestus, cx. hortensis, cx. mimeticus, culiseta longiareolata, ochlerotatus caspius s.l., oc. geniculatus and uranotaenia unguiculata. this is the first record of oc. geniculatus in the province. conclusion: due to the geographical location of the west azerbaijan province, it comprises different climatic condition which provides suitable environment for the establishment of various species of mosquitoes. the solidarity geographical, cultural and territorial exchanges complicate the situation of the province and its vectors as a threat for future and probable epidemics of mosquito-borne diseases. keywords: culicidae, arboviruses, larval habitat, mosquito-borne diseases, fauna introduction the family culicidae (diptera) comprises at least 3531 species representing 111 genera divided into two subfamilies, anophelinae and culicinae (harbach 2013). several important diseases are transmitted by them, among which some of mosquito-borne viruses such as west nile and sindbis have been reported from iran (naficy and saidi 1970, saidi et al. 1976). chinikar et al. (2010) reported a case of dengue fever in tehran, imported from malaysia moreover the possibility of the outbreak of some other mosquito-borne arboviruses such as japanese encephalitis and rift valley fever in the who eastern mediterranean region, including iran, should be considered (who 2004). other mosquito-borne diseases such as dirofilariasis (azari-hamidian et al. 2009) and many other mosquito-borne diseases are endemic in some parts of iran, including west azerbaijan province. *corresponding author: dr ali reza chavshin, e-mail: chavshin@umsu.ac.ir http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 164 several studies have been conducted to identify the fauna of mosquitoes across iran (zaim 1987, vatandoost et al. 2004, ghavami and ladonni 2005, vatandoost et al. 2006, abai et al. 2007, azari-hamidian 2007b, moosa-kazemi et al. 2009, azari-hamidian et al. 2010, azarihamidian 2011, oshaghi et al. 2011, hanafi-bojd et al. 2012, khoobdel et al. 2012, saghafipour et al. 2012, banafshi et al. 2013, soleimani-ahmadi et al. 2013). according to the most updated checklist of iranian mosquitoes, 64 species representing seven genera occur in the country (azarihamidian 2007a). in addition to morphological studies, molecular studies have been carried out in order to clarify some problematic situations (sedaghat et al. 2003, oshaghi et al. 2007, naddaf et al. 2012). oshaghi et al. (2008) recently identified anopheles superpictus grassi as a complex of three genotypes (x, y, and z). naddaf et al. (2010) reported an. fluviatilis james species u from fars province and were in doubt about the occurrence of the species (form) v in iran. besides, mehravaran et al. (2011) recorded the species u in southeastern iran. mosquito oviposition sites include different types which may differ in the point of the quantity and quality of water and environmental characteristics (machault et al. 2009). each mosquito species prefer certain breeding site for oviposition. the breeding habitat plays a crucial role for mosquito population dynamics. there is a strong association between the density and distribution of the mosquito larval stages and that of the adults. then larval control programs can reduce the health problems caused by mosquitoes (floore 2006). the province of west azerbaijan is located at the northwestern part of iran and has a common border line with other countries such as turkey, armenia, azerbaijan and iraq. the solidarity geographical, cultural and territorial exchanges complicate the situation of the province. an example of such complexity is the collapse of the soviet union and the destruction of health infrastructures in the newly independent countries, leading to resurgence and outbreaks of malaria in the region and neighboring countries such as iran (greenwood et al. 2002, lak et al. 2002, vatandoost et al. 2003). furthermore the presence of other mosquito-borne diseases such as west nile fever (ahmadnejad et al. 2011) and dirofilariasis (azari-hamidian et al. 2007) in the province indicates the importance of investigation of mosquitoes in this region. according to the results of the previous studies, 24 species of mosquitoes in six genera have been reported from the province, although there is doubt about the presence of some species (beklemishev and gontaeva 1943, zolotarev 1945, kalandadze and kaviladze 1947, lotfi 1973, saebi 1987, sedaghat et al. 2003). there is no recent study of mosquitoes in west azerbaijan province, despite of the occurrence of some mosquito-borne diseases such as west nile, dirofilariasis, and malaria in the region. the aim of this investigation was to study the fauna, composition and distribution of mosquitoes in this province. materials and methods study area west azerbaijan province is located in the northwest of iran, bordering the countries: turkey, iraq, armenia, azerbaijan, and the provinces of east azerbaijan, zanjan and kurdistan. it covers an area of 39,487 km², or 43,660 km² including lake urmia. the 12 counties of this province have been located in different climate and geographical areas such as mountainous areas near the border with iraq and turkey, plains near the aras and other rivers and the coast-line of the urmia lake (fig. 1). the geographical details of collecting localities have been presented in table 1. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 165 specimen and data collection in the present study, larvae collection was carried out from different habitats using the standard (350 ml dipper) dipping method (silver 2008) in twenty five localities of seven counties across the province (table 1). sample collections were done during may– november 2012. the larvae were transferred to the laboratory and the microscope slides of the larvae were prepared. the thirdand fourth-instar larvae were identified using the keys of shahgudian (1960) and azari-hamidian and harbach (2009). for collecting the adult mosquitoes, several methods such as hand catch, night landing catch on human, cow, total catch, and pit shelter collection were employed fortnight (silver 2008). collected samples were transferred to the laboratory and identified using the standard keys (azari-hamidian and harbach 2009). the abbreviations of the genus name have been used according to reinert (reinert 2009). physical and biological characteristics of larval habitats various physical and biological characteristics of the breeding places of larvae including habitat type (natural or artificial), water condition (standing, slow running, transient or permanent water) vegetation (presence of vegetation and the kind of vegetation) and sunlight exposure (full or partial sunlight or shaded) were recorded visually or using proper equipments. results specimen collections were carried out on 35 occasions during may–november 2012. overall, 1569 mosquitoes including 1336 larvae and 233 adults were collected from 25 localities. the details of geographical properties (latitude, longitude and altitude) of localities are presented in table 1. five genera along with 12 species were collected and identified, including: an. claviger, an. maculipennis s.l., an. superpictus, cx. pipiens, cx. theileri, cx. modestus, cx. hortensis, cx. mimeticus, cs. longiareolata, oc. caspius s.l., oc. geniculatus and ur. unguiculata (table 2). this is the first record of oc. geniculatus in west azerbaijan province. the species, ur. unguiculata and cx. mimeticus were collected only in larval stage and oc. caspius s.l., and oc. geniculatus only as adults adjacent to trees and green spaces using hand catch method. in larval stage, cx. pipiens (25%), cx. theileri (21%), cx. hortensis (13.4%) and an. maculipennis complex (11.6%), were the most prevalent species respectively but in contrast in adult stage, an. maculipennis (41.6%), cs. longiareolata (19.3%), an. claviger (18.45%), cx. pipiens (3.86%) and cx. hortensis (3.86%) were the most abundant species. however, ur. unguiculata (0.07%) was scarce species (table 2). species such as cx. pipiens, cx. theileri and an. maculipennis have a wide distribution in the study areas but some species such as oc. geniculatus, oc. caspius and ur. unguiculata have been collected from limited areas (table 3). all of the different types of habitats were occupied by mosquito larvae. the breeding places with vegetation were more favorable for larvae. most of anopheles larvae were collected from permanent, still and clear water. artificial habitats like artificial standing water (nav-jihan), slow running water (ghezelkurd, ghezel-ajam) were the most common habitats for culicinae larvae but most of anopheles larvae were collected from natural habitats like natural standing water (bazargan) and transient water (zakerloo). likewise the members of the genus culex were collected from wider range of habitats and this shows their adaptation ability to different conditions. the properties of larval habitats have been shown in table 4. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 166 table 1. geographical properties of collecting localities, west azerbaijan province, iran, 2012 location latitude longitude altitude (m) urmia nazloo 1 37°39'24.39"n 44°59'0.39"e 1358 nazloo2 37°39'0.69"n 44°59'5.50"e 1365 sero 37°43'50.12"n 44°39'33.78"e 1572 nav-jehan 37°42'51.58"n 44°39'41.83"e 1605 bavan-kanisi 37°49'28.46"n 44°44'3.31"e 2071 marmisho 37°34'45.91"n 44°37'50.47"e 1749 issar 37°33'25.95"n 45° 0'12.52"e 1466 makoo ghale-joogh 39°16'46.06"n 44°28'19.15"e 1299 baghche-joog 39°17'21.80"n 44°25'29.81"e 1411 sangar 39°18'59.73"n 44°25'53.99"e 1348 boljak 39°17'7.10"n 44°41'24.22"e 991 ghezelkurd 39°15'27.66"n 44°40'0.29"e 1012 ghezelajam 39°15'51.23"n 44°39'12.11"e 1021 bazargan bazargan 1 39°24'31.87"n 44°26'11.77"e 1400 yarim-ghiye 39°26'45.90"n 44°26'4.27"e 1409 poldasht poldasht 39°36'2.58"n 44°51'43.20"e 795 ghizil arkh 39°24'25.66"n 44°58'58.85"e 788 zakerloo 39°21'55.62"n 45° 3'16.74"e 782 sarisoo 39°20'26.52"n 44°53'58.04"e 858 naghadeh naghadeh 36°57'28.22"n 45°21'51.71"e 1313 mahabad mahabad1 36°48'53.18"n 45°44'3.86"e 1304 kavelan 36°23'45.33"n 45°40'32.42"e 1538 mahabad2 36°45'42.49"n 45°42'23.85"e 1371 sardasht sardasht pol 36° 9'20.63"n 45°32'7.05"e 994 kapran 36° 9'40.37"n 45°24'41.30"e 1562 table 2. relative abundance of adults and larvae of mosquitoes west azerbaijan province, iran species adults larvae n % n % an. claviger 43 18.45 99 7.4 an. maculipennis 97 41.6 155 11.6 an. superpictus 3 1.28 15 1.12 cx. hortensis 9 3.86 180 13.4 cx. mimeticus 0 0 7 0.52 cx. modestus 3 1.28 47 3.5 cx. pipiens 9 3.86 336 25 cx. theileri 14 6 281 21 cs. longiareolata 42 19.3 215 16 oc. caspius 6 2.57 0 0 oc. geniculatus 4 1.7 0 0 ur .unguiculata 0 0 1 0.07 total 233 100 1336 100 fig. 1. west azerbaijan province and the location of the studied counties, 1) bazargan, 2) poldasht, 3) makoo, 4) urmia, 5) naghadeh, 6) mahabad and 7) sardasht http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 167 table 3. composition and abundance of mosquitoes, west azarbaijan province, iran, 2012 county mosquitolife stage species of mosquito c x. pipien s c s. lon giareolata c x. th eileri c x. m odestu s c x. h orten sis a n .claviger a n .m acu lipen n is o c. gen icu latu s o c. caspiu s u r. an otaen ia a n . su perpictu s c x. m im eticu s urmia larvae 146 212 160 19 153 63 63 adult 5 34 3 1 26 65 makoo larvae 80 3 20 20 1 adult 2 3 3 1 6 4 6 bazargan larvae 10 1 21 adult 1 3 5 4 poldasht larvae 55 86 3 7 1 adult 3 3 naghadeh larvae 26 40 adult 7 6 16 mahabad larvae 55 5 4 27 8 5 7 adult 1 4 2 3 1 3 sardasht larvae 10 15 10 adult 9 3 total larvae (%) 336(25%) 215(16%) 281(21%) 47(3.5%) 180(13.4%) 99(7.4%) 155(11.6%) 1(0.07) 15(1.12%) 7(0.52%) adult (%) 9(3.86%) 45(19.3%) 14(6%) 3(1.28%) 9(3.86%) 43(18.45) 97(41.6%) 4(1.7%) 6(2.57%) 3(1.28%) total (%) 345(22%) 260(17%) 295(19.8%) 50(3%) 189(12%) 142(9%) 252(16%) 4(0.25%) 6(0.38%) 1(0.19%) 18(1.1%) 7(0.46%) http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 168 table 4. the characteristics of larval habitats of collecting localities, west azerbaijan province, iran, 2012 location habitat situation vegetation situation sunlight situation habitat kind collected species (abundance percentage in the location) urmia nazloo 1 slow running water out of water partial natural cx. pipiens (25%), cs. longiareolata (30%), cx. theileri (20%), cx. modestus (6%), cx. hortensis (11%), an. claviger (8%) nazloo2 transient water surface partial artificial cx. modestus (76%), an. maculipennis (24%) sero slow running water beneath of water surface full natural cx. theileri (55%), an. maculipennis (45%) nav-jehan slow running water beneath of water surface partial artificial cx. theileri (45%), an. maculipennis 25%), an. claviger (30%) bavan-kanisi standing water out of water partial artificial cx. pipiens (100%) marmisho permanent beneath of water surface shaded natural an. claviger (15%), cx. hortensis (85%) issar transient beneath of water surface full artificial cx. pipiens (20%), cs. longiareolata (60%), cx. theileri (15%), an. maculipennis (5%) makoo ghale-joogh permanent out of water partial natural cx. theileri (55%), cs. longiareolata (45%) baghchejoog slow running water out of water shaded natural cx. pipiens (40%), cx. theileri (60%) sangar standing water beneath of water surface partial natural cx. pipiens (70%), cx. theileri (15%), cx. modestus (15%) boljak standing water water surface shaded natural an. maculipennis (100%) ghezelkurd slow running water out of water partial artificial cx. pipiens (65%), cx. theileri (35%) ghezelajam slow running water beneath of water surface shaded artificial cx. theileri (80%), an. maculipennis (20%) bazargan bazargan 1 standing water water surface full natural an.maculipennis (100%) yarim-ghiye slowly running water beneath of water surface full artificial cx. theileri (80%), cx. modestus (5%), an. maculipennis (15%) poldasht poldasht permanent water surface partial natural cx. pipiens (30 %), cx. theileri (30 %), cx. modestus (20 %), an. maculipennis (20 %) ghizil arkh standing water out of water full natural cx. theileri (100%) zakerloo transient beneath of water surface partial natural an. maculipennis (95%), uranotaenia (5%) sarisoo standing water water surface shaded natural cx. pipiens (85%), an. maculipennis (15%) naghadeh naghadeh transient out of water full natural an. claviger (45%), an. maculipennis (55%) mahabad mahabad1 permanent water surface full natural cx. pipiens(35%), cx. theileri (10%), cx.modestus (10%) cx. hortensis (30%), an. maculipennis (15%) kavelan slowly running water beneath of water surface partial natural cx. hortensis (15%), an. maculipennis (20%), an. superpictus (25%), cx. mimeticus (40%) mahabad2 standing water water surface shaded natural cx. pipiens (20%), cx. theileri (25%), cx. modestus (10%), an. maculipennis (25%), an. superpictus (20%) sardasht sardasht pol slowly running water beneath of water surface shaded natural an. maculipennis (65%), an. superpictus (35%) kapran slowly running water water surface shaded natural an. maculipennis (45%), an. superpictus (30%), an. claviger (25%) http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 169 discussion this is the first specific and targeted investigation to study mosquitoes in west azerbaijan province which showed the presence of 12 species of mosquitoes in the study area. sporadic studies had been conducted in parts of the north western part of iran (beklemishev and gontaeva 1943, zolotarev 1945, kalandadze and kaviladze 1947, lotfi 1973, danilov 1975, saebi 1987, azari-hamidian et al. 2009, kazemi et al. 2010, banafshi et al. 2013). in addition to the 12 species collected and identified in this study, other 13 species such as an. algeriensis, an. plumbeus, cx. deserticola, cx. tritaeniorhynchus, cs. alaskaensis, cs. annulata, oc. dorsalis (kalandadze and kaviladze 1947), an. hyrcanus group (saebi 1987), an. marteri (unpublished data from institute of malariology, tehran university of medical sciences), an. sacharovi (kalandadze and kaviladze 1947, sedaghat et al. 2003), cs. subochrea (kalandadze and kaviladze 1947, zaim 1987), aedes vexans (kalandadze and kaviladze 1947, zaim 1987), oc. flavescens (kalandadze and kaviladze 1947, zaim 1987), were reported from this region. because of the oldness of the report of some of these species and the probable effect of the climate change during the time, the presence of some of these species in the study area is questionable and uncertain. seven genera and 15 species were identified in east azerbaijan province (abai et al. 2007) among which eight species (an. maculipennis, an. claviger, an. superpictus, cx. theileri, cx. pipiens, cs. longiareolata, oc. caspius and ur. unguiculata) were common between the two neighboring provinces and six (cx. theileri, cx. pipiens, cx. hortensis, cx. mimeticus, cs. longiareolata and oc. caspius) are common between west azerbaijan and sanandaj county in kurdistan province which is in southern neighboring of west azerbaijan (kazemi et al. 2010). the comparison of the results of this study with a recent comprehensive study carried out in kurdistan province (banafshi et al. 2013) showed that nine species (an. maculipennis, an. claviger, an. superpictus, cx. theileri, cx. pipiens, cx. hortensis, cx. mimeticus, cs. longiareolata and oc. caspius) were common between kurdistan and west azerbaijan provinces. comparing the results of this study with a similar study carried out in zanjan province (ghavami and ladonni 2005), showed that six species (an. maculipennis, an. superpictus, cx. pipiens, cx. theileri, cx. hortensis and cs. longiareolata) are common between zanjan and west azerbaijan provinces. in turkey (aldemir et al. 2010) and northern and central parts of iran such as eastazerbaijan, ardebil and kurdistan provinces, cx. theileri is the dominant and most abundant species, except for the low-lying areas bordering the caspian sea and urban areas (azari-hamidian et al. 2009). the results of this study showed that in the case of larvae collection, cx. pipiens (25%) and cx. theileri (21%) were the most abundant species respectively and widely distributed in all parts of west azerbaijan province. the presence of these two species (cx. pipiens, cx. theileri) in other studies in iran (azari-hamidian 2007b, azari-hamidian et al. 2009, kazemi et al. 2010, nikookar et al. 2010, saghafipour et al. 2012) and other neighboring areas in neighboring countries such as eastern parts of turkey and iraq (rueda et al. 2008) show a wide distribution of these species in the region. the species oc. geniculatus is recorded for the first time in west azerbaijan province but this tree-hole mosquito has not been reported from neighboring areas of iran and other neighboring countries. this species had been reported previously from northern parts of the country such as ardabil, guilan and http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 163–173 f khoshdel-nezamiha et al.: fauna and larval … 170 mazandaran provinces (zaim 1987, azarihamidian et al. 2009). the west azerbaijan province comprises different geographical areas with climatically different condition. these diverse climatic conditions can provide suitable environment for the establishment of various species of mosquitoes and this is the explanation for the richness of the composition of mosquito species in this province. some of identified species are principal vectors of several mosquito-borne pathogens such as west nile virus, rift valley virus. six species of known species in this study (an. maculipennis, cx. modestus, cx. theileri, cx. pipiens, cs. longiareolata, oc. caspius) are among the important vectors of west nile fever. according to reports of the existence of this disease in the study area (saidi et al. 1976, ahmadnejad et al. 2011) and surrounding countries (zeller and schuffenecker 2004), the existence of mentioned species should be considered and further investigations are needed. minding this fact that birds are involved in the cycle of transmission as amplifying hosts and existence of several ponds such as urmia lake, mahabad pond, aras basin and sardasht nature which are the destination of migrant birds from other countries, emerging of this disease in the region is more conceivable. picking these facts together should act like a wake for health system and more attention will turn to the mosquito-borne diseases. on the other hand, cx. pipies and cx. theileri, which have been identified in this study, reported as potential vectors of rift valley fever (zeller and schuffenecker 2004). the presence of this disease in mediterranean region (moutailler et al. 2008) indicates the need for more attention to this disease and its vectors. the results of this study also revealed the presence of the vectors of helminths such as setaria labiatopapillosa and dirofilaria immitis in the region. anopheles maculippenis and cx. theileri are well known vectors of the mentioned parasites in ardebil province (azari-hamidian et al. 2009). conclusion the results of this 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accepted 15 may 2013) abstract background: anopheles culicifacies, a major malarial vector has been recognized as a complex of five sibling species, a, b, c, d and e. these sibling species exhibit varied vectorial capacity, host specificity and susceptibility to malarial parasites/ insecticides. in this study, a pcr assay developed earlier for distinguishing the five individual species was validated on samples of an. culicifacies collected from various parts of india. methods: the samples were initially screened using the rdna-its2 region based primers which categorised the samples into either a/d group or b/c/e group. a proportion of samples belonging to each group were subjected to the mtdna-coii pcr assay for identifying individual species. results: among the 615 samples analysed by rdna-its2 pcr assay, 303 were found to belong to a/d group and 299 to b/c/e group while 13 turned negative. among 163 samples belonging to a/d group, only one sample displayed the profile characteristic of species a and among the 176 samples falling in the b/c/e group, 51 were identified as species b, 14 as species c and 41 as species e respectively by the mtdna-coii pcr assay. samples exhibiting products diagnostic of b/c/e, when subjected to pcr-rflp assay identified 15 samples as species e. conclusion: validation of the mtdna-coii pcr assay on large number of samples showed that this technique cannot be used universally to distinguish the 5 members of this species complex, as it has been designed based on minor/single base differences observed in the coii region. keywords: anopheles culicifacies, coii pcr assay, rdna-its2 pcr assay, sibling species introduction anopheles culicifacies, an important mosquito species occurring in the indian subcontinent, is reported as a complex of five sibling species, a to e. all of them except species b are vectors of malaria (sunil et al. 2004). though morphologically similar, members of the complex show ecological and behavioral differences which affect their vectorial capacity (subbarao 1998). identification of different members is possible based on the variable banding pattern in the x chromosome and chromosome arm 2, and also structural differences in the male mitotic karyotypes. but these observations require specifically staged specimens and hence cannot be applied to the entire sample collection. several pcr assays were developed based on the sequences of the d2, d3 and its2 region of the ribosomal dna, which were able to categorize the 5 members into two groups, viz, a/d group and b/c/e group. recently, a three step pcr assay was developed by goswami et al. (2006), based on the differences in the coii region of the mitochondrial dna for identifying the individual members of this species complex. it was later reduced to a two step process by manonmani et al. (2007), by replacing the its2 and rflp steps by a single its2 group diagnostic assay. *corresponding author: dr arulsamy mary manonmani, e-mail: ammanonmani@yahoo.com 154 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 this paper presents the findings of evaluation of this two step pcr assay using field specimens of an. culicifacies collected from various parts of india. materials and methods mosquito collection adult samples of an. culicifacies were collected from malkangiri district, orissa state, alwar district, rajasthan state, tiruvanamalai and ramanathapuram districts, tamil nadu state (fig. 1) (table 1). daytime resting mosquito collections were done between 6:00 to 8:00am using aspirators, while all night collections were done between 6:00pm to 6:00am by using light traps (gunasekaran et al. 1994). the collections were done from human dwellings and cattle sheds. samples of an. culicifacies from all the catches were identified based on the taxonomic keys of christophers (1933). dna extraction and pcr assays dna was extracted from the thoracic region of each individual mosquito sample using genelute mammalian genomic dna extraction kit (sigma, st. louis, mo, usa). the dna pellet was dissolved in 50µ l of te (tris/edta) buffer and stored at -20 °c. two pcr reactions were carried out on each dna sample. using the rdna-its2-pcr assay described by manonmani et al. (2007), samples were initially grouped either as a/d or b/c/e. these samples were then subjected to mtdna-coii pcr assay reported by goswami et al. (2006). samples grouped as a/d were subjected to the ad-pcr assay while those grouped as b/c/e were subjected to the bce-pcr assay. details related to the primers used in these pcr assays and the expected products are given in table 2. the amplified products were electrophoresed on an ethidium bromide stained 1.5% agarose gel, along with dna molecular weight marker (genei, bangalore, india) and visualized under uv light. amplification of the coii region the coii region of the mtdna for 5 individuals each of species b, c and e, identified cytologically was amplified using a pair of primers, coiif: 5’-agagcttctcctttaa tggaaca-3’ and coiir: 5’-caattggta taaaactatgatttg-3’ respectively. the reaction mixture comprised 4.5 mm mgcl2, 200 µ m of each dntp’s (ge healthcare uk limited, buckinghamshire, uk), 10 µm of each primer (metabion, martinsried, germany), 2.5 µ l of taq buffer and 2 units of taq polymerase (finnzyme, espoo, finland). to this mixture was added 1/100th of the dna from a whole mosquito and the reaction mixture was made up to 25 µ l volume using deionized water. these reactions were amplified in a bio-rad cycler pcr machine (biorad, california, usa). pcr conditions were: denaturation at 94 °c for 4 min. followed by 40 cycles of denaturation at 95 °c for 40 sec, annealing at 50 °c for 1 min, extension at 68 °c for 1 min and a final extension step at 72 °c for 10 min. after checking a portion of each amplification product by gel electrophoresis, the remainder of the sample was purified using qiaquick pcr purification kit (qiagen, hilden, germany). the concentration of dna was quantified and subjected to sequencing in an abi automatic sequencer (applied biosystems, california, usa), using the amplification primers to obtain sequence from both strands. pcr-rflp samples which exhibited products that are diagnostic of species b, c and e were subjected to coii pcr assay followed by ddei digestion, which is reported to seperate species e from b and c (goswami et al. 2005). each 20µl digest consisted of 17.5µ l pcr product, 2µl 10x buffer and 0.5 u of ddei enzyme (sigma, st. louis, mo, usa). for each sample, paired control reactions were set up that 155 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 contained only pcr product and 10x buffer. reactions were incubated at 37 °c overnight, followed by heat inactivation at 65 °c for 10 minutes. digested and undigested pcr products were electrophoresed and observed as mentioned earlier. results the results of the validation of the rdnaits2 pcr assay and mtdna-coii pcr assay is given in table 3. collections from tiruvanamalai district of tamil nadu represented individuals belonging to bce group in major proportion (86.8%) while those from alwar district of rajasthan represented individuals belonging to ad group in major proportion (87.7%) (table 3). all the an. culicifacies collections from malkangiri district of orissa and ramanathapuram district of tamil nadu belonged to the bce group. thirteen (2.1%) of the samples did not belong to either of the groups. about half the number of samples from each group, representing different types of collections/ habitats/ villages were randomly selected and subjected to the coii-pcr assay developed by goswami et al. (2006). among the 163 samples from tiruvanamalai and alwar districts, belonging to the ad group, one was found to be species a, 160 were species d and 2 belonged to neither. the single sample belonging to species a originated from tiruvanamalai district. the samples which belonged to the bce group when subjected to the species diagnostic mt-dna pcr assays showed that all the 3 sibling species were present in the 4 districts, though species c was seen in lesser numbers in tiruvanamalai, alwar and malkangiri districts respectively. however, samples exhibiting products diagnostic of all the 3 species were found in sizeable numbers (36.4%) (fig. 2). sequencing of the mtdna-coii region showed that polymorphism was noticed in the nucleotide positions which were diagnostic. with primer cr designed to amplify species c, it was noticed that the base ’a’ at position 171 which is the only diagnostic point differentiating species c from b was found to be ’g’ in all the species c samples sequenced by us (fig. 3). with primer er, the diagnostic point ‘t’ and ‘c’ in species b and e was found to be ‘t’ in one of our samples belonging to species e (fig. 4). with the primer bcr, the only diagnostic point ’g’ at position 334 differentiating species b and c from e was found to exhibit polymorphism by being either ’g’ or ’a’ (fig. 5). samples which exhibited products diagnostic of species b, c and e were subjected to the pcr-rflp assay. amplification of the mtdna-coii region gave a band of size 573bp. this product, when subjected to digestion with the restriction enzyme, ddei produced 3 fragments of sizes 300, 156 and 117bp respectively for species e and 2 fragments of sizes 456 and 117bp respectively for species b and c. these fragments were as per the predicted restricted sites of this enzyme. however, only 15 samples out of 64 yielded fragments corresponding to e, 15 produced fragments corresponding to species b and c and the products of 34 samples remained uncut (table 3). 156 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 fig. 1. map showing the collection sites of anopheles culicifacies s.l. 157 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 fig. 2. coii species diagnostic pcr assay for anopheles culicifacies. lane 1–3: sibling d (359bp+166bp), lane 4: sibling a (359bp), lane 5: 100bp ladder, lane 6, 11: b/c/e (248bp+178bp+95bp), lane 8: sibling c (248bp+95bp), lane 10: sibling b (248bp), lane 7, 9, 12–15: sibling e (248bp+178bp). fig. 4. er primer site of the mtdna-coii sequences for species b, c and e of the anopheles culicifacies complex. accession numbers aj518810, aj519493 and aj534646 (goswami et al. 2006). accession numbers coxiiacsl-b1 and coxiiacsl-e1 (surendran et al. 2006). accesssion numbers hq377221hq377228: mtdna coii sequences of species b, c and e generated in the current study. fig. 3. cr primer site of the mtdna-coii sequences of species b and c of the anopheles culicifacies complex. accession numbers aj518810 and aj519493 goswami et al. (2006). accession numbers hq377221-hq377225: mtdna-coii sequences of b and c generated in the current study. fig. 5. bcr primer site of the mtdna-coii sequences for species b, c and e of the anopheles culicifacies complex. accession numbers aj518810, aj519493andaj534646 (goswami et al. 2006). accession numbers coxiiacsl-b1 and coxiiacsle1 (surendran et al. 2006). accession numbers hq377221-hq377228: mtdna coii sequences of species b, c and e generated in the current study. fig. 6. its2 region based phylogenetic tree (neighbor joining method) for anopheles culicifacies showing two distinct clades of a/d and b/c/e with an out group anopheles aconitus 158 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 table 1. details of collected anopheles culicifacies samples district and state geographical co-ordinates village habitat type of collection no. tiruvanamalai tamilnadu lat: 11°55'–13°15'n long: 78°20'–79°50'e kolamanjanur cattle shed light trap 3 kolamanjanur cattle shed hand catch 141 kolamanjanur human dwelling hand catch 34 labour colony cattle shed hand catch 22 labour colony human dwelling hand catch 5 ramanathapuram tamilnadu lat: 9°05'–9°50'n long: 78°10'–79°27'e karaiyur human dwelling hand catch 13 rajakoil human dwelling hand catch 11 tharavaithoppu human dwelling hand catch 36 alwar rajasthan lat: 27°03'–28°14'n long: 76°07'–77°13'e balana cattle shed hand catch 20 balana human dwelling hand catch 27 lalpura cattle shed hand catch 109 lalpura human dwelling hand catch 101 indok cattle shed hand catch 2 sawar cattle shed hand catch 40 sawar human dwelling hand catch 25 malkangiri orissa lat: 17°45'–18°40'n long: 81°10'–82°00'e teakguda cattle shed hand catch 26 total samples collected 615 lat: latitude, long: longitude table 2. details of primers used in pcr assays s. no primer name/ sequence (5’-3’) tm value gc content pcr product size (bp) 1 5.8s atcactcggctcatggatcg 60 55 ad group: 409 bce group: 253 2 sp ad cagtgctgcaaaccaccacttat 64 45.8 3 sp bce tgttagtaggctgccggggttc 66 59.1 4 adf ctaatcgatatttattacac 48 25 species a: 359 species d: 359+166 5 adr ttactcctaaagaaggc 48 41.2 6 df ttagagtttgattcttac 45 27.8 7 bcef aaattatttgaacagtattg 46 20 species b: 248 species c: 248+95 species e: 248+1788 bcr ttatttattggtaaaacaac 46 20 9 cr aaggagtattaatttcgtct 49 31.6 10 er gtaagaatcaaattctaag 47 26.3 1–3: primers for rdna-its2 group diagnostic pcr assays (manonmani et al. 2007) 4–10: primers for mtdna-coii species diagnostic pcr assays (goswami et al. 2006) 159 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 table 3. evaluation of pcr assays for identification of individual members of anopheles culicifacies area rdna-its2 pcr assay mtdna-coii pcr assay pcr-rflp a/d b/c/e t ot al c ol le ct ed a /d gr ou p b /c /e gr ou p -v e n u m b er s p ro ce ss ed a d -v e n u m b er s p ro ce ss ed b c e b /c /e -v e b /c /e e b /c tiruvanamalai 205 19 178 8 19 1 17 1 81 25 3 17 32 4 32 7 7 ramanathapur am 60 0 58 2 0 0 0 0 43 15 9 7 11 1 11 3 1 alwar 324 284 38 2 144 0 14 3 1 27 4 1 10 12 0 12 4 1 malkangiri 26 0 25 1 0 0 0 0 25 7 1 7 9 1 9 1 6 total 615 303 299 13 163 1 16 0 2 176 51 1 4 41 64 6 64 1 5 15 -ve = negative fig. 7. d3 region based phylogenetic tree (neighbor joining method) for anopheles culicifacies showing two distinct clades of a/d and b/c/e with an out group anopheles aconitus discussion accurate identification and knowledge about the distribution of vector and non-vector members of species complexes is required for planning effective control measures. hence, development of molecular diagnostics for the individual species of the an. culicifacies complex was felt necessary as 4 of these species have been incriminated as vectors of malaria in india. goswami et al. (2006) have developed a three step pcr assay for the identify cation of 5 individual members of this species complex, which involved (1) amplification of the rdna-its2 region, (2) restriction digestion of the its2 product and (3) pcr assay based on the coii region of the mtdna. this three step pcr assay was later reduced by developing a pcr assay based on the rdnaits2 region (manonmani et al. 2007) which grouped the 5 species into the same 2 categories as obtained after the first two steps of goswami et al. (2006). in the present study this two step pcr assay was evaluated on 160 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 samples of an. culicifacies collected from different regions of india. of the 615 samples evaluated, 49.3% were categorized as a/d while 48.6% were b/c/e. selected samples (163) from the a/d group when subjected to mtdna-coii pcr assay showed that 0.6% were species a and 98.2% were species d. likewise, when 176 samples belonging to the b/c/e category were analyzed, 29%, 8% and 23.3% exhibited products diagnostic for species b, c and e respectively, while 28% exhibited products diagnostic of all the 3 species. the specificity of the coii-pcr assay in identifying the individual member within the 2 groups was low. this can be attributed to single base differences in the coii region which has been used for the design of species diagnostic assay. similar problem was encountered when single base differences of the rdna-its2 region was used for designing pcr assay for species a and c of the an. minimus complex (garros et al. 2004). the major role played by members of the an. culicifacies complex in malaria transmission in several regions of india has compelled the authors to use the minor differences in designing these primers. hence, the basic requirements such as 18–24 nucleotide length, tm value between 56–62 and gc content between 45–60% could not be met while designing the 7 species diagnostic primers (table 3). gc content, melting and annealing temperatures being dependent on one another play a crucial role in the efficiency and sensitivity of pcr assays (rychlik et al. 1990, dieffenbach et al. 1993, he et al. 1994). in the case of ad-pcr assay, species a should exhibit a single product of size 359bp while species d should produce 2 products of size 359 and 166 bp respectively. but in this study, all samples produced products characteristic of species d. goswami et al. (2006), while evaluating this assay found that the ad-pcr results did not agree with the earlier reports on the distribution of these members in kheda and sonepat, india. they attributed this to the polymorphic nature of the i1 inversion in species a in these areas and hence samples collected from these regions, whether homozygous or heterozygous for this inversion, have been identified as species d. this might be the reason for all samples being identified as species d in our study also. the i1 homozygotes could be species d or polymorphic forms of species a. hence, the ad-pcr assay will not be useful in areas where the i1 inversion is polymorphic. vasantha et al. (1991) using i1 inversion for detecting species d found that in certain areas, presence of species d becomes evident only due to the deficiency of heterozygotes. hence, it appears that cytotaxonomy also, cannot be used universally in the identification of species a and d of this species complex. in our study, sequencing of the mtdnacoii region for species b, c and e showed polymorphism in the nucleotide positions used for designing of diagnostic primers. similar observations were made by surendran et al. (2006) while characterizing species b and e of this species complex from srilanka. though they observed acrocentric and submetacentric y chromosomes in species b and e as reported by kar et al. (1999), they were not able to correlate these results with those obtained by the pcr-rflp assay (goswami et al. 2005). this was because several bases of the coii region were found to exhibit polymorphism. in particular, the base ‘c’ at position 255 in the coii gene which is the only diagnostic point differentiating species e from b as well as serving as the restriction site for the enzyme, dde i, itself exhibited polymorphism. this base which was ’t’ and ’c’ in species b and e respectively, in the samples studied by goswami et al. (2006) was found to be `c’ in both the species from srilanka. hence, the species diagnostic pcr assay and pcrrflp cannot be used with 100 % accuracy for distinguishing species b and e. while using cytotaxonomical methods for identifying species b and c of the an. 161 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 culicifacies complex, b/c heterozygotes were seen in forested and deforested villages of northern orissa, which represented interspecific hybrids obtained due to breakdown of premating barriers between species b and c (nanda et al. 2000). phylogenetic analysis of the its2 and d3 region sequences (figs. 6, 7) showed that the 5 members fell into 2 clades, one representing species b, c and e and the other representing species a and d. similar categorization has been made by dassanayake et al. (2008) and raghavendra et al. (2009) based on the sequences of the its2 and d2 regions of the rdna respectively. hence, species a and b of an. culicifacies reported by green and miles (1980) appears to be the two distinct sibling species of this malaria vector while the others namely species d and species c and e seems to have diverged very recently from species a and species b respectively. further, the fact that the members within the 2 groups have not yet developed post mating barriers (raghavendra et al. 2009) shows that the speciation process among the members of the 2 groups is not yet complete. the present study has shown that the mtdna-coii pcr assays cannot be used universally to distinguish the members of an. culicifacies complex, as has been observed by surendran et al. (2006) in srilanka as well. acknowledgements the authors are grateful to dr sl hoti, scientist `f’ and dr ar rajavel, scientist ’d’, vector control research centre, puducherry for critically reviewing the manuscript. the technical assistance rendered by mr s murugaraj and staff of the vector biology and control section is gratefully acknowledged. this study was supported by a research grant from the department of science and technology (dst), govornment of india. the authors declare that there is no conflict of interests. references christophers sr (1933) the fauna of british india. diptera, vol. iv. family-culicidae. tribe anophelini. today and tomorrow’s printers and publishers, new delhi. dassanayake rs, gunawardene yi, silva bd (2008) its-2 secondary structures and phylogeny of anopheles culicifacies species. bioinformation. 2: 456–460. dieffenbach cw, lowe tmj, dveksler ds (1993) general concepts for pcr primer design. genome res. 3: s30–s37. garros c, koekemoer ll, coetzee m, coosemans m, manguin s (2004) a single multiplex assay to identify major malaria vectors within the african anopheles funestus and the oriental anopheles minimus groups. am j trop med hyg. 70: 583–590. goswami g, raghavendra k, nanda n, gakhar sk, subbarao sk (2005) pcr–rflp of mitochondrial cytochrome oxidase subunit ii and its2 of ribosomal dna: markers for the identification of members of anopheles culicifacies complex (diptera: culicidae). acta trop. 95: 92–99. goswami g, singh op, nanda n, raghavendra k, gakhar sk, subbarao sk (2006) identification of all members of the anopheles culicifacies complex using allele-specific polymerase chain reaction assays. am j trop med hyg. 75: 454–460. green ca, miles (1980) chromosomal evidence for sibling species of the malaria vector anopheles (cellia) culicifacies giles. j trop med hyg. 83: 75–78. gunasekaran k, jambulingam p, sadanandane c, sahu ss, das pk (1994) reliability of light trap sampling for anopheles fluviatilis, a vector of malaria. acta trop. 58: 1–11. he q, marjamaki m, soini h, mertsola j, viljanen mk (1994) primers are decisive 162 j arthropod-borne dis, december 2013, 7(2): 154–163 am manonmani et al.: evaluation of the … http://jad.tums.ac.ir published online: august 31, 2013 for sensitivity of pcr. biotechniques. 17: 82–87. kar i, subbarao sk, eapen a, ravindran, j, satyanarayana ts, raghavendra k, nanda n, sharma vp (1999) evidence for a new malaria vector species, species e within the anopheles culicifacies complex (diptera: culicidae). j med entomol. 36: 595–600. manonmani am, sadanandane c, sahu ss, mathivanan a, jambulingam p (2007) rdna-its2-pcr assay for grouping the cryptic species of anopheles culicifacies complex. (diptera: culicidae). acta trop. 104: 72–77. nanda n, yadav rs, subbarao sk, joshi h, sharma vp (2000) studies on anopheles fluviatilis and anopheles culicifacies sibling species in relation to malaria in forested hilly and deforested riverine ecosystems in northern orissa, india. j am mosq control assoc. 16: 199–205. raghavendra k, cornel aj, reddy bp, collins fh, nanda n, chandra d, verma v, dash ap, subbarao sk (2009) multiplex pcr assay and phylogenetic analysis of sequences derived from d2 domain of 28s rdna distinguished members of the anopheles culicifacies complex into two groups, a/d and b/c/e. infect genet evol. 9: 271–277. rychlik w, spencer wj, rhoads re (1990) optimization of the annealing temperature for dna amplification in vitro. nucleic acids res. 18: 6409–6412. subbarao sk (1998) anopheline species complexes in south east asia. technical publication, serial number 18, world health organization, regional office for south east asia, new delhi. sunil s, raghavendra k, singh op, malhotra p, huang y, zheng l, subbarao sk (2004) isolation and characterization of microsatellites markers from malaria vector, anopheles culicifacies. mol ecol notes. 4: 440–442. surendran sn, hawkes nj, steven a, hemingway j, ramasamy r (2006) molecular studies of anopheles culicifacies (diptera: culicidae) in sri lanka: sibling species b and e show sequence identity at multiple loci. euro j entomol. 103: 233–237. vasantha k, subbarao sk, adak t, sharma vp (1991) anopheles culicifacies complex: population cytogenetic evidence for species d (diptera: culicidae). ann entomol soc am. 84: 531–536. 163 microsoft word 8-hanafi .doc iranian j arthropod-borne dis, 2007, 1(2): 4852 b farzin-nia and aa hanafi-bojd: the sand fly fauna of … 48 short communication the sand fly fauna of an endemic focus of visceral leishmaniasis in central iran *b farzin-nia1, aa hanafi-bojd2 1school of public health, qom university of medical sciences, iran 2department of medical entomology and vector control, school of public health, medical sciences/university of tehran, iran (received 22 july 2007; accepted 1 dec 2007) abstract visceral leishmaniasis due to leishmania infantum is endemic in many rural areas of iran. an investigation was carried out during april to november 2001 in ghahan rural district of qom province, in order to identify the phlebotomine sand fly fauna. 971 sand flies were totally collected from outdoor and indoor resting places by sticky traps. ten species of phlebotomine sand flies including 8 species of the genus phlebotomus (phlebotomus sergenti, p. major, p. alexandri, p. kandelakii, p. tobbi, p. brevis, p. halepensis, p. (adlerius) sp) and 2 species of the genus sergentomyia (sergentomyia pawlowsky and s. theodori) were collected from outdoor and indoor resting-places by sticky traps. high indoor density of p. sergenti (46.9%) and p. major (40.4%) was found with the peak of activity in early july. based on the results of the present study and also the related studies in other vl foci of iran, p. major can be considered as the probable vector of the disease in the area. keywords: sand flies, visceral leishmaniasis, ecology, qom, iran introduction visceral leishmaniasis (vl) caused by leishmania infantum, exists in about 70 countries, is an endemic disease in many parts of iran, especially in northwest and south of the country. it is a serious public health problem in these areas. since the first report of the disease by pouya in 1949, about 9000 cases were reported in iran (unpublished data, institute of public health research). dogs (canis familiaris) are reported as the main reservoir of vl, but wild carnivores such as jackals and foxes have been also found infected with leishmania infantum. so they may be considered as reservoirs of the infection, particularly in areas where sporadic cases of the disease were reported (nadim 1978, hamidi et al. 1982). moreover, two species of parasite, l. infantum and l. donovani, have been isolated from rodents in northwest of the country (mohebali et al. 1998). leptomonad infection of phlebotomus major, p. keshishiani, p. perfiliewi and p. kandelakii were reported in the main vl foci in iran (nadim et al. 1992, sahabi et al. 1992, seyedi-rashti et al. 1995, rassi et al. 1997). the first case of the disease was reported in 1997 from ghahan rural district, the study area, and two other cases were observed by the authors in the following years. the area is a new endemic focus of vl in our country (fakhar et al. 2004). the objective of this study was to determine the sand fly fauna of the area. *corresponding author: b farzin-nia, tel/fax: +98 251 7833361, e-mail: bfarzinnia@yahoo.com iranian j arthropod-borne dis, 2007, 1(2): 4852 b farzin-nia and aa hanafi-bojd: the sand fly fauna of … 49 materials and methods ghahan rural district (50o 90' n, 34o 44' e) is located in qom province, central iran, and has a dry climate with a total rainfall of 122 mm and altitude of 2000 m. the mean maximum and minimum of monthly temperature in january and june 2001 was reported -0.3 and 37 ºc, respetively. the relative humidity was ranged from 11.9% in september to 73.4% in january. the entomological studies were conducted in anjile village, where the vl cases were reported. from april to november 2001, sand flies were collected biweekly by 60 sticky traps from 3 outdoor (hole of rocks and caves, dog shelters) and 3 indoor (living rooms, cow shelter, stable) resting places. in this method, traps were installed after sunset and were collected before the following sunrise. sand flies were removed from the traps, rinsed in acetone and then conserved in 70% ethanol. all specimens were mounted as permanent microscopy slides, using puri's medium (smart 1965). species identification was carried out using the keys of lewis (1982), nadim and javadian (1976) and seyedi-rashti and nadim (1992). results a total of 971 adult sand flies were collected biweekly by sticky traps during april to november 2001, and the following species belonging to genera of phlebotomus and sergentomyia were identified (table 1). the outdoor species were: phlebotomus sergenti (28.6%), p. major (6.8%), p. alexandri (2.7%), p. kandelakii (2.4%), p. tobbi (0.2%), p. brevis (0.2%), p. (adlerius) sp (0.5%), sergentomyia pawlowsky (1.7%) and s. theodori (56.9%). sand flies, collected from indoor resting places were: phlebotomus sergenti (46.9%), p. major (40.4%), p. alexandri (4.3%), p. kandelakii (3.9%), p. tobbi (0.4%), p. brevis (0.2%), p. halepensis (1.6%), p. (adlerius) sp (0.9%) and s. theodori (1.4%). sand fly activity started in april and ended in october. common sand flies were p. sergenti (46.9%), p. major (40.4%) in indoor resting places and s. theodori (56.9%) and p. sergenti (28.6%) in outdoors. the sex ratio (number of males/females x 100) of p. sergenti and p. major was 431.8 and 250 in outdoors, and 338.3 and 460 in indoors, respectively. table 1. fauna and frequency of sand flies in the village of anjile, ghahan rural district, qom province, 2001 place of collection indoors outdoors total species no. % no. % no. % phlebotomus sergenti 263 46.9 117 28.6 380 39.1 phlebotomus major 226 40.4 28 6.8 254 26.2 phlebotomus alexandri 24 4.3 11 2.7 35 3.6 phlebotomus kandelakii 22 3.9 10 2.4 32 3.4 phlebotomus halepensis 9 1.6 0 0 9 0.9 phlebotomus tobbi 2 0.4 1 0.2 3 0.3 phlebotomus brevis 1 0.2 1 0.2 2 0.2 phlebotomus (adlerius) sp 5 0.9 2 0.5 7 0.7 sergentomyia theodori 8 1.4 234 56.9 242 24.9 sergentomyia pawlowsky 0 0 7 1.7 7 0.7 iranian j arthropod-borne dis, 2007, 1(2): 4852 b farzin-nia and aa hanafi-bojd: the sand fly fauna of … 48 discussion fakhar et al. (2004) introduced a new endemic focus of kala-azar in ghahan rural district, qom province. a preliminary study on vl vectors of the area showed four species: p. major, p. alexandri, p. sergenti and s. dentata (akhavan et al. 2000), although we could not find the latter species. they reported p. major as the dominant species in both indoors and outdoors. study on the sand fly fauna in kashan county, near our study area, showed 17 species (dourodgar et al. 1999). their identified species were: p. papatasi, p. sergenti, p. alexandri, p. major, p. tobbi, p. kandelakii, p. ansarii, p. halepensis, p. longiductus, p. brevis, p. jacusieli, s. sintoni, s. dentata, s. powlowskyi, s. tiberiadis, s. palestinensis, and s. clydei. two probable vectors of visceral leishmaniasis, p. major and p. kandelakii were collected from this area. regarding high density of p. major in outdoor and indoor resting places, and also based on the related studies in other vl foci of iran, this species is considered as a probable vector. another study in southern foci of the country has reported leishmania infection in this species (sahabi et al. 1992). it is the main vector of vl in greece (leger et al. 1979) and is also considered to be a vector of vl in other countries of mediterranean basin (hoogstral et al. 1969). p. major was first reported from northern part of iran (pervomaniski 1948). it has been also found in all areas where human cases of vl have been reported. based on epidemiological evidence, p. major is suspected to be the main vector of vl in iran (nadim 1978). two other species collected in this study, p. kandelakii and p. alexandri, have been reported as probable vl vectors in iran (rassi et al. 2005, azizi et al. 2006). p. kandelakii appears to be a vector of l. donovani in georgia (perfil'ev 1966) and was considered to be the main vector of vl in transcaucasia (sergiev 1979). this sand fly currently introduced as the proven vector of visceral leishmaniasis in iran with a natural infection rate of 1.1% (rassi et al. 2005). phlebotomus alexandri is introduced as the proven vector of l. donovani in china (guan et al. 1986), and is considered to be an important vector of cl in the south of the former soviet union (lewis 1982). females of this species have been found infected with flagellates in a cl area of khuzestan, iran (javadian et al. 1975) and the species is suspected of transmitting cl in tunisia (croset et al. 1978). azizi et al. (2006) reported p. alexandri infected with l. infantum in fars province, southern iran. demonstrating natural infection of p. tobbi with l. infantum shows that this species may be the most probable vector of l. infantum in cyprus where promastigotes of l. infantum zymodeme mon1 were isolated from this species (leger et al. 2000). three species of p. alexandri, p. kandelakii and p. tobbi had low density in the study area and it is unlikely to have a role in vl transmission, but complementary studies are suggested to determine the proven vector(s). acknowledgements we are grateful to our colleagues in qom medicine school, qom public health center and medical entomology department of school of public health, medical sciences/university of tehran, for their kind collaboration. this project was financially supported by qom university of medical sciences. references akhavan aa, yaghoobi-ershadi mr, mohebali m, farzinnia b (2001) a preliminary survey on vectors of visceral leishmaniasis in a 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infection in human and canine in qom province, iran. iranian j armaghan danesh 9(33): 43-51 [in persian with english abstract]. guan lr, xu yx, li bs, dong j (1986) the role of phlebotomus alexandri, sinton, 1928 in the transmission of kala-azar. bull who. 64: 107–112. hamidi an, nadim a, tahvildare-bidruni g, javadian e (1982) visceral leishmaniasis of jackals and dogs in northern iran. trans roy soc trop med hyg. 76(6): 756-757. hoogstral h, heyneman d (1969) leishmaniasis in the sudan republic, final epidemiological report. ann trop med hyg. 18: 1091-1210. javadian e, nadim a (1975) studies on cutaneous leishmaniasis in khuzestan, iran. part ii: status of sand flies. bull soc path exot. 68: 467-471. leger m, saratsiotis a, pesson b, leger p (1979) la leshmaniose en greece. resaltas de une enquet entomologic effectuee en juine 1977. ann parasit hum comp. 54: 11-29. leger n, depaquit j, ferte h. rioux ja, gantier jc, gramiccia m, ludovisi a, michaelides a, christophi n, economides p 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original article structure and antibacterial activity of chitosan from the american cockroach, the german cockroach and the mealworm beetle *ebrahim cheraghi1, majid kababian1, eslam moradi-asl2, seyed mahdi mousavi bafrouyi1, abedin saghafipour3 1department of biology, faculty of sciences, university of qom, qom, iran 2department of public health, school of health, ardabil university of medical sciences, ardabil, iran 3department of public health, school of health, qom university of medical sciences, qom, iran *corresponding author: dr ebrahim cheraghi, e-mail: e.cheraghi@qom.ac.ir (received 11 nov 2021; accepted 22 nov 2022) abstract background: owing to chitosan properties such as biocompatibility and antimicrobial activities, and several applications in biomedical field, some physicochemical and anti-bacterial properties, and the level of chitosan from three species of american cockroach, periplaneta americana (dictyoptera: blattidae), the german cockroach, blattella germanica (dictyoptera: ectobiidae) and the mealworm beetle, tenebrio molitor (coleoptera: tenebrionidae) were investigated. methods: the cuticle of adults derived from specimens was dried and grounded. the powders were demineralized as well as deproteinized after deacetylation via naoh. at last, the chitosan yields from insects were studied for antibacterial activity on gram-positive bacteria (proteus mirabilis, klebsiella pneumoniae), and gram-negative bacteria (enterococcus faecalis and staphylococcus epidermidis). the fourier transform infrared spectroscopy (ftir) was used to analyze the chitosan composition. results: the chitosan ratios of the american and german cockroaches and the mealworm beetle were 5.80, 2.95, and 1.70% per 3 g of the dried bodies respectively. the chitin dd’s for the american cockroach, the german cockroach and the mealworm beetle were 36.8%, 31.5% and 27.3%, respectively. the bactericidal activity of chitosan obtained from the american cockroach at a concentration of 1% had the greatest effect on p. mirabilis compared to other concentrations, while chitosan obtained from the german cockroach at a concentration of 0.01% had the greatest effect on k. pneumoniae compared to other concentrations. conclusion: according to the results, the anti-bacterial influence of the chitosan is based upon the insect species and chitosan concentration. probably, the variation relates to the changes in the chitin structure among the three insect species. keywords: chitosan; cockroaches; tenebrio; anti-bacterial introduction chitosan, the chitin deacetylated derivatives, is a polysaccharide with a fibrous structure enormously detected in animals such as crustacean and insect exoskeletons (1). chitin and chitosan is widely attended as a result of their useful biological characteristics like biodegradability, biocompatibility, non-antigenicity, and non-toxicity (2). its exceptionally biological characteristics (anti-microbial, anti-bacterial, coagulating activities, bio-adhesivity, and wound healing capacity) caused it to be used in cosmetics, medicine and pharmacy, agriculture, food industry, and wastewater treatment (3, 4). currently, more attention has been paid to the producing of chitin and chitosan from insect sources. firstly, insects have extensive biodiversity and show 95% of the animal group (5). thus, they, as a natural source, have a great capacity to produce chitin and chitosan. in addition, the inorganic content of insect cuticles is less than that of crustacean shells, causing their demineralization to be extremely appropriate (6). the physicochemical properties of chitosan copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 326 http://jad.tums.ac.ir published online: dec 31, 2022 have been examined in several investigations employing element analysis, differential scanning calorimetry (dsc), scanning electron microscopy (sem), and x-ray diffraction patterns (7, 8). chitosan's activity depends on the yields, molecular weight (mw), level of deacetylation (dd), and amino groups (nh2) presence (9). according to research by currently, different researches have investigated the powerful anti-microbial influence of chitosan on various microorganisms, from bacteria (10) to fungi (11), parasites (12, 13), and yeasts (14) in tests including in vivo or in vitro interactions with various chitosan (solutions, films, and composites). therefore, studies on chitosan and its antimicrobial ability have recently been important (15, 16). molecular weight (mw) plus the degree of deacetylation (dd) become essential to the chitosan activity, and also on some experimental conditions, such as temperature and ph (17). basseri et al. (18) showed the degree of dd of chitin was 31% and 32.1% for the german cockroach nymphs and adults, respectively, and 39.2% and 37.3% for the american cockroach nymphs and adults. in all cases, the weight of chitosan’s produced was almost half that of chitins. they tested solutions of chitosan at the concentration of 10mg/ml on three different bacteria: escherichia coli and pseudomonas aeruginosa, as gram-negative, and staphylococcus aureus, as gram-positive species. also, chung et al. have shown the disruption of cell structure of e. coli and s. aureus due to the binding of chitosan to microbial enzymes and nucleotides (18). however, chitosan molecules show different influences on various microorganisms (19, 20). it has been suggested that the bactericidal mechanism of chitosan depends on the existence of a positively charged molecule (nh3+ sites) interacting with the negatively charged membrane of a microbial cell, causing the ammonia group to bind as a protonated molecule to the negative residues by electrostatic forces (5). chitosan has been effectively used in various fields such as environmental applications especially in water, paper, and textile treatment for antimicrobial activity, biomedical applications such as tissue engineering, wound healing, obesity treatment, preventing vascular diseases, and food industrial applications such as food packaging film, nanocapsule and nanoparticles (1). despite different investigations on the antimicrobial influences of chitosan (16, 21, 22), no consensus has yet been reached. therefore, additional investigations are needed. considering these useful functional properties of chitosan, this study focused on chitosan from edible insect, which has not been investigated sufficiently. to investigate functional properties of edible insect chitosan from the mealworm beetle (tenebrio molitor), and to find other possible uses as a new biomaterial, compared to insects of hygiene pest such as the american cockroach (periplaneta americana) and the german cockroach (blattella germanica). this study aimed to extract the chitosan from the american cockroach, the german cockroach and the mealworm beetle, to compare their structural homology, and then to measure the antimicrobial activities of the resulting extracted chitosan’s against four strains of bacteria: proteus mirabilis, klebsiella pneumoniae, enterococcus faecalis and staphylococcus epidermidis. selection of these bacteria was due to their role as nosocomial infections and human pathogenicity. according to previous studies (23), these bacteria exist symbiotically in the body of the studied insects in laboratory and environmental conditions. materials and methods sample collection the adult cockroaches were provided from the laboratory of medical entomology, tehran university of medical science (tums), and the mealworm beetle from the laboratory of entomology, university of qom. the insects were kept in an insectary at 25±2 °c with 12h: 12h light–dark cycle. their food was dried bread, date, and water. they hungered http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 327 http://jad.tums.ac.ir published online: dec 31, 2022 for 48h so that the gut contents emptied. next, they were killed through freezing at -20 °c, and the body was washed with water. they were dried through heating at 50 °c for 24h. next, the body was mechanically ground and filtered by a 20-mesh sieve (21). at last, 3g of powder of every sample was utilized to extract chitosan. extraction of chitin and chitosan chitosan was obtained from insect processing discards via a chang proposed method (24). to deproteinize, 3g of powder of species were distinctly treated with 1m hcl at 100 °c for 24h, filtered through a 20-mesh sieve, washed with distilled water, and treated with oxalic acid for 3h at ambient temperature with moderate stirring. consequently, the demineralization process was performed through filtering the treated specimens by a 20-mesh sieve as well as washing them in distilled water. the process continued with adding 50ml of 1% sodium hypochlorite solution to each sample and locating at ambient temperature for 3h with moderate stirring to eliminate the color of the samples. extracted chitins were filtered via a 20mesh sieve, washed in distilled water, and dried overnight at 60 °c. the yields were treated via 50% naoh at 100 °c by moderate stirring for 4h, then washed in distilled water and ethanol so that the acetyl group was eliminated from chitins. the procedure was repeated three times. the chitosan was dried at room temperature and was put in a clean and dry container. the chitosan was dissolved in 1% acetic acid (sigma‐aldrich, mi, usa) to obtain a starting concentration of 10mg/ml. one gram of chitosan was dissolved in 100ml of acetic acid solution by stirring for 3h (hotplate magnetic stirrer) at 50 °c. hence, the chitosan used in the succeeding assays were dissolved in 1% acetic acid. previous studies used 1% acetic acid despite its anti-bacterial activity (22, 25). different chitosan concentrations (0.01, 0.1, and 1%) were prepared through dilution of 1% stock solution. similar procedure was done to test the antibacterial activity of commercial chitosan, was purchased from sigma-aldrich (cas-no: 9012-76-4, chemie gmbh eschenstrasse, germany). fourier transform infrared (ftir) analysis to identify the composition of chitin and chitosan, and the degree of acetylation (da), the analysis of specimens was performed via fourier-transform infrared spectroscopy (ftir) (tensor 27, bruker) at the central laboratory, university of tehran (tehran, iran) at 4,000– 500cm−1 with potassium bromide (kbr) pellets. commercial chitin and chitosan obtained from sigma aldrich –were considered criteria. the wavelength range was 500–4,000cm−1 at a resolution of 4cm−1. the absorbance of the peaks was compared with that of the reference peak at a1655/a3450 (26). furthermore, the chitin deacetylation degree (dd) was evaluated. the findings of the obtained chitosan were compared to the commercial one. the deacetylation degree (dd) was found by the following equation (27): dd (%)= 100 [(a1655 / a3450)100]/1.33 in which a1655= mean% absorbance before and after wavenumber 1655. a3450= mean % absorbance of wavenumber 3450. scanning electron microscopy (sem) scanning electron microscope (sem; zeiss dsm 960a, carl zeiss, oberkochen, germany) was utilized to test the chitin surface morphology at the central laboratory, university of tehran (tehran, iran). the samples of chitosan were ground, located on an adhesive tape as well as coated with a fine gold layer via sputter coater. the sem was performed at 20.0kv. bacterial strains the bacterial strains, including p. mirabilis (atcc 43071), k. pneumoniae (atcc 1705), e. faecalis (atcc 29212), and s. epidermidis (atcc 12228) were provided with the industrial research organization of iran. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 328 http://jad.tums.ac.ir published online: dec 31, 2022 each bacterium was inoculated into an erlenmeyer flask consisting of 100ml of sterile nutrient broth (peptone 1%, beef extract 0.5%, nacl 0.5%, ph 6) and incubated at 37 °c for 24h. sterile mueller hinton agar (mha, himedia) medium was arranged in sterile petri dishes, incubated at 37 °c for 24h as well as utilized to test antibacterial activity. anti-bacterial assays the disc diffusion method was employed in this study for antimicrobial assay (28). firstly, 20μl of freshly bacterial cultures of p. mirabilis, k. pneumoniae, e. faecalis, and s. epidermidis, equal to 0.5 mcfarland prepared, were then spread uniformly onto mueller–hinton agar plates. chitosan sample discs-prepared by impregnating 50μl of chitosan solution on sterile filter paper discs (6mm diameter), were placed on the agar plates. the plates were then incubated at 37 °c for 24h using an incubator (in55, memmert, germany). the presence of inhibition zones was measured around each disc in millimeters (mm) by a metric ruler and was considered as evidence of antimicrobial activity. the experiments for each test organism were carried out in triplicate. for each plate, filter‐paper discs soaked of acetic acid and solution of commercial chitosan were used at same concentrations as positive controls, while distilled water as a negative control. statistical analysis all steps were done in triplicate. spss (version 25, ins. usa) was utilized to analyze data. results were expressed as mean ± standard error of growth inhibition zones diameters obtained with extracted chitosan, which amount was adequate for repetitions. statistical differences of diameter of growth inhibition zones between the insects-derived chitosan, the bacterium type, and concentration of extracted chitosan were determined by analysis of variance. the lsd test was used to determine the difference among means at the level of 0.05. results chitin and chitosan extraction chitin and chitosan obtained from 3g of dried insect powder differed in terms of insect species. comparably, the chitosan yield amount was nearly half of the chitin one (fig. 1). the chitosan ratios per 3g of the dried body in the american cockroach, the german cockroach and the mealworm beetle were 5.8, 2.95, and 1.7%, respectively. the degrees of chitin deacetylation (dd) of all selected samples are calculated by ftir analysis and shown in table 2. the chitin deacetylation degree for the american cockroach, the german cockroach and the mealworm beetle was 36.8%, 31.5%, and 27.3 %, respectively; illustrating that the extracted chitosan of german cockroach is more deacetylated than the other chitosan (fig. 1). scanning electron microscopy of extracted chitosan under electron microscopic examination, the extracted chitosan’s of the american cockroach, the german cockroach and the mealworm beetle showed similar microfibrillar structure. however, commercial chitosan did not exhibit an apparent microfibrillar structure. the extracted chitosan’s of the american cockroach and the german cockroach exhibited rough and thick surface morphology more than the mealworm beetle (fig. 2). at the sem photographs, chitin of the american and german cockroaches (fig. 2a, b) markedly arranged in a microfibrillar crystalline structure was obvious compared to chitin of the mealworm beetle (fig. 2c). fourier transform infrared spectroscopy analysis based on the ftir graph, the molecules of chitin and chitosan of three groups consist of the same stretching, bending vibration bands with various infrared spectrum graphs (fig. 3), indicating decreased peaks because of the absorbing, which shows a loss of acetyl group and chitin deacetylation. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 329 http://jad.tums.ac.ir published online: dec 31, 2022 the absorption bands of spectra at 1560– 1630cm−1 (amid i stretching in c=o) and 1370– 1400cm−1 (nh2 binding) determined two prominent amide bands. the absorption band at 1010– 1030cm−1 shows c–o–c stretching vibrations existing in chitosan molecules (fig. 3). the existence of a chitosan absorption band of the c-h stretching, bending vibration along with the c–o–c stretching vibrations, particularly in the specimen from the mealworm beetle, are presented in fig. 3. the absorption bands are created via the stretching, c-h bending vibrations existing in their chitosan molecules. the broad and wide wavelength on commercial chitosan from the american cockroach, the german cockroach and the mealworm beetle (with a peak of 3249.1, 3260.9, 3250.2, and 3210.2cm−1, respectively) show the existence of hydroxyl group (o-h) in total samples, while sharp peaks at 2900.47cm−1, 2919.36cm−1, 2917.49cm−1 and 2910.12cm−1 of the commercial plus obtained chitosan from the american cockroach, the german cockroach and the mealworm beetle, respectively, show strong existence of alkanes in the specimens. this supports the chitosan correct chemical structure mainly composed of a c-c single bond. anti-bacterial activities analysis table 2 shows the antibacterial activities of chitosan obtained from the insects. the findings indicate the effect of the extracted chitosan on gram-positive and gram-negative bacteria, including p. mirabilis, k. pneumoniae, e. faecalis and s. epidermidis. also, the zone of inhibition for different concentrations of chitosan against the tested bacteria is presented in table 2. the extracted chitosan shows different levels of antibacterial activity on gram-positive bacteria versus gram-negative bacteria. the results showed that the antibacterial activity of chitosan obtained from the american cockroach with a concentration of 1% had the most significant effect on p. mirabilis (gram-negative), when was compared to standard chitosan (p= 0.000). the antibacterial activity of chitosan obtained from the american cockroach at a concentration of 1% had the most significant effect on p. mirabilis compared to other concentrations of chitosan extracted from the american cockroach (p= 0.000). the antibacterial activity of chitosan obtained from the american cockroach with a concentration of 0.1% had the most significant effect on s. epidermidis (gram-positive), when was compared to standard chitosan (p= 0.003). also, the results showed that the antibacterial activity of chitosan obtained from the american cockroach with a concentration of 1% and 0.01%, almost with the same inhibition zone, had the most significant effect on k. pneumoniae (gram-negative) compared to the concentration of 0.1% (p= 0.000). overall, the antibacterial activity of chitosan obtained from the american cockroach at a concentration of 1% compared to other concentrations had the most significant effect on p. mirabilis (gram-negative) than other bacteria (p= 0.000) (table 2). the results showed that the antibacterial activity of chitosan obtained from the german cockroach with a concentration of 1 and 0.1% had the most significant effect on p. mirabilis (gram-negative), when was compared to standard chitosan (p= 0.000). the antibacterial activity of chitosan obtained from the german cockroach at a concentration of 0.01% had the most significant effect on k. pneumoniae (gramnegative) compared to standard chitosan (p= 0.000). also, the results showed that the antibacterial activity of chitosan obtained from the german cockroach with a concentration of 0.01% had the most significant effect on s. epidermidis (gram-positive) compared to other concentrations (p= 0.002). overall, the antibacterial activity of chitosan obtained from german cockroach at a concentration of 0.01% compared to other concentrations had the most significant effect on k. pneumoniae (gram-negative) than other bacteria (p= 0.000) (table 2). however, the antibacterial activity of chitosan obtained from the mealworm beetle had http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 330 http://jad.tums.ac.ir published online: dec 31, 2022 the least effect compared to others, so that only 1% concentration had the most significant effect on p. mirabilis (gram-negative), when was compared to standard chitosan (p= 0.003) (table 2). the results showed that the antibacterial activity of chitosan obtained from the american cockroach at a concentration of 1% had the greatest effect on p. mirabilis (gram-negative) compared to other bacteria in different concentrations of chitosan obtained from insects. also, the results showed that the antibacterial activity of chitosan obtained from the german cockroach with a concentration of 0.01% had significant effect on both grampositive and gram-negative bacteria. in addition, the results showed that insect-derived chitosan has a great inhibitory effect on gramnegative compared to gram-positive bacteria (table 3, fig. 4). the mean± sem of diameter of growth inhibition zone (mm) were measured and recorded as recommended by world health organization 2003 (49). in addition, all significant values of antibacterial activity between chitosan of cockroach and beetles compared to standard chitosan are given in brackets. here, superscript a stands for the best of growth inhibition zone among bacteria types in the same concentration from the same insect's extracted chitosan, and superscript b stands for the best of growth inhibition zone among the different concentrations of the extracted chitosan in the same insect that affected on the same bacteria, and superscript c stands for the best of antibacterial activity among the same bacteria and same concentration of the extracted chitosan from different insects. the rest of the cases that are not mentioned p value, were not significant compared to standard chitosan. the experiment was conducted in triplicate. table 1. the degree of deacetylation of the insect’s chitin using infrared spectra analysis at 4,000–500cm−1 insect species a1655 a3450 dd german cockroach 0.153 0.182 36.8 american cockroach 0.126 0.149 31.5 mealworm beetle 0.175 0.181 27.3 fig. 1. the yields of chitin and chitosan obtained from 3-g insects’ powder after extraction process http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 331 http://jad.tums.ac.ir published online: dec 31, 2022 table 2. anti-bacterial activities of yield chitosan extracted from adults of insects measured based on the diameter of growth inhibition zone (mm) chitosan extracted concentration of chitosan (%) zones of growth inhibition (g-) proteus mirabilis (p value) (g-) klebsiella pneumoniae (p value) (g+) enterococcus faecalis (p value) (g+) staphylococcus epidermidis (p value) american cockroach 1 12.2±0.3 (0.000) a, b, c 10.2±0.2 (0.000) b 6.2±0.1 8.2±0.2 (0.000) 0.1 8.2±0.2 9.2±0.1 7.2±0.2 7.2±0.2 (0.003) a 0.01 10.2±0.2 (0.003) 10.2±0.2 (0.000) a 8.2±0.1 8.2±0.2 german cockroach 1 11.2±0.3 (0.000) a 9.2±0.2 (0.008) 8±0.3 8.2±0.2 (0.000) 0.1 11.2±0.1 (0.000) a 10.2±0.2 7.2±0.1 7.2±0.2 (0.003) 0.01 8.2±0.1 11.2±0.3 (0.000) a, b, c 8.2±0.2 9.2±0.2 (0.002) b, c mealworm beetle 1 9.2±0.2 (0.003) a, b 8.2±0.1 7.2±0.2 6.2±0.2 0.1 9.2±0.2 9.2±0.2 6.2±0.1 6.2±0.2 0.01 8.2±0.2 9.2±0.2 6.2±0.2 8.2±0.1 standard (commercial chitosan) 1 8.2±0.2 8.2±0.1 8.2±0.2 6.2±0.2 0.1 9.2±0.3 10.2±0.2 8.2±0.1 6.2±0.2 0.01 9.2±0.2 9±0.0 8.2±0.2 8±0.3 fig. 2. sem micrographs of chitosan extracted from adults of insects: a) standard (commercial chitosan), b) american cockroach, c) german cockroach, d) mealworm beetle http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 332 http://jad.tums.ac.ir published online: dec 31, 2022 table 3. the best of anti-bacterial activity yields the concentration of extracted chitosan from adults of insects on the gram-positive and gram-negative bacteria chitosan source gram-negative (concentration%) gram-positive (concentration%) american cockroach 12.2±0.3 (1) 8.2±0.2 (1) german cockroach 11.2±0.3 (0.01) 9.2±0.2 (0.01) mealworm beetle 9.2±0.2 (1) 8.2±0.1 (0.01) standard (commercial chitosan) 10.2±0.2 (0.1) 8.2±0.1 (0.1) fig. 3. infrared spectra of commercial and extracted chitosan. a) standard (commercial chitosan), b) american cockroach, c) german cockroach, d) mealworm beetle http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 333 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 4. comparison of the growth inhibitory effect of different concentrations of extracted chitosan from insects on gram-positive and gram-negative bacteria. a) american cockroach, b) german cockroach, c) mealworm beetle http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 334 http://jad.tums.ac.ir published online: dec 31, 2022 discussion in this research, chitin and chitosan of the american cockroach, the german cockroach and the mealworm beetle were prepared and partly determined. then, the dd was measured. afterward, bactericidal activities of the chitosan yield were studied. in the investigation, the antimicrobial activity of the obtained chitosan was varied among these insects. furthermore, the degree of polymerization and crystallinity of chitin and chitosan of the american cockroach, the german cockroach and the mealworm beetle were various. according to several previous studies on chitin yield from other insects depending on growth stage, the chitin yield ranged from 5.3 % to 36.6% as follows: seven orthoptera species contained 5.3–8.9% (29), holotrichia parallela contained 15% (30), ranatra linearis contained 15–16% (31), and cicada contained 36.6 % (32). in this study, the chitin yield from cockroaches was higher than from the mealworm beetle. physicochemical characteristics, rheological characteristics as well as surface morphology of chitosan of cicada slough, silkworm chrysalis, mealworm, or grasshopper were compared to shrimp shell chitosan. according to findings, the chitosan activities of insects are completely distinct from shrimp chitosan (5). furthermore, the chitosan anti-microbial activities are based upon dd (33). in this case, the chitosan of insects is indicated to be much viscous compared with shrimp shell chitosan having a high deacetylation degree. typically, part of chitosan bacteriostatic and bactericidal activity is based upon viscosity. low viscosity is very efficient (5, 34). chitosan dd of three insects was varied due to variation of chitosan antibacterial characteristics. the main element of the insect integument has usually been known to be an efficient substitute source having organic materials like chitin, especially the cuticle having decreased inorganic materials (35). compared to commercial chitosan’s yield from shrimp (26), the chitosan yields of the cockroaches were high within demineralization and deacetylation processes. while insects including cockroaches may be abundant and accessible chitin sources of chitin and chitosan, rearing them is a limiting factor for industrialization. the cockroach species-derived chitins illustrated the same physiological characteristics. they seem to be appropriate for chitosan production. while the chitin-chitosan yield from the american cockroach increases, the chitin /chitosan dd was somewhat high in the german cockroach-derived specimens. the variation of molecular weights of chitosan obtained from both groups leads to these findings. the finding is consistent with previous findings (21, 36). according to the literature, surface morphology is one of the most important properties that determines the efficient use of chitin and its derivatives (4). the best usage area for chitin can be determined according to its surface morphology. the number of pores in the chitin surface increased the chitin’s ability to absorb metal ions, while the chitin that has a fibrillar surface morphology can be used in textiles (29). in addition, a porous structure means the chitin can be a useful agent for tissue engineering (4). it can be seen from previous studies that the surface morphologies of chitin and its derivatives obtained from crab, krill, insects and fungi are quite different (29). in this experiment, the extracted chitosan’s of the american cockroach and german cockroaches exhibited rough and thick surface morphology with a microfibrillar crystalline structure, which was like the findings of previous studies (26, 30). at the sem photographs, chitin of the american and german cockroaches markedly arranged in a microfibrillar crystalline structure was obvious compared to chitin of the mealworm beetle. similar results can be seen in the sem photographs of the beetle chitin from cicada sloughs (32). the ftir results suggest that there was a similarity between the chemical composition and the bonding types of chitosan in the extracted chitosan’s and commercial chitosan. the present findings showed http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 335 http://jad.tums.ac.ir published online: dec 31, 2022 that the peaks at around 1560–1630cm-1 and 1370–1400cm−1, which correspond to (c= o) in the nhcoch3 group (amide i band) and (nh2) in the nhcoch3 group (amide ii band), respectively, were characteristic of chitosan. these present findings are consistent with previous reported (21, 30). the peaks displayed at around 1010-1030cm-1 were attributed to the β (1–4) glycosidic bond in the polysaccharide unit and the stretching vibrations of c‐o‐c in the glucose ring. these findings are similar to previous reports (21, 30, 36). additional broad absorption bands observed at 2900–3250cm-1 were attributed to symmetric stretching vibrations of the o‐h and alkane caused by the strong intermolecular hydrogen bonding of chitosan polysaccharides. these present findings are consistent with previous reports (21, 30). in this research, it was determined that the chitosan of groups inhibited the growth of gram-positive and gram-negative bacteria. generally, many elements can have influenced the bactericidal activity strength of chitosan, such as the molecular weight, deacetylation degree, chitosan concentration in solution, or ph of medium culture (37). its anti-bacterial effect has been said to be highly dependent upon its molecular weight in lieu of the dd (38). reduced molecular weight indicated great inhibiting influence on gram-positive, gram-negative bacteria and the yeast (39). however, chitosan anti-microbial characteristics are based upon various factors and may cause different results mentioned by several authors. thus, the bactericidal activity of chitosan is slightly debatable. chitosan is said to have high bactericidal activity on gram-positive bacteria in comparison to gram-negative ones (15, 40). conversely, some authors mentioned, owing to the hydrophilicity of chitosan, gram-negative bacteria are very susceptible to chitosan compared to gram-positive ones (37). in the current research, among the examined bacteria, gram-negative ones became exceedingly susceptible to the cockroach chitosan. it is assigned to a high deacetylation degree of chitosan. some authors have noted chitosan influence on gram-positive bacteria is greater in comparison to gram-negative ones (41, 42). the bacterial influence on gram-positive along with gram-negative bacteria is somewhat debatable. in contrast, hydrophilicity in gram-negative bacteria has been illustrated to considerably increased in comparison to in gram-positive bacteria, causing them to be susceptible to chitosan (43). the results are proven through many in vitro tests where gramnegative bacteria are significantly susceptible to chitosan, indicating enhanced morphological changes in treatment in comparison with gram-positives (44-46). chitosan from the mealworm beetle showed slight inhibition zones against bacillus cereus, listeria monocytogenes and e. coli, and also slight inhibition zone against s. aureus in antimicrobial activity test (47). this finding is consistent with our findings. also, this experimental study showed for the first time that chitosan from the mealworm beetle has antimicrobial activity against pathogenic bacteria such as on gram-positive bacteria (p. mirabilis, k. pneumoniae) and gramnegative bacteria (e. faecalis, s. epidermidis). a crucial factor is establishing the adsorbed chitosan level is the charge density on the cell surface (48). the chitosan binding to microbial dna is the additional suggested mechanism. this results in suppressing the mrna plus protein synthesis by the chitosan entry to the nuclei of the microorganisms (45). another mechanism is the metal chelation, spore component inhibition as well as binding to essential nutrients for microbial growth (15). the gramnegative bacteria cell wall is very complicated but thinner than that of gram-positive ones. it includes a semi-permeable outer membrane locating on a peptidoglycan layer suppressing the antibiotic penetration into the cell (22). it is an asymmetric lipid bilayer consisting of lipopolysaccharide (lps). interaction between chitosan and gram-negative bacteria via electrostatically interacting with the negatively charged lps changing permeability (22). http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 325–339 e cheraghi et al.: structure and … 336 http://jad.tums.ac.ir published online: dec 31, 2022 conclusions based on the results, we found the amount of chitosan yield and the degree of deacetylation depended on the species of insects. the anti-bacterial influence of the chitosan is based upon the cockroach species. the chitosan obtained from cockroaches, especially the american cockroach, showed a high impact of inhibition on the gram-negative bacteria. the variation likely is because of variations in 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case report the first report of eustigmaeus johnstoni (acari: stigmaeidae) parasitic mite of phlebotominae sand flies from iran mehdi badakhshan 1, javid sadraei 1, *vahideh moin-vaziri 2 1department of medical parasitology and entomology, college of medical sciences, tarbiat modares university, tehran, iran 2department of parasitology and mycology, faculty of medicine, shahid beheshti university of medical sciences, tehran, iran (received 10 may 2011; accepted 1 nov 2011) abstract background: stigmaeids mites have been recorded only on phlebotominae sand flies up to now. five species of eustigmaeus, and three of stigmaeus were reported on infested sandflies in different country up to the present. methods: sand flies collection was done using cdc light trap and sticky paper. the mites were isolated from infested specimens, mounted in puri’s medium and identified using reliable keys. results: a mite infested phlebotomus papatasi was observed during a study on sandflies of one of the southern provinces of iran, near to the persian gulf. several scars resulting from mite attachment were found on abdominal tergites of this female sand fly. the mites were identified as eustigmaeus johnstoni. conclusion: this parasitic mite is one of the eyeless species, which has a great distribution over the world, reported from tunisia, saudi arabia, pakistan, yemen, cyprus and palestine. but, this is the first record of this species from iran. keywords: eustigmaeus johnstoni, phlebotomus papatasi, sand flies, mite, iran introduction five species of eustigmaeus and three species of stigmaeus (both stigmaeidae), also one species of dasythyreus (dasythyreidae) have been reported as parasitic mites on insects yet. stigmaeids mites have been recorded only on sand flies eustigmaeus dyemkoumai (abbonec 1970), e. gamma, e. gorgasi and e. parasitica (chaudri 1965), e. johnstoni (zhang and gerson 1995), (shehata and baker 1996), stigmaeus smithi (mitra and mitra 1953), s. sinaei (swift 1987) and s. youngi (hirst 1926 and wood 1972) were reported as parasitic mites of different species of phlebotominae sand flies comprising phlebotomus and sergentomyia spp. mites of genus eustigmaeus berlese, 1910 (prostigmata: stigmaeidae) comprise a group of globate, red acarines whose their bodies are covered by ornamental armour. in females this dorsal armour is separated into propodosomal and hysterosomal plates, the latter being often subdivided in the males. females carry thirteen pairs variously-shaped dorsal setae, of which three pairs are often ventrally displayed: the humerals (c2) laterally and the posteriormost b1 and b2 caudally. most species have one pair of eyes, located between propodosomal setae ve and sci. the eyeless species include e. lirella, e. parasitica, e. gamma, e. gorgasi and e. johnstoni (zhang and gerson 1995). parasitic mite, e. johnstoni has a broad distribution and was reported from different countries including yemen, saudi arabia, palestine, cyprus, tunisia and pakistan (eddie et al. 2006). however, this is the first report of this *corresponding author: dr vahideh moin-vaziri, email: vmvaziri@gmail.com 94 j arthropod-borne dis, june 2013, 7(1): 94–98 m badakhshan et al.: the first report of… http://jad.tums.ac.ir published online: april 10, 2013 mite from iran. several reports from different parts of the world provide this evidence that the eustigmaeus-sand fly association is not accidental (zhang and gerson 1995). materials and methods sand fly collection was done using cdc light trap and sticky trap in july 2010. we observed a sand fly (out of 286 collected specimens) which infested with mite, during a study on the phlebotominae of zirah village (bushehr province) located south of iran, in 2010. the mites were identified using the keys of zhang and gerson (1995) and fan and zhang (2005). setal terminology follows kethley (1990) and zhang and gerson (1995). also some of the morphological characters were measured using gradient lens. all measurements are in micron. accordind to fan and zang 2005 the following characters were used to identify the eustigmaeus spp from the other genus of stigmaeidae (key to genus eustigmaeus, also key to eyeless eustigmaeus species are mentioned at the end of the article). palptibial claw prominent, sub equal to palptarsus; with 2 pairs of subcapitular setae, setae e1 and f1 situated on same shield in female (except villersiella), without genital setae, chelicerae separate, prodorsal and dorsal hysterosomal shields separate, setae sce situated on main prodorsal shield and setae d1 and e1 situated on same shield. results the infested sand fly was identified as phlebotomus papatasi (female). at least three mites (also female) were observed on the parasitized specimen, one near to the legs and two others attached on the abdomen (fig. 1a and 1b). several scars were observed resulting from mite attachment. the scars occurred only on the abdomen, particularly the anterior tergites. they had irregular outline, dark in color with a pale border (fig. 2). according to the keys, the mite, was identified as eustigmaeus johnstoni zhang and gerson, 1995 (acai: stigmaeidae) (fig. 3a, 3b, 3c, 3d and 3e). according to zhang and gerson (1995) variation in the lengths and distances between dorsal setae are evident in specimens from different countries. as table 1 shows our measurements are comparable with the extent that he was stated, but due to the small number of mite in our access, the statistical comparison was not possible and meaningful. fig. 1. eustigmaeus johnstoni, located near the legs of infested phlebotomus papatasi (a) and two others attached to its abdomen (b) iran, 2010 95 j arthropod-borne dis, june 2013, 7(1): 94–98 m badakhshan et al.: the first report of… http://jad.tums.ac.ir published online: april 10, 2013 table 1. measurements (µ ) of dorsal setae and setal distances in eustigmaeus johnstoni (female), iran, 2010 in comparison with other countries (zhang et al. 1995) yemen saudi arabia cyprus tunis pakistan iran n vi 22–32 19-23 24 21 21 25 1 ve 23-28 19-21 23 22 21 27 14 sci 18-23 15-20 21 21 19 20 15 sce 22-28 19-23 21 21 21-23 25 13 c1 22-26 19-23 21 23 21 27 14 c2 22-28 19-23 22 23 22-23 18 15 d1 19-28 19-21 21 23 21-22 26 14 d2 19-26 17-21 21 21 20-21 22 15 e1 22-28 21-23 23 21 20-21 23 14 e2 20-23 19-23 23 19 20-21 22 15 f 24-35 21-25 28 24 23-24 28 14 b1 22-32 20-26 24 21 24 24 15 b2 23-24 19-23 21 19 21 25 13 vi-vi 32-34 23-32 30 28 26-27 31 13 ve-ve 54-56 41-49 47 47 42-50 58 13 sci-sci 91-95 80-86 86 86 78-81 100 13 sce-sce 112-120 100-114 109 103 105 130 13 c1-c1 51-60 41-54 50 50 52-53 54 12 d1-d1 40-56 43-52 54 53 47-52 57 13 d2-d2 133-140 106-128 126 116 116 135 13 e1-e1 44-49 34-45 43 42 40 48 12 e2-e2 110-116 84-111 100 92 90 120 12 f-f 56-59 48-60 54 50 52-57 65 11 b1-b1 24-26 19-25 23 22 17-21 25 12 b2-b2 56-71 49-63 58 54 54 70 12 fig. 2. abdominal scars left on female phlebotomus papatasi which was infested by three eustigmaeus johnstoni mite, iran, 2010 fig. 3. parasitic mite, eustigmaeus johnstoni, on phlebotomus papatasi from iran, 2010, a. idiosoma, dorsal view, b. idiosoma, ventral view, c. upper part of idiosoma, palp and legs, d. chelicera, e. setae with long branches 96 j arthropod-borne dis, june 2013, 7(1): 94–98 m badakhshan et al.: the first report of… http://jad.tums.ac.ir published online: april 10, 2013 discussion parasitic mite, e. johnstoni has a broad distribution and was reported from different countries, however this is the first report of this parasitic mite from iran. members of stigmaeidae are soil or plant dwelling mites which are regarded as predators of other small arthropods (swift 1987). little is known about the relationship between mites and sand flies, whether this association is phoretic or parasitism? but several reports of scars, left by mites on infested sand flies (mitra and marta 1953, abbonec 1970, lewis and macfarlane 1982) strengthen the parasitic relationship. however, the nature of eustigmaeus-sand fly association remains indeterminate. there is no report of males or immature of eustigmaeus on phlebotominae sandflies. it could be postulated that development and mating of the mentioned mite were done elsewhere, probably in the habitat where sandflies breed and rest. so, additional work at different times of the year also in other areas, with investigations of sand fly breeding and resting sites are essential to provide further insight to the significance of the mite-sand fly association. also the exact nature of these parasitic mites infestation is poorly understood and further works are required to determine if such heavily infected sand flies have reduced longevity. key to eyeless eustigmaeus genus (zhang and gerson 1995) 1dorsal hysterosomal setae c1 and d1 short, not reaching the bases (alveoli) of any neighboring setae...................................... 2 dorsal hysterosomal setae c1 and d1 long, over reaching the bases (alveoli) of at least two neighbouring setae........................... 4 2distance between alveoli of dorsal setae pairs, c1-c1, d1-d1 and e1-e1 subequal........... 3 distance c1-c1 and d1-d1 subequal, both mach less than e1-e1 (setae e1 laterally displaced) ………….…… eustigmaeus gamma 3dorsal setae flat, with short barbs on distal ¾; all intercoxal setae (1a, 3a and 4a) weakly barbed…………...……….. e. lirella dorsal setae thin, with long branches throughout; intercoxal setae (1a, 3a, and 4a) with long branches………….... e. johnstoni acknowledgements the authors wish to thank dr zhi-qiang zhang and prof. leger for their kind collaboration in providing some key references. this study was financially supported by the department of medical parasitology and entomology, college of medical sciences, tarbiat modares university, also department of medical parasitology and entomology, faculty of medicine, shahid beheshti university of medical sciences. the authors declare that there is no conflict of interests. references abbonec e (1970) notes sur les acarines parasites des phlebotomus. cahiers l’office de la recherche scientifique et teqnique oter-mer. entomol med parasit. 8: 89–94. berlese a (1910) acari nuovi. manipulus v, vi. redia 6: 199–234 + plates xviii– xxi. chaudhri wm (1965) new mites of the genus ledermuelleria. acarol. 7: 467–486. eddie au, harten a, magdalena kp (2006) the mites and ticks (acari) of yemen: an annotated check-list. fauna of arabia. 22: 243–286. fan q, zhang zq (2005) fauna of new zealand. manaaki whenua press, lincoln, canterbury, new zealand. 97 j arthropod-borne dis, june 2013, 7(1): 94–98 m badakhshan et al.: the first report of… http://jad.tums.ac.ir published online: april 10, 2013 hirst s (1926) report on the acari found on or associated with sand flies in india. indian j med res. 13: 1023–1026 kethley j (1990) acarina: prostigmata (actinedida). in: dindal dl (ed) soil biology guide, vol. 1. john wiley, new york, pp. 667–756. koch cl (1836) deutschlands crustaceen, myriapoden und arachniden. ein beitrag zur deutschen fauna. 4: 9. (herrich-schäffer, regensburg). lewis dj, macfarlane d (1982) the mites of phlebotomine sandflies (diptera: psycodidae). in: canning e.u. (ed) society of protozoology. special publication, vol 1. allen press, kansas, pp. 177–183. mitra crd, mitra sd (1953) a new species of raphignathus (acarina) associated with phlebotomus in india. zeitschrift für parasitenkunde. 15: 429–432. shehata m, baker a (1996) mites infesting phlebotomine sand flies in southern sinai, egypt. med vet entomol. 10(2): 193–196. swift sf (1987) a new species of stigmaeus (acari: prostigmata: stigmaeidae) parasitic on phlebotomine flies (diptera: psychodidae). int j acarol. 13(4): 239–243. wood tg (1972) redescription of stigmaeus youngi (hirst), acari, stigmaeidae. acarol. 14(2): 163–165. zhang zq, gerson u (1995) eustigmaeus johnstoni, new species (acari: stigmaeidae), parasitic on phlebotomine sandflies (diptera: psychodidae). tijdschrift voor entomologie. 138: 297– 301. 98 microsoft word 6-dr_nasirian_1st_edite.doc iranian j arthropod-borne dis, (2008), 2(1): 37-43 h nasirian: short-term german cockroach … 37 original article rapid elimination of german cockroach, blatella germanica, by fipronil and imidacloprid gel baits h nasirian department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran (received 17 feb 2007; accepted 1 jan 2008) abstract background: baits have become popular and effective formulations against urban insect pests. compared with residual sprays toxic gel baits are used more and more frequently to control urban cockroach populations. the aim of this study was to investigate the usage of two commercially available fipronil and imidacloprid gel bait formulations against blattella germanica field infested in iran. methods: the study was carried out in an urban area at tehran from march 2004 to september 2005. the 0.05% fipronil and 2.15% imidacloprid gel baits were placed continuously in 3 residential german cockroach infested units. preand post-treatment cockroach density was assessed by visual count method. results: preand post-treatment visual count of cockroaches in treatment and control areas, and percentage reduction in cockroach density in treatment areas in comparison to control areas was showed that density reduction was increased with the 0.05% fipronil and 2.15% imidacloprid gel baits in treated areas from 1st to 9th week in comparison to control area. after 60 days, german cockroaches eliminated completely from these areas. conclusion: these results show that fipronil and imidacloprid gel baits are highly effective in field german cockroach infested after insecticide spraying control failure german cockroach infested fields where spraying of pyrethroid insecticides failed to control the situation and confirm previous reports stating that avermectin and hydramethylnon are more effective than conventional insecticides in baits against cockroaches. therefore, fipronil and imidacloprid gel baits are appropriate candidates for controlling german cockroach infested dwellings in iran where control with other insectices failed because of resistance. keywords: blattella germanica, fipronil, imidacloprid, gel bait, elimination introduction german cockroaches, blattella germanica (l.), are the most common urban cockroaches found in houses and restaurants and remain one of the most economically and medically important pest. it is omnivorous and consumes a wide range of food types and may hitchhikes into the house on food material cartons, sacks of potatoes or onions, used furniture or appliances, beer cases, etc. produce departments, pawn shops, nursing homes and other such places are constantly fighting german cockroaches (as a general rule) and are notorious for being the source of residential infestations (cochran 1999). it is proven or suspected carrier of the organisms causing diarrhea, dysentery, cholera, leprosy, plague, typhoid fever (czajka et al. 2003) and viral diseases such as poliomyelitis (prado et al. 2002). in addition, they carry the eggs of parasitic worms and may cause allergic reactions including dermatitis, itching, swelling of the eyelids and serious respiratory conditions. in addition to a major source of indoor allergens *correspondence: hassan nasirian, tel/fax: +98 21 88951393, e-mail: hanasirian@yahoo.com iranian j arthropod-borne dis, (2008), 2(1): 37-43 h nasirian: short-term german cockroach … 38 and responsible for increased incidence of asthma (roberts 1996, eggleston and luisa 2001, katial 2003), there is emphasis to the need for the control of this insect pest. sanitation is a critical step in german cockroach control therefore water residues should be eliminated as many moisture sources as possible. all cockroach food sources should be eliminated. searching for new insecticides and new methods of insecticide delivery to control the heavy infestations of german cockroach continues as another important tool. until recently, the control of german cockroach relied largely on sprays based on synthetic insecticides. with the development of baits, which can be selectively applied where the german cockroach live, the situation has now changed. compared with residual sprays, baits take advantages of long residual activity, safer application and less environmental pollution. cockroach baits that are currently available include such formulations as granular, pelleted, containerized, pastes and gels, and have been improved due to their efficient non-repellant active ingredients, highly attractive and palatable food ingredients, as well as their high moisture content. toxic gel baits are used more and more frequently to control urban cockroach populations (appel 1990, koehler et al. 1995, cochran 1999). gel baits can be selectively used in sensitive areas, such as premises used for the preparation of food, hospitals and kindergartens (benson and zungoli 1997). gel baits have been the main method for german cockroach control in the united states for at least 5-8 yr (harbison et al. 2003). gel baits are proven to be convenient to use and highly effective to german cockroach control (appel 1992, ross 1993, appel and benson 1995, kaakeh et al. 1997, appel and tanley 2000). when the active ingredient is incorporated into palatable bait, cockroaches readily consume a lethal dose from a single meal. therefore, baits were considered less likely to select for high-level cockroach resistance than insecticide sprays and other formulations. fipronil is a disruptor of the insect central nervous system via the gaba channel, acting with contact and stomach action. it blocks the gaba-gated chloride channels of neurons in the central nervous system, resulting in neural excitation and death of the insect. it is used against cockroaches, ants, termites, fleas, ticks, and mites in several formulations including sprays, baits, granules, dusts, and in flea and tick collars. it is the active ingredient in frontline, termidor, maxforce ant gel, and various other brand names (moschetti 2004). imidacloprid is an insecticide belonging to the chloronicotinyl class of compounds and its use as a new crop protection agent was first proposed in 1991 (leicht 1993). because it exerts its effects after oral ingestion, imidacloprid is also suitable for use in bait formulations (londershausen 1996). imidacloprid exerts no contact effects in gel form and does not vaporize into the surrounding atmosphere. this is of practical benefit, as it means there is no contamination of the environment by the active ingredient, and that the cockroaches must continue to have gel available to them until no more is being eaten. imidacloprid generally has low toxicity to mammals (acute and chronic), birds, and fish. the low affinity of chloronicotinyl for vertebrate relative to insect nicotinic receptors is a major factor in their favorable toxicological profile. as to its performance: good reliable control, high selectivity, quick knock-down/protection and long residual activity are key features (cox 2001). the efficacy of toxic gel baits has been widely investigated, but their toxicity is generally estimated only from mortality rates (scott 1991, ross 1993, kaakeh et al. 1997b). a number of laboratory studies have reported efficacy of fipronil and imidacloprid gel baits in control of cockroach infesiranian j arthropod-borne dis, (2008), 2(1): 37-43 h nasirian: short-term german cockroach … 39 tation but only a few field studies have been done so far. nasirian et al. (2006) reported that the fipronil and imidacloprid gel baits completely killed the german cockroaches under laboratory conditions in ingested bait method (nasirian et al. 2006). very little field information is available about the efficacy and performance of fipronil and imidacloprid gel baits against field b. germanica infestation in iran. the present study was thus conducted with the aim of evaluating the efficacy of two commercially available fipronil and imidacloprid gel bait formulations in the control of b. germanica field infested in iran. materials and methods study area and duration the study was carried out in 4 residential units in an urban area at tehran from march 2004 to september 2005. the residential units were located in different areas of tehran. of these 4 residential units, 3 were selected for treatment and 1 for control. sanitation field cockroaches infested were managed by controlling the availability of food, water, and hiding places. food and water were not left out overnight and stored so as to deny access by the cockroaches. all spilled foods, including crumbs on the floor were cleaned up. trial procedure the evaluated insecticide were imidacloprid gel bait 2.15%, bayer ag leverkusen, fipronil gel bait 0.05%, commercialized as goliath, rhone-poulenc rhodic, lyon, france. pre-treatment assessment in the field in all residential unit included in the study following pre-treatment assessment was done: visual counts the visual counts were done in the night after 24.00 h. the participant residential units were asked to close the residential units by 23.00 h and switch off the light. the lights were switched on after 24.00 h and cockroaches were counted as they ran about hiding over tables, sink, cooking areas, etc. light from a torch was thrown behind cabinets, storage areas and dish racks for counting the cockroaches. a 5 minute count of cockroaches was taken (tilak et al. 2002). visual counts more than 75, between 25 to 75, and less than 25 graded as severe, moderate, and mild infestations, respectively. insecticide treatment the 0.05% fipronil and 2.15% imidacloprid gel baits (fipronil gel 0.05%, commercialized as goliath, rhone-poulenc rhodic, lyon, france and imidacloprid gel bait 2.15%, bayer ag leverkusen) were placed continuously in 3 residential units german cockroach infested. the fipronil and imidacloprid gel baits were placed simultaneously inside split, seams, under sink and in the soft drink lids as a container where no split exist and seams by linear or point application. the gel baits repeated periodically every week. post-treatment density post-treatment density was assessed by visual count method every week up to nine weeks, since it was found to be a better indicator of cockroach infestation. the visual assessment data in treatment and control residential units were considered for calculation of the percentage of reduction in cockroach infestation in residential units using the following formula of mulla (mulla 1971). % reduction= 100(c1/t1 x t2/c2) x 100. where, c1 is the number of cockroaches in control residential unit pre-treatment; t1 is the number of cockroaches in treatment residential unit pretreatment; c2 is the number of cockroaches in control residential unit post-treatment; and t2 is the number of cockroaches in treatment residential unit post-treatment. results the 0.05% fipronil and 2.15% imidacloprid gel baits were placed continuously in iranian j arthropod-borne dis, (2008), 2(1): 37-43 h nasirian: short-term german cockroach … 40 3 residential units german cockroach infested. preand post-treatment visual count of cockroaches in the treatment and control areas and percentage of reduction in cockroach density are presented in tables 1 and 2. it was observed that 25.0, 23.6 and 26.5% reduction was achieved with the 0.05% fipronil and 2.15% imidacloprid gel baits in treated area 1, 2 and 3, respectively by 1st week in comparison to control area. percentage reduction increased in the 0.05% fipronil and 2.15% imidacloprid gel baits treated areas 1, 2 and 3, 45.6, 53.0 and 51.8 percent, respectively by week 3 post-treatment and it was 100 percent by the end of week 9 post-treatment. after 60 d german cockroaches eliminated completely from these areas. discussion extensive use of insecticides has led to the development of resistance in german cockroach to a wide range of insecticides including organochlorines, organophosphates, carbamates and pyrethroids (lee et al. 1996, cochran 1997, ladonni 2001, nasirian et al. 2006b) and consequent control failures in some field populations have been reported (cochran 1989, scott et al. 1990, atkinson et al. 1991, valles and yu 1996, dong et al. 1998, valles 1999, valles et al. 2000, wei et al. 2001). fipronil and imidacloprid are relatively new and act at new target sites, which are currently not affected by resistance than other previous insecticides that will be used for pest control especially against german cockroach. these results show that fipronil and imidacloprid gel baits are highly effective in field german cockroach infested after insecticide spraying control failure with pyrethroid group insecticides and confirm the previous investigator reports that reported the avermectins (cochran 1985), boric acid (kocak 1990) and hydramethylnon (mac donald et al. 1987) are to be more effective than conventional insecticides in baits against cockroaches, and it will be suggested that fipronil and imidacloprid gel baits are highly effective in field german cockroach infested. therefore, fipronil and imidacloprid gel baits are the appropriate candidates for controlling the infested german cockroach control failure dwellings of insecticide resistance in iran. table 1. cockroaches density estimation in control and treatment areas by visual assessment method visual count treatment with fipronil and imidacloprid gel baits duration area 1 area 2 area 3 control area pre-treatment post-treatment (wk) 115 214 189 138 1 95 180 153 152 2 68 125 95 145 3 39 75 68 103 4 25 36 29 136 5 17 25 18 175 6 12 10 13 204 7 8 5 7 193 8 3 1 2 169 9 0 0 0 139 iranian j arthropod-borne dis, (2008), 2(1): 37-43 h nasirian: short-term german cockroach … 41 table 2. comparison of reduced percentage of cockroach density in treatment vesus control areas percentage reduction in fipronil and imidacloprid gel baits treated areas in comparison to control area post-treatment weeks area 1 area 2 area 3 1 25.0 23.6 26.5 2 43.7 44.4 52.2 3 45.6 53.0 51.8 4 77.9 82.9 84.4 5 88.3 90.8 93.6 6 92.9 96.8 95.1 7 95 98.3 96.8 8 97.9 99.6 99.1 9 100 100 100 acknowledgments the author would like to thank dean of school of public health and head of medical entomology and vector control department, tehran university of medical sciences for their support to carry out this research. references appel ag (1990) laboratory and field performance of consumer bait products for german cockroach (dictyoptera: blattellidae) control. 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accepted 15 nov 2014) abstract background: dengue is amongst the most serious mosquito-borne infectious disease with hot spots in tropical and subtropical parts of the world. unfortunately, no licensed vaccine for the disease is currently available in medicine markets. the only option available is the management of dengue vector mosquito, aedes aegypti (diptera: culicidae). method: predatory potential of five odonate nymphs namely anax parthenope, bradinopyga geminate, ischnura forcipata, rhinocypha quadrimaculata, and orthetrum sabina were evaluated against the 4th instar larvae of the dengue vector mosquito, aedes aegypti, under laboratory conditions. the consumption of the mosquito larvae was evaluated at three water volume levels viz., 1 liter, 2 liter and 3 liter. results: the number of ae. aegypti larvae consumed varied significantly among the five species, and at different levels of water volume (p< 0.01). however, the interaction between odonate nymphs and the water volumes was statistically non-significant (p> 0.05). ischnura forcipata consumed the highest number of ae. aegypti larvae (n=56) followed by a. parthenope (n=47) and b. geminate (n=46). the number of larvae consumed was decreased with increasing search area or water volume, and the highest predation was observed at 1-liter water volume. conclusion: the odonate nymphs could be a good source of biological agents for the management of the mosquitoes at larval stages. keywords: biological control, dragonflies, damselflies, mosquitoes, dengue vectors introduction dengue is amongst the most serious mosquito-borne infectious disease with hot spots in tropical and subtropical parts of the world. unfortunately, no licensed vaccine for the disease is currently available in medicine markets (kovendan et al. 2012). the only option available is the management of the mosquito, aedes aegypti (diptera: culicidae), which is a vector of deadly diseases like dengue fever, chikungunya and yellow fever (khan and akram 2013). different chemical measures such as indoor residual sprays, larviciding, insecticide treated bed nets and fogging are prioritized for the management of dengue mosquitoes worldwide (zia et al. 2012), however, these measures are linked with serious environmental concerns like the development of insecticide resistance and environmental pollution (bilal et al. 2012). moreover, recent reports on the development of insecticide resistance in different mosquito species including dengue vector mosquitoes (khan et al. 2011, rathore et al. 2013) stress the need to explore alternate measures. naturally, occurring aquatic predators have been assumed a significant ecological factor in regulating different mosquito species. for example, the predators such as amphibians (ohba et al. 2010), copepods (marten and reid 2007), crustaceans (su and mulla 2002), odonates (mandal et al. 2008), water bugs (aditya et al. 2004), wolf spiders (futami et al. 2008) and *corresponding author: dr hafiz azhar ali-khan, e-mail: azhar_naturalist@yahoo.com j arthropod-borne dis, june 2016, 10(2): 253–258 w akram and ha ali-khan: odonate nymphs: … 254 http://jad.tums.ac.ir published online: january 05, 2016 backswimmers (rodriguez-castro et al. 2006) have shown their tendency to feed on and regulate different mosquito species in aquatic habitats like ponds and paddy fields (kweka et al. 2011). of these stated predators, odonates (insecta: odonata) have been explored less for their predatory potential both in the asian and world perspective (mandal et al. 2008). to the best of authors’ knowledge, aquatic predators, particularly odonate nymphs have not been explored to much extent against mosquitoes in pakistan. the odonate nymphs usually co-exist with many mosquito species immatures, and their long nymphal stage (1 year or more) and competitive predatory ability (corbet 1980), offer a good opportunity to use them as biological agents. therefore, the present study focused on the comparative evaluation of predatory potential of the different odonate nymphs against the larvae of ae. aegypti. the results presented provide a baseline for the field experiments, and possibility to include these predators in environment friendly management plans for the mosquito control. materials and methods a field collected population of ae. aegypti from lahore (31° 32′ 59 n; 74° 20′ 37 e) was reared under laboratory conditions (25± 2oc, 65± 5% rh) as described previously (khan et al. 2011). briefly, the mosquito larvae and adults were collected from artificial containers and natural habitats and reared in the laboratory by standard rearing procedures. the larvae were reared in steel trays approximately 3 inch deep and fed on tetramin (artificial diet) until the adults emerged. early-instar naiads/nymphs of five odonate species (insecta: odonata) namely anax parthenope (family aeshnidae), bradinopyga geminate (libellulidae), ischnura forcipata (coenagrionidae), rhinocypha quadrimaculata (chlorocyphidae), and orthetrum sabina (libellulidae) were collected from ponds and rice fields by using aquatic dip nets. the nymphs were identified by following fraser (1933), anjum (1997) and nesemann et al. (2011), and were kept in distilled water under the laboratory conditions. before predation experiments, the nymphs were provided chironomid larvae for feeding. a feeding bioassay was performed by following the methodology of mandal et al. (2008) with some modifications. before starting the experiment, the nymphs were starved for a period of 6 hours. a single nymph of each odonate species was introduced into water bowl (4-liter capacity) containing distilled water and one hundred 4th instar larvae of ae. aegypti. the consumption rate of the nymphs was evaluated at three different water levels viz., 1 liter, 2 liter and 3 liter, and the number of mosquito larvae consumed was noted after 24 h of the introduction of the nymphs into the bowl. the experiment was replicated at six different times, using the new nymphs and mosquito larvae. all the data on consumption rate by the odonate nymphs at three different water levels were analyzed by 2-way analysis of variance using the software statistix 8.1v (analytical software 2005) and means were compared with the least significant difference test. p< 0.005 was considered signifant. results the number of ae. aegypti larvae consumed varied significantly among the five species of odonate nymphs (f= 144.30, df= 4, 75, p<0.001 ), and at different levels of water volume (f= 18.32, df= 2, 75, p< 0.001). however, the interaction between odonate nymphs and the water volumes was statistically non-significant (f= 0.32, df= 8, 75, p= j arthropod-borne dis, june 2016, 10(2): 253–258 w akram and ha ali-khan: odonate nymphs: … 255 http://jad.tums.ac.ir published online: january 05, 2016 0.96). ischnura forcipata consumed the highest amount of ae. aegypti larvae (55.89) followed by a. parthenope (47.22) and b. geminate (46.06) (fig. 1). the number of larvae consumed by different odonate species was decreased with increasing search area or water volume. the highest consumption of the larvae was observed at 1 liter water volume (46.90) followed by 2 liter (44.56) and 3 liter (42.27) volumes (fig. 2). fig. 1. rate of consumption of 4th instar aedes aegypti larvae by different odonate nymphs fig. 2. cumulative effect of different different water volumes on the consumption rate of 4th instar aedes aegypti larvae by different odonate nymphs j arthropod-borne dis, june 2016, 10(2): 253–258 w akram and ha ali-khan: odonate nymphs: … 256 http://jad.tums.ac.ir published online: january 05, 2016 discussion in the present study, predatory potential of five different odonate nymphs has been evaluated. the predator-prey relationship could have a significant impact in an ecosystem by affecting population dynamics and energy flow through food webs. predators could affect prey populations directly through prey consumption (khan et al. 2012). the only mosquito species, which have been investigated in the present study, is ae. aegypti. recently this species along with ae. albopictus played havoc in different parts of pakistan. to manage these pests different measures have been adopted with the major focus on chemical control. resultantly, occurrence of field evolved resistance in mosquitoes and other public health pests have been reported which stressed the need to explore alternate management tools (khan et al. 2011, khan et al. 2013). in the present study, predatory potential of five different odonate nymphs has been evaluated. the results showed that the nymphs were able to consume ae. aegypti voraciously, however, increasing the volume of water had a negative effect on the consumption rate, perhaps due to the evasion tactics of the mosquito larvae (bhattacharjee et al. 2009). since ae. aegypti mosquitoes usually lay eggs and complete immature stages in small water volumes (vezzani et al. 2005), the findings of the study are of worth importance. previously, some researchers have evaluated the potential of odonate species against different mosquito species (mandal et al. 2008, kweka et al. 2011) but such studies are rare in pakistan. our results are in agreement with those of mandal et al. (2008) who evaluated different species of odonates against cx. quinquefasciatus and found that i. forcipata was the most voracious feeder of the mosquito larvae. they further reported that the volume of the water had a negative impact on predation efficiency. the negative effect of increasing water volume has also observed with hemipteran bug species (saha et al. 2008) and larvivorous fish species (ghosh et al. 2005, bhattacharjee et al. 2009). with increasing water volume, the aquatic predators possibly required more time to search, capture and ultimately consume the mosquito larvae (ghosh et al. 2006). in myanmar (sebastian et al. 1990) and india (mandal et al. 2008) the augmentative releases of different odonate species have regulated ae. aegypti and cx. quinquefasciatus mosquitoes, respectively. the lengthened developmental time of odonate nymphs (i.e. 1 year or more from egg to adult) and predation ability (corbet 1980) provide an opportunity to use these predators in the management plans designed for ae. aygypti. conclusion keeping in view the high consumption rate of the larvae per 24 h, these predators could be assumed to feed on a good number of ae. aegypti larvae during their long nymphal stage. although the species used in the present study varied in their consumption rate, all of the species could be considered for inclusion in the management plan. however, there is a need to explore the predatory potential of the species in the field and in different ecological zones. acknowledgements the authors declare that there is no conflict of interests. references aditya g, bhattacharyya s, kundu n, saha gk, raut sk (2004) predatory effij arthropod-borne dis, june 2016, 10(2): 253–258 w akram and ha ali-khan: odonate nymphs: … 257 http://jad.tums.ac.ir published online: january 05, 2016 ciency of the water bug sphaerodema annulatum on mosquito larvae (culex quinquefasciatus) and its effect on the adult emergence. biores technol. 95: 169–172. analytical software, statistix version 8.1: user's manual. analytical software, tallahassee, florida, 2005. anjum sa (1997) biosystematics of odonata naiads of the punjab by rearing technique. 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larvae (odonata: libellulidae) with community participation in yangon, myanmar. bull entomol res. 80: 223–232. su t, mulla ms (2002) spatial occurrence and hatch of field eggs of the tadpole shrimp triops newberryi (notostraca: triopsidae), a potential biological control agent of immature mosquitoes. j vector ecol. 27: 128–137. vezzani d, rubioa a, velazquez sm, schweigmanna n, wiegand t (2005) detailed assessment of microhabitat suitability for aedes aegypti (diptera: culicidae) in buenos aires, argentina. acta tropic. 95:123–131. zia k, hafeez f, ashfaq m, akram w, khan haa (2012) larvicidal action of four indigenous plant extracts against dengue vector aedes aegypti. pak entomol. 31: 93–97. j arthropod-borne dis, december 2014, 8(2): 224–227 a grzywacz et al.: first palaearctic record … 224 short communication first palaearctic record of the bird parasite passeromyia heterochaeta (diptera: muscidae) from the iranian persian gulf islands andrzej grzywacz 1, *mehdi khoobdel 2, kamran akbarzadeh 3 1chair of ecology and biogeography, faculty of biology and environment protection, nicolaus copernicus university, lwowska, poland 2health research center, baqiyatallah university of medical sciences, tehran, iran, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 31 mar 2013; accepted 25 jan 2014) abstract background: passeromyia is a muscid genus previously known from the old world afrotropical and oriental regions and eastwards from australia and the west pacific. the genus is known from nest-dwelling larvae which may be parasites of the nestlings. this study was aimed to identify of the passeromyia species in the iranian persian gulf islands. methods: the flies were collected during april 2010 to march 2011 on the 4 iranian persian gulf islands, greater tunb, lesser tunb, abu-mousa and qeshm with fly bottle trap and entomological net. results: during this sampling, 18 representatives of p. heterochaeta, a species with free-living haematophagous larvae, have been collected. the species is reported herein from greater tunb, lesser tunb, abu-mousa and qeshm islands. conclusions: this is the first palaearctic record of the species as well as the first report of the genus passeromyia from the palaearctic region. keywords: passeromyia heterochaeta, bird parasite, palaearctic, iran, persian gulf, new record introduction passeromyia rodhain and villeneuve, 1915 is a small muscid genus known from only five valid species (pont 1974). representatives of the genus occur in the old world afrotropical and oriental regions as well as australia and the west pacific (pont 1974). nests-dwelling, trimorphic larvae act as scavengers or parasites of the nestlings (pont 1974, skidmore 1985). in muscidae, larval haematophagous behaviour has also been reported in philornis meinert, 1890, but the genus is restricted to the new world. in both genera larvae may realize their behaviour as free-living saprophages or either free-living or under skin haematophages. among representatives of the genus passeromyia, p. heterochaeta (villenueve 1915) is the only species present in the afrotropical region (couri 2007). the species is widespread in this region, and to date was reported from botswana, burundi, democratic republic of the congo, kenya, nigeria, senegal, south africa, tanzania, uganda, zambia, zimbabwe (pont 1974). in the oriental region p. heterochaeta was reported from burma, china, india, indonesia (sumatra), sri lanka, taiwan (pont 1974). the biology and natural history of p. heterochaeta has been relatively well studied. larvae of p. heterochaeta pierce the skin of the nestlings and subsequently feed as a free-living hematophages, although *corresponding author: dr mehdi khoobdel, e-mail: khoobdel@yahoo.com  http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 224–227 a grzywacz et al.: first palaearctic record … 225 were also observed feeding on dead nestlings, penetrating the body and in the nostrils of the host (pont 1974). the species was reported from a wide range of hosts and detailed list of those was provided by pont (1974), nevertheless, impact of larval activity on certain host species population is unknown. this study was aimed to identify and record of the passeromyia species in the iranian persian gulf islands. materials and methods insects were collected during a sampling period from april 2010 to march 2011. the area of entomological survey included iranian persian gulf islands belonging to hormozgan province in southeastern iran: greater tunb (26o30'n–55o16'e, 10.3 km2), lesser tunb (26o14'n–55o08'e, 2.1 km2), abu-mousa (25o51'–26o19' n, 54o26'–55o19' e, 68.8 km2) and qeshm (26o32'–27o06'n, 55o15'–56o30'e, 1491 km2). flies were collected with fly bottle trap and entomological net, killed with ethyl acetate vapours, pinned, labelled and identified by the first author (ag) according to pont (1974) and couri (2007). species identification of selected specimens was verified based on the collection of the natural history museum of denmark (copenhagen, denmark). reference specimens were deposited in the collection of the health research center, baqiyatallah university of medical sciences, tehran, iran and the chair of ecology and biogeography, nicolaus copernicus university, toruń, poland. photos were taken with nikon 8400 digital camera mounted on a nikon smz 1500 stereomicroscope (nikon corp, tokyo, japan). figure was prepared with combine zp image stacking software. results during the time of an entomological survey on the iranian persian gulf islands, carried out from april 2010 to march 2011, a total of 18 specimens representing p. heterochaeta were collected (table 1). this is the first report of bird parasite species p. heterochaeta from the area of the persian gulf. representatives of passeromyia can be differentiated from other houseflies based on the following combination of characters: large truncate lower squama, bare anepimeron and prosternum, meron below posterior spiracles covered with hairs, plumose arista and bowshaped wing vein m1, long antennae and dichoptic males (pont 1974). adults of p. heterochaeta are moderately large and robust flies (body length 8–9 mm). the ground colour of thorax and abdomen is black, however the scutellum is yellow on apical half or more. passeromyia heterochaeta can be discriminated from other species of the genus by the shifting priunose pattern on the abdomen, marked vittae on the dorsal surface of thorax, and both ash-grey dusted (pont 1974). eyes are haired, halters brown, calypters whitish and wings are hyaline (fig. 1). table 1. passeromyia heterochaeta collected from the iranian persian gulf islands from april 2010 to march 2011 totaloctober...junmayaprilmonth island 21♀1♂abu-mousa 32♀1♀greater tunb 11♀lesser tunb 111♂2♀1♂, 7♀1♀qeshm 1813113total http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 224–227 a grzywacz et al.: first palaearctic record … 226 fig. 1. female of passeromyia heterochaeta, lateral view discussion soós and papp (1986) in the catalogue of palaearctic diptera defined the borders of the palaearctic region based on the already defined borders of the afrotropical and oriental regions. for a consistency we followed these limits of the palaearctic region herein, hence according to soós and papp (1986) “in north africa and the arabian peninsula the border extends in the sahara along the tropic of cancer and joins south western confines of pakistan”. thus southern iran and particularly iranian persian gulf islands are discerned as a part of the palaearctic region. summarizing, this is the first report of both the species p. heterochaeta and the genus passeromyia from the palaearctic region, hitherto unknown from this region (pont 1986). evidence of range expansions have recently been provided for some species of dipterans of primary medical and veterinary importance (e.g. grassberger et al. 2003, szpila et al. 2008, velasquez et al. 2013). some representatives of philornis, recently introduced to galápagos islands, have been revealed as causing serious mortality in birds, particularly darwin’s finches (o’connor et al. 2010). conclusion passeromyia heterochaeta larvae exhibit parasitic behaviour and the species was hitherto unknown from the persian gulf area. future studies are necessary to establish particular distribution of p. heterochaeta and its effect on the local avian fauna. acknowledgements the authors declare that there is no conflict of interest. this study was financially supported by baqiyatallah university of medical sciences, tehran, iran (project no bmsu/hrc-87.128). references couri ms (2007) a key to the afrotropical genera of muscidae (diptera). rev bras zool. 24: 175–184. grassberger m, friedrich e, reiter c (2003) the blowfly chrysomya albiceps (wiedemann) (diptera: calliphoridae) as a new forensic indicator in central europe. int j leg med. 117: 75–81. pont ac (1974) revision of the genus passeromyia rodhain and villeneuve (diptera: muscidae). bull br mus nat hist entomol. 30: 339–372. pont ac (1986) family muscidae. in: soós a, papp l (eds) catalogue of palaearctic diptera. scatophagidae-hypodermatidae, volume 11. akadémia kiadó, budapest, pp. 57–215, 1–345. o’connor ja, sulloway fj, robertson j, kleindorfer s (2010) philornis downsi parasitism is the primary cause of nestling mortality in the critically endangered darwin’s medium tree finch (camarhynchus pauper). biodiv conserv. 19: 853–866. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 224–227 a grzywacz et al.: first palaearctic record … 227 skidmore p (1985) the biology of the muscidae of the world. ser entomol 29. w. junk publishers, dordrecht, pp. 64–72. soós á, papp l (1986) catalogue of palaearctic diptera, scathophagidaehypodermatidae, vol. 11. akadémiai kiadó, budapest, pp. 6–8. szpila k, matuszewski s, bajerlein d, konwerski s (2008) chrysomya albiceps (wiedemann 1819), a forensically important blowfly (diptera: calliphoridae) new for the polish fauna. pol j entomol. 77: 351–355. velasquez y, ivorra t, grzywacz a, martinez-sanchez a, magana c, garciarojo a, rojo s (2013) larval morphology, development and forensic importance of synthesiomyia nudiseta (diptera: muscidae) in europe: a rare species or just overlooked? bul ent res. 103: 98–110. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 168 http://jad.tums.ac.ir published online: january 05, 2016 original article crimean-congo hemorrhagic fever in the one-humped camel (camelus dromedarius) in east and northeast of iran mohsen champour 1, sadegh chinikar 2, *gholamreza mohammadi 1, gholamreza razmi 1, ehsan mostafavi 2, nariman shah-hosseini 2, sahar khakifirouz 2, tahmineh jalali 2 1faculty of veterinary medicine, ferdowsi university of mashhad, mashhad, iran 2arboviruses and viral hemorrhagic fever laboratory (national reference lab) pasteur institute of tehran, iran (received 3 aug 2013; accepted 22 feb 2015) abstract background: this comprehensive study was conducted on multi-purpose one-humped camel (camelus dromedarius) sera and ticks to assess the epidemiological aspects of the crimean-congo hemorrhagic fever virus (cchfv) in northeast iran. methods: from may 2012 to january 2013, eleven cities were randomly selected in the khorasan provinces of iran as “clusters,” and at least 14 one-humped camels were sampled from each area. reverse transcriptase polymerase chain reaction was used for the detection of the cchfv genome in ticks. sera were analyzed using specific enzymelinked immunosorbent assay tests. results: four hundred and eighty ixodid ticks were collected, and the genome of the cchfv was detected in 49 (10.2%) out of 480 ticks. the cchfv genome was detected in two out of four tick species, and in tick samples from three cities in khorassan-e-jonoobi. all three provinces, and six out of eleven cities, were cchfv-specific iggpositive. in total, nine (5.3%) out of 170 one-humped camels were igg-positive. the highest rate of igg-positive samples was found in nehbandan (16.67%). conclusion: continued surveillance and strictly enforced importation and quarantine practices should be implemented to prevent human exposure and the on-going dispersal of infected ticks and livestock in these regions. it is recommended that acaricides be used to prevent cchf transmission to humans, and to reduce the tick population. in addition, care should be taken by abattoirs workers and people who work with one-humped camels. keywords: epidemiology, survey, cchfv, elisa, rt-pcr, iran introduction “crimean-congo hemorrhagic fever virus belongs to the genus nairovirus in the bunyaviridae family, and is a human pathogen that can cause a severe, and often fatal, hemorrhagic fever.” (ergonul 2006). cchf virus is the most geographically widespread tick-borne virus of medical importance (ergonul 2006). the disease is endemic in large areas of sub-saharan africa and the middle and far east, as well as in eastern europe. a significant increase of cases in countries such as kosovo, albania, turkey, iran, and greece has recently been observed (alavi-naini et al. 2006). the cchfv genome has been isolated from at least 31 different tick species in the ixodidae (hard ticks) and argasidae (soft ticks). hyalomma spp ticks are considered the most important in the epidemiology of cchfv; however, the virus has also been isolated from ticks of other genera (i.e., rhipicephalus, boophilus, dermacentor, haemaphysalis, and ixodes spp, (saijo et al. 2002, tahmasebi et al. 2010). infected animals are, however, asymptomatic (papa et al. 2009). humans in high-risk occupations *corresponding author: dr gholamreza mohammadi, e-mail: gmohamad@um.ac.ir j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 169 http://jad.tums.ac.ir published online: january 05, 2016 (e.g., slaughterhouse workers, shepherds, health care workers, and veterinarians) are prone to cchf infection (garcia et al. 2006). this study was performed to ascertain the prevalence of cchfv in ticks present on the one-humped camel (camelus dromedarius), and to estimate the prevalence of cchfv igg in the one-humped camel's sera in three provinces: khorassan-e-shomali, khorassane-razavi, and khorassan-e-jonoobi of iran. materials and methods study area the study was conducted in three provinces: khorassan-e-shomali, khorassan-erazavi, and khorassan-e-jonoobi located at 55°17′, 61°15′e and 30°24′, 38°17′n in northeastern and east of iran (fig. 1). khorassan-e-shomali is a mountainous region, with temperate, cold weather. khorassan-erazavi is a semi-desert region that has mild weather. khorassan-e-jonoobi is a semidesert region that experiences arid conditions. the average annual rainfall is approximately 300–400 mm in the northern areas (khorassan-e-shomali) and 150 mm in the central and southern areas (khorassan-erazavi and khorassan-e-jonoobi). there are approximately 25 million camels in the world, and nearly 150,000 one-humped camels are in iran, this is 0.6% of the world camel population, and 3.8% of the asian camel population (fao 2011). the majority of iran's camels are dromedaries, and they are scattered across the country’s provinces. in the khorassan provinces the camel population is 37,400 (ministry of agriculture jihad, 2002), but in the authors’ experience at present the actual number is several times greater, the majority of these are in khorassan-e-shomali and khorassan-e-jonoobi. sampling from may 2012 to january 2013, eleven cities and towns were randomly selected from three provinces: khorassan-e-shomali, khorassan-e-razavi and khorassan-e-jonoobi as “clusters,” and at least 14 one-humped camels were sampled from each cluster. from each animal, two or three ticks were collected and placed in separate sterile tubes; the tubes were labeled with the date of collection, animal number, sex, age, and area. collected ticks were sent to the laboratory and identified under a stereo microscope using general identification keys (hoogstraal 1979, apanaskevich and filippova 2007). the samples were then pooled according to the area, sex and species of tick (each pool routinely contained 1–8 ticks, but occasionally the number was greater) and immediately sent to the arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory), pasteur institute of iran, and stored at −70 °c until analysis. for the serum assay, 20 ml of blood collected from the jugular vein of each camel then the sample was labeled with the date of collection, animal number, sex, age, and area. the samples were immediately sent to a laboratory and centrifuged at 5000 rpm for 10 min. the sera were then separated and transferred into holding tubes and sent to the arboviruses and viral hemorrhagic fevers laboratory and stored at −70 °c until analysis. tick sample size calculation according to previous studies in animals, tick infectious was 10% and with assumption 5% accuracy, 95% confidence interval and design effect equal 1.5 we need at least 207 ticks. serum sample size calculation with assumption 20% seropositivity in the studied animals, 8% accuracy, 95% degree of confidence, and design effect equal 1.5, the sample size will be 144, each cluster has at least 14 sera. j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 170 http://jad.tums.ac.ir published online: january 05, 2016 molecular detection reverse transcriptase polymerase chain reaction: ticks were individually washed twice by pbs (pbs, ph 7.4) and crushed with a mortar and pestle in 200-300μl of pbs. total rna was extracted using an rneasy mini kit (qiagen, cat no. 2215716) according to the manufacturer's instructions. the extracted viral rna was stored at −70 °c until analysis. for the rt-pcr, a master mix was prepared as follows: 28 μl of rnase free water, 10 μl of buffer (5 x conc), 2 μl of dntp mixture, 2μl of enzyme mixture containing reverse transcriptase and taq dna polymerase enzymes, 1μl of primer f2 (5'tggacaccttcacaaactc-3'), 1μl of primer r3 (5'-gacaattccctacacc3'), 1μl of rnase inhibitor and 5μl of extracted viral rna as template. the f2 and r3 primers amplify a 536 bp fragment inside the s-segment of the cchfv genome, 536 bp fragment is rt-pcr target. the thermal cycling program for the rt-pcr, included 30 min at 50 °c for reverse transcription reaction (cdna synthesis), 15 min at 95 °c for activation of hot star taq dna polymerase and inactivation of reverse transcriptase, followed by 35 cycles of 95 °c for 30 sec, 50 °c for 30 sec, 72 °c for 45 sec, and a final extension at 72 °c for 5 min. for gel-based rt-pcr product analysis, 5μl of the pcr product was mixed with 1μl loading buffer (6 x conc). then, the mixture was load in agarose gel 1.5%, and visualized with ethidium bromide (chinikar et al. 2004, chinikar et al. 2010). igg-sandwich elisa for igg antibody detection, the elisa plates were coated overnight at 4 °c with mouse hyperimmune ascitic fluid diluted at 1:1000 in 0.05% tween 20-pbs containing 5% skim milk as a saturating reagent. this solution was used to dilute antigen and sera. the native or the recombinant antigen (produced in our laboratory) diluted in pbstm (pbs containing 0.05% tween and 3% skim milk) was added to the plates and the plates were incubated for 1h at 37 °c and extensively washed. serum samples diluted in pbstm were added, and the plates were incubated for 1h at 37 °c. after washing, the peroxidase-labeled anti-human or animal immunoglobulin diluted in pbstm was added to each weel and the plates were incubated for 1 h at 37 °c. the plates were then washed 3 times with pbs containing 0.5% tween (pbst). finally, hydrogen peroxide and tetramethylbenzidine (tmb) were added and the plates were incubated for 15 min at room temperature. the enzymatic reaction was stopped by the addition of sulphuric acid (4n) and the plates read by elisa reader (anathos 2020) at 450 and 620 nm. taken together, an igg-positive serum was considered as positive control and a negative serum taken as negative control in the igg elisa (garcia et al. 2006, duh et al. 2008, chinikar et al. 2010). statistical analysis data were analyzed using ibm/ spss version 20.0 statistics package. descriptive statistics (i.e. prevalence and percentages) were used to summarize the quantitative variables. location of noted research is shown on the map (fig. 1). results a total of 200 one-humped multi-purpose camels (rearing for milk, meat, riding and offspring) were examined. tick infestation was observed in 170 of them, and 480 ixodid ticks (133 females and 347 males) were collected from different regions in the khorassan-e-shomali, khorassan-e-razavi and khorassan-e-jonoobi (table 1). in the current study, only four species of hyalomma genus were observed. population frequency of h. dromedarii (90.7%) was j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 171 http://jad.tums.ac.ir published online: january 05, 2016 higher than others and h. asiaticum had the lowest frequency (0.4%). hyalomma marginatum comprised about 2.9% and h. anatolicum accounted for 6% of total collected species. h. dromedarii is the most dominant tick species of camel in the khorassan region and a one humped camel is a suitable host. the life cycle of this tick includes one, two, or three hosts. immature ticks feed on small or large mammals, depending on their life cycle. hyalomma dromedarii, h. anatolicum and h. marginatum were collected from all three provinces but in contrast, h. asiaticum only was collected from khorassan-e-razavi's one-humped camels (table 2). the cchfv genome was found in 49 (10.2%) of 480 ticks, and three (6%) of 50 pools. therefore, the tick prevalence was 10.2%. all of the cchf-positive ticks were male. the cchfv genome was detected in two out of four tick species, and of these, 42 (85.7%) belonged to h. dromedarii and 7 (14.3%) belonged to h. anatolicum (table 2). the viral genome was detected in tick samples from three cities in khorassan-ejonoobi. the positivity rates were as follows: boshroyeh, 25 out of 480 (51%), birjand, 17 out of 480 (34.7%), and nehbandan, 7 out of 480 (14.3%, table 1). sera from 170 one-humped camels were collected. all three provinces, and six out of eleven cities and towns, were igg-positive for cchfv. nine (5.3%) out of 170 camels were igg-positive, this means that the igg prevalence was 5.3%. the positivity rates for the provinces varied, and were as follows: boshroyeh, 12.5%, kanimani, 7.14 %, birjand, 8%, nehbandan, 16.67%, chehl dokhtaran, 7.14%, and sabzevar, 6.66%. the highest rate of igg-positive samples was found in nehbandan (16.67%, two out of 12 sera), khorassan-e-jonoobi. eight out of the nine positive samples were collected from female camels (table 3). fig. 1. study area in khorassan-e-shomali, khorassan-e-razavi and khorassan-e-jonoobi are shown with asterisks j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 172 http://jad.tums.ac.ir published online: january 05, 2016 table 1. positivity rates, number, and sex of ticks collected from one-humped camels from khorassan-e-shomali, khorassan-e-razavi and khorassan-e-jonoobi in east and northeast iran area no males no females total rtpcr positive nehbandan 27 9 36 7 sarayan 28 23 51 0 birjand 41 7 48 17 kanimani 26 24 50 0 boshroyeh 52 7 59 25 robatsang 32 0 32 0 quchan 28 13 41 0 sabzevar 18 13 31 0 mashhad 31 17 48 0 chehl dokhtaran 28 6 34 0 mangale 36 14 50 0 total 347 133 480 49 table 2. the sex, species and cchf-positive rate of ticks infesting one-humped camels tick spp male female total cchf positive h. dromedarii 307 128 435 42 h .marginatum 10 4 14 0 h. anatolicum 29 0 29 7 h. asiaticum 1 1 2 0 total 347 133 480 49 table 3. igg antibody-positive sera collected from one-humped camels from khorassan-e-shomali, khorassan-erazavi and khorassan-e-jonoobi area no. sera no. igg positive local positivity nehbandan 12 2 16.67 sarayan 13 0 0 birjand 25 2 8 kanimani 14 1 7.14 boshroyeh 16 2 12.5 robatsang 16 0 0 quchan 14 0 0 sabzevar 15 1 6.66 mashhad 16 0 0 chehl dokhtaran 14 1 7.14 mangale 15 0 0 j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 173 http://jad.tums.ac.ir published online: january 05, 2016 fig. 2. amplification of the s segment of the cchfv genome using rt-pcr, in tick samples from the khorasan province. (pc: positive control, nc: negative control, s1-10: samples, s1, s2, s3, s4, s5, s6, s7 and s9 are negative, s8 and s10 are positive) discussion in this study, h. dromedarii was the most dominant species of tick on one-humped camels, and this is in agreement with the results obtained by salimabadi (2010) in iran and lawal et al. (2007) in nigeria. crimeancongo hemorrhagic fever infection was detected in 49 (10.2%) of 480 tick samples and this is a higher percentage than that previously reported by salim-abadi (3.79%) in the yazd provinces of iran (2011). all the positive samples were obtained from hyalomma spp. hyalomma ticks are the primary vectors for the transmission of cchfv throughout europe, asia, the middle east, and africa (ergonul 2006). although hyalomma ticks are considered the most important vector and reservoir for the cchfv, the virus has also been reported in other tick genera (tahmasebi et al. 2010). the cchfv genome was detected in two out of the four tick species that were collected (h. dromedarii, 85.7% and h. anatolicum, 14.3%). this result is similar to that found by salim-abadi et al. (2011), therefore, these results suggest that h. dromedarii and h. anatolicum act as vectors for cchfv in one-humped camels. hyalomma dromedarii is distributed throughout the world wherever camels are present, and h. anatolicum is widely distributed throughout iran. hyalomma anatolicum transmits at least five arboviruses, and is a significant vector of cchfv to humans (nabian and rahbari 2008). telmadarraiy et al. (2010) detected the cchfv genome in rhipicephalus bursa (in one of the three ticks that were sampled), but we failed to discover r. bursa in our study. in the present study, we only found the cchfv genome in one-humped camel ticks in khorassan-e-jonoobi (birjand, boshroyeh, and nehbandan). this province has an unusual geographical location, as it borders afghanistan to the east, the sistan-va-baluchistan province of iran in the south, and the khorassan-e-razavi province of iran in the north. since 2000, the disease has infected 23 out of 31 provinces in iran: sistanva-baluchistan (283 confirmed human cases), isfahan (44 confirmed cases), fars (26 confirmed cases), tehran (17 confirmed cases), and khorasan (12 confirmed cases) (chinikar et al. 2012). notably, sistan-va-baluchistan province (south of khorassan-e-jonoobi) has not just had the highest number of cchfv cases, but cchf infections have been observed in this area since 2000 (chinikar et al. 2012), as it shares a border with two cchf-endemic countries: pakistan and afghanistan (chinikar et al. 2010). the unusual location of khorassan-e-jonoobi, which is connected in the north, south, and east to heavily infected or endemic areas of cchf, may explain why it was the only cchfvpositive area found in the study. we could not ascertain why all the reverse transcriptase polymerase chain reaction-positive ticks were male, but this may have been due to the presence of more males than females on the animals, and also in our samples (347 males vs 133 females). during the last decade, an increasing number of human cchfv infections have j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 174 http://jad.tums.ac.ir published online: january 05, 2016 been reported in various regions of iran (chinikar et al. 2008). igg-positive serum samples collected from sheep, goats, cows, and humans have been frequently reported in different parts of the country (saidi et al. 1975, chinikar et al. 2005, moradi et al. 2008). we found cchfv igg antibodies in one-humped camel sera in all three provinces studied, and these results, as well as those from a previous study by chinikar et al. (2012), may indicate that cchf is endemic to these regions, or that it has spread from neighboring countries. in total, nine (5.3%) out of 170 camels were igg positive. this finding is not in accordance with the results obtained by saidi et al. (1975), who found that cchf samples were negative in all 157 camels that they had studied, from south and southeast iran. however, williams et al. (2000) found that 17 (16%) out of 109 camels sampled in oman were tested positive for the cchf igg antibody. sistan-va-baluchistan has been the most cchfv-infected province in iran since 2000, because, as mentioned previously, it shares a border with two cchf-endemic countries, pakistan and afghanistan (izadi et al. 2006, chinikar et al. 2012). khorasan, which is connected to heavily infected or endemic areas of cchf, shares a large border area with neighboring countries, shares common pasture with herds from cchf-endemic areas, and experiences the illegal importation of animals across the border, which may explain why it is a cchf-positive area. another important issue is the presence of disease reservoirs and vectors, such as hyalomma spp., in this area. the cchfv genome has been isolated from at least 31 different tick species in ixodidae (hard ticks) and argasidae (soft ticks) (saijo et al. 2002, tahmasebi et al. 2010). as champour (2013) noted, the main tick species that affect onehumped camels in this region are h. dromedarii, h. anatolicum, h. marginatum, and h. asiaticum. hyalomma spp ticks are considered the most important species in the epidemiology of cchfv in camels in this area (hoogstraal 1979). six out of the eleven cities and towns studied yielded one-humped camel serum that was igg-positive for cchf, and it has been shown that cchf is distributed across these three provinces. we could not ascertain why almost all of the positive-igg sera samples were collected from female camels (8/9), but this may have been due to the presence of more females than males in the samples (136 females vs 34 males), possibly because female camels remained in herds longer than did males at the time of sampling. our results reveal a lower prevalence of seropositivity in one-humped camels (5.3%) than in other domestic animals (30%) in iran (chinikar et al. 2009). however, due to the fact that camels remain in herds for longer than do other animals (champour et al. 2013), and because camel pasture is very widely geographically distributed, this small percentage has a significant effect on the epidemiology of cchf. the importance of camels in the epidemiology of cchf in russia and astrakhan oblast has been previously reported by kurbanov, berezin, and chumakov (steele 1994). conclusion because cchf is a serious threat to iran, imported animals, particularly one-humped camels that carry a large number of ticks, should be inspected and treated carefully. continued surveillance and strictly enforced importation and quarantine practices will be required to prevent human exposure and the on-going dispersal of infected ticks and livestock in these regions. the use of commercially available insect repellent, and the use of clothing impregnated with permethrin, can give some protection against tick bites (telmadarraiy et al. 2010, salim-abadi et al. j arthropod-borne dis, june 2016, 10(2): 168–177 m champour et al.: crimean-congo … 175 http://jad.tums.ac.ir published online: january 05, 2016 2011). further surveys of human and animal populations, particularly of those animals imported from neighboring countries to these regions, are recommended, in order to provide a better understanding of the distribution and epidemiology of the virus in these provinces. acknowledgements the project was funded by arboviruses and viral hemorrhagic fever laboratory, (national reference lab) pasteur institute tehran-iran and ferdowsi university of mashhad, bearing registration code 3/230667. thanks to staff members of veterinary department of khorassan-e-shomali, particularly dr razavi, dr ramezani, dr mohammad mehdi ahmadi, dr hassan safaei, dr hossein janati and dr shahin ahmadi for their collaboration in sampling. the authors declare that there is no conflict of interests. references alavi-naini r, moghtaderi a, koohpayeh hr, sharifi-mood b, naderi m, metanat m, izadi m (2006) crimean-congo hemorrhagic fever in southeast of iran. j infect. 52: 378–382. apanaskevich da, filippova na (2007) larval identification of species and subspecies of the genus hyalomma (acari: ixodidae) from russia and neighboring territories. parazitologiia. 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accepted 30 dec 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: fauna and larval habitat characteristics studies on mosquitoes are important tools to identify the breeding places of the vectors and management of the control strategies. this study was done to provide data on culicidae fauna, larval habitat characteristics and identifying poten tial vectors of west nile virus in lorestan province, west of iran. methods: culicidae mosquitoes were collected at three counties and nine site stations from lo restan province, west of iran in 2017. adult mosquitoes were collected using human and animal bite collection methods, new jersey and cdc light traps and pit shelters by aspirator. larva were collected by dipping method. rt-pcr technique was employed for detection of the west nile virus among mosquito’s samples. results: 4805 mosquitoes were collected from three counties and nine sites in lorestan province during june–october 2017, including 4363 adults and 442 larvae. the most abundant species col lected from all counties in both adult and larval stages were culex pipiens (49.10%), cx. theileri (31.82%), anopheles maculipennis (11.09%), an. superpictus (2.66%), an. stephensi (2.12%), cx. perexiguus (1.89%), an. dthali (1.17%) and an. sacharovi (0.15%) respectively. west nile virus was detected in none of mosquitoes examined. conclusion: the results of this study revealed that arbovirus vectors such as cx. pipiens along with cx. theileri and cx. perexiguus are well adapted to a broad range of habitats and different climatic conditions in lorestan province. that necessitates further routine surveillance of arboviral infections. keywords: anopheles; arboviruses; culex; west nile; habitat introduction mosquitoes (diptera: culicidae) are the medically-important species for malaria, filariasis, encephalitis and some other arboviral diseases transmission (1-3). the culicidae family include 2 subfamilies, 41 genera and 3584 species (4). the most 398 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 397–404 sh moosa-kazemi et al.: determining... important genera of the family are anopheles, culex and aedes. mosquitoes are vectors of important vector-borne diseases malaria, filariasis, dengue fever, yellow fever, chikungunya, west nile virus and zika virus which are among the greatest health problems in the world (57). considering the fact that in lorestan province, iran, some vector mosquitoes of diseases such as west nile, dirofilariasis and malaria are present; study on the composition, distribution and ecology of mosquitoes have great impacts in vector control programs. these findings guide us for better planning of disease control strategies. regards to new human activities and weather change in the research area, the outcomes of our investigation should helps health services to management vectors increasing and establishment in the area, therefore, the risk of transmission of diseases by mosquitoes gets minimized. the aim of the present study has been determining of the composition, distribution and some ecological aspects of mosquitoes in lorestan province, iran, which is of high medical importance from the point of view of the arboviral vectors. materials and methods study areas lorestan province is located in western iran, between 32º 40´-32 º 34´ n latitudes and 46º50´-46º 51´ e longitudes. the province covers an area of approximately 28,308 km² with a population of around 1,800,000 people (fig. 1, table 1). this province contains 11 counties with one million people called metropolitan. out of them, khorramabad, with a population of 800,000, is ranked amongst the 40 most populous metropolitan cities of iran. the hottest months of the year are from midjuly to mid-september when temperatures range from 28 °c to 40 °c and the coldest month’s experience 1 °c around december– january, but at certain times in winter it can reach −15 °c. khorramabad county has moderate winters and hot summers. average annual rainfall is approximately 516 millimeters, the maximum rainfall has been recorded during the winter season. on the whole, the province has a semi-arid, steppe climate in the south and mediterranean climate in the north. (fig. 1, table 1). fig. 1. map of iran and locations of study areas in lorestan province, iran fig. 1. map of iran and locations of study areas in lorestan province, iran https://en.wikipedia.org/wiki/semi_arid https://en.wikipedia.org/wiki/alpine_climate 399 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 397–404 sh moosa-kazemi et al.: determining... mosquito collection and species identification for mosquito collection, nine sites with different biotopes in khorramabad, selseleh (alashtar) and chegeni counties were selected (fig. 1, table 1). in this study, the collection of mosquito larvae was performed in different habitats using the standard dipping technique (using 350 ml clark’s dippers) and whirl pack bags (8). cdc and new jersey light traps, animal and human bites and shelter pit methods were used for collecting mosquitoes. mosquitoes capture was done during june to october 2017. larvae and adult mosquitoes were identified by morphological keys (9). physical and biological characteristics of larval habitats the ecological characteristics containing geographical data of collecting sites (latitude, longitude and altitude), type of habitat (stagnant, stream, seepage and water container), vegetation situation (presence or lack of vegetation), kind of vegetation (leaf null, oryza sativa, typha latifolia, carex dipsacea and acorus calamus), turbidity, exposure to sunlight (full, partial sunlight and covered or shaded), depth, substrate type (muddy, sandy, rocky and concrete), distance from animal and human houses and physicochemical attributes such as water temperature were recorded (7-9, 10). statistical analyses the species richness (r: margalef index), unified indices (d: simpson’s diversity index and h: shannon–wiener index) and evenness (e: distribution of abundances among the species) as measures of diversity, were calculated for lorestan province and different study counties. below is the formulae and their rationale for our research: 1 s r ln n − = ( ) ( )1 1 1 1 s i i i n n d n n= − = − −∑ ( ) ( ) 1 s i i i h p ln p =  ∆ =  ∑ ( ) h e ln s ∆ = results in total, 4363 adults and 442 larvae were collected from three counties and nine sites in lorestan province during june–october 2017. the properties of geographical larval habitats (latitude, longitude and altitude) have been shown in fig. 1 and table 1. west nile virus was not detected in mosquitoes. an. maculipennis s.l., an. superpictus s.l, cx. perexiguus, cx. pipiens, and cx. theileri, were collected at both adult and larval stages (table 2). table 1. geographical characters of the collection sites, lorestan province, iran code locations county geographical situation longitude (e) latitude (n) altitude (m) topography no m robat namaki khorramabad 48° 18' 34.67'' e 33° 36' 22.79'' n 1302.76 m mountain 1 p sarzangoleh khorramabad 48° 18' 2.10'' e 33° 26' 8.29'' n 1152.29 m plain 2 d dast be zanoo khorramabad 48° 19' 12.68'' e 33° 26' 23.79'' n 1164.07 m slope 3 d cham divan chegeni 48° 0' 29.78'' e 33° 30' 19.69'' n 1003.74 m slope 4 p sharaf bostanrood chegeni 47° 56' 29.11'' e 33° 26' 37.42'' n 957.95 m plain 5 m berkeh chegeni 48° 1' 3.39'' e 33° 33' 15.83'' n 1110.27 m mountain 6 m chartakteh selseleh 48° 17' 41.56'' e 33° 47' 51.83'' n 1715.29 m mountain 7 p varnamad selseleh 48° 8' 50.07'' e 33° 44' 19.74'' n 1565.05 m plain 8 d kakareza selseleh 48° 15' 39.02'' e 33° 43' 17.51'' n 1572.64 m slope 9 table 1. geographical characters of the collection sites, lorestan province, iran 400 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 397–404 sh moosa-kazemi et al.: determining... the most abundant species collected from all counties in both adult and larval stages were cx. pipiens, cx. theileri, an. maculipennis s.l., an. superpictus s.l. , an. stephensi, cx. perexiguus, an. dthali and an. sacharovi respectively. some species such as cx. pipiens, cx. theileri, an. maculipennis s.l. have wide distribution in the study areas, but some species such as an. stephensi have been collected from limited areas (table 2). in larvae and adult stages of mosquitoes, two genera along with 7 species were collected and identified from khorramabad county. culex pipiens, cx. theileri, an. superpictus s.l., an. maculipennis s.l., cx. perexiguus, an. dthali and an. sacharovi were the most abundant species respectively (table 2). in chegeni county, two genera including 8 species were identified: cx. pipiens, cx. theileri, an. maculipennis s.l., an. stephensi, an. superpictus, cx. perexiguus, an. dthali and an. sacharovi (table 2). in selseleh county, five species in two genera were identified: cx. theileri, cx. pipiens, cx. perexiguus, an. maculipennis s.l. and an. dthali (table 2). characteristics of mosquito larval habitats khorramabad and chegeni larval sites had stagnant water while selseleh had seepage water. the characteristics of chegeni and selseleh counties were more similar than khorramabad county. both chegeni and selseleh counties had larval sites with turbid water, muddy substrate, and shallow depth. in addition, these sites were covered by sunlight with leaf-null, oryza sativa, typha latifolia, carex dipsacea and acorus calamus, vegetation. we found stagnant and clear water without vegetation with exposed to sunlight. the sites were away from human and animal houses, around more than two table 2. composition and abundance percentage of collected species from larval habitats and adult mosquitoes in the lorestan province of iran, june–october 2017 province location species larvae adult no. % no. % lorestan khorramabad an. dthali 0 0 32 1.91 an. superpictus s.l. 0 0 57 3.41 an. sacharovi 0 0 2 0.11 an. maculipennis s.l. 14 5.90 27 1.61 cx. pipiens 124 52.32 988 59.26 cx. theileri 89 37.55 535 32.09 cx. perexiguus 10 4.21 26 1.55 total 237 100 1667 100 chegeni an. dthali 0 0 23 0.88 an. superpictus s.l. 9 5.62 62 2.39 an. stephensi 0 0 102 3.94 an. sacharovi 0 0 5 0.193 an. maculipennis s.l. 24 15 466 18 cx. pipiens 87 54.37 1090 42.11 cx. theileri 34 21.25 795 30.71 cx. perexiguus 6 3.75 45 1.73 total 160 100 2588 100 selseleh an. maculipennis s.l. 1 2.22 1 0.92 cx. perexiguus 4 8.88 0 0 cx. pipiens 18 40 52 48.14 cx. theileri 22 48.88 54 50 an.dthali 0 0 1 0.92 total 45 100 108 100 total 442 100 4363 100 table 2. composition and abundance percentage of collected species from larval habitats and adult mosquitoes in the lorestan province of iran, june–october 2017 401 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 397–404 sh moosa-kazemi et al.: determining... kilometers. in such a larval breeding site, the water temperature was 23 °c. in this larval habitat, one anophelinae (an. maculipennis s.l.) and three culicinae species of cx. theileri, cx. perexiguus, and cx. pipiens were collected (tables 2, 3). according to the results, presented in tables 2 and 3, larvae of mosquitoes were observed in all different types of the habitats. in addition, our results showed that cx. pipiens, and cx. theileri, had the most distribution and adaptation to different types of larval habitats, respectively. these mosquitoes larvae were captured in most sites but, cx. perexiguus was found only in the shallow, stagnant, and turbid water, covered with plants and with a muddy substrate. in addition, the distribution of an. sacharovi was limited and only collected in the habitats with seepage, stagnant and turbid water along with muddy substrate and shallow depth at sharaf boostanrood of chegeni. an. sacharovi was found only in paddy fields, having stagnant and clean water with a muddy substrate exposed to sunlight. we found differences in the species diversity, due to simpson’s diversity index, shannon-wiener index (h’), and species richness of the mosquitoes in the study areas of lorestasn province (table 4). the species richness and the three indices were found to be minimum in selseleh county (r= 0.640; d= 0.318; h’= 0.782; e= 0.564), whereas the estimated diversity (d= 0.458; h’= 1.008), and richness (r= 0.809) were the highest in khorramabad county. discussion the present research is the first study on distribution, diversity and ecology of mosquitoes, with emphasis on cx. pipiens as potential arbovirus vectors in lorestan province, western iran. lorestan province contains diverse geographical areas with different climates. these diverse conditions can provide a suitable environment for the establishment of different species of mosquitoes that can justify the variety of mosquito species in this region. this study showed many mosquito species had ecological adaptations in this area. in spite of these eco-biological characteristics, the table 3. characteristics of mosquito larval collection sites in three counties of lorestan province of iran, june– october 2017 county type tur exp veg dhh dah depth sub t khorramabad stagnant clear exposed without >2km >2km <1m muddy 23 chegeni stagnant turbid covered leaf-null, oryza sativa, typha latifolia, carex dipsacea >2km 500m shallow muddy 9 selseleh seepage water turbid covered leaf-null, carex dipsacea and acorus calamus, >2km 200m shallow muddy 13 nd: not determined, tur: turbidity, exp: sun exposure, veg: vegetation type, dhh: distance from the nearest human houses, dah: distance from the nearest animal house, sub: substrate type, t: temperature. table 3. characteristics of mosquito larval collection sites in three counties of lorestan province of iran, june– october 2017 table 4. the species richness (r), simpson's diversity index (d), shannon-weiner diversity index (h)̕, and evenness (e) of the adult collected mosquitoes in the study areas county s r d h̕ e khorramabad 7 0.809 0.458 1.008 0.519 chegeni 8 0.763 0.490 1.353 0.650 selseleh 4 0.640 0.318 0.782 0.564 table 4. the species richness (r), simpson’s diversity index (d), shannon-weiner diversity index (h)̕, and evenness (e) of the adult collected mosquitoes in the study areas 402 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 397–404 sh moosa-kazemi et al.: determining... ecology of mosquitoes present in lorestan province is largely unknown. in the present investigation we tried to study distribution and ecology of mosquitoes in three northeastern, northwest and central regions of lorestan province. although some studies had been conducted on fauna and checklist of mosquitoes in parts of this region based on our best knowledge, no studies have been done earlier on the ecology of mosquitoes in these regions (11, 12). in the current study, two genera and eight species were collected and identified. for the first time, cx. perexiguus species was reported from chegeni and selseleh counties. some studies had reported an. dthali, an. sacharovi, an. maculipennis s.l., and an. superpictus s.l. in lorestan province (13, 14). in our study, for the first time, an. stephensi and an. sacharovi were found in chegeni, however, in previous studies the existence of these species was not reported in such areas (11-13). some investigations reported culiseata annulata and cs. subochrea in lorestan province and cs. subochrea in khuzestan province (11-13, 15). culiseta longiareolata species was reported as the most abundant mosquito in kermanshah, kurdistan and sistan and baluchistan provinces (16, 17). comparing the results of our study with a recent study carried out in western iran (10) showed that four species (an. maculipennis, an. superpictus, cx. pipiens and cx. theileri) were common between lorestan and west azerbaijan provinces. the results of our study compared with the results of research conducted in zanjan province (18), showed that four species (an. maculipennis s.l., an. superpictus s.l., cx. pipiens, and cx. theileri, were common between these two provinces. the comparison of the results of our study with a recent study conducted in kurdistan province (19) showed that four species (an. maculipennis s.l., an. superpictus s.l., cx. theileri and cx. pipiens) are common between two provinces. in turkey (20), and in the provinces of ardabil, kurdistan and west azarbaijan of iran, cx. theileri and cx. pipiens were dominant and most abundant species in the area (21) and our study is in line with such results. the climate changes and biotic and abiotic environment factors, including plants, temperature and rainfall ranges, significantly affect the type and frequency of larval mosquito habitats. the mentioned factors have impacts on the longevity, larval stages, number of mosquitoes, behavior and adult development of mosquitoes. as a result, the transmission of diseases through mosquitoes is directly affected by environmental factors (22, 23). the presence of plants as a source of sugar for mosquitoes is very important that influence both larval and adult stage development (24, 25). plants provide energy for mosquitoes. as a result, the survival rate will increase and the longevity of mosquitoes will be longer than the extrinsic incubation period of the parasite, therefore the incidence of disease increases (26, 27). in our study, four species of plants such as leaf-null, oryza sativa, typha latifolia, carex dipsacea and acorus calamus were found in relation to mosquito larval habitats. few studies have been carried out on plant species in collaboration with mosquito species in iran. in our study, various genera of mosquitoes such as anopheles and culex were found in relation with various plants. aedes vexans and ae. caspius have been reported earlier in these areas (11-13). the results of our research show that climate changes in association with human activities effect on the species distribution in the area. abundance of species of mosquitoes related to physio-chemical characteristics of larval sites. for example, culex species were captured from different larval sites, determining that culex species survived in a range of water habitats. larval habitats in this study were stagnant, stream and seepage, water container, turbid and clear water, sun exposed or covered from sunlight, with a rocky or muddy substrate and shallow depth. studies showed that some of culex larval species were found alone or along with other mosquitoes, such as anopheles and aedes (29-31), which has been observed in 403 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, dec 2021, 15(4): 397–404 sh moosa-kazemi et al.: determining... our study. the results of some studies showed that there is a significant relationship between the distance of larval habitats of anopheles mosquitoes and human and animal sites. these studies suggested that anopheles mosquitoes are found more often near human and animal houses (32). our study showed that there is a significant correlation between the distance of larval habitats of anopheles and cx theileri with animal and human dwellings due to larval sites of both species are more near to animal and human dwellings. this association was not found between the larval sites of cx. pipiens and with human and animal houses because the habitats of these mosquitoes were found at various intervals from human and animal houses. conclusion the results of this study revealed that arbovirus vectors such as cx. pipiens along with cx. theileri and cx. perexiguus are well adapted to a broad range of habitats and different climatic conditions in lorestan province. determining of distribution and full description of ecology of arboviral vectors under local eco-demographic conditions in lorestan province have provided important ecological information on establishment of important mosquito borne diseases and help with minimizing the risk of transmission of disease by mosquitoes. acknowledgements the present manuscript is a part of the results of the first author’s dissertation for fulfillment of a msph degree in medical entomology and vector control from the department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. this study was financially supported by the deputy for research, tehran university of medical sciences project no 240/1346. competing interest the authors declare no conflict of interest. references 1. tabachnick wj(1991) evolutionary genetics and arthropod-borne disease: the yellow fever mosquito. am entomol. 37, 14–26. 2. gasperi a, bellini r, malacrida a r, crisanti, dottori m, aksoy s(2012) a new threat looming over the mediterranean basin: emergence of viral diseases transmitted by aedes albopictus mosquitoes. plos negl trop dis. 6(9): e1836. 3. gould e a, higgs s(2009) impact of climate change and other factors on emerging arbovirus diseases. trans r soc trop med hyg. 103(2):10921. 4. mosquito taxonomic inventory. available: at https://mosquito-taxonomic-inventory.myspecies. info/. 5. davisle, beckham jd, tyler kl(2008) north american 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https://pubmed.ncbi.nlm.nih.gov/?term=traore%2bmm&cauthor_id=20854666 https://pubmed.ncbi.nlm.nih.gov/?term=traore%2bmm&cauthor_id=20854666 https://pubmed.ncbi.nlm.nih.gov/?term=bah%2bs&cauthor_id=20854666 https://pubmed.ncbi.nlm.nih.gov/?term=doumbia%2bs&cauthor_id=20854666 https://pubmed.ncbi.nlm.nih.gov/?term=schlein%2by&cauthor_id=20854666 investigation on mosquitoes fauna (diptera: culicidae) and probable vector of west nile virus in lor abstract keywords introduction materials and methods study areas mosquito collection and species identification physical and biological characteristics of larval habitats statistical analyses results characteristics of mosquito larval habitats discussion conclusion acknowledgements competing interest references j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 350 http://jad.tums.ac.ir published online: dec 31, 2022 original article biodiversity and spatial distribution of mosquitoes (diptera: culicidae) in kurdistan province, western iran farid sharifi1, omid banafshi1, ali rasouli1, shadi ghoreishi2, samrand saeedi1, mahmood khalesi1, afshar rezai1, eslam moradi asl3, bushra zareie4, nasrollah veisi khodlan1, *arshad veysi1 1zoonoses research center, research institute for health development, kurdistan university of medical sciences, sanandaj, iran 2cellular and molecular research center, research institute for health development, kurdistan university of medical sciences, sanandaj, iran 3department of public health, school of health, ardabil university of medical sciences, ardabil, iran 4department of epidemiology, school of public health, hamadan university of medical sciences, hamadan, iran *corresponding author: dr arshad veysi, email: arshadveysi@gmail.com (received 30 jan 2022; accepted 17 dec 2022) abstract background: mosquitoes (diptera: culicidae) have always been considered as the vector/s of viral and parasitic diseases. this study aimed to conduct a comprehensive survey on the species composition, spatial distribution, and biodiversity indices of mosquitoes in kurdistan province, western iran. methods: this study was carried out in 10 counties of kurdistan province. the immature stages of mosquitoes were collected monthly from june to september. arcgis software was used to spatial analysis and create maps. alpha diversity indices were calculated using the related formula. results: totally, 5831 larvae belonging to the family culicidae were collected. twelve species were identified including: anopheles claviger, an. maculipennis s.l, an. superpictus s.l, culiseta. longiareolata, cs. subochrea, culex hortensis, cx. mimeticus, cx. perexiguus, cx. pipiens, cx. theileri, cx. modestus and cx. territans. based on this analysis, the high-risk areas of the province are determined as anopheles in the west, culex in the north, and the culiseta in the south of the province. analyzing the alpha biodiversity indices showed baneh and sarabad had the maximum and bijar had the minimum mosquito biodiversity. conclusion: the western counties of the province are regarded as the hotspots for anopheline mosquitos. moreover, reporting of malaria cases in the past, bordering with iraq and the high traffic of travelers have made these areas as potential foci for malaria transmission. so that, routine entomological inspections are proposed to detect any suspicious vector or case entrance. keywords: mosquitoes; gis; diversity; diptera; larvae introduction mosquitoes (diptera: culicidae) have always been the focus of entomological studies due to their important role in transmitting a wide range of viral and parasitic diseases to humans or animals. more than half of the world's population lives in areas at risk of mosquito-borne diseases such as malaria, dengue fever, chikungu nya, west nile fever, japanese encephalitis, and filariasis (1). malaria is a parasitic infection transmitted by anopheline mosquitoes. it is estimated that 219 million new cases are reported worldwide, and lead to the death of more than 400,000 people annually. dengue is the most common viral disease transmitted by ae copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 351 http://jad.tums.ac.ir published online: dec 31, 2022 des mosquitoes. more than 3.9 billion people in more than 129 countries are at risk of getting dengue fever, with an estimated 96 million symptomatic cases and an estimated 40,000 deaths each year (2). malaria is also the most important mosquito-borne disease in iran, which occurs mostly in the southern provinces of the country (3, 4). seven species of anopheles are among the most important vectors in iran, including: anopheles stephensi liston, 1901, an. culicifacies sensu lato (s.l.) giles, 1901 (5), an. maculipennis s.l. meigen, 1818 (6), an. fluviatilis s.l. james, 1902, an. sacharovi favre, 1903, an. dthali patton, 1905, and an. superpictus s.l. grassi, 1899 (7, 8). the presence of west nile and sindbis viruses, which are transmitted by mosquitoes, has also been reported in the country (9–13). recently, mosquito-borne dirofilaria (d. immitis and d. repens) has been reported in iran (14). in recent years, no local transmission of malaria has been reported in kurdistan province, and only in 2020, one case was reported in the city of marivan, which was iraqi (unpublished data from department of disease control, deputy of health, kurdistan university of medical sciences). in 2013 serologically positive cases of dengue fever was reported from kurdistan province, who had not travelled abroad (15); although, there is no report of occurring the vector ae. albopictus or ae. eagypti in this province. till now no human case of rift valley fever virus has been reported in the province, though one study showed infection in sheep (16). west nile virus is a widespread mosquito-borne arbovirus in iran as it is reported in horses from at least 26 of the 31 iranian provinces including kurdistan (9). different blood-feeding arthropods including mosquitos are considered as suspected vectors of tularemia which caused by the bacterium francisella tularensis. natural infection of tularemia in the wild reservoir, rodents (17), and in human have been reported in kurdistan province (18). the latest updated checklist of iranian mosquitoes includes 70 species that represent eight or 12 genera, depending on the general classification of aedines (19, 20). zaim (21) mentioned two genera and eight species of the subfamily culicinae in kurdistan province. in previous studies conducted in the province, 18 species of the family belonging to five genera have been reported, which include an. maculipennis s.l., an. superpictus s.l., an. claviger, an. sacharovi, an. algeriensis, an. marteri, an. sergentii, coquillettidia richiardii, culex hortensis, cx. mimeticus, cx. pipiens, cx. theileri, cx. perexiguus, cx. territans, culiseta longiareolata, cs. subochrea, ae. vexans and ae. caspius (22–24). in recent years, due to the reporting of dengue vectors in the south of the country (25, 26), and on the other hand, high reporting cases of dengue fever in eastern neighbors such as pakistan, the importance of routine entomological studies has doubled in other border provinces to monitor the entrance of dengue vector/s. sharing border with iraq, made kurdistan province as one of the most important import destinations for goods from southeast asian countries. one way of entering the dengue fever vector/s into the countries is through goods such as bamboo plant and car tires, therefore, conducting entomological studies in this critical point seem necessary. the studies that have been done in the province so far are only faunistic and scattered. the purpose of this study was to conduct a comprehensive study and update the species composition of mosquitoes in all counties of the province; moreover, to analyze the spatial distribution and to determine biodiversity indices of the mosquitoes throughout the province. materials and methods study area this study was conducted in 10 counties of kurdistan province in summer 2019. it is in the west of iran between 34°44′n and 36°28′n and 45°33′e and 48°15′e. the area is 29.137 square kilometers, equivalent to 1/7 of the total area of iran. this province, which is http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 352 http://jad.tums.ac.ir published online: dec 31, 2022 located in the scattered slopes and plains of the middle zagros mountains, is limited from the north to the provinces of west azerbaijan and zanjan, from the east to hamedan and zanjan, from the south to kermanshah, and from the west to iraq. kurdistan province has 10 counties include: sanandaj, saqez, marivan, baneh, qorveh, kamyaran, bijar, divandarreh, dehgolan and sarvabad (fig. 1). according to the general population and housing census of 2016, kurdistan province has a population of 1,603, 011 (statistical center of iran). the highest and lowest average air temperatures in 2019 are belong to sanandaj with 14.8 °c and zarrineh with 8.8 °c, which they have different height from sea level. marivan with 913.1mm and qorveh with 352.7mm had the highest and lowest rainfall, respectively (statistical center of iran). collection, mounting and identification of mosquito larvae the immature stages of mosquitoes were collected monthly, for at least four months (once a month) from june to september. larval collection was conducted in an urban point and four rural points at a distance of 15–20km in all counties. in fixed urban and rural points, three fixed and three variable locations were sampled. the distribution of sampling points included urban, rural, cultivated areas, and riversides. the mean water temperature and ambient humidity were recorded during the sampling period. the larvae of the mosquitoes were collected by standard dipping technique. collected larvae were stored in lactophenol medium, and after clarification of morphological characters, they were mounted in de faure’s medium. after that, the larvae were identified using valid morphological iranian identification keys (27). additionally, the geographical coordinates of the sampling locations were recorded using a gps device. collecting larvae using ovitraps ovitrap is one of the most common techniques for collecting eggs and larvae of aedes mosquitoes. in this study buckets, which were designed in a standard way, were used. dark buckets, preferably black due to its attractiveness for aedes mosquitoes, in a volume of two liters were used. after pouring two liters of water inside the buckets, 3 wooden pedals with dimensions of 15×5cm and a thickness of one to two mm were immersed in the water, and then they were attached to the wall of the bucket with clamps (28). all the information in the labels of the traps and the exact addresses of their installation places were recorded in a separate booklet. the traps were checked twice a week according to the weather conditions of the study areas. due to evaporation of water inside the traps, water was added to them if necessary. during twice-a week check-up, the pedals were carefully examined for the presence of eggs by a handy lens. if a suspicious case of eggs was observed, the pedals were carefully removed and transported to the laboratory, and it was replaced by a new pedal. gis analysis arcgis 10.4.1 software (http://www.esri. com/arcgis) was used to spatial analysis and creating maps. after inserting geographic coordinates into the excel software, they were entered into the arcmap in arcgis 10.4.1 software. inverse distance weighted (idw) interpolation analysis was employed to prepare raster maps. data process and analysis to determine alpha diversity indices, data were inserted in microsoft office excel 2016 software using related indices, and coefficients showing below. then, the average and standard deviation of all indices were calculated (29,30), and their p-value was determined by performing the analysis of variance (anova) test in ibm spss statistics 26 software. shannon–weiner index: h': species diversity index, s: number of species, pi: proportion of individuals of each species belonging to its species of the total number of individuals. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 353 http://jad.tums.ac.ir published online: dec 31, 2022 evenness index: = h': species diversity index, s: number of species simpson’s index: n: the total number of organisms of a particular species, n: the total number of organisms of all species menhinick’s index: s: number of species, n: the total number of organisms of all species margalef’s index: s: number of species, n: the total number of organisms of all species hill: e: napier's constant, h’: species diversity index hil2: pi: proportion of individuals of each species belonging to its species of the total number of individuals. results species composition during this study, a total of 5831 larvae belonging to the family culicidae were collected from 56 localities of 10 counties of the province (table 1–2). of these, the highest number were from marivan county and the lowest number were from kamyaran county (table 2). the collected larvae belonged to three genera: anopheles 889 (15.24%), culiseta 1826 (31.31%) and culex 3116 (53.43). collecting methods through ovitraps detected only a few larvae, which is belonging to the genus anopheles, and no aedes species were identified. 12 species of culicidae were identified including: an. claviger, an. maculipennis s.l., an. superpictus s.l., cs. longiareolata, cs. subochrea, cx. hortensis, cx. mimeticus, cx. perexiguus, cx. pipiens, cx. theileri, cx. modestus and cx. territans (table 2). spatial analysis species belonging to anopheles were identified in more than 50% of the counties with high abundance. the baneh county, the western region bordering iraq, has the highest number of species of this genus, about 2–3 times higher than other counties. species of the genus culex were caught in 60% of the counties. divandere and saqez have the highest abundance and are considered as high-risk areas for this genus. species of culiseta were identified in more than 50% of the study areas, which was abundant in the central parts of the province. in general, the abundance of culiseta was higher than that of culex and anopheles (fig. 2). inverse distance weighted (idw) analysis the results of idw for anopheles species in the study area showed that there was a very extensive hotspot in the west of kurdistan province. high density of anopheles species in these areas increase the risk of malaria transmission in case of detecting vector infection. these high-risk areas locate in three counties bordering iraq. two small hotspots were determined in saqez for the genus culex, which is limited to the county itself, and is slightly extended to the north of the province. on the one hand, the genus culiseta has a medium hotspot, which exists in the south of the province and in the county of sanandaj. based on this analysis, the high-risk areas of the province are determined as for anopheles in the west, for culex in the north, and for the culiseta in the south of the province (fig. 3). alpha diversity indices based on the calculation for the simpson index, the maximum biodiversity was observed in sarvabad and the minimum in bijar. the maximum biodiversity, based on shannon-weiner index, was determined in baneh and minimum in bijar. shannon-weiner evenness index showed maximum and minimum evenness of species distribution in qorveh and bijar, respectively. for hill 1 and hill 2 index the maxhttp://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 354 http://jad.tums.ac.ir published online: dec 31, 2022 imum and minimum calculated in baneh and bijar respectively. the amount of species richness, based on margalef’s index, was maximum in baneh and minimum in bijar. based on menhinick’s index, the maximum and minimum species richness was reported in kamyaran and bijar respectively. the statistical analyzes showed that there is a significant difference between the counties in all calculated indices (table 3). table 1. geographical coordinates of mosquito larval collecting sites, kurdistan province, summer, 2019 county location latitude longitude county location latitude longitude sananadaj sanandaj 35.320776n 46.967762e bijar gharatoreh 35.8070749n 47.454365e tavrivar 35.128055n 46.983173 e sarabghamish 35.329604n 47.047870e nadri 35.7606463n 47.529502e kanimeshkan 35.237838n 46.921330 e sadeghabad 35.6553119n 47.169327e babariz 35.364240n 47.065193e khorkhoreh 35.9689153n 47.800325e doiseh 35.455993n 46.895943e salavatabad 35.9983444n 47.550961e salavatabad 35.279508n 47.126181e ghabasorkh 35.8569962n 47.408851e hasanabad 35.260319n 46.968247e mehrabad 35.9011767n 47.913803e naran 35.154100n 47.067685e sayedan 35.9409092n 47.725527e baneh nojneh 36.128394n 45.783805e khosroabad 35.5182203n 47.623157e aloot 36.025676n 45.571554e najafabad 35.7932236n 47.238271e savan 36.074269n 45.904967e ghadimkhan 35.6248274n 47.642770e ashtarabad 35.844582n 45.917856e ghamchaghai 36.1659343n 47.625592e showe 36.044065n 45.865924e dehgolan bolbanabad 35.1404632n 47.321070e marivan vilae 35.592742n 46.307395e miraki 35.4350278n 47.290693e chashniabad 35.653148n 46.031590e sis 35.2065655n 47.279559e darahtephi 35.537565n 46.099041e bagahjan 35.3491741n 47.455589e sarvabad daranakhi 35.392268n 46.233966e qorveh mehdikhan 35.3456197n 47.649151e ghalaji 35.358081n 46.282911e farhadabad 35.4154735n 47.616394e rezab 35.260049n 46.403304e ghaleh 35.1335135n 47.803795e saqez polegheshlagh 36.091621n 46.340016e majin 35.1711562n 47.946798e siyahdare 36.139224n 45.984306e bahraloo 35.195755n 48.121763e kandalan 36.256530n 46.067004e sarab 35.1344474n 47.790924e khanemiran 36.127169n 46.590750e kamyaran bovanah 34.8655297n 46.956129e cheshmesaqez 36.262425n 46.300410e shirvanah 34.7959729n 46.964364e divandarreh gavshalh 36.016072n 47.147437e kamyaran 34.7966668n 46.940417e kolah 35.789951n 47.052898e alak 34.8033522n 46.843097e hazarkaniain 35.769183n 46.813742e aghajari 35.891579n 47.128461e divandarreh 35.916419n 47.031293e http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 355 http://jad.tums.ac.ir published online: dec 31, 2022 table 2. number of species belonging to mosquitoes in the counties of kurdistan province, summer, 2019 species county s a n a n d a j b a n e h m a r iv a n s a r v a b a d k a m y a r a n b ij a r s a q e z d iv a n d e r e q o r v e h d e h g o la n total (%) an. claviger 0 95 15 0 6 0 0 4 0 0 120 (2.05) an. maculipennis s.l. 3 136 269 52 1 0 0 52 0 0 513 (8.79) an. superpictus s.l. 2 161 37 40 15 0 0 1 0 0 256 (4.39) cs. longiareolata, 885 57 125 75 86 217 58 0 1 313 1817 (31.16) cs. subochrea 1 0 8 0 0 0 0 0 0 0 9 (0.15) cx. hortensis 116 78 41 29 1 0 1 0 4 43 313 (5.36) cx.mimeticus 9 20 25 31 12 0 1 0 22 2 122 (2.09) cx.perexiguus 0 0 96 39 0 0 5 7 0 0 147 (2.59) cx.pipiens 20 56 80 13 29 0 440 96 27 8 769 (13.18) cx.theileri 7 18 573 124 16 1 103 416 153 27 1438 (24.66) cx. modestus 2 89 0 0 0 0 0 0 1 0 92 (1.57) cx.territans 0 28 0 0 0 0 40 167 0 0 235 (4.03) total 1045 738 1269 403 166 218 648 743 208 393 5831 (100) fig. 1. map of the mosquito larval collecting sites in kurdistan province, 2019 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 356 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 2. distribution of the mosquito genera belonging to culicidae in counties of kurdistan province, summer, 2019 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 357 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 3. distribution of hot spots of the mosquito genera belonging to culicidae in kurdistan province, summer, 2019 http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 358 http://jad.tums.ac.ir published online: dec 31, 2022 table 3. the alpha diversity for mosquito species in kurdistan province, summer, 2019 county s y m p so n m e a n ( s d ) p -v a lu e * s h a n o o n m e a n ( s d ) p -v a lu e s h a n e v e n m e a n ( s d ) p -v a lu e h il 1 m e a n ( s d ) p -v a lu e * h il 2 m e a n ( s d ) p -v a lu e m a r g a le f m e a n ( s d ) p -v a lu e m n a h e n ic m e a n ( s d ) p -v a lu e bane 0.36 (0.36) p < 0 .0 0 1 1.66 (0.44) p < 0 .0 0 1 0.45 (0.26) p < 0 .0 0 1 5.31 (2.84) p < 0 .0 0 1 4.40 (2.37) p < 0 .0 0 1 1.41 (0.81) p < 0 .0 0 1 0.65 (0.31) p < 0 .0 0 3 bijar 1.00 (0.02) 0.01 (0.04) 0.007 (0.026) 1.01 (0.04) 0.93 (0.26) 0.03 (1.00) 0.35 (0.09) dehgolan 0.71 (0.23) 0.53 (0.39) 0.84 (0.59) 1.80 (0.61) 3.20 (3.72) 0.53 (0.36) 0.39 (0.09) divandareh 0.50 (0.15) 0.96 (0.26) 1.14 (0.43) 2.68 (0.70) 2.13 (0.69) 0.90 (0.21) 0.43 (0.07) ghorveh 0.67 (0.23) 0.57 (0.41) 1.16 (0.71) 1.91 (0.84) 1.63 (0.64) 0.57 (0.37) 0.58 (0.13) kamyaran 0.48 (0.30) 1.01 (0.63) 0.44 (0.18) 3.18 (1.90) 2.66 (1.67) 1.09 (0.57) 0.82 (0.28) marivan 0.32 (0.08) 1.41 (0.29) 0.49 (0.11) 4.22 (1.11) 3.26 (0.78) 1.25 (0.18) 0.54 (0.17) saghez 0.61 (0.11) 0.69 (0.20) 0.34 (0.08) 2.03 (0.42) 1.68 (0.31) 0.68 (0.28) 0.40 (0.17) sanandaj 0.76 (0.15) 0.46 (0.23) 0.26 (0.18) 1.62 (0.33) 1.35 (0.25) 0.64 (.41) 0.47 (0.23) sarvabad 0.28 (0.06) 1.42 (0.28) 0.58 (0.08) 4.24 (1.07) 3.58 (0.80) 1.13 (0.40) 0.68 (0.23) *probability from kruskal-wallis test discussion a total of 5831 mosquitoe larvae were collected, which included 12 species and three genera. the highest number of species caught belonged to cs. longiareolata (31%), and the lowest number belonged to cs. subochrea. the highest number of anopheles caught was from the border county of baneh. this city has been one of the foci of malaria transmission (unpublished information, kurdistan province health center). based on the maps that, generating using arc gis software, baneh has been identified as one of the most important hot spots in terms of the existence of anopheles species. since, this county is an official border with iraq, and on the other hand, is one of the well-known border markets in the whole country, it annually receives many travelers from inside and outside the country. due to the presence of potential vector of the disease in this area, presence of a positive case of malaria may lead to a local transmission. therefore, this issue requires a special attention to borders control and applying strict quarantine laws at the borders and entry points. on the other hand, routine entomological studies are required in this county, as well. in a recent study, the highest http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 359 http://jad.tums.ac.ir published online: dec 31, 2022 number of anopheles specimens caught was reported from sarvabad city (24). the highest number of an. superpictus s.l. specimens in our study was reported from baneh city; while, in a recent study from sarvabad city (24). anopheles superpictus s.l. and an. maculipennis s.l. have been reported as the predominant species in hamedan and west azerbaijan provinces, respectively (31, 32). moreover, in east azarbaijan province, an. maculipennis s.l. has been reported as the predominant species of the genus anopheles (33). over the past few decades, an. superpictus s.l., an. maculipennis s.l. and an. sacharovi have been reported from 29, 20 and 18 out of 31 provinces of the country, respectively. anopheles superpictus s.l. is more common in the western, and an. maculipennis s.l. and an. superpictus s.l. are more common in the north and northwest of the country (34). in this study, more than 50% of the caught species belonged to the genus culex. based on idw analysis, two high-risk points for this genus were identified in north-western areas of the province. the highest number (46%) belonged to cx. theileri, and the lowest (2.9%) belonged to cx. modestus. culex theileri mostly caught from marivan city in the west of the province. this species has recently been identified as a vector of dirofilaria in the north of the country (35). in our study, seven species of the genus culex were identified. in two other studies conducted in the province, four and five species of this genus were reported, respectively. in contrary to our study, cx. modestus was not reported, and this species introduced for the first time in the province. in agreement to the result of our study, a study introduced cx. theileri as the predominant species in the province (24). in another study, the highest number of caught species, in contrast to our study, was cx. theileri (24, 36). in another study conducted in sanandaj county, the predominant species caught in larval stage was cx. pipiens, and in adult stage, like our study, was cx. theileri (22). like our study, in hamedan, the predominant species was cx. theileri (37), and in west azerbaijan, cx. pipiens was the predominant species (38). in east azarbaijan province, cx. theileri species has been the most dominant species in the region (33). in the present study, two species of the genus culiseta reported including cs. longiareolata and cs. subochrea. culiseta longiareolata had the highest abundance (31.16%) among all species. this species was predominant in sanandaj city, which previous studies in this city confirm the same results (22). in a study conducted by banafshi et al. (12), in selected areas of kurdistan province, was reported as the dominant species. in bijar, excluding cx. theileri species, all the collected species were cs. longiareolata, as the idw analysis showed a hot spot for this species in this region. similarly, cs. longiareolata is the predominant species in hamedan and west azerbaijan provinces (37, 38). in this study, no species belonging to the genus aedes were reported by dipping technique or using ovitraps. although, in previous studies a species belonging to this genus, ae. caspius, had been caught in different parts of the province as well as neighboring provinces (22, 24, 33, 36, 38). calculation of biodiversity alpha indices showed that in most of the studied areas, the value of these indices is statistically different. this issue, in addition to the effect of confounding variables such as the manner and time of sampling, can be affected by different climates in the province. different climates have created diverse ecological niches for the establishment of different species. the results of this study showed that areas with warmer climates had provided better conditions for higher biodiversity such as sarvabad city. in studies conducted in the north of the country, mazandaran province, and northwest, east azerbaijan province, similar to our study, different values were reported for biodiversity indices in the study areas (39, 40). http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 350–363 f sharifi et al.: biodiversity and … 360 http://jad.tums.ac.ir published online: dec 31, 2022 conclusion to sum up, the western counties of the province are considered the hotspots for anopheline mosquitos. moreover, reporting malaria cases in the past, bordering of these counties with iraq, and the high traffic of travelers have made these areas as potential foci of malaria transmission. although, in our study no species belonging to the genus aedes were reported, previous studies have identified species belonging to this genus. because two border counties of the province are the source of many goods importing from southeast asian countries, which are endemic to dengue fever, routine entomological checks and rigorous quarantine inspections on entry points are necessary to detect any suspicious vector entrance. acknowledgements the authors are very grateful to kurdistan university of medical sciences for its financial support of this project. 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province, northern iran. j vector borne dis. 52(1): 63– 72. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 178 http://jad.tums.ac.ir published online: january 05, 2016 original article irritability levels of field and laboratory population of culex pipiens complex in tehran to different groups of insecticides sara rahimi 1, *hassan vatandoost 1,2, *mohammad reza abai 1, ahmad raeisi 3, ahmad ali hanafi-bojd 1, fatemeh rafi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2institute for environmental research, tehran university of medical sciences, tehran, iran 3national program manager for malaria control, center for communicable diseases, ministry of health and medical education, tehran, iran (received 6 sep 2014; accepted 11 jan 2015) abstract background: the irritant effect of some insecticides can cause a proportion of mosquitoes to leave the sprayed rooms before acquiring a lethal dose, so the repeated contact al sub-lethal dose may lead to extent the resistance. methods: larvae and pupae of culex pipiens complex were collected in mass from open canals of waste water in capital city tehran and reared to obtain the first generation at laboratory. sugar-fed 2–3 days female mosquitoes were used for the experiments and compared with laboratory strain. the irritability tests of insecticides impregnated papers were measured in plastic conical exposure chambers placed which implemented at controlled conditions according to the method described by who .number of take-offs were counted during 15 minutes of exposure time. results: ddt had the most irritancy effect against field population of cx. pipiens. ddt, permethrin and deltamethrin was moderately irritable against laboratory strain, whereas, addition to three previous insecticides, malathion, cyfluthrin and propoxur should be also considered as moderately irritable insecticides for field population of. irritability level of etofenprox, fenithrothion, bendiocarb, and lambdacyhalothrin did not differ from control group. conclusion: the irritability response of mosquitoes may have a negative impact on control measures. periodical execution of irritability tests with insecticides that routinely used in vector control program is highly recommended. keywords: irritability, insecticides, culex pipiens, tehran, iran introduction culex pipiens complex has a great medical importance due to transmission of arbovirus and zoonotic diseases including dirofilaria immitis (azari et al. 2006). also its biting and nuisance causes the severe allergies in humans and other hosts, and this led to discomfort especially in urban areas (vinogradova 2000). the cx. pipiens are known as a complex species including cx. pipiens, and culex quinquefaciatus are important members of cx. pipiens complex (azari et al. 2007, harbach 2011). culex pipiens pipiens are distributed in both temperate and tropical areas whereas cx. quinquefasciatus in the tropical regions of the world (harbach 1988, smith and fonseca 2004, azari et al. 2010). culex pipiens are spread in most parts of iran, whiles cx. quinquefasciatus are scattered in the southern of country (zaim and cranston 1986, zaim 1987, azari and harbach 2009, azari-hamidian et al. 2010). vectors control is facing many problems especially occurrence of insecticide resistance. five major mechanisms have evolved to overcome toxi*corresponding authors: prof hassan vatandoost, email: hvatandoost1@yahoo.com, mr mohammad reza abai, email: abaimr@sina.tums.ac.ir j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 179 http://jad.tums.ac.ir published online: january 05, 2016 cants such as reduces penetration, metabolic resistance, target site insensitivity, excretion and behavioral change. in the behavioral change the mosquitoes are better able to resist the toxic effects of the insecticides, these changes, gradual resistance, gradually and inevitability increases the resistance genes in the population until all individuals are physiologically resistant (thomson 1947, vatandoost and borhani 2004). the use of carbamate and phosphorus insecticides, especially in agricultural fields, was led to the development of this type of resistance to insecticides (vatandoost et al. 2005a). change of vector behaviors that occurs in different forms, so that it is possible to change the relaxing habits in the indoor and because of irritancy-repellency phenomenon due to some insecticides, which makes the insect does not rest long enough on spraying surface and thus, it does not receive a lethal dose. therefore, it led to change the movement pattern of the mosquitoes into the houses and so they bitten out of the houses, avoids the insecticidetreated surfaces, occurrence of the exophagic and exophilic tendency and finally affected the results of vector control programs (vatandoost and borhani 2004). the mosquitoes could be avoided the spraying surfaces due to natural tendency and rest outside of buildings and houses (exophilic behavior). the irritation let to reduce to contact of mosquitoes with the sprayed surfaces, as well as absorption of enough dose of insecticides (quinones and suarez 1989). the different species of anopheline vectors have a different irritability level, some stay long time on insecticide-treated surfaces, while others motivated and escape immediately. some vectors may be hyper-irritable e.g anopheles gambiae that has 40–50 take-off during 15 minute’s obligatory contact with ddt, whereas some other species may have hypo-irritable such as an. albimanus that has just 2–3 take-off in similar time, and some species are moderateirritable, this phenomenon limits the residual spraying properties of the residential places against irritable species (thomson 1947, coluzzi 1963). the first studies on the irritability of anopheles mosquitoes in iran was conducted against an. maculipennis against ddt in isfahan province (eshghy 1972). the other studies on the irritability of an. stephensi and an. culicifacies was determine against different insecticides was conducted in sistan and baluchistan province (vatandoost and borhani 2004). it has been shown that resistant strains such as: anopheles gambiae and cx. quinquefasciatus demonstrate the different levels of irritability to the pyrethroids (hougard and duchon 2003). reasonable and proper use of insecticides depends on the knowledge an awareness about sensitivity and irritability of the vectors to these insecticides. many studies aimed to better understanding and effective application of the control methods of both larval and adult of cx. pipiens complex (oshaghi et al. 2007). long-term use of insecticide may increase the resistance level of mosquitoes populations due to behavioral habits of mosquitoes and diversion from indoor to outdoor resting places (thomson 1947). the irritability properties may change the mosquitoes behavior and led the leaving the indoor to outdoor which have been observed during the residual spraying of the villages with deltamethrin in endemic parts of malaria foci at southern east of iran where the an. culicifacies and an. stephensi acts as main vectors (abai et al. 1999, alipour et al. 2005). irritability is a first reaction of mosquito towards the sprayed environment, which initially increases movements of mosquitoes and finally led to away from the sprayed area, so repellency effect of insecticides is due to their irritability property (who 1970). the cx. pipiens complex is main prevalent species at southern part of capital city of tehran and easily bred in the high density at the sewage water as well as rice fields especially in the south parts where it reaches to this plateau (vatandoost j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 180 http://jad.tums.ac.ir published online: january 05, 2016 et al. 2005). different insecticides are being used for household and agricultural pest control in the capital city of iran, tehran which may lead to enhanced tolerance of house mosquito to different insecticides (vatandoost et al. 2005). due to rare studies on irritability of cx. pipiens complex this study was designed to understand the irritability level of field strain of capital city tehran compared with laboratory teh-sph strain to different insecticides. materials and methods different ages of larvae as well as pupae were collected using standard dippers from rice field and margin of open sewage canals where it reached to the plateau at the south part of the capital city of tehran. the larval colony was transported to the insectary of school of public health (sph), bred the immature using the habitat water and a few amount of flake fish food added daily to each enameled pan until adults emerged. laboratory strain of cx. pipiens (teh-sph) which was used as a reference in this study originally collected from capital city of tehran and had been colonized in sph insectary from 1960 for more than 50 years and never exposed to any insecticides. mosquitoes were maintained at 28–30 °c and 55–75 % relative humidity (rh). the light intensity in test-room was 5–8 footcandles using two 40 w florescent lamps without any natural light and the reflected light reduced to a minimum. the tests were carried out between 9.0 am and 5.0 pm. the emerged adults were fed with 5 % sucrose solution until 2–3 age mosquitoes which used for the tests. the irritability tests were carried out according the instruction of world health organization (who) and the irritability test kit (who 1970). due to exhibition of more take-offs of female mosquitoes than males (rutledge et al. 1999), so only sugar-fed female mosquitoes were individually tested for the irritability level using both teh-sph strain and field population and exposed 15 min to who’s impregnated papers inside the who’s cone and the number of take-off was counted and mean and standard error was assessed as a criteria for irritability level. due to direct correlation of mosquitoes take-off with temperature (kaschef 1968), the tests were done at insectary condition with 29–30 °c and 55–75 % of rh and the light only provided with two 40 w florescent lamps and the intensity varied between 5 to 8 foot candle for laboratory environment without any natural light. the following insecticide impregnated papers of who were employed, ddt 4 %, bendiocarb 0.1 %, propoxur 0.1%, malathion 5 %, fenitrothion 1.0 %, permethrin 0.75 %, deltamethrin 0.05 %, lambdacyhalothrin 0.05 %, etofenprox 0.5 % and cyfluthrin 0.15 % and the oil impregnated papers were used for the control group. the mean of take-off was analyzed using anova and independent t-test was separately for the treated and control groups according to field population and teh-sph strain under spss ver. 18.0. results the irritability levels of were presented in table 1,2 and figs 1, 2,3 using teh-sph and field strains of cx. pipiens complex according to different insecticides. the mean and standard error of take-off for ddt 4.0 % as an organochlorine pesticide was 11.2±1.5 and 2±0.2 respectively in both field and teh-sph strains. this insecticide should be regarded as a discriminative for the detection of irritability among mosquitoes population. from the organophosphate, fenitrothion 1.0 % and malathion 5.0 % were assessed and number of take-off per minute were 4.6±1.6 and 2.8±1.0 for the field strain and 1.3±0.7 j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 181 http://jad.tums.ac.ir published online: january 05, 2016 and 2.2±0.9 for teh-sph compared to 1.7± 0.3 for the control group. from carbamate, propoxur 0.1 % and bendiocarb 0.1 % were assessed for the irritability level against field and teh-sph strains of cx. pipiens complex. the number of take-off per minute of propoxur 0.1 % and bendiocarb 0.1 % were 4.0±1.1 and 2.8±0.2 for the field strain and 3.6±0.2 and 2.9±0.2 compared to 1.3±0.7 for teh-sph strain. the irritability of both type i (permethrin 0.75%) and type ii (deltamethrin 0.05 %, cyfluthrin 0.15% and lambdacyhalothrin 0.05%) as well as pseudo-pyrethroid (etofenprox 0.5%) were assessed. the irritability level for deltamethrin 0.05 %, cyfluthrin 0.15 %, lambdacyhalothrin 0.05 % were respectively 6.7±0.3, 4.1±0.2, 2.3±0.2 for the field strain and 3.5±1.4, 2.1±1.0 and 2.1±0.9 compared to 1.3±0.7 and 5.4±0.4 for teh-sph strain. the irritability caused by etofenprox 0.5 % as pseudopyrethroid was 2.0±0.2 and 1.5±0.8 for field and teh-sph strains. the number of takeoff per minute for permethrin 0.75 % as pyrethroids type i was 6.1±0.3 and 4.1±1.5 for the field and teh-sph strains. table 1. irritability levels of culex pipiens (tehran field strain) to different insecticides at laboratory conditions using who’ kit and the impregnated papers irritability levelstandard error (se) mean of take off in 15 minutes number of take off insecticides moderately-irritable0.511.2223ddt 4.0% moderately-irritable0.36.7133deltamethrin 0.05% moderately-irritable0.36.1121permethrin 0.75% moderately-irritable0.34.895malathion 5.0% moderately-irritable0.24.181cyfluthrin 0.15% moderately-irritable0.23.676propoxur 0.1% hypo-irritable0.22.957bendiocarb 0.1% hypo-irritable0.12.549fenitrothion 1.0% hypo-irritable0.22.345lambdacyhalothrin 0.05% hypo-irritable0.22.039etofenprox 0.5% non-irritable0.31.9538control table 2. irritability levels of culex pipiens (teh-sph strain) to different insecticides at laboratory conditions using who kit and impregnated papers irritability levelstandard deviation (se) mean of take off in 15 minutes number of take off insecticides moderately-irritable0.94.4132ddt 4.0% moderately-irritable0.94.1123permethrin 0.75% moderately-irritable0.83.5106deltamethrin 0.05% hypo-irritable0.52.265malathion 5.0% hypo-irritable0.52.163cyfluthrin 0.15% hypo-irritable0.52.164lambdacyhalothrin 0.05% non-irritable0.21.544etofenprox 0.5% non-irritable0.21.338fenitrothion 1.0% non-irritable0.21.340bendiocarb 0.1% non-irritable0.21.338propoxur 0.1% non-irritable0.21.3165control j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 182 http://jad.tums.ac.ir published online: january 05, 2016 j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 183 http://jad.tums.ac.ir published online: january 05, 2016 j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 184 http://jad.tums.ac.ir published online: january 05, 2016 j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 185 http://jad.tums.ac.ir published online: january 05, 2016 fig. 1. irritability levels of the field population compared with the teh-sph strain of culex pipiens to 10 insecticides, tehran, iran fig. 2. comparison of irritability mean of culex pipiens (field strain) to different insecticides, tehran, iran j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 186 http://jad.tums.ac.ir published online: january 05, 2016 fig. 3. comparison of irritability means of culex pipiens (teh-sph strain) to different insecticides, tehran, iran discussion in iran, many pesticides have been applied for the control of medically important arthropods in both the private and public health sectors. various vector control measures including residual spraying (irs), larviciding using bacillus thuringiensis (bti), impregnation of bed nets (itns) and long-lasting impregnated nets (llitns) have been employed in malaria foci. currently the deltamethrin is one of the most commonly insecticide used in public health against malaria vectors in iran since 1994. in recent years, the new evidence of pyrethroids resistance has been indicated with the increasing trend of tolerance among different species of the mosquitoes in iran (vatandoost and hanafi 2012). the resistance history to insecticides has been notified during past years. susceptibility tests which carried out during july-august 2000, the cx. pipiens was highly resistant to ddt 4.0 % with lc50 value reported 6.8 % and the resistance range had been varied at both north and south parts of tehran (nazari and janbakhsh 2000). expert committees of who has been classified the irritability level of mosquitoes to insecticides as hypo-irritable (2–3 take-off per minute), moderately irritable (3–30 takeoff per minute) and hyper-irritable (30–40 take-off per minute) (who 1964). in this study, for the better statistical analysis and reduction of bias, the irritability tests were carried out at uniform conditions including fixed time (9 am–5.0 pm), controlled temj arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 187 http://jad.tums.ac.ir published online: january 05, 2016 perature (29–30 °c), humidity (55–75%) and light intensity (5–8 fc) as well as the tested mosquitoes were sugar-fed, 2–3 ages and the first generation of the field population having definite strains of female cx. pipiens were used. the locality of immature sampling was fixed in south part of tehran during the mass collection. a full series of irritability tests were carried out using 10 groups of insecticides including organochlorine, organophosphate, carbamate and pyrethroids against field population of cx. pipiens in comparison with a the-sph strain. the finding concerning ddt-irritability tests revealed the moderately irritable level both for the field population (11.2±0.5) and the teh-sph strain (6.4±0.2). the take-off mean for ddt was recorded as the highest value in field strain and statistically differed from irritation caused by other insecticides (p< 0.05). furthermore ddt, a series of irritability tests was carried out on the organophosphate (malathion 5.0%), one carbamate (propoxur 0.1%) and three pyrethroids (permethrin 0.75%, deltamethrin 0.05% and cyfluthrin 0.15%) which assessed moderately-irritable with the take-off means ranged between 3.5 % and 6.7 % against both insectary and field population of cx. pipiens. the results of statistical analysis of irritability tests were shown in fig. 1 and fig. 2. ddt, permethrin and deltamethrin induced significantly more take-offs than other insecticides but mean of take-off for three latter insecticides did not differ from each other against both strains of cx. pipiens (fig. 2). irritability tests for malathion 5.0 %, cyfluthrin 0.15 and propoxur 0.1 % also revealed the moderately irritable level. the irritability of remaining insecticides including bendiocarb 0.1 %, fenithrothion 1.0 %, lambdacyhalothrin 0.05 % and etofenprox 0.5 % did not significantly differed from control group and so assessed as non-irritable level. ddt has two types of toxic effect on mosquitoes-an initial rapid knock down effect, followed by a lethal effect as well as an excito-repellent effect is shown (hodjati et al. 2003). irritability tests with the ʺbig-sized mosquitoʺ an. maculipennis showed that the mean of take-off for ddt 4.0 % was 26.4 and 19.4 for an. superpictus (eshghi 1972). under the large cage conditions ddtresistant an. stephensi were more readily irritated by the insecticide than a susceptible strain of the same species (eshghy 1977). also the similar work with another big-sized mosquitoes an. sacharovi showed that ddt had the most and deltamethrin the least irritancy effect. the average number of take offs/fly/minutes were recorded 0.8±0.2, 0.7± 0.2, 0.5±0.2, 0.5±0.3, and 0.2±0.1, for ddt, permethrin, lambdacyhalothrin, cyfluthrin and deltamethrin respectively (vatandoost and abai 2012) all the above-mentioned three species showed variability in their irritability to different pyrethroids especially deltamethrin. results from ddt irritability assays also showed that organochlorine insecticides can induce behavior-modifying actions, such as contact irritancy and spatial repellency, which reduces man-vector contact, despite evidence of insecticide resistance within the test population (achee et al. 2009). the irritability to ddt shown in some populations of an. albimanus and an. nuneztovari may reduce the effectiveness of residual applications of this insecticide by causing the mosquitoes to seek untreated surfaces and/or leave the house to rest outdoors and thus avoid a lethal dose (quinones and suarez 1989). it has been shown that an. hyrcanus had a low irritability to insecticides but high resistance to ddt and moderate resistance to propoxur, while the susceptibility to malathion and fenitrothion remained complete. it is supposed that the nature of adaptation to insecticide pressure is, to a great extent, determined by mosquito endophily or exophily (sorokin j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 188 http://jad.tums.ac.ir published online: january 05, 2016 and mingaleva 1992). the behavioral avoidance to insecticides may play a significant role in reducing the selection pressure and thus occurrence and spread of insecticide resistance (chareonviriyaphap et al. 2013). the other irritability experiments which carried out in kahnooj district, southeastern iran, showed that ddt and permethrin had the most irritancy effect on an. stephensi and an. dthali. in contrast with the our results, ddt and deltamethrin showed the least irritancy effect against an. stephensi with 0.42+0.08 and 0.77+0.12 take-offs min/adult, respectively. however lambdacyhalothrin had the least irritancy effect against an. dthali with 0.096+0.02 take-offs/min/adult. the mean number of take-offs/min/adult with permethrin showed significant difference to ddt, lambdacyhalothrin, cyfluthrin and deltamethrin (vatandoost et al. 2005b). also in the similar area, jiroft district, southeastern iran, the low take-off for deltamethrin and the higher value for ddt were shown. the average number of take-offs per min per adult was 2.09± 0.13 for ddt, 0.581±0.05 for dieldrin, 1.85±0.08 for permethrin, 1.87± 0.21 for lambda-cyhalothrin, 1.53±0.13 for cyfluthrin, and 1.23±0.1 for deltamethrin (abai et al. 2009). the irritability tests were carried out in a malaria–prone area, iranshahr and nikshahr districts which similar values were recorded for an. stephensi exposed to permethrin, deltamethrin, cyfluthrin and lambdacyhalothrin as 6.64±1.04, 3.11±0.67, 2.73±0.61 and 2.57±0.67, and an. culicifacies were 2.24±0.37, 1.44±0.38, 1.59±0.35 and 1.46±0.5, respectively (vatandoost and borhani 2004). the results of another investigation with an. culicifacies showed the most irritancy for ddt and lowest for permethrin and cyfluthrin in iranshahr and sarbaz district, southeastern iran (vatandoost et al. 2011). the other study with an. stephensi showed the most irritancy effect for lambdacyhalothrin, the moderate for cyfluthrin and the less irritancy for deltamethrin (vatandoost 2001). behavioral resistance appears more rapidly in endophilic species than exophilic ones (muirhead 1960) and the an. stephensi has more endophilicity habit than an. culicifacies and so it is assumed that they are under insecticides pressure selection (bhatia and deobhankar 1963, abai et al. 1999, alipour et al. 2005). irritability studies of resistant and susceptible strains indicated that the resistant strain is 2.8 times less irritable to permethrin in comparison with susceptible one (vatandoost 2000) and the value of irritability may be differed with mosquito strains (brown 1958). in conclusion, based on the finding, conjoint implementation of irritability and susceptibility tests in problematic area recommended for assessment the possible changing of mosquitoes behavior such as diversion to untreated indoor shelters or resting at outdoors and in long term, reinforcement of exophilic population at the areas under insecticide pressure whether indoor residual spraying or impregnation of bed nets operations. the behavioral avoidance to insecticides may play a significant role in occurrence and spread of insecticide resistance. conclusion periodically experiments on irritancy of different insecticides against mosquito will provide a clue for integrated vector management acknowledgements we acknowledge of dr a latifi, phd student for his kind assistance during the mass collection of immature stages of culicidae in tehran. we would like to extend our appreciation and thanks to mr ah hosseini, and mrs z abbasi, staff of the sph insectary for their great efforts in mass breeding of teh-sph strain. this study was j arthropod-borne dis, june 2016, 10(2): 178–191 s rahimi: irritability levels of field … 189 http://jad.tums.ac.ir published online: january 05, 2016 part of the msc thesis supported by tehran university of medical sciences, grant no. 20149. references abai mr, vatandoost h, nateghpour m (1999) field evaluation of k-othrine® for 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control. 10th report of the who expert committee on insecticides. vector resistance to pyrethoids. who tech rep ser. 191. who (1970) insecticides resistance and vector control.17th report of the who expert committee on insecticides. instructions for determining the irritability of adult mosquitoes to insecticides. who tech rep ser. 433: 158–163. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq control assoc. 3(4): 568–573. zaim m, cranston p (1986) checklist and keys to the culicinae of iran (diptera: culicidae). mosq syst. 18 (3–4): 233– 245. j arthropod-borne dis, september 2016, 10(3): 330–336 t hazratian et al.: diversity of sand flies … 330 http://jad.tums.ac.ir published online: january 06, 2016 original article diversity of sand flies (diptera: psychodidae) in endemic focus of visceral leishmaniasis in azar shahr district, east azarbaijan province, north west of iran teimour hazratian 1, hasan vatandoost 2, mohammad ali oshaghi 2, mohammad reza yaghoobi-ershadi 2, esmael fallah 1, sayena rafizadeh 3, mohammad reza shirzadi 3, mansoreh shayeghi 2, kameran akbarzadeh 2, *yavar rassi 2 1departmemt of parasitology, faculty of medicine, tabriz university of medical sciences, tabriz, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3ministry of health and medical education, tehran, iran (received 3 aug 2014; accepted 17 aug 2014) abstract background: there are nearly 1000 species of phlebotomine sand flies in 6 genera, of which only two, phlebotomus in the old world and lutzomyia in the new world are medically important. globally, leishmaniasis prevalent in 98 countries and affects estimated 12 million people with almost two million new cases per year. some rural areas of azarshahr district in east azarbaijan province have been reported to be endemic for visceral leishmaniasis. this study is the first attempt to determine the species diversity and density in a new focus of visceral leishmaniasis in azarshahr district, east azarbaijan province, iran. methods: sand flies were collected form indoor and outdoor biweekly using sticky traps. diversity index of the collected sand flies within different villages were estimated by the shannon-weaver. results: the activity of the sand flies extended from april to october with one peak in august. diversity of sand flies within study area were estimated as 0.917, 1.867, 1.339, 1.673, and 1.562 in almalodash, jaragil, segaiesh, amirdizaj, and germezgol vvillages, respectively. conclusion: identifying the diversity and seasonal abundance of the collected species is of importance for prediction of the period of maximum risk for leishmaniasis transmission and for the successful implementation of a control program. species diversity is one of the most important factors in ecological studies. keywords: diversity, density, sand flies, visceral leishmaniasis, azar shahr, iran introduction phlebotomine sand flies belong to the family psychodidae (young and duncan 1994). psychodidae family is characterized by numerous parallel veins running to wing margin and the presence of dense hairs on the wings and thorax. from 5 psychodidae subfamilies, phlebotominae is known to phlebotomine sand flies by biting mouth parts that are longer than the head, fivesegmented palps, nearly cylindrical antennae, a five-brached radial vein on the wing, and the absence of an eye-bridge (lane and crosskey 1993). generally, sand flies are distinguished from other small flies by their size (2.5–3.5mm in length), characteristic hopping flight, and the "v" position in which they hold their wings while resting (triplehom and jhonson 2005). in the old world, about 40 species of phlebotomus have been proven or suspected vectors of leishmania (rassi 2004). in iran, 44 species of the sand flies are found and the *corresponding author: prof yavar rassi, email: rassiy@tums.ac.ir j arthropod-borne dis, september 2016, 10(3): 330–336 t hazratian et al.: diversity of sand flies … 331 http://jad.tums.ac.ir published online: january 06, 2016 presence of other 10 species is doubtful. in the endemic center of northwest of iran, two species of ph. kandelakii and ph. perfiliewi and in the south of iran, three species of, ph. keshishiani, ph. alexandri and ph. major are the vectors of the disease (rassi 2007, azizi 2009). leishmaniasis is prevalent in 98 count-ries. this parasitic disease is known one of ten major diseases by who (maia et al. 2011). it has widely spread not only in tropical countries but also in some regions with very different topography (faiman 2000, who 2010). clinically, leishmaniasis is observed in four types: visceral leishmaniasis (vl), cutaneous leishmaniasis (cl), mucocutaneus leishmaniasis (ml) and difuse cutaneous leishmaniasis (dcl) (who 1994). from 4 mentioned types, only two types of visceral and cutaneous leishmaniasis are common in iran. visceral leishmaniasis is considered as the main problems in iran (rassi 2011). this disease is observed in all areas of iran sporadically but the endemic foci of the disease have been reported from ardabil, fars, boshehr and east azerbaijan provinces (rassi et al. 2005, oshaghi et al. 2009, rassi et al. 2010). some rural areas of azarshar district in east azarbaijan province have been reported to be endemic for visceral leishmaniasis with more than 34 new cases. performing the control plans without sufficient information about the fauna and ecological parameters is not possible. therefore, this study was carried out during late april to late october 2010 in rural areas of azarshahr district, east azarbaijan province, northwestern iran, to identify diversity, species composition and other ecological parameters. material and methods study area azar shahr is located 45 km far from tabriz. the geographical coordinates is 45 30΄e and 37 ̊30΄n. the height of this county from the sea level is 1368 meters. it is limited to lake of urmia from the west, to ajabshir city from the south, to osko from the north and to sahand mountains from the east. in this county, the average rainfall and the relative humidity were 303 and 49 millimeters, respectively. the mean of the annual temperature is 15.25 and the mean of monthly maxi-mum and minimum temperatures were 27.7 and 3.7 °c respectively. the population of this county has been 103952 people in 2006. selection of study villages according to reported cases of the disease, a number of villages including: segaiesh, amirdizaj, almalodash, jaragil and germezgol were selected from the county of azarshahr in east azarbaijan province. segaiesh village: it is located 14 km from azar shahr with 183 families and 633 people in 2010. amirdizaj village: it is located 16 km from azar shahr with 250 families and 1046 people. almalodash village: it is located 20 km from azar shahr with 182 family and 860 people. jaragil village: it is located 15 km from azar shahr with 110 families and 422 people. germezgol village: it is 12 km from azar shahr with 70 families and 300 people in 2010. sand fly sampling sand flies were collected using sticky traps (castor oil coated white papers 20 cm× 30 cm) biweekly from selected villages indoors (bedrooms, guest room, toilet and stable) and outdoors (rodent burrow). traps (60 papers per village) were installed in sunset and sand flies were collected during sunrise. sample collection began in late j arthropod-borne dis, september 2016, 10(3): 330–336 t hazratian et al.: diversity of sand flies … 332 http://jad.tums.ac.ir published online: january 06, 2016 april and continued until late october 2010, when sand fly activities were reduced sharply. collected sand flies were stored in 96% ethanol. in the laboratory for species identification, head and three last abdomen segments of females, head with whole body of males specimens were mounted in puri’s medium. species identification were carried out after one week using keys of sand flies (rassi and hanafi bojd 2006). determination of density the density of sand flies collected by sticky traps was calculated using the formula number of specimens / of sticky traps and numbers of specimens/ number of traps. logistic regression used to investigate the existence of a correlation between the abundance of the sand flies, and average monthly temperature in the study area using sas software (kasap et al. 2009, tarallo 2009). the density of species was corrected according to the formula (guernaoui 2009, rajider 2010) density = determination of the species diversity to calculate the species diversity in a population, there are different methods but in most resources, shannon-weaver index has been used. shannon-weaver index is shown as    s 1i pilogepih' . in this formula, s is the richness of the species and pi is the proportion of species ith in the population and log e is common logarithm. to convert it to common logarithm, it should be multiply in 2.303. in the present study, it is used shannon-weaver formula ( h'). species diversity composed of two factors, species richness and species evenness. there are many kind of species diversity indexes, a common one is the shannonweaver index: (h'=   s i 1 pilogepi) h΄= the diversity index pi= the proportion of the ith species loge= natural log s= number of species in the community results according to formula, monthly average density, number of specimens/ m2 of sticky traps were 37.60, 41.10, 40.23, 30.38 and 30.67 in almalodash, jaragil, segaieesh, amirdizaj and germezgol, respectively. corrected monthly average density, number of specimens/ number of traps, in mentioned villages were 1.47, 1.51, 1.39 and 1.40. table 1. density of sand flies in studied villages in azarshahr district, eastern azarbaijan, iran, 2010 mean density= trapsofnumber specimensofnumber 1 mean density= trapsofm specimensofnumber 2 mean density= trapsofnumber specimensofnumber village 1.4737.601.18almalodash 1.3930.380.95amirdizaj 1.5141.101.29jaragil 1.5140.231.26segaiesh 1.430.670.96germezgol to calculate the species diversity, we used the formula shannon-weaver because it has been used in most resources to calculate diversity. the results are shown in tables 2, 3, 4, 5 and 6. j arthropod-borne dis, september 2016, 10(3): 330–336 t hazratian et al.: diversity of sand flies … 333 http://jad.tums.ac.ir published online: january 06, 2016 table 2. diversity of sand flies in almalodash village in azarshahr district, eastern azarbaijan, iran, 2010 community a (almalodash) piloge piloge piproportion (pi)numberspecies -.333-.6660.54554ph. papatasi -.202-2.5260.0877ph.sergenti -.169-2.5140.0658ph.kandelakii -.046-4.6060.019ph.(adlerius) spp -.316-1.6610.19203ph.c.group -.186-2.6500.0778ph.halepensis -.078-3.9130.0219ph.longiductus -.078-3.9130.0219ph.major -.046-4.6060.019ph.mongolensis    s i pilogepih 1 454/1'1026total table 3. diversity of sand flies in amirdizaj village in azarshahr district, eastern azarbaijan, iran , 2010 community b (amirdizaj) piloge piloge piproportion (pi)numberspecies -.328-1.5560.211214ph. papatasi -.337-1.4740.229232ph.sergenti -.442-1.7440.0099ph.mesghali -.303-1.7540.173175ph.c.group -.278-1.9450.143145ph.mongolensis -.278-1.9450.143145ph.tobbi -.042-0.4110.0099ph.kandelakii -.072-4.0810.01819ph.halepensis -.124-3.2710.03839ph.andrejevi -.072-4.0180.01819ph.caucasicus -.042-4.7110.0099ph.pawlowskyi    s i pilogepih 1 918/1'1015total table 4. diversity of sand flies in jaragil village in azarshahr district, eastern azarbaijan, iran, 2010 community c (jaragil) piloge piloge piproportion (pi)numberspecies -.159-2.9010.00567ph. papatasi -.363-1.1260.313378ph.sergenti -.087-3.7330.02328ph.tobbi -.006-4.1360.01619ph.kandelakii -.035-4.9630.0079ph.major -.362-1.1870.305369ph.c.group -.110-3.3430.03239ph.hodsoni -.286-1.8840.152184ph.mongolensis -.202-2.2520.08097ph.halepensis -.035-4.9630.0079ph.longiductus -.035-4.9630.0079ph.andrejevi    s i pilogepih 1 74/1'1208total j arthropod-borne dis, september 2016, 10(3): 330–336 t hazratian et al.: diversity of sand flies … 334 http://jad.tums.ac.ir published online: january 06, 2016 table 5. diversity of sand flies in segaiesh village in azarshahr district, eastern azarbaijan, iran, 2010 community d (segaiesh) piloge piloge piproportion (pi)numberspecies -.333-0.6160.540438ph. papatasi -.049-4.5140.0119ph.tobbi -.144-3.0580.04738ph.sergenti -.342-1.4320.239194ph.c.group -.049-4.5110.0119ph.hodsoni -.144-3.050.04738ph.kandelakii -.167-2.8310.05948ph.mongolensis -.167-3.7330.02319ph.halepensis -.049-4.5110.0119ph.perfiliewi -.049-4.5110.0119ph.major    s i pilogepih 1 413/1'811total table 6. diversity of sand flies in germezgol village in azarshahr district, eastern azarbaijan, iran, 2010 community e (germezigol) piloge piloge piproportion (pi)numberspecies -.353-1.3130.269156ph. sergenti -.066-4.1360 .0169ph.ansari -.066-4.1360٠0169ph.kandelakii -.066-4.1360٠0169ph.c.group -.207-2.2480٠08348ph.halepensis -.368-.9550.385223ph.mongolensis -.113-3.4120.03319ph.tobbi -.251-2.1460.11768ph.andrejevi -.149-2.9960.05029ph.caucasicus -.066-4.1360٠0169ph.perfiliewi    s i pilogepih 1 413/1'811total due to the tables of species diversity, it is obvious that the species diversity in amirdizaj village is in highest level than other villages and the diversity of the sand flies in this village (population b) has more diversity and better results. discussion this study is the report of the results of entomology investigations on sand flies in azarshahr district. the geographical location of this district is appropriate for the population being potentially at risk for visceral leishmaniasis. according to table 1, the corrected density of sand flies in jaragil and segaiesh villages had highest level (1.51) and we found the lowest level (1.39) in amirdizaj village. the monthly activities of sand flies started from first half of june and have been continued to second half of october. the most level of density was related to august. whereas, there was a peak of temperature in this month. in another words, j arthropod-borne dis, september 2016, 10(3): 330–336 t hazratian et al.: diversity of sand flies … 335 http://jad.tums.ac.ir published online: january 06, 2016 the population of the sand flies dynamically was direct correlation to the temperature of the area but to the humidity. given the history of the emergence of the sand flies adults in the region (20 may, 2010) and the density peak of in this region, it can be determined the control programs. especially, if there is chemical control program, we can determine exact date of residual spraying reasonably and avoid unreasonable residual spraying, further costs and extreme residual spraying. among 16 species collected and identified, ph. ansarii and ph. mesghali had lowest density (0.18%). according to the studies of rassi et al. 2000 on the fauna of the sand flies in west north of iran and kaverizadeh et al. 2000 on the fauna of sand flies in ahar, all 16 species have been reported first time in azarshahr. furthermore, two species of s. hodgsoni and p. mesghali have been observed in east azerbaijan. two main factors of species diversity are: richness of species and evenness of species. richness of species is the species diversity in the population and the evenness of species is the species diversity of the people in the species. the results are shown in tables 2, 3, 4, 5 and 6. in al-baha province in the saudi arabia, a similar study has been performed (dohaandsomy 2010) with bionomics relation of phlebotomine sand flies population with temperature and species diversity in different places that its result is compatible to our results of this study. conclusion according to the results, most species diversity was observed in amirdizaj, therefore, this village can be the best choice for determine of species composition of 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geographic distribution of lutzomyia sand flies in mexico, the west indies, central and south america (diptera: psychodidae). associate publishers, gainesville fl. j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 original article baseline susceptibility of different geographical strains of anopheles stephensi (diptera: culicidae) to temephos in malarious areas of iran aboozar soltani 1, *hassan vatandoost 1, *mohammad ali oshaghi 1, ahmad ali enayati 2, ahmad raeisi 3, mohammad reza eshraghian 1, mohammad mehdi soltan-dallal 4, ahmad ali hanafi-bojd 1, mohammad reza abai 1, fatemeh rafi 1 1department of medical entomology and vector control, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran 2school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 3national malaria control department, cdc, ministry of health and medical education, iran 4department of microbiology, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran (received 16 jun 2012; accepted 20 feb 2013) abstract background: malaria still remains a public health problem in iran. there are different vector control interventions such as insecticide spraying. the present study was carried out to determine the susceptibility status of anopheles stephensi larvae to temephos as a national plan for monitoring and mapping of insecticide resistance methods: eight different localities in two main malarious provinces were determined as field collecting sites. mosquitoes were collected from the field and reared in an insectray. susceptibility assays were carried out according to the who method. the laboratory reared susceptible beech-lab strain was used for comparison. data were analyzed using probit analysis to determine lc50 and lc90 values. results: susceptibility of an. stephensi to temephos indicated that the lc50 ranged from 0.0022 mg/l to 0.0141 mg/l. although all field strains were susceptible to temephos, considerable variations in temephos resistance ratios of field strains were noticed from all the localities studied in comparison with the susceptible strain. a low level of resistance ratio was noticed in an. stephensi populations except for the chabahar strain (rr= 4.27 fold). all field-collected an. stephensi populations exhibited homogeneity to the larvicide except for bandar abbas and hormoodar village strains (p> 0.05%). conclusion: due to intensive use of temephos in the neighboring countries and occurrence of resistant to this insecticide in the main malaria vector in the region, insecticide resistance gene may evolve in the populations of an. stephensi. if temephos be applied as a larvicide it should be used judiciously for resistance management, as rotation strategy. keywords: anopheles stephensi, temephos, susceptibility, iran, larvicide resistance introduction before implementing the national malaria control program in iran in 1957, about 60% of population of the country was living in endemic areas with 30% to 40% malaria morbidity (edrissian 2006). despite the relatively successful implementation of malaria control programs in iran in recent years, malaria still remains a main health problem especially in southeastern regions including hormozgan province, sistan and baluchistan province and southern parts of kerman province with a population of 4.8 million people where more than 90% of all cases are reported from. about 68% of all malaria cases have been reported from these provinces in 2002, whereas it increased to 95% in 2007 (raeisi *corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com, dr mohammad ali oshaghi, e-mail: oshaghima@yahoo.com 56 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 et al. 2008, vatandoost et al. 2010). the presence of vector species in these regions beside tropical climate and socio-economic conditions make appropriate situation for occurrence and persistent transmission of malaria in these regions. malaria in malarious areas of iran is unstable with two seasonal peaks mainly in spring and autumn. outbreaks due to plasmodium vivax usually occur after rainy season (hanafi-bojd et al. 2012). in iran several species and biological forms of anopheles were recorded, but only 7 anopheline mosquitoes including anopheles stephensi, an. dthali, an. culicifacies s.l., an. fluviatilis s.l., an. superpictus, an. sacharovi and an. maculipennis s.l., have been confirmed as the main vectors and an. pulcherrimus reported as a suspected vector (naddaf et al. 2003, oshaghi et al. 2003a, sedaghat et al. 2003a, 2003b, vatandoost et al. 2006a, doosti et al. 2006, vatandoost et al. 2007, vatandoost et al. 2010). anopheles stephensi is a sub-tropical species and also an important vector of human malaria throughout the middle east and south asian region, including the indo-pakistan subcontinent (dash 2007, hanafi-bojd et al. 2012), with a westward extension through iran and iraq into the middle east and arabian peninsula. this species is considered to be the main malaria vector in the persian gulf area. sporozoite rates of samples from the south of iran were reported from 0.2 to 1.8% (raeisi et al. 2008). previous studies have shown an. stephensi to be the most prevalent anopheline species in the malarious areas of southern iran (hanafibojd et al. 2012). a wide range of anthropophilic indices has been reported from different geographical regions of india (tyagi and yadav 2001). the first report of resistance of this species to ddt was in 1958 from southern iran. anopheles stephensi is resistant to ddt, dieldrin, and malathion at the adult stage (edrissian 2006), although there is some indications of tolerance to pyrethroids (hanafibojd et al. 2012). there are different studies for evaluation of different control methods for this species in iran (enayati et al. 2003, vatandoost and hanafi-bojd 2005, davari et al. 2007, abai et al. 2008, rafinejad et al. 2008, soltani et al. 2008, vatandoost et al. 2008, vatandoost and hanafi-bojd 2008, vatandoost et al. 2009a, vatandoost et al. 2009b, omrani et al. 2010) resistance to ddt, dieldrin and malathion mainly in the adults of an. stephensi, have been widely distributed in persian gulf, middle-east and indian subcontinent causing operational problems for control programs (vatandoost et al. 2005). in 2006 for the first time in the middle east, resistance to the organophophate larvicide, temephos, was confirmed in the al-dhahira region (oman) in the malaria vector an. stephensi breeding in water storage tanks. the level of resistance was 2.5 times higher than who diagnostic dose (anderasen 2006). low level of larval resistance was found in pakistan (omer et al. 1980). despite of development of ddt resistance in adults of an. stephensi, the larvae showed susceptibility to ddt in south of iran. in hormozgan province, an. stephensi larvae showed susceptibility to malathion, temephos and chlorpyrifos, but resistance to fenitrothion in bandar abbas (vatandoost et al. 2004) and tolerance to fenthion in bashagard area (hanafi-bojd et al. 2012). larval control in the past had been dependent mainly on the use of chemicals such as paris green and larvicidal oils (ansari et al. 2004). at present, biological control methods using larvivorous fish and bacillus thuringiensis in addition to chemical control using organophosphorus insecticides are being used for larviciding in south of iran (soltani et al. 2008). environmental concerns shifted researches to find natural larvicides originated from plants in recent years (vatandoost and vaziri 2004, hadjiakhoondi et al. 2005, 57 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 sadat ebrahimi et al. 2005, hadjiakhoondi et al. 2006, shahi et al. 2010, sedaghat et al. 2011). temephos, an organophosphorus insecticide, has been included in the list of who as a suitable and safe mosquito larvicide that can be used in drinking water. the toxicity of this insecticide is low and unlikely present acute hazard (chavasse and yap 1997). center for disease control section of ministry of health and medical education of iran recently decided to reuse the temephos as larvicide in malaria control program. considering the incidence of resistance in an. stephensi to temephos in oman (anderasen 2006), the southern neighbors of iran, the present study was undertaken to determine the susceptibility status of an. stephensi larval stages to the temephos before the reuse of temephos in the field. materials and methods study area eight different areas in two important malarious provinces were considered as field collecting sites including: bandar abbas port, minab county and hormoodar village (near the bandar abbas) in hormozgan province, and chabahar sea port, two villages of iranshahr county (bampoor and abtar) and two villages of sarbaz county (angoori and machkor) in sistan and baluchistan province (fig. 1). bandar abbas port (54o53’–56o03’e, 26o53’– 27o31’n) is the capital of hormozgan province, a plain area with an average altitude of 9 m above sea level. the city has a hot and humid climate. maximum summer temperature can reach up to 49 oc, whereas minimum winter temperature drops to about 5 oc. average annual rainfall in 2004–2008 was 118.44 mm and mean annual relative humidity was 63.4% (www.weather.ir). in 2010 total population of bandar abbas city was 572584. about 77% of this population is living in urban area, 23% in rural area. minab (27°11′53″n 54°22′7″e ) is a county in hormozgan province that located in eastern part of the province with climatic conditions similar to the bandar abbas (fig. 1). according to the 2010 census, the county's population was 243055 in 50478 families. chabahar port (25°33′n 60°41′e) is a county in sistan and baluchistan province, with a hot and humid climatic conditions in the vicinity of oman sea and pakistan border. based on the 2011 census, the county's population was 246175 in 41532 families. the urban population is 77128, of which a majority resides in chabahar city. iranshahr (27°34′n 59°53′e) is another tropical county in western part of sistan and baluchistan province. according to the 2010 census, the county's population was 244779 in 49443 families. sarbaz (26°26′n 61°29′e) is another county in sistan and baluchistan province in iran. according to the 2006 census, the county's population was 162960 in 31449 families. mosquito strains the field collected strains of an. stephensi were reared in the insectaries of bandar abbas and iranshahr health research stations for further tests. after the establishment of the field strains in the laboratory, the first generation of mosquitoes was used for susceptibility tests. a susceptible laboratory strain of an. stephensi (beech-lab from insectarium of department of medical entomology, tehran university of medical sciences) was used to compare the susceptibility status of the field strains with. this strain has been maintained in the laboratory without exposure to insecticides for 28 years. insecticide technical grade insecticide used in the pre 58 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 sent study was temephos 90% (batch no: tem/136-229) provided by levant overseas development ltd., argenteuil, france. based on pre-tests, five concentrations of the larvicide (0.25 mg/l, 0.0625 mg/l, 0.0156 mg/l, 0.0039 mg/l and 0.00195 mg/l) were considered for susceptibility assays. bioassay consisted of five concentrations resulting 10–90% mortality. butanone 2% in absolute ethanol was used as a control. susceptibility tests susceptibility assay was carried out according to the method described by who (who 2012). the toxicity of temephos to an. stephensi, from field-collected population was determined and compared with laboratory reared susceptible beech-lab strain. late 3rd instar larvae were exposed to five doses of the larvicide. at each concentration, a total of 100 larvae in four replicates of 25 larvae were tested. two replicates of 25 larvae were used as control in each test. the larvae were fed with bemax and fish food, and mortality counts were made 24 h after exposure. moribund larvae (presenting tremors, rigidity or mobility to reach water surface on touch) were considered as dead. abbott’s formula was used to correct the observed mortality of larvae. all the data were corrected if the control mortality is between 5 and 20% (abbott 1965). data analysis data were analyzed using probit analysis (finney 1971) to determine the 50% lethal concentration values (lc50) and 90% lethal concentration values (lc90) of the field and beech-lab strains. control mortality was corrected using abbotts’ formula (finney 1971). a statistical analysis of lc50 and lc90 was based on overlap of 95% confidence intervals. resistance ratio was defined as lc50 of field strains to lc50 of lab strain. results susceptibility of an. stephensi to temephos (table 1, fig. 1) indicated that the lc50 ranged from 0.0022 mg/l in machkor population (sarbaz county, sistan and baluchistan province) to 0.0141 mg/l in chabahar port population (south of sistan and baluchistan province). the lowest lc90 was from the beechlab strain and the highest was 0.0338 mg/l from chabahar port population. according to who criteria, a 98–100% mortality rate indicates susceptibility, 80– 97% mortality rate indicates tolerance (requires confirmation of resistance with other methods) and <80% mortality suggests resistance (who 1998). results of susceptibility tests on laboratory and field strains of an. stephensi showed that the larvae were susceptible to temephos at the diagnostic dose (0.25 mg/l). although all field strains were susceptible to temephos, considerable variation in temephos resistance ratio of filed strains in comparison with susceptible strain was noticed from all the locations studied. a low level of resistance ratio was observed in the populations of an. stephensi except in that of the chabahar sea port (rr= 4.27 folds) compared to beechlab strain (p< 0.05) (table 1, fig. 2, 3). a comparatively low degree of resistance ratio to temephos (table 1, fig. 2) was obtained in the an. stephensi from all the localities studied (rr= 0.66–1.48) whereas from chabahar port, more than 4-folds resistance was noticed, compared to beech-lab strain. almost all the field-collected an. stephensi populations exhibited homogeneity to insecticide bioassay except for the population from bandar abbas port and hormoodar village (hormozgan province), where chisquare value exceeded table value at 0.05% (table 1, fig. 3). 59 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 table 1. probit regression analysis of the temephos mortality data of field collected larvae of an. stephensi, 2011 location lethal concentration(a.i.)/ppm chi-square heterogeneity (d.f.) regression coefficient (slope) resistance ratio (rr)* lc50 lc90 (95% confidence limit) (95% confidence limit) bandar abbas 0.0042(0.0037–0.0048) 0.0091(0.0075–0.0120) 29.061 (d.f.= 2) 3.8078 1.27 minab 0.0035(0.0028–0.0042 0.0159(0.0122–0.0230) 4.780 (d.f.= 3) 1.9464 1.06 hormoodar 0.0049(0.0043–0.0057) 0.0129(0.0105–0.0170) 27.846 (d.f.= 3) 3.0519 1.48 chabahar 0.0141(0.0122–0.0163) 0.0338(0.0279–0.0435) 4.025 (d.f.= 3) 3.3709 4.27 bampoor 0.0039(0.0035–0.0044) 0.0084(0.0069–0.0111) 2.740 (d.f.= 2) 3.8727 1.18 angoori 0.0026(0.0022–0.0030) 0.0082(0.0065–0.0115) 0.161 (d.f.= 3) 2.5853 0.78 abtar 0.0027(0.0022–0.0032) 0.0097(0.0076–0.0138) 1.746 (d.f.= 3) 2.2949 0.81 machkor 0.0022(0.0017–0.0025) 0.0068(0.0055–0.0097) 4.445 (d.f.= 3) 2.5657 0.66 beech-lab 0.0033(0.0030–0.0036) 0.0055(0.0048–0.0067) 962.660 (d.f.= 1) 5.8092 * rr50, resistance ratio at lc50 (rr50= lc50 of field population/ lc50 of beech-lab) fig. 1. location of anopheles stephensi collection sites from malarious areas of iran, 2011 fig. 2. temephos resistance ratio pattern in an. stephensi field strains from southern iran, 2011 60 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 fig. 3. regression lines of eight strains of an. stephensi and susceptible beech-lab strain, 2011 discussion temephos (ec 50%) has been used for years for larval control program of malaria in southern iran (edrissian 2006). many studies on the susceptibility level of an. stephensi to pesticides such as ddt and temephos has been done in iran and other countries. an. stephensi larvae from pakistan and united arab emirate were reported to be resistant to ddt (vatandoost 1996). different levels of resistance to larvicides were reported in anopheline malaria vectors worldwide. anopheles stephensi has an extensive resistance comparing to other species and is resistant or tolerant to fenitrothion, temephos and fenthion in india, fenitrothion and pirimiphos-methyl in iraq, fenitrothion, temephos, pirimiphos-methyl, chlorfoxim and foxim in iran and finally fenitrothion in pakistan (vatandoost and hanafi-bojd 2005). resistance of an. dthali to temephos also was reported (hanafi-bojd et al. 2006). in 2006 for the first time in the middle east, resistance to temephos was confirmed in an. stephensi breeding in water storage tanks in the al-dhahira region of oman (anderasen 2006), there is not confirmed report of temephos resistance of an. stephensi in iran. the results of other investigations in iran showed that this species was completely susceptible to all tested larvicides including temephos at the who diagnostic dose (vatandoost et al. 2004, vatandoost and hanafi-bojd 2005, vatandoost et al. 2005, vatandoost 2006a). in our study, a variation in toxicity levels of temephos to an. stephensi was noticed. this may be justified by a wide scope of sampling locations. different slopes of insecticide bioassay regression lines to an. stephensi from different locations shows different degree of developing insecticide resistance. table 1 show that the toxicity of temephos to an. stephensi from angoori, abtar and machkor was lower than that of susceptible beech-lab strain. similar results were reported by other researcher that some filed populations were more susceptible to insecticide than laboratory strain (ponlawat 2005, tikar et al. 2008). although the exact reason for this phenomenon is not known, overcrowding in breeding places leading to insufficient food could result in a weaker progeny. our data provided baseline information on insecticide susceptibility of an. stephensi from geographically different locations in iran. an. stephensi is still sus61 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 ceptible to temephos from all studied localities in iran and it can be used as an effective insecticide in malaria control program despite the fact that resistance to temephos has been reported from other malarious countries such as india and oman. chabahar strain of an. stephensi exhibited the highest resistance ratio to temephos compared with all other collection sites. the most probable explanation for this is that chabahar port is the nearest area to oman that resistance of an. stephensi to temephos was confirmed by anderasen (2006). therefore it could be an important alarm for developing of resistance to temephos in iranian an. stephensi. based on the results of this research, temephos can be used as a larvicide in integrated vector management in malaria control programs in the region warily. some important issues including continuous insecticide resistance status monitoring in the vectors should be considered to ensure judicious use of pesticide and implementing insecticide resistance management strategies e.g. rotation. in addition it is essential to focus on regular surveillance of malaria vectors as a routine practice in high risk malaria areas. acknowledgements this article is a part of the first author’s dissertation for fulfillment of a phd degree in medical entomology and vector control from department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. the authors are very grateful to mr m yarian from hormozgan university of medical sciences and mr a pakari and mr shahbakhsh technicians of the national institute of health research, bandar abbas and iranshahr research 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mashayekhi m, abaie mr, aflatoonian mr, hanafi-bojd aa, sharifi i (2005) monitoring of insecticides resistance in main malaria vectors in a malarious area of kahnooj district, kerman province, southeastern iran. j vector borne dis. 42(3): 100–108. vatandoost h, oshaghi m, abai mr, shahi m, yaghoobi f, baghai m, hanafibojd aa, zamani g, townson h (2006a) bionomics of anopheles stephensi liston in the malarious area of hormozgan province, southern iran. acta trop. 97(2): 196–205. 64 j arthropod-borne dis, june 2013, 7(1): 56–65 a soltani et al.: baseline susceptibility of… http://jad.tums.ac.ir published online: april 10, 2013 vatandoost h, shahi m, hanafi-bojd aa, abai mr, oshaghi ma, rafii f (2007) ecology of anopheles dthali patton in bandarabbas. iran j arthropod-borne dis. 1(1): 21–27. vatandoost h, hanafi-bojd aa (2008) laboratory evaluation of 3 repellents against anopheles stephensi in the islamic republic of iran. east mediterr health j. 14(2): 260–267. vatandoost h, khazani a, rafinejad j, khoobdel m, kebriai-zadeh a, abai mr (2008) comparative efficacy of neem and dimethylphthalate (dmp) against malaria vector, anopheles stephensi (diptera: culicidae). asian pac j trop med. 1(3): 1–6. vatandoost h, abai mr, abbasi m, shaeghi m, abtahi m, rafie f (2009a) designing of a laboratory model for evaluation of the residual effects of deltamethrin (k-othrine wp 5%) on different surfaces against malaria vector, anopheles stephensi (diptera: culicidae). j vector borne dis. 46(4): 261–267. vatandoost h, ramin e, rassi y, abai mr (2009b) stability and wash resistance of local made mosquito bednets and detergents treated with pyrethroids against susceptible strain of malaria vector anopheles stephensi. iran j arthropod-borne dis. 3(1): 19–28. vatandoost h, akbarzadeh k, hanafi-bojd aa, mashayekhi m, saffari m, elfatih mm (2010) malaria stratification in a malarious area, a field exercise. asian pac j trop med. 3(10): 807–811. who (1998) test procedure for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces, who/ cds/cpc/mal/98.12. world health organization, geneva. who (2012) malaria entomology and vector control, participant, s guide. who, geneva. 65 j arthropod-borne dis, june 2014, 8(1): 117–118 am alizadeh and n zamani: myiasis in an … 117 cases report myiasis in an 89-year-old man with non-hodgkin lymphoma afshin mohammad alizadeh 1, *nasim zamani 2 1departmentof bone marrow transplantation, taleghani hospital, shahid beheshti university of medical sciences, tehran, iran 2department of clinical toxicology, loghman hakim hospital, shahid beheshti university of medical sciences, tehran, iran (received 17 nov 2012; accepted 27 july 2013) abstract myiasis is due to the opportunistic dipterous larvae feeding on viable or necrotic tissues of the host occurring throughout the world. we report a case of oral myiasis in an immune-compromised patient suffering from nonhodgkin lymphoma. we would like to emphasize that such ectoparasitic infections may happen in immunocompromised patients and oral hygiene should be evaluated in all of these patients. keywords: lymphoma, myiasis, immune-compromised host, oral introduction myiasis is due to the opportunistic dipterous larvae feeding on viable or necrotic tissues of the host frequently occurring throughout the world. here we report a case of oral myiasis in an immune-compromised patient suffering from non-hodgkin lymphoma to emphasize the importance of paying attention to oral hygiene in immune-compromised patients. case report an 89-year-old man with the previous history of dementia and non-hodgkin lymphoma presented with sudden initiation of tachypnea, tachycardia, and decreased level of consciousness. in physical examination, coarse crackles were auscultated all over the lungs and the patient was febrile. in intraoral examination, the following scheme was seen. he was intubated and mechanically ventilated due to hypoventilation and loss of protective airway reflexes. in the laboratory evaluations, no noticeable abnormality was detected except for leukocytosis (wbc=16000) and crp of 4+. our patient was with the previous history of dementia and follicular lowgrade lymphoma of stage ia on wait and watch follow-up. on the diagnosis of myiasis, lidocaine spray accompanying with spray of hydrogen peroxide 3% were applied for killing of the larvae. the inter-denture spaces were filled with vaseline. the patient was intubated in order to prevent further migration and aspiration of the larvae. he was put on intravenous midazolam with the dose of 1 mg per hour for maintenance of unconsciousness. cefepime and clindamycin were initiated with the dose of 2g, bd and 600 mg, tds, respectively. the patient died 7 days later from sepsis. discussion myiasis is due to the opportunistic dipterous larvae feeding on viable or necrotic tissues of the host (sankari and ramakrishnan 2010). of the many types of myiasis, furuncular myiasis  *corresponding author: dr nasim zamani, e-mail: nasim.zamani@gmail.com http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 117–118 am alizadeh and n zamani: myiasis in an … 118 is the most common (diaz 2009). since oral tissues are not permanently exposed to the external environment, oral myiasis is less common than cutaneous myiasis (rossi-schneider 2007). it has previously been reported in dental extraction, nosocomial infection, drug addiction, visits to tropical countries, psychiatric patients, and conditions that cause prolonged mouth opening including senility, alcoholism, and mental retardation. fig. 1. larvae in the necrotic maxillary bone, base of the last molar tooth persistent mouth opening facilitates the deposition of the eggs by the adult fly (sankari and ramakrishnan 2010). treatment of the condition generally includes injection of lidocaine into the draining lesions, occlusive coating of vaseline (petroleum) ointment, clear fingernail polishing, tobacco tar, and surgical or vacuum extraction of the larvae in cases of unsuccessful occlusive therapy (diaz 2009). however, after removal of the larvae, it seems that the tissues recover with no subsequent complications and further need for treatment (droma et al. 2007). this case shows the development of an ectoparasitic infection in an immunocompromised patient and shows the importance of paying attention to oral hygiene in these patients. acknowledgements the authors declare that there is no conflict of interest. references diaz jh (2009) myiasis and tungiasis in: mandell gl, bennett je, dolin r (eds) principles and practice of infectious diseases, 7th edn. vol 2. elsevier press, philadelphia, usa, pp. 3637–3638. droma eb, wilamowski a, schnur h, yarom n, scheuer e, schwartz e (2007) oral myiasis: a case report and literature review. oral surg oral med oral pathol oral radiol endod. 103: 92–96. rossi-schneider t, cherubini k, yurgel ls, salum f, figueiredo ma (2007) oral myiasis: a case report. j oral sci. 49: 85–88. sankari ls, ramakrishnan k (2010) oral myiasis caused by chrysomya bezziana. j oral maxillofac pathol. 14: 16–18. http://jad.tums.ac.ir published online: december 18, 2013 a feasibility study on using the facilities of health centers for developing a laboratory network on vectors and reservoir hosts of cutaneous leishmaniasis in iran http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... review article a feasibility study on using the facilities of health centers for developing a laboratory network on vectors and reservoir hosts of cutaneous leishmaniasis in iran *mohammad reza yaghoobi-ershadi1; amir ahmad akhavan1; mohammad reza shirzadi2; seyedeh zohreh hosseini1; oscar daniel salomon3; ahmad ali hanafi-bojd1; yavar rassi1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2communicable diseases management center, iranian ministry of health and medical education, tehran, iran 3national institute of tropical medicine, puerto iguazu, misiones, argentina *corresponding author: dr mohammad reza yaghoobi-ershadi, e-mail: yaghoobi.reza@gmail.com (received 23 jun 2020; accepted 30 mar 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: cutaneous leishmaniasis is an ancient endemic disease in iran and continues to be a growing health threat to community development and the environment. this paper explains how to use the facilities of health centers for developing a laboratory network on vectors and reservoir hosts of cutaneous leishmaniasis in iran. methods: a literature search was performed of the relevant multiple databases to include studies on vectors and reservoirs of cutaneous leishmaniasis in iran. a team of experienced experts was performed. after holding several meetings, field visits and organizing workshops, the activities of laboratories were determined at three levels. results: entomological studies showed that 5 species of the genus phlebotomus and 10 species of the genus sergentomyia are active in the south, 4 species of the genus phlebotomus and one species of the genus sergentomyia in the central part and 5 species of the genus phlebotomus and 2 species of the genus sergentomyia in the north east. reservoir hosts were identified in the study areas. the tasks of laboratories were regulated at different levels. conclusion: the iranian ministry of health and medical education should prioritize the employment of capable persons in the field of medical entomology and vector control. the survival of this laboratory network depends on hiring and employing interested and persistent people. the universities of medical sciences that have the facilities to set up this network will be a very effective partner in the control of the disease in high risk areas. the results can be used in neighboring countries. keywords: laboratory network; leishmaniasis; vectors; reservoirs; iran introduction parasitic diseases of the genus leishmania are a huge burden on human health and society. their incidence is found to be prevalent in some of the poorest countries in the world, but they summon less attention than other infectious diseases like malaria, tuberculosis and aids (1). they are categorized as a neglected tropical diseases because, although they cause significant mortality, there is little effort on the part of the global community 256 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... and pharmaceutical industry to invest in research and development of better and innovative therapeutics because lack of sufficient incentives. also, the disease is found primari ly among the poorest of the poor, who have no influence over policymakers and little access to healthcare and the health-related market (1, 2). the disease is caused by more than 20 species/subspecies of leishmania protozoa. they are transmitted to humans through the bites of infected phlebotominae sand flies (3), principally belonging to two genera: phlebotomus (in the old world) and lutzomy ia (in the new world). while leishmania parasites were found in several species of a third genus sergentomyia, which feed on hu mans, none were up to now incriminated as vectors. an estimated 31 species of phlebotomus and 47 species of lutzomyia are proven vectors of human leishmaniases (4). sand flies are also known to be the vectors of other human pathogens, such as bartonella spp. (carrion’s disease), and a number of viral agents causing sand fly fever, summer menin gitis, vesicular stomatitis and chandipura vi rus encephalitis(4). both cutaneous and visceral forms of leishmaniasis are present in the eastern mediterranean region, making a significant neglected tropical disease there. re garding cutaneous leishmaniasis, both the anthroponotic form, caused by leishmania tropica, and the zoonotic form, caused by l. major with animal reservoir hosts, are endem ic in the region. cutaneous leishmaniasis is one of the priorities in the who eastern mediterranean region as it carries a large portion of the global burden. according to data reported in the global health observatory, the eastern mediterranean region reported 69.6% of the total number of cutaneous leishmaniasis cases detected worldwide in 2016 (followed by the region of the americas with 28.5% and the european region with 1.6%). of the total cases in the region, over 90% were reported from three countries: the syrian arab repub lic, afghanistan and pakistan, each of which reported more than 10 000 cases. most of the cases are due to the anthroponotic form of the disease. regarding visceral leishmaniasis, the eastern mediterranean region carries about 19% of the global burden, with the highest number of cases reported from sudan and somalia (5). cutaneous and visceral leishmaniasis are ancient endemic diseases in iran and continue to be a growing health threat to community development and the environment in the country. cutaneous leishmaniasis is endemic in two forms, anthroponotic cuta neous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl). about 20000 cases of leishmaniasis (including acl, zcl and zoonotic visceral leishmaniasis) are reported annually but the real figures could be 4–5 folds higher due to underreport ing, because of misdiagnosis, lack of active case detection and overlooking the im portance of the disease due to the lack of mor tality (6) anthroponotic cutaneous leishmani asis is still a neglected tropical disease in many parts of the country, while it was great ly reduced in many foci by malaria control measures, many foci remained active in some large and medium sized cities such as tehran, mashhad, neishabur and sabzevar in the north-east, shiraz in the south, kerman and bam in the southeast, yazd, kashan and parts of the city of esfahan in the central region (7, 8, 9). the parasite is leishmania tropica and the suspected vector is phlebotomus (paraph­ lebotomus) sergenti parrot 1917. the main reservoir host is the human, but dogs have a role as animal reservoir host and active lesions in dogs have been reported in tehran, mashhad, shiraz and neishabur (8, 9). zoonotic cutaneous leishmaniasisis is endemic in many rural areas of 17 out of 31 provinces and is a great health problem in iran. more than 85% of cases reported in the coun try are of zcl form (9, 10). rhombomys opi mus, the great gerbil, is the main animal res ervoir in foci in the northeast and central part of the country, meriones libycus, the libyan jird, is considered the principal reservoir host in some parts of the central and south of the coun try. tatera indica, the indian gerbil, is 257 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... the main reservoir host in the southeast and meri ones hurrianae, the desert gerbil, is the reser voir in the southeastern part of iranian balu chistan, neighboring pakistan (11, 12, 13) phlebotomus papatasi scopoli 1786, the most prevalent among phlebotomus genus, is the only known vector (14, 15, 16, 17). in 2019, totally 13055 cases of cutaneous leishmaniasis were reported in the country, 12567 (96.5 %) of which from 18 endemic provinces and 488 (3.5 %) from non-endemic provinces. most of the cases were from isfahan province in the central part of the country with 3127 cases and 59.1 incidence per 100000. but the highest incidence rate was in semnan province with 95.9 per 100000 populations (cdc, iranian ministry of health and medi cal education, unpublished data). fig. 1 shows the trend of reported cases of cutane ous leishmaniasis in iran in the last 36 years. the incidence of the disease varies between 48/100000 and 15.8/100000 population dur ing 1983–2019 respectively. whether the in cidence has been reduced between these years but, the burden of the disease is still high in some regions. it should be mentioned that the reduction has been due to the implementation of active rodent control operations in zcl foci and improvement of surveillance and timely treatment of the patients in acl foci. iran is facing many challengesemergencies, crises situations, outbreaks, population dis placement, limited funding and weak surveil lance, lack of proper management in some provinces, lack of community awareness and some managersall which may have led to the rising trend in cutaneous leishmaniasis cases in some regions. even if leishmaniasis was the main public health priority of iranian min istry of health, unfortunately there is not a standard method for the different aspects to study the leishmaniasis, insect vectors in the country, so in this research project the infor mation required on the matter for surveillance and control of sand flies, in order to provide to health authorities in iran the basis for a program, that could be replicated in the region. this program requires immediate instrumentation because, during the last two decades, the reported cases of leishmaniasis especially that of cutaneous leishmaniasis due to leishmania major have increased in iran, and it has spread into sites where it did not previously exist, so properly phlebotomine sand flies have received considerable attention by health authorities in recent years (9). however, the disease needs special entomological surveillance, and to achieve this goal developing a strong laboratory network is required. at the moment, laboratories in many health centers do not have facilities for entomological activities on sand flies and res ervoir hosts of cutaneous leishmaniasis, the staffs of health centers are unaware of the matter in different endemic foci of the coun try, and their duties do not involve clearly the surveillance and control of the disease. physi cians and senior technicians are needed to be trained on leishmaniasis entomology and their awareness on the matter should be increased. therefore, by conducting this research, the main aspects for developing a laboratory net work are stated to support the structuration of this initiative. to obtain a feasible program based on evidences, a search of worldwide information about the topic was performed, followed by consensual workshops with diff erent levels of technical and academic health agents, and visits to the field to assess the actual conditions and capabilities. materials and methods this study was carried out in five high risk provinces during 2019–2020 in iran. 1) databases including pubmed, web of science, literature retrieval system of the armed forces, pest management board and also at least more than 40 eligible journal arti cles on vectors and reservoirs of cutaneous leishmaniasis and its control which are in dexed in electronic databases (isi and med line) were selected for this review. 1a) goal: to summarize the experience on surveillance and control program organization on leishmaniasis worldwide; 258 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... 2) a team of experienced experts consist of key people from the tehran university of med ical sciences and ministry of health and medical education (moh) was formed. 2a) goal: to develop the first blueprint of a program of surveillance and control of leish maniasis in iran. the members of the expert team were as follows: four academic members (including profes sors and associate professors of medical en tomology and vector control from tehran university of medical sciences with personal knowledge of participants after more than 40 years of field mission on leishmaniases in iran. manager of the zoonotic department at moh two senior technicians on leishmaniasis measures were carried out by the special ized team as follows: holding a special meeting for analysis of the current situation of leishmaniasis cases. holding more than 10 meetings with the presence of team members on determining the duties of experts at different levels and having a de tailed discussion and exchange of views. 2b) to collect relevant information on the current capabilities in the high risk are as field visits to the high risk areas (provinces of esfahan, ilam, khuzestan, sistan and ba luchestan and northern khorasan. each one visited once during the months of june-november, 2019– 2020 holding meetings with the assistant direc tors of health, disease control managers and zoonotic experts in high risk provinces field visits to the facilities of laboratories at health centers in the provinces under study. 2c) to adapt the blue print to a feasible consensual program to be implemented preparation a list of equipment required for laboratories of cutaneous leishmaniasis vec tors and reservoir hosts. determination of duties for entomology senior technicians of health centers in order to detect and reveal the abundance of sand flies and rodent reservoir hosts. to identify the collection methods of sand flies and reservoir hosts, mounting and moni toring the platform of recording and flowchart of information the needs and ways of capacitation “in practice” at different levels of responsibility from professionals to field technicians in the methods of monitoring of surveillance the capacitation of decision takers, health, zoonosis and vector agents in outbreak control and integrated vectorborne diseases man agement. results and program proposal the results of literature review of the articles on the vectors and reservoir hosts of cutaneous leishmaniasis showed that there is not any network on this issue in the world except for south america. many studies have been carried out on the control of vectors in differ ent countries with various results (use of re sidual spraying, spraying rodent burrows, the use of impregnated bed nets and cur tains, etc). although many molecular stud ies have been conducted on leishmania para site, unfortunately, studies on reservoir control are very limited and very few. due to the fact that there is no effective vaccine against cuta neous leishmaniasis, so the control of vectors and reservoirs along with health education by the relevant specialists and allocating suffi cient funds, raising awareness of authorities and the community can prevent the spread of the disease. results of entomological and reservoir studies in different provinces are as follows: khuzestan province a total of 4335 sand flies, 23.63% in doors, 76.37% outdoors, collected from shush county in september 2019. eight species including p. papatasi, p. alexandri, s. sintoni, s. squamipleuris, s. iranica, s. tiberiadis, s. 259 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... dentata and s. baghdadis were collected from indoors. eleven species collected from outdoors comprising p. papatasi, p. alexandri, s. sintoni, s. tiberiadis, s. dentata, s. iranica, s. baghdadis, s. clydei, s. squamipleuris, s. the odori and s. christophersi. out of 22 rodents which were collected in the mentioned county, 8(36.4%), 12(54.5%), and 2(9.1%) were identified as nesokia indica, tatera indica and rattus rattus, respectively. isfahan province a total of 3200 sand flies (35.81%) indoors, (64.19%) outdoors were collected from 6 villages in july 2019 around the city of isfahan. phlebotomus papatasi, p. sergenti and s. sin toni were found indoors and 5 species includ ing p. papatasi, p. segenti, p. ansarii, p. mongolensis, and s. sintoni were found outdoors. phlebotomus papatasi was the dom inant species indoors and outdoors. totally, 19 rodents were captured using 25 sherman traps baited with cucumber and dates near gerbil colonies. the great majority were 16(84.2%) rhombomys opimus and 3(15.8%) meriones libycus. ilam province in total, 1071 sand flies were collected from 4 villages around the city of mehran in june 2019. phlebotomus papatasi, p. alexan­ dri, s. sintoni and s. dentata, were found in doors and species including p. papatasi, p. caucasicus, s. squamipleuris, s. clydei, s. the odori, s. dentata and s. tiberiadis found outdoors. phlebotomus papatasi was a predom inant species in both outdoors and indoors. out of 24 rodents that were caught in three villag es, 8(72/7%) and 3(27.3%) were identified as t. indica and n. indica, respectively. sistan and baluchestan province in october of 2019, 644 sand flies collected outdoors and indoors. three species: p. papatasi, p. sergenti and s. sintoni were collected indoors and 6 species comprising p. papatasi, p. alexandri, p. salehi, s. clydei, s. christophersi and s. sintoni collected outdoors. the most predominant species was p. papatasi in both places. eight reservoir hosts were captured by sherman traps around chabahar and konarak, identified as m. hurrianae (75%) and t. indi ca (25%). northern khorasan province an overall 1346 sand flies were collected from 4 villages in esferayen and bojnord in august 2019. four species including p. papatasi, p. alexandri, p. sergenti, and s. sintoni collected indoors and 6 species comprising p. papatasi, p. sergenti, p. neglectus, p. adlerius group, s. sintoni and s. sumbarica collected outdoors. the predominant species in the indoors and outdoors were p. papatasi and s. sintoni, respectively. out of 14 rodents, 6(42.9%), 5(35.7%) and 3 (21.4%) identified as r. opimus, n. in­ dica and m. libycus respectively. the tasks and activities of laboratories at each level were determined as follows: a. periphery levels (county health center laboratory) b. province and university levels (provincial health center laboratory) c. country level (national reference laboratory on vectors and reservoirs of cutaneous leishmaniasis) peripheral level the number of peripheral level labs depends on the number of counties in each prov ince. there are 452 counties in iran (statisti cal center of iran) and each of them needs one peripheral level lab. the laboratory of the county health center requires an entomology expert and two field technicians who can play an important role in collection of sand flies and rodents. peripheral level tasks are as follows: in order to have the necessary information, at least 3–4 suitable villages will be selected under the supervision of the provin cial health center laboratory for entomology (where they could have the capability to keep sand flies colonies) in infected foci of the plain region and the 260 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... same number in the mountain ous region of the county. so that the villages are considered as the real representative of the county. then, from the beginning to the end of each active season, once every two weeks, at sunset, 30 sticky traps (castor oil coated white papers, 20× 35cm) will be installed in fixed indoor places, including bedrooms, liv ing rooms, stables, barns and toilets, and 30 sticky paper traps will be installed in fixed outdoor areas, including on the base of walls, under boulders, caves and rodent burrows. they are collected the next morning, before sunrise and then preserved in 70% ethanol, in which form they are transferred to the laboratory of the county. indoor locations should be in cluded in three houses, one in the middle of the village, another in the outskirts of the village and the third between the previous two houses to show the actual activity of sand flies. mounting of sand flies in puri’s medium according to the relevant instructions. preparing samples from sand flies blood content in the gut to determine the host blood preference of vectors according to the relevant instructions and sending them to the laborato ry of the province. collection of rodents with at least 50 sherman traps up to a radius of 500 meters from the center of the village, once a season in infected foci, preparing two slides from each rodent earlobe, staining the slides and diagnosing leishmania parasites according to the relevant instructions. separation of skin and skull of collected rodents according to the relevant instructions and sending to the middle level (provincial centers). record the results on the relevant forms and send it to the coordinator of cutaneous leishmaniasis of the county. sending monthly performance reports regularly to the province level. middle level (provincial level) the provincial health center laboratory is the university reference laboratory. there are 31 provinces in iran, and each must have one lab at this level. the provincial health center must have a leishmaniasis entomology lab. with the nec essary facilities and equipment. the responsi bilities of this level are as follows: identification of mounted sand flies at the species level. calculating the species composition of sandflies and their abundance by species. drawing diagrams related to the monthly activity of sand flies by species in the indoors and outdoors. preparing the distribution map of sand flies by species in the province as reported by rural district and county. dissection of sand flies in order to determine the natural promastigote infection rate during the months of august and september as stated in instructions in county level. computer registration of the results on sandflies and reservoir hosts information based on the relevant instructions and sending it to the national level. technical supervision and quality control of all entomological laboratories of the affili ated counties by examining the mounted sam ples of sand flies and slides prepared from rodent earlobes and sending feedback. training of the laboratory staff working at leishmaniasis entomology laboratories at the county level. evaluation of vector control operations us ing standard scientific methods in the event of epidemics and outbreaks. with the base-line of vector and reservoir relative and absolute abundances, their annual dynamics, and infection rates, any anomalies in the usual patterns provide an early warning system to intensify monitoring and active search of patients, and to make focus studies in order to define precisely the where and when to perform the more effective intervention for prevention or mitigation. 261 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... country level (national level) the national reference laboratory of vectors and reservoir hosts of cutaneous leishmaniasis is responsible for management, diagnosis, supervising, laboratory, educational and re search quality review, and its tasks are as fol lows: 1close cooperation with the infectious diseases management center in order to better implement the national programme for the control of cutaneous leishmaniasis and the goals of the ministry of health. at any level but also at this level, the programme should also include a network with the medical coun terpart. 2technical supervision and quality control of all laboratories on vectors and reservoir hosts of cutaneous leishmaniasis in the coun try, by examining a given percentage of sand flies and rodents collected and identified in the province in order to confirm and send the results as feedback. 3climate correlation with abundances and infection rates, and land use change at differ ent space and time scales, including the na tional level monitoring of changes in the pat terns. 4isolation of leishmania parasites from vectors and reservoir hosts and identification by using molecular methods if necessary. an experienced senior technician will perform this goal. 5molecular identification of vectors and reservoirs if needed. 6determination the susceptibility level of the main vectors of the disease to the used insecticides by the standard method of the world health organization, if necessary. 7determination the host-feeding preference of vectors by advanced diagnostic meth ods. 8necessary recommendations regarding the time of spraying and the type of the insecticide during outbreaks and epidemics of cu taneous leishmaniasis. 9carrying out advanced research projects on the ecology of vectors and reservoir hosts and evaluation different methods of vector control and reservoirs in cooperation with the infectious diseases management center. eco-epidemiology studies to identify exposure risk and accessibility to health care according to region, gender and age. 10preparation of necessary instructions for collection methods and sampling of sand flies and reservoirs and their survey techniques. 11launching advanced diagnostic meth ods for vectors and reservoirs of the disease in provincial laboratories. 12participating in analyzing the results of fig. 1. trend of cutaneous leishmaniasis iran 1983–2019 fig. 1. trend of cutaneous leishmaniasis iran 1983–2019 262 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... activities in preparing annual reports. 13establishing close relations with prestigious international scientific institutions abroad and inside the country. 14organizing of practical and applied training workshops on the control of cutane ous leishmaniasis at different levels for man agers, general practitioners, experts and tech nicians in the country. 15periodic visits to provinces and counties in order to monitor executive operations. 16organizing of practical and applied entomology workshops of leishmaniasis and its reservoirs at different levels. discussion the results of the search of information indicate the fact, that there are no laboratory networks on vectors and reservoirs of cutaneous leishmaniasis in any country. there is only one spanish language publication from south america (18) on this issue, which cannot be generalized to our country because of the differences in the geographical and epi demiological situation of the disease between these two regions. health education is a spe cialized issue that unfortunately is not consid ered by the authorities in iranian health cen ters, especially since the methods of educating the community have changed in the last two decades and only experts can implement them. in view of the results of this study, it seems that the iranian ministry of health and medical education should prioritize the em ployment of specialized and capable person nel in the field of medical entomology and vector control so that they can better serve in the field for the country’s health system. be cause the lack of attention to the existence of a cohesive organization and experienced per sonnel can pose a serious threat in the future in endemic foci of cutaneous leishmaniasis and other neighboring provinces. it is necessary to set up and use medical entomology laboratories in the provinces as it was proposed of the mentioned network as soon as possible while having sufficient facilities and equipment. the chancellors of medical sci ences universities and health deputies of the provinces could have an important role in this matter and should pay special attention to setting up the discussed network. if any of the universities of medical sciences in the country have the facilities to set up this network in terms of laboratory equipment and tools and experienced specialists, it will table 1. cutaneous leishmaniasis cases and incidence rate/ 100000 in endemic provinces of iran, 2019 no endemic provinces geographical zones cl number cl incidence rate/100000 1 semnan near north 719 95.9 2 ilam west 470 78.7 3 golestan north-east 1188 60.9 4 isfahan center 3127 59.1 5 fars south-west 2002 40.0 6 north khorasan north-east 214 24.0 7 khorasan razavi north-east 1581 23.4 8 yazd center 260 21.4 9 qom near central plateau 293 21.3 10 khuzestan south-west 1013 20.7 11 kerman south-east 565 17.1 12 sistan and baluchestan south-east 357 12.0 13 hormozgan south 160 8.4 14 kermanshah west 117 5.9 15 south khorasan east 39 4.8 16 lorestan west 69 3.8 17 tehran north of central plateau 434 3.1 18 bushehr south 28 2.3 table 1. cutaneous leishmaniasis cases and incidence rate/ 100000 in endemic provinces of iran, 2019 263 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... undoubtedly be very effective in the control of the disease. creating a constructive and dynamic competi tive environment in this new movement and trying to create links and interaction at differ ent levels (environmenthealth-social devel opment) among provinces and between juris dictional levels (from county to national level) can be useful. it should be noted that the sus tainability of this laboratory network depends entirely on hiring and employing literate, in terested and persistent people. it should be mentioned that the results of this research can be used in afghanistan, pakistan, iraq, syria, tajikistan and turkmenistan which have a similar problem regarding the importance of leishmaniasis as a health issue and lacking a proper national network system to perform integrated management that includes vector, reservoirs and humans. in those senses, it should be mentioned that expert manpower is important to get things done, and the adequate use of already existing or created capacities is essential. it is hoped that in the near future we will see the serious efforts of our colleagues to consolidate a network, and that all respect ed managers and experts will base their work on activities of the network according to the tasks that have been defined. acknowledgements the authors are grateful to responsible persons and staffs of provincial health centers under study. the research reported in this publi cation was supported by elite researcher grant committee under award number 982714 from the national institutes for medi cal research development (nimad, tehran, iran.). thank you very much for the role of this institute in the general support of research projects. the authors declare that there is no conflict of interests. references 1. wagstaff a (2002) poverty and health sector inequalities. bull world health organ. 80(2):97 105. 2. yamey g, torreele e (2002) the world’s most neglected diseases, bmj. 325(7357):176-7. 3. magill aj (1995) epidemiology of the leishmaniasis. dermatol clin. 13(3):505-23. 4. maroli m, feliciangeli md, bichaud l, charrel rn, gradoni l (2013) phlebotomine sand flies and the spreading of leishmaniasis and other diseases of public health concern. med vet entomol. 27(2):123-47. 5. world health organization (2018) report on the international meeting on leishmaniasis among neighbouring endemic countries in the eastern mediterranean, african and european regions. amman, jordan, 23–25, september 2018. p. 47. 6. yaghoobi-ershadi mr , akhavan aa, shirzadi mr, rassi y, khamesipour a, hanafi-bojd aa, vatandoost h (2019) conducting international diploma course on leishmaniasis and its control in the islamic republic of iran. j arthropod borne dis. 13(3):234-242. 7. yaghoobi-ershadi mr ,hanafi-bojd aa, javadian e, jafari r, zahraei-ramazani a , mohebali m (2002) a new focus of cutaneous leishmaniasis caused by leishmania tropica. saudi med j. 23(3):291-4. 8. nadim a, tahvildari bidruni g (1977) epidemiology of cutaneous leishmaniasis in. iran: b. khorassan, part vi: cutaneous leishmaniasis in neishabur, iran. bull soc pathol exot filiales. 70(2):171-7. 9. yaghoobi-ershadi mr (2016) control of phlebotomine sand flies in iran: a review article. j arthropod borne dis. 10(4):429-444. 10. yaghoobi-ershadi mr, javadian e (1995) zoonotic cutaneous leishmaniasis in the north of isfahan (iran).the human infection. bull soc pathol exot. 88(1):42-5. 11. yaghoobi-ershadi mr, javadian e (1996) epidemiological study of reservoir hosts in an endemic area of zoonotic cutaneous leishmaniasis in iran. bull world health organ. 74(6):587-90. 12. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996) meriones iibycus and rhombomys opimus (rodentia: gerbillidae) are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans r soc trop med hyg. 90(5):503-4. 13. yaghoobi-ershadi mr, hanafi-bojd aa, akhavan aa, zahrai-ramazani ar, mohebali m (2001) epidemiological study in a new focus of cutaneous leishmaniosis due to leishmania major in ardestan town, central iran. acta trop. 79(2):115-21. 14. nadim a, mesghali a, amini h (1968) epidemiology of cutaneous leishmaniasis in the isfahan province of iran: iii. the vector. trans r 264 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 255–264 mr yaghoobi-ershadi et al.: a feasibility... soc trop med hyg. 62(4):543-9. 15. yaghoobi-ershadi mr, javadian e (1997) studies on sandflies in a hyperendemic area of zoonotic cutaneous leishmaniasis in iran. indian j med res. 105:61-6. 16. akhavan aa, yaghoobi-ershadi mr, hasibi f, jafari r, abdoli h, arandian mh, soleimani h, zahraei-ramazani ar, mohebali m, hajjaran h (2007) emergence of cutaneous leishmaniasis due to leishmania major in a new focus of southern iran. iran j arthropod borne dis. 1: 1–8. 17. jahanifard e, hanafi-bojd aa, nasiri h, matinfar hr, charrahy z, abai mr, yaghoobi-ershadi mr, akhavan aa (2019) prone regions of zoonotic cutaneous leishmaniasis in southwest of iran: combination of hierarchial decision model (ahp) and gis. j arthropod borne dis. 13(3):310-323. 18. surveillance of leishmaniasis transmitting insects (2015) operational manual for the community. pictographic key for identification of phlebotomine sand fly species. clarification and mounting, red argentina. argentine network for the study of leishmaniasis in argentina. 2015 eds. inme t-msal.isbn.978-987-29115-3-9, p. 81 (spanish). a feasibility study on using the facilities of health centers for developing a laboratory network on abstract keywords introduction materials and methods results and program proposal khuzestan province isfahan province ilam province sistan and baluchestan province northern khorasan province peripheral level middle level (provincial level) country level (national level) discussion acknowledgements references j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 152 http://jad.tums.ac.ir published online: june 30, 2021 original article level of pyrethroid-resistance associated with cytochrome p450 expression in german cockroach blattella germanica (blattodea: ectobiidae) in the field collected strains azimeh ghaderi; mozghan baniardalani; *hamid reza basseri department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran *corresponding author: dr hamid reza basseri, e-mail: basserih@tums.ac.ir (received 3 jun 2020; accepted 22 jun 2021) abstract background: cytochrome p450-dependent monooxygenases are a very important metabolic system involve in insecticide resistance. this study was conducted to find the association between the expression level of cytochrome p450 (cyp450) and permethrin-resistance level among four strains of the german cockroach blattella germanica (l) (blattodea: ectobiidae). methods: three field strains of german cockroach with different frequency of exposure to pesticides, and a laboratory susceptible strain were used in the present study. insecticide susceptibility bioassays were carried out to detect resistance to permethrin. the concentration of cyp450 in each strain was determined using ion-exchange hplc chromatography. biochemical assays was performed to analyse cyp450 activities. results: the resistance ratios (rr) to permethrin among three field strains were 3.29, 4.10 and 6.17-fold comping with the susceptible stain. the cyp450 activity of three field strains was 1.6, 2.4 and 2.7 times higher than in the susceptible stain. the amount of cyp450 per mg of protein was significantly different between the susceptible and t h e three resistant strains. the resistant cockroaches showed a relatively high expression of cyp450 enzymes. a strong correlation was found between permethrin resistance level and total concentration of cyp450 enzymes. conclusion: the results of current study show that more frequent usage of a pyrethroid insecticide cause the metabolic insecticide resistance to rise in german cockroach. therefore, there is a ratio correlation between resistance level and monooxygenases activities in insect. thus, the control program must be managed according to history of pesticide usage. keywords: cytochrome p450; german cockroach; insecticide resistance; permethrin introduction the german cockroach, blattella germanica (l.) (blattodea: ectobiidae), is one of the major pests of public health importance. it is a vector of various bacterial and viral diseases (1, 2) and can induce allergic responses such as asthma in humans (3-5). over the past years, the german cockroach has developed resistance to a wide range of classes of insecticides through multiple resistance mechanisms (6-9) and this has been a major barrier in the control of this pest (7, 10). cytochrome p450 monooxygenases (cyp450) have been shown to be one of the most essential enzymes in the machinery of insecticide resistance, and play a crucial role in the metabolism and structural changes of endogenous and exogenous compounds (11) such as pesticide detoxification (12). it has been shown that, monooxygenase enzymes play a significant role in the german cockroach’s resistance to pyrethroids (13-15). the cyp450 complex has a hemoprotein structure and ubiquitous enzymes. it is found in living organisms such as bacteria, plants, animals, and fungi. the cyp450 enzymes are involved in both endogenous and xenobiotic metabolic processes that occur within living organisms. in insects, these copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:basserih@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 153 http://jad.tums.ac.ir published online: june 30, 2021 enzymes are necessary for the synthesis and degradation of steroid molting hormones, juvenile hormones and pheromones and also act as terminal oxidase in the monooxygenase system (16). the level of expression of cytochrome p450 can vary at different stages of insect development. also, it has been found that there is a considerable diversity in the number of cytochrome p450 genes among insect species (17). the variation in gene expression may be due to the differences in response to inducers and the tissues in which they are expressed (18, 19). a study on the levels of resistance to permethrin and deltamethrin among three strains of the german cockroach collected from alabama revealed that p450 monooxygenases are highly involved in pyrethroid resistance. although these strains were collected from the same geographic location, their resistance mechanisms to pyrethroid were different(20). in a previous study, the levels of german cockroach resistance to pyrethroid and cross-resistance to ddt in seven field-collected strains were investigated. the synergists pbo (piperonyl butoxide) and def (s, s, s-tributylphosphorotrithioate) were found to play a role in permethrin resistance whereas chlorphenetol decreased ddt resistance in the seven collected insect populations (9, 21). another bioassay study was conducted in 2007 to investigate the resistance level of field strains of the german cockroach to pyrethroids in the north of iran. in the study, the activity of glutathione s-transferases (gsts), esterase and the monooxygenases of cytochrome p450 was assayed using eliza test and the resistance ratios of the field-collected strains ranged between 1.5 to 2.5-fold, indicating a higher tolerance in the german cockroach to pyrethroids. the authors suggested that the high resistance of the field-collected german cockroach in the area might have been caused by the extensive use of pyrethroids for pest control (22). in-depth knowledge on the role of cytochrome p450 in pesticide resistance in insects may provide imperative information for the development of novel pesticides for effective pest control. however, several studies have been conducted on pesticide resistance involving p450 in german cockroaches. the aim of the present study was to measure the content of cyp450 enzymes in the german cockroach population and to determine its correlation with the level of pesticide resistance among the different strains of this pest. the results of current study are important for the further understanding of the role of cyp450 in pesticide resistance mechanism. materials and methods cockroach strains three field-collected and one susceptible strains of the german cockroach were used in the present study. the field strains were collected from the following sites: a restaurant with insecticide application once or twice a year (strain a), sina hospital which has insecticide spraying activity more than twice a year (strain b), and shariati hospital (strain c), where pesticides are frequently and heavily used. pyrethroids (permethrin and cypermethrin) were the most common insecticides used in the insect collection sites. the field collected populations were colonized in insectary and f1 generations were used for all experiments. the susceptible strain has been reared in the laboratory without exposure to insecticides since 1975. all cockroach strains were maintained in an insectary condition at 27±2 ºc and 60±10% rh, with a photoperiod of 12:12h (l: d). all strains were provided with unlimited bread, sugar and water. only one to two-week old males with relatively homogenous physiology and body mass were used in this study. cockroaches were anesthetized briefly with co2 to facilitate handling in each test. insecticide resistance bioassays bioassay were carried out with permethrin (92%, technical grade) cis:trans 60:40, (cyanhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 154 http://jad.tums.ac.ir published online: june 30, 2021 amid agro, india). glass jar method was used according to the previous study (23). a range of serial concentrations of permethrin (technical grade) were prepared in acetone and were exposed to the insects to find a discriminating dose for the male cockroaches. in a series of knockdown experiments, a concentration of 15mg/m2 permethrin at 35min was found to be a discriminating dose for distinguishing the susceptible strain (ss) from the resistant strain (rr). subsequently, the discriminating dose (15mg/m2) was used to coat the glass jars. jars were rotated gently in a hood until the acetone completely evaporated. exposure times for each strain were computed at the discriminating dose to find the minimum (t1: to kill 5% of cockroaches) and maximum (tn: to kill 95% of cockroaches) time of exposure. the tests were performed in 4 replicates each with 10 male adult cockroaches. the cockroaches were kept for 24h and were monitored for mortality. the knockdown time (kt50) was measured and subjected to probit analysis. chromatography assays total cyp450 was purified as explained by scharf et al. (1997) (13). thirty adult male cockroaches from each strain were dissected, and their body contents (except gut contents) were transferred into eppendorf tubes and homogenized in 0.5ml homogenization buffer (sodium phosphate 0.1m ph 7.5). the homogenates were centrifuged at 11000g for ten minutes. subsequently, the supernatants were centrifuged at 106000g for 60min (l5-50 bechman ultracentrifuge). the microsomal pellets were resuspended in 20µl resuspension buffer (homogenization buffer containing glycerol 30% [v/v]). then, 40µl of solubilizing buffer (10mm sodium phosphate, 0.6% (w/v) sodium cholate, 1mm edta, 20% (v/v) glycerol, ph 7/4) was added to the yield and mixed gently by vortexing or petting on ice for 30 minutes. the mixture was then centrifuged at 106000g for 60 minutes at a degree of 4 °c to remove particulate matter. the supernatants were analyzed using high performance liquid chromatography (knauer, germany). anion-exchange analytical hplc column (shodex asahipack) was equilibrated with buffer a (20mm tris/ acetate containing 1mm edta, 20% (v/v) glycerol). in order to obtain a standard chromatogram, 20µl of recombinant expiration cytochrome p450 reductase human (purchased from sigma co.) was loaded onto the column. subsequently, 20µl of the solubilized extracted microsomes from each sample was loaded onto the column with a 20µl super loop. flow rate was measured as 1.0ml/min and optical density column effluent was detected at 350nm. a linear gradient of buffer b (buffer a + sodium acetate 0.8m) relative to buffer a (090%) was specifically used to elute cytochrome p450 proteins. the amount of cyp450 of each samples was measured with chrome gate software. biochemical assays the activity of total cyp450 was measured based on the methods described by penilla et al. (2007)(24). briefly, twenty microliters of the insect homogenate was aliquoted prior to the addition of 80μl of 0.625m potassium phosphate buffer (ph 7.2), 200μl of 3,3′,5,5′-tetramethylbenzidine (tmbz) solution (0.01g tmbz dissolved in 5ml methanol plus 15ml of 0.25m sodium acetate buffer, ph 5.0) and 25μl of 3% hydrogen peroxide to each of a 96-well microtiter plate. the plates were incubated at room temperature for a period of 2 hours and their absorbances were measured at 450nm as an endpoint in the plate reader, and the values were compared with a standard curve of recombinant expiration cytochrome p450 reductase human (purchased from sigma co.) statistical analysis cockroach knockdown data were assessed by probit analysis (throne et al. 1995) using spss. log-probit analysis was used to determine the lethal time required to knockdown http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 155 http://jad.tums.ac.ir published online: june 30, 2021 50% of a population (kt50) and the 95% confidence interval (cl). resistance ratios were calculated as the kt50 of resistant strains divided by the kt50 response value of the ss strain. linear regression of probit knockdowns was analyzed using ssps. the strains were compared using the anova; games-howel test (p< 0.01, and p< 0.001). results determination discrimination concentration in a series of knockdown experiments, a concentration of 15mg/m2 permethrin at 35min was found to be a discriminating dose for distinguishing the susceptible strain (ss) from the resistant strain (rr). insecticide resistance bioassays the results of insecticide resistance bioassays are represented in table 1. the resistance ratio at kt50 was highest i n strain c ( 6.17fold) which had the highest exposure to pesticides followed by strain b and strain a with resistance ratios of 4.10 and 3.29-fold, respectively. the probit regression analysis of knockdown times of permethrin for the susceptible strain (ss) and field-collected strains (a, b and c) are shown in fig. 1. the probit regression shows time-to-knockdown for the four strains. a high variation in time-to-knockdown was observed among the resistant strains (a, b and c), correlating with the degree of exposure to the pesticide. strain c had the highest resistance to permethrin, with kt50 and kt95 values of 52.21 and 79.76 minutes, respectively. the kt50 and kt95 values of strain b were 34.72 and 55.88 minutes, respectively, whereas, the kt50 and kt95 values for stain a were 27.79 and 44.00 minutes respectively. the resistance ratios (rr) of the three field-collected cockroaches, strains a, b and c were 3.29, 4.10 and 6.17-folds, respectively, when compared with the susceptible strain (table 1). biochemical assays the result of biochemical assay has been presented in table 2. the data presented herein show a higher cyp450 activity in the strains c and b compared with the susceptible strain. the cyp450 activity in the resistant strains was 1.6 (strain a), 2.4 (strain b) and 2.7-fold (strain c) higher than in the susceptible stain (ss). these results indicate that the monooxidase activities between strains are statistically difference (p< 0.001). chromatography assays the analysis of high-performance liquid chromatography indicated that the amount of cyp450 significantly varied between four strains. the average of cyp450 concentration obtained from susceptible, a, b, and c strains were 0.417, 0.738, 1.110 and 1.302ng/mg respectively (fig. 2). in comparative perspective of the cy450 expression and kt50 ratio, the enzyme concentration enhanced with increasing resistance levels to insecticides among strains (fig. 4). table 1. results of probit analysis of time of knockdown response of four german cockroach strains to permethrin strain y-intercept b±s.e. x2 (df) kt50 kt90 rra ss -1.297 0.15±0.21 4.72(5) 8.46 16.82 1.00 a -2.157 0.79±0.10 7.66(5) 27.79 44.00 3.29 b -2.103 0.61±0.10 6.01(5) 34.72 55.88 4.10 c -3.251 0.57±0.21 1.63(5) 52.21 79.76 6.17 susceptible strain (ss) is the susceptible laboratory strain; strains a-c are the field-collected strains. df= degrees of freedom, kt= knockdown time, x2= chi-square. a rr (resistance ratio) = kt50 of field-collected strains / kt50 of susceptible strain. b =unstandardized coefficients, s.e= standard error. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 156 http://jad.tums.ac.ir published online: june 30, 2021 fig. 1. probit analysis of knockdown time of permethrin at the discriminating dose (15mg/m2) for the four strains of the german cockroach. strain ss is the susceptible laboratory strain; strains a, b and c are the field-collected strains fig. 2. relative concentration of cytochrome p450s in susceptible strain (ss) and permethrin-resistant strains (a, b and c) of german cockroach (blattella germanica). the error bars represent means ± standard deviation. the enzyme concentrations were determined by hplc. the strains were compared using the anova; games-howel test, *p< 0.01, **p< 0.001 indicate significant difference c o n c e n tr a ti o n o f c y to c h r o m e p 4 5 0 n g /m g p r o te in ss a b c 0.0 0.5 1.0 1.5 2.0 ** * strains 0 1 2 3 4 5 6 7 8 1 10 100 p ro b it time (min) ss a b c http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 157 http://jad.tums.ac.ir published online: june 30, 2021 table 2. descriptive analysis of the results of biochemical assays performed on german cockroach strains strain mean (units/mg protein) std. deviation std. error 95% confidence interval for mean min max lower bound upper bound ss 0.48 0.03 0.01 0.44 0.53 0.46 0.52 a 0.76 0.04 0.02 0.69 0.83 0.72 0.81 b 1.17 0.02 0.01 1.14 1.19 1.15 1.19 c 1.30 0.07 0.04 1.18 1.42 1.24 1.37 susceptible strain (ss) is the susceptible laboratory strain; strains a-c are the field-collected strains. no. of repetition for each strain = 4 fig. 3. comparison of cyp450 expression and kt50 between the susceptible strain (ss) and the three permethrinresistant strains (a, b and c) of the german cockroach (blattella germanica). kt50: knockdown time of different strains of the cockroach exposed to permethrin 15mg/m2. discussion resistance to insecticides among insects has been associated with enhanced enzymatic metabolism, particularly increased activity of cytochrome p450s (25). insect cyp450 complex play an important role in the detoxification of insecticides, which can lead to increase in insecticide resistance. there is little information on the association between the level of insecticide large peak in strains b and c. these results indicate that a higher modification occurred in the expression of cyp450 which correlated with increased level of permethrin resistance in the field-collected strains. the cyp 450 content also varied between the resistant and the susceptible cockroaches (fig. 3). the highest cyp450 content was found in strain c followed by strain b. in addition, the cyp450 content in strain a was significantly different http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 158 http://jad.tums.ac.ir published online: june 30, 2021 from that of strain s (fig. 2). fig. 3 shows the relationship between the level of permethrin resistance and cyp450 concentration in the german cockroach strains. increased cyp450 content correlated resistance and the amount of enzyme expression in insects. in the present study, the amount of total of cyp450 in four strains of the german cockroach with different levels of susceptibility to permethrin was investigated in order to determine the association between resistance to insecticide and monooxygenase enzymes expression in this pest. higher level of expression of cyp450 protein is associated with excessive expression of p450 genes in insecticide-resistant insects (26). similarly, it has recently been reported that there is alteration in the pattern of expression of some important proteins associated with detoxification in pyrethroid-resistant german cockroaches. these proteins include energy-metabolism proteins, detoxification-related proteins, signal molecule-regulated proteins, kinetic-related proteins, and gene expression-related proteins. the authors also stated that the in the pattern of gene expression is associated with stress induced by the pesticide (27). it has been shown that the regulation of multiple p450 genes through both constitutive overexpression and induction mechanisms are co-responsible for permethrin resistance in mosquitoes including culex quinquefasciatus. both mechanisms provide additional metabolic support for the detoxification of permethrin, which in turn, may lead to the development of resistance (28). moreover, a previous study indicated that 8 cyp genes are responsible for cyp450 expression in culex pipiens complex (29). in the present study, we also found different concentration of cyp450 protein based on hplc chromatograms analyzing (fig. 2). the data suggest that those cockroach strains that were more resistant to permethrin, the level of expression of total cyp450 elevated (fig. 3). these evidences suggest that several genes are involved in the expression of cyp450 enzymes. this also indicates that the german cockroach can develop a complicated mechanism of resistance to insecticides over time and place. in the present study, cyp450 content significantly increased with increasing level of insecticide resistance (fig. 3). the resistance ratio in all the field-collected resistant strains was more than three-fold when compared with the susceptible strain (table 1), which correlated with an increase in the concentration of enzymes and kt50 (fig. 3). however, the concentration of enzyme and kt increased with relatively similar slope (fig. 4). this results may indicate a positive correlation exist between cyp 450 consecration and resistance level. the german cockroach strains were collected from three different locations in tehran city, but the expression pattern of cyp450 and the level of resistance of the three strains to permethrin were directly associated with frequency of insecticide use in the collection site. german cockroaches have a high potential in developing resistance towards chemical insecticides but the pattern of insecticide resistance may vary based on the geographical location. in the present study, we collected the cockroach strains from three different locations in tehran city with different frequency of permethrin application and we observed that the pattern of resistance to permethrin was quite different. thus, the use of a particular insecticide against the german cockroach on a larger scale may not be effective, and the frequency of insecticide use should also be considered even for the same area. some studies have also suggested that resistance to insecticides depends not only on the evolutionary adaptation towards insecticide pressure but also the biological conditions of the location where the cockroaches inhabit is important (30). hence, chemical control failure can probably occur in any setting as a result of insecticide resistance due to other biological factors. german cockroaches with great adaptive potential for resistance to insecticides may be scattered over a small area in urban /rural places. therefore, a similar insecticide prescriphttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 152–161 a ghaderi et al.: level of … 159 http://jad.tums.ac.ir published online: june 30, 2021 tion may not be useful on a larger scale, and an integrated resistance management programs for combating the german cockroach must be considered even within small areas. conclusion cyp450 enzymes' expression are the successful adaptation of many insect pests to protect them against insecticides. it is clear that the enhanced metabolism of the insecticide in the resistant strains of the pests is associated with the expression of various cyp450 enzymes (31). in the present study, we observed more than one cytochrome p450 isozymes which were involved in the pyrethroid (permethrin) resistance. in addition, insecticide resistance level depends highly on the biological conditions of the insect collection sites, therefore, resistance monitoring should be done based on the specific location where the cockroaches inhabit. it can be concluded that german cockroach strains, even those from the same geographic source, can develop diverse mechanisms of resistance. therefore, alternative chemical or nonchemical insecticides should be considered as a part of the integrated pest management (ipm) based on the insecticide resistance status in a particular geographical area. world health organization recommended the following insecticides for cockroach control: bendiocarb, hydramethylnon, boric acid, fenoxycarb, flufenoxuron, pyriproxyfen, hydroprene, dinotefuran, imidacloprid, 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diabetic rats seyyedeh mahbubeh mousavi 1, sohrab imani 2, saeid haghighi 3, seyyedeh elaheh mousavi 4, akbar karimi 5 1department of entomology, islamic azad university, tehran science and research branch, iran 2department of toxicology, medical sciences university, semnan, iran 3department of pharmaceutical, microbiology, pasture institute of iran, tehran, iran 4department of pharmacology, tehran university of medical sciences, tehran, iran 5departments of developmental biology sciences, islamic azad university, tehran science and research branch, iran (received 31 oct 2010; accepted 3 sep 2011) abstract background: diabetes is an important disease. this disease is a metabolic disorder characterized by hyperglycemia resulting from perturbation in insulin secretion, insulin action or both. honey bee venom contains a wide range of polypeptide agents. the principle components of bee venom are mellitin and phospholipase a2. these components increase insulin secretion from the β-cells of pancreas. this study was conducted to show the hypoglycemic effect of honey bee venom on alloxan induced diabetic male rats. methods: eighteen adult male rats weighting 200±20 g were placed into 3 randomly groups: control, alloxan monohydrate-induced diabetic rat and treated group that received honey bee venom daily before their nutrition for four months. forty eight hours after the last injection, blood was collected from their heart, serum was dissented and blood glucose, insulin, triglyceride and total cholesterol were determined. results: glucose serum, triglyceride and total cholesterol level in treated group in comparison with diabetic group was significantly decreased (p< 0.01). on the other hand, using bee venom causes increase in insulin serum in comparison with diabetic group (p< 0.05). conclusion: honeybee venom (apitoxin) can be used as therapeutic option to lower blood glucose and lipids in diabetic rats. keywords: alloxan, glucose, honeybee venom, rat introduction diabetes type 1 is mainly results from autoimmune beta cell destruction, while viral infections and chemical agents seem to be the triggers of the disease. the well-documented effect of insulin is to mediate carbohydrates, proteins and lipids storage. therefore diabetes is considered as a defect of lipids, proteins and carbohydrates metabolism in which all the body systems and organs are affected (williams and pickup 2000). the effect of insulin deficiency mainly results in elevation of cholesterol, phospholipids and free fatty acid (williams and pickup 2000). natural toxins have been traditionally used to heal diseases and honeybee venom (apitoxin) is of a great importance in this regard. the venom is composed of varieties of peptides (mellitin, apamin, secapin, tertiapin, ado lapin, and mcd peptid), enzymes (phospholipase a2, hyaluronidase, acid phosphomonoesterase, lysophospholipase), active amines (histamine, dopamine, norepinephrine, serotonin) and many other substances (son et al. 2007). bee venom and bee sting had signifi*corresponding author: dr sohrab imani, e-mail: imanisohrab@yahoo.com j arthropod-borne dis, 2012, 6(2): 136–143 sm mousavi et al.: effect of iranian honey … 137 cant effect on quality of life (wesselius et al. 2005). mellitin and phos-pholipase a2 are the most important ingredients and play a great role in irritation and allergic responses leading to anti-inflammatory and analgesic effects (mazzanti et al. 2007). mellitin as the most important ingredient in bee venom is a strong phospholipase a2 activator which is composed of 26 amino acids and makes up 50% of the dried venom. those bee venom ingredients that contain a high molecular weight, like hyaluronidase and phospholipase a2, cause the immune reactions (bomalaski et al. 1989, zalat et al. 1999). apamin, mellitin and phospholipase a2 contained in bee venom, are strong immunoregulators but their effects on diabetes have not been investigated (habermann 1972, gauldie et al. 1976). bv apamin is known as a neurotoxin from central nervus system hyperexcitability through inhibition of the axonal potassium channels (son et al. 2007, pedarzani et al. 2008). melittin,the major constituent of bee venom, has an anti-inflammatory effect through its functionality on the anterior pituitary gland, which in turns may result in stimulation of cortisol production from the adrenal gland (son et al. 2007). part of antiinflammatory effect of melittin may attribute to its interaction with cell surface moieties which seems to render the cytotoxic effects of melittin on cancer cells (mirshafiey 2007, son et al. 2007). with regard to the side effects and high prices of blood sugar lowering chemicals, achieving new therapeutic agents with low side effects seems to be necessary. nowadays diabetic's population is growing rapidly while encountering various life-threatening disease conditions that require researchers to evaluate related therapeutic, alleviative and preventive treatments. the main purpose of the study was to evaluate the effects of iranian honeybee venom (apitoxin) on blood levels of glucose and insulin in alloxan induced diabetic rat. materials and methods honeybee venom preparation procedure bee venom samples were collected from beehives using an electric shocker, on february 2010 in khuzestan, iran. the electric shocker is composed of two components: one component as shocker and the other to collect the venom and concomitant material. the collecting unit is wooden and composed of a network of wires with small gaps between them. a glass plane is inserted under the network. the shocker was supplied by a transformer to produce a light electric shock. the shocker is designed to produce a light electric shock once every few seconds. the collector panel was first located on bottom of the beehives and then on the top, to collect the desired amount of the bee venom. when the shocker was turned on, the honeybees stroked the wires and received a light electric shock and were stimulated them to sting and discharged their bee venom. alarm pheromone produced by the exited bees to crowd and discharge toxin. the shocker was turned off after 25 minutes and the collecting panel was removed from the beehive, and the dried bee venom material scratched and transferred to a proper container. to evaluate the toxin production efficiency after collecting the samples, the crystallized bee venom material weighed using a sensitive scale. animals healthy adult male lewwis rats weighting 200±20 g were maintained in darou pakhsh pharmaceutical mfg co, tehran (iran). all animals left to acclimatize for one week before the experiment. the animal room was maintained under a constant 12-h light: 12-h dark cycle and temperature of 23±3 °c and relative humidity of 70±10% throughout the experimental period. the rats were given free access to standard pellets and water. for bees to be adapted to the new environment j arthropod-borne dis, 2012, 6(2): 136–143 sm mousavi et al.: effect of iranian honey … 138 condition, all the experiments were carried out 10 days after their first residence. the research was approved by ethical committee of the university. experimental design eighteen rats were placed randomly into three groups (n= 6): control group: normal saline 0/9% injected intraperitoneally. diabetic group: this group became diabetic by injection alloxan monohydrate at 150 mg/kg intraperitoneally. bee venom-treated group: at first received alloxan monohydrate to induce diabetes and iranian bee venom (apitoxin) at 0/5 mg/kg (the best dose chosen after pretest) after diabetes confirmation intraperitoneally at fasting condition every day for four consecutive weeks (kim et al. 1999, dong et al. 2007). blood sugar test was performed in all groups. blood glucose level was measured using acua check (germany). to measure the blood glucose level, a small incision was made on the animals’ tail using a lancet and a drop of fresh blood was extracted and used for glucometery. these samples were collected in fasting condition and expressed in mg/dl. alloxan monohydrate (sigma-aldrich germany) was used to induce diabetes in rats. the drug was administrated intraperitoneally at the rate of 150 mg/kg (viana et al. 2004, antia et al. 2005). the drug administration leads to pancreas β -cells apoptosis and necrosis. the method is preferred to induce diabetes in many other animals (soto et al. 2001). following the administration a condition of hyperglycemia like diabetes type 1 appeared in rats (byunghyun and jin-woo 2001). seventy two hours after the administration, blood glucose level was measured using acua check to determine diabetic condition. in this study, blood glucose level elevation over 280 mg/dl is supposed to reveal diabetic condition (zhang and tan 2003). usual signs of diabetes including polydipsia, polyuria and weight loss were observed 6–7 days following the administration (nuraliev et al. 1992, eldemerdash et al. 2005). to measure glucose level, fasting blood samples were collected 2 weeks following bee venom administration using "stone" method. in this method, venous blood was collected from infraorbital sinus using a hematocrit tube. the animal is kept between forefinger and thumb while the tube is inserted to the orbital foramen with a rotational movement. the capillaries are usually very sensitive and fragile here and burst following a light pressure. when a few almost large drops of blood were collected, the hematocrit tube is removed. the method is suitable for repetitive blood sampling especially for primary analysis following 2 weeks of administration. the blood glucose level was determined using enzymatic kits. biochemical analysis the animals were anesthetized with ether after 16 h of fasting to collect blood for analysis. forty eight hours after the last injection, blood samples were collected from the heart and centrifuged (3000 rpm for 15 min at 4 °c) for separating the serum. the serum was then frozen at -70 °c for the biochemical analysis. then the amount of blood glucose, insulin, cholesterol and triglyceride in blood serum weredetermined.serum glucose level was measured by kinetic (enzymatic) and colorimetric methods using glucose estimation kit (pars azmoon, iran). theserum insulin levels were assayed with an elisa, irma (biosource europe sa), serum glucose, triglyceride and total cholesterol levels were determinedusingcommercial kits and enzymatic assays. statistical analysis the values were expressed as mean ±s.e.m statistical analyses were performed by one j arthropod-borne dis, 2012, 6(2): 136–143 sm mousavi et al.: effect of iranian honey … 139 way analysis of variance (anova) followed by tukey multiple comparison test. p< 0.05 were considered as significant. results bee venom preparation results: the bee venom samples were collected and panel scratched using a sterile blade and kept in clean dark vials. average amount of venom collected from three beehives (each containing estimated 10000 bees) was 147.7 mg. the average for each beehive was calculated 49.56 mg. alloxan administration result showed a significant increase in blood glucose level in (diabetic) group compared with control group after 2 and 4 weeks (p< 0.05). in addition a significant increase was observed in blood glucose level in diabetic group after 4 weeks compared with 2-week period (p< 0.05). there was a significant decline in blood glucose level in bee venomtreated group compared with diabetic group (p< 0.05). also there was a significant decline in blood glucose level in diabetic treated group after 4 weeks compared with 2-week period (p< 0.05) (fig. 1). the effect of bee venom administration on serum insulin level is illustrated in figure 2. our results showed that serum insulin level significantly decreased in diabetic group compared with controls (p< 0.05). also there was a significant increase in bee venom-treated group compared with controls (p< 0.05) (fig. 2). the effect of bee venom administration on serum triglyceride (tg) content is illustrated in figure 3. there was a significant increase in serum tg content in diabetic group compared with controls (p< 0.05). a significant decline was observed in bee venom-treated group compared with diabetic animals (p< 0.05). but no significant difference was observed in serum tg content in bee venomtreated group compared with control (fig. 3). a significant increase was observed in serum total cholesterol in diabetic group compared with controls (p< 0.05). there was a significant decline in bee venom-treated group compared with diabetic animals (p< 0.05). but no significant difference was observed in cholesterol content in bee venomtreated group compared with control group (p= 0.552) (fig. 4). fig. 1. changes of blood glucose level after injection of bee venom in diabetic rats j arthropod-borne dis, 2012, 6(2): 136–143 sm mousavi et al.: effect of iranian honey … 140 fig. 2. serum insulin levels after injection of bee venom in diabetic rats. difference between control and treated group are significant with *p< 0.05. fig. 3. triglyceride levels after injection of bee venom in diabetic rats. difference between control and treated group are significant with *p< 0.05. fig. 4. cholesterol levels after injection of bee venom in diabetic rats. difference between control and treated group are significant with *p< 0.05. discussion in our study, blood glucose level increased following alloxan monohydrate administration which led to pancreas b-cells destruction (byung-hyun and jin-woo 2001). blood glucose level decreased following bee venom treatment. this may be contributed to substances like mellitin and phospholipase a2 contained in the venom. they may play a role in diminishing inflammation of islets of langerhans and thus elevating blood insulin level. with regard to the fact that insulin regulates blood glucose level, bee venom could decrease glucose content via increasing insulin secretion (morgan and montague 1984, fujimoto and metz 1987, kim et al. 1999, simonson et al. 2000). following alloxan administration in diabetic j arthropod-borne dis, 2012, 6(2): 136–143 sm mousavi et al.: effect of iranian honey … 141 rats, blood glucose level and triglyceride (tg) content were elevated, which indicate insulin role in regulating lipid metabolism (zhang and tan 2003). insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides (frayn 1993). according to the obtained results, bee venom decreased blood tg content. one could explain the observed decline as follows: bee venom improves glycemic control and decreases blood glucose level. also glucose consumption is increased instead of lipids. acetyl coa derived from pyrovic acid enters krebs cycle which finally leads to glucose metabolism,however acetyl coa can enter tg synthesis pathway in usual condition (zhang and tan 2003). blood cholesterol level increased following alloxan administration. (yadav et al. 2004). a decline was observed in cholesterol content of bee venom -treated group (kim et al. 1999). probably cholesterol lowering effect is largely due to inhibition of its absorption in small intestine and promoting its hepatic release. the liver plays a critical role in discharging cholesterol via bile secretion (reinner et al. 1989). alloxan administration led to destruction of islets of langerhans and diminished insulin secretion in diabetic rats. treating the rats with honeybee venom (apitoxin) increased insulin secretion up to control levels. according to the published reports, mellitin polypeptide and phospholipase a2, which are two main component of the venom, promote insulin secretion. according to the literature, the observed effect is mediated by extracellular calcium and calcium channels. when these channels are opened large amounts of calcium enters the β-cells and excite them to secret insulin (morgan and montague 1984, fujimoto and metz 1987, kim et al. 1999, simonson et al. 2000). in a study on the effects of bee venom on islets of langerhans inflammation and onset of insulin dependent diabetes, kim et al. (1999) showed that intensity of inflammation and onset of diabetes declined following bee venom treatment. they also found that insulin, tg and cholesterol levels decreased in diabetic rats compared with nondiabetic animals (kim et al. 1999). according to the experiments of morgan et al. (1984), mellitin polypeptide promotes insulin secretion from islets of langerhans in vitro. the obtained results suggest that mellitin as a valuable candidate for further studies on β-cells plasma membrane role in regulating insulin secretion. the findings also indicate that mellitin can depolarize plasma membranes of β-cells and acts as a calcium transporter in the cell, which in turn promotes insulin granules secretion. the effect of mellitin on insulin secretion depends on extracellular calcium (morgan and montague 1984). simonson et al. (2000) found that mellitin may promote insulin secretion via activating phospholipase a2 in islets of langerhans. their results indicate that phospholipase a2 activation plays a role in compensating insulin resistance response in islets of langerhans (simonson et al. 2000). treatment with exogenous phospholipase a2 or mellitin promotes arachidonic acid and lysophospholipids production and insulin secretion. produced arachidonic acid and lysophospholipids may corporate in two-step insulin production mechanism. arachidonic acid produced by phospholipase a2 induction may act as a calcium transporter in to β -cells and promote insulin secretion (fujimoto and metz 1987). conclusion in this study, our results indicate that honeybee venom (apitoxin) can be used as therapeutic option to lower blood glucose and lipids in diabetic rats, however further biochemical and pharmacological studies are necessary to provide more detailed understanding of the issue. acknowledgements this study was 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egypt j biology. 1: 577. zhang x f, tan bkh (2003) effects of an ethanolic extract of gynura procumbens on serum glucose, cholesterol and triglyceride levels in normal and streptozotocininduced diabetic rats. singapore med j. 41(1): 16. j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 104 original article larvicidal activity of citrus limonoids against aedes albopictus larvae hazrat bilal 1, waseem akram 2, *soaib ali-hassan 1 1medical entomology and disease vector control, health services academy, islamabad-pakistan 2department of agricultural entomology, university of agriculture faisalabad-pakistan (received 16 nov 2011; accepted 24 sep 2012) abstract background: development of insecticide resistance occurred due to the continuous and misuse of synthetic insecticides therefore, the recent study was conducted to explore eco-friendly plant extracts that have some potential to suppress mosquito larval population. methods: who recommended mosquito larval bioassay method for insecticide was used while for the analysis of citrus oils for limonin and nomilin content hplc was used. results: among the two citrus cultivars tested as larvicide against aedes albopictus, valencia late (citrus sinensis) was the best in terms of lc50 (297 ppm), % mortality (97%) and lt50 (18.49 hours) then freutrall early (citrus reticulate) with lc50 (377.4 ppm), % mortality (88%) and lt50 (31 hours), while nomilin gave lowest lc50 (121.04 ppm) than limonin (382.22 ppm) after 72 hours of exposure. valencia late also had more limonin and nomilin (377 µ g/ml and 21.19 µ g/ml) than freutrall early (5.29 µ g/ml and 3.89 µ g/ml) respectively. conclusion: valencia late showed best results in term of lc50, lt50 and percentage mortality against aedes albopictus as it has more amount of nomilin then freutrall early, however further evaluation in the field conditions is required. keywords: limonin, nomilin, citrus, aedes larvae introduction plants have a variety of small organic molecules called secondary metabolites (sarker et al. 2005), which are required by them for interaction with the environment (kutchan and dixon, 2005). secondary metabolites from plants are confirmed to have biological activity and that can be helpful in protecting the plants from a pathogen, herbivore or competitor. secondary metabolites normally produce low concentrations of minor compounds. these metabolites show structural similarities to primary products; these secondary metabolites can be divided into the different chemical groups like alkaloids, terpenoids, phenolic, plant amines, rare amino acids and glycosides (rohloff 2003). these compounds play an important role as anti-nutritional components of food and animal feed with a number of phenolic compounds. these include cell wall phenolic components, lignification of cells and the presence of polyphenols such as condensed tannins. plant terpenoids have been studied for their activities against a number of insects (gutierrez et al. 1997). defense strategy of plant against insect pest and pathogens depends on the presence of volatile essential oils or monoterpenes (langenheim 1994). plant also used these volatile terpenoids in plant-plant interactions and serve as attractants for pollinators (tholl 2006). cyanogenic glycosides isoflavonoids and alkaloids, these soluble secondary compounds can also be toxic to animals (morris and robbins, 1997). dengue fever (df) is transmitted by aedes aegypti while, ae. albopictus is considered as maintance vector in southeast asia (cdc 2001). the incidence and geographical distribution of df has increased dramatically over the past 30 years. it is estimated that 2.5 billion of the world’s population are at risks and *corresponding author: mr soaib ali hassan, e-mail: sohaib_hassan50@yahoo.com j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 105 that 100 million cases arise annually, including 500,000 cases of dhf (peters and geoffrey, 2007). in pakistan, an outbreak of dengue hemorrhagic fever (dhf) was first reported in karachi in 1994 (chan et al. 1995). another outbreak was seen in 2005 (ali et al. 2006) while in 2010 there were 11,024 confirmed cases of df including 40 deaths and now in 2011 again dengue outbreak has occurred in punjab with 20,500 confirmed cases with 304 deaths are reported (anonymous 2011). rational control of mosquitoes lies in personal protection and community education as the most economical method in eradicating breeding sites and application of eco-friendly larvicides for the control of mosquito larvae (certin et al. 2004). synthetic insecticides are no doubt having quick actions but due to their adverse effects to the environment received wide public concern (st leger et al. 1996), like insecticide resistance (severini et al. 1993), environmental pollution, toxic hazards to human and other non-target organisms (forget 1989). to mitigate these problems, a major emphasis has recently been explored which includes the use of natural plant based products as larvicides which can provide an alternate to synthetic chemical insecticides (junwei et al. 2006). many plants contain chemicals which are helpful for the control of insects and are useful for field applications in mosquito control programmes (kalyanasundaram and das, 1985, isman 1999) such as solanum villosum berry (nandita et al. 2008) and fenugreek (halawa 2001) have shown better insecticidal effects. citrus fruits posses a wide variety of bioactive compounds with health promoting disease preventing properties that have been shown to be effective against cancer. moreover limonoids from rutaceae particularly citrus (klocke and kubo, 1982) have attracted greater concern due to their growth regulating activity (champage et al. 1992) besides having anti-carcinogenic effects (sohail et al. 2005). they have some potential as insecticide (akram et al. 2010). in view of the recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the larvicidal potential of the citrus seed crude extracts as well as limonin and nomilin against the dengue vector aedes albopictus skuse. materials and methods collection of citrus cultivars feutrall early (citrus reticulate) and valencia late (citrus sinensis) were collected from sargodha (32°5′1″n 72°40′16″e). extraction of oil the seeds of citrus cultivars were washed with tap water to remove the pulp and then dried in the oven for 48 hours at 60o c and later were grounded in an electric grinder (anex, germany). the grounded material was put in thimble and kept in extraction tube of soxhelt apparatus with extractor id 38 mm, extractor volume 85 ml and flask volume 250 ml (vogel 1978) for the extraction of oil by steam distillation method using diethyl-ether as solvent (250 ml/20 g sample). the cycle time for one sample was 4–5 hours. oil with solvent was kept at room temperature for the evaporation of solvent, leaving oil which was then collected. hplc analysis hplc apparatus comprised of a shimadzu (japan) pump (lc-10at) with a shimadzu ultraviolet (uv) detector (sdd-10av) and shimadzu degasser unit (dgu-12a). limonoids were quantified on c18 reverse phase shim pack clc-ods column, 5 μm particle size (15 cm× 4.6 mm) and detected at 210 nm. analytical or hplc grade solvents were used. methanol and acetonitrile were obtained from merck (germany) and standards (nomilin and limonin) from mp biochemicals (france). mobile phase consisted of methanol, acetonitrile and water with the following ratio j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 106 10:41:49. 5 mg of limonin and nomilin in 100 ml of mobile phase were used for the preparation of stock solution uv absorption at 210 nm with 1 ml/min flow rate was used to monitor the elution. 0.45-μm millipore filter was used for the filtration of both standards and samples and the compounds were quantified by chromatography station software csw 32, version 1. 4.11.89. the linearity of method was estimated by analyzing standards. elution was carried out isocratically and peak responses with their respective retention time were obtained by injecting twenty micro liters of each standard in hplc. filtered samples (20μl) were injected in c18 reverse phase column, eluted isocratically with acetonitrile, methanol, and water (10:41: 49). the limonoid aglycones were identified on the basis of retention times (ohta et al. 1993). colection and rearing of mosquitoes aedes albopictus larvae and pupae were collected round the year from all artificial containers and natural habitats from faisalabad (31°21′52″n 72°59′40″e ) and brought to the mosquito rearing room in biosystematics lab of department of agri. entomology, university of agriculture faisalabad, reared in steel trays of 3 inch depth and fed on tetramin® (fish food) akram et al. (2010). adult population was maintained in transparent plastic cages (70× 35× 35 cm), with cotton wicks soaked in 10% sucrose solution while females were also fed with blood of white rats every alternate day for egg development (shaalan et al. 2006). a glass beaker with strips of moistened filter paper was provided for gravid females to lay their eggs. the population was maintained at lab conditions of 30±2 oc, 75±5% rh and l14: d10 photoperiod. bioassay each replicate containing 200ml of the oil solution placed in 250ml glass beakers (who 2005). a batch of 30 early third instar larvae of the aedes albopictus were exposed in each beaker containing oil solution (mohtar et al. 1999). larvae in control were only treated with acetone. the experiment was conducted using crd (complete randomized design) under lab conditions at 30±2 oc and 75±5% relative humidity. the percentage mortality was calculated from the average of three replicates using the following formula (sumroiphon et al. 2006). percentage mortality= number of dead larvae×100 number of larvae tested data analysis abbot’s formula (abbot 1925) was used for corrected mortality and the data so obtained was analyzed by probit analysis (finney 1989) by using the software minitab-15 for dose and time mortality regression lines. corrected mortality%= observed mortality%control mortality%× 100 100control mortality% results citrus fruits contain certain compounds (limonoids) that are extremely bitter which are mainly accumulated in seeds. peaks were recorded with standards at retention time of 8.15 min (limonin) and 12.66 min (nomilin) as shown in fig 1. non-volatile collection through hplc analysis was done and the responsive peaks at specific retention time were recorded. limonin and nomilin contents in freutrall early were 5.29 and 3.89 µ g/ml respectively, while valencia late had 377 and 21.19 µ g/ml respectively as indicated in table 1. the extracted oils were used in six different concentrations (300,400,500,600,700,800 ppm). there were three replicates for each treatment, j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 107 a. b. c. d. fig. 1. hplc analysis of a) limonin, b) nomilin, c) freutrall early d) valencia late in terms of lc50 valencia late had the lowest value (368 ppm) after 24 hours when compared with freutrall early, it gave 564.7 ppm value. after 48 hours valencia late gave 333.3 ppm and freutrall early gave 465.6 ppm while after 72 hours valencia late had 297 ppm and freutrall early had 377.4 ppm values, which clearly indicated that valencia late had good results. furthermore lethality of limonin and nomilin was also evaluated; results showed that nomilin is best as shown in table 1. freutrall early and valencia late both exhibit[min.] time 0 1 2 3 4 5 [mv] vo lta ge 0 200 400 600 0. 62 0 0. 89 0 1. 15 0 1. 36 0 1. 77 0 2. 07 0 2. 32 0 2. 73 0 3. 23 0 4. 62 0 [min.]time 0 1 2 3 4 [v] vo lta ge 0.0 0.2 0.4 0.6 0.8 1.0 1. 61 0 3. 27 0 [min.]time 0 2 4 6 8 10 [v] vo lta ge 0.0 0.2 0.4 0.6 0.8 1. 17 0 1. 42 0 2. 24 0 2. 51 0 6. 47 0 7. 74 0 8. 51 0 [min.]time 0 2 4 6 8 10 12 14 [v] vo lta ge 0.0 0.2 0.4 0.6 0.8 1.0 1. 10 0 1. 89 0 3. 41 0 9. 81 0 j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 108 ed strong larvicidal activity against aedes albopictus, with respect to percent mortality, valencia late caused highest mortality (81%) after 24 hours of exposure then freutrall early (68%), while after 48 and 72 hours again valencia late exhibited highest mortality (88 and 97%) then freutrall early (74 and 88%) as shown in fig. 2. lethal time for mortality of 50% tested population was recorded at different concentration, when both citrus cultivars were compared valencia late had the lowest time (18.49 hours) to kill the 50% tested population, while freutrall early took more than a day as is clear from table 2. table 1. lethal concentration recorded at different concentration on two citrus cultivars, limonin and nomilin against larvae of aedes albopictus citrus cultivars limonin (µg/ml) nomilin (µg/ml) time lc50 (ppm) slope±se χ2 p freutrall early 5.29 3.89 24 564.7 1.17±0.17 0.44 0.97 citrus reticulate 48 465.6 1.06±0.16 0.60 0.96 72 377.4 1.31±0.17 2.57 0.63 valencia late 377 21.19 24 368 1.09±0.17 0.17 0.99 citrus sinensis 48 333.3 1.11±0.17 2.40 0.66 72 297 1.32±0.18 8.78 0.06 limonin 24 836.70 1.22±0.15 0.60 0.99 48 548.01 1.18±0.17 0.41 0.83 72 382.22 1.27±0.17 1.21 0.04 nomilin 24 289.12 1.30±0.18 5.01 0.91 48 163.28 1.33±0.17 3.22 0.52 72 121.04 1.29±0.17 8.01 0.21 table 2. lethal time of citrus cultivars against larvae of aedes albopictus citrus cultivars lt50 (hours) slope±se p χ2 feutrall early 31.07 0.41± 0.06 2.16 0.14 citrus reticulate valencia late 18.49 0.72± 0.07 7.92 0.005 citrus sinensis 2 0 3 0 4 0 5 0 6 0 7 0 8 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 m or ta lit y t im e f ro u tra l e a rly v a la n c ia la te fig. 2. percent mortality of freutrall early and valencia late against aedes albopictus larvae after 24, 48 and 72 hours j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 109 discussion plant products (rutaceae) combat problems associated with public health especially mosquitoes that provide valuable replacement of dangerous chemicals (silva et al. 2003), therefore the present study was planned and ether extract of two citrus cultivars (freutrall early and valencia late) have been selected against ae. albopictus larvae instead of synthetic insecticides as ae. albopictus is reported resistant (khan et al. 2011). plants are well known to contain a complex of chemicals with bioactive potential (farnsworth and bingel, 1977) like deterrents or attractants (fisher 1991). secondary metabolites like limonoids contain larvicidal and anti-feedant activity, therefore, the citrus oils that contain limonoids possess more potential than citrus varieties without limonoid contents (roy and saraf, 2006) as they arrest the metabolic activities of the larvae (senthilkumar et al. 2009). our results clearly indicate that nomilin is better of the two tested limonoids (limonin and nomilin) (table 1). ruberto et al. (2002) reported limonin as the best of all extracted limonoids against spodopter spp. our results on the larvicidal effect is comparable with the study of akram et al. (2010), din et al. (2011) that citrus cultivars have potential to kill the larvae of ae. albopictus. the findings of our results are also in collaboration with the findings of sumroiphon et al. (2006) who have reported that the effect of water extract of citrus seed extract showed lc50 values of 135, 319.40 and 127, 411.88 ppm against the larvae of culex quinquefasciatus and ae. aegypti. similarly bagavan et al. (2008) have reported that peel chloroform extract of citrus sinensis, leaf ethyl acetate extracts of o. sanctum and o. canum, and leaf chloroform extract of rhinacanthus nasutus against the larvae of anopheles subpictus (lc50= 58.25, 88.15, 21. 67 and 40.46 ppm, lc90= 298.31, 528.70, 98. 34 and 267.20 ppm) and peel methanol extract of citrus sinensis, ethyl acetate extracts of o. sanctum, leaf methanol extract of o. canum and r. nasutus against the larvae of cu. tritaeniorhynchus (lc50= 38.15, 72.40, 109.12, and 39.32 ppm, 90= 184.67, 268.93, 646.62, and 176.39 ppm), respectively and the extracts from peel of different citrus varieties had good larvicidal effect with lemon peel oil as the best (mwaiko et al. 1994). however further studies are required to confirm its action. acknowledgements the funds provided by higher education commission (hec) pakistan to carry out this project are highly acknowledged. the authors declare that there is no conflict of interest. references abbot sw (1925) a method of competing the effectiveness of an insecticide. j econ entomol. 18: 265–267. akram w, hafiz aak, faisal h, hazrat b, yeon kk, jong-jin l (2010) potential of citrus seed extracts against dengue fever mosquito, aedes albopictus (skuse) (diptera: culicidae). pak j bot. 42(4): 3343– 3348. ali n, nadeem a, anwar m, tariq wu, chotani ra (2006) dengue fever in malaria endemic areas. j college phy surg pak. 16: 340–342. anonymous (2011) disease early warning system and response in pakistan. weekly epidemiol bulletin special suppl. 2(3): 1. bagavan a, kamaraj c, abdul ra, elango g, abduz-zahir a, pandiyan g (2008) evaluation of larvicidal and nymphicidal potential of plant extracts against anopheles subpictus grassi, culex tritaeniorhynchus giles and aphis gossypii glover. parasitol res. 104(5): 1109–1117. j arthropod-borne dis, 2012, 6(2): 104–111 h bilal et al.: larvicidal activity of … 110 cdc (centers for disease control) (2001) information on aedes albopictus. arboviral encephalitides. available at: http: //www.cdc.gov/ncidod/dvbid/arbor/albo pic_new.html. 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(1988a, b, 1989, 2000) isolated parasites and enteropathogenic bacteria from the external body surface and gut lumen of musca domestica l., musca sorbens wiedemann and chrysomya megacephala (fabricius). as they also visit clean food, they are important mechanical transmitters of pathogens causing human diseases (reid 1953, greenberg 1973). in fly management programme chemicals are extensively used. combined use of insect growth regulator (igr) and parasitoids would yield a better reduction in fly density (srinivasan and amalraj 2003). axtell and edwards (1983) utilized cyromazine as feed additive to caged layering hens under field conditions in high rise, wide span and narrow poultry houses, the chemical effectively controlled house flies (m domestica) and soldier flies (hermatia illucens). william and berry (1980) evaluated the igr cyromazine as topical spray and feed additive for controlling the housefly m. domestica breeding in chicken manure and provided a 70% reduction of native house flies and 100% inhibition of laboratory reared house flies. the objective of this study was to evaluate the residual effect of igrs triflumuron and pyriproxyfen on the larval stages of m domestica l.in the laboratory. *corresponding author: prof dr s. sulaiman, tel: +039 2897416, e-mail: salsul@medic.ukm.my iranian j arthropod-borne dis, (2008), 2(1): 1-6 s sulaiman et al: effect of triflumuron and … 2 materials and methods the m domestica l. used was bred in the insectarium of department of biomedical science, faculty of allied health sciences, universiti kebangsaan malaysia. the two igrs evaluated were triflumuron (starycide sc480) provided by bayer malaysia sdn bhd and pyriproxyfen (sumilarv 0.5% g) supplied by sumitomo chemical company ltd tokyo japan. in each of the glass beaker of 250 ml capacity was placed 5g mice chow and vitamin b complex, 5 ml distilled water and 5 ml of triflumuron or pyriproxyfen of varying concentrations viz 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l. 25 1 st instar m. domestica l. 1 st instar larvae were placed inside each beaker using the forceps. the beakers were then covered with nettings. for the control, only mice chow and vitamin b complex with distilled water were placed in the beaker. after pupation, the pupae were collected and transferred to a small bottle and covered with cotton wool for adult emergence. the mortality of larvae and the number of formed pupae and adults emerging were recorded. pupae were allowed to emerge into adults for 10 days, if unemerged they were assumed as dead. the efficacy of the igrs triflumuron and pyriproxyfen were determined by counting the numbers of dead larvae, pupae and adults daily and removing them until mortality and adult emergence were completed. there were 8 replicates per treatment. the above experiment was repeated using 2 nd and 3 rd instar larvae of m domestica .statistical analysis with anova followed by pos hoc tests using lsd multiple comparison. results the effect of triflumuron on 1 st instar m. domestica larvae indicated adult emergence inhibition of 98.0-100% at concentrations of 0.5-2.5 mg/l and 23.5% adult emergence inhibition for the control, respectively (table 1). effect of pyriproxyfen on 1 st instar m. domestica larvae indicated adult emergence inhibition of 98.5-100% at concentrations between 0.5 to 2.5 mg/l and 18.0% adult emergence inhibition for the control respectively (table 2). there was no significant difference between triflumuron and pyriproxyfen on the housefly adult emergence inhibition when fed to the 1 st instar larvae. however, there was a significant difference between the igrs and the control (p< 0.05). the effect of triflumuron on 2 nd instar m. domestica larvae indicated adult emergence inhibition between 93.0 to 100% at concentrations of 0.5-2.5 mg/l and 19.0% adult emergence inhibition for the control (table 3). effect of pyriproxyfen on the 2 nd instar m. domestica larvae indicated adult emergence inhibition of 97.0-100% at concentrations varying between 0.5 to 2.5 mg/l and 13.0% adult emergence inhibition for the control respectively (table 4). there was no significant difference between triflumuron and pyriproxyfen on the housefly adult emergence inhibition when fed to the 2 nd instar larvae (p> 0.05). however, there was a significant difference between the igrs and the control (p< 0.05). the effect of triflumuron on 3 rd instar m. domestica larvae indicated adult emergence inhibition of 91.0-99.5% at concentrations between 0.5 to 2.5 mg/l and 6.5% adult emergence inhibition for the control respectively (table 5). effect of pyriproxyfen on 3 rd instar m. domestica larvae indicated adult emergence inhibition of 97.0100% at concentrations varying between 0.5 to 2.5 mg/l and 8.5% adult emergence inhibition for the control respectively (table 6). there was no significant difference between triflumuron and pyriproxyfen on the housefly adult emergence inhibition when fed to the 3 rd instar larvae. however, there was a significant difference between the igrs and the control (p<0.05). iranian j arthropod-borne dis, (2008), 2(1): 1-6 s sulaiman et al: effect of triflumuron and … 3 table 1. residual effect of triflumuron on 1 st instar m domestica l.larvae and emerging pupae and adults in the laboratory concentrations (mg/l) no. of l1 larvae mortality of l1 larvae (% in parentheses) no. of pupae formed (% in parentheses) adult emergence (% in parentheses) adult emergence inhibition (%) 0.5 200 98 (49.0) 102 (51.0) 4(2.0) 98.0 1.0 200 112 (56.0) 88 (44.0) 2(1.0) 99.0 1.5 200 156 (78.0) 44 (22.0) 0 (0) 100 2.0 200 140 (70.0) 60 (30.0) 1(0.5) 99.5 2.5 200 163 (81.5) 37 (18.5) 0 (0) 100 control 200 32 (16.0) 168 (84.0) 153 (76.5) 23.5 table 2. residual effect of pyriproxyfen on 1st instar m domestica l.larvae and emerging pupae and adults in the laboratory concentrations (mg/l) no. of l1 larvae mortality of l1 larvae (% in parentheses) no. of pupae formed (% in parentheses) adult emergence (% in parentheses) adult emergence inhibition (%) 0.5 200 23 (11.5) 177 (88.5) 3 (1.5) 98.5 1.0 200 26 (13.0) 174 (87.0) 1(0.5) 99.5 1.5 200 28 (14.0) 172 (86.0) 0 (0) 100 2.0 200 44 (22.0) 156 (78.0) 1 (0.5) 99.5 2.5 200 61(30.5) 139 (69.5) 0 (0) 100 control 200 22 (11.0) 178 (89.0) 164 (82.0) 18 table 3. residual effect of triflumuron on 2 nd instar m domestica l.larvae and emerging pupae and adults in the laboratory concentrations (mg/l) no. of l1 larvae mortality of l1 larvae (% in parentheses) no. of pupae formed (% in parentheses) adult emergence (% in parentheses) adult emergence inhibition (%) 0.5 200 56 (28) 144 (72.0) 14 (7.0) 93.0 1.0 200 65 (32.5) 135 (67.5) 7 (3.5) 96.5 1.5 200 80 (40.0) 120 (60.0) 2 (1.0) 99 2.0 200 93 (46.5) 107 (53.5) 3 (1.5) 98.5 2.5 200 102 (51.0) 98 (49.0) 0 (0) 100 control 200 23 (11.5) 177 (88.5) 162 (81.0) 19.0 iranian j arthropod-borne dis, (2008), 2(1): 1-6 s sulaiman et al: effect of triflumuron and … 4 table 4. residual effects of pyriproxyfen on 2 nd instar m domestica l.larvae and emerging pupae and adults in the laboratory concentrations (mg/l) no. of l1 larvae mortality of l1 larvae (% in parentheses) no. of pupae formed (% in parentheses) adult emergence (% in parentheses) adult emergence inhibition (%) 0.5 200 18 (9.0) 183 (91.5) 6 (3.0) 97.0 1.0 200 19 (9.5) 181(90.5) 5 (2.5) 97.5 1.5 200 24 (12.0) 176 (88.0) 1(0.5) 99.5 2.0 200 26 (13.0) 174 (87.0) 1(0.5) 99.5 2.5 200 26 (13.0) 174 (87.0) 0 (0) 100 control 200 14 (7) 186 (93.0) 174 (87.0) 13.0 table 5. residual effect of triflumuron on 3 rd instar m domestica l.larvae and emerging pupae and adults in the laboratory. concentrations (mg/l) no. of l1 larvae mortality of l1 larvae (% in parentheses) no. of pupae formed (% in parentheses) adult emergence (% in parentheses) adult emergence inhibition (%) 0.5 200 16 (8.0) 184 (92.0) 18 (9.0) 91.0 1.0 200 25 (12.5) 175 (87.5) 8 (4.0) 96.0 1.5 200 40 (20.0) 160 (80.0) 4 (2.0) 98.0 2.0 200 62 (31.0) 138 (69.0) 4 (2.0) 98.0 2.5 200 89 (44.5) 111(55.5) 1 (0.5) 99.5 control 200 6 (3.0) 194 (97.0) 187(93.5) 6.5 table 6. residual effect of pyriproxyfen on 3 rd instar m domestica l.larvae and emerging pupae and adults in the laboratory concentrations (mg/l) no. of l1 larvae mortality of l1 larvae (% in parentheses) no. of pupae formed (% in parentheses) adult emergence (% in parentheses) adult emergence inhibition (%) 0.5 200 11(5.5) 189(94.5) 6(3.0) 97.0 1.0 200 11(5.5) 189(94.5) 5(2.5) 97.5 1.5 200 16(8.0) 184(92.0) 1(0.5) 99.5 2.0 200 14(7.0) 186(93.0) 2(1.0) 99.0 2.5 200 15(7.5) 185(92.5) 0(0) 100 control 200 10(5.0) 190(95.0) 183(91.5) 8.5 iranian j arthropod-borne dis, (2008), 2(1): 1-6 s sulaiman et al: effect of triflumuron and … 5 discussion kelly et al. (1987) evaluated cyromazine against insecticide-resistant field strains of m domestica. no tolerance to cyromazine was found, neither was there a direct correlation between larval responses to cyromazine and adult responses to other insecticides. it was concluded that cyromazine has a good potential for the control of houseflies that exhibited high levels of resistance to other insecticides. vazirianzadeh et al. (2007) studied the effects of oral application of cyromazine and triflumuron on housefly larvae. both igrs had sufficient effect on larval mortality compared with their controls. it was concluded that cyromazine should be used in a larvicidal programme to control housefly rather than triflumuron. in the present study both triflumuron and pyriproxyfen had residual effects on all the larval stages of m. domestica. both igrs inhibit adult emergence of 98 to 98.5% when applied at the lowest concentration of 0.5 mg/l on the 1 st instar, 93 to 97% adult emergence inhibition on the 2 nd instar and 91 to 97% adult emergence inhibition on the 3 rd instar larvae. according to hatakoshi et al. (1987) s-31183 (now called pyriproxyfen) was more effective than methoprene and diflubenzuron against 4-day old larvae of m. domestica in artificial medium and more active than methoprene against eggs and 4day-old larvae of m. domestica in the chicken manure medium. kostina (1999) studied the influence of pyriproxyfen on preimaginal stages of m. domestica and aedes aegypti. the presence of 10, 20, and 40 grams preparation per square meter of surface the number of fly puparia was 62.2%, 50% and 38.7% respectively. the emergence of adults was 0.5% in the case of 10 gram per square meter, and it was completely absent in the latter cases. according to zhang and shono (1997), the toxicity of pyriproxyfen gradually increased from the early stage of 3 rd instar larva of houseflies to the white pupal stage. further studies indicated that the white pupa was the most susceptible stage to the chemical and the mortality of pupae decreased sharply after the white pupal stage. in conclusion, both triflumuron and pyriproxyfen are effective in inhibiting adult emergence of housefly m domestica and therefore should be recommended for fly control particularly in chicken farms and dumping grounds in malaysia for housefly control activities. acknowledgements we wish to thank faculty of allied health sciences, universiti kebangsaan malaysia for providing research facilities. we appreciate bayer malaysia sdn bhd for providing triflumuron and sumitomo chemical company tokyo, japan for providing pyriproxyfen. references axtell rc, edwards td (1983) efficacy and nontarget effects of larvadex as a feed additive for controlling house flies in a caged-layer poultry manure. poult sci. 62(12):2371-2377. greenberg b (1973) flies and disease, vol ii. biology and disease transmission. princeton university press, new jersey. hatakoshi m, kawada h, nishida s, kisida h, nakayama i (1987) laboratory evaluation of 2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxy] pyridine against larvae of mosquitoes and housefly. jpn j sanit zool. 38(4):271-274. kelly ja, stubbs mr, pinniger db (1987) laboratory evaluation of cyromazine against insecticide-resistant field strains of musca domestica. med vet entomol. 1(1):65-69. kostina mn (1999) influence of pyriproxyfen on preimaginal stages of musca doiranian j arthropod-borne dis, (2008), 2(1): 1-6 s sulaiman et al: effect of triflumuron and … 6 mestica and aedes aegypti. proceedings of the 3 rd international conference on urban pests, 1999 19 22 july, prague, czech republic . p. 626 reid ja (1953) notes on house-flies and blowflies in malaya. bull inst med res. fed of malaya. 7:1-26. srinivasan r, amalraj dd (2003) efficacy of insect parasitoid dirhinus himalayanus (hymenoptera: chalcidae) and insect growth regulator, triflumuron against house fly, musca domestica (diptera: muscidae). indian j med res.118:15866. sulaiman s, sohadi ar, yunus h, iberahim r (1988a) the role of some cyclorrhaphan flies as carriers of human helminths in malaysia. med vet entomol. 2:1-6. sulaiman s, aziz ah, yunus h, sohadi ar (1988b) isolations of enteropathogenic bacteria from some cyclorrhaphan flies in malaysia. malays appl biol. 17(2): 129-133. sulaiman s, sohadi ar, jeffery j (1989) human helminth parasite burdens on cyclorrhaphan flies (diptera) trapped at an aboriginal settlement in malaysia. bull ent res.79:625-629. sulaiman s, othman mz, aziz ah (2000) isolations of enteric pathogens from synanthropic flies trapped in downtown kuala lumpur. j vector ecol. 25(1): 90-93. vazirianzadeh b, jervis ma, kidd nac (2007) the effects of oral application of cyromazine and triflumuron on housefly larvae. iranian j arthropod-borne dis. 1(2): 713. william re, berry tg (1980) evaluation of cga 72662 as a topical spray and feed additive for controlling house flies breeding in chicken manure. poult sci. 59(10): 2207-2212. zhang li, shona t (1997) toxicities of pyriproxyfen to susceptible and resistant strains of houseflies. jap soc appl entomol zool. 32(2): 373-378. microsoft word 3-dr_nabian.doc iranian j arthropod-borne dis, (2008), 2(1): 16-20 s nabian and s rahbari: occurrence of soft … 16 original article occurrence of soft and hard ticks on ruminants in zagros mountainous areas of iran s nabian1, s rahbari, 1 1 department. of parasitology, faculty of veterinary medicine, university of tehran, tehraniran (received 22 may 2007; accepted 26 apr 2008) abstract background:the distribution and preferences of ticks of animals in central of iran were studied four times a year. methods: one thousand seven tick specimens were collected from different localities including isfahan, chaharmahalbakhtiary, eastazarbaijan, west azerbaijan, kordestan, kermanshah, lorestan and fars. results: most of sampled animals in this area were infested. we also encountered with a wild goat (capra hircus aegagrus) in kolah qazi national park in this part that was infested intensively by boophilus kohlsi. fifteen ixodid tick species were identified over the study period from cattle, sheep and domestic and wild goats namely b. kohlsi (3.6%) rhipicephalus sanguineus (4.5%), rh.bursa (21.9%), rh.turanicus (2.9%), dermacentor niveus (12.9%), d.raskemensis (4.1%), d.marginatus (7.3%), haemaphysalis punctata (3.5%), h. parva (0.6%), h. choldokovskyi (2%), hyalomma anatolicum excavatum (4.8%), h.anatolicum anatolicum (5.2%), h. asiaticum asiaticum (7.3%), h. marginatum marginatum (13%), and h. detritum detritum (5.9%). the only soft tick found was ornithodoros canestrinii which occurred in all localities of isfahan province but with significant differences in abundance. clear pattern of seasonality was evident for this species and it was generally present from november to march, while ixodid ticks were present throughout the year. the largest numbers of adult ixodid ticks were generally present from april to august. conclusion: the results showed that rh.bursa, d.niveus and h.marginatum marginatum are dominant tick species. keyword: tick, ruminant, iran introduction the tick studies started by delpy (1936, 1938) in iran. abbasian(1960, 1961) and mazlum (1968, 1971) described a list of adult ticks collected from domestic animals. filipova et al. (1976) presented data for 642 ixodid tick specimens taken from small size mammals, chiefly rodents in different zoogeographical zones of iran. hoogstraal (1979, 1980) studied ixodid ticks parasitizing wild sheep and goat in iran with focusing on maintaining natural foci of many hazardous diseases for human. rahbari (1995) published ecological aspects of various species of ticks encountering domestic animals in north west of iran. razmi (2002) published a list of tick species of domestic animals in north east of iran. the zagros mountains are iran's second largest mountain range. they have a length of 1500 kilometres from western iran to the southern parts of the persian gulf. the rolling hills around the mountain range are main summer pastures for animals. it seems that it is a gap of study in the recent years in these areas. therefore, the objective of this study was to determine the species and distribution of ticks infesting ruminants in zagros mountainous areas. *corresponding author: dr s nabian tel: +98 21 66924469, fax: +98 2166933222, e-mail: nabian@ ut.ac.ir iranian j arthropod-borne dis, (2008), 2(1): 16-20 s nabian and s rahbari: occurrence of soft … 17 materials and methods this study was conducted in seasonal activity of ticks from eight provinces of zagros mountainous areas consisting of isfahan, chaharmahalbakhtiary, east azerbaijan, west azerbaijan, kurdistan, kermanshah, lorestan and fars. tick sampling was performed on the whole body of 259 cattle, 334 sheep, 110 goats, and one 6 yr old dead wild goat (capra hircus aegagrus) located in kolah qazi national park in isfahan province. the collected specimens were counted and preserved in 70% alcohol in glass vial and brought to laboratory for further studies. determination was done using a stereomicroscope (zeiss) according to the identification keys (delpy 1938, hoogstraal & kaiser 1959, kaiser & hoogstraal 1963, 1964, walker et al. 2003, eastrada et al. 2004, apanaskevich & horak 2005). results in this study 1007 ticks specimens were collected from 704 animals thus mean intensity for each animal was 1.43. most of ticks were found on the host during the hot and humid months, but a few collections revealed on autumn and winter months. a total of 15 species of ixodid ticks were specified. rh. bursa was observed as the most abundant tick (21.9%), whereas rh. sanguineus (4.5%) and rh. turanicus (2.9%) were rare species and the only species from genus boophilus was b.kohlsi (3.6%). important descriptions of tick infested wild goat strongly supported that the tick species in this collection was only b. kohlsi. the most diversity of tick species determined within the genus hyalomma with four species; h. marginatum marginatum was found as a common fauna in this place (13%). the infestation to h. asi. asiaticum, h. detritum detritum, h.ana anatolicum and h.ana.excavatum were 7.3, 5.9, 5.2 and 4.8%, respectively. three species of haemaphysalis was recorded, the infestation rate of h.punctata, h. parva and h. choldokovskyi were 3.5, 0.6 and 2 % respectively. d. niveus (12.9%), d. marginatus (7.3%) and d. raskemensis (4.1%) were identified. the only soft tick found was ornithodoros canestrinii which occurred at all localities but with significant differences in abundance, clear pattern of seasonality was evident for this species and it was generally present from november to march, while ixodid ticks were present throughout the year. the largest numbers of adult ixodid ticks were generally present from april to august. discussion mazlum (1971) previously reported all species recorded in this study except b. kohlsi and rh. turanicus, rahbari et al. (2007) reported b. kohlsi as a new record for iran. aeschliman and morel (1965) have described the morphological characteristic of this species. this tick is restricted to sheep and goats and occasionally horses and is reported from syria, iraq, israel, jordan (hoogstral, and kaiser 1960, shamsuddin and mohammad 1988), western saudi arabia (hussein et al. 1988). it is also endemic to west africa (walker et al. 2003). wild goats inhabit throughout iran except in forest and other tall vegetation, although open rangeland has been grazed occasionally by wild and domestic animals but in this survey, we could not find any specimens of b kohlsi in domestic animals although the restricted home range of ibex implies that they could serve as a source of tick infestation for other animals within the same habitat. h. choldokovskyi was found in sheep pastured in surrounding area of these localities in low number, nabian and rahbari (2007) iranian j arthropod-borne dis, (2008), 2(1): 16-20 s nabian and s rahbari: occurrence of soft … 18 demonstrated it from some part of caspian sea, but delpy (1938) concluded that it was distributed in high altitude territories. h. parva is a rare species in this part, but it was also reported from caspian sea area and semi desert zones; the immature stages are frequently found on small rodents such as social vole. the adults are frequently found on sheep and goat, carnivora are also host of adult. hoogstraal et al. (1980) previously identified it from wild sheep and he believed that the range of this species extends to italy and also some parts of libya. this tick has been known to transmit theileria sergenti, and crimean-congo hemorrhagic fever virus. h. punctata was recorded throughout rocky mountain slopes of zagros mountainous areas, though, mazlum believed that it has been expanded its range in the most of provinces in iran. estrada et al. (2004) recorded that the larvae of this tick feed on small animals such as great gerbil, the nymphs also feed on small mammals and birds. it is well known vector for babesia motasi and b. major, it has also been demonstrated to carry rickettsia siberica and to cause tick paralysis. h. anatolicum anatolicum was recorded over widely scattered throughout iran. it is a vector of causative organism of tropical theileriosis and it transmits a variety of pathogenic organism such as theileria lestoquardi, th. equi, b.cabali, trypanosoma theileri and crimeancongo haemorrhagic fever virus (walker et al. 2003). we found that h. ana. excavatum is adapted to a variety of climatic conditions and was often less commonly found on livestock than h.ana.anatolicum. walker et al. (2003) described its distribution from the mediterranean, steppe climatic regions of north africa to steppe climatic regions elsewhere in its wide range including iran and turkmenistan. h. asi.asiaticum was found in all localities of this area. abbasian (1961) described it as a species from south-eastern iran near the pakistan borders. the adults parasitize camels and other domestic herbivores and also wild goats and sheep, and gazelles in semi desert and desert environments (hoogstraal 1980). the distribution of h.marginatum marginatum extended in all rolling hills of these areas with highest percentage in tick population, more or less similar pattern was observed by mazlum (1971). but hoogstraal et al. (1988) described h. detritum detritum as a tick fauna ranges from southern europe to the caspian, with outlying pockets in the mountains of golestan national park in northern part of iran. walker (2003), assumed that adults of this two-host tick infest cattle, horse, sheep, goats and camels. the immature stages feed on small mammals such as hares and rabbits and occur in the areas with mediterranean climate of many widespread areas through to central asia. according to the study of mazlum (1971) who emphasized that rh. bursa occurred as a dominant tick in most sheep areas, we also found rh. bursa as the major species in these areas. rh. sanguinus population was low. our observations demonstrated that most of sampled animals in central areas were infested with d. niveus and d. marginatus. mazlum (1971) previously determined and reported them in most mountainous areas. anaplasma phagocytophilia and borrelia burgdorferi recently isolated from d. marginatus and there is no important report of its implications of tick borne disease in sheep and goat. based on the results it is concluded that rh. bursa, h. marginatum and d. niveus are dominant tick species in this area and should be noted for their ability in transmission of infectious agents. acknowledgments we wish to express our sincere thank to dr allan walker for his kind assistance. we also iranian j arthropod-borne dis, (2008), 2(1): 16-20 s nabian and s rahbari: occurrence of soft … 19 appreciate the assistances received from iranian center of tick and tick-borne diseases. this study was supported by research council of tehran university and veterinary organization. references abbasian l (1960) a preliminary list of ticks (acarina: ixodidae) occurring in iran and their distributional data. acarologia. 2: 43-61. abbasian l (1961) records of tick (acarina: ixodidae) occurring in iran and their distributional data. acarologia. 3:546-559. aeschlimann a, morel pc (1965) boophilus geigyin.sp. (acarina: ixodoidea), a new tick from cattle of west africa. actatrop. 22:162-168. apanaskevich da, horak ig (2005) the genus hyalomma koch, 1844.ii. taxonomic status of h .anatolicum koch, 1844 and h. excavatum koch, 1844 (acari, ixodidae) with redescriptions of all stages. acarina. 13(2):181-197. chen fan my, bi d, zhang jz, huang yp (1998) detection of rickettsia sibirica in ticks and small mammals collected in three different regions of china. acta virol. 42(1): 61-4. delpy l (1938) les especes iranienes du genre haemaphysalis koch 1844. annalles de parasitologie humaine et comparee.16 (1): 1-10. delpy l (1936) note sur les ixodides du genre hyalomma (koch). annalles de parasitologie. 14 (3): 206-245. eastradapena a, bouattour a, camicas jl, walker ar ( 2004) ticks of domestic animals in the mediterranean region, a guide to identification of species. university of zaragoza. filopova na, neronov vm, farhangazad a (1976) data on ixodid tick fauna (acarina, ixodidae) of small mammals in iran (in russian). ent. obozr. 55(2): 467-479. harwood rf, maurice tj (1979) tick paralysis and toxicosis, entomology in human and animal health. macmillan publishing, america, pp.460-463. hoogstraal h, kaiser mn (1960) boophilus kohlsi n .sp. (acarina: ixodidae) from sheep and goats in jordan. j parasitol. 46: 441-8. hoogstral h, valdez r (1980) ticks (ixodoidea) from wild sheep and goats in iran and medical and veterinary implications. fieldiana zoology. 6: 1-16. hoogstraal h, kaiser n (1959) observations on egyptian hyalomma ticks (ixodoidea, ixodidae) biological notes and differences in identity of h.anatolicum and its subspecies anatolicum koch and excavatum koch among russian and other workers. identity of h.lusitanicum koch. ann ent soc am. 52(3): 243-261. hoogstral, h, wassef hy (1979) haemaphysalis (allophysalis) kopetdaghica: identity and discovery of each feeding stage on the wild goat in northern iran (ixodoidea: ixodidae). j parasitol.65 (5): 783-790. hooshmand rad p (1976) the pathogenesis anaemia in theileria annulata infection, res. vet sci. 20: 324-329. hussein hs, alkhalifa ms, diab fm, alasghah na (1988) the distribution, host range and seasonal abundance of the arabian goat tick, boophilus kohlsi (acari: ixodoidae) in saudi arabia. persian gulf journal of scientific research. 6: 1 287 kaiser mn, hoogstraal h (1964) the hyalomma ticks (ixodoidea, ixodidae) of pakistan, india and ceylon, with keys to sub genera and species. acarologia. (6): 257-386. kaiser mn, hoogstraal h (1963) the hyalomma ticks (ixodoidea, ixodidae) iranian j arthropod-borne dis, (2008), 2(1): 16-20 s nabian and s rahbari: occurrence of soft … 20 of afghanistan. j parasitol. 49(1):130139. lewis d, herbert a (1980) a large babesia of sheep from north wales. vet rec.107: 352-353. mazlum z (1971) ticks of domestic animals in iran: geographic distribution, host relation, and seasonal activity. j vet fac univ tehran, iran. 27:1, 1-32. mazlum z (1968) hyalomma asiaticum asiaticum (schulze and schlottke) 1929 its distribution, hosts, seasonal activity, life cycle and role in transmission of bovine theileriosis in iran. acarologia. 10 (3):437-442. nabian s, rahbari s, shayan p, haddadzadeh hr (2007) current status of tick fauna in north of iran. iranian j parasitol. 1(2): 12-17. rahbari s (1995) studies on some ecological aspects of tick fauna of west azarbayejan, iran. j appl anim res. 7: 189-194. razmi g,naghibi a,aslani m, fathivand m, dastjerdi k (2002) an epidemiological study on bovine babesiosis in the mashhad suburb area, province of khorasan, iran. vet parasitol. 108: 109-115. shchelkanov m, kolobukhina lv, moskvina tm, aushev id, kartoev aa, kelli ei, merkulova ln, grenkova ep, samokhvalov ei, petriaev vg, serobian ag, klimova ea, galkina iv, malyshev na, aristova va, slavskii aa, lukianova na, deriabin pg, gromashevskii vl, onishenko gg, lvov dk (2005) detection of the circulation of crimeancongo hemorrhagic fever virus in the piedmont steppes of the north caucasus. vopr virusol. 50(5): 9-15. rahbari s, nabian s (2007) the first report of rhipicephalus (boophilus) kohlsi (hoogstraal and kaiser 1960) from wild goats (capra hircus aegagrus) in iran. iranian j parasitol. 2: 53-56. shamsuddin m, mohammad mk (1988) incidence, distribution, and host relationships of some ticks (ixodoidea) in iraq. j univ kuwait ssc. 15(2): 321-330. walker ar, bouattour a, camicas jl, estradapena a, horak ig, latif a, pegram rg, preston pm (2003) ticks of domestic animals in africa, a guide to identification of species. bioscience reports,.u.k . pp.157. yin h, lu w, luo j, zhang q, lu w, dou h (1996) experiments on the transmission of babesia major and babesia bigemina by haemaphysalis punctata. vet parasitol. 2, 67(1-2): 89-98. j arthropod-borne dis, june 2016, 10(2): 141–147 s rafizadeh et al.: molecular detection of … 141 http://jad.tums.ac.ir published online: january 05, 2016 original article molecular detection of leishmania major and l. turanica in phlebotomus papatasi and first natural infection of p. salehi to l. major in north-east of iran sayena rafizadeh 1, *mehrzad saraei 2, mohammad reza abaei 3, mohammad ali oshaghi 3, mehdi mohebali 4, amir peymani 5, taghi naserpour-farivar 5, hassan bakhshi 3, *yavar rassi 3 1department of biochemistry and genetic, qazvin university of medical sciences, qazvin, iran 2department of parasitology and mycology, cellular and molecular research center, qazvin university of medical sciences, qazvin, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4department of parasitology and mycology, tehran university of medical sciences, tehran, iran 5cellular and molecular research center, qazvin university of medical sciences, qazvin, iran (received 20 sep 2014; accepted 29 sep 2014) abstract background: leishmaniasis is an important public health disease in many developing countries as well in iran. the main objective of this study was to investigate on leishmania infection of wild caught sand flies in an endemic focus of disease in esfarayen district, north east of iran. methods: sand flies were collected by sticky papers and mounted in a drop of puri’s medium for species identification. polymerase chain reaction techniques of kdna, its1-rdna, followed by restriction fragment length polymorphism were used for identification of dna of leishmania parasites within infected sand flies. results: among the collected female sand flies, two species of phlebotomus papatasi and phlebotomus salehi were found naturally infected with leishmania major. furthermore, mixed infection of leishmania turanica and l. major was observed in one specimen of p. papatasi. sequence analysis revealed two parasite its1 haplotypes including three l. major with accession numbers: kj425408, kj425407, km056403 and one l. turanica. (kj425406). the haplotype of l. major was identical (100%) to several l. major sequences deposited in genbank, including isolates from iran, (gen bank accession nos.ay573187, kc505421, kj194178) and uzbekistan (accession no.fn677357). conclusion: to our knowledge, this is the first detection of l. major within wild caught p. salehi in northeast of iran. keywords: leishmania major, l. turanica, p. salehi, phlebotomus papatasi, iran introduction leishmaniasis is an important public health disease in the world. the disease is endemic in more than 98 countries including iran (who 2010). cutaneous leishmaniasis (cl) is a worldwide public health and a social problem in many developing countries. old world cutaneous leishmaniasis is present in many endemic areas of north africa, the mediterranean, the middle east, the indian subcontinent and central asia. the species responsible for old world cutaneous leishmaniasis are mainly l. major and l. tropica, l. infantum and l. donovani can also cause localized cl but, are observed less frequently in the mediterranean areas. diffuse cl is uncommon and is caused by l. aethiopica in africa (goto et al. 2010, hotez et al. 2012). *corresponding author: professor yavar rassi, email: rassiy@tums.ac.ir, dr mehrzad saraei, email: msaraei@qums.ac.ir j arthropod-borne dis, june 2016, 10(2): 141–147 s rafizadeh et al.: molecular detection of … 142 http://jad.tums.ac.ir published online: january 05, 2016 the incidence rate of the disease is 0.7 to 1.2 million cases all over the world (alvar et al. 2012). the important factors that lieshmaniasis is a serious public health in many countries are increasing of deforestation, urbanization, human migration, and hiv/ aids (desjeux 2001). the disease, almost affects the poor people, especially those with vulnerable housing and environmental conditions (alvar et al. 2006). at the present, zoonotic cutaneous leishmaniasis is the first important vector borne disease in iran and is endemic in 17 out of 31 provinces of the country (afshar et al. 2011). the annual incidence of zoonotic cutaneous leishmaniasis has gradually increased in iran and more than 20 000 cases has been reported in 2013 (unpublished data). this increasing outbreak is in relation to humansand fly-rodent contacts, itself probably the product of the development of irrigation schemes and the spread of human populations into the habitats of the vector and the rodents that act as reservoir hosts. the causative agent of disease is l. major and the sand fly species of p. papatasi has been reported as the most important and proven vector of disease to human in several endemic foci of iran (rassi et al. 2008, 2012). esfarayen district of northern khorassan province, in the north east of iran is an important focus of zoonotic cutaneous leishmaniasis due l. major with more than 400 new cases of cl in 2012. the main objective of this study was to detection of species of leishmania parasite in wild caught sandflies as the vector(s) of disease. materials and methods study area this descriptive cross sectional study was conducted in esfarayen county, north khorasan, north east of iran. the capital of the county is esfarayen. at the 2006 census, the county's population was 119,152, in 30,307 families. in general, the northern part of the esfarayen has a temperate climate due to its proximity to mountainous areas and the south and southwest areas with hot summers and cold winters. the average annual precipitation is nine mm. the main occupations of the population are farming and raising animal. sand fly collection based on prevalence of disease with positive human cases, four villages of kalatereza, esmaeilabad, kalatehshor and hosseinabad were selected. sand flies were collected biweekly from indoors (e.g. bedroom, guest bedroom, toilet, and stable) as well as outdoors (wall cracks and crevices and animal burrows) by using sticky paper (30 papers for indoors and 30 papers for outdoors per village) during july–october 2013. all traps were installed at sunset and collected near sunrise. the sand fly specimens were washed in 96 % ethanol alcohol to get rid of the sticky materials and to preserve them. dissection of preserved sand flies was done in phosphate buffered saline (pbs) solution. the terminal segments of the abdomen containing the spermatheca and the heads of females were removed and mounted in a drop of puri’s medium and identified to species level using light microscope and key of theodor and mesghali, 1964. the remains of the bodies of the sand flies were kept individually in 96 % alcohol and stored at -20 ºc for molecular analysis. dna extraction dna of the specimens was extracted using the bioneer genomic dna extraction kit cat. no.k-3032 lot.no.1204d, (north korea), according to the manufacturer’s instructions. extraction was carried out on the remaining body of the individual sand fly and stored at 4 °c. double distilled water as a negative control and dna from l. major j arthropod-borne dis, june 2016, 10(2): 141–147 s rafizadeh et al.: molecular detection of … 143 http://jad.tums.ac.ir published online: january 05, 2016 and l. tropica, provided to the iranian institute of pasteur by the world health organization, were used as positive controls. dna amplification and pcr-rflps primary examination for infection of sand flies with leishmania species was performed using nested-polymerase chain reaction (pcr) against the mini circle kinetoplast (k)-dna using the following primers (noyes et al. 1998) csb2xf (forward): 5´-c/ga/gta/ gcagaaac/tcccgttca-3´(20bp),csb 1xr(reverse): 5´-atttttcg/cga/tttt/c gcagaacg-3´ (20 bp), 13z (forward): 5´actgggggttggtgtaaaatag-3´ (22 bp), lir (reverse): 5´-tcgcagaacgcccct-3´ (15 bp). positive samples against kdna were tested against the ribosomal internal transcribed spacer 1 (its1) region using the primers litsr (5'-ctggatcattttccgatg-3') and l5.8s (5'-tgataccacttatcgcac tt-3') followed by digestion with hae iii (el tai et al. 2000). the pcr products were run along with a 100 bp ladder on 1.2 % agarose gel containing ethidium bromide for 1 h at 80 v. the gel was observed on an ultraviolet (uv) transilluminator and then digital photographs were prepared. parasites were identified by comparison with positive controls of l. infantum, l. major and l. tropica and molecular weight markers. we added 2 μl hae iii to the its1 pcr products (20 μl) at 37 °c for 12 h with conditions recommended by the supplier (fermentas, germany). the restriction fragments were subjected to electrophoresis in 3 % agarose gel containing ethidium bromide (0.5 μg/ ml) for 3 h at 65 v and observed on a uv trans illuminator (hide and banuls 2006, oshaghi et al. 2009). results in total 2305 sand flies comprising five species (3 phlebotomus and 2 sergentomyia) were collected and identified. they included: phlebotomus papatasi (43.43%), p. salehi (0.74%), p. caucasicus (2.33%), sergentomyia sintoni (42.12%), and s. dentata (11.28%). among the collected sand flies, a total of 390 females of sand flies were surveyed to find leishmania parasites. they were p. papatasi (200/390), p. salehi (17/390), s. sintoni (120/390) and s. dentata (53/390). all specimens of p. caucasicus were male. our results showed only 3 out of p. papatasi (1.5%) and 1 out of p. salehi (5.88%) were positive to l. major, whiles one specimens of p. papatasi (0.5%) were found mix infection with l. turanica and l. major. this was observed in the kdna nested-pcr amplification assays where a ~560 bp pcr band was produced. this length of pcr in the system is assigned to l. major (fig. 1). their abdominal stages were either gravid or empty indicating there was enough time for the parasites to develop and transform to promastigote, the infective form. further analyses showed that they were positive against its1 locus and produced a band of ~340 bp in gel electrophoresis. also, its1 pcr– rflp analysis by haeiii revealed the fragments of 220 and 140 bp for infected sand flies which are characteristic of l. major. the diagnostic fragments are 200 and 60 bp for l. tropica and 200, 80 and 60 bp for l. infantum/l. donovani (figs. 2, 3). sequence analysis revealed two parasite its1 haplotypes including three l. major with accession numbers: kj425408, kj425407, km052753 and one l. turanica (kj425406). the three specimens of l. major were 100 % identical, although they were isolated from different species of p. papatasi, and p. salehi. the haplotype of l. major was identical (100 %) to several l. major sequences deposited in genbank, including isolates from iran, (genbank accession nos. ay573187, kc505421, kj194178) and uzbekistan (accession no.fn677357). also it was found to be 99 % similar to l. mexicana venezuelensis j arthropod-borne dis, june 2016, 10(2): 141–147 s rafizadeh et al.: molecular detection of … 144 http://jad.tums.ac.ir published online: january 05, 2016 from mexico (accession no. f339752). the haplotype of l. turanica was found to be identical (100%) to that of isolates of l. turanica from central asia, including iran (genbank accession no. ef413079), mongolia (accession no. aj272380), turkmenistan, (accession nos. aj272379 and aj272381), and kazakhstan (accession no. aj272382). furthermore was found to be 99 % similar to l. gerbilli from china (accession no. hq 830351). fig. 1. kdna nested pcr amplification (560 bp). l. major in p. papatasi (lane b, c), l. major in p. salehi (laned), mixed infection of l. major and l.turanica in p. papatasi (lane a), positive control of l. tropica (lane p3, 720 bp), positive control of l. major (lane p2), positive control of l. infantum (680 bp, lanep1), negative control (lane n) and (l) 100 bp molecular weight marker (fermentase) fig. 2. its1 amplification of l. major in p. papatasi (lane b,c) and p.salehi (lane d), mixed infection of l. major and l. turanica in p. papatasi (lane a), positive control of l. major (p), negative control (lane n) and (l)100 bp molecular weight marker (fermentase) fig. 3. pcr-rflp analysis of its1 region for identification of leishmania species using haeiii. (l) 100 bp molecular weight marker, (p), positive control of l. major, (n) negative control, (a,b,c) samples of infected p. papatasi to l. major, (d) infected p. salehi to l. major discussion due to the nature of zoonotic infections, the challenge of elucidating the structure of ecologicalsystems are highly complex and is very important for the effective application of control measures (reithinger et al. 2007). cutaneous leishmaniasis (cl) is an old endemic public health problem in iran and more than of 80 % cases are caused by l. major, the zoonotic (zcl) form of disease (rassi et al. 2006, 2008, 2011a, 2011b ).the incidence of cl in northern khorassan province has been reported to be around 400 per 100,000 in 2013 (unpublished data). it is more disabling disease with several endemic foci in north east of iran. entomo-parasitological survey with epidemiological data are very important for control planning against leishmaniasis disease (rassi et al. 2006). natural infection of wild caught sand flies with the same leishmania parasites in human and their anthropophily, indicate the capacity of them as the vectors (killick-kendrick 1990). molecular techniques is highly sensitive to detection of leishmania parasites in sand flies and commonly used in iran and other countries (rassi et al. 2011b, j arthropod-borne dis, june 2016, 10(2): 141–147 s rafizadeh et al.: molecular detection of … 145 http://jad.tums.ac.ir published online: january 05, 2016 2012b, killick-kendrick 1990, de bruijn et al. 1992, mukherje et al. 1997). based on animal reservoir host of zcl, the great gerbil of rhombomys opimus and sand fly species of p. papatasi are the main reservoir and vector in transmission of parasite to human in central and north east of iran. at the present study, high density of p. papatasi and its natural infection with l. major is attributed to the fact that this species plays a major role as a principally vector in the region. phlebotomus papatasi is known as a restricted vector and specifically is able to support only the development of l. major (dobson et al. 2010). in iran this species is the most predominant sand fly in and around the burrows of great gerbils as well as human places. according the results of current study we found mix infection of l. turanica an l. major in one specimens of p. papatasi. this finding is incompatible with the concept of restriction transmission of l. major by the species but is congruent with the findings of strelkova et al. 1996, parvizi and ready, 2008 and bakhshi et al. 2013 demonstrating possible transmission of both l. major /l. turanica by p. papatasi (strelkova et al. 1996, parvizi and ready 2008, bakhshi et al. 2013). detection of l. major in one specimen of p. salehi was another finding of this study. although the population and infection rate of this species to l. major appeared to be low, our results confirmed the studies of other scientists in south and south east of iran (azizi et al. 2012, kassiri et al. 2012). conclusion since p. salehi specimens were only collected from rodent burrows and this species with p. papatasi appear to occur sympatrically and simultaneously in a few zcl foci of iran, indicating that the p. papatasi has the main vector’s role in transmission of leishmania parasite to human (killick-kendrick 1990) and p. salehi is a secondary maintenance vector in the transmission cycle of infection between humans and rodents in iran (killick-kendrick 1999). acknowledgement the authors gratefully thank esfarayen health center, northern khorassan province, north east of iran for its field assistance. this study was financially supported by the school of public health, tehran university of medical sciences (project no.24217) and qazvin university of medical sciences. references alvar j, yactayo s, bern c (2006) leishmaniasis and poverty. trends in parasitology. 22(12): 552–557. alvar j, velez id, bern c, herrero m, desjeux p, cano j, jannin j, den boer m (2012) leishmaniasis worldwide and global estimates of its incidence. plos. 7(5): e35671. afshar aa, rassi y, sharifi i, abai mr, oshaghi m, yaghoobi-ershadi m, vatandoost h (2011) susceptibility status of phlebotomus papatasi and p. sergenti (diptera: psychodidae) to ddt and deltamethrin in a focus of cutaneous leishmaniasis after earthquake strike in bam, iran. iran j arthropod borne dis. 5(2): 32–41. azizi k, fakoorziba m, jalali m, moemenbellah-fard m (2012) first molecular detection of leishmania major within naturally infected phlebotomus salehi from a zoonotic cutaneous leishmaniasis focus in southern iran. trop biomed. 29(1): 1–8. j arthropod-borne dis, june 2016, 10(2): 141–147 s rafizadeh et al.: molecular detection of … 146 http://jad.tums.ac.ir published online: january 05, 2016 bakhshi h, oshaghi ma, abai mr, rassi y, akhavan aa, sheikh z, mohtarami f, saidi z, mirzajani h, anjomruz m ( 2013) molecular detection of leishmania infection in sand flies in border line of 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rafizadeh et al.: molecular detection of … 147 http://jad.tums.ac.ir published online: january 05, 2016 mohtarami f, salahi r (2008) molecular detection of leishmania major in the vectors and reservoir hosts of cutaneous leishmaniasis in kalaleh district, golestan province, iran. j arthropod-borne dis. 2(2): 21–27. rassi y, abai mr, javadian e, rafizadeh s, imamian h, mohebali m (2011b) molecular detection of leismania infection due to leishmania major and l. turanica in the vectors and reservoir host in iran. vector borne zoonotic dis. 11(2): 145–150. rassi y, sanei dehkordi ar, oshaghi ma, abai mr, mohtarami f,enayati aa, zarei z, javadian e (2012b) first report on natural infection of the phlebotomus tobbi by leishmania infantum in northwestern iran. exp parasitol. 131: 344–349. reithinger r, dujardin jc, louzir h, pirmez c, alexander b, brooker s (2007) cutaneous leishmaniasis. the lancet infec dis. 7(9): 581–596. strelkova mv (1996) progress in studies on central asian foci of zoonotic cutaneous. folia parasitologica. 43: 1–6. theodor o, mesghali a (1964) on the phlebotomine of iran. j med entomol. 1: 285–300. who (2010) control of the leishmaniases: report of a meeting of the who expert commitee on the control of leishmaniases, geneva, 22–26 march 2010. j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 51 http://jad.tums.ac.ir published online: march 31, 2023 original article genetic structure of aedes (stegomyia) albopictus populations in russia *elena shaikevich1, ludmila karan2, marina fedorova2 1vavilov institute of general genetics, gubkin str. 3, 119333 moscow, russia 2central research institute of epidemiology, novogireevskaya str., 3a, 111123 moscow, russia *corresponding author: dr elena shaikevich, e-mail: elenashaikevich@mail.ru (received 23 nov 2021; accepted 31 jan 2023) abstract background: aedes (stegomyia) albopictus was found for the first time in 2011 on the black sea coast in russia, and during 2011–2019, the species expanded over two climate zones cfa and csa. methods: here, we studied the sequence diversity of the mitochondrial cytochrome c oxidase i (coi) gene, 1317–1433bp in length. in total, 131 specimens of ae. albopictus sampled from 21 locations in russia and abkhazia were examined. results: two of the six identified mitochondrial haplotypes were detected for the first time. four coi haplotypes were shared by at least two studied local populations. the most prevalent h1 and h2 haplotypes dominated in all the sampled localities in the cfa zone. the h3 haplotype was prevalent in the csa zone. other haplotypes were rare. phylogenetic analyses, spatial isolation and limited gene flow revealed that the samples from the csa zone differed significantly from those from the cfa zone. conclusion: two spatially isolated genetic lineages exist in ae. albopictus population in southern region of russia. one lineage obtained on the seacoast and inland (in valleys of the caucasus mountains and steppe zone) is widely distributed worldwide including mediterranean populations. this confirms the hypothesis that the emergence of ae. albopictus population in southern region of russia may be associated with the terrestrial spread of mosquitoes from the wellestablished european population due to human activity. the other lineage, discovered in novorossiysk, a maritime port, is similar to ae. albopictus from the usa and japan, suggesting the independent introduction of these mosquitoes. keywords: aedes albopictus; distribution; population genetics; coi; mtdna introduction aedes (stegomyia) albopictus (skuse, 1895) is recognized as a competent vector of arboviruses, including dengue, chikungunya, zika and yellow fever viruses (1). being initially native to restricted tropical regions of southeast asia, ae. albopictus spread widely during the last 30 years in tropical and subtropical climates owing to its ecological features and human activity (2). in europe, established populations of ae. albopictus were recorded for the first time in albania (1979) and italy (1991), but currently, the species is reported to occur in countries of the mediterranean basin, balkans and central europe, including the czech republic, and germany (3–5). aedes albopictus introduction and expansion led to epidemics of chikungunya fever in italy and france and to autochthonous cases of dengue in france, croatia and spain (6–9). on the black sea coast ae. albopictus was first observed in 2011 in bulgaria, turkey, georgia, abkhazia and russia, and the following year was found in romania (4, 10–12). in russia, it was originally recorded in only one location, adler, near the abkhazia border, but in subsequent years, ae. albopictus spread along the coast, being discovered in 2015 in gelendzhik and in 2016 in novorossiysk (13–14). monitoring of the species range showed that during the period 2011–2019, ae. albopictus expanded along the coast for 400km and inland for 200km, establishing breeding populations in valleys of the caucasus mountains and in the steppe zone of the krasnodar krai (15). the widespread distribution of ae. albopic copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 52 http://jad.tums.ac.ir published online: march 31, 2023 tus in southern russia enhances the risk of autochthonous transmission of dengue, chikungunya and zika viruses in a large area on the black sea coast, especially in summer, when many tourists from different countries visit the region (16). such scenarios of disease outbreak occurred in europe and in the usa (florida) (6). the epidemic potential of ae. albopictus depends on the mosquito population’s vector competence and vectorial capacity, which are thought to vary in invading populations according to their genetic background and geographic origin (17, 18). hence, information on the genetic diversity of invasive populations is important for survey of ae. albopictus populations and disease risk assessment. currently, both nuclear and mitochondrial markers are used to analyse the genetic structure and origin of invasive ae. albopictus populations (19). the application of a nuclear marker, the its2 region of rrna genes, displayed a low level of differentiation between geographical populations, indicating that highly related sequences were distributed across native and invasive populations (19, 20). analyses of microsatellites and a panel of genome-wide distributed single nucleotide polymorphisms (snps) showed that low levels of intra-population differentiation were caused by multiple re-introductions into invaded zones, which led to the formation of local populations consisting of individuals with different origins (21–23). in europe, a high admixture and lack of geographical structure was found based on its2 and snps (20, 22). this result suggests a long-distance human-aided dispersal of ae. albopictus in europe (22). mitochondrial dna genes are often used as genetic markers to test biodiversity, ancestry and demographic changes in populations due to their uniparental (maternal) inheritance and lack of recombination (24). the cytochrome c oxidase i (coi) gene in ae. albopictus has been widely analysed not only for species identification as a barcode tool but also through phylogenetic and population studies (25–29). a short barcode fragment of 450–650bp was used to confirm discovery of ae. albopictus in turkey (10), countries of the balkan peninsula (4, 30, 31). the limited sequence length and number of individuals analysed did not reveal a high level of diversity. at the same time, it is shown that the long fragment (circa 1400bp) of coi gene can be used to analyse the population structure of ae. albopictus. a high level of polymorphism was detected among long coi sequences of ae. albopictus sampled in native areas in south asian countries (25, 26, 32–34). more than a thousand published ae. albopictus coi sequences reflects the genetic diversity of populations both in native and non-native invasive areas, highlighting the reliability of the long coi fragment to identify genetic divergence among ae. albopictus populations and facilitating the study of phylogeographic relationships and the probable origin of individuals in recently established populations (25, 27–29, 34). our aim was to study the genetic diversity and population structure of ae. albopictus in the krasnodar krai, russia, and in abkhazia. two hypotheses regarding the structure of ae. albopictus populations can be assumed. the simultaneous occurrence of the vector in the eastern black sea countries suggests a common single source of origin, from which the mosquitoes spread locally by different modes of transport. in this case, the population genetic diversity should be low, because the population has existed for less than 10 years, since 2011 (12). on the other hand, the possibility of independent importation by sea cannot be excluded, as both russia and abkhazia have ports on the black sea and then subpopulations and high genetic diversity can be expected due to multiple importations and mixing of individuals in local populations. to test these assumptions, we sampled mosquitoes in krasnodar krai, russia, and abkhazia in 2018 and sequenced a long fragment of the coi gene. we compared ae. albopictus coi haplotype composition between local populations across the studied area and with the haplotypes known for other endemic and non-endemic territories. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 53 http://jad.tums.ac.ir published online: march 31, 2023 materials and methods study area our study sites covered an area from 45° 13’n to 43°10’ n and from 38°59’e to 40°20’ e (fig. 1, table 1). according to the köppengeiger classification, most of this area lies in the cfa climate zone, defined as temperate (c) climate without dry season (f) and with a warm summer (a) (35). the caucasus mountains cross the cfa zone and form a watershed that divides the zone into three climate subzones. the climate of seacoast of abkhazia and the part of seacoast of russia is close to subtropical climate with an average january temperature of > 6 °c and annual rainfall above 1400mm and denoted as the subzone cfa-1 (table 1). the valleys located on the western and northern slopes of the mountains are characterized by a mild temperate climate (cfa-2) with an average january temperature of > 2.0 °c and annual rainfall of approximately 1000mm. the climate of the zakubanskaya plain located to the north and northwest of the caucasus mountains is mild continental (cfa-3), with average january temperatures of > 0.0 °c and approximately 600– 700mm of precipitation per year. to the west of the caucasus mountains, in novorossiysk and its surroundings, the climate is close to the dry mediterranean type (csa), with precipitation of the driest month in summer < 40mm. mosquito sampling mosquitoes were sampled at 17 sites in krasnodar krai, russia, and three sites in abkhazia in 2018 (fig. 1). the collection points were at a sufficient distance to avoid collecting siblings. the sampling sites were combined into seven groups on the basis of territory (table 1). the collections were performed in augustseptember, when the population density of mosquitoes was expected to be the highest (36). adult females were collected with aspirators and the human landing catch approach. for the collection of mosquito larvae, we inspected containers with water in cemeteries, used tires and plastic containers in yards or along roadsides, buckets and other types of containers. mosquito larvae and pupae were harvested, brought to a lab, reared to adults and identified by morphological identification keys (37). dna extraction, amplification, and sequencing each mosquito was homogenized in 300µl of saline buffer. the homogenate was centrifuged at 800g for 2min, and the supernatant was retained. total dna was extracted from 100µl of supernatant using the riboprep kit (amplisense, moscow, russia) according to the manufacturer’s instructions. pcr was carried out in a 25μl volume with an encyclo plus pcr kit (evrogene, moscow, russia). the coi gene fragments were amplified using primers 1454f and 2160r and 2027f and 2886r according to the original protocol (25). the amplification products were sequenced with forward and reverse primers for each primers pair. sequencing of amplicons was performed using an abi prism 310 sequencer and a bigdye termination kit as recommended by applied biosystems (united states). the sequences were edited manually with chromas and then aligned using mafft v7 (https://mafft.cbrc.jp). the total length of the coi gene sequences was 1317– 1433bp. the nucleotide sequences were deposited in genbank under accession numbers mz 501500-mz501561. sequences mh817490– mh817558 from our preliminary study (38) were also included in the analysis. a total 131 sequences from individual mosquitoes were used for the genetic analysis. analysis of mtdna data the length of the aligned analysed sequences was 1317bp. dna polymorphism was evaluated using dnasp v5 (39). dnasp software was used also to evaluate the values of number of migrants (nm), level of population differentiation (fst) and neutrality tests of tajima d and fu’s fs to assess the genetic differentiation among the populations and deviations from selective neutrality. a chi-squared test was used to show the significance of differences in the http://jad.tums.ac.ir/ https://www.multitran.com/m.exe?s=republic%20of%20abkhazia&l1=1&l2=2 j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 54 http://jad.tums.ac.ir published online: march 31, 2023 haplotype distributions. a blast search was performed against genbank sequences to identify genetically related published haplotypes (fig. 2). genealogical relationships among the haplotypes were constructed using a median joining (mj) network of coi haplotypes generated in network ver. 4.6 (40) with a 95% probability. to reveal the evolutionary relationships between individuals from the russian populations and from known established populations in europe, the new world and asia, our data were combined with previously published coi data: 6 haplotype sequences found in this study plus 26 genetically related sequences from genbank were used in this analysis (fig. 2). results the cytochrome c oxidase i gene diversity a 1317bp fragment of the coi gene was sequenced from the 131 mosquitos, 10–34 specimens for each of 7 localities (table 1). the likelihood of amplification of the pseudogene was rejected as no insertions, deletions or stop codons were found in any of the samples. eight polymorphic nucleotide sites and six haplotypes (h) were detected, and two haplotypes were unique (table 2). data on the nucleotide diversity (pi), the average number of nucleotide differences (k) and the observed haplotype diversity (hd) are shown in table 3. the highest values of hd (0.757) and pi (0.00182) were found in the csa zone (table 3). no obvious departure from neutrality was found in the investigated populations (table 3), although tajima’s d statistic values were slightly positive (0.89987, p> 0.10) for the local population from the csa zone and slightly negative for the local population from the cfa-2 zone (-1.21122, p> 0.10) (table 3). tajima’s d and fu’s fs test values for all populations were not significant. among the six ae. albopictus coi haplotypes discovered in our study, four were previously known in ae. albopictus populations worldwide. the coi haplotypes designated in our work as h1, h2 and h5 correspond to h03, h17 and h54, respectively (25). haplotype h3 is identical to the 26_j-wa1 (41) and h79 (27). two haplotypes, h4 and h6, are described for the first time. thus, our results show that ae. albopictus mosquitos collected in russia in 2018 are related to populations involved in the worldwide expansion of this species through temperate regions. population structure and differentiation four out of six coi haplotypes were shared by at least two local populations (table 1). in the cfa zone, haplotype h1 dominated in all the sampled localities, with the highest frequency recorded in the cfa-3 subzone (85%, 29/34), followed by cfa-2 (73%, 32/44) and cfa-1 (68%, 19/28). in the csa zone, only 20% of individuals had haplotype h1, which was significantly lower than in the cfa zone (χ2= 7.5902, p< 0.05). no significant differences were observed in the distribution of haplotype h2, which was detected in 19% of individuals. the h3 haplotype was found in 10.7% (14/131) of individuals, of which 71.4% were collected in novorossiysk and its surroundings, i.e. in the csa zone. the difference in the distribution of the h3 haplotype in the csa zone compared with that in the cfa zone was significant (χ2= 15.6415, p< 0.01). the remaining haplotypes were rare (table 1). five haplotypes, h1-h5, were detected in ae. albopictus sampled along the seacoast both in cfa-1 and csa zones, and only two haplotypes, h1 and h2, were found in individuals sampled in the steppe zone of krasnodar krai (cfa-3). no genetic differentiation (fst) was observed between the samples collected in the three cfa climatic subzones. the migration (nm) values between the cfa-1, cfa-2 and cfa-3 local populations indicate frequent gene exchange between these populations (table 4). the samples from the csa zone differed significantly from those from the cfa zone (table 4). phylogenetic analysis the network of mitochondrial haplotypes was generated from six coi haplotypes obtained http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 55 http://jad.tums.ac.ir published online: march 31, 2023 in our study and 26 identical, close related (differed by one nucleotide substitution) and connecting sequences from genbank (fig. 2). the h1 haplotype is the most widely distributed in russia and identical sequence was found in asia (japan, taiwan, and china), america (the usa and canada) and europe (albania, italy, greece and portugal). the sequence identical to h2 was also reported from china, taiwan, the usa, canada and italy. the h3 was found in japan and in the usa, whereas the h5 was revealed in the usa. the unique h4 and h6 haplotypes are connected by one mutation with h3 and h5, respectively. a total 32 coi haplotype sequences form two clusters in the network diagram. the first cluster includes the haplotypes h1 and h2 distributed in europe and in other continents. the second cluster contains h3-h6 and coi haplotypes found mainly in the northeastern united states and canada. the populations of italy are the most studied in terms of the genetic diversity of ae. albopictus, but no individuals with h3-h6 haplotypes were found here. blast search based on the short and widely studied 450bp 5' coi gene fragment revealed that haplotypes h1 and h2 are distributed globally, including local european populations in italy, albania, romania, portugal, serbia, greece, turkey and abkhazia (genbank acc. numb. jf 810659, hf912379, hq906848, mg198595600, jq 412504-6, ln808745-46, mk518354, mk 995313). this short fragment of coi sequences, barcode fragment, is considered insufficient for phylogenetic analysis of ae. albopictus, nevertheless haplotypes h3-h6 are quite different from h1 and h2 in this 5' gene fragment as well. these nucleotide and amino acid differences are shown in table 2 including variable nucleotide positions in sites 72, 103, 110 and 276. table 1. collection information and distribution of aedes albopictus mtdna coi gene haplotypes in russia and abkhazia localities climate zone/ subzone mean january temp. (t°c) annual rainfall (mm) no. of studied specimens no. of specimens with haplotypes: h1 h2 h3 h4 h5 h6 1 abkhazia cfa-1 ≥ 6,0 1200-1400 12 7 5 2 sochi cfa-1 ≥ 6,0 1200-1400 16 12 1 3 3 mountains-1 cfa-2 ≥ 2,0 1200-1000 10 8 2 4 mountains-2 cfa-2 ≥ 2,0 800-1000 34 24 6 1 1 2 5 krasnodar cfa-3 ≥ 0,0 600-800 21 19 2 6 adygeya cfa-3 ≥ 0,0 600-800 13 10 3 7 novorossiysk csa ≥ 2,0 400-600 25 5 6 10 1 3 total 131 85 25 14 1 4 2 table 2. variable sites of the aedes albopictus coi gene sequences haplotype no. of individuals variable nucleotide sites* аа** 72 103 110 276 732 861 888 1002 35 37 h1 85 t a t t a c t t i i h2 25 . . . . . t . . . . h3 14 . g . . g t . c v . h4 1 . g . . g t c c v . h5 4 . . c c . t . . . t h6 2 c . c c . t . . . t *dots denote identity with the reference sequence (as in zhong et al. 2013, genbank accession no. jq004525), and the base letters denote substitutions; **variable amino acid sites http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 56 http://jad.tums.ac.ir published online: march 31, 2023 table 3. estimates of genetic diversity for the mtdna coi region for populations of aedes albopictus climate zone / subzone no. s н hd (±sd) к pi (±sd) tajima’s d fu’s fs cfa-1 28 4 3 0. 5 (0.091) 1.048 0.0008 (0.00024) 0.05021 1.674 cfa-2 44 7 5 0.445 (0.081) 0.891 0.00068 (0.00019) -1.21122 -0.548 cfa-3 34 1 2 0.258 (0.086) 0.258 0.0002 (0.00019) 0.08512 0.555 csa 25 7 5 0,757 (0.051) 2,393 0.00182 (0,00016) 0.89987 1.609 total 131 8 6 0.534 (0.043) 1.294 0.00098 (0.00013) -0.27687 0.706 no.number of tested specimens, snumber of polymorphic sites, нnumber of haplotypes, hdhaplotype diversity, кaverage number of nucleotide differences, pinucleotide diversity (pijc), tajima's d and fu's fs statistics neutrality tests, (not significant, p> 0.10) fig. 1. map of the study area and sample sites. isohytes show annual rainfall (mm), mjt mean january temperature (t°c). the location of the region on the world map (below) is shown with a red circle http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 57 http://jad.tums.ac.ir published online: march 31, 2023 table 4. pairwise differentiation (fst, below the diagonal), and gene flow (nm, above the diagonal) among populations of aedes albopictus from different climatic zones/subzones climate zone / subzone fst (white) and nm (gray) estimates cfa-1 cfa-2 cfa-3 csa cfa-1 38.59 3.86 0.82 cfa-2 0.00644 9.72 0.55 cfa-3 0.06088 0.02507 0.34 csa 0.23388 ** 0.31392 *** 0.42646 *** the significance of differences: ns not significant; *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p< 0.001. estimates of fst: csa-cfa-1 chi2= 15,817, p-value of chi2: 0,0033 (p< 0.01) (df= 4); csa-cfa2 chi2= 28,263, p-value of chi2: 0,0000 (p< 0.001) (df= 5); csa-cfa3 chi2= 66, p-value of chi2: 0,0000 (p< 0.001) (df= 4) fig. 2. the network diagram of the aedes albopictus coi haplotypes obtained in this study and published data (25, 27, 28, 41). colors correspond to different regions discussion a high degree of genetic variability within and between populations of ae. albopictus is usually observed in endemic areas. thirty-five coi haplotypes of ae. albopictus were detected in the south asian region, 33 in malaysia, 42 in china, and 44 in lao pdr (25, 26, 32, 34). in non-endemic areas where ae. albopictus populations have recent origin and pass the bootleneck, the genetic diversity is low. in europe, 11 coi haplotypes were found in italy (25). five coi haplotypes were found in portugal, being confirmed by subsequent analyses based on ae. albopictus mitogenomes (28). in studied populations, that spread along the black sea coast of the caucasus, the genetic variability is also low that indicates its recent expansion. no significant differentiation was found among populations in cfa zone despite the climatic differences between cfa-1, cfa-2 and cfa3 subzones. the values of gene flow (nm) and http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 58 http://jad.tums.ac.ir published online: march 31, 2023 genetic differentiation (fst) tests indicate the presence of a panmictic population in the cfa zone (table 4). our conclusions are also supported by the results of the analysis of the genomes of individuals from sochi and krasnodar cities (cfa zone), which showed that despite significant differences in the allele frequency spectrum, ae. albopictus from sochi and krasnodar were closely related, had a common origin and formed a clade related to mosquitoes from italy and greece (42). the lack of genetic structure or isolation by distance were observed in many studies on ae. albopictus populations worldwide independently of whether the populations are native or invasive. these data indicate ongoing and frequent gene flow among populations and seem to be due to a combination of low natural dispersion capabilities and a high level of humanmediated spread (19). haplotypes h1 and h2 dominated in all three cfa subzones (table 1) and these haplotypes are also wide distributed both in the native regions of ae. albopictus (taiwan and china) and in the areas of introduction (the usa, canada, and europe) (25, 27, 41). these results suggest two possibilities for the origin of the russian ae. albopictus populations in the cfa zone. first, mosquitoes might have been introduced through seaports located on the black sea coast, such as sochi, russia and batumi, georgia from areas where ae. albopictus with coi haplotypes h1 and h2 are widely distributed, for example, from southeast asia, china or the usa. maritime transportation of tires and ornamental plants is known as the main dispersal mechanism of ae. albopictus (43). on the other hand, we cannot completely rule out the possibility of european origin of ae. albopictus сfa population. in countries of the black sea basin the mosquito was recorded first in 2005 in greece, then in 2011 in turkey, georgia and abkhazia suggesting its rapid and successive dispersion along the eastern black sea coast. the previous genetic studies confirmed the identity of short fragments of ae. albopictus coi sequences, 450bp barcording fragment, from greece, turkey, serbia, romania, and abkhazia (4, 10, 30, 31, 44). the 1317 bp fragments, identical to our h1 and h2 sequences, were also detected in ae. albopictus from albania, greece and italy (25, 41). thus, the emergence of the russian population can be associated with the terrestrial spread of mosquitoes due to human activity. this mechanism of ae. albopictus dispersion has been observed in france and spain and was suggested for romania (4). if the mosquitoes of the russian population are of european origin, their vector competence to dengue, chickungunya and zika viruses may be high enough for local transmission in the case of the occurrence of infected people as it took place in italy, france and other mediterranean countries (6). our results show the presence of spatial isolation and limited gene flow between populations from cfa and csa climatic zones. in csa zone the haplotype diversity is twice as high as in the cfa subzones with h3 being the most common haplotype, which is currently only found in the usa, ohaio (27) and japan, wakayama (41). haplotype h5 is also found in novorossiysk and solely in usa, new york (25). these data indicate the presence of genetic structure in russian population. the coi-based population structure has been found in portugal, china, and laos and is usually explained by climatic factors. though the presence of a genetic structure in our population can also be associated with adaptation to the dry mediterranean climate of novorossiysk, this explanation seems unlikely. the history of introduction of ae. albopictus to russia has only about 10 years, and in novorossiysk mosquitoes were recorded only in 2016. this time is not enough to accumulate adaptive mutations which is known to take a long time to occur. the introduced populations are more likely to establish if they already possess alleles advantageous in the invaded environment (45). in europe, the spread of the mosquito began after the importation in the 1990s in italy, which became the source for the rest of the european populations (46). in subsehttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 51–62 e shaikevich et al.: genetic structure of … 59 http://jad.tums.ac.ir published online: march 31, 2023 quent period, the mosquito has spread widely in the areas with a dry mediterranean climate, but haplotypes h3 and h5 distributed in novorossiysk population were found neither in neighbouring countries (turkey and greece) no in europe in general. presumably, these haplotypes are rare and either have not been introduced in europe or have not a selective advantage over other haplotypes in the specified climate. this suggests the independent introduction of ae. albopictus to novorossiysk, the largest seaport in southern russia. conclusion the key finding of this work is the existence of two spatially isolated genetic lineages in ae. albopictus population in southern region of russia. phylogenic analysis indicated that one of the lineages is similar to ae. albopictus from usa and japan whereas the second lineage is related to ae. albopictus from mediterranean countries. our data for the first time confirm the hypothesis that the emergence of ae. albopictus population in southern region of russia may be associated with the terrestrial spread of mosquitoes from the well-established european population due to human activity. this finding is important for predicting the spread of invasive ae. albopictus mosquitoes and the subsequent distribution of the medically important arboviruses that they transmit. further research is needed to confirm this hypothesis. in the future, we plan to clarify the origin of the russian populations 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https://www.cabdirect.org/globalhealth/search/?q=au%3a%22medyanik%2c+i.+m.%22 https://www.cabdirect.org/globalhealth/search/?q=au%3a%22shaikevich%2c+e.+v.%22 https://www.cabdirect.org/globalhealth/search/?q=do%3a%22parazitologiya%22 j arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 192 http://jad.tums.ac.ir published online: january 05, 2016 original article species abundance distribution of ectoparasites on norway rats (rattus norvegicus) from a localized area in southwest china *xian guo guo 1, wen ge dong 1, xing yuan men 2, ti jun qian 1, dian wu 1, tian guang ren 1, feng qin 1, wen yu song 1, zhi hua yang 1, quinn e fletcher 3 1vector laboratory, institute of pathogens and vectors, dali university (branch of yunnan provincial key laboratory for zoonosis control and prevention), dali, yunnan, china 2institute of plant protection, shandong academy of agricultural sciences, jinan, china 3département de biologie, chimie et géographie, université du québec à rimouski, rimouski, canada (received 28 july 2013; accepted 7 sep 2014) abstract background: the species of ectoparasites that live on a specific host in a geographical region form an ectoparasite community. species abundance distributions describe the number of individuals observed for each different species that is encountered within a community. based on properties of the species abundance distribution, the expected total number of species present in the community can be estimated. methods: preston’s lognormal distribution model was used to fit the expected species abundance distribution curve. using the expected species abundance distribution curve, we estimated the total number of expected parasite species present and the amount of species that were likely missed by our sampling in the field. results: in total, 8040 ectoparasites (fleas, sucking lice, gamasid mites and chigger mites) were collected from 431 norway rats (rattus norvegicus) from a localized area in southwest china. these ectoparasites were identified to be 47 species from 26 genera in 10 families. the majority of ectoparasite species were chigger mites (family trombiculidae) while the majority of individuals were sucking lice in the family polyplacidae. the expected species abundance distribution curve demonstrated the classic pattern that the majority of ectoparasite species were rare and that there were a few common species. the total expected number of ectoparasite species on r. norvegicus was estimated to be 85 species, and 38 species were likely missed by our sampling in the field. conclusions: norway rats harbor a large suite of ectoparasites. future field investigations should sample large numbers of host individuals to assess ectoparasite populations. keywords: ectoparasite, species abundance distribution, expected species estimation, norway rat, rattus norvegicus introduction the norway rat (also called the brown rat), rattus norvegicus (berkenhout 1976), is a dominant rodent species in residential areas and farmlands in many places throughout the world. in addition to destroying crops, the norway rat is also an important reservoir host of many zoonoses (yu and xu 1988, huang et al. 1995). norway rats harbor a suite of ectoparasites including fleas and sucking lice as well as chigger and gamasid mites that act as important vectors for zoonoses and vector-borne diseases. specifically, fleas are known to be vectors of the plague, murine typhus (endemic typhus), and fleaborne spotted fever. sucking lice (for example the human louse, pediculus humanus) are vectors of epidemic typhus, epidemic relapsing fever, and trench fever. gamasid mites may play a potential role in transmitting hemorrhagic fever with renal syndrome (hfrs, also called epidemic hemorrhagic fever, ehf) and chigger mites are the vectors of scrub typhus, or tsutsugamushi disease (deng et al. 1993, li et al. 1997, jin 1999, song 1999, brouqui and raoult 2006, wang and ye 2006, wu 2007, bitama et al. 2010). the species of ectoparasites that live on a specific host in a geographical region form *corresponding author: dr xian-guo guo, email: xianguoguo@yahoo.com j arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 193 http://jad.tums.ac.ir published online: january 05, 2016 an ectoparasite community. species abundance distributions describe how the abundance of a species changes over space. by using the species abundance distributions of ectoparasites sampled in the field, the expected number of species in a community can be estimated (guo 1999). it is often necessary to use this estimation procedure because it is impossible to collect all species that are present at all sites within a geographical region (guo 1999). the species abundance distributions of ectoparasitic gamasid and chigger mites living on small mammals including rodents have been quantified over a large geographical region in yunnan province of china (guo 1999, guo et al. 2006). this paper examines the species abundance distributions of ectoparasites (fleas, sucking lice, gamasid mites and chigger mites) on norway rats (r. norvegicus) in the area surrounding erhai lake, in dali prefecture of yunnan province, southwest china. in addition to quantifying the species abundance distribution of these ectoparasites, the present study also aims to estimate how many ectoparasite species norway rats (a single rodent species) harbor in this small area (only about 450 km2). materials and methods collection and identification of norway rats and ectoparasites the study site included the area surrounding erhai lake of dali (100005′e100017′e, 25036′n-25058′n). the total study area covered approximately 450 km2 at an altitude of approximately 1976 m. norway rats were captured using mouse traps (guixi mousetrap apparatus factory, guixi, jiangxi, china) at three sampling sites (xiaguan, wase and xizhou) in 2003 and 2004. mouse traps were set in residential areas (houses, barns, stables, etc.) and farmlands (paddy fields, corn fields and other crop lands) in the evening and then checked the following morning. trapped rats were placed in cloth bags and brought to the laboratory. all the rats were identified according to their morphological features (huang et al. 1995, kia et al. 2009, huang et al. 2013). at the laboratory, ectoparasites were collected and preserved in vials containing 70% ethanol (kia et al. 2009, zuo and guo 2011, zuo et al. 2011, huang et al. 2013, zhan et al. 2013). chigger mites were mainly collected from the auricles and external auditory canals of the host ears using a curette and a lancet (li et al. 1997, guo et al. 2006). all the ectoparasites were dehydrated, made them transparent, mounted on glass slides, and identified to species under microscopes (deng et al. 1993, li et al. 1997, jin 1999, wu 2007, kia et al. 2009, huang et al. 2013). voucher ectoparasite species, together with specimens of rats are deposited in the specimen repository at the institute of pathogens and vectors, dali university. basic statistical analysis we calculated the constituent ratios (cr), as well as the prevalence (p), mean abundance (ma) and mean intensity (mi) of each ectoparasite species (ritzi and whitaker 2003, storm and ritzi 2008, huang et al. 2013, zhan et al. 2013). description of species abundance distribution species abundance distributions describe the number of individuals observed for each different species that is encountered within a community. we examined the species abundance distribution using the methodology provided in preston 1948. in a semi-logarithmic rectangular coordinate system, the x-axis indicating the ectoparasite individuals was marked with log intervals based on log3m (table 4 in "results") and the y-axis was an arithmetic scale, indicating the ectoparasite species. preston’s lognormal distrij arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 194 http://jad.tums.ac.ir published online: january 05, 2016 bution model was used to fit the expected species abundance distribution curve (preston 1948, greig-smith 1983, baltanas 1992, guo 1999, guo et al. 2006) using the following equation. 2 0 )]([ 0 ^ )( rraesrs  where )( ^ rs is the expected number of species within the r-th log interval, r0 is the modal log interval (the log interval with the most species), s0 is the number of species within the r0 log interval (the modal log interval). the value of α (spread constant) was determined by calculating the coefficient of determination (r2) using the following formula (ni 1990, guo et al. 2006).         m r m r rsrs rsrs r 0 2 0 2 ^ 2 )]()([ )]()([ 1 ,     m r rs m rs 0 )( 1 )( where s(r) is the observed number of ectoparasite species in r-th log interval, m is the number of log intervals, and )( ^ rs and r are the same as presented above. total expected species estimation based on the species abundance distribution, the expected number of ectoparasite species (st) and the number of ectoparasite species likely missed in the field sampling (sm) were approximated following preston (1948) (see also greig-smith 1983, baltanas 1992, guo 1999) using the following formulas. asst /)( 0  , atm sss  sa represents the observed number of ectoparasite species collected in the field. results ectoparasites present on norway rats in total, 8040 ectoparasites (fleas, sucking lice, gamasid mites and chigger mites) were collected from 431 norway rats in a localized area (the area surrounding erhai lake of dali), yunnan province, southwest china. the collected ectoparasites were identified as comprising 47 different species from 26 genera in 10 families. of these ectoparasites, 857 fleas were identified to be 6 species from 6 genera in 3 families, 4307 sucking lice were identified to be 2 species from 2 genera in 2 families, 2531 gamasid mites were identified to be 16 species from 10 genera in 4 families, and 345 chigger mites were identified to be 23 species from 8 genera in 1 family (table 1). in total, 71.2 % of the rats were infested by ectoparasites with a mean abundance of 18.7 parasites/per examined host and a mean intensity of 26.2 parasites/per infested host. of the 4 ectoparasite taxa (fleas, sucking lice, chigger mites and gamasid mites), the majority of ectoparasite species were chigger mites, which accounted for 48.9 % of the ectoparasite species diversity. the majority of ectoparasite individuals, however, were sucking lice, which accounted for 53.6 % of the total number of parasite individuals (table 2). overall, we collected a suite of potential zoonoses vectors, including 5 species of fleas that are potential vectors of the plague and murine typhus, 6 species of gamasid mites that are potential vectors of hfrs, and 5 species of chigger mites that are potential vectors of scrub typhus (table 1). species abundance distribution of ectoparasites from norway rats (a single rodent species) in the area surrounding erhai lake of dali, the collected individuals of ectoparasites were greatly different among 47 different species of the parasites. most ectoparasite species were rare with few individuals collected and only a few parasite species were really common with abundant individuals on the rat. table 3 showed the relationship between the individuals and species of j arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 195 http://jad.tums.ac.ir published online: january 05, 2016 ectoparasites on norway rats (r. norvegicus). the species abundance distribution of ectoparasites on norway rats was fitted by preston’s lognormal distribution model (r2= 0.80). the expected curve (theoretical curve) of the species abundance distribution showed a gradually descending tendency from the rare parasite species to the dominant parasite species. the expected curve of the species abundance distribution indicated that the majority of the parasite species were rare and that fewer species were abundant (table 4, fig. 1). the equation of the expected curve equation (theoretical curve equation) was 2)27.0( ^ 13)( rers  (s0 = 13, r0 = 0, α = 0.27). total expected species estimation of ectoparasites based on asst /)( 0  and sm =st sa (preston’s method), the total expected species of ectoparasites on r. norvegicus was estimated to be 85 species. this calculation suggests that 38 species of ectoparasites were missed by our sampling. table 1. collection details of ectoparasite species, grouped by families, collected from norway rats (rattus norvegicus) in the area surrounding erhai lake of dali, yunnan province, southwest china taxonomic taxa of ectoparasites collected species and individuals 1. fleas pulicidae xenopsylla cheopis* (90) leptopsyllidae leptopsylla segnis* (182), frontopsylla spadix* (190), paradoxopsyllus custodis* (359) ceratophyllidae monopsyllus anisus* (35), macrostylophora euteles (1) 2. sucking lice hoplopleuridae hoplopleura pacifica (118) polyplacidae polyplax spinulosa (4,189) 3. gamasid mites laelapidae laelaps echidninus* (380), l. nuttalli (823), l. xingyiensis (4), l. guizhouensis (3), l. algericus (1), l. chini (1), l. turkestanicus* (1), haemolaelaps ( androlaelaps) casalis* (1), androlaelaps singularis (6), hypoaspis pavlovskii (4), haemogamasus pontiger* (5), eulaelaps stabularis (1), echinonyssus (hirstionyssus) sunci* (61) macronyssidae ornithonyssus bacoti* (1226) aceocejidae proctolaelaps pygmaeus (13) parasitidae parasitidae sp. ( unidentified genus and species) (1) 4. chigger mites trombiculidae leptotrombidium rusticum (3), l. shuqui (21), l. bambicola (1), l. shanghaense (1), l. insulare* (6), l. scutellare* (5), l. jinmai (3), l. sinicum (12), l. yui*(10), l. kitasatoi (1), l. bengbuense (48), l. taishanicum (2), l. akamushi* (36), leptotrombidium sp.(25), ascoschoengastia indica* (2), a. rattinorvegici (127), a. leechi (1), helenicula simena (24), walchia koi (5), herpetacarus hastoclavus (7); trombiculindus yunnanus (1), gahrliepia madun (3), cheladonta micheneri (1) annotation: (1) the species marked “*” are potential vectors of the plague, murine typhus (endemic typhus), hemorrhagic fever with renal syndrome (hfrs; also called epidemic hemorrhagic fever, ehf in china) and scrub typhus (tsutsugamushi disease). (2) the numbers given in brackets reflect the number of individuals collected for each ectoparasite species. j arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 196 http://jad.tums.ac.ir published online: january 05, 2016 table 2. the constituent ratios (cr), prevalence (p), mean abundance (ma) and mean intensity (mi) of the 4 taxa of ectoparasites on norway rats (rattus norvegicus) in the area surrounding erhai lake of dali, yunnan province, southwest china ectoparasite taxa species and constituent ratios (cr) parasite individuals and constituent ratios (cr) infestation prevalence (p) mean abundance (ma) mean intensity (mi) species cr (%) individuals cr (%) infested rats p (%) ma mi fleas 6 12.8 857 10.7 143 33.2 1.9 5.9 sucking lice 2 4.3 4307 53.6 127 29.5 9.9 33.9 gamasid mites 16 34.0 2531 31.5 190 44.1 5.9 13.3 chigger mites 23 48.9 345 4.3 32 7.4 0.8 10.8 all insects (fleas and sucking lice) 8 17.0 5164 64.2 221 51.3 11.9 23.4 all mites (chigger and gamasid mites) 39 82.9 2879 35.8 207 48.0 6.7 13.9 total ectoparasites 47 100.0 8040 100.0 307 71.2 18.7 26.2 table 3. the relationship between the individuals and species of ectoparasites on norway rats (rattus norvegicus) in the area surrounding erhai lake of dali, yunnan province, southwest china number of individuals number of ectoparasite species number of individuals number of ectoparasite species number of individuals number of ectoparasite species number of individuals number of ectoparasite species 1 13 10 1 36 1 190 1 2 2 12 1 48 1 359 1 3 4 13 1 61 1 380 1 4 2 21 1 90 1 823 1 5 3 24 1 118 1 1226 1 6 2 25 1 127 1 4189 1 7 1 35 1 182 1 table 4. the statistical parameters for fitting the expected curve of species abundance distribution of ectoparasites on norway rats (rattus norvegicus) in the area surrounding erhai lake of dali, yunnan province, southwest china log intervals (r) individual ranges in each log interval (i) midpoint values of each individual range (m) observed parasite species expected parasite species 0 1 1 13 13.00 1 2–4 3 8 12.09 2 5–13 9 9 9.71 3 14–40 27 5 6.75 4 41–121 81 4 4.05 5 122–364 243 4 2.10 6 365–1093 729 2 0.94 7 1094–3280 2187 1 0.37 8 3281–9841 6561 1 0.12 annotation: m is the midpoint of i, 3r =m, log3m =r. α = 0.27, r2 = 0.80. j arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 197 http://jad.tums.ac.ir published online: january 05, 2016 fig. 1. the species abundance distribution of ectoparasites on norway rats (rattus norvegicus) fitted by preston’s lognormal distribution model with the expected curve equation of 2)27.0( ^ 13)( rers  (s0 = 13, r0 = 0, α = 0.27, r 2 = 0.80 discussion infestation of ectoparasites on norway rats ectoparasites are parasites that live on the skin or body surface of their hosts, and include a variety of taxonomic taxa, including fleas, sucking lice, chewing lice, batflies, ticks, chigger mites, gamasid mites, itch mites (mange mites, scabies mites), demodex mites (vermiform mites, follicle mites), feather mites, fur mites (cheyletiellid mites) and even some occasional arthropods that are parasitic on skin (e.g. some maggots) (wilson and durden 2003, changbunjong et al. 2010, zhan et al. 2013). this paper examined 4 taxa (fleas, sucking lice, gamasid mites and chigger mites), which are the majority of ectoparasites on rodents and other small mammals (pan and deng 1980, deng et al. 1993, li et al. 1997, jin 1999, wu 2007, zuo and guo 2011, zuo et al. 2011, huang et al. 2013, zhan et al. 2013). in the present study, the sample size of 431 host animals of a single species in a localized area was larger than some previous studies which have included multiple species sampled over much wider geographical range (bengston et al. 1986, oguge et al. 2009, paramasvaran et al. 2009, changbunjong et al. 2010). from this large host sample we collected 47 species of ectoparasites. this number also exceeded the numbers observed in some previous studies using smaller sample sizes (luyon and salibay 2007, kia et al. 2009, oguge et al. 2009, paramasvaran et al. 2009, changbunjong et al. 2010). for example, from 14 species and 204 individuals of rodents and shrews in malaysia, 20 species of ectoparasites were collected and included 5 taxa (paramasvaran et al. 2009). another survey documented 6 species of ectoparasites present on 56 individuals of 4 species of rodents in philippines (luyon and salibay 2007). the present study showed that norway rats had high and diverse infestations of ectoparasites. the large host sample (431 rats) increased the probability of detecting additional uncommon ectoparasite species. therefore, a large host sample is strongly recommended in future field investigations. in addition, the larvae of chigger mites are often present on the auricles and external auditory canals of the host ears (li et al. 1997, guo et al. 2006), and they are too small to be seen by the naked eye. in our collection, both ears of each rat were carefully inspected and this allowed us to find many chigger mite species (23 species). these may have been overlooked in some previous investigations. of the 4 taxa of ectoparasites we found on the norway rat, the majority of species were chigger mites, while the majority of individuals were sucking lice (table 2). many species of chigger mites infesting such a single host species (r. norvegicus) imply that the norway rat seems to have a great potential to harbor many chigger species. in contrast, only two species of sucking lice with numerous individuals in the sample were collected from the rat and this reveals that the norway rat may be a suitable host for these sucking lice. j arthropod-borne dis, june 2016, 10(2): 192–200 xg guo et al.: species abundance … 198 http://jad.tums.ac.ir published online: january 05, 2016 some transmitting vectors (or potential transmitting vectors) of zoonoses were found from the norway rat (table 1). of these vectors, the flea xenopsylla cheopis is a important transmitting vector of the plague and murine typhus in yunnan of china (wu 2007, zuo and guo 2011), and it increases the transmission risk of the zoonoses between rodents and human beings. species abundance distribution and total expected species estimation the preston’s lognormal distribution model is an established method to fit the species abundance distribution, which aims to illustrate the relationship between species and individuals in a given community (preston 1948, greig-smith 1983, baltanas 1992, guo 1999). the expected curve (theoretical curve) of species abundance distribution for the ectoparasites on r. norvegicus showed a gradually descending tendency, and this revealed that most parasite species were rare with very few individuals in the sample, but few dominant species were abundant with numerous individuals in the sample. based on the expected curve, the total expected number of ectoparasite species on the rats was estimated to be 85 species. hence, about 38 parasite species might have been missed in our investigation. in ecology, it is important to estimate the expected total species in a given community because some species are too rare to be found in a limited sample (guo 1999). besides preston’s method asst /)( 0  , some other methods (e.g. chao1 method) can also be used to estimate the total expected species number (chao 1984, bunge et al. 1993, cowell 2009). as a simple nonparametric estimator, chao1 formula is described as   b ass at 2 2  , where st = the estimate of the total number of species, sa= the observed number of species, a = the number of rare species with one individual in the sample and b = the number of rare species with two individuals in the sample. when chao1 method was used in the present study, the total expected parasite species (st) was estimated to be 89 species (sa= 47, a= 13 and b= 2 in table 3) and the number of species missed was about 42 species, which was more than the number 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yunnan province, china. med vet entomol. 25(4): 421–427. zuo xh, guo xg, zhan yz, wu d, yang zh, dong wg, huang lq, ren tg, jing yg, wang qh, sun xm, lin sj (2011) host selection and niche differentiation in sucking lice (insecta: anoplura) among small mammals in southwestern china. parasitol res. 108(5): 1243–1251. j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 original article a field experiment to assess the rate of infestation in honey bee populations of two metarhizium anisopliae isolates on varroa destructor (acari: mesostigmata) *khodadad pirali-kheirabadi 1, jaime a teixeira-da-silva 2, mehdi razzaghi-abyaneh 3, mehdi nazemnia 4 1department of pathobiology, faculty of veterinary medicine and research institute of zoonotic diseases, university of shahrekord, shahrekord, iran 2faculty of agriculture and graduate school of agriculture, kagawa university, takamatsu, japan 3department of mycology, pasteur institute of iran, tehran, iran 4azad university of karaj branch, karaj, iran (received 19 apr 2011; accepted 22 sep 2012) abstract background: the protective effect of two isolates of an entomopathogenic fungus, metarhizium anisopliae (demi 002 and iran 437c) on the adult stage of varroa destructor was evaluated in comparison with fluvalinate strips in the field. methods: a total of 12 honey bee colonies were provided from an apiculture farm. the selected hives were divided into 4 groups (3 hives per group). the first group was the control, treated with distilled water. the other two groups were exposed to different fungi (m. anisopliae isolates demi 002 and iran 437c) and the last group was treated with one strip of fluvalinate per colony. the number of fallen mites was counted using sticky traps during a 6-day period, six days before and after treatments. a fungal suspension at a concentration of 5× 106 conidia/ml was sprayed onto the frames and the number of fallen mites was counted. results: metarhizium anisopliae demi 002 and iran 437c isolates were as effective (i.e., caused as much mite fall) as the fluvalinate strip in controlling bee colonies than no treatment. conclusion: both m. anisopliae isolates are promising candidates as agents in the control of varroa mites under field conditions. isolate demi 002 can be considered as a possible non-chemical biocontrol agent for controlling bee infestation with v. destructor in the field. in order to substantiate this hypothesis, tests are currently being performed using larger colonies and larger doses than tested in the present study in our beekeeping. keywords: apis mellifera, biological control, entomopathogenic fungi, fluvalinate, metarhizium anisopliae, varroa destructor introduction the honey bee, apis mellifera, is an important insect for honey production and crop pollination. the ectoparasitic mite of the genus varroa is currently the most serious threat to beekeeping around the world (de jong et al. 1982, anderson and trueman 2000). this parasitic mite causes weight loss, malformation, and shortens the life span of honey bees, it also serves as a disease vector (chen and siede 2007). bee mortality, due to infestation with varroa mite, reaches up to 100% in untreated colonies. miticides, especially used in the area around iran, are fluvalinate (fluvalinate, vita (europe) ltd.) and coumaphos, which remain the most cost-effective and widely used chemicals of mite control for management of honey bee colonies (ferrer-dufol et al. 1991). however, these chemicals leave *corresponding author: dr khodadad pirali kheirabadi, e-mail: khpirali@yahoo.com 15 j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 residues in wax and honey (cabras et al. 1994, wallner 1995, 1999). other chemical control agents such as flumethrin, amitraz, cymiazole and bromopropylate are also associated with toxic residues (gamber 1990, wallner 1995). considering these problems and the development of resistance in varroa mite populations to fluvalinate and coumaphos (elzen et al. 1998, elzen and westervelt 2002), it is critical to develop new and safer alternatives for better control measures. there has been an increasing interest to search for alternative sustainable control methods of varroosis in recent years. several biological control programs have been developed for varroa control and some studies have used herbal medicine such as essential oils of aromatic plants and organic acids (imdorf 1997, 1999). a relatively new alternative to varroosis control is based on biological control using entomopathogenic fungi, natural enemies of mites (chandler et al. 2001). entomopathogenic fungi are known to infect different tick species, among which beauveria bassiana and metarhizium anisopliae have received major attention (kalsbeek et al. 1995). the mechanism by which fungi infect arthropods is not fully understood, but it involves the production of some important fungal enzymes such as chitinases, proteases, lipases and also fungal structures that penetrate the cuticle (st leger et al. 1987, 1997, campos et al. 2005). fungi are widely used for the control of agricultural and forest pests and in recent years efforts have been made to evaluate the biological control potential of these fungi against important arthropod vectors of human and animal diseases (pirali-kheirabadi et al. 2007a, b). the successful biological control of varroa destructor and v. jacobsoni using entomopathogenic fungi has previously been achieved under laboratory and field assay conditions (shaw et al. 2002, kanga et al. 2006, garciafernandez et al. 2008). based on their findings and on our previous data on the pathogenicity of these fungal isolates on rhipicephalus (boophilus) annulatus (acari ixodidae) (pirali-kheirabadi et al. 2007a, b), we decided to evaluate the efficacy of two iranian isolates, demi 002 and iran 437c, of the entomopathogenic fungus metarhizium anisopliae. these isolates were used for the first time in this field trial to determine their ability to be used as promising biological control agents for varroa mite control in honey bee colonies. the aim of this study was to ultimately determine the pathogenicity of both isolates against adult stages of v. destructor, with special reference to mortality rate. materials and methods fungal isolates metarhizium anisopliae isolates demi 002 and iran 437c were obtained from the fungal culture collection of the department of mycology of the iranian research institute of plant protection, tehran, iran (table 1). fluvalinate was prepared as recommended by the manufacturer (sichuan wang's animal health co., ltd.) and used as a treatment comparison against the two fungal isolates (table 1). preparation of conidial suspension the fungi were cultured on potato dextrose agar (pda, e. merck) plates for 2 weeks at 25 °c and 70% relative humidity (rh) in the dark. to avoid bacterial contamination, an antibiotic, ampicillin (200 µ g/ ml, biotika) was mixed with 10 ml of ethanol and added to the medium after filter sterilizing. conidia were harvested by washing the plates with an aqueous solution of sterilized distilled water with 0.005% tween 80 under a laminar flow hood. the conidial suspension was filtered through four layers of sterilized (muslin) gauze to remove fungal mycelia and other debris. conidial numbers 16 j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 were determined using a neubauer camera under a light microscope (olympus cx41) at 400x magnification and the concentration of conidia was adjusted to 5× 106 conidia/ ml, which was sprayed evenly onto the frames in hives as described in more detail below. for this purpose frames were drawn out of the hives and the suspension was sprayed directly onto the frames using a microporous sprayer. treatment of colonies with conidial suspension and chemical acaricide the treatments were conducted early in the morning. both m. anisopliae isolates were used at a concentration of 5× 106 conidia/ ml (every hive received 10 ml of conidial suspension). these fungi isolates have no adverse effects on bees, animals, humans and the environment (pirali-kheirabadi et al. 2007a, b). treatment with the fluvalinate strip was carried out following the methods of kanga et al. (2006) and the number of fallen mites was counted using sticky traps during a 6-day period, even though the experiment spanned for 6 days prior to the sampling period and 6 days after. experimental design twelve bee hives of a. mellifera infested with varroa mites were identified in an apiary located in the north of iran (lavizan, tehran) (35° 38' 34'' n, 51° 21' 27'' e). the type of beehive was langstroth and the colonies had at least 4 frames within each hive. established groups were separated from the rest by about 3–4 m. the experiment was carried out in autumn, in september-october, when the temperature was between 15 and 25 °c and the rh was 50%. the mass, status and other parameters (age of queen, honey and pollen reserves and number of brood frames) were homogeneous. the selected hives were divided into 4 groups (3 hives per group). the first group was the control, which was treated with distilled water. the other two groups were exposed to different fungi (m. anisopliae isolates demi 002 and iran 437c) and the last group was treated with one strip of fluvalinate (apistan, 10% tau-apistan, 800 mg per strip, vita europe ltd. co.) per colony. the fallen mites were collected daily from each hive during the first 6 days (this experiment spanned for 12 days). statistical analysis preand post-treatment data were used in the analysis to determine the efficacy of the fungal treatments. the experiment was organized in a complete randomized block design (crbd). there were three replicates per block and data was collected daily. the total number of collected mites for each hive throughout the experiment was recorded. daily percentage of collected mites was calculated for each hive. the effect of the main factors, i.e. day and type of treatment (control, isolate type (iran 437c and demi 002) and fluvalinate) on the percentage of collected mites was analyzed using the general linear model (glm) procedure of sas. least square means (ls means) were used with the sas pdiff test (sas statistics, 9.2). the overall effects of treatments on the mean percentage of collected mites from hives before and after treatments were analyzed using the glm procedure of sas. data was expressed as lsmeans ± sem. results the total (mean/hives) number of collected mites from the control, fluvalinate, metarhizium demi 002 and iran 437c isolates was 40 (1.1±0.02), 155 (4.3±0.6), 170 (4.7±1.3) and 74 (2.05±0.05), respectively. fig. 1 shows the percentage of collected fallen mites before and after treatment within each group. the mean percentage of total collected fallen mites before treatments was not significantly different between groups (table 2). there was no statistical difference 17 j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 between collected mites before and after treatment in the control group (table 2). the mean percentage of total collected mites was higher after treatment than before within the three groups tested (table 2). however, there was no difference in the mean percentage of total mites collected after treatments among the three treatment groups (table 2). fig. 2 shows the daily changes in cumulative percentages of the collected fallen mites during the treatment for the four established groups. table 1. general characteristics of metarhizium anisopliae fungal isolates used in this study origin host strain rasht, iran chilo suppressalis iran 437 c noor, iran rhychophorus ferrugine demi 002 table 2. total collected mites and the percentages (ls mean ± sem) of collected mites (per day) before (7 days) and after (5 days) treatment within experimental groups mean daily percentages of collected mites during experiment total collected mites groups after treatmentbefore treatment 7.05±5.8aa9.24±5.8aa40control 15.5±5.8bb3.5±5.8aa155apistan® strip 12.05±5.8bb5.7±5.8aa170ma demi002 13.1±5.8bb4.9±5.8aa74m.a. iran 437 c ab values with different upper case letters within rows differ significantly (p< 0.05). abvalues with different lower case letters within columns differ significantly (p< 0.05). 0 10 20 30 40 50 60 70 80 90 100 control apista n m.a . demi002 m.a . ira n 437 c p er ce n ta ge treatment groups after treatment before treatment fig. 1. the percentages of collected fallen mites before and after treatment within each group (control: n= 40, apistan: n= 155, metrhizium anisopliae demi002: n= 170 and m. anisopliae iran 437c: n= 74) 18 j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 fig. 2. mean percentage of daily collected fallen mites from four experimental groups. (day 6= 3 days before treatment and day 7= day of treatment commencement) discussion in this experiment the basal data depended on an equal number of fallen mites, considered at the beginning of the experiment. thus, among the hive colonies selected for the experiment, the primary infestation (according to the fallen mites) was statistically negligible. therefore it could be concluded that both m. anisopliae isolates reduced the number of mites per hive at the end of the experimental period, and the mean total number of fallen mites per hive was significantly higher in iran 437c, demi 002 and the fluvalinate strip compared to the control (table 2). at the end of the experimental period, fungal treatments had significantly reduced mites, about 1.85and 4.25-fold more than the control group for iran 437c and demi 002 isolates, respectively. the number of fallen mites was 3.87-fold higher than the control using the fluvalinate strip (table 2), which showed comparable effects on mites to m. anisopliae demi 002. experiments considering more replicates in a different are with variable humidity and temperature are needed to assess the proper dosage and to improve the effect of these potential biocontrol agents. entomopathogenic fungi have been used widely for the control of agricultural and forest pests (kaaya et al. 1996), and much attention has been paid to evaluate the biological control potential of entomopathogenic fungi against the important bee parasite v. destructor (syn. acari mesostigmata) under laboratory and field conditions since 2000. therefore, the biological control of v. destructor requires natural enemies from other hosts. at present, the management of varroa is based on the use of chemical pesticides throughout the world, but resistance to the miticide fluvalinate in varroa mite populations has become widespread since 1998 (elzen et al. 1998) and resistance to coumaphos was documented since 2002 (elzen and westervelt 2002). there is a need, therefore, for alternative and sustainable forms of varroa management. the present study 19 j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 shows clearly that these fungal isolates are promising candidates for the control of v. destructor in field conditions (kanga et al. 2003, 2006). lower concentrations of fungal spores were tested in our experiment compared to those in other studies in which some isolates of m. anisopliae (shaw et al. 2002, kanga et al. 2006) and beauveria bassiana were tested against v. destructor (meikle 2006, 2008). a possible explanation for fairly different results relative to other studies may lie in the fact that in our assay we directly sprayed conidial suspensions onto the frames. in contrast, in most other studies, a strip coated with conidia was used (eg kanga et al. 2006). the other possible explanation is that isolate iran 437c truly has a powerful miticidal effect on mites at higher doses compared with the fluvalinate strip although different areas with variable humidity and temperature are needed to assess the proper dosage; these are currently being performed using more recipients and larger doses than those tested in our field experiment. the m. anisopliae fungal isolate demi 002 can control v. destructor effectively, more than isolate iran 437c. the fluvalinate strip could inhibit mites during days 8 and 9 (2–3 days) post treatment but in the case of m. anisopliae isolates demi002 and iran 437c caused most mortality during the first 72 h post treatment (3 days) (garcia-fernandez et al. 2008) (fig. 2). differences between treated groups were significant considering the sum of squares before and after treatment (p< 0.05). future research will aim to determine the most suitable method and concentration to deliver the fungus into the hives and also to determine the effect of these fungal isolates on non-target organisms. unlike miticides, it is less likely that varroa mite populations could develop resistance to fungal infections because of the multiple target receptors involved in fungal infections as compared to a miticide’s mode of action (chandler et al. 2001). acknowledgements the authors declare that there is no conflict of interest. references anderson d, trueman jwh (2000) varroa jacobsoni (acari: varroidae) is more than one species. exp appl acarol. 24: 165–189. cabras p, martini mg, floris i, spanedda l (1994) residues of cymiazole in honey and honey bees. j apicult res. 33: 83– 86. campos ra, arruda w, boldo jt, da silva mv, de barros nm, de azevedo jl, schrank a, vainstein mh (2005) boophilus microplus infection by beauveria amorpha and beauveria bassiana: sem analysis and regulation of subtilisin-like proteases and chitinases. curr microbiol. 50(5): 257–261. chandler d, sunderland kd, ball bv, davidson g (2001) prospective biological control agents of varroa destructor n. sp., an important pest of the european 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beauveria bassiana (deuteromycota: hyphomycetes) conidia on varroa mites (acari: varroidae) and on honeybee (hymenoptera: apidae) colony health. exp appl acarol. 46(1–4): 105–117. pirali-kheirabadi kh, haddadzadeh hr, razzaghi-abyaneh m, bokaie s, zare, r, ghazavi m, shams-ghahfarokhi m (2007a) biological control of rhipicephalus (boophilus) annulatus by different isolates of metarhizium anisopliae, beauveria bassiana and lecanicillium psalliotae fungi. parasitol res. 100: 1297–1302. pirali-kheirabadi kh, haddadzadeh hr, razzaghi-abyaneh, m, zare r, ranjbar-bahadori sh, rahbari s, nabian s, rezaeian m (2007b) preliminary study on virulence of some isolates of entomopathogenic fungi in different developmental stages of boophilus annulatus in iran. j vet res. 62(4): 113– 118. 21 j arthropod-borne dis, june 2013, 7(1): 15–22 kh pirali-kheirabadi et al.: a field experiment… http://jad.tums.ac.ir published online: april 10, 2013 shaw ke, davidson g, clark sj, ball bv, pell jk, chandler d, sunderland, kd (2002) laboratory bioassays to assess the pathogenicity of mitosporic fungi to varroa destructor (acari: mesostigmata), an ectoparasitic mite of the honey bee, apis mellifera. biolo control. 24: 266–276. st leger rj, cooper rm, charnley ak (1987) distribution of chymoelastases and trypsin-like enzymes in five species of entomopathogenic deuteromycetes. arch biochem biophys. 258: 123–131. st leger rj, joshi l, roberts dw (1997) adaptation of proteases and carbohydrases of saprophytic and entomopathogenic fungi to requirements of their ecological niches. microbiology. 143: 1983–1992. wallner k (1995) use of varroacides and their influence on the quality of bee products. am bee j. 135: 817–821. wallner k (1999) varroacides and their residues in bee products. apidologie. 30(2– 3): 235–248. 22 j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 148 http://jad.tums.ac.ir published online: january 05, 2016 original article rhipicephalus annulatus (acari: ixodidae) control by nigella sativa, thyme and spinosad preparations *shawky mohamed aboelhadid 1, hesham a mahran 2, hazem m el-hariri 3, khalid mohamed shokier 4 1department of parasitology, faculty of veterinary medicine, beni-suef university, egypt 2department of hygiene, management and zoonoses, faculty of veterinary medicine, beni-suef university, egypt 3department of community medicine research, national research center, egypt 4 animal health research, beni-suef branch, beni-suef, egypt (received 7 may 2014; accepted 2 nov 2014) abstract background: several compounds obtained from plants have potential insecticidal, growth deterrent or repellent characteristics. the control of hard ticks by non-chemical substances was targeted in this study. methods: the effect of 36 materials on in-vitro ticks was studied, including 2 absolute controls (water only or absolute ethyl alcohol only), 6 conventionally used spinosad preparations (aqueous solutions), 12 nigella sativa (n. sativa) preparations (aqueous and alcoholic solutions), and 12 thyme preparations (aqueous and alcoholic solutions). the engorged ticks were tested in-vitro for mortality and oviposition ability using the studied materials. results: the final mortality after 48 hours of application in n. sativa aqueous preparations began from 10.0% concentration, 1.0% to 100% by concentration preparations ≥10%. in addition, n. sativa alcoholic preparations began from 50.0% concentration, 2 % to 100% by concentration ≥5%. meanwhile, thyme aqueous and alcoholic preparations began from 70.0% concentration, 5% to 90% by concentration 10–20%. additionally, spinosad aqueous preparations and both of control preparations (water and alcohol) resulted in no mortality. all differences were statistically significant. the oviposition was stopped in n. sativa (aqueous ≥10% and alcoholic ≥5%) and in spinosad (aqueous≥25%). the aqoues dilution of the used matters killed b. annulatus larvae beginning from the concentration 5%. conclusion: nigella sativa alcohol 20% was the best of studied preparations being the lowest concentration (20%) that could achieve the highest lethal (100%) effect in shortest time (12 hours). moreover, thyme oil and spinosad could not kill 100% of adult but did on larvae. keywords: rhipicephalus annulatus, nigella sativa, thyme oils introduction rhipicephalus annulatus (formerly boophilus annulatus) represents one of the main risks for the cattle industry in egypt. tick control is more difficult due to the presence of resistant populations to all major families of acaricides (rodriguez-vivas et al. 2006). chemical control is a common strategy used in daily life. synthetic insecticides became the common used forefront of insect-controlling agents. however, the environmental threat that these chemicals pose, effects on nontarget organisms, and the resistance of insect to insecticides have all raised during the last five decades (wattanachai and tintanon 1999, amer and mehlhorn 2006). there is an increasing need to develop insecticides for controlling insect, which are more environmentally safe and also biodegradable and targetspecific against the mosquitoes. several compounds obtained from plants have potential insecticidal, growth deterrent or repellent characteristics (isman 2006, pavela 2008). *corresponding author: dr shawky m aboelhadid, e -mail: drshawky2001@yahoo.com j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 149 http://jad.tums.ac.ir published online: january 05, 2016 essential oils, secondary metabolites of the plants, are odor components that can be extracted from plant tissue by water steam distillation or supercritical fluid extraction. most of them are complex mixtures of monoand sesquiterpenes and biologically related phenolic compounds. essential oils and their volatile constituents are commonly used in the prevention and treatment of human illnesses. various essential oils (garlic oil, onion oil, etc) have also been documented to exhibit acute toxic effects against insects (aboelhadid et al. 2013). nigella sativa is an herbal plant grows in countries of the mediterranean sea area, pakistan and india. the seed of it is known as black seed, habbatusawda, and habatul baraka ‘‘the blessed seed’’. it is referred to the prophet mohammed as having healing powers. the essential oil in its extract was shown to have antihelminthic effect, antinematodal and antischistosomal (mahmoud et al. 2002). there is no available data about the application of this oil on ticks. thyme (thymus vulgaris l), a perennial labiate, is endemic to the mediterranean area. its components and quality vary with geographical distribution, growing states, and stage and method of extraction (stahlbiskup and sáez 2002). the biological activity of thyme was found to have insecticide potential of obtained extracts using both organic solvents and hydro-distillation (regnault-roger et al. 1993, isman et al. 2001, knio et al. 2008). some essential t. vulgaris chemotypes or the essential oils obtained from them had larvicidal and adulticidal efficiency. there is no previous work to investigate its effect on ticks. spinosad was first registered for agricultural/horticultural use in the late 1990s and by 1999; it was approved for use on over 100 crops in 24 different countries (thompson et al. 2000). being a natural product derived from fermentation of the microorganism saccharopolyspora spinosa (mertz and yao 1990), spinosad has several favorable characteristics for a pesticide. for example, anastas et al. (1999) reported that spinosad will not bio-accumulate volatize or persist in the environment and will degrade naturally when exposed to light. in addition, spinosad displays activity against a range of insect pests, especially those in the genera of lepidoptera, diptera and thysanoptera, and to a lesser extent the coleoptera and orthoptera (thompsonet et al. 2000). spinosad gives highly efficacious control of many caterpillars of lepidopteran insects on numerous crops, including cotton, apples, sweet corn, potatoes, fruiting/leafy vegetables and tobacco, however, spinosad is relatively non-toxic to most beneficial insects and mites (deamicis et al. 1997). the present investigation aimed to evaluate the acaricidal effect of n. sativa and thyme oils and also spinosad against adult and larvae of r. annulatus through using different dilutions and different solvents. materials and methods sampling of r. annulatus in summer 2012, engorged females ticks were carefully detached from naturally infested cattle according to walker et al. (2003). the collected ticks were kept alive in a special glass tubes closed with hydrophobic cotton a strip of filter paper soaked in 15 % nacl. in the laboratory of parasitology, faculty of veterinary medicine, beni suef university, the tubes were labeled, incubated at 28 ˚c and relative humidity of 85 % for further studies. procedure of biological studies each engorged female tick was put separately in glass tube contains a filter paper soaked in 15% nacl, was put to give a relative humidity of 85–90% (bod incubator, felpr), then the tubes were closed tightly with a cotton tampon and were labeled (anij arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 150 http://jad.tums.ac.ir published online: january 05, 2016 mal host, date, tick species), and were kept in an incubator at the same conditions during the whole period of the experiments. the tubes were observed daily to record the duration of the preoviposition and oviposition periods, daily egg output then the eggs were placed in sterile disposable syringes, identified, plugged with cotton balls and were placed in the incubator under the previously mentioned conditions to observe the hatching. the pre-hatching and hatching periods were noticing and recording. oils extraction from plants the used oils of n. sativa and thyme were obtained by cold squeezing of the plant seeds. in this work, the oils were obtained from royal herbal company. preparation of alcoholic and aqueous solutions of the used oils five concentrations of (1, 3, 5, 10 and 20%) were prepared for oils (n. sativa and thyme), were diluted by distilled water or ethyl alcohol as solvents. the control individuals were treated with ethyl or distilled water only. preparation of spinosad (tracerr) for applied on adult tick this product was used in agriculture for control of insects. different concentrations were prepared from this product according the label of the manufacturing (2 ul, 20 ul and 40 ul for 50 ml water). the higher concentrations 10%, 25 % and 50% were tested. application methods for treatment of ticks with the used oils and product: adult immersion test (ait) semi and fully engorged females of rhipicephalus (boophilus) annulatus, b. annulatus were used in the present study were to adult immersion test (ait) as described in drummond et al. (1973) and benavides et al. (1999), the engorged females of r. annulatus, after being washed in a sieve with tap water, were dried on soft absorbent paper. after that, 50 females were divided into five groups of 10 females each and were immersed in petri dishes containing the above different concentrations of oils, for 2 min. the control group was also composed of 10 engorged females that had been immersed in alcohol or distilled water for the same period. ticks were then dried in absorbent paper and placed in an incubator at 28 ˚c and 85% relative humidity. the observations of oil effects on tick mortality were recorded at 6, 12, 24 hrs and 48hs post treatment. evaluation of the acaricidal efficiency of the used materials the mortality rate of treated adults was reported by noticing the number of dead ticks for each concentration. the oviposition ability of the treated engorged females and the ability of these eggs to hatch were investigated. dead ticks were diagnosed based on three criteria, signs of cuticle darkness and hemorrhagic skin lesions, leg movement, and halted malpighian tube movement. the legs movement was tested with a paintbrush with ticks placed in an inverted position under a stereomicroscope lamp. the change of cuticle color and hemorrhagic skin lesions were demonstrated in dead ticks. failure in egg laying was then directly observed by eye (chungsamarnyart and jansawan 2001). larval immersion test the different concentrations of the used oils in aqueous solution were screened against the unfed (15 days old) larvae. the larval immersion technique was proposed by shaw (1966). a group of approximately 600 larvae, 5 replicates in each one 120 larvae for each concentration and control, were distributed on a 14 cm diameter of filter paper lying in a petri dish. five milliliters of each concentration was poured over the larvae. another 14 cm diameter filter paper was j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 151 http://jad.tums.ac.ir published online: january 05, 2016 placed over the larvae and 5 ml of the extract poured over it. after the immersion period of 5 min, the larvae were removed from the filter paper and were placed in a folded filter paper (5×10 cm) with the openings closed with a tape and then were incubated at 28 ˚c and 85% relative humidity (borges et al. 2003). statistical analysis the collected data were coded, tabulated, and statistically analyzed using spss program (statistical package for social sciences) software version 18.0. the data was independent categorical, descriptive statistics were done as number and percentage, while inferential analyses were done using chi square. the level of significance was taken at p< 0.050 is significant, otherwise is non-significant. results the effect the used materials on adult r. annulatus no lethal effect were noticed in ticks that were subjected to the 2 absolute control (water only and ethanol only) and the 6 conventionally used tracer preparations (aqueous solutions of different concentrations, 2 µl/50 ml, 20 µl/50 ml, 40µl/50 ml, 10%, 25% and 50%). none of the studying materials produced any lethal effects 6 hours after application. the highest lethal effect 12 hours after application was n. sativa aqu (50% and 100%), n. sativa alc (20%, 50% and 100%) this was significantly higher than n. sativa aqu 20% and n. sativa alc 10% that reached 90% death. the final evaluation of different materials is 48 hours after application of each material. the most lethal materials (100% death) were n. sativa aqu (10%, 20%, 50% and 100%), n. sativa alc. (10%, 20%, 50% and 100%), this was significantly higher than the lesser lethal material (90% death) produced by thyme aqu (10% and 20%) and thyme alc (10% and 20%), they also significantly higher than the lesser lethal material (70% death) produced by n. sativa aqu 5% and thyme alc 5%), they also significantly higher than the lesser lethal material (50% death) produced by n. sativa alc 3%), they also significantly higher than the lesser lethal material (10% death) produced by n. sativa aqu 3%, the other materials produced no lethal effects (table 1). the effect on egg oviposition of r. annulatus the oviposition of live ticks after treatment, the live ticks after application of n. sativa oil stopped egg ovipositin in aqu (10%, 20%, 50% and 100%), n. sativa alc (5%, 10%, 20%, 50% and 100%). by following the ability of the produced eggs from treated live ticks, the eggs hatched in the expected period of normal eggs from untreated ticks (14 days). the live ticks subjected to different concentrations of thyme oils oviposited normal eggs just after application (24 h). these eggs hatched in the mean time after incubation. at the lower concentrations of spinosad (tracerr) the ability of treated ticks to oviposition did not affect and the produced eggs were normal in quantity and time of oviposition. these produced eggs hatched in the expected time of the normal eggs from untreated ticks, while the oviposition activity of treated ticks stopped at the higher concentrations (25% and 50%) where the ticks became to shrinkage, contrcted but not dead and it did not produced eggs (table 3). the effect on larvae of r. annulatus no lethal effect was noticed in ticks that were subjected to the absolute control (water only) and n. sativa aqueous 1% and thyme aqueous 1%. the lethal effect was significantly variable among different concentrations of aqueous concentrations of n. sativa, thyme and spinosad, it reached 100% in aqueous concentrations of n. sativa≥5% and spinosad ≥20µl/50ml while none of aqueous concentrations of thyme showed that level of mortality (table3). j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 152 http://jad.tums.ac.ir published online: january 05, 2016 table 1. effect of different materials on ticks viability s material 6 hours 12 hours 24 hours 48 hours 1 water only 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 2 ethanol only 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 3 n. sativa aqu 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 4 n. sativa aqu 3% 0 (0%)a 0 (0%)a 5 (10%)b 5 (10%)b 5 n. sativa aqu 5% 0 (0%)a 25 (50%)c 35 (70%)d 35 (70%)d 6 n. sativa aqu 10% 0 (0%)a 35 (70%)d 45 (90%)e 50 (100%)f 7 n. sativa aqu 20% 0 (0%)a 45 (90%)e 50 (100%)f 50 (100%)f 8 n. sativa aqu 50% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 9 n. sativa aqu 100% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 10 n. sativa alc 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 11 n. sativa alc 3% 0 (0%)a 5 (10%)b 25 (50%)c 25 (50%)c 12 n. sativa alc 5% 0 (0%)a 25 (50%)c 35 (70%)d 50 (100%)f 13 n. sativa alc 10% 0 (0%)a 45 (90%)e 50 (100%)f 50 (100%)f 14 n. sativa alc 20% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 15 n. sativa alc 50% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 16 n. sativa alc 100% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 17 thyme aqu 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 18 thyme aqu 3% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 19 thyme aqu 5% 0 (0%)a 0 (0%)a 25 (50%)c 35 (70%)d 20 thyme aqu 10% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 21 thyme aqu 20% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 22 thyme aqu 50% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 23 thyme aqu 100% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 24 thyme alc 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 25 thyme alc 3% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 26 thyme alc 5% 0 (0%)a 0 (0%)a 25 (50%)c 35 (70%)d 27 thyme alc 10% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 28 thyme alc 20% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 29 thyme alc 50% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 30 thyme alc 100% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 31 spinosadaqu µl/50ml 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 32 spinosad aqu20µl/50ml 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 33 spinosad aqu40µl/50ml 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 34 spinosadaqu 10% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 35 spinosadaqu 25% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 36 spinosadaqu 50% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a alc: alcoholic form, aqu: aqueous form. %: percentage of died ticks in relation to studied sample (50) of each group, a,b,c,d,e,f: homogenous groups so that, similar letters are not-significantly different, while different letters are significantly different. j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 153 http://jad.tums.ac.ir published online: january 05, 2016 table 2. effect of different materials on ticks oviposition ability s material oviposition 1 water only normal 2 ethanol only normal 3 n. sativaaqu 1% normal 4 n. sativaaqu 3% normal 5 n. sativaaqu 5% normal 6 n. sativaaqu 10% stopped 7 n. sativaaqu 20% stopped 8 n. sativaaqu 50% stopped 9 n. sativaaqu 100% stopped 10 n. sativaalc 1% normal 11 n. sativaalc 3% normal 12 n. sativaalc 5% stopped 13 n. sativaalc 10% stopped 14 n. sativa alc 20% stopped 15 n. sativaalc 50% stopped 16 n. sativaalc 100% stopped 17 thyme aqu 1% normal 18 thyme aqu 3% normal 19 thyme aqu 5% normal 20 thyme aqu 10% normal 21 thyme aqu 20% normal 22 thyme aqu 50% normal 23 thyme aqu 100% normal 24 thyme alc 1% normal 25 thyme alc 3% normal 26 thyme alc 5% normal 27 thyme alc 10% normal 28 thyme alc 20% normal 29 thyme alc 50% normal 30 thyme alc 100% normal 31 spinosadaqu 2µ l/50ml normal 32 spinosad aqu20µl/50ml normal 33 spinosad aqu40µl/50ml normal 34 spinosadaqu 10% normal 35 spinosadaqu 25% stopped 36 spinosadaqu 50% stopped normal means the quantity and time of egg deposition of untreated normal tick. all ticks that were subjected to each material gave the same response (normal/ stopped) oviposition table 3. effect of different concentrations of in aqueous solution on tick larvae after 24 h (n=100 larvae in 5 replicates) s material 24 hours 1 water only 0 (0%)a 2 n. sativa aqu 1% 0 (0%)a 3 n. sativa aqu 3% 338 (67.6%)d 4 n. sativa aqu 5% 500 (100%)g 5 n. sativa aqu 10% 500 (100%)g 6 n. sativa aqu 20% 500 (100%)g 7 n. sativa aqu 50% 500 (100%)g 8 n. sativa aqu 100% 500 (100%)g 9 thyme aqu 1% 0 (0%)a 10 thyme aqu 3% 145 (29.0%)b 11 thyme aqu 5% 251 (50.2%)c 12 thyme aqu 10% 342 (68.4%)d 13 thyme aqu 20% 417 (83.4%)e 14 thyme aqu 50% 426 (85.2%)e, f 15 thyme aqu 100% 435 (87.0%)e, f 16 spinosadaqu 2µ l/50ml 446 (89.2%)f 17 spinosad aqu20µl/50ml 500 (100%)g 18 spinosad aqu40µl/50ml 500 (100%)g 19 spinosadaqu 10% 500 (100%)g 20 spinosadaqu 25% 500 (100%)g 21 spinosadaqu 50% 500 (100%)g aqu: aqueous form n: the total number of died larvae %: percentage of died ticks in relation to studied sample (500) of each group, a,b,c,d,e,f,g: homogenous groups so that, similar letters are not-significantly different, while different letters are significantly different. discussion most plants contain compounds that they use in preventing attack from phytophagous (plant eating) insects. these compounds fall into several categories, including repellents, feeding deterrents, toxins, and growth regulators. it can be grouped into five major groups, nitrogen compounds (primarily alkaloids, terpenoids, phenolics, proteinase inhibitor, and growth regulators. although the primary functions of these compounds are defense against phytophagous insects, many are also effective against mosquitoes and other j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 154 http://jad.tums.ac.ir published online: january 05, 2016 biting diptera, especially those volatile components released because of herbivory (pichersky and gershenzon 2002) these properties encourage the researchers to investigate the effect of n. sativa oil on r. annulatus. the present results investigated the ability of n. sativa oil on the adult females of r. annulatus. both alcoholic and aqueous or solution of n. sativa killed all ticks after 12 h or 24 post-treatment from the concentration 5% and 10%. the same results were obtained from the higher concentrations. regarding the oviposition of live ticks after treatment, the live ticks after application of oils were found to oviposite the normal eggs and in the normal manner. by following the ability of the produced eggs from treated live ticks, the eggs hatched in the expected period of normal eggs from untreated ticks. the n. sativa oil (nso) had many activities, antithelmintics, anticestodal effects in children (akhtar and riffat 1991), antischistosomal (mahmoud et al. 2002), trichenella spiralis (abu el ezz 2005), aspiculuris tetraptera and hymenolepis nana (ayaz et al. 2007), these changes were correlated mainly with the ability of nso to improve liver function and the immunological system of infected mice and to its antioxidant effects (mahmoud et al. 2002). the protection is also due to the ability of nso and tq to reduce the cytogenetic damage induced by schistosomiasis infection (aboul-ela 2002). the n. sativa aqueous extract could be useful in the treatment of blastocystis hominis (el wakil 2007). thymoquinone, the most abundant constituent of black seed essential oil, is the active principle responsible for many of the seed’s beneficial effects (muhtasib et al. 2006). the seed has diverse chemical composition. they contain amino acids, proteins, carbohydrates, fixed and volatile oils (khan 1999). many of the pharmacological activities have been attributed to quinine and thymoquinone (tq), constituents in the seed. mahfouz and el-dakhakhny (1960) were isolate of ‘nigellone’possess antihistaminic properties. el-fatatry (1975) reported the isolation of thymohydroquinone (thq) from n. sativa seed volatile oil. the medicinal potential of black seed (n. sativa) referred to the presence of 135 nigellimine and nigellicine (ur-rahman et al. 1985), saponins and crude fiber as well as minerals such as calcium, iron, sodium and potassium. other constituents of the volatile oil include thymol (aboutabl et al. 1986). from these constituents of n. sativa oil may be explaining the acaricidal effect of the oil on adult ticks. these components may be cause saphocation of the ticks due to immersion in the solutions. it was noticed that the crude thyme oil (100%) or 50%, 1% and 3% diluted oil by water or alcohol did not kill any ticks. the solvent (water or alcohol) has no effect on the ability of thyme oil to cause death of the treated ticks. the tick deaths reached to 90% of treated ticks at concentrations of 10% and 20% in 48hs pa. the live ticks from these concentrations oviposited normal eggs just after application by short time (24 h) but the same quantity. these eggs hatched in the mean time after incubation. the insecticidal effect was ensured through the direct toxicity on adult insects and by inhibiting reproduction. therefore, one can profit using the essential oils of t. vulgaris and t. serpyllumin additionas an effective fumigant against a wide group of agricultural pests that damages its host plant. the oils have a toxic effect on adultinsects and inhibit the reproduction through ovicidal and larvicidal effects (regnaultroger and hamraoui, 1994). this insecticidal action is also produced by othercomponents of the species such as non-volatile phenols, non-proteinic amino acids, andflavonoids (regnaultroger and hamraoui 1995). thymol was shown to be more potent than thyme oil as a deterrent factor for reducinegg lying by the mite. morj arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 155 http://jad.tums.ac.ir published online: january 05, 2016 tality percentage reached 100% with both materialsused, however, at low concentrations the effect again was more pronounced applyingthymol than applying thyme oil (el-gengaihi et al. 1996). thymus is a multiple constituent's plant its composition groups can categorized into the following groups: essential oil, glycosoides, flavonoids, flavonoid glycosides and pheoliv acids. most ofthem are complex mixtures of monoand sesquiterpenesand biologically related phenoliccompounds. various essential oils have also been documented to exhibit acute toxic effects against internal and external parasites. using spinosadin in this study revealed that the low concentrations had no effect on adult r. annulatus. the higher concentrations (25 and 50%) led to the adult engorged female tick still alive but did not able to give eggs. spinosad is primarily a stomach poison with some contact activity and is particularly active against lepidoptera and diptera. it is a neurotoxin with a novel mode of action involving the nicotinic acetylcholine receptor and apparently the gaba receptors as well (salgado 1998). the mode of action of the spinosynsis through neural functions disruption, most likely via an alteration of nicotinic receptor function (salgado and saar 2004). ploch et al. (2007) demonstrated that symptomological assessment of insects treated with spinosad and spinosyn-a has suggested that the most likely mode of action of these insecticides is via a neural mechanism. these studies reported a possibly of a secondary physiological action of spinosyns. the primary interaction was associated with activation of an a-bgt sensitive current, thus suggesting a nicotinic mode of action (crouse et al. 2007). in addition, in some specific instances, a high-potency gabaergic response was also noted (watson 2001). the results presented heredemonstrates that spinosyn-a does not appear to interact with known nicotinic or gaba target sites. the weak affinity of spinosyn-a for displacement of the voltagegated calcium channel radioligand, [3h] vpm is suggestive of an interaction of spinosyns with this channel. spinosyn j and thec17 pseudoaglycone of spinosyn-a were both less potent than spinosyn-a in the [3h] vpm assay, which is consistent with their reduced biological activity against several insect species (salgado 2005). in general, the high non-specific binding of these ligands makes them suboptimal for studying membrane bound receptors. recently, however, the utility of one spinosyn ligand, [3h] 5, 6-dihydrospinosyn-a for cloned insect da6 receptorshas been demonstrated (orr et al. 2006). davey et al. (2001) reported that spraying of 0.05% and 0.15% spinosad on tick-infested cattle achieved 86% and 87% control, respectively, of larval r. annulatus at time of treatment. besides, cetin et al. (2009) who demonstrated that spinosad provides a complete control of larval argus persicus and r. turanicus in a short exposure time. conclusion nigella sativa has acaricidal effect than the other used materials. nigella sativa alc 20 % was the best of studied preparations being the lowest concentration (20%) that could achieve highest lethal (100%) effect in shortest time (12 h). the thyme oil is of limited effect. spinosad did not affect adult ticks at the lower concentrations but at the higher ones, it stopped the egg oviposition. acknowledgements the authors are appreciated to thank the royal herbal company to supply by the extracted oils. the authors declared that this study has no any potential competing interests. j arthropod-borne dis, june 2016, 10(2): 148–158 sm aboelhadid et al.: rhipicephalus annulatus … 156 http://jad.tums.ac.ir published online: january 05, 2016 references aboelhadid sm, kamel aa, arafa wm, shokier ka (2013) effect of allium sativum and allium cepa oils 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dengue preventive practices based on precaution adoption process model among health care professionals in northwest of iran samira firooziyan1,2, roya sadeghi3, maryam sabouri4, azar tol3, ehsan rikhtehgar5, behrouz fathi2,6, *mohammad mehdi sedaghat1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2urmia health center, disease control unit, urmia university of medical sciences, urmia, iran 3department of health education and promotion, school of public health, tehran university of medical sciences, tehran, iran 4department of health education and promotion, school of public health, shahid beheshti university of medical sciences, tehran, iran 5urmia deputy for health affairs, urmia university of medical sciences, urmia, iran 6assistant professor, health economics and management department, public health school, urmia, iran *corresponding author: prof mohammad mehdi sedaghat, e-mail: sedaghmm@tums.ac.ir (received 16 june 2022; accepted 30 nov 2022) abstract background: dengue fever as a mosquito-borne disease, has rapidly spread due to climate change, globalization, and human behavior. iran is prone to dengue fever, as its vector recently has been found in the country. this study aimed to assess predictors of dengue preventive practices based on precaution adoption process model (papm) factors in west azerbaijan province, northwest of iran. methods: this cross-sectional study conducted on 405 health professionals of communicable disease sector who were interested in study participation. data-gathering instrument was an online researcher-made questionnaire consisting of demographic characteristics (11 items), questions based on papm, and dengue preventive practices (85 items). content validity and reliability of the instrument, content validity ratio, content validity index, and cronbach α were utilized, respectively. descriptive, analytical, and regression analysis using spss and stata were examined. results: regression analysis revealed that awareness of appropriate actions for dengue prevention was a stronger predictor of preventive practice in borderline and appropriate categories (β= 4.09, p< 0.001) and (β= 4.42, p< 0.001), respectively. among factors of papm, beliefs about precaution effectiveness and difficulty in borderline (β= 1.04, p= 0.04) and appropriate (β= 1.12, p= 0.03) groups had direct and significant relation with dengue preventive practice. conclusion: the highest mean score of beliefs about hazard likelihood and severity factor was related to dengue prevention. therefore, theory-based interventions that address beliefs about precaution effectiveness and difficulty can lead to assistance in acting. to improve dengue preventive practices, a well-designed promotive intervention that addresses associated factors in a context-specific manner is essential. keywords: preventive practices; dengue fever; precaution adoption process model (papm); health professionals introduction dengue is a viral mosquito-borne disease endemic in tropical and subtropical regions, transmitted by the bite of aedes mosquito vectors (aedes aegypti and aedes albopictus). climate change, globalization and trade, urbanization, travel, and human behavior have led to the spread of dengue fever and its vectors to human communities (1–3). dengue is a rapidly spreading arboviral disease, its incidence has increased 30-fold over the past 50 years while the vectors ae. aegypti and ae. albopictus have expanded their copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:sedaghmm@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 341 http://jad.tums.ac.ir published online: dec 31, 2022 geographical distribution. the disease is now endemic in 128 countries across africa, the americas, the eastern mediterranean region with several outbreaks occurring in pakistan, yemen, and saudi arabia(4–5). a virus of the flaviviridae family causes dengue. four distinct, but closely related, serotypes of the virus cause dengue. the symptoms differ in younger children and those who have the disease for the first time (3, 6). presentation varies, ranging from no symptoms to a mild fever to severe dengue hemorrhagic fever and dengue shock syndrome may threaten human life (7). the ailment presents high fever accompanied by frequent headaches, nausea, skin rashes, enlargement of lymph nodes, pain behind eyes, severe muscular, bone and joint pains, epitaxis along with leukocytopenia i.e. reduction in white blood cells count. dengue fever (df) is also characterized as bone break fever or bone crush fever due to severe pain in joints and bones (5). mild df causes fever above 40 °c, headache, rashes, eye pain, vomiting, swollen lymph nodes, and muscle and joint pain. df in severe situation can cause ecchymosis, hematemesis, severe abdominal pain, nosebleeds and bleeding in the mouth, permanent nausea, pulmonary, cardiac and hepatic problems, hypertensive shock, and death. it to df diagnose is difficult because of symptoms similar of many diseases with those of df (8). the global burden of dengue is not well recognized, but its epidemiological patterns are affecting both human health and the health economy (3). annually, about ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue hemorrhagic fever mainly in southeast asia, the pacific, and the americas (9). mortality and morbidity of severe dengue are reported at 20 percent approximately (10). there is no specific treatment for dengue fever for the infection, and management is only supportive care (6). according to the who, dengue control is technically feasible with coordinated international technical and financial support for national programs (6). preventive and vector control interventions aim to reduce dengue transmission, thereby decreasing the incidence of the infection and preventing outbreaks of the disease. primary prevention includes wearing protective clothing to reduce skin exposure, using mosquito repellents, using mosquito traps and nets, installing screens on doors and windows, eliminating unnecessary container habitats that collect water, and covering water storage containers with a fine mesh. these actions presented the most effective factors in dengue prevention and control (3, 6, 9). public health system preparedness has a very important role to decrease morbidity and mortality of dengue fever, especially during epidemics. knowledge, attitude and belief of health professionals are very crucial in this regard (11). precaution adoption process model (papm) is a stage model of health behavior, which derives from social learning theory and the health belief model also the papm has been generally used for health behavioral change in different issues such as wearing seat belts, using condoms, weight management, preventing smoking and following treatment plans (12). this model attempts to clarify how a person make a decision to take an action and how decision translate into action (13). the papm was chosen as a theoretical framework for this study because it offers a more detailed stage, especially the early stages from awareness to decision regarding health-related behaviors. factors of papm consisted of awareness (media messages about the hazard and precaution, communications from significant others, and personal experience with hazard), beliefs about hazard likelihood and severity, beliefs about personal susceptibility, beliefs about precaution effectiveness and difficulty, perceived social norms and assistance in acting (14). in the covid-19 pandemic, increasing dengue cases happened due to disruptions in the http://jad.tums.ac.ir/ https://www.sciencedirect.com/topics/medicine-and-dentistry/arthralgia https://www.sciencedirect.com/topics/medicine-and-dentistry/leukopenia j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 342 http://jad.tums.ac.ir published online: dec 31, 2022 pathogen-host-vector relationship reasoned by changes in individual behaviors in response to covid-19. lockdowns and social distancing policies could spread dengue in an endemic area. this situation could limit coverage of dengue preventive programs and delay the medical management of both diseases due to co-infection and misdiagnosis (15). it is well noted that medical staff and health workers working in health centers in endemic areas need regular training and consistent supervision to improve their knowledge of the prevention and care of dengue patients (16). west azerbaijan province is in the northwest of iran with some points of entry, which makes the province susceptible to dengue fever. the points of entry in the province include urmia international airport, razi railway border, and five land border entry and exit points. for the first time, this study aimed to assess predictors of dengue preventive practices based on (papm) factors among health care professionals in west azerbaijan province in 2022. materials and methods this cross-sectional study was conducted on 405 health professionals through an online questionnaire. health professionals employed in the communicable disease sector in health centers affiliated to west azerbaijan province and willing to study participants included in the study. ethical approval was obtained from the research ethics committees of the national institute for medical research development (ir.nimad.rec.1400.027). online consent was obtained from respondents before participation in this study. all identities of the respondents were kept anonymous, and the information in this study was kept confidential. the self-administered questionnaire was in persian, the national language of iran. an explanation of the nature and purpose of the study was presented on the first page of the online instrument. the questionnaire used in this study was a researcher-made with two parts. the first part consists of demographic characteristics and work-related variables; age, sex, marital status, job profile, work history, educational level, workplace, information about dengue disease, and the source of obtaining information related to dengue collected in this part of the questionnaire (10items). in the second part of the questionnaire items were based on papm factors including staging definition, awareness of disease symptoms, types of disease transmission, prevention, breeding places of vectors and vector control, beliefs about personal susceptibility, beliefs about hazard likelihood and severity, beliefs about precaution effectiveness and difficulty, perceived subjective norms and assistance in carrying out an action related to dengue prevention and items of preventive practices (85 items). the range score of this part of the questionnaire was zero to 24 and scoring was based on a five-point likert from absolutely agree to absolutely disagree (0–4) and a three-point likert as yes, no, or do not know. based on achieved scores in the papm questionnaire, the total score was divided into three parts inappropriate, borderline, and appropriate. this classification was performed based on zero to 100. this instrument was developed through literature review and experts’ opinions. at first, one hundred questions related to dengue preventive practices were developed. ten experts in medical entomology and vector control, and health education and promotion revised this version. in the second stage, a revised instrument based on fiveand three-point likert was developed. to assess the content validity of the revised instrument, qualitative and quantitative methods were used. in the qualitative method, experts were asked to evaluate using appropriate words and scoring the items. feedback was gained then a final revised instrument was developed. to assess content and the validity of the instrument, content validity ratio (cvr) http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 343 http://jad.tums.ac.ir published online: dec 31, 2022 and content validity index (cvi) were utilized. cvr upper than 0.62 and 0.79 based on ten experts’ opinions were approved, respectively (17). to assess face validity, thirty health professionals filled out the questionnaire and evaluated the instrument in terms of proper words, grammar, simplicity, and understanding. in this assessment, cvr were calculated at 0.97. reliability was confirmed using cronbach α (0.7) (18). the study questionnaire was completed by thirty eligible health care professionals with two weeks interval and α was calculated at 0.89 in total. reliability of staging definition, awareness about dengue, beliefs about personal susceptibility, beliefs about hazard likelihood and severity, beliefs about precaution effectiveness and difficulty, perceived subjective norms and assistance in carrying out an action related to dengue prevention, and items of preventive practices were calculated 0.73, 0.74, 0.71, 0.87, 0.93, 0.77, 0.87, 0.74, and 0.78 respectively. descriptive, analytical such as kruskal-wallis h test and mann-whitney u (using a nonparametric test based on the abnormality of data) and regression analysis using spss version 25 and stata was performed at a significant level that was set less than 0.05. results study results presented that 48.4 percent (n= 196) were in the age range 36–45 with mean years old. the mean ± standard deviation age of the participants was 38.5±7.4. about half (49%) of the participants have 11– 20 years of work history. more than half of the study participants were female (53.8%, n= 218). among demographic variables, education level revealed significant relation with dengue preventive practices (p= 0.03) (table 1). figure. 1 represented 85.9 percent of the participants who had heard about dengue and 67.9 percent of the participants who decided to adopt dengue prevention practices (fig. 1). the mean prevention practices score of participants was 20.03±2.48 out of 24. among factors of papm, beliefs about hazard likelihood and severity (20.40±3), and beliefs about precaution effectiveness and difficulty (17.93 ±2.92) had higher mean and sd. awareness about the definition of vector breeding places among types of awareness including disease symptoms, types of the disease transmission, prevention, and vector control was higher (5.72±2.64) out of 24 (table 2). after assessing normality of data, regression analysis using inter method variables evaluated by the uni variate method, variables with sig< 0.1 enter the analysis, and others are excluded (table 3). results of regression analysis revealed that awareness of appropriate actions for dengue prevention was a stronger predictor of dengue preventive practices in borderline and appropriate categories (β= 4.09, p< 0.001) and (β= 4.42, p< 0.001), respectively. among factors of papm, beliefs about precaution effectiveness and difficulty in borderline (β= 1.04, p= 0.04) and appropriate (β= 1.12, p= 0.03) categories had direct and significant relation with dengue preventive practices, respectively (table 4). http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 344 http://jad.tums.ac.ir published online: dec 31, 2022 table 1. characteristics analysis of participants in this study (n= 405) p value mean rank number (%) groups variables 0.35 211.27 140 (34.6) 21–35 age (yrs.) 195.66 196 (48.4) 36–45 207.09 69 (17) above 45 0.92 203.44 218 (53.8) female sex 202.48 187 (46.2) male 0.55 201.76 349 (86.2) married marital status 210.71 56 (13.8) unmarried 0.58 194.77 106 (26.2) health workers job profile 203.13 225 (55.6) health professionals 211.23 60 (14.8) doctor 227.93 14 (3.5) another job 0.03 259.06 8 (2) up to diploma level of education 180.85 78 (19.3) diploma 207.01 319 (78.8) academic degree 0.53 209.69 152 (37.6) 1–10 work history(yrs.) 198.99 201 (49.6) 11–20 195.22 52 (12.8) above 20 0.79 199.52 308 (76) urmia city 213.44 16 (4) piranshahr 209.04 12 (3) poldasht 221.89 18 (4.4) mako 212.66 51 (12.6) another cities in the province 0.31 204.43 290 (71.6) rural health center workplace 194.09 100 (24.7) urban health center 234.77 15 (3.7) another health sites 0.27 206 324 (80) yes have information about dengue disease 191 81 (20) no 0.23 146.50 32 (7.9) continuous education source of information 172.73 75 (18.5) academic education 173.75 56 (13.8) media 157 161 (39.8) workshop *t test for quantitative variables and anova for more than two sample groups table 2. descriptive analysis of papm factors and dengue preventive practices (n= 405) minimum maximum mean± (sd) groups variables (0, 5) 2.55±1.46 staging definition (0, 6) 4.72±1.64 disease symptoms awareness (0, 5) 2.63±1.35 type of transmission (0, 5) 3.59±1.25 prevention (0, 9) 5.72±2.64 breeding places (0, 4) 3.51±1.01 control actions (0, 16) 11.23±2.24 beliefs about personal susceptibility (0, 24) 20.40±3.95 beliefs about hazard likelihood and severity (3, 20) 17.93±2.92 beliefs about precaution effectiveness (0, 20) 8.87±3.90 beliefs about precaution difficulty (4, 20) 15.89±3.83 perceived subjective norms (6, 20) 13.23±3.35 assistance in carrying out the action (0, 21) 20.03±2.48 preventive practices http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 345 http://jad.tums.ac.ir published online: dec 31, 2022 table 3. multivariate regression analysis of preventive practices among the study participants (n= 405) p value icc** 95% β* groups variables upper bound lower bound 35–21 ־ ־ ־ ־ age (yrs.) 0.36 0.84 -0.3 0.26 36–45 0.53 1.06 -0.55 0.25 > 45 female sex ־ ־ ־ ־ 0.93 0.54 -0.59 0-.02 male married marital status ־ ־ ־ ־ 0.38 1.05 -0.4 0.32 unmarried up to diploma ־ ־ ־ ־ level of education 0.07 0.18 -3.48 -1.65 diploma 0.65 1.48 -2.34 -0.43 academic degree *linear regression coefficient **intraclass correlation coefficient table 4. multivariate regression analysis of preventive practices among study participants (n= 405) variables groups grouping by score β* icc** 95% p value lower bound upper bound staging definition 1 (0,1) 2 (2) 0.19 -0.51 0.89 0.59 3 (3–5) 0.27 -0.37 0.92 0.4 awareness disease symptoms 1 (0–4) 2 (5) 0.01 -0.57 0.61 0.94 3 (6) 0.03 -0.56 0.62 0.91 type of transmission 1 (0–1) 2 (2) 0.06 -0.62 0.75 0.84 3 (3–5) -0.15 -0.82 0 .50 0.64 prevention 1 (0–3) 2 (4) 0.13 -.35 0.63 0.58 3 (5) -0.2 -.81 0 .41 0.52 breeding places 1 (0–4) 2 (5–7) 0.03 -0.52 0.59 0.9 3 (8–9) 0.33 -0.29 0.96 0.29 control actions 1 (0–1) 2 (2–3) 4.09 2.91 5.27 0.001 3 (4) 4.42 3.24 5.60 0.001 beliefs about personal susceptibility 1 (0–10) 2 (11–12) 0.33 -0.17 0.84 0.19 3 (13–16) 0.16 -0.47 0.81 0 .60 beliefs about hazard likelihood and severity 1 (0–17) 2 (18–23) 0.34 -0.32 1.01 0.31 3 (24) 0.36 -0.49 1.21 0.40 beliefs about precaution effectiveness 1 (3–15) 2 (16–19) 1.04 0.32 1.75 0.004 3 (20) 1.12 0.38 1.86 0.003 beliefs about precaution difficulty 1 (0–7) 2 (8–9) 0.87 -0.50 0.67 0.77 3 (10–20) 0.27 -0.25 0.81 0.30 perceived subjective norms 1 (4–14) 2 (15–18) 0.04 -0.51 0.61 0.86 3 (19–20) -0.33 -1.006 0.34 0.33 assistance in carrying out the action 1 (6–11) 2 (12–14) 0.42 -0.11 0.96 0.12 3 (15–20) 0.22 -0.38 0.84 0.46 *linear regression coefficient **intraclass correlation coefficient http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 346 http://jad.tums.ac.ir published online: dec 31, 2022 yes, 85.9 yes, 29.6 yes, 32.8 yes, 39 yes, 67.9 no, 14.1 no, 70.4 no, 67.2 no, 61 no, 32.1 0 20 40 60 80 100 have you heard of dengue fever? i have never thought about dengue fever prevention practices i have not yet made a decision on preventive practices i have decided on preventative measures for dengue fever but have… i have decided to implement dengue fever prevention practices fig. 1. percentage stages of change of dengue prevention practices discussion dengue is one of the most important arthropod-borne viral diseases with widespread distribution in the tropics and subtropics (3). iran is at risk of transmission of df disease due to the presence of the main vector ae aegypti, changing rainfall and floods pattern that increases aedes mosquito larvae habitats, as well as the spread of df in some countries of the region, including pakistan and yemen (19). this study aimed at assessing predictors of dengue preventive practices based on papm factors among health care professionals in west azerbaijan province. it is important to note that there was a limited study about dengue and health education and promotion models/ theories which affected to discuss results of the present study in papm factors. among socio-demographic factors, the only level of education had a significant and direct relation with dengue preventive practices. this result was in the line with wong et al. (20), their results approved that religion, level of education, and the job had a significant as sociation with dengue preventive practices. chanyasanha et al. (21) reached a similar conclusion; they revealed that age, religion, family income, education, knowledge, and attitude were associated with preventive practices (21). the present study confirmed the findings of the study results of guard et al. (22) about the significant association of knowledge, attitude, and preventive practice (kap) regarding dengue infection among community residents in endemic areas. this result demonstrated that knowledge and awareness among different educated individuals could vary. it seems that health promotion activities should purpose at raising awareness at the community level on the importance of combining mosquito bite prevention with the elimination of breeding sites in and around high-density residence locations (23). the present study confirmed that awareness about the appropriate breeding places of vectors had the highest mean score in comparison with other related awareness; this finding is directly in line with previous findings with http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 347 http://jad.tums.ac.ir published online: dec 31, 2022 sarfraz et al. study (24). from the results, awareness of appropriate actions for dengue prevention was a stronger predictor of dengue preventive practices in borderline and appropriate categories respectively. this finding ties well with wong et al. (20), showing the importance of sufficient information of participants about dengue, many people had adequate knowledge of dengue fever and mosquitoes, but understanding of the important relationships among mosquitoes, human behavior, and disease transmission was sub-optimal. results of a qualitative assessment of community beliefs and practices about dengue revealed that participants had suffered from df and were identified through the surveillance system. also, participants with previous dengue diagnosis were more concerned about risk of the disease, had better knowledge about disease prevention (25). in another study, shuaib et al. (26) demonstrated that individuals well informed regarding taking self-protection actions using dengue prevention methods. one of the most important measures in dengue virus transmission reduction is the interruption of host-mosquito contact. individuals should cover the limbs by wearing full length pants, trousers, and full sleeved clothes. mosquito repellents or lotions as conventional measures must be utilized to protect and avoid mosquito biting during daytime hours. furthermore, coils, electric vapor mats, trap lights, insecticide treated nets and curtains and so forth should be used to prevent direct contact with mosquitoes (5). among factors of papm, beliefs about precaution effectiveness and difficulty in borderline and appropriate categories had direct and significant relation with dengue preventive practices. in accordance with our findings, siddiqui et al. (27) showed that providing health messages in interventional programs considering regular environmental sanitation can be helpful and would benefit in reducing the number of dengue mosquito vectors in residential places. it seems practical and sustainable efforts are a necessity to tailor behavioral changes in health education and promotion interventions through appropriate mass media and community clean-up campaigns among communities to dengue control in endemic areas. this study had some limitations. although this study tried to sample to reflect the characteristics of health care professionals employed in health centers of west azerbaijan province, the study findings cannot be generalized to all health care professionals in iran. furthermore, the study findings showed important associations among study variables, but the findings obtained from cross-sectional survey data with self-reported instruments cannot clarify causal-effect relationships between papm factors and the variables. this study had a novelty that explained predictors of dengue preventive practices based on papm factors among health professionals. conclusions based on study results, the highest mean score of beliefs about hazard likelihood and severity factor related to dengue prevention, providing theory-based interventions that address beliefs about precaution effectiveness and difficulty related to assistance in acting might be beneficial. to improve dengue preventive practices, well-designed promotive intervention programs that address the associated factors in a context-specific manner are essential. practical efforts should be conducted to raise awareness about dengue transmission and develop community-based sustainable vector control programs involving both the public and private sectors. acknowledgements research reported in this publication was supported by elite researcher grant committee under award number [4000586] from the national institute for medical research development (nimad), tehran, iran. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 348 http://jad.tums.ac.ir published online: dec 31, 2022 ethical considerations the study was approved by the ethical committee of the national institute for medical research development (nimad), tehran, iran, ethic no. ir.nimad.rec.1400.027. conflict of interest statement the authors declare there is no conflict of interests. references 1. ebi kl, nealon j (2016) dengue in a changing climate. environ res. 151: 115–123. 2. wesolowski a, qureshi t, boni mf, sundsøy pr, johansson ma, rasheed sb, engømonsen k, buckee co (2015) impact of human mobility on the emergence of dengue epidemics in pakistan. proc natl acad sci usa. 112(38): 11887–1192. 3. guzman mg, gubler dj, izquierdo a, martinez e, halstead sb (2016) dengue infection. nat rev dis primers. 2(1): 1– 25. 4. rasheed sb, butlin rk, boots m (2013) a review of dengue as an emerging disease in pakistan. public 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p, mukherjee a (2021) dengue virus infection: a tale of viral exploitations and host responses. viruses. 13(10): 1–20. 11. jayawickreme k, jayaweera d, weerasinghe s, warapitiya d, subasinghe s (2021) a study on knowledge, attitudes and practices regarding dengue fever, its prevention and management among dengue patients presenting to a tertiary care hospital in sri lanka. bmc infect dis. 21 (1): 1–14. 12. diclemente rj, crosby r, kegler mc (2009) emerging theories in health promotion practice and research. john wiley and sons. new jersey. 13. porche dj (2004) public and community health nursing practice: a populationbased approach. sage publications, ca. 14. glanz k, rimer bk, viswanath k (2008) health behavior and health education: theory, research, and practice:4th edition john wiley and sons, san francisco, ca. 15. wiyono l, rocha icn, cedeño tdd, miranda av, lucero-prisno iii de (2021) dengue and covid-19 infections in the asean region: a concurrent outbreak of viral diseases. epidemiol health. 43: 1–5. 16. tsheten t, clements ac, gray dj, gyeltshen k, wangdi k (2021) medical practitioner’s knowledge on dengue management and clinical practices in bhutan. plos one. 16(7): 1–15. 17. lawshe ch (1975) a quantitative approach to content validity. j pers psychol. 28 (4): 563–575. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 340–349 s firooziyan et al.: predictors of … 349 http://jad.tums.ac.ir published online: dec 31, 2022 18. george d, mallery p (2019) ibm spss statistics 26 step by step: a simple guide and reference: 16th edition, routledge, new york. 19. khoobdel m, jonaidi jafari n (2016) dengue fever, an old arboviral disease threatening the persian gulf region and southern iran. j mil med. 18(2): 181–183. 20. wong lp, shakir smm, atefi n, abubakar s (2015) factors affecting dengue prevention practices: nationwide survey of the malaysian public. plos one. 10 (4): 1–16. 21. chanyasanha c, guruge gr, sujirarat d (2015) factors influencing preventive behaviors for dengue infection among housewives in colombo, sri lanka. asia pac j public health. 27(1): 96–104. 22. guad rm, mangantig e, low wy, taylor-robinson aw, azzani m, sekaran sd, sim ms, azizan n (2021) development and validation of a structured survey questionnaire on knowledge, attitude, preventive practice, and treatment-seeking behaviour regarding dengue among the resident population of sabah, malaysia: an exploratory factor analysis. bmc infect dis. 21(1): 1–11. 23. elsinga j, schmidt m, lizarazo ef, vincenti-gonzalez mf, velasco-salas zi, arias l, burgerhof jgm, tami a (2018) knowledge, attitudes, and preventive practices regarding dengue in maracay, venezuela. am j trop med hyg. 99(1): 195–203. 24. sarfraz ms, tripathi nk, tipdecho t, thongbu t, kerdthong p, souris m (2012) analyzing the spatio-temporal relationship between dengue vector larval density and land-use using factor analysis and spatial ring mapping. bmc public health. 12: 1–19. 25. panagos a, lacy er, gubler dj, macpherson cn (2005) dengue in grenada. revista panamericana de salud pública. 17(4): 225–229. 26. shuaib f, todd d, campbell-stennett d, ehiri j, jolly pe (2010) knowledge, attitudes and practices regarding dengue infection in westmoreland, jamaica. west indian med j. 59(2): 139–146. 27. siddiqui tr, ghazal s, bibi s, ahmed w, sajjad sf (2016) use of the health belief model for the assessment of public knowledge and household preventive practices in karachi, pakistan, a dengueendemic city. plos negl trop dis. 10 (11): 1–15. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6085811/ https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=21132094 https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=21132094 microsoft word 9-dr molawi .doc iranian j arthropod-borne dis, 2007, 1(2): 53-56 h razmjou et al.: ophthalmomyiasis caused by … 53 case report ophthalmomyiasis caused by flesh fly (diptera: sarcophagidae) in a patient with eye malignancy in iran h razmjou 1 , *gh mowlavi 2 , m nateghpour 2 , s solaymani-mohamadi 2 , eb kia 2 1department of ophthalmology, school of medicine, isfahan university of medical sciences, isfahan, iran 2department of medical parasitology and mycology, school of public health, medical sciences/university of tehran, tehran, iran (received 20 aug 2007; accepted 1 nov 2007) abstract here we describe a case of ophthalmomyiasis in a male patient with basal cell carcinoma. during the operation several live and motile maggots were removed from the lesion. preliminary examination on the larvae confirmed their affiliation to the genus sarcophaga (diptera: sarcophagidae).this genus is widely distributed throughout the world and species are very difficult to identify. the authors made attempt to approach species identification by rearing larvae to the adult flesh flies, but due to shortage of adult male specimen, reliable diagnosis in the level of species was not obtained. possible interaction between ocular myiasis and malignancy concerning the case has not been addressed in this paper. keywords: ophthalmomyiasis, sarcophaga, malignancy introduction myiasis is defined as infestation of a vertebrate host by fly larvae that feed on living tissue, body fluids, or ingested foods. although myiasis in man is generally rare, many species of particularly calliphoridae, oestridae and sarcophagidae may produce myiasis (james 1947). myiasis is usually classified from an entomological or a clinical point of view. entomologically, flies may be classified into three myiasis-producing groups: obligatory, facultative, and accidental. clinically, myiasis can be classified according to the part of the body affected. cutaneous myiasis includes wound myiasis and furuncular myiasis. although it is most unusual in our country, imported cases can be found because of increased traveling and immigration (langan et al. 2004). there are more than 150,000 dipteran species. few, however, cause ocular injuries (glasgow et al. 1995). ocular involvement is responsible for 5% of all cases of myiasis and is termed ophthalmomyiasis (wilhelmus 1986). keyt was the first to describe a case of ophthalmomyiasis in 1900 (sivaramasubramanyam et al. 1968). depending on the site of infestion, ophthalmomyiasis may be categorized as external, internal or orbital. in external ophthalmomyiasis the adult fly deposits its larvae on the conjunctiva or eyelids. when the larvae penetrate the globe, internal ophthalmomyiasis occurs. in orbital myiasis the maggots invade the orbit producing severe orbital damage. human ophthalmomyiasis is caused by a number of myiasis-producing flies. host animals include cattle, sheep, horses, deer and rodents. however, human ophthalmomyiasis is commonly caused by the ovine nasal botfly, oestrus ovis (cameron et al. 1991) and the russian botfly, rhinoestrus purpureus. several cases of human ophthalmomyiasis caused by other species such as hypoderma tarandi, wohlfhartia magnifica, and cochliomyia hominivorax have been reported from various *corresponding author: dr gh mowlavi, tel/fax: +98 21 88951392, e-mail: molavig@yahoo.com iranian j arthropod-borne dis, 2007, 1(2): 53-56 h razmjou et al.: ophthalmomyiasis caused by … 54 places in the world (chodosh et al. 1992). cases of ophthalmomyiasis in hospitalized and elderly individuals as well as in debilitated people and patients with malignancies have been reported on several occasions (greenberg 1984). ocular myiasis is usually manifested as conjunctivitis in the beginning. corneal opacity and marginal ulcer with subsequent poor eyesight would occur if the parasite invaded the eyeball. in some cases, the maggot penetrates the sclera into the anterior chamber or retina, causing blindness (jiang 2002). case report a 62yearold male afghan refugee residing in iran for more than 20 years was admitted to the feyz eye specialist hospital in isfahan. he was suffering from a non – healing lesion in the left canthal area. muco-purulent discharge, redness, tearing, and itching of the left eye were mainly complained by the patient. on examination, visual acuity was 10/10 in both eyes. early observations showed a progressive malignant tumor completely affecting the orbit and ethmoidal sinus simultaneously. more investigation revealed the invading basal cell carcinoma confirmed histopathologically at the subsequent follow-up. the intensively progressive tumor degenerated the ocular appearance along with extensive necrotic tissues and severe chemosis. debriding of the lesion while the patient was under general anaesthesia, a number of live and motile maggots were found superficially and deep within the lesion. the vigorously motile larvae were mechanically removed superficially and from the canthal area as well as the ethmolidal sinus (fig. 1) .it seems that the burrowing larvae penetrated the orbital space through a 2-cm deep canal produced in the left canthal area. fig. 1. a number of third instar larvae removed from the lesion iranian j arthropod-borne dis, 2007, 1(2): 53-56 h razmjou et al.: ophthalmomyiasis caused by … 55 fig. 2. the anterior part of the larva showing hooklets fig. 3. the posterior end of the larva showing the characteristic spiracles iranian j arthropod-borne dis, 2007, 1(2): 53-56 h razmjou et al.: ophthalmomyiasis caused by … 56 agent description the larvae recovered were preliminary identified as sarcophaga sp (diptera: sarcophagidae) according to characteristic structures such as spiracle patterns and mouth parts (fig. 2, 3). the third instar larvae of the flesh flies is characterized by being densely spinulose, and by having a cephaloskeleton with a bifurcated dorsal cornua and an open peritermal ring of the posterior spiracles (zumpt 1965). in order to obtain adult flies for species identification, some of the larvae were reared on ground beef placed according to tirgari method with some modifications (tirgari et al. 2003), and the rest were preserved in 70% ethanol. the full grown larvae crawled into the soil and developed into adult fleshflies. based on above mentioned identification key, the adult flies were seen similar, in feature, to the species, s. haemorrhoidalis, but due to specimen shortage however, exact specification did not achieved. references cameron ja, shoukrey na, al-garni aa (1991) conjunctival ophthalmomyiasis caused by the sheep nasal botfly, oestrus ovis. am j ophthalmol. 112(3): 331-334. chodosh j, clarridge j (1992) ophthalmomyiasis: a review with special reference to cochliomyia hominivorax. clin infec dis. 14: 444449. glasgow bj, maggiano jm (1995) cuterebra ophthalmomyiasis. am j ophthalmol. 119(4): 512-514. greenberg b (1984)two cases of human myiasis caused by phaenicia sericata (diptera: caliphoridae) in chicago area hospital. j med entomol. 21(5): 615. james mt (1947) the flies that cause myiasis in man. u.s. dept agri misc pub no. i, washington dc. jiang cipeng (2002) a collective analysis on 54 cases of human myiasis in china from 1995-2001. chinese med j. 115(10): 14451447. langan sm, dervan p, o'loughlin s ( 2004) a moving scalp nodule in a returning traveler. br j dermatol. 151(6): 1270. sivaramasubramanyam p, sadanand av (1968) ophthalmomyiasis. br j ophthalmol. 52(1): 64-65. tirgari s, nateghpour m, javadian ah, akbarzadeh k (2003) first record of human myiasis caused by chrysomia bazziana (villeneuve) in iran (diptera, caliphoridae). iranian j publ health. 32(3): 68-70. wilhelmus k (1986) myiasis palpebrarum. am j ophthalmol. 101: 496-498. zumpt f (1965) myiasis in man and animals in the old world. butterworths, london. 267 pp. microsoft word 8-dehghani.doc iranian j arthropod-borne dis, (2008), 2(1): 49-52 r dehghani and t khamehchian: scrotum injury… 49 case report scrotum injury by scorpion sting *r dehghani1, t khamehchian2 1department of medical entomology, school of medicine, kashan university medical sciences, kashan,iran 2 department of pathology, school of medicine, kashan university medical sciences, kashan, iran (received 17 feb 2007; accepted 1 jan 2008) abstract androctonus crassicauda is the second most frequent causes of scorpion sting in south-west iran. its venom can cause sever pain, autonomic, central nervous system (cns), muscle function disturbances, and death. appropriate medical and nursing cares can lead to complete recovery with no sequel .the majority of scorpion stings are oligosymptomatic, occurring mainly on the hands and feet (about 90%). here one rare case of a scorpion sting on the scrotum is reported from kashan, central iran. keywords: scorpion sting, androctonus crassicauda, scrotum, iran introduction in iran 45000-50000 scorpion stings are reported every year (deghani 2003). according to the reports a total of 32 species of scorpions are found in iran (kovarik 1997). three species of hemiscorpius lepturus, androctonus crassicauda and mesobuthus eupeus play an important role in almost all cases of scorpion stings in iran. the black scorpion a. crassicauda (fig.1) and the gadim (the local name of h. lepturus) are considered as the most dangerous scorpions exist in sub-tropical areas of iran. scorpion stings recorded in khuzestan and hormozgan provinces have significant social hazard. about 5 species of scorpions were found in the kashan (dehghani et al. 1999). the city of kashan (33°58’28” n; 51°26’07” e, altitude ca. 850 m asl) in esfahan province (central iran), is one of the oldest cities of iran. this city is situated in a peculiar natural area. its west borders kavir salt desert (dasht-e kavīr), which is one of the seven desert plains of iran and one of the driest and the hottest parts of iran. on the east, the karkas mountains are located, while further to the west, there also is a large mountainous area, the north-eastern portion of the zagros mountains (dehghani 2003). a. crassicauda with sting being common and fatalities on record is of medical importance in the middle east. a. crassicauda is the first and most dangerous cause of scorpion sting in kashan. at serum therapy center of kashan, two-hundred cases were observed per year. thirty percent of the cases of scorpion sting in kashan and the total of mortality are caused by a. crassicauda. the rate of mortality was 1.5% (dehghani et al. 1999). androctonus genus is very toxic, with symptoms of envenomation including malignant hyperthermia, myocarditis and pulmonary edema and with many lethal stings on record (nouira et al. 1995). typical effects of the sting are pain and tenderness at the injection site, with severe neurological effects and hypertension (nouira et al. 1995). a. crassi*corresponding author: dr r dehghani, tel: +98 0913 3610919, fax: +98 0361 5550111, e-mail: dehghani37@yahoo.com iranian j arthropod-borne dis, (2008), 2(1): 49-52 r dehghani and t khamehchian: scrotum injury… 50 cauda is the second frequent of scorpion sting in south-west iran. its venom can cause severe pain, autonomic cns, muscle function disturbances and death. appropriate medical and nursing cares can lead to complete recovery with no sequel (radmanesh 1990). the majority of scorpion stings occurring mainly on the hands and feet (about 90%) are oligosymptomatic and fatality is rare. here we report a rare case of a scorpion sting on scrotum from kashan, central iran. case report a 38-yr old man with 88 kg weight, when had been wearing his work trousers was stung on his scrotum by a. crassicauda. it was associated with burning pain at the site of sting. administration of alcohol and pressuring on the sting site were used by victim without success to relieve pain. he received medical assistance one hour after the event when he suffered from diffuse erythema, edema and severe local pain on the scrotum and inguinal lymph node and when severe systemic manifestation including: sweating, thirsty, dry mouth, agitation and restlessness and local muscle spasm were observed. he did not complain of visual disturbances, sensory disturbances or weakness of his limbs. lidocaine was administered subcutaneously for the immediate relief of the pain. antivenin to scorpion sting was not administrated because it was not available at that time in the center, but he was managed with atropine in order to antagonize the muscarinic effect of ach and chlorpromazine as a tranquilizing of the agitation and restlessness. discussion envenomation by scorpion stings is a major public health problem especially in children in iran (radamnesh 1990). scorpions are held responsible for a lot of injured annually, especially in the rural areas and during the hot seasons (dehghani 2003). envenoming by the iranian scorpion a. crassicauda can cause massive discharge of catecholamine. the relationship between these effects and the clinical situation is unclear (vatanpor 2003). a. crassicauda venom can specifically stimulate the acetylcholine receptors through the body, so it can be considered as neurotoxic venom (radmanesh 1990). the classical clinical picture of scorpion stings is characterized by local pain, which may extend to regional lymph nodes, hyperemia and edema (garcia et al. 1999). systemic manifestations are meiosis, lacrimation, strabismus, increased salivation, dry mouth, thirst, increase bronchial secretion, sweating, nausea, vomiting, priapism, urinary incontinence, confusion, coma, local muscle spasm, general muscle paralysis, convulsion, more commonly in children and old people (radmanesh 1990). hypertension, hypothermia, cardiac arrhythmia and pulmonary edema may also occur. there are only three reported cases of stings on unusual areas of the body: one on the face (nishioka et al. 1992) by t. serrulatus and two on the penis (nishioka et al. 1993), both by t. serrulatus and t. trivitatus (garcia et al. 1999). in addition to unusual site of the sting, our patient showed a clinical picture of mild intensity. self-administration of alcohol may have contributed to augmenting the pain. a sting on the scrotum should be treated like stings on any other part of the body. local analgesic block with 1% lidocaine for pain relief is usually resorted to. for those cases of systemic manifestation, a specific antivenin is indicated. using prazosin for treatment of hypertension and acute pulmonary edema has been reported to be successful. in our case report the scorpion was the a. crassicauda species, and mild systemic manifestations were observed. a. crasicuda venom can increases the release of acetylcholine at the neuromuscular junction by both increasing quantal content as well as the release after single shock iranian j arthropod-borne dis, (2008), 2(1): 49-52 r dehghani and t khamehchian: scrotum injury… 51 stimulation (vatanpor 2003). in addition injection of a. crassicauda venom in animal laboratory such as rat, clinical manifestations were occurred include: paralysis, irregular pulse, twitching, rhinorrhea, salivation, hemorrhage from eyes, nosebleeds and death (dehghani et al. 2006). the venom of a. crasicuda predominantly parasympathetic effects but it is possible that under special circumstances it may also cause sympathetic activities (radmanesh 1990). the venom of a. crasicuda is a potent autonomic stimulator. severity of symptoms depends on the size of the victim, season, and duration lapse between sting and hospitalization. so therapy should disappear such effects. first aid measures should include putting ice on the sting site and elevation of the affected part. general supportive measures include the use of antihistamines, corticosteroids, and analgesics. barbiturate therapy is recommended to control seizures and excessive neuromuscular activity (carbonaro et al. 1996). we recommend that the treatment for scorpion stinging in kashan should be based on the neurotoxic effect of their venom produced in victims (dehghani et al. 1998). in many cases, the patients were stung by scorpions due to their lack of knowledge and carelessness such as in putting their hands into scorpion homes, walking barefoot, lifting up stones carelessly, and putting on their clothes and shoes without shaking them to check for scorpions (ozkan et al. 2005). in conclusion, people living in those regions where most scorpion stings were seen must be educated and informed about scorpions and their stings. fig. 1. androctonus crassicauda (scorpion from kashan) acknowledgements the authors are grateful to mr hoseini, kashan medical sciences university for his cooperation references carbonaro pa, janniger ck, schwartz ra (1996) scorpion sting reactions. cutis. 57(3):139-141. iranian j arthropod-borne dis, (2008), 2(1): 49-52 r dehghani and t khamehchian: scrotum injury… 52 deghani r (2003) the impact of thermotherapy on healing of hemiscorpius lepturus scorpion sting in mice, and its clinical and hematological features in rat; determining the fractions of the venom of native scorpion mesobuthus eupeus using tricine-sds-page [phd dissertation]. school of public health, tehran university of medical sciences, iran. dehghani r, khademi m, sayyah m (1999) a survey of scorpion stings in central iran. arch iranian med. 2(2) april, abstract. dehghani r, tirgari s, sayyah m (1998) a survey on scorpion fauna in kashan. pajouhesh va sazandegi. 38: 126–127. dehghani r, khamechian t, asadi ma (2006) surveying the effect of androctonus crassicauda venom on clinical manifestations of rat. feyz journal of kahshan university sciences. 10(2): 17-20. garcia as, freitas dg, filho of (1999) penis wound by scorpion sting. sao paulo med j rev paul med. 117(2): 85-86. kovarik f (1997) results of czech biological expedition to iran part 2. arachnida: scorpiones, with description of iranobuthus krali gen.n.et sp.n and hottentotta zagrosensis sp.n. (buthidae). acta soc zool bohem. 61: 39-52. nishioka sa, silveira pvp, ugrinovich r, de oliveira rb (1992) scorpion sting with cranial nerve involvement: letter toxicon; 30: 685. nishioka sa, silveira pvp, pereira cad (1993) scorpion sting on the penis: case report. j urol.150:1501. nouira s, abroug f, haguiga h, jaafoura m, boujdaria r, bouchoucha s (1995) right ventricular dysfunction following severe scorpion envenomation. chest. 108(3): 682-7. ozkan o, kat i (2005) mesobuthus eupeus scorpionism in sanliurfa region of turkey. j venom anim toxins incl trop dis. oct/ dec. 11(4): 479-491. radmanesh m (1990) androctonus crassicauda sting and its clinical study in iran. journal of tropical medicine and hygiene. 93: 323-326. vatanpour h (2003) effects of black scorpion androctonus crasicuda venom on striated muscle preparation in vitro. iranian journal of pharmaceutical research. 17-22. j arthropod-borne dis, december 2013, 7(2): 194–198 m soleimani-ahmadi et al.: first report of… http://jad.tums.ac.ir published online: august 31, 2013 case report first report of pharyngostomy wound myiasis caused by chrysomya bezziana (diptera: calliphoridae) in iran *mussa soleimani-ahmadi 1, 2, hassan vatandoost 3, ahmad ali hanafi-bojd 3, fatemeh poorahmad-garbandi 4, mehdi zare 5, seyed mohammad vahid hosseini 6 1department of medical entomology and vector control, school of public health, hormozgan university of medical sciences, bandar abbas, iran 2infectious diseases research center, hormozgan university of medical sciences, bandar abbas, iran 3department of medical entomology and vector control, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran 4bandar abbas health center, hormozgan university of medical sciences, bandar abbas, iran 5department of occupational health engineering, school of public health, hormozgan university of medical sciences, bandar abbas, iran 6department of pediatric, school of medicine, hormozgan university of medical sciences, bandar abbas, iran (received 27 aug 2012; accepted 15 apr 2013) abstract wound or traumatic myiasis is the infestation of animal and human orifices or wounds by dipterous larvae. it is more common in tropical and sub-tropical countries. chrysomya bezziana is a major agent of wound myiasis throughout the tropical regions of the old world. in iran many cases of human myiasis due to c. bezziana were reported from south and south-east of country. this study reports a case of wound myiasis in a 3-year-old pharyngostomized girl who referred to the pediatric hospital in bandar abbas for pharyngological follow-up. during the examination, several live and mobile larvae were removed from the lesion. the patient received antibiotics and then transferred to pediatric ward for respiratory care. the specimens were identified as c. bezziana according to the morphological characters of fully grown larvae. this is the first report of the pharyngostomy wound myiasis caused by c. bezziana in iran. this finding also confirms the results of previous studies indicating the occurrence of the old world screwworm fly, c. bezziana as a causative agent of human myiasis in the south of iran. keywords: chrysomya bezziana, pharyngostomy, wound, myiasis, iran introduction wound or traumatic myiasis is the infestation by dipterous larvae of primarily the cutaneous tissues in animals and humans, usually at the sites of orifices and wounds openings into the body (sherman et al. 2000). besides open wound, predisposing factors for wound myiasis in human include poor social condition, poor overall hygiene, advanced age, very young age, illness, mental retardation, diabetes and physical inability to prevent flies from depositing their ova (mcgraw and turiansky 2008). cochliomyia hominivorax and chrysomya bezziana, new world and old world screwworms, respectively, are two important species of screwworm flies that cause obligatory wound myiasis in humans, domestic mammals, and wild mammals (robbins and khachemoune 2010). human cases of c. bezziana myiasis are most prevalent in india and southeast asia (mcgraw et al. 2008). in iran, the first case of human myiasis due to c. bezziana was reported in 1978 (jdalayer et al. 1978). re*corresponding author: dr mussa soleimaniahmadi, e-mail: mussa.soleimani@yahoo.com 194 j arthropod-borne dis, december 2013, 7(2): 194–198 m soleimani-ahmadi et al.: first report of… http://jad.tums.ac.ir published online: august 31, 2013 cently, human cases of myiasis caused by c. bezziana in south and south-east of iran have been increased as nasal myiasis (tirgarie et al. 2003), scalp myiasis (davami et al. 2005, soleimani ahmadi et al. 2009), oral and traumatic myiasis (faramarzi et al. 2009), and orbital myiasis (khataminia et al. 2011). the present paper reports a case of pharyngostomy wound myiasis due to c. bezziana for the first time in iran. case report a 3-year-old girl who resides in a rural area in sirik district, hormozgan province, south of iran referred to the pediatric hospital in bandar abbas, in july 2011. she ingested aluminum hydroxide powder by accident. patient underwent flexible pediatric endoscopy by a gastroenterologist on arrival, which showed severe burns of oral cavity and esophagus. she received antisecretory agent, antibiotics, and hydrocortisone. physicians devised a pharyngostomy tube in right side of her neck by number 32 mushroom catheter and the esophagus was closed by a prolene mesh plug which completely was filled with cyanoacrylate glue to prevent swallowing of saliva (fig. 1). nine months after the first diagnosis, the patient referred to hospital for pharyngological follow-up. during examination of the patient, with a lateral motion of the flange of the pharyngostomy tube, many larvae observed between the tube and the pharyngeal wall. the lesion was macroscopically cleared of visible larvae by mechanical removal. the procedure had to be repeated three more times to extract all larvae. a total of 23 third-instar larvae were collected from the patient, the bed and the floor of the ward. the patient received antibiotics, anti-secretory agent and then transferred to surgical icu for respiratory care. larvae were preserved in 70% methanol and sent to the entomology laboratory, school of health, hormozgan university of medical sciences for identification. the larvae were 12–15 mm long, whitish and without prominent papilla. other morphological characters of the specimens included, open peritreme, slightly pigmented tracheal trunks, posterior margin of segment 11 with dorsal spines, anterior spiracles with 4–6 papillae, and each posterior spiracles encircled by a heavily sclerotised peritreme which is incomplete ventrally and with 3 slit-like spiracular opening at about 45 degrees to the horizontal (fig. 2). the full grown larvae were identified using the morphological characters based standard key (zumpt 1995). fig. 1. human wound myiasis caused by chrysomya bezziana in a girl with pharyngostomy after removing the larvae (original photo) fig. 2. (a) the full grown third-instar larvae of chrysomya bezzianna, (b) posterior and anterior (c) spiracles (original photo) 195 j arthropod-borne dis, december 2013, 7(2): 194–198 m soleimani-ahmadi et al.: first report of… http://jad.tums.ac.ir published online: august 31, 2013 discussion human myiasis is a rare condition in any part of the world, but is more common in the regions with a warm and humid climate. the old world screwworm, c. bezziana, is a major agent of traumatic myiasis throughout the tropical regions of the old world (spradbery 1994). chrysomya bezziana occurs throughout tropical africa, indian subcontinent, in most of the south-east asia to china, and the philippines to papua new guinea (goddard 1996). it also has been introduced to several countries on the coast of persian-gulf, including iran (jdalayer et al. 1978, tirgarie et al. 2003, soleimani ahmadi et al. 2009, faramarzi et al. 2009, khataminia et al. 2011), iraq (abdul rassoul et al. 1996, siddig et al. 2005), saudi arabia (ansari and oertley 1982, alahmed 2002), oman (spradbery et al. 1992, hall et al. 2005), bahrain (siddig et al. 2005) and united arab emirates (spradbery and kirt 1992). the old world screwworm is largely restricted to tropical and subtropical climates, being most successful under hot and wet conditions and, conversely, most sensitive to prolonged cold and dryness (sutherst et al. 1989). the tropical climate of south of iran is a factor that is suitable for growing flies. in this area the incidence of myiasis was generally low and seasonal, with most of cases reported during cooler seasons of the year when there is occasional rainfall (soleimani ahmadi et al. 2009). myiasis has become increasingly prevalent, particularly when human activity is carried out in environments with poor hygiene or near to domestic animals (romero-cabello et al. 2010). open wounds and body orifices that emit odors of natural secretions are major factors in susceptibility to myiasis, as they provide a favorable environment for the attraction and oviposition of flies (batista-da-silva et al. 2011). one major problem in myiasis is the potential transfer of bacterial pathogens. in general, larvae causing myiasis have been demonstrated to carry a broad spectrum of bacteria (sesterhenn et al. 2008). a reason for acquiring this rare miyasis infestation was that the patient had open wound which emits odors attractive for c. bezziana and stimulates the female insects to deposit eggs. another reason is that, our case lived near to livestock in rural area which provides suitable condition for occurrence of c. bezziana. moreover, poor personal hygiene and the lack of self-care ability might have played the role as predisposing factors for myiasis infestation. prevention of human wound myiasis involves control of fly populations and personal hygiene. window screens, mosquito nets, repellents, and insecticides can be used for controlling the fly populations and prevention of flies from entering houses and hospitals. in addition, prevention of this disease is accomplished by proper dressing of wound and, considering the wound hygiene. recently, human cases of myiasis caused by the old world screwworm fly, c. bezziana in south and southeast of iran have been increased (faramarzi et al. 2009, khataminia et al. 2011). this study also confirmed c. bezziana as a causative agent of human wound myiasis in the south of iran. therefore, increasing awareness of medical personnel about clinical symptoms and pro-per diagnosis and treatment of disease is necessary. acknowledgements the authors would like to appreciate the personnel of pediatric hospital of bandar abbas for providing the larval samples. the 196 j arthropod-borne dis, december 2013, 7(2): 194–198 m soleimani-ahmadi et al.: first report of… http://jad.tums.ac.ir published online: august 31, 2013 authors declare that they have no conflicts of interest. references abdul rassoul ms, ali ha, jassim fa (1996) notes on chrysomya bezziana villeneuve (diptera: calliphoridae), first recorded from iraq. bull iraq nat hist mus. 8: 113–115. alahmed am (2002) incidence of myiasis in sheep caused by chrysomya bezziana in saudi arabia. j kin saud uni agric sci. 14: 109–112. ansari ma, oertley re (1982) nasal myiasis due to bezzi’s blowfly (screwworm): case report. saud med j. 3: 275–278. batista-da-silva ja, borja ge, queiroz mm (2011) patient with tracheostomy parasitized in hospital by larvae of the screwworm, cochliomyia hominivorax. j insect sci. 11: 163. davami mh, kiani a, salimi m, farhadi e (2005) case report: myiasis of scalp due to chrysomya bezziana. iran j dermatol. 8: 311–315 (persian with english abstract). faramarzi a, rasekhi ar, kalantari m, hatam gr (2009) chrysomya bezziana as a causative agent of human myiasis in fars province, southern iran . iran j arthropod-borne dis. 3: 60–63 goddard j (1996) flies whose maggots cause myiasis in humans. in: physician’s guide to arthropods of medical 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phlebotomus papatasi, the main vector of zoonotic cutaneous leishmaniasis, to different who recommended insecticides in an endemic focus, central iran zahra saeidi1, hassan vatandoost1,2, morteza zaim1, reza ahmadkhaniha3, yavar rassi1, alireza zahraei-ramazani1, mohammad hossein arandian4, reza jafari4, yamdeu joseph hubert galani5,6, alireza sanei-dehkordi7, *mohammad reza yaghoobi-ershadi1,* amir ahmad akhavan1,2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of human ecology, school of public health, tehran university of medical sciences, tehran, iran 4esfahan research station, school of public health, tehran university of medical sciences, tehran, iran 5school of food science and nutrition, university of leeds, leeds, ls2 9jt, uk 6section of natural and applied sciences, school of psychology and life sciences, canterbury christ church university, canterbury, ct1 1qu, uk 7infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran *corresponding author: dr mohammad reza yaghoobi-ershadi, e-mail: yaghoobi.reza@gmail. com, dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir (received 29 june 2021; accepted 1 november 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: among neglected zoonotic diseases, leishmaniases caused by leishmania parasite through infected female sand fly bite, are a group of diseases found in 98 countries and territories representing a critical burden of disease worldwide. vector management plays a crucial role in reducing the burden of vector-borne diseases by who’s global plan. the objective of the current study was to assess the susceptibility status of wild phlebotomine sand flies from esfahan province, central iran, to the recommended insecticides by who. methods: sand flies were collected by mouth aspirator in matin abad desert eco-resort and were tested using who adult mosquito test kit against dichlorodiphenyltrichloroethane (ddt) 4%, deltamethrin 0.05%, malathion 5% and propoxur 0.1%. the number of knockdown sand flies were recorded during exposure time in ten minutes interval for ddt and deltamethrin and they were allowed to recover for 24 hours. knockdown time50 (kd50) and kd90 were generated for them using probit software. they were mounted and identified by valid keys. results: among the tested insecticides against female phlebotomus papatasi, ddt, deltamethrin, and malathion recorded the highest mortality rate of 100%, followed by propoxur with 92.2% mortality for a one-hour exposure. for ddt, kd50 and kd90 were calculated 21.87 and 42.93 and for deltamethrin, they were 23.74 and 56.50 minutes respectively. total sand flies exposed with ddt and deltamethrin shed their leg(s). conclusion: it is concluded that ph. papatasi from central iran is susceptible to ddt, deltamethrin, malathion, and propoxur. keywords: phlebotomus papatasi; insecticide; susceptibility; iran j arthropod-borne dis, dec 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... 367 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... introduction leishmaniases caused by parasite (protozoan) are a group of neglected zoonotic diseases (nzds) that draw more attention among all the neglected tropical diseases (ntds). there are over 20 leishmania species that are transmitted by the female phlebotomine sand flies through infected bite; a total of 98 sand fly species are identified as medically important vectors (1– 3). the most common form of the disease is cutaneous leishmaniasis (cl) that causes on the exposed parts of the body skin lesion/s mostly ulcer/s and long-life scares (2). although cl is a self-healing form of the disease, it creates permanent scars and serious disability (4). approximately 95 % of cl cases occur in the middle east mediterranean basin in the old world, and central asia and the americas in the new world, and 70% of worldwide cases are related to the eastern mediterranean region (2). in 2018 it was reported that 85 % of cases occurred in 10 countries including iran (2). according to the 2018 who report, 98 countries and territories are endemic for leishmaniasis (5). more than 200,000 new cases reported in 2018 and the disability adjusted life years (dalys) were about 260,000 in 2017(6). also cl is one of the skin ntds affecting subcutaneous tissue and skin resulting in disfigurement, disability, stigmatization, and other socio-economic problems (7). in iran, leishmaniasis is endemic in many rural areas of 18 provinces out of 31 (cdc, ministry of health and medical education, iran, unpublished data) in the way that several research groups have worked on different aspects of the disease. in addition, some international courses about the disease and its control were conducted which attracted lots of interest among different countries (8– 21). phlebotomus papatasi is the first line incriminated vector of zoonotic cutaneous leishmaniasis (zcl) in iran. studies have shown there are 48 species of sand flies, among which 30 species belong to the genus phlebotomus, and 18 species of the genus sergentomyia. four species of the family cricetidae of rodents are considered as the main reservoir host including rhombomys opimus, meriones libycus, tatera indica, and meriones hurrianae in different parts of iran (22). diseases transmitted by vectors cause a critical burden in the world, especially in tropical and neotropical areas. several important vector-borne diseases as parts of ntds or skin ntds in public health continue to need to intensify vector control interventions aimed at monitoring and reducing transmission. the who has several global plans to combat ntds for decades by the multi-intervention packages including integrated vector management (ivm) (23–28). vector control has a vital role to play in reducing the burden of vectorborne diseases. however, vector control also has proven well-known weaknesses, including the development of insecticide resistance in vectors that played a critical role in the breakdown of the eradication, elimination, and even controlling. today, there is a need to learn how to monitor and manage vector resistance in a better way (23). control methods include insecticide spray, use of insecticide-treated nets, environmental management, and personal protection (2). residual spraying for endophilc, exophilic, and peridomestic sand flies is recommended by world health organization pesticides evaluation scheme (whopes). various insecticide classes can be used for indoor residual spraying (irs), such as organochlorines (for example ddt), synthetic pyrethroids (for instance deltamethrin and lambda-cyhalothrin), organophosphates (for example malathion), and carbamates (for example propoxur) (29). although major scientific breakthroughs have been made worldwide during recent decades in the different aspects of leishmaniases diagnosis, prevention, treatment and control, morbidity and mortality of that disease still show a worrying raising trend (29). vector control with insecticides remains one of the most efficient approach to tackle the disease, and targeting 368 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... adult insects with insecticide compounds has shown good results on the spread of the disease. however, non-managed application of insecticide as harmful poisons in any way can result in long or short term toxicity. it is therefore critical to assess the susceptibility or resistance of vectors against the current or foreseen used insecticides. toxicity cannot be defined as a single molecular event, it is a cascade of events that start with “exposure”. it proceeds through “distribution and metabolism” and ends with “interaction with cellular macromolecules” and expresses with” toxic endpoint” (30). insecticides susceptibility testing has a long history worldwide. wood (1962) tested aedes aegypti against ddt 1% and 2% and dieldrin 0.1% and found females more tolerant than males, pener and wiliamovsky (1987) tested ph. papatasi, a colony originating from the jordan valley, against ddt, permethrin, and methoxychlor. they found sand flies susceptible to ddt and permethrin but tolerant to methoxychlor, el-sayed, et al (1989) worked on baseline susceptibility of ph. papatasi and mechanism of resistance by comparing the process with ddt-resistant and susceptible strains of culex quinquefasciatus and anopheles gambiae (31– 33). the susceptibility investigation on sand flies is less explored due to lack of specific protocol, and researchers who studied it had to follow the test procedure of mosquitoes. in this study, the susceptibility of sand flies as the main vector of zcl in iran was tested against ddt, deltamethrin, malathion, and propoxur, following the commonly used mosquitos’ protocol, to pave the way towards further studies on sand fly susceptibility with a specific protocol. materials and methods study area this study was conducted in matinabad desert eco-resort, matinabad village, badroud rural district, natanz county (33.753584 n, 51.990596 e), located 60 km southeastern of kashan city, esfahan province, central iran (fig. 1). this area is one of the most important endemic focus of zcl and one of the most popular touristic desert eco-resort in iran which received the peace and environment award of 2015 (34). the average annual rainfall was 15.44 mm, the average temperature was between 11.3 to 21.3 °c, and relative humidity reported fig. 1. map of study area, esfahan province, iran fig. 1. map of study area, esfahan province, iran 369 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... between 30.3–50.7% (meteorological organization esfahan province). sand fly collection sand flies were collected using a filtered mouth aspirator, most of them on a car trap inside matinabad desert eco-camp before sunset till early in the next morning around haloxylon bushes and rodent burrows, from july to september 2019. sand flies were kept in the cage with a wet towel and were transported to the sand fly insectary in esfahan health station. susceptibility tests were conducted the day after in the laboratory. sand flies were fed with cotton soaked in 10% sucrose solution, and the insects were kept at 25–28 ºc temperature, 70–90% relative humidity, and 14:10 l:d photoperiod. insecticides all who testkit tubes and impregnated papers were procured by cdc, ministry of health and medical education, iran by the who collaborating center in university sains malaysia, penang, malaysia. the choice of insecticides was based on highly recommended who insecticides at least one from each class such as organochlorine: dichlorodiphenyltrichloroethane (ddt) 4% (batch no: dd 265), organophosphate: malathion 5% (batch no: ma 234), carbamate: propoxur 0.1% (batch no: pr 123) and pyrethroid: deltamethrin 0.05% (batch no: de 527). bioassay (susceptibility) tests since there is no integrated standard protocol for susceptibility testing of sand flies, they were tested using adult susceptibility test procedures of adult mosquitoes based on who the latest protocol of 2018. (28) the who susceptibility tube test is a kind of “direct response-to-exposure” test. it measures mosquito mortality to a known standard concentration of a given insecticide, either with a discriminating concentration or with intensity concentrations.(28) control papers were prepared using ‘acetone and silicone oil’-impregnated paper (0.66 ml oil + 1.34 ml acetone) as a control for ddt and pyrethroid group and ‘acetone and olive oil’impregnated paper (0.71 ml oil + 1.29 ml acetone) as a control for organophosphate and carbamates according to the standard method of world health organization pesticides evaluation scheme (whopes) institute of research for development (ird), montpellier, france. standard procedure sand flies were offered a 10 % sucrose solution for water and energy sources and kept in insectary condition, then transferred to the tubes about one hour prior to starting the test. insecticide impregnated papers inside test tubes kept refrigerated in a plastic bag were put at room temperature about 1 hour prior to the test. all sand flies were exposed to insecticides for one-hour paralleling with control tubes for each replication. at the end of exposure time, all tubes were kept in insectary condition (t: 25–28 °crh: 70– 90%) for 24 hours to recover after exposure, with a cotton pad containing 10% sucrose on the top net. then the mortality of sand flies in both test and control tubes was read and recorded the day after (28). all sand flies that had the ability to fly were considered alive, regardless of leg losing. the number of knocked down sand flies was recorded every 10 minutes for sand flies exposed to ddt and deltamethrin. if observed mortality in control groups after 24 h recovery time ranged between 5 to 20%, mortality in the test tubes of that group should be corrected using abbott’s formula (35). if observed mortalities in control groups exceeded 20%, the entire tubes of that group were discarded. for mortality percentage calculation and correction of mortality the following formulas, adopted from who (2016) were used (28). total number of dead sand flies observed mortality *100 total sample size = ( ) ( ) % observed mortality % control mortality corrected mortality *100 100 % control mortality − = − based on the 2018 who test procedure 370 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... if the mortality recorded equal or more than 98%, the tested group will be categorized as susceptible; if the mortality ranged between 90 to 97% it shows the resistance possibility. when it happens, the test must be repeated. if the second test mortality is less than 98% the resistance is confirmed. if the mortality recorded less than 90%, we are facing a confirmed resistance. then researchers can determine the intensity of resistance or mechanism of resistance by applying following the protocol (28). sand flies testing total number of 1316 unfed female ph. papatasi sand flies have been tested. since they were wild, all fed, gravid, semi-gravid females, all males, and other species were excluded at the time of transferring to the test tubes, checking mortality, mounting and also during identification. susceptibility tests were carried out on six to fifteen replications in several rounds to obtain enough sand flies tested (at least 100 for each insecticide) with relevant enough control tube/s in each group in parallel. sand fly mounting and identification all sand flies tested after recording the mortality results, transferred to ethanol 70% for mounting and identification. they were mounted in pouri’s media and mounted sand flies’ species were identified based on valid identification keys (36, 37). knockdown effect and leg loss the number of knocked down sand flies was counted in the ddt and deltamethrin test tubes and recorded every ten minutes during the exposure time. sand flies leg loss was investigated and recorded after 24h recovery in males and females. data analysis/ knockdown curve the knock down time regression line was created for ddt and deltamethrin using probit software and data analysis was made with 95% confidence interval and the kd50 and kd90 were calculated (table 1, fig 3,4) (38) number of sand flies tested shows in table 2. results knock down time50 (kd50) and kd90 the number and percent of knock down sand flies are shown in fig. 2 and 3. the probit parameters and the kd50 and kd90 with 95% confidence interval were calculated (table 1). table 1. the parameters of probit regression line of knockdown times for wild-caught sand flies matinabad desert ecoresort, esfahan province, 2019 insecticide name a b ± se kd50, (lcl-ucl) 95% c.i. kd90, (lcl-ucl) 95% c.i. x2 (df) p value deltamethrin 0.05% -4.68 3.4 ± 0.326 19.9 46.75 12.93 (4) <0.05 23.74 56.5 27.44 75.43 ddt 4% -5.86 4.38 ± 0.495 17.74 35.81 23.78 (4) <0.05 21.87 42.93 25.69 56.84 a = y-intercept b = the slope of the line; se = standard error; kd50, 95 % ci = time causing 50 % knockdown and its 95 % confidence interval kdt90, 95 % ci = time causing 90 % knockdown and its 95 % confidence interval lcl: lower confidence limit ucl: upper confidence limit x2 = heterogeneity about the regression line df = degree of freedom p value = represent heterogeneity in the population of tested table 1. the parameters of probit regression line of knockdown times for wild-caught sand flies matinabad desert eco-resort, esfahan province, 2019 371 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... total sand flies exposed with ddt and deltamethrin shed their leg(s). additionally, sand flies exposed to these two insecticides experienced the “knockdown effect” evidently by muscle spasm, involuntary movement/move less (convulsion or erratic movement or paralysis) during the exposure time (39). it was observed that sand flies exposed with ddt had more involuntary movements and then the ones exposed with deltamethrin who were more moveless. susceptibility status the susceptibility status of female sand flies is shown in table 2. the mortality rate of sand flies exposed to propoxur has shown a possible resistance in the first round of test and according to the most recent test protocol, the test was repeated in 2 more fig. 2. the number of knockdown sand flies in ten-minute intervals during exposure time with ddt and deltamethrin. matinabad desert eco-resort, esfahan province, 2019 0 50 100 150 200 250 0 10 20 30 40 50 60 70 n u m b e r o f k n o c k d o w n d time (min) number of knockdown sand flies in ten minute interval ddt 4% deltamethrin 0.05% fig. 2. the number of knockdown sand flies in ten-minute intervals during exposure time with ddt and deltamethrin. matinabad desert eco-resort, esfahan province, 2019 fig. 3. knockdown percent of sand flies exposed to ddt and deltamethrin for one hour in tenminute intervals. matinabad desert eco-resort, esfahan province, 2019 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 pe rc en t o f k no ck do w n (± sd ) time (min) percent of knockdown sand flies in ten minute interval ddt 4% deltamethrin 0.05% fig. 3. knockdown percent of sand flies exposed to ddt and deltamethrin for one hour in ten-minute intervals. matinabad desert eco-resort, esfahan province, 2019 372 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... rounds and replications obtaining enough number sand flies. in the current study, 1248 female ph. papatasi were exposed to different standard discriminative concentrations of insecticides. one hundred and thirty-four unfed adult ph. papatasi were exposed to ddt 4% which resulted in 100% mortality, showing that this species is susceptible to ddt insecticide. for deltamethrin 0.05% and malathion 5%, 138 and 223 females were tested respectively, and both of them resulted in 100% mortality that was determined as a totally susceptible population. one hundred and five sand flies were tested against propoxur 0.1% and resulted in 90.4% mortality which was shown as resistance at the first glance. according to the 2018 who guideline, the second round of tests was done using 133 females and resulted in 99.2% mortality that was evaluated as susceptible species. while control groups were tested in parallel for each batch accordingly by recorded mortality of 1.1 and 0.9% for acetone/ silicone oil and 1.3% and 1.2% for acetone/ olive oil respectively. survival curve regression analysis was performed for ph. papatasi to estimate kd50 and kd90 fig. 4. the regression line for ddt and deltamethrin for sand flies exposed for one-hour. matinabad desert eco-resort, esfahan province, 2019   3 3.5 4 4.5 5 5.5 6 6.5 7 10 100 pr o b it time (min) ddt 4% deltamethrin 0.05% fig. 4. the regression line for ddt and deltamethrin for sand flies exposed for one-hour. matinabad desert ecoresort, esfahan province, 2019 table 2. susceptibility status of female phlebotomus papatasi to different insecticidesmatinabad desert eco-resort, esfahan province, 2019 insecticide/ concentration test control result total no. of unfed females no. of dead mortality rate (%) total no. of unfed females no. of dead mortality rate (%) susceptibility status ddt 4% 134 134 100 174 2 1.1 susceptible deltamethrin 0.05% 138 138 100 109 1 0.9 susceptible malathion 5% 223 223 100 75 1 1.3 susceptible propoxur 0.1% 1st round 105 95 90.47 75 1 1.3 propoxur 0.1% 2nd round 133 132 99.24 82 1 1.2 susceptible table 2. susceptibility status of female phlebotomus papatasi to different insecticidesmatinabad desert ecoresort, esfahan province, 2019 373 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... for ddt and deltamethrin with a 95% confidence interval. a knockdown time regression line was created for them showed in fig. 4. discussion the objective of the current research was to investigate the susceptibility/resistance of ph. papatasi to certain insecticides from various chemical classes using the who test kit. world health organization bioassay susceptibility test kit is a direct responseto-exposure test that is vital in insecticide resistance management worldwide (24). the discovery of ddt in 1939 was one of the most meaningful developments in the history of pest control. deltamethrin also was the most active insecticide ever known at the time of its discovery. continuous use of many insecticides is a potential threat in the field of emerging resistance in insects (40). wild-caught ph. papatasi in the current study was found to be susceptible to organochlorine (ddt 4%), organophosphate (malathion 5%), pyrethroid (deltamethrin 0.05%), and carbamate (propoxur 0.1%). there are studies conducted worldwide on baseline susceptibility of various species of sand flies in different countries. old world in india phlebotomus argentipes reported resistant to ddt in 1992 and in different parts of bihar they found developing resistance to ddt 4% in 2001. (41, 42) in the latter study they do their research on a different species from our study. in north africa and the middle east researchers worked on bendiocarb, cyfluthrin, ddt, malathion, permethrin, and resmethrin on phlebotomus bergeroti, phlebotomus langeroni, ph. papatasi and phlebotomus sergenti in 2001. they worked on four different species from ours and various insecticide and reported 3 least toxic insecticides in order of toxicity permethrin, malathion and ddt, with ddt being the least toxic. it has been stated that the response to three other insecticides: bendiocarb, cyfluthrin and resmethrin has not been as uniform among species (43). in italy (2002) phlebotomus perniciosus and ph. papatasi were susceptible to ddt 2%, lambda-cyhalothrin 0.06% and permethrin 0.2% (44). it agree with our study while the percentage of ddt is less than current experiment. in some parts of india and nepal border ph. argentipes in 2010–2012 reported resistant to ddt 4% and susceptible to deltamethrin 0.05% and malathion 5%. they conducted the study on different species and their result about ddt was not same as ours. it is explained that the use of ddt in irs measures for vl control could effect on sand flies susceptibility (45, 46). also, in 2012 ph. papatasi and ph. sergenti were susceptible to ddt and lambda-cyhalothrin in morroco, in parallel with our report (47). in some area of sudan populations of ph. papatasi was susceptible to ddt, permethrin, malathion, and propoxur that is agree to our research and in some area sensitive to ddt and permethrin but highly resistant to malathion and propoxur in 2012 which is probably due to anti-malaria activities during last 50 years in the area and it is on the contrary to our experiments (48). in 2015 ph. argentipes in west bengal, india reported developing resistance to ddt (49). in kerala ph. argentipes, ph. sintoni, sergentomyia bagdhadis, se. zeylonica and se. babu were susceptible to ddt and deltamethrin (50). phlebotomus argentipes in 2016 reported resistance in kala-azar endemic region and susceptible to ddt in the non-endemic region in india (51). all of these last-mentioned studies have been done by different sand flies species from ours. in two different western provinces of turkey with and without a background of insecticides use, populations of sand flies found resistant and susceptible to deltamethrin 0.05% and permethrin 0.75% respectively (2017) as a result of long term application of both insecticides in the region (52). they did not mention the species of sand flies tested. in 13 villages of bihar ph. argentipes as a different species from our region was highly susceptible to deltamethrin, 374 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... lambda-cyhalothrin, alpha-cypermethrin (2016) (53). in nepal and bangladesh ph. argentipes was highly susceptible to alphacypermethrin 0.05%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, permethrin 0.75%, malathion 5% and bendiocarb 0.1% in 60 min of exposure (2017) (54). in the last two studies they tested ph. argentipes that this species is a vector on that area but not in iran. also in iran, there are studies on the susceptibility status of sand flies. during 1985–88 seyedi rashti et al experimented on various areas of iran with the treatment background with ddt which discontinued from 1969. they expressed that sand flies from esfahan showed more tolerance against ddt in comparison to other areas. (8) but our experiments show different condition in this area now. yaghoobi ershadi and javadian found ph. papatasi tolerant to ddt 4% in borkhar county in esfahan province due to ddt or related compound application in public health or in agricultural pest control which is in contrary to our results, but susceptible to dieldrin 4% and in varzane they were susceptible to ddt 4% similar to our research results (9, 10). it is reported that ph. papatasi and ph. sergenti was susceptible to ddt 4% in kerman province. (11). it agree with our result about ph. papatasi. in 1998 a study showed that ph. kandelakii and ph. perfiliewi as a probable vector of zoonotic visceral leishmaniasis (zvl) were susceptible to ddt 4% during 1994 in ardabil province, northwest of iran (12). these species are in different area where it is a zvl foci with different vectors from zcl. in arsanjan county of fars province, ph. papatasi recorded sensitive to ddt 4% in 1999 same as current report (13). it is showed that ph. sergenti was susceptible to ddt 4% in esfahan city in 2005 it is a study on a different species in same province (14). in 2004 and 2005 in bam city, kerman province ph. papatasi and ph. sergenti were susceptible to ddt 4% and deltamethrin 0.05% similar to this reports (15). wild-caught ph. papatasi in badrood, esfahan province and their progeny were found susceptible to permethrin 0.75%, deltamethrin 0.1%, cyfluthrin 0.15% and lambda-cyhalothrin 0.05% and to ddt 4% during summer 2010 (16, 17). our research also confirm the susceptibility of them in this area to ddt and deltamethrin. another study in the same place during summer 2015 showed that there is susceptibility to cyfluthrin 0.15%, lambda-cyhalothrin 0.05%, permethrin 0.75%, and deltamethrin 0.05% same as our report and tolerant to ddt 4% unlike to our study (18). a study in north khorasan showed the development of resistance against ddt (4%) in the wild strain of ph. sergenti but susceptible to bendiocarb 0.1% and permethrin 0.75% (19). this report is about another species with various insecticide differ from our experiment. during 2016 and 17 laboratory reared of ph. papatasi were found susceptible to permethrin 0.75%, deltamethrin 0.05%, cyfluthrin 0.15%, and lambda-cyhalothrin 0.05% but resistant candidate to ddt 4% (20). this study reported likely result about deltamethrin and unlike result from current research about ddt. new world in 1997 a comprehensive study carried out on field population of lutzomyia longipalpis of venezuela against ddt 2%, propoxur 0.01 %, malathion 2%, fenitrothion l%, pirimiphos methyl l%, deltamethrin 0.06%, lambda-cyhalothrin 0.06%, and permethrin 0.2% insecticides and compered with laboratory population of reference strain and reported highly susceptible (55). the species used in this experiment is different from ours because in new world lu. longipalpis has medical importance as a vector but there is no in the old world and the concentration of deltamethrin, malathion and ddt used in their study are not same as concentration used in current study. in 2009 researchers reported two wild populations of lu. longipalpis with different exposure backgrounds susceptible to malathion, fenitrothion, lambdacyhalothrin, permethrin, and deltamethrin in brazil (56). in 2015 another study in brazil reported lu. longipalpis highly 375 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... susceptible to alpha-cypermethrin (57). also brazil located in new world and the vector is lu. longipalpis and the only common insecticide was deltamethrin. in the united state, some tests performed on laboratory populations of ph. papatasi and lu. longipalpis using cdc bottle bioassay against different concentrations of cypermethrin, deltamethrin, lambdacyhalothrin and permethrin, chlorpyrifos, fenitrothion, and malathion, bendiocarb, propoxur and ddt and they documented as susceptible population (39). same species and same insecticide tested by different methods of cdc bottle bioassay but reported the same result. in colombia in a study with the same method on lu. longipalpis, lambdacyhalothrin showed the highest degree of toxicity followed by alpha-cypermethrin and deltamethrin (58). there is another study in brazil using a modified method of who comparing laboratory population of lu. longipalpis with some population in the field with different exposure background and reported that lab-reared sand flies were more tolerant to field-collected ones against lambda-cyhalothrin (0.05%), deltamethrin (0.5%) and control was (silicone oil) (59). the sand fly species is different and also the concentration of deltamethrin is not the same. it can be observed that the only resistant phlebotomus registered in the arthropod pesticide resistance database is ph. argentipes in 23 locations of bihar state in india (60– 62). it is reported as resistance to ddt in vl endemic area of bihar and also developed resistance/ tolerant to malathion in a larger area but susceptible to deltamethrin and the wild-caught and their seven offspring’s is reported resistant to ddt (60, 61). they also experimented another species in different location and the result also is unlike to current research. in the current study, it was found that sand flies from esfahan province, were highly susceptible to deltamethrin and ddt and it was also noted that during the exposure time and counting the knockdown numbers of sand flies, those who exposed with ddt had more involuntary movement in their place but the vast majority of those who exposed to deltamethrin was moveless. pyrethroids as a major class of neurotoxic insecticide and ddt, fairly slow-acting on the protein of voltage-gated sodium channels in the cell membrane of the insect nerves. exposing insects to ddt and deltamethrin disrupts the normal process leading to paralysis and finally death. peripheral nervous system influenced by ddt causing tremors in appendages or entire body called “ddt jitters” then leads to excitatory paralysis and eventually death. deltamethrin affects both the central and peripheral nervous systems by producing repetitive discharge and cause paralysis the same as ddt but more obvious. after exposure with deltamethrin, the channels remain open and leads to abnormal hyperexcitability but “knockdown” is its sub-lethal effect (40). sand flies in response to exposure to ddt and deltamethrin manifested evident leg shedding in the current study. the same observation was made by denlinger and alexander (39, 56). sand flies with shedding legs, as a significant sub-lethal effect, will not be able to transmit the parasite as a consequence of disabling for blood-feeding (56). on the other hand, the authors reported that sand flies after shedding legs could still be capable of blood feed (39). we did not check the ability to have blood meals for leg shedded sand flies because the mortality rate was high, they were wild-caught, and we needed to identify them after keeping in alcohol and mounting. nevertheless, this will be considered in further studies. conclusion this study revealed that ph. papatasi from central iran is susceptible to ddt, deltamethrin, malathion, and propoxur. knowing about the susceptibility/ resistance of sand flies in this endemic area can play a vital role in the field of vector control and pesticide management. excessive use of insecticide with unsuitable concentration can cause resistance in vector sand flies and complicate 376 http://jad.tums.ac.ir published online: dec 31, 2021 j arthropod-borne dis, des 2021, 15(4): 366–379 z saeidi et al.: evaluation of susceptibility status... disease control. this result brings additional data to the worldwide need to assess the insecticide susceptibility status of sand flies, in order to strengthen vector surveillance and integrated vector management. we strongly recommend performing susceptibility tests on sand flies in various parts of the world as systematic monitoring and evaluating the status of leishmaniasis vectors against various insecticides, as regular or periodic susceptibility tests can ring a timely alert regarding early resistance. also doing some further tests on the resistant ones is recommended to determine the resistance intensity and mechanism according to standard protocols of who. acknowledgements the authors would like to appreciate dr nikpoor at tums for procurements of the susceptibility test kit, and standard impregnated filter papers. we also appreciate professor corbel and his team at ird, montpellier, france for their kind support and for generous sharing and training the impregnation methods and principles. many thanks to the personnel and manager of matinabad desert eco-resort particularly mr sh vaghefi for their precious cooperation and kind support. this study was conducted as a part of a phd dissertation of zahra saeidi, by financial supports of school of public health (sph) and institute of environmental research (ier), tehran university of medical sciences (tums), code of 96-04-27-35872. ethical considerations this study was conducted as a part of a phd dissertation, and it has been reviewed and approved by the school of public health (sph), tehran university of medical sciences (tums) ethics committee and has been registered with the code ir.tums. sph.rec.1396.3602. conflict of interest statement authors declare that there is no conflict of interest. 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https://www.pesticideresistance.org/display.php?page=species&arid=676 evaluation of susceptibility status of phlebotomus papatasi, the main vector of zoonotic cutaneous l abstract keywords introduction materials and methods study area sand fly collection insecticides bioassay (susceptibility) tests standard procedure sand flies testing sand fly mounting and identification knockdown effect and leg loss data analysis/ knockdown curve results knock down time50 (kd50) and kd90 susceptibility status survival curve discussion old world new world conclusion acknowledgements ethical considerations conflict of interest statement references microsoft word 9-dr sadjadi ready to go.doc iranian j arthropod-borne dis, (2008), 2(1): 53-56 t mohammadzadeh et al: a case of gingival… 53 case report a case of gingival myiasis caused by wohlfahrtia magnifica t mohammadzadeh 1, r hadadzadeh 2, f esfandiari1, *sm sadjjadi 1 1departement of parasitology and mycology, school of medicine, shiraz university of medical sciences, shiraz, iran 2 dayyer health organization, bushehr, iran (received 1 jun 2007; accepted 15 jul 2008) abstract a gingival myiasis in a four years old mental retarded boy with anorexia and weight loss is presented from southern part of iran. entomological studies on larvae showed the larvae as wohlfartia magnifica which is a rare causative agent of gingival myiasis. keywords: wohlfartia magnifica, myiasis, gingival, iran introduction myiasis broadly includes interaction between flies and warm blooded hosts. it is usually involves infestation by larval or pupal stages of the flies in the tissues of human and other vertebrate animals (larry and millikan 1999). infestation with fly larvae may occur when flies deposit eggs or first-stage larvae on the body or its apertures. the clinical aspects of myiasis vary with the regions affected, with the species of fly involved and with the number of maggots present (john and petri 2006). myiasis is more prevalent in tropical regions. in the present paper a human case with gingival myiasis due to w.magnifica is presented from south part of iran. case report an iranian four years old mental retarded boy residing in bardestan village near the dayyer port from bushehr province of iran, was referred to a number of physicians and dentists because of his anorexia and weight loss. finally, superior gingivitis and loosing the central and lateral teeth (21, 12) were observed. after central tooth extraction, larvae were observed in dental cavity. the superior gingiva was exposed and a total of 40 larvae were extracted from this area. the larvae were sent to department of parasitology and mycology, school of medicine, shiraz university of medical sciences. the larva was identified according to the key of the third stage of larva of some of the muscoid flies ( belding 1965); such that the larvae was muscoid, slender shaped with posterior spiracle with button area very slightly chitinized and the ring incomplete as well as inner slits slopping. entomological studies on larvae including the character of posterior spiracles showed the larvae as w. magnifica ( fig. 1 -2 ). *corresponding author: prof. sm sadjjadi, shiraz, p.o.box:71345-1735, iran, e-mail: smsadjjadi@sums. ac.ir iranian j arthropod-borne dis, (2008), 2(1): 53-56 t mohammadzadeh et al: a case of gingival… 54 fig. 1. wohlfahrtia magnifica whole larva detected from gingival cavity. fig. 2. posterior spiracle of the larva with button area very slightly chitinized and the ring incomplete iranian j arthropod-borne dis, (2008), 2(1): 53-56 t mohammadzadeh et al: a case of gingival… 55 discussion myiasis with different causative agents is common in domestic and wild mammals of all over the world (john and petri 2006). the most important agent of myiasis in the genus wohlfahrtia is w. magnifica, an obligate parasite of warm-blooded vertebrates in southeastern europe, southern and asiatic russia, the near east and north africa (zumpt 1965). the present case had anorexia and weight loss with mental retardation and possible mouth breathing. in the mean time he was from a hot region which is suitable for flies. on the other hand, some of his teeth had been lost. these criteria make the possibility of infection with w. magnifica which deposited its larvae in the ear, eyes or nose causing an extensive destruction of healthy tissues (noutsis and millikan 1994). some 120 to 170 larvae are deposited near the wounds or body openings of humans and other animals. the larvae feed and mature in five to seven days and then leave the wound for pupation (zumpt 1965). w. magnifica has been reported as the cause of otitis (fawzy 1991), ophthalmomyiasis (morsy and farrag 1991), vulvar myiasis (delir et al. 1999), wound myiasis (iori et al. 1999, lmimouni et al. 2004) and cutaneous myiasis (kokcam and saki 2005). in addition, w. magnifica has been previously reported in gingiva and dental cavity (sahba 1981, athari and fallah 1993, tang et al. 2003, dorma et al. 2007) as well as orotrachea (ciftcioglu et al. 1997). alcoholism, senility, mental retardation, hemiplagia and mouth breathing during sleep may facilitate the development of oral myiasis (anil et al. 1989). however, the present case could be infected during mouth breathing. necrotic tissues, present in advanced periodontal diseases will form a good substrate in which the fly can lay its eggs (zelster and lutsmann 1988). the treatment of choice is removal of larvae from the affected areas. proper oral hygiene is essential to ensure against oral myiasis (gursel et al. 2002). greater awareness on the part of physicians about clinical symptoms and relevant exposure histories would improve the expediency and efficacy of treatment for patients with myiasis (zygutiene et al. 2006). references anil s, jacob oa, hari s (1989) oral myiasis: a case report. ann dent. 48: 28-30. athari a, fallah t (1993) dental cavity myiasis due to wohlfahrtia magnifica. med j islamic republic iran. 7: 209-10. belding dl (1965) textbook of parasitology. third edition, meredith publishing company. new york. ciftcioglu n, altintas k, haberal m (1997) a case of human orotracheal myiasis caused by wohlfahrtia magnifica. parasitol res. 83(1): 34-6. delir s, handjani f, emad m, ardehali s (1999) vulvar myiasis due to wohlfahrtia magnifica. clin exp dermatol. 24: 274-280. droma eb, wilamowski a, schnur h, yarom n, scheuer e, schwartz e (2007) oral myiasis: a case report and literature review; oral surg oral med oral pathol oral radiol endod. 103(1): 92-6. fawzy af (1991) otitis media and aural myiasis. j egypt soc parasitol. 21(3): 883-5. gursel m, aldemir os, ozgur z, ataoglut (2002) a rare case of gingival myiasis caused by diptera (calliphoridae). j clin periodontol. 29: 777-780. iori a, zechini b, cordier l, luongo e, pontuale g, persichino s (1999) a case of myiasis in man due to wohlfahrtia magnifica (schiner) recorded near rome. parasitologia. 41(4): 583-5. john dt, petri wa (2006) markell and voge’s medical parasitology. 9th edition. saunders, elsevier. pp. 328-334. iranian j arthropod-borne dis, (2008), 2(1): 53-56 t mohammadzadeh et al: a case of gingival… 56 kokcam i, saki ce (2005) a case of cutaneous myiasis caused by wohlfahrtia magnifica. j dermatol. 32(6): 459-63. larry e, millikan md (1999) myiasis. clin dermatol. 17: 191-5. lmimouni be, baba ne, yahyaoui a, khallaayoune k, dakkak a, sedrati o, el mellouki w (2004) wound myiasis due to wohlfahrtia magnifica. first human case in morocco. bull soc pathol exot. 97(4):235-7. morsy ta, farrag am (1991) two cases of human ophthalmomyiasis. j egypt soc parasitol. 21: 853-5. noutsis c, millikan le (1994) myiasis. dermatol clin. 12: 729-36. sahba gh (1981) a case of gingival myiasis in man and animals in iran. the third international congress for tropical medicine and malaria, montreal, canada. tang h, wang xw, tang gh (2003) two cases with gum myiasis caused by wohlfahrtia magnifica. zhongguo ji sheng chong xue yu ji sheng chong bing za zhi. 21(1): 30. zelster r, lutsmann j (1988) oral myiasis. int j oral maxillofac surg. 17: 288-289. zumpt f (1965) myiasis in man and animals in the old world. butterworths, london, uk. zygutiene m, narkeviciute i, mudeniene v, ziliukiene j (2006) a case of myiasis due to hypoderma bovis, lithuania, 2004. euro surveill 15; 11(2): e1-2. a review on important zoonotic bacterial tick-borne diseases in the eastern mediterranean region http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... review article a review on important zoonotic bacterial tick-borne diseases in the eastern mediterranean region manijeh yousefi behzadi1,2, ehsan mostafavi 1,2, mahdi rohani4, ali mohamadi1, mozhgan ahmadinezhad1, neda moazzezy3, masoomeh shams-ghahfarokhi5, *mehdi razzaghi-abyaneh6 1department of epidemiology and biostatics, research centre for emerging and reemerging infectious diseases, pasteur institute of iran, tehran, iran 2national reference laboratory of plague, tularemia and q fever, research centre for emerging and reemerging infectious diseases, pasteur institute of iran, akanlu, kabudarahang, hamadan, iran 3molecular biology department, pasteur institute of iran, tehran, iran 4department of microbiology, pasteur institute of iran, tehran, iran 5department of mycology, faculty of medical sciences, tarbiat modares university, tehran, iran 6department of mycology, pasteur institute of iran, tehran, iran *corresponding author: prof. mehdi razzaghi-abyaneh, mrab442@yahoo.com (received 10 may 2021; accepted 03 aug 2021) copyright © 2021 the authors. published by tehran university of medical sciences.copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/ bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited.bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. abstract background: zoonotic diseases as health concerns worldwide account for more than half of the emerging infectious diseases. arachnids are powerful vectors to transmit several diseases to humans. additionally, these emerging zoonotic diseases have been a considerable health threat in the eastern mediterranean region of the who (emro) due to the large population living close to farms and international trade with nearby countries. methods: this review study is based on the reported three tick-borne diseases, lyme disease, tularemia, and q fever, from iran and other emro countries. to this end, we searched pubmed central, isi web of science, and google with the related keywords in english at any time. the reported data are then sorted by countries for each disease. results: according to the published data, 15 countries in the region have one/more emerging infectious diseases. q fever has been the most frequent infection in emro countries, while lyme was less recorded. furthermore, iran is among the countries with documented history of all three investigated diseases. conclusion: tick-borne disease is popular among emro countries, indicating that they have natural conditions for infections in animals and humans. it appears necessary to develop a disease management strategy and control programs against tick-borne diseases (tbds). moreover, the disease-resistant animal could be bred instead of susceptible livestock. therefore, research studies to control tbds should be regarded as a top priority plan. keywords: tick-borne diseases, tularemia, lyme, q fever, mediterranean region, iran introduction it is estimated that approximately one billion cases of illness and millions of deaths occur from zoonoses every year worldwide. moreover, 60% of globally reported emerging infectious diseases are zoonotic. during the last three decades, 266 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... there were more than 30 new human pathogens from which 75% had originated from animals (1, 2). the tick-transmitted infections of humans, currently considered zoonoses, involve ticks and wild and/or domestic animal hosts with the maintained pathogens in natural cycles (3). tick-borne diseases are among the crucial public health issues. there have been considerable efforts to control these infectious diseases worldwide. however, tick populations are increasing with geographic expanding ranges. they can transmit viral, parasitic, and bacterial pathogens, often harboring some agents simultaneously (4, 5). in fact, these arachnids play roles as powerful vectors transmitting several diseases, such as lyme disease, tularemia, q fever, and rickettsiosis. therefore, surveillance of ticks and possible transmittable pathogens is a practical tool for a better prevention and control program within the epidemiological surveillance framework (6). the emerging zoonotic diseases are a considerable public health threat in the eastern mediterranean region of the who (emr), which have been reported from 15 countries in the region often with bursting outbreaks in the last two decades (2). this region has been particularly prone to zoonotic infections due to the large population living rather close to animals and well-developed international trade with nearby countries (table s1) (fig. 1). therefore, the region has remained at the crossroad of repeated outbreaks from emerging infectious diseases. variable surveillance level and collaboration capacity of countries at the animal-human interface have mostly provoked these outbreaks (7, 8). these zoonotic infections result in global health concerns owing to their rapid spread possibility. moreover, they impose economic consequences according to loss of economic opportunities through livestock loss (2, 9). in this review on emerging tick-borne diseases, we focused on zoonotic bacterial infections, including q fever, tularemia, and lyme disease in emro countries based on the available reports and studies. fig. 1. eastern mediterranean region (emro) (31) fig. 1. eastern mediterranean region (emro) (31) 267 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... materials and methods we searched the databases pubmed, isi web of sciences, and google as the main international databases for the articles. the search strategy was based on the terms emro countries, thick-bone diseases, tularemia, francisella tularensis, lyme disease, borrelia burgdorferi, q fever, and coxiella burnetii, in english sources. the database search was performed in 2020 to obtain the related articles. results the description of three diseases tularemia, lyme and q fever in emro countries according to the literature reviewed is summarized in table 1. the summary of distribution of studied diseases in emro countries are shown in fig. 2. tularemia tularemia is a zoonotic infection caused by francisella tularensis, a gram-negative intracellular bacterium. this pathogen is a biological threat agent and can spread by aerosols and survive for months (39). however, no vaccine is available to prevent tularemia (40). the species f. tularensis is classically divided into four subspecies: type a (f. tularensis subsp. tularensis), type b (f. tularensis subsp. holarctica), f. tularensis subsp. mediasiatica, and f. tularensis subsp. novicida (41, 42). type a, which is most prevalent in north america, and type b, which is spread throughout the northern hemisphere, are the main causative agents of tularemia worldwide. type a usually causes more severe infection than type b, and its mortality rate is assessed 10–40 % in untreated patients (43). francisella tularensis infections have been identified in more than 250 animal species, including invertebrates and mammals (44). multiple sources of infection have been documented, such as arthropod (ticks, flies, and mosquitoes), contaminated water, aerosol droplets, animal bites, and direct contact with their products (45). the reservoir of f. tularensis has not been known yet. although outbreaks of the infection caused by type b are associated with a high rate of occurrence among lagomorphs and rodents, they do not appear to be capable of bacteria harboring between the outbreaks (32). the clinical presentation of tularemia in humans may vary from an asymptomatic level to severe diseases, which can cause death. after 3-5 days of the incubation period (ranging from 1 to 21 days), the infection often presents with flu-like symptoms, such as fever, chills, weakness, sore throats, joint pain, and headache. then, the disease can evolve to glandular, ulceroglandular, pneumonic, oropharyngeal, oculoglandular, and typhoidal (41, 43). ulceroglandular form is the most characteristic profile of the disease in which a papule develops and then becomes pustular and ulcerated at the tick bite site. tularemia usually lasts over a period of 60-90 days if the treatment is not applied there. antibiotics applied to treat the disease include streptomycin and ciprofloxacin, which usually last 10-21 days depending on the disease stage (6). table 1 summary of the three investigated tick-borne diseases features disease bacteria tick vectors fatality rate approved vaccine ref. tularemia francisella tularensis amblyomma spp. dermacentor spp. haemaphysalis spp. ixodes spp. 10-15% n/a (32-34) q fever coxiella burnetii haemaphysalis spp. ixodes spp. rhipicephalus spp. dermacentor spp. 19% q-vax® (35, 36) lyme borrelia burgdorferi ixodes spp. rare vla15 (37, 38) table 1. characteristic features of the three investigated tick-borne diseases 268 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... iran in 1973, the first positive serological tests were reported in domestic livestock and wild animals obtained from the south-eastern and north-western regions of iran. this study aimed to detect f. tularensis from a small wild mammal’s spleens, which were then examined by a passive haemagglutination test. the test revealed a positive titer on the haemagglutination obtained from an afghan hedgehog (hemiechinus megalofis) collected around zabol in eastern iran (46). tularemia was reported once in afghanistan in a porcupine in 1973 (46, 47). the first human case report was from southwestern iran (47, 48). the assessment of tularemia presence in different groups (including hunters and healthcare workers) as well as in rodents from kurdistan and lorestan provinces exhibited positive antitularemia igg antibodies (49, 50). similar results were reported from the south-east in sistan and baluchistan province (51) and chaharmahal va bakhtiari province (52). the serology assessment results of butchers and slaughterhouse workers from ten cities of sistan and baluchistan province indicated that tularemia was endemic in this region (53). the data indicate that a high prevalence of tularemia in the neighboring countries of iran, such as armenia (54), azerbaijan (55) and turkey (56), could have high potential for human infections with f. tularensis in different parts of the country. egypt in a study conducted in egypt, a total of 319 ticks, along with blood and fecal samples were taken from camels and tested for the presence of francisella spp. by pcr. moreover, serum samples from 75 camel slaughterhouse workers were screened for the presence of f. tularensis igg using the enzyme-linked immunosorbent assay. the prevalence of francisella spp. was 4.7% among the ticks; however, it was not detected fig. 2. the situation of the infection carriers in countries. the human and animal symbols are representative of the discussed infections in with picture border, without picture border and dot picture border for tularemia, q fever and lyme respectively. fig. 2. the situation of the infection carriers in countries. the human and animal symbols are representative of the discussed infections in with picture border, without picture border and dot picture border for tularemia, q fever and lyme respectively. 269 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... in blood or fecal samples from camels, even camels carrying francisella spp. positive ticks. on the contrary, seroprevalence of f. tularensis antibodies among the tested abattoir staff was estimated 9.3% with a significant prevalence among those frequently exposed to tick bites (57). sudan there was the first case report of bacteremia caused by f. tularensis from the rural area of southern sudan, a 29-year-man who presented dry cough, shortness of breath, fever, and abdominal discomfort, along with pale and dehydrated. coccobacilli were isolated by blood culture, which resulted in f. tularensis detection (48). yemen the first study on collected ticks from livestock was conducted in which 34 isolated hyalomma marginatum ticks were examined for the presence of pathogens using the molecular approach, which showed that three out of 34 samples were positive for the presence of francisella spp. (59). pakistan a total of 2280 soil samples were applied, representing several villages in punjab province, pakistan. in addition, 6.22% of domestic animals were found to be positive in the seroconversion test. francisella tularensis detection over a large geographic area indicated its extension to the enzootic range (60). in addition, the first report of f. tularensis isolation from a patient suffering from lung infection was reported in 2019 (61). q fever q fever as a zoonosis disease is caused by coxiella burnetii, a strictly intracellular, gram-negative bacterium. various species, including ticks, birds, and mammals are reservoirs of c. burnetii. this infection is mostly latent in animals, with durable bacteria shedding into the environment (62-64). q fever can remain unrecognized due to poor surveillance of the disease. human is usually infected following birth or abortion, where the birthing fluids of an infected animal contaminate the environment (65). abattoir workers, farmers, veterinarians and laboratory staff performing c. burnetii culture are at risk of q fever (66, 67). q fever may have different presentations, including an acute (febrile illness, pneumonia, or hepatitis) or a chronic disease (mainly endocarditis); however, it is often asymptomatic. the specific diagnosis of q fever is based on serology tests. igm and igg antibodies can be detected 14-21 days after infection (68, 69). iran q fever is endemic in iran like other middle east countries (70, 71). the first acute q fever case was documented in 1952 (72). in 1970, four other infected individuals were reported from shiraz city (73), from 1970 to 1973, 45 acute q fever subjects were also diagnosed from abadan city in southwestern iran (73). furthermore, 80 q fever patients were also documented from 1972 to 1976. after this time, the disease was neglected for more than thirty years, and no cases were reported until the first q fever endocarditis was reported in tehran in 2013 (74). the seroprevalence assessment of q fever among slaughterhouse workers and butchers among different parts of sistan va baluchistan province showed that q fever survived among these workers in this area (75). in a systematic review study in 2017, it was identified that the total distribution of q fever in iran was 27% in animals, mostly in goats and sheep. the contamination of dairy products was 5%. the results showed that higher positive pcr results in cattle but greater seropositivity in small ruminants. therefore, c. burnetii related antibodies or dna were frequently reported from ruminants in iran, which could be a potential threat for human health and the livestock industry (71). afghanistan q fever was detected for the first time in afghanistan in 2011 (76). the disease has 270 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... been known as an endemic threat to humans and animals in this country. moreover, q fever has been detected in american and english soldiers returning from afghanistan (77). egypt in a cross-sectional study between 2016 and 2017, it was shown that the prevalence of c. burnetii igg antibodies in sheep, goats and humans was 25.68%, 25.20% and 25.71%, respectively. in humans, the only significant deference was higher positive infected women compared to men. no difference was found between the ruminants and humans (78). in another research, the prevalence of c. burnetii by specific antibodies was evaluated in 2,699 blood samples obtained from ruminants and camels. the specific antibodies were detectable mostly in camels (40.7%) followed by cattle (19.3%), buffaloes (11.2%), sheep (8.9%) and goats (6.8%). the seropositive results were dominant in animals older than four years. in addition, 8.7% of the living people in the related areas consumed raw camel milk from which no one had knowledge about q fever. according to this study, exposure to c. burnetii was common in camels and ruminants, requiring increase of awareness among animal owners and veterinarians (79). sudan the prevalence of c. burnetii antibodies in camels and cattle sera samples collected from nine states was investigated in a study in 2015. the overall of 29.92% prevalence of c. burnetii antibodies was obtained although an overall of 64.5% prevalence rate was observed in camels (80). saudi arabia the seroprevalence of q fever in domestic livestock, including 630 sheep, 489 camels, 428 cattle and 423 goats, was assessed in 2018. the total seroprevalence was found to be 30.71%. significant differences in seroprevalence were recorded between the species, which were 51.53%, 30.67%, 34.04% and 12.38% in camels, cattle, goats and sheep, respectively. the domestic livestock and the camel were demonstrated to be the source of q fever endemicity in saudi arabia (81). in the other study, 1310 serum samples were collected to examine antibodies of c. burnetii. the prevalence of c. burnetii among tested animals was 9.2%, which was most frequent among goats (15.6%). moreover, old animals were 23 times more susceptible to c. burnetii (p< 0.01). c. burnetii infection was widespread among various ruminants of the eastern province of ksa, indicating a high risk for environmental contamination and the possible infection of humans and animals (82). pakistan according to a cross-sectional study conducted in punjab province, determination of the prevalence and distribution of c. burnetii in soil was evaluated. the results demonstrated that in 47 samples of the total 2425 soil samples, c. brunetti dna was detectable. the elisa revealed an increase of antibodies in sheep (17.9%) and goats (16.4%). the correlation between soil dna and c. burnetii antibodies in small ruminants showed that the odds of detecting these antibodies were significant in sheep [2.81 (95% ci: 1.20-7.37), p=0.02]. this investigation provided the first evidence of c. burnetii presence in the environment in punjab province, pakistan (83). qatar a sero-epidemiological study in 2005 was conducted to estimate the seroprevalence of q fever in two military groups, including one that resided in an area with the known history of q fever epidemic and personnel from central and southwest asia. the prevalence rate was found to be 7.2% and 2.1% in the first and second investigated groups, respectively. nevertheless, there were no significant risk factors for q fever seroconversion in either population (84). 271 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... tunisia the prevalence of the c. burnetii antibody among 500 sera from blood donors was tested, resulting in 26% confirmed antibodies against c. burnetii and indicating a high seroprevalence of q fever in tunisia (85). syria in an investigation conducted between 1984 and 1988, q fever was recorded in czech workers in syria. moreover, there was a belgian patient diagnosed with c. burnetii infection in this country (86). iraq a study conducted from 1984 to 1988 in iraq was identified that 42 czech workers had q fever (87). there was also an outbreak of this infection in 58% of marines in iraq in 2005 (87). furthermore, among 909 military personnel deployed in iraq, antibody tests were performed against c. burnetii, and the data showed 10% of q fever seroconversion, which was a significant infectious disease concern for the military personnel deployed in iraq (88). jordan the seroprevalence of c. burnetii was studied on-farm animals in 2019, and the data showed that on-farm biosecurity was crucially essential to reduce the transmission of the infection to humans and animals (89). another study aimed to determine the prevalence of c. burnetii antibodies in the bulk reservoir milk obtained from dairy cattle, goats and sheep in the country. the positive result rate was 62.9% of the ruminant herds, which revealed the widespread exposure of jordanian ruminants to the infection (89). lebanon in 2018, the human seroprevalence of q fever was aimed at evaluating c. burnetii antibodies among 421 serum samples. the result recorded the exposure of 37% to c. burnetii distributed in five provinces with the highest rate in two provinces of bekaa and akkar, and the lowest in mount lebanon (89). yemen according to a survey to detect c. burnetii antibodies in approximately fifty veterinarians and butchers, 50 samples were examined from which three samples were positive, one was positive for phase 1, six were equivocal, which might have indicated the disease distribution in cattle and among professionals (90). libya in 1951, an explosive outbreak of 25 cases of q fever was recorded of which 22 were positive (91). q fever was also documented in 48 czech workers in libya from 1984 to 1988 (92). morocco q fever outbreak was discovered in southern morocco in 1947 where three c. burnetii strains were isolated from ticks and identified as hyalomma savignyi gerv. these ticks were widely prevalent among different domesticated animals (91). in 1951, outbreaks of q fever were documented in six cities, and c. burnetii was isolated from gerbils and bovine ticks. in addition, 38% of infections were identified in people. moreover, the prevalence was estimated to be 55%, 45%, and 38% in goats, cows and sheep, respectively (91). furthermore, eight cases diagnosed with q fever were recorded in the medical service of the hospital of meknes in morocco in 1961 (93). presence of c. burnetii in emro countries was shown in both humans and animals through different seroprevalence studies. however, more studies on tick species are needed in this region to identify the presence of c. burnetii in ticks in order to elucidate the role of different tick species in the q fever transition and epidemiology. lyme disease lyme disease as a vector-borne disease is caused by spirochete borrelia burgdorferi. the clinical presentation may vary depending on the illness stage, including erthyma migrans, meningitis and cranial nerve palsies, carditis and arthritis. the infection 272 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... symptoms are relieved in the vast majority of individuals after proper cure application during 2-4 weeks. serologic testing is often used to diagnose the infection; however, it sometimes results in misdiagnosis on blood samples from those with fatigue or arthralgia as non-specific symptoms. b. burgdorferi is transmitted by ixodidae ticks, primarily by ixodes scapularis, the deer tick in the united states. there are also other vectors to transmit the bacteria, including ixodes persulcatus, ixodes ricinus and ixodes pacificus in asia and europe (94, 95). the experimental findings showed that in humans in which the b. burgdorferi transmission risk from ticks to humans was 25% for nymphal ticks, which had fed for at least 72 hours. this declines to 0% for nymphal ticks that had fed less than 72 hours. the treatment for the early phase of the disease is based on a simple 10-14-day course of oral antibiotics application to eradicate the infection (96). iran the caspian sea in iran is the habitat for various hard tick species like i. ricinus, the notorious vector of lyme borreliosis (lb). ixodes ricinus and other hard ticks were examined, along with small mammals and common rodents for lb (fig. 3). the ticks were collected from different mammalian hosts, including camels, sheep, dogs, goats, fig. 3. iran map by its provinces. only the names of the provinces in which the disease sample was discovered are mentioned. the human and animal symbols are representative of the discussed infections in with picture border, without picture border and dot picture border for tularemia, q fever and lyme respectively. fig. 3. iran map by its provinces. only the names of the provinces in which the disease sample was discovered are mentioned. the human and animal symbols are representative of the discussed infections in with picture border, without picture border and dot picture border for tularemia, q fever and lyme respectively. 273 http://jad.tums.ac.ir published online: sep 30, 2021 j arthropod-borne dis, sep 2021, 15(3): 265–277 m yousefi behzadi et al.: a review... and cattle. the real-time pcr sequence revealed that the borrelia dna in 14% of specimens (71 out of 501) belonged to i. ricinus and rhipicephalus ticks. nevertheless, none of the rodents nor small mammals had borrelia infection (97). iraq there was a case report from iraq, a 28-year-old army male who presented with a rash on his anterior forearm for approximately 3 days. the clinical examination did not find any evidence of a bite mark, vesicles, induration or necrosis. the diagnosis of early localized lyme disease was applied according to the erythema migrans as the unexpected solider case in 2010 (98). saudi arabia the only case report from this country was a 30-year-old male from a resident of dammam in the eastern province of saudi arabia who presented an acute skin disease with a sudden onset. a red papule on his arm appeared, and the lesion quickly spread to a large size over a short time. the patient also complained of swelling and numbness of the upper arm, along with body ache, dizziness and unsteadiness. finally, erythema migrans were diagnosed as a skin presentation of early localized lb (99). morocco the prevalence of borrelia infections was assessed from 2006 to 2011 on ticks, small mammals and thick blood films in patients. a considerable burrows proportion was infested with ticks of the o. erraticus complex with a mean of 39.5% among the whole country. borrelia infections were found in 10.2% of the ticks and 8.6% of the rodents. moreover, 102 tested patients were positive by thick blood film (100). conclusions according to the published data, it is clear that tick-borne disease is common among emro countries, indicating that they have natural favorable conditions for infections in animals and humans. thus, they require disease management strategy development and control programs against tick-borne diseases (tbds). additionally, the movements of animals owing to trade or migration should be considered for having a probable infection as a risk of zoonoses. this policy must stem from the knowledge of the pathogen, host, and tick disease triangle concerning environmental changes, global warming, and the tick habitats’ ecology and distributions. recombinant vaccines to prevent the related infections and anti-tick agents can be applied to break this triangle, particularly in the case of animals. diseaseresistant animal promotion instead of susceptible breeds can be another approach. therefore, further studies to control tbds should be regarded as a top priority plan. the weakness of this study might be the keywords as well as the publications, which are not in english and are related to some unreported countries. a systematic and analytic review on this issue in emro countries may contribute to design a standard policy to achieve safer developing trades in the region. conflict of interest the authors declare that they have no conflicts of interest relevant to this manuscript. references 1. malik mr, el bushra he, opoka m, formenty p, velayudhan r, eremin s (2013) strategic approach to 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30(1):311-28. 96. nadelman rb, nowakowski j, fish d, falco rc, freeman k, mckenna d, et al (2001) prophylaxis with single-dose doxycycline for the prevention of lyme disease after an ixodes scapularis tick bite. n engl j med. 345(2):79-84. 97. naddaf sr, mahmoudi a, ghasemi a, rohani m, mohammadi a, ziapour sp, et al (2020) infection of hard ticks in the caspian sea littoral of iran with lyme borreliosis and relapsing fever borreliae. ticks tick borne dis. 11(6):101500. 98. fisher jb, curtis ce (2010) an unexpected case of lyme disease in a soldier serving in northern iraq. mil med. 175(5):367-9. 99. selim mme, elbashier am, awad ai, borgio f (1994) erythema migrans case report from dammam central hospital. ann saudi med. 14(6):521-2. 100. diatta g, souidi y, granjon l, et al (2012) epidemiology of tick-borne borreliosis in morocco. plos negl trop dis. 6(9):e1810. a review on important zoonotic bacterial tick-borne diseases in the eastern mediterranean region abstract keywords introduction materials and methods results tularemia iran egypt sudan yemen pakistan q fever iran afghanistan egypt sudan saudi arabia pakistan qatar tunisia syria iraq jordan lebanon yemen libya morocco saudi arabia lyme disease iraq morocco conclusions conflict of interest references j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 292 http://jad.tums.ac.ir published online: january 06, 2016 original article a pictorial key for culex pipiens complex (diptera: culicidae) in iran hossein dehghan 1, javid sadraei 2, *seyed hassan moosa-kazemi 1, esmail abolghasemi 1, hassan solimani 3, ahmad jaffari nodoshan 4, mohammad hassan najafi 4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical entomology and parasitology, school of medical sciences, tarbiat modares university, tehran, iran 3yazd health training and research center, yazd, iran 4shahid sadoughi university of medical sciences and health services, yazd health center, yazd, iran (received 30 oct 2013; accepted 19 nov 2014) abstract background: the aim of this study was to design pictorial key and taxonomic literature of culex pipiens complex in iran. methods: larvae were collected using standard dipping methods in 13 randomly selected areas of bushehr, hamedan, kerman, khorasan-e-razavi, khuzistan, mazandaran, tehran, sistan and baluchistan and yazd provinces from april 2009 to october 2010. the data were analyzed using spss ver. 11.5. results: culex pipiens larvae were identified based on the seta 1 of the abdominal segments iii–iv in north and central parts of iran. this diagnostic character had some variation among the cx. quinquefasciatus collected from south of the country. the identification value of intersection of costa, subcosta and bifurcation of r2+3 of female veins, was calculated as 90–100 % for cx. pipiens. this diagnostic character was varied among the cx. quinquefasciatus specimens. the male genitalia found as the main characters to distinguish of cx. quinquefasciatus from cx. pipiens. conclusion: it is necessary more studies on the behavior and genetic variations of cx. pipiens complex in iran. keywords: pictorial key, taxonomy, culex pipiens complex, iran introduction culex pipiens complex species have been known as important vectors of medical and veterinary arthropod -borne diseases (kasai et al. 2008). some vector borne diseases such as filariasis, west nile fever, western and eastern equine encephalitis, japanese encephalitis and st louis encephalitis are transmitted by these species complex (smith 1973, vinogradova 2000, kasai et al. 2008). cx. pipiens transmits west nile virus among wild birds and plays an important role in enzootic cycles (hayes et al. 2005). some pathogens transmitted by culicine mosquitoes such as west nile and sindbis viruses, dirofilaria immitis (dog heartworm) and d. repens (dirofilariasis), and have been reported in iran (naficy and saidi 1970, saidi et al. 1976, azari-hamidian et al. 2007). moreover, potential outbreaks of some mosquitoborne arboviral diseases such as japanese encephalitis (je) and rift valley fever reported in the eastern mediterranean region (who 2004). the morphological and ecophysiological variations of cx. pipiens complex have been an important topic in extensive researches (harbach 1985, 1988, vinogradova 2000, 2003, azari-hamidian and harbach 2009, dehghan et al. 2010, 2011, 2014). because of the complicities, more than 75 synonyms have been *corresponding author: dr seyed hassan moosakazemi, e-mail: moosakazemii@tums.ac.ir j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 293 http://jad.tums.ac.ir published online: january 06, 2016 proposed for this complex species (knight and stone 1977). barr (1982) reported a wide geographical distribution, and morphological variations among the cx. pipiens and cx. quinquefasciatus populations in the world. there are variations among the diagnostic characters of these species complex that attributed to some degree of expression such as species, subspecies, variety or forms (ishii 1991). culex quinquifasciatus is distributed in the tropical areas with various hosts, whereas, cx. pipiens found in the moderate areas with host preferences of nest maker birds (vinogradova 2000). shahgudian (1960) and lotfi (1976) provided identification keys for iranian anopheles and culex species, respectively. the study on physiology and behavior of cx. pipiens, the nominotypical and molestus form, was described in iran (lotfi 1970, 1973, 1976, amirkhanian 1974, zaim and cranston 1986). zaim (1984) reported six genera and 55 species of iranian mosquitoes. some years later a checklist and systematic key was provided for iranian culicinae by zaim and cranston (1986). although, in the previous study had not been mentioned about diagnostic characters of cx. pipiens and cx. quinquefasciatus in the systematic keys (zaim and cranston 1986), but azari-hamidian and harbach (2009) addressed these characters. the present article, reviews some morphological characters of cx. pipiens complex species, collected in some parts of iran to facilitate conducting comprehensive research about systematics, ecology, medical and veterinary importance of the complex. until now, seven genera, 64 species, and three subspecies of iranian mosquitoes was reported (azarihamidian 2007a). culex pipiens complex belongs to the pipiens group, and was divided to several subgroups and subtypes (harbach 2011, 2013). culex pipiens including form molestus and cx. quinquefasciatus have been reported in iran (azari-hamidian 2007a, azari-hamidian and harbach 2009). furthermore, the larval habitat of cx. pipiens form molestus was reported by golestani (1967) in tehran city. the polygene chromosomal pattern of the autogenous tehran strain of culex pipiens molestus was described by amirkhanian in 1974. further studies about distribution of cx. pipiens have been reported in iran (zaim 1987, azari-hamidian et al. 2005, 2011, azerihamidian 2007b, moosa-kazemi et al. 2009, 2010, dehghan et al. 2013). distribution of cx. quinquefasciatus has been reported in yazd and kermanshah provinces (ghaffary 1955). more studies reported distribution of this species in southern parts of iran (zaim 1987, mousa-kazemi 2000, azari-hamidian et al. 2005, moosa-kazemi et al. 2009, azarihamidian et al. 2010, moosakazemi et al. 2010, khoobdel et al. 2012). the distribution of this species has been reported in tropical areas in south of iran based on molecular identification (azari-hamidian et al. 2010, dehghan et al 2013). morphological studies on cx. pipiens complex, revealed that, male genitalia and dv/d ratio are the main morphological characters for distinguishing adults of cx. quinquefasciatus and cx. pipiens (sundararaman 1949). the main reliable character for identification in larval stage has been known as the number of branches of seta 1 on the abdominal iii–iv segments (harbach 1988, dehghan et al. 2013). since there are scatter studies about the morphological variations and distribution of cx. pipiens complex in iran, (zaim 1987, azari-hamidian and harbach 2009, azarihamidian et al. 2010) it is necessary to obtain more accurate data on the variability of the species. on the other hand, the final decision about taxonomic status needs more complete data that will be obtained from further studies in different geographical areas. the results of this study can be useful to identify cx. pipiens complex species, as well as designing plans for vector control programs in the future. j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 294 http://jad.tums.ac.ir published online: january 06, 2016 materials and methods study area chabahar (25°17′n, 60°37′e) and nikshahr (26°04′n, 60°37′e) counties were selected from sistan and baluchistan province with tropical warm and humid climate. in southern iran, jiroft (28.5°n, 57.8°e) from kerman, borazjan (29°15′n, 51°12′e) from bushehr, ahvaz (31°19′n 48°41′e) and bostan (31°27′n 48°04′e) counties from khuzistan province with subtropical warm and humid climate were selected. yazd, zarch (54°04'n 31°59'e) from yazd and kerman (30°17′n 57°04′e) from kerman province with hot and dry desert climate were selected. for cool and moist mediterranean climate neka (36°42′n 53°33′e) county from mazandaran and mashhad (36°18′n 59°36′e) from khorasan-e-razavi province were chosen. hamedan (34°48′n 48°31′e) from hamedan, and tehran (35°45′n 51°35′e) from tehran province were selected from cold and dry climate (fig. 1). mosquito sampling and morphological studies this cross sectional study was conducted in 13 randomly selected areas in iran. larval stages of the mosquitoes were collected, using standard dipping technique from april 2009 to october 2010. the larvae were collected from different regions of the country using who standard dipping method (who 1992). the samples were transferred to the entomology laboratory, department of entomology and parasitology, school of medical sciences, tarbiat modares university. the mosquitoes’ larvae maintained in specific cage for rearing in insectarium condition (22–25 ºc, 70–75% rh). microscopic slides were used to mounting of some parts of the adult body such as wings and maxillary palps using canada balsam diluted with xylen. the caudal abdominal segment of males were removed, and placed in koh 10% for 20 to 30 minutes, then washed with distilled water and placed in ethanol 96% for dehydration (barr 1957, jakob et al. 1979). microscope slides of the samples were prepared using puri’s medium. the taxonomic figures were drawn using light zeiss microscope with a nikon drawing tube accessory long arm (9.1 inches) (22.5 cm). the morphological features were used to identify the cx. pipiens complex includes: in larval stage a. seta 1 of abdominal segments iii–iv (fig. 2) (azari-hamidian and harbach 2009). b. seta 1a–s and 1b–s in the siphon (fig. 3) (harbach 1988). c. shape of the siphon (fig. 3) (harbach 1988, azari-hamidian and harbach 2009). in adult stage (male genitalia) a. dv/d: the ratio used for adult identification, also used as a confirmation for molecular studies. dv was described as the distance between two tips of dorsal and ventral arms and d was defined the distance between two tips of dorsal arms (fig. 4) (mattingly et al. 1951, barr 1957, kamura 1959, vinogradova 2003, smith and fonseca 2004). female characters (wing venation) a. costa and subcosta intersection with r2+3 furcation (fig. 5) (harbach 1985, azari-hamidian and harbach 2009). data were analyzed using spss ver. 11.5. results overall, 304 larvae and 419 adults (177 males and 242 females) were randomly selected. the branch number of seta 1 on abdominal segments iii–iv, 1a–s tuft, 1b–s tuft and siphon shape are showed in table 1. culex pipiens larvae were identified in the samples of mashhad, tehran, neka, yazd and zarch1 areas whereas, cx. quinquefascij arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 295 http://jad.tums.ac.ir published online: january 06, 2016 atus was found in zarch 2 and kerman based on the seta 1 abdominal segments iii–iv. the findings showed variations at the mentioned character among the samples collected from borazjan, chabahar, jiroft, and nikshahr. the mean average of 1a–s tuft branches were calculated 2.7 to 4.7 and range of 2 to7 and 6.2 to 7.9, range 2 to 10 for cx. pipiens and cx. quinquefasciatus respectively. the mean average of 1b–s tuft branches were counted 2.8 to 4.4, rage 2 to7 and 6 to7.8, range 3 to 13 for cx. pipiens and cx. quinquefasciatus respectively. the siphon shape of cx. pipiens larvae were found gradually narrowing toward the end of siphon in all samples that were collected from mashhad, tehran, neka, and zarch1 whereas, this character included 84.2 % of cx. pipiens samples collected from yazd. the variation was found among the siphon shape of cx. quinquifasciatus larvae in borazjan, chabahar, jiroft, kerman, and nikshahr samples (table 1). the species of cx. pipiens were identified based on the morphological characters of female wings vegetation in hamedan, yazd and bostan, while it was found varied among the samples collected from neka, zarch, ahvaz, chabahar, nikshahr and jiroft. culex quinquefasciatus was identified in chabahar (97%), ahvaz (88.9%), and nikshahr (73.7 %) (table 2). table 3 shows the dv/d and d/v ratios. the dv/d ratio calculated -1.23–0.12 in mean average and range -0.21–0.2 in cx. pipiens. this ratio was calculated in mean average of 0.5–1.09 and range 0.33–2.37 for cx. quinquefasciatus. the d/v ratio of cx. pipiens samples were calculated in mean average of 0.95–1.35, and range of 0.7–1.75. in addition, mean average of this ratio was calculated 0.35–0.64 and the range was 0.17– 0.78 in cx. quinquefasciatus. table1. the variations of some morphological characters of culex pipiens and cx. quinquefasiatus larvae, islamic republic of iran, 2009–2010 areas no seta 1 abdominal segment iii – iv seta 1a-s seta 1b-s siphon shape single double mean max min mean max min gradually narrowing widen in middle mashhad▲ 21 0 (0%) 21(100%) 3.2 7 2 3.2 5 2 21 (100%) 0 (0%) neka▲ 29 0 (0) 29 (100) 2.7 5 2 2.8 4 2 29 (100) 0 (0) tehran▲ 50 0 (0) 50 (100) 4.3 7 2 4.4 7 2 45 (100) 5 (0) yazd▲ 19 0 (0) 19 (100) 4.7 7 3 4.4 6 3 16 (84.2) 3 (15.8) zarch1▲ 46 0 (0) 46 (100) 4.5 6 3 4.2 7 2 46 (100) 0 (0) borazjan△ 21 4 (19) 17 (81) 6.3 8 4 6.2 10 3 1 (5) 20 (95) chabahar△ 40 20 (50) 20 (50) 6.9 10 4 6.9 10 4 4 (10) 36 (90) jiroft△ 23 4 (17.4) 19 (82.6) 7.9 9 6 7.8 13 6 17 (74) 6 (26) kerman△ 16 0 (0) 15 (100) 6.7 10 4 6.3 8 4 15 (93.7) 1 (6.3) nikshahr△ 24 8 (33.3) 16 (76.7) 6.2 9 4 6 10 4 4 (16.7) 20 (83.3) zarch 2△ 15 0 (0) 15 (100) 6.3 8 2 6.5 8 5 0 (0) 15 (100) total 304 – – – – – – – – – – ▲all of the species identified as culex pipiens△all of the species identified as culex quinquefasciatus j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 296 http://jad.tums.ac.ir published online: january 06, 2016 table 2. the variations of some morphological characters of wings in the females of culex pipiens and cx. quinquefasciatus, islamic republic of iran, 2009–2010 areas no costa and subcosta intersection at or beyond level of furcation of r2+3 (cx. pipiens) before level of furcation of r2+3 (cx. quinquefasciatus) prevalence % prevalence % hamedan▲ 16 16 100 0 0 neka▲ 34 33 97.06 1 2.94 yazd▲ 5 5 100 0 0 zarch▲ 84 75 90 9 10 ahvaz△ 27 3 11.1 24 88.9 bostan▲ 9 9 100 0 0 chabahar△ 33 1 3 32 97 jiroft△ 15 14 93.33 1 6.67 nikshahr△ 19 5 26.3 14 73.7 total 242 – – – – ▲all of the species identified as culex pipiens△all of the species identified as culex quinquefasciatus table 3. the ratio variations of male genitalia in culex pipiens and cx. quinquefasciatus, islamic republic of iran, 2009–2010 areas no dv/d d/v mean max min mean max min hamedan▲ 20 0.09 0.05 -0.17 1.27 1.51 0.92 neka▲ 25 0.07 0.14 0.21 1.21 1.75 0.78 tehran▲ 9 -1.23 0.00 0.19 1.35 1.61 1.00 yazd▲ 30 0.12 0.20 0.19 0.95 1.63 0.70 ahvaz△ 24 0.90 1.54 0.47 0.35 0.55 0.25 chabahar△ 22 0.50 1.55 0.41 0.64 0.78 0.24 jiroft△ 19 1.09 2.29 0.33 0.36 0. 60 0.18 kerman△ 7 0.90 1.68 0.40 0.38 0.56 0.23 nikshahr△ 21 0.79 2.37 0.38 0.46 0.62 0.17 total 177 – – – – – – ▲all of the species identified as culex pipiens△all of the species identified as culex quinquefasciatus fig. 1. the study areas and distribution of culex pipiens and culex quinquefasciatus in different stratum of iran, 2010 j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 297 http://jad.tums.ac.ir published online: january 06, 2016 fig. 2. identification of culex pipiens and culex quinquefasciatus larvae based on abdominal segments of iii-iv fig. 3. identification culex pipiens and culex quinquefasciatus larvae based on siphon characters j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 298 http://jad.tums.ac.ir published online: january 06, 2016 fig. 4. identification of adult male in culex pipiens and culex quinquefasciatus by male genitalia and different of dorsal and ventral arms fig. 5. identification of adult female in culex pipiens and culex quinquefasciatus based on vegetation of wings discussion in present research, the seta 1 on the abdominal segments iii–iv was found the main reliable characters for identification of cx. pipiens and cx. quinquefasciatus in north and central of the country. this character was not found a reliable character for j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 300 http://jad.tums.ac.ir published online: january 06, 2016 cx. quinquefasciatus in southern parts of the country. moosa-kazemi et al. (2010) was reported the occurrence of cx. pipiens in kurdistan province, northeast of iran.this study showed that seta 1 of abdominal segments iii–iv of cx. pipiens had two branches. harbach (1988) and azari-hamidian and harbach (2009) described this character in cx. pipiens as well. our finding showed, siphonal seta 1a-s and 1b-s of cx. pipiens had range of 2–7 branches. these results are supported by previous study, knight and malek (1951) reported an average 4 and a range 2–9 branches for cx. pipiens. results of this study showed the branch number of seta 1a-s and 1b-s had a mean average of 6–7.9 and range of 2–13 for cx. quinquefasciatus. similarly, harbach (1988) reported more branches in cx. quinquefasciatus than cx. pipiens. in this study, shape of the siphon was studied among 165 larvae samples and confirmed vast most of the cx. pipiens species. as mentioned above, there are character variations among the samples which were collected from south of iran. generally, the length of siphon in cx. pipiens was reported longer than in cx. quinquefasciatus (harbach 1988, azari-hamidian and harbach 2009). according to our observations, the seta 1 branches of abdominal segments iii–iv and shape of the siphon in larval samples were found as valuable characters, which can easily used to distinguish of cx. pipiens from cx. quinquefasciatus. in our study, some characters were overlapped between cx. pipiens and cx. quinquefasciatus species, therefore, it is recommended the whole characters should be evaluated to accurate identification. culex pipiens complex is considered cosmopolitan species. although, the distribution patterns of the complex species have been reported in iran (zaim 1987, harbach 1988, azari-hamidian et al. 2010) results of this study indicated the distribution of cx. pipiens in bostan, yazd, neka, mashhad, hamedan and tehran. nevertheless, the distribution of cx. quinquefasciatus was limited in chabahar, nikshahr, jiroft, kerman, borazjan, ahvaz and zarch. the occurrence of cx. quinquefasciatus in the iranian persian gulf islands had been reported previously (azari-hamidian et al. 2010, khoobdel et al. 2012). in addition this species was reported in kermanshah, western iran (ghaffary 1954), and in bandar-e anzali northern iran (harbach 1988). there are scattered data about the species composition of cx pipiens and cx. quinquefasciatus in southern iran. as mentioned, the male genitalia reported as the main morphological characters to identification of the cx. pipiens complex species. in our research dv/d ratio of cx. quinquefasciatus was more than 0.4 (0.56–1.89, mean 1.03), but in cx. pipiens this ratio found ranges -0.14 to zero (harbach 1988). the dv/d ratio of cx. pipiens, calculated at mean average of -1.23–0.12 and range of 0.21–0.2 while, calculated at mean average of 0.5–1.09 and ranges 0.33– 2.37 for cx. quinquefasciatus. the d/v ratio was calculated at mean average of 0.95–1.35, and range of 0.7–1.75 for cx. pipiens samples collected in south of the country, whereas it was calculated at mean average of 0.35–0.64 and range of 0.17–0.78 for cx. quinquefasciatus. further supports for these results also came from some previous studies, sasa (1967) express the d/v ratio in range of 0.4–0.9 in cx. pipiens pallens and less than 0.3 in cx. quinquefasciatus and average of 1.2 in cx. pipiens form molestus. moreover, choochote (1987) reported the d/v ratio at average of 0.35 for cx. quinquefasciatus. in parallel to the present research, mohsen et al. (1995) reported d/v ratio as 1.43 for cx. pipiens form molestus and 0.337 for cx. quinquefasciatus. in recent research, dorsal arms of male genitalia of cx. quinquefasciatus described j arthropod-borne dis, september 2016, 10(3): 292–304 h dehghan et al.: a pictorial key for … 301 http://jad.tums.ac.ir published online: january 06, 2016 as narrow, sharp apex and parallel as the base toward the end. in addition, the ventral arms were flat and leaf shape. whereas, in cx. pipiens dorsal arms were described quite broad, truncate at the apex, and diverges in the base toward the end of arms, which, indicated the occurrence of cx. pipiens in north to central parts of iran. further support to these results also came from a previous study, harbach (1988) described the dorsal arms of phalosoma as divergent, broad and nearly truncate at the apex for cx. pipiens. conclusion identification of the cx. pipiens complex species using morphological key have some difficulties because of occurring variations among closely related complex species. in present pictorial key, the colors of the siphon and male’s genitalia are not referring to the color of original samples. however, the described taxonomical characters in this article should be included in other characters, which were reported in the previous literatures. the most complex species as well as cx. pipiens complex should be identified by morphological characters in the first step, although some researchers prefer molecular studies such as ace. 2 gene, microsatellite loci and coi gene for solving the morphological taxonomic problems. comprehensive studies such as phylogenic and molecular are necessary to obtain new information for identification of cx. pipiens complex species in the future. acknowledgements the authors are grateful to dr azarihamidian, school of health, guilan university of medical sciences, rasht, iran, dr hanafi-bojd school of public health, tehran university of medical sciences, tehran, iran and mr arshad veisi phd student in medical entomology, school of public health, tehran university of medical sciences for reviewing the manuscript. we would also like to express our appreciation from the people of study areas. this study has been financially supported by research committee in school of medical sciences, tarbiat modares university, id no 5278604. the authors declare that there is no conflict of interests. references amirkhanian jd (1974) the salivary gland chromosomes of culex pipiens molestus. iran j public health. 3(1): 40–53. azari-hamidian s, yaghoobi-ershadi mr, javadian e, mobedi i, abai mr (2007) review of dirofilariasis in iran. j med fac guilan univ med sci. 15(60): 102–113 (in persian). azari-hamidian s (2007a) checklist of iranian mosquitoes (diptera: culicidae). j vect ecol. 32: 235–242. azari-hamidian s (2007b) larval habitat characteristics of mosquitoes of the genus culex (diptera: culicidae) in guilan province, iran. iran j arthropod borne dis. 1: 9–20. 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2021 original article the effect of geographical and climatic factors on the distribution of phlebotomus papatasi (diptera: psychodidae) in golestan province, an endemic focus of zoonotic cutaneous leishmaniasis in iran, 2014 seyed hamid hosseini1,2, ehsan allah-kalteh3; *aiuob sofizadeh4 1school of health, tehran university of medical sciences, tehran, iran 2vector borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 3health management and social development research center, golestan university of medical sciences, gorgan, iran 4infectious disease research center, golestan university of medical sciences, gorgan, iran *corresponding author: dr aiuob sofizadeh, e-mail: a_sofizadeh@yahoo.com (received 9 feb 2018; accepted 21 jun 2021) abstract background: phlebotomus papatasi is known as the main vector of zoonotic cutaneous leishmaniasis. this study aimed to investigate the effect of geographical and bioclimatic factors on the ph. papatasi distribution. methods: a total of 34 villages were selected, and sampling was performed three times using 120 sticky traps in each selected village. all the collected species were mounted and identified their species. the densities of ph. papatasi were measured in all the villages and entered into arcmap as a point layer. the required bioclimatic and environmental variables were extracted from the global climate database and the normalized difference vegetation index was obtained from the modis satellite imagery, also, all variables entered into arcmap as raster layers, so the numerical value of each independent variable in the cell where the selected village is located in this, was extracted using spatial analyst tools and the value to point submenu. all the data were finally entered into ibm spss, and the relationship was examined between the number of collected ph. papatasi and the independent variables using spearman's correlation test. results: a total of 1773 specimens of ph. papatasi were collected. the findings of this study showed that max temperature of warmest month, temperature annual range, temperature seasonality, mean diurnal range, precipitation seasonality, mean temperature of driest and warmest quarter were positively associated with the density of ph. papatasi. conclusion: air temperature and precipitation were shown as the most significant factors in the distribution of ph. papatasi. keywords: ecology; phlebotomine sand fly; gis introduction leishmaniasis is one of the most important vector-borne parasitic diseases and appears as a significant health problem in iran. cutaneous and visceral leishmaniasis are two types of the disease in iran. the cutaneous leishmaniasis (cl) is also prevalent in two forms of anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl) in the country (1). different studies conducted in iran showed that vectors for acl and zcl were phlebotomus sergenti and phlebotomus papatasi, respectively (1-3). in golestan province as one of the most important zcl foci in iran (4-8), leishmania major is the agent and wild rodents such as rhombomys opimus and meriones libycus are reservoir hosts of zcl (9-11). in a wide range of similar studies conducted in the province, ph. papatasi was shown as the main zcl vector and the infection range of ph. papatasi to l. major was reported as 10% (11-12). further, various studies conducted in the province showed the cl incidence equal to 31.7 per 100,000 people. among them, the two counties of gonbad-e kavus and copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 226 http://jad.tums.ac.ir published online: june 30, 2021 maraveh tappeh located in the northeast of the province had the highest incidence rates of 153 and 117 per 100,000 people (13). during 2010– 2017, 360–1766 people from all age and gender groups in the province were infected with cl annually (14). compared to the other regions of the province, the vectors and reservoirs of the disease are more abundant in the northeastern region of the province and the disease has higher incidence. these differences could be reasons for the geographical and climatic susceptibility of the region to a higher prevalence of the disease (13, 15). leishmaniasis is naturally dependent on environmental factors and climate change (16), and the environment plays a significant role in the transmission of the disease (17). due to different species of agents and vectors, environmental changes can have various effects on the transmission of leishmaniasis in dif-ferent regions of the world (18). hence, environmental factors and climate change may significantly affect the growth, development, and distribution of ph. papatasi as the main zcl vector. temperature and relative humidity are known as the most significant factors associated with the distribution of ph. papatasi (19), and the climate change that has taken place in recent years has become a critical factor in the distribution of this species (17). it is also believed that the wide distribution of this species in the nature is dependent on environmental conditions and many of these conditions are measurable (20). therefore, in recent years, many studies have been carried out on finding effective factors in the distribution of ph. papatasi and several factors have been considered as essential factors in the distribution of this species. in a study conducted in golestan province (21), slope, altitude, annual mean temperature, and the normalized difference vegetation index (ndvi) were introduced as the most significant factors in the distribution of ph. papatasi. another study conducted in iran (22) also reported the mean temperature of the wettest quarter, slope, precipitation seasonality, and the precipitation of the wettest quarter as major factors associated with the distribution of this species. a study conducted in eastern mediterranean (23) revealed that land cover, mean temperature of coldest quarter, max temperature of warmest month, and min temperature of coldest month were the most critical factors for the distribution of ph. papatasi. further, in a study conducted in libya (20), the altitude from the sea level was reported as a major factor associated with the distribution of ph. papatasi. given the significance of ph. papatasi in the transmission of cl in golestan province, as well as the climate diversity of different regions in the province, the present study was carried out to examine the effect of different geographical and climatic factors (mentioned in the materials and methods section) on the distribution of ph. papatasi. materials and methods study area golestan province is one of the 31 provinces of iran, located in the north-east of the country, south of the caspian sea (53°57′56°23′ e, 36°30′-38°08′ n) and makes approximately 1.3% of iran's total area with a landmass of 20437.74 square km. this province is located between the three provinces of mazandaran, semnan and north khorasan, is bordered by turkmenistan from the north. the province is connected to the caspian sea from the east and to alborz mountains from the south. weather conditions are largely diverse in various regions of the province. as such, one can experience mountainous climates in the south part of the province, arid and semiarid climate in the north part, and mild mediterranean climate in the western and central regions. in 2019, the province had maximum and minimum air temperatures of 40 and 20 mm, respectively, maximum and minimum relative humidity of 70 and 90%, respectively, and a rainfall amount of 333ml. it can be concluded that a wide range of suitable clihttp://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/iran https://en.wikipedia.org/wiki/caspian_sea j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 227 http://jad.tums.ac.ir published online: june 30, 2021 matic and geographical conditions are available for the development of various insect species in the province (24). sand fly collection this analytical cross-sectional study was carried out from july to september 2014 by performing a three-time capture in a total of 34 villages (2–4 villages in each county). sticky paper traps coated with castor oil were used to collect sand flies. for each village, 60 indoor and 60 outdoor sticky paper traps were installed before the sunset and collected the following morning before the sunrise. all the collected sand flies were placed in acetone for two minutes and stored in 70% ethanol before transferring to the laboratory. in the laboratory, microscopic slides of the specimens were provided and the specimens were mounted in puri’s medium. species of all the sand flies were determined using relevant morpholo-gical keys (25-26). the data were analyzed using spearman's correlation test in ibm spss version 22.0. study variables in this study, we used 22 geographical and climatic variable including: alt: altitude from sea level (m) (alt), slope: slope in degrees obtained from altitude (%), bio1: annual mean temperature (°c), bio2: mean diurnal range (monthly mean (max temp-min temp)) (°c), bi03: isothermality (bio2/bio7) (×100), bio4: temperature seasonality (standard deviation × 100), bio5: max temperature of warmest month (°c), bio6: min temperature of coldest month (°c), bio7: temperature annual range (bio5bio6) (°c), bio8: mean temperature of wettest quarter (°c), bio9: mean temperature of driest quarter (°c), bio10: mean temperature of warmest quarter (°c), bio11: mean temperature of coldest quarter (°c), bio12: annual precipitation (mm), bio13: precipitation of wettest month (mm), bio14: precipitation of driest month (mm), bio15: ), bio16: precipitation of wettest quarter (mm), bio17: precipitation of driest quarter (mm), bio18: precipitation of warmest quarter (mm), bio19: precipitation of coldest quarter (mm), ndvi: normalized differentiated vegetation index. bioclimatic variables (n=19), environmental variables including altitude and slope, and ndvi were independent variables. the bioclimatic variables were obtained from the worldclime global climate database (http://www.worldclim.org/current) at a spatial resolution of 1km2. these variables were derived from long-term (1950–2000) monthly rainfall and temperature values for the development of significant biological variables. the environmental variables such as altitude and slope were obtained from a digital elevation model, and ndvi was obtained from modis satellite images in august, 2014. this study evaluated the relationship between the number of collected ph. papatasi as the dependent variable and the independent variables. the data from the sampled villages were entered into arcmap as a point layer, and each of the independent variables was entered into arcmap as a raster layer. then, each village layer was activated separately with an independent variable layer, by using spatial analyst tools and the value to point submenu, the numerical value of each independent variable in a cell, where the selected village was located, was extracted and entered into spss 22.0. eventually, due to the non-normal distribution of densities of ph. papatasi and the lack of linear regression assumptions, the relationship between the densities of ph. papatasi with the bioclimatic variables, the environmental variables, and ndvi was examined using spearman's correlation test. results in this study, a total of 1773 ph. papatasi were collected from 34 villages of golestan province, with the highest frequency in gonbad-e-kavus and maraveh tappeh counties (table 1). the findings show that max temperature of warmest month (˚c), temperature anhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 228 http://jad.tums.ac.ir published online: june 30, 2021 nual range (bio5-bio6) (˚c), temperature seasonality (±sd×100), mean diurnal range (average min/max temp) (˚c), precipitation seasonality (coefficient of variation), mean temperature of driest quarter (˚c), and mean temperature of warmest quarter (˚c) were positively associated with the density of ph. papatasi, moreover, the density of ph. papatasi showed a positive inverse correlation with ndvi, but no correlation with altitude and slope (table 2). table 1. densities of phlebotomus papatasi in different counties of golestan province, 2014 county number of selected villages number of collected ph. papatasi number of collected ph. papatasi for 60 traps maraveh tapeh 3 396 132 gonbad-e kavus 4 564 141 aqqala 2 114 57 ramiyan 2 42 21 gomishan 2 64 32 aliabad-e katul 3 156 52 azadshahr 3 138 46 gorgan 2 14 7 kalaleh 3 261 87 kordkuy 2 2 1 bandar-e gaz 2 0 0 bandar-e torkman 2 10 5 galikesh 2 6 3 minudasht 2 6 3 total 34 1773 52.1 table 2. bioclimatic and environmental variables with direct and positive correlation with densities of phlebotomus papatasi correlation variables mean±sd* correlation coefficient p direct and positive number of collected ph. papatasi 50.08±54.43 r= 0.747 p= 0.00 max temperature of warmest month (°c) 34.06±1.16 number of. collected ph. papatasi 50.08±54.43 r= 0.529 p= 0.002 temperature annual range (max temp of warmest month–min temp of coldest month) (°c) 32.01±2.17 number of. collected ph. papatasi 50.08±54.43 r= 0.523 p= 0.002 temperature seasonality (standard deviation ×100) 7.66±0.56 number of. collected ph. papatasi 50.08±54.43 r= 0.466 p= 0.006 mean diurnal range (mean of monthly (max temp–min temp)) (°c) 11.52±0.87 number of. collected ph. papatasi 50.08±54.43 r= 0.465 p= 0.006 precipitation seasonality (coefficient of variation) 57.45±8.91 number of. collected ph. papatasi 50.08±54.43 r= 0.449 p= 0.009 mean temperature of driest quarter (°c) 26.81±1.08 number of. collected ph. papatasi 50.08±54.43 r= 0.387 p= 0.026 mean temperature of warmest quarter(°c) 26.97±0.87 number of. collected ph. papatasi 50.08±54.43 r= 0.345 p= 0.045 ndvi 0.36±0.11 number of. collected ph. papatasi 50.08±54.43 r= 0.459 p= 0.007 annual precipitation (mm) 348.42±80.26 number of. collected ph. papatasi 50.08±54.43 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 229 http://jad.tums.ac.ir published online: june 30, 2021 direct and negative precipitation of driest month (mm) 6.78±4.41 r= 0.497 p= 0.003 number of. collected ph. papatasi 50.08±54.43 r= 0.552 p= 0.001 precipitation of driest quarter (mm) 23.93±13.56 number of. collected ph. papatasi 50.08±54.43 r= 0.590 p = 0.00 precipitation of warmest quarter (mm) 25.66±13.29 number of. collected ph. papatasi 50.08±54.43 r= 0.348 p= 0.047 precipitation of coldest quarter (mm) 120.6±22.66 without correlation number of. collected ph. papatasi 50.08±54.43 r= 0.018 p= 0.925 altitude 208.48±312.79 number of. collected ph. papatasi 50.08±54.43 r= 0.174 p= 0.331 slope 89.78±0.37 number of. collected ph. papatasi 50.08±54.43 r= 0.127 p= 0.481 annual mean temperature (°c) 17.20±1.15 number of. collected ph. papatasi 50.08±54.43 r= 0.123 p= 0.495 isothermality (bio2/bio7) (×100) 3.54±0.09 number of. collected ph. papatasi 50.08±54.43 r= 0.244 p= 0.164 mean temperature of wettest quarter (°c) 25.16±21.75 number of. collected ph. papatasi 50.08±54.43 r= 0.323 p= 0.067 mean temperature of coldest quarter (°c) 7.60±1.88 number of. collected ph. papatasi 50.08±54.43 r= 0.285 p= 0.108 precipitation of wettest month (mm) 60.06±13.27 number of. collected ph. papatasi 50.08±54.43 r= 0.191 p= 0.287 precipitation of wettest quarter (mm) 145.15±25.53 number of. collected ph. papatasi 50.08±54.43 r= 0.340 p= 0.053 min temperature of coldest month (°c) 2.05±1.93 *: standard deviation !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !(!( !(!(!( !( !( !(!( !( gonbad-e kavus aqqala kalaleh gorgan maraveh tappeh gomishan galikesh kordkuy ramiyan azadshahr minudasht aliabad-e katul bandar-e gaz bandar-e torkaman legend !( selected villages golestan province counties ¯ 0 30 60 90 12015 kilometers fig. 1. location of golestan province in iran and collection sites for phlebotomus papatasi table 2. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 230 http://jad.tums.ac.ir published online: june 30, 2021 discussion the highest frequency of the collected ph. papatasi was reported in gonbad-e-kavus and maraveh tappeh counties in accordance with results of a similar study conducted in golestan province, similarly reporting that the presence probability of this species was higher in these counties (21). these two counties are known as endemic focus for cl in golestan province (4-9). moreover, the incidence of cl and the number of the rodent’s active burrows (wellknown reservoir hosts of zcl) were higher in these regions than in the other regions of golestan province (7, 9, 15). temperature was shown as one of the main factors associated with the growth and development of insects such as ph. papatasi (19). the results of this study revealed that temperature significantly affected the density of ph. papatasi. the densities of ph. papatasi were significantly positively associated with factors such as max temperature of warmest month (°c), temperature annual range (bio5-bio6) (°c), temperature seasonality (standard deviation ×100), mean diurnal range (mean of max/ min temp) (°c), mean temperature of driest quarter (°c), and mean temperature of warmest quarter (°c). however, there was no significant relationship between the densities of ph. papatasi with annual mean temperature (°c), isothermally (bio2/bio7) (×100), mean temperature of wettest quarter (°c), mean temperature of coldest quarter (°c), and min temperature of coldest month (°c). it can be concluded that max and mean temperatures of driest and warmest quarters and months were significantly associated with the density of ph. papatasi. however, mean temperature of wettest and coldest quarters and months had no significant role in the density of ph. papatasi, which may be due to the effect of degree-day on the growth, development, and density of different species of insects. degree-day is a measure of the amount of heat that accumulates above a specified temperature during a 24h period (27). ac cording to results of studies on the growth of this species, a specific temperature limit is required for growth, and temperatures higher or lower than this limit will lead to a stop of growth. the maximum temperature threshold for the growth of this species is 35 °c, and the optimal minimum temperature for the growth according to different growth stages of this species is 20, 25, and 11.6 °c. as such, preoviposition activities are carried out at a temperature above 20 °c (8, 28-29). therefore, temperature in the cold seasons, in which the air temperature does not reach 20 °c, cannot have a significant relationship with the density of this species. colacicco-mayhugh mg et al. (23) found that max temperature of warmest month was one of the most critical factors associated with the density of ph. papatasi in eastern mediterranean, which is supported by the findings of our study. they also showed that mean temperature of coldest quarters and min temperature of coldest month were the most influential factors in the density of this species (23); while, in our study, there was no significant relationship between the values of these variables and the densities of ph. papatasi. further, the results of the present research showed no significant relationship between the densities of ph. papatasi with isothermality (bio2/bio7) (×100) and mean temperature of wettest quarter (°c). these variables had low effects on the density of ph. papatasi in another study conducted in golestan province (21). however, in a study conducted in iran (22), contrary to the results of this study, isothermality (bio2/bio7) (×100) and mean temperature of wettest quarter (°c) showed high impacts on the density of this species. in addition, we found no significant relationship between annual mean temperature and densities of ph. papatasi. this result is in accordance with the results of a study in iran (22), showing a relatively low correlation between this factor and the density of ph. papatasi. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 231 http://jad.tums.ac.ir published online: june 30, 2021 while in a study conducted in golestan province (21), this variable showed a significant effect on the density of this species. differences between the results of these studies and those of our study may be due to differences in the nature of these studies. all of these studies applied the maxent model as an ecological niche model, in which modeling and prediction are based on the existence of a species in a region (30) and quantity is not measured. however, the present study analyzed the effect of each variable quantity on the densities of ph. papatasi and quantity had a direct effect on the study outcomes. precipitation was another main factor associated with the density of ph. papatasi. the findings of the current study suggested that the densities of ph. papatasi were positively associated with precipitation seasonality (coefficient of variation). in other words, the increased precipitation increased the density of sand flies, which is in accordance with the results of studies conducted in iran and other countries (22, 31-32). rainfall influences the dynamics, reproduction, and breeding of vectors such as sandflies (33-34), as sandflies need a certain amount of moisture for their development and survival. however, heavy rainfalls can kill adults and immature stages of sand flies (35-38). we found no significant relationship between the density of ph. papatasi with precipitation of wettest quarter and precipitation of wettest month. however, hanafi-bojd et al. (22) and rodgers et al. (39) revealed that precipitation of wettest quarter was a major factor associated with the density of ph. papatasi. colacicco-mayhugh mg et al. (23) revealed that precipitation of wettest quarter and precipitation of wettest month were not significantly associated with the abundance of ph. papatasi, which is consistent with our study. perhaps the reason for this is that the wettest months and quarters of the year occur in winter and sand flies have no activity in this season; with the end of the wet months of the year, the population of sand flies increases, as it was pointed out in another article (40). moreover, ndvi is another factor associated with the density of ph. papatasi. the findings of our study revealed a significant reverse relationship between ndvi and the densities of ph. papatasi, as this species is more abundant in areas with lower ndvi, which is in compliance with other studies conducted in this province and in morocco county (15, 21, 41). moreover, abdel-dayme et al. (20) showed vegetation type as a major factor associated with the density of ph. papatasi. colacicco-mayhugh mg (23) also introduced land cover as a significant factor associated with the density of ph. papatasi. moreover, mollalo et al. (42) revealed a significant negative relationship between ndvi and the incidence of zcl, as zcl is more prevalent in areas with lower ndvi. therefore, it can be concluded that ndvi is an essential factor in the incidence of zcl. another factor associated with the density of ph. papatasi is elevation. accordingly, in a previous study conducted in golestan province, ph. papatasi was collected at altitudes ranging from -32m from sea level to 598m above sea level, and densities of this species were more in plain areas with a lower altitude than in areas with a higher altitude (15). in a study conducted in libya (20), it was observed that areas with an altitude lower than 600m were mostly suitable for the distribution of ph. papatasi. further, in a study conducted in pakistan (43), altitude had the highest effect on the density of cl among various examined variables and ph. papatasi was the dominant species in areas with a higher altitude. however, in this study, there was no significant relationship between altitude and the abundance of ph. papatasi, which is inconsistent with previous studies. regarding slope as another factor associated with the density of ph. papatasi, in different studies conducted in golestan province (21, 44) and iran (22), slope was introduced as a major factor in the density of ph. pahttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 225–235 s h hosseini et al.: the effect of … 232 http://jad.tums.ac.ir published online: june 30, 2021 patasi. however, the findings of the present study showed no significant relationship between slope and the densities of ph. papatasi. the density of sandflies might also depend on other environmental factors, such as soil type, land-use, or wind, which can impair their flight activity (38). in the present study, we could not assess the relationship between these factors and the density of ph. papatasi, which could be one of the limitations of the study. conclusions air temperature and precipitation 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adnan m, rasheed sb (2017) environmental risk modelling and potential sand fly vectors of cutaneous leishmanisis in chitral district: a leishmanial focal point of mount tirich mir, pakistan. trop med int health. 22(9): 1130–1140. 44. mollalo a, sadeghian a, israel gd, rashidi p, sofizadeh a, glass ge (2018) machine learning approaches in gisbased ecological modeling of the sand fly phlebotomus papatasi, a vector of zoonotic cutaneous leishmaniasis in golestan province, iran. acta trop. 188: 187–194. http://jad.tums.ac.ir/ https://www.researchgate.net/journal/1573-2959_environmental_monitoring_and_assessment j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 186 original article distribution of anopheles culicifacies and detection of its sibling species e from madhya pradesh: central india *ajay kumar sharma 1, varun tyagi 1, sompal singh 2, vijay veer 3, om prakash agrawal 4, devanathan sukumaran 1 1vector management division, defence research and development establishment, madhya pradesh, india 2national institute of malaria research, new delhi, india 3defence research laboratory, assam, india 4school of studies in zoology, jiwaji university, madhya pradesh, india (received 29 oct 2013; accepted 8 dec 2013) abstract background: anopheles culicifacies is an important vector of malaria in southeast asia, contributing to almost 70% of malaria cases in india. it exists as a complex of five morphologically indistinguishable species a, b, c, d and e with varied geographical distribution patterns. in india, 8% of the total population of madhya pradesh (central india) contributes about 30% of total malaria cases, 60% of total falciparum cases and 50% of malaria deaths. an. culicifacies is the major malaria vector in this state. vector control mainly relies on the proper identification and distribution of vector species exists in a particular area. the present study was carried out to identify the distribution of an. culicifacies sibling species in certain endemic district of central india, madhya pradesh. methods: the an. culicifacies mosquitoes collected from the study districts were identified morphologically. the genomic dna was isolated from the mosquitoes and subjected to allele specific pcr targeting d3 domain of 28s ribosomal dna. results: the mean prevalence of an. culicifacies during the study period was in the range of 8–120 per man per hour (pmh). from the study areas species b was identified from jabalpur, chindwara and hoshangabad, species c from hoshangabad only, species d from narsinghpur and khandwa and sibling species e from mandla, chindwara and hoshangabad respectively. conclusion: this is the first report to detect species e from madhya pradesh region which necessitate for reconsideration of species distribution of each an. culicifacies sibling species that would enable to develop required vector control strategies. keywords: malaria, madhya pradesh, anopheles culicifacies, sibling species, as-pcr introduction malaria is a major public health problem in tropical and subtropical countries including india and its dynamics vary from place to place. among all anopheline vectors, anopheles culicifacies (diptera: culicidae) is a principal malaria vector in rural, periurban and tribal settings (mishra et al. 2012). an. culicifacies has a wide distribution that extends from ethiopia, yemen and iran in the west via afghanistan, pakistan, india, bangladesh, myanmar and thailand, to laos and vietnam and combodia in the east (harrison 1980, van bortel et al. 1984, subbarao 1988, zaim and javaherian 1991, surendran et al. 2000, mahmood et al. 2002, vatandoost et al. 2011). to the north it is found in nepal and southern china, and in the south in sri lanka. it is responsible for about 65–70% malaria cases in india (goswami et al. 2006). an. culicifacies is a complex of 5 isomorphic *corresponding author: dr ajay kumar sharma, e-mail: ajaysharma@drde.drdo.in http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 187 types which are designated as species a and b (green and miles 1980), species c (subbarao et al. 1983), species d (subbarao et al. 1988, suguna et al. 1989, vasantha et al. 1991) and species e (kar et al. 1999) with varying biological characteristics such as feeding preference, biting activity, and susceptibility to commonly used insecticides in public health programs (joshi et al. 1988, subbarao et al. 1988, raghavendra et al. 1991, subbarao et al. 1997) which are relevant for the transmission of the disease and control. madhya pradesh is situated in the central part of india with an area of 308 thousand km2 of which forest cover 76,429 km2 (about 25% of the total land area). madhya pradesh (population 72.6 million) along with other states like orrisa (population 42 million), jharkhand (population 33 million), and chhattisgarh (population 25.5 million) contributes for more than 60% of reported (confirmed) malaria cases in india. according to national vector borne diseases control program (nvbdcp) epidemiological data for 2010 from predominantly these tribal states with a total population of 173.1 million (out of a total of the country population i. e., 1.21 billion) represent 14.3 % population show persistent malaria transmission with high api (annual parasite incidence), slide positivity rate (spr) and very high pf% (sharma 2012). madhya pradesh alone account for 6% of the total population of the country but contributes to 8.6% of the total malaria cases. malaria is complex in madhya pradesh because of vast tracts of forest with tribal settlement (20% of state population) (singh et al. 2004, anon 2007). the magnitude of the problem can be accessed from an estimate made in 1987, that 54 million individuals of various ethnic origins residing in forested area of india and accounting for 8% of the total population contributed 30% of total malaria cases, 60% of total falciparum cases and 50% of malaria deaths in the country (sharma 1996). the reasons for such a high diseases prevalence in madhya pradesh is mainly due to locations of the villages in the deep forest and is characterized by rocky undulation interspersed with ravines and foothills. another reason is the innumerable streams which flow into the river, narmada. these streams flow continuously and provide ample breeding sites covered with dense aquatic vegetation for production of number of anophelines particularly an. culicifacies (singh 2006). therefore malaria control in these areas requires specific approaches and control strategies which includes the proper surveillance for distribution of an. culicifacies members and their identification (pattanayak et al. 1994). since sibling species a, b, c, d and e of an. culicifacies are morphologically indistinguishable at any stage of life and due to practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, a molecular method using an allele-specific polymerase chain reaction (as-pcr) assay targeted to the d3 domain of 28s ribosomal dna was used to distinguish these sibling species (singh et al. 2004, goswami et al. 2006). the assay discriminates an. culicifacies species at two tier level diagnosis. firstly in d3pcr the species complex is distinguished in two groups i.e., a and d in one group and species b, c and e in the second group. in second tier involves ad-pcr assay which distinguishes species a from species d, whereas the bce-pcr assay distinguishes species b, c and e with each other. with combination of these two tier pcr assays it is possible to identify individual mosquito of the an. culicifacies complex. the present study was aimed to find the distribution of members of an. culicifacies species in various districts of madhya pradesh, india. in the earlier reports from this region species a, b, c and d were identified but species e reported from southern parts of india only was not reported from this area. noticeably in our study we encountered spehttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 188 cies e from some of the districts in co-habitation with species b which indicates that all the five members of an. culicifacies species complex occurs in this part of central india. materials and methods collection of mosquitoes the adult anopheline mosquito species were collected from different districts of madhya pradesh ie, mandla (dungaria village), jabalpur (barela village), chindwara (chakarpat and chikhla villages), hoshangabad (dhadav and padav villages), narsinghpur (chinki village) and khandwa (chighdhalia) (fig. 1 and table 1). these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria (singh 2004, 2006, 2009, sharma 2012). the collections were made during the transmission period i.e. february–march and august–september in the morning period between 0600 h to 0800 h using mouth aspirator and battery operated torch. the fed anophelines were captured at various collection sites including human dwellings, cattle sheds, mixed dwelling and random collection sites. the fed mosquitoes were captured so that f1 generation of these mosquitoes can be utilized for further use after egg lay. all adult mosquitoes were brought to the lab for their identification by using standard keys (christopher 1933, wattal and kalra 1961, das et al. 1990, nagpal and sharma 1995). each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection. from these collection the an. culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction (as-pcr) (goswami et al. 2006, singh et al. 2006). dna isolation the dna extraction was done by using method as described in our previous publication (sharma et al. 2009, 2010). each single adult mosquito was homogenized in the micro centrifuge tube by adding 100 µ l lysis buffer. the homogenate was immediately kept on ice for 10 minutes and followed by heat treatment at 65 ºc for 30 minutes. subsequently, 30 µl 5m potassium acetate was added and immediately transferred to ice for one hour followed by centrifugation at 13,000 rpm for 15 minutes at 10 ºc. to the supernatant obtained, a double volume of absolute chilled ethanol was added for precipitation of dna and kept tubes at -20 ºc for overnight. after centrifugation at 13,000 rpm for 15 minutes at 10 ºc, the precipitated dna was washed in 70% ethanol twice. the dna pallet was allowed to air dry and finally dissolved in 50 µ l te buffer. allele specific pcr (as-pcr) d3-pcr the targeted region, d3 domain of 28 s rdna, was amplified by pcr using universal primers, d3a and d3b designed for platyhelminth (litvaitis et al. 1994) and later used for an. minimus (sharpe et al. 1999) and for both an. fluviatilis and an. culicifacies (singh et al. 2004a, b). another set of allele specific primers namely aca and acb which are specific to species a/d and species b/c/e respectively were selected for design of multiplex as-pcr. the sequences for the primers used were given in the table 2 with their annealing temperatures. the amplification was performed in a total of 15 µ l of reaction mixture consisting of tris. hcl 10 mm ph 9.0, kcl 50 mm mg cl2 2 mm, dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase (mbi fermentas) and 10ng of genomic dna. reactions were performed in a (biorad pcr system icycler) thermal cycler. the pcr conhttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 189 dition consisted of initial denaturation step for 5 min at 95 ˚c followed by 35 cycles of 30 sec at 95 ˚c, 30 sec at 55 ˚c, and 60 sec at 72 ˚c. a final extension step was performed at 72 ˚c for 7 min. ad-pcr and bce-pcr a total of seven primers of which three primers adf, adr, and df were used in the ad-pcr assay differentiating sibling species a from d, and the other four set of primers bcef, bcr, cr and er were used in the bce-pcr assay for the dedifferentiating species b, c and e from each other (table 2). optimized condition for ad-pcr assay includes 35 cycles of the initial denaturation temperature at 95 ˚c for 40 s, annealing at 50 ˚c for 40 s, and extension 68 ˚c for 40 s, followed by a final extension at 72 ˚c for 10 min. the pcr reaction was comprised of adf, adr and df primers each at 25 pmol, 200µ mol/l of each of the dntp, 1.5 mmol/ l mgcl2, 20 mmol/l (nh4) so4, 75 mmol/ l tris-hcl ph 9.0 and 0.625 unit of taq dna polymerase. whereas the condition for bce-pcr assay are similar as described for the ad-pcr assay except for the primer concentration of 25 pmol bcef primer, 12 pmol bcr primer, 25 pmol er primer and 30 pmol cr primer respectively. results during the collection period a large number of anopheles mosquitoes were collected from the various collection sites (fig. 1 and table 1) and per man per hour (pmh) count estimate was also made from each site. a total of 45 houses including cattle sheds were sampled for collection of mosquitoes. the mean prevalence of an. culicifacies during the study period was found to be in the range of 8–120 pmh, with a high density during august-september (90–120 pmh) and to a low density in february–march (8– 50 pmh). the as-pcr assay using different primers, the a/d specific primer (aca) in conjunction with d3b produces 313 bp amplification product and b/c/e-specific primer (acb) forms 133 bp product with d3a. additionally, the external primers d3a and d3b form common product in all the samples with 382bp products in species a and d whereas 385bp in species b/c/e serving as positive control (fig. 2). for further distinguishing the sibling species in a/d and b/c/e individually, a total of seven primers of which three primers adf, adr, and df were used in the ad-pcr assay differentiating sibling species a from d, and the other four set of primers bcef, bcr, cr and er were used in the bce-pcr assay differentiating sibling species b, c and e from each other. in ad-pcr, the sibling species a and d produced the bands of 359 bp for d species and 359 bp and 166 bp for sibling species a. on the other hand in bce-pcr, the products are 248 bp for b, 248 bp and 95 bp for c and 248 and 178 for sibling species e respectively (fig. 3). the an. culicifacies collected from different districts from madhya pradesh ie, mandla, jabalpur, chindwara, hoshangabad, narsinghpur and khandwa respectively. the collected mosquitoes were subjected to two tier pcr assays. from the d3-pcr only narsinghpur and khandwa samples were identified in a/d group whereas all the other districts samples were identified in and identified in b/c/e group (fig. 2). results obtained from ad-bce pcr showed the presence of sibling species b from jabalpur, chindwara and hoshangabad, c from hoshangabad only, d from narsinghpur and khandwa and sibling species e from mandla, chindwara and hoshangabad respectively (fig. 3). http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 190 fig. 1. map showing different collection sites in the study fig. 2. d3-pcr showing the different bands to differentiate a/d and b/c/e sibling species of an. culicifacies fig. 3. allele specific multiplex ad-bce-pcr showing different bands for each an. culicifacies sibling species http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 191 table 1. list of collection sites and result using as-pcr assay of an. culicifacies mosquito village (district) (latitude, longitute) number of houses sampled number of an. culicifacies samples subjected species identified dungaria (mandla) (220 60’ n, 800 38’ e) 7 54 e (all) barela (jabalpur) (230 10’ n, 790 59’ e) 5 26 b (all) chakarpat, chikhla (chindwara) (220 03’ n, 780 59’ e) 8 56 b (21), e (35) dhdav, padav (hoshangabad) (220 46’ n, 770 45’ e) 10 124 b (40), c (16), e (68) chinki (narsinghpur) (220 57’ n, 790 15’ e) 7 85 d (all) chighdhalia (khandwa) (210 49’ n, 760 22’ e) 8 74 d (all) table 2. list of primer used for molecular identification of an. culicifacies sibling species sequence no. primer sequence (5’-3’) tm 1 d3a gac ccg tct tga aac acg ga 67.3 2 d3b tcg gaa gga acc agc tac ta 61.3 3 aca gcc gtc ccc ata cac tg 62.7 4 acb ccg taa tcc cgt gat aac tt 60.2 5 adf cta atc gat att tat tac ac 45.5 6 adr tta ctc cta aag aag gc 48.8 7 df tta gag ttt gat tct tac 42.9 8 bcef aaa tta ttt gaa cag tat tg 48.4 9 bcr tta ttt att ggt aaa aca ac 48.6 10 cr agg agt att aat ttc gtc t 49.3 11 er gta aga atc aaa ttc taa g 45.1 discussion the accurate identification and distribution pattern of anopheline mosquitoes is necessary for planning effective vector control strategies and for a better understanding of their potential role in malaria transmission. wattal and kalra in 1961 described 32 species of female anophelines in different regions of india. they divided india in six regions and included madhya pradesh in hyderabad region where about 28 species were described from this region (wattal and kalra 1961). in our collection we have also encountered about 11 species of anopheles from the study area which includes an. culicifacies, an. fluviatilis, an. stephensi, an. subpictus, an. annularis, an. barbirostris, an. varuna, an. jamesi, an. jeyporiensis, an. tessellates and an. theobaldi respectively. but in the study area malaria is mainly transmitted by two efficient vectors i.e., an. culicifacies and an. fluviatilis, moreover the density of an. culicifacies is very high throughout the year (singh 2006). an. culicifacies being a major vector of malaria in india is responsible for approximately 65% of total malaria cases (sharma 1998). in india all five species of an. culicifacies have been found among these species b was found throughout the country whereas species e was reported only from the southern parts of india. all these sibling species poses a number of biological variations among them. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 192 they may be different in the feeding pattern an important characteristics that influence vectorial capacity. distinct difference were observed in laboratory studies with reference to insemination rate, fecundity, longevity, oviposition, gonotropic cycle, egg hatching, larval mortality rates and adult emergence time have been observed in different species of an. culicifacies (ansari et al. 1997, subbarao et al. 1998). attempts to find morphological markers for the members of species complex have not been successful so far except the variation in spermatheca of sibling species a and b (das 1990) but this difference still need to be reconfirmed by other techniques. the classical technique of cytotaxonomic is difficult and has limited use as this requires semi gravid females only and moreover it requires highly skilled personnel. with the advent of dna based technology we are now able to differentiate members of an. culicifacies complex. the dna based technology includes pcr assay which are simple and sensitive at the same time they are applicable to all stages and either sexes of mosquitoes (goswami et al. 2006). as reported by sharma (2012) in his review that the epidemiological indices of malaria in madhya pradesh revealed a very dismal picture of malaria. an international team of experts reported a very high incidence of malaria in pregnancy (mip). for example in madhya pradesh (rural) 183,000–1.5 million per year contract malaria in pregnancy, and result in 73,000– 629,000 lost foetus and 1,500 to 12,600 maternal deaths. authors state “plausible estimate of 220,000 mip cases per year (136,000–305,000), 95,800 lost fetus (56,800–147,600) and 1,000 maternal deaths (650–1,600)” (diamond et al. 2009). in our study an. culicifacies mosquito species were collected from different malaria endemic district of madhya pradesh by using as-pcr we were able to identify four species b, c, d and e from these areas. species e was not reported earlier from these areas as this species is prevalent only in southern peninsular part of india. recently species e was reported by das et al. (2013) from odisha, eastern india also showing its vectorial role. species e is highly anthropophagic and possesses high sporozoite rate up to 20% and also known as vector in southern india and srilanka (kar et al. 1999). in our study we encountered species e from mandla, chindwara and hoshangabad districts respectively which have high disease prevalence and represent the tribal belts (singh 2004, 2006, 2009, sharma 2012). noticeably this species e was found sympatric with a non vector species b from chindwara and hoshangabad district, but in jabalpur district only species b and in mandla only species e were identified. topographically the villages under study from mandla and jabalpur are very close to each other. although species b is a poor vector of malaria in india but through examination of mitotic y chromosome morphology (kar et al. 1999), that what was reported as b on the sri lanka island is really a sympatric mixture of b and e (surendran et al. 2000). moreover these two sibling species in sri lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides (surendran et al. 2006b). it is evident from the literature that species e cannot be differentiated from species b because they have homosequential polytene chromosome arrangements. species e requires mitotic chromosome examination of male progeny and/or vectorial potential needs to be established for distinction from species b (kar et al. 1999). in the absence of either of these, identification of species e may not be accurate. after screening several enzyme systems, electrophoretic variation at the lactate dehydrogenase (ldh) locus was useful. it could group species a and d in one category and species b and c in another http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 186–196 ak sharma et al.: distribution of anopheles … 193 category (adak et al. 1994). species e showed the same ldhs allele as in species b and c (kar et al. 1999). with the identification of species e, the paradox that species b females from northern india are not vectors, but homosequential females from rameshwaram island are vectors is resolved. similar studies are needed to be carried out to see the vectorial capacity and disease transmission in the studied districts of madhya pradesh. as in mainland areas close to rameshwaram, species e were found, populations in other parts of tamilnadu state where species b has been identified also should be examined (kar et al. 1999). an. culicifacies populations identified as species b in sri lanka also should be examined immediately for y-chromosome variations and correlated with malaria infection (surendran et al. 2000, 2006a). recently, adak et al. (1997) has reported acrocentric and submetacentric y-chromosomes within species b but no epidemiological or dissection data is available that indicates that species b is a vector. thus, there is an urgent need to develop suitable markers that can differentiate species b and e and also to see the other biological characteristics of these two species in such cohabiting areas to conclude that which species is responsible for disease transmission. moreover new areas should be explored for the presence of species e as it is a potent vector of malaria. hence there is an urgent need for nationwide surveillance and identification of vector sibling species distribution once again so that modified species pattern in these areas could be established. apart from confirming their identity, distribution pattern and their differential malaria vector status, it will be important to determine the susceptibility of these sibling species to insecticides in each part of the states/ country as this affects the efficiency of vector control operations in the malaria control programs in india. conclusion in conclusion we can say that as we encountered a new sibling species e of an. culicifacies from the study sites where it was not reported earlier. existence of such modified distribution of sibling species may exist in other areas also which necessitate for reconsidering the sibling species distribution in newer area. knowledge of proper identification and distribution pattern of sibling species may further help us in development of vector control strategies. acknowledgements the authors are thankful to prof (dr) mp kaushik, outstanding scientist and director, defence research and development establishment, gwalior, madhya pradesh, india for his keen interest and providing all necessary facility to conduct this research work. sincere thanks also due to the scientists and supportive staff of vector management division for their kind cooperation for carrying out the above work. the authors 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for a new sibling species, species c. ann entomol soc am. 76: 985–986. subbarao sk, vasantha k, sharma vp (1988) response of anopheles culicifacies species a and b to ddt and hch in india: implications in malaria control. med vet entomol. 2: 219–223. subbarao sk, sharma vp (1997) anopheline species complexes and malaria control. indian j med res. 106: 164–173. subbarao sk (1998) anopheline species complexes in south-east asia. world health organization regional office for south-east asia, new delhi, india. suguna sg, tewari sc, mani tr, hiryan j, reuben r (1989) a cytogenetic description of a new species of the anopheles culicifacies species complex. genetica. 78: 225–230. surendran sn, abhayawardana ta, de silva bgdnk, ramasamy ms, ramasamy r (2000) anopheles culicifacies y chromosome dimorphism indicates the presence of sibling species (b and e) with different malaria vector potential in sri lanka. med vet entomol. 14: 437–440. surendran sn, hawkes nj, steven a, hemingway j, ramasamy 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for species d (diptera: culicidae). ann entomol soc am. 84: 531–536. vatandoost h, emami sn, oshaghi ma, abai mr, raeisi a, piazzak n, mahmoodi m, akbarzadeh k, sartipi m (2011) ecology of malaria vector anopheles culicifacies in a malarious area of sistan va baluchestan province, southeast islamic republic of iran. east mediterr health j. 17(5): 439–445. wattal bl, kalra nl (1961) region wise keys to the female indian anophelines. bull natl soc. indian mal mosq dis. 9: 85–138. zaim m, javaherian z (1991) occurrence of anopheles culicifacies species a in iran. j am mosq control assoc. 7(2): 324– 326. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 139–146 mh davami et al.: molecular survey on … 139 original article molecular survey on detection of leishmania infection in rodent reservoirs in jahrom district, southern iran mohammad hassan davami 1, mohammad hossein motazedian 2, *mohsen kalantari 3, qasem asgari 2, iraj mohammadpour 2, abdolreza sotoodeh-jahromi 1, kavous solhjoo 1, morteza pourahmad 4 1department of medical microbiology, school of medicine, jahrom university of medical sciences, jahrom, iran 2department of parasitology and mycology, shiraz university of medical sciences, shiraz, iran 3department of public health, mamasani paramedical school, shiraz university of medical sciences, shiraz, iran 4department of internal medicine, jahrom university of medical sciences, jahrom, iran (received 9 june 2012; accepted 8 oct 2013) abstract background: zoonotic cutaneous leishmaniasis (zcl) is endemic in many parts of iran. recently its incidence is considerable in different parts of jahrom district, in fars province, southern iran. the aims of our study were to investigate the prevalence of leishmania infection, and identify and characterize the leishmania species present, among the rodents by molecular methods in a new endemic focus of zcl, in an urban and rural area of the jahrom district, fars province, southern iran. methods: from may to november 2010), 55 rodents in four regions of jahrom focus were caught and checked for leishmania infection by the microscopical examination of liver, spleen, ears, and footpads’ smears. results: overall 18 meriones persicus, 15 tatera indica, 14 mus musculus, and 8 rattus rattus were caught. totally, four (16.5%) and two (13.3%) of the me. persicus and ta. indica, but only one of mu. musculus and ra. rattus were found smear-positive for leishmania amastigotes, respectively. in the nested-pcr assay 8 (14.6%) smears were found positive for leishmania major, none was found positive for any other leishmania species. sequencing based detection of leishmania confirmed the microscopic and pcr findings. all positive specimens were shown 95–96% similarity with l. major friedlin. conclusion: tatera indica and me. persicus are incriminated as the main ‘reservoir’ hosts of l. major in the rural area of jahrom, moreover, mu. musculus and ra. rattus have the minor but remarkable role in the maintenance of the disease in the urban regions of jahrom focus. keywords: rodent, leishmania, pcr, sequencing, iran introduction the leishmaniases are parasitic diseases with an extensive variety of clinical symptoms that have an effect on over 12 million people in 88 countries, mainly in the tropical and subtropical situated regions, as well as iran (javadian et al. 1976, mohebali et al. 2004, guedes et al. 2008). zoonotic cutaneous leishmaniasis (zcl) takes place in many rural endemic foci in different parts of iran (yaghoobi-ershadi et al. 2005). sandflies (diptera: phlebotominae) have been naturally found infected with leishmania major and considered as the sole vectors of leishmaniasis. moreover, gerbil rodents (muridae: gerbillinae) are the reservoir hosts of zcl in iran (azizi et al. 2011 a). rodents, as the ‘reservoir’ hosts, are the main bases in the ep*corresponding author: dr mohsen kalantari, email: kalantari22@yahoo.com http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 139–146 mh davami et al.: molecular survey on … 140 idemiology and control strategy of the zcl (pourmohammadi et al. 2008). due to high similarity of different species of the parasite, various sources of leishmania dna such as kinetoplast dna (kdna), ssu rrna, its1 and repetitive sequences have been used for molecular characterizations (van eys et al. 1992, noyes et al. 1998, parvizi and ready 2008, davami et al. 2011, ghasemian et al. 2011). recently, pcr-based assays are characteristically used to detect leishmania species in patients, vectors and reservoir hosts (rodgers et al. 1990, bulat et al. 1992, parhizkari et al. 2011). materials and methods study area fars province placed in southern iran and covers an area of about 122,400 km². jahrom district is located in the south-east of province and situated at 30° 4' 45" north, 51° 43' 29" east, and 1050m above the sea level (fars budget and planning organization 2000). due to a favorable ecological conditions for vectors and reservoirs of leishmaniasis, jahrom has always been considered as one of the foci of cutaneous and visceral leishmaniasis in south of iran (fig. 1) (davami et al. 2010, davami et al. 2011). collection and examination of rodents between may to november 2010, rodents were caught alive in wire traps on agricultural plantations, surrounding and near the houses in four regions including jahrom city and three villages in the area including mousavieh, ghotb-abad and fath-abad. each trap was set in the evening and checked in the next early morning. there were set at least 50 ‘trap-nights’ per months. each rodent was identified to species (eisenberg and redford 1999), killed by over-anesthesia and dissected, so that four impressions smears (two of the liver, spleen, ear and footpad) could be prepared. each smear was fixed in methanol, giemsa-stained and checked for amastigotes under a light microscope (mohebali et al. 2004). dna extraction all specimens were checked in a nestedpcr assay for leishmania kdna. therefore, each dry smear was scraped from the slides and mixed with 200μl of lysis buffer [1 mm edta, 50 mm tris-hcl (ph 7.6, 1% (v/v) tween 20] containing 5μl of a proteinase k solution which already had 23mg/ ml of enzyme. the specimen was incubated for 2 h at 56 ºc or 12h at 37 ºc before 75μl of a phenol: chloroform: isoamyl-alcohol solution (25:24: 1, by vol.) was added (motazedian et al. 2002). after being shaken vigorously, specimen was centrifuged at 6000× g for 10min. dna in the supernatant solution was precipitated with 300μl of cold ethanol, resuspended in 100μl of double distilled water, and stored at 4 ºc before using in the pcrassay (noyes et al. 1998). nested-pcr assay the specific and sensitive nested-pcr was used to amplify the variable area of the minicircle kdna of leishmania spp. in the rodent liver, spleen, ear and footpad as previously described by noyes et al. (1998) with slight modification (moemenbellah-fard et al. 2003, davami et al. 2011, ghasemian et al. 2011). the first-round (external) primers were csb1xr (atttttcgcgattttcgcaga acg) and csb2xf (cgagtagcagaaa ctcccgttca). a reaction mixture containing of 1.5 mm of mgcl2, 2 mm of dntp, 2.5μl of 10 x pcr buffer (boehringer mannheim, mannheim, germany), 1 unit of taq dna polymerase (cinagene, tehran), and 10pmol of each primer were used in a total reaction volume of 25μl including 5μl of dna sample. the second-round (internal) primers were 13z (actgggggttggtgtaaaa tag) and lir (tcgcagaacgcccct). the reaction mixture was used in a total volume of 30μl including 2μl of dna product of the http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 139–146 mh davami et al.: molecular survey on … 141 first round. these mixtures were amplified in a programmable thermocycler (eppendorf ag (mastercycler gradient), germany) for 5min at 94 ºc (1 cycle) followed by 30 cycles at 94 ºc for 30 seconds, 55 ºc for 60 seconds and 72 ºc for 1.5min followed by a final elongation step at 72 ºc for 5 minutes. the who reference strains of l. major (mhom/il/67/ lv561) was used as standard dna. a band of 560bp indicated that l. major kdna is present in the sample (davami et al. 2011). electrophoresis a 6μl sample of each second-round product of pcr was subjected to electrophoresis in 1.5% agarose gel, stained with ethidium bromide and visualized by ultraviolet transillumination (davami et al. 2011). sequencing the pcr products of all positive samples were purified by gel purification kit (accu prep®, cat. no. k-3035-1, bioneer, usa). the strands of amplified dna were sequenced (both forward and reverse sequencing) with the pcr primers on an automated sequencer (applied biosystems 377xl). the nucleotide homologies of the sequenced products were investigated with the tritrypdb blast programme. the characterization of consensus sequences was performed by using fasta formatted sequences aligned with the chromas programme. results the 55 rodents trapped over the study belonged to four species; totally, 18 (32.7%) me. persicus, 15 (27.3%) ta. indica, 14 (25.5%) mu. musculus, and 8 (14.5%) ra. rattus were caught from jahrom district (table 1). amastigotes were seen in the liver, spleen, ear and/or footpad smears of seven (12.7%) of the rodentsthree (16.7%) of the me. persicus, two (13.3%) of the t. indica, one (7.1%) of the mu. musculus, and one (12.5%) of the ra. rattuscaught in jahrom district (table 2). the nested-pcr results showed that 8 (14.6%) of rodentsfour (22.2%) of the me. persicus, two (13.3%) of the ta. indica, one (7.1%) of the mu. musculus, and one (12.5%) of the ra. rattuswere positive for leishmania kdna, also 10 out of the 220 smears belonged to different parts of rodents organs (liver, spleen, ear and/or footpad) were found positive, separately (table 2,3). all the kdna detected appeared to come from l. major (fig. 2). using tritrypdb sequence analysis against trypanosomatidae species, the target sequence of pcr products showed 95–96% similarity with l. major strain iranjwmaj (genbank accession no. ab67 8349.1) (fig. 3). fig. 1. map of iran, showing the locations of fars province and the city of jahrom fig. 2. the results of the nested pcr-based amplification of kdna recovered either from a negative control (lane 2), and the reference samples of leishmania major (lane 3), or positive and negative smears of liver, spleen, ear, or footpad of mus musculus (lane 4, 5 and 6), rattus rattus (lanes 7, 8), meriones persicus (lanes 9, 10 and 11), and tatera indica (lanes 12 and 13). molecular-weight markers were run in lanes 1 http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 139–146 mh davami et al.: molecular survey on … 142 table 1. the species, numbers and geographical distributions of the rodents caught in jahrom district no. and (%) of rodents caught study areas meriones persicus tatera indica mus musculus rattus rattus all species *jahrom 14 (77.8) 13 (86.7) 6 (42.9) 8 (100) 41 (74.5) fath-abad 3 (16.7) 0 (0) 3 (21.4) 0 (0) 6 (10.9) mousavieh 0 (0) 2 (13.3) 4 (28.6) 0 (0) 6 (10.9) ghotb-abad 1 (5.5) 0 (0) 1 (7.1) 0 (0) 2 (3.6) all 18 (100) 15 (100) 14 (100) 8 (100) 55 (100) * urban and suburban areas of jahrom table 2. the prevalences of leishmania infection in the rodents, as revealed by microscopy and, nested-pcr based detection of leishmania major kdna in jahrom district no. and (%) of rodents found positive meriones persicus tatera indica mus musculus rattus rattus all species study areas *ch. ** mic. pcr ch. mic. pcr ch. mic. pcr ch. mic. pcr ch. mic. pcr ***jahrom 14 1(7.1) 2(14.3) 13 0(0) 0(0) 6 0 (0) 0 (0) 8 1(12.5) 1(12.5) 41 2(4.9) 3 (7.3) fath-abad 3 2(2.67) 2(66.7) 0 0 (0) 0(0) 3 0 (0) 0 (0) 0 0 (0) 0 (0) 6 2(33.3) 2 (33.3) mousavieh 0 0(0) 0(0) 2 2(100) 2(100) 4 1 (25) 1(25) 0 0 (0) 0 (0) 6 3 (50) 3 (50) ghotb-abad 1 0(0) 0(0) 0 0 (0) 0 (0) 1 0 (0) 0 (0) 0 0 (0) 0 (0) 2 0 (0) 0 (0) all 18 3(16.7) 4 (22.2) 15 2(13.3) 2(13.3) 14 1(7.1) 1(7.1) 8 1(12.5) 1(12.5) 55 7 (12.7) 8 (14.6) *checked, **microscopy, ***urban and suburban areas of jahrom district table 3. the prevalence of leishmania infection in the studied organs of rodents, as revealed by the nested-pcr in jahrom district studied organs of rodents (no. and %) footpad ear liver spleen all species *ch. ** pos. (%) ch. pos. (%) ch. pos. (%) ch. pos. (%) ch. pos. (%) meriones persicus 18 2 (11.1) 18 1 (5.6) 18 2 (11.1) 18 0 (0) 72 5 (6.9) tatera indica 15 2 (13.3) 15 1 (6.7) 15 0 (0) 15 0 (0) 60 3 (5) mus musculus 14 1 (7.1) 14 0 (0) 14 0 (0) 14 0 (0) 56 1 (1.8) rattus rattus 8 0 (0) 8 0 (0) 8 1 (12.5) 8 0 (0) 32 1 (3.1) all 55 5 (9.1) 55 2 (3.6) 55 3 (5.5) 55 0 (0) 220 10 (4.6) *checked, **positive fig. 3. alignment analysis of the kdna of leishmania major isolated from mus musculus foot-pad, l. major strain iran j wmaj (genbank accession no. ab678349.1) and l. major isolate mhom/il/67/lv561 (accession no. af308685.1). target sequence of pcr product of mu. musculus showed 95% similarity with l. major strains, of iranjwmaj. stars indicate the different regions between the isolates http://jad.tums.ac.ir published online: april 09, 2014 iran j wmaj (accession no. ab678349.1) l. major isolated from mus musculus mhom/ il/ 67/ lv561 (accession no. af308685.1) j arthropod-borne dis, december 2014, 8(2): 139–146 mh davami et al.: molecular survey on … 143 discussion detection of the mammalian ‘reservoir’ hosts is one of the main problems in front of researchers who make an effort to evaluate the epidemiology of zcl to control the disease (pourmohammadi et al. 2008). in the present study using a specific and sensitive nestedpcr on scrapings from giemsa-stained smears of rodent tissues, the overall prevalence of rodents with leishmania infection was outcome as 14.6% that showed the higher infection than microscopical observations (12.7%). different biochemical, immunological, and molecular assays such as iso-enzyme electrophoresis, monoclonal antibodies, and pcr have been used to characterize the causative agents of leishmaniasis (fakhar et al. 2008, parvizi et al. 2008, fakhar et al. 2010, pourmohammadi et al. 2010, azizi et al. 2011b). usuallym, in the leshmanial detections molecular assays are more sensitive than the other diagnostic methods such as micriscopical examinations or culture (fakhar et al. 2011). as it is doable to use the molecular techniques to detect and identify the leishmania parasites in fixed and stained smears utilized for microscopy, recently, those methods chiefly based on nested-pcr and sequencing have been remarkably raised to detect the leishmania infections (noyes et al. 1998). in iran, rhombomys opimus, ta. indica, me. lybicus, and me. hurrianae have been reported as the major ‘reservoir’ hosts of zcl in endemic foci of the central and north-east, west and south-west, south, and south-eastern parts of iran, respectively (yaghoobi-ershadi et al. 1996, moemenbellah-fard et al. 2003, parvizi et al. 2005, asgari et al. 2007). meriones lybicus, me. persicus, me. hurrianae and ta. indica have been found positive for leishmania kdna in fars province (rassi et al. 2001, moemenbellah-fard et al. 2003, rassi et al. 2006, mehrabani et al. 2007, mehrabani et al. 2011, parhizkari et al. 2011, azizi et al. 2012). in our study, the results of rodents trapped in jahrom district of fars province, indicated that ta. indica and me. persicus were the most common rodents in that part of the province, and the results of the nested-pcr showed that 22.2% and 13.3% of mentioned rodents were found to be positive for l. major. me. persicus and ta. indica are the most important mammalian hosts of l. major in the rural areas of jahrom district. no r. opimus and me. hurrianae were caught in the present study in jahrom. nesokia indica, gerbillus nanus have been described as an accidental, main or a probable ‘reservoir’ hosts in different parts of iran (pourmohammadi et al. 2008, azizi et al. 2011a, azizi et al. 2011c, azizi et al. 2012). in our investigation any nesokia or gerbilus species were not caught. mu. musculus has been found naturally infected with l. major in fars province (parhizkari et al. 2011). in the present study, the footpad of one out of 14 (7.1%) mu. musculus were found to be both microscpical and molecular positive. recently motazedian et al. has isolated the causative agents of zcl (l. major) from ra. norvegicus in fars province (motazedian et al. 2010). in this investigation one liver of ra. rattus caught in urban area of jahrom district was found to be infected with leishmania kdna, the present report appears to be the first to describe natural infection of ra. rattus with l. major in iran. mu. musculus and ra. rattus infection with l. major may explain why, in recent years, zcl causative has been remarkably increased in some urban areas of iran (razmjou et al. 2009, davami et al. 2010). the importance of the ‘reservoir’ hosts in the epidemiology of zcl appreciated with the local sandflies feeding on that species and the infectivity of the leishmania infections http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 139–146 mh davami et al.: molecular survey on … 144 in each host species (motazedian et al. 2006). even though mu. musculus and ra. rattus were found infected with l. major, but some reports show that the prevalence of leishmania infection have been observed from those which caught from other regions of iran (parhizkari et al. 2011). there was no evidence of l. infantum or l. tropica in the tested smears. this study includes the first isolation and characterisation of l. major from iranian me. persicus and ta. indica, caught in an area where zoonotic cl has recently occurred. in jahrom, whatever the time of the year, more than half of all rodents attracted to walnut-baited traps are infected with l. major. if local sandflies take their bloodmeals from both rodents and humans, there is clearly much scope for transmission of these parasites to humans. conclusion based on observing of amastigotes in livers, spleens, ears, and footpads smears, using a high sensitive and specific nested-pcr designed for kdna of leishmania, and comparing the kdna of sequenced products with genbank that confirmed the highest homology of 95– 96% with l. major, concluded that the species isolated form rodents is l. major. acknowledgements the authors are grateful to the office of the vice-chancellor for research at jahrom and shiraz university of medical sciences for financial support, and the department of parasitology and mycology in the medical school of shiraz university of medical sciences for facilitating the pcr assays. they also thank to dr r zarei for her assistance in the identification of the rodents caught, and to dr b sarkari, for his critical review of the manuscript. the authors declare that there is no conflict of interests. reference asgari q, motazedian mh, mehrabani d, oryan a, hatam gr, owji sm, paykari h (2007) zoonotic cutaneous leishmaniasis in shiraz iran: a molecular, isoenzyme and morphologic approach. j res med sci. 12: 7–15. azizi k, davari b, kalantari m, fekri s (2011a) gerbillid rodents fauna (muridae: gerbillinae) and detection of reservoir hosts(s) of zoonotic cutaneous leishmaniasis using a nested-pcr technique in jask city in hormozgan 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and its use for detection and identification of leishmania parasites. mol biochem parasitol. 51: 133–142. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996) meriones libycus and rhombomys opimus (rodentia: gerbillidae) are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans r soc trop med hyg. 90: 503–504. yaghoobi-ershadi mr, akhavan aa, zahraeiramazani ar, jalali-zand ar, piazak n (2005) bionomics of phlebotomus papatasi (diptera: psychodidae) in an endemic focus of zoonotic cutaneous leishmaniasis in central iran. j vector ecol. 30: 115–118. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 219–223 m ganjali et al.: species diversity and … 219 short communication species diversity and distribution of ticks (acari: ixodidae) in zabol county, eastern iran *maryam ganjali 1, mansour dabirzadeh 2, masoud sargolzaie 3 1department of parasitology, faculty of veterinary medicine, university of zabol, zabol, iran 2department of microbiology, school of public health, zabol university of medical sciences, zabol, iran 3veterinary office of zabol, zabol, iran (received 1 jan 2013; accepted 4 feb 2014) abstract background: ticks are important vectors of human and animal pathogens. they are considered as main vectors for transmission of many viral, bacterial, rickettsial and parasitical pathogens. the aim of the present study was to find out species diversity of ticks, which infested the domestic ruminants in zabol county, eastern iran in 2012. methods: ticks were selected randomly from sheep, goats, cattle and camels. the ticks were collected from the body of infested animals and stored in 70% ethanol, then transported to the laboratory of zabol university of medical sciences. following examinations under stereomicroscope, ticks were identified using available taxonomic keys. results: in this study, a total number of 469 adult ticks (381 males and 88 females) were collected. ticks were classified into 2 genera and 9 species including: hyalomma dromedarii (17.3%), hy. schulzei (1.8%), hy. marginatum (0.5%), hy. anatolicum excavatum (12.60%), hy. anatolicum anatolicum (11.2%), hy. asiaticum asiaticum (11.0%), rhipicephalus sanguineus (21.2%), rh. bursa (10.2%) and rh. turacunis (13.911%). the frequency of genus hyalomma (54.6%) was higher than rhipicephalus. rh. sanguineus was the predominant tick species and accounted for 21.26% of the ticks. the ratio of males was more than the female ticks. conclusion: hyalomma and rhipicephalus species are commonly distributed in the study area. further investigations are needed to identify the role of above tick species as vectors of pathogenic organisms. keywords: epidemiology, ticks, hyalomma, rhipicephalus, iran introduction ticks are blood-sucking ectoparasites which transmit serious diseases to animals and humans. they are considered as main vectors for transmission of many viral, bacterial, rickettsial and parasitical pathogens (garcia 2007). several tick species are important in veterinary medicine as vectors of theileriosis, babesiosis and anaplasmosis. in addition, lyme disease, ehrlishiosis, babesiosis, rocky mountain fever, colorado tick fever, tularemia, q fever, spotted fever, tick paralysis and tick encephalitis are the most common diseases which have been transmitted to human by ticks. they transmit diseases, produce paralysis, weight loss and cause economic damages to livestock (wall 2001). in view of the disease relationships of ticks, rhipicephalus species are important vectors of ovine babesiosis and ovine ehrlichiosis. hyalomma anatolicum transmits theileria lestoquardi, th. annulata and crimean-congo hemorrhagic fever virus (jongejan and uilenberg 1994, chinikar et al. 2009). tick species are widely distributed around the world. the first record of hyalomma in iran was described by delpy (1937). hyalomma anatolicum anatolicum, hy. anatolicum excavatum, hy. asiaticum asiaticum, hy. detrium, hy. aegeptium, and hy. dromedarii are commonly distributed in iran (rahbari et al. 2007). *corresponding author: dr maryam ganjali, email: m_g2507@yahoo.com http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 219–223 m ganjali et al.: species diversity and … 220 nasiri et al. (2010) reported the presence of hy. marginatum (44.67%), hy. anatolicum (43.17%), hy. asiaticum (6.37%), hy. dromedarii (5.55%) and heamaphysalis sulcata (0.24%) species in ilam. salimabadi et al. (2010) carried out a study in yazd province, and reported 7 species of ticks including: hy. dromedarii, hy. marginatum, hy. anatolicum, hy. detritum, hy. asiaticum, rh. sanguineus and dermacentor marginatus. numerous studies on the distribution of tick fauna in iran have been conducted (delpy 1936, abbasian 1961, mazlum 1971, razmi et al. 2007, rahbari 2007, nabian et al. 2007, telmadarraiy et al. 2009). rehman et al. (2004) reported different species of ixodidae (hard ticks) in rawalpindi and islamabad, pakistan. five species of ticks, he. sulcata 74%, hy. anatolicum 14%, hy. anatolicum anatolicum 12%, hy. marginatum 0.1% and he. erinacei 0.1% have already been found. the prevalence of he. sulcata and hy. anatolicum was highly significant in this region. this article was conducted to collect and identify the tick species prevalent in domestic animals in zabol county. materials and methods sistan and baluchestan is located in the southeast of iran and zabol is in the north of this province with hot and dry desert climate. zabol lies on the border along with afghanistan. the latitude and longitude gps coordinates of zabol (iran) is: lat: 31.0385, long: 61.4962. this survey was carried out to investigate the prevalence of hard tick species (acari: ixodidae) on cattle in zabol county during 2012. sampling was done during the activating seasons of ticks (i.e. summer and spring). veterinary staff provided us with the latest information about infested flocks. the survey was conducted in some randomly selected villages. the number of ticks was determined according to the cochran formula: ) where, n= sample size, z= 1.96, p= 0.56 prevalence estimated, q= 1–p, d= 0.45. a total number of 469 hard ticks were collected from sheep, goats, cattle and camels. the ticks were collected from the body of infested animals and stored in 70% ethanol, then transported to the laboratory of zabol university of medical sciences. following examinations under stereomicroscope, ticks were identified using available taxonomic keys (kaiser et al. 1963, walker et al. 2007). results a total number of 469 adult ticks (381 males and 88 females) were collected from, sheep, goats, cattle, and camels. ticks were classified into 2 genera and 9 species. table 1 shows the frequency of different species of male ticks. rhipicephalus sanguineus was the predominant tick species and accounted for 21.26% of the ticks. the frequency of genus hyalomma (54.6%) was higher than rhipicephalus. the ratio of males was more than the female ticks. table 2 shows the frequency of different tick species on various hosts. the maximum number of ticks was collected from sheep and goats 253(66.4%), followed by camels 73(19.16%) and cattle 55 (14.43%). rhipicephalus species were observed in sheep and goats. moreover, hy. dromedari, hy. schulzei and hy. marginatum were commonly found on camels and rarely in cattle. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 219–223 m ganjali et al.: species diversity and … 221 table 1. subspecies and numbers of male ticks collected in zabol, during 2012 (percentage of male ticks was calculated) percentagenumberspecies 17.3266hy. dromedarii 1.837hy. schulzei 0.5252hy. marginatum 12.6048hy. anatolicum excavatum 11.2843hy. anatolicum anatolicum 11.0242hy. asiaticum asiaticum 21.2681rh. sanguineus 10.2339rh. bursa 13.9153rh. turacunis table 2. the host of collected ticks in zabol, during 2012 species host cow camel goat sheep hy. dromedarii 4 62 hy. schulzei 7 hy. marginatum 2 hy. anatolicum excavatum 17 2 23 6 hy. anatolicum anatolicum 26 13 4 hy. asiaticum asiaticum 8 27 7 rh. sanguineus 42 39 rh. bursa 19 20 rh. turacunis 23 30 total 55 73 147 106 discussion this article reports a study conducted to collect and identify the tick species prevalent in domestic animals in zabol, during year 2012. a total of 9 tick species was identified (table 1). hyalomma and rhipicephalus had the most prevalence in this area. the frequency of genus hyalomma (54.6%) was higher than that of rhipicephalus. rhipicephalus sanguineus was the predominant tick species and explained 21.26% of the ticks. hyalomma dromedarii was the second most common species. a similar study was also reported from meshkinshahr (a northwest area in iran) in 2009 by telmadarraiy, in which most of the ticks belonged to genus rhipicephalus and hyalomma (telmadarraiy et al. 2009). rhipicephalus sanguineus ticks are widely distributed around the world and one of the most common species in sheep herds in northeast of iran (razmi et al. 2007). in view of the importance of these species, hyalomma ticks are widespread in north africa, southern europe, middle east, central asia and china (durrani et al. 2009), including the species that are vectors of various diseases in humans and domestic animals. hyalomma anatolicum anatolicum is known as vectors of some pathogens as, th. lestoquardi, th. annulata and crimeancongo hemorrhagic fever virus (jongejan and uilenberg 1994). different species of ticks play an important role for the transmission of crimean-congo hemorrhagic fever (cchf) across the country. according to the http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 219–223 m ganjali et al.: species diversity and … 222 findings, geographical distribution of cchf cases corresponds most closely with the distribution of hyalomma ticks, although, some species of dermacentor and rhipicephalus genera have the ability of transmission (chinikar et al. 2009). in this study, a total number of 469 adult ticks (381 males and 88 females) were collected. the ratio of males was more than that of female ticks. the report of tick fauna in iran provided by rahbari et al. (2007) indicated that the number of ticks on each animal was low and male ticks were more than the females. nasiri et al. (2010) collected ticks from sheep in abdanan township and showed that the frequency of male and female ticks was (77%) and (23%), respectively. several studies on the distribution of tick fauna have been reported. salimabadi et al. (2010) reported 7 species of hy. dromedari, hy. marginatum, hy. anatolicum, hy. detritum, hy. asiaticum, rh. sanguineus and de. marginatus in yazd province. a study in ilam province exhibited 5 species of hy. marginatum, hy. anatolicum, hy. asiaticum, hy. dromedari and he. sulcata (nasiri et al. 2010). rehman et al. (2004) reported different species of ixodidae (hard ticks) in rawalpindi and islamabad of pakistan. the prevalence of he. sulcata and hy. anatolicum was highly significant in this region. in the above mentioned studies, the researchers reported the genus of dermacentor and heamaphysalis which were not found in our article. as was found in this article, camels are the most suitable host for hy. dromedarii, hy. schulzei and hy. marginatum. kaiser and hoogstraal (1963) emphasized that hy. schulzei is the common species in the saravan area of sistan and baluchestan, iran. abbassian-lintzen (1960–1961) and mazlum (1971) found that these ticks usually occur on camels. in ethiopia, camels were infested with hy. dromedarii (zeleke and bekele 2004). nazifi et al. (2011) reported hard ticks infestation of one-humped camels in qeshm island. hyalomma dromedarii was the predominant tick species. other hard ticks were hy. anatolicum excavatum, hy. asiaticum asiaticum, hy. marginatum, hy. impeltatum and rh. bursa. moreover, we found hy. asiaticum asiaticum which infested cattle, sheep and goats. abbasian-lintzen (1961) described it as the single species in south-eastern iran near the pakistan borders, but nabian et al. (2009) reported the presence of hy. asiaticum asiaticum ticks in all four zoogeographical zones of iran. our results showed that rhipicephalus species were in sheep and goats and that rh. sanguineus was the predominant tick species. in the present study most of samples were collected from sheep and goats because they are the dominant livestock of the surveyed area. conclusion as the incidence of the tick-borne disease increases and the geographic areas in which they are found is expanding, it becomes increasingly important to distinguish tick species, which is essential to promote tick and tick-borne disease control. the results obtained from the present study serve as the starting point for future epidemiological studies and further investigations are needed to detect the vector role of ticks in this area. acknowledgements great appreciation goes to dr sanchuli the manager of the veterinary office of zabol for his kind assistance and providing some of the ticks used in this study. this study was supported by zabol university of medical sciences. the authors declare that there is no conflict of interest. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 219–223 m ganjali et al.: species diversity and … 223 references abbasian-lintzen r (1961) records of tick (acarina: ixodidae) occurring in iran and their distributional data. acarologia. 3: 546–559. chinikar s, ghiasi sm, ghalyanchilangeroudi a, goya mm, shirzadi mr, zeinali m, haeri a (2009) an overview of crimean-congo hemorrhagic fever in iran. iran j microbiol. 1(1): 7–12. delpy l (1936) note sur les ixodides du genre hyalomma (koch). ann de parasi. 14(3): 206–45. durrani az, shakoori ar (2009) study on ecological growth conditions of cattle hyalomma ticks in punjab, pakistan. iran j parasitol. 4(1): 19–25 garcia l. sh, ms, mt, cls, f (aam) (2007) diagnostic medical parasitology. 5th ed. american society for microbiology. jongejan f, uilenberg g (1994) ticks and control methods. rev sci tech off int epiz.13(4): 1201–1226. kaiser mn, hoogstraal h (1963) the hyalomma ticks (ixodoidae) of afghanistan. j parasitol. 49: 130–139. mazlum z (1971) ticks of domestic animals in iran: geographic, host relation, and seasonal activity. j vet fac univ tehran, iran. 27(1): 1–32. nabian s, rahbari s, shayan p, haddadzadeh hr (2007) current status of tick fauna in north of iran. iran j parasitol. 2(1): 12–17. nabian s, rahbari s, changizi a, shayan p (2009) the distribution of hyalomma spp. ticks from domestic ruminants in iran. j med entomol. 23(3): 281–283. nasiri a, telmadarraiy z, vatandoost h, chinikar s, moradi m, oshaghi m, salimabadi y, sheikh z (2010) tick infestation rate of sheep and their distribution in abdanan county, ilam province, iran, 2007–2008. iran j arthropod-borne dis. 4(2): 56–60. nazifi1 s, tamadon a, behzadi ma, haddadi sh, raayat-jahromi ar (2011) onehumped camels (camelus dromedaries) hard ticks infestation in qeshm island, iran. vet res forum. 2(2): 135–138. rahbari s, nabian s, shayan p (2007) primary report on distribution of tick fauna in iran. parasitol res. 101(2): 175– 177. rehman w, ahmad khan i, qureshi ah, shujaat h (2004) prevalence of different species of ixodidae (hard ticks) in rawalpindi and islamabad pakistan. j med res. 43(2). razmi gr, glinsharifodini m, sarvish (2007) prevalence of ixodid ticks on cattle in mazandaran province, iran. korean j parasitol. 45(4): 307–310. salimabadi y, telmadarraiy z, vatandoost h, chinikar s, oshaghi m, moradi m, mirabzadehardakan e, hekmat s, nasiri a (2010) ticks on domestic ruminants and their seasonal population dynamics in yazd province, iran. iran j arthropod-borne dis. 4(1): 66–71. telmadarraiy z, vatandoost h, rafinejad j, mohebali m, tavakoli m, abdigoudarzi m, faghihi f, aboulhasani m, zarei z, jedari m, mohtarami f, azamsoulki a, salari lak sh, entezarmahdi r (2009) distribution of ticks (ixodidae and argasidae) family and susceptibility level to cypermethrin in meshkinshahr district, ardabil province, iran. ardabil uni med sci j. 9(2): 127–133. wall r, shearer d (2001) veterinary ectoparasite: biology, pathology and control. 2nd ed. blackwell science ltd. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 21 original article laboratory evaluation of toxicity of insecticide formulations from different classes against american cockroach (dictyoptera: blattidae) *ruhma syed, farkhanda manzoor, rooma adalat, abida abdul-sattar, azka syed department of zoology, lahore college for women university, lahore, pakistan (received 13 mar 2011; accepted 3 sep 2013) abstract background: the present study was designed to investigate the insecticidal efficacy of four different classes of insecticides: pyrethroids, organophosphates, phenyl-pyrazoles and neo-nicotenoids. one representative chemical from each class was selected to compare the toxicity: deltamethrin from pyrethroids, dichlorovinyl dimethyl phosphate (ddvp) from organophosphates, fipronil from phenyl-pyrazoles and imidacloprid from neo-nicotenoids. the objective of this study was to determine which of these insecticides were most effective against american cockroach. methods: these insecticides were tested for their lc50 values against periplaneta americana under topical bioassay method, using different concentrations for each chemical. results: fipronil 2.5% ec was highly effective at all concentrations applied, while ddvp 50% ec was least toxic amongst all. one way analysis of variance confirmed significant differences between mortality of p. americana and different concentrations applied (p< 0.05). conclusion: locality differentiation is an important factor in determining the range of resistance between various localities, as all three localities behaved differently in terms of their levels of resistance. keywords: insecticides, periplaneta americana, topical bioassay, resistance, efficacy introduction periplaneta americana (linnaeus) is an obnoxious and grubby domestic pest of tropical countries of the world (lee and robinson 2001). their aesthetically unappealing damage to household materials and stored products, and transmission of diseases makes them a high priority pest. it is considered as a commonly found pest of bakeries, restaurants and kitchens (gadd and raubenheimer 2000, lee and lee 2000). they are usually found in basements, steam tunnels, grocery stores and boiler rooms. they contaminate food in contact by carrying filth and microbial pathogens on their body and legs (watanabe et al. 2003, fasulo et al. 2005). as they have unsanitary mode of life, cockroaches are potent vectors of infectious pathogens (vythilingam et al. 1997). several species of enterobacteriaceae including klebsiella spp., salmonella spp. and escherichia spp. can be harbored in cuticle of p. americana (mpuchane et al. 2006). according to the findings of barcay (2004), e. coli, compylobacter spp., streptococcus spp., salmonella spp., toxoplasma gondii, staphylococcus spp. and shigella spp. are some of the medically important species of pathogens for which p. americana serves as an important vector. moreover, they may cause allergic reactions to some people by carrying the eggs of parasitic worms. some of these major allergic responses include asthma, itching, swelling of eyelids, dermatitis and severe respiratory problems (sarinho et al. 2004, gore and schal 2007, pechal et al. 2007). several diseases such as cholera, pneumonia, anthrax, tetanus, diphtheria, tuberculosis, enteric fever, gastroenteritis and food poisoning may spread *corresponding author: dr ruhma syed, e-mail: ruhmasyed@gmail.com http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 22 by p. americana (lane et al. 2001, morane 2002). kinfu and erko (2008) have reported in their studies that cockroaches are potential carriers of human intestinal parasites especially ascaris lumbricoides, entamoeba coli and e. histolytica. besides mechanically transmitting a variety of diseases, it is also responsible for causing insectophobia in some people (czajka et al. 2003). the control of p. americana can be done by applying insecticides to the hiding and resting places in the form of insecticidal dusts and residual sprays. chemical control has been the most popular and effective method so far lee and robinson (2001), but their control with insecticides is not a suitable approach because of several reasons; the most important of which is that they may develop resistance against certain frequently used insecticides (tsai and lee 2001). resistance against pesticides is a phenomenon, mainly dependant on genetic basis. exposure of a population of pest to a certain pesticide results in development of resistance against that chemical (pesticide). not necessarily all insects need to be killed during this phenomenon. hemingway et al. (1993) have reported that cockroaches have developed resistance against many groups of insecticides especially pyrethroids. much of the work regarding insecticidal efficacy has been done on german cockroach, however, very little data is available with respect to american cockroach. therefore, keeping in view the work carried out by various researchers, the present work was designed to investigate the insecticidal efficacy of four groups of insecticides (organophosphates, pyrethroids, phenyl-pyrazoles and neo-nicotenoids) on p. americana, and the susceptibility of adults to these insecticides through laboratory bioassay using topical application method. considering all the facts which demonstrate that these insecticides work as strong agents for the chemical control of p. americana, the aims of this study were to: investigate the current status of insecticide resistance in different field populations of p. americana, that is useful to find out their extent of resistance against commonly used insecticides. evaluate the toxic efficacy of four insecticides and to compare the older groups of insecticides i.e. pyrethroids and organophosphates with that of relatively new compounds i.e. neo-nicotenoids and phenyl-pyrazoles. this information will be helpful for the end users to choose better options for the management of these insects in pakistan. materials and methods experimental site the laboratory bioassays were conducted at the entomology research laboratory, lcwu, lahore, pakistan. experimental insect keeping in view their unappealing damages to house-hold materials and medical importance among all urban pests, p. americana was selected as experimental insect for testing against commonly used insecticides. these were collected from kitchens, basements, bakeries, and sewerage manholes by hand catch and trapping methods. based on the previous history of insecticidal spray frequency, three different localities were selected for the collection of cockroaches (table 1). these far apart areas were chosen to determine the degree of variability of insecticide resistance in different field populations of p. americana. collection of p. americana (l) the collection traps for cockroaches comprised of simple glass or plastic jars with butter, sugar and bread inside. the trapping jars were comprised of two parts: the upper http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 23 inside of it was coated with petroleum jelly (vaseline®) in order to avoid their escape and keep them entrapped till bioassay. the sliding lower part was used to separate them from trapping jars and cockroaches were transferred to the main collection containers placed in laboratory. considering the nocturnal habitat of cockroaches and to provide their preferred hidden places of living, a dark paper was used to cover these containers externally. they were provided with wet bread, card-board and water till the onset of bioassay. the collection containers were held at specified laboratory conditions (temperature= 28± 2 ºc and relative humidity= 75± 3%). following the methodologies described by appel et al. (1983) and abd-elghafar et al. (1990), only adult males were selected for bioassays because of their uniform weights, as compared to that of females. chemicals following technical grade insecticides and chemicals were used during bioassay: imidacloprid 5% sc, fipronil 2.5% ec, deltamethrin 2.5% sc, ddvp 50% ec (ali akbar group of chemicals, lahore), acetone (e-merck, d-6100, germany), distilled water. the choice of these insecticides was based on the fact that these chemicals have not been tested against p. americana in pakistan so far. moreover, relatively new insecticides like imidacloprid and fipronil were also needed to be investigated for their toxicity, in order to determine whether they are effective against field populations of p. americana. therefore, comparison between four groups of insecticides (in terms of lc50 values) was studied by selecting one representative from each insecticidal class. all chemical were obtained from ali akber group of chemical industries, 1-km bhoptian chowk defence road, and off raiwind road, lahore, pakistan. preparation of stock solution for toxicity tests, stock solutions for four insecticides imidacloprid 5% sc, fipronil 2.5% ec, deltamethrin 2.5% sc and ddvp 50% ec were 5.0, 2.0, 20.0 and 50.0µ l/ml respectively. known quantity from each insecticide was pipetted out and dissolved in acetone (solvent for dissolving these insecticides). following formula was used for preparing concentrations. [concentration required in µ l/ ml] x [volume required in ml] [% concentration in µ l/ ml] x 10 = ---------µ l/ml for preparing 5.0 µ l/ ml stock solution of imidacloprid 5% sc, 2.0 µ l of it was pipetted out and dissolved in acetone to make final volume up to 20.0 ml. 2.0 µ l/ ml stock solution of fipronil 2.5% ec was prepared by dissolving 1.6 µ l of it in acetone to make final volume up to 20.0 ml. for preparing 20.0 µ l/ ml stock solution of deltamethrin 2.5% sc, 16.0 µ l of it was pipetted out and dissolved in acetone to make final volume up to 20 ml. for preparing 50.0 µ l/ ml stock solution of ddvp 50% ec, 2.0 µ l of it was pipetted out and dissolved in acetone up to final volume of 20.0 ml. serial dilutions using these stock solutions, further serial dilutions were prepared to obtain five concentrations for each insecticide. serial dilutions for imidacloprid 5% sc were 1.0, 2.0, 3.0, 4.0 and 5.0µl/ml. serial dilutions for fipronil 2.5% ec were 0.05, 0.25, 0.5, 1.0 and 2.0µ l/ ml. serial dilutions for deltamethrin 2.5% sc were 1.0, 1.5, 10.0, 15.0 and 20.0µ l/ml. serial dilutions for ddvp 50% ec were 5.0, 10.0, 30.0, 40.0 and 50.0µ l/ ml. topical application method topical bioassay was used to treat the adult male p. americana according to the method described by lee et al. (1996). four http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 24 preliminary tests were performed to find out the discriminating doses. twenty insects were separated from collection containers and for easy handling during bioassay, they were exposed to 4 ºc for 1–2 minutes to keep them immobilized for few minutes. using an insulin syringe serving as micro applicator, 1.0µ l of pre-determined amount of each insecticide concentration was topically applied to the first segment of abdominal sternites. five to seven doses of each insecticide having mortality range between 1 and 100% were determined. no insecticide was applied to control group, they only received 1.0µ l acetone. clean autoclaved petri plates were used to maintain the treated cockroaches and they were provided with bread and moistened cotton plug as food source. each plate was having ten treated cockroaches which were provided with same temperature and humidity conditions as that of colony. freshly prepared concentrations were applied for the treatment, starting from low to high dilutions and three replicates were prepared for each concentration. pair of forceps was used to properly orient the immobilized cockroaches. mortality criterion was as follows: if a cockroach was unable to return back to its normal position within 2–3 minutes after being touched with forceps, it was considered dead and counted in mortality data. data evaluation and statistical analysis the data obtained from this study was subjected to mean, standard error, regression analysis and one way analysis of variance. the lc50 values for each insecticide were obtained by using epa probit analysis program used for calculating lc/ec values (version 1.5). regression analysis of variance was applied to express the trend of variation within each treatment by using microsoft office excel 2007. results with p< 0.05 were considered statistically significant for all comparisons. results table 2 represents the comparative toxicities of four insecticides against three localities of p. amercana (l). when different concentrations (1.0, 2.0, 3.0, 4.0 and 5.0µ l/ ml) of imidacloprid 5% sc were tested against p. americana, variable range was obtained according to its respective localities. lc50 for smh was 2.712 µ l/ ml, whereas comparatively low values were recorded for ktch and klpt (1.966 µ l/ ml and 1.318 µ l/ ml respectively). the regression analysis of variance further confirmed that there was an increasing trend in mean mortality of insects after 48 hours of treatment [for smh: b= 3.1± 0.9, f(1,4)= 106.78, p= 0.001, for ktch: b= 3± 2.63, f(1,4)= 3.42e+ 32, p= 3.48e49 and for klpt: b= 2.7± 0.1, f(1,4)= 729, p= 0.001) (fig. 1–3(a)]. the concentrations used for fipronil 2.5% ec were 0.05, 0.25, 0.5, 1.0 and 2.0µl/ ml for all three localities tested. it is evident from results, that there is marked difference in toxicity of this insecticide. localities 1, 2 and 3 were having their lc50 values 0.362, 0.176 and 0.092µ l/ ml respectively. the regression analysis of variance further confirmed that there was an increasing trend in mean mortality of insects after 72 hours in topical application method [for smh: b= 7.03± 9.63, f(1,4)= 12.46, p= 0.04, for ktch: b= 6.63± 8.89, f(1,4)= 11.99, p= 0.04 and for klpt: b= 5.20± 10.49, f(1,4)= 6.26, p= 0.08) (figs 1–3(b)]. when different concentrations (1.0, 5.0, 10.0, 15.0 and 20.0 µ l/ ml) of deltamethrin 2.5% sc were tested against p. americana, variable range was obtained according to its respective localities. it is evident from table 2, that lc50 for smh was 4.145µl/ ml, whereas comparatively low values were recorded for ktch and klpt (2.500µ l/ ml and 2.067µ l/ ml respectively). the regression analysis of variance further confirmed that there was an http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 25 increasing trend in mean mortality of insects at 48 hours post-treatment [for smh: b= 0.82± 4.68, f(1,4)= 33.45, p= 0.01, for ktch: b= 0.67± 4.98, f(1,4)= 21.32, p= 0.02 and for klpt: b= 0.64± 4.12, f(1,4)= 22.70, p= 0.02) (fig. 1–3(c)]. the concentrations used for ddvp 50% ec were 10.0, 20.0, 30.0, 40.0 and 50.0µl/ ml for all three localities tested. all three localities were having their lc50 values 28.556, 26.388 and 20.138 µ l/ml respectively. the regression analysis of variance further confirmed that there was an increasing trend in mean mortality of insects after 48 hours in topical application method [for smh: b= 0.5± 0.267, f(1,4)= 937.5, p= 7.65e-05, for ktch: b= 0.49± 0.367, f(1,4)= 654.82, p= 0.000 and for klpt: b= 0.44 ± 2.53, f(1,4)= 76.42, p= 0.003) (fig. 1–3(d)]. based on lc50 values, imidacloprid, deltamethrin and ddvp were having non-significant values, and tested insecticides were arranged according to following descending order of preferences: fipronil 2.5% ec> imidacloprid 5% sc> deltamethrin 2.5% sc> ddvp 50% ec. table 1. information on field collected p. americana (l.) localities used in toxicity bioassay (during 2011) localities sources n dates of collection smh (locality 1) sewerage manholes from lcwu, lahore 160 march 28–30, 2011 ktch (locality 2) kitchens and basements from residential areas in lahore cantonment and nearby localities 185 april 15–20, 2011 klpt (locality 3) bakeries and food-courts from kot lakhpat area 210 may 21–26, 2011 table 2. comparative toxicities of four insecticides against three localities of p. americana (l) in topical application method insecticides localities lc50 (µl/ml) 95% confidence limits lower upper fit of probit line rr slope ± se χ2 (df) p imidacloprid 5% sc smh ktch klpt 2.712 1.966 1.318 2.024 3.622 1.304 2.554 0.714 1.771 2.353±0.579 2.321±0.562 2.419±0.585 2.698 (4) 1.701 (4) 3.091 (4) p< 0.001 p< 0.001 p<0.001 2.00 1.50 1.00 fipronil 2.5% ec smh ktch klpt 0.362 0.176 0.092 0.220 0.554 0.092 0.275 0.038 0.156 1.552±0.294 1.508±0.289 1.483±0.305 0.435 (4) 1.960 (4) 1.646 (4) p< 0.001 p< 0.001 p< 0.001 3.93 1.91 1.00 deltamethrin 2.5% sc smh ktch klpt 4.145 2.500 2.067 2.540 5.879 1.226 3.874 0.930 3.298 1.938±0.352 1.632±0.321 1.602±0.323 3.252 (4) 2.951 (4) 2.148 (4) p< 0.001 p< 0.001 p< 0.001 2 1.21 1.00 ddvp 50% ec smh ktch klpt 28.556 26.388 20.138 24.650 32.292 22.293 30.380 16.119 23.900 6.028±1.097 5.018±0.885 4.035±0.695 3.680 (4) 4.164 (4) 6.600 (4) p< 0.001 p< 0.001 p< 0.001 1.42 1.31 1.00 http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 26 fig. 1. regression line of (a) imidacloprid 5% sc (b) fipronil 2.5% ec (c) deltamethrin 2.5% sc and (d) ddvp 50% ec on mean mortality of smh (locality 1) of p. americana (l) http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 27 fig. 2. regression line of (a) imidacloprid 5% sc (b) fipronil 2.5% ec (c) deltamethrin 2.5% sc and (d) ddvp 50% ec on mean mortality of ktch (locality 2) of p. americana (l) http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 28 fig. 3. regression line of (a) imidacloprid 5% sc (b) fipronil 2.5% ec (c) deltamethrin 2.5% sc and (d) ddvp 50% ec on mean mortality of klpt (locality 3) of p. americana (l) discussion in various studies, researchers have paid attention to an effective and powerful strategy for control of cockroaches (shafiqur-rahman and akter 2006, sulaiman et al. 2007, ahmad et al. 2010). conventional pyrethroids and carbamates which are not much effective in pest management strategies have been replaced by new chemicals like neonicotenoids and phenyl-pyrazoles. resistance against these new formulated insecticides has not been rehttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 29 ported yet. therefore, keeping in view the recent development in insecticides resistance management for the control of urban pests, this study was designed to monitor the insecticidal efficacy and resistance control strategy of these new chemicals and why these are considered as better options for cockroach control. among phenyl-pyrazole family of insecticides, fipronil is a relatively new insecticide that is found to be very effective against cockroaches and other household pests. previous studies have also explained the strong neurotoxic effects of fipronil, which demonstrate that even a very low dose of it is suitable enough to attain desirable results. studies conducted by ahmad and suliyat (2011) have reported that in the form of gel baits, it worked efficiently and even a very small amount (0.03%) of it was proved to be lethal against cockroaches. similar results were obtained by nasirian et al. (2006) who have reported that fipronil acts as powerful neurotoxicant against german cockroach. current investigation is in strong agreement with these findings, where low concentrations of fipronil were effective against adult p. americana. the toxicity data from this study explains that few hours after the topically applied fipronil, there was successive increase in mortality of treated cockroaches (same pattern observed in all three localities) that gradually got decreased up to 48 hours post treatment and then became stable after 72 hours of exposure. similar results were obtained by holbrook et al. (2003) and nasirian et al. (2006) who have reported that topical application is the most sensitive method in determining resistance ratio in field populations of cockroaches and even 0.01% of fipronil is toxic to field population. they have also reported that regression line curve of log dose is in accordance with mortality rates of treated individuals. rettich and stejskal (2008) have reported that in bait formulations, soluble concentration (sc) of fipronil is very effective against field populations of germen cockroach. american cockroach in our study has also found to be non-resistant against fipronil at low concentrations. among all the four insecticides tested, fipronil was proved to be highly effective. in various other findings, same conclusions have been reported. scott and klen (1997), wang et al. (2004) and rina et al. (2002) have presented similar results as in current study. our findings are also parallel with those of valles et al. (1997) who have concluded that fipronil is sufficient enough to kill pre-resistant populations even in nano-gram quantity. the most reasonable justification in this regard is perhaps fipronil being a new addition in phenylpyrazole class of insecticides, which has not been used frequently. that is why insects and agricultural pests have not yet developed resistance against this chemical. the results of present study have also revealed the fact that phenyl-pyrazole class is more toxic then organophosphates. ddvp, an effective member of organophosphates failed to control resistant field population of american cockroaches. kaakeh et al. (1997) supported our findings, while concluding that organophosphates are not effective enough to combat susceptible and resistant populations of cockroaches. in contrast to a few studies where organophosphates acted as neurotoxic agent against cockroaches, this chemical did not come up with satisfactory results in our study. the possible reason for this least toxic effectiveness might be the frequency and level of its application. this class is less favored by pest control industries, mainly because of its strong odor than pyrethroids (lee and lee 2004). in those parts of the world where such studies have proved organophosphate as effective class of insecticides, this chemical might not have been sprayed quite often, and its reduced frequency of application have resulted in emergence of susceptible populations of cockroaches, which are sensitive to this chemical. unless and until, an insecticide is aphttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 30 plied repeatedly again and again over a certain period of time, resistance does not develop. whenever a chemical is sprayed quite often and its active ingredient is repeatedly exposed to pests’ species, it reduces its effectiveness and as a result, resistance against that insecticide gets developed gradually. here in case of ddvp 50% ec, same phenomenon seems to have happened. this active ingredient is readily mixed in every other insecticidal spray from low to moderate quantity, it has almost lost its efficacy and the targeted species have become resistant against this insecticide. various researchers have reported the presence of high level resistance in insects against pyrethroids and have highlighted its control failure incidence (zhai and rhobinson 1996, lee et al. 1996, scharf et al. 1997, lee et al. 1997, lee 1998, lee et al. 2000). the results of present study highlight the fact that among all four insecticides used, pyrethroid (deltamethrin 2.5% sc) exhibited moderate to high level of resistance against adult p. americana. the comparison of percentage mortality of these localities at different concentrations of deltamethrin reveals that there is a marked difference in range of effective concentrations for these localities. even very low concentration (1µ l/ml) was found to be effective against localities 2 and 3 of p. americana, however, locality 1 remained almost ineffective (with almost 15% mortality rate comparable with other two). the highest concentration (20.0 µ l/ml) ended up with expected results, killing 100% of the overall tested individuals. similar results were obtained by ballard et al. (1984), scott et al. (1986), scharf et al. (1996) and lee at al. (1996) where either or two of the tested localities came up with 100% mortality rate at this concentration. the findings of shafiqur-rahman and akter (2006), however, contradict our results proving that pyrethroids are highly effective against p. americana. using cypermethrin and permethrin instead of deltamethrin, the treated individuals gave immediate mortality response after treatment with topical application. this variation in susceptibility of different localities is because of their far apart localities from where they were collected. locality 3, which proved to be highly susceptible in both toxicity bioassays, was collected from those areas which were not previously exposed to any insecticide (or their exposure level was comparatively lower to develop resistance). that is why when they were treated with deltamethrin, all of them showed mortality immediately after few hours. these results of current investigation strongly favor the concept given by lee and lee (2002), who have reported that pyrethroids exhibit low to moderate level of resistance. similar conclusion has been made recently by ahmad et al. (2009) and nasirian et al. (2009) who have reported that resistance against pyrethroids have been developed in certain species of cockroaches. same concepts of deltamethrin resistance have been put forward by choo et al. (2000) and chai and lee (2010) who have reported heavy resistance for pyrethroids (deltamethrin and cypermethrin) against both american and german cockroaches. unlike conventional pyrethroids and organophosphates which are older compounds and have given rise to resistant localities of cockroaches, imidacloprid is a relatively new chemical which have not been tested for so long in pakistan. low usage and reduced application of imidacloprid has raised its significance in controlling urban pests effectively. it’s completely different mode of action from organophosphates and pyrethroids has made it a sensible option for pest control industries (cole et al. 1993, salgado 1997). imidacloprid was found to be slow responding insecticide during toxicity bioassays, and considerable results were achieved after 48 to 72 hours of treatment. same case was reported by appel and tanley (2000). regarding toxicity of imidacloprid, kaakeh http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 31 et al. (1997) observed its transient and rapid knockdown effect in a similar manner as recorded in our study. among its different concentrations tested, the mortality response for all three localities was significantly different. this variable level of susceptibility is perhaps because of locality differentiation. locality 3 might not be previously treated with imidacloprid or any other insecticide, which is why their individuals did not have resistant genes among them. wei et al. (2001) have achieved similar results and presented the idea that cross resistance might be involved in this case. as the resistance ratio is quite low in this case, it is difficult to conclude that whether these localities are naturally resistant against imidacloprid or phenomenon of cross-resistance is involved. conclusion in the light of above mentioned findings it can be suggested that resistance against insecticides is a common phenomenon and all tested localities of p. americana populations exhibited resistance against tested insecticides. it is therefore, mandatory to establish a sensitive method for a prior detection of resistant localities. the bioassay methods adopted in this study are suitable for meeting this criterion well. moreover, dealing with discriminating doses requires extreme caution, because minor errors may possibly alter the end results and susceptible localities may wrongly be considered as resistant localities. another important aspect while establishing the discriminating doses of insecticides is the selective choice of susceptible locality. there should be completely distinguished range of doses for resistant and susceptible localities while dealing with insecticides resistance. acknowledgements our deepest acknowledgement is for the our deepest acknowledgement is for the head of zoology department, lcwu, for providing all the facilities to conduct research. the authors are grateful to the laboratory staff for providing necessary equipments and their nice cooperation throughout the research period. the authors declare that there is no conflict of interest. references abd-elghafar sf, appel ag, mack tp (1990) toxicity of several insecticide formulations against adult german cockroaches (dictyoptera: blattellidae). j econ entomol. 83: 2290–2294. ahmad i, suliyat a (2011) development of fipronil gel bait against german cockroaches, blattella germanica (dictyoptera: blattellidae). j entomol. 8(3): 288–294. ahmad i, sriwahjuningsih sa, putra re, permana ad (2009) monitoring pyrethroid resistance in field collected blattella germanica linn. 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(dictyoptera: blattidae) in malaysia. j trop med parasitol. 30: 64–67. tsai c, lee h (2001) analysis of specific adaptation to a domicile habitat: a comparative study of two closely rehttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 21–34 r syed et al.: laboratory evaluation of … 34 lated cockroach species. j med entomol. 38: 245–252. valles sm, koehler pg, brenner rj (1997) antagonism of fipronil toxicity by piperonyl butoxide and s, s, s,-tributyl phosphorotrithioate in the german cockroach (dictyoptera: blattellidae). j econ entomol. 90: 1254–1258. vythilingam i, jeffery j, oothuman p, abdul razak ar, sulaiman a (1997) cockroaches from human dwellings: isolation of bacterial pathogens and control. southeast asian j trop med pub hlth. 28: 218–222. wang c, scharf m, bennett g (2004) behavioral and physiological resistance of the german cockraoch to gel bait (blattodea: blattellidae). j econ entomol. 97(6): 2067–2072. watanabe h, kobayashi y, sakura m, matsumoto y, mizunami m (2003) classical olfactory conditioning in the cockroach periplaneta americana. zool sci. 20: 1447–1454. wei y, appel ag, moar wj, liu n (2001) pyrethroid resistance and cross-resistance in the german cockroach, blatella germinca (l). pest manag sci. 57: 1055– 1059. zhai j, robinson wh (1996) instability of cypermethrin resistance in a field population of the german cockroach (orthoptera: blattellidae). j econ entomol. 89: 332–336. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 35–42 h dehghan et al.: the ecological aspects … 35 original article the ecological aspects of culex pipiens (diptera: culicidae) in central iran hossein dehghan 1, *seyed hassan moosa-kazemi 2, javid sadraei 1, hassan soleimani 2 1department of medical entomology and parasitology, school of medical sciences, tarbiat modares university, tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 2 aug 2012; accepted 3 july 2013) abstract background: the aim was to survey the specific factors, which cause to decrease blood feeding of mosquitoes important to succeed vector control. methods: larval collection was carried out from fixed and variable breeding places of yazd county, central iran in 2009. autogeny-anautogeny, stenogamy-eurygamy, and blood preference of culex pipiens were studied using standard mosquito cages blood meal source for cx. pipiens females considered as the chickens and human and fed females were kept in insectary condition (16:8 l: d, 27±3 °c and 70±10% rh). the data were analyzed using spss ver. 11.5 soft ware. results: totally, 96 females' mosquitoes were tested for stenogamy versus eurygamy and 122 for blood preference assay. in the small cages (20× 20× 20cm) and large cage (60× 40× 60cm), the ability of mating and insemination rates were 60.0 and 67.0%, respectively. in spite of cx. pipiens fed from sucrose 5%, none of them laying eggs in 60× 40× 60 cages during the study. this finding indicated the anautogeny behavior of this species. this species was found of low tendency to human blood and almost 4 fold fed on chicken. conclusion: the occurrence of steno-eurygamy, anautogeny, and ornithophilic behaviors of cx. pipiens was noted. more studies need to be carried out about the bionomics of this species to gain more data about the ecophysiological and behavioral characteristics in other parts of iran. keywords: bionomics, behavior, mosquito, culex pipiens, iran introduction malaria, west nile and sindbis viruses, japanese encephalitis, rift valley fever as well as dirofilaria immitis (dog heart worm) and d. repens (dirofilariasis) are important medical and veterinary mosquito-borne diseases in the world (naficy and saidi 1970, saidi et al. 1976, hayes 2005, who 2008, azarihamidian et al. 2009). culex pipiens linnaeus is medically important due to bite humans and animals besides transfer d. immitis in iran (ataei et al. 2012). humans and some hosts may have allergy to mosquito bite, and this allergy can sometimes be very severe (vinogradova 2000). azari-hamidian (2007) cited sixty four species, three subspecies and seven genera of mosquitoes in iran. by now, 33 species from two anopheles and cellia subgenera, siblings, genotype and type forms are recorded in the country (moosa-kazemi et al. 2009, vatandoost and hanfi-bojd 2012). different studies have introduced eight malaria vectors including, an. stephensi (type, intermediate and mysorensis forms (vatandoost et al. 2006), an. superpictus as genotypes x, y, and z (oshaghi et al. 2008), an. culicifacies *corresponding author: dr seyed hassan moosakazemi, e-mail: moosakazemi@tums.ac.ir http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 35–42 h dehghan et al.: the ecological aspects … 36 sibling a and b (vatandoost et al. 2011), an. fluviatilis as three cryptic species s, t and u (mehravaran et al. 2011), an. maculipennis group (an. atroparvus, an. maculipennis, an. melanoon, an. messeae, an. sacharovi and an. persiensis (sedaghat et al. 2003), an. dthali (manoochehri et al. 1972), and an. pulcherrimus (zaim et al. 1993) is detected serologically positive to malaria parasite(s) in baluchistan malaria foci, southeastern of the country. five species of anophelinae have been previously reported in yazd province, central iran, including an. dthali, an. marteri sogdianus, an. multicolor combouliu, an. turkhudi liston, and an. superpictus grassi (shahgudian 1956, saebi 1987). zaim (1987) mentioned the twelve species and four genera of culex, culiseta, ochlerotatus, and uranotaenia in this area. culex pipiens distributed in the europe, the tropical and subtropical regions of asia and africa, the middle part of north america and southern america and australia (vinogradova 2000). this species is distributed in the most part of iran (zaim et al. 1985, koosha 2011). distribution of this species has close relationship with economic activities and development of new territories. the human activity on natural environment with a change in the land and underground water resources can affect the mosquito fauna and abundance in a region. some human activities created the irrigation domains marsh, dry land irrigation, channels, irrigation water storage, and various breeding places near the industrial activities (vinogradova 2000). culex pipiens complex has at least two species (dehghan et al. 2013). culex quinquifasciatus say distributed in the tropical area with the wide range of hosts whereas, cx. pipiens is found in the moderate area with the limited range of birds nest maker. culex pipiens form molestus is autogenous, and the first laying eggs perform without a blood feeding (mousakazemi 2000, vinogradova 2000). culex pipiens was reported as ornithophilous, and intend to blood feeding on birds. some reports indicated this species feeding on humans. the main host of this species in rural area was reported birds, and animals, whereas in urban area was reported as human, animal, and birds. vinogradova (2000) described stenogamy as the mating ability of mosquitoes in small spaces whereas, eurygamy as the mosquitoes need to large space. meanwhile cx. pipiens form molestus, cx. quinquefasciatus and possibly cx. pipiens pallens coquillett were described as sten-ogamous, whereas cx. pipiens, cx. australicus dobrotworsky and drummond, cx. vagans wiedemann, and cx. torrentium martini were reported as eurygamous (vinogradova 2000). there are limited studies in relation to physiology, biology, feeding behavior of cx. pipiens in yazd. the aim of this study was to obtain new data which would be useful for future mosquito control programs. materials and methods study area a descriptive cross sectional study was carried out in yazd county, central iran in summer 2009. the study took place in two randomly selected rural villages with 889 houses and 24358 populations in yazd county (54°04'n 31°59'e). the study area is located in the yazd province and bounded by isfahan in the west, south khorasan in the east, kerman and fars in the south, khorasan-erazavi and seman provinces in the north (fig. 1). the maximum and minimum mean monthly temperatures were recorded as 40.6 °c in july and -4.4 °c in january in 2009. the total annual rainfall was 62 mm, the minimum of 0.3 mm in may and maximum of 18 mm in march. the mean annual relative humidity was 37%. the main occupation of the peoples is agriculture and husbandry of cow and goats. based on available epidemiological data from yazd health cenhttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 35–42 h dehghan et al.: the ecological aspects … 37 tre, there are no villages under the entomological survey. sampling methods the specimens were collected from three fixed villages, abrand-abad, nosrat-abad and gerd-e-faramarz of shahediyeh district and two variables villages, elah abad and sarcheshmeh from zarch district. mosquitoes larvae were collected by dipping method (who 1963, who 1992). larval habitats present within 500 meter around each village were studied for mosquito larvae collection. five larval breeding places randomly selected within a 500-m radius of the study site. up to 10 dipper samples were taken at intervals along the edge of each larval habitat using a standard mosquito dipper (350 ml) depending on the size of the habitat (who 1963). mosquito identification the larvae were preserved in lactophenol and the microscope slides of the preserved larvae were prepared using liquid de faure’s medium. all samples were sent to the school of public health, tehran university of medical sciences, where the author identified the specimens using the keys of shahgudian (1960), zaim and cranston (1986), harbach (1988), azari-hamidian and harbach (2009). seta 1 of abdominal segment of iii–iv in larval stage and vein venation including intersection of costa, subcosta and bifurcation of r2+3 veins used to distinguishing of adult females of cx. pipiens complex. male genitalia shape as dorsal and ventral arms of phalosoma used to distinguishing of adult males of cx. pipiens complex. the mosquito name abbreviations are cited based on reinert (2001). mosquito rearing the larvae for each habitat were placed separately and transported to the laboratory where they were sorted by genus and instars counted and recorded. culex pipiens larvae were transferred to the laboratory of entomology and parasitology department, school of medical sciences, tarbiat modares university for rearing. de-chlorinate tap water used for rearing of larvae and prepared by stand the water in glass jar for at least 24 hours. larvae were placed in the covet and fed by available diets such as fish powder and milk baby food. pupae in batches of 30 samples were transferred to cups container covered by paper funnel and was placed into the cages. petri dish containing de-chlorinate tap water was placed in to the cages for egg-laying. cotton wool soaked in sucrose 5% used for feeding of adult mosquito. mosquitoes were checked and monitored daily. behavioral and physiological study the experiment was designed to determine the degree stenogamy or eurygamy behaviors in a 60× 40× 60cm (fig. 2a), and 20× 20× 20cm (fig. 2b) cages. the spermathecae of the females cx. pipiens were dissected for evidence of insemination (fig. 3). the larvae were reared in insectarium and pupae were separated and transferred into cups inside the cages. autogeny, anautogeny and blood preference of this species also were studied with rearing the larvae and transferred the pupae in cages. sucrose 5% and moisture cotton wool were used for feeding of this species in each cage. petri dishes with no chlorine water were used for oviposition. eurygamy, and stenogamy behaviors were studied 10 days after emerging and mating of species using the dissection of spermathecae. anautogeny and autogeny behaviors of non blood feeding mosquitoes were studied at a 16:8 l: d, 27±3 °c and 70±10% rh for 20 days. adult females were fed on chickens and human forearm for blood meal source in insectary. fifty eight female mosquitoes were used for feeding on chicken and sixty four for human blood type. (knight and malek 1951, who http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 35–42 h dehghan et al.: the ecological aspects … 38 1975, vinogradova 2000). the data were analyzed using spss ver. 11.5 soft ware. results results of stenogamy versus eurogamy showed that sperm was active and observed in 60% of dissected cx. pipiens. the ability of mating was found in cages in 20×20×20 cm sizes. the same result was found in large cages, 67% of mosquitoes were able to mating in cages 60×40×60 cm sizes (table 1). in autogeny and anautogeny study, the mosquitoes feed on sucrose 5%, none of the mosquitoes laying eggs in 60×40×60 cm cages during the 20 days study. this finding indicated the anautogeny behavior of this species. the mosquitoes exposed to chicken body almost 4 fold the mosquitoes were contact with the human arm. this finding indicated tendency to bird blood more than human one (table 2). table 1. result of the spermateca dissection of culex pipiens to determination of stenogamy versus euٍrogamy behaviors in department of entomology and parasitology insectary, school of medical sciences, tarbiat modares university, 2009 spermateca dissection cage sizes negativepositive small 20×20×20 491 552 473 564 1827total -large 60×40×60 371 392 6113 574 1734total table 2. frequency and percentage of the blood tendency of culex pipiens on human and chicken hosts in department of entomology and parasitology insectary, school of medical sciences, tarbiat modares university, 2009 ulex pipiensc replicate %fed%unfedhost 84.62215.44 chicken cage 1 7524258cage 2 79.84620.212total 27.3972.724 human cage 3 35.51164.520cage 4 31.32068.744total fig. 1. map of iran, highlighting the position of yazd province and its study area fig. 2. the mosquito cages including (a) large size, 60× 40×60 cm, (b) small size, 20×20×20 cm. the pictures were taken by a digital camera in the insectary of the department of entomology and parasitology, school of medical sciences, tarbiat modares university (original photos) http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 35–42 h dehghan et al.: the ecological aspects … 39 fig. 3. the spermatheca dissection (a) culex pipiens spermatheca, (b) cx. pipiens spermatozoids, the original photos of spermatheca dissection of cx. pipiens female were taken by a digital camera in the department of entomology and parasitology, school of medical sciences, tarbiat modares university discussion culex pipiens larvae were collected from different larval habitats such as subterranean water accumulated with sewage, livestock trough, ponds in residential houses and garden ponds. the flume water contaminated by sewage was found the main breeding places of cx. pipiens inside urban area. similarity, vinogradova (2000) cited cx. pipiens larvae found in natural and artificial breeding places with high degree of contamination by organic and industrial materials. water obtained from sewage treatment plants was created the suitable habitats of mosquito larvae outside the urban area. culx pipiens mosquitoes can be made adaptable with the larval habitats created by human. this species can be considered as an indicator for estimating the environmental conditions in a human habitat. development of human activity, changed the environment, and increased the abundance indicates ecological and physiological flexibility of this species. in the present study, 60% and 58% of cx. pipiens were able to mating in small and large cages respectively. the eurygamy behaviors of cx. pipiens were studied in cages in 100× 100× 100cm, 75× 75× 75cm and 60× 40× 60cm in russia (vinogradova 2000). in contrast, some researchers designed cages in 200× 200× 200cm sizes, and was stated a few of this species able to mating (vinogradova 2000). vinogradova (2000) stated the several populations of cx. pipiens with eurygamy behavior never did mating in cages in 20× 20× 30 sizes. we observed both steno and eurygamy behaviors. further support to these results also came from a previous study, reported by rioux (1965), which described cx. pipiens with steno-eurygamous behaviors. it should be considered, at the present, cx. pipiens populations were not observed with the pure form of eurygamy or stenogamy behavior. it seems that there are no agreements among researchers regarding to the maintenance condition of this species in insectariums. jupp (1987) reported that changed photoperiodic conditions of rearing of this species could be increased or decreased the mating rate. it should be noted that, cx. pipiens after blood meal was able to laying eggs in the 60× 40× 60cm cages, whereas, none of the individuals which fed only on sucrose 5% were able to lay eggs in the same cages. our finding indicated the anautogenous behavior of this species. similarity, rioux (1965) and vinogradova (2003) cited the anautogenous behavior in cx. pipiens populations. tendency of cx. pipiens to bird blood was more than human. further support to this results also came from a previous study, cx. pipiens was reported strongly ornithophil behavior in south-central sweden (jaenson 1990). also, tempelis (1975) cited 28% of blood feeding on human and more tendency of this species to avian blood preference. rioux (1965) expressed the ornithophilic behavior of this species. blood feeding of cx. pipiens on birds and human was reported by knight and malek (1951), and vinogradova (2003). culex pipiens has great morphological variations in all life stages and it is not easy to separate this species from cx. quinquefasciatus http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 35–42 h dehghan et al.: the ecological aspects … 40 and cx. torrentium. males of this species are distinguishable from the mentioned species using the male genitalia (harbach 1988). dehghan et al. (2011) reported the male genitalia was the only reliable character to identification of cx. torrentium, cx. quinquefasciatus and cx. pipiens. the most important diagnostic characters of cx. torrentium, cx. quinquefasciatus and cx. pipiens larvae mentioned the branch of seta 1 of abdominal segment of iii–v (harbach 1988, dehghan et al. 2010). based on the mentioned above, we used the recent characters to diagnosis of the cx. pipiens samples. conclusion in situations where frequent blood feeding on birds are common for cx. pipiens, the main complication with respect to estimating the potential for arboviral parasite transmission is that vectorial capacities will be necessary. therefore, more study about of bionomics of mosquitoes especially among the cx. pipiens needs to be studied rigorously in the future. acknowledgements the authors are grateful to dr azarihamidian, school of health, guilan university of medical sciences, rasht, for reviewing the manuscript, mr kalantari, head of zarch city council, mr rezaei, mr eslami, environmental health director of zarch and shahediyeh health center, for supporting the investigation. we also would like to express our appreciation to the people of abrand-abad, nosrat-abad, gerd-e-faramarz, elah abad, and sarcheshmeh villages in yazd county for their kind cooperation during the study. many thanks also dedicated to the efforts of the field staff of the yazd health training and research center. it is noteworthy that this study has been done by financial support of research committee in school of medical sciences, tarbiat modares university, id no 5278604. the authors declare that there is no conflict of interests. references ataei a (2012) some ecological aspects of potential vectors of dirofilariasis in the ahar county, east azerbaijan province, iran. 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cont assoc. 3: 568–573. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 72 http://jad.tums.ac.ir published online: march 31, 2022 original article epidemiological study of pediculosis among primary school children in sulaimani governorate, kurdistan region of iraq *yadgar hussein hama-karim1, pary mohammad azize2, salar ibrahim ali1, shahow abdulrehman ezzaddin3 1college of health and medical technology, chwarchra qtr 2sulaimani technical insitute, bakhtiari qtr 3college of medicine: sulimani university old campus, majid bag main street, beside smart health tower *corresponding author: dr yadgar hussein hama-karim, email: yadgar.hamakarim@spu.edu.iq (received 7 sep 2021; accepted 9 mar 2022) abstract background: pediculosis is a common parasitic public health issue. many socio-demographic and economic factors affect the pattern and prevalence of pediculosis. it is widely spread among pupils, particularly those in elementary schools. this study aimed to determine the prevalence of pediculus capitis infestation and its related risk factors in primary school children in sulaimani governorate, kurdistan region of iraq. methods: this is a cross-sectional study, overall, 2064 primary school children (aged 5–14 years) during the period from october 2019 to february 2020 were randomly examined for the presence of any stage of pediculosis capitis in both urban and suburban areas. the schools were selected by multistage sampling methods. a questionnaire was sent to the parents to get consent and fill in for every single pupil. the results were computed using the chi-square test and logistic regression analysis. results: the overall prevalence of head lice infestation was (9.16%). the study found a significant association of pediculosis capitis infestation with gender, parents’ education level, mother’s employment, number of siblings, and previous infestation. the older age of children (≥12 years old) and the higher-grade pupils (4th to 6th class) were more infested. conclusion: head lice remain a health problem for school children. it needs collaboration of many sectors for decreasing and early detection and treatment in both urban and suburban areas since they have a close rate of infestation. keywords: pediculosis capitis; risk factors; children; primary schools; sulaimani governorate introduction lice as compulsory ectoparasites have a long relationship with the human host, indicating co-evolution between hosts and parasites (1). blood sprinkling lice are wingless bugs that feed on sebaceous secretions and bodily fluids. the human body hosts three kinds of lice, including head louse, body louse, and pubic louse (2, 3). the head louse is an ectoparasite of humans, and unfed lice will die within 2 to 4 days if they are kept away from people and are denied a blood meal (4, 5). lice infection is considered as one of the world's public health problems and especially in developing countries. depending on the cir cumstances especially for school children and their developmental ages, this infection is of special importance. the distribution of lice infection occurs across the world and especially with a wide range of pediculosis has been observed in the developing countries (6, 7). in epidemiological studies in schools around the middle east countries, the prevalence of head lice in iran is 7.6% (8), syria 14.3% (9), turkey 13.1% (10), saudi arabia 64.2% (11), and jordan 20.4% (12). the status of primary school children head lice infestation in iraq varies from 3.6% in bagh copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:yadgar.hamakarim@spu.edu.iq https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 73 http://jad.tums.ac.ir published online: march 31, 2022 dad (13) to 20.08% in primary schools in kirkuk (14) and 34.7% in secondary schools in the same city (15). the highest infestation was ever reported was 56% among prisoners in erbil. in the kurdistan region of iraq, the total prevalence of the pediculus capitis infestation was approximately 14% among school children (16–19). the variance in infection rate might be attributed to a variety of variables, including eradication methods, the number of head-to-head interactions, diagnostic procedures, school head lice policy (no-nit policy), pesticide resistance, and general head lice awareness (20). direct contact with infected people is the main route of transmission of lice. in addition, the disease is transmitted indirectly through contact with infected personal items such as combs or brushes, hats, and pillows (21). also, various factors such as personal hygiene, parents' job, parent's education, hair length, and frequency of bathing per week can affect the prevalence of infection (22). the ectoparasite is found mainly in crowded places where close contacts occur, including schools, nurseries, sports facilities, playgrounds, camps, and prisons (2). the highest incidence is observed among children aged from five to thirteen years old. nevertheless, the incidence among the 24– 36-year age group is increasing due to their exposure to infected children. pediculosis is more common in younger girls and those in crowded families (23, 24). the itching scalp is already caused by lice bites, saliva, and fecal matter. depression, sleeplessness, exhaustion, educational failure, mental problem, a decline in social stigma, and allergic responses are some of the additional symptoms that might occur. because of the societal shame associated with infestations and the vast majority of cases are unreported (5, 25). no thorough study on head lice infestation has been conducted over recent years in sulaimani governorate and considering the rising trend in pediculosis frequency among children in sulaimani schools; therefore, this study was conducted to determine the prevalence of p. capitis infestation and the associated risk factors among primary school children. materials and methods study setting and participants the school-based, cross-sectional study was performed in the primary schools (classes 1–6) inside and outside the sulaimani governorate. the study was performed in the sulaimani governorate/ kurdistan region of iraq between october 2019 and february 2020. at the time of the study, 333045 children were enrolled in primary schools. sampling and data collection a pre-test of about 3000 children was examined and semi-structured questionnaires were sent to the children's parents to fill in and get their consent. only the respondent and completed forms were considered in the study. the sample size was estimated via using this formula: , where n is the sample size, z is the confidence level at 95% (1.96), p is the expected prevalence (50%), and margin of error (d), at 5%. based on this formula, the minimum sample size needed was 385 pupils; however, the sample size was increased to include all eligible pupils in the selected schools to increase the power and validity of the study. a total of 2064 primary school children (aged 5–14 years), in which 1046 boys and 1018 girls were included in this study from nine primary schools of both urban and suburban areas. multi-step, stratified, cluster sampling was used. the total number of children in the primary schools was divided into urban and suburban. inside the urban areas (sulaimani city) was also divided into western and eastern zones. the number of chosen schools was determined proportionally to the whole population. the schools were selected randomly among the areas. when a school was selected it was considered as a cluster and all the children’s heads http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 74 http://jad.tums.ac.ir published online: march 31, 2022 from the first to the sixth classes were examined. special attention was given to the back of the ears and neck area. the examination was done by the researchers and by at least nurses that manage the school health of that area. the infested child was recorded secretly and checked in the questionnaire form. the questions in the form were designed about sex, age, class, education levels of parents, mother’s workplace, family size, and income. hygiene-related questions were also added, which include frequency of head washing, combing, bathing, hair length and style, head wash materials, sharing bed and tools, and history of infestation. statistical analysis the data were analyzed in the statistical software stata v.14. categorical variables were defined as the number and percentage (%) and analyzed using the chi-square test. for finding the relationship between the head lice infestation and potential predictors, the univariate logistic regression model was first used and then a multivariate logistic regression was used to calculate the adjusted odds ratios (ors) for compromising the risk factors. any variable having a p-value less than 0.20 was chosen to be entered in the multivariate logistic analysis. in both logistic regression models besides or 95% confidence intervals (95% ci) were presented. a p-value of less than 0.05 was considered statistically significant. results head lice infestation was found in 189 school children out of 2064 one examined; the overall infestation rate was 9.16%. the infestation was significantly more common among girls (18.7%) than boys (1.82%). the mean and median age of the infested pupils were 9.4 and 9 years old, respectively. the rate of pediculosis was increasing as the age group increased as shown in (table 1). the prevalence of p. capitis was higher significantly among the 6th class and aged ≥ 12 as well as among those who had 4–6 siblings and housewife mothers, parents with primary education levels, long or wavy hair, and those with the previous infestation. however, family income, living in suburban areas were not significantly associated with head lice. also, none of the hygiene factors were found to have significant relation with head lice (table 2). univariate analysis showed that prevalence of head lice was related to sex, age group, class grade, father and mother’s education, mother’ employment, number of siblings, the income of the family, hair length, sharing the bed, and either if the child had the previous infestation of pediculosis or not (table 3). however, in the multivariate logistic regression model, 12 variables were adjusted as it is shown in (table 4). it was found that girls were 12 folds more at risk to get head lice than the boys (or= 12.21, 95% ci= 2.46 to 60.51). the odds of developing pediculosis are two times higher among the second class when compared with the first class (or= 2.18, 95% ci= 1.19 to 3.99). surprisingly, children whose fathers completed primary and secondary schools were 91% and twice more prone to have head lice than the illiterate ones (or= 1.19, 95% ci= 1.08 to 3.38 and or= 2.0, 95% ci= 1.09 to 3.67, respectively). in opposite, mothers who completed secondary school had their children were 42% less likely to have pediculosis compared to illiterate mothers (or= 0.58, 95% ci= 0.35 to 0.98). likewise, the employed mothers’ children had less likelihood of developing head lice 55% than the children whose mothers were housewives (or= 0.55, 95% ci= 0.23 to 0.88). the greater number of siblings increases the greater risk of pediculosis; however, the significance level of those who live with more than six siblings was almost on the borderline. pupils who had been infested with a previous attack of the parasite had two folds more likely to get it again than those who get it for the first time (or= 2.31, 95% ci= 1.56 to 3.41). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 75 http://jad.tums.ac.ir published online: march 31, 2022 table 1. characteristics of pediculosis in the infested and noninfested primary school children, 2019–2020 examined pupils infested pupils characteristics n (%) n (%) p value age (years) 0.023 6–8 870 (42.15) 65 (7.47) 9–11 920 (44.57) 89 (9.67) ≥ 12 274 (13.28) 35 (12.77) sex <0.001 boys 1046 (50.68) 19 (1.82) girls 1018 (49.32) 170 (16.70) school grade 0.032 1 425 (20.59) 21 (4.94) 2 346 (16.76) 34 (9.83) 3 337 (16.33) 33 (9.79) 4 345 (16.72) 36 (10.43) 5 305 (14.78) 30 (9.84) 6 306 (14.83) 35 (11.44) residency 0.891 urban 1126 (54.55) 104 (9.24) sub-urban 938 (44.45) 85 (9.06) no of siblings 0.001 0–3 1560 (75.58) 121 (7.76) 4–6 441 (21.37) 60 (13.61) ≥ 7 63 (3.05) 8 (12.70) education level of the father < 0.001 illiterate 261 (12.64) 19 (7.28) primary 723 (35.03) 85 (11.76) secondary 646 (31.30) 70 (10.84) higher education 434 (21.03) 15 (3.46) mother education level < 0.001 illiterate 372 (18.02) 43 (11.56) primary 659 (31.93) 80 (12.14) secondary 565 (27.37) 43 (7.61) higher education 468 (22.67) 23 (4.91) occupation of the mother < 0.001 housewives 1552 (75.19) 170 (10.95) work outside 512 (24.81) 19 (3.71) family income 0.137 low 178 (8.64) 21 (11.80) medium 1218 (65.13) 129 (9.58) high 495 (26.47) 39 (7.30) hair length < 0.001 short 1056 (51.16) 21 (1.99) long 1008 (48.84) 168 (16.67) hairstyle 0.018 straight 1507 (73.01) 122 (8.10) curly 158 (7.66) 21 (13.29) wavy 399 (19.33) 46 (11.53) previous infestation < 0.001 yes 234 (11.34) 52 (22.22) no 1830 (88.66) 137 (7.49) total 2064 (100) 189 (9.16) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 76 http://jad.tums.ac.ir published online: march 31, 2022 table 2. association between hygiene factors and the head louse infestation, 2019–2020 characteristics examined pupils n (%) infested pupils n (%) p value frequency of body washing per week ≤ 1 time 2 times ≥ 2 times 0.448 60 (2.91) 3 (5.0) 912 (44.19) 81 (8.88) 1092 (52.91) 105 (9.62) daily hair combing 0.753 less than once 1179 (57.12) 110 (9.33) once or more 885 (42.88) 79 (8.51) sharing beds 0.169 yes 725 (35.13) 75 (10.34) no 1339 (64.87) 114 (8.51) sharing instruments 0.254 yes 333 (16.13) 25 (7.5) no 1731 (83.87) 164 (9.47) cleansing material for hair washing 0.860 shampoo 1934 (93.70) 117 (9.20) soap 16 (0.78) 1 (6.25) both 114 (5.52) 11 (9.65) table 3. univariate logistic regression for predictors of head louse infestation among school children, 2019, 2020 predictors crude or (95% ci) p value sex male female 1 10.84 (6.69–17.56) <0.001 age 6–8 9–11 ≥ 12 1 1.33 (0.95–1.85) 1.81 (1.17–2.80) 0.006 class 1st 2ns 3rd 4th 5th 6th 1 2.10 (1.19–3.86) 2.09 (1.84–3.68) 2.09 (1.28–3.92) 2.10 (1.18–3.74) 2.49 (1.41–4.36) 0.006 father’s education level illiterate primary secondary higher education 1 1.70 (1.01–2.85) 1.55 (0.91–2.63) 0.46 (0.23–0.91) 0.007 mother’s education level illiterate primary secondary higher education 1 1.06 (0.71–1.59) 0.63 (0.40–0.98) 0.40 (0.23–0.67) <0.001 mother’s occupation housewives work outside 1 0.31 (0.19–0.51) <0.001 no. of siblings <0.001 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 77 http://jad.tums.ac.ir published online: march 31, 2022 ≤ 3 4–6 ≥ 7 1 1.87 (1.35–2.60) 1.73 (0.81–3.72) income low medium high 1 0.79 (0.48–1.29) 0.59 (0.33–1.03) 0.046 weekly head washing once 2 times > 3 times 1 4.54 (0.62–33.39) 4.87 (0.67–35.64) 0.256 combing frequency less than once once or more 1 0.95 (0.70–1.29) 0.753 cleansing material shampoo soap both 0.66 (0.09–5.04) 1.06 (0.56–2.01) 0.914 hair length short long 1 9.86 (6.21–15.66) <0.001 hairstyle straight curly wavy 1 1.18 (0.68–2.04) 0.68 (0.47–0.97) 0.015 sharing instruments no yes 1 0.78 (0.50–1.20) 0.256 sharing bed no yes 0 1.24 (0.91–1.68) 0.169 history of infestation no yes 1 3.53 (2.48–5.03) < 0.001 table 4. multivariate logistic regression for predictors of head louse infestation among school children predictors adjusted or (95% ci) p value sex male female 1 12.21 (2.46–60.51) 0.002 age 6-8 9-11 ≥12 1 1.11 (0.56–2.17) 1.80 (0.73–4.50) 0.772 0.204 class 1st 2ns 3rd 4th 5th 6th 1 2.18 (1.19–3.99) 1.86 (0.91–3.82) 1.76 (0.72–4.32) 1.55 (0.62–3.88) 1.56 (0.57–4.25) 0.012 0.091 0.218 0.352 0.384 father’s education level illiterate primary 1 1.91 (1.08–3.38) 0.026 table 3. continued ... http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 78 http://jad.tums.ac.ir published online: march 31, 2022 secondary higher education 2.00 (1.09–3.67) 0.34 (0.23–1.75) 0.025 0.540 mother’s education level illiterate primary secondary higher education 1 0.89 (0.57–1.39) 0.58 (0.35–0.98) 0.93 (0.44–1.96) 0.617 0.038 0.841 mother’s occupation housewives work outside 1 0.45 (0.23–0.88) 0.020 no. of siblings ≤3 4-6 ≥7 1 1.47 (1.01–2.13) 2.33 (0.99–5.47) 0.044 0.052 income low medium high 1 1.79 (0.48–1.29) 0.59 (0.33–1) 0.859 0.438 hair length short long 1 0.92 (0.20–4.27) 0.913 hairstyle straight curly wavy 1 1.08 (0.59–1.97) 0.99 (0.42–1.47) 0.798 0.973 sharing bed no yes 0 1.16 (0.82–1.63) 0.400 history of infestation no yes 1 2.31 (1.56–3.41) < 0.001 discussion head louse infestation is a widespread health concern that is most frequent in elementary schools worldwide, particularly in developing countries such as iraq's kurdistan region (8). it is most often affects children between the ages of 5 and 13 (4, 26). worldwide, pediculosis has seen a considerable rise in recent decades as a consequence of inappropriate application of pesticides, louse resistance to conventional pesticides, and misdiagnosis (27, 28). according to the present research, the prevalence rate of head louse infection is 9.16 percent. according to the national pediculosis association of the united states of america's definition of an epidemic, 5% of the children in the study region are infected (9); hence, based on the findings of this research, head pediculosis will be come epidemic in sulaimani governorate schools if ignored. the overall prevalence of p. capitis among primary school children in the sulaimani governorate was 9.16%. previous epidemiological studies in iraq reported different prevalence rate, 14.52% and 14% in erbil (19), 14% in najaf (17), 14.4% in kalar (29), 21.9% in tikrit (14), 3.6% in baghdad (13) and 5.4% in basrah (30). although the prevalence rate in this study is less than in other cities except for baghdad and basrah, it demonstrates that p. capitis remains a significant public health issue in the sulaimani governorate among school-aged children. population density, school head lice policies, eradication techniques, number of close contacts per person, overcrowded housing, economtable 4. continued ... http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 79 http://jad.tums.ac.ir published online: march 31, 2022 ic conditions, family income, and lack of care for head louse infection may all influence the infestation rate (20). girls had much greater infection rate than boys (16.1 and 1.82%, respectively); the association between sex and head lice was extremely significant, with a p-value of 0.002 in the final model of multivariate analysis. this finding corroborated the findings of many previous research papers (31–34). probably the disparity in head lice frequency is likely attributable to a variety of factors, including the behavioural differences between boys and girls, the appropriateness of female hair as a breeding ground for head lice, and girls have closer, prolonged, and more intimate head contacts in small groups (35, 36). although, many studies suggested that may be the long hair in females is another reason, whereas a non-significant relationship was found between long hair and pediculosis in the current study. the findings of this study are consistent with other research studies (10, 35, 37), which indicated that the prevalence of pediculosis was lower in children with educated parents than in children with uneducated parents, indicating that literacy plays a significant role in decreasing the prevalence of pediculosis. they also accord with our earlier observations, which showed that the children with mother's employment outside the home were less liable to head lice infestation. this may be due to the mothers who work outside may have higher educational level and their health awareness status could be greater. in line with many prior researches, poor family incomes are predisposed to p. capitis (38, 39). however, this finding was not significant in the last model of analysis in the current study. a significant prevalence of infestation was found among school children from households with larger siblings. larger families may pay less attention to their children's hair care, putting them at a greater risk of infestation due to interaction with their siblings and house overcrowding. close contact between siblings and a crowded household facilitates head lice transmission, particularly if another family member has been involved (40). moreover, the prevalence of p. capitis was directly associated with the history of the disease. it seems possible that the recurrent infestation is due to misdiagnosis, non-adherence to proper recommendations, poor product quality, use of alternative traditional remises such as kerosene, and/or parasite resistance to the available product (41–43). head louse infestation was more common among pupils with curly hair and long hair in the present study. however, in the multivariate analysis, they were not significant. in general, the lack of a major difference between the head louse prevalence rate and the hair length is consistent with previous publications. contrary to popular belief, hair cutting does not seem to lessen the incidence of head louse infestation in school children (44). living inside and outside the city was not made a significant difference in the rate of p. capitis infestation. this can be explained by the number of pupils and the rate of infestation in both urban and suburban schools were close. besides, the living standards, medical care system regarding head lice, and availability of anti-pediculosis agents are almost equal in both regions. the sample did not involve rural schools except one, which may have a higher index of suspicion. none of the hygiene factors were found to be related to head louse infestation in this study. some works of literature have shown that there is no significant association between head lice and inadequate cleanliness (3). head louse transmission may readily occur through head-to-head contact, despite being sanitary. the increased incidence of pediculosis among individuals who were washed more often may be attributed to severe itching owing to infestation (3). this study has some limitations. first, because the visual screening was used to identihttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 80 http://jad.tums.ac.ir published online: march 31, 2022 fy an infestation, some students with very low levels of head lice may have gone unnoticed. second, the lockdown of the school due to covid-19 did not let us cover all the schools that had been planned. therefore, the researchers could not take any private schools. also, because there were so many children who took part in this study, we didn't have access to some socioeconomic and cultural parameters. third, like other cross-sectional studies, this study was not able to detect causation. the strengths of this study could be expressed as the first community-based study in sulaimani governate. secondly, the sample size of this study was relatively high. fortunately, the examination of the students accompanied by educational advice on personal hygiene especially hair hygiene. conclusion the prevalence of p. capitis infestation among primary school children was (9.16%). it is more common among girls (16.70%) than in boys (1.82%), and more frequent in 6th class grade (11.44%) and ≥12 years old children (12.77%). the rate of infestation was almost equal among pupils who live in urban and suburban areas. parents’ illiteracy, housewife mother, crowded families, and history of infestation were considered as risk factors. for successful treatment of pediculosis, it is necessary to raise the awareness of the parents, teachers, and schools regarding the phenomenon through an educational program. acknowledgements the authors would like to thank the general directorate of education and directorate of preventive health in sulaimani for their encouragement and support. we would like to thank the nurses (school health staff in health centers) who helped us in the examination of the children and especially to mr hadi mohammad. special thanks to the teachers at the schools who were involved in contacting the pupils during the data collection. ethical considerations this study was reviewed and approved by the ethical committee of technical college of health, sulaimani polytechnic university (no. ch00026). the official permission was obtained from the general directorate of health and general directorate education, principals of the schools, class teachers, and social workers. the signed letters of informed consent before the study were obtained from the pupil’s parents. all pupils had the right not to participate in the study. conflict of interest statement the authors declare that no 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kokturk a, baz k, bugdayci r, sasmaz t, tursen u, kaya ti, ikizoglu g (2003) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2022, 16(1): 72–83 yh hama-karim et al.: epidemiological study of … 83 http://jad.tums.ac.ir published online: march 31, 2022 the prevalence of pediculosis capitis in school children in mersin, turkey. int j dermatol. 42(9): 694–698. 38. karakuş m, arici a, töz sö, özbel y (2014) prevalence of head lice in two socio-economically different schools in the center of izmir city, turkey. turkiye parazitol derg. 38(1): 32–36. 39. djohan v, angora ke, miezan s, bédia ak, konaté a, vanga-bosson ah, kassi fk, kiki-barro pcm, yavo w, menan ei (2020) pediculosis capitis in abidjan, côte d’ivoire: epidemiological profile and associated risk factors. parasite epidemiol control. 11: e00159. 40. nordin rb, che wa, ibrahim na, naing l (2006) prevalence and associated factors of head lice infestation among primary school children in kelantan, malaysia. southeast asian j trop med public health. 37(3): 536–543. 41. jones kn, english jc iii (2003) review of common therapeutic options in the united states for the treatment of pediculosis capitis. clin infect dis. 36(11): 1355– 1361. 42. gunning k, pippitt k, kiraly b, sayler m (2012) pediculosis and scabies: a treatment update. am fam physician. 86(6): 535–541. 43. feldmeier h (2014) diagnosis of head lice infestations: an evidence-based review. open dermatol j. 4(1): 69–71. 44. kassiri h, esteghali e (2016) prevalence rate and risk factors of pediculus capitis among primary school children in iran. arch pediatr. 4(1): e26390. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2016, 10(1): 87–91 s mohapatra et al.: a comparative study … 87 short communication a comparative study of serum, urine and saliva using rk39 strip for the diagnosis of visceral leishmaniasis *sarita mohapatra, jyotish c samantaray, arnab ghosh department of microbiology, all india institute of medical sciences, new delhi, india (received 12 nov 2013; accepted 1 july 2014) abstract background: immunochromatographic based rk39 antibody detection test became popular for the diagnosis of visceral leishmaiasis (vl) because of high sensitivity, rapidity, easy to interpret, and cost effectiveness. however, false positive result after complete cure of the patients is the major limitation with this test. the aim of the study to access the usefulness of non-invasive samples i.e. urine and saliva by rk39 test for the diagnosis of visceral leishmaniasis in comparison to serum. methods: seventy two clinically suspected vl patients were enrolled in the study among which 61 cases were confirmed as vl and 11 cases were included in the control group. serum, urine, and saliva samples of all the cases were tested for rk39 dip stick test. results: urine and saliva both were equally sensitive as serum for the diagnosis kala-azar. in the control group, rk39 antibody test was negative in 10 cases out 11 (91%) with saliva in comparison to 4 cases with serum (36%), thereby found to be more specific. conclusion: saliva sample found to be highly reliable for the diagnosis of vl cases by rk39 test. the test with saliva sample showed less false positive result in comparison to serum sample, thereby can be used an adjunct with serum sample for the diagnosis of kala-azar in endemic areas. keywords: visceral leishmaniasis, noninvasive samples, rk39 test introduction visceral leishmanisis (vl) is a protozoal disease manifested as prolonged fever, hepatospleenomegaly, anorexia, and weight loss. it shows a spectrum of epidemiological diversity: leishmania donovani is the causative agent of kala-azar in east africa and indian subcontinent, whereas l. infantum is found in latin america and the mediterranean basin (haldhar et al. 2011). timely and accurate diagnosis of the condition is needed for proper treatment. direct detection of amastigote forms in splenic or bone marrow aspirate is taken as the gold standard for diagnosis of vl, but these methods are invasive, carry high degree of risk for the patient and require technical expertise to detect. recently, rk39 dip stick test has gained popularity because of high sensitivity, specificity, rapidity, and easy to use (sundar et al. 1998). this can be used as field test for diagnosis of vl cases in kala-azar endemic areas (khan et al. 2010). but, the major limitation of the test is false positive result in cured patients due to persistence of antibody. urine and sputum samples from kala-azar patients proved as useful as serum for the diagnosis of vl by variety of diagnostic tests (singh et al. 2009). however, the role of saliva is not established in the diagnosis of vl patients. in this study, we compared serum, urine, and saliva samples of clinically diagnosed vl cases by rk39 dip stick test for the diagnosis of vl and also followed up to evaluate their utility in the prognosis of patients. *corresponding author: dr sarita mohapatra, e -mail: saritarath2005@yahoo.co.in http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 87–91 s mohapatra et al.: a comparative study … 88 materials and methods as per who, a vl case is defined as a person from an endemic region with fever for more than 2 weeks, spleenomegaly and confirmed positive by either rapid diagnostic test based on rk39 antigen or biopsy (huda mm et al. 2012). a total of 72 clinically suspected vl patients were enrolled in the study from 2011 to 2012 within a period of 2 year. ethical clearance for the study was not required as the samples were referred to the microbiology laboratory for the diagnostic purposes as a public health measure. sixty one out of these 72 patients were diagnosed as vl as per the above definition and included in the test group. rest 11 cases were included in the control group (n= 11), which consisted of pyrexia of unknown origin (puo) (n= 1), post kala-azar dermal leishmaniasis (pkdl) (n= 3), past history of kala-azar (n= 2), mycosis fungoidosis (n= 1), malaria (n= 3), and filaria (n= 2). serum, urine and saliva samples were collected from all patients (cases and controls) and tested for rk39dip stick test. detailed clinical history of the patients such as fever, hepatospleenomegaly, residence, weight loss, loss of appetite, complete blood count, albumin: globulin ratio, liver function test, and past history of kala-azar were obtained from all patients. serum sample was tested for rk39 test (kalaazar detecttm rapid test, inbios international, usa) as per the instruction of the kit. urine and saliva samples were tested with rk39 strip test following the similar procedure. only 11 patients out of 61 vl confirmed cases in the test group could be followed up to 3–4 months after completion of treatment and rk39 test with serum, urine and saliva samples were repeated. all the rk39 test strips were graded from 3+ (equal to or more than control) to 1+ (faint) depending on the intensity of test band in comparison with control band. statistical analysis could not be done because of small sample size. results all patients in the test group (n= 61) presented with fever for more than 2 weeks and hepatospleenomegaly during the first visit to the hospital. on examination, features like pancytopenia, anorexia, weight loss, hypoproteinemia, and reversal of albumin: globulin ratio was observed less frequently in comparison to the previous one. rk39 dip stick test with serum sample was positive in all the patients (100%) of test group (table 1). urine showed a complete concordance with serum exhibiting 100 % positivity in all the patients of the test group, whereas only 51 patients (83.6%) were found positive with saliva sample. eleven patients from the test group were followed up after the completion of treatment and tested with serum, urine and saliva samples to observe the response of treatment. all the cases in the follow up group showed positive result with serum and urine samples in comparison to 10 cases with saliva. we graded the intensity of the test band of the rk39 strip of these 11 patients before and after treatment as it indirectly correlates the disease status (table 2). the test band intensity with serum sample before and after therapy obtained to be same among these patients. the band intensity found to be reduced with the urine and saliva samples in 4 cases after completion of therapy (table 2). among the control patients (n= 11), the rk39 test with serum, urine and saliva sample found positive in 7 (63.6%), 2 (18%), and 1 (9%) case respectively (table 3). http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 87–91 s mohapatra et al.: a comparative study … 89 table 1. comparison of rk39 test with serum, urine, and saliva samples in kalaazar patients and the control group kalaazar pts. (n= 61) f/up cases (n= 11) control (n= 11) serum positive n (%) 61 (100) 11(100) 4(37) urine positive n (%) 61 (100) 11(100) 2(18) saliva positive n (%) 51 (83.6) 10(91) 1(9) table 2. gradation of test band intensity rk39 strips in the kalaazar patients before and after treatment pts. sl. no. serum serum f/up urine urine f/up saliva saliva f/up 1. 4+ 4+ 4+ 4+ 4+ 4+ 2. 3+ 1+ 1+ 1+ 1+ 1+ 3. 4+ 4+ 4+ 2+ 4+ 2+ 4. 4+ 3+ 3+ 1+ 1+ negative 5. 3+ 4+ 3+ 4+ 3+ 3+ 6. 4+ 4+ 4+ 1+ 4+ 2+ 7. 4+ 4+ 2+ 2+ 3+ 1+ 8. 4+ 4+ 4+ 4+ 4+ 4+ 9. 4+ 4+ 4+ 2+ 4+ 4+ 10. 4+ 4+ 4+ 4+ 4+ 3+ 11. 3+ 2+ 3+ 3+ 3+ 1+ *1+ and 2+: band intensity weaker than control, 3+: band intensity matches with control, 4+: band intensity stronger than control table 3. comparison of rk39 test with serum, urine, and saliva samples in control group serum urine saliva pkdl 1. 4+ neg neg 2. 1+ neg neg 3. 1+ neg neg past h/o kalaazar 1. 3+ neg neg 2. 1+ 1+ neg pyrexia of unknown origin 2+ neg neg mycosis fungoidosis 3+ 2+ 2+ malaria 1. neg neg neg 2. neg neg neg filaria 1. neg neg neg 2. neg neg neg discussion this study was designed to detect the performance of non-invasive samples for the diagnosis and prognosis of vl patients. it is known that the test band intensity of the ict strip correlates with the course of many diseases (moody 2002, sako et al. 2011). the intensity is maximum during the active phase of the disease and gradually fades after treatment. in our study, urine was more useful in comparison to saliva for the diaghttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 87–91 s mohapatra et al.: a comparative study … 90 nosis of kala-azar in the active stage of disease. during the first visit, the result of rk39 test with urine sample showed complete concordance with that of the serum sample. however, saliva was positive in 84 % cases. the urine and saliva samples showed similar results as with serum samples in follow up patients. it shows that, urine and saliva do not have much role in the determination of prognosis of vl patients. but, in the control group, the rk39 test with the saliva sample was found negative in majority (10/11, 91%) of cases in comparison to serum (4/11, 36.4 %). the test was also observed to be negative in majority of cases with urine sample. conditions such as past history of kalaazar, post kala-azar dermal leishmaniasis showed false positive result with serum sample in the control group. all these conditions found to be negative with saliva. the sensitivity and specificity of rk39 test is reported differently in different literatures. singh et al. found sputum samples to be highly specific for vl patients (2009). many literatures discussed about the false positive result of urine sample for the diagnosis of vl (khan et al. 2010). but, in this study the false positive results by urine sample was much less. the limitation of the study is that, it is comprised of less number of patients in the control group and also did not include the samples of healthy persons from endemic area. however, the results were encouraging. non-invasive samples in endemic area can be used as an adjunct to the serum samples to decrease the rate of false positivity. urine and saliva samples can also be used as part of surveillance in countries where animals behave as reservoir and help to control vl (taran et al. 2007). the role of non-invasive samples in the prognosis of the patients is also difficult to establish because of small sample size in the follow up group. to overcome these limitations and to determine their role, further studies in field with a large sample size is required. conclusion the present data highlights the usefulness of non-invasive samples such as urine and saliva as the diagnostic tools for the patients of kala-azar particularly in endemic regions. they were equally good with regard to as serum samples tested by rk39 strip. these samples can be collected easily in a non-invasive manner. hence, rk39dip stick test with noninvasive samples such as urine and saliva may be adapted in routine diagnostics along with serum samples in endemic areas for the diagnosis of vl conditions. this may reduce the rate of false positivity with serum samples. acknowledgements the authors declare that there is no conflict of interests. references khan mgm, alam ms, podder mp, jamil km, haque r, wagatsuma y (2010) evaluation of rk39 strip test using urine for diagnosis of visceral leishmanisis in an endemic area in bangladesh. parasit vectors. 3: 114. haldhar ak, sen p, roy s (2011) use of antimony in the treatment of leishmaniasis: current status and future direction. mol biol int. article id 571242, 23 pages, doi:10.406/2011/571242. huda mm, hirve s, siddique na, malaviya p, banjara mr, das p, kansal s, gurung ck, naznin e, rijal s, arana b, kroeger a, mondal d (2012) active case detection in national visceral leishmaniasis in bangladesh, india and nepal: feasibility, performance and cost. bmc public health.12: 1001. moody a (2002) rapid diagnostic tests for malarial parasites. clin microb rev. 15(1): 66–78. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 87–91 s mohapatra et al.: a comparative study … 91 sundar s, reed sg, singhvp, kumar pc, murray hw (1998) rapid accurate field diagnosis of indian visceral leishmaniasis. lancet. 351(9102): 563–565. singh d, pandey k, dsa vn, das s, kumar s, tapno rk, das p (2009) novel noninvasive method for diagnosis of visceral leishmaniasis by rk39 testing of sputum samples. j clin microbiol. 47(8): 2684–2685. sako y, tappe d, fukuda k, kobayashi y, itoh s, frosch m, gruner b, kern p, ito a (2011) immunochromatographic test with recombinant em18 antigen for the follow-up study of alveolar echinococcosis clin vaccine immunol. 18(8): 1302–1305. taran m, mohebali m, modaresi mh, mamishi s, mahmaudi m, mojarad m (2007) diagnosis of canine visceral leishmaniasis by elisa using k39 sub recombinant antigen. iran j publ health. 36(2): 1–6. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 original article mtdna cytb structure of rhombomys opimus (rodentia: gerbellidae), the main reservoir of cutaneous leishmaniasis in the borderline of iran-turkmenistan hasan bakhshi 1, *mohammad ali oshaghi 1, mohammad reza abai 1, yavar rassi 1, amir ahmad akhavan 1, mehdi mohebali 2, homa hajaran 2, fatemeh mohtarami 1, hossein mirzajani 3, naseh maleki-ravasan 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical parasitology, school of public health, tehran university of medical sciences, tehran, iran 3sarakhs health center, sarakhs, razavi khorasan province, iran (received 3 feb 2012; accepted 18 aug 2013) abstract background: great gerbils, rhombomys opimus, are the main reservoir host of zoonootic cutaneous leishmaniasis (zcl) in iran and neighboring countries. based on morphological traits two subspecies r. opimus sodalis and r. opimus sargadensis have reported in the country. however, variation in infection rate and signs to leishmania parasites, phenotype, size, and sexual polymorphisms demand more details to elucidate clearly the role of great gerbils in zcl epidemiology. methods: pcr-rflp and pcr-direct sequencing were used to analyze mitochondrial dna cytochrome b (mtdnacytb) gene structure of r. opimus collected from golestan and khorasan-e-razavi provinces in 2011 that are neighbor to turkmenistan country where zcl is endemic in both sides of the borderline. results: all of the specimens (n= 61) were morphologically or genetically similar to the typical r. opimus sodalis. however, there were 9 (1.5%) dna substitutions throughout the 583 bp of the cyt b gene of the samples sequenced comprising six dna haplotypes. maximum likelihood or neighbor joining phylogenetic trees inferred from the sequences could resolve the populations according to their subspecies as well as geographical origins. discussion: the dna polymorphisms in the great gerbils may correspond to the signs and infection rate in the animal. however, further studies are needed to match these six haplotypes with different signs and parasite sustaining following infection with l. major in the great gerbils. keywords: leishmaniasis, zcl, rombomys opimus, mtdna cytb, iran introduction leishmaniasis is considered an emerging and re-emergent disease, with an increase in its incidence in the last decades (reithinger et al. 2007). it has a global estimated prevalence of 12 million cases, with an estimative of 1.5–2 million new cases each year. at the present moment leishmaniasis occurs in 88 countries throughout europe, africa, asia and america, and 350 million people are at risk of contracting the disease (ameen 2010, who 2012). ninety percent of annual cases are reported from afghanistan, brazil, iran, peru, saudi arabia, syria, algeria and sudan (desjeux 2004). zoonotic coutaneous leishmaniasis (zcl) due to leishmania major is one of the infectious increasing diseases in iran where it almost doubled (from 11505 to 22705 cases) over a nine-year period in 2001 *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir 173 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 to 2009 (imhme 2010, oshaghi et al. 2010). leishmaniasis is endemic in turkmenistan which has a long borderline in northeast of iran, and over the years 2000–2009, it has been reported 1,562 cases of cl, mostly at the southern territories bordering iran (who 2009). some of the most important causes of this upward trend are the movement of human populations toward the reservoir as well as vector habitats and the development of irrigation project. the great gerbils, rhombomys opimus, is known to be the most important reservoir host of l. major, being transmitted by sand flies of the genus phlebotomus (yaghoobiershadi et al. 1996, yaghoobi-ershadi and javadian 1996, mohebali et al. 2004, rassi et al. 2008a, 2011, akhavan et al. 2010a,b,c, oshaghi et al. 2008 and 2010). this rodent is distributed both in the arid and semi-arid regions throughout central and south asia from kazakhstan, kyrgyzstan, turkmenistan, uzbekistan, tajikistan, iran, pakistan, afghanistan, to southern mongolia, and north-western china (sosnina 1979, mallon 1985, strelkova et al. 2001, molur et al. 2005, smith and xie 2008, shar et al. 2009, abai et al. 2010). in iran, it is widely distributed in central and northeast parts of the country (yaghoobiershadi and javadian 1996, rassi et al. 2008b, akhavan et al. 2010a,b,c, oshaghi et al. 2011). the rate of infection of r. opimus to l. major is normally high and may reach to 92.5% at endemic areas (rassi et al. 2008a). the parasite persists in the great gerbils for up to 25 months (strelkova et al. 2001). they are also known to carry the germs of bubonic plague (davis et al. 2004, zhang and dai 2008) as well as being an agricultural pest (nowak 1999). the great gerbils occupy desert to semidesert habitats, and are associated with succulent plants of family chenopodiaceae (climacoptera spp., salsola spp., suaeda spp.) (dubrovsky 1975, rogovin et al. 2003). these saline ecological biotopes are discontinuous in distribution in center, north, and northwest of iran, thus leading to a fragmentation of the populations of great gerbils. based on morphometric characteristics and phenotypic differences, four subspecies namely r. opimus opimus, r. opimus furmicolor, r. opimus sargadensis, and r. opimus sodalis have been reported within the species (goodwin 1940, etemad 1978). two subspecies of r. opimus sargadensis and r. opimus sodalis occur in iran (abai et al. 2010). r. opimus sodalis is bigger than r. opimus sargadensis. this subspecies is brown to hazelnut color, whereas r. opimus sargadensis has a yellowish color. these phenotypic variations are related to the wide geographic distribution of the great gerbils. additionally, sexual dimorphism and variation in the size of different developmental stages may make the diagnosis of the subspecies more challenging. accurate identification of reservoir host species is important in the epidemiology and control of zoonotic diseases such as zcl (meerburg et al. 2009, sakthianandeswaren et al. 2009). although great gerbils have a fundamental role in the epidemiology of zcl, not many studies have been focused on the relationship of r. opimus population structure and zcl prevalence or severity of the disease. it is shown a considerable variation in infection rate and signs to leishmania parasites. for example the rate of leishmania infection and its signs ranged from 11– 92.5% and asymptotic to scar respectively among different r. opimus populations in iran (yaghoobi-ershadi et al. 1996, mohebali et al. 2004, rassi et al. 2008a, akhavan et al. 2010b,c). in another study, it was shown that exposure of r. opimus populations to l. major resulted a wide range of reactions, out of 194 gerbils examined, l. major and l. turranica were isolated from 80 (41.23%) of the animals, 55 (68.75%) of the infected animals showed no skin lesion (asymptomatic), and only 25 (31.25%) of the animals showed skin lesions and even the rate of parasitemia 174 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 was not uniform (hajjaran et al. 2013). this seems to be due to various physiological and genetic traits that affect the host-parasite interaction. therefore, studying the genetic structure of different r. opimus populations is of fundamental importance in zcl epidemiological investigation. at least two major lineages (subspecies) exist within the species of r. opimus in iran and analysis of the mitochondrial cytochrome b (mtdna-cytb) gene, revealed a range of 1–10% genetic variation among the populations of seven districts belonging to five provinces in central and north-eastern iran (oshaghi et al. 2011). in the current study, we aimed to characterize the mtdna-cyb gene sequences of three great gerbil populations distributed in northeastern iran close to the border line with turkmenistan where zcl is endemic. materials and methods study area and rodent collection this study was conducted in three districts of northeastern part of iran including sarakhs of khorassan-e-razavi province, and gonbad-e qabus and maraveh-tappeh from golestan province in 2011. details of collection sites are shown in table 1. these districts respectively are located in the northern and far northeastern parts of the borderline of iran-turkmenistan (fig. 1). animal collections were performed using sherman live-trap (30cm× 15cm× 15cm wire mesh) baited with a mixture of walnut, cucumber, tomato, and bread dabbed with sunflower oil. approximately 30–40 live traps were used per night at each location in monthly collections. the traps were set close to burrow entrance 2–3 h before the dusk and were checked in the next morning after sunrise. the captured animals were transported to the laboratory, anesthetized using a mixture of 150mg/kg ketamine 10% and 15mg/kg xylazine 2% before 200–1000μl of blood was taken from the tail vein in tubes containing 20–30μl anticoagulants (heparin, edta, 0.29 m) and stored at -20 °c until use. we also used dbc (dna banking card) to store blood samples of the rodents. blood sample (50μl) were spotted directly on separate dbc cards and dried at room temperature as explained previously by karimian et al. (2011). morphological measurements were also recorded while the animals were anesthetized. all experiments on the rodents were performed in accord with the guidelines of the ethical board of tehran university of medical sciences, iran. species and subspecies identification the genus and species of the rodents were determined by external morphological characteristics including color, grooves on the incisor teeth, length of ears, tail, hind feet, head, body, and skull (ziaei 1996). presence of two fine grooves on the incisor teeth is the most important characteristics for r. opimus identification. the specimens with hazel-nut color, body length ranging from 300 to 320mm and a tail length of 135 to 140mm, and hind feet length of 43 to 45mm were considered to be r. opimus sodalis subspecies. specimens with brown to yellowish color, body length ranging from 200 to 250mm and a tail length of 100 to 130mm, were determined to be r. opimus sargadensis subspecies (abai et al. 2010). mtdna cytb pcr and sequencing one mm diameter disc of each dbc card containing dried blood sample were punched and then dna was extracted according to the manufacturer's guide (karimian et al. 2011) and used for pcr amplification. we also extracted dna from the blood samples (200μl blood per animal) using g-spin blood and tissue dna kit (bioneer, south korea) according to the manufacturer's instructions. a 624bp fragment of the cytochrome b gene (cytb) of the mitochondrial (mt) dna were 175 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 amplified using the protocol of (malekiravasan et al. 2009) and the primers described by kent and norris (kent and norris 2005). they primers used were unfor403 (5′-t gaggacaaatatcattctgagg-3′) and unrev1025 (5′-ggttgtcctccaattca tgtta-3′). the pcr amplification was performed in 25μl reaction mixtures containing 10mm tris, ph 8.3, 50mm kcl, 1.5mm mg cl2, 0.01% gelatin, 1.0mm deoxynucleotide triphosphates, 1.25 units of taq polymerase, 50pmol of each primer, and 5μl of the extracted dna or and one mm disk of dbc card. an initial denaturation step at 95 °c for 5min was applied prior to 35 cycles of denaturation for 1 min at 95 °c, hybridization for 1 min at 58 °c and extension for 1 min at 72 °c followed by a final extension at 72 °c for 7min. some dna of r. opimus sodalis (oshaghi et al. 2011) were prepared from the insect molecular biology lab at the school of public health and used as positive controls in pcr and pcr-rflp analysis. pcr products were analyzed by electrophoresis on 1% agarose gel stained with ethidium bromide. a subset of pcr products, representatives of populations of the three districts were selected for sequencing (table 1). sequencing was performed using an abi 3730 sequencer machine by seqlab (göttingen, germany). sequences were checked to correct ambiguities. homologies with the available sequence data in genbank was checked using basic local alignment search tool (blast) analysis software (www.ncbi.nlm.nih.gov/ blast). the cytb gene sequences of the great gerbil r. opimus available in genbank database (table 1) were retrieved and used as reference to generate multiple alignments using the clustal w software (higgins and sharp 1988) and phylogenetic analysis. pcr-rflp and phylogenetic analysis the sequences of the cytb gene of the great gerbils obtained in this study were checked by the specific restriction enzymes previously introduced by (oshaghi et al. 2011). hinfi restriction enzymes (re) could identify subspecies of r. opimus sodalis from r. opimus sargadensis. digestions were performed in 25μl mixtures containing 15μl of pcr product mixed with 2.5μl of the enzyme buffer and 5 units of hinfi re overlaid with two drops of mineral oil. the mixture was incubated at the temperature recommended by the enzyme suppliers. an aliquot (14μl) of the digestion product was mixed with 6μl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol), and electrophoresed in a 2.5% agarose gel. the gels were stained with ethidium bromide (2mg/ml) and the rflp profiles were visualized under ultraviolet light. phylogenetic analysis was performed by comparing the sequences obtained in this study plus the ones were available in genbak for r. opimus cytb gene. neighbor joining (nj) and maximum parsimony (mp) trees were constructed by the algorithm in mega 4.0 (tamura et al. 2007). robustness of the phylogenetic trees was tested with bootstrapping value. cytb sequence of meriones lybicus (ab381902) was acquired from genbank and used as an out group in this study (table 1). results rhombomys opimus subspecies a total of 61 great gerbil specimens including 25 females and 36 males were collected in this study. details of the captured specimens are shown in table 1. approximately 68 percent (n= 38) of the rodents were caught from sarakhs district. field observation revealed that number of rodent burrows in gonbadli village was twice as other villages. the genus and species of the rodents were determined by testing external morphological characteristics already explained in m and m section. comparison between the populations was performed for speci176 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 mens of the same sex. according to the classification mentioned above, we could find just one subspecies r. opimus sodalis in the study area. all of the specimens had a color like hazel-nut and the range of body length was 120–180mm without calculating tail length. the average of the body length without calculating the tail was 163mm. this is important that some of these rodents may not be adult and so their body length may be shorter than adult ones. weights of these rodents were ranged from 112 to 269 grams and the average weight was 176 grams. cytb sequence and pcr-rflp the sequences of mtdna cytb gene of all 61 r. opimus specimens captured in this study were analyzed by either sequencing or pcr-rflp assays. the pcr amplicons of all specimens showed a single band of the expected size (624bp) on the agarose gel. eleven specimens, as representatives of the populations or villages (table 1) were selected for sequencing. almost 583–586bp of results of sequencing were trustable for analysis. we found six mitochondrial cytb haplotypes among the specimens sequenced (table 2). when we added other r. opimus sodalis sequences available in genbank, totally eight haplotypes were found in the subspecies (table 2). there was a strong correlation between the haplotypes and their geographical origins. generally, the sequences were more or less similar to the sequences of typical r. opimus sodalis (accession nos: fj648772, fj648775, and aj430556) previously reported in iran. however, there were 9 (1.5%) dna substitutions throughout the 583bp of the cyt b gene of the samples sequenced. when other r. opimus sodalis sequences added to the analysis the number of substitutions rose to 12. most of the nucleotide substitutions (92%) were transition and, except one amino acid (aa) substitution, there were no aa substitution throughout the 194 base of aa in the cytb region of the populations. hinfi restriction enzyme showed three bands of 284, 212, and 128bp which is a specific rflp profile for r. opimus sodalis for all of the 61 specimens as well as the r. opimus sodalis from kalaleh population as positive control (fig. 2). phylogenetic analysis tree reconstruction on the combination of data obtained in this study plus those from genbank using neighbor-joining (nj) and maximum parsimony (mp) methods revealed similar topology for the r. opimus populations or subspecies. the trees showed two main clades resolving very well the r. opimus sodalis from r. opimus sargadensis (fig. 3). specimens of gonbad-e-qabus, maravehtappeh, and sarakhs districts were associated with the specimen of kalaleh district and the ones already known as r. opimus sodalis, altogether grouped in one clade whereas the second group included the r. opimus sargadensis (fig. 3). within the clade of r. opimus sodalis three major branches were observed, two of them contained specimens of golestan and the other one included the saraks specimens. the specimens of golestan were scattered in two distinct subclades. sarakhs specimens were positioned in between golestan specimens. except three cases, the bootstrap values of the nodes were more than 90 percent indicating high confidence in tree topology and well resolutions between the populations or subspecies. 177 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 fig. 1. study areas and the location where rhombomys opimus specimens were collected. 1, sangar, 2, gonbadli, 3, sozesh, 4, ghareh gol, 5, dashli-borun, and 6, fadavi table 1. details of rhombomys opimus specimens collected from the area study close to borderline of iranturkmenistan subspecies province (country) county village(s) no. latitude– longitude genbank a.n. r. opimus sodalis golestan (iran) gonbad-e-qabus dashli-borun, fadavi 5 6 37.3–54.5 37. 3–54.4 jx412206 jx412213-4 r. opimus sodalis golestan (iran) maraveh-tappeh ghare gol, sozesh 6 6 37.5–55.3 37.5–55.4 jx412207 jx412212 r. opimus sodalis razavi khorasan (iran) sarakhs gonbadli, sangar 22 16 36.2–60.5 36.1–61.1 jx412210-1 jx412215-6 jx412208-9 r. opimus sodalis golestan (iran) kalaleh qareh gol gharbi 37.9–55.7 fj648772* r. opimus sodalist/ sargadensis hybrid northernkhorasan (iran) kalaleh qareh gol gharbi 37.9–55.7 fj648775* r. opimus ni iran ni ni ni aj430556* r. opimus sargadensis northernkhorasan (iran) esfarayen kalateh-shur 37.0–57.1 fj648773* r. opimus sargadensis northernkhorasan (iran) shirvan hossein-abad 37.2–57.7 fj648774* r. opimus sargadensis semnan (iran) shahrood ahmadabad mayamey 35.4–52.2 36.4–55.6 fj648771* fj648770* r. opimus sargadensis semnan (iran) damghan soltanieh 36.0–54.3 fj648769* r. opimus sargadensis esfahan (iran) kashan badrood 32.6–52.0 fj648768* r. opimus sargadensis esfahan (iran) habibabad habibabad 33.3–52.5 fj648767* meriones lybicus xinjiang (china) uygur turfan 42.5–89.1 ab38190* the asterisk (*) show the specimens obtained from genbank database. ni: not indicated 178 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 fig. 2. pcr-rflp profiles (284, 212, and 128 bp) of cytb gene for rhombomys opimus sodalis digested by hinfi re. all the specimens were identified as r. opimus sodalis. m: 100 bp ladder (sinaclone, iran). specimens in lanes 1–2: sangar (sarakhs), 3–4: dashli-borun (gonbad-e-qabus), 5: reference strain of r. opimus sodalis from kalaleh (gorgan), 6–7: gonbadli (sarakhs), 8: ghare-gol (maraveh-tappeh), 9: sozesh (maraveh-tappeh), and 10–11 fadavi (gonbad-eqabus) fig. 3. phylogenetic relationship between rhomomys opimus subspecies/populations of iran based on sequence analysis of 583 bp mtdnacytb gene. sequences this study (jx412206-16) was combined with the available sequences from the genbank database (table 1). species of meriones lybicus (ab381902, ito, direct submission) was used as an outgroup. *is a hybrid specimen of r. opimus sodalis from golestan (female) with r. opimus sargadensis (male) from shahrood which showed maternal sodalis haplotype. **was a specimen migrated from golestan to shahrood (oshaghi et al. 2011). genetic distance scale is shown underneath 179 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 table 2. dna sequence comparison of about 583 bp of cytb gene of rhombomys opimus sodalis populations distributed in northeastern iran origin haplotype accession number position in the pcr product 0 0 1 1 2 3 3 3 4 4 4 5 6 8 5 6 6 4 6 8 2 6 7 4 3 1 6 9 7 0 5 4 3 5 7 9 gonbadli, sarakhs gonbadli, sarakhs gonbadli, sarakhs fadavi, golestan ghare gol, golestan gonbadli, sarakhs dashli-borun, golestan sangar, sarakhs sangar, sarakhs sozesh, golestan fadavi, golestan kalaleh, golestan shahrood/kalaleh iran i i i ii ii iii iv iv iv v vi vii viii ix jx412210 jx412211 jx412215 jx412214 jx412207 jx412216 jx412206 jx412209 jx412208 jx412213 jx412212 fj648772 fj648775 aj430556 c g t c c t c c c c a a . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . c . . . . . . . . . . . c . t . . . . . . . . a c . . . . . . . . . . a c . . . . . . . . . . a c . . . . . . . . . . . c t . . . t t . g . t . c . . . t a t . g . # . c . . g t t t . g . . . c . . . . . . t . g . a c . . . . . . . . . dots show identical sequences to the top sequence. three last row sequences derived from genbank (accession no. fj648772 and fj648775, oshaghi et al. 2011, aj430556, chevre 2005: direct submission). #: not indicated discussion in this study, a combination of morphological and molecular (pcr-rflp, pcrdirect sequencing, phylogenetic topology) characteristics were used to reveal taxonomic situation and genetic structure of the r. opimus populations scattered in the north and northeast of iran where zcl is endemic and the rodents act as the main reservoir host of the disease. this investigation revealed the presence of six haplotypes, all were of r. opimus sodalis subspecies in the study area. this finding is in agreement with the previous report indicating the presence of r. opimus sodalis in kalaleh district, an adjacent area in golestan province in north of iran (oshaghi et al. 2011). we have no access to the populations of great gerbils of turkmenistan on the other side of border line with iran; however, due to presence of similar ecological niches in those areas we suggest the presence of similar subspecies or haplotype in turkmenistan. we suggest testing the genetic structure of great gerbils which is known as the main host of leishmania parasites causing zcl in the neighboring country. all of the areas investigated in this research comprising gonbad-e-qabus, maravehtappeh, and sarakhs districts plus kalaleh district are located in northern slopes of alborz mountain chain (amc) (fig. 1). the amc in north of iran acts as a natural geographical barrier between two main subspecies and seems to be the southern limit of r. opimus sodalis in the region. this natural barrier between r. opimus sargadensis and r. opimus sodalis resulted in accumulation of mutations, independent evolution, and fragmentation between the great gerbil populations/ subspecies. considering presence of 2–10 percent intra-specific genetic variation among the populations (oshaghi et al. 2011) 180 j arthropod-borne dis, december 2013, 7(2): 173–184 h bakhshi: mtdna cytb structure … http://jad.tums.ac.ir published online: august 31, 2013 may lead to speciation in future if gene flow does not occur between them. phylogenetic analysis revealed good resolution between the populations as well as subspecies. however, presence of high rate of sequence homology between a few specimens originated from two distinct areas (sarakhs and golestan) suggested possible transportation via vehicles (train, bus, and car) or natural migration from sarakhs to golestan province and vise versa. the distance between these two locations is about 500km, however the areas in between are plain and there is no significant natural barrier to obstruct merging these populations and transportation could have been happened easily throughout the time by this active animal. this observable fact can also be explained by present of a common ancestor in those regions. this phenomenon also has been observed in the previous study indicating possible migration of r. opimus sodalis from kalaeh to the territory of r. opimus sargadensis in shahrood district (oshaghi et al. 2011). different populations of great gerbils have different rate of leishmania infection which could affect their ability to sustain l. major (yaghoobiershadi et al. 1996, mohebali et al. 2004, rassi et al. 2008a,b, akhavan et al. 2010b). leishmaniasis clinical manifestation depends upon leishmania species and host genetic background which governs generation of type of immune response (mohamed et al. 2003, salhi et al. 2008, sakthianandeswaren et al. 2009 and 2010, castellucci et al. 2012, moravej et al. 2012). variation in maintaining pathogens such as plague bacilli or rift valley fever virus between populations of a given rodent species have been reported in literature (korobitsyna 1974, arntzen et al. 1991, gora et al. 2000, mills 2005). in this study we have tested the mtdna cytb gene between great gerbil populations and found 1.5% variation between them. however, supplementary studies are needed to assess whether or not these six haplotypes are correlated to various outcoms (symtomatic to asymtomatic) and to the ability l. major parasite to infect and to remain within the great gerbils. also it is suggested testing other genes such as tnf-alpha, tnf-beta, il-4, il-10 and ifnγ among r. opimus populations. these genes could influence the host-pathogen associations and could determine the severity of the disease upon infection with l. major (kamali-sarvestani et al. 2006, salhi et al. 2008, sakthianandeswaren et al. 2009 and references herein). these studies could be used to develop accurate ecological control strategies and provide important step towards deeper understanding of zcl epidemiology particularly the host-pathogen associations among r. opimus populations. acknowledgements this research was supported by research deputy of tehran university of medical sciences. we are very grateful to mr naroyi and mr koohkan, the staff of sangar, gonbadli, nobonyad and sarakhs county health center for their helps in the field. we are also thanks mr a ataei, b shiravand, m 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otarod 3, amanollah fathnia 4, ali ahmadabadi 5, mohammad h. fallah 6, alireza zavareh 1, nargess miandehi 1, benoit durand 7, philippe sabatier 2 1viral vaccines production department, pasteur institute, tehran, iran 2timc-imag team epsp, vetagrosup, campus vétérinaire de lyon, france 3quarantine and biosafety directorate general, iran veterinary organization, tehran, iran 4department of geography, razi university, kermanshah, iran 5faculty of geography, kharazmi university, tehran, iran 6department of poultry viral diseases, razi vaccine and serum research institute, alborz, iran 7university paris est, anses, laboratory of animal health, epidemiology unit, maisons-alfort, france (received 31 may 2014; accepted 17 dec 2014) abstract background: geographic distribution of west nile virus (wnv) is heterogeneous in iran by a high circulation in the southern-western areas. the objective of our study was to determine environmental and climatic factors associated with the risk of wnv equine seropositivity in iran. methods: serological data were obtained from a serosurvey conducted in equine population in 260 districts in iran. the climate and environmental parameters included in the models were distance to the nearest wetland area, type of stable, normalized difference vegetation index (ndvi), annual mean temperature, humidity and precipitation. results: the important risk factors included annual mean temperature, distance to wetlands, local and seasonal ndvi differences. the effect of local ndvi differences in spring was particularly notable. this was a normalized difference of average ndvi between two areas: a 5 km radius area centered on the stable and the 5–10 km surrounding area. conclusion: the model indicated that local ndvi’s contrast during spring is a major risk factor of the transmission of west-nile virus in iran. this so-called oasis effect consistent with the seasonal production of vegetation in spring, and is associated to the attractiveness of the local ndvi environment for wnv vectors and hosts. keywords: west nile virus, environment, climate, ndvi, wetland introduction west nile virus (wnv) is not restricted by international borders and involves all continents, revealing adaptation of the virus with different ecological zones. mosquitoes and birds, as vector and reservoir respectively, play an important role in the virus circulation. several studies have defined the potential importance of environmental factors on the wnv-vector-host interaction, the virus replication and on the population/ habitat of the vector/reservoir (bolling et al. 2005, kinney et al. 2006). climate variation, as an environmental factor, has two different effects on ecological processes: (i) direct effects on virus/ hosts/vectors, for example, by selecting the variants sensitive, or controlling the physiology of organism such as metabolic processes and reproduction (gubler et al. 2001, reiter 2001), and (ii) indirect effect on the habitats, the ecosystems and the relationships among the organisms (stenseth et al. 2002). effect of climatic factors on human wnv infection has been studied by analyzing a spectrum of *corresponding authors: dr farzaneh ahmadnejad, e-mail: f_ahmadnejad@yahoo.com j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 318 http://jad.tums.ac.ir published online: january 06, 2016 weather factors for different states in the united states, and demonstrated a relation between these factors and incidence of human wnv cases (soverow et al. 2009). climate variations have changed the geographic and seasonal patterns of wnv with shifting toward new geographic areas and occurring earlier in the transmission season. gibbs et al. demonstrated that temperature, housing density, urban/suburban land use, and physiographic region are important variables associated with the geographic distributions of wnv in georgia. in addition, the models according climate and landscape change were used to predict the future expansion of wnv in north america (gibbs et al. 2006, chen et al. 2013). environmental factors such as elevation range, physiographic region, and percentage of vegetation cover have been evaluated to determine human wnv risk in chicago (ruiz et al. 2004). in western kenya, mosquito larval presence was associated with lower elevations, greater wetness, short distances to water, and land use (bian et al. 2006). among climatic factors, temperature is the most important extrinsic factor affecting the population and dynamic of virus/vector/ hosts. humidity is another factor, which has a direct effect on the survival of mosquitoes, by increasing vector flight and host-seeking behaviors. survival rate might be reduced when hot weather is accompanied by low humidity. rain provides the breeding sites for mosquitoes and helps create a humid environment, which prolongs the life of vectors. however, response to precipitation depends on the geographic location, season and the mosquito species (reiter 2001, ruiz et al. 2010). normalized difference vegetation index (ndvi) is a simple numerical indicator that can be used to assess vegetation density. as plants respond quickly to climate variations, ndvi is related to the climatic variations such as increase or decrease of precipitation, temperature and sunshine level (stow et al. 2004).the first evidence of wn virus circulation in iran, a country with climatic and environmental diversity, goes back in 1970s when existence of wnv antibodies among healthy human population has been demonstrated (saidi s. 1976). after a several decades gap, in a study carried out among 1054 animal sera collected from different provinces, in 2008–2009, a relatively high circulation of the virus has been found in the country. the study showed a highly heterogeneous prevalence in different provinces, so that a higher prevalence was detected in the southwestern areas while lower prevalence was in the northern areas (ahmadnejad et al. 2011). according to the last checklist, iranian mosquitoes include 64 species and 3 subspecies classified in 7 genera and 12 unverified species. the most important vector of wnv, culex spp, includes 19 species (azari-hamidian 2007). although, there are many studies on iranian mosquito fauna, there is no information about wnv vectors in the country. this study addressed the dynamic and the spatial patterns of wnv transmission by analyzing the geographical and ecology parameters affect the wnv circulation in the infected areas in iran. materials and methods study area iran is bordered by the gulf of oman, the persian gulf, and the caspian sea. it has arid or semiarid climates mostly characterized by low rainfall and high potential evapotranspiration and its location cause it to receive less than a third of the world average precipitation. the complex physical conditions of iran including topography and landscape have created a diverse climate pattern, so that it led to the formation of different ecological zones with various species of plants and animals (heshmati 2007). j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 319 http://jad.tums.ac.ir published online: january 06, 2016 furthermore, the climate is influenced by caspian sea in north, coastal areas of south of the country, mediterranean area and red sea. alborz and zagros mountains also play an important role in determining the nonuniform spatial and temporal distribution of precipitation in the whole country. iran is located at high latitudes rendering vast differences between seasons in the country. temperature in iran is intense function of altitude, latitude and moisture content of the atmosphere. the summer is extremely hot with temperatures in the interior; over 55 °c has been recorded in some places. humidity prevents temperature fluctuations in the north and south of the country (alijani 1995). the rainy period in most of the country is from november to may followed by dry period between may and october with rare precipitation. the average annual rainfall of the country is about 240 mm with maximum amounts in the caspian sea plains. collection and processing of data serological data: equine data and their geographic coordinates were obtained from a previous seroprevalence study. the sera (n= 1054) were collected from 260 stables located within 27 of 30 provinces in the country. diagnosis of wnv antibodies in the sera was performed by using plaque reduction neutralization test (prnt) (fig. 1). antibodies to wnv were detected in 249 (23.7%) of the samples (ahmadnejad et al. 2011). landscape and climatic data climatic data: we analyzed three climatic factors such as rainfall, temperature and relative humidity, which are very important factors to affect the mosquito breeding activities. mean monthly temperature, precipitation and relative humidity data of 10-year period (1996–2005) from 103 stations were obtained from iran meteorological organization (irimo). numerical layers of sampling places and meteo-stations were inputted into arcmap 9.3 software. the stations and their nearest sampling places were defined by thiessen method (fortes et al. 2005). mean yearly data was used for analysis between wnv prevalence and climatic parameters. average temperature and precipitation is given in degrees celicius and millimeter, respectively. relative humidity is expressed as a percentage. normalized difference vegetation index (ndvi) is a simple numerical indicator that can be used to analyze remote sensing measurements and assess vegetation density and whether the target being observed contains live green vegetation or not (brown et al. 2008). ndvi time series data were acquired by advanced very high resolution radiomete (avhrr) sensor onboard the national oceanic and atmospheric administrationr satellite (noaa). the images have been extracted from http://www.class.ngdc.noaa.gov based on avhrr level 1b and resolution of 1.1 km. as satellite images cannot be directly combined with other geographic information system, so geometric correction is necessary to bring the images from ground or slant range geometry into a map reference, and for this purpose, we used envi 4.3 software. composites were used to calculate ndvi values using erdas 8.7 software and using near infrared (nir) and red bands as follows: dnir dnir ndvi re re    ndvi raster files were imported into a geographic information system (gis, esri1, arcgistm 9.3). buffers of 5 and 5– 10 km around each sampled point were created with the gis software and mean ndvi was extracted within each circular buffer. j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 320 http://jad.tums.ac.ir published online: january 06, 2016 these values were exported into a dbase iv table for further analysis. three ndvi indicators were used for analysis, 1annual mean ndvi, 2seasonal ndvi differences, which is normalized differences of average ndvi for successive seasons in a 5km radius area around each stable and, 3local ndvi differences, which is normalized differences of average ndvi between two areas: a 5 km radius area centered on each stable and the 5–10 km surrounding area (fig. 1). dispersal of adult mosquitoes is an important factor in the spatial scale of transmission of vector-borne pathogens. the buffers of 5 and 5–10 km were chosen based on estimates of dispersal distances of culex mosquitoes and their blood-sucking activities on which birds (infected) at the first and then other hosts, such as equines. we assume that to bite a bird, the maximum estimated dispersal of a female mosquito is about 2.5km and similarly in another host (2.5×2=5km). the second buffer area was chosen by the same radius of 5km buffer. moreover, ndvi contrast between two buffers could be an indicator of localized mosquitoes as well as birds (venkatesan and rasgon 2010, hamer et al. 2014). distance to the nearest wetland area iranian wetlands possess a great diversity and provide a habitat for more than 140 migratory and sedentary bird species. among 42 kinds of wetlands in the world characterized by the ramsar convention, all, except one, are found in iran, indicating an immense variety of iranian wetlands. due to the extension of the country, there are a large number of wetlands with a broad range of size: area between a few to 500000 hectares (bagherzadeh-karimi and rouhani-rankouhi 2007). the data on wetlands was acquired from iran environment protection organization. for this study, we collected the data of 60 important wetlands area, of which 22 are ramsar sites. the area of these wetlands extends around 2954970.20 hectares (fig. 2). to define proximity to the wetlands, the shape files of wetlands and sampling sites of animals was imported into arcmap version 9.3. using the ‘euclidean distance’ function, from the spatial analyst extension, nearest wetland and its distance to each sampling place was calculated. statistical analysis univariate analyses were performed in order to test the relationship between the outcome variable and each explanatory variable. finally, logistic regression analyses were performed in order to identify which variables were involved in the stables seropositivity. explanatory variables used in logistic regression modeling included annual average temperature, relative humidity and precipitation, distance to the nearest wetland, annual average ndvi, normalized seasonal differences of ndvi, normalized local differences of ndvi and the type of stable (farm or club) (table 1). normalized seasonal differences of ndvi for two successive seasons s1 and s2 and for stable s was calculated as: (ndvis,s2–ndvis,s1)–(1/n) σ(ndvi*,s2–ndvi*,s1), where ndvix,y is the average ndvi value observed in a 5km radius area around stable x during season y, and n is the number of stables. normalized local differences of ndvi were calculated between two areas; a 5 km radius area centered on the stable and the 5– 10 km surrounding area. for stable x and season y, value was calculated as: (ndvix,y,0– 5km–ndvix,y,5–10km)–(1/n) σ(ndvix,*,0–5km– ndvix,*,5–10km), where ndvix,y,z is the average ndvi value observed around stable x during season y in area z, and n is the number of stables. as the previous study showed a high effect j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 321 http://jad.tums.ac.ir published online: january 06, 2016 of the type of stables on the seropositivity (ahmadnejad et al. 2011), we included this variable in the present analyses. accuracy (percent of testing sites correctly classified), sensitivity (percent of positive testing sites correctly classified), and specificity (percent of negative testing sites correctly classified) were computed for the model. in addition, the area under the receiver operating characteristic curve (auc roc) was calculated as indices of the fit of the model. all the statistical analyses were conducted using r 2.10.1, and maps were produced with a geographic information system (esri1, arcgistm 9.0). results the stables with at least one positive animal considered as positive stable and 108 of 260 stables (41.5%) were positive for wnv infection. in the training set, on univariate analysis, all the variables were associated with the presence of wnv antibody (p< 0.05). the odds ratio and 95% confidence intervals were calculated for seropositivity and all risk factors. multivariate logistic regression analyses revealed a strong relation between wnv antibody prevalence and annual mean temperature, distance to the nearest wetland, seasonal ndvi differences and local ndvi differences variables (table 1). the average minimum and maximum annual temperature were 5 °c and 27.7 °c, respectively. annual mean temperature for infected and uninfected places was 19.97 °c (95% ci 18.97–20.97) and 15.58 (95% ci 15.04–16.11), respectively. the average minimum and maximum annual humidity were 25.6% and 83.8%, respectively. annual mean humidity for infected places was 44.57% (95% ci 42.07– 47.06) and for uninfected places was 47.94% (95% ci 45.98–49.89). the average minimum and maximum annual precipitation were 50.8 mm and 1759.1 mm, respectively. annual mean precipitation for infected places was 258.74 mm (95% ci 210.58–306.91) and for uninfected places was 299.18 mm (95% ci 266.83–331.53). the risk of being wnv seropositive increased for stables located in the area with higher temperature, as 5 °c increase in mean annual temperature was associated with a statistically significant 100% higher prevalence of wnv infection. fig. 3 fig. 3 shows the aggregation of infected stables within the area with higher temperature. no significant association was observed between type of stable, annual mean humidity, precipitation, annual mean ndvi and seropositivity. it is notable that type of stable is different from their location, as the former more is indicating the management of the stable and the latter indicating geographic location. there was a significant negative correlation between distance to the nearest wetland and seropositivity of stables. the risk of being wnv seropositive for stables increased (27%) with 10km decrease of distance from the nearest wetland area (fig. 4). a logistic regression model showed an association between seasonal/ local ndvi and seropositivity. a higher significant difference between ndvi of spring and summer within a 5 km radius area around the stable increased the seropositivity four times higher. the difference of ndvi between two areas, a 5 km radius area centered on the stable and the 5–10 km surrounding area, in spring for infected stables was four times higher that of uninfected stables. fig. 5 shows annual mean ndvi in spring and distribution of the stables according to the normalized local ndvi differences. j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 322 http://jad.tums.ac.ir published online: january 06, 2016 the roc analysis resulted in an auc of 0.838, indicating the model had relatively good discrimination ability. the model computes probabilities for a cell to have a high wnv circulation status. computing sensitivity and specificity of this prediction for various probability cut-offs clearly showed that at a decision threshold of 0.4. the model had a sensitivity of 75 % and a specificity of 83.6 %. corresponding positive and negative predictive values were 76.4 % and 82.5 %, respectively (fig. 6). fig. 1. the studied ndvi indicators for the sampling stables table 1. multivariate logistic model of the presence of anti-wnv seropositive animals in iranian stables, 2008– 2009 variable odds-ratio (95% ci) p type of stable (village of club) ns2 0.07 annual mean temperature δ=5°c 2.03 (1.10-3.92) 0.03 annual mean %humidity1 <38% reference 38–46% ns 0.12 46–52% ns 0.27 >52% ns 0.34 annual mean precipitation1 <154 mm reference 154–247 mm ns 0.84 247–327 mm ns 0.67 >327 mm ns 0.71 distance to the nearest wetland area δ=10 km 0.73 (0.54–0.99) 0.05 annual mean ndvi ns 0.77 seasonal ndvi differences δspring-summer>0 2.37 (1.07–5.41) 0.04 δsummer-autumn>0 ns 0.96 δautumn-winter>0 ns 0.38 δwinter-spring>0 ns 0.92 local ndvi differences spring>0 4.26 (1.07–17.98) 0.04 summer>0 ns 0.11 autumn>0 ns 0.35 winter>0 ns 0.23 1classes bounds based on distribution quantiles 2ns= nonsignificant j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 323 http://jad.tums.ac.ir published online: january 06, 2016 fig. 2. important studied wetlands and sampling sites fig. 3. annual mean temperature and geographical distribution of wnv infected and uninfected stables in iran fig. 4. distribution of positive stables (thick line) and of negative stables (thin line) according to the distance to the nearest wetland fig. 5. annual mean ndvi in spring in iran and the geographic distribution of wnv infected and uninfected stables fig. 6. receiver operating curve of the multivariate logistic model of the presence of anti-wnv seropositive animals in iranian stables, 2008–2009 discussion the evidence presented here reveals the geographic factors associated with wnv circulation in iran, which is reported for the first time in the country. four studied factors, temperature, distance to wetlands, local and regional differences ndvi were correlated to the wnv infection in equine. j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 324 http://jad.tums.ac.ir published online: january 06, 2016 positive anomalies of the temperature in some southwestern province appear to have facilitated the mosquito abundance and, consequently, wnv infection in equine. we observe a north-south gradient of seropositivity following the gradient of annual average temperature. annual mean temperature above 22 °c was notable in which all, except three, of the stables by this temperature were seropositive. these results are consistent with previous observations made in egypt (taylor et al. 1956). in southern provinces of the country, where the climate is arid, the infection reaches a high level. in northern provinces, the situation is quite different: warm, wet summers are common, but droughts occasionally occur in late summer and early fall; and outbreaks of wnv can occur at that time. temperature in iran gets warmer from west to east and from north to south. increase of temperature from west to east is due to concentration of the mountains at western part of the country while increase from north to south is because of approaching the equator and increasing of the solar radiation angle. overall, northern and mountainous parts of the country have higher annual fluctuating and southern areas have relative stability (alijani 1995). temperature influences the mosquito life cycle, its reproduction rates, development of eggs within the mosquito (gonotrophic cycle), the vector–host contact rate and consequently, virus transmission. although, patz and et al. (2000) suggested that higher temperatures may increase or reduce survival rate, depending on the vector, its behavior, ecology, and many other factors. thus, the probability of transmission may or may not be increased by higher temperatures (taylor et al. 1956, patz et al. 2000), but it has been shown that even a small increase in temperatures can have a significant impact on transmission of wnv by mosquitoes (kilpatrick et al. 2008). effect of high temperature on increasing of mosquito abundance and vector competency, has also been experimentally proven (dohm et al. 2002). wnv is endemic in people living in the semi-arid regions, where the weather most of the year is decidedly hot and dry. cases has been regularly reported in sub-desertic areas around the mediterranean basin: (i) along the nile valley (egypt, 1950, 1994) (ii) along the syrian-african rift (israel 1951, 1998– 2000, jordan 2003), (iii) in the timimoun oasis in the central sahara (algeria 1994) (taylor et al. 1956, murgue et al. 2001). the warmer condition of these area, along with directly effects on vector and host, cause the standing water with high rich organic materials attracting the mosquito and birds, consequently increase the interaction and circulation of wnv between them (epstein and defiippo 2001). beside the vectors, temperature often plays a significant role in bird life cycles, availability of food and habitat and also in the distribution of migratory birds (greenberg and marra 2005). precipitation and humidity could have a major influence on the distribution of mosquito species. humidity increases vector flight and host-seeking behaviors and precipitation is necessary for the formation of mosquito breeding habitats (shaman et al. 2002). however, we could not find a significant relation between wnv infection and annual mean humidity/rainfall. evidences on relationship between wnv/mosquito abundance and precipitation and humidity are variable, some indicate a weak, some strong correlations (soverow et al. 2009, wan norafikah et al. 2009). seropositivity was negatively correlated to the distance to wetlands, so, near to wetland can increase infection of the animals to the virus. this is consistent with the previous studies, which showed wnv infection is more strictly linked to the wetlands with abundant bird populations, especially migratory birds. wetlands provide suitable breeding habitat for mosquitoes and birds and j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 325 http://jad.tums.ac.ir published online: january 06, 2016 consider as a natural foci of wnv infections in palearctic. outbreaks in temperate area, especially in europe, often have occurred in or near wetlands (hubalek and halouzka 1999, jourdain et al. 2007). in 1998, wnv was isolated from horses suffering from neurologic disease and residing in a large wetland area in italy. in camargue (france), wetland variations identified as important risk factors in wnv spillover in horses. (autorino et al. 2002, pradier et al. 2014). moreover, proximity to wetland increase mosquito abundance, as the first three stages of their life is aquatic dependent. iranian wetlands serve as wintering or staging area for migratory birds coming from wnv endemic area and using the west siberiancaspian-east african and central siberianindus-south asian flyways (undp 2004). the strongest association was detected between seropositivity and ndvi, at seasonal and, especially, local differences. while this relation could not be found with annual mean value of ndvi. the local ndvi’s contrast during spring is a major risk factor of the transmission of west-nile virus in iran. during the early drought season, the stable seropositivity is increasing with the contrast between the local and regional ndvi environment of the stable. this socalled oasis effect is associated to the attractiveness of the local ndvi environment for wnv’s vectors (mosquitoes and birds). this oasis effect disappeared in autumn, and the local ndvi’s contrast becomes protector (negative impact). this oasis effect is consistent with the seasonal production of vegetation in spring, which is more important compared to the national average (contrast springsummer). the higher level of seropositivity of the stables can be related to the attraction of many birds to the resources around these stables (bock et al. 2008). the extreme climatic situation of the southwestern provinces of iran encourages birds to congregate around shrinking water sites, encouraging viral circulation among birds and mosquitoes, while heat accelerates viral maturation. droughts and heat wave were found to induce wnv amplification by bringing the hosts and the vectors together. in opposition with the dogma that increasing precipitation predicts mosquito abundance, some authors hypothesize that wild populations should generate outbreaks under drought conditions. standing water pools become richer in the organic material that culex vectors needs to thrive and the mosquito predators, such as amphibians and dragonflies, are fewer in number (epstein 2004). these findings are consistent with the hypothesis that spring= nesting (in an "oasis", the birds will nest closer to the horses) and autumn = dispersal of juveniles (juveniles are going further if they leave from an "oasis"). these findings are also consistent with the fact that viral circulation is related to the presence of chicks, sedentary and still bare skin in altricial species (marra et al. 2004). high values of ndvi are related to higher photosynthetic activity and improved ecological condition. this suitable condition increases the main food supply, such as arthropods, for most migratory birds. several studies have used ndvi for assessments of wnv infection foci and its vector habitat. seasonal difference in ndvi was one the best predictive factors for determining mosquito distribution abundance in the model studied by jacob et al (jacob et al. 2009). ward et al. investigated association between ndvi and cases of equine wnv encephalomyelitis. they found that the mean ndvi in biweekly periods with reported cases was significantly higher than the mean ndvi in periods without cases (ward 2009). climatic condition of iran varies throughout the country, resulting in a complex hydrological and vegetative landscape, with arid and semiarid areas in the center, southeast and southwest and temperate rain forest in the north. although areas with high wnv j arthropod-borne dis, september 2016, 10(3): 317–329 f ahmadnejad et al.: impact of climate … 326 http://jad.tums.ac.ir published online: january 06, 2016 infection rates, such as khuzestan, have a dry climate, but their water sources are mainly from rivers originated from mountains and precipitations, which play an important role in producing oasis areas in these areas. although these results provide a new understanding of some ecological parameters effects on wnv circulation in iran, additional work is needed. arboviruses circulation is multifactorial and studies are needed to evaluate various mosquito species for their potential to transmit wnv in iran. there are many studies on mosquito fauna in iran, however there is no specific information about wnv vectors. wnv has been identified in numerous mosquito species, including members of the genera culex, aedes and ochlerotatus, but mosquitoes belonging to the culex species are the main vectors for wnv. culex pipiens, the main vector of wnv in us, has spread in different ecological zones of iran and its distribution is very similar to its climatic distribution in north, south america and africa. the virus has been isolated from cx. vishnui complex in pakistan. there are some doubtful records of this mosquito in iran; however, based on the record of this species in pakistan, it seems that cx. vishnui may occur in southeastern iran (zaim et al. 1985, hubalek and halouzka 1999, vatandoost et al. 2004, dehghan et al. 2010). if we consider iranian wnv vector within culex spp, it is speculated that other than several species may be involved in wnv circulation in south and southwestern parts of iran, where there is a high circulation of the virus. however, vector competency studies should be conducted on various species of mosquitoes in order to determine the ability of the mosquito species to transmit wnv and detection of the virus from mosquitoes. another study was conducted to determine wnv antibody and genome in different species of wild water birds captured from wetlands in iran. samples were collected from 26 different species, 15% of which were serologically positive, while no wnv viral rna-positive samples were found in this study. the majority of positive birds were common coot (fulica atra) (fereidouni et al. 2011). they did not find any positive samples from khuzestan province where the most important region for the virus circulation is. moreover, they had a few samples (n=4) from this province, all of which were birds other than common coot, maybe due to lower abundance of common coot (fulica atra) in this area. the area identified with high risk for wnv circulation may be used for entomological or epidemiological surveillance. conclusion the model indicated that local ndvi’s contrast during spring is a major risk factor of the transmission of west-nile virus in iran. this so-called oasis effect consistent with the seasonal production of vegetation in spring, and is associated to the attractiveness of the local ndvi environment for wnv vectors and hosts. further studies are needed to understand better the ecology of wnv in iran. the reservoir hosts and vectors are the keys of circulation of the virus and will be the focus of the future work. acknowledgements we thank all the personnel of ir of iran meteorological organization (irimo), especially dr ghaemi. we also thank the personnel of iran environment protection organization, especially ms elahi-rad and mr amini. 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(scorpionida: buthidae) venom mehdi khoobdel 1, taghi zahraei-salehi 2, bahar nayeri-fasaei 2, *mohammad khosravi 1, zahra omidian 3, mohammad hassan motedayen 4, abolfazal akbari 4 1health research center, baqiyatallah university of medical sciences, tehran, iran 2department of microbiology, faculty of veterinary medicine, university of tehran, tehran, iran 3department of parasitology, faculty of veterinary medicine, university of tehran, tehran, iran 4razi vaccine and serum research institute-karaj branch, karaj, iran (received 25 jun 2012; accepted 22 jan 2013) abstract background: scorpions stings are a health problem in many parts of the world. mesobuthus eupeus (buthidae) is the most prevalent species in the middle east and central asia. definition of toxicogenic and immunogenic characteristics of the venom is necessary to produce antidote. in this study, the noted properties of m. eupeus venom were evaluated. methods: venom was obtained by milking m. eupeus scorpions for lyophilization. toxicity was determined after injecting the venom to albino mice and calculating ld50. polyclonal antibodies against m. eupeus venom were obtained from immunized rabbits. the ch-sepharose 4b column was used for isolating the specific antibodies. 10 mg of the affinity-purified antibodies were conjugated with a ch-sepharose 4b column and m. eupeus venom was applied to the column. the bound fragments were eluted using hydrogen chloride (ph: 2.5). crude venom and affinitypurified fractions of the venom were analyzed by sds-page technique. results: lethal dose (ld) was 8.75, 11.5 and 4.5 mg/kg for ip, sc and iv respectively. the ld50 of m. eupeus venom was 6.95 mg/kg. the crude venom had 12 detectable bands with molecular weights of 140, 70, 50, 33, 30, 27, 22, 18, 14, 10 kda and two bands less than 5 kda. the affinity-purified venom presented eight bands. the 27 kda band was clearly sharper than other bands but 70, 18, 10 and one of the less than 5 kda bands were not observed. conclusions: contrary to popular belief, which know scorpion venom as non-immunogenic composition, the current study was shown that the most fractions of the m. eupeus are immunogenic. keywords: mesobuthus eupeus, scorpion, venom, immunogenic, toxicogenic introduction scorpions have existed on earth about 400 million years ago (ozkan et al. 2007). the scorpion stings are a major threat to human and animal health especially in tropical regions (bawaskar et al. 2012, warrell 2012). annual rate of scorpion stings is 1.2 million, and the mortality rate is about 3250 per year. children are more vulnerable to scorpion envenomation and the highest death rate is observed in this age group (chippaux and goyffon 2008). scorpions belong to the phylum arthropoda, class arachnida, order scorpiones. 1500 discribed species of scorpions are inclued 70 genera and 6 families. 50 species are dangerous for human (keskin and koc 2006) where buthidae family is the most venomous of them (shirmardi et al. 2010). iranian scorpion (sting agents) species are classified in buthidae and scorpionidae families with 16 genera and 25 species (dehgani et al. 2009). the limited number of dangerous species are found in iran (sagheb et al. 2012). mesobuthus eupeus is a species be*corresponding author: dr mohammad khosravi, e-mail: khosravi.m@ut.ac.ir 139 j arthropod-borne dis, december 2013, 7(2): 139–146 m khoobdel et al.: purification of the… http://jad.tums.ac.ir published online: august 31, 2013 longing to the buthidae family and commonly known as the lesser asian scorpion or the mottled scorpion. it was found in the middle east and central asia and is responsible for many cases of envenomation in these regions (karatas 2003, sadeghian 2003, dehghani and khamehchian 2008). mesobuthus eupeus is the most common species in iran. its venom contains several toxin fractions, which may cause a number of scorpion sting symptoms (tuuri and reynolds 2011, sagheb et al. 2012). scorpion venom consists of many biological compounds which affects vertebrate and invertebrate organisms (upadhyay and ahmad 2008). scorpion venom composes of short-chain peptides with low molecular weight (adiguzel 2010), which elicit a strong immunogenic reaction in the host (corzo et al. 2001). as yet, about 400 toxic peptides have been detected in scorpion venoms but it has been estimated that 100.000 distinct peptides exist in scorpion venom (karatas 2003). serotherapy is the only effective treatment against scorpion stings and has been an issue of discussion in the last decade (boyer et al. 2009, duarte et al. 2010). based on previous reports, approximately 42500 scorpion stings occur in iran annually (dehghani and fathi 2012). in iran, the scorpion antivenom is made through the process of injecting horses with a mixture of six different scorpion venoms including: hemiscorpius lepturus, buthotus saulcyi, b. schach, odontobuthus doriae, m. eupeus and androctonus crassicauda (razi vaccine and serum research institute, karaj, iran). many investigations were performed to improve the quality of antidote against scorpion venom. study of the immunological properties of venom is critical for antivenom development as much as better (inceoglua et al. 2006). moreover the detection of antigenic proteins is very important in the field of toxicology and parasitology (kalapothakisa et al. 2001). so development of specific antibodies against immunogenic fragments of the venom can effectively improve therapeutic alliance. gel electrophoresis, electro-focusing or liquid chromatography are used to detect protein patterns of venoms (escoubas et al. 2002, pimento et al. 2003). the current study was conducted to investigate the immunogenic and toxicogenic properties of the m. eupeus venom. materials and methods venom preparation mesobuthus eupeus scorpions were collected with uv light at night from different parts of the khuzestan province (31°19′– 32°73′n , 48°41′–49°4′ e, with an area of 63,238 km²) in south west of iran and were milked by electric stimulation at the end of the tail. the freeze-dried venom was dissolved in distilled water and then dialyzed against distilled water at 4 °c for 48 hours. after dialysis, the venom solution was centrifuged at 1500rpm for 15 minutes, and the supernatant was collected. protein assay the protein content of venoms was determined by the absorbance at 280nm with bovine serum albumin (bsa) as standard. toxicity determination all experiments were performed according to the guidelines of the ethical committee of the faculty of veterinary medicine of tehran university, iran (national ethics advisory committee 2006). for toxicity determination, increasing concentrations of the venom were injected subcutaneously (sc), intraperitoneally (ip) and intravenously (iv) to albino mice. following treatment with venom solution, animals were monitored for 24 hours, and the number of dead animals was recorded at the end of the 140 j arthropod-borne dis, december 2013, 7(2): 139–146 m khoobdel et al.: purification of the… http://jad.tums.ac.ir published online: august 31, 2013 experiment, then, ld was calculated. ld50 was determined using the spearman-kaerber method. briefly, 35 mice were divided into 7 groups of 5 mice each. appropriate venom concentrations were prepared to cover the full range between zero and 100% of induced animal mortalities. different doses (175, 160, 145, 130, 119, and 109µ g) of the venom stock solution were prepared and injected intraperitoneally (ip). an equivalent volume of buffer was injected into 5 mice as a negative control group. deaths were scored up to 24h and ld50 was then calculated. production of polyclonal antibody outbreed new zealand white male rabbits were acclimatized to room temperature at 18 °c for two weeks former to immunization. preimmune sera was attained throughout this period. the immunization plan and programmes of immunization were the alike as those detailed previously. in initial immunization, three rabbits were each injected intradermally with 250 µg of venom in 0.5 ml of pbs emulsified with 0.5 ml of complete freund’s adjuvant by a multiple injection method (10 sites/ rabbit) (inceoglua et al. 2006). these first injections were pursued by three sets of booster injection. booster injections were made at 2nd, 4th and 6th weeks with 130 µ gr of immunogen, 0.5ml of pbs and 0.5ml of incomplete freund’s adjuvant at two sites in both thighs intramuscularly. the existence of antibodies in serum was determined through immunodiffusion and ascoli's test. finally, after 10 days, the immunization blood was directly collected into sterilized glass tubes without any anti-coagulants and allowed to clot in cold. serum was pipette out and centrifugated at 1500 rpm for 10 minutes and then isolated in a sterilized vial and stored at 4 °c for bioassay tests. purification of polyclonal antibody against venom polyclonal antibody against venom was first purified by ammonium sulfate precipitation (50% saturation for the final solution) and dialyzed in pbs and then subjected to an affinity column conjugated with venom. the column was prepared by conjugating 20mg of venom with 7ml of activated ch-sepharose 4b. cyanogen bromide activation was performed by the method of cuatrecasas (march et al. 1974). antibody was eluted from the column with 0.1m glycine ph 2.5 and fractions were collected and neutralized immediately by adding an appropriate amount of 1 m tris-ph 9 to each fraction. purification of immunogenic peptides of venom the fractions, including the exact antibodies were merged, dialyzed against borate buffer ph 8.4, overnight and used for another affinity column. ten mg of this affinity purified antibody conjugated with a chsepharose 4b column and 5mg of m. eupeus venom were applied to it. the bound proteins were eluted as before. sds-page analysis of the venom the protein profiles of crude venom as well as the affinity fractions (purified venom) were analyzed by sds-page (laemmli 1970), the concentration of acrylamide was 15%. proteins were stained with 1% coomassie blue r 250. molecular mass standard (vivantis, product no: pr0602) was run in parallel in order to calculate molecular weights of the proteins. then, the gels were photographed and molecular weights of the proteins were calculated. results venom lethal dose (ld) was assessed by either subcutaneous, intraperitoneal or iv injection using 18±2g albino mice. ld was 8.75, 11.5 and 4.5mg/ kg of the body weight of albino mice for ip, sc and iv, respectively. 141 j arthropod-borne dis, december 2013, 7(2): 139–146 m khoobdel et al.: purification of the… http://jad.tums.ac.ir published online: august 31, 2013 the median lethal dose (ld50) of m. eupeus venom was 6.95mg/ kg with ip injection. proteins of the venom were determined to be between 5 and 140 kda on electrophoresis on 15% polyacrylamide gel. the crude venom had 12 detectable bands with molecular weights of 140, 70, 50, 33, 30, 27, 22, 18, 14, 10 kda and two bands less than 5 kda. the affinity-purified venom presented eight bands. the 27 kda band was clearly sharper than other bands but 70, 18, 10 and one of the less than 5 kda bands were not observed (fig. 1). fig. 1. the sds-page analysis of mesobuthus eupeus scorpion venom. from right lane 1: marker proteins (175, 130, 95, 70, 62, 51, 42, 29, 22 and14 respectively). lanes 2 and 3: electrophoretic pattern of the immunogenic fractions present in the venom (140, 50, 33, 30, 27, 22, 14, ≤5 kda) and crude venom (140, 70, 50, 33, 30, 27, 22, 18, 14, 10, ≤5, ≤5 kda) respectively. table 1. the variations of protein in mesobuthus eupeus venom protein bands (kda) 140 70 50 33 30 27 22 18 14 10 fever than 5 total number of protein bands a + + + + + + + + + + ++ 12 b + + + + + + + + 8 a) venom samples b) immunogenic fractions of venom discussion in the present investigation, we determined the in vivo toxic effects of the venom of m. eupeus. the venom of m. eupeus appears to be more toxic when injected intravenously. this phenomena could be associated to different toxicokinetics of the three injection methods. additionally, we studied the electrophoretic protein pattern of the crude venom, and immunogenic fractions of the venom. the results clearly displayed that most of m. eupeus venom fragments were immunogenic. our results also showed that scorpion toxins were proteins with various molecular 142 j arthropod-borne dis, december 2013, 7(2): 139–146 m khoobdel et al.: purification of the… http://jad.tums.ac.ir published online: august 31, 2013 weights, which induce both toxicological and immunological reactions invivo. we also developed an approach toward application and refining of the immunogenic fractions of m. eupeus venom. previous studies in iran determined 4.5 mg/kg (zayerzadeh et al. 2012) and 1.45 mg/ kg (hassan 1984) as the median lethal dose (ld50) of m. eupeus venom. another study was calculated the median lethal dose of m. eupeus venom 0.18mg/ kg via intracerebroventricular (icv) injection (ozkan and carhan 2008). in our study, ld50 of the venom was 6.95mg/ kg via ip injection. diverse studies reported various numbers of protein bands with different molecular weights for scorpion venoms. molecular weights of iurusdufoureius asiaticus specie venom were determined 14–205 kda with individual variations (turkey) (keskin and koc 2006). a similar study on tityus pachyurus specie suggested 14–97 kda venom proteins using electrophoresis (sds-page) method (latin america) (barona et al. 2004). they developed three anivenom which prominently reacted with low molecular weight fragments. the most of venom proteins molecular weights of m. eupeus were 12–112 kda (ozkan and carhan 2008). we determined protein fragments from 5 to 140 kda. one study showed that the venom of m. gibbosus consisted of 19 protein bands with molecular weight from 6.5 to 210 kda (ucar and tas 2003). protein bands with molecular weight of 28, 30, 33, 68 and 98 kda were detected in the venom of the captive male m. gibbosus from the same biotope during the summer (turkey, mugla province) (ozkan and ciftci 2010). the causes of disagreement between studies may be due to the effects of the sex, geography, and hormonal condition of scorpions, which all alter feeding manners and result in venom creation with diverse molecular weights. in the current study, 12 protein bands were detected in m. eupeus scorpion venom. variations in the biochemical and immunological contents of the various scorpion venoms must be considered to realize clinical signs, produce efficient antivenoms and determine optimal dosage (el-hafny et al. 2002, calvete 2010). recognition and comparing of the mesolys-c amino acid sequence of three major species scorpion is used for detecting phylogenetic relationships of various scorpion species. for example, mesolys-c isolated from m. eupeus of khuzestan exhibited the highest and the lowest sequence similarities with m. gibbosus and m. cyprius, respectively (eskandari and khoonmirzaei 2011). the ability of heminecrolysin to suppressing the major physiopathological effects of h. lepturus envenomation may be due to elicit high titer of specific iggs (borchani et al. 2011). the antigenicity studies of iberiotoxin of eastern indian scorpion demonstrated whole protein was not necessary to stimulate the immune system, because a small fragment of the venom protein called the antigenic determinant was adequate for eliciting the immune response (gomase et al. 2009). a study performed by garcia et al. (2003) approved this statement. gazarian et al. (2005) realized that no immunity was developed against scorpion venom during evolution. because of no evolutionary relationship between humans immunity and scorpion venom, scorpion venoms can be suitable candidates for immunogenic probes (march et al. 1974). because of completely distinct phylogenetics properties of two noted entities, any structural changes of scorpion venoms can followed and probably manipulated for inactivation of their antigenic activity (gazarian et al. 2005). contrary to popular belief, which know scorpion venom as non-immunogenic composition, the current study was shown that the most fractions of the m. eupeus were immunogenic. further investigations are necessary to explain more details of these immu143 j arthropod-borne dis, december 2013, 7(2): 139–146 m khoobdel et al.: purification of the… http://jad.tums.ac.ir published online: august 31, 2013 nogenic fractions and to detecting lesser toxicant fragments, which, improves the quality of the antidotes and 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situation in an endemic area, southeastern iran sajjad fekri 1, hassan vatandoost 2, ali daryanavard 3, mehran shahi 1, reza safari 3, ahmad raeisi 4, abdiqani sheikh omar 5, mohammad sharif 6, abdollah azizi 7, aref ahmad ali 8, aboud nasser 9, ibrahim hasaballah 10, *ahmad ali hanafi-bojd 2 1infectious and tropical diseases research center, hormozgan university of medical sciences, bandar abbas, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3hormozgan province health center, bandar abbas, iran 4malaria control program, cdc, ministry of health, tehran, iran 5malaria control program, somalia 6malaria control program, afghanistan 7health center, zarrindasht, iran 8roll back malaria program, aden, yemen 9roll back malaria program, hadramaut, yemen 10state malaria program manager, north kordofan, sudan (received 21 oct 2012; accepted 30 sep 2013) abstract background: malaria is an endemic infectious disease in southeastern parts of iran. despite years of efforts and intervention programs against malaria, transmission still occurs in jask county. methods: the epidemiological perspective of malaria in jask county was conducted by gathering data from jask county health center, during 2006–2010. a knowledge, attitude and practice study was also carried out. data analysis was conducted using spss ver. 11.5. results: a total of 2875 malaria cases were recorded, with highest and lowest numbers in 2007 and 2010, respectively. the number of cases had a decreasing trend from 1022 cases in 2006 to 114 cases in 2010. the main causative parasitic agent was plasmodium vivax. blood examination rate and slide positive rate were also decreased from 39.5% and 4.3% in 2006 to 15.6% and 1.4% in 2010, respectively. most of people interviewed in the kap study had a good knowledge about malaria transmission and symptoms but their use of the bed net for prevention was low (35%). conclusion: malaria incidence had significant reduction during the study years. the main reason for this may be due to changing environmental condition for anopheline breeding and survival because of drought. another reason may be integration of vector management by using long lasting insecticide treated bed nets, active case detection and treatment by implementation of mobile teams and increasing in financial sources of malaria control program. knowledge, attitude and practice of people were good in malaria control and prevention, but needs to do more activities for health education and awareness. keywords: malaria situation, jask county, iran introduction more than half of the world's population in approximately 100 countries is exposed to malaria. according to figures provided by the world health organization (who 2011a), 36% of the global population live in areas where there is risk of malaria transmission, 7% reside in areas where malaria has never been under meaningful control, and 29% live in areas *corresponding author: dr ahmad ali hanafi-bojd, e-mail: aahanafibojd@tums.ac.ir http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 83 where malaria was once transmitted at low levels or not at all, but where significant transmission has been re-established. in iran, the total population at risk of malaria is 2,714,648 (4% of the total population) mainly living in southeastern provinces, namely sistan and baluchistan, kerman and hormozgan (who 2011a). there are some studies in last decade focused on malaria situation analysis (hanafi-bojd et al. 2010, vatandoost et al. 2010, 2011), as well as epidemiological features and community based studies in malarious areas of southern parts of iran (rakhshani et al. 2003, salehi et al. 2010, hanafi-bojd et al. 2011a, youssefi and rahimi 2011, mckelvie et al. 2012, zoghi et al. 2012). according to the national strategy plan for malaria control, in respect to malaria status the total country has been classified in four strata: 1. areas where local transmission of malaria occurs such as areas in sistan and baluchistan, hormozgan and south parts of kerman, and occasionally some-areas in ardebil, bushehr, fars and khorassan-e-razavi provinces. 2. areas where the imported cases are found and the potential risk of malaria transmission exists such as areas in guilan, mazandaran, and golestan provinces. 3. areas where the imported cases are found, but there is no risk of malaria transmission such as yazd, kurdestan, and hamedan provinces. 4. areas where no malaria case was reported during the last three years. it seems there was no such area in iran. malaria remains an important public health concern in jask county, where transmission occurs regularly, and infected immigrants can play an important role as mobile reservoirs (personal communication with health center authorities). therefore it is important to do a situation analysis of the disease for planning the control activities in future. the aim of conducting a situational analysis is to systematically understand the malaria epidemiology of an area in a very short space of time and the health status, system and resource available for controlling the disease (who 2011b). a successful planning for the county, needs to use of the information that is already available and analyzing in such a way as to understand the problems. on the other hand, the main point of the beginning of the situational analysis is to collect the data from respective references such as, health network office, hospital, health centers, communities, meteorology department and others. these items are covered by international diploma courses on malaria planning and management, as a joint training course by who and tehran university of medical sciences (mesdaghinia et al. 2013). the general objective of this study was to facilitate the development of friendly-user implementation plan for jask county to identify malaria-relevant gaps with its possible solutions. materials and methods study area jask county (25° 64’ n, 57° 77’ e) is a sea port located in south-eastern corner of iran adjacent to the oman sea, with a surface area of 16063 km2 (fig. 1). total population of the county is 50070 (74% are living in rural areas). there are scattered inhabitants and their occupation is mainly fishing and trade. data collection in this retrospective study, the needed data for 2006–2010 were obtained from health center of jask, published papers and reports, weather forecasting organization of the city and annual reports of hormozgan official authorities. an excel databank was created and analysis was conducted using this software. a knowledge, attitude and practice (kap) study was conducted to evaluate the knowledge, attitude and practice of people who referred to http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 84 the urban health center of touhid, about malaria using structured questionnaire and interviewing. data analysis for this purpose spss ver. 11.5 was used and graphs were prepared by excel 2007. results a total of 2875 malaria cases were recorded and treated during the study period (fig. 2). plasmodium vivax is the main malaria parasite (94.92%) in the county followed by p. falciparum (5.08%). average of malaria morbidity in months of the years during the study period showed two peaks in may and november, respectively. the maximum and minimum of annual parasitic index (api) were observed in 2006 (22.1) and 2010 (2.1), respectively (table 1). most of cases were reported to be autochthonous during 2006–2009, while in 2010 the imported cases were higher. blood examination rate (ber) has increased to 2007 and then collapsed, but slide positive rate has a decreasing trend during the study period, although it shows a peak in 2008 (table 1). autochthonous cases of malaria have decreased during the study period, while the imported cases had an increasing trend (table 1). figure 2 shows the distribution of malaria cases based on rural districts of the jask county during the study period. during april-may and october-november the temperature is between 25–30 oc with a relative humidity of more than 60% (fig. 3), the suitable climatic factors for mosquito breeding and increasing longevity. based on the unpublished reports of jask health center, anopheles stephensi and an. culicifacies seems to be the main vectors in the area. these species are active during the year due to favorite climatic conditions. there was no published entomological investigation in the jask area for fauna, density, insecticide susceptibility and parasitic infection of anopheline mosquitoes. the health facilities and personnel of the county were included: one urban heath center, 6 rural health centers, one sub health center, 27 health houses, one hospital, 11 physicians, 6 public health officers and 49 health care workers (behvarz). furthermore, malaria control program has had a total of 25 mobile teams. based on the national protocol, malaria control interventions in the area are including case detection and treatment, indoor residual spraying by deltamethrin wp 10% in rural area with 90% coverage, distributing long lasting insecticide impregnated nets (llins) in rural areas with 80% coverage, as well as larviciding. data obtained from the jask county health center shows high irs coverage during the study period and distribution of llins during 2009 and 2010. these vector control activities along with other measures such as good active case detection (table 2) and increasing the financial resources of malaria control program for the study area are the main reasons for the decline of the disease. a total of 41 people (64.3% males, 35.7% females) participated in the kap study, 96.4 % under 45 years old. the education level was categorized in four levels: illiterate (10.7%), primary school (32.1%), high school (25%), and university degree (32.1%). among the respondents just 17.9% had a history of malaria infection. most of the study population (82.1%) believed that mosquito bite is the transmission route of malaria and fever is the main symptom. when they asked about the role of insecticide treated bed net (itns) for prevention malaria, 67.9% answered yes, meanwhile 60.7% was agree that water storage utensils can increase the risk of malaria transmission. the attitude of respondent showed 28.6% and 39.3% were respectively agree and strongly agree with this fact that itns can prevent malaria transmission. these http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 85 rates about using insecticide were 50% and 17.9%, respectively. in practice, 78.6% stated that they allow the spray men to spray their house, 96.4% will refer to the nearest health center as soon as their child feels feverish, but just 35.7% used itns during last year (table 3). fig. 1. location of jask county in iran fig. 2. malaria distribution map, jask county, 2006–2010 table 1. malaria indices in jask county, southeastern iran, 2006–2010 year malaria indices no. cases spr api ber autochthonous imported p. vivax p. falciparum total 2006 4.3 22.1 39.5 1000 22 1000 22 1022 2007 2.5 21.6 50.6 1013 12 954 70 1024 2008 3.5 10.6 28.1 485 27 495 11 506 2009 2 4.2 20.6 162 47 183 26 209 2010 1.4 2.2 15.6 34 80 97 17 114 spr: slide positive rate, api: annual parasitic index, ber: blood examination rate http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 86 table 2. activities of malaria control program in jask county, southeastern iran, 2006–2010 index year 2006 2007 2008 2009 2010 total no. of distributed llins 0 0 0 3560 2660 irs coverage no data 95% 93.2% 93.8% 95% active case detection 687 879 335 118 87 mobil teams 25 25 25 25 25 average of malaria cases in relation to climatologically parameters,jask district, 2006-2010 0 20 40 60 80 100 120 ja n fe b m ar ch ap ril m ay ju ne ju ly au g se p oc t no v de c month rh% rai nfal l (mm) temp (oc) ave. cas es fig. 3. average of malaria cases in relation to climatology parameters in jask county, southeastern iran, 2006–2010 table 3. kap study on malaria, jask country, 2011 question answer frequency percent gender female male 10 18 35.7 64.3 age 15-30 30-45 45< 17 10 1 60.7 35.7 3.6 martial status single married widow 9 18 1 32.1 64.3 3.6 education level illiterate primary/secondary high school university 3 9 7 9 10.7 32.1 25 32.1 job employed unemployed retired 12 15 1 42.9 53.6 3.6 history of malaria infection yes no 5 23 17.9 82.1 transmission route air mosquito bite food i don’t know 1 23 2 2 3.6 82.1 7.1 7.1 malaria symptoms fever cough other 23 4 1 82.1 14.3 3.6 using insecticide treated bed nets can prevent us from getting malaria true false i don’t know 19 6 3 67.9 21.4 10.7 http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 87 storing water in utensils can increase the risk of malaria transmission true false i don’t know 17 6 5 60.7 21.4 17.9 using insecticide treated nets can prevent malaria transmission strongly disagree disagree uncertain agree strongly agree 3 2 4 8 11 10.7 7.1 14.3 28.6 39.3 applying insecticide residual spray can prevent malaria transmission disagree uncertain agree strongly agree 4 5 14 5 14.3 17.9 50 17.9 the price of insecticide treated nets is low strongly disagree disagree uncertain agree strongly agree 1 3 12 11 1 3.6 10.7 42.9 39.3 3.6 applying insecticide residual spraying control malaria transmission, cannot contaminate our house strongly disagree disagree uncertain agree strongly agree 2 1 7 16 2 7.1 3.6 25 57.1 7.1 i allow the spray man to spray my home to prevent malaria transmission yes no don’t answer 22 3 3 78.6 10.7 10.7 if my child feels feverish, i bring him/her to the nearest health center yes no 27 1 96.4 3.6 if i see a place with stagnant water around my house, i will prefer to add oil land filling or drainage leaving 5 19 4 17.9 67.9 14.3 i used itn for prevention of malaria in the last 1year yes no 10 18 35.7 64.3 discussion both p. vivax and p. falciparum are reported from the jask county, although vivax malaria is dominant species. there is also report of malaria due to imported species p. malariae in bandar abbas, west of the study area, but this species in not common in iran (nateghpour et al. 2010b). since drug resistance is reported from malaria parasites in iran (raeisi et al. 2006, nateghpour et al. 2010a), it is suggested to do such a survey in jask county for better understanding the situation of malaria and planning control programs in the area. although there is no published report on anopheline mosquitoes of the study area, five malaria vectors including an. stephensi, an. culicifacies, an. dthali, an. superpictus and an. fluviatilis are reported to be active in bashagard, adjacent to the north of jask county (hanafi-bojd et al. 2012a,b). among these species, an. stephensi and an. culicifacies were collected during the entomological activities of jask health center. these two species are reported as main malaria vectors of south and southeastern parts of iran (vatandoost et al. 2006, hanafi-bojd et al. 2011a,b). as it can be understood from the climatology chart (fig. 3), the mosquito breeding can be done around the year, because of favorable condition. therefore, a comprehensive entomological study on the fauna of anopheline and their density during the year, their parasitic infection and susceptibility status to who recommended insecticides/ larvicides is suggested and should be included in malaria vector control activities in the area. regarding to our results, the knowledge, attitude and practice of the respondents was relatively good in malaria control and prevention, but needs to do more health education to improve their awareness. the relative good table 3. countinued… http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 88 level of knowledge may be due to living people in an endemic area for malaria. the same results were obtained by hanafi-bojd et al. (2011a) in bashagard county, north of the study area. some other studies reported an increasing trend in literacy can be a protective factor for malaria morbidity (koram 1989, masoumi et al. 2003). one-third of the respondents in a kap study in baluchistan area, east of the jask county, considered malaria as an important disease in the area, and more than 58% of them considered mosquitoes to be the cause of the malaria disease. so it can be concluded more contact with malaria rural areas resulted to more knowledge about the disease (rakhshani et al. 2001). constraints and challenges seems to be: massive population movement between iran, pakistan and afghanistan, vector control challenges at the county, inadequate skilled medical staff in malaria case management, weak inter-sectoral coordination for malaria control especially in urban areas, low socio-economical status of those who live in the area. the most important immigrants in southeastern part of the country are illegal and so they pass the unofficial borders, their exact number and infection to infectious diseases including malaria is unknown. but it is necessary to plan for detection of asymptomatic cases that may act as the reservoir of the disease (nateghpour et al. 2011, turki et al. 2012). priority areas for malaria control in the study area are: strengthening malaria surveillance system in the county, developing integrated vector management strategy, strengthening vector control activities by larviciding at the county level, capacity buildings on prompt case detection and treatment in the immigrant population, supporting cross border coordination with afghanistan and pakistan, strengthening malaria early warning and early detection system and developing malaria epidemics preparedness plan based on a powerful database that should be established for the area. therefore it is recommended to reinforce human capacity on malaria control activities at different levels of health personnel, strengthen the existing vector management program at the county level, establishing a spatial malaria database for the county and training arcgis to the malaria experts of the county to handle the database, developing and implementation a plan for malaria epidemics preparedness and response, build up malaria surveillance system with appropriate epidemic thresholds in epidemic-prone settings, support operational research in the field of malaria entomology and parasitology as well as insecticide resistance. conclusion malaria incidence had significant reduction during the study years. the main reason for this may be due to changing environmental condition for anopheline breeding and survival because of drought. another reason may be integration of vector management by using long lasting insecticide treated bed nets, active case detection and treatment by implementation of mobile teams and increasing in financial sources of malaria control program. knowledge, attitude and practice of people were good in malaria control and prevention, but needs to do more activities for health education and awareness. acknowledgements the authors are grateful to the kind personnel of the jask county health center as well as hormozgan province health center authorities, for their collaboration during this study. this work is a part of field exercise in 13th international diploma course on malaria planning and management that was held out by collaboration of world health organization, tehran university of medical sciences, ministry of health and national institute of health research (bandar abbas stahttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 82–90 s fekri et al.: malaria situation in … 89 tion as who regional malaria training centre) in 2011. the authors declare that there is no conflict of interest. references hanafi-bojd aa, vatandoost h, philip e, stepanova e, abdi ai, safari r, moh seni gh, bruhi mi, peter a, abdulrazag sh, mangal g (2010) 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pac j trop med. 3(10): 807–811. vatandoost h, rashidian a, jafari m, raeisi a, hanafi-bojd aa, yousofzai aw, daryanavard a, mojahedi ar, pakari a (2011) demonstration of malaria situation analysis, stratification and planning in minab district, southern iran. asian pac j trop med. 4: 67–71. who (2011a) the world malaria report. geneva. who (2011b) planning and managing malaria control programmes, guide for participants. global malaria programme. geneva. zoghi s, mehrizi aa, raeisi a, haghdoost aa, turki h, safari r, ahmadi kahanali a, zakeri s (2012) survey for asymptomatic malaria cases in low transmission settings of iran under elimination programme. malaria j. 126. youssefi mr, rahimi mt (2011) prevalence of malaria infection in sarbaz, sistan and bluchistan province. asian pac j trop biomed. 1(6): 491–492. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 43 original article cost effectiveness of malaria interventions from preelimination through elimination: a study in iran mohsen rezaei-hemami 1, *ali akbari-sari 2, ahmad raiesi 3, hassan vatandoost 4, reza majdzadeh 5 1department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran 2department of health management and economics, school of public health, tehran university of medical sciences, tehran, iran 3national malaria control programme manager, center for disease management, teheran, iran 4department of medical entomology and vector control,school of public health and national institute of health research,tehran university of medical sciences, tehran, iran 5school of public health, knowledge utilization research center, tehran university of medical sciences, tehran, iran (received 25 june 2012; accepted 16 sep 2013) abstract background: malaria still is considered as a public health problem in iran. the aim of the national malaria control department is to reach the elimination by 2024. by decreasing the number of malaria cases in preelimination phase the cost effectiveness of malaria interventions decreases considerably. this study estimated the cost effectiveness of various strategies to combat malaria in preelimination and elimination phases in iran. methods: running costs of the interventions at each level of intervention was estimated by using evidence and expert opinions. the effect of each intervention was estimated using the documentary evidence available and expert opinions. using a point estimate and distribution of each variable the sensitivity was evaluated with the monte carlo method. results: the most cost-effective interventions were insecticide treated net (itn), larviciding, surveillance for diagnosis and treatment of patients less than 24 hours, and indoor residual spraying (irs) respectively, no related evidence found for the effectiveness of the border facilities. conclusion: this study showed that interventions in the elimination phase of malaria have low cost effectiveness in iran like many other countries. however itn is the most cost effective intervention among the available interventions. keywords: malaria, prevention and control, iran, cost effectiveness, irs, itn introduction based on the endemicity of the disease, malaria-hit areas are classified into four main groups: control, pre-elimination, elimination and prevention of reintroduction. the measures to be adopted to fight the disease are different in each of these stages. as the incidence of the disease reduces over time across the country elimination programs mainly focus on malaria foci rather than whole region (mendis et al. 2009). the prevalence of malaria has reduced in iran during the past few years, placing iran among the countries classified as nations in the pre-elimination phase (edrissian 2006). as a result, the intervention strategies have changed upon the decline noted in the num*corresponding author: dr ali akbari-sari, e-mail: akbarisari@tums.ac.ir http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 44 ber of affected cases. at the time being, various measures, such as vector control activities, early diagnosis systems and some other complementary measures have been adopted to fight malaria in elimination phase around the world (moonen et al. 2007), but there are limited studies about their efficacy and costeffectiveness in this phase, in addition many of the previously-approved strategies including early warning system have never been adopted in the iranian health care system. many researchers have studied the costeffectiveness of different strategies adopted to fight malaria across the globe; but most of them were conducted in malaria-endemic areas. in other words, not many studies have targeted the low-endemic areas (goodman et al. 2000, yukich et al. 2008, kang et al. 2008) so using models seems necessary in this situation. developing an accurate model, therefore, can play an important role in this regard, particularly in conditions in which there is limited evidence or unapproved documents and reports. the importance of these modeling is highlighted at times when the policymakers are to decide upon setting up a program (goodman et al. 2000). the present study aims to estimate the cost as well as cost-effectiveness of main strategies to combat malaria in the pre-elimination and elimination phases in a malaria focus as a unit of malaria combat operation in iran. materials and methods interventions assessed a total of seven interventions were assessed. these interventions include: larviciding, indoor residual spraying (irs), distributing insecticide treated net (itn), set up the diagnosis and treatment in less than 24 hours, and set up the border facilities. general consideration this study was conducted in perspective of ministry of health and medical education (mohme), time period for effects was considered one year, we consider the case averted as outcome. basis for evaluating most of intervention was a focus with 500 peoples population. the interventions defined as bellow: larviciding: a focus with 200m2 water resource area, which needs 10 times larviciding operation every year. irs: a focus with 200 building, in each building there is 100m2 area needs to insecticide spraying two rounds a year. itn distribution: a focus with 100 households (each having an average of 5 members), for each household a bed net would be distributed with 5 years life time. establishment diagnosis and treatment in less than 24 hours: it means intensified activities for detecting ant treatment of malaria cases using the current structure of health care system in affected regions. set up border posts: the intervention included the establishment of a facility that has been deployed in the border areas. their task is providing diagnosis and treatment services to those who live and travel to the edge of the border. cost assessment cost for each intervention were assessed in different central and peripheral levels in five expenses groups, by using expert opinions, current evidences and documents. when a resource was used for several tasks, its cost was calculated proportionally. these five groups consist of: building and its currents expense (water, electricity power, telephone, warming and cooling), capital expenses, operational expenses, human force, and transport. in case of building we consider the rent of similar building, in addition a 3% discount rate considered for capital goods. effectiveness the cost effectiveness of the interventions was assessed based on evidence-based conhttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 45 trolled trials and meta-analyses if possible. the case averted considered as outcome variable in our study. the evidence was selected from studies in low-endemic areas and as for conditions with no reliable evidence expert panels were held. the comparison was made based on the epidemiologic status of the region (control, pre-elimination, elimination) as well as the endemicity status at the time when no strategy against the disease had been adopted (raeisi et al. 2004). sensitivity analysis considering the effect of undetermined variables of cost and efficacy on the intervention, mont carlo analysis was used to assess the sensitivity of the interventions. in this regard the triangle distribution was used to calculate the cost for each variable. the point estimation, as the most likely estimate, was considered the vertices of the triangle distribution (goodman et al. 2000) as for the maximum and minimum values, 10 percent was added to and subtracted from the point estimation value, respectively. sampling was repeated in 20000 times. results table 1 provides a list of different costs met in this study. as mentioned in the material and methods section, these interventions have been followed at different levels and therefore various numbers of individuals may have benefited from the intervention. the development of border posts and providing prophylactic treatment accounted for the highest cost per capita. figure 1 shows the combination of fees spent on different strategies. similarly, the highest fee was paid for human resources for border posts and diagnostic and therapeutic system in less than 24 hours. building charges hold the smallest share in the funds. in view of the available evidence and the expert panel, the relative risk of the protective properties of each of these strategies were determined, the highest case averted was for diagnosing and treating the patients in less than 24 hours. no evidence was found regarding the development of border posts. the decline noted in the number of affected cases was then calculated in four different scenarios based on the incidence of malaria in the region (table 2). irs (indoor residual spraying), itn (insecticide treated net), api (annual parasite incidence). the most cost-effective interventions, were the use of insecticide-treated nets, larviciding, diagnosis and treatment in less than 24 hours and indoor residual spraying (table 3, fig. 2) along with the changes made in the cost effectiveness as the incident of the disease declines over time are shown. as shown in the fig. 2, the cost per averted cases increases considerably as the number of affected cases decreases. fig. 1. cost per capita for each intervention based on its components (us dollar) irs (indoor residual spraying), itn (insecticide treated net) c os t pe r ca pi ta 12 10 8 6 4 2 0 http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 46 table 1. annual cost of studied intervention based on their components and their cost per capita (us dollar) costs building capital expense operational expense human force transport total population* per capita larviciding 49 0 24 741 370 1184 500 2.37 irs 49 12 309 74 111 555 500 1.11 itn 49 311 0 57 407 824 500 1.65 surveillance 49 0 267 2052 630 2997 500 5.99 border facilities 49 63 630 4024 0 4765 500 9.53 * this includes the proportion of whole cost needed for each interventions. irs (indoor residual spraying), itn (insecticide treated net), api (annual parasite incidence) * population under service ** cost per capita for each intervention, calculated by dividing total price by population table 2. the composition of cost for each intervention costs building specialized equipment. (life time> 1year) supply (life time< 1year) human force larviciding rent for building, cost of warming and cooling, office equipment and current expense 0 larvicide larvicidinig activity and supervision irs pump insecticide irs operation and supervision itn 200 itn 0 distribution, training and supervision surveillance 0 rdt, antimalaria drug active and passive surveillance, supervision border facilities microscope, medical equipment rdt, antimalaria drug active and passive surveillance, supervision http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 47 table 3. estimating the efficacy of the strategies in reducing the incidence of the disease and the number of affected cases (relative risk) intervention point estimate interval reference number of averted cases in different epidemiologic setting elimination api= 0.34 preelimi nation api=2.4 control, api=12.9 historical evidence api=350 larviciding 0,35 0,3–0,4 (invest and lucas 2008) 0.0595 0.42 2.2575 61.25 irs 0,12 0.10–0.14 (pluess et al. 2010) 0.0204 0.144 0.774 21 itn 0,48 0,52–0,44 (lengeler 2004) 0.0816 0.576 3.096 84 diagnosis, treatment in less than 24 hours 0,66 0,7–0,62 (carrara et al. 2006) 0.1122 0.792 4.257 115.5 table 4. the cost-effectiveness rate for case averted in each intervention intervention cost effectiveness in assumed population point estimate (ci 95%) preelimination api= 2.4 control api= 12.9 historical evidence api= 350 elimination api= 0.34 larviciding 22378.4 (20247.52– 24901) 3170.27 (2865.58– 3541.81) 589.82 (533.59– 656.1) 21.74 (19.65–24.2) irs 30582.42 (27373.73– 34550.42) 4332.51(3864.05– 4886.9) 806.05(720.8– 910.85) 29.71(26.57–33.6) itn 19891.91 (17646.76– 22617.98) 2818.02 (2497.5– 3214.54) 524.28 (465.11– 596.94) 19.32 (17.11– 21.97) diagnosis, treatment in less than 24 hours 30049.15 (28246.64– 31928.47) 4256.96 (3999.47– 4524.27) 791.99 (743.78– 841.09) 29.19 (27.45– 31.04) irs (indoor residual spraying), itn (insecticide treated net), api (annual parasite incidence) table 5. the cost-effectiveness of the intervention in other studies intervention location cost effectiveness cost per capita (us $) program phase reference itn south africa 18 -----control (goodman et al. 2001) irs mozambique 20–29 3,84 control (conteh et al. 2004) irs eritreatogo ---------1.2–6 control (yukich et al. 2008) itn eritreatogo ---------1.38–1.91 control (yukich et al. 2008) itn togo 3.26 -------control (mueller et al. 2008) larviciding sri lanka 0,50 ------control (konradsen et al. 1999) http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 48 1. 00 2. 00 3. 00 4. 00 5. 00 6. 00 7. 00 8. 00 9. 00 10 .0 0 11 .0 0 12 .0 0 13 .0 0 14 .0 0 15 .0 0 16 .0 0 17 .0 0 18 .0 0 19 .0 0 20 .0 0 21 .0 0 22 .0 0 23 .0 0 24 .0 0 25 .0 0 26 .0 0 27 .0 0 28 .0 0 29 .0 0 30 .0 0 incidence of malaria 10,000 8,000 6,000 4,000 2,000 0 dx,tx in 24 hours itn irs larviciding fig. 2. changes of cost effectiveness of different strategies with changes of malaria incidence discussion the present study suggested that the most cost effective strategies in fighting malaria were the use of insecticide-treated nets, larviciding, diagnosing and treating the affected cases in less than 24 hours and indoor residual spraying respectively. the most important point in all of these strategies is the reduction noted in the cost effectiveness as the incidence declines. as a result, the cost effectiveness of the strategies has been reported to be much lower in our study in comparison with the malaria-endemic areas. the difference is believed to increase as the incidence declines. compared with the cost-effectiveness rate of other researches (table 4), our study had the highest rate. as shown in the table, there is a considerable difference in the our cost-effectiveness rate and the cost per capita which could be due to the difference noted in the fees, effectiveness and the incidence. the latter is the main reason contributing to the difference. this also points out the considerable difference noted in the control and elimination phase. larviciding larviciding is among the strategies long been used in this regard. it had the second rank of cost effectiveness among used intervention but yet it seems so expensive in comparison with other studies (table 5). it is more frequently adopted in areas with limited water resources and obviously the urban areas. the three iranian malaria-endemic provinces are also reported to have low and scattered water resources. the adaptation of the technique in these areas, however, needs to consider its technical feasibility and efficiency. c os t pe r av er te d ca se http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 49 distributing insecticide-treated nets our study showed itn as the most cost effective intervention, and considering its better acceptance in comparison with irs, however it is less cost effective comparing with control setting in other studies (table 5). moreover, its use doesn’t need any specific expertise or skilled human resources. in addition, the longevity of its effects lowers the distribution cost. indoor residual spraying vector control is one of the main components in malaria-elimination programs (greenwood et al. 2008). this intervention had a lesser cost effectiveness and there is a high difference between our finding and similar studies (table 5), furthermore it needs some equipments which make it harder to than other intervention, despite these fact, indoor residual spraying is one of the most important tools in this regard yet. its use, however, is associated with certain challenges including resistance to the poison and not well being accepted in the society. recent improvements in the housing conditions has also lowered the acceptability of spraying, limiting the measure to places used for keeping animals. it should be added that using the techniques in these places worsens the condition through forcing the mosquitoes to move to the places where humans live. diagnosing and treating the patients in less than 24 hours the measure is among the priciest malariacontrol interventions, and this is mainly because of its high cost of human resources. during the elimination phase, while the number of vector-control activities decline that of the healthcare activities increase. referring to the who list for granting the malariaelimination certificate, a vast number of activities should be adopted in the malaria care system, each of which is pricey and time consuming (elimination 2007). their main objec tive is to develop an efficient system for rapid diagnosis and treatment of the patients and at the same time preventing from the spread of the disease in its early stages. as a result, the act, regardless of its cost, is necessary for achieving an elimination phase and hence one should benefit from the available healthcare system to lower the cost of surveillance to the lowest amount possible. for instance, using a delivery system by using motorcycle to send the samples to the laboratories equipped with microscopes can help considerably lower the cost of diagnosis and improve the accuracy of detecting the suspicious samples. this delivery system currently is used in some part the malarial region and it is successful. establishing border posts this strategy is adopted in many parts of the world and there are sufficient evidence supporting its efficacy, particularly when used in confined areas. in this regard, one could highlight the successful attempt in saudi arabia, where the country built several posts on its border with yemen and is paying for more than half of the total cost of the program (meleigy 2007). in view of iran’s long border (2000 km) with pakistan and afghanistan, the adaptation of the strategy seems rather challenging, making it a great concern in the country’s malaria elimination program (tatem et al. 2010). despite all these, establishing such posts play an important role in reducing the number of infected cases entering iran and subsequently lowering the parasite reservoir in the country. hence, despite its high cost, adopting such a measure seems necessary and more research is needed to be conducted to assess its impact. costs as mentioned in figure 1, the establishment of border posts accounted for the highest. during the elimination phase when the incident cases declines, it is expected not to see http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 50 a considerable difference between their effectiveness. as a result, the most important factor in choosing the most appropriate intervention is its technical feasibility and efficiency and its cost. for instance, who stresses that indoor residual spraying is effective only if the whole area is sprayed (najera and zaim 2001). as a result, the strategy would not be effective if less than 80–85% of the area is sprayed and in these conditions, other efficient interventions should be considered. another important point is the composition of costs (fig. 1), we can see in costs of two relatively expensive intervention, border facility and diagnosis and treatment of patients less than 24 hours the most component is human force expense, so applying multifunction staff can be considered an effective strategy to decrease their costs. the present study showed a reduction in the cost-effectiveness of the interventions as the number of affected cases decline. as a result, assessing outcomes such as mortality and the number of affected cases on their own cannot be used as an acceptable indicator of cost-effectiveness and thus different aspects of the disease should be studied. in other words, each country should develop a criteria based on its current condition. it should be noted that success of malaria elimination in many countries needs accepting financial risk which necessitate the paradigm shift to investing in malaria instead of a rapid and striking result (sachs and mcarthur 2005). malaria elimination program needs long term expenses until the disease totally eliminated (sabot et al. 2010). several studies have pointed out that the policymakers should not expect a short-or mid-term positive economic feedback from the program (sabot et al. 2010). the high cost of the program, even at the time when the incidence of the disease is low, is the main point which should be highlighted before launching a control or elimination program. it should be kept in mind that the costs may even increase in the latter condition and this is because of the high charges of intensified surveillance. hence, the policymakers should be informed that several decades may be needed before malaria is completely eliminated and thus they should be committed to support the program for long-term at the beginning (lines et al. 2007). it should be kept in mind that the only and at the same time the most important reason which caused the failure of the malaria program in 1960s was the governments’ irresponsibility regarding the program (hommel 2008). this comes while the results of a control plan are more noticeable than those of the elimination programs in the policymakers’ point of view. as a result, policymakers should be briefed regarding the cost of the program and possible forthcoming challenges. considering the decline noted in the number of malaria cases and uncertainty regarding the efficacy of other interventions in iran, the best measure for fighting malaria should be selected based on technical concerns and the cost. moreover, it is necessary to define a more comprehensive outcome rather than conventional outcome like, morbidity and mortality of malaria for convincing the policy maker to sustain the elimination malaria program. limitations the present study was based on the data gathered from the iranian mohme as well as the expert panels held to discuss specific scenarios regarding the disease. this comes while various variables such as population, area and … may affect the results in the real life and thus may negatively influence the accuracy of our study. despite all this, adopting certain modeling can play an important role in health care decision makings particularly at times when there is not much information available (janssen and martens 1997). it should also be added that the authors failed to find any evidence regarding the efficacy of establishing border posts in fighting malaria. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 43–52 m rezaei-hemami et al.: cost effectiveness of … 51 one of criteria for search strategy was finding evidence of effectiveness in low endemicity region, but yet we expect difference in efficacy in different low endemic area due to variation in climate, health system, ... we, however, had tried to reduce this bias through adopting the most conservative method. conclusion this study showed that interventions in the elimination phase of malaria have low cost effectiveness in iran like many other countries. however itn is the most cost effective intervention among the available interventions. acknowledgements we thank all experts in mohme malaria office and in sistan and baluchestan, kerman and hormozgan provinces. furthermore we especially thank mrs nikpur for her help in this study. this article was part of phd thesis in epidemiology supported by tehran university of medical sciences. the authors of study declare that they have no competing interests. references carrara vi, sirilak s, thonglairuam j, rojanawatsirivet c, proux s, gilbos v, ashley ea, mcgready r, krudsood s, leemingsawat s (2006) deployment of early 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365: 347–353. tatem aj, smith dl, gething pw, kabaria cw, snow rw, hay si (2010) ranking of elimination feasibility between malaria-endemic countries. the lancet. 376: 1579–1591. yukich jo, lengeler c, tediosi f, brown n, mulligan ja, chavasse d, stevens w, justino j, conteh l, maharaj r, erskine m, mueller dh, wiseman v, ghebremeskel t, zerom m, goodman c, mcguire d, urrutia jm, sakho f, hanson k, sharp b (2008) costs and consequences of large-scale vector control for malaria. malar j. 7: 258. doi: 10.1186/1475-2875-7-258. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 1 original article larvicidal, biological and genotoxic effects, and temperature-toxicity relationship of some leaf extracts of nerium oleander (apocynaceae) on culex pipiens (diptera: culicidae) *shaurub h el-sayed, ghada m el-bassiony department of entomology, faculty of science, cairo university, giza, egypt (received 17 mar 2013; accepted 12 july 2014) abstract background: the present study was undertaken to study the larvicidal activity of different extracts of nerium oleander leaves, and post-treatment temperaturetoxicity relationship of these extracts against culex pipiens. further, the most potent extract was used to evaluate its biological and genotoxic activities. methods: crude extracts of n. oleander leaves were prepared using water, chloroform, acetone and diethyl ether as solvents. extraction was carried out using soxhlet apparatus. bioassay test was carried out on the larvae, and the lc50 of each extract was determined. thus, newly hatched first instar larvae were treated, and the mortality count was recorded daily till pupation (accumulated mortality). the lc50 of diethyl ether extract, as the most potent extract, was used for the further biological and genotoxic studies. results: the results obtained indicated that diethyl ether extract of n. oleander leaves was the most potent extract, with lc50 of 10500 mg/l. the toxicity of the four extracts, using the lc50, at 10 °c was higher than that at 35 °c. the lc50 of diethyl ether extract significantly decreased the larval duration, pupal duration, percentage of pupation, percentage of adult emergence, longevity of females, fecundity, and oviposition activity index, whereas the growth index and the percentage of development per day of larvae and pupae were significantly increased compared to nontreated insects. moreover, treatment with this extract induced significant dominant lethality in both male and female adults. conclusion: it appears that diethyl ether extract of n. oleander leaves is potential control agent to cx. pipiens. keywords: culex pipiens, genotoxicity, larvicidal activity, nerium oleander, temperature-toxicity relationship introduction culex pipiens complex inhabits europe, mainly the extra-tropic parts of asia and africa, the middle part of north america, the southern one-third of south america and australia. it is wide spread in egypt, where both autogenous and anautogenous forms co-exist (vinogradova 2000). this mosquito transmits many diseases to man and animals including rift valley fever, lymphatic filariasis (wuchereria bancrofti), st louis encephalitis and west nile virus (vinogradova 2000). prevalence of mosquito-borne diseases is one of the world’s most health hazardous problems. in the absence of an effective vaccine/antiviral therapy, at present vector control is the only way to limit the mosquitoborne diseases (gautam et al. 2013). in this scenario, conventional pesticides such as organophosphorous and pyrethroids are generally used for mosquito control but their indiscriminate usage may cause environmental pollution, residual effect, resistance in mosquito species and adverse effects on human health. these problems forced to search for new control measures especially from plant sources, as plant-derived molecules are ecofriendly, biodegradable, target specific and development of resistance by vectors against them has not been reported so far (gautam et al. 2013). extracts of n. oleander leaves, an *corresponding author: dr shaurub h el-sayed, e-mail: sayedshaurub@yahoo.com http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 2 evergreen flowering shrub which grows in the mediterranean tropical and subtropical regions (raveen et al. 2014), have shown potential in this respect against mosquitoes (komalamisra et al. 2005, lokesh et al. 2010, kumar et al. 2012, roni et al. 2013, madhuri et al. 2013, raveen et al. 2014). the present study aimed to elucidate the larvicidal activity of water, chloroform, acetone and diethyl ether extracts of n. oleander leaves and post-treatment temperaturetoxicity relationship to cx. pipiens. further, the most potent extract was used to evaluate its biological and genotoxic activities. materials and methods insect rearing the stock colony of cx. pipiens complex was maintained in the laboratories of entomology department, faculty of science, cairo university, egypt, at 25±2 °c, 65±5% rh and 12: 12 h (l: d) photoperiod for several years without exposure to insecticides. adults were kept in wooden cages (30× 30× 30 cm) and offered 10% sucrose solution through a piece of sponge for a period of 3–4 days after emergence. females were then allowed to take a blood meal from a pigeon for 2–3 h for egg production. oviposition occurred in a plastic cup (150 ml) containing distilled water (das et al. 2007). the resulting egg rafts were picked up and transferred into plastic dishes (25× 30×15 cm) containing distilled water and covered with muslin cloth. the hatching larvae were fed daily on the tropical fish food (tetramin®). water was aerated daily using an air bubbler to avoid scum formation, and evaporated water was replaced as needed to maintain volume. preparation of stock extracts the leaves of n. oleander were collected from giza governorate, egypt, and identified in botany department, faculty of science, cairo university, in comparison with herbarium sheets of the authentic sample. the plant leaves were thoroughly washed with distilled water, shade-dried at room temperature for about two weeks and powdered finely using a blender. the resulting powder was soaked separately, for a week at room temperature, in a tightly sealed conical flask (one liter) containing water, chloroform, acetone and diethyl ether at a ratio of 1: 4 (w/ v). the solution was occasionally stirred using a magnetic stirrer and extracted using soxhlet apparatus. the extracts were concentrated under the rotary vacuum apparatus, at an evaporator, until the complete solvents evaporated (at 45 °c) to get semi-solid mass of crude extracts. the collected concentrated extracts (stock extracts) were lyophilized (at -80 °c) to obtain solid residue. the extracts were labelled and stored in a refrigerator at 4 °c till use (kumar and yadav 2013, raveen et al. 2014). larvicidal bioassay bioassay of water, chloroform, acetone and diethyl ether extracts of n. oleander leaves was performed to determine the lc50 of each extract against cx. pipiens larvae. five to six serial concentrations of each extract were prepared: 0.50 × 104, 1.00 × 104, 2.00 × 104, 4.00 × 104 and 8.00 × 104 mg/l for larvae treated with water extract, 0.25 × 104, 0.50 × 104, 1.00 × 104, 2.00 × 104 and 4.00 × 104 mg/l for larvae treated with chloroform extract, 0.25 × 104, 0.50 × 104, 1.00 × 104, 2.00 × 104, 4.00 × 104 and 8.00 × 104 mg/l for larvae treated with acetone extract, 0.25 × 104, 0.50 × 104, 1.00 × 104, 2.00 × 104 and 4.00 × 104 mg/l for larvae treated with diethyl ether extract. bioassay was carried out on the first larval instars (ibarra and federici 1987), with minor modifications. twenty five newly hatched first instar larvae were released into each 300 ml plastic cup containing 200 ml of distilled water and test concentration. the extracts were diluted directly in the rearing water (w/v) without solhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 3 vents. a parallel group of non-treated larvae was used as the control. three replicates for each concentration (total n= 75 larvae/ concentration) were undertaken. all experiments were incubated at 25 ˚c. the mortality count was recorded daily until pupation i e, accumulated mortality. effect of temperature on the toxicity of extracts to elucidate the relationship between posttreatment temperature and the toxicity of the above mentioned four extracts of n. oleander leaves to cx. pipiens larvae, two groups per extract of twenty five newly hatched first instar larvae each were released in a plastic cup, as described above, containing the lc50 of each extract (4.82 × 104, 1.91 × 104, 3.00 × 104 and 1.05 × 104 mg/l for water, chloroform, acetone and diethyl ether extract, respectively), as previously determined in the present study (table 1). the two groups were incubated at 10 and 35 ˚c, respectively. a parallel control of non-treated larvae was also run. each experiment was repeated three times. the percentage of accumulated mortality was recorded, as mentioned above. biological effect of diethyl ether extract as the present larvicidal bioassay revealed that diethyl ether extract of n. oleander leaves was the most potent extract (table 1), this extract will be used for the further experiments. twenty five newly hatched first instar larvae of cx. pipiens were treated with the lc50 of this extract (10500 mg/l) and incubated at 25 ˚c. the control experiment consisted of non-treated larvae. the experiment was repeated three times. prior to the end of pupation, the pupae were transferred into adult rearing cages for adult emergence at room temperature, as previously described. the following biological activities were determined for the survivors: larval duration, pupal duration, female fecundity (number of eggs/female), based on the cross mate treated unmated males × treated virgin females, using a fixed number, and longevity of both sexes. moreover, certain biological indices were calculated as follows: growth index= a/b, where: a= % instar survival, b= mean developmental period of the instar in days (moonis 1979), survival index= a/b, where: a= % instar survival, b= maximum survival of the instar in all treatments (moonis 1979), oviposition activity index (oai) = nt nc/ nt + nc, where: nt= number of eggs in treatment, nc = number of eggs in the control, the index values lie within the range -1 to +1 (kuppusamy and murugan 2012). they differentiated the oai according to the resulting values, where negative values indicate that more eggs were deposited in the control than in the treatment, ie the extract is deterrent. conversely, positive values indicate that more eggs were deposited in the treatment than in the control, i e the extract is attractant, % development/day= (1/a) 100, where: a= mean developmental period of the instar in days (powers and oatman 1984). genotoxic effect of diethyl ether extract to evaluate the genotoxic effect of diethyl ether extract of n. oleander leaves on cx. pipiens adults surviving larval treatment with the lc50, the following crossing combinations were tested, using a fixed number: treated unmated males × treated virgin females, normal unmated males × treated virgin females, treated unmated males × normal virgin females, and normal unmated males × normal virgin females. the latter combination was used as the control. each resulting egg raft was collected, allowed to hatch and carefully examined under a suitable magnification. twenty egg rafts per crossing combination was examined. eggs with open opercula were considered as hatched, while those with closed ones were taken as unhatched. the number of unhatched eggs per egg raft was taken as the measure for calhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 4 culating the dominant lethality, as follows: % frequency of induced dominant lethality= (a/b) 100, where: a= number of unhatched eggs in one egg raft, b = total number of eggs in the egg raft (bhinder and chaudhry 2013). statistical analysis the crude mortality obtained from the larvicidal bioassay was corrected using abbott’s formula (1925). the corrected mortality was then subjected to probit analysis (finney 1971) for determining the lc50 of each extract. in the experiments of temperature-toxicity relationship, chi-square test (χ2) was carried out for each temperature regime, assumed that the applied lc50 will give 50 % mortality at the two temperatures tested. in the experiments of biological and genotoxic effects, student’s t-test was undertaken between treated and non-treated (control) experiments. all of the analyses were carried out using statistical package social science (spss) software version 11.5 (spss 2007). significant level was set at p< 0.05. results larvicidal activity the insecticidal activity of water, chloroform, acetone and diethyl ether crude extracts of n. oleander leaves to cx. pipiens larvae revealed that diethyl ether extract was the most potent extract, with lc50 of 10500 mg/l (table 1). this extract was about 4.59, 1.82 and 2.86 folds as toxic as water, chloroform and acetone extract, respectively. in contrast, water extract was the least toxic extract, with the lc50 value of 48200 mg/l. this indicates that water extract of n. oleander leaves might be unsuitable for controlling cx. pipiens. effect of post-treatment temperature table 2 shows that exposure of the lc50 of water, chloroform, acetone and diethyl ether extracts of n. oleander leaves to 10 °c enhanced the mortality of cx. pipiens larvae by about 24.10, 39.16, 47.80 and 78.80 % of the expected mortality (50%), respectively. this enhancement was significant (p< 0.05) in case of acetone and diethyl ether extracts. on the contrary, exposure of the lc50 of water, chloroform and acetone extracts to 35 °c showed about 23.34, 28.34 and 21.68 % decline in the larval mortality, while exposure of the lc50 of diethyl ether extract to the same temperature regime increased (~3.32%) the larval mortality relative to the expected mortality. chi-square test (χ2) indicated that the change in the larval mortality at 35 °c was insignificant. biological effect of diethyl ether extract application of the lc50 of diethyl ether extract of n. oleander leaves, as the most potent extract, on the first instar larvae of cx. pipiens significantly decreased (p< 0.05) the resulting larval duration, pupal duration, percentage of pupation, percentage of adult emergence, and fecundity compared to the control (table 3). the reduction in fecundity was about 36.57 % of the control. the longevity of treated adults differed with the sex, where the longevity of males was insignificantly increased, whereas that of females was significantly shortened (p< 0.05) to about 45.58 % of the control. this treatment increased the growth index of both larvae and pupae to about 13.05 and 18.01 % of the control, respectively, and the percentage of development per day of larvae and pupae to about 40.10 and 48.26 % of the control, respectively. in contrast, the survival index of larvae and pupae was reduced to 19.00 and 20.00 % of the control, respectively. oviposition activity index was consistent with the reduction in fecundity, where it recorded about -0.22 (table 4). mutagenic effect of diethyl ether extract the egg hatchability of cx. pipiens surviving http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 5 larval treatment with the lc50 of diethyl ether extract of n. oleander leaves was significantly reduced (p< 0.05) in all the crossing combinations compared to the control, except for the combination containing treated males mated with untreated females, where such decrease was insignificant (table 5). the highest decline in egg hatchability was attained in the crossing combination containing both treated sexes, followed by that containing treated females, and then that containing treated males. in turn, the percentage of unhatched eggs was significantly increased (p< 0.05) in all the crossing combinations compared to the control, with the highest magnitude in the crossing combination containing both treated sexes, followed by that containing treated males, and then that containing treated females (table 5). table 1. toxicity of water, chloroform, acetone and diethyl ether extracts of nerium oleander leaves to culex pipiens larvae at 25 °c extract lc50 (mg/l) 95% confidence limits (mg/l) slope χ2 (d.f.) lower upper water 4.82×104 3.70×104 13.68×104 0.05 3.05 (3) chloroform 1.91×104 0.21×104 7.71×104 1.09 2.02 (3) acetone 3.00×104 0.89×104 10.09×104 2.44 1.08 (4) diethyl ether 1.05×104 0.33×104 3.35×104 3.04 2.30 (3) table 2. temperature-toxicity relationship of water, chloroform, acetone and diethyl ether extracts of nerium oleander leaves to culex pipiens larvae, previously treated with the lc50 of each extract at 25 °c extract temperature 10°c 35°c % mortality χ2 (d.f.) % mortality χ 2 (d.f.) water 62.05 2.90 (4) 38.33 2.72 (4) chloroform 69.58 7.67 (4) 35.83 4.02 (4) acetone 73.90 11.42* (4) 39.16 2.35 (4) diethyl ether 89.40 31.05* (4) 51.66 0.06 (4) * significant at p< 0.05. table 3. biological activities of culex pipiens surviving larval treatment with the lc50 of diethyl ether extract of nerium oleander leaves at 25 °c biological activity control (mean ± s e) treated (mean ± s e) larval duration (days) 8.32±0.43 5.94*±0.07 pupal duration (days) 4.36±0.26 2.94*±0.07 % pupation 95.00±0.26 76.70*±2.77 % adult emergence 96.60±1.71 76.88*±2.56 male longevity (days) 12.33±0.88 15.67±1.45 female longevity (days) 19.00±1.53 10.34*±0.40 female fecundity (no. of eggs/♀) 216.00±8.47 137.00*±8.89 * significant at p< 0.05, using student’s t-test. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 6 table 4. growth index, survival index, percentage of development per day and oviposition activity index of culex pipiens surviving larval treatment with the lc50 of diethyl ether extract of nerium oleander leaves at 25 °c experiment growth index survival index % development/day %oviposition activity indexlarvae pupae larvae pupae larvae pupae control 11.42 22.16 1.00 1.00 12.02 22.94 treated 12.91 26.15 0.81 0.80 16.84 34.01 -0.22 table 5. genotoxic effect of diethyl ether extract of nerium oleander leaves to culex pipiens male and female adults surviving larval treatment with the lc50 of the extract at 25 °c crossing combination egg hatchability ± s e (no. of hatched eggs/egg raft) % frequency of unhatched eggs ± s e control 172.00a±8.37 5.15a ±0.28 n ♂♂ × t ♀♀ 123.05b±11.76 8.94b±1.27 t ♂♂ × n ♀♀ 158.65a±12.03 11.37b±1.78 t ♂♂ × t ♀♀ 113.33b±8.68 15.91c±0.71 figures followed by different letters are significantly different from each other (p< 0.05), using student’s t-test. n= normal, t= treated. control = n ♂♂ × n ♀♀. discussion different parts of plants contain a complex of chemicals with unique biological activity (farnsworth and bingel 1977). preliminary screening is a good approach to evaluate the potential larvicidal activity of plants (sakthivadivel and daniel 2008, arivoli et al. 2012, tennyson et al. 2012), and the activity of crude plant extracts subjected further to partial purification with respective solvent washed fraction is often distributed to the complex mixture of active compounds (sakthivadivel and daniel 2003). in this context, qualitative phytochemical analysis of n. oleander leaves confirmed the presence of various phytochemicals like carbohydrates, cholesterol, protein, amino acids, alkaloids, flavonoids, tannins, saponins, cardiac glycosides, terpenoids, and phlobatinins in their aqueous extract followed by ethanol, ethyl acetate, diethyl ether and chloroform (santhi et al. 2011, kumar and yadav 2013). in comparison, the phytochemicals in methanolic leaf extract of ervatamia coronaria (apocynaceae) are alkaloids, saponins, tannins, steroids and flavonoids (mathivanan et al. 2010). the larvicidal activity of the different extracts of n. oleander leaves against cx. pipiens in the present study is supported by the findings of komalamisra et al. (2005) who reported larvicidal activity of ethanolic extract of the leaves of this plant against aedes aegypti with lc50 of 197.97 mg/l. roni et al. (2013) reported that the lc50s of water leaf extract of n. oleander against the 1st, 2nd, 3rd , and 4th larval instars of anopheles stephensi were 232.90, 273.71, 318.94, and 369.96 ppm, respectively. also, raveen et al. (2014) attained 24 h lc50 of 102.54 and 2758.87 ppm for hexane and water flower extracts of n. oleander against cx. quinquefasciatus, respectively. similarly, the larvicidal activity of the leaf extracts of other plants of family apocynaceae were reported by some authors. for instance, mathivanan et al. (2010) found that 24 h lc50 value of methanolic leaf extract of e. coronaria against cx. quinquefasciatus was 72.41 mg/l, while sakthivadivel et al. (2014) showed that 24 h lc50 of water and petroleum ether leaf extracts of wrightia tinctoria against the http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 7 same vector was 0.21 and 0.37 %, respectively. the differential responses induced by phytochemicals on various species of mosquitoes were influenced by extrinsic and intrinsic factors such as the species of the plant, the part of the plant, the geographical location where the plants were grown, photosensitivity of the compounds in the extract, the methods used for extraction, and the solvent used for extractions (sukumar et al. 1991, shaalan et al. 2005). as to the latter factor, polar solvents will extract polar molecules and non-polar solvents will extract non-polar molecules (rawani et al. 2010). although oleanders contain cardenolides, and the basis for their physiological action is similar to that of the classic digital glycosides, i e inhibition of plasma membranes na+k+atpase, there are differences in toxic and cardiological effects between the oleanders and digital cardenolides, thus the human mortality associated with oleander ingestion is generally very low, even in cases of intensive uptake (suicide attempts) (langford and boor 1996). moreover, it was pointed out that the active ingredient in the ethanolic extract of n. oleander leaves is the glycoside neriifolin which displays very slight mammalian cytotoxicity and negligible mutagenicity (el-shazly et al. 2000). these findings indicate that field application of n. oleander leaf extracts in controlling cx. pipiens should be safe. the toxicity of the extracts of n. oleander leaves were affected by post-treatment temperature in a manner similar to that reported for pyrethrin and pyrethroids ie, the toxicity increased with the decrease in post-treatment temperature (sparks et al. 1982, 1983). the relationship between temperature and the toxicity of an insecticide is a complex matter, where absorption of the insecticide, permeability through the cuticle, distribution within the insect body, detoxification and excretion are affected by the change in temperature (hinks 1985). temperature should be included as an important factor in the decision-making process in situations where comparable products from multiple insecticide classes are available (musser and shelton 2005). the increase in the toxicity of diethyl ether extract of n. oleander leaves in the current investigation after exposure to both 10 and 35 °c may indicate that this extract will be a potential agent in controlling cx. pipiens in both winter and summer. the decrease in the larval duration of cx. pipiens after treatment with diethyl ether extract of n. oleander leaves is in agreement with the results of el-sheikh et al. (2011), who reported a significant decrease in the larval duration of the same vector after treatment with diethyl ether extract of cupressus sempervirens leaves. on the contrary, prolonged larval duration of ae. aegypti was reported after treatment with methanolic extract of nerium indicum leaves (mohtar et al. 1999). the dramatic reduction in the female longevity of cx. pipiens to about half of the control (45.58%) in the present investigation might render this species less potential vector. the suppression of fecundity due to treatment with diethyl ether extract of n. oleander leaves may be attributed to the suppression of uptake of vitellogenin from the haemolymph. elevation in oostatic hormone in mosquitoes suppresses trypsin biosynthesis in the cells of the mosquito’s midgut, inhibits the action of juvenile hormone on vitellogenic follicle cells and prevents the ovary from accumulating vitellogenin from the haemolymph during yolk deposition (borovsky 1988). the latter process in mosquitoes includes a series of metabolic changes that is occurred by changing in the composition of haemolymph proteins (clements 1992). the increase in the growth index and the percentage of development per day of cx. pipiens larvae and pupae treated, starting from the first instar larvae, with the lc50 of diethyl ether extract of n. oleander leaves in http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 1–11 sh el-sayed and gm el-bassiony: larvicidal, biological … 8 the current study is suggestive of an attempt by the treated insects to avoid/tolerate the toxic effect of this extract. in this context, it was found that as an. stephensi larvae increased in age and weight, starting from the first larval instars to pupae, their tolerance to water extract of n. oleander leaves increased (roni et al. 2013). the increase in the percentage of development per day in cx. pipiens larvae and pupae treated with diethyl ether extract of n. oleander leaves might be attributed to their lower durations compared to the control, where the development per day is the inverse of the duration (powers and oatman 1984). on the other hand, the decrease in the survival index of treated larvae and pupae might reflect the decrease in the percentage of pupation and adult emergence, respectively. the oviposition activity index indicated that diethyl ether extract of n. oleander leaves was oviposition deterrent, according to the formula of kuppusamy and murugan (2012). it seems likely that diethyl ether extract of n. oleander leaves is potential mutagen for the genome of cx. pipiens, where it induced significant dominant lethality. similarly, water leaf extract of azadirachta indica, momodrica charantia, murraya exotica, and caspicum frutescens decreased the egg hatchability in cx. quinquefasciatus (bashar et al. 2011), and the organophosphate insecticides, acephate and chlorpyrifos induced significant dominant lethality in the same mosquito species (bhinder and chaudhry 2013). the genetic basis of dominant lethality is mainly the induction of structural and numerical chromosomal anomalies which tend to induce non-viable zygotes, early embryonic death, sterility and semi-sterility in the offsprings of effected parents (chaubey et al. 1999). pesticides significantly increase the cellular reactive oxygen species production which causes chemical modifications and alterations in dna and nucleoproteins, including modified bases and sugars and even strand breaks leading to chromosomal aberrations (hreljac and filipic 2009). although the second and third most highest decrease in the egg hatchability was obtained in the crossing combination containing treated females and that containing treated males, respectively, these two combinations showed the third and second most highest increase in the percentage of frequency of unhatched eggs, respectively (table 5). this apparent conflict might be due to the variations in the total number of eggs in the egg rafts, based on the formula of bhinder and chaudhry (2013). conclusion the present study explores the potential role of the different leaf extracts of n. oleander as control agents to cx. pipiens. diethyl ether extract of n. oleander leaves should reduce the population dynamics of cx. pipiens, either directly through larval kill, or indirectly through its latent effects expressed in reduction of survival, fecundity and hatchability, and induction of dominant lethality in the subsequent generation. this extract can be considered for use in future integrated management strategies of cx. pipiens. acknowledgements the authors are grateful to professor dr moataza a dorrah, department of entomology, faculty of science, cairo university, for her help during the preparation of the manuscript. the authors declare that there is no conflict of interests. references abbott ws (1925) a method of computing the effectiveness of insecticide. j econ entomol. 18: 265–267. 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http://jad.tums.ac.ir published online: june 30, 2021 original article population fluctuations and abundance indices of mosquitoes (diptera: culicid), as the potential bridge vectors of pathogens to humans and animals in mazandaran province, northern iran seyed hassan nikookar1; *mahmoud fazeli-dinan1; *ahmadali enayati2 1department of medical entomology and vector control, health sciences research center, addiction institute, school of public health, mazandaran university of medical sciences, sari, iran 2department of medical entomology and vector control, school of public health and health sciences research center, mazandaran university of medical sciences, sari, iran *corresponding authors: dr mahmoud fazeli-dinan, e-mail: fazelidinan@gmail.com, dr ahmadali enayati, email: aenayati@mazums.ac.ir (received 1 dec 2020; accepted 19 may 2021) abstract background: seasonal activity patterns of mosquitoes are essential as baseline knowledge to understand the transmission dynamics of vector-borne diseases. this study was conducted to evaluate the monthly dynamics of the mosquito populations and their relation to meteorological factors in mazandaran province, north of iran. methods: mosquito adults and larvae were collected from 16 counties of mazandaran province using different sampling techniques, once a month from may to december 2014. “index of species abundance” (isa) along with “stand ardized isa” (sisa) was used for assessing the most abundant species of mosquitoes based on the explanations of robert and hsi. pearson’s correlation coefficient (r) was used to assess the relationships between the monthly population fluctuations and meteorological variables. results: overall, 23750 mosquitoes belonging to four genera and nineteen species were collected and identified. the highest population density of mosquitoes was in july and the lowest in may. the isa/sisa indices for culex pipiens were both 1 for larvae and 1.25/0.973 for adults in total catch performed in human dwellings. for cx. tritaeniorhynchus, the isa/sisa were 1.68/0.938 in pit shelter method. a significant positive correlation was observed between population fluctuations of cx. tritaeniorhynchus and mean temperature (r: 0.766, p< 0.027). conclusions: the results indicated that the mosquitoes are more active in july, and cx. pipiens and cx. tritaeniorhynchus were the most abundant species. considering the potential of these species as vectors of numerous pathogens, control programs can be planed based on their monthly activity pattern in the area. keywords: seasonal activity; mosquitoes; abundance indices; mazandaran; caspian sea littoral introduction mosquitoes are distributed almost all over the world, except a few islands and the antarctica (1). since the earliest times, mosquito bites and habitats have been related with human diseases, and mosquitoes were the first arthropods formally convicted as intermediate hosts of vertebrate parasites in 1878 (2). they are the most important arthropod taxon in medical entomology because of their nuisance and transmission of malaria, arboviral diseases and microfilariae (3). mosquito-borne infectious dis eases are known as the most commonly transmitted diseases by vectors in terms of mortality and disability-adjusted life years (4). malaria is one of the most important anophelesborne parasitic diseases in the world. the disease has a massive burden universally, an estimated number of cases of 229 million and 409000 deaths occurred in 2019 (5). there was also approximately 96 million cases and 1091 deaths related to dengue globally. albeit, the global burden of zika, chikungunya and west copyright © 2021 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:fazelidinan@gmail.com mailto:aenayati@mazums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 208 http://jad.tums.ac.ir published online: june 30, 2021 nile is not as large as malaria and dengue (4), but their impact on health system is high, especially as several large outbreaks of the diseases occur every year and their transmission is extending to new areas (6-8). iran is in the perspective of eliminating malaria by 2025. although there were 1105 imported cases of malaria in 2019, no indigenous malaria cases were reported in iran since 2018 (5). thirty species of anopheles have been documented from iran (9), of which, seven species have been reported from mazandaran province (10). anopheles sacharovi, an. maculipennis s.l., an. fluviatilis s.l., an. stephensi, an. superpictus s.l., an. dthali, and an. culicifacies s.l. are identified as the proven malaria vectors in iran (11), while an. pulcherrimus is stated as a suspected vector (12). recently, an. hyrcanus and an. subpictus s.l. were revealed to be infected with plasmodium based on molecular analysis in northern and southern iran (13, 14). anopheles maculipennis s.l. and an. sacharovi are known to play an important role in transmission of malaria in the northern parts of the country (15). zika, dengue and chikungunya are arboviral diseases transmitted by aedes mosquitoes, especially ae. aegypti and ae. albopictus. there are no reports of zika virus in iran, while imported cases of dengue (16-18) and chikungunya (19) have lately been reported from the country. aedes aegypti and ae. albopictus are the main vectors of dengue, chikungunya and zika, worldwide. aedes aegypti had been reported in southern parts of iran in 1920–1953 (20-22), however, it retreated to arabian peninsula and northern africa since for no known reason. recently, only a few adults and larvae of ae. albopictus has been observed in sistan and baluchistan province, southeastern iran (23). knowledge about the behavior of mosquitoes is very important in the epidemiology of disease transmission and vector control. data on fluctuations of seasonal abundance of species can describe their relative risk in the transmission of diseases in human populations, and can also help in the planning and implementation of proper control programs (24). environmental changes greatly affect the habitat of mosquito species (25). meteorological factors also affect the population of mosquitoes by quantitative and qualitative changes on the larval habitats (25, 26). the combination of mosquito behavior pattern (circadian rhythmicity) with climate factors makes a foundation for determining the timing/month/season of mosquito activities (27, 28). therefore, determining the seasonal prevalence of mosquito fauna in an area is crucial for the development of effective vector control programs and updating of ecological information related to vectors of diseases in the area (25, 29). there were little published data on the seasonal abundance of mosquitoes in iran (30-32). up to now, fauna, checklist, physicochemical factors of larval habitats, co-occurrence, association, affinity and biodiversity of mosquitoes have been studied in mazandaran province (10, 33-35), however, there is no recent comprehensive study on the seasonal activity of mosquitoes in the province. therefore, this study was conducted to evaluate the monthly dynamics of the mosquito populations and their relation to climatic factors in mazandaran province. materials and methods study area mazandaran province is located on the coast of the caspian sea in northern iran, in coordinate latitude 35°47′–36°35′n and longitude 50°34′–54°10′e between the caspian sea and the alborz mountain range. the province is enclosed by golestan province in the east, guilan province in the west and tehran and semnan provinces in the south and the caspian sea to the north. the diverse nature of the province included plains, grassland, forests and rainforest with an area of approximately 23,842 square kilometers and a population of approximately 3,073,943. in the study area, the main agricultural products are rice, followed by wheat, http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 209 http://jad.tums.ac.ir published online: june 30, 2021 barley, beans, fruits and vegetables. vast hyrcanian forests, temperate climate, abundant wetlands and rice fields in the province, provide enormous adequate habitats for the development of mosquitoes. specimen and data collection mosquitoes were collected from 30 villages in 16 counties from mazandaran province, in a uniform method, once a month from may to december 2014. in each county, two villages were randomly designated for sampling mosquitoes according to topography of areas. in each village, one fixed habitat was selected for collection of larvae. larval collection was conducted by a standard 350ml dipper for 15– 20min per natural and artificial breeding sites in fixed habitats. in the larval habitat, 10–30 dips were taken based on the size of the breeding site. samples were always taken by the same individual in the morning (0800–1200 hours) or afternoon (1400–1800 hours). adult mosquitoes were collected in eight places (three human and three animal fixed and one human and one animal variable places) between 0500 and 0800 hours in selected villages by methods of total catch. before spraying, all the eaves, windows, doors and other exit points were closed and white cloth sheets were spread on the floor. after pyrethrum spraying, the room was kept closed for 10min and the knocked-down mosquitoes were then collected from the floor sheet. pit shelters (90× 150× 150cm w× l× d) were dug in shady places in each village, then small cavities, about 30cm deep were dug horizontally at each side, about 50cm above the bottom of the pit. the collections of resting mosquitoes were carried out by aspirator from the walls of the pits between 0500 to 0600 hours. cdc light traps (john w. hock company, gainesville, fl) were set about 1.5–2m above the ground in human and animal dwellings in each village. light traps were switched on at 1900 and switched off at 0600 hours local time. the mosquitoes were attracted to the light at night, and were arrived to funnel nets screen using the airflow generated by the fan motor. human-landing collections (two human baits and one collector) using mouth aspirators were performed for sampling of aedes, during the daytime between 0900 and 2100 hours, in each village. sampling teams were stationed mostly between human and animal sites near appropriate larval habitats. the mosquitoes were collected at 1h intervals and were placed in paper cups relevant to the same hour. the third and fourth instar larvae were preserved in lactophenol solution and adults were transferred to cups container with moist cotton, labeled and mounted (10). the specimens were identified by direct observation of morphological characters using valid taxonomic keys (36). the specimens are deposited at the museum of medical entomology, school of health, mazandaran university of medical sciences, sari, iran. the abbreviations of mosquito genera and subgenera are cited by reinert (37). meteorological data monthly meteorological variables containing mean temperature and rainfall were obtained from the synoptic stations of mazandaran province meteorological organization in 2014. abundance indices “index of species abundance” (isa) was used to assess the most abundant species of mosquitoes in the province based on the explanations of robert and hsi (38) with a minor modification in the formula. isa is calculated by the following formula: where ‘a’ is the number of sampling sites that the species not present in it, ‘c’ is the highest rank of the species in sampling sites plus 1, and ‘rj’ is the sum of the rankings of each species in all sampling sites, ‘k’ is the number of sampling sites. “standardized index of species abundance” (sisa) was applied as a standardized formula http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 210 http://jad.tums.ac.ir published online: june 30, 2021 for isa by converting to a scale from zero to one with the following changes: when sisa is closer to 1, it represents the most abundant species (39). statistical analysis statistical analysis was performed on data obtained from mosquito population density during the study to understand whether meteorological variables could be the cause of population fluctuations of mosquitoes in the study area. the data were calculated using spss ver. 23. the relationships between monthly population fluctuations of mosquitoes with meteorological variables were evaluated by statistical test of “pearson’s correlation coefficient (r)”. results overall, 23,750 mosquitoes (7,566 larvae and 16,184 adults) belonging to two subfamilies, four genera and nineteen species were collected and identified throughout the year during this study. the subfamily anophelinae was represented by one genus and 7 species, while the subfamily culicinae was in 3 genera and 12 species. monthly fluctuations in population dynamic of mosquitoes are displayed in (fig. 1, table 1 and 2). the highest total number of larvae (2,383) and adult (4,723) mosquitoes were observed in july and the lowest in may (fig. 1). among the larvae, an. maculipennis s.l. and an. pseudopictus were collected during each month except may and december, respectively, while an. hyrcanus was observed only during june–october. the population density of an. maculipennis s.l. reached its peak in june (n= 238). after june, the population density of the species with an irregular fluctuation decreased rapidly and the lowest density was in september (n= 5). the highest population peak of an. hyrcanus was quite similar to that of an. maculipennis s.l, while it was in july for an. pseudopictus. culex pipiens and cx. torrentium was found from may to december. the largest population density of this species was observed in july (n= 1670) and june (n= 287) respectively. culex tritaeniorhynchus was collected from may to november, with a major peak in july. culex perexiguus and cx. mimeticus were almost non-active in the first and last seasons of the year and had the maximum number in july. culiseta annulata and cs. longiareolata were almost absent during the warm seasons of the year and their highest activity peaks were recorded in october and november, respectively. anopheles marteri, an. claviger and cs. morsitans were not found in adequate numbers to draw their monthly activity patterns (table 1). among the adults, an. maculipennis s.l. was present almost throughout the year except for december, the population dynamics of this species starts in may, reaching a major peak in june and then decreases gradually. the population density of an. hyrcanus begins to increase in may, reaching its greatest peak in june and after that, decreased. the species disappeared from monthly sampling in august, and was observed with the lowest population density again in september. anopheles pseudopictus was recorded during june to november, its highest and lowest population peak was in july (n= 914) and november (n= 40), respectively. the density of an. claviger and an. sacharovi was peaked during august (n= 28) and july (n= 87), respectively. culex pipiens was successively found throughout the monthly sampling period with major peak in july. since july, the population of the species decreased gradually in august and september, then the activity increases and reaches the smaller peak in december. the population density of cx. tritaeniorhynchus among the collected mosquitoes was high in july (n= 1868), it decreased in august (n= 1288), increased again to the smaller population peak in september (n= 1820), finally decreased progressively to the end of the season. culex perexiguus, ae. vexans and cs. anhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 211 http://jad.tums.ac.ir published online: june 30, 2021 nulata had the most activity in june, october and november in the study area. the rest of the species were collected in low numbers, so it was not possible to predict a proper pattern of monthly population fluctuations for them in the study area (table 2). the highest mean temperature and rainfall was observed during the months of august and october, respectively (fig. 2). bivariate pearson’s correlation analyses of mosquito population density with meteorological variables exhibited that the population fluctuations of cx. territans (r= 0.855, p= 0.007), cs. annulata (r= 0.711, p= 0.0048), cs. longiareolata (r= 0.826, p= 0.011) and ae. vexans (r= 0.831, p= 0.011) have significant positive correlation with rainfall in the study area. however, no significant association was observed between other mosquito species and rainfall. the monthly temperature showed a significant positive correlation with the adult population fluctuations of cx. tritaeniorhynchus (r= 0.766, p= 0.027). as for other species, the temperature was not seen as an important variable in population fluctuations in the study area (table 3). the interaction between mosquito population fluctuations with mean monthly temperature and rainfall is shown in (fig. 2). the highest number and percentage of mosquitoes were collected by total catch in animal places (n:8051, 49.74%) followed by 16.82% using light trap, 14.87% by total catch in human places, 11.15% using pit shelter and 7.42% by day biting. among anophelinae, an. maculipennis s.l. (n: 1555, 86.97%) and an. pseudopictus (n: 680, 52.84%) were the most common in total catch of animal places and the least in daily bites and pit shelter, respectively, while an. claviger and an. sacharovi were collected with the highest number and percentage using pit shelter sampling method. among culicinae, cx. tritaeniorhynchus is more common than cx. pipiens using total catch in animal places and light trap, whereas the species was observed with the lowest number and percentage by total catch in human places and pit shelter than cx. pipiens. aedes vexans were collected by all sampling methods, but the species was collected with the highest percentage up to 94.5% (n: 1200) in day biting collections. further data on other species collected by each trap are shown in table 4. based on table 5, larvae of cx. pipiens, cx. torrentium and cx. tritaeniorhynchus showed values of sisa 1 (isa= 1), 0.805 (isa= 3.62) and 0.564 (isa= 6.87), respectively, whereas it was 0.550 (isa= 7.06) for an. maculipennis s.l. among adult mosquitoes, the highest sisa (0.822, 0.637) and the lowest isa (3.31, 5.71) were calculated for an. pseudopictus and an. maculipennis s.l. in total catch performed in animal shelters, respectively. sisa was 0.977 (isa= 1.25), 0.946 (isa= 1.594) and 0.933 (isa= 1.87) in association with cx. pipiens in total catch performed in human dwellings, pit shelter and total catch performed in animal shelters, respectively. it was 0.938 (isa= 1.68) in pit shelter, 0.938 (isa= 1.69) in light trap, 0.913 (isa= 2.125) in total catch carried out in animal places and 0.886 (isa= 2.25) in total catch performed in human dwellings for cx. tritaeniorhynchus. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 212 http://jad.tums.ac.ir published online: june 30, 2021 table 1. monthly changes in population fluctuations of mosquito larvae in mazandaran province, northern iran may june july august september october november december total species no % no % no % no % no % no % no % no % no % an. claviger 0 0 0 0 0 0 15 100 0 0 0 0 0 0 0 0 15 100 an. hyrcanus 0 0 16 40 2 5 9 22.5 9 22.5 4 10 0 0 0 0 40 100 an. maculipennis s.l. 0 0 181 47.14 80 20.83 30 7.81 5 1.30 14 3.64 64 16.67 10 2.60 384 100 an. pseudopictus 0 0 11 7.14 61 39.61 13 8.44 35 22.73 19 12.34 15 9.74 0 0 154 100 an. marteri 0 0 0 0 1 100 0 0 0 0 0 0 0 0 0 0 1 100 cx. pipiens 75 1.52 1197 24.27 1670 33.87 527 10.69 818 16.59 261 5.29 277 5.61 106 2.15 4931 100 cx. torrentium 1 0.16 287 46.22 154 24.8 20 3.22 17 2.74 76 12.24 53 8.53 13 2.09 621 100 cx. tritaeniorhynchus 2 0.27 4 0.53 324 43.32 139 18.58 213 28.48 58 7.75 8 1.07 0 0 748 100 cx. perexiguus 0 0 0 0 32 65.31 5 10.2 4 8.16 8 16.33 0 0 0 0 49 100 cx. territans 0 0 0 0 1 1.59 0 0 0 0 44 69.84 18 28.57 0 0 63 100 cx. mimeticus 1 1.19 0 0 54 64.29 22 26.19 1 1.19 6 7.14 0 0 0 0 84 100 cx. hortensis 1 20 0 0 0 0 0 0 4 80 0 0 0 0 0 0 5 100 cs. annulata 15 8.88 27 15.98 0 0 0 0 0 0 60 35.5 67 39.64 0 0 169 100 cs. longiareolata 0 0 89 29.57 4 1.33 0 0 0 0 119 39.53 80 26.58 9 2.99 301 100 cs. morsitans 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 100 1 100 total 95 1.26 1812 23.95 2383 31.5 780 10.31 1106 14.62 669 8.48 582 7.69 139 1.83 7566 100 table 2. monthly changes in population fluctuations of adult mosquitoes in mazandaran province, northern iran may june july august september october november december total species no % no % no % no % no % no % no % no % no % an. claviger 8 14.55 0 0 2 3.63 28 50.91 14 25.45 3 5.46 0 0 0 0 55 100 an. hyrcanus 14 9.93 95 67.38 21 14.89 0 0 2 1.42 9 6.38 0 0 0 0 141 100 an. maculipennis s.l. 20 1.12 889 49.72 652 36.47 148 8.28 44 2.46 31 1.73 4 0.22 0 0 1788 100 an. pseudopictus 0 0 54 4.19 914 71.02 109 8.47 90 6.99 80 6.22 40 3.11 0 0 127 100 an. marteri 2 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 100 an. sacharovi 0 0 1 0.89 87 77.68 22 19.65 1 0.89 0 0 1 0.89 0 0 112 100 an. superpictus 0 0 0 0 5 55.56 0 0 0 0 0 0 4 44.44 0 0 9 100 cx. pipiens 426 8.26 918 17.8 1134 21.99 612 11.87 285 5.53 420 8.14 562 10.9 800 15.51 5157 100 cx. tritaeniorhynchus 0 0 379 6.14 1868 30.26 1288 20.87 1820 29.48 607 9.84 210 3.40 1 0.01 6173 100 cx. perexiguus 23 25.56 47 52.22 15 16.67 0 0 0 0 5 5.55 0 0 0 0 90 100 cx. mimeticus 2 16.67 2 16.67 5 41.66 0 0 3 25 0 0 0 0 0 0 12 100 cx. hortensis 0 0 0 0 1 100 0 0 0 0 0 0 0 0 0 0 1 100 ae. vexans 5 0.39 0 0 11 0.87 9 0.71 4 0.31 1202 94.65 39 3.07 0 0 1270 100 ae. caspius 7 18.42 21 55.26 8 21.05 0 0 0 0 1 2.63 0 0 1 2.63 38 100 cs. annulata 1 2.04 0 0 0 0 2 4.08 0 0 2 4.08 44 89.8 0 0 49 100 total 508 3.14 2406 14.87 4723 29.18 2218 13.70 2263 13.98 2360 14.58 904 5.59 802 4.96 16184 100 http://jad.tums.ac.ir published online: june 30, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 213 http://jad.tums.ac.ir published online: june 30, 2021 table 3. pearson’s correlation coefficient (r) of mosquito population density with meteorological variables in mazandaran province, northern iran species temperature rainfall larvae r p r p an. claviger 0.394 0.335 -0.374 0.361 an. hyrcanus 0.630 0.094 -0.143 0.735 an. maculipennis s.l. 0.236 0.574 0.001 0.998 an. pseudopictus 0.548 0.159 -0.263 0.529 cx. pipiens 0.666 0.710 -0.364 0.379 cx. torrentium 0.314 0.449 0.136 0.747 cx. tritaeniorhynchus 0.667 0.071 -0.478 0.231 cx. perexiguus 0.441 0.274 -0.179 0.672 cx. territans -0.260 0.534 0.855 0.007 cx. mimeticus 0.496 0.211 -0.350 0.395 cs. annulata -0.400 0.326 0.711 0.048 cs. longiareolata -0.238 0.571 0.826 0.011 adults an. claviger 0.570 0.140 -0.546 0.162 an. hyrcanus 0.295 0.478 0.051 0.904 an. maculipennis s.l. 0.489 0.218 -0.166 0.694 an. pseudopictus 0.426 0.293 -0.289 0.488 an. sacharovi 0.444 0.270 -0.379 0.354 cx. pipiens 0.060 0.887 -0.115 0.786 cx. tritaeniorhynchus 0.766 0.027 -0.475 0.234 cx. perexiguus 0.292 0.483 -0.48 0.911 cx. mimeticus 0.584 0.128 -0.523 0.183 ae. vexans -0.086 0.840 0.831 0.011 ae. caspius 0.298 0.474 -0.066 0.877 cs. annulata -0.475 0.235 0.140 0.741 fig. 1. the total number of mosquitoes collected by month in mazandaran province, northern iran http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 214 http://jad.tums.ac.ir published online: june 30, 2021 table 4. number and percentage of adult mosquitoes collected based on sampling method sampling method total catch in human places total catch in animal places pit shelter light trap daily biting total no % no % no % no % no % no % an. claviger 11 20 20 36.36 24 43.64 0 0 0 0 55 100 an. hyrcanus 7 4.97 89 63.12 2 1.42 44 31.21 0 0 141 100 an. maculipennis s.l. 91 5.09 1555 86.97 41 2.29 101 5.65 0 0 1788 100 an. marteri 0 0 2 100 0 0 0 0 0 0 2 100 an. pseudopictus 88 6.84 680 52.84 81 6.30 438 34.03 0 0 1287 100 an. sacharovi 0 0 2 1.78 108 96.44 2 1.78 0 0 112 100 an. superpictus 0 0 9 100 0 0 0 0 0 0 9 100 cx. pipiens 1437 27.87 2225 43.14 870 16.87 625 12.12 0 0 5157 100 cx. tritaeniorhynchus 700 11.34 3371 54.60 621 10.06 1481 24 0 0 6173 100 cx. perexiguus 27 30 25 27.77 18 20 20 22.22 0 0 90 100 cx. mimeticus 1 8.33 10 83.33 0 0 1 8.33 0 0 12 100 cx. hortensis 0 0 1 100 0 0 0 0 0 0 1 100 ae. vexans 43 3.38 16 1.25 5 0.39 6 0.47 1200 9.54 1270 100 ae. caspius 0 0 13 34.21 24 63.15 0 0 1 2.63 38 100 cs. annulata 2 4.08 33 67.34 10 20.40 4 8.16 0 0 49 100 total 2407 14.87 8051 49.74 1804 43.64 2722 16.82 1201 7.42 16184 table 5. abundance indices (isa/sisa)* of the most abundant species (lower isa values and sisa closer to 1) of mosquitoes collected based on sampling method in mazandaran province, northern iran larvae adult total catch in human places total catch in animal places pit shelter light trap daily biting species sisa isa species sisa isa sisa isa sisa isa sisa isa sisa isa an. claviger 0.000 24.06 an. claviger 0.000 20.97 0.000 21.22 0.000 20.88 0.000 21.3 0.000 17.1 an. hyrcanus 0.000 14.5 an. hyrcanus 0.000 18.78 0.000 14.09 0.000 20 0.000 16.4 0.000 17.1 an. maculipennis s.l. 0.550 7.06 an. maculipennis s.l. 0.369 7.93 0.637 5.719 0.000 12.78 0.000 16 0.000 17.1 an. pseudopictus 0.273 10.81 an. pseudopictus 0.454 7 0.822 3.313 0.46 6.938 0.668 4.66 0.000 17.1 an. marteri 0.000 24.5 an. marteri 0.000 22.19 0.000 23.97 0.000 20.75 0.000 22 0.000 17.1 cx. pipiens 1 1 an. sacharovi 0.000 22.19 0.000 23.94 0.000 20.69 0.000 19.8 0.000 17.1 cx. torrentium 0.805 3.62 an. superpictus 0.000 22.19 0.000 22.44 0.000 22.06 0.000 22 0.000 17.1 cx. tritaeniorhynchus 0.564 6.87 cx. pipiens 0.977 1.25 0.933 1.87 0.946 1.59 0.696 4.34 0.000 17.1 cx. perexiguus 0.000 17 cx. tritaeniorhynchus 0.886 2.25 0.913 2.12 0.938 1.68 0.938 1.69 0.000 17.1 cx. territans 0.000 19.19 cx. perexiguus 0.000 17.53 0.000 18.03 0.000 20.78 0.000 20.6 0.000 17.1 cx. mimeticus 0.037 14 cx. mimeticus 0.000 21.13 0.000 19.19 0.000 22.06 0.000 20.9 0.000 17.1 cx. hortensis 0.000 21.69 cx. hortensis 0.000 22.19 0.000 23.94 0.000 22.06 0.000 22 0.000 17.1 cs. annulata 0.000 15.12 ae. vexans 0.000 19.75 0.000 22.63 0.000 19.78 0.000 19.8 0.1 9 cs. longiareolata 0.152 12.44 ae. caspius 0.000 22.19 0.000 23.69 0.000 19.66 0.000 22 0.000 16.1 cs. morsitans 0.000 22.69 cs. annulata 0.000 19.97 0.000 16.34 0.000 16.47 0.000 18.6 0.000 17.1 http://jad.tums.ac.ir published online: june 30, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 215 http://jad.tums.ac.ir published online: june 30, 2021 *isa: index of species abundance; sisa: standard index of species abundance fig. 2. relation between population fluctuations of larvae/adult mosquitoes and meteorological variables in mazandaran province, northern iran http://jad.tums.ac.ir published online: june 30, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 216 http://jad.tums.ac.ir published online: june 30, 2021 discussion demonstration of the monthly changes in the population abundance of mosquito species, along with meteorological parameters is crucial in understanding the biology of potential vector species for efficient mosquito control strategies. this is the first comprehensive study on this subject in mazandaran province, northern iran using the isa/sisa indices in order to identify the most abundant species. vector abundance is a key determining factor that is often used as a risk indicator of vector-borne diseases. it is quantitatively more important than vector competence (although competence is a transmission requirement). it could explain the importance of abundant species in an area and why some vectors could contribute to epidemics without considering the competence for transmission (40-41). culex pipiens is the most abundant species both in the larval and adult stages based on indices of sisa/ isa in the present study. it was noted to be a common species in other studies in different parts of iran (35, 4245), however, it should be mentioned that none of these researches reported abundance of species based on the sisa/isa indices. although cx. pipiens was collected most frequently by total catch in animal places, it was the predominant species (based on indices of sisa/isa) in total catch in human places and pit shelter compared to other species, respectively. it indicates that the species tends to be attracted to humans, animals and also shows some extent of exophily in the study area. it is possible that humans come into contact with pathogens this species may carry. such studies could establish baseline data for public health interventions in control programs, therefore, an assessment of different trapping methods is also required to adjust the design of future entomological and pathogen surveillance efforts (46). the monthly activity of cx. pipiens (larvae and adult) begins in mid-spring after diapause termination and reaches its largest peak in july and then declines with irregular fluctuations until the species disappears. it seems that the spring rains are cause for the population of the species to peak in july, while no significant association was observed between cx. pipiens and meteorological factors, probably high compatibility and access to diverse breeding places could help increasing population density of the species in early warm season. in contrast, cx. pipiens was active from may to november and june to september (29, 47). the population density of this species increased in july (47) and may to august (48), reaching its largest peak in august (47-48). culex pipiens showed an increase in population density from may to july in north-eastern croatia, then sharp decline occurred toward the end of the season (49). in northern italy, the species had maximum activity peak in july (50), that is almost consistent with the results of the present study. there is not much data about the seasonal activity of culicine mosquitoes, especially in adult stage, in iran (30). the highest activity peak of cx. pipiens was documented in mid-july in guilan province. there is no significant correlation between species population density and the meteorological data (30), which is in accordance with our research. variations in the seasonal patterns of the species in this study and findings in other regions can probably be due to the differences in the topography and climates. culex pipiens with its ornithophilic and opportunistic feeding behavior that bites both humans and animals, can be as bridge vector between birds and humans so is believed to be the principal vector of west nile virus (51). it also shows an important role in transmission of several human pathogens including st. louis encephalitis virus (slev), filarial worms as well as wildlife pathogens such as avian malaria (52). the existence of swamps for migrating birds and their active presence throughout the autumn and winter in mazandaran province and detection of virus in the species in guilan province (53) can cause a concern for http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 217 http://jad.tums.ac.ir published online: june 30, 2021 entry and the spread of the virus in the region. culex tritaeniorhynchus, the second most abundant species based on indices of sisa/isa in the study area, was first collected in may (larval stage) and june (adult stage), increased sharply in july, becoming the most abundant species, and displayed a rapid decline in december. the abundant species shifted from cx. tritaeniorhynchus to cx. pipiens in november, probably due to high adaptability of cx. pipiens to the environmental conditions in the area. in contrast, this species showed the highest peak of activity in august in belek region of turkey (29). while the peak of activity was observed in july in guilan province (30), which is in agreement with our study. culex tritaeniorhynchus is caught with the highest number by total catch in animal places which reflects the zoophilic tendencies of the species in the study area. based on the results of a study in india, it is considered to be predominantly exophilic and normally zoophilic (54). it was recorded to be the most abundant species in numerous studies in iran, without reporting any sisa/ isa indices (33-34, 44-55). culex tritaeniorhynchus is the primary vector of japanese encephalitis (je) in southern asia. it has also been observed infected with dengue fever, rift valley fever, sindbis, getah and tembusu viruses, and the filariae of both brugia malayi and wuchereria bancrofti, in many areas of eastern and southeastern asia (55-56). the species is a principle vector of rift valley fever in saudi arabia (57), wnv in asia (9) and a possible vector of japanese encephalitis in iraq (58), which indicates a high risk species for human health. among the anopheles, an. maculipennis s.l. is numerically the most abundant species in the present study, mostly sampled by total catch method in animal places, displayed to be predominantly zoophilic. this is consistent with other studies, but there is no evidence of sisa/isa indices to express the most abundant species in these studies (43, 59-61). what is interesting is that, despite having the highest numerical abundance of species, an. pseudopictus was calculated as the most abundant species in all sampling methods (except daily bites) based on isa/sisa indices. in addition, an. sacharovi also had the highest numerical abundance in the pit shelter, but it was not considered as the most abundant species based on the indices of sisa/isa. moreover, an. sacharovi was the most abundant species in the pit shelter, but only with regards to its high numerical abundance. therefore, this shows the importance of species distribution at different sites and computational value of the isa/sisa indices. anopheles maculipennis s.l. is known to be the most important malaria vector in northern and western parts and the central plateau of iran (62). moreover, there is a belief that this species can play a vector role in wnv transmission in various countries of the old world (63, 64). recently, the myxoma virus genome was detected in wild caught an. maculipennis that fed on wild rabbits by polymerase chain reaction (pcr) in southern england, uk. batai virus (batv) and anopheles associated c virus (aacv) was also identified and isolated from an. maculipennis complex in entomological surveys in germany, italy and france (65). with regards to the malaria historical records and wnv in the northern parts of iran (53, 66), understanding the population dynamics and the maximum monthly peaks of the species is important in controlling the diseases in the study area. the highest monthly activity peak of an. maculipennis (larvae and adults) is in june. in contrary to our results, an. maculipennis s.l. showed the highest peak of activity in the midjuly in guilan (30) and july–august in kalaleh county of golestan province (31), northern iran. in neighboring turkey, the species peaked in august and july–august (67-68). these differences in population patterns could possibly be due to regional ecological differences. on the other hand, some researchers believe that these discrepancies might also be due to the low collection effort involved in the studies (27) or sampling regimes (29-30, 50, 67-68). climate http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 218 http://jad.tums.ac.ir published online: june 30, 2021 change and the environment affect the abundance and distribution of vectors and their intermediate hosts (69). in the present study, monthly population fluctuations of cs. annulata, cs. longiareolata and ae. vexans are correlated with rainfall. although these species showed irregular monthly population fluctuations, they were more active in spring and autumn. culiseta annulata had a major peak in november (29), which is in accordance with our research. on the other hand, cs. longiareolata was active in july and september (67), whereas in the present study, the species had the largest peak in october, which is quite similar to that of ae. vexans. the largest peak of ae. vexans was reported in june (67) and august (68). there are evidence that ae. vexans and cs. annulata are important vectors of wnv (70-71). among the tribe aedini, ae. vexans is recorded to be the most abundant species based on isa/sisa indices. it was caught in all traps and is the most abundant collected species in day biting method compared with other species, and shows anthropophilic tendencies in the study areas. this species demonstrated high prevalence and collected by day biting method in other studies performed in different parts of iran (59, 72-74), this may be a reflection of the fact that this method has a high level of efficiency for collecting this species. however, to observe ethical values, this collection methods cannot be used freely and with extended time, and should in fact be restricted or modified in compliance with ethical standards (75). the species is known to be the main vector of tahyna virus in central europe (76), potential vector of the dog heartworm dirofilaria immitis in europe (77) and principal vectors of rift valley fever in saudi arabia (57). recently, zika virus is revealed in the salivary glands of the field-caught ae. vexans (78). it is a flood water mosquito, so, their population abundance depends on the water dynamics of temporary pools (27). rainfall as an important climatic factor may have a range of different correlation with the population of the species, from positive, negative and/ or ineffective and sometimes with a lag phase (79), a significant positive correlation between its monthly activity and rainfall was observed in our study. the current study was not designed and aimed to address the analysis of lag time between climate factors and population density of the species, however, it seems that there is probably a lag time of at least 15 days after rain at the beginning of the rainy season before the ae. vexans population jump start. in concordant with our findings, there is a lag time of at least 15 days between the peak of rainfall and abundance of the species (80-82). moreover, other studies have revealed a correlation between rainfall and the abundance of the species with a 10 days lag in the early rainy season and 20 days after the end of the rainy season (83). in the study of diallo et al. (82) rainfall patterns displayed that heavy rains between august and september 2005 had negatively impacted on the population density of ae. vexans, whereas in 2006, population peak was observed following the rainfall peak. these findings show the complexity of the relationship between climate factors and population density of mosquitoes. therefore, it is recommended to measure the seasonal activities of species over multiple years, to show a better understanding of the correlation between population frequency and climatic factors, and the impact of other variables. the highest number and population density of ae. vexans was found during early autumn, especially in october. vector-borne diseases show seasonal patterns with inter and intra-annual variability, which are partly described by climate and environmental factors (80). therefore, these results are important for health authorities in controlling mosquitoes as well as in the tourism industry for nuisance control, but more importantly, it provides a detailed estimate of the timing of risk for human populations. conclusion based on isa/sisa indices, july in which http://jad.tums.ac.ir/ j arthropod-borne dis, june 2021, 15(2): 207–224 sh nikookar et al.: population fluctuations and … 219 http://jad.tums.ac.ir published online: june 30, 2021 cx. pipiens and cx. tritaeniorhynchus have the highest population peak, is the most crucial time for efficient mosquito control program in the area study. acknowledgements we would like to thank the staff of mazandaran provincial health deputy for their direct help in the field sampling. also we express our appreciation to the people of the villages for their kind assistance with the sampling teams during the study. the study was supported by the deputy for research and technology of mazandaran university of medical sciences, mazandaran, iran by grant no. 93– 1017. the authors declare that there is no conflict of interest. references 1. rueda lm (2008) global diversity of mosquitoes (insecta: diptera: culicidae) in freshwater. hydrobiologia. 595: 477–487. 2. gary r, mullen and lance a (2019) mosquitoes (culicidae). in: woodbridge a, foster and edward dw, elsevier. pp. 261– 325. 3. suesdek l (2018) microevolution of medically important mosquitoes–a review. acta trop. 191(2019): 162–171. 4. world health organization (2019) global burden of major vector-borne diseases. available at: https://www.who.int/vectorcontrol/burden_vectorborne_diseases.pdf 5. world health organization (2020) world malaria report 2020. world health organization, geneva, switzerland. 6. campos gs, bandeira ac, sardi si (2015) zika virus outbreak, bahia, brazil. emerg infect dis. 21(10): 1885–1891. 7. van bortel w, dorleans f, rosine j, blateau a, rousset d, matheus s, leparcgoffart i, flusin o, prat c, cesaire r (2014) chikungunya outbreak in the caribbean region, december 2013 to march 2014, and the significance for europe. euro surveill. 19(13): 20759. 8. nash d, mostashari f, fine a, miller j, o'leary d, murray k, huang a, rosenberg a, greenberg a, sherman ml (2001) the outbreak of west nile virus infection in the new york city area in 1999. engl j med. 344(24): 1807–1814. 9. azari-hamidian s, norouzi b, harbach re (2019) a detailed review of the mosquitoes (diptera: culicidae) of iran and their medical and veterinary importance. acta trop. 194: 106–122. 10. nikookar sh, fazeli-dinan m, azari-hamidian s, nasab snm, aarabi m, ziapour sp (2018) fauna, ecological characteristics, and checklist of the mosquitoes in mazandaran province, northern iran. j med entomol. 55(3): 634–645. 11. edrissian g (2006) malaria in iran: past and present situation iran j parasitol. 1 (1): 1–14. 12. zaim m, subbarao s, manouchehri a, cochrane a (1993) role of anopheles culicifacies s.l. and an. pulcherrimus in malaria transmission in ghassreghand (baluchistan), iran. j am mosq control assoc. 9(1): 23–26. 13. sharifzadeh y (2014) species composition and some bionomic characters of anopheles subpictus in sarbaz district, southeastern iran. 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different solvents and evaluate for potential use to control different larval stages of mosquito species, aedes aegypti and anopheles stephensi. methods: qualitative and quantitative estimation of ingredients from go. hirsutum (bt) plant extract was carried out and their inhibitory action against mosquito larvae was determined using mosquito larvicidal assay. results: lc50 values of water, ethanol, ethyl acetate and hexane extracts for ae. aegypti were 211.73±21.49, 241.64±19.92, 358.07±32.43, 401.03±36.19 and 232.56±26.00, 298.54±21.78, 366.50±30.59, 387.19±31.82 for 4th instar of an. stephensi, respectively. the water extract displayed lowest lc50 value followed by ethanol, ethyl acetate and hexane. owing to the comparatively better activity of water extract, its efficacy was further evaluated for mosquito larvicidal activity, which exhibited lc50 values of 133.95±12.79, 167.65±11.34 against 2 nd and 3rd instars of ae. aegypti and 145.48±11.76, 188.10±12.92 against 2nd and 3rd instars of an. stephensi, respectively. crude protein from the water extract was precipitated using acetone and tested against 2nd, 3rd and 4th instars of ae. aegypti and an. stephensi. it revealed further decrease in lc50 values as 105.72±25.84, 138.23±23.18, 126.19±25.65, 134.04±04 and 137.88±17.59, 154.25±16.98 for 2nd, 3rd and 4th instars of ae. aegypti and an. stephensi, respectively. conclusion: leaves extracts of go. hirsutum (bt) is potential mosquito larvicide and can be used as a potent alternative to chemical insecticides in integrated pest management. keywords: cotton, leaves extract, mosquito, toxicity, a. aegypt introduction mosquitoes are top most insect vectors related to human health (chakkaravarthy et al. 2011). different mosquito species like anopheles stephensi, aedes aegypti and culex quinquefasciatus are widely distributed in tropical and subtropical zones and acting as vectors for pathogen of diseases like malaria, dengue, filaria, japanese encephalitis, yellow fever, chikungunya (redwane et al. 2002). according to who report 2012, an estimated 3.3 billion people were at risk of malaria in 2011 around the globe (who 2012). a total of ninety-nine countries had on-going malaria transmission. there were about 219 million cases of malaria in 2010 and an estimated 660,000 deaths, majority of them were from africa continent. analogous to that about 50 million people in the world are infected with dengue. malaria is an entirely preventable and treatable disease. report also stated that in endemic countries in the year 2011, 278 million courses of artemisinin-based combination therapies (acts) were procured by the public and *corresponding author: dr satish v patil, e-mail: satish.patil7@gmail.com http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 92 private which were 182 million in 2010, and just 11 million in 2005. this indicates that these diseases are major cause of morbidity and mortality (who 2012). drug resistance to antimalarial drugs is a major concern for the global effort to control malaria. p. falciparum resistance to artemisinins has also been detected in about four countries, therefore vector control through the use of insecticide is one of the most important measures for control of malaria. solution to control population of these mosquitoes involve the use of different organophosphate and carbamate insecticides like temephos, fenthion, malathion, distillate of crude oil, malaria larvicide oil (mlo) but their persistent use leads to development of resistance in species (raghvendra 2002). in addition, chemical insecticides few examples such as methoprene, pyriproxyfen, diflubenzuron, ddt have several disadvantages like interference in food chain, toxicity to nontarget organism, soil and water contamination, long environmental persistence. besides this, mosquito resistance to at least one insecticide used for malaria control has been identified in 64 countries around the world (who 2012). therefore, use of these insecticides needs to be restricted and search for alternatives to traditionally used pesticides is urgently needed. use of larvicidal agents from natural sources has shown to reduce the harmful effects of chemical pesticides on environment (cheng et al. 2009), therefore, mosquito larvicidal agents from plants and microorganisms can be considered as alternatives to chemical insecticides (sharma et al. 2009, tchicaya et al. 2009, patil et al. 2011, salunkhe et al. 2011). besides this, botanical insecticides are easily available, cheap and are ecofriendly. researchers have screened and reported natural products for their mosquito larvicidal potential. crude plant extracts attracted most attention as mosquito larvicidal agents. aina et al. (2009) have evaluated larvicidal potential of seed extracts from piper guineense which gave 83% inhibition of second instar larvae of an. gambiae. euphorbia hirta leaves extract in carbon tetrachloride, methanol and petroleum ether were tested and found effective on an. stephensi by sharma et al. (2009). the leaves and root extract of cissampelos mucronata and tephrosia villosa from tanzania coast region (nondo et al. 2011) leaves of morinda citrifolia (kovendan et al. 2012), were found effective against mosquito larvae. other researchers isolated active principles from crude plant extract and analysed their insecticidal potential. the pipernonaline, a piperidine alkaloid from piper longum (lee se, 2000), goniothalamin from bryonopsis laciniosa (kabir et al. 2003), methanol extract of acorus calamus rhizome (hidayatulfathi et al. 2004), mosquito larvicidal protein from solanum villosum leaves (chowdhury et al. 2008), plumbagin from plumbago zeylanica (plumbaginaceae) and saponins from cestrum nocturnum (solanaceae) (patil et al. 2011), hexane and petroleum ether extracts of citrus limletta peels kumar et al. (2012), limonin and nomilin from citrus cuiltivars (bilal et al. 2012), essential oil from plant melaleuca cajuputi (abu baker et al. 2012), pectolinaringenin from chloroform extract of clerodendrum phlomidis l. (lamiaceae) (muthu et al. 2012). gossypium hirsutum (malvaceae: malvales) is commonly known as upland cotton or mexican cotton (fig. 1). it is native from central america and mexico and an important fibre crop and most widely cultivated species. worldwide around 35 mha land is under cotton crop. india alone constitutes 9.5 mha about one quarter of global area under this crop. the country reported a little over 21% of the global cotton production in 2008–2009 and stands second in cotton production having 4.9 million tons behind china having 7.8 million tons (karihaloo and kumar 2009). however, this production is http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 93 less as compared to land under cultivation. low production is majorly attributed to attack by sap sucking pest helicoverpa armigera commonly known as bollworm. advancement in plant biotechnology with introduction of genetically modified (gm) variety of cotton known as ‘btcotton’ by mahyco seeds in collaboration with monsanto has improved situation of he. armigera attack and production of cotton in india. the bt cotton variety involves incorporation of cry1ac gene from common soil bacterium bacillus thuringiensis, which gives protection against bollworm. apart from prime source of fibre used in textiles cotton crop also reported to have some medicinal uses. leaves of cotton plant show antibacterial activity against clinically important bacteria like escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, shigella dysenteriae (omojasola and awe 2004). leaves of cotton in combination with other plant extracts are effective against malaria (igwe et al. 2012) and good remedy for uterine fibroid and cancer (hartwel 1971) and as antifertility agent (randel et al. 1992). in the present study, we have tried to extract the ingredients from leaves of go. hirsutum (bt cotton) using different solvents and evaluate for potential use to control different larval stages of mosquito species, ae. aegypti and an. stephensi. materials and methods collection of plant material the healthy and fresh leaves of go. hirsutum (bollgaurd ii), a bt cotton variety and non-bt variety (y1) were collected from local farms around the area of jalgaon (210 00′24.5″ n, 750 29′45.5″ e, elevation 218m) district. leaves were thoroughly washed 2–3 times with distilled water and shade dried for three months at 27±2 ˚c. extraction method hundred grams of shade dried leaves of three month old go. hirsutum (bollgaurd ii) and local non-bt variety (y1) were ground to make powder and were dissolved in 500ml each of different solvents (water, ethanol, ethyl acetate and hexane) in separate 1000ml capacity conical flasks and were kept at shaking condition for 48h. the mixture was then filtered through whattman filter paper so as to remove the undissolved plant materials. these extracts were then concentrated using vacuum evaporator (roteva, equitron, india) and air dried. the dried materials were weighed; stock concentrations were prepared by dissolving in respective solvents and used for their evaluation as mosquito larvicidal agents. acetone precipitation was done to obtain crude protein from the water extract of leaves, which was also inspected for its larvicidal potential. test organisms for the laboratory trial, locally collected early second, third and fourth instar larvae of ae. aegypti and an. stephensi were used as experimental mosquitoes. the larvae were kept in plastic enamel trays containing dechlorinated tap water. they were maintained as per previous report (patil et al. 2011). mosquito larvicidal bioassay stock solution (1000ppm) was prepared by dissolving 100mg of crude semisolid extract from respective solvents in 100ml of distilled water. different concentrations of these stocks were prepared by dilution in dechlorinated tap water making the final volume 100ml and were used as working stocks. the larvicidal activity was assessed by the procedure of who (1996) with some modifications and as per the previous method (patil et al. 2012 a,b). for bioassay test, 25 larvae of iind, iiird and ivth instars of ae. aegypti and an. stephensi were added in 249ml of water in http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 94 four different batches and 1.0ml of the desired extract concentration. the control was set up with dechlorinated tap water. the numbers of dead larvae were counted after 48 hours of exposure, and the percentage mortality was recorded for the average of four replicates. the experimental media, in which 100% mortality of larvae occurred, was selected for the dose-response bioassay (data not shown). dose response bioassay based on the preliminary screening results, crude extract of experimental plants were subjected to dose-response bioassay for larvicidal activity against the iind, iiird and ivth instars larvae of ae. aegypti and an. stephensi. different concentrations ranging from 100 to 500ppm were prepared and number of dead larvae was counted after 24 hours of exposure. the percentage of mortality was reported from the average of three replicates. to evaluate further partially purified protein i.e. the acetone precipitate of water extract were dried and used to evaluate toxicity against the iind, iiird and ivth instars larvae of ae. aegypti and an. stephensi. phytochemical analysis extracts from all four solvents were subjected to qualitative phytochemical analysis which was carried out to detect the presence of different metabolites as per method of kokate et al. (1999). qualitative and quantitative estimation of protein water extracts of bt and non bt cotton (go. hirsutum) leaves were precipitated with acetone, dried and dissolved in phosphate buffer (ph 7.5) and used for qualitative and quantitative analysis of proteins by method of lowry et al. (1951). statistical analysis mortality was calculated and corrected using abbott’s formula (abott 1925). the dose-response data were subjected to probit regression analysis (finney 1971). the lethal concentrations in parts per million (lc50, lc90) and the 95% confidence intervals of lc50 (upper confidence limit) and (lower confidence limit) were calculated. results differential extraction of 100 gm of dried leaves from go. hirsutum (bollgaurd ii) yielded crude extracts of 0.570gm, 0.498gm, 0.287gm, 0.345gm with the solvents water, ethanol, ethyl acetate and hexane, respectively. all extracts showed the larvicidal efficacy within 48h of exposure. mortality rate (y) was found to be directly proportional to the concentration of dose (x) indicating that mortality rate increases with the increasing dose. efficacy of leaves extracts from water, ethanol, ethyl acetate and hexane were evaluated against both the targets which showed lc50 values 211.73±21.49, 241.6±19.92, 358.07±32.43, 401.03±36.19 against ivth instar larvae of ae. aegypti and 232.56±26.00, 298.54±21.78, 366.50±30.59, 387.19±31.82 for ivth instar larvae of an. stephensi, respectively (table 1). with these values we can infer that water extract showed comparatively high potential than the other solvent extracts. water extract was further evaluated for mosquito larvicidal activity, which showed lc50 values of 133.95±12.79, 167.65±11.34 against iind and iiird instar larvae of ae. aegypti and lc50 values of 145.48±11.76, 188.10±12.92 against iind and iiird instar larvae of an. stephensi, respectively (table 2). we further purified the active component from crude water extract by using acetone precipitation technique. qualitative test of the precipitated material revealed protein as a major component. dried acetone precipitate of water extract showed protein content http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 95 of 350 µ g/gm from dry leaves. acetone extracted protein material was further solubilised in phosphate buffer (50–500ppm) and screened for mosquito larvicidal activity on iind, iiird and ivth instar larvae of ae. aegypti and an. stephensi. crude protein showed the larvicidal efficacy within 48h of exposure. the crude protein when subjected for evaluation against different larval stages showed lower lc50 values compared to water and other solvent extracts. lc50 for crude protein was 105.72±25.84, 126.19±25.65, 137.88± 17.59 and 134.04±24.04, 138.55±23.18, 154.2 5±16.98 against iind iiird and ivth instars of ae. aegypti and an. stephensi, respectively (table 3). qualitative analysis as per kokate et al. (1999) of different solvent extracts of bt leaves showed presence of alkaloids, phenolic compounds, terpenoids, tannins, saponins, flavonoids and proteins (table 4). water extract showed strong positive test for all metabolites under study. ethanol extract showed y strong positive test for terpenoids and moderate positive for alkaloids, phenolic compounds, tannins, saponins, flavonoids and devoid of proteins. ethyl acetate extract showed absence of phenolic compounds and proteins while hexane extract remarkably showed presence of only saponins (table 4). the leaves extracts of non-bt cotton variety (y1) in different solvents did not show any substantial effect on the mortality of both mosquito larvae. only the hexane extract showed 30 and 40% mortality for the both the mosquito species at 450 and 650ppm, respectively (data not shown). the different solvent extracts of this variety also showed presence of alkaloids, saponins, phenol, tannins and proteins. acetone precipitation for leaves water extract revealed protein content of 300µ g/gm but did not show any larvicidal effect. therefore, results of extracts of non-bt cotton variety are not presented in tables. fig. 1. a) bt cotton (gossypium hirsutum) plant with flower and fruit, (b) bt cotton plantation in field. table 1. larvicidal activity of differential extracts from leaves of gossypium hirsutum against ivth instar larvae of aedes aegypti and anopheles stephensi mosquito species leaves extract(btcot) lc50 ±se (mg lit-1 ) 95% fiducial limits (lcl-ucl) lc90±se (mg lit-1 ) 95% fiducial limits (lcl-ucl) regression equation aedes aegypti water 211.73±21.49 165.01253.86 528.59±52.02 448.88673.31 y=1.93+0.0143 x ethanol 241.64±19.92 201.07282.76 536.98±48.96 460.90669.48 y=1.24+0.0154 x ethyl acetate 358.07±32.43 303.74750.27±96.81 609.91y=0.811+0.0117x a b http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 96 444.33 1047.67 hexane 401.03±36.19 342.34501.14 780.38±99.93 635.451086.77 y=0.399+0.0114x anopheles stephensi water 232.56±26.00 176.31286.13 625.49±77.94 514.72866.47 y=2.18+0.0121 x ethanol 298.54±21.78 257.97347.87 603.70±57.19 515.61760.30 y=0.561+0.0150x ethyl acetate 366.50±30.59 315.32446.32 722.72±85.06 597.08974.12 y=0.418+0.0125x hexane 387.19±31.82 334.72471.24 735.99±85.30 609.77987.03 y= 0.212+0.0123 y mortality rate (significant at p< 0.05 level), x concentration (significant at p< 0.05 level), lc50 lethal concentration that kills 50% of the exposed larvae, lc90 lethal concentration that kills 90% of the exposed larvae, se standard error (all values are mean of four replicates), lcl lower confidence limit, ucl upper confidence limit table 2. larvicidal activity of water extracts from leaves of gossypium hirsutum against iind and iiird instar larvae of aedes aegypti and anopheles stephensi mosquito species larval stage (instars) lc50 ±se (mg lit-1 ) 95% fiducial limits (lcl-ucl) lc90±se (mg lit-1 ) 95% fiducial limits (lcl-ucl) regression equation aedes aegypti ii 133.95±12.79 105.21-157.26 283.64±18.12 253.49-328.26 y= 2.00+0.0222x iii 167.65±11.34 143.50-189.24 312.32±18.07 282.11-356.18 y=0.631+0.0252x anopheles stephensi ii 145.48±11.76 119.71-167.34 287.18±17.43 258.07-329.72 y=1.45+0.0237x iii 188.10 ±12.92 160.59-212.88 364.92±23.01 327.15-422.15 y=0.524+0.0234x y mortality rate (significant at p< 0.05 level), x concentration (significant at p< 0.05 level), lc50 lethal concentration that kills 50% of the exposed larvae, lc90 lethal concentration that kills 90% of the exposed larvae, se standard error (all values are mean of four replicates), lcl lower confidence limit, ucl upper confidence limit table 3. larvicidal activity of crude protein from water extract of gossypium hirsutum (bollgaurdii) against ii nd, iiird and ivth instar larvae of aedes aegypti and anopheles stephensi mosquito species larval stage lc50 ±se 95% fiducial limits lc90±se 95% fiducial limits regression equation (instars) (mg lit-1 ) (lcl-ucl) (mg lit-1 ) (lcl-ucl) aedes aegypti ii 105.72±25.84 35.50-147.30 273.28± 25.56 232.00-345.43 y=4.53+0.0127x iii 126.19±25.65 56.87-168.45 314.17±27.89 269.46-391.62 y=3.73+0.0140x iv 137.88±17.59 96.13-169.37 350.23± 30.23 302.8-432.062 y=2.29+0.0195x anopheles stephensi ii 138.23±23.18 78.18-177.71 317.83±27.09 274.38-392.16 y=3.30+0.0150x iii 134.04±04 70.56-174.23 315.33±27.05 95.71-389.51 y=3.30+0.0150x iv 154.25± 16.98 115.178-185.6 371.42±32.6 320.54-460.49 y=1.92+0.0197x mortality rate (significant at p< 0.05 level), x concentration (significant at p< 0.05 level), lc50 lethal concentration that kills 50% of the exposed larvae, lc90 lethal concentration that kills 90% of the exposed larvae, se standard error (all values are mean of four replicates), lcl lower confidence limit, ucl upper confidence limit table 1. countinued… http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 97 table 4. qualitative analysis of phytochemicals present in differential solvent extracts from leaves of bt-cotton (gossypium hirsutum) phytochemical constituent water extract ethanol extract ethyl acetate extract hexane extract alkaloids +++ ++ ++ phenolic compounds +++ ++ terpenoids +++ +++ ++ tannins +++ ++ + saponins +++ ++ +++ ++ flavonoid ++++ ++ ++ protein +++ +++ strong positive, ++ moderate positive, + less, -not detected discussion the phytochemical constituents of plant extracts represent a large source of potentially bioactive molecules. go. hirsutum (bt) is a genetically modified variety, which has ability to express a protein toxin in its tissues. the differences in the levels of toxicity in each extract depends on the insecticidal ingredients of plant extracts, solubility in different solvents which can vary significantly depending on plant species, plant part, age of plant part, solvent used for extraction, seasonal variation and target insect species. among the evaluated different solvent extracts of leaves from bt and non-bt go. hirsutum, the water extract of non-bt cotton (y1var) leaves did not show any mortality while water extract of bt cotton was found to be most effective (lc50 211.73 and 232.56ppm) against ivth instar larvae of ae. aegypt and a. stephensi respectively. water extract and precipitated protein from water extract were further studied for their potential against various instars of ae. aegypti (lc50 137.88 ppm) and an. stephensi. (lc50 154.25ppm). this manifestation might be due to the high water solubility of effective ingredients than in other solvents. in addition, it was found that protein obtained from water extract was more effective than water extract itself suggesting the major role of protein component in larvicidal action. the water extract of non-bt cotton leaves also precipitated by acetone but it did not show any significant larvicidal activity. in the present study, lc50 values of 211.73 ±21.49, 241.64±19.92, 358.07±32.43, 401.03 ±36.19 and 232.56±26.00, 298.54±21.78, 366.50±30.59, 387.19±31.82 for water, ethanol, ethyl acetate and hexane extracts of bt cotton (go. hirsutum) against the ivth instar mosquito larvae of ae. aegypti and an. stephensi, respectively. the lowest lc50 value was found for water extract displayed compared to ethanol, ethyl acetate and hexane. water extracts showed lc50 values of 133.95 ±12.79, 167.65±11.34 against 2nd and 3rd instars of ae. aegypti and 145.48±11.76, 188.10 ±12.92 against 2nd and 3rd instars of an. stephensi, respectively. further lower lc50 values of 105.72±25.84, 138.23±23.18, 126.19 ±25.65, 134.04±04 and 137.88±17.59, 154.25 ±16.98 were found for acetone precipitated crude protein against 2nd, 3rd and 4th instars of ae. aegypti and an. stephensi, respectively. our results of mosquito larvicidal capacities of bt cotton leaves extract are comparable to the investigations by other researchers on plant extracts. efficiency of leaf chloroform extract of nyctanthes arbortristis have been reported with lc50 value of 526.3, http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 98 780.6ppm (24 hours) and 303.2, 518.2 (48h) for ae. aegypti and an. stephensi (mathew et al. 2009). valencia late (citrus sinensis) was the best in terms of lc50 (297ppm), % mortality (97%) and lt50 (18.49h) then freutrall early (citrus reticulate) with lc50 (377.4 ppm), % mortality (88%) and lt50 (31 hours), while nomilin gave lowest lc50 (121.04 ppm) than limonin (382.22ppm) after 72h of exposure against aedes albopictus larvae (bilal et al. 2012). kovendan et al. (2012) reported morinda citrifolia leaf extract in hexane, chloroform, acetone, methanol, and water had values of lc50 345.10, 324.26, 299.97, 261.96, and 284.59ppm and 361.75, 343.22, 315.40, 277.92, and 306.98ppm for third instar larvae of an. stephensi and ae. aegypti respectively. isolated proteins have more potential larvicidal activity as compared to crude extracts. in the previous reports, the proteins isolated from mature leaves of solanum villosum were found to have larvicidal activity against iiird instar larvae of an. stephensi, cu. quinquefasciatus and stegomyia aegypti mosquitoes and molecular weights of the protein bands ranged between 69–109 kda (chowdhury et al. 2008). the larvicidal activities of the essential oils separated from h. persicum, f. vulgare and c. sativum plants against an. stephensi revealed that the lethal dosage (lc50) ranged between 20.10 and 120ppm (sedaghat et al. 2011). in case of m. cajuputi 5 and 10% essential oil aerosol spray shows mortality of 20.002.85, 48.05±0.37 for ae. aegypti and 22.90±4.22, 44.20±2.10 ae. albopictus (abu baker et al. 2012). the phytochemical studies indicate presence of alkaloids, phenolic compounds, terpenoids, tannins, saponins, flavonoids and protein. botanical pesticides have different mode of action like feeding and oviposotion deterrence (su and mulla 1999). phytochemicals have targeted action on midgut epithelial cells of mosquito (cohen et al. 2012). morya et al. (2007), stated that, the crude extract were more effective than individual active compound because of natural synergism that discourages the emergence of resistance in mosquito vectors. the crude extract can be obtained by simple procedure than purification of active compounds and are more effective against insect and less toxic to aquatic organism (prakash and rao 1997). use of plant products may be considered as one of the best policy in integrated pest management (ipm) for control of mosquito larvae. fibres of cotton are used in textile mills but other parts of cotton crop like leaves, roots are mostly used conventially for cooking and burning purpose in india. owing to such huge plantation of bt cotton, there is need to utilize the agro-waste into useful products. bt cotton leaves possess qualities of ideal bioinsecticide having ecofriendly, biodegradable nature, as it is less harmful to aquatic species than chemical insecticides and have low production cost, easy availability, easy utilization, and simple storage. this can even create employment opportunities to local population and can prove to be helpful to reduce the use of costly insecticides. though agricultural waste is abundant but it is remained unutilized, so greater attention is needed to be paid towards converting it into useful products (nand 1998). conclusion control of various mosquito species at various developmental larval stages is a useful measure to mitigate transmission of pathogens by mosquito vectors. in conclusion present study confirms that leaves extracts of go. hirsutum (bt) have potential mosquito larvicidal activity. phytochemical studies revealed presence of proteins that play major part in killing larval stages. this may give an additional insight for the use of genetically modified agricultural waste to control the development of mosquitoes and eventually to http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 91–101 c d patil et al.: mosquito larvicidal potential … 99 control spread of different life threatening diseases. bt cotton has added advantage of being largely cultivated worldwide for the production of fibre. the present study throws light on the additional advantage of cotton plant leaves for well-being of humans. as per our knowledge this is the first report on use of cotton (bt) leaves as mosquito larvicidal agents. further studies on extraction, characterization, size estimation of bioactive compounds in leaves and determination of mode of action of protein individually and synergistic performance of the ingredients from the extracts will be needed for recommendation as bioinsecticide in future. acknowledgements dr s v patil is thankful to rajiv gandhi science and technology commission (rgstc, mumbai, india). the authors declare that there is no conflict of interest. references abott ws (1925) a method of computing the effectiveness of an insecticide. j eco entomol. 18: 265–266. abu bakar a, 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application of microbial larvicides on malaria transmission in central côte d'ivoire. j am mosq control assoc. 25(3): 382–385. who (1996) report of the who informal consultation on the evaluation on the testing of insecticides ctd/who pes/ ic/96. 1: 69. world health organization (2012) malaria report. available at: http://www.who. int/malaria/publications/world_malaria _report_2012/report/en/index.html. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 53–59 b vazirianzadeh et al.: the first report of … 53 original article the first report of drug resistant bacteria isolated from the brown-banded cockroach, supella longipalpa, in ahvaz, south-western iran babak vazirianzadeh 1, *rouhullah dehghani 2, manijeh mehdinejad 3, mona sharififard 4, nersi nasirabadi 3 1department of medical entomology, college of health and infectious and tropical disease research centre, ahvaz jundishapur university of medical sciences, ahvaz, iran 2department of environmental health, college of health and social determinants of health (sdh),research center, kashan university of medical sciences, kashan, iran 3departments of medical microbiology, college of medicine, ahvaz jundishapur university of medical sciences, ahvaz, iran 4departments of medical entomology, college of health, ahvaz jundishapur university of medical sciences, ahvaz, iran (received 7 july 2012; accepted 30 june 2013) abstract background: the brown-banded cockroach, supella longipalpa is known as a carrier of pathogenic bacteria in urban environments, but its role is not well documented regarding the carriage of antibiotic-resistant pathogenic bacteria in iran. the aim of this study was to determine the resistance bacteria isolated from the brown-banded cockroach in ahvaz, south west of iran. methods: totally 39 cockroaches were collected from kitchen area of houses and identified. all specimens were cultured to isolate the bacterial agents on blood agar and macconky agar media. the microorganisms were identified using necessary differential and biochemical tests. antimicrobial susceptibility tests were performed for isolated organisms by kirby-bauer’s disk diffusion according to ncli guideline, using 18 antibiotics. results: from the 39 collected s. langipalpa, 179 bacterial agents were isolated, 92 of alimentary ducts and 87 of external body surfaces. isolated bacteria from cockroaches were identified as enterobacter spp., klebsiella spp., citrobacter spp., escherichia coli, salmonella spp., proteus spp., coagulase negative staphylococci, serratia marcescens, staphylococcus aureus, and bacillus species. the pattern resistance rates were determined for gram negative bacilli and gram positive cocci regarding 18 antibiotics. conclusion: the brown-banded cockroach can be involved in the spread of drug resistant bacteria and increases the possibility of contacting human environment to drug resistant bacteria. therefore, the potential of removing this insect should be improved. this is the first original report of drug resistant bacteria isolated from the brown-banded cockroach of iran. keywords: supella longipalpa, drug resistant bacteria, iran introduction cockroaches are the most abundant and important pest insects that inhabit various public places such as hospitals, food manufacturing sites and kitchens (ebelling 1978, burgess and chetwyn 1979). cockroaches have been considered as transmitters and spreaders of pathogenic bacteria in hospitals and households or residential areas (lamiaa bouamama 2005). cockroaches feed indiscriminately on garbage and sewage and so have copious opportunity to disseminate human pathogens. in addition, their nocturnal and filthy habits make them ideal carriers of various pathogenic microorganisms (allen 1978, graczyk et al. 2005, pai et al. 2005). so far numerous pathogenic bacteria, in*corresponding author: dr rouhullah dehghani, email: dehghani37@yahoo.com http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 53–59 b vazirianzadeh et al.: the first report of … 54 cluding salmonella spp., shigella spp., campylobacter spp., pseudomonas aeruginosa and k. pneumonia have been isolated from cockroaches (cotton et al. 2000). antibiotic resistance is a serious publichealth problem, reduced effectiveness of antibiotics results in greater patient mortality rates, prolonged hospitalization and increased healthcare costs. the economic impact of antibiotic resistance has been estimated between $5 and $24 billion annually in the united states alone (hall 2004). insects associated with food animals, especially cockroaches not only are important nuisance pests but also potential vectors of animal and human pathogens. organic waste in and around animal production facilities provide excellent habitats for the growth and development of these insects. because of their habitat preferences, unrestricted movement, mode of feeding, and attraction to residential areas, cockroaches have a great potential to disseminate fecal bacteria, including human and animal pathogens and antibiotic resistant strains (zurek and gorham 2008, graczyk et al. 2001). the brown-banded cockroach, supella longipalpa, is a small species of cockroach, measuring about 10 to 14,mm long. cockroaches like s. longipalpa which live in drier parts of houses can harbor and transmit the different bacteria within and between premises because of small bodies. this leads to spread the bacteria which may be pathogenic and antibiotic resistance, potentially (manweiler 1998, gibson and hunter 2009). there are insufficient data on the potential health impact of this species in the resident areas of iran. more infestation with this species has recently been reported from urban environments of iran, but its role is not well documented regarding the carriage of antibiotic-resistant pathogenic bacteria in iran. this is the first original report of drug resistant bacteria isolated from the brownbanded cockroach of ahvaz and iran. the aim of this study was to determine the resistance bacteria isolated from external surfaces and digestive tract of the brownbanded cockroaches (s. longipalpa) which were collected from kitchen area of houses of ahvaz, south west of iran. materials and methods a total of 39 cockroaches were collected from kitchen area of houses, ahvaz sw iran. the samples were transported to the medical entomology laboratory of ahvaz jundyshapur university of medical sciences (ajums) and placed in the sterile dishes in freeze-temperature for 10–15min to anaesthetize them. the cockroaches were identified after immobilization by freezing, using keys of department of entomology and nematology university of floridaas and cohran (cochran 1999). afterward, two samples were taken from each cockroach, one of external surface body with sterile cotton swabs and the other one directly from alimentary duct after dissecting the insects. all specimens were cultured to isolate the bacterial agents on blood agar and mac conky agar media (himedia india and merck germany). the microorganisms were identified using necessary differential and biochemical tests. cultured media were incubated at 37 ˚c for 24 hours. the various bacteria growth on the agar media were identified by colonial morphology on blood agar plates, gram stains characteristics. in addition, other necessary biochemical tests including: oxidase, catalase, motility, coagulase, indole, methylred, voges proskaeur (mrvp), gelatine hydrolysis, gas from glucose, h2s production and acid produced from various sugar according to standard microbiological procedures were also used for identification (forbes et al. 2002, jorgensen et al. 2005). antimicrobial susceptibility testing was performed for isolated organisms by kirbybauer’s disk diffusion according to clinical and http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 53–59 b vazirianzadeh et al.: the first report of … 55 laboratory standards institute (clsi) guideline. antibiotic disks were used and the susceptibility pattern of isolated from samples to commonly used antibiotics was then reported. the antibiotic disks (padten teb, tehran, iran) comprised ampicillin (10μg), cephalothin (30μg), ceftriaxone (30μg), ciproflexoxacin (5μg), chloramphenicol (30μg), gentamicin (10μg), tetracycline (30μg), trimethoprimsulfamethoxazole (25μg), ceftazidime (10μg), amikacin (30μg), nitrofurantoin (300μg), nalidixic acid (30μg), cefalexin (10μg), imipenem (10μg), vancomycin (30μg), cefotaxime (10μg), kanamycin (30μg), and penicillin (10μg) guidelines to determine susceptibility of utis agents (clsi 2002, mpuchane 2006). results from 39 collected s. langipalpa, 179 bacterial agents were isolated, 92 of alimentary ducts and 87 of external body surfaces (table 1). isolated bacteria from cockroaches were identified as enterobacter spp., klebsiella spp., citrobacter spp., e. coli, salmonella spp., proteus spp., coagulase negative staphylococci, s. marcescens, staphylococcus aureus, and bacillus species. the pattern resistance rates were determined for gram negative bacilli and gram positive cocci regarding 18 antibiotics. among all the isolates gram negative bacilli, from kitchen area of houses, ampicillin, cephalothin, ceftazidime, nitrofurantoin, nalidixic acid, trimethoprim-sulfamethoxazole, cefalexin and tetracycline, resistance rates were above 52.4% and cefotaxime expressed the highest susceptibility among all the isolates from the kitchen area of houses. among all the isolates gram positive cocci, from kitchen area of houses, ampicillin, amikacin penicillin, ceftazidime, nitrofurantoin, nalidixic acid, trimethoprimsulfamethoxazole, cefalexin, cefotaxime and tetracycline, resistance rates were above 53.8% and ciproflexoxacin expressed the highest susceptibility among all the isolates from the kitchen area of houses (table 2). table 1. frequency of isolated bacteria from cockroaches total no. (%)external surface no. (%)alimentary tract no. (%)isolated bacteria from cockroaches 5(2.79) 6(3.35) 18(10.10) 29(16.20) 2(2.30) 3(3.45) 9(10.35) 14(16.10) 3(3.26) 3(3.26) 9(9.78) 15(16.30) enterobacter aerogenes enterobacter cloacae enterobacter agglomerans total 25(14.00) 3(1.70) 28(15.64) 13(14.94) 1(1.15) 14(16.10) 12(13.04) 2 (2.17) 14(16.30) klebsiella pneumonia klebsiella oxytoca total 16(8.94)7(8.05)9(9.78)citrobacter freund 24(13.41)11(12.64)13(14.13)escherichia coli 5(2.79)2(2.30)3(3.26)salmonella para a 8(4.50)4(4.60)4(4.35)serratia marcescens 11(6.2) 3(1.70) 14(7.82) 6(7.00) 1(1.15) 7(8.10) 5(5.43) 2(2.17) 7(7.61) proteus mirabilis proteus vulgaris total 20(11.17)9(10.3511 (11.96)coagulase negative staphylococci 7(3.91)4(4.60)3 (3.26)staphylococcus aureus 19(10.61) 7(3.91) 26(14.53) 10(11.50) 4(4.60) 14(16.10) 9(9.78) 3(3.26) 12(13.04) bacillus cereus bacillus subtilis total 179(100)87(47.43)92(52.57)total http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 53–59 b vazirianzadeh et al.: the first report of … 56 table 2. percentages of resistant of isolated bacteria from cockroaches gram positive coccigram negative bacilliantibiotics resistance 10086.8ampicillin 44.369.5cephalothin 45.542.4ceftriaxone 19.624.7ciproflexoxacin -47.3chloramphenicol 38.844.2gentamicin 63.459.4tetracycline 57.153.6trimethoprim-sulfamethoxazole 85.3100ceftazidime 53.825.9amikacin -70.1nitrofurantoin 74.952.4nalidixic acid 63.267.9cefalexin --imipenem --vancomycin 85.313.5cefotaxime -36.5kanamycin 100-penicillin discussion the biology and ecology of s. longipalpa make it an ideal mechanical vector of bacteria to animal and human. brown-banded cockroaches are found in homes, apartments, hotels, and hospitals. they are less frequently found in stores restaurants and kitchens. they are frequently transported in furniture and will spread rapidly through an entire building. brown-banded cockroaches are generally found on ceilings, high on walls, behind picture frames, and near motors of refrigerators and other appliances. the results of this study indicated that supella longipalpa could play a great role as a mechanical carrier of bacteria. in this study, all 39 cockroaches were found to carry the 9 genera of medically important bacteria. the most frequent bacteria isolated from s. angipalpa coming from external bodies of this cockroaches was k. pneumonia followed by e. coli and e. coli was the most frequent coming of alimentary tract of s. langipalpa followed by k. pneumonia in the present study was. this is very similar to the studies of vahabi et al. in iran and bouamama et al. in morocco (bouamama et al. 2010, vahabi et al. 2011). however, the most biodiverse isolated bacteria during the current investigation was totally related to enterobacter sp.: e. aerogenes, e. cloacae and e. agglomerans, with 16.20% of all species. this is important when enterobacter spp. are known as food spoilage bacteria and are considered as cockroach transmitted bacteria in food poisoning. in this study, most of the bacteria isolated were medically important, including enterobacter spp., klebsiella spp., citrobacter spp., e. coli, salmonella spp., proteus spp., coagulase negative staphylococci, s. marcescens, staphylococcus aureus, and bacillus species. these findings agree with the results of chaichanawongsaroj et al. (2004) in thailand, which showed presence enterobacter spp., klebsiella spp., citrobacter spp., e. coli, salmonella spp., proteus spp., serratia spp., species on the periplaneta americana and blatta orientalis collected from hospital, food-handling establishments and human dwellings (bennett 1993, chaichanawongsaroj et al. 2004, salehzadeh et al. 2007). the similar bacteria have been isolated from p. americana and b. germanica in anhttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 53–59 b vazirianzadeh et al.: the first report of … 57 other study of botswana (mpuchane 2006). however, they are known as pathogenic (salmonella, shigella, b. cereus), opportunistic pathogens (pseudomonas, klebsiella, vibrio) and food spoilage species (pseudomonas, enterobacter, escherichia, erwinia) they belong to cockroach transmitted bacteria (mpuchane 2006). bacterial species, such as klebsiella spp., e. coli, staphylococcus spp., enterobacter spp., streptococcus spp., pseudomonas spp., proteus spp., bacillus spp., neisseria spp., shigella spp., and salmonella spp., have also been isolated from p. americana and polyphaga aegyptica cockroaches by other researchers from khuzestan (vazirianzadeh et al. 2011, kassiri and kazemi 2012). it is important to say that the above named bacteria have been isolated from housefly, musca domestica in khuzestan (vazirianzadeh et al. 2008). the present study indicates that brownbanded cockroach posses a possible health risk to communities proved that the isolated strains of bacteria were resistant to various antibiotics. it is well-established fact that the resistance to various antimicrobials may be due to presence of some virulence gene, involvement of secretion machinery of multi drug efflux proteins, through mutations in bacterial genome or by gaining additional genes through horizontal gene transfer or by physiology dependent resistance (mitchell et al. 2004). one of the most important problems facing global public health today is antimicrobial resistance. the problem is most horrible in developing countries, where the bacterial infections causing human disease are also those in which emerging antibiotic resistance is most evident (shears 2000, fathpour et al. 2003, kalantar et al. 2008). cockroaches are able to mechanically transfer infectious agents in unsanitary places. some bacteria that exist in the digestive tract of cockroaches can be also found in environment, that most of them belong to gram-negative bacilli of enterobacteriacea family (cloarec and rivault 1992) this organisms can cause urinary infections, poisoning, inflammation of the stomach and intestines, and abdominal cavityinfection, pneumonia or wound infections (le guyader et al. 1989, kopanic et al. 1994). therefore, elimination of cockroaches from sensitive areas, such as hospitals, is essential. in food-handling establishments and human dwellings, cockroaches must also be controlled, to maintain acceptable hygiene standards (chaichanawongsaroj et al. 2004). conclusion brown-banded cockroaches collected from kitchen area of houses may be involved in the spread of drug resistant bacteria and may increase the potential for human exposure to drug resistant bacteria. it is recommended that suitable steps must be taken to control the cockroaches and monitor the sensitivity pattern of the pathogens transmitted by the cockroaches. therefore, the potential of removing this insect should be improved. parallel to integrated pest management of cockroaches, we must go ahead to produce new antibiotic from natural resources like the filthy habitats of cockroaches which kills them (lee 2012). acknowledgements the authors gratefully acknowledge personnel of department of medical microbiology, college of medicine, ahvaz jundishapur university of medical sciences for providing, their assisting in the study. the authors declare that there is no conflict of interests. references allen bw (1987) 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region of ahvaz, sw iran. jundishapur j microbiol. 1(1): 28–31. zurek l, gorham jr (2008) insects as vectors of food borne pathogens. in wiley handbook of science and technology for homeland security. edited by: hoboken v jg, n j john. wiley and sons,inc. new york. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 201 http://jad.tums.ac.ir published online: january 05, 2016 original article chemical composition and repellent activity of achillea vermiculata and satureja hortensis against anopheles stephensi masoumeh pirmohammadi 1, *mansoureh shayeghi 1, *hassan vatandoost 1,2, *mohammad reza abaei 1, ali mohammadi 1, akbar bagheri 1, mehdi khoobdel 3, hasan bakhshi 1, maryam pirmohammadi 4, maryam tavassoli 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran 2instituet for environmental research, tehran university of medical sciences, tehran, iran 3health research center, baqiyatallah university of medical sciences, tehran, iran 4kashan university, kashan, iran (received 3 aug 2014; accepted 17 jan 2015) abstract background: one of the best ways to control the malaria disease and to be protected human against anopheles mosquito biting is the use of repellents. throughout repellents, herbal ones may be an appropriate and safe source for protection. methods: chemical constituents of achillea vermiculata and satoreja hortensis were determined by using gas chromatography-mass spectrometry. efficacy and the protection time of these plants were assessed on anopheles stephensi under the laboratory condition. results: the mean assessed protection time and efficacy for a. vermiculata was 2.16 and 3.16 hours respectively and the obtained ed50 and ed90 for this plant was 5.67 and 63 µ l/cm 2 respectively. the figured for s. hortensis was 4.16 and 5 hours respectively. ed50 and ed90 for this plant were 5.63 and 45.75µl/cm2 respectively. conclusion: results of investigation showed that s. hortensis plant has an acceptable protection time, therefore, this plant could be considered as a good herbal repellent against anopheles mosquitoes. keywords: achillea vermiculata, satureja hortensis, anopheles stephensi, repellency, protection time introduction mosquito borne diseases affect human societies by reduction in labor productivity especially in tropical and subtropical countries. the important point is this fact that all countries all over the world have a problem with insect borne diseases (govindarajan et al. 2011). anopheles mosquitoes are bloodsucking insects, responsible for transmission of malaria, filariasis and arboviruses (service 1980). there are 33 currently recognized anopheles species including sibling, biological forms and zygotypes, seven of these species have an important role in malaria transmission in iran. among these species, an. stephensi is considered as a primary vector of malaria in southern parts of the country (sedaghat et al. 2003, vatandoost et al. 2004, sedaghat et al. 2005). according to the who report, a total of 627000 people die due to malaria (who 2013). malaria is caused by plasmodium parasite. malaria is one of the most important diseases which the parasite is transmitted by female anopheles genus (who 2010). there are several methods for malaria vector control. *corresponding authors: dr mansoureh shayeghi, e-mail: mansorehshayeghi@yahoo.com, dr hassan vatandoost, e -mail: hvatandoost1@yahoo.com, dr mohammad reza abaei, e-mail: abaimr@tums.ac.ir j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 202 http://jad.tums.ac.ir published online: january 05, 2016 synthetic insecticides which are generally used have side-effects on human, animal health, and the environment. the side-effects of synthetic organophosphorus compounds on fish and other organisms in the environment are being increasingly reported. a lot of attention is being paid to natural products in vector control as they are environmentally safe, degradable and target-specific. recent studies have demonstrated that use of repellents is one of the effective ways to control the disease and to avoid anopheles bites (vatandoost et al. 2008). deet is slightly yellow oil. it is the most common active ingredient in insect repellents. it is intended to be applied to the skin or to clothing, and provides protection against mosquitoes and many other biting insects. deet was developed during world war ii. the findings bring evidence that, deet has side effects, so it has proposed to use alternative repellents for protection (karunamoorthi et al. 2010). some prefer to use natural insect repellent products. repellents of plant origin do not pose hazards of toxicity to human and domestic animals and are easily biodegradable. natural products are safe for human when compared to that of synthetic compounds (fradin 1998). the effect of some plant origin essential oils have been tested in iran (oshaghi et al. 2003, yaghoobi-ershadi et al. 2006, vatandoost and hanafi-bojd 2008, tavassoli et al. 2011, mozaffari et al. 2014 ). achillea vermiculata is a flowering plant in the family asteraceae with a height of 10– 30 cm. it is native to temperate regions of the northern hemisphere in asia, europe, and north america. it is an erect herbaceous perennial plant. the leaves have varying degrees of hairiness. the leaves are almost feathery (mozaffarian 2012). satureja hortensis has lilac tubular flowers. it grows to around 30 to 60 cm in height and has very slender, bronze-green leaves. this plant belongs to order: lamiales and family: lamiaceae. it is used in traditional medicine as a botanical treatment (mozaffarian 2012). this study was conducted to evaluate the repellent properties of two plants a. vermiculata and s. essential oil against an. stephensi in laboratory condition on animal model and also to determine chemical compositions in their essential oils. materials and methods mosquitoes rearing established colony of susceptible strain of an. stephensi obtained from the insectary of school of public health, tehran university of medical sciences, tehran, iran. mosquitoes were reared and maintained at 28±2 °c and 65±5% relative humidity (rh) under a 16:8 (l: d) photoperiod. larvae were fed on a diet of fish food and water lettuce. the adults were maintained in screen cages and fed with 10% aqueous sucrose solution as a source of energy and guinea pigs as bloodfeeding female mosquitoes for maturing the eggs. starved 5 to 8 days old females were used for the repellency tests. the sucrose solution was picking up from the cage, 12 hour before starting the experiments. collection, identification and extraction of plants fresh flowers and leaves flowers of a. vermiculata and s. hortensis were collected from armand and sheyda district which are located in chaharmahal and bakhtiari province in south-west of iran in june 2013 (fig.1,2). they were rapidly transported to the school of public health, tehran university of medical sciences. achillea vermiculata was collected from natural habitat in armand district at coordinate 31° 39.428′e 50° 46.659′n, 1136 meters above sea level. satureja hortensis was collected from natural habitat in sheyda district in ben at coordinate 32° 37.206′e 50° 42.434′n, 2219 meters above sea level. j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 203 http://jad.tums.ac.ir published online: january 05, 2016 the plant a. vermiculata was identified by experts in department of plant sciences, tehran university. also s. hortensis was identified by experts in ecotoxicology and herbarium laboratory, school of public health, tehran university of medical sciences. the flowers and leaves of a. vermiculata and s. hortensis were dried at room temperature under good ventilation. about 525 gr of dried a. vermiculata and 420 gr of dried s. hortensis were chopped into small pieces using a knife mill. the essential oil was extracted from the plants using a clevengertype water steam distillation apparatus. it took about 4 hours for extraction of the essential oils. for extraction of essential oil of each plant we used sulphate sodium anhydr. the distilled essential oils were stored in a refrigerator at 4 °c until being used in the experiments. the composition of the volatile constituents was established by gas chromatography-mass spectrometry. test method all series of the experiments of effective dose and protection time were carried out in laboratory condition. we tested these plants on white rabbits. the white rabbits (o. cuniculus) (laboratory reared albino male aged 6–8 months) were used to determine both protection time and effective dosage in insectary at the school of public health. in this investigation, the 50% concentration of essential oils was used for protection time test. for this purpose essential oils were diluted by absolute ethanol. then for estimating the protection time of plants essential oils against an. stephensi the back of male rabbits by 52cm2 were shaved in the next stage, 80µ l of 50% essential oils of two plants by sampler on the shaved back of male rabbits were applied. after 5 minutes the rabbits were placed in a box which is designed for the test. then it is placed in box containing the rabbit in a cage at dimension of 53×53×53 cm containing 150 starved 5–8 days mosquitoes. after 3 minutes of biting and probing records, we brought out the cage and we tested again 30 minutes later. these tests continued until two successive bites. this time is called protection time. we continued these tests until 10 bites. this time is called failure time (pitasawat et al. 2003). the procedure for determination of effective dosages of the repellents was adopted by the standard method of american society for testing and material (astm 2000). the testing kit was made of plexiglas cube at dimension of 4×5×18cm having four rectangular holes 4×3cm. before starting the test for determination of effective dosage, the abdomen skins of rabbits were cleaned with alcohol and the kit was fixed on the abdomen. each of 4 adjacent cells of kit was provided with 5 female 5–8 days mosquitoes that randomly selected from a cage containing 150 starved mosquitoes. circles were drawn on the rabbit's skin. the drawn circles on the abdomen skin’s of hold rabbit were treated with 50μl of essential oil diluted with absolute ethanol at 6.6, 13.2, 26.4 and 52.8µ l for a. vermiculata and 3.3, 6.6, 13.2, 26.4 and 52.8µ l for s. hortensis microliter with 4 repetitions. the same dilutions were applied on 3 holes because of prevention of contamination as well as the absolute ethanol was applied in remaining control circle. we used 5 mosquitoes for each hole. the treated circles were allowed to dry, and then test apparatus containing starved mosquitoes were fixed on the treated skin. the counts of probing and biting were recorded for 5 minutes. after each test, the mosquitoes were transferred to netted cups and the mortality of mosquitoes was recorded after 24 hours. the ed50 and ed90 values and regression parameters were analyzed using probit 79 programs and the regression lines were plotted in microsoft excel 2007. plants essential oils analysis chemical composition of a. vermiculata j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 204 http://jad.tums.ac.ir published online: january 05, 2016 and s. hortensis was analyzed using an agilent 7890–5975 gas chromatographymass spectrometer. with a hp5ms (5% phenyl methyl silox) capillary column (30m×0.25mm, film thickness 0.25μm), split ratio, 1: 1, and using a flame ionization detector. the gc was programmed at 50 °c for 0.5 min and then increased at 5 °c/min to 280 °c, and finally held with an isothermal for 3min. the injector temperature was 280 °c. the flow rate of the carrier gas was 1ml/min. the identification of compounds was performed by comparing their retention times and mass spectra with mass spectra from wiley library. results essential oil volumes by the use of clevenger-type water steam distillation, about 1849µl of essential oil of 525gr of dried a. vermiculata flowers was extracted. also about 4480µ l of essential oil of 420gr of dried s. hortensis leaves extracted. gc-mass analysis one microliter of each essential oil was injected to gc-mass. a total of 40 compounds were identified in flowers of a. vermiculata. (e)-β-damascenone with 27.4, (e)-2-hexenal with 8, eugenol with 6 and geranyl acetone with 6 percent were the major components (table 1). we just found a repellent component “camphene” of all identified components by researchers until now in this plant with 0.7%. also we identified 23 components in the leaves of s. hortensis. b-oplopenone with 57, trans-carvone oxide with 15.13 and thymol methyl ether with 13 percent were the major components (table 2). protection time the protection time of a. vermiculata essential oil against an. stephensi on animal subject provided 2.0–2.5 hours range with a mean of 2.16 hours protection and a failure time of 3–3.5 hours range with a mean of 3.16 hours. also the protection time of s. hortensis essential oil provided 4–4.5 hours range with a mean of 4.16 hours protection and a failure time of about 5 hours (table 3). significant differences of protection time and failure time between a. vermiculata and s. hortensis repellents were observed by anova (games-howel), p< 0.05. effective dose the ed50 and ed90 values of a. vermiculata essential oil were 5.67 and 63 µ l/cm2 with confidence interval ranged, 2.25-8.68 and 38.21–198.07 µ l/cm2 respectively (table 4). the ed50 and ed90 values of s. hortensis essential oil were 5.63 and 45.73 µ l/cm2 with confidence interval ranged, 3.83-7.43 and 30.92–86.55 µ l/cm2 respectively (table 4). we did not observe any significant differences between ed50 and ed90 of s. hortensis and a. vermiculata by t-test and p> 0.001 analysis. fig. 1. the plant achillea vermiculata in its natural habitat, armand district in chaharmahal and bakhtiari province, south-western of iran (original) j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 205 http://jad.tums.ac.ir published online: january 05, 2016 table 1. chemical constituents of flower essential oil from achillea vermiculata no compound composition% ri 1 isovaleric acid 0.2 833 2 (e)-2-hexenal 8 854 3 tricyclene 0.1 921 4 limonene 0.3 1029 5 methybenzoate 0.5 1093 6 linalool 3.1 1108 7 α-campholenal 2.8 1131 8 camphene 0.7 1156 9 pinocarvone 0.7 1164 10 cis-piperitol 0.7 1194 11 verbenone 0.8 1208 12 trans-carveol 4 1222 13 cis-carveol 3.7 1233 14 geraniol 1 1258 15 2e,4e-decadienal 3.1 1313 16 eugenol 6 1359 17 (e)-β-damascenone 27.4 1382 18 cis-a-bergarnotene 5.6 1416 19 trans-a-bergamotene 3 1433 20 geranyl acetone 6 1452 21 allo-aromadendrene 0.8 1462 22 gamma-gurjunene 0.3 1473 23 a-muurolene 0.6 1499 24 a-cadinene 1.2 1537 25 spathulenol 0.7 1579 26 b-oplopenone 0.4 1601 27 humulene epoxide ii 0.3 1609 28 silphiperfol-6-en-5-one 0.6 1623 29 1-epi-a-eudesmol 2 1660 30 8-cedren-13-ol 0.3 1694 31 xanthorrizol 0.3 1751 32 8-a-acetoxyelemol 2 1788 33 nootkatone 0.4 1802 34 flourensiadiol 1.8 1864 35 hexadecanol 1.7 1881 36 methyl hexadecanoate 1.9 1911 37 methyl hexadecanoate 1.8 1925 38 methyl hexadecanoate 2.2 1941 39 methyl hexadecanoate 2.6 1957 40 heneicosane 0.25 2057 99.55 table 2. chemical constituents of leaf essential oil from satureja hortensis no compound composition% ri 1 isovaleric acid 0.06 831 2 (z)-3-hexenol 2.1 856 3 trans-sabinene hydrate 0.3 1095 4 linalool 1 1109 5 α-campholenal 0.6 1132 6 borneol 1 1171 7 trans-carveol 2 1221 8 thymol methyl ether 13 1237 9 trans-carvone oxide 15.1 1277 10 undecanal 0.4 1306 11 β-caroyophyllene 3 1424 12 b-oplopenone 57 1600 13 10-epi-gammaeudesmol 0.3 1620 14 hinesol 0.6 1637 15 8-cedren-13-ol 1 1704 16 oplopanone 0.2 1726 17 8-a-acetoxyelemol 0.3 1781 18 flourensiadiol 1.04 1867 19 methyl hexadecanoate 0.12 1910 20 methyl hexadecanoate 0.2 1923 21 methyl hexadecanoate 0.2 1935 22 heneicosane 0.1 2097 23 n-docosane 0.2 2213 99.82 fig. 2. the plant satureja hortensis in its natural habitat, sheyda district in chaharmahal and bakhtiari province, south-western of iran (original) j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 206 http://jad.tums.ac.ir published online: january 05, 2016 fig. 3. dose-response lines for two botanical repellents against anopheles stephensi on animal model table 3. protection time and failure time of achillea vermiculata and satureja hortensis against an. stephensi on animal subject in laboratory condition failure time (hour)protection time (hour)districtspecies (plants) meanrangemeanrange 3.163–3.52.162–2.5lordegana. vermiculata 55–54.164–4.5bens. hortensis table 4. effective dose of achillea vermiculata and satureja hortensis essential oils against anopheles stephensi on animal subject in laboratory condition p-valueχ2 table (df) χ2 (heterogeneity) ed90 (mg/cm2) ± 95% c.l. ed50 (mg/cm2) ± 95% c.l. b ± seaplants 0.0122.3563 (38.21–198.07) 5.67 (2.25–8.68) 1.22±0.27-0.092a. vermiculata 0.0133.1345.75 (30.92–86.55) 5.63 (3.83–7.43) 1.4±0.20-1.05s. hortensis discussion application of larvicides and repellents are generally accepted as they play an important role in control of the mosquitoes. the use of botanical essential oils as repellents against vectors of malaria disease including an. gambiae and a. stephensi has been tested successfully (seyoum et al. 2002). in this study the components of a. vermiculata essential j arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 207 http://jad.tums.ac.ir published online: january 05, 2016 oil were identified. (e)-β-damascenone with 27.4, (e)-2-hexenal with 8, eugenol with 6 and geranyl acetone with 6 percent were the major components. totally 40 components were identified from this plant. in an investigation by ahmadi et al. (2011), on a. santolina, 29 components were identified from this plant which the major components were camphor, alpha-pinene, camphene and 1,8 cineole with 26.27, 10.14, 9.09 and 8.26 percent respectively. we found two same components “camphene” and “linalool” of a. vermiculata with a. santolina. also we identified 23 components in the leaves of s. hortensis. b-oplopenone with 57, trans-carvone oxide with 15.13 and thymol methyl ether with 13 percent was the major components. in an investigation by kamkar et al. (2013) (32 components were identified and reported from this plant which the major components belonged to γterpinene with 24.72%, thymol with 29.1% and carvacrol with 26.6%. in this investigation, we identified some components which had been identified in kamkar et al. (2013) study. these components are: borneol, linalool and thymol. also cis-sabinene hydrat was identified in kamkar investigation, but our investigation revealed the presence of trans-sabinene hydrate. in another investigation by tajalli et al. (2012) on this plant, the major components were thymol, carvacrol, gamma-terpinene with 48.67, 8.96, 9.16 and 9.16 percent respectively. in our investigation, we found thymol and linalool which had been identified in tajalli et al. (2012) study too. our investigation revealed that a. vermiculata essential oil can have 2.16 hours protection time and also its failure time is 3.16 hours. compared to a. vermiculata, s. hortensis could have 4.16 hours protection time and a failure time of 5 hours which is about 2 times as much as a. vermiculata’s protection time and failure time. also we revealed an ed50 and ed90 effective dose of 5.67 and 63 µ l/cm2 respectively, while for s. hortensis they were 5.63 and 45.75 µ l/cm2 respectively. the repellency effect of essential oils of some plants has been studied in iran. on a laboratory trial by vatandoost et al. (2008), the repellency of neem tree’s essential oil against an. stephensi in animal subject was determined. the ed50 and ed90 values of neem tree’s essential oils were calculated 0.159 and 1.388 mg /cm2 respectively. also the protection time and effective dose of this plant calculated 31 minutes and 65 minutes respectively. the repellency effect of essential oils of both myrtus communis and calendula officinalis had been reported against an. stephensi on human subject and the effective dose of these plants was 0.11 and 0.6 mg/ cm2 respectively. also the protection time and failure time for m. communi were 4.36 and 4.4 hours respectively. the protection time and failure time for c. officinalis were 2.15 and 3.30 hours respectively (tavassoli et al. 2011). in an investigation, the mean protection time of 50% essential oil of cionura erecta (l) provided 2.28 hours protection against an. stephensi. the figures for ed50 and ed90 values were 10.12 and 23.01ppm respectively (mozaffari et al. 2014). we estimate that the most protection time of mentioned investigations and our investigation, belongs to m. communi and s. hortensis with a protection time of 4.36 and 4.16 hours and failure time of 4.4 and 5 hours respectively. the weakest protection time and failure time belongs to neem plant by 31 minutes and 65 minutes respectively. there have been so many investigations about repellency effects of plants against mosquitoes all over the world until now (moore et al. 2002, rajkumar and jeanesabn 2007, mullai et al. 2008, karunamoorthei et al. 2010, shahi et al. 2010). finally we recommend s. hortensis as a candidate for prodution of insect repellents because of its high protection against mosj arthropod-borne dis, june 2016, 10(2): 201–210 m pirmohammadi et al.: chemical composition … 208 http://jad.tums.ac.ir published online: january 05, 2016 quitoes and also medicinal properties without any side effects. although this plant did not show any significant differences of effective dose rate with a. vermiculata. we recommend doing more investigations on this plant. conclusion according to the results it could be concluded that the plant is appropriate for the repellent formulation for mosquito control, although the field trail should be conducted in a malarious areas. acknowledgments this study was funded and supported by tehran university of medical sciences (tums), we are thankful to the staff of insectary of culicidae, department of medical entomology and vector control, school of public health, tehran university of medical sciences for the mass production of the mosquitoes for this study. we are thankful to dr salmaki, assistant professor from department of plant sciences, tehran university for detection of a. vermiculata. we are also thankful to staff of ecotoxicology laboratory and central laboratory of school of public health for identification of plants and analyzing the contents. the authors declare that there is no conflict of interests. references ahmadi z, sattari m, tabaraee b, bigdeli m (2011) identification of the constituents of achillea santolina essential oil and evaluation of the anti-microbial effects of its extract and essential oil. arak medical university journal. 14(56): 1– 10. 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report. world health organization. who (2013) malaria entomology and vector control. world health organization. yaghoobi-ershadi mr, akhavan aa, jahanifard e, vatandoost h, amin gh, moosavi l, ramazani ar, abdoli h, arandian mh (2006) repellency effect of myrtle essential oil and deet against phlebotomus papatasi, under 2965 laboratory conditions. iran j public health. 35(3): 7–13. j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 55 original article simultaneous morphological and molecular characterization of tatera indica in southwestern iran somayeh mohammadi 1, 2, *parviz parvizi 1 1molecular systematics laboratory, parasitology department, pasteur institute of iran, tehran, iran 2islamic azad university, tehran medical sciences branch, tehran, iran (received 4 aug 2014; accepted 10 dec 2014) abstract background: interest in tatera indica rodent arises mostly because it is believed that this species is survived among four subspecies reported from iran, two of which exist in khuzestan province. in addition, it might has a role as reservoir hosts of zoonotic cutaneous leishmaniasis in the transmission of leishmania major in some of the widespread asian foci including southwestern iran. methods: diagnostic morphological and molecular markers for t. indica were sought by characterizing from individual specimens, such as some taxonomic features and mitochondrial cytochrome b gene that had previously proven useful for the taxonomy of rodents. wild rodents were caught using live wooden and wire traps. the specimens were identified morphologically using external criteria and molecularly by sequencing of cyt b gene and phylogenetic analyses. results: forty one t. indica were collected and identified morphologically in khuzestan province, iran. two morphotypes of t. indica were found and classified but sequencing and phylogenetic analyses of mitochondrial cyt b gene did not support any subspecies between two morphotypes of t. indica. because all 21 sequences of both morphotypes of t. indica had no variation with only one common and novel haplotype (genbank accession no kp001566). conclusion: this is the first time that t. indica was characterized molecularly in iran. there is no molecular evidence for t. indica morphotypes or subspecies, and so a population genetics approach using several polymorphic genes might be employed using species-specific molecular markers. in addition, more specimens of t. indica species in large geographical locations should be tested. keywords: tatera indica, molecular characterization, reservoir host, zoonotic cutaneous leishmaniasis, iran introduction the indian gerbil, tatera indica (hardwicke) is believed to be the main reservoir host of zoonotic cutaneous leishmaniasis (zcl) in khuzestan province, in border of iran and iraq, southwest of iran. khuzestan province is an important focus of zcl in iran and the reports shows that the occurrence of the disease has increased through the recent years (javadian et al. 1998, kia et al. 2010, shirzadi 2012, vazirianzadeh et al. 2013). the female phlebotomus papatasi (scopoli) (diptera: psychodidae) sandfly is the proven vector of the parasitic protozoan l. major (yakimoff and schokhor) (kinetoplastida: trypanosomatidae), the causative agent of zcl in iran (parvizi and ready 2008, bordbar and parvizi 2014). tatera indica is distributed through iran, afghanistan, pakistan, india, sri lanka, turkey, iraq and syria (harrison and bates 1991, agrawal 2000, yigit et al. 2001). the genus tatera lataste, (rodentia: gerbillinae) are widespread in the africa, near east, middle east and pakistan subcontinent (colangelo et al. 2005). at the morphological taxonomic level, 12 species of genus tatera were identified of which 11 are present in africa (t. afra, t. *corresponding author: dr parviz parvizi, e-mail: parp@pasteur.ac.ir http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 56 brantsii, t. bohemi, t. guineae, t. inclusa, t. vicina, t. kempi, t. leucogaster, t. nigricauda, t. phillipsi and t. robusta) and only one species (t. indica) lives in asia (corbet 1978, corbet and hill 1991). based on morphological characters, t. indica can be considered as tatera sensu stricto and consequently all 11 african tatera species were placed in one genus of gerbilliscus thomas. molecular characterizations of genomic dna data showed that the genus of tatera is a polyphyletic taxon (chevert and dobigny 2005). of several subspecies of t. indica, four subspecies are present in iran. t. indica persica exists in sistan, t. indica scansa in kerman; and finally t. indica monticola and t. indica bailwardi in khuzestan (ellerman 1948, missone 1959, mirshamsi et al. 2007). studies on tatera genus have highlighted a complex situation by karyological studies in africa (colangelo and civitelli 2001). the main objective of this research was to characterize the t. indica populations based on taxonomic knowledge by using and comparing morphological, genetic and molecular criteria concurrently from a specific geographical location of iran where this rodent is predominant. in addition, phylogenetic analyses were employed to get the better understanding of t. indica subspecies in case of any homology at their nucleotide scales. accurate and firm identification of animal reservoir hosts of zcl are essential, requires any epidemiological research, and are crucial for predicting species-specific population, and developing control strategies. materials and methods study sites, collections and identification of rodents the active colonies of rodents were identified firstly from ten locations of khuzestan province, southern iran with an altitude of 18 meters above sea level and geographical coordinates as 31° 32′ 73″ n and 48° 69′ 40″ e, and then the rodents were trapped alive in various parts of these areas using wooden and wiry live traps (fig. 1). sampling was conducted from 10 cities in khuzestan province. however, only from 6 of them (table. 1) including 15 villages (fig. 1) t. indica rodents were presented and captured. the collection of samples was done from the colonies of rodents’ burrows located around the villages where zcl were endemic using 50 live wooden and wirytraps for each location. sampling was conducted from 10 cities in khuzestan province, however in only 6 of them t. indica rodents were present and captured. the collection of samples were done from the colonies of rodents burrows located around the villages where zcl were endemic using 50 live wooden and wire traps for each collection. rodents were caught by baiting cucumbers and dates in 2012–2013. the traps were set up early in the morning and evening then the trapped rodents were transferred to pasteur institute of iran, tehran, and maintained for morphological and molecular testing (mehrabani 2011). the morphological identification of collected specimens were employed using external criteria such as color, head and body length, body measurements, ears length, tail length, hind foot length, cranial and dental measurements and cranium. the standard external characters of body and tail were measured using a ruler and/or digital calipers. the sizes of dental characters were measured using nikon measuring microscope mm-40 (table 1, 2) (etemad 1978). dna extraction and pcr amplification of cyt b gene from rodents all the captured rodents first identified morphologically then genomic dna of t. indica was extracted using genet bio kit, http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 57 phenol-chloroform and ish-horovize methods (parvizi and ready 2008). after comparing these three dna extraction methods, ish-horovize method was found to be more sensitive than others were and selected as main dna extractions method with minor modifications. mitochondrial cytochrome b (cyt b) gene, which is a maternally inherited marker were used to characterize this rodent. for molecular characterization of rodents, extracted dna from the tissue and blood of each rodent was used to amplify a fragment of cyt b with the forward primer unfor403 (5'-tgaggacaaatatcattctgagg3') and reveres primer unrev1025 (5'ggttgtcctccaattcatgtta-3') from single rodent by pcr (kent and norris 2005). the pcr products were directly sequenced, and the sequences edited and aligned using sequenchertm v. 4.4 to permit the identification of haplotypes (= unique sequences) and their phylogenetic analysis using mega5.05 and paup* softwares (tamura et al. 2011). results collection and morphological identification of tatera indica of all rodents caught during this study a total of 41 t. indica were collected and identified morphologically from 6 different locations in khuzestan province (fig. 1). the most t. indica were captured from behbahan district with 24 out of 41, then ahvaz (9/41), dezful (4/41), shush (2/41), shushtar and ramhormoz each with one samples. morphological characterizations of t. indica showed that the rodents were in two color groups, buff black (21/41) and buff brown (20/41), total rodent length was about 33–39 cm, body length 16–20 cm, ears length 2.1–2.5 cm, tail length 16–19 cm and hind foot length 3.8–4.5 cm (table 1) (etemad 1978). three morphological characters of total rodent length, hind foot (hf), head and body (hb), ear length (el) and tail length (tl) were considered for separating t. indica from other rodent species in the region. the details of morphological characters of captured t. indica in khuzestan are summarized and shown in table 1 and 2 (fig. 2). based on the development degree of the supraorbital crests and dental wear especially upper molars, three ages group among our t. indica samples were classified (etemad 1978). the incisor teeth of t. indica are formed with a groove and upper molars (fig. 2b). by considering the colors of rodent body, two types of morphological characters of t. indica were found. the buffblack group was larger with wider snout and they had a semi wide white halo above their eyes. however, the rodents of buffbrown group were smaller, with longer snout and a narrow white halo above their eyes (table 2, fig. 3). on bone formation of t. indica, zygomatic bones and pulled bsyarh is longer than other rodents (fig. 2a). molecular characterization and identification of tatera indica cyt b gene of extracted dna from all 41 t. indica was screened and amplified by pcr. the pcr products of 21 out of 41 were sequenced, aligned and analyzed via molecular softwares (sequenchertm v. 4.4, mega 5.05). this is the first time that a molecular study is performed on iranian t. indica, which provided us with novel findings that all the sequences are similar and no variation was found among the nucleotides; therefore, all the samples from one haplotype of t. indica. cyt b sequences were identified among 21 sequences obtained from individual t. indica. only haplotype kht01 (genbank accession no. kp001566) predominated and was common in khuzestan province. after comparing our sequences with only one submitted sequence in genebank from france (genbank accession no: aj430563.1) some http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 58 similarities and few differences were observed. based on the results of our sequences along with the sequences of different rodents submitted in genebank, a phylogenic tree was constructed (fig. 4). fig. 1. location of villages and cities in khuzestan province, iran where tatera indica was sampled fig. 2. tatera indica skull: (a): zygomatic plate, (b): upper molars and upper incisors http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 59 table 1. morphological characters of tatera indica which were collected in khuzestan province, iran (tb: total rodent length, hb: head and body, tl: tail length, hf: hind foot, el: ear length) t ot al colorelhftlhbtb morphometric characters b ro w n b la ck 2.1-2.53.8-4.516-1916-2033-39 size (cm) 2. 5 2. 4 2. 3 2. 2 2. 1 4. 5 4. 3 4. 243. 9 3. 8 1918 .5 1817 .5 1716 .5 162019 .5 1817 .5 163938 .5 3837 .5 36 .5 3534 .5 33 .5 33 11001000000010100000010000100000000ramhormoz l oc at io n s an d th e n u m b er s of t . i n di ca 11010000000010001000010000000000100shushtar 21120000002000001000010000002000000shush 42201120000121021001010300001020100 dezful 241311104532001410907490501301200004970400behbahan 92722014111411030122121411011111211 ahvaz 41202115866611491511112710281191191111810101811total fig. 3. buff brown (1) and buff black (2) tatera indica rodents (b) along with their skull image (a) casing the morphologic differences of two groups http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 60 fig. 4. unrooted maximum-likelihood bootstrap tree showing the relationships of different genus and species of rodents by employing cyt b gene fragment, including rodents submitted in genbank and our tatera indica sample using mega5 software. (kht sample showed in the phylogenic tree is our tatera indica sample captured from khuzestan province, southern iran) table 2. morphological classification of tatera indica based on their colors, khuzestan province, iran t. indica morphological characters buff brown (cm)buff black (cm) 0.42-0.470.55-0.57width of rostrum 4.26-4.474.75-4.88occipitonasal length 3.91-4.144.38-4.51condylbasal length 2.18-2.412.64-2.67zygomatic width 0.60-0.760.73-0.78least interorbital width 1.53-1.561.68-1.79cranial width 1.93-1.952.13-2.11length of nasal 1.17-1.261.33-1.36length of diastema 0.75-0.760.91-0.93length of anterior palatine foramina 1.14-1.191.28-1.29length of tympanic bullae 0.61-0.660.76-0.79width of tympanic bullae 0.61-0.640.67-0.75upper cheekteeth 0.55-0.610.63-0.71lower cheekteeth 1.71-1.871.98-1.99height of skull 2.41-2.512.58-2.65length of mandible discussion bates (1988) drew attention to the importance of systematic and zoogeoghraphy of genus tatera of northeast africa and asia. the genus tatera has an extensive geographhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 61 ical distribution in africa and asia (agrawal 2000, yigit et al. 2001, mirshamsi et al. 2007). there are reports of numerous studies on morphological and karyological taxonomy of genus tetera, however only few molecular characterizations are available worldwide (mirshamsi et al. 2007). t. indica was considered the main important reservoir host of zcl in khuzestan (mohebali and javadian 2004, mehrabani et al. 2007, hajjaran et al. 2013). principally p. papatasi may have been acquiring l. major from t. indica and other rodent reservoir hosts living peridomestically (yaghoobi-ershadi et al. 2013). although different rodent species were found but they were limited and located in only one location. only t. indica is considered as prominent reservoir host, which has a significant role in maintaining leishmania parasites in different locations of zcl in khuzestan province and this, highlights the importance of current survey on t. indica. in this survey, t. indica was investigated more precisely in case of morphological features and molecular methods for the first time in iran (khuzestan province) as the foremost and dominant reservoir in this focal region of leishmaniasis (table 1, 2, fig. 2, 3). the comprehensive study in broad-spectrum ranges and distribution of t. indica was done in khuzestan province but this rodent was restricted to only six locations. the cyt b gene was employed for molecular typing and characterizing of t. indica for the first time in iran and only one cyt b gene sequence of t. indica is available from france in genbank (genbank accession no: aj430563.1) and this indicates that sufficient molecular analyzing has not been carried out yet. previous investigation of t. indica in iran were only based on morphological identification and/or on leishmania infection of this reservoir (etemad 1978, mehrabani et al. 2011, hajjaran et al. 2013) and there was not any study on determining the principle reservoir host, morphological features along with molecular systematics analyzing simultaneously. the interesting finding of this investigation was that two types of morphological features (phenotypes) of t. indica was found in khuzestan however after using cyt b gene, only one haplotype from all 21 sequences (11 buffblack and 10 buff-brown) was identified (table 2, fig. 4) it is not clear how authors identified four subspecies of t. indica in iran and classified four subspecies (mirshamsi et al. 2007). they did not give any definitive morphological or molecular taxonomic characterization for separating the t. indica subspecies. a phylogenetic analysis of the new sequence, and those previously reported for t. indica and other rodents, found no support for recognizing more than one species or subspecies (t. indica) in khuzestan province, iran (fig. 4). mitochondrial cyt b demonstrated an absence of any subspecies between two morphotypes of t. indica, which classified previously and this study in khuzestan province (mirshamsi et al. 2007, oshaghi et al. 2011). all four taxa (t. indica persica in sistan, t. indica scansa in kerman, t. indica monticola and t. indica bailwardi in khuzestan) might be good biological species and did not characterize molecularly but could showed mitochondrial introgression caused by occasional inter-breeding the same as conclusions for some sandfly sibling species are reported (testa et al. 2002, pesson et al. 2004, parvizi et al. 2010). for resolving sibling t. indica species, a population genetics approach using several polymorphic genes might be tested by considering carefully for species-specific molecular markers and more specimens of t. indica species in large geographical locations. we only could conclude if two subspecies in khuzestan province or four subspecies of t. indica exist in iran, might have good evidence for associating specific taxa with phehttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 55–64 s mohammadi and p parvizi: simultaneous morphological … 62 notypes of epidemiological importance. there is no such evidence for t. indica subspecies, and so there is no reason to give priority to resolving the species status of four siblings t. indica species. conclusion tatera indica was characterized by both cyt b molecular marker and morphological features for first time in iran. there is no any morphotypes or subspecies of t. indica was found by molecular tools. we only could conclude if two subspecies in khuzestan province or four subspecies of t. indica exist in iran, might have good evidence for associating specific taxa with phenotypes of epidemiological importance. there is no such evidence for t. indica subspecies, and so there is no reason to give priority to resolving the species status of four siblings t. indica species. acknowledgements the work was supported by the pasteur institute of iran, grant 605 awarded to dr parviz parvizi. we thank dr vazirianzadeh, amraei, kajkolah, adel spotin, ali bordbar, sahar ebrahimi, javad sameii, mehdi baghban, roozbeh taslimian for help with the field work and elnaz alaeenovin and narmin najafzadeh for help in molecular systematics laboratory. this research through a studentship to miss somayeh mohammadi, based at the pasteur institute of iran, tehran, and registered for islamic azad medical university, tehran, iran. we confirm that there are no known conflict of interests associated with this publication. references agrawal vc (2000) taxonomic studies on indian muridae and hystricidae (mammalia: rodentia). rec zool surv india. 180: 1–177. bordbar a, parvizi p (2014) high density of leishmania major and rarity of other mammals' leishmania in zoonotic cutaneous leishmaniasis foci, iran. trop med int health. 19(3): 355–363. chevret p, 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rodentia) in turkey. turk j zool. 25: 65–67. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 review article conducting international diploma course on malaria program planning and management (1996–2012) ali reza mesdaghinia 1,2, *hassan vatandoost 1,2, ahmad ali hanafi-bojd 1, reza majdzadeh 1, ahmad raeisi 1 1school of public health, tehran university of medical sciences, tehran, iran 2environmental research institute, tehran university of medical sciences, tehran, iran (received 4 aug 2013; accepted 20 aug 2013) abstract background: malaria is still a public health problem in the world. one of the main objectives of world health organization is capacity building of authorities who are involved with malaria control activities. methods: the first course was conducted in 1996 in bandar abbas training center. the course was conducted jointly by the ministry of health and medical education of iran, who-emro and school of public health, tehran university of medical sciences. in year 2002, iran has been designated as who regional malaria training center. prior to initiate the course, pre-test evaluations including 11 subjects were carried out. the examinations include multiple choice questions. different methods of teaching including lecture, laboratory, workshop, team work, field exercise and presentation were used. the duration of the course was 9 weeks. a total of 360 contact hours were taught. the main subjects were basic epidemiology and simple statistics, malaria parasitology, malaria disease management, malaria entomology, vector control, epidemiological approach, filed work and planning. the requirement for achievement of the course was to have at least 60% of the total mark for awarding the diploma certificate. the 13th course was conducted by the financial support of islamic development bank (idb). results: a total of 300 participants from 26 different countries have been graduated from these courses so far. conclusion: this course is providing the skill for decision making, how to combat against malaria in their country and is parallel to the policy of the malaria control for capacity building in malarious areas of the world. keywords: malaria, course, iran introduction malaria still a major public health problem in the world. according to who (2010) the main strategy for malaria control is prevention and treatment. these activities should focus mainly on case diagnostic and prompt treatment, vector control using larviciding, impregnated bednet, indoor residual spraying (irs), biological control, monitoring and evaluation of drug and insecticide resistance and health education to the health workers and community. malaria is still a major endemic disease in foci located in south and southeast of iran. the annual malaria cases have been reported from 66075 to 3200 during 1995–2011, indicating the sharp decline of disease (fig. 1). it is unstable with two seasonal peaks mainly in spring and autumn. these areas include the provinces of sistan and bluchistan, hormozgan and kerman. iran is going to eliminate the malaria by 2025. the achievement of malaria control in the country is attributed due to bilateral collaborating between ministry of health and scientist. school of public health, tehran university of medical sciences with long history of work on malaria and publication of several papers on different aspects of malaria including insecticide resistance moni*corresponding author: prof hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir 100 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 toring (salari lak et al. 2001, enayati et al. 2003, vatandoost et al. 2004b, 2005, davari et al. 2006, hanafi-bojd et al. 2006, davari et al. 2007, abai et al. 2008, vatandoost and abai 2008, vatandoost and zahirnia 2010, vatandoost and hanafi-bojd 2012, soltani et al. 2013), sibling species, molecular study, new record (dezfouli et al. 2003, naddaf et al. 2003, oshaghi et al. 2003, sedaghat et al. 2003, azari-hamidian et al. 2003, oshaghi et al. 2007, mehravaran et al. 2011, naddaf et al. 2012), novel methods for vector control (soltani et al. 2008, omrani et al. 2010a,b, omrani et al. 2012, chavshin et al. 2012, soltani et al. 2012), faunestic study (moosakazemi et al. 2009, oshaghi et al. 2011), use of plants for larval control (hadjiakhoondi et al. 2000a,b, hadjiakhoondi et al. 2003, oshaghi et al. 2003, vatandoost and vaziri 2004, sadat-ebrahimi et al. 2005, hadjiakhoondi et al. 2005, hadjiakhoondi et al. 2006, vatandoost et al. 2008, shahi et al. 2010, khanavi et al. 2011, sedaghat et al. 2011a,b, vatandoost et al. 2012, khanavi et al. 2013), using bednets and long lasting impregnated nets (vatandoost et al. 2006, moosa-kazemi et al. 2007, rafinejad et al. 2008, vatandoost et al. 2009, soleimani-ahmadi et al. 2012a,b, vatandoost et al. 2013), morphological studies (doosti et al. 2006, emami et al. 2007, doosti et al. 2007), malaria epidemiology (vatandoost et al. 2003, hanafi-bojd et al. 2010, vatandoost et al. 2010, hanafi-bojd et al. 2012a,b, hemami et al. 2013), ecology of malaria vectors (vatandoost et al. 2006a,b, vatandoost et al. 2007, hanafi-bojd et al. 2011a, vatandoost et al. 2011a,b, hanafibojd et al. 2012c, mehravaran et al. 2012, soleimani-ahmadi et al. 2012b, soleimaniahmadi et al. 2013), biodiversity (oshaghi et al. 2006, nikookar et al. 2012), community participation (hanafi-bojd et al. 2011b, soleimani-ahmadi et al. 2012), vector control (vatandoost et al. 2009), repellent evaluation (vatandoost and hanafi-bojd 2008), anthropophic index of malaria vectors (oshaghi et al. 2006a,b,c), training (vatandoost et al. 2004a) is designated as malaria training center by who. course contributors this course was organized jointly by the ministry of health and medical education, islamic republic of iran, the school of public health and national institute of health research, tehran university of medical sciences, world health organization, eastern mediterranean region and the islamic development bank group (idb). it provided participants with the knowledge and skills in malaria control program planning and management, through small group work, field exercises, exchange of experiences and discussion with qualified specialists. course objectives the objectives of the course was for the participants to gain sufficient knowledge and skills to be able to: analyze the malaria situation and problems and find solutions, plan, implement, manage and evaluate antimalaria programs and develop and organize a training program for capacity building for malaria control. entry requirements: the course is designed for medical officers and scientists involved in disease control, particularly malaria, which are presently, or will be in future, responsible for antimalarial control activities. this includes managers and potential managers of disease control program and provincial and district medical officers. candidates had at least a medical degree or a phd or msc in medical parasitology, medical entomology or a related subject and had a good command of the english language. time and duration of the course: the duration of this intensive course was 8–9 weeks and was practical oriented (laboratory 101 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 and field) with lectures kept to a minimum, and with emphasis on group work. a minimum length of time was devoted to each subject in the class. the minimum total structured time is 360 tutor contact hours for the entire course. sessions be held for a minimum of seven hours each week day (saturday–thursday). formal sessions were not being held on fridays. towards the end of the course, during planning, participants are required to work irregular and longer hours to complete their assignments. this course provides intensive training in planning and management of malaria control programs. it conducted entirely in english. participants were expected to be fluent in spoken and written english. this course had a maximum capacity of 24 places. selections of candidates were made in consultation between experts of course contributors. applicants prepared themselves for the course. this should include compiling data and obtaining maps and charts on the demographic, climatic, geographic, economic, social, health and malaria status of their country and area of work in their country over the past three years. this data were brought to the course as it was needed during the planning sessions where each participant individually developed a plan for malaria control for his or her country or place of work, as an exercise in planning. attendance and certification: the participants attended all sessions throughout the course and to attend the opening and closing ceremonies without exception. those participants fulfilling the attendance requirements and reaching a satisfactory standard received the diploma in malaria program planning and management (dmppm) from the tehran university of medical sciences, ir iran. follow up activities by the participants: participation in a practical, intensive training course such as this is only the first step towards better management of national malaria control programs and human resources development. after completion of the course, participants need to put to good use the knowledge, skills and competence acquired. it is hoped that governments will select participants for this course who will, upon returning to their countries or place of work, be deeply involved in antimalarial activities. this would include planning and re-planning malaria control activities, managing, supervising or implementing antimalaria action, and to pass on their knowledge and skills to other health workers. fig. 1. malaria incidence in iran (1995–2012) 102 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 fig. 2. the number of participants from 26 countries in the 15 courses (1996–2012) fig. 3. global distribution of participants from malaria courses 103 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 course contents the major subjects for study were: 1basic epidemiology and biostatistics: this session include, introduction to epidemiology, rates, ratios, proportions, prevalence and incidence, type of data, frequency tables, statistical graphs and charts, computer practice: excel, measures of central tendency and variability, normal distribution and chi-square test, health facility based epidemiological studies, surveys and data management, introduction and designing questionnaire, computer practice: ms powerpoint software, surveys and data management, sampling techniques, sample size calculation for surveys, design effect, finite population-correction, principles of surveillance system, assessing the accuracy of a test or surveillance system, computer practice: powerpoint software, implementation of rapid survey in a village near to the training centre, analysis of the surveys and conclusions. 2basic malaria microscopy: malaria parasites, life cycle of malaria parasites and blood cells morphology, morphology of erythrocytic stages of plasmodium vivax and plasmodium falciparum in thin films, laboratory practice (examining thick blood films of p. malariae, p. vivax, p. falciparum and p. ovale and counting of parasites, orphology of p. vivax and p. falciparum in thick films and counting of parasites, differential diagnosis of human plasmodia and practical notes on malaria microscopy, practical work on identification of human plasmodia in thin and thick films, antimalarial drugs and chemotherapy of uncomplicated malaria and resistance. 3malaria disease management: clinical features of malaria, and antimalarial drugs, management of uncomplicated malaria and sever malaria, what you know about diagnosis and management?, severe malaria, pathophysiology of severe malaria, guidelines for diagnosis, picture quiz, management of severe malaria, assessment of recovery, case studies, discussion on outcome of questionnaires, acs: scientific rationale and global situation, who case management manual, who malaria treatment guidelines, malaria rapid diagnostic tests, practical and theoretical aspects, qa of malaria diagnostic tests, qc of antimalarial medicines, supply chain management, acts and rdts, pharmacovigilance of acts, passive and active surveillance systems, case management indicators, definitions and expected trends, acs: scientific rationale and global situation, case reporting form, data flow and interpretation, malaria drug policy in participants country , general discussion on act. 4malaria entomology and vector control: biology and ecology of anopheline mosquitoes, study and identification of adult anopheline mosquitoes and larvae, introduction to various vector control methods especially integrated vector control, epidemiological principles (vectorial capacity) of malaria transmission in relation to vector control, who and irs strategy guidelines, classification of pesticides, introduction to various vector control methods especially integrated vector control, insecticides used for vector control including formulations and their use, judicious use of insecticides, pesticide management, study and identification of adult anopheline mosquitoes and larvae collected from the field, indicators for monitoring and evaluation of vector control, scaling up of interventions-itns financing, distribution and networking, inter-sectoral coordination/collaboration, multiple use of intervention, vector control needs assessments through community participation and role of primary health care-group work, vector resistance to insecticides, concepts, mechanisms and genetics, practical work for itn impregnation, spray equipments, training of spraying team, measurement of pesticide, vector control: important factors for the se104 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 lection of appropriate insecticides and equipment, interpretation of results of adult and larval susceptibility tests, methods of testing mosquitoes for susceptibility to insecticides for adult and larvae and the safe use of equipment-observation and practice in the field, capacity strengthening in vector control, areas of operational research in vector control, impregnated mosquito nets-important principles and methods, mosquito rearing, fogging machine, biological control of mosquito. 5epidemiological approach to malaria control: the natural history of malaria in the human host-group discussions, social and economic aspects, introduction completion of questionnaire, indicators for malaria control, drug resistance, vector control, control of malaria, general discussion on outcome of questionnaires, application of the epidemiological approach in malaria control of participants’ workplace, general program management for disease control. 6situation analysis field exercise: effective small team work, organization of the field work, situation analysis field exercise, some guidance on an analysis of the situation, assessing community participation in malaria control, visit to district health authorities for briefing on primary health care structure and general health situation, accomplish tasks identified in the work plan, evening review the findings of the day, analyze data, discuss the work plan and write up report, spend the day discussing outcome of situation analysis, agree upon stratification, and describe in detail, discuss work plan, continue writing the report, presentation of situation analysis by four participants followed by discussion. 7planning and management of malaria control programs: introduction to planning and basic principles, situation analysis, stratification, selection of malaria control measures, formulation of disease reduction objectives, development of approaches to achieve objectives, setting operational targets, field teams brainstorm raw data requirements for the field exercise, primary health care and malaria control, writing the plan, support activities and milestones, preparation of individual plans, situation analysis, program budgeting, the research and development approach, preparation of individual plans, stratification, presentation of the malaria situation analysis, stratification, objectives, approaches, activities and targets by colleagues and senior faculty examiner. overall a total of 15 courses have been conducted so far and from them 300 participants graduated from the courses coming from 26 different countries including, russia, turkey, south africa, saudi arabia, iraq, oman, egypt, jordan, syria, sudan, djibouti, somalia, afghanistan, botswana, pakistan, zambia, zimbabwe, kenya, namibia, uganda, gambia, sierra leon, bangladesh, yemen, nigeria and iran (figs.2,3). according to the experience of the school of public health, we also conducted the international course on management and control of leishmaniasis. all the participants who wish to take part in these courses are able to have access to the announcement in the whoemro as well as tehran university of medical sciences websites. all the alumni of the past participant are available at the site of international affairs, school of public health, tehran university of medical sciences. acknowledgments the authors would like to appreciate the internal and external course tutors including: prof k mohamad, prof r majdzadeh, prof k holakouie naieni, dr a rahimi, dr m osouli, dr m rezaei, prof gh edrissian, dr m nateghpour, dr a motavali-haghi, dr h jabbari, prof e saebi, dr andrea bosman, prof e javadian, prof h vatandoost, prof y rassi, dr h edallat, dr hr basseri, dr h mousakazemi, dr ma oshaghi, eng mr abai, 105 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 eng k akbarzadeh, dr aa hanafi-bojd, dr a raeisi, dr m ostowar, dr f gholami, dr a rashidian, dr m jafari, dr a akbari-sari, prof p beales, dr h atta, dr g zamani, dr m warsame, dr belajev, dr kondrachin, dr sadrizadeh, prof h keshavarz, prof mr eshraghian, prof h azordegan, dr h zeraati, dr f abedi, dr a daryanavard, dr f malek, dr n safi, dr r safari, mrs m mashayekhi, dr a mnzava, mr g mohseni, prof mm gouya, dr ah madani. also thank to the course facilitators including: who representatives in iran, dr el bashier sallam, prof m rezaian, prof m mohebali, dr eb kia, dr b rokni, dr gh moulavi, dr a fallahi, dr mousavi, eng m sheshbaradaran, eng v verdizadeh, mrs fasihi, mrs hassan-zadeh, eng hassanpour, eng ar salehi, eng e shojaizadeh, eng b poya, mr a zomoredian, mr sa mirzakhanlo, mrs rezai, mrs ramazani, mrs riazi, mr m sadeghi, mr m shiri, mr m naseri, mrs m masoudi, mr a rahimi, mr seyed-somea, mr heidari, mrs charedar, mrs ariaepour, mr hosseini, mrs rafi, eng aboulhassani, mrs h samimi, mr bayat, mr a pakari, mr h javedan, mr h shabkhiz, mrs e torabi, mrs r kamyabi, mr a darvishi, dr m ranjbar, mrs f nikpour, mrs l faraji, dr s bozorgzadeh, dr h malikul. according to the announcement of the course, all the tuition fees for the participants are being paid by world health organization as well as idb for capacity building of malaria managers. there is no conflict of interest. references abai mr, mehravaran a, 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akbarzadeh k (2007) deltamethrin impregnated bed nets in a malaria control program in chabahar, southeast baluchistan, ir iran. iran j arthropodborne dis. 1(1): 43–51. moosa-kazemi sh, h vatandoost, h nikookar, m fathian (2009) culicinae (diptera: culicidae) mosquitoes in chabahar county, sistan and baluchistan province, coutheastern iran. iran j arthropod-borne dis. 3(1): 29–35. naddaf sr, oshaghi ma, vatandoost h, assmar m (2003) molecular characterization of anopheles fluviatilis species complex in the islamic republic of iran. east mediterr health j. 9(3): 257–265. naddaf sr, oshaghi ma, vatandoost h (2012) confirmation of two sibling species among anopheles fluviatilis mosquitoes in south and southeastern iran by analysis of cytochrome oxidase i gene. j arthropod-borne dis. 6(2): 144–148. nikookar sh, moosa-kazemi sh, oshaghi ma, vatandoost h, kianinasab a (2010) species composition and diversity of mosquitoes in neka county, mazandaran province, northern iran. iran j 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control in an endemic area, southeast of iran. acta trop. 123(3): 146–153. soleimani-ahmadi m, vatandoost h, shaeghi m, raeisi a, abedi f, eshraghian mr, madani a, safari r, shahi m, mojahedi a, poorahmadgarbandi f (2012c) vector ecology and susceptibility in a malaria-endemic focus in southern islamic republic of iran. east mediterr health j. 18(10): 1034–1041. soleimani-ahmadi m, vatandoost h, hanafibojd aa, zare m, safari r, mojahedi a, poorahmad-garbandi f (2013) environmental characteristics of anopheline mosquito larval habitats in a malaria endemic area in iran. asian pac j trop med. 6(7): 510–515. soltani a, vatandoost h, jabbari h, mesdaghinia ar, mahvi ah, younesian m, hanafi-bojd aa, bozorgzadeh s, abai mr, pakari a, shabkhiz h ( 2008) use of expanded polystyrene (eps) and shredded waste polystyrene (swap) beads for control of mosquitoes. iran j arthropod-borne dis. 2(2): 12–20. soltani a, vatandoost h, jabbari h, mesdaghinia ar, mahvi ah, younesian m, hanafi-bojd aa, 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(9): 722–726. vatandoost h, sanei dehkordi a, sadeghi sm, davari b, karimian f, abai mr, sedaghat mm (2012) identification of chemical constituents and larvicidal activity of kelussia odoratissima mozaffarian essential oil against two mosquito vectors anopheles stephensi and culex pipiens (diptera: culicidae). exp parasitol. 132(4): 470–474. vatandoost h, mamivandpoor h, abai mr, shayeghi m, rafi f, raeisi a, nikpoor 111 j arthropod-borne dis, december 2013, 7(2): 100–112 ar mesdaghinia et al.: conducting international … http://jad.tums.ac.ir published online: august 31, 2013 f (2013) wash resistance and bio-efficacy of alpha-cypermethrin long lasting impregnated nets (llin-interceptor®) against anopheles stephensi using tunnel test. j arthropod-borne dis. 7(1): 31. world health organization: world malaria report. insecticide resistance monitoring geneva, switzerland 2010, p. 204. 112 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 69 original article aerobic microbial community of insectary population of phlebotomus papatasi naseh maleki-ravasan 1, *mohammad ali oshaghi 1, sara hajikhani 2, zahra saeidi 1, amir ahmad akhavan 1, mohsen gerami-shoar 3, mohammad hasan shirazi 2, bagher yakhchali 4, yavar rassi 1, davoud afshar 2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of pathobiology, school of public health, tehran university of medical sciences, tehran, iran 3department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 4department of industrial and environmental biotechnology, national institute of genetic engineering and biotechnology, tehran, iran (received 28 apr 2013; accepted 1 oct 2013) abstract background: microbes particularly bacteria presenting in the gut of haematophagous insects may have an important role in the epidemiology of human infectious disease. methods: the microbial flora of gut and surrounding environmental of a laboratory strain of phlebotomus papatasi, the main vector of zoonotic cutaneous leishmaniasis (zcl) in the old world, was investigated. biochemical reactions and 16s rdna sequencing of the isolated bacteria against 24 sugars and amino acids were used for bacteria species identification. common mycological media used for fungi identification as well. results: most isolates belonged to the enterobacteriaceae, a large, heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. enterobacteriaceae groups included edwardsiella, enterobacter, escherichia, klebsiella, kluyvera, leminorella, pantoea, proteus, providencia, rahnella, serratia, shigella, tatumella, and yersinia and non enterobacteriaceae groups included bacillus, staphylococcus and pseudomonas. the most prevalent isolates were proteus mirabilis and p. vulgaris. these saprophytic and swarming motile bacteria were isolated from all immature, pupae, and mature fed or unfed male or female sand flies as well as from larval and adult food sources. five fungi species were also isolated from sand flies, their food sources and colonization materials where candida sp. was common in all mentioned sources. conclusion: midgut microbiota are increasingly seen as an important factor for modulating vector competence in insect vectors so their possible effects of the mirobiota on the biology of p. papatasi and their roles in the sandflyleishmania interaction are discussed. keywords: symbiont, microflora, bacteria, fungi, phlebotomus papatasi, leishmaniasis introduction symbiont microorganisms of vector insects are involved in many aspects of the host life including nutrition, reproduction, tolerance to environmental perturbations, maintenance and/or enhancement of host immune system homeostasis, defense, speciation, mucosal barrier fortification, xenobiotic metabolism, and pathogen transmission ability (o’neill et al. 1997, baumann et al. 2000, volf 2002, bourtzis and miller 2003, rio et al. 2004, dillon and dillon 2004, weiss and aksoy 2011). the gut of sand flies is a site where most leishmania’s entire life cycle takes place, thereby resident gut bacteria could possibly *corresponding author: prof mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 70 have a role in modulating the parasite development, either enhancing or inhibiting it. in nature, sand flies feed on different kinds of sugars, so having the chance to acquire contaminating microorganisms, such as bacteria and fungi that can eliminate the leptomonad infection or even kill the sand fly (schlein et al. 1985, killick-kendrick and killick-kendrick 1987, schlein and yuval 1987, cameron et al. 1995). yeasts and bacteria could reduce the infection rate of l. major in phlebotomus papatasi (schlein et al. 1985). p. papatasi is of particular interest as a vector of zoonotic cutaneous leishmaniasis (zcl) to humans in the old world. the conventional vector control measures such as application of insecticide has got undesirable effects on the environment, human health, and the emergence of insecticide resistance in sand flies (alexander and maroli 2003). the larvae of sand flies live in contaminated soils of domesticated animal shelters, termite mounds and rodent burrows and are exposed to a variety of different soil microbes that could be ingested alongside of food (feliciangeli 2004, mukhopadhyay et al. 2012). moreover, gravid p. papatasi attracts to the oviposition sites contaminated by frass and certain soil bacteria (radjame et al. 1997, wasserberg and rowton 2011). therefore it is expected that the gut of sand flies will support a range of microorganisms. these microbes could be genetically manipulated to express an antiparasitic molecule and then be reintroduced back into the sand fly gut through larval breeding places. this is a new vector borne disease control method called paratransgenesis that leads to reduce pathogen transmission by an insect vector (chavshin et al. 2013). this method has been successfully applied for a few insect vectors such as rhodnius prolixus, vector of trypanosoma cruzi the causative agent of the chagas disease in central america (beard et al. 2001), glossina morsitans, the vector of african sleeping sickness (aksoy et al. 2008, pontes and dale 2011), and anopheles stephensi, a vector of malaria in asia (wang et al. 2012). boulanger et al. (2004) demonstrated that bacteria present in the digestive tract of p. duboscqi induced the secretion of antimicrobial peptides with a significant antiparasitic activity. dillon et al. (1996) and volf et al. (2002) demonstrated that the maximum prevalence of bacteria in p. papatasi and p. duboscqi is recorded two days after blood feeding. high diversity in the bacterial gut microbiota, associated with different populations of p. argentipes and lutzomyia longipalpis, was registered using culture-dependent methods (gouveia et al. 2008, hillesland et al. 2008). notwithstanding these studies, there are a few reports available on the micro flora of p. papatasi (dillon et al. 1996, guernaoui et al. 2011, mukhopadhyay et al. 2012). gut flora is depended upon the environment they live. male and female sand flies feed on natural sugars, such as nectar, sap, and aphid and coccid secretions (young et al. 1980, killickkendrick and killick-kendrick 1987). these sugars are the main sources of carbohydrates for adults sand flies. furthermore, females also feed on blood. sand fly larvae are terrestrial and feed on soil organic matter (feliciangeli 2004). it is thus supposed that the diversity in their feeding behavior, as well as conditions that pre-imaginal stages encounter during their development, affect gut microbiota and could impact their overall capacity to sustain leishmania development. sand flies gut bacterial flora has been investigated on the isolated or pooled guts via culture of bacterial gut content and were identified by the use of classical bacteriology, cloning, and ribotyping (16s rdna sequencing) (rajendran and modi 1982, hillesland et al. 2008, gouveia et al. 2008, chavshin et al. 2012). fungi (yeasts) have been used as larva nutrient (e.g. wermelinger and zanuncio 2001) or entomopathogen (warburg 1991) of http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 71 sand flies. however, there is a little information about fungi as part of gut flora of sand flies (schlein et al. 1985). the aim of the present study was to examine different aerobic microbial community (bacteria and fungi) in the gut, food sources and colonization materials of a laboratoryreared colony of p. papatasi (isfahan strain) in the school of public health, tehran university of medical sciences (sph-tums). results of this study may lead to identify appropriate candidate/s for paratransgenesis approach. materials and methods sand fly insectary sand flies of badrood strain originally collected from badrood (33° 44´ n, 52° 2 e), natanz district, isfahan province, central iran, were reared under laboratory condition at 24–27 °c, 80% rh and 14:10 (l: d) photoperiod in the sand fly insectary of sphtums, following the method of modi and tesh for mass rearing of p. papatasi with some modifications (modi and tesh 1983). we have examined microbial infestation of a variety of sand fly biological specimens as well as non-biological materials used in the rearing of sand flies in the laboratory. the biological specimens included sand fly eggs, 1–4 instar larvae, pupae, unfed males, honey fed males, unfed females, honey fed females, blood and honey fed females, white mouse auricle, and white mouse muzzles. tested non-biological materials were larval chow as immature sand fly food sources, saturated sucrose and honey solution 50% as mature sand fly food sources, and sea sand (merck), plaster (chalk), and soil as colonization materials (table 1). isolation of bacteria isolation of sand fly guts was conducted in a sterile environment on a sterile glass slide. before dissection, each fly was surface sterilized for 1min in 70% ethanol. bacterial microflora was isolated from each sand fly by one of the two following methods. for adults, the gut from each sand fly was microdissected and homogenized in test tubes containing 5cc brain heart infusions (bhi) broth. for immature sand flies, the whole body of each individual larva homogenized in bhi broth. sterile cotton swabs (presenting in a sterile test tube) were used to swab the area around the auricle or nose of white mouse where sand flies choose to take blood meal. the infested swabs then were merged in bhi broth. for larval chow, sucrose, honey solution, sea sand, plaster and soil weights equal to 0.7 micro tubes volume were poured in the medium. bhi broth was chosen as a nonselective medium to promote growth a diverse range of microbes including nutritionally fastidious bacteria and even fungi. the complete transparent test tubes were incubated aerobically at 37 °c overnight. after 24 hours, opaque test tubes considered as positive and sub cultured in bhi agar medium overnight at the same condition. the colonies with different phenotype were sub cultured sequentially to obtain single colony of the microbes. pure cultures for each microbe were used for further identification procedure. a test tube containing bhi broth open near the dissection area constituted our sterility control during the dissection process. bacterial identification procedure all of the isolates were differentiated by standard gram staining and morphotypes. macconkeys agar, a special selective medium for gram negative bacteria which can differentiate those bacteria capable to ferment lactose was used for further confirmation. biochemical properties of the gram negative rods belonging to the entrobacteriaceae were investigated by “ds-dif-entro-24” kit. this kit is produced for the identification http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 72 and differentiation of 81 species and subspecies corresponded to 25 genera of the family entrobacteriaceae within 24 hours. this ready to use kit has been designed for the performance of 24 biochemical tests including simmons citrate, malonate, esculine, lysine, arginine, ornithine, hydrogen sulphide, phenylalanine, voges-proskauer, indole, glucose, βgalactosidase, lactose, mannitole, sucrose, inositole, sorbitol, arabinose, maltose, adonitol, trehalose, ramnose, dulsitol and urease. the interpretation of the results was based on numerical probabilistic identification using easybac software package and the distance method. gram-positive isolates that were not able to grow on the macconkeys agar medium, tested with manual laboratory examinations such as oxidase, catalase, coagulase, novobiocin susceptibility tests and mannitol medium. to test the accuracy of the ds-difentro-24 kit, three bacterial isolates from adult (fu), larvae (34l1) and white mouse muzzle (mn2moz) were selected randomly and subjected for molecular ribotyping following pcr amplification and pcr-direct sequencing of a subset of about 1500bp of 16s rrna gene according to weisburg et al. (1991) protocol. homologies with the available sequence data in genbank was checked by using basic local alignment search tool (blast) analysis software (www.ncbi.nlm. nih.gov/blast). isolation of fungi for initial isolation of fungi we followed overnight bhi broth test tubes. in the subsequent subcultures we used subaru dextrose agar with chloramphenicole medium and samples were incubated at 25–27 °c for a week. positive samples were examined macroscopically and then microscopic slides were prepared using the lactophenol cotton blue wet mount. chrome agar candida, a chromogenic differential culture medium, used for isolation and identification of the yeast species in incubation conditions of 35 °c for 2 days. corn meal agar medium plus tween 80 were used for discrimination of candida albicans from non-albicans species. results biochemical identification of bacteria totally 25 bacterial species were isolated comprising 20 species of entrobacteriaceae, one species of pseudomonadaceae, one species of bacillaceae, two species of staphy lococcaceae, and one non-identified species in the phelebtominae insectary of sphtums. details of the bacterial isolates from different stages of sand flies (egg to adults), food sources, and colonization materials are listed in table 1. molecular identification of the bacteria three isolates of 34l1, mn2moz and fu, that were biochemically identified as proteus vulgaris, p. vulgaris and bacillus sp. respectively, were identified accordingly as p. vulgaris, p. penneri and b. flexus based on partial sequences of 16s rrna. their sequences with length of 882, 995 and 1279 bp were deposited in genbank with accession numbers of jq928898-jq928900 respectively. isolation of fungi five fungal species were identified including three molds species of cladosporium sp., penicillium sp., and aspergillus flavus that were isolated from a honey and blood fed female, rhizopus sp. from saturated sucrose, and one yeast species, candida sp., from different sources including honey and blood fed females, unfed male, second instar larvae, sea sand, soil, larval chow, saturated sucrose, and honey (table 1). although c. albicans formed chlamydospores on corn meal agar medium however mycelia have been observed on the medium. so we encountered candida sp. other than c. albicans. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 73 table 1. summary of the isolated bacteria from the sand fly insectary and their sources identified by ds-dif-entro-24 kit (*based on 16srrna ribotyping) sources species e gg l ar va e 1 l ar va e 2 l ar va e 3, 4 p u p ae u n fe d m al e h on ey f ed m al e u n fe d f em al e h on ey a n d b lo od f ed f em al e s an d l ar va l c h ow s u cr os e h on ey a u ri cl e of w h it e m ou se m u zz le o f w h it e m ou se c h al k s oi l found in sand flies, insects or other sources [notes] bacillus sp (*b. flexus) x x p. papatasi (mukhopadhyay et al. 2012), macrotermes carbonarius (tay et al. 2010) plants (sanchez-gonzalez et al. 2011), seaweed (singh et al. 2011) staphylococcus aureus x x p. argentipes (hillesland et al. 2008), cockroaches (al-bayati et al. 2011), human skin, somewhen systemic infections (foster 2005) staphylococcus saprophyticus x p. papatasi (mukhopadhyay et al. 2012), p. argentipes (hillesland et al. 2008), musca domestica (butler et al. 2010) not identified bacterium x ***** pseudomonas aeruginosa x p. papatasi (mukhopadhyay et al. 2012), p. argentipes (hillesland et al. 2008), [pathogen on human, insects and plants (silby et al. 2011)] shigella dysenteriae x x cockroaches (salehzadeh et al. 2007, al-bayati et al. 2011), musca domestica (bolaños-herrera 1959) edwardsiella ictaluri x [pathogen on fish (santander et al. 2010)] kluyvera ascorbata x ips typographus (muratoğlu et al. 2011) proteus vulgaris x x x x x cockroaches (al-bayati et al. 2011), ips typographus (muratoğlu et al. 2011) klebsiella oxytoca x x ips typographus (muratoğlu et al. 2011) bactrocera cacuminata (thaochan et al. 2010) klebsiella planticola x ips typographus (muratoğlu et al. 2011) providencia rettgeri x [pathogens on humans and drosophila melanogaster (galac and lazzaro 2011)] bactrocera cacuminata (thaochan et al. 2010) klebsiella pneumonia ozaenae x x cockroaches (al-bayati et al. 2011), bactrocera cacuminata (thaochan et al. 2010) leminorella grimontii x anopheles stephensi (rani et al. 2009) enterobacter amnigenus x x x anopheles dirus (khampang et al. 2001) proteus mirablis x x x x x x x blow fly maggots (erdmann 1987, fleischmann et al. 2004), lucilia sericata (ma et al. 2012), calliphora vicina (greenberg et al. 1970), cochliomya hominivorax (erdmann and khalil 1986), lucilia cuprina (mohd masri et al. 2005), musca domestica (greenberg 1959), parasitoide wasps, itoplectis (bucher 1963), cockroaches (al-bayati et al. 2011) escherichia coli x anopheles gambiae (dong et al. 2009) cockroaches (al-bayati et al. 2011) enterobacter gergoviae x x pectinophora gossypiella (kuzina et al., 2002) pantoea agglomerans x x x anopheles gambiae (dong et al. 2009) escherichia blattae x x cockroaches (burgess et al. 1973) tatumella ptyseos x x [pathogen on human and pineapple (marín-cevada et al. 2010, hollis et al. 1981)] serratia plymuthica x manduca sexta (toth-prestia and hirshfield 1988) yersinia pseudotuberculosis x [ubiquitous bacteria affecting humans, animals and fleas (hinnebusch 2005)] rahnella aquatilis x x pacific coast wireworm,( lacey et al. 2007), southern pine beetle (vasanthakumar et al. 2006), toxoptera aurantii (sevim et al. 2012) providencia alcalifaciens x [pathogens on humans (galac and lazzaro 2011)] http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 74 discussion we could isolate and identify 25 bacteria and five fungi species from the sand fly strain and its rearing materials. majority of these microbes including 20 of the bacteria and four of the fungi species, were isolated from sand fly guts. many species of candida could be found in the gut of a variety of organisms including c. albicans in mammalian and insect as endosymbionts (nguyen 2007, suh 2008). although bacterial micro flora of some laboratory reared sand flies have been pointed out in a few studies but the source of this flora is the first time that marked and studied in details. in this study we tested microbial infestation of all fields and materials used in sand fly colonization procedure. in this study the correctness of the biochemical kit were examined against 16s-rrna ribotyping. on general there was a great concordance between the results of two methods: both methods identified 34l1 and mn2moz isolates as proteus vulgaris. it is noteworthy that p. penneri is formerly known as p. vulgaris biogroup 1. however, the molecular method could identify the fu isolate up to species level (b. flexus) whereas the biochemical method identified it up to genus level (bacillus sp) concluding that the ribotyping is more specified than biochemical one. although we used a nonselective medium (bhi broth) to promote growth a wide range of bacteria and fungi, however, due to not using various media and culture conditions (e.g. anaerobic condition) the medium generally favored in growth of gram negative enterobacteriaceae. similarly, almost all of the studies analyzing the sand fly guts for bacterial communities have also relied on culture dependent techniques in their analyses where enterobacteriaceae constituted the majority of their findings (dillon et al. 1996, hillesland et al. 2008, mukhopadhyay et al. 2012). even the studies that have implemented molecular tools used these tools only in the identification and analysis of isolated pure colonies from plate culture, not in the initial isolation of bacteria from the guts (gouveia et al. 2008, hillesland et al. 2008). the molecular tools used in both studies were implemented in the identification of bacterial colonies obtained by culturing, thereby limiting the findings to the small proportion of cultivable microbes. taking into account the limitations of culture dependent techniques makes these findings incomplete. a correlation between the type of microbial gut flora detected and the area inhabited by the sand fly has been addressed by hillesland et al. 2008, where flies collected from the same region harbored almost the same kinds of bacteria. therefore, it was suggested that gut flora diversity more or less is a reflection of the environment where the sand fly resides (hillesland et al. 2008). for example, bacillus megaterium that is present in biofertilizers widely used in the state of bihar, india, was isolated from the guts of a number of sand flies inhabiting that area. another example was that of brevibacterium linens, the bacterium used in cheese ripening industry that was also isolated from the gut of sand flies collected from regions known to be involved in dairy preparations (hillesland et al. 2008). both these bacteria were proposed as candidates for use in a paratransgenesis model, being already employed in biotechnological operations without concerns about their safety (hillesland et al. 2008). mukhopadhyay et al. 2012 carried out a survey to study the abundance of different natural gut flora of p. papatasi in different habitats (sheep shed, rabbit hole, chick/sheep shed, human dwellings and lab colony) of tunisia, turkey, india and egypt. they found variation in the species and abundance of gut flora in sand flies collected from different habitats. howhttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 75 ever b. flexus and b. pumilus were common in most of the sites examined. b. flexus is capable to grow under highly alkaline conditions that are the case for phlebotomine sand flies (panizzi and parra 1991) as alkaline amylase producers was found in our study from midgut of unfed female as well. in our study we found staphylococcus saprophyticus from the saturated sucrose used as adult’s phytophagic meal. this bacterium which has already isolated from p. argentipes (hillesland et al. 2008), musca domestica (butler 2010) and p. papatasi (mukhopadhyay 2012) is a very strong oviposition inducer for gravid p. papatasi (radjame 1997). mukhopadhyay et al. 2012 could success in introducing of bacillus flexus, b. pumilus, b. licheniformis, b. megaterium and b. subtilis as candidate species for paratransgenesis due to these bacteria ability in induction of sand fly oviposition behavior and real function of those as symbionts and not merely as environmental contaminants. the presence of p. mirabilis and p. vulgaris nearly in all our analyzed samples raises questions about the nature of the interaction between this microorganism and sand flies. proteus species are found in the digestive tracts of many animals including insects (guentzel 1991, rozalski 1997). a field survey of bacteria from the digestive tracts of newly emerged house flies breeding in horse manure showed the predominant flora to consist of p. vulgaris, p. mirabilis, aerobacter aerogenes, and citrobacter freundii. a similar census from flies bred in the laboratory recovered p. vulgaris, p. mirabilis, p. morganii, p. rettgeri, e. coli, and aerobacter aerogenes (greenberg 1959). interestingly, in maggots, the bacterium p. mirabilis secrete antibacterial toxins (including phenylactic acid and phenylactaldehyde) that kill other microbes but do not harm the maggots (erdmann 1987, fleischmann et al. 2004). p. mirabilis is also highly resistant to the action of antimicrobial peptides, such as polymyxin b (pm), protegrin, and the synthetic protegrin analogi b-367 (mccoy 2001). ma et al. 2012 found that p. mirabilis interkingdom swarming signals attract blow flies. they obtained p. mirabilis from the salivary glands of the blow fly lucilia sericata, this strain swarmed significantly and produced a strong odor that attracts blow flies. greenberg et al. (1970) demonstrated that microorganisms ingested by the blowfly calliphora vicina are rapidly eliminated if p. mirabilis is also present in the ingested material. several pathogenic microorganisms have been killed by brief exposure to 15 days old p. mirabilis culture broth (erdmann and khalil 1986). greenberg (1968) referred to the active anti-bacteria constituents as “mirabilicide.” it was also reported that the symbiotic relationship between p. mirabilis and the screwworm could protect the larvae from other harmful bacteria by bactericidal agents secreted by p. mirabilis (erdmann and khalil 1986). interestingly, complete sterility of lucilia cuprina maggots for the purpose of debriding intractable wounds was achieved in all cases, except that p. mirabilis was consistently found. however, the presence of this microorganism was considered beneficial (mohd-masri et al. 2005). to what extent the bacterium could play a similar role during sand fly larval development remains unknown and has to be investigated. a few studies have examined the impact of the gut microbiota on the establishment of human pathogens and parasites in their insect vectors. the possibility that colonization resistance is involved in suppressing medically important parasites such as plasmodium and leishmania in their dipteran vectors has been discussed (dillon et al. 1996, pumpuni et al. 1996). although a recent study suggesting a dose dependent inhibitory effect of gut bacteria on leishmania promastigotes (muniaraj et al. 2008), however, more investigation need to find the most effective bacteria or bacterium which can be used as bio-agent for combating leishmania http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 69–81 n maleki-ravasan et al.: aerobic microbial … 76 parasites. an indigenous biota (not transient one) is present in all individuals of the species and maintains stable climax communities (dillon and dillon 2004) also in some cases an indigenous species may colonize only in the presence of other members of the microbial community (dillon and charnley 2002). indigenous bacteria modulate expression of genes involved in several important intestinal functions including nutrient absorption, mucosal barrier fortification, and xenobiotic metabolism (dillon and dillon 2004). regarding to these criteria, herein we can nominate proteus spp. as indigenous isolates of p. papatasi. however, whether or not, the resident gut microbiota as a micro ecological factor can regulate the prevalence of sand flies with transmissible infections need to be investigated. koch and schmid-hempel (2011) showed that the presence microbiota protects bee hosts against a widespread and highly virulent natural parasite, crithidia bombi. their results emphasize the importance of considering the host microbiota as an “extended immune phenotype” in addition to the host immune system itself and provide a unique perspective to understanding bees in health and disease. metacyclic promastigotes are highly adapted for transmission and early survival in the vertebrate host (kamhawi 2006). messy condition of sandfly midgut and many interactions will determine the triumphant. peritrophic matrix, enzymes, temperature, ph, oxygen pressure, insect immune system, vertebrate host immune system, sugar meals, blood meals, leishmania, bacteria, fungi and many putative unknown internal/external factors are determinants in these relationships (young et al. 1980, killick-kendrick and killickkendrick 1987, kamhawi 2006, bates 2007, bates 2008, oshaghi et al. 2009, weiss and aksoy 2011). as major contestant, microbiota and leishmania parasites interactions make it possible to introduce a sand fly as a vector or non-vector rather than other intrinsic and extrinsic criteria. although midgut bacteria are increasingly seen as an important factor determining vector competence in mosquitoes and there are some recently examples against or assist this point (mourya et al. 2002, xi et al. 2008, meister et al. 2005, cirimotich et al. 2011, rodrigues et al. 2011). recent studies have shown the capacity of endogenous bacteria to decrease viral and parasitic infections in mosquito and tsetse fly vectors by activating their immune responses or directly inhibiting pathogen development (cirimotich et al. 2011). this could be happen in the sand fly gut and this in turn may lead to a reduction in leishmania infection within the sand fly host. conclusion midgut microbiota are increasingly seen as an important factor for modulating vector competence in insect vectors so the possible effects of the mirobiota on the biology of p. papatasi and their roles in the sandflyleishmania interaction were discussed. however a more in depth research on the interactions between sandfly and their midgut residing bacteria and leishmania is required. acknowledgements this article is a part of the first author’s dissertation for fulfillment of a phd degree in medical 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jamal3, fizza maryam3, *muhammad faheem3 1school of science, monash university, subang jaya, malaysia 2yusra institute of pharmaceutical sciences, yusra medical and dental college, islamabad, pakistan 3department of biological sciences, national university of medical sciences, rawalpindi, pakistan *corresponding author: dr muhammad faheem, e-mail: muhammad.faheem@numspak.edu.pk; faheem08@live.com (received 15 mar 2022; accepted 24 dec 2022) abstract background: since 1952 when zika virus (zikv): a flavivirus, was first discovered in humans, it has not received enough scientific research compared to some of the other members of the family flaviviridae; like dengue virus (denv). however, this has not stopped the virus from infecting the human population globally. in particular, the glob al spread of zikv has led to a surge in observational studies. methods: regarding recently published zikv-related literature, we are not aware of any reviews strictly focusing on zikv from the perspective of observational studies. therefore, we reviewed recently published observational studies exploring the global spread of zikv and its association with congenital zikv infection (czi) and clinical manifestations in adults. online databases including google scholar, pubmed and elsevier were used for retrieving relevant studies. results: zikv cases have been reported in different parts of the world, with certain regions reporting more cases than the rest, like brazil. zikv causes a wide spectrum of diseases and disorders including microcephaly, developmental disorders, and guillain-barre syndrome to name a few. furthermore, czi in neonates mainly manifests into neurological disorders and diseases, whereas zikv in adults’ targets various organs. conclusion: zikv poses a serious threat to human population and observational studies provide a different perspective on the damaging capabilities of zikv in real-life settings. moreover, there are gaps in the literature regarding zikvrelated-complications that future experimental studies need to address. these complications include in-utero transmission, guillain-barre syndrome, cross-reactivity, sexual transmission, along with its persistence in the male reproductive tract. keywords: zika virus; arbovirus; flavivirus; congenital zika infection introduction zika virus (zikv) is a mosquito-borne flavivirus first discovered in 1947 in uganda in monkeys (1). and then, shortly after, in humans in 1952 in the united republic of tanzania and uganda. importantly, zikv was isolated in gabon from the asian tiger mosquito aedes albopictus (2), which is a rapidly expanding aedes species living in close contact with urban human populations. it typically bites during the daytime but showcases its peak activity at dusk and dawn (3). at present, zikv has received far less attention in the literature than other mosquito-borne flaviviruses like dengue virus (denv). nevertheless, it is considered an emerging virus because of its global spread during the last decade. furthermore, zikv cases have been reported in people from different walks of life, including laboratory personnel getting infected while conducting experimental studies on the virus (4). moreover, its pathogenic potential is reminiscent of that of denv. according to the reported zikv cases, zika virus infection (zvi) causes various symptoms and complications in both younger (including neonates) and older patients. they range from general ones like copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:muhammad.faheem@numspak.edu.pk mailto:faheem08@live.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 263 http://jad.tums.ac.ir published online: dec 31, 2022 fever, myalgia, and conjunctivitis to more specific ones, like guillain-barre syndrome (gbs) and central nervous system (cns) complications like microcephaly in neonates. along with cases of zvi, the number of observational studies on zvi from around the world have surged in recent years. therefore, using these observational studies, we provide a new perspective on zikv. the literature we review here provides new insight into the following topics: 1) zikv spread around the world, 2) congenital zikv infection (czi), and 3) clinical manifestations of zvi in adults. we also discuss the gaps in knowledge about the zvi and what areas future research should explore. materials and methods observational studies published in recent years reporting the global spread of zikv, as well as its health implications on human population were searched for on multiple online databases including google scholar, elsevier and pubmed. studies that fit this description were included in this review. results background of zvi in humans mode of entry zikv enters the human body through the epidermis or dermis after the bite of the aedes mosquito (fig. 1). at this point, the host defense system is mainly under the control of skin fibroblast cells and epidermal keratinocytes. however, zikv deceives them quickly as they behave highly permissive toward the zvi (4). as a result, viral rna copies rapidly increase, and viral replication occurs in skin fibroblasts causing the virus to dominate (5). as zikv concentrates on its replication, it initiates apoptosis in numerous epidermal keratinocytes to deceive antiviral immune responses. as a result, immune cells divert their attention towards disseminating dying cells and zikv proliferates. type i and iii interferons are critical antiviral immune responses in keratinocytes, but their stimuli are kept inactive in zvi. therefore, the dendritic or other immune cells in the dermal or epidermal regions implement no initial resistance (5, 6). pathogenesis according to zikv pathogenesis, summarized in fig. 1, after replication the virus either starts showing its effects on the skin or causes fever, myalgia, or conjunctivitis. the adaptive immune system controls these pathological conditions and produces protective antibodies against them. nevertheless, zvi has many unanswered questions including its inutero or transplacental transmission and teratogenic effects on the fetus in the form of, for example, microcephaly. moreover, the association between gbs and zvi is not wellunderstood. in gbs demyelination of nerve cells occurs, causing muscle weakness in adults, also leading to tingling of limbs or even paralysis (7, 8). this uncovering disturbs signal transduction within the body and creates a neurological complication. the gathering of t-cells, macrophages, leukocytes, and other inflammatory cells near the blood brain barrier is seen in zvi (9), which is dangerous for the integrity of bbb, and in this infectious atmosphere the virus can slip through the loose junctions of bbb (10, 11) (fig. 2). also, there is a need to understand the increased pathogenicity of zikv by cross-reacting with antidenv antibodies. if zikv threatens to infect the human body in the presence of denv antibodies, the antibodies tend to cross-react with zikv triggering the phenomenon of antibodydependent enhancement (ade) (12). after binding of the antibody-antigen complex to the fcγr receptor, it facilitates the viral entry through the cell membrane and viral replication occurs (12). sexual transmission of zikv is another question to answer, since the transmission of zikv is possible through sexual fluids, but the mechanism is still unknown. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 264 http://jad.tums.ac.ir published online: dec 31, 2022 immune response viral envelope proteins mediate the entry of flaviviruses while several host surface receptors or attachment factors facilitate the viral entry. the receptors mediating zikv entry are dc-sign, axl, tyro3, and tim-1 (13). tim-1 may act as an attachment factor and facilitate viral entry by accumulating virions on the cell surface and enabling their contact with axl (14, 15). after entering host cells and replicating, zikv has to protect itself from being recognized as pamps (pathogen associated molecular pattern), which can trigger an immune response instantly. pattern recognition receptors like rig-i, mda-5 and tlr3 are active in zikv pamp recognition, in which tlr3 is considered the pioneer in initiating immune response (16). prrs, including tlr3 and tlr7, initiate specific pathways that lead to the expression of various cytokines including type i interferons (17). the expression and role of type iii interferons are still unknown in zvi. in contrast, transcription levels of irf 7 rise during the infection, which is a precursor for initiating expression of interferon stimulating genes after attaching to the promoter region of its genes, i.e., interferon-stimulated response element (isre). the exact mechanism by which tlr3 generates antiviral responses in zvi is still unknown. however, the activation of these receptors in cns regions can be harmful. it may leave lifelong complications like microcephaly in infected individuals (18). numerous interferon (ifn) stimulated genes like oas2, mx1 and isg15 are also unregulated during a zvi. in addition to this, cxcr3 ligands also play an essential role in recruiting t-cells and other leukocytes to the site of infection and boosting immune response (19). interferons share downstream pathways among themselves and other proinflammatory cytokines, which creates a sense of cytokine redundancy against invading viruses and list them among the first line of defense antivirals. experiments have shown that the production of type i and type ii ifns are not limited to irf3 pathway activation, and other unknown corridors of activation and expression also exist (20). autophagosome-like vesicles are present in infected fibroblasts during zvi, but the protein degraded in these autophagosomes is reused by ingenious viruses for their replication and proliferation (21, 22). thus, though autophagy promotes viral replication in zvi, it also evades the antiviral immune response by either limiting the replicating process within mammalian cells or by providing additional amenities for membrane structures (23, 24). however, the exact mechanisms by which zikv controls the host cells for its replication are yet to be determined. zikv: findings from observational studies using "observational studies" as an umbrella term, we have reviewed different case reports, cross-sectional studies, cohort studies, case-control studies and case series in this review concerning the zvi. the findings from these studies are distributed into three sections to help establish their relevance to the following topics: a) the global spread of zikv, b) association between zvi during pregnancy and congenital disabilities and c) zvi in adults. global spread cases of zvi have been reported by numerous countries worldwide, especially in recent years. however, in terms of observational studies, the amount and nature of literature produced vary from country to country, with brazil contributing the most (26). however, it is imperative to highlight zikv-related findings from different parts of the world to understand the extent of its spread (fig. 3). in one such study from costa rica, zikv spread in the region through sexual transmission was investigated, since the impact of sexual contact patterns and their alteration on zikv spread has not yet been explored. nevertheless, by way of deleting sexual contacts in a heterosexual network model, the study showed that deleting sexual contacts can curb http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 265 http://jad.tums.ac.ir published online: dec 31, 2022 the spread of zikv. study results also showed that females were infected more by zikv than males (27). among countries that have underreported the spread of zikv, mali is one of them. therefore, to better understand the extent to which zikv exists in mali, a serological survey was conducted in two ecoclimatic regions (warm semiarid and tropical savannah). samples were collected from 637 blood donors in 2013 and later in 2016 from 793 asymptomatic volunteers aged above 15. results showed an overall seroprevalence of 12%, which increased with age, but gender did not play a role in the prevalence of zikv. moreover, the same study detected an undiscovered outbreak from the latter part of the 1990s in 18% of the participants in the warm semiarid region using immunological markers. on the other hand, zikv only reached endemic proportions annually in the tropical savannah, with only 2.5 % of the population infected (28). furthermore, in seroprevalence studies, the population of vietnam was evaluated for seroprevalence of zikv antibodies during and following the zikv epidemic of 2016. the number of collected serum samples was 879 in total from 801 participants between 2017 (january) and 2018 (july). moreover, the area surrounding the district of krong bruk, where the samples from random participants were collected, showed zikv presence. also, the prevalence of zikv antibodies had been around 1.1% since at least 2016, suggesting a low protective immunity towards zikv in the region, while a limited circulation and seroprevalence also hinted at a limited outbreak among the population (29). mexico has also been a target region for observational studies to better understand the natural history of zikv in the region, namely the city of tapachula in the mexican state of chiapas, generally considered hyperendemic for dengue virus (denv). the zikv outbreak in mexico around 2016 was followed by the recording of the frequency of zvi, chikungunya and denv cases from 2016– 2018. zikv frequency increased and then decreased, but denv kept on increasing throughout the study (30). other investigated zikv outbreaks worldwide include the 2016 outbreak in puerto rico by williamson et al. (31) where the participants in the study were all zikv-positive blood donors. furthermore, the study estimated that zikv infected 21% of the population of puerto rico during the outbreak. congenital zika infection congenital disabilities associated with czi are the most studied area in terms of observational studies, compared with other aspects of the zvi. to better understand the frequency of czis, a surveillance study by morris et al. (26) evaluated congenital zikv syndrome (czs) occurrence in the caribbean and the whole of latin america from outbreaks between 2015–2017. the study relied on surveillance reports for infectious diseases to measure two main factors: czs cases per 1,000 births and czs cases per 1,000 births to women infected with zikv during pregnancy. the study also investigated whether these reports were complete or errors in reporting occurred. forty-seven countries contributed data from central and south america, the caribbean, and mexico. pan american health organization (paho)/world health organization (who) epidemiology reports were used in the study to assess the presence of czs. by the start of 2018, all 47 countries reported 548,623 cases suspected for zikv and 239, 063 confirmed cases to paho/who, with 80 percent of the patients reported from 25 countries being suspected cases of zikv. confirmed czs cases were 3,617, reported by 25 countries, with brazil making up 82% of these cases (2,952) and colombia 7% (248 cases). for the czs cases per 1,000 births, brazil and caribbean communities of st. martin, puerto rico, martinique, grenada, and guadeloupe reported the highest prevalence of czs with more than 0.5 per 1,000 births. http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 266 http://jad.tums.ac.ir published online: dec 31, 2022 on the other hand, incomplete reports and underreporting started to appear when analyzing the data for czs births per 1,000 births to zikv-infected pregnant women, with venezuela, in particular, being responsible for massive czs underreporting. according to the same study, by counting czs cases concerning total births instead of absolute numbers, the disease burden can be better illustrated. furthermore, the study concluded that data enumerating the pregnant women infected with zikv is helpful for identifying any inaccuracies in the reporting of czs cases (26). brazil brazil reported the most czs cases during the outbreaks of 2015–2017 (26). consequently, the need for understanding the association between zvi during pregnancy and cross-reactive immunity to other viruses of the same family arose. therefore, numerous observational studies focusing on these topics have surfaced recently. one of these studies aimed to study pregnant women infected by zikv during the 2015–2016 outbreak, who gave birth to microcephalic infants. a total of 50 women participated and underwent serological testing. as a result, they all showed immunity toward denv but still gave birth to microcephalic infants (32). before the 2015 zikv outbreak in brazil, reports of microcephaly were rare, and zikv exposure during pregnancy has been reported as one of its significant causes. moreover, the role of inflammation is not well understood during the pathogenesis of microcephaly, as this could prove to be of assistance in deriving therapeutic strategies. vinhaes et al. (34), in a case-control study, tried to shed some light on this phenomenon. a total of 50 participants took part in the study (14 normocephalic and 22 microcephalic newborns exposed to zikv, and 14 newborns that were healthy and not exposed to zikv). the selected participants were also part of a previous surveillance of neonates for czi, conducted in 2016 (33). therefore, plasma samples from these subgroups were measured for inflammatory biomarkers. in summary, the inflammation level did not increase due to microcephaly (34). moreover, in terms of microcephaly caused by czi, the impact of zikv exposure during twin pregnancies is not well understood. sobhani et al. (35) explored this concept in a prospective cohort study. the study involved 244 pregnancies (out of which only 5 were twin pregnancies) with maternal zikv. the selected 4 out of 5 twin pregnancies did not have any co-infection and participated in the study. the researchers evaluated placental, maternal, and neonatal samples by pcr testing. three out of 8 neonates showed abnormal outcomes, and after a long-term follow-up on six of the infants only three showed any abnormalities caused by zikv. besides, infant outcomes and placental findings also hinted at a discordance between co-twins exposed to zikv during pregnancy. therefore, the researchers concluded that it is necessary to evaluate each twin separately for vertical transmission of zikv. along with microcephaly, prenatal zikv exposure and other adverse pregnancy outcomes including small-for-gestational-age, low birth weight, prematurity, and fetal death have also been studied. for example, in a study by clemente et al. (36), a total of 574 pregnant women were tested for zikv during the outbreak of 2015–2017. in addition, collection of urine samples from the women and the infants upon birth and rt-pcr tests were conducted. in total, 44 women tested positive for zikv during pregnancy. as for the 409 neonates tested during the first ten days following birth, 19 tested positive. in conclusion, zikv exposure during pregnancy did not contribute to an increased risk of adverse outcomes like smallfor-gestational-age, low birth weight, prematurity or fetal death. however, it was discovered that compared to zikv-negative neonate, the risk of developing microcephaly and disproportionate microcephaly was fivehttp://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 267 http://jad.tums.ac.ir published online: dec 31, 2022 fold and ten-fold higher in zikv-positive infants, respectively. as established, czi is associated with microcephaly, but to further explore this phenomenon, coutinho et al. (37) conducted the largest brazilian cohort study of zvi during pregnancy. they investigated adverse pregnancy outcomes associated with maternal zvi. the participating pregnant women were symptomatic, and the study also included their infants. in addition, researchers performed prenatal and early neonatal screening. zvi had occurred in the participants during the zikv outbreak in brazil from 2015–2016. moreover, the total number of infected pregnant women enrolled in the study was 511 (confirmed zvi through pcr), as well as 513 fetuses as two were twins. pregnancy losses and/or congenital disabilities associated with zikv happened in only 42 infected mothers. microcephaly or any other cns malformations were detected in 1 stillbirth out of 4 in total, and in 19 live births out of 489. also, it was found that fetal abnormalities were 14 times more likely to manifest when the mothers contracted zvi before reaching the gestational age of 11 weeks. the study concluded that zikv leads to shortterm adverse pregnancy outcomes less often than previous reports have shown. however, in the long term, although the 470 livebirths showed no signs of microcephaly or any other neurological deformity within three months of their birth, a considerable number presented with subclinical findings related to eye, cns imaging, and neurological symptoms. regarding improved diagnosis of czs, there are also suggestions for measuring the degree of musculoskeletal alterations in the patients. these alterations are highly prevalent in czs patients and can be categorized into type 1: low incidence of musculoskeletal complications, type ii: progressive deformities, and type iii: most severe and prevalent deformities present simultaneously. furthermore, this can allow orthopedic surgeons to treat their patients according to the severity of their condition (38). even though czi is associated with microcephaly, it is also imperative to pay equal attention to screening normocephalic asymptomatic births for czi. a study conducted in salvador, brazil, explored this concept during the 2016 microcephaly outbreak. the study used 151 newborns comprising both normocephalic (119 in total) and microcephalic newborns (32 in total) suspected of czi. moreover, using serological tests (to check for igg and igm antiglobulin) and quantitative reverse transcriptase-polymerase chain reaction (rtqpcr), presence of zikv was confirmed. as a result, 17 normocephalic newborns were detected with czi, proving that asymptomatic neonates can also have czi. thus, emphasizing the need for prenatal and neonatal screening, especially in endemic regions, for zikv (33). along with screening normocephalic births for czi, it is equally important to understand the impact of intrauterine zikv exposure on asymptomatic children later in life. this was investigated by abtibol-bernardino et al. (39) in a case series study where the researchers looked for minor neurological disorders caused by zikv exposure later in life. the study included 26 participants (25–42 months age range), all non-microcephalic children, who had underwent intrauterine exposure to zikv. they were subjected to neurological examination and the bsid-iii test (bayley scales-iii) which assessed their cognition, language, and motor performance. in total, 64.5% (17/26 children) performed satisfyingly, however, the language domain was impaired the most in eight children. at the same time, five children had severe neurological impairment (epilepsy, spastic hemiparesis, autism, and progressive sensorineural hearing loss). in total, the majority of the children showcased mild alteration. in another study of a similar nature, 194 normocephalic neonates had undergone prenatal exposure to zikv and were then investigated for neurological defects within 3 and 24 months of their birth. bayley scales of infant and toddler development screening test-3rd http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 268 http://jad.tums.ac.ir published online: dec 31, 2022 edition was used to perform the investigation and scores for the cognitive domain were in the typical range. however, the slower development of communication skills hinted at delayed neurodevelopment in older age (40). in contrast, signs of cns infection have also been studied in children with non-congenital zvi. like in the study by bentes et al. (41), where 11 children with non-congenital zvi participated and zikv rna was detected using real-time rt-pcr tests in cerebrospinal fluid samples. upon follow-up assessment of the patients, one-quarter of the subjects required antiepileptic medication, whereas two of the participants went on to experience delay-in-development/learning difficulties. other parts of the world with brazil contributing the most in the way of observational studies concerning czi, still, there are significant findings from other parts of the world that need equal attention. nevertheless, some of the conclusions we review here support and add to the findings made in brazilian studies regarding czi. at this point, the global spread of czis has been established (26). even in southeast asia, where zikv spread has not received any coverage since at least 2017, the lao people's democratic republic reported their very first case of czi-related microcephaly in 2020 (42). regardless, southeast asia as a whole, dating back to 1997 until 2015, has not reported enough data concerning the zikvand-related adverse pregnancy outcomes. in the case of thailand however, between 1997– 2015 prevention of perinatal transmission of hiv and hbv was studied. the samples from the study were stored and later used by ngogiang-huong et al. (43) in an unmatched casecontrol study to explore the relationship between adverse outcomes of pregnancy and the presence of zikv antibodies in the subjects. the participants included a case group of pregnant women and a control group without adverse pregnancy outcomes, and their infants. all the women had either hiv or hbv. none in the case group had zikv igm, and the same was true for their live-born neonates. as for zikv igg, prevalence in the case group averaged at 29% (24% case, 34% control). furthermore, the authors did not report any zikv-related infections in the neonates, or zikv-related adverse pregnancy outcomes in any of the women who participated in this 18-yearlong study. the study further suggested that zikv immunity is common among pregnant women in thailand. the zikv epidemic in colombia during 2015–2017 has also contributed to the literature on czi. various aspects were studied, like the brain or eye defects found in neonates born to mothers with laboratory confirmed zikv infection between the 2015 and 2016 outbreaks. moreover, these defects were found more common during the outbreak than shortly before or after it (44). one of the studies also investigated the link between the epidemic prevalence of cns defects/ microcephaly cases and czi based on the biological specimens accumulated from maternal and infant/fetal sources. it was discovered that out of 858 reported cases of cns defects and/or microcephaly, 503 (58%) could be attributed to czi, but the strength of evidence fluctuated from limited to strong (45). however, according to a preliminary report from 2016 on the colombian zikv epidemic, it was reported that the zvi during the third trimester of pregnancy did not cause any structural deformities in fetuses. these observations were based on the data collected from different types of patients infected by zikv, including a subgroup of pregnant women. according to the same report, zvd was reported twice as much in females than in males, aligning with previous findings from brazilian studies (46). apart from the immediate manifestation of czi shortly after birth, studies in brazil reported asymptomatic births with czi which developed a broad spectrum of neurological disorders later in life. similar observational studies have been conducted in other countries http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 269 http://jad.tums.ac.ir published online: dec 31, 2022 as well. in one of these studies, conducted in response to the zikv outbreak in french territories in the americas in 2016, the effects of in-utero exposure to zikv on children's neurodevelopment in their early childhood was studied. compared to other aspects of czi, this topic is less understood, with most of the literature focusing on congenital disabilities. the study involved toddlers (24 months old) born normocephalic during the 2016 zikv outbreak, and the researchers, between june 2018 and august 2019, assessed their neurodevelopment. the assessment involved the use of ages and stages questionnaire-iii (asq) to measure communication, gross motor, fine motor, problemsolving, and personal-social skills as the five dimensions of overall development, as well as the modified checklist for autism on toddlers (m-chat) to observe behavior, and the french macarthur inventory scales (ifdc) for the acquisition of french language. the results were collected and reported by the parent for the 156 toddlers who underwent in-utero exposure and for the 79 who did not. it was found that 24 (15.4%) of the zikv-exposed toddlers and 20 (25.3%) without the zikv exposure scored below the reference ranges in their asq results. however, the overall difference in neurodevelopment was minimal between toddlers exposed to zikv and those who weren’t at 24 months of age (47). czs encompasses a broad spectrum of developmental disorders in children. these include mobility, postural control, and social skills. many observational studies have hinted at the development of these disorders either at birth or soon after. however, very few studies have attempted to develop possible forms of therapy to help cure these disorders. in a case report from norway, first of its nature, researchers focused on a single child with czs at the age of 17-18 months and observed the impact of intensive physical therapy intervention for six weeks, at home. according to the findings presented by the study, among other measures adopted to evaluate the outcome, mobility, postural control, and social skills of the child were improved due to physical therapy, but further research is re quired to validate these findings (48). fig. 1. pathogenesis of zikv in humans and the unanswered questions surrounding it. adapted from "the role of ilc2s in asthma pathogenesis", by biorender.com (2023). retrieved from https://app.biorender.com/biorendertemplates http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 270 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 2. immune response during viral entry and infection of cns: cns is covered by bbb and other immune cells for protection against pathogenic microbes. with the bbb in a resting state (1), viruses can enter the cns by infecting peripheral nerves and traveling by anterograde axonal transport into the cns, by infecting host immune cells in the periphery and using these cells as "trojan horses" to carry them across the bbb, or by directly infecting bbb endothelial cells (2). viral pamps then activate microglia, astrocytes, and oligodendrocytes (3). microglia and astrocytes produce a range of antiviral/proinflammatory cytokines, including type-i ifns, il-6, tnf-α, il-12, il-1α, and il-1β (3). astrocytes also produce mmp-3 and mmp-12, resulting in the up-regulation of adhesion molecules on endothelial cells (3). interactions between adhesion molecules and neutrophils contribute to bbb breakdown via the production of mmp-9 and the disassembly of the tight junctions (4). dcs are seen in the cns within several days and migrate to draining lymph nodes where they activate and expand virus-specific t cells (5). chemokines produced by astrocytes are responsible for recruiting virus-specific cd4+ and cd8+ t cells and ascs to the cns (6). cd8+ t cells produce ifn-γ and lytic molecules, including granzyme b and perforin, to eliminate the virus from astrocytes, while ifn-γ controls viral replication in oligodendrocytes (7). virus-specific antibodies control virus replication in cells such as neurons via complement– independent, non-cytolytic mechanisms. these antibodies inhibit virus budding and replication, viral rna transcription, and cell-to-cell virus spread (25). adapted from "blood brain barrier (transverse)", by biorender.com (2023). retrieved from https://app.biorender. com/biorender-templates http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 271 http://jad.tums.ac.ir published online: dec 31, 2022 fig. 3. global spread of zikv according to reviewed observational studies. adapted from "global presence (world map)", by biorender.com (2023). retrieved from https://app.biorender.com/biorender-templates zikv in adults compared to czi, zvi is also prevalent in adults and in most cases its symptoms are more apparent and abundant compared to younger patients. therefore, revision of the current definition of paediatric zikv provided by who and pan american health organization (paho) is needed as age is an essential factor to consider when describing the clinical implications of zikv. furthermore, diagnosing zikv in children is more challenging as the symptoms are mild and often non-specific. therefore, attending clinicians might need to perform molecular and serological tests to confirm the diagnosis of zikv (49). like czi, zvi in adults has been investigated more in brazil than in any other country. these investigations have focused on the effects of zikv on different parts of the human body, like the male reproductive system, specifically, the seminal fluid. this is because zikv can be transmitted sexually but how the virus persists in the male reproductive tract is not yet well understood. therefore, this was investigated in a case study during the 2016 zikv epidemic in brazil involving a 33-year old semen donor. the donor was followed before, during, and after the zvi. after the infection, there was delayed damage in the form of reduced progressive motility and concentration of the sperm. also, eight months after the virus became undetectable in the body fluids, the rapid directional motility of the sperm remained reduced. however, the complete clearance of zikv from semen could not be achieved and is a matter of concern, something to be investigated by future studies (50). guillain-barré syndrome (gbs) is another way in which zvi manifests itself in adults. however, the association between zvi and gbs is unknown. during the zikv outbreaks in brazil, a rise in gbs cases was recorded and as a response, an observational cohort study was conducted by leonhard et al. (51) which recruited 71 (36 females and one child aged 9) gbs patients between december 2014 and february 2017. the median age of the patients was 46, with a total of 48 patients confirmed with a recent arbovirus infection (25 of them for zikv). it was found that most of the patients with a recent arbovirus infection had a senhttp://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 272 http://jad.tums.ac.ir published online: dec 31, 2022 sorimotor, demyelinating gbs. furthermore, an observational study by ferreira et al. (52), conducted during brazil's 2015–2016 zikv outbreak, established a similar association between zikv and gbs. the study investigated neurological disorders caused by zikv and other arboviruses, individually and by dual infection. a total of 201 adults participated (median age: 48), with 106 females. in conclusion, a wide variety of neurological diseases were caused by zikv, including gbs, which also overlapped with other arboviruses like chikungunya. among findings from other countries, crossreactivity of zikv antibodies with denv is another topic not completely understood. however, in a case report from the united states, a school-aged female child returning from the dominican republic presented with symptoms including vomiting, fever, abdominal pain, and hypovolemic shock. the death of the child occurred in less than 24 hours following hospitalization. she had an early acute first denv infection, confirmed by performing pcr on serum, plasma, and spinal fluid samples. moreover, no igg antibodies were present against denv nonstructural protein 1 (ns1) for any of the four known serotypes of denv. however, immunity toward a previous zvi existed due to antibodies against the zikv ns1 envelope. therefore, the researchers came to believe that perhaps the presence of immunity towards zikv triggered the antibody-dependent enhancement (ade) phenomenon, as it has been reported in the past that zikv antibodies tend to cross-react with denv. therefore, this was the first case of such nature in the united states where zikv immunity probably contributed to the triggering of the ade phenomenon, eventually leading to the fatality of the patient (53). with that in mind, there are also reports of zikv vaccination boosting preexisting immunity towards flaviviruses, as demonstrated when tested on a denv-experienced individual, and protecting against denv and zikv (54). regarding the association between zvi and different diseases, a connection was established between acute anterior uveitis (inflammation of the middle layer of the eye) and an acute systemic zvi in a study conducted in the french west indies in guadeloupe in the 2016 zikv outbreak. an ophthalmic investigation was performed in 62 adults with red-eye who had tested positive for zvi through serology. half of the patients who presented with red-eye and a zikv-positive profile had anterior uveitis. the uveitis was mainly associated with a rise in intraocular pressure and was bilateral. therefore, the study suggested that patients with red-eye who test positive for acute systemic zvi should undergo an ophthalmic examination to diagnose them with hypertensive anterior uveitis (55). discussion zikv is a major threat to public health in various countries around the world (26–31). the virus itself is transmitted to humans through the aedes mosquito (4). zikv penetrates the skin (4-6), causing a wide variety of symptoms, disorders, and diseases. these range from general symptoms like fever, rash, myalgia and conjunctivitis, to name a few, to more serious conditions like gbs and microcephaly (7, 8). these disorders can be further distributed between adults and infants, with the virus manifesting into specific diseases in each age group. so far, most of the published research concerning zikv has involved experimental studies that have provided valuable insight into the innerworkings of this virus and how it causes infection in humans (4–25). however, to grasp the severity and real-life implications of the zvi on different age groups, observational studies are necessary. most of the data collected from the observational studies involve the effects of zikv on neonates (32–41, 43–48), whereas only a limited number of studies have highlighted its effect on adults (49–55). furthermore, most http://jad.tums.ac.ir/ j arthropod-borne dis, dec 2022, 16(4): 262–277 t saleem et al.: zika virus from … 273 http://jad.tums.ac.ir published online: dec 31, 2022 of these observational studies regarding zikv have emerged from south america, particularly brazil (32–41). since the 2015 zikv outbreak, brazil has reported a vast number of zvi cases with most of them involving neonates. czi in neonates causes neurological disorders and diseases like microcephaly. moreover, it was reported that the infected infants had undergone in-utero exposure to zikv during pregnancy, resulting in czs. whereas in adults, observational studies have shown zvi to cause multiple conditions like gbs, acute anterior uveitis and cross-reactivity of zikv antibodies with denv. however, a gap in literature exists concerning all these areas. especially, the association between gbs and zikv, the sexual transmission of zikv, and the effects of crossreactivity between zikv and anti-denv antibodies require further investigation. conclusion in this review, we attempted to enlighten the reader about the threats that zikv poses to the human population. zikv was presented in the light of observational studies rather than experimental studies to stress its damaging capabilities in real-life settings. in that, we highlight the damage it is capable of in infants and adults alike, worldwide, with brazil being one of the countries reporting the most zikv cases and zikv-related observational studies. the reviewed research covers multiple disorders and diseases caused by zikv, including microcephaly, developmental disorders in children, gbs, cross-reactivity of denv and anti-zikv antibodies, and acute anterior uveitis. it is concluded that neurological disorders/ diseases are most prevalent in neonates with czi, while the clinical manifestations of zikv in adults involve different organs. we also highlight gaps in the literature regarding zikvrelated-complications that future experimental studies need to address. these include in-utero transmission or guillain-barre syndrome or its persistence in the male reproductive tract, cross-reactivity, and sexual transmission. acknowledgements we would like to 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worldwide. cases of human infestation have been reported worldwide. in this study we report infestation in three members of a family referred with pruritus and allergic dermatitis rash. they have collected very small animals and carried them to the laboratory which later was confirmed as d. gallinae. they claimed that they had been bitten with this ectoparasite. this is the first case report of human infestation owing to d. gallinae from iran. keywords: dermanyssus gallinae, infestation, human introduction the poultry red mite, dermanyssus gallinae is one of the most economically deleterious ectoparasite of laying hens and sometimes in broiler birds worldwide. recent surveys and sample collection from norway have confirmed the enzootic nature of d. gallinae in poultry farming worldwide (sparagano et al. 2009). the parasite infestation already existed in the farm. dermanyssus mites are blood feeders and are responsible for anemia and welfare problems in birds, dermatitis in humans and egg downgrading as well as blood spotting which imply a huge economic, welfare, epizootic and epidemiological problems for birds and human populations. on average the dermanyssus infestation rate was between 60% and 65% for cage, free-range and organic systems while it was around 54% for barn production systems (sparagano et al. 2009). dermanyssus gallinae can also feed on some species of mammals, including humans, causing dermatitis and skin lesions. ectoparasitic diseases have been reported in travelers returning from both developed and developing nations. ectoparasitic diseases afflict the skin and its appendages and orifices, especially the scalp, facial, and pubic hairs, external ears, nares, orbits and eyelids, and genitourinary and rectal orifices. the red chicken mite can also cause pruritic dermatitis usually on the back of the hands and forearms in poultry workers and can transmit both st. louis encephalitis and western equine encephalitis (james et al. 2006). case report three members of a family referred to parasitology department of razi institute complaining pruritus, reported itching all over their body with intensified itching of hands, forearms, back of neck, and chest intensifying particularly at evening hours. in physical examination common findings of usual pruritus dermatitis such as erythematous maculopapular rash covered with bloody  *corresponding author: dr mohammad abdigoudarzi, e-mail: m.abdi@rvsri.ir http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 119–123 m abdigoudarzi et al.: human infestation with … 120 crust due to violent itching have been noted (fig. 1, 2). other than above mentioned signs of infestation, they physically appeared normal and no other medical examinations or tests were done. their medical history and records showed no chronic condition. the specimens were identified through microscopic examination in the parasitology department of razi ins. as d. gallinae (fig. 3, 4). d. gallinae is characterized by long styliform chelicerae, tibia ii–iv with one seta and was discriminated from a near member (ornithonyssus silviarum) (di-palma et al. 2012). the family members were informed of the findings and control measures to protect themselves from additional infestations and to protect relation of birds or birds' nests was emphasized. fig. 1. skin rashes over forearm of (mother) due to infestation by dermanyssus gallinae (original) fig. 2. erythematous maculopapular rash were covered with bloody crust due to violent itching on the back of neck (son), due to infestation by dermanyssus gallinae (original) fig. 3. dermanyssus gallinae (female) charcterized by long styliform chelicerae (arrow, very thin elements) (original) fig. 4. dermanyssus gallinae (female) charcterized by long styliform chelicerae (arrow, very thin elements) (genitoventral shield), (anal shield) and mesostigmatal pore between coxa 3 and coxa 4 (original) discussion three members of a family referred to parasitology department of razi institute due to suffering from pruritus dermatitis. they carried few samples of ectoparasites with themselves. on the basis on medical and physical examination, history of medical illness, parasitological studies on the samples, form of skin rashes and rolling out of other differential diagnosis such as pediculosis and scabies, d. gallinae infestation was confirmed. all family members were infested. this co-infestation has been reported by other studies. for example akdemir et al. showed that similar signs had been seen in all members of the infested household (akdemir 2009). the referred cases had skin rashes over http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 119–123 m abdigoudarzi et al.: human infestation with … 121 forearm (mother), chest and back of neck (son). erythematous maculopapular rash was covered with bloody crust due to violent itching specially in forearm of mother. other studies have showed that d. gallinae infestations afflict the skin (causing intensified itching of body (akdemir 2009) and finally pruritic dermatitis usually on the backs of the hands and forearms (neva et al. 1994). scalp (causing scalp pruritus), face, pubic hairs, external ears (causing otitis externa (rossiter 1997), nares, orbits and eyelids, and genitourinary and rectal orifices (james et al. 2006). dermanyssus gallinae was first identified by de geer in 1778, and first report of human infestation was reported by willian in 1809. in 1828, saint-vincent spotted this parasite on the skin of a human, but first observation of its feeding on human blood was published by williams in 1958. now it seems that human d. gallinae is a worldwide infestation. cases of human d. gallinae infestation have been reported from denmark, france, japan, montenegro, morocco, norway, serbia, the netherlands, uk, egypt, turkey etc. (dogramaci et al. 2010). this is the first reported case of d. gallinae infestation from human in iran. these cases are of significance because most dermatologists have difficulty identifying ectoparasitosis, such as those that arise within new or atypical conditions. these cases may be misdiagnosed with other pruritus and treated with antihistamines and topical corticosteroids with temporary relief of the symptoms for long time (dogramaci et al. 2010). d. gallinae has role as vectors for some bacteria such as salmonella, spirocheta, ricketsia, pasteurella besides being ectoparasitic activities (vaiente et al. 2007). otitis externa is only occasionally occurred occupational in origin and infestations of the ear are even less common. two cases of occupational otitis externa due to infestation with d. gallinae, the red poultry mite, are reported occurring in poultry workers (rossiter 1997). although a marked clinical dermatitis is common in some individuals that become closely associated with the bird mite, d. gallinae, the literature presents little evidence that this mite will ingest human blood and many investigators think this species will never ingest it. the invasion of a new york (williams 1958) city apartment by these mites resulting in attacks on the occupants was described in a famous study. the finding of mammalian erythrocytes in the digestive tract of mites collected from that apartment (williams 1958), some of which were taken from the bed of the occupants, as well as the appearance of fresh blood splotches on the bed sheets resulting from crushed mites which had recently fed, offer some factual evidence that d. gallinae may, on occasion at least, partake of human blood. an older research article (williams 1958), had clearly documented human blood was ingested by d. gallinae, which was contrary to what many had previously thought, the bird mites do not feed on humans, but only on birds. cutaneous manifestations of avian mite bites are not well recognized by physicians or patients. clinical signs and symptoms are usually caused by bites from avian mites that have infested domestic poultry or birds nesting in or near human habitation. pruritic dermatitis visible with papules and vesicles which sometimes is mistaken for scabies or pediculosis may be chronic or recurrent with erythematous maculopapular or papulovesicular lesions (personal communication). in the present case physical examination common findings of usual pruritus dermatitis such as erythematous maculopapular rash covered with bloody crust due to violent itching have been noted. treatment of bird mite infestation consists of removing the old nests. treatment of http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 119–123 m abdigoudarzi et al.: human infestation with … 122 the patients is symptomatic. epizoonosis belongs in the differential diagnosis of pruritus, infestation with bird or chicken mites is one of the possibilities (prins et al. 1996). human costs are difficult to establish but cases of dermatitis related to d. gallinae are now more and more obvious while workers in some countries had to be paid 3 times more in recent years to work with d. gallinae infested birds (sahibi et al. 2008). keeping pigeons, love birds and other birds is common in iran. besides, the poultry sector in iran is much expanded and still developing. an important part of hens is still kept by small holders, so, it seems that many people may expose to poultry ectoparasites like d. gallinae. more studies on epidemiology of this ectoparasite in different exposed groups are suggested. pruritic dermatitis cases would be more frequent than the reports made to medical centers as in this case, and cutaneous reactions resulting from avian mites are generally gone unnoticed. it may be necessary to collect a detailed surveillance on referred patients to hospitals upon such symptoms. it is also concluded that existence of birds inside or nearby human inhabited places should be 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caused by the mite, dermatophagoides (acarina, epidermoptidae) proceedings of the entomological society of washington vol. 53, no. 1. vaiente mc, chauve c, zenner l (2007) experimental infection of salmonella enteritidis by the poultry red mite, dermanyssus gallinae. vet parasitol. 146: 329–336. watson cr (2003) human infestation with bird mites in wollongong, commun dis intel. 27: 259–261. williams rw (1958) an infestation of a human habitation by dermanyssus gallinae (degeer, 1778) (acarina: dermanyssidae) in new york city resulting in sanguisugent attacks upon the occupants. am j trop med hyg. 7: 627–629. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, 2012, 6(2): 144–150 sr naddaf et al.: confirmation of two … 144 original article confirmation of two sibling species among anopheles fluviatilis mosquitoes in south and southeastern iran by analysis of cytochrome oxidase i gene *saied reza naddaf 1, mohammad ali oshaghi 2, hassan vatandoost 2 1department of parasitology, pasteur institute of iran, tehran, iran 2department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran (received 5 jun 2012; accepted 11 dec 2012) abstract background: anopheles fluviatilis, one of the major malaria vectors in iran, is assumed to be a complex of sibling species. the aim of this study was to evaluate cytochrome oxidase i (coi) gene alongside 28s-d3 as a diagnostic tool for identification of an. fluviatilis sibling species in iran. methods: dna sample belonging to 24 an. fluviatilis mosquitoes from different geographical areas in south and southeastern iran were used for amplification of coi gene followed by sequencing. the 474–475 bp coi sequences obtained in this study were aligned with 59 similar sequences of an. fluviatilis and a sequence of anopheles minimus, as out group, from genbank database. the distances between group and individual sequences were calculated and phylogenetic tree for obtained sequences was generated by using kimura two parameter (k2p) model of neighborjoining method. results: phylogenetic analysis using coi gene grouped members of fars province (central iran) in two distinct clades separate from other iranian members representing hormozgan, kerman, and sistan va baluchestan provinces. the mean distance between iranian and indian individuals was 1.66%, whereas the value between fars province individuals and the group comprising individuals from other areas of iran was 2.06%. conclusion: presence of 2.06% mean distance between individuals from fars province and those from other areas of iran is indicative of at least two sibling species in an. fluviatilis mosquitoes of iran. this finding confirms earlier results based on rapd-pcr and 28s-d3 analysis. keywords: anopheles fluviatilis, sibling species, cytochrome oxidase i, taxonomy introduction despite a considerable progress in malaria control in iran over the past few years that led to significant reduction of cases, the disease still remains a major health problem in south and southeastern parts of the country. inherent problems of drug resistance (zakeri et al. 2008, afsharpad et al. 2012) and insecticide resistance of anopheles vectors (enayati et al. 2003, vatandoost et al. 2005, hanafi-bojd et al. 2012) are aggravated by continuous influx of imported cases, mostly with plasmodium falciparum, from neighboring countries of afghanistan and pakistan (zakeri et al. 2010), making control of disease much more difficult in these areas. one of the key elements in fighting malaria is accurate identification of malaria vectors. the latest checklists of iranian mosquitoes include 28 anopheles species, identified mostly on the basis of morphological features, and a few by dna-based approaches (azarihamidian 2007). seven species namely an. stephensi, an. culicifacies, an. dthali, an. fluviatilis, an. superpictus, an. sacharovi, and an. maculipennis are known to be responsible for transmission of malaria in the country (manouchehri et al. 1992, sedaghat and harbach 2005). some important malaria vectors in iran are assumed to be members of species *corresponding author: dr saied reza naddaf, email: snaddaf_2001@yahoo.com j arthropod-borne dis, 2012, 6(2): 144–150 sr naddaf et al.: confirmation of two … 145 complexes or species groups, which are often difficult to distinguish morphologically. application of dna-based approaches has resolved some cryptic species in iranian complex species including an. culicifacies, an. maculipennis, an. superpictus, and an. fluviatilis, however, the identity of some members are still doubtful or were refuted later (azari-hamidian 2007, naddaf et al. 2010). molecular taxonomy of an. fluviatilis in iran has received great attention over the past decade. this, to great extent, is due to introduction of molecular markers such as its2 and 28s-d3 genes for discriminating the members of this complex species in india (manonmani et al. 2001, singh et al. 2004). biology, variation in behaviors, and role of this species in malaria transmission in different geographical areas of iran has been extensively reviewed by others (eshghi et al. 1976, manouchehri et al. 1976, edalat 1997– 1998, hanafi-bojd et al. 2012). in an early study comparison of its2 sequence of iranian specimens from various localities in south and southeastern iran revealed only species y, which is presumably species t (naddaf et al. 2003), however, rapd-pcr analysis of same specimens revealed two distinct patterns, separating representatives of fars province from other areas (naddaf et al. 2002, naddaf et al. 2003). analysis of 28s-d3 gene from same populations corroborated rapd results, fars province specimens showed to be identical to species u in india, while individuals from other areas exhibited heterozygocity at the only base pair position that identifies species u and t (naddaf et al. 2010). in addition, in a separate study based on 28s-d3 analysis, species t and species u were reported from jiroft of fars province and chabahar of sistan va baluchestan province, respectively (mehravaran et al. 2011). the aim of this study was to evaluate cytochrome oxidase i (coi) gene alongside 28s-d3 as a diagnostic tool for identification of an. fluviatilis sibling species in iran. coi gene sequences have been extensively used for population studies and resolving evolutionary relationship among closely related species groups of insects (lunt et al. 1996) and anopheline mosquitoes (krzywinski and besansky 2003). variations in this fragment have been exploited as dna barcodes for identifications of culicidae mosquitoes including an. fluviatilis (cywinska et al. 2006, kumar et al. 2007). materials and methods mosquitoes dna the dna samples used in this study were obtained from an. fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars, hormozgan, kerman, and sistan va baluchestan provinces. the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously (naddaf et al. 2002, naddaf et al. 2003, naddaf et al. 2010). the details for dna samples used in this study are shown in table 1. pcr and sequencing of dna all the dna samples were initially subjected to allele specific (as)-pcr based on 28s-d3 gene as described by singh et al. (2004). the coi gene was amplified using universal primers, ubc6 (5′gga gga ttt gga aat tga tta gtt cc -3′) and ubc9 (5′-ccc ggt aaa att aaa ata taa act tc-3′), designed by simon et al. (1994) and later used by sedaghat (2003). the pcr reaction conditions were as outlined by singh et al. (2004) with minor modifications. each 25µ l reaction contained 20 pmol of each primer, 2mm mg cl2, 10mm tris-hcl, 50mm kcl, 150µm of dntps, 1u of taq, and 2µ l of dna. pcr products were purified using a gel band purification kit (pharmacia, piscataway, nj, usa) according to manufacturer’s recommendations and later sequenced using the j arthropod-borne dis, 2012, 6(2): 144–150 sr naddaf et al.: confirmation of two … 146 same primers as used for amplification at seqlab laboratory in germany. the sequences were manually edited and corrected using bioedit software, version 7.1.3.0 (hall 1999) and fragments of 474 bp length were selected for analysis. the coi sequences of our specimens were aligned with 59 similar sequences of an. fluviatilis, and one sequence of an. minimus as outgroup from genbank database using clustal x software (thompson et al. 1997). the distances between groups and between individual sequences were calculated, and phylogenetic tree for iranian sequences was generated using the kimura two parameter (k2p) model of neighbor-joining method in a complete deletion procedure using mega 4 software (tamura et al. 2007). the robustness of the topologies was estimated through 1000 bootstrap replications. the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729. results all the dna specimens from fars province yield only a product of approximately 375 bp length indicative of species u, whereas specimens from hormozgan, kerman and sistan va baluchestan provinces amplified two bands of 375 bp and 128 bp length. phylogenetic analysis using coi gene grouped individuals from fars province in two distinct clades separate from other iranian individuals representing populations of hormozgan, kerman, and sistan va baluchestan (fig. 1). within group mean distances for iranian and indian individuals were 1% and 1.09%, respectively. the mean distance between iranian and indian groups was 1.66%, while the value between fars group and the group comprising other iranian members was 2.06%. the indian group exhibited the same distance (2.06%) with fars group. the highest distance (3.09%) among iranian individuals was between specimens 930 from fars province and 398 from sistan va baluchestan province. six individuals belonging to two clades (87, 97, 655, and 928) and (92) from fars province showed 100% identity. in addition, ten individuals from other geographical areas including four from koveh (170, 172, 173, and 186) and one from minab (121) in hormozgan province, two from abchekan (392–393) in sistan va baluchestan province, and three from kahnouj in kerman province (815, 836, and 839) were 100% identical. table 1. details for dna samples used in this study no. province specimen id collection area identification on its2/ref. 28s-d3/ref. 1 hormozgan 117 siahoo/koveh np np 2 hormozgan 164 siahoo/koveh t (*) h (**) 3 hormozgan 169 siahoo/koveh np np 4 hormozgan 170 siahoo/koveh np np 5 hormozgan 172 siahoo/koveh np h (**) 6 hormozgan 173 siahoo/koveh t (*) h (**) 7 hormozgan 186 siahoo/koveh np np 8 hormozgan 120 minab/tombe basat np np 9 hormozgan 121 minab/tombe basat np np 10 kerman 815 kahnouj/ condor garmaei np h (**) 11 kerman 834 kahnouj/manoujan np h (**) 12 kerman 836 kahnouj/manoujan np h (**) 13 kerman 839 kahnouj/manoujan np h (**) j arthropod-borne dis, 2012, 6(2): 144–150 sr naddaf et al.: confirmation of two … 147 14 sistan va baluchestan 392 daman/abchekan np np 15 sistan va baluchestan 393 daman/abchekan np np 16 sistan va baluchestan 398 daman/abchekan t(*) h (**) 17 fars 87 kazeroun/pirzabs np np 18 fars 91 kazeroun/pirzabs np np 19 fars 92 kazeroun/pirzabs t (*) u (**) 20 fars 97 kazeroun/pirzabs np np 21 fars 655 kazeroun/islamabad np np 22 fars 926 khesht/chiti t (*) u (**) 23 fars 928 khesht/chiti t (*) u (**) 24 fars 930 khesht/chiti np np np= not performed, ref. = reference, h= heterozygosity at the nucleotide position of 28s-d3 gene that identify species t and u. * naddaf et al. 2003, ** naddaf et al. 2010. fig. 1. phylogenetic tree based on coi sequences. the scale bar corresponds to a 0.005 distance and the accession numbers of gene sequences are shown in parentheses. discussion accurate identification of malaria vectors is not only one of the most basic requisite for success of malaria control programs, but also has become an intriguing issue for understanding speciation process and evolution of anopheles mosquitoes. in absence of cytotaxotable 1. countinued… j arthropod-borne dis, 2012, 6(2): 144–150 sr naddaf et al.: confirmation of two … 148 nomic evidence, rapd-pcr methodology and variation in 28s-d3 gene have resolved two potential sibling species in an. fluviatilis mosquitoes of iran. here we report further evidence for the occurrence of these two sibling species by coi analysis of mitochondrial dna from the same specimens. anopheles fluviatilis james is a complex of cryptic species; cytotaxonomic studies of polythene chromosomes has revealed three reproductively isolated species in india known as s, t, and u (subbarao et al. 1994). application of the first dna-based method using its2 gene identified two putative species of x and y that are presumably equivalent to species s and t, respectively (manonmani et al. 2001). later, a complete as-pcr assay based on variations of 28s-d3 gene against chromosomally examined specimens identified all the members of the complex (singh et al. 2004). analysis of its2 gene revealed a single species in an. fluvialitis mosquitoes of iran. however, the same specimens displayed variations in 28s-d3 gene; the individuals from fars exhibited similarity with species u in india whereas individuals from others areas showed heterozygocity at the single nucleotide position that identifies species u and t. the identity of an. fluviatilis complex in iran became complicated as the heterozygocity in 28-d3 was not reflected in its2 fragment and individuals from fars province exhibited dual identity of t and u based on its2 and 28s genes, respectively (chen et al. 2006, naddaf et al. 2010). kumar et al. (2007) identified 62 mosquitoes species, including an. fluviatilis s.l. among members of the family culicidae from india by coi analysis. the variation between an. fluviatilis sibling species were not addressed in their study, however, k2p genetic distances between different species of culicidae were reported to be >2%. in our study, the mean distance between iranian and indian populations was 1.66%, whereas the value between fars group and the group comprising other iranian individuals was 2.06%. the indian group exhibited the same distance (2.06%) with fars group. the distance between most individuals from fars province and individuals from other areas was >2%. two individuals (91–92) from fars province exhibited almost equivalent distance with all other members including those (930, 926, 928, 655, 87, and 97) from the same province. no variation was seen over 154 amino acids shared by all 24 specimens. there was a sequence in genbank from bandar abbas (accession no. jf966741, unpublished) that showed a high divergence from other sequences and appeared as an out group beyond an. minimus in early phylogenetic trees and hence was excluded. the results of present study were almost concordant with earlier results obtained by rapd-pcr methodology and 28s-d3 analysis (naddaf et al. 2002, naddaf et al. 2010). conclusion this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for resolving closely related taxa of an. fluviatilis complex species. analysis of more identified specimens of an. fluviatilis mosquitoes (by its2 and 28s-d3 genes) from india, iran, and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species. acknowledgments we gratefully acknowledge technical assistance of our colleague dr n piazak from the department of parasitology, pasteur institute of iran. this study was supported by a grant from the pasteur institute of iran (no 292). the authors declare that there is no conflict of interest. j arthropod-borne dis, 2012, 6(2): 144–150 sr naddaf et al.: confirmation of two … 149 references afsharpad m, zakeri s, pirahmadi s, djadid nd (2012) molecular monitoring of plasmodium falciparum resistance to antimalarial drugs after adoption of sulfadoxine-pyrimethamine plus artesunate as the first line treatment in iran. acta trop. 121(1): 13–18. azari-hamidian s (2007) checklist of iranian mosquitoes (diptera: culicidae). j vector ecol. 32(2): 235–242. chen b, butlin rk, pedro pm, wang xz, harbach re (2006) molecular variation, systematics and distribution of the anopheles fluviatilis complex in southern asia. med vet entomol. 20(1): 33–43. cywinska a, hunter ff, hebert pd (2006) identifying canadian mosquito species through dna barcodes. med vet entomol. 20(4): 413–424. edalat h (1997–1998) ecology of anopheles fluviatilis, the main malaria vector, and its role in epedimiology of malaria. 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accepted 29 mar 2023) abstract background: a combined morphological and molecular survey was performed to determine the agent of human linear dermatitis paederus fabricius, 1775 (coleoptera: staphylinidae, paederinae) species composition in mazandaran province in the caspian sea coast in northern iran, where most of linear dermatitis cases of the country occurred. methods: altogether, 397 paederus specimens were collected from may to august 2021 and classified using morphological characters and its2-rdna sequence analysis. results: morphological investigation revealed that all the specimens were paederus fuscipes. its2 polymerase chain reaction (pcr) direct-sequences and the profiles of restriction fragment length polymorphism (rflp) derived from digestion of pcr products by hinfi, hpaii, and sali enzymes were identical confirming the morphological results, implying that all specimens belonged to a single taxon. conclusion: paederus fuscipes (fabricius, 1775) is considered the dominant taxon and responsible for linear dermatitis in mazandaran province. to our knowledge, we have provided the first molecular typing of paederus beetles at the species level, suggesting that its2-rdna characterization is an alternative tool for species discrimination of paederus spp. keywords: paederus fuscipes; rove beetle; linear dermatitis; its2-rdna; iran introduction rove beetle (staphylinidae) is the prime family of coleoptera order with more than 6.000 species (1, 2). paederus fabricius, 1775, is categorized into the tribe paederini and subfamily paederinae, presently encompasses around 490 species (3). according to herman (2001), paederinae subfamily is the third largest subfamily within the staphylinidae family, and it is represented about 6.000 species (4). in the palaearctic region, the genus paederus is included 85 species and subspecies. some rove beetle are scavenger (5) or predator and act as biological agents in control of pests (6). paederus beetles are nocturnal and attracted by incandescent and fluorescent lights. as paederus species turn towards particularly fluorescent light, they encounter with people. these insects that can enter homes at nights from an open window or underneath of door, excrete toxic substance in their hemolymph when they are disturbed or rushed. the hemolymph of some species within the paederus has long been recognized to be an irritation as, once released, it leads to human linear dermatitis and conjunctivitis (7). the signs are as a result of a toxic amide substance, named copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:moshaghi@sina.tums.ac.ir mailto:oshaghima@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 95 http://jad.tums.ac.ir published online: march 31, 2023 pederin (c25 h45 o9 n, ld50: 0.14mg/kg rat i.p.) (8) and makes up roughly 0.025% of an adult p. fuscipes weight. after pederin touches the skin, this substance to be proportional to its amount may cause important results from a simple rash to large wounds. at least 20 species of paederus beetles have been associated with linear dermatitis (9). along with causing rigorous skin lesions, their ordinary substructure accounts for similar antitumor and antiviral activities, cytotoxicity and interruption of dna metabolism which are primarily based on obstruction of eukaryotic protein biosynthesis (10– 13). at the same time owing to have the ability of inhibiting protein and dna synthesis and being a very strong toxic substance, pederin has been habited in tumor studies in current years. the secretion or production of pederin depends on the activities of a paederus bacterial endosymbiont (pseudomonas sp.) (14–18). the production of pederin is mainly limited to adult female beetles which protects the beetles against predators (14, 15, 19). males and larvae only accumulate pederin gained maternally (i.e., through eggs) or by ingestion (13). systematics of paederus species is rather problematic and is relied on the male main and secondary sexual morphological characters (20). this has ended a very complex history for paederus taxonomy and has reformed it intensely (21) and some species are regarded as synonyms of each other and or lowered to a single subspecies/species (22 and references herein). recent advances in molecular methods have provided an extensive range of molecular markers for taxonomy and systematics of insects (23–30). the ribosomal dna (rdna) of insect genome has many advantages comprising known pcr primers, easy to use, a high interspecies mutation rate, and low intraspecies mutation rate due to homogenization has acknowledged particular attention for taxonomic and systematic studies of insects (24, 31–39). earlier studies on the ecology, geographical distribution, medical importance, and fauna of paederus beetles in iran showed presence of fourteen paederus subspecies or species in the country, in which six species p. littoralis ilsae, p. fuscipes, p. balachowskyi, p. balcanicus, p. riparius, and p. duplex are reported in caspian sea coast (mazandaran, guilan, and golestan provinces) (22). however, due to problems in paederus species identification, various lists of species have been reported for a same region. in north of country, for example, three species of p. pietschmanni (synonym of p. mesopotamicus), p. fuscipes, and p. spectabilis have been reported by janbaksh and ardalan (1977) in mazandaran province at the caspian sea shore (40). later, majidi-shad et al. (1989) registered three species of p. riparius, p. littoralis and p. fuscipes, from the same region (41). afterwards, p. balcanicus, p. fuscipes, and p. kalalovae were reported from the same region by nikbakhtzadeh and tirgari (2008) (42). ultimately, three species of p. balachowskyi (synonym of p. mesopotamicus), p. balcanicus, and p. fuscipes, was reported from the same region by nikbakhtzadeh et al. (2012) (22). this discrepancy and inconsistency stimulated us to test genetic variation of its2-rdna, as a powerful molecular marker, of paederus specimens collected from the province to unravel systematic status of the paederus beetles in the area. materials and methods study area and paederus collection mazandaran province (mp) is 23,833km2, situated on the southern coast of the caspian sea; clockwise it is bounded by the, tehran, alborz, golestan semnan, qazvin, and guilan ps. city of sari is the capital and the largest city of the province. the province is geographically partitioned into two parts: the mountainous areas, and the coastal plains. the alborz mountain range borders the coastal belt and plains of the caspian sea. there is often snowstorm throughout most of the seasons in the alborz regions, which run correspondence to the caspian sea's southern coast, partitioning the province into http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 96 http://jad.tums.ac.ir published online: march 31, 2023 many remote valleys. the province includes 15 counties including sari, babol, amol, behshahr, babolsar, chaloos, tonekabon, ramsar, juybar, qaem shahr, savad kooh, neka,mahmood abad, noshahr, and noor (fig. 1). adult paederus samples were collected by mouth aspirator on various vegetation (particularly rice) in early morning or afternoon and under soils at warm hours of daytime. in some areas, an ultraviolet black light was used for specimen collection from dusk to midnight at night as previously described by nikbakhtzadeh and tirgari (2008) (42). the collections were done mostly in rice farms in 33 sites belong to the 15 mp’s counties (fig. 1, table 1). the rice fields were selected randomly, and sampling was conducted throughout the growth time from may to august 2011. the captured specimens were kept in 70% ethanol till morphological and molecular identification. morphological identification the pictorial keys of coiffait (1982) were used to determine the specimens to genus level (20) and then we send them to the specialist (dr sinan anlaş laboratory) in turkey for species identification. the specimens were identified on the base of habitus and sound structure of male basal and secondary sexual characters (fig. 2). dna extraction a subset (n=66) of paederus specimens’ representative of the populations and counties, were chosen for dna analysis. total body of individual samples was used for genomic dna extraction using dneasy® blood and tissue kit (qiagen, hilden, germany), according to the manufacturer’s instructions. preceding to dna extraction, the specimens were frozen and then pounded in the kit supplied buffer and extraction followed based on the manufacturer's directions. dna amplification the primer pair its3f (5′-gcatcgatgaagaacgcagc-3’) and its4r (5′tcctccgcttattgatatgc-3’) was used to amplify its2 (internal transcribed spacer number 2) region of rdna gene of the specimens. the primer pair has already been proposed by white et al. (1990) and utilized for some coleopteran (43). pcr amplification cocktail included 5μl 10× pcr-buffer, 120μm of each dntps, 50pmol of each primer, 2μl (around 100ng) of template dna, and 2.5u of taq polymerase (sinaclon, iran) in a 25μl reaction volume. pcr amplification was accomplished with an eppendorf thermal cycler (germany). the cycling program was: 3min denaturation at 94 °c followed by 35 cycles of 1min at 94 °c, 1min at 52 °c, and 2min at 72 °c, and a final 7min chain elongation. well-characterized dna samples and double distilled water (ddh2o) were used as positive and negative controls. sequencing and pcr-rflp a subset (n=17) of the specimen pcr products were refined from gels by using a gel purification kit and used for sequencing. an abi 3730 sequencer machine by bioneer (south korea) was used for sequencing the samples. the ambiguities of resultant sequences were verified and their homologies with the accessible sequence data in genbank was blasted by using basic local alignment search tool (blast) analysis software (www.ncbi.nlm.nih.gov/blast). to thrifty in time and cost of sequencing, the rest of specimens were tested by restriction enzyme fragment polymorphism (rflp) assay. to do this, the its2 sequences attained in present study was tested to prepare its physical map and to choose restriction/digestion enzymes by using the nebcutter program (44) (fig. 3). restriction/digestion enzymes were selected according to their sites on the pcr product, costs, profiles, and obtainability in the marketplace. digestion or cutting of pcr products was accomplished in 25μl of a solution containing 15μl of pcr product associated with 2.5μl of enzyme buffers and 5 units of individual restriction enzymes (hinfi, hpaii, and sali) overlaid with two droplets of mineral oil. the blend was nursed at the temperature suggested by the http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 97 http://jad.tums.ac.ir published online: march 31, 2023 enzyme supplier. an aliquot part (14μl) of the restricted product was blended with 6μl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol), loaded on to a 2% agarose gel, and electrophoresed. gels were contaminated with ethidium bromide (2mg/ml) and the rflp outlines were envisioned under ultraviolet light. results species identification and pcr-rflp during the study period, a total of 397 adult paederus specimens were gathered by hands or black-light trap. except one specimen of philonthous cf. sp which belongs to staphylininae subfamily, (tribe staphylinini, subtribe philonthinina), all the specimens were classified morphologically as paederus fuscipes (fig. 2). this species was the predominant taxon representing 99.75% (396) of the total number of collected rove beetles. a subsection of the morphologically distinguished p. fuscipes were laid open to its2-rdna pcr amplification. a 495bp fragment was amplified for all tested specimens. the its2 pcr products of a total of 17 morphologically identified p. fuscipes were selected based on their geographical origin for sequence characterization. the specimens were sequenced for both strands and the consensus data were submitted to genbank database with genbank identification numbers (ids): kc414867kc414883 (table 1). the amplified fragment comprises 126bp of 3’ end of 5.8s ribosomal dna gene, 312bp internal transcribed spacer number 2 (its2), and 57bp of 5’ end of 28s ribosomal dna gene. the sequences were relatively high (58.4%) gc content. among the sequences, there was no substitution or indel throughout the 495bp of the fragment indicating identical sequences. afterward, a blast search on the sequences showed that there were some matching its2 sequences for p. fuscipes species in genbank including a specimen from south of iran (genbank id: km086334) with 98.75% identity), and a specimen from china (genbank id: mz172410) with 96.34% identity. also, there were some homologous sequences with 89.45% maximum identity belonged to p. mesopotamicus from turkey (genbank id: kc414866) and with 85.18% maximum identity belonged to p. littoralis from south of iran (genbank id: km098054). sequence analysis of p. fuscipes its2 fragment revealed appropriate restriction sites for hpaii (cc↓gg) at 146, sali (g↓tcgac) at 221, and hinfi (g↓antc) at 310 nucleotide positions (fig. 3). restriction digestion of the its2 pcr products gave two fragments of 310/185, 146/349, and 221/274bp for hinfi, hpaii, and sali respectively for all the 396 morphologically identified p. fuscipes. fig. 1. map of the area study (mazandaran province) and the collection sites (stars) of paederus specimens http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 98 http://jad.tums.ac.ir published online: march 31, 2023 fig. 2. details of paederus fuscipes (a)habitus; (b)aedeagus in ventral view (c)aedeagus in dorsal view (d)aedeagus in lateral view. scale bars: 1.0mm (a); 0.2 mm (figs. b-d) fig. 3. physical map of 495bp fragment of its2-rdna region of paederus fuscipes. the hpaii, sali, and hinfi restriction sites at 146, 221, and 310 nucleotid positions are shown by arrows http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 99 http://jad.tums.ac.ir published online: march 31, 2023 table 1. details of morphologically identified paederus fuscipes specimens collected in this study location (township) date 2011 site no. further identification method genbank accession number mahmood-abad may zardab, hezarpey-shomali abolhassan-abad 11 12 sequencing pcr-rflp kc414874 june may ahlmarestaghe-shomali ahlmarestaghejonobi 11 12 pcr-rflp pcr-rflp kc414875 july mirdeh-olya, daboye-shomali 27 pcr-rflp na amol may bisheh-mahaleh 13 sequencing kc414873 june roodbar 12 pcr-rflp na june aghozin, linkooh 10 pcr-rflp na july osko-mahaleh, mirza kochakkhan jungle, linkooh 13 pcr-rflp na april marzango 21 sequencing kc414878 ghaem-shahr june haji-kola, balanjan 9 pcr-rflp na june ahangar-kola, noukandeh 14 sequencing kc414872 noor june banafsheh, mianrood 12 sequencing kc414877 babol june markazi kati tayebi, babolkenar 11 sequencing kc414871 july mansour-kandeh, feyziyeh 8 pcr-rflp na babolsar june sorkh-dasht, bahmanmir 17 pcr-rflp na location (township) date 2011 site no. further identification method genbank accession number sari june jafar-abad, banaft 15 pcr-rflp na july taher-abad, roodpeye-shomali 16 sequencing kc414879 fereydoon-kenar june shahrae-daryasar, barikrood 10 sequencing kc414880 juybar june seraj-mahaleh, siahrood 11 pcr-rflp na neka june ghaleh-sare-olya, peyrajeh 12 pcr-rflp na behshahr june abbas-abad, kouhestan 11 sequencing kc414876 gelogah june mosayeb mahaleh, tooska, spring 16 sequencing kc414870 shirgah june kati-lateh 14 pcr-rflp na pool-sefid july pool-sefid 10 sequencing kc414881 zirab july zirab 12 pcr-rflp na alasht july larzaneh 11 sequencing kc414882 noushahr july kajour, khachak 9 pcr-rflp na chalous july namak abrood, kalarestake-gharbi 8 sequencing kc414869 tonekaboun july kelarabad, yalbandan, 10 sequencing kc414868 ramsar july janate-roodbar, grasma-sar 8 sequencing kc414883 abbas-abad july kelar-abad 11 pcr-rflp na royan aug. noor 10 sequencing kc414867 discussion to our knowledge, this is the pioneer molecular analysis at population level aiming at identifying paederus beetles in literature. based on the results of present study, the paederus spp population in mazandaran province in the caspian sea shore almost exclusively consists of p. fuscipes. in the other hand, previous studies (22, 41, 42) reported the presence of five more species including p. kalalovae, p. littoralis, p. balcanicus, p. riparius, and p. balachowskyi (synonym of p. mesopotamicus) in the province. this discrepancy may be due to the collection method we used in this study. the validity of our findings requires a thorough process from design to using various sample collection methods. however, the present study http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 100 http://jad.tums.ac.ir published online: march 31, 2023 failed to consolidate presence of other five species in the province. thus, p. fuscipes can be regarded as the main local causative agent of linear dermatitis. it is worth mentioning that p. kalalovae (one of the five species mentioned above) and p. fuscipes is treated as synonym species in the modern systematics of staphylinidae (45, 46). paederus fuscipes has been found in north, centre and south of iran (22, 40, 47‒54). it has also an extensive geographical dispersal globally in the old-world countries (20, 55, 56). species classification of the rove beetles is mostly dependent on the examination of the genitalia and demands a distinguished level of proficiency particularly for doubtful species. this results in struggle to identify and confines their examination, management, and control. in current study the its2 of rdna region was used for species identification of the rove beetle. this locus is a recognized powerful molecular indicator for species diagnostic assay. it is proven that inter-specific variation in this moderately fast evolving region is higher than intraspecific sequence variation (38, 57‒60). further studies are now necessary to clarify the paederus species composition of in the country using type specimens or morphologically distinguished species. nikbakhtzadeh et al. (2012) reported 14 subspecies and species of the genus paederus, belonged to five subgenera in the country (22). it is also suggested to examine sequence variation of its2 region or other genes such as mitochondrial dna cytochrome oxidase subunit one (mtdna coi), 28s, topoisomerase i, and wingless, in paederus spp to provide a molecular key for distinguishing of the iranian paederus species. there is a substantial report on application of coi in species identification, systematics, and population genetics of beetles in the literature (32, 36, 57, 61, 62). there are a scarce existing coi sequence data of p. ruficollis (jx416589), p. littoralis (ans: dq155980; jx416591), p. fuscipes (dq156010), and p. riparius (jx 416588) in genbank database. species identification can be performed by using rflp outlines on pcr amplicons or developing speciesspecific primers to supply species-specific band/ s. bazrafkan et al. (2016) demonstrated that molecular typing using mtdna coi gene followed by rflp was useful to differentiate between two species p. littoralis (=syn: p. lenkoranus, p. ilsae) and p. fuscipes, and recommended pcr-rflp of mtdna coi for distinguishing of other paederus species, which morphologically are identical or very challenging to be distinguished (63). conclusion the composition of the local paederus population in mazandaran province in north of iran consist typically of p. fuscipes and seems to be the main source of linear dermatitis in the region. according to our estimation, this dermatitis is probably seen in almost every place of mp at caspian sea coast and in adjacent regions during hot and humid summer months, it is confused with various dermatoses thus detailed research must be done. due to difficulties and doubtful morphological identification of paederus species, more detailed morphological and molecular systematic and faunal investigations need to be done in the future to determine the paederus fauna of mp and iran more accurately, and the number of species confirmed. acknowledgements this work was supported financially by tehran university of medical sciences, iran. ethical considerations this study was performed based on tehran university of medical sciences (tums) ethical committee guideline. http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/nuccore/jx416589.1 http://www.ncbi.nlm.nih.gov/nuccore/jx416591.1 http://www.ncbi.nlm.nih.gov/nuccore/jx416588.1 http://www.ncbi.nlm.nih.gov/nuccore/jx416588.1 j arthropod-borne dis, march 2023, 17(1): 94–104 a heydari et al.: molecular characterization of … 101 http://jad.tums.ac.ir published online: march 31, 2023 conflict of interest statement the authors declare there is no conflict of interests. references 1. tokareva a, koszela k, ferreira vs, yamamoto s, żyła d (2023) the oldest case of paedomorphosis in rove beetles and description of a new genus of paederinae from cretaceous amber (coleoptera: 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accepted 27 july 2013) abstract background: there is a little or no data available on the natural babesia bovis (b. bovis) infection in water buffaloes (bubalus bubalis) comparing to the available one for cattle. this study was conducted to investigate the natural b. bovis infection in water buffaloes in comparison to crossbred cattle under field conditions in egypt. methods: a total of 35 buffaloes and cattle were clinically and laboratory investigated from march to june 2008. twenty-nine buffaloes and cattle out of 35 were naturally infected with b. bovis and showed signs of bovine babesiosis. three cows and three buffaloes showed no clinical signs and were free from external, internal, and blood parasites served as control group. results: babesia bovis-infected cattle showed typical signs of bovine babesiosis while b. bovis-infected buffaloes showed a milder form (less severe) of the clinical signs. advanced cases of cattle showed dark brown to dark red (coffee-color) urine, hemoglobinuria and nervous manifestations while these manifestations were not detected in the infected buffaloes. hematological changes in both species however, these changes were less significant in buffaloes than those reported in cattle. conclusion: this paper documents the first description of natural b. bovis infection in water buffaloes which were found to be more likely to be tolerant than cattle to the natural clinical infection with b. bovis and its subsequent haematological changes. our finding may lead to a better understanding of the disease pattern of b. bovis infection under field conditions in buffaloes. keywords: babesia bovis, natural infection, water buffaloes, clinical signs, hematology introduction bovine babesiosis is economically the most important tick-borne disease of cattle worldwide including areas of australia, africa, south and central america (bock et al. 2004). in addition, the united states is continuously under threat of reintroduction of the vector and the disease (bock et al. 2004). under natural conditions, babesia bovis transmitted by the tick rhipicephalus microplus, although transmission may occur by other tick species (papadopoulos et al. 1996). the life cycle of b. bovis has two phases. in the vertebrate host they multiply by merogony in erythrocytes while in ticks by sporogony (susan and asa 1999). the disease is the most prevalent in tropical and subtropical countries, affecting cattle industries causing a major economic impact worldwide (böse et al. 1995, nayel et al. 2012). costs due to babesiosis are the results of high mortality, ill-thrift, abortions, loss of milk/meat production and draft power and from control measures such as acaricide treatments, purchase of vaccines and therapeutics (bock et al. 2004). it was estimated that losses and control of babesiosis and anaplasmosis *corresponding author: dr yasser mahmmod, e-mail: yasser@sund.ku.dk, yasserpcr@gmail.com http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 1–9 y mahmmod: natural babesia bovis… 2 in kenya, zimbabwe, tanzania, south africa, china, india, indonesia and philippines cost 5.1, 5.4, 6.8, 21.6, 19.4, 57.2, 3.1 and 0.6 million us dollars annually, respectively (bock et al. 2004). kaufmann (1996) reported that the mortality rates in cattle infected by b. bovis without treatment could reach 70– 80%. the diagnosis of ruminant babesiosis is generally based upon the microscopic examination of giemsa-stained blood smears and clinical signs in acute cases. previous studies provide information on the relative susceptibility of various breeds of cattle to babesia infection (bock et al. 1997). in egypt, bovine babesiosis is caused mainly by b. bigemina and b. bovis and considered as the most important and endemic parasitic disease affecting cattle (nagati 1947, adham et al. 2009). bovine babesiosis has a significant impact on meat and milk production and consequently, on livestock management (adham et al. 2009). the rapidly changing patterns of demand for cattle and its products point to cattle production being an important and increasing component of the egyptian agriculture economy which required improving cattle health. egyptians farmers cross between the holstein-friesian breed and a native local breed known as baladi cattle breed (bos taurus) to improve production and disease resistance. water buffaloes (bubalus bubalis) represent an important source of various human needs, such as meat, horns, hides, milk and milk products, leather, land plowing, and transportation of people and crops (somparn et al. 2004). due to the fact that water buffaloes are raised together with cattle, among which bovine babesiosis is highly prevalent (iseki et al. 2010), they might be potential carriers for babesia parasites. babesia bovis infection was experimentally investigated in splenectomised buffaloes (mahmoud and abou-zeina 2008). yao et al. (1997) reported the clinical findings on buffaloes after experimental infection with cryopreserved b. bovis parasites. however, efforts to furnish information about natural infection with b. bovis in water buffaloes as well as crossbred cattle are necessary for better understanding of disease pattern under uncontrolled field conditions and subsequently, implementation the suitable policy for treatment and control. the objective of the present study was to investigate the natural b. bovis infection in water buffaloes in comparison to crossbred cattle under field conditions in egypt. materials and methods animals and sampling blood samples were collected from the jugular vein into edta-containing tubes from 35 animals (23 cattle and 12 buffaloes) of both sexes and aged 2–5 years, and were originating from different villages: el-aslogy, shobk basta and tel basta around zagazig city, sharkia province. these animals were divided into 2 groups, the field-exposed (diseased) group comprised of 20 cattle and 9 water buffaloes which was examined at the veterinary teaching hospital, faculty of veterinary medicine, zagazig university, during the period from march to june 2008 and resulted to have persistent fever, anemia and anorexia. the control group (3 cows and 3 buffaloes) was carefully examined clinically and parasitologically and found healthy and free from external, internal, and hemoparasites. the common available foods for animals under the present study were mainly consisted of barseem (trifolium alexandrinum), rice or wheat straw and concentrate mixture (1–2 kg/head/day). crossbred cattle were resulted from crossbreeding between the imported holstein-friesian breed cattle and egyptian baladi cattle breed (bos taurus). samples collection, handling and examination of cattle and buffaloes under the current study were done after approval of animals’ owners. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 1–9 y mahmmod: natural babesia bovis… 3 clinical examination animals were subjected to clinical and hematological examinations at veterinary teaching hospital, faculty of veterinary medicine, zagazig university. the filed-exposed group showed various degrees of bovine babesiosis such as high fever (>40 °c), anorexia, hemoglobinuria (bloody urine), anemia, and jaundice. they were also infested with ticks to various degrees. the control group was examined thoroughly for presence of any abnormal clinical changes and external parasites, and was thoroughly examined by different laboratory techniques such as direct smear, flotation, sedimentation and barmen’s techniques and blood film to confirm the absence of any internal parasites and/or hemoparasites (rosenberger 1990). microscopic examination thin blood films were prepared immediately after taking the blood samples directly from the ear vein in the field to allow these smears to dry by air then fixed by using methanol for about 3–5min, allow them to dry by air after fixation step then stained with giemsa stain diluted at 8% with distilled water for about 30–45min. they were dried by air and examined on olympus microscope using oil immersion lens at x1000 magnification (kelly 1984). blood film was examined for b. bovis at 1/4–1/2 inch from the end of the film by visually scanning from one side of the film to other (cross–sectional method) to give constant and representative examination. each blood film and at least twenty microscopic fields of each slide were examined twice before being considered negative. hemogram parameters approximately 5ml of blood was taken from the jugular vein of all animals with a syringe containing edta. the blood samples were subjected to hematological parameters analysis (schalm et al. 1975, coles 1986), that is, red blood cell (rbc) and white blood cell (wbc) counts were made with improved neubauer hacmocytometers, hemoglobin concentration (hb) by sahli's haemoglobinometer and packed cell volume (pcv%) by microhematocrit tubes. differential wbc counts were performed on thin blood smears by the battlement technique. statistical analysis the obtained data were statistical analyzed by mean of computer based statistical program, spss (borenstein et al. 1997). data were analyzed using student’s t-test to compare the mean data between groups. the results obtained were expressed as mean ±sd. differences were considered statistically significant based on p< 0.05. results clinical findings cattle infected with b. bovis showed typical clinical signs of babesiosis, table 1. briefly, highly rise in body temperature (40– 41.5 °c), conjunctival and vaginal mucous membranes were anemic and the clinical severity was ranged from paleness in mild cases to severe yellow discoloration (icterus) in more progressive cases, dark brown to dark red (coffee-color) urine, hemoglobinuria was common sign in cattle with severe clinical manifestation and accelerated heart and respiratory rates. some cases showed nervous manifestations in advanced stages such as incoordination and head pressing. various degrees of tick infestations were present around groins, horns, inter-mandibular space, and ears. water buffaloes infected with b. bovis showed a milder form (less severe) of clinical signs of infection in comparison to the clinical signs appeared on b. bovis-infected cattle. these clinical signs were in the form of highly rise in body temperature (40–41.5 http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 1–9 y mahmmod: natural babesia bovis… 4 °c), and conjunctival and vaginal mucous membranes were mainly anemic and pale in color and loss of body condition. icterus, hemoglobinuria and nervous manifestations were not detected/ observed in the affected buffaloes. hematological findings giemsa-stained blood smears from b. bovis infected animals showed intra-erythrocytic piroplasms of b. bovis that were in the form of pyriform or pear-shaped, fig. 1. blood smears from b. bovis progressive cases of cattle showed severe hemolytic anemia with abnormalities in cell size (anisocytosis) and cell shape (poikilocytosis) of erythrocytes, fig. 2. giemsa-stained blood smears from b. bovis uninfected cattle and buffaloes showed no parasites or erythrocytic changes. the control group resulted to be healthy on clinical and laboratory examination and free from external, internal and hemoparasites. the mean values of rbcs, hemoglobin amount, pcv %, wbcs, and differential leucocytic count are listed in table 2. briefly, the important findings can be summarized as follows; there is a clear significant difference in the haematological parameters between b. bovis-infected buffaloes and b. bovis-infected cattle in comparison to control group at p-value (≤ 0.01) and (≤ 0.001), respectively. haematological changes for b. bovis-infected buffaloes were less significant than their changes for b. bovis-infected cattle. table 1. clinical findings of babaesia bovis (b. bovis)-infected cattle and buffaloes in comparison to control group under natural field conditions parameters field-exposed animals (n= 29) control group (n= 6) b. bovis infected cattle (n= 20) b. bovis infected buffloes (n= 9) temperature (°c) 40.8 (40.3–41.4) 40.6 (40.2–41.1) 38.5 (38.1–38.8) appetite anorexia anorexia normal haemoglobinuria present in advanced acute cases no haemoglobinuria straw yellow mucus membranes varied from paleness in mild cases to severe yellow discoloration in progressive ones paleness and anemic of mucous membranes bright red, moist and no lesions icterus marked and characteristic no no nervous signs incoordination, head pressing no no body condition thin/emaciation and anemic weak to moderate good lymph nodes normal normal normal respiration exaggerated/ accelerated exaggerated/ accelerated normal giemsa-stained blood film intraerythrocytic piroplasms of b. bovis in the form of pyriform or pear-shaped advanced cases showed severe haemolytic anemia with anisocytosis and poikilocytosis intraerythrocytic piroplasms of b. bovis in the form of pyriform or pear-shaped no parasites, normal rbcs http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 1–9 y mahmmod: natural babesia bovis… 5 table 2. hemogram findings of b. bovis-infected cattle and buffaloes in comparison to control group under natural field conditions (mean ±s.e) parameters field-exposed animals (n= 29) control (n= 6)b. bovis infected buffaloes (n= 9) b. bovis infected cattle (n= 20) rbcs 1 (106 /ml) 3.4±0.1 a ** 2.6±0.2 b *** 6.9±0.9 c hb 2 (g/dl) 7.3±0.2 a ** 5.5±0.3 b *** 12.7±1.2 c pcv 3 (%) 24.6±1.1 a ** 18.1±1.5 b *** 36±1.3 c wbcs 4 (103 mul-1) 7.3±0.2 a* 7.2±0.5 a * 9.3±0.5 b lymphocytes (103 mul1) 4.1±0.1 a* 3.8±0.5 a * 4.9±0.8 b monocytes (103 mul-1) 0.46±0.07 a 0.44±0.9 a 0.46±0.01 a neutrophils (103 mul-1) 3.1±0.4 a 2.9±0.5 a 3.2±0.6 a eosinophils (103 mul-1) 0.2±0.01 a 0.2±0.02 a 0.2±0.04 a basophils (103 mul-1) 0.02±0.001 a 0.02±0.002 a 0.02±0.003 a *values with different superscripts are significantly different from each other (*p≤ 0.05, **p≤ 0.01, ***p≤ 0.001). 1 rbcs= red blood cells, 2 hb= hemoglobin, 3 pcv= packed cell volume, 4 wbcs= white blood cells fig. 1. giemsa-stained blood smear showing intraerythrocytic pyriform (pear-shape) of babesia bovis in pairs fig. 2. giemsa-stained blood smear showing severe hemolytic anemia with abnormalities in cell size (anisocytosis) and cell shape (poikilocytosis) of erythrocytes from advanced cases of cattle naturally infected with babesia bovis discussion babesia bovis is one of the most important blood parasites affecting cattle and buffaloes and in its acute forms, it lowers the productive performance of the affected animals (talkhan et al. 2010, ziapour et al. 2011). most of the previous studies described the clinical findings of b. bovis infection in cattle of different breeds. to the best of our knowledge, this is the first study which investigating thoroughly the clinical and hematological pictures of natural b. bovis infection in water buffaloes under uncontrolled field conditions. the reported clinical findings of b. bovis infection in cattle come in agreement with what was previously described by brown and torres (2008), georgi et al. (1990) and kaufmann (1996). the demonstrated high fever could be attributed as response to the effect of unspecific toxic substances produced during the metabolism of babesia on thermoregulatory (radostits et al. 2000). it was notable that water buffaloes identified as b. bovis-infected showed a milder fgdfdd http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 1–9 y mahmmod: natural babesia bovis… 6 form (less severe) of clinical signs of b. bovis infection in comparison to the clinical signs appeared on b. bovis-infected cattle. this variation was represented in appearance of icterus, hemoglobinuria and nervous manifestations in clinically infected cattle while they were not reported in infected buffaloes. this finding may propose that buffaloes may have more tolerance to clinical infection with b. bovis than cattle. tolerance means that the host is infected by the pathogen, but suffers little adverse effect (fao 2007). it could be argued that buffaloes may have acquired natural immunity/tolerance to some extent against b. bovis infection. genetic variations within the host between cattle and buffaloes may explain the variation in their susceptibility. this finding is inconsistent with the experimental findings by yao et al. (1997) who found that b. bovis produces acute, often fatal, infections in buffaloes. the proportion of buffaloes identified as b. bovis-infected was (31.1%) while the proportion of cattle identified as b. bovis-infected was (68.9%) within the same period of the study. this finding could suggest that water buffalos have more tendencies to be carriers (apparently healthy) than showing clinical manifestations. this finding supported by ferreri et al. (2008) who noticed that water buffaloes seem to be unapparent carriers of the parasite. the marked anemia and hemoglobinuria in cattle could be attributed to the severe haemolytic process associated the presence of babesia piroplams inside the erythrocytes and destruction of large numbers of these erythrocytes by the parasite resulting in hemoglobinaemia and consequently hemoglobinuria (georgi et al. 1990, fujinaga 1981), the physical effect of parasite multiplication (wright 1981), the increase of phagocytosis of erythrocytes by activated macrophages (shoda et al. 2000, court et al. 2001), the production of an anti-erythrocyte antibody (goe′s et al. 2007) and the increase in the erythrocytic membrane permeability (alkhalil et al. 2007). hematological findings showed a significant decrease in the rbcs, wbcs counts, hb concentrations and pcv% in the b. bovis-infected animals in comparison to the control group, these observations were similar to what were reported by col and uslu (2007) and durrani et al. (2006). it seems that the immune response to the babesial antigen causes a significant lymphocytosis. this comes in agreement with what was described previously by schalm (2000). hematological changes resulted from b. bovis infection in buffaloes are less significant than the hematological changes of b. bovis infection in cattle, table 2. this finding reflected clinically on b. bovis-infected buffaloes which showed a milder form of clinical picture of b. bovis infection than b. bovis-infected cattle. the hemolytic anemia due to the breakdown of erythrocytes membranes leading to release of hemoglobin and manifested by the presence of free hemoglobin resulting in the discoloration of the plasma (sowemimo-coker 2002). extensive lipid peroxidation in biological membranes causes disturbances of its structural integrity, loss of fluidity, decrease in membrane potential, and increased permeability to ions (gutteridge 1995). these changes lead to rupture of the membrane and release of cell contents (halliwell and chirico 1993). babesia parasite (alkhalil et al. 2007), and b. bovis (aikawa et al. 1985) dramatically alters the permeability of its host erythrocytes to various organic solutes. b. bovis infection is associated with impairment of blood parameters and subsequently, hematological examination may be a useful tool for confirmation the clinical diagnosis of bovine babesiosis. conclusion water buffaloes showed a milder form of b. bovis infection than cattle suggesting that http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 1–9 y mahmmod: natural babesia bovis… 7 buffaloes may be more tolerant to the clinical infection with b. bovis than cattle. hematological changes as a result of b. bovis infection in buffaloes are less significant than hematological changes of b. bovis infection in cattle. b. bovis infection might be associated with severe clinical and hematological changes especially in cattle, which might be of bad prognosis. with respect to the study population, future studies should consider a larger sample size for cattle and buffaloes for the robustness of the findings. recent molecular techniques such as pcr showed many advantages with regard to the sensitivity and specificity for detection and surveillance of hemoparasites (nayel et al. 2012, hüe et al. 2013, ybañez et al. 2013). hence, it would be advisable for future studies to use such techniques for investigating the b. bovis infection in buffaloes. acknowledgements this study was accomplished and funded by faculty of veterinary medicine, zagazig university, egypt. author is grateful to prof farouk elbalkemy and dr ahmed abdelaal, department of animal medicine, faculty of veterinary medicine, zagazig university, for their support. special thanks for prof ahmed badwy, department of parasitology, faculty of veterinary medicine, zagazig university for his help in the lab examination. the author of this paper has not a financial or personal 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anophelism in … http://jad.tums.ac.ir published online: august 31, 2013 original article anophelism in a former malaria area of northeastern spain *rubén bueno-marí, ricardo jiménez-peydró laboratorio de entomología y control de plagas, instituto cavanilles de biodiversidad y biología evolutiva, universitat de valència-estudi general, valencia, spain (received 6 oct 2012; accepted 14 may 2013) abstract background: a field study on diversity and distribution of anophelines currently present in a past endemic malaria area of spain was carried out in order to identify possible risk areas of local disease transmission. methods: multiple larval sites were sampled from june to october of 2011 in the region of somontano de barbastro (northeastern spain). the sampling effort was fixed at 10 minutes which included the active search for larvae in each biotope visited. results: a total of 237 larval specimens belonging to four anopheles species (anopheles atroparvus, an. claviger, an. maculipennis and an. petragnani) were collected and identified. conclusions: malaria receptivity in the study area is high, especially in the area of cinca river valley, due to the abundance of breeding sites of an. atroparvus very close to human settlements. although current socio-economic conditions in spain reduce possibilities of re-emergence of malaria transmission, it is evident that certain entomological and epidemiological vigilance must be maintained and even increased in the context of current processes of climate change and globalization. keywords: malaria, mosquitoes, entomological surveillance, vector borne diseases, spain introduction malaria was a widespread disease in europe until the second half of 20th century. the parasitosis was particularly devastating between xvi and xix centuries in southern europe due to the boom of irrigation techniques based on long flooding periods, mainly rice cultivation. although the disease is currently considered eradicated from europe, it must be noted that cycles of malaria transmission still are relatively common in countries like georgia, azerbaijan, kyrgyzstan, tajikistan, uzbekistan and turkey (who 2010). moreover, the increasing of imported malaria cases in last decades together with the high presence of anophelines in many southern europe regions (romi et al. 1997, ponçon et al. 2007, bueno marí and jiménez peydró 2012) has enabled the appearance of few autochthonous or probable autochthonous malaria cases, as recently has occurred in countries like italy (baldari et al. 1998, romi et al. 2012), greece (kampen et al. 2002), france (doudier et al. 2007) or spain (santa-olalla peralta et al. 2010). from the fifteen anopheles species reported in spain (bueno marí et al. 2012a), only two were considered primary malaria vectors: an. atroparvus van thiel, 1927 and an. labranchiae falleroni, 1926. an. labranchiae was found to be abundant in a restricted area of the contiguous alicante and murcia provinces (southeastern spain) in 1946 (clavero and romeo viamonte 1948), but had disappeared by 1973 (blázquez and de zulueta 1980) probably due to abandonment of rice cultivation in this area (eritja et al. 2000). on the other hand, an. atroparvus still is well distributed in different regions of ibe*corresponding author: dr rubén bueno-marí, email: ruben.bueno@uv.es 147 j arthropod-borne dis, december 2013, 7(2): 147–153 r bueno-marí and r jiménez-peydró: anophelism in … http://jad.tums.ac.ir published online: august 31, 2013 rian peninsula (encinas grandes 1982, bueno marí 2011). anopheles labranchiae and an. atroparvus are considered the most important eastern and western mediterranean malaria vectors respectively. the aim of this study was to analyze the diversity and distribution of anophelines currently present in a past endemic malaria area of northeastern spain in order to identify possible risk areas of local disease transmission. materials and methods study area the study area selected was the somontano de barbastro region (n 42º02’47’’/ e 0º07’23’’), which is situated in huesca province (northeastern spain) (fig. 1). this region is a transition zone between high mountain area (pyrenees) and vast plain area (monegros) that is characterized approximately by 15 ºc of average annual temperature and 400–700 mm of annual rainfall. in this region malaria was a common disease during xviii century, even have been described several epidemic episodes as occurred between 1783 and 1785 with hundreds of deaths (nieto callén and bosch ferrer 1991). when malaria was present, two different areas could be distinguished in terms of disease morbidity and mortality: for one side lowland territories adjacent to cinca river where the anthroponosis was very intense and on the other side the rest of the region where malaria incidence was very low. the area of cinca river valley has been traditionally associated with various types of irrigated crops and the presence of an. atroparvus was supposed to be very frequent according to old registers of malaria incidence. the study area is also characterized by a mountainous region at north with several small rivers and streams, all of them tributaries of cinca river, and a dry region dedicated to the development of rainfed crops at south. sampling methods and species identification multiple larval sites were sampled using the standard dipping method (service 1993) from june to october of 2011. the sampling effort was fixed at 10 minutes which included the active search for larvae in each biotope visited (bueno marí 2010). for small larval habitats such as tree holes or small containers, the sampling was done by emptying or pipetting the contents for immature stages. tree holes are particularly interesting since an. plumbeus stephens, 1828 is a strictly dendrolimnic species frequent in the palearctic region and has been recognized as a minor vector for human malaria in europe since the beginning of the 20th century (shute 1954, krüger et al. 2001, bueno marí and jiménez peydró 2011a, schaffner et al. 2012). data were recorded from all identifiable aquatic environments across the three different regions (eastern region of cinca river valley, northern mountainous region and southern region of rainfed crops) of the study area. the sampling effort was fixed at 10 minutes, which included the active search for larvae in each biotope visited (bueno marí 2010). larval exemplars collected were identified at specific level according to the taxonomic criteria of schaffner et al. (2001) and bueno marí (2010). fig. 1. situation of study area 148 j arthropod-borne dis, december 2013, 7(2): 147–153 r bueno-marí and r jiménez-peydró: anophelism in … http://jad.tums.ac.ir published online: august 31, 2013 results a total of 237 anopheles larval specimens were collected and identified (table 1). four anophelines were recorded: an. atroparvus, an. claviger (meigen 1804), an. maculipennis meigen, 1818 and an. petragnani del vecchio, 1939. besides these anophelines collections, nine species of mosquitoes were also caught in the study area, namely culex hortensis ficalbi, 1889 (n= 23), cu.impudicus ficalbi, 1890 (n= 27), cu. modestus ficalbi, 1889 (n= 11), cu. mimeticus noè, 1899 (n= 67), cu. pipiens linnaeus, 1758 (n= 481), cu. territans walker, 1856 (n= 85), culiseta annulata (schrank 1776) (n= 5), cu. longiareolata (macquart 1838) (n= 133) and ochlerotatus caspius (pallas 1771) (n= 48). none mosquito exemplar was collected in tree holes. table 1. information about anopheline records species coordinates n coordinates w/e altitude (m) biotope region nº exemplars an. atroparvus 41°58' 12.6'' 0°10' 15.3''e 273 river margin cinca valley 6 an. atroparvus 41°58' 11.1'' 0°10' 12.4''e 266 river margin cinca valley 3 an. atroparvus 42°03' 54.3'' 0°12' 58.5''e 323 river margin cinca valley 13 an. atroparvus 42°07' 09.5'' 0°13' 47.1''e 355 river margin cinca valley 9 an. atroparvus 42°07' 10.8'' 0°13' 49.5''e 372 river margin cinca valley 7 an. atroparvus 42°11' 28.9'' 0°09' 14.6''e 568 river margin cinca valley 10 an. atroparvus 42°11' 11.4'' 0°09' 15.1''e 557 river margin cinca valley 11 an. atroparvus 42°08' 23.3'' 0°01' 31.3''e 447 river margin mountainous 5 an. atroparvus 42°08' 59.1'' 0°01' 40.7''e 463 river margin mountainous 14 an. atroparvus 42°15' 01.2'' 0°05' 13.2''w 637 river margin mountainous 8 an. atroparvus 42°10' 26.2'' 0°04' 26.5''w 628 container mountainous 2 an. atroparvus 41°56' 49.8'' 0°02' 05.2''w 409 container crops 5 an. atroparvus 41°55' 22.7'' 0°05' 37.8''w 348 irrigation channel crops 9 an. atroparvus 41°57' 42.2'' 0°04' 03.6''e 315 temporal puddle crops 7 an. atroparvus 41°56' 35.6'' 0°03' 36.6''e 301 rice field crops 45 an. atroparvus 41°56' 56.9'' 0°03' 42.8''e 306 temporal puddle crops 12 an. claviger 42°14' 09.9'' 0°02' 37.5''o 672 temporal puddle mountainous 2 an. maculipennis 42°03' 54.3'' 0°12' 58.5''e 323 river margin cinca valley 4 an. maculipennis 42°07' 10.8'' 0°13' 49.5''e 372 river margin cinca valley 2 an. maculipennis 42°04' 39.1'' 0°02' 14.6''e 375 river margin crops 9 an. maculipennis 42°08' 23.3'' 0°01' 31.3''e 447 river margin mountainous 6 an. maculipennis 42°08' 59.1'' 0°01' 40.7''e 463 river margin mountainous 5 an. maculipennis 42°09' 54.4'' 0°01' 36.3''e 479 river margin mountainous 7 an. maculipennis 42°15' 01.2'' 0°05' 13.2''o 637 river margin mountainous 3 an. maculipennis 42°08' 47.9'' 0°03' 34.2''o 565 river margin mountainous 6 an. petragnani 42°10' 28.1'' 0°03' 25.1''e 574 container mountainous 12 an. petragnani 42°09' 54.0'' 0°1' 59.1''o 372 container mountainous 6 an. petragnani 42°14' 09.9'' 0°02' 37.5''o 672 temporal puddle mountainous 9 149 j arthropod-borne dis, december 2013, 7(2): 147–153 r bueno-marí and r jiménez-peydró: anophelism in … http://jad.tums.ac.ir published online: august 31, 2013 discussion the presence of an. atroparvus in the cinca river valley has been confirmed decades after. the occurrence of an. atroparvus is particularly interesting not only all along cinca river, but also in irrigation channels and flooding areas such as small plots transformed in rice fields where high densities of an. atroparvus were found. it is important to note that in the neighboring region of monegros, where large territories are flooded artificially and used for rice cultivation, a probable autochthonous case of malaria was diagnosed in 2010 presumably transmitted by an. atroparvus (bueno mari et al. 2012b). this is probably the first indigenous case of the disease in spain since 1961. it is very difficult to compare the entomological data obtained in the study area with other neighboring regions, since current researchers about mosquito fauna in these regions are practically nonexistent. however, if we compare the epidemiological situation of the region of somontano de barbastro with the available information of other bordering territories such as the region of monegros, we can conclude that an. atroparvus densities are clearly higher in the second one, mainly because rice fields are strongly colonized by the species. despite both regions are characterized by high extensions of dry environments, the abundance of rice fields in monegros region makes this area more suitable the development of an. atroparvus. at phenological level, it is important to note that larval exemplars of an. atroparvus were collected during all the months of the study (june-october) which means that the species has a multivoltine cycle and overlapping of generations is quite frequent as has been suggested by other authors for northern spain areas (encinas grandes 1982). moreover, also following the criteria of encinas grandes (1982) it is likely than our last collections of october corresponds to future over wintering females, since the activity of the species has been traditionally circumscribed between april and september months for northern spain, although local climatology could obviously alter this situation. although it has been shown that adults of an. atroparvus can migrate distances close to 12 km in search of optimal hosts for feeding (kaufmann and briegel 2004), we must highlight that breeding sites of the species detected in our study have been mainly situated near from human settlements. from an epidemiological point of view, of course the proximity of vectors and humans is a very important factor in order to evaluate the possibilities of a disease outbreak. infectivity tests carried out on european populations of an. atroparvus showed that this species can transmit asian strains of plasmodium vivax (grassi and feletti 1890), but is refractory to african strains of pl. falciparum (welch 1897) (ramsdale and coluzzi 1975). however, more recent studies have shown the ability of an. atroparvus to generate oocysts of p. faliciparum (marchant et al. 1998), but not to complete the sporogony. despite an. atroparvus was the main responsible in the maintenance of malaria endemicity in much of europe during centuries, the vectorial role of the species was particularly interesting in northern countries due to its endophagic behavior and semiactive overwintering females (huldén et al. 2005). endemic northern malaria reached to 68°n latitude in europe during the 19th century, where the summer mean temperature only irregularly exceeded the lower limit of 16 °c needed for sporogony of p. vivax (garnham 1988). this temperature conditions should have caused that malaria transmission have mainly occurred in indoor conditions due to transmission of sporozoites throughout the winter by semiactive hibernating mosquitoes (huldén et al. 2005), since it is well known that in 150 j arthropod-borne dis, december 2013, 7(2): 147–153 r bueno-marí and r jiménez-peydró: anophelism in … http://jad.tums.ac.ir published online: august 31, 2013 warm conditions the overwintering females of an. atroparvus can take several blood meals (encinas grandes 1982). therefore northern malaria existed in a cold climate by means of summer dormancy of p. vivax hypnozoites in addition to the indoor feeding activity of overwintering females of an. atroparvus and also probably other species of maculipennis complex (huldén et al. 2005, bueno marí and jiménez peydró 2011b). with regards to the collections of an. claviger and an. maculipennis, their aquatic stages were mainly found in fresh or slightly brackish water in the mountainous area away from anthropised environments. although these bioecological aspects, together with their zoophilic tendency, indicate a minor role in malaria transmission, it must be noted that both species have been related with several vivax malaria outbreaks in some eastern mediterranean countries (gramiccia 1956, coluzzi et al. 1964, schaffner et al. 2001). respect to an. petragnani, the species was only found in small biotopes distributed in the mountainous area. an. petragnani is a strictly zoophilic species very frequent in low anthropised environments of southern europe (bueno marí and jiménez peydró 2011c). consequently, an. petragnani is not considered as an important malaria vector. this information coincides with an absence of data about the vectorial status of the species in scientific literature. ultimately we can conclude that malaria receptivity is high, especially in the area of cinca river valley, due to the abundance of breeding sites of an. atroparvus very close to human settlements. although an. atroparvus is also present in the other areas, the shortage of suitable larval biotopes for the species in the dry area of crops and the low percentage of humans that lives and can serve consequently as a regular hosts for an. atroparvus in the mountainous area, provokes that the epidemiological interest of these areas would be less in comparison with the area of cinca river valley. all this information fit perfectly with old descriptions of malaria outbreaks in the region (nieto callén and bosch ferrer 1991). in any case we must highlight that malariogenic potential of the study area is low due to the low vulnerabity of the country. vulnerability is a key factor to assess about possible re-emergence of disease and can be defined by the number of gametocyte carriers (malaria patients) during the suitable period for malaria transmission. in spain the imported malaria cases (mainly tourists and immigrants arrived form endemic regions) reported yearly by national ministry of health are around 400 cases. moreover, malaria is a notifiable disease in spain, so these patients are quickly identified and treated in order to minimize the possible dispersion of parasite by anophelines. despite current socio-economic conditions in spain which reduce possibilities of re-emergence of malaria transmission (bueno marí and jiménez peydró 2008), it is evident that certain entomological and epidemiological vigilance must be maintained and even increased in the context of current processes of climate change and globalization. acknowledgements dr rubén bueno marí wishes to thank to centro de estudios del somontano de barbastro (cesb) and culture commission of barbastro council for the grant of the xxii research award lópez novoa to him because this was a key factor to carry out this study. moreover authors want to acknowledge to instituto aragonés de gestión ambiental (inaga) for the grant of insects capture permissions in natural enclaves of the study area. finally we want to recognize the important task of alberto bernués-bañeres and francisco alberto chordá-olmos (phd students) in the field part of the study. the authors declare that there is no conflict of interest. 151 j arthropod-borne dis, december 2013, 7(2): 147–153 r bueno-marí and r jiménez-peydró: anophelism in … http://jad.tums.ac.ir published online: august 31, 2013 references baldari m, tamburro a, sabatinelli g, romi r, severini c, cuccagna p, fiorilli g, allegri mp, buriani c, toti m (1998) introduced malaria in maremma, italy, decades after eradication. the lancet. 351(9111): 1246–1248. blázquez j, zulueta j (1980) the disappearance of anopheles labranchiae from spain. parassitol. 22(1–2): 161–163. bueno marí r (2010) bioecología, diversidad e interés epidemiológico de los culícidos mediterráneos (diptera: culicidae). servei de publicacions de la universitat de valència, valencia. bueno marí r (2011) el anofelismo de la comunidad valenciana: un ejemplo de estudio del potencial malariogénico de españa. bol asoc esp ent. 35(1–2): 47–83. bueno marí r, jiménez 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(1954) indigenous p. vivax malaria in london believed to have been transmitted by anopheles plumbeus. mon bull minist health publ health lab serv. 13: 48–51. who (2010) centralized information system for infectious diseases, malaria, denmark. 153 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 original article wash resistance and bioefficacy of alpha-cypermethrin long lasting impregnated nets (llin-interceptor®) against anopheles stephensi using tunnel test hassan vatandoost 1, hossein mamivandpoor 1, *mohammad reza abai 1, mansoreh shayeghi 1, fatemeh rafi 1, ahmad raeisi 2, fatemeh nikpoor 2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of malaria, cdc, ministry of health and medical education, tehran, iran (received 23 nov 2011; accepted 2 feb 2013) abstract background: the long-lasing insecticide impregnated nets (llins) is considered as an effective tools for malaria vector control. the aim of this study was to evaluate the residual efficacy of alpha-cypermethrin long lasting impregnated nets (llin-interceptor®) against anopheles stephensi using tunnel test. methods: the wash-resistance of interceptor® nets were assessed under laboratory conditions using tunnel test. females of an. stephensi were released into the tunnel and then they were provided blood meals from guinea pigs. bed nets were washed according to the standard procedure up to 20 times. the bioefficacy indicators such as inhibition of bloodmeal from experimental animal, knockdown, irritancy rate, survival rate, entry index and mortality were calculated. results: it induced 90–100% mortalities in the population of an. stephensi up to 15 washes. the kt50 values reduced from 73.47 to 26.30 minutes in unwashed in comparison to one washed, respectively. the mean of mortality rate of blood-feeding inhibition and entry indexes was reached to 91.6%±2.8, 87.0±3.4 and 24.9±2.8 respectively after 20 washing. conclusion: this net could provide a good personal protection against malaria vectors and could induce relatively high mortality, inhibit the blood-feeding as well as reduce the entry rates of female mosquitoes even after several washes. keywords: alpha-cypermethrin, llins, anopheles stephensi, tunnel test introduction according to the recent publication of who (2012) bednet impregnated with pyrethroids especially long lasting one is highly recommended for control of malaria. this intervention also is carrying out in iran. the southeastern part of the iran remains as malaria-endemic area. annual parasite incidence (api) was reported as 7 per 1,000 populations. the disease mainly occurs in three south-eastern provinces. in 2002, 68% of positive cases belonged to the hormozghan, sistan and baluchistan and kerman provinces whereas in 2009 it increased to 90% (raeisi et al. 2008). there are several interventions to control malaria such as diagnosis and effective treatment of malaria cases and vector control, including the use of insecticide treated nets (itns), and indoor residual spraying (irs) (abai et al. 2008, raeisi et al. 2008). an important innovation touring the past decade is the widespread introduction of insecticidetreated mosquito nets (itns and llins) for protection against malaria transmission. *corresponding author: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir 31 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 various methods for mosquito control have been suggested by who. pyrethroids are recommended for impregnation of mosquito nets due to high efficacy at low dosage, rapid knock-down effect and low mammalian toxicity (who 2007). incorporation of pyrethroids into conventional netting fibers (polyester, polypropylene) is a new method for preparing the llins. α-cypermethrin is a cyano-group pyrethroid recommended for impregnation of bednet modeled on the natural defensive compound produced by chrysanthemums. the main challenge was to incorporate α-cypermethrin, in a polymer so that it would migrate to the surface at a steady rate over several years (duchon et al. 2005). the who pesticide evaluation scheme (whopes) recommended five long-lasting insecticidal nets (llins) for the prevention of malaria: olyset®, permanet® 2.0, netprotect®, duranet® and interceptor® (who 2005). comparative efficacy of itns and llins had been carried out in order to optimize the different bioassay tests and calibration of technique in iran (vatandoost et al. 2006 a,b,c, kayedi et al. 2008, rafinejad et al. 2008, vatandoost et al. 2009a,b). the objective of this study was to determine the wash resistance of interceptor® (llins) based on employing bioefficacy indicators using tunnel test device against the main malaria vector, anopheles stephensi. materials and methods long lasting insecticidal mosquito net (llins) alpha-cypermethrin, interceptor® was provided by basf chemical company, ludwigshafen, germany. this polyester mosquito net has 100 deniers. alpha-cypermethrin at the dosage of 200 mg a.i./m2 was mixed in a resin coating the netting fibers. the insecticide is progressively released from the resin and the net retains efficacy after repeated washings. the batches of 25×25 cm nets were provided for using in tunnel test between small compartment and large compartment. an untreated similar net type used for control. washing procedure net samples (25×25 cm) were individually introduced into one liter beakers containing 0.5 liter deionized water, with 2 g/l soap savon de marseille “le chat®”. the soap was added and fully dissolved just before introduction of net samples. beakers were immediately placed into a water bath at 30 ºc and were shacked for 10 min at 155 rounds per min. the machine is the same as recommend by who (2005). the samples were then removed and rinsed twice for 10 minutes in clean, deionized water in the same shaking conditions. nets were dried at room temperature and stored at 30 ºc in the dark. a piece of 25×25cm was used for tunnel test prior and after every washing (eg, 0x, 1x, 2x, 3x, 6x, 8x, 15x, 20x ). this is following of who standard washing procedure (who 2005). the net was washed and dried once a week. the netting sample had nine holes with 1 cm diameter and the nets were fixed on a cardboard frame. the nets were replicated as appropriate number. mosquitoes the beech strain of an. stephensi which is susceptible to insecticides is used. the breeding was carried out at 29±1 ºc and 60±10% relative humidity at 16:8 h light-dark photoperiod. the larvae were fed with enriched wheat germ and the adults received a 10% sucrose solution. guinea pigs were used for blood-feeding of female mosquitoes. at each replicate of the biological efficacy of the nets, a total of 100 sugar-fed of 5–8 days females were used. the sucrose solution was picked up from the cage, 12 hour before starting the experiments. the female mosquitoes were introduced into the releasing chamber (big compartment) of the tunnel 32 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 at 18:00 and then results were observed at 09:00 in the following day in the morning. the experiments were carried out at 28.0±1 ºc and 55.0±10% relative humidity. tunnel test laboratory tests were carried out in cubic glass tunnel device which simulated the experimental hut conditions, as described by elissa et al. 1995, chandre et al. 2000, hougard et al. 2003, corbel et al. 2004, who 2005, duchon et al. 2007. the tunnel dimension was 25 cm in width, 25 cm in height and 60 cm in length. the width, height and length were respectively 25, 25 and 40 cm fitted with a metal cubic cage covered with untreated polyester netting for releasing of mosquitoes. the small compartment, 25x 25x 20 cm was designed for placing guinea pigs. the mosquitoes can entered into small compartment passing the 9 circulars of 1 cm diameter holes on both treated and untreated nets (fig. 1). one separate tunnel with untreated netting was used as a control. mosquitoes were free to fly in the tunnel. they also had a free choice contact with torn netting prior passing throughout the hole to reach the bait. live mosquitoes were transferred into disposable cups with cotton pad of 5% sucrose solution. indicators and statistical analysis the mosquitoes which were not able to fly and attached to the nets or corner of glass walls of the tunnel apparatus considered as knock-down. the knockdown rate was taken into consideration the number of unable mosquitoes to fly during 1-64 minutes after releasing in the compartments of tunnel apparatus. the probit analysis of finney was employed based on knockdown data at logarithmic times (finney 1971) for calculating kt50. the following morning, the live mosquitoes were recaptured using glass aspirator. they were transferred to a cupboard cups covered with net and maintained 24 hour for calculating the mortality rate. the dead mosquitoes were removed from each compartment after 15 hours and then scored separately at both big and small compartments. overall mortality was measured by pooling the immediate and delayed mortalities. if the control mortality was between 5 and 20%, the percentage mortality was corrected using abbott’s formula (abbott 1925). the inverse of the rate was calculated as the survival rate including both living blood-fed and unfed mosquitoes. the blood-feeding rate was defined as the number of mosquitoes which took a bloodmeal and may remain in small compartment or return to at the opposite side. the inhibition of blood feeding was assessed by comparing the proportion of blood-fed females (alive or dead) in treated or control tunnels. during the experiments, a proportion of bloodfed mosquitoes came back from the host chamber to releasing chamber was considered as returning rate. this phenomenon may be related to a behavioristic response to find a place having the greatest distance from treated nets. the proportion of mosquitoes (dead or alive and blood-fed or unfed) which passed through the netting was also recorded as the entry index. by comparison with the control group, any change in the entry index was considered as the indication for the repellent effect of the treated net. the significant differences between entry index, inhibition of blood feeding and mortalities at various washing conditions were analyzed using pearson and likelihood ratio chisquare tests at minitab 15.0 software. results knockdown rate one hour knockdown rate of females to unwashed, up to 20-washed is shown in table 1. the knockdown rates were always lower than mortality rates in the treatment group (fig. 2). the knockdown rates had the decreasing trend from unwashed to 20 washes (fig. 2). kt50s were 73.4, 26.3, 45.3, 72.4, 33 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 138.6, 90.0, 52.5, and 211.8 minutes respectively to different washing conditions and the confidence interval of kd50s are conjointly presented (fig. 3). mortality tests the total mortality rates ranged from 89.0 to 99.0% in unwashed and up to 15 times with the mean of 89.0±3.1%.the figures was ranging between 0 and 4.0% on serial washed untreated nets (table 3). the mortality slightly drop to 75.0±4.3% at 20 washed treated net (fig. 3). the 80.0% mortality rate of an. stephensi was indicated as a cut-off point for the decision making the reduction of alphacypermethrin bioefficacy (duchon et al. 2005). the mortality rates of an. stephensi at releasing chamber were 75.0% and 90.0% on unwashed and one-washed, respectively, which correlated with related number mosquitoes which repelled and remained at big compartment (table 3). the mortality rates in bloodfed and unfed are separately compared in control and treatment group. blood feeding indicators inhibition of blood-feeding was ranged between 80.0–99.0% in the treatment group, including unwashed and all nets which were washed 1–20 times (table 4). a drop to 64.0% was considered on 20 washes. the inhibition of blood-feeding was negligible. it is varied from 0–10.0% for untreated netting. during this experiment, a total of 800 hungry females were tested on unwashed and all nets washed 1–15 times, 49 mosquitoes (6.1%) succeed to pass throughout the holes of treated nets toward the guinea pig in small chamber. they took blood and returned toward releasing chamber and survived after 24 hours of exposure. the blood feeding rate (individual protection) was reached to the highest level (36.0%) after 20-washing. survival rate survival rate of an. stephensi from washed to unwashed treatment were 1.0 to 11.0% (table 4). this criteria was increased to 25.0% after 20-washing. the figure was 96.0 to 100% in the control group. entry index the proportion of hungry mosquitoes that succeed passing (entry index) on unwashed and one-washed ranged between 25.0 to 10.0% .taking a blood meal varied from 7.0 to 1.0%, whereas in the control group the entry index was 54.0 to 96.0% and 96.0 up to 100%, respectively. the variation of entry index and taking a blood-meal in the treatment group were 16.0–39.0% and 8.0–36.0%. the figures in the control group were 71.0– 89.0% and 90.0–100% respectively during the test on 2-washed up to 20-washed nets (table 3). individual and community protection rates individual protection and community rates were ranged between 80.0–99.0% and 91.0– 99.0% respectively in unwashed and all nets washed for 1–15 times (table 4). a drop to 64.0 and 79% were considered on 20 washes. the individual protection and community rates were negligible and varied from 0–14.0% and 0–10% for untreated netting. irritancy effect the irritancy effect was ranged from 62.0– 90.0% and 4.0–46.0% in treated and control groups (table 3). the highest and lowest values of irritancy effect were 61.0±4.9% and 90.0±3.0% which for 20 and 1 washes respectively. 34 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 table 1. comparison of knockdown and mortality rates of an. stephensi exposed to long lasting alphacypermethrin treated nets number of washes total mosquito tested knockdown rate (%) after 1 hour at big compartment (mortality rate after 24 h) knockdown rate (%) after 1 hour at small compartment (mortality rate after 24 h) total knockdown rate (%) after 1 hour (mortality rate after 24 h) ratio of knockdown rate / mortality rate unwashed 100 52 (75) 4 (24) 56 (99) 0.6 1 100 87 (90) 9 (9) 97 (99) 1.0 2 100 60 (73) 9 (18) 69 (91) 0.8 3 100 40 (60) 15 (29) 55 (89) 0.6 6 100 37 (84) 8 (11) 45 (95) 0.5 8 100 30 (83) 5 (13) 35 (96) 0.4 15 100 45 (74) 8 (15) 53 (89) 0.6 20 100 32 (56) 2 (19) 34 (75) 0.5 table 2. probit analysis on knockdown (kt50) of an. stephensi and parameters of regression lines of kt50 at different washing conditions of long-lasting alpha-cypermethrin treated nets p-valueχ 2 (heterogeneity)kt50 with 95% c.i. b± seanumber of wash table (df)calculated <0.0516.721.216 33.3574 73.4729 119.3313 1.1043±0.235-2.06080 <0.0516.741.625 15.0470 26.3098 31.1217 1.9350± 0.413-2.74801 <0.0516.719.593 27.9765 45.2653 58.4710 1.6496±0.286-2.73132 <0.0516.720.885 37.7958 72.4025 105.4589 1.5188±0.319-2.82463 >0.0516.77.385 83.9697 138.5827 298.6072 0.9605±0.117-1.05726 >0.0516.71.625 69.4992 90.0128 133.6082 2.0853±0.270-4.07538 >0.0516.73.358 43.4574 52.5295 66.9256 1.9756±0.189-3.398915 >0.0516.73.989 116.2165 211.8473 562.8679 0.9507±0.127-2.211420 35 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 fig. 1. tunnel test apparatus 0 20 40 60 80 100 120 unwashed 1 washed 2 washed 3 washed 6 washed 8 washed 15 washed 20 washed no. of washing k no ck do w n ra te (% ) fig. 2. knock down rate of an. stephensi exposed to unwashed and repeated washed alpha-cypermethrin nets using tunnel test method 0 20 40 60 80 100 unwashed 1-washed 2-washed 3-washed 6-washed 8-washed 15-wahsed 20 -washed washing m or ta lit y ra te (% ) treatment control c u cut-off point fig. 3. mortality rates of an. stephensi exposed to unwashed and serial washed long-lasting alpha-cypermethrin treated nets with control group 36 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 table 3. indicators for bioefficacy of alpha-cypermethrin treated nets (t) against an. stephensi at both big and small compartments of tunnel test apparatus compared to untreated netting as control group (c) number of washing type of test entry index % ± se irritant effect (%) (at big compartment) ± se ratio of mosquito dispersion at compartments (b/s ratio) mortality rate (%) at small compartment (dead no.) mortality rate (%) at big compartment (dead no.) total mortality rate (%) ± se 0 t 25.0±4.3 75.0±4.3 3.0 96.0 (24) 100 (75) 99.0±1.0 c 54.0±5.0 46.0±5.0 0.9 0 (0) 8.7 (4) 4.0±2.0 1 t 10.0±3.0 90.0± 3.0 9.0 90.0 (9) 100 (90) 99.0±1.0 c 96.0±2.0 4.0±2.0 0.1 1.1 (1) 25 (1) 2.0±1.4 2 t 27.0±4.4 73.0±4.4 2.7 66.7 (18) 100 (73) 91.0±2.9 c 80.0±4.0 20.0± 4.0 0.3 0 (0) 0 (0) 0 3 t 38.0±4.9 62.0±4.9 1.6 76.3 (29) 96.8 (60) 89.0±3.1 c 77.0±4.2 23.0±4.2 0.3 0 (0) 8.7 (2) 2.0±1.4 6 t 16.0±3.7 84.0±3.7 5.2 68.8 (11) 100 (84) 95.0±2.2 c 71.0±4.5 29.0±4.5 0.4 1.4 (1) 0 (0) 1.0±1.0 8 t 17.0±3.8 83.0±3.8 6.4 76.5 (13) 100 (83) 96.0±1.9 c 76.0±4.2 24.0±4.3 0.3 0 (0) 0 (0) 0 15 t 21.0±4.1 79.0±4.1 4.9 71.4 (15) 93.7 (74) 89.0±3.1 c 83.0±3.8 17.0±3.8 0.2 0 (0) 0 (0) 0 20 t c 39.0±4.9 89.0±3.1 61.0±4.9 11.0±3.1 2.9 0.1 48.7 (19) 0 (0) 91.8 (56) 9.1 (1 ) 75.0±4.3 1.0±1.0 0 20 40 60 80 100 unwashed 1-washed 2-washed 3-washed 6-washed 8-washed 15-wahsed 20 -washed washing en try in de x (% ) treatment control fig. 4. comparison of entry index of an. stephensi at compartment having long-lasting alpha-cypermethrin treated torn nets compared to untreated torn nets (control group) 37 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 table 4. bioefficacy indicators of an. stephensi exposed to alpha-cypermethrin treated nets at laboratory condition n o f n et s w as h ed t yp e te st t ot al n te st ed small compartment big compartment individual protection rate (%) ± se community protection rate (%) ± se inhibition of blood feeding (%) ± se mortality rate (%) ± se survival rate (%) ± se alive (n) dead (n) alive (n) dead (n) uf ff uf ff uf ff uf ff 0 t c 100 100 0 0 1 54 18 0 6 0 0 0 0 42 75 0 0 4 93.0±2.6 0 99.0±1.0 4.0±2.0 99.0±2.5 4.0±2.0 99.0±1.0 4.0±2.0 1.0±1.0 96.0±2.0 1 t c 100 100 0 0 1 95 9 1 0 0 0 2 0 1 90 1 0 0 99.0±1.0 4.0±2.0 99.0±1.0 4.0±2.0 99.0±1.0 0 99.0±1.0 2.0±1.4 1.0±1.0 98.0±1.4 2 t c 100 100 0 0 9 80 15 0 3 0 0 0 0 20 70 0 3 0 85.0±3.6 0 91.0±2.9 0 85.0± 3.6 0 91.0±2.9 0 9.0±2.7 100 3 t c 100 100 0 1 9 76 29 0 0 0 2 1 0 20 60 2 0 0 91.0±2.9 14.0± 3.5 91.0±2.9 4.0 ±2.0 91.0±2.9 4.0±2.0 89.0±3.1 2.0±1.4 11.0±3.1 98.0±1.4 6 t c 100 100 0 0 5 70 9 1 2 0 0 9 0 20 83 0 1 0 92.0±2.7 10.0±3.0 95.0±2.2 10.0±3.0 92.0±2.7 10.0±3.0 95.0±2.2 1.0±1.0 5.0±2.2 99.0±1.0 8 t c 100 100 4 0 0 76 5 0 8 0 0 2 0 22 83 0 0 0 92.0±2.7 2.0±1.4 100 2.0±1.4 92.0±2.7 2.0±1.4 96.0±1.9 0 4.0±2.0 100 15 t c 100 100 1 2 5 81 0 0 15 0 5 5 0 12 74 0 0 0 80.0± 4.0 7.0±2.6 95.0±2.2 7.0±2.6 80.0± 4.0 7.0±2.6 89.0±3.1 0 11.0±3.1 100 20 t c 100 100 1 0 19 89 4 0 15 0 3 7 2 3 56 1 0 0 64.0±4.8 8.0±2.7 79.0±4.1 8.0±2.7 64.0±4.8 8.0±2.7 75.0±4.3 1.0±1.0 25.0±4.3 99.0±1.0 38 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 0 20 40 60 80 100 unwashed 1-washed 2-washed 3-washed 6-washed 8-washed 15-wahsed 20 -washed washing in di vd ua l p ro te ct io n (% ) treatment group control group individual protection rate 0 20 40 60 80 100 unwashed 1-washed 2-washed 3-washed 6-washed 8-washed 15-wahsed 20 -washed washing c om m un ity p ro te ct io n (% ) treatment group control group community protection rate fig. 5. individual and community protection rates provided by long-lasting alpha-cypermethrin treated nets against an. stephensi compared to untreated netting 39 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 discussion there are several reports on the different aspect of malaria vector in iran. the main malaria vectors in refractory malaria area are an. culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus, and an. pulcherrimus which are known to be the malaria vectors (vatandoost et al. 2002, enayati et al. 2003, naddaf et al. 2003, davari et al. 2006, 2007, doosti et al. 2006, 2007, oshaghi et al. 2006a, b, abai et al. 2008, omrani et al. 2010, vatandoost and zahirnia 2010, hanafibojd et al. 2011, 2004a,b, 2005, 2006a,b,c,d, 2008, 2009a,b, 2010). aopheles sahacrovi and an. maculipennis are considered as malaria vector in northern part of the country (salarilak et al. 2002, sedaghat et al. 2003a,b). in this study the efficacy and wash-resistance of interceptor® long-lasting nets (llins coated with alpha-cypermethrin) were assessed by employing bioefficacy indicators using tunnel test method. several indicators were defined including knockdown rate, kd50, kd90, mortality tests, blood feeding indicators, assoli of blood feeding, survival rate, individual and community protection rates, irritancy effect and entry index. each indicator reflect the conditions which may occurred when the mosquitoes exposed to long-lasting or conventional nets. our results indicated that kd50 and kd90 of mosquitoes during the exposure to longlasting nets are considered as important indicator due to positive correlation with mortality rate of mosquitoes. the ratio of knockdown rate to the mortality rate showed a range of 0.4–1.0% at different washing conditions (table 1). knockdown rates had the decreasing trend from unwashed to 20 washes (fig. 2). one hour knockdown rates did not correlate with 24 h mortality rates (pearson's correlation coefficient= 0.485, p= 0.224). the kt50 trend showed an increase after progressive washes of long-lasting alpha-cypermethrin treated nets but there was an unexpected result in knockdown rates, which may be related to the presence of enough space at big compartment to avoid the mosquitoes to pick up from treated net. this variation in knockdown rate may be related to the activation of fendozin®, a textile new auxiliary that binds the potent long-lasting alpha-cypermethrin treated nets in a special coating to the fibers of the net. the washing of treated nets was led to slowly releasing of alpha-cypermethrin and rapidly knocks down, kills or repels the mosquitoes as they come in contact with the treated nets. kt50 also increased after progressive washing. the mortality of mosquitoes exposed to longlasting alpha-cypermethrin treated nets which was washed 1 to 15 times, was high (89.0 to 99.0%). the mortality rate of an. stephensi that could pass overnight (15 h) from unwashed torn long-lasting alpha-cypermethrin treated nets toward to guinea pig place in the small compartment showed 96.0 and 90.0% mortality on unwashed and one-washed treated nets, which correlated with 25.0 and 10.0% of related entry index. the greater part of the mortality rates was occurred in the mosquito releasing chamber both on unwashed and all serial washes. it could be concluded that majority of the repelled mosquitoes were died without success in probing host placed at small compartment. previous studies have found that a significant impact of washing (20 times) on long-lasting alpha-cypermethrin treated nets was observed on mortality (56%) but not on kd (97%) (duchon et al. 2005). our finding exhibited that the ratio of mosquitoes at the big compartment had always higher ranging 2.1–10 folds of small compartment both at unwashed nets and all washed. this ratio is always below 1.0% in control group. when the ratio of mosquitoes at big to small compartments was decreased, it could be expected the increasing of passing the mosquitoes through holes of treated nets toward the guinea pig as a blood feeding host placed 40 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 at small compartment. a previous laboratory work on long-lasting alpha-cypermethrin treated nets against an. stephensi showed that after 3 washes, the mortality rate was 97.5% which concordance with 118.6 mg/m2 as well as the mortality was 15% after 20 standard washes with the residue of 61.2 mg/m2 (vatandoost et al. 2011). in the absence of insecticide, the inhibition of blood-feeding on host is negligible (0–10%). this is attributed on exposing of skin for mosquitoes as well as amount of chemical attractants (eg odors) of host. our result indicated that inhibition of blood-feeding of mosquitoes was high (80–99%). a decreasing trend with increasing of washing times was observed. the previous studies also found the higher inhibition of blood-feeding of mosquitoes even after 30 standard washings of long-lasting alpha-cypermethrin treated nets. also the blood feeding inhibition of mosquitoes in the alpha-cypermethrin treated houses was statistically significant (p< 0.001) (sharma et al. 2010). during the other field trial on longlasting alpha-cypermethrin treated nets, high mortality (above 95%) and high blood feeding inhibition (above 90%) were observed on an. gambiae s.s as well as for the other mosquito species. also the latter study showed a blood-feeding increased in an. gambiae with the number of washes and was significantly lower (duchon et al. 2005). our findings showed that in control group, a significantly higher percentage of blood fed females was observed (p< 0.001). our results may indicate that long-lasting alpha-cypermethrin treated nets (llins) have higher individual protection (>80%) up to 15 washes as well as higher community protection regardless of how many times the nets have been washed. a previous work on long-lasting alphacypermethrin treated nets resulted lower entry index and higher mortality. the greatest reduction in blood feeding (4.8%) was achieved this insecticide (mosha et al. 2008). in a previous field trial, the personal protection of long-lasting alpha-cypermethrin treated which had been washed 20 times was almost 100%. however nets did not strongly reduce the entry rate of female mosquitoes, but kill almost all of them. these results suggested that the nets could have a strong mass effect of mosquito populations if used at large scale (duchon et al. 2005). survival rates of an. stephensi which exposed to long-lasting alpha-cypermethrin treated nets showed an expected increasing trend toward to higher washing times, whereas this rate showed a nearly fixed trend in the control group. the entry index is reflecting the rate of mosquitoes that succeed in passing through torn nets try to enter the compartment where the host for blood feeding is available. the results from this study showed that the entry index was suppressed from the killing effect of long-lasting alphacypermethrin treated nets. there was a positive correlation between entry index and taking a blood meal (pearson's correlation coefficient =0.784 and p=0.02). in a field study on long-lasting alpha-cypermethrin treated nets induced high mortality of an. gambiae that entered the hut. this fell significantly with the number of washes (0 washes: 93%, 20 washes: 79%, 30 washes: 66%) (duchon et al. 2005). our findings indicated that both entry index and blood-feeding rates increased trend from unwashed toward 20 washes. the entry index of mosquitoes that could pass overnight (15 h) through the holes of unwashed and one-washed treated nets was 25.0 and 10.0% which correlated with 24.0 and 9.0% mortality respectively (pearson's correlation coefficient=0.682 and p=0.01). with the decrease of α long-lasting alphacypermethrin treated residue due to repeated washing, the entry index increased. the are several reports on the bioefficacy of bednet worldwide (gimnig 2005, kayedi et al. 2007, 2009, gunasekaran and vaidyanathan 2008, oxborough et al. 2009). 41 j arthropod-borne dis, june 2013, 7(1): 31–45 h vatandoost et al.: wash resistance and… http://jad.tums.ac.ir published online: april 10, 2013 the irritant effect of long-lasting alphacypermethrin treated nets was 75.0 and 90.0% on unwashed and one-washed nets. irritancy effect of treated nets against an. stephensi was high. there is a significant reduction in entry rate of mosquitoes in alpha-cypermethrin treated houses in the absence of irs (p< 0.01). also the total excito-repellency rate of all the mosquitoes in houses with alpha-cypermethrin nets and untreated nets was 29.9 and 4.8% respectively (sharma et al. 2010). during another field study on application of long-lasting alpha-cypermethrin treated bednets, the entry rates of the female mosquitoes were significantly reduced (15–30%) (duchon et al. 2005). llins could be employed as a vector control intervention and the first field trials in india indicated that alpha-cypermethrin llin was effective in contributing to reducing the burden of malaria in the affected communities (banek et al. 2010). in conclusion, our laboratory trial on interceptor® long-lasting nets (llins coated with alpha-cypermethrin) could provide a good personal protection against main malaria vector an. stephensi and could inhibit the bloodfeeding of mosquitoes as well as induce relatively high mortality and reduce the entry rates of female mosquitoes. we propose the hut and field trail on the effect of this kind of llin for measuring the impact and outcome indicators in the malarious area of south-eastern part of the country. acknowledgments the authors are grateful to iranian ministry of health and medical education, malaria department for providing interceptor® longlasting nets for our experiments. we are thankful to the staff of insectary, department of medical entomology and vector control, school of public health, tehran university of medical sciences for mass production of mosquitoes for this study. this study has been funded and supported by tehran university of medical sciences (tums). the authors declare that they have no competing or conflict interests. references abai mr, mehravaran a, vatandoost h, oshaghi ma, javadian e, mashayekhi m, mosleminia a, piyazak n, edallat h, mohtarami f, 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and planning in minab district, southern iran. asian pac j trop med. 4: 67–71. yates a, n'guessan r, kaur h, akogbeto m, rowland m (2005) evaluation of ko-tab 1-2-3®: a wash-resistant 'dip-ityourself insecticide formulation for long-lasting treatment of mosquito nets. malaria j. world health organization (2005) guidelines for laboratory and field testing of long-lasting insecticidal mosquito nets. who/cds/whopes/gcdpp/2005.11. world health organization (2007) who global malaria programme: position statement on itns. who, geneva. world health organization (2012) malaria entomology and vector control. participants' guide. who. switzerland, geneva. 45 j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 1 http://jad.tums.ac.ir published online: march 31, 2023 review article worldwide status of insecticide resistance of aedes aegypti and ae. albopictus, vectors of arboviruses of chikungunya, dengue, zika and yellow fever tahereh sadat asgarian1, *hassan vatandoost1,2, ahmad ali hanafi-bojd1,2, fatemeh nikpoor2 1department of vector biology and control of diseases, school of public health, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir (received 19 aug 2022; accepted 24 dec 2022) abstract background: controlling of aedes aegypti and ae. albopictus, vectors of five important mosquito-borne diseases, is known as the most effective method to prevent the transmission of arboviruses to humans, but the emergence of insecti cide resistance is threat for control and prevention of vector borne diseases. a better understanding of mosquito resistance to insecticides will help to develop more effective methods to control insecticide resistance in mosquito vectors. methods: worldwide geographical distribution of insecticide resistance in ae. aegypti and ae. albopictus by the available papers and map of the data for carbamates, organochlorines, organophosphates, pyrethroids, microbial and insect growth regulator insecticides were reviewed. article data published up to december 2022 were investigated by searching the following databases: "google scholar", "pubmed", "scopus", "sid" and "web of knowledge". results: the results showed that the susceptibility and resistance status of ae. aegypti and ae. albopictus to insecticides in the world is very diverse. conclusion: due to the importance of ae. aegypti and ae. albopictus in the transmission of mosquito-borne arboviruses, resistance management should be given more attention worldwide to prevent insecticide resistance in the arbovirus vector and replace the new approach for vector control. keywords: insecticide; resistant; aedes; arboviruses; world introduction among arboviruses that are widespread worldwide, chikungunya, dengue, zika and yellow fever are widely transmitted by aedes spp. (1, 2). aedes aegypti mosquitoes originated from africa, but they are also found in tropical, subtropical, and temperate parts of the world (3, 4). aedes albopictus has adapted to urban, suburban, and rural regions. this species is a forest species that has spread from asia to africa, america, and europe through the trade of used tires (5). dengue is the fastest mosquito transmitted disease, it has increased more than 15 -fold since 2000 and has affected more than 129 countries. because vaccines or drug treatments exist for only a small number of vector-borne pathogens, the primary method for controlling many vector-borne diseases is direct vector control (6). chemical control by organic or inorganic insecticides is one of the methods of mosquito control and as part of integrated management. insecticides used for mosquitoes, that are neurotoxic: organochlorines, organophosphates, carbamates, and pyrethroids (7). insecticide resistance is a growing topic around the world that limits the effectiveness of control methods against vector mosquitoes. the data on insecticide resistance in aedes is varying. insecticides have an important role in the control of dengue fever, but the resistance of mosquitoes copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:vatando@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 2 http://jad.tums.ac.ir published online: march 31, 2023 reduces the effectiveness of these interventions in some countries (8). according to the world health organization (who), resistance to insecticides is a major risk in interventions to control vector-borne diseases. it is essential to identify the geographical regions where vector resistance to insecticides exists and can make it difficult to control the vector, and to improve the induction of innovative tools for vector control. a better identifying of resistance of insecticides helps to formulate a global measure to control insecticide resistance in disease vectors. materials and methods published articles were searched by terms “resistance”, “aedes aegypti”, “aedes albopictus”, “pyrethroid”, “organochlorines”, “organophosphate”, “carbamate”, “igr”, and “kdr”, “p450”, “monooxygenase”, “glutathione”, or “esterase”. data were extracted from articles published up to december 2022. the terms in the following databases: pubmed, web of knowledge, scopus, google scholar, and sid were reviewed. the who guideline was considered for insecticide resistant level. results resistant status of aedes aegypti and ae. albopictus to organochlorines insecticides in africa resistance of two aedes species to dichlorodiphenyltrichloroethane (ddt) has been reported from each country where susceptibility testing has been performed. in ghana (9, 10), cape verde, and senegal (11) high ae. aegypti resistance to ddt was reported. experiments showed one ae. aegypti population from gabon and two populations of ae. albopictus from cameroon were resistant to ddt (12, 13). also, in another study adult bioassays of three field populations of ae. albopictus and ae. aegypti from cameroon showed different susceptibility levels from 68.75% to 100% against 4% ddt (14). in central african republic ae. aegypti was resistant to ddt and ae. albopictus had a sensitive population and the rest of the populations were tolerant (15). in lagos state of nigeria all populations of ae. aegypti showed resistance to ddt (16), while in kwara state, nigeria, ae. aegypti was completely susceptible to this insecticide (17). highest resistance to 4% of ddt was observed in populations of ae. aegypti from northern nigeria, while the knockdown rate in dieldrin was very high (18). two population types of ae. aegypti (white and brown populations) examined in cote d’ivoire, both populations were resistant to ddt (19, 20). nine populations tested of ae. aegypti from senegal were resistant to ddt (21) (fig. 1). in asia in thailand, resistance, or increased tolerance to dieldrin has been reported, and there is resistance to ddt, although ddt is not used in aedes control programs. ddt has been used for agriculture in thailand since 1934 was banned in agriculture in 1983. in health and for indoor spraying, it has been used against malaria vectors since 1949, and since 2000, its use in health has been ceased due to its destructive effects on the environment and the development of physiological resistance in other mosquitoes (22–27). in malaysia, ae. aegypti and ae. albopictus showed resistance to ddt. aedes aegypti was susceptible to dieldrin except in one area. dieldrin resistance was reported in ae. albopictus except in kuala lumpur and two other areas. the higher resistance of ae. albopictus to dieldrin was due to the ecology of this species and its breeding sites, which was close to plants and in agricultural areas, and therefore exposed to dieldrin because it was still used in agriculture to control soil pests (28, 29). the adult ae. aegypti from jharia, bihar were the first case of ddt resistance in india (30). in india ddt resistance in field collected ae. aegypti has reported from http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 3 http://jad.tums.ac.ir published online: march 31, 2023 goa (31) and jharkhand (32). ddt and dieldrin resistance have been reported in all indian ae. aegypti. aedes albopictus from western bengal has shown resistance to ddt. aedes aegypti females’ exposure with 0.4% dieldrin resulted in 48% and 100% mortality, while 4% dieldrin for same exposure period showed 88% and 100% mortality (33–38). in china all ae. albopictus adults tested populations showed resistance to ddt. urban ae. albopictus had a higher level of resistance to ddt than strains collected from rural areas (39–42). in laos ae. albopictus was resistant to ddt (43). there was high ddt resistance in adult of ae. aegypti in myanmar (44). in philippines ae. aegypti and ae. albopictus larvae were sensitive to lindane, dieldrin, and ddt (45). also, there was ddt resistance in vietnam (46–48). in japan all strains of ae. albopictus were highly resistant to ddt (49). in phnom penh, cambodia papers impregnated with ddt at 4% and dieldrin at 0.4 and 4% were used for the bioassays of adult ae. aegypti. the mortality rates were reported 0% for 4% ddt and 87.6± 2.1% and 97.8±4.3, for 0.4% and 4% dieldrin%, respectively (50) (fig. 1). in america the development of resistance to ddt in mexico and many parts of the americas was due to the widespread use of this insecticide during the 1950s and 1960s (51). in the americas ae. aegypti resistance to ddt was initially found in trinidad in 1955, and it was susceptible to dieldrin. now, all parts of the caribbean have ae. aegypti populations that are resistant to ddt and dieldrin. from french guiana to the bahamas, the situation is uniform regarding this resistance (52). ddt resistance has been confirmed in ae. aegypti caribbean populations (53) and in two florida populations and one new jersey population (54). there were 45 dengue epidemics in cuba in 1977, 1981, 1997 and 2002 and ae. aegypti resistance to ddt has been seen in this country (55, 56). in colombia resistance to ddt was seen in all mosquito populations tested, although this insecticide did not use for vector control in any of the study areas (57–59). in venezuela low mortality in larvae ae. aegypti was exhibited by field strains toward ddt and confirmed the presence of high resistance to this insecticide (60). after exposure of adults of ae. aegypti from santo domingo, dominican republic to discriminating concentrations of ddt, it was found that wild populations were resistant to this insecticide (61). in brazil eradication programs from 1950 to 1970 might have led to spread of ddt resistance (62). all of populations of ae. aegypti from peru were resistant to ddt (4%), after 1 hour of exposure with papers treated (63) (fig. 1). in australia and oceania adult bioassays detected significant resistance to dieldrin and ddt in ae. aegypti population from townsville city, despite ddt has not been used for adult control in this city for minimum 12 years, and dieldrin is not an adulticide (64). susceptibility to ddt of ae. aegypti and ae. albopictus populations seems to have decreased in papua new guinea (65) (fig. 1). resistant status of aedes aegypti and ae. albopictus to organophosphates insecticides in africa complete susceptibility to temephos, fenitrothion and malathion has been reported from both vectors in cameroun, gabon (12, 13), and mayotte (66). in central african republic larvae of both species were sensitive to temephos and fenitrothion and only one population of resistant to fenitrothion was reported (15). in cape verde ae. aegypti was sensitive to fenitrothion (11), but it shows resistant to temephos and sensitivity to malathion (67). in dakar, senegal also its sensibility to fenitrothion was reported (11). organophosphates (0.8% and 5% malathion, 0.05% pirimiphos-methyl and 1% fenitrothion) were used for bioassays of ae. aegypti http://jad.tums.ac.ir/ https://www.google.com/search?sa=x&bih=588&biw=1213&hl=en&q=oceania&stick=h4siaaaaaaaaaongvulqz9u3mm0rsn_eamwt8plhpweprulrtl5jvohics7il3fnk8ksqrqs42kdsnikuljgmngwsbl7j6cm5mumagbnmpsdtaaaaa j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 4 http://jad.tums.ac.ir published online: march 31, 2023 from senegal. all the ae. aegypti populations tested were susceptible to 5% malathion, while only two populations showed susceptibility to 0.8% malathion. resistance in all populations were reported for 0.05% pirimiphos-methyl. louga, mbour and barke´dji strains showed resistance to 1% fenitrothion (21). the ae. aegypti populations in northern nigeria were susceptible to fenitrothion in 2018 and 2019, with mortality of 98.6% and 100%, respectively. but, in 2020, mortalities of 93% observed and moderate resistance were registered (18) (fig. 2). in asia in thailand ae. aegypti and ae. albopictus resistance to temephos was demonstrated in some areas. aedes aegypti showed susceptibility to malathion. all strains of ae. aegypti were resistant to temephos except strains of nakhon ratchasima. aedes albopictus was susceptible to both insecticides (68). in other study the larvae of both species were resistant to temephos and resistance or tolerance to malathion and fenitrothion has been reported in different parts of north and south of thailand, while in central and eastern parts of thailand, were still susceptible to fenitrothion (24). temephos has been used against ae. aegypti larvae in thailand since 1950, and malathion, fenitrothion, and pirimiphos methyl have been used for irs and fogging (22–24, 26, 68, 69). in sri lanka ae. aegypti and ae. albopictus were sensitive to malathion. although this insecticide has been widely used in the control of malaria and filariasis in endemic areas (70). also, in 2020 reported that adult ae. aegypti mosquitoes from sri lanka were susceptible to malathion 5%. for temephos the resistance ratios (rr50) varied between 0.69 and 3.93 (71). in one study in malaysia, ae. aegypti was sensitive to malathion and fenitrothion (29), in 2015 was sensitive to this insecticide, except in kuala lumpur and ae. albopictus was resistant to malathion except in kuala lumpur. no resistance was observed in larvae to temephos, and various populations were susceptible or tolerant, although it has been widely used since the 1970s and during the 1998 global pandemic (28). chen et al. reported greater resistance of ae. aegypti to temephos than ae. albopictus (72). in india the susceptibility of two species was reported to malathion in 1993 (73). in other studies, ae. aegypti adults were tolerant to fenitrothion and sensitive to malathion, also the larvae were sensitive to three tested insecticides including temephos, fenitrothion and malathion (32–34). resistance of this species to dichlorvos was also reported (74). results from the studies in 2015 showed that only one ae. aegypti population had high levels of resistance to temephos and the rest also ae. albopictus were susceptible (35, 74). in 2017 ae. aegypti was reported sensitive to temephos except one population. in this study, exposure of ae. aegypti with 1% fenitrothion for 30 and 60 minutes caused 40% and 100% mortality, respectively (36). in 2018, larvae of ae. aegypti was susceptible to temephos (37). in a recent study in west bengal all populations ae. aegypti were reported sensitive to malathion (75). in china, resistance to malathion was clear in ae. aegypti in hainan province but ae. albopictus was susceptible (76). aedes albopictus larvae were resistant to temephos and adult was sensitive to dichlorvos, with 100% mortality (41), also adult populations of ae. albopictus in the four districts were all sensitive to malathion, but high resistance to temephos was registered (42). in cambodia there is larval resistance to temephos in endemic areas. in the first study on the sensitivity of two ae. aegypti populations to temephos, in one population, the larvae showed some degree of resistance to temephos and the other population was sensitive (77). in 2022, moderate ae. aegypti resistance was observed for temephos in phnom penh, cambodia. also, papers impregnated with fenitrothion at 1%, malathion at 0.8% and pirimiphos-methyl at 0.2%, were used for the bioassays of adult ae. aegypti. organophosphate insecticides showed high mortality rates (50). development of mild resistance of ae. aehttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 5 http://jad.tums.ac.ir published online: march 31, 2023 gypti to malathion was found in jakarta, indonesia. south denpasar mosquitoes had the highest resistance to malathion, which was significantly different from the resistance reported in north denpasar (78–80) and populations of ae. aegypti in banjarmasin, kalimantan, indonesia were susceptible to malathion 5% (81). in vientiane laos, based on larval bioassay, the wild population of ae. aegypti strain was relatively resistant to temphos (82). in other study in laos all seven population of ae. albopictus but one was resistant to malathion and three of populations showed resistance to temephos and the rest were tolerant (43). in philippines ae. aegypti and ae. albopictus larvae were reported sensitive to malathion (45). in singapore, both species were reported to be sensitive to pirimiphos methyl (83). in another study, all ae. aegypti populations were sensitive to pirimiphos methyl, and all populations of ae. albupictus were resistant to pirimiphos methyl but southeastern populations were tolerant (84, 85). in another study, ae. aegypti larvae were reported to be sensitive to temephos, although resistance was possible (86). aedes aegypti from dili, timor-leste was sensitive to malathion (87), also in vietnam was sensitive to malathion (46–48). in bangladesh ae. aegypti mosquitoes varied in susceptibility to malathion (50μg/bottle) (88) (fig. 2). in america high levels of resistance to chlorpyrifos in ae. albopictus has been found in alabama and florida (89). none of the eight populations of us ae. albopictus larvae were resistant to temephos, also ae. albopictus resistance to malathion was reported in florida and new jersey (54). levels of malathion and temephos resistance were observed in ae. aegypti caribbean populations. guyana, jamaica, and suriname ae. aegypti populations showed only slight resistance to the larvicide. in most caribbean countries, where malathion has been used sporadically for 20 to 30 years to control adults, there is little resistance to this insecticide (90) and low levels of temephos resistance were confirmed in grand cayman (53). in united states ae. albopictus from florida, california, three different north carolina populations and ae. aegypti from texas were susceptible to malathion. conversely, ae. albopictus from north carolina and texas were resistant to the same malathion doses (91). mortality of ae. aegypti mosquitoes tested in jamaica with malathion, was from 84 to 90% at 30 minutes of exposure and 100% with increasing exposure time to 45 minutes (92). in 2019, ae. aegypti strains from new mexico were susceptible to the chlorpyrifos (93). in mexico, the ae. aegypti populations showed less resistant to chlorpyrifos than to pyrethroids (94). resistance to fenthion, fenitrothion, and temephos, has been found in cuba (55, 56). on the island of martinique (caribbean), ae. aegypti populations were resistant to naled (organophosphate) that showed insecticide resistance reduced its effect when applied by ultra-low volume (ulv) thermal fogging (95). there is evident that the organophosphate insecticide resistance (malathion, fenthion and temephos) is prevalent in trinidad and tobago larval strains of ae. aegypti (96). in cuba, venezuela, costa rica and jamaica, ae. aegypti was sensitive to malathion despite its widespread use in vector control programs in these countries (97). resistance to organophosphates is found throughout latin america due to its intense use to control larvae, including colombia, cuba, martinique, costa rica, havana, brazil, bolivia, french guiana, argentina, french polynesia, and the caribbean. but resistance to temephos is lower in west africa. the susceptibility of adults and larvae of ae. aegypti to insecticides from santo domingo, dominican republic, was evaluated. hatched larvae from eggs collected from ovitraps were resistant to temephos. adults were resistant to malathion (61). larvae of ae. aegypti from cuba and other latin-american countries were investigated for organophosphate insecticide resistance, including malathion, pirimiphos methyl, temephos, fenthion, fenitrothion http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 6 http://jad.tums.ac.ir published online: march 31, 2023 and chlorpyriphos. all the strains showed resistance to temephos except nicaragua strain with moderate resistance to temephos. the highest resistant ratio (rr) value to temephos was in the havana city strain followed by panama, costa rica, peru, jamaica, and venezuela. aedes aegypti larvae were susceptible to malathion in all of the strains, and the same results obtained for fenthion and fenitrothion, but peru strain showed moderate resistance to fenthion, and the havana city strain had high resistance to fenthion and moderate to fenitrothion. high resistance to pirimiphos methyl has been reported in panama, costa rica and venezuela; however, santiago de cuba, jamaica and peru showed moderate resistance to this chemical. the santiago de cuba strain had the highest chlorpyriphos, resistant ration of 50 followed by costa rica and jamaica, with havana city, panama, nicaragua, and peru showing susceptibility to chlorpyriphos. the venezuela strain showed moderate resistance to chlorpyriphos (98). aedes aegypti mosquitoes from peru were exposed for 1 hour with malathion (5%) and pirimiphos-methyl (0.25%) papers, and for 2 hours with fenitrothion (1%) papers. the chosica population showed resistance to fenitrothion and pirimiphos-methyl and developing resistance to malathion. while the punchana and piura populations were susceptible to malathion and showed resistance to other evaluated insecticides (63). in southern ecuador ae. aegypti was resistant to malathion and had a mortality rate below 80% (99). in brazil from 1999–2000 until 2010–2011, the effectiveness of temephos on ae. aegypti was high, resulting in larval mortality of more than 80% throughout brazil. the frequency of temephos-resistant individuals in the insect population increased steadily during each biennial survey, indicating a significant reduction in the effectiveness of temephos. this trend reached high levels (less than 50% mortality) in about half of the country in early 2004–2005 (100). in colombia mosquitoes were susceptible to malathion. a high frequency of fenitrothion resistance was reported in all ae. aegypti mosquitoes (58), also in 2019 no evidence of malathion resistance was found in ae. aegypti (59). in venezuela high mortality was obtained with fenitrothion and fenthion against adults and larvae ae. aegypti in field strains, which suggested the absence of any appreciable amount of resistance to these insecticides, but resistance to malathion, temephos, chlorpyrifos, and pirimiphos-methyl was observed (60). larvae of ae. aegypti mosquito from the guadeloupe and saint martin islands were resistant to temephos and malathion compared with the susceptible bora bora strain. for the first time in guadeloupe, mosquito populations of guadeloupe and saint martin were resistant (weakly) to malathion (101). aedes aegypti resistance to temephos has been observed in colombia (102), and the caribbean (90). result of resistance of ae. aegypti in brazil show resistance to at least one of the organophosphates (temephos, fenitrothion, malathion) tested in all populations in the states of rio de janeiro and espírito santo (103). a study in the city of curitiba, state of paraná in brazil reported that temephos could be used for control larvae of ae. aegypti (104). in sao paulo, ae. aegypti was resistant to temephos and susceptible to malathion. it was suggested that malathion be used in ulv instead of cypermethrin in the dengue fever control program in sao peteo, and fenitrothion be used in residual spraying (105). the larvae of all studied ae. aegypti populations from different brazilian regions were resistant to temephos (106, 107). aedes aegypti larvae from northeastern brazil showed the highest levels of resistance to temephos, also adults from northeastern showed the lowest levels of susceptibility to malathion (108), and adults from recife brazil were susceptible to malathion (109). in tocantins state in brazil all evaluated populations of ae. aegypti were resistant to temephos (110). resistance to fenitrothion for the four populations of ae. aegypti distributed along the french guiana exhibited resistance to fenitrothion (111). a high level of resistance to temephos was reported from ae. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 7 http://jad.tums.ac.ir published online: march 31, 2023 aegypti populations in martinique (french west indies) (112). in this study the resistance level of juazeiro do norte population was much less (rr=7.2), which is only slightly lower than the 2003 level (rr=10.2), because temephos has not been used for at least seven years to control aedes and has been replaced by bacillus thuriginensis (bti). the increased resistance levels to temephos in crato and barbalha populations, indicate that the resistance management programs in juazeiro do norte should be done in neighboring cities. these results also show that even if temephos was replaced in juazeiro do norte, the recovery of susceptibility was slow (113). the results of bioassays with temephos, that has been used for decades against ae. aegypti larvae in brazil caused all the evaluated populations to be resistant to temephos (110) (fig. 2). in europe exposure of ae. aegypti from pau´l do mar (madeira) to fenitrothion given 100% mortality that indicate mosquito susceptibility to this insecticide but ae. aegypti from funchal (madeira) was resistant to malathion and fenitrothion (114). aedes albopictus adults from the swiss-italian border region were susceptible malathion (109) (fig. 2). in australia and oceania investigating the state of sensitivity to insecticides in ae. aegypti from townsville showed that mosquitoes were resistant to malathion and fenthion and susceptible to other organophosphates, temephos, fenitrothion and chlorpyrifos (64). aedes aegypti and ae. albopictus were susceptible to malathion in papua new guinea (65) (fig. 2). resistant status of aedes aegypti and ae. albopictus to carbamates insecticides in africa in cameroon, resistance of ae. aegypti to bandiocarb and tolerance of ae. albopictus to this insecticide were reported (13). in 2021 it was reported this species was susceptible to bendiocarb (14). in central african republic both vectors were sensitive to propoxur and only one resistant population to propoxur was reported (15). in kwara state, nigeria, mortalities from exposure of ae. aegypti to bendiocarb showed that all the mosquito samples tested were resistant (17). in northern nigeria, ae. aegypti mosquitoes were resistant to 0.1% propoxur (18). in cape verde low sensitivity ae. aegypti to propoxur was reported and in dakar, senegal mortality of ae. aegypti with propoxur was 87.2% (11). in cote d’ivoire white and brown populations of ae. aegypti were resistant to propoxur (19, 20). in senegal, apart from the matam populations of ae. aegypti were susceptible to 0.1% bendiocarb, and all other populations were resistant to bendiocarb and propoxur (21) (fig. 3). in asia various levels of tolerance/ resistance to propoxur and resistant to bendiocarb and propoxur in ae. aegypti has been reported in parts of central and southern in thailand (24, 26, 69). aedes aegypti resistance to bendiocarb and propoxur was also reported from malaysia as well as ae. albopictus resistance (28, 29). in india, ae. aegypti females were exposed to 0.1% propoxur for 30 and 60min and mortality after 24h was 96% and 100%, but in other study ae. aegypti was resistant to propoxur, although this insecticide has not been used to control vectors in india. resistance has been due to accidental exposure or to intersection resistance (36, 37), also ae. albopictus from subhimalayan areas of west bengal exhibit severe resistance against propoxur (38). in china, ae. albopictus adult was sensitive to propoxur (41), and resistant to bendiocarb (42). in indonesia ae. aegypti was resistant to bendiocarb. developed resistance, with about 80–90% mortality, was observed against bendiocarb 0.1% (80, 81). papers impregnated with bendiocarb at 0.1% and propoxur at 0.1% were used for the bioassays of adult ae. aegypt in phnom penh, cambodia, results showed high mortality rates http://jad.tums.ac.ir/ https://www.google.com/search?sa=x&bih=588&biw=1213&hl=en&q=oceania&stick=h4siaaaaaaaaaongvulqz9u3mm0rsn_eamwt8plhpweprulrtl5jvohics7il3fnk8ksqrqs42kdsnikuljgmngwsbl7j6cm5mumagbnmpsdtaaaaa j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 8 http://jad.tums.ac.ir published online: march 31, 2023 with carbamate (50). complete susceptibility to bendiocarb was reported in populations of ae. aegypti from bangladesh (88) (fig. 3). in america in the states of alabama and florida, propoxur cross-resistance levels were evaluated in ae. albopictus. propoxur showed the least toxicity to ae. albopictus. the results showed that propoxur tolerance levels were absent or very low in all strains (89). in new jersey, pennsylvania, and florida ae. albopictus was sensitive to propoxur (54). in colombia all of ae. aegypti field-collected strains were susceptible to propoxur (59). in venezuela ae. aegypti resistance to propoxur was reported (60). aedes aegypti adults from santo domingo, dominican republic, were resistant to propoxur (61) (fig. 3). in europe aedes aegypti from two localities in madeira (funchal and pau´l do mar) was resistant to bendiocarb (114). after 1h exposure and 24 h holding period to bendiocarb, ae. albopictus field populations from the swiss-italian border region were susceptible (109) (fig. 3). in australia and oceania in australian 1995 strain of ae. aegypti was significantly less susceptible to bendiocarb than the 1989 strain, and both strains showed high resistance to propoxur (64). aedes aegypti from the madang population, papua new guinea was susceptible to bendiocarb and ae. albopictus also were found to be susceptible to bendiocarb (65) (fig. 3). resistant status of aedes aegypti and ae. albopictus to pyrethroids insecticides in africa both the aedes species are present in central africa, but ae. albopictus is more influential and was first reported in 2009. a study in this area found that both vectors were resistant to deltamethrin, and their mortality was less than 90% (15). in ghana, ae. aegypti was resistant to deltamethrin, and lambda-cyhalothrin but was sensitive to permethrin (9). in other study some populations were resistant to permethrin and some tolerant (10). in mayotte both species had highly sensitive to deltamethrin. no resistance to deltamethrin has been recorded on the island, although deltamethrin has been used on the island since 1984 (66). in cameroun complete susceptibility of both species was reported to deltamethrin except in yaounde, where ae. albopictus populations showed a mortality rate of about 80% (12), but in 2017 both species were resistant to deltamethrin and decreased sensitivity to permethrin was observed (13). in 2021 ae. aegypti and ae. albopictus mosquitoes from cameroun were exposed to 0.05%, and 0.75% permethrin. aedes albopictus populations showed full susceptibility to permethrin and deltamethrin insecticides, but ae. aegypti was resistant to these insecticides (14). in a study conducted in agricultural and non-agricultural areas of lagos state, nigeria, all mosquitoes from both sites were susceptible to permethrin, but about deltamethrin the most populations in non-agricultural areas were resistant and mosquitoes collected from agricultural areas were reported sensitive (16). in another study, ae. aegypti was completely sensitive to permethrin (17). adult bioassays of ae. aegypti in northern nigeria were conducted by pyrethroids: 0.25% and 0.75% permethrin, 0.03% and 0.05% of deltamethrin, lambda-cyhalothrin, and α-cypermethrin, and 0.15% cyfluthrin. populations were susceptible to cyfluthrin but highly resistant to other type i and ii pyrethroids (18). in dakar, senegal, ae. aegypti populations was susceptible permethrin. about deltamethrin mortality was 94.5% and for lambda-cyhalothrin 81.6%. in cape verde ae. aegypti was sensitive to deltamethrin, permethrin, and lambda-cyhalothrin (11). in another study, ae. aegypti was resistant to deltamethrin, and cypermethrin (67). bioassays of pyrethroids (0.25% and 0.75% permethrin), http://jad.tums.ac.ir/ https://www.google.com/search?sa=x&bih=588&biw=1213&hl=en&q=oceania&stick=h4siaaaaaaaaaongvulqz9u3mm0rsn_eamwt8plhpweprulrtl5jvohics7il3fnk8ksqrqs42kdsnikuljgmngwsbl7j6cm5mumagbnmpsdtaaaaa j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 9 http://jad.tums.ac.ir published online: march 31, 2023 0.05% deltamethrin, 0.03% and 0.5% lambdacyhalothrin, 0.05% alpha-cypermethrin), by standard who test kits for adults of ae. aegypti from senegal showed that susceptibility to 0.75% permethrin was observed in all populations except in three, and one population had suspected resistance to 0.25% permethrin. only populations of the southern regions were susceptible to type ii pyrethroids (21). in dar es salaam, tanzania all sites showed lower susceptibility to deltamethrin, permethrin and resistance to lambda-cyhalothrin (115). in cote d’ivoire white populations of ae. aegypti were resistant to deltamethrin and brown populations were susceptible (19, 20) (fig. 4). in asia in japan ae. albopictus in nagasaki showed high tolerance to pyrethroids. the cause for this may be due to the widespread use of ddt in the 1950s (116). most studies on pyrethroid resistance in ae. aegypti in southeast asia are from thailand. the first outbreak of dengue fever in thailand was in 1958 (117). since 1950, carbamates, organophosphates, and organochlorines have been used for control. synthetic pyrethroids have been used since 1992, low price of pyrethroids, their quick knockdown effect and relative safety for humans due to low toxicity to mammals has increased their use (22). increased tolerance or resistance to pyrethroids has been reported from ae. aegypti and ae. albopictus in thailand, such as deltamethrin and permethrin, which are widespread throughout thailand, resistance to cypermethrin in parts of northern and western thailand, and resistance to bioallethrin, bioresmethrin, and alpha-cypermethrin in larvae and adults. mortality of ae. aegypti with lambda cyhalothrin was 100% and had tolerated to mosquito coils. aedes aegypti has shown behavioral resistance and avoidance to alpha-cypermethrin, deltamethrin, permethrin, tetramethrin, cyphenothrin in thailand (26, 68, 118–120). in malaysia, many studies have been conducted on the resistance of the two species to insecticides. the first study on pyrethroid resistance was in 2001. the ae. aegypti urban strain had the highest resistance to permethrin, cyphenothrin, lambda-cyhalothrin, and deltamethrin. aedes albopictus was sensitive to permethrin and deltamethrin (121–123). the first study on mosquito coils against ae. aegypti was in 1996, that mortality was minimal (124). in 2017, in a study was reported that mosquito coils had low insecticidal activity against ae. aegypti and therefore may have little protection for mosquito bites (125). in malaysia was reported ae. aegypti resistance to permethrin (29). in the other study across malaysia, ae. aegypti was resistant to permethrin and deltamethrin (28). in india, ae. aegypti and ae. albopictus resistance to pyrethroids such as permethrin has been reported in delhi and kerala (73). in 2001 ae. aegypti adults was sensitive to deltamethrin, permethrin, and lambda cyhalothrin in delhi (33). in another study a total of five different pyrethroid compounds were tested against ae. aegypti females with exposure period of 5 and 15min in madurai west city in tamil nadu state, they were found invariably susceptible to all tested pyrethroid compounds. based on the results, the efficacy in descending order of pyrethroids was cyfluthrin > permethrin > deltamethrin > lambda cyhalothrin and > etofenprox (36). in recent studies in west bengal, all populations ae. aegypti except one site were sensitive to deltamethrin and alpha-cypermethrin (75), and two ae. albopictus populations, from sub-himalayan districts were severely resistant to permethrin (38). in china, ae. albopictus is resistant to deltamethrin, permethrin, cypermethrin, and beta-cypermethrin, and resistance to pyrethroids is greater than resistance to other common insecticide groups (40–42). in cambodia, there is ae. aegypti adult resistance to the two main insecticides including permethrin (nets) and deltamethrin (fumigation) in all regions (126). the pyrethroids alpha-cypermethrin at 0.03%, bifenthrin at 0.2%, cyfluthrin at 0.15%, deltamethrin at 0.03%, etofenprox at 0.5%, lambda-cyhalothrin at 0.03% and permethrin at 0.25% were used for the bioassays of http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 10 http://jad.tums.ac.ir published online: march 31, 2023 adult ae. aegypti. the adult mortalities were 0 % for etofenprox, 1±1.9% for permethrin, 3.1± 4.1% for bifenthrin, 4.2±8.3% for lambdacyhalothrin, 10.2±7.1% for deltamethrin, 11± 4.5% for alpha-cypermethrin and 35±11% for cyfluthrin and there were low mortality rates with all the tested pyrethroid insecticides (50). in indonesia, ae. aegypti larvae were sensitive to pyrethroids (78, 127). aedes aegypti adult resistance was reported to more than one pyrethroids of cypermethrin, permethrin and d-allethrin (78). in other study, adult ae. aegypti resistance to permethrin and deltamethrin and high levels of resistance to alphacypermethrin and lambda cyhalothrin has been reported (128). in 2017, all ae. aegypti adults tested in jakarta were resistant to deltamethrin, permethrin, and lambda cyhalothrin, with a mortality rate of less than 90%. resistance to permethrin was higher than the others (79). also, resistance of ae. aegypti to deltamethrin, permethrin, lambda cyhalothrin, cyfluthrin in denpasar, bali has been reported (80). in banjarmasin, kalimantan, indonesia, all ae. aegypti populations showed different degree of resistance to 0.15% cyfluthrin 0.05% deltamethrin, 0.05% lambda-cyhalothrin, and 0.75% permethrin, with mortalities less than 90% (81). in laos high level of resistance against permethrin, and high susceptibility to deltamethrin was reported in ae. aegypti (129). in the other study seven populations of ae. albopictus were sensitive to permethrin and deltamethrin (43). in myanmar high pyrethroid resistance (permethrin and deltamethrin) has been reported in the larvae and adult of ae. aegypti (44). in singapore, ae. aegypti is the primary vector of dengue virus. the first case was reported in the 1960s (130). aedes aegypti resistance to permethrin, cypermethrin, and deltamethrin has been demonstrated in singapore (83-85). in other two studies, ae. aegypti larvae were resistant to permethrin and etofenprox (86), and ae. aegypti adult with permanet® had less than 80 % mortality (131). aedes aegypti from dili, timor-leste, has become resistant to permethrin, lambda-cyhalothrin and resmethrin (87). in the first insecticide resistance investigation of ae. aegypti in several locations of central highlands and nam bo in vietnam, the mosquitoes were susceptible to pyrethroids such as deltamethrin in the central and northern regions. in the southern and central mountainous regions ae. aegypti was resistant to deltamethrin, permethrin, lambda-cyhalothrin and alpha-cypermethrin. this is due to the long use of pyrethroids in the control of malaria and dengue fever and its use in agriculture, especially in the southern and central regions (46). in the next study, ae. aegypti's sensitivity to permethrin, lambda-cyhalothrin, deltamethrin, and alphacypermethrin was reported in many parts of the north and center but was resistant to these insecticides in southern and central vietnam (47). in 2009, it was reported that, ae. aegypti sensitivity to d-allethrin has decreased. mosquitoes were sensitive to pyrethroids in northern and central vietnam but were resistant in southern and central vietnam (132). in 2016 and 2003 also, ae. aegypti resistance was reported to permethrin and lambda-cyhalothrin (48, 133). also, populations of ae. albopictus tested from vietnam exhibited high-level resistance to permethrin (134). in 2010, study of pyrethroid susceptibility of larvae ae. albopictus collected from nagasaki city, japan, indicated populations of ae. albopictus tolerant to pyrethroids spread widely in nagasaki (116). in other study in this city insecticide susceptibility tests were performed on ae. albopictus adults and larvae of f1 colonies collected from nagasaki city, japan. the results were compared with those of several such colonies collected from other locations in japan. the larvae collected from nagasaki city, also from other locations in japan were resistant to dt80-allethrin and more than half of the adults of the nagasaki and fukuoka colonies showed resistance to permethrin (135). susceptibility of adult ae. aegypti mosquitoes from sri lanka to deltamethrin 0.05%, permethrin 0.75% were evaluated by the standard who mosquito bihttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 11 http://jad.tums.ac.ir published online: march 31, 2023 oassay protocol. resalts revealed resistance in all ae. aegypti populations for both permethrin (10–89%) and deltamethrin (40–92%) (71). in bangladesh ae. aegypti mosquitoes were exposed to diagnostic dose of 15μg/ bottle for permethrin, and 10μg/bottle for deltamethrin. high levels of resistance to permethrin were reported in ae. aegypti, while sensitivity to deltamethrin was different between populations (88) (fig. 4). in america high resistance to pyrethroids were observed in ae. aegypti. all grand cayman ae. aegypti populations survived after one hour of exposure to pyrethroid-impregnated papers (53). in the usa adult mortality after a 24h exposure to the pyrethroid insecticides (deltamethrin, prallethrin, and phenothrin) at discriminating doses showed that, all the field populations tested were susceptible (99–100% mortality) (54). in florida, california, north carolina, and texas, vectors were resistant to low doses of etofenprox. ae. aegypti was also resistant to high doses in texas. six populations of ae. albopictus showed susceptibility to bifenthrin and two populations ae. aegypti and ae. albopictus were resistant and possible resistance was shown in one ae. albopictus population. seven aedes spp. populations were susceptible (100%) to permethrin and one population was resistant. possible resistance was shown in one ae. albopictus population. all tested ae. albopictus were susceptible (100%) to phenothrin and one population of ae. aegypti was resistant. six ae. albopictus populations were susceptible to the low dose of deltamethrin. at the high deltamethrin dose, seven ae. albopictus populations were susceptible and two ae. albopictus and ae. aegypti populations showed possibility of resistance (91). aedes aegypti from new mexico showed high pyrethroid resistance and kdr mutation f1534c in the para gene (93). in texas/mexico border cities all populations of ae. aegypti showed resistance to permethrin, deltamethrin and sumithrin, although none of these insecticides are commonly used for vector control activities in this region (136). all ae. aegypti mosquitoes tested in jamaica were resistant to permethrin (92). laboratory bioassay on the island of martinique (caribbean) showed that ae. aegypti populations were strongly resistant to pyrethrins and deltamethrin therefore insecticide resistance decreased the efficacy of space sprays for adult mosquito and dengue control (95, 137). there is evidence of resistance to permethrin in populations in baja california north, south and in quintana roo, south of mexico and some states of northeast mexico due to use for more than 10 years in mexico for control of ae. aegypti (138-140). the results obtained from veracruz state mexico showed that the field strains of ae. aegypti were resistant to most of the pyrethroids analyzed including cypermethrin, deltamethrin, δ-phenothrin, α-cypermethrin, d-phenothrin, z-cypermethrin, λcyhalothrin, bifenthrin, and permethrin and suggested that populations in the state of veracruz were under the strong selection pressure, causing from the continuous use of permethrin for more than a decade (141). in cuba, ae. aegypti resistance was reported to deltamethrin, lambda cyhalothrin, beta-cypermethrin, cypermethrin, and cyfluthrin (55, 56). aedes aegypti from jamaica was resistant to permethrin with types of the mode of mechanism (92). one study on mosquitoes collected from eight latin american countries showed that larvae from the havana city strain were resistant to all the pyrethroids, the highest rr50 was to deltamethrin, as well as lambda cyhalothrin, betacypermethrin and cyfluthrin showed high resistance. larvae from santiago de cuba, peru and venezuela showed high deltamethrin rr50 values as well as havana city (98). populations of ae. aegypti from peru were exposed for 1h to papers treated with alpha-cypermethrin (0.05%), cypermethrin (0.05%), deltamethrin (0.05%), etofenprox (0.5 %), lambda-cyhalothrin (0.05%), and permethrin (0.75%). results showed ae. aegypti from chosica was resistant to alpha-cypermethrin, cypermethrin, etofenprox, also developing resistance http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 12 http://jad.tums.ac.ir published online: march 31, 2023 to lambda-cyhalothrin, and susceptibility to deltamethrin were reported. but the punchana and piura populations were resistant to all insecticides (63). in tocantins state in brazil the data showed different resistance to deltamethrin among the samples of ae. aegypti (110). in southern ecuador ae. aegypti was resistant to deltamethrin and alpha-cypermethrin (99). in study of insecticide resistance status of ae. aegypti from colombia, susceptibility to deltamethrin and cyfluthrin was reported in all mosquito populations studied. six populations were resistant to lambda-cyhalothrin. four field populations showed resistance to permethrin, two populations were also resistant to lambdacyhalothrin. pyrethroid resistance was not found in populations from the state of antioquia. resistance to etofenprox was reported in all mosquito populations, however, there is no record of using this insecticide for dengue control programs at any of the study sites (58). in other study the most of field populations were resistant to permethrin. the resistance to deltamethrin was observed only in one site and lambda cyhalothrin resistance in three sites (59). the lambda cyhalothrin resistance has been commonly detected in colombia (142). in venezuela ae. aegypti moderate levels of resistance to permethrin and lambda cyhalothrin was reported (60). aedes aegypti in different brazilian regions were resistant to deltamethrin, cypermethrin, permethrin (104–108, 113, 143). in brazil ae. albopictus adult mortality rates with permethrin and λ-cyhalothrin were close to 100% (109). evaluation of insecticide resistance in ae. aegypti populations the case of tocantins state in brazil showed different state of resistance to deltamethrin among the samples (110). in one study, severe resistance to deltamethrin with weak knockdown effect and low mortality was observed in french guiana ae. aegypti populations (111). aedes aegypti adults from santo domingo, dominican republic, were reported resistant to permethrin and deltamethrin (61). tests done with adult mosquitoes of ae. aegypti reported moderate resistance status to deltamethrin from guadeloupe and saint martin islands (101) (fig. 4). in europe in italy resistance to α-cypermethrin and permethrin in adult ae. albopictus has been reported. resistance to permethrin was seen from ferrara province in emilia-romagna and from bari province in puglia while the field-population from athens (greece) showed possibility of resistance. resistance to α-cypermethrin was reported for the mosquitoes in ferrara province venezia province and rome but four italian populations were susceptible. eight italian populations, and the albanian one, showed susceptibility to deltamethrin but the greek laboratory colony was resistant (144). in the swiss-italian border region ae. albopictus adult mortality rates with permethrin and λ-cyhalothrin were close to 100% (109). in spain, 5 populations of ae. albopictus were studied, four populations were sensitive to deltamethrin and permethrin. in the case of cypermethrin, two populations of ae. albopictus were sensitive to this toxin and two populations were tolerant and one population was reported to be resistant (145). aedes aegypti from funchal was reported to be resistant to cyfluthrin and permethrin. resistance to pyrethroids was also seen in the pau´l do mar population. pyrethroids mortality rates were between 2% (permethrin), and 88% (cyfluthrin) (114). populations of ae. albopictus tested from italy exhibited high-level resistance to permethrin (134) (fig. 4). in australia and oceania results a study in 1999 showed that there was no evidence of resistance to synthetic pyrethroids (deltamethrin and permethrin) (64). experiments on queensland mosquitoes with pyrethroids (bifenthrin, deltamethrin and lambda-cyhalothrin) did not show ae. aegypti resistant, and bioassays showed only weak tolerance. there was no evidence of kdr mutations in queensland ae. aegypti. in cairns south, a low resistance to lambda-cyhalothrin was rehttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 13 http://jad.tums.ac.ir published online: march 31, 2023 ported in a sample of ae. aegypti at 24h after exposure. obvious resistance to bifenthrin has not been detected in field populations of ae. aegypti from northern queensland (146). study of resistance of ae. aegypti and ae. albopictus in papua new guinea clearly show high pyrethroid resistance: deltamethrin and lambda cyhalothrin in both madang and port moresby ae. aegypti populations. the ae. aegypti population in port moresby show a higher level of deltamethrin resistance than madang where susceptibility to lambda-cyhalothrin has been greatly reduced (65) (fig. 4). resistant status of aedes aegypti and ae. albopictus to insect growth regulator (igrs) and microbial insecticides in africa in cameroon, gabon (12) and central african republic (15) larvae of both species were susceptible to bacillus thuringiensis israelensis (bti). in mayotte complete susceptibility to bti, and igrs including spinosad, diflubenzuron, pyriproxyfen, and methoprene was reported (66). in cape verde also was observed sensitivity to diflubenzuron and bti (67) (fig. 5). in asia in china, ae. albopictus larvae from all studied areas were sensitive to bti and hexafluoromoron, but in one population showed high resistance to pyriproxyfen and moderate resistance to pyriproxyfen (42). in singapore, larval mortality due to bti was 100% (86). in malaysia susceptibility of field ae. aegypti larvae to bti was reported (147), but ae. aegypti and ae. albopictus populations from 12 states in malaysia showed a different state of resistance to five igrs (pyriproxyfen, methoprene, cyromazine, novaluron and diflubenzuron). aedes albopictus was susceptible against cyromazine, pyriproxyfen, and methoprene. field-collected ae. aegypti showed high susceptibility to diflubenzuron, cyromazine, and novaluron (148). susceptibility of a wild larval population of ae. aegypti against bti, diflubenzuron, pyriproxyfen and spinosad was tested in vientiane laos. with bioassays of larvae was found that the wild ae. aegypti strain was moderately resistant to spinosad and susceptible to the other insecticides. based on field bioassays, all tested insecticides remained above the who acceptable larvicidal threshold after 28 weeks (82). in cambodia for larvae, moderate ae. aegypti resistance was reported for spinosad (rr90 < 5.6) but, there was no resistance against bti (rr90 < 1.6) (50) (fig. 5). in america the susceptibility ae. albopitus to imidacloprid, spinosad, and bti was investigated in alabama and florida. these results reported that mosquitoes collected from field were much more susceptible to spinosad than to the other insecticides tested in the study (89). of the eight usa populations of ae. albopictus, none were resistant to the larvicides tested (bti, spinosad, methoprene, and pyriproxyfen) but reduced susceptibility ae. albopictus to the igrs pyriproxyfen and methoprene was observed in florida and new jersey (54). the larvae of all studied ae. aegypti populations from different brazilian regions were susceptible to diflubenzuron and bti (106, 107, 109). a tolerance to igrs and full susceptibility to spinosad and bti was reported in martinique. in experiments pyriproxyfen and bti had 28and 37-weeks activities in permanent breeding containers, whereas under field conditions they could not control ae. aegypti populations after four weeks. but diflubenzuron and spinosad were effective for 16 weeks, therefore these chemicals can be alternatives to bti and temephos to control resistant ae. aegypti (112, 149). in french guiana, ulv spraying with bt was caused 100% mortality in ae. aegypti populations (111) (fig. 5). in europe the ae. albopictus populations from the canton of ticino in southern switzerland, and the como area in northern italy were all sushttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 14 http://jad.tums.ac.ir published online: march 31, 2023 ceptible to bti. the efficacy of lysinibacillus sphaericus, another entomopathogenic bacterium, was tested against ae. albopictus and showed good activity. the activity a mixture of bti and l. sphaericus crystals “vectomax®”, was also studied to determine if these insecticidal components are effective for controlling ae. albopictus. in this study ae. albopictus was also susceptible to diflubenzuron (109) (fig. 5). mechanisms of insecticide resistance in aedes aegypti and ae. albopictus the mode of action of insecticides is mostly on the nervous system of insects. target-site mutations and metabolic resistance are two main resistance mechanisms in ae. aegypti and ae. albopictus. in the mutation at the target site, which is one of the worst resistance mechanisms in insects, the insect makes the insecticides ineffective by changing the structure of the targets of the insecticides. pyrethroids and ddt act on voltage-gated sodium channels, and the mutation in the amino acid sequence of this gene causes the sensitivity of the channels to prevent the binding of pyrethroids and ddt to decrease. the use of pyrethroids with cross-resistance mechanisms (especially kdr mutations) existing in ddt caused to the rapid rise of pyrethroid resistance. also, resistance to acetylcholinesterase (ache) is current in aedes and is related to organophosphate and carbamate insecticides. mutations in the gene coding for the neurotransmitter acetylcholinesterase number one (ace-1) decrease the inhibition effect of the insecticide on the enzyme. metabolic resistance is very current in mosquitoes and has been observed against all insecticides used for vector control. mosquitoes have enzyme systems that protect them from foreign compounds, and some of these enzymes can break down insecticides before they reach their site of action. in metabolic resistance, the enzymes that detoxify the insecticide are overexpressed or the enzyme's affinity for the insecticide is altered by amino acid substitution. three of the most important of these enzymes are cytochrome p450 monooxygenases, glutathione s-transferases (gsts) and esterases (62, 150, 151). bti bacterial larvicide due to the presence of an intact endotoxin complex and synergy among individual toxins, especially the presence of cyt1a, it has the lowest risk of developing resistance. bti plays an important role in reducing the development of resistance and maintaining sensitivity in other biolarvicids. binary toxins from bacillus sphaericus have many advantages in controlling mosquito larvae. laboratory data and field studies worldwide showed that a combination of bti with b. sphaericus, the best way to increase larvicidal activity is to prevent resistance and restore susceptibility to b. sphaericus. in the case of igr insecticides, the risk of developing resistance is low. late 4th instar larvae and pupae with lower internal juvenile hormone titers are more susceptible to juvenile hormone analogs such as methoprene and pyriproxyfen in the transfer from late 4th instar larvae to pupae and adults. in aquatic habitats, different ages of mosquito larvae coexist, with very different levels of internal rejuvenation hormones. this phenomenon leads to sublethal exposures, tolerance and even the development of resistance (152). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 15 http://jad.tums.ac.ir published online: march 31, 2023 fig. 1. world mapping of organochlorine insecticide resistance in aedes aegypti and aedes albopictus up to december 2022 fig. 2. world mapping of organophosphates insecticide resistance in aedes aegypti and aedes albopictus up to december 2022 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 16 http://jad.tums.ac.ir published online: march 31, 2023 fig. 3. world mapping of carbamates insecticide resistance in aedes aegypti and aedes albopictus up to december 2022 fig. 4. world mapping of pyrethroids insecticide resistance in aedes aegypti and aedes albopictus up to december 2022 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 1–27 ts asgarian et al.: worldwide status of … 17 http://jad.tums.ac.ir published online: march 31, 2023 fig. 5. world mapping of insect growth regulator (igrs) and microbial insecticides insecticide resistance in aedes aegypti and aedes albopictus up to december 2022 conclusions who recommended bendiocarb, propoxur, ddt, fenitrothion, malathion, pirimiphos-methyl, á-cypermethrin, bifenthrin, cyfluthrin, deltamethrin, etofenprox, lambda cyhalothrin has been recommend as indoor residual spraying for mosquito control. alpha-cypermethrin, cyfluthrin, deltamethrin, deltamethrin, etofenprox, lambda cyhalothrin, permethrin are being used for impregnation of bednets. fenitrothion, malathion, pirimiphos-methyl, bioresmethrin, cyfluthrin, cypermethrin, cyphenothrin, d,dtrans-cyphenothrin, deltamethrin, d-phenothrin, etofenprox, lambda cyhalothrin, permethrin, resmethrin for thermal fog and space spraying. fuel oil, b. thurigiensis, chlorpyrifos, diflubenzuron, fenthion, methoprene, novaluron, pyriproxyfen, pirimiphos-methyl, and temephos are recommended as larvicides. because of importance of ae. aegypti and ae. albopictus in transmission mosquito-borne arboviruses, resistance management programs and strategies should be further considered in worldwide to prevention from 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s (ed): insecticides resistance. intech europe, rijeka, croatia, pp. 135–154. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 204 original article anaplasma infection in ticks, livestock and human in ghaemshahr, mazandaran province, iran nasibeh hosseini-vasoukolaei 1, mohammad ali oshaghi 1, parviz shayan 2, hassan vatandoost 1, farhang babamahmoudi 3, mohammad reza yaghoobi-ershadi 1, *zakkyeh telmadarraiy 1, fatemeh mohtarami 1 1department of medical entomology and vectors control, school of public health, tehran university of medical sciences, tehran, iran 2deptment of parasitology, faculty of veterinary medicine, university of tehran, iran 3department of infectious diseases, mazandaran university of medical science, sari, iran (received 4 may 2013; accepted 25 jan 2014) abstract background: anaplasmosis is an important issue for animal breeders in terms of economic losses as well as a health concern to human. ticks are considered as the main vector of this disease. lack of documented information about anaplasma species in iran was the scope of this study to determine the population of ticks and the presence of anaplasma in ticks, domestic ruminants and also human beings in northern iran. methods: a total of 101 unengorged hard ticks, 78 domestic ruminants and 40 human blood samples collected from ghaemshahr, mazandaran province, northern iran were tested by nested pcr against 16s rrna gene of anaplasma species. results: positive pcr was found in 50 ticks, 28 sheep, 2 cattle, one goat, and 10 human specimens. sequence analysis of the pcr products confirmed presence of a. ovis in two rhipicephalus sanguineus and two ixodes ricinus ticks, one human and 4 sheep samples. moreover one boophilus annulatus tick and one sheep sample were infected with a. bovis. furthermore one sample of sheep was infected with a. centrale. conclusion: this study is the first report of tick infection to a. ovis, a. bovis and human infection to a. ovis in iran. the result of this study is a survey of anaplasma infections from ticks, domestic animals and human in iran which help to have appropriate prevention measures for anaplasmosis. keywords: anaplasma, human, iran, livestock, tick introduction anaplasmosis, a disease caused by various species of anaplasma, poses important economic constraints to animal breeders. besides the costs of the additional veterinary care, anaplasmosis causes abortion in animals, reduction of milk production, body weight, and frequently leads to death (stuen et al. 2003). members of the genus anaplasma are obligatory intracellular gram negative bacteria that infect blood cells of mammals. six anaplasma species are currently recognized (dumler et al. 2001). vertebrates are main reservoirs of the anaplasma bacteria, however in many cases bacteria from the genus anaplasma cause diseases in domestic animals and human. anaplasma ovis invades and reproduces within erythrocytes. this bacterium induces acute anemia in sheep and goats (splitter et al. 1956). anaplasmosis in cattle is caused by a. bovis infecting monocytes (uilenberg 1993), or by a. marginale and a. centrale which parasitize and replicate in red blood cells (kuttler 1966). anaplasma bovis is reported mostly from cattle, but also detected in small ruminants *corresponding author: dr zakkyeh telmadarraiy, e-mail: ztelma@yahoo.co.in http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 205 which could be a reservoir of this bacterium (goethert et al. 2003). ixodid ticks play an important role in maintaining anaplasma species in nature. it is evidenced that various species of ixodes, dermacentor, rhipicephalus and amblyomma genera are the main vectors of the anaplasma bacteria in different regions of the world. rhipicephalus sanguineus, a common tick vector for anaplasma, has been reported from india, the united states, all regions of africa, and around the mediterranean basin (stafford 2007). agriculture and animal husbandry is the main activities of people in mazandaran province, northern iran, and anaplasmosis is one of the major veterinary health problems there. however, there have been only a few studies to detect tick anaplasmosis infections in the country (bashiribod et al. 2004, spitalska et al. 2005). these two studies reported a. phagocytophilum and ehrlichia ovina infection in ticks in north and counter parts of iran. due to the lack of documented information about anaplasma species in ticks, animals and also human beings in the country and having found clinical features and laboratory findings similar to anaplasmosis in some shepherds in suburban areas of ghaemshahr county in northern iran during recent years, we conducted the present study to understand more about the anaplasma infections in iran. materials and methods sampling the study was carried out in ghaemshahr county in north of iran, in which a number of suspected cases of anaplasmosis were reported (mahmoudi 2004). the collection site was villages of suburban forest area in ghaemshahr in which the climate is subtropical with cold winters and moderate summers. about 425 domestic ruminants (361 sheep, 54 goats and 10 available cattle) from 18 herds of nine villages in ghaemshahr were inspected for tick infestation. the whole body of each animal was inspected and the ticks were manually removed from animals’ body. the sampling was done through 2008 in a period corresponding to seasonal tick activity. collected ticks from infested animals were kept in dry plastic tubes containing few fresh grass leaves covered by a lid containing several minute holes. tubes were labeled and conditioned under room temperature for a few days, and then they were dispatched to the laboratory. the purpose of this procedure was to maintain ticks alive inside the tubes until the laboratory taxonomic identification. ticks species identification was done by using the criteria key described by hoogstraal (hoogstraal 1979). totally 323 ticks were collected from which 101 unengorged ticks were selected for pathogen detection by pcr examination. blood samples were additionally taken from both domestic ruminants and corresponding voluntary shepherds. blood samples were taken from jugular vein of sheep and goat and from caudal vein of cattle. a total of 78 blood samples including 65 from sheep, nine from cattle and four from goats were collected, from which 38 were from animals with tick infestation and 40 from animals without any tick infestation. forty samples were taken from median cubital vein of corresponding voluntary shepherds from different age groups. all animals and humans had not any clinical signs. all tissue had been obtained with consent given according to the institutional guidelines. dna extraction and pcr amplificationsequencing dna extraction carried out from ticks and blood samples using the g-spin™ genomic dna extraction kit (intron biotechnology, korea) and i-genomic blood http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 206 dna extraction mini kit (intron biotechnology, korea), respectively following the manufacturer protocol. anaplasma dna was detected by means of nested pcr with primers derived from the 16s rrna gene of anaplasma species. pcr was performed in 20 µl reaction mixture containing 10 mm tris-hcl (ph 9.0), 30 mm kcl, 1.5 mm mgcl2, 250 mm each dntp, 0.5 mm each sense and antisense primers, 1 u taq dna polymerase and 2 µl of dna. the conditions for pcr included an initial denaturation at 94 ˚c for 3 min followed by 35 cycles of amplification (1 min denaturation at 94 ˚c, 1 min annealing at 57 ˚c and 1 min elongation at 72 ˚c) (rar et al. 2008). the primer pair used for pcr were ehr1 (5´-gaacgaacgct ggcggcaagc-3´) and ehr2 (5´-agta [t/c]cg[a/g]accagatagccgc-3´). nested pcr was performed with 2 µ l of the first pcr reaction using primer pair ehr3 (5´-tgcataggaatctacctagtag-3´) and ehr4 (5´-ctaggaattccgctatcc tct-3´) and resulted in a 524-bp product. the pcr procedure was with exception of annealing temperature (60 ˚c), the same as described above (rar et al. 2008). as positive control we used anaplasma dna obtained from department of parasitology, faculty of veterinary medicine, university of tehran (noaman et al. 2009), and double distilled water as negative control was used. the pcr analysis was performed on agarose gel using ethidium bromide and uv condition. the pcr products were directly subjected to sequencing by seqlab (gmbh, germany). the nucleotide sequences were then blasted by blastn (http://www.ncbi.nlm. nih.gov/blast) program. results blood and ticks infected by anaplasma species overall 102 (24%) out of 425 inspected animals and in particular 28.25% (88/361) sheep, 22.22% (12/54) goats and 20% (2/10) cattle were infested with ticks. altogether 323 ticks were collected categorized into four genera and six species: rhipicephalus sanguineus (266, 82.35%), r. bursa (1, 0.31 %), ixodes ricinus (49, 15.17%), boophilus annulatus (4, 1.24%), haemaphysalis punctata (1, 0.31%) and h. numidiana (2, 0.62%). rhipicephalus sanguineus with 82.35% was observed as the most abundant tick species found in the study area. totally 101 unengorged ticks were examined by nested pcr for presence of anaplasma species. a 524-bp 16s rrna gene fragment of anaplasma species was identified in 49.5% of examined ticks (50 out of 101), including r. sanguineus (34/54, 62.96%), i. ricinus (14/39, 35.9%), b. annulatus (1/4, 25%), and h. punctata (1/1, 100%). similarly, blood samples from ruminants also contained anaplasma dna. genome of anaplasma species was detected in 39.74% (31/78) blood samples, and in particular 43.08% (28/65) sheep, 22.22% (2/9) cattle, and 25% (1/4) goats were infected (table 1, fig. 1). thirty eight out of 78 tested blood samples, belonged to the ruminants infested with ticks. nineteen out of 38 (50%) were infected with anaplasma, and in 16 cases, anaplasma dna was detected in both the tick and its host simultaneously. of 78 blood samples, 40 were collected from ruminants without tick infestation; in 12/40 (30%) infection to the bacteria were revealed. this study also is the first which tested human blood by molecular methods for the presence of anaplasma species in iran. blood samples from 40 people between 15 and 78 years old were examined by nested pcr with the same primers. dna of anaplasma species was detected in 25% (10/40) blood samples, and in particular six of 22 (27.27%) female and four of 18 (22.22%) male. most of the infected cases http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 207 were from shepherds older than 40 years old and in contact with livestock for most of their lives (table 1, fig. 1). sequencing a number of positive pcr samples against anaplasma in ticks separated from sheep (5), sheep blood (6), and human blood (1) were sequenced by the forward and reverse primers and the consensus sequences were submitted to genbank. all nucleotide sequences were registered in genbank under accession numbers jf51 4503, jf514504, jf514505, jf514506, jf49 5135, jf514507, jf514510, jf514511, jf51 4512, jf514513, jf514508 and jf514509. among the specimens, two r. sanguinus (jf514510, jf495135), two i. ricinus (jf514 503, jf514511), three sheep (jf514504, jf 514505, jf514506) and one human (jf5145 07) with 484 bp length were 100% identical to a. ovis present in genbank database. one specimen originated from sheep (jf514512) was identical to a. ovis except for one nucleotide transmission (c/t). nucleotide sequences of a. bovis determined in one female b. annulatus tick (jf 514508) and one sheep (jf514513) were identical to each other. also one blood sample from a female sheep was infected with a. centrale (jf514509). the present study for the first time demonstrated the presence of a. ovis in human, r. sanguineus and i. ricinus, and a. bovis in b. annulatus in iran. table 1. results of nested pcr for detection of anaplasma species in ticks, domestic animal and human blood samples collected from ghaemshahr, iran, 2008 male total ticks rhipicephalus sanguineus 11/ 16 (68.75) 34/ 54 (62.96) rhipicephalus bursa 0 0/1 (0) ixodes ricinus 1/6 (16.67) 14/39 (35.9) boophilus annulatus 0/1 (0) 1/4 (25) haemaphysalis punctata 1/1 (100) 1/1 (100) haemaphysalis numidiana 0 0/2 (0) domestic ruminants sheep 9/15 (60) 28/65 (43.08) goat 0 1/4 (25) cattle 0 2/9 (22.22) human age groups < 20 years 0/3 (0) 1/5 (20) 20-30 years 1/4 (25) 1/7 (14.29) 30-40 years 0/3 (0) 0/8 (0) 40-50 years 1/5 (20) 3/9 (33.33) 50-60 years 1/2 (50) 3/6 (50) > 60 years 1/1 (100) 2/5 (40) http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 208 fig. 1. nested pcr amplicons of a 524-bp anaplasma 16s rrna gene fragment from biological samples (tick and blood). m, 100-bp size marker (fermentase, germany), lane 1, negative (water) control, lane 2, positive control, lane 3–7, anaplasma positive samples of tick, sheep, cattle, goat and human blood, respectively discussion the main vectors of the anaplasma bacteria are ticks, especially the genera ixodes, dermacentor, rhipicephalus and amblyomma. during our study six species of hard ticks were identified: r. sanguineus, r. bursa, i. ricinus, b. annulatus, h. punctata, and h. numidiana. half of these species are known as anaplasma vectors. out of 323 collected ticks from animals, a significant number of 266 r. sanguineus were identified. this species stands out as being the most prevalent tick species comprising 82.35% of the ticks collected from domestic ruminants in ghaemshahr. a total of 101 unengorged ticks were assayed by nested pcr for presence of anaplasma species. most of (34/50, 68%) the infected ticks belonged to r. sanguineus species. therefore r. sanguineus with a high level of infestation of domestic ruminants, and the prevalence of anaplasma dna (62.96%) could be the most abundant vector of anaplasma species in ghaemshahr. the nucleotide sequence determination deriving from one male and one female r. sanguineus were identical to that of the gene of a. ovis (jf495135, jf514510). this is in agreement with the results of other researches indicating the role of r. sanguineus in anaplasma transmission. previous study in africa had shown presence of a. platys dna in a female r. sanguineus (sanogo et al. 2003). in a study in turkey a. ovis 16s rrna gene fragment was detected in two r. sanguineus ticks (aktas et al. 2009). in the present study a. ovis 16s rrna was detected in two i. ricinus ticks making this tick species as a probable vector of a. ovis in ghaemshahr. the tick i. ricinus has been reported from europe and northern africa (sarih et al. 2005), and was evidenced to be the vector of anaplasma and in particular a. phagocytophilum in europe (parola et al. 2005, silaghi et al. 2008, aktas et al. 2009, portillo et al. 2011). in iran, i. ricinus ticks were found only in caspian sea regions in north part of iran (rahbari et al. 2007, razmi et al. 2007). in ghaemshahr, iran a. phagocytophilum 16s rrna was detected in 5 (5.1%) of tested i. ricinus ticks. it showed a high probability of human granulocytotropic ehrlichiosis (hge) in some stock-farmer patients from ghaemshahr suburban areas with similar clinical and laboratory findings to he (bashiribod et al. 2004). in brazil, a. marginale dna was detected in b. microplus ticks. b. annulatus could experimentally transmit this bacterium to calves (shimada et al. 2004). we detected a 524 bp 16s rrna gene fragment of a. bovis in one female b. annulatus collected from sheep in ghaemshahr. we could detect anaplasma sp. in one h. punctata tick collected from a sheep. h. punctata was experimentally able to transmit babesia major to calves. anaplasma phagocytophilum and a. bovis was detected in h. megaspinosa and h. longicornis in japan and korea, respectively (oh et al. 2009, yoshimoto et al. 2010). we examined two groups of blood samples obtained from animals with and without tick infestation. fifty percent (19/38) of samples from animals with tick infestation were anaplasma positive compared to 30% (12/ http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 209 40) positive samples taken from animals without any ticks on their bodies. these results demonstrate the importance of ticks in anaplasma transmission to domestic animals in the studied area. a few studies on the infectivity of animal blood samples to anaplasma have been done in iran; in khorasan province, north east of iran 80.3% of sheep and 38.92% of goats blood smears were infected with a. ovis (razmi et al. 2006). in isfahan province in the center of iran, 16s rrna gene fragment of a. marginale was detected in 50 % of cattle without any clinical signs (noaman et al. 2009). in the present study 39.7% of blood samples were infected with anaplasma. these blood samples were obtained from domestic ruminants without any clinical signs showing their role as reservoirs of anaplasma. during our study, a. ovis and a. bovis dna was detected in sheep blood samples. a. bovis is a bacterium detected mainly in cattle and small mammals (goethert et al. 2003), but in our study was detected in sheep. these results make sheep also as potential reservoirs of this bacterium. of 40 blood samples taken from people from different age groups 15–78 years, 10 (25%) were infected. there was not any significant difference between males and females showing equal cooperation of women and men in livestock husbandry in studied area. most of the infected cases were from shepherds older than 40 years old and in contact with livestock for most of their lives. one blood sample obtained from a 55-yearold woman without any clinical symptoms was infected with a. ovis. some researches in cyprus showed anaplasma spp. infections in humans (psaroulaki et al. 2008, chochlakis et al. 2009). in another laboratory testing of human blood samples by universal primers against all anaplasma species in cyprus, a. ovis 16s rrna was found in one human blood sample taken from a 27-year-old woman with thrombocytopenia and elevated levels of transminses (chochlakis et al. 2010). in iran, a. ovis was identified in sheep (spitalska et al. 2005, razmi et al. 2006). since sheep are reservoirs of a. ovis, infection of humans with this pathogen may occur, but transmission of a. ovis to humans is uncertain. r. sanguineus and i. ricinus are dominant tick species in sheep in northern iran (rahbari et al. 2007, hosseini vasoukolaei et al. 2010). results revealed that r. sanguineus highly infested domestic ruminants and a. ovis dna was detected in this tick species. ixodes ricinus role as a vector of a. phagocytophilum to human has been suggested (bashiribod et al. 2004). however, potential role of r. sanguineus and i. ricinus as vectors of a. ovis to human is unknown. conclusion this study is the first to report molecular detection of a. ovis from human in iran, whereas does not show a. ovis human pathogenicity. however, further epidemiological, clinical and pathological investigations are needed to understand a. ovis potential to infect human. infection of ticks, ruminants and humans to anaplasma species demonstrated the potential endemicity and circulation of the pathogen among different tick species found in the region. ticks of r. sanguineus are probably the main vector of anaplasma in northern part of iran. application of control measures with emphasizing on control of r. sanguineus could prevent anaplasma transmission in the region. acknowledgements this study was funded by the school of public health of tehran university of medhttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 204–211 n hosseini-vasoukolaei et al.: anaplasma infection … 210 ical sciences (grant no. 8768-63-02-88). we are thankful to staff of ghaemshahr hospital for their sundry precious contribution. the authors declare that there is no conflict of interests. references aktas m, altay k, dumanli n, kalkan a (2009) molecular detection and identification of ehrlichia and anaplasma species in ixodid ticks. parasitol res. 104: 1243–1248. bashiribod h, kazemi b, eslami g, bigdeli s, bandehpour m, rahbarian n, ramezani z (2004) first molecular detection of anaplasma phgocytophilum in ixodes ricinus ticks in iran. j med sci. 4: 282–286. chochlakis d, koliou m, ioannou i, tselentis y, psaroulaki a (2009) kawasaki disease and anaplasma sp. infection of an 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of domestic animals. oxford pergamon press, oxford, pp. 293–332. yoshimoto k, matsuyama y, matsuda h, sakamoto l, matsumoto k, yokoyama n, inokuma h (2010) detection of anaplasma bovis and anaplasma phagocytophilum dna from haemaphysalis megaspinosa in hokkaido, japan. vet parasitol. 168(1–2): 170–172. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 102 short communication species composition and ecological aspects of immature mosquitoes (diptera: culicidae) in bromeliads in urban parks in the city of são paulo, brazil walter ceretti-junior 1, rafael de oliveira christe 1,2, marco rizzo 3, regina claudia strobel 3, marco otavio de matos junior 4, maria helena silva homem de mello 4, aristides fernandes 1, antônio ralph medeiros-sousa 1, gabriela cristina de carvalho 1, *mauro toledo marrelli 1,2 1department of epidemiology, school of public health, university of são paulo, são paulo, brazil 2tropical medicine institute, university of são paulo, são paulo, brazil 3office of parks and green areas, department of the environment and green areas, são paulo city hall, são paulo, sp, brazil 4laboratory for research into and identification of synanthropic fauna, zoonosis control center, health surveillance coordination unit, municipal health department, são paulo city hall, são paulo, sp, brazil (received 26 apr 2014; accepted 6 dec 2014) abstract background: bromeliads can be epiphytic, terrestrial or saxicolous and use strategies to allow water to be retained in their leaf axils, where various arthropods can be found. these include mosquitoes, whose larvae are the most abundant and commonly found organisms in the leaf axils. the objective of this study was to look for immature forms of mosquitoes (the larval and pupal stages) in bromeliads in municipal parks in são paulo and to discuss the ecological and epidemiological importance of these insects. methods: from october 2010 to july 2013, immature mosquitoes were collected from bromeliads in 65 municipal parks in the city of são paulo, brazil, using suction samplers. the immature forms were maintained until adult forms emerged, and these were then identified morphologically. results: two thousand forty-two immature-stage specimens belonging to the genera aedes, culex, trichoprosopon, toxorhynchites, limatus and wyeomyia were found in bromeliads in 15 of the 65 parks visited. aedes albopictus was the most abundant species (660 specimens collected), followed by culex quinquefasciatus (548 specimens) and cx. (microculex) imitator (444). the taxa with the most widespread distribution were ae. aegypti and toxorhynchites spp, followed by ae. albopictus and cx. quinquefasciatus. conclusion: bromeliads in urban parks are refuges for populations of native species of culicidae and breeding sites for exotic species that are generally of epidemiological interest. hence, administrators and surveillance and mosquito-control agencies must constantly monitor these microenvironments as the presence of these species endangers the health of park users and employees as well as people living near the parks. keywords: mosquitoes, bromeliads, municipal parks, culicidae, breeding sites, immature, brazil introduction members of the family bromeliaceae can be epiphytic, terrestrial or saxicolous. the taxon is essentially neotropical, with more than 3,100 described species distributed in 58 genera. in brazil, where 40 % of these species are found, the highest diversity is in the atlantic forest (givnish et al. 2011). bromeliads use strategies that allow water and organic debris to be retained in their leaf axils, forming rosettes or tanks from which they acquire nutrients. these structures also act as biotopes for various organisms. central and lateral bromeliad tanks provide a permanent habitat for many terrestrial arthropods that have aquatic immature stages (frank et al. 2004), such as insects of the order diptera, whose larvae are the most abundant and commonly *corresponding author: dr mauro toledo marrelli, e-mail: mmarelli@usp.br http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 103 found organisms in these biotopes (derraik and heath 2005). among the most common dipterans, the culicids include species whose immature forms are specialists in colonizing these microhabitats as well as species that are occasional visitors (frank and lounibos 2009). the family culicidae currently has around 3,500 valid described species worldwide (harbach 2014). many culicid species are of great importance in public health because the blood-feeding habits of the females are associated with allergies and the transmission of pathogens (consoli and lourenço-de-oliveira 1994, forattini 2002). the genera aedes, anopheles and culex include important vectors of diseases such as malaria, dengue and filariasis in various regions of the planet (who 2013a, bhatt et al. 2013, who 2013b). in addition, a number of mosquito-borne diseases, such as the west nile virus, chikungunya and yellow fever were or are public health problems in many countries (weaver 2013). brazil has a great variety of culicids, 490 species having been described to date (wrbu 2014). in brazil, malaria, which is transmitted by anopheles, and dengue, which is transmitted by ae. aegypti, are together responsible for thousands of cases of morbidity. also worthy of note is lymphatic filariasis, which is transmitted by cx. quinquefasciatus in some cities in the northeast of the country, as well as various arboviruses that circulate in natural environments and cause sporadic cases of epizootics or even human infections such as saint louis encephalitis, mayaro encephalitis, equine encephalitis, rocio encephalitis and wild yellow fever (forattini 2002, oliveiraferreira et al. 2010, viana and ignotti 2013). the city of são paulo, located in southeastern brazil, is the largest city in south america and has more than 11 million inhabitants. mosquito-borne diseases in the city include dengue, outbreaks of which occur every year and which was responsible for 2,617 confirmed autochthonous cases in 2013 (são paulo 2014a). malaria outbreaks due to plasmodium vivax and p. malariae in the atlantic forest have been detected on the edge of the city of são paulo (couto et al. 2010, duarte et al. 2013). the ilhéus virus was isolated in birds in the tietê ecological park, in the municipality of são paulo (pereira et al. 2001). in regions of the atlantic forest close to the city of são paulo, antibodies for various arboviruses that cause different types of encephalitis were detected in birds (ferreira et al. 1994) and there have been sporadic cases of wild yellow fever in humans and primates, the virus responsible for the disease having been isolated in mosquitoes from the species haemagogus leucocelaenus (souza et al. 2009). as urbanization continues apace, many cities around the world strive to preserve their green areas (koh and sodhi 2004). the construction, preservation and rehabilitation of green areas in parks or plazas is intended to provide leisure facilities for the population, preserve remnants of flora and fauna and reduce the environmental and social stress caused by urbanization (mcintyre 2000, mckinney 2008). the city of são paulo currently has a hundred public parks, many of which contain fragments of the atlantic forest and representatives of native or exotic flora and fauna (são paulo 2014b). the diversity of culicids found in some of these green fragments has been investigated in a variety of studies (urbinatti et al. 2001, taipe-lagos and natal 2003, montes 2005, ribeiro et al. 2012). a recent preliminary survey in fifty-nine urban parks in the city of são paulo found that some medically important species of culicids, such as ae. albopictus, ae. fluviatilis, ae. scapularis, cx. nigripalpus, and cx. quinquefasciatus, are quite common in these environments (medeiros-sousa et al. 2013). these and other species maintain their populations using a variety of natural or artificial containers found in these green fragments that allow their immature forms to develop. bromeliads can act as breeding grounds for many species of epidemiologic http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 104 interest in the city as they are very common in parks, where they occur naturally and in some cases are planted as part of the landscape. the aim of this study was to investigate the species composition and ecological aspects of mosquitoes collected in bromeliads in urban parks in são paulo to determine how important these plants are for the maintenance of populations of native or exotic culicid species, with a particular emphasis on species of epidemiological interest. materials and methods the city of são paulo (23.54° w 46.63° s) is located in the atlantic forest in the southeast of brazil at an altitude of 750 m above sea level and with a mean relative humidity of 78 % (fig. 1). the climate is considered temperate tropical, and there is reduced rainfall in the winter. mean annual temperature is 20.7 °c, winters are mild (15 to 21 °c) and summer temperatures are moderately high (22 to 27 °c). mean annual rainfall is 1,376 mm, and most of the rainfall is concentrated in the summer months (ibge 2014). the species composition of immature culicids in fifteen parks with bromeliads in the city of são paulo was investigated over thirty-four months. data collected by medeiros-sousa et al. (2013) between october 2010 and february 2011 were used, together with data from collections made between march 2011 and july 2013. bromeliads planted in the ground (terrestrial bromeliads) and bromeliads on tree trunks (epiphytic bromeliads) at heights ranging from 1.0 to 5.0 m were sampled. immature specimens were actively sought out in the central and lateral leaf axils of bromeliads using suction samplers (fig. 2). the extracted contents were then transferred to small recipients (400ml) and analyzed for the presence of mosquito larvae and pupae. the larvae and pupae collected were taken to the public health entomology laboratory at the faculty of public health, university of são paulo, where they were maintained until the emergence of adult forms, which were identified morphologically based on lane (1953), consoli and lourenço-deoliveira (1994) and forattini (2002), as well as by comparing them with standard specimens in the collection at the faculty of public health, university of são paulo. the abbreviations for genera and subgenera used here follow the standardization proposed by reinert (2009). after all the individuals collected had been identified morphologically, the number of individuals in terrestrial and epiphytic bromeliads in each park broken down by species was recorded in tabular form (table 1). the frequency with which each species was found in terrestrial and epiphytic bromeliads was calculated to determine whether there was an association between culicid species and type of bromeliad (table 2). results overall, 2,042 immature-stage specimens belonging to 6 genera (aedes, culex, trichoprosopon, toxorhynchites, limatus and wyeomyia) and 17 species were found in 15 of the 65 parks visited (fig. 1, table 1). table 1 shows the number of specimens of each species collected and the type of bromeliad (epiphytic or terrestrial) in which collections were made. sixty-six of the specimens collected were classified in the taxon tx. spp and five in wy. spp either because they died before the adult form emerged or because of limitations in the keys for morphological identification, which were out of date. the only clear association between culicid species and type of bromeliad (table 2) was for cx. quinquefasciatus, one of the most abundant species, which was found exclusively in terrestrial bromeliads. the bromeliads found in the parks belonged to the genera aechmea, alcantarea, edmundoa, neoregelia, nidularium, quesnelia and vriesea, all of which are http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 105 common in the atlantic forest. aedes albopictus was the most abundant mosquito species collected (660 specimens or 32.35% of the total), followed by cx. quinquefasciatus (548 specimens or 26.86%) and culex (microculex) imitator (444 or 21.76%). another epidemiologically important species collected was ae. aegypti (122 or 5.98%). the other species together accounted for only 13.05% of the total number collected (table 1). the taxa with the widest distributions were ae. aegypti and tx. spp., found in eight parks, followed by ae. albopictus (seven parks) and cx. quinquefasciatus (five parks). the parks with the highest species richness were anhanguera park, ibirapuera park and santo dias park, each of which had eight species, followed by previdência park with six species, piqueri park with five and burle marx park with four. no more than three species were recorded for any of the other parks (table 1). the largest numbers of specimens independently of species were collected in piqueri park (1,019) and santo dias park (632), which together accounted for almost three quarters of all the specimens collected and had the largest number of bromeliads. ibirapuera park and park mario covas, where 136 and 102 individuals were collected, respectively, also accounted for a significant number of the sample population. the remaining parks together accounted for just 151 specimens. fig. 1. location of the fifteen urban parks in the city of são paulo, brazil, where immature stages of culicids were found in bromeliads between october 2010 and july 2013 fig. 2. collection of immature mosquitoes in leaf axils of bromeliads using suction samplers in urban parks in the city of são paulo, brazil, between october 2010 and july 2013. (a) terrestrial bromeliad, (b) epiphytic bromeliad http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 106 table 1. number of immature mosquitoes collected by species for each urban park and type of bromeliad (terrestrial or epiphytic) collected between october 2010 and july 2013 in the city of são paulo, brazil taxon a n h an gu er a b u rl e m ar x c ar m o c ol in as d e s ão f ra n ci sc o c h ic o m en d es g u ar ap ir an ga ib ir ap u er a l aj ea d o m ár io c ov as n ab u co p iq u er i p re vi d ên ci a r od ri go d e g as p er i s an to d ia s s ev er o g om es t ot al f re q u en cy ( % ) t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic t er re st ri al e p ip h yt ic aedes (stegomyia) aegypti (linnaeus, 1762) 3 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 2 0 0 0 72 19 2 2 0 7 0 13 0 0 122 5.97 aedes (stegomyia) albopictus (skuse, 1895) 16 0 7 0 0 0 0 0 0 5 0 0 28 14 0 0 0 0 0 0 90 467 2 7 0 0 3 21 0 0 660 32.3 aedes (ochlerotatus) fluviatilis (lutz, 1904) 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0.3 culex (culex) chidesteri dyar, 1921 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0.05 culex (culex) quinquefasciatus say, 1823 15 0 0 0 0 0 0 0 0 1 0 0 70 0 0 0 100 0 0 0 362 0 0 0 0 0 0 0 0 0 548 26.8 culex (microculex) imitator imitator theobald, 1903 0 0 0 0 0 0 1 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 78 362 0 0 444 21.7 culex (microculex.) group imitator (theobald, 1903) 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 13 23 0 0 40 1.9 limatus durhami theobald, 1901 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 1 0 0 0 0 0 0 8 0.4 trichoprosopon (trichoprosopon) pallidiventer (lutz, 1905) 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 3 0.15 toxorhynchites bambusicola (lutz , neiva, 1913) 1 0 0 0 0 0 0 0 0 0 0 0 3 2 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 2 9 0.4 toxorhynchites solstitialis (lutz, 1904) 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0.1 toxorhynchites spp. theobald, 1901 9 0 29 0 0 0 0 2 0 0 5 0 8 0 0 0 0 0 0 1 0 0 0 8 0 0 1 3 0 0 66 3.2 wyeomyia (phoniomyia) davisi (lutz, 1904) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 52 0 0 59 2.9 wyeomyia (miamyia) oblita (lutz, 1904) 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0.15 wyeomyia serratoria (dyar , nunez tovar, 1927) 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 2 0.1 wyeomyia (phoniomyia) cf galvaoi (correa, ramalho, 1956) 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 51 0 0 64 3.1 wyeomyia. spp theobald, 1901 0 0 4 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0.2 total no. of individuals 49 0 41 0 0 3 1 2 0 7 14 0 119 17 1 0 102 0 0 1 533 486 6 19 0 7 106 526 0 2 2,042 100 total no. of species 8 4 3 2 3 2 8 2 2 1 5 6 1 8 1 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 107 table 2. number and frequency of culicids in decreasing order of abundance in terrestrial and epiphytic bromeliads collected between october 2010 and july 2013 in urban parks in the city of são paulo, brazil species n. in terrestrial bromeliads frequency in terrestrial bromeliads n. in epiphytic bromeliads frequency in epiphytic bromeliads total no. of specimens aedes (stegomyia) albopictus (skuse, 1895) 146 0.22 514 0.77 660 culex (culex) quinquefasciatus say, 1823 547 0.99 1 0.002 548 culex (microculex) imitator imitator theobald, 1903 82 0.18 362 0.81 444 aedes (stegomyia) aegypti (linnaeus, 1762) 80 0.65 42 0.34 122 toxorhynchites spp theobald, 1901 52 0.78 14 0.21 66 wyeomyia (phoniomyia) cf. galvaoi (correa, ramalho, 1956) 13 0.20 51 0.79 64 wyeomyia (phoniomyia) davisi (lutz, 1904) 7 0.11 52 0.88 59 culex (microculex.) group imitator (theobald, 1903) 15 0.37 25 0.62 40 toxorhynchites bambusicola (lutz , neiva, 1913) 4 0.44 5 0.55 9 limatus durhami theobald, 1901 7 0.87 1 0.12 8 aedes (ochlerotatus) fluviatilis (lutz, 1904) 6 1.0 0 0.0 6 wyeomyia spp theobald, 1901 5 1.0 0 0.0 5 trichoprosopon (trichoprosopon) pallidiventer (lutz, 1905) 3 1.0 0 0.0 3 wyeomyia (miamyia) oblita (lutz, 1904) 3 1.0 0 0.0 3 toxorhynchites solstitialis (lutz, 1904) 1 0.5 1 0.5 2 wyeomyia serratoria (dyar , nunez tovar, 1927) 1 0.5 1 0.5 2 culex (culex) chidesteri dyar, 1921 0 0.0 1 1.0 1 total no. of individuals per type of bromeliad 972 0.48 1,070 0.52 2,042 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 108 discussion this study, the first to investigate immature mosquitoes in bromeliads in municipal parks in the city of são paulo, brazil, found seventeen species or groups from the genera aedes, culex, trichoprosopon, toxorhynchites, limatus and wyeomyia in these plants. the findings were similar to those of previous studies except that we failed to find species from the subgenus kerteszia of anopheles, a group of mosquitoes found almost exclusively in bromeliads, some species of which are considered vectors of “bromeliad-malaria” in the atlantic forest of south and southeast brazil (consoli and lourenço-de-oliveira 1994, marques et al. 2012, ribeiro et al. 2012). this absence could be due to the urban or peri-urban nature of the parks visited, as this subgenus is considered a bioindicator of preserved environments (dorvillé 1996). of the sabethini tribe, which includes mosquito species with diurnal habits usually associated with less anthropic environments, only species of the genera limatus, trichoprosopon and wyeomyia were found. of these species, limatus durhami tends to be adapted to anthropic environments (consoli and lourenço de oliveira 1994), and immature forms of this species have been reported in artificial breeding sites such as waste tires near houses (zequi et al. 2005). like most other culicidae, the sabethini are potential vectors of several arboviruses and have been found carrying the pixuna, bussuquara, wyeomyia, ilhéus and yellow fever viruses (forattini 2002). in our survey, we recorded ae. aegypti and ae. albopictus in eight and seven parks, respectively. these species are the main vehicles of dengue and chikungunya virus in various parts of the world but are considered exotic species in brazil (bhatt et al. 2013). they use the same breeding sites in the urban environment, although ae. albopictus is also often found inhabiting rural and natural environments. the finding of ae. albopictus and ae. aegypti in bromeliads demonstrates the high ecological valence of both species. according to crovello and hacker (1972), the use of bromeliads by ae. aegypti may be considered a remnant of primitive behavior of the species, as outside its original environment (northwest of africa) this species is found strictly only in synanthropic environments, is highly dependent on clean water in containers associated with intradomicile environments and exhibits anthropophilic behavior (forattini 2002). aedes albopictus is considered the wilder and more exophilic of the two species, with the potential to disperse through various habitats, and is often found breeding in phytotelmata, such as those in bromeliads and bamboos (natal et al. 1997). our results indicate that in urban parks ae. albopictus is better able to colonize bromeliads, a finding that may be related to the fact that these environments possess similar characteristics to those of rural and wild environments despite being embedded in an urban landscape. another aedeini registered was ae. fluviatilis, a suspected vector in the transmission of arboviruses to humans that is found in wild or modified environments and is associated with small, usually temporary rupiculous breeding sites with little shade (forattini 2002). in the present study, only six specimens were found (in ibirapuera park), showing that, although not their preferred breeding sites, bromeliad phytotelmata may be used by this species for the development of its immature form. culex quinquefasciatus is a recognized vector of the worm that causes bancroftian filariasis infection, wuchereria bancrofti. this cosmopolitan mosquito species is also associated with the transmission of west nile virus in the united states, canada and mexico (murray et al. 2010). the species is adapted to environments that have been changed by anthropogenic activities, mainly in areas where the disorderly use of urban spaces is associated with a lack of inhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 109 frastructure and failures in sewage and drainage systems, resulting in the accumulation of dirty still waters, which favor the proliferation of this culicid (morais et al. 2006). although the species is characteristic of large breeding sites, it has high ecological valence and may occupy small natural and artificial containers, such as bromeliads, tree holes, bamboo and tires (consoli and lourenço de oliveira 1994, forattini 2002). our results suggest that this species is found more abundantly in terrestrial bromeliads in parks in the city of são paulo. the subgenus microculex theobald, 1907 of culex displays a strong preference for breeding in natural containers, especially bromeliads and tree hollows, the former typically used by species cx. (mcx) imitator (muller and marcondes 2006). in the present study, cx. imitator was recorded in five parks, but in high abundance only in santo dias park, where it was found in terrestrial and epiphytic bromeliads. the genus toxorhynchites includes species that typically develop in bromeliads, tree holes and bamboo internodes and rarely in artificial breeding sites such as tin cans, barrels and tires (lopes 1997). the adults are exclusively phytophagous and are considered to be the only culicid that is harmless to man and other vertebrates. their larvae are equipped with powerful jaws adapted to predation of smaller immature aquatic arthropods, including other culicids. for this reason, species of this genus have been suggested as tools for biological control in the context of integrated pest management (nyamah et al. 2011). bromeliads are considered highly selective breeding sites, and some species of culicids, such as those of the genera wyeomiya and trichoprosopon, are closely associated with these plants (mocellin et al. 2009). the spatial distribution of these culicids is therefore quite restricted. the bromeliads found in parks may represent ecological refuges for these species, which may have been introduced in these green areas unintentionally when these plants were used for ornamentation. previous studies have shown that various species of culicids are present in conservation areas and public parks in the city of são paulo (urbinatti et al. 2001, taipelagos and natal 2003, montes 2005, ribeiro et al. 2012, medeiros-sousa et al. 2013). contributing to this body of knowledge, our study has shown that bromeliads in urban parks in the city of são paulo not only act as refuges for populations of native species of culicids, but are also used for breeding by exotic and epidemiologically important species such as ae. aegypti, ae. albopictus and cx. quinquefasciatus, which have a high ecological valence, are synanthropic and play an important role as vectors of human diseases. these green areas constitute highly favorable niches, as they increase the supply of resources (food and shelter), provide protection against natural enemies and physical changes in the environment (such as changes in temperature and rainfall) and may improve the survival of species that can exploit small and isolated remnants of native habitats and therefore tolerate a greater degree of urban development than species that are more sensitive to the same transformations in environments occupied by humans (mcintyre 2000). conclusion the bromeliads present in urban parks of the city of são paulo not only act as refuges for populations of native species of culicids but also allow exotic and epidemiological important species such as ae. aegypti, ae. albopictus and cx. quinquefasciatus, to use these habitats for proliferation of their populations. it is important for administrators and mosquito control and surveillance agencies to constantly monitor this microenvironment, since the presence of these species endangers the health of park users and employees as well as people living near the parks. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 102–112 w ceretti-junior et al.: species composition … 110 acknowledgements we would like to thank the fundação de amparo à pesquisa do estado de são paulo (biota program, project 2010/51230-8) for providing financial support. we are also grateful to the field team from the department of epidemiology in the faculty of public health, university of são paulo; to the staff at the office of parks and green areas, department of the environment and green areas, são paulo, and to the staff at the zoonosis control center, são paulo city hall, who helped collect and identify the specimens. we extend our special thanks to m bicudo de paula for assistance with the identification of the specimens and to dr pr urbinatti for assistance with the fieldwork and for the pictures. the 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published online: june 27, 2015 j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 337 http://jad.tums.ac.ir published online: january 06, 2016 original article biocontrol of the brown-banded cockroach, supella longipalpa f. (blattaria: blattellidae), with entomopathogenic fungus, metharhizium anisopliae mona sharififard 1, mohammad saeed mossadegh 2, *babak vazirianzadeh 3,4, seyed mahmood latifi 5 1department of medical entomology and vector control, school of health, ahvaz jundishapur university of medical science, ahvaz, iran 2department of plant protection, college of agriculture, chamran univercity, ahvaz, iran 3health research institute, infectious and tropical diseases research center, ahvaz jundishapur university of medical sciences, ahvaz, iran 4department of medical entomology, ahvaz jundishapur university of medical sciences, ahvaz, iran 5department of statics and epidemiology, school of public health, ahvaz jundishapur university of medical science, ahvaz, iran (received 13 may 2013; accepted 17 jan 2015) abstract background: considering to the high distribution of cockroaches as urban pests, the efficacy of different formulations of metarhizium anisopliae strain iran 437c were assessed against the brown-banded cockroach, supella longipalpa f. under laboratory and field conditions. methods: metarhizium anisopliae isolates were screened with immersing adults of the brown-banded cockroachs in aqueous suspension of 108 conidia ml-1 followed by surface or bait treated with different doses of the most virulent isolate against the nymphs. then formulations of conidia oil-in-water were examined versus cockroach nymphs using different plant oils and paraffin. then they were evaluated and compared with aqueous suspension and control group. on a large-scale, the sunflower oil-in-water formulation of conidia was sprayed at houses using a hand sprayer. results: metarhizium anisopliae iran 437c was the most virulent isolate against the brown-banded cockroach, causing 100% mortality in adults at seven days post-exposure. inoculated bait with this isolate was not enough pathogenic against the cockroach even at two weeks after treatment. treated surface with conidia as aqueous suspension or oil-inwater formulation was more effective than the bait formulation against the cockroach caused 39.4–97.2% mortality compared with 2.5% mortality in control group after two days. spraying the conidia formulated with sunflower oil was an effective formulation causing 76.1% reduction in the cockroach density on the third day post treatment in the houses. conclusion: the oil-in-water formulation of m. anisopliae iran 437c could be recommended as a promising alternative for cockroach control. keywords: supella longipalpa, metarhizium anisopliae, entomopathogenic fungus, biocontrol introduction cockroaches are recognized as cosmopolitan and trouble-maker insect pests in the different premises such as: homes, restaurants, hospitals, warehouses, offices, and other buildings regarding stores of food materials (stankus et al. 1990, nasirian et al. 2006, vazirianzadeh et al. 2014). they move among different premises and transmit the pathogenic and non-patho genic microorganisms from waste material to the food and kitchen stuffs. in this way, they carry bacterial disease agents such as cholera, leprosy, plague, typhoid fever, viral diseases such as poliomyelitis and parasitic worms and protozoa disease agents (stankus et al. 1990, vazirianzadeh et al. 2014). in addition, they are considered as insect pests, which cause allergic reactions includ*corresponding author: dr babak vazirianzadeh, e-mail: babakvazir@yahoo.co.uk j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 338 http://jad.tums.ac.ir published online: january 06, 2016 ing dermatitis, itching, swelling of the eyelids, and more serious respiratory conditions like asthma by their feces and disintegrated dead bodies (stankus et al. 1990, savoldelli and luciano 2005). the brown-banded cockroach, supella supellectilium (longipalpa) (f.) (blattaria: blattellidae) as a small species of cockroach, measuring about 10 to 14 mm long, carries a variety of microorganisms (le guyader et al. 1989, ehdae 2014). it is a vector of pathogenic bacteria in urban environments and hospitals (rivault et al. 1994, ehdae 2014, vazirianzadeh et al. 2014). supella longipalpa is also reported as an allergen source (eggleston and arruda 2001, savoldelli and luciano 2005). high amounts and numbers of chemical insecticides are used annually to reduce cockroach infestations in urban areas. this fact leads to development of resistance in these filth pests especially the german cockroaches. they are a major threat to chemical and pest control industries worldwide (lee et al. 1996, nasirian et al. 2006, ladonni et al. 2013). in all field-collected strains of iran, cockroaches have showed different level of resistance to different insecticides of organochlorine, organophosphorus and carbamat insecticides. therefore, application of these insecticides should be stopped as a recommendation (nasirian et al. 2006, ladonni et al. 2013). insecticide resistance, environmental concerns, and increased optimal costs of new insecticides have promoted searching for safer compounds with novel modes of action and environmentally safe biological products such as biocontrol agents (pachamuthu et al. 2000). biological control uses one organism to control another. where insecticides are undesirable or it is not necessary to eliminate cockroaches, biological control program is considered. cockroaches as animal pests like almost all animals have natural enemies with no exception. biological control plays an important role in managing cockroach populations. natural cockroach enemies include long list agents which entomopathogenic fungi are one of them. metarhizium species are known to attack a wide range of arthropods: greater than 200 species in over 50 families. these include many species of agricultural, medical and veterinary importance. metarhizium anisopliae is a fungus that grows naturally in soils throughout the world and causes disease in various insects. m. anisopliae does not appear to infect humans or other animals and is considered safe as an insecticide. the disease caused by the fungus is sometimes called green muscardine disease because of the green color of its spores (freimoser 2003, bischoff et al. 2009). microbial control agents, such as entomopathogenic fungi, can be effective and serve as alternatives to broad-spectrum chemical insecticides (zimmermann 1993, lacey et al. 2001). earlier studies have shown the efficacy of different m. anisoplaie isolates in cockroach control, especially the german cockroach, blattella germanica, (gunner et al. 1991, kaakeh et al. 1996, pachumathu et al. 1999, zurek et al. 2002, quesada-moraga et al. 2004, lopez and alves 2011). there are rare studies about control of the brown-banded cockroach by entomopathogens. evaluation of conidia-dust formulation of m. anisopliae strain iran 437 c exhibited high performance against this cockroach (sharififard et al. 2014). this fillith pest has recently become wide distribution in the city of ahwaz, khuzestan province, south west of iran, especially in apartments and hospitals (vazirianzadeh et al. 2014, sharififard et al. 2014). the current research was undertaken to evaluate different formulation of the most virulence isolate of m. anisopliae including aqueous suspension, inoculated bait and oilin-water formulations against the brownbanded cockroach, s. longipalpa, under laborj arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 339 http://jad.tums.ac.ir published online: january 06, 2016 atory and field conditions. materials and methods insects this study was carried out from april 2011 to march 2012 in medical lab of ahvaz joundishapur university of medical sciences (ajums) as small scale and in apartment houses as field scales. the brown-banded cockroaches originally were collected from kitchen area of human dwellings. they were reared in plexiglas containers (25 cm high× 17 cm diameter) and maintained at 27±2 °c, 50±5% rh for a photoperiod of 12:12 (l: d) h. they were fed on bread or dry crumbled saghe talaei biscuits and water. pieces of facial tissue were provided as harborage. cockroaches were anesthetized with extreme cold to facilitate handling. fungus four strains of the entomopathogenic fungus, m. anisopliae (m. anisopliae strain iran 437c, m. anisopliae strain iran 715c, m. anisopliae strain 1018, and m. anisopliae strain rhynchophorus), were provided from the fungi collection of the plant protection institute of iran. they were cultured on sday plates, kept at 27 ˚c and in a photoperiod condition of 12:12 (l: d) h. sporulating cultures were harvested by scraping dry conidia from the surface of the culture plate with a scalpel (talwar 2005). experiments virulence screening of metarhizium anisopliae isolates aqueous suspensions of four isolates of m. anisopliae were prepared and the concentrations were adjusted to 108 conidia ml1. in order to determine the virulence of fungal isolates against the brown-banded cockroach, a group of 20 adults (10 males and 10 females) in 4 replicates were dipped in the conidial suspension for 10 sec and were then placed on damp filter paper in plexiglas containers. the control group was dipped in sterile distilled water. all containers were incubated at 27±1 °c, 75±5% rh and a photoperiod of 12:12 (l: d) h. the cockroaches were fed during the test and mortality was recorded daily for a week. cockroach cadavers were collected, surface sterilized, and transferred to sterile petri dishes containing damp filter paper. true mortality was taken to occur for those cadavers on which fungal sporulation were visible (butt and goettle 2007). surface treated with aqueous suspension stock aqueous suspension of m. anisopliae isolates iran 437c with tween 80 (0.01 percent) was prepared and diluted serially to give the concentrations of 5×108, 108, 5×107, 107 and 5×106 conidia ml-1. whatman filter paper (no.1, 8cm diameter) was dipped into the suspension and then placed in the bottom of a glass jar (with 600 ml capacity which 2 ml of suspension was nearly absorbed by a filter paper). the last instar cockroach nymphs (3 and 4 instars) were released simultaneously into each glass jar and confined to the floor. the control group was exposed to a surface treated with distilled water. the experiment was repeated four times. cockroaches were fed on bread and water during the trial period. mortality was recorded daily for a week (butt and goettle 2007). bait inoculated with the most virulent isolate attractive bait consisting of bread wetted in beer was used in this test. this bait was recommended for cockroach live trapping by cochran, (1999). ten grams of the bait in a petri dish was inoculated with 1ml of the conidia suspension of m. anisopliae isolate iran 437c with 5 doses of 5×106, 107, 5×107, 108 and 5×108 conidia ml-1. twenty large brown-banded cockroach nymphs (3 and 4 instars) were released into plexiglas containers (25 cm high×17 cm diameter) and alj arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 340 http://jad.tums.ac.ir published online: january 06, 2016 lowed to adapt for one day before the introduction of treated baits. after that, inoculated bait was introduced to each container and remained as the only food source for 72 h. treated bait was then replaced with untreated food. the control group was fed on untreated bait. there were four replicates for each treatment. mortality was recorded daily for two weeks. evaluation of conidia oilin-water formulation against supella longipalpa oil-in-water emulsions were prepared with sesame oil, sunflower oil, coconut oil, and aqueous suspension of 2.5%, 5%, and 10% (v/v). the surfactant tween 80 was added to each formulation at a concentration of 50 percent of the amount of oil. all compounds were previously mixed and agitated at 250 rpm (albernaz et al. 2008) for 20 min at room condition. then a conidia suspension was added to give the defined concentration (108 conidia ml-1). large instar cockroach nymphs (3 and 4 instars) were released into glass jars (600 ml beakers) and exposed directly to 2.5 ml of different oil-inwater or aqueous formulations using a handheld pressure sprayer. the nozzle of the sprayer was held 45 cm away from and at a right angle to the application surface. the control group was exposed to distilled water. there were four replicates for each treatment. the treated cockroaches were maintained at 27±1 ºc, with a photoperiod of 12:12 l: d, and rh: 45±5 percent. field evaluation of the fungus the sunflower oil-in-water formulation of conidia (proportion of 10%) of m. anisopliae strain iran 437c (equal to 109 spore ml-1 10 times the laboratory concentration) was selected for further evaluation at house-scale, because it showed the highest efficiency against the brown-banded cockroach. the method of thavara et al. (2007) with a little modification was used to evaluate cockroach infestations in houses. eight houses with brown-banded infestation were selected and surveyed for cockroach density using sticky traps before and after treatment (four houses for treatment and four houses for control). after the preliminary survey, to assess the degree of cockroach infestation, the sticky traps were placed in each house and left there for a night. one sticky trap per 10 cm2 was located at the selected house. then, all the traps were collected and the cockroaches caught in each trap were counted. after the initial infestation evaluation, the conidia oil-in-water formulation at the standard dosage of 40 ml/m2 for residual treatment (rozendal 1997) was applied by a hand-held pressure sprayer. the treatment was carried out only once in each house. cockroach density was assessed on the seventh day after treatment by sticky trap. the average number of collected cockroaches per house (mean no.) and standard error of the mean (se) were calculated. the percentage of reduction in the number of cockroaches following treatment at each treated site was calculated by mulla’s formula (mulla et al. 1971): reduction (%)= 100[(c1/t1)×(t2/c2)] 100 where: c1= average number of cockroaches per house at the control site (pre-treatment), t1= average number of cockroaches per house at the treated site (pre-treatment), c2= average number of cockroaches per house at the control site (post-treatment), t2= average number of cockroaches per house at the treated site (post-treatment). statistical analysis firstly, all mortality data was transferred to percentages. mean and standard error mortality for each dose of conidia per each method were calculated. kruskal-wallis and mann-whitney tests and probit analysis were used to compare the treatments and to create the toxicity values of conidia doses (ld50, ld90, lt50 and confidence intervals), j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 341 http://jad.tums.ac.ir published online: january 06, 2016 respectively. the significant level tests were p< 0.05. the spss version 16 and sas software were used for data analysis. on the field treatment, the percentage of reduction in cockroach infestation was calculated using mulla’s formula (mulla et al. 1971). results virulence screening of metarhizium anisopliae isolates as shown in table 1, m. anisopliae strain iran 437c was the most virulent isolate against s. longipalpa. it caused 82% and 100% mortality in adults in the third and seventh day after treatment, respectively. the mortality percentage of this isolate was significantly different from the other isolates (p= 0.0001). green moscardin were observed on the all sterilized cockroach cadavers (fig. 1). therefore, this more virulent isolate was selected for further evaluation. surface treated with aqueous suspension results showed that cockroach mortality rates increased by an increase in the concentration of conidia suspension. mortality rates of the treated cockroach nymphs ranged from 34.2 to 97.8% at seventh day post-exposure at the lowest and the highest doses, respectively. it was also significant at the second day post-exposure and ranged from 9.2%–65.3% (table 2). the mortality rates were not significantly different at the doses of 2×107 and 4×106 conidia cm-2 on the seventh day, and they did not differ on the fifth and seventh day at the dose of 2×107 conidia cm-2. the average mortality rate of the control group was significantly different from the treated groups and was always lower than 5%. the ld50 and ld90 values were 7.7×106 and 108 conidia cm-2 at third day post-exposure but reduced to 5.6×105and 2.7×106 conidia cm-2 at seventh days post-exposure (table 3). calculation and comparison of lethal time values (lt50) also showed a significant differences between applications of different doses of conidia, because there was nearly no overlap in their confidence intervals. the shortest time for killing cockroach nymphs was recorded when they were exposed to 2×107 conidia per cm2, although the mortality rates using this dose was not significantly different with 4×106 conidia per cm2 on the seventh day. the other obtained lt50s with their confidence intervals according to the treated surface using 2×107, 4×106, 2×106 and 4×105conidia/cm2 were 1.4(1.1–1.7), 2.6 (2.3–2.8), 2.9(2.6–3.2), 11.4(8.2–14.9) days after a week, respectively. bait inoculated with the most virulent isolate bait inoculated with fungus did not show high performance against the cockroach nymphs, even at the highest conidia dose when compared with untreated bait. although mortality was recorded up to two weeks, the average mortality rate did not exceed to 20 percent, even at the highest dose. the ld50 and ld90 were not calculated, since the highest mortality rate was lower than 50 percent (table 2). the various concentration of the conidia oil-in-water formulation the oil-in-water formulation of conidia with different proportions increased the mortality rates of the brown-banded cockroach nymphs compared with the aqueous formulation and the control group. significant differences in mortality rates were obtained due to the treatments with formulated and nonformulated conidia or the control group. mortality percentages of different oil formulations were significantly different from those of the aqueous suspension or the control group (pvalue= 0.003, pvalue= 0.003 and pvalue= 0.005 and pvalue= 0.002 for sesame, coconut, paraffin, and sunflower oils, respectively). there were no significant differences, however, between the mortality rates of three concentrations of each oil j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 342 http://jad.tums.ac.ir published online: january 06, 2016 (pvalue= 0.151, pvalue= 0.165, pvalue= 0.123 and pvalue= 0.012 for sesame, coconut, paraffin, and sunflower oils respectively). comparison of the same proportion of different oils didn’t show any difference between the mortality rates of the 10 percent proportion of all oils, but the differences were significant in 5 (pvalue= 0.017) and 2.5 (pvalue= 0.016) percent concentrations. the highest level of mortality rate was obtained when cockroach nymphs were exposed to 10 percent sunflower oil concentration (97.2%), although there were no significant differences between proportions of 5% and 10% of sesame, sunflower, and paraffin oil concentrations. coconut oil formulation performed the lowest mortality compare to the other oil formulations and the lowest mortality rate was observed in the aqueous suspension treatment (39.4%) (table 4). the observed mortality in all conidia oil formulations and aqueous suspension treatments were significantly different from the control group. field evaluation of the fungus twelve houses were surveyed for cockroach infestation during the preliminary inspection before treatment, and totally eight houses were selected because of their relatively severe cockroach infestation. the average number of cockroaches collected at each house ranged from 15.2–27 cockroaches per house prior to treatment. on the seventh day after treatment with the oil-inwater formulation of m. anisopliae, the cockroach density decreased to 4.6–7.5 cockroaches per house with average density reduction of 76.1 percent using the formula of mulla et al. (1971). in the control houses, the average number of cockroaches was 24.5 cockroaches per house pre-treatment, and then it increased to 30.5 cockroaches per house after seven days (table 5). table1. virulence screening of metarhizium anisopliae isolates on adult supella longipalpa by immersing in conidia aqueous suspension (108 conidia ml-1) m. anisopliae isolates % mortality mean± se after 3 days after 5 days iran 437c 82± 2.3 100± 0.0 iran 1018c 56±3.6 76±1.9 iran 715 39±1.5 67±4.2 rhynchophrus 28±2.7 45±3.1 fig. 1. the brownbanded cockroach with green muscardin (left: male, right: female) j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 343 http://jad.tums.ac.ir published online: january 06, 2016 table 2. cumulative mortality of supella longipalpa nymphs exposed to different concentrations of metharhizium anisopliae isolate iran 437c presented as treated surface and treated bait (2011–2012) treated surface conidia/cm2 % mortality mean ±se after 2 days after 3 days after 5 days after 7 days 2×107 65.3±4.6 88.9±3.9 97.8±0.27 97.8±0.27 4×106 38.9±3.8 64.4±7.1 81.1±8.6 93.6±10.8 2×106 28.2±3.1 59±3.7 72.5±2.6 83.1±3.2 4×105 9.2±0.83 20.8±1.6 30.8±0.83 34.2±2.2 control 0.0 0.0 0.0 3.3±2.9 inoculated bait conidia g-1 % mortality mean ±se after 5 days after 7 days 2.5×108 3.7±2.4 18.7±2.4 5×107 2.5±1.4 13.7±2.4 2.5×107 0.0 5±2.04 table 3. probit analysis of metharhizium anisopliae (iran 437c) against nymphs of supella longipalpa presented as treated surface (conidia per cm2) (2011–2012) post exposure day ld50 (95% ci) ld90 (95% ci) pvalue (probit model) pvalue(x 2) 2 7.7×106(5.2×106–1.1×107) 108(6.1×107–2.2×108) 0.0001 0.179 3 2×106(8.5×105–3.2 ×106) 1.7×107(107–3.3×107) 0.0001 0.015 5 9.5×105(2.9×105–1.8×106) 6.6×106(4.2×106–1.1×107) 0.0001 0.072 7 5.6×105(5.3×105– 1.7×106) 2.7×106(8.8×105–1.7×107) 0.0001 0.001 ci: confidence intervals table 4. supella longipalpa nymphs mortality rates exposed to different concentration of metharhizium anisopliae strain iran 437c formulations presented as treated surface at 2 days post exposure (108 conidia/ ml-1) (2011–2012) conidia-formulations concentration of oil (%) mortality means (%)±se (2 days post exposure) 10 96.1±2.5 sesame oil 5 94±2.2 2.5 89.1±2.8 10 85.2±3.3 coconut oil 5 84±3 2.5 75.5±3 10 94.4±2.7 paraffin oil 5 95.4±2.1 2.5 91.8±3.4 10 97.2±1.7 sunflower oil 5 93.6±0.88 2.5 86.1±4.3 aqueous suspension 0.01 39.4±8.8 control 2.5±1.6 j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 344 http://jad.tums.ac.ir published online: january 06, 2016 table 5. field evaluation of metharhizium anisoplae strain iran 437c as oil-in-water formulation containing 10% sunflower oil applied against supella longipalpa on seventh day after exposure (2011–2012) pre-treatment post-treatment house mean± se a mean± se b reduction c (%) 1 20.7±3.2 7.5±0.87 71.2 treatment 2 15.2±1.5 6±1.4 68.7 3 25±1.4 4.7±2.1 85.1 4 27±2.9 7±1.2 79.4 1 26±2.2 34±3.1 control 2 22±4.2 29±2.6 3 23±2.5 27±5.2 4 27±3 32±1.4 a and b: mean number of cockroach per house pre-treatment and posttreatment c: average percent reduction of cockroach in each house. discussion in the current study, m. anisopliae strain iran 437c was the most effective isolate causing 100% mortality rate in adult brownbanded cockroach at seven days after exposure using 108 conidia ml-1 in screening of the fungus isolate. the ability to produce the high performance of conidia and its high virulence were the reasons which this isolate was selected for the brown-banded cockroach control. this fungus isolate exhibited high virulence against s. longipalpa as conidia – dust formulation in dose ranges of 6.6×1053.3× 107 conidia per cm2 which led to 45– 97.5% mortality using treated surface bioassay method with survival time range of 3.4 to 6.7 days (sharififard et al. 2014). other studies have shown different efficiencies of m. anisoplaie in the control of cockroaches. according to gunner et al. (1991), the m. anisoplae strain pa-2 required≈6 week to produce 90% mortality in the german cockroaches. kaakeh et al. (1996) reported 26–30 d to achieve 90% or higher mortality in german cockroaches with the m. anisopliae strain esc-1 using the contact method. pachumathu et al. (1999) reported 4.18×108 spore ml-1 as ld50 of m. anisopliae, applying 1µ of the spore solution as a topical assay. the average mortality of german cockroaches with m. anisopliae using a 4µ l suspension of concentrations ranging from 4.2×106 to 4.2×109 spores ml-1 was determined to be 42.3 to 93.3% in the treated insects, whereas 13.6% mortality was observed in the untreated cockroaches. the ld50 value was recorded as 1.4×10 7 spore ml-1 (quesada-moraga et al. 2004). we observed 97.8% and 93.6% mortality rates in the brown-banded cockroach nymphs exposed to a surface treated with 2×107 and 4×106 conidia cm-2 of m. anisopliae strain iran 437c, respectively, at seven days postexposure with ld50 and ld90 values of 5.6×105 and 2.7×106 conidia cm-2 respectively. lopez and alves (2011) reported 73.9% and 76.1% adult cockroach mortality rates in the german cockroach nymphs at nine and fifteen days after exposure to a surface inoculated with 6.5×106 conidia cm-2 of m. anisopliae. adult cockroach mortality rates were 96.1% and 100% at the mentioned times and doses. they also reported 9.4% and 28% mortality in adults at nine and fifteen days after exposure to bait inoculated with m. anisopliae at 5×109 conidia g-1. the lt50 values of the brown-banded j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 345 http://jad.tums.ac.ir published online: january 06, 2016 cockroach nymphs were obtained 1.4 and 2.6 days using 2×107 and 4×106 conidia cm-2 in this study. mean survival times of nymph and adult of b. germanica exposed to 6.5×106 conidia cm-2 on a treated surface were 6.5 and 5.6 days (lopes and alves, 2011). quesada-moraga et al. (2004) reported lt50= 5.3 days for 4.2 ×10 9 spores ml-1 for the german cockroach by topical application of spore solution. the differences in results can be attributed to the cockroach species, virulent of the fungus isolate, the bioassay methods, conidia dose and even the exposure time. we stopped data recording on the seventh day, whereas lopes and alves (2011) continued their study to the fifteenth day. it seems that the brown-banded cockroach is more susceptible to control agents than the german cockroach, so it takes a shorter time and lower doses of entomopathogenic fungi to kill them. our results showed that bait inoculated with conidia could not be a very effective method for the brown-banded cockroach control. the successful development of a mycoinsecticide ultimately depends on the availability of a virulent strain, an optimized and economic production system, and a suitable formulation to optimize its application, efficacy, and storage characteristics as well as persistence after application (kassa 2003). oil-based formulations of fungal propagules are known to enable the infection of various insect pests at low humidity (albernaz et al. 2009). performance evaluation of oil-inwater formulations of m. anisopliae showed very promising results, because it increased cockroach mortality in shorter lethal time. using oil formulation in arid areas (relative humidity less than 35 percent) enhances the efficacy of entomopathogenic fungi compared with aqueous formulation (kassa 2003). sesame oil was more effective in increasing the efficacy of m. anisopliae against the brownbanded cockroach, though all tested oils were compared with the aqueous formulation. conclusion in this study, we used a sunflower-oil formulation (proportion of 10%) for cockroach control in houses. mulla's formula was used to assess the degree of cockroach infestation before and after treatment. the reduction in the number of cockroaches was significant (76.1% reduction on the seventh day post-treatment). although there is a time lag between treatment and cockroach death compared with the rapid effect of chemical insecticides, this time lag can be reduced by combining entomopathogenic fungi with a sublethal dose of chemical insecticides (sharififard et al. 2011a). it may also be acceptable to produce where insects have become too resistant to such chemicals, and this can lead to a reduction in the use of them. other research on the m. anisoplaie isolate iran 437c has shown high virulence against m. domestica, as a widespread vector of many human pathogens (sharififard et al. 2011a). additionally, this isolate was highly pathogenic in low humidity environments (sharififard et al. 2012). therefore, it can be developed as a mycoinsecticide for the safer control of houseflies and brown-banded cockroaches because of its high virulence, the effectiveness in conidial production, and the high pathogenicity in low ambient humidity. acknowledgements the authors wish to acknowledge ahvaz jundishpur university of medical sciences, ahvaz, iran for financial support and the iranian plant protection institutes for providing the fungus isolates. the authors declare that there is no conflict of interests. j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 346 http://jad.tums.ac.ir published online: january 06, 2016 references albernaz das, tal mhh, luz c (2009) enhance ovicidal activity of an oil formulation of the fungus metarhizium anisopliae on the mosquito aedes aegypti. med vet entomol. 23: 141–147. bischoff jf, rehner sa, humber ra (2009) a multilocus phylogeny of the metarhizium anisopliae lineage. mycologia. 101(4): 512–530. butt tm, goettel ms (2007) bioassays of entomopathogenic fungi. in: navon a, ascher krs (eds): bioassay of microbes and nematodes,. cabi publishing, wallingford, oxfordshire, uk, pp. 141–195 cochran dg (1999) cockroaches, their biology, distribution and control. switzerland world health organization. who/cds/cps/whopes/ 99.3. p. 83. eggleston pa, arruda lk (2001) ecology and elimination of cockroaches and allergens in the home. j allergy clin immunol. 107: 422–429. ehdae b (2015) stisolation and identification bacteria from cockroaches of ahvaz (sw of iran) hospitals and determination of their susceptibility to antibiotics. 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[phd thesis], department of agricultural microbiology, university of agricultural sciences, dharwad, india. thavara u, tawatsin a, bhakdeenuan p, wongsinkongman p, boonruad t, bansiddhi j, chavalittumrong p, komalamisra n, siriyasatien p, mulla ms (2007) repellent activity of j arthropod-borne dis, september 2016, 10(3): 337–348 m sharififard et al.: biocontrol of the … 348 http://jad.tums.ac.ir published online: january 06, 2016 essential oils against cockroaches (dictyoptera: blattidae, blattellidae, and blaberidae) in thailand. southeast asian j trop med public health. 38(4): 663–673. vazirianzadeh b, dehghani r, mehdinejad m, sharififard m, nasirabadi n (2014) the first report of drug resistant bacteria isolated from the brown-banded cockroach, supella longipalpa, in ahvaz, south-western iran. j arthropodborne dis. 8(1): 53–59. zimmermann g (1993) the entomopathogenic fungus, metarhzium anisopliae and its potential as a biological agent. pestic sci (37): 375–379. zurek l, wes watson d, schal c (2002) synergism between metarhizium anisopliae (deuteromycota: hyphomycetes) and boric acid against the german cockroach (dictyoptera: blatellidae). j biol control. 23: 296–302. j arthropod-borne dis, december 2013, 7(2): 185–187 s wiwanitkit and v wiwanitkit: ckikungunya virus … http://jad.tums.ac.ir published online: august 31, 2013 short communication ckikungunya virus infection and relationship to rainfall, the relationship study from southern thailand *somsri wiwanitkit, viroj wiwanitkit wiwanitkit house, bangkhae, bangkok, thailand (received 24 july 2012; accepted 6 apr 2013) abstract there are several important mosquito borne infectious disease in the tropical countries. chikungunya virus infection is an important arbovirus in this group. we hereby used a standard medicogeographical analysis to assess the correlation between prevalence of chikungunya virus infection and rainfall in the endemic area of thailand, the southern region. in this work, the correlation was poor but significant. the result is concordant with a similar report from the retrospective analysis from the local center of disease control. several other factors can affect the pattern of chikungunya virus infection in the studied endemic area. keywords: ckikungunya virus, infection, relationship, rainfall, thailand introduction there are several important mosquito borne infectious disease in the tropical countries. chikungunya virus infection is an important arbovirus in this group (thiboutot et al. 2010). in thailand, as a tropical country, this disease can be seen and is still the important local public health problem (halstead 1966). it is classified into the group of hemorrhagic fever and can manifest with fever, arthralgia and hemorrhage (halstead 1966, chhabra et al. 2008). in fact, the mosquitoes and mosquito-borne viral diseases are common in thailand. millions of vector mosquitoes can be seen in thailand in any areas (gratz 1993). due to proper climate, hot and humid, the breeding of mosquito is very good in thailand and this is the main cause of high prevalence of the mosquito-borne viral diseases (gratz 1993). the reported prevalence of mosquito-borne viral diseases in thailand can reach 62/100,000 in some outbreak period of dengue infection (hemungkorn et al. 2007). focusing on chikungunya disease, it is the second common disease (the most common is dengue infection) (pulmanausahakul et al. 2011). the basic climatogeography in the country might help promote the distribution of this infection. the authors hereby used a standard medicogeographical analysis to assess the correlation between prevalence of chikungunya virus infection and rainfall in the endemic area of thailand, the southern region. materials and methods the objective of this work was to study on the relationship between the rainfall, an important geographical parameter, and the prevalence of chikungunya virus infection in thailand. since the complete data on other parameters are not available at present, the author studied only a single parameter in this preliminary report. the data on the prevalence of disease (thai cdc. available *corresponding author: dr somsri wiwanitkit, email: somsriwiwan@hotmail.com 185 j arthropod-borne dis, december 2013, 7(2): 185–187 s wiwanitkit and v wiwanitkit: ckikungunya virus … http://jad.tums.ac.ir published online: august 31, 2013 at: http://epid.moph.go.th: accessed 1 may 2010) and rainfall (royal irrigation department thailand. available at: http://www.rid. go.th/bid/bid.html: accessed 1 may 2010) were collected and further processed based on the already published protocol in the previous studies reported from the authors’ laboratory (the protocol can be assessed via the cited references) (wiwanitkit 2006a, wiwanitkit 2006b). briefly, after matching between data from the same geographical area was done, the already matched primary data were used for mathematic correlation assessment and a predictive map was further generated based on the derived correlation. to avoid the duplication, the complete details can be seen in the previous authors’ publications (wiwanitkit 2006a, wiwanitkit 2006b). fig. 1. predicted prevalence of japanese encephalitis based on the rainfall distribution in southern region of thailand. this figure is created based on the results of the present study. the least square equation plot rainfall (x) versus prevalence (y) is y= 0.8x+0.6 results averages for prevalence and rainfall were 0.4 (/100,000) and 9.1 (inches) respectively. the derived least square equation plot prevalence (y) versus rainfall (x) was y= 0.8x+ 0.6 (r= 0.54, p< 0.05). the predicted prevalence of chikungunya virus infection based on the rainfall distribution in thailand is shown in fig. 1. discussion mosquito borne arboviral infection becomes the focused problem at present due to many remergings and new emergings in several countries (weaver and reisen 2010). climate factor is mentioned as an important factor contributing to change of the disease epidemiology (weaver and reisen 2010). anyamba et al. (2012) recently detected that “in southeast asia, chikungunya outbreaks were negatively correlated (p< 0.05) with drought conditions, but positively correlated with warmer-than-normal temperatures and rainfall.” the study on climate factor and relationship to chikunguya disease epidemiology can be interesting. in this work, the correlation was poor but significant. the result is concordant with a similar report from the retrospective analysis from the local center of disease control (ditsuwan et al. 2011). this confirms that there are several other factors that can affect the pattern of chikungunya virus infection in the studied endemic area (ditsuwan et al. 2011). indeed, this fact can be confirmed based on the data that there are sporadic outbreaks of disease in some years (such as the recent outbreak in 2009) despite there is no change of the pattern of rainfall in thailand. in fact, chikungunya can be classified as a mosquito-borne viral zoonotic disease. the finding on relationship between rainfall and disease cannot completely imply in epidemi186 j arthropod-borne dis, december 2013, 7(2): 185–187 s wiwanitkit and v wiwanitkit: ckikungunya virus … http://jad.tums.ac.ir published online: august 31, 2013 ology. many obvious factors can alter the epidemiology of mosquito-borne diseases especially in zoonotic ones. some mosquitoes can fly into distance area; hence, the rainfall cannot be the single factor in the epidemiology of this disease. acknowledgements the authors declare that there is no conflict of interest. references anyamba a, linthicum kj, small jl, collins km, tucker cj, pak ew, britch sc, eastman jr, pinzon je, russell kl (2012) climate teleconnections and recent patterns of human and animal disease outbreaks. plos negl trop dis. 6(1): 1465. chhabra m, mittal v, bhattacharya d, rana u, lal s (2008) chikungunya fever: a re-emerging viral infection. indian j med microbiol. 26(1): 5–12. ditsuwan t, liabsuetrakul t, chongsuvivatwong v, thammapalo s, mcneil e (2011) assessing the spreading patterns of dengue infection and chikungunya fever outbreaks in lower southern thailand using a geographic information system. ann epidemiol. 21(4): 253–261. gratz ng (1993) lessons of aedes aegypti control in thailand. med vet entomol. 7(1): 1–10. halstead sb (1966) mosquito-borne haemorrhagic fevers of south and southeast asia. bull world health organ. 35(1): 3–15. hemungkorn m, thisyakorn u, thisyakorn c (2007) dengue infection: a growing global health threat. biosci trends. 1(2): 90–96. pulmanausahakul r, roytrakul s, auewarakul p, smith dr (2011) chikungunya in southeast asia: understanding the emergence and finding solutions. int j infect dis. 15(10): e671–6 thiboutot mm, kannan s, kawalekar ou, shedlock dj, khan as, sarangan g, srikanth p, weiner db, muthumani k (2010) chikungunya: a potentially emerging epidemic? plos negl trop dis. 4(4): e623 weaver sc, reisen wk (2010) present and future arboviral threats. antiviral res. 85(2):328-45. wiwanitkit v (2006a) an observation on correlation between rainfall and the prevalence of clinical cases of dengue in thailand. j vector borne dis. 3(2): 73–76. wiwanitkit v (2006b) correlation between rainfall and the prevalence of malaria in thailand. j infect. 52(3): 227–230. 187 microsoft word dr sulaiman rtl.doc iranian j arthropod-borne dis, (2008), 2(2): 7-11 s sulaiman et al: evaluation of bifenthrin … 7 original article evaluation of bifenthrin and acorus calamus linn. extract against aedes aegypti l. and aedes albopictus (skuse) *s sulaiman, dsf abang kamarudin, h othman department of biomedical science, faculty of allied health sciences, university kebangsaan malaysia, jalan raja muda abdul aziz, 50300 kuala lumpur, malaysia (received 10 jan 2009; accepted 12 may 2009) abstract background: bifenthrin and acorus calamus linn extract were evaluated against dengue vectors in the laboratory. methods: both bifenthrin and acorus calamus linn crude hexane extract were bioassayed against the adults and larval stages of dengue vectors aedes aegypti l. and aedes albopictus(skuse) in the laboratory. results: the a. calamus crude hexane extract exhibited a larvicidal activity against 4th-instar ae. aegypti larvae with lc50 and lc90 values of 0.4418 and 11.3935 ppm respectively. the plant crude extract exhibited against ae. albopictus larvae with a higher lc50 and lc90 values of 21.2555 ppm and 36.1061 ppm, respectively. there was a significant difference on the effect of a. calamus extract on both aedes spp. larvae (p< 0.05). however, bifenthrin showed a significant difference on larvicidal effect to that of a. calamus hexane extract on both aedes spp (p< 0.05). in testing the adulticidal activity, this plant extract exhibited the lc50 and lc90 values of 17.4075 and 252.9458 ppm against ae .aegypti and a higher lc50 and lc90 values of 43.9952 and 446.1365 ppm respectively on ae. albopictus. there was no significant difference on the effect of a. calamus extract on both aedes spp adults (p> 0.05). conclusion: bifenthrin however showed a significant difference on both aedes spp adults (p< 0.05). with the wide availability of a. calamus in malaysia, it could be utilized for controlling dengue vectors. keywords: acorus calamus, hexane extract, aedes aegypti, aedes albopictus, bifenthrin introduction in southeast asia, aedes albopictus (skuse) has been incriminated as a secondary vector of dengue fever and ae. aegypti (linn.) as the principal vector of dengue viruses(russell et al. 1969, chan et al. 1971, jumali et al.1979, harinasuta 1984). the dengue viruses may produce occasional fatal diseases, usually among children (rudnick and chan 1965, harinasuta 1984). chemicals derived from plants offer promise in future mosquito control programs (sukumar et al.1991).the search for new environmentally safe, target specific insecticides is being conducted all over the world. to find new modes of action and to develop active agents based on natural products, efforts are being made to isolate, screen and develop phytochemicals possessing pesticidal activity (mulla and su, 1999). there is an urgent need for economically feasible natural and biodegradable compound for the control of mosquito vector. plants may be a source of alternative agent to replace the more expensive synthetic insecticides for mosquito control. thus, the objective of this study was to compare the efficacy of the plant extract acorus calamus linn (fam. araceae) compared to the synthetic pyretroid bifenthrin 80 sc against ae. aegypti and ae. albopictus in the laboratory. *corresponding author: prof dr s. sulaiman, tel: +03 92897416, fax: +603 26929032, e-mail:salsul@ medic. ukm.my iranian j arthropod-borne dis, (2008), 2(2): 7-11 s sulaiman et al: evaluation of bifenthrin … 8 material and methods extraction the rhizome of acorus calamus was grinded to smaller size to enhance extraction yield and was extracted using the soxhlet apparatus for at least 20 h with hexane. the filtrate was then evaporated to dryness under vacuum pressure. the crude hexane extract of a. calamus was bioassayed against the 4th instar larvae and adults of ae. aegypti and ae. albopictus. insecticide the insecticide bifenthrin 80sc was supplied by fmc pt bina guna kimia, semarang, indonesia. bioassay against adults aedes aegypti and aedes albopictus the bioassay was conducted according to who standard procedures (who 1981a) with some modifications. twenty adult mosquitoes of ae. aegypti and ae. albopictus laboratory colonies, 2-5 d old were exposed for 1 h to filter paper (15x16cm) impregnated with varying concentrations of a. calamus extract and bifenthrin 80sc in the range of 1.0 ppm to 8.0 ppm for testing against ae. albopictus and 0.3125 ppm to 5.0 ppm for ae. aegypti. each concentration was diluted in 0. 1% acetone for bifenthrin and control impregnated papers; the stock solution for a. calamus extract was prepared by diluting the crude extract with tween 20 and 0.1% hexane. the knockdown was recorded for each 1, 3, 5, 10, 20, 30 and 60 min. all mosquitoes were then transferred to holding tubes with clean filter papers. the mosquitoes were fed with cotton pads soaked in 10% sucrose solution. each experiment conducted was in duplicate. after 24 h the mortality rate of the adult mosquitoes were recorded. the experiment was repeated three times and analyzed by using probit analysis program (raymond, 1985) and spss software. bioassay against 4th instar larvae of ae. aegypti and ae. albopictus the bioassay was conducted according to who (1981b) with some modification. twenty-five ae. aegypti and ae. albopictus larvae were exposed in each of 600 ml glass beakers containing 250 ml of prepared a. calamus extract and bifenthrin 80 sc in varying concentrations. the a. calamus and control were diluted in 0.1% hexane; bifenthrin 80sc and control were diluted in 0.1% acetone. after 24 h the mortality of the larvae were recorded. the experiment was conducted in duplicate and repeated three times. if the control mortality was between 5% and 20%, the percentage mortalities were corrected by abbott’s formula: % test mortality-%control mortality x 100 100-% control mortality lc50 and lc90 values were determined by probit analysis (raymond 1985) and spss software. results table 1 indicated the lc 50 and lc90 values of bifenthrin on 4th-instar ae. aegypti larvae of 0.0034 and 0.0101 ppm; ae. albopictus with lc50 and lc90 values of 0.1360 and 0.5110 ppm, respectively. there was a significant difference on the effect of bifenthrin to both ae. spp. (p< 0.05).the values of lc50 and lc90 of a. calamus extract on 4th instar ae. aegypti larvae were 4.4418 and 11.3935 ppm and ae. albopictus with lc50 and lc90 values of 3.1330 and 21.2555 ppm respectively. there was also a significant difference on the effect of a. calamus extract on both ae. aegypti and ae. albopictus larvae (p< 0.05). bifenthrin showed a significant difference on larvicidal effect to that of a. calamus extract to both ae. spp. (p< 0.05). table 2 indicated the lc50 and lc90 values of bifenthrin on ae. aegypti adults of iranian j arthropod-borne dis, (2008), 2(2): 7-11 s sulaiman et al: evaluation of bifenthrin … 9 0.7020 and 2.3287 ppm; ae. albopictus with lc50 and lc90 values of 2.4267 and 4.2532 ppm respectively. there is a significant difference on the effect of bifenthrin to both ae. spp adults (p< 0.05). the crude extract of a. calamus indicated the lc50 and lc90 values of 17.4075 and 252.9458 ppm on ae. aegypti; and 43.9952 and 446.1365 ppm on ae. albopictus, respectively. however, there was no significant difference on the effect of a. calamus extract on both ae. spp. (p> 0.05). thus, a higher dosage is needed for a. calamus extract to be effective as an adulticide compared to bifenthrin. table 1. the lc50 and lc90 of bifenthrin and acorus calamus extract on aedes aegypti and ae. albopictus 4th-instar larvae in the laboratory table 2. the lc50 and lc90 of bifenthrin and acorus calamus extract on aedes aegypti and ae. albopictus adults in the laboratory treatment lc50 (ppm) lc90 (ppm) slope±se lc50 (ppm) lc90 (ppm) slope±se aedes aegypti aedes albopictus bifenthrin 0.7020 2.3287 2.4610±0.2994 2.4267 4.2532 5.2596±0.7134 acorus calamus extract 14.4075 252.9458 1.1028±0.1212 43.9952 446.1365 1.2742±0.1303 discussion hidayatulfathi et al. 2004 evaluated methanol extracts of some malaysian plants for larvacidal activities against mosquitoes. the methanol extract of a. calamus linn. showed a high degree of toxicity to all mosquito species of anopheles maculatus theobald, culex quinquefasciatus say, ae. aegypti (l.) and ae. albopictus (skuse) with lc50 of 39.15-58.29 µg/ml. hidayatulfathi et al. (2005) using the hexane fraction showed the highest larvicidal effect on ae. aegypti 4th instar larvae with lc50 value of 1.88 ppm and the lc90 value of 10.76 ppm respectively. the present study using hexane fraction indicated lc50 of 0.4418 ppm and lc90 value of 11.3935, respectively against ae. aegypti 4th instar larvae, while the lc50 and lc90 values on ae. albopictus 4th instar larvae were 21.2555 and 36.1061 ppm, respectively. choochote et al. (2005) found that the volatile oil of curcuma aromatica (fam: zingiberaceae) possessed a significantly higher larvicidal activity against 4th instar larvae of ae. aegypti than that of hexane extracts with lc50 values of 36.30 and 57.15 ppm, respectively. choochote et al. (2004) also found that apium graveolans (fam. apiaceae) seed extract possessed larvicidal activity against 4th instar ae. aegypti larvae with ld50 and ld95 values of 81.0 and 176.8 mg/l (ppm), respectively. thomas et al. (2004) also conducted the laboratory bioassay of ipomoea treatment lc50 (ppm) lc90 (ppm) slope±se lc50 (ppm) lc90 (ppm) slope±se aedes aegypti aedes albopictus bifenthrin 0.0034 0.0101 2.6937±0.2575 0.1360 0.5110 2.2290±0.2642 acorus calamus extract 4.4418 11.3935 3.1330±0.3571 21.2555 36.1061 5.5701±0.6731 iranian j arthropod-borne dis, (2008), 2(2): 7-11 s sulaiman et al: evaluation of bifenthrin … 10 cairica (fam. convolvulaceae) essential oil against ae. aegypti larvae and found that the lc50 and lc90 values were 22.3 and 92.7 ppm, respectively. thus, the present study indicated that a. calamus extract is more effective than extracts of curcuma aromatica, apium graveolans and ipomoea cairiaca as a larvicide against ae. aegypti 4th instar larvae evaluated by the above authors. hidayatulfathi et al. (2004) found that using the hexane fraction from methanol extract of a. calamus rhizome against ae. aegypti adults was the most effective, exhibiting lc50 and lc90 values of 0.04 mg/cm2 and 0.09 mg/cm2, respectively. for litsea elliptica the methanol fraction also displayed good adulticidal property with lc50 and lc90 values of 0.11 mg/cm2 and 6.08 mg/ cm2, respectively. sulaiman et al. (2005) evaluated a. calamus extract and bifenthrin in the field at high rise flats in kuala lumpur. the impact of both plant extract and insecticide on field populations of ae. aegypti and ae. albopictus was monitored weekly. a. calamus extract showed adulticidal effect causing 93.9% (inside flats) to 94.9% (outside flats) adult ae. aegypti mortalities compared to bifenthrin with 98.3% (inside flats) and 99.1% (outside flats) adult mortalities. in the control group, the adults of ae. aegypti mortalities were 19.2% (inside flats) and 18.2% (outside flats), respectively 24 h after ulv spraying. choochote et al. (2004) evaluated the adulticidal efficacy of the crude seed extract of apium graveolans against ae. aegypti and found a slightly adulticidal potency of this extract with ld50 and ld95 values of 6.6 and 66.4 mg/cm², respectively. choochote et al. (2005) also tested for adulticidal activity of hexane-extracted curcuma aromatica against ae. aegypti females, it was found to be slightly more effective with the lc50 value of 1.60µg/mg compared to volatile oil with lc50 value of 2.86 µg/mg, respectively. in conclusion, although bifenthrin has more toxic effect on ae. spp larvae and adults than acorus calamus extract, but in search for botanical insecticide, a. calamus extract could be utilized for dengue vector control. the wide availability of this plant in tropical area could be exploited for vector control usage. acknowledgements we wish to thank faculty of allied health sciences, universiti kebangsaan malaysia for providing research facilities. we appreciate the ministry of science technology and innovation for giving the grant irpa 06-02-02-0030 ea098 to conduct this study. references chan yc, ho bc, chan kl (1971) aedes aegypti (l.) and aedes albopictus (skuse) in singapore city.5. observations in relation to dengue haemorrhagic fever. bull who. 44: 651-58. choochote w, chaiyasit d, kanjanapothi d, rattanachanpichai e, jitpakdi a, tuetun b, pitasawat b (2005) chemical composition and anti-mosquito potential of rhizome extract and volatile oil derived from curcuma aromatica against aedes aegypti (diptera:culicidae). j vector ecol. 30(2): 302-309. choochote w, tuetun b, kanjanapothi d, rattanachanpichai e, chaithong u, chaiwong p, jitpakdi a, tippawangkosol p, riyong d, pitasawat b (2004) potential of crude seed extract of celery, apium graveolens l., against the mosquito aedes aegypti (l.) (diptera: culicidae). j vector ecol. 29(2): 340-46. harinasuta c (1984) mosquito-borne diseases in southeast asia.mosq-borne dis bull. 1: 1-11. hidayatulfathi o, sallehuddin s, ibrahim j (2004) adulticidal activity of some malaysian plant extracts against aedes aegypti linnaeus. trop biomed. 21(2): 61-67. iranian j arthropod-borne dis, (2008), 2(2): 7-11 s sulaiman et al: evaluation of bifenthrin … 11 hidayatulfathi o, sallehuddin s, shafariatul ai, zaridah mz (2005) studies on the larvicidal,adulticidal and repellent activities of acorus calamus linn. against aedes aegypti linn. (diptera: culicidae). anns med entomol. 14(1): 6-13. jumali, gubler dj, nalim s, eram s, saroso js (1979) epidemic dengue haemorrhagic fever in rural indonesia iii entomological studies. am j trop med hyg. 28: 717-724. mulla ms, su t (1999) activity and biological effects of neem products against arthropods of medical and veterinary importance. j am mosq control assoc. 15: 133-152. rudnick a, chan yc (1965) dengue type 2 virus in naturally infected aedes albopictus mosquitoes in singapore. science. 149: 638-639. russell pk, gould dj, yuill tm, nisalak a, winter pe (1969) recovery of dengue4 viruses from mosquito vectors and patients during an epidemic of dengue haemorrhagic fever. am j trop med hyg. 18: 580-583. sukumar k, perich mj, boobar lr (1991) botanical derivatives in mosquito control: a review. j am mosq control assoc. 7: 210-237. raymond m (1985) log probit analysis: basic programme of microcomputer. cahiers orstrom serie entomol med parasit. 22(2): 117-121. sulaiman s, hidayatulfathi o, abdullah arl, pawanchee za, shaari n, wahab a (2005) evaluation of acorus calamus extract and bistar® (bifenthrin) against dengue vectors in high rise flats in bandar baru sentul,kuala lumpur, malaysia. anns med entomol. 14(2): 42-47. thomas tg, rao s, lal s (2004) mosquito larvicidal properties of essential oil of an indigenous plant, ipomoea cairica linn. jap j infect dis. 57(4): 176-177. world health organization (1981a) instructions for determining the susceptibility or resistance of adult mosquitoes to organochlorine, organophosphate, and carbamate insecticides. establishment of base-line. who/ vbc/81.805 world health organization (1981b) instructions for determining the susceptibility or resistance of mosquito larvae to insecticide.who/vbc/81.807. j arthropod-borne dis, september 2016, 10(3): 426–430 k pirali-kheirabadi et al.: a first report of … 426 http://jad.tums.ac.ir published online: january 06, 2016 case report a first report of infestation by pseudolynchia canariensis in a herd of pigeons in shahrekord (southwest of iran) khodadad pirali-kheirabadi 1, *amir dehghani-samani 2, nader ahmadi-baberi 3, vida najafzadeh 4 ¹department of pathobiology, faculty of veterinary medicine, shahrekord university, shahrekord, iran ²department of clinical sciences, faculty of veterinary medicine, shahrekord university, shahrekord, iran 3expert of the faculty of veterinary medicine, shahrekord university, shahrekord, iran 4under graduated student of faculty of veterinary medicine, shahrekord university, shahrekord, iran (received 3 aug 2013; accepted 1 aug 2015) abstract background: pigeons (columba livia) have been kept as pet and reared for food in several countries including iran. ectoparasites are regarded as the basic causes of retardation in growth, lowered vitality and poor conditions of the birds. pseudolynchia canariensis a hippoboscidae fly is one of the important ectoparasites of pigeons and is responsible for the transmission of pathogens to birds and humans same as pathogenic protozoan haemoproteus columbae. methods: a herd of domestic pigeons contained 50 pigeons in shahrekord, southwest iran was evaluated clinically infested by ectoparasites. ectoparasites were removed. the samples were collected and then referred to the laboratory of parasitology of shahrekord university, shahrekord, iran. results: usin diagnostic key for diptera fly, these flies were find p. canariensis. this is a rare report of infestation of pigeons herd by p. canariensis in iran. the infestation rate was 40% that rate of infestation in pipers was more than females and in females was more than males. conclusion: the rate of infested pipers was more than adults that maybe the less potential of pipers in removing of ectoparasites is reason of this higher rate. keywords: domestic pigeons, pseudolynchia canariensis, iran introduction pigeons and doves have colonized suburban and urban areas of iran. their interaction with humans and other domestic and wild animals portends them as a potential carrier of zoonotic pathogens and parasites same as haemoproteus columbae (dehghani-samani et al. 2013). pigeons constitute a major source of infection and transmission of diseases to human, same as yersiniosis (hubbert 1972), salmonellosis (muller 1965), western equine encephalomyelitis (fothergill et al. 1938), cryptococcosis (newberry et al. 1967), chlamydiosis or ornithosis (terskikh et al. 1961) and q fever (syrucek and raska 1956). several health problems can affect pigeons, but parasite infections play a major role. ectoparasites are regarded as the basic causes of retardation in growth, lowered vitality and poor conditions of the birds. they can affect bird health directly by causing irritation, discomfort, tissue damage, blood loss, toxicosis, allergies and dermatitis which in turn reduce the quality and quantity of meat, initiate excessive preening which interrupts feeding, as the birds spend much time preening rather than being involved in other essential life activities and egg production (wall and shearer 2001, mullen and durden 2002). *corresponding author: dr amir dehghani-samani, e-mail: amir@stu.sku.ac.ir, amirds2008@gmail.com j arthropod-borne dis, september 2016, 10(3): 426–430 k pirali-kheirabadi et al.: a first report of … 427 http://jad.tums.ac.ir published online: january 06, 2016 pseudolynchia bequaert, 1926 a hippoboscid fly is responsible for the transmission of a pathogenic protozoan in pigeons (dehghani samani et al. 2013), hippoboscid flies are also considered potential carriers of parasites, mainly of chewing lice and mites that infest birds (harbison et al. 2009). pseudolynchia is a genus containing five species. of these, only pseudolynchia brunnea latreille, 1812 is endemic to the american continent (bequaert 1955). pseudolynchia canariensis macquart, 1840 presents a large dorsoventrally flattened body, with a small head located immediately adjacent to the prothorax (yamauchi et al. 2011). hippoboscidae flies are ubiquitous and a vector for haemoproteus sp among pigeons and doves of the order columbiformes, whereas p. canariensis is the vector of h. columbae among columba livia pigeons, both in the case of natural and experimental infections (graciolli and carvalho 2003). they can be found in all biogeographical regions in the world and usually infect birds and some mammalian orders (freitas et al. 2002). pseudolynchia canariensis feeds for 20 to 80minute about twice a day (arcoverde et al. 2009). the life history of p. canariensis has features that make it amenable to study in the lab. eggs are hatched in uterus in the female and then three stages of larvae feed from milk glands (harwood and james 1979). the larvae pupate and female flies deposit puparia in the substrate in or around pigeon nests (arcoverde et al. 2009). the flies will reproduce on captive birds, depositing puparia under layers of newspaper lining pigeon cages. female p. canariensis produce their first puparium six days after their first blood meal; they produce one puparium about every two days thereafter (herath 1966, klei 1971). the present investigation was concentrated on the occurrence of p. canariensis infestation in the herd of domestic pigeons. the aim of this report was to presentation of infestation with adult stage of a rare ectoparasite named p. canariensis, reported in southwest of iran. we focused on some of keys that help us for identification of this fly's species. materials and methods study area is located in shahrekord, southwest iran at 2000 meters above sea level with very cold climate. it is beside the southwest mountains, far from the sea, (31°59′ 59″n 50°39′42″e), the temprature in winter is less than 0 °c usually. at first we received information about a sever infestation of a domestic pigeons herd with diptera on july 2012, in visit of the herd of domestic pigeons with 50 pigeons (17 pigeons were male, 18 female and 15 pipers), all of the pigeons were infested. clinical examination revealed several flies on the surface of the skin of all of the pigeons. twenty samples were collected carefully from different parts of the body of birds, from down and contour feathers of skin, trunk, rump and nape feathers, quill feathers of wing and tail, and samples referred to the laboratory of parasitology for diagnosis. the identification was carried out under a microscope, according to identification keys of hutson (1984). results our macroscopic and microscopic graphs show that wings of collected flies had only one cross-vein. palpi distinctly more than twice as long as broad (fig.1), median length of scutellum is about the distance between bases of scutellar setae; posterior margin of scutellum is more or less straight (fig. 2), male mid metatarsus had ventral peg-like setae near base. samples were identified and confirmed as p. canariensis (fig. 3). j arthropod-borne dis, september 2016, 10(3): 426–430 k pirali-kheirabadi et al.: a first report of … 428 http://jad.tums.ac.ir published online: january 06, 2016 fig. 1. head, mouth appendix and palpi of pseudolynchia canariensis fig. 2. ventral view of abdomen of pseudolynchia canariensis fig. 3. pseudolynchia canariensis removed from columba livia discussion for the first time we report the infestation by p. canariensis of domestic pigeons in shahrekord with very cold climate in southwest of iran. infestation by p. canariensis is reported from many locations in world but there is a few reports of this fly from different climates of iran. same as our study, radfar et al. (2012) observed a higher prevalence of p. canariensis on adult individuals of c. livia captured in the municipality of khorasan, in the iranian semiarid region. a low intensity of infestation of p. canariensis on adult individuals of c. livia is expected, as they acquire a higher level of immunity against parasites (merila et al. 1995). the prevalence of c. livia parasites was studied on the canary islands, where p. canariensis was observed in 36% of studied pigeons (adlard et al. 2004). in the state of minas gerais, in southeastern brazil, a research study with free-living pigeons (c. livia domestica) showed that all pigeons were infested with p. canariensis (oliveira et al. 2000). in this report, we showed the infestation by p. canariensis in pigeons only. we did not study on the seasonal distribution of this fly but the prevalence and intensity of infestation by p. canariensis were positively correlated to temperature, both being followed by autumn and least in winter. the increased prevalence of ectoparasites during summer months can be attributed to the requirement of optimum temperatures for the development of parasites and decreased resistance of birds to the parasites in high temperatures resulting in heavy infestations (nadeem et al. 2007). in this report high infestation by p. canariensis occurred in summer in iran same as nadeem et al. (2007) report. j arthropod-borne dis, september 2016, 10(3): 426–430 k pirali-kheirabadi et al.: a first report of … 429 http://jad.tums.ac.ir published online: january 06, 2016 conclusion this report shows that pigeons can have high prevalence of ectoparasite infestation even if they kept in metal cages and near the humans. parasitic infestations are harmful to their host; also, infested pigeons may transfer ectoparasites and other organisms to other hosts and humans. therefore, we should care about this risk and find new effective ways to ward of these ectoparasites from our pets. acknowledgment the authors are grateful to the deputy of research, university of shahrekord for financial support of this project. the authors declare that there is no conflict of interests. references adlard rd, peirce ma, lederer r (2004) blood parasites of birds from southeast queensland. ornithology. 104: 191–196. arcoverde ar, rodrigues af, daemon a (2009) feeding and breeding aspects of pseudolynchia canariensis (macquart, 1839) (diptera: hippoboscidae) under laboratory conditions. parasitology research. 104: 277–280. bequaert jc (1955) the hippoboscid flies or louse flies (diptera) of mammals and birds. part ii. taxonomy, evolution and revision of american genera and species. entomol am, new series. 35: 233– 416. dehghani-samani a, pirali-kheirabadi kh, dehghani-samani a (2013) prevalence and rate of parasitemia of haemoproteus columbae in columba iivia domestica in southwest of iran. iran j parasitol. 8(4): 641–644. fothergill l, john d, dingle h, farber s, connerley ml (1938) human encephalitis caused by the virus of the eastern variety of equine encephalomyelitis. new england j med. 219: 411. frittas fl de, oliveira mb de, oliveira ab (2002) gastrointestinal parasites of captive wild birds in pernambuco state, brazil. parasitol latinoam. 57: 50–54. graciolli g, carvalho cjb (2003) hippoboscidae (diptera: hippoboscoidea) in the state of paraná, brazil: keys, hosts and geographic distribution. rev bras zool. 20: 667–74. harwood rf, james mt (1979) entomology in human and animal health. seventh edition. macmillian, new york, new york, usa, pp. 319–341. harbison cw, jacobsen mv, clayton dh (2009) a hitchhiker’s guide to parasite transmission: the phoretic bahaviour of feather chewing chewing lice. int j parasitol. 39: 569–575. herath prj (1966) colonizing pseudolynchia canariensis on hosts other than the pigeon, columba livia. dissertation. wayne state university, detroit, michigan, usa. hubbert wt (1972) yersiniosis in mammals and birds in the united states. am j trop med and hyg. 21: 458–463. hutson am (1984) keds, flat-flies and batflies. diptera, hippoboscidae and nycteribiidaehandbooks for the identification of british insects. 10(7): 1–40. klei tr (1971) studies on haemoproteus columbae of the pigeon, columba livia, and the intermediate host pseudolynchia canariensis. [dissertation]. wayne state university, detroit, michigan, usa. merila j, bjorklund m, bennett gf (1995) geographic and individual variation in haematozoan infections in the greenfinch, carduelis chloris. can j zool. 73: 1798–1804. mullen gr, durden la (2002) medical and veterinary entomology. academin press, london. j arthropod-borne dis, september 2016, 10(3): 426–430 k pirali-kheirabadi et al.: a first report of … 430 http://jad.tums.ac.ir published online: january 06, 2016 muller g (1965) salmonella in bird faeces. nature. 207(5003): 1315. nadeem m, khan mn, iqbal z (2007) determinants influencing prevalence of louse infestations on layers of district faisalabad (pakistan). brit poul sci. 485: 546–550. newberry mw, jinks j, walter e, john w, chandler j, fred et (1967) epidemiological study of cryptococcus neoformans. ann int med. 67: 724–732. oliveira pr, mundim mjs, cabral dd (2000) a survey of parasitic fauna in domestic pigeons (columba livia domestica) in uberlândia, mg, brazil. vet not. 6: 53–56. radfar mh, asl em, seghinsara hr, dehaghi mm, fathi s (2012) diversity and prevalence of parasites of domestic pigeons (columba livia domestica) in a selected semiarid zone of south khorasan, iran. trop anim health pro. 44: 225–229. syrucek l, raska k (1956) q fever in domestic and wild birds. bull world health organ. 15(1–2): 329–337. terskikh ii, chel’tsov-bebutov am, kuborina ln, keleinikov aa (1961) study on ornithosis in birds and its natural focal occurrence. vopr virusol. 6(2): 141–146. wall r, sherarer d (2001) veterinary ectoparasites: biology, pathology and control. 2nd edn. wiley blackwell, london, p. 304. yamauchi t, tsuda y, sato y, murata k (2011) pigeon louse fly, pseudolynchia canariensis (diptera: hippoboscidae), collected by dry-ice trap. j am mosq control assoc. 27(4): 441–443. j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 383 http://jad.tums.ac.ir published online: january 06, 2016 original article molecular assay on crimean congo hemorrhagic fever virus in ticks (ixodidae) collected from kermanshah province, western iran maria mohammadian 1, sadegh chinikar 2, *zakkyeh telmadarraiy 1, hassan vatandoost 1, mohammad ali oshaghi 1, ahmad ali hanafi-bojd 1, mohammad mehdi sedaghat 1, mehdi noroozi 3, faezeh faghihi 4, tahmineh jalali 2, sahar khakifirouz 2, nariman shahhosseini 2,5, firoozeh farhadpour 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2arboviruses and viral hemorrhagic fevers laboratory (national reference laboratory), pasteur institute of iran, tehran, iran 3department of virology, school of public health, tehran university of medical sciences, tehran, iran 4cellular and molecular research center, iran university of medical sciences, tehran, iran 5who collaborating centre for arbovirus and haemorrhagic fever reference and research, department of virology, bernhard nocht institute for tropical medicine, hamburg, germany (received 25 dec 2013; accepted 10 mar 2015) abstract background: crimean-congo hemorrhagic fever (cchf) is a feverous and hemorrhagic disease endemic in some parts of iran and caused by an arbovirus related to bunyaviridae family and nairovirusgenus. the main virus reservoir in the nature is ticks, however small vertebrates and a wide range of domestic and wild animals are regarded as reservoir hosts. this study was conducted to determine the infection rate of cchf virus in hard ticks of sarpolezahab county, kermanshah province, west of iran. methods: from total number of 851 collected ticks from 8 villages, 131 ticks were selected randomlyand investigated for detection of cchf virus using rt-pcr. results: the virus was found in 3.8% of the tested ticks. hyalommaanatolicum, h.asiaticum and rhipicephalus sanguineus species were found to have viral infection, with the highest infection rate (11.11%) in rh. sanguineus. conclusion: these findings provide epidemiological evidence for planning control strategies of the disease in the study area. keywords: ixodidae, cchfv, kermanshah, iran introduction crimean congo hemorrhagic fever (cchf) is a viral disease with approximate mortality rate of 30% in humans (ergonul 2006). the disease can be transmitted via contact with blood or secretions of infected animals and tick bite or manipulation and squishing of cchf infected ticks. human to human transmission i.e. nosocomial infection is another main way for the disease transmission (hoogstraal 1979, charrel 2004, appannanavarand mishra 2011). cchf disease has a worldwide dissemination and is considered to be an endemic disease in many countries in asia, africa and europe continents (charrel 2004, appannanavar and mishra 2011). until now cchf virus has been detected in 31 species of several species of hard and soft ticks (hoogstraal 1979, linthicum and bailey 1994, papa et al. 2002). the disease was first reported in iran during 1970 (chumakov1972), and now is considered as an *corresponding author: dr zakkyeh telmadarraiy, email: ztelma@yahoo.co.in j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 384 http://jad.tums.ac.ir published online: january 06, 2016 endemic disease in many parts of the world. previous studies confirmed cchf cases. in iran, many studies were conducted on disease carriers; however in 1978, the virus was separated from soft tick larvae, ornithodoros lahorensis (sureau 1980) for the first time. since then, many studies were conducted in different regions of iran to find the cchf infection in ticks. based on previous studies, cchf infection was detected in 5 genera of soft and hard ticks including hyalomma, rhipicephalus, dermacentor, haemaphysalis and ornithodoros (shirani et al. 2004, telmadarraiy et al. 2007). a diverse range of infection rates has been reported in these ticks from 0.2 to 33.3% (shirani et al. 2004, moradi et al. 2008, nasiri 2008, tahmasebi et al. 2010, telmadarraiy et al. 2007, 2010, 2014, salim-abadi et al. 2011, chinikar et al. 2012, faghihi et al. 2015, sarifinia 2012, karimi 2013, mehravaran et al. 2013, champour et al. 2014). kermanshah province contains a big population of nomads in west of the country, so it is an important region for the legal and illegal import/export of domestic animals from iraq, the neighbor country at the borderline of sarpole-zahab city. crimeancongo hemorrhagic fever is reported from some parts of iraq and seems to be an endemic disease there, where during 1979– 1981, 63 cases of the disease were reported within number of 48 mortalities. studies over 50% of iraqi goat, sheep, and horse sera were positive for the presence of antibodies, in another sero-survey 29% of all animal breeders tested in iraq were also reported to be positive for those antibodies (defens pest managment information analysis center armed forces glen section walter reed army medical center washington 1999). according to the national monitory system of iraq, 0–6 of cchf cases were annually reported during 1998–2009, and in 2010, 11 confirmed cases of the disease with 36% mortality and 28 suspected cases with 4% mortality were reported (majeed et al. 2012). presence of more than 140,000 livestock in the county makes it as a major area of animal husbandry in kermanshah province. adjacent plains and mountainous areas are the major locations for nomad migration, which have large herds of livestock. sarpolezahab has a long borderline with iraq; make it a suitable area for legal/illegal livestock export/import trades between two countries. so risk of the disease transmission exists in both sides of the borderlines of the two neighboring countries where it can be passed from one side to another side of the border, periodically. this study was aimed to investigate cchf virus infection rate in ticks of domestic animals, as the main vectors of the disease. materials and methods study area kermanshah province is located at the western region of iran. sarpole-zahab county located at the western margin of the province, in coordinates of 34o27’40” n and 45 o 51’46” e, with an area of about 1.271 square kilometers. sarpole-zahab has a relatively warm and semi-arid climate with the mild winters and hot summers. the county has mountainous, plain, and foothill topographic areas. in this study, eight villages of sarzal, ghalee vari, mela kabob, salman tape, berimov and, anzal, dare balut and sare baghe golin located in different geographical locations of the county were selected randomly (table 1). tick specimen collection from the livestock was conducted as described below during the years 2012–2013 (fig. 1). based on statistical analysis of available data, 131 tick specimens were selected to determine the cchf infection rate in ticks of the area study. j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 385 http://jad.tums.ac.ir published online: january 06, 2016 sample collection and preparation tick specimens were collected seasonally based on the species diversity, type of host animals and geographic location of the area study. samples were maintained individually in labeled tubes and transferred to the laboratory of school of public health, tehran university of medical sciences in cool boxes. the specimens were identified to species level using the known morphologically keys (hoogstraal 1979, walker et al. 2003). 128 specimens of the identified ticks were selected in random and transferred in cold chain to the arbovirus and viral hemorrhagic fevers laboratory (national ref. lab) at pasture institute of iran for molecular detection of cchf virus. rna extraction and rt-pcr for rna extraction, each tick was washed twice with pbs 1x and then crushed by a mortar and pestle in 300 µ l of pbs buffer. rna extraction was performed using rna easy mini kit (qiagen, germany) based on the protocol recommended by the manufacturer. the extracted rna was dissolved in rnase-free water and kept at -70 ˚c until use. rt-pcr reaction was performed using one-step rt-pcr kit (qiagen, germany) based on the protocol. in each pcr reaction, 5µ l of the extracted rna and 1µ l of each specific primer (forward: 5’-tggacacc ttcacaaactc-3’and reverse: 5’-gac aattccctacacc-3’) were added to amplify the small segment (s-segment) of the virus (chinikar et al. 2004). at next step, 5 µ l of rt-pcr products were mixed with 1µ of loading buffer and the mixture was loaded on 1.5% agarose gel for electrophoresis. dna bands were stained with ethidiumbromide and were visualized under uv trans illuminator (chinikar et al. 2008, 2010). sequencing and sequence analysis rt-pcr products were sequenced by abi genetic analyzer 3130 machine using big dye terminator v3.1 cycle sequencing kit and specific primers (chinikar, shahhosseini et al. 2013).the partial sequences around 500 bp of the s-segment were used for phylogenetic analysis (table 2). the multiple alignments were performed using clustal w, for seven sequences of current study and some sequences from gen bank. phylogenetic tree was drawn by maximum-likelihood method with kimura 2-parameter model using mega 5.2 software. bootstrap method with replications of 1000 was used for assessing confidence in phylogenetic tree results. results a total number of 851 ticks were collected and identified in this study. tick infestation rate was accounted as much as 84.2%, 10.53% and 5.27%, in sheep, cows and goats respectively. three genera of ixodid ticks including 10 species hyalommaanatolicum, hy. asiaticum,hy. dromedarii, hy. marginatum, hy. detritum, hy. sp, rhipicephalus sanguineus, rh. bursa, rh. sp and haemaphysalissulcata were identified. a subsetof 131 ticks (15.4%) out of 851 ticks was examined to detect cchf virus transcripts (fig. 2). rt-pcr amplification of s-segment of cchf virus produced a pcr band of 536bp (table 1). the results of rtpcr showed an infection rate of 3.8% (n=5) among the tested specimens. the infected species were found to be hy. anatolicum (4.1%), hy. asiaticum (4.54%) and rh .sanguineus (11.11%). the infected ticks were collected from cow and sheep. molecular results showed cchf virus genome in 6.38% (3/47) and 2.85% (2/70) of ticks from cowand sheep, respectively, while all ticks collected from goat were negative (fig .3). phylogenetic analysis the phylogenetic relationship of the isolated cchf sequences from ticks of sarj arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 386 http://jad.tums.ac.ir published online: january 06, 2016 pole-zahab with genbank available sequences was drawn using maximum likelihood method and kimura2 parameter (fig. 4). all the isolates were clustered in asia i clade, closely to matin and sr3 strains of pakistan and newly released strain from afghanistan (ölschläger et al. 2011). overall mean distance computation of this study isolates show just 0.3% divergence between them. fig. 1. map of the study area in kermanshah province, iran fig. 2. rt-pcr products of cchf s-segment (536 bp band) found in tick specimens collected in sarpole-zahab county, kermanshah province. lad: 100 bp ladder, pc: positive control, nc: negative control, s1, s2, s3, s4, s5, s6, s7 and s9: negative samples; s8 and s10: positive samples. sh ee p co w go at 0 20 40 60 80 negative positive host n o of t ic ks t es te d fig. 3. details of cchf infected ticks and their animal hosts in sarpole-zahab county, kermanshah, western iran j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 387 http://jad.tums.ac.ir published online: january 06, 2016 fig. 4. maximum likelihood phylogenetic tree retrieved from 500bp of cchfv partial s-segment sequences obtained in this study (cchfm14, cchfm16, cchfm18, cchfm22 and cchfm24) and the available data from genbank. only boot strap values more than 70% are shown. j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 388 http://jad.tums.ac.ir published online: january 06, 2016 table 1. details of the tick species were collected and examined for the presence of cchf virus genome, sarpole-zahab county, western iran animal location hy. anatolicum (tested /+) hy. asiaticum (tested/+) hy. dromedarii (tested/+) hy. marginatum (tested/+) hy. detritum (tested/+) hy. sp (tested /+) rh. sanguineus (tested /+) rh. bursa (tested/+) rh. sp (tested/ +) ha. sulcata (tested/+) total sheep sz 7/0 3/0 0/0 0/0 1/0 0/0 0/0 1/0 0/0 0/0 12/0 gv 6/0 0/0 0/0 0/0 0/0 1/0 8/0 0/0 1/0 1/0 17/0 mk 5/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5/0 st 7/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 7/1 bv 3/0 2/0 4/0 2/0 2/0 0/0 1/1 0/0 0/0 0/0 14/1 an 1/0 2/0 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 4/0 db 4/0 0/0 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5/0 sbg 1/0 3/0 0/0 0/0 2/0 0/0 0/0 0/0 0/0 0/0 6/0 cow sz 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 gv 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 2/0 mk 7/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 7/0 st 3/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 3/0 bv 2/0 0/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 4/0 an 9/1 4/1 5/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 18/2 db 6/0 4/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 12/0 sbg 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/1 goat sz 3/0 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5/0 gv 0/0 3/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 3/0 mk 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 st 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 bv 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 2/0 an 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 db 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 sbg 2/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 2/0 total 73/3 23/1 15/0 2/0 5/0 1/0 9/1 1/0 1/0 1/0 131/5 sz: sarzal, gv: ghaleevari, mk: mela kabob, st: salman tape, bv: berimovand, an: anzal, db: darebalut, sbg: sarebaghegolin. j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 389 http://jad.tums.ac.ir published online: january 06, 2016 table 2. details of sequence data used in this study isolate name or accession number country city host isolated reference cchf14 iran sarpole-zahab sheep this study cchf16 iran sarpole-zahab sheep this study cchf18 iran sarpole-zahab cow this study cchf22 iran sarpole-zahab cow this study cchf24 iran sarpole-zahab cow this study hm452305 afghanistan ns human ölschläger et al. 2011 aj538198 pakistan karachi human hewson et al. 2004 ay366379 iran sistan-baluchistan human chinikar et al. 2004 ay366378 iran sistan-baluchistan human chinikar et al. 2004 dq446213 iran ns ns direct submission dq446212 iran ns ns direct submission ay366377 iran sistan-baluchistan human chinikar et al. 2004 ay366374 iran sistan-baluchistan human chinikar et al. 2004 ay366373 iran sistan-baluchistan human chinikar et al. 2004 dq446214 iran ns ns direct submission u88414 ns ns ns direct submission ay366376 iran sistan-baluchistan human chinikar et al. 2004 ay366375 iran qom human chinikar et al. 2004 af527810 pakistan ns ns direct submission gu456725 iran hamedan tick chinikar et al. 2010 gu456728 iran hamedan tick chinikar et al. 2010 gu456724 iran hamedan tick chinikar et al. 2010 gu456727 iran hamedan tick chinikar et al. 2010 gu456726 iran hamedan tick chinikar et al. 2010 ay905662 pakistan quetta ns burt and swanepoel 2005 ay905663 pakistan quetta ns burt and swanepoel 2005 ay905661 pakistan quetta ns burt and swanepoel 2005 ns: not stated j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 390 http://jad.tums.ac.ir published online: january 06, 2016 discussion our results in this study showed the presence of cchf virus in different regions of sarpol-e-zahab city, kermanshah province. this infection was confirmed in hy. anatolicum, hy. asiaticum, r. sanguineus. findings of this study are consistent with the results obtained other groups in different regions of the country (tahmasebi et al. 2010, telmadarraiy et al. 2010). although hyalomma ticks are the main carriers of the virus in africa, asia, europe and the middle east (swanepoel et al. 1987, whitehouse 2004, ergönül 2006) the virus was also detected in other genera of both soft and hard ticks (whitehouse 2004) as we found in r. sanguineus. in other studies conducted in western part of iran, cchf virus infection was reported in hy. marginatum, hy. anatolicum, hy. detritum, hy. dromedarii, hy. asiaticum, haemaphysalispunctata, r. sanguineus, and r. bursa, (moradiet al. 2008, tahmasebi et al. 2010, sharifinia 2012, fakoorziba et al. 2012, nasiri unpublished data). according to our previous study conducted in ardabil province, northwest of iran, viral infection was detected in hy. schulzei, hy. marginatum, hy. aegyptium and r. bursa (telmadarraiy et al. 2010). studies conducted in west part of iran showed that the genome of cchf virus exists in different species of three genera of hard ticks including hyalomma, rhipicephalus and haemaphysalis. the infection rate of ticks in this study was 3.8%. this rate has been reported from 0.2 to 33.3% in previous studies in iran (shirani et al. 2004, telmadarraiy et al. 2006, 2007, 2010, 2014, moradi et al. 2008, nasiri 2008, tahmasebi et al. 2010, salim-abadi et al. 2011, chinikaret al. 2012, fakoorziba et al. 2012, sarifinia 2012, karimi 2013, mehravaran et al. 2013, champour et al. 2014). ticks of hyalomma genus showed an infection rate of 3.36%, while the species of this genus were found to be infected 1.57% to 20% to cchf virus in other studies (shiraniet al. 2004, telmadarraiy et al. 2006, 2007, 2010, 2014, moradi et al. 2008, nasiri 2008, tahmasebi et al. 2010, salim-abadi et al. 2011, chinikar et al. 2012, fakoorziba et al. 2012, sarifinia 2012, karimi 2013, mehravaran et al. 2013, champour et al. 2014). although 9.09% of rhipicephalus ticks were infected in this study, other groups reported the infection rate between 1.8% to 55% (telmadarraiy et al. 2006, 2010, moradiet al. 2008, tahmasebi et al. 2010, karimi 2013), therefore it can be concluded that although the hyalomma ticks are usually introduced as the vectors of cchf, this potential exists in other genera as well. comparison of results obtained from different regions of the county showed that ticks collected from central regions were more infected than southern and northern regions. the reason of such infection may be due to the condensation of livestock and the quality of breeding management including poor hygienic conditions of livestock breeding sites. people of sarpol-e-zahab area work in high risk professions in close contact with ticks and animals’ tissue and blood. therefore, it is instructed to prevent these people from being subjected to different ticks infected with the virus and or infected animals’ blood or tissue. this study indicates that cchf must be considered as a critical health problem in health centers of sarpolezahab as well as kermanshah and other neighboring provinces, and appropriate strategies must be used for controlling carrier ticks. future research should be focused on the population of carriers and their infection rate, presence of the virus in domestic animal populations and also humans in other regions of the province in order to present a better picture of the dissemination and epidemiology of the virus in the province. j arthropod-borne dis, september 2016, 10(3): 383–393 m mohammadian et al.: molecular assay on … 391 http://jad.tums.ac.ir published online: january 06, 2016 conclusion the prevalence of infected to cchf was higher in plain region of the county rather than mountain region. teaching and prevention programs are recommended to people who are in close contact with ticks and animals’ tissue and blood to prevent from infection. this study indicates that cchf must be considered as a critical health problem in health centers of sarpole-zahab and other neighbouring county, and appropriate strategies must be 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6(3): 13–18. walker ar, bouattur a, camicas j, estradapena a, horak i, latif a, pegram r, preston p (2003) ticks of domestic animals in africa: a guide to identification of species. bioscience reports edinburgh. pp. 572–577 whitehouse ca (2004) crimean-congo hemorrhagic fever. antiviral research. 64: 145–60. j arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 211 http://jad.tums.ac.ir published online: january 05, 2016 original article evaluation of isotope 32p method to mark culex pipiens (diptera: culicidae) in a laboratory chongxing zhang 1, 2, guihong shi 1, yuqiang zhao 1, dongmei yan 1, huaiju li 1, hongmei liu 1, *itsanun wiwatanaratanabutr 3, *maoqing gong 1 1shandong institute of parasitic diseases, shandong academy of medical sciences, jining, shandong, people’s republic of china 2collaborative innovation center for the origin and control of emerging infectious diseases, taishan medical university, taian, shandong 271000, china 3department of plant production technology, faculty of agricultural technology, king mongkut's institute of technology ladkrabang, bangkok 10520, thailand (received 16 jan 2014; accepted 20 may 2015) abstract background: the aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32p and determine the optimal concentration of it. methods: an isotonic physiological saline solution, containing different concentration of radioactive isotope 32plabeled disodium phosphate (na2h 32po4) was injected into rabbits via the jugular vein in the laboratory. emerged cx. pipiens were marked after feeding on rabbit. at the same time, the labeled conditions of emerged cx. pipiens were also measured by placing feces of no. 6 rabbit into containers with mosquito larvae and pupae inside. results: according to the label condition of cx. pipiens after taking blood and the effect of different dosage na2h 32po4 on rabbit health, the optimal concentration of radioactive isotope was determined, that is, 0.1211 mci/kg. by placing feces of no. 6 rabbit into containers with mosquito larvae and pupae inside, the emerged mosquitoes were also labeled. therefore, feeding mosquitoes on the animal injected with radioactive na2h 32po4 was more practical for detecting and tracing mosquitoes. conclusion: the method was less time-consuming, more sensitive and safer. this marking method will facilitate post-bloodmeal studies of mosquitoes and other blood-sucking insects. keywords: radioactive isotope, mark, culex pipiens, rabbit introduction mosquitoes, the most important group of nuisance pest insects, due to their diversity and abundance, demonstrated vector competence and frequent infection in nature, they are regarded as one of the most important vectors of diseases (sardelis et al. 2002, molaei et al. 2006, burkett-cadena et al. 2008a, b). tracking the movement of mosquitoes in their natural habitat is critically important for understanding their basic biology, demography, ethology and vector-borne disease control as well as prevention. a reliable method for marking is critical important to study mosquito behavioral characteristics. animal marking have been used since 218 bc when fisher and peterson used banding to distinguish ownership of birds (fisher and peterson 1964). unfortunately, for marking insects, most marking techniques for vertebrate, such as bands, brands, tattoos, tags, notches, paints are not practical because they are tedious, time-consuming, heavy and costly (southwood 1978, basavaraju et al. 1998). insect marking for scientific studies dates back to 1920, since then, a variety of marking techniques, paints, dyes etc. were used to in studies of insect population dynamics (geiger et al. 1919, dudley and searles 1923). *corresponding author: dr maoqing gong, e -mail: maoqingg@yahoo.com, dr itsanun wiwatanaratanabutr, e-mail: kwitsanu@kmitl.ac.th j arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 212 http://jad.tums.ac.ir published online: january 05, 2016 methods to mark mosquitoes have included dyes (welch et al. 2006, midega et al. 2007), paints (trpis and hausermann 1986, niebylski and meek 1989, service 1993, bellini et al. 2010, ciota et al. 2012, liu et al. 2012, verhulst et al. 2013), dusts (reisen et al. 1978, reisen et al. 1992, russell et al. 2005), trace elements (anderson et al. 1990, holbrook et al. 1991, solberg et al. 1999, wilkins et al. 2007), and radioactive isotopes (jenkins 1949, abdel-malek 1966, lindquist et al. 1967, hood-nowotny et al. 2006, hamer et al. 2012). however, for study on the behavioral characteristics of post bloodmeal, existing techniques tend to be labor intensive, as they require rearing mosquitoes, marking them in large quantities, and then inspecting large numbers of individuals to detect recaptures (walker et al. 1987). furthermore, compared with natural populations, rearing mosquitoes, marking them in large quantities by using artificial methods, and releasing them may change their behavior (reisen et al. 2003, silver 2008). for study the behavior characteristics after blood meal, these methods are not ideal. however, the problem is how to mark breeding mosquitoes without inhibiting their normal biology, and with long-term retention after blood meal, it is still bothering most biological scientists. until now, only zhang et al. (2014) marked adult mosquitoes by feeding them on cow injected with isotope 32p and subsequent ecological investigations. in some studies, successfully labeled mosquitoes by feeding them on radioactive animal blood (hassett and jenkins 1951), use of large bait animals for marking wild population of adult anophels aquasalis injected with a dose of 1.7 curies of 32p (bruce-chwatt 1956). however, such high dosage would be dangerous to the animal (winteringham london meeting, 1953). the aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32p and determine the optimal concentration of it. injection of rabbits with na2hp 32o4 and blood feeding of culex pipiens before experimenting, 6 healthy rabbits (2 kg) were selected and physically examined by veterinarian. the injection method of normal saline solution to the rabbits was according to smith et al. (1951), i e. an isotonic buffered saline solution, containing different concentration of na2hp 32o4, was injected intravenously of rabbits. the dosage for no. 1, 2 rabbits, no. 3, 4 rabbits, no. 5 rabbit and no. 6 were 0.2 mci, 0.4 mci, 0.8 mci and 1.7 mci, respectively. for the negative control, 1 healthy rabbits was injected with isotonic buffered saline solution without na2hp 32o4.then 20 to 50 emerged female cx. pipiens fed on these rabbits at 6, 12, 24, 48, 72 h and 120 h after injected with na2hp 32o4. at the same time, 0.2 ml of blood was extracted from rabbits, as well as at 16th and 32nd days for radioactivity level measure. every batch of cx. pipiens’ radioactivity levels were measured at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after blood meal. measuring methods the measurement of radioactivity was conducted using a liquid scintillation counters (model ysj-76). before injection of 32p to rabbits, to normalize background radiation, radioactivity level of 30 emerged adult cx. pipiens reared in the lab was measured. mosquitoes tested were anesthesia with ether, placed in a β bell counter tube of the vitriol chambers for 1 minute, as for the rabbits blood test, 0.2 ml of blood was dipped on the paper in the tinfoil sample plates, counts that exceeding 50% of the background was as the standard that were labeled. placing feces of no. 6 rabbit in tap water to mark mosquitoes at the 2nd day after no. 6 rabbit was inj arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 213 http://jad.tums.ac.ir published online: january 05, 2016 jected with na2h 32po4, up to 5 g feces was placed into a container with 300 ml of tap water and cx. pipiens’ larvae and pupae inside. as the negative control, placed 5 g feces of the rabbit without injected na2hp 32o4 into the water. radioactivity levels of emerged mosquitoes were measured. ethics clearance the experimental project was reviewed and approved by the ethical committee of shandong academy of medical sciences (jinan, shandong). urine and feces of the rabbits were collected and sent to the institute of radiation medicine, shandong academy of medical sciences for appropriate processing to prevent spread of the isotope. the half-life of 32p was 14.3 d. results conditions of mosquitoes radioactively labeled after blood feeding radioactivity level of emerged adult cx. pipiens and rabbits blood was measured, the background was determined as was ～12–13 counts per minute (cpm), i e counts that exceeded 50% of background (>20 cpm) were considered positive. within 5 days after no. 1 to 5 rabbits were injected with na2h 32po4, 579 female cx. pipiens fed on the rabbits, among the 222 mosquitoes blood feeding, except one mosquito was fewer than 20 cpm, the others 221 mosquitoes were labeled no matter how much dosage of na2h 32po4 was injected. the more dosage the rabbits injected with na2h 32po4, the higher radioactive levels of cx. pipiens after blood feeding. the radioactive levels started to decrease from the 48 hours (2nd day) after injection. compared with radioactive level at 48 h, though the radioactive level of labeled mosquitoes was higher than background at 72 h, 96h and 120 h, radioactive level decreased to a lower level (figs. 1, 2, 3). radioactivity levels of rabbit blood after injection of na2hp 32o4 the radioactive level decreased very fast in the blood of rabbits. at 6 hour the radioactive level was 100%, the radioactive level decreased 26–48%, 60–75% and 95–97% at the 2nd, the 5th and the 32nd day, respectively (table 1). effect of na2h 32po4 on rabbit health no. 1 to 4 rabbits showed good health, have no change on body temperature and body weight. on the 2nd day, no. 5 rabbit abortion, and gave birth to 2 died bunnies, the radioactive level was 15 mr / h at 5 cm away from these died bunnies. the no. 5 rabbit presented no abnormalities and was dissected at the 143rd day after injected with na2h 32po4, no pathological changes were found in the internal organs. the radioactivity level of liver was measured, which decreased to similar level of background. however, no. 6 rabbit showed diarrhea, appetite loss, weight loss and other symptoms. at the 31st days, and no special lesions were found in internal organs after dissection. it still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc (table 2). conditions of mosquitoes radioactively labeled after placing feces in the container at the 2nd day after no. 6 rabbit was injected with na2h 32po4. after 5 g feces of no. 6 rabbit was placed into a container, at the 2nd and 7th day, there were total 39 adult cx. pipiens mosquitoes emerged, among them, 22 (56.5%) were marked, their cpm was 21～875, the average was 323. j arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 214 http://jad.tums.ac.ir published online: january 05, 2016 counts per minute (cpm) of mosquitoes after blood-feeding (hours) 0 20 40 60 80 100 120 2 h 6 h 12 h 24 h 48 h 72 h 96 h 120 h cpm 6 12 24 48 72 120 fig. 1. levels of radioactivity of culex pipiens after fed on no. 1, 2 rabbits after injection of 32p (0.2 mci) legend 6, 12 , 24, 48, 72, 120: mosquito fed time after injection 32p into rabbit (hours) counts per minute (cpm) of mosquitoes after blood-feeding (hours) 0 50 100 150 200 250 2 h 6 h 12 h 24 h 48 h 72 h 96 h 120 h cpm 6 h 12 h 24 h 48 h 72 h 120 h fig. 2. levels of radioactivity of culex pipiens after fed on no. 3, 4 rabbits after injection of 32p (0.4 mci) legend 6, 12, 24, 48, 72, 120: mosquito fed time after injection 32p into rabbit (hours) counts per minute (cpm) of mosquitoes after blood-feeding (hours) 0 100 200 300 400 500 2 h 6 h 12 h 24 h 48 h 72 h 96 h 120 h cpm 6 24 48 72 120 fig. 3. levels of radioactivity of culex pipiens after fed on no. 5 rabbits after injection of 32p (0.8 mci) legend 6, 24, 48, 72, 120: mosquito fed time after injection 32p into rabbit (hours) j arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 215 http://jad.tums.ac.ir published online: january 05, 2016 table 1. radioactivity levels of rabbit blood after injection of 32p (1.7 mci) at different time rabbit no. dosage (mci) radioactivity level of rabbit blood after injection of 32p 6h 12h 24h 2 days 3 days 5 days 16 days 32 days c p m c p m d ec re as e% c p m d ec re as e% c p m d ec re as e% c p m d ec re as e% c p m d ec re as e% c p m d ec re as e% c p m d ec re as e% 1, 2 0.2 2709 1922 29 1701 37 1413 48 992 63 681 75 226 92 84 97 3, 4 0.4 4155 3383 19 2926 30 2383 43 1820 56 1199 71 317 92 120 97 5 0.8 5701 5189 9 5290 7 4234 26 3681 35 2300 60 783 86 261 95 6 1.7 9284 1,426 85 cpm = pulse per minute, do not measured table 2. radioactivity levels in no. 6 rabbit tissue after injection of 32p (1.7 mci) 31 days tissue weight (mg) cpm cpm per tissue liver 275 5,263 19,138 muscle 295 5,586 19,000 spleen 197 3,118 15,827 heart 336 5,170 15,387 small intestine 205 2,419 11,800 lung 270 2,685 9,944 kidney 306 2,628 8,588 background radiation= 22 cpm 215 j arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 216 http://jad.tums.ac.ir published online: january 05, 2016 discussion we could see that the more dosage the rabbits injected with na2h 32po4, the higher radioactive levels of cx. pipiens after blood feeding. the radioactive levels started to decrease from the 48 hours (2nd day) after injection. compared with radioactive level at 48 h, though the radioactive level of labeled mosquitoes was higher than background at 72 h, 96 h and 120 h, radioactive level decreased to a lower level. the more injection of na2h 32po4 into rabbits, the higher radioactivity level in the blood and the slower decrease, was also consistent with the labeling condition of cx. pipiens (figs. 1, 2, 3). no. 1 to 4 rabbits showed good health, but as for the no. 5 rabbit injected with 0.8 mci of na2h 32po4, on the 2 nd day, abortion, and gave birth to 2 died bunnies. at the 143rd day, the no. 5 rabbit presented no abnormalities, no pathological changes were found in the internal organs. the radioactivity level of liver was similar to background. however, no. 6 rabbit injected with 1.7 mci of na2h 32po4 showed diarrhea, appetite loss, weight loss and other symptoms. at the 31st days, it still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc. (table 2). therefore, the appropriate concentration for not only marking mosquitoes but also no harm to rabbits was not more than 0.4 mci. stable isotopes occur naturally in the environment, are safe and non-invasive, pose no health or environmental risks (hood-nowotny and knols 2007). in medical research, most stable isotopes are non-toxic and are routinely used for mosquito feeding trials, in which human adults are ‘labelled up’ through supplementary feeding with stable isotopes, may be useful for host seeking behavior and repellent testing, etc., in ‘real’ environments. several tracers were studied, such as 60co, 89sr, 65zn, 131i, 45ca and 32p, whereas 32p was the most applied radioisotope for tagging due to its short half-life, safety, activity and easy of detection (o’brien and wolfe 1964). one of the earliest examples of using inorganic 32p labelled ae. aegypti mosquitoes was reported by hasset and jenkins (1949). hasset and jenkins (1951) also performed a detailed study of the conditions affecting mosquitoes labelled with 32p and compared stages, 32p concentrations and age. the filarial larvae, setaria digitata linstow was marked after adult mosquitoes (armigerea obturbans walker) fed on cows or men infected with microfilaria, that larvae of mosquitoes were reared in water containing 1 μc of 32p/ml (dissanaike et al. 1957). toxicity to the insect was also a serious problem to be considered in many studies (quarterman et al. 1955). the radioisotopes 45ca and 131i were very toxic when fed to adult houseflies at 1 μc/ml of milk, whereas 32p was satisfactory (quarterman et al. 1954). by using 15n-labeled potassium nitrate and 13c-labeled glucose to mark larval mosquitoes, there were no consistent effects of isotopic enrichment on immature mosquito survival or adult mosquito body size (hamer et al. 2012). although fluorescent dyes or powders are also suitable for marking mosquitoes (takken et al. 1998, mccall et al. 2001, pates 2002, lapointe 2008, baber et al. 2010, bellini et al. 2010), and no effect of these dyes and powders on performance of ant (pogonomyrmex owyheei), mountain pine beetle (dendroctonus ponderosae), grasshopper (melanoplus spp.), ae. aegypti, anopheles sinensis in some studies (porter and jorgenson 1980, linton et al. 1987, mcmullen et al. 1988, narisu et al. 1999, liu et al. 2012, valerio et al. 2012). others have found a reduced longevity, behavioural response or survival of opine parasitoids (diachasmimorpha spp.), weevils, ae. aegypti, asian citrus psyllid (diaphorina citri) and codling moths (laspeyrej arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 217 http://jad.tums.ac.ir published online: january 05, 2016 sia pomonella) (sheppard et al. 1969, moffitt and albano 1972, reinecke 1990, messing et al. 1993, nakata 2008, verhulst et al. 2013). as for the fluorescent marker, be caused of the restrictions of the retention, most studies researches primarily focus on dispersal of nulliparous female mosquitoes during the initial host-seeking event and sometimes a second host-seeking event (hamer et al. 2012). as for trace element, mosquitoes were successfully marked after feeding on hosts injected with trace element rb and cs (anderson et al. 1990, solberg et al. 1999). marking with trace elements has many advantages over other insect-marking procedures. they are not radioactive, safe for workers and for the environment. as for insects marked with an element, no tags, paints, dyes, dusts, or visible marks were found left to alter insects behavior or interactions with other insects (hagler and jackson 2001). however, a limitation to use of trace elements as insect markers in large fields is that the detection of elements can be difficult, expensive, and time-consuming, requires technical expertise and expensive detection equipment (akey and burns 1991). some trace elements are not retained very well in certain insect species, for example, rb could be detected for only 2–6 days after marking aphids (guillebeau et al. 1993) and adult lygus lineolaris (fleischer et al. 1986). high concentrations of trace elements can adversely affect development, survival, increased adult deformity, reduced pupation, eclosion, egg production and fecundity of certain insects (stimmann et al. 1973, hayes 1989, knight et al. 1989, van steenwyk et al. 1992). naturally, occurring stable isotope markers are useful, as they do not require the premarking of individuals (hood-nowotny and knols 2007). labelling a distinct portion of an ecosystem with stable isotopes is a useful, minimally invasive method to study insect dispersal from an ecophysiological perspective (macneale et al. 2004, 2005). stable isotopes occur naturally in the environment, unlike painting, dusting, etc., stable isotope methods are non-invasive and samples require only minimal preparation following collection, which makes the cost of the process as a completely comparable to methods such as polymerase chain reaction (hood-nowotny and knols 2007). other advantages are the analysis costs (depending on the isotope and the matrix, the cost per sample may range from us$ 5–100.00), shipping stable isotope samples is simple, safe and inexpensive (iaea 2009). it would cost between $150– 250 to label 1 000 000 anopheles mosquitoes with 13c-labelled glucose in the larval stages (hood-nowotny et al. 2006). it is possible to trace the fate of labelled sperm into female spermatheca in studies of male mosquitoes labelled with 13c (helinski et al. 2007). these stable isotope markers meet the usual criteria for use in insect studies: retention, no effect on behavior, durability, easily applied, clearly identifiable, and not expensive (hagler and jackson 2001). a labelled blood source also provides an easily identifiable point source for post feeding dispersal studies. tracing of labelled blood to determine resource allocation to the eggs or other tissues could also provide useful physiological information. it was possible to mark large numbers of mosquitoes by providing blood meals from a host fed injected with a stable isotope substance. conclusion na2h 32po4 was injected via the jugular of rabbit vein; these rabbits were bitten by emerged female cx. pipiens, so mosquitoes were successfully marked. the appropriate dosage (not only can label mosquito but also have no ill effects on rabbits) of na2h 32po4 was 0.1 mci/kg. the emerged adult cx. pipiens mosquitoes were also can be marked by placing rabbit feces or na2h 32po4 into j arthropod-borne dis, june 2016, 10(2): 211–221 ch zhang et al.: evaluation of isotope … 218 http://jad.tums.ac.ir published online: january 05, 2016 container. the technique was less time-consuming, more sensitive, and safer, can be used to study post-blood meal dispersal of mosquitoes and other blood-sucking insects. the short half-life of 14 days of 32p, however, was a disadvantage in studies where prolonged observations were necessary. acknowledgements we gratefully acknowledge the support and technical assistance provided by mr tianbao fan. the project sponsored by shandong natural science foundation (grant no. zr2014yl038). the scientific research foundation for the returned overseas chinese scholars, state education ministry, joint research and development project under the twenty first session of the sino-thai scientific and technical cooperation (grant no. 21-rd-05). shandong academy of medical sciences foundation (2013–2016). the 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(diptera: culicidae). j med entomol. 24: 485–493. welch ch, kline dl, allan sa, barnard dr (2006) laboratory evaluation of a dyed food marking technique for culex quinquefasciatus (diptera: culicidae). j am mosq control assoc. 22: 626–628. wilkins ee, smith sc, roberts jm, benedict m (2007) rubidium marking of anopheles mosquitoes detectable by field-capable x-ray spectrometry. med vet entomol. 21: 196–203. j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 222 http://jad.tums.ac.ir published online: january 05, 2016 original article time series analysis of meteorological factors influencing malaria in south eastern iran afshin ostovar 1, *ali akbar haghdoost 2, abbas rahimiforoushani 1, ahmad raeisi 3, reza majdzadeh 4 1epidemiology and biostatistics department, school of public health, tehran university of medical sciences, tehran, iran 2research center for modeling in health, institute for future studies in health, kerman university of medical sciences, kerman, iran 3malaria control office of moh and me, tehran university of medical sciences, tehran, iran 4knowledge utilization research center, tehran university of medical sciences, tehran, iran (received 6 oct 2013; accepted 21 feb 2015) abstract background: the malaria early warning system is defined as the use of prognostic variables for predicting the occurrence of malaria epidemics several months in advance. the principal objective of this study was to provide a malaria prediction model by using meteorological variables and historical malaria morbidity data for malaria-endemic areas in south eastern iran. methods: a total of 2002 locally transmitted microscopically confirmed malaria cases, which occurred in the minab district of hormozgan province in iran over a period of 6 years from march 2003 to march 2009, were analysed. meteorological variables (the rainfall, temperature, and relative humidity in this district) were also assessed. monthly and weekly autocorrelation functions, partial autocorrelation functions, and cross-correlation graphs were examined to explore the relationship between the historical morbidity data and meteorological variables and the number of cases of malaria. having used univariate auto-regressive integrated moving average or transfer function models, significant predictors among the meteorological variables were selected to predict the number of monthly and weekly malaria cases. ljung-box statistics and stationary r-squared were used for model diagnosis and model fit, respectively. results: the weekly model had a better fit (r2= 0.863) than the monthly model (r2= 0.424). however, the ljungbox statistic was significant for the weekly model. in addition to autocorrelations, meteorological variables were not significant, except for different orders of maximum and minimum temperatures in the monthly model. conclusions: time-series models can be used to predict malaria incidence with acceptable accuracy in a malaria early-warning system. the applicability of using routine meteorological data in statistical models is seriously limited. keywords: malaria, models, statistical, time-series, iran introduction successful implementation of programmes to combat malaria has led to a significant decrease in cases in iran in recent years (edrissian 2006, raeisi et al. 2009). iran is currently in the ‘elimination phase’ in terms of malaria control programmes, and the malaria control office of the centre for disease control of the ministry of health and medical education has developed a programme toeliminate malaria in iran by the year 2025 (raeisi 2009). the malaria early warning system (mews) is defined as the use of prognostic variables for predicting the occurrence of malaria epidemics several months in advance. the three categories of indicators that are used in the system to detect the epidemic at its early stage are (1) indicators of vulnerability, such as immune status, malnutrition and population displacement, (2) transmission risk indicators, such as an abnormal increase of rainfall, and (3) early detection in*corresponding author: dr ali akbar haghdoost, email: ahaghdoost@gmail.com j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 223 http://jad.tums.ac.ir published online: january 05, 2016 dicators, such as reports of an excessive number of cases (thomson and connor 2000). a combination of the above variables can be used to predict epidemics before they occur, with varying accuracy. in fact, there is a balance between the timeliness and accuracy of predictions made with this method. as we get closer to the occurrence of the epidemic, the predictions become more accurate, but there is shorter lead time for taking action (matola and magayuka 1987). the relationship between predictor variables and occurrences of malaria epidemics is very complex, and the connection between some variables, such as meteorological factors, and malaria, has been investigated in some previous studies (abeku et al. 2002, abeku et al. 2003, bouma 2003, abeku et al. 2004, teklehaimanot et al. 2004a, pascual et al. 2006, medina et al. 2007, pascual et al. 2008, haghdoost et al. 2008, paaijmans et al. 2009, yamana and eltahir 2010, ermert et al. 2011). however, the impact of these variables is also very complex, and they interact with one another. therefore, using these relationships to predict epidemics is fraught with limitations. moreover, due to the complexity of the biological, statistical and mathematical models offered for use at a peripheral level, and their limited validity, based on the period and place of data on which they are built, such models cannot be practically used in malaria control programmes (hay et al. 2002b, cox and abeku 2007). although these facts have been taken into consideration, efforts to provide an early warning system for malaria epidemics by monitoring meteorological variables have nevertheless been undertaken in recent years (teklehaimanot et al. 2004b). however, the results have shown that such attempts can lead to unreliable predictions, especially if these systems are used in areas where an association between the disease and climatic conditions has not been established (hoshen and morse 2004). of all the epidemiologic models, time-series models, especially univariate auto-regressive integrated moving average (arima), are currently frequently used for prediction, as well as for the investigation of the relationship between prognostic factors and malaria. these models use the autocorrelation that exists in malaria incidence in consecutive time units, as well as the association between malaria incidence and meteorological variables, to make predictions. the principal objective of this study was to provide a model to predict the occurrence of malaria by using meteorological variables and cases that have occurred in the past 5 years at district level in the malaria-endemic, south-eastern provinces of iran. materials and methods study area this study was conducted in the minab district, which is an area of approximately 7500 square km, located in hormozgan province in southern iran. the majority of this district is flat, with a primarily hot and dry climate, and in 2006 its population was approximately 257,000. the public health system, which is responsible for the malaria surveillance system, included two urban and 10 rural health centers. figure 1 shows a map of the district and its subdivisions. calendar the jalali calendar was used in this study. on the basis of this calendar, a year consists of 365 days made up of 12 months, in which the first 6 months are all 31 days in length, the next 5 months each have 30 days, and the 12th month has 29 days, but 30 days once every 4 years. march 21 is the first day of the year in the jalali calendar. malaria data malaria surveillance system data covering a 6-year period from 1382 to 1387 (march 21, j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 224 http://jad.tums.ac.ir published online: january 05, 2016 2003 to march 20, 2009) were used. individual records data were used, because the malaria surveillance system was based on paper forms during the study period. individual records of malaria patients included their demographic characteristics, including their full name and parents’ names, age and gender, epidemiological data, including parasite types (p. vivax, p. falciparum and mixed), surveillance type (passive or active), epidemiological status (local transmission or imported), treatment outcome and the date of diagnosis. all malaria cases had been confirmed microscopically by expert examiners. meteorological data data from meteorological stations for the period from 2000 to 2009 were received from the iran meteorological organization (http:// irimo.ir). the minab district has a synoptic meteorological station and a climatology meteorological station, both of which are located in the political center (i.e., minab). figure 1 shows the name and location of the meteorological stations in the district of minab. the climatology meteorological station of kargah-asminon in the southern part of the district was inactive during the period covered by this study, therefore no data from this station were available. as shown in fig. 1, there are also three meteorological stations, a synoptic station and two climatology stations, in the neighborhood. data used for the analysis are the mean data from all five meteorological stations. time-series models the forecasting menu of ibm spss statistics 19 was used for data analysis. the dependent variable in the analysis was the total cases of local transmission in the desired time period (months or weeks) in the first 5 years of the study, that is, years 1382 to 1386 (2003– 2008). the independent variables included the total rainfall (in ml), the minimum mean temperature, the maximum mean temperature, the dry bulb mean temperature, the wet bulb mean temperature (all in ºc), the relative humidity (in percent) and the effect of the maximum temperature interaction (as representative of temperature variables) with the relative humidity. expert modeler was used to build the model. appropriate date variables were defined for the consideration of seasonal terms in the model by using the define date menu from the data tab in spss (for the monthly models), or by using special syntax (for the weekly models). outliers in the data were automatically found, and their effects were controlled. in case other independent variables, except in cases of malaria in the past, was remained in the model, arima models with transfer function (tf) and otherwise univariate arima model was fitted to the data. normalized bic and stationary r-squared indicators were used to determine the fitness of the model, and the ljung-box q statistic was used to determine its suitability. models with a significance value of more than 0.05 were considered suitable. the autocorrelation function (acf) and partial autocorrelation function (pacf) graphs of the residuals were assessed to ensure that they were not correlated. the non-significant independent variables were automatically removed from the model by the expert modeler. to assess other possible models, the acf and pacf graphs, as well as cross-correlation function (ccf) graphs between the dependent variable and the predictor variables, and trend analysis and seasonal decomposition charts were initially examined. suitable arima models, fitting the description of the data, were then created. finally, the models with the best fit, with respect to the above criteria, were selected. the files of the selected models were stored as xml data and applied to the year 1387 (almost 2008) data. minitab 16 statistical software and microsoft excel 2010 were also used to sketch the graphs. j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 225 http://jad.tums.ac.ir published online: january 05, 2016 results malaria data the total number of malaria cases in the years 1382 (2003) to 1387 (2008) was 2,239. of these, 2002 were locally transmitted. a total of 98.8% of cases of malaria that occurred in the minab district during this time were due to p. vivax. the majority of the malaria cases were observed during the months of september and october (sahrivar and mehr in the jalali calendar). the number of cases in the months of january, february, march and april (day, bahman, esfand and farvardin in the jalali calendar) was very low. the increase in the number of cases begins in may. figure 2 shows the annual temporal distribution of malaria cases in the district of minab. as shown in fig. 3, temporal distribution was different in different years, and the highest number of cases was recorded in the year 1382 (2003). in the years 1382 (2003) to 1384 (2005), the number of cases peaked once in a year, but during the years 1385 (2006) to 1387 (2008), two peaks per year were observed. a significant increase in the number of cases was shown in december 1386 (2007), which was not observed in the other years studied. meteorological variables there was a considerable annual variation in rainfall, but a more or less comparable seasonal pattern, with a minimum of 78mm in the year 1387 (2008) and a maximum of 366mm in the year 1383 (2004) (fig. 2). changes of temperature and humidity had a seasonal pattern during the year, which was accompanied by an increasing trend during the years of study. as indicated in fig. 3, the peak average maximum temperature increased over the years. however, the peak average minimum temperature decreased during this time. change of humidity also followed a seasonal trend. however, unlike the changes in temperature, changes in humidity were not regular. figure 3 shows the changes that occurred during the study period. autocorrelation analysis evaluation of the acf graphs shows that there was a significant positive correlation between the numbers of monthly malaria cases with those that occurred in the previous 1 month (fig. 4, top left graph). these positive correlations were for 5 weeks for the weekly data (fig. 4, bottom left graph). on the basis of the pacf graphs, these correlations were 2 months for the monthly data and up to 6 weeks for the weekly data, after controlling for the effects of other lag times (fig. 4, top right of the chart (monthly) bottom right (weekly)). cross-correlation analysis assessment of the monthly meteorological variables data in the cross-correlation graphs in figure 5 indicated that there was no significant correlation between the total rainfall and the number of malaria cases in the monthly data. although a lag time of 6 to 10 months, and the maximum correlation at a lag time of 7 months (top left graph), were non-significant positive correlations, significant positive correlations were found between the monthly number of malaria cases and the mean monthly temperature at a lag time of 1 to 4 months (top middle graph), and significant negative correlations were observed for the relative humidity variable at a lag time of 4 and 5 months (top left graph). the weekly graph was similar to the monthly graph in terms of cross-correlation between the number of malaria cases and rainfall, temperature and humidity variables. the contrasting point in the graph corresponds to the rainfall, where a number of positive and negative correlations were significant at different lag times (fig. 5, bottom graphs). j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 226 http://jad.tums.ac.ir published online: january 05, 2016 time series analysis table 1 shows the details of the monthly and the weekly time. as can be observed, the optimum model [arima (0, 0, 1) (0, 1, 0)] did not fit well for the monthly data, (stationary r2= 0.424). none of the predictor variables remained in the arima model created for monthly data, while the maximum temperature and the minimum temperature were the two significant variables in the weekly data arima model [arima (2,0,1) (0,0,0)]. although the weekly model gave better predictions than the monthly model (stationary r2= 0.863), the ljungbox test for this model was significant. figure 6 shows the cases predicted in comparison with the observed cases in the best-fit arima monthly model (top left) and arima weekly model (top right). as seen in the graph of the monthly model, the prediction was not made in the first year (1382–2003), due to the seasonal order in the model. in this model, the predictions made in the second year were in excess of the number of cases actually observed, due to a peak in the first year. the number of cases predicted in the sixth year (1387–2008) was also nowhere near the number that was actually observed. acf and pacf charts for the residuals follow white noise pattern in the monthly model (fig. 6, bottom left), which shows that there was no autocorrelation among the residuals, and therefore that this model was suitable. although the weekly model could reliably predict the weekly amount, on the basis of what is shown in figure 6 (top right), the acf and pacf graphs do not follow the white noise pattern for the weekly model (fig. 6, bottom right). fig. 1. minab districts’ health centers and meteorological stations j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 227 http://jad.tums.ac.ir published online: january 05, 2016 fig. 2. monthly malaria case count and rainfall in minab, years 1382-1387 (2003-2008) j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 228 http://jad.tums.ac.ir published online: january 05, 2016 fig. 3. monthly malaria case count and temperature (°c) and relative humidity (%) in minab, years 1382–1387 (2003–2008) fig. 4. acf graph (left) and pacf graph (right), monthly (top) and weekly (bottom) number of locally transmitted malaria case count j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 229 http://jad.tums.ac.ir published online: january 05, 2016 fig. 5. cross-correlation graphs between monthly (top) and weekly (bottom) malaria case count and rainfall (left), mean dry temperature (middle), and relative humidity (right) fig. 6. graphs of the observed and predicted cases for the monthly (top left) and the weekly (top right) prediction models, and acf and pacf graphs of residuals for the monthly (bottom left) and the weekly (bottom right) models table 1. comparison of the time-series models of locally transmitted cases in minab, years 1382–1386 (2003– 2007) level (time interval) model predictors (s) fit statistics (s) model statistics model parameter(s) (ljung-box q) parameter estimate (sig.)statistics sig. district (monthly) arima (0,0,1)(0,1,0) no stationary r2: 0.424 normalized bic: 7.357 2.200 1.000 ma lag1 sd 1 -0.883 (0.000) district (weekly) arima (2,0,1)(0,0,0) max. t min. t stationary r2: 0.863 normalized bic: 3.752 52.906 0.000 ar lag2 ma lag1 max. t num lag0 den lag2 min. t num lag0 0.751 (0.000) -0.999 (0.000) -0.644 (0.001) -0.518 (0.005) 1.000 (0.000) discussion meteorological variables based on the results of the statistical model used, rainfall is not a reliable predictor for the occurrence of malaria. the cross-correlation j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 230 http://jad.tums.ac.ir published online: january 05, 2016 graphs show that the 6 and 7 months’ delay in incidence of malaria had an insignificant positive correlation with the monthly total rainfall (fig. 5). this variable is absent in both of the monthly and weekly models. below average rainfall in the district and centralisation of rainfall in the months of autumn and winter, in which the average temperature is below the minimum requirement for the development of malaria parasites in the mosquito, decreases the model’s capacity to use this variable as a predictor that is potentially effective in parasite and vector development. another important point is that the data on rainfall in the meteorological stations may not be generalised to the entire region in the district. other variables, such as altitude, slope and soil type, have a considerable effect on the extrapolation of these data. the type and extent of vegetation and cultivation, as well as product types and sources of ground/ surface water interact with the effect of rainfall. conversely, since the possibility of other water sources for the development and breeding of the vectors, in addition to the rainfall, is greater in urban areas, the effect of rainfall could be different in urban and rural populations (teklehaimanot et al. 2004a). the fact that the developmental stages of malaria vectors require water cannot be denied. in addition, environmental water contributes to providing relative humidity, and also increases the longevity of adult mosquitoes (martens et al. 1995). however, this environment is not necessarily provided by the rainfall. stagnant water from agriculture, drinking water pipes, or even water dripped from air conditioners, can provide the right environment for the breeding of vectors. conversely, heavy rain can reduce malaria transmission by washing away the breeding sites in the short-term. many positive correlations between rainfall and malaria incidence, or epidemics, have been reported (loevinsohn 1994, lindblade et al. 1999, kilian et al. 1999, gomezelipe et al. 2007, tian et al. 2008), and other studies have also shown a positive correlation between rainfall and vector density (koenraadt et al. 2004, koenraadt et al. 2006, kristan et al. 2008, yamana and eltahir 2010). however, due to the complex relationship between the rainfall and entomological variables and malaria incidence, the efforts to build statistical models using rainfall to predict malaria have not been successful in many cases (abeku et al. 2003, cox and abeku 2007, briet et al. 2008, haghdoost et al. 2008, zhang et al. 2010, loha and lindtjorn 2010). effective vector control measures, especially larviciding, after the creation of breeding sites following rainfall, can also reduce the impact of rainfall on the incidence of malaria (briet et al. 2008). in addition, minab river (a permanent river in the district) may act as a permanent breeding place for vectors and reduce the effect of rainfall as well as high temperature in transmission. another difference between our results and those obtained in other studies is the time interval (lag time) between the rainfall and its effect on the incidence of malaria. in most previous studies, the impact of this variable has been considered with a monthly lag time, but in this study the highest positive correlation between rainfall and malaria can be seen with a lag time of 6 to 7 months. this difference could be associated with the difference between the climate of the minab district assessed in this study and the climates examined in other studies. the majority of the previous studies were conducted in tropical areas with a high average rainfall throughout the year, while other studies have been conducted in temperate regions, with a higher average and a longer period of rainfall over a year, compared to minab. the j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 231 http://jad.tums.ac.ir published online: january 05, 2016 effect of rainfall on the incidence of malaria in areas where it is very hot (like minab) is shorter than in regions with lower temperatures (due to evaporation of the surface water and drying of breeding sites (teklehaimanot et al. 2004b), so the effect of rainfall could indirectly be through the increase of the water level of rivers, surface water level, ground water level, and extent of cultivation. this might explain the relatively long delay observed with regard to the effect of rainfall on malaria incidence. the annual mean of the maximum, the minimum, and the average temperature was negatively correlated with the incidence of malaria in the district studied. since high temperature is fatal to malaria vectors, to some extent this correlation is biologically justified in areas with very high temperatures. similar results have also been reported in a previous study (teklehaimanot et al. 2004a). however, the increase in mean annual temperature (especially at the lowest temperature) can increase incidence by increasing the annual transition period, which is in contrast with the results obtained in this study. since the transmission peak is at the end of summer in the district of minab, a temperature rise in this period can cause the delay and shortening of the transmission period, which could be considered as an explanation for this negative correlation. in this study, the temperature variable with 2 months’ lag time shows the maximum correlation with malaria cases (fig. 6). this delay is reported as being 1 month in a study which was also conducted in iran (haghdoost et al. 2008). time delay for the effect of temperature on malaria incidence was reported as being 2 months in a study carried out in tibet (huang et al. 2011), and as being 1 month in studies conducted in china (zhang et al. 2010, bi et al. 2003). temperature variables (the minimum and the maximum temperature) were also significant in the weekly model. despite the undeniable importance of the effect of temperature on the development of parasite and vector, the applicability of using this variable to predict the incidence of malaria is uncertain. further, a justifiable correlation between malaria cases and temperature was not demonstrated in the present study. numerous studies have been conducted to examine this correlation and to use it to predict incidence of malaria, and a variety of variables, such as the maximum temperature, the minimum temperature and the mean temperature, or a combination of these variables, was used for prediction. a positive correlation between temperature and malaria, or its successful use in prediction, has been reported in many previous studies (kiang et al. 2006, gomez-elipe et al. 2007, haghdoost et al. 2008, zhou et al. 2010, wangdi et al. 2010). however, in some other studies, use of these variables has not been successful in predicting the incidence of malaria (loha and lindtjorn 2010). relative humidity in the 4th and the 5th months prior to the current month showed a significant correlation with malaria incidence (fig. 7). however, this correlation was negative. a negative correlation between humidity and the incidence of malaria was reported in a study, conducted in kahnouj (iran) (haghdoost et al. 2008). since relative humidity of 50% to 60% is necessary for vector activity, and thereafter the incidence of malaria, the effect of interaction between humidity and temperature could explain this negative correlation. however, in the present study there was no significant correlation between average monthly temperature and humidity. interaction effect of temperature and humidity variables was entered in timeseries models to control this effect. however, this variable was not present in any of the weekly or the monthly models (table 1). with the exception of a single study in iran (haghdoost et al. 2008), a negative corj arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 232 http://jad.tums.ac.ir published online: january 05, 2016 relation between humidity and malaria has not been reported in any previous studies. a positive correlation has been reported in some studies (loha and lindtjorn 2010, huang et al. 2011), while in others, the correlation between humidity and incidence or epidemic of malaria was not significant (bi et al. 2003, zhang et al. 2010). it appears that further epidemiological and entomological studies are required to investigate the causes of the negative correlation that was found in the present study. in short, meteorological variables do not appear to explain a significant amount of the variation in malaria incidence. although there is a known biological association between malaria and these variables, it seems that the limitations in measurement are hindering the showing of this relationship in the statistical models. in addition, their use is not of much help in predicting incidence of malaria. data from meteorological stations are hardly generalizable to areas that are far from the stations with high dispersion. however, a microclimate, with different climatic conditions, has made it possible for the vectors to dwell and reproduce in any climatic condition; therefore the impact of climate variables on the incidence of malaria is more complex. the impact that interactions of climate variables make on each other, and the interaction between other variables affecting the incidence of malaria and the climate variables, is another factor that adds to the complexity of this correlation. some studies have suggested that additional variables, such as socio-economic factors, should be used in malaria prediction models (hay et al. 2002b, small et al. 2003, abeku et al. 2004, teklehaimanot et al. 2004a, craig et al. 2004, zhou et al. 2004, gething et al. 2010). since the vast majority of cases of malaria in this study are p. vivax malaria, consideration of relapse cases is highly important. such cases can show the association between meteorological variables and incidence of malaria as being weaker than it actually is. in fact, as explained previously with regard to removing the imported cases from this study, the incidence of relapse cases has no correlation with climate variables and therefore should be excluded. a high relapse rate can create a serious problem in the assessment of the correlation between malaria and meteorological variables. a study reported an annual relapse rate of 16.8% and a biennial relapse rate of 24.5% in kahnouj, iran, using epidemiological methods (haghdoost et al. 2006a). if extended to other parts of the country, including the minab district, this relapse rate is very significant and therefore a serious obstacle to the approach of using statistical models to predict p. vivax malaria through meteorological variables. however, a serious limitation to the present study is the inability to recognize and delete the relapse cases. the results of previous studies examining the correlation between malaria incidence and meteorological variables, and the possibility of using this correlation in an early warning system to predict malaria incidence, have shown different, and sometimes conflicting, results. although the reason for this heterogeneity is not clear, biological, social and economic variables effective in the incidence of malaria could explain these contrasting results (ostovar et al. 2012). these studies have been conducted in various regions/climatic conditions (tropical, temperate, highlands etc.) and endemicity (stable malaria, unstable malaria). there was also variation in the type of malaria parasite investigated. in most studies, only cases of p. falciparum malaria have been examined, but in some cases, both, or only p. vivax parasites have been assessed. the sources of the climate data and the area across which the data were generalised also varied in different studies. j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 233 http://jad.tums.ac.ir published online: january 05, 2016 while some studies used ground meteorological station data, others used satellite data. the number of malaria cases in the past the past temporal records of the number of malaria cases are the most important variables of the models in this study. these variables were significant in both the monthly and the weekly models, and have had a considerable effect on model fit. in fact, the number of previous malaria cases may reflect the interaction of all the current factors effective in malaria incidence, including meteorological, social and economic variables. however, the results of a study in an unstable malaria region in ethiopia have shown that previous incidence of malaria cannot alone suitably predict the incidence of malaria now (abeku et al. 2002). based on the acf and pacf graphs in the present study, the current incidence of malaria shows a significant correlation with the incidence of malaria in the last 2 months. in addition, in the time-series models, one moving average (ma) order in the monthly model, and two ma order with one autoregression (ar) order in the weekly model remain. the results of this study are consistent with those obtained by a number of other studies (haghdoost et al. 2008, briet et al. 2008, loha and lindtjorn 2010). most previous studies were conducted based on monthly data, due to unavailability of weekly data. however, a number of studies have suggested weekly (githeko and ndegwa 2001, teklehaimanot et al. 2004, abeku et al. 2004, gomez-elipe et al. 2007) or decade (10-day interval) (haghdoost et al. 2008) data. however, the models obtained from the weekly and the monthly data were not compared with each other in any of these studies. when considering the number of previous malaria cases, since there are positive correlations between the number of malaria cases and 1 or 2 months’ lag time in the monthly models, and 4 to 5 weeks’ lag time in the weekly models, is there sufficient lead time for the mews to apply malaria control measures? if the answer is in the negative, as it was in the huang study (huang et al. 2011), then there will be serious limitations to the use of these models in practice. the present study has a number of limitations. first, the data obtained from the surveillance system were not qualitatively and quantitatively adequate for the analysis of further districts, and are therefore limited to minab. second, it is doubtful that the data obtained from meteorological stations located in the center of the entire district of minab can be extended, due to the fact that a large area of the district has a low number of meteorological stations. finally, the time period allocated to building the model in this study was relatively short. despite all these limitations, this study can be considered as a step toward clarifying both the relationship between meteorological variables and the incidence of malaria in iran, and the statistical models to consider for use in prediction. conclusion the statistical models can be used in line with a mews to predict malaria incidence, while the time-series models can also be used, due to their acceptable accuracy. it appears that the contribution of the meteorological variables is not significant in explaining the variations in the incidence of malaria, and that the monitoring, recording and use of these data in a malaria surveillance system, in order to predict an early warning of incidence, cannot be of any help in making predictions. however, this study has limitations that make its conclusion uncertain, therefore further entomological and epidemiological studies are required to better understand the j arthropod-borne dis, june 2016, 10(2): 222–237 a ostovar et al.: time series analysis … 234 http://jad.tums.ac.ir published online: january 05, 2016 relationship between meteorological variables and the incidence of malaria. acknowledgements the authors would like to acknowledge the invaluable contribution by dr reza safari, manager of disease control department of deputy for health of hormozgan university of medical sciences, for his help in accessing the data and mr gholamreza khatami for his help in cleaning and preparing meteorological data for analyses. this paper is a part of the phd thesis in school of public health, tehran university of medical sciences. the authors declare that there 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germany 4department of plant systematics, university of bayreuth, 95440 bayreuth, germany (received 24 may 2007; accepted 28 oct 2007) abstract blister beetles (coleoptera: meloidae) are the main natural source of cantharidin, but the compound titre is depended on several factors including, age, sex and mating status of the insects. in order to eliminate such uncertainty factors in physiological and chemical studies deuterium labelled cantharidin (d2c) with no natural abundance is normally introduced into the beetles’ body to use it as a model for studying the cantharidin behaviour in vivo. experiments were achieved on mylabris quadripunctata (col.: meloidae) from southern france and the beetles were exposed to an artificial diet containing a defined amount of d2c. on the other hand, because of the high similarity between the two compounds they cannot be well quantified by gas chromatography. in order to remove the burden, mrm technique was used for the first time which could successfully create well-defined cantharidin and d2c peaks and hence a precise measurement. mrm technique was examined using a gc-ms varian saturn which collected ms/ms data of more than one compound in the same time window of the chromatogram. it is especially useful when coeluting compounds have different parent ions, i.e. m/z 84 for d2c (coeluting isotopically-labelled compound) and m/z 82 for cantharidin (beetle-originated compound). using the routine gc-ms runs, measurement accuracy may be significantly reduced because the d2c peak is covered by the cantharidin huge peak while mrm could reveal the two coincided peaks of cantharidin and d2c. therefore mrm is hereby introduced as the method of choice to separate cantharidin from d2c with high sensitivity and thus provide a precise base of quantitation. keywords: cantharidin, labelled cantharidin, meloidae, gc-ms/ms, mrm introduction cantharidin (c10h12o4), mainly found in blister beetles (coleoptera: meloidae), is among the most widely known insect natural products (mccormick and carrel 1987, dettner 1997). it is highly toxic to most animals (ld50 for human 10-60 mg/kg and intraperitoneal mouse ld50 1.0 mg/kg) (dettner 1997). its reputation principally derives from descriptions of its physiological activities as an aphrodisiac and a blistering agent for humans and livestock. cantharidin aphrodisiac property is initialized by inhibition of phosphodiesterase and protein phosphateses (pps) activity and stimulation of ßreceptors (sandroni 2001). current studies on the cantharidin are focused on the site of production in producing beetles, cellular detoxification of cantharidin, in vivo biosynthesis and the intermediate compounds as well as the pps inhibitory properties (knapp et al. 1998, mccluskey et al. 2001, mccluskey et al. 2002, liu et al. 1995) which all require the precise measurement of cantharidin as a key ele*corresponding author: dr mr nikbakhtzadeh, tel: +98 21 88011001, fax: +98 21 88013030, e-mail: nikbakht_m@excite.com iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 20 ment. similarly, we had to precisely measure the titre of the compound in different organs of both sexes in order to study the cantharidin pharmacodynamics in male and female of mylabris quadripunctata. cantharidin titre in blister beetles is depended on several factors including, age, sex and mating status (mccormick and carrel 1987, carrel et al. 1993). therefore to eliminate such uncertainty factors, deuterium labelled cantharidin (d2c) in which a single hydrogen atom in each of the two angular methyl group has been replaced by deuterium atoms is introduced into the beetles’ body. because of the high structural similarity to cantharidin, d2c is hence used as a model to predict the cantharidin behaviour in vivo. since d2c has no natural abundance, tracing and exact measurement of the compound is possible, however because of the very close structural similarity, they cannot be well separated by gas chromatography (fig. 1 and 2). d4c synthesizing is on the other hand difficult and at the same time purifying the very low yield of the product is another technical challenge. since quantitation is based on the peak area of a single compound, the precise measurement of these two chemicals by gc-ms was faced with a technical limitation. in order to create well-separated peaks, a kind of ms/ms technique called mrm (multiple reaction monitoring) was used for the first time in this study to precisely separate cantharidin from d2c and increases the quantitation accuracy. materials and methods field collection of beetles and transport to laboratory specimens of mylabris quadripunctata (linné, 1767) were collected manually from st. jéan du gard, department gard, southern france, while sitting on flowers or stems of different shrubs of the families of astraceae, compositeae and leguminoseae. the material was directly transported to the university of bayreuth, germany, where the work was carried out. voucher specimens are being kept at the tök ii, university of bayreuth, germany. administration of the labelled compound the beetles in the lab were fed for three days with an artificial diet (selander 1986) to which 5 µl of a chloroform based d2c (conc. 2000 ng/µl) had been added. after complete evaporation of the solvent, the beetles were exposed to the diet containing a defined amount of d2c (nikbakhtzadeh et al. 2007). sample preparation and extraction the measurement of the total titre of cantharidin and d2c, hydrolysis and extraction of the dissected tissues were carried out according to a well defined protocol (holz et al. 1994). quantitative gc-ms normal gc-ms runs were performed by a gc 6000, vega series 2, carlo erba gas chromatograph equipped with a ht8 (8% phenyl polysiloxane-carborane, non-polar) bounded phase fused silica capillary column (sge: ft 0.25 µm, id 0.32 mm, od 0.43 mm, length: 25 m) which in turn connected to a finnigan mat ion trap detector (itd). constant elium pressure was 53 mbar and injector temperature defined as 230 °c. electron impact ionization (ei 70 ev) provides mass spectra with a characteristic fragmentation of cantharidin: the base peak with m/z 96 and two other fragments of m/z 128 and 67 (m+: 197). d2c provides mass spectrum with m/z 98, 130, 69 (m+: 199). these provide an absolute proof if the beetle extracts truly bear the two compound of cantharidin and its isotopically labelled standard, d2c (fig. 3). to analyse the data, the software it data system, version 4.00 and gcq data software package 1997, version 2.2 (build 173), finnigan corporation were used. mrm technique was examined using a gcms varian saturn 2000.40 equipped with a zb5 capillary column coated with 5% phenyl polysiloxane (phenomenex: ft 0.25 µm, id 0.25 mm, length: 60 m). a 1079 injector was used and samples (1 µl) were injected splitlessly. iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 21 trap and transfer line were kept at 175 °c and 260 °c, respectively. mass spectra were taken at 70 ev (ei mode) at 1 scan sec-1. data were processed by a saturn® gc/ms workstation package, saturn view ™ version 5.2.1, 19891998, varian associates, inc. helium at constant pressure served as the carrier gas (1.8 ml min-1). the elution of compounds was programmed from 40 °c (2 min) to 130 °c at 100 °c min-1, then to 195 °c at 3 °c min-1, followed by a rapid heating to 250 °c at 100 °c min-1 kept for 2 min prior to cooling. authentic cantahridin (purity 98%, sigma-aldrich, uk) served as standards for identification and calibration. results mrm collects ms/ms data on more than one compound in the same time window of the chromatogram. it is especially useful when coeluting compounds have different parent ions, i.e. m/z 84 for d2c (coeluting isotopically-labelled compound) and m/z 82 for cantharidin (beetleoriginated compound). after mrm, several compounds could be directed towards different channels based on the defined parent ion which makes quantitation not only easier but more reliable (fig. 4). cantharidin and d2c viewed and independently integrated by designating the appropriate channels in a file selection dialog (fig. 5). it was even possible to send up to 10 different parent ions in each chromatographic segment. our experiments indicated that the beetle feeding was inhibited if the d2c titre of the artificial diet was higher than 2000 ng/µl, so only a very low amount of d2c could be accumulated in each internal organ. using the routine gc-ms runs, measurement accuracy may be significantly reduced because the d2c peak -less than 50 kcountsis covered by the cantharidin huge peak with over 600 kcounts (fig. 6) while mrm could reveal the two coincided peaks of cantharidin and d2c (fig. 6). therefore mrm is hereby introduced as the method of choice to separate cantharidin from the deuterium labelled cantharidin (d2c) with high sensitivity. fig. 1. cantharidin-d2c total ion chromatogram based on a gc 6000, vega series 2 gas chromatograph, injection vol.: 1 µl, scan range: 900-1050, int: 5156, 100%: 16991. chromatogram appeared at scan 981 (rt 16:22) iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 22 fig. 2. auto quantitative peak integration in gc 6000, vega series 2, carlo erba gas chromatograph by the it data system (version 4.00). the two compounds of cantharidin and d2c are integrated in one peak and the peak area which is the basis of the qualitative measurement indicates the total amount of these two compounds together. abbreviations: exp rt= expected retention time, act rt= actual retention time. fig. 3. ei mass spectra of cantharidin-d2c mixture with base peaks at m/z 128, 96 and 67 for cantharidin and 130, 98 and 70 for d2c, according to a gc 6000, vega series 2 gas chromatograph, 100%= 1805 iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 23 fig. 4. mrm setting shows different channel options based on the defined parent ion of cantharidin (m/z 82) and d2c (m/z 84) fig. 5. three windows of the same segment indicate channel one at 1st window (d2c characteristic peak), channel two at the 2nd window (cantharidin peak) and both peaks have been finally shown together in the last window iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 24 fig. 6. mrm of the introduced d2c into mylabris quadripunctata. the bigger peak belongs to cantharidin. in normal gcms, these two peaks cannot be individually observed and the calculation shows the total amount of the both compounds. discussion however hplc has sometimes been used by the workers (mccormick and carrel 1987) to find cantharidin, it was widely replaced by gc since 1980s and has proved to be a more reliable method in both detecting and quantifying cantharidin of the insect-derived materials (capinera et al. 1985, carrel et al. 1986). gc is in particular more attractive because of the quick sample extraction and derivatization. low vapour pressure and the good thermal stability of cantharidin on the other hand have made gas chromatography the method of choice. using authenticated cantharidin in gc analyses, former researchers had no applied restriction to confirm detection of cantharidin in biological samples. their data were easily confirmed by a ms detector which provided the molecular fragment information. much of the recent studies require not only the detection of cantharidin trace amounts, but a very precise quantitation. it is known that cantharidin is mainly produced by male blister beetles and transfer to females in large quantities along with sperm during copulation (dettner 1997, nikbakhtzadeh et al. 2007). studying the cantharidin pharmacodynamics in the field collected materials is therefore challenging because most of the collected beetles have previously copulated and their original reservoir of the compound has been dramatically altered. one alternative could be working with the laboratory cultured beetles, although rearing of blister beetles and especially their feeding in captivity was difficult with an uncertain yield (selander 1986). in the absence of such laboratory controlled beetles, the only robust tool to study the cantharidin movement and tissue accumulation and also researches on the compound’s site of production is introducing the very similar mimic of cantharidin, d2c, into the tissues of the living beetles of both sexes (holz et al. 1994). using the ei-mass spectra, holz and their co-workers (holz et al. 1994) iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 25 easily traced cantharidin and its isotopically-labelled compound by a gc-ms machine, however they had no intention to make a quantitative analysis. the exact quantitation of cantharidin and d2c which is based on the precise separation of their chromatogram peaks in gas chromatography is very important if the labelled compound is going to be applied in any biological studies. we used three gc-ms machines if there was a difference in the accuracy of their normal gc-ms runs, but a considerable miscalculation was observed in all these runs because cantharidin and d2c coelute on the gc column and their chromatogram peaks have almost the same retention time. modification of the technical parameters such as column type, oven programme, carrier gas and its velocity were also examined whether they had any sharpening effect on the coeluting cantharidin/d2c, but no well-defined peak was obtained. as an alternative to the normal ms, we hereby suggest mrm technique which proved to be of high precision in the quantitation of d2c when the compound was present in a cantharidin rich biomaterial, e.g. tissues of a blister beetle. in this way, the peak area of a well defined chromatogram is independently integrated and the total amount of a compound calculated. it should be noted that although d2c has two mass units more than cantharidin, it is eluted earlier because the deuterium-replaced hydrogens affect the spatial angles of the molecule which in turn increases the molecule interaction to the internal film of the compact capillary column. acknowledgements the authors express their immense gratitude to elisabeth helldörfer and andrea beran, department of animal ecology ii, university of bayreuth for their technical supports. we are also thankful to andreas jürgens who kindly cooperated with us to analyse the insect materials by a varian gc-ms. this project has been financially supported by dfg within grako 678, university of bayreuth, bayreuth, germany. references capinera jl, gardner dr, stermitz fr (1985) cantharidin levels in blister beetles (coleoptera: meloidae) associated with alfalfa in colorado. j econ entomol. 78(5): 10521055. carrel je, doom jp, mccormick jp (1986) identification of cantharidin in false blister beetles (coleoptera: oedemeridae) from florida. j chem ecol. 12(3): 741-747. carrel je, mccairel mh, slagle aj, doom jp, brill j, mccormick jp (1993) cantharidin production in a blister beetle. experientia. 49(2): 171-174. dettner, k (1997) inter and intraspecific transfer of toxic insect compound cantharidin. in: dettner k, bauer g, volkl w (eds): vertical food web interactions. springer verlag, berlin, pp. 115-145. holz c, streil g, dettner k, dütemeyer j, boland w (1994) intersexual transfer of a toxic terpenoid during copulation and its parental allocation to developmental stages: quantification of cantharidin in cantharidinproducing oedemerids (coleoptera: oedemeridae) and canthariphilous pyrochroids (coleoptera: pyrochroidae). z naturforsch. 49c: 856-864. knapp j, boknik p, huke s, gombosova i, linck b, lüss h, müller fu, müller t, nacke p, schmitz w, vahlensieck u, neumann j (1998) contractility and inhibition of protein phosphatases by cantharidin. gen pharmacol. 31(5): 729-733. liu xh, blazsek i, comisso m, legras s, marion s, quittet p, anjo a, wang g, misset jl (1995) effects of norcantharidin, a protein phosphatase type-2a inhibitor, on the growth of normal and malignant hematopoietic cells. eur j cancer. 31a: 953-963. iranian j arthropod-borne dis, 2007, 1(2): 19-26 mr nikbakhtzadeh et al.: a novel approach to the … 26 mccluskey a, keane ma, walkom cc, bowyer mc, sim atr, young dj, sakoff ja (2002) the first two cantharidin analogues displaying pp1 selectivity. biol med chem lett. 12(3): 391-393. mccluskey a, walkom c, bowyer mc, ackland sp, gardiner e and sakoff ja (2001) cantharimides: a new class of modified cantharidin analogues inhibiting protein phosphatases 1 and 2a. biol med chem lett. 11: 2941-2946. mccormick jp, carrel je (1987) cantharidin biosynthesis and function in meloid beetles. in: prestwitch gd and blomquist gj (eds): pheromone biochemistry, academic press, orlando, florida, pp. 307-350. nikbakhtzadeh mr, dettner k, boland w, gäde g, dötterl s (2007) intraspecific transfer of cantharidin within selected members of the family meloidae (insecta: coleoptera). j insect physiol. 53(9): 890-899. sandroni p (2001) aphrodisiacs past and present: a historical review. clin autonom res. 11(5): 303-307. selander rb (1986) rearing blister beetles. insecta mundi. 1(4): 209-220. microsoft word dr sh salari lak rtl.doc iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 28 original article seasonal activity of ticks and their importance in tick-borne infectious diseases in west azerbaijan, iran sh salari lak 1 , *h vatandoost 2 , z telmadarraiy 2, r entezar mahdi 1 , eb kia 2 1department of epidemiology, orumyeh university of medical sciences, iran 2 school of public health & institute of health research, tehran university of medical sciences, iran (received 1 nov 2008; accepted 12 may 2009) abstract background: west azerbaijan is considered as a main region for domestic animal breeding. due to importance of herd as a main host and ticks as a vector of relapsing fever and cchf, a comprehensive study was undertaken in the region. methods: outdoor, indoor collection as well as ticks stick to the animals’ body were collected and identified. the study was conducted during the whole seasons in 2004-2005. results: during four seasons a total of 2728 ticks of two families (ixodidae and argasidae) were collected comprising 7 genera of 5 hard ticks and two genera of soft ticks including haemaphysalis, hyalomma, rhipicephalus, boophilus and dermacentor. the soft ticks were ornithodoros and argas. these 7 genera included 18 species. the main species were haemaphysalis inermis, h. punctata, h. sulcata, h. numidiana, h. concinna, hyalomma marginatum, hy. anatolicum, hy. detritum, hy. dromedarii, hy. asiaticum, hy. schulzei, h. aegyptium, rhipicephalus bursa, r. sangiuneus, dermacentor marginatus, boophilus annulatus, ornithodoros lahorensis, and argas persicus. frequency of ticks during different seasons was different. a pyrethroid insecticide, cypermethrin, which is widely used for tick control was tested against soft ticks. the test method was based on who recommendation. at the ld50 level a. persicus needs more concentration than o. lahorensis. conclusion: ornithodoros and argas are the more prevalent soft ticks in the region. distribution and prevalence of hard ticks was varied in different seasons. results of this study will provide a clue for vectors of tick-borne diseases in the region for local authorities for implementation of tick control. keywords: tticks, iran, borrelia introduction ticks are important to human and veterinary medicine, they act as vectors of bacterial, protozoal, rickaettsial, spirochaetal and viral diseases of humans, domestic stock and companion animals. as ectoparasites with irritating bites, they cause extensive harm to their hosts due to blood loss, damage to the skin and anorexia leading to reduction in growth. they are considered as agents of 'tick paralysis' in man and animals, probably due to the secretion of toxic substances in their saliva. lesions can be caused by ticks (dermatophilosis) in cattle, goats and sheep. ticks and tick-borne diseases affect animal and human health worldwide and are the cause of significant economic losses. approximately 10% of the currently known 867 tick species act as vectors of a broad range of pathogens of domestic animals and humans are also responsible for damage directly due to their feeding behavior (jongejan and uilenberg 2004). there are several works on biology, distribution, systematics of hard and soft ticks in iran (janbakhsh and ardelan 1970, abbassian-lintzen 1960, brumpt 1935, deply 1936, baltazard et al. 1952, maghami 1968, mazlumi 1968). the aim of this study was to find fauna of hard & soft ticks (families: ixodidae *corresponding author: dr. h. vatandoost, tel: +98 21 88951393, fax: +98 21 88951393, email: hvatandoost @yahoo.com iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 29 & argasidae) in west azerbaijan province and susceptibility level of soft ticks to currently used insecticide, cypermethrin. material and methods study area west azerbaijan province is located in north west part of iran (37.5528 ° n 45.0759° e, fig. 1). in the study area the total numbers of 20 villages were selected randomly and survey conducted in these villages. tick collection tick collection were carried out in animal, human dwelling and poultry shelter from cracks, crevices, ceiling and floor in 30 min. ticks were collected, from animal body of sheep, cow, goat, calf and buffalo. after collection they were transferred into the holding tubes. all the specimens were identified by morphological characteristics. susceptibility of soft ticks to cypermethrin the susceptibility of different strains of soft ticks including, a. persicus and o. lahorensis to cypermethrin by topical application method was determined. the o. lahorensis strains were collected from bijar, kurdistan province, takab, western azerbaijan province and meshkinshahr, ardebil province respectively. the a. persicus strains were collected, from khoramabad, lorestan province, and takab, western azerbaijan province, in 2004 from iran and tested with cypermethrin. mortality data from the replicates were pooled and the dose-response was assessed by probit analysis, using appropriate statistical package. results during the study period among 20 villages the total number of 2728 ticks were collected and identified. table 1 shows the number of soft and hard ticks in whole study area. from the table it can be concluded that the frequency of ticks in spring was more than other seasons, the least was observed in summer. table 1 indicate the number of soft and hard ticks separately, the population of hard ticks in winter was lower than other seasons, in contrasts the hard ticks was more prevalent during autumn. in spring all 18 species of hard and soft ticks were collected (table 2). among 881 specimens a. persicus has the highest prevalence. in spring hyalomma was more prevalent than other hard tick genus. the species of hy. anatulicum had the highest frequency among hard ticks. in summer only 8 species of ticks were collected. in spring a. persicus also considered the most prevalent species. out of 16 species of hard ticks only 6 species were collected, mainly comprise the genus hyalomma. during the summer which is hot season in the region the distribution of all collected ticks was normally equal. from table 3 it can be concluded that the different species of hyalommma is active and soft ticks also present. during autumn a total of 628 ticks were collected and identified. they belong to the families of ixodidae and argasidae and soft ticks o. lahorensis and a. persicus, among which the h. concinna, was more prevalent (table 4). the pattern and occurrence of ticks during winter was different, o. lahorensis had the highest activities. among 744 collected ticks, this species comprises 78% of all species, remaining (22%) includes h. inermis, h. punctata, h. sulcata, a. persicus, h. concinna (table 5). in the topical application bioassay, the average ld50 of o. lahorensis bijar, west azerbaijan, meshkin shahr, and a. reflexus lorestan, and west strains were 0.03, 0.04, 1.7, 0.7 and 1.7 µg/ticks, respectively and the steep slopes of dose-response curves indicated that the field populations of these soft tick strains were homogenous in response to cypermethrin. iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 30 table 1. total number of collected ticks in different seasons in west azerbaijan, iran spring summer autumn winter total season hard ( 427) soft (454) hard (159) soft (316) hard (486) soft (142) hard (92) soft (652) hard (1164) soft (1564) no. collected 881 475 628 744 2728 table 2. distribution of ticks in spring in west azerbaijan, iran number species male female total 1-h. inermis 2h. punctata 3-h. sulcata 4h. numidiana 5h. concinna 6hy. marginatum 7hy. anatulicum 8hy.detritum 9hy. dromedarii 10hy. asiaticum 11hy. schulzei 12hy. aegyptium 13r.bursa 14r. sangiuneus 15d. marginatus 16b. annulatus 17o. lahorensis 18a.persicus total 5 7 3 18 1 37 47 10 23 5 2 0 5 1 1 0 40 10 38 9 28 31 53 13 36 16 4 1 6 2 1 1 45 17 41 27 29 68 100 23 59 21 6 1 11 3 2 1 110 317 881 table 3. distribution of ticks in summer in west azerbaijan, iran number species male female total 1hy.marginatum 2hy. anatolicum 3hy. detritum 4hy. dromedarii 5hy. asiaticum 6hy. schulzei 7o. lahorensis 8a.persicus total 22 37 19 4 11 14 7 12 7 2 10 14 29 49 26 6 21 28 41 275 475 iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 31 table 4.distribution of ticks in autumn in west azerbaijan, iran number species male female total 1-h. inermis 2h. punctata 3-h. sulcata 4h. numidiana 5h. concinna 6hy.marginatum 7o. lahorensis 8a. persicus total 15 22 1 6 43 2 14 17 1 31 334 0 29 39 2 37 377 2 37 105 628 table 5. distribution of ticks in winter in west azerbaijan, iran number species male female total 1-h. inermis 2h. punctata 3-h. sulcata 4h. concinna 5o. lahorensis 6a. persicus total 77 9 3 3 0 0 0 0 77 9 3 3 582 70 744 fig. 1. study area in west azerbaijan province, iran iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 32 discussion among soft ticks the two genus ornithodoros and argas exhibit the more prevalence in the region. the host preference of hard ticks is mainly observed on sheep, poultry, cow and goat, respectively. the soft ticks, ornithodoros was mainly on sheep, calf and cow, respectively. hen was found the most favorable host for argas. the hard ticks were more prevalent during spring and autumn but soft tick mainly occurs in winter. genus hy. anatulicum and hy. dromedarii in spring, ornithodoros in autumn, hy. detritum and hy. marginatum in summer were more prevalent. hy. schulzei was found mainly in spring. in soft ticks genus argas had minimum activity during winter. the ranking order of hard ticks in terms of frequency during the year was haemaphysalis, hyalomma, rhipicephalus, dermacentor and boophilus. dermacentor and boophilus had the lowest frequency in the area which is collected only in spring. tilecoo (1997) in takab of west azerbaijan province, found o. tholozani. in another study piazak et al. in 1991 found that the genus hylaomma, rhipicephalus, haemaphysalis, and boophilus live in some parts of west azerbaijan which is parallel to our results, they also emphasized that genus dermacentor was active during the spring. tavakolli et al. (1987) carried out a similar study in lorestan province and found that genus o. lahorensis was active during the winter which is parallel to our study, but dermacentor life time occurs mainly in spring. from the results it can be concluded that the variation among genus was wider in west azerbaijan than lorestan province. he was not able to collect boophilus. in similar study in adjacent province to the west azerbaijan, of the 1,421 ticks collected from adrebil, northwestern iran, 45.9%, 40.3% and 13.8% were of the o. lahorensis, o. tholozani and a. persicus species, respectively. the prevalence of ticks was the highest in khandabil village. the ticks collected from three villages were found to be infected with borrelia (arshi, et al. 2002). a case of meningitis from borreliasis is found in ardebil province (majidpour, 2003). telmadarraiy et al. (2004) described the fauna of ticks in west azerbaijan and found 15 species of hard and soft ticks. vatandoost et al. (2003) conducted a survey in a borreliasis foci of iran in hamadan province and reported that the a. persicus, a. reflexus, o. tholozani, o. canestrinni and o. lahorensis were present in the region. o. tholozani was found infected by borrelia persica. (agighi et al. 2007) explained the situation of borreliasis in qazvin province. they found that o. tholozani was infected with b. persica and o. erraticus by b. microti. in a similar study a total of 5938 soft and hard ticks were collected from different habitats, mostly human dwellings and stables of semnan province. the rate of infection among o. tholozani ticks by borrelia persica was 36.6% (nekoui, 1999). in our study we were not able to collect the borreliasis vectors, i.e. o. tholozani and o. erraticus, found in disease foci in iran. robinson & spradling (2006) cited the main important tick borne disease transmitted by different ticks in iran and neighboring countries of caucasus region. they referred to bunya fever by different species of haemaphysalis specially h. punctata; crimreancongo hemorrhagic fever by the bite of hy. marginatum, hy. anatulicum, hy. detritum, hy. dromedarii, hy. schulzei, hy. impleltatum, hy. asiaticum and o. lahorensis; dhori virus fever transmitted by the bite of hy. dromedarii, hy. marginatum and d. marginatus; boutanneuse by the bite of r. sangiuneus, dermacentor spp, haemaphysalis spp, hyalomma spp, and boophilus spp; astarakhan fever by the bite of r. pumilio; tick-borne relapsing fever transmitted by o. tholozani, o. erraticus and o. asperus; lyme disease which its primary vector is ixodes ricinus and i. persulcatus and other ixodes species. tick iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 33 borne relasping fever (tbrf) is a notifiable disease in iran. in the last ten years a total of 1415 cases have been reported from the entire country. the highest prevalence was observed in year 2002 with incidence of 0.41/ 100.000 population. ardabil province was the first ranked infected area (625 out of 1415). the ranking order of prevalence was seen in hamedan, zanjan, kurdestan and qazvin provinces, respectively (dr. masoumi asl, ministry of health, personal communication). from our results it can be concluded that nearly main species which are emphasized earlier (robinson & spradling 2006) are exist in the study area, in addition o. tholozani play an important role in relapsing fever transmission in adjacent region. the possibility of contact between human, animal and ticks in this area may creates a high risk of acquiring different diseases such as bunya fever, cchf, dhori virus and boutanneuse. both soft ticks (argasidae) and hard tick (ixodidae) play an import role for transmission of disease to human (gray, 2002; cunha, 2000). our recent work in ardebil province indicated the presence of virus in the genera of hyalomma, rhipicephalus and ornithodoros. around 50% of host animals were igg positive to the cchf virus (unpublished documents). we propose further serological and molecular works for clarification of infectivity of ticks, host and reservoir for disease transmission cycle confirmation. it is found that cypermethrin is effective against soft ticks which is mainly find in whole seasons, so that application of pyrethroid insecticides with periodically monitoring of insecticide resistance is recommended for tick control in the region as indoor residual treatment. for control of hard ticks which are more prevalent in autumn and spring dipping method could be an appropriate measure for this purpose. acknowledgments the authors would like to appreciate the collaboration of all the staff of the department of medical entomology. this research was partially financed by the orumyeh university of medical sciences, iran. references abbassian-lintzen r (1960) a preliminary list of ticks (acarina: ixodidae) occurring in iran, and their distributional data. acarologia. 2: 43-61. aghighi z, assmar m, piazak, n, javadian e, seyedi rashti ma, kia eb, rassi y, vatandoost h (2007) distribution of soft ticks and heir natural infection with borrelia in a focus of relapsing fever in iran. iranian j of arth-borne dis. 1(2):14-18. arshi sh, majidpoor a, sadeghi h, and asmar m (2002) relasping fever in ardabil, a northwestern province of iran. arch of iranian med. 5(3): 141-145. baltazard m, bahmanyar m, pournaki r, mofidi ch. and chama m (1941) urnithodorus tartakovsky olenev 1931 et borrelia (spirochaeta) latychevi sofiev. note préliminaire. ann parasit humaine et compare, 1952. 27: 311-328. brumpt e (1935) presentation de deux ornithodotus canstrinii bir 1895, vivants originaires d isphahan (perse). bull soc path exot. 28: 51-53. cunha ba (2000) tick borne infectious disease, diagnostic and management. marcel dekker, inc. publication, new york. pp. 283. deply l (1936) notes sur les ixodidés du genere hyaloma (koch). ibid. 14: 206-245. gray js, kahl o, lane sr, stanek g. lyme borreliosis (2002) biology and control. cabi publishing book. pp.368. janbakhsh b, ardelan a (1970) the ticks of sporadic cases of relapsing fever in iran, 5th proceeding iranian plant protection congress. iranian j arthropod-borne dis, (2008), 2(2): 28-34 sh salari lak et al: seasonal activity of … 34 jongejan f, uilenberg g (2004) the global importance of ticks. parasitol. 129 supplement: s3-14. maghami g (1968) internal parasite of livestock in iran. institute of razi. 20: 81 83. majid-pour a (2003) a case of borrelia meningitis. arch of iranian med. 6(3): 222223. mazlumi z (1968) hyalomma asiaticum asiaticum schulze and schlottke, its distribution, hosts, seasonal activity, life cycle, and role in transmission of bovine theileriosis in iran. acarologia. 10: 437442. nekoui h, assmar m, amirkhani sa, pyazak n (1999) distribution of ticks and their association with borrelia in semnan province. iranian j of pub health. 28(1-3): 103-109. piazak n (1991) preliminary studies on the presence of lyme disease in iran through the study of ixodes ricinus population. msc thesis. school of public health & institute of health research, tehran university of medical sciences. robinson rg, spradling ms (2006) vectorborne disease in iran. washington dc publication usa.available in internet. http://www.afpmb.org/pubs/dveps /iran.pdf. tavakoli m (1997) survey on geographical distribution of ticks in lorestan province (western iran). msc dissertation. tarbiat modarres university, iran. telmadarraiy z, bahrami a, vatandoost h (2004) a survey on fauna of ticks in west azerbaijan province, iran. iranian j of pub health. 33: 65-69. tilecooh r (1997) investigation on the infection of ornithodoros tholozani in takab, west azerbaijan province, iran, msc dissertation. school of public health & institute of health research, tehran university of medical sciences. vatandoost h, ghaderi a, javadian e, zahir nia ah, rassi y, piazak y, kia eb, shaeghi m, telmadarreiy z, aboulghasani m (2003) distribution of soft ticks and their infection with borrelia in hamadan province, iran. iranian j of pub health. (1): 22-24. 6. dr_rassi rtl iranian j arthropod-borne dis, (2009), 3(1): 41-45 l tajedin et al: study on ectoparasites … 41 original article study on ectoparasites of rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis in endemic foci in iran l tajedin1, *y rassi1, ma oshaghi1, z telmadarraiy1, aa akhavan1, mr abai1, mh arandian2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2 esfahan health training and research center, institute of public health, tehran university of medical sciences, isfahan, iran (received 1 july 2009; accepted 28 july 2009) abstract background: ectoparasites of mammalian hosts play an important role for transmission of diseases from the host reservoirs to human. the aim of this study was to determine the species composition and infestation parameters for parasitic arthropods associated with rhombomys opimus. methods: ectoparasites of r. opimus were collected from seven endemic district of zcl in iran including shirvan and sfaraysen in north khorasan province, kalaleh in golestan province, damghan and shahrood in semnan province, and badrood and habibabad in isfahan province. the areas of study were mainly desert and plain. rodents were captured using sherman life traps during active seasons from may to november 2008. captured rodents were transported to laboratory and their ectoparasites were picked up using brushing against the fur of the rodents. ectoparasites were stored in 70% ethanol for their preservation and then identified based on morphological characters. results: ectoparasites belonged to one flea species of xenopsylla nuttalli and one mite species of ornithonussus bacoti. the flea species with 75.3% was more common than the mite. o. bacoti might play an important role in transmission of rat mite dermatitis among r. opimus colony. conclusion: results will provide an essential clue for combating zoonotic diseases in the region. keywords: rhombomys opimus, ectoparasites, xenopsylla nuttalli, ornithonussus bacoti, iran. introduction several studies confirmed the presence of the rodent ectoparasites in iran (hagghi et al. 2000, shayan 2006, hanafi-bojd et al. 2007, telmadarraiy et al. 2007). rodents are the largest order of mammals, representing 43% of specific global mammalian diversity, and including 443 genera and 2021 different species (saluzzo et al. 1999). rodents together with arthropod ectoparasites can play an important role in distribution of the arboviruses, streptococcal infections, choriomeningitis, plague, tularemia, leptospirosis and spirochaetosis (manson 2005). rhombomys opimus is considered as the main reservoir host of zoonotic cutaneous leishmaniasis and plague. knowledge on reservoir host and their ectoparasites will provide a clue for control planning of diseases in a given areas. there are a few documented papers on ectoparasites of r. opimus in different regions of iran. the great gerbil, r. opimus constructs highly complicated and deep burrows and has one of the most abundant and richest flea as*corresponding author: prof yavar rassi, e-mail: rassiy@sina.tums.ac.ir iranian j arthropod-borne dis, (2009), 3(1): 41-45 l tajedin et al: study on ectoparasites … 42 semblages among rodents (zagniborodova 1960, 1968). xenopsylla nuttalli was found on hemiechinus auritus, vulpes vulpes, rhombomys opimus, gerbill nanus, meriones persicus, m eriones meridianus and nesokia indica in iran; so, this flea is ectoparasite of rodents and wild mammals (asmar 1979). the tropical rat mite commonly occurs on rats throughout the world, particularly in tropical and subtropical regions, but also in some temperate areas. it is an ectoparaisite of rats, and attacks people living in rat-infested buildings. its bite may cause irritation and sometimes painful dermatitis. it is an important pest of laboratory animals, particularly rats, mice, and hamsters, sometimes deteriorating their health or even causing death by exsanguinations (baker et al. 1956, vatandoost et al. 2002). in iran, ornithonyssus bacoti was reported on other wild rodent including: microtus socialis, meriones persicus; so this mite is general ectoparasite for extremely wild rodent (asmar 1979, telmadarraiy et al. 2005). however, a few studies on the ectoparasites of rodents and other small mammals have been documented, e.g. study on ectoparasites of domestic animals in iran (maghami 1968), ectoparasites of rodents in semnan (eghbali 1991), report of a new species of myocoptid mites on calomyscus spp. in iran (bochkov et al. 1999), and the study of ectoparasites of nesokia indica and meriones spp. in ardestan (moniri et al. 2000). the aim of this study was to determine the species composition and infestation parameters for parasitic arthropods associated with r. opimus. materials and methods study areas based on distribution if r. opimus in iran, seven districts including kalaleh (golestan province), esfarayen, shirvan (northern khorassan province), shahrood, damghan (semnan province), badrood and habibabad (isfahan province) were selected for this study (fig. 1). fig. 1. the map of study areas located in zoonotic cutaneous leishmaniasis foci iranian j arthropod-borne dis, (2009), 3(1): 41-45 l tajedin et al: study on ectoparasites … 43 rodent collection the gerbils were live captured at different localities of above-mentioned areas, using sherman traps, baited with roasted walnut, cucumber, tomato and placed close to active burrows. they were set up monthly in the early morning and evening from spring to autumn 2008. collection of ectoparasites captured rodents were transported to the laboratory at the department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran and their ectoparasites were picked up using brushing against the fur of rodents. ectoparasites were stored in 70% ethanol for their preservation and identification. specific identification systematic keys were used for species identification. identification of ectoparasites and rodents preparing of microscopic slid of ectoparasites were carried out as follows: specimens were washed with water several times, and cleared with koh (%10) for 1–24 h. then, they were washed with distilled water. in next stage all specimens were placed in 5% acetic acid for 0.5–1 h and washed with distilled water. then they were transferred into 50% ethanol for 1 h followed by ethanol 70%, 90% and pure ethanol for 1-24 h, respectively. specimens were then placed in clove juice for 1–24 h. in this stage, only fleas were maintained in xylol for 1-24 h. finally, specimens were fixed on microscope slides using canadabalzam mounting material. different criteria of morphology of rodents were used for r. opimus identification. field operation, site selection, active reconnaissance, trap timing, trapping method, trap baiting, trap collection, biometrics measurement, data recoding, ectoparasites collection laboratory operation, preparation of rodent skulls, mounting of ectoparasite, identification using valid keys, confirmation of some species by expertise institute was carried out according to standard methods provided (kranatz 1975, etemad 1978, asmar 1979). results totally 50 r. opimus were captured and 97 specimens of ectoparasites were observed. two species of ectoparasites were identified including xenopsylla nuttalli (flea) and ornithonussus bacoti (mite) (fig. 2). xe. nuttalli was dominant (75.3%) collected specimen fleas were found in all parts of the study areas. the most frequency of xe. nuttalli (58.9%) was in badrood county. the highest frequency of xe. nuttalli was 12.33% in habibabad, 58.9% in badrood, 6.85% in esfarayen, 5.48% in shirvan, 2.74% in shahrood, 4.1% in damghan and 9.59% in kalaleh. the most frequency of ornithonyssus bacoti (54.16%) was found in esfarayen county. this species was not observed in kalaleh, habibabad and badrood. the frequency of or. bacoti was 54.16% in esfarayen, 20.83% in shirvan, 12.5% in damghan and 12.5% in shahrood (fig. 3). fig. 2. ectoparasites collected from r. opimus in the study area: a: xenopsylla nuttalli and b: ornithonyssus bacoti a b iranian j arthropod-borne dis, (2009), 3(1): 41-45 l tajedin et al: study on ectoparasites … 44 0 0 13 5 3 3 0 0 2 4 6 8 10 12 14 habibabad badrood esfarayen shirvan shahrood damghan kalaleh fig. 3. frequency and geographical distribution of ornithonyssus bacoti species collected from r. opimus in the study area. discussion rhombomy opimus has an important role in the cycle of zoonotic diseases such as plague and zoonotic cutaneous leishmaniasis (zcl) in iran, afghanistan and china (parvizi et al. 2003, yaghoobi-ershadi et al. 2003, 2004, rassi et al. 2006, 2007, faulde et al. 2008) xe. xenopsylla nuttali was previously reported from isfahan, sabzevar, kurdistan and golestan. (zagniborodova 1960, 1968). in this study we report the presence of this species in isfahan, golestan, northern khorasan and semnam. it seems that this flea is adapted on this rodent and regardless of the rodent different geographical habitats it can be found wherever the rodent present. the differences of prevalence ectoparasites in this area did not show relationship to the locality of their hosts. r. opimus was captured in different seasons but seasonal changes had no effect on the abundance of ectoparasite. ectoparasites such as lice and ticks were not found in this study and this was attributed to the technique used. there was no significant relationship between host body weight and abundance of ectoparasites. some of the ectoparasites that are identified in this study are potentially vectors of veterinary and medical importance diseases. mites of o. bacoti were not found in isfahan and golestan provinces. these two provinces do not have similar topography as well as climatic characters. however, this mite has been found in other provinces similar to either isfahan or golestan. more investigations need to clarify the reasons of the biased distribution of the species in indifferent localities in iran. this mite is a pest of rats (tenorio et al. 1980) and called the tropical rat mite. there is one report indicating the association of this mite with cases of skin dermatitis in humans (baker et al. 1956). overall, we have documented the presence of two ectoparasites species distributed in wide geographical ranges from northeastern to central regions of iran. the area of study is forming some important cutaneous leishmaniasis foci in iran. acknowledgments we thank mr r jafari, isfahan health training and research center, for technical assistance. this research was financially supported by grant no. 87-03-27-7836 from the school of public health, tehran university of medical sciences. the authors declare that they have no conflicts of interest. iranian j arthropod-borne dis, (2009), 3(1): 41-45 l tajedin et al: study on ectoparasites … 45 references asmar m, piazak n, karimi y (1979) fleas: pictorial identification key of fleas of iran. publication of pasteur insitute of iran, tehran. bochkov a, malikov v, arbabi m (1999) trichoecius calomysci sp. (acari: myocoptidae), a new mite species from iran. folia. parasitol. 46: 316–18. eghbali m (1991) study of rodents and their ectoparasites in semnan. [ms thesis]. school of public health, tehran university of medical sciences, iran. etemad a (1978) mammals of iran, rodents and identification key. natural resource protection and human environment association press, tehran. faulde m, schrader j, heyl g, amirih m, hoerauf a (2008) zoonotic cutaneous leishmaniasis outbreak in mazar-e sharif, northern afghanistan: an epidemiological evaluation. med microbiol j. 298(5-6): 543–50. hanafi-bojd aa, shahi m, baghaii m, shayeghi m, razmand n, pakari a (2007) a study on rodent ectoparasites in bandar abbas: the main economic southern of iran. j environ health sci eng. 4(3): 173-176. kranatz gw (1975) a manual of acarology. o.s.u. book stores inc, corvallis, oregon state. maghami g (1968) external parasites of livestock in iran. arch inst razi. 20: 81-83. moniri v, sahabi z, vatandoost h, et al. (2000) ectoparasites of nesokia indica and meriones spp. in ardestan. the 14th iranian plant protection congress, isfahan university of technology, iran. pp. 305–306. parvizi p, benlarbi m, ready pd (2003) mitochondrial and wolbachia markers for the sandfly phlebotomus papatasi: little population differentiation between peridomestic sites and gerbil burrows in isfahan province. iran. med vet entomol. 17(4): 351–62. saluzzo jf, dodet b (1999) emergence and control of rodent-borne viral diseases. elsevier science, paris. shayan a, rafinejad j (2006) arthropod parasites of rodents in khorram abbad district, lorestan provincen of iran. iranian j publ health. 35(3): 70–76. telmadarraiy z, vatandoost h, mohammadi s, akhavan aa, abai mr, et al. (2007) determination of rodent ectoparasite fauna in sarpole-zahab district, kermanshah province, iran. iranian j arthropodborne dis. 1(1): 58–62. tenorio jm, goff ml (1980) ectoparasites of hawaiian rodents (siphonaptera, anoplura and acari). special publication department of entomology bishop museum, honolulu. vatandoost h, hanafi bojad aa (2002) ectoparasites of medical and veterinary importance. tehran university of medical science publications, tehran. yaghoobi-ershadi mr, akhavan aa, zahrairamazani ar, abai mr, ebrahimi b, vafaei-nazhad r, hanafi-bojd aa, jafari r (2003) epidemiological study in a new focus of cutaneous leishmaniasis in the islamic republic of iran. east mediterr health j. 9(4): 816–826. yaghoobi-ershadi mr, jafari r, hanafi-bojd aa (2004) a new epidemic focus of zoonotic cutaneous leishmaniasis in central iran. ann saudi med. 24(2): 98–101. zagniborodova en (1960) fauna and ecology of fleas on the western turkmenistan. in: fenyuk bk (ed): problems of natural nidi and epizootiology of plague in turkmenistan, turkmenian anti-plague station and allunion sci anti-plague inst microb, saratov, pp. 320–334. zagniborodova en (1968) long-term study of the ecology of fleas of the great gerbil in the southern central karakum desert. in: fenyuk bk (ed): rodents and their ectoparasites, saratov univ press, saratov, pp. 78–86. j arthropod-borne dis, june 2013, 7(1): 66–70 s rafizadeh et al.: epidemiology of… http://jad.tums.ac.ir published online: april 10, 2013 original article epidemiology of scorpionism in iran during 2009 sina rafizadeh 1, javad rafinejad 2, *yavar rassi 2 1department of deputy of strategic planning, reference health laboratories research center, deputy of treatment, ministry of health and medical education of health, tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 20 nov 2012; accepted 19 dec 2012) abstract background: scorpion sting is a major health problem in iran. the aim of current study was to measure the incidence rates of scorpion stings, mortality, recovery, and affected age groups. the results of treatment with and without anti venom also were considered in the entire country during 2009. methods: all the data were collected from emergency section of different hospitals and then were analyzed by related software. the responsibility of such data collection and surveillance is related to the department of violence and injury, ministry of health and medical education of iran. results: a total incidence of 59.5/100000 was found for the 12-month period. during the study period the most and the least cases were reported from khuzestan and mazandaran provinces with incidence of 541 and 0 per 100000 respectively. totally 40220 anti venom vials were used, i.e., the ratio of 91 vial/ 100 affected cases. the stings occur mainly in rural areas (57.7%). young people with the age group of 15–24 years old were the most victims of stings. the mortality and recovery rates of cases who had received anti venom less than 6 h of stings were calculated as 0.01% and 99.9% respectively. conclusion: the high incidence of scorpion stings in iran especially in khuzestan suggests the necessity of preventive programmes for decreasing the incidence. such programmes could start by community educating in the high prevalent areas. in addition prompt and local treatment is particularly important for infants and pre-school children. keywords: epidemiology, scorpion, iran introduction scorpions are venomous arthropods, members of the class arachnida. these animals are found in all continents except antarctica, and are known to cause problems in tropical and subtropical regions. scorpion stings and their consequences represent an important cause of emergency, in particular pediatric, in many countries (chippauxa et al. 2008). the epidemiology of the scorpionism in the world is poorly investigated. accidents caused by scorpion stings are a relatively common event in subtropical and tropical countries and can cause lethal envenomation in humans, especially in children (ismail 1995). the clinical symptoms in scorpion sting are typically varied, depending on factors such as scorpion’s species, amount of injected venom, season, age and physical conditions of injured patient that may be weak as brief local responses or as dangerous as severely physiological changes that are led to death (magurie 2001, solegland et al. 2004). the iranian scorpion fauna consists of over 44 named species from 23 genera in two families, buthidae and scorpionidae. in iran, similar to other parts of the world, there are a few known species of scorpions responsible for severe envenoming. at least seven *corresponding author: prof yavar rassi, e-mail: rassiy@tums.ac.ir 66 j arthropod-borne dis, june 2013, 7(1): 66–70 s rafizadeh et al.: epidemiology of… http://jad.tums.ac.ir published online: april 10, 2013 species have been implicated in envenoming of humans and considered medically important. hemiscorpius lepturus (hemiscorpionidae), is the most medically important scorpion in iran (zarei et al. 2009). even though scorpions are abundant in many parts of iran, envenomation arising from scorpion stings is not fully defined from the clinical signs, symptoms and epidemiological aspects. so the aim of this study was to appraisal these aspects of the topic. materials and methods the initial data including age, sex, signs and symptoms, site of biting, and final outcome of the patients were derived and recorded in a previously prepared questionnaire by emergency section of hospitals of 42 universities of medical sciences from the whole parts of iran and then were analyzed by related software, excell 2009, excell maritime carriers ltd, usa. results all together, 44366 cases of scorpionism were recorded during 2009. of these, 42.3 % was related to urban and remaining in rural districts. the males with 51.8 % were the most affected gender. the age group of 15– 24 years was by far the most commonly affected group (21.8%), however, the least number of patients with 5.1% belonged to the age group of more than 65 years. table 1 shows age and sex distribution of patients stung by scorpion in iran in 2009. of 44336 scorpion stung, 8998 (19.2%) observed on head and trunk, 18898 (40.3%) on hand and 18991(40.5%) on leg areas (table 2). the average incidence of scorpionism was estimated 59.5 per 100000, however, the highest and the lowest affected cases were observed in khuzestan and mazandaran provinces with incidences of 541 and 0 per 100000 individuals, respectively. other four provinces including hormozghan, sistan and baluchistan, boshehr and ilam with incidences of 153.9, 136.1, 127 and 123 per 100000 persons were the next important scorpionism priority within the country respectively. the times elapsed between sting and injection of anti venom were less than 6 hour for 56.6% of cases, 6–12 hour for 21% of cases, more than 12 hours for 11% of cases and the rest of individuals had not received any anti venom. totally 40220 anti venom vial were used, indicates the coverage of 91 vial/100 affected cases. the yellow scorpion was easily identified by the patients (54.6%). moreover, stings were occurred during the night. the mortality and recovery rates of cases with and without anti venom, was evaluated (0.03, 99.97%) and (0.11, 99.89%) respectively. according to results, the mortality rate of affected cases was 0.04% (19/44366). table 1. epidemiological characteristics of cases stung by scorpions in iran, 2009 age (yr) male female totally % 0–4 1656 1296 2952 6.7 5–9 1756 1428 3184 7.2 10–14 1830 1675 3505 7.9 15–24 4899 4770 9669 21.8 25–34 4788 4659 9443 21.3 35–44 3360 3211 6571 14.8 45–54 1868 1743 3611 8.1 55–64 1654 1500 3154 7.1 >65 1153 1120 2273 5.1 totally 22064 21402 44366 100 67 j arthropod-borne dis, june 2013, 7(1): 66–70 s rafizadeh et al.: epidemiology of… http://jad.tums.ac.ir published online: april 10, 2013 tai* (hours) patients (n) % recovery (n) % mortality (n) % <6 25052 56.47 25048 56.46 4 0.009 6-12 8690 19.58 8686 19.57 4 0.009 >12 5248 11.84 5243 11.83 5 0.010 wai** 5376 12.11 5370 12.10 6 0.012 totally 44366 100 44347 99.96 19 0.04 *time of anti venom injection after stung **without anti venom injection table 2. features of scorpion stings among the studied cases in iran, 2009 characteristics patients (n) % sting site head and trunk 6477 14.6 hand 18898 42.6 leg 18991 42.8 discussion envenomings by scorpion stings are also an important, yet neglected, health issue in many parts of the world, particularly in the extreme northern and southern parts of africa, the middle east, southern states of usa, mexico and parts of south america, and the indian sub-continent. the true incidence of scorpion sting envenoming is not known because many cases do not seek medical attention. however, it has been estimated that there are approximately 1 million stings per year. in mexico alone, 250,000 scorpion stings are reported yearly, but fatalities have declined from 2,000 to less than 50 per year following widespread distribution of antivenoms (who 2007). in tunisia 40,000 stings, 1,000 hospital admissions and 100 deaths are reported each year. there is a high incidence in other parts of northern africa, the middle east (notably iran), india and latin america (who 2007). in this study 44366 scorpion stings were registered in 2009 and in comparison with 2002 (37535 cases), we observed notably increasing on incidence of scorpion sting in iran. different studies have shown varied age distribution for scorpion stings. children from 9 to 15 years old were more frequently affected (54.1%) than other age groups (38.8% for 3 to 8 years old, and 7.1% for 0 to 2 years) (adiguzel et al. 2007). this result was not the similar with our study. the age groups of 15–24 years were by far the most commonly affected. in the present study, male victims predominated over females (51.8% versus 48.2%). several authors assign similar results to more activity displayed by boys than by girls (hernandez 1997). however, there was no difference in severe cases between the sexes. other studies indicated that males, with their outdoor activities had more contact with scorpions than females (16–18), while other reports indicated that females accounted for the majority of victims (bergman 1997, forrester 2004, ozkan 2006). in the current study, hands and legs were the most affected parts of the body (85.4 %), a frequency that is not very different from those reported by other authors who showed that hands, arms, feet and legs were more frequently stung than other body parts (farghly 1999, gordillo 2000, silva et al. 2000, al-sadoon 2003, de roodt et al. 2003). table 1. countinued… 68 j arthropod-borne dis, june 2013, 7(1): 66–70 s rafizadeh et al.: epidemiology of… http://jad.tums.ac.ir published online: april 10, 2013 our study also showed that the majority of scorpion stings occur in july and august, during the hot season of the year. this is in accordance with data from the medical literature (abroug 1994, touloun 2001, soulaymani 2004). conclusion the present paper is the first comprehensive epidemiological study on injuries related to scorpions’ sting in iran. the high incidence of scorpion stings in iran especially in khuzestan suggests the necessity of preventive programmes for decreasing the incidence. such programmes could start by educating those affected by scorpion stings on how to avoid further stings. in addition early and local treatment is particularly important for infants and preschool children. works on behaviour of scorpions in the region and different control measure are also recommended. acknowledgements the authors are grateful to staffs of emergency section of hospitals of 42 univesities of medical sciences from the whole parts of iran for their contribution and help in registration and sending of data. the authors declare that there is no conflict of interest. references abroug f, nouira s, saguiga h (1994) envenimations scorpioniques: avancées chimiques, physiopathologiques et thérapeutiques [monograph]. pp. 1–68. abroug f, nouira s, saguiga h (1994) envenimations scorpioniques: avancées chimiques, physiopathologiques et thérapeutiques. paris: édition arnetteblackwell, (monographie, éditée par la société de réanimation de langue française). al-sadoon mk, jarrar bm (2003) epidemiological study of scorpion stings in saudi arabia between 1993 and 1997. j venom anim toxins incl trop dis. 9(1): 54–64. adiguzel s, ozkan o, inceoglu b (2007) epidemiological and clinical characteristics of scorpionism in children in sanliurfa, turkey. toxicon. 49(6): 875–880. bergman nj (1997) clinical description of parabuthus transvaalicus scorpionism in zimbabwe. toxicon. 35(5): 759–771. chippauxa jp, goyffonb m (2008) epidemiology of scorpionism: a global appraisal. acta trop. 107: 71–77. de roodt ar, garcia si, salomon od, segre l, dolab ja, funes rf, de titto eh (2003) epidemiological and clinical aspects of scorpionism by tityus trivittatus in argentina. toxicon. 41(8): 971–977. farghly wm, ali fa (1999) a clinical and neurophysiological study of scorpion envenomation in assiut, upper egypt. acta paediatr. 88(3): 290–294. forrester mb, stanley sk (2004) epidemiology of scorpion envenomations in texas. vet hum toxicol. 46(4): 219–221. gordillo me, bugliolo ag, delloni a (2000) escorpionismo en pediatria. arch argent pediatr. 98(5): 296–303. hernandez ihf (1997) picadura de alacran. epidemiologia. 11(1): 1–2. ismail m (1995) the scorpion envenoming syndrome. toxicon. 33(7): 825–858. maguire jh, spielman a (2001) ectoparasite infestations and arthropod bites and stings. in: braunwald e, fauci as, kasper dl, hauser sl, longo dl, jameson jl, eds. harrison's principles of internal medicine. 15th ed. mcgrawhill, new york, usa. ozkan o, adiguzel s, yakistiran s, cesaretli y, mehmet o, karaer z (2006) androctonus crassicauda (olivier 1807). scorpionism in the saniliurfa prov69 j arthropod-borne dis, june 2013, 7(1): 66–70 s rafizadeh et al.: epidemiology of… http://jad.tums.ac.ir published online: april 10, 2013 inces of turkey. turk parazitol derg. 30(3): 239–245. silva rlm, andrea m, amorim tk (2000) envenomation by tityus stigmurus (scorpiones: buthidae) in bahia, brazil. rev soc bras med trop. 33(3): 239–245. solegland me, fet v (2003) high-level systemathics and phylogeny of extent scorpions (scorpions: orthosterni). euscorpius. 11: 157–175. soulaymanibencheikh r, semlali i, ghani a, badri m, soulaymani a (2004) implantation et analyse d'un register des piqûres de scorpion au maroc. revue santé publ. 3(16): 487–498. touloun o, slimani t, boumezzough a (2001) epidemiological survey of scorpion envenomation in southwestern morocco. j venom anim toxins. 7(2): 199–218. world health organization (2007) report of a consultative meeting on rabies and envenomings: a neglected public health issue. who. zarei a, rafinejad j, shemshad k, khaghani r (2009) faunistic study and biodiversity of scorpions in qeshm island (persian gulf). iran j arthropod-borne dis. 3(1): 46–52. 70 j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 501 http://jad.tums.ac.ir published online: october 04, 2016 original article evaluation of oxalic acid treatments against the mite varroa destructor and secondary effects on honey bees apis mellifera *noureddine adjlane 1, 2, el-ounass tarek 3, nizar haddad 4 1department of biology, university m’hamed bougara of boumerdes, boumerdès, algeria 2laboratory of biology, and physiology animal, ens kouba, algiers, algeria 3institut technique des elevages, baba ali, alger, algérie 4department of bee research, national center for agriculture research and extension, baqa’a, jordan (received 22 sep 2013; accepted 13 july 2015) abstract background: the varroa destructor varroasis is a very serious parasite of honeybee apis mellifera. the objective of this study was to evaluate the effectiveness of varroa treatment using organic acid (oxalic acid) in algeria identifying its side effects on bee colonies. methods: treatment was conducted in one apiary consisting 30 colonies kept in langstroth hives kind. oxalic acid dripped directly on bees 5ml of this solution of oxalic acid per lane occupied by a syringe. three doses were tested: 4.2, 3.2 and 2.1% oxalic acid is 100, 75 and 50 g of oxalic acid dehydrate in one litter of sugar syrup (1water to1 surge) concentration. results: the percentage of average efficiency obtained for the first dose was 81%, 72.19% for the second dose, and 65% for third one, while the dose of 100 g oxalic acid causes a weakening of honey bee colonies. conclusion: the experiments revealed that clear variation in the treatment efficiency among colonies that this might be related to brood presence therefore in order to assure the treatment efficiency oxalic acid should be part of a bigger strategy of varroa treatment. keywords: varroa destructor, apis mellifera, oxalic acid, effectiveness, algeria introduction varroa disease is a parasitic mite of adult and brood stages of honeybees due to an external parasitic mite, varroa destructor anderson and trueman, 2000. varroa is responsible for an outbreak in apis mellifera since its transfer from the asian honeybee, a. cerana, the original host (colin 1999). it is considered as the major factor responsible for colony losses worldwide (martin et al. 2012, nazzi et al. 2012). parasitism of the bee by this mite causes deformation and weakness of the young workers. heavy infestation causes death before the emergence of nymphs and the birth of mutilated bees (boecking and genersch 2008). varroa heavily decrease the general weight up to 30% of individuals (bowen-walker and gunn 2001), the total hemolymph volume and its protein content (bowen-walker et al. 1999). it was estimated to be between 10% and 50% reduction in total protein of parasitized nymphs (dandeu et al. 1991). the work of yang and cox-foster (2005) clearly show that the varroa weakens the immune system of bees and makes it more susceptible to viral and bacterial infections. varroa by its role as a vector injects the virus it carries directly into the hemolymph of the honeybee. on the other hand, as an activator through the bite of varroa allows the activation of certain viruses present in a latent state in the hemolymph of the honeybee (tentcheva et al. 2004). in algeria, the varroa mite has become a *corresponding authors: dr noureddine adjlane, e-mail: adjlanenoureddine@hotmail. com j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 502 http://jad.tums.ac.ir published online: october 04, 2016 major concern of a beekeeper since the discovery of the first cases of infestation with varroa in the country. many acaricide were used to fight against this disease (adjlane et al. 2012). today, chemical control with strips containing pyréthriniode (apistan ®, bayvarol ®) can be used to a limited extent because these products are no longer optimal efficiency due to mites resistant against these products (adjlane et al. 2013). it is becoming crucial to provide beekeepers with new treatments for control varroa mite, oxalic acid is considered as one the natural products that became widely used as alterative treatment. in addition, it was used for this purpose since the early '80s. in early tests, a reduction of 20 to 30% was observed in varroa infestation after spraying it on each side of populated frame with honeybees (charrière and imdorf 1995). however, this method of treatment was criticized due to the bees disrupting after the frames moving, the danger oxalic acid vapours on the beekeepers respiratory system in addition to being a very laborious work and time-consuming charriére et al. (1998). however, the sugar role in the oxalic acid treatment is not clear, it is proposed that the sugar addition increase the solution oral absorption by honeybees and improves its adhesion to their bodies. many experiments worldwide were conducted to test the efficiency of this acid in varroa treatment (nanetti et al. 1995, nanetti and stradi 1997, imdorf and charriere 1998, higes 1999, gregorc and planinc 2001, 2002, moosbeckhofer et al. 2003, nozal et al. 2003, hatjina and haristos 2005, bacandritsos et al. 2007, girişgin and aydin 2010, giovenazzo and dubreuil 2011, mert and yucel 2011, gregorc and planinc 2012). these studies showed that the population density, amount of brood, climatic conditions, honey availability, migratory beekeeping, hive management and the development of varroa population do directly influence the treatment efficiency. thus, there are no scientific studies under algerian conditions. the aim of this work was to study the effectiveness of oxalic acid against varroa mites, and to determine the side effects of the application of this treatment on bee colonies materials and methods the experiment was conducted in the northcentral algeria in the region of boumerdes, this province is one of the wettest regions in the country. annual rainfall varies between 500 mm and 1300 mm per year (36° 46 '00 "n, 3° 29' 00" e) (fig.1). the relief of boumerdes is divided into several physical units: the plains and valleys to the north, the hills and plateaus in the middle part and the mountains south. winter temperatures vary between 8 °c and 15 °c. they climb to 25 °c in may to an average of 28 °c to 32 °c in july and august. the average relative humidity is high enough, it varies between 75–90%. thirty apis mellifera intermissa colonies kept in standard langstroth hives. in each colony, a mobile bottom board was installed with wire screen to count dead mites and to avoid mite removal by honeybees. the natural mite mortality was equal for the thirty honeybee colonies. the application of oxalic acid is produced by the drip method (imdorf et al. 1997), dripped directly on bees 5 ml of this solution of oxalic acid per lane occupied by a syringe (imdorf et al. 1997, brødsgaard et al. 1999). oxalic acid is an organic compound with the formula h2c2o4. it shows in the form of colourless crystals of oxalic acid dehydrate (71.4% active ingredient). oxalic acid dehydrate is partially soluble in water (12.5% by weight at 25 °c) and some organic solvents (ethanol, diethyl ether). it has a molecular weight of 126,07g / mol, a density of 1.653, a melting point at 101.5 °c and sublimed at 150 °c (inrs 2005). the colonies were treated with three different concentrations: 4.2, 3.2 and 2.1% oxj arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 503 http://jad.tums.ac.ir published online: october 04, 2016 alic acid is 100, 75 and 50 g of oxalic acid dehydrate in one litter of sugar water (1.1). for each colony, two applications were made at weekly intervals. dead varroa were collected and counted every 2 days throughout the trial period using diapers greased covering the bottom of the hive. to determine the number of mites remaining after treatment with oxalic acid, a control treatment with apivar was made two weeks later. the effectiveness of oxalic acid is calculated as follows: the strength of each treated colony was estimated to analyze the possible impact of oxalic acid on the development of the colony. the data was analysed using spss version 5.0 (chicago, il, usa) and analysis of variance (anova). results efficacy results and mortality of varroa during treatment with oxalic acid and control (apivar) for the three experimental groups are shown in tables 1, 2, 3 and fig. 1. treatment with oxalic acid applied by the dropby-drop method gives an efficiency of the dose used. the solution of 100 g of oxalic acid was a higher compared to the other two doses (70 and 50 g) efficiency. the most efficient dose was 100 g of oxalic acid is 81% (table 1), while 70 g of oxalic acid efficiency rate varies between 57 and 87% (table 2), the dose of 50 g of oxalic acid was 65% (table 3). statistical analysis revealed a significant difference between the three treatments (f= 7.87, df= 10, p= 0.002). fig. 1. the test area on the map of algeria table 1. determination of the effectiveness of oxalic acid applied by drip for colonies of group i (100g of oxalic acid in 1 liter of syrup) (august-september 2012-boumerdes area) number of colony number of dead varroa (oxalic acid) (a) number of dead varroa (apivar®) (b) total number of dead varroa (a+b) (a) effectiveness of oxalic acid (b) / (a+b)*100 1 345 271 616 78,50 2 245 251 496 86,34 3 167 15 182 91,65 4 390 114 504 77,31 5 401 175 576 69,54 6 290 65 355 81,65 7 189 61 250 75,45 8 365 178 543 67,20 9 423 54 477 88,65 10 289 19 308 93,76 j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 504 http://jad.tums.ac.ir published online: october 04, 2016 table 2. determination of the effectiveness of oxalic acid applied by drip for colonies of group ii (70g of oxalic acid in 1 liter of syrup) (august-september 2012-boumerdes area) number of colony number of dead varroa (oxalic acid) (a) number of dead varroa (apivar®) ( b ) total number of dead varroa ( a+b ) effectiveness of oxalic acid (c) / (a+b ) *100 11 411 194 605 67,87 12 309 124 433 71,23 13 233 115 348 66,89 14 245 54 299 81,87 15 523 387 910 57,44 16 298 166 464 64,12 17 203 57 260 77,98 18 453 93 545 83,09 19 314 179 493 63,65 20 234 33 266 87,65 table 3. determination of the effectiveness of oxalic acid applied by drip for colonies of group iii (50g of oxalic acid in 1 liter of syrup) (august-september 2012-boumerdes area) number of colony number of dead varroa (oxalic acid) (a) number of dead varroa (apivar®) ( b ) total number of dead varroa ( a+b ) (d) effectiveness of oxalic acid (e) / (a+b ) * 100 21 198 102 300 65,90 22 311 247 558 55,65 23 382 150 532 71,78 24 254 160 414 61,34 25 412 275 687 59,89 26 256 101 357 71,65 27 201 113 314 63,87 28 399 290 689 57,88 29 421 93 514 81,90 30 345 229 574 60,1 0 10 20 30 40 50 60 70 80 90 100 100g oa 70 g oa 50 g oa dose of oxalic acid e ff ec ti ve n es s o f o xa lic a ci d ( % ) fig. 2. the average efficiency obtained for each treatment. different letters indicate a significant difference between the three groups (<0.05, newman-keuls test at the 5% level) (august-september 2012-boumerdes area) j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 505 http://jad.tums.ac.ir published online: october 04, 2016 discussion charriere and imdorf (2001) reported in trials conducted by dripping on colonies without brood in switzerland showed an average 97% efficiency for the solution of 45 g of oxalic acid, against 96% efficiency for the treatment of 30 g of oxalic acid. in slovenia, the efficacy of oxalic acid treatments between 8 august and 16 september was 41% (gregorc and planinc 2012). a lower dose of 10–15 ml/hive (3.5%) showed a good acaricidal effect (98%) when smaller colonies were treated (moosbeckhofer 2001). gregorc and planinc (2001) treated colonies with brood three times in the summer by applying 4.1–5.2% oxalic acid dehydrate in varying sugar solutions w/w corresponding to approximately 4.5–5.9% oxalic acid dehydrate w/v. an efficacy of 39–52% was achieved. in this experiment, the efficiency is related to the presence of a quantity of brood in the colonies. in northern algeria, the brood is present almost throughout the year but with a very small amount in july–august. indeed, several authors recommend applying this treatment period of absence of brood (nanetti et al. 1995, nanetti and stradi 1997, imdorf and charriere 1998, higes 1999, gregorc and planinc 2001, 2002) as oxalic has no effect on varroa staying in sealed brood cells acid. investigations from canada in a climatic region comparing with central europe confirmed the results of autumn treatments: 2.8 % oxalic acid dihydrate solution (40–50 ml/ hive) killed about 55% of the mites, 3.5% about 90% in colonies with remaining brood (nasr et al. 2001). efficiency of up to 100% was reached only in broodless artificial swarms (büchler 1998). experts recommend treatment with oxalic acid using spray application compared to treatment with dripping mode. this treatment guarantees a very good efficacy against varroa coupled with good tolerance to bees. indeed, several trials reported efficiency greater than 95% for the spray application of 30 g of oxalic acid in one litter of water (colin 1997, imdorf et al. 1997, charriére et al. 1998). the mode of action of treatment with oxalic between passes and the amount of frames allows the bees to absorb the acid solution, and therefore the treatment penetrates and flows in their hemolymph. treatment method using (oxalic acid+sugar) is reported to be efferent due to adding sugar, and it reduces the variation among colonies (charriere and imdorf 1999). regarding the influence of these treatments on the development of colonies, no abnormalities were detected immediately after treatment with oxalic acid. studies with a higher concentration of 5% described doubled bee mortality in autumn, bad overwintering (reduced colony strength) of treated colonies and impaired spring development (charrière 2001). colonies treated twice with 3% oxalic acid dihydrate solution (7.3 and 6.4 g per comb side, one gram corresponds here approximately to one millilitre) resulted in high bee mortality averaging 170 dead bees after each administration and queen loss in one colony (nanetti et al. 1995). the effect of oxalic acid on bee larvae was investigated by gregorc et al. (2004). a solution of 6.5 g oxalic acid dihydrate/ 50 g sugar/ 100 ml water sprayed on honeybee larvae (0.121 mg/ larvae) affected the columnar cells of the midgut, leading to necrosis. in order to determine the tolerance of bees to these treatments, and avoid a possible weakening of colonies post treatment with oxalic acid, monitoring of treated colonies is performed by repeated checks the strength of the colony during autumn and winter. in general, we observed a significant drop in bee populations during the winter. the colonies treated with oxalic acid solution of 100 g group are weakened by 25%. however, the j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 506 http://jad.tums.ac.ir published online: october 04, 2016 colonies of the second group treated with a solution of 70 g of oxalic acid have lost 15% of their bee populations. with the third processing, distribution, there has been a weakening of 10%. it is therefore apparent that the higher the oxalic acid content increases, winter losses more significant. although different experimental conditions make direct comparisons difficult, assessments made in switzerland and germany tends to a higher loss of bees during winter of 45 g oxalic acid solutions. lower concentrations have provided better wintering colonies (nanetti 1999). higes et al. (1999) showed that oxalic acid has a negative effect on the development of the brood; these authors reported that three queens treated colonies died after applying a solution of 45 g of oxalic acid by spraying. the bad weather and low temperatures prevent persistent bees collect pollen and nectar pollen insufficient reserves generate a slow evolution of colonies (imdorf et al. 1996). the number of individuals populating the colony is a direct function of floristic conditions, they even dependent on climatic conditions (vandame 1996). from january, all colonies showed a slight recovery in parallel with the improved weather conditions, and thus flora. however, there were much colonies in group 2 (70 g of oxalic acid) that had resulted in better recovery from the first group. conclusion the experiments revealed clear variation in the treatment efficiency among colonies that this might be related to brood presence therefore in order to assure the treatment efficiency oxalic acid should be part of a bigger strategy of varroa treatment. the solution of 100 g of oxalic acid appears to be the most efficient comparing to the other two doses but it was poorly tolerated by the honey bees while the (70 and 50 g of oxalic acid) solutions where less efficient in varroa treatment. therefore, under the similar conditions we recommend the treatment with 70 g of oxalic acid. acknowledgements the authors thank the association of beekeepers boumerdes for its material and technical support during this study. we also would like thank the usaid grant no. tamou-11-m32-035, entitled: “impact of the varroa destructor infestation and its viral load on honeybee colonies in the middle east and north africa” for facilitating the collaboration between the jordanian and the algerian research teams. the authors declare that there is no conflict of interest. references adjlane n, doumandji se, haddad n (2012) situation de l'apiculture en algérie: facteurs menaçant la survie des colonies d'abeilles locales apis mellifera intermissa. cah agric. 21: 235–41. adjlane n, doumandji se, haddad n (2013) varroa destructor resistance to fluvalinate in algeria. trends entomol. 12: 123–125 anderson dl, trueman jwh (2000) varroa jacobsoni (acari: varroidae) is more than one species. exp appl acarol. 24: 165–189. bacandritsos n, papanastasiou i, saitanis c, nanetti a, roinioti e (2007) efficacy of repeated trickle applications of oxalic acid in syrup for varroosis control in apis mellifera: influence of meteorological conditions and presence of brood. vet parasitol. 148: 174–178. boecking o, genersch e (2008) varroosisthe ongoing crisis in beekeeping. j verbr lebensm. 2: 221–228. bowen-walker pl, martin sj, gunn a (1999) the transmission of deformed wing j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 507 http://jad.tums.ac.ir published online: october 04, 2016 virus between honeybees (apis mellifera l.) by the ectoparasitic mite varroa jacobsoni oud. j invertebr pathol. 73: 101–106. bowen-walker pl, gunn a (2001) the effect of the ectoparasitic mite, varroa destructor on adult worker honeybee (apis mellifera) emergence weights, water, protein, carbohydrate, and lipid levels. entomol exp appl. 101(3): 101–112. büchler r (1998) überprüfung der bienenverträglichkeit von oxalsäure an kunstschwärmen, allg. dtsch imkerztg. 11: 21–23. charrière jd (2001) optimisation of the oxalic acid trickling method and bee tolerability of different winter treatments: trials in liebefeld during the last 3 years. the 6th meeting of the european group for integrated varroa control, 22–23 june 2001, york, united kingdom, pp. 12–23. charrière j.d, imdorf a, fluri p (1998) potentiel et limites de l’acide oxalique pour lutter contre varroa. rev suisse apic. 95: 331–316. charrière j-d, imdorf a (1995) comment faire à la recrudescence des varroa résistants? 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[phd dissetation]. université claude bernard, lyon 1, france. j arthropod-borne dis, december 2016, 10(4): 501–509 n adjlane et al.: evaluation of oxalic … 509 http://jad.tums.ac.ir published online: october 04, 2016 yang x, cox-foster dl (2005) impact of an ectoparasite on the immunity and pathology of an invertebrate: evidence for host immunosuppression and viral amplification. proc nat acad sci. 102: 7470–7475. j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 12 original article assessment of recombination in the s-segment genome of crimeancongo hemorrhagic fever virus in iran *sadegh chinikar 1, nariman shah-hosseini 1, saeid bouzari 2, mohammad ali shokrgozar 3, ehsan mostafavi 4, tahmineh jalali 1, sahar khakifirouz 1, martin h groschup 5, matthias niedrig 6 1arboviruses and viral hemorrhagic fevers laboratory (national reference lab), pasteur institute of iran, tehran, iran 2department of molecular biology, pasteur institute of iran, tehran, iran 3national cell bank of iran, pasteur institute of iran, tehran, iran 4department of epidemiology, pasteur institute of iran, tehran, iran 5friedrich-loeffler-institut, federal research institute for animal health, greifswald-insel riems, germany 6robert koch institute, berlin, germany (received 21 may 2014; accepted 29 june 2014) abstract background: crimean-congo hemorrhagic fever virus (cchfv) belongs to genus nairovirus and family bunyaviridae. the main aim of this study was to investigate the extent of recombination in s-segment genome of cchfv in iran. methods: samples were isolated from iranian patients and those available in genbank, and analyzed by phylogenetic and bootscan methods. results: through comparison of the phylogenetic trees based on full length sequences and partial fragments in the ssegment genome of cchfv, genetic switch was evident, due to recombination event. moreover, evidence of multiple recombination events was detected in query isolates when bootscan analysis was used by simplot software. conclusion: switch of different genomic regions between different strains by recombination could contribute to cchfv diversification and evolution. the occurrence of recombination in cchfv has a critical impact on epidemiological investigations and vaccine design. keywords: recombination, phylogenetic, diversity, crimean congo hemorrhagic fever, iran introduction crimean congo hemorrhagic fever (cchf) is caused cchf virus (cchfv), which is a real threat for human life. cchfv is a tick-borne virus in the family bunyaviridae, genus nairovirus (kinsella et al. 2004). endemic regions of cchf have been reported in africa, the middle east, eastern europe and western asia where the vector and/ or reservoir ticks of hyalomma spp. are distributed (elevli et al. 2010, mehravaran et al. 2013, champour et al. 2014a). in the last 10 years, turkey, bosnia, and iran have been reported the most frequent outbreaks of cchf worldwide (chinikar et al. 2012a). clinical symptoms of cchf include headache, high fever, back pain, joint pain, stomach pain and vomiting (swanepoel et al. 1989). an infected tick remains infected throughout its life and transmits the infection to large vertebrates. livestock play a significant role in virus amplification because the animals become viremic for seven days (champour et al. 2014b). *corresponding author: dr sadegh chinikar, e-mail: sadeghchinikar@yahoo.com http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 13 the viral genome is segmented, comprising three circular single strands of negative sense rna (flick et al. 2003). the small (s) segment (approximately 1.7 kb) has a single open reading frame encoding the nucleocapsid (n) protein. in contrast, the medium (m) segment (approximately 5 kb) encodes a large polyprotein, which is processed into the two surface glycoproteins (gn and gc) and several non-structural proteins. the large (l) segment encodes a single l protein of approximately 460 kda, which is probably the viral polymerase (nuttall et al. 2001). to date, different phylogenetic analysis studies on cchf virus s segment have revealed that strains from different regions of the world cluster into several distinct phylogenetic groups. the genetic diversity of cchfv can be explained by mutation, reassortment and recombination (chinikar et al. 2004, deyde et al. 2006). although mutations are among the most important reasons for increasing rna virus genome diversity, rna reassortment, mixing of the genetic material of a species into new combinations, of segmented viruses is also a critical mechanism for rapid novel virus creation. in this regard, one recent paper reported convincing evidence for genome reassortment in cchfv (hewson et al. 2004). concerning recombination in cchfv, a preliminary study reported the possibility of recombination in cchfv, without providing clear evidence (chare et al. 2003). the results of other studies depicted evidence of potential recombination among s segment in cchfv (deyde et al. 2006). the only solid evidence of recombination in s-segment of cchfv was eventually demonstrated by lukashev (2005). there have been several articles on the molecular epidemiology of cchfv employing a phylogenetic approach, but only few studies discussed the reasons behind the observed genetic diversity (chinikar et al. 2010, chinikar et al. 2004). given the potential importance of recombination, it is important to determine its frequency for cchfv. according to the results of lukashev study, we decided to focus our recombination analysis only on the s-segment by analyzing the extent of recombination events in sequences from samples which were isolated from iranian patients. materials and methods viral extraction, rt-pcr and sequencing initially, viral rna was extracted from 140 μl of serum using a qiaamp rna mini kit, according to of the manufacturer's instructions (qi agen gmbh, hilden, germany) (yashina et al. 2003). specific primers for amplification of full-length cchfv genome (s-segment) were designed by clc main workbench software version 5.0. pcr products were amplified using one-step rtpcr, according to rodriguez et al. (rodriguez et al. 1997). the amplified products were visualized by ethidium bromide agarose gel staining (yadav et al. 2012). the pcr products were then sequenced using big dye terminator v3.1 cycle sequencing kit with modified sanger sequencing method by abi genetic analyzer 3130 (chinikar et al. 2013a). sequence alignments and phylogeny analysis to compare the topology of phylogenetic tree in different regions of s-segment genome of cchfv, full-length sequences of 6 cchfv genome from iranian patients in this study (khorasan-e-razavi 72, kerman 43, zahedan 19, tehran 65, isfahan 78 and gilan 69), in addition to sixteen full length sequences available from genbank at www. ncbi.nih.gov used as role model and compared with phylogenetic trees based on regions of s-segment genome including nucleotides 1–200, 600–800 and 1000–1200 (table 1). all sequences were aligned with the clustal w software program version http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 14 1.6. phylogenetic tree was generated by the neighbor-joining (nj) with kimura 2-parameter distance using mega 5 software. bootstrap confidence limits were based on 1000 replicates. similarity plotting and bootscaning simplot software calculates and plots the percent identity of the query sequence to a panel of reference sequences and identifies the potential recombination among analyzed sequences in a sliding window, which is moved across the alignment in steps. to obtain evidence of recombination, a similarity plotting based on isolated sequences in this study (khorasan-e-razavi 72, kerman 43, zahedan 19, tehran 65, isfahan 78 and gilan 69) was performed by simplot version 3.5.1 software. a boot-scanning analysis in simplot version 3.5.1 was undertaken to determine potential recombination between query genes and cchfv sequences from other parts of the world. both simplot and bootscanning analysis were performed with kimura (2parameter), and window and step sizes of 200 and 10 bp (lole et al. 1999, worobey and holmes 1999). results the phylogenetic tree based on full length sequence created using the nj algorithm showed the same topology and strongly supported the same phylogenetic groups as the mj tree (data not shown). the phylogenetic tree based on nj demonstrated that tehran 65, kerman 43 and khrazavi 72 sequences grouped within clade-iv (asia-1). the zahedan 19 sequence fell in clade-iv (asia-2) and showed 82 % similarity to the isolated strain niv112143 (jn572089). meanwhile, gilan 69 and isfahan 78 sequences fell within clade-v (europe) (fig. 1a). the phylogenetic trees based on region of nucleotides 1–200 showed a completely different topology in comparison to full length sequence. firstly, ex-afghanistan and khrazavi 72 strains formed a separate group together. besides, baghdad-12 constructed an out group and did not fall in clade-iv (asia-1). secondly, in contrast to full length sequence of oman strain which located in clade-iv (asia-1), the initial region of oman strain (nt 1–200) formed an out group in clade-iv (asia-2). in clade v, turkey strain (200310849) was an out group with 99 % bootstrap values (fig. 1b). given the phylogenetic tree of s-segment fragment including nucleotides 600–800, out groups were different. in this region, ex-afghanistan and khrazavi 72 strains formed an out group within clade-iv (asia-2), while full length sequence of these isolates belong to clade-iv (asia-1). in contrast to baghdad12 strain which was remained as out group, oman isolate fell in its original clade-iv (asia-1) (fig. 1c). in phylogenetic tree based on the region from 1000–1200 nt, the out group was china (79121) and dubai (616). in this region, isolate gilan69 was an out group within clade v (fig. 1d). evidence for recombination by examining the points at which the similarities between query (zahedan 19, kerman 43, tehran 65, gilan 69, khrazavi 72 and isfahan 78) and reference sequences increased or decreased, we could tentatively identify recombination breakpoints along the s-segment of cchfv (results for similarity plotting are not shown). figures show a boot-scan analysis on a window size of 200 nucleotides and moving in steps of 10 nucleotides along the alignment. boot-scanning analysis of zahedan 19 provided evidence for viral recombination. thus, we speculate that the recently isolated zahedan19 suggesting a mosaic pattern comprising cchfv isolates dubai (dubai 616) and india (niv 112143) (fig. 2a). moreover, evidence of recombination was http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 15 observed in kerman 43 isolate. in this regard, recombination was occurred between isolated cchfv strains from pakistan, matin and sr3 (fig. 2b). boot-scanning analysis of tehran65 showed different cross-over site between pakistani cchfv strains matin and sr3. moreover, two possible cross-over sites at nucleotide positions 600 and 630 were observed between matin strain and baghdad 12 strain, while tehran 65 was considered as query sequence (fig. 2c). analysis for gilan 69 was performed to confirm mosaicism and determine recombination breakpoints. in this regard, bootscanning of the gilan 69 sequence with two russian sequences (kashmanov and drosdov) and two sequences from turkey (200310849 and kelkit 06) revealed the presence of several points of crossover (fig. 2d). the location of the recombination event in isolate khorasan-e-razavi 72 was determined by using the simplot program and bootscanning analysis. isolate khorasan-erazavi 72 was compared with four representative isolates of cchfv (sr3, oman, baghdad 12 and ex-afghanistan) on the full genome. comparison of these data indicates that some fragments of the query cchfv genome (khorasan-e-razavi 72) are prone to be replaced by representative isolates, as a result of recombination (fig. 2e). the result of recombination analysis, which is depicted in fig. 2f, shows that isfahan 78 is a mosaic between kosovo (1917), russia (drosdov) and turkey (200310849) sequences, and subsequently evidence of recombination was detected. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 16 fig. 1. continued… http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 17 fig. 1. a) phylogenetic relationships between iranian isolated sequences: zahedan 19, kerman 43, tehran 65, gilan 69, khorasan-e-razavi 72 and isfahan78, and respective representative cchfv from genbank based on full length sequence. b) phylogenetic tree based on nucleotides 1–200. c) phylogenetic tree based on nucleotides 600–800. d) phylogenetic tree based on nucleotides 1000–1200. the virus strain and the geographic origin are given for each isolate. the sequences obtained from this study are shown by asterisk symbol. the numbers above the branches indicate the bootstrap values in percentages (of 100 replicates) table 1. crimean congo hemorrhagic fever virus isolates used in this study, with associated countries of origin, collection date and genbank accession numbers code of virus isolate location year of isolation clade no/ name (s segment) genbank accession no s segment iran-gilan69 iran (northwest) 2012 v/eur kj027521 iran-isfahan78 iran (central) 2013 v/eur kj027522 iran-kerman43 iran (southeast) 2013 iv/asi-1 kj196326 iran-khrazavi72 iran (northeast) 2012 iv/asi-1 kj485700 iran-tehran65 iran (north) 2011 iv/asi-1 kj566219 iran-zahedan19 iran (southeast) 2012 iv/asi-2 kj676542 niv 112143 india 2011 iv/asi-2 jn572089 sct ex afghanistan afghanistan 2012 iv/asi-1 jx908640 baghadad12 iraq 1979 iv/asi-1 aj538196 china79121 china 1979 iv/asi-2 af358784 kosovo 1917 kosovo 2009 v/eur jn173797 matin pakistan 1976 iv/asi-1 af527810 fig. 1. continued… http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 18 pakistansr3 pakistan 2000 iv/asi-1 aj538198 drosdov russia 1967 v/eur dq211643 kashmanov russia 1967 v/eur dq211644 ard15786 senegal 1972 i/w.afr-1 dq211640 hodzha uzbekistan 1967 iv/asi-2 ay223475 ap92 greece 1975 vi/greece dq211638 oman oman 1997 iv/asi-1 dq211645 dubai 616 dubai 1979 iv/asi-2 jn108025 kelkit06 turkey 2006 v/eur gq337053 200310849 turkey 2003 v/eur dq211649 table 1. continued… position window: 200 bp, step: 10 bp, gapstrip: on, reps: 100, kimura (2-parameter), t/t: 2.0, neighbor-joining position window: 200 bp, step: 10 bp, gapstrip: on, reps: 100, kimura (2-parameter), t/t: 2.0, neighbor-joining bootscan-query: iran (kerman 43)* bootscan-query: iran (zahedan 19)* % o f p er m u te d t re es % o f p er m u te d t re es http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 19 fig. 2. continued… position window: 200 bp, step: 10 bp, gapstrip: on, reps: 100, kimura (2-parameter), t/t: 2.0, neighbor-joining position window: 200 bp, step: 10 bp, gapstrip: on, reps: 100, kimura (2-parameter), t/t: 2.0, neighbor-joining bootscan-query: iran (gilan 69)* bootscan-query: iran (tehran 65)* % o f p er m u te d t re es % o f p er m u te d t re es http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 20 fig. 2. the location of the recombination event in iranian isolates as query: zahedan 19(a), kerman 43(b), tehran 65(c), gilan 69(d), khorasan-e-razavi 72(e) and isfahan 78(f) was determined by using the bootscanning analysis. bootscaning was conducted with simplot 3.5.1 software. the window size was 200 bp, with a step size 10 bp fig. 2. continued… bootscan-query: iran (khrazavi 72)* bootscan-query: iran (khrazavi 78)* position window: 200 bp, step: 10 bp, gapstrip: on, reps: 100, kimura (2-parameter), t/t: 2.0, neighbor-joining position window: 200 bp, step: 10 bp, gapstrip: on, reps: 100, kimura (2-parameter), t/t: 2.0, neighbor-joining % o f p er m u te d t re es % o f p er m u te d t re es http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 21 discussion given that major histocompatibility complex (mhc) can appear virus particles with molecular weight more than 10 kda on the surface of host cells and encoding nucleoproteins by s-segment has approximately 53 kda weight, these appeared proteins recognize as the predominant antigen inducing a high immune response (garcia et al. 2006). thus, recombination in s-segment may provide new antigenic epitopes to the virus provided that recombination occurred in the coding regions of nucleoproteins and subsequently may deceive immune response (alcami and koszinowski 2000, weber and elliott 2002). recombination is being recognized increasingly as a potentially important reason of creating and shaping genetic diversity in rna viruses (worobey and holmes 1999). this aspect of genomic analysis is rarely investigated for cchfv and there is only one previously solid documented record of recombination in cchfv (lukashev 2005). the small (s) segment of cchfv (approximately 1.7 kb) has a single open reading frame encoding the nucleocapsid (n) protein. this protein plays a role in encapsidating the viral rna to form ribonucleoprotein complexes (rnp). the n protein is also involved in a range of interactions with other molecules including viral rna, viral polymerase, other viral proteins, host proteins as well as forming multimers with themselves (han and rayner 2011). through comparison of the phylogenetic trees based on full length sequences and partial fragments in the s-segment genome of cchfv, genetic switch was evident, due to recombination event. the phylogenetic results obtained in this study are in accordance with lukashev, who reported that phylogenetic grouping according to short fragments of s-segment genome are not usually reliable and the possibility of recombination should be taken into consideration (lukashev 2005). to obtain more solid evidence of recombination in this study, bootscan analysis was used by simplot software. as a result, evidence of multiple recombination events was detected in query isolates. by ignoring the presence of recombination in s-segment genome of cchfv, phylogenetic data based on partial sequences cannot be reliable due to misinterpretation. thus, this study strongly recommends using full sequence or at least long length sequences for future molecular epidemiological studies to obtain more precise phylogenetic trees. conclusion this report provides evidence of recombination in the s-segment of cchfv. switch of different genomic regions between different strains by recombination could contribute to cchfv diversification and evolution. thus, recombination may impact on investigations of virus taxonomy, phylogenetic investigations and vaccine design. this is of utmost important to countries where multiple cchfv variants are circulating. acknowledgements this paper is based on the results of corresponding author's phd thesis. we thank all members of the arboviruses and viral hemorrhagic fevers laboratory (national reference lab), pasteur institute of iran, and also keyhan azadmanesh, and ms niknam and mohsen chiani for technical assistance. this research has been financially supported by pasteur institute of iran. the authors declare that there is no conflict of interests. references alcami a, koszinowski uh (2000) viral mechhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 12–23 s chinikar et al.: assessment of recombination … 22 anisms of immune 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dt (2012) genetic characterization and molecular clock analyses of the crimean-congo hemorrhagic fever virus from human and ticks in india, 2010–2011. infect genet evol. 14: 223–231. yashina l, vyshemirskii o, seregin s, petrova i, samokhvalov e, lvov d, gutorov v, kuzina i, tyunnikov g, tang yw (2003) genetic analysis of crimean-congo hemorrhagic fever virus in russia. j clin microbiol. 41: 860–862. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, june 2014, 8(1): 102–107 s bokaie et al.: prevalence and epizootical … 102 short communication prevalence and epizootical aspects of varroasis in golestan province, northern iran saeid bokaie 1, *laleh sharifi 2, majid mehrabadi 3 1department of epidemiology, veterinary medicine faculty, university of tehran, tehran, iran 2immunology, asthma and allergy research institute, tehran university of medical sciences, tehran, iran 3graduated from azad islamic university, garmsar branch, garmsar, iran (received 20 nov 2011; accepted 15 may 2013) abstract background: the varroa destructor mite is considered as a major pest of honey bees apis mellifera. the rapid spread of varroa mites among bee colonies may be due to several factors, including drifting of infested bees, movement of bee swarms, and robbing of weakened colonies. disease spread and predisposing the infested bees to other diseases lead to high economic losses in beekeeping industries. the aim of this study was to determine the prevalence of and evaluate some managing factors in golestan province in iran in 2008. methods: according to the records of agricultural research center, 80 infested beekeeping centers identified and a questionnaire consists of managing factors for each center has been designed. all data were recorded and analyzed by spss software to calculate χ2 test. results: among 80 apiculture centers, 72 centers (92%) were infested to varroa and hive density of 90.6% of the centers was 31–60 hives in one center (p= 0.324). all of the apiculture centers had more than 6 km distance to nearest beekeeping center (p= 0.687). amongst bee keepers 15(93.8%) had low literacy level (p= 0.479) and 26(89.7%) had 5–10 years experience in beekeeping (p= 0.953). conclusion: we can conclude that because of the high prevalence of the disease, the usual methods of prevention are not effective. this high prevalence emphasizes that we are very far from a solution for varroa infestation and extra researches on mite biology, tolerance breeding, and varroa treatment is immediately required. keywords: apis mellifera, varroa destructor, honey bee, iran introduction the new worldwide distributed and hemophagous mite varroa destructor is considered as a main pest of honey bees apis mellifera. before year 2000, v. destructor was understood to be varroa jacobsoni (anderson and trueman 2000) which successfully shifted from the original host, a. cerana to the western honey bee, a. mellifera. (rath 1999). the details of the host shifting phenomenon are unclear. varroa destructor acts as a vector of different bee viruses. until now 18 different viruses have been identified in honey bees (chen and siede 2007). numerous of bee viruses can be transmitted by v. destructor such as acute bee paralysis virus (abpv), israeli acute paralysis virus (iapv), kashmir bee virus (kbv), sacbrood virus (sbv), and deformed wing virus (dwv) (boecking and genersch 2008). before the appearance of varroa mites in a. melifera, viral diseases were minor troubles for honey bee health (allen et al. 1986, bailey and ball 1991, bowen-walker et al. 1999, yue and genersch 2005). at a short time v. destructor has worldwide geographical distribution and we can not find a country free of this disease rather than australia. the economic burden of this ectoparasite is high. disease *corresponding author: dr laleh sharifi, e-mail: lalehsharifi@yahoo.com http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 102–107 s bokaie et al.: prevalence and epizootical … 103 distribution and predisposing the infested bees to other diseases lead to high economic losses in beekeeping industries. in addition, varroa mite may intensify the problems of pollination in future time (de la rua et al. 2009). varroa females start to reproduce by entering the brood cells of last-stage worker or drone larvae, normally within 20–40 hours before the cells are sealed (boot et al. 1992). about 60 h after the bee cell is capped; the adult female mite puts her first egg and can produce over 10 progeny (sammataro et al. 2000). the adult female mite and progeny feed on the hemolymph of pupae from a single feeding site (kanbar and engels 2003). all reproduction of varroa occurs in the brood cells, and only the adult females survive after the bee emerges. some immature females, eggs (rarely), and males are left and removed by the nurse bees when the bee emerges. varroa mites suck the hemolymph from adults and developing pupae of honey bees, thereby weakening the bees and reduction their life length. the rapid spread of varroa mites among bee colonies is due to a number of factors such as drifting of diseased bees, movement of bee swarms, and robbing of weakened colonies (de jong 1997). in addition, migratory beekeeping practices and the importation of infested bees lead to rapid distribution of varroa mites (sammataro et al. 2000). application of synthetic acaricides has been the main way for controlling the pest. but the intensive use of many chemical substances against the mites resulted in the increase of resistance and decrease of their efficiency (milani 1999) and contamination of products such as honey and beeswax (wallner 1999). the problems of chemical acaricides encourage the scientists to find new and safer ways control of varroa. natural products such as essential oils offer a highly desirable alternative to synthetic products. these substances are used increasingly because they are generally inexpensive and have fewer health hazards to both man and honeybees (isman 2000). various alternative ways for managing the mite have been investigated (imdorf et al. 1995, fries 1997, thomas 1997, calderone 2005). most of them are suggesting nonchemical methods for reaching lower occurrence of mite infestation in beehives (imdorf et al. 1999). one of these non-chemical methods is use of formic acid that has received great consideration because of its activity against v. destructor (calderone 2000, currie and gatien 2006). based on the veterinary organization protocol in iran the bee hives with ≤ 5% infestation of v. destructor must be treated with standard treatments such as formic acid®, api life var®, apiguard®, apistan® and apivar®. the objectives of this study were to determine the prevalence of v. destructor and evaluating the effect of the hygienic factors in infestation rate in golestan province of iran. this province with temperate and wet macro climate is one of the most important sites of beekeeping industry in iran. materials and methods all beekeeping centers were identified in golestan province according to the information of the agricultural research center. due to protocol of the iranian veterinary organization, varroa sampling was done every season and 5% of hives in each beekeeping center were investigated. beehives with less than 5 honey-combs were excluded from sampling. a questionnaire consisted of managing and hygienic factors were filled for each beekeeping center. questions included distance to the nearest apiculture center, altitude of apiculture center from the sea level, use of guard wall around the beekeeping center, migration situation, numbers of hives, the height of hives from the ground, distance between hives, disinfection procedures, and http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 102–107 s bokaie et al.: prevalence and epizootical … 104 methods to provision of pollen, wax, apiculture equipments, queen and water supplying. literacy and experience levels of bee keepers were asked also. all data were recorded and analyzed by spss software version 15 to calculate χ2 test and fisher exact test. p values less than 0.05 were considered as significant level. results among 80 apiculture centers, 72 centers (92%) were infested by varroa mite. the hive density of 90.6% of centers was 31–60 hives/ center (p= 0.324). all of the apiculture centers had more than 6 km distance to nearest beekeeping center (p= 0.687). fire have been used to disinfect the equipments in 54 (90%) of studied centers (p= 1.000). fifty eight infected centers (90.6%) had migrated beehives (p= 0.657). amongst beekeepers 15(93.8%) had low literacy level (p= 0.479) and 26(89.7%) had 5–10 years experience in beekeeping (p= 0.953). in table 1 the association between varroa infestation of beekeeping centers and the altitude of apiculture center from the sea level, the height of beehives from the ground, distance between beehives and use of guard wall around the beekeeping center, have shown. the methods for provision of pollen, wax, apiculture equipments, queen and water supply have been shown in table 2 and their relations with varroasis are in the same table. table 1. the relations between varroasis and the altitude of apiculture center, the height of hives, distance between hives and use of guard wall variablesvarroasis positive negative p value altitude of apiculture center ≤800m 47(90.4%) 5(9.6%) p= 1.000 >800m 25(89.2%) 3(10.8%) height of hives from the ground surface 10–25cm 69(89.6%) 8(10.4%) p= 0.0284* 26–40cm 3(100%) 0(0%) distance between hives ≤50cm 63(90%) 7(10%) p= 1.000 >50cm 9(90%) 1(10%) use of guard wall yes 19(79.2%) 5(20.8%) p= 0.0485* no 53(95%) 3(5%) *p< 0.05 is significant table 2. the relation between varroasis and the use of pollen in apiculture, source of pollen, water, wax, equipments, and queen variables varroasis positive n (%) negative n (%) p value use of additional pollen yes 62(89.8) 7(10.2) p= 0.00011* no 10(91) 1(9) purchase pollen from other apiculture centers yes 65(91.5) 6(8.5) p= 0.2201 no 7(77.7) 2(22.3) purchase wax from other apiculture centers yes 58(89.2) 7(10.8) p= 1.000 no 14(93.3) 1(6.7) purchase second hand equipments from other apiculture centers yes 24(92.3) 2(7.7) p= 1.000 no 48(88.9) 6(11.1) purchase queen from other apiculture centers yes 32(84.2) 6(15.8) p= 0.1414 no 40(95.2) 2(4.8) source of water river 63(88.7) 8(11.3) p= 0.0008* non river 9(100) 0(0) *p< 0.05 is significant http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 102–107 s bokaie et al.: prevalence and epizootical … 105 discussion according to the results, this high prevalence of infestation shows the high spread of v. destructor in beekeeping centers in golestan province. this occurrence is similar to other sites of the world that had reported, the eastern coastal region of the ussr in 1952, pakistan in 1955, japan in 1958, china in 1959, bulgaria in 1967, paraguay in 1971, germany in 1977 (ruttner and ritter 1980), united states in 1987 (de guzman and rinderer 1999). today, v. destructor has global distribution, but according to the reports published by australian government (http:// www.daff.gov.au/qis/quarantine/pests-diseases/ honeybees) it has not yet been found in australia. in this study we showed that heights of hive from the ground level associated to varroais prevalence. distance from the ground surface can affect the humidity of brood cells and influence the mite reproduction. we found a significant relation between varroasis and use of additional pollen for hives in beekeeping centers. in addition we found that the infestation rate of bee-keeping centers that supply their water from the river are less than centers which provide water from other routs also use of guard wall around the apiculture center was related to lower infestation rate of varroa. the micro-climatic conditions inside the colony are affected by outside factors including temperature, humidity or the accessibility of pollen and nectar. this may influence the proportion of non-reproducing mites (eguaras et al. 1994, garcia-fernandez et al. 1995, kraus and velthuis 1997, moretto et al. 1997). according to the non-significant results of the effect of bee-keepers’ literacy and experience level in prevention of disease, inefficiency of hygienic factors such as having suitable distance to other beekeeping centers and decreasing hive density in a center (number of hives), the altitude of apiculture center from the sea level and distance between beehives, it can be concluded that the usual methods of varroa prevention are not effective. however the ways to provision pollen, bee wax, equipments, and queen had not any influence on varroa infestation rate. use of varroa tolerant honey bees and chemical and biological methods of varroa treatments are suggested as important controlling ways. the control of mite reproduction is considered the most effective tool for the host to prevent the growth of a varroa population within the colony (fries et al. 1994). however further exploration of mite biology in preparing control measures as well as using tolerant types and treatment especially by nonchemical substances are instantly needed. conclusion because of the high prevalence of the disease (92%), the usual methods of prevention are not effective and we should consider the new methods for varroa control. acknowledgements we would like to thank mr. gholam abbas badrkhani, for providing helpful comments to carry out this research project. the authors declare that there is no conflict of interest. references allen mf, ball bv, white rf, antoniw jf (1986) the detection of acute paralysis virus in varroa jacobsoni by the use of a simple indirect elisa. j apicult res. 25: 100–105. anderson dl, trueman jwh (2000) varroa http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 102–107 s bokaie et al.: prevalence and epizootical … 106 jacobsoni (acari: varroidae) is more than one species. exp appl acarol. 24: 165–189. bailey l, ball bv (1991) honey bee pathology. academic press, london. boecking o, genersch e (2008) varroosis – the ongoing crisis in bee keeping. j consum protect food safety. 3(2): 221–228. boot wj, calis nm, beetsma j (1992) differential periods of varroa mite invasion into worker and drone brood cells of honey bees. exp appl acarol. 6: 295–301. bowen-walker pl, martin sj, gunn a (1999) the transmission of deformed wing virus between honeybees (apis mellifera) by the ectoparasitic mite varroa jacobsoni oud. j invertebr pathol. 73: 101–106. calderone nw (2000) effective fall treatment of varroa jacobsoni (acari: varroidae) in colonies of the honey bee (hymenoptera: apidae) with formic acid in a northern climate. j econ entomol. 93: 1065–1075. calderone nw (2005) evaluation of drone brood removal for the management of varroa destructor (acari: varroidae) in colonies of the honey bee apis mellifera l. (hymenoptera: apidae) in the northeastern usa. j econ entomol. 98: 645–650. chen yp, siede r (2007) honey bee viruses. adv. virus res. 70: 33–80. currie rw, gatien p (2006) timing acaricide treatments to prevent varroa destructor (acari: varroidae) from causing economic damage to honey bee colonies. can entomol. 138: 238–252. de guzman li, rinderer te (1999) identification and comparison of varroa species infesting honey bees. apidologie. 30: 85–95. de jong d (1997) mites: varroa and other parasites of brood. in: morse rm, flottum pk (eds.), honey bees pests, predators, and diseases, 3rd ed. the a. i root company, medina, oh, pp. 281–327. de la rua p, jaffe r, dall´olio r, muñoz i, serrano j (2009) biodiversity, conservation and current threats to european honeybees. apidologie. 40: 263–284. eguaras m, marcangeli j, oppedisano m, fernández n (1994) seasonal changes in varroa jacobsoni reproduction in temperate climates of argentina. bee sci. 3: 120–123. fries i, camazine s, sneyd j (1994) population dynamics of varroa jacobsoni: a model and a review. bee world. 75: 5–28. fries i (1997) organic control of varroa. in: munn p, jones r (eds) varroa! fight the mite. ibra, cardiff. garcia-fernandez p, rodriguez rb, orantesbermejo fj (1995) influence of climate on the evolution of the population-dynamics of the varroa mite on honeybees in the south of spain. apidologie. 26(5): 371–380. imdorf a, bogdanov s, ibáñez ochoa r, calderone n (1999) use of essential oils for the control of varroa jacobsoni oud. in honey bee colonies. apidologie. 30: 209–228. imdorf a, charriere jd, maquelin c, kitchenmann v, bachofen b (1995) alternative varroa control. federal dairy research institute, apiclutural department, liebefeld. isman mb (2000) plant essential oils for pest and disease management. crop prot. 19: 603–608. kanbar g, engels w (2003) ultrastructure and bacterial infection of wounds in honey bee (apis mellifera) pupae punctured by varroa mites. parasitol res. 90: 349–354. kraus b, velthuis hhw (1997) high humidity in the honey bee (apis mellifera l.) brood nest limits reproduction of http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 102–107 s bokaie et al.: prevalence and epizootical … 107 the parasitic mite varroa jacobsoni oud. naturwissenschaften. 84: 217–218. milani n (1999) the resistance of varroa jacobsoni oud. to acaricides. apidologie. 30: 229–234. moretto g, gonçalves ls, de jong d (1997) relationship between food availability and the reproductive ability of the mite varroa jacobsoni in africanized bee colonies. am bee j. 137: 67–69. rath w (1999) co-adaptation of apis cerana fabr. and varroa jacobsoni oud. apidologie. 1999. 30: 97–110. ruttner f, ritter w (1980) das eindringen von varroa jacobsoni nach europa im rückblick. allg deut imkerz. 14: 130– 134. sammataro d, gerson u, needham g (2000) parsitic mites of honey bees: life history, implications, and impact. annu rev entomol. 45:519–548 thomas hu (1997) practical aspects of alternative varroa control methods. in: munn p, jones r (eds) varroa! fight the mite. ibra, cardiff, pp. 22–30. wallner k (1999) varroacides and their residues in bee products. apidologie. 30: 235–248. yue c, genersch e (2005) rt-pcr analysis of deformed wing virus in honeybees (apis mellifera) and mites (varroa destructor). j gen virol. 86: 3419–3424. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, march 2016, 10(1): 92–97 sk borthakur et al.: occult dirofilariosis … 92 short communication occult dirofilariosis in dogs of north eastern region in india *sonjoy kumar borthakur 1, dilip kumar deka 2, saidul islam 2, prabhat chandra sarmah 2 1department of parasitology, college of veterinary sciences and ah, central agricultural university, selesih, aizawl, mizoram, india 2department of parasitology, college of veterinary science, assam agricultural university, khanapara, guwahati-781 022, assam, india (received 2 mar 2014; accepted 28 june 2014) abstract background: the north eastern region in india is endemic for canine heartworm disease but in clinics accurate diagnosis is some times difficult. the aim of the present study was to determine the prevalence of occult infections for heartworm disease in canine in two geographical regions of north eastern india. methods: a total of 782 numbers of three categories of dogs namely, working dogs of military and paramilitary forces, pet dogs and stray dogs were screened for the presence of heartworm infection from august 2011 to july, 2012 in guwahati (assam) and aizawl (mizoram). conventional, immunological and molecular techniques were followed for this epidemiological study. the criteria to determine the occult heartworm cases were based on the differences between heartworm positive cases in pcr test and antigen elisa test. results: the findings revealed an overall 22.69 percent occult case. the working dogs had highest prevalence (60%) followed by pet (29.16%) and stray dogs (17.75%). conclusion: the highest percentage of occult heartworm infection was present in working dogs maintained under military or paramilitary forces. keywords: dirofilaria immitis, occult infection, india introduction india’s dog population is estimated over 25 million and 80 % of this population are either partially restricted or community (stray), or feral (unrestricted) dogs (menezes 2008). again, 17 % of indian households were reported to own a pet or domesticated dog (sudarshan et al. 2006). among the disease producing agents in canines, “helminths” are one of the predominating group of which dirofilaria immitis is established as the most pathogenic filarial nematode in terms of disease pathogenicity. the nematode releases unsheathed microfilariae (mf) into blood. mosquitoes like culex, aedes, anopheles, mansonia etc are incriminated as intermediate host of this parasite (bemrick and sandholm 1966, ludlam et al. 1970, lok 1988, arellano et al. 2002). in india, prevalence of this parasite in dogs has been earlier reported (borthakur et al. 2006, megat abd rani et al. 2010, bhattacharjee 2011, borthakur et al. 2011). another filarid, dirofilaria repens, a parasite of subcutaneous tissue of dogs and other canids. this parasite has been reported from many parts of the world including india (megat abd rani et al. 2010). dirofilaria repens accidentally affects humans and several human cases have been reported from india, mostly from south india (senthilvel and pillai 1999, sekhar et al. 2000, nadgir et al. 2001, sabu et al. 2005) and also reported from assam (nath et al. 2010). laboratory diagnosis of dirofilariasis in live animals is always in forefront in terms of simple demonstration and identification of microfilariae, serology and molecular techniques. several commercial elisa based test kits are available to diagnose heartworm in dogs but these kits are not widely used in *corresponding author: dr sonjoy kumar borthakur, email: sanjoy_barthakur@rediffmail.com http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 92–97 sk borthakur et al.: occult dirofilariosis … 93 our country where heartworm test in every dog is not mandatory. dna based techniques provide an alternative approach which is very sensitive and accurate for identification of the filarial parasites (favia et al. 1996, nuchprayoon et al. 2005, rishniw et al. 2006). sometimes microfilariae in circulating blood of heartworm infected dogs are not seen and such condition is termed as “occult infection”. the reason for occult infection of d. immitis in dog with no microfilariae in blood is due either to male or female worm infection, single worm infection, presence of immature females during pre-patent stage of development, geriatric infection and ectopic infection. numerous side effects associated with occult infections are severe cough, dyspnoea and crackles. immune mediated allergic pneumonitis sometimes associated with occult infections produces eosinophilia besides pneumonic sign and symptoms. the present communication is based on the reports of epidemiological study of heartworm disease in north eastern states of india. materials and methods study areas the study was undertaken systematically for a period of one calendar year from august, 2011 to july, 2012, in dogs from guwahati and aizawl. guwahati, a city of assam is located at the latitude of 26°11'0" n and longitude of 91°44'0" e having annual rainfall of 1500–2600 mm with an average altitude of 52 mts msl and aizawl, the capital city of mizoram state of north east located at 23°43'27" n and 92°43'2" e having annual rainfall of 2400–2962 mm with an average altitude of 1132 mts msl. both the cities are separated by surface distance of 550 km. selection of dogs three categories of dogs were selected for the epidemiological study, working dogs of military and paramilitary force, pet dogs and stray dogs. pet dogs of different breeds and paramilitary dogs mostly of labrador and german shepherd breeds brought to the teaching veterinary clinical complexes (tvcc) of the college of veterinary science, assam agricultural university, khanapara and the college of veterinary sciences and ah, central agricultural university, selesih, aizawl, mizoram during the study period were taken for the study. the stray dog population consisted of local non-descript street dogs of either sexes captured from different parts of the city for sterilization by local non-governmental organization like peoples for animal (pfa) and just be friendly (jbf). additionally stray dogs brought for slaughter at aizawl were examined during slaughter. altogether 782 dogs were examined. dogs exhibiting specific clinical signs of heartworm diseases were also recorded. three categories of dogs like working (103), pet (266) and stray (413) totaling 782 numbers were examined. dogs under the study were of either sexes or a total of 488 dogs from different localities of guwahati and 294 dogs from aizawl formed the entire base of study during the programme. blood sampling blood sampling from hospital dogs was done at clinics soon after presenting the animals by the owners for clinical investigation. in case of stray dogs, dog shelters were visited time to time and samples were collected. approximately 5 ml of blood was drawn from the cephalic vein collected in disodium salt of ethylene diamine tetra acetic acid (na2edta) vacuum tubes and stored at 4 ºc until further use. parasitological investigation the prevalence study for d. immitis was conducted on the basis of conventional wet blood film method and knott’s concentration technique (kct), immunological with a commercially available elisa test kit http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 92–97 sk borthakur et al.: occult dirofilariosis … 94 (snap® 4dx, idexx labs. inc., westbrook, usa) and molecular techniques targeting to amplify the its-2 region of filarial worms rdna developed by rishniw et al. (2006) was followed. isolation of genomic dna from blood was carried out using the dneasy blood and tissue kit (quiagen® kit, catalogue no 51104) and the protocols follows as per handbook provided by the manufacturer. the primer utilized was referred to as pan filarial primers, forward: didr f1 5'-agt gcg aat tgc aga cgc att gag-3' and reverse: didr r1 5'-agc ggg taa tca cga ctg agt tga -3' were utilized to amplify and differentiate d. immitis, d. repens, brugia malayi, b. pahangi, acanthocheilonema (dipetalonema) reconditum and a. dracunculoides. to amplify the targeted ribosomal gene of d. immitis the pcr reaction mixture was consisted as 2.5 µ l taq polymerase buffer (10x), 01 µ l dntp (10mm), 0.5 µ l mgcl2 (50mm), 0.75 µ l of each forward and reverse primer (60pm), 0.5 µ l taq polymerase, 3.0 µ l template dna and by making the final volume upto 25.0 µ l with nfw. the cycling condition used for amplifying the targeted product consisted of a initial denaturing step at 94 °c for 2 min and 32 cycles of denaturing (30 s at 94 °c), annealing (30 s at 60 °c) and extension (30 s at 72 °c), a final extension (7 min at 72 °c) and a soak at 4 °c in a technee-5000 thermal cycler (bibby scientific). the confirmation of the amplified products were made by gel electrophoresis of the pcr product in 1.5 % agarose gel stained with ethidium bromide and visualized under gel doc (dnr bio-imaging system, minilumi). since pcr test can be utilized to detect up to one microfilaria per 250 µ l of tested blood sample, hence, differences between heartworm positive cases in pcr test and antigen detection test (snap®4dx) was considered as criterion for calculation of number of occult cases. results overall, 782 dogs were examined to detect d. immitis infection. the parasitological investigations were carried out by employing conventional, serological and molecular methods on 3 different categories of dogs like, working dogs maintained by military and paramilitary forces, pet dogs and stray dogs. the main objective of the study was to determine the occult dirofilariasis due to heartworm disease. the study revealed an overall 22.69 percent occult case. the working dogs had highest prevalence (60%) followed by pet (29.16%) and stray dogs (17.75%) (table 1). table 1. occult cases of dirofilaria immitis dog category numbers examined numbers found +ve by ag elisa (snap 4dx) numbers found +ve by specific pcr (mff dna) occult cases of d. immitis* % occult cases (1) (2) (3) (4) stray dogs 413 107 88 19 17.75 pet dogs 266 24 17 7 29.16 working dogs 103 10 4 6 60.0 overall 782 141 109 32 22.69 * values in column (3) is obtained from values of column (1)-(2) http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 92–97 sk borthakur et al.: occult dirofilariosis … 95 discussion the present findings of higher occult cases recorded in working and pet dogs in comparison to stray dogs might be due to the fact that, owners of pet and working dogs are very much concerned about the health status of their animals. hence, there is regularity in their health check-up by a professional often necessitated for anthelmitic medication with a preference for an endecticidal drug like ivermectin. this endecticide has microfilaricidal activity, thereby reducing circulating microfilariae. on the other hand, stray dogs are seldom taken care of with such type of medications. earlier, borthakur et al. (2006) recorded 35.80 % dog affected with occult dirofilariasis in mizoram. their study was based on necropsy finding of confirmed heartworm cases and subjecting the blood sample from the same cases for kct. grieve et al. (1986) could record up to 61.9 percent occult dirofilariasis in infected dogs, who conducted the study with necropsy findings and elisa test in freeport, grand bahamas. similarly, lai et al. (2001) from central taiwan, alves et al. (1999) from the city of receife, pernambae, brazil, labarthe et al. (1997) in the state rio de janerio, brazil, and yildiz et al. (2008) from kirikkale, turkey could record 25.53, 57.1, 34.64 (n= 127) and 27.46 (n=172) percent occult infection in dogs, respectively. these authors had their conclusion based on antigen detection test by elisa and kct. further, they opined that abusive use of microfilaricidal agent contributed substantially to high rate of occult dirofilariasis. recently, malmasi et al. (2011) reported 17.7 % occult infections in southern coasts of the caspian sea based on 200 numbers of dogs surveyed by antigen test and modified knott’s test. in a more recent study, conducted in guwahati, bhattacharjee (2011) reported 7–37.5 percent occult cases of dirofilariasis. dogs with 50–100 mature worms that received little exercise may never show signs of heartworm disease, also, one may not be able to find microfilaria in the blood of such subjects (nayar 1990). it is generally known that, an infection may be occult due to presence of either one sex of the parasite or due to longer pre-patent period (180 days) leading to amicrofilaremic. conclusion the highest percentage of occult heartworm infection was present in working dogs maintained under military or paramilitary forces. acknowledgements authors acknowledge dean, college of veterinary science, assam agricultural university, khanapara, guwahati-781 022 for providing facilities to carry out the research programme. further, the receipt of snap® 4dx kits from idexx corporation, usa, in the form of gift is gratefully acknowledged. the authors declare that there is no conflict of interests. references alves lc, silva lv, faustino mag, mccall jw, supakonderj p, labarthe nw, sanchez m, caires o (1999) survey of canine heartworm in the city of recife, pernambuco, brazil. mem inst oswaldo cruz, rio de janeiro. 94: 587–590. arellano pjl, andrade ma, lopez aj, carranaza c, muro a (2002) helminths and the respiratory system. arch. bronconeumol. 42: 81–91. bemrick wj, sandholm ha (1966) aedes vexans and other potential mosquito vectors of dirofilaria immitis in minnehttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 92–97 sk borthakur et al.: occult dirofilariosis … 96 sota. j parasitol. 52: 762–767. bhattacharjee k (2011) studies on haemoparasitic infections of dog with special reference to dirofilaria immitis. (phd dissertation). assam agricultural university, khanapara, guwahati-781 022, assam, india. borthakur sk, sarma k, rajkhowa tk, das mr, rahman s (2006) dirofilaria immitis infection in dog. j vet parasitol. 20: 167–169. borthakur sk, ali ma, patra g (2011) clinical, haematological and biochemical studies on dirofilaria immitis in dog. j vet parasitol. 25: 63–66. favia g, lanfrancotti a, della torre a, cancrini g, coluzzi m (1996) polymerase chain reactionidentification of dirofilaria repens and dirofilaria immitis. parasitol. 113(6): 567–571. grieve rb, glickman lt, bater ak, grieve mc, thomas cb (1986) canine dirofilaria immitis infection in a hyperenzootic area: examination by parasitologic findings at necropsy and by two serorodiagnostic methods. am j vet res. 7: 329–332. labarthe n, almosny n, guerrero j, duquearaújo, am (1997) description of the occurrence of canine dirofilariasis in the state of rio de janeiro, brazil. mem inst oswaldo cruz, rio de janeiro. 92: 47–51. lai ch, ting ch, tung kc, wang js (2001) variation in the prevalence of dirofilariasis in stray dogs from central taiwan. j chinese soc vet sci. 27: 69–73. lok jb (1988) dirofilaria sp: taxonomy and distribution. in: dirofilariasis. boreham, pfl and atwell rb (eds), crc press, florida, pp. 1–28. ludlam kw, jachowski la, otto gf (1970) potential vectors of dirofilaria immitis. j american vet med assoc. 157: 1354–1359. malmasi a, hosseini sh, aramoon m, buhunar a, seiff ha (2011) survey of canine dirofilaria immitis infection in caspian provinces of iran. iran j vet res. 12: 340–343. megat abd rani pa, irwin pj, gatne m, coleman gt, mclnnes lm (2010) a survey of canine filarial diseases of veterinary and public health significance in india. parasites and vectors. 3: 30. menezes r (2008) rabies in india. canadian medical assoc j. 178: 564. nadgir s, tallur ss, mangoli v, halesh lh, krishna bv (2001) subconjunctival dirofilariasis in india. southeast asian j trop med public health. 32: 244– 246. nath r, gogoi r, bordoloi n, gogoi t (2010) ocular dirofilariasis. ind j pathol microbiol. 53: 157–159. nuchprayoon s, junpee a, poovorawan y, scoot al (2005) detection and differentiation of filarial parasites by universal primer and polymerase chain reaction-restriction fragment length polymorphism analysis. am j trop med hyg. 73(5): 895–900. rishniw m, barr sc, simpson kw, frongillo mf, franz m, dominguez-alpizar jl (2006) description between six species of canine microfilariae by a single polymerase chain reaction. vet parasitol. 135: 303–314. sabu l, devada k, subramanian h (2005) dirofilariosis in dogs and humans in kerala. ind j med res. 121: 691–693. senthivel k, madhavan pillai k (1999) a case of subcutaneous dirofilariosis in a woman in kerala. ind vet j. 76: 263– 264. shekar hs, srinivasa h, batru rr, mathai e, shariff s, macaden rs (2000) human ocular dirofilariasis in kerala southern india. ind j pathol microbiol. 43: 77–79. sudarshan mk, mahendra bj, madhusudhana http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 92–97 sk borthakur et al.: occult dirofilariosis … 97 sn, ashwoath narayan dh, rahman a, rao ns, x-meslin f, lob d, ravikumar kg (2006) an epidemiological study of animal bites in india: result of a who sponsored national multicentric rabies survey. j commun dis. 38(1): 32–39. yildiz k, duru sy, yagci bb, ocal n, gazyagci an (2008) the prevalence of dirofilaria immitis in dogs in kirikkale. türkiye parazitoloji dergisi. 32: 225–228. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 445 http://jad.tums.ac.ir published online: october 04, 2016 original article serological and molecular detection of dirofilaria species in stray dogs and investigation of wolbachia dna by pcr in turkey *sami simsek 1, ayse turkan ciftci 2 1department of parasitology, faculty of veterinary medicine university of firat, elazig, turkey 2parasitology and bee diseases laboratory, veterinary control and research institute, elazig, turkey (received 14 apr 2014; accepted 10 mar 2015) abstract background: dirofilaria immitis and dirofilaria repens are the most common species of filarial nematodes described in the dogs. a single-step multiplex pcr was applied to detect and differentiate simultaneously and unequivocally d. immitis and d. repens on dna extracted from canine peripheral blood and besides to detect the seroprevalance of d. immitis by elisa in elazig province, turkey. a pcr detection of the wolbachia, which plays an important role in d. immitis biology and contributes to the inflammatory pathology of the heartworm, was also applied for the first time in turkey. methods: a total of 161 whole blood and sera samples were collected from stray dogs and stored at -20 °c until used. after dna extraction, all samples were processed with dirofilaria primers by multiplex-pcr and wolbachia primers by conventional pcr besides elisa for serology. the amplification was performed using a set of primers designed on a portion of the small subunit ribosomal rna gene of the mitochondrion (12s rdna). results: three of the examined dogs (1.8%) were found to be infected with only d. immitis, one (0.6%) with d. repens and three (1.8%) with both parasites. besides, 10 out of 161 dogs (6.2%) were found infected with wolbachia sp. finaly, the seroprevalence of dirofilariosis in the examined dogs was found to be 3.7% (6/161). conclusion: although dirofilariosis is not a serious problem in the region, the stray dogs still continue to be a source of infection. keywords: dirofilaria immitis, dirofilaria repens, wolbachia, multiplex-pcr, elisa introduction dirofilariosis, caused by dirofilaria immitis, is found world-wide, but the most endemic areas are those with high temperatures and appropriate mosquito vector populations. dirofilaria immitis typically inhabits the right ventricle and pulmonary arteries of dogs. “this vector-borne parasite can cause patent infections in dogs, cats and wild canidae” (dillon 2000). it is one of the most pathogenic nematode parasite of dogs. adult heartworms may cause clinical signs ranging from mild cough to congestive heart failure, intravascular hemolysis and pulmonary thromboembolism which are often fatal if untreated (soulsby 1986). dirofilaria immitis in dogs can be diagnosed through careful morphological examination of circulating microfilariae, detection of circulating antigens, histochemical or immuno-histochemical staining of circulating microfilariae or, more recent-ly, through molecular approaches. morphological identification of circulating microfilariae, however, is not always easy and is potentially misleading (rishniw et al. 2006). dirofilaria repens, a filarial parasite of canids, is transmitted by mosquitoes. the adult worms are observed mainly in the subcutaneous tissue of dogs, and produce microfilariae that circulate in the blood stream of infected dogs. dignosis of it can be done by blood smear evaluation for the presence of microfilariae, serologic detection *corresponding author: prof dr sami simsek, email: ssimsek@firat.edu.tr j arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 446 http://jad.tums.ac.ir published online: october 04, 2016 antigen or antibodies and detection of microfilarial dna by pcr (lee et al. 2004). dirofilaria immitis is transmitted by several culicid mosquito species belonging to a wide range of genera, including culex, aedes, ochlerotatus, anopheles, armigeres and mansonia (cancrini et al. 1995). aedes vexans and culex pipiens were detected as the potential vectors of d. immitis in turkey (yildirim et al. 2011). for the first time, cytochrome c oxidase i (coi) sequences were obtained from iranian specimens of an. hyrcanus, an. pseudopictus, cx. theileri and oc. caspius s.l. only culex theileri were found naturally infected with third-stage (infective) larvae of d. immitis (azari-hamidian et al. 2009). dna-based diagnostic tests for d. immitis and d. repens infections have been shown to overcome some deficiencies of parasitological and serological diagnosis, and specific and sensitive polymerase chain reaction (pcr)based assays have been reported (mar et al. 2002, rishniw et al. 2006). the usefulness of different pcr methods for the identification of dirofilaria spp microfilaria in dog blood (the definitive host) has been reported in recent publications (gioia et al. 2010, simsek et al. 2011, giangaspero et al. 2012, latrofa et al. 2012). dirofilaria immitis is one of the several species of parasitic nematodes that hold the obligate symbiont bacteria wolbachia spp. large colonies of wolbachia live in the subdermal lateral cords of both female and male nematodes, as well as in the reproductive structures of females (mchaffie 2012). the aim of the current study was to performe a single-step multiplex pcr to detect and differentiate d. immitis and d. repens on genomic dna isolated from dog blood and also detect the seroprevalance of d. immitis by elisa. the amplification was performed using a set of primers designed on a portion of the small subunit ribosomal rna gene of the mitochondrion (12s rdna). the other aim of this work was to pcr detection of the wolbachia which is play an important role in d. immitis biology and contributes to the inflammatory pa-thology of the heartworm. materials and methods samples collection a total of 161 whole blood and sera samples were obtained from stray dogs in elazig province of eastern turkey within 2010. these dogs had been captured from suburbs by the local authorized for the aim of spaying and during this procedure the blood samples were acquired under anesthesia. the blood and sera samples were stored in -20 °c untill use and age, breed and genders were recorded. dna (gdna) isolation, pcr amplification and sequencing the blood samples were removed from freezer and waited at room temperature untill thawed. then 1 ml blood sample was putted into an eppendorf tube and centrifuged during 5 min by 5000 rpm for sink to the bottom of possible microfilaria. supernatant was removed and prior to gdna isolation pellet was digested overnight at 56 °c with 600 µ l lysis buffer of the kit to which 20 µ l proteinase-k (20 mg/ml) (sigma, usa) were added. the tubes were incubated at 56 °c for overnight and the kit procedure was followed and at the last step the pellet was resuspended in 80 µ l sterile distilled water, and the gdna samples were stored at -20 °c until use. the multiplex-pcr reactions for d. immitis and d. repens were performed using two sets of primer in the same reaction. general primer pairs 12sf (5’-gttccagaataatcggcta-3’) and 12srdeg (5’-attgacggatg(ag)tttgtacc-3’) were used previously designed on the 12s rdna region (casiraghi et al. 2004). besides we used a j arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 447 http://jad.tums.ac.ir published online: october 04, 2016 specific forward primer for d. immitis (12sf2b 5’-tttttacttttttggtaatg3’) and a specific reverse primer for d. repens (12sr2 5’-aaaagcaacacaaataa (ca)a-3’) previously designed by gioia et al. (2010). the pcr reactions were carried out in a total volume of 50 µ l containing 5 µ l of genomic dna for each sample amplification, 5 µ l of mgcl2, 1.25 mm of each dntp, 5 µ l 10 x pcr buffer, 0.5 iu taq dna polymerase and 20 pmol of each primers. the thermal profile used was 92 °c for 1 min; 40 cycles of 92 °c for 30 s, 52 °c for 45 s, 72 °c for 1 min and final elongation step at 72 °c for 10 min. the amplified products were separated by electrophoresis in 2% agarose gel with a trisboric acid–edta (tbe, ph 8.3) buffer at 90 v for 45 min. following electrophoresis, the amplified products were visualized with ethidium bromide (0,5 µ g/ml) staining for 45 min at room temperature. dirofilaria immitis genomic dna positive control sample was extracted from microfilariae present in the blood of infected dogs (gifted from another research group) (yildirim et al. 2007). another gdna control sample was extracted from an adult d. repens parasite (this worm was gifted by luigi venco (veterinary hospital “citt`a di pavia”, viale cremona pavia, italy). extracted dna was also tested for the presence of wolbachia using a pcr-based assay and the gene primer wsp. a specific primer sets (forward 5'-tggtccaataa gtgatgaag aaactagcta-3', reverse 5'-aaaattaaacgctactccagcttct gcac-3') previously described by zhou et al. (1998) were used for the amplification of gdna. the pcr mixtures were composed of 5 µ l of 10x pcr buffer, 5 µ l of mgcl2, 125 µ m of each dntps, 20 pmol of each primers, 0.2 µ l (5 iu) taq-dna polymerase and 5 µ l of genomic dna was used for each pcr reaction. the reactions were performed on a pcr thermal cycler (thermo electron corporation, waltham, ma, usa) under the following conditions: 94 °c for 3 minو 40 cycles of 94 °c for 1 min, 52 °c for 1 min and 72 °c for 1 min with a final extension at 72 °c for 5 min. pcr products were analyzed on 1.4% agarose gels stained by ethidium bromide and visualized under ultraviolet light. randomly selected six dirofilaria and two wolbachia samples were sequenced for confirmation of the pcr results. serological analysis filarcheck (agrolab, italy) kit was used for working the dog sera for serological analysis. the test is based on a sandwich elisa technique. microplate wells were coated with a monoclonal antibody against the circulating antigen of d. immitis. canine serum was added into the wells. if the serum contained the antigen, wells gave blue colour otherwise colorless. statistical analysis the data were evaluated by spss 15.0 programme using of 2x2 fischer’s exact test and pearson’s chi square test. results multiplex-pcr reaction showed the expected amplification products of app-roximately 500 bp for the genus dirofilaria, 327 bp for d. repens and of 204 bp for d. immitis. (fig. 1). wolbachia surface protein pcr amplified 630 bp band as shown in fig. 2. the results of the pcr assay according to the ages and gender of filarial agents and wolbachia are shown in table 1. thirty five male dogs were examined by multiplex-pcr and the prevalance values were 5.7% for d. immitis, 2.8% for d. repens and 2.8% for mixed infection (both d. immitis and d. repens). on the other hand, 126 female dogs were examined by pcr and only one case was d. immitis (0.8%) and two cases were j arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 448 http://jad.tums.ac.ir published online: october 04, 2016 d. repens (1.5%). there was no any mix infection in female dogs. among the 161 samples screened by the elisa, 6 samples (3.7%) tested positive for the d. immitis. there was no significant difference in the number of positive d. immitis infection among female dogs (4 out of 126, 3.2%) and male dogs (2 out of 35, 5.7%). only 2 out of the 69 dogs belonging to the 0–1 yrs old group were positive (2.9%), while 4 out of 71 dogs belonging to the 2–4 yrs old group were positive (5.6%). a total of 21 dogs belonging to the >4 yrs old group showed no seropositivity of d. immitis infection. fig. 1. multiplex-pcr bands of samples. m: molecular weight marker (100 bp), 1: positive control of mix infection (500 bp, 327 bp and 204 bp), 2: positive control of dirofilaria repens (500 bp and 327 bp), 3: positive control of dirofilaria immitis (500 bp and 204 bp), 4: only d. repens detected sample, 5, 6, 7: only d. immitis detected samples, 8, 9, 10: mix infected samples. table 1. positivity of filarial agents and wolbachia according to ages and gender inspected dog (n) only d. immitis only d. repens mix wolbachia n % p n % p n % p n % p gender male 35 2 5.7 0.119 1 2.8 0.217 1 2.8 0.523 4 11.4 0.226 female 126 1 0.8 2 1.5 6 4.7 p p p p ages 0-1 69 3.875 0.144 1.276 0.528 2.628 0.269 3 4.3 1.098 0.5772-4 71 3 4.2 1 1.4 2 2.8 6 8.4 4> 21 1 4.7 1 4.7 total 161 3 1.8 1 0.6 3 1.8 10 6.2 j arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 449 http://jad.tums.ac.ir published online: october 04, 2016 fig. 2. pcr bands of wolbachia surface protein. m: molecular weight marker (100 bp), 1, 2: positive samples (630 bp), 3: positive control discussion adult d. immitis, inhabit the right ventricle of the heart the pulmonary arteries where they cause canine heartworm disease while the adults d. repens usually inhabit the subcutaneous tissue. in addition, it is wellknown that d. immitis and d. repens produce microfilariae that circulate in the blood of dogs (soulsby 1986). dirofilaria immitis occurs worldwide in tropical, sub-tropical and temperate climates however d. repens occurs in the oldworld, in particular, throughout the mediterranian sub-region, south asia and sub-saharan africa (cringoli et al. 2001). vector borne pathogens are sensitive to climatic condition, and there are some evidence that climate change may increase the incidence and dentensity of the diseases transmission (purse et al. 2005). by altering the global environment, climate change has significant potential to intensify the vector borne diseases (khasnis and nettleman 2005). dirofilaria immitis vectors are mosquitoes of culicidae family with nearly 70 species susceptible for developing of parasite and thus considered potential vectors (vezzani and carbajo 2006). aedes albopictus is reported as the primary potential vector of d. immitis in italy (cancrini et al. 2003). whereas, cx. theileri was detected as a vector of d. immitis in portugal (santa-ana et al. 2006). there are limited study about vectors of dirofilaria species in turkey. yildirim et al. (2011) determined that ae. vexans and cx. pipiens are the main potential vectors for d. immitis in central turkey. in the current study we could not investigate the potential vectors of dirofilaria species. several studies have been published regarding the distribution and prevalence of d. immitis in dogs in turkey. it was first reported in a dog the year of 1951 in turkey (guralp 1981). tasan (1983) detected microfilaraemia in 53/283 (18.7%) stray dogs in elazig. the prevalence was recorded as 1.52% in istanbul (oncel and vural 2005), 9.6% in kayseri, (yildirim et al. 2007), 8.1% in erzurum (simsek et al. 2011) besides 12.3%, 18.3%, 10.5% and 14.8% in sakarya, kocaeli, mersin and ankara, respectively (simsek et al. 2008). these different prevalence rates may reflect different testing methodologies or true regional differences. the prevalence of d. immitis in dogs has been determined traditionally by postmortem inspection, detection of microfilariae and serological testing. however, dogs with occult heartworm infections are amicrofilaraemic. in addition, some antiparasitic treatments such as macrolides may render an infected dog amicrofilaraemic for 6–9 months (hoover et al. 1996). thus, serological and microfilarial examinations should be applied together for screening d. immitis in dogs. dna based diagnostic tests for d. immitis infections have been shown to overcome some deficiencies of parasitological and serological diagnosis, and specific and sensitive polymerase chain reaction (pcr)based assays have been reported (rishniw et al. 2006). the current study describes a quick and accurate molecular method for the j arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 450 http://jad.tums.ac.ir published online: october 04, 2016 simultaneous detection of the d. immitis and d. repens for the first time in turkey. although there have been some records about variability between age and dirofilariosis prevalence (montoya et al. 1998, song et al. 2003, fan et al. 2003), some authors (rowley 1981, martin and collins 1985) have reported that age has no effect on dirofilariosis and it can be occur in all ages dog. while the others (song et al. 2003, fan et al. 2001) have stressed that ages and positivity, have positive relation and especially 3–7 ages group have higher risk than the others. fan et al. (2001) found the lowest prevalence in 1–3 ages (6.3%) and following 3–6 ages (14.1%) while the highest rates has been found in up to 6 ages (23.7%). simsek et al. (2008) determined the highest percent 3-5 ages dog (17.5%) while there was no positivity in up to 6 ages. in the current study, all dogs that were defined only d. immitis by pcr were in 2–4 ages group (4.2%). similarly, the elisa seropositivity was 2.9% in 0–1 ages and 5.6% in 2–4 ages dog. a possible explanation for higher seroprevalence of d. immitis infection in older dogs might be due to their longer exposure to the risk factor like mosquito (fan et al. 2001). selby et al. (1980) also indicated that the age of dogs was an important risk factor and determined by time of exposure in the endemic area. in the present study, elisa and pcr positivity were higher in male than female dogs. similarly, montoya et al. (1998) indicated that heartworm infection was more common in male dogs than female dogs, and the generally higher infection rate in male dogs had been postulated to be due to their stronger attraction to mosquitoes. however, simsek et al. (2008), reported 10.7% for males and 14.4% for females. more male dogs live in the outdoor, due to their use in defence of property. they are, therefore, more likely to be bitten by mosquitoes (montoya et al. 1998). however, all studied stray dogs in this work were living in outdoor. thus, living conditions are not unique on the prevalence. we believe that, some individual parameters like hormonal changes and immune deficiencies in female dogs may more tend to dirofilariosis. canine heartworm disease is generally diagnosed by antigen testing for d. immitis, and/or identification of microfilariae in the blood of infected dogs. however, some other filariae, including dipetalonema reconditum, d. repens and approximately 1% of d. immitis infestations, can produce persistent microfilaremias with negative heartworm antigen tests (rishniw et al. 2006). thus, serological and molecular techniques should be use as combined. in this study, the seroprevalence with elisa was 3.7% while the positivity was 1.8% by pcr. this differences might be related with some possible cross reactions with the other nematodes, single-sex adults and/or possible treatment of microfilaria by macrolids. dirofilaria repens is transmitted by mosquitoes. the adult parasites are observed in the subcutaneous tissues of dogs and produce microfilariae that circulate in the perifer blood of infected dogs. it is less remarkable than d. immitis due to the lower pathogenicity (soulsby 1982). dirofilaria repens was detected first time in turkey in 1962 (merdivenci 1970). tasan (1984), necropsied 120 dogs and found the occurence of d. repens as 2.5% in elazig province of turkey. whereas, yildirim (2004) examined a total of 300 dog blood by modified knott and membrane filtration tests and no detected any d. repens micofilaria in ankara. in the current study, one of the examined dogs (0.6%) was infected with only d. repens by pcr and three of them (1.8%) were infected with both d. immitis and d. repens. these rates are close to reported by tasan (1984). microfilariae of d. repens are difficult to discriminate from d. immitis, since they have similar morphology. staining of microfilaria was widely used for disj arthropod-borne dis, december 2016, 10(4): 445–453 s simsek and at ciftci: serological and molecular … 451 http://jad.tums.ac.ir published online: october 04, 2016 criminate the both species. in the last years, pcr analysis was reported to be quite sensitive and specific for the differentiate the species (lee et al. 2004). gioia et al. (2010) designed a single-step multiplex pcr was based on the amplification of a partial 12s rrna gene of the mitochondrion with a mix of general and species-specific filarial primers in a single reaction. we also used the same primers for the amplification of d. immitis and d. repens 12s rrna genes by pcr in a single tube. thus, the simultaneous detection of both d. immitis and d. repens in naturally infected dogs has been achived for the first time in turkey. wolbachia is an intracellular alphaproteobacteria endosymbionts sheltered in a broad range of insects and nematodes (pfarr and hoerauf 2007). according to reports based on dna amplification, one in five of the arthropods are infected with wolbachia, rendering this bacterium the most ubiquitous intracellular symbiont yet described (bourtzis 2008). dirofilaria immitis and d. repens harboured the wolbachia endosymbiont (kozek 2005). we amplified the wsp (wolbachia surface protein) gene by pcr and detected 6.2% (10/161) positivity in the current study. there are very limited study about this subject in turkey. sarali et al. (2009) collected 150 dogs blood from izmir and aydın provinces and detected the prevalences as 12.3% for both d. immitis and wolbachia sp. in the current work, we determined the wolbachia in 6 samples together with d. immitis and d. repens and in 4 samples without dirofilaria spp as well. in this instance, either those 4 samples were infected with any dirofilaria species and the pcr could not detect or those dogs had another residence for wolbachia in the dogs. it is widely accepted that wolbachia is released into the tissues of the infected host following worm death and that bacteria derived molecules provoke innate inflammatory responses (saint andre et al. 2002). thus, doxycycline treatment may reduce wolbachia levels in adultworms and less severe pathology as well. conclusion this is the first study on the detection of dirofilaria species using of multiplex-pcr in turkey. besides, it was attentioned to neglected filarial nematod which is d. repens in turkey and obtained actual prevalence data about d. repens, d. immitis and wolbachia, as well. besides, the seroprevalence of d. immitis was determined by elisa. those results have been shown that canine dirofilariosis still prevalent and there is no effective reduction yet. acknowledgements this study was supported by a grant from the scientific and technological research council of turkey (tubi̇tak project no: 110o934). the authors declerate that there is no conflict of interest. references azari-hamidian s, yaghoobi-ershadi mr, javadian e, abai mr, mobedi i, linton ym, harbach re (2009) distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern iran, with the first finding of filarial larvae in naturally infected local mosquitoes. med vet entomol. 23: 111–121. bourtzis k (2008) wolbachia-based technologies for insect pest population control. adv exp med biol. 627: 104–113. cancrini g, pietrobelli m, frangipane di regalbono a, tampieri mp, della torre a (1995) development of 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immitis in dogs from kayseri province, turkey. res vet sci. 82: 358–363. yildirim a, inci a, duzlu o, biskin z, ica a, sahin i (2011) aedes vexans and culex pipiens as the potential vectors of dirofilaria immitis in central turkey. vet parasitol. 178: 143–147. zhou w, rousset f, o’neill s (1998) phylogeny and pcr-based classification of wolbachia strains using wsp gene sequences. proc r soc lond b. 265: 509–515. j arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 586 http://jad.tums.ac.ir published online: october 04, 2016 original article epidemiological study of endemic relapsing fever in hamadan province, west of iran *mansour nazari, ali najafi department of medical entomology, school of medicine, hamadan university of medical sciences, hamadan, iran (received 13 nov 2014; accepted 6 oct 2015) abstract background: endemic relapsing fever remains under diagnosed in our area according to a low index of suspicion among clinicians, as well as its difficult diagnosis. the goal of this study was to present the epidemiological aspects of the disease in western iran. methods: in this analytical-descriptive cross-sectional study, the epidemiological and clinical aspects of relapsing fever were investigated in hamadan province, western iran from 1999 to 2013. a confirmed patient was defined as a person who had both febrile illness and detected spirochetes by wright-giemsa or dark-field microscopy in a peripheral blood smear. for the statistical analysis, the statistical software spss was used. results: during the study period, 276 cases of relapsing fever were recorded that 146 were male. due to the age group distributions, most of the patients aged less than 20 yr. patients noticed from april through march, most cases were reported in september (53 cases, 19.2%). considering time trend of the mentioned disease between 1999 and 2013 showed an increasing trend of disease from 1999 to 2003 (from 2.5% to 21.0%), while the prevalence of disease had a decreasing trend after than from 21.0% in 2003. conclusion: the rate of endemic relapsing fever is similar in both male and female genders, but its prevalence reduced by increase of age. the trend of the changes in prevalence of the mentioned disease has shown to be downward in recent years probably due to improving health policies especially among children and adolescents and particularly in rural areas. keywords: endemic relapsing fever, tick, borrelia, epidemiology, iran introduction relapsing fever, is characterized by recurrent acute episodes of fever. these are followed by periods of which differ in their durations. relapsing fever is an infection spread by the vectors of lice and ticks (jump et al. 2004). tick-borne relapsing fever is mostly caused by borrelia species generally called ‘asian or asiatic tbrf’ (goubau 1984, fukunaga et al. 1996, trape et al. 2013). the geographical distribution of the borellia is mostly related to eurasia. the most important agent is b. persica, which causes the large majority of human cases and has the widest distribution (dschunkowsky 1913, baltazard et al. 1950, colin de verdière et al. 2011). in eurasia, dschunkowsky (1913) was the first person who described the tick-borne relapsing fever (tbrf in the ardebil region of iran, and because of this reason it was called borrelia persica (euzeby 1997). however, in the same area, at early time in1882, tholozan also published the first clinical description of a case of tbrf, which he called ‘fie `vre re´currente asiatique’, and which was transmitted to humans by an ornithodoros tick (theodorides 1998). in the following decades, cases of relapsing fever were reported and described all over the middle east region and were attributed mainly to b. persica, but other bacteria were also reported (nicholson 1919, adler et al. 1937, kalra *corresponding author: dr mansour nazari, email: ynazari@yahoo.com j arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 587 http://jad.tums.ac.ir published online: october 04, 2016 and rao 1951, davis and hoogstraal 1954, babudieri 1955). tick-borne relapsing fever is an endemic disease in canada (southern portion of british columbia), mexico, central and south america, central asia, africa, the mediterranean region, and russia (vial et al. 2006, cutler et al. 2009, platonov 2011, diatta et al. 2012). finally, tbrf has a mortality rate of less than 2% (in treated patients) to 4– 10% (in untreated individuals) (dworkin et al. 2002). borrelia duttoni (causes tbrf), is found in east africa and transmitted by the soft tick o. moubata (carlisle 1906). for the first time, b. persica was isolated and separated from the ardabil, northwestern iran, from the blood smear of a patient by dschunkowsky and luhs in 1913. the vector of b. persica was first found in iran in 1879 and after that, it was named as o. tholozani (rodhain 1998, arshi et al. 2002, aghighi et al. 2007). tick-borne relapsing fever is acquired by at least 15 different borrelia species (blevins et al. 2008). in iran, tbrf transmission to humans are mainly attributed to three species of argasidae including, o. tholozani, o. erraticus and o. tartokovyskyi. furthermore, b. persica, b. microti, b. latyschevi, and b. baltazardi are the major transmitting agents of tbrf in iran (vatandoost et al. 2003). in iran, principal foci of tbrf are almost mountainous provinces of north-west and west parts, and is reported from various parts situated in south and center of the country (baltazard et al. 1948, rodhain 1976, aghighi et al. 2007, rafinejad et al. 2012). people in endemic regions should aware of tick infected areas and rodents and use repellents and protective costume to impede tick bites (dworkin et al. 2002). this disease is almost not diagnosed in our area due to a low index of suspicion among clinicians as well as its difficult diagnosis. the aim of this study was to present the epidemiological aspects of the disease in hamadan province, western iran, during 1999–2013. materials and methods in this analytical-descriptive cross sectional study, the epidemiological and clinical aspects of relapsing fever were investigated in hamadan province, western iran (fig. 1) from 1999 to 2013. the county´s populations at the 2011 were 525794, 169352, 13711, 75445, 27645 and 20349 in hamadan, malayer, razan, nahavand, bahar and kabudarahang, respectively. demographical, clinical, and geographical information included in the disease reporting forms and records of relapsing fever cases were requested from the health department of the hamadan university of medical sciences. a confirmed patient was defined as a person who had both febrile illness and detection of spirochetes by wright-giemsa or dark-field microscopy in a peripheral blood smear. results were presented as mean±standard deviation (sd) for quantitative variables and were summarized by frequency (percentage) for categorical variables. continuous variables were compared using non-parametric kruskal-wallis h test whenever the data did not appear to have normal distribution or when the assumption of equal variances was violated across the study groups. categorical variables were, on the other hand, compared using chi-square test. for the statistical analysis, the statistical software spss version 16.0 for windows (spss inc., chicago, il) was used. p-values of 0.05 or less were considered statistically significant. results during the study period, 276 cases of relapsing fever were recorded that 146 j arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 588 http://jad.tums.ac.ir published online: october 04, 2016 (52.9%) were male and 130 (47.1%) were female with overall mean age of 15.89±13.69 yr. incidence rate was calculated 3.3/10000 population. regarding age group distributions, most of patients aged less than 20 yr and the prevalence of the disease in patients aged less than 10 yr was 41.3%, in those aged 10 to 20 yr was 31.9%. the prevalence of disease was considerably lowered in older than 40 yr (4.7%). there has been a downward trend in the prevalence of relapsing fever by increasing age. according to the occupational status, 37.3% were rancher followed by other jobs (table 1). there was no difference between the genders in terms of mean age, age distribution, and clinical manifestations, however occupational status was different between them (p < 0.01) (table 1). regarding clinical manifestations, most patients suffered from classic symptoms of the mentioned disease including sweating, abdominal pain, cough, fever, chilling, headache, nausea, and vomiting. jaundice was only revealed in 8.7% and photophobia and eosinophilia were found only in 8.7% of them (table 1). the death due to tbrf in none of the patients in this study was reported. interestingly, more than half of the infected cases were found out in a city named razan (58.3%), followed by kabudrahang (37.3%), while the prevalence of disease in hamadan city as the center of province was only 2.2% (table 2). most cases were reported in september (53 cases, 19.2%) (fig. 2). almost cases were diagnosed during the summer (147 cases, 53.3%). considering time trend of the mentioned disease between 1999 and 2013 showed an increasing trend of disease from 1999 to 2003 (from 2.5% to 21.0%), while the prevalence of the disease had a decreasing trend after than from 21.0% in 2003 to 1.1% in 2010 (fig. 3). the trend of the changes in the prevalence of relapsing fever by gender was shown with the peak of occurrence in 2003 as 29 (19.9%) in men and in 2002 as 34 (26.2%) in women (fig.4). in addition, assessing the age proved that the occurrence of disease within this time showed no significant changes in the trend of the age of disease occurrence (fig. 5). fig. 1. map of study area in hamadan province, western iran j arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 589 http://jad.tums.ac.ir published online: october 04, 2016 table 1. baseline characteristics of tick-borne relapsing fever of hamadan province, western iran (1999–2013) total (n= 276) male (n= 146) female (n= 130) p-value age (yr) 15.89±13.69 14.55±12.50 17.40±14.82 0.088 1–10 114 (41.3) 63 (43.2) 51 (39.2) 11–20 88 (31.9) 51 (34.9) 37 (28.5) 21–30 35 (12.7) 13 (8.9) 22 (16.9) 31–40 26 (9.4) 15 (10.3) 11 (8.5) older than 40 13 (4.7) 4 (2.7) 9 (6.9) occupation < 0.001 rancher 103 (37.3) 54 (37.0) 49 (37.7) farmer 7 (2.5) 6 (4.1) 1 (0.8) housekeeper 22 (8.0) 0 (0.0) 22 (16.9) students 72 (26.1) 47 (32.2) 25 (19.2) age under the occupation 72 (26.1) 39 (26.7) 33 (25.4) clinical symptoms 0.128 classic symptoms 228 (82.6) 121 (82.9) 9 (6.2) classic symptoms plus jaundice 24 (8.7) 107 (82.3) 15 (11.5) classic symptoms plus photophobia and eosinophilia 24 (8.7) 16 (11.0) 8 (6.2) table 2. distribution of participants of tick-borne relapsing fever according to different regions of hamadan province, western iran (1999–2013) region total (n= 276) male (n= 146) female (n= 130) hamadan 7 (2.5) 5 (3.4) 2 (1.5) malayer 1 (0.8) 0 (0.0) 1 (0.8) razan 161 (58.5) 83 (57.2) 78 (60.0) nahavand 3 (1.1) 1 (0.7) 2 (1.5) bahar 1 (0.4) 1 (0.7) 0 (0.0) kabudarahang 103 (37.5) 56 (38.6) 47 (36.2) fig. 2. number of tick-borne relapsing fever cases by months in hamadan province, western iran (1999–2013) j arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 590 http://jad.tums.ac.ir published online: october 04, 2016 fig. 3. number of tick-borne relapsing fever cases of different years in hamadan province, western iran (1999– 2013) fig. 4. the trend of the changes in the prevalence of tick-borne relapsing fever by gender in hamadan province, western iran (1999–2013) fig. 5. the trend of the mean age of patients of tick-borne relapsing fever within study period in hamadan province, western iran (1999–2013) j arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 591 http://jad.tums.ac.ir published online: october 04, 2016 discussion tick-borne relapsing fever is known as endemic disease in hamadan province. our study has some important findings. a total number of 276 cases were investigated. in the study in kurdistan province, the tbrf cases included 50.7% women and 49.3% men (kassiri et al. 2014) and in ardebil province, iran, a total of 391 (49% females, 51% male) patients were studied from 1998–2001 (arshi et al. 2002), which is similar to our findings. in the study of masoumi-asl et al. (2009), the tbrf cases included 55% women and 45% men, that results of our study is opposed. in a study in the united states from 1997 to 2000, the patients included 235 men (52%) and 178 women (40%), data regarding gender was missing for 37 (8%) (dworkin et al. 2002), that is similar with our study. in this study, most prevalence of the disease was 41.3%, among the age groups of 1– 10 years. in a study by croche santander et al. (2013), the median age of patients was 11 years. although the distribution of the disease was similar in both male and female genders (p> 0.05), its prevalence was reduced by increase of age. masoumi-asl et al. (2009) reported that there were 33% of the cases younger than 5 years old, 18% of 6-10 years old and 27% of 11-20 years old. in the study of rafinejad et al. (2012), in kurdistan province, iran, 2000–2004, most frequencies of tbrf based on age groups were observed as 43.3%, 17.5% and 17.5% in 10–19, 20–29 and 0–4 years old, respectively. in a new highland endemic focus of western iran, most (91%) of the patients were reported to be young people (moemenbellah-fard et al. 2009). in this study, the rate of infection in rancher, farmer, housekeeper, students and age under the occupation was similar to the finding of rafinejad et al. (2012) on tbrf in kurdistan province, western iran. this information represented that all job groups are at risk of obtaining infection but the majority of cases in our study was rancher. the most frequent clinical presentations showed fever, chills, headache, vomiting, myalgia and abdominal pain (croche et al. 2013). the most common clinical manifestations of tbrf in the recent study were classic symptoms (82.6%), followed by classic symptoms plus jaundice and classic symptoms plus photophobia and eosinophilia (8.7%, 8.7%), which is similar to other researches performed in this field. in the study of moemenbellahfard et al. (2009) in western iran, cases of tbrf was almost characterized by recurring episodes of fever, chills and headache. in the investigation of arshi et al. (2002) in ardabil, northwestern province of iran, the most prevalent clinical manifestations were fever (93.3%), chills (86.2%) and headache (85.9%). in a study in montana, all patients had fever and other clinical manifestations co-related with tbrf (such as headache, rash, vomiting and myalgia) (schwan et al. 2003). similar to other countries, tbrf prevalent is frequent in warm months. however, the disease also occurs during the winter (cutler 2010). in our study, nearly 53.3% were occurred in the summer, and the frequencies were from april to march. cases of rf are reporting from warm provinces in south of the country as well. in masoumi-asl et al. investigation, the peak of the tbrf prevalence was in summer and the disease occurred during all months of the year with the maximum reported from june through november (2009). in the united states, patients were reported during all months of the year: 71% cases were occurred from june to september; the majority of cases diagnosed in july (24%) and august (23%) (dworkin et al. 2002). the majority cases of tbrf were reported in the year 2002 and 2003 (n=115, 41.7%). the trend of the disease was downj arthropod-borne dis, december 2016, 10(4): 586–594 m nazari and a najafi: epidemiological study… 592 http://jad.tums.ac.ir published online: october 04, 2016 ward in recent years probably due to improving health strategies especially among children and adolescents and particularly in rural areas. this significant trend was also shown in both genders, but the changes of this trend were independent to patients' age. this disease still has resistant foci of infection, where control can be a major healthcare difficulty. relapsing fever can be acquired by travelers and transmitted to regions where the disease is not epidemic (colebunders et al. 1993, dworkin et al. 2002). after eco-challenges or in association with military training or activities such as camping or caving, provided potential hosts and natural disease ecologic cycles coincide (sidi et al. 2005, wyplosz et al. 2005). although many would complain this limited disease, impact is not a threat to public health, the lack of consideration of relapsing fever as a potential cause of clinical findings is a cause for concern. according to epidemiological distribution, tbrf is endemic in canada (southern portion of british columbia), mexico, central and south america, central asia, africa, the mediterranean region, and russia (colebunders et al. 1993). these mentioned diseases are mostly reported from areas of war, famine, mass migrations, or overcrowding (dworkin et al. 2008). conclusion the distribution of relapsing fever was similar in both male and female genders, but its prevalence showed a reduction in the number of victims by the increase of age. the trend of the changes in prevalence of disease has shown a downward prognosis in recent years probably due to improving health strategies especially among children and adolescents and particularly in rural areas. our study proved that the epidemiological sections of the disease is consistent with previous reports from other regions, but further studies are required to assess its epidemiological parts correctly in different areas of our country. acknowledgements the authors are grateful to the vice chancellor of research in hamadan university of medical sciences for the financial assistance (project no. 9206261850). the authors declare that they have no competing interests. references adler s, theodor o, schieber h (1937) observations on tick-transmitted human spirochaetosis in palestine. ann trop med parasitol. 31: 25–35. aghighi z, assmar m, piazak n, javadian e, seyedi rashti ma, kia eb, rassi y, vatandoost h (2007) distribution of soft ticks and their natural infection with borrelia in a focus of relapsing fever in iran. iran j arthropod borne dis. 1(2): 14–18. arshi s, majidpoor a, sadeghi homayoun ed, asmar m, emdadi d, derakhshan mh (2002) relapsing fever in ardabil, a 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durand p, sokhna c, rogier c, renaud f, trape jf (2006) incidence of tick-borne relapsing fever in west africa: longitudinal study. lancet. 368: 37–43. wyplosz b, milhaila amrouche l, baixench mt, bigel ml, berardi grassias l, fontaine c, hornstein m, izri a, baranton g, postic d (2005) imported tick borne relapsing fever, france. emerg infect dis. 11: 1801–1803. j arthropod-borne dis, june 2013, 7(1): 8–14 mr aflatoonian et al.: the emergence of … http://jad.tums.ac.ir published online: june 10, 2013 original article the emergence of anthroponotic cutaneous leishmaniasis following the earthquake in southern villages of bam district, southeastern iran, 2010 mohammad reza aflatoonian 1, *iraj sharifi 1, somayeh poursmaelian 1, maryam hakimi-parizi 1, nasser ziaali 2 1leishmaniasis research centre, kerman university of medical sciences, kerman, iran 2department of parasitology and mycology, school of medicine, kerman university of medical sciences, kerman, iran (received 16 mar 2011; accepted 15 apr 2012) abstract background: the objective of this study was to assess the epidemiological characteristics of a new emerging focus of cutaneous leishmaniasis (cl) in southern villages of bam district, southeastern iran, 2010. methods: a house-tohouse census survey of 5544 individuals were interviewed and physically examined for the presence of active lesions or scars. diagnosis was confirmed by direct smears, cultures and identification by pcr. the data were entered into a computer and spss ver. 15. results: overall, 1.2% of the inhabitants were infected, 0.5% active and 0.7% scars and females were more significantly infected (1.7%) than males (0.8%), (p= 0.003). all age groups were equally affected. most of the lesions were on the face and majority had single lesion. most of the cases appeared from 2006 to 2008 during the cl epidemic in the city of bam. pcr indicated l. tropica as the causative agent. conclusion: the presence of non-immune individuals along with suitable ecological conditions could induce a new emerging focus of acl in villages. keywords: anthroponotic cutaneous leishmaniasis, leishmania tropica, emergence, iran introduction leishmaniasis consists of a spectrum of clinical manifestation ranging from simple self-limiting cutaneous lesions to disfiguring and fatal visceral forms (postigo 2010). the disease endemicity extends to over 88 countries, but its public health impact remains grossly neglected. the overall prevalence is estimated 12 million, with 2 million new cases occur annually and the population at risk is 350 million. cutaneous leishmaniasis (cl) comprises 1.5 million new cases each year and 90% are limited to 8 countries including afghanistan, algeria, iran, iraq, saudi arabia, syria, brazil and peru (who 2009). two epidemiological forms are present in iran, anthroponotic cutaneous leishmaniasis (acl) due to leishmania tropica is a wellknown disease in bam (nadim and aflatoonian 1995) where field trials of killed l. major against l. tropica have recently been conducted (sharifi et al. 1998b). in bam district, most of the cases are confined to the city, the reservoir is human and the main vector is phlebotomus sergenti. zoonotic cl (zcl) due to l. major presents in a frequency of 5 to 10% where gerbils are the main reservoir and p. papatasi, the vector (nadim and aflatoonian 1995, sharifi et al. 1997, aghasi and sharifi, 2003, oshaghi et al. 2008), although, zcl cases have been reported predominantly from southern and central iran (akhavan et al. 2007, rassi et al. 2008, parvizi et al. 2010, yaghoobi-ershadi et al. 2010). epidemics of cl have been occurred after the earth quake of december 2003, where the number of recorded cases increased to *corresponding author: prof iraj sharifi, e-mail: iraj.sharifi@yahoo.com 8 j arthropod-borne dis, june 2013, 7(1): 8–14 mr aflatoonian et al.: the emergence of … http://jad.tums.ac.ir published online: june 10, 2013 almost 4folds two years following the earth quake (sharifi et al. 2011). several risk factors such as human behaviors, ecological disturbances, environmental changes, and population displacement, due to natural disasters and human intervention influence on the emergence and spread of acl (ashford 2000, patz et al. 2000, desjeux 2001). the incidence of cl cases has been significantly increased. simultaneously, local health authorities detected cl cases in these villages of bam district. the selection of theses villages was based on the newly reported cl cases by passive case detection in early 2010. there has not previously been report of any case of cl from these areas. during june to august in 2010 the cl cases have been occurred and a survey team was appointed to carry out an epidemiological investigation and to identify the causative agent. the objective of this study was to assess the magnitude of a newly emerged focus in southern villages of bam district, kerman province, southeastern iran. due to different strategic measures between acl and zcl this study is highly needed for planning a suitable future control method. materials and methods study area this study was carried out in nezamshahr county, 50 km south to the city of bam, the center of bam district, kerman province, southeastern iran. the altitude is 1032 meters above sea level and the main crops are palm trees, orchard of oranges and alfalfa. the county is hot in summer (40–45 ○c) and rather moderate in winter (15–25 ○c). the yearly rainfall is about 40mm with relative humidity of 25%. sampling and population this work was carried out as descriptive cross-sectional study from june to august 2010. a census survey of 5544 individuals was carried out by house-to-house visits. diagnosis and culturing a list of households was obtained from the bam health center. a team including an experienced physician, two health assistants and a driver performed interviewing and physical examination. a whole body was examined for the presence of active lesions or scars. suspected active lesions were scraped with a sterile blade. a questionnaire was completed for each individual, recording age, sex, the location and number of the cl lesions, place of residence and the year of contraction. the samples smeared on to glass slides, fixed in methanol, stained using giemsa and examined by a light microscope for presence of amastigotes. at the same time, samples from 15 patients were inoculated into novy–macneal–nicolle (nnn) culture media, transported on ice to kerman and leishmaniasis research centre for further characterization of the isolates by pcr. the culture media were maintained at 24±1 ○c for 7days, then transferred into rpmi 1640 medium (gibco, uk) containing 15% heat inactivated fetal calf serum (fcs), penicillin (200 units/ml) and streptomycin (200 mg/ ml) and incubated at 24±1 ○c. cultures were checked weekly for the growth of promastigotes for a period of 4 weeks. of 26 samples we could extract dna from 15 isolates. the remaining 11 samples were either culture negative or lost their viability through being transferred from the rural health clinic to school of medicine in kerman. although, the direct smear preparations were available, no attempt was made to scrape and extract dna from these smears, since we intended to identify only a representative sample. oral consent of the inhabitants was obtained. the infected subjects received proper medication free of charge. the data were entered into a computer and spss ver. 15 and χ2test were used to determine any significant difference between disease prevalence and demographic characteristics. statistical significance was at p< 0.05. 9 j arthropod-borne dis, june 2013, 7(1): 8–14 mr aflatoonian et al.: the emergence of … http://jad.tums.ac.ir published online: june 10, 2013 molecular characterization dna was prepared from 15 randomly selected clinical isolates. the extraction protocol followed relatively the method described elsewhere (mahboudi et al. 2001). a pair of primers, upstream (5'tcgcagaacgcccctac c3') and downstream (5'aggggttggtgt aaaataggc3') specific for conserved sequences of kdna of leishmania species was used. the differentiation was based on the size of the products. two leishmania species provided amplified fragments of about 800 bp for l. tropica and 620 bp for l. major. l. tropica (mhom/ir/02/mash2) and l. major (mrho/ ir/75/er), kindly provided from tehran university of medical sciences were used as positive and distilled water as negative controls. the pcr was carried out applying a stepup program as follows, initial denaturation for 3 min at 95 ○c, followed by 30 cycles of 94 ○c for 1 min, 62 ○c for 1 min at 72 ○c, with a final extension for 7 min at 72 ○c. results a total of 5544 inhabitants aged 1–96 years (mean, 26.3 years, sd±18.3), comprising 2647 females (47.8%) and 2897 males (52.2%) were interviewed and physically examined for presence of active lesions or scars (table1). in both sexes most of the individuals (24.4%) were in the age group 21–30 years and the lowest (12.5%) in 31–40 years. overall, 1.2% of the population had lesions, 0.5% active and 0.7% scars (table 2). there was a significant difference between females (1.7%) and males (0.8%) in terms of the prevalence of active lesions and scars (p< 0.005). the age distribution of the cl infection is presented in fig. 1. all age groups were affected, although the number of affected subjects was generally low and some increase was observed in the age group 11-20 years, though with no significant difference. most of the lesions were on the face (37%) followed by legs (31%), hands (27%) and others (5.0%). the majority had one lesion (88%), and 6% had 2 or ≥3 lesions each. the average number of lesions was 1.2, equally distributed among the sex. the households had frequent history of travelling to the city of bam and the lesions were merely restricted to the endogenous inhabitants in this locality. there are 4 main villages in this county; taraz was the most infected (29.3%), followed by nezamabad (28.4%), bagh-e-balla (25.5%) and momenabad (16.8%). the first case started as sporadic during the winter of 2005, however, the cases increased significantly, there after. random selection of 15 isolates by pcr method indicated l. tropica the sole causative species (fig. 2). table 1. distribution by sex and age of 5544 inhabitants examined for cutaneous leishmaniasis in southern villages of bam, southeastern iran, 2010 age (yr) female male total no. percent no. percent no. percent ≤10 565 21.3 592 20.4 1157 20.9 11-20 586 22.1 630 21.7 1216 21.9 21-30 615 23.2 737 25.5 1352 24.4 31-40 325 12.3 370 12.8 695 12.5 >40 556 21.1 568 19.6 1124 20.3 total 2647 100.0 2897 100.0 5544 100.0 10 j arthropod-borne dis, june 2013, 7(1): 8–14 mr aflatoonian et al.: the emergence of … http://jad.tums.ac.ir published online: june 10, 2013 table 2. distribution of anthroponotic cutaneous leishmaniasis by sex and type of lesion in southern villages of bam, southeastern iran, 2010 active lesion scar sex no. / percent no. (percent) no. (percent) female 44/1.7 16(0.6) 28(1.1) male 23/0.8 10(0.3) 13(0.5) total 67/1.2 26(0.5) 41(0.7) fig. 1. distribution of anthroponotic cutaneous leishmaniasis by age in southern villages of bam, southeastern iran, 2010 fig. 2. gel electrophoresis of the leishmania speciesspecific pcr products using kdna marker extracted from promastigotes grown in culture media. lane 1: 100bp ladder marker, lane2: negative control, lane3: leishmania major positive control, lane4: leishmania tropica positive control, lane 5, 6, 7, 8, 9: leishmania tropica isolates obtained from the cases with anthroponotic cutaneous leishmaniasis in southern villages of bam, southeastern iran 11 j arthropod-borne dis, june 2013, 7(1): 8–14 mr aflatoonian et al.: the emergence of … http://jad.tums.ac.ir published online: june 10, 2013 discussion cutaneous leishmaniasis is still a major health problem in iran, where mainly nonhealing forms of leishmaniasis recidivans and non-responsive clinical forms to pentavalent animony drugs are present (esfandiarpour et al. 2007, sharifi et al. 2010). the disease has a dynamic epidemiology with a complex life cycle and diverse transmission pattern. it can change under the influence of climate, ecology, human behavior, presence of suitable vectors and reservoirs (ashford 2000, patz et al. 2000, desjeux 2001). these factors singly or in combination have been accompanied by global increases in morbidity and mortality from emergent leishmania species (patz et al. 2000, desjeux 2001). the prevalence data showed that all age groups were affected, which indicated that this emergence was a new occurrence among a non-immune population. in addition the data suggest that females were more than twice susceptible (odd ratio= 2.1) to the infection than males. the households, frequently travel to the city of bam, where they contract the infection, however, there is no clear explanation for such a sex distribution. it might be due to individual risk factors and more exposure of females to the source of infection (desjeux 2001, fazaeli et al. 2009). the clinical features of the cl cases in terms of the number and the location of the lesions observed in this study are consistent with those of acl reported previously from the city of bam (nadim and aflatoonian 1995, sharifi et al. 1998a, aflatoonian and sharifi 2006) or elsewhere (zahraei-ramazani et al. 2007). more over molecular characterization of the pcr of a number of isolates proved that l. tropica was responsible for this emergence. the devastating earthquake of bam in december 2003 demolished almost 90% of the health infrastructures, sacrificed 30000 and left million tons of construction and raw bricks around the houses. although, the health conditions and infrastructures have been considerably improved, but various risk factors are still around, creating a suitable condition for vector breeding and transmission of the causative agent. in spite of remarkable efforts and implementation of various approaches to reduce transmission and control the disease unfortunately, the cl cases have been increased to epidemic proportion after the earthquake especially during 2006–2008 in the city of bam. in bam district, cl control has been integrated with that of other infectious diseases. the disease has not been reported from this county before. occupation is mainly restricted to farming and agricultural activities. movement of the villagers, both men and women back and forth to the city of bam, more frequently after the earth quake, where the disease is highly endemic, for various purposes is common. therefore, due to presence of a suitable anthropophyllic vector (aghasi and sharifi 2003), the villagers become a highly sensitive reservoir for transmission of acl endemic to their former home grounds. the current expansion of villages and urbanization constitute other contributory factors (patz et al. 2000, desjeux 2001). similar risk factors have contributed to the emergence of new acl foci in sudan (el-safi and peters 1991), morrocco (guessous-idrissi et al. 1997, ramaoui et al. 2008), israel (jacobson et al. 2003), pakistan (kolczinski et al. 2004) and new zcl foci in iran and abroad (ashford et al. 2000, yaghoobiershadi et al. 2001, fazaeli et al. 2009, razmjou et al. 2009, sharifi et al. 2011). in conclusion, the presence of non-immune population along with suitable ecological conditions could induce a new emerging focus of acl for human, beyond the original focus. further epidemiological studies are required to identify the main vectors and strains of the leishmania species involved in this focus. 12 j arthropod-borne dis, june 2013, 7(1): 8–14 mr aflatoonian et al.: the emergence of … http://jad.tums.ac.ir published online: june 10, 2013 acknowledgements this work was approved by the vice chancellor for research and received financial support from the kerman university of medical sciences and health services (project no. 89/24). we are grateful to the 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zahraei-ramazani ar, abdoli h, akhavan aa, aghasi m, arandian mh, ranjbar aa (2010) sand fly surveillance within an emerging epidemic focus of cutaneous leishmaniasis in southeastern iran. iran j arthropod-borne dis. 4: 17–23. zahraei-ramazani ar, yaghoobi-ershadi mr, mokhtari ar, akhavan aa, abdoli h, arandian mh (2007) anthroponotic cutaneous leishmaniasis in nonendemic quarters of a central city in iran. iranian j publ health. 36: 7–11. 14 j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 349 http://jad.tums.ac.ir published online: january 06, 2016 original article wolbachia endobacteria in natural populations of culex pipiens of iran and its phylogenetic congruence mohsen karami 1, *seyed hassan moosa-kazemi 1, *mohammad ali oshaghi 1, hasan vatandoost 1, mohammad mehdi sedaghat 1, ramazan rajabnia 2, mostafa hosseini 3, naseh maleki-ravasan 4, yousef yahyapour 2, elaheh ferdosishahandashti 5 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2infectious diseases & tropical medicine research center, babol university of medical sciences, babol, iran 3department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran 4malaria and vector research group, biotechnology research center, pasteur institute of iran, tehran, iran 5department of advanced technologies in medicine (satim), medical biotechnology,tehran university of medical sciences, tehran, iran (received ١٥ apr 2015; accepted 3 oct 2015) abstract background: wolbachia are common intracellular bacteria that infect different groups of arthropods including mosquitoes. these bacteria modify host biology and may induce feminization, parthenogenesis, male killing and cytoplasmic incompatibility (ci). recently wolbachia is being nominated as a bio-agent and paratransgenic candidate to control mosquito borne diseases. methods: here we report the results of a survey for presence, frequency, and phylogenetic congruence of these endosymbiont bacteria in culex pipiens populations in northern, central, and southern parts of iran using nested-pcr amplification of wsp gene. results: wolbachia dna were found in 227 (87.3%) out of 260 wild-caught mosquitoes. the rate of infection in adult females ranged from 61.5% to 100%, while in males were from 80% to 100%. the blast search and phylogenetic analysis of the wsp gene sequence revealed that the wolbachia strain from iranian cx. pipiens was identical to the wolbachia strains of supergroup b previously reported in members of the cx. pipiens complex. they had also identical sequence homology with the wolbachia strains from a group of distinct arthropods including lepidopteran, wasps, flies, damselfly, thrips, and mites from remote geographical areas of the world. conclusion: it is suggested that wolbachia strains horizontally transfer between unrelated host organisms over evolutionary time. also results of this study indicates that wolbachia infections were highly prevalent infecting all cx. pipiens populations throughout the country, however further study needs to define wolbachia inter-population reproductive incompatibility pattern and its usefulness as a bio-agent control measure. keywords: culex pipiens, wolbachia, cytoplasmic incompatibility, nested-pcr, iran introduction mosquitoes including culex pipiens complex with global distribution are vectors of arboviral pathogens and parasites such as west nile, st louis, sindbis, wuchereria bancrofti, dirofilaria immitis, d. repens, plasmodium relictum, and p. gallinaceum (vinogradova *corresponding author: dr seyed hassan moosakazemi, email: moosakazemii@tums.ac.ir, dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 350 http://jad.tums.ac.ir published online: january 06, 2016 2000, pawelek et al. 2014). among the ‘neglected’ mosquito-borne diseases, lymphatic filariasis continues to be a hazard to over a billion people in 83 countries (o'connor et al. 2012). culex pipiens is a species complex and comprise cx. quinquefasciatus and cx. pipiens in south and north america, asia and africa, as well as cx. globocoxitus and cx. australicus in australia (farajollahi et al. 2011). culex pipiens and cx. quinquefasciatus are distributed in most parts of iran ranging from north to south (zaim 1986, azarihamidian 2007, nikookar et al. 2010, khoshdel-nezamiha et al. 2013, banafshi et al. 2013, dehghan et al. 2013, 2014). the raising of resistance to current insecticides by insect vectors (hemingway and ranson 2000), the progress of drug resistance in parasites (talisuna et al. 2004) and lack of clinical cures or vaccines for many vector borne diseases have led researchers to develop urgently new and advanced approaches to control of the diseases. paratransgenesis, as a new approach, direct towards reducing vector competence through genetically manipulated symbionts (coutinhoabreu et al. 2010). transformed symbionts are distributed across the insect population via transovarial or transstadial transmision routs (durvasula et al. 1997, chavshin et al. 2012, 2014, 2015, maleki-ravasan et al. 2015). symbionts currently aimed at in paratransgenesis include fungi (rasgon 2011), symbiont bacteria of triatomine bugs (durvasula et al. 1997, durvasula et al. 1999, durvasula et al. 2008), tsetse flies (cheng and aksoy 1999), sandflies (maleki-ravasan et al. 2015) and mosquitoes (favia et al. 2007, chavshin et al. 2014), and densoviruses infecting an. gambiae and ae. aegypti mosquitoes (ward et al. 2001, ren et al. 2008). recently, paratransgenesis have been successfully employed to reduce vector competence of the triatomine bug, rhodnius prolixus, vector of trypanosoma cruzi, the causative agent of chagas disease (durvasula et al. 1997), and anopheles gambiae and an. stephensi, two main malaria vectors (rasgon 2011, wang and jacobs-lorena 2013). these data showed that the genetically manipulated symbionts could interfere with the development of the parasites in the vectors and provide the groundwork for the use of genetically modified symbionts as a potent tool to battle vector borne diseases. the bacterium of wolbachia pipientis is an intracellular organism and inherited maternally. it is established in more than 20% of all insects and a vast majority of other arthropods as well as filarial nematodes (werren 1997a, dobson 2004, lo and evans 2007). recent studies imply that 20–76% of investigated insects give shelter to wolbachia (hilgenboecker et al. 2008), as well as many arachnids, terrestrial crustaceans, and mites (cordaux et al. 2001, gotoh et al. 2003, rowley et al. 2004). this unique endosymbiont species was originally found in cx pipiens but later molecular studies have discovered a number of phylogenetically diverse strains within the species (lo et al. 2007). this endosymbiont bacterium has significant effects on its arthropod hosts and nominated as a bioagent to control important arthropod pests. wolbachia is the cause of various modifications in insect reproductive arrangement, comprising male-killing, feminization, cytoplasmic incompatibility (ci), and parthenogenesis (werren et al. 2008). when ci occurs, sperm and eggs are not able to produce feasible progeny (werren 1997b, clark et al. 2003, beckmann and fallon 2013). infected females relative to uninfected ones, participate more in offspring production, which permit wolbachia to take up by all of host individuals even if it cases fitness costs (field et al. 1999). the bacterium also can be used as a vector for delivering desirable genetic modifications in insect populations (werren 1997b). as reviewed by werren (1997a), wolbachia have potential roles in j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 351 http://jad.tums.ac.ir published online: january 06, 2016 the rapid speciation of their hosts. also as a pandemic endosymbiont, wolbachia can be recruited to control of a large number of human infectious diseases (slatko et al. 2014). in filarial nematodes comprising wuchereria bancrofti, brugia malayi, brugia timori and onchocerca volvulus that infect humans, wolbachia are obligated for proper development, fertility and survival, whereas in arthropods, although they can affect development and reproduction, but are not required for host survival. so wolbachia have been a target for drug discovery against filariasis. in vivo/ vitro experiments indicate that antibiotics such as doxycycline and tetracycline can kill both adults and immature nematodes through depletion of wolbachia (foster et al. 2013, taylor et al. 2014). it is also shown that, wolbachia spp where naturally infected or artificially introduced into vector population can affect and decrease the mosquitoes competence carrying of viruses, such as yellow fever, chikungunya, dengue, west nile, as well as ones transmitting of the plasmodium protozoans and filarial nematodes (bourtzis et al. 2014). due to the fact that wolbachia is an obligate endosymbiont that cannot be cultured exterior their hosts, recognition of infection has been based vastly on amplification of wolbachia dna using pcr. until now a number of loci including wsp, 16s rdna, coxa, ftsz, hcpa, gatb, groel, fbpa, glta and dnaa genes have been studied and evaluated in the phylogenetic studies (zhou et al. 1998, ravikumar et al. 2011). the sequences from wolbachia surface protein (wsp) gene were extremely mutable and could be used to recognition and to re solve the phylogenetic relationships of different wolbachia strains (zhou et al. 1998). in the present study we used a nested pcr assay to detect and investigate the prevalence of wolbachia endobacteria using the partial genomic nucleotide sequence of wsp gene in twelve field populations of culex pipiens in various geographical regions across iran ranging from north to south. results of this study will provide fundamental background for understanding ecology, distribution, and potential utility of wolbachia as bio-control agent of cx. pipiens. materials and methods study areas the study was conducted in twelve locations belong to three provinces of iran, mazandaran in the north (six locations), isfahan in the center (3 locations) and hormozgan in the south (3 locations) of the country (fig. 1). live larvae, pupae, and adult mosquitoes were collected from different biotypes including plane, jungle, riverside, rice field and human dwellings. mosquito collection adult mosquitoes were collected in human dwellings monthly for a period of five months (june to late october, 2014) by handcatch collection method using mouth aspirator. also live larvae and pupae were collected from mosquito breeding sites locating in plane, jungle, riverside and rice field using dipping method, transferred to insectary, and allowed them to grow till adult emergence. adult specimens were keyed to species level using standard morphological keys (zaim 1986, azari-hamidian and harbach 2009). the male and female mosquito specimens belong to cx. pipiens were selected and stored individually at -20 ºc for further molecular investigations. double distilled water and mix of 10 adult male and female specimens of anopheles maculipennis were collected from mazanderan province and used as negative controls. dna extraction and pcr totally 260 (120 males and 140 females) cx. pipiens specimens originated from different biotopes from north to south of iran j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 352 http://jad.tums.ac.ir published online: january 06, 2016 were randomly subjected to genomic dna extraction. genomic dna of an. maculipennis ss was extracted and used in all pcr assays as negative control. total dna of individual mosquitoes was extracted using collins dna extraction method (collins et al. 1987). previously a pcr based method for the classification of wolbachia has been described (zhou et al. 1998). in that method, group-specific wsp pcr primers have been used to identify wolbachia strains without the need to clone and sequence individual wolbachia genes. here in detection of wolbachia infection in the mosquitoes was performed by a nested-pcr assay on the basis of zhou introduced primers. initially, a set of primers including 81f: 5'–tggtcca ataagtgatgaagaaac–3' and 691r: 5'– aaaaattaaacgctactcca–3' were recruited to amplify 632 bp of partial sequence of the wsp gene. the pcr product of the first step was applied as a template for second step. in the second step, another pairs of the primers, 183f: 5'–aaggaaccg aagttcatg–3' and 691r: 5'–aaaaa ttaaacgctactcca–3', were used to amplify a 501 bp fragment. the pcr amplification was performed using maxime pcr premix kit (i-taq) cat. no. 25026 in 20 μl reaction mixtures containing 2.5 μl of 10 μm both forward and reverse primers and 5 μl (~0.5 μg) of genomic dna and 2.5 μl pcr product for the first and second step of nested-pcr reactions respectively. an individual specimen of anopheles maculipennis s.s. was used as dna extraction and pcr negative controls. the pcr conditions were set as an initial denaturation at 95 °c for 5 min, followed by 35 cycles of denaturation at 94 °c for 1 min, annealing at 55 °c for 1 min, and extension at 72 °c for 1 min, followed by a final extension at 72 °c for 7 min. pcr products were visualized on a 1% agarose gel containing ethidium bromide and using an uv transilluminator. wsp gene sequencing and analyzing representative specimens with clear and sharp wsp gene amplicons of the twelve cx. pipiens populations were sequenced via the same amplification primers by bioneer company (s. korea). the consensus of confident sequences was analyzed using ncbi (nucleotide collection) database. the wsp gene sequences determined in this study were subjected to molecular phylogenetic analysis together with 44 wsp gene sequences of wolbachia from various arthropod host species retrieved from the genbank database (table 1). a multiple alignment of the wsp sequences was generated by the program package clustal w (thompson et al. 1994). phylogenetic trees were constructed using the neighbor-joining method embedded in mega5 software. bootstrap tests were performed with 1,000 replications. statistics analyzing wolbachia infection data in culex pipiens specimens were analyzed using spss 22.0 and chi square (χ2) test to make comparisons and evaluate variation in infection rates between the males and females and among the twelve populations. the p-value more than 5% was considered as significant. results wolbachia detection in cx. pipiens the infection of wolbachia in different cx. pipiens populations was detected by the nested-pcr assay using wsp gene. the amplicons of first and second runs of nestedpcr assay were ~ 650 and 500 bp respectively (fig. 2). wolbachia infection rate results of the study demonstrated that in total, 227 (87.3%) out of 260 individual adult mosquitoes belonged to 12 distinct populations were positive against wsp gene (table 2). all the infected mosquitoes were j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 353 http://jad.tums.ac.ir published online: january 06, 2016 found to harbor a single wpip strain. infection rate in adult females and males were 61.5–100% and 80–100% respectively. there were no significant differences between total infection rates of either sexes (female= 89.2%, male = 85.7%, df= 1, p> 0.05) or zones (df= 3, p> 0.05). wolbachia wsp sequences seven nested–pcr products the wsp gene of wolbachia found in different iranian populations of cx. pipiens were successfully sequenced and submitted to genbank (accession numbers (ans): km401551–7). the nested primers we used were only able to amplify fragments from infected specimens and not from uninfected an. maculipennis ss hosts. the sequences were a-t rich (61%) with only 39% gc content. the blast results indicated that all the wsp sequences of wolbachia detected from the iranian cx. pipiens were 100% identical to each other and to the wolbachia strains found in other members of the cx. pipiens complex including cx. pipiens, cx. pipiens form molestus, cx. pipiens (syn. pallens), and cx. quinquefasciatus from remote geographical areas of the world (table 3). since the wolbachia strain that infects cx. pipiens complex belongs to pip group of b supergroup (wpipb) (zhou et al. 1998, pidiyar et al. 2003), we can conclude that the wolbachia strains from iranian cx. pipiens specimens belongs to wpipb strain. in addition, the sequences of wolbachia wsp gene of iranian cx. pipiens were 100% identical to the wsp gene of wolbachia strains found in divers insect or arthropod groups particularly to the order of lepidoptera comprising 18 different butterfly and moth species, as well as to wasps, thrips, damselflies, aedes mosquito, threestriped fruit fly, leaf-mining fly, and mite. these wolbachia host species belong to geographically remote regions of asian, european, and african countries (table 3). a comparison of the wsp sequences from the arthropod hosts showed up to 30.67% genetic diversity between taxa, in which the wsp sequence from bedbug was the most diverged one. phylogenetic analysis for phylogenetic analysis a subset of the wolbachia strains identified in this study were combined with a 44 available sequence data of other wolbachia strains from genbank. these sequences belonged to twenty different arthropod hosts of wolbachia including mosquitoes (culex and aedes), fruit flies, blow flies, sand flies, tsetse flies, leaf mining flies, bed bugs, thrips, damselflies, plant hoppers, crickets, termites, butterflies, moths, wasps, ants, beetles, pill woodlouse, spiders, and mites (table 1). phylogenetic tree was constructed using neighbor-joining method, based on the 445–511 bp of wsp sequences (fig. 3). the length variation between sequence data was due to insertion or deletion (indels) events. we also used dirofilaria immitis wsp sequence as an out-group in the analysis. phylogenetic analysis showed that wolbachia strains from iranian cx. pipiens specimens were clustered with wolbachia strains of other members of the cx. pipiens complex such as cx. pipiens, cx. pipiens (syn. pallens), cx. pipiens form molestus and cx. quinquefasciatus (fig. 3). they also associated with wolbachia strains found in distinct groups of arthropods not obtained from the same insect genus, family, or even order. in other word, wolbachia strains obtained from the same insect genus or families were not clustered into distinct groups but were scattered throughout the phylogenetic tree. except for the congenic clusters of mosquitoes, sand flies, and tsetse flies, there were no other congenic clusters indicating little congruence between wolbachia phylogeny and host systematics. the phylogenetic analysis revealed six main clades for the wsp sequences of wolbachia strains analysed (fig. 3). the first clade was comj arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 354 http://jad.tums.ac.ir published online: january 06, 2016 posed of all mosquitoes (eight culex spp and two aedes spp) and ten wsp sequences from lepidopteran, wasp, thrips, damselfly, threestriped fruit fly, leaf-mining fly, leaf beetle, and mite, all belonged to the known supergroup b of wolbachia. the second lineage was composed of nine wsp sequences from blowfly, plant hopper, cricket, moth, wasp, fire ant, flour beetle, and mite. eleven wsp sequences from fruit flies, sand flies (2 species), tsetse flies (2 species), termite, moth, wasps (2 species), ant, and spider, constituted an isolated lineage. the wsp sequences from one of each wasp, plant hopper, and moth formed a distinct clade. most of strains of second and third clades belong to the known supergroup a of wolabachia. notably the bedbug and one termite wsp sequences associated together and formed a well-defined clade, and finally pill wood louse constituted a diverse clade well separated from other five clades. except for four nodes with 57– 71% support, all of the nodes had very high (82–100) bootstrap support values (fig. 3). fig. 1. map of study areas for collection of culex pipiens specimens in iran. nos, 1–2: ramsar, 3–4: amol, 5–6: behshahr in mazandaran province, 7: vinicheh, 8: dizicheh, 9: dorcheh in isfahan province, 10: hormodar, 11: siahoo, and 12: shamil in hormozgan province fig. 2. species-specific nested-pcr products (~ 500 bp) of wolbachia wsp gene of culex pipiens specimens. lanes: m, 1 kbp molecular weight marker (fermentas), 1–2: mazandaran provine, 3: isfahan provine, 4–5: hormozgan provine, 6: anopheles maculipennis as negative control j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 355 http://jad.tums.ac.ir published online: january 06, 2016 table 1. description of wolbachia strains used for phylogenetic analysis in this study no wolbachia strain host common name accession number references 1 wpip/b culex pipiens mosquito km401552 this study 2 wpip/b cx. pipiens mosquito km401553 this study 3 wpip/b cx. pipiens mosquito km401556 this study 4 wpip/b cx. pipiens mosquito jx474753 direct submission 5 wpip/b cx. pipiens (syn. pallens) mosquito af216860 direct submission 6 wpip/b cx. pipiens form molestus mosquito hg428761 (pinto et al. 2013) 7 wpip/b cx. quinquefasciatus mosquito af020060 (zhou et al. 1998) 8 wpip/b cx. quinquefasciatus mosquito kj140126 direct submission 9 walbb/b aedes albopictus mosquito af020059 (zhou et al. 1998) 10 wpip/b ae. punctor mosquito aj311040 (ricci et al. 2002) 11 w alba/a ae. albopictus mosquito af020059 (zhou et al. 1998) 12 wno/b drosophila simulans fruit fly af020074 (zhou et al. 1998) 13 wmel/a d. melanogaster fruit fly af020072 (zhou et al. 1998) 14 waus/a glossina austeni tsetse fly af020077 (zhou et al. 1998) 15 wmors/a g. morsitans morsitans tsetse fly af020079 (zhou et al. 1998) 16 n.s protocalliphora sialia blow fly dq842482 (baldo et al. 2006) 17 wpak-b1 hydrellia pakistanae leaf mining fly af217718 (jeyaprakash and hoy, 2000) 18 papa01/a phlebotomus papatasi sand fly eu780683 (parvizi et al. 2013) 19 turk 07 ph. mongolensis sand fly kc576916 (parvizi et al. 2013) 20 wcon/b tribolium confusum flour beetle af020083 (zhou et al. 1998) 21 n.s chelymorpha alternans leaf beetle dq842458 (baldo et al., 2006) 22 wori/b tagosodes orizicolus plant hopper af020085 (zhou et al. 1998) 23 wstri/b laodelphax striatellus plant hopper af020080 (zhou et al. 1998) 24 f cimex lectularius bed bug dq842459 (baldo et al. 2006) 25 wdei/b trichogramma deion wasp af020084 (zhou et al. 1998) 26 wtde-heb t. dendrolimi wasp jx027991 direct submission 27 wkue/a spalangia cameroni wasp af289668 direct submission 28 n.s encarsia formosa wasp dq842471 (baldo et al. 2006) 29 wnpan/a nomada panzeri red wasp kc798315 (gerth et al. 2013) 30 a solenopsis invicta fire ant dq842483 (baldo et al. 2006) 31 a formica truncorum ant af326978 (wenseleers et al. 2002) 32 wcaub/b ephestia cautella moth af020076 (zhou et al. 1998) 33 wcaua/a ephestia cautella moth af020075 (baldo et al. 2006) 34 b ostrinia scapulalis moth dq842481 (baldo et al. 2006) 35 ns eurema hecabe butterfly ab285478 (narita et al. 2007) 36 ns udaspes folus butterfly jn236179 (salunke et al. 2012) 37 ns agriocnemis femina damselfly ay173939 (thipaksorn et al. 2003) 38 ns gryllus firmus cricket dq842474 (baldo et al. 2006) 39 a incisitermes snyderii termite dq842475 (baldo et al. 2006) 40 f coptotermes acinaciformis termite aj833931 (baldo et al. 2006) 41 ns hercinothrips femoralis thrips ab245521 direct submission 42 ns nephila clavata spider ef612772 direct submission 43 ns oxyopes sertatus spider ef612771 direct submission 44 ns eriovixia cavaleriei spider dq778738 direct submission 45 ns tetranychus urticae two-spotted spider mite aj437290 direct submission 46 ns bryobia berlesei mite jn572865 (ros et al. 2012) 47 ns armadillidium vulgare pill woodlouse dq842457 (baldo et al. 2006) 48 outgroup dirofilaria immitis nematode aj252062 (bazzocchi et al. 2000) ns: not stated. j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 356 http://jad.tums.ac.ir published online: january 06, 2016 table 2. prevalence of wolbachia pipientis infection in the culex pipiens collected from north, center and south of iran, 2014 province location biotope males tested (% p +) females tested (% p+) total (% p+) mazandaran (north) amol 1 plane 10(90) 13(61.5) 74 amol 2 jungle 10(80) 10(100) 90 behshar 1 plane 10(100) 10(100) 100 behshar 2 jungle 10(90) 10(90) 90 ramsar 1 plane 10(90) 10(80) 85 ramsar 2 jungle 10(100) 14(100) 100 isfahan (center) dizicheh rice fields 10(90) 10(90) 90 vinicheh rice fields 10(80) 10(70) 75 dorcheh rice fields 10(100) 15(100) 100 hormozgan (south) shamil date groves 10(80) 13(61.5) 70 siahoo riverside 10(80) 10(90) 85 hormoodar date groves 10(90) 15(86.7) 88 total 120(89.2) 140(85.7) 87.3 (260) table 3. details of arthropods have identical wolbachia wsp sequences with the iranian culex pipiens arthropod group species accession number country reference mosquito culex pipiens cx. pipiens form molestus cx. pipiens (syn. pallens) cx. quinquefasciatus cx. quinquefasciatus cx. quinquefasciatus cx. quinquefasciatus cx. quinquefasciatus cx. quinquefasciatus aedes punctor jx474753 hg428761 af216860 kj140126 eu194487 af397413, af397412 ay462861 am999887 aj311040 turkey ns china china india india india taiwan ns italy direct submission (pinto et al. 2013) direct submission direct submission direct submission direct submission direct submission (tsai et al. 2004) (klasson et al. 2008) (ricci et al. 2002) butterfly udaspes folus hypolimnas bolina castalius rosimon eurema hecabe ypthima asterope papilio demoleus zizeeria knysna colotis amata pseudozizeeria maha leptidea sinapis pararge aegeria polygonia calbum hypolimnas bolina jn236179 jn236180 jn236182 jn236189 jn236192 jn236193 jn236194 jn236195 jn236205 kc137222 kc137224 jn093149 aj307076 india india india india india india india india india ns ns ns fiji (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (salunke et al. 2012) (russell et al. 2012) (russell et al. 2012) (kodandaramaiah et al. 2011) (dyson et al. 2002) moth corcyra cephalonica epirrita autumnata spodoptera exempta corcyra cephalonica acraea encedon kc844060 jx310335 jn656943 ay634679 aj271198 china ns tanzania china tanzania direct submission (kvie et al. 2012) direct submission direct submission direct submission wasp trichogramma chilonis t. dendrolimi t. brassicae t. dendrolimi t. japonicum tropobracon schoenobii ay311486 jx027991 af452646 dq017751 kc161917 af481194 china china china china china ns direct submission direct submission direct submission direct submission direct submission (kittayapong et al. 2003) thrips hercinothrips femoralis ab245521 japan direct submission damselfly agriocnemis femina coenagrionidae sp ay173939 kc161926 ns china (thipaksorn et al. 2003) direct submission j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 357 http://jad.tums.ac.ir published online: january 06, 2016 fruit fly bactocera diversa af295353 ns (jamnongluk et al. 2002) leaf-mining fly hydrellia pakistanae af217718) ns (jeyaprakash and hoy 2000) mite bryobia berlesei jn572865 france (ros et al. 2012) ns: not stated. fig. 3. the phylogenetic tree inferred from 445–511 bp of wsp sequences of wolbachia pipientis hosts using the neighborjoining method embedded in mega 5.0. c.p1–3 (culex pipiens from this study), c.p4 (culex pipiens), c.pm (culex pipiens form molestus), c.q and c.q2 (culex quinquefasciatus), c.pa (culex pipiens, syn.: pallens), a.a (aedes albopictus), d.m (drosophila melanogaster), d.s (drosophila simulans), g.m (glossina morsitans morsitans), g.a (glossina austeni), p.s (protocalliphora sialia), p.p (phlebotomus papatasi), p.m (phlebotomus mongolensis), t.c (tribolium confusum), ch.a (chelymorpha alternans), l.s (laodelphax striatellus), t.o (tagosodes orizicolus), c.l (cimex lectularius), t.d (trichogramma deion), t.dr (t.dendrolimi), s.c (spalangia cameroni), e.f (encarsia formosa), n.p (nomada panzeri), s.i (solenopsis invicta), , f.t (formica truncorum), e.c1–2 (ephestia cautella), o.s (ostrinia scapulalis), e.h (eurema hecabe), g.f (gryllus firmus), i.s (incisitermes snyderii), c.a (coptotermes acinaciformis), n.c (nephila clavata), ox.s (oxyopes sertatus), e.c (eriovixia cavaleriei), t.u (tetranychus urticae), a.v (armadillidium vulgare), a.f (agriocnemis femina), h.f (hercinothrips femoralis), b.b (bryobia berlesei), a.p (aedes punctor), u.f (udaspes folus), h.p (hydrellia pakistanae), and d.i (dirofilaria immitis). the bootstrap values are shown as numbers on the nodes table 3. continued… j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 358 http://jad.tums.ac.ir published online: january 06, 2016 discussion this is the first report on wolbachia infection from cx. pipiens populations of iran. in our study, 260 specimens of cx. pipiens collected from the 12 villages were individually assayed for wolbachia, and the overall rate of infection was determined to be 87.3%. this result is in agreement with previous study conducted in south west iran revealed 100 percent wolbachia infection in cx. quinquefasciatus specimens (behbahani 2012). in california, wolbachia infection frequency in cx. pipiens complex during 1999 and 2000 was 99.4% (rasgon and scott, 2003). also sunish et al. (2011) found an overall prevalence of 91.2% wolbachia infections in cx. quinquefasciatus mosquitoes from south india. study of chen et al (2013) revealed that three cx. pipiens (syn. pallens) populations of china were all infected with wolbachia. this rate was reported between 10–100% in members of cx. pipiens complex mosquitoes from the upper rhine valley in germany and cebu city in philippines (mahilum et al. 2003). in this study we found no wolbachia infection in an. maculippenis ss specimens which is in concurrence of study of rasgon and scott (2004) who tested five genera of mosquito (aedes, anopheles, culiseta, culex, and ochlerotatus) for wolbachia, and infections was only detected in members of the cx. pipiens complex. also study of kittayapong et al. (2000) detected wolbachia infection in all main disease vector genera excluding anopheles. in our study, the percentage prevalence in adult males was 80–100%, while in females were 61.5–100%. however the difference was not significant between males and females. in contrast, in the study of sunish et al (2011) the rate of wolbachia infection in females of cx. quinquefasciatus was found slightly higher than in males but like our study it was not statistically significant. this study showed no sequence variation in wsp gene of wolbachia from cx. pipiens populations across geographical regions of iran, which is similar to the results of morais et al. (2012) which showed that both cx. quinquefasciatus and cx. pipiens × cx. quinquefasciatus hybrids collected brazil and argentina were infected with a single wolbachia strain. the genetic similarity detected among wolbachia samples in the culex mosquitoes from geographically scattered regions may be explained by either wolbachia host-endosymbiont specificity (werren et al. 2008) or recently wolbachia infection in culex populations (morais et al. 2012). high sequence homology and close phylogenetic relationships of wolbachia strains from mosquitoes, spider, wasp, mite, damselfly, butterfly, thrips, fruit fly, and leaf mining fly indicate that wolbachia endosymbionts not only are maternally transmitted through host generations by vertical transmission but also horizontally transfer between unrelated host organisms (i.e. shift host species or “jumping”) (van meer et al. 1999, baldo et al. 2005). although the mechanisms of jumping are still unclear, it is believed that parasitoids may involve (heath et al. 1999, huigens et al. 2000, noda et al. 2001, kikuchi and fukatsu 2003). recombination in wsp gene of wolbachia strains has been evidenced by other researchers (werren and bartos 2001, jiggins 2002, reuter and keller 2003). for example, werren and bartos (2001) reported recombination within supergroup b, occurring between the two wolbachia strains of a parasitoid wasp and the fly it parasitizes. more recently it is shown that hypervariable regions of wsp gene of wolbachia strains have got a complex mosaic structure, suggesting a clear intragenic recombination of segments among several divergent strains, both within and j arthropod-borne dis, september 2016, 10(3): 349–365 m karami et al.: wolbachia endobacteria … 359 http://jad.tums.ac.ir published online: january 06, 2016 between the arthropod supergroups (baldo et al. 2005). the phylogenetic analysis of wsp sequences of wolbachia from 20 different arthropod hosts scattered the sequences into five main clades that in some parts, topographically matched well with the tree of zhou et al. (1998). based on wolbachia ftsz gene sequences, two major supergroups a and b were reported within the wolbachia strains (werren and jaenike 1995) where the type strain from cx. pipiens was placed within supergroup b. in the tree we obtained in this study, two main clades represent supergroups a and b (fig. 3). in addition to the wolbachia strains from mosquitoes, the strains from spider, wasp, mite, damselfly, butterfly, thrips, fruit fly, and leaf mining fly also placed in supergroup b. interestingly the wolbachia strain from bedbug was associated with the one from termite of supergroup f or h. as reviewed by lo et al. (2007), currently the genus wolbachia was divided into eight taxonomic supergroups (a to h) where a and b are the two major groups established in arthropods, c and d are found in filarial nematodes, e infecting springtails and f contains wolbachia bacteria that infect termites and filarial species. supergroup g and h were reported in spiders and termites respectively. in addition other divergent lineages, such as those from various flea species and the filarial nematode dirofilaria repens, might be added to the list of supergroups. therefore, as more sequence information becomes available the number of clades, groups, or supergroups might be increased. for example, in our analysis the wolbachia from woodlouse construct a single clade and might be considered as a separate clade. conclusion in this study we found a single wolbachia strain from cx. pipiens populations across the country. although it is suggested that a large set of compatible wolbachia strains are always locally dominate within mosquito populations (duron et al. 2011), however, several studies have showed that some wpip strains are reciprocally incompatible but also that some others, although genetically distinct, are fully compatible (duron et al. 2006, duron et al. 2007, atyame et al. 2011). therefore, it is worth to test cytoplasmic incompatibility (ci) between the iranian populations. in case of having ci, it can be used as a form of sterile-insect technique (sit), to suppress, to replace, or to reduce the survival of mosquito populations and thereby control them or reduce their ability to transmit the infection (townson 2002). acknowledgements tehran university of medical sciences (tums) financially supported this work (grant no. 22738). also it is noteworthy that this research has been done by support of the babol university of medical sciences (bums). special thanks to roghayeh pourbagher, seyedeh narges mousavi kani, zeinab abedian, and seyed mohsen aghajanpour mir for helping in cellular and 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of iran (diptera: culicidae). mosq syst. 18: 233–245. zhou w, rousset f, o'neil s (1998) phylogeny and pcr-based classification of wolbachia strains using wsp gene sequences. proc biol sci. 265(1395): 509–515. j arthropod-borne dis, sm omrani et al.: microsporidium infecting … short communication microsporidium infecting anopheles superpictus (diptera: culicidae) larvae *seyed-mohammad omrani 1, seyedeh-fatemeh moosavi 2, kourosh manouchehri 2 1department of medical parasitology, school of medicine, shahrekord university of medical sciences, shahrekord, iran 2cellular and molecular research center, shahrekord university of medical sciences, shahrekord, iran (received 7 june 2014; accepted 4 jan 2015) abstract background: microsporidia are known to infect a wide variety of animals including mosquitoes (diptera: culicidae). in a recent study on the mosquito fauna of chahar mahal and bakhtiari province, at the central western part of iran, a few larvae of anopheles superpictus were infected with a microsporidium-resembled microorganism. current investigation deals with the identification of the responsible microorganism at the genus level. methods: fresh infected larvae were collected from the field. after determining the species identity they were dissected to extract their infective contents. wet preparations were checked for general appearance and the size of the pathogenic microorganism. fixed preparations were stained with geimsa and ryan-blue modified trichrome techniques to visualize further morphological characters. the obtained light microscopy data were used in the identification process. results: the infected larvae were bulged by a whitish material filling the involved segments corresponding to a microsporidium infection. bottle-shaped semioval spores ranged 4.33±0.19×2.67±0.12 and 4.18±0.43×2.45±0.33 micron in wet and fixed preparations, respectively. they were mostly arranged in globular structures comprised of 8 spores. these data was in favor of a species from the genus parathelohania in the family ambliosporidae. conclusion: this is the first report of a microsporidium infection in an. superpictus. the causative agent is diagnosed as a member of the genus parathelohania. further identification down to the species level needs to determine its ultrastructural characteristics and the comparative analysis of ss rrna sequence data. it is also necessary to understand the detail of the components of the transmission cycle. keywords: microsporidia, mosquito, parathelohania, anopheles superpictus, iran introduction mosquitoes are of major health concern not only because of their irrititating bites but also because of the capability to transmit lifethreatening diseases to human being and his livestock (tolle 2009). while this is so, the effective control of mosquitoes is still a farreaching dream (ranson et al. 2010). apart from the resistance problem, one of the main obstacles is the scarcity of control measures, which are specific and also safe to use (kamareddine 2012). in this context, the discovery of a new event on the natural enemies of mosquitoes may be a promising occurrence in the field of finding environmental-friendly control measures to overcome these troublesome insects. microsporidia are single celled intracellular eukaryotic fungi parasitizing all groups of animals (corradi et al. 2009, wittner et al. 1999). to date more than 1300 species of these microorganisms have been described in the literature (vávra et al. 2013). while 14 species are known to infect humans, at least 150 more species have been recognized to parasitize 14 genera of mosquitoes (canning 2001, andreadis 2007). based on these evi*corresponding author dr seyed mohammad omrani, e-mail: omranism@skums.ac.ir j arthropod-borne dis, sm omrani et al.: microsporidium infecting … dences, it has been stated that all of the mosquito species could be possibly the host of at least one microsporidium agent (andreadis 2007). microsporidia are transmitted horizontally and vertically between and within hosts by means of a specific form called spore. the spores are very small and characterized by their unique invading behavior and the exclusive ultrastructure (wittner et al. 1999). while the monomorphic forms (eg anncaliia and vavraia) produce only one spore type, the polymorphic ones (eg amblyospora and parathelohania) have a complex life cycle and produce different types of spores. the latter are generally more host specific and commonly there is an obligatory intermediate copepod host in their life cycle (becnel et al. 2005). microsporidia can be recognized by light microscopy techniques such as geimsa and different versions of trichrome staining (garcia 2002). further verification can be achieved by transmission electron microscopy (wittner et al. 1999). the molecular information on the ss rrna sequence is an asset and facilitates phylogenetic analysis (andreadis 2007). anopheles superpictus grassi (diptera: culicidae) is a major malaria vector in asia (zahar 1974). it has been incriminated in the stable malaria as its longevity frequently fits for the completion of the parasite sporogonic cycle (macdonald 1957). this mosquito is also known to transmit malaria in iran with a widespread distribution (saebi 1987, edrissian 2006). recently, in a study on the mosquito fauna of chahar mahal and bakhtiari province, at the central western part of iran, a few lactophenol-preserved larvae of an. superpictus were found disfigured by the swelling of their thorax and/or first abdominal segments (fig. 1). close observation showed that the multiple packets of a tiny microorganism have been infiltrated in the affected areas. this picture was suggestive of a microsporidium infection (andreadis 2007). thus, a preliminary study was conducted to identify the responsible microorganism at the genus level. materials and methods a few trips were arranged to collect fresh specimens from the field. the locality was the same area in kiar district of chahar mahal and bakhtiari province where the infected larvae of an. superpictus were previously discovered in 2012. anopheline larvae were collected with the aid of a dipper and an eyedropper based on their surface swimming behavior. close observation of the larvae in a small black pan was used to detect the whitish discoloration of body segments as the primary sign of microsporidium infection. in laboratory, larval identities were checked with the aid of a valid local taxonomic key (azari-hamidian and harbach 2009). the infected larvae were observed under a low power stereomicroscope and dissected to remove their infective contents. wet preparations were prepared by adding a small sample from the extracted tissues to a drop of tap water. simple pressure with the tip of a pencil was applied on the coverslip to test if it is possible to extrude polar tube. other preparations were prepared by the gentle spreading of the infective material on microscope slides. the air-dried specimens were fixed in absolute methanol for 10 minutes. for geimsa staining 1:14 v/v concentrate solution was buffered at 7.41 by distilled water and used for 20 minutes. for trichrome staining the modified procedure previously introduced by ryan et al. 1993) was followed. all of the preparations were viewed under the 1000-x power of a conventional compound microscope. spore measurements were performed at random for 50 spores in both wet and fixed preparations. j arthropod-borne dis, sm omrani et al.: microsporidium infecting … the morphological characters proposed by andreadis (2007) and hazard and anthony (1974) were used to identify the microsporidium genus. results the infected larvae were recollected from the same locality previously discovered in chahar mahal and bakhtiari in 2012 (fig. 2). in a typical larva the first 3 abdominal segments was the primary site of infection. at times, the thorax was also affected. the involvement of the whole abdomen was relatively rare. the infected larvae were sluggish and tended to remain more silent at the bottom of the pan once they were physically stimulated by an eyedropper. close observation of the involved segments under a stereomicroscope showed that they were filled with the globular masses of a fine opaque material indicative of a microsporidium infection (fig. 3). in wet preparations, the in situ stirring spores ranged 4.33±0.19×2.67±0.12 micron. they were seemed semioval and dually refractive (fig. 4). the posterior, transparent and larger half had a rounder border. the anterior, smaller and opaque half was seemed to be a bit blunt and composed of heterogenous components with a small projection into the aforementioned section. this was conferring a bottle shape appearance to the spores. the refractive surrounding border was in favor of the presence of a tick wall around the body. in the stained specimens, spores were ranged 4.18±0.43×2.45±0.33 micron. the blurred internal structures were a little apparent (fig. 5). the posterior and anterior light spots could be attributed to the posterior vacuole and anterior sporoplast, respectively. the spores were frequently arranged in spherical aggregates comprised of 8 spores (fig. 6). immature spheres were blue tint containing multiple purple nuclei. simple pressure applied on wet preparations was not able to extrude the polar filaments of spores. overall, these features suggested the presence of a microsporidium infection from the genus parathelohania. fig. 1. apparent infection the thorax and first 3 abdominal segments in a lactophenol-preserved larva of anopheles superpictus fig. 2. one of the typical breeding places in which the infected anopheles superpictus larvae were collected j arthropod-borne dis, sm omrani et al.: microsporidium infecting … fig. 3. infected 4th instar larvae of anopheles superpictus. heavy infection of first 3 abdominal segments (above) multiple globular masses of the infective material in thorax (below) fig. 4. microsporidia in wet preparations from infected anopheles superpictus larva fig. 5. geimsa (above) and ryan-blue modified trchrome staining (below) of the microsporidia in anopheles superpictus larva fig. 6. typical clusters of parathelohania sp spores in anopheles superpictus larva. (above) geimsa and (below) ryan-blue modified trichrome stained specimens j arthropod-borne dis, sm omrani et al.: microsporidium infecting … discussion this study indicates that the detected infection in the an. superpictus larva has been caused by a microsporidium species from the genus parathelohania. to our knowledge, this is the first report of microsporidium infection in an. superpictus. it is also the first report of natural microsporidium infection in a mosquito species from of iran. parathelohania, with at least 22 species, is the second largest polytypic genus next to amblyospora in the family amblyosporidae (andreadis 2007). this genus which was firstly described by codreanu in 1966, redescribed later by anthony and hazard in 1974 (hazard and anthony 1974). the type species is parathelohania legeri (synonym: thelohania legeri) which was discovered in anopheles maculipennis meigen sl about 110 years ago (andreadis 2007). the latest described species, p. tomski, p. divulgata, p. sibirika, p. formosa and p. teguldeti have been reported by simakova and pankova from russia in 2004 (simakova and pankova 2004). all of these microsporidia were isolated from anopheline mosquitoes. in reality, anopheline mosquitoes constitute the principal hosts for the genus parathelohania (hazard and anthony 1974). parathelohania chagrasensis and p. barra are the only species described from aedomyia and ochlerotatus mosquitoes (pillai 1968, hazard and oldacre 1976). vertical and horizontal transmission is a common feature in parathelohania and normally there is an intervening obligatory copepod host in the life cycle (becnel et al. 2005). in the infected larvae, the first 3 abdominal segments were primarily involved (fig. 3). this region corresponds with the middle part of the midgut, which is highly alkaline. a series of digestive enzymes are maximally active in this portion of the gut (clements 1992). perhaps, from here the germinated spores access to their target cells in the fat tissues and progressively extend to invade other areas. while the severe overgrowth of spores kills male larvae, the infection in females is less pronounced. these larvae transmit their infection to the adulthood during which the vertical transmission will occur (andreadis 2007). the extrusion of the polar tube is a reliable sign of a microsporidium spore (wittner and weiss 1999). in this study, the application of simple mechanical pressure was not able to extrude the polar tube. this might be regarded as an exception to the general recommendation proposed by andreadis (andreadis 2007). therefore, the chemical stimulation of the spores should be considered in future attempts. geimsa staining is a routine technique in the diagnosis of microsporidia infection in mosquitoes (andreadis 2007). in the present study, with this method the central structures of the spores were deeply stained (fig. 5). however, a better shadow of the internal structures was obtained by ryan-blue modified trichrome technique (fig. 5). at times, it was possible to detect the position of posterior vacuole and/or anterior poloroplast as one or two light spots in the stained spores. since the introduction of the weber-green trichrome staining technique, various modifications are proposed by different authors (garcia 2002). while a few have addressed the quality of the visualization of the morphological characters, the others have tried to improve the practical items, or both. this study suggests that the trichrome staining might be considered as a preferred technique in those situations in which more details of the spores are aimed at. in the present study, the size of spores overlaps with the spores of p. anophelis, p. evansae, p. periculosa, p. indica, p. tomski, p. divulgata and p. formosa (hazard and anthony 1974, garcia and becnel 1994, j arthropod-borne dis, sm omrani et al.: microsporidium infecting … simakova and pankova 2004). while the first 3 species have been discovered in the nearctic region, the remainder has been described from the palearctic region. it is clear that the spore size cannot be used as a reliable method for species differentiation. instead, with the aid of electron microscopy techniques, comparisons that are more accurate would be possible. in some occasions, the definitive identification of a microsporidium species needs a good knowledge on its ecological attributes and the ultrastructural detail of developmental stages in the hosts (andreadis 2007). on the other hand, molecular information on the ss rrna sequence is a valuable tool in the phylogenetic analysis of the microsporidium species (vossbrinck et al. 2005). the checklist of the culicidae of iran contains the name of 64 species (azarihamidian 2007). a number of these mosquitoes might be considered as the potential host for microsporidia infection. this is because there is at least one report of natural microsporidium infection in these species from elsewhere (castillo 1980). these mosquitoes include an. hyrcanus (pallas), an. maculipennis meigen s.l, an. subpictus grassi s.l and an. messeae falleroni, ochlerotatus caspius (pallas) s.l, aedes vexans (meigen), culex modestus ficalbi, cx. pipiens linnaeus, cx. quinquefasciatus say, cx. territans walker and cx. theileri theobald. the vital consequences of artificial infection with anncaliia algerae (synonym: nosema algerae, synonym: brachiola algerae) on an. stephensi liston has been explored in laboratory, as well (haq et al. 1981). in the past, at least two extensive countrywide mosquito larval surveys have been performed in iran (saebi 1987, zaim 1987). although there is no report of microsporidium infection in these studies, it is still possible that new cases of microsporidium infection could be found with more intensive larval surveys. there are few reports, which declare that some microsporidia species are connected with human infections (cheney et al. 2000, coyle et al. 2004). however, the research on the application of these microorganisms in the biological control of mosquitoes is going on (solter and becnel 2007). mathematical modeling is also in favor of their effectiveness in the control of mosquitoes (koella et al. 2009). recent efforts have been focused on species with polymorphic development and maximum phylogenetic distance to human parasitizing microsporidia (andreadis 2007). although parathelohania is a genus with average characters, the possibility of the utilization of these pathogens in the control of specific mosquitoes cannot be completely excluded. conclusion the first report of an. superpictus larval infection with a microsporidium species from the genus parathelohania is presented here. diagnosis was made based on gross morphological characters in light microscopy. although the definitive diagnosis of the species needs specific ultrastructural and molecular data, current ecological attributes predicts that it is likely a new microsporidium species. acknowledgements the authors are grateful to dr james j becnel, united states department of agriculture, agricultural research service, gainesville, united states, for his kind contribution on the verification of the microsporidium genus identified here. the authors also wish to thank mrs simin taghipour, shahrekord university of medical sciences, school of medicine, shahrekord, iran, for her helpful assistance in the laboratory works. dr shahyad azari-hamidian, school of health, guilan university of medical sciences, rasht, iran, is appreciated for reviewing the manuscript. j arthropod-borne dis, sm omrani et al.: microsporidium infecting … this paper is a part of the results of the project no. 1820-74-01-1392, which is financially supported by shahrekord university of medical sciences. the authors declare that there is no conflict of interest. references andreadis tg (2007) microsporidian parasites of mosquitoes. j am mosq control assoc. 23: 3–29. azari-hamidian s (2007) checklist of iranian 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thelohania henneguy 1892 and descriptions of two new species of parathelohania from anopheline mosquitoes, us department of agriculture, agricultural research service. hazard ei and oldacre s (1976) revision of microsporida (protozoa) close to thelohania: with descriptions of one new family, eight new genera, and thirteen new species, us department of agriculture, agricultural research service. kamareddine l (2012) the biological control of the malaria vector. toxins. 4: 748–767. koella jc, lorenz l, bargelowski i (2009) microsporidians as evolution-proof agents of malaria control? adv parasitol. 68: 315–327. macdonald g (1957) the epidemiology and control of malaria. london, oxford university press. pillai j (1968) thelohania barra n. sp., a microsporidian parasite of aedes (halaej arthropod-borne dis, sm omrani et al.: microsporidium infecting … des) australis erichson, in new zealand. z angew entomol. 64: 395–398. ranson h, n’guessan r, lines j, moiroux n, nkuni z, corbel v (2010) 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[phd dissertation] school of public health, tehran university of medical sciences. tehran, iran [persian]. simakova a, pankova t (2004) microsporidia of genus trichoctosporea (microspora: thelohaniidae) from blood-sucking mosquitoes (diptera: culicidae) of western siberia. vestnik of tomsk state university (supplement). 10: 116–121. solter l, becnel j (2007) entomopathogenic microsporidia. in: lacey l and kaya h (eds) field manual of techniques in invertebrate pathology. springer, dordrecht, pp. 199–221. tolle m (2009) mosquito-borne diseases. curr probl pediatr adolesc health care. 39: 97–140. vávra j, lukeš j (2013) microsporidia and “the art of living together”. adv parasitol. 82: 253–319. vossbrinck c, debrunner-vossbrinck b (2005) molecular phylogeny of the microsporidia: ecological, ultrastructural and taxonomic considerations. folia parasitol. 52: 131–142. wittner m and weiss lm (1999) microsporidia and microsporidiosis. asm press, washington dc. zahar a (1974) review of the ecology of malaria vectors in the who eastern mediterranean region. bull world health organ. 50: 427–440. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq cont assoc. 3: 568–573. j arthropod-borne dis, 2012, 6(2): 151–155 z yazici et al.: the first investigation of … 151 short communication the first investigation of west nile virus in horses using real time rt-pcr in middle black sea region in turkey *zafer yazici 1, harun albayrak 1, emre ozan 2, semra gumusova 1 1department of virology, faculty of veterinary medicine,ondokuz mayis university, samsun, turkey 2virology laboratory, regional veterinary control and research institute, ministry of agriculture and rural affairs, samsun, turkey (received 4 jan 2011; accepted 13 sep 2011) abstract background: west nile virus (wnv) is a mosquito-borne disease that can cause fatal infection in mammals including humans, dogs, horses, birds and reptiles. although west nile virus is an asymptomatic infection, especially it can cause neurologic disorders in humans and horses. the aim of this study was to the investigate virological presence of wnv in horses in the black sea region of turkey using real time rt-pcr (rrt-pcr). methods: totally, 120 horse sera were collected equally from 4 provinces in middle black sea region of turkey and investigated for wnv presence by taqman based rrt-pcr. results: wnv nucleic acid was not detected in any horse serum sample. conclusion: although obtained result indicated no evidence of wnv–rna in horses, black sea region of turkey is one of the suitable places for the wnv infection. for this reason, our research will continue for the determination of the viruses in vectors and susceptible animals such as horses, dogs, etc. keywords: horses, rt-pcr, sera, west nile virus introduction west nile virus (wnv) is a positive sense single stranded rna virus of the flavivirus genus in family flaviviridae (monath and heinz 1996) and is classified in the japanese encephalitis serocomplex group together with st louis encephalitis (sle), murray valley encephalitis (mve), japanese encephalitis (je) and kunjin (kun) viruses (petersen and roehrig 2001). west nile virus is an important pathogen for vertebrates including humans, horses, dogs birds and reptiles (castillo-olivares and woods 2004). birds are considered the main reservoir hosts of wnv, and migratory birds play an important role in its spreading (rappole et al. 2000). the natural cycle of wnv typically involves ornithophilic culex mosquitoes feeding on avian hosts (apperson et al. 2004). horses are highly susceptible to wnv infection and mortality in these animals can be observed during wnv outbreaks (castillo-olivares and wood 2004). the latest outbreaks of wnv were characterized by an increased proportion of neurological disease in both humans and horses (petersen and roehrig 2001). mortality rates among clinically affected horses have been estimated around 38%, 28%, 44% and 42% during outbreaks in the usa, france (2000), morrocco and italy (1998) respectively (tber abdelhaq 1996, cantile et al. 2001, murgue et al. 2001, ostlund et al. 2001). west nile virus has a wide geographical distrubution that includes countries of europe, asia, africa, australia, and america (hubalek and halouzka 1999, savage et al. 1999, hayes et al. 2005). since the summer of 1999 during which wnv was introduced in new york *corresponding author: dr zafer yazici, e-mail: zyazici@omu.edu.tr j arthropod-borne dis, 2012, 6(2): 151–155 z yazici et al.: the first investigation of … 152 city, the distribution of wnv has expanded to include 46 states of the united states, 7 provinces of canada, mexico, and probably a number of the caribbean islands (kramer et al. 2001, blitvich et al. 2004). serological evidence of arboviral infection based on the detection of haemagglutination-inhibiting (hai) antibodies was reported in turkey in the 1970s (ari et al. 1972, radda et al. 1973). subsequently, serological identification of wnv was reported in humans and different mammalian species including; sheep, ass-mule, cat, cattle (meco 1977, ozkul et al. 2006, ozer et al. 2007). on the other hand, there is only one virological datum reported in turkey (arpacı et al. 2009) the presence of wnv in neighboring countries (bulgaria and greece) of turkey was reported in mammalian species and mosquitoes (katsarov et al. 1980, koptopoulos et al. 1980). additionally wetland ecosystems such as river deltas or flood plains and migration routes of migratory birds are mainly the appropriate foci of wnv infections (komar 2000, ozer et al. 2007) and middle black sea region of turkey involves river deltas and migratory routes of birds. all these data suggest that wnv might be present in our region, so the aim of this study was to investigate the presence of wnv in horses, which is the most susceptible animal to infection in the black sea region of turkey using real time rt-pcr. materials and methods sera blood sera samples were collected from randomly selected 120 horses from 4 different provinces (samsun, sinop, amasya and tokat ) of central black sea region, turkey between march and august 2007 (fig. 1) and transported to the laboratory at 4 °c. all sampled horses were older than 1 year old. the blood samples were centrifuged at 2000 rpm for 10 min at 4 ○c. the sera were subsequently separated into vials (eppendorf germany), then inactivated at 56 ○c for 30 min and stored at 20 ○c until tested. rna extraction and taqman based rrtpcr assay viral rna was extracted from 350 µl of the horse sera by using the magna pure lc rna isolation kitiii (roche, mannheim, germany). purified nucleic acid was eluted in 50 µl elution buffer and stored at -70 °c until used. the samples were tested by real time rt-pcr for wnv-rna detection using the oligonucleotide primers and taqman probe targeting wnv e gene designed by lanciotti et al. (2000). the taqman probes were labeled at the 5’ end with the fam reporter dye and were labeled at the 3’ end with the quencher dye tamra (table 1). for the taqman assay, 2 µl of rna combined with 50 pmol of each primer and 10 pmol of the famand tamra-labeled probe in a 50 µl total reaction volume by using the onestep rt-pcr kit (qiagen, hilden, germany) and amplified in a lightcyler@2.0 real time pcr system (roche diagnostics, ger-many).the following cycling times and tem-peratures were applied: 1 cycle of 42 °c for 30 min and 94 °c for 2 min and 40 cycles of 94 °c for 30s, 55 °c for 30s, and 72 °c for 20s. results samples tested using taqman-based real time rt-pcr for the presence of wnvrna but all samples were negative (table 2). j arthropod-borne dis, 2012, 6(2): 151–155 z yazici et al.: the first investigation of … 153 table 1. oligonucleotide primers and probe used in the taqman-based rrt-pcr assay primer genome position sequence (5’-3’) rrt-pcr product size (bp) wnenv-forward 1160–1180 tcagcgatctctccaccaaag wnenv-reverse 1209–1229 gggtcagcacgtttgtcattg 70 wnenv-probe 1186–1207 tgcccgaccatgggagaagctc table 2. distribution and results of serum sample according to provinces provinces number of tested horse sera results of real time rt-pcr for wnv number of positive sera samsun 30 sinop 30 amasya 30 tokat 40 total 120 fig. 1. location of provinces where sera collected from horses in the black sea region of turkey discussion the transmission cycle of wnv involves several species of mosquitoes (primarily culex spp) and various species of birds. mosquitoes become infected with wnv when they feed on a bird carrying the virus in its blood. after 10 to 14 days, the virus can be transmitted to another bird, person, or other animal such as horse, dog, cats that the mosquito bites. during blood feeding the mosquito injects the virus, contained in its saliva, into the bird, animal, or person and may cause illness. horses and humans, are considered dead-end or incidental hosts for the virus because they do not produce enough virus to reinfect a mosquito and maintain the transmission cycle. however, they are capable of showing clinical symptoms (bunning et al. 2002). various test methods can be used for the diagnosis of wnv. virus isolation can be attempted from cerebrospinal fluid (csf), blood or tissues in cell cultures such as vero, rk-13 cells or mosquito cell lines (ostlund et al. 2001). real time rt pcr methods were designed to specifically detect wnv. this methj arthropod-borne dis, 2012, 6(2): 151–155 z yazici et al.: the first investigation of … 154 od has demonstrated greater sensitivity than traditional rt-pcr methods and gives advantages over classical virological methods (porter et al. 1993, lanciotti et al. 2000). the presence of wnv has been reported with virological and serological study results in many european countries (hubalek and halouzka. 1999, zeller et al. 2004). in turkey, wnv had been identified only serologically until 2009 (meco 1977, ozkul et al. 2005, ozer et al. 2007). subsequently, arpaci et al. (2009) reported the first time virological identification of wnv from graft-versus-host disease patient using rt-pcr in turkey. however wnv has not been virologically identified from animal and vectors in turkey. in this study, wnv-rna was not detected in screening horses in a middle black sea region of turkey. however, the reasons of these negative results can depend on many factors such as the quantity of the virus in the material; region’s environmental features i.e. having cold weather conditions in winter which provides unsuitable circumstances for the presence of mosquitoes all around the year in northern turkey. in conclusion, although obtained result indicated no evidence of wnv–rna in horses, black sea region of turkey is one of the suitable places for the wnv infection. for this reason, our research will continue for the determination of the viruses in vectors and susceptible animals such as horses, dogs, e.g. acknowledgments the authors declare that there is no conflict of interests. references apperson cs, hassan hk, harrison ba, savage hm, aspen se, farajollahi a, crans w, daniels tj, falco rc, benedict m, anderson m, mc millen l, unnasch tr (2004) host feeding patterns of established and potential mosquito vectors of west nile virus in the eastern united states. vector-borne zoonotic dis. 4: 71–82. ari a (1972) studies on activity and ecology of arboviruses in turkey. turk hij tek. biyol derg. 32: 134–143. arpaci f, cetin t, kubar a, ozturk m, kuzhan o, komurrcu s, ozturk b, ataergin s, ozet a (2009) west nile infection in a patient with acute graft versushost diseases. haemotologica. 94(suppl 2): 687. blitvich bj, fernandez-salas i, conterascordero jf (2004) phylogenetic analysis of west nile virus, nuevo leon state, mexico. emerg infect dis. 10: 1314– 1317. bunning ml, bowen ra, cropp b, sullivan kg, davis bs, komar n, godsey ms, baker d, hettler dl, holmes da, biggerstaff bj, mitchell cj (2002) experimental infection of horses with west nile virus. emerg infect dis. 8: 380–385. cantile c, del piero f, di guardo g, arispici m (2001) pathologic and immunohistochemical findings in naturally occuring west nile virus infection in horses. vet pathol. 38: 414–421. castillo-olivares j, wood j (2004) west nile virus infection of horses. vet res. 35: 467–483. hayes eb, komar n, nasci r, montgomery s, o’leary dr, campell gl (2005) epidemiology and transmission dynamics of west nile diseases. emerg infect dis. 11: 1167–1173 hubalek z, halouzka j (1999) west nile fever–a reemerging mosquito-borne viral disease in europe. emerg infect dis. 643. katsarov g, vasilenko s, vargin v, butenko s, tkachenko e (1980) serological studies on the distribution of some arj arthropod-borne dis, 2012, 6(2): 151–155 z yazici et al.: the first investigation of … 155 boviruses in bulgaria. problems of infec and parasitic dis. 8: 32–35. komar n (2000) west nile viral encephalitis. rev sci technol. 19: 166–176. koptopoulos g, papadopoulos o (1980) a serological survey for tick-borne encephalitis and west nile viruses in greece. zentralbl bakteriol. 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hospitals, east azerbaijan province, iran in 2013 afshin salehi 1, hassan vatandoost 2, teimour hazratian 3, alireza sanei-dehkordi 4,5, hossein hooshyar 6, mohsen arbabi 6, yaser salim-abadi 7, reza sharafati-chaleshtori 8, mohammad amin gorouhi 9, *azim paksa 6 1college of paramedics, kashan university of medical sciences, kashan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3departmemt of parasitology, faculty of medicine, tabriz university of medical sciences, tabriz, iran 4department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 5social determinants in health promotion research center, hormozgan university of medical sciences, bandar abbas, iran 6department of parasitology, school of medicine, kashan university of medical sciences, kashan, iran 7rafsanjan university of medical sciences, rafsanjan, iran 8research center for biochemistry and nutrition in metabolic diseases, school of medicine, kashan university of medical sciences, kashan, iran 9faculty of health, kerman university of medical sciences, kerman, iran (received 7 jan 2014; accepted 24 june 2015) abstract background: insecticide resistance is one of the serious problems for german cockroach control program. this study was conducted to determine the bendiocarb and carbaryl resistance mechanisms in german cockroaches using the piperonyl butoxide (pbo). methods: bioassay tests were conducted with 4 to 6 different concentrations of both insecticides with four replicates of 10 susceptible strain cockroaches per concentration to determine of discriminative concentration. after determining discriminative concentration, the result was compared to wild strain. the levels of susceptibility and resistance ratio (rr) and synergism ratio (sr) were calculated for each five wild strains. moreover resistance mechanisms in wild strains were determined using pbo synergist in vivo. results: hospital strains showed different levels of resistance to bendiocarb and carbaryl compared to susceptible strain. the bendiocarb and carbaryl resistance ratios ranged from 2.11 to 7.97 and 1.67 to 2 at ld50 levels, respectively. the synergist pbo significantly enhanced the toxicity of bendiocarb and carbaryl to all strains with different degrees of synergist ratio, 1.31, 1.39, 3.61, 1.78, 1.62 and 2.1 fold for bendiocarb, 1.19, 1.18, 1.12. 1.29, 1.45 and 1.11fold for carbaryl, suggesting monooxygenase involvement in bendiocarb and carbaryl resistance. conclusion: the synergetic effect of pbo had the highest effect on bendiocarb and resistance level was significantly reduced, which indicates the important role of monoxidase enzyme in creating resistance to bendiocarb. piperonyl butoxide did not have a significant synergistic effect on carbaryl and did not significantly break the resistance. keywords: german cockroach, resistance mechanism, carbaryl, bendiocarb introduction german cockroach is one of the most important pests in terms of hygiene with a global spread. this insect is allergenic and lives in residential places, restaurants, hotels, hospi*corresponding author: dr azim paksa, email: a.paksa@yahoo.com j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 406 http://jad.tums.ac.ir published online: january 06, 2016 tals, food warehouses, and can transmit many pathogens such as bacteria, viruses, fungi, protozoa and parasite eggs to human through mechanical methods or their digestive tract. hence, it is necessary to control this insect (nasirian 2007, behmanesh et al. 2010, fakoorziba et al. 2010, nejati et al. 2012). development of resistance to insecticides in german cockroach due to excessive consumption and long-term insecticide spraying is one of the serious problems for controlling this pest (fahiminia et al. 2010, nasirian 2010). many synergistic studies have been conducted in the world to study resistance mechanism of german cockroach to phosphorous, carbamate and pyrethroid insecticides (payne et al. 1984, lee et al. 1996, shinji kasai et al. 1998, el-merhibi et al. 2004, chai and lee 2010). synergistic studies with piperonyl butoxide (pbo) indicate the role of monoxidase system in creating resistance to these insecticides (hemingway et al. 1993, valles et al. 1994, hodgson and levi 1998, tozzi 1998). increasing resistance of german cockroach to organochlorine, organophosphate, carbamate, and pyrethroid insecticides has been reported (cochran 1990, ahad and hollingworth 2004). the resistance mechanisms of german cockroach to phosphorous, carbamate and pyrethroid insecticides have been determined using pbo and s, s, s-tributyl phosphorotrithioate (def) synergists (metcalf 1976, rust 1987, scott et al. 1990, cochran 1990, dong and scott 1992, lee et al. 1999). in iran, german cockroach is resistant to pyicom, diazinon, actylic, permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin and propoxur (ladonni 1993, 1997). resistance mechanisms to permethrin, ddt, chlorpyrifos, malathion have been determined using pbo, def and dmc synergists (ladonni and sadegheyani 1998, paksa et al. 2011, limoee et al. 2011). synergists are chemicals which lack pesticide effect but increase pesticide properties of their active ingredient. piperonyl butoxide prevents activity of p-450 cytochrom. if resistance is due to increase of metabolism using mfo, one can overcome resistanceusing pbo, which is mfo inhibitor. synergists are widely used in insecticides containing active ingredient of pyrethrin, pyrethroids and carbamates. without pbo, enzymes interfering in mechanism of the insect’s body particularly enzymes of p-450 cytochrom group can detoxify active ingredient of insecticide before affecting. adding pbo to an insecticide reduces the dose of active ingredient required for production of the desired effect and increases the toxicity of insecticide (hodgson and levi 1998). carbamate insecticides act in this way: when a nerve is stimulated, concentration of sodium and potassium ions changes inside and outside the nerve, which leads to neurotransmission. neurotransmission from one nerve to another requires neurotransmitters such as acetylcholine, which acts in central nervous system (cns) and in neuromuscular junction. carbamate insecticides inhibit acetyl cholinesterase and in the absence of this enzyme, acetylcholine accumulates in synapses and neural transmission continues in synapses or neuromuscular junctions, which disrupts harmony and finally results in insect death (talebi-jahromi 2006). in iran, no study has been conducted on resistance mechanisms of german cockroach to bendiocarb and carbaryl. therefore, the present study was conducted to investigate and determine resistance mechanisms of german cockroach to these poisons in vivo in tabriz city using pbo synergist. conducting this study and knowing resistance mechanisms in this pest, we can manage defeating this pest, and the phenomenon of resistance in this pest compared with insecticide of carbamate group particularly bendiocarb and carbaryl. j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 407 http://jad.tums.ac.ir published online: january 06, 2016 materials and methods in this experimental study, five wild strains of german cockroach were trapped or manually collected from behbood, sina, shafa, taleghani and noor hospitals in tabriz during summer 2013. then, they were taken to laboratory and housed along with sensitive strains in special glass containers containing bread, starch, sugar and water under laboratory conditions at 27±2 ˚c and humidity of 60±10% and lighting period of 12 h. chemicals used in this study included formulation of technical insecticides such as bendiocarb 97% ai, (technical grade), carbaryl 98% ai, (technical grade), (cyanamid agro, india), piperonyl butoxide synergist (pbo) 94% ai, (zeneca, haslemere, uk), a monooxygenase inhibitor for synergism study. co2 was used as anesthetic and acetone as solvent. internal surface of the test containers was measured in order to calculate the amount of insecticide needed (schraft et al.1995). standard dilutions of insecticide with aceton were poured at the bottom of the glass container. the solvent was evaporated through constant rotation of the container, so a uniform layer of exterminator remained on the internal wall of the container. in this way, the desired concentration was used based on mg/m2. after specifying the concentrations for sensitive strains, the level and mechanisms of resistance for wild strains were determined using piperonyl butoxide in vivo. synergistic tests were conducted in vivo using pbo synergist concurrently with bendiocarb and carbaryl using cochran contact method (cochran 1990). these tests were performed in 4 replicates each with 10 male adult cockroaches. for each synergistic test, two controls were assigned to acetone and synergist. the data were analyzed using probit software (finny 1972). for this purpose, ld50 was calculated with confidence level of 95%. to calculate resistance ratio (rr), ld50 of resistant strain was divided by ld50 of sensitive strain and ld50 of insecticide was divided by ld50 (insecticide+ synergist) to calculate synergistic rate (sr). results resistance to bendiocarb all five strains of german cockroach collected from hospitals showed different degrees of resistance to bendiocarb with confidence level of 95% based on resistance ratio (rr) compared with the sensitive strain (table 1). lethal dose 50 of bendiocarb was calculated between 38.44 and 306.21 mg/m2 for sensitive strain and five collected strains with confidence level of 95% (table 1). resistance ratio of bendiocarb was obtained between 2.11 and 7.97 for five collected strains. the highest resistance ratio for bendiocarb was found 7.97 in strain of behbood hospital (table 1). resistance to carbaryl different levels of resistance to carbaryl were observed in all strains of german cockroach collected from hospitals (table 2). ld50 of carbaryl was calculated between 280.87 and 560.81 for sensitive strain and five collected strains with confidence level of 95% (table 2). rr of carbaryl was obtained between 1.67 and 2 for five collected strains. the highest resistance ratio (rr) for carbaryl was found 2 in strain of behbood hospital (table 2). synergistic effect of piperonyl butoxide on bendiocarb as for the synergistic effect of pbo on bendiocarb, ld50 for sensitive strain and five collected strains was obtained between 25.76 and 220.90 and the highest resistance ratio (rr) for bendiocarb was found to be 7.51 in strain of behbood hospital (table 3). j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 408 http://jad.tums.ac.ir published online: january 06, 2016 by studying the synergistic effect, synergism ratio of bendiocarb was calculated 1.31, 1.39, 3.61, 1.78, 1.62 and 2.12 fold in sensitive strain and strains from behbood, sina, shafa, taleghani and noor hospitals, respectively and the highest synergism ratio of carbaryl (sr) was calculated 3.61 in sina strain (table 3). synergistic effect of piperonyl butoxide on carbaryl lethal dose 50 of carbaryl+pbo for all collected strains was obtained between 235.77 and 480.64 and the highest (rr) for carbaryl was found to be 2.04 in noor hospital. as for synergistic effect, synergism ratio (sr) of carbaryl was calculated 1.19, 1.18, 1.12, 1.29, 1.45 and 1.11 fold in the sensitive strain and strains from behbood, sina, shafa , taleghani and noor hospitals, respectively and the highest synergism ratio (sr) of carbaryl (sr) was calculated 1.45 in taleghani strain (table 4). table 1. lethal dose values for bendiocarb on susceptible and five hospital-collected strains of german cockroach strain n y-intercept slope(se) x 2(df) ld50 , 95% c.l rr susceptible 200 -9.17 5.78±0.778 5.89 (3) 38.44 1 behbood-h 200 -8.79 3.54±0.501 2.94 (2) 306.21 7.97 sina-h 200 -3.93 1.1±0.265 5.71 (3) 92.96 2.42 shafa-h 200 -4.92 2.58±0.305 3.03 (3) 80.92 2.11 talaghani-h 200 -4.86 2.54±0.301 4.9 (3) 81.32 2.12 nour-h 200 -6.10 2.72±0.434 4.37 (2) 175.61 4.57 rr: resistance ratio h: hospital n: number table 2. lethal dose values for carbaryl on susceptible and five hospital-collected strains of german cockroach strain n y-intercept slope(se) x 2(df) ld50 , 95% c.l rr susceptible 200 -11.33 4.63±2.015 20.06 (3) 280.87 1 behbood-h 200 -45.84 16.68±2.780 4.05 (2) 560.81 2 sina-h 200 -69.88 25.86±11.740 18.28 (2) 504.06 1.8 shafa-h 200 -41.90 15.68±2.592 2.10 (2) 469.1 1.67 talaghani-h 200 -48.0365 17.71±2.145 1.680 (3) 514.96 1.83 nour-h 200 -93.09 34.14±10.015 9.45 (2) 533.47 1.9 rr: resistance ratio h: hospital n: number j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 409 http://jad.tums.ac.ir published online: january 06, 2016 table 3. toxicity bendiocarb with and without piperonyl butoxide to five bendiocarb resistant strains of the german cockroach compared to the susceptible strain insecticide alone insecticide + pbo strain n ld50 , 95% c.l rr n y-intercept slope(se) x2(df) ld50 , 95% c.l sr rr susceptible 200 38.44 1 200 -8.37 5.70±0.753 2.50 (2) 29.42 1.31 1 behbood-h 200 306.21 7.97 200 -9.33 3.98±0.990 7.57 (2) 220.90 1.39 7.51 sina-h 200 92.96 2.42 200 -4.13 2.92±0.444 2.61 (2) 25.76 3.61 0.88 shafa-h 200 80.92 2.11 200 -8.69 5.24±0.708 1.36 (2) 45.46 1.78 1.55 talaghani-h 200 81.32 2.12 200 -6.62 3.89±0.493 5.08 (2) 50.11 1.62 2.81 nour-h 200 175.61 4.57 200 -9.69 5.05±1.917 13.68(2) 82.65 2.12 2.12 h: hospital n: number resistance ratio: ld50 of resistant strain/ ld50 of susceptible strain synergist ratio: ld50 of insecticide alone/ ld50 of insecticide + pbo table 4. toxicity carbaryl with and without piperonyl butoxide to five carbaryl resistant strains of the german cockroach compared to the susceptible strain (sus) insecticide alone insecticide + pbo strain n ld50 , 95% c.l rr n y-intercept slope(se) x2(df) ld50 , 95% c.l sr rr susceptible 200 280.87 1 200 -12.42 5.23±2.404 32.03 (3) 235.77 1.19 1 behbood-h 200 560.81 2 200 -65.17 24.36±3.526 3.78 (2) 472.89 1.18 2.01 sina-h 200 504.06 1.8 200 -46.90 17.69±4.519 6.34 (2) 448.22 1.12 1.9 shafa-h 200 469.1 1.67 200 -70.68 27.61±7.532 7.41 (2) 362.98 1.29 1.53 talaghani-h 200 514.96 1.83 200 -49.51 19.42±2.535 2.15 (2) 354.42 1.45 1.5 nour-h 200 533.47 1.9 200 -67.09 25.02±7.164 9.34 (2) 480.64 1.11 2.04 h: hospital n: number resistance ratio: ld50 of resistant strain/ ld50 of susceptible strain synergist ratio: ld50 of insecticide alone/ ld50 of insecticide+pbo j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 410 http://jad.tums.ac.ir published online: january 06, 2016 discussion the results of pbo synergistic tests on bendiocarb insecticide showed that resistance level was reduced in all tested strains. synergism rate (sr) for bendiocarb on susceptible, behbood, sina, shafa, taleghani and noor hospitals of strain was 1.31, 1.39, 3.61, 1.78, 1.62, 2.12 fold, respectively. based on the obtained results in strain of sina hospital and noor hospital, the synergetic effect of pbo on bendiocarb insecticide showed the highest effect and resistance level was significantly reduced, which indicates the important role of monoxidase enzyme (mfo) in creating resistance to bendiocarb exterminator. in strain of behbood, taleghani and shafa hospitals, the synergistic effect of pbo was found to be low to medium. these results indicated that monoxidase was not among the main factors of resistance but it was a cofactor of resistance compared with bendiocarb. pbo did not have a significant synergistic effect on carbaryl insecticide and did not significantly break the resistance. this result shows that monoxidase enzyme does not have a main role in creating resistance to carbaryl in this strain. a major part of resistance was created probably due to mechanisms such as reduced cuticle penetration, esterase and other factors created due to fighting with german cockroach. synergism rate (sr) for carbaryl no susceptible, behbood, sina, shafa, taleghani and noor hospitals of strain were 1.19, 1.18, 1.12, 1.29, 1.45, 1.11 fold respectively. in strains of shafa and taleghani hospitals, the synergistic effect of pbo on carbaryl, these two are different. these results are consistent with our previous studies on resistance mechanisms among german cockroach strains in vivo in tehran (limoee et al. 2006). besides, the results of the present study are in line with those of another study on resistance mechanisms in some populations of the german cockroaches from kashan (paksa et al. 2014). according to reierson, 10-fold resistance measured by topical application is the critical point above which operational control failures are likely to occur while; resistance ratio at 5x and below may still achieve a good control of the german cockroach population. thus, the low resistance ratios of the three strains to chlorpyrifos compared with the susceptible strain probably reflect a relatively low levels of resistance to this compound because these strains have not been selected by chlorpyrifos for several generations. hence, bendiocarb and specially chlorpyrifos may still provide adequate control of these strains (reierson et al. 1998).the findings show involvement of monooxygenase in cyfluthrin resistance. in 1987, first rust recognized resistance to diazinon and bendiocarb insecticides in different strains of german cockroach (rust 1987). a study was conducted by hemingway in 1993 to investigate resistance of german cockroach to chlorpyrifos and propoxur compared with sensitive strain. thirteen out of 14 strains showed resistance to chlorpyrifos and 12 strains showed resistance to propoxur. synergistic studies with pbo also showed role of monoxidase system in creating resistance to these insecticides (hemingway et al. 1993). in this study, an increase in activity of esterase was observed in strains resistant to a broad spectrum of organophosphates. lee et al. collected 12 strains of german cockroach from malaysia in 1996 to provide direct evidence for mechanisms involved in resistance of german cockroach to propoxur, chlorpyrifos, cypermethrin and permethrin and performed synergistic tests after performing sensitivity tests and specifying their resistance ratio. in these studies, pbo and def synergists reduced resistance to propoxur, which j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 411 http://jad.tums.ac.ir published online: january 06, 2016 indicated involvement of monooxygenase resistance (lee et al. 1996). however, these synergists did not affect resistance to cypermethrin and permethrin. scott et al. conducted a study in 1990 to determine sensitivity of 4 strains of german cockroach and the effect of synergists on increasing toxicity of pyrethrin, propoxur, malathion, deltamethrin, chlorpyrifos and bendiocarb insecticides using topical application method and showed that bendiocarb insecticides along with pbo and def synergists were applied in kenly strain (resistant to bendiocarb and permethrin) and rugers strain (resistant to permethrin) and as a result of this test, resistance was reduced indicating the presence of hydrolytic and oxidase metabolism in creating resistance in the above strains (scott et al. 1990). lee et al. collected 23 strains of german cockroach in 1999 and calculated their sensitivity to propoxur, baygon, bendiocarb and chlorpyrifos. resistance in pesticides of carbamate group was incompletely removed by pbo and def synergists and probably, resistance was controlled by monooxygenase and esterase. resistance to phosphorous insecticides is neutralized by def (lee et al. 1999). generally, pbo reduces resistance level of but it does not remove resistance completely in the present study like the studies conducted in the world, which indicated enzymes of cytochrom p540 and monooxygenase as the factor helping create resistance to bendiocarb and carbaryl in the above strains. monooxygenase p540 interferes in resistance of b. germanica to carbamate insecticides and confirms the present study which is conducted for the first time in iran (rust 1987, scott et al.1990, hemingway et al. 1993, lee et al. 1996, lee et al. 1999). to succeed in controlling german cockroach in management of resistance to exterminators, it seems vital to recognize resistance in the early stages and the mechanism of pest resistance using bioassay tests. such tests measure the sensitivity of insects. therefore, knowing of resistance in order to control pests desirably is based on measurement of pest resistance before its occurring or development in conventional insecticides or new compounds. considering the obtained results of this study and receiving information about the resistance mechanisms, it is recommended not to use bendiocarb and carbaryl exterminators or to use them periodically and in places which have not been sprayed with them, or use new insecticides such as spinosad or poisonous baits with different mechanism of action from those of carbamate group such as poisonous baits of imidacloprid and fipronil (nasirian 2007, nasirian et al. 2011). furthermore, it will not be possible to manage resistance to insecticides unless resistance mechanisms neutralizing the effect of insecticide inside body of german cockroach are studied comprehensively. studies that are more comprehensive are recommended in this field to investigate other mechanisms such as reduced penetration of cuticle and insensitivity to acetylcholinesterase. conclusion all five collected strains showed different levels of resistance compared with sensitive strain. as a result, rr of all strains was significant compared with sensitive strain. piperonyl butoxide significantly increased toxicity of bendiocarb and carbaryl almost in all strains at different degrees, but resistance was not completely removed by pbo. these results, reduction of resistance, show involvement of monoxidase enzyme in creating resistance to these insecticides. considering that resistance was not removed by pbo synergist, other mechanisms such as reduced cuticle penetration and insensitivity to acetylcholinesterase may be involved. j arthropod-borne dis, september 2016, 10(3): 405–414 afshin salehi et al.: detection of bendiocarb … 412 http://jad.tums.ac.ir published online: january 06, 2016 acknowledgements we would like to thank vice-chancellor of research, kashan university of medical sciences for supporting this study. this work is supported by a research grant from the vice secretary of research of kashan university of medical sciences (grant no. 93126). references ahad m, hollingworth rm 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december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 528 http://jad.tums.ac.ir published online: october 04, 2016 original article evaluation of some plant essential oils against the brown-banded cockroach, supella longipalpa (blattaria: ectobiidae): a mechanical vector of human pathogens mona sharififard 1, farhad safdari 2, amir siahpoush 3, *hamid kassiri 1 1department of medical entomology and vector control, school of health, ahvaz jundishapur university of medical sciences, ahvaz, iran 2health center of khuzestan province, ahvaz jundishapur university of medical sciences, ahvaz, iran 3department of pharmacognosy and herbal medicine and natural product research center, school of pharmacy, ahvaz jundishapur university of medical sciences, ahvaz, iran (received 27 apr 2014; accepted 10 june 2015) abstract background: essential oils, as secondary plant compounds, present a safer alternative to conventional insecticides in insect control programs. so five essential oils including eucalyptus, mint, yarrow, oregano and rosemary oils were evaluated against the brown-banded cockroach supella longipalpa. methods: evaluation was done against the 3rd and 4th instar nymphs using three bioassay methods; continuous contact toxicity, fumigant toxicity and repellent activity. the study was done in the laboratory of medical entomology, during april 2012 to september 2013. results: mortality rates by the lowest concentration (2.5%) of rosemary, oregano, yarrow, eucalyptus and mint oils were 100%, 62.2%, 45 %, 36.2% and 5.2% at 24 h after exposure respectively. rosemary oil was determined as the most toxic oil because of 100 % mortality rate at the concentration range of 2.5% to 30%. the lowest fumigation effect using 50 µl /l air was recorded from mint oil with 97.2 % mortality after 24 h, while the other oils caused 100% mortality. the most repel activity was related to oregano oil which showed 96.5–99.1% repellency at the concentration range of 2.5–30% with a residual effect lasting at least a week after treatment. conclusion: oregano oil could be used as a potential repellent against s. longipalpa. also, all five essential oils could be used as the safe compounds for surface treating or fumigation in cockroach control programs while rosmary and oregano oils exhibited the most toxicity. keywords: essential oil, supella longipalpa, mechnical vector introduction natural contamination of cockroaches with wide range of pathogenic organisms including about 40 species of bacteria, nearly 12 species of pathogenic helminthes, the second largest group of vertebrate pathogens, and also viruses, protozoa and fungi affecting man and other vertebrate animals have been reported by numerous studies (le guyader et al. 1989, rivault et al. 1994, baumholtz et al. 1997, cochran, 1999, eggleston and arruda 2001, savoldelli and luciano 2005). often their movement between waste and food materials led to acquire, carry, and mechanically transfer of these pathogens. as proven or suspected carries, cockroaches play a prominent role in caring and distributing of organisms causing ddiarrhea, dysentery, cholera, leprosy, plague, typhoid fever, and viral diseases such as poliomyelitis are carries by cockroaches. they also carry the parasitic worms such as taenia, shistosoma, ascaris and may cause allergic reactions, including dermatitis, itching, swelling of the eyelids, and more serious respiratory conditions (stankus et al. *corresponding author: dr hamid kassiri, email: hamid.kassiri@yahoo.com j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 529 http://jad.tums.ac.ir published online: october 04, 2016 1990, savoldelli and luciano 2005). the brown-banded cockroach, supella longipalpa known previously as s. supellectilium as a nearly cosmopolitan cockroach, has recently become a hygiene problem in the city of ahvaz, southwestern iran and it seems to be the dominant species of dwellings particularly in apartments (vazirianzadeh et al. 2013, sharififard et al. 2014). this is small cockroach measuring 10–14 mm in length with pronotum of male is rather uniformly dark with lateral edges and definitely lacks of the two parallel stripes of blatella germanica. the adult males appear to be very slender with its wing extending beyond the tip of the abdomen. adult females have short wings which expose the considerable portion of their stout abdomen. the common name derives from presence of two dark-colored transverse bands on mesonotal and abdominal terga (cochran 1999). this cockroach species carries a variety of microorganisms (le guyader et al. 1989), and is a vector of pathogenic bacteria in urban environments (rivault et al. 1994). it is also reported as an allergen source (eggleston and arruda 2001, savoldelli and luciano 2005). twenty nine bacterial species were isolated from s. longipalpa caught in the hospital (le guyader et al. 1989). its movement from one department to the others, inside the hospital increases potential bacterial contamination risks, for some of the species such as acinetobacter and pseudomonas are dangerous for some kind of patients (le guyader et al. 1989). the cockroach infestations are common particularly in dwellings without proper ventilation in warm climates, hospitals, and restaurants and in business establishments with a relatively high ambient temperature and humidity (schal et al. 1984, le guyader et al. 1989, baumholtz et al. 1997, phillips and appel 2010). the brown-banded cockroach needs nearly much less relative humidity to complete its life cycle and spread the infestation. it may be the main reason for more distribution of this cockroach species in ahwaz city, as a warm area, in recent years compared with same species, the german cockroach. conventional insecticides are used as main tool to control cockroach infestations but there are many concerns about the harmful side-effects of these chemical compounds. also the insecticide use is restricted in places such food preparation areas, restaurants, storage buildings and apartments. these restrictions of chemical insecticide application increase demand for safer alternatives against cockroach infestations (savoldellis and suss 2005, phillips and appel 2010). different level of resistance to many compounds of chemical insecticides including organochlorine, organophosphorus and carbamat insecticides have been documented in many fieldcollected strains of cockroaches from iran. so, application of these insecticides should be stopped and replaced with other safer compounds (nasirian et al. 2006, nasirian 2010, ladonni et al. 2013). essential oils, as secondary plant compounds responsible for the aromatic characteristics of plants, present the potential alternative to conventional insecticides (isman 2000, 2006, omara et al. 2013). plant extracts and essential oils are reported to have a wide range of activity against insect and mite pests, plant pathogens, fungi and nematodes (isman 2006). recent reports have highlighted antimicrobial, antifungal, anticancer and insecticidal properties of plant essential oils (isman 2000, 2006). they have fumigants, antifeedant and repellent effects as well as inhibiting the reproduction in cockroaches and other insects (omara et al. 2013). they could be used in areas where chemical insecticides are prohibited. the repellent effect of essential oils has been reported against many insect pests such as cockroaches, termites, mosquitoes, ticks, ants and houseflies (chen et al. 2002). numerous j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 530 http://jad.tums.ac.ir published online: october 04, 2016 studies have demonstrated the toxicity and repellency of essential oils against cockroaches (ahmad et al. 1995, apple et al. 2001, jang et al. 2005, thavara et al. 2007, ferrero et al. 2007, ling et al. 2009, tunza et al. 2009, phillips et al. 2010, phillips and appel 2010, zhu et al. 2012, manzoor et al. 2012, omara et al. 2013). consider to cockroaches role in transmission and distribution of many human pathogens, their resistance to many chemical insecticides, the side-effects of insecticide usage in human dwellings, high distribution of the brown-banded cockroach in ahvaz city during recent years, and eventually in order to finding a safe alternative for chemical insecticides, the present study was done to evaluate toxicity and repellency of following five essential oils, eucalyptus oil (eucalyptus sp), mint oil (mentha piprita), yarrow oil (achillea millefolium), oregano oil (origanum vulgare) and rosemary oil (rosmarinus officinalis) against this cockroach species. material and methods cockroach the brown-banded cockroaches were reared in plexiglas containers and maintained at 27±2 °c, 50±5% rh, and the photoperiod of 12:12 (l: d) h. they were fed with dry crumbled biscuits, bread and water. pieces of facial tissue were provided as a harborage and surface sticking of ootheca. cockroaches were anesthetized by chilling to facilitate handling. the cockroach colony had been established at least 2–3 years before the study. the study was done in the laboratory of medical entomology, department of medical entomology and vector control, ahvaz jundishapure university of medical sciences during april 2012 to september 2013. essential oils five essential oils including, eucalyptus sp (eucalyptus oil), mentha piprita (mint oil), achillea millefolium (yarrow oil), origanum vulgare (oregano oil) and rosmarinus officinalis (rosemary oil) used in the tests were extracted from fresh or dried plant foliage by the hydrodistillation method using clevenger apparatus in herbal and natural product research center of ahvaz jundishpapur university of medical science. plant material was placed in a 2-liter round bottomed flask with distilled water (100 ml for 75g dry material and 400 ml for 200 g fresh material) and the essential oil was extracted by water distillation. the distillation period was 1 h for fresh samples and 1 h 15 min for dried samples (charles and simon 1990). bioassays contact toxicity: the bioassay method of who (world health organization) (1975) was used to determine susceptibility or resistance of the cockroaches to essential oils. essential oils were prepared in acetone as the solvent (v/v) at concentrations of 2.5%, 5%, 10%, 15% and 30%. glass jars (600 ml) were treated uniformly with 2.5 ml of each concentration and left under room conditions to dry. the top inner surfaces of jars were smeared with a thin layer of butter to restrict cockroach movement within the jars. groups of thirty 3rd and 4th nymph instars were anesthetized by chilling and transferred to plastic cups. after recovery, they were transferred to treated jars and left to continuous exposure to treated surface. control group was exposed to surface treated with acetone. the mortality was calculated 24 h after recovering period. fumigant toxicity: groups including thirty 3rd and 4thnymphs instars were released in 1lit glass jars with a 1-cm diameter cotton ball treated with 50 µ l pure essential oil. to prevent direct contact of cockroaches with essential oil, it was injected to the center of each cotton ball by micro sampler. control group was exposed to cotton ball treated j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 531 http://jad.tums.ac.ir published online: october 04, 2016 with acetone. the mortality was calculated 24 h after recovering period. repellency test: the method applied that of ferrero (2007) and manzoor (2012) with some modifications. circular white filter paper no 1 (15 cm diameter, whatman) was divided in to two approximately equal pieces. acetone was used as the solvent. one half was treated with 1 ml essential oil solution using a micro sampler and the other half was left untreated. the oils were assayed at the following 5 concentrations: 2.5, 5, 10, 15 and 30% (v/v). after solvent evaporation, filter paper was used to cover the floor of cylindrical plexiglas jars. thirty nymphs (3rd and 4th instars) were released into the center of each jar and distribution of the nymphs was calculated 24 h after exposure. for the control group, one half of the filter paper was treated with acetone and the other half was left untreated. all experiments were done with four replicates. data analysis means of mortality percentages and standard errors in contact and fumigant toxicity were calculated using spss 16 software. repellency values (rv) were determined using this formula: repellency (%)= 100-(t×100)/n where t stands for the number of cockroaches located in the treated area and n stands for the total number of cockroaches used (thavara et al. 2007). analyses by anova and comparison of mortality and repellency percentage means was done by tukey’s test (p< 0.05), using spss software (chicago, il, usa). results contact toxicity concentrations of 30% and 15% of the five essential oils caused 100% mortality against the cockroach nymphs using continuous exposure method. mortality rates were 100%, 62.2%, 45%, 36.2% and 5.2% respectively at the concentration of 2.5% for the essential oils of rosemary, oregano, yarrow, eucalyptus and mint after 24 h (table 1) which were significantly different (p< 0.0001). furthermore, significantly difference was noted between effectiveness of the essential oils with comparison of the total means of mortality (p< 0.0001) against the brown-banded cockroach. rosemary oil was the most toxic oil against s. longipalpa because it caused 100% mortality of the cockroach nymphs at all concentrations (table 1). the next most effective oils were oregano and eucalyptus oils because they killed 100% of the nymphs at the concentration range of 5–30%. at the concentration of 2.5% nymph mortality means were reduced to 62.2% and 36.2% with oregano and eucalyptus oils respectively which showed significantly difference (p< 0.0001). so, oregano oil was more effective than eucalyptus oil for the brown-banded cockroach (table 1). mortality means of the cockroach nymphs varied from 45% to 100% by yarrow oil which were significantly different (p< 0.0001). mint oil caused 100% mortality at concentrations range of 10–30% but mortality reduced to 24.7% and 5.2% at the treatments of 5% and 2.5% respectively. so, mint oil was clearly determined as the least effective oil against the cockroach at lower concentrations compared to the other tested oils but its effect was similar to that of other oils at higher concentrations. all nymphs in the control group remained live 24 h after exposure, and even after a week. fumigant toxicity no mortality was observed in the control group during 24 h test period. fumigation of all the five essential oils caused 97.2% to 100% mortality in the brownbanded cockroach nymphs at 24 h after exposure or even earlier. mint oil exhibited lower fumigant efj arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 532 http://jad.tums.ac.ir published online: october 04, 2016 fect compared to the other essential oils and it caused a mean mortality rate of 97.2% in nymph population that was not significantly different with the other oils (p> 0.05). repellency effect all tested essential oils showed high repellency against the brown-banded cockroach at the prepared concentrations (table 2). evaluations for repellent activity were different according to the essential oil and the concentration. the highest and the lowest repellent effects were recorded in oregano oil and eucalyptus oil respectively compared with the control group. repellent effect of oregano oil at different concentrations ranged from 96.5 to 99.1% which were not significantly different together (p> 0.05), while the highest repellency (99.1%) was observed at the lower concentration of 2.5% (table 2). in the repellency test with oregano oil, nearly all the released cockroaches were concentrated at the same place in untreated area and they were not close to the treated area even after a week. eucalyptus oil was determined as the least repellent compound, it caused 27.7–49.8% repellency at different concentrations against the brown-banded cockroach compared to the other oils. its repel effects were a little significantly different (p< 0.05) at different concentrations. mint oil caused 59.1–68.8% repellency against the brown-banded cockroach and that repellency of this oil at lower concentration (2.5%) was not significantly different with higher concentrations (p> 0.05). repellent effect of yarrow oil was varied from 79.3 to 92.8% that was not significantly different (p> 0.6). the most repellent activity was related to the concentration of 5% that was 92.8% but decreased to 79.3% at the concentration of 2.5% while the different between repellency at the concentration of 2.5% and 5% was not significantly (p= 0.149). the repellent effect of rosemary oil was tested with only two concentrations. while the highest effect was recorded at the lower concentration of 2.5% (94.3%) but it was not differ significantly with the concentration of 5% (86.2%). the difference was significant between the repellency of the essential oils with control group (p< 0.0001). table 1. contact toxicity of essential oils against supella longipalpa at different concentrations at 24 h after recovering period. (department of medical entomology, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 essential oil concentrations (%) (acetone was used as solvent) mortality means (%)±se rosemary oil 30 15 10 5 2.5 100 100 100 100 100 oregano oil 30 15 10 5 2.5 100 100 100 100 62.2±1.4 eucalyptus oil 30 15 10 5 2.5 100 100 100 100 36.2±1.5 j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 533 http://jad.tums.ac.ir published online: october 04, 2016 yarrow oil 30 15 10 5 2.5 100 100 93±3 79.9±3.3 45±2.9 mint oil 30 15 10 5 2.5 100 100 100 24.7±4.1 5.2±1.9 control (treated with acetone) 100 0 table 2. repellency effects of plant essential oils against the brown-banded cockroach, s. longipalpa at 24 h after recovery period (department of medical entomology, ahvaz jundishapure university of medical sciences during april 2012 to september 2013. essential oil concentrations (%) (acetone was used as solvent) repellency means (%)±se origanum vulgare 30 15 10 5 2.5 96.5± 3.5 96.8 ± 3.8 96.03± 2.4 98.8± 1.2 99.1± 0.9 eucalyptus sp 30 15 10 5 2.5 27.7± 3.9 33.3 ± 3.1 43.2± 2.8 51.7. ± 0.9 49.8± 4.9 mentha piprita 30 15 10 5 2.5 63.3± 4.7 60.1 ± 3.8 59.01± 2.8 68.8± 3.2 63.3± 3.7 achillea millefolium 30 15 10 5 2.5 86.7 ± 4.9 84.2± 3.5 83.3± 4.5 92.8± 1.7 79.3± 3.9 rosmarinus officinalis 30 15 10 5 2.5 86.2± 4.3 94.5± 0.87 control (treated with acetone) 100 6.6± 2.3 discussion there are many studies on evaluation of essential oils against other species of cockroaches, but this is the first time to evaluate the efficacy of these components against the brown-banded cockroach, s. longipalpa. the tested essential oils showed contact toxicity, fumigant toxicity and repellent activity against the brown-banded cockroach compared to table 1. continued… j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 534 http://jad.tums.ac.ir published online: october 04, 2016 control group during this study. results of continuous contact toxicity showed considerable effect at different concentrations, but yarrow oil was determined as the most toxic oil. it caused 100% mortality even at the lowest concentration of 2.5%. comparison contact toxicity of rosemary, oregano, yarrow, eucalyptus and mint oils at the lowest concentration (2.5%) showed mortality rates of 100%, 62.2%, 45%, 36.2% and 5.2% respectively at 24 h after treatment. these mortality rates were significantly different together and with control group and confirmed that rosemary oil was the most toxic oil against the brown-banded cockroach nymphs. the highest repellency was related to oregano oil and the most repellent activity was observed at lower concentrations of 2.5% that was 99.1% with a residual effect lasting at least a week after treatment. oregano and rosemary oils were more efficient at the concentration of 2.5% with 99.1% and 94.5% repellency. yarrow, mint and eucalyptus oils showed the most repellent effects at the concentration of 5% but the differences were not significant with the repel activity of 2.5% (p values were 0.15 and 0.97 and 0.99 for yarrow, mint and eucalyptus oils respectively). repellency of these oils was 92.8%, 68.8% and 51.7% at the concentration of 5% and it was 79.3%, 63.3% and 49.8% at the concentration of 2.5%. so it could be concluded that the essential oil concentration of 2.5% is the favorite concentration to recommend for repellency against the brown-banded cockroach for further evaluation. many previous studies have evaluated the efficacy of different essential oils against cockroaches. evaluation of repellency and fumigant toxicity of clove (syzygium aromaticum) and sesame (sesamum indicum) oils against the american cockroach (periplaneta americana) showed complete repellency (100%) against first nymph at concentration of 2% for clove oil and 6% for sesame oil. same result was obtained against fourth nymph at concentration of 10% of sesame oil after 48 h. while clove oil completely repelled all fourth nymphs after 24 h at concentration of 8 %. for adult stage, the greatest repellency percentages were recorded by clove oil (90.00± 5.77%) and sesame oil (83.33±3.33%) after 48 h at a concentration of 10% (omara et al. 2013). the repellency value of clove oil at concentration of 8 % against the fourth instar nymphs of the american cockroach is nearly close to our repellency value of oregano oil at concentration ranges of 2.5–10% which repel 96.03–99.1% of the brown-banded cockroach nymphs after 24 h and event lasted for a week. both essential oils have considerable repellency against cockroaches but the cockroach species, essential oil type led to a little difference in the obtained results. toxicity and repellent evaluation of eucalypltus citriodora, mentha arvensis and cymbopogen citratus against p. americana. c. citratus exhibited the maximum toxicity and repellency with 20% to 100% toxicity between 2 to 24 h intervals, 100% repellency and 70–100% fumigation after 24 h exposure. minimum repellency ranged from 11–67% was observed from eucalyptus oil against p. americana (manzoor et al. 2012). the minimum repellency against s. longipalpa was also recorded for eucalyptus oil in our study that ranged from 27.7–51.7%. differences in the obtained result could be probably related to difference in cockroach species. in another study, the essential oil of citrus hystrix exhibited 100 % repellency effect against p. americana and blatella germanica, and also about 87.5% against neostylopyga rhombifolia under laboratory conditions. the essential oil caused an 86% reduction in cockroaches with a residual effect lasting a week after treatment in the field (thavara et al. 2007). these results are also similar to repellency values of oregano oil that continued for at least a week or even 10 days after treatment. j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 535 http://jad.tums.ac.ir published online: october 04, 2016 besides, all five essential oils showed nearly complete fumigant toxicity (97.2– 100%) against the brown-banded cockroach at the concentration of 50 µ l per 1 lit air of pure oil. apple et al. (2001) investigated that fumigant toxicity of mint oil at the concentration of 50 µ l per 1 lit air of pure oil killed 100% of both p. americana and b. germanica after 24 h and caused 92.3–100% repellent effect on both cockroach species during each day of the 14day experiment (appel et al. 2001). repel activity of mint oil ranged from 59.1–68.8% against s. longipalpa with the concentration of 2.5–30% and the fumigant toxicity was 97.2% at 24 h after exposure in our study. it seems that the fumigant toxicity of mint oil in the mentioned concentration is not significantly difference against the three cockroach species whereas the repellent activity shows considerable difference. this difference could be related to cockroach species or essential oil component. clove oil provided highly fumigant toxicity against nymphs and adults of p. americana after 24 and 48 h, respectively. complete mortality (100%) was recorded at a concentration of 7.5 µ l/l of air for first nymph, 10 µ l for fourth one and 17.5 µ l for adults after 48 h of fumigation (omara et al. 2013). in the another study, the fumigant toxicity of 12 essential oil components including: carvacrol, 1,8-cineole, trans-cinnamaldehyde, citronellic acid, eugenol, geraniol, s-(-)-limonene, (-)-linalool, (-)-menthone, (+)-alphapinene, (-)-beta-pinene, and thymol was determined against adult male, adult female, gravid female, and large, medium, and small nymphs of the german cockroach, b. germanica. 1,8-cineole was the most toxic essential oil component to adult males and females, gravid females, and large nymphs, with lc50 values of 6.8, 8.4, 5.3, and 11.4 mg/liter air at 24 h, respectively (phillips and appel 2010). evaluation of fumigant toxicity of some plant essential oils by tunza et al. determined that allyl isothiocyanae (monoterpen oil component) was the most toxic compound, followed by essential oil of allium sativum against b. germanica (tunza et al. 2009). while all five essential oils exhibited complete fumigant toxicity against s. longipalpa which is comparable with the findings of other studies but it is necessary to test lower concentrations. the findings of current study are confirmed by all the mentioned studies in that essential oils can be favorite alternative to conventional chemical insecticides to control cockroach infestations especially in situations where the use of chemical insecticides has many harmful side effects. differences in the obtained results could be probably related to difference in cockroach species, type of essential oil, bioassay method and exposure time. plant essential oils offer the potential repellent agents to cockroach infestations in hidden and hard-to-reach areas and to eliminate such infestations (steltenkamp et al. 1992, ngoh et al. 1998). this strategy would increase the efficacy of non-repellent, insecticide treated areas because cockroaches in the hidden areas will be get out and they will exposed to such treated areas and finally killed (steltenkamp et al. 1992). besides, essential oils could be applied as a safe treatment and used to treat surfaces for food preparation in order to deter cockroach infestations (steltenkamp et al. 1992). essential oil applications may also be effective around cockroach infested places that cannot be treated with chemical insecticides, such as food stores, sensitive equipment, clothes lockers and in beds (koehler et al. 1995). the repellent effects of essential oils could be applied as a flushing agent during inspections of cockroach infestation in order to determine the degree of an infestation. conclusion given that s. longipalpa can be found in j arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 536 http://jad.tums.ac.ir published online: october 04, 2016 all parts of the buildings such as clothes lockers, beds, furniture and cabinets and that ootheca stick in hard-to-reach or obscure places, application of chemical insecticide spraying in such places is hard and unacceptable by the people. however, the use of essential oils presents a safe alternative for indoor application against cockroaches because of less toxicity to humans and no toxic residues. oregano oil showed considerable repellent effect against s. longipalpa, so, it could be recommended as a potential repellent compound for further evaluation on a larger scale and under field conditions. contact and fumigant toxicity of the five selected essential oils were considerable but additional study is required for the practical application and developing favorable formulation of them against the brownbanded cockroach and also other cockroach's species. acknowledgments the paper is issued from a research project with project no. u90200 and financial support was provides by ahvaz jundishapure university of medical sciences. we appreciate experts of the herbal medicine and natural product research center of ajmus for providing the essential oils. the authors declare that there is no conflict of interests. references ahmad fbh, mackeen mm, ali am, mashirun sr, yaacob mm (1995) repellency of essential oils against the domiciliary cockroach, periplaneta americana. int j trop insect sci. 16: 391–93. apple ag, gehret jm, tanley mj (2001) repllency and toxicity of mint oil to american and german cockroaches (dictyoptera: blattidae and blattellidae). j agric urban entomol. 18(3): 149–156. baumholtz ma, parish lc, witkowski ja, nutting wb (1997) the medical importance of cockroaches. int j dermatol. 36(2): 90–96. charles dj and simon je (1990) comparison of extraction methods for the rapid determination of essential oil content and composition of basil. j amer soc hort sci. 115(3): 458–462. chen f, zungoli pa, benson e (2002) screening of natural insecticides from tropical plants against fire ants, termites, and cockroaches. final report, clemson university, clemson. available at: http://www.clemson.edu/ipm/ reports/02chen ferrero aa, sanchez chopa c, werdin gonzalez jo, alzogaray ra (2007) repellence and toxicity of schinus molle extracts on blattella germanica. fitoterapia. 78(4): 311–14. isman m b (2000) plant essential oils for pest and disease management. crop protect. 19: 603–608. isman mb (2006) botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. ann rev entomol. 51: 45–66. jang y s, yang y c, choi d s, ahn y j (2005) vapor phase toxicity to marjoram oil compounds and their related monoterpenoids to blattella germanica. j agric food chem. 53: 7892–7898. koehler pg, patterson rs, owens jm (1995) chemical systems approach to german cockroach control. in: rust mk, owens jm, reierson da (eds) understanding and controlling the german cockroach. oxford university press, new york, pp. 287–324. le guyader a, rivault c, chaperon j (1989) microbial organisms carried by brown-banded cockroaches in relation to their spatial distribution in a hospital. epidem inf. 102: 485–492. ling i, sulaiman s, othman h (2009) evaluaj arthropod-borne dis, december 2016, 10(4): 528–537 m sharififard et al.: evaluation of some … 537 http://jad.tums.ac.ir published online: october 04, 2016 tion of piper aduncum linn. essential oil (family: piperaceae) against periplaneta americana (l.). iran j arthropod-born dis. 3(2): 1–6. manzoor f, munir n, ambreen a, naz s (2012) efficacy of some essential oils against american cockroach periplaneta americana (l.). j medicine plant research. 6(6): 1065–1069. ngoh sp, choo lew, pang fy, huang y, kini mr, ho sr (1998) insecticidal and repellent properties of nine volatile constituents of essential oils against american cockroach, periplaneta americana (l.). pestic sci. 54: 261–268. omara sm, al-ghamdi km, mahmoud m, sharawi se (2013) repellency and fumigant toxicity of clove and sesame oils against american cockroach (periplaneta americana (l.). afr j biotechnol. 12(9): 963–970. phillips ak, appel ag (2010) fumigant toxicity of essential 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wongsinkongman p, boonruad t, bansiddhi j, chavalittumrong p, komalamisra n, siriyasatien p, mulla ms (2007) repellent activity of essential oils against cockroaches (dictyoptera: blattidae, blattellidae, and blaberidae) in thailand. southeast asian trop med public health. 38(4): 663–673. tunza h, kubilay er m, isikber aa (2009) fumigant toxicity of plant essential oil and selected monoterpenoid components against adult german cockroach, blattella germanica (l.) (dictyoptera: blattellidae). turk j agri. 33: 211–217. vazirianzadeh b, dehghani r, mehdinejad m, sharififar, nasirabadi n (2014) the first report of drug resistant bacteria isolated from the brown-banded cockroach, supella longipalpa, in ahvaz, south-western iran. j arthropod-borne dis, june 2014. 8(1): 53–59. who (1975) tentative instruction for determining the susceptibility or resistance of cockroaches to insecticides. world health organization, switzerland. who/vbc/75. zhu wx, zhao k, chu ss, liu zl (2012) evaluation of essential oil and its three main active ingredients of chinese chenopodium ambrosioides (family: chenopodiaceae) against blattella germanica. j arthropod-borne dis. 6(2): 90–97. j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 original article sand flies of the subgenus adlerius (diptera: psychodidae) in an endemic focus of visceral leishmaniasis and introduction of phlebotomus (adlerius) comatus as a new record for iran ali reza zahraei-ramazani 1, dinesh kumar 1, *mohammad reza yaghoobi-ershadi 2, abdollah naghian 2, reza jafari 3, mohammad reza shirzadi 4, hamid abdoli 3, hassan soleimani 5, niloofar shareghi 3, maryam ghanei 3, mohammad hossein arandian 3, ahmad ali hanafi-bojd 2 1department of zoology, centre of advanced study, banaras hindu university, india 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3esfahan health research station, national institute of health research, esfahan, iran 4communicable disease management center, ministry of health and medical education, tehran, iran 5yazd health research station, national institute of health research, yazd, iran (received 12 dec 2012; accepted 22 jan 2013) abstract background: sand flies of subgenus adlerius has a wide geographical distribution in iran and are mostly found in wild form in mountainous areas. they are always considered as probable vectors of visceral leishmaniasis. the objective of this study was to determine the adlerius species and its composition in an endemic focus of zoonotic visceral leishmaniasis in northwest of the country. methods: sand flies were collected from 6 different areas of azarbaijan-e-sharqi province using sticky paper traps from august to september which is active season for sand flies in this area, in 2009. the flies were mounted and identified. the length of third antennal segments, ascoid, labrum, coxite, surstyle, style, aedeagus, genital filament, genital pump, width of style, and the end of aedeagus were measured and the number of costal hairs group was also counted as the morphological characters. results: a total of 30 adult sand flies, (26 males and 4 females) including phlebotomus halepensis (46.8%), p. longiductus (13.3%), p. balcanicus (23.3%), p. comatus (3.3%), and adlerius spp. (13.3%) belong to subgenus adlerius were identified respectively in 6 counties. one p. comatus male was captured in front of a cave located in the hillside of a mountain covered with the vegetation in varzeqan area. conclusion: the presence of at least 5 species of the subgenus adlerius in azarbaijan-e-sharqi province, an endemic focus of zoonotic visceral leishmaniasis in iran, shows that the risk of parasite transmission among man and reservoir animals is high during the active season of sand flies. p. comatus is a new record for iran and needs to be added to the list of iranian phlebotomines of subgenus adlerius. keywords: phlebotomus (adlerius) comatus, phlebotomine sand flies, visceral leishmaniasis, new species, iran introduction visceral leishmaniasis (vl) is a fatal protozoan disease caused by leishmania donovani complex (kinetoplastidae: trypanosomatidae) transmitted by the bites of phlebotomine sand flies (diptera: psychodidae) of the genus phlebotomus in the old world and lutzomyia in the new world. it is a serious illness that gives rise to epidemics and causes high mortality if left untreated. visceral leishmaniasis appears as an opportunistic disease associated with the hiv infection and in other immune suppressed patients (desjeux and alvar 2003). *corresponding author: dr mohammad reza yaghoobi-ershadi, email: yaghoobia@tums.ac.ir 1 j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 leishmania infantum nicolle, 1908 can also cause cutaneous lesions (who 2010). there are at least 7 endemic foci of zoonotic visceral leishmaniasis (zvl) in iran as follows: in iran, some parts of fars province in the south and ardabil province in the northwest are indicated as the highly endemic areas while some parts of azarbaijan-e-sharqi province in the northwest, saveh and qom in the central, khorramabad area of lorestan province in the west, some parts of khuzestan province in the southwest and khorassan-e-shomali area in the northeast are known as the low endemic areas. in all foci, kala-azar is caused by l. infantum. dogs, foxes and jackals are main reservoir hosts (yaghoobi-ershadi 2012). kala-azar is endemic in four counties of azarbaijan-e-sharqi province including ahar, sarab, kaleybar and azarshahr. five species of sand flies including p. kandelakii, p. perfiliewi, p. keshishiani, p. neglectus and p. alexandri are considered as probable vector species of zvl in iran (yaghoobi-ershadi 2012). the subgenus adlerius nitzulescu includes about 20 species which some are suspected or proven vectors of zvl in the old world, as follows: phlebotomus balcanicus in armenia and georgia, p. longiductus in china, kazakhestan, kyrgyzstan, ukrine and uzbekistan, p. halepensis in syrian arabic republic and georgia (who 2010). the main key to species identification of subgenus adlerius of iran was prepared by seyedi-rashti and nadim (1992). they believed that females of the subgenus are unidentifiable. so the key is based on male morphology as follows: 1coxite with less than 29 hairs. subterminal tubercle of aedeagus 19–28 μm from tip. aedeagus short and thick. p. brevis coxite with more than 30 hairs. aedeagus long. 2 2aedeagus with rectangular subterminal notch, coxite with 50 hairs or less (39– 40). p. halepensis subterminal tubercle of aedeagus not rectangular. coxite with more than 50 hairs. 3 3coxite with 50–80 hairs. genital filaments very long 8–15 times length of pump. p. longiductus coxite with 69–105 hairs. genital filament 8 times length of pump. p. balcanicus in the last 3 decades zvl has become an endemic disease in azarbaijan-e-sharqi province, northwest of the country. the objective of this study was to determine the adlerius species and their population size in an endemic focus of zvl in northwest of the country. materials and methods study area the azarbaijan-e-sharqi province is located in the northwest of iran (38.0766 o n, 46.2800 o e) bordering armenia and the republic of azerbaijan countries and the provinces of ardabil, western azarbaijan, and zanjan (fig. 1). it covers an area of approximately 47830 km2, with altitude range of 1310 m in plain locale to 3722 m in highland locale. it has a cool and dry climate being in the main a mountainous region. temperatures run between -17 °c in the winter and up to 40.6 °c in the summer. the annual precipitation is about 255 mm. it has a population of 3691270 people (sci, 2006). sand flies collection sand flies were collected monthly from 6 different areas including tabriz, ahar, sharabianesmaeilabad, varzeqan, kaleibar and marand in azarbaijan-e-sharqi province using 50 sticky paper traps (castor oil coated white papers 15×21 cm) from the beginning (august) to the end (september) of the active season in 2009. traps were installed before sunset and collected before sunrise. collected sand flies 2 j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 were removed from sticky papers using needle dipped and stored in 75% ethanol. females of sand flies belong to the subgenus adlerius are more or less morphologically identical and there is no key for identification so only male specimens were selected for morphological study (seyedi-rashti and nadim 1992). mounting the head and genitalia of individual male sand flies were cut off within a fresh drop of sterile saline on a clean slide then they were transferred to a drop of puri’s media between a slide and cover slip (smart et al. 1965). identification of specimens was carried out after 24 h using the keys of theodor and mesghali 1964, artemiev 1973, lewis 1982, seyedi-rashti and nadim 1992. morphometric measurements morphometric measurements were done by olympus microscope (ch-2) and photographs were taken by camera of leica microscope (model: dm 2000). the specimen characters on the length of third antennal segments, ascoid, labrum, coxite, surstyle, style, aedeagus, genital filament, genital pump, and also the width of style were measured with different magnifications. in addition, for the p. comatus male, the end of aedeagus was measured. number of costal hairs group, a very important morphological character, for all adlerius specimens were counted. the entire given measurements are in micrometer (μ.m). results adult sand flies belong to genus phlebotomus were collected and identified. males were significantly larger numbers than females from the area sampled (30:4). thirty four phlebotomus sand flies belong to the subgenus adlerius (30 males and 4 females) were identified. phlebotomus halepensis (46.8%), p. longiductus (13.3%), p. balcanicus (23.3%), p. comatus (3.3%), and females of adlerius spp. (13.3%) have been captured during this study in 6 areas of the azarbaijan-e-sharqi province (table 1). phlebotomus halepensis was present in the all areas. the relative abundance of this species was 46.8%, higher than of the other species in comparison. the most captured adlerius specimens, including p. halepensis, p. longiductus and p. balcanicus, were related to varzeqan area. the altitudinal distribution and abundance of all adlerius species collected from the study areas are shown in table 1. phlebotomus halepensis was found at an altitude range of 1200–1670 m, p. longiductus was captured from two different altitudes at 1200 and 1670 m, while p. balcanicus and p. comatus were only trapped at 1670 m. as the table shows different species of subgenus adlerius have been collected at altitude of 1670 m in varzeqan area. it seems that the highest altitudes are preferred for breeding places of these phlebotomine sand flies. we found only one phlebotomus comatus male from varzeqan area, which was a new record for iran. it was collected in front of a cave located in the hillside of a mountain covered with the vegetation in varzeqan area. the humidity was measured as normal (60%). table 2 shows the measurements of the species. in this specimen, the coxite is wide with dense group of about 176 hairs. the hairy spots are located almost the middle of the coxite. the paramere is pigmented at the end. aedeagus with subterminal tooth at 23 μ.m from the end. the length of genital filament is 931 μ.m and f/p=6.5. figure 2 shows photos of some important characters on the head eg, antennal segment 3, clypeus, epipharynx and palp and also the genitalian parts at the end of the abdomen eg, genital filament, genital pump, length and width of aedeagus, dense group of 176 hairs on coxite and pigmented paramere. 3 j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 table 1. sand flies of subgenus adlerius and their relative abundance (%) in 6 areas of azarbaijan-e-sharqi, an endemic province of visceral leishmaniasis in northwest of iran row area altitude* species of subgenus adlerius (male) p. halepensis p. longiductus p. balcanicus p. comatus adlerius sp. (female) total no. % no. % no. % no. % no. % no. 1 2 3 4 5 6 tabriz ahar mianeh varzeqan kaleibar marand 1200 m 1367 m 1300 m 1670 m 1240 m 1334 m 1 3 1 6 2 1 25 100 25 40 100 50 3 0 0 1 0 0 75 0 0 6.7 0 0 0 0 0 6 0 1 0 0 0 40 0 50 0 0 0 1 0 0 0 0 0 6.7 0 0 0 0 3 1 0 0 0 0 75 6.6 0 0 4 3 4 15 2 2 total 14 46.8 4 13.3 7 23.3 1 3.3 4 13.3 30 *(statistical center of iran, 2006) fig. 1. map of azarbaijan-e-sharqi province, showing sand fly sampling areas 4 j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 table 2. the comparison of morphometric measurements (μ.m) on males of phlebotomus (adlerius) comatus from iran and afghanistan country iran present study afghanistan (seyedirashti and nadim) * number of coxal setae 176 ascoid 3 length 49 labrum length 270 270–300 clypeous length 180 cibarium teeth unarmed unarmed palp1 length 60 palp2 length 175 palp3 length 190 coxite length 450 420 surstyle length 450 style length 220 200 style width 50 aedeagus length 170 aedeagus end subterminal tooth a3 length 399 310–370 genital filament 931 genital pump 144 *(seyedi-rashti and nadim 1992) fig. 2. phlebotomus (adlerius) comatus artemiev male: 1a, antennal 3(×400), 1b, pharynx and pharyngeal armature (×400), 1c, epipharynx (labellum, maxilla) and palp (×400), 1d, genital filament, genital pump, surstyle (×100), 1e, dense group of 176 hairs on coxite (×1000), 1f, length and width of aedeagus, paramere (×630), 1g, style and the seta(×400), 1h, pigmented paramere (×400), 1i, clypeus(×400) 5 j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 discussion most species of the subgenus adlerius were considered as subspecies of phlebotomus chinensis (lewis 1978). theodor and mesghali (1964) supposed that some of the subspecies may have to be made into species. after the examination of numerous specimens from afghanistan, central asia and caucasus it was concluded that the supposition of these scientists was right (artemiev 1973). males of all described adlerius species from iran can be easily identified according to antennal formula, position of the coxal tuft and its number of setae and shape of aedeagus, but females are much more uniform. prior to this study six species including p. halepensis, p. brevis, p. longiductus, p. balcanicus, p. turanicus and p. salangensis were recorded from iran (seyedi-rashti and javadian 2008, akhundi et al. 2012). whether p. kabulensis has also been recorded in the past (kasiri et al. 2000) but it has not been confirmed by senior entomologists in the country yet. some morphomethric measurements have been processed to characterize another new species for the list of phlebotomine sand flies of iran (table 3). artemiev revised the adlerius of afghanistan in 1978. he caught and identified 44 males of p. comatus from northern and central afghanistan including kala-inaw (badghis), khulm, bamyan and gorband valley. he mentioned that it is a species of rocky mountain (1000–2600 m) and it seems to be moderately thermophilic and hydrophilic. he described that male of p. comatus has antennal formula 2/3–5, 1/6–15, coxite very wide, with dense group of 165(126203) hairs, distal border of the hairy spot at 0.47 (0.41–0.51) of coxite, paramere with wide pigmented end, aedeagus rather short, with subterminal tooth a 20 (16–24) μ.m from the end, genital filaments 1040 (800–1280) μ.m long, f/p=8.2 (6.9-9.4). seyedi-rashti and nadim (1992) mentioned that in afghan specimens of p. comatus, the measurements are as follows: the length of antennal segment 3, 310–370 μ.m, labrum 270–300 μ.m, coxite 420 μ.m and style 200 μ.m (table 3). the iranian male specimen of p. comatus has 176 numbers of coxal hairs. it is in the range record numbers of the afghan specimen by artemiev in 1978. antenna 3 observed to be longer than flies recorded by seyedi-rashti and nadim (1992) but labrum is in the range record of the latter authors. the length of coxite is larger and style length is also bigger than which was described by seyedi-rashti and nadim in 1992. according to the literature p. halepensis was recorded by theodor and mesghali from tabriz county, azarbaijan-e-sharqi province in 1964 and from sarab county by mesghali in 1961. during an entomological study of visceral leishmaniasis p. halepensis, p. brevis, p. longiductus and p. balcanicus were recorded in kaleybar county at the same province in 2000 (rassi et al. 2000). our captures and identifications showed that p. halepensis, p. longiductus and p. balcanicus are common species in this province. phlebotomus brevis which had recorded from the area previously was not found in this study. phlebotomus halepensis and p. brevis have wide distribution in iran at altitudes between 800 and 1700 m above sea level mostly in mountainous regions. phlebotomus longiductus and p. balcanicus have limited distribution in comparison of p. halepensis and p. brevis in the country. these four species are present in sympatry in the northwest of iran (ardehali et al. 1995). we caught the iranian specimen of p. comatus from varzeqan area in elevation of 1670 m. its coxite is also wide, paramere with wide pigmented ends. in comparison with other species aedeagus is short with subterminal tooth at 23 μ.m from the end and it is in the range record numbers of artemiv’s 6 j arthropod-borne dis, june 2013, 7(1): 1–7 ar zahraei-ramazani et al.: sand flies of the… http://jad.tums.ac.ir published online: april 10, 2013 specimens. the ratio of f/p in iranian specimen is also in the range of his record from afghanistan. it should be mentioned that varzaqan is the mining center for base metal mining as well as gold and farming is the predominant occupation in the rural areas. recently spruce trees have also been planted as the start of a forestry industry at the same place. more investigations are needed to reveal the distribution of p. comatus in iran and its role in the transmission of l. infantum in the study area remains to be specified. acknowledgments authors thank the leishmaniasis staff of esfahan, meshkinshahr and yazd health research stations, national institute of health research for their contribution in sand fly collections. the study was funded by deputy of research of tehran university of medical sciences through project number: 5146-2701-86. the authors declare that there is no conflict of interest. references akhundi m, parvizi p, baghaei a, depaquit j (2012) the subgenus adlerius nitzulescu (diptera: psychodidae, phlebotomus) in iran. acta trop. 122: 7–15. ardehali sv, rezaei hr, nadim a (1995) leishmania parasite and leishmaniasis. academic press center, tehran, iran, pp. 208 (in persian). artemiev v mm (1973) sand flies (diptera, psychodidae, phlebotomine) of afghanistan, malaria and leishmania institute, afghanistan. 174 pp. desjeux p, 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sciences, isfahan, iran 2department of genetics and molecular biology, school of medicine, isfahan university of medical sciences, isfahan, iran 3center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran 4cellular and molecular biology research center, department of parasitology, school of medicine, shaheed beheshti university of medical sciences, tehran, iran (received 15 apr 2007; accepted 14 jul 2008) abstract background: leishmania, needs to detoxify the macrophage derived potent peroxides (h2o2). tryparedoxin pathway contains tryparedoxin peroxidase (tsa or tryp). the aim of the study was to detect the full-length gene sequence and its encoded protein of the lmtryp6 gene (eu251502), and comparison the gene sequence with lmtryp6 (lmjf15.1140), another previously reported member of this gene family. methods: l.major (mrho/ir/75/er) promastigotes were cultured, dna and rna were extracted and the interested gene was amplified using pcr and rt-pcr methods. pcr/ rt-pcr fragments were purified and cloned first in ptz57r/t and then in pet15b expression vector. the expressed protein was verified using western blot method. characterization of the expressed protein was performed bioinformatically. results: molecular evaluation revealed that the cloned lmtryp6 gene (eu251502) encoded a predicted 184 amino acid long protein with a theoretical isoelectric point of 6.1101. alignment showed a number of changes in amino acid composition including the replacement of highly conserved trp177 by cys in lmtryp6 (abx26130). conclusion: so far no study has been done on this group, i.e. tryp6 gene, from tryparedoxin peroxidase family. the low homology with lmtryp6 (lmjf15.1140) and vast array of differences observed in the gene under study (lmtryp6; eu251502) could open new windows in the field of anti-leishmania combat. based on its important role in the viability and successful establishment of the parasite in the host organism it looks to be very good candidate for vaccine development and any other sort of novel drug development. keywords: tryparedoxin peroxidase, l. major, peroxiredoxin, tryp6, cloning introduction leishmania, similar to other aerobic organisms, is exposed to reactive oxygen species (ros) and reactive nitrogen species (nos) producing by macrophage as a part of defense mechanisms against invading parasites. ros damage various cellular components, including membrane lipids and nucleic acids, leading to parasite death. leishmania possesses a unique dithiol trypanothione (n1n8bisglutathionylspermidine) pathway which eliminates h2o2 (nogoceke et al. 1997, mcgonigle et al. 1998, muller et al. 2003). this pathway consists of a cascade of low molecular weight thiol specific oxidoreductases acting in order *corresponding author: dr rasoul salehi, tel: +98 311 7922487, e-mail: r_salehi@ med.mui.ac.ir iranian j arthropod-borne dis, (2008), 2(1): 7-15 g eslami et al: cloning and expression of … 8 of trypanothione reductase (tr), trypanothione (t [sh] 2), tryparedoxin (txn), and tryparedoxin peroxidase (txnpx) in a nad (p)h dependent manner to detoxify peroxides. as shown in genedb (www.genedb.org), the genes encoding tryparedoxin peroxidase in l. major comprise tryp1 (tryparedoxin peroxidase), tryp2 (tryparedoxin peroxidase, txnpx, pxn1, tsa), tryp3 (tryparedoxin peroxidase, txnpx, pxn3, tsa), tryp4 (tryparedoxin peroxidase, tsa, txnpx, pxn), tryp5 (tryparedoxin peroxidase, txnpx, pxn, tsa), tryp6 (tryparedoxin peroxidase, txnpx, pxn, tsa), and tryp7 (tryparedoxin peroxidase) that present on chromosome 15 in a tandem array. tryps 1, 3, 5 and 7 encode a predicted protein with 199 amino acids whereas tryps 2, 4 and 6 code a predicted protein with 191 amino acids (levick et al.1998). tryparedoxin peroxidase is a potential target area of specific trypanocidal agent (nogoceke et al. 1997) due to its unique antioxidant system in trypanosomatidae which is highly crucial for survival of parasite in an unfavorable oxidative environment (zarley et al. 1991, wilson et al. 1994). also, other functions have been attributed to tryparedoxin peroxidase including protection of the mitochondrial genome from direct or indirect peroxide-mediated damage (harder et al. 2006). recently, its role in arsenite resistant (lin et al. 2005) and metastasis (walker et al. 2006) are defined. the association of tryparedoxin peroxidase with metastasis in leishmania strengthens the link between parasite virulence and antioxidant defence (walker et al. 2006). peroxiredoxins are shown to possess peroxinitrite reductase activity and participate in detoxification of ros (chen et al. 1998, bryk et al. 2000). therefore, due its involvement in vast array of biological phenomenon, tryparedoxin peroxidase aimed as a target for investigation. to our knowledge there is no report on tryp6 gene sequence from l. major (mrho/ir/75/er), as an approved iranian isolate which is used for leishmanization and preparation of old world experimental leishmania vaccine and leishmanin (khalil et al. 1988, nadim and javadian 1998, sharifi et al. 1998, momeni et al. 1999, bahar et al. 2000, khamesipour et al. 2005). in this study the full-length gene sequence and its encoded protein of the lmtryp6 gene (eu251502) is reported, the gene sequence was also compared with lmtryp6 (lmjf15.1140), another previously reported member of this gene family. materials and methods parasites l. major promastigotes (mrho/ir/75/ er) were grown at 26±1º c in rpmi 1640 medium (sigma, usa) supplemented with 10% fetal calf serum (fcs, sigma), 100 u/ml penicillin g and 100 µg/ml streptomycin (hendricks et al.1978, ozbilgin et al. 1995, castro et al. 2004). dna extraction dna extraction was done based on method described by eisenberger and jaffe (1999) with a minor modification. sds (10%) was used instead of triton-x 100 (1%). the proteinase k and rnase a were added at the same time and incubated for 24 h. the extracted dna sample was quantified and analyzed by agarose gel electrophoresis. rna extraction rna extraction was performed using rnxtm solution (cinnagen) according to the manufacturer instruction. the sample was quantified and analyzed by agarose gel electrophoresis under rnase free condition. cdna synthesis using revertaidtm first strand cdna synthesis kit (#k1621, fermentas), cdna was synthesized according to the manufacturer instruction. primers sense and antisense oligonucleotide primers were designed based on the nucleotide seiranian j arthropod-borne dis, (2008), 2(1): 7-15 g eslami et al: cloning and expression of … 9 quence data of lmtryp6 gene (lmjf15. 1140) obtained from genebank. the sequences of sense and antisense primers used in this study are: 5′-atgtcctgcggtaacgccaag-3′ and 5′-ttacttgttgtggtcgaccttcatgc-3′. pcr, rt-pcr and sequence analysis pcr amplification was performed using l. major genomic dna or cdna as template. pcr master mix contained 10mm trishcl ph 8.3, 50mm kcl, 1.5mm mgcl2, 0.2mm each dntps, 20 pmol of each primer and 0.5 unit of taq polymerase (fermentas). thermal cycling was applied as follow: 94º c for 5 min as initial denaturation, 30 cycles with 94º c for 45 seconds, 63º c for 45 seconds and 72º c for 45 seconds. the final 1 cycle of 72º c applied for 20 min. the pcr product was analyzed by agarose gel and the bands contained amplified products were purified using high pure pcr product purification kit (#11732668001, roche). cloning of lmtryp6 in ptz57r/t this step was performed using inst/ aclonetm pcr product cloning kit according to the manufacturer instruction. the recombinant plasmid was transformed into e. coli xl1-blue. to confirm the ligation reaction success, restriction digest was performed on isolated plasmids and the size of the linearized recombinant vector was assessed by agarose gel electrophoresis. cloning of lmtryp6 in pet15b the insert of ptz57r/t was isolated using restriction enzyme digestion and ligated into pet15b expression vector using t4 dna ligase. the recombinant dna was transformed into e. coli bl21. the in-frame cloning was verified by sequence analysis of the isolated insert from purified vector. after this verification step, in vitro protein expression of lmtryp6 was carried out. expression of lmtryp6 protein in vitro the cultured bacteria harboring recombinant plasmids were induced to express by adding iptg (final concentration 1mm) into the culture medium. the protein was purified after 2, 4, 6 and 24 h post induction and analyzed by sds-page and western blot. characterization and molecular analysis the interest cloned gene and its encoded protein was analyzed separately using software tools accessible at ncbi, srs, swiss_prot and predict protein server. results amplification the pcr and rt-pcr products showed exactly equal size of lmtryp6 gene on agarose gel (fig. 1). cloning of lmtryp6 gene into ptz57r/t cloning of lmtryp6 gene was confirmed by restriction enzyme digestion using bamhi and ndei (fig. 2). cloning of lmtryp6 gene into pet15b the 555 bp fragments were cut from ptz57r/t vectors and cloned in the dephosphorylated pet15b. the recombinant pet15b plasmids were transferred into e. coli bl21 in order to propagating through bacteria. isolated recombinant dna plasmids were confirmed by restriction enzyme digestion (fig. 3) and sequencing. in vitro expression of lmtryp6 protein iptg induced recombinant lmtryp6 protein (abx26130) was expressed in e. coli bl21, analyzed and characterized using sds-page (fig. 4) and western blot methods (fig. 5). characterization and molecular analysis molecular characterization of the amplified lmtryp6 gene at dna level showed that the coding region of lmtryp6 (eu251502) contained 555 bp. a deletion in non-conserved sequence of the gene towards its cterminal domain was seen. the homology of lmtryp6 gene (eu251502) with lmtryp6 (lmjf15.1140) was 79.6%. the coding region of dna sequence was g_c rich (62.7%) and iranian j arthropod-borne dis, (2008), 2(1): 7-15 g eslami et al: cloning and expression of … 10 codon usage was biased for g or c occupancy in the third base position (97.3 %). 44.56% of the amino acids were encoded by ten codons uuc, cug, auc, ccg, aac, aag, gac, gag, cgc, and ggc. fig. 1. agarose gel electrophoresis of pcr products (555bp) resulting from dna (line 1) and cdna (line 3) amplification of the lmtryp6 gene (gene bank accession no eu251502). as shown in this fig., both amplicons have the same size. line 3 shows 100bp ladder. fig. 2. double digestion of recombinant t-vector by bamhi and ndei enzymes. line 1 shows 555 bp insertion cutting from t-vector (2886bp). line 2 shows a 1kb ladder. fig. 3. double digestion of pet15b by bamhi and ndei restriction enzymes. line 2 shows the linearized 5708 bp pet15b plasmid and the cut off 555 bp fragment insert from plasmid. line 1 shows a 1kb ladder iranian j arthropod-borne dis, (2008), 2(1): 7-15 g eslami et al: cloning and expression of … 11 fig. 4. sda-page analysis of extracted lmtryp6 protein (gene bank accession no. abx26130) from induced bl21 bacteria harboring recombinant plasmids. line 1, extracted protein from culture containing bl21 bacteria with no plasmid, as control; line 2, extracted protein form culture containing bl21 bacteria harboring recombinant pet15b before inducing; line 3, extracted protein from culture containing bl21 bacteria, two hour after inducing; line 4, extracted protein from culture containing bl21 bacteria, four hour after inducing; line 5, extracted protein from culture containing bl21 bacteria, six hour after inducing; line 6, extracted protein from culture containing bl21 bacteria, an overnight after inducing. lines 3-6 have additional band that was not in lines 1 and 2. it was considered as recombinant protein but for verifying, the two hour induced culture containing bl21 bacteria harboring recombinant plasmids were analyzed using western blot. line 7 shows a protein size marker. fig. 5. western blot analysis of extracted lmtryp6 protein (abx26130) from two hour induced culture containing bl21 bacteria harboring recombinant plasmids. line1, the extracted protein from culture containing bl21 bacteria with no plasmids, as control; line2, the extracted protein from two hour induced culture containing bl21 bacteria harboring recombinant plasmids; line 3, the extracted protein from culture containing bl21 bacteria harboring recombinant pet15b plasmid without any inducing. line 2 show expressed lmtryp6 (abx26130). iranian j arthropod-borne dis, (2008), 2(1): 7-15 g eslami et al: cloning and expression of … 12 discussion due to the nature of leishmania genome which is comprised of intron free genes, the products of pcr and rt-pcr, as was expected, showed the identical size of 555bp (fig. 1), on the other hand, its size was smaller than lmtryp6 (lmjf15.1140; 573bp). as shown in alignment with lmtryp6 (lmj15. 1140), a homology 79.6% between lmtryp6 (eu251502) and lmtryp6 (lmj15.1140) was seen. there is a deletion in nonconserved sequence towards c-terminal domain of lmtryp6 (eu251502) (531-548; 3.1% of the gene). the cai (codon adaptation index) for lmtryp6 (eu251502) was 0.897, and for lmtryp6 (lmj15.1140) was 0.814. this index is also a useful criterion to predict the level of expression of a gene. therefore, lmtryp6 protein (abx26130) encoded by lmtryp6 gene (eu251502) would categorized as an abundantly expressed protein. on the basis of its abundance and high level of expression, it is concluded that the expressed protein is of vital importance in the life cycle of parasite. based on differences between the cloned gene and its encoded hypothetical protein compared to those reported previously, they were proposed and accepted in gene bank as new gene and its predicted protein with accession no eu251502 and abx26130, respectively. the predicted protein encoded by this gene contained 184 amino acids with molecular weight of 20547.56 daltons and an isoelecterical point (pi) of 6.1101. its homology with lmtryp6 protein (lmjf15. 1140) was 74%. based on the protein sequence, it is clearly a member of the 2-cys peroxiredoxins. the characteristic feature of this category is the presence of 2 highly conserved redox active-cysteine residues: the peroxidatic cysteine (cys52) and the resolving cysteine (cys173) present in the valine-cysteine-proline motif. as shown in alignment, the conserved tryptophan (trp) 177 in some other 2cys tryparedoxin peroxidases is replaced by cys in lmtryp6. as far as our knowledge concern, there are two conserved tryptophans, trp87 and trp177, in all remaining tryparedoxin peroxidases (montemartini et al. 1999). these two tryptophan residues contribute to the activation of the active-site cysteines (cys52, cys173) in other 2-cys peroxiredoxins. trp87 most probably interacts with cys52, while trp177 might similarly interact with cys173 in the c-terminal domain (montemartini et al. 1999, chauhan and mande 2002, koshkin et al. 2004). the same consideration seems true for trp177 with respect to cys173 (montemartini et al. 1999). based on another explanation, his169 and his 182, located in the vicinity of the cys173 may be involved in stabilizing the thiolate form (choi et al. 1998), as one or both of these histidines might take part in trp177 activities. on the other hand, in similar nonparasite models, the third cys residue is highly reactive and readily participates in non-specific intermolecular disulfide bond which is involved in catalytic activity of enzyme (guimaraes et al. 2005). chauhan and mande (2002) suggested that in 3 cys models, the n-terminal cys, cys52, is capable of forming a disulphide linkage with both c-terminal cysteines, i.e. conserved cys173 and the additional cys located at the position of 177 in the protein under study. it is also shown that the n-terminal cys, cys52 in our case, assumes a central role in catalysis, the two c-terminal cys, cys173 and cys177 in our case, are also important for peroxidase activity. after peroxide reduction, the n-terminal cys, cys52, changes to sulfenic acid (soh), which is in turn attacked by the sulfhydryl group of the resolving cysteine, one of the c-terminal cys, either cys173 or cys177 (chauhan and mande 2002, koshkin et al. iranian j arthropod-borne dis, (2008), 2(1): 7-15 g eslami et al: cloning and expression of … 13 2004). a possible proposed scenario might be the attack of two conserved cys, cys52 and cys173 in lmtryp6 (abx26130), by the sulfhydryl group of cys177 to form a cys173-cys177 disulfide. as a consequence, the disulfide is in the flexible c-terminal arm of a single monomer, free to move and highly exposed for subsequent reduction by an external thiol (guimaraes et al. 2005). the aforesaid accounts could be summarized as presented in table1. in conclusion, an attractive molecular target was studied. further studies are needed for using this molecule in analyzing of vaccination experiments, designing of drugs to fight an important human pathogen and investigating the proposed theories. table 1. difference between lmtryp6 (abx26130) and lmtryp6 (lmjf15.1140) conserved substitution of conserved trp177 with cys177 deletion of 6 amino acids from c-terminal (178-183) non-conserved adaptive changes with no phenotypic expression: ile8→leu8, glu16→asp16, val18→met18, ser25→thr25, ile29→val29, trp38→tyr38, leu46→met46, ser49→thr49, val56→ile45, leu69→ile69, leu74→ile74, ala75→ser75, ala83→ser83, ser111→ala111, ser115→ala115, glu120→asp120, leu130→val130, his136→ asn136, val144→ile144, val149→ile149, leu157→ile157, leu160→val160, phe163→leu163, lys168→glu168 changes with the same polarity persistence: ser14→glu14, ser62→asp62, val63→ala63, ser64→lys64, thr99→pro99, ser122→asp122, gln123→ser123, ser152→asn152 changes with phenotypic expression: ser10→his10, glu17→val17, ala58→gln58, asn67→ala67, cys71→thr71, ile78→cys78, leu89→ser89, gln90→val90, met138→lys138, thr143→ile143 acknowledgements we are very grateful to research council, isfahan university of medical sciences for financial support, miss bandehpour, cellular and molecular biology research center, shaheed beheshti university and dr. mansour salehi, isfahan university of medical sciences for their help and valuable suggestions, stuffs of genetics and parasitology departments, isfahan medical school for their help and technical assistance. references bahar k, dowlati y, shidani b, alimohammadian mh, khamesipour a, ehsasi s, hashemi-fesharki r, ale-agha s, modabber f (1996) comparative safety and immunogenicity trial of two killed leishmania major with or without bcg in human volunteers. clin dermatol. 14 (5): 489-495. bryk r, griffin p, nathan c (2000) peroxynitrite reductase activity of bacterial peroxiredoxins. nature. 407(6801): 211–215. castro h, sousa c, novais m, santos m, budde h, cordeiro-da-silva a, flohe l, tomas am (2004) two linked genes of leishmania infantum encode 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leishmania donovani chagasi promastigotes. role of hydroxyl radical and protection by heat shock. j clin invest. 88: 1511–21. j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 147 original article chemical composition, larvicidal and repellency properties of cionura erecta (l.) griseb. against malaria vector, anopheles stephensi liston (diptera: culicidae) ehssan mozaffari 1, mohammad reza abai 1, mahnaz khanavi 2, *hassan vatandoost 1, mohammad mehdi sedaghat 1, abbas moridnia 1, mahsa saber-navaei 2, alireza saneidehkordi 1, fatemeh rafi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of pharmacognosy, faculty of pharmacy, tehran university of medical sciences, tehran, iran (received 30 june 2013; accepted 1 oct 2013) abstract background: application of plant derivatives have been suggested as alternative sources for mosquito control. methods: the root essential oil and methanol extract of cionura erecta (l.) griseb was tested under laboratory conditions for larvicidal and skin repelleny activities against anopheles stephensi. the chemical compositions of essential oils were analyzed using gas chromatographymass spectrometry. results: among the five concentrations tested, the 320 ppm of essential oil and 1280 ppm of methanolic extract had the most toxic effects yielding 100% mortality. the lc50 values of c. erecta for both essential oil and methanolic extract were 77.30 and 250.38 ppm, respectively. a total of 19 compounds were identified in essential oil of root. the major components were detected in root essential oil including cedren-9-one (7.89%), alpha cadinol (5.67%), eugenol (4.02%) and alpha muurolene (3.58%). the protection time of 50% solution of essential oil against bites of an. stephensi was 2.28 hour on white rabbit and the ed50 and ed90 values of the essential oil were 10.12 and 23.01 ppm respectively. conclusion: the findings suggest that c. erecta oil has a potential source as larvicidal and repellency properties against an.stephensi. keywords: cionura erecta, extract, essential oil , anopheles stephensi, larvicidal effect, repellency introduction the mosquitoes are the important vectors of human diseases and can be transmissed malaria, dengue fever, yellow fever and filariasis. they also bother the people both inside and outside places (lehane 1991). malaria is one of arthropod-borne disease and approximately 83000 deaths are reported annually in the world (who 2012). at the present, this parasitic disease is one of the main health problems in iran. several chemical compounds have been used against malaria vectors as larvicides including organophosphates, insect growth regulator and microbial dervied compounds (ghosh et al. 2012). use of synthetic insecticides is causing various problem such as environmental pollution, insecticide resistance and toxic hazards to humans and animals (aktar et al. 2009, kunz and kemp 1994, vatandoost et al. 2005). therefore using plant derivatives have been suggested as alternative sources for mosquito control. they are selective, safe and biodegrade to break down readily in soil and are not stored in plant or animal tissue (isman 2000, 2006). the various extracts of local plants have been investigated against an. *corresponding author: prof hassan vatandoost, email: hvatandoost1@yahoo.com http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 148 stephensi (hadjiakhoondi et al. 2000, 2003, 2005, vatandoost and vaziri 2004, 2012, sedaghat et al. 2010, 2011a,b, vatandoost et al. 2012, khanavi et al. 2013). personal protection using repellents is one of the effective methods for preventing mosquito-borne diseases by reducing man–mosquito contact. the most common insect repellent is deet (n, n-diethyl-m-toluamide) moreover, there are several reports about of its toxicity against the skin, nervous and immune systems (fradin et al. 1998, katz et al. 2008, nerio et al. 2010). insect repellents from natural sources are a good and safe approach for personal protection against the mosquito bites (fradin et al. 1998). the repellent effect of the plant essential oil has been examined against mosquito species in iran (oshaghi et al. 2003, yaghoobi-ershadi et al. 2006, vatandoost and hanafi-bojd. 2008, tavassoli et al. 2011). cionura erecta is a woody-based plants 50–100cm high and up to 2m wide perennial with numerous herbaceous rambling stems, often twining at the tips, with copious poisonous milky juice and is widespread in the mediterranean region which is classified in the family apocynaceae. this plant known as poisonous composition which traditionally used for killing pest animals, therefore we used of it. previous studies indicated that safranal, (z)-3hexenyl benzoate, heneicosane were obtained as major components from the essential oil of c. erecta in turkey (myrianthopoulos et al. 2007). this study was aimed at assessing the potential of plant essential oil and methanolic extract for possible use as larvicidal or repellency activites against an. stephensi under laboratory conditions and to determine the chemical composition of the essential oil. materials and methods mosquito rearing the tested mosquitoes were the colony of an. stephensi which obtained from the insectary of school of public health, tehran university of medical sciences, iran, and maintained at 29±1 ºc with a photoperiod of 12 hours light and 12 hours dark in 60±10% relative humidity. the enriched wheat germ was used as food source. larvae of an. stephensi were continuously available for the larvicidal and repellency experiments. starved 7 to 10 days old females were used for the repellency tests and the early fourth-instars larvae used for the larval bioassays. plant materials the fresh branch and root of c. erecta collected in august 2011 from rural areas located in western part of ilam province, iran (33º 46' n, 46º 11' e at elevation 1195m) (fig. 1). the specimens was identified and authenticated by the department of pharmacognosy, faculty of pharmacy, tehran university of medical sciences. essential oil isolation fresh roots (500g) of c. erecta were subjected to hydrodistillation using a modified clevenger-type (pyrexfan®) apparatus for 3 hour, the oil obtained was separated from water and dried over anhydrous na2so4 and transferred into airtight vials at 5 °c. analysis of essential oils chemical composition of c. erecta was analyzed using an agilent 7890–5975 gas chromatography-mass spectrometer. with a hp5ms (5% phenyl methyl silox) capillary column (30m×0.25mm, film thickness 0.25μm), split ratio, 1: 1, and using a flame ionization detector. the gc was programmed at 50 °c for 2min and then increased at 5 °c/min to 280 °c, and finally held with an isothermal for 3min. the injector temperature was 280 °c. the flow rate of the carrier gas was 1ml/min. the identification of compounds was performed by comparing their retention times and mass spectra with mass spectra from wiley library. additional identification was achieved by comparing linear retention indices, relative to n-alkanes, to those from literature (adams http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 149 2001). details on the identification of volatile compounds were reported in previous paper (myrianthopoulos et al. 2007). methanolic extract of plant the branch and root of c. erecta were airdried at room temperature and 100g of plant were submitted to percolation separately with methanol (80%) during 3 days, and this procedure were repeated for three times successive and totally last nine day at laboratory temperature (22 to 25 °c). the extracts were next evaporated in a rotary evaporator (heidolph persia®). larvicidal tests the essential oil and methanol extract first dissolved in absolute ethanol (99.0%) and methanol (99.0%) respectively. the 400ml glass beakers were used for the treatment or untreated experiments. the third or early fourth instar larvae were exposed to 10, 20, 40, 80 and 160ppm and 40, 80, 160, 320, 640, 1280 ppm of both essential oil and methanolic extract respectively according to standard who procedure (who 1981). repellency tests the white rabbits (oryctolagus cuniculus) (laboratory reared albino male aged six months) were used to determine both protection time and effective dosage. the 25, 50 percent and pure essential oil of c. erecta was prepared using absolute ethanol as well as this solvent used for untreated group against an. stephensi on the shaved back of male rabbits with 4 repitations. the procedure for determination of effective dosages of the repellents was adopted by the standard method of american society for testing and material (astm 2000). the testing kit was made of plexiglas cube at dimension of 4×5×18cm having four rectangular holes 2 ×3cm. before starting the test for determination of effective dosage, the abdomen skins of rabbits were cleaned with alcohol and the kit was fixed on the abdomen. the eligibility of the rabbits for repellency tests was 10 landings or probes within 30 seconds. each of 4 adjacent cells of kit was provided with 5 female 7–10 days mosquitoes that randomly selected from a cage containing 150 starved mosquitoes. five circles were drawn on the rabbit's skin. the drawn circles on the abodemen skin’s of holded rabbit were treated with 25µ l of essential oil diluted with absolute alcohol at 2, 4, 8, 16, 32ppm with 4 repitations. the serial dilutions were applied on 3 holes as well as the absolute ethanol was applied in remaining control circle. the treated circles were allowed to dry, and then test apparatus containing starved mosquitoes were fixed on the treated skin. the counts of probing and biting were recorded at 1 minute intervals up to 5 minutes. after each test, the mosquitoes were transferred to netted cups and the mortality of mosquitoes was recorded after 24 hours .the ed50 and ed90 values and regression parameters were analyzed using probit 79 program and the regression lines were plotted in microsoft excel 2007. ethical approval animal experiments were performed after obtaining institutional animal ethical committee’s approval from tehran university of medical sciences. results gc-mass analysis the hydrodistillation of the c. erecta root gave oil in 0.16% (w/w) yield on fresh weight material. a total of 19 compounds was 36.4% in roots of c. erecta were identified (table 1). the major components in root oil were cedren9-one (7.89%), alpha cadinol (5.67%), eugenol (4.02%) and alpha muurolene (3.58%) respectively. mosquito larvicidal activity the larvicidal activities of both essential http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 150 oil and methanol extract of the c. erecta root against an. stephensi larvae under laboratory conditions are shown in table 2. among the five concentrations tested, the dosages of 320 ppm and 1280ppm of essential oil and methanolic extract were respectively found to be the most toxic with 100% larval mortality. the essential oil c. erecta extracted with root and showed the higher toxicity than methanolic extract against the larvae. the lc50 and lc90 values of c. erecta essential oil were 77.30 and 199.58ppm, and for methanolic extract were recorded 250.38 and 490.00ppm, respectively. effective doses the ed50 and ed90 values of c. erecta essential oil were 10.12 and 23.01 ppm with confidence intervals ranged, 7.89–13.9 and 16.12–50.37 respectively (table 4). protection time the 25, 50 percent and pure essential oil c. erecta against an. stephensi on animal subject were provided 2.0–3.15 hours protection. the repellent failure time was ranged 2.5–4.25 hours (table 3). effective doses the ed50 and ed90 values of c. erecta essential oil were 10.12 and 23.01ppm with confidence interval ranged, 7.89–13.9 and 16.12–50.37 respectively (table 4). protection time the 25%, 50% and 100% essential oil c. erecta against an. stephensi on animal subject were provided 2.0–3.15 hours protection. the repellent failure time was ranged 2.5– 4.25 hours (table 3). table 1. chemical constituents of root essential oil from cionura erecta no. compounds composition% ri 1 2,4 decadienal 1.3 1307 2 dimethyl phenyl acetate 1.25 1315 3 eugenol 4.02 1360 4 beta elemen 0.332 1391 5 alpha humulene 0.21 1457 6 trans caryophyllene 0.71 1473 7 alpha muurolene 3.58 1483 8 delta cadinene 1.52 1404 9 caryophyllene oxide 1.38 1540 10 viridiflorol 0.55 1546 11 silphiperfolenone 0.85 1551 12 trans cadinene 0.64 1569 13 alpha cadinol 5.67 1577 14 eudesmol 1.78 1584 15 gama epoxy elemen 0.69 1598 16 cedren-9-one 7.89 1633 17 isolongifolene-5-one 2.15 1644 18 tetradecanol 1.34 1648 19 cadalene 0.58 1677 36.44 *ri: retention indices determined on hp-5 column http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 151 fig. 1. the plant c. erecta in its natral habitat, ilam provinc, west of iran (original) table 2. lc50 and lc90 values of essential oil and methanolic extract of cionura erecta roots against larvae of an. stephensi type of extraction a b ± se lc50 (ppm) ± 95%c.l. lc90 (ppm) ± 95%c.l. χ2 (heterogeneity) χ2 table (df) p-value essential oil -5.87 10.11 ± 0.34 69.28 77.30 86.40 169.71 199.58 244.91 5.419 * 11.345 (3) 0.01 methanol extract 4.40 10.54 ± 0.34 229.078 250.38 273.66 434.03 490.00 571.00 11.999 * 13.277 (4) 0.01 * no heterogeneity table 3. protection and failure times of essential oil of cionura erecta against anopheles stephensi on abdomen of albino rabbits at laboratory condition concentration of essential oil protection time (h)± sd failure time (h)±sd 25% 2.01±.95 2.5±.5 50% 2.28±15.6 3.25±17 100% 3.15±17.3 4.25±17.3 tables 4. effective doses of essential oils cionura erecta (l.) roots against an. stephensi on albino rabbits a b ± se ed50 ( mg/cm 2) ± 95%c.l. ed90 ( mg/cm 2) ± 95%c.l. χ2 (heterogeneity) χ2 table (df) p-value -3.59 3.575 ± 0.784 7.89 0.01012 13.89 16.12 0.02301 50.37 3.048* 13.277 (3) 0.01 * no heterogeneity discussion application of larvicides and repellents are generally accepted as the playing a significant role in control of the mosquitoes. in this study, major constituents of root essential oil of c. erecta were evaluated. cedren-9-one (7.89%), alpha cadinol (5.67 %), eugenol (4.02%) and alpha muurolene (3.58%) were found as main compounds. the chemical ingredients of c. erecta essential oil was reported comprised 72 compohttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 152 nents, from which the main one considered as safranal (16.8%), (z)-3-hexenyl benzoate (6.1%), heneicosane (5.7%) linalool (4.8%) and tricosane (4.4%) (myrianthopoulos et al. 2007). some constitute was not found in our study. according to the larvicidal assay, the essential oil and methanol extract of c. erecta were effective against an. stephensi with lc50 and lc90 values of 77.30ppm and 250.38 ppm, respectively. the bioassay of different herbal extracts has been stidied against an. stephensi larvae in iran. there is a report about the efficacy of the essential oil and methanolic extract of eucalyptus camaldulensis against an. stephensi in which, the lc50 and lc90 values were found 89.85ppm and 397.75 ppm, respectively (sedaghat et al. 2010). the larvicidal activity of azadirachta indica extract against an. stephensi were gained 0.35 ppm and 1.81ppm respectively for lc50 and lc90 values (vatandoost and vaziri 2004). also the lc50 and lc90 of cupressus arizonica essential oil have been reported respectively 79.30ppm and 238.89ppm against an. stephensi (sedaghat et al. 2011b). the larvicidal activity of three plants from the family apiaceae have been studied and the lc50 values of three essential oils ranged from 20.10 to 120.95ppm (sedaghat et al. 2011a). in the other study, the efficacy of kelussia odoratissima essential oil was evaluated at dose of 10ppm induced 100% larval mortality, against larvae of both an. stephensi and cx. pipiens (vatandoost et al. 2012). the repellency effect of the c. erecta essential oil against an. stephensi was first evaluated under laboratory conditions. the mean protection time of 50% essential oil of c. erecta provided 2.15 hours protection against an. stephensi. the figures for for ed50 and ed90 values were 10.12 and 23.01ppm respectively. the repellency effect of essential oils of many plants has been evaluated against an. stephensi (ansari et al. 2000, prajapati et al. 2005, klun et al. 2006, rajkumar and jebanesan 2007, mullai et al. 2008, pandey et al. 2009, govindarajan et al. 2011, prabhu et al. 2011). the repellency effect of essential oils of both myrtus communis and calendula officinalis had been reported and the ed50 values were 0.11 and 0.6 mg/cm2, respectively on human subjects (tavassoli et al. 2011). other laboratory trial revealed the repellency of 3 chemical and herbal repellents against an. stephensi. the ed50 value of neem tree’s essential oil was 0.191 mg/cm2 against field strain of mosquitoes (vatandoost et al. 2008). the results indicated both the repellency of essential oil as well as the larvicidal activity of c. erecta extract against an. stephensi. conclusion results of this study will provide a clue for possible use of plants for control of mosquito-borne disease. acknowledgments this study was funded and supported by tehran university of medical sciences (tums); grant no. 90-04-27-16617. we are thankful to the staff of insectary of culicidae, department of medical entomology and vector 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against two mosquito vectors anopheles stephensi and culex pipiens (diptera: culicidae). exp parasitol. 132: 470–474. vatandoost h, vaziri vm (2004) larvicidal activity of a neem tree extract (neemarin) against mosquito larvae in the islamic republic of iran. east mediterr health j. 10: 573–581. vatandoost h, zahirnia ah (2010) responhttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 147–155 e mozaffari et al.: chemical composition … 155 siveness of anopheles maculipennis to different imagicides during resurgent malaria. asian pacific j trop med. 3: 360–363. who (1981) instruction for determining the susceptibility or resistance of mosquito larvae to insecticides. world health organization-vbc. who (2012) world malaria report. world health organization. yaghoobi-ershadi mr, akhavan aa, jahanifard e, vatandoost h, amin gh, moosavi l, ramazani ar, abdoli h, arandian mh (2006) repellency effect of myrtle essential oil and deet against phlebotomus papatasi, under laboratory conditions. iran j public health. 35: 7–13. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 113 http://jad.tums.ac.ir published online: january 05, 2016 review article measures to control phlebotomus argentipes and visceral leishmaniasis in india deanna c. bublitz 1, *richard m. poché 1, rajesh garlapati 2 1genesis laboratories, 10122 ne frontage road, wellington, co 80549, usa 2genesis laboratories india private limited, patna, bihar, india (received 11 apr 2015; accepted 26 oct 2015) abstract visceral leishmaniasis is a deadly parasitic disease that is transmitted via the bite of a female sand fly, phlebotomus argentipes. the highest burden of this disease is in northern india. in 2005, india embarked on an initiative with nepal, bangladesh, and the world health organization to eliminate visceral leishmaniasis by 2015. with the goal of 1 case in 10,000 people still unmet, it is prudent to evaluate the tools that have been used thus far to reduce vector numbers and cases of the disease. herein, we present a review of studies conducted on vector-control strategies in india to combat visceral leishmaniasis including indoor residual spraying, insecticide-treated bed nets, environmental modification, and feed-through insecticides. this review suggests that the quality of indoor residual spraying may enhance control measures while a combination of spraying, nets, and feed-through insecticides would best confront the diverse habitats of p. argentipes. keywords: visceral leishmaniasis, leishmania donovani, phlebotomus argentipes, sand flies, vector control introduction leishmaniasis is a parasitic disease that can manifest in three forms: 1) mucosal, 2) cutaneous, and 3) visceral (who 2010, maroli et al. 2013). visceral leishmaniasis (vl) is the most severe form caused by the protozoan flagellate, leishmania donovani (east africa and indian subcontinent) and l. infantum (also known as l. chagasi, found in europe, north africa, and latin america). as this review focuses on elimination efforts in india, we will only refer to l. donovani in this article. globally, the annual incidence rate is approximately 200,000–400,000 cases, the majority of cases are present in bangladesh, nepal, and india. furthermore, twothirds of those cases occur in india where vl (also known as kala-azar; black fever) is endemic in the states of uttar pradesh, jharkhand, west bengal, and bihar (alvar et al. 2006, joshi et al. 2008, alvar et al. 2012). reports have noted the annual incidence in india as 146,700–282,800 cases with a mortality rate of at least 2.4%. however, other studies involving active searches at the village level have discovered mortality rates from 10–20%, partly due to a delay in diagnosis (alvar et al. 2012). vl carries a mortality rate over 90% when left untreated (desjeux 1996, jeronimo et al. 2006). transmission of l. donovani occurs via the bite of a female phlebotomus argentipes sand fly. of the nearly 50 species of sand flies present in india, p. argentipes is the only one known to transmit vl in this country (kumar et al. 2012). once the parasite is in the human body it rapidly invades macrophages and eventually moves in this way to the liver, spleen, and lymph nodes (chappuis et al. 2007). symptoms include: fever lasting weeks to months, splenomegaly, hepatomegaly, and anemia (desjeux 1996, guerin et al. 2002, chappuis et al. 2007). of patients that recov*corresponding author: dr richard m. poché, email: richard@genesislabs.com j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 114 http://jad.tums.ac.ir published online: january 05, 2016 er, an estimated 5–10% go on to develop a rash known as post kala-azar dermal leishmaniasis (pkdl), this can occur anywhere from several months to several years following treatment (ramesh and mukharjee 1995, rahman et al. 2010). pkdl patients do not experience other symptoms outside of the rash, however, they are thought to serve as a reservoir for l. donovani where feeding sand flies can acquire the parasite (addy and nandy 1992, rahman et al. 2010). as humans are the only known reservoir for l. donovani in india, pkdl patients are another concern for vl elimination efforts. visceral leishmaniasis is a poverty-associated disease linked to poor housing and sanitary conditions and malnutrition, these factors have led to a number of difficulties regarding treatment and elimination (cerf et al. 1987, thakur 2000, boelaert et al. 2009, singh et al. 2010, picado et al. 2014). easy access to medical care in the rural vl-endemic regions of india is still limited, meaning patients are not routinely identified or wait until the disease has progressed to more severe symptoms before seeking treatment. moreover, until recently treatment regimens required medication injections over the course of 20–28 days leading to poor compliance rates (clem 2010, moore and lockwood 2010, stockdale and newton 2013). in addition to hurdles with medical treatment, increased sand fly density has been associated with certain types of housing, owning livestock, and nearby vegetation (ranjan et al 2005, singh et al. 2010, poché et al. 2011, poché et al. 2012, perry et al. 2013, malaviya et al. 2014, picado et al. 2014). the risk of exposure to p. argentipes is even higher for some, as many people in these poor regions of india live in close proximity to their cattle, keeping them inside their dwellings (singh et al. 2010, perry et al. 2013, malaviya et al. 2014). as a by-product of ddt spraying for malaria elimination programs in the 1950s and 1970s, vl was nearly eliminated. however, when ddt spraying ended, sand fly numbers and cases of vl rose again, leading to several major epidemics (kishore et al. 2006, thakur 2007, ostyn et al. 2008). in 2005, the governments of bangladesh, nepal, and india began a concerted vl elimination effort, with a target goal of 2015 for elimination (1 case in 10,000 people). indoor residual spraying (irs) and insecticide-treated bed nets (itn) have been the main forms of control regimens tried in india, while environmental modification (evm) and feedthrough insecticides (fti) have been less studied. despite significant efforts to control sand flies and treat infected persons, the country remains a bastion for this disease. as such, we sought to review the current practices regarding vector/vl control programs in india. a literature search was performed using the term “india” in combination with the following keywords: phlebotomus argentipes, sand fly, kala-azar, and/or visceral leishmaniasis. the following paper is a summation of the articles collected in hopes of highlighting what has worked, what has not, and what can be learned as we move forward towards elimination goals. control strategies indoor residual spraying irs has been the main line of defense against vector-borne diseases in india. in the 1950s and again in the 1970s there were aggressive irs initiatives to eliminate malaria (kishore et al. 2006, thakur 2007, ostyn et al. 2008). during these years, households in malaria-endemic regions, many of which were the same as vl-endemic areas, were sprayed with ddt to cull mosquitoes. sand fly populations declined in kind, and with them, cases of vl. however, at the end of those campaigns, with no systematic irs programs, sand fly populations and cases of j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 115 http://jad.tums.ac.ir published online: january 05, 2016 vl quickly rebounded (kishore et al. 2006, thakur 2007, ostyn et al. 2008). since then, irs using ddt has been employed to fight vl transmission in india with mixed results. currently, the national vector borne disease control programme of india dictates that irs should be performed on all homes and cattle sheds in endemic regions. they recommend two annual applications, the first to occur in february/march and the second in may/june (nvbdcp 2014). applied properly, irs has been shown to dramatically impact p. argentipes density (joshi et al. 2009). in that carefully controlled study involving all homes in six clusters in each of four villages, there was a 72.4% decline in sand fly numbers. other studies that have assessed the efficacy of irs in india have instead highlighted a number of issues limiting its success in controlling sand flies and vl. huda et al. monitored local irs programs for control of vl in bangladesh, nepal, and india (2011). in india, (though it was not unique for some of these issues) they found functional pumps and spare parts were lacking and 23.5% of the pumps were leaking. spraying staff were not adequately trained and as such, proper mixing of ddt was done only 29.4% of the times observed. additionally, proper distance and swath coverage during spraying were maintained only 49% and 58.8% of the time, respectively (huda et al. 2011). these results are echoed in a similar report that found the same issues with equipment, training, mixing, and distance from surface, in addition to storage and quality issues for the ddt (chowdhury et al. 2011a). this group also noted that the ddt residue levels on the walls varied at the village level from 66%– 90% of the intended concentration while at the household level, the concentration varied as much as 9.1% to 330% (chowdhury et al. 2011a). furthermore, spraying must be applied to >80% of the homes in an area for mass effect (cdc 2012). according to huda et al. the reported coverage rate in their study was only 64% (2011). another point of concern with irs in india is resistance of p. argentipes to ddt. resistance to ddt was documented for the district of samastipur in bihar, india as early as 1990 (mukhopadhyay et al. 1992). in general, p. argentipes remains largely susceptible to ddt with the majority of flies succumbing to the insecticide (singh et al. 2001, dhiman et al. 2003, joshi et al. 2009). however, select districts are showing signs of this trend shifting (kishore et al. 2004). huda et al. documented only a 54% mortality rate after 24 h for sand flies exposed to walls sprayed with ddt while another group found up to 70% were killed when exposed to surfaces that had been sprayed 2 weeks prior (chowdhury et al. 2011a). it is possible that the results obtained by huda and chowdhury et al. may have been partly due to improper storage, mixing, spraying, and/or active ingredient concentration which were all problems documented by those studies (chowdhury 2011a, huda 2011a). a controlled study involving irs in three states in india found mortality rates for p. argentipes ranging from 31–89%, indicating moderate to significant levels of resistance to ddt, even with proper use (singh et al. 2012). despite these many problems recorded by groups studying irs, in all of the aforementioned studies, post-treatment sand fly abundance was significantly reduced in the short term (chowdhury et al. 2011a, huda et al. 2011, singh et al. 2012). in fact, joshi et al. demonstrated a negative effect on sand fly numbers up to five months post-treatment (joshi et al. 2009). both nepal and bangladesh employ pyrethroids for their irs regimens and have shown them to be effective (joshi et al. 2009, chowdhury et al. 2011a, chowdhury et al. 2011b). some work has been conducted using different insecticides in india. in one report they found sand flies were resistant to ddt but not deltamethrin j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 116 http://jad.tums.ac.ir published online: january 05, 2016 (dhiman et al. 2003). a more recent study documented that deltamethrin was effective in nearly 100% of the locations tested in india (singh et al. 2012). the government of india is currently testing the efficacy of synthetic pyrethroids on sand fly control in bihar (nvbdp 2014). a comprehensive table of susceptibility studies can be found in the review by ostyn et al. (2008). while irs can aid in controlling endophilic sand flies, it does not address outdoor transmission of vl. in india, p. argentipes are peridomestic, found in cattle enclosures as well as vegetation (poché et al. 2011, perry et al. 2013, poché et al. 2012). another matter that is infrequently addressed in studies regarding irs is the health effects to humans, animals, and the environment as a result of frequent and long-term irs. the stockholm convention on persistent organic pollutants has banned ddt (van den berg 2009). however, there is yet no consensus on whether ddt exposure leads to deleterious health effects among humans (sharpe and stewart 2004, beard 2006). regardless, there are well documented studies regarding its toxicity in birds and a variety of aquatic species (blus 2003, sparling 2010, beckvar and lotufo 2011). while further work is needed to verify them, these data indicate that alternative irs compounds may be more efficacious in controlling p. argentipes abundance. research looking at effects on human health due to long-term exposure would still need to be addressed. ultimately, these data suggest that with proper execution, irs could be an even more effective tool against vl in india but is not sufficient as a standalone given that p. argentipes is found outdoors in significant numbers. bed nets bed nets, in particular, itns and longlasting insecticide nets (llins), have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl. itn/llins have been effective against other vector-borne diseases, including cutaneous leishmaniasis (lengeler 2004, kulkarni et al. 2007, wilson et al. 2014) but there are mixed results when it comes to sand flies in india. a trial using untreated nets found that the number of female, blood-fed p. argentipes declined by 85% following the introduction of the nets (picado et al. 2009). however, the authors of that study note that they lacked concurrent controls and thus, blood-feeding rates could potentially be attributed to changes in environmental factors (temperature, humidity, precipitation) or changes in host availability (i.e. an increase in domestic animals). even so, other groups in bangladesh and nepal have found similar results (bern et al. 2000 and 2005). outside of that one study, all others have utilized itns or llins in india. the report by joshi et al. investigated the usefulness of irs, llins, and evm to mitigate sand fly density in india, nepal, and bangladesh. when llins were in place, there was a village-wide reduction in p. argentipes numbers by 43.7% in india when measured 5 months post-intervention (joshi et al. 2009). a subsequent village-wide study in india noted a 25% decline in sand fly density (picado et al. 2010a). in that study, 16 clusters were enrolled, of those, 10 were used for sand fly capture studies. the reduction appeared to be at the community level and not displacement as p. argentipes abundance did not increase in cattle enclosures (picado et al. 2010a). however, this observation does not exclude other environmental refuges, such as vegetation (poché 2011, poché 2012). as a part of the same study by picado et al. they also investigated rates of vl in these same villages and found no protective effect of llins on the rate of seroconversion (picado et al. 2010b). nearly 20,000 people were enrolled in the seroconversion study. over the course of 24 months, incij arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 117 http://jad.tums.ac.ir published online: january 05, 2016 dence of l. donovani infection was found to be 5.4% in the intervention group and 5.5% in the control group while clinical vl rates were 0.38% and 0.40% in the intervention and control groups, respectively. one explanation for this result was that llins do not prevent outdoor transmission (picado et al. 2010b). an earlier study of 48 homes and mixed dwellings (homes shared with livestock) in bihar compared two types of llins to two types of untreated nets and looked at sand fly densities. at all time-points, up to 9 weeks post-treatment, there were no differences in p. argentipes abundance between the groups (dinesh et al. 2008). it should be noted, that during the course of the aforementioned studies, unplanned irs took place, and in both cases there was no noticeable effect on the number of sand flies captured in either study (dinesh et al. 2008, picado et al. 2010a). furthermore, when the statistical models used to analyze the vl seroconversion data were adjusted for irs, no changes were found (picado et al. 2010b). while the number of studies has been limited, the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh (chowdhury et al. 2011b, mondal et al. 2013). the potential reasons for this are many, including: study design, environmental factors (temperature, humidity, flooding etc.), and susceptibility to insecticides p. argentipes in bangladesh may be more susceptible, given their history of irs campaigns is more recent compared to india (bern et al. 2006, picado et al. 2010a, chodhury et al. 2011b). as breeding sites are yet unknown, and sand flies have been associated with vegetation in india, these remain confounding factors when accounting for differences in intervention strategies (poché et al. 2011, poché et al. 2012). non-compliance and net quality are another concern. in the studies by picado and joshi, care was taken to inspect the nets and ensure compliance (joshi et al. 2009, picado et al. 2010a, 2010b). even so, compliance with nets can be difficult as sand flies are much smaller than mosquitoes requiring finer mesh that people find stifling to sleep under in the heat of summer (ostyn et al. 2008, perry et al. 2013). while in theory, the impregnated insecticide should provide repellent activities, thus allowing for a larger mesh, efficacy in the field has not been shown (picado et al. 2010a, 2010b). moreover, 93% of 1,217 people surveyed in 2009–2011 in vl-endemic regions reported sleeping outside at some point during the hottest months of the year (perry et al. 2013). assuming this trend holds true throughout other vl-afflicted areas, this is a serious hurdle to be faced in regards to the efficacy of irs, itns, and evm as none mitigate transmission that occurs outside of a person’s dwelling. environmental modification typical dwellings in the regions of india afflicted by vl are often made of mud and thatch, or brick/plaster. some have earthen floors while others have brick or cement (perry et al. 2013, malaviya et al. 2014). in all cases, cracks and crevices where sand flies can rest and hide are prevalent. all home types are susceptible to habitation by p. argentipes, but thatched homes in particular cater to high densities (malaviya et al. 2014). evm is a method little studied when compared to irs and itns as a means to control vl in india. evm has generally meant alterations to the home or surrounding environment by means of covering or filling in cracks and crevices in walls and floors. a pilot study involving 15 homes saw a reduction in sand fly numbers using a mud and lime plaster mix to seal cracks in homes and cattle enclosures (kumar et al. 1995). the same well-controlled study comparing the efficacy of irs and itns in india, nepal, and bangladesh found a 42% decline in sand fly abundance five months after the walls of j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 118 http://jad.tums.ac.ir published online: january 05, 2016 homes and cattle enclosures were plastered with a mud and lime mixture. the negative effect on sand fly populations may be due to the lime ph or limiting available moisture, thus, inhibiting breeding of p. argentipes, though breeding sites for these flies have not been confirmed (kumar et al. 1995, sharma and singh 2008, joshi et al. 2009). indeed, a study in bangladesh saw no effect on sand fly populations when crevices were filled with mud (chowdhury et al. 2011b). importantly, another study in india found mudplastered walls themselves to be risk factor for vl (ranjan et al. 2005). however, while mud/lime mixes are effective, this method requires continual maintenance and is costly compared to irs and itns (das et al. 2008). but like irs and itn, it is only effective against the sand flies that are inside homes; populations that reside in outdoor enclosures and vegetation would remain a source of vl. feed-through insecticides a newer, relatively untested, yet promising addition to the vector-control arsenal is the use of ftis. several compounds including ivermectin, fipronil, and imidacloprid have been tested in rodents to cull p. papatasi, a vector for cutaneous leishmaniasis (mascari et al. 2008, wasserberg et al. 2011, mascari et al. 2013, derbali et al. 2014). in these reports, the agents have been effective at controlling adult and larval sand flies when fed on blood or feces from treated animals, respectively. in regards to p. argentipes, ivermectin and fipronil as well as diflubenzuron and eprinomectin were tested as ftis in rats (ingenloff et al. 2013). in that study, fipronil at 150 ppm was shown to result in the quickest mortality and its effects had greater longevity than the other three compounds, even when they were used at greater concentrations. fipronil has been the only fti further evaluated for control of p. argentipes. cattle and other livestock act as a major source of blood meals for female sand flies. blood-meal analysis in one study found that 39.3% of the flies tested had fed on some form of livestock (garlapati et al. 2012). so as to target p. argentipes that are feeding off of cattle and therefore, possibly residing outdoors, a controlled study involving cattle dosed with 0.5, 1.0, 2.0, and 4.0 mg/kg body weight of fipronil was conducted (poché et al. 2013). that investigation demonstrated that fipronil as an fti is effective, even at the lowest dose, at killing both adult and larval sand flies. the majority of adult sand flies had a 100% mortality rate within four days when fed on cattle up to 21 days posttreatment for all but the lowest dose (which had a ~22% mortality rate at 21 days posttreatment). nearly all of the flies that were fed on days 1, 3, and 5 post-treatment succumbed on the same day. for larval p. argentipes, the mean-time to death when fed on feces from cattle 1 or 3 days post-treatment was 4.5 days, larva fed on feces with the lowest dose (0.5 mg/kg) on day 1 posttreatment had 100% mortality within 5.5 days while the highest dose (4.0 mg/kg) was 4.0 days. additionally, for all doses tested, 100% mortality of larval sand flies was achieved by day 15.5 when fed on dung collected 21 days post-treatment (poché et al. 2013). given that this method is untested under field conditions, conclusions regarding its effect on p. argentipes abundance or vl transmission cannot be drawn at this time. while no effects to cattle health were noted in the aforementioned study, future work will need to be done to assess the health of cattle treated long-term. moreover, at the lowest dose, fipronil can remain in the animal for up to 35 days but it is at concentration below international allowable limits (poché et al. 2013). regardless, future studies should also address milk consumption by humans from these cattle. studies regarding the efficacy of fipronil as an fti are ongoing and if they j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 119 http://jad.tums.ac.ir published online: january 05, 2016 prove positive, this method would be a valued addition to irs and itns as it would better address exophilic p. argentipes, making control measures more comprehensive. conclusion with humans as the only known reservoir for vl in india, rapid diagnosis and treatment would go a long way in controlling epidemics. however, if pkdl patients serve as a source of l. donovani, vector-control programs will also be needed to reach the goal of elimination. in this way, surveillance and reporting programs may need to be reevaluated as under-reporting of vl remains an issue within india (mubayi et al. 2010). taking into account the diverse feeding and living habits of p. argentipes, it is likely that a combination of control measures will be necessary to eliminate vl in india. irs, when executed properly can be highly effective (joshi et al. 2009). switching insecticides may be beneficial with mounting evidence of increased resistance of p. argentipes to ddt coupled with potential health effects on humans, animals, and the environment (blus 2003, van den berg 2009, sparling 2010, huda et al. 2011, singh et al. 2012, cohn et al. 2015). in the end, the efficacy of irs is dependent on organized government programs that coordinate the spraying schedule and train the technicians, which can be either a benefit or hindrance depending on the resources available to these groups (joshi et al. 2009, chowdhury et al. 2011a, huda et al. 2011). even still, irs works well to curb adult p. argentipes abundance. efforts should also be placed on identifying breeding grounds. to this end, irs could be focused and effective at eliminating both adult and larval flies. insecticide treated nets have shown limited success in india despite reports of their usefulness in other countries in combating sand fly densities (dinesh et al. 2008, picado et al. 2010a, 2010b). while there is some level of personal protection from sand flies afforded to people who use bed nets, more work would need to be done to confirm that the cost of these initiatives is validated. itns can often be more effective control measures against disease-transmitting vectors as proper use is in the hands of the affected persons instead of an outside program. that being said, non-compliance in india could be a problem as many people report sleeping outdoors during the hot summer months (perry et al. 2013). long-lasting insecticide nets can have repellent and insecticidal properties for years, making them a relatively cheap supplement to irs, however, given the impoverished state of most of the afflicted regions, nets may still need to be provided by government or non-profit groups. the most expensive of the three most studied intervention strategies is evm (das et al. 2008). a mud and lime mixture to seal cracks and crevices in walls and floors has shown some negative effects on sand fly abundance, yet these studies have been limited (kumar et al. 1995, joshi et al. 2009). given the cost and need for continual maintenance, evm is a strategy that may be best left for use on a case-by-case basis rather than a district-wide, vector-control measure. the state of dwellings in vl-endemic villages is a by-product of the greater issue of region-wide poverty, if that issue were better addressed, evm would be a moot point. the last strategy covered in this report is fti. this relatively new addition to vl vector-control has been effective at killing both adult and larval p. argentipes under controlled settings (inglenoff et al. 2013, poché et al. 2013). at this time there is no known resistance by sand flies to fipronil and the tactic of dosing cattle begins to address outdoor transmission of vl. however, ftis would still rely on proper usage by individuals and like itns, would carry similar benefits and risks of proper use. similar to j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 120 http://jad.tums.ac.ir published online: january 05, 2016 irs, ftis will result in the potential for some human, animal, and environmental exposure. although, reported residue levels of fipronil in milk is below international standards (poché et al. 2013), long-term studies have not been conducted. work would need to be done to monitor for any adverse effects to humans, animals, and the environment, following long-term use of ftis to treat cattle. should ftis prove effective under field conditions, they would begin to fill the gap in available treatments that target sand flies outside of homes and cattle enclosures. similar to fti and irs is the use of natural botanicals as insecticides in place of synthetic chemicals. one study demonstrated that a 2% concentration of neem oil mixed with either coconut or mustard oil was effective at repelling sand flies from human subjects in india (sharma and dhiman 1993). while no work has been done regarding the use of potential biochemicals as insecticides in india, studies conducted in other countries on different sand fly species showed that various plant-derived compounds were effective at killing adult and larval stages of the insects (dinesh et al. 2014). although many of the tested biochemicals only had a ~50% mortality rate, this area of research is still relatively unexplored and may be a useful alternative to synthetic insecticides. with millions of homes that would require intervention, vector-control programs need to balance rapid efficacy with longterm cost to ensure that if a treatment measure is terminated the country does not experience the rapid resurgence as occurred at the end of ddt irs in the 1970s. one year of irs costs on average, $5.90 per household which is more than llins ($4.50/house/ year) but less than evm ($8.70/house/year), and those costs have likely only gone up since that report was conducted (das et al. 2008). there is a real need for thorough costeffectiveness studies using combinations of control measures and disease prevalence/ sand fly population scenarios. research documenting the direct and indirect costs, estimated expenditures for effective training and supervision of staff, as well as equipment maintenance would help to make informed decisions on the best use of resources for this endeavor. moreover, modelling has been done to estimate what percent of the sand fly population would need to be culled in order for vl to be eliminated. in that report, if sand fly life expectancy was reduced (eg via irs, itns, or ftis), there would need to be a 67% decline in abundance in order for vl to be eliminated (stauch et al. 2014). of the research conducted to date, only the fti report and some of the irs studies meet that threshold (chowdhury et al. 2011a, singh et al. 2012, poché et al. 2013). further focusing of efforts and resources could be done by implementing remote sensing and gis data. preliminary work done in brazil and india modeling both climate and land data has been used to try and identify vector habitat as well as regions that may be the focus of vl outbreaks due to various weather and geo-environmental factors (bhunia et al. 2013). while standardization of analysis methods and data acquisition are still needed, gis and remote sensing could greatly help to target high-risk areas before an outbreak occurs. vector-control and vl-transmission studies should begin to focus on combination intervention strategies as well as enhanced public education programs. if the public are unaware of the risk, the symptoms, and treatments for vl, control programs will struggle (singh et al. 2006, siddigui et al. 2010, malaviya et al 2013). from what is known at this point, irs is efficacious in quickly curbing adult sand fly populations, itns may have some benefit for personal protection and might also be useful if irs is not performed correctly 100% of the time. lastly, fti is deleterious to both adult and j arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 121 http://jad.tums.ac.ir published online: january 05, 2016 larval p. argentipes and has the potential to disrupt populations of exophilic sand flies. what has been lacking outside of the one study by picado et al. (2010b), are studies that attempt to link a decline in sand fly abundance with a subsequent drop in cases of vl. a broader review of 84 studies in 22 countries involving all forms of human leishmaniasis found that only 35% measured leishmania infection as an outcome (stockdale and newton 2013). this is an issue afflicting many vector/disease-control studies (wilson et al. 2015). future studies should concentrate their efforts on making this connection. while a decline in sand fly numbers should in theory correlate to a decline in vl infections, this link has yet to be demonstrated clearly by the research performed to date. future work investigating the connection between reduced sand fly abundance and vl infection rates as well as shifts in parasite availability and sand fly feeding behaviors should be a priority. in combination, these tactics may work to bring sand fly numbers down quickly in the short-term, allowing for vl-patient identification and treatment. currently, the state of bihar is beginning tests for use of synthetic pyrethroids in place of ddt for irs. the national roadmap laid out by the government of india emphasizes case detection and treatment as well as surveillance for pkdl. while they state the need for integrated vector management, irs remains the mainstay for vector control (nvbdp 2014). ongoing work with sand fly breeding site identification, and implementation of more novel technologies like gis and remote sensing will greatly aid in curbing this disease. the global initiative to eliminate vl in all endemic regions has been strong, however a coordinated effort between groups employing the various control tactics will be vital to see the elimination goal met. once caseloads have been brought to 1:10,000, the use of irs, itns, ftis and all of the aforementioned technologies and trainings should be evaluated to determine which would be best for continued control measures. acknowledgements we would like to thank all of the researchers who have worked on this issue for many years whose work is represented herein. we apologize for any studies we may have left out of this report. we would also like to thank christopher bublitz for his careful editing of this manuscript. additionally, we extend our appreciation to the people of india that allow this work to happen in their homes. references addy m, nandy a (1992) ten years of kalaazar in west bengal, part i: did postkala-azar dermal leishmaniasis initiate the outbreak in 24-parganas? bull world health org. 70(3): 341–346. alvar j, yactayo s, bern c (2006) leishmaniasis and poverty. trends parasitol. 22(12): 552–557. alvar j, vélez id, bern c, herrero m, desjeux p, cano j, jannin j, den boer m, who leishmaniasis control team (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7(5): e35671. beard j (2006) ddt and human health. sci total environ. 355(1-3): 78–89. beckvar n, lotufo gr (2011) ddt and other organohalogen pesticides in aquatic organisms. in: beyer wn, meador jp (eds) environmental contaminants in biota: interpreting tissue concentrations. vol 2. crc press, new york, pp. 47–101. bern c, joshi ab, jha sn, das ml, hightower a, thakur gd, bista mb (2000) factors associated with visceral leishj arthropod-borne dis, june 2016, 10(2): 113–126 d bublitz et al.: measures to control … 122 http://jad.tums.ac.ir published online: january 05, 2016 maniasis in nepal: bed-net use is strongly protective. am j trop med hyg. 63(3-4): 184–188. bern c, hightower aw, chowdhury r, ali m, amann j, wagatsuma y, haque r, kurkjian k, vaz le, begum m, akter t, cetre-sossah cb, ahluwalia ib, dotson e, secor we, breiman rf, maguire jh (2005) risk factors for kala-azar in bangladesh. emerg infect dis. 11(5): 655–662. bern c, chowdhury r (2006) the epidemiology of visceral leishmaniasis in bangladesh: prospects for improved control. indian j med res. 123(3): 275–288. bhunia, gs, kesari s, chatterjee n, kumar v, das p (2013) the burden of visceral leishmaniasis in india: challenges in using remote sensing and gis to understand and control. isrn infect dis. 2013: article id 675846. blus lj (2003) organochlorine pesticides. in: hoffman dj, rattner ba, burton ga jr, cairns j jr. 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phlebotomus papatasi salivary gland antigens in central iran nasibeh hosseini-vasoukolaei 1, ahmad-reza mahmoudi 2, ali khamesipour 3, mohammad reza yaghoobi-ershadi 1, shaden kamhawi 4, jesus g. valenzuela 4, mohammad hossein arandian 5 , hossein mirhendi 6, shaghayegh emami 2, zahra saeidi 1, farah idali 7, reza jafari 5, *mahmood jeddi-tehrani 2, *amir ahmad akhavan 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2monoclonal antibody research center, avicenna research institute, acecr, tehran, iran 3center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran 4laboratory of malaria and vector research, national institute of allergy and infectious diseases, national institute of health, rockville, usa 5esfahan health research station, national institute of health research, tehran university of medical sciencesesfahan, iran 6department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 7reproductive immunology research center, avicenna research institute, acecr, tehran, iran (received 11 nov 2014; accepted 6 dec 2014) abstract background: sand fly saliva helps parasite establishment and induce immune responses in vertebrate hosts. in the current study, we investigated the modulation of phlebotomus papatasi salivary gland antigen expression by seasonal and biological factors. methods: sand flies were grouped according to physiological stages such as unfed, fed, semi-gravid, gravid, parous, nulliparous, infected or non-infected with leishmania major and based on the season in which they were collected. salivary gland antigens (sgas) were analyzed using sds-page and the antibody response against sgas in rhombomys opimus was determined by elisa and western blot. results: the highest protein content was found in the salivary glands of unfed sand flies. the saliva content was higher in parous compared to nulliparous, in summer compared to spring, and in leishmania-infected compared to non-infected flies. the salivary gland lysate (sgl) electrophoretic pattern variations were observed among sand flies with various physiological stages particularly from 4–9 protein bands of 14–70 kda. the sgl of unfed and gravid flies had extra protein bands compared to fed and semi-gravid sand flies. there was missing protein bands in sgl of parous compared to nulliparous; and in summer compared to spring collected flies. rhombomys opimus serum reacted strongly with an antigenic band of around 28 kda in the sgl of all sand fly groups. conclusion: certain biological and environmental characteristics of wild populations of vector sand flies affect the protein content and antigenicity of saliva. this might have an important implication in the design of vector-based vaccines. keywords: antibody response, phlebotomus papatasi, rhombomys opimus, salivary gland antigens, iran introduction zoonotic cutaneous leishmaniasis (zcl) is a neglected tropical disease of public health importance in many rural areas of iran (yaghoobi-ershadi et al. 1996, 2001, 2003, 2010). leishmania major is the causative agent, phlebotomus papatasi is the main *corresponding authors: dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir and prof mahmood jeddi-tehrani, e-mail: mahjed@avicenna.ac.ir http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 40 vector and rhombomys opimus (great gerbil) is the major reservoir host of the disease in esfahan province, which is a hyperendemic zone of zcl in central iran (yaghoobiershadi et al. 1995, akhavan et al. 2010a, b, yaghoobi-ershadi 2012). the incidence rate of zcl in esfahan province is reported around 2400 cases per year (communication from the esfahan center for public health) and is considered an underestimate of the actual incidence. saliva of phlebotomines consists of different molecules that are necessary for a sand fly to take successfully a blood meal (ribeiro 1987). additionally, previous exposure to sand fly saliva indirectly affects the establishment of leishmania in vertebrate hosts (oliveira et al. 2013). mice previously exposed to saliva by injection or by uninfected sand fly bites showed both a humoral and a cellular immune response against salivary antigens that protected them against l. major infection (belkaid et al. 1998, 2000, kamhawi et al. 2000). importantly, immunization of mice with defined molecules from saliva of vector species also conferred a strong protection against l. major infection (valenzuela et al. 2001, oliveira et al. 2008, gomes et al. 2012). this suggests that sand fly salivary components may be considered as candidates for a cocktail vaccine against leishmania infection. in the esfahan hyperendemic focus of zcl, the most abundant sand fly species is p. papatasi (yaghoobi-ershadi and javadian 1997, 1999). of relevance, antibodies against saliva of this vector species were demonstrated in the main animal reservoir of l. major in this area, r. opimus (akhavan 2011). differences in the antigenic components of the salivary gland lysate (sgl) of various sand fly species, sex, and age have been reported (volf et al. 2000). in the esfahan hyperendemic focus, vertebrate hosts are bitten by p. papatasi with various physiological characteristics and under diverse environmental conditions. it is therefore important to address the effect, if any, of the variability of vector salivary gland components on l. major infections and the clinical outcome of the disease. the aim of the current study was to determine the composition of salivary gland antigens (sgas) of p. papatasi with respect to certain seasonal and biological factors in vector populations in the esfahan hyperendemic focus, and to further characterize the p. papatasi sgas reacting with r. opimus antibodies. the composition of the sgas was studied with respect to physiological aspects of the collected sand flies comprising unfed, fed, semi-gravid, gravid, parous, nulliparous, infected or non-infected with l. major, and with respect to the season in which they were collected. materials and methods study area this investigation was undertaken during 2012–2013 in the three villages of parvanehaliabadchi, habibabad and abbasabad, esfahan province, central iran (fig. 1). the villages of habibabad and parvaneh-aliabadchi are located 25–40 km north of the city of esfahan (32°39′ 35" n, 51°40′ 17"e) at an altitude of around 1550 m and abbasabad is located 5 km from badroud city (33° 42′ n, 52° 2′ e) at an altitude of 1056 m, in the foothills of karkas mountains. the biotope of the selected areas is desert with hot summers and cold winters. in 2013, the maximum and minimum monthly relative humidity in esfahan city was 81 % and 9.1 % in november and july, respectively. the minimum monthly temperature was -5.3 °c in december and the maximum was 39.8 °c in july. in badrood district, the minimum and maximum monthly temperatures were -2.9 °c in december and 43 °c in july, respectively. the maximum relative humidity was 62 % in december and the minimum was 20 % in http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 41 february. total annual rainfall was 84.6 mm in esfahan city and 77.5 mm in badrood district (esfahan metrological organization). sand fly collection and rearing phlebotomines were aspirated from resting places throughout the selected study villages during the active season of sand flies in 2012–2013. sand flies were identified according to morphological characters using a valid systematic key (seyedi-rashti and nadim 1992). females of p. papatasi were separated from other species for inclusion in the study and categorized into ten groups according to certain seasonal and biological factors: accessory glands status, parous and nulliparous, unfed, fed, semi-gravid and gravid. two groups of sand flies were collected throughout spring and summer and analyzed according to their l. major-infection status. salivary gland antigens of parous versus nulliparous, l. major-infected versus non-infected, spring versus summer collection and unfed versus fed, semi-gravid and gravid were compared. sand fly colonies were reared at the department of medical entomology and vector control, tehran university of medical sciences and the esfahan center for public health according to the methods of modi and tesh (1983) and killick-kendrick and killick-kendrick (1991) with modifications. phlebotomus papatasi colonies were reared on a 14:10 ld photoperiod, at 26–28 ºc and around 80 % relative humidity. adult sand flies were fed on 20 % sucrose and females were blood fed on a white small balb/c anesthetized with ketamine hydrochloride (60 mg/kg) and xylazine (5 mg/kg). preparation of salivary gland lysates of phlebotomus papatasi sand fly salivary glands were dissected in cold phosphate buffered saline (pbs), ph 7.2, and stored in fresh pbs at -20 ºc until used. salivary glands were disrupted by three cycles of freeze/thaw in liquid nitrogen and boiling water just before use. the sgl was centrifuged at 18000 g for 10 min, and the supernatants were used for experiments (volf and rohousova 2001, rohousova et al. 2005, akhavan 2011). the concentration of sgas was determined using the bca protein assay kit according to the manufacturer’s instructions (pierce biotechnology, rockford, usa). standards were prepared from bovine serum albumin (bsa) in sodium azide saline. animal sera great gerbils were collected from badrood rural district using sherman live traps. collections were carried out during the active sand fly season when the gerbils are supposedly repeatedly bitten by sand flies. animals were anesthetized with ketamine hydrochloride (60 mg/kg) and xylazine (5 mg/kg) and the isolated sera were kept at -20 ºc until use (akhavan 2011). hrpconjugated anti-rhombomys opimus antibody production rhombomys opimus antibodies were purified from animal sera by hitrap protein g chromatography. the antibodies were then injected intramuscularly in the hind legs of rabbits and the induction of anti-r. opimus antibodies was checked using elisa. antir. opimus antibodies were purified from rabbit sera and conjugated to horseradish peroxidase (hrp) then the titer of hrpconjugated anti-r. opimus antibodies was determined by elisa (akhavan et al. 2011). anti-phlebotomus papatasi saliva antibodies assessed by elisa anti-saliva antibodies were measured by elisa. sgl was prepared from 2–6 day old sand flies. elisa wells were coated with 50 μl sgl (equal to 0.5 gland per well) in carbonate-bicarbonate buffer (0.01 m, ph 9.6) overnight at 4 ºc. wells were washed three http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 42 times with pbs-tween 1x buffer. each well was treated with 50 μl r. opimus serum and incubated for 1 hour at 37 ºc. after 3 washes, 50 µ l of hrp-conjugated anti-gerbil antibodies (1: 1000 in pbs-tween) was added to each well, and incubated for 1 hour at 37 ºc. the wells were washed and 50 μl of substrate (3, 3’, 5, 5’-tetramethylbenzidine, tmb) added to each well and incubated for 15 minutes at room temperature. the stopping solution (20% h2so4) was added and the optical density measured by an elisa reader at 450 nm. negative sera were obtained from lab-bred r. opimus not bitten by any sand fly. the cut-off value was calculated by adding two standard deviations to the mean optical densities of negative controls. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) salivary glands were removed from collected sand flies. a sgl prepared from 10– 14 pooled glands was loaded into each lane to separate and visualize proteins or glycoproteins. sgl from 8 salivary glands were loaded into each well for western blots. electrophoresis was carried out at room temperature under reduced conditions on 13 % tris-glycine gels of 1mm thickness and a 100v constant voltage, using mini-protean iii (biorad, munich, germany). prestained protein ladders (pageruler, fermentas) were used and the gels were silver stained according to the methodology of heukeshoven and dernick (1985). western blot analysis after sds-page, one part of the gel was silver-stained and the second part was electrotransferred to pvdf (polyvinylidene difluoride) membrane (roche, pore size 0.45 µm) using a mini protean tetra cell (biorad) under constant voltage 100v for 75 min. the pvdf membrane was blocked using 5 % skim milk in pbs-tween and incubated overnight at 4 ºc. the pvdf membrane was treated with r. opimus positive serum, which was detected by elisa. the second part of pvdf membrane was treated with naïve r. opimus negative serum as a control. hrp-conjugated anti-r. opimus antibodies were added and the membrane was incubated for 1 hour at room temperature. positive bands were visualized using luminata forte (millipore, billerica, ma) immunochemical staining (akhavan 2011). detection and identification of leishmania species in phlebotomus papatasi dna was extracted from individual sand flies using exgenetm tissue sv (plus) kit (geneall biotechnology, korea). nested pcrs were done on individual sand flies using specific primers: outer forward primer (5′-aaa ctc ctc tct ggt gct tgc3′), outer reverse primer (5′-aaa caa agg ttg tcg ggg g-3′), inner forward primer (5′aat tca act tcg cgt tgg cc3′), inner reverse primer (5′-cct ctc ttt ttt ctc tgt gc-3′) (akhavan et al. 2010a). pcr-rflp was carried out to confirm the identity of the leishmania species in positive samples (akhavan et al. 2010b). additionally, pcr products of a limited number of specimens were sequenced for species identification. the composition and protein concentration of sgas were compared between infected and non-infected sand flies regardless of the physiologic state of the sand flies. ethical consideration the protocol was approved by the ethics committee of tehran university of medical sciences (no. 18511/16.5.2012). results protein concentration of sand fly salivary gland proteins the average protein content per pair of glands was 0.2, 0.1, 0.1 and 0.1µ g for unfed, fed, semi-gravid and gravid sand flies, rehttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 43 spectively. the protein content per pair of glands for sand flies collected in the spring and summer was <0.1 and 0.2 µ g, respectively. in sgl of parous and nulliparous sand flies, the protein content was 0.1 and <0.1 µ g per pair glands, respectively. the protein content in l. majorinfected and in non-infected sand flies was 0.1 and <0.1 µ g per pair of glands, respectively. leishmania infection in collected phlebotomus papatasi sand flies nested pcr and rflp analyses were used to detect and identify leishmania infection in p. papatasi sand flies. overall, 49 (32.2%) out of 152 p. papatasi females were infected with l. major. of those, 44 (28.9%) were infected with l. major alone, 1 (0.7%) sand fly was infected with l. major and l. turanica and 4 (2.6%) were infected with l. major, l. turanica and l. gerbili. three (2%) sand flies were infected with l. turanica and l. gerbili. determination of the presence of antiphlebotomus papatasi saliva antibody in rhombomys opimus serum serum samples were obtained from 13 r. opimus, collected during the active sand fly season when the gerbils are supposedly repeatedly bitten by sand flies and tested for antibodies against saliva of p. papatasi by elisa. all of the 13 gerbil sera examined were positive. phlebotomus papatasi salivary gland protein profile on polyacrylamide gels, the number of visualized protein bands was different between sand fly groups varying in the status of their accessory glands and physiological stages as well as by their collection seasons and presence or absence of leishmania infection. overall, 4–9 protein bands were observed with molecular weights ranging from 14 to 70 kda. the electrophoretic pattern of sgl of all ten groups of p. papatasi is shown in figure 2. the sgl of parous and nulliparous separated into 5 and 6 major protein bands with molecular masses of 14 to 70 kda, respectively, and one faint band of about 30 kda in both parous and nulliparous flies. the difference was a missing protein band from sgl of parous flies with molecular weight of around 42 kda (fig. 2a). in the sgl electrophoretic profile of unfed, fed, semi-gravid and gravid groups of sand flies, 3 major protein bands with molecular weights of 14–17 kda were observed. the groups of unfed and gravid flies differed from fed and semi-gravid flies in showing the extra protein bands in their electrophoretic profiles. in sgl of gravid sand flies 2 faint bands with molecular weights of around 28 and 42 kda and in unfed sand flies one faint band with around 28 kda were also visible. the sgl of unfed and gravid flies differed also in the intensity of bands of 14– 17 kda. these protein bands in unfed and gravid groups of sand flies were stronger than fed and semi-gravid groups (fig. 2b). the sgl of sand flies collected throughout spring separated into 6 strong proteins bands with molecular weights of 14–42 kda and one weak band of around 36 kda. sand flies collected throughout summer had a sgl pattern of 5 protein bands with molecular weights ranging from 14 to 30 kda. the sgl of both spring and summer groups of sand flies, showed 5 protein bands of around 14–30 kda. the difference was in the presence of 2 protein bands of around 36 and 42 kda in sgl of spring flies which were missing from sgl of flies collecting in summer (fig. 2c). the sgl profiles of l. major-infected and non-infected sand flies were similar with 5 proteins bands ranging from 14–30 kda. just a little difference was seen in the intensity of mentioned protein bands, which were a bit stronger in non-infected sand flies (fig. 2d). http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 44 rhombomys opimus antibody response to phlebotomus papatasi salivary proteins the antibody response of r. opimus against sgas of the ten different sand fly groups was determined by western blot analysis (fig. 3). negative serum did not react with any of the sgas in the ten sand fly groups. the positive serum reacted variably to the sgas of the different groups. western blot analyses revealed 6–9 antigenic bands with molecular masses of 14–72 kda. the immunoreactions were stronger with sgas around 17, 22, 24 and 28 kda in the sgl of parous flies and with sgas around 14, 22, 24 and 28 kda in the sgl of nulliparous flies, respectively (fig. 3a). the serum reacted with 9 sgas of unfed, fed, semi-gravid and gravid sand flies. the strongest reactions were observed against antigens of gravid sand flies. a strong reaction was observed against a 28-kda antigen in unfed, fed, semi-gravid and gravid sand flies (fig. 3b). the r. opimus serum recognized 6 antigenic bands, five common antigens were between 14–28 kda in sgl of both spring and summer collected sand flies. one antigenic band of 44 and one band of 69 kda was specifically recognized in spring and summer collected sand flies, respectively (fig. 3c). anti p. papatasi serum from r. opimus reacted similarly with sgl of leishmania infected and non-infected sand flies. six antigenic bands from 14 to 44 kda and one faint band of 68 kd were recognized. the serum reactions differed only in intensity with non-infected flies recognizing a 22 kda and a 24 kda antigen more strongly than infected flies (fig. 3d). fig.1. geographical location of abbasabad, habibabad and parvaneh-aliabadchi in esfahan province, central iran fig. 2. sds-page analyses of salivary gland antigens of sand flies collected from esfahan province, central iran. a: parous(p) and nulliparous(n) groups, b: unfed(u), fed(f), semi-gravid(sg) and gravid(g) groups, c: spring(sp) and summer(su) collections, d: infected(in) and non-infected(no), m: prestained protein marker http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 45 fig. 3. western blot analyses of salivary gland antigens (sgas) collected from esfahan province, central iran. a: sgas of parous(p) and nulliparous(n) groups, b: sgas of unfed(u), fed(f), semi-gravid(sg) and gravid(g) groups, c: sgas of spring(sp) and summer(su) collected sand flies, d: sgas of infected(in) and non-infected(no) sand flies, m: prestained protein marker discussion salivary glands composition depends on various characteristics of sand flies such as sex, age, generation and geographical location (volf et al. 2000, ben hadj ahmed et al. 2010, akhavan 2011). mice pre-immunized with salivary gland extract (sge) of f 29 laboratory-bred female p. papatasi were protected against l. major co-inoculated with the same type of sge while the mice pre-immunized with sge of wild-caught or f1 sand flies were not protected (ben hadj ahmed et al. 2010). however, in field conditions, humans naturally exposed to p. duboscqi sand fly bites reacted with a th1-like response to the bites of colonized sand flies, suggesting that humans react in the same manner to colonized and field-collected sand fly salivary proteins (oliveira et al. 2013). the composition of sand fly salivary glands is therefore an important element of leishmania infectivity, where the amount of sgas may affect their antigenicity and the outcome of infection with l. major. in this study, the salivary protein content differed in the various physiological stages of sand flies. the highest content of sgl protein was seen in unfed sand flies. our results also showed that the number and the intensity of protein bands in the sgl of unfed sand flies was more than of fed and semi-gravid sand flies. this might be due to the deposition of saliva as an obligatory part of the sand fly blood feeding strategy, reducing the amount of saliva in blood fed flies. compatible with this, the reactivity of antigens was stronger in the sgl of unfed and gravid sand flies than in semi-gravid and fed sand flies. the protein content of saliva is not constant and varies with age, reaching the full electrophoretic pattern in 3–5 days flies (volf et al. 2000), but in the sand flies collected from the field, age is not defined, very young sand flies as well as very old ones have very low amounts of sga proteins. this may account for the overall five to ten times lower salivary gland protein content observed in this study compared to the 1ug reported for 4-day-old colonized p. papatasi (volf et al. 2000, valenzuela et al. 2001). the protein content of sand fly saliva also varies by geographical location and in different species (volf and rohousova 2001). the salivary protein content in p. papatasi from http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 46 a cyprus colony was 0.51 µ g per gland, for p. papatasi from a turkish colony was 0.33 µg/gland, for p. duboscqi was 0.78 µg/gland, for p. halepensis was 0.41 µ g/gland, and for p. sergenti from a turkish colony was 0.23 µ g/gland and for lutzomyia longipalpis was 0.18 µ g/gland (cerana et al. 2002). in our study, the salivary protein content was higher in parous compared to nulliparous sand flies, in summer compared to spring collected sand flies, and in leishmania-infected compared to non-infected sand flies. nulliparous sand flies collected in this study might also be too young with their glands containing an insufficient amount of saliva. additionally, the protein content of sand fly salivary glands collected in the summer was higher than in spring-collected sand flies, which seems to be due to environmental conditions. in the study area, the climate conditions change between seasons, in summer, the relative humidity is lower and the relative temperature is higher compared to spring, and there is almost no rainfall during summer. in a previous study, the effect of ecological characteristics on salivary gland gene expression was determined; expression levels of five salivary gland genes of p. papatasi were up regulated in september due to a water deficiency, which resulted in a reduction of sugar sources for sand flies (coutinhoabreu et al. 2011). in the current study, the climate variations supposedly change the type and the amount of vegetation in the study area which effect on the phytophaghy of sand flies. in sgl of sand flies collected through spring there were 2 more protein bands of around 36 and 42 kda compared to those collected in the summer, showing the influence of ecological factors. the higher amount of saliva in infected sand flies in the current study might be explained by the fact that infected sand flies probe longer to obtain a blood meal and need to inject more saliva during blood meal to benefit from the biological and immunomodulatory effects of saliva (kamhawi 2006). in the current study, the sgl of the different groups of p. papatasi showed 4–9 protein bands with molecular weights ranging from 14 to 70 kda. in a study performed by volf and rohousova (2001) from 5 to 8 prominent protein bands with molecular weights from 28 to 50 kda were recognized. in another study sds-page analysis showed 14 major protein bands with 12–70 kda molecular weights (rohousova et al. 2005). in a previous study in our lab, 7 major protein bands of p. papatasi salivary gland lysate with molecular masses of around 1240 kda and 3 faint bands of 20, 55 and 65 kda were observed (akhavan 2011). this difference might reflect geographical distance of p. papatasi subpopulations (hamarsheh et al. 2009) or be due to the generation of sand fly (ben hadj ahmed et al. 2010). in our study western blot analysis of r. opimus serum revealed 6–9 antigenic bands with molecular masses of 14–72 kda compared to 8 antigenic bands from p. papatasi collected from borkhar and sejzi rural district in iran (akhavan 2011), 4–9 major antigenic bands for colonized p. papatasi scopoli against balb/c mice (volf and rohousova 2001) and 4–6 major antigenic bands for colonized p. papatasi from turkey against balb/c mice (rohousova et al. 2005). these differences may be because the reservoir species was not the same and highlight the specificity of the immunogenicity of sgas in various hosts. the antigenic profile of spring and summer collections was different by one band only which seems to be due to environmental factors. the serum reactions with sgl of leishmania infected and non-infected sand flies were similar and the difference was only in the strength of the reaction which may indicate that leishmania infection do not affect saliva components. importantly, the r. opimus serum reacted strongly with an http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 39–49 n hosseini-vasoukolaei et al.: seasonal and … 47 antigenic band of around 28 kda in the sgl of all sand fly groups collected in this study. further studies are needed to confirm the immunogenicity of this protein in a larger sample of r. opimus to assess its potential as a marker of exposure to p. papatasi. this protein may be ppsp32, a protein that is highly recognized by humans bitten by p. papatasi (marzouki et al. 2012). conclusion in our study, some faint protein bands in the sgl profile reacted strongly with the serum of r. opimus. conversely, some major protein bands in the sgl profile reacted weakly in the western blot analysis. this should be considered in saliva-based vaccine development. this study shows that certain biological and environmental characteristics of wild populations of vector sand flies affect the protein content and antigenicity of saliva. this might have an important implication in the design of vector-based vaccines. acknowledgements this research was supported by research deputy of tehran university of medical sciences, project no. 18511/16.5.2012 and avicenna research institute project no.910206018. we are very grateful to dr shabani for his valuable comment and ms babaei, ms balaei, mr hadavi for their technical assistance, from avicenna research institute, acecr. we also thank ms. shareghi, esfahan health research station, national institute of health research, tums, for her assistance in the project. we are grateful to ms. ahmadi and ms. bolandian, school of public health, tums, for their assistance in sand fly rearing. the authors declare that there is no conflict of interests. references akhavan 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yaghoobi-ershadi mr, javadian e (1997) studies on sandflies in a hyperendemic area of zoonotic cutaneous leishmaniasis in iran. indian j med res. 105: 61–66. yaghoobi-ershadi mr, akhavan aa (1999) entomological survey of sandflies (diptera: psychodidae) in a new focus of zoonotic cutaneous leishmaniasis in iran. acta trop. 73: 321–326. yaghoobi-ershadi mr, hanafi-bojd aa, akhavan aa, zahrai-ramazani ar, mohebali m (2001) epidemiological study in a new focus of cutaneous leishmaniasis due to leishmania major in ardestan town, central iran. acta trop. 79: 115–121. yaghoobi-ershadi mr, akhavan aa, zahraeiramazani ar, abai mr, ebrahimi b, vafaei-nezhad r, hanafi-bojd aa, jafari r (2003) epidemiological study in a new focus of cutaneous leishmaniasis in the islamic republic of iran. east mediterr health j. 9: 816–826. yaghoobi-ershadi mr, hakimiparizi m, zahraei-ramazani ar, abdoli h, akhavan aa, aghasi m, arandian mh, ranjbar aa (2010) sand fly surveillance within an emerging epidemic focus of cutaneous leishmaniasis in southeastern iran. iran j arthropod borne dis. 4: 17–23. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod borne dis. 6: 1–17. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, december 2016, 10(4): 602–607 cd patil et al.: vorticella sp: prospective … 602 http://jad.tums.ac.ir published online: october 04, 2016 short communication vorticella sp: prospective mosquito biocontrol agent chandrashekhar devidas patil, chandrakant prakash narkhede, rahul khushal suryawanshi, *satish vitthal patil school of life sciences, north maharashtra university, jalgaon, maharashtra, india (received 14 nov 2014; accepted 10 nov 2015) abstract background: considering the disadvantages of chemical insecticides, we aimed to evaluate vorticella parasites for control of mosquito larvae of anopheles stephensi and aedes aegypti at different larval stages. methods: vorticella sp infected mosquito larvae were crushed in the 0.85% saline and homogenized well to get vorticella in suspension. the effects of vorticella sp infections on larval development were investigated by inoculating protozoan on different larval instars of an. stephensi and ae. aegypti and observed under light microscope. lethal time of the vorticella infected larvae at different stages was calculated. results: first and 2nd larval instars of both an. stephensi and ae. aegypti did not show signs of infection by vorticella sp., whereas 3rd instars of an. stephensi showed more vorticella infection than those of ae. aegypti. however, 4th larval instars of both mosquitoes were heavily infected with vorticella parasite which was responsible for sluggish movements of larvae and eventually death. moreover, parasites (vorticella spp) were responsible for more than 90% reduction in adult emergence for both infected an. stephensi and ae. aegypti. conclusion: this study provides insights for mosquito larvicidal action of surface parasite vorticella on different larval stages of an. stephensi and ae. aegypti. it could be suggested as a potential candidate in mosquito biocontrol programs. keywords: anopheles stephensi, aedes aegypti, vorticella, biocontrol introduction pathogenic agents of diseases like malaria, dengue fever, yellow fever and filariasis are mainly transmitted by mosquitoes and therefore are responsible for human mortality and morbidity throughout the tropics (who 2004). as there is less development in drugs controlling pathogenic agents, mosquito vector control has been recognized as an effective tool for controlling the spread of these tropical diseases. several synthetic chemical insecticides such as organophosphate, temephos, etc. have been used during the past several decades for the mosquito control. however, their use has been greatly impeded due to development of insecticide resistant vectors (who 2013). besides the use of chemical insecticides raised issues of environmental pollution which impart harmful effects on non-target animals (rao et al. 1995). due to such reasons, there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. bacillus thuringiensis subsp israelensis (bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. although bti based strategies represent an environmentally friendly approach to mosquito suppression with the potential for area-wide implementation, it is still lagging behind chemical insecticides, presumably due to its high cost and geometrical variation in bti activity. in addition, the development of bti resistance has made it difficult to control insect pests with bti alone (margot et al. 2011). several insect control agents have been with*corresponding authors: dr satish vitthal patil, e-mail: satish.patil7@gmail.com j arthropod-borne dis, december 2016, 10(4): 602–607 cd patil et al.: vorticella sp: prospective … 603 http://jad.tums.ac.ir published online: october 04, 2016 drawn for economic or regulatory reasons resulting in greater selection pressure and more rapid resistance development to the remaining materials (phillip et al. 2011). thus, it is necessary to search new alternatives to develop promising biological control against mosquitoes. although various biocontrol measures are in vogue, their effective control is yet to be highlighted. in continuation of our previous studies (chandrashekhar et al. 2011a, 2011b, rahul et al. 2011, suryawanshi et al. 2014, 2015) on searching of insecticidal agents for mosquito borne disease control, this study was conducted to isolate new insecticidal agents against mosquito larvae. various samples of dead mosquito larvae were collected from different mosquito breeding sites. we found extracellular protozoan parasite associated with these larvae. the parasite was identified as vorticella, a genus of peritrich protozoan. it is one of the very first protists seen by antony van leeuwenhoek who observed it through the lens of his simple microscope in the late seventeenth century. vorticella has a bell shaped body with a cilia lined in oral cavity at one end and a long stalk on the other. vorticella reproduces asexually by mitotic cell division (noland and finley 1931). they are sessile fresh water forms reported from different habitats like moist soil, mud and plant roots (neumann and martinoia 2002). in this study we aimed for i) collection of the vorticella sp in suspension form ii) collection of mosquito larvae from local breeding sites, iii) evaluation of vorticella as a future biocontroling agent for mosquito larvae iv) calculation of lethal time of mosquito larvae for vorticella suspension and v) to study the target larval selection by vorticella sp. materials and methods aedes aegypti and an. stephensi larvae (1st, 2nd, 3rd and 4th instar) were collected from nearby marshy places or stagnant ponds of jalgaon area, at geographical coordinates of 21°2′54″n, 76°32′3″e, elevation 209 m and used as experimental mosquitoes. key factor for primary identification was swimming behavior of aedes and anopheles species (e.g. anopheles larvae rests parallel while aedes larvae attach to water surface with their body pointing downwards). from such samples, few larvae (5–10 depending on population size) were taken randomly for identification purpose. identification of mosquito larvae was done by observing body parts like antenna, cephalon, mesothorax, metathorax and abdominal segments (du bose and curtin 1965, ramachandra 1984, subbarao et al. 1988, amerasinghe 1990). for maintenance, the larvae were kept in plastic trays containing dechlorinated tap water. the experiments were carried out at 28± 2 °c and 75–85% relative humidity under 14:10 light and dark cycles. larvae were fed with a diet of finely ground brewer's yeast and dog biscuits (3:1) (chandrashekhar et al. 2011a, suryawanshi et al. 2014). larvae were observed under microscope (labomade lb341), heavily infected larvae were identified by presence of attached parasites all over the body surface, more populated at the junction of all segments and sluggish movements of infected larvae. five heavily infected mosquito larvae were selected. the larvae were further crushed in the 0.85% saline (5 ml) and homogenized well to prepare vorticella suspension. the presence of vorticella in the suspension was confirmed by observation under microscope. this suspension (5 ml) was inoculated into glass trays each containing the test 300 mosquito larvae (each instars) of an. stephensi or ae. aegypti in one liter tap water containing glass tray and observations were made after each 24 h. moreover, the effects of vorticella sp infections on larval development were investigated by inoculating protozoan on the different larval instars of an. stephensi and ae. aegypti. j arthropod-borne dis, december 2016, 10(4): 602–607 cd patil et al.: vorticella sp: prospective … 604 http://jad.tums.ac.ir published online: october 04, 2016 similar experiment was done for observing the preference of mosquito species by vorticella by infecting vorticella suspension to the mixture of both an. stephensi and ae. aegypti larvae at their 4th larval instar. mortality was corrected using abbott's formula (abbott 1925). the dose–response data was subjected to probit regression analysis (finney 1971). lethal time 50 (lt50) (the time at which 50% larvae died) of the larvae at different stages was calculated by probit analysis. results after 24 h, around five to ten vorticella sp stalk bodies could be observed on first segment and 20–25 stalk bodies on last anal segment of only 4th instar larvae of an. stephensi. only a few stalked bodies of parasites were observed on 3rd instar larvae. while no any infection was observed on 1st and 2nd larval instars of an. stephensi. similarly, ae. aegypti larvae were not infected by protozoan up to 48 h and only few stalk bodies were observed on the first segment of 4th instars after 50 h after which infection was found to increase during the progression of life cycle. in contrast, the whole body surfaces of an. stephensi larvae were infected by the long stalked vorticella sp up to 48 h (fig. 1). anopheles stephensi larvae did not showed abnormal changes in motility behavior for the first 48 h. however, the larvae became sluggish and then immotile after 48 h. during this stage, larvae showed movement of only upper mouthpart and siphon. when mixed larval populations of anopheles and aedes were inoculated with vorticella the parasites preferred only anopheles larvae for infections. however, they heavily infect the aedes larvae after the death of anopheles larvae. the effects of vorticella sp infections on larval development were investigated by inoculating the parasite into different larval instars of an. stephensi and ae. aegypti. the 50% of 3rd and 4th instars population of an. stephensi larvae were killed after 72 h and 59 h, respectively (lt50). the 1st and 2nd instars larvae of both the mosquito species did not show any mortality up to72 h. the 3rd instar of ae. aegypti showed the infection but did not cause mortality. however, 4th instar larvae showed 50% mortality after 84 h. only first segment of larvae was infected by parasites (table 1). in adult immergence assay, out of the total 300 larvae, 244 larvae of ae. aegypti died within 84 h, 56 went to pupal stage and 23 adult mosquitoes were emerged. upto 255 out of 300 an. stephensi larvae were killed, and 43 developed to pupal stage and 17 emerged to adult mosquitoes. the emergence rate of control an. stephensi and ae. aegypti without vorticella treatment was 73% and 76.1%, respectively (table 2). only 18.66% and 10% larvae of an. stephensi and ae. aegypti attained pupal stages, respectively and died thereafter. only 5.86% and 7.43% emerged as adult mosquitoes (table 2). however, the emerged mosquitoes died within 4–5 days after their transfer and culture on the artificial diet. table 1. effect of vorticella sp infection on mortality of mosquito larvae mosquito species larval instars stage infection lt50 (hours) aedes aegypti i ii iii iv nil nil d ++ ---82.3±8.6 j arthropod-borne dis, december 2016, 10(4): 602–607 cd patil et al.: vorticella sp: prospective … 605 http://jad.tums.ac.ir published online: october 04, 2016 anopheles stephensi i ii iii iv nil nil +++ ++++ --72±7 59±8.5 lt50= the time at which 50% larvae died d= detectable ++= infected only first segment of larvae and very few vorticella parasites were observed +++= vorticella parasites infected the whole larval body and many vorticella parasites were observed ++++= very high infection throughout larval body and larvae became sluggish table 2. effect of vorticella sp infection on emergence of adult mosquitoes mosquito species measure 4th instars larval mortality (number/ 300) pupae emerged (number/300) adults emerged (number/300) aedes aegypti controla testb 39.3±4.04 244.67±15 260±15 56.6±9 228.3±12.6 22.3±5 anopheles stephensi controla testb 29.6±4.5 255.7±10 271.6±17.5 43.6±6 219.3±17.7 17.6±5 acontrollarvae without vorticella infection btestlarvae with vorticella infection fig. 1. infection of parasite (vorticella spp) to the 4th larval instars of anopheles mosquito (10x magnification) discussion vorticella spp. are largely known as bacterial feeders and have also been reported to parasitize nematodes with the help of their integument, in rana pipiens and rana sylvatica tadpoles it cause tegumentary impairment while in fresh water prawns acrobrachiumrosen bergii vorticella sp infect the eye stalk, antenna, uropod and egg masses (tonguthai 1997). ciliates and vorticella sp have been found occasionally infecting mosquito larvae (muspratt 1945, micks 1950, 1955, schober 1967). vorticellids of vorticella can cause infection in mosquito species at risk (micks 1950). in the present study, the effects of vorticella sp on all larval instars of an. stephensi table 1. continued… j arthropod-borne dis, december 2016, 10(4): 602–607 cd patil et al.: vorticella sp: prospective … 606 http://jad.tums.ac.ir published online: october 04, 2016 and ae. aegypti were investigated. the study on life cycle of the infected an. stephensi and ae. aegypti larvae has revealed the inhibition of larval growth, development and adult emergence of both the test mosquito species. micks (1950) reported vorticella infection in anopheles and death of 7000 infected larvae. however, the mechanism behind this inhibition is still unknown since more than 74 years of first report. the foot of the stalk attaches to a substrate, such as a rock, plant, or even an aquatic animal. the stalks of these organisms are capable of contracting at astounding speeds. the mechanism by which the adhesive pad of v. convallaria adheres to so many different substrates is also unknown. the organism secrets a some sort of “glue”, probably a biopolymer. it may be possible that organism secretes some biochemical substances with these glue to fix itself on substrate and those substances may damaging some surface sensory system or causing pore formation in larval body. either it may also be possible that the metabolic and secretary products of vorticella are toxic to the mosquito larvae, polluting its natural environment. as per schober (1967) this association is benefited by vorticella as it may be using host as a transportation vehicle to spread in new environment, while as per mick (1955) the larval death may be apparently due to the inability of the infected larvae to remain on the water surface, thereby interfering with respiration. previously it was observed that vorticella infection was found only in anopheles and it did not show either infection or mortalities in aedes aegypti. in the present study, although vorticella sp has first preference to anopheles but it can attack other mosquito species like ae. aegypti. the observations from this study can be important to design strategy to control of mosquito larvae. further studies on non-target organisms and persistence in different environment will be needed for use of vorticella sp as potential biocontrol agent in the mosquito larval habitat. conclusion the vorticella sp found in the present study showed potential of infection and mortality of an. stephensi and ae. aegypti mosquito larvae. being major aquatic living species, vorticella sp alone can be valuable for devising easy, effective and eco-friendly strategy for mosquito biocontrol. this may be a new species and need to do the morphological study to be identified. acknowledgements authors are thankful to ugc-sap and dst-fist, government of india, for providing financial support to the school of life sciences. the authors declares is no conflict of interests. references abbott ws (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–266. amerasinghe fp (1990) a guide to the identification of the anopheline mosquitoes (diptera: culicidae) of sri lanka. i. adult females. ceylon j sci (biol sci). 21(1): 1–16. bose dwp, curtin tj (1965) identification keys to the adult and larval mosquitoes of the mediterranean area. j med entomol. 1(4): 349–355. chandrashekhar dp, satish vp, bipinchandra s, rahul bs (2011a) insecticidal potency of bacterial species bacillus thuringiensis sv2 and serratia nematodiphila sv6 against larvae of mosquito species aedes aegypti, anopheles stephensi, and culex quinquefascitus. parasitol res. 110(5): 1841–1847. chandrashekhar dp, satish vp, bipinchandra s, rahul bs (2011b) prodigiosin produced by serratia marcescens nmcc46 j arthropod-borne dis, december 2016, 10(4): 602–607 cd patil et al.: vorticella sp: prospective … 607 http://jad.tums.ac.ir published online: october 04, 2016 as a mosquito larvicidal agent against aedes aegypti and anopheles stephensi. parasitol. 109(4): 1179–1187. finney dj 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insecticidal potency of novel compounds on multiple insect species of medical and veterinary importance. pest manag sci. 67: 26–35. rahul bs, satish vp, chandrashekhar dp, bipinchandra s (2011) larvicidal potential of silver nanoparticles synthesized using fungus cochliobolus lunatus against aedes aegypti (linnaeus, 1762) and ano pheles stephensi liston (diptera: culicidae). parasitol res. 109(3): 823–831. ramachandra rt (1984) the anophelines of india. indian medical council, new delhi. rao dr, mani tr, rajendran r, joseph as, gajanana a, reuben r (1995) development of high level of resistance to bacillus sphaericus in a field population of culex quinquefasciatus from kochi, india. j am mosq control assoc. 11: 1–5. schober h (1967) observations on culex pipiens larvae infected with vorticella sp. mosq news. 27: 523–525. subbarao sk, adak t, sharma vp. (1988) cytotaxonomy of certain malaria vectors of india. in: service mw (eds): biosystematics of haematophagous insects, clarendon press, oxford, pp. 25–37. suryawanshi rk, borase hp, chandrashekhar dp, narkhede c, patil sv (2014) screening of rubiaceae and violaceae extracts for mosquito larvicidal potential. nat prod res. 15: 1–6. suryawanshi rk, patil cd, borase hp, narkhede cp, salunke bk, patil sv (2015) mosquito larvicidal and pupaecidal potential of prodigiosin from serratia marcescens and understanding its mechanism of action. pest biochem physiol. 123: 49–55. tonguthai k (1997) diseases of the fresh water prawn, macrobrachium rosenbergii. aquatic animal health research institute newsletter. available from: http://www.agriaua.ait.ac.th/aahri/seaa dcp/aahrt/newsletter/art6.htm. who (2013) world malaria report 2012. available at http://www.who.int/malaria/ publictions/world_malaria_report_2012/ en/. who (2004) the world health report: changing history. available at: http://www. who.int/whr/2004/en/. j arthropod-borne dis, june 2013, 7(1): 23–30 m koosha et al.: lectin activity in… http://jad.tums.ac.ir published online: april 10, 2013 original article lectin activity in gut extract of culex pipiens mona koosha 1, mohammad reza abai 1, mandan abolhasani 1, soroor charedar 2, *hamid reza basseri 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical parasitology, school of public health, tehran university of medical sciences, tehran, iran (received 15 june 2011; accepted 13 june 2012) abstract background: the role of lectins is important in interaction between pathogens and mosquito vectors. this study was performed to identify agglutinin activities of protein molecules on the midgut of culex pipiens. methods: culex pipiens was reared in insectray condition and the midguts of males and females (blood fed and unfed) were dissected separately in tris-hcl buffer. the extracts of midguts were applied for hemagglutinin assay against red blood cells of rabbit, mouse, rat, dog, horse, sheep, guinea pig, cow, human (a, b, ab, o groups). then, the rbcs with relatively high agglutinin activity were chosen for carbohydrate inhibition assay. d (+) glucose, d (+) galactose, d (+) mannose, d (-) fructose, d (-) arabinose, l (-) fucose, lactose, n-acetyl-d-glucosamine, n-acetyl-dgalactosamine, sialic acid were used to specify carbohydrate binding lectin. results: the highest agglutinin activities were found against sheep and rabbits rbcs. sexual diversity of agglutinin activities was observed among midgut extraction of males and females. in addition, variation in agglutinin activity of blood fed and unfed female mosquitoes were detected. the lectin activity was inhibited highly with glucose, galactose, fucose and fructose but less inhibitor activities was observed by arabinose, n-acetyl-d-galactosamine, nacetyl-d-glucosamine, lactose and mannose. conclusion: the secretion of hemagglutinins (lectins or lectin-like molecules) in the digestive system depends on the type of food in the gut. this suggests that emptying of the gut in preparation for protein rich food probably starts the secretion of hemagglutinins. keywords: mosquitoes, culex pipiens, lectin, hemagglutination activity, midgut introduction lectins are defined as carbohydrate-binding proteins or glycoproteins of non immune origin which agglutinate cells and/or precipitate glyco-conjugates (goldstein and poretz 1986). lectins in insects with distinct sugar specificities involve in recognition and protective roles in immune defense against microbial pathogens. the agglutinins against vertebrate erythrocytes have been reported in various insects (basseri 2002). insect hemagglutinins are lectin or lectin-like molecules that are ubiquitous, non-enzymatic carbohydrate binding proteins or glycoproteins and once bound to erythrocytes or other cells, usually cause their agglutination, and may also precipitate glycol conjugates (stebbins and hapner1986, ingram 1997). most studies have concentrated on the hemagglutination activity (ha) in the gut of hematophagous insects especially dipteran (rudin and hecker 1989, volf 1993, grubhoffer et al. 1994, volf et al. 1994, 1995, 1998). the role of lectins are important in the life cycle of some parasitic protozoa carried by mosquitoes, sand flies, tsetse flies, and many other bloodsucking insects (ingram *corresponding author: dr hamid reza basseri, email: basserih@tums.ac.ir 23 j arthropod-borne dis, june 2013, 7(1): 23–30 m koosha et al.: lectin activity in… http://jad.tums.ac.ir published online: april 10, 2013 and molyneux 1991, basseri 2002, basseri et al. 2008). these molecules in the gut and hemolymph cause the establishment of infection and parasitic development (ibrahim et al. 1984, mello et al. 1999). in addition, anti-parasite agglutinins have been detected in midgut extracts of trypanosome vectors (ibrahim et al. 1984, wallbanks et al. 1986). unfortunately there is not enough information about the nature of lectincarbohydrate in insect-vector–parasite interaction. in spite of the fact that there are specific mechanisms between lectin receptors on surface of parasites of vector tissue which can ensure the success or failure of infection (rudin 1991, billingsley 1997, basseri et al. 2008), some investigators suggest that interactions between parasites and vector gut walls may be mediated by the carbohydrates on the surface of parasites. besides the lectins in the vector gut, as characteristic carbohydrate markers have been identified on the surface of parasites such as trypanosoma and leishmania (schottelius 1982a, 1982b, 1982c). the midgut lectins have been identified from such vectors as rhodnius prolixus (pereira et al. 1980) glossina austain (ibrahim et al. 1984) phlebotomus papatasi (wallbanks et al. 1986) and anopheles gambiae (mohamed and ingram 1994). culex pipiens is usually the most common pest mosquito in urban as well as rural areas of iran (azari-hamidian 2007, dehghan et al. 2010). the purpose of present study was to detect the hemagglutinin activity in the midgut extraction of males and females of cu. pipiens. furthermore, to find changes in hemagglutination activity in blood fed and unfed female mosquitoes. moreover, specific carbohydrates of lectins were surveyed to characterize the lectins in the midgut. the mosquito lectin has been partially characterized for further study and their functional roles. materials and methods insect rearing and sample preparation the laboratory strain of the cx. pipiens was used. the mosquitoes were reared for more than 48 years in the insectray. during the present study they were occasionally allowed to feed on laboratory guinea pigs and reared under a photoperiod of 12:12 day/night at 28±2 ºc and 50–60% relative humidity. the adult females including fed and unfed females as well as males were then applied separately for midgut dissections. preparation of gut mosquitoes gut were dissected separately in tn buffer (20mm tris hcl, 0.15m nacl, ph=7, 5mm cacl2). the guts were collected and washed with the buffer and homogenate using mechanical homogenizer in cold condition. then, the homogenate samples were centrifuged at 10,000g for 15min, three times. the supernatant were kept in -80 °c until use. protein assay the concentration of midgut proteins was estimated as discussed by bradford (1976) and in order to obtain standard curve, serial dilution of different concentrations of bovine serum albumin (bsa) was used. preparation of erythrocyte blood from rabbit, mouse, rat, dog, horse, sheep, guinea pig, cow, human (a, b, ab, o groups) were prepared in 3.8% (w/v) trisodium citrate. in order to prepare red blood cells, whole bloods were washed three times in tn buffer at 1500g for 5min each to remove serum and gain rbcs. finally a 2% (v/v) suspension of rbc was prepared and kept at +4 °c until use for hemagglutination assay and also hemagglutination inhibition assay. 24 j arthropod-borne dis, june 2013, 7(1): 23–30 m koosha et al.: lectin activity in… http://jad.tums.ac.ir published online: april 10, 2013 hemagglutination assay five microlitre of each midgut extract was serially diluted in tn buffer (as suggested by uhlir et al. 1996) in the v-bottom wells of micro titration plates. then 5 microlitre of 2% mentioned erythrocytes suspension was added to each well. the titer of hemagglutination activity was determined under stereomicroscope after 60 min incubation at room temperature. unagglutination described as rbcs with clear dot on the bottom of the well, and agglutinatinated targets formed a diffuse mat. all experiments were repeated three times. the controls contained tn buffer and 2% bsa. finally, the erythrocyte which had the highest dilution activities visually was chosen for next experiments. the reciprocal value of the highest dilution with positive reaction was scored as the titer. hemagglutination inhibition assay (hia) the hia was performed to determine the inhibitory activities of different carbohydrates against the midgut lectin activities as follows: d (+) glucose, d (+) galactose, d (+) mannose, d (-) fructose, d (-) arabinose, l (-) fucose, lactose, n-acetyl-d-glucosamine, nacetyl –d-galactosamine, sialic acid (all form sigma). the stock solutions of carbohydrates were prepared in nacl/tris/ca 2+ buffer at 0.2m stored at -20 ºc until use. for each inhibition (5μl) aliquots of buffer contain carbohydrate was added in each microlitre wells, followed by 5 microlitre of gut extracted adjusted subsequently to titer of 1: 4096. these were mixed gently by shaking and incubated for 60 min at room temperature. finally, 5 microlitres of 2% rbcs of sheep or rabbit suspension was added into each well and left the microplates for 1 h at room temperature to incubate the mixtures. these tests were done three times. well without lectin or inhibitors were considered as control. enzyme treatment of rbcs in order to expose more receptors of rbcs to extracted lectins, all rbcs were treated with trypsin. equal volume of 2% above rbcs, trypsin (2 mg/ml) solution prepared in tris-hcl, were mixed together and incubated at 37 ºc for 25 min. the rbcs were then washed three times with buffer and adjusted to 2% suspensions. treated or untreated rbcs were incubated with gut extract at room temperature for 30 min. subsequently, hemagglutination titers were assessed. results hemagglutination assay the hemagglutination patterns of whole proteins from the midguts of cx. pipiens against a range of erythrocytes were determined (fig. 1). the highest activates occurred against sheep erythrocytes (titer of 256) followed by rabbits (titer of 64). the less activity was observed against guinea pig erythrocytes (titer≤ 16). thus, sheep erythrocyte was used for inhibition assay. trypsinization assay modification of rabbit’s erythrocyte membranes by trypsin treatment increased the agglutinin activities more than eight times indicating more lectin molecules present in the extracted proteins. appearance of hemagglutinating activity at sex and fed condition the hemagglutination activity of midgut extracts of male and female mosquitoes was different (table 1) while the proteins of males' midgut showed less agglutinin activities rather than females. in addition, fed females of cx. pipiens demonstrated two times more agglutinin activity than unfed females (table 1). 25 j arthropod-borne dis, june 2013, 7(1): 23–30 m koosha et al.: lectin activity in… http://jad.tums.ac.ir published online: april 10, 2013 carbohydrate inhibition assay as shown in table 2, the hemagglutination inhibition of sugars for extracted proteins of midgut was dissimilar. the lectin activity was inhibited highly by glucose followed by galactose, fucose and fructose but less inhibitor activities were observed by arabinose, n-acetyl-d-galactosamine, n-acetyld-glucosamine, lactose and mannose. table 1. effect of induction and blood meal feeding and compartment with mosquito sex on lectin hemagglutination activity (ha) protein concentration (mg/ml) ha (with candidate rbcs) (titer) fed 0.17 32 unfed 0.06 16 male 0.15 8 the number in last column represents endpoint titers expressed as the reciprocals of the dilutions (1/n). all samples were prepared three times and the assay repeated table 2. effects of inhibitory sugars on agglutinin activities of protein on midgut extract of culex pipiens inhibitor titer d(+) glucose >256 d(+) galactose >128 d(+) mannose >64 l(+) fucose >128 lactose >64 n-acetyl-d-glucosamine >64 n-acetyl-d-galactoseamine >64 l(+) arabinose >64 d(+) fructose >128 fig. 1. agglutination activities of midgut extract of culex pipiens against different erythrocytes fig. 2. comparison between trypsin enzyme treated and untreated rbc agglutinin activities against midgut extract of culex pipiens discussion the present study shows that hemagglutination activity exists in midgut of adults of both sexes. in addition, this activity was higher among fed females. the hemagglutination activities were described in digestive systems of six species of phlebotomine sand flies (volf and killick-kendrick 1996). they showed that higher hemagglutination activity in the gut of 26 j arthropod-borne dis, june 2013, 7(1): 23–30 m koosha et al.: lectin activity in… http://jad.tums.ac.ir published online: april 10, 2013 females and characterized the lectins in the females' midgut. the results of their study showed that, the agglutinin activity was more than 50 times higher in unfed females than in males. similarity, blood meal increased agglutinin levels in the midgut extraction of cx. quinquefasciatus against escherichia coli as well as rabbit rbcs (ayaad 2009). presence of variation in lectin activity between males and females is typical only for hematophagous nematocera (suborder of dipterous insects), presumably because only the females of these insects take blood meals. this presumption is supported by findings in glossinae, in which both sexes take blood and where there are no significant differences between lectin activities of males and females (ingram and molyneux 1988, 1991). however, our results indicated that the secretion of hemagglutinins (lectins or lectinlike molecules) in the digestive system depends on the type of food in the gut. interestingly, trypsinization increased binding of agglutinin of midgut extracted proteins with rabbit erythrocytes indicating there are more hidden lectins molecule receptors. though, fasting adults have very high hemagglutination activity, which may be due to the degradation of gut cells and presence of innate lectin molecules in the epithelial cells. on the other hand, the high activity in the rest of abdomen may be correlated with the presence of symbionts. generally, in adults of mosquitoes, symbionts might use ‘host’ reserves for their own growth and start to act like pathogens inside the body. in this case, haemocytes, which presumably produce the lectins or hemagglutinins, act to eliminate infection. this concept is supported by the observation the levels of hemagglutinins in females taking blood meals are uncharged. however, our results is different with those presented by gelbic and olejnicek (2004) though they stated that hemagglutination activity in the midgut of cx. pipiens complex is not dependent on a blood meal or the uptake of protein food. apparently, the variation in results may be due to different population used. we showed the effect of geographical populations of anopheles stephensi on agglutinin activities of the mosquitoes' midgut (basseri et al. 2004). similarly, grubhoffer and noriega (1995) and also grubhoffer et al. (1997) reported significant increases in hemagglutination activity in female aedes aegypti after the ingestion of protein food and volf and palanova (1996) obtained similar results in phlebotomids. mohamed et al. (1992) did not find differences either between sexes or after blood fed of an. gambiae. according to the present results, hemagglutination activity correlates with some digestive processes and sex of the mosquitoes. it has been shown that haemagglutinins in the gut of cx. pipiens may separate food from liquid of blood meal or nectar and retard the passage food through the gut (gelbic and olejnicek 2004). interestingly, significant increase of hemagglutination titer was observed in females after blood meal. this suggests that the empty gut probably starts the secretion of hemagglutinins to prepare for protein-rich food. male mosquitoes do not suck blood, and therefore the filling of the gut does not start the reaction in the same way as in the females. hemagglutination assay on the midgut of ae. aegypti showed that the agglutinin might not be protein (lectin) but a glycan (olejnıcek et al. 2000). however, further species of culicinae need to be investigated for induction of gut hemagglutination activity because different responses in different mosquito genera or even species can be expected (nayar and knight 1997). the mosquito midgut represents one of the most challenging environments for many microorganism born diseases such as plasmodium, arboviruses and fungus (tajedin et al. 2009, chugh et al. 2011, cox et al. 2011). the ability of a panel of lectins to potentiate the uptake of a variety of microorganisms is espe27 j arthropod-borne dis, june 2013, 7(1): 23–30 m koosha et al.: lectin activity in… http://jad.tums.ac.ir published online: april 10, 2013 cially important in terms of nonself recognition in invertebrate immune system (rattclife and whitten 2004, yoshida et al. 2007). however, agglutinin activity may be important for the elimination of infections, as well as for the processing of food and the utilization and transportation of nutrients. in conclusion, the secretion of hemagglutinins (lectins or lectin-like molecules) in the digestive system of cx. pipiens depends on the type of food as well sex. acknowledgements we received no external funding sources for this study. the authors would like to appreciate very much for kind collaboration of staff in the department of medical entomology and vector control, school of public health, tehran university of medical sciences. the authors declare that there is no conflict of interest. references ayaad th (2009) isolation of midgut agglutinin of culex quinquefasciatus (diptera: culicidae). egypt acad j biolog sci. 2(2): 55–68. azari-hamidian (2007) larval habitat characteristics of mosquitoes of the genus culex (diptera: culicidae) in guilan province, iran. iran j arthropod-borne dis. 1(1): 9–20. basseri hr (2002) role of lectins in interaction between parasites and the important insect vectors. iranian j publ health. 31: 69–74. basseri hr, doosti s, akbarzadeh k, nateghpour m, whitten mm, ladoni h (2008) competency of anopheles stephensi mysorensis strain for plasmodium vivax and the 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accepted 30 oct 2013) abstract background: mosquitoes are well known as vectors of several disease causing pathogens. the extensive use of synthetic insecticides in the mosquito control strategies resulted to the development of pesticide resistance and fostered environmental deterioration. hence in recent years plants become alternative source of mosquito control agents. the present study assessed the larvicidal and oviposition altering activity of six different plants speciesalstonia scholaris, callistemon viminalis, hyptis suaveolens, malvastrum coromandelianum, prosopis juliflora, vernonia cinerea against aedes albopictus mosquito in laboratory. methods: leaf extracts of all the six plants species in five different solvents of various polarities were used in the range of 20–400ppm for larval bioassay and 50,100 and 200ppm for cage bioassay (for the study of oviposition behavior) against ae. albopictus. the larval mortality data were recorded after 24 h and subjected to probit analysis to determine the lethal concentrations (lc50), while oai (oviposition activity index) was calculated for oviposition altering activity of the plant extracts. results: vernonia cinerea extract in acetone and c. viminalis extract in isopropanol were highly effective against aedes albopictus larvae with lc50 value 64.57, 71.34ppm respectively. acetone extract of p. juliflora found to be strong oviposition-deterrent which inhibited >2 fold egg laying (oai-0.466) at 100ppm. conclusion: vernonia cinerea and c. viminallis leaf extracts have the potential to be used as larvicide and p. juliflora as an oviposition-deterrent for the control of ae. albopictus mosquito. keywords: larvicide, oviposition activity, aedes albopictus, plant extracts, vector management introduction mankind and mosquitoes must have lived in close association since our ancestors first evolved. the ‘asian tiger mosquito’ ae. albopictus (skuse) (deptera: culicidae) is an important vector of dengue, chikungunya and other viruses (pialoux et al. 2007, rezza 2012). approximately 3.5 billion people live in dengue endemic countries located in the tropical and subtropical regions of the world (who 2011). the invasion of aedes mosquito is very close to human habitat and the preferable breeding sites are small water bodies near the vicinity of human, like water tanks, waste pots, tyres etc., therefore a need has arisen to control their reproduction by using such means, and those are not harmful to living environment unlike using synthetic insecticides. the extensive use of synthetic insecticides in the mosquito control strategies resulted to the development of pesticide resistance and fostered environmental deterioration. hence in recent years plants become alternative source of mosquito control agents. the use of plant products for vector control has several appealing features as they are easily degradable, less hazardous and rich stock house of chemicals of diverse biological activity and economical as well as practical in *corresponding author: dr ruchi yadav, e-mail: ruchi.drdo@gmail.com  http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 175 application. therefore botanicals have attracted considerable interest in integrated pest management programs in the recent time (koul and walia 2009). plant products especially the secondary metabolites can be used, either as insecticides for killing larvae or adult mosquitoes, as repellents for protection against mosquito bites or as oviposition deterrents, depending on the type of activity they possess (sukumar et al. 1991). the control of mosquitoes at larval stage is considered as an efficient way in the integrated vector management (rutledge et al. 2003). furthermore altering the behavior of mosquitoes for oviposition may also reduce the breeding of mosquitoes. some plant metabolites may also inhibit the egg laying of female mosquito which can be used as oviposition deterrents in integrated vector management. many studies have been carried out to investigate the oviposition altering behavior of plants secondary metabolites against mosquitoes (prajapati et al. 2005, waliwitya et al. 2009). the present investigation has been carried out to explore the larvicidal and oviposition activity of six different plants viz. alstonia scholaris, callistemon viminalis, hyptis suaveolens, malvastrum coromandelianum, prosopis juliflora, vernonia cinerea. alstonia scholaris (apocynaceae) is an evergreen tree, native to the indian subcontinent and southeast asian countries. the plant is used in ayurvedic, unani and siddha/tamil types of alternative medicinal systems (khare 2007). callistemon viminalis (myrtaceae) is an ornamental shrub, widely distributed to asian countries. it has been reported to possess antimicrobial properties and extensively used for intestinal illness (delahaye et al. 2009). jiji et al. (2012) investigated the insecticidal activity of this plant against mosquito larvae. hyptis suaveolens (lamiaceae) is an aggressive annual weed, showed effective repellency against adult an. gambiae mosquito (palsson and jaenson 1999). malvastrum coromandelianum (malvaceae) has been reported for its antimicrobial activity (islam et al. 2010) and larvicidal property against cx. quinquefasciatus (nazar et al. 2009). prosopis juliflora (fabaceae) is a drought resistant, wide spread exotic weed in semi arid areas of india and it is well reported for insecticidal activities against mosquitoes (senthilkumar et al. 2009). vernonia cinerea (asteraceae) is an annual weed and is extensively used in indigenous medicine as stomachic and for cold, asthma and bronchitis (kirtikar and basu 2000). further more this plant is also possesses insect antifeedent property (tandan et al. 1999). several studies on the bioactivity of these plants have been documented in literatures, both the larvicidal and oviposition altering activities of these plants against ae. albopictus mosquito are not yet explored extensively. materials and methods plant material the green leaves of six plant species alstonia scholaris, callistemon viminalis, hyptis suaveolens, malvastrum coromandelianum, prosopis juliflora, vernonia cinerea, were collected during september-december, 2011 from the gwalior region (lat. 26.2215º n, long. 78.1780º e) madhya pradesh, india. each specimen was identified with the help of expert from school of studies in botany, jiwaji university, gwalior, india. plant material dried under shade for 15 days. plant extraction dried plant leaves were powdered by using an electric blender. powder was separately subjected to solvent extraction (solvents of different polarity-hexane, methanol, acetone, isopropanol, di-methyl-sulphoxide) with soxhlet apparatus (make-borosil). totally, 200gm of powdered material in 500ml solvent was extracted for 8h at boiling point range 50–80 ºc. the extracts were filtered through a buchner funnel with whatman filhttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 176 ter paper number 1. crude extracts were dried using rotary evaporator, stored in glass vials. stock solution of each plant extracts were prepared (as per who 2005 norms with slight modifications) in their respective solvents except of dried hexane extract, which was dissolved in acetone (hexane has its own larval toxicity). test solutions of five concentrations20, 50, 100, 200, 400ppm, were prepared by diluting the stock solution with distilled water. mosquitoes the aedes albopictus mosquitoes were taken from the colony, being maintained in our laboratory (defence research and development establishment, gwalior, india). cyclic generations of vector mosquitoes were reared at 27±2 °c room temperature and 70± 5% relative humidity. larval stages were maintained in bowls (2.5l) and fed on yeast powder. adult mosquitoes were reared in wooden cages (30x30x30 inches) and were provided cotton soaked with 10% sugar solution. females were offered rabbit blood once in a week. moist filter paper was kept in beaker inside the cage for female mosquito to lay their eggs. larval bioassay bioassay for the larvicidal activity was carried out as per who (2005) procedure. a total of 20 early third instar larvae of ae. albopictus were introduced in glass beakers (250ml) containing 100ml tap water. one ml of test solutions of different concentrations was introduced in beakers for treatment along with control which was treated with the respective solvents (acetone is used for hexane extract). four replicates were set up for each test concentration and larval mortality was recorded after 24h. to determine the lethal concentration for each plant species, data were analyzed by probit analysis (finney 1971) using polo pc software. the corrected mortality was estimated by abbott’s formula (1925) and the lc50 values were calculated. oviposition activity oviposition altering activity of acetone extract of four easily available plantsc. viminalis, h. suaveolens, p. juliflora and v. cinerea was studied against ae. albopictus mosquitoes. three concentrations50, 100 and 200 of test solution of each plant extract were prepared in acetone solvent for the treatment. oviposition behavior of mosquitoes, in four replicates for each concentration was tested using dual choice oviposition bioassay according to xue et al. (2003) method with few modifications. a total of 20 gravid females were released in a cage of size 60x60x45 cm and two white plastic bowl of 500ml capacity were filled with 100ml distilled water, one as control and another as treatment bowl and the filter paper strip of size 30x 5cm was placed inside each bowl for oviposition. treatment bowl contained test solution and control bowl was treated with acetone. thirty minutes after treatment, bowls were placed in cages in diagonal position approx. fifteen inches apart from each other. egg laying in treated as well as control bowls were observed after 24h of treatment. oviposition activity index (oai) was estimated using (karmer and mulla 1979), oai= (nt-nc)/ (nt+nc), where nt is number of eggs laid in treatment, nc-no. of eggs laid in control bowls. results larvicidal effect the toxicity of crude extracts of all the six plants were evaluated against third instar larvae of ae. albopictus and the results of larval susceptibility are presented in table 1 and fig. 1a–f. alstonia scholaris extract showed larval mortality with almost similar lc50 values, ranged from 394.26–473.78ppm for all the solvents. it caused only 35% mortality in hexane extract at highest concentration (fig. 1a). callistemon viminalis extract http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 177 exhibited variations in larval mortality for different solvents. isopropanol, acetone and methanol extracts were highly effective (lc50 values71.34, 110.35, 115.33 respectively) as compare to hexane and dmso extracts. larvae of ae. albopictus were almost equally susceptible towards h. suaveolens and m. coromandelianum with very high range of lc50 in each solvent extract. methanol extract of p. juliflora was relatively more effective with lc50 value80.22ppm as compared to other solvent extracts and it showed 90% larval mortality at highest concentration of 400ppm. vernonia cinerea was comparatively more effective among the plant used for larval toxicity, as it exhibited maximum mortality (45–90% larval mortality at 200ppm) in all the solvent extracts except dmso extract (55% mortality at 400ppm). results revealed that acetone extract of v. cinerea was most effective with lc50 value64.57ppm. according to fig. 2 among all the solvents used for extraction, acetone and methanol extracts were most effective as compared to the other solvent extracts. isopropanol extract exhibited moderate larval mortality followed by hexane and dmso extract of all the plants tested. oviposition activity four plant species were selected for the evaluation of oviposition activity. callistemon viminalis extract showed attractancy towards the gravid females with maximum egg laying in treated container as compared to untreated (table 2). in case of h. suaveolens and v. cinerea, egg laying was not significantly different in treated and untreated containers, however p. juliflora was found to be effective in preventing egg laying at 50 and 100ppm concentration with almost equal oai value -0.460 and -0.466 (table 2 and fig. 3). fig. 1a. larvicidal activity of alstonia scholaris leaf extract in different solvents against iiird instar larvae of ae. albopictus. fig. 1b. larvicidal activity of callistemon viminalis leaf extract in different solvents against iiird instar larvae of ae. albopictus http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 178 table 1. lc50 and x 2 value of larval bioassay of six plant extracts using different solvents plant species solvent for extraction lc50 (95% lower and upper limit) x 2 value alstonia scholaris *hexane 1.195 isopropanol 394.26 (242.21-853.65) 0.183 methanol 473.19 (339.31-754.13) 0.785 acetone 459.81 (328.81-732.33) 0.883 dmso 443.78 (267.28-1113.98) 0.048 callistemon viminalis hexane 308.77 (199.34-619.48) 0.534 isopropanol 71.34 (40.92-111.50) 0.503 methanol 115.33 (62.24-244.27) 0.593 acetone 110.35 (76.56-161.71) 1.094 *dmso 0.512 hyptis suaveolens hexane 689.69 (402.39-1989.57) 0.436 isopropanol 349.77 (237.62-570.54) 0.613 methanol 310.47 (208.72-556.74) 0.435 acetone 258.39 (177.51-422.71) 0.670 dmso 569.73 (349.95-1543.41) 1.437 malvastrum *hexane 0.030 coromandelianum isopropanol 410.22 (279.35-707.06) 0.372 methanol 337.27 (249.12-478.590) 1.419 acetone 371.45 (255.76-607.49) 0.843 *dmso 0.302 prosopis juliflora hexane 393.07 (253.17-916.88) 0.014 isopropanol 218.15 (132.65-435.48) 1.340 methanol 80.22 (57.84-114.94) 0.353 acetone 130.74 (83.33-227.46) 0.738 dmso 244.67 (139.29-606.93) 0.880 vernonia cinerea hexane 250.36 (154.93-508.61) 0.320 isopropanol 162.10 (114.33(252.41) 0.192 methanol 198.27 (141.83-286.98) 2.071 acetone 64.57 (45.68-90.04) 0.549 * dmso 0.667 *lc50 could not accessible due to less mortality fig. 1c. larvicidal activity of hyptis suaveolens leaf extract in different solvents against iiird instar larvae ofae. albopictus http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 179 table 2. oviposition activity of acetone extract of selected plants against aedes albopictus females plant species conc. (ppm) mean no. of eggs laid (±se) (treatment) (control) oai (oviposition activity index) callistemon viminalis 50 94.00(17.66) 39.66 (10.47) 0.406 100 133.75 (11.79) 86.66 (8.81) 0.213 200 125.25 (25.5) 100.00 (23.62) 0.112 hyptis suaveolens 50 101.25 (18.3) 131.00 (16.09) -0.128 100 77.00 (12.70) 121.00 (14.97) -0.22 200 115.75 (30.17) 97.33 (4.631) -0.086 prosopis juliflora 50 36.00 (10.49) 98.33 (27.28) -0.46 100 55.25 (17.221) 152.00 (35.15) -0.466 200 87.00 (38.31) 133.00 (11.01) -0.209 vernonia cinerea 50 54.25 (15.63) 75.00 (15.69) 0.160 100 101.50 (16.17) 91.00 (36.66) 0.054 200 102.25 (37.65) 95.66 (32.06) 0.332 fig. 1d. larvicidal activity of malvastrum coromandelianum leaf extract in different solvents against iiird instar larvae of ae. albopictus fig. 1e. larvicidal activity of prosopis juliflora leaf extract in different solvents against iiird instar larvae of ae. albopictus fig. 1f. larvicidal activity of vernonia cinerea leaf extract in different solvents against iiird instar larvae of ae. albopictus http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 180 fig. 2. comparative larvicidal activity of solvent extracts (alsa. scholaris, cal-c. viminalis, hyp-h. suaveolens, mal-m. coromandalianum, pro-p juliflora, ver-v. cinerea) at different concentration against ae. albopictus larvae. fig. 3. oviposition activity index (oai) values for selected plant extracts at different concentration. discussion the present study focused on the larvicidal as well as the oviposition altering activity of crude plants extracts. among all the plants v. cinerea was comparatively more effective than other plants against the larvae of ae. albopictus. in a study arivoli et al. (2011) had reported the larvicidal property of this plant in ethyl acetate extract against cx. quinquefasciatus larvae with an lc50 value of 1.63 g/l. vernonia cinerea is a member of asteraceae family which possesses various types of phytochemical compounds (flavonoids, sesquiterpines, thiophene derivatives) those have been found to be toxic to insects including mosquito larvae (ribeiro et al. 1994). significant results in larval mortality were also observed for c. viminalis. in the preliminary study jiji et al. (2012) reported the larvicidal efficacy of aqueous extract of this plant against culex quinquefasciatus. khambay et al. (1999) isolated two compounds viminadione a and viminadione b from hexane extract of c. viminalis and viminadione a found to be effective against house flies, aphids and thrips. the present study revealed that the methanolic leaf extract of p. juliflora was effective against larvae of aedes mosquito. senthilkumar et al. (2009) also evaluated the larvicidal and adulticidal efficacy of 10% http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 181 leaves extracts of p. juliflora and showed that it was quite effective (lc509.3 mg/l) against larvae of an. stephensi. recently bansal et al. (2012) studied the methanol extract of p. juliflora and reported lc50 value 128 ppm against ae. aegypti larvae. hyptis suaveolens is well known for its repellent property against mosquitoes. palsson and jaenson (1999) observed that h. suaveolens provided approximately 84% protection for 2 hrs against an. gambaie. although it is good repellent but not showed significant larvicidal activity. our findings agree with the results of cavalcanti et al. (2004) who showed lc50 value 261ppm, while contradicts with amusan et al. (2005), who revealed high mortality rate (80%) in ethanol extract of h. suaveolens at conc. 0.9ppm against ae. aegypti larvae. moderate larval mortality was observed in m. coromandelianum and a. scholaris plant extracts. nazar et al. (2009) has reported similar mortality rate in methanolic extract of m. coromandelianum against cx. quinquefasciatus larvae. kaushik and saini (2009) while screening some plants for larvicidal activity against ae. aegypti, found that acetone extract of a. scholaris showed its efficacy with the lc50 value 239.9ppm. in the present investigation acetone extract of v. cinerea, isopropanol extract of c. viminalis and methanol extract of p. juiflora showed maximum efficacy, while dmso and hexane extracts of all the plant species did not cause significant toxic effect on larvae. the dose dependent response in larval mortality was noticed in the present study. larvicidal activities of crude plant extracts varied according to the plant species and solvents used. crude extract in these solvents might have some complex mixture of biocidal active compounds, including phenolics, terpenoides, flavonoids and alkaloids which may jointly or independently contribute to produce mortality of larvae. phytochemicals limonin and nomilin from some citrus plants were reported to have larvicidal activity against mosquitoes (bilal et al. 2012). acetone extract contains maximum quantity of phenols and flavonols, while methanol extract contains flavones, terpenoids, tannins and polyphenols (tiwari et al. 2011) and these phytochemicals are reported for their insecticidal activity. the higher activity of the methanol and isopropanol extracts as compared to the hexane and dmso extract can be attributed to the presence of higher amounts of polyphenols. different solvent types can significantly affect the potency of extracted plant compounds (karmegam et al. 1997) because of the polarity range. roots extract of derris elliptica in different solvent was evaluated for its larvicidal activity against ae. aegypti, indicating that among the crude residues acetone extract was the most effective (ameen et al. 1985). the mode of action and site of effect for larvicidal phytochemicals has received little attention. ray et al. (1999) and david et al. (2000) found that botanical derivatives primarily affect the midgut epithelium and secondarily affect the gastric caeca and the malpighian tubules in mosquito larvae. phytochemicals can also influence the ovipositional behavior of mosquitoes. present study reported that among four plants tested for oviposition behavior, p. juliflora exhibited significant results and suggested the repellency towards gravid females with 3 fold egg laying in untreated bawls at 100ppm. h. suaveolens and v. cinerea did not show any significant difference in oviposition whereas c. viminalis showed slightly attractancy at 50ppm with almost 2 fold egg laying in treated bowls. this suggests that these extracts produce some volatile compounds, which may act as repellents or attractants at specific concentrations. aedes is a container breeder mosquito, follows visual and olfactory cues to find appropriate oviposition sites and then both physical and chemical factors of water, to assess the suitability of potential larval habitats in http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 182 order to maximize the fitness of offspring (sumba et al. 2004). rajkumar and jebanesan (2005) found that 0.01, 0.025, 0.05, 0.075 and 0.1 % ethanol leaf extract of solanum trilobatum reduced egg laying by an. stephensi, from 18–99% in treated containers. the oviposition deterrent activity of four essential oils derived from chamomile (matricaria recutita), sesame (sesamum indicum), jojoba (simmondsia chinensis) and ginger (zingiber officinalis), were evaluated against the gravid females of the filarial vector culex pipiens under laboratory conditions at three doses (0.5, 5 and 50ppm) exhibited potent deterrent activity against gravid female mosquitoes with various degrees of repellency (ranging from 48.73–100%) (el-gendy and shaalan 2012). tawatsin et al. (2006) studied 18 essential oils and reported their oviposition deterrent activity against ae. aegypti ranging from 16.6 to 94.7%. many plants from different families possess promising phytochemicals for mosquito control which are much economical and environmental friendly (sivagnaname and kalyanasundaram 2004, bakar et al. 2012). these phytochemicals like phenolics, alkaloids and terpenoids exist in plants extracts may jointly or independently contribute to the mosquitoes as larvicidal/oviposition deterrents/ repellents. further more use of plant products in vector control is target specific, efficacious, safe and appropriate for use at the commu nity level, widely available at no or limited cost and withstand the risk of vector developing resistance against them. in the present study potentially effective plants included some weeds, this may also contribute to assess the possibility of using large biomass of weeds available in the wastelands of northern india as potential insecticides in spite of using other medicinal or cultivable plants. for improving the potency and stability of the plant products, in depth investigation on the active fraction or compound of these plant species are necessary to be further elucidated. conclusion our results revealed that crude extract of v. cinerea and c. viminalis were significantly effective as larvicide and p. juliflora as oviposition deterrent among the tested plants. these plants can be used as natural insecticides in integrated vector control programs. acknowledgements the authors are thankful to prof (dr) mp kaushik director defence research and development establishment (drde), gwalior, for providing necessary research facilities and support in the study. the authors declare that there is no conflict of interest. references abbott sw (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–267. amusan aas, idowu ab, arowolo fs (2005) comparative toxicity of bush tea leaves (hyptis suaveolens) and orange peel (citrus sinensis) oil extract on larvae of the yellow fever mosquito aedes aegypti. tanzan health res bull. 7(3): 174–178. ameen m-ul, shahjahan rm, khan hr, chowdhury aka (1985) larvicidal effect of indigenous derris elliptica root on aedes aegypti (diptera: culicidae). int j entomol. 1: 39–43. arivoli s, tennyson s, martin jj (2011) larvicidal efficacy of vernonia cinerea (l.) 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http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 174–185 r yadav et al.: differential larval toxicity … 185 xue r, barnard dr, ali a (2003) laboratory evaluation of 18 repellent compounds as oviposition deterrents of aedes albopictus and as larvicides of aedes aegypti, anopheles quadrimaculatus, and culex quinquefasciatus. j am mosq control assoc. 19(4): 397–403. http://jad.tums.ac.ir published online: april 09, 2014 1.dr yaghoobi new rtl!! iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 1 original article high performance thin layer chromatography analysis of deltamethrin residue on the impregnated bed nets during a leishmaniasis control program in iran sh moosa-kazemi 1, m shayeghi 1, *mr yaghoobi-ershadi 1, h vatandoost 1, mt sadeghi 1, e javadian1, m motabar 1, mr hosseini 2, m abtahi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 4 aug 2009; accepted 5 sep 2009) abstract background: the control of leishmaniasis, a tropical neglected disease, has been concern of iranian health authorities due to the increasing number of cases during the last two decades. the objective of this study was to determine deltamethrine residue on the impregnated bed nets using hptlc technique in a leishmaniasis control program in iran. methods: during this experimental study, a total of 130 small pieces of polyester netting were sewn to top, upper, and lower sides of some bed nets and then were impregnated with deltamethrin .the treated bed nets were distributed in isfahan and mashhad areas in april 2003. the samples were cut randomly after impregnation intervals. deltamethrin was extracted using acetone from samples and the extract was applied for spotting onto plates. the plates were developed with n-hexane: ethyl acetate, 90+10(v/v), as a mobile phase in a camage chamber. the qualifying of residue was observed in uv cabinet with λ=254 nm wavelength. data were analyzed using spss version 11.5. and stata version 8. a three way anova was used to compare the means of deltamethrin residue in each area, group and measuring time. the kruskal-wallis test was used to compare the means of residue for each of these factors with the control separately. results: the retardation factor of deltamethrin was calculated 0.50±0.02. the residues of deltamethrin persisted well on impregnated nets at least for 15 weeks after impregnation. no significant difference could be detected in the loss of residue of insecticide in comparison to measuring times and positions of sampling pieces on the bed nets in these areas. conclusion: based on the results of the present study the use of hptlc technique is recommended instead of other chromatographic methods for analysis of insecticide residue on the impregnated bed nets. keywords: leishmaniasis control, impregnated bed nets, insecticides, deltamethrin, hptlc, iran introduction cutaneous leishmaniasis due to leishmania major (clm) is still a great and increasing public health problem in many rural areas of 17 out of 30 provinces of iran (moinvaziri et al. 2007). cutaneous leishmaniasis caused by leishmania. tropica (clt) is also a very old endemic disease in many foci and thousands of cases occur in large cities and small towns in the country (yaghoobi-ershadi et al. 2005). in recent years, considerable attention has been paid to vector control due to the increase of cases and resurgence of the diseases in some non-endemic areas of iran. bed nets impregnated with pyrethroid insecticides have been used successfully against phlebotomine sand flies worldwide (yaghoobi ershadi et al. 2006, moosa-kazemi et al. 2007). deltamethrin has proved to be one of the most *corresponding author: prof mohammad reza yaghoobiershadi, e-mail: yaghoobi.reza@gmail.com iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 2 effective insecticides, especially under different climatic conditions, and it has already the most widely used insecticide for the effective impregnated bed nets for effective long term control of vector-borne diseases (lengeler et al. 1998). residue of insecticides on impregnated bed nets may be decreased during the transmission season of diseases and interrupt the process of control, so the quantifying of insecticides, residues as a quality control method on netting materials treatment is of great important (bami 1981). application of chromatography for the separation, detection, and qualitative determination of pesticides, other organic chemicals, and related compounds are reviewed by several researchers. analysis is involved in a variety of samples, such as food, agricultural and dairy products. there are many analytical methods such as gas chromatography (gc), gas-chromatography/mass spectrometry (gc/ms), high-performance liquid chromatography (hplc), high-performance thin-layer chromatography (hptlc). quantitative hptlc offers a viable alternative to gas chromatography or high performance liquid chromatography in terms of simplicity of operation, the availability, reproducibility, quantification at any time with changed conditions, calibration for quantitative analysis, cost effectiveness, and flexibility (ludwick et al. 1977). although hptlc method have been previously used for separation, detection and determination of residue of pesticides on soil, water, etc (chen et al. 1996, lekic et al. 2002, haiqun et al. 2005, sherma 2005), but there are few work using this method for quantification of pesticide on impregnated bed nets (ibs) in the world. hptlc had not been used for chemical analysis of insecticides residues on impregnated bed nets during the vector-borne diseases control programs in iran yet. in this study, this method was utilized in the course of a cutaneous leishmaniasis control programme using ibs and impregnated curtains (ics) in the country to analyze the insecticide residue during 2003-2004. materials and methods the experimental investigation was conducted in the field and under laboratory circumstances. the field trial includes the selection of the study areas, bed net preparation, impregnation by deltamethrin, and bed net sampling. a laboratory study includes extraction, clean up, spotting, chromatogram development, detection and qualifying by uv, quantitative detection by cats4 software of hptlc. the field study was conducted from june to september 2003 in two places, habibabad in the rural district of borkhar, isfahan province (32°38 n, 51°29 e) central iran, and shaghayegh located in aab-o-bargh area in the south west of mashhad city (36°16 n, 59°35 e), khorasan-e-razavi province, the northeast of the country. in 2003, the maximum and minimum mean monthly temperatures of isfahan were 39.1 °c and -2.8 °c in july and december, respectively. the total annual rainfall was 124 mm with minimum 5.4 mm in march and maximum 49.6 mm in january. the minimum mean monthly relative humidity was 12% (august and september) and the maximum was 89% (january). in the same period, the maximum and minimum mean monthly temperatures of mashhad were 35.5 °c and -6 °c in july and december; respectively .the total annual rainfall was 276.3 mm. the minimum mean monthly relative humidity was 32% (july and august) and the maximum was 93% (january) in the city. the white mosquito nets of multifilament polyester fiber 75-denier strength, 156-mesh size (12×13 holes/inch 2) in single (70×180× 150 cm), double (130×180×150 cm) and family sizes (190×180×150cm) were manufactured by a factory in tehran. they were impregnated with deltamethrin (k-othrine® sc 5, oms 1988, agrevo marseille, france) at the target dose of 25 mg a.i/m2, as described by who in 2002. a total of 130 small pieces in 5cm× 5cm size of netting of similar type were randomly sewn to some bed nets (top, upper, and iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 3 lower side) and were impregnated by deltamethrin (who, 2002).the impregnation of the bed nets in both areas were carried out in april 2003. the nets and netting pieces received a code number and all the information such as the time of impregnation, size of bed nets were recorded in a special form. to assess the chemical residue analysis and quality of treatment, 3 to 10 pieces of the netting were cut randomly before use and after 3, 9, 12 and 15 weeks of impregnation in borkhar area. the same numbers of the netting were sampled randomly before use and after 3, 8, 12 and 15 weeks of impregnation in aab-o-bargh area. samples were put in small sterile vials individually and coded. the small vials were sealed by para film and kept in 4° c temperature for the specific analysis (gupta et al. 1998). a total of 130 samples (106 as deltamethrin-treated nets and 24 as untreated nets) were sent for chemical analysis to pesticide evaluation laboratory at the department of medical entomology and vector control, tehran university of medical sciences, iran. the standard analytical grade of deltamethrin (99%) was purchased from accustandard company (tehran). acetone, hexane, ethyl acetate, methanol (analytical-grade), and 20 cm ×20 cm aluminum foil backed silica gel 60 f254 hptlc plates (layer thickness, 0.10-0.20 mm) were from merck, darmstadt, germany. linomat iv applicator, chromatography tank, uv cabinet, tlc scanner 4, an external pc running wincats software (version 1.1.2) were from camag, muttenz, switzerland. absorbance was measured at wavelength of λ= 254 nm by means of a deuterium lamp (gupta et al. 1998, sherma 2005). for extraction of deltamethrin residue from fabrics, 20 ml pure acetone was added to each vials containing 25 cm2 pieces of netting. the insecticide residue was extracted by 1 h shaking and then allowed to laid 1 h just before analysis to ensure extraction of a representative quantity of deltamethrin (sherma 2005). the extracts were evaporated until the final solution reach to 1ml. three replicate extractions were performed for each treatment. the spotting on a plate was performed by an applicator and capillary tubes. volume of each spot was 10 µl and the distance between spots was 1 cm. ten mg of deltamethrin standard was mixed with 90 ml of pure acetone solution to produce 10% concentration. then the solution stored in glass-stopper bottles at 4 °c. ten µ l of extracted and standard solutions were spotted separately on a plate as a stationary phase. different extract solutions from treatment and control netting samples as well as standard solution were used. after spotting and drying the spots, the plate was put in a vertical tlc chamber. a mixture of nhexane: ethyl acetate, 90+10(v/v) was used as a solvent (mobile phase) (sherma 2005). the plate was placed in the chamber saturated with vapors of the developing solvent. then it was withdrawn from the chamber and the mobile phase was evaporated after 30 min. after drying, the plate was irradiated at 254 nm wavelength in uv cabinet and retardation factor (rf) value was calculated. the retention factor, or rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent (sherma 2005). a retention factor is calculated and stored in the database for each of the samples in accordance with the following formula: finally, the quantitative of insecticide residue were scanned by tlc scanner 4 (camag), with 207 nm wavelength, using cats4 software according to external standard calibration (gupta et al. 1998, sherma 2005). the data were analyzed by using spss 11.5 and stata 8.0. a three-way anova test was used to compare the means of deltamethrin residue in each areas for three groups, top, upper, and lower sides of samples in five measuring times. the kruskal-wallis test was used to compare the means of deltamethrin re iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 4 sidue for each of these factors with the control samples separately. the amount of insecticide residue was considered significantly, when the p value was less than 0.05 (zar 1996). results the retardation factor (rf) value of deltamethrin was 0.50±0.02. table 1 shows the residue of deltamethrin in the net samples detected by hptlc technique before use and 3, 9, 12 and 15 weeks after impregnation in borkhar area. the average initial uptakes of the deltamethrin by top, up and lower sides of net samples were 26.50, 26.70, and 26.80 mg a.i./ m2, respectively. the mean uptake of insecticide was the greatest in the lower side sample for the first measuring time (26.70±1.63) followed by the second measuring time (25.89± 2.80) after 3 weeks of impregnation. non-significant difference was observed in the residue analysis of top, up and lower side of samples in all sampling time using a kruskal-wallis test. table 2 presents the results of the residue analysis of the netting samples taken randomly from the impregnated bed nets before use and after 3, 8, 12 and 15 weeks of impregnation in aab-o-bargh area. the average initial uptake from top, up and lower side of the impregnated net samples indicated that deltamethrin suspension concentrate (dsc) formulations were 28.2, 27.80 and 27.11 mg a.i./m2, respectively. the average and variation of insecticides residue were greatest for various side samples at 27.70± 0.44 of the first measuring time followed by top and lower sides of samples at 27.05±11.90, 27.01± 7.70 mg a.i./m2 of the second sampling time, respectively. non-significant difference was observed in the loss of chemical activity of top, up and lower sides of net materials, in each sampling time. statistical analysis show that there are non-significant differences between two study areas, the situation of samples and duration of sampling in terms of deltamethrin residues by anova test. table 1. results of the tlc analysis of deltamethrin impregnated bed net samples (mg a.i/m2) in borkhar (isfahan province), iran position of piece top upper side lower side weeks after treatment control (mg/m2) no. mean sd se no. mean sd se no. mean sd se just after impregnation 0.0 2 26.50 3.80 2.70 2 26.70 1.50 1.10 2 26.80 1.50 1.10 3 0.0 5 25.61 3.83 1.7 4 25.52 2.74 1.37 4 26.56 10.91 5.45 9 0.0 5 25.24 8.59 3.54 3 25.258 3.27 1.88 3 24.82 2.50 1.44 12 0.0 4 25.00 5.21 2.60 3 25.17 1.39 0.8 3 24.69 4.69 2.71 15 0.0 3 24.58 3.08 1.77 3 25.12 3.23 1.86 3 24.48 1.269 0.73 table 2. results of the tlc analysis of deltamethrin impregnated bed net samples (mg a.i./m2) in mashhad (khorasan-e-razavi province), iran position of piece top upper side lower side weeks after treatment control (mg/m2) no. mean sd se no. mean sd se no. mean sd se just after impregnation 0.0 2 28.20 16.40 11.60 2 27.8 12.10 8.60 3 27.10 9.8 8.02 3 0.0 5 27.05 26.60 11.90 3 26.88 10.11 5.84 3 27.01 13.34 7.70 8 0.0 4 26.06 13.45 6.72 4 26.10 3.91 1.95 4 25.54 8.80 4.40 12 0.0 3 25.46 3.24 1.87 3 25.37 3.96 2.29 4 25.45 3.58 1.00 15 0.0 3 24.60 2.21 1.28 4 25.16 10.78 5.39 3 24.58 2.45 1.42 iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 5 discussion this is the first report on the chemical analysis of the deltamethrin impregnated bed nets using hptlc technique in relation to clm and clt endemic areas of iran. there are several reports for quantification of pesticide on different samples (ludwick et al. 1977, haiqun cao et al. 2005). quantifying the insecticides deposit as a quality control in fabric treatment is very important. deltamethrin was one of the photo stable synthetic pyrethroids to be developed for its stable residual activity, and specifically recommended by who to use for impregnation of mosquito nets. this insecticide with other alpha cyano compounds tend to be rather more toxic to insects and low toxicity to human, for this reason, remarkable small quantities are required on a net (miller je 1994, alten et al. 2003). the susceptibility status of phlebotomus papatasi and ph. sergenti to this insecticide using who standard method indicated that the both species were susceptible to deltamethrin in borkhar and abobargh areas (yaghoobi-ershadi et al. 2006, moosakazemi et al. 2007). as mentioned above, bed nets made of polyester with variation in size, weight, and different absorption capacities were used in this study. the difference between initial concentration of deltamethrin before used and after 3 weeks of impregnation of the bed nets in both areas was not significant. it is noteworthy that in the same field study, the initial uptake of the deltamethrin on bed net was high and dropped to nine-tenth of the original activity after 6 months (alten et al. 2003). in this study, the uptake of deltamethrin was closed the target dose after 15 weeks of impregnation in borkhar and aab-o-bargh areas, where the uptake were 24.72±1.45 and 24.78±2.69 mg a.i./m2, respectively. in the same study persistent of deltamethrin on treated nets was reported for 1 year, even with washed once or twice (njunwak et al. 1991, miller 1994,). bioassay test is simple and quick enough to use as a simple measure to show the effectiveness of impregnated nets and indirectly represents the amount of the deposit of deltamethrin on impregnated bed nets .it should be mentioned that in the same time a field study was carried out in the borkhar area in 2003, and the mortality rate of p. papatasi (wild-caught, fed females) after 3 min of exposure time followed by 24 h recovery time was100%. the mortality of the species was 100% until the end of week 12. the mortality of control sand flies never exceeded 3.5% (yaghoobiershadi et al. 2006). at the same time, bioassay test showed that p. sergenti was susceptible to deltamethrin impregnated bed nets and this insecticide remained effective for 3 months and more in abo-bargh area (moosa-kazemi et al. 2007). chemical residue analysis correlated well with bioassay results. the efficacy of deltamethrin-impregnated bed nets and curtains of this study confirmed findings of another similar recent trial which was carried out in the colombia (alexander b et al. 1995), however, alexander et al. mentioned that the effectiveness of pyrethroid on the bed nets persisted at least 4 months (alexander et al. 1995). in the field study, the chemically detectable of deltamethrin was decreased approximately half the target dose by 3 months and to a third by 5 months (jawara et al. 2001). such a remarkable low residual activity is believed to be mainly due to the different netting or materials, poor dipping and impregnation techniques, dust and dirt in the area, as well as washing habits of residents, which affects the residue activity of insecticide. in the other study, it was found that 14% of nets had been washed within one month and 76% within three months after impregnation (jawara et al. 1998).washing habits decreased insecticides concentration of treated bed nets (snow et al. 1987, lindsay et al. 1991, lines 1996). it should be mentioned that at the same time, washing habits of residents were questioned through the projects and few of the impregnation bed nets were washed in borkhar iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 6 and mashhad areas (yaghoobi-ershadi et al. 2006, moosa-kazemi et al. 2007). in conclusion, the low cost and long persistence of deltamethrin on the impregnated bed nets and curtains (up to three months without washing) means that it can be used for preventing and controlling of the vector-borne diseases such as leishmaniasis in the other parts of iran and neighboring countries. in the forthcoming control program the use of hptlc technique and quality control of ibs, ics and llitns can be recommended instead of other chromatographic methods. acknowledgements authors wish to extend their sincere thanks to the staff of the leishmaniasis laboratory at the isfahan training and health research center during the field studies and to dr h ladonni for his valuable comments on the manuscript. the authors are also indebted to other colleagues from mashhad and isfahan provincial health centers for their kind assistance in this program. we thank all inhabitants for their kind cooperation to carry out the intervention. this study was financially supported by diseases management center, iranian ministry of health and medical education, and the institute of public health research, academic pivot for education and research, tehran university of medical sciences: project id no: 941.82.53. references alexander b, usma mc, cadena h, quesada bl, solartic y (1995) phlebotomine sand flies associated with a focus of cutaneous leishmaniasis in valle del cauca. colombia. med vet entomol. 9: 273-278. alten b, caglar ss, kaynass s, simsek m (2003) evaluation of protective efficacy of k-o-tab impregnated bed nets for cutaneous leishmaniasis control in southeast anatolia-turkey. j vector ecol. 28: 53-64. bami hl (1981) pesticides in criminal poisoning. j forensic sci soc. 21: 1527. chen zm, yh wang (1996) review chromatographic methods for the determination of pyrethrin and pyrethroid pesticide residues in corps, foods and environmental samples. j chromatogr. 754: 367-395. gupta s, handa sk, sharma kk (1998) a new spray reagent for the detection of synthetic pyrethroids containing a nitrile group on thin-layer plates. talanta. 45: 1111–1114. haiqun cao, yongde yue, rimao hua, feng tang, rong zhang, wei fan, haiyan chen (2005) hptlc determination of imidacloprid, fenitrothion and parathion in chinese cabbage. j planar chromatogr. 18: 151–154. jawara m, pinder m, cham b, walraven g, rowley j (2001) comparison of deltamethrin tablet formulation with liquid deltamethrin and permethrin for bed net treatment in the gambia. trop med int health. 4: 309-316 . jawara m, mcbeath j, lines jd, pinder m, sanyang f, greenwood bm (1998) comparison of bednets treated with alphacypermethrin, permethrin or lambdacyhalothrin against anopheles gambiae in the gambia. med vet entomol. 12: 60–66. lekic m, mijanovic m, pujic z (2002). thin layer chromatography of pesticides-a review of application. pharmacia 13: 39-42. lengeler c, armstrong schellenberg j, d'alessandro u, binka f, cattani j (1998) relative versus absolute risk of dying reduction after using insecticide-treated nets for malaria control in africa. trop med int health. 3(4): 286-290. lindsay sw, hossain im, bennett s, curtis cf (1991b) preliminary studies on the insecticidal activity and wash-fastness of twelve pyrethroid treatments impregnated iranian j arthropod-borne dis, (2009), 3(1): 1-7 sh moosa-kazemi et al: high performance thin… 7 into bedneting assayed against mosquitoes. pestic sci. 32: 397–411. lines j (1996) the technical issues. in: lengeler c, cattani j, de savigny d (eds) net gain: a new method for preventing malaria deaths. world health organization, geneva, pp. 17–52. ludwick ag, lau aldrich nk, ludwick lm (1977) simple and sensitive technique for detecting organo chlorine pesticides on thin layer chromatogram. j assoc of anal chem. 60(5): 1077–1080. miller je (1994) relative efficacy of three pyrethroid insecticides for treating mosquito bednets. pestic outlook. 5: 23-25. moin-vaziri v, depaquit j, yaghoobi-ershadi mr, oshaghi ma, derakhshandeh-peykar p, ferté h, kaltenbach m, bargues md, léger n, nadim a (2007) intraspecific variation within phlebotomus sergenti parrot (1917) (diptera: psychodidae) based on mtdna sequences in islamic republic of iran. acta trop. 102(1): 29–37. moosa-kazemi sh, yaghoobi-ershadir mr, akhavan aa, abdoli h, zahraei-ramazani ar, jafari r, houshmand b, nadim a, hosseini m (2007) deltamethrin-impregnated bed nets and curtains in an anthroponotic cutaneous leishmaniasis control program in northeastern iran. ann saudi med. 27(1): 6–12. njunwa k.j, lines, jd magesa sm, mnzava aep, wilkes tj, kivumbi k, curtis cf (1991) trial of pyrethroid impregnated bednets in an area of tanzania holoendemic for malaria part i. operational methods and acceptability. acta trop. 49: 87–96. sherma j (2005) thin-layer chromatography of pesticides-a review of applications for 2002-2004. acta chromatographica 15: 5-30. snow rw, rowan kr, greenwood bm (1987) a trial of insecticide-treated bed nets in the prevention of malaria in gambian children. trans r soc trop med hyg. 81: 563-567. who (2002) instructions for treatment and use of insecticide treated mosquito nets. who/cds/whopes/gcd. yaghoobi-ershadi mr, zahraei-ramazani ar, akhavan aa, jalali-zand ar, abdoli h, nadim a (2005) rodent control operations against zoonotic cutanious leishmaniasis in rural iran. ann saudi med. 25(4): 309–312. yaghoobi-ershadi mr, moosa kazemi sh, zahraeii ramazani ar, akhavan aa, jalali zand ar, hossaini m, nadim a, houshmand b (2006) evaluation of deltamethrin-impregnated bed nets and curtains for control of zoonotic cutaneous leishmaniasis in a hyperendemic area of iran. bull soc pathol exot. 99(1): 43–48. zar jh (1996) biostatistical analysis. prentice hall, upper saddle river, new jersey. paper on stability revised iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 19 original article stability and wash resistance of local made mosquito bednets and detergents treated with pyrethroids against anopheles stephensi *h vatandoost, e ramin, y rassi, mr abai department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 7 jun 2009; accepted 5 sep 2009) abstract background: we aimed to evaluate different fibres of bednets impregnated with various pyrethroids. the stability of insecticide on the bednet was measured using different methods of washings as well as local made detergents. methods: the entire test was carried out according to the who-recommended methods. in addition, the impact of the numbers of washes on the stability of the insecticides was determined. permethrin 10% (ec), deltamethrin 10% (sc), lambdacyhalothrin 2.5% (cs) and cyfluthrin 5% (ew) were used at the recommended dosages. three different local detergents were used. two kinds of washing methods (shaking, no shaking) were used and in each method four kinds of washings, i.e. no wash, one wash, two washes and three washes was done. the main malaria vectors, anopheles stephensi, which is susceptible to all insecticides (beech strain), was tested with impregnated bednets in 3 minutes exposure time and the mortality was measured after 24 hours recovery period. knock-down was measured as well using appropriate statistical methods. results: lambdacyhalothrin has saved its insecticidal impact after being washed, whereas, deltamethrin has lost its activity faster than other insecticides. tow other insecticides had moderate effect. golnar soap detergent has least effect on the durability of insecticides, but the shoma had the most. whit increasing the times of washing, insecticidal effects was decreased , but shaking had no influence on the decreasing of the quality of insecticidal impact. conclusion: results will be useful for local people who wish to use pyrethroid-impregnated bednets with their own local made detergent and bednets. keywords: pyrethroid, malaria vector, bednet. anopheles stephensi introduction different methods for mosquito control have been proposed by investigators. an important innovation during the past decade is the widespread introduction of insecticidal pyrethroidtreated mosquito nets for protection against malaria transmission. pyrethroids are today the only insecticides recommended for the treatment of mosquito nets. this is due to the rapid knockdown effects and high insecticidal potency of pyrethroids at low dosages combined with relative safety for human contact and domestic handling. for the treatment of net, who lists the insecticide products which have been passed the whopes including alpha-cypermethrin 10% sc, cyfluthrin 5% ew, deltamethrin 1% sc and wt 25%, etofenprox 10% ew, lambdacyhalothrin 2.5% cs, permethrin 10% ec (who 1997, 2002, zaim et al. 2000). the concentration, which is recommended, depends on texture of net, but the different dosages have been proposed for their effectiveness against malaria vectors. due to operational use of itns for malaria control is now advocated as a component of roll back malaria initiative led by the who. iran has been classified into four different strata for malaria epide*corresponding author: dr hassan vatandoost, e-mail: hvatandoost@yahoo.com iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 20 miology and vector control (raeisi et al. 2004). the disease is a major health problem in southeast of iran. it is unstable with two seasonal peaks mainly in spring and autumn. outbreaks usually occur after rainy season. southeastern of iran includes the provinces of sistan and baluchistan, hormozgan and the tropical areas of kerman provinces are characterized by "refractory malaria" (manou-chehri et al. 1999). in this part of the country six anopheline mosquitoes including anopheles culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus, and an. pulcherrimus are known to be the malaria vectors (zahirnia et al. 1998, vatandoost 2001, zahirnia et al. 2001, naddaf et al. 2003, enayati et al. 2003, vatandoost and moinvaziri 2004, vatandoost and borhani 2004, vatandoost et al. 2004, vatandoost et al. 2005, vatandoost et al. 2005, hanafi-bojad and vatandoost 2006, vatandoost et al. 2006 a, b, oshaghi et al. 2006 a, b, davari et al. 2006). according to the national strategic plan the use of impregnated bed net and recently long lasting impregnated nets (llitn) is the one important objective (ministry of health, unpublished data). in this study, we aimed to evaluate different fibres of bednets impregnated with various pyrethroids. the stability of insecticide on the bednet was measured using different methods of washings as well as local made detergents. materials and methods the supply/material requirements for insecticide treatment of nets the supply were water, insecticide formulation, measuring device for insecticide (micropipette) and water (measuring cylinder), container to dip the net (s) and to collect insecticide drips after dipping, rubber gloves and protective clothing including a face-mask, spray equipment when nets are sprayed. pyrethroid insecticides suspension concentrate formulation (sc) of deltamethrin (%10) from aventis company at the dosage of (25 mg/m2), emulsifiable concentrate formulation (ec) of permethrin (10%) from aventis company at the dosage of (500 mg/m2), suspension concentrate formulation (sc) of lamabdacyhalothrin (2.5%) from zeneca company, at the dosage of (20 mg/m2), emulsifiable oil in water formulation (ew) of cyfluthrin (5%) from bayer company at the dosage of (50 mg/m2) were used at the recommended dosages. detergents local made detergents such as wash machine powder named shoma, handle clothes wash powder named rakht and handle clothes wash soap named golnar were used through the study. nets local made, nylon net with mesh size of 156 holes per square inch were used and impregnated with recommended dosage of pyrethroids. there was no information about iso and stability on the local made net. bioassay tests for determination of biological efficacy of pyrethroids on treated mosquito nets there are three methods recommended by who as follows: test using who holding and exposure tubes as used for adult susceptibility test, test using who cones, test using the netting apparatus with wire frame. in this test, we followed the bioassay test with cones methods as described below (who 2005). composition of test kit: conical chambers of transparent plastic,8.5 cm in diameter at the bas and 5.5 cm high, two glass (or plastic) aspirator tubes of 12 mm internal diameter, together with 60 cm of tubing, and mouthpiece, one role of self-adhesive plastic tape or one sheet of label tag, instruction sheet, 3 sheets of log-probit papers for plotting regression line for calculating lt50, using variable times with constant concentration, counter, to count the mosquito while releasing in the cones or calculating the knock-down). iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 21 treatment of netting: pieces of 25 x 25 cm of netting material will be fold and put in a disposable plastic petri dish and a pipette on the net will drop carefully and homogeneously required diluted formulation. netting will be then soaked carefully for few seconds with fingers, protected by plastic gloves, so that all insecticide solution will be absorbed and nothing left in the petri dish. net sample will be left in the same dish to dry. ideally the treated nets shall be used in 1 to 3 d, but not later than 1 wk after treatment. separate petri dishes are used for treatment of each net, and later disposed off properly. net samples will be treated the day before bio-assay and stored at the refrigerator (who 2005) (fig.1). fig. 1. treatment of netting (original) bioassay one to 3 d after treatment of the netting materials and 24 h after each washing, as well as after dipping insecticide treated nets, the netting sample was subjected to standard who bioassays (3 minute exposure under who cones, using 1 to 3 d old, non blood fed, standard susceptible anopheles females (beech strain). four cones gently fitted on the net. five female mosquitoes introduced at a time in each cone with 8 replicates per net sample (40 mosquitoes tested). time interval between each set of a “4 cone” was as brief as possible. mosquitoes from the first 4 cones, tested and were grouped in one plastic cup (n=20). knock down (kd) was recorded at regular interval of time during the 20 to 30 min, following exposure, starting once the fourth cone (in each set) was transferred to the cup and ending when about 80% of mosquitoes were kd, stopping in any case after 60 min. sucrose provided to each cup, added on a cotton plug. in addition to kd rate at 60 min post-exposure, mortality recorded after 24 h (fig. 2). fig. 2. bioassay test in conical (original) study the physical removal of the insecticide during washing washing with no shaking: insecticidetreated net samples individually introduced in 1 l beakers containing 0.5 litter deionised water, with 2 gram/litter detergent added just before and fully dissolved and left for 20 min without any shaking. water maintained at 30 °c during exposure to detergent and rinsing. then, samples were removed and rinsed twice for 10 min in clean still water (without shaking). solution changed and beaker decontaminated after every wash (who 2005, fig. 3). wash with shaking: net samples individually introduced in 1l glass bottles containing 500 ml deionised water, with 2 g/l detergent added just before and fully dissolved. bottle immediately introduced into a water bath at 30 °c and shacked for 20 min at 155 movements per minute. they immediately rinsed twice during 10 min at the same agitation speed (fig. 4). in both cases, ph of the washing bath aciranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 22 curately measured. comparisons made between no wash, 1-, 2-, and 3-times washed nets. fig. 3. washing of nets with different detergents (original) fig. 4. washing of nets with shaking (original) test condition tests carried out ideally at 25±2 °c and 70-80% relative humidity (rh); never at temperature higher than 30 °c. interpretation of the test results percentage mortality was recorded after 24 h recovery period on the report form. if the control mortality was between 5-20%, the percentage mortality was corrected by abbott’s correction. for example if the mortality in the control was 10% and mortality at discriminating concentration was 80%, then the corrected mortality would be 78%. if control mortalities exceed 20%, the results should be recorded and test should be repeated again. for calculating kd50 the probit analysis as described by finney (1971) was used. statistical methods were used for data analysing using spss programme. there were significant difference between variables when p< 0.05%. results results of tests with permethrin, deltamethrin, lambdacyhalothrin and cyfluthrin are tabulated in tables 1-4. results of permethrin exhibited that there was significant difference between mortalities washed by rakht detergent, by shaking and no shaking methods and two type of washing (p< 0.01). the times of washing also affect on the stability of permethrin. washing impregnated bednets whit deltamethrin by rakht detergent, exhibited that no shaking method was similar in mortality rate with other washing method, whereas, in no shaking method, a significant difference was observed between no wash and other washing times (p< 0.01). washing procedure using lambdacyhalothrin with rakht detergent, exhibited that all washing stages had significant distinction. additionally, in no shaking method, a significant difference was observed between no wash and three washes. in shaking method, a significant difference was observed between different washes (p< 0.01). cyflutrhin impregnated nets whit rakht detergent, showed significant difference in the mortality rate, either in different stages of washing or in both two methods. washing the same nets whit soap detergent, only washing in no shaking method, had a significant difference in mortality rate between no wash and other stages of washing (p< 0.01). iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 23 table 1. mortality rate of an. stephensi in contact to impregnated bednets with permethrin (500 mg/m2) (ec) (%10) in process of washing whit three detergents in different stages of washing with two different kinds of washing mortality ± se shoma soap rakht detergent kind of wash 50 ± 6.8 59.32 ± 6.4 72.4 ± 5.9 no wash 19.23 ± 5.5 91.93 ± 3.5 72.2 ± 6.1 one wash 15.25 ± 4.7 96.77 ± 2.2 81.66 ± 5 two washes 13.33 ± 4.4 93.33 ± 3.2 82.45 ± 5 three washes no shaking 61.67 ± 6.3 67.27 ± 6.5 51.72 ± 6.6 no wash 38.33 ± 6.3 94.83 ± 2.9 49.1 ± 6.7 one wash 36.67 ± 6.2 89.83 ± 3.9 67.85 ± 6.2 two washes 21.67 ± 5.7 78.69 ± 5.2 68.42 ± 6.2 three washes shaking table 2. mortality rate of an. stephensi in contact to impregnated bednets with deltamethrin (25 mg/m2) (sc) (%10) in process of washing whit three detergents in different stages of washing with two different kinds of washing mortality ± se shoma soap rakht detergent kind of wash 57.63 ± 6.4 54.24 ± 6.5 48.27 ± 6.6 no wash 62.07 ± 6.4 52.54 ± 6.5 16.7 ± 4.8 one wash 42.37 ± 6.4 20.69 ± 5.3 16.1 ± 4.9 two washes 36.06 ± 6.1 5 ± 2.8 11.86 ± 4.2 three washes no shaking 88.33 ± 4.1 54.17 ± 7.2 47.27 ± 6.7 no wash 17.24 ± 5 25.45 ± 5.9 16.67 ± 4.8 one wash 8.93 ± 3.8 23.73 ± 5.5 15 ± 4.6 two washes 8.33 ± 3.6 6.78 ± 3.3 13.33 ± 4.4 three washes shaking table 3. mortality rate of an. stephensi in contact to impregnated bednets with lambdacyhalothrin (20 mg/m2) (ec) (%2.5) in process of washing detergents in different stages of washing with two different kinds of washing mortality ± se shoma soap rakht detergent kind of wash 91.80 ± 3.5 91.67 ± 3.6 79.31 ± 5.3 no wash 100 ± 0 100 ± 0 78.9 ± 5.4 one wash 100 ± 0 100 ± 0 78.69 ± 5.2 two washes 100 ± 0 100 ± 0 56.36 ± 6.7 three washes no shaking 95 ± 2.8 89.47 ± 4.1 83.05 ± 4.9 no wash 100 ± 0 100 ± 0 71.19 ± 5.9 one wash 100 ± 0 100 ± 0 62.59 ± 6.3 two washes 100 ± 0 100 ± 0 37.29 ± 6.3 three washes shaking table 4. mortality rate of an. stephensi in contact to impregnated bednets with cyfluthrin (50 mg/m2) (ec) (%5) in process of washing detergents in different stages of washing with two different kinds of washing mortality ± se shoma soap rakht detergent kind of wash 96.49 ± 2.4 80 ± 5.2 89.65 ± 4 no wash 86.67 ± 4.4 63.33 ± 6.2 87.27 ± 4.5 one wash 83.93 ± 4.9 51.66 ± 6.4 81.35 ± 5.1 two washes 60.34 ± 6.4 53.33 ± 6.4 82.14 ± 5.1 three washes no shaking 83.87 ± 4.7 76.67 ± 5.4 89.83 ± 3.9 no wash 33.33 ± 6.1 75.86 ± 5.6 85.96 ± 4.6 one wash 15 ± 4.6 67.24 ± 6.2 84.74 ± 4.7 two washes 16.67 ± 4.8 63.33 ± 6.2 82.54 ± 4.8 three washes shaking iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 24 discussion to determine the resistance of impregnated bed nets with different pyrethroids using washing, shaking and no shaking procedure, the malaria vectors, an. stephensi (beech strain) was used. four pyrethroids i.e.; permethrin, deltamethrin, lambdacyhalothrin and cyfluthrin were employed. local made bednet was impregnated with pyrethroids as mentioned above. three main important detergents, which are used frequently in the country, were used for washing. results showed that there was significant difference between mortalities of mosquito when using permethrin and washed by rakht detergent, shaking and no shaking methods and two type of washing (p< 0.01). this difference has been observed especially in one wash which shaking method; the insecticide removed from the nylon nets. as it is shown in tables there was no significant difference in mortality among different stages of washing in no shaking method, but there was significant differences between one wash whit two wash or three wash in shaking method (p< 0.05). while washing the impregnated bednets with permethrin using soap, there had been a significant reduction only between two washes in shaking and no shaking methods (p< 0.05). there is a significant difference between washing and no washing stages in no shaking method (p< 0.01). the results also observed in shaking method with increasing in mortality rate (p< 0.01). a difference has been observed between one wash and two wash using shoma (p< 0.01). it is observed the permethrin stability on nylon bednets using shoma is maintained and shaking method had no effect on its reduction, however the number of washing up to three times was effective. the stability of permethrin is reduced whit two washes using soap, although shaking had no effect. impregnated bednets washed with rakht using deltamethrin caused no significant difference in mortality rate, whereas, a significant difference has been observed between no wash and other washing times (p< 0.01). rakht detergent reduced deltamethrin efficacy, a significant decrease has been observed in mortality in two washes and three washes compared whit no wash stage (p<0.01). in shaking method, a significant decrease in all stages of washing was met in comparison to no wash stage. (p< 0.01). therefore, soap was able to decrease the mortality ratio in impregnated bednets with deltamethrin. when deltamethrin impregnated bednets was washed by shoma detergent, an obvious reduction was seen in each stages (p< 0.01). more reduction in mortality also observed using shaking. shaking method whit shoma decreased the mortality. it was clear that use of soap decreases the stability of this insecticide, however shaking had no effect. washing the lambdacyhalothrin impregnated bednets with rakht exhibited all washing stages had significant effect. in shaking method, a significant difference has been observed between no wash, two and three washes (p< 0.01). soap and shoma detergents resulted an increase up to 100% in mortality. the stability of lamdacyhalothrin insecticide has been reduced by the use of rakht detergent. the number of washing and shaking method was also effective. cyflutrhin impregnated nets which was washed with rakht, no significant difference was observed in the mortality rate, in any time of washing and methods. washing the same nets whit soap, only washing in no shaking method, had a significant difference in mortality rate (p< 0.01). shoma, exhibited a significant difference between one wash, wo washes and three washes (p< 0.01). a significant difference in mortality ratio in no wash, two and three wash stages in no shaking method was observed (p< 0.01). iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 25 in conclusion, washing by shoma detergent decreased the mortality rate in an. stephensi and more intensive decreases were observed in shaking method. a decrease in the stability of cyfluthrin was observed using shoma. in addition, both shaking and number of washing has more effect. rakht was not able to decreased the stability of cyfluthrin. residual effect of pyrethroid impregnated bednets reduced due to uv light, dust, fogging, weather condition, washing method, type of insecticide (rozendaal 1989). kayedi et al (2008) found that washing is more effective than uv light for degradation of insecticide on net. many fields research exhibited that if pyrethroid impregnated bednets was not exposed to washing, the residual effect of insecticide will be remained up to 6 to 12 months. this residual effect depends on type and quantities of insecticide used as well as type and characteristics of bed net. in practice, washing methods, type of detergents, the numbers and method of washing, are not as the same as in different parts of the world. it is determined that remaining soap on nets will demolish the molecular structure of pyrethroids (who 1989). for instance, the number of washing in surinam is weekly (rozendaal, 1989), once every two weeks in gambia (snow 1987) and annually in china (zuzi 1987) was documented. results of bioassay in different parts of the world are vary. this difference was due to formulation of insecticide, species of anopheles, susceptibility level of mosquito, the time of exposure, and texture of bednet, and type of test. (yaghoobi et al. 2006, kazemi et al. 2007). in this study, results showed that stability of permethrin impregnated bednets using shoma decreased and the numbers of washings up to three times caused more instability. it should be noted that the method of washing (shaking and no shaking had no significant effect on this phenomenon. the studies for measuring the washing effects on the stability of permethrin showed that washing with washing-machine powder and soap detergent in 50 ˚c, significantly removed the effectiveness of permethrin. the results of bioassay test using who holding tubes in 30 second exposure, the mortality rate in no wash, one wash, two washes, and three washes were 100%, 95%, 10% and 0%, respectively. the results of washing in cold water were significantly different. the net samples had been impregnated with permethrin at 63% g/m2, showed even 100% mortality after 10 times of washing with soap and cold water (schreck et al. 1978). during the tests of nylon bednets impregnated with permethrin after seven weeks of impregnation against an. sinensis, the kd50, and kd 95% were estimated, 13' 51" and 21' 1" respectively, and knock-down starting time was seven minute after exposure (jinijiang et al. 1988). the next step of this study was focused on washing of impregnated nets with deltamethrin. results showed that stability of deltamethrin on nets, decreased significantly by using soap an method of washing had not effect, but increasing the numbers of washing up to three times had significant effect. washing of deltamethin impregnated bed net with soap increased the kd50 . miller et al (1991) showed that net impregnated with deltamethrin washed three times, indicated no signs of the insecticide residues at the end of the processing. our study revealed that washing of lambdacyhalothrin impregnated bednets by rakht reduced the effectiveness of insecticide. the number and type of washing had significant effect on mortality. millers et al. (1991) showed that washing impregnated bednets with lambdacyhalothrin for 3 times decreased the effectives of insecticide. rafinejd et al. (2008) used three methods of bioassay tests against an. stephensi. they found that permanet® was more efficient than olysetnet® net. results on itns showed that deltamethirn and permethin were more effective than bifenthrin and etofenptorx iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 26 result of this study showed that a decrease in the stability of cyfluthrin by using shoma, also both shaking and number of washing has been effective, while using rakht detergent are not able to decrease the stability of cyfluthrin. finally statistical analysis to test the interaction of four pyrethroids, three detergents and 2 types of washing showed that the stability of permethrin using shoma and deltemethrin using soap with two type of washing decreased. the stability of lambdacyhalothrin with rakht and shaking method reduced. the stability of cyfluthrin using shoma has significantly reduced. it should be emphasized that the number of washing had negative effect. results of tests revealed that lambdacyhalothrin more than other insecticides has saved its insecticidal effect after being washed, on the contrary, deltamethrin lost its effectiveness faster than other insecticides. permethrin and cyfluthrin had moderate effect. washing by soap had no more effect on the residue of insecticides than other two detergents. increasing in the numbers of washing had significant effect on insecticidal activity. with increasing the number of washing these figures was more significant. shaking has no overall influence on the decreasing of the quality of insecticidal effect. monitoring and evaluation of impregnated bednet in the field condition for adverse effect of washing using different detergents is vital for implementation of any impregnated bednet. acknowledgments the authors would like to appreciate very much for kind collaboration of all staff of department of medical entomology and vector control, school of public health and institute of health research, tehran university of medical sciences. this project is financially supported by who and tehran university of medical sciences. references abbott ws (1965) a method of comparing the effectiveness of an insecticide. j eco entomol. 18: 265–267. davarai b, vatandoost h, ladonni h, shaeghi m, oshaghi ma, basseri hr, enayati aa, rassi y, abai mr, hanafi bojd aa, akbarzadeh k (2006) comparative efficacy of different imagicides against different strains of anopheles stephensi in the malarious areas of iran, 2004-2005. pakistan j biological sci. 9(5): 885–892. enayati aa, vatandoost h, ladonni h, townson h, hemingway j (2003) molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito anopheles stephensi. med vet entomol. 17(2): 138–144. jinjiang xu, meiluao z, xintu l, rangen g, shi xian p, shuyou l (1988) evaluation of permethrin-impregnated mosquito nets against mosquito in china. who/ vbc/88-96. hanafi-bojd aa, vatandoost h, jafari r (2006) susceptibility status of an. dthali and an. fluviatilis to commonly used larvicides in an endemic focus of malaria, southern iran. j vector borne dis. 43: 34–38. kayedi mh, lines jd, haghdoost aa, vatandoost mh, rassi y, khamisabady k (2008) evaluation of the effects of repeated hand washing, sunlight, smoke and dirt on the persistence of deltamethrin on insecticide-treated nets. trans r soc trop med hyg. 102: 811–816. li z, jingjiang x, banquan l, taihua z, mingxin l (1987) mosquito nets impregnated whit deltamethrin against malaria vectors in china. who/vbc/87-93. manouchehri av, zaim m, emadi am (1992) a review of malaria in iran, 1957–1990. j am mosq control assoc. 8(4): 381-385. iranian j arthropod-borne dis, (2009), 3(1): 19-28 h vatandoost et al: stability and wash resistance … 27 miller je, lindsay sw, armestrong jrm (1991) experimental hut trials of bednets impregnated with synthetic pyrethroid or organophosphate insecticide for mosquito control in the gambia. med vet entomol. 5: 465–476. moosa-kazemi sh, yaghoobi-ershadir mr, akhavan aa, abdoli h, zahraei-ramazani ar, jafari r, houshmand b, nadim a, hosseini m (2007) deltamethrin impregnated bed nets and curtains in an anthroponotic cutaneaous leishmaniasis control programme in north-eastern iran. ann saudi med. 27(1): 6–12. naddaf sr, oshaghi ma, vatandoost h, asmar m (2003) molecular characterization of the anopheles fluviatilis species complex in iran. who eastern mediterr hlth j. 9(3): 257–265. oshaghi ma, moradi mt, taghilo b (2004) specific detection of malaria parasites using nestedpcr in individual mosquitoes and infected bloods in chabahar and iranshar, iran. hakim. 7(3): 24–31. oshaghi ma, taghilo b, moradi mt, vatandoost h (2004) mosquitoes of anopheles culicifacies complex, species a and b in baluchistan using mtdna pcrrflp assay: the first report of species b from iran. hakim. 7(1): 35–41. oshaghi ma, chavshin ar, vatandoost h, yaaghoobi f, mohtarami f, noorjah n. 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43–48. zaim m, aitio a, nakashima n (2000) safety of pyrethroid-treated mosquito nets. med ve entomol. 14(1): 1–5. zaim m, manouchehri av, motabar m, emadi am, nazari m, pakdad k, kayedi mh, mowlaii g (1995) anopheles culicifacies in baluchistan, iran. med vet entomol. 9(2): 181–186. zahirnia ah, vatandoost h, nateghpour m, javadian e (1998) insecticide resistance/ susceptibility monitoring in anopheles pulcherrimus (diptera: culicidae) in ghasreghand district, sistan and baluchistan province, iran, 1997. hakim. 1(2): 97-106. zahirnia, ah, taherkhani h, vatandoost h (2001) observation of malaria sporozoite in anopheles culicifacies (diptera: culicidae) in ghasreghand district, sistan and baluchistan province. hakim. 4(2): 149– 153. world health organization (1997) guidelines on the use of insecticide-treated mosquito nets for the prevention and control of malaria in africa. ctd/mal/afro/97.4. world health organization (1998) test procedure for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces, who/cds/cpc/mal/98.12. world health organization (1989) the use of impregnated bednets and other material for vector-borne diseases control. who/ vbc/89-91. world health organization (2001) specifications for netting materials, roll back malaria, who/cds/rbm/2001.. world health organization (2002) malaria vector control, decision making criteria and procedures for judicious use of insecticides, by; d ja najera and dr m zaim. who/cds/whopes/2002.5. world health organization (2005) guideline for laboratory and field testing of longlasting insecticidal mosquito nets. who/ cds/whopes/gcfdpp/2005.11. j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 65 original article physicochemical characteristics of larval habitat waters of mosquitoes (diptera: culicidae) in qom province, central iran mohammad reza abai 1, abedin saghafipour 2, hossein ladonni 1, nahid jesri 3, saeed omidi 4, *shahyad azari-hamidian 4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2qom provincial health center, qom university of medical sciences, qom, iran 3department of watershed and rangeland management, yazd university, yazd, iran 4school of health, guilan university of medical sciences, rasht, iran (received 20 sep 2014; accepted 18 oct 2014) abstract background: mosquitoes lay eggs in a wide range of habitats with different physicochemical parameters. ecological data, including physicochemical factors of oviposition sites, play an important role in integrated vector management. those data help the managers to make the best decision in controlling the aquatic stages of vectors especially using source reduction. methods: to study some physicochemical characteristics of larval habitat waters, an investigation was carried out in qom province, central iran, during spring and summer 2008 and 2009. water samples were collected during larval collection from ten localities. the chemical parameters of water samples were analyzed based on mg/l using standard methods. water temperature (°c), turbidity (ntu), total dissolved solids (ppm), electrical conductivity (µ s/cm), and acidity (ph) were measured using digital testers. thermotolerant coliforms of water samples were analyzed based on mpn/100ml. data were assessed by kruskal-wallis test and spearman correlation analysis. results: in total, 371 mosquito larvae were collected including 14 species representing four genera. some physicochemical parameters of water in emamzadeh esmail, qomrood, qom city, and rahjerd showed significant differences among localities (p< 0.05). the physicochemical and microbial parameters did not show any significant differences among different species (p> 0.05). there was no significant correlation between the abundance of larvae and the different physicochemical and microbial parameters (p> 0.05). conclusion: the means of ec, tds, and phosphate of localities and species were remarkably higher than those of the previous studies. other parameters seem to be in the range of other investigations. keywords: anopheles, culex, culiseta, ochlerotatus, larvae, oviposition site introduction mosquitoes (diptera: culicidae) lay eggs in a wide range of habitats with different physicochemical characteristics. the temperature of larval habitat water has a great influence on the development of the aquatic stages of mosquitoes (muirhead-thomson 1951). while some subarctic aedine species larvae are able to develop at 1.1°c, the development of many mosquito species critically decrease below 14–16 °c. on the other hand, few species are able to breed and survive at 42 °c in tropical areas, while the temperature above 30 °c decreases larval development and 37–38 °c is fatal for many temperate species (muirhead-thomson 1951, clements 1992). most of mosquito larvae develop in fresh water (a salinity of up to 2 parts per thousand). however, nearly 5 % live in brackish (a salinity between fresh water and sea water 34.5 per thousand) or saline waters (very rich in soluble salts). some species occur in both fresh and brackish waters, even closely *corresponding author: dr shahyad azarihamidian, e-mail: azari@gums.ac.ir http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 66 related species may be found in different larval habitats for example fresh and brackish waters for the maculipennis group species and fresh, brackish, and saline habitats for an. gambiae giles complex species (clements 1992). in nature different mosquito larvae species were found in a wide range of ph from 3.3 to 10.5 and some species were reared in ph from 2 to 9 in the laboratory. many species larvae were found in both acid and alkaline habitats. though ph effects on the distribution of some species, there is no evidence that ph is a limiting factor (clements 1992). organic matter and pollution, for example ammonium ion, which has a range between 2 to 5 mm in sewage, can restrict larval breeding and few species survive in heavily polluted waters. anopheline larvae mainly develop in clean water and seldom are found in polluted habitats, which seem to be favorable for some culicines (muirheadthomson 1951, clements 1992). malaria is the most important mosquitoborne disease in iran, especially in southern and southeastern areas, and seven species of the genus anopheles meigen are known as proven vectors in the country, an. culicifacies giles s.l., an. dthali patton, an. fluviatilis james s.l., an. maculipennis meigen s.l., an. sacharovi favre, an. stephensi liston, and an. superpictus grassi (hanafi-bojd et al. 2011). the mosquito fauna of iran includes seven genera and 64 species (azarihamidian 2007a). qom province is located in the central plateau of iran where the risk of malaria infection is lower than the southern and southeastern areas of the country. however, the situation of the province increases the risk of transmission, because many passengers/ pilgrims visit the province from different parts of the country including malarious areas (farzinnia et al. 2010). the province is classified in the stratum 2 (with the imported cases of malaria and potential transmission) of the national malaria strategy plan for malaria control in iran (raeisi et al. 2004). in total, 448 cases of malaria were recorded in qom province during 2001–2008. the trend of disease shows decline in the province like many other iranian provinces. total number of the cases has been decreased from 151 in 2001 to 22 in 2008. though nearly all cases are imported from outside of the province with mostly non-iranian origin, one indigenous case was found in 2004 (farzinnia et al. 2010). after 2008, the cases were 16, 24, 49, and 54 from 2009 to 2012, respectively (unpublished data from department of disease control, deputy of health, qom university of medical sciences). macan (1950) found an. multicolor combouliu larvae in “darya-i-namak” (namak lake, salt lake, or qom lake). macan (1950) also showed an. superpictus around qom in the distributional maps. farzinnia et al. (2010) reported an. claviger (meigen) for the first time in the province. saghafipour et al. (2012) found 14 species representing four genera in qom province including 12 new provincial records. in total, 15 species and 4 genera are found in the province. there is little information about physicochemical characteristics of larval habitats in iran. in the most of previous investigations, the collecting data of larval habitats included only water temperature and/or ph (eg macan 1950, lotfi 1976, azari-hamidian et al. 2004, azari-hamidian 2005, 2006, 2007b, 2011). yaghoobi-ershadi et al. (2001) investigated the bionomics of an. sacharovi in ardebil province, northwestern iran, with a note on salinity, based on calcium bicarbonate and sodium sulfate, for the larval habitats of species. ghanbari et al. (2005) studied some physical and chemical factors of oviposition sites including turbidity, electrical conductivity (ec), temperature, ph, total hardness, calcium, chloride, sulfate, nitrate, phosphate, and nitrite in iranshahr, southeastern iran, where eight anopheline species including an. culicifacies s.l., an. stephensi, an. dthali, an. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 67 hyrcanus (pallas) (most probably misidentification), an. superpictus, an. turkhudi liston, an. multicolor, and an. pulcherrimus theobald were collected. hanafi-bojd et al. (2012) investigated larval habitats, including the temperature, ph, total hardness, ec, and dry residue of waters, and biodiversity of anophelines in bashagard, southern iran. ecological data, such as physicochemical factors of oviposition sites, larval habitat characteristics, species composition, and active season play an important role in integrated vector management (ivm). those data help the managers to make the best decision in controlling the aquatic stages of vectors especially using source reduction through environmental manipulation and modification in addition to chemical and biological controls. to study some physicochemical features of habitat water of mosquito larvae, this investigation was carried out in qom province, central iran. materials and methods study area qom province is bounded by tehran province in the north, isfahan province in the south, semnan province in the east, and markazi province in the west with an area of approximately 11,240 square kilometers (0.68 % total area of iran). the center of the province, qom city, is almost 880 m above sea level. the province with arid climate has about 150 mm annual rainfall and is located between 34o09'–35o11' n latitude and 50o06'– 51o58' e longitude and formally includes one county (qom) and five districts, jafarabad, kahak, khalajestan, markazi (qom), and salafchegan (fig. 1). specimen and data collection larval collection was carried out from different habitats using dipping method (350 ml dipper) in ten localities of four districts, kahak district (emamzadeh esmail and dastgerd), khalajestan district (ahmadabad, agholak, dastjerd, and ghahan), markazi district (qom city and qomrood), and salafchegan district (ghal-e-cham and rahjerd) during spring and summer 2008 and 2009. the larvae were preserved in lactophenol and the microscope slides of the preserved larvae were prepared using de faure’s medium. the third-and fourthinstar larvae were identified using the key of azari-hamidian and harbach (2009). the mosquito name abbreviations follow reinert (2009). physicochemical analysis of water of larval habitats the samples of water of larval habitats in ten aforementioned localities were collected and the chemical factors including alkalinity, total hardness (both based on caco3), calcium (ca), chloride (cl), fluoride (f), nitrite (no2), nitrate (no3), phosphate (po4), and sulfate (so4) were analyzed based on mg/l using standard methods (rice et al. 2012). moreover, other physicochemical parameters which were tested, their units, and digital testers, are as follow: water temperature (°c) (cyberscan, singapore), turbidity (ntu) (aqualytic, germany), total dissolved solids (tds) (ppm) (cyberscan, singapore), electrical conductivity (ec) (µs/cm) (cyberscan, singapore), and acidity (ph) (cyberscan, singapore). thermotolerant coliforms of water samples were analyzed as a biological (microbial) parameter that shows habitat pollution with human sewage and feces based on mpn (most probable number)/100 ml. statistical analysis the means of physicochemical and microbial parameters of the water samples of the species were compared by kruskal-wallis test of nonparametric analysis and the relation of physicochemical and microbial parameters to abundance was assessed by spearman correlation analysis using spss software (version 17 for windows, spss inc. chicago, il). http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 68 results in total, 371 mosquito larvae were periodically collected from nine localities in four districts of qom province during 2008–2009 and morphologically identified including 14 species representing four genera (table 1). no larva was collected from the river of qom city. all larvae were collected from natural habitats including ground pools, stream edges, riverbeds, and river edges (fig. 2). the physicochemical and microbial parameters of water of different localities were showed in table 2 and 3. phosphate in emamzadeh esmail, turbidity, ec, tds, total hardness, chloride, and nitrate in qomrood, ec, tds, sulfate in qom city, and nitrite in rahjerd were significantly higher than other localities (p< 0.05), however temperature, ph, alkalinity, calcium, and fluoride did not show any significant difference among different localities (p> 0.05) (table 2). the physicochemical and microbial parameters did not show significant differences among different species (p> 0.05) (table 4 and 5). there was no significant correlation between the abundance of larvae and the different physicochemical and microbial parameters (p> 0.05) (table 6). most of physicochemical and microbial parameters for an. claviger and an. marteri senevet and prunnelle and all culicine species were presented for the first time in iran (table 4 and 5). table 1. the distribution and composition of the mosquito larvae in nine localities of qom province, iran, spring– summer 2008–2009 species locality n % a gh olak a h m ad ab ad d astgerd d astjerd e m am zad eh e sm ail g h ah an g h al-e-ch am q om rood r ah jerd an. claviger 66 135 1 16 10 228 61.5 an. marteri 2 2 0.5 an. superpictus 12 20 32 8.6 an. turkhudi 7 7 1.9 cx. arbieeni 1 1 0.3 cx. hortensis 3 2 1 3 9 2.4 cx. mimeticus 9 1 1 11 3.0 cx. modestus 6 6 1.6 cx. pipiens 13 13 3.5 cx. territans 7 3 10 2.7 cx. theileri 1 4 1 6 1.6 cs. longiareolata 34 5 2 41 11.1 cs. subochrea 1 1 2 0.5 oc. caspius s.l. 3 3 0.8 total 77 175 30 1 25 25 10 24 4 371 100 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 69 fig. 1. map of iran in which the position of qom province and its five districts is highlighted, 1. jafarabad, 2. kahak, 3. khalajestan, 4. markazi, and 5. salafchegan (*qom city) fig. 2. different types of larval habitats in qom province, iran, spring–summer 2008–2009, a, b, and c) ground pools, d) stream edge, e) river bed, f) river edge (original photos) a b a c d e f http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 70 table 2. the physicochemical parameters of water in qom province, iran, spring–summer 2008–2009, *no larva was collected, a values in a row which are significantly different in habitats where include larvae (n=9) and b in all samples (n=10) (at p=0.05) physicochemical parameters locality mean±sd (larval habitats, n=9) mean±sd (total, n=10) a gh olak a h m ad ab ad d astgerd d astjerd e m am zad eh e sm ail g h ah an g h al-e-ch am q om rood q om c ity* r ah jerd temperature (°c) 20.4 17.5 21.8 17.5 22.0 20.4 22.7 22.7 15.3 20.4 20.60±1.98 20.07±2.51 acidity (ph) 7.5 7.3 7.7 7.3 7.7 7.5 7.3 7.1 7.7 7.0 7.37±0.24 7.41±0.25 turbidity (ntu) 1.0 0.1 0.3 0.1 1.9 1.0 0.2 6.0a 0.5 0.2 1.20±1.89 1.13±1.80 electrical conductivity (µs/cm) 2070 1827 1005 1827 815 2070 2240 4240a 8820b 392 1831.77±1110.74 2530.60±2445.44 total dissolved solids (ppm) 1030 914 502 914 407 1030 1120 2120a 4380b 197 914.88±555.05 1261.40±1214.31 alkalinity (mg/l) 300 540 230 540 250 300 530 410 300 210 367.77±138.90 361.00±132.70 total hardness (mg/l) 799.6 428.4 238.0 428.4 266.5 799.6 534.7 1294.7a 875.8 180.8 552.30±357.58 584.65±352.31 calcium (mg/l) 323.6 649.5 30.4 649.5 30.4 323.6 620.8 220.8 83.7 26.6 319.46±267.23 295.89±262.74 chloride (mg/l) 154.9 364.8 229.9 364.8 129.9 154.9 389.8 1479.5a 329.8 24.9 365.93±435.91 362.32±411.14 fluoride (mg/l) 0.96 1.05 0.28 1.05 0.41 0.96 0.95 0.75 1.23 0.17 0.731±0.349 0.781±0.365 nitrite (mg/l) 0.003 0.008 0.001 0.008 0.006 0.003 0.004 0.004 0.004 0.012a 0.0054±0.0033 0.0053±0.0032 nitrate (mg/l) 0.0 1.0 2.2 1.0 1.2 0.0 1.8 9.8a 0.4 0.0 1.88±3.07 1.74±2.93 phosphate (mg/l) 0.43 0.39 1.53 0.39 2.74a 0.43 0.48 0.48 0.23 0.12 0.776±0.834 0.722±0.805 sulfate (mg/l) 784 400 134 400 184 784 832 832 1445b 37 487.44±325.67 583.20±431.24 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 71 table 3. the microbial parameter of water in qom province, iran, spring–summer 2008–2009 (*no larva was collected, n= not determined) microbial parameter locality mean ± s d (l arval h ab itats, n = 8) m ean ± s d (t otal, n = 9) a gh olak a h m ad ab ad d astgerd d astjerd e m am zad eh e sm ail g h ah an g h al-e-ch am q om rood q om c ity* r ah jerd thermotalerant coliforms (mpn/100ml) n 0 75 0 1 1 0 0 1 5 0 1 1 0 0 0 93 2 9 7 .87± 49 6 .3 6 2 6 4 .77± 47 4 .8 0 table 4. the physicochemical parameters of habitat water of mosquito larvae in qom province, iran, spring–summer 2008–2009 physicochemical parameters (mean±sd) species (number of occurrence) a n . claviger (5) a n . m arteri (1) a n . su perpictu s (2) a n . tu rkh u di (1) c x. arbieen i (1) c x. h orten sis (4) c x. m im eticu s (3) c x. m odestu s (1) c x. pipien s (1) c x. territan s (2) c x. th eileri (3) c s. lon giareolata (3) c s. su boch rea (2) o c. caspiu s s.l. (1) temperature (°c) 19.7± 2.2 20.4 21.9± 0.1 21.8 21.8 20.0± 1.8 21.4± 8.8 22.7 22.7 18.9± 2.0 20.7± 2.8 19.4± 1.6 21.5± 1.6 22.7 acidity (ph) 7.3± 0.1 7.0 7.7± 0.0 7.7 7.7 7.5± 0.1 7.6± 0.1 7.1 7.1 7.4± 0.1 7.3± 0.3 7.2± 0.2 7.3± 0.2 7.1 turbidity (ntu) 0.4± 0.4 0.2 1.1± 1.1 0.3 0.3 0.6± 0.4 1.0± 0.8 6.0 6.0 0.5± 0.6 2.6± 3.0 0.4± 0.4 3.5± 3.5 6.0 electrical conductivity (µs/cm) 2006. 8± 178.2 392 910.0 ± 134.3 1005 1005 1743. 0± 505.1 1296.6 ± 676.4 4240 4240 1948.5 ± 171.8 2294.0 ± 1759.6 1429. 6± 906.8 3155.0 ± 1534.4 4240 total dissolved solids (ppm) 1001. 6± 88.0 197 454.5 ± 67.1 502 502 869.0 ± 259.7 335.6± 193.7 2120 2120 972.0± 82.0 1147.0 ± 879.9 713.6 ± 451.1 1575.0 ± 770.7 2120 alkalinity (mg/l) 442.0 ± 129.6 210 240.0 ± 14.1 230 230 342.5 ± 135.7 260.0± 36.0 410 410 420.0± 169.7 400.0± 145.2 416.6 ± 120.1 355.0± 77.7 410 total hardness (mg/l) 598.1 ± 188.9 180.8 252.2 ± 20.1 238.0 238.0 566.4 ± 280.2 434.7± 316.3 1294.7 1294.7 614.0± 262.4 663.2± 552.8 469.6 ± 311.4 1047.1 ± 350.0 1294.7 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 72 physicochemical parameters (mean±sd) species (number of occurrence) a n . claviger (5) a n . m arteri (1) a n . su perpictu s (2) a n . tu rkh u di (1) c x. arbieen i (1) c x. h orten sis (4) c x. m im eticu s (3) c x. m odestu s (1) c x. pipien s (1) c x. territan s (2) c x. th eileri (3) c s. lon giareolata (3) c s. su boch rea (2) o c. caspiu s s.l. (1) calcium (mg/l) 513.4± 173.6 26.6 30.4± 0.0 30.4 30.4 331.7 ± 259.9 128.1 ± 169.2 220.8 220.8 486.5 ± 230.4 300.2 ± 317.1 333.2 ± 311.5 272.2 ± 72.6 220.8 chloride (mg/l) 285.8± 119.9 24.9 179.9 ± 70.7 229.9 229.9 226.1 ± 98.9 171.5 ± 52.0 1479.5 1479. 5 259.8 ± 148.4 658.0 ± 721.0 181.5 ± 171.5 817.2 ± 936.6 1479.5 fluoride (mg/l) 0.99± 0.05 0.17 0.34± 0.09 0.28 0.28 0.81± 0.35 0.55± 0.36 0.75 0.75 1.00± 0.06 0.73± 0.32 0.72± 0.48 0.85± 0.14 0.75 nitrite (mg/l) 0.005± 0.002 0.012 0.003 ± 0.003 0.001 0.001 0.003 ± 0.002 0.003 ± 0.002 0.004 0.004 0.005 ± 0.003 0.006 ± 0.002 0.007 ± 0.004 0.003 ± 0.000 0.004 nitrate (mg/l) 0.7± 0.7 0.0 1.7± 0.7 2.2 2.2 0.8± 1.0 1.1± 1.1 9.8 9.8 0.5± 0.7 4.0± 5.0 0.3± 0.5 4.9± 6.9 9.8 phosphate (mg/l) 0.42± 0.03 0.12 2.13± 0.85 1.53 1.53 0.69± 0.55 1.56± 1.15 0.48 0.48 0.41± 0.02 1.20± 1.33 0.31± 0.16 0.45± 0.03 0.48 sulfate (mg/l) 640.0± 219.9 37 159.0 ± 35.3 134 134 525.5 ± 317.6 367.3 ± 361.7 832 832 592.0 ± 271.5 472.0 ± 329.9 407.0 ± 373.5 808.0 ± 33.9 832 table 5. the microbial parameter of habitat water of mosquito larvae in qom province, iran, spring–summer 2008–2009 microbial parameter (mean±sd and range) species (number of occurrence) a n . claviger (4) a n . m arteri (1) a n . su perpictu s (2) a n . tu rkh u di (1) c x. arbieen i (1) c x. h orten sis (3) c x. m im eticu s (3) c x. m odestu s (1) c x. pipien s (1) c x. territan s (1) c x. th eileri (3) c s. lon giareolata (3) c s. su boch rea (1) o c. caspiu s s.l. (1) thermotalerant coliforms (mpn/100ml) 3 .7 5 ± 7 .50 (0– 1 5) 9 3 5 8 7 .50 ± 72 4 .7 8 (7 5–1 1 00 ) 7 5 7 5 3 0 .0 0± 3 9.6 8 (0–7 5 ) 3 9 6 .66 ± 60 9 .8 4 (1 5–1 1 00 ) 1 1 0 0 1 1 0 0 0 7 3 3 .33 ± 63 5 .0 8 (0– 11 0 0) 3 6 .0 0± 4 9.9 2 (0–9 3 ) 1 1 0 0 1 1 0 0 table 4. continued … http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 73 table 6. spearman correlation coefficient between physicochemical and microbial parameters of habitat waters and larvae abundance in qom province, iran, spring–summer 2008–2009, sig. (2-tailed), n=9 (for thermotalerant coliforms, n=8) (p> 0.05) physicochemical and microbial parameters abundance temperature (°c) -0.137 0.725 acidity (ph) 0.532 0.140 turbidity (ntu) 0.174 0.655 electrical conductivity (µs/cm) 0.030 0.940 total dissolved solids (ppm) 0.030 0.940 alkalinity (mg/l) -0.030 0.940 total hardness (mg/l) 0.131 0.737 calcium (mg/l) 0.076 0.845 chloride (mg/l) -0.080 0.838 fluoride (mg/l) 0.190 0.625 nitrite (mg/l) -0.453 0.220 nitrate (mg/l) -0.051 0.896 phosphate (mg/l) 0.233 0.546 sulfate (mg/l) -0.004 0.991 thermotalerant coliforms (mpn/100ml) 0.056 0.896 discussion in the present study, 371 mosquito larvae representing 14 species in four genera were collected from qom province. all species except for an. claviger and an. superpictus were collected for the first time in the province (saghafipour et al. 2012) (table 1). anopheles multicolor, recorded in qom province already (macan 1950), was not found in the present investigation. some physicochemical parameters of water in emamzadeh esmail (phosphate), qom city (ec, tds, and sulfate), and rahjerd (nitrite) showed significant differences among localities, however qomrood included much more significantly different parameters (turbidity, ec, tds, total hardness, chloride, and nitrate) (p< 0.05) (table 2). it is noteworthy that no larva was found in the river of qom city where three parameters (ec=8,820 µ s/cm, tds=4,380 ppm, and sulfate=1,445 mg/l) were significantly higher than those of other localities (p< 0.05) (table 2). some aforementioned water parameters have a public health importance and are used as an indicator of drinking-water quality (who 2008). http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 74 khamala (1971) found that sedimentary solids, suspended solids, dissolved organic matter, total nitrogen, and ph did not have significant correlation with the density of mansonia africana (theobald) and ma. uniformis (theobald) larvae in kenya, while sasikumar et al. (1986) showed that na, k, and ph had significant correlation with the density of ma. uniformis and ma. annulifera (theobald) larvae, however ca, mg, and rainfall showed no significant relation in india. in iran, ghanbari et al. (2005) showed the significant correlation of seven physicochemical factors including ph, total hardness, nitrate, phosphate, ec, calcium, and sulfate with five species an. culicifacies s.l., an. stephensi, an. superpictus, an. turkhudi, an. multicolor. though some of aforementioned factors were found to be predictor species, none of them was predictor for all anopheline species. that might show biological differences of the species. hanafi-bojd et al. (2012) noted the temperature, ph, total hardness, ec, and dry residue of larval habitats in bashagard where eight species an. culicifacies s.l., an. dthali, an. stephensi, an. superpictus, an. fluviatilis s.l., an. turkhudi, an. moghulensis christophers, and an. apoci marsh were collected, however they did not calculated any correlation. both ghanbari et al. (2005) and hanafi-bojd et al. (2012) mentioned the figures of habitats in general and none of them provided the exact values (means) of physicochemical features for each species. piyaratne et al. (2005) found a positive correlation of an. culicifacies s.l. and an. varuna iyengar abundances only to temperature and calcium, respectively. surendran and ramasamy (2005) observed a significant correlation of an. culicifacies species e abundance to dissolved oxygen. abdel-hamid et al. (2011) found that there is a positive correlation between larval density and temperature for total collected species including three more prevalent ones: cx. antennatus (becker), cx. pipiens linnaeus, and cx. perexiguus theobald and a negative correlation between density and ph for total collected species and for cx. pipiens. however the correlation was positive for cx. antennatus and cx. perexiguus in el gharbia governorate, egypt. ibrahim et al. (2011) found that temperature, amonia, and nitrate are the best predictor for larval density in qalyubiya governorate, egypt, where they collected nine species in which cx. pipiens was the most prevalent (64.7%). however, no correlation was found between larval density, ph, and dissolved oxygen. among the physicochemical parameters of the present investigation, the means of ec, tds, and phosphate of localities (1,831.77, 914.88, and 0.77, respectively) (table 2) and species (table 4) were remarkably higher than those of surendran and ramasamy (2005) (ec: 715.7), piyaratne et al. (2005) (ec: 534.7 and 828.8, tds: 265.3 and 407.7, phosphate: 0.03 and 0.22), and ghanbari et al. (2005) (ec: 256, phosphate: 0.07). other parameters seem to be in the range of other investigations (muirhead-thomson 1951, khamala 1971, sasikumar et al. 1986, clements 1992, ghanbari et al. 2005, piyaratne et al. 2005, surendran and ramasamy 2005, abdel-hamid et al. 2011, ibrahim et al. 2011, hanafi-bojd et al. 2012). as it is obvious, some available data are contradictory and there is not enough information about physicochemical parameters of larval habitats for many mosquito species. in addition to the biological differences of different species, the same species has a range of tolerance and sometimes show different correlation with physicochemical parameters. thus, the present data is too basic for a general assessment and conclusion. the most important limitation of the present study is the limited numbers and occurrences of different species because of the arid climate of qom province. this phenomenon caused that the distribution of some parameters were not normal (table 2) and the occurhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 75 rences of the species ranged from only one to five (tables 4 and 5). that is why kruskalwallis test and spearman correlation coefficient of nonparametric analysis were used to assess data. though according to the present physicochemical and microbial parameters, there is no significant difference among different species (tables 4 and 5) and the parameters did not show correlation with the abundance of larvae (table 6), they may change with more sampling and using parametric analysis. conclusion in the present investigation, the means of ec, tds, and phosphate of localities and species were remarkably higher than those of the previous studies. other parameters seem to be in the range of other investigations. more samplings of habitat waters (with or without larvae for comparing) in different climates and topographical regions (to collect more diversified species) are recommended. acknowledgements the authors are grateful to dr a akbari, mh babakhani, f abedi-astaneh, qom provincial health center, qom university of medical sciences, qom, for their kind collaborations. the authors also thank dr sm omrani, department of medical parasitology, school of medicine, shahrekord university of medical sciences, shahrekord, and shadi azari-hamidian for reviewing the manuscript. this study was financially supported by the institute of public health research, academic pivot for education and research, tehran university of medical sciences: project no.: 241.83.77. this investigation also was financially supported by qom university of medical sciences. the authors declare that there is no conflict of interests. references abdel-hamid ym, mostafa aa, allam km, kenawy ma (2011) mosquitoes (diptera: culicidae) in el gharbia governorate, egypt: their spatial distribution, abundance and factors affecting their breeding related 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health association, american water works association, water environment federation, cenveo publisher services, richmond. saghafipour a, abai mr, farzinnia b, nafar r, ladonni h, azari-hamidian s (2012) mosquito (diptera: culicidae) fauna of qom province, iran. j arthropod-borne dis. 6: 54–61. sasikumar ps, suryanarayanan p, thomas c, kalyanaraman k, prasad rs (1986) influence of certain physic-chemical factors upon the larval population of mansonia mosquitoes (culicidae: diptera) in trivandrum city, india. proc indian acad sci (anim sci). 95: 549– 555. surendran sn, ramasamy r (2005) some characteristics of the larval breeding sites of anopheles culicifacies species b and e in sri lanka. j vector borne http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 65–77 mr abai et al.: physicochemical characteristics … 77 dis. 42: 39–44. who (2008) guidelines for drinking-water quality. second addendum to third edition. vol. 1. recommendations. world health organization, geneva. yaghoobi-ershadi mr, namazi j, piazak n (2001) bionomics of anopheles sacharovi in ardebil province, northwestern iran during a larval control program. acta trop. 78: 207–215. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, december 2014, 8(2): 124–131 m alizadeh et al.: a review of myiasis … 1 review article a review of myiasis in iran and a new nosocomial case from tehran, iran mahbobeh alizadeh 1, gholamreza mowlavi 2, faranak kargar 3, mehdi nateghpour 2, *kamran akbarzadeh 4, maryam hajenorouzali-tehrani 5 1baharloo hospital, tehran university of medical sciences, tehran, iran 2department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 3shahid rajaei heart hospital, faculty of medicine, tehran university of medical sciences, tehran, iran 4department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 5department of pediatric dentistry, torabinejad dental research center, school of dentistry, isfahan university of medical sciences, isfahan, iran (received 20 feb 2013; accepted 25 june 2014) abstract background: due to various climatic conditions in different parts of iran, presenting of various kinds of human myiasis is expected. despite of a few case series, most papers related to myiasis are case reports originated from various parts of iran. this study discusses on different clinical features of myiasis in iran and description of one case from tehran as a representative to nosocomial infection in iran. methods: the information needed for this descriptive study was derived mainly from the digital library of tehran university of medical sciences. the larvae have been identified with observing on posterior spiracles, spines of their body and anal tubercles. results: total number of reported myiasis cases from iran is 77 which can be categorized clinically as furuncular, wound, ophthalmic, auricular, nasopharyngeal, oral, intestinal and genitourinary. based on parasitological features, all myiasis agents in iran are belonging to calliphoridae, sarcophagidae, oestridae and syrphidae flies. the case which is reporting in this paper can be mentioned as representative of nosocomial myiasis which it seems to be underreported because of some medicolegal reasons. conclusion: low number of investigations on various aspects of human myiasis, as well as incuriosity to report of the cases in disease reporting system of health minister, made the myiasis as a neglected disease. keywords: myiasis, diptera, nosocomial, hospital, iran introduction the parasitic relation which is induced with dipteran true fly larvae including feeding on ingested food, body materials and living/dead tissues of their hosts has been defined as myiasis disease (zumpt 1965). various species of true flies can cause myiasis disease around the world (john and petri 2006). this kind of infestation in human is more spread in humid and warm geographical conditions (maturo et al. 2007). thus various kinds of the human myiasis are expected to be found in most parts of iran due to their climatic conditions. there is one fruncular myiasis from iran in literature. that was an imported case on thighs of an iranian 40 yr old man who came back from africa after finishing his business trip. presence of larvae made red pruritic lesions on his body (mostafavizadeh et al. 2003). various cases of wound myiasis have been reported from various parts of iran which occurred in different clinical settings such as head skin (talari et al. 2004, davami et al. 2005), scalp (soleimani-ahmadi et al. 2009), and basal cell carcinoma of head (asilian *corresponding author: dr kamran akbarzadeh e-mail: akbarzadehk@yahoo.com 124 http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 124–131 m alizadeh et al.: a review of myiasis … 125 and andalib 2012) as well as the wound around pharyngostomy operation (soleimaniahmadi et al. 2013). lucilia sericata (diptera: calliphoridae) and chrysomyia bezziana (diptera: calliphoridae) were identified as agents of wound myiasis in iran. aalam described some cases of ophthalmomyiasis in his phd thesis (ghassemi 1989). ophthalmomyiasis is infestation of human eyes and orbits tissues with larvae of myiasis agents which usually cause by the botfly oestrus ovis (diptera: oestridae) (akbarzadeh 2012). this kind of myiasis has been reported from various parts of iran (minar 1976, ferdowsi and rostami 2007, soleymani et al. 2012, akbarzadeh 2012). auricular myiasis is another kind of the disease which infects human ears. there are a few cases of auricular myiasis in iran which were occurred by ch. bezziana (talari et al. 2002, yaghoobi et al. 2005) and lu. sericata (akbarzadeh 2012). majority of myiasis cases reported in the literature from iran were nasal myiasis originated from various parts. the agents of these cases were ch. bezziana, lu. sericata and erystalis tenax (diptera: syrphidae) (tirgari et al. 2003, salimi et al. 2010a,b, ghafori et al. 2011, babamahmoodi et al. 2012, youssefi et al. 2012). oral/gingival myiases also have been reported from various parts of iran which affect on jaws or on mucosal tissues in oral cavities. the agents of these disease were obligatory myiasis producing flies, oe. ovis (diptera: oestridae) and wohlfahrtia magnifica (diptera: sarcophagidae) (athari and fallah 1993, pour eslami and fallah 1996, hakimi and yazdi 2002, mohammadzadeh et al. 2008, moshref et al. 2008, tamizi et al. 2008, faramarzi et al. 2009). the pharyngeal manifestation with larvae of oe. ovis is more prevalent among shepherds and ranchers of fars province (akbarzadeh 2012). there is only one comprehensive study on treatment of 33 cases of respiratory and non-respiratory manifestations of human pharyngeal myiasis in fars province (masoodi and hosseini 2003). intestinal myiasis or accidental myiasis is due to eating of food contaminated with egg or larvae of true flies (mandell et al. 2010). this kind of myiasis is very rare in iran and has the only two reports. sarcophaga haemorrhoidalis (diptera: sarcophagidae) and er. tenax cleared as responsible for these two cases (khalili et al. 2007, youssefi et al. 2010). urogenital myiasis is also not common and there are two case reports from iran. larvae of obligatory myiasis agents, ch. bezziana and wo. magnifica were observed in these two cases (jdalayer et al. 1978, salimi et al. 2010b). in addition to their medical importance as mechanical vectors of parasitic disease agents (kassiri et al. 2012) dipteran true flies can make myiasis in hospital environment called nosocomial myiasis (nazni et al. 2011). despite of low environmental sanitation in few hospitals, there are some features to make hospitalized patients easily accessed for flies (franza et al. 2006). all of the nosocomial myiasis infections in iran were nasal myiasis which occurred by facultative myiasis agents, lu. sericata (diptera: calliphoridae) and wohlfahrtia nuba (diptera: sarcophagidae) (mowlavi et al. 2011, maleki et al. 2012). after occurring of fatal nosocomial myiasis in one case in a hospital in tehran, this study was conducted to describe this case as a representative for this indifferent nosocomial infection. on the other hand all documented reports related with different kinds of myiasis disease in iran have been collected and analyzed with the aim of declaring the status of this dangerous disease and showing the research gap in this issue. materials and methods the information needed for this study inhttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 124–131 m alizadeh et al.: a review of myiasis … 126 cluding papers, case reports, case series and student dissertations were derived mainly from the digital library of tehran university of medical sciences. regardless of number of papers or scientifically reports, all reported cases of various kinds of myiasis from iran were counted and stratified in two ways, based on anatomical and parasitological features. the data was analyzed based on the number of cases, age grouping, gender, percent of death and of nosocomial infection, and spatial distribution of cases. a case of nosocomial myiasis infection from one hospital in tehran has been described as a representative sample of more underreported cases of such disease in hospitals. the larvae which originated from this case were sent to the dipterology laboratory, department of medical entomology and vector control in tehran university of medical sciences. some of the larvae has preserved in 70% ethanol and remained alive. live larvae placed in the jar on a piece of fresh cow meat to complete the larval cycle and emerge the adults for confirming identification of samples. identification of larvae has been done using the key of zumpt (1965). results total number of myiasis cases in all published documents from iran was 77. based on the clinical/anatomical features, it is cleared that majority of cases (52%) were oral myiasis (fig. 1). categorizing of age groups of reported cases shows that there are two peaks (fig. 2). i.e. most of the patients were between 21–40 years old (41.2%), while another peak indicates people with more than 61 years old. despite of near 6.5% of non-identified/ not-reported agents, majority of the myiasis cases were due to oe. ovis (diptera: oestridae) (65%) (fig. 3). human myiasis reports have been documented from 16 out of 31 provinces of iran. it is cleared that more than 62% of all cases are reported from fars province (fig. 4). analysis of the documented cases based on gender showed 80.5% were male and 19.5% were female. about 3.9% of all cases were nosocomial infections in which one case (1.3%) has been reported as fatal nosocomial myiasis. case presentation a 36 year old woman from zanjan province was referred to baharloo hospital in tehran with fever, weakness, general body pain, vomiting and vaginal secretions at the end of 2012. she remarked that the symptoms have started since 6 weeks ago. she had iud but few days before admission in hospital, it had been removed. in physical examination, pale conjunctiva was observed and systolic heart murmur was auscultated. severe mitral regurgitation without vegetation was reported in her echocardiography. other significant paraclinical results were: hb: 8, wbc: 14000, crp: 93 and esr: 84. after a few days of starting empirical antibiotic therapy for covering both sbe/pid (sub acute bacterial endocarditis/pelvic inflammatory disease), and while complementary diagnostic process was planning, she started to clinical improvement and fever was discontinued. in order to correcting of anemia, she received packed cell and several hours later, she fell down in progressive respiratory distress and then respiratory arrest occurred (probably due to pulmonary edema or transfusion related acute lung injury), therefore cpr was performed and after intubation she transferred to icu. after one month of admission, while she was in comatose state and she had sepsis with hospital acquired gram negative organisms, during suction process, crawling fly larvae were observed from pharynx and tracheal trunk. she died one day later. the larvae were sent to the department of medical parasitology, tehran unihttp://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 124–131 m alizadeh et al.: a review of myiasis … 127 versity of medical sciences, for further investigations and identification. the agent of this case was lu. sericata (diptera: calliphoridae). some of the basic characters which have been obvious in the sampled larvae were completed perithremal ring, absence of accessory oral sclerite on cephalopharyngeal skeleton, absence of short process on dorsal and lateral surface on the larval body (zumpt 1965). fig. 1. number of reported cases in iran divided by anatomical features fig. 2. age groups of reported myiasis cases in iran fig. 3. dipteran species which have reported as myiasis agents in iran fig. 4. spatial distribution of documented myiasis cases in provinces of iran discussions clinically, myiasis may be categorized as based on the involved tissues or cavitites of body organs (mandell et al. 2010). literature review showed that except of furuncular myiasis, all clinical forms of the disease have reported to be originated from various parts of iran. myiasis agents can be categorized as obligatory, facultative and accidental forms (mullen and durden 2002). all parasitological kinds of myiasis disease have been reported from iran. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 124–131 m alizadeh et al.: a review of myiasis … 128 nosocomial myiasis is not common even in endemic areas (sinclair et al. 2011) but most of these rare cases will not report officially because of some reasons (joo and kim 2001). based on this study the rates for nosocomial myiasis infections in iran are about 4% and the proved mortality rate is 1.3%. most of hospital originated myiasis cases were due to facultative and accidental myiasis (joo and kim 2001). the agent of the case of this study was lu. sericata which is a facultative myiasis fly. however it is proved that nosocomial myiasis caused by facultative agents can be fatal (mowlavi et al. 2011). a questionnaire based study in fars province cleared that more than 88% of the target community who are involved in sheep and goat breading industries were infected at least one time with oral myiasis (akbarzadeh et al. 2012a). based on high number of people involving with these industries, it cleared that total oral myiasis cases in iran would be more than the 77 reported cases in literature. oestrus ovis is usually known as the agent of oral myiasis. about 65% of all reported cases in iran were produced by this species. it can be resulted that this fly can be mentioned as the common human myiasis agent in the country. various climatic condition in different parts of iran has provided suitable conditions for presence of various potentially myiasis agents. more than 62% of all myiasis reports were from fars province. this issue may be due to comprehensive studies with actively search for this disease and its agents (akbarzadeh et al. 2012a,b, akbarzadeh 2012) as well as a valuable study on treatment of 33 oral myiasis cases (masoosdi and hosseini 2003) in this province. conducting synchronized studies in various parts of iran may change the features of geographical distribution of myiasis cases in iran. neither human nor animal myiasis diseases have been defined in governmental reporting systems of iran. therefore the exact status of both diseases, their distribution in the country and their causative agents remain obscure (akbarzadeh et al. 2012a, akbarzadeh 2012). a few reasons can be mentioned for this gap even in scientific literature of iran. there are no more emphasizing on definition of myiasis diseases and identifications on their causative agents in medical colleges. due to high volume of basic knowledge which should be learnt by medical students, the basic information about myiasis disease remained unconsidered. identification of myiasis agents (dipteran larvae) are more difficult especially for the larvae of sarcophagidae (except of wo. magnifica and wo. nuba) as well as some species of muscidae (amendt et al. 2010, pinto et al. 2011). temporary rearing is essential for precise identification of the myiasis agents. this process is not possible in pathological laboratories and need subdivision cooperation between them and entomological laboratories in medical universities which is not occur in most cases. conclusion results of this review will provide useful information about distribution of myiasis agents in iran for preventing the disease in man and animals. improving new vision to this disease, conducting new efforts for situation analysis and providing suitable programs for control and treatment of the disease is highly recommended. acknowledgement the authors wish to thanks to ms neda mirsepahi for her kind cooperation and also want to make gratitude to lab technicians and nurses who helped us in accomplishing this work. the authors also wish to thanks dr aa hanafi-bojd for kindly editing of the manuscript. the authors declared that there is no conflict of interest. http://jad.tums.ac.ir published online: april 09, 2014 j arthropod-borne dis, december 2014, 8(2): 124–131 m alizadeh et al.: a review of myiasis … 129 references akbarzadeh k (2012) estimation of geographical distribution, biodiversity and species richness of myiasis inducing flies in fars province. 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http://jad.tums.ac.ir published online: august 31, 2013 original article molecular characterization of leishmania infection from naturally infected sand flies caught in a focus of cutaneous leishmaniasis (eastern iran) mohammad akhoundi 1, 2, ahmad baghaei 2, *jérôme depaquit 1, parviz parvizi 2 1université de reims champagne-ardenne, anses, ea4688-usc «transmission vectorielle et épidémiosurveillance de maladies parasitaires (vecpar)», faculté de pharmacie, département de la parasitologie, reims, france 2pasteur institute of iran, department of parasitology, molecular systematic laboratory, tehran, iran (received 15 feb 2012; accepted 16 march 2013) abstract background: cutaneous leishmaniasis due to leishmania major is a serious and increasing problem affecting many rural areas of 17 out of 31 provinces in iran. little is known about sand fly fauna and leishmaniases in eastern iran and no study has been carried out in sarbisheh county. the aim of this study was to determine sand flies composition and probable leishmania infection to find the probable vectors of leishmaniasis in sarbisheh district. methods: sand flies were caught using both sticky papers and cdc light traps in august 2010. they were identified morphologically and analyzed for leishmania infection by amplification of its-rdna. results: totally, 842 specimens were caught and 8 species recorded. they belonged to the genera phlebotomus and sergentomyia: p. (phlebotomus) papatasi, p. (paraphlebotomus) sergenti, p. (pa.) caucasicus, p. (pa.) mongolensis, p. (pa.) jacusieli, s. (sergentomyia) dentata, s. (se.) sintoni and s. (sintonius) clydei. all collected females were processed for leishmania dna detection by pcr amplifying of internal transcribed spacer1 (partial sequence), 5.8s (complete sequence) and its2 (partial sequence) fragments. thirteen females were positive for leishmania dna. the sequencing of the 430 bp amplicons indicated that 9 p. papatasi and 3 females belonging to the caucasicus group carried l. major dna whereas one p. sergenti carried l. tropica dna. conclusion: phlebotomus papatasi and p. sergenti are, like in several places, the probable vectors of cutaneous leishmaniases in this emerging or unknown focus of cutaneous leishmaniases. keywords: leishmania major, leishmania tropica, its-ribosomal dna, iranian sand fly introduction cutaneous leishmaniases (cl) due to leishmania major yakimoff and schokhor, 1914 and l. tropica wright, 1903 (kinetoplastida: trypanosomatidae) occur serious increasing health problems in iran, affecting mainly rural areas in 17 out of 31 provinces in iran (yaghoobi-ershadi 2012). the most important foci are due to l. major. they are endemic and are located in turkemen sahara and lotf abad in north east of iran, abardejh, esfahan and yazd districts in center of iran, fars and sistan v baluchestan provinces in south and south east, ilam and khuzestan provinces in south west of the country (nadim and seyedi-rashti 1971, javadian et al. 1998, yaghoobi-ershadi et al. 2005, nekouie et al. 2006, oshaghi et al. 2010). in iran, as in many foci located all over the world, the main vector of l. major is p. papatasi scopoli, 1786 (nadim and seyedirashti 1971, yaghoobi-ershadi et al. 2005, parvizi and ready 2008). however, other species belonging to the subgenus phlebotomus or females belonging to the caucasicus group have also been reported as vectors of l. major, the latter especially as secondary vectors in areas where p. papatasi is not recorded, or at low densities (nadim and *corresponding author: prof jérôme depaquit, email: jerome.depaquit@univ-reims.fr 122 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 seyedi-rashti 1971, yaghoobi-ershadi et al. 1994). l. tropica is mainly transmitted by p. sergenti parrot, 1917 (oshaghi et al. 2010). despite the report of several new foci of cutaneous leishmaniases in iran and the potential spread of the disease in the country according to the iranian authorities, leishmaniases were unknown in the province of khorasan-e-jonoubi till now. according to the report of the ministry of health, 417 new cases of cl were reported from khorasan-ejonoubi in 2009. an increase of 4% was recorded in 2010 compared to 2009 (iran ministry of health and medical education 2010). in the present study, phlebotomine sand flies were sampled in sarbisheh county bordering afghanistan (fig. 1). according to our knowledge, no entomological or epidemiological study has been carried out in this region. the aim of this study was to carry out a pilot study on the sand fly species composition in the prospected area and to have a look on their leishmania infection. materials and methods phlebotomine sand flies sampling was carried out from 18th to 27th august, 2010, in sarbisheh county in east of iran, 58˚48' n and 32˚34' e, at an altitude of about 1800 meters above sea level (fig. 1). the climate in this area bordering afghanistan is mild and dry. the highest temperature in summer, 40 degrees above zero and the lowest in winter, 23 degrees below zero. the normal annual rainfall is 228 mm in sarbisheh county. during the collection period, the average minimum and maximum temperatures were 31˚c and 39 ˚c while the mean humidity was 54%. in this cross-sectional study, sampling was done in rural regions of sarbisheh county during 10 summer nights. we selected two catching methods: sticky papers and cdc miniature light traps. the goal of our study was not to collect data of the relative abundance of the species, that could be done by using sticky papers, but to mix two trapping methods in order to maximize the opportunity to catch all the species from a prospected location, photophilic or not. sticky papers consist of white sheets 21x 29.7 cm coated with castor oil placed in different habitats and various biotopes: indoors, animal shelters, and outdoors (scuppers, wall cracks, burrows, and vegetation). they were put on the ground with a stick, or rolled into cones and placed in the interstices of stone walls, in walls made of clay, or placed vertically in cracks, crevices and large boulders. cdc miniature light traps were put in the same locations. all these traps were installed before sunset and remained functional throughout the night until the next morning. sand flies specimens were stored in 96% ethanol and kept in refrigerator (-20 ˚c) for further analysis. after recording the sampling data and locations, sand fly specimens were washed in 1% detergent then in sterile distilled water. each specimen was then dissected in fresh drop of sterile normal saline by cutting off the head and genitalia with sterilized entomological needles, then were mounted in berlese medium and identified using the identification key of theodor and mesghali (1964). we considered the females of p. caucasicus marzinovsky, 1917 and p. mongolensis sinton, 1928 indistinguishable (artemiev and neronov 1984, parvizi et al. 2010). all these females were identified as “caucasicus group”. thorax, abdomen, legs and wings were stored in the sterile 1.5 ml microtube, then frozen and defrosted twice to break up tissue using a sampler tips or pestle, with grinding mix. then sds mix was used to denature proteins associated with the dna, and then ice cold 8 m potassium acetate was added to effectively remove the sds-bound proteins from solution. cell debris and proteins were separated from the dna by centrifugation and the dna in 123 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 the supernatant was precipitated over night at -20 ˚c in 96% ethanol. following ethanol precipitation, the dna was dissolved in 1xte (10 mm tris–hcl, 1 mm edta ph 8.0) and stored at 4 ˚c. leishmania dna was detected in phlebotomine sand flies by amplification of the first internal transcribed spacer of the ribosomal dna (partial sequence), 5.8s ribosomal rna gene (complete sequence) and internal transcribed spacer 2 (partial sequence) that able to detect l. major and l. tropica. this fragment was amplified using the forward (its1f) and reverse (its2r4) primers (parvizi and ready 2008). the length of pcr band was 430 bp for l. major and l. tropica. double distilled water and dna from l. major and l. tropica were used as negative and positive controls for each batch of pcr. standard pcr was performed in a 45 µl volume using extracted dna solution 5 µ l, 10x buffer 4 µ l, mgcl2 2.4 µ l, dntps 4 µ l, taq polymerase 0.4 µ l, ddw 28.6 µ l and 0.3 µ l from each forward (its1f: 5′gcagctggatcattttcc-3′) and reverse (its2r4: 5′-atatgcagaagagaggaggc-3′) primers and according to the pcr thermocycler program for leishmania parasite (parvizi and ready 2008). amplicons were analyzed by electrophoresis in 1.5% agarose gel containing ethidium bromide. pcr products were sequenced both directions directly using the its1f and its2r4 primers which used for dna amplification. sequences compared to homologous sequences in genbank thanks to the nucleotide-nucleotide basic local alignment search tool (blast: www.ncbi.nlm.nih.gov/blast). strains were considered as identified at the species level when their sequence showed ≥99 % homology with a sequence deposited in genbank. sequences were also aligned with bioedit v7.0.0 software (hall 1999) for comparison. results a total of 842 sand flies were collected: 574 by sticky papers and 268 by cdc miniature light traps (table 1). five species belong to the genus phlebotomus: phlebotomus (phlebotomus) papatasi scopoli, 1786, p. (paraphlebotomus) sergenti parrot, 1917, p. (pa.) caucasicus marzinovsky, 1917, p. (pa). mongolensis sinton, 1928 and p. (pa.) jacusieli theodor, 1947 and also 3 species belong to the genus sergentomyia: s. (sergentomyia) dentata sinton, 1933, s. (se.) sintoni pringle, 1953 and s. (sintonius) clydei sinton, 1928 (fig. 2). the captured specimens by sticky papers show that p. papatasi is the most abundant species in this area (33%) followed by p. sergenti (22%) and s. sintoni (13%). the other species have relative abundances less than 10% (table 1). the genus phlebotomus (77.9%) is more abundant than the genus sergentomyia (22.1%). the sex ratios show more males than females: 1.1 for the genus phlebotomus and 1.9 for the genus sergentomyia. the majority of these caught female specimens are unfed and gravid. of 268 specimens caught by cdc miniature light traps, the most prevalent sand fly species was p. papatasi (45%) followed by p. sergenti (26%). the sex ratios show more females than males (1.05) for the genus phlebotomus and fewer females than males (0.75) for the genus sergentomyia. most of the sand flies were collected from outdoor places (463: 55%) and in animal shelters (320: 38%). a few specimens have been caught indoors (59: 7%): p. papatasi, p. sergenti, caucasicus group and s. sintoni. of the specimens caught outdoors, 21 p. caucasicus/caucasicus group, 19 s. clydei, 21 s. sintoni and 14 s. dentata were captured in rodent burrows. the females consisted of 368 out of 842 (43.7%) caught specimens. nine out of 151 (6%) female p. papatasi and three out of 60 124 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 (5%) female caucasicus group were infected by l. major and one out of 90 (1.1%) female p. sergenti tested for leishmania dna was also positive for l. tropica infection using the standard pcr method. each one of these specimens produced 430 bp band (fig. 3). there was not any infected sample of female sergentomyia sand flies in this study. fig. 1. villages prospected in sarbisheh county (eastern iran) fig. 2. phlebotomus jacusieli caught in sarbisheh. female pharynx (a) and spermathecae (b), male coxite and style (internal view) (c) 125 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 table 1. caught samples of sand fly from different rural regions and various habitats of sarbisheh county in eastern iran county village h ab it at t ra p t yp e t ot al phlebotomus paraphlebotomus sergentomyia p. papatasi p. sergenti p. caucasicus p. mongolensis caucasicus group p. jacusieli s. sintoni s. dentata s. clydei ♂ ♀ ♂ ♀ ♂ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ sarbisheh gongan a s 6 5* 4 1 3 3 1 _ _ 4 2 1 1 2 1 34 c 2 4 2 3 _ _ 1 _ _ 1 1 _ 2 _ _ 16 h s 2 1 1 _ _ _ _ _ _ 2 _ _ _ _ _ 6 c 1 1 1 _ _ _ _ _ _ _ _ _ _ _ _ 3 o s 9 6* 4 3 2 3 3 2 _ 6 2 2 1 2 1 46 c 2 5 2 4 1 2 _ _ _ 1 1 _ 3 1 _ 22 zusk a s 5 6 4 5 2 1 2 _ _ 2 1 1 1 1 1 32 c 2 _ 3 3 _ _ 1 _ _ 1 _ _ 1 1 1 13 h s _ 1 2 1 _ _ _ _ _ 1 _ _ _ _ _ 5 c 1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1 o s 8 4 6 7 2 4 3 2 1 4 1 2 _ 2 _ 46 c 4 4 4 3 2 2 1 _ _ _ 1 1 1 _ _ 23 mud a s 6 3 2 3 3 2 2 _ _ 3 2 _ _ 2 2 30 c 3 2 3 2 _ _ _ _ _ 1 1 _ 1 _ 1 14 h s _ _ _ _ _ _ _ _ _ 2 _ _ _ _ _ 2 c 2 1 _ _ _ _ _ _ _ _ _ _ _ _ _ 3 o s 7 4 6 3 2 3 4 2 _ 2 3 2 1 3 1 43 c 5 5 2 3 1 1 1 _ _ 1 1 1 _ _ _ 21 janat abad a s 5 3 5 3 1 2 2 _ _ 2 1 1 1 1 1 28 c 4 7 2 2 _ _ 1 _ _ _ _ 1 _ 1 _ 18 h s 3 2 2 _ _ _ 1 _ _ _ _ _ _ _ _ 8 c 1 _ _ 2 _ _ _ _ _ _ _ _ _ _ _ 3 o s 8 5* 6 5 1 3 3 _ _ 3 2 3 1 2 _ 42 c 3 5 2 3 1 1 1 _ _ 1 _ 1 _ _ _ 18 sarbisheh a s 4 6 3 1 2 2 2 _ _ 3 1 1 _ 1 2 28 c 3 3 3 2 1 _ 1 _ _ 1 _ 1 _ _ _ 15 h s 3 3 1 _ _ _ _ _ _ 1 _ _ _ _ _ 8 c 1 1 1 1 _ _ _ _ _ _ _ _ _ _ _ 4 o s 9 4 5 6** 1 2 3 _ _ 4 2 1 2 2 1 42 c 2 5* 3 2 1 1 2 _ _ 2 _ 1 _ 1 _ 20 salm abad a s 5 4 4 1 _ _ 3 _ _ 3 1 2 _ 2 _ 25 c 3 4 2 1 1 1 1 _ _ 1 1 _ _ _ _ 15 h s 3 _ _ 1 _ _ 1 _ _ 1 _ _ _ _ _ 6 c 1 _ 1 _ _ _ _ _ _ _ _ _ _ _ _ 2 o s 4 7* 5 6 2 2 6* 1 1 3 2 1 1 2 1 44 c 3 5* 2 2 1 1 1 _ _ 2 _ _ _ _ 1 18 asadieh a s 7 9* 3 2 1 1 4* _ _ 2 1 2 1 _ 1 34 c 4 8 1 _ 1 1 2 _ _ _ _ _ _ _ 1 18 h s 2 2 _ _ _ _ 1 _ _ 1 _ _ _ _ _ 6 c 1 1 _ _ _ _ _ _ _ _ _ _ _ _ _ 2 o s 11 9* 8 7 2 3 5* 2 1 4 1 2 1 2 1 59 c 6 6* _ 2 1 1 1 _ _ 1 _ _ _ 1 _ 19 total 161 151 105 90 35 42 60 9 3 66 28 27 19 29 17 842 a: animal shelter, h: house, o: outdoor, s: sticky paper, c: cdc miniature light trap *: includes specimens infected by l. major **: includes specimen infected by l. tropica 126 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 fig. 3. gel electrophoresis profile of the standard pcr based amplification products. the bands correspond to molecular weight marker (lane1), control negative (lane 2), reference strains of l. major (lane 3) and l. tropica (lane 4), p. papatasi (bir-733: lanes 5 and bir-803: lane 6), caucasicus group (bir-211: lane 7, bir-341: lane 8 and bir-624: lane 9) and p. sergenti (bir-797: lane 10) discussion little is known about sand fly fauna in khorasan-e-jonoubi province. several investigations have previously been carried out on the sand flies fauna in khorasan district e.g. in meshed, lotf abad, esfarayen and neishabur counties (mesghali et al. 1967, nadim et al. 1971, javadian et al. 1976, nadim and tahvildar-biruni 1977). nadim et al. (1971) reported a list of sand fly species caught in the mountains and plains of khorasan district. they consisted of 12 species of phlebotomus and 9 sergentomyia. out of the mentioned sand flies, some species were found only in the mountains and the caves (p. major, s. pawlovskyi). others species were found both in mountains and in flat areas: p. mongolensis, p. caucasicus, p. mofidii, p. ansarii, p. kandelakii in the north khorasan, p. kazeruni, p. eleonorae, s. christophersi, s. tiberiadis and s. mervinae near the central desert in the south of the central desert in the south of khorasan district. nadim and seyedi-rashti (1972) have studied the sand fly fauna of the western part of the khorasan district and recorded nine phlebotomus and 10 sergentomyia species: p. papatasi, p. sergenti, p. caucasicus, p. alexandri, p. kazeruni, p. jacusieli, p. eleonorae, p. major, p. chinensis, s. sintoni, s. dentata, s. mervynae, s. grekovi, s. sumbarica, s. squamipleuris, s. pawlovskyi, s. clydei, s. tiberiadis and s. christophersi. moreover, p. papatasi, p. sergenti, females belonging to the caucasicus group (p. mongolensis and p. caucasicus), s. clydei and s. sintoni have been found infected by promastigotes (nadim and seyedi-rashti 1972). our findings are in accordance with the previous investigations carried out in this district (nadim et al. 1971, nadim and seyedi-rashti 1972). since the 70’s, no investigation has been carried out in this province and the sarbisheh county has never been prospected. nine p. papatasi females and three females belonging to the caucasicus group carried l. major dna according to the blast process. the sequences obtained from the present study have been deposited in genbank under accession numbers jn541326 to jn541337. these are identical or highly sim127 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 ilar to several sequences deposited in genbank, including isolates from iran and friedlin references (friedlin fr796423, iran ay260965, ay283793, ef413075, gq402544, gq402543, gq466354 and gq466350). the sequence of its-rdna obtained from the female p. sergenti (accession number: jn541338) is similar (99% homology) to those l. tropica that previously have been deposited in genbank from two regions of iran: shiraz (hm060588, hm060589, hm060590) (oshaghi et al. 2010) and kaleybar (eu604811, eu604813) (parvizi and ready 2008). investigation on the reservoirs of zoonotic cutaneous leishmaniasis (zcl) was previously carried out by seyedi-rashti and nadim (1967) in two regions of khorasan district: meshed (and its suburbs) and lotf abad. they mentioned rhombomys opimus as the main reservoir of cl in the endemic focus of the rural type in lotf abad region. later, rodent reservoirs of cl were studied in meshed, lotf abad, sarakhs and esferayen districts (nadim and seyedi-rashti 1972, javadian et al. 1976) and rhombomys opimus was again reported as the main reservoir. the reservoirs of zcl in khorasan-e-jonoubi province due to insufficient studies in mentioned rural regions are not very clear. it seems that r. opimus is the main reservoir in this region as well as khorasan district. in our study, 3 infected specimens by l. major have been captured in rodent burrows. two of them were p. papatasi and the third one belonged to the caucasicus group. sarbisheh county is located in the north of sistan v balouchestan province, in border of afghanistan. this area is separated from the central parts of iran by deserts (dasht-e-kavir in north-east and dasht-e-loot in south-east). several investigations have been carried out in sistan and balouchestan both on vectors and reservoirs of cutaneous leishmaniases in some regions of this province (kassiri et al. 2011a, 2011b, 2012). kassiri et al. (2011a) reported five species as proven (p. papatasi) or probable (p. salehi, p. sergenti, p. alexandri and p. keshishiani) vectors of cutaneous and visceral leishmaniasis in this province. they showed the role of p. papatasi and p. salehi in maintenance and transmission of l. major to humans and reported meriones hurrianae and tatera indica as the probable reservoirs of zcl (kassiri et al. 2011b, 2012). to our knowledge, no study has been carried out on the leishmaniases in the province of khorasan-e-jonoubi. however, it seems that illegal immigration with low sanitary conditions occurs from afghanistan and pakistan, two countries where cl are endemic (bhutto et al. 2009, ruiz postigo 2010) to sarbisheh county. consequently, there is a potential risk of leishmaniasis outbreak in this area, according to the presence of numerous p. papatasi and p. sergenti, in this area. several investigations show various ratios of females p. papatasi infected by l. major in following districts: 19.8% in shiraz (oshaghi et al. 2010), 15.6% in badrood (yaghoobiershadi et al. 2005), 6.5% in baft (oshaghi et al. 2008), 11% in damghan (rassi et al. 2011), 12.5% in shahrood (abaei et al. 2007), 22.1% in abardejh (nekouie et al. 2006), 12.7% in rafsanjan (yaghoobi-ershadi et al. 2010) and 2.1% in chabahar (kassiri et al. 2012). our findings indicates 9 out of 151 (6%) p. papatasi carrying l. major dna, constitute a relatively low level. concerning the females belonging to the caucasicus group, 4.2 to 7.5% of the specimens were infected by l. major in several studies carried out in borkhar, ahar, damghan and shahrood districts (yaghoobiershadi et al. 1994, rassi et al. 2004, abaei et al. 2007, rassi et al. 2011). in the present study, 3 out of 60 (5%) of females belonging to caucasicus group have been found positive for l. major dna, ranking these specimens in the mean of reported range in the mentioned areas of the country. 128 j arthropod-borne dis, december 2013, 7(2): 122–131 m akhoundi et al.: molecular characterization of … http://jad.tums.ac.ir published online: august 31, 2013 cutaneous leishmaniasis due to l. tropica occurs in a vast distribution areas of the world. its cycle varies between localities and generally does not require a sylvatic reservoir. several surveys report the isolation of l. tropica from p. sergenti, its classical vector (al-zahrani et al. 1988, oshaghi et al. 2010). however, in the middle-east, an atypical focus transmitted by p. (adlerius) arabicus with reservoirs (hyraxes) has been recorded very close to classical focus (svobodova et al. 2006). in eastern africa, the subgenus larroussius can also been implicated in the transmission of l. tropica (lawyer et al. 1991). in iran, l. tropica cl was first described in tehran by schlimmer several decades before the discovery of the parasite. some of the most important foci of this disease are tehran in center, meshed in north-east, shiraz and kerman in south of iran (nadim and seyedi-rashti 1971). our pilot study reports a sand fly inventory showing that p. papatasi and p. sergenti are abundant species. the detection of l. major and l. tropica from the latter shows their local role in the transmission of the disease in emerging or previously unknown foci of cutaneous leishmaniases. there is lack of knowledge in the prospected county about the local reservoirs and human prevalence and incidence. we suggest starting new studies at different periods of the year in 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focus of zoonotic cutaneous leishmaniasis in central iran. j vect ecol. 30(1): 115–118. yaghoobi-ershadi mr, javadian e, tahvildarebidruni gh (1994) the isolation of leishmania major from phlebotomus (paraphlebotomus) caucasicus, in isfahan province, islamic republic of iran. trans r soc trop med hyg. 88(5): 518–519. 131 j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 50 http://jad.tums.ac.ir published online: march 14, 2017 original article effect of climatic conditions and water bodies on population dynamics of the dengue vector, aedes aegypti (diptera: culicidae) *shabab nasir 1, farhat jabeen 1, sadia abbas 1, iram nasir 2, mustapha debboun 3 1department of zoology, government college university, faisalabad, pakistan 2department of statistics, government college university, faisalabad, pakisatn 3harris county public health and environmental services, houston, tx, usa (received 13 feb 2015; accepted 21 nov 2015) abstract background: the population of mosquitoes is mainly influenced by the biotic and abiotic factors. although aedes aegypti was reported until 1960’s in the punjab, pakistan, the population has increased dramatically since 2009 and caused severe epidemics in 2011 due to heavy floods and rains. thus, this study was carried out to know the effect of biotic and abiotic factors on the population of aedes aegypti. methods: mosquitoes were collected from fresh, sewage, and rain water ponds, fish ponds, rice fields, tyres, tree holes, and manmade storage containers present in and around residential homes twice during every winter (october– february), summer (march–june) and monsoon season (july–september) from marked rural areas. results: more mosquitoes were collected in 2010 and 2011 due to floods than other years with heavy rains. high population (52.4%) was recorded during the rainy season due to high temperature (28–36 °c) and high relative humidity (up to 75%), while low population was recorded during the winter due to low temperature (< 5 °c) and low relative humidity (< 22%). specimens were recorded indoors when outside temperature was below freezing point. ae. aegypti was largely collected from tyres and urban areas mostly during the rainy season from small water containers. years, months, seasons, temperature and relative humidity were statistically significant concerning the population dynamics of mosquitoes. conclusion: abiotic factors (temperature & relative humidity) along with habitat have significant impact on population dynamics of mosquitoes. keywords: population dynamics, climatic conditions, dengue vector, aedes aegypti introduction mosquitoes act as vectors for many vector-borne diseases (vaughn et al. 2009) such as malaria, dengue and yellow fever (snow et al. 2005). they are found all over the world except in antarctica (gadahi et al. 2012). anopheles, culex and aedes are three medically important genera of mosquitoes. aedes mosquitoes are recognized as daytime biters, widely distributed in more than 100 countries of the world (gratz 2004). aedes aegypti and ae. albopictus are two main mosquito species involved in dengue transmission to humans (knudsen 1995). dengue was a sporadic disease during the previous century. however, currently it has become a major health problem throughout the world due to the modern means of transportation, trade, population growth, mobility and environmental change. twenty two thousand deaths are reported annually worldwide mainly in children and young adults (haile-mariam and polis 2009, vaughn et al. 2009). recent epidemic of dengue in pakistan has been attributed to heavy rains and floods, deterioration of health, and sanitation principles at all stages of the community (anonymous 2011). in 1994, dengue fever (df) and dengue hemorrhagic fever (dhf) were first rec*corresponding authors: dr shabab nasir, e-mail: flourenceshabab@yahoo.com j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 51 http://jad.tums.ac.ir published online: march 14, 2017 orded in karachi, pakistan (chan et al. 1994). in 2011, about two decades later from its first record in pakistan, dengue appeared in its epidemic form in the punjab province with 100 confirmed cases daily (khawar 2011). the hotspot of the epidemic was the capital city lahore where more than 131 people dead and over 12,000 people infected according to the punjab health department (anonymous 2011). mosquitoes have a well-adjusted life, particularly by inhabiting quiet pools, underground buildings, and seepage areas near any bodies of water. water bodies are different from each other in respect to chemical properties of water (acidity or alkalinity, freshness, salty or brackish) and the type or amount of vegetation present in them (foster and walker 2002). thus, there is no doubt some mosquito species use more than one kind of habitat. however, generally many mosquitoes can be grouped according to their preference for water bodies such as permanent, flood, temporary or container water (vanwambeke et al. 2007). since dengue fever is a viral infection, and no vaccine or treatment is available for people, entomologists are key researchers who had better understand the vector and its life cycle to develop an integrated mosquito management program (rathor 1996, gratz 2004). the study of the biology of the vector along with the climatic conditions suitable for his life cycle is vital for entomologists who help in the timely application of mosquito management system against dengue vectors (sharma et al. 2005). container location and capacity, water source and temperature, all can vary seasonally. aedes aegypti breeds in standing water generally in artificial containers. the amount of rainfall, temperature, and relative humidity are very important climatic factors that affect the population of aedes mosquitoes especially ae. aegypti (scott et al. 2000). due to these reasons, we decided to explore the role of the climate, water bodies (size and location), physical and chemical properties of water, environmental abiotic factors and distance from human population on the population dynamics of ae. aegypti. materials and methods this study was conducted from 2009 to 2013 involving the collection of ae. aegypti from rural and urban areas of the punjab (31.0000° n, 72.0000° e and 139m above sea level) (anonymous 2014) and identification of key ecological factors affecting the population dynamics and seasonal abundance of ae. aegypti. punjab is the most populous province with an area of 79,284 square miles. almost 70% of its population consists of farmers who live in villages or around the big cities. its boundaries meet with jammu and kashmir in the northeast, indian punjab and rajasthan in the east, province sindh in the south, baluchistan and kpk (khyber pakhtunkhwa) in the west and islamabad and azad kashmir in the north. it lies on the margin line of the monsoon climate. its climate is mixed, some areas are too cold like murree (up to -4 °c) and some are hot like multan (up to 47 °c). the average annual precipitation is low, but sometimes-heavy rains occur due to monsoon airs during july–september that become the cause of heavy floods (anonymous 2015). mosquitoes were collected randomly from forests, fields, parks, and residential areas (in and around the houses). since ae. aegypti prefers the small artificial containers, we collected from buckets, waste water near houses, seepage pools, dairy and poultry farms (harrel et al. 2011), used tyres, broken vases, bottles, irrigation channels, cemented tanks, bamboo sticks (bashar et al. 2005), tyre shops, and tree holes (akram and lee 2004). adults were collected with aspirators and collection nets, while larvae and pupae with plastic dippers. j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 52 http://jad.tums.ac.ir published online: march 14, 2017 seasonal abundance collection (adults with aspirators and collection nets, while larvae and pupae with plastic dippers) was done seasonally to examine the population fluctuations in different areas of the punjab, pakistan (fig. 1). therefore, the year was divided into three quarters based on seasons, i.e. summer (march to june), rainy or monsoon (july to september) and winter (october to february) (mohiuddin 2007). global positioning system (gps) was used to record the height from sea level and coordinates (longitude and latitude) of marked sample sites during the survey. temperature, relative humidity and ph were measured with a multi-meter (elmetro poland, model # cpc 401) while the location of sites were recorded with magellan gps (explorist, 660). after the adult mosquitoes were killed using the poison bottle, the genera were separated and counted at the site to identify the seasonal abundance (reisen and milby 1986). the larvae were placed in 500ml plastic jars filled with water from the source to keep them alive during transport to the vector biology laboratory, government college university and faisalabad, pakistan and reared in rearing cages. after the adults emerged they were killed, pinned, and identified to species level with the help of available taxonomic keys (barraud 1934, darsi and pradhan 1990). specimens were identified using the microscope (nikon binocular microscope eclipse, model # e-100). biotic and abiotic factors biotic (flora and fauna present in the water) and abiotic factors including the physical condition of water (clear, turbid, and foul), ph, temperature and relative humidity (rh) (akram and lee 2004, harrel et al. 2011) have been sampled from each site. we also recorded the location of each site as urban or rural and the distance of each site from the residential areas. after anova, chisquare test was applied on significant factors and then logistic regression was applied on highly significant factors effecting the population. statistical analysis we used mathematica 7 to analyze the data for chi-square value to determine the relationship between different variables affecting the population of ae. aegypti. logistic regression model was also used for significant factors from chi-square test to identify the key factors affecting the population of ae. aegypti. a stepwise forward approach was applied, for this model (vanwambeke et al. 2007). results aedes aegypti mosquitoes were collected from different rural and urban areas of the central punjab (lahore, faisalabad, sargodha, sheikhupura and gujranwala) and upper punjab (rawalpindi, islamabad and murree) but not from northern punjab (multan, dera ghazi khan, bahawalpore and rahim yar khan). they were mostly sampled from latitude (max 033º 45.403, min 29º 57.859) and longitude (max 074º 45.403, min 072º 57.859) with elevation (max 1218m, min. 144m). maximum population was collected from lahore during the rainy season and minimum from faisalabad during the winter season. populations of ae. aegypti from 810 water container sites, 83 sites (10.25%) were positive for aedes mosquitoes. populations of ae. aegypti showed a great variation in their density during all seasons (p-value= < 0.001). maximum population (52.4% of the total collected population) was collected during the rainy season followed by the summer season (41.7%) while the lowest percentage (5.9%) was found during the winter. similarly, maximum population (42.7%) was recorded during 2011 due to heavy rains j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 53 http://jad.tums.ac.ir published online: march 14, 2017 followed by 35% in 2010, likely due to heavy floods in the province. water quality had significant influence on population density (p-value< 0.001). most populations were collected from turbid water (46.1%) followed by turbid foul (33.8%), clear (17.4%) and clear foul (2.7%). in addition, most of the population (97.8%) was found in standing water than in flowing water (2.2%). color of the water was also significant (p= 0.04) in relation with population density since most of the population of ae. aegypti was found in colorless medium (29.6%). in addition, light had a significant (p< 0.002) impact on population density with 60.0% population exposed in shady medium (60.0%) (table 1). there were more breeding sites during 2010 and 2011 than other years due to more rainfall and floods. aedes aegypti was mostly collected from small containers such as buckets or cans and used tyres near or around residential areas (fig. 2). abiotic factors had a significant impact on the population of ae. aegypti. more rain fell in 2011 resulting in an increase of its population. thus, more rainfall in 2011, heavy floods in 2010, and the increase of the relative humidity in these years resulted in an increase of the population of ae. aegypti and more numbers were collected (fig. 3). in addition, more breeding sites were observed during the rainy seasons, particularly in 2010 and 2011. a little effect was observed on the population dynamics of ae. aegypti because it was mostly collected from artificial water containers that were mostly found in the residential areas (fig. 2). habitat of ae. aegypti the majority of ae. aegypti population was sampled in artificial containers (44.8% in tyres, 37.8% in tanks and cans). the remaining 17.4% has been collected in natural containers such as water bodies and none from natural tree holes (fig. 2). locality also showed a significant effect on the population level (p= 0.019). the population was recorded from urban areas (78.1%) than rural (21.9%). among the flora, algae, grass, spirogyra, dragonflies, damselflies, frog tadpoles, and water bugs were recorded but we did not assess their role. logistic regression analysis the seasons had a significant role in ae. aegypti population dynamics as seen in the rainy season (or: 0.287, p= 0.003) than the summer (or: 0.052, p= 0.005). location also had a significant effect on the future population dynamics of this mosquito as found in the rural environment (or: 0.496, p= 0.048) (table 2). table 1. key parameters for the presence of aedes aegypti in punjab area, pakistan, 2009–2013 characteristics population of aedes aegypti p-value 2009 2010 2011 2012 2013 % age seasons ˂0.001 winter 5 7 12 7 6 5.9 summer 37 61 72 46 41 41.7 rainy 49 75 92 57 52 52.4 habitat ˂0.001 tyres 38 66 79 49 44 44.8 water bodies 18 24 29 19 17 17.4 others (tree holes, cans, tanks and any other) 35 53 66 42 38 37.8 water quality ˂0.001 clear 18 23 30 19 17 17.4 turbid 41 67 81 51 46 46.1 j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 54 http://jad.tums.ac.ir published online: march 14, 2017 turbid foul 30 49 59 37 33 33.8 clear foul 2 4 5 3 3 2.7 water condition 0.100 standing 89 141 169 108 97 97.8 flowing 2 2 5 2 2 2.2 light ˂0.002 exposed 27 43 59 35 31 31.6 exposed shady 57 87 101 66 60 60.0 shady 7 13 14 9 8 8.4 ph 0.370 ≤ 7.00 0 0 0 0 0 0 7.01–8.00 15 34 43 25 22 22.5 8.01–9.00 57 79 93 62 56 56.2 ˃9.01 19 30 38 23 21 21.3 area size (m) ˂0.001 ≤ 1.00 55 78 89 60 54 54.4 1.01–10.00 3 6 8 5 4 4.1 10.01–100.00 2 3 4 2 2 2.2 ˃ 100 31 56 73 43 39 39.3 location 0.019 urban 67 106 146 86 78 78.1 rural 24 37 28 24 21 21.9 temperature (°c) ˂0.001 ≤ 10 3 8 12 6 6 5.6 11–20 3 10 29 11 10 10.3 21–30 20 35 60 31 28 28.2 31–40 63 87 70 59 53 53.9 ˃41 2 3 3 3 2 2.0 relative humidity (%) ˂0.001 ≤ 30 4 4 5 4 3 3.2 31–40 20 33 40 25 22 22.8 41–50 29 47 67 39 35 35.0 ˃51 38 59 62 42 39 39.0 distance from houses (m) 0.160 ≤ 25 72 109 135 85 77 77.5 26–100 18 31 34 22 20 20.3 ˃100 1 3 5 3 2 2.2 table 2. logistic regression as predictor for the presence of aedes aegypti, punjab, 2009–2013 variables odds ratio (or) 95% confidence interval of or p-value lower upper seasons summer 0.052 0.069 0.437 0.005 rainy 0.287 0.195 0.798 0.003 light exposed 0.593 0.197 1.899 0.395 exposed shady 0.292 0.101 0.865 0.33 turbid water 0.998 0.998 1.454 0.067 location rural 0.496 0.259 1.106 0.048 distance from houses ≤ 25 8.097 2.098 15.90 0.001 26-100 16.097 3.978 78.09 0.012 table 1. continued… j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 55 http://jad.tums.ac.ir published online: march 14, 2017 fig. 1. location of selected districts of the punjab for the collection of aedes mosquitoes, 2009–2013 fig. 2. number of aedes aegypti collected from different visited habitats in punjab, 2009–2013 j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 56 http://jad.tums.ac.ir published online: march 14, 2017 fig. 3. the relationship between abiotic factors during different seasons from 2009–2013 and the population of aedes aegypti discussion during this study, the population of ae. aegypti was collected from 83 sites (out of 810) and showed a great variation during the entire year with the majority collected during the rainy season rather than the summer one. small breeding habitats around or inside the residential areas, old tyres and exposed shady water pools were the sites chosen and preferred by ae. aegypti. water temperature ranging from 21 to 38 °c and a relative humidity higher than 35% supported the population density. however, the maximum population density was observed at 32–38 °c with relative humidity of about 50%. aedes aegypti population was generally more supported in the urban environment than the rural one. other scientists who also studied the distribution patterns according to seasons had similar results as ours showing that the cool season put a greater stress on the survival of adults and larvae than on the eggs (preechaporn et al. 2007). population density was flexible throughout the year depending on the larval breeding habitats. rainfall plays an important role in the population density of aedes (ndiaye et al. 2006). our results showed that aedes mosquitoes were collected during the rainy as well as summer season and these circumstances were mostly found in areas where the elevation was higher and ecological conditions were mild (alto and juliano 2001, preechaporn et al. 2007). in bangladesh, mosquitoes were collected from all sampling sites in the rural areas (bashar et al. 2005). the domestic water storage containers (drinking water jars, buckets, and tanks) followed by tyres were the regular productive containers in our study area. the number of containers primarily filled with rainwater increased during the rainy season, generally in the used tyres. therefore, this is a possible reason why ae. aegypti population did not reach low levels at a time when rainfall was limited. as the people manage j arthropod-borne dis, march 2017, 11(1): 50–59 sh nasir et al.: effect of climatic … 57 http://jad.tums.ac.ir published online: march 14, 2017 these containers, they also manage the production of mosquitoes in these containers unintentionally. these results are in agreement with the results of previous studies (koenraadt et al. 2004, barbazan et al. 2008). mainly, a few key types of containers play important role for a large proportion of the pupal and adult production (koenraadt et al. 2004, barbazan et al. 2008). however, the control measures such as placement of lids on water containers, use of larvicides or biological control agents, removal of discarded and unused containers have decreased the mosquito population density. container capacity, water temperature, source of water, and container location, all of which could vary seasonally (koenraadt et al. 2004, kay and nam 2005) have been reported as key ecological factors affecting population of ae. aegypti. a study in samoa island revealed that the most productive containers during the dry and wet seasons were the buckets and used tyres (lambdin et al. 2009). during this study, they also noted that the knowledge of environmental variables such as habitat density, water source, seasonal variations and neighborhood conditions played a significant role in container productivity. the typical rainfall system in the punjab, normally during july–august was associated with a peak of aedes population. a better understanding of the environmental factors affecting the water container productivity is a key part of adaptive vector control efforts (scott and morrison 2008). work is being carried out in tropical regions to investigate the effect of source reduction on the population of ae. aegypti (arunchalam et al. 2010). we can understand the population dynamics of ae. aegypti more consistently and design a better effective vector control program by integrating the knowledge of breeding site density and the ecology of larval breeding sites. conclusion environmental factors and breeding sites had a strong influence on the population dynamics of ae. aegypti mosquito. although the population was recorded during the entire year but most of the population was recorded during the rainy season. in addition, man’s activities had a strong relationship 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dengue, dengue hemorrhagic fever, japanese encephalitis, west nile encephalitis, st. louis encephalitis, tick-borne encephalitis). in: mandell gl, bennett je, dolin r (eds) principles and practice of infectious diseases, 7th ed. elsevier churchill livingstone, philadelphia, pp. 2133–2156. chap. 153. j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 original article molecular epidemiological study of cutaneous leishmaniasis in the focus of bushehr city, southwestern iran *mohammad reza yaghoobi-ershadi 1, farideh shahbazi 1, mohammad darvishi 2, amir ahmad akhavan 1, reza jafari 3, mohammad khajeian 2, yavar rassi 1, hassan soleimani 4, mohammad reza shirzadi 5, ahmad ali hanafi-bojd 1, hossein darabi 6, mohammad hossein arandian 3, alireza sanei-dehkordi 1, mansour heidari 7 1school of public health, tehran university of medical sciences, tehran, iran 2deputy of health services, bushehr university of medical sciences, bushehr, iran 3esfahan health research station, national institute of health research, esfahan, iran 4yazd health research station, national institute of health research, yazd, iran 5communicable diseases management center, ministry of health and medical education, tehran, iran 6the persian gulf tropical medicine research, bushehr university of medical sciences, bushehr, iran 7department of medical genetics, school of medicine, tehran university of medical sciences, tehran, iran (received 8 june 2013; accepted 4 aug 2013) abstract background: cutaneous leishmaniasis (cl) represents the most frequent vector borne parasitoses in iran. the objective of this study was to determine the epidemiological features of cl including human infection and the reservoir host in the city of bushehr, bushehr province, iran during 2010–2011. methods: studies on human infection was carried out on 2962 school children aged 7–14 years old from 60 primary schools and among 400 households with a total population of 1568 in four infected districts of the city in december 2010. serosity materials from patients on glass slides were collected for molecular identification of causative agent. rodents were caught by sherman traps and examined for identification of the parasite. results: prevalence of scars and ulcers among the inhabitants were 5.86% and 0.12% respectively. molecular study indicated the presence of two coexisting species: leishmania major and l. tropica among patients. the scar rate was 1.24% but no ulcers were seen among the students. nineteen rodents were caught and identified as tatera indica (47.4%) and rattus norvegicus (52.6%). specimens from 7 t. indica and 9 r. norvegicus were examined by two techniques, microscopic examination and nested-pcr. out of 7 t. indica, 14.3% were infected with l. major and 42.9% with l. turanica by nested-pcr. out of 9 r. norvegicus 22.2% were infected with l. turanica and 11.1% with l. gerbilli. conclusion: based on this survey l. major and l. tropica are the causative agents of the disease among patients and t. indica plays a predominant role in the dissemination of l. major in the city. keywords: molecular epidemiology, cutaneous leishmaniasis, l. major, l. tropica, tatera indica introduction cutaneous leishmaniasis (cl) is the first most important vector borne disease at present in iran (yaghoobi-ershadi 2010, 2012). it has been neglected as a major public health problem because it is not a fatal disease. it is still the cause for considerable morbidity of a vast number of people in the endemic foci and characterized by chronic skin lesions followed by permanent scars and deformation of the infected area (yaghoobi-ershadi 2002). for only this reason in urban and rural areas it is politicized and health authorities are usually questioned on the matter in the islamic parliament of iran. *corresponding author: dr mohammad reza yaghoobi-ershadi, e-mail: yaghoobi.reza@gmail.com 113 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 cutaneous leishmaniasis has been epidemic during the years 1988, 1997 and 2008 in the city of bushehr (health center of bushehr province, unpublished data). it is one of the free trade-industrial zones of the country and the bushehr nuclear power plant which is unique in terms of its technology in the middle east is located 12 km, southeast of the city along the persian gulf, so lots of people travel around and some make several trips in a year for business. if the disease does not receive considerable attention by the health authorities, it may spread into other parts of the country which are free from cl. however the epidemiological aspects of cl have not been examined in the city yet and there is no accurate data on the prevalence, reservoir (s) and vector(s) of the disease. the objective of this study was to determine for the first time the epidemiological features of cl including human infection and the reservoir hosts in the city during 2010–2011. materials and methods study area the city of bushehr located in a plain running along the coastal region on the persian gulf coast of southwestern iran and is the administrative center of its province. field studies were carried out over a period of 22 months (from september 2009 to end of june 2011) in the city of bushehr (latitude: 28˚ 55̕ 30˝ n, longitude 50˚ 50̕ 17˝ e, altitude: 5 m above sea level) (fig. 1). the city had a population of 221016 in 2011, while this was 133753 in 1991 with an increase about two folds in the last two decades. the area has a hot desert climate though it does receive more rainfall than most cities on the persian gulf. the rain is confined to the period from november to may, when temperature is pleasantly mild and is extremely erratic. the long summer from april to october is brutally hot, humid and completely rainless. in 2010, the maximum and minimum mean monthly temperature was 39 and 12.1 °c in august and february respectively, and the total annual rainfall was 4.29 mm with a minimum of 0.1 mm in may and 2.45 mm in february. the minimum mean monthly relative humidity was 58 % (december) and the maximum was 74 % in january (bushehr meteorological organization, unpublished data). population studies the study was carried out in two population groups: (1) school children and (2) the population of the infected parts of the city (to obtain data on human infection rate in all age groups). for the first group, a list of all the elementary schools was obtained from the department of education. one hundred and twenty classes were selected by cluster sampling technique. each class was visited and in each class, a list was prepared from all the school children and they were questioned and examined for the presence of ulcer(s) or scar(s). for each case having ulcers or scars a form was completed to record the necessary information such as name, address, age, sex, number of ulcers or scars, site of ulcer(s) or scar(s), date and place of acquiring the disease, etc. smears were prepared from scraping of the edge of the ulcer fixed in methanol, stained with giemsa and examined under a light microscope for the presence of amastigotes. serocity materials from some cl patients on glass slides were used for molecular identification of causative agent. all the school children were visited in december 2010. for the second group, four infected districts of the city were selected, called tangak in the south, sangi, in the city center, chaharmahalleh in the north and imamzadeh in the southwest of the city. one hundred households from each district whose buildings were located near each other were visited and all members of the households 114 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 examined by coincidence of visiting schools in the selected parts of the city. the λ2 test was used to determine any statistical significant difference in disease prevalence between males and females of school children and inhabitants of the infected districts. collection and examination of rodents colonies of rodents were identified and caught using 35 sherman traps baited with cucumber and dates monthly. in the laboratory they were identified by morphological characters (etemad 1978) then regardless of the presence of lesions, impression smears were prepared from the ear lobes of the animals (edrissian et al. 1982, mohebali et al. 2004) fixed in methanol and stained by the standard giemsa method, and examined carefully under the light microscope (1000 x) for detection of leishmania amastigotes during september and december of 2011. sample collection twenty specimens were collected from suspected cl patients who were referred to the health center of bushehr. the tissue samples from patients and rodents were placed in labeled micro tubes, stored in 70% ethanol (pbs) at -20 °c until further examination. dna extraction the smears from suspected cases of leishmaniasis were used, all the slides were washed with absolute ethanol and after drying washed three times in cold sterile pbs (ph 7.2). the smear on the slides was scrapped off and collected in the 1.5 ml microtubes. genomic dna was extracted and purified using qiagen extraction kit (qiagen, germany, cat.no.69504) according to the manufacturer’s manual with the minor modification of increasing incubation time to increase the yield of dna in the final step. dna was stored at -20 °c until analysis. before submitting the tissue from rodents to the dna extraction procedure described for the slides, the tissue was subjected to 13 freeze/ thaw cycles, using liquid nitrogen and boiling water, to disrupt the tissues, and treated as described above. the concentration of extracted dna was measured by nanodrop (thermo fisher scientific, usa). molecular assays primer design for amplification of its2 primers designed previously and used to amplify a 230 bp product in l. major, a 215 bp product in l. tropica, a 206 bp in l. gerbili and a 141 bp in l. turanica across the internal transcribed spacer 2. the external primers, leish out f (5′-aaa ctc ctc tct ggt gct tgc-3′) and leish out r (5′-aaa caa agg ttg tcg ggg g-3′), and internal primers, leish in f (5′-aat tca act tcg cgt tgg cc-3′) and leish in r (5′-cct ctc ttt ttt ctc tgt gc3′) were selected to distinguish among the parasite species in a nested pcr system (akhavan et al. 2010a). nested-pcr we used nested-pcr to identify the leishmania species. conditions and parameters for pcr were as previously described with the minor modification (akhavan et al. 2010). all samples were tested in 25 µ l amplification reaction mixtures with 12.5 µ l of the master mix (taq dna polymerase, 2x master mix red, amplicon, germany), 1.8 µ l of primers( each primer: 10 pmol), 10.7 µ l h2o, and 1 µ l of template dna (20 ng). the first-round pcr was performed based on the following conditions: initial denaturation at 95 °c for 5 min; followed by 35 cycles including denaturation at 95 °c for 30 s, annealing at 56 °c for 30 s, and extension at 72 °c for 45 s, and a final extension at 72 °c for 5 min. the second-round (nested) pcr was performed as the same first round exception for annealing at 58 °c for 30 s. at the end, 10 μl of the reaction mix was analyzed by 2.5% agarose gel electrophoresis. 115 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 additionally, for all pcr reactions one negative control without dna and one positive control with standard dna were included to confirm the results of two rounds of nested-pcr. the pcr products of the negative and positive controls of the firstround pcr were used as negative and positive controls in the second round (respectively). finally, 10 µ l of the pcr products were loaded on 2.5% (w/v) agarose gels, and stained with ethidium bromide to visualize by electrophoresis. initially, itspcr was confirmed with standard dna of reference strains l. major (mrho/ir/75/er), l. gerbilli (mrho/cn/60/gerbilli) and l. turanica (mrho/su/1983/marz-051) l. tropica (mhom/ir/o4/mash10) as positive controls and distilled water were used as negative controls (akhavan et al. 2010a, 2010b). rflp-pcr analysis the results of nested-pcr were confirmed by enzymatic analysis using the mnl1 enzyme. pcr products (20 μl) were digested with mnli 2 μl at 37 °c for 4 h without prior purification using conditions recommended by the supplier (fermentas life sciences, germany). the restriction fragments were subjected to electrophoresis in 3% agarose gel containing ethidium bromide for 3 h at 65v and visualized on a uv transilluminator. results altogether, 60 primary schools with 2962 school children (1533 boys and 1429 girls) from 7 to 14 years of age were visited in december 2010. the overall scar rate was 1.24% but no ulcers were seen among them (table 1). this means a medium endemicity of the disease which has recently become endemic, otherwise a higher scar rate should be observed. in children with scar, 81.9% were recorded with only one and 18.1% with two scars. hands, legs, and face were the most affected parts of the body with 34.9, 34.9 and 27.9% of scars respectively. a study of prevalence among 400 households with a total population of 1568 in four infected districts of the city showed 5.86% for scars and 0.12% for ulcers. the scar rate was higher than what was seen for school children. the scar rate was 3.92% for individuals under 10 years of age and 6.16% for those above 10 years old. males and females were equally infected. out of 38 individuals with scar cases, 2 had contracted the disease in shiraz and one in khuzestan (south of the country), one in gilan-e-gharb (western iran) and the rest in the city of bushehr. prevalence of ulcers among the inhabitants was 0.12 % and the infected age group was 25+ with a rate of 0.24%. no active case was seen less than 24 years of age. a 45 year old man presented with approximately 3 month history of ulcer on the right lower leg. he was staying in the district of imamzadeh, south of the city. the other case was also 45 year old with 6 month history of ulcer on his right hand and he was staying at the same district of the city. both of them had contracted the disease in 2010 in bushehr city. out of 13 specimens, 2 specimens were positive by microscopic examination. leishmania dna was found in 8 specimens collected from 13 (61.5%) suspected cl patients. the visualized obtained bands in 2 infected specimens of human indigenous cases were similar to the standard strain of l. major, which was equal to 231 bp and in 6 specimens were similar to the standard strain of l. tropica, which was equal to 215 bp (fig. 2). treatment was provided for the 8 subjects with molecular diagnosis of leishmaniasis. nineteen rodents were caught and identified as tatera indica (47.4%) and rattus norvegicus (52.6%). specimens from 7 t. indica and 9 r. norvegicus, captured from the city were examined by two diagnostic techniques, direct (microscopic) examination and nested-pcr. all specimens were negative by 116 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 direct examination but out of 7 t. indica, 14.3% were infected with l. major and 42.9 % with l. turanica by nested-pcr. out of 9 r. norvegicus 22.2% were infected with l. turanica and 11.1% with l. gerbilli. we also found mixed natural infections with l. gerbilli and l. turanica in 11.1% of r. norvegicus by the same molecular method (table 2, fig. 3–5). the study showed that t. indica acts as the animal reservoir host in the city of bushehr. fig. 1. map of the city of bushehr, showing the geographical location and study sites table 1. the prevalence of scar rate among the school children (both sexes) of primary schools in the city of bushehr, december 2010 age (yr) no. observed no. of scars % no. of ulcers % 7 665 8 1.2 0 0 8 520 2 0.38 0 0 9 622 4 0.64 0 0 10 618 16 2.58 0 0 11 519 3 0.57 0 0 12 15 4 26.6 0 0 13 2 0 0 0 0 14 1 0 0 0 0 total 2962 37 1.24 table 2. natural leishmania infection rates of rodents by nested-pcr in the city of bushehr, iran, sept. 2010–jan. 2011 rodent species no. of examined leishmania species l. turanica l. major l. gerbilli l. gerbilli + l. turanica r. norvegicus t. indica 9 7 22.2 (2/9) 42.9 (3/7) – 14.3 (1/7) 11.1 (1/9) – 11.1 (1/9) – figures in parentheses are numbers of positive/no of examined rodents and figures at the top of them are percent of positive` 117 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 fig. 2. nested-pcr amplification of dna extracted from reference strains and giemsa-stained smears lane m, 100 bp dna ladder (fermentas), lane 1–4, reference strains, leishmania major, leishmania gerbilli, leishmania turanica, leishmania tropica, respectively, lane 5, leishmania major isolated from giemsastained smears of human, lane 6, leishmania tropica isolated from giemsa-stained smears of human, lane 7, mixed infection of leishmania major and leishmania tropica isolated from giemsa-stained smears of human. fig. 3. nested-pcr amplification of dna extracted from rodents lane m, 100 bp dna ladder (fermentas), lane 1, leishmania turanica isolated from tatera indica (skin samples), lane 2–3, leishmania turanica isolated from rattus norvegicus (skin samples), lane 4–5, negative sample, lane 6, leishmania major isolated from tatera indica (skin sample), lane 7, negative sample, lane 8, leishmania turanica (reference strain), lane n, negative control(distilled water). fig. 4. nested-pcr amplification of dna extracted from rodents lane m, 100 bp dna ladder (fermentas), lane 1, reference strain, leishmania major, lane 2, leishmania major isolated from tatera indica (skin sample), lane 3, mixed infection of leishmania gerbilli and leishmania turanica isolated from rattus norvegicus (skin samples), lane 4, leishmania gerbilli isolated from rattus norvegicus (skin sample), lane 5, negative sample, lane 6, leishmania gerbilli isolated from rattus norvegicus (skin sample), lane n, negative control (distilled water). fig. 5. restriction products of nested-pcr amplicons after digestion with mnl1 lane m, 100 bp dna ladder (fermentas), lane1, leishmania major, lane 2, leishmania gerbilli, lane 3, leishmania turanica. 118 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 discussion a study based on our detailed observation of school children and also local people showed scars of cl in all age groups with a maximum of 8.9% among 15–19 years old indicated that the disease is prevalent with a low endemicity in the city of bushehr. the disease agents are l. major and l. tropica but in order to determine the dominant causative agent, more specimens from patients should be examined in the coming years. statistical analysis in the school survey and also community data (patients with scars) showed non-significant differences by sex (p< 0.005). it should be mentioned that there are some difficulties in using scars to assess past infection. scars may be missed, become less detectable through time or may have causes other than leishmaniasis. based on this survey at the present time the indian gerbil (t. indica) and the norway rat (r. norvegicus) are well established in the infected districts of the city. rattus norvrgicus exists in coastal areas near the sea, in garbage dumps and in sewer systems but t. indica whether is essentially a field rodent, occurs in sandy parts where the city of bushehr has extended its expanse. it is found near human dwellings under bushes and appears to be the main reservoir host of zoonotic cutaneous leishmaniasis in the city. 3.1% of the animals had l. major infection. this rodent species has been also reported as the main animal reservoir host in other foci of bushehr province such as dashti and dashtestan counties and also khuzestan, and ilam provinces with leishmanial infection rate between 2.3–9% in iran and also in subsaharan africa (javadian et al. 1998, hamzavi et al. 2000). gerbils infected by l. major having a great effect in the transmission cycle of cutaneous leishmaniasis due to l. major (clm) so it is important to accurately assess the rate of l. major infection in important reservoirs (gramiccia and gradoni 2005). the results of the current study shows that l. major and l. turanica are circulating in t. indica populations and l. turanica, l. gerbilli and also mixed natural infections with l. turanica and l. gerbilli in r. norvegicus populations of bushehr city. in previous studies l. turanica has been isolated in rhombomys opimus in sabzevar northeast of the country (yaghoobi-ershadi et al. 2004) and mixed natural infections with 3 species, l. major, l. gerbilli, and l. turanica in r. opimus in esfahan area in central part of iran (akhavan et al. 2010a, 2010b). leishmania turanica was also isolated from nesokia indica in an area lies on the border between iran and iraq in 2009 (hajjaran et al. 2009). leishmania turanica was also reported as the dominant species in r. opimus populations in hypoendemic, mesoendemic and hyperendemic foci of zcl in turkmenistan and uzbekistan in 2001 (srelkova 1996, strelkova et al. 2001). based on the results l. major and l. tropica are the causative agents of the disease among patients and t. indica plays a predominant role in the dissemination of l. major in the city of bushehr. the occurrence of cl in the city of bushehr seems to be the results of expansion of the city and urbanization, constructing of buildings nearby rodent colonies, increase of nonendemic people in south pars projects, bushehr military complex and the bushehr nuclear power plant. the people take a trip more than one month in summer during the active season of sand flies to other zoonotic cutaneous leishmaniasis or anthroponotic cutaneous leishmaniasis foci of the country and all are exposed to the infected bites of sand flies. any residual insecticide spraying is not recommended during the next year due to the low prevalence of the disease in the city. if the present epidemiological situation for 119 j arthropod-borne dis, december 2013, 7(2): 113–121 mr yaghoobi-ershadi et al.: molecular epidemiological … http://jad.tums.ac.ir published online: august 31, 2013 breeding of the vector are changed (example setting up big gardens in different parts of the city and so forth and the disease becomes epidemic in the future, deltamethrin 0.025 (g/m) spraying in the houses with active cases and also their neighboring houses and in districts with high density of phlebotomus sergenti or p. papatasi should be applied only once during the active season of sand flies and repeated once again two weeks before the beginning of sand fly activity in the next year. infected stray dogs should be found and eliminated if any. destruction of rodent burrows and using 2.5% zinc phosphide baits or coumavec (a mixture of coumatetralyl 0.5% and etofenprox 0.5%) within 500-meter circle of houses is recommended (yaghoobi-ershadi et al. 2000, yaghoobi-ershadi et al. 2005, veysi et al. 2012). active and passive case detection and rapid treatment should be provided for the positive subjects. intensive health education programs have to be strongly supported in order to promote awareness among the exposed population. regular epidemiological studies to address risk factors and transmission patterns are also 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10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 259 http://jad.tums.ac.ir published online: january 05, 2016 short communication clinicopatho-biochemical alterations associated with subclinical babesiosis in dairy animals amrita sharma, *lachhman das singla, ashuma, balvinder kaur batth, paramjit kaur department of veterinary parasitology, college of veterinary science, guru angad dev veterinary and animal sciences university, ludhiana, india (received 13 apr 2014; accepted 16 sep 2014) abstract background: present investigation is based on the clinicopatho-biochemical alteration related to natural tick borne bovine babesiosis in punjab state. methods: blood samples from jugular vein of 542 bovines (cattle 466 and buffaloes 76) having history of tick infestation, fever, hemoglobinurea or anemia were collected and tested for babesia bigemina by blood smear examination and pcr targeting 18s rrna gene to distinguish clinically and subclinically infected groups. further the hemato-biochemical parameters were correlated with the status of infection. results: overall, of the 542 samples tested 16.42 % were positive by pcr while only 1.66 % by blood smear examination. the trend of molecular prevalence was found to decrease from north-eastern towards western punjab. analysis of the hematobiochemical alterations showed significant decrease in the levels of rbc, hb, pcv, and mcv with significant increase in tbil, mch and mchc levels. conclusion: as the transmission of b. bigemina is transovarian, presence of even few infected rhipicephalus (boophilus) microplus ticks on a subclinically infection can be the nidus of infection for whole herd, causing severe economic losses, at the same time significantly affecting the physiology of carrier animal. keywords: babesiosis, dairy animals, haemato-biochemical, prevalence, pcr introduction bovine babesiosis, caused by babesia bigemina, is an important tick-borne disease in the tropical and subtropical counties, transmitted by rhipicephalus (boophilus) microplus. india suffers losses of about 57.2 million us dollars annually due to babesiosis and anaplasmosis in livestock (mcleod and kristjanson 1999). microscopic techniques like giemsa stained blood smear is the most appropriate for the diagnosis of acute babesiosis but the low sensitivity of these method do not permit to identify the carrier animals (juyal et al. 1994, almeria et al. 2001, singh et al. 2003). serological tests, though employed for epidemiological survey of babesiosis, lack discrimination between previous and current exposu re to infection (passos et al. 1998). molecular tests, such as pcr assays, are highly specific and sensitive method over the existing diagnostic techniques for the diagnosis of subclinical infections (figueroa et al. 1992). the molecular tests can depict the status of active infect in exposed animals and further their correlation with haematobiochemical parameters will provide an insight into the pathogenicity of subclinical infection primarily diagnosed by pcr assay. the present study was therefore undertaken to diagnose subclinical cases of babesiosis by pcr based molecular diagnosis in relation to the haemato-biochemical parameters to study the pathogenicity induced by latent infection of b. bigemina. materials and methods sampling frame the province of punjab covers a total *corresponding author: dr lachhman das singla, e-mail: ldsingla@gmail.com j arthropod-borne dis, june 2016, 10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 260 http://jad.tums.ac.ir published online: january 05, 2016 area of 50,362 square kilometers between 29"30’n to 32"32’n latitude and 73”55’e to 76”50’e longitudes. the study area was divided into three regions: western punjab (western and western plain zone), central punjab (central plain zone) and north-eastern punjab (submountain and undulating zone) (fig. 1). to study the status of molecular prevalence of the babesiosis caused by b. bigemina, the expected prevalence to be 50% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples was considered. the number of samples thus calculated was adjusted for finite population (thrusfield 2005) and correlated with 542 samples (cattle 466 and buffaloes 76) collected from month of may to october, 2011 from jugular vein of dairy animals having history of tick infestation, fever, hemoglobinurea or anemia from punjab state (india) also including the samples from the outbreaks of b. bigemina in central and north-eastern regions. about 5ml of blood was collected aseptically from the jugular vein of each animal in edta coated vials for dna isolation and serum separation, respectively. the extracted dna and collected sera were stored at -20 °c for further analysis. clinical and subclinical diagnosis animals diagnosed positive by giemsa stained thin blood smears technique were considered clinically positive cases. for subclinical diagnosis, dna was extracted using dna isolation kit (hipura blood genomic dna miniprep purification spin kit) as per the protocol of the manufacturer. the bg3/ bg4 set of oligonucleotide primer was used to amplify 18s ribosomal rna gene (ellis et al. 1992). the nucleotide sequence of the primer was bg3 5’ tagttgtatttcagc ctcgcg 3’ and bg4 -5’ aacatccaa gcagctahtttag 3’. the pcr reaction mixture (25µ l) mixture constituted of 12.5µ l of kapa 2g tm fast hot start ready mix (2x containing kapa2g fast hot start dna polymerase, kapa 2g fast hot start pcr buffer, 0.2 mm dntp each, 1.5 mm mgcl2), 3 µ l of 25 mm mgcl2, 1.5 µ l of 10 pmol bg3/bg4 primers, 1.5 µ l depc-treated water and 5 µ l of dna template. the reaction was carried on in automated thermocycler (eppendrof, master cycler personal) on the following programme: initial denaturation at 95 ˚c (5min), 30 cycles of denaturation at 95 ˚c (30 sec), annealing at 57 ˚c (1min), and extension at 72 ˚c (1.5 min) with final extension at 72 ˚c for 10 min. the amplified pcr products were separated by electrophoresis on 1% agarose gel and visualized under uv transilluminator for detection of 689 bp amplified product. haemato-biochemical analysis the hematology was done on advia 2120 hematology system (siemens health care diagnostic inc. deerfield, il, usa) and serum biochemical profile was drawn on vtros dt 6011 system chemistry using ortho-clinical diagnostics kit (johnson and johnson company). nucleotide sequence analysis the 689 bp product obtained by the bg3/ bg4 primers specific for b. bigemina pcr were custom sequenced from xcelris genomics, ahmedabad, india. the nucleotide sequences were subjected to blastn analysis (altschul et al. 1990) for determining the similarity with the sequences present in the nucleotide database. statistical analysis chi-square test was applied to evaluate association of disease prevalence with various districts under study. one-way analysis of variance (anova) was applied to various haematological and biochemical parameters to determine the variance in these parameters using spss software. agreement between the results of the two techniques was analyzed through cohen's kappa coefficient. j arthropod-borne dis, june 2016, 10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 261 http://jad.tums.ac.ir published online: january 05, 2016 results diagnosis of infection out of total 542 blood samples (466 cattle and 76 buffaloes), pcr based (fig. 2) diagnosis revealed 89 animals (16.42%, 95% ci= 13.73–19.1) positive for babesia that included 83 cattle (17.81%, 95% ci= 14.81– 20.81) and 6 buffaloes (7.89%, 95% ci= 2.66–13.12). the relative prevalence of b. bigemina by pcr amplifying 689 bp fragment for 18s rrna gene showed highest prevalence of the disease in north-eastern region (64.28%, 95% ci= 51.79–76.78) (fig. 1) and lowest in western region (3.89%, 95% ci= 1.45–6.32) of punjab. overall overall percentage of animals positive for b. bigemina b. bigemina by giemsa-stained thin blood smears was 1.66 % (9/542), the higher in cattle 1.73 % (8/463) than in buffaloes 1.31 % (1/76). chi-square test showed significant difference in the prevalence of the infection as revealed by blood smear examination and pcr. kappa contingency test indicated a slight agreement between blood smear examination and pcr assay with latter being highly sensitive for the detection of latent infections. nucleotide sequence analysis the 689bp sequence showed homology (99%) with b. bigemina isolate brco2 18s ribosomal rna gene (genbank no. fj 426361.1). haemato-biochemical alteration anaemia haematological parameters (fig. 2) showed significant (p< 0.05) decrease in the tec, hb, pcv and mcv, while significant increase (p< 0.05) in mch and mchc in clinically and subclinically infected animals as compared to non-infected healthy control group (fig. 3). liver function biochemical alterations a significant increase (p< 0.05) was seen in alkp and ast of clinically infected group while the increase as non-significant in subclinically infected animals as compared to non-infected control group. the increase in alt levels was non-significant (p< 0.05) in both the infected groups (fig. 4) when compared with the non-infected healthy controls. kidney function biochemical alterations there was no significant difference in the level of creatinine and bun in the three groups under study, however the level of total bilirubin had increased significantly in the infected groups as compared to non-infected group (p< 0.05) (fig. 5). blood cellular and biochemical response significant response in terms of decrease in wbc, tp, glo and plt was seen only in clinically infected animals (p< 0.05), while the alterations in albumin level was non-significant in both the infected groups as compared to non-infected control group (fig. 6). fig. 1. geographic distribution clinical and subclinical babesiosis of bovine babesiosis in punjab j arthropod-borne dis, june 2016, 10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 262 http://jad.tums.ac.ir published online: january 05, 2016 lane m, molecular size marker 100 bp plus, lane p positive control and n negative controls, lane a–d showing amplified b. bigemina genomic dna from the blood of animals positive for infection, lane e showing no amplification of b. bigemina genomic dna from the blood of animal negative for infection. fig. 2. agarose gel (1.5%) electrophoresis showing amplified dna (689bp) from babesia bigemina targeting ssu rrna gene using primer bg3/bg4 fig. 3. hematological alterations indication anemia in clinically and subclinically infected animals (rbc (red blood cells): 106cells/ µl, hb (hemoglobin level): g/dl, pcv (packed cell volume):%, mcv (mean corpuscular volume: fl, mch (mean corpuscular hemoglobin): pg, mchc (mean corpuscular hemoglobin concentration): g/dl). bars with different alphabets a, b and c differ significantly for the parameter j arthropod-borne dis, june 2016, 10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 263 http://jad.tums.ac.ir published online: january 05, 2016 fig. 4. liver function biochemical alterations in clinically and subclinically infected animals (alkp (alkaline phosphatase), ast (aspartate aminotransferase), alt (alanine aminotransferase) fig. 5. kidney function biochemical alterations in clinically and subclinically infected animals (crsc (cretinine), tbil (total bilirubin), bun (blood urea nitrogen) j arthropod-borne dis, june 2016, 10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 264 http://jad.tums.ac.ir published online: january 05, 2016 fig. 6. blood cellular and biochemical response in clinically and subclinically infected animals (wbc (white blood cells), tp (total protein), glo (globulin), alb (albumin), plt (platelets) discussion in the clinically positive (9) cases the marked clinical symptoms of high fever (39.4–40.5˚c), history of hemoglobinurea, jaundice, icterus, tachycardia, weight loss and decrease in milk yield were recorded. the blood smear of these animals revealed typical pyriform bodies of b. bigemina at an acute angle inside the erythrocytes. apart from the typical forms other pleomorphic forms b. bigemina were also observed. previously, parasitological prevalence of babesiosis due to b. bigemina in cattle is reported to be 5.94% in punjab (aulakh et al. 2005). however, the status of subclinical infection is essential to be drawn as this kind of infection can be the source of infection to other animals of the herd as well as may flair in the condition of stress. thus, it has been observed that the molecular approaches based on nucleic acids such as polymerase chain reaction (pcr) assays offer greater sensitivity and specificity over the existing diagnostic tests (fahrimal et al. 1992) which proved to be true in the present study also. the trend of geographic distribution of babesiosis can be corroborated with the availability of favorable conditions of high humidity and soil moisture content (ghai et al. 2008) and higher population of tick vector, r. microplus (singh et al. 2000), in northeastern parts are punjab as it covers the sub mountain and undulating zones. ticks were observed on 78.6% of the affected cattle and 9.4% of the affected buffaloes. population of the ticks was mainly constituted by r. (boophilus) microplus followed by hyalomma anj arthropod-borne dis, june 2016, 10(2): 259–267 a sharma et al.: clinicopatho-biochemical … 265 http://jad.tums.ac.ir published online: january 05, 2016 natolicum anatolicum, correlating the fact that incidence of b. bigemina is a tick borne pathogenesis. above all the major fraction of sampling was done from cattle and they primarily considered susceptibly to the infection (vohara et al. 2012). findings of hematobiochemical profiling indicate that the subclinical infection of babesiosis in bovines is also responsible for causing anemia. the anemia was normo to hyperchromic and microcytic type. marked anemia has been documented in clinical cases of bovine babesiosis in previous studies (sharma et al. 2000, aulakh et al. 2005) also. a number of factors contribute to anemia in babesiosis including lyses of erythrocytes by emerging parasites (dwivedi et al. 1976), indiscriminate phagocytosis of infected and non-infected erythrocytes by activated macrophages system (ruprah 1985), suppression of erythopoitietic activity of bone marrow (pandey and misra 1987) and antigen antibody mechanism (ferris et al. 1967). non-significant difference was seen in glucose level among the three groups, when compared with the normal range (42–75 g/dl) (latimer et al. 2005). the apparent increase in the levels of ast, alt, alkp, bun and creatinine signifies harmful effect of toxic metabolites of babesia sp. on liver cells lead to impairment and alterations of the liver and kidney enzymes. hussein et al. 2007 reported the significant increase in aspartate aminotransferase, alanine aminotransferase (alt) and gamma glutamyltransferase (ggt) in babesiosis. a significant increase in total bilirubin in subclinically infected group hemolytic crisis of babesiosis (panday and misra 1987) and hepatic damage (yeruham et al. 2003) was seen. overall, the degenerative changes in the internal organs as indicated by biochemical response may be due to anemic hypoxia. infiltration of circulating immune complexes may further complicate the problem (aulakh et al. 2005). conclusion the comparison of prevalence of bovine babesiosis by pcr and giemsa stained thin blood smear in dairy cattle of punjab, revealed highest prevalence in north-eastern region of punjab by both the techniques. as the transmission of b. bigemina is transovarian, the presence of rather few infected ticks on even a subclinically infected animal may acts as nidus of infection for the whole herd, posing severe economic losses and pathognomic effects on the animal as indicated by altered vital parameters contemplated though significant decrease in the level of rbc, hb, pcv, and mcv with significant increase in tbil, mch and mchc levels. acknowledgements thanks are due to the dean, college of veterinary science and director of research, guru angad dev veterinary and animal sciences university for providing financial support to carry out the research work under rkvy 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http://jad.tums.ac.ir published online: january 05, 2016 anand districts of gujarat, india. vet world. 5: 223–224. yeruham i, avidar y aroch i, hadani a (2003) intra-uterine infection with babesia bovis in a 2-dayold calf. j vet med b infect dis vet public health. 50: 60–62. microsoft word 5-dr khoobdel.doc iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 28 original article arthropod borne diseases in imposed war during 1980-88 *m khoobdel1, a mehrabi tavana1, h vatandoost2, mr abaei2 1health research center, baqiyatallah university of medical sciences, iran 2departement of medical entomology and vector control, school of public health, tehran university of medical sciences, iran (received 15 sep 2007; accepted 9 feb 2008) abstract background: personnel of military forces have close contact with natural habitat and usually encounter with bite of arthropods and prone to be infected with arthropod borne diseases. the imposed war against iran was one of the most important and the longest war in the middle east and even in the world and military people faced various diseases. the aim of this study was to review prevalence of arthropod borne diseases and to collect relevant information and valuable experiences during the imposed war. methods: the present survey is a historical research and cross-sectional study, focused on arthropod fauna, situation of different arthropod borne diseases and also the ways which military personnel used to protect themselves against them. the information was adopted from valid military health files and also interviewing people who participated in the war. results: scabies, cutaneous leishmaniasis, sandfly fever and pediculosis were more prevalent among other arthropod –borne diseases in iran-iraq war. measures to control arthropods and diseases at wartime mainly included: scheduled spraying of pesticides, leishmanization and treatment of patients. conclusion: although measures used during the war to control arthropods were proper, however, due to needs and importance of military forces to new equipment and technologies, it is recommended to use deltamethrin-impregnated bed net, permethrin treated military uniforms and various insect repellents in future. keywords: arthropods, iran, iraq, war, insects, leishmania, scabies introduction military forces face to arthropod borne diseases all over the world because of their missions, type of activities and their frequent contact with insects in comparison with other people (debboun et al. 2001, faulde and uedelhoven 2006). all of the armies in the world face to some species of insects and related diseases (deparis et al. 2004), dependent on zoogeographical regions and also arthropods fauna there, thus they have different strategies to protect their military forces from damage of arthropods (barnard 2000). for example, in french army, diseases such as malaria, dengue fever and cutaneous leishmaniasis are important (deparis et al. 2000, meynard et al. 2001) but african, american and european army forces which serve in african region, in addition to malaria and leishmaniasis also involve yellow fever, trypanosomiasis and onchocerciasis (fall et al. 2001). incidence rate of leishmaniasis among french military forces that were resident in new guinea was 3.2% in 1999 (lightburn et al. 2002). in south american countries also malaria, leishmaniasis, yellow fever, and lymphatic filariasis are important (souza-santos and car*corresponding author: dr m khoobdel, e-mail: khoobdel@yahoo.com, fax: +98 21 88057023 iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 29 valho 2000). foreign militaries that enter to these regions face to such diseases, for example australian soldiers that were in east timor, introduced dengue fever to australia after return back (kitchener and leggat 2002). in iran, several arthropod borne diseases are endemic, because it is located at two zoogeographic regions, i.e. oriental and palearctic (service 1996). among arthropod borne diseases, malaria, cutaneous and visceral leishmaniasis, scabies, pediculosis, papatasi fever and scorpion sting are significant in iran (khoobdel et al. 2003). armies have strategies to recognize arthropod borne diseases that exist specially in their regions and in addition to identifying species and fauna of arthropods, they study about ecology of arthropods to have better control (guessan et al. 2001, faulde et al. 2003, 2006). for instance, french armies have developed some procedures to protect military forces from arthropod borne diseases including: use of military clothes (uniforms) which are permethtin-impregnated and using insect repellents on exposed skin and utilization of deltamethrin-impregnated tents (deparis et al. 2004). during the imposed war against iran, soldiers faced with several arthropod borne diseases. lack of facilities and occurring some problem, it was not feasible to provide such a perfect study in that time. this study aimed at reviewing of the state of arthropod borne diseases and control methods during imposed war using available documentary information and interviews with health personnel, to retrieve useful information and highlight the problems and future needs of iranian military forces to keep away from arthropod borne diseases. materials and methods in this descriptive cross-sectional and historical study, information of wartime from several sources was accumulated. furthermore, we interviewed health personnel that combated at northwest and west of iran about our research subject. west battle field comprised of war regions of 4 west provinces of iran including ilam, kermanshah, kurdistan and west azerbaijan. at first, we developed a questionnaire including 70 questions about several aspects of arthropods and related diseases as vectors. then we used from documentary sources and verbal history for completion of questionnaires. finally information accumulated from these two sources, classified separately and compared with each other. x2 test was used for data analysis. documentary sources sources used in this research included the reports and files about several aspects of arthropods and to assess quality of health personnel action in west war regions. these documents have remained since wartime. the other documentary sources included letter reports of health revenue. there is one transcript at army files now. others included student thesis and scientific articles printed in iranian journals and articles presented in seminars. in addition, study pictures and films existed in several file-keeping units were used. verbal history sources in interview section, statistical and target society included all of health personnel that were active during the war especially at last 3-4 yr of war. we interviewed with all 80 possible members of the population mentioned above and we did census. results during iran-iraq war, the most important problems or diseases due to arthropods were included: scabies (30%), cutaneous leishmaniasis (24%), sandfly fever (15%), mosquito bites (10%), pediculosis (9%), malaria (8%) and others (4%). iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 30 as a whole, 87 active persons at war time were found through introduction of health personnel. among them we interviewed with 46 persons (88%) of west war region health personnel. the average of their attendance in heath affairs of west war regions was 4.5 yr. their academic degrees were mostly diploma and junior college (75% of cases) at wartime. with assessment of files or articles and other documents, only 53 questions (%76) were replied in interviews. according to various evidences arthropods were serious problems to soldiers and diseases such as malaria, leishmaniasis, scabies, sandfly fever and scorpion sting were prevalent. furthermore, prevalence of infestation to flea, louse, bug and other insects, especially at warm seasons has occurred. during the war, identification of medically important arthropods was not perfected completely. discussion malaria malaria is endemic in iran and local endemic regions of that exist at east regions of iraq with common boundary along with iran. despite of that, during the war fortunately, only 100 cases of malaria have reported among iranian soldiers (mehrabi tavana and purtagi 2000). in west and south west war regions, 9 species of anopheles mosquitoes have been identified comprising 5 known vector species of malaria in iran as follows (maleki 1985): anopheles stephensi an. superpictus an. sacharovi an. maculipennis an. dthali leishmaniasis according to the information in the files and data obtained from interviews, cutaneous leishmaniasis at west war regions especially mehran, dehloran, sumar and marivan was prevalent. in 1989, 80% of soldiers that fought in dehloran region had cutaneous leishmaniasis (ranjbar kermani 1989). after war, studies showed that incidence of leishmaniasis in mehran region was 220 per 1000 (dehestani 1994). the type of cutaneous leishmaniasis at west war regions was probably zoonotic cutaneous leishmaniasis (zcl) (ranjbar kermani 1989, dehestani 1994). several species of sandflies in kermanshah, javan rood, paveh, dehloran and marivan regions have been identified (vahabi 1998). leishmania parasites have been isolated from two species, phlebotomus papatasi and p. alexanderi. the reservoirs of cutaneous leishmaniasis in west war regions were rodents such as nesokia and tatera. in mehran region, tatera indica is reservoir of cutaneous leishmaniasis and parasite species at this region is leishmania major (dehestani 1994). during the war at south west regions such as mucian, fakkeh, sumar, eine khosh, dasht abbas, abu gharib, shoosh, bostan, dasht-e-azadeghan and sharhani, 11 species of sandflies have been captured and furthermore 4 other species of sandflies have been identified at other cities of khoozestan province. natural promastigote infections have seen in p. papatasi, p. alexanderi and sergentomyia sintoni species. p. papatasi is known as vector of diseases between human and animals (reservoirs). natural contamination to leishmania have been seen among reservoirs at rodents such as tetera indica, nesokia, meriones libycus and some of dogs, at this region (javadian and nadim 1989). during that time, researches showed that at khoosestan region, zcl have been dominant and the most important reservoir of cutaneous leishmanisis was tetera indica (husainidoost and javadian 1989). sandfly fever sand fly or papatasi fever is one of the arboviral diseases which are endemic in iran iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 31 and transmitted by sandfly (p. papatasi). during the war, the disease was prevalent among soldiers in west and southwest regions. during the last years of war, isoenzymes assess in mehran and gilangharb regions resulted in detecting of two serotypes that cause sandfly fever include cessil and naple serotypes (mehrabi tavana et al. 2000). in 1988, an epidemic disease was seen among 1000 soldiers with clinical manifestation of fever, chills, painful joints, thus physicians were suspected to dengue fever and sandfly fever (rezaeimoghadam 1989). in spite of existence of vectors of dengue fever in iran (some species of culex and aedes), we did not have any reports of that during and after the war. scabies scabies has been the most important illness after intestinal disease (like diarrhea) among soldiers during the iran-iraq war (heidarpur 1996). according to evidences in documentary sources during the war, scabies also was prevalent among iraqi soldiers. in addition to its prevalence in all seasons, sometimes it was epidemic among soldiers in ilam, mahabad, sardasht and kermanshah regions. sarcoptes scabiei, hominis variety, was isolated from soldiers infected with scabies during the war. this mite is known as causative agent of scabies in iran. pediculosis infestation to louse was prevalent during the war especially in kurdistan and kermanshah regions but there is no report of typhus at that time (mehrabi tavana et al. 1998). all of three species of human louse (pediculus capitis, p.humanus and phthrius pubis) had been prevalent among soldiers in west regions. plague kurdistan is potentially the most important focal regions for plague in iran. among 7 rodent species of meriones exist in iran, 4 species have been found in kurdistan and among them, meriones persicus is very effective in persistence of plague in that region (asmar and nekuei 1996a). according to the studies of pasteur institute in iran, during the war there were no yersinia pestis in 1800 rodents and 36000 fleas (asmar and nekuei 1996b). despite that fact, according to the wartime documents and expression of health personnel, outbreak of fleas has been reported in some operations among soldiers. myiasis myiasis is the infestation of live human and vertebrate animals with fly (dipterous) larvae, which, at least for a certain period, feed on dead or living tissue or ingested food of the host (zumpt 1965). during the war because of bullet injuries and wounds on soldier's bodies, traumatic myiasis was prevalent, especially when they were unconscious and could not repel flies. wohlfahrtia magnificica, lucilia sericata and other calliphora species invaded wounded soldiers. according to documents, some of the war martyrs had been infested with calliphora and sarcophaga species (ghasemi 1988). in addition to insects that have discussed above, other insects also were harmful to soldiers including musca domestica, stomoxys calcitrans and cimex lectularius. mosquitoes and scorpions have been harmful during war, but unfortunately they have not been identified. according to the reports, sometimes soldiers had such an entomophobia that could not sleep at all. arthropod control methods in imposed war chemical control this has been done in military regions periodically during epidemics and outbreaks of insects. for example, in kurdistan war regions, rifle pits of soldiers received insecticide spray in winter. insecticides that were used included ddt, lindane, permethrin, malathion, ficam, propoxur, temephos. in outbreak of pediculosis, ddt and later permethrin powder was used on the head of iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 32 soldiers in middle years of the war. methyl bromide ampoule was used for sterilization of clothes that were contaminated to lice, fleas and scabies. the contaminated clothes were put in plastic bags and then a methyl bromide ampoule was broken on them, inside the bags, to kill insects and other arthropods that mentioned above (interviews and documents). repellents during the 8 yr of war, repellent ointments such as indian "odimus" and german "autan" ointments were used (topical application on face and hand), to protect soldiers from insects bites. at that time a kind of insect repellent, called "trench pomade" (a mixture of deet+dmp) was widely used to protect military personnel, which had a satisfactory result (mehrabi tavana et al. 2000). on the basis of interviews, trench pomade was useful and its protection time was about 3-4 h, but it did not protect completely, especially against mosquitoes which bite overnight. on the other hand, this pomade had some undesirable effects including: oily hand and face. also, sometimes soldiers used other substances such as: iraqi repellents, smoky agents, gas oil and lemon juice on skin to repel insects (interviews). physical control a number of various physical methods have been used during the war, mosquito net at low rates, clearance of environment, during of stagnant waters around residence of military forces, covering of sewage sources and using of disinfectants such as kerolin to sterilize sewages and using of metal-nets for buildings windows are examples of health care affairs during the war (interviews and documents). biological control health personnel have used "gambusia affinis" for biological control of anopheles larvae in sanandaj, marivan, javanrood and gilan gharb regions (interviews and documents). gambusia fish were released into the pools and stagnant waters to control anopheles and culex larvae by hunting them. according to interviews this method worked successfully in the region applied. chemoprophylaxis most of military personnel in the regions where malaria was prevalent were given chemoprophylaxis. also soldiers who were came from high-risk regions such as sistan and baluchestan province consumed anti malaria drugs for precaution (razeghifam 2002). for this purpose they took daraprime tablet (25 mg weekly, for 4 weeks) and chloroquine. in the case of malaria infected patients, they were transferred backward for treatment. at wartime 1,483,760 daraprime and chloroquine tablets have been distributed among soldiers (razeghifam 2002). leishmanization leishmanization is intradermal inoculation of l. major promastigotes into covered sites of body (often thigh or arm). this is applied on healthy soldiers. wounds were found on the inoculated regions and were recovered spontaneously because of stimulation of immune system. during the war because of high prevalence of cutaneous leishmaniasis and failure in control procedures of sandflies, more than 200,000 soldiers were under leishmanization from 1982 to 1989. assessment of this procedure demonstrated that about 87% of them did not suffer from leishmaniasis. in affected persons, duration of the disease was short and wounds were smaller (mahmodzadeh 2002). unfortunately this procedure had some side effects such as large wounds in 23% of inoculated persons and treatment course was long (nadim et al. 1991). glucantim and pentostam were used to treat persons affected by leishmaniasis. scorpion sting to prevent scorpion sting tobacco solved in water and used around military tents and trenches, although they believed this method iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 33 was effective against scorpions but we did not find any scientific document. during imposed war treatment of scorpion stings was done by injection of polyvalent serum that was composed of antitoxin of 5` important species of scorpions in iran (except for hemiscorpius lepturus). now polyvalent serum has been reevaluated and includes 6 antitoxins of scorpion species which covers h. lepturus too. scabies γ -benzene and benzyl benzoate ointment have been used to treat scabies. to achieve this purpose, centers to spend convalescence have been prepared in kermanshah, mehran, orumieh and sanandaj regions in back lines of war regions and soldiers received treatment there (for 4-5 d). also their clothes were disinfected with methyl bromide ampoule or exposed to sun or cold rooms to kill mites that caused scabies (interviews and documents). assessment of health files (documentary sources) and interview (verbal history) showed that during the war, less scientific studies were conducted about arthropod species of medical importance. thus, most of studies have been performed after finishing of the war at regions. it seems that it is necessary to study the fauna of medically important arthropods in each region separately. results show that most problems caused for soldiers by arthropods were cutaneous leishmaniasis, scabies, sandfly fever and pediculosis, respectively. however, above mentioned diseases decreased the combat ability of the forces but had not caused heavy casualties for soldiers. in some cases they were exposed to severe epidemics because of being weakened and affected with unknown disease in iraq region. by war prolongation and increment in the intensity of army combat which weakened the military health system and also by iranian troops progress and their entrance to some parts of iraq and encountering to probable diseases; there was this concern that iranian troops face new problems and severe epidemics of arthropod borne diseases; in authorities opinion by efforts of health personnel of war these problems have been controlled. but today we do not know that they were really successful in their control or not, because there is no valid evidence to prove their success. on the other hand, continuous military operations and heavy casualties makes it impossible to exactly estimate the success of health personnel in control of diseases. according to official reports, in addition to arthropod borne diseases, in most of cases only insect biting (especially mosquito biting) causes problems for soldiers. to prevent mosquito biting trench pomade widely have been used. according to a recent laboratory study, protection time (pt) of trench pomade is about 4.5 h (khoobdel et al. 2007). this shows that soldiers to prevent mosquito bites need more effective repellents with longer pt and higher quality. usually in each region, there are some specific diseases. diseases such as malaria, leishmaniasis, scabies and sandfly fever are specific diseases which are endemic in iran and adjacent countries. because of prevalence of pediculosis and bed bugs and other insects among military forces, education of individuals about personal protection technique against medically important arthropods is essential. finally the results indicated that scabies, cutaneous leishmaniasis, sandfly fever and pediculosis were more prevalent in the imposed war. during imposed war various methods of vector control have not been used completely (khoobdel et al. 2003, 2005), therefore it is recommended to use personal protection methods such as delthamethrin-impregnation bed net, various insect repellents, permethrin iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 34 treated military uniforms and 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[msc dissertation]. school of public health, tehran university of medical sciences, iran. razeghifam a (2002) role of health engineering in control of disease in iraniraq war. the 2nd iranian military health congress, 2002 november 1820, baqiyatallah university of medical sciences. pp.140-146. rezaeimoghadam f (1989) 1000 case of fever and chill in the gilangarb region (west of iran). the 1st iranian military health congress, 1989 september 1820, health department of armed forces chief office, iran. p. 70. iranian j arthropod-borne dis, (2008), 2(1): 28-36 m khoobdel et al: arthropodborne diseases in… 36 schreck ce (1977) techniques for the evaluation of insect repellent: a critical review. ann rev entomol. 22:101-9. service mw (1996) medical entomology. hall and chapman. pp. 200-202. souza-santos r, carvalho ms (2000) spatial analysis of aedes aegypti larval distribution in the ilho do governador neighborhood of rio de janeiro, brazil. cad saude publica. 1631-1642. vahabi a (1998) determination of distribution and seasonal activity of phlebotominae in kermanshah province (iran). [msc dissertation]. school of public health, tehran university of medical sciences, iran. zumpt f (1965) myiasis in man and animals in the old world. london, butterworths. pp. 267. j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 119 original article a modified trap for adult sampling of medically important flies (insecta: diptera) kamran akbarzadeh 1, *javad rafinejad 1, 2, jamasb nozari 3, yavar rassi 1, mohammad mehdi sedaghat 1, mostafa hosseini 4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2center for solid waste research, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of plant protection, faculty of agronomy sciences, college of agriculture and natural resources, university of tehran, karaj, iran 4department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 13 dec 2011; accepted 7 may 2012) abstract background: bait-trapping appears to be a generally useful method of studying fly populations. the aim of this study was to construct a new adult flytrap by some modifications in former versions and to evaluate its applicability in a subtropical zone in southern iran. methods: the traps were constructed with modification by adding some equipment to a polyethylene container (18× 20× 33 cm) with lid. the fresh sheep meat was used as bait. totally 27 adult modified traps were made and tested for their efficacies to attract adult flies. the experiment was carried out in a range of different topographic areas of fars province during june 2010. results: the traps were able to attract various groups of adult flies belonging to families of: calliphoridae, sarcophagidae, muscidae, and faniidae. the species of calliphora vicina (diptera: calliphoridae), sarcophaga argyrostoma (diptera: sarcophagidae) and musca domestica (diptera: muscidae) include the majority of the flies collected by this sheep-meat baited trap. conclusion: this adult flytrap can be recommended for routine field sampling to study diversity and population dynamics of flies where conducting of daily collection is difficult. keywords: trap, diptera, calliphoridae, sarcophagidae, iran introduction the insect order of diptera (the true flies) is one of the most species-rich, anatomically varied and ecologically innovative groups of organisms, contributing 10–15% of known animal species. an estimated 150,000 species of diptera have been described (groombridge 1992, thompson 2005). diptera outrank other insect orders in terms of medical and veterinary significance, being responsible for the transmission of a wide variety of pathogens such as viruses, bacteria, fungi, protozoan and metazoan parasites in humans and animal (banjo et al. 2005, forster et al. 2007, forster et al. 2009). the invasion of host tissues by fly larvae, called myiasis, is the most widespread form of host invasion by arthropods (mullen and durden 2002) and is commonly noted in veterinary medicine and can occur in humans even in the west’s sanitized society (byrd and castner 2001). myiasis is the infestation of live human and *corresponding author: dr javad rafinejad, e-mail: jrafinejad@yahoo.com j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 120 vertebrate animals with dipterous larvae, which at least for a certain period on the host’s dead or living tissue, liquid body substances, or ingested food (zumpt 1965). human myiasis is a rare condition in any part of the globe, but is more common in the regions with a warm and humid climate (maturo et al. 2007). the first case was reported by minar when he recovered oestrus ovis larva from the eye of an iranian woman in tehran (minar 1976). from that time, varieties of case reports and case series have been reported in iran (tirgari et al. 2003, mohammadzadeh et al. 2008, faramarzi et al. 2009, soleimani ahmadi et al. 2009, salimi et al. 2010a,b). some valuable surveys with topic of fauna of potentially myiasis producing flies has been done in tehran (capital of iran) which various species of blowflies, flesh flies (khoobdel et al. 2008), muscidae and fanniidae (khoobdel and davari 2011) have reported. despite of these scattered published data, status of human and animal myiasis in iran is remained obscure. study on various populations of medically important flies as causative agents of myiasis and vectors of various pathogens should be more emphasized. the first step for a comprehensive study of fly population is conducting appropriate design using improved equipments. bait trapping appears to be a generally useful method of studying fly populations (suenaga and kurahashi 1994, hall 1995). bait traps have no chemical reagents, do not harm the environment, and can be used both for shortand long-term control (suenaga and kurahashi 1994). they can be used also for studying population dynamics of flies. systematically confirmation of identification process needs the presence of adult and larvae of the same sample. therefore, adult flies collected by traps should be reared for acquiring the larvae (zumpt 1965). adult flytraps usually are using for trapping and studying of adults. this study has been conducted to design and to evaluate an adult flytrap to collect adult flies and to prepare a breeding place for egg laying, fixing, and storing larvae simultaneously. materials and methods study area fars is one of the 31 provinces of iran, located in the southern part of the country. due to topographic characters, there are three distinct climatic regions in this province. the first part is hilly area in north and northwest of the province, with considerable vegetation covering. this part has a moderate temperature with 400–600 mm of precipitation annually. the second part is central part of the province with a relatively moderate temperature and hot and dry weather in summer. the average of annual precipitation in this part is around 200–400 mm. the third part is the lowland of south and southeast regions of the province with moderate temperature in winter and very hot wet weather in summer. the average rainfall of this part is below 200 mm annually. three cities in each geographical region were selected for evaluation of the trap including abadeh, arsanjan and sepidan cities located in north part, shiraz, kazerun and neyriz cities located in central part and darab, jahrum and lar cities from southern part. some meteorological information of three geographical parts of fars province in june 2011 has been shown in table 1. observations on applicability of the trap were performed during june 2010. three villages were randomly selected around each city and one trap hanged in each village. one modified trap was hanged in or around of sheep breeding place in each selected villages. totally 27 adult modified traps were hanged in the selected cities of the province. the sampling sites have been pointed by gps device (garmin 76csx®). all points have been shown in fig. 1 using google earth software. using of this kind of flytraps for fly mass j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 121 population control has been done in abadeh city. one city was set one trap for hanging it in his yard, situated in the city. he brought back the trap with collected flies. trap construction the trap consists of a polyethylene container (18× 20× 33 cm) with lid (fig. 2) with some modifications from satl arya company®. round openings with 8 cm diameter was prepared in each of four sides of the container. each opening should be placed in first half of the height of container. each opening was equipped with wiry funnel prepared on each side of the container (fig. 3). the opening at the end of funnel has 1 cm diameter to prevent escaping of attracted flies. a rectangular vegetable plastic netted dish used as bait chamber. the dish had large mesh to permit falling down of any larval instars. bait chamber placed in the inner of the container in front of funnels and fixed by means of hard cupper wires on the lowest screw of 3–4 screws, which has used for fixing the wiry funnels (fig. 3 and 4). all four funnels ended toward the middle of bait chamber. bottom of the trap filled with 2% formaldehyde and 0.02% chlorhexidine mixed solution (fig. 5). about 20–30 gr of sheep fresh meat were used as bait for the traps. the traps were hanged in suitable parts of the villages away from animal and children disturbing (fig. 5). flies attracted to odors of bait and entered the trap via one-way funnel. they laid egg after feeding on the bait. a few of the fullygrown larvae became pupa in the bait (meat) or around its chamber. adults, which appeared from these pupae (as f1), were found alive in the trap or were fallen down in the solution in bottom of trap. majority of fullygrown larvae fell down and fixed in the solution placed in bottom of the trap. collecting of samples from the traps was carried out monthly. the collected samples transferred the laboratory of medical entomology. adult flies in each batch of samples were washed with 70% ethanol and dried out on tissue paper for carful identification. larvae were washed out with 70% ethanol and preserved in 70% ethanol. morphological identification identification of the collected adult and larval specimens was based on morphological identification keys (james 1947, zumpt 1965, ishijima 1967, whitworth 2006, amendt et al. 2010, whitworth 2010). results the traps collect various groups of adult flies and maintain their f1 larvae. some species of calliphoridae, sarcophagidae, muscidae, and faniidae families were collected during the study period. numbers of adult flies, which have been collected by these traps, have been shown in table 2. most of the trapped flies were female and majority of them laid their eggs or larvae on the meat bait. due to condition of the inner condition of the trap, none of the eggs hatched. it was shown that all of the first instar larvae of both from hatched eggs or from mother flies (in sarcophagidae) were not able to complete their larval cycle. therefore, various larval instar of different families would be found in bottom of the traps. the identification of first and second instar larvae of most of dipteral families is somehow difficult. the identified samples of larvae, which were conserved in the liquid of trap bottom, have been shown in table 3. from table 2 and 3, it is concluded that calliphora vicina (diptera: calliphoridae), sarcophaga argyrostoma (diptera: sarcophagidae) and musca domestica (diptera: muscidae) were the highest number of collected adults or larvae. j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 122 table 1. meteorological information of three geographical parts of fars province in june 2011 part of province min temp max temp mean temp min humidity max humidity mean humidity precipitation northern 20.74 35.13 27.94 10% 33% 18% 0 central 22.58 39.42 31 10 33 21% 0 southern 25.28 43.32 40.3 12 44 28% 0 table 2. number of adult flies, trapped by the modified adult fly trap, in each geographical regions of fars province, iran, june 2010 region calliphoridae species n n per trap sarcophagidae species n n per trap muscidae species n n per trap faniidae species n n per trap northern part calliphora vicina 90 3.3 sarcophaga sp 25 0.9 musca domestica 83 3 lucilia sericata 16 0.6 sarcophaga argyrostom 34 1.3 muscina stabulans 39 1.4 sarcophaga variegata 2 0.07 hydrotea sp 70 2.6 wohlfahrtia sp 1 0.04 subtotal 106 3.9 subtotal 64 2.37 subtotal 192 7.1 central part calliphora vicina 10 0.37 sarcophaga sp 104 musca domestica 30 1.1 fania canicula ris 3 0.11 lucilia sericata 2 0.07 sarcophaga argyrostom 39 musca autumnali s 2 0.07 sarcophaga aegyptica 5 wohlfahrtia sp 2 subtotal 12 0.44 subtotal 150 5.5 subtotal 32 1.18 subtotal 3 0.11 southern part calliphora vicina 3 0.11 sarcophaga sp 63 2.3 musca domestica 48 1.8 lucilia sericata 2 0.07 sarcophaga argyrostom 11 0.41 muscina stabulans 2 0.07 chrysomyia albiceps 8 0.3 sarcophaga aegyptica 7 0.26 chrysomyia rufifacies 1 0.04 wohlfahrtia sp 8 0.3 wohlfahrtia nuba 2 0.07 subtotal 14 0.52 subtotal 91 3.37 subtotal 50 1.85 number of fly per trap was 4.86, 11.24, 10.13, and 0.11 for members of calliphoridae, sarcophagidae, muscidae and fanniidae respectively (table 2). regionally muscidae has the greatest number of flies per trap in northern part of the province that was 7.1, however, in central and southern parts of the province the highest number of fly per trap was 5.5 and 3.37 respectively as for sarcophagidae. due to unknown medically importance of some of the collected samples such as anthomyiidae and hypoboscidae, specimen identification to genus and species were not performed. these flies might be entered the traps accidentally or wind oriented. for finding the applicability of this kind of traps for collecting synanthropic flies, only one trap was hanged in a house in city center of abadeh city. this trap equipped with fresh sheep meat as well. results of the identification of the collected flies in this trap have shown in table 4. j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 123 table 3. number of fly larvae, collected by modified adult fly trap, in any geographical regions of fars province, iran, june 2010 region calliphoridae species n sarcophagidae species n muscidae species n noerthern part calliphora vicina 235 sarcophaga sp 96 musca domestica 212 lucilia sericata 85 sarcophaga argyrostom 155 unknown 21 unknown 8 unknown 32 wohlfahrtia sp 5 central part calliphora vicina 88 sarcophaga sp 338 musca domestica 178 lucilia sericata 24 sarcophaga argyrostom 198 unknown 22 unknown 5 sarcophaga aegyptica 28 unknown 12 southern part calliphora vicina 30 sarcophaga sp 112 musca domestica 136 lucilia sericata 10 sarcophaga argyrostom 46 unknown 11 unknown 11 sarcophaga aegyptica 34 wohlfahrtia sp 28 unknown 8 total 496 1092 580 table 4. number of adult flies was trapped by a single modified trap in abadeh city, fars province, iran, june 2010 region calliphoridae n sarcophagidae n muscidae n northern part calliphora vicina 68 sarophaga sp 11 musca domestica 471 lucilia sericata 15 sarcophaga argyrostoma 5 muscina stabulans 103 fig. 1. study area showing distribution of sample sites for evaluating of the modified adult flytrap. data imported from gps garmin 76csx® to gis software 9, fars province, iran, 2010 j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 124 fig. 2. a modified adult fly trap, evaluated as an applicable trap for collecting various families of diptera, fars province, iran 2010 (original) fig. 4. bait chamber of the modified adult flytrap, placed in lower level in front of round openings, tehran university of medical sciences, 2010 (original) fig. 3. round openings of the modified adult flytrap with 8cm in diameter, provided for one-way entering flies, tehran university of medical sciences, 2010 (original) fig. 5. a hanged meat baited modified adult flytrap with collected adult flies in kazerun station of research, school of public health, tehran university of medical sciences (original) discussion trapping systems can be used for research monitoring (hwang and turner 2005) and control of fly populations (hall 1995, baz et al. 2007, gerry et al. 2007). rearing of larvae is the way (zumpt 1965, amendt et al. 2010) to have adult and larvae of the same sample, j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 125 which is difficult, need more time and sometimes not successful. adult trapping process is the way to find medically important flies (hall 1995) somewhere the larval population reduced by chemical controls. it has been observed that the modified adult flytraps are useful for collecting adults and larvae of flies simultaneously. systematic observation needed to connect the adults and larvae after collecting them in the traps. due to difficulties in systematic of the flies, their identification and completing the systematic relationship between larvae and adults should be done with experts. idea of the modified flytrap derived from the original trap introduced by suenaga and kurahashi (1994) which has bottom filled with soil in most of the volume. presence of soil in their trap prepares suitable conditions for completing the f1 progeny from the entering flies. their flytrap is suitable for studying of population dynamic of flies. the modified flytrap which has been evaluated in this study has bottom filled with solution of 2% formaldehyde and 0.02% chlorhexidine which comprising about one third of all volume. the surveyed modified flytrap is suitable for faunestic and diversities phenomenon. the first problem in working with flies is correct identification. morphological identification of some flies such as female and larvae of sarcophagidae (guo et al. 2010) and immature stage of muscidae (amendt et al. 2010) are very difficult. therefore, in the case of female samples of family sarcophagidae (sarcophaga sp and whohlfahrtia sp) their genus has reported merely. due to the same reason some of larval samples have left as unidentified. in field studying and routinely use of the modified adult flytrap, comparing of the results of adults and larval samples will lead to a precise identification. with any sampling process, there will be adults beside of their larvae, which very desired for systematically identifications. presence of identified or semi-identified adults beside unknown larval samples will help for identification by limiting the range of searching. for example, it can be postulated that unknown larvae in central part of fars province (table 2), could be calliphora vicina or lucilia sericata with high probability. new techniques such as molecular identifications can be employed for identification of adults and larvae. the tabled data were the only one sampling process for presenting the applicability of this kind of traps for collecting adults and relevant larvae simultaneously. the calliphora vicina (diptera: calliphoridae), sarcophaga argyrostoma (diptera: sarcophagidae) and musca domestica (diptera: muscidae) formed the majority of the flies collected by this sheepmeat baited trap. by changing the baits, some desired flies can be collected (boonchu et al. 2003). it has been observed that by changing the place of traps the number of trapped flies as well as their species will be changed (table 1 and 3). temperature is probably the most influential environmental factor in the life history of populations, particularly in organisms with short life cycles such as insects (levine and levine 1991, oshaghi et al. 2009, shiravi et al. 2011). the highest number of fly per trap was as for sarcophagidae (11.24%). regional analysis revealed that sarcophagid flies also had the greatest number of fly per trap in central and southern parts. these relatively higher rates may be due to of the higher temperature in these two regions in comparison with traps hanged in northern part. to find significant difference more sampling and comparing various temperatures in any seasons nis required. wind direction and visual cues are also important to attract more flies (boonchu et al. 2003). by considering the bionomics of any species and its behavior, the trap can be focused for collecting specific species. pheromones or j arthropod-borne dis, 2012, 6(2): 119–128 k akbarzadeh et al.: a modified trap for … 126 pheromone based baits (geden et al. 2009) can be used for collecting exactly the target species. the modified trap used for this study was enough big and able to collect high number of adult flies. this character can be used for control of fly population when large number of traps can be hanged up around the specific places. it can be recommended as an environmental friendly fly control. for this purpose, education, finding suitable baits as well as appropriate places for local use in various geographical conditions is needed. the highest number of m. domestica in sheep breeding places was 83, but this was 479 for the single trap. these results also showed the synanthropic behavior of this fly as it confirmed by figueroa-roa and linhaes (2004). it is so cleared that finding of one trap is not enough for showing scientifically the synanthropic behavior of m. domestica and need more research in this province. in conclusion, this kind of adult fly trap can be recommended for routine field sampling for studying the diversity of flies as well as study of population dynamics where conducting of daily collection is difficult. this kind of flytrap can be applied also for long-term faunestic study of flies. acknowledgements the authors wish to 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accepted 28 dec 2015) abstract background: protecting the environment from chemical hazards of synthetic insecticides along with offering of new breeding areas for vectors by urbanization indicate the trial of natural insecticides. methods: the acetone extracts of anethum graveolens, ocimum basilicum and thymus vulgaris were tested for their insecticidal effect on culex pipiens adults and larvae in different concentrations depending on the technique used. results: the extracts were significantly effective in all models used with basil being the best in all tested three techniques (lc50= 0.064) in larval feeding, (lc50= 0.330) in cdc bottle assay and (lc50= 13.148) in adults feeding (p< 0.05). conclusion: the results recommend the eco-friendly studied extracts as candidates for controlling cx. pipiens the lymphatic filariasis vector. keywords: culex pipiens, anethum graveolens, ocimum basilicum, thymus vulgaris, insecticides introduction climatic changes can cause expanding the range of geographical distribution and dynamics of vector-borne diseases. global warming and changing sea level may lead to increase in transmission intensity and extending spatial distributions of these diseases (hongoh et al. 2012) by influencing the development rate, longevity of insects and vector reproduction rates, creating new challenges in these insects control in areas where they were never recorded before. anthropogenic factors with changes in landscaping, rising in housing and settlements, extensive tropical urbanization, colonization, increase in environmental pollution and changing lifestyle patterns offer new breeding areas for vectors development (tatem et al. 2006, weaver and reisen 2010, carvalho et al. 2014). insecticides overuse led to several ecological drawbacks over the past years. the toxicity, development of resistance phenomenon and the residual effects of these insecticides are the main concern of scientists. the urging need for developing environmental friendly insecticides is rising (kebede et al. 2010). several studies on botanicals potential as insecticides are ongoing with the rise of green insecticides concepts and awareness of these safe, specific, biodegradable, eco-compatible components (park et al. 2005, khater and shalaby 2008, kumar et al. 2012). mosquitoes are a major public health threat as they play a vital role in transmitting serious human diseases to hundreds million people annually (akinkurolere et al. 2011). mosquitoes were reported up to 2551–2528 bc also the invention of mosquito nets were attributed to the ancient egyptians as herodotus reported in 445–440 b.c. (arnold 1995). culex pipiens is a worldwide mosquito causing nuisance and transmitting many dangerous diseases as west nile virus, st louis en*corresponding author: dr noha a. elleboudy, e-mail: nohaleboudy@gmail.com j arthropod-borne dis, march 2017, 11(1): 147–155 ea el zayyat et al.: bioefficacy of some … 148 http://jad.tums.ac.ir published online: march 14, 2017 cephalitis virus, filarial worms and avian malaria (farajollahi et al. 2011). with the emergence of cx. pipiens resistance to many insecticides, control is becoming more difficult providing a flourishing chance to these diseases (zayed et al. 2006, knio et al. 2008) essential oils are volatile oils with strong aroma giving distinctive odor, flavor and scent to a plant. they are secondary metabolites characterized by being complex mixtures of organic compounds (okunowo et al. 2013). usage of essential oils as a whole usually have higher efficacy than using its separate metabolites (mourey and canillac 2002, bassole´ and juliani 2012) as synergism of all compounds of the essential oil seems to be important (zoubiri and baaliouamer 2011). despite being safe for domestic insect control strategies, few products based on plant essential oils are commercially available (koul et al. 2008, sutthanont et al. 2010). from the ancient pharaohs, egyptians were familiar with aromatic plants and included their use in religion, cosmetics, embalming and medicinal purposes. in addition, the first record of essential oil distillation was from egypt (fakhry 2004). egypt has a variety of flora, among which anethum graveolens (dill), ocimum basilicum (basil) and thymus vulgaris (thyme) were chosen to conduct this study being available in local market, cheap and widely used in traditional medicine. this study aimed at determining the insecticidal effect of three indigenous plant extracts, commercially available in egypt for domestic use, to control culex pipiens adults and larvae. materials and methods collection of plants materials anethum graveolens, oc. basilicum and th. vulgaris were purchased from local market, washed with distilled water to remove dust particles and identified by department of botany, faculty of science, ain-shams university staff members. preparation of plant extracts the whole plants including leaves and stems were dried for 7v10 days in the shade at 27–37 °c. the dried plants were powdered using a commercial electric blender then soaked in acetone 99% (biochem. company, egypt) at a ratio of 1:4 for three days. then the extracts were sieved and filtered through a buchner funnel with sterile whatman filter paper number one, acetone was evaporated using rotator evaporator apparatus (hs-2005s, jisico co., ltd. korea) in central laboratories, faculty of science, ain-shams university, egypt. the extracts were concentrated under reduced pressure 22–26mm hg at 45 °c and the crude extracts residue were kept in dark bottles, labeled and preserved in the refrigerator at 4 °c until further use (akinkurolere et al. 2011). insects rearing laboratory reared colony of cx. pipiens free from insecticides and pathogens obtained from mosquitoes research department, research institute of medical entomology, ministry of health and population, dokki, giza was maintained starting from egg rafts. larvae used in the tests were reared in a plastic cup containing dechlorinated water, under the standard conditions of 28±2 °c temperature and 70±5% rh and l12/d12 photoperiod and fed daily on tetramine (tropical fish food) until the larvae transformed into the pupal stage. the larvae were reared until adults in insectaries. adults were maintained on a 10% sugar solution and females were allowed to feed periodically on guinea pig blood for 2–3h every two days to obtain protein used principally for egg production (kovendan et al. 2012). larvicidal bioassay the assay was done on healthy late third instar larvae, three replicates for each test were used. larvae were collected with a pasture pipette, placed on a filter paper to rej arthropod-borne dis, march 2017, 11(1): 147–155 ea el zayyat et al.: bioefficacy of some … 149 http://jad.tums.ac.ir published online: march 14, 2017 move excess water then placed in cups containing 250ml dechlorinated tap water to which different concentrations of the tested extracts were added (0.02g/l, 0.05g/l, 0.1 g/l, 0.2g/l and 0.3g/l) then readjusted according to obtained results. three controls cups containing water were used in the presence of the larvae semi synthetic diet. all cups were covered with muslin cloth for protection. the observed mortality was recorded at the end of 72h, larvae were considered dead if there is no sign of any movement even after mild touch with a glass rod (kumar et al. 2014). adulticidal bioassay adaptation of cdc bottle bioassays technique was done to evaluate insecticidal properties of the extracts as follows: 250ml wheaton bottles with screw lids were properly cleaned and dried then they were coated with 1ml of (0.2g/l, 0.4g/l, 0.6g/l, 0.8g/l) of the tested extracts by swirling assuring complete coating of the bottle and its cap. one ml of absolute alcohol was added to the control bottle handled as before. forty mosquitoes, divided among four replicate bottles were introduced using an aspirator into each bottle with the tested extract concentrations (brogdon 2014). each bioassay included a control bottle with 10 mosquitoes. mortality was assessed after 2h then after 24h after the mosquitoes' introduction and mosquitoes were considered dead if they could no longer stand (aïzoun et al. 2013). the experiment was done under normal room conditions. feeding toxicity was done according to allan (2011) for cx. pipiens adults using randomly selected male and female, 3–6 days after emergence, starved for 24h, and placed in cages 0.5× 0.5× 0.5m (10/cage), baits were presented in a 1cm length of cotton wick soaked with 200μl of 10% sucrose containing the tested extracts doses (10g/l, 20g/l, 40g/l) and placed on top of the mesh of each cage and removed after 2h. the insects with distended abdomens indicating ingestion of a sugar meal were transferred to a new cage (10/cage) and maintained. a cotton pad soaked with glucose only solution was used as a control for each extract. mortality was assessed up to 72h after the treatment performance. adults that did not respond to pin prick were be considered dead. percentage of mortality was corrected using of abbott’s formula (abbott 1925) if needed. x100 all statistical tests were performed using the spss version 22, 2013 (14· 0, chicago, il, usa). a probit regression model to predict the probability of percentage of insect mortality and logarithmic concentration of the extracts was done. z-scores were estimated. pearson goodness-of-fit of chi-square test to determine the applicability of the resultant regression models was done. lc50, lc90 with their 95 per cent confidence limits were determined. the toxicity index was calculated as follows, llc50 of the most effective compound ×100 divided by the llc50 of the compound used and the most toxic compound was given 100 units on the toxicity index scale according to rawi et al. (2011). the probability of error at 0.05 was considered significant, while at 0.01 and 0.001 were considered highly significant throughout the whole study. results evaluation of insecticidal properties of three indigenous plants was done using different an. graveolens, oc. basilicum and th. vulgaris crude extracts concentrations on both larvae and adults of cx. pipiens mosquito. the results revealed the effectiveness of the used extract different doses to obtain mortality and suggested the potentiality of their usage in cx. pipiens control. the results of j arthropod-borne dis, march 2017, 11(1): 147–155 ea el zayyat et al.: bioefficacy of some … 150 http://jad.tums.ac.ir published online: march 14, 2017 larvicidal activity of extracts by feeding technique after 72h are shown in table 1 and 2). adulticidal activity of the tested extracts by feeding technique after 72h is shown in table 3 and 4 and by cdc bottle bioassay after 24 h is shown in table 5 and 6. plate (1) shows scanning electron microscopy of cx. pipiens larva induced by basil extract after 72h while plate (2) for cx. pipiens larva induced by basil extract by cdc technique after 24h. table 1. probit analysis of larvicidal activity of the tested extracts against culex pipiens by feeding technique after 72h plant parameter mortality rate standard error z sig 95% confidence interval pearson goodness of fit test lower bound upper bound chisquare sig thyme slope 1.057 0.228 4.641 .000 0.611 1.503 0.725 0.867 intercept 1.220 0.204 5.991 .000 1.016 1.424 dill slope 1.428 0.192 7.442 .000 1.052 1.804 2.319 0.509intercept 1.642 0.217 7.554 .000 1.424 1.859 basil slope 0.996 0.190 5.257 .000 0.625 1.368 1.589 0.662 intercept 1.187 0.211 5.615 .000 0.976 1.398 table 2. larvicidal activity of the tested doses of extracts against culex pipiens by feeding technique after 72h plant concentration (g/l) log concentration observed responses expected responses residual probability thyme 0.02 -1.699 15 15.935 -0.935 0.398 0.05 -1.301 24 22.590 1.410 0.565 0.1 -1.000 28 27.392 0.608 0.685 0.2 -0.699 30 31.518 -1.518 0.788 0.3 -0.523 34 33.512 0.488 0.838 dill 0.02 -1.699 14 13.000 1.000 0.217 0.05 -1.301 26 24.883 1.117 0.415 0.1 -1.000 30 35.091 -5.091 0.585 0.2 -0.699 46 44.413 1.587 0.740 0.3 -0.523 50 48.883 1.117 0.815 basil 0.02 -1.699 12 12.263 -0.263 0.307 0.05 -1.301 17 18.262 -1.262 0.457 0.1 -1.000 26 23.026 2.974 0.576 0.2 -0.699 28 29.893 -1.893 0.747 0.3 -0.523 33 32.500 0.500 0.812 j arthropod-borne dis, march 2017, 11(1): 147–155 ea el zayyat et al.: bioefficacy of some … 151 http://jad.tums.ac.ir published online: march 14, 2017 table 3. probit analysis of adulticidal activity of the tested extracts against culex pipiens by feeding technique after 72h table 4. adulticidal activity of the tested doses of extracts against culex pipiens by feeding technique after 72h plant concentration (g/l) log concentration observed responses expected responses residual probability thyme 10 1.000 9 8.514 0.486 0.284 20 1.301 14 15.016 -1.016 0.501 40 1.602 22 21.514 0.486 0.717 dill 10 1.000 7 7.201 -0.201 0.240 20 1.301 18 17.573 0.427 0.586 40 1.602 26 26.183 -0.183 0.873 basil 10 1.000 14 12.883 1.117 0.429 20 1.301 16 18.221 -2.221 0.607 40 1.602 24 22.953 1.047 0.765 table 5. probit analysis of adulticidal activity of the tested extracts by cdc bottle bioassay after 24h plant parameter mortality rate standard error z sig 95% confidence interval pearson goodness of fit test lower bound upper bound chisquare sig thyme slope 1.903 0.571 3.334 .001 0.785 3.022 0.215 0.643 intercept -2.475 0.754 3.281 .001 -3.229 -1.721 dill slope 3.066 0.636 4.823 .000 1.820 4.311 0.042 0.837 intercept -3.772 0.824 4.579 .000 -4.595 -2.948 basil slope 1.496 0.568 2.632 .008 0.382 2.610 1.063 0.303 intercept -1.674 0.741 -2.26 .024 -2.415 -0.933 plant parameter mortality rate standard error z sig 95% confidence interval pearson goodness-of-fit test lower bound upper bound chisquare sig thyme slope 2.750 0.570 4.825 .000 1.633 3.868 3.383 0.184 intercept 0.988 0.233 4.246 .000 0.755 1.221 dill slope 2.964 0.595 4.984 .000 1.799 4.130 2.465 0.292intercept 0.906 0.232 3.911 .000 0.674 1.138 basil slope 3.247 0.592 5.480 .000 2.085 4.408 1.011 0.603 intercept 1.561 0.262 5.967 .000 1.300 1.823 j arthropod-borne dis, march 2017, 11(1): 147–155 ea el zayyat et al.: bioefficacy of some … 152 http://jad.tums.ac.ir published online: march 14, 2017 table 6. adulticidal activity of the tested doses of extracts against culex pipiens by cdc bottle bioassay after 24h plant concentration (g/l) log concentration observed responses expected responses residual probability thyme 0.2 -0.699 7 5.252 1.748 0.175 0.4 -0.398 10 13.729 -3.729 0.458 0.6 -0.222 19 19.418 -0.418 0.647 0.8 -0.097 25 22.941 2.059 0.765 dill 0.2 -0.699 5 3.654 1.346 0.122 0.4 -0.398 9 11.765 -2.765 0.392 0.6 -0.222 17 17.941 -0.941 0.598 0.8 -0.097 24 21.958 2.042 0.732 basil 0.2 -0.699 8 7.182 0.818 0.239 0.4 -0.398 16 18.183 -2.183 0.606 0.6 -0.222 25 23.994 1.006 0.800 0.8 -0.097 27 26.811 0.189 0.894 discussion natural insecticides are of growing interest worldwide being eco-friendly as with climatic changes and urbanization offering new breeding areas for vectors, it is becoming mandatory to meet the needs for screening of plants potential insecticidal effect. plants phytochemical compounds have demonstrated a promising potential for insecticidal activity. the toxicological safe nature of most essential oils and its availability in developing countries with huge biodiversity and complex mixtures of its constituents resulting in more slowly development of resistance may ultimately have their greatest impact in future insects control programs (koul et al. 2008). the usage of plant crude extracts as a whole besides being less expensive, it advances the synergistically of its active compounds complex mixtures providing greater bioactivity compared to its purified individual constituents (cavalcanti et al. 2004). several studies reported the botanical insecticidal effect on culex mosquito with variation depending on geographical origin of the plant, the plant parts processed, type of solvent used and the culex species tested (jeyabalan et al. 2003). the mortality effect of plant extracts evaluated in this study varied according to the plant, concentration of the extract and the used technique. highly significant linear predictions from the resultant probit models were obtained as shown by z-scores. there was a non-significant difference on comparing the observed and expected response by chi-square goodness of fitness test (tables 1, 3 and 5) so assumptions made by this study were reasonable and the choice of models were appropriate and the frequency counts distributed identically across different populations. dose response relationship of the tested extracts showed increase in the mortality rate with increasing the tested doses of extracts with non-significant difference between the observed value of mortality and the predicted value, the residual (tables 2, 4 and 6) indicating the appropriateness of the used models. the mechanism of toxicity of essential oils on insects needs further study to be clarified. comparing these results to other studies on same extracts may vary in results as variation of plant essential oil composition according to its indigenous origin, target mosquito species tested, method tested, different exposure times and extract concentrations used (kumar et al. 2011). in larvicidal assay of the tested extracts by feeding technique after 72h, basil was j arthropod-borne dis, march 2017, 11(1): 147–155 ea el zayyat et al.: bioefficacy of some … 153 http://jad.tums.ac.ir published online: march 14, 2017 highly effective (lc50= 0.064) followed by thyme (lc50= 0.070) and dill (lc50= 0.071). concerning adulticidal activity of the tested extracts by feeding technique after 72h basil was highly effective (lc50= 13.148) followed by dill (lc50= 16.996) and thyme (lc50= 19.967) and regarding adulticidal activity of the tested extracts by cdc bottle bioassay, no mortality recorded after 2h but after 24h basil was highly effective (lc50= 0.330) followed by thyme (lc50= 0.437) and dill (lc50= 0.495) according to the lowest lc50 and in return the highest toxicity indices. so ocimum basilicum was the most effective extract tested on cx. pipiens larvae and adults in accordance to other studies as aarthi and murugan (2010), belong et al. (2013) and govindarajan et al. (2013). conclusion the integrated usage of indigenous an. graveolens, oc. basilicum and th. vulgaris in control of cx. pipiens would probably help in reducing the magnitude of vector borne diseases. they provide promising results as insecticides besides being cheap, available and easily handled. further studies on the mechanism of killing, effect on insect development, physiology and metabolism as well as field trials are recommended. acknowledgements special thanks to dr/ azza abdel fattah mostafa, professor of insecticides, research institute of medical entomology, for her assistance during insect bioassay techniques. moreover, to dr/ amany samy el-hefny, senior researcher, plant protection research institute, agricultural research center for her assistance during plant extraction processes. the authors declare that there is no conflict of interests. references aarthi n, murugan k (2010) larvicidal and repellent activity of vetiveria zizanioides l, ocimum basilicum linn and the 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1laboratory of veterinary parasitology, college of veterinary medicine and bio-safety research centre, chonbuk national university, jeonju, republic of korea 2department of medicine surgery and obstetrics, faculty of veterinary and animal science, hajee mohammad danesh science and technology university, dinajpur, bangladesh (received 2 aug 2014; accepted 13 sep 2015) abstract background: the aim of the present study was to induce salivation in haemaphysalis longicornis to increase saliva production and to characterize the collection of proteins present in the collected saliva using on-chip-electrophoresis. methods: saliva of adult female engorged h. longicornis was collected by treatment with 0.2% dopamine hydrochloride. all protein samples were characterized by sds-page electrophoresis using a microfluidic high sensitivity protein assay 250 kit by 2100 bioanalyzer (agilent technologies, usa) under non-reducing conditions. results: the average salivary protein concentration was 0.169 µ g/µl/tick and saliva secretion decreased with increased time of tick detachment from the host. saliva secretion volume increased to 3.56 µl in the group of ticks with a body weight between 301–350 mg as compared to higher and lower body weight groups. on-chip-electrophoresis results show 13 distinct bands ranging from 9.9 to 294 kda. conclusion: based on molecular weight, the putative salivary proteins are comprised of proline-rich proteins, triabin, apyrase members of the 12-kda protein family, platelet inhibitors and anti-inflammatory proteins as tick saliva contains anti-inflammatory components. keywords: haemaphysalis longicornis, induction, on-chip-electrophoresis, salivation introduction “ticks are ectoparasitic, blood-feeding arthropods that transmit pathogens to wild and domestic animals and rank second only to mosquitoes as vectors of human disease” (zhang et al. 2011). haemaphysalis longicornis is the most predominant species in korea (>91.8%) and the prevalence of tickborne pathogens by taqman pcr and species-specific pcr was frequent for some bacteria, including anaplasma platys, ehrlichia chaffeensis and rickettsia sp. in h. longicornis ticks for the most part (kim et al. 2006). haemaphysalis longicornis also known vector of q fever caused by rickettsia, russian spring-summer encephalitis caused by a virus, and theileriosis and babesiosis caused by protozoa species (battsetseg et al. 2002). h. longicornis is also known to transmit coxsackie-like virus (hoogstraal 1973), powassan encephalitis virus (hoogstraal 1973), khasan virus (lavov et al. 1978), and borrelia species (hao et al. 2011). in addition to the pathogens they carry, ticks themselves causes severe toxic conditions including tick paralysis, various tick toxicoses, irritation, and tick bite allergies. losses due to tick infestations can be considerable. in 1974, australian husbandry losses due to the cattle tick (boophilus microplus) were estimated at 62 million usd (springell 1983) and brazil economically loses around 2 billion usd per year (grisi et al. 2002). *corresponding authors: dr myung jo you, email: tick@chonbuk.ac.kr j arthropod-borne dis, march 2017, 11(1): 10–18 ms islam and mj you: drug induced … 11 http://jad.tums.ac.ir published online: march 14, 2017 during tick infestation, the tick secretes bioactive substances that modify the host’s physiological and immunological reactions. macromolecules present in the saliva consist of lipids and proteins, which alter physiological changes at the feeding site thereby affecting pathogen transmission. tick salivary glands produce a large proportion of these substances that are secreted into the host during blood feeding. based on proteomic studies, many tick salivary proteins with specific functions have been identified (hovius et al. 2008). tick salivary immunosuppressive proteins interfere with the host’s innate and adaptive immune responses, which may result in more efficient transmission of several tick-borne pathogens (zhang et al. 2011). the study of tick saliva is important to understand tick biology as tick saliva plays a special physiological role in pathogen transmission. due to the importance of tick saliva in inflammation, pathogen transmission, immunity and hemostasis, the isolation and characterization of saliva molecules responsible for these effects is the most important to understanding host-pathogen immune reactions in vector borne diseases. however, the amount of protein in tick saliva is very limited and the isolation of protein components from the saliva is a big challenge. to overcome this challenge, researchers have studied salivary glands in lieu of saliva for the molecular characterization of protein compounds. salivary proteins are classified as concealed or exposed. by default, studies with salivary glands lack assessment of exposed proteins. the purpose of the present study was to induce salivation in h. longicornis to increase saliva production and to characterize the collection of proteins present in the collected saliva using on-chip-electrophoresis. results of this may help researchers to identify tick proteins as potential candidates for further studies aimed to develop novel tick control strategies. materials and methods ticks the jeju strain of the hard tick h. longicornis, collected form jeju island in korea has been maintained on rabbits in our laboratory since 2003. engorged female ticks were used for the analysis of salivary proteins. at least 150 clean h. longicornis adults were placed on each ear of 4–5 week old female new zealand white rabbits (samtako, korea) and the ticks were allowed to attach and feed to repletion. engorged female ticks dropped from the rabbit ear were assessed for body weight and kept in plastic tubes at 27 °c under humid conditions. the chonbuk animal care and use committee approved all rabbit studies and the protocols are in agreement with the ethical principles for animal research. saliva collection dropped engorged female h. longicornis were carefully collected and sterilized by wash in 70% ethanol for 5 min. the ticks were then rinsed in at least three changes of sterile distilled water. the ticks were gently ventrally attached to a slide using doublesided bonding tape for tick immobilization and observation under a stereo zoom microscope (nikon smz-u, japan). a 3–5 μl aliquot of 200 mg/5 ml dopamine hydrochloride solution (hunguhsu pharma, korea) in 0.2% phosphate-buffered saline, ph 7.4, was introduced into the posterior to fourth coxae in the region of epimeral and anal plates each tick using a micro-fine specially made glass needle (fig. 1). tick saliva was harvested from the tick mouthparts using a glass microtube and stored at -80 °c until further use. if the tick failed to produce saliva by 5 min post-injection, the tick was re-injected. if the tick failed again within 5 min of the second injection, it was discarded from the study. saliva was collected from 120 out of 150 ticks. total protein concentration of the saj arthropod-borne dis, march 2017, 11(1): 10–18 ms islam and mj you: drug induced … 12 http://jad.tums.ac.ir published online: march 14, 2017 liva was determined using a bio-rad protein assay based on the method of bradford (biorad laboratories, hercules, ca, usa). on-chip-electrophoresis all protein samples were characterized by sds-page electrophoresis using a microfluidic high sensitivity protein assay 250 kit and a 2100 bioanalyzer (agilent technologies, usa) under non-reducing conditions. all proteins were observed as single bands in the electropherograms. the protocol for on-chip-electrophoresis was followed per the manufacturer’s instructions with slight modification to improve results with diluted labelled protein. briefly, six different dilution samples were loaded onto six chips (fig. 4a, b). tick saliva was divided as three groups based on dilution as sample 1, no dilution, sample 2, 1:10 dilution with te buffer and sample 3, 1:100 dilutions with te buffer. each of three sample were duplicated to load in six different well of agilent high sensitivity protein 250 chip and analyzed. recording data and analysis twenty ticks were divided into five groups based on body weight at less than 250 mg, 251–300 mg, 301–350 mg, 351–400 mg, and more than 400 mg. on the day of detachment, the ticks were treated with dopamine as described for saliva collection and the data were recorded to determine the relationship between body weight and salivation. saliva was collected for five consecutive days. the data were subjected to anova with sas (ver.8.x). duncan's multiple range tests was used to determine significant differences among the various durations or ticks body weight. results the measured protein concentration was found to be 0.169 µ g/µ l. this method of saliva collection used dopamine to stimulate salivation and yielded over 1.74 μl of saliva per adult engorged female tick. the maximum saliva production was found from the ticks that were treated immediately after detachment from the tick. saliva production decreased with an increase of time after detachment. at day 1 post detachment, the average saliva production was 3.56 μl saliva per adult engorged female tick while at day 5 the average production was only 0.55 μl per adult engorged female tick (fig. 3). in addition, salivation significantly (p< 0.001) increased with an increase in body weight from less than 251–350 mg body weight group of engorged ticks. the 301–350 mg tick group body weight produced the highest volume of saliva at an average of 3.7 μl/tick. however, the ticks that weighed more than that showed a decrease in saliva volume as ticks of more than 400 mg body weight produced on an average 2.57 μl of saliva per engorged female (fig. 2). a total of 13 protein bands were identified based on the on-chip-electrophoresis analysis. in the electrophoretogram, the strong signals were at 109 kda (948 pg/ µ l), 9.9 kda (900 pg/µ l), 190 kda (332 pg/ µ l), 12 kda (327 pg/µ l), 67 kda (311 pg/µ l), and 10.3 kda (213 pg/µl). other protein bands identified were at 13.6, 24.3, 28.6, 251.0, 294.1, 108.2, 68.1, and 110.4 kda (fig. 4 and table 1). fig. 1. collection of saliva from engorged adult female haemaphysalis longicornis after injecting with 0.2% dopamine j arthropod-borne dis, march 2017, 11(1): 10–18 ms islam and mj you: drug induced … 13 http://jad.tums.ac.ir published online: march 14, 2017 table 1. protein bands and their relative concentration found from saliva of adult female engorged haemaphysalis longicornis no size [kda] relative concentratin (pg/µl) 1 9.9 900.1 2 10.6 68.4 3 12.0 327.5 4 13.6 166.2 5 24.3 120.5 6 28.6 119.7 7 67.0 311.6 8 108.2 31.7 9 109.9 948.2 10 110.4 49.9 11 190.0 332.7 12 251.0 68.8 13 294.1 56.2 fig. 2. body weight vs. saliva production from engorged adult female haemaphysalis longicornis. ticks are collected after spontaneous detachment between 5 to 7 days. values presented are the mean ±sd. **identifies significance between two groups (p< 0.001) fig. 3. saliva production vs. detachment time (day) from engorged adult female haemaphysalis longicornis threehost tick values presented are the mean ±sd. **identifies significance between two groups (p< 0.001). s al vi a pr od uc ti on ( μl ) body weight (mg) s al vi a pr od uc ti on ( μl ) detechment from host (day) j arthropod-borne dis, march 2017, 11(1): 10–18 ms islam and mj you: drug induced … 14 http://jad.tums.ac.ir published online: march 14, 2017 fig. 4. on-chip-electrophoresis. a. gel view. b. electropherogram with the peak relative to the protein bands shown in column 4 (rfu, relative fluorescence units) discussion tick hemolymph, salivary glands, and saliva are important tools for the study of tick-borne pathogen transmission, adaptation, growth, and multiplication in both the tick and the host (labuda and nuttall 2004, horka et al. 2009, socolovschi et al. 2009). haemaphysalis longicornis adult tick length (unengorged) is approximately 2.65 mm (2.7–3.4 mm) with a breadth of 1.8 mm (1.4– 2.0 mm). although body weight increases after engorgement, there is no detectable change to the size of the mouthpart. for this reason, researchers typically use salivary glands to collect protein extracts instead of saliva for proteomic analysis of h. longicornis (lvov et al. 1978, mulenga et al. 1999, wang et al. 2001 oliveira et al. 2011, jinlin et al. 2013, wikel 2013). when collecting saliva, it is critical to inject the tick so the salivary glands are not ruptured or lost in the tick remains. saliva contains additional proteins originating from the salivary gland cells that are not present in tick salivary gland extract itself. many chemical stimulatory techniques have been used for saliva collection, the most common of which are dopamine and pilocarpine (kramer et al. 2008, patton et al. 2012, oliveira et al. 2013). although the hemolymph and other tick fluids rapidly degrade dopamine, it has selective organ action. other stimulatory agents such as pilocarpine have been explored for saliva collection; however, all were shown to affect salivary composition as their action has affects on multiple cell types (ribeiro et al. 2004). in this study, we used dopamine to reduce the chance of changing saliva composition. for the first time, we successfully collected saliva from h. longicornis and characterized that collected saliva via on-chipf lu or es ce nc e (r el at iv e f lu or es ce nc e u ni t, r f u ) protein weight (kda) j arthropod-borne dis, march 2017, 11(1): 10–18 ms islam and mj you: drug induced … 15 http://jad.tums.ac.ir published online: march 14, 2017 electrophoresis analysis. our collected saliva induced by dopamine was ash in color and produced a precipitate following centrifugation at 10,000 × g for 1 min, which is similar to the collected saliva by oliveira et al. (2013). the color change may be due to dopamine itself, most likely because dopamine triggers the phenoloxidase pathway. the protein concentration of these saliva samples was on an average 0.169 μg/μl for engorged h. longicornis females. the concentration of saliva induced by dopamine found in ornithodoros moubata ticks was 0.33 μg/μl for females (wang et al. 2001) and 0.184 μg/μl for rhipicephalus sanguineus ticks (oliveira et al. 2011).our saliva concentration results resemble the protein concentrations found for r. sanguineus, which is predictable as these parasites are almost identical physiologically. saliva collection by our method yielded approximately 1.74 μl per tick with an average high production on day 1 at 3.56 μl and an average low production at 0.55 μl. lucas et al. (2014) found an average of 0.8 μl saliva per fully engorged r. microplus. saliva production decreased in engorged ticks with an increase of time to treat with dopamine. this may be due to degeneration of the salivary gland after detachment from the host. in ticks, the salivary gland is also responsible for hydrodynamic equilibrium (benoit and denlinger 2010) and before detachment from the host ticks excretes more water. due to this, our collected volume of saliva was higher compared to partially fed females as they were found to secrete 0.1 μl saliva per tick (lucas et al. 2014). on-chip-electrophoresis is a good approach for saliva characterization. the commercial agilent high sensitivity protein 250 kit and bioanalyzer 2100 (agilent technologies, usa) system were used for saliva protein determination. this technique is a combination of chip microfluid separation technology and the fluorescent detection of proteins. it automatically performs all the steps of gel-based electrophoresis including sample separation, staining, imaging, band detection, and data analysis. in our electrophoresis results, intense bands were identified at 9.9, 10.3, 10.6, 12, 67, 109, and 190 kda. proline-rich proteins form a major fraction of salivary proteins (approximately 20–30% of total), and the molecular weight of proline-rich proteins (acidic and basic) is usually between 10–40 kda while large glycosylated proline-rich proteins range from 60–70 kda (schwartz et al. 1995). the major sources of salivary prolinerich proteins (prps) are the salivary glands (bishop et al. 2002). a 9.9-kda salivary protein was found in t. infestans (charneau et al. 2007) and a 10.6 kda protein was isolated from t. infestans as a triabin protein. triabin belongs to the family of lipocalins that are extracellular transport proteins and have platelet inhibitor function (andersen et al. 2005). the 67-kda protein corresponds to tick salivary apyrases known to inhibit adp and collagen. these properties indicate that apyrases could play an important role during tick feeding (mans et al. 1998). the 12-kda protein is another characteristic protein family exclusive to both hard and soft ticks that was previously found only in ixodes scapularis, o. coriaceus, and o. moubata (francischetti et al. 2009). the proteins that resemble the 10.6, 12, and 67 kda proteins are likely platelet inhibitors and anti-inflammatory proteins, as tick saliva is known to contain anti-inflammatory components (chmelar et al. 2011, fontaine et al. 2011). when oliveira et al. (2013) treated r. sanguineus with pilocarpine, they found rabbit host proteins in the collected saliva. none of our salivary proteins collected via dopamine treatment matched any rabbit host protein sequences. even still, saliva collected by the dopamine technique has some limitations as only 0.2% dopamine was used and further dose dependent studies need to be conducted to optimize saliva proj arthropod-borne dis, march 2017, 11(1): 10–18 ms islam and mj you: drug induced … 16 http://jad.tums.ac.ir published online: march 14, 2017 duction. an additional problem is the color change of the saliva, most likely due to activation of the phenoloxidase pathway, which could be verified by adding phenoloxidase inhibitors such as dtt or edta to the saliva. lastly, proteomic studies are required to characterize further the protein bands identified in this study. conclusion as most of the protein bands identified in this study do resemble tick salivary proteins, this method could be a useful tool for the primary identification key of h. longicornis antigens for vaccine development. acknowledgements this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education (nrf-2015r 1d1a1a01059607). the authors declare that there is no conflict of interests. references andersen jf, gudderra np, francischetti im, ribeiro jm (2005) the role of salivary lipocalins in blood feeding by rhodnius prolixus. arch insect biochem physiol. 58(2): 97–105. battsetseg b, lucero s, xuan x, claveria f, byambaa b, battur b, boldbaatar d, batsukh z, khaliunaa t, battsetseg g, igarashi i, nagasawa h, fujisaki k (2002) detection of equinebabesia spp. gene fragments in dermacentor nuttalli olenev 1929 infesting mongolian horses, and their amplification in egg and larval progenies. j vet med sci. 64(8): 727–730. benoit jb, denlinger dl (2010) meeting the challenges of on-host and off-host water balance in blood-feeding 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online: march 14, 2017 schwartz ss, zhu wx, sreebny lm (1995) sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human whole saliva. arch oral biol. 40(10): 949–958. socolovschi c, mediannikov o, raoult d, parola p (2009) update on tick-borne bacterial diseases in europe. parasite. 16(4): 259–273. springell ph (1983) the cattle tick in relation to animal production in australia. wld anim rev. (fao). 36: 1–5. wang h, kaufman wr, cui ww, nuttall pa (2001) molecular individuality and adaptation of the tick rhipicephalus appendiculatus in changed feeding environments. med vet entomol. 15(4): 403–412. wikel s (2013) ticks and tick-borne pathogens at the cutaneous interface: host defenses, tick countermeasures, and a suitable environment for pathogen establishment. front microbiol.19(4): 337. zhang p, tian z, liu g, xie j, luo j, zhang l, shen h (2011) characterization of acid phosphatase from the tick haemaphysalis longicornis. vet parasitol. 182 (2–4): 287–296. j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 original article notes on the iran caddisflies and role of annulipalpian hydropsychid caddisflies as a bio-monitoring agent naseh malekei-ravasan 1, abbas bahrami 1, mansoreh shayeghi 1, mohamad ali oshaghi 1, masomeh malek 2, allah bedasht mansoorian 3, *hassan vatandoost 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of biology, tehran university, tehran, iran 3department of parasitology, school of public health, tehran university of medical sciences, tehran, iran (received 20 nov 2012; accepted 9 feb 2013) abstract background: eco-faunistic studies are inevitable step in environmental researches. aquatic organisms like caddisflies are known as biological indicators for water quality assessment and water resource management. they have special role for energy flow in the freshwater habitats as food web and food chain among aquatic creatures. methods: in addition to an extensive literature review on iran caddisflies, a field study was carried out in lavasan river flows in north east of tehran to collect aquatic insects using d-frame nets and or direct search on stone beneath. the water quality was measured using analytical method. results: literature revealed record of 62 trichopterid species in the country comprising 14 families. the most abundant species belonged to the hydropsychidae. herein we report presence of the annulipalpian hydropsyche sciligra h malicky, 1977 in the study area. habitat water quality of h. sciligra resembled human drinkable water. however presence of snail, physa acuta and fish capoeta buhsei in the water sampling area indicated inferior quality. conclusion: from ecological point of view caddisfly larvae are predators of most important medical vectors like mosquitoes, blackflies and midges. also they are useful and important indicator for monitoring physicochemical effects in the nature, so that they can be used for bio-monitoring program. from medical point of view, wing hairs or other body parts of caddisflies can be inhalant and contact allergens in trichopterists and in sensitive individuals who come in contact. key words: caddisfly, hydropsyche, faunistics, bio-monitoring, iran introduction trichoptera, is one of the largest insect orders presenting in aquatic ecosystems. the order includes 3 suborders spicipalpia, annulipalpia and integripalpia each one containing four, eight, and 33 families, respectively. the aquatic larvae assemble a portable case (integripalpia, and two of four families of “spicipalpia”) or fixed retreat (annulipalpia), except for those of a pair of “free-living” spicipalpian families. the net, retreat, case, and pupal structures of caddisfly larvae that filter fine organic matter or larger particles are among the most intricate and complex structures built by insects, at least among the non-social orders (holzenthal et al. 2007). trichoptera and lepidoptera, concoct the amphiesmenoptera super order (kristensen 1991). their presenting and archaic lineages diversification ascribe to the triassic and the middle jurassic respectively. the earliest prototrichopteran dates to the mid-early permian (kristensen 1997). so, trichoptera depict a significant and important branch on the tree of life, whose present distribution and bioge*corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir 71 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 ography is reflective of those past events that have affected the distribution and diversification of earth’s entire freshwater biota. for instance extinction of an endemic alpine caddis fly, rhyacophila angelieri (rhyacophilidae) was predicted with reduced melt water inputs (brown et al. 2007). the role of trichopteran larvae in trophic dynamics and energy flow in the freshwater habitats seems to be inevitable (resh and rosenberg 1984). both of habitats and inhabitants of these ecosystems affected strongly and environmentally intimidated. trichoptera are applicable and important for monitoring physicochemical effects, and are widely used in biomonitoring programs in developed countries (lenat 1993, resh and unzicker 1975, dohet 2002). hydropsychidae curtis (1835), with about 1,500 described species, is the 3rd largest family in trichoptera and the most diverse of the net-spinning annulipalpians. five subfamilies are currently recognized: arctopsychinae, macronematinae, hydropsychinae, diplectroninae, and smicrideinae. hydropsychinae contains 19 prevalently genera from all biogeographic regions, which the largest genera include hydropsyche pictet (275 species), ceratopsche ross and unzicker, often considered a subgenus of hydropsyche (100 species), and cheumatopsyche wallengren (260 species) (schefter 1996, holzenthal et al. 2007). hydropsychid larva exhibits a large spectrum of tolerance values and is used in biomonitoring programs throughout the world. for example, north carolina biotic index (ncbi) tolerance values for hydropsychids range from 0.0 for h. carolina ranks to 8.8 for h. betteni ross (on a scale of 0–10, with 0= least tolerant of pollution) (lenat 1993). one of the major environmental issues that the world faces today is the water crisis (aldhous 2003). industrial activities and urbanization have raised pollution in rivers, streams, and lakes in developing countries. most sewage enters waterways without adequate treatment, and the water quality is being degraded (langergraber and muellegger 2005). loss of freshwater for human consumption is correlated with cease to existing aquatic biodiversity. a critical step is to describe aquatic biota to help developing countries build efficient, fast, and inexpensive techniques to diagnose and monitor negative perturbations to water quality. the use of aquatic organisms as indicators of water quality is a standard method in management of water resources. the pros and cones of the use of macro-invertebrates in biological monitoring were outlined. among the pros is that the taxonomy of many groups is very well known and identification keys are available. this pro is true for the united states and europe, and australia, but lack of taxonomic knowledge and identification manuals is the greatest barrier to the development of biological monitoring studies in other regions like our country (resh 1995, bonada et al. 2006). the iranian trichoptera fauna mostly was studied by non-autochthonous researchers (schmid 1959, malicky 1986, mirmoayedi and malicky 2002, mey 2004, malicky 2004, chvojka 2006) based on collections of 62 species from the 23 provinces of iran. despite the evolutionary, ecological, environmental, and adorable importance of the trichoptera, there is a little knowledge about ecology with emphasizing habitat characteristics of iranian trichoptera. so, to cover this brevity firstly we collected some caddisflies, then listed families presenting in iran with emphasizing hydropsychidae family species and finally measured water quality of surrounding larval habitat and some ecological features of lavasan district, tehran, capital of iran. materials and methods this bi-seasonal (summer/winter) study was conducted on lavasan river runs in northeast of tehran capital of iran. the rampant climate of the region is cold mediterranean. water 72 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 temperature was measured using thermometer during sampling. we collected immature stages of the trichoptera by d-frame nets and/or removing river floor stones from three different habitat types run, riffle and stream bank locating in the way of the running water where larvae stick their retreat under/beside of the stones. the retreats and probably harboring immature insect preserved in 70% ethanol and transferred to the school of public health (sph) laboratory. the key morphological characters of the extracted immature trichoptera plus retreats general feature were examined under microscope (olympus szx12) through taken high resolution photos. water samples were collected in summer 2010, prior the seasonal rains start. the specimens were taken in autoclavable pyrex bottle w/cap 250 ml, previously acidwashed and rinsed with copious amounts of distilled water. electrical conductivity (ec) and total dissolved solids (tds) was measured using ec/tds hach co150 meter. the ph was determined using suntex ts-2 ph meter. color and turbidity of the water samples were examined with hach data logging spectrophotometer 2010. the data was analyzed to establish a relationship between the water quality and trichoptera fauna. results totally seven caddises and only one larva (without retreat) was collected during two referral season in summer and winter 2010. larval and pupal characteristics of h. sciligra are shown in figures 1 through 11 and 12 through 16 respectively. faunistic investigation was carried out and reinforced using two information resources including available published data on iranian trichoptera and the trichoptera world checklist database search. the collected retreats were identified as hydropsychid sp. (fig. 12). with regard to being lethargic collected larva in the winter season, probably trichoptera overwinter in larval stage in this region. hydropsychidae larvae of this family are easily separated from others by the hard sclerotized plates on the top of each thoracic segment and highly branched gills on venter of abdominal segments (fig. 1,2). each branched gills have 30 or more filaments on venter of thoracic segments and on most abdominal segments (fig. 6). larvae of hydropsyche distinguished from other hydropsychid genera by pair of large sclerites in inter-segmental fold posterior to prosternal plate of prosternum (pescador and rasmussen 1995). according to the world checklist database (morse 2010) there are 62 trichopterid species in iran comprising 14 families. suborders, families, number of species, case/retreat making, feeding type and biotic index values (rbp) of iran caddisflies are shown in table 1. on the basis of mentioned source 18 reported iranian hydropsychidae members are presented in table 2. in addition to the caddisflies we collected two invasive following biota that may have impact on the results explanations. bladder snail, physa acuta draparnaud 1805 (gastropoda: physidae) this snail has cosmopolitan distribution and has been spread through human agency around the world (dillon et al. 2002). it is prevalent in lentic waters, especially in rich, disturbed and/or artificially eutrophic environments. p. acuta is a “weedy” or r-selected species (dillon 2000). its rapid maturation, high reproductive rate, and ease of culture have made it the “fruit fly of malacology”. p. acuta is easily the most successful physid, having been dispersed worldwide and found in a variety of few habitats. it is a generalist and reproduction selected. it can survive well under harsh conditions, as long as they are shortlived. other life-cycle characteristics are high 73 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 proliferation rates, high passive dispersal capacities and high tolerance to polluted water (bernot et al. 2005). a high adaptability to changing environmental characters is thought to enhance invasiveness of p. acuta (kefford and nugegoda 2005). capoeta buhsei kessler, 1877 cyprinidae (synonym: varicorhinus nikolskii derjavin, 1929) capoeta was reviewed by karaman (1969) who recognized seven species that two of them, c. buhsei and c. fusca are restricted to iran. c. buhsei, ray-finned fish, a benthopelagic species in freshwater systems, is found in the lake namak basin, iran. c. buhsei has been assessed as least concern due to its relatively large range, and a lack of widespread threats in the area (bianco and banarescu 1982, coad 1998, devi and boguskaya 2009, krupp and schneider 1989). analyzing water quality water quality of lavasan river in the summer 2010 was measured and its mean physicochemical characteristics at the time of sampling were as follow: ec 376 µ s/cm, ph 8.43, tds 188 mg/l, ntu 13.7 ntu, water temperature (at noontime and 50 cm depth) 22.2 ºc. table 1. suborders, families, number of species, case/retreat making, feeding type and biotic index values (rbp) of iran caddisflies are shown suborder family no of species case/retreat making feeding group biotic index values (rbp) a n n u li p al p ia ecnomidae 2 fixed retreat predator, collectors. 0.0 hydropsychidae 18 fixed retreat collector (filterer) 4.0 polycentropodidae 2 fixed retreat collector/shredder/predator 6.0 pseudoneureclipsidae 2 fixed retreat grazers 0.0 psychomyiidae 8 fixed retreat collector (gatherer) 2.0 philopotamidae 3 fixed retreat collector (filterer) 3.0 in te gr ip al p ia leptoceridae 5 portable case shredder/collector/predator 4.0 sericostomatidae 1 portable case shredder/collector (gather) 3.0 lepidostomatidae 3 portable case shredder 1.0 limnephilidae 5 portable case scraper/shredder 4.0 s p ic ip al p ia glossosomatidae 2 portable case scraper/collector (gatherer) 0.0 hydroptilidae 9 portable case, fixed retreat, free-living piercer/scraper/shredder 4.0 hydrobiosidae 1 free-living predator 0.0 rhyacophilidae 1 portable case predator/scraper 0.0 data for biotic index values (rbp) column from hilsenhoff wl (1988) 74 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 table 2. 18 type species records of hydropsychidae from iran and neighborhood countries subfamily taxon distribution region reference diplectroninae diplectrona vairya iran albania baharestan mirmoayedi and malicky, 2002 olah, 2010 hydropsychinae cheumatopsyche processuata iran,india,pakistan,myanmar and vietnam ***** mirmoayedi and malicky, 2002 olah et al 2008 olah and johanson, 2008 hydropsyche consanguinea iran iran iraq chalus, damavand, azarbaijan-e-gharbi (takht soleyman), lorestan (lenje abad) chvojka, 2006 mirmoayedi and malicky, 2002 al-zubaki and al-kayalt, 1987 hydropsyche demavenda (syn: h. integrate) iran china damavand mirmoayedi and malicky, 2002 huang et al 2005 hydropsyche djabai iran iran turkey azarbaijan-e-gharbi (takht soleyman), minoudasht, golestanforst, damavand mirmoayedi and malicky, 2002 chvojka, 2006 sipahiler, 2004 hydropsyche mahrkusha iran turkey turkey mazandaran, alborzgebirge, chalus-karadj assalemhashtpar, moghan mirmoayedi and malicky, 2002 sipahiler, 2004 sipahiler, 2007 hydropsyche ressli iran turkey alborz valley, chalus mirmoayedi and malicky, 2002 sipahiler, 2007 hydropsyche sakarawaka (syn: h. remmerii) iran chalus-karaj mirmoayedi and malicky, 2002 hydropsyche sciligra (syn: h. gracilis) iran turkey caucasus north site of alborz, chalus, makou, qazvin, minoudasht mirmoayedi and malicky, 2002 sipahiler, 2007 ivanov, 2011 hydropsyche supersonica iran iran gilan (gichob), nowshahr mirmoayedi and malicky, 2002 chvojka, 2006 hydropsyche iokaste iran amolchalus mirmoayedi and malicky, 2002 hydropsyche bujnurdica iran bojnurd botosaneanu, 1998 potamyia psamathe iran bushehr chvojka, 2006 cheumatopsyche flavellata iran iran turkey tehran (eyn varzan), minoudasht, rasht, fars (firuz abad, khollar), khuzestan (izeh) mey, 2004 chvojka, 2006 olah, 2010 cheumatopsyche persica iran iran turkey hormozgan (khoshangan), khuzestan (si mili, izeh) azarbaijan-e-gharbi (chuplu), kermanshah (khosrow abad), fars (posht chenar, firuz abad), lorestan (bavineh), zanjan (sorkheh dizaj), chaharmahal va bakhtiyari and kerman mey, 2004 chvojka, 2006 olah, 2010 hydropsyche esfahanica iran isfahan mey, 2004 hydropsyche lundaki iran hamadan (alanje) chvojka, 2006 hydropsyche masula iran masuleh river olah and ka johanson, 2008 *****= unknown 75 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 fig. 1. free living larva fig. 2. larval sclerites (found in the retreat) fig. 3. larval anal projections fig. 4. prosternum fig. 5. anterior part of the body showing trochantin and long antennae fig. 6. abdominal gills, with up to 10 filaments fig. 7. dorsum of abdomen showing club hairs 76 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 fig. 8. distinctly yellow vshaped mark on the frontoclypeus fig. 9. two large brown smudges underside view of head capsule fig. 10. robust prothoracic leg fig. 11. sclerites on the abdominal sternum viii and ix fig. 12. retreat containing pharate adult (late pupa) of h. sciligra fig. 13. extracted pharate adult fig. 14. pupal anal projections 77 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 fig. 15. features on the abdominal segments. fig. 16. features with more resolution on the abdominal segments discussion faunistic studies of geographical areas, such as countries, states, watersheds, bio-geographical regions, or even conservation areas, are a very important resource for environmental studies. ecologists, taxonomists and natural resource managers benefit from these data. iran caddisflies were collected from 23 provinces including azarbaijan-e-gharbi, azarbaijane-sharqi, ardebil, esfahan, fars, gilan, mazandaran, golestan, tehran, alborz, qazvin, hamadan, hormozgan, kerman, kermanshah, khuzestan, kordestan, lorestan, sistan va baluchestan, bushehr, north khorsan, chaharmahal bakhtiari and zanjan provinces mostly by non-autochthonous researchers. caddisfly species of iran are more prevalent along the alborz and less along zagros mountain ranges. from the literatures the northern fauna of iran clearly close to the europeanmediterranean fauna, whereas, in the southern part of the country there is a gap which connects africa and south asia (malicky 1986). in this study we collect specimens from tehran province and evaluated some morphological characters like head, thorax, legs, and abdomen accessories on both larva and late pupa body but we don’t find any similarity in introduced species in manuals and published hydropsychidae keys. except for chetotaxi standpoint that our collecting larva due to lacking minute spines and scale hairs but having club hairs on dorsum of abdomen (fig. 7) was very similar to the hydropsyche (ceratopsyche) sparna (pescador and rasmussen, 1995). although all collecting specimens in our study were identified as hydropsyche sciligra (h malicky 1977 synonym: h. gracilis av martynov, 1909) by vladimir d. ivanov, an expert trichopterologists from russia, however, further molecular investigations are under processing to complete species taxonomy confirmation. this species were reported only from iran, turkey and caucasus and there is no any report from other part of the world (morse 2010). nevertheless, the iranian caddisfly fauna is still incipient, and data are lacking from many regions. insect faunas present in the fluvial water bodies provide a valuable resource to increase our understanding of both succession patterns of ecosystem development at individual sites and spatial comparisons between sites of a similar age (amoros et al. 1987) morphological traits can be as predictors of niche requirements so appraisal of the aquatic macro-invertebrates will provide indication of water quality. different macro-invertebrates have different tolerances to pollution. among the 4 categories of the water bugs sensitivity to pollution, caddisflies larvae together with alderflies and water mites are categorized as sensitive bugs (hunter-central rivers waterwatch). family hyropsychidae, are actually tolerant of poor water quality, but would be counted as indicators of good water quality with other trichoptera in an 78 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 ephemeroptera-plecoptera-trichoptera (ept) index (voshell 2002). water quality determines the ‘goodness’ of water for particular purposes. our collecting water was colorless, odorless liquid, and maybe with an insipid taste. on the basis of who guidelines these samples were generally clean even drinkable (who geneva, 2008). indian recommended standard for drinking water quality parameters for ph, turbidity (ntu), tds (mg/l) and ec (µ s/ cm) are 6.5–8.5, 5, 500 and 400 respectively (is: 10500 1991). three cases of our results fall into these categories except for tds that was 2.5 times less than introduced value. ec and tds of presented in this study is reagent of freshwater rivers characteristics. meanwhile we collected some snails and some fish against these guidelines indicating inferior quality of water and its contamination with human and animal excretions. probable reasons for these controversial findings may be due to ecological changes as a result of growing urbanization in lavasan river upstream. another less important reason may be related to geographical condition of the place. lavasan district have a temperate climate and because of vicinity to tehran metropolis, where many mansions and country houses belonging to wealthy families were built there in recent year. so reliability of aquatic insects versus water samples as measures of aquatic ecosystems safety must be noticed with more subtlety. as we see, in one hand both the literature review (brown et al. 2007) and current study indicate that caddisflies are good agent for bio-monitoring and on the other hand organisms like the snail (p. acuta) and the fish (c. buhsei) couldn’t be a suitable indicator due to high adaptability to changing environmental characters so these vicissitudes must be explored through monitoring of physicochemical characteristics. salinity, a measure of the dissolved salts in the water, is measured as either tds or as ec. a sample’s ec can be converted to tds and vice versa. sources of salinity include urban and rural run-off containing salt, fertilizers and organic matter. land use issues related to high levels of salinity include clearing of vegetation and the resultant rise in the water table, excessive irrigation, groundwater seepage and runoff containing dissolved solids from industry, sewage, and agriculture and storm water. while an appropriate concentration of salts is vital for aquatic plants and animals, salinity that is beyond the normal range for any species of organism will cause stress or even death to that organism (huntercentral rivers waterwatch). the accepted taste threshold for tds is 500–1000 mg/l. very low tds can leave water tasting flat. high tds is associated with scaling corrosion and possibly unhealthy levels of salt. for non drinking purpose up to 1800 mg/l can be managed with frequent check on taps and infrastructure for scale build up (who, geneva 2008). the units of turbidity from a calibrated nephelometer are called nephelometric turbidity units (ntu). to some extent, how much light reflects for a given amount of particulates is dependent upon properties of the particles like their shape, color, and reflectivity. suspended solids usually enter the water as a result of soil erosion from disturbed land or can be traced to the inflow of effluent from sewage plants or industry. turbidity measurements also take into account algae and plankton present in the water (hunter-central rivers waterwatch). who establishes that the turbidity of drinking water shouldn't be more than 5 ntu, and should ideally be below 1 ntu. therefore, low ntu values indicate high water clarity, while high ntu values indicate low water clarity. (who, geneva 2008). ph varies naturally within streams as a result of photosynthesis. geology, soils types and different runoff affect ph. extreme values of ph can cause problems for aquatic fauna. a 79 j arthropod-borne dis, june 2013, 7(1): 71–82 n malekei-ravasan et al.: notes on the iran… http://jad.tums.ac.ir published online: april 10, 2013 ph range of 6.5–8 is optimal for freshwater (hunter-central rivers waterwatch). temperature of a waterway is significant because it affects the amount of dissolved oxygen in the water. the amount of oxygen that will dissolve in water increases as temperature decreases. water at 0 °c will hold up to 14.6 mg of oxygen per liter, while at 30 °c it will hold only up to 7.6 mg/l. temperature also affects the rate of photosynthesis of plants, the metabolic rate of aquatic animals, rates of development, timing and success of reproduction, mobility, migration patterns and the sensitivity of organisms to toxins, parasites and disease. life cycles of aquatic organisms are often related to changes in temperature. temperature ranges for plants and animals can be affected by manmade structures such as dams and weirs and releases of water from them (hunter-central rivers waterwatch). while net-spinning and retreats maker caddisflies are filter feeders and use in biomonitoring programs, free-living ones that build neither cases nor nets are predaceous on the most important medical vectors including mosquitoes, blackflies and midges and use in biological control programs. larvae of hydrophilidae feed on mosquitoes larvae and are therefore relatively important predators of mosquitoes (hintz 1951, nielson and nielson 1953). the 2–3 cm long larvae of phrynganea sp and limnephilus sp have often observed capturing larvae of the snowmelt mosquitoes (culicidae: culiseta and aedes) (becker et al. 2010). rhyacophila larvae are generally predaceous, feeding on simuliidae (black fly) larvae, chironomidae (midge) larvae and pupae, and the pupae of other caddisflies (thut 1969, wiggins 2004). caddisflies normally are not considered medically important insects. they can cause allergic responses (asthma and dermatitis) in sensitive individuals who come in contact with wing hairs or other body parts. trichopterists who collect adult caddisflies by aspiration may receive significant exposure to wing hairs. the literature suggests that hypersensitivity to caddisflies may be quite common among allergy patients. the range of reported responses is broad, ranging from minor annoyance to near incapacitation. some patients sensitized by exposure to caddisfly antigens have developed cross-reactivity to shellfish and stings of venomous insects. this could result in life-threatening situations for individuals with a predisposition for severe ige-mediated reactions (anaphylaxis) due to contact with arthropod proteins (david e. bowles) acknowledgements we would like to thank dr vladimir d ivanov, a thoroughbred trichopterologist, from the department of entomology, faculty of biology, sent petersburg state university for identifying and providing some good advises. we also express gratitude to dr mahmood ali mohammadi from department of environmental health engineering, sph, tums for testing of water quality. references aldhous p (2003) the world's forgotten crisis. nature. 422: 251–253. amoros c, roux al, reygrobellet jl, bravard jp, pautou g (1987) a method for applied ecological studies of fluvial hydro systems. regul rivers 1: 17–36. becker n, petric d, zgomba m, boase c, madon m, dahl c, kaiser a (2010) mosquitoes and their control. springer, heidelberg, dordrecht, new york. bernot rj, kennedy ee, lamberti ga (2005) effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail physa acuta. environ toxicol chem. 24: 1759–1765. bianco pg, banarescu p (1982) a contribution to the knowledge of the cyprinidae of iran (pisces, cypriniformes). cybium. 6(2): 75–96. 80 j arthropod-borne 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smith tp (2002) populations of the european freshwater pulmonate physa acuta are not reproductively isolated from american physa heterostropha or physa integra. invertebr biol. 121(3): 226–234. dillon rtjr (2000) the ecology of freshwater molluscs. cambridge university press, cambridge, united kingdom. dohet a (2002) are caddisflies an ideal group for the biological assessment of water quality in streams? nova supplementa entomologica (proceedings of the 10th international symposium on trichoptera). 15: 507–520. hintz hw (1951) the role of certain arthropods in reducing mosquito populations of permanent ponds in ohio. ohio j sci. 51: 277–279. hilsenhoff wl (1988) rapid field assessment of organic pollution with a familylevel biotic index. j north am benthol soc. 7: 65-68. holzenthal rw, blahnik rj, prather al, kjer km (2007) order trichoptera kirby, 1813, (insecta), caddisflies. zootaxa. 1668: 639–698. hunter-central rivers waterwatch, water quality parameters and indicators, fact 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accepted 27 sep 2014) abstract background: tropical rat mite (ornithonyssus bacoti) is reported from many parts of the world and is considered important in transmitting rickettsial pathogens. there have been scanty reports on prevalence of this parasite from india. following a recent report of o. bacoti infestation in a laboratory mice colony from nilgiris, tamil nadu, india, attempts were made to detect the parasite in its natural reservoir, ie the domestic and peridomestic rats (rattus rattus). methods: the national centre for disease control, coonoor is involved in screening plague in domestic and peridomestic rats in nilgiris and erstwhile plague endemic areas of southern india. the parasite samples were identified based on the morphological characteristics attributable to o. bacoti and as per description of published literature. results: seven mite samples identified as o. bacoti based on morphological characteristics were isolated incidentally from domestic and peridomestic rodents in and around the hilly districts of nilgiris, southern india, during the routine plague surveillance programme. the identification was based on the morphological characteristics attributable to o. bacoti observed under a low power microscope. conclusion: in india, this is probably the first record of isolation of o. bacoti from domestic and peridomestic rodents. prevalence of such parasite in domestic and peridomestic rats necessitates further investigation on monitoring and surveillance of rickettsial diseases in the locality, as these parasites are considered to be potential vector of transmitting rickettsial pathogens. keywords: ornithonyssus bacoti, india, tropical rat mite introduction ornithonyssus bacoti hirst, 1931 (tropical rat mite), an ectoparasite belongs to the family macronyssidae, is an obligate, bloodfeeding parasite commonly associated with wild rat throughout the world. the mite is found mostly in tropical and moderate climate zone which causes rat mite dermatitis in human (beck and fölster-holst 2009). although the status of rat mites as vectors in naturally occurring human infections has not been proven, yet there are considerable numbers of publications indicating that o. bacoti experimentally transmits rickettsia akari (rickettsial pox), francisella pestis (plague), coxsackie virus, francisella tularensis (tularemia), and trypanosoma cruzi (chagas disease) and there is evidence that o. bacoti specimens are positive for coxiella burnetii (q fever), hantavirus, borrelia sp, bartonella sp, and rickettsia sp as reviewed by watson (2008). the definitive diagnosis of o. bacoti requires the detection of the parasite which is more likely to be found in the environment of its host (cages, litter, corner or cracks of living area) than on host’s skin itself (beck *corresponding author: dr pranab jyoti bhuyan, e-mail: pranabjyoti333@rediffmail.com http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 98–101 pj bhuyan and aj nath: record of tropical … 99 and fölster-holst 2009). there has been scanty report of this parasite from india. however, the recent report by nath et al. (2013) from nilgiris in a breeding facility of swiss mice necessitated the search for the parasite in its reservoir, i.e. the domestic and peri-domestic rats, which are under plague surveillance activity of national centre for disease control, coonoor, nilgiris. this study reveals the detection of o. bacoti from domestic and peri-domestic rats in nilgiris hills, tamil nadu during the routine surveillance of plague from rodents and is probably the first of its kind in this topography. materials and methods during the routine surveillance of plague in and around nilgiris district, the domestic and peridomestic rats were collected by wonder trap (live trapping, multiple catch). the dead rodents were excluded from the routine surveillance of plague as per standard procedure. the rats were classified as domestic and peridomestic are as per criteria provided by agarwala et al. (2005). the rats were euthanized following standard protocol and searched for ectoparasite by combing on a white tray. during january-august, 2013, our branch of ncdc received 401 rats, out of which 323 belonged to rattus rattus (based on morphology as per standard literature). the collected mite samples were preserved in 10 % formalin and later examined in the laboratory and confirmed as o. bacoti according to entomology walker key (walker 1997). nilgiris is a hilly district located in the northwestern part of tamil nadu state bordering kerala and karnataka, southern india. it lies between the latitude of 11˚10’ n and longitude of 76˚5’ situated in altitude ranging from 900 to 2600 meters above m s l (mean sea level) with 56.42 % forest cover. the nilgiris district has an annual rain fall of 1,200 mm, average daily temperature ranges between 5–28 oc. these peculiar land configurations, prevailing climatic conditions, abundance of varieties of rodents and ectoparasites harboring on such rodents might attributes to the endemicity of various disease in such locality. nilgiris district has long been recognized as a potential plague endemic area which was attributed to the existence of complex factors of climate and a broad spectrum of rodent and flea fauna. results all the seven mite samples in the present study were identified as o. bacoti based on their morphological characteristics attributable to o. bacoti, such as elongated, oval and hairy body, caudally tapered dorsal shield etc. (engel et al. 1998, watson 2008, beck and fölster-holst 2009). the gnathosoma has chelicera and pedipalp. figure 1 depicts a female o. bacoti mite observed under 10x objective of a light microscope (nikon). all the mite samples were recovered from the domestic and peridomestic rattus rattus (n=323). the parasite could not be found in the other rodent species, ie bandicoota bengalensis (n=78) collected from the same areas during the said collection period. fig. 1. an adult female ornithonyssus bacoti under 10x objective http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 98–101 pj bhuyan and aj nath: record of tropical … 100 table 1. some characteristic features that aid differentiation of ornithonyssus bacoti from other confusing parasites viz. d. gallinae and o. sylviarum morphological features o. bacoti d. gallinae o. sylviarum hairiness very hairy few hair few hair dorsal plate caudally pointed dorsal plate caudally rounded dorsal plate dorsal plate with a conical end. anal plate anal plate with cranial anus anal plate with caudal anus anal plate with caudal anus discussion ornithonyssus bacoti is prevalent worldwide except arctic and antarctic regions as a parasite of wild, commensal and laboratory rodents or of humans (engel et al. 1998, abai et al. 2002, sabol-jones et al. 2005, beck and fölster-holst 2009, radhar and vazirianzadeh 2009). it is reported from india as occupational disease in laboratory personnel and veterinary students (tikaram et al. 1986) and recently, in laboratory mice colony (nath et al. 2013). although r. norvegicus and r. rattus are known to be the preferred hosts, infestation of 10 other rodent species has been reported (engel et al. 1998). however, they might infest a wide range of hosts, including domestic and wild animals and birds (watson 2008) when the preferred hosts are not available. this parasite is more likely to be found in the environment of its host, viz., in the cages, in the litter, or in corners, or cracks of the living area, than on the hosts’ skin itself (beck and fölster-holst 2009). this might justify the very low record of its recovery from the trapped animals in the present study. just as most representatives of the macronyssidae tropical rat mites are active at night and seek dark hiding places during the daytime. at night the parasites search for their preferential hosts (norway rat, house rat, mouse, gerbil and hamster) to feed on blood (beck and fölster-holst 2009). however, the record of this parasite from domestic and peridomestic rodents in a locality is a potential threat of transmitting zoonotic diseases. the tropical rat mite dermatitis in human is a self-limiting disease but a condition for diagnostic dilemma which leads to underreporting of suspected number of unrecognized infestation. certain morphological structures (e.g. hairiness, caudally pointed scutum [dorsal plate], typical form of the anal plate with a cranial anus) allow for differentiation of the tropical rat mite from other mite species like red bird mite (dermanyssus gallinae) and the nordic bird mite (o. sylviarum) (beck and fölster-holst 2009) which are enlisted in table 1. recently, there was a report of an outbreak of o. bacoti infestation in the laboratory mice colony of from the nilgiris district (nath et al. 2013). therefore it is obvious that the parasite is being harbored by some rodents. but, available literature suggests that there is no record of prevalence of o. bacoti in rodents in this particular geographical area. this report, probably, is the first of its kind about the prevalence of this parasite from the domestic and peridomestic rodents in this topography. therefore, the epizootiology and the diseases transmitted by such parasites in the community under risk should be studied extensively. conclusion with the evidence of an outbreak of o. bacoti infestation in laboratory mice colony http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 98–101 pj bhuyan and aj nath: record of tropical … 101 in the nilgiri hills of southern india it is also recorded from the domestic/ peridomestic rodets in the same area. prevalence of such parasite necessitates further investigation on monitoring and surveillance of rickettsial diseases in the locality, as these parasites are considered to be potential vector of transmitting rickettsial pathogens. acknowledgements there is no conflict of interest. references abai mr, motabar m, vatandoost h, javadian e (2002) the first case report of rat mite dermatitis due to ornithonyssus bacoti in southern iran. proceedings of 4th international conference on urban pest, 2002 july 7–10, port-city of charleston, south carolina, united states of america, pp. 471–472. agarwala sp, lal s, ichhpujani rl, mittal v, singh j (2005) plague control in india. national institute of communicable disease, directorate general of health services, ministry of health and family welfare, govt of india, new delhi. beck w, fölster-holst r (2009) tropical rat mites (ornithonyssus bacoti)-serious ectoparasites. j dtsch dermatol ges. 7: 1–4. engel pm, welzel j, maass m, schramm u, wolff hh (1998) tropical rat mite dermatitis: case report and review. clin infect dis. 27: 1465–1469. nath aj, venkataramana kn, fölster-holst r, beck w (2013) epizootiology, treatment and control of tropical ratmite infestation in a breeding colony of swiss mice under temperate climate of nilgiris hill-india. anim sci reporter. 7(3): 114–120. rahdar m, vazirianzadeh b (2009) a case report of tropical rat mite infestation ornithonyssus bacoti (dermanyssidae: acarina) in ahvaz, sw iran. jundishapur j microbiol. 2(2): 78–80. sabol-jones m, karolewski b, byford t, cole js (2005) ornithonyssus bacoti infestation and elimination from a mouse colony. contemp top lab anim. 44: 27–30. watson j (2008) new building, old parasite: mesostigmatid mites-an ever-present threat to barrier rodent facilities. ilar j. 49(3): 303–309. http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, december 2016, 10(4): 608–612 f temocin et al.: sandfly fever … 608 http://jad.tums.ac.ir published online: october 04, 2016 case report sandfly fever with skin lesions: a case series from turkey fatih temocin 1, *tugba sari 2, necla tulek 3 1department of infectious diseases and clinical microbiology, yozgat hospital, yozgat, turkey 2department of infectious diseases and clinical microbiology, buldan chest diseases hospital, buldan/denizli, turkey 3clinic of infectious diseases and clinical microbiology, ankara training and research hospital, ankara, turkey (received 13 apr 2015; accepted 17 feb 2016) abstract sand fly fever (sf) is an arthropod-borne viral disease, also known as “phlebotomus fever”, ‘’mosquito fever’’, three-day fever or “papatacci fever”. it is transmitted by phlebotomus papatasi, starts with acute onset of high fever, and lasts for three days. we present first cases in a different district of turkey with the clinical findings of fever, myalgia-arthralgia, headache, gastrointestinal symptoms such as diarrhoea and nausea-vomiting and skin lesions (in two of them). all the patients were treated symptomatically and discharged with complete cure. these cases are indicating that sand fly fever is more common than we thought. it should be considered in the differential diagnosis in patients presenting with fever, arthralgia-myalgia and skin lesions, especially it is important to be aware of this disease in travellers returning from endemic areas. keywords: sandfly fever, phlebotomus fever, skin lesions introduction sand fly fever (sf), also known as phlebotomus fever, three-day fever or pappataci fever, is an arthropod-borne disease caused by different types of phlebovirus belonging to family bunyaviridae and transmitted by phlebotomine sand flies (dionisio et al. 2003). sand fly fever occurs in subtropical countries, particularly in the countries around the mediterranean sea, and cases have been reported from the balkans, russia, iraq, iran, pakistan, india and some parts of south america (eitrem et al. 1991, saidi et al. 1977, tavana 2007). in the mediterranean area, the common serotypes of phleboviruses are sand fly fever sicilian virus (sfsv), sand fly fever naples virus (sfnv) and toscana virus (tosv) (izri et al. 2008). in endemic areas, infections occur during the summer due to the activity of the sand fly vectors (dionisio et al. 2003, konstantinou et al. 2007). furthermore, the disease may occur among travellers and troops going to endemic countries and among immigrants from endemic regions (eitrem et al. 1990, 1991, imirzalioglu et al. 2006, ellis et al. 2008, nissen et al. 2011). infections with sand fly fever are generally mild, but acute encephalitis may present with toscana virus. the incubation period is between three and six days, and then high fever (39–40 °c) develops afterwards (threeday fever) (dionisio et al. 2003, konstantinou et al. 2007). patients present with influenza-like symptoms, including headache, myalgia, photophobia, retro-orbital pain, arthralgia, fatigue, chills and abdominal discomfort, and they usually recover within a few days to weeks. significant leukopenia, thrombocytopenia and elevation of serum aspartateaminotransferase (ast) and alanine-aminotrans*corresponding authors: dr tugba sari, e-mail: drtugba82@gmail.com j arthropod-borne dis, december 2016, 10(4): 608–612 f temocin et al.: sandfly fever … 609 http://jad.tums.ac.ir published online: october 04, 2016 ferase (alt) levels are characteristic laboratory findings (dionisio et al. 2003, konstantinou et al. 2007). given that the symptoms of sand fly fever are non-specific and may mimic a broad spectrum of infectious diseases and haematological malignancies, difficulty in differential diagnosis and misdiagnosis is frequent. recently, sand fly fever and crimean-congo haemorrhagic fever (cchf) have become important public health problems in some regions in turkey although their vectors are different. these two diseases are characterised by fever, arthralgia-myalgia headache, gastrointestinal symptoms such as diarrhoea, nausea and vomiting, and abnormalities in liver enzymes and haematological test results (mcgraw hill 2007). distinguishing one disease from the other has become important because of the different transmission, prognosis and management of the diseases. although hives on skin due to insect bites may occur in cases with sand fly fever, primary skin lesions have not been described previously. in this paper, we present the first cases of sand fly fever infection with skin lesions misdiagnosed as cchf in yozgat province, turkey. we aim to increase awareness about sand fly fever and its unusual findings. yozgat is a province in central anatolia located in an endemic area for cchf. the city has not been reported as the endemic areas for sand fly fever, and thus the other purpose is to emphasise that sand fly fever may be more common than we thought. case report four patients from yozgat province in 2015 were admitted to the infectious disease unit of yozgat state hospital, yozgat province, turkey, with clinical findings of fever, myalgia-arthralgia, headache, gastrointestinal symptoms such as diarrhoea, nausea and vomiting, and skin lesions (superficial erythematous lesion in two patients). the patients dealt with livestock, lived in a rural endemic area for cchf, but had no history of tick bites. in the physical examination, all patients had fever >38.3 °c, and their system evaluations revealed normal findings except for the dermatologic lesions (fig. 1). laboratory findings revealed leukopenia (2100 cell/µl–4500 cell/µl), thrombocytopenia (80000 cell/µ l–110000 cell/µ l), elevated ast (65 iu/l–218 iu/l), elevated alt (72 iu/l–305 iu/l), elevated creatinine phosphokinase (268 u/l–1441 u/l) and elevated c-reactive protein (35 mg/l–50 mg/l) in all patients. the patients were hospitalised with presumptive diagnosis of cchf because of haematological findings accompanying high fever. during hospitalisation, additional microbiological analyses were performed for differential diagnosis. stool samples were obtained from three patients suffering from diarrhoea. direct microscopic examination and culture of the stool samples did not reveal any features. gruber–widal agglutination tests for salmonella and tube agglutination tests for brucella were negative. the clinical and laboratory findings of the patients are presented in table 1. the patients were tested for possible cchf, borrelia burgdorferi and sand fly fever. although the same vector is involved in cutaneous leishmaniasis, we did not perform any test for cutaneous leishmaniasis but preferred to follow-up on the lesions of the patients when we considered the clinical findings and abnormal laboratory values. serum samples were sent to the virology reference laboratory of public health institution of turkey in ankara. all serum samples were negative for cchf real-time polymerase chain reaction (rt-pcr) and cchf igm. borrelia burgdorferi and also tested by a commercial mosaic immunofluorescence test (ift) (euroimmun, germany) for the detection of specific igm and igg antibodies against sfsv, sfnv, and tosv. sfv-igg antibodies were positive for the sfsv serotype in two patients, and sfvj arthropod-borne dis, december 2016, 10(4): 608–612 f temocin et al.: sandfly fever … 610 http://jad.tums.ac.ir published online: october 04, 2016 igm antibodies were positive for the sfsv serotype in all patients. sfsv by rt-pcr was positive in all patients. all patients were treated symptomatically and discharged following complete recovery, including that of skin lesions. the diagnosis of lyme disease and cutaneous leishmaniasis was excluded clinically. table 1. symptoms, clinical and laboratory findings of patients patient number 1 2 3 4 gender/age (yr) female/24 male/26 male/27 male/28 fever 38.3 39.7 38.8 39.1 headache yes yes no no myalgia/arthralgia yes yes yes yes diarrhoea no yes yes no nausea/vomiting no yes yes no skin lesion yes yes no no wbc count (109/l) 2100 2700 3200 4500 platelets ( 109/l) 80000 82000 98000 110000 crp (mg/dl)* 38 35 42 50 ast/alt (i.u)* 65/86 92/72 218/305 124/99 ck (mg/dl)* 743 1441 268 648 sfv-igm + + + + sfv-igg + + pcr result + + + + wbc, white blood cell, crp, c-reactive protein, ast, aspartate aminotransferase; alt, alanine aminotransferase, ck, creatine kinase, sfv, sand fly virus, *: normal values: ck 21–232 mg/dl, crp <5 mg/dl, ast 15–37 mg/dl, alt 30–65 mg/dl. fig. 1. superficial erythematous lesions of patients discussion although sand fly fever has been known for a long time, it remains a significant public health problem in some parts of world (tesh et al. 1976, dionisio et al. 2003). the prevalence of sand fly fever is related to the distribution of its vector. after world war ii, human cases and outbreaks were reported from many countries, mainly around the mediterranean area, the middle east and central asia (saidi et al. 1977, darwish et al. 1987, tavana 2001, konstantinou et al. 2007). infected phlebotomine sand flies remain infected through their lifetime, and the virus may be transmitted transovarially, which leads to the further spread of the disease. the distribution of the disease may not be limited to the reported areas and may be underdiagnosed in many regions, even in endemic regions (depaquit et al. 2010). sand fly fever viruses cause a variable clinical picture involving asymptomatic diseases, mild and self-limited diseases and central nervous system complications (dionisio et al. 2003 and imirzalioglu et al. 2006). none of the clinical and laboratory findings is distinctive. therefore, most infectious diseases, such as influenza, viral haemorrhagic j arthropod-borne dis, december 2016, 10(4): 608–612 f temocin et al.: sandfly fever … 611 http://jad.tums.ac.ir published online: october 04, 2016 fever, brucellosis and salmonellosis, are determined in differential diagnosis depending on the regional endemic diseases. conversely, sand fly fever is a neglected disease. as it is usually not suspected, diagnosis is difficult and may lead to unnecessary and expensive laboratory tests especially in travel-related conditions. recently, sand fly fever outbreaks and cases have been reported in turkey, especially in the mediterranean region and in central anatolia. all three serotypes (sfsv, sfnv and sand fly turkey virus) have been identified (ergunay et al. 2011). in this report, the first cases of sand fly fever in a distinct city were described. fever, myalgia– arthralgia, headache and gastrointestinal symptoms such as diarrhoea, nausea and vomiting were the most common symptoms in our cases as previously reported. laboratory findings included leukopenia, thrombocytopenia, elevation of alt and ast levels and elevation of creatinine phosphokinase (konstantinou et al. 2007, ergunay et al. 2011). as yozgat is an endemic region for cchf, we had to consider presumptive diagnosis. moreover, we had to distinguish it from lyme disease because of the appearance of lesions on the leg of the first patient. similar findings have also been presented in previous studies, but skin lesions have not been reported (eitrem et al. 1990, simsek et al. 2007, konstantinou et al. 2007, semenza and menne 2009, torun et al. 2010, carhan et al. 2010, ergunay et al. 2011). cutaneous leishmaniasis is another disease to distinguish. the detection of igm antibody is a reliable method for the diagnosis of an acute sand fly fever infection, but cross-reactions can occur among sfv serotypes (dionisio et al. 2003). we confirmed the diagnosis by pcr. some recommendations to be considered for sand fly fever prevention. these recommendations include conduct public awareness and education campaigns, avoid contact with animals and apply repellents in endemic areas, in addition to being awareness of sand fly fever signs and symptoms (tavana 2015). conclusion sand fly fever may be common in all subtropical countries and may present with unusual symptoms, as shown in this case report. physicians should keep it in mind during differential diagnosis of cases with compatible clinical manifestations, even in the presence of dermatological lesions in travellers returning from endemic areas. acknowledgements we acknowledge the contributions of all research team members who have played crucial role in data acquisition. each author has contributed important intellectual content during manuscript drafting or revision. the authors declare that there is no conflict of interests. references carhan a, uyar y, ozkaya e, ertek m, dobler g, dilcher m, wang y, spiegel m, hufert f, weidmann m (2010) characterization of a sandfly fever sicilian virus isolated during a sand fly fever epidemic in turkey. j clin virol. 48: 264–269. darwish ma, feinsod fm, scott rm, ksiazek tg, botros ba, farrag ih, said se (1987) arboviral causes of non-specific fever and myalgia in a fever hospital patient population in cairo, egypt. trans r soc trop med hyg. 81(6): 1001–1003. depaquit j, grandadam m, fouque f, andry p, peyrefitte c (2010) arthropod-borne viruses transmitted by phlebotomine sand flies in europe: a review. euro surveill. 15(10): 19507. j arthropod-borne dis, december 2016, 10(4): 608–612 f temocin et al.: sandfly fever … 612 http://jad.tums.ac.ir published online: october 04, 2016 dionisio d, esperti f, vivarelli a, valassina m (2003) epidemiological, clinical and laboratory aspects of sand fly fever. curr opin infect dis. 16: 383–388. eitrem r, vene s, niklasson b (1990) incidence of sand fly fever among swedish united nations soldiers on cyprus during 1985. am j trop med hyg. 43: 207–211. eitrem r, niklasson b, weiland o (1991) sand fly fever among swedish tourists 1991. scand j infect dis. 23: 451–457. ellis sb, appenzeller g, lee h, mullen k, swenness r, pimentel g, mohareb e, warner c (2008) outbreak of sand fly fever in central iraq. mil med. 173: 949–953. ergunay k, saygan mb, aydogan s, lo mm, weidmann m, dilcher m, sener b, hascelik g, pınar a, us d (2011) sand fly fever virus activity in central/ northern anatolia, turkey: first report of toscana virus infections. clin microbiol infect. 17(4): 575–581. imirzalioglu c, schaller m, bretzel rg (2006) sand fly fever naples virus serotype toscana infection with meningeal involvement after a vacation in italy. dtsch med wochenschr. 131: 2838– 2840. izri a, temmam s, moureau g, hamrioui b, de lamballerie x, charrel rn (2008) sand fly fever sicilian virus, algeria. emerg infect dis. 14: 795–797. konstantinou gn, papa a, antoniadis a (2007) sand fly-fever outbreak in cyprus: are phleboviruses still a health problem? travel med infect dis. 5: 239–242. mcgraw hill (2007) arthropod borne and rodent-borne viral diseases in: melnick and adelberg’s medical microbiology. 24th ed. new york, pp. 511–524. nissen nb, jespersen s, vinner l, fomsgaard a, laursen a (2011) sand fly virus meningitis in a danish traveller returning from tuscany. ugeskr laeger. 173: 2505–2506. saidi s, tesh rb, javadian e, sahabi z, nadim a (1977) studies on the epidemiology of sand fly fever in iran. ii. the prevalence of human and animal infection with five phlebotomus fever virus serotypes in isfahan province. am j trop med hyg. 26(2): 288–293. semenza jc, menne b (2009) climate change and infectious diseases in europe. lancet infect dis. 9: 365–375. simsek fm, alten b, caglar ss, ozbel y, aytekin am, kaynas s, belen a, kasap oe, yaman m, rastgeldi s (2007) distribution and altitudinal structuring of phlebotomine sand flies (diptera: psychodidae) in southern anatolia, turkey: their relation to human cutaneous leishmaniasis. j vector ecol. 32: 269–279. tavana am (2001) the seroepidemiological studies of sand fly fever in iran during imposed war iran. j public health. 30 (3–4):145–146. tavana am (2007) minireview on sand fly fever. j entomol. 4: 401–403. tavana am (2015) sand fly fever in the world. ann trop med public health. 8: 83–87. tesh rb, saidi s, gajdamovic s j, rodhain f, vesenjak-hirjan j (1976) serological studies on the epidemiology of sand fly fever in the old world. bull world health org. 54: 663–674. torun edis c, yagcı caglayık d, uyar y, korukluoglu g, ertek m (2010) sand fly fever outbreak in a province at central anatolia, turkey. mikrobiyol bul. 44: 431–439. chryzomya iranian j arthropod-borne dis, (2009), 3(1): 60-63 a faramarzi et al: chrysomya bezziana as … 60 case report chrysomya bezziana as a causative agent of human myiasis in fars province, southern iran a faramarzi, ar rasekhi, m kalantari, *gr hatam department of parasitology and mycology, school of medicine, shiraz university of medical sciences, iran (received 13 jun 2009; accepted 1 sep 2009) abstract myiasis is the invasion of body tissues of humans or animals by the larvae of the diptera or two-winged flies. the various forms of myiasis may be classified from clinical or entomological point. this study describes the existence of chrysomya bezziana (diptera: calliphoridae) cases as a causative agent of myiasis in 18 and 87 year-old men in two different regions in fars province. to our knowledge, this is the first observation of mentioned species in this province. keywords: chrysomya bezziana, myiasis, iran introduction myiasis can be defined as invasion of the organs and tissues of human or vertebrate animals with dipterous larvae (rohela et al. 2006). dipterous larvae belonging to the family calliphoridae are able to invade to humans’ tissues or cavities (granz et al. 1975). they are usually saprophagous, feeding on carcasses, but the eggs can also be laid upon livestock and humans, or either on wounds, sores or areas contaminated with faeces or urine, where the feeding of larvae causes debilitating myiases (green et al. 2004). they have been reported as a secondary myiasis producer in livestock operations (byrd and allen 2001). myiasis appears to be fairly common but underestimated in many rural areas. in some areas of fars province in the south of iran and, the cases of myiasis caused by this family specially belonging to chrysomya bezziana have been increased (navidpour et al. 1996; radmanesh et al. 2000, masoodi et al. 2004). this study presents two patient cases of myiasis infected by c. bezziana. the importance of this study was to report the new species of myiasis in fars province in the south of iran. case report two cases with the following characteristics were investigated in this study. an 18 yr old boy from lamerd county was one of the victims of variable myiasis cases causes in fars province. he was referred to khalili hospital (affiliated to shiraz university of medical sciences) by his mother. he was a known case of congenital cerebral palsy with quadriplegia and mental retardation associated with severe failure to thrive. he was referred due to an oral lesion and poor feeding because of his inability to feed orally. his family was in low economic status with poor hygiene. in physical examination, he looked like a 5 yr old boy due to severe failure to thrive. he had diffused erythematous, a tender hard palate and a superficial ulcerative mucosal lesion between the peripheral aspect of the palate and the upper gum, associated with odynophagia, lethargy and dehydration. a total of 33 worm-like organisms were detected in the peripheral part of his palate (fig. 1a). during the hospital course, after local and supportive therapy condition of the patient, he improved and was discharged from hospital. *coresponding author: dr gholam reza hatam, e-mail: hatamghr@sums.ac.ir iranian j arthropod-borne dis, (2009), 3(1): 60-63 a faramarzi et al: chrysomya bezziana as … 61 another case was an 87 yr old addict man with cardiovascular as well as peripheral vascular disease based on the longstanding diabetes mellitus from shiraz, southern iran. he also referred to hospital with chief complaint of severe itching of both lower extremities. the patient had history of both feet vascular ulcers for which receiving pentoxiphiline at a dose of 1000 mg/day and also antianginal drugs. in both cases, the different stages of isolated larvae were cultured in blood agar (ba) medium. the growth rates of them were followed up at 37 oc incubator, and the metamorphosis of the pupa to the adult stage were followed in glass flasks containing ba medium (kalantari et al. 2006). diagnosis of myiasis causing larvae is based on the form of spiracles, cephalopharyngeal skeleton, shape and size of developed larvae: the posterior spiracles (fig. 1c), do not have a distinct button and the numbers of lobes on the anterior spiracles are six, which would give a definitive identification of c. bezziana. finally, the robust spine bands (fig. 1b), are other indicative criteria of c. bezziana (belding 196), as well as characteristics of reared adults. in the subfamily chrysomyinae, body-length varies between 8 and 12 mm, abdomen and thorax is shining metallic, dorsum of the first and the second abdominal tergites are black. body is metallic green or blue and abdominal tergites include narrow dark bands along posterior margins. legs are black or partly dark-brown; wings are hyaline with the infuscated base (fig. 2) (whitworth 2006). based on the mentioned diagnostic keys and comparing the whole shape of the larvae (fig. 1b), posterior spiracles (fig. 1c), cephalopharyngeal skeleton (fig. 1d), and also adult characteristics, they were diagnosed as chrysomya bezziana (diptera: calliphoridae). fig.1. (a): the patient's mouth part infested by chrysomya bezziana larva, (b): c. bezziana in the third stage of larvae (125×), (c): posterior spiracle of c. bezziana in the third stage of larvae (125×), (d): cephalopharyngeal skeleton of c. bezziana in the third larval stage (125×) (original photos) iranian j arthropod-borne dis, (2009), 3(1): 60-63 a faramarzi et al: chrysomya bezziana as … 62 fig. 2. details of the adult of chrysomya bezziana found in this study: (a): whole body (40×), (b): wing (100×), thorax (125×), (d): head (125×) (original photos). discussion a number of species in calliphoridae family are responsible for causing myiasis in human and animals (zumpt 1965, granz et al. 1975). human myiasis due to this family have been reported from the united states of america (hall et al. 1986; alexis and mittleman 1988; miller et al. 1990) and canada (alikhan and ali-khan 1975). they have also been reported as the agents of urogenital myiasis in pakistan (jabbar-khan and jabbar-khan 1985). lucilia sericata (diptera: calliphoridae) has caused wound (talari et al. 2004) and auricular myiasis (yaghoobi et al. 2005) in the central parts of iran. a rare case of internal myiasis caused by cynomyopsis cadaverina has been reported from shiraz, southern iran (kalantari et al. 2006). an oral mucosal myiasis has been caused by oestrus ovis in hamadan, situated 336 km south west of tehran (hakimi and yazdi. 2002). in shiraz, a case of gingival myiasis due to wohlfahrtia magnifica has been reported (mohammadzadeh et al. 2008). c. bezziana (diptera: calliphoridae) has been reported from khoozestan province, southeastern, iran (navidpour et al. 1996; radmanesh et al. 2000). however, to our knowledge this is the first observation of c. bezziana as the causative agent of oral and wound myiasis in fars province. references alexis jb, mittleman re (1988) an unusual case of phormia regina myiasis of the scalp. am clin pathol. 90: 734–737. ali-khan fe, ali-khan z (1975) a case of traumatic dermal myiasis in quebec caused by phormia regina (meigen) (diptera: calliphoridae). can j zool. 53: 1472–1476. belding dl (1965) textbook of parasitology. third edition, meredith publishing company. new york. byrd jh, allen jc (2001) the development of the black blow fly, phormia regina (meigen). forensic sci int. 120: 79–88. iranian j arthropod-borne dis, (2009), 3(1): 60-63 a faramarzi et al: chrysomya bezziana as … 63 granz w, schneider d, schmann h (1975) human myiasis in middle europe. z gesamte inn med. 38: 293–301. green pwc, simmonds msj, blaney wm, khambay bps (2004) effects of plantderived compounds on larvae of a blowfly species that causes secondary myiasis: laboratory studies. phytother res. 18: 538-541. hakimi r, yazdi i (2002) oral mucosa myiasis caused by oestrus ovis. arch iranian med. 5: 194–196. hall rd, anderson pc, clark dp (1986) a case of human myiasis caused by phormia regina (diptera: calliphoridae) in missouri, usa. j med entomol. 23: 578–579. jabbar-khan ma, jabbar-khan r (1985) a case of urogenital myiasis of infant caused by maggots of the black blow-fly, phormia regina (maigen) (diptera: calliphoridae), in karachi, pakistan. asian med j. 28: 54–57. kalantari m, rezanezhad h, sherafat-mola aa, asgari q, sadjjadi sm (2006) evaluation of larval myiasis growth rate cynomyopsis cadaverina (diptera: calliphoridae) on mueller hinton agar and blood agar media. 2nd iranian congress of medical entomology and vector control, 2002 may 16–18, tehran iran, p. 84. masoodi m, keramatalah h (2004) external ophthalmyiasis caused by sheep botfly (oesterus ovis) larva: a report of 8 cases. archives of iranian medicine. 7(2): 136 –139 miller kb, hribar lj, sanders lj (1990) human myiasis caused by phormia regina in pennsylvania. j am podiatr med assoc. 80: 600–602. mohammadzadeh t, hadadzadeh r, esfandiari f, sadjjadi sm (2008) a case of gingival myiasis caused by wohlfahrtia magnifica. iranian j arthropod-borne dis. 2(1): 53–56. navidpour s, hoghooghi-rad n, goodarzi h, pooladgar (1996) an outbreak of chrysomya bezziana in khoozestan province, iran. vet rec. 139: 217. radmanesh m, khataminia gh, eliasi p, korai mk, ebrahimi a (2000) chrysomya bezziana-infested basal cell carcinoma destroying the eye. int j dermatol. 39: 455–462. rohela m, jamaiah i, amir l, nissapatorn v (2006) a case of auricular myiasis in malaysia. southeast asian j trop med public health. 37: 91–94. talari sa, sadr f, dorrodgar a, talari mr, gharabagh as (2004) wound myiasis caused by lucilia sericata. arch iranian med. 7(2): 128–129. whitworth t (2006) keys to the genera and species of blow flies (diptera: calliphoridae) of america north of mexico. proc entomol soc. 108(3): 689–725. yaghoobi r, tirgari s, sina n (2005) human auricular myiasis caused by lucilia sericata: clinical and parasitological considerations. acta med iran. 43: 155–157. zumpt f (1965) myiasis in man and animals in the old world: a textbook for physicians, veterinarians and zoologists. butterworths publishing company, london. j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 180 http://jad.tums.ac.ir published online: sep 30, 2022 review article scabies as a neglected tropical disease in iran: a systematic review with meta-analysis, during 2000–2022 *mehdi khoobdel1, shahyad azari-hamidian2,3, ahmad ali hanafi-bojd4,5, hasan bakhshi6, amirsajad jafari7, mohammad moradi1 1health research center, lifestyle institute, baqiyatallah university of medical sciences, tehran, iran 2research center of health and environment, school of health, guilan university of medical sciences, rasht, iran 3department of medical parasitology, mycology and entomology, school of medicine, guilan university of medical sciences, rasht, iran 4department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 5zoonoses research center, tehran university of medical sciences, tehran, iran 6vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 7department of basic sciences, school of veterinary medicine, shiraz university, shiraz, iran *corresponding author: dr mehdi khoobdel; e-mail: khoobdel@yahoo.com (received 25 june 2022; accepted 31 aug 2022) abstract background: scabies is referred to the infestation of skin by an ectoparasite, sarcoptes scabiei. having considerable financial consequences, this disease is a public health concern in several countries. in this review, we aimed to determine the current status of scabies in different provinces of iran. methods: google scholar, pubmed, scopus, science direct, scientific information database (sid), sci-explore, civilica, magiran, iranian research institute for information science and technology and elmnet databases were searched to find the related data in the time period within 2000–2022. to have a better insight into the status of prevalence of scabies in iran, a meta-analysis and meta-regression was performed. results: a total of 943 relevant studies were retrieved from the databases, and 62 eligible studies met all the needed criteria for inclusion in this systematic review. scabies was investigated and reported in at least 22 and 21 provinces of iran respectively. most of the studies were conducted in tehran, razavi khorasan, hormozgan, fars and guilan provinces. positive samples of scabies belonged to humans, sheep, goats, dogs, rabbits, mice and gazelles. meta-analysis showed that the overall estimated presence of scabies in iran during 2000–2022 was 7% (95% ci 4.7–10.3%, p< 0.001). conclusion: infestations due to s. scabiei in iran occur in different geographical locations and different climates. as a neglected tropical disease, the literature about the burden of scabies in iran is inadequate. the present review highlights the importance of development of comprehensive strategies for the diagnosis and control of scabies, especially the provinces with high infestation rates. keywords: scabies; sarcoptosis; sarcoptes scabiei; ectoparasites; iran introduction ectoparasites, such as fleas, lice, ticks and some species of mites, flies and hemipteran bugs, use vertebrates as feeding and breeding grounds to survive and complete their life cycles. scabies, caused by an obligate microscopic parasitic mite, sarcoptes scabiei (linnaeus, 1758) (acarina: sarcoptidae), also named sar coptic mange in cases referring to animals, is a contagious and common skin parasitic disease (1). the members of the genus sarcoptes generally are referred to as scabies mites. scabies is also known as sarcoptosis based on the standardized nomenclature for parasitic diseases (snopad) (2). scabies is considered to copyright © 2022 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 181 http://jad.tums.ac.ir published online: sep 30, 2022 be a neglected ectoparasite progressing to secondary bacterial skin infection that may lead to serious complications including septicemia, renal disease and rheumatic heart disease, resulting from the burrowing of s. scabiei in the epidermis of humans and animals (3). adult s. scabiei digs burrow tunnels and lays 2 to 3 eggs each day. despite several reports of scabies in different countries, little is known about the biology and host-parasite interactions of s. scabiei (4). the disease is spread around the world and is a public health concern in several countries and is mainly related to poverty and overcrowding. more than 200 million people all around the world suffer from scabies (5). global prevalence estimate ranges from 0.2% to 71% with an average of 5–10% in children. meanwhile, s. scabiei infects more than 100 mammalian species, including domestic animals, leading to cross-species transmission (6). scabies covers a wide range of clinical manifestations, presenting three main forms: classic, nodular, and contagious crusted variant also called norwegian scabies, containing millions of s. scabiei on a single infested individual (7). scabies is a disease of public health importance and is formally designated as a neglected tropical disease (ntd) by world health organization (who) in 2017, to encourage the efforts for its eradication (8). having considerable financial consequences, this disease is a public health concern for underprivileged communities in several low and middle-income countries, and also in some developed countries (9). transmission of s. scabiei occurs through skin-to-skin contact with a person who is infested with the mite. this skin disease is more prevalent among vulnerable groups such as children and young people (7). people infested with s. scabiei require rapid identification and treatment, as a misdiagnosis subsequently may lead to an outbreak and an increased economic burden (10). infestation in the elderly and people with immunodeficiency disorders often manifests as general dermatitis that affects many parts of the body, accompanied by scaling and sometimes blistering and cracking of the skin. scabies is usually transmitted through close contact with an infected person. the disease is more common among people that are forced to live together as a group. different studies have shown a significant relationship between the prevalence of the disease and densely populated areas (11). hence, the spread of this ectoparasite is more likely in crowded families, soldiers, and school children. less frequently, contaminated clothing and bedding can transmit the mite to other people. to date, different studies have shown the prevalence of scabies in iran. however, a comprehensive review and analysis on published data seems to be essential in order to have a better insight on the distribution of scabies in different hosts in all provinces of iran, to understand the latest situation of the disease in the country, and development of a national program by health authorities for regular diagnosis and control of the disease in provinces with high infestation rates. in this systematic review and meta-analysis, the latest available literature on the prevalence of s. scabiei in humans and hosts in different provinces of iran was investigated. materials and methods searching strategy the present systematic review and metaanalysis was accomplished on the basis of the preferred reporting items for systematic reviews and meta-analyses (prisma) statement. a comprehensive literature search was conducted using databases: google scholar, pubmed, scopus, science direct, scientific information database (sid), sci-explore, civilica, magiran, iranian research institute for information science and technology and elmnet from 2000 until may 2022. selected keywords used for the search were based on previously published studies comprising: scabies, sarcoptes scabiei and http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 182 http://jad.tums.ac.ir published online: sep 30, 2022 iran. in addition, all provinces of iran were searched specifically with a similar protocol. the search was conducted in both english and persian languages with similar keywords. paper selection studies’ quality assessment were developed according to cocopop structure [co (condition)= infestation by mites; co (context)= provinces of iran; pop (population)= animals and human]. inclusion and exclusion criteria author’s names, publication year, host, geographical region, positive samples and sample sizes were retrieved and then added to predesigned forms. all animal and human studies reporting infestation with the mite in different parts of iran with respect to publication year were included. papers with no full-text availability, from other countries, review articles, unpublished data, as well as studies conducted before 2000 were excluded. meta-analysis and meta-regression random model was used to calculate pooled prevalence with 95% confidence intervals. cochran q test (p< 0.05 shows statistically significant heterogeneity) and i2 test [< 25% (low), 30% < x < 60% (moderate), and > 75% (high) heterogeneity] were used to determine heterogeneity. also, a forest plot was employed to visualize the heterogeneity among the studies. in addition, heterogeneity was explored through meta-regression using some moderators. egger’s and begg's tests (p> 0.05 indicate a reasonable publication bias) as well as funnel plot and trim and fill test were applied to investigate the publication bias. the subgroup analysis was conducted according to the publication year, host and geographical location. in this meta-analysis, each row of table 1 was considered a separate study. data were analyzed with comprehensive meta-analysis version 3 software (biostat, usa). results search output a total of 943 relevant studies were retrieved from the databases, of which 140 studies were screened and 132 full-text articles were assessed for eligibility. furthermore, 62 eligible studies met all the needed criteria for inclusion in this systematic review. in the final step, 44 studies were included in the metaanalysis (fig. 1). our results show that during the period of 2000 to 2022, scabies was studied and reported in humans, sheep, dogs, goitered gazelles (gazella subgutturosa), white rabbits, house mice, and second-hand clothes, in at least 22 and 21 provinces, respectively. most of the studies were conducted in tehran (10), razavi khorasan (8), hormozgan (6), fars (6) and guilan (5) provinces (table 1). the studied provinces included tehran (10 studies, reporting scabies in humans, sheep, dogs and second-hand clothes), razavi khorasan (8 studies, reporting scabies in humans, sheep and dogs), hormozgan (6 studies, reporting scabies in human), fars (6 studies, reporting scabies in humans and dogs), guilan (5 studies, reporting scabies in humans and dogs), isfahan (4 studies, reporting scabies in humans), khuzestan (4 studies, reporting scabies in humans and dogs), west azerbaijan (4 studies, reporting scabies in humans, sheep, goats and white rabbits), mazandaran (4 studies, reporting scabies in humans and dogs), bushehr (3 studies, reporting scabies in humans and goitered gazelles), kermanshah (3 studies, reporting scabies in humans and dogs), kerman (3 studies, reporting scabies in humans and house mice), hamadan (3 studies, reporting scabies in humans), alborz (2 studies, reporting scabies in humans), east azerbaijan (2 studies, reporting scabies in humans and white rabbits), ardabil (1 study, reporting scabies in humans), qazvin (1 study, no evidence of scabies in dogs), golestan (1 study, reporting scabies in humans), kurdistan, lorestan, qom, and sistan and baluchistan (1 study, reporting scabies in humans), and http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 183 http://jad.tums.ac.ir published online: sep 30, 2022 two studies in the country without specifying the details of the rates of infestation in each province, where the positive cases of humans were found. on the other hand, the status of mite infestation was not clear in chahar mahal and bakhtiari, ilam, north khorasan, south khorasan, zanjan, semnan, kohgiluyeh and boyer-ahmad, yazd, qazvin and markazi provinces (fig. 2). pooled effect size, heterogeneity and bias meta-analysis random effect model showed that the total prevalence of mite infestation in iran during 2000–2022 was 7% (95% ci 4.7%–10.3%, p< 0.001). a forest plot determined the prevalence of infestation rate across the country (fig. 3). results showed a strong significant heterogeneity (q= 4859.445, df= 47, i2= 99.03%, p< 0.001). begg and mazumdar rank correlation test as well as egger's test of the intercept illustrated that the publication bias was not substantial (2-tailed p> 0.05). in line with the results of both egger and begg’s tests, funnel plot showed symmetry in the presence of negligible publication bias (fig. 4). duval and tweedie’s trim and fill test initially trims the asymmetric studies from the left-hand side to locate the unbiased effect (in an iterative procedure), and then fills the plot by re-inserting the trimmed studies on the left as well as their imputed counterparts to the right of the mean effect. subgroups’ meta-analysis the subgroup analysis was conducted according to publication year, host, and geographical location (table 2). in terms of publication year, subgroup meta-analysis indicated a higher prevalence of infestation in published literature from 2015 until the present time (8.1% 95% ci: 0.046–0.139) in comparison to years before this time point (6.2%; 95% ci: 0.037– 0.103). location-based analysis suggests that the highest (14.6%; 95% ci: 0.072–0.272) and lowest (3.9%; 95% ci: 0.025–0.059) prevalence belonged to south and north, respectively. hosts’ subgroup analysis showed the prevalence of infestation was higher among animal (8.5%; 95% ci: 0.053–0.134) in comparison to human cases (6.2%; 95% ci: 0.036–0.104). meta-regression analysis a meta-regression was performed, pooling all studies across location (geographical latitude), publication year, and sample sizes. a significant heterogeneity was associated with sample size, according to the method of the moments test (p= 0.000), with a r2 of 39% (fig. 5). in terms of publication year, method of moments showed that some heterogeneity can also be related to the publication year (r2= 13%; p= 0.133), however this was not statistically significant. an increase in the publication year was in line with an increase in the prevalence, which indicates that recently published studies had reported higher infestation rates in comparison to older ones (but this relationship was not also statistically significant) (fig. 5). maximum likelihood model was assessed to perform the meta-regression for the location moderator and the geographical latitude. r2 showed that a very small heterogeneity might be justified due to latitude. though, this was not statistically significant (4%; p= 0.06). table 1. details of investigations carried out and reported the occurrence of scabies in iran, during 2000–2022 province description prevalence host year reference (s) positive/total (%) 1 alborz prevalence of scabies among prisoners 31/1404 2.2 human 2006 (12, 13) 2 ardabil frequency of scabies# 51/na na human na (14) 3 azerbaijan, east ectoparasites of white rabbits 9/50 18 white rabbit 2020 (15) a case of scabies in a sheepherder* 1/1 100 human 2014 (16) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 184 http://jad.tums.ac.ir published online: sep 30, 2022 table 1. continued ... 4 azerbaijan, west ectoparasites of white rabbits 9/50 18 white rabbit 2020 (15) ectoparasites of sheep and goat flocks 2/77 2.6 sheep 2006 (17) ectoparasites of sheep and goat flocks 0/119 0 goat 2006 (17) scabies among police forces # na/na na human 2002 (18) ectoparasites of goats 12/403 2.97 goat 2011 (19) 5 bushehr study on the sarcoptic mange of gazelle 7/8 87.5 gazella subgutturosa 2007 (20) scabies in primary schools 84/3913 2.14 human 2001 (21) scabies among police forces # na/na na human 2002 (18) 6 fars a 40-year-old man* 0/1 0 human 2021 (22) scabies in hiv/aids individuals 4/240 1.66 human 2018 (23) scabies in people 51/203 25.12 human 2012 (24) a case of crusted scabies* 1/1 100 human 2018 (25) a case with low-vision problem* 0/1 0 human 2021 (22) scabies in dogs 16/16 100 dogs 2005 (26) 7 golestan scabies among primary school children # na/na na human 2001 (27) 8 guilan ectoparasites of stray dogs* 1/35 2.85 dog 2016 (28) prevalence of skin diseases among prisoners 95/2100 4.5 human 2003 (29) scabies among police forces# 194/na na human 2002 (18) scabies among primary school children 50/3656 1.36 human 2003 (30) skin disorders among elder patients 19/440 4.31 human 2013 (31) 9 hamadan prevalence of scabies among prisoners 10/384 2.6 human 2015 (32) parasitic investigation among patients 0/124 0 human 2006 (33) scabies in west of iran# 7/na na human 2014 (34) 10 hormozgan study on primary school children 15/480 3.1 human 2021 (35) prevalence of scabies among prisoners 38/67 56.71 human 2007 (36) cases of skin diseases were studied 277/6841 4.04 human 2005 (37) prevalence of scabies in soldiers 95/763 12.45 human 2002 (38) scabies among police forces # 262/na na human 2002 (18) scabies in human community # 122/na na human 2018 (39) 11 isfahan an 85-year-old man* 1/1 100 human 2021 (40) a family, all infected 5/5 100 human 2018 (41) scabies in patients 817 /2899 28.18 human 2009 (42) scabies in patients 28/129 21.7 human 2017 (43) 12 kerman scabies in patients # na/87 na human 2017 (44) prevalence of scabies among prisoners 32/2851 1.12 human 2000 (45) scabies in house mouse 2/2 100 mus musculus 2018 (46) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 185 http://jad.tums.ac.ir published online: sep 30, 2022 13 kermanshah ectoparasites fauna of dogs 21/137 15.33 dog 2017 (47) scabies in hiv infected prisoners 9/79 11.39 human 2003 (48) scabies in west of iran# 170/na na human 2014 (34) 14 khuzestan two cases of norwegian scabies 2/2 100 human 2019 (49) severe case in a 4 month old child* 1/1 100 human 2008 (50) scabies among police forces# 263/na na human 2002 (18) scabies in companion dogs 7/126 5.55 dog 2012 (51) 15 kurdistan scabies among police forces# na/na na human 2002 (18) 16 lorestan scabies among police forces# na/na na human 2002 (18) 17 mazandaran ectoparasites of stray dogs 2/15 13.33 dog 2016 (28) scabies in primary school children 225/10737 2.09 human 2003 (52) scabies among patients 89/1140 7.8 human 2007 (53) groin pruritus in female patients 1/115 0.8 human 2010 (54) 18 qazvin ectoparasites of stray dogs 0/20 0 dog 2016 (28) 19 qom scabies among patients 68/411 16.54 human 2015 (55) 20 razavi khorasan suspected patients 375/ 1814 20.67 human 2014 (56) infestation in dogs 10 /460 2.17 % dog 2020 (57) scabies in a herd of sheep 10/75 13.33 sheep 2008 (58) genital dermatoses 36/355 10.1 human 2016 (59) scabies among police forces # na/na na human 2002 (18) a case of disseminated scabies infection* 1/1 100 human 2005 (60) scabies in a rehabilitation center 21/110 19.09 human 2019 (61) dermatological study on domestic doges 17/316 5.06 dog 2013 (62) 21 sistan and baluchistan scabies among police forces# na/na na human 2002 (18) 22 tehran prevalence of skin related diseases 26/3120 0.83 human 2021 (63) study on dermatological emergencies 132 /2539 5.2 human 2017 (64) study on sheep sarcoptic mange 278/ 5603 4.96 sheep 2009 (65) infestation in dogs 37/143 25.9 dog 2012 (66) scabies among police forces # na/na na human 2002 (18) a case of crusted scabies * 1/1 100 human 2020 (67) ectoparasites of second-hand clothes 6/800 0.75 na 2021 (68) a case of norwegian scabies* 1/1 100 human 2002 (69) a case of scabies* 1/1 100 human 2006 (70) a case of crusted scabies with brain astrocytoma* 1/1 100 human 2010 (71) 23 iran scabies among soldiers 66/58850 0.11 human 2003 (72) scabies in iran’s army 5277/na# na human 2014 (73) *: these studies were not included in the meta–analysis due to the sample size less than 2 #: these studies were not included in the meta–analysis due to the insufficient data table 1. continued ... http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 186 http://jad.tums.ac.ir published online: sep 30, 2022 fig. 1. flowchart of the data selection in terms of sarcoptes scabiei infestation in iran, during 2000–2022 fig. 2. human cases of scabies reported in different provinces of iran; further to investigations conducted during 2000–2022, scabies has been reported in humans in at least 21 provinces http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 187 http://jad.tums.ac.ir published online: sep 30, 2022 table 2. prevalence of sarcoptes scabiei infestation in iran according to year, host, and location, during 2000–2022 subgroup variable number of studies prevalence (95% ci) i2 (%) heterogeneity (q) p value location north 16 0.039 (0.025–0.059) 97.431 583.954 0.195 south 11 0.146 (0.072–0.272) 98.026 506.709 0.000 west 9 0.059 (0.029–0.118) 87.016 61.614 0.000 east 6 0.097 (0.051–0.177) 95.202 104.218 0.000 center 6 0.142 (0.011–0.716) 99.784 2310.669 0.000 host human 30 0.062 (0.036–0.104) 99.349 4453.800 0.000 animal 18 0.085 (0.053–0.134) 94.052 285.794 0.000 year 2015 30 0.062 (0.037–0.103) 99.357 4511.214 0.000 2015 18 0.081 (0.046–0.139) 94.679 319.480 0.000 fig. 3. forest plot of the prevalence of sarcoptes scabiei infestation in iran during 2000–2022; total prevalence of mite infestation in iran during 2000–2022 was 7% (95% ci 4.7%–10.3%, p< 0.001) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 188 http://jad.tums.ac.ir published online: sep 30, 2022 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 0.0 0.5 1.0 1.5 2.0 s ta n d a rd e rr o r logit event rate funnel plot of standard error by logit event rate fig. 4. funnel plot of standard error by logit event rate for sarcoptes scabiei infestation in iran during 2000–2022; most of the studies are distributed at the top of the graph in a balanced way, which indicates a negligible publication bias fig. 5. meta-regression chart showing the logic event rate of sarcoptes scabiei infestation in iran according to sample size during 2000–2022; a significant heterogeneity was associated with sample size (the number of mites) http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 189 http://jad.tums.ac.ir published online: sep 30, 2022 discussion scabies caused by s. scabiei may be one of the first reported human diseases with a well-known etiology (74). such an infestation significantly impacts the quality of life in vulnerable people with adverse sequelae, especially in low-resource settings (9). to our knowledge, the present meta-analysis is the first large-scale study that examined the prevalence of scabies in different provinces in iran. further to our review, the occurrence of scabies has been reported in at least 21 provinces out of 31 provinces. pooled prevalence of mite infestation in iran was calculated as 7% (95% ci 4.7%–10.3%, p< 0.001). meanwhile, the subgroups’ metaanalysis showed that the prevalence of infestation was higher in the south (14.6%; 95% ci: 0.072–0.272) and center [14.2%, 95% ci: 0.011–0.716] of iran. also, meta-regression analysis suggested that lower latitude have higher prevalence of mite infestation, suggesting that the humidity and climate may be associated with the spread of s. scabiei (but the latter result was not statistically significant). a previous investigation has shown that the mites have better survival and fertility rates in humid conditions (75); however, some other studies have concluded that cold weather favors the population dynamic of mites (76, 77). meta-regression illustrated that an increase in the publication year was in line with an increase in the prevalence which indicates that recently published studies had reported higher infestation rates in comparison to older ones. this may be justified due to the improvements in the knowledge of health workers or health system. during the period of the present review (2000–2022), scabies was recorded in humans, sheep, dogs, goitered gazelles, white rabbits, house mice and second-hand clothes in iran; however, there are some records of scabies in cattle, cats, horses and pigs in older documents (78). also, there are few unpublished documents on scabies in camels in the country (79). in the present study, based on the searching criteria and the eligible documents, the presence of scabies was shown in 21 provinces of iran (out of 31); thus, there were not eligible documents for final analysis for 10 remaining provinces including chahar mahal and bakhtiari, ilam, north khorasan, south khorasan, zanjan, semnan, kohgiluyeh and boyer-ahmad, yazd, qazvin and markazi. on the other hand, there are some documents on the occurrence of scabies in some of the aforementioned provinces such as ilam (80), qazvin (79) and zanjan (73) that did not meet our criteria. following by an increased occurrence of scabies in some countries in europe, asia and africa, scabies cases have recently been increased in turkey (81) and saudi arabia (82). in contrast with the results of our meta-regression analysis, in turkey, located in the northwest of iran, the prevalence of scabies was reported as 10.9%, showing that there was an increase in the cases of scabies both before and during the covid-19 pandemic. this situation was considered an epidemic of scabies (83, 84). studies on scabies during the covid-19 pandemic in iran was limited to a few investigations (published during the pandemic), showing a prevalence of 0.83% in patients referred to a hospital in tehran (63), 0.75% in secondhand clothes studied in tehran (68), and 3.1% in primary school children in hormozgan (35), suggesting that the pandemic has not resulted in a higher frequency of scabies compared to former investigations. in iraq, alsamarai (2008) has reported scabies in a dermatology clinic in tikrit with a frequency of 1.1% among 1,194 studied patients. among the positive cases, prisoners were identified as the high-risk group (85). different studies have shown that scabies is more frequent among prisoners in iran with 56.71% in hormozgan (36), 2.2% in alborz (12, 13), 11.39% in kermanshah (hiv infected) (48), 1.12% in kerman (45), 4.5% in guilan (29) and 2.6% in hamadan (32) provinces. furthermore, a study http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 190 http://jad.tums.ac.ir published online: sep 30, 2022 carried out on the incidence of scabies in 1,300 displaced people who visited the dermatology clinics and health care centers in duhok governorate, located in the kurdistan region of iraq bordered with kermanshah and kurdistan provinces of iran, the prevalence of scabies was reported in 45% of studied people (86). in saudi arabia, located in the south of iran, high temperature is explored as a leading factor for the occurrence of scabies (87). in iran, khuzestan, bushehr and hormozgan are among the provinces with a high temperature. the prevalence of scabies in khuzestan province in companion dogs has been reported as 5.55% (51). furthermore, in hormozgan province, the prevalence of scabies among primary school children, prisoners, people with skin diseases, and soldiers, have been reported as 3.1% (35), 56.71% (36), 4.04% (37) and 12.45%, respectively (38). in bushehr province, an investigation on gazelles (gazella subgutturosa) showed that 7 of 8 studied animals were infested with s. scabiei (20). another study showed that 2.14% of 3,913 of primary school children in this province were positive for s. scabiei (21). previous findings in pakistan have identified "inadequate bathing", "infrequent changing of clothes", "low education", "sharing beds, clothes, towels" and "being away from the barracks" as the most important risk factors for scabies (88). prevalence of scabies in soldiers has been studied in some provinces of iran: soleymani ahmadi et al. (38) have reported a prevalence of 12.45% in 763 studied soldiers in hormozgan province. another large-scale investigation on 58,850 soldiers conducted in iran showed a prevalence of 0.11% (72). meanwhile, 5,277 positive cases among iran’s army (the prevalence rate was not identified) were reported in 2014 (73). in another investigation carried out by tufail and khan in 2021 on patients who visited a hospital in pakistan, they showed that the risk of scabies was higher in crowded places. furthermore, the risk was lower in people having acceptable living and hygiene conditions (89). meanwhile, molecular analysis of s. scabiei in pakistan has shown the presence of different varieties of mites in different hosts and different geographic regions (90), suggesting the possibility of the occurrence of different varieties of s. scabiei in different regions of iran. in most parts of the meta-analysis the results indicated high heterogeneity (i2> 80%). subgroup meta-analysis of separate moderators, also revealed high heterogeneity. we tried to justify this heterogeneity by meta-regression. accordingly, it was concluded that almost 50% of the reported heterogeneity might be related to different sample sizes, locations and publication year. also, some other factors may be the reason behind the high heterogeneity that were not included in this meta-regression (for example different detection methods, populations of interest, and seasons). publication bias is a considerable problem for the integrity of a meta-analysis, as it illustrates overestimated and underestimated effects. conclusion with an overall estimated presence of scabies in iran during 2000–2022 as 7% (95% ci 4.7–10.3%, p< 0.001), s. scabiei infestation is considered a health concern. in the first step, diagnosis and treatment of the infestations caused by this species are among the key factors for prevention of its distribution. meanwhile, scabies is preventable by identification of risk factors that should be characterized in different regions and different groups of people. the literature on the identification of scabies, molecular analysis of s. scabiei collected in different groups of people in different provinces, and the burden of scabies in iran is not adequate. the present systematic review highlights the need for development of a national program for regular diagnosis of scabies in high-risk places such as schools, prisons, and regions with inadequate health facilities, and the importance of adopting effective strategies for control and prevention of scabies in the provinces with high infestation rates. http://jad.tums.ac.ir/ j arthropod-borne dis, sep 2022, 16(3): 180–195 m khoobdel et al.: scabies as a … 191 http://jad.tums.ac.ir published online: sep 30, 2022 acknowledgements we highly appreciate dr zakkyeh telmadarraiy (tums) for her scientific support. conflict of interest statement we declare that we have no conflict of interest. references 1. moroni b, rossi l, bernigaud 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biodiversity of scorpions in qeshm island (persian gulf) a zarei1, *j rafinejad2, k shemshad3, r khaghani4 1 departement of biology, qeshm azad university, qeshm, iran 2 department of medical entomology and vector control, school of public health tehran university of medical sciences, tehran, iran 3department of entomology, science and research branch, islamic azad university, tehran, iran 4department of medical parasitology and entomology, college of medical sciences, tarbiat modarres university, tehran, iran (received 17 nov 2008; accepted 1 sep 2009) abstract background: scorpions are known as the most widespread poisonous creatures that cause casualties and death to human. they are distributed in tropical and subtropical areas. among the sixteen iranian island in the persian gulf, qeshm island is the biggest and is located in the focus of the important internal and international commercial marinelines. furthermore, thousands of tourists also visit the island every year for its natural beauties and the siteseeings. the present research study was carried out during 1998-99 throughout qeshm island in order to know the species of scorpions, their abundance, the distribution and control strategy in order to reduce the scorpion stings and to safeguard the visitors as well as the local population. methods: collections were made randomly during the night. samples were searched by black light and then collected by forceps. a total of 102 scorpions were captured. results: the scorpions were identified as buthotus jayakari numbered 42 specimens (41.17%) and b. leptochelys numbering only 7 specimens (6.86%) belonging to the family buthidae. androctonus crassicauda was collected in the most parts of the island and considered as the dominant species. the sex ratio was 1:1.53 in favor of females. b. leptochelys was rare species and by far restricted in its distribution. conclusion: considering the high population of a. crassicauda and its wide distribution, it is regarded to be responsible for the majority of scorpion stings in qeshm island. kaywords: scorpion, fauna, distribution, iran introduction scorpions are classified as arachnids. fossils indicate that scorpions have originated in the sea and emerged on the land during the middle silurian period, 425 to 450 million years ago. scorpions have a wide geographic distribution, from tropical to temperate, including deserts, savannas, tropical forests, mountains over 5500 m in altitude, and the intertidal zone (polis 1990). scorpions hide under stones, bark, wood or other objects on the ground where they wait or search for prey. their foods comprise small insects, spiders, centipedes, earthworms, and other scorpions. once they capture their prey, they use the large pincers to crush and draw it toward the mouth. the body juices of the prey are eaten by the scorpion. scorpions are active at night and this behavior helps scorpions manage temperature and water balance, important functions for survival in dry habitats. many species dig burrows in the soil. they detect and capture prey by the sense of touch. *corresponding author: dr javad rafinejad, e-mail: jrafinejad@yahoo.com iranian j arthropod-borne dis, (2009), 3(1): 46-52 a zarei et al: faunistic sttudy and … 47 they also have a well-developed sense of hearing. some of these habitats, e.g. arid deserts, are of extreme environmental conditions, and scorpions have a range of adaptations for life in such harsh environments and they are most common and diverse (jeram 1990). scorpions are nocturnal arthropods that have minimal surface activity and prefer to leave their burrows during periods surrounding the new moon (warburg and polis 1990). totally, there are over 1500 species of scorpions worldwide which have been recorded although doubtless others await discovery. of the 1500 species of scorpion only about 50 species were stated to have a sting which can be dangerous to humans (mullen and stockwell 2002, ozkan and karaer 2003). although scorpions has wide distribution in world but the majority distribute in altitude 23º to 38º (kjellesvig-waering 1986, farzanpay 1988, kovarik 1997) as well as iran which is located in altitude 25 to 40 ºn and scorpions are highly active (kamali 1984, akbari 1996, pouladgar 1999). so far there is no any documented study about distribution of scorpions specious in qeshm island. therefore, this investigation was carried out in the island regarding to identify the species as well as their distribution. so based on the results of this study, a control strategy cab be planned to reduce scorpion stings. materials and methods qeshm island is the biggest island of the persian gulf and located in the strait of hormuz in the south coast of iran and east of the persian gulf (26° 50' n 56°0' e). it has an area of over 1295 km2 and almost 100 kilometers long. qeshm city is located in the northeast of the island. this island has hot and humid weather. the surface is mostly rocky and barren with a small human population. qeshm island is also a free trade zone, which has made the island important to the regional trade. on what little cultivated land there is, dates and melons are grown. in order to determine the study areas the island divided to 20 regions and then 10 regions were selected by clustered random sampling. the flora of the studied areas was rich and the population density was high. in each studied area specimen collection was carried out. the studied areas were dargahan, laft village, gourzin, tabl, doulab, basaeedoo port, doostkoo, namakdan, salkh, sibe deraz, souza and qeshm city (table 1). the most scorpions are nocturnal, hiding under rocks, in crevices, or within burrows during the day, and coming out after sunset. the scorpion possesses a greenish glow. unique to the scorpions is that the cuticle fluoresces under uv light, helping the nightly collection and observation of these interesting animals. collections were made during the night searches using the black light (russel 1968). diurnal study was carried out by searching under the stones, in piles of the rocks or masonry, in piles of the wood and under the trees’ barks. after collection, specimens were transferred in to the holding tubes including alcohol. for species identification several parameters including morphology, biology and ecology, date, place, habitats were considered. identification was carried out by morphological characters according to the national standard taxonomic key identification (farzanpey 1988). the data were analyzed, using microsoft excel. results scorpion's fauna and sex ratio in qeshm island in this study, 102 scorpions were collected including buthidae and two genera buthidae vachon, 1949 [hottentotta birula, 1908] and buthacus birula, 1908 including b. jayakari with 42 specimens (41.17%) (simon, 1880), buthacus leptochelys with 7 specimens (6.86%), hemiscorpius leptorus with 3 specimens (2.94%) [hemprich and ehrenberg 1829], a. crassicauda with 35 specimens (34.31%) iranian j arthropod-borne dis, (2009), 3(1): 46-52 a zarei et al: faunistic sttudy and … 48 and mesobuthos eupeus with 15 specimens (14.70%). b. jayakari is not a digger species. the average body length of adults is 13 cm. telson, the 3 latest segment of tail, two movable and immovable fingers, prefemur of pedipalp, carapace, anterior margins of carapace and posterior part of mesosoma (apart from the latest segment of mesosoma) have blackish color. other parts of the body are yellowish. the highest length of this scorpion among 42 collected specimens recorded as 11.8 cm. in males pectines had 36 to 39 teeth and in females pectines had 32 to 36 teeth (fig. 1.). hemiscorpius. leptorus belongs to the family scorpionidae and is the county’s most dangerous scorpion. this scorpion is not digger species and found in bare areas. h. leptorus has sexual dimorphism. the average body length of adults is 5 cm and in males 7.5 cm. the color of the body varies from light yellow to the dark yellow. legs and cheliceres are light, movable and immovable fingers are brown to red color. brown patches are seen on the latest segment of the legs. a. crassicauda is not a digger species as well. the average body length of adults is 12 cm. body’s color varies widely from brown, black to an olive color. pectins, chelicera, end of legs and ventral surface of the body are light yellow (fig. 2 and 3). mesobuthos eupeus is not a digger species. the average body length of adults is 6 cm. the body’s color varies from yellow to a dark yellow. dark and brown patches are seen on the dorsal surface of the body (fig. 4). buthacus leptochelys is also not a digger species. the average body length of adults is 7 cm. color of dorsal surface varies from the yellow to a dark yellow. carapace has no granule but according to its distribution it has smooth or disturbed granule carapace. lateral eyes are small. the males have has30 to 32 pectinated teeth while the females have 25 to 27 teeth (fig 4). one hundred two specimens were captured from qeshm island. b. jayakari with 42 (41.17%) specimens was the most abundant in the studied area and h. lepturus with 3 specimens had the lower frequency. a. crassicauda had the same distribution in all over the island. notes on the captured scorpions sex ratio on a. crassicauda, was 1:1.32 and show that females of a. crassicauda are more prevalent. the sex ratio was correlated to the population density of the adult males but the frequency of courtship attempts on females negatively correlated to the longevity of females. higher population density of males limits the density of adult females (hailey and willemsen 2000). three out of the 7 collected b. leptochelys were male and the rest were female. this research was carried out in 13 regions of qeshm island. a. crassicauda had a uniform distribution in all parts of the island but b. leptochelys was only collected from tablvi. and dargahan and had less distribution in comparison to the b. jayakari. ramkan and souza with 93 (25.07%) scorpions collected had the highest frequency while the region from parke zaitoon to simin beach with 9 specimens (2.43%) showed the least frequency of scorpions. most scorpions were catched in the regions with a rich flora. only in one place that had poor vegetation flora the frequency of scorpions were high. the reason of this fact is that, there were much polluted and population density of insects that are the food of scorpions were high. there was not seen any scorpions in the coastal areas. the nearest collection point was 6 meters away from the coastal line (salakh and holor to ramchah area). table1. prevalence of different species of scorpions collected from qeshm island, iran prevalence (%) no species 41.17 42 buthotus jayakari 6.86 7 buthacus leptochelys 2.94 3 hemiscorpius leptorus 34.31 35 androctonus crassicauda 14.70 15 mesobuthos eupeus 100 102 total iranian j arthropod-borne dis, (2009), 3(1): 46-52 a zarei et al: faunistic sttudy and … 49 fig. 1. mesobuthos eupeus fig. 2. buthacus leptochelys iranian j arthropod-borne dis, (2009), 3(1): 46-52 a zarei et al: faunistic sttudy and … 50 fig. 3. buthotus jayakari fig. 4. androctonus crassicauda iranian j arthropod-borne dis, (2009), 3(1): 46-52 a zarei et al: faunistic sttudy and … 51 disscussion qeshm island scorpion fauna is comprised of a.crassiauda and b. leptochelys, b. jayakari, h. leptorus, m. eupeus. a. crassiauda and b. leptochelys, which belong to the family buthidae. these two species had the most prevalent among the collected specimens. this study showed that b. jayakari and a. crassiauda were more prevalent in the areas with rich flora of vegetation. b. jayakari has been recorded from hormozgan (hengam island, aboomousa, tonbe-bozorg and kish island) and fars province (polis 1990). androctonus crassiauda has wide distribution in all parts of iran from the northern to the southern provinces such as khuzestan (ahwaz, omidiyeh, bostan, sousangerd, mahshahr, khoramshahr and abadan), semnan, kerman, kurdistan (marivan), boushehr, ilam province (mehran, ivan and dehloran), west azerbaijan (chaldoran, khoy, makoo, oshnoviyeh, oroomiyeh), kermanshah (javanrood, sarpolzahab, qasr-eshirin) and khorasan (tibad, khaf, qaen, birjand and nehbandan) provinces (russel 1968). buthacus leptochelys is more prevalent in arid areas such as khuzestan (susa, mahshahr, sousangerd, ramhormoz and ahwaz), hormozgan (qeshm island), and boushehr (boushehr) provinces (kjellesvig 1986). previous studies have shown that h. leptorus has wide distribution in khuzestan (ahwaz, dezfoul, izeh, masjid-soleiman, ramhormoz and susa), semnan, fars (the city of shiraz), kurdistan (marivan), hormozgan (the city of bandar abbas), boushehr, ilam (mehran, ivan and dehloran), lorestan (poldokhtar and aligoudarz) and kermanshah (javanrood, sarpolzahab, paveh, qasr-eshirin) provinces (russel 1968). mesobuthos eupeus has been recorded from khuzestan (susa, mahshahr and abadan), kurdistan (marivan, saqqez, baneh), hormozgan (the city of bandar abbas), golestan (gorgan), tehran (varamin and barghan mountains), ilam (mehran, ivan and dehloran), west azerbaijan province (chaldoran, khoy, makoo, oshnoviyeh, oroomiyeh, piranshahr and sardasht), kermanshah (javanrood, sarpol zahab and qasr-e-shirin) and khorasan ( tibad, khaf, qaen, birjand and nehbandan cities) provinces (russel 1968). these regions prepared the best thermal microclimate in comparison to the other parts of qeshm island. this condition attracts insects and the other arthropods which are the food source of the scorpions. manmade modifications such as trash and debris have altered the situation for the benefit of the scorpions and careful inspection and cleaning-up of the polluted area needed to decrease their population. presence of b. jayakari was also reported from the other neighboring islands such as aboomousa and kish (khaghani 2005) but in the mainland this species was just once reported in some southern cities (akbari 1996). regarding the high prevalence of b. jayakari in qeshm island, it is concluded that this island offers the best ecological condition for the activity of this species. in agricultural landscapes, the dominant vegetation associations will generally provide a useful basis for the activity of scorpions. these associations tend to have dominant species and characteristic structural attributes. these associations also tend to correlate with the soil type and the other land features. control of scorpions using different method such as remove all trash, logs, boards, stones, bricks and other objects from around the home, keep grass closely mowed near the home, prune bushes and overhanging tree branches away from the house, tree branches can provide a path to the roof for scorpions is necessary specially in villages. acknowledgements the authors would like to appreciate mr. mohammad mohebi, eng. maad moshtaghi (sama university), eng. hosseini and iranian j arthropod-borne dis, (2009), 3(1): 46-52 a zarei et al: faunistic sttudy and … 52 eng. jabarzadeh, tutors of the qeshm university for their helpful comments, friendly collaboration, helping us on the scorpion taxonomy and providing the assistance to carry out the current research. this research was in part supported by a tarbiat modarres research grant to the senior author. the authors declare that they have no conflicts of interest. references akbari a (1996) study on geographical distribution of scorpions in south parts of iran. pazhohesh va sazandegi. 34: 115–112 [in persian]. andrew j jeram (1990) book-lungs in a lower carboniferous scorpion. nature. 343: 360–361. farzanpay r (19288) a catalogue of the scorpion occuring in iran, up to january 1986. revue arachnologique. 8(2): 33–44. hailey a, willemsen re (2000) population density and adult sex ratio of the tortoise testudo hermanni in greece: evidence for intrinsic population regulation. journal of zoology. 251: 325–338. kamali k (1984) an introduction to khuzestan important scorpions. faculty of agriculture press, chamran university. agriculture science journal. 35 pp [in persian]. kaghani r, tirgari s, omrani gh, rafinejad j, mousavi e (2005) faunestic study and distribution of scorpions in kish island, iran (persian gulf). modares journal of medical sciences. 8(1): 7– 12 [in persian]. kjellesvig-waering en (1986) a restudy of the fossil scorpion of the world. paleontographica americana; on. 55. ithaca, n. y.: paleontological research institute.. kovarik f (1997) results of the czech biological expedition to iran. part 2, arachnida: scorpiones, with description of iranobuthus krali gen.n. et sp.n. and hottentotta zagrosensis sp.n. (buthidae). acta soc zool bohem. 61: 39–52. mullen g, stockwell sa (2002) medical and veterinary entomology. academic press., amsterdam. ozkan o, karaer z (2003) the scorpions in turkey turk hig. deney biyol derg. 60: 55–62. pouladgar a (1999) study on the prevalence of different species of scorpions in khuzistan province. pazhohesh va sazandegi. 40: 175–177 [in persian]. polis ga (1990) the biology of scorpion. stanford university press, stanford, california. russel fe (1968) scorpion collecting. toxicon. 6: 307–308. warburg mr , polis ga (1990) behavioral responses, rhythms, and activity patterns. in: polised ga( ed): the biology of scorpions. stanford university press. stanford, california, pp 224–246. flve a (راه���� �� � در��ن ) ���� � دارو�� � و� وس در �����ارد � �� �� �� ا�#" �! و� وس �ا�$ا� %&�'a ء���� �� �.� و -,�+* ��ارد (�س / �$د�0 )٧د#2�را1��� ( ���و�4 2"�4 �4 �+��ر��8ي وا56� آ$ �� : ه ف ومبتال ويروس به منظور درمان بيماران ي استفاده از داروهاي ضدمايي قابل تجديد نظر درخصوص چگونگفراهم آوردن راهن نحوه برخورد راهنما ،اين . با منشاء خوكي) h1n1) a عفونت با ويروس آنفلوانزايدر برابر ي داروييهمچنين پروفيالكس خوكي را شامل با منشاءهمچنين موارد تماس نزديك با عفونت آنفلوانزاي بيماران قطعي ، موارد متحمل يا مشكوك ورويي با دا . شود �ارد >&��4 �� و� وس '�&%�ا�$ا� � =� �>a (h1n1) ء���� �� �.�/ virus) (swine-origin-influenza a 0� =� 4 ��رد<���&< �� � s-oiv :از >?�ر<� تائيد . تائيد آزمايشگاهي آن توسط مركز مديريت بيماريهاي واگير ميباشدبا همراه اني گهناتب دار فردي با بيماري تنفسي :يا تعداد بيشتري از تست هاي ذكرشده در زير است يك آزمايشگاهي براساس مثبت شدن 1. real-time pcr or pcr rt-pcr= .يا كشت سلول هاي انسانيدر تخم مرغ جنين دار كشت ويروس .2 �� 4��&< !� ��a#� رد�� =� �>s-oiv : آنتي ژن هاي شايع يعني ( h1 & h3منفي از نظر و aمثبت از نظر گونه آنفلوانزاي فردي با بيماري تنفسي تب دار حاد و .باشد مي rapid testيا influenza pcr براساس تست )فصلي aودرگردش آنفلوانزاي �� 4��&< !� �����رد �� =� �>s-oiv : :كه آغاز آن به يكي از صورت هاي زير باشداست به صورت بيماري تنفسي ناگهاني تب دار بوده است s-oivمورد قطعي روز پس از يك تماس نزديك با فردي كه 7طي ز عالئم فوق در اآغ .1 -s كه داراي يك يا تعداد بيشتري از موارد قطعي)جامعه اي ( روز پس از مسافرت به منطقه اي7 طيآغاز بيماري در .2 oiv است . تنيز در آن ديده شده اس s-oiv ساكن منطقه اي باشد كه يك يا تعداد بيشتري از موارد قطعي عفونت فرد .3 � �+��ر��bc 4ا� 2 dدور: . ميباشدمسري روز بعد از آغاز عالئم بيماري 7از يك روز پيش از بروز عالئم تا (h1n1) ويروس آنفلوانزاي خوكيمورد قطعي �د�+� =� �رت <�f *س �$د�0 �! ا��): ي يا مورد مشكوك به بيماري در كل دوره سرايت پذيري بيماري ياكمتر با مورد قطع) متر1,5 (داشتن تماس از فاصله حدود ���86�� �g&�> ر���+� =� �>(acute respiratory illness, iri) :شروع اخير حداقل دو مورد از موارد زير ميباشد . )با يا بدون تب قطعي يا احساس گرما( ، گلو درد ، سرفه يا آبريزش از بينياحساس گرفتگي 6 وd ه�ي =� �> / c : در جامعه شامل افراد باضعف سيستم ايمني خوكي در معرض خطر زيادي باشد يعني ي ا آنفلوانز عوارضفردي كه از لحاظ ابتال به افراد بيماران قلبي–) آسم و برونشيت( من بيماران ريوي مز–زنان حامله بخصوص در سه ماهه دوم و سوم – سال 5كودكان زير ض همانند پاين عوا . سال65 نارسايي كليه و كبد و افراد باالي –زير درمانهاي ضعيف كننده ايمني مانند شيمي درماني ها . عوارض آنفلوانزاي فصلي هستند �"i��ت /�ص � ا� .�د.�ن rayeنت بوده يا مورد قطعي عفونت هستند به علت احتمال بروز سندرم سال يا كمتر از آن كه مشكوك به عفو18براي افراد براي كاهش ) peptobismol –وت ساب ساليسيالت مانند بيسم. ( ي آسپرين قدغن است تجويز آسپرين يا فراورده هاي حاو . ها استفاده كرد nsaidاستروئيدي تب ميتوان از ساير تب برها مانند استامينوفين يا ساير داروهاي ضد التهابي غير j� وس :�و�g� 4?4 �! داروه�� � و� به داروهاي ضد ويروس مهاركننده نورآمينيداز همانند زاناميوير فعال با منشاء خوكي a (h1n1)ويروس آنفلوانزاي . آمانتادين وريمانتادين مقاوم ميباشدآدامانتان ماننداين ويروس به ضد ويروس خانواده دارويي . واوسلتاميوير حساس است ��4 ( در��ن � و� و�2&< �� � �� ����� ، �1��#iارد ا�� �.��ا�$ا� /%&�'(: طيف ، به آخرين اطالعات مبني بر ميزان اثربخشي دارو ان آنتي ويرال ميتواند با دستيابيتوصيه هاي مربوط به استفاده از درم براي ضد ويروستوصيه ميشود درمان . حساسيت ويروس به دارو تغيير نمايد و ، عوارض جانبي داروهاي ضد ويروسماريباليني بي .نفلوانزاي خوكي درنظر گرفته شود احتمالي يا مشكوك به عفونت با ويروس آ، موارد قطعي يا pcrتنها .ض خطر هستند بايستي در تقدم باشددرمان بيماران بستري يا بيماراني كه از لحاظ ابتال به عوارض بيماري در معر ستهاي و تكارايي تست هايي مانند تست آنتي ژن سريع . خوكي را اثبات كند aكشت ويروس ميتواند عفونت با ويروس آنفلوانزاي احتمال ابتال به از نظر اپيدميولوژيك كسي كه . است %70تا% 90خوكي aايمونوفلونورسانس براي شناسايي ويروس آنفلوانزاي يك نتيجه شود ويا pcrداشته باشد ونمونه خلط وي پس از دو روز از بروز عاليم باليني خوكي را aي عفونت ويروس آنفلوانزا . خوكي نميباشدaمنفي حاصل از تست سريع آنتي ژن يا ايمونوفلنورسانس ، ردكننده عفونت آنفلوانزاي ران بستري يا بيماران يوير بايستي هرچه زودتر پس از احراز عاليم بيماري شديد در بيمااوسلتامدرمان آنتي ويرال با زاناميوير يا فصلي ، اثر داروهاي ضد aعالئم بيماري آغاز شود براساس شواهد حاصل از مطالعات برروي آنفلوانزاي سرپايي با ضعف ايمني و تواند اثرات مفيدي همانند كاهش مرگ ومير وكاهش زمان مي ساعت اول شروع بيماري بيشتر است و پس از آن48ويروس در . روز ميباشد 5مدت زمان توصيه شده براي درمان . بستري در بيمارستان را داشته باشد ميزان س به دارو واطالعات حاصله درمورد حساسيت ويروتواند براساس آخرين ميدستورالعمل هاي استفاده از دارويي آنتي ويرال خوكي در بالغين وكودكان aدوز پيشنهادي داروي ضد ويروس براي عفونت آنفلوانزاي . دارو دستخوش تغيير گردد اثربخشي )1جدول . ( ميباشد aباالي يك سال همانند درمان آنفلوانزاي ول m48 در��ن. ١m وس d داروه�ي � و�p !+f��r+"آqg ��دوز <c� آ� �ا�$اي %&�' � m q2و ��4 و�&<a (h1n1) �ع دارو � /u2 dو 6 q�دوز در�� qgآ"+rو c� دوز آ� oseltamivir adults 75-mg capsule twice per day for 5 days 75-mg capsule once per day 15 kg or less 60 mg per day divided into 2 doses 30 mg once per day 15-23 kg 90 mg per day divided into 2 doses 45 mg once per day 24-40 kg 120 mg per day divided into 2 doses 60 mg once per day children ≥ 12 months >40 kg 150 mg per day divided into 2 doses 75 mg once per day zanamivir adults two 5-mg inhalations (10 mg total) twice per day two 5-mg inhalations (10 mg total) once per day children two 5-mg inhalations (10 mg total) twice per day (age, 7 years or older) two 5-mg inhalations (10 mg total) once per day (age, 5 years or older) 2mg/kg دوبار در روز ودر كودكان يك تا دوازده سال به علت متابوليسم سريع تر 1mg/kgدر بالغين دوز اسلتاميوير * .است روزانه و 75mgدوز اسلتاميئيرنصف مي شود يعني درمان mg/kg 30ارسايي كليه در كليرانس كرآتينين زير در ن** .يوير به علت عدم جذب سيستميك در نارسايي كليه تغيير نمي كند دوز زانام. يك روز در ميان 75mgپيشگيري بطور cdc ي آمريكاستفاده از اوسلتاميوير براي كودكان زير يك سال اخيراً وبه دنبال درخواست مركز مديريت بيماريهاا زير يك سال براساس سن ميباشد دكانودوز دارو در ك. به تاييد رسيده است (fda)اورژانس توسط سازمان غذا و داروي امريكا .) مالحظه فرمائيد را2جدول ( فصلي مقاوم h1n1خصوصاً مناطقي كه داراي گردش ويروس ( درمناطقي كه هنوز از نظر آنفلوانزاي فصلي فعال هستند : توجه هاي غير شديد فصلي سال هاي مانند ژاپن و نروژ كه حاصل كاربرد وسيع اين داروها در سرما خوردگيبه اوسلتاميوير ميباشند براي درمان ) اوسلتاميوير و آمانتادين يا (ممكن است استفاده از زاناميوير يا تركيبي از اوسلتاميوير وريمانتادين ) .قبل است empiric )روفيالكسي دارويي بيماران آنفلوانزاي يا پ) درمان وسيع الطيف بدون تعيين حساسيت داروييa (h1n1) با . ترجيح داده شودخوكي ء منشا ول mاي در��ن . ٢ � �� d داروي � و� وq2 ُا��#%2+p !+f�دوز < آ�دآ�ن آ�y از ��gxل �ار/5p *2 ت z d p !+f�> q�روز٥دوز در�� <3 months 12 mg twice daily 3-5 months 20 mg twice daily 6-11 months 25 mg twice daily و�g."+r ��داروه�� � و� وس c: بمنظور (يوير توصيه شده اند خوكي ، هريك از داروهاي اوسلتاميوير يا زانام aبراي پيشگيري از عفونت با ويروس آنفلوانزاي ). را مالحظه بفرمائيد 1جدول ايي با دوز دارو آشن . روز پس از يك تماس مشخص با مورد قطعي دوره آنفلوانزاي خوكي توصيه ميشود10درمان پروفيالكسي دارويي تا دوره 7قبل تا طي يك روز ( تماس در دوره عفونت زايي با بيمار پروفيالكسي پس از تماس بايستي براي افرادي درنظر گرفته شود كه اگر تماس با فرد بيماري باشد .ي به ضعف ايمني كه در باال ذكر شد باشند گروه ها وجزو.داشته اند) روز بعد از بروز عالئم باليني . روفيالكسي دارويي ضرورت نخواهد داشتپروز قبل آغاز شده است، 7كه عالئم وي بيشتر از ، پيشگيري بايد.) با ضعف ايمني كه جدا سازي آن ها از بيماران ممكن نباشدفقط در بيماران( براي پروفيالكسي قبل از تماس . ادامه يابد خوكي a آنفلوانزاي مورد قطعي روز پس از آخرين تماس با 10به مدت قبل از تماس احتمالي آغاز شود و مقاومت دارويي به اسلتاميوير در موارد د كثر موار وبايد توجه داشت كه استفاده شودميوير ميتواند براي پروفيالكسي ااوسلتا ) 3جدول ( .پروفيالكسي اتفاق افتاده و از پيشگيري هاي دارويي بي مورد جز در موارد ضعف ايمني بايد اجتناب كرد اي � �� d داروي � و� وq2 ُا��#%2+p !+f� ول ٣. دوز <m وr+"آqg آ�دآ�ن آ�y از ��gxلc� آ� �ار/5p *2 d p !+f�> q�ت دوز در�� z qgآ"+rو cروز١٠ <3 months not recommended unless situation judged critical due to limited data on use in this age group 3-5 months 20 mg once daily 6-11 months 25 mg once daily : توصيه شودزيرپروفيالكسي دارويي با هريك از داروهاي اوسلتاميوير يا زاناميوير بايستي به افراد برونشيت – بخصوص آسم افراد دچاريك بيماري طبي مزمن( فردي كه از لحاظ ابتالء به عوارض آنفلوانزا پرخطر تلقي ميشود -1 يك تماس نزديك كه سال وزنان حامله 5 ، كودكان زير سال 65 با سن باالي افراد، -يي ارگان ها نارسا– ديابت كنترل نشده ).عي يا مشكوك به بيماري داشته اندخانگي با مورد قط ، متحمل يا مشكوك بد حال، در طي تماس نزديك با مورد قطعيدر دوره بيماري زايي كاركنان مراكز بهداشتي يا درماني كه -2 .ز وسايل حفاظت فردي مناسب استفاده نكرده اندا :در نظر گرفته شودبراي افراد زير پروفيالكسي دارويي با هريك از داروهاي اوسلتاميوير يا زاناميوير ميتواند سال 65كساني كه يك بيماري طبي مزمن دارند ، افراد باالي ( فردي كه از لحاظ بروز عوارض آنفلوانزا پرخطر تلقي نمود -1 ) فرد مشكوك به بيماري داشته اند سال وزنان حامله كه يك تماس نزديك خانگي با5كودكان زير كه يك ) كودكان داراي يك بيماري مزمن مشخص( كودكان مدرسه اي يا مهدكودكي وپرخطر از لحاظ بروز عوارض آنفلوانزا -2 . به بيماري داشته اندبا مورد قطعي ، محتمل يا مشكوك) صورت به صورت( تماس نزديك داراي يك بيماري طبي مزمن افراد( ميشوند كاركنان مراكز بهداشتي درماني كه از لحاظ بروز عوارض آنفلوانزا پرخطر تلقي-3 خوكي aموارد قطعي آنفلوانزا ي كار مي كنند كه در آنجا مراقبت از يودر بخش ها) زنان حامله سال و 65خاص ، افراد باالي . ر را برعهده دارنداتب دحاد صورت مي گيرد يا اينكه خود اين افراد كار مراقبت از بيماران دچار بيماري تنفسي . وتماس با بيماران ثابت شده با بيماري فعال ر صورت ضعف ايمني و عدم امكان جدا سازي د -4 ?+. :� در��ن < درموارد شديد ابتالي انساني آنفلوآنزاي پرندگان از اسلتاميوير با دوز دو برابر معمول يا كاربرد همزمان اسلتاميوير و آمانتادين .استفاده شده است �د.�ن ز� �0 �2ل .: a ويروس آنفلوانزاي خصوصيات بيماري انساني. اين كودكان از لحاظ بروز عوارض آنفلوانزاي فصلي پرخطر محسوب ميشوند دست مطالعه ميباشد و هنوز مشخص نيست كه آيا شيرخواران در مقايسه با كودكان بزرگتر وبالغين درخوكي در حال حاضر هنوز اطالعات محدودي در مورد بي ضرر بودن استفاده از اوسلتاميوير يا . از لحاظ بروز عوارض آنفلوانزا ريسك باالتري دارند يا خير از اسلتاميوير براي كودكان زير يك سال وجود با احتياط ير يراي كودكان زير يك سال در دسترس است و مجوز استفاده زاناميو .اطالعات موجود نيز براساس مطالعه بر روي اثرات اسلتاميوير در درمان آنفلوانزاي فصلي برست آمده است. دارد استفاده از در خصوص است وانجمن بيماريهاي عفوني امريكا اخيراً ند كه بروز عوارض جانبي نادرناين اطالعات بيان ميك مطالعات گذشته نگر محدود بر روي بي ضرر "اسلتاميوير در كودكان زير يك سال مبتال به آنفلوانزاي فصلي بيان داشته است كه از آنجايي كه . "را نشان نداده استبودن واثربخشي اسلتاميوير در گروه بيماران كم سن وسال ، تاكنون توكيستيه قابل توجهي aريسك مرگ ومير شيرخواران در آنفلوانزا باالست بنابراين احتمال مي رود كه اوسلتاميوير در درمان عفونت ويروس آنفلوانزاي دن بي ضرر بوموردكاركنان مراكز بهداشتي درماني توجه داشته باشند كه به دليل كمبود اطالعات در . باشدخوكي سودمند اوسلتاميوير در حين استفاده از اين دارو در شيرخواران بدحال مبتال به عفونت آنفلوانزاي خوكي يا شيرخواراني كه تماس اژودوز . ونيتورينگ دقيق شيرخوار از نظر بروز عوارض دارويي ضروري است م. ا مورد قطعي بيماري داشته اند نزديك ب !%��i ز��ن: داروها هستند كه مبين عدم انجام مطالعات براي اثبات بي c گروه در آمريكا جزواوسلتاميوير وزاناميوير در رابطه با حاملگي، ود اين با وج . يعني بي ضرر در حاملگي قرار داده است bولي استراليا آن ها را جزو گروه . ين داروها در حاملگي استضرر بودن ا شده ن حامله يا نوزاد متولد شده از آنها گزارش خانم هايتاكنون گزارشي از عوارض جانبي استفاده از اوسلتاميوير و زاناميوير در بدليل فعاليت سيستميك دارو . حاملگي نبايد بعنوان يك مورد منع مصرف براي اوسلتاميوير وزاناميوير در نظر گرفته شود واست ر مشخص شده داروي انتخابي در پروفيالكسي كمت. آنفلوانزا نسبت به زاناميوير ارجح است دچار درمان زنان حامله اوسلتاميوير در سيستميك محدود ، براي اين منظور ترجيح داده شود ولي عوارض تنفسي وابسته به زاناميوير ممكن است بدليل جذب. است يباشد خصوصاً در خانم هاي باريسك بيماريهاي تنفسي بايستي مد نظر قرار زاناميوير كه بدليل روش استفاده استنشاقي آن م به اضافه با توجه به اطالعات به دست آمده از پاندمي در آمريكا و ساير گشور هاي با ابتالي زياد زنان حامله بخصوص در سه .گيرد .بيشتري از افراد عادي قرار دارند ماهه دوم و سوم به علت ضعف ايمني نسبي در معرض عوارض شديد و مرگ و مير توصيه قوي به شروع هرچه زودتر اسلتاميوير در خود july 2009 31به همين علت سازمان بهداشت جهاني در دستورالعمل ساعت 48 جديد را مطرح مي كند بخصوص در h1n1زنان حامله اي كه شرايط اپيدميولوژيك آن ها احتمال ابتال به انفلوانزاي وارض شديد مانند پنوموني و باال رفتن ريسك مرگ تا از ع. ل شروع عاليم بدون منتظر شدن براي تاييد آزمايشگاهي مي كند او كمتر شدن تاثير دارو بازهم موثر است وچنانچه بيمار ساعت با وجود48شروع درمان پس از . جنين وسقط جنين پيشگيري شود .ري مراجعه كرد دارو ي اسلتاميوير باز بايد به كار برود ساعت از شروع بيما48ححامله پس از جزو اولين گروه هايي هستند كه بايد جديد در دسترس قرار گرفت زن هاي حامله h1n1هر زمان كه واكسن آنفلوآنزاي .واكسينه شوند �ارض دارو�� <: ض عمده گوارشي وخفيف وشامل تهوع دل درد و گاهي عوارممانعت كننده نورآمينيداز كم عارضه اند ودر اسلتاميوير داروهاي اسهال كه بعد از دو روز درمان معموال قطع ميشوند عوارض نادر و گاه شديد حساسيت هاي پوستي از كهير تا سندرم استيونس ه در عوارض توهم آژيتاسيون و حتي تشنج بيشتر در نوجوانان ديده شده است ك– منگي -جانسن و عوارض عصبي شامل گيجي .شديد دارو بايد قطع شود زاناميوير عوارض عمومي كمي دارد ولي در بيبماران آسمي وبرونشيتي مي تواند حمالت شديد آسم وبرونكواسپاسم بدهد كه در .زنان حامله آسمي شديد تر است ��� ه�� ���c ادر��ن$�'� �! د�?�ل '�&% ويثروس ودر صورت تشديد بيماري ونارسايي تنفسي استفاده از درمان با داروهاي ضد : پنوموني هاي ويرال -1 respirator. كامال مانند پنوموني هاي باكتريال پس از آنفلو،نزاي فصلي وعلل شايع پنوموكوك و سپس : پنوموني هاي باكتريال -2 ل تركيب سفالوسپورين هموفيلوس آنفلوآنزا در بچه ها ودرنهايتد استافيلوكوك طاليي است كه درذ درمان دو عامل او درمورد پنوموني هاي استافيلوكوكي از . به كار ميرود ....) اريترومايسين يا آزيترومايسين يا ( نسل سوم و ماكروليد كلوگزاسيلين ئو سفازولين در استافيلوكوك هاي حساس به دارو ها تا وانكومايسين در استافيلوكوك هاي مقاوم به كار .رود مي پوشش استافيلوكوك و گرم منفي هاي مقاوم بيمارستاني بايد icuهاي ايجاد شده در بيماران بستري در در پنوموني -3 ./مد نظر باشد �+��ر�� p م r `a"< *+b1�� در: �ن ار�mع �! � ا.$ در����+2��� اد ���b1 .! ا�rا �yg� ا� � دار� اگر همراه حال عمومي بد باشد خصوصاً) روز3بيش از (ي تب باال بمدت طوالن – dyspneaتنگي نفس – difficulty in breathing نشانه هاي ديسترس تنفسي – chest painدرد يا احساس فشار در قفسه سينه يا شكم – (dizziness)سرگيجه ناگهاني – )confusion( گيجي – استفراغ شديد يا مداوم – زا در ابتدا ولي عود ناگهاني تب وسرفه شديد بهبود عالئم آنفلوان – �+��ر� در .�د.�ن� p م r `a"<: �ن ار�mع �! � ا.$ در����+2��� �د.��� .! ا�. �yg� ا� دار� ؟ � )تنفس تند يا نفس دشوار(نشانه هاي ديسترس تنفسي – تغيير رنگ كبود يا خاكستري پوست – عدم نوشيدن مايعات به ميزان كافي – راغ شديد يا مداوماستف – )كودك به دشواري قابل بيدار كردن باشد يا اساساً نتوان وي را بيدار نمود(اختالل سطح هشياري – عدم تمايل براي بازي كردن يا عدم تعامل كودك با اطرافيان – تحريك پذيري شديد درحدي كه كودك تمايلي به آغوش والدين نداشته باشد – ا ولي عود ناگهاني تب وسرفه شديد بهبود عالئم آنفلوانزا در ابتد – �ل p+�+��� '����#�د�*� r: �ل '���#�د�*gd.: ���+�+p ل�� r ��:ا��#%2+ �لgd.4� p و �� :ا��#%2+ : ���ل p+�+��� زا���+� r ا��1�8�� :زا���+ j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 194 http://jad.tums.ac.ir published online: may 27, 2017 original article a retrospective study on scropionism in iran (2002–2011) rouhullah dehghani 1, *javad rafinejad 2, 6, behrooz fathi 3, morteza panjeh shahi 4, mehrdad jazayeri 4, afsaneh hashemi 5 1social determinants of health (sdh) research center and department of environmental health, kashan university of medical sciences, kashan, iran 2department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran 3department of pharmacology, school of veterinary medicine, ferdowsi university of mashhad, mashhad, iran 4health center, kashan university of medical sciences and health services, kashan, iran 5veterinarian medicine, head office of veterinary khorassan razavi province, mashhad, iran 6center for solid waste research (cswr), institute for environmental research (ier), tehran university of medical sciences, tehran, iran (received 2 aug 2014; accepted 7 may 2016) abstract background: scorpion sting is a common medical emergency in iran. the epidemiological features and control of such cases vary from south to north. this review will provide new information about the epidemiology of scorpion stings in different provinces of iran. methods: in this descriptive retrospective study, data on scorpionism including incidence rates, mortality as well as locality from 2002 to 2011were collected. results: overall, 433203 victims of scorpion stings had been referred to health centers from of all of the 31 provinces. the incidence of scorpion stings in 100000 populations was from 54.8 to 66. the highest rate of scorpion stings occurred among the 25–34 yr old group. the highest incidence of scorpion stings during 2011 was observed in khuzestan province and the lowest number in mazandaran province. the peak number of human cases (scorpion stings) was recorded during may to august. conclusion: scorpion stings in iran are high. it is necessary that physicians and health care professionals should be familiar with local scorpions, especially those that are potentially more dangerous than others, the effective treatment protocols, and supportive care as well as their control of scorpionism keywords: scorpion stings, epidemiology, iran introduction venomous animal bites and stings are one of the important health concerns in rural regions in many parts of the world (warrell 1992). scorpions form a large group of poisonous creatures found throughout the warm and temperate regions of the world. nearly all medically significant species of scorpions are located in buthidae family. those considered most dangerous are native to africa, asia and the middle east (dehghani and arani 2015). the outcomes of scorpion sting may cause only localized or general pain but may also cause more severe manifestations such as shock, kidney damage and cardiac muscle injury (white 2000). scorpions are an evolutionarily old group of animals and some 2000 species have been described from around the world (dupre 2012). in iran, the scorpion fauna has had a long-standing interest from scientist’s worldwide long ago paying attention of the scientists worldwide in terms of sys*corresponding authors: dr javad rafinejad, email: jrafinejad@tums.ac.ir j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 195 http://jad.tums.ac.ir published online: may 27, 2017 tematic, biology and ecology. dispensation information of species is reviewed based on scientific literature until 2012. scorpion stings were observed in all over iran, and there are 51 species of scorpions found in different parts of the country, but only ten species are important (dehghani et al. 2012). like with most creatures that use venom only for defense, scorpions seldom sting their prey, as a substitute using their pedipalps and crushing chelicera to acquire food (vetter and visscher 1998). within scorpion fauna of iran, the buthidae family is the more frequent, with 82% of all the genera and 88.5% of all the species. among these, the androctonus genera have the main number of known medically significant species. the number species of two others families is alike. the hemiscorpiidae family is, with 2 genus (9%) and 3 species (5.75%). in this family, the medically chief genus is hemiscorpius with the main number of notorious species. lastly, scorpionidae is another family with 2 genus (9%) and 3 species (5.75%). the south and southwest of iran with about 95% species of scorpions are the most heavily occupied areas in the country (navidpour et al. 2008a,b). khuzestan province is highlighted for its scorpions and scorpion sting amongst the provinces of iran (vazirainzadeh et al. 2012). khuzestan with 19 species of scorpions is one of the most significant areas in terms of scorpion sting problem in the south west of iran. in iran, medical importance, epidemiology and geographic distribution of scorpions have been reported (dehghani 1998, navidpour et al. 2008a, dehghani et al. 2012). due to the significance of scorpion stings and the shortage of epidemiological data about this public health difficulty, the study was carried out to collect new data concerning scorpion stings in iran. this would allow the system to arrangement strategies to decrease and scorpion stings among the inhabitants of all provinces in iran. the purpose of this project was to conduct a retrospective study to describe the incidence and geographic location of scorpion stings in iran provinces, and to assess the magnitude and distribution of the problem in order to optimize prevention and treatment. materials and methods this descriptive and retrospective study was carried out in 2012. data was gathered based on information of ministry of health and medical education from the files of outpatient or hospitalized persons referred to the health centers and hospitals of all provinces for the last decade. the data of scorpion stings was assessed from the epidemiological aspects including: gender and ages of scorpion sting victims, antivenin therapy, the time of scorpion sting, subjected parts of body, and environmental circumstances such as rural or urban habitats. the recorded data were evaluated prospectively and statistically analyzed using excel's simple statistical functions, and then interpreted and presented in the form of tables and graphs geographical information on iran iran is the eighteenth biggest country of the world. its borders are limited to the north, caspian sea, in the northwest, armenia and azerbaijan, the east, afghanistan and pakistan, in the west, iraq and turkey, in the northeast, turkmenistan, and ultimate the waters of the persian gulf and the sea of oman in the south. iran's district is 1,648,000 square km. iran has three separate geographies: (a) the foremost mountain ranges comprise the zagros mountains in the west and south, and the elburz mountains in the north. most of these mountains are upper than 2,440 meters. some peaks are higher than 4,268 meters in the zagros and 5,486 meters in the elburz, including qolleh-ye damavand at 5,671 meters. (b) most of the rest of the country consists of a flat terrain that contains several j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 196 http://jad.tums.ac.ir published online: may 27, 2017 closed basins and two salt deserts, the dashte kavir and the dasht-e lut. a lot of the flat terrain has internal drainage and is distinguished by many irregular streams, discontinuous salt lakes, and wet salt flats. (c) lesser flat plains are situated the length of the caspian sea, persian gulf, and sea of oman (http://en.wikipedia.org/wiki/geography_of_ iran). iran has various climates with very warm summer and cold, with some snow, winter. winter season is usually the rainy for the entire country. the northwest is generally the coldest and among the rainiest parts of the country. the winters in kurdistan province and west and east azarbaijan and ardabil provinces and can be harsh: temperatures from time to time drop as low as -20 oc. snow often remains until near the beginning spring, or even later in the mountains. however the littoral regions have moderately a different weather. the caspian sea shoreline is moist all year round and provides a difference with the aridness of highland. the temperature can vary typically. in the summer, temperatures differ from 50 oc in the south to 1 oc in the northwest. mean winter and summer are 5.9 oc and 37.8 oc respectively. rainfall also varies actually, range from less than 50 mm in the southeast to about 2000mm in the caspian region. the annually mean is about 250mm (reference http://iranto.ca/en/index.php/about-iran/geography-and-climate). results during 2002–2011, a total of 433201 scorpion-stung patients referred to the health center and hospitals of all provinces (fig. 1). the incidence of scorpion stings in 100000 of population was from 54.8 to 66 during 2002–2011 (fig. 2). the highest incidence of scorpion sting cases were reported in 2008 (47510) and the lowest in 2004 (36806) in all provinces of iran. out of 433201 scorpion sting cases, 433003 cases (99.95%) recovered, however deaths (0.05%) were recorded during the study period (fig. 3). the highest mortality rate of scorpion stings were occured during 2004 (29), however the lowest mortality rate of scorpion stings were reported in 2002 (14) among all provinces of iran highest prevalence of scorpion sting and its resulting death has been recorded from khuzestan and the lowest in mazandaran provinces, respectively (figs. 4 and 5). only about 55.5% of scorpion stings and 38.1% the mortalities occurred in khuzestan province. the highest of scorpion stings after khuzestan were reported in fars, hormozgan, kohgiloye and boyerahmad, kerman and the rest of provinces of iran, respectively. based on recorded data, the rate of incidences in different provinces has increased from north to south. the incidence of scorpion sting in iran has been calculated as 62.02 persons in 100000 during one year (march 22nd 2011–march 21st 2011). in general, most victims of scorpion stung people were in rural (24683: 52%), and 46 % (21803) from urban and the rest (729:2%) were unknown areas of iran. wholly, the number and percent of stung women and men of scorpion-stung patients were 24339 (52%) and 22896 (48%), respectively. the highest rate of scorpion stings victims are related to the 15–34 yr old (43.5%) followed by 5–14 (14.3%) and 35–44 (13.5%). the lowest rate of scorpion stings were reported among the population more than 65 yr old (5.3%) (fig. 6). scorpion sting were seen in all months, the highest incidence of scorpion sting cases were in july (16.3%) and the lowest in december (1.2%) in iran (fig. 7). legs and hands were exposed by scorpion more than the other parts (80%), followed by head and trunk with 18%, the rest (2%) were unknown. out of 47,235 scorpion sting cases, 47,214 cases (99.95%) recovered, however deaths (0.05%) were recorded during the 2011. totally, 16% of victims recovered using convenience treatj arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 197 http://jad.tums.ac.ir published online: may 27, 2017 ments without scorpion antivenin serum. however, the rest were treated by scorpion antivenin serum including intra-muscular (32849:86%) and intra-venin (5540:14%) injections and convenience treatments. the interval hours after stings and injections were recorded as: survival was 60.9 after 0–6 h, 11.8% after 6–12 h and 6.3% after more than 12 h. out of 21 deaths, 11 cases were not received any antivenin, and the rest were received antivenin, 10 cases of deaths occurred during 6 to 12 h and more than 12 h, after scorpion antivenin serum was injected. fig. 1. number of scorpion stings in iran during 2002–2011 fig. 2. incidence of scorpion sting in iran during 2002–2011 j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 198 http://jad.tums.ac.ir published online: may 27, 2017 fig. 3. number of human cases of scorpion stings resulted to death in iran during 2002–2011 fig. 4. spatial distribution map of scorpion sting cases in different provinces of iran, 2002–2011 j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 199 http://jad.tums.ac.ir published online: may 27, 2017 fig. 5. percentage of scorpion stings according to provinces in iran during 2002–2011 fig. 6. frequency of scorpion stings according to age groups in iran during 2002–2011 j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 200 http://jad.tums.ac.ir published online: may 27, 2017 fig. 7. number of scorpion stings, based on month in iran during 2002–2011 discussion the present study showed that the incidence of scorpion stings in 100000 of population was from 54.8 to 66 during 2002–2011. these variations were likely due to the difference of climatologic factors and preventive measures. the mean rate of sting incidences in iran was 61.2 per 100,000 populations during the study period. in the world incidence annually, 1.2 million people are estimated with around 3250 deaths per year. base on chippaux and goyffon 2010, the mean rate of sting incidences in the world in per year per 100,000 population is about 17.14 (chippaux and goyffon 2010). it shows scorpion sting incidence in iran which is higher than the global average. the important matter is that the scorpions stung every year with a resembling rate 54.8 to 66 during whole period of 2002–2011 (table 1). the highest of scorpion stings were reported in khuzestan and the lowest in mazadaran. this agrees with the results of dehghani and fathi (2012), labaf ghasemi (1999) and rafizadeh et al. (2013) (labaf ghasemi 1999, dehghani et al. 2012, rafizadeh et al. 2013). there was a difference between frequency of males (48%) and females (52%) among the patients referred to the health centers and hospitals, with scorpion stings. it means that the females were at higher risk of scorpion stings than males in all provinces of iran. this amount is agreed with the results of vazirianzadeh et al. 2012, shahbazzadeh et al. 2009). our results are not in agreed with the results in saudi arabia (al-sadoon 2003, jarrar 2008) who reported that scorpion stings were higher in males than in females. the highest rate of scorpion stings occurred among the 15–34 yr old people, in accordance with the findings in kashan, central of iran (deghani et al. 2010), ahvaz, south west of iran (emam at al. 2008). the highest incidence of scorpion sting cases during 2011 occurred in summer (44.16%). this is in agree j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 201 http://jad.tums.ac.ir published online: may 27, 2017 with the studies in iran (rafeeazadeh et al. 2009, deghani et al. 2010, dehghani and fathi 2012a), saudi arabia (shahbazzadeh et al. 2009, jarrar and al-rowaily 2008) and in turkey (emam et al. 2008, ozkan and kat 2005). they have reported that 49.7–93.4% of scorpion sting cases occurred in summer. in general, a total of 84% scorpion stung persons received antivenin. this antivenin is prepared in razi vaccine and serum research institute, iran in a 5ml hexavalent vial of 6 species including hemiscorpius lepturus, androctonus crassicauda, mesobuthus eupeus, odontobuthus doriae, hottentotta saulcyi and hottentotta schach (sanaei-zadeh 2014). the most important health-threatening scorpions in iran is androctonus crassicauda from buthidae family and hemiscorpius lepturus, which belongs to the hemiscorpiidae family. they are described as potentially dangerous to humans. other important species of the buthidae family are: androctonus crassicauda, compsobuthus matthiesseni, orthochirus spp, mesobuthus eupeus, odontobuthus doriae, hottentotta schach, ho. saulcyi, mesobuthus caucasicus and apistobuthus pterygocercus (malhotra et al. 1978, radmanesh and shaffiee 1989, radmanesh 1990a,b, mashak et al. 2000, pipelzade 2007, dehghani and khamechian 2008, dehghani et al. 2009, jalali 2010, dehghani and fathi 2012, dehghani et al. 2012). due to variability of scorpion venoms, the severity of envenoming is species dependent. therefore, determination of the species responsible for sting is critical and can affect the clinical procedures of patient’s treatment. we recommend that the treatment for scorpion stinging in iran should be based on the neurotoxic and cytotoxic effect of their venom produced in victims. it is necessary to distinguish all the native scorpion species, especially those medically important, in every region and determine their life, behaviors, mode of actions, and their venom properties (dehghani et al. 2009, dehghani and fathi 2012). it is recommended to consider the possible connection between any stung patient and dangerous species of the region, for this it will be useful to investigate about the species of scorpion from the victims’ relative or those who accompany the patient. in addition, it is necessary that physicians and health care professionals are familiar with native scorpions, especially those that are potentially more dangerous than others, the effective treatment protocols, and supportive care. conclusion scorpion stings have a seasonal pattern and mostly take place during the warmer months of the year. therefore, formation of professional reinforcement staff and regular visits to high risk regions during this time can significantly reduce the risk of scorpion stings. moreover, due to the rate of stings is higher in rural regions, training the rural people in public places like schools, mosques, and also by influenced persons will reduces the number of stings incidents. acknowledgements the authors gratefully acknowledge of non-communicable diseases management department of ministry of health and medical education or his assistance in this study. the authors declare that there is no conflict of interests. references al-sadoon mk, jarrar bm (2003) epidemiological study of scorpion stings in saudi arabia between 1993 and 1997. j venom anim toxins incl trop dis. 9: 54–64. chippaux jp, goyffon m (2010) epidemiology of scorpionism: a global apprasial. acta trop. 107: 71–79. j arthropod-borne dis, june 2017, 11(2): 194–203 r dehghani et al.: a retrospective study … 202 http://jad.tums.ac.ir published online: may 27, 2017 dehghani r, khamechian t (2008) scrotum injury by scorpion sting. iran j arthropod-borne dis. 2(1): 49–52. dehghani r, djadid nd, shahbazzadeh d, bigdelli s (2009) introducing compsobuthus matthiesseni (birula 1905) scorpion as one of the major stinging scorpions in khuzestan, iran. toxicon. 54 (3): 272–275. deghani r, 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hosseinzadeh m, zarean m, moravej sa (2012) an epidemiological and clinical study on scorpionism in hospitalized children in khuzestan, iran. j arthropod-borne dis. 6: 62–69. vetter rs, visscher pk (1998) bites and stings of medically important venomous arthropods. int j dermatol. 37: 481–496. warrell da (1992) recent advances in toxinology research. in: gopalakrishnakone p, tan ck, (eds). national university of singapore, singapore. pp. 121–153. white j (2000) bites and stings from venomous animals: a global overview. there drug monit. 22(1): 65–68. 2-hashareh dr pourfallah rtl 88 10 3 iranian j arthropod-borne dis, (2009), 3(2): 7-11 fpourfallah et al: evaluation of serum levels… 7 original article evaluation of serum levels of zinc, copper, iron, and zinc/copper ratio in cutaneous leishmaniasis *f pourfallah1, s javadian1, z zamani1, r saghiri1, s sadeghi1, b zarea2, sh faiaz1, f mirkhani1, n fatemi1 1department of biochemistry, pasteur institute of iran, tehran, iran 2qom central hospital, qom, iran (received 19 jun 2009; accepted 28 oct 2009) abstract background: the purpose of this study was to evaluate the levels of zinc (zn), copper (cu), iron (fe) and zinc/ copper ratio in the serum of patients with cutaneous leishmaniasis in qom province, center of iran. methods: serum levels of zinc and copper were determined by flame atomic absorption spectrophotometer and serum iron concentration was measured by using an auto analyzer. the study group consisted of 60 patients with cutaneous leishmaniasis and the control group of 100 healthy volunteers from the same area who were not exposed to cutaneous leishmaniasis. result: there were no statistically significant differences in age and body mass index between the two groups. serum zn (p< 0.001) and fe (p< 0.05) levels were lower in patients with cutaneous leishmaniasis than the control group. we also found serum cu concentration (p< 0.05) in the patient group was significantly higher than that of the control group. however, zinc/ copper ratio (p< 0.001) was lower in patients with cutaneous leishmaniasis than in the control group. conclusion: our data indicated that zn/cu ratio was significantly lower in patients with cl as compared to the controls. earlier reports suggest that, this ratio imbalance could be a useful marker for immune dysfunction in leishmaniasis. there was also strong association of zn, cu and fe with cl. it suggests the use of blood zinc, copper, iron concentration and the copper/zinc ratio (zn/cu), as a means for estimating the prognosis of cl. keywords: cutaneous leishmaniasis, zn, cu, fe. zn/cu ratio, iran introduction leishmaniasis is a group of diseases caused by several species of the genus leishmania, a protozoa transmitted by the bite of a tiny insect vector, the sandfly. the four clinical patterns of the disease in the human host are: cutaneous leishmaniasis (cl), diffuse cutaneous leishmaniasis (dcl), mucocutaneous leishmaniasis (mcl), and visceral leishmaniasis (vl).the annual incidence of cl is 1 to 1.5 million cases. iran is known endemic foci of cl (desjeux et al. 1996). trace elements are needed for many metabolic and physiological processes in the human body (mertz et al. 1981). alterations in iron (fe), zinc (zn) and copper (cu) levels in the sera change during inflammation and infections. these are associated with elevated levels of acute phase proteins, such as ceruloplasmin (cousins et al. 1985, barber et al. 1988). zinc is a component of more than 200 enzymes, involved in various activities, such as metabolic functions, immunity and wound healing (tudor et al. 2005). copper is an essential nutrient that is widely spread in food and water. it is a part of several metalloenzymes that is required for oxidative metabolism, including cytochrome oxidase, feroxidase, amino oxidase, superoxide dismutase, ascorbic acid oxidase and ty*corresponding author: dr fatemeh pourfallah, e-mail: fpourfallah@ yahoo.com iranian j arthropod-borne dis, (2009), 3(2): 7-11 fpourfallah et al: evaluation of serum levels… 8 rosinase (panemangalore and bebe 1996). iron plays a role in the oxygenation of tissues as it is incorporated in the heme structure of hemoglobin (peralta et al. 1999). in this study, we investigated the association of essential trace elements such as zinc (zn), copper (cu) and iron (fe) levels in serum of patients with cutaneous leishmaniasis. materials and methods the subjects of this case-control study were collected from the central hospital of qom, center of iran and a known endemic foci of cutaneous leishmaniasis. the study group consisted of 60 leishmaniasis patients (28 f, 32 m; mean of ages= 33.9±17.2 yr), and the control group consisted of 100 healthy volunteers (63f, 37m; mean of ages= 36.5± 16.3 yr) from the same area who were not exposed to cutaneous leishmaniasis. all individuals gave a written consent, and this study was reviewed and approved by human subjects ethical community of the pasteur institute of iran. the patients were interviewed with structured questionnaire requesting information related to various criteria. the age, height and weight of all of the groups were recorded. the patients were selected based on clinical symptoms confirmed by laboratory diagnosis. it was based on the presence of parasites in geimsa-stained smears that were prepared with material aspirated from borders of skin lesions and tissue imprints from biopsy. the patients should not have taken any antimonial treatment before the blood testing and had not any other or secondary infections. both patient and control groups had the same socio-economic status and no mineral supplement should have been taken prior to the test. standard methods were used to determine height (cm) and weight (kg). body mass index (bmi) was calculated by kg/m2. five ml of the venous blood was drawn after overnight fasting, from all individuals and transferred into acid-washed test tubes without any addition of anticoagulants. the blood samples were allowed to clot at room temperature for about one hour, then, the blood samples were centrifuged at 3000 rpm for 15 min at room temperature to separate the sera. hemolytic sera were discarded. sera were aliquoted into the eppendorf tubes and stored at -20 °c until they were tested in the biochemistry department of pasteur institute of iran. analysis of copper and zinc were measured by using flame atomic absorption spectrometry (thermo jarrel ash, germany) according to the method of kirgbright et al. serum samples were diluted by deionized water. different concentrations of trace elements were prepared for calibration of standard graphs. absorbances were read at 324.7 nm and 213.9 nm, for copper and zinc, respectively. for accuracy, the standard solutions were run for every 10-test sample. serum samples were run in triplicate, and individual values were averaged (kirgbright et al. 1980). serum iron concentration was measured by using an auto analyzer (technicon, ra1000, usa) with commercial kit (shim enzym, iran) the spss software package (windows version 14, spss, chicago, ill, usa) was used for all statistical analyses. the mean and standard deviation (sd) were used for reporting and a p-value of <0.05 was considered significant. result table 1 shows the patient and control group were similar in age, height, weight body and body mass index (table 1). when patients with cutaneous leishmaniasis were compared to control group, levels of serum zn (p< 0.001), fe (p< 0.05) and zn/cu ratio (p< 0.001) were significantly lower than the control group (table 2). however, serum cu concentration iranian j arthropod-borne dis, (2009), 3(2): 7-11 fpourfallah et al: evaluation of serum levels… 9 was higher in patients with cutaneous leishmaniasis than the control group (p< 0.05) (table 2). the mean duration of the disease was 34±27 d and there was no significant differences when it was compared these trace elements before and after this time. table 1. physical characteristics of patients with cutaneous leishmaniasis and control group patient group no.=60 mean ±sd control group no.=100 mean ±sd p value* age (yr) height (cm) weight (kg) body mass index (kg/m2222) duration of disease(day) 33.4±17.2 165±5 62.2±12 22.5±4.1 34±27 36.5±16.4 167.6±7 64.2±11 22.43±3.7 na p> 0.05 p> 0.05 p> 0.05 p> 0.05 na *the mean difference is not significant at p> 0.05, na negative table 2. comparison of serum zinc, copper, iron concentrations and zinc/copper ratio in patients with cutaneous leishmaniasis and control group patient group no.=60 mean±sd control group no.=100 mean±sd p value* serum zn (µg/dl) serum cu (µg/dl) serum fe (µg/dl) zn /cu 98.58±19.7 133.65±9.1 119.78±41.6 0.7385±0.14 126.38±40.2 127.3±13.4 143.56±54 1.008±0.35 p<0.001 p<0.05 p<0.05 p<0.001 *the mean difference is significant at p< 0.05. discussion trace elements play a part in the synthesis and structural stabilization of both protein and nucleic acid. hence, imbalances in the optimum levels of trace elements may adversely affect biologically processes, and are associated with many diseases (muralidhar et al. 2004). determinations of trace elements in various diseases have been carried out for many years, but there are a few studies of these elements in cutaneous leishmaniasis. in this study, we found that serum zn concentration was significantly lower in patients with cl (table 2) and malnutrition was not a problem, because bmi, weight, and height were not statistically changed in patients and normal individuals (table 1). decreasing serum zn levels is due to several reasons, mainly, synthesis of methallothionein (mt) in liver, and other tissues. methallothionein binds 7 g atoms of zn per mol and serves to draw zn away from freecirculating pools and it is induced by il-1 in vivo (svenson et al. 1985, rofe et al. 1996). in our study, we also observed that cu levels were significantly higher in patients sera than normal individuals (table 2). increased serum cu is associated with an increase in the synthesis of the copper-binding protein ceruloplasmin, (cillarie et al. 1989, liew et al. 1990). it was demonstrated that il-1, but not tnf-α, induced hypercupremia when injected into the preoptic anterior hyiranian j arthropod-borne dis, (2009), 3(2): 7-11 fpourfallah et al: evaluation of serum levels… 10 pothalamus (klassing et al. 1987). we also found that serum iron concentrations were significantly lower in patients with cl as compared to controls (table 2). iron has a major role in chronic inflammatory diseases (barollo et al. 2005). lin et al, demonstrated in vitro that iron chelation effectively blocked nf-kappa b activation and upregulates tnf-α and il-6 genes in a model of cholestatic liver injury, suggesting a basic role for iron in the activation of the inflammatory process (lin et al. 1997). kocygit observed similar results, in which the cl patients had significantly lower zn and fe level and higher serum cu level as compared to the control subjects (kocyigit et al. 1998). the authors claimed that the changes could be a part of defense strategies of organisms and were induced by il-1, tnf-α, and il-6 (kocyigit et al. 2002). another study in agreement with our data, demonstrated that serum cu concentration was found significantly higher in patients with acute and chronic cutaneous leishmaniasis than those of control group. however, zn and fe levels were lower in patients with acute (p< 0.001) and chronic cutaneous leishmaniasis than in the control group (faryadi and mohebali 2003). our data indicate that zn/cu ratio were significantly lower in patients with cl as compared to the controls. earlier reports suggest that, this ratio imbalance could be a useful marker for immune dysfunction in leishmaniasis (weyenbergh et al. 2004). different studies were carried out on the effect of zn on immune function. oral znso4 was administered to mice with cl and reported to be effective. it had been reported that deficiencies of trace elements can change the immune function from cellular th1 to humoral th2 prematurely and oral znso4 seemed effective (sprietsma et al. 1997). in vitro sensitivities of promastigotes and axenic amastigotes of both l. major and l. tropica to zinc sulfate were studied by najim et al. (1998). the efficacy of 2% znso4 both as an oral and intralesional injection was studied in treatment of cl but they showed inadequate therapeutic value (yazdan panah et al. 2003, khatami et al. 2005). our data indicate that zn/cu ratio was significantly lower in patients with cl as compared to the controls. earlier reports suggest that, this ratio imbalance could be a useful marker for immune dysfunction in leishmaniasis (weyenbergh et al. 2004). the findings of such studies indicate a strong association of zn, cu and fe and the copper/zinc ratio with cl. a strategy can be devised to use blood zinc, copper, iron concentration and the copper/zinc ratio (zn/cu) as a means for estimating the prognosis of cl. acknowledgements the authors wish to thank pasteur institute of iran for financial support and qom central hospital for their technical help. the authors declare that they have no conflicts of interest. references barber ef, cousins rj (1988) interleukin-1 stimulated induction of ceruloplasmin synthesis in normal and copper-deficient rats. j nutr. 118: 375–381. barollo r, d'inc m, scarpav, et al. (2005) effects of iron manipulation on trace elements level in a model of colitis in rats. world j gastroenterol. 11(28): 4396-399. cillarie e, dieli m, maltese e, et al. (1989) enhancement of macrophage il-1 production by leishmania major infection in vitro and its inhibition by ifn. j immunol .143(6): 2001-2005. cousins rj (1985) absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. physiol rev. 65: 238-241. desjeux p (1996) cutaneous leishmaniasis: iranian j arthropod-borne dis, (2009), 3(2): 7-11 fpourfallah et al: evaluation of serum levels… 11 public health aspects and control. clin dermatol. 14: 417-423. faryadi m, mohebali m (2003) alterations of serum zinc, copper and iron concentrations in patients with acute and chronic cutaneous leishmaniasis. iranian j publ health. 32 (4): 53-58. khatami a, firooz a, gorouhi f, et al. (2005) treatment of acute old world cutaneous leishmaniasis: a systematic review of the randomized controlled trials. journal of the american academy of dermatology. 57(2): 335-340. klassing kc, laurin de, penk rk, et al. (1987) immunological mediated growth depression in chicks: influence of feed intake, corticosterone and interleukin1. j nutr. 117: 16291637. kirgbright gf (1980) atomic absorption spectroscopy, elemental analysis of biological materials. vienna technical report series. int atomic agency. 197: 141-165. kocyigit o, erel ms, gurel s, et al. (1998) alterations of serum selenium, zinc, copper and iron concentrations, and some related antioxidant enzyme activities in patients with cutaneous leishmaniasis. biol trace element res. 65: 271–281. kocyigit a, gur s, erel o, et al. (2002) associations among plasma selenium, zinc, copper, and iron concentrations and immunoregulatory cytokine levels in patients with cutaneous leishmaniasis. biol trace elem res. 90: 47-55. liew fy, parkinson c, millott s, et al. (1990) tumor necrosis factor (tnf-α) in leishmaniasis. tnf-α mediates hostprotection against cutaneous leishmaniasis. immunology. 69(4): 570-73. lin m, rippe ra, niemela o, et al. (1997) role of iron in nf-kappa b activation and cytokine gene expression by rat hepatic macrophages. am j physiol. 272: 1355-1364. mertz w (1988) the essential trace elements. science. 213: 1332-1338. najim ra (1998) zinc sulfate in the treatment of cutaneous leishmaniasis: an in vitro and animal study. mem inst oswaldo cruz. 93: 831-833. panemangalore m, bebe fn (1996) effect of high dietary zinc of plasma ceruloplasmin and erythrocyte superoxide dismutase activities in copper-depleted and repleted rats. biol trace elem res. 55(1-2): 111-26. peralta v, cuesta mj, mata i,et al. (1999) serum iron in catatonic and noncatatonic psychotic patients. biol psych. 45(6): 788-90. rofe am, philcox jc, coyle p (1996) trace metal, acute phase and metabolic response to endotoxin in metallothioneinnull mice. biochem j. 314: 793-797. shanker ah, parasad as (1998) zinc and immune function: the biological basis of altered resistance to infection. aj nutr. 68 (suppl): 447s-63s. sharquie kb (2001) oral zinc sulfate in the treatment of acute cutaneous leishmaniasis. clin exp dermatol. 26: 21-26. soleimani m (2003) oral zinc sulfate in the treatment of acute cutaneous leishmaniasis. iranian journal of dermatology. 2: 20-24 (in persian). sprietsma je (1999) zinc-controlled th1/th2 switch significantly determines development of diseases. med hypotheses. 49: 1-14. svenson klg, hallgren r, johansson e, lindh u (1985) reduced zinc in peripheral blood cells from patients with inflammatory connective tissue disease. inflammation. 9(2): 189-199. tudor r, zalewski pd, ratnaike rn (2005) zinc in health and chronic disease. j nutr health aging. 9(1): 45-51. weyenbergh v, santana g, d'oliveira a, et al. (2004) zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study. bmc infect dis 4: 50-59. j arthropod-borne dis, december 2016, 10(4): 483–492 y salim-abadi et al.: high insecticides … 483 http://jad.tums.ac.ir published online: october 04, 2016 original article high insecticides resistance in culex pipiens (diptera: culicidae) from tehran, capital of iran yaser salim-abadi 1, mohammad ali oshaghi 1, ahmad ali enayati 2, mohammad reza abai 1, *hassan vatandoost 1, mohammad reza eshraghian 3, hossein mirhendi 4, ahmad ali hanafi-bojd 1, mohammad amin gorouhi 1,5, fatemeh rafi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 3department of biostatistics, school of public health, tehran university of medical sciences, tehran, iran 4department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 5department of environmental health and medical entomology, school of health, kerman university of medical sciences, kerman, iran (received 23 nov 2014; accepted 04 agu 2015) abstract background: during recent years transmission of dirofilaria immitis (dog heart worm) by culex pipiens and west nile virus have been reported from iran. the present study was preformed for evaluating the susceptibility status of cx. pipiens collected from capital city of tehran, iran. methods: four insecticides including: ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05% and cyfluthrin 0.15 % according to who standard methods were used for evaluating the susceptibility status of cx. pipiens from tehran moreover for comparison susceptibility status a laboratory strain also was used. bioassay data were analyzed using probit program. the lethal time for 50% and 90% mortality (lt50 and lt90) values were calculated from regression line. results: the susceptibility status of lab strain of cx. pipiens revealed that it is susceptible to lambdacyhalothrin, deltamethrin, cyfluthrin and resistant to ddt. moreover cyfluthrin with lt50=36 seconds and ddt with lt50=3005 seconds had the least and most lt50s. field population was resistance to all tested insecticides and ddt yielded no mortality. conclusion: highly resistance level against all who recommended imagicides were detected in field populations. we suggest more biochemical and molecular investigations to detect resistance mechanisms in the field population for further decision of vector control. keywords: susceptibility status, culex pipiens, tehran introduction mosquitoes known as the main groups of arthropods in medical and public health due to their role in transmission of malaria, filariasis, several types of encephalitis and also many arboviral diseases (horsfall 1955, service 2003, mullen 2009). in the culicidae family, culex genus and specifically cx. pipiens complex members including: cx. pipiens pipiens, cx. quinquefasciatus, cx. p. pallens, cx. pipiens form molestus act as efficient vectors for sindbis virus, west nile virus, equine encephalitis, st louis, oropouche, rift valley fever,moreover cx. pipiens transmit plasmodium relictum that *corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 483–492 y salim-abadi et al.: high insecticides … 484 http://jad.tums.ac.ir published online: october 04, 2016 causing bird malaria. its distribution is wide so that this species approximately present in all continent of the world (mitchell et al. 1980, vinogradova 2000, smith and fonseca 2004, savage et al. 2007, mullen 2009, strickman and fonseca 2012). in iran during recent years transmitting of dirofilaria immitis (dog heart worm), west nile and sindbis viruses by culex mosquitoes have been reported (naficy and saidi 1970, azarihamidian et al. 2007, azari-hamidian et al. 2009). culex genus habituated capital city of tehran in sewage system of the houses, where there are different insecticides which have been used for controlling of household and agricultural pests such as: permehrin, bioallethrin, dursban, pirimiphos-methyl, malathion, chlorpyrifos-methyl, allethrin, propoxur. i addition different reagents also exist in the sweage systems. we postulated that resistance to insecticides is as a result of continous exposure to different group of insecticides which may indirectly cause selection pressure on the susceptibility of mosquitoes mainly breed in wastewater habitats (horsfall 1955, lotfi 1976, golestani 1976, lines 1988, vatandoost et al. 2004, calhoun et al. 2007). here upon the evaluation of susceptibility level of mosquitos for monitoring of resistance to insecticides is necessary and for this approach in iran in recent years the most studies about evaluating susceptibility level of mosquitos to common insecticides have been on anopheles mosquitos (enayati et al. 2003, vatandoost and borhani 2004, vatandoost and hanafibojd 2005, vatandoost et al. 2005, hanafibojd et al. 2006, shahi et al. 2006, abai et al. 2008, hanafi-bojd et al. 2010, oshaghi et al. 2011, hanafi-bojd et al. 2012, soltani et al. 2013) and there are just a limited study about susceptibility status of culex genus in iran for example in the performed studies in caspian sea coast about susceptibility level of cx. pipiens complex results indicated that this species is resistant to ddt and susceptible to dieldrin (lotfi et al. 1975). nazari and janbakhsh in 2000 reported that cx. pipiens in the southern area of tehran is resistance to ddt (nazari and janbakhsh 2000). in 2004 the susceptibility level of laboratory and field collected strains of cx. quinquefasciatusin a medically important member belong to cx. pipiens complex to different insecticides evaluated, ddt resistant was observed in both laboratory and field collected strains (vatandoost et al. 2004). according to the most performed study around the world it seems that this species approximately is resistant to many insecticides or have multiple insecticide resistances (davidson 1964, mukhopadhyay et al. 1993, ben cheikh et al. 1998, bisset et al. 1999, martinez-torres et al. 1999, corbel et al. 2007, tantely et al. 2010, toma et al. 2011, jones et al. 2012, pocquet et al. 2013). the present study was preformed for evaluating susceptibility status of cx. pipiens of tehran city. the result of this study can be useful for future chemical control programs in the study area. materials and methods study area this study was conducted in tehran city (35° 41′ 46″ n, 51° 25′ 23″ e), tehran province, iran. tehran is the capital of iran and also is iran's largest city (fig. 1). mosquito strains and adult susceptibility test in this study four insecticides including: ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, cyfluthrin 0.15% were used for evaluating susceptibility status of cx. pipiens. moreover for comparison susceptibility status a laboratory strain also was used. all tested species were reared in the insectary of school of public health, tehran university of medical sciences under the standard condition. data analysis bioassay data were analyzed with probit j arthropod-borne dis, december 2016, 10(4): 483–492 y salim-abadi et al.: high insecticides … 485 http://jad.tums.ac.ir published online: october 04, 2016 program (finney 1971). for correction mortality, when control mortality is greater than 5% but less than 20%, then the observed mortality was corrected using abbott's formula (abbott 1965). by method of finney the lethal time for 50% and 90% mortality (lt50 and lt90) values and their 95% confidence interval and probit regression line parameters were determined for both strains (lab and field population). the regression line belong to each insecticides after different exposure times were plotted using microsoft excel (ver. 2013). results the result of susceptibility test for lab and filed strains of cx. pipiens has been summarized in table 1 and 2. lab strain exhibited different lt50 valuses to different insecticides. cyfluthrin with lt50=36sec, lambdacyhalothrin (lt50=79), deltamethrin (lt50= 326) and ddt (lt50=3005) had the lowest to highest lethal time (table 1 and 3). although this result for field population indicated that ddt exhibited no mortality. cyfluthrin with lt50=27minutes, lambdacyhalothrin (lt50= 111 minutes) and deltamethrin (lt50= 182 minutes) had lowest to highest lt50 (table 2 and 3). the result also showed that among these insecticides, lab strain is susceptible to lambdacyhalothrin, deltamethrin, cyfluthrin and resistance to ddt according to who criteria that suggested (98-100% mortality indicates susceptibility, 90-97% mortality indicates resistance candidate (more investigation is needed or requires confirmation of resistance with other methods) and <90% mortality suggests resistance) (who 2013). and also cyfluthrin with lt50=45sec and ddt with lt50=3005 were the most and least effect (table 1,3 and fig. 2,3). fig.1. the map of iran and location of tehran city table 1. probit regression line parameters of lab strain of culex pipiens exposed to different insecticides insecticides a b ± se lt50 , 95% c.i. (second) lt90, 95% c.i. (second) x2 (df) p value ddt 4% -9.21 2.64± 0.31 2647 7061 3.56(3) >0.053005 9156 3455 13701 lambdacyhalothrin 0.05% -2.50 1.31±0.18 43 510 2.41(2) >0.0579 752 120 1302 deltamethrin 0.05% -4.17 1.6± 0.16 255 1443 5.85 (4) >0.05326 1937 406 2872 cyfluthrin 0.15% -1.84 1.17±0.18 14 308 3.60(4) >0.0536 448 64 725 a= y-intercept, b= the slope of the line, se= standard error, ci= confidence interval, x2= heterogeneity about the regression line, df= degree of freedom, p> 0.05 = represent no heterogeneity in the population of tested mosquitos. j arthropod-borne dis, december 2016, 10(4): 483–492 y salim-abadi et al.: high insecticides … 486 http://jad.tums.ac.ir published online: october 04, 2016 fig. 2. regression lines for lab strain of culex pipiens exposed to different insecticides table 2. probit regression line parameters of field population of culex pipiens exposed to different insecticides insecticides a b ± se lt50 , 95% c.i. (minute) lt90, 95% c.i. (minute) x2 (df) p value lambdacyhalothrin 0.05% -7.58 3.71±0.36 99 214 5.34(2) >0.05111 245 122 293 deltamethrin 0.05% -4.36 1.9± 0.35 152 511 0.37 (2) >0.05 182 838 234 2322 cyfluthrin 0.15% -2.29 1.59±0.23 21 117 3.47(2) >0.05 27 172 33 336 ddt 4%* *no mortality after 24 hours exposure j arthropod-borne dis, december 2016, 10(4): 483–492 y salim-abadi et al.: high insecticides … 487 http://jad.tums.ac.ir published online: october 04, 2016 fig. 3. regression lines for field population of culex pipiens exposed to different insecticides table 3. mortality rate and susceptibility status of culex pipiens (lab and field population ) exposed to different insecticides at one hour exposure and 24 hours recovery period insecticides mr±eb* resistance status** lab strain field population lab strain field population lambdacyhalothrin 0.05% 100 20±2 s r deltamethrin 0.05% 98±1 18±3 s r cyfluthrin 0.15% 100 66±3 s r ddt 4% 55±3 0 r r *mortality rate±error bar **r resistance, s susceptible discussion in the present study four insecticides including: ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05% and cyfluthrin 0.15% were used for evaluatin the susceptibility status of cx.pipiens according to who criteria lab starin was resistant to ddt and filed strain exhibited resistant to all insecticides used. in the both strains a highly level of resistance to ddt were determined and also the lt50 for ddt >deltamethrin > lambdacyhalothrin >cyfluthrin respectively. in the two previous studies conducted in tehran, resistant to ddt have been reported in cx. pipiens and cx. quinquefasciatusin and these results was in parallel to our finding (nazari and janbakhsh 2000, vatandoost et j arthropod-borne dis, december 2016, 10(4): 483–492 y salim-abadi et al.: high insecticides … 488 http://jad.tums.ac.ir published online: october 04, 2016 al. 2004). it seems that cx. pipiens in the most part of the world has been resistant to ddt (mukhopadhyay et al. 1993, nazni et al. 2005, corbel et al. 2007, sarkar et al. 2009, jones et al. 2012, pocquet et al. 2013). in the study conducted by nazni et al. (2005) mosquitoes from two field sites in kuala lumpur exhibited resistance to ddt with no mortality 24h after exposure, and this result was same to our result about field population (nazni et al. 2005). in another study, high frequencies of resistance to ddt in cx. quinquefasciatus from benin have been reported and mortality rate ranged from 5 to 54% (corbel et al. 2007). in northeastern india cx. quinquefasciatus which caught from 7 different field sites of study area this species was resistance to ddt in all sites and its mortality rate varied from 11.9 to 50.0% (sarkar et al. 2009). result of susceptibility test in northwest and southeastern part of iran indicated that this species is highly resistant to ddt (ataie et al. 2015, fathian et al. 2015). in the current study, field population also was resistance to lambdacyhalothrin, deltamethrin and cyfluthrin and in some same studies this result also reported, for example resistance to lambdacyhalothrin and cyfluthrin reported in the southeastern part of iran and resistance to lambdacyhalothrin in northwestern part of iran (ataie et al. 2015, fathian et al. 2015). in cx. quinquefasciatus from wete on pemba island in zanzibar resistance to deltamethrin and lambdacyhalothrin also have been reported (jones et al. 2012). in thailand, cx. quinquefasciatus belong to the baan suan strain was highly resistant to deltamethrin even its mortality was very lower than our results (sathantriphop et al. 2006). resistance to deltamethrin also reported by chen et al. (2010), so that all the six surveyed cx. pipiens pallens populations strains were resistance to deltamethrin and also their mortality ranged from 20.2% to 78.6% (chen et al. 2010). in some studies resistance to the others group of insecticides like organophosphates and carbamate also have been reported (bisset et al. 1999, corbel et al. 2007, tantely et al. 2010, toma et al. 2011, ataie et al. 2015 fathian et al. 2015). for example in the both study that performed in northwestern and southern part of iran, cx. pipiens showed resistance to propoxur (ataie et al. 2015, fathian et al. 2015). tolerance to deltamethrin (with mortality=86%) in culex populations from kilimani, unguja island in zanzibar also reported by jones et al. (2012). although this populations was susceptible to lam-bdacyhalothrin. moreover in this study culex from the nearby site of tibirinzi in pemba was relatively susceptible to deltamethrin and also lambdacyhalothrin (jones et al. 2012). in the same previously study that performed by vatandoost et al in 2004, field population of cx. quinquefasciatus which collected from sewage system of tehran after evaluating their susceptibly status to insecticides result showed that this species is susceptible to cyfluthrin and also have tolerance to lambdacyhalothrin and deltamethrin. routine use of pesticides in household and agricultural pest control might have developed this enhanced tolerance to insecticide in the wastewater mosquito, cx. quinquefasciatus (vatandoost et al. 2004). conclusion in the present study, in the population whitch collected from field highly resistance to all insecticides exhibited it might be due to pollution of wastewater with chemical substances findings of this 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organization, geneva, switzerland. j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 original article detection of a new strain of wolbachia pipientis in phlebotomus perfiliewi transcaucasicus, a potential vector of visceral leishmaniasis in north west of iran, by targeting the major surface protein gene *parviz parvizi 1, farzaneh fardid 1, 2, somaieh soleimani 1,2 1molecular systematics laboratory, department of parasitology, pasteur institute of iran, tehran, iran 2department of microbiology, islamic azad university of qom-branch, qom, iran (received 18 jan 2012; accepted 7 july 2012) abstract background: wolbachia pipientis is maternally inherited endoparasitic bacterium belonging to the α-proteobacteria, infecting 20–75% of all insect species including sand flies. the wolbachia surface protein (wsp) was employed as an appropriate marker for strain typing. the objective of our research was to find the possibility of detection of w. pipientis in phlebotomus perfiliewi transcaucasicus. methods: individual sand flies were screened for the presence of w. pipientis. the obtained sequences were edited and aligned with database sequences to identify w. pipientis haplotypes. results: two haplotypes of w. pipientis were found in p. perfiliewi transcaucasicus. the common haplotype of w. pipientis was found to be identical to the sequences of those submitted in genbank. new strain (haplotype) of w. pipientis was found novel. the sequence of new strain of w. pipientis occurs in p. perfiliewi transcaucasicus is very different from those already submitted in genbank. conclusion: finding one genetically modified new strain of w. pipientis in p. perfiliewi transcaucasicus, now we can conclude that further documents and studies need to reach the role of cytoplasmic incompatibility of w. pipientis through wild sand fly populations to drive a deleterious gene into and to reduce the density of natural populations of sand flies. keywords: wolbachia pipientis, phlebotomus perfiliewi transcaucasicus, leishmania infantum, kala-azar, iran introduction phlebotomus perfiliewi transcaucasicus transmits ‘infantile visceral leishmaniasis’ (ivl) in northwest of iran to the east of turkey which has a mediterranean climate (nadim et al. 1978, parvizi et al. 2008, franco et al. 2011, mahamdallie et al. 2011). the intracellular rickettsia-like bacterium wolbachia pipientis hertig has been detected in phlebotomine sand flies (diptera: psychodidae, phlebotominae) using pcr to amplify a fragment of the major wolbachia surface protein (wsp) gene (zhou et al. 1998, cui et al. 1999, ono et al. 2001, benlarbi and ready 2003, kassem et al. 2003, parvizi et al. 2003). wolbachia, are gram negative, polymorphic and wide spread bacteria belonging to the family anaplasmataceae within the order rickettsiaceae, related to α-proteobacteria that infect reproductive tissues of many arthropods and nematodes. in addition, these bacteria have been found in approximately 80% of insect species all over the world (zhou et al. 1998, cui et al. 1999, benlarbi and ready 2003, kassem et al. 2003, parvizi et al. 2003). wolbachia are unique endosymbionts that is maternally inherited, intracellular rickettsia like bacteria which spread themselves to next generation, by transovarian transmission (weeks et al. 2002, benlarbi and ready 2003). 46*corresponding author: dr parviz parvizi, e-mail: parp@pasteur.ac.ir j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 this transmission is known as vertical transmission. wolbachia has been implicated in causing reproductive manipulations on its hosts. they induce a number of reproductive abnormalities that appear in their host phenotypes including cytoplasmic incompatibility (ci), parthenogenesis, feminization, male killing (curtis and sinkins 1998, cui et al. 1999, ono et al. 2001, weeks et al. 2002). cytoplasmic incompatibility is a common observed phenotype of wolbachia, when infected populations of same species cross to each other, results may appear to be signs of incompatibility, unidirectional incompatibility or bidirectional incompatibility that can be completed or partial (curtis and sinkins 1998, kassem et al. 2003). in case of an infected male mates with an uninfected female (or infected with another strain of wolbachia, this cross is incompatible and no offspring will be produced (unidirectional ci). in case of male and female populations are infected with more than one strain of wolbachia, bidirectional incompatibility will be observed (braig et al. 1998, werren 1998, cui et al. 1999, weeks et al. 2002, kassem et al. 2003). there are four known forms of parthenogenesis that have been observed in different types of insect species (thelytoky, pseudogamy, automixis, apomixis). they have different aspects but there was the same one in which female individual produces offspring without participation of male partner in mating. parthenogenesis in insects can cover a wide range of mechanisms. wolbachia induces a particular type of parthenogenesis in some species, called thelytoky parthenogenesis. by this action, wolbachia causes duplication in gametes in some insects therefore the resulting off spring would be all female, they also carry the wolbachia infection (anonymous 1997). in addition, wolbachia causes embryonic mortality of male zygotes in some arthropods. in addition, wolbachia has horizontal transmission between different species of arthropods (o’ neill et al. 1992, breeuwer and jacobs 1996, braig et al. 1998). this transmission has been observed in groups of parasitic wasps that they were not infected with wolbachia naturally, due to predating and feeding from a species of drosophila, after a short period of time within the host cells of mentioned wasps, they have become infected with the same strain that was present in drosophila species (rousset and solignac 1995). in addition, wolbachia was found in isopods, mites and nematodes. the following reasons can describe research on wolbachia: the wide spread of bacteria, manipulations on its hosts and its role to make speciation, the affect of bacteria of host’s fertility and being a potential natural enemy or a vector. useful genes can be synthesized by genetic engineering and then transferring both into wolbachia and insect populations for biological control which might cause decreasing arthropod transmitted diseases in human as a secondary reservoir (curtis and sinkins 1998, turelli and hoffmann 1999). the selection of wolbachia surface protein (wsp) gene has been due to free availability of this protein at the surface of the bacteria, ease of its identification, diversity of this gene in various genus and the possibility of usage of this gene for the purpose of studying the evolutional relationship and phylogenetic proximity of these groups of bacteria (werren 1997, bandi et al. 1998, werren 1998, sinkins and o’neill 2000). the wolbachia surface protein (wsp) gene is useful marker for strain typing (stouthamer et al. 1999, baldo et al. 2006). there are no reports of the bacterial wsp gene being isolated and being sequenced from the same individual specimens of phlebotomus perfiliewi transcaucasicus, in order to investigate the number of strains of w. pipientis infecting wild 47 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 populations of this insect. we have reached to beneficial consequences. this is now reported of p. perfiliewi transcaucasicus from an endemic of (ivl) in iran, and it is an essential piece of information for detecting w. pipientis through the wild populations of this sand fly (benismail et al. 1987, seccombe et al. 1993, benlarbi and ready 2003, parvizi et al. 2009). materials and methods sand flies were collected from three locations of sarab and kaleybar in the northwest of iran in azerbaijan province as well as meshkin shahr in ardabil province using cdc traps and sticky papers. sand flies were dissected, head and genital termination were kept for identifying of species based on morphological characters, thorax and abdomen were stored at -80 °c until further operations for dna extraction followed pcr assays (parvizi et al. 2003, parvizi and ready 2008). about 550 base-pairs (bp) (minus primers) of the wsp fragment of wolbachia surface protein were amplified by pcr using the primer pair wsp 81f (forward) (5´tggtccaataagtgatgaagaaa c3´) and wsp 691r (reverse) (5´aaaaa ttaaacgctactcca3´), pcr amplification was carried out according to the protocol of benlarbi and ready (2003). a 20l pcr reaction mixture consisted of 2l 1x promega buffer, 2l mgcl2, 0.5l of each dntp, 1l of each primer, 0.2l taq dna polymerase (promega) and 2l of sand fly genomic dna. the pcr amplification was carried out with the following thermal profile using a geneamp® pcr system 9700 thermal cycler (pe applied biosystems): 2 min denaturation at 94 °c, 35 cycles of denaturation at 94 °c for 30 sec, annealing at 50 °c for 30 sec, extension at 72 °c for 1.5 min, and a final extension at 72 °c for 10 min (benlarbi and ready 2003, parvizi et al. 2003). after amplification, the samples were fractionated by horizontal submerged gel electrophoresis, using 1.5% agarose gels and dna size markers (promega pcr markers g316a, or bioline hyper ladder iv). dna fragments were visualized by ethidium bromide staining, then excised and purified using a geneclean ii kit (bio 101 inc) before cycle sequencing each strand. the sequences obtained were edited and aligned with database sequences using sequencher tm v. 3.1 software (gene codes corp.) to identify unique sequences (=haplotypes), which were analyzed phylogenetically using paup* software (swofford 2002). results five species of larrossius subgenus and six species of adlerius subgenus identified from three locations of sarab, kaleybar in north eastern of azerbaijan province and meshkin shahr in ardabil province in northwest of iran. majority of sand fly species identified in these locations belonged to phlebotomus and paraphlebotomus subgenera and sergentomyia genus. abundance of some species of larrossius subgenus and adlerius subgenus were very low but due to their importance in ivl transmission we tried to detect wsp gene of wolbachia to all sand fly species (table 1). in total 12 out of 183 sand flies were found infected with wolbachia including 11 out of 41 p. perfiliewi transcaucasicus and one p. kandelakii. two out of seven p. perfiliewi transcaucasicus were found infected with wsp gene in sarab region in north east of azerbaijan province. seventy three sandflies species belonged to other larrossius and adlerius species were examined for wolbachia but no positive specimen was found for wsp gene in this focus. 48 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 nine out of 24 p. perfiliewi transcaucasicus were found infected with wsp gene in kaleybar region in north east of azerbaijan province. twenty two sandflies species were be longed to other larrossius and adlerius species which were tried to detect wolbachia but no positive wsp gene was found in this region. three of p. perfiliewi transcaucasicus were examined but no infection found with wsp gene in meshkin shahr in ardabil province in north west of iran. one out 26 p. kandelakii was found infected with wsp gene in meshkin shahr in ardabil province but the band of pcr product in agarose gel was too weak to sequence. in total (12/183) 6.5% of all screened sand flies were positive with wolbachia wsp gene that (2/81) 2.5% of these specimens were male and (10/102) 9.8% were female (table 2). only 9/11 (80%) positive pcr products of wolbachia wsp gene in p. perfiliewi transcaucasicus contained enough dna for direct sequencing. one haplotype of the wsp gene were recognized by aligning of new sequences comparison with homologous ones from genbank. the common haplotype of w. pipientis was found to be identical to the sequences of those submitted in genbank (genbank accession number eu780684), and it predominated in iranian sand flies infected with this species (7/11 infections). new strain of w. pipientis (genbank accession number (jx488735) was found novel (2/11 infections). this new strain differs pairwise by 36 to 120 bp nucleotide positions from those haplotypes of w. pipientis submitted in genbank (genbank accession numbers af237882, ay288297, hm 563686, hm775090) (fig. 2). fig. 1. locations of iranian provinces, cities and villages where larrossius and adlerius group sand fly species were sampled. (villages: 1sheghlan, 2bastam lou, 3safar lou, 4aslanbagh lou, 5sarma lou, 6aylily, 7shekhm lou, 8olou gheshlagh, 9oliurdy, 10abdolrazagh, 11aghamir lou, 12molan, 13jou aghaj, 14ajoudan abad, 15agh miun, 16sahzab, 17hasan jan, 18sanzigh, 19razligh, 20ghalajough, 21dowlat abad, 22arzanagh, 23alni, 24ourkandi, 25mizan, 26mouyil, 27ghassabeh, 28aghbolagh, 29ghourt tappeh) 49 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 a rd ab il p ro vi n ce e as t a za rb ay ej a n p ro v in ce m es h k in sh ah r k al ey b ar s ar ab p .k an de la ki i p .t ob bi p .p er fi li ew i tr an sc au ca si cu s p .k es h is h ia n i p .m aj or p .s im ic i p .b re vi s p .h al ep en si s p .l on gi do ct u s p .b al ca n ic u s c h in en si s g ro u p table 1. wolbachia infections in two subgenus species using wsp gene in three endemic visceral leishmaniasis locations in north west of iran province / region subgenus larrossius adlerius region species total ghalajough 5 2 2 1 3 13 razligh 2 2 6 10 sahzab 1 6 (2+ve) 7 (2+ve) sanzigh 2 3 1 6 agh miun 1 3 4 arzanagh 1 5 5 6 17 hasan jan 2 2 4 8 dowlat abad 4 2 7 1 1 15 total 14 5 14 (2+ve) 0 2 7 1 21 0 1 15 80 (2+ve) sheghlan 2 1 1 1 5 bastam lou 1 1 safar lou 1 1 aslanbagh lou 3 4 7 sarma lou 1 1 2 aylily 6 (4+ve) 6 (4+ve) shekhm lou 1 (1+ve) 3 4 (1+ve) olou gheshlagh 4 (2+ve) 4 (2+ve) oliurdy 2 1 3 abdolrazagh 4 (2+ve) 2 6 (2+ve) aghamir lou 1 1 molan 1 2 1 4 jou aghaj 1 1 ajoudan abad 1 1 total 10 0 24 (7+ve) 0 0 0 0 0 1 0 11 (2+ve) 46 (9+ve) alni 1 3 2 6 ourkandi 2 3 5 10 mizan 3 5 3 11 mouyil 9 9 ghassabeh 2 1 3 aghbolagh 4 (1+ve) 2 6 (1+ve) ghourt tappeh 6 3 3 12 total 26 (1+ve) 0 3 0 5 0 0 7 6 0 10 57 (1+ve) total 29 village 50 (1+ve) 5 41 (9+ve) 0 7 7 1 28 7 1 36 (2+ve) 183 (12+ve) 50 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 p. papatasi (eu780683) agctactacgttcgtttgcaatacaacggtgaatttttacctcttt-tcacaaaagttga 59 p. perfiliewi agctactatgttcgtttgcaatacaacggtgaaattttacctctttat-acaaaagttga ********.************************.************.*.*********** p. papatasi (eu780683) tggtgctacaggtgctaagaagactgcagatactgctacaactact-gacctttata-aa 117 p. perfiliewi tggtattacaaatg-taa-cag---gta-a-a--g--aaaagga-tagtccctta-acaa ****..****..**.***..**...*.*.*.*..*..*.**..*.*.*.**.***.*.** p. papatasi (eu780683) gcttcttttatggctggtggtggtgcatttggttataaaatggacgacatcagggttgac 177 p. perfiliewi g-atcttttatagctggtggtggtgcatttggttataaaatggacgacattagagttgat *..********.**************************************.**.*****. p. papatasi (eu780683) gttgaagggctttattcgcagctaagc-aaggatgca-cttgct-gtagctcctactcca 234 p. perfiliewi gttgaagggctttactcacaattg-gctaaagatacagct-g-tagtaaatacttctgaa **************.**.**..*..**.**.***.**.**.*.*.***..*.**.**..* p. papatasi (eu780683) gcaa-t-t-gcagacagtttaacagcaatttcagggctagttaacgtttattacgatata 291 p. perfiliewi acaaatgttgcagacagtttaacagcattttcaggattggttaacgtttattacgatata .***.*.*.******************.*******..*.********************* p. papatasi (eu780683) gcaattgaagatatgcctatcactccatacattggtgttggtgttggtgcagcatatatt 351 p. perfiliewi gcgattgaagatatgcctatcactccatacgttggtgttggtgttggtgcagcatatatc **.***************************.****************************. p. papatasi (eu780683) agcaca-cctttggcaactgctg-tg-agt-a--g-tcaaaatggtaaatttgcttttgc 404 p. perfiliewi agca-atcctt---caaaagctgatgcagttaaagatcaaaaagg-a--tttggttttgc ****.*.****...***..****.**.***.*..*.******.**.*..****.****** p. papatasi (eu780683) tggtcaagcaagagctggtgtt 426 p. perfiliewi ttatcaagcaaaagctggtgtt *..********.********** fig. 2. alignment of the single wsp gene sequence of w. pipientis isolated from iranian p. papatasi with the genbank sequence eu780683 reported by parvizi et al. (2009). nucleotide differences are marked by a point, not a star table 2. wolbachia detection in sand flies screened by pcr using wsp gene in north west of iran (+ve = wolbachia positive) location no. female sand flies screened by pcr no. +ve female sand flies +ve female sand flies% no. male sand flies screened +ve male sand flies +ve male sand flies% total sand flies total +ve sand flies total +ve sand flies % kaleybar 39 9 23.1 7 0 0 46 9 19.6 sarab 29 0 0 51 2 3.9 80 2 2.5 meshkin shahr 34 1 2.9 23 0 0 57 1 1.7 total 102 10 9.8 81 2 2.5 183 12 6.5 51 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 discussion recently, wsp gene has been used to improve phylogenetic resolution within the species clade of w. pipientis, which was divided into four groups (a–d) and 12 subgroups (zhou et al. 1998, ono et al. 2001). the groups a and b are concordant with those identified by 16s rdna for the strains of w. pipientis from insects, mites and crustaceans, whereas groups c and d harbor the strains from filarial nematodes. populations of p. perfiliewi transcaucasicus from iran were screened and for the first time, w. pipientis were found in this sand fly. two haplotypes including, one new haplotype were obtained from wsp gene (fig. 2). the common widespread strain (genbank id: eu780683, ay288297) was the a-group strain of w. pipientis (wpap) but the new haplotype was indistinguishable from that of the agroup strain of w. pipientis (wpap) previously isolated from p. papatasi originating from israel/west bank (af237883 (ono et al. 2001) and india (genbank accession number af237882 (ono et al. 2001), as well as from spain and iran (benlarbi and ready 2003, parvizi et al. 2003). from the three regions under study, 183 sand flies were selected in order of their genus and diversities of types and regions. wolbachia infection was detected in 1 case out of 57 samples in meshkin shahr (1.7%), 9 cases out of 46 samples in kaleybar (19.6%), and 2 cases out of 80 samples in sarab (2.5%) out of subgenus of larroussius “wolbachia surface protein genes”. larroussius and adlerius subgenus species were first identified as male because only males have good morphological characters of the head and abdominal terminalia for differentiation of species but females do not have this advantage. so it is obvious that this females are mates of that detected male geneses that were taken from this area (the males of these three species were taken from the given three regions, same as females) (parvizi et al. 2003, akhondi et al. 2012). out of adlerius subgenus, only two cases in female sand flies were infected by wolbachia which had no morphological characters for identification of genus (as mentioned above), but the males had good morphological characteristics for identification. as in these regions, male sand flies of adlerius subgenus “p. longiductus, p. halepensis, p. brevis, p. balcanicus, p. simici” have been found, the two cases of detected wolbachia in female sand flies of adlerius subgenus in kaleybar region, could be of any of the above five mentioned types. we would like to mention that all collected sand flies of above 5 mentioned species from kaleybar were not examined because it was not the purpose of this paper. the selection of wolbachia surface protein gene has been due to free availability of this protein at the surface of the bacteria, ease of its identification, diversity of this gene in various genus and the possibility of usage of this gene for the purpose of studying the evolutional relationship and phylogenetic proximity of these groups of bacteria (werren 1997, bandi et al. 1998, werren 1998, sinkins and o’neill 2000). there is a natural wolbachia infection in sand flies. cytoplasmic incompatibility is the recognized phenotype in sand flies which have been shown to be infected with wolbachia (mcgraw et al. 2002, rasgon 2003). moreover, it has been suggested that wolbachia can prevent the carriage and transferring of parasites and viruses via infected insects. in addition, it is a unique unknown manner which allows wolbachia to be a good selection and used as a transgene of the target genes and controlling the leishmaniasis which has been recently adapted among the population of sand flies (breeuwer and jacobs 1996, werren 1998, sinkins and o’neill 2000, brownstein et al. 2003). 52 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: april 10, 2013 as the existence of wolbachia in many sand flies which carry cutaneous leishmaniasis agents of urban and rural types is not known, it is suggested that the existence of this bacteria in other types of carriers of leishmaniasis is surveyed (dobson et al. 2002). wolbachia can be used as a transferring gene or transgene in insects. this can be done by designing and synthesizing of target genes by genetic engineering techniques and then to transfer them into wolbachia genome the point at which proper genes of wolbachia can be released to the mass of insects. it appears that the use of this technology would be very useful for the purpose of biologically control and combat against varieties of parasites and viruses of the region by employing arthropods (werren 1998, dobson et al. 2002, mitsuhashi et al. 2002, rasgon 2003). acknowledgements the work was supported by the pasteur institute of iran, grant 463 awarded to dr parviz parvizi. the collections of sand flies were made possible by the assistance of the centre of health service in kaleybar and sarab, azerbaijan province as well as meshkin shahr in ardabil province. we thank mehdi baghban for help with the field work and elnaz alaeenovin for help in molecular systematics laboratory. this research was msc studentships to farzaneh fardid and somaieh soleimani, based at the pasteur institute of iran, tehran, and registered for islamic azad university, qom, iran. the authors declare that there is no conflict of interest. references akhoundi m, parvizi p, baghaei a, depaquit j (2012) the subgenus adlerius nitzulescu (diptera, psychodidae, phlebotomus) in iran. acta trop. 122: 7–15. anonymous (1997) wolbachia pipientis overview. national science, available at: http://www.wolbachia.sols.uq.edu.au. baldo l, bordenstein s, wernegreen jj, werren jh (2006) widespread recombination throughout wolbachia genomes. mol biol evol. 23: 437–449. bandi c, anderson tc, genchi c, blaxter ml (1998) phylogeny of wolbachia in filarial nematodes. proc r soc lond b. 265: 2407–2413. ben ismail r, gramiccia m, gradoni l, helal h, ben rachid ms (1987) isolation of leishmania major from phlebotomus papatasi in tunisia. trans r soc trop med hyg. 81: 749. benlarbi m, ready pd (2003) host-specific wolbachia strains in widespread populations of phlebotomus perniciosus and p. papatasi (diptera: psychodidae), and prospects for driving genes into these vectors of leishmania. bull entomol res. 93: 383–391. braig hk, zhou w, dobson sl, o’neill sl (1998) cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont wolbachia pipientis. j bacteriol. 180: 2373–2378. breeuwer jaj, jacobs g (1996) wolbachia: intracellular manipulators of mite reproduction. exp appl acarol. 20: 421–434. brownstein js, hett e, o’neill sl (2003) the potential of virulent wolbachia to modulate disease transmission by insects. j invert pathog. 84: 24–29. curtis cf, sinkins sp (1998) wolbachia as a possible means of driving genes into populations. parasitology. 116: 111–115. cui l, chang sh, stickman d, rowton e (1999) frequency of wolbachia infection in laboratory and field sand fly (diptera: psychodidae) populations. j am mosq control assoc. 15: 571–572. dobson sl, fox cw, jiggins fm (2002) the 53 j arthropod-borne dis, june 2013, 7(1): 46–55 p parvizi et al.: detection of a new … http://jad.tums.ac.ir published online: 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as a mechanism for introducing transgenes into arthropod populations. insect mol biol. 8: 243–255. weeks ar, reynolds kt, hoffmann aa (2002) wolbachia dynamics: what has (and has not) been demonstrated? trends ecol evol. 17: 257–262. werren jh (1997) biology of wolbachia ann rev entomol. 42: 587–609. werren jh (1998) wolbachia and speciation. in endless forms: species and speciation. eds, harvard d, berlocher s. oxford university press: oxford. pp. 245– 260. zhou w, rousset f, o’neill s (1998) phylogeny and pcr-based classification of wolbachia strains using wsp gene sequences. proc r soc lond b. 265: 509–515. 55 j arthropod-borne dis, march 2017, 11(1): 139–146 ke benallal et al.: presence of phlebotomus … 139 http://jad.tums.ac.ir published online: march 14, 2017 original article presence of phlebotomus perniciosus atypical form in algeria *kamel eddine benallal 1, razika benikhlef 1, rafik garni 1, brahim gassen 2, jean-pierre dedet 3, zoubir harrat 1 1laboratoire d’eco-epidémiologie parasitaire et génétique des populations, institut pasteur d’algérie, algeria 2direction de la santé publique de tamanrasset, algeria 3université montpellier 1 et chru de montpellier, centre national de référence des leishmanioses, département de parasitologie-mycologie, france (received 2 nov 2014; accepted 23 apr 2016) abstract background: phlebotomus perniciosus and phlebotomus longicuspis are two phlebotomine sand fly species morphologically similar and differing in males only by the shape of the copulatory valves which are bifurcated in p. perniciosus, tip long and tapered in p. longicuspis. methods: a count of the median coxite setae was carried out on 208 specimens from the collections of dedet and of parrot, identified previously as p. longicuspis and on 38 p. perniciosus male sand flies captured during the year 2012–2013, in order to seek the presence of atypical p. perniciosus form. results: the analysis revealed the presence of 33/246 (13%) atypical p. perniciosus previously confused with p. longicuspis species and whose distribution is mainly located in the semi-arid and arid bioclimatic regions. conclusion: this study proved for the first time the presence of atypical form of p. perniciosus in algeria. keywords: phlebotomus perniciosus, phlebotomus longicuspis, atypical phlebotomus perniciosus, leishmaniasis, algeria introduction in algeria, the first sand fly specimens were reported by foley and leduc (1912). since then, several taxonomic studies were conducted on different species and how they might influence transmission of leishmaniasis in algeria. the recent entomological surveys have reported the presence of phlebotomus (transphlebotomus) mascittii grassi, 1908, p. (larroussius) chadlii females in the northeast and p. (paraphlebotomus) kazeruni in the extreme south of algeria (berdane-brouk et al. 2011, benallal et al. 2013). in the mediterranean basin, phlebotomus (larroussius) perniciosus newstead, 1911 is the most common phlebotomine sand fly, related to humid and arid bioclimatic stages. its distribution spreads out up to the saharan edge of tassili and hoggar, with higher abundance in the humid areas (rioux et al. 1967, dedet et al. 1984, berdjane-brouk et al. 2012). in algeria, it is the main vector of leishmania infantum mon-1, the parasite that causes the majority of the visceral leishmaniasis (vl) cases in humans and dogs (izri et al. 1990). this species is often collected in sympatry with a closely related species, ph. (larroussius) longicuspis nitzulescu, 1930. parrot considered p. longicuspis as a potential vector of l. infantum in the arid areas (parrot et al. 1941) and recently, this species was found naturally infected with this parasite (berdjane-brouk et al. 2012). females of the larroussius subgenus are differentiated mainly by the aspect and the dilatation of the *corresponding author: dr kamel eddine benallal, e-mail: benallalkamel4@yahoo.fr j arthropod-borne dis, march 2017, 11(1): 139–146 ke benallal et al.: presence of phlebotomus … 140 http://jad.tums.ac.ir published online: march 14, 2017 distal spermathecal ducts (léger et al. 1983, killick-kendrick et al. 1990). however, males are distinguished by the morphology of the copulatory valves (aedeagus), with bifurcated apex in p. perniciosus (pn) (fig. 1b) and with a long, slightly curved, single pointed apex in p. longicuspis (lc) (fig. 1c) (parrot 1936 a and b). previous studies on p. perniciosus males based on the number of median coxite setae highlighted atypical forms of p. perniciosus (pna) confused with p. longicuspis mainly in spain (morrillas-marquez et al. 1991, collantes and martinez-ortega 1997, martinezsanchez et al. 2000), in morocco (benabdennbi and pesson 1998, pesson et al. 2004, guernaoui et al. 2006, boussa et al. 2008) and recently in tunisia (ghrab et al. 2006, boudabous et al. 2008). then isoenzyme analysis has permitted distinguishing these morphs at hexokinase (hk) locus thus as molecular biology using random amplified polymorphic dna (radp) technique. the result of the three techniques has allowed including pna with p. perniciosus species (benabdennbi et al. 1999, martinez-sanchez et al. 2000, pesson et al. 2004, boudabous et al. 2012). parrot and durand-delacre (1947) reported that a p. longicuspis males collected in beniounif (oran sahara) displayed a copulatory valves more curved than usual, and in 2012, we identified a male of the subgenus larroussius collected in tamanrasset (extreme south of algeria) which exhibited the same features (curved copulatory valves with a single point). this work aimed whether atypical forms of p. perniciosus in algeria are present using the morphometric technique. materials and methods in order to investigate the presence of atypical p. perniciosus males in algeria, a retrospective study was done on 208 specimens previously identified as p. longicuspis belonging to the two collections, of louis parrot (specimens collected between 1934 and 1958) and of jean-pierre dedet (specimens collected between 1973 and 1975). in addition, other samples of the same species (p. perniciosus and p. longicuspis) were captured in animal shelters between 2012 and 2013 by sticky traps in different localities of the country (setif, oum-bouaghi, blida, ghardaïa and tamanrasset) and included in the study. only males of p. perniciosus and p. longicuspis were slide mounted with canada balsam after treatment in naoh 20% (abonnenc 1972). in all cases, examination of copulatory valves morphology following the key of dedet (1984) was associated to a counting of the median coxite setae using a motic 210 camera. the details of the examined specimens and their place of trapping according to bioclimatic zones are summarized in (table 1). morphometric data (number of median coxite setae) were compared using an analysis of variance with t test of student with excel software version 2007. results overall, 246 males belonging to p. perniciosus and p. longicuspis were analyzed. within the 208 males of the two collections previously identified as p. longicuspis and 38 collected in field, 33 sand flies presented curved copulatory valves characteristic of p. perniciosus atypical male (fig. 1a). the counted setae number of these males varied between 10 and 16 setae (fig. 2). the number of setae allowed classifying the atypical males with p. perniciosus species that possessed between 10 and 19 coxite setae (fig. 2) and forked copulatory valves (fig. 1b). however, the number of median coxite setae of p. longicuspis varied between 18 and 32 (fig. 2) with long, slightly curved, single point (fig. 1c). the number of specimens j arthropod-borne dis, march 2017, 11(1): 139–146 ke benallal et al.: presence of phlebotomus … 141 http://jad.tums.ac.ir published online: march 14, 2017 examined and their setae mean numbers (+/standard deviation) are summarized in (table 2) with the minimal and maximal numbers of setae. the student’s t-test showed significant differentiation between p. perniciosus and p. longicuspis populations (p< 0.05) (table 2). the distribution of pna form is very marked in northwest part of algeria (tlemcen and sidi bel-abbes districts) and remained scarce in the rest of regions whereas the abundance of p. perniciosus decreased once moved southward over the country (fig. 3). table 1. sand flies origin and numbers of specimens studied, according to bioclimatic stages bioclimatic zone region (n) collection site gps coordinate tizi ouzou* 30 36°32'20.88"n 4°26'28.55"e humid blida (c) 4 36°33'22.45"n 3° 3'17.06"e blida** 7 / béjaïa* 7 36°37'40.21"n 5°20'42.62"e subhumid boumerdes* 5 36°43'47.24"n 3°32'17.61"e alger** 35 / sétif (c) 26 35°41'27.74"n 5°25'43.75"e arid el bayadh* 1 33°10'3.76"n 0°28'27.47"e naâma* 6 32°29'31.70"n 0°28'4.65"o ain defla* 1 36°15'47.17"n 2°12'39.07"e bouira* 3 36° 8'47.98"n 3°49'58.83"e oran* 7 35°41'54.90"n 0°38'23.72"o oum bouaghi(c) 6 35°52'30.79"n 7° 6'48.95"e semi-arid relizane* 3 35°42'59.07"n 0°45'21.03"e sidi-bel-abbes* 3 35°14'25.07"n 0°14'43.24"o tlemcen* 22 34°38'27.30"n 1°33'41.14"o tlemcen** 34 / batna** 20 / constantine** 1 / béchar* 1 31°55'33.51"n 1°50'19.16"o béchar** 7 / ghardaïa (c) 1 32°28'55.09"n 3°42'3.59"e saharan tamanrasset (c) 1 22°53'23.07"n 5°21'33.07"e tamanrasset** 5 / biskra* 4 35°10'48.41"n 6° 0'32.26"e biskra** 6 / total 246 (*) collection of dedet, (**) collection of parrot, (c) collected, (/) data not available and (n) sample size. j arthropod-borne dis, march 2017, 11(1): 139–146 ke benallal et al.: presence of phlebotomus … 142 http://jad.tums.ac.ir published online: march 14, 2017 table 2. comparison of the average numbers of median coxite setae by student test “t” of phlebotomus perniciosus and phlebotomus longicuspis species number of measure setae number min max p. perniciosus 96 13.67±3.39 10 18 p. longicuspis 150 24.33±5 18 32 student’ s test 20.69 ddl= 13 p= 0.000 1385 fig. 1. copulatory valves of phlebotomus perniciosus and phlebotomus longicuspis. a) atypical form of phlebotomus perniciosus, b) typical form of phlebotomus perniciosus, c) typical form of phlebotomus longicuspis, 1) curved form of copulatory valves, 2) forked form and 3) slightly curved, single point. bar = 10 µ m fig. 2. frequency of median coxite setae for phlebotomus perniciosus atypical (pna), phlebotomus perniciosus (pn) and (lc) phlebotomus longicuspis. j arthropod-borne dis, march 2017, 11(1): 139–146 ke benallal et al.: presence of phlebotomus … 143 http://jad.tums.ac.ir published online: march 14, 2017 fig. 3. distribution map of phlebotomus perniciosus and phlebotomus longicuspis in algeria. lc: p. longicuspis, pn: p. perniciosus, pna: atypical p. perniciosus. discussion the two collections conserved in pasteur institute of algeria, collection of parrot which counts 114 species distributed over 16,482 slides, most species belong to the african continent (164 species from 16 countries), to the european continent (36 species from 08 countries) and to the middle east and asia (29 species from nine countries), and that of dedet which counts 4,689 slides with 15 species originating only from algeria. despite the small number of specimens reviewed due to the improper mounting of some specimens which did not allow performing a correct counting of the median coxite setae our results provide, for the first time, the evidence for atypical forms of p. perniciosus in algeria and thus correct the identification of several specimens identified as p. longicuspis. the number of median coxite setae remains a powerful tool in the morphological identification since it was previously used to distinguish between p. ariasi/ p. chadlii and p. neglectus/ p. syriacus of larroussius subgenus (benabdennbi and pesson 1998). it has allowed the description of p. perniciosus atypical male form in morocco, spain and tunisia (morrillas-marquez et al. 1991, benabdennbi and pesson 1998, ghrab et al. 2006). the bioclimatic and geographic repartition of the different analysed sand flies highlighted a different distribution between pn and pna forms. however, pn are more abundant in the north and northeast of algeria. this form occupies humid and sub humid bioclimatic zones in concordance with the previous entomological surveys conducted in mila and jijel by berchi et al. (2007). nevertheless the abundance of pna is rather from the northwest to the great south, thus occupying semi arid, arid and saharan bioclimatic zones. in morocco, pna repartition spreads out from northeast to the south at the moroccoj arthropod-borne dis, march 2017, 11(1): 139–146 ke benallal et al.: presence of phlebotomus … 144 http://jad.tums.ac.ir published online: march 14, 2017 algerian coast (boussa et al. 2008), whereas in tunisia its distribution is higher to south in the arid and semi arid areas (boudabous et al. 2012). consequently, in maghreb the repartition of pna obeys to the same bioclimatic and geographic conditions and seems to be linked to arid climate. further investigations in libya where distribution of p. perniciosus is limited in north africa (benabdennbi and pesson 1998) should give more details about p. perniciosus morphs distribution. the repartition of p. longicuspis is large and this species is found from the north up to tamanrasset in the extreme south of algeria. recently, its distribution area has won more ground since it has been reported also in burkina faso (depaquit et al. 2005). nonetheless, lc density remains very low comparing to p. perniciosus and it is found in humid to saharan bioclimatic stages (dedet et al. 1984). conclusion association of median coxite setae to the standard morphological criteria revealed the presence of 33/246 (13%) of atypical p. perniciosus until now confused with p. longicuspis species mainly located in the semi arid and arid areas. the morphometric tool allowed us to draw a new distribution map of pn, pna and lc in algeria and to add the atypical form of p. perniciosus into the list of algerian phlebotomine fauna. further biochemical, molecular and morphometric studies should be done in order to highlight the status of the different p. perniciosus morphs of algeria, and also it is important to check the competence of pna females to develop and to transmit leishmania sp parasite. acknowledgements 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10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 577 http://jad.tums.ac.ir published online: october 04, 2016 original article chemical compositions of the peel essential oil of citrus aurantium and its natural larvicidal activity against the malaria vector anopheles stephensi (diptera: culicidae) in comparison with citrus paradisi alireza sanei-dehkordi 1,2, *mohammad mehdi sedaghat 3, hassan vatandoost 3, mohammad reza abai 3 1department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 2social determinants in health promotion research center, hormozgan university of medical sciences, bandar abbas, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 29 dec 2014; accepted 3 nov 2015) abstract background: recently, essential oils and extracts derived from plants have received much interest as potential bioactive agents against mosquito vectors. methods: the essential oils extract from fresh peel of ripe fruit of citrus aurantium and citrus paradisi were tested against mosquito vector anopheles stephensi (diptera: culicidae) under laboratory condition. then chemical composition of the essential oil of c. aurantium was analyzed using gas chromatography-mass spectrometry (gc–ms). results: the essential oils obtained from c. aurantium, and c. paradisi showed good larviciding effect against an. stephensi with lc50 values 31.20 ppm and 35.71 ppm respectively. clear dose response relationships were established with the highest dose of 80 ppm plant extract evoking almost 100% mortality. twenty-one (98.62%) constituents in the leaf oil were identified. the main constituent of the leaf oil was dl-limonene (94.81). conclusion: the results obtained from this study suggest that the limonene of peel essential oil of c. aurantium is promising as larvicide against an. stephensi larvae and could be useful in the search for new natural larvicidal compounds. keywords: citrus aurantium, citrus paradisi, essential oil, larvicidal activity, anopheles stephensi introduction mosquitoes are very significant vectors from the medical entomology's point of view. they are responsible for the transmission of many diseases to man and animals such as malaria, dengue, yellow fever, encephalitis or filariasis (eldridge 2000). human malaria, as a mosquito-borne disease, caused by parasitic protozoa plasmodium consider as the most important vector-borne disease, which is transmitted only by females of anopheles mosquitoes (lehane 1991). malaria is still an endemic disease in certain foci located in south and southeast of iran (manouchehri et al. 1992, sedaghat et al. 2003 a,b, vatandoost et al. 2012). in this part of the country six anopheline mosquitoes including an. stephensi, are known as the main malaria vectors (manouchehri et al. 1976, sedaghat and harbach 2005). anopheles (cellia) stephensi liston 1901 is an important malaria vector with wide distribution in the arabian peninsula and the indian subcontinent. it has also distributed in in khuzestan, fars, kerman, hormozgan, sistan va baluchestan and southern *corresponding author: dr mohammad mehdi sedaghat, e-mail: sedaghat@hotmail.co.uk j arthropod-borne dis, december 2016, 10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 578 http://jad.tums.ac.ir published online: october 04, 2016 kermanshah provinces in iran (manouchehri et al. 1976, vatandoost et al. 2006, hanafi et al. 2011). several methods have been applied for control of the vectors of the disease including using synthetic pesticides as larvicides or imagicides in the malaria control program. organophosphate compounds are the most common chemical larvicides which are used in control of anopheles. however, their toxicity to fish and other non-target organisms and the environment are increased (mittal et al. 1991, pinkney et al. 1999). also resistance of anopheles mosquitoes to these compounds has appeared in many areas (vatandoost and borhani 2004, vatandoost et al. 2004). a lot of attention is being paid to the plant extracts or their essential oils as an alternative source of mosquito larval control agents (isman 2000, sedaghat et al 2011a, b, vatandoost et al. 2012). citrus aurantium linnaeus (rutaceae) which called as bitter orange or marmalade orange is too sour, but the juice of ripe fruit is used as a condiment in iran. the peel of c. aurantium is often used in marmalade and dried peel is used in different food and drinks. the flowers are used in tea and its essential oil is used in perfumes and orangeflower water, which is used to flavor sweets (kiple and ornelas 2000). the dried whole fruit or peel of the fruit is used in asian and western herbal medicine to treat digestive problems (wichtl 1994). the hydrolate of the flowers has been used for treatment of mild depression, sedation and as a heart tonic for many years in iran (zargary 1986, ayenechi 1991). the studies on biologic effects of c. aurantium indicated potential mosquito repellent, larvicidal and insecticidal activities of this plant (cetin et al. 2006, sumroiphon et al. 2006, yoon et al. 2009). citrus paradisi macfadyen (rutaceae) or grapefruit like other citrus fruits contains many phytochemicals which contribute to a healthy diet (fellers et al. 1990). it is used in persian traditional medicine to treat infected injuries, some digestive problems, cold and helps to lower cholesterol. recent studies have shown the potential of c. paradise to favorably affect metabolic syndrome, lipid and sugar metabolism (fujioka et al. 2006, goldwasser et al. 2010, ogura et al. 2011). its seed extract has shown the antimicrobial properties against bacteria and fungi (von et al. 1999). the objective of this work was to study the effect of the peel essential oils of ripe fruits of c. aurantium and c. paradisi against fourth instar larvae of an. stephensi under laboratory conditions and determine the chemical composition of c. aurantium essential oil. materials and methods collection of plant materials fresh plants samples of ripe fruit of c. aurantium and c. paradisi were collected in october 2012 from babol, iran (52º 41’e, º36 32’n, elevation: -5 m above sea level) and jiroft (57º 45’e, 28º 37’n, elevation: 650 m above sea level) respectively. the plants were identified and authenticated and the voucher specimen was deposited at vector biology laboratory, department of medical entomology, tehran university of medical sciences, iran. extraction of essential oils the peel essential oils of fresh (50 g) of c. aurantium and c. paradisi were hydrodistilled using a clevenger-like apparatus (model: british pharmacopoeia, manufactured by pyrexfan company, iran and mantle model em manufactured by bibby scientific company, united kingdom) for 3 hours at 70 °c. the yields were averaged over four experiments and calculated according to fresh weight of the plant materials. the obtained oil was dried over anhydrous na2so4 and transferred into an airtight amber-colored vial at 4 °c for further experimentation by j arthropod-borne dis, december 2016, 10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 579 http://jad.tums.ac.ir published online: october 04, 2016 gas chromatography–mass spectrometry (gc and gc-ms). gc and gc/ms analysis of essential oil gc analysis was carried out using an hp6890 gas chromatograph equipped with flame ionization detector and an hp-1 capillary column (30 m× 0.25 mm i.d., film thickness 0.25 μm) and split ratio, 1:25. the gc settings were as follows: initial oven temperature was held at 40 °c for 1 min, rising to 250 °c at 5 °c/min. the injector temperature was maintained at 250 °c. the detector temperature was at 230 °c. the carrier gas used was helium at a flow rate of 1 ml/min. gc-ms was performed on agilent technology 5973 mass selective detector connected with an hp 6890 gas chromatograph. the oil of c. aurantium was analyzed using an hp-1ms (fused silica) with the same column and temperature programmed as above. the ms operated at 70 ev ionization energy. quantitative data were obtained from the electronic integration of the flame ionization detector (fid) peak areas. determination of oil composition identification of the oil components were assigned based on retention indices which were calculated by using retention times of n-alkanes that were injected after the oil at the same chromatographic conditions. the compounds were identified by comparison of their relative retention indices and with those in the literature. in addition, computer searching followed by matching of the mass spectra data with those stored in the computer library. the percentage of each component is presented in table 1. mosquito culture fourth instar larvae anopheles stephensi was used in this study. all larvae of an. stephensi were obtained from laboratory culture of department of medical entomology, tehran university medical sciences (tums). the anopheles colony was maintained at 27 °c with 12: 12 light and dark photoperiod in 60±10% relative humidity. larvicidal bioassay tests of mosquito larval activity were conducted with reference to the standard method recommended by the world health organization (who 2005). since the essential oils do not dissolve in water ethanol 99.0% was used as co-solvent. different concentrations of the essential oils in distillated water and the co-solvent were prepared. the oil-ethanol-water solution was gently stirred for 30 seconds with a glass rod to ensure a homogeneous test solution and each glass beaker was left at room temperature. after 15 minutes 20 larvae were taken with a fine mesh strainer and transferred gently to a 400 ml glass beaker. control group included batches of mosquitoes from the colony exposed to water and the solvent alone. the larvae were exposed to the concentrations of 10, 20, 40, 80 and 160 ppm of essential oil in distilled water for 24 hours at room temperature. in the control beakers only 1 ml of solvent was added to each beaker. mortality was recorded after 24 h of exposure while during the test no food was given to the larvae. each treatment was done with five replicates. statistical analysis toxicity and activity were reported as lc 50 and lc90, representing the concentrations in ppm that killed 50% and 90%, respectively of larvae in 24 h. the lc50 and lc90 values and their 95% confidence intervals of each essential oil were calculated by log concentration-probit equation (finney 1971) using the spss 16.0 probit procedure. controls with mortality between 5–20% were corrected using abbott’s formula (abbott 1925). when mortality in controls exceeded 20%, test results were rejected or repeated. comparison of the lc50 and lc90 values were analyzed using anova test with spss verj arthropod-borne dis, december 2016, 10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 580 http://jad.tums.ac.ir published online: october 04, 2016 sion 17.0. differences between means were considered significant at p≤ 0.05. result yields and chemical constituents of essential oil the yields of peel essential oils of c. aurantium and c. paradisi were 0.7±0.12% and 0.85±0.14% (w/w) based on fresh weight respectively. the chemical composition of c. aurantium peel essential oil is presented in table 2. a total of 21 compounds were identified representing about 98.62% of the oil. the results revealed that terpenoids in the oil were predominant. the main constituents in the c. aurantium peel essential oil were dl-limonene (94.81%), β-myrcene (1%) and α-pinene (0.65%) respectively. larvicidal activity of essential oils the lc50 and lc90 values of c. aurantium and c. paradisi oils against an. stephensi larvae were 31.20 ppm and 73.83 ppm, 35.71 ppm and 70.23 respectively (table 1). both peels essential oils at the 80 ppm concentrations killed more than 90% of the fourth instars larvae (fig. 1). the mortality rates in the control groups were lower than 5% in all concentrations, no correction were applied. the probit regression lines of an. stephensi exposed to different interval concentrations of a c. aurantium and c. paradisi extractions are shown in fig 2. the statistical test (anova) showed that there was no significant statistical difference in mortality rate among two essential oils (p≤ 0.628 ). table 1. probit regression line parameters of anopheles stephensi to peel essential oil extraction of citrus aurantium and c. aurantium at different interval concentrations species a b±se lc50 , 95% c.i. lc90, 95% c.i. x2 (df) heterogeneity p-value c. aurantium -5.12 3.43±0.28 28.17 31.20 34.62 63.30 73.83 90.08 3.15 (2) > 0.05 c. paradisi -5.51 3.55±0.29 32.28 35.71 39.62 82.03 70.23 100.47 2.54 (2) > 0.05 a= y-intercept, b= the slope of the line, se= standard error, lc50, 95% ci= lethal concentration causing 50% mortality and its 95% confidence interval, lc90, 95% ci= lethal concentration causing 90% mortality and its 95% confidence interval, x2= heterogeneity about the regression line. fig. 1. percentage of larval mortality of anopheles. stephensi after treatment with citrus aurantium and c. paradise j arthropod-borne dis, december 2016, 10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 581 http://jad.tums.ac.ir published online: october 04, 2016 fig. 2. probit regression line of anopheles stephensi exposed to different interval concentrations of a citrus aurantium in comparison with c. paradisi essential oils table 2. chemical constituents of peel essential oil from citrus aurantium concentration (%)ki acompoundn 0.30909α-pinene1 0.65947β-pinene2 1.00958β-myrcene3 94.81997dl-limonene4 0.191016trans-ocimene5 0.011029gamma-terpinene6 0.041039linalool oxide7 0.1310431-octanol8 0.021044trans-linalool oxide9 0.011045isoterpinolene10 0.021054nonanal11 0.441057α-terpinolene12 0.321234linalyl acetate13 0.031235sabinene hydrate acetate14 0.031338neryl acetate15 0.131358geranyl acetate16 0.031386trans-caryophyllene17 0.081453germacrene-d18 0.061536nerolidol19 0.061929palmitic acid20 0.261930trans-oleic acid21 98.62total concentration (ppm) j arthropod-borne dis, december 2016, 10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 582 http://jad.tums.ac.ir published online: october 04, 2016 discussion during past five decades using of chemical insecticides against vector mosquitoes have been developed the resistance in vectors to the insecticides and also hazards to the environment (mittal 1991, gunasekaran et al. 2004, vatandoost et al. 2012). although chemical larval control is considered as a major component in malaria prevention strategies, this part of control has side-effects on human and animal health and also the environment (sedaghat et al. 2010, vatandoost et al. 2012). in order to reduce the dependency on chemical insecticides, alternative methods including botanical insecticides for the control of vectors are considered. certain plant’s essential oils or extracts have been found effective for mosquito larval control. it is important to identify chemical constituents of the indigenous plans and their efficacies of essential oils as natural larvicides. the yield of oil obtained of c. aurantium was 0.7%. it was nearly as same as reported from pakistan (siddique et al. 2011) but less than as reported in egypt (hifnaway et al. 2004). gas chromatography–mass spectrometry of peel essential oil of c. aurantium revealed the presence of 21 components. in this study, major constituent of peel essential oil of c. aurantium was dl-limonene, 94.81 % of the oil. in previous studies, various constituents of the oil of c. aurantium were reported. although limonene is the most abundant constituent in the oils obtained from all similar studies, its percentage were varied, based on the origins of the plant (caccioni et al. 1998, boussaada and chemli 2007, moraes et al. 2009, hosni et al. 2010). the result is in agreement with the results obtained from other study in iran (hosni et al. 2010). the other constituents of the peel oil were αpinene (0.30%) and β-pinene (0.65%), while the results obtain from a study in pakistan αpinene and β-pinene were reported 0.476% and 0.176% respectively (siddique et al. 2011). the result of the larval bioassay tests showed that essential oils of c. aurantium and c. paradise have a same level of bioactivity against an. stephensi larvae. previous studies have demonstrated the same level of larvicidal activity against mosquito larvae. the lc50 of the essential oil of c. paradise were 47.3 ppm and 85.1 ppm for ae. aegypti and ae. albopictus, respectively (moralessaldañaet al. 2007). in another study reported by boussaada and chemli, the content of limonene in tunisian c. aurantium from 87 % to 92.2% on fresh weight basis (boussaada and chemli 2007). the results nearly similar from the study of caccioni et al. (1998) which reported limonene (94.3%) myrcene (1.88%) as the main constituents of c.s aurantium. in another study on the peel oil composition of brazilian c. aurantium, limonene (97.5–98%), myrcene (1.2–1.45%) and octanol (0.34–0.54%) were found as the main constituents (moraes et al. 2009). the results of constituents of the peel oil of c. aurantium obtained from this study are similar with all prior studies. our previous studies on the same strain of an. stephensi larvae revealed that the efficacy of eucalyptus camaldulensis, cupressus arizonica, coriandrum sativum and heracleum persicum oils were less than the efficacy of c. aurantium and c. paradise oils, while the efficacy of foeniculum vulgare and kelussia odoratissima are more than the two citrus (sedaghat et al. 2010, 2011ab, vatandoost et al. 2012). according to the classification of vatandoost et al. (2012) the level of larvicidal activity of c. aurantium and c. paradise demonstrated them as active plants. if we accept cheng et al. (2003) suggestion, these plants should be considered as very active. based on both above classifications these two plants j arthropod-borne dis, december 2016, 10(4): 577–585 a sanei-dehkordi et al.: chemical compositions … 583 http://jad.tums.ac.ir published online: october 04, 2016 need more attention as they lied in active or very active categories. results on the larval mortality of c. aurantium and c. paradise oils against an. stephensi confirm their potential to control of the mosquito populations. it seems that the presence of a high amount of dl-limonene in c. aurantium oil, could demonstrate its efficacy against an. stephensi larvae. in brief, this study clearly illustrated the potential use of c. aurantium and c. paradise as natural mosquito larvicides. conclusion essential oils from aromatic plants are the complex mixture of constituents with several usages in health and medical sciences. the essential oils of c. aurantium and c. paradise oils show larvicideal activity 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sadr 4 1 department of parasitology, school of medicine, kashan university of medical sciences, iran 2 department of parasitology, pasteur institute, tehran, iran 3 department of parasitology, school of public health, tehran university of medical sciences, iran 4 department of internal medicine, school of medicine, kashan university of medical sciences, iran (received 23 nov 2008; accepted 12 may 2009) abstract background: considering the prevalence of leishmaniasis in iran and many side effects associated with pentavalent antimony compounds use in its treatment, this study was designed to evaluate the effect of artemisia sieberi essence on the experimental ulcers of cutaneous leishmaniasis on balb/c mice. methods: this experimental research was performed to determine the effect of various concentrations of artemisia essence in balb/c mice previously infected with active leishmania major promastigote. a total of 50 infected balb/c mice were randomly divided into 5 groups. three groups (30 mice) were used in the experimental conditions and the others were assigned as the control groups. the experimental groups received 1%, 3% and 5% of artemisia, respectively. one of the control groups received ethanol 80% and the other received no treatment. the drug was administered by dropping the liquid on the top lesions, three times daily for maximum of 30 d. every 10 days the ulcers diameter were measured and sampled for amastigote in all groups. ulcers diameter changes were determined by statistical tests. results: after 30 days, diameter of cl lesions increased in 1%, 3% and 5% artemisia concentrations and the control groups. ulcers got bigger with the more concentration. treatments could not reduce the diameter or caused small lesions. in addition, the mice direct smears in microscopic studies were positive. conclusion: to find the effective concentration and the mechanism of the effectiveness of the drug, further investigations with less concentrates of a. sieberi essence are recommended. keywords: cutaneous leishmaniasis, leishmania major, artemisia sieberi, balb/c introduction cutaneous leishmaniasis (cl) represents a common health problem and standard treatments are often ineffective or yield poor cosmetic results (berman 2003). cl is caused by leishmania species belonging to the leishmania tropica complex. leishmaniases are transmitted by sand flies belonging to the genus phlebotomus (service 1996, hepburn 2001, markell and voge 2006). it is found in most tropical and subtropical countries, but 90% of the estimated 1.5 million new cases each year occur in afghanistan, brazil, iran, peru, saudi arabia and syria, where it is often associated with poverty (hepburn 2001). this ailment affects around 12 million people in 88 countries (who 1990). it is estimated that there are about 2-3 million new cases each year. it is also considered that presently there is a population of 350 million of people facing the risk of infection (who 1990, iwu et al. 1994). cutaneous leishmaniasis incidence average is 0.28 in each 1000 people *corresponding author: abbas doroodgar, tel: +98 361 5550021,fax: +98 361 5551112, e-mail: adoroudgar@kaums.ca.ir iranian j arthropod-borne dis, (2008), 2(2): 42-47 a doroodgar et al: treatment of cutaneous 43 in iran. about 20000 cl cases are reported from different parts of iran annually and estimated the rural number of cases exceeds this statistics (mohebali 1996). the classic treatment is pentavalent antimonials. the disadvantages of the antimonials are their requirement for intramuscular or intravenous injection each day for 20-28 days, their toxicity, and the recent development of resistance in same regions such as india (berman 2003). traditional treatment of cl is a common habit of natives in many endemic areas including many parts of iran (weigel et al. 2003). natural extract of different plants such as euphorbia spp., gossypium herbacium, and berberis vulgaris are directly used on skin lesions as well as on the parasite in nnn medium (fata et al. 2006). artemisinin is an aromatic herb found in the extract of some medicinal plants such as a. sieberi (tyler et al. 1988, farzaneh et al. 2006, baldi and dixit 2007). this plant is an endemic species of iran, which grows in many regions. in order to evaluate the effect of a. sieberi essence on the experimental ulcers of cl on balb/c mice, this study was undertaken over a 12-month period. material and methods this experimental study was done in kashan university of medical sciences in 2006. this study was considered ethically approved by ethic committee of deputy of research of kashan university of medical sciences. seventy small laboratory white mice were inoculated subcutaneously by 0.1 ml liquid phase culture containing at least 5x l06 promastigotes of standard l. major (mhom/ 64/ir/er75) (obtained from tehran university of medical sciences). after 4 weeks, nodules and ulcers appeared on 50 inoculated mice. fifty balb/c mice were divided into 5 groups. three groups (30 mice) were used in the experimental conditions (group a) and the others as control (group b). the stems and leaves of a. sieberi, obtained from kashan city, central iran, were prepared, washed, dried, and extracted by soxhlet apparatus and solved in ethanol 80% in the barij essence pharmaceutical company. one, 3 and 5 percent concentrations of hydro alcoholic essence of a. sieberi were used for cl lesions treatment. before using essence, diameters of each lesion were measured. different concentrations of the essence of a. sieberi were dropped on cl lesions of three groups of mice, three times a day, for 30 d. the placebo group (control group a) received ethanol 80% which was the solvent of the essence and the other group received no treatment (control group b). the increase or decrease of diameters of each lesion was measured by metric caliber. at the end of each 10 d treatment period the ulcers diameter were measured and sampled for amastigote. direct stained smear by giemsa was prepared from the lesions of experiment and control groups at days 10th, 20th and 30th after treatment. after the end of the treatment period, mice in the experimental and control groups were followed for 1 month. the results were analyzed by mean± sem (standard error of mean). anova and lsd tests were used to examine the changes. a probability level of p< 0.05 was statistically considered significant. results the results showed that after the end of treatment period, diameter of cl each lesion increased in 1, 3 and 5 percent of artemisia essence concentrations and the control groups. statistically significant increase in the size of ulcers observed in 3% and 5% concentrations and in the control groups (p< 0.05). treatments could not reduce the diameter or caused small lesions to disappear completely. these changes are shown in table 1 and fig. 1. iranian j arthropod-borne dis, (2008), 2(2): 42-47 a doroodgar et al: treatment of cutaneous 44 the examinations showed that using more concentrations of the essence caused more increase in diameter of cl lesions (p< 0.001). direct giemsa stained smears prepared from the lesions of experiment mice were positive for leishman bodies and parasites were isolated from skin lesions. ten days after treatments, secondary infections of the lesions in treated mice were seen and developed until the end of treatment period. the figure shows that although the concentrations of 1%, 3% and 5% of plant drug could not reduce the size of the lesions or reduce the number of parasites, the increase diameter ulcers was less in treatments compared with control groups. further analysis was performed comparing the amount of diameter lesions decrease in each treatment with control groups. the results of analysis showed that in 1% concentration group compared to ca and cb control groups, the size of ulcers was decreased 7 and 8.9 times, respectively. in the 3% concentration group comparing to ca and cb groups, the decrease of the diameter lesions was seen 1.2 and 1.6 times, respectively. in 5% concentration group comparing to ca and cb groups the decrease was seen 1.5 and 1.9 times, in that order. one month after the treatment period, the sizes of ulcers in remained mice increased in the experimental and control groups and the mice direct smears in microscopic studies were positive. table 1. average diameter increased of cl ulcers by different concentrations of artemisia sieberi diameter groups before treatment (mm) after treatment (mm) increase (%) 1% 9.72 10.29 5.9 3% 10.67 14.2 33.1 5% 8.79 11.28 28.3 ca 7.53 10.63 41.2 cb 7.59 11.57 52.4 control groups: ca= receiving ethanol 80% (placebo group), cb= received no treatment. 5.9 33.1 41.2 28.3 52.4 0 10 20 30 40 50 60 1 3 5 ca cb groups d ia m et er in cr ea se (% ) fig. 1. average diameter increased of cutaneous leishmaniasis ulcers of mice inoculated by leishmania major treated by different concentrations of artemisia sieberi compared to control groups: ca= receiving ethanol 80% (placebo group), cb= receiving no treatment. iranian j arthropod-borne dis, (2008), 2(2): 42-47 a doroodgar et al: treatment of cutaneous 45 discussion artemisinin is a sesquiterpene lactone presents in a number of clinically important medicinal plants, including a. annua (sweet wormwood) and a. sieberi (arab et al. 2006, baldi and dixit 2007). artemisinin containing plants are used medicinally in virtually all traditional medical systems, and have a history of usage in chinese medicine dating back to thousands years. artemisinin has demonstrated significant antimalaria activity against plasmodium falciparum, and p. vivax (rita bilia et al. 2002). although widely used throughout the world as an anti-malarial, new studies show that this herb becomes cytotoxic in the presence of ferrous iron, and iron influx is naturally high in cancer cells. case reports show effectiveness against a wide variety of cancers (rowen 2002, lai et al. 2005). on the other hand, studies showed some artemisia species have antiviral and antiparasitic activity such as essential oil of the a. arborescens against hsv-1 and hsv2 and active principle santonin of a. nilagirica against trichinella spiralis larvae (sukul et al. 2005, saddi et al. 2007). due to artemisia species effects in pathogenic agents, the extract of a. sieberi effect in the leishmania parasites in murine model was examined in this paper. however, we used the leaves of a. sieberi since is known that, it contained artemisinin (arab et al. 2006). on the other hand, this plant is a common table vegetable all over iran and abundant in kashan as well as desert areas and access to this plant is very inexpensive, therefore essence of a. sieberi was used. there is evidence that show aqueous extract and essential oil of a. herba-alba asso has the strongest leishmanicidal activity in vitro. in one study, aqueous extract and essential oil of artemisia herba-alba asso were tested for their antileshmanial activity again leishmania tropica and leishmania major. the strongest leishmanicidal activity was observed with the essential oil at 2 µg/ml as versus the other two strains tested. the aqueous extract showed an antileshmanial activity at 4 µg/ml (hatimi et al. 2000). in another study, methanolic demonstrated that extract of a. aucheri inhibited the l. major parasite multiplication at doses of 150, 300 and 450 µg/ml at 48 and 72 h of culture. doses of 600 and 750 µg/ml showed the same effect at 24, 48 and 72 h of culture (p< 0.05) (sharif et al. 2006). the results of the studies mentioned above are not in agreement with the results of this study. this difference of finding may be due to the nature of studies conducted before. while the previous researches mentioned in this paper used in vitro approach, this study used in vivo method to examine the effectiveness of this treatment. however, it was concluded that a. sieberi essences were ineffective on l. major ulcers of balb/c mice. the present results shows, even though 1, 3 and 5 percent of concentrations of plant drug could not reduce the size of the lesions or cause small lesions to disappear completely and reduce the number of parasites, it could reduce the size of ulcers in treatment compared with the control groups. the result of therapeutic effect of artemisia herba-alba extract against cutaneous leishmaniasis by l. major in small white mice (out-bred) was negative and herb drug could not reduce the diameter of ulcers or the number of parasites (babaee khou et al. 2007). the size of ulcers was increased in all cases. this result is similar with that of our study. there is several reason indicated lack of affect of a. sieberi essences on l. major and increase in the size of ulcers with the more concentration. they include ineffective major of active components of a. sieberi, essences and the other ingredients, genetic of reservoir host and effective genes on cellular immunity reactions, and genetic of species agent of the disease. meanwhile, the number of parasite inoculated and the lesions seconiranian j arthropod-borne dis, (2008), 2(2): 42-47 a doroodgar et al: treatment of cutaneous 46 dary fungi and bacterial infections development are also effective in treatment process. thus, probably cytotoxic components in the plant drug are effective. the major components of a. sieberi are camphor (54.7%), camphene (11.8%), 1, 8-cineol (9.9%), b-thujone (5.7%), a-pinene (2.5%) (ghasemi et al. 2007). in another study on a. herba-alba (from kashan area), comphor (42.5%), 1, 8-cineol (18.3%) and camphenes (8.7%) were recognized (bamoniri 2004). in conclusion, to find the effective concentration and the mechanism of the effectiveness of the drug, further investigations with less concentrates of a. sieberi essence are recommended. acknowledgements we wish to thank of vice chancellor research for providing research facilities. we appreciate of dr hossein hooshyar, dr mansour sayyah 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(received 3 nov 2015; accepted 5 dec 2015) abstract background: malaria is still a health problem in iran. there are several vector control activities, including indoor residual spraying, using insecticide treated nets and larviciding including temephos. in addition nuisance mosquitos are prevalent in the urban areas. so that evaluation of this species to larvicide will provide a clue for management of vector control activities. methods: two mosquito species were used in this study: anopheles stephensi were collected from kazeroun and culex pipiens from tehran, capital of iran. all the tests were carried out according to the who method. all the test kis was provided by who. results: results showed a lc50= 0.0523 and lc90=0.3822 mg/l for an. stephensi. the figure for cx. pipiens was 0.1838 and 0.8505 mg/l respectively. conclusion: monitoring of insecticide resistance to temephos should be evaluated regularly for management of vector control. keywords: temephos, anopheles stephensi, culex pipiens, evaluation, efficacy introduction mosquito-borne diseases are from the main public health problems around the world, especially in tropical and semi-tropical areas. among them malaria, dirofilariasis, dengue, west nile, yellow fever, and some other viral diseases have the main morbidity. malaria occurs in tropical and some temperate regions of the world with an annual morbidity of 300–500 million cases and 574000 deaths in 2013 (who 2014). iran is going to eliminate the disease by 2025 although local transmission happens in some areas in south and southeast. anopheles stephensi is one of the main malaria vectors is coastal lowlands of southern iran (hanafi-bojd et al. 2011). west nile and dirofilariasis are diseases which can be transmitted by cx. pipiens, a worldwide distributed mosquito species. both diseases are potentially transmittable in iran (azari-hamidian et al. 2009, ahmadnejad et al. 2011). the first line in malaria control program in iran is indoor residual spraying to interrupt the transmission cycle, followed by distributing long lasting insecticide impregnated nets (llins) and larviciding. the latter is doing by biological larvicides in current years, although temephos, an organophosphate compound, has been used for years in mosquito larvae control. there are reports of resistance to some insecticides such as ddt, diledrin and malathion in an. stephensi, as well as some indications of resistance to pyrethroids in current years (davari et al. 2006, hanafi-bojd et al. 2012, vatandoost and hanafi-bojd 2012), although current studies have confirmed full susceptibility of this species to malathion (iranpour et al. 1993, vatandoost et al. 2005, hanafi*corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com,vatando@tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 510–518 mr abai et al.: laboratory evaluation of … 511 http://jad.tums.ac.ir published online: october 04, 2016 bojd et al. 2012). by the way, larvicides tests showed resistance/tolerance to fenthion and fenitrothion in this species, but susceptibility to temephos (vatandoost et al. 2004a, 2005, vatandoost and hanafi-bojd 2005, hanafi-bojd et al. 2012, soltani et al. 2013). in insecticide resistance management it is necessary to evaluate used insecticides/ larvicides periodically to monitor susceptibility status of the vectors. considering the extensive use of insecticides in public health sector as well as agriculture and urban pests, physiological and behavioral resistance is a big issue that must be determined in mosquito vector species. organophosphates are using widely in the agriculture sector, while they are using for larviciding against mosquitoes, it is possible to develop resistance in some vectors. a study on different geographical strains of an. stephensi in iran showed considerable variations in temephos resistance ratios collected from different localities (soltani et al. 2013). culex pipiens is known as urban pest nuisance around the world and has a wide distribution in the northern and central parts of iran (azari-hamidian 2007). it can transmit dirofilariasis and has high resistance levels to different insecticides (maraghi et al. 2006, azari-hamidian et al. 2009). this species usually breeds in waste waters which are contaminated with different detergents, insecticides, industrial pollutants, oil compounds etc. therefore it is possible to develop resistance to insecticides and/or larvicides in this mosquito, as it has been reported in recent years (vatandoost et al. 2004b). temephos, a most widely used organophosphate insecticide, has been included in the list of world health organization (who) as a suitable and safe mosquito larvicide that can be used even in drinking water for controlling of the most mosquito vectors. the toxicity of this insecticide is low and unlikely to present acute hazard for human (who 2006). in 2006 for the first time in the middle east, resistance to temephos was confirmed in an. stephensi breeding in water storage tanks in the al-dhahira region of oman (anderasen 2006). during last three decades temephos has considered as a safe larvicide (lc50= 8600 mg/l) in vector control programs (pierce et al. 1989). world health organization suggested 1 ppm of this compound for larviciding in drinking water bodies. it is effective for 3 months in this concentration (bang et al. 1972), and this is confirmed by several studies against different mosquito species (mulla et al. 2004, thavara et al. 2004, 2005). also larval control in natural breeding places such as riversides has achieved by temephos ec 1% (shililu 2001, parvez and al wahaibi 2003). laboratory tests on this formulation resulted to 15 weeks residual and larvicidal effect against aedes aegypti (chen et al. 2006). with due attention to the long history of insecticide/larvicides resistance in anopheles stephensi and culex pipiens in iran and other countries it is necessary to screen the susceptibility status of these species at regular intervals using who standard bioassay tests and to map the spatial and temporal changes in their level of susceptibility/resistance. therefore this study was aimed to determine the susceptibility of these species to temephos in two sentinel sites in iran. materials and methods mosquito collection and rearing two mosquito species were used in this study: an. stephensi mysorensis collected from kazeroun county, south of iran and cx. pipiens collected from tehran, the capital city of iran. collected larvae were transferred to the insectary (kazeroun station, school of public health, tehran university of medical sciences for an. stephensi and department of medical entomology and j arthropod-borne dis, december 2016, 10(4): 510–518 mr abai et al.: laboratory evaluation of … 512 http://jad.tums.ac.ir published online: october 04, 2016 vector control, school of public health, tehran university of medical sciences for cx. pipiens) and fed on fish flakes (tetramine). the specimens were reared under 28±1 °c temperature and 14:12 l: d photoperiod to late 3rd early 4th instars, the suitable physiological ages for performing tests. susceptibility tests standard who method (1981, 2005) was followed for the tests using who standard kit and 1.25, 6.25, 31.25 and 156.25 mg/l concentration were used to provide the final concentration of 0.005, 0.025, 0.125 and 0.0625 when added 1 cc of each concentration to 249 cc of tape water. the obtained results were corrected by abbotts’ formula when mortality rate in control group was 5– 20 percent (abbott 1925) and analyzed by probit (finney 1971) to obtain lc50, lc90 and regression line. for each larvicides concentration 4 replicates of 25 larvae were used and 2 replicated for control. after 24 hrs all larvae without movement on the water surface were considered as dead. results table 1 shows the results of mortality of an. stephensi to temephos. we obtained lc50= 0.0523 and lc90=0.3822 for this species (tables 1 and 2). regression line is presented in fig. 1. it shows y= 1.9014+ 1.4837x. for cx. pipiens a mortality rate of 3–99% was obtained in different concentrations (table 3). lc50 and lc90 for this species were calculated as 0.1838 and 0.8505 ppm (table 4). regression line is presented in fig. 2. it shows y=1.4171+1.9264 x. table 1. mortality rate of anopheles stephensi larvae (kazeroun strain) against who standard concentrations of temephos, 2014 concentration (ppm) replicates no. of tested no. of dead mortality rate (%) observed probit mortality expected probit mortality 0.005 4 100 8 8 3.595 3.487 0.025 4 100 26 26 4.357 4.524 0.125 4 100 77 77 6.555 5.561 0.625 4 100 93 93 6.476 6.599 control 2 50 0 0 table 2. lethal concentration values obtained from regression analysis of temephos against anopheles stephensi larvae of kazeroun strain, 2014 a b ± se lc50 (ppm) ± 95%c.l. lc90 (ppm) ± 95%c.l. λ2 (heterogeneity) λ2 table (df) p-value 1.9014 1. 4837 ± 0.128 0.0411 0.0523 0.0664 0.2695 0.3822 0.6001 3.906 * 5.991 (2) 0.001 * no heterogeneity j arthropod-borne dis, december 2016, 10(4): 510–518 mr abai et al.: laboratory evaluation of … 513 http://jad.tums.ac.ir published online: october 04, 2016 table 3. mortality rate of culex pipiens larvae (tehran strain) against who standard concentrations of temephos, 2014 table 4. lethal concentration values obtained from regression analysis of temephos against culex pipiens larvae of tehran strain, 2014 * heterogeneity fig. 1. mortality regression line of temephos against anopheles stephensi of kazeroun strain, 2014 concentration (ppm) replicates no. of tested larvae no. of mortality mortality rate (%) observed mortality probit expected mortality probit 0.005 4 100 3 3 3.119 1.984 0.025 4 100 8 8 3.595 3.331 0.125 4 100 11 11 3.773 3.677 0.625 4 100 99 99 7.327 6.024 control 2 50 0 0 a b ± se lc50 (ppm) ± 95%c.l. lc90 (ppm) ± 95%c.l. λ2 (heterogeneity) λ2 table (df) p-value 1.4171 1.9264 ± 0.174 0.1493 0.1838 0.2285 0.6186 0.8505 1.3039 11.113 * 5.991 (2) 0.001 j arthropod-borne dis, december 2016, 10(4): 510–518 mr abai et al.: laboratory evaluation of … 514 http://jad.tums.ac.ir published online: october 04, 2016 fig. 2. mortality regression line of temephos against culex pipiens of tehran strain, 2014 discussion our study showed lc50 value of 0.0523 for an. stephensi in kazeroun area, southern iran. previous studies on this species from different localities of southern regions of the country have reported 0.001613 mg/l (vatandoost and hanafi-bojd 2005) and 0.0022– 0.0141 mg/l (soltani et al. 2013). other studies on diagnostic dose of temephos (0.25 mg/l) found an. stephensi was susceptible to this larvicide in iran (vatandoost et al. 2004a, hanafi-bojd et al. 2012). by the way as it shown in table 1, in concentration of 0.625 mg/l only 93% mortality has occurred. considering diagnostic dose for this species, kazeroun strain is resistant to temephos. by the way these findings should be confirmed by more studies with different batches of who standard kits for temephos on more larval population collected from the same area. resistance of an. stephensi to this larvicide has previously reported from oman country (parvez and al wahaibi 2003). anopheles stephensi has been reported susceptible to temephos in india with lc50 range of 0.008–0.015 (tikar et al. 2011, singh et al. 2014). study on an. labranchiae in morocco found low resistance to temephos in a sentinel site. they revised the diagnostic dose of temephos for this species (faraj et al. 2010). a more recent study in iran showed that the altered enzymes are responsible for temephos resistance in an. stephensi (soltani et al. 2015). although temephos is not used in national malaria program in last years but the study area has different rice fields and citrus gardens, and farmers use organophosphate insecticides to control pests. this may affect susceptibility j arthropod-borne dis, december 2016, 10(4): 510–518 mr abai et al.: laboratory evaluation of … 515 http://jad.tums.ac.ir published online: october 04, 2016 of an. stephensi as well. integrated pest management (ipm) in agriculture sector should be considered as an important priority parallel to integrated vector management (ivm) in health sector to overcome vector/pests resistance to pesticides. our results on cx. pipiens also showed lc50 (0.1838 mg/l) was more than diagnostic dose (0.02 mg/l) recommended by who for this species. it means resistance to temepho. other studies on temephos in cyprus and tunisia against cx. pipiens reported lc50 ranges of 0.00076–0.310 and 0.0021– 0.015, respectively (ben cheikh et al. 1993, vasques et al. 2009). dose response tests of the technical temephos against cx. pipiens in india showed lc50 of 0.01 mg/l in this species (badawy et al. 2015). this value for culex quinquefasciatus was 0.0000473 mg/l in another study in that country (dorta et al. 1993). in tunisia esterases were found to be contributed to temephos resistance by their increased activity (ben cheikh et al. 2008). although there is no national program to control this species, but because wastewaters are the main breeding places for cx. pipiens and they are contaminated with different pollutants, insecticides, detergents, etc this species has developed resistance to these compounds to save itself. a longitudinal on cx. pipiens in martinique showed that resistance ratio for temephos was 8.6 to 42folds during 1991–1999 comparing 2.9 to 4.6-folds in 1990. they found organophosphate resistance was associated with decreasing of susceptible genotypes at ester and ace-1 foci, as well as to an allele replacement at the easter locus (yebakima et al. 2004). our results on lc50 of cx. pipiens were higher than the above mentioned studies in different countries. therefore, it is necessary to do biochemical and molecular studies to find the reason for resistance to temephos. in conclusion, it is recommended to do temephos 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article insecticide susceptibility status of wild-caught sand fly populations collected from two leishmaniasis endemic areas in western turkey *mehmet karakus 1, bayram gocmen 2, yusuf özbel 3 1department of zoology, institute of science, ege university, bornova, izmir, turkey 2department of zoology, science faculty, ege university, bornova, izmir, turkey 3department of parasitology, medical school, ege university, bornova, izmir, turkey (received 30 aug 2014; accepted 2 feb 2016) abstract background: in turkey, vector control programs are mainly based on indoor residual spraying with pyrethroids against mosquitoes. no special control program is available for sand flies. most insecticide susceptibility tests were done for mosquitoes but not for sand flies. we therefore aimed to determine the insecticide susceptibility against two commonly used insecticides; deltamethrin and permethrin, on wild-caught sand fly populations collected in two geographically separated leishmaniasis endemic areas. methods: insecticide susceptibility of wild-caught sand flies to deltamethrin (0.05%) and permethrin (0.75%) using ready-to use impregnated insecticide papers of who was investigated in 2010 based on knockdown time using standard who tube-test kit and procedures. sand flies used in this study were collected from villages of aydin (bascayır) and mugla (tepecik). results: the resistance and early resistance were detected on the sand fly population from mugla province against deltamethrin and permethrin, respectively. however, populations from aydin province were sensitive to both insecticides. conclusion: the resistance against deltamethrin and permethrin was detected on sand fly population in mugla province where both insecticides have been applied for long time while no resistance was found in the insecticide free area, aydin province. these findings can be an indicator for showing the ability for developing the insecticide resistance in sand flies. because of the presence and dominancy of vector sand fly species of leishmania infantum (phlebotomus neglectus, p. tobbi) in both study areas, the systematic monitoring for resistance of sand fly populations and more attention are needed by the authorities involved in control programs for sand fly-borne diseases. keywords: sand flies, insecticide susceptibility, pyrethroids, phlebotomus neglectus, turkey introduction phlebotomine sand flies (diptera: psychodidae) transmit leishmania (kinetoplastida: trypanosomatidae) parasites that can cause severe, lethal clinical form, visceral leishmaniasis (vl), and moderate skin disease, cutaneous leishmaniasis (cl). in turkey, l. infantum causes vl in all over turkey and cl in east part of mediterranean region and transmitted by phlebotomus neglectus and p. tobbi while l. tropica causes cl and transmitted by p. sergenti in southeastern region and p. similis in western part of turkey (ozbel et al. 2000, ok et al. 2002, toz et al. 2013). in recent years, more than thousands of active ingredients of the pesticides are in use for insect control in many developed and developing countries as well as in turkey (tomlin 1997, koçak 1998). since 1957, insecticides have been applied heavily for pest or vector control especially in the areas where malaria cases are seen (curtis 1962). insecticide usage data shows that agricultural use of pesticides has reached 30000 tons/yr in *corresponding authors: dr mehmet karakus, email: mehmetk1986@yahoo.com j arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 87 http://jad.tums.ac.ir published online: march 14, 2017 turkey (durmusoglu et al. 2010). for this reason, monitoring of insecticide resistance is a necessary element of any medium-scale or large-scale deployment of an insecticidal intervention. vector control measures using insecticides have been mainly applied against mosquitoes but they are also affecting other insect vectors indirectly. improper, disorganized and uniform usage of the insecticides for vector control has led to the development of insecticide resistance in insects as well as different vector arthropods in tropical and subtropical countries (singh et al. 2012). the pyrethroids are the only insecticide class used for the insect control in europe and turkey and widespread use of a single class of insecticide increases the risk that insects (mainly mosquitoes) can develop resistance to it. the development of insecticide resistance in the insect vector has been threatened the effectiveness of these control measures (kishore et al. 2006, singh et al. 2012). routine monitoring of insecticide resistance in the natural populations of vectors is necessary and helps us to detect early resistance and improve effectiveness of operational control strategies (aizoun et al. 2014). in turkey, control measures against leishmaniasis include disease notification and treatment of patients, but not specific vector control. insecticides have been mainly used for mosquito control by indoor residual spraying and therefore many studies have been conducted on insecticide resistance in the malaria vectors (curtis 1962, kasap et al. 2000, lüleyap et al. 2000, aldemir et al. 2005, abdallah et al. 2008, himeidan et al. 2011). testing of insecticide susceptibility in leishmaniasis vectors is the first important step in insecticide resistance surveillance in a particular endemic area. so far, insecticide susceptibility status of sand flies has not been studied in turkey. thus, the present study was undertaken to investigate the susceptibility status of phlebotomine sand flies to two insecticides, deltamethrin and permethrin, which are currently and commonly used for insect control in turkey. materials and methods study sites this study was conducted in two villages from two provinces where they have been subjected with different histories of insecticide exposure. insecticides for mosquito control program have been actively used for long time in the first study area, tepecik village in mugla province, while no exposure of insecticides in the second study area, bascayir village in aydın province. both study areas are endemic for cutaneous leishmaniasis in human and canine leishmaniasis in dogs, located in western part of turkey and had similar environmental/climatic condition and geographic features (table 1, fig. 1). sand fly collection sand flies were collected using cdc light traps from both villages between june and september 2010. totally, 20 cdc light traps were set up in each village at 7 pm. and collected at 7 am. following day. traps were placed mostly in animal barns. alive sand flies from the light traps were released into a plexiglas cage by mouth aspirator and a 10% sucrose solution soaked cotton was placed on the cage. then they were transported to the leish-bio-lab in ege university faculty of medicine, izmir and kept in appropriate conditions (25±2 ˚c and 70±10% relative humidity). insecticides and bioassay tests permethrin and deltamethrin at concentrations 0.75% and 0.05% respectively were used with an exposure time of 60 min. the choice of these two insecticides was justified by their widely use in the formulations in turkey. who test-kit tubes and impregnatj arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 88 http://jad.tums.ac.ir published online: march 14, 2017 ed papers were procured from collaborating center of who in malaysia. all the susceptibility tests were done according to standard who testing protocols on unfed female sand flies using at least 20 specimens (not yet identified). the sand flies were transferred into the exposure tubes and were gently transferred to the holding tube after one-hour exposure period and fed with 10% sugar solution placed in the top of the holding tube. control test tubes carrying control papers were also held parallel to each set of tests. all the tests were ignored if the mortality was higher than 20% in the control group. the test was done in five replicates for each insecticide. during these bioassays, laboratory condition was stabilized at 27±2 ˚c and 80±10% rh as stated on whopes (who 1981). the specimens were kept for several hours in the lab and all tests were started at between 06:00 and 07:00 pm. the resistance status of sand fly specimens was determined according to the latest who criteria (who 2013) as follows, (a) mortality rates between 98–100% indicate full susceptibility, (b) mortality rates between 90–97% require further investigation, (c) mortality rates < 90%, the population is considered resistant to the tested insecticides. knockdown rates were recorded as indicated in whopes starting from 10 min, 15 min, 20 min, and then 10 min intervals up to 1 h for determining the exact knockdown time (kdt) of the populations, which is important for the detection of early resistance. kdt50, kdt95 and kdt100 values were also noted for both insecticides. following the testing procedures, all sand fly specimens were dissected, mounted and identified according to the keys and descriptions presented by (perfil’ev 1968, lewis 1987, killick-kendrick et al. 1991). data analysis data analysis was made using log-probit analysis software (probit v1.5). this software is able to calculate of kdt50, kdt95 and kdt100 and their confidence intervals (finney 1971). results sand fly fauna of the study sites totally, 486 sand fly specimens used in the study were dissected, mounted and identified. sand fly fauna of both study areas was very similar as follows: tepecik village in mugla province, 3 phlebotomus (64% p. tobbi, 30% p. papatasi, 5% p. neglectus/syriacus) and one sergentomyia (1% s. minuta) species, bascayir village in aydın province, 4 phlebotomus (79% p. tobbi, 9% p. neglectus/syriacus, 6% p. papatasi, 2% p. alexandri) and 2 sergentomyia (2% s. minuta, 2% s. dentata) species were found (table 2). insecticide susceptibility tests the test results were evaluated according to the who standards, and resistance/early resistance was detected on sand fly specimens collected in first study area (mugla) against both insecticides while the specimens caught in the second study area (aydin) were susceptible to both insecticides. different death and knockdown rates were found in the sand flies from both areas. the relative susceptibilities of the two sand fly populations to tested insecticides were comparable. first study area (tepecik village, mugla province) for the sand fly specimens collected in mugla province, 90% of death rate for deltamethrin (0.05%) was noted by the end of 24 h. first knockdown effect was observed by the end of 10 min and 10 of 100 specimens was noted alive but not able to fly after 24 h (table 2, 3, fig. 2). same results were obtained for permethrin (0.75%) and 93.3% of death rate was noted by the end of 24 h. no knockdown effect was noted in first 10 min after exposure. j arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 89 http://jad.tums.ac.ir published online: march 14, 2017 second study area (bascayir village, aydın province) for the sand fly specimens collected in aydın province, 99% of death rate for deltamethrin was noted by the end of 24 h. bascayir village population was found to be susceptible to deltamethrin. the first knockdown effect was observed in 3 min after exposure and 99 out of 100 specimens dead by the end of 24 h (table 2, 3, fig. 2). same results were obtained for permethrin (0.75%) and 100% of death rate was noted by the end of 24 h. bascayir village was susceptible to permethrin (0.75%) by the terms of who. first knockdown effect was observed in 3 min after exposure and all specimens died in 24 h. control group one hundred sand fly specimens per study area were used for control and none of the specimens was died during the experiment except physical deaths. abbott’s formula was not used in this study for the correction of mortality rates because of the mortality rates in all controls was always less than 5%. knockdown time (kdt) kdt50 and kdt95 knockdown times for diagnostic doses of both insecticides have calculated for sand fly populations in both study area. no significant knockdown rate for diagnostic doses of insecticides were recorded for the specimens collected in 1st study area while delayed knockdown times (>45 min) were detected for the specimens collected in 2nd study area (table 3). table 1. demographic information of study sites environmental characteristics of the areas studied study area 1 (tepecik village, muğla) study area 2 (bascayir village, aydin) latitude 37° 08' 06.46'' n 37° 58' 26.74'' n longitude 27° 38' 42.96'' e 28° 04' 21.98'' e altitude (m) 82 359 population 369 1614 climate type mediterranean mediterranean average temperature in summer (2010) 28.45 ˚c 23.61 ˚c average temperature in winter (2010) 11.32 ˚c 11.73 ˚c annual mean rainfall (2010) 1050 mm 670 mm fig. 1. map showing the location of both study sites where sand flies were collected j arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 90 http://jad.tums.ac.ir published online: march 14, 2017 table 2. sand fly species used in the study and the results of insecticide susceptibility tests insecticide exposed area (tepecik village, mugla) insecticide free area (bascayır village, aydın) number of sand fly specimen 240 246 sand fly species used in the tests fauna (%) phlebotomus tobbi p. papatasi p. neglectus/syriacus p. alexandri sergentomyia dentata s. minuta susceptibility rate (% in 24 hours) and who status against deltamethrin against permethrin 90 – resistance 93.3 – early resistance 99 – susceptible 100 susceptible observations first death at the end of 24 h in 1 h 10% of the total specimens was alive but not able to fly in 5 min all dead fig. 2. number of knocked down sand flies and knock down times of sand flies after exposure with deltamethrin (del) and permethrin (per) with 10 min intervals in two areas (bascayir and tepecik) j arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 91 http://jad.tums.ac.ir published online: march 14, 2017 table 3. the results of insecticide susceptibility tests and knockdown times of wild-caught sand flies in the study areas study area insecticide no. of sand flies exposed mortality rate (%) kdt50 min kdt95 min kdt100 min who status tepecik village, muğla deltametrin 0.05% 100 90 37,5 40,7 >45 t permethrin 0.75% 105 93.3 34,6 36,1 >42 t control 40 1 bascayir village, aydin deltametrin 0.05% 100 99 18,6 38,6 >35 s permethrin 0.75% 106 100 22,4 35,9 >33 s control 45 1 t: tolerance (resistance that needs to be confirmed), s: susceptible, *the knockdown rates and mortality rates were given as an average of 5 repeated tests discussion in turkey, after plasmodium vivax malaria reached the elimination level, leishmaniasis, especially cutaneous form, remained most serious and important vector-borne disease reported from all geographical regions of the country. according to turkish ministry of health official reports, more than 46.000 cl cases were reported between 1990 and 2010 (gürel et al. 2012) and 2200 cl and 40 vl cases were recorded in the year 2013 (ministry of health of turkey 2012). the control of leishmaniasis is only based on free treatment of vl and cl cases in turkey. however, indoor residual spraying, using pyrethroids have been mainly used for mosquito or malaria control programs in most of the regions in the country and it affects other insects including sand flies. the insecticide application activity can be disparate in provinces from one to another according to the endemicity of vector-borne diseases and/ or importance for tourism. the present study was undertaken because no data were available about the susceptibility of sand flies to who recommended insecticides in turkey. here, we reported the results of the first preliminary study on insecticide susceptibility of wild-caught sand fly populations in two leishmaniasis endemic areas with different history of insecticide application activities. the principle of the who bioassay is to expose insects to a given dose of insecticide for a given time to assess susceptibility or resistance. in turkey, insecticide susceptibility tests were mainly done for different mosquito species but no single study was conducted on phlebotomine sand flies. for this reason, we compared our findings with different bioassays, conducted on sand flies in different countries. although, deltamethrin 0.05% is the discriminating concentration given for anopheles, it is not obvious to extrapolate this to sand flies. in brazil, bioassays with 0.05% deltamethrin were used and a clear difference between the insecticide susceptibility of two sand fly populations was observed (alexander et al. 2009). in that study, the sand fly population with no history of previous insecticide exposure, 25 min was determined as lt50 and all sand flies died after one hour. in the sand fly population exposed to sand fly control measures using pyrethroids since long time, lt50 was significantly higher (40 min) and the mortalj arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 92 http://jad.tums.ac.ir published online: march 14, 2017 ity was only 62% after 1 h (alexander et al. 2009). there are several studies for determining insecticide susceptibility of p. papatasi populations, main vector of l. major causing cl in a wide geographical area in the old world (afshar et al. 2011, faraj et al. 2012, hassan et al. 2012, saeidi et al. 2012). in sudan case, p. papatasi was sensitive (kdt95: 20.16 min) to permethrin in rahad game reserve and white nile areas while it was highly resistant (kdt95: 193.93 min) in surogia village (hassan et al. 2012). the lt50 value of deltamethrin (0.05%) against p. papatasi populations in iran was recorded as 13.6 min (afshar et al. 2011). in the present study, p. papatasi was representing 5% among wildcaught sand flies but in general, kdt values for both insecticides were higher in the sand fly population from mugla province. in particular, deltamethrin and permethrin provided 100% knockdown after 45 min in mugla province while it was 35 min in aydin province. the identification of sand flies indicated that the dominant species was p. tobbi in two study areas. p. tobbi is one of the proven vectors of l. infantum in turkey (svobodová et al. 2009) as well as in the old world. our results of knockdown times reveal that p. tobbi has resistance that needs to be confirmed especially for mugla province. prolonging death times and knockdown times are the strongest evidence of upcoming resistance on the insecticides and insect populations with extended kdt values are more prone to develop resistance to insecticides that used in the area (martinez-torres et al. 1998, chandre et al. 1999, kamgang et al. 2011). our results clearly showed that kdt values (table 3) of the sand fly population from mugla province are much longer than aydin population and therefore the sand fly populations in former area was more prone to develop resistance in compare to latter. upcoming resistance can be explained by long exposure to insufficient or ineffective doses of insecticides. short kdt values of sand fly population in aydın province supports the idea that these sand flies were not exposed to pyrethroid-based insecticides previously. the ready-to use impregnated insecticide papers of who have been used to test the susceptibility mainly in mosquitoes as well as sand flies (hassan et al. 2012). the results of the tests at different durations of exposure indicated that wild-caught sand fly populations from aydin province were fully susceptible to both insecticides used, whereas the early resistance was detected in the population of mugla province. developing resistance to permethrin and deltamethrin in mugla population can be attributed to long time usage of insecticides for mosquito control purposes. conclusion the early resistance against deltamethrin and permethrin were detected on sand fly population in mugla province where both insecticides have been applied for long time while no resistance was found in the insecticide free area, aydin province. these findings can be an indicator for showing the ability for developing the insecticide resistance in sand flies as also pointed out in previous studies. because of the presence and dominancy of vector sand fly species for l. infantum in both study areas, the systematic monitoring for resistance of sand fly populations needs to be accepted as a public health issue. these results clearly pointed out the more attention are needed by the authorities involved in control programs for sand fly-borne diseases. another important point is the needs create devices and guidelines (by who or expert committee) for applying insecticide susceptibility tests using sand flies because of the tubes prepared for mosquitoes are not actually fit for sand flies. j arthropod-borne dis, march 2017, 11(1): 86–94 m karakus et al.: insecticide susceptibility … 93 http://jad.tums.ac.ir published online: march 14, 2017 acknowledgements we special thank to dr suha kenan arserim for his helps during bioassays. the authors declare that they have no competing interests. references abbott ws (1987) a method of computing the effectiveness of an insecticide. j am mosq control assoc. 3: 302–303. abdallah h, matamboa ts, koekemoera ll, mnzavae ap, hunta rh, coetzeea m (2008) insecticide susceptibility and vector status of natural populations of anopheles arabiensis from sudan. trans r soc trop med hyg. 102: 263–271. afshar aa, rassi y, sharifi i, abai mr, oshaghi ma, yaghoobi-ershadi mr, vatandoost h (2011) susceptibility status of phlebotomus papatasi and p. sergenti (diptera: psychodidae) to ddt and deltamethrin in a focus of cutaneous leishmaniasis after earthquake 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ozbel y, oskam l, ozensoy s, turgay n, alkan mz, jaffe cl, ozcel ma (2000) a survey on canine leishmaniasis in western turkey by parasite, dna and antibody detection assays. acta trop. 74: 1–6. perfil’ev pp (1968) phlebotomidae (sandflies) in: o theodor (eds): fauna of ussr. vol. 3 no: 2 wiener bindery ltd, jerusalem. saeidi z, vatandoost h, akhavan aa, yaghoobi-ershadi mr, rassi y, sheikh z, arandian mh, jafarib r, sanei dehkordia ar (2012) baseline susceptibility of a wild strain of phlebotomus papatasi (diptera: psychodidae) to ddt and pyrethroids in an endemic focus of zoonotic cutaneous leishmaniasis in iran. pest manag sci. 68: 669–675. singh rk, mittal pk, dhiman rc (2012) insecticide susceptibility status of phlebotomus argentipes, a vector of visceral leishmaniasis in different foci in three states of india. j borne dis. 49: 254–257. svobodová m, alten b, zídková l, dvořák v, hlavačková j, myšková j, seblová v, kasap oe, belen a, votýpka j, volf p (2009) cutaneous leishmaniasis caused by leishmania infantum transmitted by phlebotomus tobbi. int j parasitol. 39: 251–256. tomlin rs, forrest l, pu mm, kim mh (1997) discourse semantics. in: ta van dijk (eds): discourse as structure and process.vol.1, london: sage, pp. 63–111. toz so, culha g, zeyrek fy, ertabaklar h, alkan mz (2013) a real-time its1pcr based method in the diagnosis and species identification of leishmania parasite from human and dog clinical samples in turkey. plos negl trop dis. 7(5): e2205. who (2013) test procedures for insecticide resistance monitoring in malaria vector mosquitoes. 1(1): 1–40. j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 226 http://jad.tums.ac.ir published online: may 27, 2017 original article evaluation of larvicidal activity of essential oil from leaves of coccinia grandis against three mosquito species shahid iqbal mohammed, kishor sukhlal vishwakarma, *vijay laxminarayan maheshwari school of life sciences, north maharashtra university, jalgaon, india (received 2 mar 2015; accepted 7 may 2016) abstract background: to study the chemical constituents and larvicidal activity of essential oil extracted from the leaves of coccinia grandis against three mosquito species. methods: essential oil was extracted by hydro distillation using clevenger apparatus and was analyzed for chemical constituents by gas chromatography-mass spectrophotometry (gc-ms). larvicidal activity was recorded after 12 and 24h of post-exposure against three mosquito species, anopheles stephensi, aedes aegypti and culex quinquefasciatus. dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical re gion. the lc50 and lc90 values for three mosquito larvae were calculated by probit analysis. results: the gc-ms analysis revealed that essential oil contains 23 different constituents. out of these 23 constituents, major constituents identified were n-tetracosane (39.18%), n-eicosane (30.04%), tetratriacotane (2.97%), 7-octadecanal (2.81%), and tricosane (2.31%). essential oil from leaves of coccinia grandis exhibited significant larvicidal activity against an. stephensi with lc50 and lc90 values 39.41ppm and 123.24ppm, respectively. this was followed by ae. aegypti and cx. quinquefasciatus with lc50 and lc90 values of 48.20ppm, 131.84ppm and 52.80ppm, 135.48ppm, respectively after 24h of exposure. conclusion: the results could be useful in developing a cost effective, ecofriendly, region specific and practical strategy for the control of mosquito vectors. keywords: coccinia grandis, essential oil, mosquito, larvicidal, gc-ms introduction mosquitoes are responsible for a number of human health problems causing illness and death throughout the world in both children and adults. they are vector for many diseases such as malaria, filariasis, dengue, japanese encephalitis, chikungunya and west nile virus infection in tropical and subtropical countries (anupam et al. 2012). malaria is a deadly disease and globally about 3.3 billion peoples are at the risk of it. about 198 million cases of malaria and 0.58 million deaths occurred globally in 2013 (who 2014). of the six malarial vector species, anopheles stephensi is the main mosquito vector responsible for malaria in urban areas of india (senthilkumar et al. 2009). dengue fever is caused by mosquito vector species, ades aegypti in its epidemic areas affecting millions of people and thousands of deaths per year all over the world (service 1996). similarly, culex quinquefasciatus is a vector for lymphatic filariasis, commonly known as elephantiasis, in india. lymphatic filariasis is caused by the worms wuchereria bancrofti, brugia malayi and br. timori of which, first is found to be more endemic in indian subcontinent. according to who (2009) more than 1.3 billion people spread over 72 different countries worldwide are threatened by it. collectively, these mosquito mediated diseases are responsible for long term suffering, morbidity and high socio economic burden on society (ramaiah et *corresponding author: prof vijay laxminarayan maheshwari, e-mail: vlmaheshwari@rediffmail.com http://jad.tums.ac.ir/ http://en.wikipedia.org/wiki/wuchereria_bancrofti http://en.wikipedia.org/wiki/wuchereria_bancrofti http://en.wikipedia.org/wiki/brugia_malayi http://en.wikipedia.org/wiki/brugia_timori http://en.wikipedia.org/wiki/brugia_timori j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 227 http://jad.tums.ac.ir published online: may 27, 2017 al. 2000, intirach et al. 2012). efficient control of these diseases would require a two-pronged strategy, (i) prompt treatment with effective medicines and (ii) vector control based prevention strategies. injudicious use of medicines, particularly antimalarial drugs, has resulted in development of resistance by the malarial parasite and enhanced casualties in endemic areas (intirach et al. 2012). mosquito management therefore, offers a better and practical alternative for controlling diseases mediated by them. mosquito adulticides, causing temporary reduction in population are good but do not offer a lasting solution. mosquito larvae being delicate, less mobile and more concentrated in their natural habitat offer a much simpler and efficient point of intervention and control (rutledge et al. 2003, dharmagadda et al. 2005). however, there are reports of development of resistance and behavior changes in adult mosquitoes and larvae towards these chemicals (mittal et al. 2004, who 2006). moreover, they adversely affect the environment, causing soil, air, water pollution and harm the beneficial non target organisms (dharmagadda et al. 2005). plant extract including essential oils or insecticides from botanical origin are attractive alternatives because they contain high amount of various bioactive compounds, many of which are selective and have little or no harmful effects on non-target organisms and environment (rutledge et al. 2003, dharmagadda et al. 2005). essential oil is natural volatile substances found in many plants. essential oils isolated from plant are generally a mixture of many constituents, primarily biologically active monoterpenes (govindrajan 2010). traditionally, they have been used for flavor enhancement in food, odorants in fragrances, pharmaceuticals and confectionary industries (zhu et al. 2001). of late, they have received considerable attention as potentially active, human and environment friendly bio insecticides (cheng et al. 2003). there are several reports on larvicidal activity of essential oil from neem, basil, citronella, lemon, eucalyptus, pine etc (cheng et al. 2003, amer and mehlhorn 2006, dua et al. 2009). the resistance against plant derived insecticides has not been reported so far (kannathasan et al. 2011). coccinia grandis (family cucurbitaceae) is a unique tropical plant, commonly known as ‘little gourd’, growing abundantly and widely all over the india. it is a fast growing perennial climbing shrub with white flowers. it grows several meters long and forms dense mat that readily cover shrubs and small trees. it is well known for its hypoglycemic activity (ajay 2009, munasinghe et al. 2011). the present study was focused on the chemical constituents and larvicidal activity of coccinia grandis leaf essential oil against vectors of malaria (an. stephensi), dengue (ae. aegypti) and filariasis (culex quinquefasciatus). to the best of our knowledge it is the first report on the larvicidal activity of coccinia grandis leaf essential oil against the three mosquito species. materials and methods collection of plant material and extraction of essential oil plant material of coccinia grandis was collected from eklagna village jalgaon. [20° 58ʹ 54.3ʹʹ n, 075° 27ʹ 09.5ʹ e (elevation: 199m)] maharashtra, identified at botanical survey of india, pune and a specimen voucher number msmi-1 was deposited in the school of life sciences, north maharashtra university jalgaon. the collected fresh leaves were cut in to small pieces and extraction was done using clevenger apparatus for 6h (singh et al. 2008). the extracted essential oil was subjected to dryness over anhydrous sodium sulfate (na2so4) to remove traces of moisture. the physical characteristics of extracted essential oil were recorded, percentage average yield was calculated and it was stored at 4 ºc in amber-colored bottle in refrigerator until further analysis. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 228 http://jad.tums.ac.ir published online: may 27, 2017 gc and gc-ms analysis gas chromatography mass spectroscopy (gc-ms) analysis of essential oil was performed using jeol gcms-mate-ii model gas chromatograph-mass spectrometer equipped with an aoc-20i auto injector and hp-5 capillary column (30m x 0.25mm id x 0.25μm coating thickness) column. the injector temperature was set at 280 °c, and the oven temperature was initially set at 40 °c then programmed to increase up to 300 °c at the rate of 10 °c/min and finally held at 200 °c for 5 min. helium gas was maintained at a flow rate 1.0ml/min as a carrier gas. one microliter of the sample diluted with acetone in 1:10 ratio was injected in the split mode. the percentage of constituents in essential oil of leaves was calculated by the gc peak areas. data handling was made through jeol software and the compounds were identified based on the comparison of their retention time (rt) and mass spectra of wiley, nist library data of the gc-ms. mosquito larvicidal assay mosquito larvicidal activity was performed against mosquito larvae of species an. stephensi, ae. aegypti and cu. quinquefasciatus. larvae of an.s stephensi [21000’14.3n, 075029’39.8e (elevation: 207m)], ae. aegypti [2101’01.2n, 075029’52.3e (elevation: 192m)] and cu. quinquefasciatus [21000’53.5n, 0750 29’39.8e (elevation: 185m] were collected from local breeding areas of jalgaon, india and identified using the microscopic examination as per theodore et al. (2005). the collected mosquito larvae were brought to laboratory and maintained at 25–30 °c with 80–90% relative humidity and 12 h/d/night cycle in plastic trays containing dechlorinated water. mosquito larvae were fed with 10% sterile sucrose solution and pet biscuits. the mosquito larvicidal activity was performed according to standard procedure recommended by who (1981). the extracted dried and pre weighed essential oil was dissolved in 1ml of acetone and from this different concentrations were made such as 3.125, 6.25, 12.50, 25, 50 and 100ppm in distilled water. twenty five early fourth instar stage larvae of each of the three species of mosquito were used for larvicidal assay in 200ml beakers and three replicates were maintained for each concentration used. during the experiment, no food was given to the larvae. statistical analysis the larval mortality rate was calculated after 12 and 24 h of exposure time. the lethal concentrations, lc50 and lc90 and their 95% confidence limit of the lower and upper levels were calculated by probit analysis using statistical software stats direct 2.8.0. results the essential oil yield from fresh and finely cut leaves of coccinia grandis was 0.14gm% (w/w). the yield was calculated after drying (removing the moisture) over anhydrous sodium sulfate (na2so4). the essential oil after dryness gave a slightly sticky clump with light yellow color and a characteristic odor. the gc-ms profile of the essential oil from leaves of co. grandis is shown in fig. 1. the various constituents of essential oil, their retention time and percent composition in order of elution from the column are given in the table 1. the gc-ms profile shows a total of 23 constituents accounting for 99.60% of total oil. the two major constituents of essential oil from leaves of co. grandis were n-tetracosane (39.18) and n-eicosane (30.04%). six constituents (peak number 4, 6, 10, 13, 18 and 19) were present between 2–3 percent were as the percentage composition of remaining ranged between 0.1–2 percent (table 1). the essential oil extracted from leaves of co. grandis shows promising larvicidal activity against three mosquito species an. stehttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 229 http://jad.tums.ac.ir published online: may 27, 2017 phensi, ae. aegypti and cu. quinquefasciatus, (table 2). the lc50 and lc90 values against early fourth instar larvae of an. stephensi, after 12 and 24 h of exposure were calculated to be 72.60 and 169.90 and 39.41 and 123.24ppm, respectively. similarly, lc50 and lc90 values against early fourth instar larvae of ae. aegypti after 12 and 24h of exposure were calculated to be 83.25 and 191.60 and 48.20 and 131.84ppm, respectively. the values were marginally higher with early fourth instar larvae of cx. quinquefasciatus than the other two species under identical conditions (table 2). table 1. chemical composition of essential oil of the leaves of coccina grandis. peak no. retention time (min) chemical compounds percentage 01. 35.95 e,e,z-1,3, 12nanodecatriene-5,14-diol 0.81 02. 37.76 heneicosane 1.34 03. 39.52 phytol 1.35 04. 41.17 1-heptatriacotanol 2.06 05. 42.83 17-pentatriacontene 1.19 06. 44.29 tricosane 2.31 07. 45.16 1dodecanol, 2-coctyl1.37 08. 45.96 2,5-furandione, 3-dodecyl 0.96 09. 47.00 tetrapentacosane 1.98 10. 47.19 2-dodecen-1-yl(-)sucinic anhydride 2.08 11. 48.77 n-eicosane 30.04 12. 49.21 octasane 1.37 13. 50.69 7-octadecanal 2.81 14. 51.06 hexatriacontane 1.23 15. 52.77 n-tetracosane 39.18 16. 54.74 1,3 o-triacotanediol 1.09 17. 55.41 z-14-octadecen-1-ol acetate 0.98 18. 55.96 pentadeachal 2.09 19. 57.13 tetratriacotane 2.97 20. 57.38 triacotane 0.11 21. 62.62 meissyl alcohol 0.13 22. 62.76 palmitic acid 1.23 23. 64.63 myristic acid 0.92 total 99.60% http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 230 http://jad.tums.ac.ir published online: may 27, 2017 table 2. larvicidal activity of coccinia grandis leaf essential oil after 12 and 24 h of exposure period on larvae of anopheles stephensi, aedes aegypti and culex quinquefasciatus mosquito species time concentration (ppm) % of mortality±se lc50 (lcl– ucl)a lc90 (lcl– ucl)a x2 (df=4)b an. stephensi after 12h 3.125 8.0±6.89 72.60 (43.12– 106.50) 169.90 (90.05– 265.96) 12.60 6.25 18±5.77 12.5 25±1.25 25 35±1.52 50 42±1.55 100 60±0.57 after 24h 3.125 17±0.21 39.41 (12.07– 67.619) 123.24 (43.276– 212.45) 28.581 6.25 21±0.82 12.5 41±1.00 25 55±1.52 50 66±1.20 100 75±1.85 ae. aegypti after 12h 3.125 7.0±0.33 82.35 (25.73– 145.96) 191.60 (35.89– 370.94) 27.077 6.25 11±0.87 12.5 26±0.21 25 38±1.52 50 43±0.57 100 51±0.29 after 24h 3.125 14±1.20 48.20 (19.25– 78.66) 131.84 (49.23– 223.96) 27.862 6.25 17±0.88 12.5 33±0.86 25 51±0.68 50 61±1.15 100 71±0.26 cx. quinquefasciatus after 12h 3.125 5.0±0.28 100.40 (36.18– 175.94) 217.39 (54.32– 413.03) 19.41 6.25 10±0.39 12.5 20±0.57 25 31±1.20 50 36±0.21 100 41±0.28 after 24h 3.125 12±0.13 52.805 (23.92– 83.50) 135.48 (55.54– 224.83) 25.756 6.25 16±0.57 12.5 29±0.39 25 47±1.20 50 59±0.92 100 69±0.96 adegree of freedom lcl lower confidence level, ucl upper confidence level a95% confidence level http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 231 http://jad.tums.ac.ir published online: may 27, 2017 fig. 1. gas chromatography–mass spectrometry profile of essential oil obtained from leaves of coccinia grandis discussion several authors have reported different compositions of essential oils obtained from different plant species (govindrajan 2010, senthilkumar and venkatesalu, 2010, zhu et http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 232 http://jad.tums.ac.ir published online: may 27, 2017 al. 2011, 2012, rajkumar et al. 2011, intirach et al. 2012, liu et al. 2012 and senthilkumar et al. 2013). the plants show significant variations, both in terms of number and percentage composition, of different constituents in essential oil and it appears to be the characteristics of a particular plant. the excellent larvicidal activity of the essential oil of co. grandis against three species of mosquitoes could be either due to the major components, i.e. n-tetracosane and eicosane or synergistic action of the major and minor components present in it and is difficult to pin point at this stage. tetracosane is an alkane hydrocarbon and use of hydrocarbons as pesticidal agents is reported (siddique et al. 2004). similarly, there are evidences indicating larvicidal and antimicrobial activities of the other major component, eicosane (akpuaka et al. 2013, manas et al. 2014). the results show significantly improved bioefficacy against one of the mosquito species, an. stephensi compared to earlier report which showed lc50 and lc90 values of 93.3 and 192.6ppm, respectively (as against the 39.41 and 123.24ppm in present study) (rajkumar et al. 2011). similarly, the lc50 and lc90 values against ae. aegypti have been found to be 47.54 and 86.54ppm for mentha piperita, 40.50 and 85.33ppm for zingiber officinale, 115.60 and 193.30ppm for cu. longa and, 148.50 and 325.70ppm for oc. basilicum, respectively (kalaivani et al. 2012) compared to 48.20 and 131.84ppm for co. grandis in the present study. the lc50 and lc90 values of the co. grandis leaf essential oils against larvae of cx. quinquefasciatus were marginally better than essential oils of acorus calamus reported by senthilkumar and venkatesalu (2012). the results are also in agreement with several other previous reports where the major components of essential oils have shown excellent larvicidal or insecticidal activities, eg plectranthus amboinicus leaf essential oil (senthilkumar and venkatesalu 2010), clausena anisata leaf essential oil (govindrajan 2011), feronia limonia leaf essential oil (senthilkumar et al. 2013). similarly, intirach et al. (2012) studied essential oils of six different plant families and demonstrated their larvicidal activity against laboratory colonized an. cracens mosquito. a careful observation of these representative studies indicated that there was no common thread in terms of chemical constituents, in these essential oils. the composition and major and minor components of essential oil are characteristics of particular plant and, at the best may be represented in the other members of same family. the composition and larvicidal activity of essential oil of a plant may vary as a function of age of plant, geographical location and season. the observed variations in the efficacy of essential oils from various plants against different vectors could be due to different chemical compositions and/or synergistic action of major and minor components in them (senthilkumar and venkatesalu 2012). the natural diversity of essential oils in the indigenous plants thus offer good opportunity of developing a cost effective, ecofriendly, region specific and practical strategy for the control of mosquito vectors either independently or as a part of integrated vector management strategy. though there are no reports of insecticide/larvicide resistance in the study area, the same is well documented in african countries for anopheles species against all the approved four classes (organochlorines, pyrethroids, carbamates and organophosphates) of insecticides (kristan et al. 2003). of the four classes, resistance to pyrethroids and its mechanisms in an. gambiae: the most important malarial vector in africa has been extensively studied. it was found out that the insect develops resistance to insecticide either by altering its binding site, by point mutations, or by detoxifying it enzymatically before it reaches the target site (tielong et al. 2014). pyrethroids are the insecticides of choice for http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 226–235 si mohammed et al.: evaluation of larvicidal … 233 http://jad.tums.ac.ir published online: may 27, 2017 mosquito control primarily because of their superior human and environment safety records (adedayo et al. 2012). besides, the use of insecticide mixtures and their periodic rotation, integrated vector management, involving biopesticides/essential oils of plant origins could be the answer for preventing/ delaying development of resistance in mosquitoes (brogdon and allister 1998). the present study is a step forward in the direction, demonstrating the larvicidal potential of essential oil of a locally available plant against three most common mosquito species. the chemical analysis shows a different set of major and minor components in the essential oil than the earlier reported studies which can be gainfully utilized further. conclusion results of this study will be helpful in developing cost effective, ecofriendly, region specific and practical strategy for the control of mosquito-borne diseases. acknowledgements one of the authors (sim) acknowledges the fellowship from ugc, new delhi under its manf (maulana azad national fellowship for minorities) scheme. financial support from ugc, new delhi and dst, new delhi for strengthening the research facilities in the school under the sap-drs and fist programmes, respectively are gratefully acknowledged. the authors declare that there is no conflict of interests. references adedayo oo, emmanuel ti, muyiwa ko, adedapo oa, judith bo, olubunmi ao, taiwo sa (2012) evidence of carbamate resistance in urban populations of anopheles gambiae s.s. parasit vectors. 5: 116. ajay ss (2009) hypoglycemic activity of coccinia indica (cucurbitaceae) leaves. int j pharm tech res. 1(3): 892–893. akpuaka a, ekwenchi mm, dashak da, dildar a (2013) biological activities of characterized isolates of n-hexane extract of azadirachta indica a. juss (neem) leaves. new york sci j. 6: 119–124. amer a, mehlhorn h (2006) “larvicidal effects of various essential oils against aedes, 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resistance measurements. parasit vectors. 7: 54. world health organization (1981) instruction for determining the susceptibility or resistance of mosquito larvae to insecticideas. who-vbc 81. 807: 1–6. world health organization (2009) global programme to eliminate lymphatic filariasis. wkly epidemiol rec. 84: 437–444. world health organization (2014) world malaria report. who, geneva. world health organization (2006) guidelines for the treatment of malaria. who-wc. 770: 12–13. zhu bcr, henderson g, chen f, fei h, laine ra (2001) evaluation of vetiver oil and seven insect-active essential oils against the formosan subterranean termite. j chem ecol. 27(8): 1617–1625. http://jad.tums.ac.ir/ j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 546 http://jad.tums.ac.ir published online: october 04, 2016 original article genetic analysis of aedes aegypti using random amplified polymorphic dna (rapd) markers from dengue outbreaks in pakistan hafiz muhammad ashraf 1, *muhammad kashif zahoor 1, shabab nasir 1, humara naz majeed 2, sarwat zahoor 3 1department of zoology, government college university faisalabad, islamabad, pakistan 2department pharmaceutical and biological chemistry, school of pharmacy, university college london, london, uk 3centre of agricultural biochemistry and biotechnology (cabb), university of agriculture faisalabad, faisalabad, pakistan (received 14 june 2014; accepted 28 july 2015) abstract background: keeping in view the havoc situation of dengue fever in pakistan, the current study was designed to demonstrate the genetic variations, gene flow and rate of migration from lahore and faisalabad. methods: the larvae were collected from both natural and artificial breeding places from each collection site. the adult mosquitoes were collected by means of sweep net and battery-operated aspirator. dna extraction was performed using tne buffer method. ten genelink-a series rapd primers were used for pcr amplification and the data was analyzed through popgene. results: the number of amplification products produced per primer varied from 8-12, ranging from 200 to 2000 bp with an average of 10.0 bands per primer. the percentage of polymorphic loci amplified by each primer varied from 22.5 to 51%. the upgma dendrogram demonstrates two distinct groups from faisalabad and lahore populations. the genetic diversity ranged from 0.260 in faisalabad to 0.294 in lahore with a total heterozygosity of 0.379. the gst value for nine populations within lahore was 0.131 (nm= 3.317), whereas for nine populations in faisalabad gst value was 0.117 (nm= 3.773). the overall genetic variation among eighteen populations showed gst= 0.341 and nm= 1.966. conclusion: the genetic relatedness and nm value show that ae. aegypti populations exhibit intra-population gene flow both in faisalabad and lahore. although, both cities show a distinct pattern of genetic structure; however, few areas from both the cities show genetic similarity. the gene flow and the genetic relatedness in few populations of lahore and faisalabad cities need further investigation. keywords: aedes aegypti, dengue, genetic diversity, rapd markers introduction aedes aegypti and ae. albopictus, mosquitoes are responsible to transmit dengue fever virus which is a major health concern in tropical and subtropical areas (pinheiro and corber 1997, fradin and day 2002, gibbons and vaughn 2002). world health organization (who) reported that about 2.5 billion people (more than 40%) are at risk of dengue fever around the world and about 50 million new cases of dengue would be estimated mated to occur every year (who 2009, 2014). dengue disease is considered endemic in more than 100 countries, with southeast asia as a major affected region (itrat et al. 2008, khan et al. 2010, raheel et al. 2011, fatima et al. 2011, idrees and ashfaq 2012). aedes aegypti originated from africa and became spread to the southeast asia. with the passage of time, it established here and now it has become a major vector of dengue **corresponding author: dr muhammad kashif zzahoor, e-mail: kashif.zahoor@gcuf.edu.pk j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 547 http://jad.tums.ac.ir published online: october 04, 2016 in the region (who 1997, fraga at al. 2003). better road infrastructure and good transportation facilities caused dispersal of ae. aegypti. it can easily find breeding places in human dwelling areas; a calamity of living style of people in the sub-continent. in addition, it can withstand desiccation, which also serves as a factor for its success (raheel et al. 2011, sajid et al. 2011). thus, the spread mosquito and thereby the virus, has led to a resurgence of dengue (cologna et al. 2005). the climatic change and use of insecticides cause appreciable effect on the genetic population structure of ae. aegypti (huber et al. 2002, paupy et al. 2005, scarpassa et al. 2008). this vector has been considered as homogeneous species, however, it shows both morphological and genetic variations (morlais et al. 2003). due to epidemiological importance, various molecular studies have been conducted on ae. aegypti using different dna markers (ayres et al. 2002, gorrochotegui et al. 2002, ravel et al. 2002, ayres et al. 2003, fraga et al. 2003, beebe et al. 2005, herrera et al. 2006, bracco et al. 2007, rasheed et al. 2013). random amplified polymorphic dna (rapd) and rflp markers are most commonly used molecular markers to elucidate the genetic variations in ae. aegypti (severson et al. 1999, 2002, fulton et al. 2001, ayres et al. 2003, julio et al. 2009). unfortunately, the genetic analysis of ae aegypti has been neglected in southeast asia despite of its serious damaging aspect of dengue outbreaks. in pakistan, dengue fever has emerged as havoc for the last few years and has become one of the key public health priorities. from the first diagnosed case in 1994, now the virus has rapidly spread across the country. the dengue fever has shaped a cyclic pattern with high infection rate in the monsoon season and low in winter season. more than 2,000 people were reported with dengue infection in 2010. over 14,000 cases were reported with more than 300 deaths from lahore alone in november 2011, likewise, 11,500 people were affected until september 2012 (gilani 2012, sajid et al. 2012). the latest outbreak resulted in 18,000 cases across the country including significant number of confirmed cases reported from lahore and faisalabad (siddiqui et al. 2009, gilani 2012, sajid et al. 2012). according to who reports, majority of infected people belonged to lahore area in punjab, pakistan (who 2013). to counter this threat and to devise effective control measures, there is essentially a dire need of time to know maximum about dengue and its vector. despite the fact of not significant ecological differences in both cities, more reported cases of dengue reflect the severity of disease in lahore. therefore, it was hypothesized genetic variation might exist in ae. aegypti populations in lahore. thus, the current study was designed to analyze genetically the ae. aegypti populations to demonstrate the genetic variations, gene flow and rate of migration from lahore and faisalabad, punjab using random amplified polymorphic dna (rapd) markers. materials and methods mosquito collection eighteen populations were collected from faisalabad and lahore (fig. 1). the adult mosquitoes were collected with the help of sweep net and battery-operated aspirator (herrel et al. 2001, shortall et al. 2009, florencio et al. 2014). the larvae and pupae of mosquitoes were collected from both natural and artificial breeding places (tree holes, stagnant water, tires, waste material, lawns, homes) by using dipper from each collection site (naeem-ullah et al. 2010, nikookar et al. 2010). the collected samples were preserved in 70% alcohol in labelled vials and stored at 4 °c for dna extraction (zahoor et al. 2013). dna extraction dna was extracted from individual mosj arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 548 http://jad.tums.ac.ir published online: october 04, 2016 quitoes from each sample using salt extraction method. the mosquitoes were homogenized in 400 μl of tne buffer (tris-nacledta), and then 100 µ l of 20 µ g/µ l of proteinase-k and 40 µ l of 20% sodium dodecyl sulphate (sds) were added. the homogenates were incubated at 55 ˚c for one hour, 300 μl of 5m nacl was added and vortexed. the mixture was centrifuged at 15,000 rpm for 10 min and the supernatant was shifted to separate eppendorf tube. dna was precipitated by adding of 300-400 µ l isopropanol or ice-cold 100% ethanol, kept at -21 ˚c for 1 hour, and then centrifuged at 15,000 rpm for 10 min. dna pellet was washed with chilled 70% ethanol, air-dried and re-suspended in 50 µ l of sterile water (d3h2o). the dna concentration was estimated by measuring optical density (od) at 260nm and its quality was checked on 1% agarose gel electrophoresis (ashraf 2013, zahoor et al. 2013, bibi 2015). rapd-pcr analysis ten gene link-a series rapd primers were used for pcr amplification (table 4). each pcr reaction was carried out in a final volume 25 µ l, approximately 100 mg of genomic dna, 3 mm of mgcl2, 20 pmol of primer, 2.5 µ l buffer, 1.0 units of taq dna polymerase and 0.3 mm of each dntps. pcr program comprised of 35 cycles with initial denaturing of dna at 94 ˚c for 5 min, denaturation at 94 ˚c for 1 min, primer annealing at 36 ˚c for 1.5 min, extension at 72 ˚c for 1.5 min, final extension at 72 ˚c for 10 min and then hold at 4 ˚c until the tubes were removed. the pcr products were run on 1.6% agarose gel electrophoresis at 80 voltages for one hour (ashraf 2013, zahoor et al. 2013). statistical analysis of data the fingerprints examined under ultra violet transilluminator and photographed using (syngene) gel documentation system. the size of amplified bands or loci was compared against the dna marker. all the fragments scored as present (1) or absent (0) for each sample. ambiguous bands were not scored. the bands counted by starting from top to the bottom in all lanes. the rapd markers were analyzed using the following assumptions: (1) the rapd alleles follow the mendelian pattern of segregation, (2) monomorphic fragments are homologous (co-migrate), (3) loci are independent from each other and (4) and mosquito populations are in hardy-weinberg equilibrium as described by ayres et al. 2003. a dendrogram was constructed using upgma method (unweight pair-group mean analysis) (nei 1978). effective migration rates (nm) were estimated based on inbreeding indices (gst), where nm= 0.5(1gst)/ gst (ayres et al. 2002). the genetic analysis was performed through popgene (version 1.32) software. results aedes aegypti populations were analyzed by rapd-pcr using 10 oligonucleotide primers (genelink, table 4). four primers were further selected which produced distinct and easily detectable bands (table 1). the reproducible bands over repeated pcr reactions were used for analysis. the number of fragments per primer varied from 8–12, ranging from 200 to 2000 bp size, with an average of 10.0 bands per primer (table 1 and fig. 1). various amplified fragments differentiated the populations of ae. aegypti from lahore and faisalabad (fig. 2). the percentage of polymorphic loci amplified by each primer varied from 22.5 to 51% (fig. 3). total 55 polymorphic loci were scored in all samples, with an average 5.5 polymorphic loci per primer (data not shown). four primers were selected for further due to their reproducible and distinct banding pattern (table 2). the heterozygosity, gene flow and the variation j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 549 http://jad.tums.ac.ir published online: october 04, 2016 among eighteen populations were estimated using nei’s analysis. the genetic diversity ranged from 0.260 in faisalabad to 0.294 in lahore with a total heterozygosity of 0.379. the gst value for nine populations within lahore was 0.131 (nm= 3.317), whereas for nine populations in faisalabad gst value was 0.117 (nm= 3.773), respectively. the overall genetic variation among eighteen populations showed gst= 0.341 and no. of migrants nm= 1.966 (table 3). cluster analysis the upgma dendrogram based on similarity matrix of nei’s genetic distances demonstrates two distinct groups from faisalabad and lahore populations (fig. 4). one group (a) shows seven populations from main faisalabad city, ie. nazim abad, gulfishan colony, university of agriculture faisalabad (uaf) jail road, government college university faisalabad (gcuf), awami colony, narwala road, national institute for agriculture and biology (niab). the second group (b) represents four populations from lahore, i.e. govt. muhammadan anglo oriental college (mao college), qila gujersing, uc69 and uc 81(a). there are three subgroups in group c, comprising of two populations from faisalabad (c2, rescue office jhang road and aari jhang road) and four populations from lahore (c3, uc 73(a) and ghawal mandi, and and c4, mazang chungi and uc73 (b), respectively). the bise lawrence road shows that both the groups a and b were originated from lahore. the subgroup a5 and a4 comprising of uaf jail road, gulfishan colony and gcuf found to be closely related population groups in faisalabad the subgroup a3 consists of populations from narwala road and niab. the genetic similarity could be speculated due to the transportation of mosquitoes in these areas. the subgroup b2 consists of closely related populations from gujersing area and mao college. this subgroup shows more genetic similarity to the mosquito population from uc69 site. the subgroup b1 represents relatedness to the subgroup b2, thereby shows more genetic similarity to gujersing area and mao college mosquito populations. within the main group c, there are three subgroups converged at c1 (c2, c3 and c4). the subgroup c3 shows that uc 73(a) is closely related to the mosquitoes from gawal mandi area. similarly, the subgroup c4 represents that mazang chungi and uc 73(b) are more closely related to each other. interestingly, the main group c also contains populations from faisalabad. the subgroup c1 consists of areas from the main high way of jhang road, rescue office and aari. the collected mosquito populations show a genetic similarity to lahore populations. the rapd primer (a-04) produced fragments, which showed genetic variation in ae. aegypti populations from both lahore and faisalabad. the genetic relatedness in faisalabad populations and the nm value for an estimate of gene flow shows that the dengue vector ae. aegypti populations exhibit gene flow among different populations. similarly, ae. aegypti populations from lahore also show intra-population gene flow (table 3). although, both cities present a distinct pattern of genetic structure, however, few areas from both the cities show that ae. aegypti populations are genetically related to each other (fig. 4). j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 550 http://jad.tums.ac.ir published online: october 04, 2016 table 1. localities from lahore and faisalabad selected for sampling of ae. aegypti populations sr. no. samples from faisalabad district sr. no. samples from lahore district sampling areas code sampling areas code 1 rescue office jhang road f1 10 uc 73 (a) l1 2 awami colony tariq road f2 11 uc 81 (a) l2 3 aari jhang road f3 12 ghawal mandi l3 4 gulfishan colony f4 13 mazang chungi area l4 5 nazimabad f5 14 qila gujersing l5 6 narwala road f6 15 bise lawerence road l6 7 gcuf f7 16 uc69 l7 8 uaf jail road f8 17 uc 73 (b) l8 9 niab f9 18 mao college l9 table 2. primers used in rapd analysis showing size and number of amplified fragments obtained (no.) from ae. aegypti populations from lahore and faisalabad sr. no. primers nucleotide sequence size (bp) no. 1 a-03 5’agtcagccac 3’ 200–1500 12 2 a-04 5’aatcgggctg 3’ 350–1850 11 3 a-06 5’ggtccctgac 3’ 375–1200 9 4 a-18 5’aggtgaccgt 3’ 210–1300 8 table 3. summary of nei’s analysis of gene diversity among between aedes aegypti populations from lahore and faisalabad through rapd markers populations genetic variation gst gene flow nm heterozygosity ht lahore 0.131 3.317 0.294 faisalabad 0.117 3.773 0.260 lahore, faisalabad 0.341 1.966 0.379 table 4. rapd primer sequence (genelink) and their codes sr. no. primer name (code) primer sequence gc contents 1 gl decamer a-03 (a-03) agtcagccac 60% 2 gl decamer a-04 (a-04) aatcgggctg 60% 3 gl decamer a-05 (a-05) aggggtcttg 60% 4 gl decamer a-06 (a-06) ggtccctgac 70% 5 gl decamer a-08 (a-08) gtgacgtagg 60% 6 gl decamer a-10 (a-10) gtgatcgcag 60% 7 gl decamer a-11 (a-11) caatcgccgt 60% 8 gl decamer a-12 (a-12) tcggcgatag 60% 9 gl decamer a-18 (a-18) aggtgaccgt 60% 10 gl decamer a-19 (a-19) caaacgtcgg 60% j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 551 http://jad.tums.ac.ir published online: october 04, 2016 fig. 1. map showing different collection sites from lahore and faisalabad (faisalabad: 73°74 e and 30°31.5 n, lahore: 74°01-74°39′ e and 31°15′-31°45′ n) fig. 2. amplification profile of aedes aegypti populations with primers a-04 from lahore and faisalabad. (l1-9: lahore samples, f1-9: faisalabad samples, lane m= dna marker (size 1kb) j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 552 http://jad.tums.ac.ir published online: october 04, 2016 fig. 3. percentage of polymorphic loci amplified by each rapd primer fig. 4. dendrogram based on nei’s genetic distances among aedes aegypti populations from lahore and faisalabad. (scale bar: branch length, *f/l: faisalabad/lahore) f1: rescue office jhang road l1: uc 73 (a) f2: awami colony tariq road l2: uc 81 (a) f3: aari jhang road l3: ghawal mandi f4: gulfishan colony l4: mazang chungi area f5: nazimabad l5: qila gujersing f6: narwala road l6: bise lawerence road f7: gcuf l7: uc69 f8: uaf jail road l8: uc 73 (b) f9: niab l9: mao college j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 553 http://jad.tums.ac.ir published online: october 04, 2016 discussion aedes aegypti is one of the most important mosquito vectors of viral agents in world, due to its role in transmitting chikungunya virus (chikv) and dengue virus (denv) (thavara et al. 2001, bosio et al. 2005). the genetic diversity has been reported in ae. aegypti populations (gorrochotegui-escalante et al. 2000, souza et al. 2001, ayres et al. 2002, franco et al. 2002). many dna techniques such as rapd, microsatellites and mitochondrial dna markers have been widely used to detect variation in geographically and genetically isolated populations (ballinger-crabtree et al. 1992, apostol et al. 1994, ayres et al. 2003, paduan et al. 2006). in pakistan, few studies have been conducted on barcoding of mosquitoes through mitochondrial coi gene and genetic structure of ae. aegypti populations using microsatellite markers (ashfaq et al. 2013, rasheed et al. 2013). however, still genetic analysis of ae. aegypti populations has not been performed yet, particularly in the dengue outbreaks. thus, the present study reinforced the significance of genetic analysis of ae. aegypti populations in dengue affected areas in pakistan. the level of genetic diversity was revealed among various mosquito species and multiple haplotypes were reported by ashfaq et al. (2013) in pakistan. however, they mainly focused on biodiversity and distribution of mosquitoes in kpk and punjab provinces. furthermore, rasheed et al. 2013 used 13 microsatellite markers and found low genetic diversity in mosquitoes collected from 13 different cities. low level of genetic diversity with less differentiation was revealed and considered due to better road infrastructure. although present results are consistent to their resultsو however, based on the amplified fragments in our findings, it is suggested that ae. aegypti populations are genetically more diverse as previously reported in pakistan. in addition, the genetic similarity in populations at the border area of faisalabad to few of lahore populations supports the idea of transportations (rasheed et al. 2013). the heterozygosity in ae. aegypti populations from faisalabad and lahore (ht. 0.311) found similar to the results obtained from brazil using 27 rapd loci (ht= 0.390) (ayres et al. 2003) and to the average heterozygosity of 21 loci (ht= 0.388) in brazil, 57 loci in puerto rico (ht= 0.354) (apostol et al. 1996) and in 131 loci of 20 ae. aegypti populations from mexico through rapd markers (franco et al. 2002). gorrochoteguiescalante et al. (2000) also distinguished populations of ae. aegypti from ten cities of the mexican northeast coast, using 60 rapd polymorphic loci. the gene flow and genetic variations were observed within populations separated within a range of 90 to 250 km. while, lahore-faisalabad ae. aegypti populations correspond to ~100 km distance. thus, the polymorphic pattern in our findings showed genetic variations by distance (55 out of 70 loci, data not shown). the genetic variation (gst = 0.341, nm= 0.966) and polymorphism (51%) found in the present study were consistent to those reported in argentina (gst = 0.249, nm= 0.75) (souza et al. 2001) and brazil (gst = 0.317, nm= 0.54, hs= 0.274) (ayres et al. 2003). santos et al. (2011) found high level of polymorphism and distinguished 52 markers in the four populations through rapd analysis, ranging in size from 300 to 2072 bp. their percentage of polymorphic loci varied from 82.69 to 94.23. in contrast, we amplified loci ranged from 200 to 2000 bp with 22.5 to 51 percentage of polymorphism, which is closer to the genetic variability and 50% similarity in ae. aegypti populations as demonstrated by hiragi et al. (2009) using rapd markers. however, current findings showed maximum similarity (0.8846) and genetic distance (0.045) among ae. aegypti j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 554 http://jad.tums.ac.ir published online: october 04, 2016 populations (data not shown). a population differentiation from faisalabad (gst = 0.131) and lahore (gst = 0.117) with higher degree of gene flow (nm= 3.317 faisalabad, nm= 3.773 lahore) showed similarity to a low population differentiation (gst = 0.208) with higher degree of gene flow (nm= 1.90) as described by paduan et al. (2006) in brazil indicating both intraand inter-population genetic variability. current findings agree to the study of de sousa et al. (2001). they successfully used rapd markers to estimate the level of polymorphism and genetic relatedness in five populations of ae. aegypti from argentina and puerto rico. however, contrary to the present study, heterozygosity and genetic distance values were found significant at all loci suggesting that the population from puerto rico was different from argentina populations. subsequently, de patarro et al. (2013) assessed the genetic variability through rapd markers in five ae. aegypti populations from brazil and usa. a total of 165 polymorphic dna loci were generated with six primers compared to 55 loci using ten primers in the present study. the mean value of inter-population genetic diversity (gst) ranged from 0.044 to 0.289 suggesting that genetic variation existed in ae. aegypti populations. similarly, we found genetic diversity (gst) ranged from 0.260 in faisalabad to 0.294 in lahore. ocampo and wesson (2004) found genetic variation in ae. aegypti populations in colombia. the genetic heterogeneity ranged from 19% to 60% revealed through rapd markers closer to our findings. in addition, the genetic distance and migration rate (nm) demonstrated a moderate genetic differentiation among ae. aegypti populations which is consistent to the current findings of genetic differentiation in ae. aegypti populations existed in faisalabad and lahore. despite of rapd, other dna markers are also being used to analyse genetically mosquitoes. paupy et al. (2012) examined genetic structure and geographical origin of ae. aegypti in bolivia and found polymorphism at nine microsatellite loci and in two mitochondrial dna regions (coi and nd4). similar to the present work, two genetic lineages were obtained after genetic analysis of mtdna sequences. one lineage was restricted to rural localities and the other was found closely related to african ae. aegypti populations. similarly, caldera et al. (2013) genetically analyzed two populations of ae. aegypti from dengue outbreak areas in colombia using mitochondrial gene nd4. the genetic differentiation with a limited gene flow (nm= 1.40) indicated that both the populations of ae. aegypti are genetically distinct. hlaing et al. (2010) also found genetic heterogeneity in ae. aegypti populations using microsatellite markers in myanmar, cambodia, thailand, sri lanka and nigeria. the genetic variation even in a distance of 500 m and genetic similarity in distant locations due to dispersal were observed. in parallel, costa-da-silva (2005) also found a distinct genetic pattern in ae. aegypti populations in peru using mitochondrial dna. furthermore, yáñez et al. 2013 tested 51 samples of ae. aegypti populations from peru using mitochondrial nd4 gene and identified five haplotypes of ae. aegypti grouped in two lineages. this variability occurred mainly due to migration of this vector and partly through human activity. thus, transportation has been considered as a main source of spread of mosquito borne diseases, however, other factors like use of insecticides and elimination of larval habitats in and around dwellings had a demonstrable impact on gene flow and genetic structure of ae. aegypti populations (wallis et al. 1984, failloux et al. 1995, paupy et al. 2000, lerdthusnee and chareonviriyaphap 2002) which needs to be further investigated on larger scale using gene specific molecular markers. j arthropod-borne dis, december 2016, 10(4): 546–559 hm ashraf et al.: genetic analysis of … 555 http://jad.tums.ac.ir published online: october 04, 2016 conclusion the genetic relatedness and nm (number of migrants) value show that ae. aegypti populations exhibit intra-population gene flow between faisalabad and lahore. although, both cities show a distinct pattern of genetic structure, however, few areas from both the cities show genetic similarity. it is also concluded that ae. aegypti populations are genetically more diverse as previously reported in pakistan. acknowledgements the facilities provided by department of zoology, government college university faisalabad (gcuf) and the financial support from hec project are highly acknowledged to conduct this research work. the authors are also thankful 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iqbal a, awan fs (2013) molecular characterization of scarab beetles (scarabaeidae: coleoptera) using rapd markers. pak j life soc sci. 11: 238–243. j arthropod-borne dis, december 2016, 10(4): 454–461 s bazrafkan et al.: discrimination of … 454 http://jad.tums.ac.ir published online: october 04, 2016 original article discrimination of paederus fuscipes and paederus littoralis by mtdna-coi pcr-rflp sahar bazrafkan 1, hassan vatandoost 1, abbas heydari 1, hassan bakhshi 1, somayeh panahi-moghadam 1, saedeh hashemi-aghdam 1, fatemeh mohtarami 1, abbas rahimiforoushan 2, sinan anlaş 3, *mansoreh shayeghi 1, *mohammad ali oshaghi 1, seyed mohammad abtahi 4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2isfahan university of medical sciences, isfahan, iran 3celal bayar university alaşehir, vocational school, department of entomology, alaşehir, manisa, turkey 4epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 26 oct 2014; accepted 11 jan 2015) abstract background: linear dermatitis is endemic in iran where most cases occur in the caspian sea coast and fars province. the disease is caused by beetles of the genus paederus which are active from early spring to beginning of autumn although its incidence rises from may to august. the classic taxonomy of paederus spp. is based on the male genitalia that is very complex and needs expertise. in this study, we report a dna-based method to discriminate paederus fuscipes and paederus littoralis (=syn: p. lenkoranus, p. ilsae). methods: type specimens were collected from north and south of iran. molecular typing of the species was performed using restriction fragment length polymorphism (rflp) analysis of polymerase chain reaction (pcr)-amplified fragments of mtdna-coi. results: sequence analyses of the data obtained in this study showed significant dna polymorphisms. there were 89 substitutions between coi sequences of the two species. the mtdna-coi fragment comprises several useful species-specific restriction sites comprising haeiii that could result in distinctively different species-specific pcr– rflp profiles. the haeiii enzyme cuts the 872 bp pcr amplicon of p. littoralis into 737 and 100 bp and two small nonvisible bands whereas it does not cut p. fuscipes amplicon into fragments. conclusion: this study demonstrates that molecular typing is useful method and allows one to differentiate between two species and is recommended for discrimination of other paederus species, which morphologically are indistinguishable or very difficult to be distinguished. keywords: paederus, linear dermatitis, mtdnacoi, pcr-rflp, molecular typing introduction the genus paederus fabricius (staphylinidae: coleoptera) is represented by 622 species worldwide and 85 species and subspecies in the palaearctic region. the hemolymph of some species within the genus paederus is nuisance as, once released, it causes linear dermatitis and conjunctivitis in humans. the symptoms are due to a toxic amide substance, which has been named pederin (pavan and bo 1953) and makes up approximately 0.025% of an insect's weight (for p. fuscipes). most cases of linear dermatitis in iran occur in the caspian sea shoreline and fars in south of iran (nikbakhtzadeh and tirgari 2008). systematics of paederus beetles at species *corresponding author: dr mansoureh shayeghi, email: mansorehshayeghi@yahoo.com, dr mohammad ali oshaghi, email: moshaghi@sina.tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 454–461 s bazrafkan et al.: discrimination of … 455 http://jad.tums.ac.ir published online: october 04, 2016 level is rather difficult and is based on the morphology of male primary and secondary sexual characters (coiffait 1982). this makes them difficult to identify and limits their study and management. consequently, this has made a very complicated history for paederus taxonomy and has changed it dramatically and some species are treated as synonyms of each other and or downgraded to a single subspecies/species (nikbakhtzadeh et al. 2012 and references herein). for example p. lenkoranus scheerpeltz (scheerpeltz 1957) p. littoralis ilsae (bernhauer 1932) and p. ilsae (coiffait 1982) have been recently considered synonymous (nikbakhtzade 2012). paederus fuscipes fuscipes curtis (1826) was formerly known by the synonymous names of p. iliensis coiffait (1970) and p. kalalovae roubal (1932). these older species are downgraded to a single subspecies of p. fuscipes in the current systematics of staphylinidae. previous studies on the fauna, geographical distribution, ecology and medical importance of paederus beetles in iran revealed presence of 14 paederus species or subspecies in the country (nikbakhtzadeh et al. 2012). however, due to difficulties in paederus species discrimination, different lists of species have been reported for an identical region. in north of iran, for example, janbaksh and ardalan (1977) reported three species of p. fuscipes, p. pietschmanni (synonym of p. mesopotamicus), and p. spectabilis in mazandaran province at the caspian sea coast. majidi-shad et al. (1989) reported three species of p. fuscipes, p. riparius, and p. littoralis from the same region. later on, p. fuscipes, p. kalalovae and p. balcanicus were reported from the province by nikbakhtzadeh and tirgari (2008). finally, nikbakhtzadeh et al. (2012) reported three species of p. fuscipes, p. balachowskyi (synonym of p. mesopotamicus), and p. balcanicus from the same area. in southern part, nikbakhtzadeh (2002, 2008) reported p. ilsae and p. iliensis coiffait from fars province. in 2012, p. littoralis ilsae bernhauer (=syn: p. lenkoranus scheerpeltz, p. ilsae) and p. fuscipes fuscipes curtis (=syn: p. iliensis coiffait, p. kalalovae roubal) were reported from that area. recent advances in dna-based technology have made a wide range of molecular characteristics and markers available for taxonomic and systematic studies of insects. one region of the insect genome including beetles that has received particular attention is the mitochondrial dna (mtdna). mitochondrial dna with a fast mutation rate has significant variation in sequences between species. a 658 bp region (the folmer region) of the mitochondrial cytochrome c oxidase subunit i (coi) gene was proposed as a potential barcode (hebert et al. 2003). the mtdna genes have many advantages including a relatively fast mutation rate, easy to use and known pcr primers. the discrepancy and inconsistency in the number of species in iran plus difficulties in systematics of paederus species encouraged us to test sequence variation of mtdna-coi, to introduce a molecular marker to discriminate p. fuscipes and p. littoralis, the two sympatric and common species in the mediterranean basin including southern part of iran. materials and methods paederus specimens and morphological identification adult specimens were collected by aspirator on rice plants and weeds in early morning or afternoon and under clays at hot hours of day time. collection of p. fuscipes and p. littoralis were performed mostly in rice fields in various locations of mazanderan and fars province during the growing season from may to august 2011. the specimens were preserved in 70% ethanol and were sent to the insect molecular biology laboratory of the school of public health, tehran univerj arthropod-borne dis, december 2016, 10(4): 454–461 s bazrafkan et al.: discrimination of … 456 http://jad.tums.ac.ir published online: october 04, 2016 sity of medical sciences, iran for molecular characterization. the pictorial key of coiffait (1982) was used to identify the specimens to genus level. the specimens were then identified to species level based on the habits and morphology of male primary and secondary sexual characters by the turkish expert, s. anlaş. dna extraction paederus specimen representative different populations of both species were selected for dna analysis. total genomic dna was extracted from total body of individual samples using dneasy® blood and tissue kit (qiagen, hilden, germany), according to the manufacturer’s instructions. the specimens were frozen prior to dna extraction and then grinded in the kit supplied buffer and extraction followed according to the manufacturer's directions. dna amplification the coi (cytochrome oxidase subunit 1) region of mtdna gene (mitochondrial) was amplified with the primer pair c1-j-2183 (5′caacatttattttgattttttgg-3′) and tl-2-n-3014 (5′ccattgcatctgccatatta-3′). these primers have already been introduced by simon et al. (1994) and used for some colleopteran. pcr amplification reaction conditions were: 5 μl 10× pcr-buffer, 120 μm of each dntps, 50 pmol of each primer, 2 μl (about 100 ng) of template dna, and 2.5 u of taq polymerase (sinaclone, iran) in a 25 μl reaction volume. pcr amplification was performed with an eppendorf thermal cycler (germany). the cycling parameters were: 2 min initial denaturation at 94 °c followed by 5 cycles of 30 sec at 94 °c, annealing at 45 °c for 40 sec and extension at 72 °c for 1 min and 35 cycles of 94 °c at 30 sec, annealing at 51 °c for 40 sec and extension at 72 °c for 1 min. the final extension step was 72 °c for 10 min (www.dnabarcodes2011.org). double distilled water was used as negative and wellcharacterized dna samples were used as positive controls. sequencing and pcr-rflp the pcr products of coi fragment were purified from gels by using a gel purification kit, and subjected to sequencing. sequencing was performed using an abi 3730 sequencer machine by bioneer (south korea). the resultant sequences were checked to correct ambiguities. homologies with the available sequence data in genbank was checked by using basic local alignment search tool (blast) analysis software (www.ncbi.nlm. nih.gov/blast). the coi sequences obtained in this study was checked to obtain its physical map and to select restriction enzymes by using the nebcutter program (vincze et al. 2003). restriction enzymes were selected based on their positions (beginning, middle, and last part of pcr product), profiles, costs, and availabilities in the market. digestion of pcr products was performed in 25 μl of a solution containing 15 μl of pcr product mixed with 2.5 μl of enzyme buffers and 5 units of haeiii restriction enzyme overlaid with two drops of mineral oil. the mixture was incubated at the temperature recommended by the enzyme supplier. an aliquot (14 μl) of the digestion product was mixed with 6 μl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol), loaded on to a 1% agarose gel, and subjected to electrophoresis. gels were stained with ethidium bromide (2 mg/ ml) and the rflp profiles were visualized under ultraviolet light. results species identification and pcr-rflp in this study, a total of 154 adult paederus specimens were collected from the two iranian provinces. the p. littoralis specimens and a subset of the morphologically-identified p. fuscipes were subjected to mtdnaj arthropod-borne dis, december 2016, 10(4): 454–461 s bazrafkan et al.: discrimination of … 457 http://jad.tums.ac.ir published online: october 04, 2016 coi pcr amplification followed by pcrdirect sequencing. sequencing was performed for both strands and the consensus data were submitted to genbank with accession numbers (ans): kf724713, kf724714, km07 1620, km071621, km071622, km071623, km071624, km071625, km071626, km07 1627, km071628 and km071629. there were minor intra-species sequence variations between individuals of both species; however, there were considerable dna polymorphisms between the two species. subsequent sequence analysis confirmed identical 872 bp pcr amplicons for both species. an alignment of the 805 bp of the coi region from these sequences is shown in fig. 1. the amount of sequence similarity between the two species was 88% and 98% at dna and amino acid level respectively. subsequently, a blast search on the sequences revealed that there were corresponding coi sequences for p. fuscipes and p. littoralis species in genbank respectively with 96% and 98% maximum identity. sequence analysis of the coi fragments of both species revealed a number restriction sites for discrimination of the species. among them, an appropriate restriction site for haeiii (gg↓cc) gave two distinct profiles of 737/100/23/12 bp fragments for p. littoralis and an intact 872 bp for p. fuscipes (fig. 2). of course, for p. littoralis, only the two 737 and 100 bp bands were visible because the small ones (23 and 12 bp) were not visible in the gel. p.fuscipes ttacctggatttggaataatttctcatattatctcttacagaagtggaaaacaagaaact 60 p.littoralis ttaccaggatttggaataatttctcatatcatttcttacagaagagggaaacaagaaact ***** *********************** ** *********** ** ************ 60 p.fuscipes tttggagcaattgggataatttatgctatgcttgcaattggtttattaggttttattgta 120 p.littoralis tttggggcaataggaataatttatgctatattagcaattggtttattaggttttattgtt ***** ***** ** ************** * ************************** 120 p.fuscipes tgagctcatcatatatttactgtcggaatggacattgatactcgagcttactttacatca 180 p.littoralis tgagcccatcatatatttacagtaggtatagatattgatacacgagcttattttacctca ***** ************** ** ** ** ** ******** ******** ***** *** 180 p.fuscipes gccacaatagtaattgctgttccaactggaattaaggtttttagatgaataggaacaatt 240 p.littoralis gcaactatagtaattgctgtacctacaggaattaaagtatttagttgaatagcaacaatt ** ** ************** ** ** ******** ** ***** ******* ******* 240 p.fuscipes tatggtggaaatttaaattttagcccaccaataatctgaagtttagggtttgtattttta 300 p.littoralis tatggaggaaatttaaattttagacccccaataatttgaagattaggttttgtattttta ***** ***************** ** ******** ***** ***** ************ 300 p.fuscipes tttactgtcggaggattaactggagtaattttagctaattcatcaattgatattgtatta 360 p.littoralis tttactgtaggaggattaacaggagtgattttagctaattcatcaattgatattgtttta ******** *********** ***** ***************************** *** 360 p.fuscipes catgacacatattatgttgtagctcattttcattatgtcttatcaataggggcagtattt 420 p.littoralis catgatacttactatgtagtagctcactttcactatgttttatcaataggagctgttttt ***** ** ** ***** ******** ***** ***** *********** ** ** *** 420 p.fuscipes gctattatagcagggttagtacaatgatacccaatatttattgggttaatattaaacgaa 480 p.littoralis gctattatagcaggattagtgcaatgatttccaatattcattggattaatattaaatgaa ************** ***** ******* ******** ***** *********** *** 480 p.fuscipes aaatatttaaaaattcaatttttaattatatttattggggtaaatttaacttttttccct 540 p.littoralis aaatacttaaaaatccaatttttaattatatttattggggtaaatttaacattttttcct ***** ******** *********************************** ***** *** 540 p.fuscipes caacattttttaggtttatcaggaataccacgtcgatattcagattacccagatgcttac 600 p.littoralis caacattttttaggattatcaggaatacctcgtcgatactcagattaccctgatgcttat ************** ************** ******** *********** ******** 600 p.fuscipes acaatatgaaatgtaatttcatctattggatcaataatttcatttattggaattatattc 660 p.littoralis acaatatggaacgtaatttcatctattggatcaataatttcatttattggaattatattc ******** ** ************************************************ 660 p.fuscipes tttttatgaattatttgagaaagatttatttcaatacgaaaaattattggagctccaatc 720 p.littoralis tttttatgaattatttgagaaagatttatttctatacgaaaaattattggggccccaatt ******************************** ***************** ** ***** 720 j arthropod-borne dis, december 2016, 10(4): 454–461 s bazrafkan et al.: discrimination of … 458 http://jad.tums.ac.ir published online: october 04, 2016 p.fuscipes ccaccaacagcattagaatgaatgcattcataccccccatcagaacacacctattctgag 780 p.littoralis cccccaactgctttagaatggatacattcttatccaccctcagagcatacttactcagaa ** ***** ** ******** ** ***** ** ** ** ***** ** ** ** ** ** 780 p.fuscipes ttgccttttataacaattaagttct 805 p.littoralis ttaccttttataacaattaaattct ** ***************** **** 805 fig. 1. alignment of 752 bp of mtdna-coi sequences of paederus fuscipes and paederus littoralis. stars show the conserved position and gaps or dots indicate substitutions. the haeiii restriction site (ggcc) for p. littoralis is shown at position of 705–708. fig. 2. digestion profiles of mtdna-coi pcr products (872 bp) with haeiii in paederus fuscipes and paederus littoralis. lane 1 (872 bp) for p. fuscipes and lane 2 (737 and 100 bp) for p. littoralis, m: 100 bp molecular weight marker (sinaclon, iran). discussion to our knowledge, this is the first molecular investigation aiming at discrimination of paederus fuscipes and paederus littoralis by mtdna-coi in literature. according to the present results, p. fuscipes and p. littoralis specimens can be easily distinguished by mtdna-coi pcr-restriction fragment length polymorphism (rflp) using haeiii. this assay could end up the discrepancy, inconsistency, and difficulties present in systematics of paederus species, allowing studies regarding their distribution, biology, and behavior to proceed, as well as better understanding of their role in linear dermatitis and antiviral and antitumor activities of the pederin presents in their hemolymph. correct species identification is crucial in entomological surveys and application of control measures for the species that are morphologically indistinguishable or difficult to be distinguished. targeting correct species is particularly important where more than one species live sympatric which is the case for p. littoralis and p. fucipes in south of iran (nikbakhtzadeh et al. 2012). paederus littoralis has been collected from different parts of fars province (nikbakhtzadeh et al. 2012). on the other hand, p. fuscipes has a worldwide geographical distribution and has been reported from different countries in africa, asia and europe (coiffait 1982, smetana 2004). this species is reported from central and southern iran and is the most frequent species in north of iran (janbakhsh and ardalan 1977, tirgari and nikbakhtzadeh 2002, nikbakhtzadeh and tirgari 2008, anlas and newton 2010, nikbakhtzadeh et al. 2012). nowadays many researchers have been focused on medicinal insects such as paederus species as well as horseflies, blister beetles and american cockroaches that have been well known due to their effects against various pathogens such as viruses and bacteria as well as diseases such as thrombosis and cancer (richter et al. 1997, witczak et al. 2012, mosey et al. 2012). pederin of paedefig. 1. continued… j arthropod-borne dis, december 2016, 10(4): 454–461 s bazrafkan et al.: discrimination of … 459 http://jad.tums.ac.ir published online: october 04, 2016 rus species has been used in cancer studies in recent years. pederin and its common substructure accounts for similar antiviral and antitumor activities, cytotoxicity and disruption of dna metabolism that are mainly based on inhibition of eukaryotic protein biosynthesis (piel 2002). the production of pederin relies on the activities of endosymbiont bacteria (pseudomonas species) within paederus (kellner 2002, piel et al. 2004). the manufacture of pederin is largely confined to adult female beetles which protects the beetles against predators (kellner and dettner 1996, kellner 2002). larvae and males only store pederin acquired maternally (i.e., through eggs) or by ingestion (piel 2002). the species commonly causing linear dermatitis are beetles of p. fuscipes in asia (anlaş and çevik 2008). also amount of pederin and its related compounds might be variable in different species (kellner and dettner 1996). therefore correct species identification is essential to study the biological effect of the molecule. in this study we used the coi gene of mtdna genome, which is a known powerful molecular marker for molecular identification of various organisms. however, in addition to coi, other interest marker such as its2 (rdna), wingless, topoisomerase i, and 28s might be useful in order to develop a molecular key for discrimination of paederus species. there are considerable reports on using coi gene in species diagnosis, population genetics, and systematics of beetles in the literature (e.g. andreev et al. 1998, gallego and galián 2001, becerra 2004, chatzimanolis et al. 2010, germain et al. 2013). also there are a few available coi sequence entries of p. littoralis, p. ruficollis, p. riparius, p. moesopotamicus, and p. fuscipes in genbank database. species discrimination can be achieved by using rflp profiles on pcr products or designing species specific primers to produce species specific product. however, pcr-rflp method is reasonably cheap, fast, and user friendly and has been used frequently in many laboratories involving species identification including insects and the microbes they transmit (clark et al. 2001, mukabana et al. 2002, armstrong and ball 2005, oshaghi et al 2006a and 2006b, greenstone 2006, oshaghi et al 2008, 2009, 2010, kato et al. 2010, oshaghi et al 2011). conclusion further molecular studies using type or morphologically well-known species are now required to verify the species composition of paederus in iran and other countries in europe, asia, and africa. by performing molecular typing of paederus species in the future, we expect that the paederus fauna of iran and other countries will be identified more accurately. acknowledgements this work was supported financially by tehran university of medical sciences, iran. references andreev d, breilid h, kirkendall l, brun lo, ffrench-constantm rh (1998) lack of nucleotide variability in a beetle pest with extreme inbreeding. insect molec biol. 7: 197–200. anlaş s, çevik ie (2008) faunistic studies on pederinae (coleoptera: staphylinidae) in manisa province, turkey. mun ent zool. 3: 665–674. anlaş s, newton af (2010) distributional checklist of the staphylinidae 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wings of phlebotomus sergenti populations in central morocco fatima-zahra abou-elaaz1, *denis sereno2, oumnia himmi1, mohamed ghamizi3, *souad guernaoui4 1geophysics, natural patrimony and green chemistry research centre (geopac), geo-biodiversity and natural patrimony laboratory (geobiol), scientific institute, mohammed v university in rabat, morocco 2intertryp, parasite infectiology and public health research group, mivegec, ird, montpellier university, montpellier, france 3water, bidiversity and climate change laboratory, fssm, museum of natural history of marrakesh, cadi ayyad university, marrakesh, morocco 4biotechnology, conservation and valorisation of natural resources laboratory, faculty of sciences dhar el mahraz, sidi mohamed ben abdellah university, fez, morocco *corresponding authors: prof souad guernaoui, e-mail: souad.guernaoui@usmba.ac.ma; dr denis sereno, e-mail: denis.sereno@ird.fr (received 27 sep 2022; accepted 14 feb 2023) abstract background: phlebotomus sergenti, the proven vector of leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis, is widely distributed in morocco. previous works using molecular markers (internal transcribed spacer 2 rdna and cytochrome b mtdna) hypothesized the existence of multiple closely related populations of sand fly species (cryptic species) that would exhibit distinct vectorial capacities. this work studies morphotypic diversity using traditional and geometric morphometry analyses carried out on ph. sergenti's wings from central morocco, where active l. tropica transmission occurs for 30 years. methods: descriptive characteristics (size and shape) of the right wings were measured in ph. sergenti’s specimens collected from fourteen stations in central morocco. both traditional and geometric morphometry methods were used to analyse geographic variations in ph. sergenti wing’s size and shape. results: these analyses support the existence of distinct ph. sergenti populations, enlightening significant phenotypic variations of ph. sergenti’s wings, regarding their size and shape, depending on geographic origin. in addition, traditional and geometric morphometric analyses of the wing’s length, centroid size, β, ɵ, and γ distances allowed clear discrimination of ph. sergenti sub-populations. conclusion: these data pinpoint the adaptative ability of ph. sergenti to local environmental conditions. additional studies are now required to further shed light on the genetic structure of ph. sergenti populations in morocco. keywords: phlebotomus sergenti; wing traditional morphometry; wing geometric morphomery; morocco introduction sand flies (diptera, psychodidae) are vectors of human pathogenic micro-organisms, including parasites of the genus leishmania, bacteria (bartonella bacilliformis), and various arboviruses belonging to the genera phlebovirus and vesiculovirus (1–3). they represent, therefore, a public health scourge. in morocco, the phlebotomine fauna encompasses 24 species. six species are proven, or suspected vectors involved in visceral or cutaneous leishmaniases (4). variability in genetic, biochemical, morphometric, or reproductive features between allopatric populations of the subfamily phlebotominae are frequent and probably favored by their large copyright © 2023 the authors. published by tehran university of medical sciences. this work is licensed under a creative commons attribution-noncommercial 4.0 international license (https://creativecommons.org/licenses/bync/4.0/). non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:souad.guernaoui@usmba.ac.ma mailto:denis.sereno@ird.fr https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 37 http://jad.tums.ac.ir published online: march 31, 2023 geographic distribution and the limited dispersal capacity of populations with reproductive activity near their breeding site (5). these would lead to speciation processes not detectable with dichotomic keys (6). phlebotomus sergenti, parrot, 1917, is a proven leishmania tropica, vector, the causative agent of anthroponotic cutaneous leishmaniasis (acl) (1, 7–9). phlebotomus sergenti is distributed from the southern to the western mediterranean basins. it is replaced in the northeast of this region by ph. similis perfiliev, 1963 (10). its distribution extends eastward into iran, the former ussr, pakistan, and india. such geographic distribution vastly exceeds that of l. tropica (10). the genetic diversity of ph. sergenti investigated with internal transcribed spacer 2 (its2) rdna and/or cytochrome b (cyt b) mtdna lightened the possibility of closely related sand fly species (cryptic species) that may have distinct vectorial capacities (11-15). phlebotomus sergenti is widely distributed throughout morocco, with a preference for semi-arid habitats (16, 17). in acl foci, ph. sergenti is closely associated with the human habitat and involves a domestic and peridomestic transmission of l. tropica (17, 18). the analysis of the population's genetic structure of ph. sergenti from moroccan foci of taza, azilal, and essaouira, using cyt b mtdna, disclosed the presence of three mitochondrial lineages with a striking genetic diversity in the azilal focus (12). geometric morphometry (gm) has been applied to analyze morphological variations of insect wings (19). it has a taxonomic value for specimens belonging to puzzling morphological taxa (20) and highlights spatio-temporal dynamics of some phlebotomus populations; ph. tobbi adler and theodor 1930, ph. papatasi (scopoli, 1786), ph. ariasi tonnoir 1921 and ph. sergenti (21–25). in this work, traditional and geometric morphometry analyses were carried out on ph. sergenti's wings from central morocco, where active l. tropica transmission occurs for 30 years (26). materials and methods study area the studied area locates in central morocco (fig. 1), where cutaneous leishmaniases (cl) are widespread (26); the first cl case due to l. tropica was reported in 1986 in azilal (27). the environment is diverse, with altitudes varying from 491m to 1650m above sea level. it encompasses the tadla plain, the phosphate plateau, and the high and middle atlas mountains (fig. 1, table 1). the climate is continental, with a wet season (november to march) and a dry season (april to october). the annual average temperature is 18 °c but ranges from 2 °c to 40 °c. the temperatures recorded during the capture period are given in table 1. precipitation varies from 100mm to 1200mm. béni mellalkhénifra region endow the natural forest of holm oak (quercus rotundifolia), junipers (juniperus phoenicea), thuja (tetraclinis articulata), pines (pinus halepensis, pinus pinaster), cedar (cedrus atlantica), poplar (populus nigra), and carob tree (ceratomia siliqua). this region also has agricultural activity; cereals, olives, citrus fruits, sugar beets, grenadines, almonds, apples, carobs, sesame, and vegetables (28). sandflies sampling and identification sampling was carried out using sticky paper traps placed in domestic, peridomestic, and wild biotopes in fourteen stations between may and october 2017. traps were left in the field for a single night to avoid the deterioration of wings. in the laboratory, each specimen was dissected under a binocular. we first separated the wing for each male, and then the head and genitalia were dissected and mounted in canada balsam. next, we identified specimens by examining the morphology of the external genitalia. identification was performed using published keys and descriptions (10, 29). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 38 http://jad.tums.ac.ir published online: march 31, 2023 wings preparation well-preserved wings of specimens identified as ph. sergenti were prepared following the described protocol (30), with modifications by reducing the incubation time to 10min in 5% koh, in methylene blue to 10min. this is sufficient to visualize veins and ribs without damaging the wings. after treatment, wings are mounted in canada balsam on marked slides and photographed with a digital camera (leica microsystem ch) connected to a microscope (leica) in the museum of natural history of marrakech cadi ayyad university, morocco. measurements measures were carried out using the gryphax 12.0 software (jenoptik) (31). in addition, pictures were processed with tps-util 32 version 1.74 and tps-dig 2 version 2.30 software (32). we considered the 17 landmarks already used in previous studies of other sand fly species (21, 22). these landmarks are located at the wing veins’ intersections with their margin and the crossroads of the transverse veins with the prominent veins, as depicted in fig. 2. the size of ph. sergenti wings is given by measures of length (5–17), width (1–9), and centroid size (cs) (fig. 2). the cs is defined by the square root of the sum of the squared distances between the centre of the configuration of landmarks and each landmark. it was extracted from each matrix using morphoj 1.07a version 1.8.0_251 software (33). the analysis of the wing shape is based on the coordinates of the 17 landmarks and seven distances α (2-12), β (12-11), δ (1-11), ɛ (312), ɵ (4-11), π (10-11) and γ (11-14) (5, 25). data analysis traditional morphometry traditional morphometry aims to describe the wing’s morphology and delineate quantitative morphological characteristics that allow discrimination of ph. sergenti subpopulations. firstly, we performed a descriptive analysis of the length, width, cs, and seven distances. then, the mean, standard deviation, median, mode, and variation ratio were calculated. next, the normality and variance homogeneity assumptions were analysed for each variable using normalized plots. secondly, for each character, the student t-test was used to compute the significance of the differences at a significance level of 0.05. finally, the link between each measure (size, shape) (quantitative variable) and station (qualitative variable) was examined by estimating the correlation ratio (r) that is used for allometric analysis to test the link between the wing’s shape and size (cs, length, width). univariate and bivariate statistical analyses were performed using microsoft excel 10. in addition, a typology of wing morphs (size, shape) according to the geographical origin was made using canonical analysis, carried out with the statistica software (34). geometric morphometry the geometric morphometric analysis allows the visualization and comparison of the geometric configurations of ph. sergenti wings. first, ward's method (35) is used to construct the hierarchical classification dendrograms for wing size and shape, using past 3.23 software (36). then, the ph. sergenti wing geometric configurations were investigated using a generalized procrustes analysis (gpa) (37). after gpa, shape variables were measured and analyzed by the principal component of the “relative warps” scores calculated using the past 3.23 software (36). results this study used descriptive and geometric morphometry analyses of ph. sergenti wings. it investigates the size and shape of a set of stations from central morocco differing in their ecological characteristics. ninety-two wings of ph. sergenti were collected in 14 stations in central morocco (table 1). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 39 http://jad.tums.ac.ir published online: march 31, 2023 wing size and shape as described in the material and methods section, the wing's size measured is presented in supplementary data table 3. overall, in males, the length of the right wings is 2137.01±124.05µm, the width is 738.17±69.78 µm, and the cs is 3236.79±160.66µm. the shape of ph. sergenti wings is characterized by seven distances: α, β, δ, ɛ, ɵ, π, and γ (supplementary data table 4). the wing shape varies mainly in the β (12-11), ɵ (4-11), and γ (11-14) distances. all measurements are log-normally distributed. allometric analysis was performed to assess the link between wing size and shape by estimating the correlation coefficient. as a result, a positive correlation is recorded particularly between, on the one hand, the 3 distances ɵ, α, and β, and on the other hand, the size parameters, mainly the cs and the width, as shown in table 2. wings variability analysis to investigate the spatial variations of ph. sergenti’s wing parameters from stations where the ph. sergenti density is low, we gathered samples from stations 2, 3, 4, 5, 7, 10, 12, 13, and 14 into the label “other stations” as shown in (figs. 3 and 4). the size of the right wing shows significant differences linked to their geographic origin (fig. 3), as demonstrated by the correlation ratios computed between the origin and the length, width, or cs (0.68 and 0.56 and 0.74, respectively). similarly, the seven distances characterizing the wing shape showed differences according to the stations (fig. 4), with β, ɵ, and γ as the most variable distances. the correlation ratios between the collection site and the distances α, β, δ, ɛ, ɵ, π and γ are 0.39, 0.49, 0.37, 0.39, 0.55, 0.31 and 0.46, respectively. wings of ph. sergenti of afourer exhibit peculiar characteristics, as shown in figs. 3 and 4. differences were noticed, especially for the cs, β, and ɵ distances. furthermore, the cs in afourer is significantly different from all other stations, even the neighbouring ones. the student t-test are; bzou (t= -10,626), foum jamaa (t= -23,531), tiski (t= -4,281), ksiba (t= -10,747). as for results from the descriptive analysis, canonical analyses of the size and shape of ph. sergenti wings, illustrated in figure 5, point to substantial variation according to the wings' geographical origin (sampling site). canonical analysis of the size (fig. 5a) discloses that the plane (1-2) explains 92.57% of the total inertia (canonical variate 1: 76.43% and canonical variate 2: 16.14%). these results indicate that the wing size differs from station to station. first, specimens from afourer and tiski are grouped on the right of the canonical analysis, followed by those from bzou, foum jamaa, and the other stations (stations 2, 3, 4, 5, 7, 10, 12, 13, and 14). for the wing shape, the projection in the plane (1-2) explains 63.06% of the total inertia (canonical variate 1: 43.23% and canonical variate 2: 19.83%) (fig. 5b). as for the parameter of wing size, the canonical analysis reveals gradual variations according to the stations. the hierarchical single-link classification trees for the measured parameters of size (fig. 6a) and wing shape (fig. 6b) demonstrate a clustering according to the geographic origin, which again reinforces the results of the canonical analysis. but, again, and particularly for wing size, two groups stand out; the populations of afourer and tiski with smaller wings according to measured parameters vary from those of foum jamaa, bzou, or the other stations. likewise, the mean configurations of the wing shapes vary according to the geographical origin, with marked differences in the position of landmarks 2, 9, 10, and 11, as presented in fig. 7. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 40 http://jad.tums.ac.ir published online: march 31, 2023 table 1. stations sampled for phlebotomus sergenti in central morocco during the period may–october 2017 *lack of data. **(26) cl: cutaneous leishmaniasis table 2. correlation coefficient (r) between phlebotomus sergenti wing’s parameters (shape and size) during the period may–october 2017 shape size cs length width ɵ 0.69 0.54 0.70 α 0.55 0.32 0.66 β 0.47 0.30 0.48 ɛ 0.34 0.26 0.28 π 0.32 0.14 0.27 δ 0.26 0.08 0.29 γ 0.08 0.19 0.0007 table 3. morphometry of phlebotomus sergenti male’s right-wing collected in the study region in central morocco during the period may–october 2017 numerical parameters length width centroid size mean±sd (µm) (min-max) (µm) 2137.01±124.05 (1880-2340) 738.17±69.78 (598-935) 3236.79±160.66 (2855-3668) mode (µm) 1990.00 700.00;725.00; 740.00 3136.00;3149.00; 3181.00 median (µm) 2170.00 725.50 3205.00 variation ratio (%) 5.80 9.45 4.96 stations n° longitude (w) latitude (n) altitude (m) temperature (°c) mean (min-max) urbanization cl cases** (2009 to 2015) specimens number afourer 1 06°30´00´´ 32°13´00´´ 491 26 (18-34) urban 433 13 ait imloul 2 06°38´18´´ 32°11´00´´ 1118 * rural * 6 aguelmouss 3 05°50´35´´ 33°09´30´´ 1217 * rural * 2 béni ayyat 4 06°34´01´´ 32°12´31´ 535 * rural 37 1 béni mellal 5 06°22´32´´ 32°20´32´´ 507 27 (23-31) urban 90 2 bzou 6 07°03´18´´ 32°05´09´´ 429 26.5 (19-34) rural 489 11 el kbab 7 05°31´01´´ 32°44´27´´ 1198 * rural * 1 el ksiba 8 06°01´58´´ 32°33’54´´ 1003 * urban 5 8 foum jamaa 9 06°59´26´´ 31°37´54´´ 813 24.5 (17-32) rural 386 21 tagzirt 10 06°12´01’’ 32°26´08´´ 594 * rural 33 3 tiski 11 06°46´12´´ 32°06´44´´ 677 25.5 (18-33) rural 178 16 zaouiat cheikh 12 05°55´01´´ 32°38’38´´ 798 * urban 202 1 zaouiat ahansal 13 06°06´15´´ 31°49´59´´ 1629 * rural 2 3 benssarou 14 06° 09´42´´ 32°21´16´´ 1650 * rural * 4 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 41 http://jad.tums.ac.ir published online: march 31, 2023 table 4. numerical characteristics of the distances α, β, δ, ε, ɵ, π and γ of phlebotomus sergenti male’s right-wing collected in central morocco during the period may–october 2017 numerical parameters α β δ ɛ ɵ π γ mean±sd (µm) (min-max) (µm) 540.19±70.91 (399-782) 375.61±86.86 (230-835) 173.84±54.86 (108-502) 367.30±60.60 (255-787) 604.79±44.55 (510-690) 260.5±37.65 (200-488) 621.92±104.47 (385-984) mode (µm) 510.00; 525.00 420.00 118.0;144.0; 161.0;171.0; 182.0; 203.0 320.00; 350.00 560.00; 600.00 243.00 515.00; 560.00; 610.00 median (µm) 530.00 359.00 168.0 360.00 605.00 253.00 610.00 variation ratio (%) 13.13 23.12 31.55 16.50 7.36 14.45 16.80 fig. 1. location of the sampling area for phlebotomus sergenti populations in central morocco (source google earth) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 42 http://jad.tums.ac.ir published online: march 31, 2023 fig. 2. location of the 17 landmarks used in this study on the right-wing of phlebotomus sergenti fig. 3. boxplots of the lengths, widths, and centroid sizes of phlebotomus sergenti male’s right wings in the various station of central morocco during the period may–october 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 43 http://jad.tums.ac.ir published online: march 31, 2023 fig. 4. boxplots representation of the distances α, β, δ, ε, ɵ, π, and γ of the right wings of phlebotomus sergenti’s males in each station of central morocco during the period may–october 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 44 http://jad.tums.ac.ir published online: march 31, 2023 fig. 5. canonical analysis of the size variations (a) (92.57% of the total inertia) and the shape (b) (63.06% of the total inertia) of phlebotomus sergenti’s male right wings depending on the geographical origin of central morocco during the period may–october 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 45 http://jad.tums.ac.ir published online: march 31, 2023 fig. 6. hierarchical classifications tree of phlebotomus sergenti right-wings size (a) and shape (b) of central morocco fig. 7. superposition of the right-wing landmarks (mean measures) of phlebotomus sergenti males from afourer (a), tiski (b), el ksiba (c), bzou (d), foum jamaa (e), and other stations (f). (arrows of a given color show variations in the position of a given landmark) of central morocco during the period may– october 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 46 http://jad.tums.ac.ir published online: march 31, 2023 discussion phlebotomus sergenti is a proven and primary vector of l. tropica in saudi arabia, morocco, iran and israel (1, 7–9, 38). previous studies have considered ph. sergenti a species complex regarding the tremendous genetic diversity recorded over its distribution (11–13, 39). on the other hand, the cross-mating study showed that there is no reproductive barrier between ph. sergenti from different geographical areas (24), and no statistically important differences were found in wing morphology for all local populations of ph. sergenti, suggesting that the barriers are not sufficient to stop gene flow among local populations of sand flies (25). despite this ambiguous context, no morphotype indicator is currently available to probe ph. sergenti population diversity. therefore, the wing's geo-morphometric analyses would help shed light on the population's diversity of ph. sergenti. this has been used successfully on several sand fly species, like ph. tobbi, ph. papatasi, or ph. ariasi (21–23, 30), and has provided evidence of the separation of ph. papatasi populations between the southern and northern slopes of the high atlas mountains in morocco (21). this was further delineated via a genetic analysis of these populations (40). therefore, we applied descriptive and geometric morphometry analyses to probe ph. sergenti populations' diversity in central morocco for the first time. the morphometric parameters of ph. sergenti’s wings sampled in central morocco show significant variability. the size is variable in length, width, and cs. the shape also showed substantial variations, particularly at the distances; β, ɵ, and γ. in addition, allometric analyses demonstrate a strong link between size and shape variability. a correlation is recorded between cs or wing width on one side and ɵ, α or β, on the other side. descriptive analyses point to a link between ph. sergenti wing’s size and shape and the geographical origin. length, width, cs, and the seven distances characterizing the wing’s shape differ significantly between stations, with correlation ratios higher than 50%. population from afourer locality is notably different, with relatively smaller wings. multivariate analyses further supported these results. canonical studies reveal, in addition to the variability, the existence of a gradual variation in size and shape (92.57% and 63.06% of the total inertia, respectively). this population’s succession in the factorial plan could testify to a cline variation of the measured parameters. the results of the geometric analyses corroborate those of the descriptive ones. hierarchical classifications trace the gradual variation and define two types based on the geographic origin; the foum jamaa and bzou types mainly differ from the afourer and tiski types. relatively smaller wings characterize these last two populations. in addition, superimpositions of the average configurations of ph. sergenti male right wings indicate significant variations in landmarks 2, 9, 10, and 11. previous works have focused on the relationship with altitude regarding the origin of the variability of sand fly wings in the natural environment. however, altitude is not an ecological factor, but it can express a variety of climatic factors. in this study, we conducted a bi-monthly sampling to integrate variations in connection with climatic factors in central morocco. in areas where ph. sergenti is abundant, it was active for only two months, july and august, during the dry period. therefore, no significant temporal variation in wing parameters (size and shape) was recorded, probably due to the short active period. wings of ph. ariasi (france), ph. papatasi and ph. tobbi (turkey) showed variations in response to an altitudinal gradient (22, 23, 30). but, even via an indirect effect, altitude cannot explain the recorded gradual variation in ph. sergenti from central morocco. the individualized populations of afourer and tiski http://jad.tums.ac.ir/ j arthropod-borne dis, march 2023, 17(1): 36–50 fz abou-elaaz et al.: descriptive and … 47 http://jad.tums.ac.ir published online: march 31, 2023 locate in the same altitudinal zone as bzou and foum jamaa. furthermore, the altitudinal structuring of sand flies observed in the high atlas mountains concerned ph. papatasi, ph. ariasi, and the species of the ph. perniciosus complex (41). the density and distribution of ph. sergenti did not follow any altitudinal gradient (41). altitude can also act indirectly through the slope effect. this has been observed in ph. papatasi on the southern and northern slopes of the moroccan high atlas (21) and in ph. tobbi (22) and ph. ariasi (23). but this slope effect cannot be involved in the variation we detected in ph. sergenti; the stations compared to afourer, bzou, tiski, and foum jamaa are located on the same high atlas mountains slope. moreover, the stations which show ph. sergenti wing variations are located in the same zone of the high atlas mountains, at a maximum of 60km apart. therefore, they are subject to a similar climate, and considering the low dispersal potential of male sand flies, this adds evidence for a microenvironmental adaptation. this phenomenon is also reported in ph. ariasi in the oiselette massif in the south of france (23). furthermore, local habitat adaptation of ph. sergenti populations was observed in the western moroccan high atlas mountains and other l. tropica foci. although ph. sergenti is a widely distributed species, its abundance is linked to a particular microhabitat where shelters are present (areas in basins) and where the dwellings (construction material) are not made of cement (17). 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accepted 5 april 2009) abstract background: an epidemiological study was carried out on the vector(s) and reservoir(s) of cutaneous leishmaniasis in rural areas of kalaleh district, golestan province during 2006 2007. methods: totally 4900 sand flies were collected using sticky papers and were subjected to molecular methods for detection of leishmanial parasite. results: phlebotomus papatasi was the common species in outdoor and indoor resting places. employing pcr technique showed only 1 out of 372 p. papatasi (0.3%) was positive to parasite due leishmania major. sixteen rodent reservoir hosts were captured by sherman traps and identified as rhombomys opimus. microscopic investigation on blood smear of the animals for amastigote parasites revealed 6(37.5%) infected rodents. infection of these animals to l. major was then confirmed by pcr against rdna loci of the parasite. conclusion: this is the first molecular report of parasite infection of both vector (p. papatas) and reservoir (r. opimus) to l. major. the results indicated that p. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and r. opimus was the most important host reservoir for maintenance of the parasite source in the area. keywords: cutaneous leishmaniasis, vector, reservoir, iran introduction there are several reports indicating occurrence of cutaneaous leishmaniasis due to leishmania major (clm) in iran (yaghoobiershadi et al.1996, rassi et al. 2006). based on animal reservoir host, there are four foci of disease in our country (rassi et al. 2006) the first one has been located in central and northeast of iran, where rhombomys opimus and phlebotomus papatasi play important roles as reservoir and vector of the disease (seyedi-rashti et al. 1967, javadian et al. 1976, yaghoobi-ershadi et al. 2001). the second focus of zoonotic cutaneous leishmaniasis is located in the west and southwest of iran, where tatera indica replaced with r. opymus as a reservoir and p. papatasi as a vector. (javadian et al. 1988). baluchistan pprovince, in the southeast of iran is considered as the third focus of zcl. in this region meriones hurrianae has been approved as a natural reservoir host (seyedi-rashti and nadim 1984). from the reported evidences, *corresponding author: e-mail: rassiy@sina.tums. ac.ir iranian j arthropod-borne dis, (2008), 2(2): 21-27 y rassi et al: molecular detection of…. 22 it is apparent that the most rural areas of fars province in southern iran can be considered as the zcl focus where m. libycus is the primary and main reservoir host of the disease, while r. opimus and t. indica were absent and p. papatasi is considered as the proven vector of zcl (rassi et al. 2006, rassi et al. 2007). kalaleh district from golestan province in northern iran is a zcl focus and this study was performed to put light through the epidemiology of the disease in the region. the main objectives were to determine the sand flies species responsible for most transmission of l. major to human, as well as to determine the main reservoir hosts of the disease in the study area. material and methods study area the study was carried out in 3 villages (ghare ghol-e-gharbi, sozesh and allah nour) of maraveh tapeh district (55° 57 e, 37° 54 n) at an altitude 228 m above sea level and 90-100 km far from kalaleh city. the weather is hot in the summer and cold in the winter. it receives an average of 311.3 mm of rain per year. the temperature ranges between 2.5-36.6° c and the ratio humidity ranges between 2585%. the total population of the district was about 156939 people in 2007 (unpublished data). the major activities of the population are agriculture and animal farming. collection of sand flies sand flies were collected from indoors (bedroom, guestroom, toilet) as well as outdoors (rodent burrows, wall cracks) biweekly using sticky traps. three above mentioned villages were selected and 180 sticky traps were set up during the sand flies activity period (may november). sand flies were rinsed from the sticky traps and mounted in a drop of puri’s medium and identified after 24 h using a valid key (nadim and javadian 1976, rassi et al. 2006 a). sand flies dna extraction dna was extracted through ish-horowicz (ready et al., 1991). individual female sand flies were homogenized with a sealed pasture pipette in 1.5 ml tubes. then 100 µl lysis buffer [0.1m tris-hcl ph 7.5; 0.6m nacl; 0.1 edta] and 10 µl [0.8m trishcl ph 9.0: 0.27m edta] were added and incubated at 65 °c for half an hour before 30 µl acetate potassium (8 mol) were added. after short centrifuge it was cooled for 45 min and transferred to new tube and added 350 µl cold pure ethanol and stored at -20°c for 24 h. the tube was centrifuged at 13000 rpm for 30 min then emptied the solution until dried and washed by 500 µl ethanol (75 °c) and the te buffer was added. semi-nested pcr for detection of leshmania infection in sand fly semi-nested pcr was employed for detection of kinetoplast dna (kdna) of l. major in sand fly specimens. the primers were: linr4 (forward):5´-ggggttggtgtaaaataggg-3´ (20bp); lin19 (reverse): 5´cagaacgcccctacccg-3´ (17bp), and lin17 (reverse): 5´-tttgaacgggatttctg-3´ (17bp). positive samples were tested by pcr method against its gene using the primers of its1 (forward): 5´tccgtaggtgaacctgcgg-3´ and its2 (reverse): 5´gctgcgttcttcatcgatgc -3´. pcr amplification was followed by rflp technique using haeiii enzymes for final species identification of the parasite. collection of rodents rodents were captured by setting the sherman live traps. traps were baited with roasted walnut, cucumber, tomato and placed in the active burrows. the traps were set up early morning and evening in december, february, may, and july. in order to approve the infectivity of rodents by the parasites, their ears were examined and an impression smear was taken and stained by geimsa staining method. the iranian j arthropod-borne dis, (2008), 2(2): 21-27 y rassi et al: molecular detection of…. 23 presence of the parasite was checked under microscope. samples from infected rodents were inoculated subcutaneously at the base of tail of balb/c. procedure of species identification by pcr method is described previously. dna extraction from positive smears of rodents total dna was extracted from positive smears by digestion in 100 µl pbs buffer and the tube was centrifuged at 10000 rpm for 10 min, then 300 µl lysis buffer and 30 µl proteinase k added. the tube was incubated for 24 h at 37 °c before adding 300 µl sacharin phenol. after adding this solution the tube was centrifuged at 9300 rpm for 5 min. after transferring upper phase to new tube, 300 µl phenolchloroform should be added and was centrifuged at 10000 rpm for 5 min. again transferred the upper phase to new tube and washed by pure chloroform. thirty µl mgcl2 and 1000 µl ethanol were added to upper phase and stored at -20° c for 2 h before was centrifuged at 10000 rpm for 10 min and washed down phase by 70% ethanol with te and was centrifuged at 10000 rpm for 10 min and the te buffer was added. pcr-rflp for detection of leishmania infection in rodents pcr-rflp was employed for detection and identification of l. major in rodent specimens by the method explained by dweik et al. (2007). the primers were: ir1 5´-gct gta ggt gaa cct gca gca gct gga tca tt-3´ and ir2 5´-gcg ggt agt cct gcc aaa cac tca ggt ctg -3´ (cupolillo et al. 1995). reference strains of l. infantum (mcan/ir/96/lon49), l. tropica (mhom/ ir/89/ard2) and l. major (mhom/ir/54/ lv39) were used as positive controls. all were obtained from the medical parasitology laboratory, the school of public health, tehran university of medical sciences, iran. pcr production was followed by rflp technique using haeiii enzymes for final identification of the parasite. results sand flies totally 4900 sand flies were collected, including p. papatasi (41.2%), p. mongolensis (5.8%), p. caucasicus (3.6%), p. caucasicus group (7.3%), p. sergenti (2.2%), p. alexandri (2%), p. (adlerius) sp. (0.02%), p. brevis (0.02%), p. kazeruni (1.5%), s. sintoni (36%), s. clydei (3%) and s. sogdiana (0.02%). three hundred and seventy two p. papatasi specimens were examined by seminested pcr for leishmania infection. species-specific amplification of l. major dna was found in one (0.3%) of the p. papatasi giemsa stained prepared for detection of promastigote (fig.1). furthermore its amplification by nested pcr primers followed by rflp technique confirmed the dna of parasite in the infected p. papatasi sample (fig. 2 & 4). the infected p. papatasi was collected from bed room with parous and empty abdomen position. rodents during this study 16 rodents were captured and identified. all of them were r. opimus. although all collected animals were examined for parasite infection under light microscope, amastigotes were only found in smears of 6(37.5%) of them. each sample from infected rodents was inoculated subcutaneously at the base of tail of one balb/c. results from inoculation of parasite from infected rodents, revealed the presence of amastigotes in the nodules and ulcers of the experimentally mice after 35 d of inoculation period. parasites infection was observed in both males and females animals. isolated parasites from infected rodents were identified as l. major using pcr followed by rflp technique (figs. 3, 4). iranian j arthropod-borne dis, (2008), 2(2): 21-27 y rassi et al: molecular detection of…. 24 fig. 1. kdna pcr amplification of l.major in p.papatasi using semi-nested pcr m: molecular size marker, 1: negative control, 2: sample of p.papatasi, 3: positive control l.major fig. 2. its amplification of l.major in p.papatasi using nested pcr primers m(marker) , 1(l.major standard), 2(blank), 3 (sample of p.papatasi) iranian j arthropod-borne dis, (2008), 2(2): 21-27 y rassi et al: molecular detection of…. 25 fig. 3. its-rdna amplification of leishmania parasite in r.opimus 1-6(infected r.opimus), 7(l.major standard), 8(negative control), m (marker) fig. 4. pcr-rflp analysis of its region for identification of leishmania species using haeiii l.major standard (1), p.papatasi (2), l.tropica standard (3), l.infantum standard (4), and rhombomys opimus (5) iranian j arthropod-borne dis, (2008), 2(2): 21-27 y rassi et al: molecular detection of…. 26 discussion ecology and epidemiology of leishmaniasis are important measures for management and planning of the disease control. the entomological survey accompanied by epidemiological data is a major component for combating against disease. several epidemiological and entomological finding including anthropophily, common infection of the sand-flies with the same leishmania parasite that found in man in the same places, suggested the capacity of sand-fly as a vector (killick-kendrick et al. 1990). for further confirmation, molecular techniques (pcr) have been employed too. the highly sensitive technique of pcr has been used for detecting leishmaniain sandflies in the world (mukherjee et al. 1997), iran (azizi et al. 2006, rassi et al. 2006, rassi et al. 2007) and india (de bruijn et al. 1992). results of our study revealed that the high density of p. patasti in indoor resting places and infectivity with l. major is attributed that this species can play a major role as a principle vector in the region. another important finding of this survey was confirmation of r. opimus as the principal reservoir of zcl in rural regions of kalaleh district. this rodent has been also reported as a main reservoir in the other foci of disease (such as isfahan and khorassan provinces) in iran (yaghoobi-ershadi et al. 1996, javadian et al. 1976, seyyedi-rashti et al. 1967). this great gerbil, a colonial, burrowing rodent, is a common species in the arid desert and steppe regions of central asia. this species also exist in the southern territories of the former u.s.s.r. (i.e. turkmenistan, uzbekistan, kazakhstan, tajikistan) and neighboring countries where zcl caused by l. major is endemic and considered as an important public health problem, therefore r. opimus is considered the principal mammalian host of the parasite (strekova et al. 2001). human activities close to r. opimus burrows, the presence of high density of p. papatas in the rodent burrows and indoors as well as proximity of human habitat to r. opimus colonies lead to a rise in human contact with the disease agents and possible appearance of a new focus of leishmaniasis in the region. acknowledgements the author would like to appreciate the kind collaboration of health center staff of kalaleh district. this study was financially support by the school of public health, tehran university of medical sciences: project no.: 5416. references azizi k, rassi y, javadian e, motazedian mh, rafizadeh s, yahoobi ershadi mr, mohebali m (2006) phlebotomus (paraphlebotomus) alexandri: a probable vector of leishmania infantum in iran. ann trop med parasitol. 100(1): 63-68. cupolillo e, grimaldi jr, momen h, beverley sm intergenic region typing (irt) (1995) a rapid molecular approach to the characterization and evolution of leishmaniasis. mol biochem parasitol. 73: 145-155. de bruijn mh, barker dc (1992) diagnosis of new world leishmaniasis: specific detection of species of the leishmania brasiliensis complex by amplification of kinetoplast dna acta tropica. 52:5-58. dweik a, schönian g, mosleh im, karanis p (2007). evaluation of pcr-rflp (based on its-1 and hae iii) for the detection of leishmania species, using greek canine isolates and jordanian clinical material. ann trop med parasitol. 101 (5): 399-407. iranian j arthropod-borne dis, (2008), 2(2): 21-27 y rassi et al: molecular detection of…. 27 javadian e, nadim a, tahvidare-bidruni gh, assefi v (1976) epidemiology of cutaneaous leishmaniasis in iran. b: khorassan area, part v; report on a focus of zoonotic cutaneaous leishmaniasis in esferayen. bull soc pathol exot. 69:140143. javadian e (1988) reservoir host of cutaneous leishmaniasis in iran. the xiith international congress for tropical medicine and malaria, amsterdam, the netherlands. p.52. killick-kendrick r (1990).phlebotomus vectors of visceral leishmaniasis, a review. med vet entomol. 4:1-24. mukherjee s, hassan mq, ghosh a, bhattacharya a, adhya s (1997) short report: leishmania dna in phlebotomus and sergentomyia species during a kala-azar epidemic. american j trop med hyg. 57:423-425. nadim a, javadian e (1976) key for the species identification of sand flies (diptera phlebotominae) of iran. iranian j publ hlth. 5 (1): 25-28. rassi y, javadian e, amin m, rafizadeh s,vatandoost h, motazedian h (2006) meriones libycus is the main reservoir of zoonotic cutaneous leishmaniasis in south islamic republic of iran, east med hlth .12 (3-4): 475-7. rassi y, gassemi mm, javadian e, rafizadeh s, motazedian h, vatandoost h (2007) vectors and reservoirs of cutaneous leishmaniasis in marvdasht district, southern islamic rassi y, hanafi bojd a (2006a) phlebotomine sandflies, vectors of leishmaniasis.pp.251 ready pd, lainson r, shaw jj, souza aa (1991) dna probes for distinguishing psychodopygus wellcomei from psychodopygus complexus (diptera: psychodidae). mem inst oswaldo cruz. 86: 41-49. seyedi-rashti ma, nadim a (1967) epidemiology of cutaneous leishmaniasis in iran. b. khorassan area, part i; the reservoir. bull soc pathol exot. 60: 510-518. seyedi-rashti ma, nadim a (1984) cutaneous leishmaniasis in baluchistan, iran. abstracts of and poster volum.xi, international congress for tropical medicine and malaria. calgary, canada. sep. 16(22): 124. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996) meriones libycus and rhobmomys opimus (rodentia: gerbellidae) are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans r soc trop med hyg. 90: 503-4. strekova mv, elisev ln, ponirovsky en, dergacheva ti, annacharyava dk, perokhin i, evans da (2001) mixed leishmanial infections in rhombomys opimus: a key to the persistence of leishmania major from one transmission season to the next. ann trop med parasitol. 95(8): 811-19. yaghoobi-ershadi mr, hanafi-bojd, aa, akhavan ar, zahrai-ramazani ar mohebali m (2001) epidemiological study in a new focus of cutaneous leishmaniasis due to leishmania major in ardestan town, central iran. acta tropica. 79: 115-21. j arthropod-borne dis, september 2016, 10(3): 366–371 e ebrahimzade et al.: ectoparasites of … 366 http://jad.tums.ac.ir published online: january 06, 2016 original article ectoparasites of stray dogs in mazandaran, gilan and qazvin provinces, north and center of iran *elahe ebrahimzade, roohollah fattahi, mohammad bagher ahoo department of parasitology, faculty of veterinary medicine, university of tehran, iran (received 26 jan 2014; accepted 14 july 2015) abstract background: the aim of the present study was to determine the prevalence of ectoparasite infestations in stray dogs in mazandaran, gilan and qazvin provinces in fall and winter in 2013(december to march). methods: seventy dogs in 2013, from these provinces were examined for ectoparasite infestation and diagnosis of them based on parasitological methods and identification keys was done. results: the rate of infestation in these areas was 100%, 68.5% and 93.3% respectively. fleas were the most common ectoparasites on dogs in this study followed by lice, ticks, flies and mites respectively. the isolated arthropods were fleas in 77.5%, lice in 50%, ticks in 8.6%, flies in 6.8% and mites in 5.1% of infested dogs. the ectoparasite of the dogs included 4 flea species: ctenocephalides canis (29.8%), c. felis (19.9%), pulex iritans (2.9%) and xenopsiella cheopis (0.7%), 1 louse species: trichodectes canis (41.3%), 1 tick species: rhipicephalus sanguinus (0.7%), 1 fly species: hippobosca sp. (1.1%) and 1 mite species: sarcoptes scabiei (3.6%). conclusion: fleas and lice were the most common ectoparasites in stray dogs of the studied area. some ectoparasites transmit important human disease, therefore regular monitoring of them is a major concern to control the arthropods and arthropods-borne diseases. keywords: ectoparasite, stray dogs, mazandaran, iran introduction ectoparasites are important cause of pruritic and non-pruritic skin disorders in dogs and can cause hypersensitivity or death depending on the host nutritional and immunological condition and intensity of parasitic infestation. ectoparasites play an important role not only as pests but also as vectors of various infectious diseases of humans, livestock, pets and wild animals. they can transmit different pathogens like viruses, bacteria, protozoa or act as intermediate hosts for filarids and cestodes (fuehrer et al. 2012). ticks, after mosquitoes, are the second most important arthropods that may transmit pathogens to animals and human beings. moreover, fleas can transmit pathogens, such as the agent of flea-born rickettsioses, murine typhus and bubonic plague. they can also serve as intermediate hosts for some species of cestodes (xhaxhiu et al. 2009). chewing lice are active arthropods that can produce intense irritation with secondary bacterial infections (wall et al. 1997, jafari shoorijeh et al. 2008). trichodectes canis are highly host specific, but they can act as an intermediate host for dipylidium caninum that may affect humans, especially children (scott et al. 2001, mosallanejad et al. 2011). some studies regarding etoparasites on dogs have been done in iran (shoorijeh et al. 2008, mosallanejad et al. 2011, jamshidi et al. 2012, bahrami et al. 2012) but information about ectoparasites on stray dogs is still lacking. the aim of this study was to determine the prevalence of ectoparasite infestations in *corresponding author: dr elahe ebrahimzade, e-mail: eebrahimzade@ut.ac.ir j arthropod-borne dis, september 2016, 10(3): 366–371 e ebrahimzade et al.: ectoparasites of … 367 http://jad.tums.ac.ir published online: january 06, 2016 stray dogs in mazandaran, gilan and qazvin provinces, iran. materials and methods seventy stray dogs were inspected from december to march 2013, for the presence of ectoparasites in mazandaran, gilan and qazvin provinces, iran. their sex and age were recorded. the age was estimated by dental formulary. the studied animals were divided into three groups based on the age (<3, 3–5 and >5 years old). the body of the dogs was completely examined and the ectoparasites were removed by using forceps, combing or brushing from the animals and stored in 70% ethanol. areas of the skin with dermatologic lesions were scraped deeply with an oily scalpel blade until capillary bleeding was visible. deep otic swab specimens were obtained from all dogs for detection of ear mite. skin scrapings and ears swabs were placed in 10% potassium hydroxide and gently heated for 30 min. then, the material was centrifuged, and the sediment was microscopically examined for mites. the ectoparasite species were identified under a stereomicroscope, according to the identification keys (hopkins and rothschild 1953, smit. 1958, benton 1980, wall and shearer 2001). data analysis a chi-square test was used to determine any significant associations between rate of infestation and age and sex. analyses were conducted using spss software version 16 (chicago, il, usa) with a probability (p) value of <0.05 as statistically significant. results fifty-eight out of 70 examined dogs (82.8%) were positive for ectoparasites. the rate of infestation in qazvin, gilan and mazandaran provinces were 100%, 68.5% and 93.3% respectively. fleas were the most common ectoparasites on dogs in this study. the isolated arthropods were fleas in 45 (77.5%), lice in 29 (50%), ticks in 5 (8.6%), flies in 4 (6.8%) and mites in 3 (5.1%) infested dogs (table 1). in this survey, 4 species of flea consist of ctenocephalides canis (29.8%), c. felis (19.9%), pulex iritans (2.9%) and xenopsiella cheopis (0.7%), one species of louse, tick, fly and mite including: trichodectes canis (41.3%), rhipicephalus sanguinus (0.7%), hippobosca sp. (1.1%) and sarcoptes scabei (3.6%) were found, respectively (table 2). triple infestation was observed in 8.6% of the infested dogs (40% of these with flea+lice+tick, 40% with flea+lice+fly and 20% with tick+lice+mite). 39.6% of dogs exhibited double infestation (73.9% of these with lice+fleas, 8.7% with ticks+lice, 8.7% with fleas+flies and 8.7% with fleas+mites). 51.8% presented single infestations with lice or fleas. the analysis of different infestation types showed that the most of these hosts had single infestation, followed by double and triple infestation. the mean number of fleas, lice, ticks, flies and mites on each infested dogs was 9.9, 11.9, 1.2, 2.5 and 10, respectively. according to the host age, the highest prevalence of infestation was observed in <3 years old (88%) followed by 3-5 (77.7%) and >5 years old (70.5%), however no significant differences were observed between infestation rate and age groups of dogs (p= 0.267). ectoparasitic infestations were recorded on 33 male dogs (89.1%) and 25 female dogs (75.7%). no statistically significant difference was observed between ectoparasite infestation and host gender (p= 0.137) (table 2). ectoparasites distribution of the studied dogs was 36% around the neck, 20% on backside portion, 25% on abdomen and 19% on other body sites. j arthropod-borne dis, september 2016, 10(3): 366–371 e ebrahimzade et al.: ectoparasites of … 368 http://jad.tums.ac.ir published online: january 06, 2016 table 1. the number of stray dogs infested by different ectoparasites based on each studied areas area examined infested flea lice tick fly mite qazvin 20 20 15 13 2 4 0 gilan 35 24 20 9 1 0 1 mazandaran 15 14 10 7 2 0 2 total 70 58 45 29 5 4 3 table 2. number and percent of ectoparasite species on infested dogs in the studied areas ectoparasite male female total n (%) ctenocephalides canis 106 145 251 (29.8) c. felis 70 98 168 (19.9) trichodectes canis 126 221 347 (41.3) pulex irritans 8 16 24 (2.9) xenosylla cheopis 3 3 6 (0.7) rh. sanguinus 6 0 6 (0.7) hippobosca sp. 4 6 10 (1.1) sarcoptes scabei 12 18 30 (3.6) discussion in the present study, eight species of ectoparasite were isolated from stray dogs. the total prevalence of ectoparasites in this survey was 82.8%. this finding is almost similar with the findings of tesfaye et al. (2011) have recorded a prevalence of 88.6% in dogs in ethiopia and identified 8 species of ectoparasites in their study. different studies have been done to detect ectoparasites on dogs in various parts of the world. xhaxhiu et al. (2009) determined nine species of arthropods with a prevalence of 79% in dogs in albania. kumsa and mekonnen (2011) identified six species of ectoparasites with a prevalence of 99.5% in dogs in ethiopia. bahrami et al. (2012) have reported seven species of ectoparasites in 44.26% of the dogs from ilam province. mosallanejad et al. (2011) found eight species of ectoparasites in companion dogs in ahvaz district, 36 out of 126 dogs (28.57%) were positive for ectoparasites. nine and seven species of arthropods have been reported in pet dogs in tehran and shiraz province, with a prevalence of 36.4% and almost 25%, respectively (shoorijeh et al. 2008, jamshidi et al. 2012). fleas were the most common ectoparasites in this survey and c. canis was the most common flea, followed by c. felis, p. irritans and x. cheopis. these fleas have been reported in iran by many investigators (shoorijeh et al. 2008, mosallanejad et al. 2011, bahrami et al. 2012, jamshidi et al. 2012). in the study that was carried out by xhaxhiu et al. (2009) on dogs in albania, the most common ectoparasites were fleas. they identified three species of fleas consist of c. canis, c. felis and p. irritans. in addition, c. canis was more predominant than c. felis and p. irritans. examination of dogs in greece revealed the presences of four species of fleas consisting of c. canis, c. felis, p. irritans and x. cheopis, the first species was the most frequent that was found on the dogs (koutinas et al. 1995). c. canis was the only species found on dogs in rural areas (gonzález et al. 2004, fuehrer et al. 2012). in other studies c. canis was not observed on dogs (alberto ca˜ nón-franco et al. 2010, j arthropod-borne dis, september 2016, 10(3): 366–371 e ebrahimzade et al.: ectoparasites of … 369 http://jad.tums.ac.ir published online: january 06, 2016 slapeta et al. 2011). beresford-jones (1981), dryden and rust (1994, usa) and alca´ıno et al. (2002) noted that within flea species, c. canis is the most predominant species on dogs in rural areas, whereas c. felis is the most common in urban areas. since our study was done on stray dogs in rural and urban areas, this could explain the presence of both these two flea species. in our study, female fleas were more abundant than males, which is in agreement with other findings. one reason of this matter could be related to greater ability of females to evade capture during host grooming (durden et al. 2005, gracia et al. 2008, tavassoli et al. 2010). in this survey, lice were ranked in the second frequency of ectoparasites. the overall prevalence of lice was 50% in infested dogs. mosallanejad et al. (2011) determined only one louse species, heterodoxus spinigera and that was the most common ectoparasites on dogs in their study in ahvaz province. in the present study, only one louse species was observed, including trichodectes canis. this finding is agreement with xhaxhiu et al. (2009) in albania, jafari shoorijeh et al. (2008) in shiraz province iran. in the study carried out by jamshidi et al. (2012) 5.6% and 4% of ectoparasites were t. canis and linognathus setosus respectively. bahrami et al. (2012) observed lice species including heterodoxus spinigera and l. setosus on dogs from ilam province. ticks were as the third prevalent (8.4%) species in this study. we collected six ticks from infested dogs that all of them were rh. sanguineus. this specie has been reported on dogs in different parts of iran. (shoorijeh et al. 2008, mosallanejad et al. 2011, jamshidi et al. 2012, bahrami et al. 2012). in nigeria, adamu et al. (2012) identified ticks as the main species of ectoparasites in dogs, with a prevalence of 47% and rh. sanguineus was predominant (24.3%). in this survey, the mean number of ticks was very low, almost one tick on each infested dogs. the reason can be related to sampling season, ticks are abundant in the warm and humid weather (adamu et al. 2012). moreover, ticks are vectors of serious pathogens. rhipicephalus sanguineus can transmit the etiological agents of canine babesiosis and canine monocytic ehrlichiosis (dantas-torres 2008, rene et al. 2012). one species of fly (hippobosca) and mites (sarcoptes scabiei) were detected. these species also observed by mosallanejad et al. (2011) in khuzestan province. s. scabiei was also reported by (jamshidiet al. 2012) in tehran province. xhaxhiu et al. (2009) in albania and shoorijeh et al. (2008) identified hippobosca sp. on dogs in shiraz province. thompson et al. (1993) have assessed the prevalence of ectoparasites from aboriginal communities in australia. they have presented rh. sqanguineous and t. canis as ectoparasite of dogs. there was no evidence of flea infestation in their study (thompson et al. 1993). ectoparasites from dogs belonging to people in resource-poor communities in south africa has studied that harbored hard ticks belonging to 6 species (haemaphysalis leachi, rh. sanguineus, amblyomma hebraeum, rh. appendiculatus, rh. evertsi evertsi and rh. simus), fleas (c. felis, echidnophaga gallinacea) and lice (h. spiniger) and myiasis, caused by the larvae of cordylobia anthropophaga (brysona et al. 2000). conclusion high infestation with different arthropods were observed on stray dogs in center and north of iran. ectoparasites play an important role not only as pests but also as vectors of various infectious diseases of humans, livestock, pets, and wild animals, then regular monitoring of them is an important concern to control the 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university of medical sciences and phd research scholar, department of zoology, centre of advanced study, banaras hindu university, varanasi, india (received 20 nov 2011; accepted 14 may 2013) abstract background: cockroaches are among the medically important pests found within the human habitations that cause serious public health problems. they may harbor a number of pathogenic bacteria on the external surface with antibiotic resistance. hence, they are regarded as major microbial vectors. this study investigates the prevalence and antibiotic resistance of gram-negative pathogenic bacteria species isolated from periplaneta americana and blattella germanica in varanasi, india. methods: totally, 203 adult cockroaches were collected form 44 households and 52 food-handling establishments by trapping. bacteriological examination of external surfaces of pe. americana and bl. germanica were carried out using standard method and antibiotics susceptibility profiles of the isolates were determined using kirby-bauer disc diffusion methods. results: among the places, we found that 54% had cockroache infestation in households and 77% in foodhandling establishments. there was no significant different between the overall bacteria load of the external surface in pe. americana (64.04%) and bl. germanica (35.96%). however the predominant bacteria on cockroaches were klebsiella pneumonia, escherichia coli, enterobacter aerogenes, and pseudomonas aeruginosa. however, kl. pneumoniae and ps. aeruginosa were the most prevalent, drug-resistant strains were isolated from the cockroaches with 100% resistance to sulfamethoxazole/ trimethoprim and ampicillin. for individual strains of bacteria, escherichia coli was found to have multi-resistance to four antibiotic tested, citrobacter freundii four, enterobacter aerogenes and proteus mirabilis to three. conclusion: cockroaches are uniformly distributed in domestic environment, which can be a possible vector for transmission of drug-resistant bacteria and food-borne diseases. keywords: periplaneta americana, blattela germanica, garm-negative, bacteria, antimicrobial resistance introduction cockroaches have become most vital pest, are commonly found in association with man in areas where food is prepared and stored, such as restaurants, kitchens, bakeries, and grocery stores (rivault et al. 1993a, vythilingam et al. 1997). these insects live in different environments, like sewage pipes, latrines, garbage, wall slits, baseboards and filthy places, as they are attracted by food, organic waste and fluids regularly discharged in such sites (graczyk tk et al. 2001, william j. bell et al. 2007, allotey j et al. 2009). ). there are two cockroach species commonly found infesting in domestic area, these are the german cockroach (blattella germanica), and the american cockroach (periplanets americana) (rivault et al. 1993a, gratz 1999, graczyk et al. 2005, mpuchane et al. 2005, bell et al. 2007). the *corresponding author: dr alireza zahraeiramazani, e-mail: azahraei@sina.tums.ac.ir http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 11 german cockroach is more prevalent inside the home while the american cockroaches are common around the home and associated with water drainage systems and water pipes (gratz 1999, graczyk et al. 2005, mpuchane et al. 2005, bell et al. 2007, allotey et al. 2009). cockroaches have always been associated with different sites of domestic environments, by food, organic waste and fluids that are regularly discharged in such sites (rivault et al. 1993b, mpuchane et al. 2005). recently more than 40% of cockroach populations are found in domestic areas, because of uncontrolled of domestic sewage, poor sanitation and environment pollution which makes ideal environment for increase the cockroaches, population (gratz 1999, graczyk et al. 2005). furthermore, their feeding mechanisms and filthy breeding habits make them ideal carriers of various pathogenic microorganisms (chaichanawongsaroj et al. 2004, graczyk et al. 2005). more than 100 species of bacteria have been isolated from or passed through cockroaches, which were carried on their rough body parts, cuticle, gut, vomits and feces (cruden and markovetz 1987, allotey et al. 2009, cloarec et al. 1992). cockroaches are mechanical carriers or perfect vector for harbouring and transmitting virulent bacteria associated with food born infections. some of them demonstrate resistance to antibiotics (paul et al. 1992, rivault et al. 1993, mpuchane et al. 2005). transmission could occur by cockroach regurgitation or fecal pellet deposition into human foodstuffs (cochran 1982, bell et al. 2007). although recently several studies of cockroaches in domestic environments have shown the presence of microorganisms spread on their external surfaces, such as enterobacter sp., escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and several other potential pathogens (oothuman et al. 1989, le guyader et al. 1991, prado et al. 2002, graczyk tk et al. 2005, saitou k et al. 2009). although studies with bl. germanica showed that when ps. aeruginosa has been demonstrated to multiply in the gut and excretion of the bacteria continued up to 114 days (fotedar et al. 1993), also salmonella typhi, shigella dysenteriae and toxigenic strains of es. coli can be retained in the gut of cockroaches for up to several days (stek 1982). periplaneta americana harboured more bacteria species than the bl. germanica (fotedar et al. 1992a, rivault et al. 1993, prado et al. 2002, pai et al. 2003). in hospitals, cockroaches have been considered to be a possible carrier of nosocomial infections, especially the transmission of drug-resistant bacteria (fotedar et al. 1991, cotton et al. 2000, prado et al. 2002, saitou et al. 2009), also they play a critical role in transmitting food-borne diseases; such as diarrhea, dysentery, cholera, tuberculosis, and typhoid fever (fotedar and banerjee 1992, graczyk tk et al. 2005, saitou k et al. 2009). cockroaches are possible vectors for carrying tem-type beta-lactamases producing gramnegative bacteria, most often found in es. coli and kl. pneumonia, which showed ampicillin and penicillin resistance (fotedar r 1991, cotton et al. 2000, bradford pa 2001, paterson dl 2003). the domestic pollution and poor sanitation gives the favorable condition to growth and development of population of cockroaches in food preparation areas and multi-family dwellings, the diversity of micro-organisms that they are associated and the fact that some of these bacteria highly resistance to recent antibiotics, which is indicates one of the positive health risk for the human beings (paul et al. 1992, rivault et al. 1993, prado et al. 2002, graczyk tk et al. 2005, saitou k et al. 2009). the high population density with poor disposal of left-over’s, lack of proper sanitation and ideal temperature conditions in varanasi city could serve as a source of cockroaches’ infestation. with that in mind, this study was an attempt to isolate and identify gram-negative pathogenic bacteria from pe. americana http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 12 and bl. germanica in households (kitchen, living room, toilets and bed rooms) and foodhandling establishments (canteens, bakeries, grocery stores, food storage stores, small food processing plants and restaurants), in varanasi city in india. after isolating bacteria from the trapped cockroaches we determined to find their antibiotic resistance patterns, and also we highlighted spatial distribution, diversity and composition of bacteria in domestic cockroaches. materials and methods study area the city of varanasi is located in the middle ganges valley of north india, in the eastern part of the state of uttar pradesh, along the left crescent-shaped bank of the ganges river. the varanasi urban agglomeration consist of seven urban sub-units covers an area of 112.26km2. the urban agglomeration is stretched between 82° 56’e 83° 03’e and 25° 14’n-25° 23.5’n, being located in the indo-gangetic plains of north india (indian meteorology department 2010). varanasi experiences a humid subtropical climate with large variations between summer and winter temperatures. summers are from early april to october, with intervening monsoon. the temperature ranges between 32 °c–46 °c in the summers. winters in varanasi from december to february and temperatures below 5 °c are not uncommon. the average annual rainfall is 1,110mm (indian meteorology department 2010). the catchment’s area consists of 29 towns, with total a population of 3,138,670 (census of india 2001). however, the area under varanasi municipal council (nagar nigam) has a population of 1,100,748 (census of india 2001). also approximately 138,000 people in the municipal area live in slums (census of india 2001). due to the high population density the city produces about 350 million litters per day of sewer and 425 tons per day of solid waste (planning division, central pollution control board, archived, 2006). the solid wastes are disposed in one landfill site and sewage is drains into nearest river (planning division, central pollution control board. archived 2006). sampling and identification of microorganisms in order to select the target study are a stratified random sampling procedure were used. ten places (banaras hindu university and lanka, durgakund and bhelpura, gadowalia, sigra, cantonment railway station, city railway station, dlw, sunderpur, ganga ghats around area, kotwalia) were sampled from the varanasi city according to the geographical layout of the area. in the months of april and may 2011, 203 cockroaches were collected from 44 households (kitchen, living room, toilets and bed rooms) and 52 foodhandling establishments (canteens, bakeries, grocery stores, food storage stores, small food processing plants and restaurants), in varanasi city. these households and foodhandling establishments were selected randomly, in addition the occupants were did not use pesticides in the recent. cockroaches were caught in food-baited pitfall traps describe by patterson (1989). traps were kept on the floor under bead, pantry, cupboards, storage racks and bench for two or three consecutive nights. each trapped cockroaches was placed in a sterile test tube and was subsequently taken to the laboratory. trapped cockroaches were frozen at 0 °c for 5min, and then species of cockroaches were identified under an olympus szx12 dissecting stereo microscope according to harwood and james (harwood et al. 1979). bacterial isolation, identification and enumeration of enterobacteriaceae from external surfaces two milliliters of sterile normal saline (0.9 %) were added to test tube containing one cockhttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 13 roach, and the tube was thoroughly shaken for 2 minute to isolate micro-organisms from the external surface. aliquots (0.01ml) of the washed were then separately inoculated onto the blood agar and macconkey agar plates and incubated overnight at 37 ºc. bacterial colonies of were identified by morphological appearance, macroscopic examination, gram staining, and biochemical tests according to national standard method describe by health protection agency (health protection agency 2007, 2010). the overall load of bacteria carried by each insect was counted and expressed as number and also as a colony forming unit (cfu). antibiotic susceptibility tests the antibiotic susceptibility of ps. aeruginosa, kl. pneumoniae, es. coli, proteus mirabilis, citrobacter freundii, enterobacter aerogenes species to 12 important antibiotics were tested by the kirby-bauer disk diffusion method according to the recommendations of the clinical laboratory standards institute (clsi 2010). these antibiotics (becton dickinson, cockeysville, md, usa) included ampicillin (10g/ disk), gentamicin (10g/ disk), ciprofloxacin (5g/ disk), ofloxacin (5g/ disk), chloramphenicol (30g/ disk), tetracycline (30g/ disk), sulfamethoxazole/ trimethoprim (25g/ disk), cephalothin (30g/ disk), ceftazidime (30g/ disk), imipenem (10g/ disk), piperacillin (100g/ disk), and cefoperazone (75g/ disk). statistical analysis data on the concentration of the gram negative bacteria was entered into excel and transformed into log10 colony-forming units (cfu) of both cockroach species. rates were compared using the chi square test. p< 0.05 was considered to be statistically significant. results cockroach infestation among the 44 households studied, 28(54%) had cockroaches infestation: 16(57%) with pe. americana, 5(18%) with bl. germanica, and 7 (25%) with both species. totally 79 adult cockroaches were collected from households (fig. 1): 52 pe. americana, 27 with bl. germanica. in households, most of the pe. americana were collected from (66.7%) kitchen, also 16.4% in bed room, 9.8% in dining room and less than 3% in toilet and living room. as well as the higher rate of bl. germanica was found in the storage room (41.3%), sleeping room (25.1%) and kitchen (22.6%). the occurrence of bl. germanica in dining room and living room was 4.3 and 6.7%, respectively. a total 52 foodhandling establishments studied, 40(77%) had cockroaches infestation (fig. 2): 21(53%) with pe. americana, 8(20%) with bl. germanica, and 11(28%) with both species. totally 124 adult cockroaches were collected from food-handling establishments: 52 pe. americana, 46 with bl. germanica. in the higher rate of pe. americana were collected from bakeries (36.3%) and grocery stores (42.5%). the collection rate of pe. americana obtained in food storage stores, restaurants and small food processing plants were 12.7, 2.3 and 6.2%, respectively. bl. germanica was highly founded in the food storage stores (28.3%), small food processing plants (18.1%), restaurants (7.6%), also bakeries (16.3%) and grocery stores (29.7%). bacterial isolation among the 203 cockroaches consisting of pe. americana and blattela germanica collected from the households and food-handling establishments, eight different pathogenic species of gram-negative belonging to glucose non-fermenting gram-negative rods and enterobacteriaceae were identified from the cuticles and gust of the cockroaches analyzed (table 1). moreover, 197 (97.04%) cockroaches of pe. americana and blattela germanica were found to carry one or more species of bacteria on the external surface. there was no significant different between http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 14 the overall bacteria load of the external surface in pe. americana (64.04%) and bl. germanica (35.96%), (p> 0.05). the bacterial species were most frequently in food-handling establishments, however few bacterial species were identified from samples in households. among these bacterial pathogens, four bacterial species appeared frequently, isolated in highest numbers from cockroaches trapped around the food-handling establishments, as compared with the households (table 1). these bacteria were klebsiella pneumoniae, escherichia coli, and enterobacter cloacae, which are potential pathogens. although there was no significant different between the overall bacteria load of the external surface in cockroaches found in the food-handling establishments (60.08%) and households (39.92%), (p> 0.05) (table 1). antibiotic resistance the results of the antibiotic sensitivity tests showed that, among the 6 gram-negative pathogenic bacteria species studied, had resistance to 8 of 12 antibiotics (66.66%) were tested (table 2). including ps. aeruginosa and proteus mirabilis had resistance to 7 of 12 antibiotics tested, kl. pneumoniae and enterobacter aerogenes to 4 and extendedspectrum beta lactamase (esbl), escherichia coli to 7 and citrobacter freundii to 6 (table 2). however, kl. pneumoniae and ps. aeruginosa were the most prevalent, drug-resistant strains were isolated from the cockroaches with 100% resistance to sulfamethoxazole/trimethoprim and ampicillin. for individual strains of bacteria, es. coli was found to have multi-resistance to four antibiotic tested, citrobacter freundii four, enterobacter aerogenes and proteus mirabilis to three (table 2). an overall sensitivity to imipenem, cefoperazone, ciprofloxacin and ofloxacin also was observed. table 1. gram-negative bacteria isolated from periplaneta americana (n= 130) and blatella germanica (n= 73) collected in households and food-handling establishments bacteria cockroaches periplaneta americana blatella germanica households (n= 52) food-handling establishments (n= 78) households (n= 27) food-handling establishments (n= 46) no* % mean load* no* % mean load* no* % mean load* no* % mean load* klebsiella pneumoniae 7.0 13.5 4.2 x 107 17.0 21.8 3.9 x108 7.0 25.9 4.1 x107 11.0 23.9 3.7 x108 escherichia coli 9.0 17.3 3.5 x 106 14.0 17.9 4.0 x107 5.0 18.5 3.9 x106 8.0 17.4 4.1 x106 enterobacter aerogenes 7.0 13.5 3.2 x 105 5.0 6.4 3.1 x104 3.0 11.1 3.9 x106 6.0 13.0 3.9 x106 enterobacter cloacae 5.0 9.6 3.6 x104 11.0 14.1 3.5 x106 2.0 7.4 3.0 x105 5.0 10.9 3.4 x106 salmonella spp 6.0 11.5 3.4 x 106 5.0 6.4 3.3 x105 4.0 14.8 3.8 x105 5.0 10.9 4.1 x105 citrobacter freundii 5.0 9.6 4.0 x 104 9.0 11.5 4.3 x105 3.0 11.1 3.9 x105 4.0 8.7 4.2 x104 proteus mirabilis 6.0 11.5 3.1 x 104 7.0 9.0 3.4 x105 2.0 7.4 3.1 x104 3.0 6.5 3.6 x 105 pseudomonas aeruginosa 7.0 13.5 4.3 x 106 10.0 12.8 4.0 x108 1.0 3.7 4.1 x106 4.0 8.7 3.8 x 105 total 52.0 100.0 78.0 100.0 27.0 100.0 46.0 100.0 *one insect may simultaneously harbour more than one species of bacteria. *mean concentration of gram negative bacteria (cfu) http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 15 fig. 1. distribution (%) of the cockroaches infestation at households fig. 2. distribution (%) of the cockroaches infestation at foodhandling establishments table 2. antimicrobial resistance of gram-negative bacteria isolated from cockroaches collected in households and food-handling establishments gram-negative bacteria antimicrobial resistance (%) amp pip cp crp te sxt caz cfp imp gm cip ofx pseudomonas aeruginosa (n=22) 100 0 60.1 50 51.6 100 0 0 0 28.6 0 0 klebsiella pneumonia (n=42) 100 0 0 0 52.3 95.4 0 0 0 24.5 0 0 escherichia coli (n=36) 58.4 13.7 53.4 10.3 82.1 22.3 8.6 0 0 0 0 0 citrobacter freundii (n=21) 46.7 7.3 0 6.3 24.1 23.4 15.3 0 0 5.6 0 0 enterobacter aerogenes (n=21) 78.6 0 0 0 50.1 91.3 0 0 0 17.5 0 0 proteus mirabilis (n=18) 66.3 21.3 20.1 12.4 27.4 37.8 0 0 0 7.3 0 0 abbreviations: amp: ampicillin, pip: piperacillin, cp: cephalothin, crp: chloramphenicol, te: tetracycline, sxt: sulfamethoxazole/ trimethoprim, cfp: cefoperazone, caz: ceftazidime, imp: imipenem, gm: gentamicin, cip: ciprofloxacin, ofx: ofloxacin. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 16 discussion cockroaches are among the most notorious pests, having nocturnal and filthy habits, which do not only contaminate food by indiscriminate deposition of faecal materials and cause food poisoning but also transmit bacteria and other pathogenic microorganisms in infested areas (kopanic 1994, czajka et al. 2003, william j et al. 2007). these are of major concern for human health due to their capacity to act as a potential mechanical vector for transmitting more than 50 disease causing microorganisms (cloarec et al. 1992, rivault et al. 1993a, william j et al. 2007). in this study, there were 8 species of human pathogenic gram-negative bacteria isolated from the external bodies of two species of cockroaches (pe. americana and bl. germanica) from the domestic area in varanasi city. the bacterial species most frequently identified from cockroaches are gram-negative bacilli, especially in the family enterobacteriaceae (fotedar et al. 1991, cloarec et al. 1992, rivault et al. 1993). in agreement with those studies, we also collected seven different species of bacteria belonging to family enterobacteriaceae, kl. pneumoniae, es. coli, en. aerogenes, en. cloacae, salmonella spp., citrobacter freundii, and proteus mirabilis, carried by the cockroaches from households and food-handling establishments. these species can cause urinary tract and wound infections, typhoid, diarrhoea, pneumonia, gastroenteritis and respiratory infections, among others diseases in humans (graczyk et al. 2005, williams et al. 2010). however, we found that makeup of the microbial population of the pe. americana was considerably higher than the bl. germanica, but there was no significant different between the overall bacteria load on the external surface in these two species of cockroaches. it may depend with their size and as well as the sanitation condition of domestic environment (william et al. 2007). however the predominant bacteria on cockroaches were kl. pneumonia, es. coli, en. aerogenes, and ps. aeruginosa, as same with other numerous investigations (rivault et al. 1993a, mpuchane 2005, pai et al. 2005). these results coincide with those found by pai et al. (2005), there were no relative differences between the species of bacterial strains found in pe. americana and bl. germanica. it was assumed that, cockroaches were mechanically transmitting pathogens obtained in the environment and were capable of traveling while harboring these bacteria. cockroaches are associated with an outbreak of food-poisoning, gastro-enteritis and dysentery (mackerras and mackerras 1949, rueger and olson 1969, burgess and chetwyn 1981, s mpuchane 2005). in urban area of varanasi, we found that distribution of these bacteria among the households and food-handling establishments was no significantly different. however, most of the pe. americana collected from kitchen, bakeries and grocery stores, where as higher rate of bl. germanica founded in the storage room, small food processing plants and restaurants. these results are in conformance with those found by mpuchane et al. (2005). cockroaches, especially species that come in contact with faeces like german cockroaches may transmit bacteria responsible for food poisoning (cochran 1982, rivault et al. 1993b). they are attraction to human and animal faeces, rotting food, secretions from corpses, sputum, pus, and the like gives them a well-earned disgust factor among the general public (rivault et al. 1993b, s mpuchane et al. 2005, william et al. 2007, sudershan et al. 2009). these finding suggest the, these bacteria species are uniformly distributed among the both places and especially this two cockroaches species are importance of transmitting food-borne diseases. cockroaches may act as reservoirs for antibiotic resistant virulent bacteria found on dohttp://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 17 mestic environment, which may cause serious health problems for human beings (pai et al. 2005, ahmad 2011). similar with this, in our study 6 gram-negative bacteria strains, obtained from the households and food-handling establishment environments, showed resistant to antibiotics, ampicillin, tetracycline and sulfamethoxazole/ trimethoprim. although, we have found that lower activity of ampicillin and tetracycline against these bacterial strains, kl. pneumoniae, ps. aeruginosa and es. coli, may essentially due to the natural resistance of the bacterial species (navarro risueño et al. 2002). moreover, the antibiotic resistant strain of kl. pneumoniae bacteria that cause pneumonia has been found in patients and cockroaches in a new delhi hospital (fotedar 1991). also, the less ssusceptibility of es. coli and citrobacter freundii against ceftazidime was may due to the extended-spectrum b-lactamases enzyme activity (emery et al. 1997). but the result of the test was not agreement with pai hh (2005). however, the bacteria isolated from the cockroaches were relatively resistant to gentamicin. the results of were not in agreement with pai et al. (2005), but it coherence with those found by marinésia (2006). also recently we have found that ps. aeruginosa isolated from wound infection patients at different ward of sri sundaralala hospital, in a banaras hindu university, showed resistant to all commonly use antibiotics (anupurba s 2006, basu s 2009). this low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes, inadequate use of antibiotics and the low permeability of the bacterial cellular envelopes (poole 2004, basu 2009). we observed that activity of imipenem and ciprofloxacin against gram-negative bacteria, showed 100% sensitivity. the results are in accordance to those found by pai et al. (2005). based on our study, cockroaches are uniformly distributed among the places of households and food-handling establishments in varanasi area. we also determined that both species of pe. americana and bl. germanica, have equal potential to carry the pathogenic bacteria as a vector in their external surface. moreover, bacteria species were studied showed relative resistance to antibiotic tested in our study. it may possible threat for human health. conclusion most of the domestic area in varanasi city, especially food-handling establishments and human dwellings, are highly polluted with domestic pollutants, which play a key role for the transmission of a wide range of pathogenic bacteria and also provide ideal conditions for the breeding and multiplication of cockroach. acknowledgements this research was supported in part by a summer research project grant (fss/20102011/3625) from vice chancellor research found, banaras hindu university, varanasi, india. we are grateful to all who facilitated our field work; special appreciation is also given to dr tanittha chatsuwan, faculty of medicine chulalongkorn university, for her special guidance in bacterial identification. also special thank is given to coordinator industrial microbiology, centre of advanced study in botany, faculty of science, banaras hindu university, for enamors support in various issues. the authors declare that there is no conflict of interest. references allotey j, mpuchane s, gashe ba, simpanya m, matsheka i (2009) trapping of blattella germanica (l) populations in human dwellings in gaborone, botswana. j appl zoo res. 20(2): 175–188. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 10–20 dl wannigama et al.: prevalence and … 18 anupurba s, bhattacharjee a, garg a, sen mr (2006) antimicrobial susceptibility of pseudomonas aeruginosa isolated from wound infections. indian j dermatol. 51: 286–288. aqeel ahmad, anuradha ghosh, coby schal, ludek zurek (2011) insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. bmc microbiology. 11:23doi:10.1186/14712180-11-23. basu s, ramchuran panray t, bali singh t, gulati ak, shukla vk (2009) a prospective, descriptive study to identify the microbiological profile of chronic wounds in outpatients. ostomy wound manage jan. 55(1): 14–20. bradford pa (2001) extended spectrum lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. clin microbial rev. 48: 933–951. burgess nrh, chetwyn kn (1981) association of cockroaches with an outbreak of dysentery. trans r soc trop med hyg. 75: 332–333. census of india (2006) (provisional). 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http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 60 original article evaluation of repellency effect of essential oils of satureja khuzestanica (carvacrol), myrtus communis (myrtle), lavendula officinalis and salvia sclarea using standard who repellency tests *mohammad hassan kayedi 1, ali akbar haghdoost 2, ali salehnia 3, kiumars khamisabadi 4 1razi herbal medicines research center and department of parasitology, school of medicine, lorestan university of medical sciences, khorramabad, iran 2research center for modeling in health, kerman university of medical sciences, kerman, iran 3khorraman herbal medicines pharmaceutical company, khorramabad, iran 4national health center, tehran university of medical sciences, tehran, iran (received 21 feb 2012; accepted 30 sep 2013) abstract background: using special lotions and repellent sprays on skin is one of the effective methods to prevent arthropods biting which was verified in this study. methods: essential oils of four plants (satureja khuzestanica, salvia sclarea, lavendula officinalis and myrtus communis) were separately extracted by clevenger used hydro distillation method. then separated solutions with 10%, 20% and 40% concentrations of essential oils of plants in 99.6 % ethanol were prepared. who guidelines for efficacy testing of mosquito repellents for human skin were used on different concentrations of essential oils of plants, traditional repellents (deet, 50% and 33%) as positive control, and ethanol 99.6% and naked hands as negative controls. results: in negative control groups, the number of bits were comparable (p= 0.42) and had decreasing time trends (naked hands p= 0.011, ethanol p< 0.001). in all time points, minimum bites were observed in traditional repellents and it was significantly less than the other groups (p< 0.001). the time trend in the number of bites in the other groups was positive and showed minimum number of bites in time zero in all groups. we also found that the concentration of repellents had association with the number of bites. the maximum and minimum numbers of bites were observed with 10% and 40% concentrations respectively in all groups. conclusion: essential oils of salvia sclarea, lavendula officinalis and myrtus communis have repellency effect, even with 10% concentration of essential oils. keywords: malaria, repellents, anopheles stephensi, essential oil, plants introduction using of special lotions and repellent sprays on skin is one of the effective methods to prevent arthropods biting. however, research to find more effective repellents with minimum side effects is a very attractive research field for scientists with different backgrounds. as an example, deet (n, n-diethyl-3-methyl benzamide) is one of the most effective repellents used worldwide for six decades (katz et al. 2008). however, similar to other repellent, it might have irritant effects mainly on sensitive skins or other side effects (antwi et al. 2008, katz et al. 2008). in addition, the maximum efficacy of these substances varies between one up to twelve hours based on their ingredients and concentrations (katz et al. 2008). until now the environmental protection agency’s has approved deet, picaridin, mgk-326, mgk-264, ir 3535, oil of citronella, and oil of lemon eucalyptus as insect *corresponding author: dr mohammad hassan kayedi, e-mail: kayedi78@yahoo.co.uk http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 61 repellent (ir) ingredients for application to the skin (katz et al. 2008). based on the above explanation, continuously search is needed to find more potent materials with minimum side effects and longer durability. among these studies, research on the repellency effect of essential oils and national products of plants has a very unique position. satureja khuzestanica, salvia sclarea, lavendula officinalis and myrtus communis grow in west of iran, across zagros mountains and other parts of the country. insecticidal and larvicidal effects of different doses of essential oils of s. khuzestanica and m. communis have been reported (uehleke and brinkschulte-freitas 1979, traboulsi et al. 2002, yang et al. 2004, yi et al. 2006, cetin et al. 2007, martinez-romero et al. 2007, knio et al. 2008, amer and mehlhorn 2009). other researchers have stated antimicrobial, antifungal and antiparasite effects of mentioned plants and essential oils of two other plants (l. officinalis and s. sclarea) that grow in iran (hayder et al. 2004, shahidi-bonjar 2004, sonboli et al. 2006, pinto et al. 2006, ben arfa et al. 2006, appendino et al. 2006, mahdi et al. 2006, burt et al. 2007, cristani et al. 2007). repellency effect of essential oil of l. officinalis has been reported (nazemi rafi and moharamipour 2008). local people in some districts of lorestan province in west of iran (zagross mountains), use dried leafs of local plant “s. sclarea” inside their pillows. they believe that the plant protect them from biting of insects during night (ali salehnia, personal communication). although scattered evidence is available about the repellency effects of the mentioned four substances, we did not find any studies that compare their effects using standard methodology. so, based on above explanations we evaluated repellency effect of essential oils of 4 mentioned plants using standard who repellency tests. materials and methods extraction method l. officinalis (ostoqudoos, local name, family: lamiaceae) is a decorative plant and is used for decorations of parks and gardens. we bought dried leafs and flowers of mentioned plant from hamedan city in west of iran (latitudes 34.8000 and longitudes 48. 5167). m. communis (moort, local name, family: myrtaceae) was collected around the khorramabad city in west of iran (latitudes 33.4841 and longitudes 48.3525). this plant grows in west of iran. the name, species and family of selected parts of plants were confirmed by academic staffs of research center of agricultural department, lorestan province (shahla ahmadi, personal communication). leafs and flowers of l. officinalis and leafs of m. communis were dried and grind. essential oils of plants were separately extracted by clevenger (model bp, british pharmacopoeia, manufacturer ashke shisheh company, iran and mantle model h610 manufacturer fater company, iran) used hydro distillation method at 60 ºc. duration of extraction was 1 hour for each 0.5cc essential oil. sodium solfate was used for dehydration (stahl-biskup and saez 2002, okoh et al. 2010, gavahian et al. 2012). all above procedures were carried out in laboratories of razi herbal medicines research center, lorestan university of medical sciences. we extracted respectively 3cc, 1.3cc and 0.5cc pure (about 100%) essential oils of 100gr of dried flowers and leaves of l. officinalis and dried leaves of m. communis. essential oils of s. khuzestanica (carvacrol, family: lamiaceae), and s. sclarea (salvia, family: lamiaceae) were bought from khoraman pharmaceutical company, khorramabad, iran. this company culture s. khuzestanica and extracts its essential oil (carvacrol) for medicine marketing. s. khuzestanica grows in west of iran (south of lorestan and north http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 62 of khuzestan provinces) and s. sclarea grows in west of iran. the mentioned company extracted respectively 3cc and 0.5cc pure (about 90% to 94%) essential oils of 100 gr of dried leaves of s. khuzestanica and s. sclarea (ali salehnia, personal communication). all essential oils were kept in 4 ºc. repellency tests who guidelines for efficacy testing of mosquito repellents for human skin were used (who 2009). separate solutions with 10%, 20% and 40% concentrations of essential oils of plants in 99.6% ethanol were prepared. traditional repellents (deet, 50% n, n-diethyl-3-methyl benzamide) and 33% n, n-diethyl-3-methyl benzamide and negative control (pure ethanol, 99.6%) were bought respectively from nomad, turnpike lane, london n8 opx, medical limited and school of pharmacy, shiraz university of medical sciences in iran. the essential oils, traditional repellents as gold standard repellents (positive controls) and negative controls were rubbed on right and left forearms of four eligible volunteers from elbow to wrist and their hands were protected by gloves to make it unattractive to mosquitoes. the whole forearm exposed in a mosquito cage (50cm× 50cm×50cm) with 50, 2 or 3 days old females of susceptible strain of an. stephensi reared in insectarium, unfed and hungry. hands were kept inside the cage for 45 seconds and numbers of bites were recorded. this procedure was repeated 3 times. negative controls (naked hands and hands rubbed with pure ethanol) and positive controls (deet, 50% n, n-diethyl-3-methyl benzamide and shiraz 33% n, n-diethyl-3methyl benzamide) were used in this experiments as well. all tests were repeated every hour up to five hours after the first tests. the used mosquitoes for tests changed in every test (who 2009). repellency tests were started at 5.30am and continued to 10.30am. we used 3 cages of an. stephensi per day each cage had 50 mosquitoes. tests were carried out in a half-dark room (27 ºc and 70% humidity) near insectary. the tests were carried out in bandar abbas health research site affiliated to national institute of health, tehran university of medical sciences. this research has been approved by ethical committee of lorestan university of medical sciences. statistical analysis with respect to the ethical considerations, we minimized the number of humans that take part in the project, and the first author of the paper personally participated in the trial. however, in order to maximize the statistical power of our analysis and according to who guidelines for efficacy testing of mosquito repellents for human skin, we repeated each trail three times, with six measurements over 5 hours. the data were computerized using spss version 11.5. the number of bites in each experiment was the main dependent variable. we checked the distribution of the number of bites in each concentration and each group by comparing the 95% confidence intervals of their skewness and kurtosis with their values in a normal distributed variable; since we did not find any significant difference in these analyses, we used parametric tests to compare the number of bits groups and concentrations. we used repeated measure anova to assess the effect of time and also the type of repellents. in addition, the linear trends in the number of bites were computed using regression model. in all statistical analysis, 0.05 was considered as significant level. results the repellency effects of nomad (deet, 50% n, n-diethyl-3-methyl benzamide) and shiraz (33% n, n-diethyl-3-methyl benzamide) in all tests were comparable up to 5 hours http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 63 (p= 0.571), so we decided to incorporate these two traditional groups to improve the power of statistical tests as gold standard positive control (dta). based on the results of few first tests with essential oil of s. khuzestanica, side effects were considerable, mainly the burning, reddening of skin, and irritation for several hours after rubbed on forearms, so essential oil of s. khuzestanica was dropped from repellency tests. figure 1 shows the marginal means of the number of bites per session (adding together the results of different concentrations based on the results of multivariate analysis) of an. stephensi up to 5 hours classified by the type of repellents and control groups. based on these findings, in negative control groups, the number of bits were comparable (p= 0.42) and had decreasing time trends (naked hands p= 0.011, ethanol p< 0.001), started from around 30 bites in time zero, reached to around fourteen in negative control group (naked hands), and six in another negative control group (ethanol, 99.6% ) after five hours. in all time points, minimum bites were observed in dta group (less than 2 bites) and it was significantly less than the other groups (p< 0.001). the time trend in the number of bites in the other groups was positive and showed minimum number of bites in time zero in all groups (less than 2 bites). however, this trend in l. officinalis was more prominent (the average of bites in time five was 9.8, p= 0.001), while the corresponding numbers in s. sclarea and m. communis were less than four (p= 0.04 and p= 0.011) respectively. the regression coefficients for the effect of time (which shows the average of changes in the number bites between two consecutive measures) were 0.29, 0.40, 0.58, and 0.20 for s. sclarea, m. communis, l. officinalis and dta (as positive control) respectively. while these coefficients for negative controls (naked hands and pure ethanol) were -2.88 and -3.60. there were significant difference in the number of bites between negative controls and other repellents (p< 0.001). in time five, the difference between negative controls and m. communis was significant (p= 0.04). however, the other groups had significant difference only with negative control groups in time five (p< 0.05). we also found that the concentration of repellents had association with the number of bites (fig. 2). the maximum and minimum numbers of bites were observed with 10% and 40% concentrations respectively in all groups. in salvia and m. communis groups, only the difference between 10% and 40% concentrations were significant (p= 0.032 and p= 0.015 respectively). while in l. officinalis even the difference between the minimum and maximum concentrations had borderline p-value (0.063). fig. 1. the time trends in the number of bites per session classified by the type of repellents and control groups http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 64 fig. 2. comparison between numbers of bites in different concentrations classified by the type of repellents discussion our results showed that the essential oils of m. communis, l. officinalis and s. sclarea have repellency effects for an. stephensi. although by time, their repellency effect decreased but even after five hours, their effects were more than the effects in control groups. we found a sharp decreasing trend in the number of bites in negative control groups. this decreasing trend might be explained by this fact since we started our experiments very early morning and continued up to 10.30am. however, this decreasing trend was not observed in the other groups which might be due to the attenuation in the repellency effects of these substances. by time, the repellency effect of essential oils of plants was reduced but even after five hours still they had significant effect more or less comparable with the effect of dta which is a known and standard repellent. although the maximum effect was observed in samples with 40% concentration, even the effects of samples with 10% concentration were significant and comparable with higher concentrations. this means that for further studies 10% concentration can be used to assess the repellency effects of these substances or even similar ones. these findings are consistent with a similar study carried out by tavassoli et al. (2011). although their methods was different with our methods, they reported that repellency efficiency of 50% concentration of essential oil of m. communis (myrtle) was moderate and lower than deet. other researchers have studied repellency effects of other plants in iran. oshagi et al. (2003) evaluated repellency effect of extracts and essential oils of citrus limon burm, (lemon) and melissa officinalis, (balm) using an. stephensi in laboratory on animal and human in comparison with deet. they showed significant differences between oils and extracts (p< 0.05) and reported that difference between deet and lemon oil was not significant. they recommended lemon essential oil as an alternative to deet. in another study vatandoost and hanafi-bojd (2008) compared the repellency effect of 3 topical repellents (permethrin, deet and neem tree extract) against an. stephensi in iran. they reported that:” major heterogeneity of response was observed using deet. although neem was http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 65 the least effective agent, extracts of locally produced neem oil offer a promising repellent against mosquito biting”. repellency effect of eucalyptus based plant has been shown by researchers in different trials in the bolivian amazon (moore et al. 2002), china (yang and ma 2005) and guinea bissau (palsson and jaenson 1999). ansari et al. (2005) reported that pine oil (pinus longifolia, family: pinaceae) which is used by people in india for protection against mosquito bites provide 100% protection against an. culicifacies up to 11 hours and 97% protection against culex quinquefasciatus for nine hours. they recommended that aromatic oil of citronella and lemon eucalyptus may be promoted as a repellent. two investigators reported repellency effect of neem tree extract from trials in iran (vatandoost and hanafi-bojd 2008) and bolivian amazon (moore et al. 2002). assessment of the repellency effects of essential oils of plants is a very attractive field for entomologists around the world (palsson and jaeson 1999, curtis et al. 2002, oshaghi et al. 2003, seyoum et al. 2003, omolo et al. 2004, innocent et al. 2008, vatandoost and hanafi-bojd 2008). many researchers have carried out trials on repellency effect of different plants. in a study 41 plant extracts and 11 oil mixtures were evaluated using aedes aegypti, an. stephensi and culex quinquefasciatus on human volunteers. they reported litsea (litsea cubeba), cajeput (melaleuca leucadendron), niaouli (melaleuca quinquenervia), violet (viola odorata), and catnip (nepeta cataria) that have been most effective oils of plants with 100% repellency against three mentioned mosquitoes and up to 8 hours protection time (amer and mehlhorn 2006). investigators reported repellency effect of six plant species that grow in the kenyan coast (odalo et al. 2005), three plant species that grow in western kenya (seyoum et al. 2003) and six plant species that grow in the kenya (odalo et al. 2005). their insecticidal and larvicidal effects have been documented, for example, the essential oils of s. khuzestanica and m. communis had significant larvicidal and insecticidal effects (traboulsi et al. 2002, amer and mehlhorn 2006, yic g et al. 2006, cetin et al. 2007, martinez-romero et al. 2007). nonetheless, their toxic effects might be a point of concern particularly after high dose of oral consumption (uehleke and brinkschulte-freitas 1979). jaenson et al. (2006) reported that: lavandula angustifolia (lavender oil) and pelargonium graveolens (geranium oil) have 100% repellencies against host-seeking nymphs of ixodes ricinus when diluted to 30% in 1.2 propanediol. his results were similar to our results with l. officinalis. a study that was carried out in iran by nazemi and moharamipour (2008), showed the repellency effect of l. officinalis on red flour beetle, tribolium castaneum. they showed that after five hours the repellency effect was still considerable and significant, also they showed positive association between the concentration and repellency effect. these results were consistent with our results in the present study. conclusion we found that the different concentrations (40%, 20% and 10%) of essential oils of s. sclarea, l. officinalis and m. communis had considerable repellency effect on an. stephensi even with low concentration and after five hours. these findings might open windows for formulating new repellent with less side effect and longer durability. acknowledgements the authors would like to thank the deputy of research affairs of lorestan university of medical sciences, for funding the research, dr bahram delfan, lorestan university of medical sciences for his suggestions on herbs http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 66 and their collaborators abbas pakari, hassan jawdan and mossa hatami in bandar abbas health research center affiliated to national health center, tehran university of medical sciences for their help in repellency tests and marzieh rashidi our collaborator in razi herbal medicines research center, lorestan university of medical sciences for her assistance in extraction and preparation of essential oils of plants and other collaborators for their assistance during experimental studies. the authors declare that there is no conflict of interest. references amer a, mehlhorn 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(diptera: culicidae). pest manag sci. 58(5): 491–495. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, june 2014, 8(1): 60–68 mh kayedi et al.: evaluation of repellency … 68 tavassoli m, shayeghi m, abai mr, vatandoost h, khoobdel m, salari m, ghaderi a, rafi f (2011) repellency effects of essential oils of myrtle (myrtus communis), marigold (calendula officinalis) compared with deet against anopheles stephensi on human volunteers. iran j arthropod-borne dis. 5(2): 10–22. uehleke h, brinkschulte-freitas m (1979) oral toxicity of an essential oil from myrtle and adaptive liver stimulation. toxicology. 12(3): 335–342. vatandoost h, hanafi-bojd aa (2008) laboratory evaluation of 3 repellents against anopheles stephensi in the islamic republic of iran. east mediterr health j. 14(2): 260–267. who (2009) guidelines for efficacy testing of mosquito repellents for human skin. who/cds/ntd/whopes/2009.4. yang yc, lee hs, clark jm, ahn yj (2004) insecticidal activity of plant essential oils against pediculus humanus capitis (anoplura: pediculidae). j med entomol. 41(4): 699–704. yang p, ma y (2005) repellent effect of plant essential oils against aedes albopictus. j vector ecol. 30(2): 231–234. yi cg, choi br, park hm, park g, ahn yj (2006) fumigant toxicity of plant essential oils to thrips palmi (thysanoptera: thripidae) and orius strigicollis (heteroptera: anthocoridae). j econ entomol. 99(5): 1733–1738. http://jad.tums.ac.ir published online: december 18, 2013 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 24 original article ecological status of phlebotomine sandflies (diptera: psychodidae) in rural communities of northeastern algeria nacira kabbout 1, 2, djemoi merzoug 1, 2, *haroun chenchouni 3 1laboratoire des ressources naturelles et aménagement des milieux sensibles, larbi ben m’hidi university, oum el bouaghi, algeria 2department of nature and life sciences, faculty of exact sciences and nature and life sciences, larbi ben m’hidi university, oum el bouaghi, algeria 3department of nature and life sciences, faculty of exact sciences and nature and life sciences, university of tebessa, tebessa, algeria (received 16 june 2013; accepted 19 sep 2014) abstract background: algeria is among the most affected mediterranean countries by leishmaniasis due to its large geographic extent and climatic diversity. the current study aimed to determine the ecological status (composition and diversity) of phlebotomine sandfly populations in the region of oum el bouaghi (northeast algeria). methods: an entomological survey was conducted during the period may–october 2010 in rural communities of oum el bouaghi. catches of sandflies were carried out using sticky traps in both domestic and peri-domestic environments of 16 sites located beneath two bioclimatic areas, sub-humid and semi-arid. most of these sites have visceral and/or cutaneous leishmaniasis cases. results: a total of 1,363 sandflies were captured and identified. they belong to two genera, phlebotomus and sergentomyia, and five species. the species phlebotomus perniciosus, p. perfiliewi and sergentomyia minuta were constants. phlebotomus longicuspis was common and p. papatasi was accidental in the study sites. p. perniciosus and p. perfiliewi are the two possible species that contribute in leishmaniasis transmission across the study area due to their high densities (96 and 49 specimens/m²/night, respectively); these two species dominate other species in all study sites. conclusion: findings emphasize the key-role played by p. perniciosus, p. perfiliewi and s. minuta in outlining site similarities based on sandfly densities. the study confirms that the more susceptible sites to leishmaniasis, which hold high densities of these sandflies, were located south of the study area under a semi-arid climate. keywords: phlebotomine sandflies, leishmaniasis, ecological aspects, algeria introduction phlebotomine sandflies, vectors of leishmaniasis, are nematoceran diptera of the family psychodidae where they constitute the subfamily phlebotominae that includes about 900 species widely distributed over tropical and temperate regions, of which no more than 70 have been implicated in leishmaniasis transmission (ready 2013). these insects have been studied with considerable attention and they took a major global importance because of their role in the transmission of pathogens responsible for bartonellosis, arboviroses and leishmaniasis (dolmatova and demina 1971, tesh 1988, depaquit et al. 2010). according to kamhawi (2006), about 350 million people are at risk of contracting the leishmaniasis and some 2 million of new cases occur each year, mostly in developing countries. in algeria, 22 species of sandflies are recorded and identified, with 10 belong to the genus sergentomyia frança and parrot 1920 *corresponding author: dr haroun chenchouni, e-mail: chenchouni@gmail.com http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 25 and 12 to the genus phlebotomus rondani 1840 (belazzoug 1991). the geographical distribution of sandfly species seems to be closely related to the type of climate, and consequently the distribution of leishmania transmitted by those species, which have established host-specificity in the relationship of vector–parasite such as p. papatasi scopoli 1786 with leishmania major yakimoff and schokhor 1914 and p. perniciosus newstead 1911 with leishmania infantum nicolle 1908. this feature outlines clinical aspects of leishmaniasis worldwide and allows defining risk areas corresponding to the vector in question. like all mediterranean countries, both canine (lcan) and human (hl) leishmaniasis affect algeria. the first type concerns the entire country with parasitic prevalence that varies from one region to another (harrat et al. 1995). the hl is prevalent in two distinct forms, visceral leishmaniasis (vl) and cutaneous leishmaniasis (cl). the latter is observed in three clinical aspects caused by three different parasites: (i) in the steppe and saharan regions rife the zoonotic cutaneous leishmaniasis (zcl) of l. major mon-25 where phlebotomus papatasi is the vector (sergent et al. 1926, izri et al. 1992) and gerbillidae rodents, especially meriones shawi and psammomys obesus are the main reservoir of the disease (belazzoug 1983, belazzoug 1986, izri et al. 1992, harrat and belkaid 2003, boudrissa et al. 2012), (ii) the zcl caused by l. infantum, where the responsible zymodemes are mon-1, mon24, mon-80 (belazzoug 1982, belazzoug et al. 1985, harrat et al. 1995, harrat et al. 1996) is located in northern algeria under sub-humid and semi-arid bioclimatic conditions where it is transmitted by p. perfiliewi parrot 1930 (izri and belazzoug 1993) and domestic dogs are the main reservoir (maroli et al. 1988, benikhlef et al. 2001, benikhlef et al. 2004), (iii) the third form is the zcl due to l. killicki wright 1903 mon-301 discovered in 2005 in southern algeria at ghardaia (harrat et al. 2009) and a new variant enzymatic of l. killicki, mon-306 that has been recently identified in annaba at the extreme northeastern of algeria (mansouri et al. 2012). regarding the human visceral form (vl), the responsible zymodemes are mon-1, mon-24, mon-33, mon-34, mon-78 and mon-80 (harrat et al. 1996). while the proven vector is p. perniciosus (parrot et al. 1930, ben ismail et al. 1987, izri et al. 1990, belazzoug 1992, izri et al. 1992), the main reservoir is the dog, which was found infested with the zymodemes mon-1, mon34, mon-77 and mon-24 (benikhlef et al. 2001, benikhlef et al. 2004). these zoonotic diseases were observed in 41 of 48 provinces in the country, which are undergoing in the last decades an epidemiological upheaval with outbreaks of new infection foci of l. major. the distributional range of these foci is extending to coastal areas in the north, since it crossed the barrier of the tellian atlas range for the first time in 2004 (boudrissa et al. 2012). a marked increase in the number of cases of both cutaneous and visceral leishmaniasis, such as those recorded from 2000 to 2004, where 30,541 cases of cl and 584 cases of vl were reported at national scale. this increase in infections is probably explained by the extension of classical foci on one hand and the emergence of new foci across the country on the other hand (insp 2011). similarly, the department of health and population (dhp) at the region of oum el bouaghi (oeb) in northeastern algeria reported increasing numbers of cl and ivl “infantile visceral leishmaniasis”, where these diseases are widespreading on the majority of municipalities. within this context, the present study focuses on the determination of ecological status of sandfly populations in the region of oeb, particularly by answering the followhttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 26 ing questions: what is the composition and diversity of the sandfly fauna of the area studied? what are their densities and frequencies of occurrence in different localities of the region? what are the spatial similarities of sandfly species composition and density of these communities? this systematic and ecological study aims to provide information on phlebotomine populations to guide the establishment of control and preventive programs against leishmaniasis in this region through delineating the relationships between the spatial distribution of these insects and that of leishmaniasis. materials and methods study area and sampling sites the territory of the province of oum el bouaghi is located between 35°24' to 35°14' north for latitude and 5°59' to 7°56' east for longitude, with an altitude ranging between 700 and 1000 m above sea level. located between the tellian atlas to the north and the saharan atlas to the south, it occupies a central position at the region of hauts-plateaux in east algeria. administratively, the province of oeb consists of 12 sub-provinces “daïra” and 29 municipalities, with a total area of 6,783 km² (fig. 1). the general climate is continental mediterranean semi-arid. the summers are hot and dry and winters are cold. the utilized agricultural area (uaa), with an area of 361,688 ha, represents 62.36 % of the total agricultural area, including 35,520 ha of irrigated lands. this outlines it high potential for agricultural development, including wheat and barley that are cultivated without irrigation on these vast high plains. in this region, the study concerned 16 rural sites located under semi-arid and sub-humid bioclimatic zones (fig. 1). among these sites, some affected by the one or the other form of leishmaniasis (cl and lv), otherwise the two forms at a time, other sites until 2010 were free of these diseases. trapping techniques entomological surveys were conducted at each site at irregular intervals between may and october 2010 using the method of sticky traps. each trap consists of a sheet of extra white paper (21 × 29.7 cm) impregnated with purified castor oil. this method allows the census of insects and exploring resting and egg-laying habitats that stretch over large areas. it is very interesting in quantitative sampling, because it significantly reduces the personal coefficient inherent for manual techniques (rioux et al. 1967). at each study site, a variable set of traps (5–20 traps) were installed simultaneously in domestic and peri-domestic habitats: (i) domestic habitats consist mainly of the courtyards where people generally spend the night during the hot season. the oily adhesive papers “sticky traps”, strained or in garland were hung on trees, outside housewindows and all high openings, (ii) in the peri-domestic habitats and in various locations spread around homes (stables, halfcovered sheepfolds, rocks, ruined chambers used to collect manure or straw stock), sticky traps were placed on the walls, hanging to the roof, put in tight and sometimes profound spaces, or left between rocks. various domestic animals are raised there: dairy cows, sheep, goats, donkeys, dogs, cats, chickens and guinea fowls. placed at any time of the day, traps were retained in the selected locations and then recovered after one to three nights of sampling. the number of traps retrieved with catches varied in each sampled point. because some traps were lost or destroyed, sometimes they were taken by the wind or eaten by goats and/or rodents. therefore, the actual number of traps was that recovered intact with insects. because the number of http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 27 traps was not the same on all the sampling points, the catches in different localities, the density of sandflies (d) was standardized and calculated as the number of specimens caught total surface of traps retrieved / number of sampling nights. with the help a fine brush, captured sandflies were collected and preserved in ethyl alcohol at 95° in tubes to proceed to the identification of species. the identification was carried out according to identification keys of the phlebotomine sandflies of algeria (dedet et al. 1984, belazzoug 1991). data analyses to study characteristics of sandfly populations of each site and to enable their comparison between the study sites, the following parameters and ecological indices were calculated: – relative abundance (ra), expressed by the ratio between numbers of specimens of a species i and the total number of specimens caught in the site ×100. – degree of presence or occurrence (c)= number of sites containing species i / total number of study sites ×100. according to occurrence value, sandfly species were classified into four groups: constant species are present in 50 % or more of study sites, common species are those whose occurrence varies between 25 and 49 %, accidental species are present in 12.5–24 % of samples, very accidental species have an occurrence of less than 12.5 % (neffar et al. 2015). – density (d): the density of sandfly species was expressed by the number of specimens captured per square meter of sticky traps per night (specimens/m²/night). – species richness (sr): total number of species per site (spellerberg and fedor 2003). – jaccard similarity index (cj) was used to compare species richness of sandflies between sampled sites taken in pairs. given two sites, a and b, cj was estimated as: cj= c(a+b–c). where a and b: the total number of species present in site a and b, respectively, c: the number of species found in both sites a and b (magurran 2004). – the specific biodiversity of study sites was measured by simpson's diversity index (is), which expresses the relationship between the number of species and the number of specimens simultaneously: is= 1/(σ pi²), where pi is the proportion of the species i in a given site (pi= ra/100). the values of this index range from 1 to sr (total number of sandfly species of the site in question), with larger values indicating a higher species diversity. – evenness (e): e= (is–1)/(sr–1) was computed to estimate organizational distribution of sandfly population within each site community. site e varied from 0 (signifying the dominance of one species) to 1 (all species populations equitably distributed) (spellerberg and fedor 2003). statistical analyses to test the null hypothesis that there is no difference in the number and density of caught sandflies as well diversity indices (sr, is, e) between the sixteen sampled sites, pearson’s chi-square test (χ²) was carried out at a threshold alpha = 0.05. agglomerative hierarchical clustering (ahc) was applied to cluster the sampled sites according to their similarities of sandfly species densities based on euclidian distance. the technique used in agglomeration was ward’s method. after that, a descriptive statistical analysis, using correspondence analysis (ca), was performed to determine segregation degree of sandfly species (considering their densities) upon the 16 surveyed sites. results species abundance and spatial occurrence a total of 1363 specimens of sandflies were collected and morphologically classihttp://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 28 fied into five species as follows: phlebotomus (larroussius) perniciosus dominated with 651 caught specimens (47.8%), followed by sergentomyia (sergentomyia) minuta rondani 1843 caught with 385 specimens (28.2%), p. (l.) perfiliewi parrot, 1939 with 301 specimens (22.1%). whereas p. (l.) longicuspis nitzulescu 1930 and p. (p.) papatasi were slightly trapped with 1.5 % and 0.4 % of total captures, respectively. densities of the trapped sandflies were very uneven, the most important were recorded in sites of oued nacer, henchir tomghani, rhia, boughiel, ksar sbahi, and ain fekroun with respectively 22.73, 18.76, 17.36, 16.40, 16.29, 15.10 specimens/m²/night. whereas densities of captures (specimens m²/ night) were low in others study sites such as in lehmimet (9.03), bordj benzaoui (8.65), ain eddis (6.25), and sidi rghis (1.32) (table 1). phlebotomus perniciosus was the most constant species, which showed the widest geographic distribution due to its high frequency of occurrence (c= 100%). caught in all surveyed sites, it was followed by p. perfiliewi that was widespread too. indeed it was observed in eleven sites on sixteen that equals c= 68.75%. the third species that had an important presence was s. minuta (c= 62.50%). while p. longicuspis was poorly represented (4.14% of the genus p. and only 3.2 % of overall specimens), but it was sprinkled over seven of the sixteen study sites, its degree of presence c= 43.8%. the lowest occurrence frequency was recorded for p. papatasi that was captured in three sites (c= 18.75%), which makes it an accidental species in the region. diversity and similarity generally, species richness per site was relatively low, it varied between one species (sr= 1) in three sites and sr= 5 in oued nacer and ksar sbahi. the spatial distribution of species richness values was uneven along the study area. southern sites were richer in species number than sites located in the north of oeb. simpson’s index showed a slight diversity in all sites. this was confirmed by evenness (e) values calculated in the study sites, which may be grouped into three categories: the first (0≤ e< 0.37) includes a single dominant species; in the second (0.37≤ e< 0.5), two species co-dominated, and finally sites where evenness varied between 0.5 and 0.8, including three species co-dominated (table 2). the chi-squared test revealed no differences (p> 0.05) between the study sites for values of all parameters of diversity (sr, is, e). values of jaccard's index fluctuated between 0.20 and 1. in 32.1 % of pair similarities, resemblance value between sites was high than 0.50. the highest value of similarity reached 1 in 18 of 240 comparison cases, where the high values of similarity were recorded between sites with high numbers of species. inversely the dissimilarity was higher between the sites of low sr, where 8.75 % of cases showed similarity values less than 0.33 (table 3). the sixteen sampled sites were clustered according to euclidean distances into four different classes: (i) the first group gathered sites located in the north of oeb, basically under sub-humid climate, including bordj benzaoui, boughiel, oued nacer, and ain fekroun, (ii) the site of henchir tomghani was plotted as a single class, (iii) the 3rd class included three sites located at the periphery of oeb region, namely ksar sbahi, souk naamane and rhia, and (iv) the fourth cluster was represented by the rest of the study sites (fig. 2a). from the point of view of species contribution in the segregation of clusters of the sampled sites, p. perniciosus had an important contribution to the distinction between the four clusters, especially for the second cluster. whilst the density of p. perfiliewi characterized especially cluster 1, the density of s. minuta singularized the http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 29 third cluster of sites. however, densities of p. longicuspis and p. papatasi had slight contribution in cluster differentiation (fig. 2b). correspondence analysis (ca), applied to sandfly species densities in relation with different sampled sites, was represented on the factorial symmetric plot of two axes f1 × f2 with the maximum of inertia (88.3% hereafter). the ca showed that the x-axis (f1= 58.81%) separated sites of ksar sbahi, souk naamane and rhia, which were characterized by s. minuta. whereas y-axis (f2= 29.49%) distinguished two groups of sites, the first included basically sites located under sub-humid climatic conditions (i.e. bordj benzaoui, boughiel, oued nacer, ain fekroun and oued gacem) characterized by p. perfiliewi and p. papatasi, the second gathered the eight left sites that are marked by high densities of p. perniciosus and p. longicuspis (fig. 3). thus, these sites (i.e. ain beida, ain eddis, bhir el chergui, blala, henchir tomghani, lehmimet, menchar, sidi rghis) are the more susceptible for the disease. overall, while the x-axis separated site groups according to the degree of variability in sandfly species densities, the y-axis highlighted the involvement of climatic characteristics in the distinction between two sets of sites, one located under hub-humid climate and the other under semiarid climate. relationship between species of subgenus larroussius and leishmaniasis cases to identify and confirm the relationship between the occurrence of leishmaniasis cases registered by the direction of health and population (dsp, 2013) at different localities of oeb for the period between 1998 and 2009 and densities of phlebotomine sandflies captured at their surroundings, we calculated rates and proportions of different species of the subgenus larroussius, whose vector role was known and confirmed. results of these calculations revealed a close relationship and a concordance between the high presence of p. perniciosus and p. perfiliewi and the occurrence of leishmaniasis cases in the study sites (table 4). africa algeria 7°00’e6°40’e6°20’e 7°20’e 35 °4 0’ n 36 °0 0’ n 4 1 8 2 9 5 6 1. bordj benzaoui 2. boughiel 3. oued nacer 4. henchir tomghani 5. ksar sbahi 6. oued gacem 7. souk naamane 8. blala 9. rhia 10. bhir el chergui 11. ain beida 12. sidi rghis 13. lehmimet 14. ain eddis 15. menchar 16. ain fekroun 3 1011 12 13 15 1416 7 0 20 km10 sub-humid semi-arid climate types s a m p l e d s i t e s w i l a y a o f o u m e l b o u a g h i fig. 1. geographic and bioclimatic locations of the study sites in the region of oum el bouaghi (northeastern algeria) http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 30 table 1. distribution of phlebotomine species caught by sticky traps in 16 rural sites of oum el bouaghi region (northeast algeria) during the period may–october 2010 study sites sticky traps phlebotomus perniciosus phlebotomus perfiliewi phlebotomus longicuspis phlebotomus papatasi sergentomyia minuta all sandfly species area (m²) nights n ra d n ra d n ra d n ra d n ra d n d s1 3.12 1 5 18.52 1.60 22 81.48 7.05 – – – – – – – – – 27 8.65 s2 4.94 2 58 35.80 5.87 97 59.88 9.82 – – – – – 7 4.32 0.71 162 16.40 s3 2.64 3 87 48.33 10.98 84 46.67 10.61 3 1.67 0.38 2 1.11 0.25 4 2.22 0.51 180 22.73 s4 6.13 1 100 86.96 16.31 6 5.22 0.98 – – – – – – 9 7.82 1.47 115 18.76 s5 2.64 4 44 25.58 4.17 36 20.93 3.41 1 0.58 0.09 2 1.16 0.19 89 51.75 8.43 172 16.29 s6 3.60 1 18 45.00 5.00 18 45.00 5.00 1 2.50 0.28 – – – 3 7.50 0.83 40 11.11 s7 3.16 3 5 4.42 0.53 – – – – – – – – – 108 95.58 11.39 113 11.92 s8 4.88 2 68 95.77 6.97 1 1.41 0.10 1 1.41 0.10 – – – 1 1.41 0.10 71 7.27 s9 4.32 3 77 34.22 5.94 – – – 2 0.89 0.15 – – – 146 64.89 11.27 225 17.36 s10 10.08 2 111 79.29 5.51 16 11.43 0.79 – – – 1 0.71 0.05 12 8.59 0.60 140 6.94 s11 2.00 3 33 63.46 5.50 2 3.85 0.33 11 21.15 1.83 – – – 6 11.54 1.00 52 8.67 s12 1.52 1 2 100 1.32 – – – – – – – – – – – – 2 1.32 s13 1.44 1 9 69.23 6.25 4 30.77 2.78 – – – – – – – – – 13 9.03 s14 1.60 1 10 100 6.25 – – – – – – – – – – – – 10 6.25 s15 1.60 1 12 100 7.50 – – – – – – – – – – – – 12 7.50 s16 1.92 1 12 41.38 6.25 15 51.72 7.81 2 6.90 1.04 – – – – – – 29 15.10 chi² value (df = 15) 520.83 247.50 35.13 714.65 475.00 71.50 86.43 184.70 15.52 23.80 13.60 2.83 1328.75 769.30 110.35 935.21 41.56 p-value <0.001 <0.001 0.002 <0.001 <0.001 <0.00 1 <0.0 01 <0.001 0.414 0.069 0.559 0.999 <0.001 <0.001 <0.001 <0.001 <0.001 total n 651 301 21 5 385 1363 percentage of n 47.8 22.1 1.5 0.4 28.2 100 total of d 95.95 48.68 3.87 0.49 36.31 185.30 mean d 6.00 3.04 0.24 0.03 2.27 11.58 mean of ra 59.26 22.38 2.21 0.19 15.96 occurrence (%) 100 68.75 43.8 18.8 62.5 occurrence class constant constant common accidental constant n: number of caught specimens, ra: relative abundance (species % per site), d: species density (specimens/m²/night) (see fig. 1 for site codes). table 2. spatial distribution of diversity indices of sandfly species and their dominance in the study sites at oum el bouaghi area (northeast algeria) during 2010 surveyed sites sr is e dominant species (ra in %) bordj benzaoui 2 1.43 0.43 p. perfiliewi (81.5) boughiel 3 2.12 0.56 p. perfiliewi (59.9), p. perniciosus (35.8) oued nacer 5 1.92 0.23 p. perniciosus (48.3), p. perfiliewi (46.7) henchir tomghani 3 1.00 0 p. perniciosus (86.9) ksar sbahi 5 2.77 0.29 s. minuta (51.7), p. perniciosus (25.6), p. perfiliewi (20.9) oued gacem 4 2.12 0.37 p. perniciosus (45.0), p. perfiliewi (45.0) souk naamane 2 1.00 0 s. minuta (95.9) blala 4 1.08 0.02 p. perniciosus (95.8) rhia 3 1.85 0.42 s. minuta (64.9), p. perniciosus (34.2) bhir chergui 4 1.50 0.16 p. perniciosus (79.3) ain beida 4 2.22 0.40 p. perniciosus (63.5), p. longicuspis (21.2) sidi rghis 1 1.00 0 p. perniciosus (100) lehmimet 2 1.78 0.78 p. perniciosus (69.2), p. perfiliewi (30.8) ain eddis 1 1.00 0 p. perniciosus (100) menchar 1 1.00 0 p. perniciosus (100) ain fekroun 3 2.27 0.63 p. perfiliewi(51.7), p. perniciosus(41.4) 2 15 9.17 11.16 9.56 p 0.868 0.741 0.846 (sr: species richness, is: simpson index, e: evenness, ra: relative abundance) http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 31 table 3. matrix of proximity applied for sandfly similarity between the 16 sampled sites considered in pairs. the values are referred to the number of shared species (above the diagonal) and jaccard’s similarity index “cj” (under the diagonal) sites s1 (2) s2 (3) s3 (5) s4 (3) s5 (5) s6 (4) s7 (2) s8 (4) s9 (3) s10 (4) s11 (4) s12 (1) s13 (2) s14 (1) s15 (1) s16 (3) s1 2 2 2 2 2 1 2 1 2 2 1 2 1 1 2 s2 0.67 3 3 3 3 2 3 2 3 3 1 2 1 1 2 s3 0.40 0.60 3 5 4 2 4 3 4 4 1 2 1 1 3 s4 0.67 1.00 0.60 3 3 2 3 2 3 3 1 2 1 1 2 s5 0.40 0.60 1.00 0.60 4 2 4 3 4 4 1 2 1 1 3 s6 0.50 0.75 0.80 0.75 0.80 2 4 3 3 4 1 2 1 1 3 s7 0.33 0.67 0.40 0.67 0.40 0.50 2 2 2 2 1 1 1 1 1 s8 0.50 0.75 0.80 0.75 0.80 1.00 0.50 3 3 4 1 2 1 1 3 s9 0.25 0.50 0.60 0.50 0.60 0.75 0.67 0.75 2 3 1 1 1 1 2 s10 0.50 0.75 0.80 0.75 0.80 0.60 0.50 0.60 0.40 3 1 2 1 1 2 s11 0.50 0.75 0.80 0.75 0.80 1.00 0.50 1.00 0.75 0.60 1 2 1 1 3 s12 0.50 0.33 0.20 0.33 0.20 0.25 0.50 0.25 0.33 0.25 0.25 1 1 1 1 s13 1.00 0.67 0.40 0.67 0.40 0.50 0.33 0.50 0.25 0.50 0.50 0.50 1 1 2 s14 0.50 0.33 0.20 0.33 0.20 0.25 0.50 0.25 0.33 0.25 0.25 1.00 0.50 1 1 s15 0.50 0.33 0.20 0.33 0.20 0.25 0.50 0.25 0.33 0.25 0.25 1.00 0.50 1.00 1 s16 0.67 0.50 0.60 0.50 0.60 0.75 0.25 0.75 0.50 0.40 0.75 0.33 0.67 0.33 0.33 site codes (given in the same order as in table 1 and table 2) of the first row are associated with species richness “sr” values table 4. relationship between species of the subgenus larroussius collected in oeb region (may–october 2010) and leishmaniasis cases (1998–2009) study sites type of leishmaniasis total of catch total within larroussius number among larroussius (%) p. perniciosus p. perfiliewi p. longicuspis bordj benzaoui cl + lcan 27 27 05 (18.51) 22 (81.49) – boughiel cl + lcan 162 155 58 (37.41) 97 (62.59) – oued nacer cl 180 174 87 (50.00) 84 (48.28) 03 (1.72) henchir tomghani cl + ivl 115 106 100 (94.33) 06 (5.67) – ksar sbahi cl + ivl 172 81 44 (54.32) 36 (44.44) 01 (1.24) oued gacem ivl 40 37 18 (48.65) 18 (48.65) 01 (2.70) souk naamane – 113 05 05 (100) – – blala – 71 70 68 (97.14) 01 (1.43) 01 (1.43) rhia ivl 225 79 77 (97.47) – 02 (2.53) bhir chergui ivl 140 127 111 (87.40) 16 (12.60) – ain beida ivl 52 46 33 (71.74) 02 (4.35) 11 (23.91) sidi rghis – 02 02 02 (100) – – lehmimet – 13 13 09 (69.23) 04 (30.77) – ain eddis – 10 10 10 (100) – – menchar cl 12 12 12 (100) – – ain fekroun cl 29 29 12 (41.38) 15 (51.72) 02 (6.90) (cl: cutaneous leishmaniasis, vl: visceral leishmaniasis, lcan: canine leishmaniasis, ivl: infantile visceral leishmaniasis) http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 32 souk naamane ksar sbahi rhia oued nacer bordj benzaoui boughiel ain fekroun henchir tomghani sidi rghis oued gacem lehmimet ain eddis blala menchar bhir chergui ain beida 0 70 140 210 280 euclidean distance cluster 4 cluster 1 cluster 3 cluster 2 s t u d y s it e s (a) (b) 0 3 6 9 12 15 p. perniciosus p. perfiliewi p. longicuspis p. papatasi s. minuta phlebotomine sandfly species cluster 1 cluster 2 cluster 3 cluster 4 0 3 6 9 12 15 p. perniciosus p. perfiliewi p. longicuspis p. papatasi s. minuta phlebotomine sandfly species cluster 1 cluster 2 cluster 3 cluster 4 c lu s t e r c e n t r o id (s pe ci m en s/ m ²/ ni gh t) fig. 2. agglomerative hierarchical clustering (ahc) displaying similarities (euclidian distance, linkage rule: ward’s method) among sandfly densities captured from 16 sites in oum el bouaghi (northeast algeria). (a): dendrogram of the ahc, (b): variation of cluster centroids following phlebotomine sandfly densities s1 s2 s3 s4 s5s6 s7 s8 s9 s10 s11 s12 s13 s14 s15 s16 p. perniciosus p. perfiliewi p. longicuspis p. papatasi s. minuta -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 1.5 2 f 2 (2 9. 49 % ) f1 (58.81 %) study sites sandfly species fig. 3. factorial plot of the classification analysis (ca) applied to the distributions of densities of sandfly species on sampled sites of oum el bouaghi (north algeria) in 2010. (codes of the study sites are reported in fig. 1) http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 33 discussion the sampling of sandflies that was carried out using sticky traps has revealed the presence of five species of the 22 species already known in algeria (dedet et al. 1984). these five species belong to two genera: phlebotomus (4) and sergentomyia (1), the latter is characterized by its herpetophilic feeding behaviour (dedet et al. 1984, berchi 1990). at oeb, genus sergentomyia is represented by the species s. minuta sub-species parroti adler and theodor 1927 (ra= 28.24%, c= 62.50%) that dominates in rural peridomestic habitats of ksar sbahi and souk naamane. landscapes of these habitats are formed of hedges of prickly pear plantations (opuntia ficus-indica), which are rich environments by lacertidae species. although sergentomyia minuta has been found negative for human and cow infection during the analysis of blood meals (yaghoobi et al. 1995, dancesco 2008), some species of the genus sergentomyia (sergentomyia dubia, s. magna, s. schewtzi) are suspected in the transmission of lcan in senegal because of their high presence inside and around homes and in the narrow circle of the dog (senghor et al. 2011). in fact, this suspicion was confirmed when three females of the genus sergentomyia were found naturally infected by l. infantum; it is s. dubia, s. magna and s. schwetzi, highlighting the distribution significantly associated with the prevalence of dogs in the focus of mont-rolland (senghor et al. 2011). these findings challenge the dogma that states that only the genus phlebotomus is responsible for the transmission of leishmaniasis in the old world and criminalize genus sergentomyia in the epidemiology of this zoonosis. moreover, the high occurrence of species belonging to genus sergentomyia has been reported in several countries, including france and tunisia (croset 1969), algeria (dedet et al. 1984, belazzoug 1986), tunisia (ghrab et al. 2006), and senegal (senghor et al. 2011). among the five species collected at oeb, four belong to the genus phlebotomus, proven vector of hl in the old world. these species are distributed on two sub-genera larroussius and phlebotomus. the subgenus larroussius is proven vector of l. infantum in the mediterranean region (killick-kindrick 1985, killick-kindrick 1990). while the subgenus phlebotomus is represented by a single species, p. papatasi, which is the main vector of l. major mon-25 (killickkindrick 1990, izri et al. 1992, harrat and belkaid 2003). this species, that forms only 0.4 % of total, is found in three sites with low occurrence (c= 18.75%), which gives it an accidental status, thus its role in the transmission of cl in the area is very remote. the dominance of species of subgenus larroussius (ra= 71.38% of the total catches, ra= 99.48% of the genus phlebotomus), reflects the significant abundance and wide geographical distribution of these species. the species p. perniciosus (ra= 47.76% of the overall results, ra= 66.90% of the subgenus larroussius) is not only omnipresent in all study sites (c= 100%), but reported dominant in 11/16 of studied sites. this reflects its high ecological valence and its adaptive capacities in the region. moreover, its distributional area matches that of hl and lcan (addadi and dedet 1976). regarding this species, our results confer with those reported par berchi et al. (2007) where p. perniciosus was more abundant in catches carried out in peri-domestic areas of mila and jijel. the role of this species in the transmission of ivl and lcan is confirmed in several regions of the mediterranean basin (bettini et al. 1986, ben ismail et al. 1987, maroli et al. 1988, izri et al. 1990, izri et al. 1992, belazzoug 1992, janini et al. 1995). http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 34 in addition, the species p. perfiliewi is largely widespread and constant in the study area (ra= 20.46%, c= 68.75%), where it co-dominates next to p. perniciosus. indeed, the species is reported as dominant in two sites in the sub-humid bioclimatic area (bordj benzaoui and boughiel) and ain fekroun in semi-arid climate. it is the proven vector of l. infantum in algeria (izri and belazzoug 1993, benikhlef et al. 2004) and italy (maroli et al. 1988). as for p. longicuspis, an endemic species in algeria and north africa (dedet et al. 1984, killick-kindrick 1999), is very abundant with p. perniciosus in peri-domestic environments (berchi et al. 2007). it was captured around dog locations (croset 1969), where its feeding behaviour is very similar to that of p. perniciosus (croset et al. 1970). the males of this species have been confused with p. perniciosus (berchi et al. 2007, boussaa et al. 2007). at oem, p. longicuspis is represented by only 43 specimens (ra= 3.15%) of which 40 were identified as males. its role in the epidemiology of leishmaniasis is not excluded, as all species of the subgenus larroussius in the mediterranean basin are anthropophilic and endophilic. indeed, these species sting indoors seven times they do outside (izri et al. 1993, killick-kindrick 1999). the noteworthy presence of p. perniciosus and p. perfiliewi, proven vector species of leishmaniasis, could explain the sporadic cases of vl and cl in oeb. their presence in domestic and peri-domestic environments (such as courtyards, stables, rooms and exterior walls of abandoned houses) and in the immediate vicinity of homes confirm their both endo-exophilic and anthropozoophilic characters. moreover, their important frequencies represent a potential risk factor and at the same time a valuable bio-indication of endemicity. as the risk of disease spreading increases with the increase of vector densities, thus the larger sandfly densities are, the higher the rate of people who could be infected is important. according euzeby (1984), a minimum density of 10 to 15 sandflies/m² is required to maintain the endemic of leishmaniasis. this threshold is reached at eight surveyed sites in oeb, where the highest densities were observed at oued nacer, henchir tomghani, rhia, boughiel, ksar sbahi, and ain fekroun with 22.73, 18.76, 17.36, 16.40, 16.29, 15.10 specimens/m²/night, respectively. however, medium or low density of sandflies in the other study sites is very probably due to the phenology of species during the study period as well as local climatic conditions, including temperature, precipitation and wind speed, which play the role of limiting factor for many flying insects (compton et al. 2002). overall, the exploration of some localities in the region of oeb revealed a low diversity (five species), compared with the 22 known species in algeria. very similar results were denoted in eastern algeria, where only four species have been reported in two classic foci of hl and lcan, in constantine (moulahem 1998) and mila (messai 2011), which both are adjacent areas to oeb. in skikda at northern algeria, bouleknafed (2007) reported the presence of five species with occurrence frequencies and densities very close to our results. in oeb, the distribution of abundance and density of sandfly species is very uneven, with significant differences between the sampled locations (significant χ² test). indeed some species are found in large numbers while others are represented only by few specimens (relatively low value of evenness). this variability of species frequencies is well known, where it was particularly evident based on observations on 30,000 specimens in tunisia (croset 1996) and in the kabylia in north algeria (izri et al. 1990). our findings clearly show that p. perniciosus, p. perfiliewi and s. minuta are the most http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 24–38 n kabbout et al.: ecological status of … 35 consistently captured species. in addition, p. perfiliewi usually accompanies p. perniciosus around habitations and even indoors. the absence of p. perfieliwi is spotted in areas where sandfly catches are so low. the distribution and/or occurrence of p. perniciosus matches regions that are almost all involved in the one or the other form of leishmaniasis, or both types at once (as the case of ksar sbahi and henchir tomghani). the presence of sandflies in abundance increases susceptibility to leishmaniasis in localities in question. for example, the region of oued gacem, that was until 2010 free of leishmaniasis, was stated positive for p. perniciosus and p. perfiliewi in peri-domestic environments; has just know two cases of ivl, one in 2012 and the other in 2013 (dsp, 2013). it is likely that p. perniciosus represents the main vector of leishmaniasis in the study area, as it has been reported in the mediterranean countries (bettini et al. 1986, maroli et al. 1988, izri et al. 1990, izri et al. 1992). however, the high frequency of p. perfiliewi (20.46%) with a presence in 68.75 % of the study sites next to p. perniciosus suggests that more than one species may be implicated in the transmission of hl and lcan (izri et al. 1992). indeed, p. perfiliewi is proven vector of l. infantum mon-24, the causative agent of zcl and lcan in algeria (izri and belazzoug 1993, benikhlef et al. 2004). conclusion phlebotomus perniciosus and p. perfiliewi are the most likely species that transmit leishmaniasis in the region of oeb due to their high density and constant spatial occurrence. their massive presence in domestic and peri-domestic environments is a potential risk factor and a valuable bio-indication for an effective control against these zoonotic diseases. the present study is a contribution to the ecological study of sandflies of eastern algeria and at the same time an initiation to the study of entomological aspects of leishmaniasis in the region of oeb. acknowledgements we gratefully acknowledge dr said chawki boubidi and dr abdelkarim boudrissa for their valuable helps in the identification of phlebotomine sandflies. the authors declare that there is no conflict of interest. this study did not receive any funding. references addadi k, dedet jp (1976) epidémiologie des leishmanioses en algérie. 6 recensements des cas de leishmanioses viscérales infantiles entre 1965 et 1974. bull soc pathol exot. 69: 68–75. belazzoug s (1982) une 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glob ecol biogeogr. 12(3): 177–183. tesh rb (1988) the genus phlebovirus and its vectors. ann rev entomol. 33(1): 169–181. yaghoobi-ershadi mr, javadian e, tahuildarbidruni gh (1995) leishmania major mon26 isolated from naturally infected phlebotomus papatasi (diptera: psychodidae) in isfahan province, iran. acta trop. 59(4): 279–282. http://jad.tums.ac.ir published online: june 27, 2015 microsoft word 8-hashareh dr alavian epidemiological rtl.doc iranian j arthropod-borne dis, (2009), 3(2): 50-54 sm alavinia et al: some epidemiological aspects… 50 short communication some epidemiological aspects of cutaneous leishmaniasis in northern khorasan province, iran *sm alavinia 1, k arzamani 1, mh reihani 2, j jafari 3 1educational development center, deputy of education, northern khorasan university of medical sciences, iran 2 center of disease control, deputy of health, northern khorasan university of medical sciences, iran 3 center of disease control, health center of esfarayen, northern khorasan university of medical sciences, iran (received 27 apr 2009; accepted 16 nov 2009) abstract background: cutaneous leishmaniasis (cl) is a widespread tropical infection which has a high incidence rate in iran. the aim of this study was to determine some epidemiological aspects of disease in northern khorasan province, iran. methods: from 2005 to 2008, data concerning 1453 patients with cl were collected and analyzed from the different districts of health service registry of the province. the statistical analyses were carried out with spss version 12.0 for windows. results: the highest number of patients was in 2005 with 555 cases in this year. 19.3% of patients aged younger than 5-year and 57.5% were older than 15 year. 37.8% had only one lesion, and 40.5% had at least three lesions. the highest incidence of disease was observed in jajarm district in 2005 (381.1 per 100,000), and the lowest incidence rate was in farouj district in the same year. the disease was observed in all months of the year with the highest incidence rate from september to november. conclusion: although jajrm is a known foci in iran, based on our knowledge there is no report in the literature on assessing the prevalence of cl in this region in recent years. we also found that the disease is endemic in northern khorasan province. keywords: cutaneous leishmaniasis, epidemiology, iran introduction cutaneous leshmaniasis (cl) is a widespread tropical infection caused mainly by two species of leishmania, l. tropica and l. major, transmitted by phlebotomus sandflies (oumeish 1999, bailey lockwood 2007). although cl is endemic in 88 countries in africa, asia, europe and north and south america (herwaldt 1999), 90% of all cases occur in six countries including afghanistan, brazil, iran, peru, saudi arabia, and syria (desjeux 1996). there are an estimated numbers of 12 million cases worldwide, with 1.5-2 million new cases added each year (who 1984, 1990). in the recent years, cl has emerged as a significant cause of morbidity and social stigma in war-torn countries such as afghanistan (reithinger et al. 2005). this disease is still a great health problem in iran. the prevalence of infection has been reported as 1.8% to 37.9% in different provinces of iran. (yaghoobi ershadi et al. 2002, talari et al. 2006). several new foci have been reported in recent years, indicating the potential spread of disease in iran (razmjou et al. 2009). the pre*corresponding author: dr seyed mohammad alavinia, email: malavinia2000@yahoo.com iranian j arthropod-borne dis, (2009), 3(2): 50-54 sm alavinia et al: some epidemiological aspects… 51 valence of disease is high in some provinces of iran, including isfahan (nadim et al. 1968, nadim and faghih 1968, salimi 2000), shiraz (moaddeb et al. 1993), khorasan (javadian et al.1967), khuzestan and kerman (nadim and seyedi-rashti 1971). the aim of this study was to evaluate the incidence and some epidemiological aspects of cutaneous leishmaniasis in northern khorasan province during 2005 2008. materials and methods from 2005 to 2008, 1453 patients infected with cutaneous leishmaniasis were collected from health service registry of six districts of northern khorasan province, iran. the patients were diagnosed clinically, paraclinically or both and the diagnosis were confirmed by a physician. a questionnaire about the name, sex, habitation area, date of onset, date of diagnosis, number of lesions, and location of the lesions was completed for each patient. the statistical analyses were carried out with spss version 12.0 for windows. results table 1 shows the basic characteristics of the study population. the mean age of the study population was 23.2 yr (sd= 18.7) with 19.3% younger than 5 yr, and 57.5% older than 15 yr. the most cases were observed in 2005 with 555 cases. there was a significant difference between percentage of male and female with cutaneous leishmaniasis (p< 0.001). 37.8% of patients had one lesion, and 40.5% had at least 3 lesions. 42.9% of the patients had lesion on their face. table 2 shows the distribution of disease stratified by districts in northern khorasan province from 2005 to 2008. jajarm had the highest (45.4%) and farouj had the lowest number of the disease (1.4%) during these years. fig.1 shows the incidence rate of the disease for each district in the study population. the disease was reported in all months of the year with the highest rate from september to november (fig. 2). table1: baseline characteristics of 1453 patients with cutaneous leishmaniasis in northern khorasan province, iran: 3/20059/2008 n 23.2 ± 18.7 1453 age (yr) (mean ± sd) 42.9 624 female (%) 37.8 21.7 40.5 549 315 589 number of lesions (%) 1 2 >=3 60.8 39.2 570 884 location (%) urban rural 28.3 644 44.3 397 27.3 412 diagnosis clinical laboratory both table 2. distribution of 1453 patients with cutaneous leishmaniasis in different districts of northern khorasan province from 3/2005-9/2008 3/2005-3/2006 3/2006-3/2007 3/2007-3/2008 3/2008-9/2008 district n % n % n % n % bojnourd 75 13.5 45 11.8 54 12.3 12 15.4 esfarayen 235 42.3 90 23.6 150 34.2 16 20.5 jajarm 218 39.3 214 56.2 196 44.6 31 39.7 shirvan 22 4.0 22 5.8 24 5.5 12 15.4 maneh & samalghan 4 0.7 4 1.0 6 1.4 2 2.6 farouj 1 0.2 6 1.6 9 2.1 5 6.4 total 555 100.0 381 100.0 439 100.0 78 100.0 iranian j arthropod-borne dis, (2009), 3(2): 50-54 sm alavinia et al: some epidemiological aspects… 52 30.7 68.0 23.9 179.4 368.1 13.7 4.2 1.8 0 50 100 150 200 250 300 350 400 in ci d en ce country province bojnourd esfarayen jajarm shirvan maneh&sam alghan farouj location fig. 1. incidence rate of the cutaneous leishmaniasis in 1453 patients from different districts of northern khorasan province compared with incidence rate in iran: 3/2005-9/2008 fig. 2. month distribution of 1375 patients with cutaneous leishmaniasis in northern khorasan province3/20053/2008 iranian j arthropod-borne dis, (2009), 3(2): 50-54 sm alavinia et al: some epidemiological aspects… 53 discussion the results showed that northern khorasan province is an endemic area of cutaneous leishmaniasis. we also assessed the prevalence of this disease in details in jajarm as a known focus in iran. some limitations should be taken into account in this study. first of all, we used the data from health service archive. it is possible that some patients with cutaneous leishmaniasis are visited and treated by physicians out of the province. based on our knowledge the number of these cases is low and can be ignored. secondly, since this disease is a selflimited one, some patients might have been cured without any medications. unfortunately the information about the number of these patients is not available; therefore it is possible that the incidence of the disease were underestimated in the present study. thirdly, as we used the data about the patients, finding the relationship between the disease and the potential risk factors were not possible. age distribution of the disease through this province showed that 19.3% of the patients are under the age of 5 yr. analyzing the incidence of disease in iran showed more than 2 times higher incidence in this province. the most likely reason is an increase in human– sandfly contact. this is attributed to the development of villages and the spread of the human population into the habitats of the local vectors. although another study has introduced esfarayen district as a focus of cutaneous leishmaniasis (javadian et al. 1976), we found jajarm district as an active focus of the disease in iran. we found a significant sex difference for the incidence of cutaneous leishmaniasis. the low incidence of the disease among females could be due to the way of dressing among women in iran. another reason is that men normally work outside, considering the reservoirs of the disease, we expect higher incidence of the disease among men. the month distribution of the disease through the province reflects the rural type of the disease, which is more prominent in two districts (esfarayen and jajarm). the month distribution assessment of the disease in each districts showed also some urban foci of disease in the province. these two patterns of distribution of disease are caused by the duration of the disease, which is much longer in the urban type (nadim 1969). therefore, the highest number of patients could be found in all month in urban type. it could be concluded that for successful measures against the cutaneous leishmaniasis in northern khorasan province, we should pay attention more in two districts with high incidence rate, esfarayen and jajarm. acknowledgements the authors are grateful for the support of the deputy of health of northern khorasan university of medical sciences who provided data for this paper. the authors declare that they have no conflicts of interest. references bailey ms, lockwood dn (2007) cutaneous leishmaniasis. clin dermatol. 25(2): 20311. desjeux p (1996) leishmaniasis, public health aspects and control. clin dermatol. 14(5): 417-23. herwaldt bl (1999) leishmaniasis. lancet. 354(9185): 1191-9. javadian e, nadim a, tahvildare-bidruni g, assefi v (1976) epidemiology of cutaneous leishmaniasis in iran: b. khorassan part v: report on a focus of zoonotic cutaneous leishmaniasis in esferayen. bull soc pathol exot filiales. 69(2): 140-3. iranian j arthropod-borne dis, (2009), 3(2): 50-54 sm alavinia et al: some epidemiological aspects… 54 javadian e, nadim a, tahvildari a, assefi v (1967) epidemiology of cutaneous leishmaniasis in korassan iran. bull soc path exot. 69: 140-143. moaddeb a, gettner s, ardehali s (1993) studies on the causative agent of cutaneous leishmaniasis in shiraz iran. iran j med sci. 18: 28-33. nadim a, mesghali a, amini h (1968) epidemiology of cutaneous leishmaniasis in the isfahan province of iran. trans r soc trop med hyg. 62(4): 543-9. nadim a, faghih m (1968) the epidemiology of cutaneous leishmaniasis in isfahan province of iran. trans r soc trop med hyg. 62: 534-542. nadim a, seyedi-rashti ma (1971) a brief review of the epidemiology of various types of leishmaniasis in iran. acta medica iranica. 8: 99-106. nadim a, rashti as, faghih a (1969) epidemiology of cutaneous leishmaniasis in iran: b. khorassan. 3. human infection. bull soc pathol exot filiales. 62(4): 70210. oumeish oy (1999) cutaneous leishmaniasis: a historical perspective. clin dermatol. 17(3): 249-54. razmjou s, hejazy h, motazedian mh, baghaei m, emamy m, kalantary m (2009) a new focus of zoonotic cutaneous leishmaniasis in shiraz, iran. trans roy soc trop med hyg. 103: 727-730. reithinger r, reithinger r, mohsen m, wahid m, bismullah m, quinnell rj, davies cr, kolaczinski j, david jr (2005) efficacy of thermotherapy to treat cutaneous leishmaniasis caused by leishmania tropica in kabul, afghanistan: a randomized controlled trial. clin infect dis. 40(8): 1148-55. salimi m (2000) a clinical and epidemiological comparison on the cutaneous leishmaniasis in the city and villages of isfahan. iranian j publ health. 2(4): 214219. talari sa, shajari g, talaei r (2006) clinical finding of cutaneous leishmaniasis as a new focus of iran. internet j infec dis. volume 5, number 2. world health organization (1990) control of leishmaniasis. report of who expert committee, technical report series. 793: 159 geneva, switzerland. world health organization (1984) report of who expert committee. the leishmaniasis, who technical report series. 701: 2-4. yaghoobi-ershadi mr, hanafi-bojd aa, javadian e, jafari r, zahraei-ramazani ar, mohebali m (2002) a new focus of cutaneous leishmaniasis caused by leishmania tropica. saudi med j. 23: 291-4. microsoft word 7-drrahdar.doc iranian j arthropod-borne dis, (2008), 2(1): 44-48 m rahdar et al: a case report of sarcoptes… 44 case report a case report of sarcoptes scabiei infection in ahwaz, iran *m rahdar, b vazirianzadeh, s maraghi department of mycoparasitology, medical school, ahwaz joundi-shapour university of medical sciences, iran (received 13 jan 2007; accepted 5 feb 2008) abstract the prevalence of sarcoptes in rural areas has been investigated by many investigators. sarcoptes infestation in areas with poor education and hygienic is higher than urban areas. in this study, one severe case of sarcoptes scabiei infestation is described from a four months old child. confirmation of scabies was done by clinical examination of child and by the identification of the mite by microscopically examining scrapings from the scabies lesions. the prevalence of scabies is high in children in rural regions, suggesting that it is a serious public health problem. this phenomenon may be due to the fact that certain environmental conditions such as overcrowding, poor personal hygiene, poverty, and ignorance, which are conducive to the spread of scabies, tend to be quite common in rural regions. control programs should be put in place and implemented in an integrated manner, by reducing overcrowding, and by improving health education, personal hygiene, treatment and surveillance among high-risk populations. keywords: sarcoptes, child, rural, iran introduction most human ectoparasites live on the surface of their host and depend on host to complete their life cycle. the most common ectoparasitic infestations of medical importance in humans include pediculosis, scabies, myiasis, and tungiasis (bornstein et al. 2001). scabies is a contagious skin disorder and one of the most common itching dermatoses in the world especially in less developed countries. epidemiologic survey of scabies periodically done in many countries is a reflection of general status of public health in the community. their hosts are invertebrates as well as vertebrates (bornstein et al. 2001). the incidence of scabies was typically higher in rural areas than in cities. the most cases of scabies were noted in children and teenagers between 6 and 15 yr of age (lydden 2005). living in colonies and public places may cause increase in infectious diseases. prisons are places with high-density population and particularly where social circumstances such as overcrowded housing predispose a community to the spread of the mite (shamsaddini et al. 2000). infestation is transmitted through a direct contact with an infected person or animal, rarely also via objects, underwear, or bed linen. the adult female mite is 0.3-0.5 mm long and has 4 pairs of legs. a female of s. scabiei can survive around 30 d in the host's body penetrating into the stratum corneus of the skin and laying eggs in the burrow. outside the host it can survive from 3 to 10 d comprising, along with the other invasive stages the larvae and nymphs. (lydden 2005). *corresponding author: dr m rahdar, tel: +98 611 3337543-50 internal 2593, fax: +98 611 3332036, e-mail: mrahdar2002@yahoo.com iranian j arthropod-borne dis, (2008), 2(1): 44-48 m rahdar et al: a case report of sarcoptes… 45 she leaves behind a trail of debris, eggs, and feces (scybala), which induces an immunologic response. the female can lay up to 90 eggs in her 30-d lifespan. the larvae hatch in 3-4 d; they mature to adult forms over the next 2 weeks and continue the cycle. the average patient is infected with 10-15 live adult female mites at any given time (camila et al. 2006). a wide range of clinical manifestations may be seen in scabies, from classic pruritic papules and burrows to secondary features such as impetigo. pruritus, the main clinical manifestation, is caused by the phenomenon of hypersensitivity to the debris, eggs, and feces, rather than by direct effects of the mite. the primary lesions appear 3-10 d after exposure to the mite. these lesions include burrows, papules, vesicles, and pustules. nocturnal pruritus is characteristic of scabies infestation. main presenting features include rash and intense itching. bullous lesions are a less frequent (ansarin et al. 2006). crusted scabies is a form of massive infestation by sarcoptes scabiei observed in patients with immunosuppression and observed in children with immunosuppression or mental illness. (crusted norwegian) scabies induced by use of topical corticosteroids. patients with norwegian scabies can be infected with hundreds to millions of adult female mites (camila et al. 2006). the usual reference method for definitive diagnosis is identification of the mite with microscopic examination of skin scrapings (alain et al. 2006). in infants and young children, 75% of mites can be found on the hands and feet, making this the best site to examine for a burrow (camila et al. 2006). the treatment of choice is permethrin 5% lotion. alternative drug therapy includes precipitated sulfur in 6% petrolatum, lindane, crotamiton, and ivermectin. topical antibiotics may be used to treat secondarily infected lesions (camila et al. 2006). the lotion should be applied over the entire body, including the face and scalp in infants. it should be left on for 8-12 h and then rinsed. reapplication 1 wk later is advised. topical antihistamines and topical corticosteroids may be used to control pruritus until symptoms resolve. in this study we reported a case in severe condition of sarcoptes scabiei in four months old child in abdolkhan village north ahwaz, khuzestan province, iran. materials and methods a four-month old child with severity of infestation referred to the laboratory by special clinician. she lives in abdolkhan rural region in 50 km north of ahwaz. at first she was taken to general medical and was treated for skin hypersensitivity with antihistamine and some corticosteroid drugs. the lesion of the skin was not cured and the infected area was more scattered (fig.1, 2). after she was visited by specialist she was referred to laboratory for sarcoptes infestation. in scraped tissues of patient was found many adults sarcoptes scabiei and their eggs and nymphs in various stages (fig. 3,4) discussion scabies infestation is one of the contagious skin diseases with worldwide distribution. there are many articles about scabies infestation and all of them showed prevalence in rural areas with poor hygienic and education is than urban areas. the prevalence was age dependent, with children under five years accounting for 77%, peaking to 86% among the 5 to 9-yr-olds, and steadily declining with an increase in age. lesions were found on almost all parts of the body, but lesions were more commonly located on the fingers, legs, hands, face, belly, and genitalia. sarcoptes scabiei var hominis was recovered from 84 (67%) of the 125 skin scrapings examined (terry et al. 2001). iranian j arthropod-borne dis, (2008), 2(1): 44-48 m rahdar et al: a case report of sarcoptes… 46 in one study that performed in the students of primary schools in somea-sara in 2000-2001, 3656 students were selected and examined. fifty out of 3656 students (1.36%) were suffered from scabies. the prevalence of scabies was 1.0% in urban schools and 1.61% in rural schools, 1% in male and 1.7% in female students (golchai et al. 2003). sarcoptes infection also has been seen in prisons where hygienic conditions are poor. the rate of infection among prisoners was 1.2% (shamsaddin et al. 2000). the prevalence of disease in urban area with crowding and poor sanitation also was high. in bushehr port, in a cross-sectional study, 3913 students (1951 girls & 1962 boys) of elementary schools of bushehr port were examined. 84 (2.1%) of the examined students were infested with scabies. there was no difference for scabies infection between the girls and boys (arjomandzadeh et al. 2001). in other study in turkey, infestation was found in 5(0.4%) of 1,134 children with scabies (ciftci et al. 2006). the infestation can also be found in hospitals. an outbreak of scabies occurred in a ward of a local hospital in barbastro (huesca, spain), between november 2002 and january 2003 (larrosa et al. 2003). infection with increased risk of acquiring ectoparasitic infestation is occurred among the homelesses (estrada et al. 2003). the prevalence of scabies is high in children in the displacement camps, suggesting that it may be a serious public health problem not only in these camps, but also in the entire country. sarcoptes infection was recovered from 84 (67%) of the 125 skin scrapings examined (terry et al. 2001). infestations were more frequent in children with mothers whose education levels were low. this indicates the necessity of an improvement in the economic and sociocultural status of the community and the promotion of hygiene concepts and practices in order to improve health of children (ciftci et al. 2006). this may be due to the fact that certain environmental conditions like civil unrest, overcrowding, poor personal hygiene, poverty, and ignorance, which is conducive to the spread of scabies, is characteristically present among the rural children. control programs should be put in places and implemented in an integrated nature, by reducing overcrowding, and by improving health education, personal hygiene, treatment and surveillance among high-risk populations. fig. 1. the lesion of sarcoptes scabiei of 4 month old child (original) iranian j arthropod-borne dis, (2008), 2(1): 44-48 m rahdar et al: a case report of sarcoptes… 47 fig. 2. the lesion of sarcoptes scabiei of 4 month old child (original) fig. 3. adult female sarcoptes scabiei from scrabbled skin of 4 month old child (original) fig. 4. the embrionated eggs of sarcoptes scabiei under skin of 4 month old child (original) iranian j arthropod-borne dis, (2008), 2(1): 44-48 m rahdar et al: a case report of sarcoptes… 48 references ansarin h, jalali mh, mazloomi s, soltaniarabshahi r, setarehshenas r (2006) scabies presenting with bullous pemphigoid-like lesions. dermatol online j jan. 27; 12(1):19. arjomandzadeh s, tahmasebi r, jokar mh, khatmi sm, zarenejad m, abdolazadeh-lavari h (2001) prevalence of pediculosis and scabies in primary schools of bushehr; 1999-2000. south iramed jl.1(4): 46-41. bornstein s, morner t, samuel wm (2001) sarcoptes scabiei and sarcoptic mange. in: wm samuel, mj pybus and aa kocan, (editors), parasitic diseases of wild mammals (second ed.), iowa state university press, ames, pp. 107– 119. camila kj, giuseppe mu, hengge mk, casatelli jr (2006) scabies. www. emedicine specialties> pediatrics> dermatology. ciftci ih, karaca s, dogru o, cetinkaya z, kulac m (2006) prevalence of pediculosis and scabies in preschool nursery children of afyon, turkey. korean j parasitol. 44(1):95-8. dupuy a, dehen l, bourrat e, lacroix c, benderdouche m, dubertret l, morel p, feuilhade de chauvin m, petit a (2006) accuracy of standard dermoscopy for diagnosing scabies. j am aca of dermato. available online 13 november 2006. pages 1279-94. estrada b (2003) ectoparasitic infestations in homeless children. seminar. pedia infect dis.14(1):20-4. golchai j, zargari o, gholipour m, karbasi m (1382) the prevalence of scabies in the students of primary schools in somea-sara in 2000-01: an observational cross-sectional study. ira j derma. 25(7): 32-29. larrosa a, cortes-blanco m, martinez s, clerencia c, urdaniz lj, urban j, garcia j (2003) nosocomial outbreak of scabies in a hospital in spain. euro surveill. 8(10):199-203. lydden polley (2005) navigating parasite webs and parasite flow: emerging and re-emerging parasitic zoonoses of wildlife origin. int j parasit. 35, issues 11-12. shamsaddini s, nasiri kashani m , sharifi i, khajeh karimoddini m , pourlashkari m (1379) prevalence of infectious skin diseases in the central prison of kerman. iranian j dermatol. 13(4): 25-19. terry bc , kanjah f, sahr f, kortequee s, dukulay i, gbakima aa (2001) sarcoptes scabiei infestation among children in a displacement camp in sierra leone. pub health. 115(3):208-11. j arthropod-borne dis, march 2016, 10(1): 50–54 y goz et al.: ticks and fleas infestation … 50 original article ticks and fleas infestation on east hedgehogs (erinaceus concolor) in van province, eastern region of turkey *yaşar goz 1, ali bilgin yilmaz 1, abdulalim aydin 2, yalçın dicle 3 1department of parasitology, yüzüncü yıl university, school of health, van, turkey 2department of parasitology, hakkari university, school of health, hakkari, turkey 3department of microbiology, muş alparslan university, school of health, mus, turkey (received 14 june 2014; accepted 23 sep 2014) abstract background: ixodid ticks (acari: i̇xodidae) and fleas (siphonaptera) are the major vectors of pathogens threatening animals and human healths. the aim of our study was to detect the infestation rates of east hedgehogs (erinaceus concolor) with ticks and fleas in van province, eastern region of turkey. methods: we examined fleas and ticks infestation patterns in 21 hedgehogs, collected from three suburbs with the greater of number gardens. in order to estimate flea and tick infestation of hedgehogs, we immobilized the ectoparasites by treatment the body with a insecticide trichlorphon (neguvon®-bayer). results: on the hedgehogs, 60 ixodid ticks and 125 fleas were detected. all of the ixodid ticks were rhipicephalus turanicus and all of the fleas were archaeopsylla erinacei. infestation rate for ticks and fleas was detected 66.66 % and 100 %, respectively. conclusion: we detected ticks (r. turanicus) and fleas (a. erinacei) in hedgehogs at fairly high rates. since many ticks and fleas species may harbor on hedgehogs and transmit some tick-borne and flea-borne patogens, this results are the important in terms of veterinary and public health. keywords: tick, flea, hedgehog, turkey introduction hedgehogs are small, nocturnal animals which have bodies covered with spine. some ectoparasites of hedgehogs such as ticks and fleas have zoonotic importance (khaldi et al. 2012). hedgehogs may act as reservoir hosts for some diseases for instance salmonellosis, leptospirosis and pulmonary capilloriois (mc carthy and moore 2000, riley and chomel 2005, marie et al. 2012). ticks and fleas are blood feeding arth ropods that act as vector for transmitting many bacterial, viral, rickettsial and parasitic infection among both domestic and wild ani mals as well as between animals and humans (mullen and o'canner 2002). ticks are the most important ectoparasites affecting wild life as they transmit many important diseases in the wildlife, among the livestock and humans (mclean 2008, castellaw et al. 2011). ticks are vectors carrying a number of diseases named tick-borne diseases including lyme disease, rocky mountain spotted fever, tickborne meningoencephalitis, babesiosis and crimean-congo hemorrhagic fever (cchf) (service 2012). also several flaviviruses are transmitted by ixodes ticks, that cause encephalitis and haemorrhagic fever among humans (capinera 2010). ticks are common in eurasia and africa and are usually found in similar habitat to that of hedgehogs (causey et al. 1970). hedgehogs are a fre-quent host of hard tick i. hexagonus (gern et al. 1991, beichel et al. 1996, gern et al. 1997). in switzerland, an enzootic transmission cycle *corresponding author: dr yaşar göz, e-mail: yasargoz38@mynet.com.tr http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 50–54 y goz et al.: ticks and fleas infestation … 51 of b. burgdorferi involving hedgehogs and i. hexagenus, another tick vector, has been described in urban environment (gern et al. 1997). fleas such as ticks transmit many diseases to humans and animals, viral, bacterial, rickettsial and protozoal diseases, in addition some helminths are transmitted by fleas (boris 2008). fleas can transmit yersinia pestis, rickettsia typhy, r. felis, bartonella henselae, myxoma virus and some helminthic diseases, such as hymenolepis nana and dipylidium caninum (tapeworms). most species of fleas are host specific. rodents are predominant host and 74 % of fleas feed on rodents (otranto and wall 2008, capinera 2010). the aim of our study was to detect the infestation rates of east hedgehogs (erinaceus concolor) with ticks and fleas in van province, eastern region of turkey. materials and methods study area hedgehogs were captured around the city of van (38° 28' n 43° 20' e) in the, eastern region of turkey. the city is located around lake van and has 370.000 inhabitants. the study was conducted between june 2013– sebtember 2013 in three different localities. animal capture and collection of ticks and fleas hadgehogs were captured between june and sebtember 2013 with the aid of spotlight nightly walks through parts of the study area. in order to estimate flea and tick infestation of hedgehogs, we immobilized the ectoparasites by polluting the body with a insecticide including trichlorphon (neguvon®-bayer). immobilized fleas were removed from the hedgehogs by gently shaking the animal over a sheet of paper and then all the fleas counted. we collected all ticks from hedgehogs and conserved them in 70 % ethanol for post identification. ticks were identified according to karaer et al. (1997) and fleas were identified according to dinçer (1971). results throughout the investigation, 60 ixodid ticks and 125 fleas in total were collected from 21 hedgehogs, collected seven hedgehoges each locality, from three different localities. all the collected ticks belonged to rhipicephalus turanicus species and all fleas belonged to archaeopsylla erinacei species (fig. 1, fig. 2). other ectoparasites, apart from this kind of species, were not observed in this investigation. rates of the tick and fleas infestations were 66.66 % and 100 % respectively. table 1 and table 2 shows lo-calities where hedgehogs were obtained and the numbers and gender distribution of the fleas and ticks that were collected from these hedgehogs. fig. 1. a) dorsal view of male rhipicephalus turanicus, b) spiracular plate of male rhipicephalus turanicus, c) dorsal view of female rhipicephalus turanicus, d) spiracular plate of female rhipicephalus turanicus fig. 2. a) female archaepsylla erinacei, b) male archaepsylla erinacei, c), d) adult ticks on the hedgehog http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 50–54 y goz et al.: ticks and fleas infestation … 52 table 1. locality names and numbers of hedgehogs locality n of hedgehog kasımbağı village 7 gölağzı neighborhood 7 çınarlı neighborhood total 7 21 table 2. numbers and gender distribution of fleas and ticks obtained from hedgehogs locality kasımbağı village gölağzı neighborhood çınarlı neighborhood ectoparasite fleas ticks fleas ticks fleas ticks f m f m f m f m f m f m 4 3 3 2 2 3 2 0 1 3 1 5 6 1 0 0 4 2 0 0 5 2 4 2 3 3 4 0 6 1 1 3 5 1 1 3 3 5 4 1 3 4 3 3 4 0 0 0 3 4 0 0 6 0 3 1 6 2 2 0 4 0 0 3 3 3 0 0 3 5 0 0 3 4 2 0 0 2 1 1 0 3 3 2 female= 26 male= 20 total= 46 female= 13 male= 6 total= 19 female= 24 male= 15 total= 39 female= 10 male= 8 total= 18 female= 24 male= 16 total= 40 female= 11 male= 12 total= 33 discussion one of the most important arbovirus transmitted by ticks is crimean-congo hemorrhagic fever viruse (cchfv) believed to be survived mainly by hyalomma ixodid ticks in the nature. ticks and fleas are important vectors of several patogens and majority of this patogens are zoonotic. but cchfv has been isolated in nature among at least 30 tick species including i. ricinus, r. sanguineus, r. turanicus, r. bursa and dermacentor spp. ticks in addition to h. marginatum (estradapeña et al. 2007, nijhof et al. 2007, randolph et al. 2008). ticks in the genus rhipicephalus belongs to family ixodidae (hard ticks). rhipicephalus turanicus is a three-host tick species. adult of r. turanicus normally infest cattle, sheep and dogs in mediterranean region but it can also effect humans (chochlakis et al. 2014). its immature forms generally infest hedgehogs, gerbils and murid rodents (estradapeña et al. 2004). rhipicephalus turanicus is a vector of q-fever and north asian tick typhus caused by rickettsia sibirica (i̇offeuspensky et al. 1997). some studies performed on hedgehogs ticks, prevalence of r. turanicus infestation was detected respectively 67.70 % in urmia city and 5.26 % tabriz city of iran (gorganifirouzjaee et al. 2013, nematollahi et al. 2014), and 77.80 % tokat city, central anatolia region of turkey (bursali et al. 2013). rate of the tick infestation in present study (66.66%) was detected approximately similar to studies of gorgani-firouzjaee et al. 2013 and bursali et al. 2013. archaeopsylla erinacei is the hedgehogs fleas. it is also hosted by cats and dogs other than hedgehogs and it may be transferred http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 50–54 y goz et al.: ticks and fleas infestation … 53 from hedgehogs through direct contact. adult animal fleas may attact and bite humans. human cases with fleabite eruption reported in some countries (pomycal 1985, naimer et al. 2002). prevalence of a. erinacei in hedgehogs was detected 55.90 % in iran, neighboring country of turkey (gorgani-firouzjaee et al. 2013). our result (100%) was very higher than this study. conclusion we detected ticks (r. turanicus) and fleas (a. erinacei) in hedgehogs at fairly high rates. many ticks and fleas species may harbor on hedgehogs and transmit some tick-borne and flea-borne patogens. therefore, hedgehogs sharing the same habitates with humans in especially urban and suburban regions may be transmission of some tick-borne and fleaborne diseases from hedgehogs to humans. at least, hedgehogs ticks and fleas can attack to humans and consequently some traumatic lesions can occur in humans. acknowledgements the authors declare that there is no conflict of interest. references beichel e, petney tn, hassler d, brückner m, maiwald m (1996) tick infestation patterns and prevalence of borrelia burgdorferi in ticks at a veterinary clinic in germany. vet parasitol. 65: 147–155. boris rk (2008) functional and evolutionary ecology of fleas: a model for ecological parasitology. cambridge 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4(11): 911–913. nematollahi a, helan ja, golezardy h, zaboli n, nourizi m, azari m (2014) parasitic fauna of east european hedgehogs (erinaceus concolor) and their pathological aspects in iran. advances in zoology and botany. 2(1): 1–5. nijhof am, bodaan c, postigo m, nieuwenhuijs h, opsteegh m, franssen l, jebbink f, jongejan f (2007) ticks and associated pathogens collected from domestic animals in the netherlands. vector borne zoonotic dis. 7: 585–595. otranto d, wall r (2008) new strategies for the control of arthropod vectors of disease in dogs and cats. med vet entomol. 22(4): 291–302. pomykal j (1985) a case of infestation of humans with fleas archaeopsylla erinacei (siphonaptera: pulicidae). folia parasitol. 32(4): 348. randolph s, asokliene l, avsic-zupanc t, bormane a, burri c, gern l, golovljova i, hubalek z (2008) variable spikes in tick-borne encephalitis incidence in 2006 independent of variable tick abundance but related to weather. parasit vectors. 1: 44. riley py, chomel bb (2005) hedgehog zoonoses. emerg infect dis. 11: 1–5. service mw (2012) medical entomology for students. cambridge university press, cambridge uk. http://jad.tums.ac.ir published online: june 27, 2015 microsoft word dr soltani rtl.doc iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 12 original article use of expanded polystyrene (eps) and shredded waste polystyrene (swap) beads for control of mosquitoes a soltani 1, *h vatandoost 1, h jabbari 2, ar mesdaghinia1, ah mahvi 1, m younesian 1, aa hanafi-bojd 1, s bozorgzadeh 3, mr abai 1, a pakari 1, h shabkhiz 1 1department of medical entomology and vector control, school of public health and institute of health research, tehran university of medical sciences, iran 2 research center for environmental health, tehran, iran 3 chabahar free trade zone, sistan and baluchistan province, iran (received 17 nov 2008; accepted 12 may 2009) abstract background: mosquitoes transmit several diseases to human. there are several measures for control of larvae. as part of integrated vector management (ivm) program, the utility of floating layers of polystyrene beads (eps) is a potential alternative in habitats of mosquito larva. eps beads prevent oviposition of mosquito as well as killing the immature stages by forming a tick layer on the water surface. they are cheap, environmentally safe and do not need frequent application and remain on the surface of water for long time. the objective of the current study was to asses the effectiveness of two types of polystyrene beads of (eps) and (swap) for control of mosquito larvae under laboratory conditions. methods: anopheles stephensi and culex quinquefasciatus were used for experimental purposes. in each tray 250 larvae of late 3rd and early 4th instars were introduced. the experiment was conducted on 4 replicates for an. stephensi, cu. quinquefasciatus and combination of both. emerging of adult mosquitoes were calculated every day until the end of experiments. results: mortality rate and inhibition of emerge (ie) for cu. quinquefasciatus, an. stephensi and combination of both species was 97.8%, 100% and 99.07%, respectively using eps. in average, eps was able to kill 98.9% of larvae. the figures with swap were 63%, 91.05% and 72.65%, respectively. the average mortality for mosquitoes was 75.57% conclusion: eps and swap beads can be very effective and practical for elimination of an. stephensi and cx. quinquefasciatus under the laboratory conditions. keywords: polystyrene beads, mosquitoes, anopheles stephensi, culex quinquefasciatus, iran introduction mosquitoes belonging to the family of culicidae transmit some of the worldwide important diseases such as malaria, filariasis, yellow fever and dengue hemorrhagic fever. these diseases are rising and some of them re-emerged in many tropical and subtropical areas (who 1999). therefore, approaches to reduce the incidence of vector-borne diseases by controlling mosquito populations are highly warranted (subbiah and brij kishore 2006). urbanization with poor sanitation increases the number of breeding places for mosquitoes, especially culex spp, such as pit latrines, cess pits, cesspools, disused wells, blocked open drains, open septic tanks, polluted puddles, uncovered overhead tanks (das and rajagopalan 1989, nathan et al. 1996). in some parts of iran due to the lack of water pipe system, villagers use the stored water for drinking and washing. these kind of breeding places have ideal conditions for anopheles, aedes *corresponding author: dr h vatandoost, tel/ fax: +98 21 88951393, e-mail: hvatandoost@ yahoo.com iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 13 and culex mosquito habitats. re-emerge of malaria epidemic have occurred in the cities of iran in the recent years (edrissian 2006). mosquito control with the use of insecticides in some areas is not very effective because of vector resistance, exophilic vector behavior, environmental concern and low community acceptance (malaviya et al. 2006). additionally only few number of insecticides have been recommended for control of mosquitoes in potable water. many populations of mosquito vectors of diseases have developed resistance to synthetic organic insecticides (organochlorines, organophosphates, carbamates and pyrethroids) that have been used mostly during the last half of the 20th century. thus interest in alternate strategies as well as integrated vector management (ivm) have been increased in recent years (who 1982a, b, who 1986). therefore, methods of control are effective to produce the desired results, including the use of bio-control agents and environmental management. one of the effective non-chemical methods for control of mosquito’s larvae is usage of expanded polystyrene (eps). these agents eliminate/minimize the mosquito breeding places leading to decrease of adult population. an alternative and plentiful source of expanded polystyrene is the waste material destined for landfill sites, packaging, from perishable foodstuffs and shipping electrical and other appliances. unexpanded polystyrene beads are produced by the petrochemical industry with pentane in solid solution in each bead. industrially, the beads are heated with steam to make eps beads, which are then heated and pressed to form blocks or slabs for packaging, insulation (curtis et al. 1989, sivagnaname et al. 2005). also shredded waste polystyrene (swap) is produced from this products and a suitable alternative to use of commercially available, swap appears to offer an effective, cheep and readily available alternative to eps for mosquito controlling purposes. the transport of large volumes of eps beads over long distances is cost prohibitive and thus expansion using locally available resources is suggested (curtis and feachem 1981). a 2 cm thick layer of 2 mm beads is sufficient to eliminate mosquito breeding. floating layers of 2 mm expanded polystyrene were found to be most effective for suffocating anopheles larvae and pupae. they must be placed in sacks or dumps to take them to the pits where they are needed. if piled loose in an open trunk, they would blow away before reaching their destination. storage of beads for quite a long time would result in failure of the beads to expand to the maximum when boiled due to loss of pentane; hence it has been suggested that beads should be boiled soon after procurement from the production firm (madurai and nadu 1995). in iran there are several methods for control of mosquito larvae in breeding places. use of some organophosphate insecticides, patchy application of bacillus thuringiensis, and introducing of larvivorus fishes are the main control measures. the bionomics of malaria vectors have been studied in the region (vatandoost and moinvaziri 2004, vatandoost et al. 2004, vatandoost et al. 2006, hanafi-bojd et al. 2006). the objective of the present study was to evaluate the efficacy of local made industrial beads for mosquito control under laboratory conditions. material and methods mosquito strains an. stephensi is the main malaria vector in hormozgan province. the filed strain of bandar abbas was collected and reared in the insectray of bandar abbas health research center, southern iran related to tehran university of medical sciences, school of public health. cu. quinquefasciatus also was collected from the field and maintained in the insectary. iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 14 eps and wasp eps and swap were purchased from the national petrochemical company (npc), in iran. experimental design sample size was designed according to who methods (who 2005) with some minor modifications. a total of 36 trays of 35× 25×10 cm were used. a total of 250 larvae of mosquito (250 anopheles or 250 culex or mixed 125 culex+125 anopheles, based on designed pattern) at the late 3rd and early 4th instars were released into each tray. based on kind of treatment, four replicates were used for each species (fig. 1). temperature in the insectaria was 27±3o c, with a period of 14-10 h light-dark. relative humidity was maintained at 70-80%. larvae were fed by fish flakes. feeding and water volume were prepared for all trays as required. subsequently the eps and swap were added to the water surface of treated trays to make a layer of 0.5-1 cm. one group of 1000 larvae was used as control for each species. fig. 1 shows the design of the experiment. group1 group (ii) group (iii) anopheles : culex anopheles + culex swap eps control fig. 1. schematic diagram of the tests performance the fish food was added daily to the rearing trays, and then the surface of treated water was covered with the eps or swap. the rearing trays were covered with net to prevent the adult mosquito escaping during the test period. mosquitoes were emerged daily and trapped under the net cover. this allowed us to catch them using a mouth aspirator and to identify the genus. adult emergence was calculated daily up to 20 d. at the end of experiments all the dead and alive stages of mosquitoes were counted and recorded. after emerging all mosquitoes, the dead adults, pupae and larvae from the polystyrene layers were counted and subjected to statistical analysis. statistical analysis the larval mortality data were corrected according control mortality by the abbott’s formula (abbot 1925). inhibition of emerge (ie%) or mortality of mosquitoes is calculated using the following formula: where t= percentage emergence in treated trays and c= percentage emergence in the control. if adult emergence in the control is less than 80%, the test should be discarded and repeated (mulla and darwazeh 1975). where the percentage is between 80% and 95%, the data are corrected using abbott’s formula. anova was carried out with spss 11.5. means were compared with post hoc tests (lsd), (p< 0.05). results results of mosquitoes mortality based on life stages are given in table 1. from a total of 5750 out of 9000 larvae in all rearing trays, the highest number of mortality (2973) was recorded in eps, followed by swap (2362), and control (415) rearing trays. iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 15 if the control mortality was between 5% and 20%, the mortalities of treated samples were corrected according to abbott’s formula. results of adult mosquitoes collected live using mouth aspirator based on used materials, are showed in fig. 2 during 20 d of the study period. we can observe differences between three methods clearly. in another step of the experiment, we assessed and compared the survival means by post hoc tests (lsd) based on untreated control and treated samples. details of daily emergence based on mosquito groups and the tested materials are presented in fig. 2. as shown in the table1, the adult emergence in the control is 86.17% (mortality 13.83%), therefore our treatment results were corrected using the abbott's formula. fig. 3 has compared percentage emergence of mosquitoes with 95% confidence intervals for each types of polystyrene, species of mosquitoes and control group. table 1. mortality of mosquitoes in treated samples and untreated control eps group mosquito species larva pupa adult total %mr corrected mr(ie%) * culex 981 0 0 981/1000 98.1 97.8 anopheles 998 2 0 1000/1000 100 100 anopheles+culex 984 8 0 992/1000 99.2 99.07 total 2963 10 0 2973/3000 99.1 98.96 swap group mosquito species larva pupa adult total %mr corrected mr * culex 379 25 269 673/1000 67.3 63.0 anopheles 549 83 293 925/1000 92.5 91.05 anopheles+culex 402 26 336 764/1000 76.4 72.65 total 1330 134 898 2362/3000 78.73 75.57 control group mosquito species larva pupa adult total %mr culex 109 7 0 116/1000 11.6 anopheles 146 16 0 162/1000 16.2 anopheles+culex 137 0 0 137/1000 13.7 total 392 23 0 415/3000 13.83 * percentage of mortality (ie%) is corrected by abbott’s formula mr: mortality 0 100 200 300 400 500 600 700 800 900 1000 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 day 10 day 11 day 12 day 13 day 14 day 15 day 16 day 17 day 18 day 19 day 20 control swap eps fig. 2.number of mosquitoes collected by aspirator from different rearing trays in treated and control experiments iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 16 0 0.8 86.3 23.6 83.8 7.5 1.9 32.7 88.4 0 10 20 30 40 50 60 70 80 90 100 eps swap control eps swap control eps swap control culex anopheles anopheles + culex a a b a b c b c c the mean difference is significant at the 0.05 level fig. 3. comparing the emergence of mosquitoes in two types of polystyrene discussion there are several reports concerning use of polystyrene beads for elimination of mosquito emergence in the world in the cess pits, flooded cellars, soakage pits, water tanks, gem pits and industrial complex (rietre 1978, chavasse et al. 1995, curtis and minjas 1985, curtis 1994, maxwell et al. 1990, reuben et al. 2001, chandrahas and sharma 1987, yapabandara and curtis 2002, dua et al. 1989, rieter 1985, sharma 1985, chandrahas et al. 1993). the layer of this product reduces the density of mosquito by suffocating larvae and pupae and inhibiting mosquito egg-laying in water surface. our results showed that utility of eps and swap beads can be completely effective and practical for elimination of an. stephensi and cx. quinquefasciatus under the laboratory conditions. larval mortalities increased significantly (p< 0.05) with using both polystyrene forms and the mortality was greater in those larvae treated by eps than swap (p> 0.05). mortality rate for cx. quinquefasciatus, an.stephensi and combination of both species was 97.8%, 100% and 99.07%, respectively using eps. in average eps was able to kill 98.9% of mosquitoes. observations of the present study show that eps beads induced 98.9% mortality in the tested mosquitoes, representing 98.61% and 0.34% in the larval and pupal stages, respectively. the figures for swap were 63%, 91.05% and 72.65%. the average mortality for mosquitoes was 75.57%. the swap beads caused 75.57% mortality: 42.55% larvae, 4.29% pupae and 28.73% in newly emerged adults that were trapped in swap beads. inhibition of emerge (ie) for eps obtained 100%, 97.8 and 99.07 in the case of an. stephensi, cx. quinquefasciatus and an. ste% e m er ge nc e iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 17 phensi+cx. quinquefasciatus, respectively. the ie for swap was calculated 91.05, 63.0 and 72.65 in an.stephensi, cx. quinquefasciatus and an.stephensi+cx. quinquefasciatus, respectively. similar studies showed a mortality rate of 72.3-100% (curtis et al. 1989, sivagnaname 2005). the observed mortality using eps imply that this material provides an appropriate and unique floating layer on the water surface, however the swap beads due to its variability in beads’ diameter and irregular shapes are not able to cover water surface completely, mortality and inhibition of emerge is less than that of eps. this factor leads to adult emergence in swap treated trays. although a relatively high number of mosquito larvae could converted to adults, but most of them were trapped among layers of swap beads consequence of trapping and dead. some of emerging adults showed morphological abnormalities such as wing, leg, head, thorax and abdomen malformations at rearing trays treated by swap. difference in survival rate of anopheles and culex mosquitoes is another important point. the emergence rate for an. stephensi was 46.31%, among which 22.49% of them were trapped among swap beads and resulted to dead, so the survival rate for this species was evaluated as 23.82%. concerning cx. quinquefasciatus, 75.91% of larvae completed their development cycle and changed to adult stage, while 39.27% of them were dead and therefore the survival rate was calculated as 36.27%. the comparison between the survival rates of these two mosquito species showed that culex has more relative ability to survive than anopheles (1.5fold). this ability may be is attributed to the morphological and physiological characteristics and respiratory system of the culex larvae that allow them to breathe easier than anopheles under the covering layers on water surface. additionally culex mosquitoes are able to survive in organic polluted water bodies. parallel to our study, nathan et al. (1996) conducted laboratory and filed trail on the use of commercially available, expanded-polystyrene beads for the control of cx. quinquefasciatus. they were not able to gain 100% mortality in the population of mosquitoes in the laboratory. they concluded that the irregular shape of the swap particles greatly reduced their capacity to spread over the water surface and the interstitial air spaces also permitted respiration and development of mosquito larvae. under field conditions and in wet pit latrines the control sustained for two months. efficacy of agnique mmf, a monomolecular film formulation, was tested against immature of an. stephensi in india. simulated field trials carried out in cement tanks showed 100% inhibition of adult emergence for up to 3 weeks. in tanks and wells it produced 75% reduction of late instars and 100% reduction of pupae on day 1 (batra et al. 2006). beehler and defoliart (1991) used eps for control of aedes triseriatus in the laboratory and the field. eps reduced the emergence of adults by preventing normal eclosion from the pupae. in the field, tree holes containing eps had significantly fewer larvae than controls. bekheit et al. (1991) compared the oils and expanded polystyrene beads for the control of mosquito breeding in egypt. results revealed the superiority in effectiveness of beads over oil for the control of mosquito breeding. mosquito catches from cesspits treated with polystyrene beads dropped to zero soon after application of the beads and remained so throughout five months observation period. a field trial of the use of eps to control the breeding of mosquito larvae in household septic tanks was conducted by chang et al. (1995). one week after treatment, the breeding of cx. quinquefasciatus and ae. albopictus was reduced by 100% and 68.7%, respectively. for both species combined, a 57.25% reduction in the adult emergence rate was achieved. no adult was caught in the emergence trap one month after treatment. iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 18 data analysis of our results for mosquito’s survival showed that there are significant differences between two polystyrene forms (p< 0.05). using eps, a significant difference observed between all mosquitoes (anopheles, culex, and their combination) as compared to the control group (p< 0.05), additionally there was significant difference between two species (p= 0.012). similarly, in the swap tests a significant difference observed between all species and control (p< 0.05). therefore, the present study clearly indicates that eps beads could provide excellent larval control for mosquitoes. in addition, it was found that both materials had high larval control and both of them could be used in the control of mosquitoes. eps beads could be used as one of the promising vector control methods to eliminate/ minimize the mosquito breeding sources in ivm programs, while swap appears to offer good prospects as an alternative to expanded-polystyrene beads for inexpensive, long-term mosquito control in selected habitats. in total, regardless of efficacy of the method in the control of mosquito larval stage under laboratory conditions, the extensive field studies in the area (iran) are recommended to evaluate and may be introduce this method as an easy and inexpensive way in mosquito control, especially in non-drinking water sources. trials of using polystyrene beads for control of mosquito have been conducted in several parts of the world (curtis et al. 2002). the method greatly and substantially reduced the vector population. utility of eps and swap as a component of integrated vector management with other supportive measures could assist considerably the process of elimination of mosquito-borne disease. further study is required to the practicality of using these products in community-based, mosquito-control programs. acknowledgements authors are grateful to center for environmental research (cer), tehran university of medical sciences, for financial support of this research project. references abbott ws (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–267. batra cp, mittal pk, adak t, subbarao sk (2006) efficacy of agnique mmf monomolecular surface film against anopheles stephensi breeding in urban habitats in india. j am mosq contr assoc. 22(3): 426–432. beehler jw, defoliart gr (1991) potential use of scrap expanded polystyrene beads for the control of aedes triseriatus. j am mosq contr assoc. 7(2): 299–300. bekheit ss, el agroudy rm, mikhail mw, ibrahim sh, moneim mm (1991) small scale field trial with polystyrene beads for the control of mosquito breeding. j egypt soc parasitol. 21(1):179-182. chandrahas rk, sharma vp (1987) smallscale field trials with polystyrene beads for the control of mosquito breeding. indian j malariolo. 24: 175–180. chandrahas rk, nayak hk, kar i, ravindran j, eapenn a (1993) involvement of voluntary agencies in malaria control in madras city. in: sharma vp, editor. community participation in malaria control. delhi: malaria research centre. 211-229. chang ms, lian s, jute n (1995) a small scale field trial with expanded polystyrene beads for mosquito control in septic tanks. trans roy soc trop med hyg. 89(2): 140– 141. chavasse dc, lines jd, ichimori k, majala ar, minjas jn, marijani j (1995) mosquito control in dar el salaam. ii. impact of expanded polystyrene beads and pyriproxyfen treatment of breeding sites on culex quinquefasciatus densities. med vet entomol. 9(2): 147-154. iranian j arthropod-borne dis, (2008), 2(2): 12-20 a soltani et al: use of expanded polystyrene… 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sri lanka. acta trop. 81: 211-223. razi microsoft word 10dr edalat rtl.doc iranian j arthropod-borne dis, 2010, 4(1): 72–76 m salimi et al.: human urogenital myiasis … 72 case report human urogenital myiasis caused by lucilia sericata (diptera: calliphoridae) and wohlfahrtia magnifica (diptera: sarcophagidae) in markazi province of iran m salimi1, d goodarzi2, mh karimfar3, *h edalat4 1department of parasitology, school of medicine, arak university of medical sciences, iran 2department of urology, school of medicine, arak university of medical sciences, iran 3department of anatomy, school of medicine, ilam university of medical sciences, iran 4department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran (received 10 feb 2010; accepted 22 feb 2010) abstract we report a case of human urogenital myiasis in an 86-year-old rural man with a penil ulcer and numerous alive and motile larvae from urethra and glans penis. entomological studies on adult flies showed the larvae were lucilia sericata and wohlfahrtia magnifica. the clinical presentation and treatment strategies are discussed. keywords: lucilia, wohlfahrtia, urogenital, myiasis, iran introduction myiasis can be defined as the invasion of organs and tissues of human being or other vertebrate animals by dipterous larvae, which feed upon the living, necrotic or dead tissues for at least a period of time, or in the case of intestinal myiasis, they feed on the host's ingested food (service 1986). the various forms of myiasis may be classified from an entomological or a clinical point of view. entomologically, flies may be classified in three groups: obligatory or specific, facultative or semi-specific and accidental. clinically, myiasis can be classified according to the part of body that is being invaded. dermal and subdermal myiasis includes wound or traumatic myiasis. in furuncular myiasis a boil-like condition is produced. nasopharyngeal myiasis, including aural and ocular myiasis, involves invasion of the head cavities such as nose, eyes, and ears. some myiasis involves invasion of the alimentary tract or the urogenital system (kettle 1990). we report two species, lucilia sericata (meigen 1826) and wohlfahrtia magnifica (schiner 1862) that cause urogenital myiasis, which belong to family of calliphoridae and sarcophagidae. adult l. sericata has metallic green or copper green color with an 8–10 mm diameter, hairy back, and hairless squama wings (fig.1), and presents usually around butcher shops and slaughterhouses. it makes noise and bothers the residents. the female lays eggs in meet, fish, and animal corpse, infected wounds of humans or animals and excrement (service 1986, daniel et al. 1994). service 1986 has reported that the developmental stage of lucilia takes 10–23 days. between 8-12 h, the eggs transform into a conical larva, and complete peritreme of poste*corresponding author: dr hamideh edalat, email: edalat@tums.ac.ir iranian j arthropod-borne dis, 2010, 4(1): 72–76 m salimi et al.: human urogenital myiasis … 73 rior respiratory spiracles. after 4–8 days, larvae develop and drop on to soil and after 614 days transform in to adult flies. in humans, the myiasis due to l. sericata was reported in 1826 by meigen, that for the first time separated parasites from mouth, eyes and paranasal sinuses of a hospital patient (daniel et al. 1994). since then, many cases of human myiasis have been reported, by lucilia. ncluding myiasis in skin tumor and nosocomial myiasis (minar et al. 1998), aural myiasis in france (bahji et al. 2002), wound and auricular myiasis in iran (talari et al. 2004, yaghoobi et al. 2005), tracheostomy wound myiasis (franza et al. 2006). wohlfahrtia magnifica (sarcophagidae) are grayish and have three distinct black lines on the dorsal surface of the thorax (fig. 2). the dark markings on the abdomen are usually present as roundish lateral spots and triangular-shaped dark makings along the mid line and sometimes the dark mark are so large as to be more or less confluent making the abdomen appear mainly black (service 1986). their females are larviposit at moist body openings and at fresh wounds or scratches. larvae can even penetrate thin, unbroken skin. the larvae burrow in to the subcutaneous tissue to feed; inducing the formation of a boil-like cyst around groups of larvae with a small pore opening to the outside. a gravid wohlfahrtia female produces 120–170 larvae. in a host, the larvae grow rapidly and can cause considerable tissue destruction, after about 1 week larvae drop to the ground to pupate and can overwinter in this stage in humans (mullen et al. 2002). wohlfahrtia magnifica has been reported as the cause of otitis (fawzy 1991), ophthalmomyiasis (morsy et al. 1991), vulvar myiasis (delir et al. 1999), wound myiasis (iori et al. 1999, lmimouni et al. 2004), gingival myiasis (dorma et al. 2007, mohammadzadeh et al 2008), otomyiasis (uzunl et al. 2004), cutaneous myiasis (kokcam et al. 2005) and oral myiasis (droma et al. 2007). according to our survey, this is the first observation of urogenital myiasis in markazi province of iran. case report the patient was an 86 yr old rural man, who was well and healthy till 3 days before coming to the hospital and had developed penile ulcer, arak valeye asr larvae discharged from urethra and glans penis. physical examination revealed one painful black necrotic ulcer with numerous alive and motile larvae in the ventral of glans penis and distal of urinary meatus with erythematous and edematous border. contact dermatitis was seen in the same area but urinary meatus was not seen. skin and subcutaneous tissues destroyed the copora cavernosa. the patient was hospitalized for removal of the larvae, debridement of the necrotic tissue in the penile ulcer with surgical scalpel. some larvae was removed from the ulcer and sent to the lab of hospital for identification, then ulcer was cleaned using normal saline and povidone-iodine and the patient has taken antibiotic therapy with ampicillin and gentamycin for 7 days. the ulcer was cleaned with normal saline and povidone-iodine for two days after debridement, his skin repaired, and the patient discharged by urethral catheter and antibiotic therapy with cotrimoxazole. after one week, he was re-examined and the complaints of the patient disappeared after antibiotic and antiseptic treatment of the infested area. most of the larvae that were sent to the lab of valeye asr hospital were dead, only two larvae developed to adult flies, these adult specimens were sent to the department of parasitology and entomology, school of medicine of arak. the adults were identified as l. sericata and w. magnifica by the available keys (james 1947, khoobdel et al. 2008). the adults were deposited in the museum of insects, department of entomology, school of medicine, arak university of medical sciences. iranian j arthropod-borne dis, 2010, 4(1): 72–76 m salimi et al.: human urogenital myiasis … 74 fig. 1. adult of lucilia sericata emerged from the larva isolated from a patient in arak 2009 (original) fig. 2. adult of wohlfahrtia magnifica emerged from the larva isolated from a patient in arak, 2009 (original) discussion urogenital myiasis is due to deposition of fly larvae in the human genitourinary system. various species of flies are able to provoke urogenital myiasis, including fannia scalaris, chryzomya bezziana and sarcophaga spp. (werner 1975, ramalingam et al. 1980, cilla et al. 1992). more scattered cases have been reported in the world including the united states of america by dermatobia hominis (massey et al. 2002), in turkey by psychoda albipennis (taylan 2004), in india by chryzomya bezziana (wadhwa 2006), and also from spain by eristalis tenax (gonzalez et al. 2009). in iran only one case of huiranian j arthropod-borne dis, 2010, 4(1): 72–76 m salimi et al.: human urogenital myiasis … 75 man urogenital myiasis by chryzomya bezziana was reported (jdalayer 1978). myiasis should be considered as occupational disease among farmers and shepherd. in this present case, our patient was an elderly shepherd lived in a rural area. he stayed outdoor near sheep flock, where a considerable quantity of flies are usually present, possibly when he was urinating outdoor in the field, the female of wohlfahrtia deposits larvae at moist body opening (penis), then larvae burrow in to the penis to feed, inducing skin and subcutaneous tissue destroyed the copora cavernosa and then penis intestates with larvae of lucilia and wohlfahrtia caused penil ulcer. ulcer cleaned after debridement and the patient had taken ampicillin, gentamycin and cotrimoxazole. urogenital myiasis in this patient was due to poor hygienic condition that might have played a role in attracting the female flies of lucilia and wohlfahrtia. our case illustrates the importance of hygiene and sanitation in rural area with high fly population. it is also noteworthy that urogenital myiasis caused by larvae of blowfly (l. sericata) and flesh fly (w. magnifica) is uncommon in human, and this is the second report of human urogenital myiasis in iran. acknowledgments the authors would like to thank professor s tirgari and dr khoobdel for kind cooperation of identification and confirmation of specimens. we are also thanking to dr. mohammad ali oshaghi (dept. of medical entomology, tums) for his helpful comments and careful editing of the manuscript. the authors declare that they have no conflicts of interest. references antonio j, tavares, rodrigo barros, luciano a, favorito (2007) urgen penectomy in a patient presenting with epidermoid carcinoma of the penis associated to myiasis. international braz j urol. 33(44): 521–522. bahji m, sibiti m, agoumi a, sahibi h, asermouh a (2002) bilateral aural myiasis caused by lucilia cerisata. med trop. 62(5): 565–566. catts ep, mullen, g (2002) myiasis (muscoidea, oestroidea). in: mullen g, durden l (eds): med vet 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world. a textbook for physicians, veterinarians and zoologists. butterworth co, london. microsoft word 3dr nateghpoure rtl.doc.doc iranian j arthropod-borne dis, 2010, 4(1): 24–30 m nateghpour et al.: molecular monitoring of … 24 original article molecular monitoring of plasmodium vivax infection after radical treatment in southeastern iran *m nateghpour1, 2, s ayazian mavi1, h keshavarz1, s rezaei1, f abedi3, gh edrissian1, a raeisi4 1department of parasitology and mycology, school of public health, tehran university of medical sciences, iran 2national institute of health research 3hormozgan university of medical sciences, bandar abbas, iran 4center of infectious disease management, ministry of health, treatment and medical education, tehran, iran (received 21 feb 2010; accepted 19 apr 2010) abstract background: the aim was to evaluate the relapse risk of vivax malaria in patients who received radical treatment in hormozgan province, a malarious area located on southeast of iran. methods: a total of 95 symptomatic vivax malaria infected patients were enrolled in urban health centers of bandar-abbas, minab, bandar-jask and bashagard districts of hormozgan province, southeast of iran from january 2008 to march 2009 for consideration as a caseseries study. dna was extracted from parasite infected whole blood samples. a polymorphic region of plasmodium vivax merozoite surface protein 1 (pvmsp1) was selected and a pcr method was employed for all the samples to amplify the specific variable gene fragment. the obtained fragments in primary and secondary samples were sequenced. both nucleotide and amino acid sequences of the samples were investigated for returned patients. results: 3.2% of the patients experienced a second attack between 83-199 days after the initial episode of infection. alignment of nucleotide and their deduced amino acid sequences between pair sequences of primary and secondary isolates revealed 8 and 6 dissimilarities respectively for the first case, and 9 and 7 dissimilarities for the second case. although microscopical examination of recurrent thick blood smear of the third patient confirmed new p. vivax infection, the venous blood sample was accidentally missed. sequencing results of primary and returned isolates 1p, 1s, 2p, 2s and 3p in this study showed an identity with bp13, t117, bp13, tc28 and chesson genotypes respectively. conclusion: the returned (secondary) isolates may account to be for the sake of reinfection. keywords: p. vivax, relapse, re-infection, recrudescence, pvmsp1, iran introduction among the four species of human plasmodia, plasmodium vivax and p. ovale are associated with relapse episodes. precise discrimination between such phenomenon and re-infection or treatment failure cases in malarious areas particularly in transmission seasons cannot be easily performed unless employing new and advanced bio-molecular techniques that are accounted to be relatively helpful. although p. vivax is known as the cause of benign tertian malaria increasing evidences suggest that life-threatening complications of the parasite are more common than previously thought. p. vivax is the major agent of malaria morbidity in asia and the second most prevalent plasmodium in the most of malarious regions (wernsdorfer 1988, mendis et al. 2001, hay et al. 2004). widely distri*corresponding author: dr mehdi nateghpour, e-mail: nateghpour@sina.tums.ac.ir,tel: +982188989130 iranian j arthropod-borne dis, 2010, 4(1): 24–30 m nateghpour et al.: molecular monitoring of … 25 bution of p. vivax and existence of relapse behavior in the parasite emphasize the importance of considering the relapse phenomenon particularly after radical treatment. a number of studies have been carried out to explore the nature of relapse using various methods. it has been known that manifestation of relapse in p. vivax depends on variation of the parasite strains (collins et al. 1996). the chesson strain an example of tropical zone strains relapses with short intervals almost one month during the year (coatney et al. 1950). the st. elizabeth strain of p. vivax with 6 to 14 mo latent period belongs to temperate zone strains that are the second pattern of relapse activity (manson 1975, krotoski 1985). the manifestation of relapse in this pattern renewed during long intervals (collins and sullivan 1996). in addition to the two main mentioned patterns of relapse activity, an intermediate form also has been reported (adak et al. 1998). beside the relapse activity in p. vivax, renewed attack is also suspected as a reinfection or treatment failure in malarious areas such as iran located in middle east region. in 2008, approximately 11460 malaria cases were microscopically diagnosed in iran, which 90% of them were detected as p. vivax (cdc iran, 2008). a retrospective epidemiological study based on the biodata surveillance forms including radical treatment data at a limited part of southeastern iran showed that the relapse risk of p. vivax were 16.8% and 24.5% depending on one or two years after primary attack respectively (haghdoost et al. 2006). since radical treatment of vivax malaria is precisely employed on the malarious areas of iran results of the study stimulated us to evaluate the relapse risk of vivax malaria in hormozgan province, a malarious area of southeastern iran using p. vivax merozoite surface protein 1 (pvmsp1), as a useful genetic marker for genotypingp. vivax isolates (craig and kain 1996, zakeri et al. 2006). materials and methods study sites and sample collection this study was conducted in urban health centers of bandar-abbas, minab, bandar-jask and bashagard districts of hormozgan province, southeast of iran from january 2008 to march 2009 for consideration as a caseseries study. the first three districts have hot and humid weather with year round transmission of malaria parasites, but two main peaks are on may to august and october to november. bashagard district has temperate climate in winter with average temperature of 13.5˚ c (min.5˚ c and max.22˚ c) and warm weather in summer with average temperature of 36˚ c (min.32˚ c and max.40˚ c).transmission pattern of malaria in bashagrd is, more or less, the same as above-mentioned districts. plasmodium vivax and anopheles stephensi are the predominant species of parasite and vector in those areas respectively. a total of 95 symptomatic vivax malaria infected patients after preparing an informed consent from each of them or their parents, were enrolled for the consideration. pregnant women, patients below 4 yr of age and those who were unavailable for the follow up duration (9 months) were excluded from the study. ethical permission for the study was approved by tehran university of medical sciences ethical committee. a 2-ml venous blood from each patient was sampled into an edta contained tube. prior to sampling, all isolates were diagnosed as p. vivax by light microscopical examination of geimsa-stained thick blood smears. all samples were frozen and stored at -20˚ c until dna purification. following blood sampling the patients received 25 mg/kg chloroquine divided over three days and 0.75 mg/kg primaquine from day 3 onwards weekly for 8 consecutive weeks as a standard radical treatment of vivax malaria. the enrolled patients were followed up by microscopical examination of their blood thick smears bimonthly iranian j arthropod-borne dis, 2010, 4(1): 24–30 m nateghpour et al.: molecular monitoring of … 26 during next nine months for probable renewed manifestation. moreover, the registered patients were asked to contact us if they felt fever or any other symptoms at any time. parasite density for each sample was measured as the number of parasite per microlitre of blood in thick blood smear. genomic dna purification dna was extracted from p. vivax infected whole blood samples using a commercial genomic dna purification kit (qiagen) according to the manufacturer's instructions. polymerase chain reaction (pcr) amplification a polymorphic region of pvmsp1, where widely has been used for molecular analysis of relapse cases (craig and kain 1996, kirchgatter and del portillo 1998, chen et al. 2007, imwong et al. 2007) was selected and a pcr method was employed for all the samples to amplify a specific variable gene fragment. this gene fragment is located between two interspecies conserved blocks named: icb5 and icb6. the mentioned blocks are usually used as genetic marker for differentiation of p. vivax species. one pair of oligonucleotid primers, designed previously (craig and kain 1996, kirchgatter and del portillo 1998), were synthesized according to the mentioned conserved sequences as follows: 5΄tactacttgatggtcctc-3΄; 5΄ccttctggtacagctcaatg-3΄. pcr amplification were performed in 20µl of reaction mixture containing 2 µl premix (titan hot tag bioatlas), 20 pmol of each primer , 5 µl dna template and 9 µl distilled water. the amplification was composed of an initial denaturation for 5 min at 94˚ c, which followed with 35 cycles of 94˚ c for 30 seconds, 58˚ c for 1 min and 72˚ c for 1 min and 30 seconds. the reaction was stopped after a final extension process for 10 min at 72˚ c. pcr products were analyzed by performing gel electrophoresis on a 1% agarose gel using 100 bp dna marker (fermentase). after visualization of the ethidium bromidestained gels under uv light, the products were estimated to be approximately 680 bp. the obtained fragments were sequenced by seqlab in germany (www.seqlab.de). the resultant sequences of pcr fragments were analyzed in ncbi (ncbi, nih, usa). both nucleotide and amino acid sequences for each pcr product of primary and secondary infections have been considered for returned patients. nucleotid sequences of the pvmsp1 have been submitted to the national centre of biotechnology information gene bank and are available for public access under accession numbers gq403477, gq403478, gq403480 and gq403481 for the bandarask isolates and gq403479 for the bashagard isolate. results during the nine months follow up three cases (3.2%) of 95 p. vivax infected patients experienced a second attack on 83-199 days after the initial episode of infection respectively (table 1). the amplified segment of pvmsp1 block 5 gene in returned patients displayed identical size approximately 680 basepairs for both primary and secondary isolates (fig. 1). all five sequenced isolates in both directions revealed four distinct genotypes for pvmsp1. the rate of homology between primary and secondary isolates in each case at nucleotide and amino acid level is tabulated in table 2. sequencing results of 1p, 1s, 2p, 2s and 3p isolates, which were named pvjask-1 to pv-jask-4 and pv-bashagard respectively in genebank, showed an identity with bp13, t117, bp13, tc28 and chesson genotypes respectively in checking with genebank (ncbi, nih, usa). four isolates pvjask-1 to pv-jask-4 showed close similarity to salvador ι (sal-1) allelic type. alignment of nucleotide and their deduced amino acid between paired sequences iranian j arthropod-borne dis, 2010, 4(1): 24–30 m nateghpour et al.: molecular monitoring of … 27 of primary and secondary isolates revealed 8 and 6 dissimilarities for the first case, and also 9 and 7 for the second case in sequential characters respectively. two nucleotide insertions encoding q (caa) occurred in pvjask-1 and pv-jask-3 alleles at the beginning of the p-type part of sal-1 like sequence. moreover, in both alleles a p to q (cca to caa) and a h to q (cac to caa) substitutions was observed in amino acid sequence. in each of pv-jask-2 and pv-jask-4 alleles a single nucleotide polymorphism (snps) has occurred in p-type part of sal-1 like sequence, v to a (gta to gca). in pv-jask-1, pvjask-2, pv-jask-3 and pv-jask-4 alleles i to t (atc to acc), a to v (gcc to gtc) and e to q (gaa to caa) single nucleotide polymorphisms emerged at the same region of the gene. although microscopical examination of secondary thick blood smear of the third returned patient confirmed new p. vivax infection, the venous blood sample was accidentally missed. table 1: characteristics of returned patients and their paired primary/ secondary samples patient isolate day/month/year age (year ) area of aquisition parasitemia/µl of blood 1p 01/07/2008 11 bandar jask 13848 1r 05/11/2008 15467 2p 25/08/2008 9 bandarjask 308 2r 16/11/2008 231 3p 14/06/2008 11 bashagard * 3r 01/01/2009 27 * th e rate of parasitemia was not known. table 2. the rate of homology at nt∞ and aaδ level no patient homology at nt level homology at a.a level 1p (pv-jask-1) 1 1s(pv-jask-2) 98% 97% 2p(pv-jask-3) 2 2s(pv-jask-4) 98% 96% ∞nt : nucleotide, δ aa : amino acid fig. 1. size polymorphism of both paired isolates. m fermentase marker, p as primary infection and r as recurrent infection. c+ as positive control. for 3r we could not extract dna from prepheral blood smear. 1000 500 iranian j arthropod-borne dis, 2010, 4(1): 24–30 m nateghpour et al.: molecular monitoring of … 28 discussion understanding the type of reappearing p. vivax, the main causative plasmodium for malaria outside africa, at the same patient is a crucial recognition for combating vivax malaria at the endemic areas. such phenomenon will be more important when we encounter with the new infection after a course of radical treatment. relapses are a feature of p. vivax with at least one episode of recurrence because of activating dormant liver parasites (hypnozoites). they usually cause new clinical manifestations with a blood schizogonic pattern that are needed to be treated. beside of the relapse, reinfection (resulting from a new infected mosquito biting) and recrudescence (originating from drug resistant asexual blood stage parasites) subjects attract a number of relevant studies. reports released from some malarious areas indicate many relapses with different features of recurrence (craig and kain 1996, rowland and durrani 1999, gogtay et al. 2000, leslie et al. 2004, haghdoost et al. 2006, chen et al. 2007). this study was designed to perform a comparative molecular analysis of paired primary and secondary p. vivax isolates that appear in the same patient following an 8 wk radical treatment. three out of 95 patients experienced second episode of vivax malaria within 83-199 days after primary attack. dna sequences analysis of two paired primary and secondary p. vivax isolates collected from two patients displayed that they were not identical. with referring to the results obtained in this study we encounter with four generic explanations for these results: 1. the first probability is the recrudescence phenomenon. the new manifestation of p. vivax after primary attack in returned patients occurred on day 83 that usually is beyond the range of returning time for drug resistance at asexual blood-stage as defined (pukrittayakamee et al., 2004), unless we imagine the emergence of some primaquine-resistant hypnozoites, but owing to lack of reasonable facts the imagination remains unclear. on the other hand, the patients were living in the malarious endemic areas where transmission of malaria potentially may occur over the year. therefore, differentiation between recrudescence and reinfection may not be possible without some complementary considerations. 2. long incubation period, this phenomenon is one of the complicated propositions in p. vivax studies. inoculation of mixed genotypes of p. vivax (multiple clone infections) with different incubation periods by vector(s) or multiple bites happening over a long period can confuse among real relapse, successive infections released from liver because of different incubation periods and reinfection. besides the reason of endemicity, effective radical treatment of patients in this study seriously decreases the implementation of emergence of p. vivax with different incubation periods. 3. relapse phenomenon is another cause to explain the obtained results. although relapse is put usually at the first line attribution to new infections of p. vivax particularly if a similar genotype would be considered between primary and secondary infections (craig and kain 1996), some authors believe that parasites involved in relapse infection often bear a genotype different from those present at primary infection (imwong et al. 2007, koepfli et al. 2009). results obtained in this study may at the first view indicate to relapses, but employing precise radical treatment against p. vivax and also endemicity of malaria in the studied areas weaken such indication. 4. reinfection is the fourth conception that draws a line of explanation about the reappearance of p. vivax in this study. in spite of either 14-day or 8-week radical treatment of vivax malaria at the endemic malarious areas those recurrences occur after day 30 or day 74 can be assumed mostly to be reinfection unless reasonable facts refuse the assumption. according to our results, the nearest and latest recurrences occurred on 83 and iranian j arthropod-borne dis, 2010, 4(1): 24–30 m nateghpour et al.: molecular monitoring of … 29 199 days after primary attack and other reasons mentioned above we adopt the reinfection of p. vivax as a resultant for both returned patients in this study. leslie et al. in a comparative study between 14-day and 8week p. vivax malaria treatment using primaquine as an antirelapse drug showed 1.8% and 5.4% treatment failure respectively (leslie et al. 2008). the latter failure is comparable with the results obtained in our study. the authors did not document how they could distinguish treatment failures from those that are new infections. indeed, it is difficult to differentiate between either relapses and new infections or recrudescence by using current genotyping methods without including a number of aspects such as mixed genotyping markers and epidemiological features. although in this study recurrent venous blood sample of the third patient was lost accidentally, dna sequencing of the primary sample surprisingly revealed a chesson genotype p. vivax from bashagard area with warm dry and temperate climates in summer and winter respectively. this is the first report to our knowledge of chesson type p. vivax from such climate. some authors, as cited above, believe that the hypnozoites induce relapses are similar to those parasites cause primary attack (craig and kain 1996), but the question is that how we can differentiate between real relapses and reinfections or recrudescences. on the other hand, if the parasites in primary infections and relapses are genotypically identical why they follow different patterns of emergence particularly if genotype-specific immunity phenomenon that develops against primary bloodstage parasites (imwong et al. 2007) would be accounted. more combined studies including epidemiological and genotyping considerations are needed to clarify the questions that have been released in this field. acknowledgments we would like to thank mr. gh mohseni, mrs. e torabi, mr. a parekar, mrs. m shahrokhi, mr. a ramezanpour, mr. a ahmadi and mr. m safari for their technical cooperation and specific thanks to dr. h. hajjaran, mrs. a. motevalli, dr. r. safari and mrs. l. farivar for their usefull contribution in the study. this study was supported financially by center of infectious disease management in iran and tehran university of medical sciences. the authors declare that they have no conflicts of interest. references adak t, sharma vp, orlov vs (1998) studies on the plasmodium vivax relapse pattern in delhi, india. am j trop med hyg. 59: 175–179. cdc of ministry of health & medical education (2008) annual report of malaria cases. i.r.iran chen n, aullif a, rieckmann k, gatton m, cheng q (2007) relapses of plasmodium vivax infection result from clonal hypnozoites activated at predetermined intervals. j infect dis 195: 934–941. coatney gr, cooper wc, 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(la.) major, p. (la.) perfiliewi and p. (la.) tobbi. the objective of the present study was to identify and distinguish the females of p. perfiliewi, p. major and p. tobbi, in this district. methods: adult sand flies were collected with sticky papers, cdc light traps, and aspirator in 2006. individual sand flies of this four species from thirty different locations were characterized morphologically and by comparative dna sequences analyses of a fragment of mitochondrial gene cytochrome b (cyt b) and nuclear gene elongation factor 1 alpha (ef-1α). pcr amplification was carried out for all three species p. major, p. perfiliewi and p. tobbi in the subgenus larroussius. results: phylogenetic analyses of p. major populations in this study displayed two different populations and genetic diversity. spermathecal segment number, pharyngeal armature and other morphological characters of these three species were examined and found to present consistent interspecific differences. conclusion: according to our findings, the phylogeny of cyt b and ef-1α haplotypes confirms the relationships between p. major, p. tobbi and p. perfiliewi as already defined by their morphological similarities. keywords: phlebotomus, larroussius, cytochrome b, elongation factor-1α, morphology, iran introduction the blood-feeding female of phlebotomine sand flies (diptera: psychodidae, phlebotominae) include natural vectors of protozoa of the genus leishmania (kinetoplastida: trypanosomatidae), which are the parasitic causative agents of mammalian leishmaniasis (killick-kendrick 1990). there are approximately 700 species of phlebotominae sand flies divided among 6 genera, of which only two, i.e. phlebotomus in the old world (ow) and lutzomyia in the new world (nw) are medically importance (lane 1987, lewis 1982, lane 1993, sharma and singh 2008). only 10% of these phlebotominae sand flies act as disease vector. further, only 30 species of these are important from public health point (sharma and singh 2008). a total of about 21 leishmania spp. have been identified to be pathogenic to human (singh *corresponding author: dr yavar rassi, e-mail: rassiy@sina.tums.ac.ir iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 23 2006). however, recent studies based on molecular data have provided evidence that the genus phlebotomus is not monophyletic (depaquit et al. 1998). furthermore, rispail and leger (1998a) have recently revised the definition of morphological characters states. in the mediterranean region, species of the subgenus larroussius are the main vectors of leishmania infantum, which cause visceral leishmaniasis (vl) in humans and dogs (killick-kenrick 1990, guernaoui et al. 2005). the subgenus is readily identified by the development long extension of the spermathecal neck in females (rispail 1990). description of the subgenus larroussius created by nitzulescu (1931) with p. major annandale, 1910 as the type species (perfiliev 1968, lewis 1982). among 27 species of phlebotominae sand flies in the ow (artemiev and neronov 1984, rispail and leger 1998a, rispail and leger 1998b, secombe et al. 1993), at least 12 species of the subgenus larroussius are proven or probable vectors of leishmaniasis (killick-kendrick 1990). the subgenus larroussius is closely related to the subgenera transphlebotomus and adlerius, and the proliferation of species within larroussius and adlerius is probably recent (rispail and leger 1998b, rispail and leger 1991). although, identification of male specimens of larroussius are not very difficult, but determination of some females of this subgenus has been considered impossible based on morphological characters. leger et al. (1983) showed that three sympatric species of larroussius in greece could readily be separated by the morphology of base of the spermathecal ducts. killick-kendrick et al. (1991) separated 13 species of larroussius by the morphology of the base of the spermathecal ducts. recent studies showed that molecular tools could help resolve phylogenetic relationships between species of subgenus larroussius (esseghir et al. 1997, esseghir et al. 2000, muccio et al. 2000, pesson et al. 2004, parvizi and assmar 2007). there are four species of subgenus larroussius including p. kandelakii, p. perfiliewi, p. major and p. tobbi in meshkinshahr district, north western iran. the first species is a proven vector and the second is the probable vector of viscerotropic leishmania spp. in this area (rassi and javadian, 1998, rassi and javadian 1999, rassi et al. 2001a, rassi et al. 2001b, rassi et al. 2005). the objective of the present study was to identify and distinguish the females of p. perfiliewi, p. major and p. tobbi. they are similar in morphological characters and indistinguishable. we analyzed the sequences of cytochrome b (mtdna) and elongation factor-1α (ndna) genes of these three species and compared with morphological characteristics. complete phylogenetic information is available for these genes and they are useful for the study of molecular systematic and speciation. material and methods study area meshkinshahr district (48° 17′ n, 38° 15′ e) is located at 1890 masl in ardebil province, iran. the district occupies the northern foothills of the sabalan mountains, which rise to an altitude 4881 masl. temperature varies from -27 °c in winter to 41 °c in summer. the human population was 156141 in 2006 and the principal economic activity is sheep farming. sand fly sampling sand fly sampling was carried out from jun-october 2006 (during the period of peak activity), in 30 villages distributed throughout the meshkinshahr district. sand flies were collected once every 15 d from indoor habitats (bedroom, stable, toilet, bath room, hen nest, hay loft and store room) and outdoor habitats (yard, rodent burrow, stone and wall iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 24 crevices, fox burrows and dog kennel, rubble and riverbanks) using sticky papers, cdc light trap and aspirator. sand fly specimens were stored in 96% ethanol. dissection, mounting and morphological identification after recording the sampling data and locations, sand fly specimens were washed in 1% detergent then twice in sterile distilled water. each specimen was then dissected in fresh drop of sterile normal saline by cutting off the head and abdominal terminalia with sterilized forceps and single used mounted needles. the remainder of the body was stored in the sterile eppendorf microtubes. specimens were mounted in puri’s or berlese’s medium and identified using the identification keys of theodor and mesghali (1964), perfiliev (1968), and lewis (1982). morphological characters, which used in this study, included pharyngeal armature, spermathecal segments number, length of spermathecal neck, palpal and ascoids formula. first and second are more important characters. dna extraction dna was extracted from the dissected thorax and attached anterior abdomen of individual sand flies using the method of ishhorowiz (ready et al. 1991). in the 1.5 ml microtubes, the thorax plus anterior abdomen of each sand fly was frozen and defrosted twice to break up tissue using a sampler tips or pestel, with grinding mix. then sds mix was used to denature proteins associated with the dna, then ice cold 8m koac was added to remove effectively the sds proteins from solution. cell debris and proteins were separated from the dna by centrifugation then the dna in the supernatant was precipitated over night at -20 °c in 96% ethanol. following ethanol precipitation, the dna was dissolved in 15µl 1x te (10mm trishcl, 1mm edta ph= 8.0) and stored at -20 °c. pcr amplification of cyt b and ef-1α for cyt b one pair primers were used. cb3-fc (forward) (5′-ca(c/t) attcaacc (a/t)gaatgata-3′) with cb-r06 (reverse) (5′-tatctaatggtttcaaaaca attgc-3′) to amplify an overlapping 3′ fragment of 499 bp without primers (cb3 fragment). also for ef-1α one pair primers were used, ef-f05 (forward) (5′-cctgg acatcgtgatttcat-3′) with ef-f08 (reverse) (5′-ccaccaatcttgtagacatcctg-3′) to amplify of 454 bp without primers. the pcr reaction conditions were identical for both cyt b and ef-1α. 2µl 10x pcr buffer, 1.2 µl mgcl2, 0.15µl primers (f and r), 2µl dntps, 1.5µl dna with the reaction volume completed to 20 µl by distilled water, followed by initial denaturation of cyt b at 94 °c for 3 min. pcr consisted of 35 cycles of denaturation at 94 °c for 30 sec, annealing1 at 40 °c for 30 sec, annealing 2 at 44 °c for 30 sec, extension at 72 °c for 90 sec and then final extension at 72 °c for 10 min. for ef-1α, initial denaturation at 94 °c for 3 min. pcr consisted of 35 cycles of denaturation at 94 °c for 30 sec, annealing at 48°c for 30 sec, extension at 72 °c for 30 sec and then final extension at 72 °c for 10 min. direct sequencing of pcr products one hundred nanograms of each purified dna sample was cycle-sequenced using an abi parsim® big dye™ terminator cycle sequencing ready reaction kit (version 2.0) and abi 373/377 sequencing systems (abi, pe applied biosystems), with 3.2 pmol of the same primers that were used for pcr. aligning and phylogenetic analysis of dna sequences dna sequences from both strands were aligned and edited using sequencher demo 4.7 and bioedit softwares. multiple or pairwise sequence alignments of dna were used with clustal w ppc: clustalw version 1.7. phylogenetic analyses were done using iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 25 parsimony paup. relationships were inferred based on genetic distances using the neighbor joining (nj) option with default settings. results species composition of subgenus larroussius out of the 1743 sand fly specimens collected, 660 specimens (37.9%) belonged to the subgenus larroussius, including: phlebotomus (la.) kandelakii (31%), p. (la.) major (1.5%), p. (la.) tobbi (1.5%), p. (la.) perfiliewi (1.7%) and p. (la.) major group (2.1%). aligning and phylogenetic analysis of larroussius dna sequences pcr amplification of cyt b and ef-1α was successfully achieved for all 3 species of the subgenus larroussius. seven sequences for cyt b and four for ef-1α were compared for p. major. comparison of pairwise genetic similarity or score of sequences showed 87%-100% similarity for cyt b and 96%-100% similarity for ef1α. also 2.7% genetic diversity for cyt b and 2.8% for ef-1α was observed. there were two haplotypes for cyt b from seven sequences (table 1) and for ef1α, there were two haplotypes within four sequences (table 2). the neighbor-joining (nj) phylogram for cyt b showed two lineages, and each of these had subgroups with short branches. one of the lineages had one haplotype from sand flies from the same habitat (stone crevices) but different locations (ur kandi, mueel, agh daragh). the second lineage had one haplotype from sand flies from different habitats (rodent burrow and fox burrows) and locations (ghurt tappeh, alni, niaz suee) (fig. 1). in addition, phylogenetic tree for ef-1α showed two lineages and only one haplotype had subgroups. one of the lineages had one haplotype from sand flies from the same location (ur kandi) but different habitats (stone crevices, bedroom, and hen nest). the second lineage had one haplotype from one specimen in rodent burrow (fig. 2). seven sequences for cyt b and three for ef-1α were compared for p. tobbi. comparison of pairwise genetic similarity of sequences indicated the 100% similarity for both cyt b and ef-1α sequences. genetic diversity and unique haplotype for cyt b and ef1α were not observed. for cyt b and ef-1α one haplotype was obtained (table 1 and 2). the neighbor-joining (nj) phylogram for both cyt b and ef-1α showed one lineage for all specimens in the same location and habitat (fig. 1 and 2). six sequences for cyt b and three for ef-1α were compared for p. perfiliewi. comparison of pair wise genetic similarity of sequences indicated 100% similarity for both cyt b and ef-1α sequences. in this species genetic diversity for cyt b and ef-1α were not observed (table 1 and 2). phylogenetic tree for both cyt b and ef-1α showed one lineage for all specimens in the different location and habitats (fig. 1 and 2). the neighborjoining (nj) phylogram in combination of cyt b and ef-1α for seven specimens showed two lineages for p. perfiliewi and p. tobbi and one lineage for p. major (fig. 3). identification of the female larroussius species using morphological characters in the present study, the morphological characteristics of the three species female of larroussius were described as follows: phlebotomus major and phlebotomus neglectus palpal formula: 1, 4, 2, 3, 5 and the formula of ascoids: 2/3-8, 1/9-5. pharyngeal armature has occupied ⅓ of pharynx space and punctiform, arranged in several rows and anterior elements have serrated margin (fig .4). spermatheca with 14-16 segments and length of spermatheca neck ⅔ of spermatheca capsule (fig. 5). iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 26 phlebotomus tobbi palpal formula: 1, 4, 2, 3, 5 or 1, (2, 4), 3, 5 and the formula of ascoids: 2/3-8, 1/915. pharyngeal armature has occupied over ½ of pharynx space and punctiform, arranged in several concave irregular rows of large dots and anterior part of pharynx have a fine dots (fig. 6). spermatheca with 11-13 segments. length of spermatheca neck as long as spermatheca capsule (fig. 7). phlebotomus perfiliewi palpal formula: 1, 4, 2, 3, 5 and the formula of ascoids: 2/3-9, 1/10-15. pharyngeal armature has occupied over ½ of pharynx space and punctiform, arranged in several concave regular rows of large dots (fig. 8). spermatheca with 17-20 segments. length of spermatheca neck ½ of spermatheca capsule (fig. 9). identification of the male p. major and p. neglectus using morphological characters among 26 male specimens in this species 17 specimens of p. major (65.4%) with 20-30 ventrally directed and long hairs stand densely on coxite and palpal formula 1, 4, (2, 3), 5 (fig. 10 and 11). in 9 specimens of p.neglectus (34.6%) with less than 20 ventrally directed hairs, widely spaced and sparser and paplal formula 1, 4, 2, 3, 5 (fig. 12 and 13). discussion according to the results of previous studies, the vectors of viscerotropic leishmania spp. in ow mainly belong to the subgenera larroussius, adlerius, euphlebotomus, synphlebotomus and paraphlebotomus. in the subgenus larroussius, p.perniciosus in algeria, france, italy, malta, spain, p.ariasi in france, spain, italy, p. perfiliewi in italy, east mediterranean, north of africa, greece, azerbaydzhan, tunisia, p. tobbi in cyprus, east mediterranean, sicil, p. kandelakii in afghanistan, russia, and p. neglectus in greece, albania, portugal (killick-kendrick 1990). in north west of iran p. kandelakii and p. perfiliewi transcaucasicus are proven and probable vector of viscerotropic leishmania spp. (rassi et al. 2005). table 1. sequences comparison of cyt b-mtdna gene in the subgenus larroussius species total sequences pairwise genetic similarity (%) total aplotype unique haplotypes genetic diversity (%) p. major 7 87 – 100 2 0 2.7 p. tobbi 7 100 1 0 0.3 p. perfiliewi 6 100 1 0 0 table 2. sequences comparison of ef-1α-ndna gene in the subgenus larroussius species total sequences pairwise genetic similarity (%) total aplotype unique haplotypes genetic diversity (%) p. major 4 96-100 2 0 2.8 p. tobbi 3 100 1 0 0 p. perfiliewi 3 100 1 0 0 iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 27 table 3. collected sand flies of the subgenus larrossius in different location and habitats in meshkinshahr district, iran iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 28 fig. 1. neighbor-joining phylogenetic tree for dna haplotypes of cyt b (mtdna) of the subgenus larroussius sand flies species 0.1 tobbiaf161210 tobbiaf161212 msh899tobbi msh1657tobbi msh1481tobbi msh1292tobbi msh903tobbi msh551tobbi msh480tobbi msh721perfiliewi msh738perfiliewi msh756perfiliewi msh795perfiliewi msh812perfiliewi msh836perfiliewi perfiliewiaf161201 msh1201major msh297major msh619major msh1397major neglectusaf161191 neglectusaf161193 msh861major msh007major msh52major p.caucasicusfj217390 p.sergentieu980367 iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 29 fig. 2. neighbor-joining phylogenetic tree for dna haplotypes of ef-1α (ndna) of the subgenus larroussius sand flies species 0.01 msh297major msh312major msh317major majoref416834 neglectusaf160802 neglectusaf160801 msh522major msh721perfiliewi msh738perfiliewi msh836perfiliewi perfiliewiaf160805 msh903tobbi msh1292tobbi tobbiaf160810 p. caucasicusef416836 iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 30 fig. 3. neighbor-joining phylogenetic tree for dna haplotypes of combination of cyt b and ef-1α of the subgenus larroussius sand flies species. 0.01 msh-836-p. perfiliewi msh-738-p. perfiliewi msh-721-p. perfiliewi msh-903-p. tobbi msh-1292-p. tobbi msh-297-p. major msh-522-p. major p. caucasicus ef416836 iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 31 fig. 4. pharyngeal armature of p. major fig. 5. spermatheca of p. major fig. 6. pharyngeal armature of p. tobbi fig. 7. spermatheca of p. tobbi fig. 8. pharyngeal armature of p.perfiliewi fig. 9. spermatheca of p.perfiliewi iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 32 fig. 10. palpal formula of p.major fig. 11. coxite hairs of p.major fig. 12. palpal formula of p.neglectus fig. 13. coxite hairs of p.neglectus according to the finding of the present investigations, the phylogeny of cyt b and ef-1α haplotypes confirms the morphological relationships among the three species p. perfiliewi, p. major and p. tobbi in the subgenus larroussius. the males of these three species have a several diagnostic morphological characters, whereas the females of these species show very similarities in morphology of spermathecal segment number, pharyngeal armature and other characteristics. although morphological characteristics are the most practical methods for species distinguishing, new molecular techniques are very useful to resolve problems of identification in the cases with morphologically similarities. access on the genetic diversity and molecular systematic of the larroussius sand flies species not only useful to find the taxonomic status of them, but also indicates the ecological and geographical differences. in our analysis, all of the cyt b and ef1α sequences, the monophyly of the subgenus larroussius was confirmed, in concordance with the morphologically and molecularly based phylogenies of rispail and leger (1991, 1998b) and esseghir et al. (1997). all of the branches of the parsimony and distances trees had strong support and showed identical relationships and indicated the validity of many of characters in inferring evolutionary relationships. the trees were also topologically similar to the parsimony tree of esseghir et al. (1997). on morphological characteristics, the present study confirmed the observations of iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 33 perfiliev (1968), lewis (1982), leger et al. (1983), and killick-kendrick et al. (1991). phylogenetic analyses of p. major populations showed 2 lineages in different locations such as rodents burrow and stone crevices (table 3). according to our molecular and morphological finding, it seems that there are both p. major and p. neglectus in meshkinshahr district. this is the first report of p. neglectus in this area as well as in iran (table 3) further studies needs to resolve the taxonomic status of this species. p. major has many geographical variants the females of which have conventionally been distinguished by differences in pharyngeal armature. adler (1933) stressed the tendency of p. major to form geographical races. he showed (1946) that they differ mainly in the form of the pharyngeal armature of the females. he also mentioned ecological differences between the races and stresses that some races may have a different importance in the epidemiology of vl. our study on phylogenetic analyses of p. tobbi populations showed one lineage with single haplotype for either cytb or ef-1α gene. p. tobbi was first found in iran and israel, and was described as a variety of p. perniciosus. parrot (1934) reported that it is a single species with the name of p. tobbi. this was accepted also by theodor (1948). phlebotomus tobbi is mainly found in burrows and is rather common in transcaucasia and it seems to be identical with the sand flies from iran and southwest asia. therefore, p. tobbi is considered a separate species. phlebotomus perfiliewi is the probable vector of vl in the north west of iran. in the present study, this species had one lineage with a single haplotype. phylogenetic tree retrieved from cytb or combination of cytb and ef-1α showed close relationship of p. perfiliewi with p. tobbi. perfiliev (1966) recognized three subspecies of p. perfiliewi (perfiliewi, galilaeus and transcaucasicus) which were distinguished by minor morphological differences in the aedeagus of the male. lewis (1982) listed the same three subspecies but commented that the distinction of the male galilaeus from transcaucasicus uncertain. artemiev and neronov (1984) raised galilaeus and transcaucasicus to level species. the spermatheca of galilaeus and perfiliewi are indistinguishable, suggesting that these allopatric sand flies are taxonomically very close and are perhaps two subspecies (lewis, 1982). to find an exact distinction and identification of the female species of larroussius, needs more investigations in different parts of the world as well as iran. acknowledgements the author would like to appreciate very much for kind collaboration of health of center staffs of meshkin-shahr district. this study was m.sc thesis and financially was supported by the school of public health. tehran university of medical science: project no.: 240. 6129. references artemiev mm, neronov vm (1984) distribution and ecology of sandflies of the old world (genus phlebotomus). moscow: instituteof evolution, morphology and ecology of animals. 207 pp. [in russian]. depaquit j, perrotey s, lecintre g, tillier, s, ferte h, kaltenbach m, leger n (1998) systematique moleculaire des phlebotominae: etude pilote. paraphylie du genre phlebotomus. 321: 849-855. esseghir s, ready pd, killick-kendrick r and ben-ismail r (1997) mitochondrial haplotypes and geographical vicariance of phlebotomus vectors of leishmania major. insect mol biol. 6: 211-225. iranian j arthropod-borne dis, (2009), 3(2): 22-35 a absavaran et al: identification of sand flies… 34 esseghir s, ready pd, killick-kendrick r and ben-ismail r (2000) speciation of phlebotomus sandflies of the subgenus larroussius coincided with the late miocence-pliocence aridification of the mediterranean subregion. biol j linn soc. 70: 189-219. guernaoui s, pesson b, boumezzough a, pichon g (2005) distribution of phlebotomine sandflies, of the subgenus larroussius, in morocco. med vet entomol. 19: 111-115. killick-kendrick r (1990) phlebotomine vectors of leishmaniasis: a review. med vet entomol. 4: 1-24. killick-kendrick r, tang y, killick-kendrick m, sang dk, sirdar mk, ke l, et al. 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(ed.), proceeding of the first international symposium on phlebotominae sandflies. parasitologia 33(suppl.1): 485-492. rispail p, leger n (1998a) numerical taxonomy of old world phlebotominae (diptera: psychodidae). 1. consideration of morphological characters in the genus phlebotomus rondani and berte 1840. mem inst oswaldo cruz. 93(6): 773-785. rispail p, leger n (1998b) numerical taxonomy of old world phlebotominae (diptera: psychodidae). 2. restatement of classification upon subgeneric morphological characters. mem inst oswaldo cruz. 93(6): 787-793. secombe ak, ready pd, huddleston lm (1993) a catalogye of the old world phlebotominae sandflies (diptera, psychodidae, phlebotominae). occasional papers on systemat entomol. 8:1-57. sharma u, singh s (2008) insect vector of leishmania: distribution, physiology and their control. j vector borne dis. 45: 255 278. singh s (2006) new developments in diagnosis of leishmaniasis. indian j med res. 123:311-330. theodor o (1948) classification of the old world species of the subfamily phlebotominae (diptera: psychodidae). bull ent res. 39:85-115. theodor o, mesghali a (1964) on the phlebotominae of iran. j med entomol 1(3): 285-300. j arthropod-borne dis, september 2017, 11(3): 427–432 h bilal et al.: citrus seed oils efficacy … 427 http://jad.tums.ac.ir published online: september 08, 2017 short communication citrus seed oils efficacy against larvae of aedes aegypti *hazrat bilal 1, waseem akram 2, soaib ali hassan 1, sadrud din 3 1medical entomology and disease vector control, health services academy, islamabad, pakistan 2department of agriculture entomology, university of agriculture, faisalabad, pakistan 3water, agriculture and technology transfer program, kabul, afghanistan (received 21 sep 2012; accepted 29 nov 2016) abstract background: dengue fever is a serious public health issue in pakistan for many years. globally plants have been reported to contain compounds with insecticidal properties. these properties have been demonstrated more recently on the larval stages of mosquitoes. therefore, citrus cultivar seeds were evaluated for larvicidal potential against the primary dengue vector aedes aegypti. methods: extraction of oil was done by a steam distillation method and oils were evaluated according to who guidelines for larvicides 2005 for evaluation of insecticidal properties of citrus seed extracts against mosquito larvae. result: among the citrus cultivar seed oil, rough lemon (citrus jambhiri) had the lowest lc50 value (200.79ppm), while musambi (c. sinensis var musambi) had the highest lc50 value (457.30ppm) after 24 h of exposure. conclusion: citrus cultivars have some larvicidal potential but c. jambhiri had the greatest potential against a. aegypti larvae. further small-scale field trials using the extracts of c. jambhiri will be conducted to determine operational feasibility. keywords: citrus cultivars, aedes aegypti larvae introduction dengue fever (df) and dengue hemorrhagic fever (dhf) are serious public health concerns in many developing countries, including pakistan. over 2.5 billion people over 40% of the world's population are now at risk from dengue. who currently estimates 50–100 million dengue infections occurred worldwide every year (1). in pakistan, the first case of dhf was observed in karachi in 1994, (2) and 11,024 confirmed cases of df including 40 deaths were reported in 2010 (3). in 2011, a subsequent dengue outbreak occurred with 22,778 confirmed cases and 353 deaths (4). the majority of the cases and deaths occurred in lahore, punjab province while there was also epidemic in khyber paktunkhua (kpk) during 2013 that resulted in 23 deaths apart from the kpk province, sporadic cases have also been reported from the provinces of punjab, sindh, and balochistan (5). unlike yellow fever, there is no vaccine for dengue fever. however osorio et al. (6) is developing a vaccine, which is still in the preclinical stage. therefore, the only current effective approach to control dengue is through vector control. this is done mainly through integrated vector management programmes using insecticides, environmental management and public awareness (7, 8). due to the toxic effects and resistance to synthetic insecticides (9), vector control managers are facing problems in controlling mosquitoes. therefore, it is necessary to develop safe alternative insecticides, which require minimum care (10). to overcome these problems, concentration has been shifted steadily to the use of bi*corresponding author: mr hazrat bilal, e-mail: hazratbilal@hsa.edu.pk j arthropod-borne dis, september 2017, 11(3): 427–432 h bilal et al.: citrus seed oils efficacy … 428 http://jad.tums.ac.ir published online: september 08, 2017 opesticides as adulticides (11), larvicides (12), and repellents (13) providing alternatives to synthetic chemicals. many investigators have reported on the efficiency of biopesticides (plant extracts) against mosquito larvae (14). recent studies have encouraged the investigation of insecticidal properties of botanicals. muthukrishnan and puspalatha (15) evaluated the larvicidal effects of extracts from rhinacanthus nasutus (acanthaceae), solanum suratense (solanaceae), calophyllum inophyllum (clusiaceae), samadera indica (simaroubaceae) and myriophyllum spicatum (haloragaceae) against aedes aegypti, culex quinquefasciatus and anopheles stephensi and concluded that they are environmentally safe, degradable and target specific (16). in the view of an increased interest in the development of plant-based insecticides as alternatives to synthetic insecticides, this study was conducted to assess the larvicidal potential of citrus cultivars against the dengue vector a. aegypti. materials and methods collection and rearing of mosquitoes aedes aegypti larvae were collected from old tire shops of sargodha (32.0836° n, 72.6711° e) and, reared in 3-inch deep steel trays, and were reared in the insectary of biosystematics lab university of agriculture faisalabad-pakistan. larvae were fed on tetra-min® fish feed until adult emergence (17). adults were maintained in well-aerated cages (70× 35× 35cm), where the mosquitoes were provided with cotton wicks soaked in 10% sucrose solution. females were also fed blood from white mice every alternate day (18). a petri dish with landing pad was provided to lay their eggs. the population was maintained at set conditions of 27±2 oc, 75±5% rh and l14:d10 photoperiod. extraction of oil the seeds of citrus cultivars were washed to remove the pulp, oven dried for 48 hours at 60 ˚c and later ground in an electric grinder. a thimble was used to hold the grounded material and kept in an extraction tube of soxhelt apparatus with extractor id 38mm, extractor volume 85ml and flask volume 250ml (19) for the extraction of oil by steam distillation method using diethyl-ether as solvent (250ml/ 20g sample). the cyclic time of extraction for each sample was 4–5h. bioassay six different concentrations increasing by 100ppm from 300–800ppm of extracted oils were used with three replicates for each treatment, each replicate containing 200ml of the oil solution placed in 250ml glass beakers. batches of 30 late 3rd and early 4th instar larvae of the a. aegypti were placed in each beaker (20). control beakers were treated with diethyl-ether only. the experiment was conducted using completely randomized design (crd) under lab conditions at 27±2 ˚c and 70±5% relative humidity. data analysis abbot’s formula (21) was used to correct for mortality, and this data was analyzed by probit analysis (22) using minitab® statistical software (23) software to create a dose mortality regression line. in the control treatment, if mortality rates were between 5% to 20% then percent mortality was corrected by abbot’s formula as follows: % corrected mortality= % observed mortality% control mortality × 100 100-% control mortality results the efficacy of citrus seed oils against the late 3rd and early 4th instar larvae of a. aegypti was expressed in terms of lc50 as shown in table 2. among the oils tested, rough lemon had the lowest lc50 value (200.79ppm) which is highly significant (0.001), followed by valencia late (213.02ppm), chakutra (221.40) j arthropod-borne dis, september 2017, 11(3): 427–432 h bilal et al.: citrus seed oils efficacy … 429 http://jad.tums.ac.ir published online: september 08, 2017 and narangi (248.16ppm). musambi had the highest lc50 value (457.30ppm) followed by freutrall early (337.63ppm), kinnow (321.60 ppm), succari (316.60ppm) and red blood orange (286.41ppm) after 24h of exposure. table 1. following citrus cultivars were collected from sargodha sr.# common name botanical name 1 chakutra (citrus grandis) 2 kinnow (citrus reticulate) 3 musambi (citrus sinensis var musambi) 4 narangi (citrus mitis) 5 red blood orange (citrus sinensis) 6 rough lemon (citrus jambhiri) 7 feutrell (citrus reticulate) 8 valencia late (citrus sinensis var valencia late) 9 succari (citrus sinensis var succari) table 2. lc50values of citrus seed extract against late 3 rd and early 4th instar larvae of aedes aegypti after 24 hours of exposure citrus extracts lc50* (ppm) 95% fl (lfl**-ufl***) slope ± s.e χ2 p chakutra (citrus grandis) 221.40 155.29-268.40 1.20±0.23 5.86 0.19 kinnow (citrus reticulate) 321.60 266.89-379.10 1.09±0.15 1.15 0.76 musambi (citrus sinensis) 457.30 412.25-512.18 1.22±0.16 1.10 0.77 narangi (citrus mitis) 248.16 195.98-275.87 1.38± 0.25 6.74 0.12 red blood orange (citrus sinensis) 286.41 262.64-305.54 3.38±0.30 5.44 0.23 rough lemon (citrus jambhiri) 200.79 167.62-230.80 1.75±0.22 10.72 0.001 feutrell early (citrus reticulate) 337.63 298.24-397.30 1.06±0.14 0.55 0.87 valencia late (citrus sinensis) 213.02 138.20-161.42 1.01±0.16 2.20 0.43 succri (citrus sinensis) 316.60 267.00-351.10 1.04±0.14 2.04 0.60 *lc50 ie, lethal concentration (ppm) to kill 50% population of the subjected organism ** lower fiducial limit *** upper fiducial limit discussion mosquito borne diseases are one of the most important public health problems in the developing countries like pakistan. use of synthetic insecticides are the best option to control mosquito larvae but resistance, environmental problems etc are the some concerned problems which can be overcome by the use of plant essential oils and extracts as a part of integrated vector management (ivm). a variety of plants is well known to contain chemicals with bioactive potential (24) as deterrents and attractants (25). the ether oils of different citrus cultivars (table 2) have been studied as natural insecticides against a. aegypti larvae instead of synthetic insecticides as they are reported resistant to the mosquitoes especially a. albopictus (26). rough lemon (c. jambhiri) had the lowest lc50 value (200.79ppm) against a. aegypti larvae. this is well supported by the findings of akram et al. (17) in a. albopictus larvae. other citrus seed oils also have some larvicidal effect as reported by hafeez at al. (27) din et al. (28) against a. albopictus. sumroiphon et al. (29) j arthropod-borne dis, september 2017, 11(3): 427–432 h bilal et al.: citrus seed oils efficacy … 430 http://jad.tums.ac.ir published online: september 08, 2017 reported the effects of citrus seed extract against the larvae of a. aegypti and culex quinquefasciatus. extracts of c. sinensis (30) and c. bergamia (31) have been analyzed for the toxicity against the larvae of a. aegypti and found quite effective. bagavan et al. (32) reported the peel chloroform extract of c. sinensis, leaf ethyl acetate extracts of ocimum sanctum, o. canum and leaf chloroform extract of rhinacanthus nasutus as possible insecticides against the larvae of anopheles subpictus and methanol extract of citrus sinensis peel, methanol extract of o. canum leaves, and ethyl acetate extracts of r. nasutus and o. sanctum against the larvae of culex tritaeniorhynchus. conclusion our results indicated that out of the 9 citrus seed oils, rough lemon (c. jambhiri) had good larvicidal potential against late 3rd and early 4th instar larvae of a. aegypti in terms of lc50. further studies should be done to investigate the larvicidal potential of rough lemon oil as well as other seed oils for the control of aedes mosquitoes under field conditions. acknowledgements the funds provided by higher education commission (hec) pakistan to carry out this project are highly acknowledged. thanks are extended to ms kirsten duda, liverpool school of tropical medicine for reviewing the manuscript. the authors declare that there is no conflict of interest. references 1. world health organization (2014) dengue and severe dengue fact sheet no 117. 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accepted 22 feb 2010) abstract we report a case of human nasal myiasis caused by flower fly larva in a 14-year-old rural girl in central province of iran. entomological studies on larva showed the larva as eristalis tenax which is a rarely cause of nasal myiasis. this is the first reported case of e. tenax larva causing human nasal myiasis in iran. keywords: eristalis tenax, nasal myiasis, iran introduction myiasis is a pathogenic condition found in live humans and animals caused by various species of dipteran larvae (zumpt 1965). myiasis is usually classified from an entomological or a clinical point of view. entomologically, flies may be classified in to three myiasis-producing groups: obligatory, facultative, and accidental. clinically, myiasis can be classified according to the part of the body affected (langan et al. 2004). in iran, many cases of human myiasis have been reported. the first case was reported by minar when he recovered oestrus ovis larva from the eye of an iranian woman in tehran (minar 1976). myiasis is repeated several times as ophthal myiasis (janbakhsh et al.1977), urogenital myiasis (jdalayer et al. 1978), orbit myiasis (khataminia and elyasi 1996), pharyngeal myiasis (karimi and vahidi 1999), ear myiasis (talari et al. 2002), oral mucosa myiasis (hakimi and yazdi 2002), wound myiasis (talari et al. 2004) and auricular myiasis (tirgarie et al. 1977, yaghoobi et al. 2005). recently, ophthalmomyiasis in a 62-yr old male afghan from isfahan iran by sarcophaga (razmjou et al. 2007), gingival myiasis by wohlfahrtia magnifica in a 4 yr old boy from bushehr, iran (mohammadzadeh et al. 2008) and human extensive head skin myiasis by chrysomya bezziana in 5 yr old boy in iran (soleimani et al. 2009). the present case is of nasal myiasis caused by larva of e. tenax for the first time in iran. case report the patient was a 14 yr old rural girl. she was suffering from myiasis since 5 months ago, who developed coryza, nasal grip, mild dyspenea and coughing. she had taken antibiotic, antihistamine, and topical corticosteroid, but none of them had sufficient effects. radiographs have showed only minimal septal *corresponding author: ms salimi, e-mail: m_salimi37@yahoo.com iranian j arthropod-borne dis, 2010, 4(1): 77–80 m salimi et al.: first report of human … 78 deviation and minimal opacity in left frontal and maxillary sinuses. finally during several sneezing pulpy mass of the left nasal, released and she referred to infectious specialist, the larva was preserved in 70% methanol by physician and sent to the parasitological laboratory, faculty of medicine, university of arak, for identification. our precise identification indicated that the larva is the rat-tailed maggot (eristalis tenax) according to the following morphological characters of larva: referring to the long telescopic, threesegmented respiratory tube at their posterior end. (mullen et al. 2002). fig. 1 is a picture of the larva of the relevant specimen. fig. 1. larva of eristalis tenax isolated from the nose of the patient (original photo) discussion nasal myiasis is an infection of nasal cavities by larvae of diptera. it is a common disease in tropical and developing countries (aydin et al. 2006). parasites are quite mobile and their clinical signs vary. symptoms always appear abruptly. the main symptoms are a foreign body sensation and itching in the throat, being followed by cough and then other various respiratory and nasal manifestations such as nasal discharge, sneezing, laryngospasm, dyspnea and stridor (wolfelschneider and wiedemann 1996, massodi and hosseini 2004). many cases of nasal myiasis have been reported world wide, caused by several different species including oestrus ovis in algeria (favier 1958), and france (delhaes et al. 2001), phaenicia sericata in 2 comatose patients in arizona (beckendorf et al. 2002), chrysomya bezziana in a 80 yr old woman in malaysia (lee et al. 2005), cochliomyia hominivorax in french guiana (coppie et al. 2005), drosophila melanogaster in a 33 yr old in turkey (aydin et al. 2006), sarcophaga in a 16 yr old girl in turkey (meral türk et al. 2006), in a 70 yr old female patient presenting malignant nasal neoplasia and myiasis (manfrim et al. 2007) and in a 70 yr old female in korea, caused by lucilia sericata (tae-soo kim et al. 2009). the terms flower flies refer to their common habit of visiting flower for nectar and pollen and larvae of e. tenax called rat tailed maggot referring to the long, telescopic, three-segmented respiratory tube at their posterior and by which they breathe at the water surface (mullen et al. 2002). the larva of e. tenax develops in sewage, liquid, excrements and organic materials in decomposition, including carcasses, and occasionally, they can be found in relatively clean water (james 1947). the first case of myiasis by e. tenax was reported by hall (hall 1918).since then, many more cases of human myiasis have been reported by e. tenax, which include intestinal myiasis (lakshminaraynana et al. 1975), rectal myiasis (hira 1977), gastrointestinal myiasis (kun et al. 1998), intestinal myiasis (aguilera et al. 1999, whishwilson 2000, dubois et al. 2004), human pseudomyiasis in brazil (garciazapata et al. 2005), urinary myiasis in a 58 yr old woman in turkey (mumcuoglu et al. 2005) human myiasis by e. tenax in brazil (fernandes et al. 2009), and accidental genital myiasis by e. tenax in a 27 yr old woman in chile (gonzalez 2009). this current study, illustrates several interesting points. it shows that, our case lived in rural area, in a poor hygienic condition and lack of awareness was considered the predisposing factors for larval infestation in this pairanian j arthropod-borne dis, 2010, 4(1): 77–80 m salimi et al.: first report of human … 79 tient. it is also noteworthy that nasal myiasis caused by larva of e. tenax is uncommon in human. myiasis of this type has not been reported previously from iran and this case is the first report of human nasal myiasis caused by e. tenax in iran. acknowledgments we appreciate dr ma daneshmand from sina med laboratory for sending this specimen. the authors declare that they have no conflicts of interest. references aguilera a, cid a, regueiro bj, prieto jm, noya m (1999) intestinal myiasis caused by eristalis tenax. j clin microbial. 37 (9): 3082. aydin e, uysal s, akkuzu b, can f (2006) nasal myiasis by fruit fly larvae: a case report. eur arch otorhinolaryngol. 263 (12): 1142–1143. beckendorf r, klotzs a, hinkle n, barthelomew w (2002) nasal myiasis in an intensive care unit linked to hospitalwide mouse infestation. arch intern med. 1626): 638–640. couppie p, roussel m, rabarison p, sockeel mj, sainte marie d, marty c, carme b(2005) nosocominal myiasis to cochliomyia hominivorax: a case in french uiana. int j dermatol. 44(4): 302–303. delhaes l, bourel b, pinatel f, cailiez jc, 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beetle (coleoptera:… 35 original article scarab beetle (coleoptera: scarabaeidae) fauna in ardabil province, north west iran g mowlavi1, e mikaeili, 2 i mobedi1, eb kia1, l masoomi2, *h vatandoost3 1department of medical parasitology and mycology, school of public health and institute of public health research, tehran university of medical sciences, iran 2 department of biology, faculty of science, mohaghegh ardebil university, ardebil, iran 3 department of medical entomology and vector control, school of public health and institute of public health research, tehran university of medical sciences, iran (received 23 july 2008; accepted 12 may 2009) abstract background: dung beetles of coleoptera associated to undisturbed cattle droppings in pastures present great diversity and abundance. dung beetles also play an important role for transmission of some helminthes to human and cattle. this study was made to survey the biodiversity and abundance of these beetles in ardebil province, western iran. methods: according to the field study all beetles attracted to fresh cow dung in five areas of ardebil province including namin, ardabil, meshkinshahr, neer and sarein were collected and identified. they were collected during summer 2007 from june to september, with general peaks appearing to be correlated with temperature mainly at 11 a.m to 15 p.m. the samples were identified using appropriate systematic key results: a total of 231 specimens belonging to 9 beetle genera and at least 15 species were identified as euoniticellus fulvus, sisyphus schaffaer, euonthophagus taurus, copris lunaris, chironitis pamphilus, gymnopleurus coriarus, euonthophagus amyntas, caccobius schreberi, onthophagus speculifer, onthophagus furcatus, aphodius, lugens, aphodius fimetarius, a. scrutator, geotrupes spiniger and g. stercorarius the most abundant and diverse subfamilies were coprinae, geotrupinae, and aphodiinae. conclusion: we found 15 species of dung beetles occurred in the region. the prevalence of each species is varied depending on location. some of them play an important role for helminths transmission of veterinary and public health importance. the finding will provide a clue for pasture management as well as public health monitoring and surveillance of the disease transmitted by dung beetles. keywords: cattle dung, dung beetle, diversity, iran introduction dung beetles have intense contact with animal and human feces. this biological behavior provides them a constant access to parasites’ eggs in human and animal excretions. due to this behavior they play an important role as an intermediate host for numbers of helminths such as gongylonema sp., sprirocerca and acanthocephalans (eberhard and busillo 1999, mowlavi et al. 2006, eslami and farokhzadegan 1972, eslami and nabavi 1976, hall 1929). cattle dung pats naturally dropped in pastures are normally colonized by a diversified arthropod community which has been studied in detail in several places .the coprophagous fauna is composed mainly by diptera and coleoptera, the family scarabaeidae, which are considered the most important agents promoting dung pat decay (anderson et al. 1984). *corresponding author: h vatandoost, tel: +98 21 88951393, fax: +98 21 88951393, e-mail: hvatandoost @yahoo.com iranian j arthropod-borne dis, (2008), 2(2): 35-41 g mowlavi et al: scarab beetle (coleoptera:… 36 several studies have been done to determine the role of dung beetles in recycling the dung and to assess the possibility of using them as decomposing agents in the dung decaying process and in the control of dung breeding arthropod pests, mainly diptera (ridsdill-smith, 1981, anderson et al. 1984, roth et al. 1988). this study aimed to determine dung beetle diversity, abundance, and species richness and presents data on diversity and abundance of cattle dung beetles collected during a four month period in five areas of ardebil province. material and methods geographical information ardebil province is located about 70 km from the caspian sea and 25 km from republic of azerbaijan’s border. it has an average altitude of 1263 m and total area of 18,011 km2. its geographical coordinates are 38o15’n, 48o17’e.the province has a great diversity. the maximum temperature reaches to 35 °c during the hot summer months. the winters are bitter cold, with a temperature plummeting to -25 °c (provincial health center unpublished data) (fig.1). dung beetle collection a total of 231 dung beetles were collected manually from cattle pastures, mostly from fecal deposits and the adjacent surface soil, in rural areas of 5 different towns including namin, neer, meshkinshahr, sarein, and ardebil city, during the summer 2005. the sample size was calculated according to the previous studies based on cluster sampling. the beetles were collected directly by forceps from fresh dung and then transferred into the small plastic container and subsequently into the laboratory for species identification as well as infectivity to the helminths. the collection was carried during the warmer month (june-september) in 2005. species identification all the collected specimens were pined and transferred to the plant pathology research institute, ministry of jihad, iran for species identification and comparing them with mounted specimens. several morphological characteristics such as antennal segmentation, head shape, horn, ventral sclerites of the abdomen, elytra, claws of tarsi, body shape, middle and posterior tibiae, mesotibia, pygidium, were used for species identification (barari 2001, borror et al. 1989, booth et al. 1990.). results the collected dung beetle specimens, belonging to 9 genera and 15 species .they were collected from cow dung in different parts of the province. out of 231 collected dung beetle, onthophagus taurus (34.19%) were identified as most prevalent species while o. amyntas (0.86 %), o. speculifer (0.86%) and o. furcatus (0.86%) were the least prevalent (see fig.2). among all area, the meshkinshahr and sarein had the most and least ones, i.e. 45.02% and 3.89%, respectively. copris lunaris were present in high numbers in namin while aphodius lugens were numerous in neer. in overall, meshkinshahr and sarein had the highest and lowest diversity, respectively. eight species were collected in meshkinshahr including: sisyphus schafaferi, o. taurus, c. lunaris, chironitis pamphilus, o. amyntas, caccobius schreberi, aphodius lugens, and geotrupes stercorarius (see fig.3). iranian j arthropod-borne dis, (2008), 2(2): 35-41 g mowlavi et al: scarab beetle (coleoptera:… 37 fig. 1. map of study area located in ardebil province, north west of iran euoniticellus fulvus sisyphus schaffaer euonthophagus taurus copris lunaris chironitis pamphilus gymnopleurus coriarus euonthophagus amyntas caccobius schreberi onthophagus speculifer onthophagus furcatus aphodius lugens aphodius fimetarius aphodius scrutator geotrupes spiniger geotrupes stercorarius fig. 2. percentage of collected beetles in study area iranian j arthropod-borne dis, (2008), 2(2): 35-41 g mowlavi et al: scarab beetle (coleoptera:… 38 0 5 10 15 20 25 30 35 40 45 na mi n ar da bil sa re in m es hk ins ha hr ne er study areas pe rc en ta ge fig. 3. abundance of collected beetles in different parts of study areas discussion taxonomically, dung beetles group belongs to the order coleoptera, family scarabaeidae, and comprises about 5000 species worldwide including 12 tribes (gonzález-maya and mata-lorenzen 2008). however, there may be many more dung beetles because thousands of existing species have not yet been described or discovered. dung beetles are of high ecological and economic importance due to the many ecosystem services that they provide. the burial or removal of animal waste reduces microand macro-invertebrate parasites, facilitates nutrient cycling, contributes to soil conditioning and aeration and aids seed dispersal (spector 2006). no studies have addressed the ecology, fauna, community structure and succession pattern of the dung beetles associated with the herbivorous mammals in ardebil province. the occurrence of suitable habitats and favorable climate conditions in iran benefit the maintenance of scarabaeoides dung beetles in nature. in a study carried out by molavi et al. (2009), they were able to found larval stages of gongylonema spp, and rhabditis spp internally through the beetle dissections.in similar survey numbers of other free living nematodes such as rhabditis spp. as well as pelodera spp .have been observed externally on the body surface of the beetles overall, meshkinshahr and sarein had the highest and lowest diversity, respectively. eight species were collected in meshkinshahr (45.02%). one factor that might explain a higher overall diversity of dung beetles in meshkinshahr and neer is mammal diversity. another contributing factor might be the more moderate climate conditions there. two species were collected in sarein. low diversity and abundance in sarein may be related with loose of enough mammals. mammalian fauna in ardebil province includes a wide variety of wild and domestic species but the accurate data on mammal species is currently not available for each of the above mentioned sites. however, the main mammals in the study area are cows. dung beetles are to a large extent adapted to man-made habitats. the practice of keeping large herbivores such as cows and horses in pastures has probably increased the abundance of several species substantially. the 'suitability' of dung is also crucial because different species can have varied preferences for dung that iranian j arthropod-borne dis, (2008), 2(2): 35-41 g mowlavi et al: scarab beetle (coleoptera:… 39 are guided by their life cycle requirements. for example, the moisture content of dung is an important factor for some species (gittings and giller, 1997). some species of dung beetles prefer specific habitats (grassland or forest) or certain soil types (sand or clay). some species feed on dung of only one species of animal, while others are not so limited. some of beetles in current study were reported from other provinces in iran explain including caccobius schreberi (l.) (from golestan and tehran), onthophagus furcatus fab. (from golestan, kordestan, and tehran), onthophagus speculifer sols (from azarbayjane gharbi and fars), oniticellus fulvus (goez.) (from fars, gilan, golestan, and kordestan), chironitis pamphilus (men.) (from azarbaijane gharbi, chaharmahal bakhtiari, kohkiluyeh boyerahmad, kordestan, mazandaran, and tehran) and geotrupes spiniger marsh (from gilan) (barari 2001). the diversity of local dung-beetle communities is primarily influenced by vegetation cover, soil type and moisture, and resource (dung) availability (doube 1987, davis 2002). since dung beetles have different preferences for dung of different mammals based on texture, the structure of dung-beetle communities is influenced by the local mammalian fauna. many studies in tropical south america and southeast asia have demonstrated that deforestation caused by human activities (e.g. logging, pasture clearing, and plantation cutting) reduces the diversity of dung-beetle communities. deforestation has sometimes decreased the numbers of mammals producing dung for beetles (estrada & coates-estrada 2002). this suggests that although vegetation cover may be most important for determining the structure of dungbeetle communities. mammalian fauna may substantially influence the diversity of dungbeetle communities. there are several reports of biodiversity of dung beetles in the world (kanda et al. 2005, duräes et al. 2005). in a study in india the abundance of small rollers (sisyphus) and the low presence of large rollers (gymnopleurus) in pitfall traps and succession studies indicated the dominance of smaller rollers in elephant dung of the region(sabu et al 2006). in conclusion, dung beetles have an important function in farmland ecosystems. their role in dung breakdown and as valuable prey for vertebrate predators should not be underestimated, because of their economic importance; their use as biodiversity indicators is the important ecological and conservation plant factor. results of this study show the diversity of dung beetles in the ardabil province for the first time and this finding will help local authority for pasture management utilizing integrated pest management (ipm) principles as well as veterinary importance of these insects. a complete round the year survey considering the differences in fauna of the beetles in relation to mammals’ fauna may provide more information on the community ecology of the dung beetles. additionally, further sampling effort is needed in the area to obtain a comprehensive pool and distribution of potential species. however this first list represents an important advance in the knowledge of this important group of indicator species in the region. scientists believe that, while it is important to maintain the advantages of controlling livestock pests by using veterinary chemicals, the adverse side effects of these on dung fauna are of considerable concern. acknowledgments the authors would like to appreciate the kind collaboration of mrs. farzaneh zahabioon, mr. ali rahimi and miss neda mirsepahi. the research was financially supported jointly by school of public health & institute of health research, tehran university of medical sciences and by mohaghegh ardabil university, ardabil, iran. iranian j arthropod-borne dis, (2008), 2(2): 35-41 g mowlavi et al: scarab beetle (coleoptera:… 40 references anderson jr, merrit rw, loomis ec (1984) the insect-free cattle dung fouling of rangeland pastures. j econ entomol. 77: 133-141. barari h (2001) insects of iran. plant pests & diseases research institute, insect taxonomy research department. publ. no.7. booth rg, cox ml, madge rb (1990) guides to insects of importance to man coleoptera. international institute of entomology (an institute of cab international). the natural history museum, oxon, uk. 1990, p 384. borror dj, triplehorn ca, johnson nf (1989) an introduction to the study of insects. saunders coll publication. 9, p: 875. davis alv (2002) dung beetle diversity in south africa: influential factors, conservation status, data inadequacies and survey design. african entomol. 10: 5365. duräes r, martins w p, vaz-de-mello fz (2005) dung beetle (coleoptera: scarabaeidae) 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dung beetle (coleoptera: scarabaeidae) in south-western australia on the survival of the bush fly, musca autumnalis walker (diptera: muscidae) in dung pads. bull entomol res. 71: 425-433. roth jp, macqueen a, bay de (1988) scarab activity and predation as mortality faciranian j arthropod-borne dis, (2008), 2(2): 35-41 g mowlavi et al: scarab beetle (coleoptera:… 41 tors of the buffalo fly haematobia irritans exigua, in central queensland. southwest entomol. 13: 119-125. sabu tk, vinod kv, vineesh pj (2006) guild structure, diversity and succession of dung beetles associated with indian elephant dung in south western ghats forests. j insect sci.17:1-12. spector s (2006) scarabaeine dung beetles (coleoptera: scarabaeidae: scarabaeinae): an invertebrate focal taxon for biodiversity research and conservation. coleopterists’ bul. 60(5): 71-83. j arthropod-borne dis, june 2017, 11(2): 338–343 m s makki et al.: identification of hymenolepis … 338 http://jad.tums.ac.ir published online: may 27, 2017 short communication identification of hymenolepis diminuta cysticercoid larvae in tribolium castaneum (coleoptera: tenebrionidae) beetles from iran mahsa sadat makki 1, gholamreza mowlavi 1, farideh shahbazi 1, mohammad reza abai 2, faezeh najafi 1, bibi razieh hosseini-farash 3, salma teimoori 1,4, hamid hasanpour 1, *saied reza naddaf 5 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran 3department of parasitology and mycology, research center for skin disease and cutaneous leishmaniasis, school of medicine, mashhad university of medical sciences, mashhad, iran 4centre of excellence for therapeutic proteins and antibody engineering, department of parasitology, faculty of medicine, siriraj hospital, bangkok, thailand 5department of parasitology, pasteur institute of iran, tehran, iran (received 15 jan 2015; accepted 8 aug 2016) abstract background: hymenolepis diminuta is a cestod of rodents and rarely infects humans. infection in humans is via ingestion of infected insects. this study was aimed to detect h. diminuta cysticercoids in red flour beetles, tribolium castaneum, and cockroaches originated from different regions of iran. methods: the red flour beetles and cockroaches were collected from local bakeries in five cities including tehran, ahvaz, kazerun, and sabzevar during 2010–2011. some beetles and cockroaches were colonized in insectary and adults from f1 generation were fed on h. diminuta eggs. both laboratory-infected and field-collected samples were dissected and examined for cysticercoids. detection of h. diminuta dna in t. castaneum beetles was performed by targeting a partial sequence of ribosomal gene. results: except the beetles from ahvaz, all specimens were negative for cysticercoid by microscopy. of the four dissected beetles from ahvaz, one harbored 12 cysticercoids. also, 110 (52%) of laboratory-infected beetles showed infection with an average of 12–14 larvae. none of the cockroaches was infected. two beetles from ahvaz, including the remainder of the microscopic positive specimen, yielded the expected amplicon in pcr assay. the h. diminuta dna sequences generated in this study were identical and matched 97–100% with similar sequences from genbank database. conclusion: lack of infection in the majority of beetles may reflect a low rat infestation rate in those areas, alternatively, the examined specimens might not have been the representative samples of the t. castaneum populations. keywords: hymenolepis diminuta, cysticercoid, tribolium castaneum, iran introduction human hymenolepiasis is caused by two cestodes, hymenolepis nana, and h. diminuta (wiwanitkit 2004, magalhaes et al. 2013). there is a single report on mixed infection of the third species h. microstoma with h. nana in remote communities in the northwest of western australia (macnish et al. 2003). the tapeworm, h. nana is the primary cause of human infection, whereas h. diminuta rarely infect humans, and so far, only a few hundred cases have been reported (tena et al. 1998, wiwanitkit 2004). however, with increased awareness of the disease and im*corresponding author: dr saied reza naddaf, e-mail: snaddaf_2001@yahoo.com j arthropod-borne dis, june 2017, 11(2): 338–343 m s makki et al.: identification of hymenolepis … 339 http://jad.tums.ac.ir published online: may 27, 2017 provement of laboratory techniques, more cases become apparent. although the identified cases are mostly children, the disease can be seen in every age group (cohen 1989, tena et al. 1998, marangi et al. 2003). unlike, h. nana, transmission of h. diminuta requires an arthropod intermediate host to complete its life cycle. when h. diminuta fertilized eggs are excreted in the stool of an infected definitive host, they are ingested by various arthropods. once inside the insect body, the oncospheres are released from the eggs, penetrate the intestinal wall of the host and develop into cysticercoid larvae. soon after the ingestion of infected arthropod by the mammalian host, the cysticercoid larvae are released in the stomach, and small intestine and their life cycle is completed. the maturation occurs within 20–25d, and the adult worms can reach an average of 30cm in length. humans are accidentally infected via ingesting the arthropods carrying cysticercoids. various arthropod species including flour beetles, moths, earwigs, and flea larvae may serve as the intermediate host. the red flour beetle, tribolium castaneum, is an efficient intermediate host of this cestode (bisseru 2013). it is a worldwide stored food pest and attacks grain products including flour, cereals, pasta, biscuits, beans, and nuts. the adults are longlived and may live for more than three years. this study was aimed to detect h. diminuta cysticercoids in t. castaneum beetles and cockroaches collected from local bakeries in five cities of iran by microscopy and pcr assay. we also discuss the susceptibility of these beetles along cockroaches to h. diminuta infection under laboratory condition. materials and methods collection of tribolium castaneum beetles bakeries in different cities including tehran in upper center, ahvaz in the southwest, iranshahr in the southeast, kazerun in south, and sabzevar in the northeast of the country were searched for red flour beetles during 2010–2011. the collected samples were kept in tubes containing flour at 28–30 °c and humidity of 20–40%. the collected insects were identified to the species based on morphological features using diagnostic keys (bosquet 1990). similarly, some cockroaches were collected from the same bakeries in tehran. hymenolepis diminuta eggs rattus norvegicus rats were captured from rat-infested areas in tehran. the animals were euthanized, sacrificed, and mature worms were collected from the intestine. posterior gravid proglottids of the worms were removed and dissected in normal saline to free the eggs. laboratory-infected beetles flour beetles, t. castaneum, and american cockroaches, periplaneta americana, were reared in the insectary at 28–30 °c with relative humidity of 20–40% and light and dark cycle of 12h/12h. the adult progenies from the f1 generations were allowed to feed on dough contaminated with h. diminuta eggs. after three weeks, the cockroaches and beetles were dissected in normal saline and examined for cysticercoids under a stereomicroscope with a magnification of 40x (makki et al. 2011). identification of cysticercoids in beetles by microscopy we used 154 field-collected flour beetles (30 specimens from tehran, 60 from iranshahr, 30 from kazeroun and sabzevar each, and four from ahvaz), 50 field-collected american cockroaches from tehran, 210 laboratory-infected beetles, and 40 american and 70 german laboratoryinfected cockroaches in this study. all the insects were dissected in normal saline and examined for cysticercoid larvae under a stereomicroscope with a magnification of 40x. pcr and sequencing dna extraction was performed on individj arthropod-borne dis, june 2017, 11(2): 338–343 m s makki et al.: identification of hymenolepis … 340 http://jad.tums.ac.ir published online: may 27, 2017 ual and pools of 20–50 beetles of laboratoryinfected beetles as well as field-collected samples by phenol-chloroform method followed by ethanol precipitation as described by others (ballinger-crabtree et al. 1992). a partial ribosomal dna sequence of h. diminuta (spanning the 3’ end of the 18s rrna gene, internal transcribed spacer 1 (its1), 5.8s, its2 and the 5’end of the 28s rrna gene) was targeted using the primers hf1 5’-gcggaaggatcattacacgttc-3’ and hr1 5’-gctcgactcttcatcgatccacg-3’ designed by others (macnish et al. 2002). the 25µ l reaction mixture contained 20pmol of each primer, 2.5mm mgcl2, 10mm tris-hcl, 50mm kcl, 200μm of dntps, 1u of taq polymerase, and 3μl of dna. all amplifications were programmed for an initial denaturation step for 5 min at 94 °c, followed by 30 cycles of 94 °c for 30s, 63 °c for 40s, and 72 °c for 45s, with a final step of 10min at 72 °c. for fieldcollected samples, the annealing temperature was reduced to 61 °c and the cycles increased to 32. in all amplifications, dna from h. diminuta and h. nana adult worms were included as positive controls and dna of adult taenia sp. as the negative control. the pcr products were run on 1% agarose gel, stained with ethidium bromide and visualized under uv. amplicons from one fieldcollected beetle and h. diminuta and h. nana adult worms were sequenced in both directions using the same primers used for amplification and compared with similar sequences from genbank database. the data for sequences were submitted to genbank database with accession numbers kj917784 and kj917785-7 for h. nana and h. diminuta, respectively. results microscopy except for t. castaneum beetles from ahvaz, all the specimens were negative for h. diminuta cysticercoids by microscopy. out of four dissected beetles from ahvaz, one harbored 12 h. diminuta cysticercoids (fig. 1). also, of the 210 t. castaneum beetles fed on food contaminated with the tapeworm eggs, 110 developed cysticercoids with an average of 12–14 larvae in insects. none of the laboratory-reared cockroaches fed in similar fashion turned positive. pcr and sequencing pcr amplification of rdna gene from h. nana and h. diminuta adult worms yielded the expected bands of 645bp and 750bp, respectively. the 750bp band diagnostic for h. diminuta was observed in the pools of laboratory-infected t. castaneum beetles. our pcr method could detect the dnas equivalent to one and three cysticercoids in pools of 20 and 50 non-infected beetles, respectively. dnas from 35 individual and ten pools of 20-50 beetles collected from different areas were negative for h. diminuta. however, dna samples of two individual beetles from ahvaz including the remainder of the microscopic positive specimen yielded the expected 750bp amplicon, implying the presence of h. diminuta in the insects (fig. 2). the h. diminuta ribosomal dna sequences generated in this study were identical and showed 97%100% homology with three h. diminuta gene sequences from genbank database (accession numbers, af461125, jn258039, and jn258038) over 94%–100% of nucleotides. fig. 1. an h. diminuta cysticercoid derived from a t. castaneum beetle collected from ahvaz, iran. j arthropod-borne dis, june 2017, 11(2): 338–343 m s makki et al.: identification of hymenolepis … 341 http://jad.tums.ac.ir published online: may 27, 2017 fig. 2. amplification of a partial ribosomal dna sequence (spanning the 3’ end of the 18s rrna gene to the 5’end of the 28s rrna gene) from hymenolepis spp. 1, h. nana adult worm, m, 100bp marker, 2, h. diminuta adult worm, 3, infected beetle, 4, cysticercoid larvae, 5, taenia sp, 6, negative control. discussion the rat tapeworm, h. diminuta, is a cestode of rodents. in iran, it is commonly found in rats and mice (kia et al. 2001, meshkekar et al. 2014, yousefi et al. 2014). recently, it was found in tatera indica from southeast of the country (nateghpour et al. 2015). this species may also affect pet animals like squirrels (callosciurus prevosti) (d’ovidio et al. 2015) and laboratory rats (sreedevi et al. 2015). hymenolepis diminuta infection in humans is very rare, but with increased awareness of the disease and the ability to identify different species of the genus hymenolepis, more cases are becoming apparent. infection of h. diminuta has recently been reported from various countries including turkey, tamil nadu, sri lanka and india (kalaivani et al. 2014, sinhabahu et al. 2014, tiwari et al. 2014, kılınçel et al. 2015). in iran, the last human infection was identified in a child in 2008 (mowlavi et al. 2008). infection with this worm is commonly light and self-limiting and deworming can be achieved with praziquantel as the drug of choice (karuna and khadanga 2013). the flour beetles belonging to the genera tribolium and tenebrio are among the known intermediate hosts of this worm. they are adapted to survive in arid environments and are highly resistant to insecticides. the susceptibility to h. diminuta infection may vary among the members of these two genera, and with their life stage. in samples collected from a riding stable in quebec, the intensity of cysticercoids in tenebrio obscurus was higher than t. molitor and adults were more susceptible to infection than larvae (rau 1979). also, t. castaneum was more vulnerable to the infection than t. confusum (yan and norman 1995). in the present study, all the t. castaneum beetles from different cities, except ahvaz, were negative for h. diminuta cysticercoid by microscopy and pcr assay. examination of limited specimens from ahvaz revealed infection in two beetles. this finding suggests a high infection rate among the beetles, which can maintain the enzootic cycle. an earlier study in this city revealed high helminthic infection rates, particularly with spirurids, among r. norvegicus rats. out of 72 dissected rats, 8 (11.1%) were infected with h. diminuta (kia et al. 2001). in the present study, 52% of the t. castaneum beetles fed on dough contaminated with h. diminuta eggs developed cysticercoids, this reflects their high susceptibility to hymenolepiasis, and their potency to serve as an efficient intermediate host. unlike t. castaneum beetles, in none of the cockroaches fed in similar manner cysticercoids developed. cockroaches have shown to be refractory to h. diminuta infection due to encapsulation of larvae by the insect hemocytes (tu and lai 2006). prior infection of cockroaches with moniliformis moniliformis may predispose them to h. diminuta infection. in cockroaches, a membranous envelope engulfs the acanthocephalan m. moniliformis and protects it from the hemocyte attack in the arthropod host (holt 1989). the hatched oncospheres of h. diminuta may penetrate this envelope and, once inside, utilize its protective function to develop. j arthropod-borne dis, june 2017, 11(2): 338–343 m s makki et al.: identification of hymenolepis … 342 http://jad.tums.ac.ir published online: may 27, 2017 conclusion lack of infection in the majority of beetles may reflect a low rat infestation rate in those areas, alternatively, the examined specimens might not have been the representative samples of the t. castaneum populations. further studies with inclusion of more samples and other potential intermediate arthropod hosts are required to elucidate the biology and life cycle of h. diminuta in urban rats. acknowledgements the authors are very grateful to animal husbandry workers, neda mirsepahi and ali rahimi from school of public health, tehran university of medical sciences, for their kind assistances during this research. references ballinger-crabtree me, black wc, miller br (1992) use 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american cockroach, periplaneta americana. j invertebr pathol. 92(2): 73–78. wiwanitkit v (2004) overview of hymenolepis diminuta infection among thai patients. medgenmed. 6(2): 7. yan g, norman s (1995) infection of tribolium beetles with a tapeworm: variation in susceptibility within and between beetle species and among genetic strains. j parasitol. 81(1): 37–42. yousefi a, eslami a, mobedi i, rahbari s, ronaghi h (2014) helminth infections of house mouse (mus musculus) and wood mouse (apodemus sylvaticus) from the suburban areas of hamadan city, western iran. iran j parasitol. 9 (4): 511–518. j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 95 http://jad.tums.ac.ir published online: march 14, 2017 original article synthesis and comparison of in vitro leishmanicidal activity of 5(nitroheteroaryl)-1, 3, 4-thiadiazols containing cyclic amine of piperidin-4-ol at c-2 with acyclic amine analogues against iranian strain of leishmania major (mrho/ir/75/er) *azar tahghighi 1,2, alireza foroumadi 2, susan kabudanian-ardestani 3, seyed mohammad amin mahdian 1 1malaria and vector research group, biotechnology research center, pasteur institute of iran, tehran, iran 2faculty of pharmacy and pharmaceutical sciences research center, tehran university of medical sciences, tehran, iran 3department of biochemistry, institute of biochemistry and biophysics, university of tehran, tehran, iran (received 2 nov 2014; accepted 6 feb 2016) abstract background: cutaneous leishmaniasis (cl) is endemic in many tropical and subtropical regions of the world. due to the prolonged duration of therapy, adverse effect and resistance to current drugs in the treatment of cl, the discovery of novel, efficient, and safe leishmanicidal drugs is required. the aims of the present study was to synthesis of new compounds based on the active compounds of 5-(5-nitrofuran-2-yl)and 5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazole bearing the linear amino alcohol of 3-aminopropan-1-ol in the c-2 position of thiadiazole ring and evaluation of their activity against the promastigote and amastigote forms of leishmania major. methods: reaction between the solution of 5-(5-nitro heteroaryl)-2-chloro-1, 3, 4-thiadiazole and piperidin-4-ol in absolute ethanol was performed and the resulting products were evaluated against promastigotes form of l. major with mtt assay and amastigote form of l. major in murine peritoneal macrophages. in addition, the toxicity of these compounds was assessed against mouse peritoneal macrophages with mtt assay. results: new synthetic compounds 5a-b showed moderate in vitro antileishmanial activity against l. major promastigotes with ic50 values of 68.9 and 27µ m, respectively. these compounds have also demonstrated a good antiamastigote activity in terms of amastigote number per macrophage, the percentage of macrophage infectivity and infectivity index. conclusion: novel cyclic compounds 5a-b were synthesized and exhibited less antipromastigote and antiamastigote activity compared to linear analogues. keywords: leishmania major, thiadiazole, nitrofuran, nitrothiophen, superimpose introduction leishmaniasis is an endemic parasitic disease and a major public health problem in more than 90 countries around the world (cdc 2013). it is estimated that 1.3 million people are affected by this disease annually and the overall population at risk is 310 million people (who 2010). there are four forms of leishmaniasis: visceral leishmaniasis (vl) or kala azar, which is typically lethal if left untreated, mucocutaneous leishmaniasis (mcl), which is a mutilating disease, diffuse cutaneous leishmaniasis (dcl) related to defective immune system and cutaneous leishmaniasis (cl) that produces serious skin lesions (singh and sivakumar 2004). the current chemotherapy against all forms of leishmaniasis including parental pentavalent antimonial compounds remains the primary *corresponding authors: dr azar tahghighi, email: atahghighi2009@gmail.com j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 96 http://jad.tums.ac.ir published online: march 14, 2017 therapy, followed by making use of other drugs like amphotericin b, paramomycin, pentamidine and orally miltefosine (monzote 2009). however, making use of them is limited owing to toxicity and the high cost of treatment (chawla and madhubala 2010, tiuman et al. 2011). in addition, the development of clinical resistance and an increasing incidence of leishmaniasis/ aids co-infection in some regions is a serious health problem (molina 2003). as such, the design of new, efficient, cheap and safe drugs for the treatment of leishmaniasis is imperative. 5-(nitroheteroaryl)-1, 3, 4-thiadiazole containing a cyclic (a) and acyclic (b) amine in c-2 position, depending upon the type of substituent, have shown a promising antileishmanial activity (fig. 1). furthermore, different substitutions in c-2 position in 1, 3, 4-thiadiazole ring have been able to affect potency and physicochemical properties. on this basis, to achieve a novel anti-leishmanial agent, the new derivatives were synthesized to introduce other groups on c-2 amine of thiadiazole ring such as, 4-aroylpiperazine segment (c), piperazinyl-linked benzamidines substituent (d), n-[(1-benzyl-1h-1,2,3triazol-4-yl)methyl] moiety (e) and various 2-thioacetamides substituent (f) (fig. 1). these different attachments to 1, 3, 4-thiadiazole rings causes changes the bioresponses, depending upon the type of substituent and position of attachment (foroumadi et al. 2008, tahghighi 2011, 2012, 2013, marznaki 2013, vosooghi 2014). recently, given the importance of this position, several 5-(5-nitrofuran-2-yl)and 5-(5-nitrothiophen-2-yl)-1, 3, 4-thiadiazoles possessing acyclic amines in the c-2 position of thiadiazole ring were synthesized and theirs in vitro activity was evaluated against the promastigotes and amastigotes forms of l. major. two compounds 1a-b with linear substitution of 1propanol amine exhibited a promising antileishmanial activity against l. major promastigotes (fig. 1) (tahghighi et al. 2013). concerning these findings and simple reaction for preparation these compounds, the aim of this study was to synthesize cyclic analogues of these linear amino alcohols, examine anti-leishmanial activity against promastigote and amastigote forms of l. major and evaluate their cytotoxicity activity on macrophages. materials and methods chemistry all chemical reagents and solvents were purchased from merck company and used without further purification. the key intermediate 2-chloro-1, 3, 4-thiadiazole 4a-b was prepared by starting from 5-nitrofurfurilidine diacetate or 5-nitrothiophene-2-carboxaldehyde (tahghighi et al. 2011, 2012, 2013, marznaki 2013, vosooghi 2014). the melting points of compounds were determined using a kofler hot-stage apparatus. the ir spectra were obtained on a shimadzu 470 spectrophotometer using kbr dicks. 1h nmr spectra were recorded on a varian unity 500 and 400 spectrometer and chemical shifts (δ) were reported in parts per million (ppm) relative to tetramethylsilane (tms) as an internal standard. the mass spectra were run on an agilent 6410 lc-ms or a finigantsq-70 spectrometer (finigan, usa) at 70 ev. merck silica gel 60 f254 plates were used for analytical tlc. compounds 1a-b has been described in our previous work (tahghighi et al. 2013). general procedure for the synthesis of compounds 5a-b a solution of 2-chloro-1, 3, 4-thiadiazole 4a-b (1.1mmol) and appropriate cyclic amine of piperidin-4-o l (1mmol) in absolute ethanol (7ml) was refluxed until the reaction was completed (4h). then, the solvent was evaporated under reduced pressure and the residue was purified using silica gel column chroj arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 97 http://jad.tums.ac.ir published online: march 14, 2017 matography, eluting with appropriate solvent (fig. 2). 1-(5-(5-nitrofuran-2-yl)-1, 3, 4-thiadiazol-2yl)piperidin-4-ol (5a) the resulting product was purified using a short silica gel column eluting with ch3 cooc2h5 followed by ch3cooc2h5 containing 2% methanol. the compound was obtained as a yellow solid (yield: 78%). mp 147.7–149 °c. ir (kbr, cm-1): 3414, 1734, 1535, 1497 and 1344. 1h nmr (500 mhz, dmso-d6) δ: 7.84 (d, 1h, j= 3.25 hz, furan), 7.35 (d, 1h, j= 3.25 hz, furan), 3.80 (m, 3h, ch2 and ch), 3.41 (t, 2h, ch2), 3.33 (brs, 1h, oh), 1.85 (m, 2h, ch2), 1.51 (m, 2h, ch2). ms (esi): 296.8 [m + h +]. 1-(5-(5-nitrothiophen-2-yl)-1, 3, 4-thiadiazol2-yl)piperidin-4-ol (5b) the resulting product was purified using a short silica gel column eluting with ch3 cooc2h5 followed by ch3cooc2h5 containing 2% methanol. the compound was obtained as an orange solid (yield: 73%). mp160–162 °c. ir (kbr, cm-1): 3298, 1736, 1536, 1494 and 1354. 1h nmr (400 mhz, cdcl3) δ: 7.86 (d, 1h, j= 4.4 hz, thiophen), 7.16 (d, 1h, j= 4.4 hz, thiophen), 4.05 (brs, 1h, oh), 3.91 (m, 3h, ch2 and ch), 3.49 (t, 2h, j= 9.3 ch2), 2.03 (m, 2h, ch2), 1.73 (m, 2h, ch2). ms (esi): 312.8 [m+ h +]. antileishmanial activity against leishmania major promastigotes the antileishmanial activity of compounds 5a-b was performed using mtt assay (dutta et al. 2005). the promastigote form of parasite (vaccine strain mrho/ir/75/er, obtained from pasteur institute, tehran, iran) was grown in blood agar cultures at 25 °c. the growth curve of the l. major strain was determined daily under light microscope and counting in a neuberger’s chamber. then, parasites (2× 106/ml) in the logarithmic phase were incubated with different concentrations (12.5, 25, 50 and 75µ g/ml) of test compounds for 24 h at 25 °c. a negative control (dmso without any test compounds with culture medium), and positive control (with glucantime and fluconazole) were used on same plate. after incubation, the media was renewed with 100μg/well of mtt (0.5mg/ml) and plates were further incubated for 4 h at 37 °c. then, the plates were centrifuged (2000rpm× 5min× 4 °c) and the obtained pellets were dissolved in 200μl of dmso. the optical density (od) of samples due to cleavage of the tetrazolium salt mtt [3(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide] into a colored product formazan by the parasite was measured by elisa plate reader at a wavelength of 492nm. two or more independent experiments in triplicate were performed for each compound. the ic50 values (the concentration required to inhibit 50% growth of promastigotes after 24h) were calculated by linear regression analysis, expressed in mean ± sd. antileishmanial activity against leishmania major amastigotes compounds 5a-b was evaluated for their activity against amastigote form of l. major in murine peritoneal macrophages. briefly, the abdominal cavity of balb/c mice was torn then washed to inject 5 ml of phosphate buffered saline (pbs) by a sterile syringe and collected cells. the cell suspension was centrifuged (2000rpm× 10min× 4 °c). mouse peritoneal macrophages were plated in rpmi 1640 supplemented with 10% of heat-inactivated fetal bovine serum, 2mm glutamine, 100u/ml penicillin (sigma) and 100µ g/ ml streptomycin. macrophages were placed on sterile glass cover slips in 24-well plates (1× 106/well). after 1h, non-adherent cells were removed by washing with rpmi 1640, the stationary phase promastigotes in rpmi 1640 were added (2×106 parasites/well, three parasites/ macrophage) to macrophage monolayer and the plates were kept at 37 °c in a co2 j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 98 http://jad.tums.ac.ir published online: march 14, 2017 incubator for 2h. extracellular parasites were removed by washing and then new media containing ic50 concentration of the drug were added. two sets of experiments were carried out for each compound at 24h. following these procedures, cells were fixed with methanol, stained with giemsa stain (sigma) and the infectivity index was determined by multiplying the percentage of macrophages that had at least one intracellular parasite by the average number of intracellular parasites per infected macrophage (100 cells were examined/well) (tanaka et al. 2007). toxicity against macrophages the toxicity of compounds 5a-b was assessed against mouse peritoneal macrophages plated in 96-well plates at 2× 105 cells/ well. after cell adherence, the medium was removed and replaced by the media containing different concentrations of each compounds. the plates were incubated for 24 h at 37 °c in a humidified incubator with 5% co2. dmso without any test compounds was used as a negative control. cell viability was determined by mtt colorimetric assay (kiderlen et al. 1990). two independent experiments in triplicate were performed for determination of toxicity of each compound. the cc50 (cytotoxic concentration for 50% inhibition) were calculated by linear regression analysis. overlay study of compounds the 3d structures of the ligands of table 1 were drawn using hyperchem software (version 7.0) and subsequently energy minimized using semiemperical method, am1 level of theory. then, energy minimized molecules were superimposed using atoms selection. in this way, one of the molecules was opened and atoms selected, then the second molecule was opened and the same atoms selected and tow molecules were superimposed by overlay option of hyperchem program. results chemistry the intermediate 2-chloro-1, 3, 4-thiadiazole 4a-b was obtained from 5-nitrofurfurilidine diacetate or 5-nitrothiophene-2-carboxaldehyde [7–12]. the target compounds 5a-b were synthesized in high yield by the reaction of 5-(5-nitro heteroaryl)-2-chloro-1, 3, 4-thiadiazole 4a-b and appropriate piperidin4-ol in refluxing absolute ethanol (fig. 2). all of the synthesized compounds were characterized by ir, 1h nmr and lc-ms. biological activity we synthesized novel 5-(nitroheteroaryl)1, 3, 4-thiadiazols containing acyclic amines in c-2 and evaluated antileishmanial activity of these compounds against the promastigote and amastigote stage of l. major. the compounds of 3-(5-(5-nitrofuran-2-yl)-1, 3, 4thiadiazol-2-ylamino) propan-1-ol 1a and 3(5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2ylamino) propan-1-ol 1b showed ic50 values of 18±0.2 and 3±0.41µ m, respectively, against the promastigotes form of l. major and , in contrast, the analogue of 5-nitrothiophen 1b showed 6-fold more potent than its 5-nitrofuran counterpart 1a. this finding indicated that different responses take place due to o/s replacement in the scaffold. the previous compounds 1a-b was also evaluated against the amastigotes form of l. major. these compounds decreased the number of intracellular amastigotes per macrophage, the percentage of macrophage infectivity and infectivity index compared with the control group. the in vitro cytotoxic activity of the compounds 1a-b demonstrated toxicity against mouse peritoneal macrophages (cc50 values of 62.33 and 42.16µ m, respectively). furthermore, the compound 1b displayed the highest selectivity index (si= 14.05) (table 1). j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 99 http://jad.tums.ac.ir published online: march 14, 2017 moreover, we synthesized two cyclic compounds 5a-b similar to these linear compounds and evaluated their activity against the promastigote and amastigote forms of l. major. fluconazole and meglumine antimonate (glucantime®) were used as reference drugs. these compounds have a medium anti-promastigote activity with ic50 values less than 70μm. furthermore, these compounds were evaluated for their activity against amastigote form of l. major in murine peritoneal macrophages (fig. 3) and thiophen analogues 1b and 5b exhibited higher activity against amastigotes as showed by amastigote number per macrophage, the percentage of macrophage infectivity and infectivity index compared with furan analogues 1a and 5a (fig. 3a, 3b and 3c, respectively). in addition, the cc50 values for these compounds 5a-b against mouse peritoneal macrophages were determined using mtt assay (table 1). they were toxic to macrophages (cc50< 80 µ m) and that the compound 1a had the highest selectivity index (si= 14.05). overlay study the superimposition studies of the target compounds 5a-b and lead molecules 1a-b in their energy-minimized conformations and without hydroxyl group's selection revealed that the thiophen or furan and thiadiazole ring, nitro group and amine group in c-2 position of thiadiazole ring at these compounds overlaid completely on each other whereas hydroxyl groups located in different positions (fig. 4 a1, b1). the superimposition of these compounds with hydroxyl group selection showed that there was a weak overlay between target and lead molecules (fig. 4 a2, b2). x s nn n hno2 x s nn nhr no2 x = o, s r = alkyl, aminoalkyl, oxyalkyl, .... x s nn no2 x = o, s y = ch2, nh, nr, o n = 1, 2 n y na b 1a-b a: x = o b: x = s o s nn no2 r = alkyl, morpholine, cyclic amidine, .... x s nn no2 x = o, s r = halophenyl, halothiophen n c d n ar o n n nh nhr o s nn no2 r = alkyl, halide, .... e n h n n n r o s nn no2 r = halophenyl, nitrophenyl, isoxazolyl, benzothiazolyl, .... s o h n r ( ) n f oh ( ) 3 ( ) fig. 1. general structures of antileishmanial compounds (a) bearing a cyclic amine (b) containing acyclic amine (cf) with aromatic substitutions and (1a-b) containing hydroxypropyl amine at the c-2 position of thiadiazole ring j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 100 http://jad.tums.ac.ir published online: march 14, 2017 x s nn n oh no2 x s nn cl no2 x s nn nh2 no2 x ch(ococh3)2 no2 a, b c d 2a-b a: x = o b: x = s 3a-b a: x = o b: x = s 4a-b a: x = o b: x = s 5a-b a: x = o b: x = s fig. 2. synthetic route to compounds 5a-b. reagents and conditions: a) thiosemicarbazide, etoh, hcl, reflux, (b) nh4fe(so4)2.12h2o, h2o, reflux; c) nano2, hcl, cu, d) piperidin-4-ol, etoh, reflux table 1. in vitro antileishmanial activity of thiadiazole derivatives 1a-b and 5a-b against promastigote form of leishmania major x s nn r no2 compounds r x anti-promastigote activity ic50 (µm) cytotoxicity cc50 (µm) a sib 1a hn oh o 18 ± 0.2 62.33 3.46 1b hn oh s 3 ± 0.41 42.16 14.05 5a n oh o 68.9 ± 0.107 78.54 1.14 5b n oh s 27 ± 0.12 63.55 2.35 glucantime 68.44 c fluconazole 941.1 ± 4.98 acytotoxicity was evaluated against mouse peritoneal macrophages. bselectivity index (si) cc50/ ic50. cthe ic50 of glucantime was in mm. j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 101 http://jad.tums.ac.ir published online: march 14, 2017 fig. 3. the in vitro activity of selected compounds against intramacrophage amastigotes of leishmania major. (a) the mean number of amastigotes per macrophage after treatment with selected compounds for 24h. (b) the percentage of infected macrophages after treatment. (c) infectivity index of macrophages cultured 24 h in presence of selected compounds. the infectivity index was determined by multiplying the percentage of macrophages that had at least one intracellular parasite by the average number of intracellular parasite per infected macrophage (100 cells were examined/well). a b c j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 102 http://jad.tums.ac.ir published online: march 14, 2017 fig. 4. a1: overlay between compounds 1a and 5a without hydroxyl selection. a2: overlay between compounds 1a and 5a with hydroxyl selection. b1: overlay between compounds 1b and 5b without hydroxyl selection. b2: overlay between compounds 1b and 5b with hydroxyl selection. discussion the cyclic analogues of 5-(5-nitrofuran-2yl)and 5-(5-nitrothiophen-2-yl)-1, 3, 4-thiadiazole-2-amines bearing piperidin-4-ol at the c-2 position of thiadiazole ring were synthesized and evaluated in vitro against promastigote and amastigote forms of l. major. these novel compounds, which were compared with their linear analogues, exhibited less antileishmanial activity against promastigote form of l. major. our previous studies demonstrated that the c-2 substituent in 5-(nitroheteroaryl)-1, 3, 4-thiadiazoles is the most flexible site for chemical change and is an area where it determines the potency and physicochemical properties of these synthetic compounds. accordingly, several series of these derivatives were synthesized by our research group with different linkers and substitution groups connect to these linkers. all of the compounds were evaluated against l. major with mtt assay. it seems that in these compounds c-f (fig. 1), the type of aryl ring and the linker are the important factors for their antileishmanial activity. in the compound (c), aryl group with a piperazinyl methanone linker were the most suitable functions for antileishmanial activity. the best compound in this series has 2chlorophenyl substitution (ic50 value of 10.73µ m). in the 5-nitrofuran-2-yl-1, 3, 4thiadiazol-2-yl) piperazin-1-yl) benzamidine (d), the maximum antileishmanial activity observed with n-propyl substitution on benzamidine (ic50 value of 10µ m). in other series, 5-(5-nitrofuran-2-yl)-1, 3, 4-thiadiazol2-amines were synthesized by introducing n-[(1-benzyl-1h-1, 2, 3-triazol-4-yl)methyl] moiety as a new functionality on the c-2 amine of thiadiazole ring (e). the most active compound of this series has p-methyl substitution on phenyl ring (ic50 value of 12.2µ m). hence, 5-(5-nitrofuran-2-yl)-1, 3, 4-thiadiazole structure bearing 2-mercaptoacetamide linker (f) were prepared and displayed in vitro activity against promastigotes and amastigotes of l. major. in this series, 3, 4-dimethoxyphenethyl substitution was caused to suitable activity against promastigote form of l. major with ic50 value of 19.1µ m. the comparison of antileishmanial activity of compounds c-f with compounds 1a-b was showed linear substitution and thiophen ring at compound 1b are the best substitutions at c-2 and c-5 positions of thiadiazole ring. j arthropod-borne dis, march 2017, 11(1): 95–104 a tahghighi et al.: synthesis and comparison of … 103 http://jad.tums.ac.ir published online: march 14, 2017 the presence of aryl group on linker is not necessary for increase antileishmanaial activity. however, in the compounds with aryl substitution, type of this substitution affected the antileishmanial activity. furthermore, the compounds without aryl substitution were synthesized with a simple reaction (fig. 2) and their activity was compared with their corresponding analogues. as shown in table 1, the substitution of the linear hydroxypropyl in the c-2 position of 1, 3, 4-thiadiazole ring increased the in vitro activity against promastigotes whereas the attachment of cyclic group of piperidin-4-ol exhibited lower antipromastigote activity. linear substitution 1a-b seems to have played an important role in the mode of action of these compounds whilst cyclic substitution 5a-b has caused a steric hindrance around the c-2 position. it may have prevented the binding of the compounds to the parasitic macromolecular target. however, 5-nitrothiophene derivatives 1b and 5b with ic50 values of 3 and 27μm were more active than the corresponding 5-nitofuran analogues and, as such, these findings confirmed the fact that thiophen analogues were more potent than furan analogues at compounds 1a-b and 5a-b. besides, thiophen analogues exhibited better antiamastigote activity compared to furan analogues. concerning low potency of compounds 5a-b compared with their linear analogues, it is suggested that likewise piperidinol ring in the 2-position of thiadiazole ring may be responsible for decreased interaction between ligand and target macromolecule in amastigote form. the differences in the superimposition studies can help to explain why the target compounds 5a-b have lower activity. the compounds 1a and 1b have 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4-thiadiazol-2ylthio) acetamides derived from 5-nitrofuran. med chem res. 24: 891–900. who (2010) control of the leishmaniases: report of a meeting of the who expert committee on the control of leishmaniases, geneva, 22–26 march, 2010. j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 372 http://jad.tums.ac.ir published online: january 06, 2016 original article behavioral response of aedes aegypti mosquito towards essential oils using olfactometer *ashish uniyal 1, sachin n tikar 1, murlidhar j mendki 1, ram singh 1, shakti v shukla 2, om p agrawal 3, vijay veer 1, devanathan sukumaran 1 1vector management division, defence r and d establishment jhansi road, gwalior, india 2fragrance and flavour development center, g. t. road, makrand nagar, kannuaj, india 3school of studies in zoology, jiwaji university, gwalior, india (received 23 aug 2014; accepted 6 dec 2014) abstract background: aedes aegypti mosquito is responsible for transmitting human diseases like dengue and chikungunya. personal or space protection with insect repellents is a practical approach to reducing human mosquito contact, thereby minimizing disease transmission. essential oils are natural volatile substances from plants used as protective measure against blood-sucking mosquitoes. methods: twenty-three essential oils were evaluated for their repellent effect against ae. aegypti female mosquito in laboratory conditions using y-tube olfactometer. results: the essential oils exhibited varying degree of repellency. litsea oil showed 50.31%, 60.2 %, and 77.26% effective mean repellency at 1 ppm, 10 ppm and 100 ppm respectively, while deet exhibited 59.63%, 68.63%, 85.48% and depa showed 57.97%, 65.43%, and 80.62% repellency at respective above concentrations. statistical analysis revealed that among the tested essential oils, litsea oil had effective repellency in comparison with deet and depa against ae. aegypti mosquito at all concentration. essential oils, deet and depa showed significant repellence against ae. aegypti (p< 0.05) at all 3 concentration tested. conclusion: litsea oil exhibited effective percentage repellency similar to deet and depa. the essential oils are natural plant products that may be useful for developing safer and newer herbal based effective mosquito repellents. keywords: essential oils, aedes aegypti, repellent, y-maze olfactometer, flight orientation introduction mosquito borne diseases such as malaria, japanese encephalitis and dengue are a major concern in both developed and developing countries. dengue and chikungunya are important diseases transmitted by aedes aegypti mosquito which lives in urban habitats and breeds mostly in man-made and natural containers (julian 2009, who 2012). its peak host seeking activity is early in the morning and in the evening before dusk (who 2012). these diseases have many economical and sociological adverse effects on human, domestic animal populations (who 2013) and dengue fever is one of the main causes of child mortality in asia and africa (gupta et al. 2012). since there is no effective vaccine available for the control of these deadly diseases (norashiqin et al. 2008, gu et al. 2009), mosquito control programs are essential to prevent spread of these diseases. insect repellents are used as a personal protection that can provide a practical and economical means of preventing mosquito-borne diseases. the most common mosquito repellent deet (n,n diethyl-m-toluamide), a gold-standard of synthetic repellent, which is currently available in the market has shown repellency for 6 to 8 hours against mosquito (yap 1986, kweka et al. 2012) and other blood sucking insects (browne et al. 1997). even *corresponding authors: mr ashish uniyal, e-mail: ashish_uniyal5@gmail.com j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 373 http://jad.tums.ac.ir published online: january 06, 2016 though deet is mostly used as a mosquito repellent, but it has the side effects on human being such as skin irritation, affects the central nervous system, mucous membranes and mild toxic (maibach and johnson 1975, reuveni and yagupsky 1982, phasomkusolsil et al. 2010, miot et al. 2011) and strong solvent for plastic and other synthetic products (kazembe et al. 2012). every year more than 29 billion mosquito coils are sold for personal protection which contain pyrethrum daisy a plant product obtained from the flower of asteraceae family basically used for repellent and insecticidal purpose against blood sucking insects (who 1998, lawrance et al. 2004). due to these adverse effects, attempts are made to find safe and ecofriendly repellents derived from plant extracts/ materials. several characteristics such as insecticidal, repellent and growth reducing properties are found in plant essential oils. the major families, which contain essential oils as insecticidal and repellent properties, include lamiceae, asteraceae, myrtaceae and lauraceae. plant essential oil from myrtaceae family has effective repellent activity against mosquitoes (tapondjou et al. 2003, maia et al. 2011). however, labiatae, apiaceae, lamiaceae contain insect repellent, antifeedent activity and insecticidal properties (regnault-roger et al. 1994, vasilakoglou et al. 2007). plant family poaceae, rutaceae also showed effective repellent activity against mosquitoes (regnault-roger 1997, maia et al. 2011). essential oil from plant family such as geraniaceae, oleaceae, piperaceae, cupressaceae and burseraceae also exhibited repellent activity against blood sucking mosquitoes (amer and mehlhorn 2006). according to moore et al. (2006) plant families such as lamiaceae, myrtaceae and poaceae are best known species as insect repellent. however, pmenthone-3,8diol (pmd) from eucalyptus maculate citrodon a lemon eucalyptus and citronella oil from cymbopogon nardus are effective natural repellent preferred by various users (curtis et al. 1987, trigg et al. 1996, trigg 1996, trongtokit et al. 2005, hsu et al. 2013) and no adverse effect reported since 1984 (us epa 1999) essential oils are the alternative source against synthetic repellent because they are non-toxic for human and other organisms (das et al. 2003, tarek et al. 2012). essential oils from several plants have shown effective repelling against mosquitoes (barnard 1999). natural plant based repellents have also demonstrated good efficacy against some mosquito species in tests examining as adulticidal activity (yang et al. 2005, manimaran et al. 2012), larvicidal activity (ansari et al. 2000, adebajo et al. 2012), repellent activity (amer and mehlhorn 2006, gleiser et al. 2011) and adulticidal, repellent, larvicidal, oviposition deterrent activity (prajapati et al. 2005). the effectiveness of any plant essential oil as repellent depends on several factors such as quality, type of repellent, mode of action, temperature, humidity, biting response of mosquito, volatility, methods of extractions (tawatsin et al. 2001). factors affecting the quality of essential oils include plant species, cultivating conditions, and maturation of harvested plants, plant storage, plant preparation and methods of extraction (tawatsin et al. 2001, norashiqin et al. 2010). behavioral studies on essential oils are very important research to identify effective plant oils and their constituent responsible for exhibiting repellent effects against mosquitoes and there is a need to develop eco-friendly, safe, cost effective repellants for blood sucking insects. in the present study, an attempt has been made to study repellency of 23 various essential oils obtained from fragrance and flavour development center, kannuj, u p india, against ae. aegypti mosquitoes in la boratory condition. j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 374 http://jad.tums.ac.ir published online: january 06, 2016 material and methods test insect the test mosquitoes ae. aegypti were reared in the laboratory conditions in wooden cages (750 x600 x600 mm) for feeding and egg laying on a filter paper strip in a plastic container/bowl containing 250 ml of water. cotton with 10% sugar solution was provided for nourishment and the female mosquitoes were fed on rabbits for blood meal initially for 2 days and then at every alternative days. the eggs were collected and transferred to a bowl containing two liters of water, for rearing of hatched larvae up to adult stage. brewer’s yeast powder was provided as food for larvae and water was changed on alternate days. the pupae were collected and kept in small cages (550 x450 x 50 mm) covered with cotton cloth for emerging into the adult. aedes aegypti (five to six days old) adults were drawn from the stock colony maintained at 27±2 ˚c and 70±5% rh for all the evaluations. essential oils and synthetic repellents twenty-three essential oils as mentioned in table 1 were obtained from the fragrance and flavour development center (ffdc), kannuj, uttar pradesh, india. the synthetic repellent n, ndiethyl-m-toluamide (deet) 98.5% pure was purchased from sigma aldrich chemicals and n, n-diethyl phenyl acetamide (depa) 99% pure was synthesized by chemists from synthetic chemistry division of drde gwalior. flight orientation the repellent behavior response of ae. aegypti exposed to 23 essential oils was studied using ymaze olfactometer is shown in fig. 1 described by erler et al. (2006) slightly modified. it is made up of glass tube having internal diameter of 2.5 cm and 45 cm length from main arm containing one arm for testing repellent and the other arm for control. the olfactometer was kept on table, pressurized air was flow continuously into the olfactometer at the rate of 1.5 l/min. filter paper strips (1cmx 5cm) were loaded with 100 µ l of different concentration viz, 1 ppm, 10 ppm, 100 ppm of individual essential oils, deet, and depa dissolved in isopropanol. deet and depa were used as positive controls and the paper strips treated with solvent isopropanol alone was used as control. after the test oil was applied on treated paper, it was fixed in one arm and solvent treated paper in another arm of ymaze olfactometer as control. twenty ae. aegypti females (5 to 6 days old) were used for each test. the mosquitoes were released into the main arm located at the other end of the olfactometer has a circular tube (1 inch) having circular (1cm diameter) access hole for introduction of mosquitoes and cotton was used the keep the access hole closed. numbers of mosquitoes present in test and control arm of y -maze were counted after three minutes of exposure and all mosquitoes were removed soon after completion of experiment. after completion of one experiment the treated and control were interchanged, for each assay at different concentrations six replication were taken for test mosquito. the experiment was carried out during day time from 1000 hrs to 1600 hrs at room temperature 27±2 ˚c, relative humidity 70±5% with light intensity of 120– 125 lux (bioassay room was illuminated by three 36 w fluorescent lamps). six replicates of tests were conducted for each treatment and for each replicate a new set of twenty female mosquitoes were used. after every treatment, y-maze and set up connections were washed with acetone and dried soon to avoid any interference of other essential oils scents. j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 375 http://jad.tums.ac.ir published online: january 06, 2016 data analysis the percent repellency of essential oils was calculated as per erler et al. (2006). where pr= (c-t)/(c+t) x 100 where c= number of insects in control arm and t= number of insects in treated arm, insects that remained in the main arm were not taken into account. the data were subjected to statistical analysis for comparison analyzed by using statistical software (sigma stat v2.03). the values for repellency and mean were analyzed using one way anova for determination of variance ratio and least significant difference (lsd). results the results obtained from behavioral response of ae. aegypti to essential oils using y-maze olfactometer are present in table 2. at 1 ppm concentration, litsea, geranium and rosewood exhibited significantly higher repellency (50.31%, 48.74% and 46.00%) in comparison with the other oils and similar repellency was observed for deet (59.63%) and depa (57.97%) (f= 16.948, df= 149, p< 0.001). at higher concentration of 10 ppm, litsea (60.26%), rosewood (59.22%) and geranium (56.84%) also showed significant repellency over other oils but similar to that of deet (68.63%) and depa (65.432%) (f= 18.238, df= 149, p< 0.001) followed by lemon scented (45.12%). a further increase in the concentration to 100ppm also showed similar trend, where litsea (77.26%), rosewood (66.90%) and geranium (66.11%) exhibited significantly higher repellence’s of mosquitoes over other oils but lesser effects than deet (85.48%) and depa (80.62%), (f= 12.677, df= 149, p< 0.001) followed by lemongrass (57.49%). based on the repellency of essential oils against ae. aegypti mosquitoes, the order of effective percentage repellency can be arranged as follows, litsea > rosewood > geranium > lemongrass > lemon scented > camphor > citronella > galbanum > dill > cinnamon > basil > frankincense > lavender > black pepper > thyme > rosemary > jasmine > catnip > peppermint > chamomile > juniper > amyris > tagetes. fig. 1. ‘y’ maze olfactometer showing behavioural bioassay assembly air flow meter and regulating unit j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 376 http://jad.tums.ac.ir published online: january 06, 2016 table 1. list of essential oils obtained from different plant sources used for the repellent study against aedes aegypti mosquitoes using “y” maze olfactometer (source of oils: fragrance and flavour development center, kannuj, u p, india) name of material plant name family origin of plant distillation part used amyris amyris balsamifera rutaceae haitai, jamaica steam wood basil ocimum basilicum lamiaceae india steam fresh plant black pepper piper nigrum piperaceae india steam seed camphor cinnamomum camphora lamiaceae china steam leaves catnip nepeta cataria lamiaceae france, canada steam leaves chamomile anthemis nobilis asteraceae france, italy steam leaves cinnamon cinnamamomus zeylanicum lauraceae sri lanka steam bark citronella cymbopogon winterianus poaceae indonesia, central nepal steam leaves dill anethum graveolens apiaceae hungary steam seed frankincense boswellia carteri burseraceae somalia steam tree resin galbanum ferula galbaniflua apiaceae turkey steam tree resin geranium pelargonium graveolens geraniaceae south africa, egypt steam leaves, stalk jasmine jasminum grandiflorum oleaceae india, south asia hydro flower juniper juniperus communis cupressaceae india steam fruit lavender lavendula angustifolia lamiaceae france steam flower lemon grass cymbopogon citrates poaceae southeast asia. hydro, steam leaves lemon scented eucalyptus citriodora myrtaceae australia steam leaves litsea litsea cubeba lauraceae china steam fruit peppermint mentha piperita lamiaceae india steam leaves, flower rosemary rosmarinus officinalis lamiaceae spian, tunisia steam shrub rosewood aniba rosaeodora lauraceae brazil steam wood tagetes tagetes minuta asteraceae south america steam flower thyme thymus serpyllum labiatae europe and north africa steam leaves j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 377 http://jad.tums.ac.ir published online: january 06, 2016 table 2. repellency of 23 essential oils against aedes aegypti mosquitoes using “y” maze olfactometer in comparison with synthetic insect repellents n, n-diethyl phenyl acetamide (depa) and n, ndiethyl-m-toluamide (deet) s. no compound mean% repellency ± se 1 ppm 10 ppm 100 ppm 1 litsea 50.31 ± 5.03a 60.26 ± 2.91a 77.26 ± 2.94ab 2 rosewood 46.00 ± 4.85ab 59.22 ± 2.56a 66.90 ± 4.23b 3 geranium 48.74 ± 4.55a 56.84 ± 2.97ab 66.11 ± 2.16bbc 4 lemongrass 21.45 ± 3.83cd 43.60 ± 3.15c 57.49 ± 1.83c 5 lemon scented 33.99 ± 2.49bc 45.12 ± 2.91bc 55.39 ± 3.8c 6 camphor 23.12 ± 3.35c 41.5 ± 2.98c 54.21 ± 1.73c 7 citronella 24.56 ± 3.18c 34.22 ± 3.03c 52.95 ± 4.75c 8 galbanum 13.54 ± 2.95d 30.77 ± 3.37d 51.38 ± 3.04c 9 dill 8.10 ± 2.56d 33.29 ± 2.38cd 51.04 ± 2.98c 10 cinnamon 13.75 ± 4.23d 27.67 ± 3.62d 50.82 ± 2.3c 11 basil 20.29 ± 3.66d 39.88 ± 3.5c 50.56 ± 5.14c 12 frankincense 23.70 ± 4.67c 40.68 ± 3.73c 50.46 ± 2.29c 13 lavender 19.62 ± 3.17d 28.74 ± 2.42d 49.92 ± 4.06c 14 black pepper 22.22 ± 4.14c 33.90 ± 2.67c 48.96 ± 4.29c 15 thyme 20.11 ± 3.11d 36.14 ± 4.16c 48.61 ± 2.6c 16 rosemary 18.78 ± 5.22d 31.07 ± 3.29d 46.01 ± 2.49c 17 jasmine 15.70 ± 3.35d 24.55 ± 3.32de 46.08 ± 3.85c 18 catnip 20.34 ± 5.53d 34.16 ± 5.21c 44.30 ± 3.63c 19 peppermint 27.64 ± 6.02c 34.72 ± 2.8c 42.15 ± 3.55c 20 chamomile 11.65 ± 4.39d 30.31 ± 2.7d 40.73 ± 3.86c 21 juniper 11.04 ± 4.38d 29.98 ± 4.46d 39.92 ± 3.11cd 22 amyris 12.28 ± 3.28d 20.85 ± 3.43e 31.37 ± 3.75d 23 tagetes 13.94 ± 3.67d 16.65 ± 3.16e 30.52 ± 5.29d 24 depa 57.97 ± 2.8a 65.43 ± 2.84a 80.62 ± 2.48a 25 deet 59.63 ± 2.28a 68.63 ± 2.53a 85.48 ± 2.3a mean percentage repellency ±se, data followed by the different letters are significantly different (p< 0.05, by one-way anova and least significance difference). the values showed by the same letter are statistically non-significant (p> 0.05) discussion essential oils are composition of volatile components having minor constituents contain pleasant fragrance which are responsible for mosquito repellency and inhibit the orientation of blood sucking insects (campbell et al. 2010). host-seeking insects, orient to a host by using chemical stimuli precede visual and thermal stimuli emanating from the host. different mosquito species develop different host preferences, and it is generally assumed that host selection and discrimination is mainly based on olfactory cues (takken 1991). results obtained in the present study using olfactometer bioassay showed that volatile essential oils exhibited concentration dependent spatial repellency against ae. aegypti. litsea, rosewood and geranium oil showed effective repellency against ae. aegypti mosquito but the effectiveness of essential oils was not superior over synthetic insect repellents deet and depa. the result of litsea oil repellent efficacy was supported by vongj arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 378 http://jad.tums.ac.ir published online: january 06, 2016 sombath et al. (2012) against armigeres, culex and aedes, likewise, amer and mehlhorn (2006) against ae. aegypti, an. stephensi and cx. quinquefasciatus. they have also supported the repellent efficacy of geranium and rosewood. in this study, deet and depa were used as positive controls and the effective repellent activities of deet against mosquitoes as compared with other essential oils are supported by kazembe et al. (2012). the effective repellent activity of deet and depa against ae. aegypti and an. stephensi mosquito was reported by debboun and wagman (2004). hu et al. (2011) reported that zcitral and limonene as the predominant component of litsea oil extracted from the plant litsea cubeba an evergreen tree found in japan, southern china, and some parts of southeast asia. however, rosewood oils from the plant aniba rosaeodora contains linalool and 1,8-cineole as the main chemical constituent in rosewood oil (guilherme et al. 2007). moreover, geranium oil from pelargonium graveolenes reported to contains β-citronellol as main component (campbell et al. 2010). essential oils contain more than 20 to 80 minor and major highly volatile chemical constituents of which the major components showed effective repellent against ae. aegypti mosquito (campbell et al. 2010). plant oil which contains limonene, linalool, citronellol showed effective repellent activity against different mosquito species (barnard 1999, tawatsin et al. 2001, kline et al. 2003, kang et al. 2009, hsu et al. 2013). due to high volatile property, essential oil exhibited effective but short duration of protection against mosquitoes. synthetic repellent has low rate of vaporization and more effective than essential oils but they cause adverse effect on human health (maibach and johnson 1975, choochote et al. 2007). plant based repellents are safe, nontoxic and ecofriendly. therefore most of the repellent manufacturers use different substances, chemicals or natural products as fixative compounds such as vanillin, salicylic acid, coconut oil, mustard oil with essential oils for reducing the rate of evaporation of volatile components and for improvement of long lasting repellency against mosquitoes (tawatsin et al. 2001, das et al. 2003, kongkaew et al. 2011, sritabutra et al. 2013). however, amer and mehlhorn (2006) found that several essential oils namely litsea, niaouli and catnip prepared in vanillin demonstrated good repellent efficacy against different mosquito species as compared to synthetic chemical deet. moreover, adeniran and fabiyi (2012) using formulated cream base lemongrass oil against ae. aegypti mosquito. whereas, several essential oils with synthetic chemicals as formulated cream, spray, lotion showed effective repellency against ae. albopictus, cx. nigripalpus and ochlerotatus triseriatus were reported by barnard and xue (2004) and formulated neem cream exhibited effective repellency against aedes, culex and anopheles mosquitoes (dua et al. 1995). hence, essential oils are alternative source as a mosquito repellent as compared to synthetic mosquito repellent. present olfactometer bioassay elicited sensory response of aedes mosquito in the absence of any skin emanations. however, mosquito response to essential oils applied on skin may give a different result. behavior of mosquito towards oils in the presence of skin emanations and other unidentified human odor components were found different (bernier et al. 2005, hao et al. 2012). although many essential oils have been found to be potently repellent, because of the high volatility of major constituents of most oils, very few have found potential in personal protection. on the other hand, they have shown great potential in space protection. using an olfactometer test is quick and effective way to evaluate the behavioural responses of mosquitoes towards volatile j arthropod-borne dis, september 2016, 10(3): 372–382 a uniyal et al.: behavioral response of … 379 http://jad.tums.ac.ir published online: january 06, 2016 stimuli of essential oil. these effective essential oils can be used as plant based product for provided a protection against various mosquitoborne diseases. there is a need for promoting the use of herbal products because of their safety to individual and communities. conclusion based on the above studies on repellent study of essential oils, litsea, rosewood and geranium showed nearly effective repellency like synthetic repellent deet and depa against ae. aegypti female mosquitoes. our research is continued in this line of work for searching effective 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04, 2016 original article some new records of culicoides species (diptera: ceratopogonidae) from iran *mohammad abdigoudarzi department of parasitology, razi vaccine and serum research institute, alborz, iran (received 22 nov 2014; accepted 14 mar 2015) abstract background: biting midges of the genus culicoides act as vectors for important diseases affecting humans and both wild and domestic animals. collection of adult culicoides specimens in the near vicinity of vertebrate hosts is the major part of any bluetongue surveillance plan. there are old records of culicoides species dated from 1963, 1968 and 1975. therefore, it was decided to collect different ceratopogonids members using a light trap. methods: one night catching using light traps with a suction fan was performed at representative sites (25 places) located in north western provinces (ardebil, eastern azerbaijan, western azerbaijan and zanjan) of iran (suspected farms for clinical records of bluetongue virus or serodiagnosis of the bluetongue virus). samples were detected and identified primarily and were sent to a reference center for final verification. results: seven culicoides species including (culicoides circumscriptus, c. flavidus, c. longipennis, c. pulicaris, c. puncatatus, c. nubeculosus, and three species from culicoides (oecacta) are under study in reference laboratory in poland and c. puncticollis were confirmed from iran. conclusion: morphological and explanation of each species was regarded in this study. in comparison to old record, there are four new records of culicoides species from iran and one species is regarded suspected for viral transmission. keywords: ceratopogonidae, culicoides, iran introduction biting midges of the genus culicoides latreille, 1809 act as vectors for important diseases affecting humans and both wild and domestic animals. there is an important problem regarding these insects in european countries then it forcing international offices to get instant records of related disease from public sectors. medical and veterinary importance of culicoides species is regarded by different scientists. bluetongue virus was initially isolated from c. obsoletus midges in cyprus (mellor and pitzolis 1979), and african horse sickness virus from a mixed pool of this species and pulicaris group midges in spain (mellor 1990). culicoides obsoletus has been confirmed as bluetongue vector in northern and southern europe. culicoides obsoletus can also cause an allergic response to its bite in sheep and goats (connan and lloyd 1988). ceratopogonid midges are small nematocerous diptera, the female members have biting mouthparts and mandibles, which work rather as a pair of scissors. they are mostly one to two millimeters long, the largest british species spans four millimeters or less. the body is stout and the wings are held flat and folded over the abdomen when at rest. they can easily be mistaken for members of the chironomidae, indeed, they were formerly included in this family. they may be distinguished, however, by the short front legs (in the chironomidae the front legs are usually much longer than the others), the wing venation and by the biting *corresponding author: dr mohammad abdigoudarzi, e-mail: m.abdi@rvsri.ac.ir j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 475 http://jad.tums.ac.ir published online: october 04, 2016 mouthparts (non-biting mouthparts in the chironomidae). the wing venation is simple with (usually) two radial cells. in most species the wings are clear, without markings, but many species of culicoides (and of some species of other genera) have easily recognizable patterned wings (some chironomidae have patterned wings, but then the venation is different .) a good formal diagnosis of the family is given in (downes and wirth 1981), and a key to the british genera of ceratopogonidae is provided in boorman and rowland (1988). mesghali (1963) recorded 22 species from the genus culicoides from iran. his collection methods was using aspirator collecting flies resting on the body of livestock, collecting flies attracted to the light from the window, collecting midges when there was a screening plan for collection of anopheles members by total catch (insecticide spray) method in malaria campaign, stick trap and light trap. he also noted that except c. puncticollis and c. similis the rest of midges were recorded as new records on that time. the reason of that study by mesghali was the induction of african horse sickness in 1959 in southern parts of iran, which the vector is culicoides members where 60% up to 80% of horses died after the disease. twenty-six species have been recorded by navai and mesghali (1968). from 26 species of culicoides reported on that time, 14 species were recorded for the first time in iran: c. bulbostylus, c. flavidus, c. flavisimilis, c. ibericus, c. koreensis, c. longipennis, c. nagahanai, c. odibilis, c. pallidicornis, c. pallidipennis, c. pictimargo, c. pulicaris, c. punctatus, and c. similisbaghdadensis. navai's collection methods were light trap, tent trap and adults emerged after pupa collection from aquatic habitats. later, two new species, c. mesghalii and c. shahgudiani were recorded from the persian gulf area of iran (navai 1973). culicoides members from south-west asia have been studied by navai (1977). there are records of culicoides vector members from the nearby country like turkey (dik et al. 2006). the distribution pattern of culicoides spp. is according to iranian geographical coordination (northern latitude 40°, southern latitude 25°) (western longitude 44° and eastern longitude 63.5°). due to warming up of the north hemisphere, the spread of culicoides species has been moved from 40◦ to 43◦ (northern latitude) (djuricic et al. 2003). in any bluetongue surveillance system, the principal aim is to capture adult culicoides in the near vicinity of vertebrate hosts, and to employ a powerful trap (to enhance surveillance sensitivity at low culicoides population levels and, furthermore, to increase the number of captured midges for virus isolation studies) (goffredo and meiswinkel 2004). the results of a new serological study in iran, showed that the bluetongue virus seroprevalence of sheep in west-azerbaijan (64.86%) was the highest and lowest prevalence was seen at qom (12.1%) (khezri and azimi 2013). according to above old records and the importance of the study of ceratopogonids in iran, screening culicoides spp. for blue tongue control in sheep, increased warming up of the north hemisphere and its effect on distributional pattern of culicoides spp, it was decided to collect different ceratopogonids members using light trap. a collection method as one night catching on site using light traps with a suction fan was performed at representative sites in northwestern provinces of iran (suspected farms for clinical records of bluetongue virus or serodiagnosis of the bluetongue virus). materials and methods new collection field trips were designed according to bluetongue seropositive records in livestock in north western provinces of iran. j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 476 http://jad.tums.ac.ir published online: october 04, 2016 collection method (one night catching on site using light traps with a suction fan) (sanyo–ik-150) was performed at representative sites in (ardebil, eastern azerbaijan, western azerbaijan and zanjan) (table 1) (suspected farms for clinical records of bt virus or serodiagnosis of the bt virus). from sep 2004 up to early oct 2005, (it was done mostly on late august and early september (late summer time in iran) adult members of culicoides were collected and transferred to the laboratory near ice pack in an isolator box (at arrival time they were fresh and intact for better inspection). culicoides members were separated and have been mounted on slides using a clearing agent then hoyer'medium was applied and different characters including wing patterns and venation was regarded using identification key (rawlings 1996). accidentally collected ceratopogonids during an agricultural research on insect pests were sent to razi institute and were studied too. parallel samples were sent to dr ryszard szadziewski (poland) and dr shahin navai (germany). results from september 2004 up to october 2005, culicoides specimens from 25 different regions were collected by light trap. different members of chironomidae, psychodidae, sciaridae and cecidomyiidae families from diptera have been confirmed, forcipomyia (ceratopogonidae) also noted and some lepidoptera and hymenoptera were recorded. ceratopogonidae members were selected. culicoides spp. were separated and were studied under an anatomical microscope. nine culicoides species including (culicoides circumscriptus, c. flavidus, c. longipennis, c. pulicaris, c. puncatatus, c. nubeculosus, and three species from culicoides (oecacta) were confirmed from iran. data for geographical location, time range and type species and number of samples are included (table 1 and 3). fig. 1. culicoides pulicaris (wing pattern is distinctive), (original photo) table 1. data for different species of culicoides collected according to bluetongue seropositive records from livestock in iran province place time range collected insect type ardbil khalkhal road, heleh abad 14th sep. 2005 culicoides (culicoidea) punctatus (meigen, 1804), 7 f culicoides (oecacta) sp (under verification) b, 3 f culicoides (culicoidea) punctatus (meigen, 1804), 8 f culicoides (oecacta) sp (under verification.)c, 3 f culicoides (beltranmyia) circumscriptus kieffer, 1918, 1 m, chironomidae 6 f, j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 477 http://jad.tums.ac.ir published online: october 04, 2016 cecidomyiidae 4 specimens eastern az. 21st -22nd aug. 2005 6th -13th sep. 2005 eastern az. marand, yamchi, amir abad 6thsep. 2005 culicoides (monoculicoides) nubeculosus (meigen, 1830) 3 f, 3 m, culicoides (oecacta) sp (under verificationt.)c, 2 f jolfa, galin ghiye 6thsep. 2005 culicoides (monoculicoides) nubeculosus (meigen, 1830) 4 f, chironomidae 8 specimens western az. poldasht, vet. office 11th aug. 2005 chironomidae, 10 f culicoides (oecacta) sp (under verification) c, 4 f 12th aug. 2005 chironomidae, 1 f culicoides (oecacta) sp i(under verification ) a, 1 f culicoides (oecacta) sp indet. c, 4 f western az. 14th -18th sep. 2004 12th -14th june 2005 10th -12th aug. 2005 not included zanjan 9th -11th june 2005 16th -17th aug. 2005 4th oct. 2005 not included zanjan tarom, gilvan 4th oct. 2005 culicoides (beltranmyia) circumscriptus kieffer, 1918, 3 f culicoides (oecacta) sp (under verification.)a, 1 f chironomidae (2), psychodidae (1), sciaridae (1), cecidomiidae (1), ceratopogonidae (forcipomyia) (1 f) culicoides (oecacta) sp (under verification.)c, 1 m table 2. different recorded culicoides species from iran abdigoudarzi (2008)navai and mesghali (1968)mesghali (1963) culicoides circumscriptus culicoides flavidus culicoides longipennis culicoides pulicaris culicoides puncatatus culicoides nubeculosis *culicoides (oecacta) species a *culicoides (oecacta) species b *culicoides (oecacta) spcies c culicoides circumscriptus culicoides flavidus culicoides flavisimilis culicoides grisescens culicoides neliophilus culicoides ibericus culicoides kurensis culicoides longipennis culicoides nagahani culicoides odililis culicoides pauidicornis culicoides pallidicornis culicoides pictimargo culicoides pictipennis culicoides pulicaris culicoides puncatatus culicoides puncticillus culicoides caspius gutzevich, 1959 culicoides circumscriptus kieffer, 1918 culicoides dendrophilus amosova, 1957 culicoides firuzae dzhafarov, 1958 culicoides grisescens edwards, 1939 culicoides halophilus kieffer, 1924 culicoides heliophilus edwards, 1921 culicoides kurensis dzhafarov, 1960 culicoides maritimus kieffer, 1924 culicoides parroti kieffer, 1922 culicoides omogensis arnaud, 1956 culicoides pictipennis staeger, 1839 culicoides puncticollis becker, 1902 culicoides riethi kieffer, 1914 culicoides saeous kieffer, 1922 culicoides sejfadinei dzhafarov, 1922 culicoides schultzei enderlin , 1908 table 1. continued… j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 478 http://jad.tums.ac.ir published online: october 04, 2016 culicoides riethi culicoides saevus culicoides schuttzei culicoides bulbostylus culicoides seifadinei culicoides similisbaghda culicoides dansis culicoides subfacipennis culicoides turkmensicus culicoides similis carter, ingram and macfi, 1920 culicoides simulator edwards, 1939 culicoides sinanoensis tokunga, 1937 culicoides subfascipennis kieffer, 1919 culicoides turkmenicus guttzevich, 1959 *note: three new species are under verification table 3. different species and number of samples (final verification from dr szadziewski) no. location no. of specimens final verification (dr. szadziewski) 1iran-az. gharbi -poldashtvet.service20584(11thaug.2005) 10 sample 1. chironomidae, 10 females. 2iran-az. gharbi -poldasht vet. service 20-5-84(11th aug. 2005) 6 sample 2. culicoides (oecacta) new species are under study, 4 females. 3iran-az. gharbi -poldasht vet. service 20-5-84(11th aug. 2005) 6 sample 3. chironomidae, 1 female. culicoides (oecacta) sp new species are under study a, 1 female culicoides (oecacta) sp new species are under study, 4 females. 4iran-zanjantaromgilvan-abbar 12-7-84(4th oct.2005) 4 sample 4. culicoides (beltranmyia) circumscriptus kieffer, 1918, 3 females culicoides (oecacta) sp new species are under study, 1 female. 5iran-zanjantaromgilvan-abbar 12-7-84(4thoct.2005) 6 sample 5. chironomidae 2 psychodidae 1 sciaridae 1 cecidomyiidae 1 forcipomyia (ceratopogonidae) 1 female culicoides (oecacta) new species are under study c, 1 male. 6iranardbilkhalkhal-road heleh abad 23-6-84-1(14thsep.2005) 10 sample 6. culicoides (culicoidea) punctatus (meigen, 1804), 7 females culicoides (oecacta) new species are under study, 3 females. 7iranardbilkhalkhal-road heleh abad23-684-2(14th sep.2005) 11 sample 7. culicoides (culicoidea) punctatus (meigen, 1804), 8 females culicoides (oecacta) new species are under study, 3females. 8iranardbilkhalkhal-road heleh abad23-684(14thsep.2005) 12 sample 8. culicoides (beltranmyia) circumscriptus kieffer, 1918, 1 male chironomidae 6 females cecidomyiidae 4 specimens. 9iranaz. sharghimarandamir abad 15-6-84(6th sep.2005) 8 sample 9. culicoides (monoculicoides) nubeculosus (meigen, 1830, 3 females, 3 males culicoides (oecacta) new species are under study, 2 females. 10 iranaz. sharghimarandjolfa-galin ghieh 15-6-84(6th sep.2005) 12 sample 10. culicoides (monoculicoides) nubeculosus (meigen, 1830, 4 females chironomidae 8 specimens. total 85 table 2. continued… j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 479 http://jad.tums.ac.ir published online: october 04, 2016 discussion mesghali, 1963, recorded 22 species from the genus culicoides latreille, 1809 from iran (mesghali 1963). navai and mesghali, 1968 recorded 26 species from iran (navai and mesghali 1968). navai's collection methods were light trap, tent trap and adults emerged after pupa collection from aquatic habitats (table 2). regarding above data and precise attention to these records and after taking the advancement of synonymy of c. circumscriptus with c. puncticollis twenty-two recorded species from mesghali (1963) should be reduced to twenty-one and there are eleven shared recorded species between data from navai and mesghali (1968) and mesghali (1963). totally, regarding past and present studies there are forty recorded culicoides species from iran. in a study on culicoides species in portugal 66% of collected specimens in summer were c. imicola, but c. obsoletus and c. pulicaris were highly collected during winter. the authors of this study in portugal believed that c. pulicaris could preserve the virus of african horse sickness by overwintering and act as a reservoir of this virus (capela et al. 2003). entomological investigation of the presence of culicoides species in bosnia and herzegovina was conducted in 2007. during the investigation, 2,256 culicoides midges were collected and only one species (culicoides obsoletus meigen, 1818) was identified (omeragic et al. 2009). in a study by talavera et al. (2011), newly recorded species are: (1) c. yemenensis, new record for europe, (2) c. coluzzii and c. sejfadinei, being new records for the iberian peninsula, and (3) c. pseudopallidus, which is new record for spain. culicoides sejfadinei was recorded from iran by mesghali (1963) and navai and mesghali (1968) too. this species was not included in new collection data from iran, and then additional collection studies were done. accidentally collected ceratopogonids during an agricultural campaign in (arak, iran) was regarded and finally six females from culicoides puncticollis were confirmed in this study (alikhani, unpublished data). nine culicoides species including (c. circumscriptus, c. flavidus, c. longipennis, c. pulicaris, c. puncatatus, c. nubeculosus, and three species from culicoides (oecacta) were confirmed from iran. data for geographical location, time range and type species and number of samples are included (table 1 and 3). there are morphological and explanation of each species as follows: 1culicoides (beltranmyia) circumscriptus kieffer, 1918 culicoides circumscriptus and c. festivipennis were seen as dominant species especially in organic matter rich mud near the water reservoirs. c. circumscriptus was found in a wide variety of habitats (uslu and dik 2007). this species has been reported by mesghali (1963) and navai and mesghali (1968) (table 2). it has not been confirmed as a vector for bluetongue virus. this species could be distinguished regarding the color pattern of the wing and wing venation. 2culicoides flavidus as a new species of the genus culicoides found in the valley of the araks river (armenias lowest elevation is found in the araks river valley) is described (dzhafarov 1959). culicoides flavidus dzhafarov, belongs to the group of species with nonspotted wings. it is similar to the recently described c. firuzae from the same locality, the characteristic differences being the light yellow color of the entire body, including the mesonotum and scutellum, and the different structure of the male hypopygium. j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 480 http://jad.tums.ac.ir published online: october 04, 2016 3culicoides longipennis (khalaf 1957) = culicoides flavisimilis dzhafarov the wings are greyish with prominent pale spots. it closely resembles c. sahariensis, but in that species the pale spot at the base of cell m2 overlaps vein m1. in male, the posterior part of the aedeagus of c. longipennis terminates in a few bristles, not in a point as in either c. sahariensis or c. similis. 4culicoides pulicaris (linnaeus 1758) seventeen collected samples during present study were sent to dr navai and this species was confirmed by her. the collection place was ardbil, khalkhal, heleh abad and date of collection was (14th sep 2005). there was also one record of c. pulicaris from azerbaijan sharghi, marand, jolfa and date of collection was (6th sep 2005) (fig. 1). 5culicoides punctatus (meigen 1804) = culicoides pulicaris kieffer = culicoides kasachstanicus shakirzjanova the tips of wing veins m1, m2 and cu1 typically end at the wing tip in small pale spots, distinguishing this species from c. pulicaris, but the distinction is not always clear-cut. both of these species are variable in the extent and intensity of the wing markings. 6culicoides nubeculosus (meigen 1830) = culicoides puncticollis goetghebuer 1912 this is one of the largest of the british culicoides. the female may be distinguished from others of the subgenera by the dark markings on a pale ground, the dark second radial cell, the single ovoid spermatheca, and the presence of a yellowish spot in the centre of the scutellum. it is very similar to c. puncticollis, but in that species, the spermatheca is sausageshaped and the male aedeagus is different. 7culicoides obsoletus (meigen 1818) in the western palaearctic region, c. obsoletus is by far the most commonly encountered species on farms and stables and can be present in high abundance (light trap catches can exceed 30 000 individuals/ night). the above species feed on a wide range of mammals including humans, cattle, horses, sheep and deer. it also feeds on avian hosts. they are commonly known as the 'garden midge' due to occasional presence in semi-urban habitats. the entomological survey of vectors should be limited to high-risk areas in the western provinces of iran. trapping site should be selected by several criteria, such as farm type (sheep or cattle), average temperature, average rainfall and humidity or vicinity to (stand water). the choice of trapping sites should be made as either cattle, sheep, goats or horses must be present (large livestock holdings are preferred) (>10 animals, livestock to be located in the near vicinity of the light trap all night; stabling can be of any type (but must be open) priority should be given to farms where conditions such as pools of water or mud are found, created either naturally (rain) or by irrigation or overflows. conclusion ten culicoides species including (c. circumscriptus, c. flavidus, c. longipennis, c. pulicaris, c. puncatatus, c. nubeculosis, culicoides (oecacta) species a, culicoides (oecacta) species b, culicoides (oecacta) species c and c. puncticollis were confirmed from iran. there are four new records of culicoides species from iran and c. pulicaris is regarded suspected for viral transmission. acknowledgments the author would like to thank dr shahin navai (natural history museum at berlin, germany and dr ryszard szadziewski (poland) for their kindness and effort on final verification of specimens. in addition, j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 481 http://jad.tums.ac.ir published online: october 04, 2016 the author would like to thank dr r kargar for his kind scientific support, mrs s rivaz for her laboratory practice help as well as all the colleagues from western-azarbaijan, eastern-azarbaijan, ardbil and zanjan provinces for their kind help for field collection trips and this article was the result of a confirmed research project granted at razi vaccine research ins. (supported by razi institute), (grant no. 2-024-250000-18-00083048). the authors declare that there is no conflict of interests. references abdigoudarzi m (2008) the study of culicoides spp. as problem vectors of blue tongue virus to ruminants in ardebil, east azerbaijan, west azerbaijan and zanjan provinces of iran, final report of research published by ministry of j-eagriculture (in persian). boorman j, rowland c (1988) a key to the british genera of ceratopogonidae (diptera). entomol gaz. 39: 65–73. capela r, purse bv, pena i, wittman ej, margarita y, capela m, romão l, mellor ps, baylis m (2003) spatial distribution of culicoides species in portugal in relation to the transmission of african horse sickness and bluetongue viruses. med vet entomol. 17(2): 165–177. connan rm, lloyd s (1988) seasonal allergic dermatitis in sheep. vet rec. 123: 335–337. dik b, yagci s, linton ym (2006) a review of species diversity and distribution of culicoides latreille, 1809 (diptera: ceratopogonidae) in turkey. j nat hist. 40(32–34): 1947–1967. djuricic b, nedic d, lausevic d, pavlovic m (2003) the epizootiological appearance of bluetongue in the central balkans. in abstract book of office international de epizooties oie international symposium on bluetongue, 26–29 october, italy, p. 32. downes ja, wirth ww (1981) chapter 28: ceratopogonidae. in: mcalpine jf, peterson bv, shewell ge, teskey hj, vockeroth jr, wood dm. manual of nearctic diptera, agriculture canada monograph 27. 1: 393–421. dzhafarov sm (1959) a new species of culicoides latr. from the araks valley. ent rev. 38: 421–422. goffredo m, meiswinkel r (2004) entomological surveillance of bluetongue in italy: methods of capture, catch analysis and identification of culicoides biting midges. vet ital. 40(3): 260–265. khezri m, azimi sm (2013) epidemiological investigation of bluetongue virus antibodies in sheep in iran. vet world. 6(3): 122–125. mellor ps, pitzolis g (1979) observations on breeding sites and light trap collections of culicoides during an outbreak of bluetongue in cyprus. bull entomol res. 69: 229–234. mellor ps (1990) the replication of bluetongue virus in culicoides vectors. curr top microbiol. 162: 143. mesghali a (1963) heleidae (diptera) of iran, some records of midges of the genus culicoides. bull soc pathol exot. 56: 1063–1069. navai s, mesghali a (1968) ceratopogonidae (diptera) of iran. ii. more records of culicoides latreille, 1809. j nat hist. 2: 241–246. navai s (1973) culicoides (diptera: ceratopogonidae) from the persian gulf area of iran. 2 new species, c. mesghalii and c. shahgudiani. bull soc pathol exot filiales. 66(1): 195–204. navai s (1977) biting midges of the genus culicoides (diptera: ceratopogonidae) from south-west asia [phd thesis]. college park, university of maryland (md), usa. j arthropod-borne dis, december 2016, 10(4): 474–482 m abdigoudarzi: some new records of … 482 http://jad.tums.ac.ir published online: october 04, 2016 omeragic j, vejzagic n, zuko a, jazic a (2009) culicoides obsoletus (diptera: ceratopogonidae) in bosnia and herzegovina-first report. parasitol res. 105: 563–565. rawlings p (1996) a key, based on wing patterns of biting midges (genus culicoides latreille diptera: ceratopogonidae) in the iberian peninsula, for use in epidemiological studies. graellsia. 52: 57–71. talavera s, munoz-munoz f, pages n (2011) new insights on diversity, morphology and distribution of culicoides latreille 1809 (diptera: ceratopogonidae) from northeast spain. ann soc entomol fr. (n.s.): int j entomol. 47: 1–2, 214–231. uslu u, dik b (2007) description of breeding sites of culicoides species (diptera: ceratopogonidae) in turkey. parasite. 14: 173–177. microsoft word 3dr oshaghi rtl_2_.doc iran j arthropod-borne dis, 2010, 4(2): 19–25 a barmaki et al.: study on presence of … 19 original articles study on presence of borrelia persica in soft ticks in western iran a barmaki1, j rafinejad1, h vatandoost1, z telmadarraiy1, f mohtarami1, sh leghaei2, *ma oshaghi1 1dept. of medical entomology and vector control, school of public health, tehran university of medical sciences (tums), tehran, iran. 2health management office of qazvin province, qazvin university of medical sciences, qazvin, iran (received 25 apr 2010; accepted 5 dec 2010) abstract background: a molecular survey was conducted to investigate the presence of pathogenic borrelia persica species causing the tick borne relapsing fever (tbrf) in takistan district qazvin province, western iran. methods: a number of 1021 soft ticks were collected from 31 villages including previously reported infected and none-infected tbrf cases and individually examined for the presence of b. persica dna by conventional pcr targeting the 16s rrna. results: a total of 1021 soft ticks of three species of ornithodouros tholozani (120: 11.75%), o. lahorensis (461: 45.15%) and argas persicus (440: 43.1%) were collected and tested against borrelia infection. soft ticks were more prevalent (67%) in infected areas than none infected areas. the rate o. tholozani in infected areas was much greater (29 times) than none infected areas. ninety seven percent of soft ticks in none infected areas were of o. tholozani. sixteen (16.7%) ticks of tested (n=95) o. tholozani were infected with b. persica. three (1.3%) out of 205 soft ticks of o. lahorensis were positive for borrelia sp., and no infection was observed in a. persicus. taqi rflp analysis and sequence analysis of the positive pcr products showed the presence of b. persica. the rflp analysis showed that the positive ticks of o. lahorensis were infected with unknown borrelia species. conclusion: this study showed that although there were no tbrf cases in takisan, but still infected o. tholozani, the known vector of tbrf, presented in the region. control measures needs to be fulfilled in thakisan. keywords: borrelia persica, tick borne relapsing fever (tbrf), pcr-rflp, takistan, iran introduction the tick borne relapsing fever (tbrf) is an acute infectious disease and one of its basic properties is the recurring attacks of fever and chill. this disease is one of the important hygiene problems in middle east and central asia (karimi et al. 1979, karimi 1980, barbour and hayes 1986, arshi et al. 2002). the disease is reported from north and south america, africa, asia and europe. the epidemiology of the disease in each region depends on the relationships between the tick and borrelia species as well as environmental condition in its distribution area. reservoir hosts are usually wild rodents (rebaudet and parola 2006). feeding of a tick on an infected vertebrate is the natural route of tick infection. the pathogen is transmitted to another vertebrate host during tick feeding in the next life stage. the development of the pcr has offered a new dimension in the diagnosis of infectious diseases. capable of amplifying minute amounts of dna into billions of copies in just a few hours, pcr facilitates the sensitive and specific detection of dna or rna of pathogenic organisms. pcr for the diagnosis of infectious diseases has been directed pri*corresponding author: dr mohammad ali oshaghi, email: moshaghi@sina.tums.ac.ir iran j arthropod-borne dis, 2010, 4(2): 19–25 a barmaki et al.: study on presence of … 20 marily toward the detection of pathogens for which conventional diagnostic techniques are either too insensitive or too slow such as borrelia spp. there are many research studies involving pcr in the diagnosis of borrelia spp. particularly b. burgdorferi infections in clinical samples (schwartz et al. 1997, cerar et al. 2008). the pcr method have been used successfully for identification of b. burgdorferi in hard ticks of ixodes ricinus (horka et al. 2008) and b. persica in soft ticks of o. tholozani (oshaghi et al. 2010a, b). tick-borne relapsing fever is one of the endemic diseases in iran and its prevalence was considerable in the past, but the incidence rate of it decreased to 0.06/100,000 in 2006 (asl et al. 2009). the cause of this disease in iran is mainly borrelia persica dschunkowski 1912, that conveyed by the bite of the ornithodoros tholozani soft tick (karimi 1980, masoumi asl et al. 2009). however, other species such as b. latshywii, b. microtti and b. balthazardi are responsible for the spread of the disease in certain areas of the country (karimi 1980). qazvin province is amongst the most important foci of tbrf in iran (asl et al. 2009). the province covers 15821 km² between 45-48 to 50-50 east of greenwich meridian of longitude and 35-37 to 36-45 north latitude of the equator (aghighi et al. 2007). takistan district with almost 180,000 populations, is located in west of the province. in takistan district there are no data regarding b. persica circulation in enzootic sites, nor the presence of these bacteria in ticks, even if there were human case records (mhme). on average, five cases of tbrf had been reported from takistan, however, since 2007, no report of tbrf has been evidenced in the district. since the risk of infection to humans with borrelia depends on outdoor recreational activity, on the density of tick populations, and on the infection of the ticks with borrelia. therefore, data describing the prevalence of borrelia in ticks can be used to assess the risk of tbrf for public health (rauter and hartung 2005). in this study we tried to assess the potential risk of tbrf by testing the presence of b. persica in soft ticks, particularly o. tholozani, from takistan that had previously witnessed some outbreaks of tbrf transmitted by this tick species. materials and methods study area surveys were performed in takistan district of qazvin province in north-west iran. it covers 2430 km² and the terrain of the survey areas consists of 4 zones with 133 villages (vs) distributed in different mountainous, hilly, and plain regions. it has a temperate to dry climate with annual precipitation of about 232 mm. the temperature ranges from -20 to 40° c, with an average of 14° c. the study was performed in all previously infected (ten) and 21 none infected villages. these 21 villages were randomly selected from mountainous (8), hilly (6), and plain (5) areas. specimen collection this survey was conducted from august 2007 to july 2008. ticks were actively searched directly by eye and help of torch light and collected from cracks, crevices, ceiling, and floor of human dwelling, poultry and animal shelters or rodent burrows. process of collection took 30 min each time and the specimens transferred into the holding tubes. all ticks were handled with forceps. all the specimens were identified on the basic of their morphological characteristics (zaim and shayeghi 1989, estrada-pen˜a et al. 2004). dna extraction dna extraction from ticks was performed as previously described (cao et al. 2000). briefly, each tick was placed into a microtube and mechanically disrupted with sterile scissors in 50 µl dna extraction buffer (10 mm tris/ hcl ph 8.0, 2 mm edta, 0.1 % sds, and iran j arthropod-borne dis, 2010, 4(2): 19–25 a barmaki et al.: study on presence of … 21 500 µg proteinase k ml–1). the sample was incubated for 2 h at 56 ° c, and then boiled at 100 °c for 10 min to inactivate proteinase k. after centrifugation, the supernatant was transferred to a fresh sterile microtube and purified by extracting twice with an equal volume of phenol/chloroform before use in pcr. 16s rrna gene pcr a conventional pcr was performed with primers designed to amplify the 16s ribosomal rna (16s rrna) gene of the borrelia spp. primers rec4 (5’-atg cta gaa act gca tga-3’) and rec9 (5’-tcgtctgagtccccatct-3’) were used as previously described (ras et al. 1996). theses primers are expected to yield a 523-bp pcr products regardless of borrelia species. pcr amplification were performed in a volume of 30 µl containing 3 µl 10xpcr buffer (containing 100 mm tris/hcl, 500 mm kcl, 15 mm mgcl2), 0.3 µl taq dna polymerase (5 u µl–1), 0.3 µl dntp mix (10 mm) (all from bioneer, s. korea), 17.4 µl deionized water, 5 µl dna template and 2 µl of each primer (10 µm). the cycling conditions for amplification involved 5 min denaturation at 95 °c, followed by 35 cycles of 94 °c for 60 s, 45 °c for 60 s and 72 °c for 120 s, and a final extension at 72 °c for 7 min. in parallel with each amplification, a positive control (previously extracted dna of standard b. persica from pasteur institute of iran) and two negative controls including previously extracted dna of standard b. microtti from pasteur institute of iran and distilled water were included. all the pcr products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and visualized under uv light. to minimize contamination, dna extraction, the reagent setup, amplification, and agarose gel electrophoresis were performed in separate rooms. rflp analysis in order to identify the species of borrelia spp. infecting the ticks, the positive pcr products were analyzed by rflp as described by oshaghi et al. (2010). previously extracted dna of standard b. persica was used as positive controls. for each positive sample, 10-15 µl amplified dna was digested at 65 °c overnight with endonuclease taqi (fermentas) according to the manufacturer's recommendations. electrophoresis was conducted in 2% agarose gel at 100 v for 5 min and then 80 v for 1 h. the gels were stained with ethidium bromide, and visualized under uv light. a 100 bp dna ladder marker (cinagen, fermentas) was used as a molecular size marker. rflp profile of b. persica was identified by digestion of 523-bp into 324 and 199-bp according to oshaghi et al. (2010). 16s rdna pcr products of b. microtti was used in parallel to compare the rflp profiles of both species. dna sequencing of pcr product in order to confirm the species of borrelia spp. infecting the ticks, a sample from those pcr products that showed unique rflp profile was selected for pcr-direct sequencing. the nucleotide sequences were determined by a dideoxynucleotide cycle sequencing method with an automated dna sequencer (seqlab, germany). the sequence obtained in the present study was compared with the previously published sequences deposited in genbank using the blast program from the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov/blast). the accession number of the 16s rrna gene sequence obtained in this study is eu914141. results tick prevalence and distribution a total of 1021 soft ticks of three species of ornithodouros tholozani (120: 11.75%), o. lahorensis (461: 45.15%) and argas persicus (440: 43.1%) were collected and tested against borrelia infection. soft ticks were more prevalent (67%) in infected areas than iran j arthropod-borne dis, 2010, 4(2): 19–25 a barmaki et al.: study on presence of … 22 none infected areas. the rate o. tholozani in infected areas was 29 times more than none infected areas. 97% of soft ticks in none infected areas were of o. tholozani. on average, number of females was twice as males. ornitodorous lahorensis with 45.15% was the most common species in the study area. among ten previously infected villages, o. tholozani specimens were found only in four villages of mehin, meshkin, andagh, and ghalat. in none previously infected villages, different species of ornitodorous spp. were present in ten out of 21 villages, though o. lahorensis with 97.9% was the most prevalent soft ticks. borrelia persica prevalence in ticks dna of b. persica was detected by a conventional pcr specifically targeting the 16s rrna gene. a total of 344 soft ticks including 95 o. tholozani, 205 o. lahorensis, and 44 a. persicus were selected based on the collection sites and individually examined by pcr against borrelia genome. the target region (16s rdna) was successfully amplified for 5.5% (19/344) of the investigated ticks. sixteen (16.7%) ticks of o. tholozani were infected with b. persica. three (1.3%) soft ticks of o. lahorensis were also positive for borrelia sp., but no infection was found in a. persicus. so, two borrelia species, b. persica and borrelia sp. (unknown) were identified by pcr-rflp: b. persica was the most frequently detected species (16/19, 84.2%). all of the infected ticks with b. persica collected in the sites previously tbrf was reported whereas 100% of ticks infected with borrelia sp. were found in none previously tbrf reported areas. infection rates of ticks with respect to the area of sample collection are shown in table 1. taqi rflp and sequence analysis of the amplified products from o. tholozani ticks specimens resulted in unique profile. on the basis of taqi rflp patterns of positive controls of b. persica the profiles obtained in this study consist of restriction pattern for b. persica species (fig. 1). fig. 1. pcr-rflp profile of 523bp of 16srdna gene of borrelia persica (no. 2) and b. microtti (no. 3) digested by taqi restriction enzyme (cinnagen). no. 1: molecular size marker (100 bp cinnagen), b. persica pcr was broken down into two fragments of 324 bp and 199 bp and b. microtti into three fragments of 205, 199, and 119 bp. 500 bp iran j arthropod-borne dis, 2010, 4(2): 19–25 a barmaki et al.: study on presence of … 23 table 1. pcr-based borrelia spp infection rate in o. tholozani and o. lahoriansis in takistan o. tholozani o. lahoriansis infected (%) infected (%) area studies no. tested b. pesrica b. sp no. tested b. pesrica b. sp infected 96 16 (16.6) 0.0 (0) 95 0.0 (0) 0.0 (0) clean 4 0.0 (0) 0.0 (0) 130 0.0 (0) 3(2.3) total 100 16 (16) 0.0 (0) 225 0.0 (0) 3(1.3) discussion in this study we confirmed the presence of b. persica in 16.7% of the soft tick o. tholozani in the previously reported tbrf areas of takistan district. this infection rate is higher than the prevalence of 3.7% in the same tick species that were previously collected in qazvin, neighbor district of takistan (aghighi et al. 2007). this is mainly attributable to sensitivity of pcr methods in comparison with microscopic following xenodiagnosis (rafinejad et al. 2010). in addition, in this study we could use the pcr technique for either dead or alive, and 4 different development stage of ticks. however, this prevalence is comparable to the overall prevalence average in iran and other countries. this rate was 3.7% in hamadan (ghaderi 2001), 8.8% in qazvin (aghighi et al. 2007), 20% in different areas of iran (rafiei and rak 1986), 36.6% in semnan (nekoui et al. 1999), and 2-40% in other parts of the world (assous and wilamowoski 2009). these differences in infection rates among localities could be attributable to geographical and seasonal variations of infected ticks or to different sampling approaches and examination methods. among these factors, the examination method is more critical since the classical method, which is mainly based on xenodiagnosis, is extremely laborious, slow, expensive, and requires preparation of the samples. also we should consider the fact that the density of b. persicai in field ticks is very low and hard to be detected by the classical methods. in takistan, people are involved with poultry and animal husbandry and there is close relationship between people and animals. also there is an abundant small wild animals such rodent and canine fauna, thus it can be suggested that b. persica or other borrelia spp. are maintained particularly in enzootic cycles involving the wild and domestic mammals in the region. the two soft ticks of o. lahorensis and a. persicus, which are mainly distributed in takistan, lack any b. persica spirochetes. in spite of high prevalence (41.15%) of o. lahorensis in the area, it was not infected with b. persica. instead, it was found infected with an unknown borrelia species. similarly, other investigators have already revealed infection of o. lahorensis with unknown borrelia in iran (arshi et al. 2002) and nablos in lebanon. the results of our research and previous studies on infection of o. lahorensis to borrelia sp needs further investigations to identify the spirochetes presence in this particular soft tick. argas persicus, with 43% prevalence, which are mainly distributed in takistan (43%), was not found infected with any kind of borrelia. these diversities in vectorial capacity of different tick species is attributable to the highly specific borrelia-tick interactions as each species of borrelia is only transmitted by one or a few closely related species of ticks. for example, o. tholozani is the main vector of b. persica, and o. erraticus is vector of b. iran j arthropod-borne dis, 2010, 4(2): 19–25 a barmaki et al.: study on presence of … 24 microtti (assmar et al. 2002, masoumi asl et al. 2009). in azerbaijan, o. verrucosus is a vector of b. caucasica, which causes a very severe tbrf. in iran and the countries of central asia of the former ussr, o. tartakowskyi is the vector of b. latyshewii, which causes a very mild rf without apparent clinical relapses (parola and raoult 2001). ornithodouros sonrai is recognized as the only vector of b. crocidurae causing human relapsing fever in west africa (vial et al. 2006). the mechanisms responsible for this strict species specificity in transmission of one species of spirochete by only one species of tick are not known (schwan and piesman 2002). in conclusion, pathogenic b. persica infection in o. tholozani ticks poses a potential health threat both to local residents and to tourists and animals in this area. it will be necessary to alert public health officials and clinicians about the existence of b. persicapositive in ticks in previously infected areas of takistan district to continue vector control measures. acknowledgments this work was supported by a grant from tehran university of medical sciences (tums) (grant no. 86-03-27-6268) to m. a. oshaghi. we thank from manager of health office of qazvin province and takistan health center. we also thank eng, taghi satvat, mrs s. charedar from tums for technical assistance. the authors declare that they have no conflicts of interest. references aghighi z, assmar m , piazak n, javadian e, 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(in persian). j arthropod-borne dis, december 2016, 10(4): 493–500 ma gorouhi et al.: current susceptibility … 493 http://jad.tums.ac.ir published online: october 04, 2016 original article current susceptibility status of anopheles stephensi (diptera: culicidae) to different imagicides in a malarious area, southeastern of iran mohammad amin gorouhi 1, *hassan vatandoost 1, *mohammad ali oshaghi 1, ahmad raeisi 2, ahmad ali enayati 3, hossein mirhendi 4, ahmad ali hanafi-bojd 1, mohammad reza abai 1, yaser salim-abadi 1,5, fatemeh rafi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2national malaria control department, cdc, ministry of health and medical education, iran 3school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 4department of parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 5school of health, rafsanjan university of medical sciences, rafsanjan, iran (received 22 nov 2014; accepted 10 agu 2015) abstract background: anopheles mosquitoes are an important group of arthropods due to their role in transmission of malaria. the present study was conducted for determination of susceptibility status of anopheles stephensi to different imagicides collected from malarious area in chabahar city, iran. methods: in the present study seven insecticides including: ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5% were tested based on who method. regression line was plotted for each insecticide using mortality of different exposure times. bioassay data were analyzed using probit software and the lethal time for 50% and 90% mortality (lt50 and lt90) values were calculated. results: the susceptibility levels of field strain of an. stephensi to the discriminative dose of different imagicides were determined 100, 98, 96, 89, 82 and 62% for etofenprox, permethrin, deltamethrin, lambdacyhalothrin, cyfluthrin and ddt, respectively. our finding indicated that an. stephensi is resistant to ddt, lambdacyhalothrin and cyfluthrin, and susceptible to etofenprox and permethrin and candidate of resistant to deltamethrin based on who criteria. conclusion: our findings indicated that an. stephensi is resistant to ddt and some pyrethroid insecticides which can be developed due to application of insecticides in health and agriculture. these results can provide a clue for future chemical control program in the study area. keywords: susceptibility test, anopheles stephensi, chabahar, pyrethroid resistance introduction mosquitoes as a big group of arthropods play an important role in transmission of many diseases to human such as malaria, filariasis, yellow fever, dengue fever (horsfall 1955, tabachnick 1991, service 2003, azarihamidian 2011). some species of anopheles mosquitoes are vectors of malaria in different parts of the world. for example, anopheles stephensi liston (diptera: culicidae) is the main malaria vector in eastern mediterranean region and south of asia continent (zahar 1974,vatandoost et al. 2006). in iran there are some species of malaria vectors including: an. stephensi, an. dthali, an. culicifacies, an. fluviatilis, an. superpictus s.l., an. sacharovi, an. maculipennis complex (naddaf et al. 2003, azari-hamidian 2011, mehravaran et al. 2011, oshaghi et al. 2011). *corresponding author: dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir, dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 493–500 ma gorouhi et al.: current susceptibility … 494 http://jad.tums.ac.ir published online: october 04, 2016 before initiating of national malaria control program in 1957, malaria cases were reported from most parts of iran, since then, due to implementing of many continuous interventions, malaria confined to south eastern parts of the country including sistan va baluchestan, hormozgan and southern parts of kerman provinces (edrissian 2006, vatandoost et al. 2011). for controlling of malaria, vector control is one the most important approach which focuses on chemical control of mosquitoes. up to now different group of insecticides including: organochlorines (ddt, dieldrin and bhc), organophosphates (pirimiphos-methyl, malathion), carbamates (propoxur) and pyrethroids (lambdacyhalothrin, delthamethrin) in different forms of application such as indoor residual spraying (irs), insecticide treated nets (itns) for adult stage and some organophosphates for larviciding were used in malariaous area of iran (salim abadi et al. 2010, hanafibojd et al. 2012, vatandoost and hanafibojd 2012). resistance of anopheles spp to ddt and pyrethroid insecticides were reported from different countries around the world like china, turkey, india, some countries of africa and latin america (kasap et al. 2000, hargreaves et al. 2003, syafruddin et al. 2010, lol et al. 2013, soltani et al. 2013, chang et al. 2014). in iran many researches have evaluated susceptibility status of malaria vectors against different insecticides (vatandoost and hanafi-bojd 2005, hanafi-bojd et al. 2006, vatandoost et al. 2006, vatandoost et al. 2011, vatandoost and hanafi-bojd 2012). approximately in all previous conducted studies on an. stephensi in iran, resistance to ddt and susceptibility to pyrethroids have been reported, but in 2012 first indication of resistance to pyrethroid compounds was reported from south eastern parts of the country (vatandoost and hanafi-bojd 2012). resistances to ddt, mainly in the adult stage of an. stephensi, have been widely distributed in middle-east and indian subcontinent causing operational problems for control programs (who 1985, who 1992). this study aims to monitor susceptibility status of main malaria vector, an. stephensi, to some insecticides in chabahar city, sistan va baluchestan province, iran. materials and methods study area this study was performed in chabahar seaport (25o 25/ n, 60 o 45/ e), sistan va baluchestan province of iran during april to june 2013 (fig. 1). mosquito sampling and rearing collected larvae from the study area were transferred to the insectary for rearing under standard conditions (temperature= 25–29o c, photo-period=12:12 hours (light: dark) and humidity=50–70%). emerged adult mosquitoes were fed with 10% aqueous sucrose solution. adult susceptibility test adult susceptibility tests were carried out according to the current world health organization method (who 2013). for each insecticide mortality rate in various times also were calculated and then regression line to each insecticide plotted using microsoft excel (version. 2013). insecticide impregnated papers the following insecticides impregnated papers were supplied according to who test procedure including: ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5%. mineral oil, and silicon oil impregnated papers were used for organochlorine insecticides and pyrethroids as control, respectively (who 1981, who 2013). j arthropod-borne dis, december 2016, 10(4): 493–500 ma gorouhi et al.: current susceptibility … 495 http://jad.tums.ac.ir published online: october 04, 2016 statistical analysis results were analyzed by using of probit program (finney 1971). in case of mortality, when the control mortality was between 5% to 20% it was corrected by abbott’s formula (abbott 1925). error bars for each mortality were calculated based on statistical method at α=5%. the lethal time for 50% and 90% mortality (lt50 and lt90) values and their 95% confidence interval also probit regression line parameters were determined with finney method and then the regression line of all insecticides were plotted using mi crosoft excel (version. 2013). results the results of susceptibility test for each insecticides are shown in tables 1,2. mortality rate and lethal time for 50% mortality (lt50) of different insecticides were calculated. our finding indicated that etofenprox, deltamethrin, lambdacyhalothrin, permethrin, cyfluthrin and ddt have the lowest to highest lt50 value respectively (fig. 2). fig. 1. the map of chabahar city representing rural districts, sistan and baluchistan province (study area), iran table 1. probit regression line parameters of anopheles stephensi exposed to different insecticides insecticide a b±se lt50 , 95% c.i. (second) lt90, 95% c.i. (second) x2(df) p value etofenprox 0.05% -2.68 1.33± 0.14 75 626 5.11 (3) >0.05104 957 138 1749 permethrin 0.75% -5.61 2.22±0.21 277 984 2.89 (2) >0.05335 1266 401 1775 cyfluthrin0.15% -3.79 1.3±0.12 656 5121 1.97(4) >0.05812 7805 1010 14160 j arthropod-borne dis, december 2016, 10(4): 493–500 ma gorouhi et al.: current susceptibility … 496 http://jad.tums.ac.ir published online: october 04, 2016 lambdacyhalothrin 0.05% -3.25 1.36±0.12 185 1414 0.04(2) >0.05246 2146 324 3791 deltamethrin 0.05% -2.69 1.22±0.15 101 1194 0.95(2) >0.05159 1785 221 3277 ddt 4% -4.47 2.5±0.33 2820 7560 6.99(3) >0.053240 10200 3840 16560 a= y-intercept, b= the slope of the line, se= standard error, ci= confidence interval, x2= heterogeneity about the regression line, df= degree of freedom, p> 0.05 =represent no heterogeneity in the population of tested mosquitos. table 2. mortality rate and susceptibility status of anopheles stephensi exposed to different insecticides chabahar, southeastern iran, 2013 insecticide mr±eb* resistance status** deltamethrin 96±3.8 rc lmbdacyhalothrin 89±2.8 r cyfluthrin 82±3.5 r permethrin 98±1 s etofenprox 100 s ddt 62±4.8 r control *mortality rate±error bar **r resistance, rc resistant candidate, t tolerance, s susceptible fig. 2. regression lines of anopheles stephensi exposed to different insecticides (field population), 2013 table 1. continued… j arthropod-borne dis, december 2016, 10(4): 493–500 ma gorouhi et al.: current susceptibility … 497 http://jad.tums.ac.ir published online: october 04, 2016 discussion in the current study seven insecticides including: ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and etofenprox 0.5% were used to determine susceptibility status of an. stephensi collected from chabahar city. based on who criteria that suggested (98– 100% mortality indicates susceptibility, 90– 97% mortality indicates resistance candidate (more investigation is needed) and less than 90% mortality suggests resistance (who 2013). results indicated that species is resistant to ddt, cyfluthrin and lambdacyhalothrin, however, susceptible to permethrin and etophenprox. the indication of resistant to deltamethrin at the early stages of evolution has also be documented. our findings reveal that an. stephensi is resistant to ddt which is in line with previous researches results that have been performed in our study area (vatandoost and hanafi-bojd 2012, fathian et al. 2015). majority of susceptibility tests which performed during the past decade in different malarious area revealed resistance to ddt in southern part of iran (borhani 2004, vatandoost et al. 2005, vatandoost et al. 2006) as well as in the most distribution area of an. stephensi in the world (rathor et al. 1980, thavaselvam et al. 1993, tikar et al. 2011, chang et al. 2014, singh et al. 2014). furthermore, there are many resistance reports to ddt in other species of anopheles mosquitoes from different part of the world (hemingway and ranson 2000, hemingway et al. 2002, zahirnia et al. 2002, lak et al. 2002, balkew et al. 2006 , raghavendra et al. 2010, tikar et al. 2011, vatandoost et al. 2011, nardini et al. 2013, wang et al. 2013). in the present study resistance to cyfluthrin and lambdacyhalothrin were indicated and these findings are in line with previous research results that have been conducted in the same area (vatandoost and hanafi-bojd 2012). on the other hand our finding about cyfluthrin susceptibility status is not in concordance with another research that has been performed previously in the same area by fathian et al. (2015) that showed this species is susceptible to cyfluthrin. (fathian et al. 2015). it may be due to different sampling localities. resistance of an. stephensi to pyrethroid compounds were reported from its different distribution regions, for instance in the study was performed by rathor et al. (2013) in punjab province of pakistan, resistance to three commonly used pyrethroids, permethrin, lambda-cyhalothrin, and deltamethrin were indicated from the majority of test localities (rathor et al. 2013). in the present study an. stephensi was susceptible to etophenprox and permethrin that these findings are in parallel with other previous conducted researches results (vatandoost et al. 2005). in the current study deltametthrin was indicated as resistant candidate so that more investigation is needed. molecular and biochemical assays for this species as a main malaria vector must be conducted for accurate evaluating of resistance status of pyrethroid insecticides specially those commonly used in malaria control program. conclusion in the present study an. stephensi was found resistant to ddt and some pyrethroid insecticides. this enhanced resistance status may be due to previous chemical control programs against malaria vectors, such as irs/itns or insecticide application in agriculture. however, more investigation for determination of resistance mechanisms is necessary. furthermore regular monitoring of resistance status by standard bioassay tests and other complementary methods especially in active foci of malaria transmission is suggested. j arthropod-borne dis, december 2016, 10(4): 493–500 ma gorouhi et al.: current susceptibility … 498 http://jad.tums.ac.ir published online: october 04, 2016 acknowledgements this article is a part of the first author’s dissertation for fulfillment of a phd degree in medical entomology and vector control from department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. this study was financially supported by the deputy for research, tehran university of medical sciences. authors are grateful for dr amini head of chabahar health center and all his staff for their kind collaboration. the authors declare that there is no conflict of interest. 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vector control. who tech rep ser 818. zahar a (1974) review of the ecology of malaria vectors in the who eastern mediterranean region. bull world health organ. 50(5): 427. zahirnia a, vatandoost h, nateghpour m, djavadian e (2002) insecticide resistance/ susceptibility monitoring in anopheles pulcherrimus (diptera: culicidae) in ghasreghand district, sistan and baluchistan province, iran. iran j public health. 31(1–2): 11–14. j arthropod-borne dis, june 2016, 10(2): 268–271 f demirel-kaya et al.: a case of extensive … 268 http://jad.tums.ac.ir published online: january 05, 2016 case report a case of extensive wound myiasis caused by lucilia sericata (diptera: calliphoridae) in a patient with maxillary sinus squamous cell carcinoma, in turkey *filiz demirel-kaya 1, ömer orkun 2, ayşe çakmak 2, a çağkan i̇nkaya 3, murat öcal 1, sibel erguven 1 1department of medical microbiology, hacettepe university faculty of medicine, ankara, turkey 2department of parasitology, ankara university faculty of veterinary medicine, ankara, turkey 3department of internal medicine, infectious diseases unit, hacettepe university faculty of medicine, ankara, turkey (received 8 apr 2014; accepted 10 aug 2014) abstract the larvae causing myiasis can lead extensive tissue destruction, invasion into deep tissues and secondary infections. poor hygiene, low socioeconomic condition and presence of open wounds are the most important predisposing factors. this case report describes destructive wound myiasis in a 58-year-old male patient diagnosed with maxillary sinus squamous cell carcinoma who lives in a rural area in ankara, turkey. approximately 200 larvae were collected and identified as lucilia sericata by morphological examination. myiasis should be considered especially when the patient has open extensive lesions such as malignant wounds. keywords: myiasis, squamous cell carcinoma, lucilia sericata introduction myiasis is defined as the parasitic infestation of tissues and organs in living vertebrates with dipterous larvae which at least for a certain period, feed on the host’s living or dead tissue, mucosa, liquid body substance or digested food (hope 1840, zumpt 1963). myiasis is seen more typically in tropical and subtropical countries. low socioeconomic status, poor sanitation, advanced age and vascular disorders are the most important risk factors for myiasis (francesconi and lupi 2012). the clinical manifestation of myiasis depends on the genus and species of fly, the degree of invasion by larvae, the stage of the fly, number of larvae and the site of invasion. skin involvement is the most seen clinical manifestation. cutaneous myiasis includes furuncular, migratory and wound myiasis (zumpt 1963, francesconi and lupi 2012). a broad range of flies can cause myiasis in humans, especially members of oestridae, calliphoridae and sarcophagidae families are reported (scholl 2002). lucilia sericata flies belonging to the calliphoridae family are very common flies in the temperate zone of northern hemisphere. they can cause myiasis in humans and domestic animals (lane and crosskey 1993). diagnosis of myiasis depends on the demonstration of larvae on the patient’s tissues or organs. it is important to make the correct identification of the larvae for planning the treatment and promoting preventing measures (francesconi and lupi 2012). case presentation in this report, a case of maxillary sinus carcinoma complicated with myiasis was presented. a 58-year-old male patient living *corresponding author: dr filiz demirel kaya, email: filiz.kaya@hacettepe.edu.tr j arthropod-borne dis, june 2016, 10(2): 268–271 f demirel-kaya et al.: a case of extensive … 269 http://jad.tums.ac.ir published online: january 05, 2016 in a rural area with low socioeconomic condition, admitted to the hacettepe university department of emergency medicine, ankara, turkey in july 2013, because of a suppurated, massive destructive lesion containing numerous larvae covering left half of his face. the patient had poor general condition. he was diagnosed maxillary squamous cell carcinoma five years ago, and catatonic schizophrenia. he had been operated four times because of his tumour before. the tumoural lesion of the patient was covered the left maxillary-orbital region and nose and there were a great number of larvae moving in his tumour lesion (fig. 1). approximately 200 larvae were removed from his wound by mechanical and the lesion was rinsed using hydrogen peroxide solution. some of the larvae delivered to the parasitology laboratory. after morphological examination the larvae washed in distilled water and killed in alcohol 70 % and they were taken to the ankara university veterinary faculty department of parasitology for identification. the morphological characters of cephalo-pharyngeal skeleton, anterior spiracles and slits of the posterior spiracles were examined. measured length of 10–15 larvae were about 12 to 16 mm (fig. 2). the anterior spiracles with 7–9 lobes, the posterior spiracles with narrow and complete peritreme, straight and parallel 3 slits and cephalopharyngeal skeleton without accessory oral sclerite were observed by microscopically (fig. 3). we also observed inner, median, outer and anal tubercles on the posterior segment of the larvae and the distance between the tubercles on the top side of the posterior cavity was equal. the larvae were identified as 3rd instar of l. sericata. because of all the larvae were dead we could not rear the larvae to adult stage. the patient’s white blood cell count and c-reactive protein values were found high so meropeneme therapy was started. he had severe protein-energy malnutrition because of unabling to eat and a dietary supplement was intended. the patient was taken to the department of otorhinolaryngology for palliative operation for his tumour. excision of mass, total left maxillectomy, total parotidectomy, partial mandibulectomy and rinectomy was performed. fig. 1. extensive destructive lesion of the maxillaryorbital region of the face with a lot of larvae inside fig. 2. macroscopic aspect of a larva j arthropod-borne dis, june 2016, 10(2): 268–271 f demirel-kaya et al.: a case of extensive … 270 http://jad.tums.ac.ir published online: january 05, 2016 fig. 3. a) cephalopharyngeal skeleton, b) anterior spiracle, c-d) posterior spiracle conclusion lucilia sericata flies belong to calliphoridae family which can cause myiasis in humans and domestic animals. the adult flies of the l. sericata species are metallic green and they are known as greenbottles. females can deposit their eggs in neglected and suppurating wounds. the larvae hatch in a few days and feed on the tissues and exudates (lane and crosskey 1993). in humans, first case of myiasis due to l. sericata was reported in 1826 by magen from mouth, eyes and paranasal sinuses of a patient. in literature there are several reports of cases with myiasis by l. sericata. talari et al. (2004) reported a wound myiasis in a heroin addicted patient. kılıç et al. (2011) presented a postoperative wound myiasis caused by l. sericata in a woman who had breast cancer surgery. myiasis is one of the most undesirable conditions in cancer patients with malignant cutaneous wounds and a rare complication of squamous cell carcinoma. sesterhenn et al. (2009) reported a case of wound myiasis in an extensive skin metastasis of oral squamous cell carcinoma and they identified the larvae as lucilia sp. gabriel et al. (2008) presented a case of myiasis in a patient with extensive head and neck squamous cell carcinoma. pessoa et al. (2011) reported myiasis infestation in a patient with invasive oral squamous cell carcinoma with ulcerated necrotic wound. in all of these patients, open neglected wounds and poor hygiene are the most important predisposing factors leading the infestation with larvae. living in a rural area, advanced age and low socioeconomic conditions are also risk factors (demirel-kaya et al. 2014). psychiatric disorder, low socio-economic level and poor hygiene are the most important predisposing factors that lead to the development of myiasis in this patient as well as the presence of an open, extensive neglected wound. it should be kept in mind that the cancer patients with open wounds may develop myiasis especially in the summer months and larvae can cause progressive wound infection. sometimes the presence of larvae is useful for the wounds. maggot therapy is the medically use of sterile fly larvae in treatment of resistant wounds. larvae can debride and clean wounds and promote healing. l. sericata is usually used for the maggot therapy (falch et al. 2009). it has been also demonstrated that some of the enzymes and substances secreted by 2nd and 3rd instar of the l. sericata larvae have bacteriostatic and bactericidal effects and by this way the larvae can disinfect the lesions. (polat et al. 2012). acknowledgements the authors declare that there is no conflict of interest. references demirel kaya f, orkun o, cakmak a, inkaya ac, erguven s (2014) human cutanous myiasis caused by larvae of j arthropod-borne dis, june 2016, 10(2): 268–271 f demirel-kaya et al.: a case of extensive … 271 http://jad.tums.ac.ir published online: january 05, 2016 sarcophaga spp. in a diabetic patient. mikrobiyol bul. 48(2): 356–361. falch bm, de weerd l, sundsfjord a (2009) maggot therapy in wound management. tidsskr nor laegeforen. 129(18): 1864– 1867. francesconi f, lupi o (2012) myiasis. clin microbiol rev. 25(1): 79–105. gabriel jg, marinho sa, verli fd, krause rg, yurgel ls, cherubini k (2008) extensive myiasis infestation over a squamous cell carcinoma in the face. med oral patol oral cir bucal. 13(1): e9–11. hope fw (1840) on insects and their larvae occasionally found in the human body. trans r soc entomol. 2: 256–271. kılıç k, arslan mö, kara m (2011) a postoperative wound myiasis caused by lucilia sericata (diptera: calliphoridae) in a woman in kars. turkiye parazitol derg. 35: 43–46. lane rp, crosskey rw (1993) diptera causing myiasis in man in: lane rp and crosskey rw (eds): medical insects and arachnids, chapman and hall, london, pp. 429–451. pessoa l, galvao v (2011) myiasis infestation in advanced oral squamous cell carcinoma. bmj case rep. doi:10.1136/ bcr.04.2011.4124. polat e, cakan h, aslan m, sirekbasan s, kutlubay z, ipek t, ozbilgin a (2012) detection of anti-leishmanial effect of the lucilia sericata larval secretions in vitro and in vivo on leishmania tropica: first work. exp parasitol. 132(2): 129– 134. scholl pj, catts ep, mullen gr (2002) myiasis.in: mullen gr and durden la (eds): med vet entomol. 2nd ed. elsevier, usa, pp. 309–338. sesterhenn am, pfutzner w, braulke dm, wiegand s, werner ja, taubert a (2009) cutaneous manifestation of myiasis in malignant wounds of the head and neck. eur j dermatol. 19(1): 64–68. talari sa, sadr f, doroodgar a, talari mr, gharabagh as (2004) wound myiasis caused by lucilia sericata. arch iranian med. 7(2): 128–129. zumpt f (1963) the problem of intestinal myiasis in humans. s afr med j. 37: 305–307. j arthropod-borne dis, june 2017, 11(2): 204–210 r khaghani et al.: efficacy of aloe vera … 204 http://jad.tums.ac.ir published online: may 27, 2017 original article efficacy of aloe vera cream in the treatment of paederus dermatitis in mice ramin khaghani 1, *iraj mirzaii-dizgah 2, mostafa ghasemi 1 1department of medical parasitology, school of medicine, aja university of medical sciences, tehran, iran 2department of physiology, school of medicine, aja university of medical sciences, tehran, iran (received 2 aug 2014; accepted 7 may 2016) abstract background: dermatitis caused by paederus beetle involves many people around the world, especially iran. the symptoms include redness, itching and severe irritation. this study evaluated the effectiveness of the aloe vera cream on the treatment of dermatitis caused by paederus beetles. methods: forty male 6–8 weeks balb/c mice were randomly divided into four groups of 10 mice. after removing the mice’s back hair, the backs of mice were marked by a circle with a diameter of 3 mm. the paederus beetles were collected from babol in mazandaran province, northern iran and transferred to the animal lab of aja university of medical sciences, tehran, iran. the end of abdominal segment paederus was cut with scissors and hemolymph content was pushed by forceps on the circle. only hemolymph of one paederus applied to the back of each mouse. groups 1, 2, 3 and 4 were treated with the base (vehicle), dexamethasone 0.1%, aloe vera 0.5% and aloe vera 2% creams respectively. after 2 days, dermatitis appeared. then the mentioned creams were applied on the mice once a day. the wound area was measured every day. dermatitis surface area under curve (auc) of each mouse was calculated for 17 days after induction of dermatitis. statistical analysis of anova was used. results: application of aloe vera 0.5% and 2% significantly reduced the healing duration and dermatitis area in comparison with the vehicle and dexamethasone cream (p< 0.05). but dexamethasone had no significant effect on the healing of dermatitis as compared to vehicle. conclusion: aloe vera may clinically effective in the treatment of paederus dermatitis. keywords: paederus, dermatitis, aloe vera introduction the genus paederus is related to family staphyllinidae, order coleoptae, class insecta and consists of over 622 species distributed all around the world (kerdel-vegas and goihman-yahr 1966, zargari et al. 2003). adult rove beetles are between 7–10mm long and 0.5mm wide. these beetles have a black head, a red thorax, upper abdomen, elytral and a lower abdomen (frank and kanamitsu 1987, george and hart 1990). many species of the rove beetle genus paederus contain the hemolymph toxin named pederin (frank and kanamitsu 1987). this substance causes itching and skin lesions in humans. with their hemolymph penetrated into the skin, these re sults in an affliction called dermatitis linearis. the severe cytotoxic effects of pederin are based on blocking of protein synthesis and inhibition of mitosis in eukaryotic cells (brega et al. 1968). this dermatitis is more frequent on the uncovered parts of the body like face, neck and hands. the acute lesions become crusted within a few days and heal completely in about 2 to 3 weeks and may leave a hyperpigmentation (kerdel-vegas and goihmanyahr 1966, gelmetti and grimalt 1993). recently, the production of pederin is depended on the activities of an endosymbiont (pseudomonas species) within paederus. *corresponding author: dr iraj mirzaii-dizgah, email: emirzaii@razi.tums.ac.ir j arthropod-borne dis, june 2017, 11(2): 204–210 r khaghani et al.: efficacy of aloe vera … 205 http://jad.tums.ac.ir published online: may 27, 2017 the manufacture of pederin is mostly confined to adult females. larva and males store pederin in very small quantities compared to females (piel 2002). aloe vera (family: liliaceae) has been used in traditional medicine since ancient times. it is one of the most useful herbs in the world and the medicinal part is the leaves. aloe vera leaves contain various essential nutrients for the body, such as amino acids, b family vitamins, etc. it also has pharmacological effects like antioxidant, wound healing, antifungal, antibacterial and immunomodulatory effects (rodriguez-bigas et al. 1988, west and zhu 2003, hu et al. 2005, tai-nin chow et al. 2005, olaleye et al. 2005, rosca-casian et al. 2007, habeeb et al. 2007, maenthaisong et al. 2007). aloe vera also has anti-inflammatory properties (reynolds and dweck 1999, vogler and ernst 1999). compounds existing in the inner gel including salicylates, magnesium lactate, bradykinin, thromboxane inhibitors, sterols and a beta linked acetyl mannan (acemannan) have demonstrated anti-inflammatory activity (vázquez et al. 1996, talmadge et al. 2004). the mucilaginous polysaccharides in the pulp of aloe vera leaf are the major ingredient that induces regeneration. emodin as a derivative of anthraquinones produced by pericyclic cells on the surface of aloe leavescan also promote the healing of rats’ excisional wounds by stimulating regeneration of cells (eshun et al. 2004, tang et al. 2007). these researches support the claim that the healing properties of aloe are essentially result of the synergistic mode of action of many bioactive ingredients of aloe leaves (dagne et al. 2000). the purpose of the present study was to determine the efficacy of aloe vera in the treatment of dermatitis caused by paederus beetles. materials and methods animals forty male balb/c mice (6–8 weeks old) were obtained from pasteur institute of iran. animals had free access to food and water and were kept at room temperature and with an artificial light cycle of 12-h light and 12-h dark. all animal care and procedures and handling were performed under supervision of animal care and use committee of the aja university of medical sciences. chemicals dexamethasone acetate (cas number: 464-92-6) was purchased from chemos gmbh (regenstauf, germany). all other chemicals used in this study were of analytical grade and obtained from merck (darmstadt, germany). aloe vera the pure spray-dried aloe vera powder was purchased from anamis company (alborz, iran). this product consists of the inner gel of the leaves of aloe vera. paederus collection and identification the paederus beetles were collected from babol in mazandaran province of iran and transferred to the animal lab of aja university of medical sciences, tehran, iran. identification was made by specialists and by using the key and illustrations of coiffait (1982). paederus beetles were identified as paederus fuscipes that together with pa. kalalovae are the predominant species along the southern shores of the caspian sea (nikbakhtzadeh and tirgari 2008). dermatitis induction and treatments forty male 6–8 weeks balb/c mice were randomly divided into four groups (10 mice in each group). each mouse was shaved on the back (2cm× 2cm) and the skin was gently wiped with distilled water. the backs of mice were marked by a circle with a diameter of 3 millimeters. the end of abdominal segment of adult female paederus beetles was cut with scissors and hemolymph content was j arthropod-borne dis, june 2017, 11(2): 204–210 r khaghani et al.: efficacy of aloe vera … 206 http://jad.tums.ac.ir published online: may 27, 2017 push by forceps on the circle. only hemolymph of one paederus applied to the back of each mouse. after 48 h the dermatitis appeared clearly (fig. 1) and its surface area was measured and recorded as per square millimeter (mm2). groups 1, 2, 3 and 4 were treated with the base cream (as vehicle group), dexamethasone 0.1 % cream, aloe vera 0.5% cream and aloe vera 2% cream respectively. drug doses had been shown in a previous study to be adequate to elicit a response (babaee et al. 2012) preparation of the creams dexamethasone and aloe vera powder were formulated in an aqueous cream containing polysorbate 80 (5w/v), isopropyl myristate, stearyl alcohol (20v/v), liquid white paraffin (15v/v), purified water (100ml), methylparaben (0.18w/w) and propylparaben (0.02w/w). preparation process carried out under conditions in which dexamethasone and aloe vera powder were not heated. the creams contained aloe vera powder, dexamethasone cream and base cream were prepared according to similar protocol, under the same conditions and with the same chemicals. dermatitis surface area measurement to determine the surface areas of dermatitises, the mice were observed every day. a photograph was taken of each mouse’s dermatitis with a single lens, 14.1mp digital camera (sx30 is, canon, japan). for calibrating the magnification of photos, camera’s lens was held at a distance of 20cm from the dermatitis and a caliper was placed at the level of mice’s back. the wound area was measured per mm2 every day by analysis of images using image j (image processing and analysis in java, us national institutes of health) and adobe photoshop cs5 software. dermatitis surface area under curve (auc) of each mouse was calculated for 17d after induction of dermatitis (dermatitis surface in mm2 × time between observations in day). statistical analysis values are expressed as mean± sem. oneway analysis of variance, anova (post hoc tukey test) was used to determine significant differences among groups and p< 0.05 was considered a significant difference. statistical analysis performed using spss software (version 15, spss inc., chicago, il, usa). results in the second day after dermatitis induction, there was no significant difference in the dermatitis surface area values among groups. the mean ( sem) dermatitis surface areas were 19.610.43mm2, 19.890.43mm2, 19.34 0.46mm2 and 19.50.51mm2 for group 1 to 4, respectively. a one-way anova indicated that the means auc of the dermatitis surface was significantly different among groups (p< 0.05, fig. 2). post-hoc analysis showed that aloe vera treatment dose dependently decreased the auc of the dermatitis surface as compared to vehicle and dexamethasone treatments (p< 0.05). there was no significant difference between vehicle and dexamethasone treatments and also between two doses of aloe vera creams (p> 0.05). aloe vera creams (0.5% and 2%) significantly reduced the healing period of dermatitis in comparison with dexamethasone and base creams (groups 1 and 2) (p< 0.05, fig. 3). but there was no significant difference between two aloe vera creams. dexamethasone cream and vehicle had no significant difference in reduction of healing duration of dermatitis (fig. 3). healing time ranged from 14 to 17 days in vehicle group, 15 to 17 days in dexamethasone 1% treated group, 13 to 16 days in aloe vera 0.5% treated group and 12 to 14 days in aloe vera 2% treated group. j arthropod-borne dis, june 2017, 11(2): 204–210 r khaghani et al.: efficacy of aloe vera … 207 http://jad.tums.ac.ir published online: may 27, 2017 fig. 1. dermatitis induced by paederus hemolymph on the back of mouse. *# *# 0 200 1 a u c (m m 2 .d ay ) dexamethasone 0.1% vehicle aloe vera 0.5% aloe vera 2% fig. 2. effect of treatment with base cream (vehicle), dexamethasone 1% or aloe vera cream 0.5 and 2% on dermatitis surface changes for 17 day observations (as indicated by area under curve (auc)) following exposure to paederus’ hemolymph in balb/c mice. data are expressed as mean  sem. *and # different from vehicle and dexamethasone 0.1% treated, respectively, p< 0.05. *# *# 0 3 6 9 12 15 18 1 h ea lin g pe rio d (d ay ) dexamethasone 0.1% vehicle aloe vera 0.5% aloe vera 2% fig. 3. effect of treatment with base cream (vehicle), dexamethasone 1% or aloe vera cream 0.5 and 2% on the days passed for completing healing induced by paederus’ hemolymph in balb/c mice. data are expressed as mean  sem. *and # different from vehicle treated (vehicle) and dexamethasone 0.1 % treated, respectively, p< 0.05. discussion dermatitis caused by paederus beetle involves many people around the world, especially in iran. the symptoms include redness, itching and severe irritation. in this study the efficacy of the aloe vera cream on the treatment of dermatitis caused by paederus beetles was investigated. aloe vera creams (0.5 and 2 percent) decreased the healing period of dermatitis and auc of the dermatitis surface. thus it seems that aloe vera creams (0.5 and 2 percent) have capability to promote dermatitis healing more effectively than dexamethasone and base creams. up to now, numerous studies have been performed on the effects of aloe vera. although there are many studies that have shown the efficacy of different aloe vera preparation on various dermatologic disorders such as fungal and bacterial infections, incision and burn wounds, skin inflammations and many others (rodriguez-bigas et al. 1988, vázquez et al. 1996, reynolds and dweck 1999, vogler and ernst 1999, yagi et al. 2002, rosca-casian et al. 2007, maenthaisong et al. 2007), but its effect on dermatitis linearis caused by pederin existing in paederus’ hemolymph was unknown. “aloe vera mouthwash was effective in healing of the wound and reducing the inflammation of the mucous membrane of the mouth” (mansour et al. 2013). the topical application of aloe vera improves the total quality of life score in patients with oral lichen planus (salazar-sánchez et al. 2010). furthermore, its gel has been effective in healing of the first and second degree burning wounds without any side effects (maenthaisong et al. 2007) and also in cesarean wound healing (tarameshloo et al. 2012, molazem et al. 2014). aloe vera 2% oral gel is effective in decreasing the recurrent aphthous stomatitis patients' pain score and wound size and decreases the aphthous wound healing period too (babaee j arthropod-borne dis, june 2017, 11(2): 204–210 r khaghani et al.: efficacy of aloe vera … 208 http://jad.tums.ac.ir published online: may 27, 2017 et al. 2012). aloe vera reduces inflammatory cytokines (such as il-18), inf-α neutrophil chemoattractant, and malondialdehyde as indicator of oxidative stress (werawatganon et al. 2014). many of the biological activities of aloe vera such as immunostimulation, anti-inflammatory effects, wound healing, promotion of radiation damage repair, anti-bacterial, antiviral, anti-fungal, anti-diabetic and anti-neoplastic activities, and stimulation of hematopoiesis and anti-oxidant effects have been attributed to the polysaccharides contained in the gel of the leaves. these biological activities should be assigned to a synergistic action of the compounds contained therein rather than a single chemical substance (hamman 2008). results of our study showed that there was no significant difference between dexamethasone cream and vehicle in reducing the dermatitis healing duration. this is in agreement with qadir et al. report (2006) that combination of topical steroids and oral antibiotics have better effect than topical steroids alone on treatment of paederus dermatitis in sierra leone patients. cortisone or antihistamine preparations have no beneficial effect (deneys and zumpt 1963). as dexamethasone is 25 times more potent than cortisol in its glucocorticoid effect, it seems that due to anti-inflammatory and immunosuppressant effects of glucocorticoids, without antibiotic medication, these medications can be even harmful by favoring secondary infections. conclusion aloe vera seems to be clinically effective in the treatment of paederus dermatitis. it may be considered as an alternative or supplementary medicine for patients with this disease. further investigation on human and prolonged follow-up period is proposed in order to confirm the efficacy of aloe vera in the treatment of paederus dermatitis in human. acknowledgments we gratefully 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66 short communication hard ticks on domestic ruminants and their seasonal population dynamics in yazd province, iran y salim abadi1, *z telmadarraiy 1, h vatandoost 1, s chinikar 2, ma oshaghi1, m moradi 2, e mirabzadeh ardakan 2, s hekmat 2, a nasiri 1 1department of medical entomology, school of public health, tehran university of medical sciences, iran 2pasteur institute of iran, tehran, iran (received 9 oct 2009; accepted 19 may 2010) abstract background: ticks are the main vectors for transmission of different pathogens to human and animals. this survey was performed to find out distribution of ticks, which infested the domestic ruminants in yazd province, central iran during year 2008-2009. methods: a total number of 30 villages from both mountainous (20%) and plateau (80%) regions of the province were selected randomly. ticks were colleted from the body of infested animals and transported to the laboratory of medical entomology, school of public health, tehran university of medical sciences and then were identified to space level using valid identification key. results: a total of 583 hard ticks were collected. the ticks were classified into three genera and 7 species including: hyalomma dromedarii (55.92%), hy. marginatum (13.20%), hy. anatolicum (9.78%), hy. detritum (4.98%), hy. asiaticum (3.94%), rhipicephalus sanguineus (11.84%), and dermacentor marginatus (0.34%). the highest seasonal activities occurred in summer. the prevalence of the ixodidae ticks was more evident in plateaus area in yazd province. among the hosts including: cow, goat, sheep and camel, the ticks that collected from camel was more prevalent. the ratio of male was more than female ticks. hyalomma. dromedarii was the predominant tick species and accounted for 55.92% of the ticks. conclusion: some of the collected ticks may play an important role for transmission of vector borne disease to human; therefore, the results of this study will provide a clue for vectors of tick-borne diseases in the region for local authorities for implementation of disease control. keywords: ixodidae, ticks, iran introduction there are several works on biology, distribution and systematics of hard and soft ticks in iran. (brumpt 1935, deply 1936, 1938, baltazard et al. 1952, abbassian lintzen 1960, maghami 1968, mazlumi 1968, janbakhsh and ardelan 1970, filopova et al. 1976, robinson and spradling 2006, aghighi et al. 2007, telmadarraiy et al. 2007). ixodid ticks are parasitizing wild as well as domestic animals such as sheep and goat in iran and maintaining natural foci of many hazardous diseases for human (hoogstraal and wassef 1979, hoogstraal and valdez 1980). ecological aspects of various species of ticks encountering domestic animals in north west of iran also reported (rahbari 1995). the distribution of soft ticks, argasidae, from human dwellings, poultries, and animal shelters in hamadan province was reported (vatandoost et al. 2003). fauna of hard and soft ticks (families: ixodidae and argasidae) in west azerbaijan province highlighted (telmadarraiy et al. 2004). there are also some reports on distribution of tick fauna in iran (nabian and rahbari 2008, rahbari et *corresponding author: dr zakkyeh telmadarraiy, email: ztelma@yahoo.co.in iranian j arthropod-borne dis, 2010, 4(1): 66–71 y salim abadi et al.: hard ticks on domestic… 67 al. 2008). seasonal activity of ticks and their importance in tick-borne infectious diseases in west azerbaijan has been reported (salari lak et al. 2008). different species of ticks play important role for transmission of the disease through the country. tick-borne relapsing fever is an acute febrile and endemic disease in iran. during 1997–2006, a total of 1415 cases have been reported from the entire country (arshi et al. 2002, masumi asl et al. 2009). crimean-congo haemorrhagic fever (cchf) is a viral haemorrhagic fever also considered as a public health problem in iran. the virus is found in the cattle and ticks (telmadarraiy et al. 2010). the objective of this study was to determine the species and distribution of ticks infesting domestic ruminants in yazd province of iran. materials and methods study area yazd is located in center of iran. in the study area the total numbers of 30 villages were selected randomly and survey was conducted in these villages. tick collection tick collections were carried out on domestic ruminants. ticks were collected from sheep, cow, goat, and camel. the collected specimens were transferred into the holding tubes and counted, then were identified by morphological characteristics using the key identification guide of kaiser and hoogstraal (1963). results a total of 583 ticks were collected from 30 villages. they were collected from, sheep, goat, cattle, and camel. all of the ticks belonged to the family ixodidae. the identified tick specimens belonged to seven species, including three genera of hyalomma, dermacentor and rhipicephalus. from which 82.84% comprise the hylaomma species. hylaomma dromedarii was the most abundant of the total ticks. dynamic of population of tick revealed that they were more prevalent during summer and spring. the figure of density and activity of ticks in winter were low, i.e.; 12.69% of the total ticks collected (table 1). the occurrence of hard ticks was more noticeable in plateau area (table 2). table 3 shows frequency of different hard ticks’ species on various hosts. the camel was found the most appropriate host for ticks. the relative frequencies of sex of the tick were 57% male and 34% female. table 1. numbers of hard ticks in different seasons collected in yazd during 2008–2009 seasons species spring summer autumn winter total percentage hy. dromedarii 98 110 53 65 326 55.92 hy . marginatum 31 36 8 2 77 13.20 r. sanguineus 59 10 0 0 69 11.84 hy. anatolicum 6 41 7 3 57 9.78 hy. detritum 0 10 19 0 29 4.98 hy. asiaticum 8 6 5 4 23 3.94 d. marginatus 0 0 2 0 2 0.34 total 202 213 94 74 583 100 iranian j arthropod-borne dis, 2010, 4(1): 66–71 y salim abadi et al.: hard ticks on domestic… 68 table 2. topographical preference of hard ticks collected in yazd, 2008–2009 mountain plateau species number percentage number percentage hy. dromedarii 0 0 326 55.92 hy . marginatum 1 0.17 76 13.03 r. sanguineus 0 0 69 11.84 hy. anatolicum 1 0.17 56 9.61 hy . detritum 0 0 29 4.98 hy . asiaticum 0 0 23 3.94 d. marginatus 2 .34 0 0 total 4 0.68 579 99.32 table 3. the host of collected ticks in yazd during 2008–2009 host species cow camel sheep goat hy. dromedarii 17 306 3 0 hy. marginatum 34 12 31 0 r. sanguineus 0 0 59 10 hy. anatolicum 32 9 12 4 hy. detritum 8 0 21 0 hy. asiaticum 14 3 6 0 d. marginatus 0 0 0 2 total 105 330 132 16 discussion the findings of the current study show the occurrence of seven species of hard ticks in yazd province. the population frequencies of the species of genus hyalomma were higher than the others. hylaomma. dromedarii was the most frequent species. the host preference of main hard ticks was camel, sheep, cow, and goat, respectively. sex ratio of ticks were 57% male and remaining female. the occurrence of hard ticks was more noticeable in plateaus and this attributed the geographical condition of yazd province which is located in the plateau. the hard ticks were more prevalent during summer and spring respectively. in parallel to our study, results of tick fauna in lorestan province revealed that genera hylaomma, was active during the summer (tavakoli 1997). study in west azerbaijan province exhibited seven species of hard ticks of rh. bursa, hy. aegyptium, hy. schulzei, boophilus annulatus, d. niveus, haemaphysalis. sulcata, h. inermis (telmadarraiy et al. 2004) which were not found in our study. seven species of ticks including hy. marginatum, hy. detritum detritum, hy. anatolicum anatolicum, rh. bursa, h. sulcata, d. marginatus and b. annulatus have been reported form east azerbaijan (piazak 1991). nabian et al (2007) emphasized that hy. marginatum occurred as a dominant tick in the north of iran, they described thirteen hard tick species of hy. anatolicum anatolicum, hy. marginatum, hy. detritum, h. punctata, h. parva, h. concinna, h. choldokovsky, ixodes ricinus, rh. sanguineus, rh. bursa, b. annulatus, d. niveus, and d. marginatus. result of the study in west azerbaijan revealed occurrence of several species of ticks including h. inermis, h. punctata, h. sulcata, h. numidiana, h. concinna, hy. marginatum, hy. anatolicum, hy. detritum, hy. dromedarii, hy. asiaticum, hy. schulzei, h. aegyptium, rh. bursa, rh. sangiuneus, d. marginatus, b. annulatus, ornithodoros lahorensis, and argas persicus. frequency of ticks during different seasons was different (salari lak et al. 2008). results of meshkinshahr district reported the most frequency of the genera hyalomma and iranian j arthropod-borne dis, 2010, 4(1): 66–71 y salim abadi et al.: hard ticks on domestic… 69 rhipicephalus, the seasonal activity of ticks was in spring and summer (telmadarraiy et al. 2009). ticks collected from sheep in abdanan township also confirm the high prevalence of genus hyalomma (nasiri et al. 2009). in ethiopia, camel were infested with hy. dromedarii (zeleke and bekele 2004). nabian et al. (2009) found seven hyalomma species on goat, sheep, cattle and camels in different zoogeographical zones of iran including: hy. anatolicum, hy. excavatum, hy. asiaticum, hy. marginatum, hy. detritum, h. schulzei and hy. dromedarii. the geographical distribution and ecological preferences of haemaphysalis in domestic animals in iran were studied (rahbari et al. 2007). they found seven species of haemaphysalis on cattle, sheep and goats. based on our findings, it is concluded that hy. dromedarii and hy. marginatum are dominant ticks species in this area and camel was the most suitable host. the species of hy. dromedarii distributed from north africa to the south as far as senegal, mali, chad, sudan, and kenya; canary islands. turkey (eastern), palestine, syria, iraq, saudi arabia, yemen, oman, armenia, azerbaijan, iran, afghanistan, pakistan, kirghizia (fergana valley), uzbekistan, turkmenistan, tajikistan, india, and china (xingjian) (hoogstraal 1956). adults parasitize livestock, preferring camels. life cycle of this tick can be one-host, two-host, or three-host. immature ticks feed on small or large mammals, dependant upon life-cycle. the distribution of hy. marginatum contains morocco, algeria, tunisia, northwestern libya; portugal, spain, france (extreme southern), italy, former yugoslavia (bosnia, herzegovina, croatia, macedonia, montenegro, and serbia), albania, greece, cyprus, bulgaria, romania, moldova, ukraine, and russia to the north as far as rostov and volgograd oblasts; turkey, syria, palestine, egypt (sinai), iraq, iran, georgia, armenia, azerbaijan, kazakhstan, tutrkmenistan, kirghizia, uzbekistan, tajikistan, afghanistan, pakistan, india, and china. principal hosts of adults are all kinds of livestock. immature ticks feed on birds, hares, and hedgehogs. life cycle is two-host. in conclusion, hyalomma genus that is the principal vector of crimea-congo hemorrhagic fever (cchf) in the world, was found as the highest genus in yazd province, and prevention measures should be considered to reduce its population in order to prevent cchf epidemics. acknowledgments great appreciation to dr aa hadadzadeh, the manager of veterinary organization of yazd province and dr h poormirzaei, engineer n salim for their kind collaboration for conducting this study and also dr. hadian the manager of veterinary office of tabas county. we would like to thank due to collaboration of all the staff of the veterinary offices of yazd city. the authors declare that they have no conflicts of interest. this study was supported by tehran university of medical sciences grant no. 10446. references abbassian-lintzen r (1960) a preliminary list of ticks (acarina: ixodidae) occurring in iran, and their distributional data. acarologia. 2(1): 43–61. aghighi z, assmar m, piazak n, javadian e, seyedi rashti ma, kia eb, rassi y, vatandoost h (2007). distribution of soft ticks and their natural infection with borrelia in a focus of relapsing fever in iran. iranian j 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[msc dissertation]. tarbiat modarres university, iran. telmadarraiy z, bahrami a,vatandoost h (2004) a survey on sauna of ticks in west azerbaijan province, iran. iranian j publ health. 33(4): 65–69. telmadarraiy z, nasirian h, vatandoost h, abuolhassani m, tavakoli m, zarei z, banafshi o, rafinejad j, salarielac s, faghihi f (2007) comparative susceptibility of cypermethrin in ornithodoros lahorensis neuman and argas persicus oken (acari: argasidae) field populations. pak j biol sci. 10(23): 4315– 4318. telmadarraiy z, vatandoost h, rafinejad j, mohebali m, tavakoli m, abdigoudarzi m, faghihi f, aboulhasani m, zarei z, jedari m. mohtarami f, azamsoulki a, salari lak sh entezarmahdi r (2009) distribution of ticks (ixodidae and argasidae) family and susceptibility level to cypermethrin in meshkinshahr district, ardabil province, iran. ardabil uni med sci j. 9(2): 127–133 telmadarraiy z, ghiasi sm, moradi m, vatandoost h, eshraghian mr, faghihi f, zarei z, haeri a, chinikar s (2010) a survey of crimean-congo haemorrhagic fever in livestock and ticks in ardabil province, iran during 2004-2005. scand j infect dis. 42(2): 137–141. vatandoost h, ghaderi a, javadian e, zahir nia ah, rassi y, piazak y, kia eb, shaeghi m, telmadarreiy z, aboulghasani m (2003) distribution of soft ticks and their infection with borrelia in hamadan province, iran. iranian j publ health. 32 (1): 22–24. zeleke m, bekele t (2004) species of ticks on camels and their seasonal population dynamics in eastern ethiopia. trop animal hlth and produc. 36(3): 225–231. microsoft word 4-hashareh dr rahbari rtl.doc iranian j arthropod-borne dis, (2009), 3(2): 18-21 s rahbari et al: haematophagus mites in … 18 original article haematophagus mites in poultry farms of iran s rahbari, *s nabian, h ronaghi department of parasitology, faculty of veterinary medicine, university of tehran, iran (received 25 apr 2009; accepted 12 nov 2009) abstract background: blood sucking mites are important avian ectoparasites which being found on bird species worldwide. their presence are problematic for the producers either through potential direct effects on weight gain, egg production, sperm production in roosters or as nuisance pests on worker handle hens and eggs. the aim of this study was pointing out of the status of haematophagus mites. methods: eight caged layer and four breeder flocks were visited, monitoring for the presence of chicken mites performed by removing and examining debris from poultry house, infested nesting material collected into zip lock plastic bags and at least 20 birds were also randomly selected to examine the presence of chicken mites. mites obtained from each population were mounted in hoyer,s medium on microscope slides and identified. all eight caged layer and four breeder flocks were inspected, which were infested with chicken blood feeding mites. results: massive infestations of dermanyssus gallinae were common with huge numbers of parasites on birds, cages and the conveyor belts for egg. only one farm from mazandaran province was infested to ornithonyssus bursa. conclusion: dermanyssus gallinae was the most prevalent blood feeder mite in the breeder and caged layer flocks in iran, while o. bursa was reported as a first record, which found only in a breeder flock in mazanderan province. it seems that its presence is limited into the area which affected by both warm and humid environmental conditions. keywords: dermanyssus gallinae, ornithonyssus bursa, poultry, iran introduction the common avian ectoparasites are the blood sucking mites, which is being found on bird species world wide (walter and proctor 1999). these mites live on the host and within the nesting material, from where they infect and feed on chicks in the nest (burtt et al. 1991). blood feeder mites are from the genera dermanyssus (de geer, 1778) and ornithonyssus (berless1888), because of their life cycle, the population thus can become very high on birds, their presence are problematic for the producers either through potential direct effects on weight gain, egg production or sperm production in roosters or as nuisance pests on worker, particularly people handle hens and eggs (hogsette et al. 1991). rafie et al. (1966) indicated that poultry houses are often affected by d. gallinae, so far, there was not any report on o. bursa in iran. the aim of this study was pointing out of the status of haematophagus mites. materials and methods geographical information this study was conducted in eight caged layer (white leghorn) and four breeder (ross 308) farms in seven provinces (guilan, mazandaran, zanjan, ghazvin, markazi, ghom and tehran) based on claiming of the farmers for presence of chicken blood feeding mite during six yr ago. monitoring monitoring for the presence of chicken mites performed by removing and examining *corresponding author: dr s nabian, e-mail: nabian@ ut.ac.ir iranian j arthropod-borne dis, (2009), 3(2): 18-21 s rahbari et al: haematophagus mites in … 19 debris from roosts, slats and nesting boxes, infested nesting material collected into zip lock plastic bags and at least 20 birds were also randomly selected throughout a house and examined the vent area under bright light. when large mite populations were encountered in flocks, heavily miteinfested feathers were plucked from at least 3 to 5 individual hens. the feathers were placed into plastic bags and then transported to the laboratory to confirm identification. species identification mites obtained from each population were mounted in hoyer,s medium on microscope slides and identified with the aid of key developed by baker (1999).we did not generate data on number of mites present and therefore no analyses using density of mites could be performed. the use of presence/absence of mites was determined to be sufficient to indicate a level of parasite load that could affect the birds. results all eight caged layer and four breeder flocks were inspected, found to be infested with chicken blood feeding mites. one of them was infested with o. bursa. it matched with the description which described by baker (1999). the remained flocks were infested with d. gallinae. it can be distinguishable from o. bursa by the anus which is situated on posterior of the anal plate whereas in o. bursa, the anus is on the anterior half of this plate (fig.1) ornithonyssus, bursa (berlese) (arachnida: acari:macronyssidae) can be differentiated from o. sylviarum (canestrini and fanzago, 1877) (mesostigmata: macronyssidae) by the shape of its dorsal plate which gradually tapers to a blunt posterior end, in addition its sternal plate consists of three pairs of setae (fig. 2). dermanyssus gallinae was the most prevalent blood feeder mite in the breeder and caged layer in all inspected flocks, while o. bursa was reported as a first record only in a breeder flock in mazandaran province. fig. 1. microscopic photograph showing anal plate of dermanissus. gallinae (a) and ornithonyssus. bursa (b) fig. 2. microscopic photograph showing sternal plate of ornithonyssus. bursa discussion dermanyssus gallinae currently is the most important ectoparasite affecting egg layers in temperate and tropical countries, whereas, o. bursa is distributed throughout the warmer regions of the world (lancaster and meisch 1986). rafyi et al. (1966) showed that poultry houses are often affected by d. gallinae. in this study, we showed the presence of d. gallinae in breeder and egg layer flocks and o. bursa only in one of the breeder flocks. our literature research recovered no publications with record of tropical fowl mite hence it means this is the first report of this mite in iranian j arthropod-borne dis, (2009), 3(2): 18-21 s rahbari et al: haematophagus mites in … 20 iran. d. gallinae has been revealed in humid and dried area while o.bursa has been shown only in the north part of iran. the tropical fowl mite (o. bursa) is distributed throughout the warmer regions of the world and associated mainly with poultry, pigeon and sparrows (william et al. 1976). their presence is affected by both host and environmental conditions; being negatively correlated with the quality of an individual bird (darolova and schleicher 1997) and positively correlated with humidity (walter and proctor 1999) as well as the density of breeding sites (poiani 1992). the bite of haematophagus mites is irritating to man and some individuals react to the bite with prolonged itching and painful dermatitis (burtt et al. 1991). besides having direct effects upon their hosts, poultry mites can transmit viral, riketsial and protozoan diseases among birds (kettle 1995, sonenshine 1993). d. gallinae has been recently reported to be an experimental vector of salmonella enterititis (valiente et al. 2005). it is also a vector of equine encephalitis virus among poultry (durden 1993). several authors showed its relationship to different poultry pathogens such as avian borelliosis, chicken pox virus, newcastle virus, agent of pullorum disease and fowl typhoid and the agent of fowl cholera (zeman et al.1982). o. bursa can spread disease even if each mite bites only a single host. this is because some diseasecausing pathogens, such as avian borelliosis, can be passed vertically from a female mite to her offspring (bowman 1995). it can be concluded that dermanyssus gallinae is the most prevalent and important pest of poultry in iran but it seems that o. bursa is limited to north part of the country. public health aspects of these parasites should be considered. acknowledgements we thank all the farmers for the valuable assistance of providing the specimens. this work was supported by university of tehran central excellence. the authors declare that they have no conflicts of interest. references baker as (1999) mites and ticks of domestic animals: an identification guide and information source. british natural history museum: london. bowman dd (1995) parasitology for veterinarians (6thedn).w.b. saunders. burtt jrh, chow w, babbitt ga (1991) occurrence and demography of mites of tree swallow, house wren, and eastern bluebird nests. in: loye, j.e, zuk, m. (editors), birdparasite interactions.; 104-122, oxford university press, oxford. darolova a, hoi h, schleicher b( 1997) the effect of ectoparasite nest load on the breeding biology of the penduline tit remiz, pendulinus. ibis. 139: 115-120. durden la (1993) laboratory transmission of equine encephalomyelitis virus to chickens. medical entomology. 30: 281-285. hogsette ja, butler, jf, miller wv, hall d (1991) ornithonyssus sylviarum (canestrini and fanzago) (acari: macronyssidae) misc. publ. entomol soc am. 76: 1-62. kettle ds (1995) medical and veterinary entomology (2nd edn), cab international. lancaster jl, meisch, mv (1986) arthropods in livestock and poultry production. john wiley and sone: new york. poiani a (1992) ectoparasitism as a possible cost of social life: a comparative analysis using australian passerines (passeriformes), oecologia. 92: 429-441. rafyi a, alavi nainy a, rak h (1966) les especes do mites recontres en iran, veterinary faculty letter. 23: 39-44. sonenshine de (1993) biology of ticks, (vol. 2). oxford university press: england. iranian j arthropod-borne dis, (2009), 3(2): 18-21 s rahbari et al: haematophagus mites in … 21 valiente moro, c, shauve c, zenner l (2005) vectorial role of some dermanyssoid mites (acari, mesostigmata, dermanyssoidea, parasite. 12(2): 99-109. walter de, proctor, hc (1999) mites: ecology, evolution and behavior. university of new south wales ltd: sydney. william a, phillis hl, cromroy l, denmark ha (1976) new host and distribution records for the mite genera dermanyssus, ornithonyssus and pellonyssus (acari: mesostigmata: laelapoidea) in florida, the florida entomologist. 59(1): 89-92. zeman p, stika v, skalka b, bartik m, dusbabek f, lavickova m (1982) potential role of dermanyssus gallinae (de geer,1778) in the circulation of the agent of the agent of pullurosis-typhus in hens. folia parasit. 29: 371-374. j arthropod-borne dis, march 2017, 11(1): 156–160 s sadeghi: mediterranean recluse … 156 http://jad.tums.ac.ir published online: march 14, 2017 short communication mediterranean recluse spider, loxosceles rufescens (araneae: sicariidae) from charkhab cave, southern iran *saber sadeghi, meysam dashan, mohammad javad malek-hosseini department of biology, faculty of sciences, shiraz university, shiraz, iran (received 21 sep 2014; accepted 27 jan 2016) abstract background: the best-known dangerous spiders belong to the six genera. the genus loxosceles or violin spiders are well known for their ability to cause skin necrosis or loxoscelism. all loxosceles species have medical importance due to their necrotizing venom. the present article reports the occurrence of l. rufescens in charkhab cave, south of iran (larestan). methods: the specimens were collected from the charkhab cave using handling forceps, paintbrush and aspirator and preserved in 96% ethanol. results: loxosceles rufescens, a medically important spider, is recorded from charkhab cave in fars province (southwest of iran). identification of l. rufescens was performed based on external morphology and the features of male genitalia. conclusion: presence of l. rufescens in south of iran especially in a cave confirmed that this species is a widely distributed species in iran. therefore, cavers or cave visitors should be aware of this poisonous spider in caves. keywords: arachnida, loxosceles, iran introduction spiders with more than 45,000 described species in the world (shahi et al. 2013) are among the better-studied arthropods. however, approximately 200 species from 20 genera of worldwide spiders can cause severe human injuries, with dermonecrosis, systemic toxicity, and death. therefore, less than 0.5% of them are actually dangerous for humans (diaz 2004). the most known dangerous spiders belong to the six genera: latrodectus (black widow spiders), steatoda (false widow spiders), loxosceles (recluse spiders), atrax (funnelweb spiders), phoneutria (banana spiders), cheiracanthium (foliage spiders) (yigit et al. 2008). the recluse spiders (sicariidae: loxosceles) or mediterranean fiddle-back spiders are cosmopolitan, but most commonly in the tropics. although previously placed in the family loxoscelidae (gertsch 1949, gertsch and ennik 1983) or scytodidae (gertsch 1967), sicariid spiders are now considered a member of family sicariidae (plantick et al. 1991). loxosceles rufescens (dufour 1820) occurs around the entire mediterranean region, in asia, north africa, south and central america (platnick 2013). several species (troglophile) have been recorded from caves, houses, subterranean areas, rural and urban area (fischer et al. 2009). although some unidentified species of the genus loxosceles reported from iran during 1994 to 2013 (goodarzi 1994, moradmand and jäger 2011, kashefi et al. 2013, and mirshamsi et al. 2013), the first record of mediterranean recluse spider, l. rufescens, in iran was presented by zamani and rafinejad (2013) from tehran province. shahi et al. 2013 also reported a loxoscelism from bandar abbas in south of iran. all loxosceles species have medical importance due to their necrotizing venom, based on swanson and vetter study *corresponding author: mr saber sadeghi, e-mail: ssadeghi@shirazu.ac.ir, sabersadeghi@yahoo.com j arthropod-borne dis, march 2017, 11(1): 156–160 s sadeghi: mediterranean recluse … 157 http://jad.tums.ac.ir published online: march 14, 2017 (2006) “spiders of the genus loxosceles cause necrotic dermatologic injury through a unique enzyme, sphingomyelinase d, found only in one other spider genus and several bacteria”. tambourgi and et al. (2000) also noted, “envenomation causes dermonecrosis and complement (c)-dependent intravascular hemolysis”. typical symptoms start 2–6 h after the bite and the reactions to bites can be variable from mild to severe, and occasionally leads to death. “symptoms can include fever, rash, nausea, vomiting, hemolytic anemia, bloody urine, renal failure, and shock” (hufford 1977). in iran, the araneae fauna of iranian caves has been poorly studied. marusik et al. (2014), published a survey of spider species from shirabad cave, golestan province, north of iran, and the first troglobite species of iranian spiders trilacuna qarzi malek hosseini and grismado, 2015, was reported by malek hosseini et al. (2015). some sporadic studies such as moradmand and jäger (2011) and zamani et al. (2014, 2015) have been conducted so far. the present contribution reports the occurrence of l. rufescens in charkhab cave in south of iran (larestan). it may help to cover a very small part of information gap of our knowledge of the situation of this important genus in iran and aware caverns about l. rufescens presence in this cave and possibly other caves. materials and methods study area charkhab cave is in larestan area, south of fars province, 70 km south of larestan (fig. 1) in hormoud protected area, near villages of zad mahmood and ahveh with warm and dry climate surrounded by mountains (27º 32' 44” n: 55º 20' 21” e), altitude: 639 m a.s.l. this cave is one of 51 explored caves in fars with valuable habitants especially spider species. the cave was visited on jan. 8, 2014. collection and identification the walls and floor of the cave were precisely searched for cavernicoles. the specimens were collected by authors during a fieldtrip on january 2014 using common methods such as nets, forceps and soft paintbrush then, they preserved in 96% ethanol and transported to our entomology lab. in shiraz university. some photos were taken by a digital camera (canon ds126251). the spiders were distinguished based on external morphology and features of the male genitalia using available keys. finally, our description was confirmed by dr ym marusik and dr rs vetter, two world specialists of the spiders. the specimens were deposited in the zoological museum, collection of biology department of shiraz university (zmcbsu). results laboratory works and separating specimens from samples clarified that 5 adult males and 3 females were collected. loxosceles rufescens or fiddle back spider unlike most other spiders that have eight eyes, has six eyes as three pairs in a nearly triangular arrangement (fig. 2). a distinctive “violin” shaped dark pattern is present on the cephalothorax of this species. however, using this overly simplified diagnostic identifier will lead to mistakes because some loxosceles species have almost no pigmentation in the violin area and other species have dark maculae on the dorsal body surfaces. a particular diagnostic character of the genus (loxosceles) is existence of six eyes, which arranged in nontouching pairs in a u-shaped pattern. most spiders have eight eyes, as a hallmark for this medically important group. the tibia of males’ palpus is short, thick, and narrow at base and not very prolonged (fig. 3). the embolus length is about as long as the width of the globular bulb. j arthropod-borne dis, march 2017, 11(1): 156–160 s sadeghi: mediterranean recluse … 158 http://jad.tums.ac.ir published online: march 14, 2017 fig. 1. sampling localities of genus loxosceles and species loxosceles rufescens in iran reported up to 2015 : ilam province, (loxosceles sp) moradmand and jäger 2011. ▲: tehran province (35°43'n, 51°25'e), zamani and rafinajad 2013. : golestan province (36°50΄19″n, 54°26΄05″e), kashefi et al. 2013. : khorasan province (36°16΄24 95″n, 59°34΄ 75″e), mirshamsi et al. 2013. : fars province, laresthan (27º 32' 44″n, 55º 20' 21″e), present study. sf: hormozgan province, bandar abbas (27 º 11' 0″n, 56°16΄36.0″e), shahi et al. 2013. fig. 2. loxosceles rufescens, arrangement of eyes in three pairs fig. 3. loxosceles rufescens, male palpus, lateral view fig. 4. loxosceles rufescens, male, charkhab cave discussion the loxoscelinae is a small subfamily with about 120 species in a single genus. loxosceles rufescens (fig. 4) is native to the europe and north africa and then spread to other regions by human activity. it occurs in mediterranean area and middle east to western russia, besides has been introduced to madagascar, southern asia, australia, atlantic and pacific islands. in north and south of america, they have also been reported from many states (green et al. 2009). some species have also been introduced into human habitations, where they have been able to establish permanent populations. altj arthropod-borne dis, march 2017, 11(1): 156–160 s sadeghi: mediterranean recluse … 159 http://jad.tums.ac.ir published online: march 14, 2017 hough, this species have been reported from some parts of iran (mirshamsi et al. 2013, zamani and rafinejad 2013) but the present report is the first record of the species from an iranian cave. this cave has recently been discovered and is habitat of many bats, a kind of snake (not captured) and a variety of arthropods. loxosceles rufescens was observed throughout the cave especially at the hypogean part. this species have only been collected from charkhab cave up to now, it probably is partly due to high temperature and dry weather of the area. however, it does not mean that this species cannot spread to other caves. therefore, cavers or cave visitors should be aware of potential dangers of the poisonous spiders in caves because there is no evidence-based effective therapy for loxoscelism at present time. conclusion the presence of l. rufescens in a cave in south of iran confirms that this species is also a cave adapted species in iran, but not distributed widely because we found it only in one of more than seventeen investigated caves by authors and their collaborators. however, cavers and other visitors of caves have to take care because of some dangerous spiders with poisonous venom. our collected specimens were morphologically a little different from other areas of the world thus more studies are needed to reveal their taxonomic and toxicological differences. acknowledgements the authors are grateful to dr yuri m marusik (turku, finland) and dr richard s vetter (department of entomology, university of california riverside) for useful comments and confirmation of the species. we are also thankful mr alamdar shafiei, mr akbar shafiei and ms atefeh shafiei (pardis-e larestan mountaineering group), miss m tahami and z shamsoddini for their help in caving and sampling. we also appreciate “iran national science foundation” (insf) who supported this study. the authors declare that there is no conflict of interests. references diaz hj (2004) the global epidemiology, syndromic classification, management, and prevention of spider bites. am j trop med hyg. 71: 239–250. fischer ml, bazilio s, verginia t, dos santos b, grosskopf cb (2009) diagnóstico da ocorrência de aranhas do gênero loxosceles heineken e lowe, 1832 (araneae, sicariidae) no município de união da vitória, paraná, biotemas. 22 (1): 155–159. gertsch wj, ennik f (1983) the spider genus loxosceles in north america, central america, and the west indies (araneae, loxoscelidae). bull am mus nat hist. 175: 264–360 gertsch wj (1967) the spider genus loxosceles in south america (araneae, scytodidae). bull am mus nat hist. 175: 264–360. gertsch wj (1949) american spiders. d van nostrand, new york. goodarzi hr (1994) an introduction to the identification and classification of iranian spiders. [msc thesis]. plant pest and disease research institute, tehran, iran. greene a, breisch nl, boardman t, pagac jr bb, kunickis e, howes rk, brown pv (2009) the mediterranean recluse spider, loxosceles rufescens (dufour) an abundant but cryptic inhabitant of deep infrastructure in the washington, dc area (arachnida: araneae: sicariidae). am entomol. 55(3): 158–169. harvey m (1996) the first record of the fiddleback spider loxosceles rufescens (arane j arthropod-borne dis, march 2017, 11(1): 156–160 s sadeghi: mediterranean recluse … 160 http://jad.tums.ac.ir published online: march 14, 2017 ae: sicariidae) from western australia. rec aus mus. 18: 223–224. hufford dc (1977) the brown recluse spider and necrotic arachnidism: a current review. j arkansas med soc. 74(3): 126– 129. kashefi r, ghassemzadeh f, kami hg, mirshamsi o (2013) new data on spider fauna from golestan province, iran (arachnida, araneae). prog biol sci. 3(1): 7–22. malek hosseini mj, grismado c, sadeghi s, bakhshi y (2015) description of the first cave dwelling species of the spider genus trilacuna tong and li from iran (araneae: oonopidae). zootaxa. 3972 (4): 549–561. marusik ym, nadimi a, omelko mm, koponen s (2014). first data about cave spiders (arachnida: araneae) from iran. zool middle east. 60(3): 255–266. mirshamsi o, hatami m, zamani a (2013) new record of the mediterranean recluse spider loxosceles rufescens (dufour 1820) and its bite from khorasan province, northeast of iran (aranei: sicariidae). iran j anim biosystem. 9 (1): 83–86 moradmand m, jäger p (2011) a review on the huntsman spider genus spariolenus simon, 1880 (araneae: sparassidae: heteropodinae) in iran with description of four new species. zootaxa. 2910: 46–62. platnick ni, coddington ja, forster rr, grsiwold ce (1991) spinneret morphology and the phylogeny of haplogyne spiders (araneae, araneomorphae). am mus novitates. 3016: 1–73. platnick ni (ed.) (2013) the world spider catalog, version 13.5. amnh. available at: http://research.amnh.org/iz/spiders/ catalog, on 16 july 2013. salahi m (2009) iran caves. ney publishing co. tehran. shahi m, shahi a, khademi z, zamani ar, nakhaii ar, rafinejad j (2013) loxoscelism: a case report from bandar abbas in south of iran. hormozgan med j. 18: 5. swanson dl, vetter rs (2006) loxoscelism. clin dermatol. 24(3): 213–221. tambourgi dv, morgan bp, de andrade rm, magnoli fc, van den berg cw (2000) loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis. blood. 95(2): 683–691. yigit n, bayram a, ulasoglu d, danisman t, corakocal l, sancak z (2008) loxosceles spider bite in turkey (loxosceles rufescens, sicariidae, araneae). j venom anim toxins. 14(1): 178–187. zamani a, rafinejad j (2013) first record of the mediterranean recluse spider loxosceles rufescens (araneae: sicarridae) from iran. j arthropod-borne dis. 8 (2): 228–231. zamani a, nikmaghdam z, allahdadi m, gassemzadeh f, mirshamsi o (2014) new data on the spider fauna of iran (arachnida: araneae). zool middle east. 60(4): 362–367. zamani a, mirshamsi o, marusik ym, moradmand m (2015) the checklist of the spiders of iran. version 2015. available at: http://www.spiders.ir. microsoft word 1akhavan-rtl.doc iranian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 1 original article emergence of cutaneous leishmaniasis due to leishmania major in a new focus of southern iran aa akhavan 1, * mr yaghoobi-ershadi 1, f hasibi 1, r jafari 2, h abdoli 3, mh arandian3, h soleimani 2, ar zahraei-ramazani 3, m mohebali 4, h hajjaran 4 1dept. of medical entomology and vector control, school of public health and institute of public health research 2 yazd health research and training center 3 isfahan health research and training center, medical sciences/university of tehran, tehran, iran 4 dept. of medical parasitology, school of public health and institute of public health research (received 29 sep 2006; accepted 14 feb 2007) abstract reports from the health center of kerman province, southern iran showed an increasing of cutaneous leishmaniasis cases in orzuieh rural district, southwest of the province in 2003. the report encouraged the team to carry out an epidemiological survey in the district during 2003-2004. the objectives were to determine the ecology of sand flies, potential reservoir hosts and human infection. a total of 1075 sand flies were collected by sticky traps and 7 species of sand flies were identified. they comprised 3 species of the genus phlebotomus (p. papatasi, p. mongolensis and p. bergeroti) and 4 species of the genus sergentomyia (s. sintoni, s. clydei, s. tiberiadis and s. baghdadis). p. papatasi was the predominant species of the genus phlebotomus in indoors (90.3%) and outdoors (50.2%). susceptibility tests on p. papatasi with ddt 4%. showed that the species was susceptible to this insecticide. a total of 13 rodents consist of tatera indica (76.9%) and nesokia indica (23.1%) were collected. a study of prevalence among 2441 inhabitants in four villages showed a rate of 1.1% for active lesions and 10.4% for scars during novemberdecember 2003. in a separate study examination of 1662 school children aged 6-12 years old showed 1.14% for ulcers and 14.7% for scars at the same time. the leishmania parasites were isolated from man and characterized as leishmania major using rapd-pcr method. it seems that cutaneous leishmaniasis due to l. major (clm) has been prevailed in the district. keywords: sand flies, epidemiology, leishmania major, leishmaniasis, vector, iran introduction cutaneous leishmaniasis due to leishmania major (clm) is still a great and increasing public health problem in many rural areas of 15 out of 30 provinces of iran (yaghoobi-ershadi, et al. 2005 ). at a particular geographical location where the disease has been sporadic in the past, it has grown to epidemic portions. it has been spread into sites where it did not previously exist (yaghoobi-ershadi et al. 2001a). cutaneous leishmaniasis due to l. tropica (clt) and also sporadic cases of clm and visceral leishmaniasis (vl) were reported from kerman province during the last 3 decades (institute of public health research, unpublished data). recently a new focus of cutaneous leishmaniasis (cl) has been found in some villages of orzuieh rural district, southwest of kerman province, southern iran. in 2002, a total of 100 cases were officially reported by passive case detection from baft county and most of them were from orzoieh rural district, kerman province (kerman health center, unpublished data). although cl is a notifiable disease in iran but the real figures seem to be 4-5 folds. the region is *corresponding author: prof mr yaghoobi-ershadi, tel: +98 21 88973901 (ext: 2114), fax: +98 21 88951393, e-mail: yaghoobi@maktoob.com iranian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 2 the main pivot of agriculture of kerman and it is famous as the granary of the province. this attracts many none immune workers and they may be exposed to bites of infected sand flies during the active season. however the epidemiological aspects of cl in this district had not been examined so we carried out ecological and epidemiological investigations for the first time in this focus for the implementation of future control measures. the objectives of this study were to determine the prevalence of the disease, to study the reservoirs, ecology of sand flies, the vectors and also the nature of the parasite in this new focus. materials and methods study area field studies were conducted over a period of 12 months from august 2003 to september 2004 in four villages, dowlat-abad, soltanabad, shahmaran and vakilabad in orzuieh rural district, baft county, kerman province (56.2456.38° e , 29.13-29.15° n), southern iran. the choice of these villages was based on the increased number of the reported cases of cl by passive case detection in 2002 (kerman health center, unpublished data). the area has a desert climate, altitude between 1050-1095, hot in summer and quite cold in winter. in 2003, the maximum and minimum mean monthly temperatures were 33.1 °c and -0.4 °c in july and february, respectively. the total annual rainfall was 309 mm with a minimum of 3 mm in july and maximum of 120.9 mm in april. the minimum mean monthly relative humidity was 26% (june) and the maximum was 56% (january). wheat, indian corn, cotton, sesame plants, alfalfa, onion, sunflowers, watermelon, date, citrus fruits, pistachio, and pomegranate are the common crops in the region. the orzuieh river runs among the infected villages. population studies one hundred and sixteen households of each village whose buildings were located near each other were examined in december 2003, and corresponding forms were completed for each household during a houseto-house visit. the presence or absence of scars or ulcers of cl were indicated on the forms. we also examined and questioned all the students of the school children in the surveyed area simultaneously. each individual was examined for scar(s) or ulcer(s) and the date and place of acquiring the disease, age, sex, number of ulcer(s) or scar(s), site of ulcer(s) or scar(s) were recorded. smears were prepared from scrapings of the edge of the ulcer, then fixed in methanol, stained with giemsa, and examined under a light microscope for the presence of amastigotes. the x2 –test using spss 11.5, was used to determine statistically significant differences in disease prevalence between females and males and among different age groups in the community. the graphs were also prepared by excel®. isolation of the parasites from patients samples from two patients, with no traveling history to the other foci of clm, were taken from ulcers and inoculated subcutaneously at the base of the tail of 6 balb/c mice. parasites were reisolated from infected mice and cultured in nnn plus lit medium containing 200 iu penicillin per ml, incubated at 20-21 °c and monitored every 4 days, from day 4 for growth. all positive cultures were subcultured every 15 days in rpmi medium containing 10-20% fcs. after growing the parasites, leptomonads in logarithmic phase were cryopreserved in -196 °c and then in proper time rapd-pcr technique with the appropriate primers was used for identification of parasites at protozoology unit, department of medical parasitology, school of public health, tehran university of medical sciences, with the cooperation of school of medicine, shiraz university of medical sciences (mohebali et al. 2002, motazedian et al. 2002). collection and examination of rodents small mammals were caught by 20 live traps baited with cucumber and tomato, 16 times during different seasons in 2003 and 2004. in the laboratory, 4 impression smears were prepared from the ears of each mammal (edrissian iranian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 3 et al. 1982), fixed in methanol and stained by the giemsa method and examined carefully under the light microscope. collection of sand flies sand flies were collected from indoor (bed rooms, warehouse, toilets, and hall) and outdoor (rodent burrows) fixed places, using 30 sticky traps in each (castor oil coated white paper 20 x 32 cm) from sunset to sunrise and also by aspirator from indoors (6.00 to 10.00 am) four times in june, august, september, and october in 2004. for species identification, sand flies were mounted in puri’s medium (smart et al. 1965) and identified after 24 h using the morphological characters (theodor and mesghali 1964). then they were counted and segregated by sex. in order to determine natural promastigote infections of sand flies, some unfed, blood fed, semigravid and gravid female sand flies of rodent burrows were collected by sticky traps and examined in a fresh drop of sterile saline (9/1000) for the presence of promastigotes in alimentary canal in august and september 2003-2004. the graphs were prepared by excel®. susceptibility test the susceptibility status of phlebotomus papatasi to ddt was studied in the field surveys during september 2004 following the who standard method (who 1981) and using impregnated papers supplied by who. results a study of prevalence among 2441 inhabitants from 495 households in four villages showed a rate of 1.11% for active lesions during novemberdecember 2003 (table 1). the most highly infected age group was 0-4 yr with a rate of 3.35%. children under 10 yr of age had a rate of 2.02% for active lesions. the rate was 0.93% for those more than 10 yr old. chisquare analysis indicates that there is significant differences in number of individuals with active lesions among different age groups (x2= 15.858, df= 5, p< 0.007). the prevalence of scars was calculated to be 10.37% (table 1) .the scar rate was 6.55% for individuals under 10 yr of age and 11.11% for those over 10 yr of age. the proportion of males and females were 50.5% and 49.5% respectively. significant differences in number of individuals with scars were also observed among different age groups (x2= 40.307, df= 5, p< 0.0001). at the same time, altogether, 14 primary schools with 1662 students (885 boys and 777 girls) from 6 to 12 yr of age visited. the overall scar rate was 14.68% and the prevalence of active lesions was 1.14% (table 2). most of the patients had one active lesion and most of them were observed on the hands, legs and face. microscopic slides prepared by scraping the edges of the lesions of all patients (inhabitants of households and students of primary schools), contained parasites presumed to be leishmania major based on existence of a large vacuole in the cytoplasm. treatment was provided for 46 subjects with a parasitological diagnosis of leishmaniasis. examination of two isolates from human indigenous cases identified them as l. major. a total of 13 rodents consist of t. indica (76.9%) and n. indica (23.1%) were collected and examined for leishmanial infection during augustseptember 20032004 but all were found to be negative. during june-october 2004 a total of 1075 sand flies (310 from indoors and 765 from outdoor resting places) were collected and identified. the following 7 species were found in bedrooms, storerooms and toilets: p. papatasi (90.3%), p. mongolensis (0.3%), p. bergeroti (0.7%), sergentomyia sintoni (5.1%), s. clydei (0.7%), s. tiberiadis (0.7%) and s. baghdadis (2.2%) (fig.1). in rodent burrows: p. papatasi (50.2%), p. mongolensis (0.1%), s. sintoni (35%), s. clydei (11.5%), s. tiberiadis (2.8%) and s. baghdadis (0.4%) were collected (fig.2). nine p. papatasi and 1 s. sintoni in the vicinity of rodent burrows and 45 p. papatasi from indoors were collected and dissected in september 2003. none of them were found to be infected with promastigotes. a total of 92 p. pairanian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 4 patasi and 2 s. sintoni were collected from the vicinity of rodent burrows again in the area and dissected in september 2004. none of them appeared to be infected. the sex ratio, i.e. number of males per 100 females, of p. papatasi collected by sticky traps, in indoors and outdoors was calculated to be 156.9 and 193.13, respectively. in case of s. sintoni it was found 45.4 and 57.7 in indoors and outdoors respectively. a total of 107 fed p. papatasi were collected by aspirator from indoors in four villages in first half of september 2004. the mortality rate for ddt 4.0% after 6 0.00 min exposure time followed by 24 h recovery time was 100%. it is concluded that p. papatasi strain of orzuieh is susceptible to ddt. table 1. the prevalence of cutaneous leishmaniasis by age among 495 families (both sexes) in the study area, novdec 2003 age group (yr) no. observed no. with scars % no. with active lesions % 0-4 179 4 2.24 6 3.35 5-9 218 22 10.09 2 0.92 10-14 379 64 16.89 1 0.26 15-19 444 53 11.94 2 0.45 20-24 273 36 13.19 1 0.37 25+ 948 74 7.81 15 1.58 all groups 2441 253 10.37 27 1.11 table 2. the prevalence of active lesions and scar rate by age among the students (both sexes) of primary schools in the study area, novdec 2003 age (yr) no. observed no. with scars % no. with active lesions % 6 41 0 0 0 0 7 284 21 7.39 3 1.06 8 300 43 14.33 1 0.33 9 329 47 14.29 3 0.91 10 348 51 14.66 5 1.44 11 356 79 22.19 6 1.69 12 4 3 75 1 25 total 1662 244 14.68 19 1.14 iranian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 5 90.3 0.7 0.3 5.1 2.2 0.7 0.7 0 10 20 30 40 50 60 70 80 90 100(%) p. pa pa ta si p. be rg er ot i p. m on go len sis s. sin to ni s. ba gh da di s s. cly de i s. tib er iad is species of sandflies fig. 1. the fauna of sand flies from indoor resting places, orzuieh rural district, southwest of kerman province, southern iran, 2003 and 2004. 50.2 0.1 35 11.5 2.8 0.4 0 10 20 30 40 50 60 (%) p. pa pa tas i p. mo ng ole ns is s. sin to ni s. cly de i s. tib er iad is s. ba gh da dis species of sandflies fig. 2. the fauna of sand flies from rodent burrows, orzuieh rural district, southwest of kerman province, southern iran, 2003 and 2004. iranian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 6 discussion this study was carried out on some epidemiological aspects of cl in the study area for the first time in iran. l. major isolated and characterized from the lesions of the patients with no history of traveling to the other clm foci of iran. the same species of leishmania parasite has been isolated from p. papatasi, p. caucasicus, rhombomys opimus, meriones libycus, tatera indica and human in other parts of the country (akhavan et al. 1998 and 2003, javadian et al. 1998, rassi et al. 2001, yaghoobi-ershadi et al. 1994, 1995, 1996a, 1996b, 2001a, 2001b, 2001c, 2002). tatera indica is the common gerbil in orzoieh rural district. because of the high proportion of t. indica (76.9%) in the district, it seems this species may play a role as the reservoir host in the focus. natural leishmanial infection of this species was also recorded from khuzistan and ilam provinces of iran (javadian et al. 1988, 1998). this is the first study that reports 7 species of sand flies from baft county and p. bergeroti, p. mongolensis and s. clydei are also new records for kerman province. p. papatasi was the predominant species of the genus phlebotomus in indoors (90.3%) and outdoors (50.2%). regarding the existence of p. papatasi as peridomestic species in indoors and based on the isolation and characterization of l. major from this species in some other foci of clm in central iran (yaghoobiershadi 1995, 2001, 2005a) and also from patients and m. libycus in fars province nearby the study area (rassi et al., 2001) it seems that probably this species acts as the vector to man and also among rodents. over the last decade, cases of clm have been reported from the west and southwest of the central desert and even from the south of the country (neiriz, estahban, lar, darab, jahrom, kharame, sarvestan, arsanjan, hadjiabad and kahurestan (yaghoobi-ershadi et al. 2005b). it seems that orzuieh is also extension of these foci. cutaneous leishmaniasis due to l. major (clm) has been prevailed in the district. p. papatasi and t. indica are the probable vector and reservoir respectively. the occurrence of this outbreak of clm in the study villages seems to be the result of development of agro-industrial activities, instruction of buildings nearby colonies of gerbils and their venturing inside houses, sleeping of inhabitants in courtyards at night during active season of sand flies. following the experience gained from a research project on clm control, central iran (yaghoobi-ershadi et al. 2005b), destroying of gerbils with zinc phosphide mixed with wheat grains and vegetable oil (2.5%) within a radius of 500 meters of houses once a month during may, june, july and september in the first year and once every two years in the coming years is suggested for the control of the disease. acknowledgements sincere thanks are extended to the leishmaniasis staff of isfahan health research and training center and also the staff of the disease control office, kerman health center for their excellent assistance in achieving this study. this project received financial support from deputy for instruction affairs of school of public health, medical sciences/university of tehran. references akhavan aa, yaghoobi-ershadi mr, mehdipour d, abdoli h, farzinnia b, mohebali m, hajjaran h (2003) epidemic outbreak of cutaneous leishmaniasis due to leishmania major in ghanavat rural district, qom province, central iran. iranian j publ health. 32(4): 35-41. akhavan aa, yaghoobi-ershadi mr, 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27(1-2): 55-60. mohebali m, motazedian mh, parsa f, hajjaran h, yaghoobi-ershadi mr (2002) identification of leishmania species from different parts of iran using a random amplified polymorphic dna in humans, animal reservoirs and vectors. med j ir iran. 15(34): 243-246. motazedian mh, noyes h, maingon r (2002) leishmania and sauroleishmania: the use of random amplified polymorphic dna for identification of parasites from vertebrates and invertebrates. exp parasitol. 83(1-4):1006. rassi y, jalali m, javadian e, motazedian mh (2001) confirmation of meriones libycus (rodentia: gerbillidae) as the main reservoir host of zoonotic cutaneous leishmaniasis in arsanjan, fars province, south of iran (19992000). iranian j publ health. 30(3-4): 143-44. smart j, jordan k, whittick rj (1965) insect of medical importance. 4th ed. adlen press. oxford. theodor o, mesghali a (1964) on the phlebotominae of iran. j med ent. 1(3):285-300. world health organization (1998) instructions for determining the susceptibility of resistance of adult mosquitoes to organochlorine, organophosphate and carbamate insecticides established of the base-line. 1981. who/vbc/ 81/805. yaghoobi-ershadi mr, akhavan aa, zahraeiramazani ar, jalali-zand ar, piazak n (2005a) bionomics of phlebotomus papatasi (diptera: psychodidae) in an endemic focus of zoonotic cutaneous leishmaniasis in central iran. j vect ecol. 30(1): 115-118. yaghoobi-ershadi mr, zahraei-ramazani ar, akhavan aa, jalali-zand ar, abdoli h, nadim a (2005b) rodent control operations against zoonotic cutaneous leishmaniasis in rural iran. ann saudi med. 25(4): 309-312. yaghoobi-ershadi mr, hanafi-bojd aa, akhavan aa, zahraei-ramazani ar, mohebali m (2001a) epidemiological study in a new focus of cutaneous leishmaniasis due to leishmania major in ardestan town, central iran. acta trop. 79: 115-121. yaghoobi-ershadi mr, akhavan aa, mohebali m (2001b) monthly variation of leishmania major mon-26 infection rates in phlebotomus papatasi (diptera: psychodidae) from rodent burrows in badrood area of iran. med j ir iran. 15(3): 175-178. yaghoobi-ershadi mr, hanafi-bojd aa, akhavan aa, jafari r, mohebali m (2001c) the animal reservoir hosts of cutaneous leishmaniasis in two endemic foci in yazd province. j shahid sadoughi unv med sci. 9(2): 38-43. yaghoobi-ershadi mr, jafari r, zahraeiramazani ar, akhavan aa, mohebali m. (2002) ecology of phlebbotomine sandflies in an endemic focus of cutaneous leishmaniasis in isfahan county. j shahid sadoughi unv med sci. 9(4): 42-48. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996a) meriones libycus and rhombomys opimus (rodentia: gerbillidae) are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans r soc trop med hyg. 90: 503-504. yaghoobi-ershadi mr, javadian e (1996b) epidemiological study of reservoir hosts in an endemic area of zoonotic cutaneous leishmaniasis in iran. bull wld health org. 74(6): 587-590. yaghoobi-ershadi mr, javadian e, tahvildarebidruni gh (1995) leishmania major iranian j arthropod-borne dis, (2007), 1(1): 1-8 aa akhavan et al.: emergence of cutaneous… 8 mon-26 isolated from naturally infected phlebotomus papatasi (diptera: psychodidae) in isfahan province, iran. acta trop. 59: 279-282. yaghoobi-ershadi mr, javadian e, tahvildarebidruni gh (1994) the isolation of leishma nia major from phlebotomus (paraphlebotomus) caucasicus in isfahan province. islamic republic of iran. trans r soc trop med hyg. 88: 518-519. j arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 595 http://jad.tums.ac.ir published online: october 04, 2016 short communication evaluation of some plant fruit extracts for the control of west nile virus vector culex pipiens (diptera: culicidae) samed koc, ozay hasan evren, *huseyin cetin department of biology, faculty of science, akdeniz university, antalya, turkey (received 22 feb 2015; accepted 12 oct 2015) abstract background: the extracts of different parts of plants were found very effective against various pests. the aim of this research was to determine the insecticidal activity of fruit methanol extracts obtained from melia azedarach (meliaceae), phoenix theophrasti (arecaceae), styphnolobium japonicum (fabaceae) and pyracantha coccinea (rosaceae) against the larvae of culex pipiens (diptera: culicidae). methods: the fruits of test plants were collected from the campus of akdeniz university, antalya, turkey in 2013. a series of concentrations of the extracts ranging from 62.5–1000 ppm were tested against second instar larvae. results: only the extracts of me. azedarach and ph. theoprasti showed significant larvicidal activity against cx. pipiens and the lc50 values of these extracts were found to be 169.48 and 220.60 ppm, respectively. this is the first research investigating the insecticidal or larvicidal activity of ph. theophrasti, st. japonicum and py. coccinea extracts on mosquitoes. conclusion: the methanol extract of fruits of me. azedarach and ph. theophrasti showed significantly higher larvicidal activity against cx. pipiens. keywords: larvicidal, melia azedarach, phoenix theophrasti, pyracantha coccinea, styphnolobium japonicum introduction mosquitoes are important organisms known being vectors of many fatal or neurologic diseases such as malaria, dengue and west nile virus (wnv) infections. fortunately, most people infected with some of these diseases have no or develop a fever with other symptoms. however, mosquitoes know no borders and more than one million people worldwide die from mosquito borne diseases every year (becker 2008). some insecticide groups can be applied to control adults (adulticides) or larvae (larvicides) of mosquitoes. insecticides used for larviciding include, chitin synthesis inhibitors, diflubenzuron, novaluron and triflumuron, juvenile hormone analogs pyri proxyfen and methoprene, bacterial products (bacillus thuringiensis subsp israelensis and b. sphaericus), spinosyns (spinosad) (cetin et al. 2005, 2006, 2007). these larvicides were highly effective against different mosquito species in the genus of anopheles, culex and aedes but in some conditions some of them have negative effects on non-target organisms (lawler et al. 1999, ser and cetin 2015). in addition, many researchers observed resistance toward larvicides in field populations (sharma et al. 2003, wirth 2010). researches about botanical insecticides and acaricides have grown dramatically in recent years and essential oils and extracts of aromatic plants were found effective against different instars of arthropod pest species (koc et al. 2012). most plant essential oils and extracts obtained from flowers, fruits and leaves are complex mixtures that contain active constituents such as alcohols, aldehydes, esters, ketones, phenols and terpenes (gu et al. 2009). the use of botanical compounds *corresponding authors: dr huseyin cetin, email: hcetin@akdeniz.edu.tr j arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 596 http://jad.tums.ac.ir published online: october 04, 2016 extracted from aromatic plants may be an alternative to conventionally used insecticides to control of mosquitoes (sukumar et al. 1991). therefore, in this study the toxic effects of fruit extracts of four plant species; melia azedarach l (meliaceae), phoenix theophrasti greuter (arecaceae), styphnolobium japonicum (l) (fabaceae) and pyracantha coccinea max joseph roemer (rosaceae) were investigated against larvae of culex pipiens l (diptera: culicidae). this mosquito is found worldwide and is vector of different pathogenic organisms that cause serious diseases (i.e. wnv and filariasis). in turkey, cx. pipiens is abundant in many cities and developed resistance to some insecticide groups. materials and methods plant materials and their extractions tested plants, me. azedarach, ph. theophrasti, st. japonicum and py. coccinea were taxonomically identified by the second author. the fruits of them were collected from the campus of akdeniz university, antalya, turkey in 2013. fruit samples were deposited in the insecticide test laboratory of biology department, faculty of science, akdeniz university. fruits of each species were separated from their stalks. after that, fruit materials were dried at 25 °c about 2 weeks and ground to fine powder using blender. extractions of the samples were carried out by using methanol for 2 days at 25 °c. then filtered using a whatman no.1 filter paper and dried under vacuum rotary evaporator. target mosquito species culex pipiens used in the studies originated from arapsuyu, antalya, and was collected from a pool in august 2011. the larvae were reared at 12 h dark: 12 h light photoperiod, 60±10% relative humidity, and 26±2 °c temperature in an insectary in the biology department, akdeniz university. the second instars larvae were used for bioassays. experiments of larvicidal activities larvicidal activity of the methanol extracts of me. azedarach, ph. theophrasti, st. japonicum and py. coccinea against cx. pipiens was assessed (oz et al. 2013). for experiments, first 2 gr of each extracts were dissolved in 25 ml distilled water. then a series five concentrations (62.5, 125, 250, 500 and 1000 ppm) were prepared and controls in 500 ml tap water in containers. after approximately 5 min, 10 larvae taken on an egg tray with water were transferred gently to the test medium by tapping. four replicates of each concentrations and controls were run at a time. mortality was recorded after 24-, 4872and 96-h of exposure, during which pellet fish food was given to the larvae. all experiments were conducted at 26±2 °c and 60±10% relative humidity with 12 h dark: 12 h light photoperiod. dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical region. moribund larvae were those incapable of rising to the surface (within a reasonable period) or showing the characteristic diving reaction when the water was disturbed. larvae were also observed for discoloration, unnatural positions, uncoordination or rigor. statistical analyses corrected means of percentage mortalities were calculated using abbott`s formula. the percentage values were transformed to ensure normality and variance homogeneity using an arcsine transformation. the data was subjected to analysis of variance (anova) and the means compared with duncan’s multiple range tests (p< 0.05). the lc50 and lc90 values were calculated from percent mortality data by using probit analysis (finney 1971). results the percent mortality values of four plant fruit methanol extracts against second j arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 597 http://jad.tums.ac.ir published online: october 04, 2016 instar larvae of cx. pipiens are shown in table 1. in our experiments, low mortalities were observed in the extracts of st. japonicum (14.45%) and py. coccinea (6.42%) after 96 h exposure. these results are not significantly different from the controls (p< 0.05) (table 1). the highest mortalities were achieved by me. azedarach and ph. theoprasti. the me. azedarach extract caused 100% mortality at the concentration of 1000 ppm after 96 h exposure. ph. theoprasti also demonstrated significant larvicidal activity and in the same concentration, mortality rate was 83.49%. there was a remarkable increase in mortality (19.95 to 83.49%) when concentrations were increased (62.5 to 1000 ppm) (p< 0.05). for 125 ppm of the extracts of me. azedarach and ph. theoprasti more than 39% larvae died only after 96 h of exposure (table 1). the lc50 values against the second instar larvae of cx. pipiens were 220.60 ppm for ph. theophrasti extract, 169.48 ppm for the me. azedarach extract (table 2) (fig. 1). fig. 1. concentration-response lines for most effective test extracts j arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 598 http://jad.tums.ac.ir published online: october 04, 2016 table 1. larvicidal activity of the fruit extracts of phoenix theophrasti, melia azedarach, styphnolobium japonicum and pyracantha coccinea (percent mortalities ± standard errors) concentrations (ppm) times after exposure (hours) 62.5 125 250 500 1000 control phoenix theophrasti 24 0±0 aα, aβ 0±0 a, a 0±0 a, a 0±0 a, a 5.0±2.89 b, a 0.83±0.83 a, a 48 0±0 a, a 2.5±2.5 a, a 2.5±2.5 a, a 5.0±5.0 a, a 5.0±2.89 a a 1.67±1.12 a, a 72 7.5±4.79 a, ab 27.5±11.09 b, ab 27.5±10.31 b, b 35.0±6.45 b, b 40.0±4.08 b, b 3.33±1.88 a, a 96 19.95±7.14 a, b 42.2±12.21 b, b 53.21±2.75 bc, c 66.97±4.49 c, c 83.49±3.18 d, c 9.17±2.6 a, b melia azedarach 24 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0.83±0.83 a, a 48 7.5±4.79 a, a 0±0 a, a 7.5±4.79 a, a 20.0±4.08 b, ab 20.0±7.07 b, a 1.67±1.12 a, a 72 27.5±14.36 ab, a 25±11.9 ab, ab 32.5±6.29 ab, b 40±12.25 b, b 80±7.07 c, b 3.33±1.88 a, a 96 33.94±11.89 ab, a 39.45±17.12 bc, b 53.21±9.4 bc, c 66.97±7.79 c, c 100±0 d, c 9.17±2.6 a, b stypnolobium japonicum 24 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0.83±0.83 a, a 48 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 1.67±1.12 a, a 72 0±0 a, a 2.5±2.5 ab, a 0±0 a, a 5.0±2.89 ab, a 10.0±5.77 b, a 3.33±1.88 ab, a 96 2.98±2.98 a, a 3.21±2.91 a, a 5.96±3.44 a, b 3.21±2.91 a, a 14.45±7.08 a, a 9.17±2.6 a, b pyracantha coccinea 24 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 0.83±0.83 a, a 48 0±0 a, a 0±0 a, a 0±0 a, a 0±0 a, a 2.5±2.5 a, a 1.67±1.12 a, a 72 5.0±2.89 a, b 2.5±2.5 a, a 5.0±2.89 a, a 5.0±2.89 a, a 10.0±4.08 a, a 3.33±1.88 a, a 96 0.46±0.26 a, a 0.46±0.26 a, a 6.19±3.31 a, a 6.42±3.18 a, a 6.42±5.51 a, a 9.17±2.6 a, b α: means within a line followed by the same lower case letter are not significantly different duncan’s multiple range test (p< 0.05). β: means within a column followed by the same capital letter are not significantly different duncan’s multiple range test (p< 0.05). j arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 599 http://jad.tums.ac.ir published online: october 04, 2016 table 2. the lethal concentration 50 and lethal concentration 90 values (ppm) of fruit extracts of phoenix theophrasti, melia azedarach, styphnolobium japonicum and pyracantha coccinea on culex pipiens second-instar larvae in the laboratory conditions plant species lc50 lc90 x 2 p-value ph. theophrasti 220.60 1813.67 1.42 0.69 me. azedarach 169.48 1098.82 6.84 0.07 st. japonicum 39054.36 1792194.42 3.50 0.32 py. coccinea 32110.12 808652.32 6.19 0.10 discussion the calculated lc50’s were higher or lower to other comparable studies on the toxicity of me. azedarach to mosquitoes. these differences may be attributed to many factors such as, method of extraction (extraction solvent, extraction time, extraction temperature), parts of plant (flowers, leaves, fruits), vegetation period, origin of the pest species. the higher lc50 values were determined for st. japonicum and py. coccinea extracts, since significant mortalities were not determined even at the highest exposure concentration (1000 ppm) (table 2). the meliaceae family has been known as a source for pesticides and the efficacy of neem products on different pest species was reported (singh et al. 2006, ndione et al. 2007). melia azedarach has insecticidal effects on different mosquito genus including aedes and culex (wandscheer et al. 2004, ndione et al. 2007). the lc50 value of aqueous extracts of fruits was 2035 ppm for cu. quinquefasciatus say third and fourth instar larvae (ilahi et al. 2012). during our research the lowest dose of fruit extract (62.5 ppm) caused 33.94% mortality and the highest concentration (1000 ppm) caused 100% mortality. mode of action of me. azedarach extract on mosquito larvae may explain that extract causing serious damage to the gastro intestinal system cells. rm al-mehmadi and al-khalaf (2010) showed that changes in the midgut, included separation of the epithelial cells from the basement membrane with damage of the peritrophic membrane causes that the mixing of the gut contents with the hemolymph caused the mortality. fruit extracts of this species have also different effects on insects such as changes of behavior, antifeedant, inhibit the chitin or hormone synthesis, reduced fecundity etc. (schmidt et al. 1998, gajmer et al. 2002). according to our literature survey, no researches have been carried out so far on the insecticidal properties of ph. theophrasti, st. japonicum and py. coccinea against pest organisms. to our knowledge, this is the first research showed the insecticidal or larvicidal activity of ph. theophrasti against cx. pipiens. fruit extracts of ph. theophrasti caused high mortalities as much as me. azedarach extract except the highest concentration (p< 0.05). a series of experiments aimed at clarifying the mode of action of ph. theophrasti should be done on mosquito larvae. the various solvent extracts (e.g. acetone, methanol, chloroform) of different parts (flowers, leaves, fruits) of many plant species were found very effective against various pests (kalyanasundaram and das 1985, govindarajan and sivakumar 2014). most of them contain active components of which have toxic effects on insect biology (cecilia et al. 2014). conclusion the methanol extract of fruits of me. azedarach and ph. theophrasti showed sigj arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 600 http://jad.tums.ac.ir published online: october 04, 2016 nificantly higher larvicidal activity against cx. pipiens. our results suggest that the fruit extracts of ph. theophrasti have the potential to be used as an alternative product for the control of mosquitoes. extracts of both plants may be useful to control larvae of mosquitoes in their breeding sites. however further studies should be done to identify the active ingredients of the extracts responsible for insecticidal activity. acknowledgements the authors are thankful to akdeniz university, scientific research projects committee unit, antalya, for financial report. the authors declared that there is no conflict of interest references becker n (2008) influence of climate change on mosquito development and mosquito-borne diseases in europe. parasitol res. 103(1): 19–28. cecilia kf, ravindhran r, gandhi mr, reegan ad, balakrishna k, ignacimuthu s (2014) larvicidal and pupicidal activities of ecbolin a and ecbolin b isolated from ecbolium viride (forssk.) alston against culex quinquefasciatus say (diptera: culicidae). parasitol res. 113(9): 3477–3484. cetin h, dechant p, yanikoglu a (2007) field trials with tank mixtures of bacillus thuringiensis subsp israelensis and bacillus sphaericus formulations against culex pipiens larvae in septic tanks in antalya, turkey. j am mosq control assoc. 23: 161–165. cetin h, yanikoglu a, cilek je (2005) evaluation of the naturally-derived insecticide spinosad against culex pipiens l. (diptera: culicidae) larvae in septic tank water in antalya, turkey. j vec ecol. 30: 151–154. cetin h, yanikoglu a, cilek je (2006) efficacy of diflubenzuron, a chitin synthesis inhibitor, against culex pipiens larvae in septic tank water. j am mosq control assoc. 22: 343–345. finney dj (1971) probit analysis. cambridge university press, london. gajmer t, singh r, saini rk, kalidhar sb (2002) effect of methanolic extracts of neem (azadirachta indica a. juss) and bakain (melia azedarach l.) seeds on oviposition and egg hatching of earias vittella (fab.) (lepidoptera: noctuidae). j appl entomol. 126: 238–243. govindarajan m, sivakumar r (2014) ovicidal, larvicidal and adulticidal properties of asparagus racemosus (willd.) (family: asparagaceae) root extracts against filariasis (culex quinquefasciatus), dengue (aedes aegypti) and malaria (anopheles stephensi) vector mosquitoes (diptera: culicidae). parasitol res. 113(4): 1435–1449. gu hj, cheng ss, huang cg, chen wj, chang st (2009) mosquito larvicidal activities of extractives from black heartwood-type cryptomeria japonica. parasitol res. 105(5): 1455–1458. ilahi i, khan i, tariq m, ahmad i (2012) larvicidal activities of different parts of melia azedarach linn. against culex quinquefasciatus say. (diptera: culicidae). j basic appl sci. 8: 23–28. kalyanasundaram m, das pk (1985) larvicidal and synergistic activity of plant extracts for mosquito control. indian j med res. 82: 19–23. koc s, oz e, aydın l, cetin h (2012) acaricidal activity of the essential oils from three lamiaceae plant species on rhipicephalus turanicus pom. (acari: ixodidae). parasitol res. 111: 1863– 1865. lawler sp, jensen t, dritz da, wichterman g (1999) field efficacy and nontarj arthropod-borne dis, december 2016, 10(4): 595–601 s koc et al.: evaluation of some plant … 601 http://jad.tums.ac.ir published online: october 04, 2016 get effects of the mosquito larvicides temephos, methoprene, and bacillus thuringiensis var. israelensis in florida mangrove swamps. j am mosq control assoc. 15(4): 446–452. mehmadi rm, al-khalaf aa (2010) larvicidal and histological effects of melia azedarach extract on culex quinquefasciatus say larvae (diptera: culicidae). j king saud univ sci. 22(2): 77–85. ndione rd, faye o, ndiaye m, dieye a, afoutou jm (2007) toxic effects of neem products (azadirachta indica a. juss) on aedes aegypti linnaeus 1762 larvae. african j biotech. 6 (24): 2846–2854. oz e, koc s, dincdusen o, mammadov r, cetin h (2013) larvicidal activity of cyclamen (myrsinaceae) extracts against the larvae of west nile virus vector culex pipiens l. (diptera: culicidae). asian pac j trop med. 6: 449–452. ser ö, cetin h (2015) toxicity of mosquito larvicides on non-target mosquito predator insect backswimmer (notonecta sp.). fres environ bull. 24: 311–316. schmidt gh, rembold h, ahmed aai, breuer am (1998) effect of melia azedarach fruit extract on juvenile hormone titer and protein content in the hemolymph of two species of noctuid lepidopteran larvae (insecta: lepidoptera: noctuidae). phytoparasitica. 26: 283–291. sharma rs, sharma sn, kumar a (2003) susceptibility status of japanese encephalitis vectors in kurnool and mehboobnagar districts of andhra pradesh, india. j comm dis. 35(2): 118–122. singh g, kaura v, singha d (2006) lethal and sublethal effects of different ecotypes of melia azedarach against plutella xylostella (lepidoptera: plutellidae). int j trop insect sci. 26 (2): 92– 100. sukumar k, michael jp, boobar lr (1991) botanical derivatives in mosquito control: a review. j am mosq control assoc. 7: 210–237. wandscheer cb, duque je, da silva ma, fukuyama y, wohlke jl, adelmann j, fontana jd (2004) larvicidal action of ethanolic extracts from fruit endocarps of melia azedarach and azadirachta indica against the dengue mosquito aedes aegypti. toxicon. 44 (8): 829–835. wirth mc (2010) mosquito resistance to bacterial larvicidal toxins. open toxinol j. 3: 126–140. j arthropod-borne dis, march 2017, 11(1): 166–170 t hazratian et al.: pharyngeal myiasis caused … 166 http://jad.tums.ac.ir published online: march 14, 2017 case report pharyngeal myiasis caused by sheep botfly, oestrus ovis (diptera: oestridae) larva, tabriz, east azarbaijan province, iran: a case report *teimour hazratian 1, ali tagizadeh 2, mohammad chaichi 3, madineh abbasi 1 1departement of parasitology, school of medicine, tabriz university of medical sciences, tabriz, iran 2departement of emergency medicine, school of medicine, tabriz university of medical sciences, tabriz, iran 3departement of infectious and tropical diseases, school of medicine, tabriz university of medical sciences, tabriz, iran (received 18 dec 2014; accepted 3 aug 2016) abstract myiasis is caused by the larvae of flies infesting animal or human tissues and organs. this report aims to present a case of pharyngeal myiasis caused by the larvae of oestrus ovis (diptera: oestridae). a 55-yr old drug addict living in the shahindeje village of western azerbaijan province, northwestern iran was referred to the emam reza hospital in tabriz, having a medical history of chronic obstructive pulmonary disease (copd) and hospitalized due to respiratory distress, 20 days ago. he was intubated with a mechanical ventilator (mv) because of his respiratory distress condition. there was an evidence of the presence of pulmonary nodules in his lungs following diagnosis, and a ct scan revealed a cavity in his lung. during the nasogastric intubation procedure, a larva was seen emerging from the patient’s mouth by one of the staff of the intensive care unit of the hospital. a laboratory diagnosis was performed in the entomology department of the school of medicine, tabriz university of medical sciences. interestingly, larvae of o. ovis were identified and confirmed following the laboratory proceedings. keywords: myiasis, pharyngeal myiasis, oestrus ovis, iran introduction propounded by hope in 1840, myiais originated from a greek word known as “miya” which means fly (sharma et al. 2008). myiasis is a term used to describe a medical condition in which the tissues and organs of human or animals are infested by the larvae of flies (masoodi et al. 2000). these fly larvae periodically obtain food from their hosts’ dead or living tissues, body fluids or directly ingest solid food (laner and crosskey 1993), which may result in various clinical manifestations. the sheep nasal botfly, o. ovis, is one of the remarkable causative agents of human myiasis (verstrynge and foets 2004). the family oestridae encompasses a wider range of species in which the larvae of all included species are obligate parasites of domestic animals or wildlife. unexpected intrusion of human tissues by a number of these parasites gives rise to severe pathological consequences. the development of oestrinae larvae occur in the nasopharyngeal cavities of a host of mammalian species. first-stage larvae of o. ovis are deposited by the females into the nostrils of susceptible domestic mammals. though uncommon, the larvae are occasionally transferred into the eyes, the mouth, or the external ear of man, especially in people who are in close contact with sheep and goats. notably, patients complain being hit in the eye by a wandering insect or object. in critical situations, migration of the larvae to the nasal cavities ensues, leading to an abscess formation, pain and headache. in human nasal cavities, the growth of fly larvae into a third stage hardly occurs. when *corresponding author: dr teimour hazratian, e-mail: hazratiant@tbzmed.ac.ir j arthropod-borne dis, march 2017, 11(1): 166–170 t hazratian et al.: pharyngeal myiasis caused … 167 http://jad.tums.ac.ir published online: march 14, 2017 these larvae spread to the throat, swallowing of food becomes challenging, however these symptoms do not last long (richard and crosskey 1996). classification of myiasis may be accomplished in two wasy, entomological or clinical. in entomology, myiasis is classified according to the parasitic characteristics of the fly larvae. these include obligatory, facultative and accidental parasitic fly larvae. clinically, however, myiasis is classified based on the anatomical region in which infestation occurs (langan et al. 2004). key examples of the clinical classifications include ophthalmomyiasis (janbakhsh et al. 1977), urogenital myiasis (jdalayer et al. 1978), orbit myiasis (khataminia et al. 1999), pharyngeal myiasis (karimi and vahidi 1999), ear myiasis (talari et al. 2002), auricular myiasis (tirgary et al. 1977), oral mucosa myiasis (hakimi and yazdi 2002) and wound myiasis (talari et al. 2004). in addition, clinical classification of myiasis may be described as primary or secondary. rarely seen in humans but frequent in cattle (called bicheiras), primary myiasis results from the invasion of living tissue-feeding (biophagous) fly larvae. comparatively, secondary myasis develop when necrobiophagous fly larvae invade and feed on dead tissues, commonly seen in humans in which the patients suffer necrotic cavity lesion (shinohara et al. 2004). the life cycle of the sheep bot fly begins from the fertilization of eggs in the female which later hatch into 1mm larvae in the female’s body. subsequently, a few larvae are directly released in a tiny drop of mucus into the nostrils of the animal host, after which the larvae migrate through the mucosa of the nostrils into the nasal sinuses. the maturation of the larvae continues at this stage following their growth and molting into the second larval stage, reaching a full length of 20mm with characteristic dark-striped segments. upon full maturation, the larva prepares itself for pupation in the ground after it emerges and falls from the nostrils. temperature plays a crucial role in the determination of the period of larval growth. in warmer weather conditions, larval maturation may be completed within 25–35d, while this may take a much longer time in colder climates up to 10 months. the temperature-dependent, pupal development stage is completed within 3–9 weeks, followed by the emergence of winged adults from the soil and subsequent mating. during the first few weeks (normally 2–4 weeks) after nuptial flight, the adults do not feed but may take up water (capelle and kenneth 2012). human infestation of the disease has been recognized extensively over the years (pampiglione et al. 2012). the disease is readily associated with shepherds having close contact with sheep, although a few more cases have been recorded in non-native individuals who harbor the disease and import them into their localities (masoodi and hosseini 2004). the disease is medically curable, or corrected with some medication (gregory et al. 2004). hospital-acquired nosocomial myiasis in patients is unusually observed, and oral myiasis occurs at any place, though not very common in developed countries (reuler et al. 1985). the underlying symptoms of oral myiasis condition entail traumatized face, mouth bleeding, distinctive lesions and debilitation of the palate (bhatt and jayakrishnan 2000). globally, nosocomial myiasis are evident in intensive care units, however, only a few cases are documented due to underreporting of the disease (joo and kim 2001). a study by najjari et al. and yaghoobi et al indicated the presence of several cases of myiasis in icu patients in iran (najjari et al. 2014), (yaghoobi et al. 2005). there have been a recent report projecting the rate of nosocomial myiasis infections as 4%, and a mortality rate of 1.3% (alizadeh et al. 2014). this novel case report highlights a case of pharyngeal myiasis caused by the larva of o. ovis for the first time in emam reza hospital, tabriz, eastern azerbaijan, iran. j arthropod-borne dis, march 2017, 11(1): 166–170 t hazratian et al.: pharyngeal myiasis caused … 168 http://jad.tums.ac.ir published online: march 14, 2017 case report a 55yr old addicted iranian man residing in the shahindeje villages of west azerbaijan province, northern iran and referred to the emam reza hospital of tabriz with clinical history of chronic obstructive pulmonary disease (copd) for 4yr was hospitalized due to respiratory distress, 20 days ago. urgent treatment was given, including nasogastric intubation with a mechanical ventilator (mv) due to his respiratory distress condition. pulmonary nodules were observed in his lungs following diagnosis, and a ct scan revealed cavities in his lungs. during the nasogastric intubation procedure, a larva was seen emerging from the patient’s mouth by an icu staff of the hospital, 30 days after hospitalization. this patient having a clinical history of copd finally died a few moments later. laboratory diagnosis was performed in the entomology department of the school of medicine, tabriz university of medical sciences. interestingly, larvae of o. ovis were identified and confirmed following the laboratory proceedings. precisely, the larva of o. ovis was identified as follows; the first segment (head) carried a highly sclerotized tissue surrounding the opening of the respiratory canal followed by 5 rides and two caudal swellings. myiasis of this type has not been previously reported from tabriz. this case report is the premier report of human pharyngeal myiasis caused by o. ovis. the majority of myiasis cases reported in the literature from iran were nasal myiasis originating from various areas. the agents of these cases were chrysomyia bezziana, lucilia sericata and erystalis tenax (diptera: syrphidae), (youssefi et al. 2012). lucilia sericata (diptera: calliphoridae) and ch. bezziana (diptera: calliphoridae) have been identified as causative agents of wound myiasis in iran (alizadeh et al. 2014). ophthalmomyiasis is an infestation of human eyes and orbital tissues with fly larvae belonging to the botfly species of o. ovis, and auricular myiasis is caused by ch. bezziana and lu. sericata (akbarzadeh 2012). however, oral myiasis arises from the infestation of obligatory parasitic fly larvae of o. ovis (diptera: oestridae) and wohlfahrtia magnifica (diptera: sarcophagidae) (alizadaeh et al. 2014). ingestion of whole flies leads to intestinal myiasis or accidental myiasis in human. in iran, two fly species of sarcophaga haemorrhoidalis and er. tenax are the main causative agents of intestinal myiasis (khalili et al. 2007, mandell et al. 2010). another type of myiasis, the urogenital myiasis, rarely occurs in iran, and two cases of this type have been reported so far in the country in which the larvae of ch. bezzizna and wo. magnifica were detected (salimi et al. 2010). all nosocomial myiasis infections recorded in iran are of the nasal type caused by facultative myiasis agents, lu. sericata (diptera: sarcophagidae) (mowlavi et al. 2011). this current study illustrates several interesting facts. first, the dwelling of the patient in poor hygienic conditions of a rural setting was a predisposition factor for larval infestation. second, lack of awareness also contributed to this condition in the patient. fig. 1. the pair of sharply curved mouth hooks and stout spines of the larva of oestrus ovis according to the laboratory diagnosis j arthropod-borne dis, march 2017, 11(1): 166–170 t hazratian et al.: pharyngeal myiasis caused … 169 http://jad.tums.ac.ir published online: march 14, 2017 fig. 2. posterior spiracles in larvae of oestrus ovis without 3 distinct slits and not divided into several plates conclusion this is by our knowledge the first report of an imported case of pharyngeal myiasis caused by o. ovis in northwestern iran. myiasis of this type has not been previously reported from tabriz, and this case of human pharyngeal myiasis caused by o. ovis is primarily reported for the first time in iran. the larva belonged to the species of o. ovis according to the morphological characters of larva in which the first segment was characterized by a strongly sclerotized tissue surrounding the outlet of the respiratory canal, completely followed by 5 rides and two caudal swelling. acknowledgements we would like to thank the staff of emam reza hospital med laboratory for the provision of the specimen, and we appreciate the contributive effort of mrs afsaneh dowlatkhah in the department of parasitology, school of medicine, tabriz university of medical sciences. the authors declare that there is no conflict of interest. references akbarzadeh k (2012) estimation of geographical distribution, biodiversity and species richness of myiasis inducing flies in fars province. tehran university of medical sciences, school of 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entomology, school of public health, tehran university of medical sciences, tehran, iran 2department of biology, payame noor university, tehran, iran 3department of photobiology, school of public health, tehran university of medical sciences, tehran, iran (received 21 jan 2014; accepted 14 apr 2015) abstract background: antimicrobial peptides play a role as effectors substances in the immunity of vertebrate and invertebrate hosts. in the current study, antimicrobial peptide was isolated from the haemolymph of the american cockroach, periplaneta americana. methods: micrococcus luteus as gram-positive bacteria and escherichia coli as gram-negative bacteria were candidate for injection. induction was done by injecting both bacteria into the abdominal cavity of two groups of cockroaches separately. the haemolymphs were collected 24 hours after post injection and initially tested against both bacteria. subsequently, the immune induced haemolymph was purified by high performance liquid chromatography (hplc) to separate the proteins responsible for the antibacterial activity. results: the non-induced haemolymph did not show any activity against both bacteria whereas induced haemolymph exhibited high activity against m. luteus but did less against e. coli. two fractions showed antibacterial activity against m. luteus. finally the molecular weight of the isolated antibacterial proteins were determined as 72 kda and 62 kda using sds-page. conclusion: induced haemolymph of american cockroaches has the ability to produce peptides to combat against gram-positive bacteria when an immune challenge is mounted. further work has to be done to sequence of the protein, which it would be advantageous. keywords: american cockroach, antibacterial protein, isolation, micrococcus luteus, escherichia coli introduction insects exhibit an amazing evolutionary success that can be explained by a variety of reasons (jarosz 1996, lazzaro 2008, gao and zhu 2012), among which the fact that their potent immunity play a major role in defense against bacteria (hoffmann et al. 1996, wilson et al. 1999, lamberty et al. 2001, lazzaro 2008). based on habitat of cockroaches, they are always exposed to potentially pathogenic microorganisms and parasites, but only a few encounters result in infection (gillespie and kanost 1997). antimicrobial peptides play an essential role in fighting against invading pathogens in insects, especially those that lack an adaptive immunity (toke 2005). normally due to microbial infection, antimicrobial peptides are synthesized in fat body or certain haemolymph cells of insects or body injury, and then rapidly released into haemolymph to kill microorganisms (brivio et al. 2006, yu et al. 2010, yakovlev 2011). however, insects count on cellular and humoral mechanisms to fight against pathogens and subse*corresponding author: dr hamid reza basseri, e-mail: basserih@tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 519–527 hr basseri et al.: isolation and purification of … 520 http://jad.tums.ac.ir published online: october 04, 2016 quently, innate immunity, which is being dominant in the final category. insects are remarkably resistant to bacterial infections by detecting of bacteria, a complex genetic cascade is activated, which eventuates in the production of a series of antibacterial peptides and is released into the haemolymph (eleftherianos et al. 2006, eleftherianos et al. 2007). these antimicrobial peptides are mostly small, amphipathic, cationic molecules (gao and zhu 2013). they have an effect on membrane of microbial cell changing permeability or by breakdown bacteria membrane (toke 2005, dai et al. 2008, huang et al. 2008). in addition, insect peptides may affect the synthesizing of dna or protein as well as the protein folding of the bacteria (otvos 2000, huang et al. 2008, shen et al. 2010, bang et al. 2012). insects can synthesize some antimicrobial peptides such as cecropins, which exhibit anticancer activity (ye et al. 2004). some insects can synthesize inducible antibacterial peptides such as lysozyme which is also constitutive like lipopolysaccharide (lps)-binding protein which was isolated from the haemolymph of the american cockroach (periplaneta americana) (ha lee et al. 2007, fiolka 2008). this protein acts as an opsonin (jomori and natori 1992, hashimoto et al. 2009, kim et al. 2010). generally, five major groups of antibacterial peptides have been introduced (hultmark 1993) including cecropins, insect defensins, attacin-like (glycine-rich) proteins, proline rich peptides and lysozymes. the mechanisms of some these peptides have been studied extensively (sawa and kurahashi 1999, imler and bulet 2005, wang et al. 2009). american cockroach spends most of its time in sewage, sewer pipe. these environments usually contain high density of bacteria. therefore, it is likely to defend itself against invading pathogens by means of antimicrobial compounds. the purpose of the current study was to isolate and purify an antimicrobial protein from the haemolymph of american cockroach. in this study, we isolated and purified an antimicrobial protein in immune induced haemolymph of p. americana which may open up new way of research to detect new antimicrobial pathogens. materials and methods insect rearing and haemolymph collection american cockroaches, p. americana, were maintained in an insectary at 25±2 °c with a 12h light/dark ratio, and fed on dried bred, date and water. to collect non-induced haemolymph, two groups each included 30 adults and final instars cockroaches were anaesthetized with co2. the ventral surface of sternum of each insect was sterilized with 70% ethanol, and the coxal membranes of legs were punched with still needle. the exuded haemolymph from the wounds was immediately collected, centrifuged at 1800×g for 10 minutes. for collecting of induced haemolymph, 100 μl of m. luteus or e. coli (106 cells/ml) was injected into the abdominal cavity of each cockroach. based on preliminary time optimization, the insects were anesthetized 20 hours after injection and the haemolymph was collected using sterile syringe, and then centrifuged. all collected samples were transferred into clean and chilled eppendorf tubes containing few crystals of phenyl thiourea in order to prevent melanization. finally, the supernatants and pallets were kept in -20 °c until used. the protein concentration of all samples was analyzed by bradford method before being used. bacterial strains in order to screen the antibacterial activity of the heamolymph compounds on and based on evidences mentioned in previous studies (jomori and natori 1990, serja et al. 2003), two strains of bacteria, one gram positive and one gram negative bacteria were j arthropod-borne dis, december 2016, 10(4): 519–527 hr basseri et al.: isolation and purification of … 521 http://jad.tums.ac.ir published online: october 04, 2016 chosen. the bacteria were collected from persian type culture collection, science and industrial research organization of iran. the bacteria strains used for screening antimicrobial peptides were nonpathogenic bacteria, atcc 9341recently named as kocuria rhizophila and e. coli atcc25922 which is susceptible to all of the antibiotic, and microccus leteus atcc 9341 is resistance to all antibiotic except chloramphenicol, doxy cycline, hydramycinand tetracline. antibacterial assay the antibacterial assays were done by diffusion disc method. sterile petri dishes received 20 ml of melted luria burtenii medium, ph 7.0. after solidification of the medium, the agar surface was inoculated with 0.1 ml (106 cells/ml) of the test bacterial strain and spread with the help of a glass rod. sterile paper discs soaked with 20 μl of the haemolymph (at concentration of 1.21mg/ml) was places on the medium. as control, paper discs soaked with 20 µ l of non-induced haemolymph (at concentration of 1.34 mg/ml) were used. the plate was incubated overnight at 37 °c, and the diameters of the clear zones were recorded. the assay was carried out three times. reverse phase-high performance liquid chromatography (rp-hplc) the antibacterial compounds from haemolymph was purified by semi-preparative high performance liquid chromatography (rphplc) (knauer, germany) under the following conditions: flow rate =1 ml min-1, stationary phase = spherisorb c18 column (waters, usa, ods2 column 5 µ m, 250 mm×4.6mm), mobile phase = acetonitrile in water containing 0.1% trifluoroacetic acid (tfa), detector wavelength at 230 nm. the fractions were eluted in an elution range of 20% to 80% acetonitrile in water for 40 min. the fractions were concentrated by freezedried, redissolved in 50 ml of apyrogenic water and then the antimicrobial activity of all fractions against both bacteria was tested using disc diffusion assay. separation of antibacterial agents by sds-page the eluted protein was applied to sdspage as discussed by laemmeli (1970). the protein was mixed with 5l of 5x sdspage sample buffer under non-reducing conditions and then heated for 10 minutes at 100 c. the sample (20 l) was then centrifuged at 10000g for 5 min to remove debris before loading into the gel. the supernatant was applied to sds-page electrophoresis in a 10% polyacrylamide gel to analyse the eluted protein. electrophoresis was performed at a constant voltage of 200v for ca, 45 min using the bio-rad mini-protean ii apparatus (bio-rad laboratories ltd, hemel hempstead, hertforshire, uk). after separation, the gel was fixed by 30-min-long gentle shaking in 10% acetic acid, 50% methanol (v/v) and visualized by staining with coomassie brillant blue r-250. results antibacterial assay the whole non-induced haemolymph of cockroach did not show any antibacterial activity against m. luteus and e. coli. on contrary, the induced haemolymph showed high antibacterial activity against m. luteus and less activity against e. coli (fig. 1a, b). purification of the antibacterial peptides the haemolymph with antibacterial activity subjected to semi-preparative reverse phase-high performance liquid chromatography (rp-hplc) for separation and purification of the peptides. fifteen different peaks were obtained and the fractions collected (fig. 2). all fractions were freezedried and redissolved in 50 ml of apyrogenic j arthropod-borne dis, december 2016, 10(4): 519–527 hr basseri et al.: isolation and purification of … 522 http://jad.tums.ac.ir published online: october 04, 2016 water and then separately subjected to antibacterial susceptibility test against both m. luteus and e. coli. factions 2 and 3 showed high antibacterial activity against only m. luteus (fig. 3). no activity was observed against e. coli. to remove any possible impurity, the active fractions were reloaded onto rp-hplc, tested for antibacterial activity and then subjected to sds-page. two protein bands were observed at 60 kda and 72 kda on the polyacrylamide gel (fig. 4). fig. 1. antibacterial activity of whole haemolymph against micrococcus luteus (a) and escherichia coli (b) where ‘s’ indicates treatment and ‘b’ control (non-induced haemolymph). the clear zone around the discs indicates antibacterial activity fig. 2. rp-hplc chromatogram of periplaneta americana heamolymph induced with micrococcus luteus. the absorbance was measured at 230 nm. the antibacterial activity of all fractions was separately tested against m. luteus. only fractions 2 and 3 showed antibacterial activity. fractions 2 and 3 were concentrated and reloaded onto rp-hplc to remove any possible impurity fig. 3. antibacterial activity of fraction 2 (a) and 3 (b) against micrococcus luteus (a). the fractions were concentrated by freeze-dried, redissolved in 50 ml of apyrogenic water j arthropod-borne dis, december 2016, 10(4): 519–527 hr basseri et al.: isolation and purification of … 523 http://jad.tums.ac.ir published online: october 04, 2016 fig. 4. sds-page of fractions 2 and 3 l2 shows molecular mass of whole heamolymph proteins and l1 and l3 show molecular weight of fraction 2 and 3 respectively discussion insects have many systems that work together to limit the spread of bacteria and other pathogens. they can synthesis antimicrobial peptides within their body. they have pattern recognition proteins that can bind to the external surface of bacteria or other pathogens (vilcinskas 2013). in this study the non-induced haemolymph and the induced haemolymph of american cockroach were screened for the antimicrobial activity against two gram negative and gram-positive bacterial strains. the non-induced haemolymph did not show inhibitory activity against any of the tested bacterial strains. it does not indicate that peptides are absent but they may be present in lesser quantity so that no detectible action in vitro studies is seen. it has been stated that the adult american cockroach can generate an adaptive humoral immune response to pathogens (karp 1985, george et al. 1987, faulhaber and karp 1992). in this scenario, defensive peptides or proteins play a main and crucial in insect humoral immune response against invading microorganisms (leclerc and reichhart 2004, levy et al. 2004, mak et al. 2010). generally, each insect species may possess an individual set of antimicrobial peptides synthesized in response to non-self recognition (engstrom 1999). for example, drosophila melanogaster metchnikowins peptides have no activity against gram-negative bacteria but they inhibit growth of m. luteus (imler and bulet 2005, rahnamaeian et al. 2009, rahnamaeian and vilcinskas 2012). we found similar result by injecting gramnegative bacteria, e. coli and gram-positive bacteria, m. luteus. although, whole haemolymph of immune cockroaches showed antimicrobial activity against e. coli (fig. 1) but we could not find these activities in any fraction. it seems that they are not directly involved in killing e. coli, and some molecules may be involved in signaling mechanism to remove the bacteria. another possibility is that the quantity of antimicrobial activity against e. coli may too less to see visible action. various insect species, which bacteria injected into the haemocoel, elicit the synthesis of a number of peptides and proteins, which are individually or cooperatively active against the foreign microorganisms (cociancich et al. 1994, vilcinskas 2013). induction is a common process in many insect species (cociancich et al. 1994). in the present study, induction of such peptide(s) was done by injecting e. coli or m. luteus into the abdominal cavity of american cockroaches. the immune induced peptides were active against tested bacterial strains and this result suggests that peptides are produced to combat bacterial infection. however, constitutive and inducible proteins may be present in haemolymph of some insects and may act as signaling molecules such as lysozyme (royet and dziarski 2007, royet et al. 2011, bosco-drayon et al. 2012). we found two proteins with antibacterial activity and then they were subjected to nonreducing sds-page to determine the size j arthropod-borne dis, december 2016, 10(4): 519–527 hr basseri et al.: isolation and purification of … 524 http://jad.tums.ac.ir published online: october 04, 2016 of proteins. the molecular weight of proteins was 60 and 72 kda (fig. 4) and these separated proteins allow us to observe antibacterial activity of them separately. the purified proteins are predominantly active against the gram-positive bacteria; it suggests that the antibacterial activity of the peptides is related to the cell wall of the bacteria. it may be assumed that the proteins identified in this study might play an important role in their self-defense against bacterial infection in american cockroaches individually or cooperatively. however, further studies are needed to work out the combined effect of peptides. at this point, it is also important to remember the development of resistance to ordinary antibiotic like gentamicin, penicillin and so on, by variety of infectious bacteria. it is also believed that antimicrobial peptides will be assumed in the near future as an alternative for the nowadays-classical antibiotics (małgorzata et al. 2007). the advantages of antimicrobial peptides are many viz, selectivity, fast killing, broad antimicrobial spectra and lack of resistance development (matsuzaki 1999, papo and shai 2005). however, the present result is preliminary and future study will be done in other methods to confirm the antimicrobial property of peptides. conclusion the main idea of this research is to isolate new antimicrobial peptides from haemolymph of american cockroaches, periplaneta americana and to study its activity against these two a gram-positive and gram-negative bacteria. we found that induced haemolymph of american cockroaches has the ability to produce peptides to combat against gram-positive bacteria when an immune challenge is mounted. before immune challenge, 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(diptera: calliphoridae). zh evol biokhim fiziol. 47(6): 461–468. ye js, zheng xj, leung kw, chen hm, sheu fs (2004) induction of transient ion channel-like pores in a cancer cell j arthropod-borne dis, december 2016, 10(4): 519–527 hr basseri et al.: isolation and purification of … 527 http://jad.tums.ac.ir published online: october 04, 2016 by antibiotic peptide. j biochem. 136: 255–259. yu y, park jw, kwon hm, hwang ho, jang ih, masuda a, kurokawa k, nakayama h, lee wj, dohmae n, zhang j, lee bl (2010) diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acidtype peptidoglycan in insects. j biol chem. 285: 32937–32945. j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 394 http://jad.tums.ac.ir published online: january 06, 2016 original article perceived risk of dengue in ones’ living environment as a determinant of behavior change through social mobilization and communication: evidence from a high risk area in sri lanka *hasini banneheke 1, 2, sarath paranavitane 3, vathsala jayasuriya 4, sarath banneheka 5 1specialist in medical parasitology, faculty of medical sciences, university of sri jayewardenepura, nugegoda, sri lanka 2international medical university, kuala lumpur, malaysia 3central medical centre (pvt) ltd, nugegoda, sri lanka 4specialist in community medicine, faculty of medical sciences, university of sri jayewardenepura, nugegoda, sri lanka 5department of statistics, faculty of applied sciences, university of sri jayewardenepura, nugegoda, sri lanka (received 8 feb 2014; accepted 30 nov 2014) abstract background: the aim of this study was to assess community knowledge and behavioral impact of the social mobilization and communication strategy applied in a dengue high-risk area in sri lanka. methods: a group of adults visiting selected primary care facilities in colombo district were interviewed to collect socio-demographic data, attributes of knowledge regarding dengue and the responsive behaviors adopted by them following the dengue control program though the media and social marketing campaigns. these attributes were classified as `good ', `fair,' or `poor' by developing a composite scale for analysis and interpretation of data. results: the primary source of information was television in the majority. the overall knowledge of the disease, vector and control methods was poor. the overall level of contribution to dengue control activities was good. conclusion: awareness of the disease and its complications had not contributed to favorable behavior changes. while the social mobilization and behavior change campaign in sri lanka had low impact on knowledge and behaviors, a better understating of community perceptions of df and how these perceptions are formulated within the social and cultural context; would be useful to improve uptake. this knowledge would be valuable for program planners to strengthen dengue control activities in sl and other similar settings across the region. keywords: dengue control, social mobilization, behavior change, sri lanka introduction dengue ranks as the most important mosquito borne viral disease in the world endemic in over 100 countries in the world affecting 40% of the world population. according to who estimates, 50–100 million cases of dengue infections with 500000 cases of dengue hemorrhagic fever (dhf) and 22,000 deaths occur annually in the world (who 2011).. in asian countries, dhf is a leading cause of serious illness and death among children (who 2011) and case fatality rates vary between 0.5 and 3.5%. dengue fever (df) has now become the most dreaded and significant communicable disease affecting the south asian tropical island of sri lanka (sl) characterized by cyclical epidemics in urban centers, spread to rural areas and presence of multiple circulating virus serotypes. despite extensive efforts by the government of sl strengthening the dengue management, surveillance and control activities through a national plan of action (kyu et *corresponding authors: dr hasini banneheke, e-mail: hasini.banneheke@gmail.com j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 395 http://jad.tums.ac.ir published online: january 06, 2016 al. 2005), there had been a steady increase in the incidence of dengue in the country (annual health bulletine 2012, national plan of action for prevention and control of dengue fever 2005). in 2014 (up to november), there were 39521 cases of suspected cases of dengue reported to the national surveillance system. out of this 56.40% were reported from the western province which includes colombo district (quarterly epidemiology bulletin 2014). these epidemics cause significant morbidity and mortality and its control and management constraints an already over-burdened medical and financial resources. traditional programs for dengue control and vector control used vertical approaches to reduce the source of transmission including physical (e.g. destruction or other physical manipulation of water-holding containers), biological (e.g. use of bacteria), and chemical (e.g. use of larvicides, spraying with systemic insecticides) control methods. however, these efforts have only resulted in short-term control as the areas become reinfested in a short period. one of the recent strategies initiated in sl was ‘behavioral changes through effective communication (social mobilization)’ as recommended by who for dengue control. during this campaign, public awareness activities were carried out to disseminate messages regarding the spread of the disease and the responsibility of the community with special emphasis on removal of mosquito breeding sites and environmental management. electronic media coordinated the broadcasting of messages with the aim of improving knowledge, attitude, skills and practices towards eradicating dengue and to obtain community support towards control activities. despite being in effect for over 5 years, these strategies have not been examined from the user’s perspective to determine if they have received the intended messages and or adapted the desired behaviors. in the absence of an effective vaccine for disease control, control of the disease transmitting vectors aedes aegypti and ae. alobopictus mosquitoes is the mainstay of control with regards to this major public health problem in many parts of the world (erlanger et al. 2008). vector control is effective in reducing mosquito populations, when interventions use a community-based, integrated approach (erlanger et al. 2008). however, as vector control is highly dependent on human behavior and actions, a major obstacle to effective implementation of vector control has been the inability to mobilize and coordinate the resources needed to achieve and sustain behavioral impact among populations at risk of df/dhf (parks et al. 2004). social mobilization and communication strategies have been used for a number of years in an effort to achieve such behavioral changes among communities. social mobilization is the process of bringing together all feasible and practical inter-sectoral social allies to raise people’s awareness of and demand for dengue prevention and control, to assist in the delivery of resources and services, and to strengthen community participation for sustainability and self-reliance. social marketing is based on an appeal to the individual through mass media and other methods. evidence from other settings such as colombia, honduras, and indonesia has demonstrated the usefulness of this method in dengue control. in columbia when high school students were trained to assist as community-based health educators, a steady decline was reported in the number of houses with ae. aegypti larvae (kroeger et al. 1995) and in honduras, a method for cleaning large cement washbasins and metal drums was shown to have a significant impact on aedes larval populations (fernandez et al. 1998). moreover, in malaysia, (crabtree et al. 2001) an integrated social moj arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 396 http://jad.tums.ac.ir published online: january 06, 2016 bilization and communication campaign motivated householders in johor bahru district to seek prompt treatment, to destroy larval breeding sites, and to organize voluntary teams to inspect and control larval breeding sites in public spaces. other methods that have been effective include a participatory action research project involving community meetings, community-based production of communication materials, and door-to-door education aimed at household behaviors, particularly in the disposal of nonessential containers. for dengue control in sri lanka, national dengue control unit (ndcu) established in 2005 under the ministry of health, takes the lead in providing technical guidance for social mobilization focusing on health issues in df/dhf control activities in collaboration with the epidemiological unit and other special units such as anti malaria campaign and anti filariasis campaign at central level. the national action plan set up centrally is executed at district level by regional malaria officers, medical officers, entomologists and their teams in the ministry of health (epidemiological bulletine sri lanka 2008). according to the 2009 national action plan, a media campaign was developed with the objective of achieving sustained community action for prevention and control of dengue. the strategies employed include, dissemination of comprehensive, accurate, clear and consistent messages co-ordinated through print media, television (tv) and radio regarding characteristics of the disease and the vector, spread of the disease, responsibility of community for prevention and control. specific tasks such as declaration of mosquito control weeks, cleaning of schools and work environments are done with media seminars and mass media publicity activities. health education message to the parents through schoolchildren. health education in the form of seminars, distribution of leaflets, display of billboards by the road side and advertisements in electronic and printed media were used to deliver the messages. red notices were displayed on publicly visible places at premises that tested positive for dengue larva as another health education message. health care workers and volunteers were also being deployed for periodical inspection for mosquito larva and to provide health education for residents on a house-tohouse basis. proper disposal of refuse and reduction of sources of refuse were the main emphasis of the social mobilization campaign. electronic and print media were used to reinforce the messages and obtain public support before or during outbreaks. ndcu provides funds and technical assistance directly or through non-governmental organizations such as who, especially to those areas at high risk of dengue, and also reviews the control programmes at grass root level while continuing surveillance at the national level. the objective of this study was to assess community knowledge and behavioral impact of the social mobilization and communication strategy applied in a dengue highrisk area in sri lanka. materials and methods this descriptive cross-sectional study was carried out in 2013 in a dengue high-risk area within the capital province of the country; the colombo district. although mass media approaches used by the national dengue control program is expected to reach all parts of the country, the extent to which it reaches rural areas which have less access to and coverage of radios/tv’s and electricity. thus to increase the likelihood of the health messages reaching all of the target population and to obtain a more homogenous population with regards to socio-economic status. three hundred and forty nine randomly selected adults over 18 years visiting sej arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 397 http://jad.tums.ac.ir published online: january 06, 2016 lected primary care facilities were recruited. a pre-tested interviewer-administered structured questionnaire was used to collect socio-demographic data, attributes of knowledge regarding dengue (as disseminated by the dengue control program though the media and social marketing campaigns) and the responsive behaviors adopted by participants. questions collected responses about awareness of disease characteristics, epidemiology, symptomatology, spread of the disease and their perceived responsibility for prevention, actions and measures adopted for vector control. the extent to which the attributes were internalized by the participants was determined by assessing their perception of morbidity and mortality of the condition as well as the perceived risks of dengue in their own living environments. these attributes were classified as `good ', `fair,' or `poor' by developing a composite scale using several aspects of each assessed element. epidata 3.0 software was used for the data entry and error detection. descriptive statistics were derived using spss 16.0 software program. ethical clearance was obtained from the ethics review committee of faculty of medical sciences, university of sri jayewardenepura, sri lanka. results socio-demographic characteristics of the 349 participants are given in table 1 showing the almost equal representation of both sexes and married and unmarried adults. respondents were asked to list their sources of information regarding df/dhf as different strategies were used for dissemination of health messages, and results are shown in table 2. table 3 summarizes the respondents’ awareness of characteristics of df/dhf and its symptomology. when messages are disseminated, the effectiveness of them can be measured by the translation of knowledge into practice. information regarding the vector, its habits, and measures taken up by individuals was assessed during the survey (table 4). only 20% of the study participants knew the biting times (dusk and dawn) of dengue mosquitoes. elimination of breeding places is one of the main strategies promoted to control vector density in this high-risk area. although the respondents adopted a range of these strategies, the proportion that used each of these methods was varied. regular inspection of surroundings and removal of breeding sites was rarely or never adhered to by 20%. less effective methods to prevent mosquito-human contact such as use of bed nets at night and insecticide sprays were reported as strategies used to prevent dengue by most respondents. the composite variable created by amalgamating responses to dengue control activities are also shown in table 4. participants’ knowledge regarding the epidemiological characteristics of df and dhf, perceptions of its significance in their own living environment and its symptomology were assessed and the results are given in table 5. perceptions regarding seriousness of df and dhf (in terms of morbidity and mortality caused by them) and level of impact on one’s participants’ living area are given in table 5. the overall knowledge regarding epidemiological significance of df in living environment was derived by amalgamating responses to the above questions (table 5). comparison of knowledge and practices the composite variables created for knowledge and contribution to dengue control activities were compared to determine the extent to which knowledge is translated to practice by this community. as shown in table 6 and 7, higher proportions of those with good knowledge and average knowledge of df, had relatively good and fair contribuj arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 398 http://jad.tums.ac.ir published online: january 06, 2016 tions to dengue control activities. however, a larger proportion of those with poor knowledge of df, contributed poorly to the control activities. a similar trend was seen when knowledge of dhf and contribution to dengue control activities were compared. perceived significance of dengue in one’s own living environment was considered an important factor in respondents’ contribution to control activities (table 8) and it was seen that a larger proportion of those who perceived dengue as highly significant or somewhat significant made good and fair contributions to control activities. significantly, 69 % of those who considered df to be of low risk contributed poorly to the control activities compared to 7.5% of those who considered it highly significant and 11.7% of those who perceived it to be somewhat significant. table 1. socio-demographic characteristics of the participants variable number (n=349) percentage (%) age group (range 18–72 yr) < 30 169 48.4 31–65 155 44.4 > 65 25 7.2 gender male 181 51.9 female 168 48.1 civil status unmarried 163 46.7 married 186 53.3 educational level primary or less 31 8.9 primary to ordinary level 98 28.1 advanced level (a/l) qualified 176 50.4 higher education 44 12.6 standard of living low 109 31.2 middle 159 45.6 high 81 23.2 employment status student 60 17.2 house wife 34 9.7 employed/ retired 182 52.2 currently unemployed 73 20.9 table 2. primary source of information source number (n=349) percentage (%) television 178 51.0 newspapers/ magazines 85 24.4 radio 29 8.3 family and friends 27 7.7 health staff 5 1.4 other (web/public lectures etc) 25 7.2 table 3. knowledge of characteristics and symptoms of ddf/dhf (n= 349) characteristics /symptom number (%)* characteristics /symptom number (%)* cause of dengue fever virus parasite bacteria fungus 218 (62.5) 93 (26.6) 32 (9.2) 6 (1.7) life threatening symptoms breathing difficulty cold extremities signs of dehydration do not know 161 (46.1) 107 (30.7) 92 (26.4) 109 (31.2) j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 399 http://jad.tums.ac.ir published online: january 06, 2016 symptoms of df high fever headache muscle or joint pain skin rash pain behind the eyes nausea/vomiting abdominal pain do not know 292 (83.7) 187 (53.6) 125 (35.8) 113 (32.4) 101 (28.9) 97 (27.8) 63 (18.1) 22 (6.3) symptoms of dhf high fever skin bruising/bleeding patches flushed appearance nose/gum bleeding vomiting blood dark stools abdominal pain do not know 226 (64.8) 194 (55.6) 149 (42.7) 131 (37.5) 110 (31.5) 97 (27.8) 62 (17.8) 47 (13.5) overall knowledge of df good average poor 106 (30.4) 63 (18.1) 180 (51.5) overall knowledge of dhf good average poor 112 (32.1) 41 (11.7) 196 (56.2) *total does not add up to 349 (or 100%) as multiple responses are given table 4. knowledge and practices on vector and vector control activities (n=349) responses of the participants number (%) knowledge type of mosquito aedes species anopheles species culex species 190 (54.4) 101 (28.9) 58 (16.6) biting habits day time dawn and dusk night time 242 (69.3) 70 (20.1) 37 (10.6) possible preventive measures avoid mosquito bites (human-vector contact) avoid contact with dengue patients (human-human contact) getting a vaccine for dengue 254 (72.8) 66 (18.9) 29 (8.3) practices personal protective measures (human-vector contact) use of mosquito repellents mosquito nets when sleeping during the day clothes to cover extremities avoid areas with high vector density none 215 (61.6) 165 (47.3) 133 (38.1) 119 (34.1) 14 (4.0) eliminate breeding places inspect surroundings remove clean water collections keep surroundings clean, remove potential breeding sties clean surroundings-schools change water collections (tanks, vases) clean surroundings-workplace destroy /remove possible water collectors cover water collections/containers change water in bird baths add salt to water containers other measures 284 (81.4) 240 (68.8) 223 (63.9) 205 (58.7) 187 (53.6) 148 (42.4) 141 (40.4) 140 (40.1) 123 (35.2) 69 (19.8) 4 (1.1) table 3. continued… j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 400 http://jad.tums.ac.ir published online: january 06, 2016 destroy breeding places often sometimes rarely never 150 (43.0) 130 (37.2) 57 (16.3) 12 (3.4) sleep under mosquito net (night time) often sometimes rarely never 205 (58.7) 87 (24.9) 30 (8.6) 27 (7.7) use insecticide sprays often sometimes rarely never 86 (24.6) 123 (35.2) 77 (22.1) 63 (18.1) contribution to dengue control activities (composite variable) good fair poor 80 (22.9) 207 (59.3) 62 (17.8) *total does not add up to 349 (or 100%) as multiple responses are given table 5. knowledge regarding epidemiology and significance of the disease epidemiology and significance of the disease number (%) (n= 349) morbidity and mortality of dengue fever very high somewhat high moderate low do not know 167 (47.9) 107 (30.7) 60 (17.2) 8 (2.3) 7 (2.0) morbidity and mortality of dengue hemorrhagic fever very high somewhat high moderate low do not know 256 (73.4) 66 (18.9) 10 (2.9) 6 (1.7) 11 (3.2) significance of the disease in own residential area highly significant somewhat significant moderate and low level of significance do not know 54 (15.5) 244 (69.9) 19 (5.4) 32 (9.2) overall rating of epidemiological significance of df in living environment (composite variable) highly significant somewhat significant not significant 187 (53.6 ) 111 (31.8) 51 (14.6) *total does not add up to 349 (or 100%) as multiple responses are given table 4. continued… j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 401 http://jad.tums.ac.ir published online: january 06, 2016 table 6. contribution to dengue control activities compared to overall knowledge of df contribution to dengue control activities overall knowledge of df number (%) good average poor good 32 (30.2) 32 (50.8) 16 (8.9) fair 72 (67.9) 30 (47.6) 105 (58.3) poor 2 (1.9) 1 (1.6) 59 (32.8) total 106 (100.0) 63 (100.0) 180 (100.0) chi= 84.16, df= 4, p< 0.001 table 7. contribution to dengue control activities compared to overall knowledge of dhf contribution to dengue control activities (composite variable) overall knowledge of dhf number (%) good average poor good 55 (49.1) 14 (34.1) 11 (5.6) fair 52 (46.4) 24 (58.5) 131 (66.8) poor 5 (4.5) 3 (7.3) 54 (27.6) total 112 (100.0) 41 (100.0) 196 (100.0) chi90.6, df= 4, p< 0.001 table 8. contribution to dengue control activities by epidemiological significance of dengue in the living environment contribution to dengue control activities overall rating of epidemiological significance of df in living environment number (%) highly significant somewhat not significant good 68 (36.4) 9 (8.1) 3 (5.9) fair 105 (56.1) 89 (80.2) 13 (25.5) poor 14 (7.5) 13 (11.7) 35 (68.6) total 187 (100.0) 111 (100.0) 51 (100.0) chi137.8, df= 4, p< 0.001 discussion in the absence of an effective treatment and/or a vaccine for dengue, for the time being, the only methods for preventing and controlling are prompt diagnosis of cases of fever, appropriate clinical management, reducing human-vector contact, and controlling larval habitats. the who recommends social mobilization and communication for behavioral impact based on the combi (communication-for behavioral-impact) model (who 2005) and this has been adopted in the sri lankan national plan of action for dengue prevention and control since 2009 (national plan of action for prevention and control of dengue fever 2005). according to the results of our survey, it was seen that j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 402 http://jad.tums.ac.ir published online: january 06, 2016 common knowledge regarding df/dhf has reached majority of this community through television, radio and newspapers, similar to the effect of such social marketing programs in other countries (hairi et al. 2003, pérezguerra et al. 2005, matta et al. 2006, itrat et al. 2008). considering the life style of urban sri lankans who watch television as the major if not sole leisure activity, this medium could be utilized better to deliver the health education massages. however, it is important to maximize the way it is being done by utilizing peak hours and by using short messages. meanwhile residents living in remote areas with no access to television, would require alternative modes for delivery of health education messages. the participants overall knowledge of the disease, vector and control methods were low and respondents continued to use methods that are not effective for the dengue mosquito vector control including, use of mosquito nets at night and smoke to drive them away. significantly, a proportion of this community completely lacked awareness of some aspects of the disease and failed to take any personal protective measures or participate in any of the control activities. one of the reasons may be due to fact that health messages had not been done in a receptive manner with clear ‘dos’ and ‘don’t’ messages. since there is no ‘one for all’ type of health education process for dengue, effectiveness can be based on the characteristics of the educator and method of delivery per se. a uniform massage which is simplified to suit the educational level and age of the recipients, can be effective health educational material. overall, improvement of the quality of presentation may enhance the accessibility to a larger target group. although many of the respondents participated in activities aimed at eliminating breeding sites, a proportion of them continued to have a nonchalant approach to this essential dengue control activity despite the social marketing and community mobilization campaign. when the overall level of contribution to dengue control activities were assessed here, a significant proportion showed a poor response similar to findings from other studies. as previously acknowledged by the who and others, an informed and educated individual did not necessarily create a behaviorally responsive community member concerning dengue control activities. the study setting, colombo district is a high-risk area in sl where dengue is endemic with frequent outbreaks, and majority of respondents considered df to be of high morbidity and mortality and recognized its significance in their own residential area. however, majority (69.9%) thought it is only somewhat significant and 15% of them did not consider it significant in their own residential area, or did not know of its significance. short message services (sms) and social media have become important and effective modes of communication in many societies across world. as most telecommunication services have the access to a large pool of customers sms and social media can be used to alert or alarm the communities at risk when the cases of dengue are rising or an epidemic is impending. effective and sensitive videos shared via social media could also be used to create awareness and sensitization. often research into kap regarding dengue fails to assess the respondents’ perceived personal risk, which is important for developing an effective behavioral change communication package. as we have shown here, despite the overt risks in this setting, a significant proportion believed their own personal risks to be low. this highlights the need for dengue control programs to be informed by formative research. also local social and cultural contexts may be important in determining individuals’ uptake and responses to messages. therefore, periodical reviews existing programs and health education strategies are an urgent j arthropod-borne dis, september 2016, 10(3): 394–404 h banneheke et al.: perceived risk of … 403 http://jad.tums.ac.ir published online: january 06, 2016 need in dengue high risk areas. the web and social media can also be used to gather public perceptions about exiting strategies and programs. most respondents were aware of the basic facts regarding the dengue vector, lifethreatening symptoms of df and were able to list a few common symptoms of df and dhf as demonstrated in previous similar studies in this setting. although it was previously believed that awareness of the disease and its complications would encourage communities to adapt responsive behaviors, it is evident from this study that such knowledge does not necessarily lead to favorable behavior changes. in our study, although the community was aware of the principals of prevention, the level of involvement in control activities were low. it was demonstrated that the perceived risk of df in their own living environment was important, as almost 70% of those who considered it to be of low risk contributed poorly to the control activities. conclusion this study demonstrated for the first time the importance of assessing the community perceived risk of the disease in their own living environment as a determinant of behavior changes concerning dengue control activities in colombo district, a high-risk area for df/dhf in sri lanka. although this study was based on respondents’ self-assessments, self-reported behaviors, and probably an over-reporting of acceptable behaviors, it nonetheless reveals the importance of their own perceptions about personal risks and this is likely to be accurate. formative research that provides a better understating of the community perceptions of df/dhf, its personal significance in a living environment and how these perceptions are shaped and formulated within the local social and cultural context would be useful for program planners and practitioners to strengthen the dengue control activities not only in this setting but in other similar settings across the region. acknowledgements the authors declare that there is no conflict of interests. the study was a self-funded project. references annual health bulletin (2012) ministry of health, colombo, sri lanka. avaialable at: http://www.health.gov. lk. accessed on 21/11/2014. crabtree 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organization (2005) dengue, dengue haemorrhagic fever, dengue shock syndrome in the context of the integrated management of childhood illness. available at: www.who.int. accessed on 01/09/2013. world health organization (2011) media centre, dengue and severe dengue fact sheet 117, world health organization, geneva, switzerland. available at: www.who.int. accessed on 17/12/2013. j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 78 original article application of flumethrin pour-on on reservoir dogs and its efficacy against sand flies in endemic focus of visceral leishmaniasis, meshkinshahr, iran mohammad reza jalilnavaz 1, mohammad reza abai 2, *hassan vatandoost 2, 3, mehdi mohebali 4, amir ahmad akhavan 2, zabihollah zarei 5, sayena rafizadeh 6, hassan bakhshi 2, *yaver rassi 2 1tehran university of medical sciences, international campus (tumsic), tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3institue for environmental research, tehran university of medical sciences, tehran, iran 4department of medical parasitology, school of public health, tehran university of medical sciences, tehran, iran 5public health research of meshginshahr, school of public health, tehran university of medical sciences, tehran, iran 6ministry of health and medical education, tehran, iran (received 13 aug 2014; accepted 24 aug 2014) abstract background: visceral leishmaniasis (vl) is one of the most important parasitic zoonotic diseases in the world. domestic dogs are the main domestic reservoirs of vl in endemic foci of iran. various methods, including vaccination, treatment of dogs, detection and removal of infected dogs have different results around the world. general policy on control of canine visceral leishmaniasis is protection of them from sand fly bites. the aim of this study was evaluation of pour-on application of flumethrin on dogs against blood-feeding and mortality of field-caught sand flies. methods: once every 20 days from may untill september 2013, the treated and control dogs were exposed with field caught sandflies for 2 hours under bed net traps. after the exposure time, both alive and dead sand flies were transferred in netted cups to the laboratory. the mortality rate of them was assessed after 24 hours. the blood-fed or unfed conditions were determined 2 hours after exposure to the dogs under stereomicroscope. results: the blood feeding index was varied from 12.0 to 25.0 % and 53.0 to 58.0 % for treated and control dogs respectively (p< 0.0001). the blood feeding inhibition was 75.0–87.0 % and 41.0–46.0 % for the control and treated dogs (p< 0.0001), respectively.the total mortality rate was 94.0–100 % and 19.0–58.0 % respectively for the treated and control groups (p< 0.001). conclustion: application of pour-on flumethrin on dogs caused 90–100 % mortality until 2.5 month and inhibited the blood-feeding of sand flies. keywords: visceral leishmaniasis, flumethrin pour-on, sand fly, control, iran introduction visceral leishmaniasis (vl) is the vectorborne disease and has great importance in public health due to the fatality among children. although there is an increasing interest to vl but there is not enough approach for control measures. there are various epidemiological features of vl which indicating the needs for defining different control measures (who 1990). kala-azar is a mediterraneanform in iran which the leishmania infantum isolated from vl patients (mohebali et al. 2005), the sand flies species from subgenera larroussius and paraphlebotomus acts as vectors (azizi et al. 2006) and the dogs and wild canids are considered as principal reservoirs (edrissian et al. 1993, mohebali et al. 2002). asymptomic infected dogs are suspected as the main reservoir host for sand *corresponding authors: prof hassan vatandoost, email: hvatandoodt1@yahoo.com, prof yavar rassi, email: rassiy@tums.ac.ir http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 79 flies to establish the transmission cycle to humans (moshfeh et al. 2009). the confirmed endemic foci of vl are distributed in different provinces and districts of iran including: ardebil province, east azerbaijan province (kaleibar, ahar, azarshahr districts), fars province (ghir-karzin, jahrom) semnan province, bushehr province (poshtkooh), qom province, kerman province (baft) and alborz (savejbolagh district) (mohebali 2013). moreover, the sporadic cases are reported from different parts of iran annually. the isolated parasite from animal reservoir was identified as l. infantum lon49 using biochemical assay (isoenzyme) in ardabil and azarbaijan provinces. the isolated parasite is exactly the same strain which previously isolated from human cases, so it can certainly be considered that the canine family is the most important reservoirs for humans (mohebali et al. 2002). due to the lack of suitable vl vaccine, the interruption of the transmission cycle seems to be the most effective (ramezani et al. 2009). at the present, the common control strategies between humans and carnivores vl (zvl) due to l. infantum is limited to early diagnosis and treatment with expensive and potentially toxic pentavalent antimonial drugs (killick-kendrick et al. 1997). treatment of infected dogs with meglumine antimobiate (glucantime) may be led to decrease of sensitivity of l. infantum to these drugs (gramiccia et al. 1992). flumethrin is one of the pyrethroids of type ii. there is no record for using of flumethrin on dogs as intervention method against control of sand flies in vl foci around the world. continuous efforts for the control of leishmaniasis and the applicability of these studies will be needed more than ever in the new and the old world. as surely as the vectors could be spread and transmitted the parasite from dogs to man (braga et al. 1998). at the present, the methods that can prevent the dogs against visceral leishmaniasis could not be prevented spreading of infected sandflies (reithinger et al. 2004). prophylactic soaps, shampoos and sprays with base of pyrethroids have been used on dogs with different success around the world (ashford 1989). yet, any protective measure which could protected completely the dogs against vl, is not introduced (christopher 1996). in the endemic area of the vl foci, it is recommended that all seropositive dogs are eliminated from infected areas. in southern europe the prevalence rate of vl is about 40 % but the elimination of dogs has not been accepted in these communities. in the most endemic areas, the health policy administration system is based on the elimination of seropositive dogs (dietze et al. 1998), but due to resource and financial limitations and failure of availability of necessary supplies are not be realized with suitable methods (david et al. 1998). in europe, the chemical treatment of dogs using antimonial components have been used for dog treatment, but this method is not recommended due to drug resistant of the parasites among dogs. the drug therapy of vl among dogs may led to temporary treatment of infected dogs (gramiccia et al. 1992, alessandro et al. 1997) but this drugs do not prevent recrudesce of vl. important notice that the use of these drugs does not stop the infection of sand flies that feed on infected dogs (alvar et al. 1994). application of residual insecticides is advisable for the control of endophagic sandflies in human dwelling, stables, poultries (marcondes and nascimento 1995, jin et al. 2004) but are not applicable for the exophagic sandflies which include most of vl vectors that their resting places is outdoor (jin et al. 2004). the present study was carried out in order to evaluate the pour-on formulation of flumethrin on owned dogs in meshkinshahr district during the activity of sandflies and effects of this method was assessed based on blood-feeding and mortality of exposed sand http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 80 flies treated dogs. materials and methods totally 118 dogs of the same race and gender were selected. due to higher age (over one year) 3 dogs was excluded the study. the blood samples was prepared on filter paper in order to detect the positive cases of vl among selected dogs using direct agglutination test (dat) which was done in laboratory of medical parasitology, school of public health, tehran university of medical sciences, iran. before application of flumethrin, the parazyquantel and albendazole was administered to the dogs in order to protect the research team and it is recommended to the family to bury the dogs' feces up to 48 hours in a sanitary manner. the location of experiments in mazrae-khalaf village was selected in front of the house where the cattle stables as well as the nest of sheep dog placed adjacent to it. the bed net size of 2 x 2 x 2 meters was installed and a sheet was spread on top of dogs for collection of knockdown sand flies. the live sand flies were collected using suction tube from indoor and outdoor early morning as well as evening and transferred to the laboratory and fed on 5 % sucrose solution during three successive days. the sugar pad was removed 12 hours before test. at the dusk (19:30 pm) which is synchronize with activity of sand flies, first the control dogs and then the treated dogs were anesthetized with xylazine and transferred to the inside of each net. after this stage, 150 live sand flies were released inside each bed net and the environmental condition including temperature and humidity was recorded. subsequently the exposed sand flies were transferred to the paper caps covered with net according to physiological conditions eg blood-fed or unfed. the blood feeding condition was observed immediately after 2 hours after experiments. the mortality rate of sand flies was calculated after 24 hours. data were analyzed using paired t-test under spss ver. 18. results 1direct agglutination test (dat): before pour-on application of flumethrin, titer of the serum in dogs was determinted indicating negative. for determination of flumethrin effect on vector, two indicators were considered in relation to blood feeding and viability of exposed sand flies compared to control group. 2blood feeding index of sandflies: the blood-feeding index (bfi) of sand flies were 345 out of 620 (55.6%) and 105 out of 614 (17.1%) from 20 up to 80 days post-treatment in control and treated groups (table 1). the bfi was varied from 56.5 to 53.5 % and from 12.3 to 25 % respectively in control and treatment groups (fig. 1). the difference between bfi in control and treated groups was significant (p< 0.0001). 3the effects of application of pour-on flumethrin on inhibition of bloodfeeding (ibf) of sand flies: application of pour-on flumethrin during the peak of blood-feeding activity of sand flies were assessed at four interval times posttreatment (fig. 2). the ibf of sand flies on treated dogs was 75.0 to 87.7 % and on untreated dogs was 46.5–41.3 % respectively. the ibf of sand flies was stasistically differenent between treated and control dogs (p< 0.0001). 4estimation of mortality rate: the mortality rate of unfed and blood-fed sand flies was studied. the total number of http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 81 dead blood-fed sand flies was 115 out of 620 in treated dogs and 100 out of 614 respectively in control and treated groups. the variation of mortality rate in the dead blood-fed sandflies during four interval times was 25–32 % and 15–38 % respectively in control and treated groups (table 2, fig. 3). the mortality rate of dead unfed sand flies was varied from 5 to 51 % and 94.8 to 100 % respectively in control and treated groups. the difference in total mortality rate in control and treated sand flies was significant (p< 0.001). 5estimation of the combined effect of flumethrin pour-on: the combined effect of inhibition of blood feeding and mortality rate of sand flies exposed to treated dogs compared with control group. the combined effect was varied between 94.8 to 100 % and 19.5 to 53.3 % respectively in control and treated groups. the difference of combined effect between four interval times of observation was not significant either treated neither group nor control except for 20 days in control group (p< 0.001). table 1. blood-feeding index (bfi) of sand flies after application of pour on flumethrin on dogs using bed net trap, meshkinshahr, ardabil province, 2013 days after spraying flumethrin kind of test total number of sandflies tested number of bloodfeeding sandflies blood-feeding index 20 control 154 87 56.5 test 152 38 25.0 40 control 155 91 58.7 test 154 27 17.5 60 control 156 84 53.9 test 153 21 13.7 80 control 155 83 53.6 test 155 19 12.3 total control 620 345 55.7 test 614 10 17.1 table 2. mortality rate of blood-fed and unfed sand flies after application pour-on flumethrin on dogs compared to control group using bed net traps, meshkinshahr, ardabil province, 2013 days after application type of test number of sand flies tested number of dead blood-fed sand flies number of dead unfed sand flies total number of dead sand flies mortality rate 20 control 154 25 5 30 19.5 test 152 38 114 152 100 40 control 155 30 61 91 58.7 test 154 27 127 154 100 60 control 156 28 56 84 53.9 test 153 20 131 151 98.7 80 control 155 32 51 83 53.6 test 155 15 132 147 94.8 total control 620 115 173 288 46.5 test 614 100 504 604 98.4 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 82 fig. 1. blood-feeding index (bfi) of sand flies after application of pour on flumethrin in dogs using bed net traps, meshkinshahr, ardabil province, 2013 fig. 2. inhibition of blood-feeding rate (ibf) of sand flies after application of pour-on flumethrin on dogs, in bed net trap, meshkinshahr, ardabil province, 2013 fig. 3. mortality rate of sand flies after application pour-on flumethrin on dogs using bed net traps, meshkinshahr, ardabil province, 2013 http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 83 discussion in the north west, of iran ph. kandelakii, ph. perfiliewi, ph. tobbi and at the southern ph. keshishiani, ph. alexandri, ph. major are the responsible for the transmission of the disease (nadim et al. 1992, sahabi et al. 1992, rassi et al. 1997, 2000, 2005, 2012, azizi et al. 2006, 2008, oshaghi et al. 2009). according to the census of iranian center for control of disease, during the 1988 to 2005, the total number of recorded cases of vl in the country was 2056 cases which 624 of them (30%) related to ardabil province (mohebali 2013). in a seroepidomiological study to determine the prevalence of vl between the years of 2002 to 2005, the more positive cases related to the northwest, especially ardabil province (mohebali et al. 2006). in the foci of vl in iran, the domestic dogs act as the the main reservoir (mohebali et al. 2002). at the present, the control approach of vl due to l. infantum is reserved to early detection and treatment of the patients with expensive and potentially toxic of pentavalent antimonial drugs. in some countries, where the disease is endemic situation, the main control strategies has been established for indoor residual spraying as well as reservoir population control (infected dog culling). in brazil, in spite of residual spraying of indoor places and annually elimination of the dogs, the trend of zvl has been increased over the past 20 years (marcelino et al. 1998). methods for the elimination of dogs was questionable due to availability of facilities as well as social acceptance (reithinger et al. 2004). treatment of leishmania-infected dogs with anti-leishmania drugs was not effective due to recrudescence of vl (up to 74.0%) (mohebali et al. 2002). while waiting for a vaccine against leishmania, the new ways switch to interrupt the vl transmission, by passing the dogs from livestock deltamethrin impregnated bath (david et al. 1998, changfa etl al. 1994, guanghua et al. 1994) as well as topical application of pyrethroid lotions. epidemiological effects of topical application of insecticide on dogs, depends on not only reducing the number of sand flies blood-fed on dog , but also survival rate of sand flies infected with leishmania (reithinger et al. 2001). the main way for the control of vl is protection human and dosmetic reservoir from bite of sand flies (poli et al. 1997). despite the nature of vl epidemiology which the special species of sand flies are responsible for transmission of the disease from infected dogs to others including human. nevertheless due to specific behavior of the vectors, control of vl is based on control of reservoir population. in this relation, different methods including vaccinations, treatment of dogs, separation of infected dogs has been used with different results in the vl foci around the world (ashford et al. 1998, reithinger et al. 2001). in the recent decade, the application of deltamethrin impregnated collars has been successfully used with high social acceptance as prevention measure for the dogs in different countries. the latter method has the repellency and killing effects against sand flies and so protected the dogs against vl) ramezani et al. 2009). by application of pour-on flumethrin, the feeding rate of sand flies on treated dogs was 3.3 folds lower than control. the inhibition of feeding rate on treated dogs was 1.8 fold higher than controls respectively. the difference between total mortality of treated and control groups was highly significant (p= 0.0001). in this study, the immediate mortality rate of sand flies was 5 fold more than control group during 2 hours exposure time. the delayed mortality rate of sand flies was known as a best indicator for the evaluation. the combined effect of pour-on flumethrin was 1.3 more than control group. the delayed mortality rate in blood-fed sand flies http://jad.tums.ac.ir published online: june 27, 2015 j arthropod-borne dis, march 2016, 10(1): 78–86 mr jalilnavaz et al.: application of flumethrin … 84 was 94.8 % compared to 27.5 % in control group. there was no mortality of sand flies between unfed sand flies in control group. conclusion it seems the age of dogs is an important factor for pour-on application of flumethrin. the field observation showed high efficacy of flumethrin in younger dogs than elder. it also appears that inhibition of blood feeding or combined effects of flumethrin is prior to killing effect. according to the results, this method could be a good alternative for control of sand flies of vl with high social acceptance by dog owners. acknowledgements the authors thank and appreciate behvarz of health houses of ahmadabad, urkandy and mazrae-khalaf villages, meshkinshahr district for helping the researchers during the field operation. this study was supported by tehran university of medical sciences, international campus (tumsic), tehran, iran. the authors declare that there is no conflict of interests. references alessandro p, sabrina s, grazia g, paolo b, fiancesca m (1997) comparison of minusidine (paromorningein) and sodium stibogluconate for treatment of canine leishmaniasis. vet parasitol. 71: 263–271. alvar j, molina r, san andres m, tesoruo m, nieto j, vitutia m, gonzalez f, sam andres mu, cuggiv j, odriyuez f, sainz a, escaccna c, anez n, tang y (1994) canine leishmaniasis: clinical, parasitological and entomological follow-up 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(dip: psychodidae) populations in iran vahideh moin-vaziri 1, *mohammad ali oshaghi 2, mohammad reza yaghoobi-ershadi 2, pupak derakhshandeh-peykar 3, mohammad reza abaei 2, fatemeh mohtarami 2, ali reza zahraei-ramezani 2, aboulhassan nadim 4 1department of parasitology and mycology, shahid beheshti university of medical sciences, tehran, iran 2department of medical entomology and vector control, school of public health and institute of public health research, tehran university of medical sciences, tehran, iran 3department of medical genetics, school of medicine, tehran university of medical sciences, tehran, iran 4department of epidemiology and biostatistics, school of public health and institute of public health research, tehran university of medical sciences, tehran, iran (received 26 nov 2014; accepted 17 feb 2015) abstract background: phlebotomus sergenti s.l. is considered the most likely vector of leishmania tropica in iran. although two morphotypesp. sergenti sergenti (a) and p. sergenti similis (b)-have been formally described, further morphological and a molecular analysis of mitochondrial cytochrome oxidase i (mtdna-coi) gene revealed inconsistencies and suggests that the variation between the morphotypes is intra-specific and the morphotypes might be identical species. methods: we examined the sequence of the its2-rdna of iranian specimens of p. sergenti s.l., comprising p. cf sergenti, p. cf similis, and intermediate morphotypes, together with available data in genbank. results: sequence analysis showed 5.2% variation among p. sergenti s.l. morphotypes. almost half of the variation was due to the number of an at microsatellite repeats in the center of the spacer. nine haplotypes were found in the species constructing three main lineages corresponding to the origin of the colonies located in southwest (sw), northeast (ne), and northwest-center-southeast (ncs). lineages ncs and ne included both typical p. cf sergenti and p. cf similis and intermediate morphotypes. conclusion: phylogenetic sequence analysis revealed that, except for one iranian sample, which was close to the european samples, other iranian haplotypes were associated with the northeastern mediterranean populations including turkey, cyprus, syria, and pakistan. similar to the sequences of mtdna coi gene, its2 sequences could not resolve p. sergenti from p. similis and did not support the possible existence of sibling species or subspecies within p. sergenti s.l.. keywords: phlebotomus sergenti, p. similis, leishmania tropica, its2-rdna, iran introduction “the phlebotomine sand fly phlebotomus sergenti s.l. parrot, 1917 originally described from algeria in 1917, has a broad range of distribution which covers areas of the southern mediterranean (morocco, algeria, tunisia), the western mediterranean” (portugal, spain, sicily), middle east, arabia, iran, afghanistan, pakistan, and northern parts of india. flies of this species have been incriminated as the main vector of cutaneous leishmaniasis due to leishmania tropica (clt) throughout iran and other clt foci in the world (nadim et al. 1971, al-zahrani et al. 1988, guilvard et al. 1991, yaghoobi-ershadi et al. 2002, oshaghi et al. 2010). it has an extensive geographical distribution, wider than *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 463 http://jad.tums.ac.ir published online: october 04, 2016 that of the parasite. the presence of this sand fly in l. tropica free areas and the differences in the transmission patterns of the clt could be related to the existence of intraspecific variability or even cryptic vector species (depaquit et al. 2002, yahyia et al. 2004). intraspecific variability has been shown in some morphological and molecular characteristics of various populations from different countries. depaquit et al. (2002) studied the intraspecific variability of the internal transcribed spacer 2 (its2) of 12 populations of p. sergenti s.l. from ten different countries. accordingly, two branches could be identified: one was related to the northeastern mediterranean area (cyprus, syria, and turkey) and pakistan, and the second related to southwestern of the first one including north africa, egypt, and morocco. these branches are in accordance with postulated migration routes of p. sergenti s.l. along the tethys sea during the miocene era. the sand flies belonging to these two different branches seem to differ in ecology, host preferences, and possibly also in vectorial capacity (depaquit et al. 2002). morphological and mtdna characters of p. sergenti s.l. were also studied on 28 iranian populations and a few samples from greece, morocco, lebanon, turkey, pakistan, and syria (moin-vaziri et al. 2007). according to this study, based on the number of setae and the width of basal lobe of coxite, three morphotypes were identified as a, b and c, with some intermediate forms. sequence analysis of mtdna revealed a 6–7% genetic variation among the populations studied. however, there was no consistency between the morphotypes and the genotypes. morphotype a was considered as p. sergenti sergenti, morphotype b, was identified as p. sergenti similis, and morphotype c had an elongated style in comparison with p. sergenti sergenti. in another molecular study on p. sergenti s.l. populations of spain and morocco using rdna its2 and mtdna cytb sequences (baron et al. 2008), a high genetic diversity including five ribosomal and 16 mitochondrial haplotypes was found within 25 specimens. the authors suggested testing the vectorial capacity of those haplotypes. based on these studies, it is judicious to consider the potential existence of sibling species within this taxon. if sibling species within p. sergenti s.l. were proven, it would have important implications in epidemiology as well as in experimental studies. however, having found several intermediate morphotypes as well as sympatric ecological niche of morphotypes a, b and c, postulated us to test the gene flow between the sympatric morphotypes of this taxon using sequence analysis of the its2 region of rdna gene. this multicopy gene involves homogeneization processes usually called molecular drive (dover 1982) and has provided resolution in several studies at the taxonomic level for the larroussius and paraphlebotomus subgenera (dover 1982, depaquit et al. 2002). the use of its2 region has many advantages including, high and low mutation rhythm at interspecies and intraspecies level respectively, speed and ease of use, multiple target sites, predefined marker systems, known pcr primers, pre-existing knowledge of them in some sand fly species (di muccio 2000, depaquit et al. 2002). this study was conducted to verify the molecular variation of iranian p. sergenti populations, and to compare them with available data in genbank. materials and methods sand fly collections and morphological identification the geographical locations from which p. sergenti s.l. was sampled are shown in fig. 1. sand flies were collected from different provinces using sticky traps and aspirators. specimens caught by sticky papers were j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 464 http://jad.tums.ac.ir published online: october 04, 2016 washed in a bath of acetone before being stored. they were stored in 96% ethanol at 4 °c until morphological and molecular identification. only male specimens were selected for the study of morphological and molecular variability since their characters tends to be more reliable. the head and genitalia of individual male sand flies were cut off within a drop of ethanol, cleared in boiling marc-andré solution, and mounted between slide and cover slide in berlese fluid and morphologically identified based on external and internal characters of the head and genitalia according to the known identification keys (theodor and mesghali 1964). morphometric measurements were performed to determine morphotypes of specimens as explained by moin-vaziri et al. (2007). the body related to the specimen was stored dried in a vial at -20 °c before dna extraction. dna extraction based on ecological conditions of the location where specimens were collected and on the morphological differences noted by means of morphometric analysis, a few specimens from each morphotype (moin-vaziri et al. 2007) and ecological condition were chosen for molecular investigation. genomic dna was extracted from the thorax, wings, legs and abdomen of either individual sand flies using the qiamp dna mini kit (qiagen, germany) (depaquit et al. 2002) or ish-horowicz with small modification as described by rassi et al. (2011). pcr amplification pcr was used to amplify a fragment of 480–516 bp containing the its2 of sand fly rdna (depaquit et al. 2002). pcr were performed in a 50 µ l volume using 5 µ l of extracted dna solution and 50 pmol of each of the two primers of c1a: 5'-cct ggt tag ttt ctt ttc ctc cgc t-3' and jts3 : 5'-cgc agc taa ctg tgt gaa atc-3'. the pcr mix contained (final concentrations) 10 mm tris hcl, ph 8.3, 1.5 mm mgcl2 , kcl 50 mm, triton x 100 0.01%, 200 µ m dntp each, and 0.25 µ l (1.25 units) of taq dna polymerase (eurobio). initial denaturation at 94 °c for 5 min was followed by 35 cycles of denaturation at 94 °c for 30 sec, annealing at 62 °c for 1 min. and extension at 72 °c for 1 min with a final elongation time of 10 min at 72 °c. amplicons were analysed by electrophoresis in 1.5% agarose gel containing ethidium bromide. sequencing and comparative sequence analysis purification of the pcr product was made by agarose-gel fractionation, using the perfect prep gel cleanup (eppendorf, germany). direct sequencing of both dna strands was performed by qiagen (hilden, germany) and the department of parasitology (university of valencia, spain) using the primers used for dna amplification. sequences were edited and aligned to identify haplotypes (=unique sequences) by means of the clustalw software package (www. ebi.ac.uk/clustalw) and manually adjusted, if necessary. they were analyzed using the neighbor-joining (nj) method provided in the must software package (philippe 1993). available sequences of p. sergenti and p. similis were retrieved from genbank and used for phylogenetic analysis (table 1). results morphometric analysis morphological investigation on specimens of 28 populations of p. sergenti sl collected from 11 provinces of iran revealed three main morphotypes (a, b and c) and a few intermediate forms (moin-vaziri et al. 2007). morphotype a was similar to p. sergenti parrot (1917). morphotype b was related to p. cf similis, according to perfiliev (1968) and morphotype c represents specimens with j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 465 http://jad.tums.ac.ir published online: october 04, 2016 a curved basal lobe of coxite without a style as globulous as that of p. sergenti. in addition to the three main morphotypes, some intermediate forms were identified among the collected samples. these morphotypes were found sympatric in several provinces of the country. however, the proportion of each morphotype varied within each region. morphotype a was the most frequent in all collection sites of the country. molecular analysis the size of rdna-its2 fragments amplified was about 480 bp, from which 278 nucleotides were attributed to its2, 130 bp to 5.8s and 72 bp to 28s genes. however, in addition to the main pcr product, there was an additional product, close to the main amplicon, in all amplification. despite of gel purification before sequencing, these unwanted products affected the results of sequencing. by the way, we could obtain a total of 249–253 bp length, including most part (218–222 bp) of its2 and 31 bp of 28s, from 11 specimens comprising 5, 4, and 2 individuals of morphotype a, b and intermediate form a/c respectively. the sequences were submitted to genbank (accession numbers: ef434818-ef434828). comparative its2 sequence analysis of 11 p. sergenti s.l. individuals showed 13 (5.2%) polymorphic sites, from which 46% was due to indel (insertion/deletion) and 54% was due to substitutions. all polymorphic sites were located in its2 region and 28s region was identical in all specimens. the region was at rich, with an average of 75%. there were two variable microsatellite regions in the its2 aligned part of the specimens, which showed variable repeats of a poly (at) microsatellite. in the first variable microsatellite sites, two specimens contain 9, three specimens had 8, and six other specimens comprised 7 repeats of at (fig. 2). other entries from genbank also showed variable number of at repeats in this site. in the second polymorphic microsatellite, variations between the iranian specimens were due to two single nucleotide insertions/deletions (indels) and one variable repeats of a poly (at) microsatellite. lower degrees of variation in the poly (at) microsatellite also have been observed in non-iranian entries (fig. 2). based on the sequence alignment, nine different haplotypes (nominated as haplotype i–ix) have been identified, three haplotypes (vii–ix) in the south west (sw), two haplotypes (i and vi) in the north east (ne) and 5 haplotypes (i–v) in the north-center-south (ncs) populations (table 1, 2, fig. 3). a sample of haplotype i was positioned in ne lineage. these haplotypes differed in 1–11 nucleotide positions. the length of the segment sequenced varied between 249 to 253 bp. the its2 ribosomal haplotype i with three repeats was the most frequently haplotype among all haplotypes. distance analysis of the its2 sequences indicated three main lineages (fig. 3), which supports correlations between geographical distributions of those populations. these lineages were so-called sw, ne, ncs lineages which included haplotypes of south-west, north-east and a mix of haplotypes extended from northwest to the central to the southeast of the country, respectively. when we added the its2 sequence data of other p. sergenti and p. similis populations as representatives of other parts of the world for phylogenetic analysis, except for one iranian sample which was close to the european samples, all of the iranian haplotypes were associated with the north-eastern mediterranean populations including turkey, cyprus, syria, and also pakistan (fig. 4). the p. similis populations from iran were associated with other p. sergenti populations, however, the european p. similis populations clustered separately. this might be due to independent accumulated mutations in distinct geographical populations resulted in separate branch j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 466 http://jad.tums.ac.ir published online: october 04, 2016 in the tree. the phylogenetic tree deduced from combination of the its2 and the 381 bp of mtdna cytb-nadh1 sequences obtained in our previous study (moin-vaziri et al. 2007) revealed similar tree and did not resolve p. segenti from p. similis (data are not shown). table 1. details of phlebotomus sergenti sand flies and their sequence data used in this study specimen code ( no. assigned in fig. 1) locality morphotype haplotype its2 genbank ac. no cytb genbank ac. no reference iran-ser(a)-1 tehran, iran a i ef434822 dq840367 moin-vaziri et al. 2007 iran-sim(b)-1 tehran, iran b v ef434821 dq840363 moin-vaziri et al. 2007 iran-sim(b)-2 mashhad, iran b i ef434825 dq840345 moin-vaziri et al. 2007 iran-seri(a/c)-3 neishabur, iran a/c vi ef434820 dq840352 moin-vaziri et al. 2007 iran-serii(a/c)-3 neishabur, iran a/c i ef434819 dq840359 moin-vaziri et al. 2007 iran-ser(a)-4 izeh, iran a viii ef434828 dq840384 moin-vaziri et al. 2007 iran-ser(a)-5 bushehr, iran a ix ef434826 dq840391 moin-vaziri et al. 2007 iran-sim(b)-6 bandar-eabbas, iran b vii ef434824 dq840395 moin-vaziri et al. 2007 iran-ser(a)-7 bam, iran a iii ef434818 dq840376 moin-vaziri et al. 2007 iran-sim(b)-7 bam, iran b iv ef434827 dq840375 moin-vaziri et al. 2007 iran-ser(a)-8 iranshahr, iran a ii ef434823 dq840397 moin-vaziri et al. 2007 malta-similis* malta af462334 depaquit et al. 2002 greece-similis1* greece af462333 depaquit et al. 2002 greece-similis2* greece af218324 depaquit et al. 2000 portugal-sergenti* portugal af462327 depaquit et al. 2002 turkey-sergenti* turkey af462332 depaquit et al. 2002 syria-sergenti* syria af462328 depaquit et al. 2002 cyprus-sergenti* cyprus af462323 depaquit et al. 2002 pakistan-sergenti* pakistan af218323 depaquit et al. 2000 spain-sergenti1* spain s-c eu980387 baron et al. 2008 spain-sergenti2* spain s-b eu980386 eu980375 baron et al. 2008 spain-sergenti3* spain s-a/m-b eu980385 eu980375 baron et al. 2008 spain-sergenti4* spain af462324 depaquit et al. 2002 italy-sergenti* italy af462330 depaquit et al. 2002 morocco-sergenti1* morocco m-c eu980384 eu980371 baron et al. 2008 morocco-sergenti2* morocco m-a eu960382 eu980370 baron et al. 2008 egypt-sergenti* egypt af462329 depaquit et al. 2002 israel-sergenti* israel af462325 depaquit et al. 2002 p. papatasi iran ef408801 depaquit et al. 2007 j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 467 http://jad.tums.ac.ir published online: october 04, 2016 table 2. comparison of nucleotide characters of phlebotomus sergenti of iran at polymorphic sites of the rdna its2. gap (indel) shows by – specimen code 11 19 23 24 50 75 76 77 78 140 146 147 148 iran-ser(a)-8 a t t a t t a t a g a iran-ser(a)-7 a t t g t t a t a g a iran-sim(b)-7 c t c g c t a g a iran-sim(b)-1 a t t g t t a g a iranserii(a/c)-3 a t t g t g a iran-ser(a)-1 a t t g t g a iran-sim(b)-2 a t t g t g a iranseri(a/c)-3 a t t g t t a g c a t iran-ser(a)-4 a c t g t g c a t iran-ser(a)-5 a c t g t a c a t iran-sim(b)-6 a t t t t a a fig. 1. collection sites of phlebotomus sergenti s.l. for this study, (1) tehran, (2) mashhad, (3) neishabur, (4) izeh, (5) bushehr, (6) bandar-e-abbas, (7) bam, (8) iranshahr. dark spots represent clt endemic foci in iran j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 468 http://jad.tums.ac.ir published online: october 04, 2016 fig. 2. microsatellite region in its2-rdna sequences of different populations of phlebotomus sergenti s.l. from iran and other parts of world retrieved from genbank (marked with *). a: morphotype a, b: morphotype b, a/c: intermediate forms of morphotype a and c, ser, p. sergenti, sim, p. similis 468 http://jad.tums.ac.ir published online: october 04, 2016 j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 469 http://jad.tums.ac.ir published online: october 04, 2016 fig. 3. neighbor-joining tree inferred from 253 bp of its2-rdna sequences including 222bp of its2 and 31 bp of 28s of phlebotomus sergenti s.l. populations from iran. a, morphotype a, b, morphotype b, a/c, intermediate form of morphotype a and c, ser, p. sergenti, sim, p. similis j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 470 http://jad.tums.ac.ir published online: october 04, 2016 fig. 4. neighbor-joining tree inferred from 253 bp of its2-rdna sequences including 222 bp of its2 and 31 bp of 28s of phlebotomus sergenti s.l. populations from iran and other available data from genbank originated from europe, africa, and asia. a, morphotype a, b, morphotype b, a/c, intermediate form of morphotype a and c, ser, p. sergenti, sim, p. similis, p. papatasi (an: ef408801) has been used as an outgroup discussion results of this study showed a high diversity between specimens of p. sergenti s.l. in iran where we found nine ribosomal haplotypes. however, the rate of genetic variation (3%) within the its2 locus between the iranian populations is half of the rate (6%) that was previously observed in the mitochondrial (cytb-nadh1) sequences (moin-vaziri et al. 2007). it seems that mtdna sequences are more appropriate for the study of the intraspecific variability of p. sergenti s.l. in a more limited geographical environment. j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 471 http://jad.tums.ac.ir published online: october 04, 2016 the high diversity observed in its2 is in agreement with previous studies indicating different ecological, morphological, and molecular variation among iranian p. sergenti populations. previous studies revealed at least three morphotypes (a, b and c). however, in this study like previous study, we have not found any correlation between genotypes, ecotypes, or morphotypes and the results obtained here do not support the presence of sibling species (p. sergenti and p. similis) within the taxon. however, results of this study are in conflict with a phylogenetic analysis of nuclear ribosomal dna of depaquit et al. (2002) that showed the monophyly both of p. sergenti s.l. and p. similis and they were not sister species. their result confirmed a study previously carried out, using morphological and morphometric approaches for examining the status of the two species by the same investigators (depaquit et al. 1998). their worldwide attempt showed allopatric situation of the two taxa at that time. according to our findings, it seems that this group (morphotypes) cannot be considered as two different species because firstly we have found many intermediate morphological and genetically forms among the specimens, which indicate possible gene flow and lack of reproductive isolation between them. secondly, finding identical genotypes (100%) among different morphotypes that are p. sergenti and that p. similis in mtdna (moinvaziri et al. 2007) and its2 sequences support our morphological findings. considering the molecular drive characteristic of its2rdna strongly indicated that these two morphotypes are not isolated reproductively. moreover having found these two taxa sympatric in most provinces of iran differs from taxonomic conception of subspecies. according to our data, we encountered with different morphologically populations of p. sergenti s.l. in iran. in light of our results, it is too early to come to a final decision on taxonomic status of the species. more molecular, morphological, and hybridization studies between the two taxa, particularly between geographically distinct populations is necessary. in iran, clt is endemic in 14 foci located in 8 provinces restricted to large and medium sized cities in different parts of the country (yaghoobi-ershadi 2012).we identified three its2 lineages of p. sergenti which is in agreement to the ones identified using the mtdna loci (moin-vaziri et al. 2007). lineage ne and ncs correspond to the main foci of clt in the country which included both typical and intermediate morphotypes of p. cf sergenti and p. cf similis. in other hand sw lineage exist in clean area of clt. these findings warrant studies to examine if clt is due to differences in the vectorial capacity of the p. sergenti s.l. lineages (ne/ ncs versus sw) or other ecological and epidemiological factors are involved. conclusion phylogenetic sequence analysis revealed that most iranian haplotypes were associated with the northeastern mediterranean populations. similar to the sequences of mtdna coi gene, its2 sequences could not resolve p. sergenti from p. similis and did not support the possible existence of sibling species or subspecies within p. sergenti s.l.. more molecular studies on other genes or hybridization should be done to clarify the status of different morphotypes of mentioned species in iran. moreover, with regard to the importance of the epidemiology of leishmaniasis, further studies need to be performed on the possible role of these three morphotypes in the transmission of l. tropica. acknowledgements authors are grateful to the staff of the leishmaniasis laboratory at isfahan training and health research centre, and also aa j arthropod-borne dis, december 2016, 10(4): 462–473 v moin-vaziri et al.: its2-rdna sequence … 472 http://jad.tums.ac.ir published online: october 04, 2016 akhavan, aa hanafi-bojd, k akbarzadeh, m aghasi, h alipour, r jafari, z zare, m nazari, f mohtarami, and f yaghoobi during the field and laboratory studies. the authors are greatly also indebted to dr b hooshmand, m boutry, c grimplet, p colson, and f muller for their kind assistance in this program. our sincere thanks to dr sixte blanchy for his help on various aspects, especially for organising a close cooperation with our french colleagues at the university of reims champagne-ardenne. this study was financially supported by institute of public health research, academic pivot for education and research, tehran university of medical sciences: project id no: 241.82.72. the authors declare that there is no conflict of interests. references 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mohammad ali oshaghi*1 j arthropod-borne dis, september 2017, 11(3): 363–370 f talebzadeh et al.: insect fauna of … 363 http://jad.tums.ac.ir published online: september 08, 2017 original article insect fauna of human cadavers in tehran district fahimeh talebzadeh 1, masoud ghadipasha 2, jaber gharedaghi 2, naser yeksan 2, *kamran akbarzadeh 1, *mohammad ali oshaghi 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2iranian legal medicine organization, tehran, iran (received 15 july 2015; accepted 28 aug 2017) abstract background: entomological data can provide valuable information for crime scene investigations especially in post mortem interval (pmi) estimation. this study performed to determine insect fauna of human corpses in tehran district. methods: insect specimens were collected from 12 human cadavers during spring and summer 2014 and were identified using morphological characteristics. results: four fly species including two blowflies chrysomya albiceps and lucilia sericata (calliphoridae), one flesh fly sarcophaga argyrostoma (sarcophagidae), and one phorid fly megaselia scalaris (phoridae) and a beetle dermestes maculatus (dermestidae) was observed on the human cadavers. chrysomya albiceps was the most dominant species on the corpses temporally and spatially. conclusion: chrysomya albiceps was the most dominant insect species on human cadavers in the area study spatiotemporally. the data make c. albiceps as a valuable entomological indicator for pmi estimation in tehran and other parts of the country. however, further biological and ecological data such as its behavior, life tables, and consistent developmental time should be investigated when establishing a pmi in the region. keywords: forensic entomology, blow fly, post-mortem interval, chrysomya albiceps, iran introduction various insects and arthropods attract to the specific decomposition stage of human or animal carcass while majority of them colonize for only a limited time (1). forensic entomology families of the order diptera have the greatest importance because of their exclusive behavior in early arriving and making abundant larvae on human cadavers (2, 3). members of the family calliphoridae are the first group of insects that attack a corpse within minutes after death (3, 4). additionally, members of the family sarcophagidae often colonize in corpse around the world especially in tropical and subtropical countries (3). thus, they are also very useful in crime scene investigations especially in post mortem interval (pmi) estimation (3). pmi calculations are used for various goals including criminal items, trace of transport of the corpse after death, correlation defiant with crimes by the dna analysis of slain tissues in the midgut content of larvae, pharmacology, kid, and olds afflicting (5). determination of corpse arthropod fauna is one of the basic and inevitable information for estimating of pmi (6). in addition to arthropod fauna, the replacement (succession) of arthropods on corpse can provide important data for pmi estimation in some cases (7, 8). for pmi calculation from less than one week to so many years after death, fundamental information about insect succession plays an important role (9). successional samples of arthropods on human cadavers are related to the geographic regions of the study areas (2, 10-12). study on the particular arthropod fauna *corresponding authors: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir, dr kamran akbarzadeh, e-mail: kakbarzadeh@tums.ac.ir http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/post_mortem_interval mailto:moshaghi@sina.tums.ac.ir j arthropod-borne dis, september 2017, 11(3): 363–370 f talebzadeh et al.: insect fauna of … 364 http://jad.tums.ac.ir published online: september 08, 2017 and stage in various decomposition phases and the relationship between them can be used for estimating the pmi ranges (8). no recent information on arthropod and insect fauna and their spatio-temporal distribution was available for tehran, iran. the aim of the present study was to investigate the fauna of insect species attracted to human cadaver in tehran district and to acquire knowledge of their temporal and spatial distribution in the region. materials and methods tehran with 18909km2 and altitude of 1200 m above sea level, allocates about 2.1% of total area of the country. tehran has a hot summer, cold winter and brief spring and autumn. this province locates among mazandaran, qum, alborz and semnan provinces from north, south, west, and east, respectively. the sampling process was performed during seven months from the beginning of spring to mid autumn 2014. all procedures were performed in accordance with the terms of the iran human (scientific procedures) act project license and were approved by the tehran university of medical sciences ethical review committee. this process was done immediately after registering any human corpse in the kahrizak autopsy hall of tehran legal medicine organization (kah-tlmo). among the human cadavers (25–50 per day) referred to the kah-tlmo, the cases with any kind of arthropods on them were used for arthropod sampling. all of the collected specimens were put individually in labeled vials based on the collection time and sites of the body. the corpse characteristics were recorded including age, sex, location, cause of death, estimated pmi, latitude and longitude, and the arthropod developmental stage (table 1). to estimate the pmi, medical or scientific evidence other than entomological data such as decomposing phase, body color, decaying of various organs were used to determine pmis of the corpses. all the vials were transferred to the medical entomology laboratory of school of public health, tehran university of medical sciences (sph-tums) in a usual cold chain. all the collected specimens were washed with normal detergents and counted. some of the live immature stages of the arthropod were reared to achieve their adult stage while some of them were killed in boiling water before preserving in 70% etoh. the specimens were morphologically identified to species level using the known morphological keys (13-18). main morphological characters used for identifications of the flies were setae on meron, general body color, hairs on greater ampulla, anterior spiracle color, color and hairs on calypters. whenever it was necessary, the shape and appendages of male genitalia of the specimens were checked to confirm species identification, particularly for sarcophagidae family. some of the mature stages of collected arthropods on the corpses were preserved either in 70% etoh or pinned and deposited in medical entomology and zoology museum of sph-tums. results totally 12 human corpses with arthropod specimens were referred to the kahrizak autopsy hall. age distribution of the referred corpses was between 23 to 86 years old. details of the collected human cadavers have been shown in table 1. medical or scientific evidence other than entomological data determined pmis ranged from 3 days to six months for the corpses that could increase arthropod diversities on the cadavers (table 1). various life stages of insects (egg, larvae, and adult) have been observed on the collected cadavers (table 1). overall, 4129 arthropod specimens belong to four fly and one beehttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 363–370 f talebzadeh et al.: insect fauna of … 365 http://jad.tums.ac.ir published online: september 08, 2017 tle species have been collected and identified during the study. the flies comprised two calliphoridae species of c. albiceps (wiedemann 1819) and l. sericata (meigen 1826), one sarcophagidae species s. argyrostoma (robineaudesvoidy 1830), and one phoridae species m. scalaris (loew 1866). the beetle species was d. maculatus degeer 1774 (coleoptera: dermestidae). chrysomya albiceps was the dominant species sampled either in outdoor or in indoor adventured human cadavers whereas l. sericata and d. maculatus was related only to the outdoor places. sarcophaga argyrostoma was related mostly to indoor places. megaselia scalaris was the rarest species due to its merely one specimen in all of the study cases. in outdoor locations, the most representative species were c. albiceps (75%,) l. sericata (12%), s. argyrostoma (3%), and in indoor locations, c. albiceps (85%), and s. argyrostoma (15%). megaselia scalaris (0.39%) and d. maculatus (7%) were collected in smaller numbers in the study area. table 1. details of the human cadavers and the arthropods found on them in tehran, iran in 2014 case no gender age (yr) location location cause of death pmi estimation (d) development stage species 1 male 50 outdoor modares highway unknown 21–28 larvae c. albiceps l. sericata 2 male 68 indoor africa st. heart failure 7–10 larvae s. argyrostoma 3 male 53 indoor dinmohammadi st. unknown 14–21 larvae c. albiceps adult m. scalaris 4 male 60 indoor sattar-khan st. unknown 14–21 larvae c. albiceps 5 male 60 70 outdoor saeedi highway unknown 30–60 larvae c.albiceps l. sericata 6 male 86 indoor khorassan square heart disease 4–7 larvae c. albiceps 7 male 42 indoor vali-asr st. drug abuse 3–4 larvae c. albiceps egg s. argyrostoma 8 male 65 indoor dastvareh st asphyxiation with co 3–5 larvae c. albiceps 9 female 29 indoor sarbaz st. unknown 3–4 larvae s. argyrostoma 10 male 24 outdoor fasham lacvasan hanging on tree 21–28 pupae s. argyrostoma 11 male 59 outdoor damavand road unknown 90–180 larvae d. maculatus adult 12* male 35 outdoor not defined drowned in the lake not clear larvae c. albiceps *: the dead body originated from azerbaijan country. discussion in this study, five insect species were observed on human cadavers in tehran district. except for c. albiceps and l. sericata, the other three species including s. argyrostoma, d. maculatus, and m. scalaris are new report for arthropod fauna on human cadavers in iran. previous reports have shown presence of various species of blowflies and flesh flies (1921) on human cadavers. in addition to calliphor idae and sarcophagidae, some species of muscidae and fanniidae were also involved in human or animal corpse decomposition in tehran (21). chrysomya albiceps with 86.09% was the most common species on human cadaver in tehran districts which is in agreement with the another result (19, 20) in tehran. the abundance of this species on human corpses rehttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 363–370 f talebzadeh et al.: insect fauna of … 366 http://jad.tums.ac.ir published online: september 08, 2017 ported 58% (19) and 64% (20) which are a little less than that of reported in this study. usually, c. albiceps is one of the most dominant species during hot and dry seasons in other geographical areas (22, 23). this species has been also found dominantly in schoenly trap equipped with rabbit carcasses in north of iran (24) and pig carrion in european urban habitats (25). this species has been reported repeatedly in other faunestic studies done with installed meet baited traps in fars (26) and tehran (27, 28) province. in the present study, s. argyrostoma was found as the second foremost species in indoor places. this species is very important in forensic entomology (29) and has been reported repeatedly on animal carcasses (30). this species shows different behaviors on human cadavers in various regions around the world. it has been mentioned mostly as indoor species in switzerland (31) and poland (29). nevertheless, in germany, this species has been considered as an exclusively outdoor species (32). in this study, this species has been found in one outdoor and three indoor discovered cadavers. at least one of these three indoor cadavers has died certainly at home. this species has also reported in faunestic investigations in the installed meat baited traps in tehran (28), fars province (26) and in persian gulf islands (33) and in modified schoenly trap with rabbit carcasses in north of iran (24). we found an individual specimen of m. scalaris of phoridae in indoor location. in indoor places, phoridae flies can move in rooms with locked doors and windows due to their comparatively small size, and lay their eggs earlier than calliphoridae (34-36). this makes phoridae flies a better forensic entomological indicator for estimation of pmi than calliphoridae larvae in enclosed places and concealed environments (36, 37-39). this species might be the merely criminal entomological evidence accessible if the corpse is blocked or hidden in a habitation that is difficult for other larger insects to gain access (34). moreover, some species of phoridae family as well as m. scalaris have been mentioned as indicators for buried bodies named coffin fly (34, 40-42). megaselia scalaris has small size and enables to find carrion buried within the ground being found in coffins. they can move through the smallest openings and are able to dig about six feet deep (half a meter in a four-day period) in order to reach buried carrion and lay eggs on carrion to provide nutrition for the emerged larvae (43). however, this species is classified as secondary forensic insect because they favor older rotting cadavers (34). forensic entomological evidence comprising m. scalaris has been used in court as a tool to prove "time of neglect" or lack of care of elderly patients by caretaker (1). moreover, phorid flies are active in cold season while most blowflies are inactive due to low temperature (42). in cases of myiasis, some phorid species such m. scalaris may infest living humans or animals (44, 45). in the present study, the larvae of beetle d. maculatus have been observed on an exposed human cadaver in late decomposition stage. this species also has been reported on human cadavers in germany (46, 47). some adults of this species have been trapped in modified schoenly traps equipped with rabbit carcasses in north of iran (24). dermestes maculatus is a cosmopolitan species with common name of leather beetle (3). larvae of this species sometimes act as predator of fly larvae on dead bodies (48). therefore, observing of this species may not be limited in late stages of decomposition (3). however, this species along with other beetles can be used in forensic investigations especially in late stages of decomposition (49). estimation of pmi is urgent and essential for solving the mysteries of death investigations (50). the importance of entomological evidence can be mentioned as well as autopsy (51). majority of the pmi estimations in the 12 surveyed cadavers in tehran were more http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/carrion https://en.wikipedia.org/wiki/coffin j arthropod-borne dis, september 2017, 11(3): 363–370 f talebzadeh et al.: insect fauna of … 367 http://jad.tums.ac.ir published online: september 08, 2017 than 3 days. however, lack of accurate pmi estimation was due to lack of entomological investigations in those crime scenes. conclusion determination of insect fauna is the first fundamental step for pmi estimation of any specific region. as next step, calculating degree-day requirements and life tables of the species especially c. albiceps is highly recommended for establishing forensic entomology. the characters of c. albiceps make this species as the best forensic insect candidate for pmi estimation in tehran and other parts of iran. although preliminary studies on temperature requirement of c. albiceps has been done in iran (27) but it warrants to be continued to determine exact life table, behavior, and consistent developmental time of this species alongside with other fly species in various environment situation before establishing pmi in the region. acknowledgements this study was supported by tehran university of medical sciences grant number 25702 as well as 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association forensic entomology london, uk, p. 7. 48. braack leo (1981) visitation patterns of principal species of the insect complex at carcasses in the kruger national park. koedoe. 24: 33–49. 49. kulshrestha p, satpathy dk (2001) use of beetles in forensic entomology. forensic sci int. 120: 15–17. 50. greenberg b, kunich jc (2002) entomology and the law flies as forensic indicators. cambridge university press, cambridge, united kingdon 2002 356 pp., isbn 0-521-80915-0. 51. haskell nh, lord wd, byrd jh (2001) http://jad.tums.ac.ir/ https://books.google.com/books?id=ztslaqaamaaj https://books.google.com/books?id=ztslaqaamaaj https://www.ncbi.nlm.nih.gov/pubmed/?term=ghavami%20mb%5bauthor%5d&cauthor=true&cauthor_uid=26623439 https://www.ncbi.nlm.nih.gov/pubmed/?term=djalilvand%20a%5bauthor%5d&cauthor=true&cauthor_uid=26623439 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4662799/ j arthropod-borne dis, september 2017, 11(3): 363–370 f talebzadeh et al.: insect fauna of … 370 http://jad.tums.ac.ir published online: september 08, 2017 collection of entomological evidence during death investigations. in: byrd jh, castner jl (eds) forensic entomology the utility of arthropods in legal investigations. crc press, boca raton fl, pp. 81–120. http://jad.tums.ac.ir/ 6dr moosakazemi rtl 89 10 1_2_ iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 42 original articles the morphological variations of culex pipiens larvae (diptera: culicidae) in yazd province, central iran h dehghan1, j sadraei1, *sh moosa-kazemi2 1department of medical entomology and parasitology, school of medical sciences, tarbiat modares university, tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 6 sep 2010; accepted 5 dec 2010) abstract background: culex pipiens complex shows variations in morphological and biological characters including different biological forms and has medical and veterinary importance. because of having morphological variations, sometimes it is not easy to separate this species from cx. quinquefasciatus and cx. torrentium. the aim of this study was to identify the culex pipiens complex species in order to use in control programs in the future. methods: this study was carried out in two randomly selected rural villages in yazd county, eastern iran using dipping technique from april to october 2009. the data were analyzed using spss software version 16. results: average of siphon index in fourth-instrar larvae was 3.86±0.03, the minimum and maximum were calculated 2.43 and 5.14, respectively. siphon/saddle index was measured as average, minimum and maximum 3.2±0.2, 2.78, and 4.42 respectively. in our study, only 4 specimens had single seta 1 on segments iii and vi (2.5%) and the remaining beard double seta (97.5%). the maximum 3-6 branches seta 1a-s and 1b-s (95%) were observed on siphon. conclusion: more populations of culex pipiens from different areas of iran need to be studied to gain complete information about the taxonomy and ecology of the species in the country. keywords: culex pipiens complex, larvae, taxonomy, iran introduction culex pipiens linnaeus shows a wide morphological and biological variations and well known as a broad research topic by many researchers (knight and malek (1951). this complex includes several members of the same morphological form and has different variations in physiology and behavior. the species group includes more than 75 binomial names due to the complexity of synonyms (knight 1978). barr (1982) believes that the concept of classical complex for the geographical populations. he mentioned cx. pipiens and cx. quinquefasciatus as a simple geographical population, which has a great distribution. ishii (1991) expressed that there are variation among the characteristics of this complex and should not considered being independent species. there are some degree of intra species such as subspecies, variety and form among the members of this complex (vinogradova 2000). culex pipiens, cx. quinquefasciatus and cx. pipiens form molestus are the most common members of this complex and are widespread in the world (cornel et al. 2003). culex pipiens has wide distribution from europe, next to tropical regions of asia and africa till central part of north america, and one third of south australia and south america. culex quinquifasciatus has been distributed in tropical areas and has overlap extensively with cx. pipiens (vinogradova 2000). *corresponding author: dr seyed hassan moosa kazemi, e-mail: moosakazemi@tums.ac.ir iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 43 hybrid forms of this species have been distributed in australia, africa, the middle, and far east of asia, north and south america. the habitat of cx. pipiens form molestus is temperate climates while cx. quinquefasciatus considered cosmotropical species (bourguet et al. 1998). culex pipiens has more adaptation to larval habitats such as the pit containing high degree of waste water up to cavity include dishes of fresh water and presented as the most common species in the subgenus culex in southwestern asia (harbach 1988). in iran, cx. pipiens and its form molestus and cx. quinquefasciatus are recorded (azari-hamidian 2007). culex pipiens (including form molestus) has a wide distribution in most areas of iran and cx. quinquefasciatus is recorded in seven southern provinces of the country (zaim 1987). culex pipiens has the major role in human nuisance due to biting. wuchereria bancrofti and brugia malayi transmitted by cx. pipiens and distributed in tropical and subtropical areas of africa, asia, australia, and pacific islands. some arbovirus infections have been transmitted by cx. pipiens such as west nile fever, sindbis, japanese encephalitis, st louise encephalitis, western equine encephalitis, rift valley fever, tahyna, and oropouche. plasmodium gallinaceum and p. relictum, causes of avian malaria, are transmitted by this species (vinogradova 2000). culex pipiens form molestus is separated from cx. pipiens mainly by ecological and physiological characters. larvae of this species have been found in underground habitat including the storage of water, septic tanks and sanitary space. culex pipiens larval habitat is open space accumulated with artificial water. the main and reliable characters for diagnosis in larval stage are siphon index, siphon/saddle index, the branch number of seta 1a-s and 1b-s and seta 1 of the abdominal segments of iii and iv, and the shape of siphon (harbach 1988, azari-hamidian and harbach 2009). the siphon index has been used widely for separating the members of this complex. the index is defined as the ratio of siphon length to its basal width. many researchers have been reported considerable variations in cu. pipiens and cx. pipiens form molestus, also harbach (1988) has rejected this character as diagnostic feature for these forms. the siphon index of cx. pipiens larvae changes based on geographical and ecological variations (vinogradova 2000). in addition, considerable variations have been cited, for example siphon index of species are grown in contaminated water are shorter than the species reared in the fresh water. nevertheless, there are considerable ranges due to length of siphon inside the complex species (harbach 1988). changes in habitats, locations, sex composition is reflected in larval siphon and can be used for diagnosis analysis among the complex species (vinogradova 2003). the average of siphon index is correlated to the certain types of larval habitats in ground or underground places. the average index siphon not only correlated with larval habitats, but also has relation to geographical distribution. this index for species in above ground habitats is stronger than other species based on comparing larval habitats. culex torrentium shows close relationship to the members of cx. pipiens complex based on morphological characters, even some researchers proposed it as a member of cx. pipiens complex (vinogradova 2000, smith and fonseca 2004). harbach (1988) classified cx. torrentium with cx. vagans in the trifilatus subgroup of the pipiens group. this species with cx. pipiens complex is sympatric in europe and some areas of asia including iran (azari-hamidian et al. 2009). according to harbach (1988), the most important diagnostic characters of cx. torrentium, cx. quinquefasciatus and cx. pipiens larvae are the branch of seta 1 of abdominal segment of iii–v. cx. quinquefasciatus and cx. pipiens are sympatric in some iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 44 parts of the world that their distributions have overlap situation (jupp 1978). since the morphological variations and biological diversity of culex pipiens can be found in different populations, each study possibly obtains more accurate data of the variability of cx. pipiens complex to provide a more reasonable discussion about the applied taxonomic issue. it is evident that the final decision about the taxonomic status needs more complete information which should be obtained from different ways among the complex populations. the aim of this study was to identify the culex pipiens complex species in order to use in control programs in the future. materials and methods study area a study was carried out from april to october 2009 in yazd province, iran. this study was performed in zarch and shahediyeh, two randomly selected rural villages in yazd county (54°04'n–31°59'e). the province is bounded by isfahan province in the west, south khorasan province in the east, kerman and fars provinces in the south, razavi khorasan and seman provinces in the north (fig. 1). in 2009 the maximum and minimum mean monthly temperatures were 41° c in july and -4.4 °c in january, respectively. the total annual rainfall was 62 mm, the minimum 0.3 mm in may and maximum 18 mm in march. the mean annual relative humidity was 37%. the main occupations are agriculture and husbandry of cow and goat. based on the available epidemiological data from yazd health center, there are no villages under the entomological survey. based on this information, two rural areas with 889 houses and 24358 populations were selected. mosquito sampling and morphological studies in the present study, two cities of shahediyeh and zarch were selected and sampling was carried out in selected larval habitats. the larvae of each habitat collected by dipping technique, were separately transported to the laboratory of medical entomology, school of public health, tehran university of medical science, iran and identified using the keys of zaim and cranston (1986) and azari-hamidian and harbach (2009). in order to morphometric studies, the samples of culex pipiens larvae were separated and sent to the entomology laboratory, department of entomology and parasitology, school of medical sciences, tarbiat modarres university. the fourth-instar larvae were preserved in lactophenol and the microscope slides were prepared using d’faure’s medium. the diagnostic characters of larvae were measured carefully using a microscope equipped with a glass lens dial. the data were recorded in specific forms according to sampling method, location, and date of collection and were analyzed using spss software ver. 16. results the siphon index was measured in 176 fourth-instar larvae. the average siphon index was 3.86±0.03, the minimum were calculated 2.43 and the maximum 5.14. this index had the most frequency in 159 specimens (90%) in the range of 3.3–4.8. siphon/ saddle index of 176 larvae was measured as average, minimum and maximum 3.2±0.2, 2.78, and 4.42 respectively. this index showed the most prevalence in 174 larvae (99%) with the range of 2.7–3.95. in addition, the larval abdominal seta 1 on segments iii and iv and its branches were studied. this character was analyzed in 159 larvae, only 4 had single seta (2.5%) and the rest bifid seta (97.5%) indicating the presence of culex pipiens in the area. the number of the branches of seta 1a-s and 1b-s was 3–8 and the most prevalence branches were 3–6 (95%) on siphon. the most numiran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 45 ber of seta on the siphon was 8–9. in our research, the siphon was gradually narrows in 96% of the samples from base toward the apex. these features of cx. pipiens larvae are presented in table 1. table 1. the variations of some morphological characters of siphon in culex pipiens larvae, yazd province, central iran, 2009–2010 character n distinctiveness prevalence % single 4 2.5 seta 1 abdominal segment iii and iv 159 double 155 97.5 3 22 14 4 52 33 5 52 33 6 24 15 7 6 4 siphon seta 1a-s and 1b-s 158 8 2 1 6 12 9.5 8 85 67 9 18 14 siphon seta number 127 10 12 9.5 gradually narrowing 173 96 siphon shape 181 wide in the middle 8 4 fig. 1. map of iran indicating the location of yazd county situated in the center of yazd province iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 46 siphon index fig. 2. the range of siphon index in culex pipiens, yazd province, iran, 2009–2010 fig. 3. the range of siphon/saddle index in culex pipiens, yazd province, iran, 2009–2010 iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 47 discussion culex pipiens complex is cosmopolitan species. the distribution of the member of this complex in iran is not completely known using reliable characters for identification and a few studies have been performed in this field (zaim 1987, azari-hamidian and harbach 2009, azari-hamidian et al. 2009, azari-hamidian et al. 2010). the larval chaetotaxy and siphon characters are very important in the taxonomy of the genus culex. in our research three related characters including: siphon index, siphon/saddle ratio, and chaetotaxy were studied. siphon index was studied in 176 samples and was obtained in average 3.86 and the range of 2.43–5.14. siphon index was calculated as 2.8–4.8 in 97% of the samples and shown with a scattering in fig. 2. in similar study in egypt, this index was reported as 3.7 in average and the range 2.7–4.7 for cx. pipiens (knight and malek 1951). harbach (1988) reported the average 4.58 and the range 3–5.8 for cx. pipiens. ishii (1991) stated the overall ratios of less than 4 as indicator for mosquito larvae developed in pollutant water, whereas high ratio greater than 4.4 for the specimens caught in larval habitats with fresh water. on the other hand, there are some reports about relation between the average of siphon index and underground or above ground larval habitats. culex pipiens found in above ground habitats, whereas cx. pipiens form molestus chooses underground habitats. the range of siphon index was reported 4.8–6.2 for 80% of cx. pipiens populations and was found 3.3–4.4 for 90% of the cx. pipiens form molestus populations (vinogradova 2000). considering the samples collected in yazd province from above ground in all larval habitats, siphon index was compared based on the average amount of water pollution with organic matter. the results of the finding showed that the average of the index was 3.65 in 63 samples found in sewage water near the households. the average index was 4 in 113 specimens collected from the cistern water pool near homes and livestock shelters. the diversity of this index shows relation to larval habitats with fresh water to contaminated water with high levels of organic matter. the siphon/saddle index and seta 1 of abdominal segment of iii–iv are the main characters for the reliable differentiation of cu. pipiens from cx. quinquefasciatus (harbach 1988, azari-hamidian and harbach 2009). moosa-kazemi et al. (2010) identified cx. pipiens using these characters in kurdistan province. in our study, the average rate of the siphon/saddle index was 3.2 with the maximum 4.42, and the minimum 2.78. the diversity of this index was low and in 99% of the samples was calculated as a range between 2.7–3.95 (fig. 3). the average of this index was reported 4.08 and the range 3.48– 4.63 for cx. pipiens and was mentioned the range 2.77–3.41 for cx. quinquefasciatus by harbach (1988). the ratio of this index was reported more than 3.45 for cx. pipiens and less than 3.45 for cx. quinquefasciatus by azari-hamidian and harbach (2009). in our study, we found the present of double seta 1 of abdominal segment of iii–iv in 96.9% of the samples. in parallel to harbach (1988) and azari-hamidian and harbach (2009), this character attributed to culex pipiens. siphonal seta 1-s of culex pipiens complex, especially 1a-s and 1b-s, was used for identification, particularly cx. pipiens and cx. quinquefasciatus. in our study, siphonal seta 1a-s and 1b-s were observed in 158 samples, and was found range 3–8 branches, whereas 95% of the samples have 3–6 branches. harbach (1988) cited the variation of the number of branches and mentioned the more branches of seta in cx. quinquefasciatus than cx. pipiens. in agree with the present research, iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 48 knight and malek (1951) reported an average 4 and a range 2–9 branches in cx. pipiens. in our study, seta 1-s was counted in 127 larval samples and 8–9 setae were observed in 103 samples (81%). the most number of tuft setae (10 setae) were counted in 12 samples (table 1). harbach (1988) mentioned four pairs of seta 1-s in cx. pipiens, sometimes fifth tuft was observed on one or both sides of siphon. shape of siphon was studied in relation to siphon width in the middle and its base in 181 larvae. this character in the middle was less than in the base in 173 larvae samples, and was wide in the middle of others (table 1). in general, siphon of cx. pipiens is longer than in cx. quinquefasciatus (harbach 1988, azari-hamidian and harbach 2009). this feature can be used for the differentiation of cx. pipiens and cx. quinquefasciatus. in conclusion, our study indicated that siphon/saddle index is specific diagnostic character, in contrast, siphon index has relative diagnostic value due to more scattering range (fig. 2 and 3). according to our observations, branch of seta 1 of abdominal segment of iii–iv and shape of siphon in larval samples are valuable characters which can easily separate culex pipiens and cx. quinquefasciatus. according to the results reported previously, some characters were overlap in cx. pipiens and cx. quinquefasciatus, therefore to determine the species it is recommended studying all characters together and then making a decision about such a complex species. based on our study, cx. pipiens larvae were detect in yazd area and in order to determination of the distribution of the complex species more study should be carried out in other areas of iran. . acknowledgements the authors are grateful to dr akhond, phd student in biostatistics, school of medical sciences, tarbiat-modares university. the authors appreciate mrs akbari, noruzi, and ghasemi for their collaborations in the laboratory. also the authors thank mr solaimani, yazd health training and research center, mr kalantari, head of zarch city council, mr rezaei, mr eslami, environmental health director of zarch and shahediyeh health center, for supporting the investigation. dr azari hamidian, school of health, guilan university of medical sciences, is appreciated for reviewing the manuscript. we also would like to express our appreciation to the people of abrand-abad, nosrat-abad, gerd-e-faramarz, elah abad, and sarcheshmeh villages in yazd county for their kind cooperation during the study. many thanks also dedicated to the efforts of the field staff of the yazd health training and research center. this study was financially supported by grant of tarbiat modares university of medical sciences and partly by tehran university of medical sciences. the authors declare that they have no conflicts of interest. references azari-hamidian s (2007) checklist of iranian mosquitoes (diptera: culicidae). j vect ecol. 32: 235–242. azari-hamidian s, harbach re (2009) keys to the adult females and forth-instar larvae of the mosquitoes of iran. zootaxa. 2078: 1–33. azari-hamidian s, yaghoobi-ershadi mr., javadian e, abai mr, mobedi i, linton y-m, harbach re (2009) distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern iran, with the first finding of filarial larvae in naturally infected local mosquitoes. med vet entomol. 23: 111–121. azari-hamidian s, linton y-m, abai mr, ladonni h, oshaghi ma, hanafi-bojd aa, moosa-kazemi sh, shabkhiz h, pakari a, harbach re (2010) mosquito (diptera: culicidae) fauna of the iranian islands in the persian gulf. j nat hist. 44: 913–925. iran j arthropod-borne dis, 2010, 4(2): 42–49 h dehghan et al.: the morphological variations … 49 barr ar (1982) the culex pipiens complex. in steiner wwm, tabachnick wj, rai ks, narang s (eds): recent developments in the genetics of insect disease vectors. vol. 1. champaign, illinois, stipes. pp. 551–572. bourguet d, fonseca d, vouch g, dubois mp, chandre f, severini c, raymond m (1998) the acetylcholinesterase gene ace: a diagnostic marker for the pipiens and quinquefasciatus forms of the culex pipiens complex. j am mosq control assoc. 14: 390–396. cornel aj, mcabee rd, rasgon j, stanich ma, scott tw, coetzee m (2003) differences in extent of genetic introgression between sympatric culex pipiens and culex quinquefasciatus (diptera: culicidae) in california and south africa. j med entomol. 40: 36–51. harbach re (1988) the mosquitoes of the subgenus culex in southwestern asia and egypt (diptera: culicidae). contrib amer ent inst. 24: 1–237. ishii t (1991) integrated study on the culex pipiens complex. akaieka newsletter. 14: 35–40 jupp pg (1978) culex (culex) pipiens pipiens linnaeus and culex (culex) pipiens quinquefasciatus say in south africa: morphological and reproductive evidence in favor of their status as two species. mosq syst. 10: 461–473. knight kl (1978) supplement to a catalog of the mosquitoes of the world (diptera: culicidae). thomas say foundation, college park, md. knight kl, malek aa (1951) a morphological and biological study of culex pipiens in the cairo area of egypt. sot fouad ent bull. 35: 175–185. moosa kazemi sh, karimian f, davari b (2010) culicinae mosquitoes in sanandaj county, kurdistan province, western iran. j vector borne dis. 47: 103–107. smith jl, fonseca dm (2004) rapid assays for identification of members of the culex (culex) pipiens complex, their hybrids, and other sibling species (diptera: culicidae). am j trop med hyg. 70: 339–345. vinogradova eb (2000) culex pipiens pipiens mosquitoes: taxonomy, distribution, eecology, physiology, genetics, applied importance and control. pensoft publishers, sofia-moscow. vinogradova eb (2003) ecophysiological and morphological variations in mosquitoes of the culex pipiens complex (diptera: culicidae). acta soc zool bohem. 67: 41–50. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq control assoc. 3: 568–573. zaim m, cranston ps (1986) checklist and keys to the culicinae of iran (diptera: culicidae). mosq syst. 18: 233–245. j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 124 http://jad.tums.ac.ir published online: march 14, 2017 original article molecular identification of leishmania species in a re-emerged focus of cutaneous leishmaniasis in varamin district, iran mahmoodreza behravan 1, *vahideh moin-vaziri 1, ali haghighi 1, nourina rahbarian 1, 2, niloofar taghipour 1, alireza abadi 3, homa hajjaran 4 1department of medical parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 2cellular and molecular biology research center, shahid beheshti university of medical sciences, tehran, iran 3department of social medicine and health, school of public health, shahid beheshti university of medical sciences, tehran, iran 4department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran (received 28 feb 2015; accepted 9 apr 2016) abstract background: cutaneous leishmaniasis (cl) is one of the most important neglected tropical diseases and a major public health challenge in iran caused by leishmania spp and transmitted by phlebotomine sand flies. the number of cl cases has shown an increasing pattern all over the country, including the district of varamin, southeast of tehran, iran. this study aimed to identify the leishmania spp isolated from cl patients using molecular methods in varamin during 2012–2013. methods: exudate materials collected from the swollen edge of the skin lesions of 44 parasitological positive cl patients by disposable lancet. they were referred to varamin health center by physician. the samples were subjected to molecular method for leishmania species identification. results: the digestion pattern of restriction enzyme revealed that 37 (84.1%) cl patients were infected with l. major and 7 (15.9%) were infected with l. tropica. they were mostly male than female. more than half of the patients (58%) had multiple lesions, and they were mostly observed on extremities, 34.1% on legs and 29.5% on hands. lesions were mostly of wet ulcerative type. conclusion: dominancy of l. major provides more evidence that varamin district probably could be considered as zoonotic cutaneous leishmaniasis (zcl) areas. more investigation on other epidemiological aspects of disease is needed. keywords: cutaneous leishmaniasis, pcr-rflp, leishmania tropica, leishmania major, iran introduction leishmaniasis is one of the most devastating neglected tropical diseases with a complicated ecology (alvar et al. 2012). disease is a sand fly borne infection caused by the blood parasite leishmania, more than 20 species of parasites are transmitted via the infective bites of different species of sand flies (subfamily phlebotominae) (alvar et al. 2012). leishmaniasis has been clinically categorized into three main forms: visceral (vl), cutaneous (cl), and mucocutaneous (mcl) (reithinger et al. 2007). endemic transmission of the disease has been reported from different tropical and subtropical countries with 350 million people at risk, the overall prevalence is about 12 million worldwide with yearly estimated incidence of 0.2–0.4 million cases of vl and 0.7–1.2 million cases of cl (alvar et al. 2012). iran is a main endemic region of cl in the *corresponding author: dr vahideh moin-vaziri, e-mail: vmvaziri@gmail.com j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 125 http://jad.tums.ac.ir published online: march 14, 2017 middle east and north africa (mcdowell et al. 2011). cutaneous leishmaniasis is endemic in two forms, anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl). anthroponotic form is still of great importance in many parts of the country, including some large and medium sized cities such as tehran, mashhad, neishabur and sabzevar in the north-east, shiraz in the south, kerman and bam in the southeast, yazd, kashan and parts of the city of esfahan in the central region (yaghoobiershadi et al. 2010, yaghoobi-ershadi 2012). the parasite is l. tropica and the vector is supposed to be phlebotomus (paraphlebotomous) sergenti parrot 1917. the main reservoir host is human but dogs have a role as animal reservoir host (yaghoobi-ershadi 2012). zoonotic form is endemic in many rural areas of 17 out of 31 provinces of iran (yaghoobiershadi 2012). rodents are considered as the main reservoirs of disease, rhombomys opimus is the main animal reservoir in foci located in the north-east and central part of the country, meriones libycus in some parts of central and south of the country, tatera indica in the southeast and m. hurrianae in southeastern part of baluchistan, neighboring pakistan (akhavan et al. 2010, azizi et al. 2012, yaghoobi-ershadi 2012, hajjaran et al. 2014). phlebotomus papatasi scopoli 1786, the most prevalent species among phlebotomus genus, is the only known vector (yaghoobi-ershadi 2012). from 1983 to 2012, cases of all types of leishmaniasis in iran reached to 569164, which 99.5% of that was cl. in 2012, totally 20947 cases were recorded all through the country and highest incidence rate was observed in ilam, fars and khorassan razavi provinces in that year (shirzadi et al. 2015). official reports showed an increasing trend in number of cl cases in varamin district, from 18 cases in 2010 to 32 cases in 2011 (communicable disease report, unpublished data). varamin is located 35km, southeast of tehran, iran. a purely parasitological study was done in varamin by the same authors (behravan et al. 2015), but parasite identification at species level was not done. leishmania species are not morphologically distinguishable. identification of leishmania parasites is essential for precise prognosis of the disease as well as making proper decision regarding control and probably treatment (hajjaran et al. 2014). therefore, this study was conducted to characterize the causative agents of disease by molecular tools. in recent years, different molecular methods with different genetic markers have been developed for parasite identification (schonian et al. 2003, parvizi et al. 2008, parvizi and ready 2008, azizi et al. 2012). internal transcribed spacer-1 (its1) of the small subunit ribosomal dna was known as a reliable marker for detection of leishmania parasite. different digestion patterns which were produced by haeiii (bsuri) enzyme can clearly differentiate the parasite (hajjaran et al. 2011, alnahhas and kaldas 2013, hajjaran et al. 2013). this method also was used in current study to identify leishmania species in cl suspected patients in varamin district. materials and methods study area the present study was conducted in varamin (35°19′27″n, 51°38′45″e), located 35 km southeast of tehran, iran (fig. 1). sampling and dna extraction this study was done on cl suspected patients who were referred to varamin health center laboratory during 2012–2013 from 11 different areas mentioned in figure and table 1. patient’s information (age, sex, living area, ulcer duration and number and site of lesions) was recorded by a special questionnaire. j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 126 http://jad.tums.ac.ir published online: march 14, 2017 ethic clearance was obtained from research ethical committee of shahid beheshti university of medical sciences (approval number: sbmu.rec.1392.253). smears were prepared from exudate materials of swollen edge of lesions of patients collected by sterile lancet, then fixed in methanol, stained by giemsa and examined under a light microscope for the presence of amastigotes. grading of leishmania parasites was obtained by average parasite density as follows: 4+ (1–10 parasites/fields), +3 (1–10 parasites/10 fields), 2+ (1–10 parasites/100 fields), 1+ (1–10 parasites/1000 field) according to who protocols (who 1991). exudate materials from ulcer of cl suspected patients also were used for molecular identification. dna was extracted by using the bioneer dna extraction kit (bioneer, republic of korea) in accordance with the manufacturer’s instructions. dna extracts were stored at -20 ˚c until being used. its1 amplification and enzymatic digestion its1 was amplified by using specific primers, litsr (forward: 5′-ctggatcatttt ccgatg-3′) and l5.8s: (reverse: 5′-tga taccacttatcgcactt-3′). the amplification was carried out by using the pcrready premix (roche, germany) in 25µ l total reactions comprising 10 µ l premix, 2µ l forward and reverse primers (10 pmol), 1µ l dna template and 13µ l double distilled water. iranian reference strains of l. major (accession number: jn860745) and l. tropica (accession. noumber: ef653267) were used as positive standard controls to monitor the reactions. the pcr conditions consisted of one initial denaturing cycle at 95 ˚c for 5 min, followed by 35 cycles of 94 ˚c for 30s, 47 ˚c for 30 s, 72 ˚c for 45 s. this was followed by a final extension cycle at 72 ˚c for 7min. these set of primers amplify a fragment of about 300–360bp of leishmania genome (schonian et al. 2003, hajjaran et al. 2013). the pcr products were visualized after staining with ethidium bromide by 1.2% agarose gel electrophoresis in uv transluminator. molecular identification of leishmania species was achieved by rflp analysis. ten microliters of the pcr product were added to 2µ l of the enzyme buffer and 1µ l of the haeiii (bsuri) enzyme (fermentas, life sciences, germany), this mixture was incubated at 37 ˚c for 10 minutes, as recommended by the manufacturer. the cut site of enzyme was gg↓cc, producing different patterns based on leishmania species. digestion products were separated by using 3% agarose gels and visualized after staining by ethidium bromide. results totally, 44cl parasitologically positive patients were selected for molecular examination. the positive microscopic slides were scored for leishman body. the grading of leishmania amastigotes numbers were as follow, 8% (1+), 42% (2+), 42% (3+) and finally 8% (4+). as table 1 indicates the patients affected by cl were mostly from bagherabad (27.3%) and kheir-abad (22.7%), nearly located in north-west of varamin district near to pakdasht. among the patients, 63.6% were male. patients ranged in age from 1 to 75yr old, the parasite infected all age groups except children under 1yr old and only 3 were above 65yr. the most highly infected age group was 25–39yr old (29.5%). most of the patients (71%) had multiple lesions over exposed parts of body, even with 20 lesions in one case. hands, legs and facial areas were the most affected parts of the body with 34.1%, 29.5% and 15.9%, respectively. lesions were mostly of wet ulcerative type. the size of the most lesions was more than 1cm (fig. 2). the average duration of lesions was 5 months; most of patients (99%) had no history of treatment. j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 127 http://jad.tums.ac.ir published online: march 14, 2017 molecular outcomes using the litsr/l5.8s primers set, a single product (300–360bp) has been amplified in all 44 samples (100%) (fig. 3), the same as what observed for standard strains of parasite. after using the restriction enzyme, totally two bands of 220bp and 140bp were observed in 37 (84.1%) samples which was similar to what expected for l. major as it could be seen also in standard sample. they were mostly from bagher-abad (27.3%), kheir-abad (22.7%) and varamin (18.2%) and the rest was scattered among 8 remained areas (table 1). besides, in 7 cases (15.9%), three bands of 200, 60 and 40bp were observed, comparing to standard strains, they are representative of l. tropica (fig. 4). these cases were distributed in different areas as follows, 2 cases in bagher-abad, 2 in kheirabad, 2 in varamin and just one case in reyhan-abad. table 1 showed more detailed in formation for leishmania spp which were identified by pcr-rflp. table 1. number of cases and distribution of cutaneous leishmaniasis agents based on study areas, varamin district, iran, 2012–2013 study area cases no. (%) pcr-rflp results no. l. major l. tropica bagher-abad 12(27.3) 10 2 kheir-abad 10(22.7) 8 2 varamin 8(18.2) 6 2 deh-sharifa 3(6.8) 3 0 ahmad-abad 2(4.5) 2 0 vali-abad 2(4.5) 2 0 mohammadabad 2(4.5) 2 0 omr-abad 2(4.5) 2 0 reyhan-abad 1(2.3) 0 1 charm-shahr 1(2.3) 1 0 javad-abad 1(2.3) 1 0 total 44 (100) 37 7 fig. 1. geographical location of varamin, (inset is map of iran), showing the study area and distribution of leishmania major and leishmania tropica isolated from patients during 2012–2013. numbers on the map refer to the villages of: 1: bagher-abad, 2: kheir-abad, 3: varamin, 4: deh-sharifa, 5: ahmad-abad, 6: vali-abad, 7: mohammad-abad, 8: omr-abad, 9: reyhan-abad, 10: charm-shahr, 11: javad-abad j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 128 http://jad.tums.ac.ir published online: march 14, 2017 fig. 2. representative pictures of skin lesions caused by leishmania major in cutaneous leishmaniasis patients, varamin, iran, 2011–2012. fig. 3. electrophoresis of leishmania dna amplified with primers litsr and l5.8s from exudate materials of lesions of suspected patients to cutaneous leishmaniasis, varamin district, iran, 2011–2012, line 1–9: samples of patients with cl, line 10: l. major (accession number: jn860745), line 11: 100bp ladder marker (fermentas) and line 12 negative control. fig. 4. digestion pattern of its1 amplicons of leishmania spp of cutaneous leishmaniasis patients of varamin district, by using haeiii enzyme line 1: 50 bp ladder marker, line 2: sample of patients (l. tropica), line 3: l. tropica (accession. noumber: ef653267), line 4: l. major (accession. noumber: jn860745), line 5 samples of patients (l. major), line 6: 100 bp ladder marker discussion recently, the reported cases especially that of cl due to l. major have increased (yaghoobi-ershadi et al. 2010, yaghoobiershadi 2012, hajjaran et al. 2014), and they were reported in areas known to be non-endemic or regions with decreasing trend over the passing years. although varamin is one of the old known foci of cl (ardehali et al. 1994), but comprehensive data on cl was not available. a preliminary and purely parasitological study was done by the same authors who reported confirmed parasitological cases during 2012–2013 (behravan et al. 2015). the evidence obtained by mentioned study indicates that varamin district potentially encountered with zcl. therefore, its1-pcrrflp was conducted to identify the parasite species in patients. entomological survey for vector incrimination was done in studied area by the same authors (data under assay). j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 129 http://jad.tums.ac.ir published online: march 14, 2017 totally among 44 cl patients, frequency of male was higher than of female. some other endemic regions of the country have the same pattern, such as damghan and kashan (rafati et al. 2007, talari et al. 2006, khosravi et al. 2013). it could be due to more contact of men with vectors based on the type and time of their work compared with women. conversely, in kerman province, cl was distributed more significantly in females (sharifi et al. 2008). some studies have shown an equal distribution of zcl infection among two sexes (fakoorziba et al. 2011). although most age groups were at risk of the disease, but based on obtained results, the prevalence of cl was higher in age group 25–39yr old. in known zcl endemic regions of iran, the highest risk group is often children less than 15yr old, in kerman (< 10yr old), fars province (≤ 10 yr old), damghan (10–19 yr) and part of isfahan ( under 1 yr of age) (rafati et al. 2007, sharifi et al. 2008, nateghi rostami et al. 2013). the effect of age might be actually influenced by disease endemicity and immune reactions of the host. in this study, most of the lesions appeared on the extremities, this pattern is common in zcl foci such as damghan (rafati et al. 2007) and orzoieh district (sharifi et al. 2008). over half of the patients in current study had more than one lesion similar to what observed by other researchers in zcl foci in iran (rafati et al. 2007, sharifi et al. 2008). in cl due to l. major, multiple lesions are common (yaghoobiershadi et al. 2003). it could be because sand flies usually have a discontinuous blood-sucking habit, may bite several times and cause the development of several lesions on the skin (yaghoobi-ershadi et al. 2003, yaghoobiershadi et al. 2010, nateghi rostami et al. 2013). all 44 available exudate materials which were confirmed positive by microscopic method were positive also by molecular tools (100%), which could be dedicated that the applied genetic marker and molecular method is quit useful, although it was not the aim of this study. leishmania major as the main causative agent of zcl was identified in most lesions (84.1%), which was not far from our expectation. about 80% of cases reported in the country are of zcl form (yaghoobi-ershadi 2012). same observations were reported from neighboring provinces of varamin such as semnan and qom, which l. major was identified as the dominant causative agents of leishmaniasis in both areas (rafati et al. 2007, nateghi rostami et al. 2013). one the most important factors in leishmaniasis spreading are migration and travelling (yaghoobi-ershadi 2012). varamin is the passage-way to the several large cities, especially religious city which caused a lot of people travel around. as a matter of fact, varamin located very close to the capital of iran, is one of the most important agricultural and animal husbandry axis, which caused it to be a good site for job seekers and consequently a lot of migration happen which increase the potential risk of disease. conclusion the achieved results, specifically dominancy of l. major, indicate that varamin district could be regarded as one of the zcl region in iran, but to identify all epidemiological aspects of disease, a comprehensive epidemiological study is highly recommended. acknowledgements this study was funded by the research council of shahid beheshti university of medical sciences (grant no. 11119-91-11392). the authors have great thanks to health center staff of varamin, mr amini and mr hoobar and mr farnoosh. the authors highly appreciate the participated patients to the current study. the authors declare that there is no conflict of interest. j arthropod-borne dis, march 2017, 11(1): 124–131 m behravan et al.: molecular identification … 130 http://jad.tums.ac.ir published online: march 14, 2017 references akhavan aa, yaghoobi-ershadi mr, khamesipour a, mirhendi h, alimohammadian mh, rassi y, arandian mh, jafari r, abdoli h, shareghi n, ghanei m, jalali-zand n (2010) dynamics of leishmania infection rates in rhombomys opimus (rodentia: gerbillinae) population of an endemic focus of zoonotic cutaneous leishmaniasis in iran. bull soc pathol exot. 103: 84–89. al-nahhas sa, kaldas rm 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yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod borne dis. 6(1): 1–17. j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 27 http://jad.tums.ac.ir published online: march 14, 2017 original article disappearance of anopheles minimus and anopheles dirus from certain malaria endemic areas of assam, india *kavita yadav 1, sunil dhiman 1, bipul rabha 1, diganta goswami 1, pk saikia 2, vijay veer 1 1medical entomology, defence research laboratory, tezpur, assam, india 2zoology department, gauhati university, guwahati, assam, india (received 2 july 2014; accepted 17 nov 2015) abstract background: orang primary health centre (ophc) and balipara primary health centre (bphc) of assam (india) report mosquito borne diseases annually. current study was performed to ascertain the prevalence of known malaria and japanese encephalitis (je) vectors and their possible role in disease transmission. methods: malaria epidemiological data for 2006–2010 and je data for 2008–2013 of assam, india were obtained from the health authority. mosquitoes were collected using cdc light traps and identified morpho-taxonomically. results: plasmodium falciparum cases (81.5%, 95% ci= 72.0–91.1) were statistically higher in ophc (p< 0.0001, t= 8.0) during the recent years. there was 4.4 folds rise in the confirmed acute encephalitis syndrome (aes) and 3.2 folds increase in the confirmed je cases during 2013 as compared to 2008. altogether 9,218 mosquito specimens (ptnd= 153.6), comprising of 44.1% anophelines (ptnd= 67.7), 42.3% culicines (ptnd= 65.0) and 9.5% mansonia (ptnd= 14.6) were recorded. in bphc, anopheles vagus was recorded in high density (p< 0.0001), whereas culex quinquefasciatus was the predominant je vector (p= 0.04). in ophc, among the known malaria vectors, the density of anopheles annularis was significantly high (p< 0.0001). however culex bitaeniorhynchus was the predominant known je vector (p< 0.0001) followed by cx. quinquefasciatus. conclusion: even in the absence of known efficient vectors, many anopheles species are still involved in malaria transmission. there was disappearance of anopheles minimus and anopheles dirus and establishment of an. annularis, an. vagus and an. philippinensis/nivipes mosquitoes in study area. keywords: mosquito vectors, malaria, japanese encephalitis, ecology, india introduction mosquitoes spread pathogenic agents of malaria, japanese encephalitis (je), dengue, lymphatic filariasis and chikungunya in many countries. the data on prevalence of known mosquito vectors constitutes vital and useful information to control the mosquito-borne diseases. despite concerted vector borne diseases intervention efforts in india during 2014, approximately 1.07 million confirmed malaria cases and 535 deaths have been reported, while 1,661 confirmed cases and 293 deaths have been attributed to je (nvbdcp 2014). mosquitoes are remarkably adaptable insect group, which continue to successfully coexist with human being and survive by feeding on human host and his domestic animals in addition to plant nectar. assam is the largest state (population wise) in northeast region of india, where malaria transmission is endemic. in india, an. minimus theobald, 1901, an. dirus peyton and harrison, 1979, an. fluviatilis james, 1902 and an. culicifacies giles, 1901 have been recognised as potential malaria vectors, while an. annularis van der wulp, 1884, an. philippinensis ludlow, 1902 and an. varuna iyengar, 1924 play limited role in malaria transmission (dev et al. 2003, prakash et al. 2004, bhattacharyya et al. 2010, dhiman et al. 2011, 2012). je outbreaks are common in northeastern states *corresponding author: dr kavita yadav, e-mail: kavitanami@gmail.com j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 28 http://jad.tums.ac.ir published online: march 14, 2017 including assam and occur mainly during rainy season. sixteen mosquito species have been incriminated as je vector in india, of which culex tritaeniorhynchus giles, 1901 and culex vishnui theobald, 1901 subgroup has been reported as prominent je vectors in the endemic areas (kanojia 2007, saxena et al. 2008, dhiman et al. 2013). many culicine and mansonia species, namely, cx. vishnui, cx. bitaeniorhynchus giles, 1901, cx. gelidus theobald, 1901, ma. uniformis theobald, 1901, ma. annulifera theobald, 1901 and ma. indiana edwards, 1930 are well known vectors of je and reported in many parts of assam and other north-eastern states of india (saxena and dhole 2008, dhiman et al. 2009, 2013). udalguri and sonitpur districts of assam are highly endemic for malaria and contribute considerably to the malaria cases in the state. a recent study conducted in udalguri district has indicated that, the number of health centres having annual parasitic index (api) of > 5 and more than 30% of malaria cases due to p. falciparum welch, 1897 were increased in the recent years. the study exhibited that 11 health centres were malaria hot spots, of which 9 were part of orang primary health centre (ophc) (yadav et al. 2012). similarly, in sonitpur district 10 health centres including balipara have been identified as malaria hot spots, which have an extremely high malaria risk (nath et al. 2013). the forestlands of both the districts have remained the areas of intense malaria parasite reservoir providing foci for re-infection in the other neighbouring areas. moreover, ecological changes due to deforestation have brought some changes in geo-climate that has significantly influenced the mosquito vector ecology and diseases transmission (nath et al. 2012). the dynamic distribution and transmission of malaria and je in assam poses a serious epidemiological challenge due to various socio-economic, geo-political and environmental factors (dev et al. 2003, 2010, dhiman et al. 2011, rabha et al. 2012, yadav et al. 2014). information on vector entomology is an essential component in disease management, which depends upon the knowledge of vector species density and composition. many studies have been conducted in various parts of the state, but anthropogenic ecosystem modifications in the past few years might have influenced the known malaria and je vectors composition. therefore, it is inevitable to update data on prevalence of vector mosquitoes for reviewing vector control strategies. the present study was undertaken during april 2012 to august 2013 in malaria endemic primary health centres of udalguri and sonitpur districts of assam to generate information on known mosquito vector prevalence. materials and methods study area current study was conducted in randomly selected four sentinel survey sites each (table 1) in ophc of udalguri district and balipara primary health centre area (bphc) of sonitpur district. ophc (92 ̊ 07’–92 ̊ 22’ e longitude and 26 ̊ 33’–26 ̊ 56’ n latitude) situated at 105.2 meters, is dominated by various ethnic tribes primarily engaged in agriculture and tea cultivation. the climate is sub-tropical humid and experience an average annual rainfall of about 2,000mm, while the temperature and relative humidity varies between 34.5 °c to 13.5 °c and 65 to 90% respectively. study area has many small rivers, scattered tea gardens and vast paddy fields, which create suitable breeding ecology for mosquito vectors. bphc (92 ̊ 38’–92 ̊ 59’ e longitudes and 26 ̊ 41’–27 ̊ 02’ n latitude, 74.7 meters) is dominated by different ethnic groups, including bodo, nepali, aadivasi and assamese, with agriculture based very low socio-economic status. the average temperature ranging from 15 °c to 35 °c, about j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 29 http://jad.tums.ac.ir published online: march 14, 2017 1,900mm average rainfall and 55–90% relative humidity plays a major role in determining the climate of the area. there are many rivers and large spreads of tea meadows and paddy fields. the prevailing climatic conditions are conducive for the breeding and proliferation of different vector mosquitoes. a recent study conducted in sonitpur district using normalized difference vegetation index (ndvi) has shown that the forest-covered area was 1.2 folds decreased in two years (nath et al. 2012). malaria and japanese encephalitis situation in the area malaria epidemiological data for the years 2006–2010 were collected from the district malaria office of the concerned district and analysed to understand the malaria situation during preceding years. japanese encephalitis data of assam for the years 2008–2013 was obtained from the nvbdcp and depicted in table 2. collection and identification of known mosquito vectors mosquitoes were collected from dusk to dawn (1800–0600 h) with 6-volt battery operated miniature light traps (centres for disease control, atlanta, usa) and indoor resting using who aspirator tubes. the mud plastered kuchha houses and bamboo houses having a thatched roof and adjacent to the cattle sheds were selected for the study. the study area had numerous small ponds and irrigation channels, while number of domestic animals such as pig, fowl and duck were also available. smoking and burning was prevented during the operation of the traps. the traps were installed near unscreened windows in the rooms at about 2 m above the ground and kept on throughout the night until removed in the early morning hour. the collected mosquitoes were etherised (if alive) and identified to species complex level with the help of standard keys (barraud 1934, wattal and kalra 1961). densities of known vector mosquitoes were calculated in terms of mean numbers of mosquito of a species caught per trap night and expressed as per trap night density (ptnd) of that particular species. data analysis the collected mosquitoes were expressed in per trap night density (ptnd), whereas known vector density for each species was presented as mean±sem. comparison of mosquitoes was performed using anova followed by tukey krammer test of multiple comparison. ptnd of known malaria and je vectors was compared using unpaired students’ “t” test. results past malaria and japanese encephalitis situation malaria situation during the past years (2006–2010) in bphc and ophc is shown in fig. 1. the slide positivity rate (spr) in both the locations was similar in the recent years (p= 0.8, t= 0.3), however cases attributed to p. falciparum malaria in ophc were 81.5% (95% ci= 72.0–91.1) and found to be statistically higher than in bphc (p< 0.0001, t= 8.0). further, the average annual parasitic index (api) during the years 2006–2010 was 6.9± 12 (95% ci= 3.6–10.3) in ophc as compared to 2.4±0.5 (5% ci= 1.1–3.7) in bphc (p= 0.01, t= 3.4). past encephalitis data suggests that as compared to 2008, there has been 4.4 folds increase in the confirmed acute encephalitis syndrome (aes) cases and 3.2 folds increase in the confirmed je cases in 2013. however, confirmed deaths due to aes showed 2.7 folds while due to je showed 4.1 increases during the last six years. known malaria and japanese encephalitis vectors abundance a total of 9,218 mosquito specimens were collected in the current study using cdc light j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 30 http://jad.tums.ac.ir published online: march 14, 2017 traps and aspirators in 60 trap nights (ptnd= 153.6). of the total mosquitoes, anophelines were 44.1% (ptnd= 67.7), culicines were 42.3% (ptnd= 65.0), mansonia were 9.5% (ptnd= 14.6) while remaining 4.1% (ptnd= 6.3) corresponded to other mosquito species belonged to stegomyia, neomelaniconion, coquillettidia and armigeres. in bphc, total 7,201 (ptnd= 180.0) mosquitoes constituting 37.9% anophelines (ptnd= 68.2), 45.3% culicines (ptnd= 81.6) and 12.0% mansonia (ptnd= 21.7) were reported during the study. the density of anophelines and culicines mosquitoes was statistically higher than the other mosquitoes, however no significant difference was found among both of them (p= 0.8, t=0.3). among the reported malaria vectors in india and neighbouring bangladesh, present study found an. annularis, an. culicifacies, an. philippinensis and an. vagus doenitz, 1902 mosquitoes. anopheles vagus (58.1%, ptnd= 37.7 of the total known malaria vectors) was recorded in significantly high density (f= 18.4, p< 0.0001), but did not differ statistically from an. annularis (p= 0.2, t= 1.6). among the known je vectors, cx. quinquefasciatus say, 1823 was predominant (f= 2.6, p= 0.04) and accounted for 49.5% of the total je vectors (ptnd= 44.6). malaria and je vectors density obtained in bphc is depicted in table 3. there was no statistically difference between malaria and je vectors density (p≥ 0.3, t≤ 0.6) in bphc throughout the study period. in ophc, of the total 2,017 (21.9%) mosquitoes in 20 trap nights (ptnd= 100.9), 66.2 % were anophelines, 47.6% were culicines, while 1.8% corresponded to mansonia species. the ptnd was highest for anophelines (66.8) followed by culicines (31.8). density of anophelines was statistically higher than the other mosquitoes (f= 748.6, p< 0.0001). among the known malaria vectors, an. culicifacies, an. vagus, an. fluviatilis and an. annularis were prevalent, however the density of an. annularis was significantly high (f= 180.3, p< 0.0001). culex bitaeniorhynchus was the predominant known je vector (f= 92.1, p< 0.0001) followed by cx. quinquefasciatus. malaria and je vector density in ophc is shown in table 4. malaria vector density was statistically higher than the je vectors in ophc (f= 15.1, p< 0.0001). between bphc and ophc, the ptnd of known je and malaria vectors (fig. 2) between both the study areas were similar (p≥ 0.4, t≤ 1.0). (bsrblood smear rate, sprslide positivity rate, % pfpercent plasmodium falciparum, apiannual parasitic index) fig. 1. past malaria situation depicting blood smear rate (bsr), slide positivity rate (spr), % plasmodium falciparum and annual parasitic index (api): (a) in bphc, (b) in ophc b a j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 31 http://jad.tums.ac.ir published online: march 14, 2017 (malmalaria, bphc/ophcbalipara/orang primary health centre) fig. 2. per trap night density of known malaria and japanese encephalitis vectors in four sentinel collection sites (s1–s4) in bphc and ophc areas table 1. global positioning system coordinates of the sentinel survey locations (s1-s4) in both study areas of orang primary health centre (ophc) and balipara primary health centre (bphc) of assam (india) study area survey site gps location balipara primary health centre (bphc) s1 92°46'43.0" e 26°41'18.6" n s2 92°47'36.2" e 26°42'02.3" n s3 92°47'39.6" e 26°40'35.3" n s4 92°48'09.7" e 26°41'31.8" n orang primary health centre (ophc) s1 92°16'15.8"e 26°38'23.8" n s2 92°15'49.9"e 26°41'45.4" n s3 92°17'32.4"e 26°41'52.0" n s4 92°19'38.3"e 26°41'55.3"n table 2. past acute encephalitis syndrome and japanese encephalitis situation in assam year acute encephalitis syndrome (aes) japanese encephalitis (je) reported cases deaths (%) reported cases deaths (%) 2008 319 99 (31.0) 157 33 (21.0) 2009 462 92 (19.9) 218 46 (21.1) 2010 469 117 (24.9) 142 40 (28.2) 2011 1319 250 (19.0) 489 113 (23.1) 2012 1343 229 (17.1) 463 100 (21.6) 2013 1388 272 (19.6) 495 134 (27.1) j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 32 http://jad.tums.ac.ir published online: march 14, 2017 table 3. known malaria and japanese encephalitis vectors in balipara primary health centre area species density (mean±sem) f (p) malaria vectors an. vagus 377.3±81.5 18.4 (< 0.0001)an. annularis 241.5±15.9 an. culicifacies 15.3±6.0 an. philippinensis 15.5±2.5 je vectors cx. quinquefasciatus 446.3±270.3 f= 2.6 (p= 0.04) cx. vishnui 175.3±89.9 cx. bitaeniorhynchus 120.8±100.2 cx. whitmorei 25.5±4.2 cx. gelidus 8.5±2.9 ma. uniformis 131.8±115.7 ma. indiana 30.3±19.2 ma. annulifera 44.8±33.6 table 4. known malaria and japanese encephalitis vectors in orang primary health centre area species density (mean±sem) f (p) malaria vectors an. vagus 156.0±11.7 180.3 (< 0.0001) an. annularis 162.8±6.7 an. culicifacies 1.8±0.5 an. fluviatilis 3.5±0.6 je vectors cx. quinquefasciatus 63.8±7.7 92.0 (< 0.0001) cx. bitaeniorhynchus 90.5±6.5 cx. whitmorei 0.6±0.6 cx. gelidus 3.3±0.8 ma. indiana 0.8±0.4 ma. annulifera 1.8±0.8 discussion vector borne diseases control programmes have always stressed that up-to-date knowledge of spatial distribution and diversity of mosquito vectors across the endemic areas is inevitable for planning and implementing the effective intervention measures. in the current study, known malaria and je vectors were collected in two ecologically distinct endemic primary health centres of assam, where malaria and je transmission is supported by many efficient mosquito vectors. anopheles dirus, an. fluviatilis and an. minimus mosquito species have been considered as important malaria vectors, however in north-eastern states and neighbouring country bangladesh the role of an. annularis, an. culicifacies, an. philippinensis and an. vagus in malaria transmission is determined (prakash et al. 2004, alam et al. 2010, bhattacharyya et al. 2010, dhiman et al. 2012). the present study has indicated that many known malaria vectors were abundant in both the study areas, however could not report even a single specimen of established vectors an. minimus and an. dirus (dev et al. 2003, das et al. 2004, sarma et al. 2012). both an. minimus and an. dirus are known for uninterrupted spread of malaria in the region, but the surprising results of current study indicate that the other malaria vectors of comparatively lesser epidemiological importance might have taken over malaria transmission j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 33 http://jad.tums.ac.ir published online: march 14, 2017 in absence of the well-established vectors. in bphc of assam an. annularis was found harbouring both p. falciparum and p. vivax grassi and feletti 1890 and its density outnumbered the density of an. minimus during malaria season (dhiman et al. 2012). anopheles culicifacies, although comes in lower counts but have strong anthropophilic character and malaria transmission potential in the suburbs of the region (dhiman et al. 2012). further, an. nivipes theobald, 1903 and an. vagus have also been found positive for sporozoite in different malaria endemic areas of northeastern india (prakash et al. 2004, bhattacharyya et al. 2010). presently, an. annularis and an. vagus were recorded in large number in areas where malaria is still endemic and many cases are reported annually (rabha et al. 2012, yadav et al. 2012, nath et al. 2013). although both these species are primarily considered zoophilic and exophilic in nature, but they have been considered to be opportunistic in the host selection for blood meal and have been thought to maintain malaria transmission in the region (prakash et al. 2004, dhiman et al. 2012). large number of specimens corresponding to these two species was collected indoor resting which indicates that both of these might be shifting exophilic and exophagic behaviour to endophilic and endophagic. in assam-meghalaya border, an. annularis prefers resting indoors and a considerable proportion feed on human blood (dhiman et al. 2014). in the recent years, there has been tremendous deforestation, and new resettlements are coming up rapidly in the study area. the forestlands, which provide favourable breeding habitats for malaria vectors an. dirus and an. minimus, have been reduced significantly during last few years (nath et al. 2012). therefore, disruption in the ecology of these two vectors might have persuaded other anopheline species such as, an. annularis, an. vagus and an. philippinensis/nivipes to establish themselves as major species owing to the vast paddy cultivation in the area. among the je vectors cx. bitaeniorhynchus, cx. quinquefasciatus and cx. vishnui were recorded in large density, whereas cx. gelidus was recorded in very low number. these vectors have been reported from other areas of assam and found associated with the je transmission in the region (dhiman et al. 2013). in the past few years, the je is emerging as serious vector borne disease in the entire north-east region, where reported cases and deaths during recent years has increased to many folds (nvbdcp 2013). however, the vector abundance of potential je vectors has not been monitored regularly (dhiman et al. 2013). je outbreaks are common during the rainy season and occur at regular intervals in different parts of northeast region including study area. therefore, data on population dynamics of je vectors is important for focused control measures implementation. mosquito density builds up during the epidemics, however abundance, survival and longevity of vector mosquitoes directly influence the dynamics of disease transmission annually. the study area reports high incidence of malaria throughout the year. various ethnic tribes having poor economic condition and their socio-cultural customs and beliefs make malaria vector control difficult (dhiman et al. 2011, rabha et al. 2012, yadav et al. 2014). malaria vector density was high in ophc, which corroborates the high number of reported malaria cases. the api reported in ophc is about 2.8 folds, while the percent p. falciparum is about 2.3 folds higher than in bphc. higher density of known vectors has been found associated with increase in disease incidence across many endemic settings (alam et al. 2012, dhiman et al. 2012). although not much je cases are reported in ophc as compared to bphc, but the tribal villages in the ophc area have high pig density, which could serve as reservoir of je virus, and also the high density of known je vectors may sprout the disease j arthropod-borne dis, march 2017, 11(1): 27–35 k yadav et al.: disappearance of … 34 http://jad.tums.ac.ir published online: march 14, 2017 during favourable transmission conditions. ecological changes might have involved in the replacement of well-established mosquito species by other species. deforestation and irrigation projects resulted in change of mosquito species involved in malaria transmission in sri lanka and thailand (amerasinghe et al. 1991, prothero 1999). conclusion the present study is limited in its scope and reveals that variety of little known malaria and je vectors are maintained in the study area, while some well-known vectors were disappeared or maintained at very low density. we have not attempted to incriminate any known malaria and je vectors, but suggest that even in the presence of comprehensive vector control measures some little known vectors might have been playing a leading role in disease transmission. high density of known vectors may increase the risk of increasing insecticide resistance thereby circumventing the protection from insecticides. further investigation on breeding ecology and insecticide susceptibility status of commonly used insecticides is important to provide information for adopting suitable control measures. acknowledgements authors are thankful to the local health staff for assisting in the collection of malaria and je epidemiological data. the help rendered by the villagers during the study is also solicited. the authors declare no conflict of interests. references alam ms, chakma s, khan wa, glass ge, mohon an, norris lc, podder mp, norris de, ahmed s, haque r, sack da, elahi r, sullivan david j jr (2012) diversity of anopheline species and their plasmodium infection status in rural bandarban, bangladesh. 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http://jad.tums.ac.ir published online: october 04, 2016 original article spatial analyses of the relation between rodent’s active burrows and incidence of zoonotic cutaneous leishmaniasis in golestan province, northeastern of iran aioub sofizadeh 1, *hassan vatandoost 2, *yavar rassi 2, ahmad ali hanafi-bojd 2, sayena rafizadeh 3 1department of medical entomology and vector control, school of public health,tehran university of medical sciences, international campus (tumsic), tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3ministry of health and medical education, tehran, iran (received 24 nov 2014; accepted 19 aug 2015) abstract background: zoonotic cutaneous leishmaniasis (zcl) is one of the most important vector-borne diseases in iran. wild rodents play as a reservoir. the main aim of this study was to determine spatial analyses of the relationship between rodent’s active burrows and incidence of zcl in golestan province, north east of iran. methods: the cross-sectional study was conducted in 59 rural districts in golestan province. spatial distribution of rodent’s active burrows, human cases of zcl and incidence of disease were collected, using geographical information systems (gis). the relationship of them were analyzed by sperman test, spss software version no.13. results: the most number of rodents’ active burrows, human positive cases (100 persons) and high incidence of disease (35/1000) were observed in korand rural district of gonbad-e kavoos county. there was significant correlation between the number of rodents active burrows with incidence rate of disease (0.470, p< 0.001) as well as the number of cases in each districts (0.465, p< 0.001), there is high correlation between higher incidence rate and human positive cases in districts with number of rodents’ active burrows. conclusion: vicinity of wild rodents’ burrows to villages plays an important role in transmission of zcl to humans. keywords: cutaneous leishmaniasis, rodent burrows, spatial analysis, golestan, iran introduction the world health organization considers leishmaniasis as one of the most neglected tropical diseases which has received little attention and resources despite its serious impacts on both the economic developments and quality of life (who 2012). leishmaniasis is one of the most important vectorborne disease and public health problem in iran that transmitted by sandflies to human and other animals. iran is facing both form of leishmaniasis: cutaneous and visceral. the main visceral foci are located in ardabil (northwest) and fars (southwest). there are also foci with low endemicity in other parts of the country. cutaneous leishmaniasis is the main vector-borne disease in the country with an average of more than 22,000 cases in the last decade (karimi et al. 2014), about 80% of them are zoonotic cutaneous leishmaniasis (zcl), the endemic foci of this type are in rural areas of 17 out of 31 province (yaghoobiershadi 2012, karimi et al. 2014). zcl is a disease that primarily uses animals such as rodents as reservoir hosts (mirzaei et al. 2011). humans are an accidental host that can be involved in the transmission cycle of leishmania parasites (rouhani et al. 2014). geographically, zcl is widely distributed in af*corresponding author: dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums. ac.ir, dr yavar rassi, e-mail: rassiy@tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 569–576 a sofizadeh et al.: spatial analyses of … 570 http://jad.tums.ac.ir published online: october 04, 2016 rica, the middle east, central asia, and the rajasthan area of india (rouhani et al. 2014). in iran based on animal reservoir host, there are four foci of disease in our country: the first one has been located in central and northeast of iran, where rhombomys opimus and phlebotomus papatasi play important roles as reservoir and vector of the disease. the second focus of zcl is located in the west and southwest of iran, where tatera indica replaced with r. opimus as a reservoir and p. papatasi as a vector. baluchistan province, in the southeast of iran is considered as the third focus of zcl. in this region meriones hurrianae has been approved as a natural reservoir host. from the reported evidences, it is apparent that the most rural areas of provinces in central and southern iran can be considered as the zcl focus where m. libycus is the primary and main reservoir host of the disease, while r. opimus and t. indica were absent and p. papatasi is considered as the proven vector of zcl (yaghoobi-ershadi et al. 2001, rassi et al. 2006, rassi et al. 2008, rassi et al. 2011, azizi et al. 2011, kassiri et al. 2013). golestan province is one of the well-known foci of zcl in iran and two counties of this province are famous: gonbad-ekavoos and maraveh tapeh that located in north and northeastern of this province. in one study carried out in gonbad-ekavoos county, 4% of population have acute ulcer and 78% scar and in other study in maraveh tapeh county the prevalence of acute ulcer and scare rate were 3.03% and 63.7% respectively (sofizadeh 2007, cherabin et al. 2012, sofizadeh et al. 2013). many researchers have argued that r. opimus and m. libycus are reservoir hosts of zcl and p. papatasi is the main vector of this disease in golestan province (rassi et al. 2008, rouhani et al. 2014). the main aim of this study was to determine relation between rodents’ active burrows and incidence of zcl in golestan province using spatial analysis. materials and methods study area a cross-sectional study was carried out in golestan province from february 2013 to march 2014. golestan province is located (37-38°n and 54-58° e) in northeastern of iran and is bounded by caspian sea and mazandaran province in the west, semnan province in the south, north khorasan province in the east and a borderline with turkmenistan in the north (fig 1). most parts of golestan province are plain and more than 2/3 of the plain area has arid and semiarid climates and 1/3 of others have a mild climate. the area of the province is 20437.74 km2 (1.3 areas of iran) with 1823117 population. there are 14 counties, 27 districts, 60 rural districts, 25 cities and 1732 villages in golestan province. the main agricultural products are alfa alfa, rice, watermelon and cotton. maximum and minimum of temperatures were recorded as 40.8 and -02 °c respectively and the mean annual relative humidity was recorded as 74%. the total annual rainfall was 772 mm and the minimum precipitation in august and maximum in february. collection of data estimation of rodent burrows three villages were randomly selected in all rural districts (60) of golestan province and the number of active rodents’ burrows were counted in area of one hectare. the average number of active rodents’ burrows in each districts was estimated. estimation of human positive cases and incidence rate of the disease demographic data of all patients were registered. furthermore, all villages with positive human cases in 2013 were obtained regardless of the number of cases reported from each village maps of the spatial distribution of the disease were prepared using gis software. j arthropod-borne dis, december 2016, 10(4): 569–576 a sofizadeh et al.: spatial analyses of … 571 http://jad.tums.ac.ir published online: october 04, 2016 the number of human positive cases in each rural districts of the province, as well as the incidence of disease were calculated. for calculating, the incidence rate of the disease, we used population at risk in the denominator. all cases with scares were removed from the denominator. analyses of data the relationship between the number of rodents’ active burrows with incidence rate and the number of positive cases of disease in rural districts were calculated using spss software version 13 and sperman test. results among all counties of golestan province, the highest incidence rate of the disease were observed in moravehtappeh (121.5/100000) and gonbad kavoos (99.3/100000) respectively (table 1). these two counties were endemic foci of cl in golestan province. in the rest of counties, the incidence of disease were estimated less than 30 /100000. the highest (53.8%) and lowest (11.5%) of cl cases were observed in the age groups of ≥ 14 and 1≤ years old respectively (table. 1). it should be noted that, 60.2 % of patients were male. in the assess the existence of rodent’s active burrows in a radius of 300 meters from the villages, they were found only in the rural districts of gonbad kavoos and moravehtappeh counties. in the rest of studied districts, there were not rodent’s active burrows, or were located at a distance of one kilometer of the villages. however, the maximum number of rodent’s active burrows was observed in rural district of korand in gonbad kavoos county (fig. 2). the number of positive cases as well as the incidence of disease were calculated 0-100/100000 and 0-35/100000 in the different rural districts of golestan province respectively. it must be mentioned, similar to rodent’s active burrows, the most positive cases and the highest incidence of disease were found in rural district of korand in gonbad kavoos county (fig. 2, 3). based on the statistical analysis of data, there were significant difference between the number of rodent’s active burrows with the number of positive cases (0.465, p< 0.001) as well as, the incidence of disease (0.470, p< 0.001) (tables 2,3) . the most positive cases were found in the northern villages of plain areas in october and november (figs. 4, 6). there was no positivecases of disease in southern villages with mountainous condition (fig. 5). there was a significant positive correlation between the number of rodent’s active burrows and incidence of disease as well as the number of positive cases in studied areas (0.470, p< 0.001). according to fig. 6. the greatest number of cases were occurred in october and november. table 1. demographic characters of zcl in golestan province (2013) travel to endemic area (%) incidence rate (per 100000) number of cases districts 36(97.3%)28.7229237aghghala 18(100%)12.8382518aliabad 8(80%)10.1292510azadshahr 2(100%)3.8178142bandar-e gaz 2(100%)2.515472bandar-eturkaman 12(100%)17.7184512galikesh 9(100%)14.040789gumishan 210(62.9%)99.39175334gonbad-e kavoos j arthropod-borne dis, december 2016, 10(4): 569–576 a sofizadeh et al.: spatial analyses of … 572 http://jad.tums.ac.ir published online: october 04, 2016 35(89.8%)8.6678839gorgan 10(100%)8.49906510kalaleh 5(80%)6.8934145kordkooy 52(75.4%)121.525969maravehtappeh 2(100%)2.450532minoodasht 24(100%)27.2900924ramiyan 57331.74271573total (in golestan province) number (%)age group 66 (11.5%)>1 117(20.4%)1-6 82(14.3%)7-14 308(53.8%)<14 gender 345(60.2%)male 228(39.8%)female 573 (100%)total (in golestan province) fig. 1. location of study area, golestan province in iran fig. 2. distribution of rodent’s active burrows in golestan province, 2013 fig. 3. number of positive cases of zcl in golestan province, 2013 fig. 4. incidence of zcl in golestan province, 2013 table 1. continued… j arthropod-borne dis, december 2016, 10(4): 569–576 a sofizadeh et al.: spatial analyses of … 573 http://jad.tums.ac.ir published online: october 04, 2016 fig. 5. spatial distribution of zcl cases in golestan province (2013) fig. 6. trend of zcl cases in golestan province (2013) discussion based on literature review of cutaneous leishmaniasis in golestan province, the causative agent of disease (zcl) was l. major and p. papatsi has been reported as the principal vector to human. two rodents of r. opimus and m. lybicus were main and secondary reservoir host of disease respectively (rassi et al. 2008, parvizi and hedayati 2010, roshanghalb and parvizi 2012, bordbar and parvizi 2014). since the sand flies are often active in a distance of 200 meters from its habitats (rassi and hanafi bojd 2006), therefore, the presence of rodents’ burrows, one kilometer from the village, are considered as one of the risk factors of disease transmission. based on the results of our study, the active rodents’ burrows were only observed in rural districts of maraveh tappeh and gonbad-e kavoos counties. the previous studies have demonstrated that two species of r. opimus and m. libycus, are chosen the plains with soft soil to build their colonies under bushes and along streams (nadim et al. 2009), so, in the rural district of palizan to centroid qazanqaya village (56°15'28.686"e 37°55'31.132"n) due to the rocky nature of area as well as , in the rural districts of shalami to centroid arab qari haji village (55°45'8.727"e 37°41'23.994"n) and golidagh to centroid golidagh village (55°56'41.944"e 37°38'41.094"n) due to the mountainous nature of the region, no rodent burrows were observed. we found several active burrows with high population of rodents in both rural districts of the korand to centroid korand village (55°31'5.842"e 37° 57'51.47"n) and atrak to centroid dashliburun village (54°48'45.653"e 37°37'57.254"n) located in gonbad-e kavoos county. in the rural districts of agh-abad to centroid aghabad village (55°14'26.891"e 37°18'14.929"n) and sultan ali to centroid sultan ali village (55°3'7.15"e 37°14'4.863"n) in some villages, the rodents were active with low abundance . in the tamran rural district to centroid tamer ghare ghozi village (55°29'55.568"e 37°29'51.35"n) located at the kalaleh county, there was no rodent burrows at a distance of one kilometer from the villages. also, in the rest counties of golestan province, no rodent’s active burrows were observed nearby villages. based on our study, there were high correlation between the number of rodent’s active burrows with the number of positive cases (0.465, p< 0.001) as well as, the incidence of disease (0.470, p< 0.001). this was due to the abundance of active wild rodents as the reservoir of disease and vicinity of j arthropod-borne dis, december 2016, 10(4): 569–576 a sofizadeh et al.: spatial analyses of … 574 http://jad.tums.ac.ir published online: october 04, 2016 their colonies to human settlements. therefore, high number of positive cases as well as high incidence of disease were observed in the rural districts of moravehtappeh (moravehtappeh county) and korand and also atrak in gonbad kavoos county. it should be noted that, two rural districts of aghabad and sultan-ali in gonbad kavoos county were in the second rank of positive human cases. all positive cases in other rural districts of the province were imported cases, due their business trip to endemic areas. evaluation forms for these patients revealed that in most counties, the patients had a history of traveling to endemic areas of disease including rural districts of maraveh tapeh, atrak or korand. some patients had also at least one travel to the provinces of semnan, khorasan and isfahan. for example, the number of 20 cases of cutaneous leishmaniasis had been reported from the district of south fenderesk to centroid dar kalateh village (54°57'38.738"e 36°57'58.499"n) located at the county of ramian, they were workers of brick kilns, who had traveled to the province of semnan in the early spring and had returned to their homes in the early autumn of 2013. the highest incidences of the disease were found in the counties of maravehtappeh and gonbad kavoos counties respectively, while the high number of positive cases were observed in the second county. because the most population of the maraveh tappeh county (43.1%) were living in endemic areas and were at greater risk of the disease, while in the county of gonbad-e kavoos county about 15% of the population were living in endemic areas. the hightest (53.8%) and lowest (11.5%) of cl cases were observed in the age groups of >14 and <1 respectively (table 1). whereas most patients (53.8%) were adults over 14 years old, the diseases have hypo-endemic condition in this area. these results were similar to studies conducted in maraveh tapeh and gonbad-e kavoos countis in this province (cherabin et al. 2012, sofizadeh et al. 2013) and in the rural districts of damghan, and kasahan in the provinces of semnan and isfahan respectively (doroodgar et al. 2009, mohammadi azni et al. 2010). the lowest morbidity rate of disease (11.5%) was observed in age group of ≤1 years old. this age group had the lowest presence in outdoors and considering to hypoendemic of disease in studied areas (sofizadeh et al. 2013), they have received less infected bites. our study showed, the males are infected more than females (60.2%). because, the males are busy in agriculture tasks outside of home during the nights of summer and attending to active peak of sand flies (sofizadeh et al. 2009) they have received more infected bites. the greatest number of cases were occurred in the months of october and november and coincided with the second peak of sand flies in september (sofizadeh 2009). due to direct relation between the number of rodent’s burrows and the incidence of disease, we propose to use different methods of reodent control as complementary measure for the control of disease. conclusion results of current study indicated the direct relationship between the burrows of rodents and zcl prevalence. therefore the presences of rodents as the reservoir host of disease play an important role on prevalence as well as incidence rate of disease. considering this, control of rodents will have an important role in controlling the disease. the other point that we need to pay attention, do actions that take more than a kilometer distance between human and the rodent’s life places. acknowledgment this study is the results of ph.d thesis in the j arthropod-borne dis, december 2016, 10(4): 569–576 a sofizadeh et al.: spatial analyses of … 575 http://jad.tums.ac.ir published online: october 04, 2016 field of 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(persian). who (2012) a human rights-based approach to neglected tropical diseases. geneva: world health organization, 2008. available at: http://www.who.int/tdr/ publications/tdrresearch-publications/humanights/en/index.html [accessed on september 3, 2012]. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod-borne dis. 6: 1–17. yaghoobi-ershadi mr, hanafi-bojd aa, akhavan ar, zahrai-ramazani ar, mohebali m (2001) epidemiological study in a new focus of cutaneous leishmaniasis due to leishmania major in ardestan town, central iran. acta trop. 79: 115–121. microsoft word 4dr hanafi rtl.doc iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 31 original article malaria situation analysis and stratification in bandar abbas county, southern iran, 2004–2008 *aa hanafi-bojd1, h vatandoost1, e philip2, e stepanova3, ai abdi4, r safari5, gh mohseni5, mi bruhi6, a peter7, sh abdulrazag8, g mangal9 1department of medical entomology & vector control, school of public health, tehran university of medical sciences, iran 2torit state hospital, ministry of health, eastern equatoria state, government of south sudan 3martinoskvy institute, moscow, russia 4ministry of health, somalia 5province public health centre, hormozgan university of medical sciences, bandar abbas, iran 6senior evaluator of malaria control program, sindh province, pakistan 7acting director general for malaria control program, upper nile statemalakal, state ministry of health,government of south sudan 8ministry of health, republic of sudan 9health center of khost province, afghanistan (received 5 apr 2010; accepted 19 may 2010) abstract background: the aim of this study was that the past five years data were collected to analyze the situation of malaria and health facilities in this area for better understanding malaria problem and to find solutions. methods: in this retrospective study data of the last 5 years were obtained from health center of bandar abbas, published papers and reports, weather forecasting organization of the city and annual reports of hormozgan official authorities. an excel databank was created and analysis was conducted using this software. results: according to the national health system, bandar abbas also has referral net work system from periphery to the district health center. the maximum and minimum annual parasitic index (api) were observed in 2005 (1.31) and 2008 (0.17), respectively. the prevalence of cases in villages was more than city, except for 2008. more than 97.6% of indigenous malaria cases were caused by plasmodium vivax, although p. falciparum, p. ovale and mix infection were also reported. anopheles stephensi, an. dthali and an. fluviatilis are the main malaria vectors in rural area, while only the first species is distributed in the urban area. conclusion: according to results and many variables including api, bandar abbas is divided in two strata. from the situation analysis of bandar abbas it is postulated that the main activities of this district could be accuracy of data, and malaria vector control. keywords: malaria stratification, iran introduction malaria disease is a worldwide problem that can be found in vast area of the world, where great portions of population are at risk. unfortunately number of people lost their live because of this disease, and still it is one of the main problems in global health. the disease is transmitted by a variety of female anopheline mosquitoes (who 2009). iran is one of the countries that face this problem. before starting any malaria control program in iran about 60% of population of the country was living in endemic areas *corresponding author: mr ahmad ali hanafi-bojd, e-mail: aahanafibojd@tums.ac.ir iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 32 with 30 to 40% malaria morbidity (edrissian 2006). iran has been classified into four different strata according to the epidemiology of the disease (raeisi et al. 2004). at present, malaria problem in iran is mostly concentrated in southern part. more than 90% of cases are reported from sistan and baluchistan, kerman and hormozgan provinces in south-east of the country (moosa-kazemi et al. 2007). it is unstable with two seasonal peaks mainly in spring and autumn. outbreaks due to p. vivax usually occur after rainy season (manouchehri et al. 1992). in this part of the country six anopheline mosquitoes including anopheles culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus are known proven vectors. anopheles pulcherrimus was mentioned as suspected malaria vector (jalali-moslem 1956, manouchehri et al. 1972, eshghi et al. 1976, manouchehri et al. 1992, zahirnia et al. 2001, edrissian 2006). the national strategy on malaria was revised in 2006, with the goal of eliminating of plasmodium falciparum malaria in 3–4 yr and only introduced cases of p. falciparum might occur, further reducing the number of autochthonous p. vivax malaria in a period of 7 yr. in the third stage of the new strategy, the objective will be a drastic reduction of local transmission of p. vivax in the residual and active malaria foci. at the end of the third stage only 500–700 autochthonous cases could be reported in the country per year. reported studies in 2008 show that out of 11460 malaria cases of iran, 8% was due to p. falciparum, while 90% were infected by p. vivax (minsitry of health 2008). in who malaria report 2009, iran showed evidence of a sustained decrease in the number of cases associated with wide scale implementation of malaria control activities. this country is classified as in the pre-elimination stage (who 2009). the aim of conducting a situational analysis is to systematically understand the malaria epidemiology of an area in a very short space of time and the health status, system and resource available for controlling the disease. in succession to planning to go to the district, there is need to use of the information that is already available and analyzing in such a way to understand the problems. the main objective of the situational analysis is to collect the data from respective references such as: health network office, hospital, health centers, communities, metrology department and others for conducting anti-malarial measures. bandar abbas is the most important seaport of iran in northern part of the persian gulf. this city has thousands of passengers from different parts of iran, as well as shipmen and immigrants from malarious areas of different countries. regarding to favorable conditions for malaria transmission, the city can be an important focus for the disease in southern iran. the aim of this study was that the past five years data were collected to analyze the situation of malaria and health facilities in this area for better understanding malaria problem and to find solutions. materials and methods study area hormozgan province covers an area of 71139.62 km2. it is located in south of iran and north of the persian gulf. bandar abbas is the capital city of the province. it is located in southern part of this province between 54o 53’–56 o 03’ e and 26 o 53’–27 o31’ n on flat ground with an average altitude of 9 m above sea level. the nearest elevated area is geno mountain, 17 km north of bandar abbas. the city has a hot and humid climate. maximum temperature in summers can reach up to 49° c while in winters the minimum temperature drops to about 5° c. the average of total annual rainfall was 118.44 mm during 2004–2008 and the mean annual relative humidity was 63.4% (www.weather.ir). in 2008 total population was 521657, from which 47% female and 53% male. about 77% iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 33 of population of this district is living in urban area and 23% in rural area. foreign population in this year was 15087 (management and programming organization 2008). data collection this study was a retrospective survey conducted using data obtained from health network of bandar abbas, as well as published documents on weather, population, history of malaria and previous studies on malaria in this area. the data bank was created in excel software. situation analysis and stratification was conducted based on the obtained data. results health system and general health profile health care and public health services in iran are provided through a national wide net. this network consists of a referral system starting from primary care center in the periphery going through secondary hospital in the capital and tertiary hospital in major cities. the public sector provide primary, secondary and tertiary health services and some of these services such as prenatal care and vaccination are free of charge (table 1). the private sector plays a significant role in the health care focused on secondary and tertiary health care in urban areas. there are also many non-governmental organizations acting in health issue in iran. they provide activities in special field like children with cancer, diabetes and thalassemia. there is one university for medical sciences education in bandar abbas and health network is working under vice chancellor for health. bandar abbas health facilities during our study period are showed in table 1 (management and programming organization 2004-2008). malaria situation cases: based on our findings the malaria cases in bandar abbas city are mainly coming from rural areas and from other countries such as afghanistan and pakistan, but in rural area most of cases are due to local transmission. all these cases are diagnosed by taking blood and then examined microscopically, and then reported and treated in the public centers. all these activities are free of charge. usually the cases from the health house reach the health centers within same day and about 80% of slide positive cases (slide examination take less 30 min) get treatment within 24 h according to national guideline. in those areas which are not covered by primary health care system, the anti-malaria activities carried by mobile team. the private sectors can investigate the cases but should refer them to the public sectors for treatment. bandar abbas is divided into 19 areas for malaria detection and treatment (fig. 1). during 2004–2008 a total of 1519 cases were reported to be positive out of 140620 taken slides (table 2). most of cases were iranian (79.3%). the disease morbidity increased up to 2005 and after that had a decreasing trend to 2008. plasmodium vivax was the main causative agent of malaria in bandar abbas (97.69%), followed by p. falciparum (2.17%). there was also one mixed infection case and a report of malaria due to p. ovale detected in an african football player came to bandar abbas in 2008. cases reported in rural area of bandar abbas (64.3%) were more than urban area (35.7%). most of cases (70.7%) had over 14 yr old. males (64.5%) were infected more than females (35.5%). the transmission season was mainly between april and november (fig. 2). vectors previous studies in the area show that hormozgan province has five anopheles vector species, the most important of them are anopheles stephensi, an. fluviatilis and an. dthali. anopheles stephensi is the main malaria vector in bandar abbas region, mainly can be iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 34 found in south urban part, flat and the coastal areas. it considers being endophilic, but biting outdoor during summer months. its seasonal activity started at beginning of may reach the peak in august and gradually decrease up to december in the mountainous area, but in the coastal areas it is active all the year with two peaks one in the april–may and second in the august up to september (manouchehri et al. 1976a). another study reported the peak of activity in september in mountainous and october in coastal regions. this species is absent during the cold winter months, but has high density during spring and autumn in mountainous and coastal region, respectively (vatandoost et al. 2006). the larva can be found in different bodies of water. in rural areas, it is found in pool streambeds at the margin of the streams, in the seepage and marshy area with gentle flow water, animal hoof print, and around the seepage marsh area (vatandoost et al. 2004, vatandoost et al. 2006). sporozoite rates for an. stephensi were reported 0.2–1.8% from southern part of iran (jalali-moslem 1956). anopheles dthali plays a secondary role as malaria vector in bandar abbas after an. stephensi. it is found mainly in siahoo mountainous area, north of bandar abbas city. this anopheles has two peaks of activity during autumn (december-february) and absent during cold winter and hot weather (june-august). the mosquito is resting outdoor in animal house as well as human dwelling (vatandoost et al. 2007). in rural area of bandar abbas, the anthropophilic index and sporozoite rate in salivary glands for this species is reported 25% and 1.4%, respectively (manouchehri et al. 1972). the larvae of an. dthali are found in mineral water in high salinity with temperature of 13–28o c and ph of 6.9–8. the larva is most abundant in september and october which is the end of hot season in bandar abbas (vatandoost et al. 2007). anopheles fluviatilis is also secondary vector with an. dthali, distributed in mountainous area from the east to west of hormozgan province. this species is exophilic, exophagic and zoophilic (naddaf et al. 2003) with larval habitat in slow moving water on margin of river, stream with or without vegetation with high dissolved oxygen and pits around springs. the biting of this species on human bait started from 18.00 h and continued until 04.00 h, but most of bites on human and cow bait took place in the first half of the night, and there was no bite after 04.00 h (manouchehri et al. 1975a). seasonal activity of an. fluviatilis showed two peaks in november and may. this species was active throughout the night with one peak of blood meal, 22:00–23:00 h on animal and 24:00– 01:00 h on human (edalat and moosa-kazemi 2005). sporozoite rate of this species is reported between 1.7–11% in south of iran (eshghi et al. 1976). vector control the past program for malaria eradication in iran was based on vector control using very large quantities of ddt started in 1949 which resulted in dramatic reduction in malaria incidence. vector population recovery happens mainly due to resistant to ddt in anopheles stephensi as well as ddt lack of selectivity affecting target population of mosquito (edrissian 2006). therefore, other insecticide belongs to organophosphate, organochlorine, carbamate and pyrethroid were used in following years. however, extensive use of chemical insecticides against mosquito vectors for about four decades has resulted in resistance to ddt, dieldrin and malathion (maouchehri et al. 1992, edrissian 2006). susceptibility tests of insecticides against adult mosquitoes using standard impregnated papers provided by who showed an. stephensi was resistance to ddt, dieldrin and malathion, while an. dthali and an. fluviatilis iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 35 are susceptible (mofidi et al. 1958, mofidi et al. 1960; manouchehri et al. 1975b, manouchehri et al. 1976b, vatandoost 2004; vatandoost and vaziri 2004; vatandoost and borhani 2004; vatandoost et al. 2005). in recent studies, it was found that the field samples of an. stephensi from bandar abbas were resistant to ddt, dieldrin and fipronil, and susceptible to other insecticides including malathion in bandar abbas (davari et al. 2006, vatandoost et al. 2006,). there is also a newly report of tolerance to ddt and dieldrin from kerman province (abai et al. 2008). study on susceptibility status on an. fluviatilis to diagnostic dose of 10 insecticides including ddt 4%, dieldrin 4%, malathion 5%, fenitrothion 1%, propoxour 0.1%, bendiocarb 0.1%, permethrin 0.75%, deltamethrin 0.05%, lambda-cyhalothrin 0.05% and cyfluthrin 0.15% showed 100% mortality in all tests (shahi et al. 2006). evaluation of standard solutions of 5 larvicides against these 3 species from bandar abbas showed they were susceptible (vatandoost et al. 2004, vatandoost and hanafibojd 2005, hanafi-bojd et al. 2006). now the main measures in iran for malaria vector control are: indoor residual spraying using lambda-cyhalothrin wp 10% and deltamethrin wp 10% in malarious areas, larviciding by chlorpyrifos-methyl, bacillus thuringiensis and some larvivorous fish like aphanius dispar and gambusia affinis and distributing llints. at present, b. thuringiensis and larvivorous fish are using for larviciding in the area as well as impregnated mosquito nets. this measure have been welcomed by people at various level, it was been introduced in 2003 as itns and nowadays llitns are used in malarious areas of iran (moosa-kazemi et al. 2007). in bandar abbas district, the main measures for vector control are indoor residual spraying (irs), larviciding and llitns. during the period of our study, the vector control activity was restricted to some villages of mountainous area. treated mosquito nets were used in may 2005 as itns when epidemic started in the area, but now llitns are using (personal communications). they are distributed according to the following priorities: villages without electricity, active foci and high population movement; villages with electricity, active foci and high population movement; villages with only one of the above mentioned factors; villages without electricity. larviciding using b. thuringiensis is another vector control measure in the area. it is also limited to the infected villages of mountainous area, north of bandar abbas. in addition some other measures are conducted for larval control. treatment and drug resistance in iran there is no recommended policy for chemoprophylaxis to the pregnant women and travelers. quinine, chloroquine, fansidar, primaquine, artesunate, clindamycin, doxycychline and coartem are recommended for malaria treatment in iran. they are used in different combinations based on severity of the disease, age of patient and plasmodium type. there is also special recommendation for pregnant women (saebi et al. 2006). several studies were carried out on drug resistant in the southern part of iran. the results of studies during 1968–1976 (manouchehri et al. 1973, sutoso et al. 1978) by in vivo test showed that p. falciparum was sensitive to chloroquine, but in 1983 (edrissian et al. 1985) in vivo and in vitro tests show chloroquine resistant with rate of 5.1% which increased to 51.1% by 1996 (edrissian et al. 1999). this rate in bandar abbas was 32.5% in 1986, 64.8% in 1994–1996 and 68% to 84% by 1997 (edrissian 2006). iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 36 table 1. health facilities in bandar abbas, 2004–2008, bandar abbas county health facilities 2004 2005 2006 2007 2008 teaching hospitals 2 4 4 4 4 total district hospitals 11 10 10 10 11 private hospitals 7 6 6 6 7 governmental hospitals 4 4 4 4 4 urban health centers, polyclinics 54 58 66 73 66 rural health centers 21 21 17 23 19 health houses 128 131 106 106 107 laboratories 33 33 36 37 39 pharmacies 36 32 42 45 46 table 2. malaria information in bandar abbas county, hormozgan province, 2004–2008, bandar abbas county nationality sex age groups species residence place y ears a t risk population iranian n on-iranian m ale f em ale <5 5-14 14< p. vivax p . falciparum m ix p. ovale t otal slides t otal cases a p i sp r a b e r r e-checked slides p ositive from re-checked city village 2004 453658 160 147 243 64 7 60 240 309 1 0 0 41174 307 0.68 0.75 9.07 21414 28 147 160 2005 471816 556 64 362 258 28 166 426 609 11 0 0 34462 620 1.31 1.79 7.30 15465 26 164 456 2006 479040 355 30 213 172 35 118 232 378 7 0 0 27819 385 0.8 1.38 5.80 9203 17 110 275 2007 514450 95 24 81 38 4 20 95 116 3 0 0 17247 119 0.23 0.68 3.35 15078 32 53 66 2008 521657 38 50 81 7 3 4 81 75 11 1 1 19918 88 0.17 0.44 3.81 13260 11 68 20 total -1204 315 980 539 77 368 1074 1484 33 1 1 140620 1519 -1.08 -74420 114 542 977 api = annual parasite incidence, spr = slide positive rate, aber = annual blood examination rate iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 37 table 3. stratification of malaria in bandar abbas, hormozgan province, iran 2004–2008, bandar abbas county variables stratum i stratum ii geography mountainous coastal vector an. stephensi an. dthali, an. fluviatilis mostly an. stephensi water source pools, rivers, and containers water pipe, rivers, swage population movement no present people behavior during transmission season sleeping out side sleeping in side api 1%< <0.5% fig. 1. malaria registration centers of bandar abbas, hormozgan province, iran, bandar abbas county fig. 2. malaria cases in different months of year during 2004–2008, bandar abbas, hormozgan province, iran iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 38 fig. 3. malaria stratification in bandar abbas, hormozgan province , iran 2004–2008, bandar abbas county discussion there was no considerable change in the number of health facilities during the study period (table 1). the reduction of health houses that are the first step of the referral system is due to newly geographical changes that pass them to neighbor cities. there was a dramatic increase in malaria cases of 2005 compared to 2004. as showed in table 2, during 2005–2006 the cases in rural area were more than two times higher than urban areas. there is a limited population movement in the rural area, so this epidemic occurred due to local transmission and this is attributed to the weakness of surveillance system in that years. as it can be seen in table 2, the total slides taken decrease year by year. also the amount of insecticides used during epidemic was very limited, e.g. only 400 kg of pyrethroid insecticides and 510 kg bacillus thuringiensis as larvicide were applied during the epidemic of 2005 (management and programming organization 2005), and the control program started after the first peak of the disease. we found that the entomological data in the area are poor and not enough and reliable for designing a control program. this careless may be due to case reduction in previous years as well as poor entomological data. after this epidemic, different control activities such as llints, irs and larviciding were strengthened parallel to case detection and treatment resulted to drop positive cases in the area, so that in 2008 the local transmission that usually occurs in rural area was about one third of reported cases in urban area. reports of health services showed most of cases in bandar abbas city in this year were imported. major determinates of malaria stratification in bandar abbas were: geography, vector, water sources, population movement, people behavior and api. stratification is done based on the above determinates which are illustrated in table 3 and fig 3. so bandar abbas were divided into two strata. from the situation analysis of bandar abbas we found the main problems of this township are: inaccuracy of data and malaria vector control activities. for elimination of malaria in bandar abbas it is suggested to do the following activities: proper case management by early detection and prompt treatment, routine evaluation of drug resistance, biweekly iranian j arthropod-borne dis, 2010, 4(1): 31–41 aa hanafi-bojd et al.: malaria situation analysis… 39 entomological survey during the transmission season in the high risk area, annual survey on susceptibility status of vectors to insecticides/larvicides, free distribution of the llitns to at risk groups including travelers and refugees, improving of housing condition at the rural areas, conducting training for entomological survey as larvicidal personnel, spraying workers and response team for epidemics, treatment of breeding site by effective larvicides and biological measures, participation of school children and community in environmental management by drainage and filling the major mosquito sources and small water resources, site selection (establishment of population away from reach of mosquitoes) and introduce of animal shelter between breeding site and human houses for exophilic mosquitoes, personal protection using insect repellents, early detection of epidemics and improvement of data collection, training of staff on computerization, data collection and excel management for updating malaria database, study the health aspect of any project by health authorities and anti malaria services and seek assistant to investigate advantages and benefits, evaluation of knowledge, attitude and practice of people in high risk area by kap study, and training though mass media (tv, radio) and leadership (community leaders, schools, religious leaders). acknowledgements this study is result of teamwork and field exercise study on malaria situation analysis and stratification, in the 12th international diploma course on malaria planning and management, conducted during november 2009january 2010 in who regional malaria training centre in bandar abbas, southern iran. we are thankful from the staff of this centre. we would like to thank dr masoodi, the director and ms m mehranzadeh the focal malaria person in bandar abbas health center for their kind collaboration and data providing. the authors declare that they have no conflicts of interest. references abai mr, mehravaran a, vatandoost h, oshaghi ma, javadian e, mashayekhi m, mosleminia a, 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iran. eastern med health j. 10: 573–578. vatandoost h, borhani n (2004) susceptibility level and irritability of synthetic pyrethroids against main malaria vectors in the endemic areas of iran. acta med iran. 42 : 247–255. who (2009) world malaria report 2009. who press, world health organization, geneva. 66 pages. zahirnia ah, taherkhani h, vatandoost h (2001) observation of malaria sporozoite in anopheles culicifacies (diptera: culicidae) in ghasreghand district, sistan and baluchistan province. hakim. 4: 149–153. j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 105 http://jad.tums.ac.ir published online: march 14, 2017 original article repellency effect of essential oils of some native plants and synthetic repellents against human flea, pulex irritans (siphonaptera: pulicidae) *mohammad bagher ghavami 1, fahimeh poorrastgoo 1, behrooz taghiloo 2, jamshid mohammadi 1 1department of medical entomology and vector control, faculty of medicine, zanjan university of medical sciences, zanjan, iran 2zanjan health center, zanjan university of medical sciences, zanjan, iran (received 5 jan 2016; accepted 7 feb 2016) abstract background: fleas are important vectors of human and animal disease, and control measures for protection against their bites and flea-borne diseases are necessary. methods: the essential oils (eos) of four native medicinal plants, ziziphora tenuiore, myrtus communis, achillea wilhelmsii and mentha piperita were isolated by hydrodistillation technique and analyzed by gc-mc. the repellent activity of eos and synthetic compounds, deet and permethrin, were assayed on human subjects against field collected fleas. the effective doses of 50% and 90% of eos and synthetic compounds were estimated by probit analysis of dose and response regression line. results: analysis of eos revealed about 19 major components. all oils were found to be more repellent (ed50 range= 208–955µ g cm-2) than deet and permethrin (ed50 range= 27–182x 103µ g cm-2). thyme and myrtle oils showed high repellent activities and among the total detected terpenes, thymol (36.26%) and αpinene (32.5%) were the major components of those oils respectively. conclusion: low repellent potency of deet and permethrin against fleas might be related to flea olfactory system and further molecular and electrophysiological studies are required to conceive new ideas for the discovery and development of the next generation of repellents. based on high repellent activity of thyme and myrtle essential oils against pulex irritans further studies should be staged to develop their appropriate effective formulations. likewise, field trials should be carried out to evaluate the operational feasibility and dermal toxicity over a long period. keywords: pulex irritans, ziziphora tenuiore, myrtus communis, deet, repellents introduction the human flea, pulex irritans linnaeus, is the most invasive species worldwide with great medical importance and intolerable nuisance. the control of this vector has received considerable attention since ancient times. the most effective measure to control human flea lies in adult stage using adulticides (service 2012). although application of synthetic insecticides can be effective, their intensive use has caused concerns regarding their impact on human and environmental health leading to development of resistance in the flea populations like other vectors (hemingway and ranson 2000). in addition to adulticides, personal protection including the use of repellents can decrease contact between human and fleas. n, n-diethyl-3-methylbenzamide (deet) has been considered as one of the most effective synthetic repellents against various groups of vectors and still remains as “the gold standard” among currently available insect repellents (swal et al. 2014, debboun et al. 2014 ). however, there has been no report concerning the repellency of deet on human flea. in addition, there have been case reports of deet toxicity especial*corresponding author: dr mohammad bagher ghavami, email: ghavami@zums.ac.ir j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 106 http://jad.tums.ac.ir published online: march 14, 2017 ly among children and the elderly (qui 1998, sudakin 2003). moreover, genotoxic effects of this compound have been found previously (manikhan et al. 2012). in addition to deet, permethrin, a synthetic pyrethroid, is characterized by a high level of potency against a wide range of vectors, rapid reactivity, excellent photostability, resistance to weathering, low mammalian toxicity (yu 2014) and high potency in tick and insect bite prevention (faulde et al. 2003, 2006). however, skin irritation, including itching, swelling, redness, mild burning and stinging may occur after treatment with permethrin. moreover, arthropods show differential responses to these products, indicating the possibility of adaptation and emerging resistance or insensitivity (klun et al. 2004, stanczyk et al. 2010, pellegrino et al. 2011, sfara et al. 2011, del fabbro and nazzi 2013). thus, there is a growing demand to evaluate natural repellents to develop suitable agents for prophylactic treatment in integrated pest management. plant essential oils (eos) specially their monoterpen components; exhibit a variety of biological activities against a wide spectrum of insect pests. they can adversely affect the growth and reproduction rate, behavior trait of insect pests and act as contact insecticide, fumigant, repellent and antifeedent agent. these compounds have a low risk profile on the environment, mammals, and humans and might comprise natural alternatives for conventional insecticides (bakkali et al. 2008, koul et al. 2008). fresh and dried shoots of myrtle, mint, thyme, yarrow and their eos and extracts are widely used in cosmetic, food, gum, toothpaste and pharmaceutical industries. these plants have also been commonly used in folk medicine for treatment of colds, pains and as spices and herbal tea as well (naghibi et al. 2005, bakkali et al. 2008, saeidnia et al. 2011). moreover, these plants proved to be toxic to different species of insects and ticks (maia and moore 2010, george et al. 2008, 2009, 2014, pitarokili et al. 2011, yahoobiershadi et al. 2011, tavassoli et al. 2011, koc et al. 2012, lupi et al. 2013). for human flea, however, little work has been done on eos repellent activity. moreover, despite the great need in human health area for fleas prophylaxes by natural products, the current knowledge concerning repellent action of eos on human flea is very limited. therefore, in the present study the chemical properties and components of some eos were evaluated. additionally a comparative study was conducted for detection of flea repellent activity of these eos. materials and methods plant materials the shoots of native medicinal plants, thyme (ziziphora tenuiore), mint (mentha piperita), yarrow (achillea wilhelmsii) and myrtle (myrtus communis) were collected from different areas of zanjan and fars provinces during may–jun 2013 and 2014. collected herbs were dried in shade for 8 days at room temperature until they became brittle. air-dried samples were powdered in electric vegetable chopper and subjected to hydro distillation in a clevenger type apparatus for about 3h. the extracted oils were dried over anhydrous sodium sulfate and refrigerated at 4 ˚c in dark glass bottles until the experiment. gas chromatography and gc mass analysis the gas chromatography (gc) analysis was carried out with an hp series ii 7890n instrument equipped with flame ionization detector and hp-5-ms capillary column (30m× 0.25mm, 0.25µm film thickness). injector and detector temperatures were set at 260 ˚c and 270 ˚c respectively. thermal program of gc oven started with 60 ˚c for 4min, then rose to 60–225 ˚c at a rate of 3oc/min. helium was the carrier gas at a flow rate of 1ml/min. diluted samples (1:50 in diethyl ether) of 1µ l were injected manually in the split less mode. j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 107 http://jad.tums.ac.ir published online: march 14, 2017 quantity analysis of eos components was performed using the same condition with gc hp series ii equipped with hp gc 5975c mass selective detector in the electron impact mode 70ev. the oil components were identified by comparison of the retention time with those of authentic samples in mass spectra obtained from literature and the library of the gc-ms system. human subjects the study was conducted on 6 volunteers (four males and two females) who were aged between 28 to 50 years old. they were nonsmokers, non alcoholics and non-allergic to insect bites or herbal oils. they also had no contact with lotions, perfumes, coils or perfumed soaps on the days of assay. all volunteers signed an formal consent form after having received a full explanation of the test objectives, procedures and foreseeable risk to subject. the research protocol was approved by the ethics committee of zanjan university of medical sciences. flea collection alive samples of fleas were collected by human baits from animal shelters of khodabandeh and mahneshan districts of zanjan province from september 2013 to october 2014. volunteers in white overalls and white ladies stockings, walked around the houses for 3–5 minutes and attracted fleas were suctioned by electrical aspirator and transferred to laboratory in 2 liters erlenmeyer flasks. preparation of solutions dilutions of each essential oil and deet (aldrich usa, d100951-500g, n, n-diethyl3-methylbenzamide, lot# mkbh0428v, p code: 1001075635, density: o.97g/cm3) with grade reagent ethanol (et) were made to stock in v/v at 85% concentration. from which five serial dilutions (42.75%, 21.37%, 10.68% and 5.34% concentrations) were prepared. also a 20% permethrin (united phosphorus ltd, delhi india, with 96% active ingredient, 42:58 cis: trans ratio) solution was prepared by mixing 2.1g of permethrin with 10ml of grade reagent acetone and 5 serial dilutions (in acetone) were created. repellency tests the bioassay was performed by the standard protocols for testing mosquito and tick repellents (dautel 2004, bissinger 2010, maia and moore 2011, krober et al. 2013, who 2013, debboun 2014). about 60 unfed fleas were kept in laboratory condition (25±2 ˚c and 60% rh) and transferred to 20″×6″ clear cylinder glass vase. for testing, volunteers used the left arm for treatment and the right for control. five hundred µ l of test materials were applied to the treated area of left hand (from elbow to wrist, area approximately 500cm2) and allowed to dry for 5 minutes. the same area of right arm was applied with five hundred µ l of et. after drying of treated area, both arms were covered with white ladies stockings and volunteers kept their hands (left as treatment and right as control) in glass jars about 3 minutes. the control hand was exposed before the treatment. samples of fleas from different parts of arms (hand and wrist to elbow) were collected and recorded separately. these samples were preserved in et and observed under stereomicroscopy at 30×. samples of pulex irritans were recorded and other species of fleas were excluded from study. the percentage of repellency in each hand was calculated by the following formula: (tc-tb)/ (tc+tb)× 100, where tc= the number of fleas in hand of arm and tb is the numbers in the area between elbow to wrist. the percentage of repellency in treatment groups was corrected with control group by abbot’s formula. the data were subjected to probit analysis and the median effective dose (ed50) and 95 % effective dose (ed90) with 95 confidence j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 108 http://jad.tums.ac.ir published online: march 14, 2017 interval exposed in microgram of repellent per square of centimeter of skin area were estimated. significant differences between effective dose values were determined by comparing the confidence intervals of the values. results water distillation of the dried shoots of the examined plants furnished 0.8–1.2% (w/ w) of white to yellowish essential oils with strong pleasant odors were identified. forty seven constituents were determined in the thyme essential oil representing 97.98% of total content. the main constituents were found to be thymol (36.26%), geraniol (11.61%), carvacrol (4.96%) and 1,8 cineol (3.26%) (table 1). a total of twenty compounds were identified in the oil of myrtle, representing around 99.15% of the oil totally. alpha pinene (32.5%), 1,8 cineol (24.15), limonene (15.5%) linalool (10.6%) and linalyl acetate (5.5%) were the major constituents in the essential oil of myrtus communis (table 1) according to total 87.43% of the achillea wilhelmsii oil, eighty constituents were identified in the essential oil of this plant. the main components were camphen (19%), dimethyl heptatriene (10.15%), borneol (7.3%), artemisia alcohol (5.3%), menthen (4.56%) and 1,8 cineol (3.86%). there were fifty eight compounds in the oil of mentha pipertta, comprising 98.26% of the total weight. menthen (26.66%), piperiton (19.18%), piperiton oxide (19.07%), 1,8 cineol (8.5%) and trans caryophylen (3.29%) were the major compounds of essential oil of this plant (table 1). during the study, 4529 samples of fleas were collected by human-bait catch. human flea was the commonest species and 94.3% of samples were identified as pulex irritans. the frequencies of other fleas were 3.8% and 1.9 % for ctenocephalidescanis and c. felis respectively. the bioassay tests were carried out with 4271 samples of alive human flea. all tested essential oils in five concentrations showed differences in repellency with chemical compounds. the thyme essential oil provided highest repellency followed by myrtle, yarrow and mint essential oils (table 2). the eds50 (with 95% confidence intervals) of thyme and myrtle essential oils were 229 (208–257) and 295 (269–338)μg/cm2 respectively. in these plants the ed90 (with 95% confidence intervals) of essential oils were 776 (660–933) and 1172 (988–1412) μg/cm2 respectively (table 3). yarrow essential oil showed moderate repellency against human flea. in this plant the ed50 and eds90 (with 95% confidence intervals) of essential oil were 457 (371–630) and 2217 (1399–4673)μg/cm2 respectively. mint essential oil provided slight repellency. the ed50 and ed90 (with 95% confidence intervals) of mint essential oil were 776 (660–855) and 5717 (3890–9120)µ g/cm2 respectively. permethrin and deet compounds showed low repellency in comparison with botanical essential oils. the ed50 of permethrin and deet were estimated 7760 and 51000µ g/ cm2 respectively (table 3). the related dose response lines and regression equations for essential oils and synthetic compounds are represented in table 3 and fig. 1. j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 109 http://jad.tums.ac.ir published online: march 14, 2017 table 1. relative percentage of major components of essential oils of each plant extract no compound name lri (min) myrtus communis achillea wilhelmsii mentha piperita ziziphora tenuiore 1 α pinene 6.37 32.5 2.59 0.73 0.71 2 limonene 10.24 15.5 0 0 0 3 1,8 cineol 10.36 24.15 3.86 8.6 3.16 4 γ tepinene 11.55 0 0.33 0.11 3.33 4 artemisia alcohol 12.86 0 5.39 0 0 5 linalooll 13.42 10.6 3.2 0.88 2.26 6 dimethyhepta 14.67 0 10.15 0 0 7 camphor 15.29 0 19 0.48 0.69 8 borneol 16.38 0 7.30 0.35 3.26 9 menthen 17.55 0 4.56 26.66 2.41 11 α -terpinol 17.7 0 1.17 0.44 2.36 12 linalyl acetate 21.14 5.5 0 0.25 0 13 geraniol 21.45 0 0 0 11.61 14 thymol 23.35 0 0.8 0.22 36.26 15 carvacrol 23.57 0 0.26 0 7.98 16 piperiton 24.99 0 0 19.18 0 17 piperiton oxide 26.11 0 0 19.07 0 18 geranyl acetate 26.68 0 0 0.40 4.72 19 trans caryophylen 27.83 0 0 3.29 0 total 88.25 57.76 76.8 78.75 total compound (no) 20 80 58 47 total percentage 99.15 87.43 98.26 97.98 lri*= linear regression indices on hp-5 ms column relative to c9–c23 n alkanes , identification based on comparison of mass spectra obtained from the library of the gc-ms system and from literature. table 2. repellent activity (in percentage) of some essential oils and synthetic compounds against pulex irritanson human volunteers dosage (µ g/cm2) study groups ziziphora tenuiore myrtus communis achillea wilhelmsii mentha piperita permethrin deet 50 3.2 3 3.1 3 2 2 100 17.7 17.5 12.5 15 7.2 5.2 200 48 36 22 20 8.2 6.2 400 74 63 46 32.5 9.3 7.3 800 87.5 80 72 48.4 12.4 10.4 1600 98 96 87 69 18.5 14.6 j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 110 http://jad.tums.ac.ir published online: march 14, 2017 fig. 1. dose response lines for botanical and chemical repellent activity against pulex irritanson human subjects. table 3. parameters of probit analysis on botanical and chemical compounds and their repellency against pulex irritanson human volunteers compound a b ed50 (95 % ce) (µ g/cm2) ed90 (95 % ce) (µ g/cm2) n x2 p ziziphora tenuiore -5.78 2.44 229 (208–257) 776 (660–933) 828 101.7 <0.01 myrtus communis -5.36 2.16 295 (269–338) 1172 (988– 1412) 895 99.6 <0.01 achillea wilhelmsii -5.0 1.88 457 (371–630) 2217 (1399–4673) 169 102.1 <0.01 mentha piperita -4.32 1.49 776 (660–955) 5717 (3890–9120) 822 68.7 <0.01 permethrin -2.3 0.51 26.9 x 103 7.76x 106 362 15.0 <0.01 deet -2.74 0.50 182 x 103 51 x 106 710 10.8 0.03 discussion evaluation of botanical compounds is essential to identify effective repellents for vector control programming and protection of populations against vector nuisance and vector borne diseases. the insecticidal and repellent activities of various herbal derivatives have been demonstrated in many studies (neiro et al. 2010, bisinger and roe 2010, lupi et al. 2013). however, only a few reports present the repellency of plant products against other groups of nuisance vectors especially fleas (george et al. 2009). in our investigation among examined plants, the essential oil of three plants, thyme, myrtle and yarrow, showed excellent to good repellency against all tested fleas. the oils of these plants were rich in volatile terpenes, thymol, α–pinene, 1, j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 111 http://jad.tums.ac.ir published online: march 14, 2017 8 cineol, camphor, limonene, linalool, and geraniol. in spite of effective repellency of these compounds, the major components of mentha piperita, menthen and piperiton, showed low efficacy against fleas. findings of previous studies have proved that, thymol and α–pinene are the most toxic compounds (trabouli et al. 2002) and have effective repellent activity against mosquitoes and ticks (pandy et al. 2009, jordan et al. 2012, meng et al. 2015). high repellency of thyme and myrtle oils in our study might be associated with these components. nevertheless, further laboratory and field studies are required to prove repellent efficacy of these compounds. although the amounts of limonene, geraniol, linalool and camphor in above-mentioned essential oils were high in our study, they showed low potency of repellent activity against fleas. furthermore, 1, 8 cineol which had low repellency against insects, showed different biological activities against mosquitoes and ticks in previous studies (elseedi et al. 2012). in contrast to recent studies synthetic compounds, deet and permethrin, which have shown effective repellency against mosquitoes, could not repel human flea. low efficacy of these compounds on pulex irritans compared to other vectors might be related to their difference in mode of action (dikens and bohbot 2013, daniel et al. 2014), molecular characteristics of odorant binding proteins (bohbot and dikens 2012) and odorant receptors of fleas (ditzen et al. 2008). this study encourages further electrophysiological and molecular studies on human flea to conceive new ideas for the discovery and development of the next generation of repellents. thyme and myrtle oils showed high repellent activity among the tested oils against human flea on human subjects. the repellent effect of ziziphora tenuiore essential oil showed the highest repellency (lowest ed50 value, 208µ g/cm2 and ed90 910µ g/cm 2). according to our literature review, there is no published paper available on the repellent activity of ziziphora tenuiore essential oil. majority of published documents were in favor of thyme essential oil repellency against mosquitoes and only a few of them were conducted against ticks (zhu et al. 2006, park et al. 2012). in previous studies the lowest value of ed50 seen in laboratory study of origanium majorans, a species of thyme group, against nymphs of ixodesricinus was 15µ g/cm2 (zhu et al. 2006). thyme oil also showed highest repellency against labbred aedes aegypti (ed50= 23µ g/cm 2) and culex pipiens (ed50= 468µ g/cm 2). our ed50 and ed90 estimated values for myrtle oil on human subject were 290 and 1172µg/cm2 respectively. in the laboratory study of tavassoli et al. (2011) the ed50 and ed90 values for myrtle essential oil against lab-bred anopheles stephensi were calculated as 110 and 540µ g/cm2 respectively. moreover, in a laboratory study on animal model, using k and d apparatus against lab reared phlebotomus papatasi, yaghoobi-ershadi et al. (2006) reported the ed50 and ed90 values of myrtle oil 114 and 671µ g/cm2 respectively. interestingly, in our study the ed50 and ed90 values of myrtle oil are very close to these studies. based on the repellent result against pulex irritans we recommend ziziphora tenuiore and myrtus communis essential oils for further studies to develop appropriate effective formulations. field trials should be carried out to evaluate the operational feasibility and dermal toxicity over a long period. it is important to determine whether widespread application of these repellents would produce an overall reduction of vector biting. conclusion based on our findings the two essential oils, thyme and myrtle, are preferable in terms of j arthropod-borne dis, march 2017, 11(1): 105–115 mb ghavami et al.: repellency effect of … 112 http://jad.tums.ac.ir published online: march 14, 2017 repellency effect against human flea. further electrophysiological and molecular studies are recommended to identify the mode of action of these oils and nature of flea olfactory system in order to develop new generation of repellents. measures to determine the appropriate formulation of recommended repellents and comprehensive field studies to evaluate their potency in bite reduction and possible adverse effects are necessary. acknowledgment this work was financially supported by zums. the authors would like to express gratitude to dr bighlari, chief of zums and dr faghihzadeh, deputy of research and information technology of zums for their valuable help. we would like to thank staff of zanjan, mahneshan, and khodabandeh, health centers for their kind collaborations. the authors also thank dr yazdineghad for confirmation of herbal identification, mr zargharan (behavar chemical co) for donating the technical grade permethrin, mr salimi and mr mehreghan for excellent assistance in laboratory studies and members of animal house for providing laboratory animals. we also express our gratitude to two 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who/cds/whopes/gcdpp/2000.5. yaghoobi-ershadi mr, akhavan aa, jahanifard e, vatandoost h, amin gh, moosavi l, zahraei-ramazani ar, abdoli h, arandian mh (2000) repellency effect of myrtle essential oil and deet against phlebotomus papatasi, under laboratory conditions. iranian j publ health. 35(3): 7–13. yu sj (2014) the toxicology and biochemistry of insecticides.second edition. crc press j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 78 http://jad.tums.ac.ir published online: march 14, 2017 original article a comparative study of nesting sites and burrowing habits of two iranian burrowing scorpions babak vazirianzadeh 1,2, *amir jalali 3, mostafa chrom 4, alireza mohammady 5, hassan vatandoost 6, forozan panahi 5 1social determinants of health research center, ahvaz jundishapur university of medical sciences, ahvaz, iran 2depatrment of medical entomology, ahvaz jundishapur university of medical sciences, ahvaz, iran 3depatrment of pharmacology and toxicology, school of pharmacy and toxicology research centre, ahvaz jundishapur university of medical sciences, ahvaz, iran 4depatrment of soil science, school of agriculture, ahvaz university of shahid chamran, ahvaz, iran 5depatrment of medical entomology, ahvaz jundishapur university of medical sciences, ahvaz, iran 6depatrment of medical entomology and vector control, school of public health and national institute of health research, tehran university of medical sciences, tehran, iran (received 2 jul 2013; accepted 27 dec 2016) abstract background: the different features of scorpions can be successfully described by their nesting and burrowing behaviors. there is little information about burrowing activity of iranian scorpions. methods: the current study was performed to compare the burrowing behavior between two burrowing iranian scorpions, scorpio maurus and odonthubutus bidentatus by describing 30 nests of each species regarding collecting the scorpions. results: scorpio maurus and o. bidentatus have a tendency to make nest with elliptical, round-like entrance and oval shape with arch at the top, respectively. there was not any significant difference between nest entrance properties of two scorpions. one-way anova test showed that the height and diameter of two species nests were not significantly different. a pearson correlation also showed a relative strong direct relationship between height and diameter of s. maurus nests than o. bidentatus. this correlation was not significant in the case of o. bidentatus. the results provided additional habitat information of scorpions. conclusion: the nests morphology characteristics of two iranian scorpions including shape, depth, length and diameter depend are different from each other based on the following factors: species, soil texture, soil moisture and region conditions. keywords: nesting sites, odonthubutus bidentatus, scorpio maurus, scorpions, habits introduction scorpions are terrestrial arthropods with generally non-social habits. the majority of scorpions are nocturnal, particularly species inhabiting arid desert regions. the various degrees of adaptation to different ecological conditions have been reported for scorpions (prendni and wheeler 2005, fet and soleglad 2005, cala-riquelme and colombo 2010). therefore, this group of animals is subject to different investigations concerning their ecological, physiological and biochemical adaptations in adjusting to harsh conditions found in their habitats. in spite of many different ecological and biological aspects regarding to scorpions, there are a little information in this regard. one of these ecological aspects, not known properly, is burrowing habit. burrowing habit pro*corresponding authors: dr amir jalali, e-mail: amjalali@hotmail.com j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 79 http://jad.tums.ac.ir published online: march 14, 2017 vide important facilities for scorpions such as protection against predation, increased availability of food and ideal microclimate for their lives (shehab 2011, hembree et al. 2012). scorpions live under rocks, logs and plants during the day or in cracks and burrow their shelters and nests (cala-riquelme and colombo 2010). “normally, a majority of scorpion species are able to make some changes in their places and less able to make their nests through burrowing” (cala-riquelme and colombo 2010, 2000, shehab 2011). there are six burrowing scorpion species in iran including s. maurus (scorpionida: scopionidae), o. bidentatus, o. doriae, o. odonthodorous, apisthobothus petrigosercus and a. susanei (scorpionida: buthidae) (farzanpey 1987, navidpour 2008). scorpio maurus is seen generally in plains close to wheat fields or in deep holes of deserts. this specie has been recorded in different iranian provinces such as khuzestan, kurdistan, fars, azarbaijan, qazvin, isfahan and bushehr (farzanpey 1988, dehghani 1991). odonthubutus bidentatus, is another digger scorpion found mostly in the desert and low altitude regions. this species was formerly called o. odonturus (farzanpey 1987). the geographical distribution showed that o. bidentatus is restricted to the southwest regions of iran, mainly khuzestan. this species is not well investigated but in recent years, more investigations were focused on the other members of the genus, o. doriae (dehghani et al. 1991, jalali et al. 2007, navidpour 2008, masihipour et al. 2009, jalali et al. 2010). this scorpion, distributed in khuzestan, has potentially been considered as one of the medically important scorpion in the region. however, its sting potency is roughly equivalent to that of o. doriae, with a decreased risk for significant neuromuscular compromise. the haftgel area is recognized in terms of having high frequency of s. maurus and o. bidentatus in south west of iran, khuzestan (farzanpey 1987, vazirianzadeh and samie 2006). therefore, this area was selected as an appropriate area for this study. the purpose of this study was to investigate and provide more information regarding soil texture and soil relative humidity (sutured percentage), identification and drawing the patterns of the nest making habits and nesting activities of two iranian burrowing scorpions. this issue is an important help to provide the suitable life conditions of both species in laboratory and scorpion breeding studies in future researches. materials and methods sampling and trapping the scorpion samples were collected initially from haftgel (31° 26′ 35.85″ n, 49° 31′ 38.93″ e). the scorpions were selected through a particular nest sampling procedure. firstly, the elliptical or round holes were recognized in none resident fields of haftgel as nests of scorpions. then the nests were filled with a calcium carbonate suspension in order to move scorpion into the opening and solid formation the way of nest. next to scorpion observation, the back entrance of the nest was blocked and scorpion trapped in particular glasses (open door and porous). the scorpion samples were transferred to medical entomology laboratory, department of medical entomology, school of public health, ahvaz jundishapur university of medical sciences (ajums), ahvaz, iran and identified according to common iranian scorpion keys (farzanpey 1991, navidpour et al. 2008). thirty nests of each species were observed during this study, which dimensions of them were measured. determination of physical structure and texture of soil in order to determine the physical structure and type of soil texture, the sampling was carried out randomly in the ranges of j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 80 http://jad.tums.ac.ir published online: march 14, 2017 10–20, 20–40 and 40–60cm deep of the nests from two places. this sampling was performed using excavator cylinders (oger®). the properties of soil samples including type of soil, percentage of ingredients and moisture content of different depths were identified using hyderometery mechanical analysis procedure (day 1982). this identification was performed in the soil science department, school of agriculture, shahid chamran university, ahvaz, iran. nests pattern identification in order to measure and draw the picture of the nest tunnels and determination and preventing of soil texture disassembling throughout the tunnel route, a molage method with masonry plaster modeling was used. after initial recognition, half to one-liter slurry solution with usual viscosity was used for each nest. subsequently, the entrance of the nest at ground was turned into cone-shape with around soil. the gypsum solution was poured slowly in a stage into the entrance of nest. the aforesaid formats were firmed and molage was made after 30 to 60min. molage dimensions were measured and obtained patterns were drawn. furthermore, the diameter and heights of nests were measured by a caliber. the density of distribution nests was determined using two ways: 1) using the number of nests in a 1m2 wooden quadrant for each species ten quadrants were applied in the each habitats belonged to each species in two different sites where the definite scorpions were collected. 2) numbers of counted nests in five min (same as above aforementioned procedure). statistical analysis statistical analysis between nests was carried out by using one way anova and pearson correlation. minitab13 statistical software was used to determine the nests characteristics and relations between two species. results both species were collected from same parts of the studied field. mechanical analysis (hydrometery tests) of examined soils indicated that the type of textures in the 10–20, 20–40cm and 40–60cm depths of scorpion nests were loam, silt-loam and loam, respectively. hydrometery tests evaluated the contents of humidity of soil (sutured percentage) as 32.11%, 37.93% and 36.34% in the 10–20, 20–40 and 40-60cm depths of the nests, respectively. the structure of the studied area in the 60 cm depth was calcareous and rocky sections. the results are performed in the table 1. diameter and height variables of s. maurus and o. bidentatus nests openings are presented in table 2 and 3, respectively. the means of s. maurus and o. bidentatus height and diameter nests were determined as 1.08± 0.31 and 1.02±0.25cm and 1.78±0.42 and 1.8±0.4cm, respectively. in addition, oneway anova test showed that the height and diameter of two species nests were not significantly different (p> 0.05). pearson correlation also showed a relative strong direct relationship between height and diameter of s. maurus nests (r= 0.67). this correlation was not a strong significant in the case of o. bidentatus (r= 0.43). scorpio maurus has a tendency to make nest with elliptical and round-like entrance. their nests are began with more or less a sharp slope and then screw in the opposite direction to end finally to a sloping horizontal. scorpio maurus lives at the end of the nest. the length of s. maurus nests were measured 21–34cm (three nests were dissected), approximately (fig. 1). odonthubuthus bidentatus nests had oval shape with an arch at the top. the nest was built first with a slow slope and then followed with deeper degree and finally completely became horizontal until to end. the j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 81 http://jad.tums.ac.ir published online: march 14, 2017 length of o. bidentatus nests was measured 35–55cm, approximately (fig. 2) (5 nests were dissected). the density of nests in the studied area was measured 0.4 nests of s. maurus and 0.6 nests of o. bidentatus in 1m2 and counted 19 nests in 5 min related to both species. the results also opined that the majority of nests are in a spiral manner special regarding s. maurus nests. the entrapping was done in the early morning when the scorpions more movement and activity to come back to their nests after night promenade. the majority of s. maurus scorpions were inside of their nests (75% of trapped scorpions, or 22 s. maurus were trapped). the observations showed that the 60% of o. bidentatus nests were empty at the entrapping times (12 o. bidentatus were trapped). totally 30 nests of each species were observed. the molage procedure was performed for 3 nests of s. maurus and 5 nests of o. bidentatus. pictures of all 3 s. maurus nests are performed as fig. 1 and only 1 picture of o. bidentatus is performed as fig. 2 because the 5 nests were in very similar shape and way. table 1. soil texture and physical properties of haftgel area, 2010 % of sutured humidity% of sand% of clay% of loamsoil classdepth of soil (cm) 32.11391348loam10-20 37.93272350silt.loam20-40 36.34351847loam40-60 table 2. inlet height and diameter of scorpio maurus nests height of nest diameter of nest height of nest diameter of nest height of nest diameter of nest 1.38 1.94 1.59 2.08 0.97 1.97 0.67 1.14 0.98 1.71 1.12 2.31 0.83 1.72 1.12 1.68 1.19 1.79 0.79 1.19 1.29 1.86 0.94 1.33 1.38 2.19 0.95 1.34 0.71 1.18 0.87 1.76 1.05 1.89 1.53 2.29 1.39 2.1 0.81 1.98 0.44 0.92 1.22 2.19 0.83 1.21 1.96 2.15 1.19 1.34 1.12 2.33 1.21 2.33 1.22 2.33 1.09 1.48 0.85 1.91 table 3. inlet height and diameter of odonthubutus bidentatus nests height of nestdiameter of nestheight of nestdiameter of nestheight of nestdiameter of nest 1.931.322.221.072.181.24 1.821.121.110.502.281.10 2.081.081.530.771.501.14 2.131.381.821.242.031.14 2.101.141.160.871.731.40 2.271.061.161.141.720.94 2.161.351.760.761.520.90 1.600.611.340.772.620.52 1.260.72.501.461.851.16 1.340.821.531.052.041.04 j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 82 http://jad.tums.ac.ir published online: march 14, 2017 fig. 1. patterns of scorpio maurus nests in haftgel, south west of iran (original picture) fig. 2. patterns of odonthubutus bidentatus nests (35–55cm) in haftgel, south west of iran (original picture) discussion the iranian scorpions are classified into 3 categories of non-burrowing (hemiscorpius lepturus), semi-burrowing (mesobuthus eupeus and andrectonus crassicauda) and burrowing scorpions (s. maurus, o. bidentatos, o. odonthodorous, a. petrigosecus and o. doriae) according to the pattern of nest building habits (vazirianzadeh and tirgari 1989). this paper describes the architectural and surficial morphologies of burrows produced by s. maurus as well as o. bidentatos. this study was carried out in order to identify the burrowing habitat, nesting features and then linked to environmental conditions including soil composition, density and moisture content. the type of soil texture in the area of study was determined as a light class of soil including clay loam and sandy loam. this is in line with this fact that the high density of nests was related to the lightness of soil texture (rutin 1996). the moisture (sutured humidity) was raised from 32.11% in 10–20cm to 37.93% in the depth of 20–40cm due to raising the clay content from 13% to 23%. this finding is in consent to previous study in terms of c. jonesii burrowing habit regarding increasing clay content in the soil structure (harington et al. 1978). this may explain that deep of nests in the study area usually was distributed in 20–35cm depth range regarding both species. burrow depth approaching 75cm have been reported for s. maurus in the sahara desert (cloudsleythompson 1965). this implies that nests are not very deep in the calcareous and rocky sections and the drilling attempts had been stopped at the current depths of 20–35cm (rutin 1996). the depth of o. doriae nest has been reported 20.5±5.5cm (dehghani et al. 1991). it means that burrowing habits is different even between two species of one genus. however, this may be due to the soil structure of the region of study. the density of nests area was measured 0.4 nests in 1m2 related to s. maurus or counted 19 nests in 5 min (regardless of species) and this rate was 0.6 regarding to o. bidentatus. consequently, the soil texture of this area may provide more facilities for drilling activity of o. bidentatus than s. maurus but the number of trapped the latter species was greater. it is presumed that there were more factors determined the density values of both species including size of scorpion j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 83 http://jad.tums.ac.ir published online: march 14, 2017 bodies and shape of their pedipalps or other drilling organs. this is in accordance with hadley (1974) and hembree et al. (2012), who explained that burrowing habit in scorpions was closely related to their taxonomic position and morphological modifications. the nests of s. maurus had been built using more engineer methods with more spirals in the shapes than nests of o. bidentatus, in the current study. any artificial breeding activity for each of these species in natural enclosed piece of land, that may be called scorpion zoo park, need to be created separately with particular structure of soil texture. however, in the current case this is an advantage which both species can live in an area with the same soil texture. khuzestan province is a desert and plain region with a semi-arid climate and almost no raining occur from mid or late of may to end of september (vazirianzadeh 1989). the soil texture nests of both species were sandy loam in 10–20cm depth and clay loam in 20– 40cm depth. the soil of area had gypsum normally formed following raining. therefore, this condition made to reduce the chance of survival a burrowing than a non-burrowing scorpion in this province. due to the changing weather conditions with dry, warm days and cold nights, nests were often made spiral shape. this condition had caused nests built with multi steps. each step was used by the scorpions to do their rests at a special time due to the different conditions during 24 h (hadley 1974). in the term of entrapping in the early morning, acquired results showed that o. bidentatus scorpions were engaged in hunting activities, in contrast to the s. maurus scorpions, which were present at their nests at the same time. therefore, human contact with o. bidentatus was most likely than s. maurus in the early morning. furthermore, the majority of o. bidentatus nests was seen in intact areas far from residential region and was less observed by the residents than s. maurus scorpions. among 990 envenomed individuals by scorpion stings in khuzestan, 0.4% and 0.1% of cases were related to s. maurus and o. bidentatus, respectively (vazirianzadeh and samie 2006) distribution and profiles of nests are presented in table 1 and 2. the entries show a round hollow nest belongs to s. maurus. odonthubutus bidentatus nests were equipped with an elliptical hollow and a small arch in the upper edge. a single elliptical surface opening leads to a single narrow and flat tunnel that ends in a vertically expanded chamber. this instruction plays important role in creating awareness and precautions about scorpion nests in human communities. education also helps human to understand where to access dangerous scorpion as well as how to escape them. although there was not any significant different between nest entrance properties, stronger correlation between nest entrance properties of s. maurus than o. bidentatus was seen. this correlation may be assumed as a different manner of burrowing habitats between two species in the current study. however, different nest entrance properties between two species, individually, may be implicated as a presentation of different age sizes. this is a positive phenomenon in growing two species of scorpions in scorpion zoo parks. this adaptation makes possibility to build artificial nests in provided area, because interrupt different generation shows that parent and offspring scorpions can tolerate each other. therefore, it is taken that the burrow of s. maurus was more complex than the typical burrow of o. bidentatus and the nests morphology (including shape, depth, length and diameter) depends on the following factors: species, soil texture, soil moisture and region weather conditions (hembree et al. 2012). finally, there were some differences between two mentioned scorpions in burrowing activities. j arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 84 http://jad.tums.ac.ir published online: march 14, 2017 the difference between the 2nd and the 3rd patterns of s. maurus is on the depth of the burrowing activities. the presented fact related to the humidity rate of soil, which caused to create different pattern of burrowing activity, by the scorpion is the different between 2nd and 3rd patterns of s. maurus. the depth of 2nd pattern was shorter than the depth of the 3rd pattern. in the 2nd pattern the scorpions had reached in the lesser depth to enough soil humidity, then the depth of 2nd pattern was shorter than the two other patterns. conclusion the nests morphology characteristics of two iranian scorpions including shape, depth, length and diameter depend are different from each other based on the following factors: species, soil texture, soil moisture and region conditions. acknowledgements this work was supported by a grant from student research office, deputy of research of ajums. the authors declare that there is no conflict of interests. references cala-riquelme f, colombo m (2011) ecology of the scorpion, microtityus jaumei in sierra de canasta, cuba. j insect sci. 11: 86. clousley-thompson jl (1965) the scorpion. science j. 1: 35–41. day pr (1982) particle fractionation and particle-size analysis. in: r.h. miller rh and keeney dr (editors). chemical and microbiological properties. soil science society of american inc. pp. 545–567. dehghani r ,tirgari s, droudgar a (1991) study of nest architecture .of scorpion odontobuthus doriae in esfahan province. feyz. 1(2): 81–86 (persian with english summary). farzanpey r (1987) scorpion knowledge. iran academic press (persian). farzanpey r (1988) a catalogue of the scorpions occurring in iran, up to january 1986. revue arachnologique. 8: 33–44. fet v, soleglad me (2005) contributions to scorpion systematic. i. on recent changes in high-level taxonomy. euscorpius. 31: 1–13. hadley nf (1974) adaptational biology of desert scorpions. i . arachnol. 2: 11– 23. harington a (1978) burrowing biology of the scorpion cheloctonus jonesii pocock (arachnida: scorpionida: scorpionidae). j arachnology. 5: 243–249. hembree id, johnson lm, tenwalde rw (2012) neoichnology of the desert scorpion hadrurus arizonensis: burrows to biogenic cross lamination. palaeontologia electronica. 15(1). jalali a, moazen s, babaee m, dadashzade s, droudi a (2010) the pharmacokinetics of iranian scorpion odonthubuthus doriae venom and the available antivenom. j venom res. 1: 48–53. jalali a, vatanpour h, hosseininasab z, rowan eg, harvey al (2007) the effect of the venom of the yellow iranian scorpion odontobuthus doriae on skeletal muscle preparations in vitro. toxicon. 50: 1019–1026. navidpour s, kovarík f, soleglad me, fet v (2008) scorpions of iran (arachnida, scorpiones). part i. khuzestan province. euscorpius. 65: 1–41. masihipour b, zare am, navidpour sh, taghavi moghadam a, akbari a, rabiei h (2009) identification and purification of the three toxins from odotobuthus bidentatus, lourenco and pezj arthropod-borne dis, march 2017, 11(1): 78–85 b vazirianzadeh et al.: a comparative study of … 85 http://jad.tums.ac.ir published online: 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(persian). 5dr.basseri rtl_2_ iran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 35 original articles mosquito vector biting and community protection in a malarious area, siahoo district, hormozgan, iran kh shahandeh1, *hr basseri 2, a pakari 3, a riazi 2 1social development and health promotion research center, tehran university of medical sciences, tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3institute of public health, bandar-abbas center of health research and education, tehran university of medical sciences, tehran, iran (received 3 jul 2010; accepted 3 aug 2010) abstract background: use of bed-net continues to offer potential strategy for malaria prevention in endemic areas. local communities are indispensable during design and implementation stages. methods: a cross-sectional study of 192 randomly selected inhabitants was carried out in malarious zone, siahoo direstrict, hormozgan province, southern iran. in addition, we monitored human landing periodicity of main malaria vectors and as well as self-protection of inhabitant in the study area for a period of one transmission season between april to october 2006. results: the biting activities were seen throughout the whole night for three malaria vectors, anopheles fluviatilis, an. stephensi and an. dthali, and an. fluviatilis exhibiting bimodal peaks, the first at midnight (0:00−1:00) and the other before dawn (5:00−6:00 am) but the maximum biting activity of an. stephensi was occurred at second quarter of night (11:00−12:00 pm). the majority of interviewers (83.3%) knew that malaria was transmitted by mosquitoes and 70.3% of them stated that bed-net is the best control measures. most subjects (62%) did not have a mosquito net. conclusion: study subjects were aware of an association between mosquito bite and malaria transmission. health workers at different levels of the health care delivery system should disseminate relevant information about selfprotection to help community members to be involved more in malaria control. keywords: malaria, community protection, mosquito bite, iran introduction in spite of more than 45 yr malaria control programming, malaria remains prevalent in southern and southeastern iran. during the last six yr, 15000 to 25000 cases have been reported each year in iran and more than 85% of them occurred in the south and southeast of the country (department of communicable disease control, personal communication). the annual parasite index (api) is seven per 1000 inhabitants in the endemic area (raeisi 2006). several factors, such as presence of insecticide resistance among vectors (dinparast-jadid et al. 2006), parasite drug resistance (edrissian et al. 1993, edrissian 2006) and socio-economical problems (banguero 1984, raeisi 2006), have made eradication in this area impossible. however, several main inhibitor factors have been defined in hormozagan province, which restricted malaria control programs in that particular area (masoumi asl et al. 2003). generally, prevention of malaria through better knowledge and awareness is the appropriate way to keep malaria disease away. in addition, knowledge of host feeding pattern *corresponding author: dr hamid reza basseri, email: basserih@sina.tums.ac.ir iran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 36 and resting behavior of mosquito vectors are important for understanding the host-vector relationship and dynamic of disease transmission and for development of control strategies (singh 1998, chaves et al. 2010). the predominant mosquito species in the southern iran are anopheles stephensi, an. culicifacies and an. fluviatilis which play the major role in malaria transmitting (zaim et al. 1993, vatandoost et al. 2003, basseri et al. 2008). vector control activities in south and southeast of iran are mainly restricted to indoor residual spraying (irs) in selected endemic localities with pyrethroids and larviciding with bacillus thuringiensis, (ministry of health and medical education, iran 2008). in addition recently by planning malaria elimination program, self-protection using bed net has been more considered. with this background, this study is proposed to find the sleeping behavior of resident and the ratio of man-vector contact in the area where exophilic vector such as an. fluviatilis is highly active and therefore using indoor residual spraying is so limited. although, it has been planned to used long lasting impregnated bed net in south of iran by health authorities in local provinces, therefore, the results of this study can show more strengthens and weakness of using bed net in that particular area. materials and methods study area this study was performed in siahoo district located in about 90 km north of bandarabbas city, hormozgan province (25◦24'–28◦57 'n and 52◦41'–59◦15' e) over a period of six months, during 2007 and 2008. the temperature reaches to maximum 35° c in highlands and to 40° c in plan areas during summer but it rarely decline to 5-10° c during winter. generally, the area has a subtropical climate and is prone to seasonal malaria transmission. hormozgan comprises three regions of differing geography, the coastal region in the south, a mountainous region in the north, and a rural plateau or plains region in the centre. siahoo is a rural county located in mountainous area with a total population about 10000. it is an agricultural region irrigated by rivers, deep wells, and cement pools, which are the main of breeding sites for mosquitoes. the annual rainfall ranged from 80 to 100 mm. features contributing perennial transmission in the areas are mainly due to population movement and specially form west border of province, sistanbaluchistan. the malaria vectors are very much potent with high anthropophilic index and frequent man biting habits and above all presence of varying degree of resistance to insecticides and drugs. sampling technique a cross-sectional study was designed for the study area. ten percent of target groups including residents, who attended to malaria clinics in siahoo health centers, were selected by systematic random sampling. then they interviewed to obtain knowledge, attitude and behavior of them about mosquito biting and self-protection against it and malaria transmission. structure interview forms were obtained from participants. the structure interview form was explained to local staff in advance. the completed forms and records were then checked and collected by technicians at the health research center of bandar-abbas (institute of public health, tehran university of medical sciences). data collection; methods, instruments used, measurements: structure questions consisted of open and closed questions and designed in several parts, including participants' details, house structure, sleeping behavior, mobility, self-protection, history of malaria infection and treatments used, facilities, access to health services. the interviews were conducted in the native accent and dialects by questioners. the validity of iran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 37 questions was checked in statistics department, school of public health, and tehran university of medical sciences. data were analyzed under supervision of the statistics department, school of public health. ethical approval for the research was obtained from the tehran university of medical sciences research ethics committee. informed consent was obtained from all participants themselves. anonymity was assured to the participants, and it was explained to them that transcripts would not have identifiable features and would be kept in a secure location. mosquito collection (mosquito landing catch) activity of malaria vectors was surveyed by human landing catch by interval two weeks during seasonal activity of mosquitoes, from 10th of april to 20th of october 2006. human landing catch was carried out continuously during whole night from sunset since sunrise (19:00–06:00 h) by trained staff using oral aspirators (service 1976). two human volunteers of native people acted as baits while wearing their normal clothing. the exposed body surfaces were searched and the mosquitoes that were attempting to bite were collected using oral aspirators by insect collectors. during human landing collection, collector workers shift every two hours. the hourly collection of mosquitoes were kept and brought to laboratory alive. they were anesthetized by chloroform and identifies based on species keys of smart (2003). finally, the human biting rate was calculated directly from human landing catches as the average number of bites per person per night. results demographical information and knowledge of respondents in this study, the valid questionnaire was 192 respondents. the mean age of subjects was 31 yr (range 12 to 69 yr; standard deviation 11 yr; median 30 yr). near one third of respondents were females and totally, less than one-fifth (19.2%) of participants were illiterate. the demographic characteristics of the interviewees are showed in table 1. mosquito bite was mentioned to be the main 'cause' of malaria by 83.3% of the respondents. other mentioned causes were dirty water (10.9%), and food (0.5%) and 3.6% of respondents said they do not know (table 2). when asked about the ways of preventing malaria, 70.3% recognized using mosquito nets, 89% drying stagnant water around their homes, 4.2% using screens on doors and windows and 68.7% insecticides as control measures. practice for biting protection majority of respondents (62 %) reported that not having bed net. generally, all respondents with bed net indicated that they used bed nets more often from beginning of april to end of september but them majority of them used nets from evening and night and only 4.1% of those who owner of bed net, used it during whole night (table 3). near 40% of total interviewers stated that they always used bed-net and only a few of them sometime slept under bed-net. the majority of respondents do not have mosquito screen on windows (75%) and doors (99%). mosquito biting periodicity biting activities of the mosquito vectors are shown in fig. 1. anopheles fluviatilis, an. stephensi and an. dthali were highly active during whole night. the mosquitoes started biting continuously from sunset to sunrise (fig. 1). anopheles fluviatilis was predominantly captured on human baits followed by an. stephenis and an. dthali at relatively lower population. landing rate of an. fluviatilis with average of 11.2 mosquitoes per human bait per night was slightly higher than others were. the average number of an. stepehnsi captured on human bait per night was 4.1 and for an. dthali was 1.5. the peaks of landing for an. fluviatilis and an. stephensi was iran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 38 occurred at approximately midnight followed with a short peak before sun rise but an. dthali started landing earlier at begging of sunset and its activities declined at midnight. table 1. main demographic characteristics of the study sample (participants) characteristic no. % age (years) <15 18 9.4 16-25 63 32.8 26-45 81 42.2 46+ 30 15.6 sex ratio males 125 65.1 females 67 34.9 education level illiterate 31 16.1 literate 161 83.9 table 2. knowledge of participants about transmission route and control measures variable frequency % route of transmission mosquitoes 160 83.3 dirty water 21 10.9 polluted food 1 0.5 other incorrect 3 1.6 don’t know 7 3.6 control measures using mosquito net 135 70.3 using door/window screen 8 4.2 taking drugs regularly 10 5.2 drying stagnant water 171 89.0 using insecticides 132 68.7 others 22 11.4 table 3. mosquito biting behaviors practiced by participants variable frequency % availability of mosquito net have mosquito net 73 38.0 frequency of net use always 65 33.9 sometimes 8 4.1 time of net use night 27 14.0 evening and night 46 24.0 use of screens on windows 25 13.0 on doors 1 0.5 0 0.5 1 1.5 2 2.5 18-19 19-20 20-21 21-22 22-23 23-24 24-01 01-02. 02-03. 03-04. 04-05. 05-06. time m e a n o f m o s q u it o b it e s p e r h e u m a n b a it an. fluviatilis an. stephensi an. dthali fig. 1. biting activities of main malaria vectors during night on human bait in siahoo district, hormozgan. iran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 39 discussion although the ratio of literacy among participants was relatively high, it is necessary to provide people with adequate information about the role of mosquito vectors in transmission and incidence of malaria in their area and its complications, to increase public awareness about the importance of malaria vector. however, the study revealed that in siahoo, community comparatively recognized the role of mosquitoes in malaria transmission and knowledge of malaria was moderate but the malaria prevention activity by people was relatively poor. nevertheless low level of educational status, rampant ignorance about the cause, treatment, and prevention of malaria, poor socio economic background contributes a lot to the cause of malaria but fortunately, adequate health care services can make the disease infection declined in the region (banguero 1984). the biting behavior of any mosquito species is a biologic characteristic and could vary among species. the biting activities of an. fluvialitis and an. stephensi were seen throughout the whole night from dusk to dawn. thus, the residents in siahoo region are exposed to the vector bites even before bedtime or using bed-net. anopheles fluvialitis and an. stephensi are principal vectors in south slop of zagrus mountain chain (manouchehri et al. 1976, vatandoost et al. 2006). anopheles fluvialitis has been recognized as a wild malaria vector with high anthropophilic index (nanda 2000, basseri et al. 2005). in contrary, an. stephensi is a domestic mosquito with endophilic and endophgic behaviour in hormozgan province (vatandoost et al. 2006). in the present study, an. fluviatilis was predominant among the mosquitoes collected in the human landing catches. controlling of this species is ecologically and logistically challenging. the options for the control of this species are very limited but self-protection methods such as using insecticide integrated bed-net could be more convenient way to control malaria transmission. in addition, human-vector contact could be reduced by a adjusting the time of bed-net use (binka 1998). thus insecticide integrated bed-net can be targeted approach of adopting personal protective measures in siahoo area while an. fluviatilis avoid feed or resting in indoor places (edalat 1998). however, these measures need to be encouraged among the native population (stephens 1995). they could be sensitized and motivated by health workers to adopt such measures. the key to implementation of personal protective measures are health education and socioeconomic development, which can cause the annual infective biting rate and reduce transmission potentials. we conclude that the biting periodicity of an. fluvialitis is bimodal peaks and the peak biting activity of this mosquito coincides with the sleeping time of inhabitant. the risk of acquiring an infection based on parity status could be elucidated towards the dusk since dawn hours, as shown by the bimodal peaks of biting activity of mosquitoes, namely, a minor peak at midnight. therefore human-vector contact could be reduced by a time and space targeted approach of adopting personal protective measures such as protective (yadav et al. 1999, takken 2002). in conclusion, regular and periodic intervention in the form of health education to bring an attitudinal and behavioral change and provision of better health care services in the siahoo area can bring qualitative change in control of malaria in the region. acknowledgements the authors would like to thank personnel of bandar-abbas training and research center for their kind assistances and institute of public health research. we appreciate the kind cooperation of siahoo health auiran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 40 thorities and bandar-abbas health center. this work received financial support school of public health, tums, iran. the authors declare that they have no conflicts of interest. references banguero h (1984) socio economic factors associated with malaria in colombia. soc sci med. 19: 1099–1104. basseri hr, doosti s, akbarzadeh k, nateghpour n, whitten mma, ladoni h (2008) competency of anopheles stephensi mysorensis strain for plasmodium vivax and the role of inhibitory carbohydrates to block its sporogonic cycle. malar j. 7: 131. basseri hr, moosakazemi s, yosafi m, mohebali m, hajaran h (2005) anthropophily of malaria vectors in kahnouj district, south of kerman, iran. iran j public health. 34: 27–37. binka fn, indome f, smith t (1998) impact of spatial distribution of permethrinimpregnated bed nets on child mortality in rural northern ghana. am j trop med hyg. 59(1): 80–85. chaves lf, harrington lc, keogh cl, nguyen am, kitron eu (2010) blood feeding patterns of mosquitoes: random or structured? frontiers in zoology, 7:3, available at: http://www.frontiersinzoology.com/content/7/ 1/ 3. djadid nd, forouzesh f, karimi m, raeisi a, hassan-zehi a, zakeri s (2007) monitoring pyrethroid insecticide resistance in major malaria vector anopheles culicifacies: comparison of molecular tools and conventional susceptibility test. iran biomed j. 11(3): 169–176. edrissian gh, afshar a, sayedzadeh a, mohsseni g, satvat mt (1993) assessment of the response in vivo and in vitro of plasmodium falciparum to sulphadoxine-pyrimethamine in the malarious areas of iran. j trop med hyg. 96: 237–240. edalat h (1998) ecology of anopheles fluviatilis, the main malaria in southern iran and its role in the epidemiology of malaria during 1997-1998. phd thesis, school of public health. tehran university of medical sciences. edrissian gh (2006) malaria in iran: past and present situation. iran j parasitol:1 (1): 1–14. manouchehri av, javadian e, eshighy n, motabar m (1976) ecology of anopheles stephensi liston in southern iran. trop geog med.28: 228–232. masoumi-asl h, motabar m, zamani gh, naserinejad d, vatandoost h (2003) a case-control study of determinative factors on malaria morbidity in minab, jask and roodan counties, in hormozgan province, southern iran, 2001. iran j public health. 32(3): 14–18. nanda n, yadav rs, subbarao sk, joshi h, sharma vp (2000) studies on anopheles fluviatilis and anopheles culicifacies sibling species in relation to malaria in forested hilly and deforested riverine ecosystems in northern orissa, india. j am mosq control assoc. 16: 199–205. raeisi a (2006) malaria elimination in iran, progress achievements and challenges. 6th meeting of national malaria programme managers. cairo, egypt, 3–6 july service mw (1976) mosquito ecology: field sampling methods. john wiley and sons, new york. singh n, singh mp, saxena a, sharma vp, kalra nl (1998) knowledge, attitude, beliefs and practices (kabp) study related to malaria and intervention strategies in ethnic tribals of mandla (madhya pradesh). curr sci. 75: 1386–1390. smart j (2003) a handbook for the identification of insects of medical importance. 2nd edition, biotech books, new delhi, india. stephens c (1995) knowledge of mosquitoes iran j arthropod-borne dis, 2010, 4(2): 35–41 kh shahandeh et al.: mosquito vector biting … 41 in relation to public and domestic control activities in the cities of dar es salaam and tanga. bulletin of the world health organization. 73(1): 97–104. takken w (2002) do insecticide-treated bednets have an effect on malaria vectors? trop med int health, 7(12):1022–1030 vatandoost h, oshaghi ma, abaie mr, shahi m, yaaghoobi f, baghaii m, hanafi bojd aa, zamani g, townson h (2006) bionomics of anopheles stephensi liston in the malarious area of hormozgan province, southern iran 2002. acta trop. 97(2): 196–203. yadav sp, tyagi bk, ramanath t (1999) knowledge, attitude and practice towards malaria in rural communities of the epidemic prone thar desert, northwestern india. j com dis. 3(2): 127–136. world health organization (1992) entomological field techniques for malaria control, part i and ii learner and tutor’s uidc, geneva, switzerland: world health organization. zaim m, subbarao sk, manouchehri av, cochrane ah (1993) role of anopheles culicifacies s.l. and an. pulcherrimus in malaria transmission in ghassreghand (baluchistan), iran. j am mosq control assoc. 9(1): 23–26. j arthropod-borne dis, december 2017, 11(4): 446–452 o páez-rondón et al.: revalidation of … 446 http://jad.tums.ac.ir published online: december 30, 2017 original article revalidation of synonymy between nesotriatoma flavida and n. bruneri (hemiptera, reduviidae, triatominae) oscar páez-rondón 1,2, fernando otálora-luna 2, *elis aldana 3 1unidad de articulación comunitaria, centro multidisciplinario de ciencias, instituto venezolano de investigaciones científicas, mérida, república bolivariana de venezuela 2laboratorio de ecología sensorial, centro multidisciplinario de ciencias, instituto venezolano de investigaciones científicas, mérida, república bolivariana de venezuela 3laboratorio de entomología “herman lent”, departamento de biología, facultad de ciencias, universidad de los andes, mérida, república bolivariana de venezuela (received 6 june 2016; accepted 17 oct 2017) abstract background: we analyzed the external morphology and the external male genitalia of specimens of nesotriatoma flavida of a laboratory colony founded with specimens from guanahacabiles in pinar del río province in the west of cuba in 1980. this species was at first identified as different from n. bruneri and then later considered synonymous. methods: we proposed to revise the morphological characters with which these species were considered as different and then later synonymous, such as the fossula spongiosa or spongy grooves, length of the first antenna segment, relationship length of eye to interocular distance, the form of the phallosome, phallosome support, and the endosome processes. results: the results of the analyses of these characters in the specimens of our colony, and of the corresponding works where the separation and synonymy of these species has been proposed, allow us to sustain the revalidation of the synonymy between n. flavida and n. bruneri. conclusion: based on the body external morphology and the male external genitalia of n. flavida it is concluded that n. flavida and n. bruneri are synonymous species. keywords: chagas’ disease, phallosome, morphology, taxonomy introduction a new genus was describes, nesotriatoma, in which he includes nesotriatoma flavida (initially described as triatoma flavida) collected in the western region of the island of cuba and also a new species nesotriatoma bruneri, collected in the eastern region of this island (1). nesotriatoma flavida has a short first segment of the antenna that does not reach the peak of the clypeus, well developed spongy grooves on the front legs of the females, and eyes as wide as a third of the interocular distance. nesotriatoma bruneri describes as having a long first antenna segment, reaching or passing the peak of the clypeus, no spongy grooves on the front legs of the females, and the width of the eyes as more than half the in terocular distance. however, later proposes synonymy between n. bruneri and n. flavida, given the plasticity of the characters with which was diagnosed both species (2). some authors (3) propose as not valid the synonymy proposed earlier (2), based on morphological differences in the external genitalia of the male and include both species in the triatoma genus, already considered as synonymous with nesotriatoma in (2-4). the separation of both species and their inclusion are accepted in the triatoma genus (5). posteriorly, the separation of the two species is maintained but based on phylogenetic analyzes include them in the nesotriatoma genus (6). in a checklist of the current valid *corresponding author: dr elis aldana, e-mail: elis.aldana@gmail.com, aldana@ula.ve j arthropod-borne dis, december 2017, 11(4): 446–452 o páez-rondón et al.: revalidation of … 447 http://jad.tums.ac.ir published online: december 30, 2017 species of the subfamily triatominae also consider valid nesotriatoma genus (7). this genre is composed by the species n. flavida, n. bruneri and n. obscura (flavida complex). however, recently (8) by genetic analyzes published an article questioning the specific status of n. bruneri because this specie presented the same cytogenetic characteristics of n. flavida and an extremely low genetic distance (0.004) (8). the specimens with proposed the separation of n. flavida and n. bruneri based on the morphological characters of the external genitalia of the males insects were sent by dr w torrealba (3). “dr torrealba sent us 5 specimens from the corresponding group. one specimen was identified as a typical t. flavida female captured in a human dwelling, and 4 specimens (two males and two females) captured in an animal burrow (?) of pinar del río province, guanahacabiles in the west of cuba on 4-11-1979”. with individuals from the same locality and captured on the same date a colony was established at the universidad de la habana, cuba and with individuals of this colony was founded the colony of n. flavida at our laboratory in mérida, venezuela whose members were used in this work for analyses of external morphological characteristics and of the male external genitalia, with the aim of verifying whether the triatomines that form the colony were n. flavida or n. bruneri, in view of the polemic in the taxonomic status of these species. materials and methods entomologic material nine males and nine females of n. flavida from the laboratory of entomology “herman lent” (hlel), venezuela, were examined, being maintained at 28 °c, 50% relative humidity, and fed hen blood. this colony was founded on 18-6-1980 with eggs, 3 females, 2 males, and 5 fifth instar nymphs brought by dr scorza from the colony identified as triatoma flavida in the universidad de la habana, cuba. morphological analyses the analysis of the external morphology of the adult body was carried out following the taxonomic keys (4), and the descriptions of both species (1, 5). the analysis of the morphology of the external male genitalia was done following the terminology employed and descriptions of the external male genitalia of both species (3-5). both morphological studies were observed with a leica m205a stereoscopic microscope, the images were taken with a cmos camera (eos rebel t3, canon). the genitalia were dissected at the level of the two final abdominal segments, and processed in the following fashion: koh (eka, chemicals) at 10% in a mortar and heated without boiling until soft, later treated according to treatment 1: 36h in phenol (90%, honeywell riedel-dehaën) and 48h in guaiacol (scharlau chemicals, s.a.), or treatment 2: 72h both in the phenol and in the guaiacol. finally, they were mounted in dpx (industrias químicas erba, c.a.) on a microscope slide and observed under a stereo microscope (leica m205). the pictures were taken with a cmos camera (leica ec3) coupled to this microscope. the drawings of the borders both of the silhouette of the phallosome as well as its support in the male external genitalia were done using a raster graphics editor (photoshop cs6, 13.0 x64). results external morphology of the body general color: clear brown. body and corium are practically hairless, hairs scarce and very short. femur: variable: from completely dark or completely light, or the proximal half dark and the distal light, with the presence or absence of denticles on the front legs. tibias j arthropod-borne dis, december 2017, 11(4): 446–452 o páez-rondón et al.: revalidation of … 448 http://jad.tums.ac.ir published online: december 30, 2017 and distal parts of the end of the femur have a light color (figs. 1, 2). spongy grooves underdeveloped, present on the front legs of both males and females (fig. 3). pronotum is dark, except for the humerus, anterolateral angles, and the submedian carinae, which are of a yellow color. pronotum has prominent anterolateral and humeral angles, anterior lobes with very noticeable lateral and discal tubercles and with yellow-edged halos, a butterfly figure at the union of the two lobes. on the front edge of the scutellum disk, 1+1 prominent tubercles pointed forwards and touching the back edge of the pronotum. median region of the scutellum has noticeable transverse grooves (fig. 4). hemelytron is covering laterally part of the tergites and reaching the seventh urotergite, extensively spotted towards the back part and clearly defined dark stains on the front portion (fig. 5). proportion eye width: interocular distance 1:2 (fig. 6). third rostrum segment is shorter than the others. first antenna segment does not overshoot the peak of the clypeus (fig. 7). morphology of the male external genitalia phallosome lengthy, oval, and with concave base when the genitalia are maintained 36h in phenol and 48h in guaiacol (figs. 8, 9, 10(3)). phallosome lengthy, hexagonal, and concave base when maintained 72h in both phenol and guaiacol (figs. 11, 12, 13(3)). the process of the endosome extends approximately 75% of its length from the base of the support of the phallosome, with a lengthy, very wrinkled and grooved appearance when the genitalia are maintained 36h in phenol and 48h in guaiacol (fig. 8) as well as when maintained 72h in both the phenol and the guaiacol (fig. 11). the phallosome support long, with cylindrical base, side edges converging towards the outer end of the base under either of the treatments of the genitalia (figs. 14, 15(3)). median process of pygophore simple and pointed (fig. 16). table 1. comparison of external morphological characters of the body and of the external male genitalia of nesotriatoma flavida and nesotriatoma bruneri character (1) n. flavida (4) (3) n. flavida present work n. flavida n. bruneri n. flavida n. bruneri first antenna segment size sh l sh sh sh sh spongy groove distribution f:an f:an m:an, md; f:v m:an, md; f:v m:an, md; f:an, md m:an; f:an spongy groove degree of development d n/d n/d n/d sd sd ratio eye width: interocular distance 1:3 1:2 1:3 1:2 1:2 1:2 general body color brown and ochre mottled brown light hazel light brown dark mottled hazel light hazel form of distal protuberances of anterior pronotal lobe sm, rd, prm prm rd, subc n/d n/d prm form of lateral protuberances of anterior pronotal lobe sm, rd, prm prm rd, subc n/d n/d prm presence of denticles in anterior and median femurs a p p p p v form of phallosome n/d n/d n/d l, hx, bscc l, ov, bscc tr1:l, ov, bscc tr2:l, hx, bscc j arthropod-borne dis, december 2017, 11(4): 446–452 o páez-rondón et al.: revalidation of … 449 http://jad.tums.ac.ir published online: december 30, 2017 form of phallosome support n/d n/d n/d l, bscy, brlpr l, bscy, brlcv tr1:l, bscy, cvle tr2:l, bscy, cvle form of endosome processes n/d n/d n/d l, al sh, w l, w median processes of pygophore n/d n/d n/d n/d n/d s, pn sh: short, l: long, f: female, m: male, an: anterior, md: median, v: variable, d: developed, n/d: not described, sd: slightly developed, sm: small, rd: round; prm: prominent, subc: subconic, a: absent, p: present; hx: hexagonal, bscc: concave base, ov: oval, bscy: cylindrical base, prle: parallel lateral edges, cvle: convergent lateral edges, al: aliform, w: wrinkled, s: simple, pn: pointed, sth: smooth, tr1: treatment 1, tr2: treatment 2. numbers in parenthesis correspond to citations in references. figs. 1–7. external morphology of the body of nesotriatoma. flavida. 1: dorsal view of complete male body. 2: femur and tibia of the female. 3: spongy groove on the distal end of the foreleg tibia. 4: dorsal view of the pronotum and scutellum of the female. 5: dorsal view of the scutellum and hemelytrons of the male. 6: dorsal view of the male head. 7: ventral view of male head figs. 8–16. morphology of external male genitalia. 8: ventral view of phallosome, treatment 1 (see materials and methods). 9: phallosome silhouette, treatment 1. 10: phallosome silhouette of n. bruneri according to (3). 11: ventral view of phallosome, treatment 2 (see materials and methods). 12: phallosome silhouette, treatment 2. 13: phallosome silhouette of n. flavida according to (3). 14: phallosome support, a: treatment 1, b: treatment 2. 15: phallosome support, a: n. flavida, b: n. bruneri, according to (3). 16: median processes of pygophore. ph: phallosome, pren: endosome process, sph: phallosome support, prp: median process of pygophore. discussion the characters: length of the first antenna segment in relation to the clypeus, presence of spongy grooves, and ratio eye breadth to interocular distance as features that distinguish n. flavida from the new species he calls n. bruneri (1), all of which later (2) considers so variable that they do not justify the distinction between the species, and so he considers them synonymous. the external male genitalia of n. flavida was described as a quadrangular phallosome, phallosome support with parallel edges, semicircular vesica, and long endosome processes, while those of n. bruneri is described as being an oval phallosome, convergent edges of the phallosome support, ovoidal vesica, and short endosome processes (3). examining the illustration was provided (fig. 13), the phallosome of n. flavida was not quadrangular, but rather hexagonal (figs. 11, 12). the endosome process extends from the middle part of the phallosome to the vesica in n. flavida, and indicated it in n. bruneri, but it is indistinguishable in such illustration (not showed). although we observed that the phallosome support is pointed in both specimens: n. flavida described in our work (figs. 14a,b) and in n. bruneri described, according to these authors the phallosome support is blunt in n. flavida and more pointed in n. bruneri (fig. 15a,b) (3). although we observed spongy grooves in female and males, the presence of spongy grooves varies according to whether the legs are frontal or medial and according to sex, the authors did not describe this structure table 1. continued… j arthropod-borne dis, december 2017, 11(4): 446–452 o páez-rondón et al.: revalidation of … 450 http://jad.tums.ac.ir published online: december 30, 2017 in n. bruneri. they described the interocular distance as being double the eye width in both species (3). finally, we observed that the first antenna segment does not reach the peak of the clypeus in any of the species (fig. 1). in our opinion the distinction between n. flavida and n. bruneri proposed (3) is poorly based, the description of the phallosome silhouette is erroneous, the imprecise illustration and distinction of the vesica as semicircular in n. flavida and ovoidal in n. bruneri, the poor illustration of the endosome process in n. bruneri, the imprecision with which the endosome process is considered “large”, and the absence in that paper of a comparative analysis between the species based on those characters that first distinguished the species and then later considered them synonymous given the phenotypical plasticity of the length of the first antenna segment with respect to the clypeus, the spongy grooves, and the relation between eye width and interocular distance (1, 2). in the present work it was found that the specimens identified as n. flavida selected from the colony in our laboratory, formed from the same group of insects collected in guanahacabiles in pinar del río province in the west of cuba (3), present the following features: 1) the first antenna segment does not overshoot the clypeus peak like that described in n. flavida, and in n. flavida and n. bruneri (1,3), 2) spongy grooves poorly developed, present in the forelegs of both males and females, as describe for n. bruneri (3), and differing from what observes as well developed grooves in the female and absent from the males of n. flavida, and missing in both sexes of n. bruneri, (1) and also differing from what observe in finding the grooves present on the front and middle legs of the males and variable presence on the forelegs of the n. flavida female (3), 3) ratio eye width–interocular distance 1:2, similar to what describes for n. bruneri, for n. flavida and n. bruneri, and differing from the description in for n. flavida (1:3) (1,3), 4) when the genitalia were treated 36h in phenol and 48h in guaiacol, the phallosome silhouette is found to be oval, similar to that described for n. bruneri (3), (fig. 10), while on the other hand, phallosome has a hexagonal silhouette when treated 72h in phenol and 72h in guayacol, similar to that described as “quadrangular” in n. flavida (fig. 13), 5) phallosome support base cylindrical, pointed peak and convergent edges irrespective of the phenol and guaiacol treatments, similar to the cylindrical support base described for n. flavida (3), (fig. 15a), and to the pointed peak and convergent edges of n. brunei by these same authors (fig. 15b), and 6) endosome processes long and wrinkled in either of the different times in phenol and guaiacol, for n. bruneri (3) (table 1). in the present work, differences were found in characteristics of the external male genitalia according to as to how they were treated in phenol and guaiacol, which exemplifies the importance of a detailed description of the treatment protocols of the genitalia, an aspect not treated (3). our proposal for the revalidation of the synonymy between n. flavida and n. bruneri is based on the following facts: 1) according to our results and the corresponding ones (1, 3), the morphological characteristics such as length of the first antenna segment, presence of spongy grooves, ratio eye width: interocular distance, general body color, and denticles of femur are quite variable, 2) in our results the form of the male external genitalia varies according to the hours of treatment in phenol and guayacol, showing characters like either what described for n. flavida or for n. bruneri (3). based on the sequence of the gene 16s rrna, was found differences (ca. 1.37%) between n. flavida and n. bruneri (6). although there is no agreement about the amount of difference at the sequence level that would constitute a proof of distinction between species, the differences at the sequence level of the 16s rrna gene (not a protein coding gene but an rna structural gene with a slow rate of change), would not alone permit sustaining the j arthropod-borne dis, december 2017, 11(4): 446–452 o páez-rondón et al.: revalidation of … 451 http://jad.tums.ac.ir published online: december 30, 2017 thesis of different species. this kind of evidence is valid if it lends support to other characters like morphological, ecological, behavioral, etc. since there is no evidence of these differences, the distinction based solely on the 16s rrna gene sequence (6) is for the moment insufficient to classify n. flavida and n. bruneri as different species. the genetic variability of n. flavida and n. bruneri were analyzed by means of the rapd-pcr technique and found differences between the species (9). this technique is not valid to separate species, but rather is used for polymorphism analysis or intraspecific variation, and so found does not invalidate the synonymy between these two species. by means of an antenna phenotype discriminating analysis manage to separate n. flavida from n. bruneri (10), but since they do not find differences upon comparing the same sex between the two species, the separation should not be taken as definitive, but rather subject to later molecular, ecological, and morphological analyses to clarify the taxonomic status of both species. given the ambiguity of the differences found with the discriminating analysis of the antenna phenotype reported by these authors, we also consider that their results do not constitute evidence that invalidates the synonymy between n. flavida and n. bruneri. based on the foregoing exposition, we consider that maintaining the synonymy between n. flavida and n. bruneri is more solidly justified than considering them different species. the synonymization of n. bruneri with n. flavida is of utmost importance to public health in cuba because the country ceases to have four species of triatomine [bolbodera scabrosa, n. bruneri, n. flavida, t. rubrofasciata] and has only three species now, being n. flavida with wide geographic distribution. furthermore, the synonymization allows clarifying evolutionary questions, as for example the occupation of the antilles by this species associated with a species of rodent about 14.8 to 18.8 ma (7, 11). conclusion based on the body external morphology and the male external genitalia of n. flavida it is concluded that n. flavida and n. bruneri are synonymous species. acknowledgements to cdchta-ula project c-1932-1503-a. the authors declare that there is no conflict of interest. references 1. usinger rl (1944) the triatominae of north and central america and west indies and their public health significance. pub health bull. 288: 81–83. 2. usinger rl (1946) notes on cuban triatominae. pan-pacific ent. 22: 19–20. 3. lent h, jurberg j (1981) as especies insulares de cuba do genero triatoma laporte (hemiptera, reduviidae). rev brasil biol. 41: 431–439. 4. lent h, wygodzinsky p (1979) revision of the triatominae (hemiptera: reduviidae), and their significance as vectors of chagas disease. bull am mus nat 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arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 61 short communication malaria situation and anopheline mosquitoes in qom province, central iran *b farzinnia1, a saghafipour2, mr abai3 1department of environmental health, school of public health, qom university of medical sciences, qom, iran 2health center of qom province, qom university of medical sciences, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran (received 10 mar 2010; accepted 27 dec 2010) abstract background: the aims of this study was to analysis the current situation of malaria and to find the distribution of anopheline mosquitoes, as probable vectors of the disease, in qom province, central iran. methods: this study was carried out in two parts. first stage was data collection about malaria cases using recorded documents of patients in the province health center, during 2001–2008. the second stage was entomological survey conducted by mosquito larval collection method in 4 villages with different geographical positions in 2008. data were analyzed using excel software. results: of 4456 blood slides, 10.9% out were positive. most of cases were imported from other countries (90.4%), mainly from afghanistan (56.5%) and pakistan (16.3%). slide positive rate showed a maximum of 16.9% and a minimum of 2.9% in 2008 and 2007, respectively. plasmodium vivax was causative agent of 93.75% of cases, followed by p. falciparum (6.25%). more than 15 years old age group contained the most malaria reported cases (66.7%). two anopheles species, an. superpictus and an. claviger were collected and identified. this is the first report of anopheles claviger in qom province. conclusion: malaria is in the control stage in qom province. the rate of local transmission is very low (only 1 case), shows anopheles superpictus, as the main malaria vector of central part of iran, can play its role in malaria transmission in the area. keywords: malaria, iran, epidemiology introduction malaria is one of the most important infectious diseases in tropical and subtropical regions of the world. the last data show 225 million cases of malaria resulted to about 781000 death in 2009 (who 2010). malaria eradication program that started in 1956 in iran changed to malaria control program from 1980, restricted the local transmission of this disease, so that at present it occurs only in 3 southeastern provinces of sistan and baluchistan, hormozgan and kerman provinces and 95% of cases in 2007 reported from these provinces (raeisi et al. 2009). multiplicity of malaria vectors and their various behaviors, resistance of the main vector an. stephensi liston as well as an. culicifacies to some insecticides, absence of suitable transportation roads to some remote villages, structure of living houses, socioeconomic conditions of people, immigration from malarious neighbor countries (afghanistan and pakistan) and some other operational problems are the present problems for malaria control program in iran. according to the latest checklist of iranian mosquitoes (azari-hamidian 2007) there are 28 anophe*corresponding author: mr babak farzinnia, e-mail: b-farzinnia@muq.ac.ir iran j arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 62 les species in iran, 7 out of them are well known as malaria vectors in the country: an. stephensi, an. culicifacies, an. fluviatilis and an. dthali in the southern part, an. maculipennis and an. sacharovi in north and north-west, and an. superpictus in central part, although it is distributed in nearly all parts of iran, except for coastal area of caspian sea (saebi 1987, manouchehri et al. 1992, edrissian 2006). during the 1994–95 an outbreak of plasmodium vivax malaria appeared in the northwest parts of the country in parsabad, ardebil province (arshi et al. 2002, edrissian 2006). anopheles sacharovi was is responsible for malaria maintaining in this area (yaghoobi-ershadi et al. 2001). at present the disease in this area is under control. besides, the risk of re-emergence of malaria exists in other parts of the country. iran showed evidence of a sustained decrease in the number of cases, more than 50% since 2000, associated with wide scale implementation of malaria control activities and is classified in the pre-elimination phase of malaria (who 2010). qom province located in the central plateau region of iran, where have lower risk of malaria infection compare to southern/southeastern parts. situation of this province increases its risk for malaria, because many passengers/pilgrims from malarious area visit this province and stay for sometime. the aims of this study was to analysis the current situation of malaria and to find the distribution of anopheline mosquitoes, as probable vectors of the disease, in qom province, central iran. materials and methods study area qom is one of the 30 provinces of iran, between 50o 06’–51 o 58’ e and 34 o 09’–35 o 11’ n with 11,237 km², covering 0.89% of the total area in iran. it is in the northern part of the country, and its provincial capital is the city of qom (fig. 1). based on the last census in 2005, this province had a population of approximately 2,000,000 out of which 91.2% resided in urban areas and 8.8% in rural vicinities. the province contains one city, five counties, nine rural districts, and 256 villages. the climate of qom province varies between a desert and semi-desert climate, and comprises mountainous areas, foothills and plains. due to being located near an arid region and far inland, it experiences a dry climate, with low humidity and scanty rainfall. the minimum and maximum temperatures in 2008 were -1.9 in january and 37.4 in june, respectively. the annual rainfall in this year was 114.9 mm. relative humidity was ranged between 10.2 in june and 79.3 in february (iran meteorological organization). fig. 1. position of qom province in iran (left) and its geographical situation (right) iran j arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 63 malaria information all data about malaria cases during 20012008 were obtained from disease prevention unit, qom province health center. a databank was created in excel software. entomological survey in order to determine the anopheline mosquito fauna of qom province, 4 villages with different geographical positions and weather conditions were selected: agholak (qahan district), ahmadabad (dastjerd district), rahjerd (qomrood district) and emamzadeh esmail (kahak district). mosquito larval collection was conducted biweekly during april to november 2008, using dipping standard method of who. larvae were collected from riversides which had small vegetation, and conserved in lactophenol medium. in the laboratory, the specimens were mounted in de faure’s medium and identified using illustrated keys for iranian mosquitoes (shahgudian 1960, azari-hamidian and harbach 2009). results during the study period, 448 malaria cases were detected and reported in qom province. out of them 420 cases were infected by plasmodium vivax, while 28 cases were due to p. falciparum (table 1). based on recorded data only one case was due to local transmission and others were imported. this case was an iranian man who lived close to 15 families of foreign religious sciences students. most of cases observed among passengers who came to qom from other countries (90.4%) and different parts of iran for pilgrimage and also studying the religious sciences. afghani patients had the highest rate of infection (56.5%), followed by pakistani peoples (16.3%) and other nationalities such as sudanese, yemeni and so on (17.6%). reported cases were higher in males (63.8%) than females (36.2%). analysis of cases based on age showed 11.6%, 21.6% and 66.8% were in 0-4, 5-15 and more than 15 yr old age groups, respectively. most of cases were reported in august and september. plasmodium vivax was dominant parasite species and the trend of the disease had a decreasing pattern (fig. 2). during april–november 2008, a total of 223 anopheles larvae were collected and identified from collection stations. anopheles claviger was dominant species (78.9%) and an. superpictus had the second frequency (21.1%). the activity of these species started from june and finished at december with a peak at september for an. claviger (fig. 3) and august for an. superpictus (fig. 4). anopheles claviger is more distributed than an. superpictus in qom province, and found in three rural districts of dastjerd, qahan and kahak, while an. superpictus was only collected from kahak. in qomrood district we found only culicinae larvae. 0 20 40 60 80 100 120 140 160 2001 2002 2003 2004 2005 2006 2007 2008 year n o. p.vivax p.falciparum fig. 2. malaria cases based on parasitic species, qom province, 2001-2008 iran j arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 64 apr may jun jul aug sep oct nov dec jan feb mar months n o. pe r 10 di pp er dastjerd qahan kahak qomrood fig. 3. monthly activity of anopheles claviger larvae in qom province, 2008 apr may jun jul aug sep oct nov dec jan feb mar months n o. p er 10 di pp er dastjerd qahan kahak qomrood fig. 4. monthly activity of anopheles superpictus larvae in qom province, 2008 iran j arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 65 table 1. malaria information in qom province, 2001–2008 nationality sex age groups species residenceplace y ears a t risk population iranian non-iranian male f em ale <5 5–14 14< p. vivax p . falciparum m ix t otal slides t otal cases a p i a sp r a b e r a v i a f i city village 2001 917080 15 136 83 68 29 43 79 149 2 0 972 151 0.00016 0.155 0.0010 0.153 0.002 105 46 2002 942045 9 105 59 55 15 36 63 97 17 0 815 114 0.00012 0.139 0.0008 0.119 0.020 56 58 2003 967193 4 47 36 15 4 5 42 45 6 0 741 51 0.00005 0.068 0.0007 0.060 0.008 43 8 2004 991993 3 54 47 10 0 3 54 57 0 0 661 57 0.00005 0.086 0.0006 0.085 0 45 12 2005 1018997 4 18 16 6 1 0 21 22 0 0 233 22 0.00002 0.094 0.0002 0.094 0 18 4 2006 1046737 3 16 17 2 0 6 13 19 0 0 500 19 0.00002 0.038 0.0004 0.038 0 4 15 2007 1074475 4 8 8 4 1 1 10 10 2 0 404 12 0.00001 0.029 0.0003 0.024 0.005 12 0 2008 1102948 1 21 20 2 2 3 17 21 1 0 130 22 0.00002 0.169 0.0001 0.161 0.008 21 1 total --43 405 286 162 52 97 299 420 28 0 4456 448 -----304 144 api = annual parasite incidence, aspr = annual slide positive rate, aber = annual blood examination rate avi = annual vivax incidence, afi = annual falciparum incidence iran j arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 66 discussion in the entomological survey we found an. superpictus and an. claviger were active in the study area. saebi (1987) reported only an. superpictus from qom area, so this is the first report of an. claviger for qom fauna. a study on malaria situation in kashan city, close to qom reported three anopheline mosquitoes, an. superpictus, an. claviger and an. molticolor in that area (doroudgar et al. 1999). the human blood indexes (hbis) in an. superpictus in iran was reported 4.9 (edrissian et al. 1985). this show an. superpictus has antropophilic behavior, although it is an exophilic species. this species is introduced as malaria vector in central plateau of iran and can play its role in local transmission of malaria in qom province under favorite conditions. the activity peak of this anopheles in the area is the same with malaria disease occurrence. so it can be an alarm for malaria transmission in the area. natural sporozoite infections of an. claviger have been recorded in syria, cyprus, mesopotamia, and taranto in italy (muir and keilany 1972). more studies are recommended on this species in iran to clear its antropophilic index and probable role in malaria transmission. results of this survey show malaria is at control stage in qom province, like many other provinces of the country. trend of this disease was decreased up to 2007, but increased after that in 2008 (fig. 2). although all reported cases in this province were imported, the history of malaria in this province shows one indigenous case in 2004. annual parasite index (api) decreased from 0.00016 in 2001 to 0.00001 in 2007, while annual blood examination rate (aber) is also decreased from 0.0010 to 0.0003 during this period (table 1). this show the surveillance system is not alert and only passive slides were taken. this may be due to low importance of malaria in qom province. most of patients were non-iranian, so it is recommended to take blood slide from all foreign students that mainly came from endemic countries for malaria, to improve the surveillance system of malaria detection and treatment. qom province has thousands passengers from all parts of the country as well as other countries like afghanistan, pakistan, yemen, sudan, djibouti and other malarious countries, so imported cases can play their role as malaria parasite reservoirs in the area and local transmission can occurs in suitable conditions. results of this study show only 0.2% of recorded cases were due to local transmission. survey on the epidemiology of malaria in kashan province, bordered with qom, showed 95% of positive cases were afghani immigrants (doroudgar et al. 1999). a similar study in hamadan province, western of iran, shows 2.56% of reported cases during a 20 yr period were due to local transmission (fallah et al. 2003). in addition, in mazandaran province, northern iran, during 19992003; 13.7% of cases had no history of travel to other parts of the country (najafi et al. 2006). results of a demographic study on malaria in kohgiloye and boyer ahmad province, west of iran, showed 62.7% of cases were local iranian peoples (moshfe et al. 2003). this shows local transmission is occurred in this province, maybe due to favorable conditions for malaria vectors in that area. although the probability of local transmission of malaria in qom province is very low, it is not impossible. so we recommend improving the surveillance system for case detection and treatment by active slide preparing among foreign students of religious sciences as well as pilgrims coming from malarious countries. iran j arthropod-borne dis, 2010, 4(2): 61–67 b farzinnia et al.: malaria situation and … 67 acknowledgements the authors would like to thank dr a akbari, dr a abbasi, dr b mahmoudi and mrs f abedi astaneh their kind assistance and help. this study was financially supported by deputy of research, qom university of medical sciences. the authors declare that they have no conflicts of interest. references arshi sh, sadeghi h, mohebali m, sezavar sh, javan-roozegar a, emdadi d, sepahram v, amini-sani n (2002) malaria epidemiology in ardabil province, 19992000. ardabil university journal. 2(5): 28– 33. azari-hamidian s (2007) checklist of iranian mosquitoes (diptera: culicidae). j vec ecol. 32(2): 235–242. azari-hamidian s, harbach re (2009) keys to the adult females and fourth-instar larvae of the mosquitoes of iran (diptera: culicidae). zootaxa. 2078: 1–33. doroudgar a, dehghani r, hooshyar h, sayyah m (1999) epidemiology of malaria in kashan. j med faculty guilan univ med sci. 8(31–32): 52–58. edrissian ghh (2006) malaria in iran: past and present situation. iranian j parasitol. 1(1): 1–14. edrissian ghh, manouchehri av, hafizi a (1985) application of enzymelinked immunosorbent assay (elisa) for determination of the human blood index in anopheline mosquitoes collected in iran. j am mosq control assoc. 1(3): 349–352. fallah m, mirarab sa, jamalian sf, ghaderi a, zolfaghari a (2003) epidemiology of malaria in hamadan province during a 20 year period, 1980–2001. j behbood. 17(7): 36–44. manouchehri av, zaim m, emadi am (1992) a review of malaria in iran, 1957–1990. j am mosq control assoc. 8: 381–385. moshfe a, abolghasem hosseini sh, karimi z (2003) demographic pattern of malaria in kohgiloyeh and boyerahmad province in 1996-2003. j armaghan danesh. 8(31): 27–39. muir da, keilany m (1972) anopheles claviger meigen as a malaria vector in syria. who/ mal/72.757. najafi n, ghasemian r, farahmand m (2006) epidemiology of malaria in mazandaran province during 1999–2003. j mazandaran univ med sci, 50(15): 125–132. raeisi a, nikpoor f, ranjbar kahkha m, faraji l (2009) the trend of malaria in i.r.iran from 2002 to 2007. hakim res j. 12(1): 35–41. saebi me (1987) morphological study on anopheline larvae and their distribution in iran [phd dissertation]. school of public health, tehran university of medical sciences, iran. shahgudian er (1960) a key to the anophelines of iran. acta med iran. 3: 38–48. who (2010) world malaria report 2010. world health organization, geneva. 203 pp. yaghoobi-ershadi mr, namazi j, piazak n (2001) bionomics of anopheles sacharovi in ardebil province, northwestern iran during a larval control program. acta trop. 78: 207–215. j arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 1 http://jad.tums.ac.ir published online: march 14, 2017 original article comparative efficacy of topical pertmehrin, crotamiton and sulfur ointment in treatment of scabies *celestyna mila-kierzenkowska 1, alina woźniak 1, ewa krzyżyńska-malinowska 2, lucyna kałużna 3, roland wesołowski 1, wojciech poćwiardowski 4, marcin owcarz 5 1the chair of medical biology, collegium medicum of nicolaus copernicus university, bydgoszcz, poland 2department of cosmetology, torun higher school of business, toruń, poland 3department of cosmetology and aesthetic dermatology, collegium medicum of nicolaus copernicus university, bydgoszcz, poland 4institute of food technology, faculty of chemical technology and engineering, university of technology and life sciences, bydgoszcz, poland 5ludwik rydygier voivodship polyclinical hospital, toruń, poland (received 26 feb 2015; accepted 13 sep 2015) abstract background: scabies is an ectoparasitic infection, which occurs because of direct skin-to skin contact. the ideal treatment modality is still unclear and further research on this topic is warranted. the aim of the study was to compare the efficacy and safety of the topical scabicides: permethrin, crotamiton and sulfur ointment. methods: fifty four patients with diagnosed scabies were randomly divided into three treatment groups. the first group received 5% permethrin cream twice with one week interval, the patients from the second group were given crotamiton lotion for two days twice with one week interval, while the third group received 10% sulfur ointment for two or three weeks. all patients were followed up at 1, 2 and 4 weeks intervals. results: at one-week follow up the cure rate was significantly higher at permethrin-treated group when compared to crotamiton group (p< 0.001) and sulfur group (p< 0.001). at the end of two-week interval, the cure rate at permethrin group was 100%, while at crotamiton group, 66.7% and in sulfur group 38.9% (p< 0.001). at 4-week follow up the applied treatment was effective in all studied individuals. conclusion: the topical application of permethrin, crotamiton and sulfur was equally efficacious at 4-week follow up, however permethrin cream showed faster improvement at first and second follow up. acquiring permethrin is considered as expensive option and crotamiton lotion seems to be cost-less alternative to this cream. keywords: acaricides, skin diseases, parasitic infection introduction scabies is a contagious parasitic dermatitis that occurs among humans and other animals. the disease is caused by a tiny and usually not directly visible parasite, the mite sarcoptes scabiei (goldust et al. 2013a). scabies appears worldwide and is considered significant public health problem in the developing world. there are over 300 million cases of scabies reported annually worldwide (arif maan et al. 2015, thomas et al. 2015). the prevalence of scabies ranges from 2.2% in european and middle eastern countries to 71% in papua new guinea and the highest incidence of this infection is observed in the pacific and latin american regions as well as in aboriginal communities in northern australia (mclean 2013, romani et al. 2015). scabies affects regardless age, gender, race and social class, however, risk factors include poverty, poor nutritional status, homeless, dementia and poor *corresponding author: prof celestyna milakierzenkowska, e-mail: celestyna@o2.pl j arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 2 http://jad.tums.ac.ir published online: march 14, 2017 hygiene (shimose and munoz-prize 2013). the transmission occurs via direct skin-to-skin contact with an infected individual and it usually takes 15 to 20 min of close contact for successful transfer the mite to another person (chosidow 2012). the symptoms of scabies usually emerge up to 4 weeks following initial infestation (shimose and munoz-prize 2013). human scabies is characterized clinically by pruritus with nocturnal exacerbation and scabietic nodules, and visible skin burrows can be the pathognomic lesions of scabies (hicks and elston 2009). classic locations of burrows are the interdigital spaces of the hand, the flexural surface of the wrist, elbows, genitalia, axillae, umbilicus, belt line, nipples, buttock, and penis shaft. among pediatric population, scabies can also affect the head, neck, face, palm and soles (andrews et al. 2009). there are few methods to diagnose the scabies to be sufficiently sensitive, cost effective and convenient. the “gold standard” for diagnosis of human scabies with 100% specificity is the identification of mites, eggs, or feces from scrapings of infested skin or by identification of mite burrows (walter et al. 2011). this method relies on physically locating parasite on the host, so it can have low sensitivity when mites are low in number. alternate diagnosis method for scabies include polymerase chain reaction (pcr), microscopic examination of koh prepared skin scrapings and dermoscopy, but they are still not easily applied to clinical or public health settings (fukuyama et al. 2010, park et al. 2012, golant and levitt 2012). no commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive (rampton et al. 2013). treatment of scabies is a relevant issue in infectious dermatology and is as important as making correct diagnosis. scabies is commonly treated with various medications called acaricides, but the treatment of choice is still controversial and the search for ideal scabicide is ongoing. an ideal medication should be effective against adults and eggs, easily applicable, non-irritating, non-toxic and economical. the mainstay of treatment of scabies is topical application of scabicidal agents, like permethrin 5% cream, lindane 1% lotion or cream, benzyl benzoate 10% and 20% lotion or emulsion, crotamiton 10% cream, precipitated sulphur 2–10% ointment, ivermectin 0,8 % cream and others (karthikeyan 2005). nowadays, oral administration of ivermectin is being increasingly used (mounsey and mccarthy 2013). despite the availability of effective therapeutics, treatment failures still occur, mostly secondary to application error or failure to decontaminate fomites (golant and levitt 2012). the aim of the study was to compare the efficacy and safety of topical 5% permethrin cream vs. crotamiton lotion and 10% sulfur ointment in the treatment of scabies. materials and methods this single blind, randomized trial was conducted in patients with newly diagnosed scabies, of either gender, who were older than 18 years of age and voluntary agreed to participate in the study. diagnosis of the disease was based on clinical symptoms and clinical history. for inclusion, the patients had to satisfy the four criteria, like the demonstration of classical burrows, presence of typical scabietic lesions at the classical sites (fig. 1), complaint of nocturnal pruritus and family history of similar illness. exclusion criteria included age under 18 years, history of allergy to any of the studied drugs, pregnancy or lactation, women planning for conception in near future as well as history of severe systemic disorders, like cardiac disorders, nervous system disorders, psychiatric illness and immunosuppressive disorders. participants with abnormal kidney and liver function and known chronic infectious diseases were also excluded. j arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 3 http://jad.tums.ac.ir published online: march 14, 2017 before entry to the study, patients were given a physical examination and their history of infestations; antibiotic treatment and other information were recorded. participants with any other coexisting skin disease, which could interfere with subsequent monitoring of scabies and patients with atypical presentations, as crusted scabies were excluded from the trial. none of the patients participating in the trial had been treated with pediculicides, scabicides or other topical agents in the month preceding the trial. the studied group consisted of total 54 patients (22 males and 32 females) from 19 to 83 years of age. the patients were allocated to any of three treatment groups with simple randomization. all participants received detailed information about proper administration of the used drug. the patients included into the first group (8 men and 10 women) received single application of 5% permethrin cream at day 1 and they were asked to put a thin layer of cream to all areas of the body including the face and the scalp. the cream was washed off after 8 hours and the single application was repeated 1 week later if live mites were seen during one-week follow up. the second group (7 men and 11 women) was given topical crotamiton lotion and they were told to apply the drug to the entire skin surface, rinse off after 24 hours and then reapply for an additional 24 hours. the procedure was repeated at all individuals twice, with one-week interval. when there was no cure in two weeks, 2nd treatment was given with 10% sulfur ointment. the third group (7 men and 11 women) received 10% sulfur ointment for the period of two or three weeks (if there were no cure at 2-week follow up). ten percent precipitate sulfur in petroleum base was used and as other topical drugs, it was thoroughly rubbed into the skin over the whole body covering neck to toe. the patients were asked to wash off the ointment after 24 hours and then reapply the drug every 24 hours for two (or three) weeks with a bath taken between each application. the treatment was given to both patients and their close family members, even without symptoms, at the same time. the participants of the study were also asked not to use any antipruritic drug or any other topical medications. on entry of the study baseline, clinical parameters were comparable and the number of patients in each treatment group who were graded as having mild, moderate or severe infestation was not statistically different. the clinical evaluation after treatment was made by experienced investigator who was blinded to the treatments received. patients in all the three groups were followed up at intervals of 1, 2 and 4 weeks to assess compliance and to examine clinically the patients to evaluate efficacy and safety. at each assessment, the investigator recorded the count of the lesions and grading of pruritus both subjectively and objectively by the patients as described on the first visit. any adverse events were also recorded. the patient was considered as “cured” in case of the absence of new lesions, clinical improvement in the skin lesions and the improvement in the pruritus assessed by the visual analogue scale. “re-infestation” was defined as a cure at 2 weeks but development of new lesions at 1-month followup. treatment would be considered as failure if at the end of 4 weeks there was no improvement in the skin lesions and pruritus. the study was approved by the appropriate bioethics committee (number kb 135/ 2014) and written informed consent was obtained from all the patients. the percentage of improvement was compared between groups using the χ2 test followed by post-hoc tukey’s test and p< 0.05 was considered significant. spss software (ibm spss statistics 21) (chicago, il, usa) was used for all the analysis. results at one-week follow up in permethrin treated j arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 4 http://jad.tums.ac.ir published online: march 14, 2017 group the treatment was effective in 11 of 18 patients (61.1%) and in none of patients in crotamiton and sulfur group (fig. 2). thus, at first follow up the cure rate was significantly higher after the single application of 5% permethrin cream when compared to the use of crotamiton lotion (p< 0.001) and 10% sulfur ointment (p< 0.001). at the end of twoweek interval, the cure rate at permethrin group increased to 100% (after repeating application at remained 7 patients who had infestations at one-week follow up) and after two weeks of treatment none of patients included to this group still had severe itching and skin lesions. in crotamiton group, at the second follow-up, the cure rate was 66.7% (12 of 18 patients) compared with cure rate of 38.9% (7 of 18 patients) in the sulfur ointment group (p< 0.001). after two weeks of treatment the remaining 17 patients (6 in crotamiton group and 11 in sulfur group), who still manifested with scabietic lesions were treated with 10% sulfur ointment for the next week and the infestation was cured at all of them after three weeks of treatment (fig. 2). hence, at 4-week follow up the applied treatment was effective in all studied individuals. all treatment modalities studied in this paper were considered cosmetically acceptable and well tolerated by all patients. none of participants experienced allergic reactions and no major adverse events were observed in any of the 3 groups. the main adverse event was skin dryness, reported by patients treated with sulphur ointment longer that two weeks, but it was not serious and did not affect compliance. none of the patients experienced worsening of infestation during the study, but at two patients treated with sulfur ointment re-infestation occurred at one-month follow-up. fig. 1. characteristic skin lesions in adult with classic scabies 0 10 20 30 40 50 60 70 80 90 100 after 7 days of treatment after 14 days of treatment after one month of treatment % o f c ur ed p at ie nt s permethrin crotamiton sulphur fig. 2. the cure rate at studied patients with scabies treated with 5% permethrin cream, crotamiton lotion and 10% sulphur ointment j arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 5 http://jad.tums.ac.ir published online: march 14, 2017 discussion our study compared the use of 5% permethrin cream with that of crotamiton lotion and 10% sulfur ointment. the obtained results demonstrate that 5% permethrin cream is the most effective drug at 1 week follow up in treating scabies, what is in accordance with previous studies that have reported excellent cure rates with permethrin. in our study single application of permethrin resulted in improvement at 61.1% of patients, while in a study carried by usha and gopalakrishnan (2000) even a higher number of patients (97.8%) showed clearance of lesions at oneweek follow up as compared to our results. bachewar et al. (2009) found that at one week follow up, permethrin had significantly better cure rate than ivermectin, but at the end of two-weeks treatment this finding was reversed. taplin et al. (1990) in a double-blinded, randomized study compared crotamiton 10% cream and permethrin 5% cream for the treatment of scabies in children 2 months to 5 years of age. two weeks after a single overnight treatment, 30% children were cured with permethrin, in contrast to only 13% of subjects treated with crotamiton. four weeks after treatment the efficacy in favor of permethrin was still statistically significant and this agent also demonstrated greater effectiveness in reducing pruritus and secondary bacterial infections. a single overnight topical application of 5% permethrin cream is superior to a single dose of oral ivermectin (usha and gopalakrishnan 2000). permethrin being both miticidal and ovicidal appears to be more effective than for example crotamiton, which though is effective for adult stages of the mite, is not known to kill eggs and larvae. thus, single application of crotamiton may be not accurate to eradicate the parasite and a second dose is needed within 1 to 2 weeks for 100% cure. the study of pourhasan et al. (2013) demonstrated, in turn, that in the treatment of scabies 5% permethrin cream was as effective as 10% crotamiton cream at two week follow-up. in our study at the second follow-up the cure rate for permethrin group was 100% and in crotamiton group 66.7%, while after 4 weeks, considered the definitive point for evaluating the efficacy of applied treatments, 5% permethrin cream was found to be as effective as crotamiton lotion and 10% sulfur ointment in treating scabies. this is in accordance with few reports about the comparable efficacy of treatment with permethrin cream and other scabicides, like topical 1% ivermectin (goldust et al. 2013b), topical tenutex emulsion (containing among others disulfiram and benzyl benzoate) (goldust et al. 2013c) as well as oral ivermectin (ranjkesh et al. 2013). sharma and singal (sharma and singal 2011) in randomized double-blind controlled study evaluated the efficacy and safety of topical 5% permethrin and oral ivermectin in single and two dose regimens in treatment of scabies. their study revealed that all three-treatment modalities had the equal efficacy at the end of 4 weeks of treatment. moreover, chhaiya et al. (2012) reported that topical ivermectin as 1% lotion is as effective as 5% permethrin, but significantly more effective than oral ivermectin. on the other hand, abedin et al. (2007) found that mass treatment of scabies with 2 doses of oral ivermectin in an endemic pediatric population is more efficacious than single topical application of 5% permethrin. oral ivermectin produced also significantly better cure rate than lindane 1% lotion at 4 weeks follow-up (mohhebipour et al. 2013). the authors suggest that oral ivermectin can be alternative treatment at patients with scabies, for whom topical therapies can cause serious cutaneous and systemic problems. ivermectin is generally considered as effective as permethrin and more effective than other medications, such as lindane, benzyl benzoate, j arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 6 http://jad.tums.ac.ir published online: march 14, 2017 crotamiton and malathion (panahi et. 2015). moreover, miyajima et al. (2015) described a novel method for scabies treatment called “whole-body bathing method”. in this method, the patients would bathe themselves in a fluid containing ivermectin at an effective concentration. the selection of a drug is often based on the personal preference of physician, local availability and cost for the patient, rather than on medical evidence (wolf and davidovici 2011). in the present study, a 100% cure rate was obtained at all three studied groups after one month of treatment what suggest that all applied modalities were equally efficacious. however, permethrin cream has more rapid onset of action as at the first follow up, the patients from permethrin group reported better improvement than the patients included to the crotamiton and sulfur ointment group. permethrin is a first-line acaricide in many countries due its high effectiveness against mites and low mammalian toxicity. one treatment with permethrin cream is usually effective in eradicating scabies, but some experts recommend retreatment one week later (strong and johnstone 2007). the significant limitation in the use of permethrin in treatment of scabies is its cost, as it is the most expensive drug of all topical scabicides (roos et al. 2001). crotamiton (crotonyl-n-ethyl-o-toluidine) as 10% lotion or cream is approved for use in adults with scabies (pourhasan et al. 2011). the best results seem to be obtained when the drug is applied twice daily for five consecutive days after bathing and changing clothes. goldust et al. (2014) demonstrated that such application of crotamiton was as effective as single dose of invermectin at two-week follow up. however, some authors do not recommend crotamiton because of the lack of efficacy and toxicity data (meinking 1999). sulfur, used as an ointment (2%-10%), is the oldest scabicide in use (karthikeyan 2005). topical sulphur ointment is messy, malodourous, stains clothing, and in hot and humid climate, it may lead to irritant dermatitis (taplin and meinking 1988). however, it has the advantage of being very cheap and may be the only alternative at patients whose financial state dictates the choice of this antiscabietic drug due to its low cost. moreover, it is recommended as a safe agent in a treatment of infants, children and pregnant women (chosidow 2012). sharquie et al. (2012) evaluated the therapeutic regimen of 8% and 10% topical precipitated sulfur in petrolatum ointment for single day, three successive nights or three successive days at total 97 patients with scabies and they revealed that single-day application was much less effective than three-days treatment. in the group of participants treated for single day, only 42.4% participants responded to management, while in group who received sulfur ointment for three successive nights the response was observed in 90.6% and in those who received the treatment for three successive days it was 96.9% of patients. however, at patients who received only single dose of sulfur ointment fewer side effects were observed. in our study, 10% sulfur ointment was effective at 6 of 18 patients after two-weeks treatment and in remained 11 of patients after three weeks of daily application. although this drug is well tolerated in most of the patients the major problem of the topical scabicides, life sulfur ointment, is the requirement for repeated application because of their relative low efficacy. on the other hand, the cost is still the lowest. our study also evaluated the safety of application of various topical agents in treatment of scabies. oberoi et al. (2007) investigated the effect of topical application of 1% lindane lotion and 5% permethrin cream on oxidant-antioxidant balance in blood of patients with scabies and they found that permethrin, in contrary to lindane, showed no significant alteration in oxidative stress markers. the medication applied at studied paj arthropod-borne dis, march 2017, 11(1): 1–9 c mila-kierzenkowska et al.: comparative efficacy … 7 http://jad.tums.ac.ir published online: march 14, 2017 tients of all three groups were well-tolerated by most of the patients and no serious adverse effects were observed in the course of treatment suggesting that all studied agents are safe and non-toxic. conclusions the topical application of 5% permethrin cream, crotamiton lotion and 10% sulfur ointment was equally efficacious at one-month follow up. nevertheless, complete clearance of scabietic lesions occured earlier in pertmehrintreated group than in crotamiton and sulfur groups. no side effects and no re-infections were observed both after administration of permethrin and crotamiton. although permethrin seems to be the most effective drug in the treatment of scabies, its administration is much more expensive for patients that other common creams. thus, we conclude that crotamiton may be a cost-effective alternative to permethrin with acceptable cure rate in the treatment of sarcoptes scabiei infection. acknowledgements we thank to all the volunteers who participated in the study and we declare no financial support for the project. the authors declare that there is no conflict of interests. references abedin s, narang m, gandhi v, narang s (2007) efficacy of permethrin cream and oral ivermectin in treatment of scabies. indian j pediatr. 74(10): 915– 916. andrews rm, mccarthy j, carapetis jr, currie dj (2009) skin disorders, including pyoderma, scabies, and tinea infections. pediatr clin n am. 56: 1421–1440. arif maan ma, arif maan ms, sohail am, afif m (2015) bullous scabies: a case report and 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walton sf, carson cf, naunton m, baby ke (2015) scabies: an ancient global disease with a need for new therapies. bmc infect dis. 15: 250–255. usha v, gopalakrishnan nair tv (2000) a comparative study of oral ivermectin and topical permethrin cream in treatment of scabies. j am acad dermatol. 42(2): 236–240. walter b, heukelbach j, fengler g, worth c, hengge u, feldmeier h (2011) comparison of dermoscopy, skin scraping and the adhesive tape test for the diagnosis of scabies in a resource poor settings. arch dermatol. 147: 468–473. wolf r, davidovici b (2011) treatment of scabies and pediculosis: facts and controversies. clin dematol. 28: 511–518. j arthropod-borne dis, march 2017, 11(1): 19–26 m sefikogullari et al.: increased levels of … 19 http://jad.tums.ac.ir published online: march 14, 2017 original article increased levels of vegf-a and hif-1α in turkish children with crimeancongo hemorrhagic fever murat sefikogullari 1, ali kaya 2, huseyin aydin 3, enver sancakdar 3, veysel kenan celik 3, *gokhan bagci 3 1pediatric clinic, private samandağ medical center, hatay, turkey 2department of pediatrics, faculty of medicine, cumhuriyet university, sivas, turkey 3department of biochemistry, faculty of medicine, cumhuriyet university, sivas, turkey (received 22 feb 2015; accepted 23 sep 2015) abstract background: crimean-congo hemorrhagic fever (cchf) is a disease characterized by serious course, including acute viral fever, ecchymosis, thrombocytopenia, liver dysfunction and high rate of mortality. hypoxia inducible factor-1α (hif-1α) and vascular endothelial growth factor-a (vegf-a) play an important role both in the inflammatory process and plasma leakage. the aim of this study was to define hif-1α and vegf-a serum levels obtained from cchf patients and control group and to investigate whether these factors were correlated with the pathogenesis of this disease. methods: thirty cases younger than 17yr confirmed by rt-pcr and/or elisa for cchf were included in this study. thirty age and sex matched healthy peoples were enrolled as controls. blood samples collected from the patient and control groups. serum levels of hif-1α and vegf-a were measured with elisa. results: levels of hif-1α and vegf-a were statistically significantly increased in cchf patients compared to the control group (p< 0.05). a significant positive correlation was found between the levels of hif-1α and vegf-a in the patient group (p< 0.01). the levels of alt, ast, ck, aptt, wbc and thrombocyte count were significantly higher in the patients than in the control group (p< 0.001). a positive correlation was found among the levels of ast and ck from biochemical parameters and vegf and hif-1α in the patient group (p< 0.05) conclusion: hif-1α and vegf-a might play an important role in cchf pathogenesis. keywords: crimean congo hemorrhagic fever, vegf, hif-1α, sepsis, children introduction crimean-congo hemorrhagic fever (cchf) is a serious disease occurring in case of infection by tick-borne virus (nairovirus) genus from bunyaviridae family (ergonul 2008). it begins with fever, severe headache, nausea, weakness, vomiting and progresses with bleeding in different parts of the body. “the bleeding findings emerge as subcutaneous bleeding, nasal bleeding, gingival bleeding and visceral bleeding” (bakir et al. 2005). mononuclear phagocytic cells, hepatocytes and endothelial cells are known as main target of cchf virus (whitehouse 2004, ergonul et al. 2004). cchf cases have been reported from more than 30 countries of asia, south-eastern europe and africa until now (hubalek and rudolf 2012). in turkey, annually more than 1,000 human cchf cases are reported (maltezou et al. 2010). like sepsis, endothelial damage plays an important role in the pathogenesis of cchf. endothelium may be indirectly targeted by virus mediated host derived soluble factors or directly by viral factors (schnittler and feldmann 2003). hypoxia inducible factor (hif-1α) is an oxygen sensitive transcription factor facilitating oxygen distribution and cellular adaptation to oxygen deprivation. hif1α activates transcription of genes, which en*corresponding authors: dr gokhan bagci, email: gokhanbagci@hotmail.com.tr j arthropod-borne dis, march 2017, 11(1): 19–26 m sefikogullari et al.: increased levels of … 20 http://jad.tums.ac.ir published online: march 14, 2017 code angiogenic growth factors, including vascular endothelial growth factor (vegf), angiopoietin 1 (angpt1), (angpt2), placental growth factor (pgf) and platelet-derived growth factor b (pdgfb), playing a critical role in angiogenesis. hif-1α regulates expression of certain genes related with erythropoiesis, energy metabolism, angiogenesis, cell proliferation and apoptosis (ferrara et al. 2003, podar and anderson 2005, manalo et al. 2005). vascular endothelial growth factor (vegfa), a member of platelet-derived growth factors super family is specific for the endothelial cells and has important effects. blood vessel formation is regulated mainly by vascular endothelial growth factor (vegf-a) in health and disease. human vegf gene family consists of five members of vegf a-e, among which vegf-a is also commonly referred to as vegf (ferrara 2003). vegf-a was also known as vascular permeability factor (vpf) due to its substantial effect on increasing of vascular permeability, resulting in a leakage out of blood vessels. one of the main aberrations in pathogenesis of cchf is vascular dysfunction resulting in leakage driven hemorrhagic manifestations (dvorak et al. 1995, hoeben et al. 2004, ergonul 2006). in this study, we aimed to demonstrate whether vegf-a and hif-1α was correlated with pathogenesis of sepsis in serum samples obtained from cchf patients and control group. materials and methods study design thirty patients diagnosed with cchf in department of pediatrics, faculty of medicine, cumhuriyet university, from 2010 to 2011 and 30 healthy controls were enrolled in the study. all of the patients with cchf used to live in the same endemic region (sivas and neighbor’s cities) which showed high prevalence in cchf. blood samples collected from the hospitalized patients with presumed diagnosis of cchf were sent to refik saydam hifzisihha center, virology laboratory. thirty patients having positive cchfv rna with reverse transcriptase-polymerase chain reaction (rt-pcr) and/ or positive cchfv-specific igm with elisa were included in the study. thirty age and sex matched healthy individuals who had not any infectious or metabolic diseases were enrolled as the controls. this study was approved by the local ethics committee of cumhuriyet university, faculty of medicine. measurement of hif-1α and vegf-a levels blood samples of 5 ml were collected from patient and control groups. the samples were centrifuged at 3000 rpm for 5 min and obtained serums were portioned and kept at -80 °c until the analysis time. then these samples were cooled to the room temperature at the same time and hif-1α and vegf-a levels were measured through elisa method. human/mouse total hif-1 alpha cell-based elisa (r and d systems, inc.) and vegf-a human (biovendor) kits were used to measure the levels of hif-1α and vegf-a. microelisa (sandwich) method was performed using full automatic triturus device. statistical analysis all statistical analyses were conducted with ibm spss statistics for windows, version 20.0 (chicago, il, usa) computer program. descriptive statistics were expressed as arithmetic mean (min-max). mann–whitney u test was performed to determine the significance of independent continuous variables. for bivariate correlation analysis, pearson’s or spearman’s test was used. in evaluation of the categorical data, chi-square test was used in the statistical analyses. p< 0.05 values were considered as statistically significant for all the tests. j arthropod-borne dis, march 2017, 11(1): 19–26 m sefikogullari et al.: increased levels of … 21 http://jad.tums.ac.ir published online: march 14, 2017 results demographic features and laboratory findings of the controls and patient groups are given in table 1. mean age was found as 11.30±4.41 in the patients and 10.40±4.49 in the controls. nineteen of the patients were male (63.3%) and 11 were female (36.7%), whereas 20 (66.7%) of the controls were male and 10 (33.3%) were female. the differences between the controls and patient groups in terms of age and gender were not significant. a statistically significant increase was observed in the levels of hif-1α and vegf in the patients compared to control groups (p< 0.05, table 2). a significant positive correlation was found between the levels of hif-1α and vegf-a in patient group (p< 0.01, table 2). a positive correlation was found among the levels of ast and ck from biochemical parameters and vegf and hif-1α (p< 0.05), while this was not significant in alt (p= 0.086, table 3). the levels of alt, ast and ck were statistically significantly higher in cchf patients than in the control group (p< 0.001). there was a significant increase in the levels of aptt in coagulation tests compared to controls (p=0.022), increase in the prothrombin time and inr was not statistically significant (p> 0.05 table 1). wbc (p< 0.05) and thrombocyte counts (p< 0.001) were statistically lower in the patients than in the control group, while no significant difference was observed in the amount of hemoglobin. table 1. comparison of demographic and laboratory findings of the patients and controls variable control (n=30) cchf (n=30) p age (mean±sd) 10.40±4.49 11.30±4.41 0.05 age range 2-17 2-17 0.05 male (%) 20 (66.7%) 19 (63.3%) 0.05 female (%) 20 (66.7%) 19 (63.3%) 0.05 alt (iu/l) 35 (12-48) 83 (28-335) <0.001 ast (iu/l) 38 (11-54) 171 (26-357) <0.001 ck (iu/l) 107 (68-172) 899 (140-1235) <0.001 prothrombin time (s) 14.4 (9.6-14.0) 15.9 (9.6-20.1) >0.05 aptt (s) 34.7 (13.5-45.7 42.4 (23.8-55.1) 0.022 inr 1.2 (0.8–2.2) 1.3 (0.9–2.4) >0.05 wbc (x109 cells/l) 5.7 (3.5–7.0) 3.17 (1.2-9.0) <0.05 hemoglobin (g/dl) 14.2 (8.3-15.8) 13.6 (11.8-15.8) >0.05 thrombocyte count (x103 cells/l) 176 (69-214) 110 (38-152) <0.001 alt: alanine aminotransferase ast: aspartate aminotransferase ck: creatine kinase aptt: activated partial thromboplastin time inr: international normalized ratio wbc: white blood cell table 2. serum levels of vegf-a and hif-1α in the patient and control groups patients control p vegf-a (pg/ml) 312.66±87.31 150.70±78.48 0.001 hif-1α (pg/ml) 195.93±63.49 68.20±36.11 0.001 j arthropod-borne dis, march 2017, 11(1): 19–26 m sefikogullari et al.: increased levels of … 22 http://jad.tums.ac.ir published online: march 14, 2017 table 3. hif-1α and vegf-a correlation between biochemical parameters in patients with cchf hif-1α vegf-a variable correlation coefficient p correlation coefficient p alt 0.181 0.086 0.162 0.103 ast 0.256 0.020 0.266 0.022 ck 0.214 0.012 0.292 0.048 vegf-a 0.969 <0.01 alt: alanine aminotransferase ast: aspartate aminotransferase ck: creatine kinase discussion we aimed to demonstrate whether hif-1α and vegf, related to sepsis and viral infection, were correlated with pathogenesis of cchf disease. despite numerous studies conducted about cchf, pathogenesis of the disease is not fully understood. there was a relationship between sepsis and viral infection, and hif-1α and vegf (irwin et al. 2009). in infected humans by the cchfv, damaging of endothelial cells and vascular leakage may be a direct result of an immune mediated indirect effect (schnittler and feldmann 2003). a common characteristic of viral hemorrhagic fevers is viruses to enter macrophages and dendritic cells, making a cytopathic effect. vegf produced by monocytes and endothelial cells is known to play an important role in breakdown of coagulation, leukocyte adhesion, angiogenesis and increased vascular permeability (burt et al. 1997, geisbert and jahrling 2004). it is known as a deteriorated coagulation, leukocyte adhesion, angiogenesis and increased vascular permeability in cchf patients (bodur et al. 2010). clinical symptoms of cchf are similar to sepsis and endothelial structure of vessels is retrograded (elson et al. 2001). the correlation of sepsis and viral infection with hif-1α and vegf-a was shown (kilani et al. 2004, zinkernagel et al. 2007). at hypoxic conditions, hypoxia-inducible factor (hif) regulates expression of certain genes. in in vivo and in vitro studies, oxygen tension affects virus production either up regulating or down regulating viral replication depending on virus type and method used for to study viral replication. viral replication is modulated mainly by hif-1α in normal and hypoxic conditions (morinet et al. 2013). hif-1α is known to activate transcription of the genes including vegf and other angiogenic growth factors and plays a critical role in the development of angiogenesis in hypoxic conditions (manalo et al. 2005). hif1α mrna expression was suppressed and conversely associated with disease severity in sepsis (schaefer et al. 2013). hif-1α expression was statistically significantly higher in the shock patients compared to the controls and the reasons of shock were sepsis (78%), hemorrhage (18%), and cardiac dysfunction (4%) (textoris et al. 2012). in sepsis, microvascular permeability closely associates with the increased plasma vegf concentration (pickkers et al. 2005). ros– hif-1α–vegf pathway is partly responsible for hypoxia-induced permeability (irwin et al. 2009). the levels of vegf increased in sepsis, and this increase correlated with severity of the disease and mortality (van der j arthropod-borne dis, march 2017, 11(1): 19–26 m sefikogullari et al.: increased levels of … 23 http://jad.tums.ac.ir published online: march 14, 2017 flier et al. 2005, yano et al. 2006, karlsson et al. 2008). inhibition of vegf signaling contributes the development of sepsis-induced organ dysfunction via blocking endothelial survival and increasing apoptosis. furthermore, in pathological situations like sepsis and cancer, vegf participates to mobilizing of endothelial progenitor cells (jesmin et al. 2012). the main target of sepsis-induced events is generally endothelium, so ability to ameliorate damaged endothelium is one of the main determiners of clinical outcome in septic patients. the angiogenic factors and their soluble receptors have a dual role and seem to play both beneficial and harmful effects during sepsis development and therapy. while normal levels of vegf is required for protection of endothelial function, both extremely high or low levels of vegf have disruptive effect on endothelial barrier (zhang et al. 2013). in our study, a strong correlation was seen between the levels of hif-1α and vegf-a in cchf patients (table 3). besides, a positive correlation was found between hif-1α and vegf-a, suggesting that angiogenesis could be triggered and, pathogenesis of the disease could be influenced. accordingly, we recommend that comprehensive studies related to angiogenesis should be conducted in cchf patients. despite the absence of studies that investigated hif-1α levels in patients with cchf in the literature, the studies that evaluating vegf levels have increased in recent years. all of the studies that investigate serum levels of vegf have been conducted with turkish cchf patients. serum levels of vegf was statistically higher in the adult patients having cchf compared to the controls (bakir et al. 2013), and in another study, levels of vegf was found statistically higher in fatal cchf patients compared to the non-fatal patients (ozturk et al. 2010). in contrast to these findings, level of vegf was significantly decreased in cchf patients (bodur et al. 2010). our study was not consistent with the study by bodur et al. while it was consistent with the other two studies. in studies conducted with other hemorrhagic fevers such as dengue hemorrhagic fever (dhf) (tseng et al. 2000, srikiatkhachorn et al. 2007, furuta et al. 2012, del moral-hernández et al. 2014) and hemorrhagic fever with renal syndrome (hfrs) (ma et al. 2012), also vegf level was statistically significantly higher in patient groups. consequently, all of these studies indicate that vegf regulates vascular permeability and vegf is a good marker of the severity of the infection by viral diseases such as cchf and dhf. our study has some limitations. first, we could not linked the levels of hif-1α and vegf-a together with disease severity. second, hif-1α and vegf-a serum levels were not compared between patients with survival and non-survival. lastly, we did not evaluate the levels of vegf-a and hif-1α in another viral disease -especially the other hemorrhagic feversor patients with sepsis. increased levels of the hif-1α which is the main transcriptional factor of the regulation of oxygen homeostasis and vegf-a which has a crucial role in the regulation of angiogenesis and vascular permeability suggested that these parameters might contribute to the development of vascular endothelial damage. nevertheless, because of the insufficient information about molecular mechanism on pathogenesis of cchf, our results indicate that vegf and hif-1α should be one of the markers need to be focused on the elucidation on the pathogenesis of cchf disease. conclusion significant higher levels of hif-1α and vegf-a in the cchf patients than in conj arthropod-borne dis, march 2017, 11(1): 19–26 m sefikogullari et al.: increased levels of … 24 http://jad.tums.ac.ir published online: march 14, 2017 trols, suggested that hif-1α and vegf-a expression might be effective on the pathogenesis of cchf patients, especially on the occurrence of endothelial damage and disruption of coagulation. combined use of hif-1α inhibitors with antiviral agents may be useful for the treatment of cchf patients. further comprehensive studies on this issue are recommended. acknowledgements the authors of study declare that they have no competing interests. references 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10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 429 http://jad.tums.ac.ir published online: october 04, 2016 review article control of phlebotomine sand flies in iran: a review article mohammad reza yaghoobi-ershadi department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 22 aug 2016; accepted 17 sep 2016) abstract leishmaniasis has long been known as a significant public health challenge in many parts of iran. phlebotomus papatasi and p. sergenti are the vectors of zoonotic cutaneous leishmaniasis and anthroponotic cutaneous leishmaniasis respectively, and 5 species of sand flies including p. kandelakii, p. neglectus, p. perfiliewi, p. keshishiani and p. alexandri are considered as probable vectors of zoonotic visceral leishmaniasis. a literature search was performed of the relevant multiple databases from 1966 to 2013 to include studies on sand flies, vector control, leishmaniasis, phlebotomus. sand fly control in iran began in 1966 by iranian researchers, and long-term evaluation of its effects was completed in the study areas of the country. herein, a review of vector control strategies in iran to combat leishmaniasis including indoor residual spraying, application of chemicals in rodent burrows, impregnation of bed nets and curtains with insecticides, the use of insect repellents, impregnation of dog collars and the susceptibility of sand fly vectors to various insecticides has been summarized thus far. the investigation of the behavioral patterns of the adults of different sand fly species, introduction of biological insecticide agents, the use of insecticidal plants and other novel strategies for the control of sand fly populations have received much attention in the areas of studies, hence should be recommended and improved since they provide optimistic results. keywords: leishmaniasis, phlebotomus, sand flies, vector control, iran introduction leishmaniasis remains a significant public health problem over a wide geographical area in iran. cutaneous and visceral leishmaniasis is historical endemic disease in the country. cutaneous leishmaniasis (cl) occurs in two forms, anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl). leishmania major, the causative agent of zcl, is endemic in many rural areas of 17 out of the 31 provinces of iran. the principal vector of zcl has been identified as phlebotomus papatasi which has a wide range of distribution, and four species of rodents belonging to the family cricetidae involving rhombomys opimus, meriones libycus, tatera indica and m. hurrianae are considered as the main reservoir hosts in different parts of iran. over the last three decades, the increasing number of zcl cases in the traditional foci and their transmission to new foci in the western, southern, southwestern (near the central desert), and northeastern regions of iran has been of major concern to the health authorities (yaghoobi-ershadi and javadian 1995, yaghoobi-ershadi et al. 2001, 2003, akhavan et al. 2007). anthroponotic cutaneous leishmaniasis has been a very old endemic disease in many parts of iran. the burden of the disease was greatly reduced in many foci following the introduction of antimalarial measures in iran, however, its foci remained active in some large and medium-sized cities such as tehran, mashhad, neishabur, and sabzvar in the northeast, shiraz in the south, kerman and bam in the southeast (nadim and tahvildari-bidruni 1977, *corresponding author: prof mohammad reza yaghoobi-ershadi, e-mail: yaghoobia@tums.ac.ir, yaghoobi.reza@gmail.com j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 430 http://jad.tums.ac.ir published online: october 04, 2016 yaghoobi-ershadi et al. 2002). leishmania tropica, recognized as the agent of acl, is currently prevalent in 14 foci located in 8 provinces. the vector responsible for the maintenance of l. tropica is p. sergenti, and human serve as the main reservoir host of the disease, however infected dogs play a crucial role as animal reservoir host of the parasite (yaghoobi-ershadi et al. 2002, 2008). leishmania infantum, the causative agent of zoonotic visceral leishmaniasis (zvl), has been reported sporadically from all areas of the country, however, seven endemic foci are located in the southern, southwestern, western, northwestern and northeastern regions of the country (nadim et al. 1978, nadim 2008). leishmania tropica has also been known to cause the disease in immunosuppressed patients (mohebali 2013). five species of sand flies including p. kandelakii, p. neglectus, p. perfiliewi, p. keshishiani, p. alexandri (yaghoobi-ershadi 2012) are considered as probable vectors of zvl. dogs, foxes, jackals and wolves have been found infected in different parts, but dogs are the main reservoir host (navid-hamidi et al. 1982, nadim 2008). the mean annual number of leishmaniasis cases has been projected as almost 20,000 in iran during 1983–2014. nearly eighty percent of these cases are of the zcl form, 0.5% classified as zvl, and the rest are acl (yaghoobi-ershadi and javadian 1995, yaghoobi-ershadi et al. 2001, akhavan et al. 2007). based on proven epidemiological studies, the provinces of ilam in the west, fars in the south and khorassanerazavi in the northeast have recorded the highest incidence of cl, ranging between 59.9–98.8 cases per 100,000 inhabitants (shirzadi et al. 2015). official publications have revealed the existence of 48 species of sand flies in the country, 30 species belonging to the genus phlebotomus, and 18 speicies of the genus sergentomyia (yaghoobi-ershadi 2012, zahraei-ramazani et al. 2013, 2015). the practical application of sand fly control measures has been appraised in most old world situations (alexander and maroli 2003). indoor residual spraying (irs) with ddt (dichlorodiphenyltrichloroethane) against malaria vectors in india during the 1950s and again in the 1970s was effective in reducing the density of p. argentipes and visceral leishmaniasis (vl) cases, but after the cessation of irs, sand fly population and incidence of vl increased (kishore et al. 2006, thakur 2007). indications of the impact of ddt spraying on sand flies were obtained from a trial conducted in a few coastal villages in ramanathapuram district, tamil nadu of india during 1953–1954. no sand flies could be collected from villages sprayed with ddt at a rate of 1g/m2 and 2g/m2 for seven and 21 months respectively (who 1980). ddt and bhc (benzene hexochloride) have been used extensively in the former soviet union for sand fly control (perfiliev 1966). in a field trial in china, a village sprayed at a dosage of 1.58gr ddt/m2 against p. chinensis in 1951 was protected by a subsequent reduction of sand fly up to eight years, while in another village sprayed in the same year at a dosage of 0.128gr gamma hch/m2, the sand fly density showed an annual increase until it reached the level of density recorded in an unsprayed area in 1956. in a further trial a mixture of both insecticides was applied in a village in 1953, and very few sand flies were observed for six years after spraying (wang and wu 1959). in palestine, irs with ddt provided more than 50 days of protection against sand flies (jacusiel 1947). during 2003–2004, a residual spraying program combined with the distribution of permethrin-treated bed nets in the thi qar governate of iraq resulted in a significant reduction in vl cases, however, no information was reported on the insecticides used or any impact of treatment on the density of sand flies (jassim et al. 2006). j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 431 http://jad.tums.ac.ir published online: october 04, 2016 methods the electronic databases including pubmed, web of science, literature retrieval system of the armed forces pest management board, google scholar and medline were searched from 1966 to 2013 using the term iran in combination with the keywords: sand flies, vector control, leishmaniasis, phlebotomus. a special emphasis was performed to refer to published original articles within the last 3 decades in international scientific journals that deal with vector control of sand flies in iran. control strategies indoor residual spraying leishmaniasis control has been a matter of interest by different researchers at the institute of public health, university of tehran, in coincidence with the beginning of epidemiological studies on cl in 1965. table 1 summarizes the comparison results for the intervention against leishmaniasis vectors in iran during 1966–2010. the effect of antimalarial spraying on sand fly populations and the incidence of zcl has been evaluated in esfahan province by nadim and amini in 1970. they found that ddt spraying applied at a dosage of 2g/m2 in april in both 1966 and 1967 reduced zcl incidence, but the transmission of the disease was not interrupted. as the incidence in 19 sprayed villages was 3.8 per 1000 in the first year, the incidence in the second year was calculated to be 2.7 per 1000. in the control villages, the corresponding incidence was highlighted as 14.4 and 10.6 per 1000 respectively. collection of sand flies in sprayed rooms, with the exception of the ones collected which gave negative results, while sticky paper traps placed in the same rooms gave positive results from june to the end of season, indicated that p. papatasi has invaded the sprayed rooms. additional evidence was collated from another group of sprayed villages, which showed that the number of sand flies collected from nearby rodent burrows was similar to that recorded before spraying. the abundance of sand flies, and the consequent exposure of people to their bites as they sleep on roofs, could explain how transmission persisted despite house spraying. long-term evaluation of the effect of antimalarial ddt house spraying on the incidence of zcl was completed during 1966– 1971 in esfahan, central iran, the results of which were summarized by seyedi-rashti and nadim (1973) and given in more details by the same authors in 1975. the following groups of villages were kept under surveillance (monthly or every three months) since 1963 for the detection of zcl cases from which the annual incidence was calculated: group i, 16 villages sprayed annually with ddt throughout 1966–1969, group ii, three villages also sprayed annually with ddt during 1966–1967, and group iii, four villages kept without spraying for comparison. the annual incidence per 1000 population in group i which was 8.2 and 4.6 respectively in 1964 and 1965 before spraying decreased to 0.7 in 1969 i.e. after four years of spraying; following the cessation of spraying in 1970, the incidence increased sharply to 15, after which a further increase was recorded in 1971. in group ii, which was surveyed under irs for two consecutive years, the incidence decreased from 15.9 in 1965 to 2.9 and 5.7 in 1966 and 1967 respectively, but steadily increased after the discontinuation of spraying, reaching 17.5–19.7 during 1969–1971. in group iii (comparison group) the annual incidence per 1000 ranged between 9–21 during 1964–1970 reaching 46.3 in 1971 when an epidemic of zcl broke in one of the villages. analysis of the age distribution of children's cases in previously sprayed and comparison villages indicated that once spraying was stopped, transmission was vigorously resumed through the abunj arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 432 http://jad.tums.ac.ir published online: october 04, 2016 dance of rodent reservoirs and sand flies in the vicinity of villages, striking the children who were protected during the years of spraying. from these observations, it was concluded that house spraying cannot be considered a permanent measure for the control of zcl in an endemic area, and the feasibility of alternative control measures involving reservoir hosts and immunization should be sought. nadim et al. (1977) indicated that two rounds of antimalaria spraying with malathion (2g/m2) reduced the level of endemicity of zcl greatly in khuzestan province, southwest of iran, thus epidemiological studies have become difficult. the absence or scarcity of sand flies resting in sprayed rooms was taken as an indication of their susceptibility to insecticides as noted by nadim and amini in 1970. from his previous fauna survey in bandar abbas and jask areas in southern iran under the application of ddt, dieldrin and later malathion spraying, mesghali (1965) inferred that p. papatasi hitherto has not shown any insecticide resistance, but the lc50 became higher. although they were absent in premises freshly sprayed with malathion, they were present outdoors. almost the same remarks were repeated in a paper dealing with p. salehi in baluchestan, southeast of iran by mesghali and seyedi-rashti in 1968. in neishabur, nadim and tahvildar-ebidruni (1977) showed that ddt spraying at a rate of 2gr/m2 in and around houses of acute cases was highly effective on the control of p. sergenti but the endemicity remained at a little bit higher level than in the period before the outbreak. in 1970 during an epidemic of acl at janatabad district, northwest of tehran, the population of p. sergenti was effectively suppressed by house spraying with ddt 75% wp, at a rate of 2g/m2 and the transmission of the disease was halted (west health center of tehran, 1970). as the result of an earthquake (6.6 on the richter scale) in the city of bam (kerman province, southeastern iran) on december 26, 2003, nearly all buildings were destroyed and a suitable condition was obtained for the breeding of p. sergenti, and there was the danger of an outbreak of acl in this old endemic focus due to the: extent of the destruction, premature warning, raining in several times after the earthquake, movement of a large number of non-immune individuals from neighboring and other areas into the affected area and the destruction of all health facilities. according to the suggestions of leishmaniasis committee of iranian ministry of health, residual spraying with delthamethrin at a rate of 25mg/m2 was applied on tents and in the remaining indoors twice, the first in may and the second in late august, 2004. space spraying with symprator (a combination of cypermethrin+ tetramethrin and piperonyl butoxide) was also applied in different parts of the city once a week. interestingly, the comparison of the density of p. sergenti before and after the occurrence of the earthquake showed that irs with delthamethrin and space spraying with symprator had reduced the density of the main vector significantly. in early july coincident with the first peak, the density of this species was zero. in late august coincident with second peak of the species, the density had reduced more than 8 fold in outdoors and 14 fold in indoors (institute of public health, unpublished data). susceptibility tests by who standard method on p. sergenti, collected in the city showed that the mortality for diagnostic doses of ddt 4% and deltamethrin 0.025% were 100%, which indicates that the field population of the species is susceptible to both insecticides. according to the reports of the disease management center, the cases of leishmaniasis had reduced sharply in comparison with the cases of the previous year (2003) from april to october. although we were expecting to have a serious epidemic of acl in the city. j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 433 http://jad.tums.ac.ir published online: october 04, 2016 in 2005, irs was applied with deltamethrin, 0.025g/m2 against p. sergenti in the houses with ulcers and their neighboring houses in the city of yazd, central iran. the operations discontinued the transmission of acl (heath center of yazd province, 2005). in order to control acl in the endemic focus of dehbakri, a village of kerman province, southeastern iran, irs with deltamethrin wp 50% was applied at 25mg/m2 against p. sergenti in april 2010. the reduction of the density of the species, the percent of bloodfed and gravid sand flies and also the incidence of the disease were significant in the treated area (aghaei-afshar et al. 2013). a field trial which was carried out during 1991–1993 in 80 villages of meshkinshahr county, ardebil province, northwest of iran, reduced the incidence of zvl due to the combined application of irs with ddt 75% wp 2g/m2 in houses with human or canine cases against p. kandelakii, along with the culling infected dog and the introduction of health education. the incidence was 190/ 100000 in 1991 which dropped to 123/ 100000 in 1992. the difference was statistically significant (p˃ 0.05). taking into consideration that 75% of cases in the area were children between 1–4 age group, the incidence in this age group was compared before and after intervention. the incidence before intervention was 440/100000 which was reduced to 71/100000 at the end of the study (p˃ 0.05) (bokaei 1994). indoor residual spraying is a cost effective method of controlling of p. sergenti in acl foci during epidemic scenarios in iran. in temperate regions one round of spraying with one of available insecticides is needed but in tropical areas the second round of spraying should be carried out in mid or late august in urban areas. although spraying of houses and shelters is unlikely to be effective in zcl and zvl foci but one round of irs is recommended in epidemic conditions. table1. comparison results for the intervention against leishmaniasis vectors by indoor residual spraying in iran, 1966–2010 referenceresultsimpact measurement main vectordosage (gai/m2) insecticideyearlocality nadim and amini 1970, seyedi-rashti and nadim 1973 with discontinuation of spraying incidence increased reduced zcl incidence p. papatasi2ddt (one round) 1966– 1967 esfahan nadim et al. 1977long term studies did not carry out as abovep. papatasi2malathion (2 rounds) 1976khuzestan mesghali 1965as aboveas abovep. papatasi2malathion1965bandar-abbas and jask nadim and tahvildare-bidruni 1977 endemicity remained at a low level reduced acl incidence p. sergenti2ddt1976neishabur health center of yazd province 2005 as above transmission of acl reduced sharply p. sergenti0.025 mg/m2 deltamethrin2005yazd j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 434 http://jad.tums.ac.ir published online: october 04, 2016 west health center of tehran 1970 acl cases disappeared transmission of acl discontinued p. sergenti2ddt1970janatabad (tehran) institute of public health 2004 reduced acl incidence sharply reduced density of sand fly vector p. sergenti25 mg/m2 deltamethrin2004bam aghaei-afshar et al. 2013 endemicity remained at a low level reduced density, percent of blood fed and gravid sand flies p. sergenti25 mg/m2 deltamethrin2010dehbakri bokaei 1994incidence of vl reduced reduced density of the vector p. kandelakii2ddt1991meshkinshahr applying chemicals in rodent burrows in 1972, in five villages with a total population of 1471 in the infected area of esfahan, central iran rodent burrows were dusted with 75% ddt powder within a radius of 300 meters around the villages, once every month (june, july, august and september) at a rate of 0.5g/m2 in each burrow with a hand dusting equipment. dusting of the burrows had no effect on the density of p. papatasi, the main vector of l. major in the area. this could be due to the loose nature of the soil within the area such that the insecticide dust is quickly buried under soil upon falling to the ground, having no effect on sand flies coming out from the depth of the burrows (seyedi-rashti and nadim 1974). during 1972–1973 coincident with the active season of sand flies, rodent control operations within 300 meters of the houses using poisoned bait consisting of 12–15 grams wheat containing 2.5% zinc phosphide was carried out against zcl once every month from early june through september in 5 villages of borkhar county, esfahan province, central iran. the method was very effective in destroying the rodent reservoirs, but it was ineffective in the reduction of the incidence of the disease. its effect on the sand flies was not evaluated in the area (seyedi-rashti and nadim 1974). in 1974, rodent control by carbon monoxide (co) was carried out against zcl at an area of 25 hectare in the village of adermanabad, borkhar county, esfahan province. although it was very effective on the control of the population of great geribil, rhombomys opimus, its effect on the reduction of the density of p. papatasi and incidence of the disease was not evaluated (deputy of health, esfahan university of medical sciences, 1974). in 1978, the effect of insecticide spraying inside the rodent burrows against p. papatasi was evaluated in borkhar area, esfahan province, central iran. about 8000 rodent holes in an area of 38 hectare were sprayed by ddt 75% wp (4.3gr in each hole), in two rounds, the first in late june and the second round in early august. monitoring the density of sand flies before and after the spraying showed that although the operations could reduce the population of sand flies, the density returned to its normal level after about a week. this could be due to the curving nature of rodent burrows and the resulting lack of access to the depth of holes which are considered the best resting place and oviposition of sand flies. hence, this method has no robust effect in the reduction of sand fly population for a longer time (moosavi 1979). yaghoobi-ershadi et al. (2000) conducted table 1. continued… j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 435 http://jad.tums.ac.ir published online: october 04, 2016 a field trial for the control of zcl during 1997–1999 in the rural district of badrood, esfahan province. rodent control operations were carried out within a radius of 500 meters from the houses baited with 12–15 grams of wheat containing 2.5% zinc phosphide in each burrow. the evaluation showed that the control program reduced the incidence of zcl 12 fold in the treated village compared to the control at the end of the first year of operation. comparison of the density of p. papatasi, in the treated and the control area indicated that the program was effective in the reduction of the density of this species in rodent burrows of the treated area. at the end of july, coincident with the first peak of p. papatasi its density was 261 per 30 traps in the control area which reduced to 75/30 traps in the treated area. in the second peak (end of september) the density was 717/30 traps and 199/30 traps in the control and treated areas respectively. the operation was also effective in disturbing the sex ratio of the species which was 172 and 21.9 in the control and treated areas respectively. there was also significant difference between fed and unfed p. papatasi. it means that in the intervention village the ratio decreased to one-third in comparison with the control area. the method was ineffective on the density of p. papatasi in indoors in the study area (motavalli-emami 1998). a comparative study was carried out on the effectiveness of comavec and zinc phosphide against the great gerbil, r. opimus in a hyperendemic focus of zcl in central iran during 2011–2012. the reduction rate of rodent holes in intervention areas with comavec and zinc phosphide was calculated to be 48.5% and 58.2% respectively and the incidence of zcl reduced significantly in the treated areas. comavec seems to be effective on the outdoor density of p. papatasi, the main vector of l. major (veisi et al. 2012). in another study at the same time, a field evaluation of phostoxin and zinc phosphide was also conducted against zcl in four villages around the city of esfahan, central iran. the incidence of the disease decreased to 19.2 and 11.4 per thousand in areas treated with phostoxin and zinc phosphide respectively and the density of p. papatasi in outdoors of the village treated with phostoxin was lower than indoors but its density in the village treated with zinc phosphide was higher in outdoors (akhavan et al. 2014). impregnated bed nets and curtains nadim et al. (1995) evaluated the use of deltamethrin (k-othrin) impregnated bed nets, 25mg per ml for the control of acl in a restricted area in the city of bam, kerman province southeast of iran where, it was very hot during the day in summer but rather good weather at night. the results showed that the number of active cases decreased 2.6 fold in the intervention area but the transmission was not interrupted. non-significant difference was observed between the density of p. sergenti in indoors of the intervention and control areas. during 2003–2004 in a study which was conducted in the hyperendemic area of cl due to l. major (borkhar, esfahan province, iran) efficacy of deltamethrin impregnated bed nets and curtains, 25mg a.i/m2 polyester mesh size 156 holes inch2 combination with a health education program were assessed in relation to zcl control (yaghoobi-ershadi et al. 2006a). the results indicated that the nets could afford a definite personal defence against sand fly bites and interrupted the transmission of zcl in the intervention area. non-impregnated bed nets and curtains did not give any protection against the disease. there was no significant difference in monthly density of p. papatasi in indoors and outdoors among the villages (p˃ 0.05). moosa-kazemi et al. (2007) carried out a field evaluation of deltamethrin-impregnated bed nets and curtains with 25mg/m2, in combination with a health education program against j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 436 http://jad.tums.ac.ir published online: october 04, 2016 acl in the city of mashhad, northeastern iran. the area has a cooler climate in summer where people sleep in rooms at night. these nets provided very good protection against sand fly bites and subsequently reduced the transmission of acl (4.5 fold) but there was non-significant differences in the reduction of the density of p. sergenti among different months. non-impregnated bed nets and curtains did not provide any protection against the disease. bioassays confirmed that the nets treated with deltamethrin remained effective for more than 3 months. high performance thin layer chromatography (hptlc) analysis of deltamethrin residue on the impregnated bed nets showed that the residue of the insecticide persisted well on impregnated nets at least 15 weeks after impregnation (moosakazemi et al. 2009). a large scale installation of deltamethrin impregnated screens and curtains, 25mg/m2, combination with passive and active case surveillance and treatment was assessed in the southeastern iranian city of bam during 2008– 2010. the city which was devastated in 2003 earthquake is known historically as a major endemic focus of acl in the country. comparison of cumulative incidence between the intervention and control areas before and after installation indicated a significant differential reduction in the cumulative acl incidence in the intervention area. the authors believed that the use of impregnated screens and curtains provided a good barrier in the way of sand flies entering the buildings (noazin et al. 2013). insect repellents during the iran-iraq war (1980–1988) cl due to l. major was prevalent among the soldiers, inhabitants and emigrants in the west of the country especially in the provinces of khuzestan, ilam, kermanshah, and some parts of kurdestan (marivan). every year more than 10 thousand cases were reported among the soldiers and emigrants. based on official reports in 1982 more than 3000 military personnel referred to provincial health center of khuzestan for treatment. the density of p. papatasi was in a high level, as in a study on hourly activity of sand flies, the number of biting was reported 120 in each hour during the night around the city of dezful, khuzestan province. the available repellents such as odomos (12% n, n-diethyl-benzamide) and autan (deet 25%) were used as topical application on face and hands by military personnel to protect them from sand fly bites. at the same time, a kind of insect repellent called "trench pomade" which was a mixture of diethyl-toluamide (deet 25%) and dimethyl-phthalate (dmp 5%) in the ratio of 5:1 respectively and had been produced at the school of pharmacy, tehran university of medical sciences was widely used by soldiers which provided 11h protection against the bites of p. papatasi. about one million tubes of trench pomade was produced monthly and 4000 tubes were distributed every day among the personnel. each soldier was delivering one tube of the pomade twice in each month (javadian e, school of public health, tums, personal communication). the vehicle of this cream is propylene glycol and is still used by military personnel in the borderlines of the country. repellency effect of the plant myrtle, myrtus communis essential oil and deet against p. papatasi, the main vector of zcl was evaluated under laboratory and field conditions during 2004–2005. the modified k&d (klun and debboun) apparatus with wirtez method was employed in dose-response tests (calculation of ed50 and ed90 values). significant difference was observed between ed50 of deet and myrtle essential oil (p˂ 0.05). deet was found to be more effective as a repellent than myrtle essential oil against p. papatasi (yaghoobi-ershadi et al. 2006b). to determine the protection time of myrtle essential oil and deet at the laboratory, tests j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 437 http://jad.tums.ac.ir published online: october 04, 2016 were carried out on 9 volunteers for 10 hours in 18 days. the mean protection time for myrtel essential oil and deet on p. papatasi was calculated to be 51min and 10h respectively. significant difference was observed between the protection time of them (p˂ 0.0001). to find out the protection time of the repellency in the field, tests were carried out on 9 volunteers in the village of matinabad, natanz (esfahan province) during 18 nights. the mean protection time of myrtle essential oil 20% and deet 20% were calculated to be 110 minutes, 28 seconds and 10 hours respectively. significant difference was observed between the protection time of them (p˂ 0.0001). to calculate the protection percent of myrtle essential oil 20% and deet 20% a total of 3981 female p. papatasi were collected on the treated volunteers by myrtel essential oil (1381 flies) and control (2600 flies). no flies were collected on volunteers against p. papatasi for 1h following application to human volunteers but it decreased to 50% after 4h. the percentage of protection of myrtel essential oil 20% and deet 20% were calculated 46.41% and 100% correspondingly after 10h during 18 nights. the mean number of bites on the treated legs with myrtel essential oil was 7.69+_4.51 bites human-h, whereas those figures on the control were 14.33+_3.95 bites/human-h. a significant difference was observed between the protection percent of myrtel essential oil and deet (p˂ 0.0001) (jahanifard 2006). saberi et al. (2011) studied the efficacy of deet repellent pen against zcl in a military area in esfahan, central iran. the trial was carried out on 430 high school students. the results showed non-significant efficacy on the prevention of zcl. asilian et al. (2003) evaluated the efficacy of permethrin-impregnated uniforms for the prevention of zcl among 324 iranian soldiers in the hyperendemic area of esfahan. the uniforms were impregnated with permethrin and its concentration was 850mg/ m2. statistical analysis showed non-significant difference between the incidence of zcl in the permethrin group and that in the control group (p˂ 0.05) and did not protect volunteers from sand fly bites. impregnated dog-collars a pilot field study was implemented on the effects of deltamethrin-impregnated dog collars against sand fly bites in azar-shahr district, an endemic focus of zvl in eastern azarbaijan province, north-west iran in 1999. each dog was fitted with a plastic collar (length 48cm, weigh 20g) containing deltamethrin 40mg/g and the protective value of collars were tested against wild caught p. papatasi. for each test, the dog was caged in a net with 70–100 wild caught sand flies overnight. the results showed that the successful blood-feeding by p. papatasi was significantly reduced about 80% (halbig et al. 2000). a matched community–based trial undertook to investigate the epidemiological impact of applying collars to all domestic dogs in the counties of kalaybar and meshkin-shahr in north-west iran during 2000–2001. the area is considered as the main focus of zvl in the country. eighteen villages including nine treated and nine as control were selected. leishmania infantum zymodeme mon-1 is the causative agent of the disease and the probable sand fly vector is p. kandelakii in the area. dog collars consisted of 65cm strip of white polyvinyl chloride weighing 25g, impregnated with deltamethrin 40mg/g. the authors reported that serconversion rate in children was 1.49% in the intervention villages and 2.4% in control villages. leishmanian skin test conversion was also lowered but not significantly. the seroconversion rate in dogs of the intervention villages was also significantly reduced (mazloumi-gavgani et al. 2002). the efficacy of deltamethrin-impregnated j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 438 http://jad.tums.ac.ir published online: october 04, 2016 dog collars (scalibor) was evaluated against p. papatasi in an endemic focus of zvl in bojnord county, north khorasan province, northeastern iran during 2006–2007. in this experiment 6 healthy dogs with similar age, sex, weight and race were selected and randomly classified in two groups as treated and untreated. at the beginning of the transmission season, 3 dogs received collars and the other group was considered as control group. two weeks after fitting the collars, one of the treated and untreated dogs were exposed to the bites of wild caught p. papatasi in the evening for 2h under the bed net biweekly. in order to assess the mortality of sand flies, they were recaptured at the end of the exposure time and transferred into cups and maintained at optimal condition for 24h. data analysis revealed that the blood-feeding index of p. papatasi was between 13.4–27.6% for dogs with collars and 54.2–59.3% for the dogs of control group which showed significant difference (p˂ 0.005). the index of bloodfeeding inhibition were calculated to be between 51.3–66.2% and 37%-44.8% for collared and collarless dogs respectively and showed a significant difference (p˂ 0.01). the range of combined effects (inhibition of blood-feeding and mortality of p. papatasi) was between 72.37–86.62% for collared dogs and 40.74–45.83% for control dogs (ramazani awal et al. 2009). in 2013, the pour-on formulation of flumethrin on dog (with same race, sex and age) was evaluated against bloodfeeding and mortality of sand flies in endemic focus of zvl in meshkinshahr, northwest of iran. before the application of flumethrin, direct agglutination test (dat) was used for determining the positive or negative cases of dogs. the treated and control dogs were exposed with wild sand flies for 2h under the bed net (2x2x2 meters) once every 20 days during may-september. both alive and dead sand flies were transferred in netted cups to the laboratory and their mortality was assessed after 24h. the blood-fed or unfed conditions of sand flies were determined 2h after exposure to the dogs under stereomicroscope. the results showed that the feeding rate of sand flies on treated dogs was 3.3 fold lower than control and the inhibition of feeding rate on treated dogs was 1.8 fold higher than control. the difference between total mortality of treated and control groups was highly significant (p= 0.0001) (jalilnavaz et al. 2016). susceptibility of sand flies to insecticides studies on the susceptibility level of p. papatasi to insecticides started in 1970 and continuing in some foci of zcl in the country. seyedi-rashti et al. (1970) evaluated the susceptibility of field population of p. papatasi strain from unsprayed area of mashhad by who standard method in northeastern iran in august 1970. the baseline lc50 value for ddt and dieldrin was calculated to be 0.47% and 0.54% respectively (seyedi-rashti 1971). in susceptibility tests carried out on this species from the esfahan area, central iran the lc50 for ddt was 0.47% in september 1976 (soltani 1977), indicating no development of resistance. another survey was performed by who standard method on wild caught p. papatasi from human dwellings in mashhad (northeast), esfahan (central iran) and khuzestan (southwest) during 1985–1986. the results showed that the lc50 value for ddt had increased to 2.3% and 3% in mashhad and esfahan respectively, disclosing the existence of selection pressure in the lack of public health insecticide use (seyedi-rashti et al. 1992). yaghoobi-ershadi (1993) provided evidence of tolerance of p. papatasi to 4.0% ddt following a standard who technique in esfahan. in this program 40 series of susceptibility tests were carried out on a total of 1248 fed p. papatasi from 4 villages in the rural district of borkhar. lt50 and lt90 values were calcuj arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 439 http://jad.tums.ac.ir published online: october 04, 2016 lated to be 18.12 and 63.31min respectively. the mortality rate after one hour exposure to 4.0% ddt and the 24h recovery period was 88.8%. when the exposure time increased to 90 and 120min, the mortality rate was 95.2% and 98.8% respectively. appearance of tolerance on p. papatasi in the area was due to the irregular and excess use of chlorinated insecticides in agriculture. the mortality rate of this species after one hour exposure to 4.0% dieldrin was 100%. at the same time 32 series of tests were conducted on 1112 p. papatasi against ddt 4.0% in two villages located in the rural district of varzaneh (73–98km, southeast of esfahan). lt50 and lt90 values was calculated to be 20.51 and 43min respectively and the mortality rate after one hour exposure and 24h recovery period was 98.61% so it was found to be susceptible to ddt 4.0% (yaghoobi-ershadi and javadian 1993). in 2001, the tests with who technique which was performed in the rural district of borkhar (esfahan) showed that the susceptibility of p. papatasi to ddt 4.0% has been returned because the mortality rate after one hour exposure was calculated to be 100%. this was achieved due to the low use of chlorinated insecticides in agriculture, during the last 9 years in the area (yaghoobi-ershadi, tehran university of medical sciences, unpublished data). during 1993–2001 the same study was carried out in the provinces of fars (southwest), kerman (southeast) and badrood area in esfahan and lt50 ranged between 5.5–29.7 minutes and lt90 between 18.5–58 minutes respectively. phlebotomus papatasi was found to be susceptible to ddt in all of these areas (aghasi 1996, yaghoobi-ershadi and akhavan 1999, rassi et al. 2000). susceptibility tests which were performed again during 2002–2013 on p. papatasi against ddt 4%, deltamethrin 0.05% and 0.025% and propoxur 0.1% by who standard method in sabzevar (northeast), esfahan (central), bam, baft and dehbakri (southeast) showed that the field population of the species was still susceptible to these insecticides in different studied areas (yaghoobi-ershadi et al. 2003, 2007, akhavan et al. 2007, aghaei et al. 2011, saeidi et al. 2011). several susceptibility tests which have been undertaken on p. sergenti, the main vector of leishmania tropica in the cities of mashhad (northeast), kerman and bam (southeast) during 1970–2010 showed that it was susceptible to ddt, dieldrin and deltamethrin (aghasi 1996, yaghoobi-ershadi and akhavan 1999). in 1994, susceptibility tests were conducted on p. kandelakii and p. perfiliewi, the probable vectors of zvl in meshkinshahr and germi counties, ardebil province (northwest), iran. the results revealed that the both species were susceptible to the insecticides (rassi and javadian 1998). concluding remarks the national strategy arranged by the iranian government emphasizes case detection and treatment for acl and zvl. results of many scientific works in the country state that, whether integrated vector management is needed, irs remains the main support for vector control in epidemics which provides a cheaper and more practical solution to prevent cases of leishmaniasis. house spraying with insecticides, although being able to combat an outbreak, cannot be used on a longterm basis in urban areas to maintain transmission at a low level. well organized town planning expansions based on environmental protection measures such as a lack of waste management or open sewage, the presence of idle land among the new construction homes, inadequate garbage collection and sanitation, crowded suburban areas with residents of low socio-economic levels and with existence with domestic animals are the key to prevention of leishmanisis in urban areas. as they provide a good source for high phlebotomine density and favorable conditions for disease transmission. impregnated bed nets and curtains may j arthropod-borne dis, december 2016, 10(4): 429–444 mr yaghoobi-ershadi: control of phlebotomine … 440 http://jad.tums.ac.ir published online: october 04, 2016 provide the best way out in rural areas where transmission is largely occurs outdoors. the critical studies such as biting behavior of different sand fly species, their resting and breeding places, the type of plants which provide sugar meal, potential reservoir hosts along with community participation and education should be studied in leishmaniasis foci which will help in the battle against sand fly vectors and reduce the cost of intervention. new methodological advances, such as geographical information systems and remote sensing can make a positive contribution to these efforts. increasing the awareness of physicians and medical staff on the ecology and control of sand flies and close collaboration between health authorities and researchers are required to achieve the best control measures. health education activities focused on sand fly control among inhabitants living in foci of leishmaniasis, early reporting of active cases by individuals, disease mapping of zcl, acl, zvl and qualified staff for vector control are critical points in effective prevention and control of leishmaniasis. acknowledgements sincere thanks are extended to professor kp chang (chicago medical school/rfums. usa) for his encouragement the author to write the present review and prof a nadim (department of epidemiology and biostatistics, school of public health, tehran university of medical sciences) for kindly commenting on an earlier draft of the manuscript. i also would like to thank all scientists who have worked on this matter for ages whose work is pointed out here. 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the subgenus adlerius (diptera: psychodidae, phlebotomus) in iran. j arthropod-borne dis. 9(1): 84– 97. j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 560 http://jad.tums.ac.ir published online: october 04, 2016 original article faunal distribution and seasonal bio-ecology of naturally infected sand flies in a new endemic zoonotic cutaneous leishmaniasis focus of southern iran kourosh azizi 1, hayedeh parvinjahromi 1, *mohammad djaefar moemenbellah-fard 1, bahador sarkari 2, mohammad reza fakoorziba 1 1research centre for health sciences, department of medical entomology and vector control, school of health, shiraz university of medical sciences, shiraz, iran 2basic sciences in infectious diseases research center, shiraz university of medical sciences, shiraz, iran (received 29 sep 2012; accepted 28 july 2015) abstract background: cutaneous leishmaniasis is a major health problem in iran in spite of implementation of control program. this infectious disease caused morbidity in less than 27000 people in 2010. this study was set to determine some ecological aspects of sand flies in fasa district, fars province, southern iran during 2011–2012. methods: a total of 4792 sand flies were captured by means of sticky paper and cdc miniature light traps in 10 selected villages from the beginning to the end of the active season, from which 1115 specimens were captured for abundance study and 3677 specimens captured for monitoring monthly activities in fasa. after species identification, extracted dna was processed for detection of leishmania parasite infection in sand flies. results: twelve species (6 phlebotomus, 6 sergentomyia) were identified. the most common sand fly was p. papatasi (82.4%) which represented 86.6% of sand flies from indoors and 82.7% from outdoors. the monthly activity of the species extended from april to the end of november. there were two peaks in the density curve of this species, one in june and the second in september. natural infection to l. major was detected in p. papatasi (25 out of 130 sand flies, 19.2%). conclusion: phlebotomus papatasi is considered as a main vector of zoonotic cutaneous leishmaniasis in fasa, fars province, south of iran. keywords: sand flies, leishmania, vector, phlebotomus, iran introduction leishmania parasites can induce a range of clinical manifestations exhibited from selfhealing localized dermal lesions to chronic non-healing diffuse muco-cutaneous forms (davami et al. 2010, shirian et al. 2011). human cutaneous leishmaniasis (cl) remains a pressing public health problem in many countries of the eastern mediterranean region, including iran. it is the most important and widely known malady after malaria in iran (moemenbellah-fard et al. 2012). it is a group of clinically complex sand fly-borne skin infections caused by different species of the blood flagellate protozoan parasites in the genus leishmania (kinetoplastida: trypanosomatidae). about 0.7–1.3 million cases of cl occur in 88 countries annually (desjeux 2001). cl appears to be a major health concern, which, despite its notifiable status, necessitates urgent action to contain its increasingly reported cases (abai et al. 2007, azizi et al. 2010, 2012a-d, fakoorziba et al. 2011). more than a quarter of all cl cases (n= 26,824) registered in 2010 within the eastern mediterranean region were iranians (postigo 2010). this skin lesion is reported from many old world and new world countries with over 90% of cases occurring only in seven coun*corresponding author: dr mohammad djaefar moemenbellah-fard, e-mail: momenbf@sums.ac.ir j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 561 http://jad.tums.ac.ir published online: october 04, 2016 tries including iran, afghanistan, saudi arabia, syria, brazil, nepal and peru (desjeux 2004). the clinical signs and symptoms of cl in humans are in two forms: dry or anthroponotic (anthroponotic cutaneous leishmaniasis or acl) and wet or zoonotic (zoonotic cutaneous leishmaniasis or zcl) forms. leishmaniasis includes parasitic infections caused by the interaction of a wide variety of vectors and reservoirs spread throughout the world except australia (ashford 2000). leishmania-like parasites were shown in kangaroos of australia (rose et al. 2004). leishmaniasis is mainly known as a neglected disease in tropical regions. hematophagous females of some sand flies (diptera: psychodidae, subfamily phlebotominae) are the natural vectors of leishmania parasites in fars province (oshaghi et al. 2010, azizi et al. 2013). the sand fly, p. papatasi, which is the main incriminated vector of l. major, is mostly associated with colonies of different rodent reservoir hosts distributed in various regions of iran (moemenbellah-fard et al. 2003, azizi et al. 2011, 2012c, parvizi et al. 2013, akhoundi et al. 2013, davami et al. 2014). human cl is endemic in more than half of the 31 iranian provinces. the majority of cl cases in iran are caused by l. major (akhoundi et al. 2013). zcl is widespread in the central, southern, eastern and western provinces of iran. the county town of fasa is one of the most important endemic foci of zcl in fars province, southern iran, with 1088 cases in 2007 and 185 cases in 2011. the main aim of this investigation was to capture and identify the vectors of zcl in this focus using nested pcr. therefore, faunal distribution and seasonal activity of naturally infected sand flies were investigated in this new endemic zcl focus of southern iran. to the best of our knowledge, this is the first study on the infection of sand flies with leishmania parasites in this county of southern iran. materials and methods study area this investigation was conducted in fasa county (29°24´n, 54°15´e), fars province, southern iran (fig. 1). it is about 4,000 km2 in area and 1,370-meter altitude above the sea level. the total population of this county town was about 220,000 people in 2010. the climatic condition is very hot in summer and cold dry during winter. the main activities of the people are agriculture and farming. sand fly collection sand flies were collected from ten villages, selected based on history of cl prevalence, differential topography, and the mere presence of vectors and reservoirs of infection. sand flies were caught biweekly in two villages (fedeshkooyeh and miandeh) from fixed sites indoors (bedrooms, sitting rooms, toilets and stables) and outdoors (rodent burrows), using 30 sticky traps (castor oil-coated white papers measuring 20 cm×30 cm) from the beginning to the end of the active season (may-september) to determine sand fly monthly activity. each trap set on one night was taken as a "trap-night" and there were 120 "trap-nights"/month/village. for vector faunal study, eight villages at different points of the county town of fasa were selected randomly. sand flies were collected three times during the active season by using sticky paper and cdc traps. trapped sand flies were removed from sticky papers with needles, washed in absolute acetone and stored in 70% ethanol. for species identification, sand flies were mounted in puri's medium (smart 1965). they were identified using the criteria set in the keys of theodor and mesghali (1964), and then mounted and segregated by sex. the middle body segments (apart from heads and last abdominal segments) of some unfed parous female sand flies with the midguts were kept in 70% ethanol for dna extraction and subsequent pcr processing. j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 562 http://jad.tums.ac.ir published online: october 04, 2016 dna extraction each female sand fly was transferred to a microtube for dna extraction as described elsewhere (azizi et al. 2008). the sand fly sample with dna was added to a microtube containing 100 μl lysis buffer [50 mm trishcl (ph 7.6), 1 mm edta, 1% (v/v) tween 20] and 12 μl of a proteinase k solution (20 μg/ml), in a 1.5 ml tube (motazedian et al. 2002). the tube was incubated for 24 h at 37 °c before 200 μl of a phenol: chloroform: isoamyl alcohol mixture (25:24:1, by vol.) was added. it was stored at room temperature for 5 min then it was centrifuged at 15,000 rpm for 15 min. the supernatant solution with dna was then taken with sampler and 200 μl cold absolute ethanol was added. it was centrifuged at 15,000 rpm for 5 min at 4 °c, then it was evicted on a floated solution in the tube and then stored at 37 °c and 50 μl double distilled water was added and then stored at -20 °c, until it could be tested for leishmania kdna. pcr amplification the pcr used to amplify the variable area of the minicircle kinetoplastic dna of any leishmania in sand fly is described elsewhere (aransay et al. 2000). the primers were csb1xr (cga gta gca gaa act ccc gtt ca) and csb2xf (att ttt cgc gat ttt cgc aga acg) for the first round and lir (tcg cag aac gcc cct) and 13z (act ggg ggt tgg tgt aaa ata g) for the second round. they were designed within the conserved area of the minicircle kinetoplast containing the conserved sequence blocks 3 and 2. each 25 μl reaction mixture contained 0.5 μm of each deoxynucleoside triphosphate, 1 mm mgcl2, 1 unit taq. polymerase (cinnagen, tehran, iran), 0.5 μm csb1xr, 0.5 μm csb2xf primers, 5 μl dna extract, 2.5 μl pcr buffer and 0.3 μl dna polymerase. pcr reaction mixture was set at 94 °c for 5 min, followed by 30 cycles, each of 30 s at 94 °c, 1 min at 55 °c and 1.5 min at 72 °c, and then a final extension at 72 °c for 5 min in a thermocycler (eppendorf ag: humburg, germany). for the second round of pcr, only 1 µ m of each of 13z and lir primers were used. one μl of the first round products with 1/9 dilution (by vol.) were used as templates for the second round of pcr. five μl of final products were subjected to electrophoresis on 1.5% (v/v) agar gel stained with ethidium bromide and visualized by uv transillumination. the size of each band was estimated by comparison with the size of reference strains of l. infantum (mcan/ir/ 96/lon49), l. tropica (mhom/ir/89/ard2) and l. major (mhom/ir/54/lv39). a band of 560 bp indicated that l. major kdna was present (azizi et al. 2012d). results a total of 4792 sand flies were captured by means sticky paper and cdc miniature light traps in 10 selected villages, from which 1115 specimens were counted for abundance study and 3677 specimens were monitored for monthly activities (table 1). of all specimens caught, 3250 (67.8%) male and 1542 (32.2%) female were identified. furthermore, 1108 (23.1%) from internal sites and 3684 (76.9%) were captured from external sites. twelve phlebotomine species were morphologically identified as sand flies fauna (6 phlebotomus, 6 sergentomyia) involving p. papatasi, p. sergenti sl, p. caucasicus, p. alexandri, p. mongolensis, p. bergeroti, s. theodori, s. dentata, s. antennata, s. clydei, s. baghdadis, and s. sintoni. the most abundant of these sand flies was p. papatasi, which was thus considered the dominant species too. sergentomyia baghdadis and p. sergenti sl ranked second and third dominant after p. papatasi in this area. phlebotomus papatasi was also the dominant species both indoor (86.7%) and outdoor (80.8%), and s. baghdadis (5.8%) was the dominant species in j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 563 http://jad.tums.ac.ir published online: october 04, 2016 outdoor-based observations of monthly activity study. the activity of these insects started in may and ended in september (fig. 2), and the peaks activity were twice a year (mid-june and mid-august). the kinetoplast dna of the parasite l. major was detected in 26 (17.3%) out of the 150 female phlebotomine sand flies with nested pcr (table 2). twenty-five (19.2%) out of the 130 p. papatasi sand flies were infected with l. major (fig. 3 and 4). table 1. fauna and relative abundance of sand flies collected in fasa, fars province, southern iran, 2011 sand fly species indoor n (%) outdoor n (%) male n (%) female n (%) total n (%) p. (phlebotomus) papatasi p. (phlebotomus) bergeroti p. (paraphlebotomus) alexandri p. (paraphlebotomus) sergenti sl p. (paraphlebotomus) caucasicus p. (paraphlebotomus) mongolensis s. (sergentomyia) theodori s. (sergentomyia) dentata s. (sergentomyia) antennata s. (sergentomyia) sintoni s. (parrotomyia) baghdadis s. (sintonius) clydei 267 (86.7) 4 (1.3) 9 (2.9) 8 (2.6) 3 (0.97) 1 (0.3) 5 (1.6) 3 (0.97) 0.0 0.0 7 (2.3) 1 (0.32) 652 (80.8) 5 (0.61) 19 (2.35) 25 (3.09) 11 (78.6) 0.0 21 (2.7) 8 (1) 1 (0.1) 2 (0.2) 47 (5.8) 16 (2) 687 (74.8) 0.0 17 (60.7) 22 (66.7) 14 (100) 1 (100) 5 (19.2) 3 (27.3) 1 (100) 2 (100) 21 (38.9) 11 (64.7) 232 (25.2) 9 (100) 11 (39.3) 11 (33.3) 0.0 0.0 21 (80.8) 8 (72.7) 0.0 0.0 33 (61.1) 6 (35.3) 919 (82.4) 9 (0.8) 28 (2.5) 33 (2.95) 14 (1.25) 1 (0.1) 26 (2.3) 11 (1) 1 (0.1) 2 (0.2) 54 (4.8) 17 (1.5) total 308 (27.62) 807(72.38) 779 (69.9) 336 (30.1) 1115 (100) fig. 1. local map of the study area, fasa, in fars province of southern iran. the numbers 1–10 refer to sampling sites: 1-amirhajiloo, 2-sheshdeh, 3-jelian, 4-vaselabad, 5-miandeh, 6-fedeshkooyeh, 7-sahrarood, 8-kooshkeghazi, 9-maghaberi, 10-vakilabad j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 564 http://jad.tums.ac.ir published online: october 04, 2016 table 2. pcr-based rate of infection among selected sand fly species in fasa, fars province, southern iran, 2011 sand fly species tested infected (%) p. (phlebotomus) papatasi p. (paraphlebotomus) sergenti s. (sergentomyia) theodori s. (parrotomyia) baghdadis 130 5 10 5 25 (19.2) 0 (0) 1 (10) 0 (0) total 150 26 (17.3) fig. 2. seasonal activity of sand flies collected in fasa, southern iran, during 2011 fig. 3. nested pcr gel electrophoresis of reference strains with four distinct primers (csbixr, csb2xf, lir and 13z) in 1.5% agar gel stained with ethidium bromide. lane 1: 100 bp marker, lane 2: leishmania. tropica, lane 3: l major and lane 4: l. infantum standards fig. 4. electrophoresis results produced by nested pcr of female sand fly specimens with four primers (csbixr, csb2xf, lir and 13z) in 1.5% agarose gel stained with ethidium bromide. lane 1: 100 bp marker, lane 2: leishmania tropica, lane 3: l. infantum, lane 5: negative control (male sand fly), lanes 6, 8, 9, 12, and 14: parasite-free female phlebotomus papatasi, lane 15: uninfected female sand fly p. sergenti, lanes 4, 7, 10, 11, 13 and 16: sand fly p. papatasi infected with the parasite l. major 0 200 400 600 800 1000 1200 1400 1600 1800 5th m ay 20th may 5th june 20th june 5th july 20th july 5th aug 20th aug 5th sept 20th sept n u m b e r o f sa n d fl ie s annual cycle p. papatasi total no j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 565 http://jad.tums.ac.ir published online: october 04, 2016 discussion control of leishmaniasis necessitates studies on the epidemiology and ecology of this disease vectors and reservoir hosts. the entomological survey and epidemiological study are two important components of control against the spread of infectious agents. common infection with leishmania parasites in sand fly and human in the same place are important features for introducing the main vectors (killick-kendrick 1990). fasa district is one of the most endemic cutaneous leishmaniasis foci of fars province. in spite of control program, some 3224 people were affected by this disease during the period of 2006–2013 (khosravani et al. 2014) and the disease foci still exist in this district. this study was the first on vectors of leishmaniasis in this area. the present study found 12 species of sand flies (6 phlebotomus and 6 sergentomiya). based on these results, p. papatasi was the most abundant both indoors and outdoors. p. papatasi was introduced as the dominant species in this province (salehi 1997, kalantari 2003, azizi et al. 2008, 2010, davami et al. 2010). phlebotomus alexandri was one of the identified species in this study, previously reported to be a visceral leishmaniasis vector in some parts of iran (azizi et al. 2008, bakhshi et al. 2013). phlebotomus sergenti sl was also identified in this study which is effective in the transmission of cutaneous leishmaniasis in iran (moin-vaziri et al. 2007, oshaghi et al. 2010). sand fly activity from mid-may to the beginning of november had two peaks in late june and september and p. papatasi was the dominant species which was modulated with the activity design of other sand flies in this area and was almost consistent with the activity of sand flies in zcl foci in iran (yaghoobi-ershadi et al. 2001, abai et al. 2007). molecular (pcr) method was used to detect leishmania infection in sand flies. some 150 unfed parous females were examined by nested pcr techniques and proved their infection to be l. major in 19.2% of p. papatasi species. previous reports had proved l. major infection in p. papatasi in iran (rassi et al. 2011). this method was used since it was very easy compared to other methods if it was set up in the laboratory, large number of species can be examined in a short time. this method could not distinguish between amastigotes (those parasites without flagella in human) and promastigote (flagellated form of parasite in sand fly body) (azizi et al. 2012b). in order to ensure transmission of parasite by sand flies, it is essential to examine parous or empty stomach female sand fly (molyneux and ashford 1983). a single infection of s. theodori was found in this study. natural promastigote infection of other species of sergentomyia like s. dentata sand flies have previously been reported from ardebil province, northwest of iran (rassi et al. 1997), though this finding does not represent an evidence of this sand fly being a vector. evaluation of infection in sand flies was tested by pcr. this method has as high sensitivity and specificity as elisa method used before (maleki-ravasan et al. 2009). the sand flies that were used in this study had mostly digested blood. according to our studies, a consistent pattern of abundance and fauna in this region occurs with other zcl foci in iran. phlebotomus papatasi is the main cutaneous leishmaniasis vector in this district. epidemiologic study on reservoirs and human infection are essential for providing proper control program. the personal protection including the use of long-lasting insecticidal nets (llin), installing screens on windows and doors, using repellents, and environmental improvements such as waste fertilizers and trash as well as rodent control in houses are j arthropod-borne dis, december 2016, 10(4): 560–568 k azizi et al.: faunal distribution and … 566 http://jad.tums.ac.ir published online: october 04, 2016 the most important strategies in the leishmaniasis control program. conclusion phlebotomus papatasi is considered as a main vector of zoonotic cutaneous leishmaniasis in fasa, fars province, south of iran. acknowledgements the authors appreciate the improvements to this article meticulously proposed by the anonymous peer reviewers. the present paper was extracted from the results of an approved student msc thesis in medical entomology (contract no: 89-5280 dated 13th august 2011) conducted by the second author, ms hayedeh parvinjahromi. it was financially supported by shiraz university of medical sciences (sums). thanks are due to the vice-chancellor for research and technology at sums, for permitting the use of facilities at the university. we are also indebted to ms dabagh for help 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[msc thesis]. school of medical sciences, tarbiat modarres university, tehran, iran. (persian). shirian s, oryan a, hatam gr, daneshbod k, daneshbod y (2012) molecular diagnosis and species identification of mucosal leishmaniasis in iran and correlation with cytological findings. acta cytol. 56(3): 304–309. smart j (1965) a handbook for identification of insects of medical importance, 4th edition. british museum (natural history), london. theodor o, mesghali a (1964) on the phlebotominae of iran. j med entomol. 1: 285–300. yaghoobi-ershadi mr, hanafi-bojd aa, akhavan aa, zahraei ar, mohebali m (2001) epidemiological study in a new focus of cutaneous leishmaniasis due to leishmania major in ardestan town, central iran. acta trop. 79: 115– 121. giriş j arthropod-borne dis, december 2017, 11(4): 463–468 f duygu et al.: brucellosis in … 463 http://jad.tums.ac.ir published online: december 30, 2017 original article brucellosis in patients with crimean-congo hemorrhagic fever fazilet duygu 1, *tugba sari 1, turan kaya 2, nermin bulut 3 1public health institution of turkey, ankara, turkey 2tokat state hospital, department of infectious diseases and clinical microbiology, tokat, turkey 3gaziosmanpasa university, faculty of medicine, department of microbiology and clinical microbiology, tokat, turkey (received 15 may 2016; accepted 3 dec 2017) abstract background: crimean-congo hemorrhagic fever (cchf) is a fatal zoonotic viral disease caused by infection with a tick-borne virus of the genus nairovirus. in this study, we investigated the incidence of brucellosis in patients diagnosed with cchf. methods: overall, 169 patients hospitalized with an initial diagnosis of cchf were included in 2011 in tokat/ turkey. immunoglobulin m (igm) antibodies and/or pcr results were used in the laboratory diagnosis of cchf, while plate and standard tube agglutination (sta) tests were used to diagnose brucellosis. results: overall, 120 patients (79%) with positive pcr tests were diagnosed with cchf. five (4.16%) were also diagnosed with brucellosis based on the positive plate and sta test results. four patients (2.36%) had negative cchf pcr and positive sta test results. conclusion: brucellosis and cchf can mimic each other and that all patients with cchf or brucellosis should be screened for both conditions. keywords: crimean-congo hemorrhagic fever, haemorrhagic fever, brucellosis, zoonosis introduction crimean-congo hemorrhagic fever (cchf) is a viral disease occurring in different regions worldwide and may be life threatening due to the manifestations of fever accompanied by hemorrhage. it is a zoonosis caused by nairoviruses transmitted via hyalomma ticks (1, 2). this disease, observed in more than 30 countries, including countries in asia, europe, and africa, results in a mortality rate of 5.4–80% (3, 4). tokat province in northern turkey is in an endemic area for cchf, which is also endemic in the middle and eastern parts of the country. cchf is characterized by fever, malaise, sweating, anorexia, and arthralgia, and various nonspecific symptoms following an incubation period lasting less than a week. as the disease progresses, it may potentially manifest disseminated intravascular coagulation (dic) and shock (5). brucellosis caused by brucella bacteria represents a zoonosis transmitted to humans via the body fluids and secretions of animals such as sheep, goats, cows, buffalos, and pigs and via dairy products made with contaminated milk. after a 2–3 week incubation period, nonspecific symptoms that include fatigue, lack of appetite, muscle and joint pain, and subfebrile fever may occur. depending on the part of the body affected by the infection, different clinical characteristics may manifest (6, 7). as the symptoms of brucellosis are nonspecific, differential diagnoses are difficult and misdiagnoses are frequent. this trial designed to assess the incidence of brucellosis among patients with a preliminary diagnosis of cchf and the clinical conditions resulting from the co-existence of the two zoonoses. *corresponding author: dr tugba sari, e-mail: drtugba82@gmail.com j arthropod-borne dis, december 2017, 11(4): 463–468 f duygu et al.: brucellosis in … 464 http://jad.tums.ac.ir published online: december 30, 2017 materials and methods this is a prospective cohort study implemented from apr 2011 and jul 2011 in tokat, turkey. one hundred and sixty-nine inpatients monitored at the tokat state hospital with a preliminary diagnosis of cchf were enrolled in the trial. all the biochemical analyses were performed by auto-analyzer and complete blood counts were performed by automatic hemocounter at central laboratory of our hospital. the public health institution of turkey routinely submitted case definition forms and serum samples obtained from the suspected cases (the first sample) to the national reference laboratory. all patients preliminarily diagnosed with cchf based on the case definition criteria were hospitalized and followed up, as required by the regulations. diagnosis of cchf and brucellosis requires satisfaction of the following criteria 1) compatible clinical picture, 2) diagnosis of cchf based on detection of cchf igm antibodies by elisa and/or cchfv rna with rreverse transcription polymerase chain reaction assays and direct sequence analyses (8, 3), 3). all patients underwent a brucella rose-bengal slide agglutination test, with blood cultures performed for patients with a positive result based on a standard tube agglutination (sta) test and/ or coombs’ tests (titers≥1/160) (9, 10). we used a commercial kit (cromatest, knickerbocker laboratories, barcelona, spain) for the sat. the coombs’ test used anti-human gamma globulin sera (ortho diagnostic systems, madrid, spain) to detect blocking antibodies. in cases of positive sera, we performed serial tube dilutions that ranged from 1:10 to 1:1240. the bactec 9050 blood culture system (becton, dickinson and company, usa) was used to culture brucella. the histories and physical examination results of the patients diagnosed with brucellosis based on the clinical and laboratory findings were recorded. the age, gender, white blood cell and platelet counts, and the aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels of the patients were recorded. statistical analyses were conducted using spss ver. 17.0 for windows (inc., chicago, il, usa). qualitative data were expressed as number (percentage) and mean comparisons for continuous variables were performed using independent-group t-tests. results ninety patients (53.2%) were men, and the mean age of the patients was 32.8±9.21yr. the brucella sta test was positive for nine of the 169 patients (5.02%). these nine patients were living in rural areas. they all had a history of stockbreeding and consumption of unpasteurized milk products. none had a history of brucellosis. one hundred and twenty patients with positive cchf pcr results were diagnosed with cchf (79%). five of those diagnosed with cchf also had positive brucella slide and tube agglutination test results (4.16%). all patients had general symptoms of infection and fever (100%). one of the patients had sacroiliitis (11.1%) and two had splenomegaly (22.2%). four of the 169 patients hospitalized with an initial diagnosis of cchf was found to be cchf pcr negative and brucella sta positive (2.36%). all patients had thrombocytopenia and leukopenia. two patients had anemia. five patients had elevated alt levels and eight patients had elevated ast levels. the demographic characteristics and laboratory results of the patients are presented in table 1. j arthropod-borne dis, december 2017, 11(4): 463–468 f duygu et al.: brucellosis in … 465 http://jad.tums.ac.ir published online: december 30, 2017 table 1. demographic characteristics and laboratory results of the patients diagnosed with brucellosis patient number 1 2 3 4 5 6 7 8 9 age (yr) 62 23 35 46 22 42 40 46 52 gender male male male male female male female female male living area of the patients rural rural rural rural rural rural rural rural rural contact with ticks yes no yes yes yes yes yes yes yes milk* yes yes yes yes yes yes yes yes yes alt u/l 55 36 27 127 40 75 65 128 69 ast u/l 76 66 30 164 98 82 57 164 72 wbc 1 count (109/l) 2400 2300 2700 2000 1600 3300 4300 2000 5200 wbc 2 (109/l) 2100 1800 2000 2000 1600 3200 3500 2000 4500 hb 12.2 11.6 13.9 14.9 13.8 14 11.2 14.9 13.6 platelets 1 (109/l) 60 000 115000 139000 59000 70000 112000 112000 59000 120000 platelets 2 (109/l) 42 000 61000 58000 55000 26000 90000 87000 59000 79000 cchf pcr positive positive positive positive positive negative negative negative negative brucella sta 1/1280 1/640 1/1280 1/320 1/640 1/320 1/1280 1/640 1/1280 blood culture sterile sterile sterile sterile sterile sterile sterile sterile brucella spp. *consumption of unpasteurized milk and milk products alt: alanine aminotransferase, ast: aspartate aminotransferase, wbc 1: initial white blood cell count on referral, wbc 2: lowest white blood cell count, hb: haemoglobin, platelet 1: initial platelet value, platelet 2: lowest platelet value, sta: standart tube agglutination. normal values: ast: 15–37 iu/l, alt: 30–65 iu/l, wbc: 4. 800–10.800mm3, hb: 12–17g/dl, platelet: 150000–400000mm3 discussion this trial showed that zoonotic diseases can coexist due to similar modes of transmission and that brucellosis should not be ruled out in patients with cchf. in this trial assessing the incidence of brucellosis in patients with cchf, five patients had both of the diseases (4.16%). the pcr results of the four patients with a preliminary diagnosis of cchf were negative, these four patients were diagnosed with brucellosis. we believe the patients with a preliminary diagnosis of cchf should also be investigated for brucellosis. cchf is a zoonotic disease transmitted by hyalomma ticks (1). brucellosis is a zoonosis transmitted via body secretions of animals with brucellosis and by the consumption of milk and dairy products not boiled or pasteurized (11). farmers, shepherds, veterinarians, butchers, and laboratory staff are at risk for transmission (12). both diseases are more commonly observed in people who deal with stockbreeding and live in rural areas. the most common patient complaints upon presentation with cchf include fever, fatigue, diffuse body pain, and lack of appetite. patients less commonly present with haemorrhage and rash compared to nausea and/ or vomiting, diarrhoea, and abdominal pain (1). brucellosis is a disease that can involve any organ and tissue and therefore may manifest with different symptoms and findings (6, 12). because the symptoms of brucellosis are nonspecific, differential diagnoses are difficult and misdiagnoses are frequent. patients most commonly present with fever, fatigue, sweating, joint pain, and lack of appetite. as the disease may involve various organs, the physical examination findings can also be varj arthropod-borne dis, december 2017, 11(4): 463–468 f duygu et al.: brucellosis in … 466 http://jad.tums.ac.ir published online: december 30, 2017 iable (6). the most common findings in brucellosis are fever, splenomegaly, hepatomegaly, lymphadenomegaly, and arthritis. this disease may involve all the systems and may manifest with complications. osteoarticular involvement is most common (13). in this trial, all patients developed at least two of the general infection signs of fever, fatigue, and lack of appetite. leukopenia, thrombocytopenia, and anaemia may develop in cchf (14). similar laboratory findings can also be seen in brucellosis as a result of bone marrow suppression (13). the negative cchf pcr results and the positive brucella sta test results obtained for four of the patients admitted with a preliminary diagnosis of cchf with symptoms of fever, general infections signs, and bicytopenia who presented due to tick contact demonstrated the importance of performing brucellosis tests in patients monitored for suspected cchf. cchf is seasonal and occurs between apr and sep, while brucellosis may occur in any season. therefore, we believe that seasonal features should also be considered in establishing a diagnosis and that these two diseases could co-exist during the summer. the diagnosis of cchf is based on virus isolation in the cell culture, serologic methods (immunofluorescence assays [ifas], elisas) and reverse transcription pcr (1). the most common method of diagnosing brucellosis is the standard tube agglutination (sta) test. standard tube agglutination test is an inexpensive, convenient method with a reported sensitivity of 94%. the gold standard in diagnosis is the growth of bacteria in culture (15). all the patients were diagnosed based on pcr and/or igm results. as the patients were hospitalized with a preliminary diagnosis of cchf, the blood cultures could not be obtained routinely. one of the patients for whom blood culture was conducted after a brucellosis diagnosis was established exhibited brucella growth in the blood culture. debate on the treatment of cchf is on going. while some publications show ribavirin is beneficial, others indicate its lack of efficacy (16-19). ribavirin treatment has been shown not to reduce mortality in the treatment of cchf (20). in this trial, ribavirin was not administered to patients diagnosed with cchf; rather, symptomatic treatment was administered. double or triple combinations of doxycycline, streptomycin, and rifampicin are recommended to treat brucellosis (12). in this trial, all these three drugs were administered to the patients with osteoarticular involvement. other patients were given doxycycline and rifampicin. all patients recovered. brucellosis can show great similarity with hematologic and zoonotic diseases, such as cchf (21-23). conclusion brucellosis should be considered in the differential diagnosis of pancytopenia, treatment-resistant immune thrombocytopenia, and viral hemorrhagic disease, especially in countries where brucellosis is endemic. 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and crimean-congo hemorrhagic fever coinfection in an endemic area. mikrobiyol bul. 50(2): 322–327. https://www.ncbi.nlm.nih.gov/pubmed/?term=soares-weiser%20k%5bauthor%5d&cauthor=true&cauthor_uid=20626907 https://www.ncbi.nlm.nih.gov/pubmed/?term=thomas%20s%5bauthor%5d&cauthor=true&cauthor_uid=20626907 https://www.ncbi.nlm.nih.gov/pubmed/?term=thomson%20g%5bauthor%5d&cauthor=true&cauthor_uid=20626907 https://www.ncbi.nlm.nih.gov/pubmed/?term=thomson%20g%5bauthor%5d&cauthor=true&cauthor_uid=20626907 https://www.ncbi.nlm.nih.gov/pubmed/?term=garner%20p%5bauthor%5d&cauthor=true&cauthor_uid=20626907 https://www.ncbi.nlm.nih.gov/pubmed/20626907 https://www.ncbi.nlm.nih.gov/pubmed/?term=tanir%20g%5bauthor%5d&cauthor=true&cauthor_uid=25467988 j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 182 http://jad.tums.ac.ir published online: may 27, 2017 original article utility of mtdna-coi barcode region for phylogenetic relationship and diagnosis of five common pest cockroaches saedeh sadat hashemi-aghdam 1, golnaz rafie 1, sanaz akbari 1, *mohammad ali oshaghi 2 1deptartment of biology, damghan branch, islamic azad university, damghan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 16 mar 2015; accepted 17 feb 2016) abstract background: cockroaches are of vital importance medically and hygienically as they can disperse human pathogenic agents and are especially responsible for food contamination and spreading of food borne pathogens. in this study, part of mtdna-coi gene of five common pest cockroaches was tested for diagnostic and phylogenetic purposes. methods: we have described barcode region of mtdna-coi gene of five cockroach species: blattella germanica, blatta orientalis, periplaneta americana, shelfordella lateralis, and supella longipalpa, along with the development of a pcr-rflp method for rapid detection and differentiation of these health pest species. results: the pcr generates a single 710 bp-sized amplicon in all cockroach specimens, followed by direct sequencing. alui predicted from the sequencing data provided different rflp profiles among five species. there was a significant intra-species variation within the american cockroach populations, but no genetic variation within other species. accordingly, phylogenetic analysis demonstrates common monophyly for cockroach families in agreement with conventional taxonomy. however s. longipalpa (ectobiidae) diverged as an early ancestor of other cockroaches and was not associated with other ectobiidae. conclusion: the pcr-rflp protocol might be useful when the conventional taxonomic methods are not able to identify specimens, particularly when only small body parts of specimens are available or they are in a decaying condition. mtdna-coi gene shows potentially useful for studying phylogenetic relationships of blattodea order. keywords: cockroach, mtdna-coi, pcr-rflp, molecular marker, phylogeny introduction cockroaches are considered one of the most successful groups of animals. because cockroaches are so adaptable, they are almost found around the world with most species living in tropical and equatorial regions (schal and hamilton 1990). cockroaches are synanthropic and endophilic, exhibit communicative behavior, attracted to dirt and filth that could contain human pathogens and human food products, and harbors these pathogens. cockroaches are present in homes, groceries, food warehouses, restaurants, hotels, hospitals as well as in sewer systems and rubbish bins. these biological characters and their ecological association with humans positioned them in the list of dirty species by fda as the greatest health hazard risk for transmitting human diseases through food products (olsen et al. 2001, sulaiman et al. 2011). cockroaches may transmit four strains of poliomyelitis virus, viruses of encephalitis, yellow fever, coxsakie, and the eggs of seven species of pathogenic ringed worms. moreover, twelve species of adult cockroaches are known as intermediate host for invertebrates, some species of bacteria including: salmonella, staphylococcus, strep*corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 183 http://jad.tums.ac.ir published online: may 27, 2017 tococcus, escherichia coli, proteus, klebsiella, serratia and some protozoa such as: giardia, balantidium, entamoba histolytica, trichomonas and some of the pathogenic fungi such as aspergillus. cockroaches coexist with approximately 150 species of bacteria, 60 species of fungi, six species of yeast, 90 species of protozoa, 45 species of pathogenic ringed worms and some of the other worms (pai et al. 2003, mlso et al. 2005, salehzadeh et al. 2007, sookrung et al. 2008, karimizarchi and vatani 2009, fakoorziba et al. 2010, akbari et al 2014). they also are a principal contributor to allergies (arruda et al. 2001, arlian 2002). in most regions of the world, residual insecticide sprays are applied into cracks and crevices of filthy places to control cockroaches (limoee et al. 2006). based on the current classification, cockroaches belong to the order blattodea that comprises three superfamilies of corydioidea, blaberoidea, and blattoidea (beccaloni and eggleton 2013). overall, 7570 living species of blattodea are currently recognized, of which 4641 are cockroaches (beccaloni 2007) and 2929 are termites (krishna et al. 2013). about 30 out of 4641 cockroach species are more associated with human habitations where some species such as periplaneta americana (american), b. germanica (german), b. orientalis (oriental) are well known as pests (valles et al. 1999, schal and hamilton 1990). in addition, shelfordella lateralis (turkestan) and supella longipalpa (brown-banded) are among the rapidly replacing known pest species in some places of the world. shelfordella lateralis is well distributed in central asia, the caucasus mountains, and northeastern africa. recently it has been rapidly replacing the common oriental cockroach in urban areas of the southwestern usa (kim and rust 2013) and north of iran (oshaghi, personnel observation). supella longipalpa (brown-banded) is a worldwide pest (charlton et al. 1993) and one of the most recent cockroaches has been gradually replacing the common german cockroach and to form breeding colonies in many parts of the world including iran (laddoni, personnel observation). cockroach species identification is traditionally based on morphological characteristics, however, morphology is not always a suitable method for these types of studies and sometimes it suffers from deficiencies particularly when only small body parts of the specimen are available or they are in a decaying condition. consequently, today, in addition to morphological, molecular markers, specifically dna-based molecular markers are being widely used in animals systematic. molecular data contributes to resolve the systematic issues in different organisms including cockroaches (lazebnaya et al. 2005, mukha et al. 2007, pechal et al. 2008, hashemi-aghdam and oshaghi 2014). however, the relationships within blattodea order are still uncertain in the literature. studying of the barcode region of cytochrome oxidase subunit i gene of mitochondrial dna (mtdna-coi) has been proposed as a modern systematic tool applied in evolutionary and population study on species delimitation and taxonomy. this gene is one of the largest genes in the metazoan mitochondrial genome and its adaptability is more than the other mitochondria genes. it is also one of the most important molecular markers used for molecular taxonomy and systematic of living things and microorganisms (hebert et al. 2003, karimian et al. 2014). of different segments of the coi gene, dna barcode is selected and used as a standardized region in molecular systematic in literature. in fact, barcoding region provides a common source of dna sequence for identification and taxonomy of organisms (hollingsworth et al. 2011), whereby scientists can compare living organisms. as of february 2013, the barcode of life data systems database (http://www.boldsystems.org) included almost 2,000,000 barcode sequences from over j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 184 http://jad.tums.ac.ir published online: may 27, 2017 160,000 species of animals, plants and fungi. in this study, we designed a pcr-rflp assay from the barcode region of mtdna coi sequences generated from five cockroach species, and attempted to infer the phylogenetic position of those species in the blattodea more reliably. this diagnostic and inferring phylogeny might be a valuable tool for cockroach species identification for entomological studies and control measures strategies. materials and methods sample collection and dna extraction periplaneta americana, b. orientalis, b. germanica, sh. lateralis and su. longipalpa were trapped between march and august 2012– 2013 from different locations such as dwelling, hospital, confectionary, park and insectarium in towns located in center, north and northwest of iran using live-catch trap, box matches and hand catch. the specimens were anesthetized in cold and then preserved in 70% alcohol. details of the specimens used in this study are described in table 1. the specimens were morphologically identified according to the available identification keys (pratt and stojanovich 1966, cochran 1999). fifty-seven different specimens belong to the five species (comprising 14 american cockroaches, 17 german cockroaches, ten brown-banded cockroaches, eight oriental cockroaches, and eight turkistan cockroaches) were used. genomic dna was extracted from the hind leg (femur) for larger cockroaches whereas for small ones such german and brown-banded cockroaches the entire body was used. for dna extraction, the cockroach femur or entire body was isolated and dried at 37 °c in an incubator. samples were subsequently placed in liquid nitrogen for 2–5 min and then homogenized with autoclaved glass pestle. dna was extracted from the resultant homogenate using the method described by collins et al. (1987) and stored at -20 °c until used. pcr-direct sequencing a fragment of 710bp of the coi mitochondrial region designated as the barcoding region was amplified by pcr using primers 5΄-ttaaacttcagggtgaccaaaaaatca-3' (hco2198) and 5΄-g gtcaacaaatcataaagatattgg-3΄ (lco1490) (folmer et al. 1994). pcrs were carried out in 20μl reaction using 2μl genomic dna. the pcr reaction consisted of 2µ l premix (intron®, south korea), and 18µ l of a solution containing 200 nm each primer, and 2μl (50 ng) genomic dna. the premix is a premixed solution containing hotstartaq dna polymerase, pcr buffer, and deoxynucleoside triphosphates (dntps), with a final concentration of 1.5mm mgcl2 and 200 mm each dntp. the reactions were run with an initial denaturation of 2 min at 94 °c and then followed first by 5 cycles at 94 °c for 40 sec, 45 °c for 40 sec, 72 °c for 1 min and then 35 cycles at 94 °c for 40 sec, 51 °c for 40 sec, 72 °c for 1 min, with a final extension 72 °c for 5 min in a peqlab thermocycler machine. the pcr products were electrophoresed on 1% agarose gel with tae buffer (40mm tris-acetate and 1mm edta, ph 8.0), and stained with ethidium bromide (etbr) at a concentration of 0.5µ g/ml, along with a 100-bp ladder (sinaclon, iran). nucleotide sequencing and phylogenetic analysing the amplified products of mtdna coi gene for individuals of the five cockroach species were sequenced bi-directionally and consensus nucleotide sequences were obtained. multiple alignments of the nucleotide sequences were performed using clustalw2 program (http://www.ebi.ac.uk/tools/msa/clustalw2). blast (http://blast.ncbi.nlm.nih.gov/blast.cgi) software was used in order to confirm the accuracy of sequences, discovering their identity and rate of similarity with available data in genbank. the obtained sequences in this study plus a subset of mtdna-coi sequencj arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 185 http://jad.tums.ac.ir published online: may 27, 2017 es of other cockroach species available in genbank were used for phylogenetic analysis. the sequence data contained 16 species belonged to four families of cryptocercidae, blattidae, ectobiidae (blattellidae) and corydiidae (polyphagidae) (table 1). the sequence of myzus persicae (accession no. jx844381) was used as an out-group. phylogenetic tree inferred using the neighbour-joining method embedded in clustalw2 program and plotted using software program tree view (http:// taxonomy.zoology.gla.ac uk/rod/rod.html). pcr-rflp based on sequence variation among the five species, alui restriction enzyme was selected by manufacturer’s nebcutter v2.0 program (http://tools.neb.com/nebcutter) and a physical map provided (fig. 1b). for restriction fragment assays, 15µ l of the pcr products was digested in a 25µ l reaction mixture containing 10 u of alui (vivantis) and 2.5µ l of the appropriate restriction buffer at 37 °c overnight, following the instructions of manufacturer. the digested products were fractionated on a 2.5% agarose gel and visualized by ethidium bromide staining under ultraviolet light. results mtdna-coi gene and phylogenetic analysis result of pcr amplification revealed that the primers could amplify a unique 710bp fragment of the coi gene for all of the five cockroach species. of the 57 pcr amplified products of the specimens, the mtdna-coi nucleotide sequences were generated for 26 specimens. of these, 14 sequences were generated for p. americana, three sequences each for b. germanica, s. lateralis, s. longipalpa, and b. orientalis specimens. the nucleotide sequences have been deposited in the genbank database under accession nos. jq267476 to jq267498 and kj787108. on average, a high a-t content (65.7%) was evident in the amplified mtdnacoi gene fragment of the species. however, the a-t contents did not show significant variations at the intraor inter-species levels. a significant inter-species genetic diversity was observed among the five cockroach species characterized at the mtdna-coi gene. the most divergence rate (22.26%) was observed between s. longipalpa and p. americana and the least rate (11.57%) was seen between american and oriental cockroaches. however, the most genetic difference among the species was lowered to 0.0–9.1% at amino acid level indicating that most of the substitutions were silent or synonymous (table 2). generally, the brown-banded cockroaches with the lowest similarity rate at dna (80%) and amino acid (92.86%) levels were the most diverged species among the species (table 2). additionally, based on mtdna molecular clock (2% per my), s. longipalpa is suggested to have diverged from other species about 10 my. at the intra-species level, a considerable genetic polymorphism (2.62%) was detected among the american cockroach populations whereas no genetic variation has been noticed within other species. phylogenetic relationships among 16 species belonged to four cockroach families cryptocercidae, blattidae, ectobiidae (blattellidae) and corydiidae (polyphagidae) were estimated based on the 648bp dna sequences of the mitochondrial cytochrome oxidase subunit i (coi) gene. a cladogram inferred using the neighbor-joining method indicated monophyly of each of the four families (fig. 1). however, s. longipalpa of ectobiidae (blattellidae) was diverged early and formed a separate branch distinct from other members of ectobiidae. the phylogenetic tree showed that all species of that blattidae grouped together and formed a main clad. in addition, they were sister group of ectobiidae (blattellidae). the cladogram suggested that s. longipalpa was the basal species and the early ancestor of other cockroaches. following s. longipalpa, cryptocercidae was the basal families of j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 186 http://jad.tums.ac.ir published online: may 27, 2017 other cockroaches. pcr-rflp based on nucleotide sequence analysis of the mtdna-coi gene, a pcr-rflp assay was developed for the diagnosis and differentiation of the five cockroach species. it was accomplished by digesting the pcr product (710bp) with alui restriction enzymes. alui cuts the pcr–amplified mtdna-coi fragment of the five species at unique positions: at 251 and 455 to produce three bands of 251, 204 and 255bp for s. lateralis, at 167, 251, 392, and 612 to produce five bands of 167, 84, 141, 220 and 98bp for p. americana, at 251 and 275 to produce three bands of 251, 24, and 435bp for b. orientalis, at seven restriction sites to produce eight bands of 296, 15, 24, 33, 24, 220, 50 and 48bp for b. germanica, at seven cut sites and therefore produces eight bands of 65, 54, 39, 138, 39, 132, 87 and 156 bp for s. longipalpa (fig. 2). although some bands had approximate molecular weights (such as 255 and 251bp for s. lateralis and 132 and 138bp for s. longipalpa) or the bands with less than 100bp length were not easily visible in agarose gel, still a distinctive and visible rflp profile with alui restriction enzyme was obvious after restriction digestion for each of these five species (fig. 2a). fig. 1. phylogenetic tree obtained by comparing 630 bp sequences of mtdna-coi gene of cockroaches. g.b: genbank. myzus persicae with accession no. jx844381 is used as outgroup j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 187 http://jad.tums.ac.ir published online: may 27, 2017 table 1. details of sequence data used for cockroach phylogenetic analysis in this study. ns: not stated species common name family accession origin reference number cryptocercus relictus relict cryptocercidae jx144941 ns direct submission periplaneta japonica japanese blattidae jq350708 ns direct submission periplaneta fuliginosa smoky brown blattidae ab126004 ns direct submission periplaneta americana american blattidae jq267476 iran: kashan this study periplaneta americana american blattidae jq267480 iran: tabriz this study periplaneta americana american blattidae jq350707 ns direct submission pelmatosilpha guyanae n.s. blattidae eu253833 ns direct submission blatta orientalis oriental blattidae jq267490jq267492 iran: tabriz this study blatta orientalis oriental blattidae eu253827 ns direct submission shelfordella lateralis turkestan blattidae jq267493jq267494 iran: assalem this study shelfordella lateralis turkestan blattidae jq267495 iran: tehran this study phyllodromica iberica n.s. ectobiidae am600690 spain: zaragoza direct submission phyllodromica subaptera n.s. ectobiidae am600683 spain: granada direct submission parcoblatta pensylvanica pennsylvania wood ectobiidae gu013646 canada:ontario direct submission blattella bisignata double-striped ectobiidae jx233805 ns direct submission blattella germanica german ectobiidae jq267496jq267498 iran: tehran this study blattella germanica german ectobiidae eu854321 china direct submission supella longipalpa brown-banded ectobiidae kj787108 iran: tehran this study supella longipalpa brown-banded ectobiidae eu253834 ns direct submission eupolyphaga sinensis n.s. corydiidae jf700164 ns direct submission polyphaga sp n.s. corydiidae jq267475 iran: tehran direct submission therea petiveriana desert corydiidae eu253835 ns direct submission myzus persicae peach-potato aphid aphididae jx844381 china direct submission fig. 2. profile of alui pcr-rflp (a) and physical map (b) of mtdna-coi (barcode region) for five cockroach specis.1: shelfordella lateralis, 2: periplaneta americana, 3: blatta orientalis, 4:blattella germanica, 5: supella longipalpa a b j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 188 http://jad.tums.ac.ir published online: may 27, 2017 table 2. genetic similarity rates of mtdna-coi gene among five cockroach species of p. americana (american), b. orientalis (oriental), s. lateralis (turkestan), b. germanica (german), and s. longipalpa (brown-banded) at dna (648 bp, left and below asterisks) and amino acid (210 bases, right and above asterisks) levels american oriental turkestan german brown-banded american *** 98.57 100 96.19 92.38 oriental 88.43 *** 98.57 95.71 91.90 turkestan 87.65 88.27 *** 96.19 92.38 german 82.10 83.49 82.56 *** 92.86 brown-banded 77.74 80.53 80.53 79.44 *** discussion in this study, we developed a pcr-rflp method and defined a unique restriction enzyme (alui) for the first time that can rapidly detect and differentiate the five pest species of cockroaches: p. americana, b. orientalis, b. germanica, sh. lateralis, and s. longipalpa. using a simple conventional pcr and just one single restriction enzyme (alui) for differentiating of five cockroach species is the advantages of this method in comparison with the only previous study that used two restriction enzymes and a more complicated nested-pcr for discrimination of four cockroach species (sulaiman et al. 2011). pcrrflp technique is a very cheap and rapid method for species identification of many organisms. this molecular assay was used to identify many species of organisms such as rodents (oshaghi et al. 2011a), parasites (oshaghi et al. 2009, 2010, 2011b), anopheles (oshaghi et al. 2008, mehravaran et al. 2011) and to determine blood type within insect guts (chavshin et al. 2006, malekiravasan et al. 2009). this will provide a cost-effective solution that so many specimens can be studied with no need to provide dna sequencing. in this method, using a set of conserved primers, a tiny amount of dna can be amplified and then be identified by one restriction enzyme that overcomes lack of ample amount of cockroach body parts. in addition, this method can be used to determine identity of museum specimens, unknown samples and malformed specimens or residues of cockroach body parts in food samples. analysis of the data revealed a high interspecies variation (11.56–22.26%) in the barcoding region of mtdna coi gene across these five cockroach species, suggesting the gene fragment is a good molecular marker for the development of a diagnostic tool for the detection of food pests in food samples. dna barcoding has been used as a shared resource of dna sequences for identification and taxonomic clarification of many organisms, including birds (hebert et al. 2004), sea turtles (vargas et al. 2009), eutherian mammals (luo et al. 2011), fishes (mabragana et al. 2011) and insects (barrett and hebert 2005, smith et al. 2006, wilson 2010). in the light of dna barcoding, researchers can compare different kinds of organisms and have access to their genetic information providing evidence of interpreting systematic situation. hebert et al. (2003) proposed the use of coi gene as an original source for species identification in the animal kingdom. additionally, they demonstrated that species-level assignments could be obtained by creating a comprehensive coi. in addition to coi, many researchers used various genes including rrna (kambhampati et al. 1995, mukha et al. 2000, lazebnaya et al. 2005), coii (maekawa and matsumoto 2000), ets (mukha et al. 2002, mukha et al. 2005), nts (mukha et al. 2007), its1 (pechal et al. 2008) and complete mitochondrial genome (yamauchi et al. 2004, zhang et al. 2010, cameron et al. 2012, xiao et al. j arthropod-borne dis, june 2017, 11(2): 182–193 ss hashemi-aghdam et al.: utility of mtdna … 189 http://jad.tums.ac.ir published online: may 27, 2017 2012, chen 2013) for systematic classification of different species of cockroaches. the inferred relationships among cockroach families (s. longipalpa + (s. longipalpa + (blattidae + (cryptocercidae + (corydiidae + ectobiidae)))) is partly in agreement with some previously published analyses (kambhampati 1995, maekawa and matsumoto 2000, lo et al. 2007). furthermore, s. longipalpa has been introduced as the early ancestor of other cockroaches by the phylogenetic trees inferred from both amino acid and dna sequence data where it did not group with ectobiidae or indeed nest within cockroaches. it is in controversy with earlier morphologybased phylogenetic hypotheses as well as molecular study using a combination of four mitochondrial (16s and coi+coii) and nuclear (18s and 28s ribosomal subunits) loci indicating monophyletic topology of blaberoidea superfamily (djernæ et al. 2002). a wider sampling of blaberoidea including blaberidae is highly required in order to test the topological situation of s. longipalpa. conclusion the results of this study demonstrate the utility of the mtdna-coi gene as a valuable and powerful molecular marker in unraveling medically important cockroach species, particularly when morphological 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2-azarihamidian-rtl, gally proof, last version.doc iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 9 original article larval habitat characteristics of mosquitoes of the genus culex (diptera: culicidae) in guilan province, iran s azari-hamidian school of public health, guilan university of medical sciences, rasht, iran (received 17 oct 2006; accepted 28 mar 2007) abstract an investigation was carried out during april to december 2000 to study mosquito fauna and ecology in guilan province of northern iran. the mosquito larvae were collected by dipping method and larval habitat characteristics recorded according to hydro-ecological features. in total, 3937 larvae of the genus culex from 92 larval breeding sites were collected. six species of the genus culex; cx. mimeticus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus, cx. hortensis, and cx. territans were identified in the province and respectively comprised 10.3%, 47.2%, 2.2%, 31%, 6.5%, and 2.8% of the samples. most of the larvae were collected from the natural habitats (75.6%) such as river edges (6.5%), riverbed pools (28.2%), rain pools (47.8%), stream edges (9.4%), grasslands (1.9%), marshes (2.8%), and hoof-prints (3.4%) and others from artificial habitats (24.4%) including rice fields (32.1%), irrigation channels (7.1%), wells (16.4%), discarded concrete tubes (33.1%), discarded tires (11.0%), and agricultural water-storage pools (0.3%). the ecology of cx. pipiens and cx. tritaeniorhynchus, which are the most prevalent species and potentially involved in the transmission of many pathogens to humans and domesticated animals, must be extensively studied. keywords: culex, ecology, iran, larva introduction the genus culex l. includes 25 subgenera and at least 751 species in the world fauna (service 1993, reinert 2001). certain species of the genus culex are involved in the transmission of the various arboviral and filarial diseases to humans and domesticated animals and/or are important for their biting nuisance in different parts of the world (cranston et al. 1987, service 1993). naficy and saidi (1970) reported sindbis virus in iran as well as west nile virus in iran and guilan province of northern iran. siavashi and massoud (1995) reported human cutaneous dirofilariasis in guilan province. there is no information about the vectors of these diseases in iran. the possibility of some culicine borne arboviral outbreaks like japanese encephalitis (je) and rift valley fever in the who eastern mediterranean region, including iran, is noteworthy (who 2004). the species cx. tritaeniorhynchus giles was mentioned as the vector of je in iran and iraq (harbach 1988). based on the last classification of the tribe aedini (reinert et al. 2004) and the genus anopheles meigen (harbach 2004), iranian mosquitoes include nine genera and near to seventy species (zaim and cranston 1986, azari-hamidian et al. 2002b, sedaghat and harbach 2005, azarihamidian et al. 2006, doosti et al. 2006). while providing exact iranian mosquito checklist needs more investigations, 24 species of the genus culex have been recorded so far in iran (gutsevich 1943, lotfi 1970, 1973, 1976, danilov 1975, zaim and cranston 1986, harbach 1988). the old records of cx. apicalis adams in the palaearctic region by different investigators, corresponding author: s azari-hamidian, tel/fax: +98 131 3229599, e-mail: azari@gums.ac.ir iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 10 including iran by gutsevich (1943), were misidentifications. this species is very similar to cx. territans walker and occurs only in the nearctic region (cranston et al. 1987). cx. impudicus ficalbi was recorded in iran only by lotfi (1970, 1973, 1976) and was not mentioned in the checklist of iranian culex by zaim and cranston (1986). zaim and cranston (1984) noted that the records of cx. vishnui theobald in iran were misidentifications and the correct name is cx. pseuodovishnui colless. also, culex vagans wiedemann and cx. torrentium martini are hard differentiated from cx. pipiens l. in the larval stage (zaim and cranston 1986, harbach 1988), so the records of these species based on the larval stage (lotfi 1970, 1976) are doubtful. of course, danilov (1975) recorded cx. torrentium in rasht, northern iran. cx. univittatus theobald in southwestern asia and iran was considered to refer to cx. perexiguus theobald. the former species is an afrotropical one that occurs only in yemen republic in south-western asia (harbach 1988). at last, zaim and cranston (1986) mentioned 19 species of the genus culex in iran. almost half of the iranian mosquito species (31 species) and seven genera have been reported in guilan province (danilov 1975, harbach 1988, azari-hamidian et al. 2002b, sedaghat et al. 2003, gholizadeh et al. 2004). there are more faunistic data about the genus culex than other culicine mosquitoes in iran, but the information on their ecology is not great. dow (1953) presented some notes on mosquitoes including six species of the genus culex in different areas of iran such as larval breeding places and associated species. golestani (1967) studied cx. pipiens (as form molestus forskal) in the sewage wells of tehran. lotfi (1970, 1973, 1976) briefly noted some ecological aspects of the culex species that she found in iran including larval breeding places and water temperature and ph. yaghoobi-ershadi et al. (1986) showed the characteristics of the larval breeding sites and associated species of mosquitoes including eight species of the genus culex in minab, hormozgan province (southern iran). zaim (1987a) found six species of this genus in kashan, isfahan province (central iran), and noted some aspects of their adult and larval ecology. zaim (1987b) reviewed the distribution and larval habitat characteristics of the iranian culicinae including 19 species of the genus culex. mousakazemi et al. (2000) stressed the fauna and ecology of mosquitoes including cx. pipiens, cx. theileri theobald, and cx. perexiguus in zarrin-shahr and mobarakeh area of isfahan province. azari-hamidian et al. (2002a) studied mosquito fauna in rasht county of guilan province, northern iran, and presented some breeding places and associated species of cx. mimeticus noe, cx. pipiens, cx. theileri, and cx. tritaeniorhynchus. the ecological data about culex larvae in iran are mostly based on the little information of faunal records, only yaghoobi-ershadi et al. (1986) and zaim (1987b) exactly studied the larval habitat characteristics of the genus, especially based on the specimens of southern iran. there is little information about the ecology of this genus in northern iran. this article is the first that specializes in some aspects of the ecology of culex larvae such as their larval habitat characteristics, water temperature, and associated species in northern iran. materials and methods study area guilan province in the caspian sea littoral of northern iran, between caspian sea and alborz mountain chains has coastal, plain, foothill, and mountainous areas with an area of approximately 14700 square kilometers. the province has a temperate climate and relatively warm humid summer, and is located between 36o34´38o27´ n latitudes and 48o34´-50o36´ e longitudes. with about 1200 mm annual rainfall, guilan province has the highest rainfall in iran. the vast forests of hyrcania and temperate climate of this province provide ideal conditions for mosquitoes to breed. iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 11 specimen and data collection guilan province includes 16 counties. in each county one fixed and three variable sites (including different topographical areas) were randomly selected and larval collection was carried out for 15-20 minutes in the sites during the spring, summer, and autumn seasons in 2000. larvae were collected from natural breeding sites such as river edges, riverbed pools, rain pools, marshes, grasslands, and tree holes and from artificial breeding sites such as rice fields, irrigation channels, wells, discarded tires, and buckets by means of dipper, pipette, and bucket. physical and biological characteristics of the larval habitats such as habitat situation (permanent or transient, standing or running), habitat kind (natural or artificial), vegetation situation (with or without vegetation), bottom type, sunlight situation (full or partial sunlight or shaded), water situation (clear or turbid), and water temperature were recorded. taxonomic note despite collecting and identifying anopheles hyrcanus (pallas) and an. pseudopictus grassi in the adult stage in guilan province (dow 1953, azari-hamidian et al. 2002b), these species have not been differentiated from each other in the larval stage in the present investigations and mentioned only as an. ’hyrcanus’ in this article. different species of an. maculipennis complex have been recorded in the province according to egg pattern and polymerase chain reaction (pcr) (dow 1953, azari-hamidian et al. 2002b, sedaghat et al. 2003, gholizadeh et al. 2004), but they have not been differentiated in the larval stage either and are cited as an. maculipennis meigen sensu lato. the abbreviations for mosquito genera and subgenera follow reinert (2001) and reinert et al. (2004). results a total of 6656 larvae including 1547 anopheline larvae (23.2%) and 5109 culicine larvae (76.8%) were collected from 127 larval breeding sites on 55 occasions. among 127 larval breeding sites 14 (11.0%) included only the subfamily anophelinae, 37 (29.2%) included only the subfamily culicinae, and 76 (59.8%) included both subfamilies anophelinae and culicinae. anopheline larvae were found in 90 breeding sites (70.8%) and culicine larvae in 113 (88.9%). during this study, 3937 larvae of the genus culex were collected from 92 larval breeding sites (72.4% of the total breeding sites and 81.4% of the culicine breeding sites) on 44 occasions (80.0% of the total). six species of the genus culex; cx. (culex) mimeticus, cx. (cux.) pipiens, cx. (cux.) theileri, cx. (cux.) tritaeniorhynchus, cx. (maillotia) hortensis ficalbi, and cx. (neoculex) territans were found among the samples. the larvae were collected from many different natural habitats (75.6%) such as river edges (6.5%), riverbed pools (28.2%), rain pools (47.8%), stream edges (9.4%), grasslands (1.9%), marshes (2.8%), and hoof-prints (3.4%) and artificial habitats (24.4%) including rice fields (32.1%), irrigation channels (7.1%), wells (16.4%), discarded concrete tubes (33.1%), discarded tires (11.0%), and agricultural waterstorage pools (0.3%). abundance percentages of these species are shown in table 1. details of the larval habitat characteristics of the six species are shown in table 2. the association occasions of the culex species with other collected species are shown in table 3. the association percentages of the species with other mosquito larvae are shown in table 4. culex (culex) mimeticus noe cx. mimeticus larvae were collected on 12 occasions (21.8% of the total) from 14 larval breeding sites (11.0% of the total and 12.3% of the culicine breeding sites) in june, july, august, september, october, and november 2000. maximum and minimum breeding site water temperatures were respectively 28 oc and 12 oc, and the mean temperature was 21.6 oc (for 13 larval breeding sites). culex (culex) pipiens l. cx. pipiens larvae were collected on 28 occasions (50.9% of the total) from 40 larval breeding sites (31.4% of the total and 35.3% of iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 12 the culicine breeding sites) in april, may, june, july, august, september, october, and november 2000. maximum and minimum breeding site water temperatures were respectively 32 oc and 12 oc, and the mean temperature was 20.1 oc (for 39 larval breeding sites). culex (culex) theileri theobald cx. theileri larvae were collected on 7 occasions (12.7% of the total) from 10 breeding sites (7.8% of the total and 8.8% of the culicine breeding sites) in may, june, july, and august 2000. maximum and minimum breeding site water temperatures were respectively 33 oc and 20 oc, and the mean temperature was 25.2 oc (for 10 larval breeding sites). culex (culex) tritaeniorhynchus giles cx. tritaeniorhynchus larvae were collected on 25 occasions (45.4% of the total) from 44 breeding sites (34.6% of the total and 38.9% of the culicine breeding sites) in june, july, august, september, october, and november 2000. maximum and minimum breeding site water temperatures were respectively 33 oc and 12 oc, and the mean temperature was 23.5 oc (for 44 larval breeding sites). culex (maillotia) hortensis ficalbi cx. hortensis larvae were collected on 7 occasions (12.7% of the total) from 15 breeding sites (11.8% of the total and 13.2% of the culicine breeding sites) in may, august, and october 2000. maximum and minimum breeding site water temperatures were respectively 31 oc and 12 oc, and the mean temperature was 19.6 oc (for 15 larval breeding sites). culex (neoculex) territans walker cx. territans larvae were collected on 8 occasions (14.5% of the total) from 11 breeding sites (8.6% of the total and 9.7% of the culicine breeding sites) in april, june, july, august, september, and october 2000. maximum and minimum breeding site water temperatures were respectively 32 oc and 16 oc, and the mean temperature was 23.0 oc (for 11 larval breeding sites). table 1. composition and abundance of the genus culex larvae in guilan province, iran, apr to dec 2000 category no. percentage in family (%) percentage in subfamily (%) percentage in genus (%) cx. mimeticus 405 6.08 7.93 10.29 cx. pipiens 1858 27.92 36.37 47.19 cx. theileri 88 1.32 1.72 2.24 cx. tritaeniorhynchus 1220 18.33 23.88 30.99 cx. hortensis 256 3.85 5.01 6.50 cx. territans 110 1.65 2.15 2.79 total 3937 59.15 77.06 100 culicinae 5109 76.76 culicidae 6656 iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 13 table 2. larval habitat characteristics and occurrence percentages of larvae of the genus culex in guilan province, iran, apr to dec 2000 larval breeding site characteristics and habitats cx. mimeticus (%) cx. pipiens (%) cx. theileri (%) cx. tritaeniorhynchus (%) cx. hortensis (%) cx. territans (%) culex (%) a) habitat situation 1. permanent 0.49 19.05 0.74 4.3 3.64 9.67 2. transient 99.51 80.95 100 99.26 95.7 96.36 90.33 3. slow-running water 0.25 9.31 0.49 1.17 2.73 4.73 4. standing water 99.75 90.69 100 99.51 98.83 97.27 96.27 b) vegetation situation 1. without vegetation 7.40 68.14 1.14 24.18 76.95 51.82 46.89 2. with vegetation 92.60 31.86 98.86 75.82 23.05 48.18 53.11 2a) out of water 14.40 98.64 83.90 72.21 35.59 13.20 67.28 2b) water surface 1.51 0.66 2c) beneath of water surface 86.13 35.97 19.54 38.48 66.10 86.80 47.53 c) bottom type 1. mud 22.47 82.19 84.09 55.24 59.45 57.8 62.73 2. sand 15.06 16.57 39.37 15.6 11.32 3. gravel 62.47 17.81 15.91 28.19 1.18 26.6 25.95 d) water situation 1. turbid 2.83 0.25 1.35 2. clear 100 97.17 100 99.75 100 100 98.65 e) sunlight situation 1. full sunlight 99.75 35.67 95.45 84.09 36.33 6.36 57.79 2. partial sunlight 0.25 12.43 3.3 28.12 35.46 9.74 3. shaded 51.19 4.55 12.61 35.55 58.18 32.47 f) habitat kind 1. natural habitat 88.15 67.30 78.62 80.59 84.77 98.18 75.57 1a. river edge 14.85 0.85 8.95 17.98 12.04 6.53 1b. riverbed pool 83.19 8.29 66.40 26.15 30.41 53.70 28.25 1c. stream edge 0.28 13.40 11.81 2.78 9.43 1d. grassland 13.60 4.36 1.85 1e. marsh 6.35 2.76 1f. rain pool 1.68 63.36 20 48.62 51.61 31.48 47.78 1g. hoof-print 7.75 0.11 3.40 2. artificial (man-made) habitat 11.85 32.70 21.38 19.41 15.23 1.82 24.43 2a. rice field 100 7.81 100 66.19 94.87 50 32.08 2b. rice irrigation channel 32.38 7.08 2c. well 24.56 5.13 50 16.36 2d. discarded concrete tube 50.72 33.13 2e. discarded tire 16.91 11.04 2f. water-storage pool 1.43 0.31 iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 14 table 3. the association occasions of culex larvae with different mosquito larvae in guilan province, iran, apr to dec 2000 table 4. association percentages of culex larvae with different mosquito species larvae in guilan province, iran, apr to dec 2000 species t otal occasions a n. claviger a n.’hyrcanus’ a n. m aculipennis s.l. a n. plum beus a n. superpictus a e. vexans c x. m im eticus c x. pipiens c x. theileri c x. tritaeniorhynchus c x. hortensis c x. territans c s. longiareolata c s. m orsitans c s. annulata u r. unguiculata cx. mimeticus 14 12 10 2 3 4 5 1 1 1 1 1 cx. pipiens 40 2 13 14 1 1 4 4 9 3 12 4 5 3 1 3 cx. theileri 10 6 8 2 2 8 1 1 cx. tritaeniorhynchus 44 1 31 30 5 1 5 12 8 3 4 3 2 cx. hortensis 15 4 4 2 2 1 1 4 1 4 1 5 2 cx. territans 11 4 5 5 2 1 5 3 1 species association frequency (%) cx. mimeticus an.’hyrcanus’, an. maculipennis s.l., cx. tritaeniorhynchus 41.98 an.’hyrcanus’, an. maculipennis s.l. 16.05 an.’hyrcanus’, an. maculipennis s.l., an. superpictus, cx. tritaeniorhynchus 12.34 an. maculipennis s.l. 11.85 cx. hortensis, cx. pipiens 9.38 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens 7.16 an.’hyrcanus’, an. superpictus, ur. unguiculata 0.74 an.’hyrcanus’, cx. pipiens, cx. territans, cx. tritaeniorhynchus 0.25 an.’hyrcanus’, ae. vexans, cx. pipiens, cs. annulata, cs. morsitans 0.25 total 100 cx. pipiens alone 31.16 an. maculipennis s.l. 11.95 an.’hyrcanus’, cx. hortensis, cx. tritaeniorhynchus 10.55 cx. tritaeniorhynchus 6.08 an.’hyrcanus’, an. maculipennis s.l., cx. theileri, cx. tritaeniorhynchus 5.49 an.’hyrcanus’, ae. vexans, cs. annulata 4.41 an. claviger, an.’hyrcanus’, an. maculipennis s.l., ae. vexans 3.93 cx. tritaeniorhynchus, cs. longiareolata 3.40 an.’hyrcanus’, an. maculipennis s.l., cx. mimeticus 3.34 an. plumbeus 2.91 an.’hyrcanus’, an. maculipennis s.l., cx. territans 2.31 cx. hortensis, cs. annulata 2.26 an. maculipennis s.l., cx. tritaeniorhynchus 2.26 ae. vexans 1.72 an.’hyrcanus’ 1.61 an.’hyrcanus’, ae. vexans, cx. mimeticus, cs. annulata, cs. morsitans 1.45 an. claviger, cx. territans 0.97 an.’hyrcanus’, an. maculipennis s.l., cx. tritaeniorhynchus 0.97 an. maculipennis s.l., cx. territans 0.86 an.’hyrcanus’, an. maculipennis s.l., cx. theileri, cx. tritaeniorhynchus, cs. longiareolata 0.7 cx. territans 0.59 an.’hyrcanus’, cx. mimeticus, cx. territans, cx. tritaeniorhynchus 0.38 cs. longiareolata 0.32 an.’hyrcanus’, an. maculipennis s.l., cx. theileri 0.16 an.’hyrcanus’, an. superpictus, cx. hortensis, cx. tritaeniorhynchus 0.11 cx. mimeticus, cx. hortensis 0.11 total 100 iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 15 table 4. continue… cx. theileri an.’hyrcanus’, an. maculipennis s.l., cx. tritaeniorhynchus 37.50 an.’hyrcanus’, an. maculipennis s.l., an. superpictus, cx. tritaeniorhynchus 19.32 an.’hyrcanus’, cx. hortensis, cx. tritaeniorhynchus 18.18 an. maculipennis s.l., cx. tritaeniorhynchus 11.36 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens 3.41 an. superpictus, cx. tritaeniorhynchus 3.41 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens, cx. tritaeniorhynchus 3.41 an. maculipennis s.l. 2.27 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens, cx. tritaeniorhynchus, cs. longiareolata 1.14 total 100 cx. tritaeniorhynchus an.’hyrcanus’, an. maculipennis s.l. 30.90 an.’hyrcanus’ 11.97 cx. pipiens 9.51 alone 6.64 an.’hyrcanus’, an. maculipennis s.l., cx. theileri 5.90 an.’hyrcanus’, cx. hortensis, cx. theileri 5.24 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens, cx. theileri, cs. longiareolata 4.92 an.’hyrcanus’, an. maculipennis s.l., an. superpictus, cx. mimeticus 3.61 an.’hyrcanus’, an. maculipennis s.l., cx. mimeticus 3.36 an.’hyrcanus’, an. maculipennis s.l., an. superpictus, cx. theileri 3.11 an. maculipennis s.l., an. superpictus 2.95 an.’hyrcanus’, an. superpictus, cx. hortensis, cx. pipiens 2.30 an. maculipennis s.l., cx. pipiens 2.13 an. superpictus, cx. theileri 1.89 an.’hyrcanus’, ae. vexans 1.56 an.’hyrcanus’, an. maculipennis s.l., cx. hortensis 1.15 an.’hyrcanus’, an. maculipennis s.l., cx. territans 0.82 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens, cx. theileri 0.74 an.’hyrcanus’, cx. mimeticus, cx. pipiens, cx. territans 0.49 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens 0.41 an. claviger, an. maculipennis s.l., cx. territans 0.16 an.’hyrcanus’, cx. hortensis, cx. pipiens 0.16 cx. pipiens, cs. longiareolata 0.08 total 100 cx. hortensis cs. longiareolata 30.47 an. claviger 14.85 ae. vexans 14.46 an. maculipennis s.l. 14.06 an. claviger, cs. longiareolata, cs. annulata 7.81 an. claviger, cs. longiareolata 6.25 an. claviger, an. superpictus, cs. longiareolata 3.52 an.’hyrcanus’, cx. pipiens, cx. tritaeniorhynchus 2.34 an.’hyrcanus’, cx. theileri, cx. tritaeniorhynchus 1.56 an.’hyrcanus’, an. superpictus, cx. pipiens, cx. tritaeniorhynchus 1.56 alone 1.17 cx. pipiens, cs. annulata 0.78 cx. mimeticus, cx. pipiens 0.78 an.’hyrcanus’, an. maculipennis s.l., cx. tritaeniorhynchus 0.39 total 100 iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 16 table 4. continue… discussion results of this study revealed that the genus culex species laid their eggs in many different natural and artificial habitats. in parallel to the present research, service (1993) stressed this genus is, by far, the largest, most common, and most important genus of the tribe culicini. in these investigations, the species of this genus were the most abundant species of mosquitoes in the studied area (table 1). cx. mimeticus larvae are found in pooled streams, springs, and pools (horsfall 1955). dow (1953) collected the larvae in shallow pebbly pools in northern iran. lotfi (1970, 1976) found it in river edges and riverbed pools with water temperature 18-24 oc and ph 6-7.5. some other breeding places of this species are pools in swampy ground, irrigation ditches, and animal hoof-prints (harbach 1988). zaim (1987b) found this species mostly in natural habitats (88.7%), and in man-made ones such as overflow water and rice fields. azari-hamidian et al. (2002a) observed it only in natural habitats. this phenomenon was nearly seen in this study either, and a few cx. mimeticus larvae (11.8%) found only in rice fields as artificial habitat (table 2). harbach (1988) mentioned an. cinereus theobald, as an. hispaniola (theobald), an. sergentii (theobald), and an. superpictus larvae with cx. mimeticus, and azari-hamidian et al. (2002a) collected it with an. ’hyrcanus’. an. cinereus does not exist in iran and an. sergentii occurs only in southern iran, but other species were collected with cx. mimeticus in the current research (table 4). cx. pipiens larvae have been found in many different natural and artificial habitats including underground ones such as flooded cellars, drains, wells, septic tanks, underground train systems, abandoned mine tunnels, and coal mines (horsfall 1955, cranston et al. 1987, harbach 1988, service 1993). the wide variety of the breeding places can explain why this species is one of the most abundant and common mosquito species in iran as well as guilan province (table 1 and 2). in iran, dow (1953), lotfi (1970, 1976), and yaghoobi-ershadi et al. (1986) found this species mostly in natural habitats. also, zaim (1987b) collected it mainly in natural habitats (63.7%), but mousakazemi et al. (2000) collected it only in rice fields (artificial habitat) and azari-hamidian et al. (2002a) found it mainly in man-made habitats (83.7%). in the present study, the species was collected mainly in natural larval sites (67.3%), but it was collected from man-made ones (32.7%) more than any other culex species (table 2). it seems that the most favorable breeding places of cx. pipiens in cities are the house ponds, wells, and the sewage wells (golestani 1967, lotfi 1976, zaim 1987a). lotfi (1976) noted the water temperature and ph for cx. pipiens 18-27 oc and 7-8 (once 6.2), respectively. dow (1953) found cx. cx. territans an. claviger 26.36 an.’hyrcanus’, an. maculipennis s.l., cx. tritaeniorhynchus 26.36 an.’hyrcanus’, an. maculipennis s.l. 15.45 cx. pipiens 11.82 an.’hyrcanus’, an. maculipennis s.l., cx. pipiens 9.09 an.’hyrcanus’, cx. mimeticus, cx. pipiens, cx. tritaeniorhynchus 2.73 an. claviger, an. maculipennis s.l., cx. tritaeniorhynchus 2.73 ae. vexans, cs. morsitans 1.82 an. claviger, an.’hyrcanus’, ae. vexans 1.82 an. maculipennis s.l., cx. pipiens 0.91 an. claviger, cx. pipiens 0.91 total 100 iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 17 pipiens larvae with an. claviger and cx. quique fasciatus say, yaghoobi-ershadi et al. (1986) with cx. perexiguus, as cx. univittatus, cx. theileri, cx. tritaeniorhynchus, cx. deserticola kirkpatrick, and cs. longiareolata, and azarihamidian et al. (2002a) with an. ’hyrcanus’, an. maculipennis s.l., ae. vexans, and cx. tritaeniorhynchus. among these species, cx. quiquefasciatus, cx. perexiguus, and cx. deserticola were not found in guilan province in the current study, but cx. pipiens was with other species (table 4). cx. pipiens may be associated with many species and share some larval sites with cx. torrentium (cranston et al. 1987). these species are difficult to be differentiated from each other and cx. vagans in the larval stage (zaim and cranston 1986, harbach 1988). there is little information about these two species in iran. cx. theileri is found in a wide range of breeding sites such as streams, irrigation ditches, swamps, different pools, springs, disused wells, open cisterns, and seepage water (harbach 1988). lotfi (1970, 1976) found it in seepages, small ponds, grassy ponds, rice fields, and agriculture water-storage pools with water temperature 1627 oc and ph 7-8 (once 5.4) in iran. dow (1953) and yaghoobi-ershadi et al. (1986) found it in different natural and artificial habitats. zaim (1987b) noted that the species was mainly in natural habitats (82.1%) and mousakazemi et al. (2000) collected it from rice fields in isfahan province more than any other mosquito species. azari-hamidian et al. (2002a) found it only in natural habitats (pools). in the present study, cx. theileri was often collected from natural breeding sites (78.6%) and a few larvae (21.4%) were found only in rice fields as man-made ones (table 2). the larvae of cx. theileri were collected with an. ’hyrcanus’, an. maculipennis s.l., an. superpictus, cx. hortensis, cx. pipiens, cx. tritaeniorhynchus, cx. quiquefasciatus, cx. perexiguus, cx. deserticola, cs. longiareolata, oc. caspius s.l., and ur. unguiculata in different areas of iran (dow 1953, yaghoobi-ershadi et al. 1986, azari-hamidian et al. 2002a) and harbach (1988) mentioned that the larvae often found alone or with cx. pipiens, cx. perexiguus, or cx. antennatus (becker). in the current investtigation, cx. theileri larvae were not observed alone and among these species cx. perexiguus, cx. quiquefasciatus, cx. deserticola, and cx. antennatus were not found in guilan province and oc. caspius s.l. was found only in the adult stage (azari-hamidian et al. 2002b), but the species was associated with other species except for ur. unguiculata (table 4). cx. tritaeniorhynchus is typically found in rice fields, flood waters, and marshy areas and some other natural or artificial habitats (harbach 1988). lotfi (1970, 1976) found this species in rice fields, ponds, river edges, and stream edges with water temperature 19-33 oc and ph 6.4-7. zaim (1987b) found it mostly (76%) in man-made breeding sites especially rice fields (68.5%), more than any other culex species in iran, but azari-hamidian et al.(2002a) found it mainly (83.9%) in natural habitats in rasht county of northern iran. this phenomenon was observed in this investigation either, and the species mainly found in natural habitats (80.6%) (table 2). the reason probably is that there are many different natural breeding sites in guilan provine because of great annual rainfall, but other areas of iran mainly have less annual rainfall and natural breeding sites are fewer. horsfall (1955) mentioned many species with cx. tritaeniorhynchus larvae. some of them have been recorded in iran but was not found in this study such as: an. marteri senevet and prunnelle, an. sergentii, cx. bitaeniorhynchus giles, and cx. vagans. dow (1953) found cx. tritaeniorhynchus with an. ’hyrcanus’ and an. melanoon hackett (as an. subalpinus hackett and lewis) of the maculipennis complex, and yaghoobiershadi et al. (1986) with cx. pipiens, cx. theileri, cx. sinaiticus kirkpatrick, cx. quiquefasciatus, cx. perexiguus, cx. deserticola, oc. caspius s.l., and ur. unguiculata in southern iran and azari-hamidian et al. (2002a) with an. ’hyrcanus’, an. maculipennis s.l., ae. vexans, and cx. pipiens in northern iran. among these species cx. sinaiticus, cx. quiquefasciatus, cx. perexiguus, and cx. deserticola were not found iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 18 in this study and oc. caspius s.l. was found only in the adult stage (azari-hamidian et al. 2002b). cx. tritaeniorhynchus was associated with other species except for ur. unguiculata (table 4). cx. hortensis is found in algal mats, seeps, brackish pools, domestic containers, and cement vats (horsfall 1955). dow (1953) found it in the irrigation ditches, small, spring like pools of the river banks and shallow pools in the river beds. zaim (1987b) found the species mostly (93.5%) in natural habitats. in the present study, this species was mainly (84.8%) found in natural habitats (table 2). lotfi (1976) collected this species in seepages and agriculture waterstorage pools with water temperature 15-26 oc and ph 6-7.5 and associated species were cx. theileri and cx. pipiens in northern iran and cx. theileri and cx. quinquefasciatus in southern iran. dow (1953) found cx. hortensis with an. maculipennis s.l., an. superpictus, cx. theileri, and cs. longiareolata in northwestern iran. zaim (1987a) stressed that the species was found mainly with an. maculipennis s.l., cs. longiareolata, and cs. annulata. in this study, this species was with all of these species except for cx. quiquefasciatus that it was not found in guilan province (table 4). cx. territans is mostly found in natural swamps, ponds, and pools (horsfall 1955). lotfi (1973, 1976) found it in ponds with water temperature 23-27 oc and ph 7-7.5. zaim (1987b) found it in small permanent pools. there are few records of utilization of artificial habitats including containers such as barrel for this species (cranston et al. 1987). it is noteworthy that this species was found in man-made habitats (1.8%) such as rice fields and wells as well as natural ones (98.2%), in this study (table 2). cranston et al.(1987) noted this species is often in company with an. claviger. this phenomenon was exactly observed in this investigation and most of cx. territans larvae (26.3%) were associated with this species (table 3 and 4). horsfall (1955) listed many species with cx. territans larvae. among them, an. claviger, ae. vexans, cx. pipiens, cx. quinquefasciatus, and cs. morsitans are in iran. in the present study, cx. quinquefasciatus was not found in guilan province, but other species were associated with cx. territans (table 4). during this investigation all reported species of the genus culex in guilan provoince were collected (zaim 1987b, azari-hamidian et al. 2002b), except for two records. danilov (1975) reported cx. torrentium from rasht in guilan province. the best character for identification of this species from cx. pipiens complex is the characters of the male hypopygia (cranston et al. 1987, harbach 1988). there is a unique record of cx. quinquefasciatus in enzeli of guilan province by harbach (1988). this is out of the usual distribution of this species that occurs in central and southern iran (zaim 1987b). there is no more information about this record (professor re harbach, personal communication). the ecology of cx. pipiens and cx. tritaeniorhynchus, which are involved in the transmission of many pathogens to humans and domesticated animals and making nuisance, yet to be extensively studied. larval habitat water temperature, ph, electric conductivity (ec), turbidity, and different organic and inorganic compounds are items which should be considered in forthcoming studies. all specimens of this study are deposited in the museum of medical entomology at the school of public health, guilan university of medical sciences. acknowledgements the author is grateful to professor j minar, institute of parasitology, czech republic, for studying specimens, professor jf reinert, center for medical, agricultural and veterinary entomology, usa, for providing reprint of his articles, professor j jezek, natural history museum of prague, czech republic, professor i. gelbic, institute of entomology, czech republic, professor f rettich, national institute of public health, czech republic, professor re harbach, the natural history museum, uk, for their important correspondence and assistance. iranian j arthropod-borne dis, (2007), 1(1): 9-20 s azari-hamidian: larval habitat characteristics… 19 the author also wishes to thank professor h vatandoost, department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran, for reviewing the manuscript. this study was financially supported by guilan university of medical sciences. references azari-hamidian s, yaghoobi-ershadi mr, javadian e (2002a) the distribution and larval habitat characteristics of mosquitoes (diptera: culicidae) in rasht county (guilan province, 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khosravi 1, mansour mayahi 2, farnoosh kaviani 3, mohammad nemati 4 1department of pathobiology, faculty of veterinary medicine, shahid chamran university of ahvaz, ahvaz, iran 2department of clinical sciences, faculty of veterinary medicine, shahid chamran university of ahvaz, ahvaz, iran 3student of veterinary medicine, faculty of veterinary medicine, shahid chamran university of ahvaz, ahvaz, iran 4razi reference laboratory of scorpion research, razi vaccine and serum research institute, karaj, iran (received 19 dec 2015; accepted 20 dec 2017) abstract background: many elements such as immunosuppressive, chemotactic and anti-inflammatory peptide that could effect on human and animals physiologic system were determined in venom. this study evaluated the use of mesobuthus eupeus scorpion venom fractions as an immunomodulator. methods: the venom fractions collected from khuzestan province in south west of iran were purified by ion exchange chromatography. elution of the bounded elements was done by using a linear gradient of sodium chloride (0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5 and 2 molar). the fractions were analyzed by bradford spectrophotometric and sds-page method. after treatments of chicken with venom fractions and sheep red blood cell (srbc), direct haemagglutination test in microtiter plate was used for the determination of the chicken srbc antibody titer. results: the fraction released by nacl 1.25m had the highest protein concentration. the highest and lowest antibody titer was determined at the fifth (nacl 0.75 molar) and seventh fraction (nacl 1.25 molar), respectively. conclusion: different protein profile of isolated fractions, were associated with various effect on immune response. both enhancing and suppressing of the chicken humoral immune response to srbc were observed after m. eupeus faction’s venom treatment. it is due to biological functions of venom components. purification of these elements would provide the new agents for immune responses manipulation. keywords: venom, mesobuthus eupeus, immune response introduction scorpion venom contains biological compounds as short-chain peptides, bioactive substances such as enzymes, nucleotides, lipids, mucoprotein, mucopolysaccharides, biogenic amines, neuroactive peptides, protease inhibitors, phospholipase, hyaluronidase (1) and other unknown compounds, which could affect on the physiologic system of vertebrate and invertebrate organisms (2). only 400 peptides out of the expected 100000 peptides of venom have toxic effects on human and animals (3). envenomated organism produces various mediators, which contains both pro and anti-inflammatory cytokines (4). scorpion venom could induce local and systemic inflammatory responses. the local effects can lead to the activation of vascular endothelium, increase the vascular permeability and leukocytes migration to the affected tissues. the systemic inflammation persuades the acute phase *corresponding author: dr mohammad khosravi, e-mail: dr.khosravim@gmail.com j arthropod-borne dis, december 2017, 11(4): 497–503 m khosravi et al.: the effects of … 498 http://jad.tums.ac.ir published online: december 30, 2017 response (5). the consequence of this inflammatory reaction is undergoing by a variety of factors such as duration of the stimulus and the balance between the inflammatory and anti-inflammatory mediators (4). different methods, used for fractionation, purification, analysis of the structure and characterization of toxins have made it possible to clarify the components of venoms. identification of toxins or others biologically important peptide were the outcome of these efforts (6). many elements such as immunosuppressive (7) chemotactic (8, 9) and anti-inflammatory peptides were determined in venom and are as potential therapeutic agents (10). scorpion’s venom has been used in medicine for relation of pain, osteoporosis, neurological diseases, homeostasis and rheology, cancer and autoimmune disease (11, 12). scorpions were divided into 13 families and about 1400 species and subspecies (13). the medical importance scorpion species, belonging to the family buthidae mainly, androctonus, buthus, mesobuthus, buthotus, parabuthus, and leirus (4). the m. eupeus venom could affect on cytokine releases with both pro and anti-inflammatory properties (14, 15). venom components that affect on immune response could be used as immunomodulatory agents. we investigated the possibility of using different m. eupeus scorpion venom fractions as immunomodulator like elements for elevation or suppression of immune response. materials and methods venom mesobuthus eupeus scorpions were collected from khuzestan province in south west of iran (31°19′–32°73′n, 48°41′–49°4′e) milked by electric stimulation. the total protein concentration was measured using the usual bradford spectrophotometric method with bovine serum albumin (bsa) as standard. chickens forty adult rass 308 chickens were selected and kept in the isolation facility. feed and water were provided during the experiment. experimental procedures were according to the guidelines of the animal care. purification of venom fractions the venom fractions were purified by ion exchange chromatography. anion-exchange chromatography was performed using diethylaminoethyl cellulose (deae-c) column (sigma, product number: d3764) at a flow rate of 1ml/min. the column-stabilizing buffer was 0.05 molar tris-hcl, ph 8.6. tow milliliter of m. eupeus venom were dissolved in stabilizing buffer and loaded on the column (12 mg/ml). after washing away of the unbound components, elution of the bound elements was done by using a linear gradient of sodium chloride (0.1, 0.25, 0.5, 1, 1.25, 1.5, 1.75 and 2 molar) in 3ml of stabilizing buffer. all the fractions were dialyzed overnight against distilled water, ph 7.2 at 4 °c. the protein concentrations of the collected fractions were measured by bradford spectrophotometric method (accu reader, serial no: 96501575) with bsa as standard. sds-page the protein profiles of crude venom, as well as the purified fractions, were analyzed by sds-page (16), the stacking and resolving gel concentration was 4% and 11%, respectively. samples were denatured by boiling in loading buffer containing sds and β-mercaptoethanol prior to loading onto the gel. proteins were stained with 1% coomassie blue r 250. molecular mass standard (vivantis, product no: pr0602) was run in parallel in order to calculate molecular weights of the proteins. j arthropod-borne dis, december 2017, 11(4): 497–503 m khosravi et al.: the effects of … 499 http://jad.tums.ac.ir published online: december 30, 2017 immunomodulatory analysis of the purified fractions the purified fractions were diluted to 200 μg/ml with pyrogen-free water. the 0.5ml of each fraction injected subcutaneously at breast region of four adult chickens. the sheep red blood cells (srbc) were prepared by centrifugation of ethylenediaminetetraacetic acid (edta) anticoagulated whole blood, followed by 3 times washing with normal saline. the suspension of fresh 2% srbc was prepared and the 0.5ml injected in intra-muscularly route. the clinical symptoms were recorded during the test periods. two ml of the venous blood were collected from the injected chickens 5 and 10 d after the injections. the serum was separated and kept at minus 20 °c until use. hemagglutination test direct hemagglutination test in microtiter plate used for the determination of the srbc antibody titer. serum samples were serially diluted in a microtiter plate and 1% srbcs were added. after mixing, the plates were incubated at 37 oc for 45min and examined for hemagglutination. the test repeated 3 times for each samples and antibody titer was determined as the equal of the maximum dilution that exhibiting hemagglutination. results ion exchange chromatography the total protein concentration of the eluted fractions has been measured and as shown in table 1, the eluted fraction by nacl 1.25m had the highest percentage of protein. sds-page venom fractions had a different protein profile (fig. 1). proteins of the venom had 15 detectable bands, which were between ≤ 5 and ≥ 175 kda on 12% polyacrylamide gel electrophoresis. hemagglutination test the fifth fraction (nacl 0.75) had the lowest hemagglutination titer, this was less than the control titer. the seventh fraction (nacl 1.25) had the highest anti srbc titer (table 2). table 1. the total protein concentration of the venom fractions which eluted by a linear gradient of sodium chloride elution (molar) tris 0.05 nacl 0.1 nacl 0.25 nacl 0.5 nacl 0.75 nacl 1 nacl 1.25 nacl 1.5 nacl 2 protein concentration (mg/ml) 4.2 1.27 1.20 1.61 3.95 3.8 4.3 2.05 0.82 table 2. the hemagglutination titer against sheep red blood cells (srbc) in chickens which treated with venom fractions or phosphate buffered saline (pbs) fraction (molar) pbs srbc 1 2 3 4 5 6 7 8 9 5 days titer 0 10 10 20 30 20 5 30 60 20 10 10 days titer 0 20 20 40 40 30 10 40 160 40 20 j arthropod-borne dis, december 2017, 11(4): 497–503 m khosravi et al.: the effects of … 500 http://jad.tums.ac.ir published online: december 30, 2017 fig. 1. the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of mesobuthus eupeus scorpion venom. the lane m: marker proteins (175, 130, 95, 70, 62, 51, 42, 29, 22, 14 and 10, respectively). the lanes 1 to 9 are eluted fractions by 0.05 m trishcl and 2, 1.5, 1.25, 1, 0.75, 0.5, 0.25 and 0.1 m of nacl, respectively discussion the isolated fractions of m. eupeus venom have immunomodulatory effects. the humoral immune response to the srbc was a sensitive endpoint to evaluate drug modification of the humoral immunity (17). the cooperation and interaction of antigen presenting cells, t helper and b cells are involved in the production of the anti-srbc (17). both inductions and suppression of the humoral immune response to srbc were observed in treated chickens with venom fractions. the six fractions out of nine fractions increase the anti srbc titer, from 1.5 to 4 fold. several proinflammatory mediators, such as leukotrienes, phospholipases a2, prostaglandins, kinins, h2o2, no production and activation of complement system, were increased in scorpion envenomated peoples (18–20). the fractionated extracts of venom may improve the phagocytic efficacy of the pmn cells and stimulate innate immune response. in addition, venom induces the release of cytokine and activation of endogenous immunological and inflammatory mediators (18). moreover, scorpion venoms could enhance the release of different inflammatory mediators which cause leukocytosis and raise the cytokines levels such as il1b, il6, il8, il10, tnfa and no (21, 22). a mixture of peptide with diverse potential for induction of pro-inflammatory mediator can exist in m. eupeus venom. production of ifn-𝛾 and il-4 enhance the effect of venom on innate and humoral immune responses (23). improved humoral response to srbc was reported for the administration of nnav (23). the elevation of th1 and higher proinflammatory response were induced by bothrops venom (24). the venom of hemiscorpius lepturus and androctonus crassicauda scorpion stimulate the monocytes immune response by il12 production (25, 26). an inhibitory effect on humoral immune response of chickens to srbc was observed on one fraction of m. eupeus venom. the m. eupeus venom was contained effective antiinflammatory mediators (14). in addition, the other venom has suppressive effects on immune system. significant inhibition of the immune responses and interfering with the synthesis of immunoglobulin g were observed after viper snakes venom treatment (27, 28). some of venom derived peptides have immunosuppressant properties and used for the treatment of autoimmune diseases and the organ transplantation (29). in addition, mucopolysaccharides of venom inhibit nitrous oxide and interleukin human chondrocytes (1), hence have anti-inflammatory properties. the naja naja atra venom had anti-inflammatory effects (30). the cobratoxin had anti-inflammatory and inhibitory effect on activation of nf-𝜅b (31). both proand anti-inflammatory cytokines were produced after scorpion envenomation (22, 32). the ifn-γ, tnf-α, gm-csf il-1α, il-6 and il-10 levels were increased in envenomed peoples (22, 33). the b. erythromelas and c. d. cascavella venom have significant immunomodulatory effects. however, b. erythromelas enhance a proinflammatory profile and that c. d. cascavella venom j arthropod-borne dis, december 2017, 11(4): 497–503 m khosravi et al.: the effects of … 501 http://jad.tums.ac.ir published online: december 30, 2017 has anti-inflammatory effects (34). the venom of t. serrulatus contains substances with immunomodulatory effects (35). conclusion different protein profiles of isolated fractions of m. eupeus venom are associated with various effects on the immune response. both enhancing and suppressing of the chicken humoral 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http://www.ncbi.nlm.nih.gov/pubmed/?term=qin%20zh%5bauthor%5d&cauthor=true&cauthor_uid=21841815 j arthropod-borne dis, december 2017, 11(4): 539–540 m panigrahi and a panigrahi: curbing malaria: … 539 http://jad.tums.ac.ir published online: december 30, 2017 letter to the editor curbing malaria: a new hope through clustered regularly interspaced short palindromic repeats (crispr) technology manjit panigrahi 1, *ansuman panigrahi 2 1animal genetics division, indian veterinary research institute, izatnagar, bareilly, uttar pradesh, india 2department of community medicine, kalinga institute of medical sciences, kiit university, bhubaneswar, india dear editor the term genome refers to the total genetic composition of an organism or species. the normal genes transcribe and translate into functional gene products, which in turn bring about a normal phenotype. genomics, the study of genomes has revealed the significant level of diversity in the human genome attributed not only to single nucleotide polymorphisms (snps) but structural variations as well. it also allows us to explore fundamental details at the molecular level. using molecular techniques, researchers have easily identified many molecular markers within a given species’ genome. however, the application of such knowledge in medical science and treatment of diseases is still in its infancy (1). the main challenge in genomics today is to understand the role of molecular aberrations in various diseases and to apply such knowledge to control the diseases. the ability to perturb the genome in a precise and targeted fashion is crucial for understanding genetic contributions to biology and disease. recently, a new form of genome editing known as crispr (clustered regularly interspaced short palindromic repeats) has shown promising results. the crisprs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea (2). it allows researchers to make very precise changes to the genome in cells of various organisms. the crispr and cas (crisprassociated) proteins function as the rna based adaptive immune system in bacteria and archaea (3) and crispr sequences give bacteria a physical record of the viruses that bacteria come across. the protein associated with crispr system i.e. cas9 is programmed to find and bind to specific sites in the virus genome, directed by special guide molecules made of rna (4, 5). also, crispr can add or delete base pairs at specifically targeted dna loci and have been used to cut as many as five genes at once (6). by delivering the cas9 protein and appropriate guide rnas into a cell, the organism's genome can be cut at any desired location (7). malaria parasites are unicellular organisms residing inside the red blood cells. although the genomes of many malaria parasites have been sequenced, the functions of approximately half of the genes are still unknown. thus, editing genes in malaria parasite genomes is still inefficient (8). recently through gene drive, a method for stimulating biased inheritance of particular genes to alter entire populations of organisms, crispr can be incorporated in mosquito populations to aid in malaria control (9, 10). briefly, a package of genes including the crispr system i.e. rna-guided cas9 endonuclease and guide rnas along with the malaria resistance gene (gene controlling the immune response of mosquitoes against malaria) will be inserted into the germline cells that produce sperm/egg. the rna guided cas9 endonuclease cuts *corresponding author: dr ansuman panigrahi, e-mail:dr.ansuman3@gmail.com http://en.wikipedia.org/wiki/archaea http://en.wikipedia.org/wiki/cas9 http://en.wikipedia.org/wiki/endonuclease j arthropod-borne dis, december 2017, 11(4): 539–540 m panigrahi and a panigrahi: curbing malaria: … 540 http://jad.tums.ac.ir published online: december 30, 2017 the genome at desired site and the broken strands of dna trigger the cell’s repair mechanisms. while repairing the damage to the original gene, both the resistance gene and the crispr system will be copied to it. then these engineered mosquitoes carrying the resistance gene and the crispr system will be allowed to mate with other wild-type mosquitoes. consequently, these desired genes will be preferentially inherited to the offspring. the crispr genes would produce their molecules inside the offspring and again cut the original gene from the wild type to copy over the resistance gene and the crispr system. this way, the crispr system can effectively make the same edit in every generation and finally create a mosquito population inherently resistant to the plasmodium parasite. the crispr technology is very young and thus need more exhaustive studies in order to thoroughly evaluate the utility of this system, including the potential for off-target effects and our ability to accurately predict the ecological consequences of these interventions. nevertheless, researchers have high hopes for wiping out malaria from the world through this newer, highly efficient genetic approach. references 1. gonzaga-jauregui c, lupski jr, gibbs ra (2012) human genome sequencing in health and disease. annu rev med. 63: 35–61. 2. grissa i, vergnaud g, pourcel c (2007) the crisprdb database and tools to display crisprs and to generate dictionaries of spacers and repeats. bmc bioinformatics. 8: 172. 3. wiedenheft b, sternberg sh, doudna ja (2012) rna-guided genetic silencing systems in bacteria and archaea. nature. 482(7385): 331–338. 4. bhaya d, davison m, barrangou r (2011) crispr-cas systems in bacteria and archaea: versatile small rnas for adaptive defense and regulation. annu rev genet. 45: 273–297. 5. terns mp, terns rm (2011) crispr-based adaptive immune systems. current opinion in microbiology. 14(3): 321–327. 6. pennisi e (2013) the crispr craze. science. 341(6148): 833–836. 7. mali p, esvelt km, church gm (2013a) cas9 as a versatile tool for engeneering biology. nat methods. 10(10): 957– 963. 8. zhang c, xiao b, jiang y, zhao y, li z, gao h, ling y, wei j, li s, lu m, su xz, cui h, yuan j (2014) efficient editing of malaria parasite genome using the crispr/cas9 system. mbio. 5(4): e01414-14. 9. esvelt km, smidler al, catteruccia f, church gm (2014) concerning rnaguided gene drives for the alteration of wild populations. elife. 3: e03401. 10. mali p, yang l, esvelt km, aach j, guell m, dicarlo je, norville je, church gm (2013b) rna-guided human genome engineering via cas9. science. 339(6121): 823–826. j arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 538 http://jad.tums.ac.ir published online: october 04, 2016 original article canine visceral leishmaniasis in wild canines (fox, jackal, and wolf) in northeastern iran using parasitological, serological, and molecular methods mehdi mohebali 1,2, kourosh arzamani 3, *zabiholah zarei 1, behnaz akhoundi 1, homa hajjaran 1, saber raeghi 3, zahra heidari 1, seyed mousa motavalli-haghi 1, samira elikaee 1, ahmad mousazadeh-mojarrad 3, zahra kakoei 1 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran 3vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran (received 30 nov 2014; accepted 8 nov 2015) abstract background: although many studies had been conducted on various aspects of canine visceral leishmaniasis (cvl) in domestic dogs in the endemic areas of iran, investigations on cvl in wild canines are rare. methods: this is a cross-sectional study was conducted from december 2012 to 2013 in northeast of iran where human vl is endemic. wild canines were trapped around the areas where human vl cases had been previously identified. wild canines were collected and examined both clinically and serologically using direct agglutination test (dat). microscopically examinations were performed in all the seropositive wild canines for the presence of the amastigote form of leishmania spp. some leishmania sp. which had been isolated from the spleens of wild canines, were examined analyzed by conventional pcr and sequencing techniques using α-tubulin and gapdh genes. results: altogether, 84 wild canines including foxes (vulpes vulpes, n=21), jackals (canis aureus, n=60) and wolves (canis lupus, n=3) were collected. four foxes and seven jackals showed anti-leishmania infantum antibodies with titers of 1:320–1:20480 in dat. furthermore, one fox and one jackal were parasitologically (microscopy and culture) positive and l. infantum was confirmed by sequence analysis. conclusion: the present study showed that sylvatic cycle of l. infantum had been established in the studied endemic areas of vl in northeastern iran. keywords: canine visceral leishmaniasis, wild canines, iran introduction visceral leishmaniasis (vl) is one of the most important infectious diseases in human and canines. mediterranean type of vl which caused by leishmania infantum is commonly seen in children less than 10 years old. domestic and wild canines are known animal reservoir hosts and some genus and species of sandflies are the main vectors of the disease (who 2010). wild canines including fox, jackal and wolf were infected by l. infantum and it seems that these carnivores have the potential role in sylvatic transmission cycle of l. infantum in endemic areas of vl particularly in villages located in mountainous regions, where the transmission cycle was established (who 2010). determination of prevalence of canine visceral leishmaniasis particularly in endemic areas is necessary to define control measures for zoonotic visceral leishmaniasis (tesh 1995). based on annual reports of bojnurd health centre from northeastern iran, 164 cases of human vl were *corresponding author: mr zabiholah zarei, e-mail: z-zarei@farabi.tums.ac.ir j arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 539 http://jad.tums.ac.ir published online: october 04, 2016 microscopically diagnosed during two last decades which are higher than the average reported vl cases of iran (arzamani 2012). based on a sero-epidemiological study that was done on 1385 children up to 12 year ages, vl was known as an endemic disease in some areas of north khorasan province from northern northeastern iran (mohebali et al. 2011). in cause because some of limitations in diagnosis and reporting of vl in the studied areas, it seems that real numbers of vl are being higher than registered cases. considering that epidemiological aspects on sylvatic cycle of vl are unknown in endemic areas of the disease thus, this study was conducted on wild canines. the results of this study can be help to health authorities to make special managements for prevention and control of the disease. materials and methods study areas this cross-sectional study was conducted for a period of 1 year from 2012 to 2013. seven villages in north khorasan province were selected, where human vl had been reported in the last 10 years. altogether, 21 foxes (vulpes vulpes), 60 jackals (canis aureus) and 3 wolves (canis lupus) were trapped around the villages after obtaining necessary permits from the directorate general for the environment (fig. 1). serological test all suspected canines were physically examined by a veterinary doctors and then blood samples (2 ml) were taken from them and processed 4–10 h after collection. the collected blood samples were centrifuged at 800 g for 5–10 min, and the sera were separated and stored at -20 ºc until tested by dat. the leishmania infantum antigens were prepared in the leishmaniasis lab. of protozoology unit at the school of public health of tehran university of medical sciences. the procedure for making dat antigen were mass production of promastigotes of iranian strain of l. infantum [mcan/ir/07/mohebgh. (genbank accession no fj555210)] in rpmi1640 medium (biosera, south america) plus 10% fetal calf serum (biosera, south america), following tripsinization of the parasites, staining with coomassie brilliant blue r-250 (sigma, usa) and fixing with formaldehyde 1.2% (harith et al. 1989, edrissian 1996a, mohebali et al. 2005, 2006). all collected serum samples were tested by dat. samples were diluted from 1:40 to give end-point titers of 1: 20480. one negative and one positive serum controls were included in each plate daily for comparing of the agglutination phenomena among all examined sera in each 96 wells of each v shaped plate. the titer was defined as the highest dilution at which agglutination was still visible, as blue dot, compared to negative control wells, which showed clear blue dots. two individuals read the tests independently. specific antibodies against leishmania infantum at a titer of 1:320 were considered as positive based on previous studies (edrissian 1996a, boelaert et al. 1999, mohebali et al. 2005). parasitological study parasitological examinations were performed in symptomatic canines (ie hair shedding, skin lesions and cachexia) with dat positive results (≥ 1:320) after their euthanization with ketamin and acepromizine. microscopical smears were prepared from any skin lesion, liver, spleen and large lymph nodes of all autopsied canines. all of the prepared smears were fixed with absolute methanol, stained with giemsa 10% and examined microscopically for the demonstration of amastigote forms of leishmania spp. biopsy specimens were collected aseptically from the spleen and liver of the infected caj arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 540 http://jad.tums.ac.ir published online: october 04, 2016 nines, then cultured into novy mac-neal and nicolle (nnn) culture media (prepared from nutrient agar containing 10% whole rabbit blood overlaid with normal saline containing 100–200 ui/ml penicillin g and 1 mg/ml streptomycin). the cultures were incubated at 23 °c for up to six weeks and examined weekly for the demonstration of promastigotes. molecular characterization some leishmania spp. isolated from infected wild canines, were checked by conventional pcr using α-tubulin and gapdh genes.dna was isolated from cultured promastigotes and giemsa-positive slides (amastigotes) using a commercial dna extraction kit (roche diagnostics gmbh, mannheim, germany, lot no: 13779500 high pure pcr template preparation) according to the manufacturer’s instructions. pcr was done with α-tubulin and gapdh (glyceraldehyde 3-phosphate dehydrogenase) genes (sheppard and dwyer 1986, kazemirad et al. 2013) gene and primers used in this study summarized in table 1. amplication was conducted using the pcr premix (roche) in a 25 µ l total reaction volume. 12 µ l master mix, 1 µ l of each primer (10 pmol), 1 µ l dna and for the rest distilled water were used. the both amplicons, 154 bp for α-tubulin and 119 bp for gapdh, were analysed on 2% agarose gels and visualized by uv light after staining with gelred stain. parasite species were determined by comparing the profiles of the samples with those of the reference species (fig. 2). the results were compared with standard species of l. infantum (mcan/ir/97/lon49), l. tropica (mhom/su/74/k27) and l. major (mrho/ir/75/er) at the school of public health, tehran university of medical sciences. the pcr products of one fox and one jackal samples were purified using an accuprep gel purification kit (bioneer, deajeon, korea), then sequenced (mwg-biotech, ebersberg, germany) by the primers employed in the pcr. sequence alignments were constructed using the program clustalw version 1.83. (http://www.ddbj.nig.ac.jp/search/clustalwe. html). clustalw alignment and phylogenetic analysis with the construction of a gene tree were performed using the tamura 3-parameter model. ethical approval the trial was reviewed and approved by ethical committee from vice-chancellor for research, tehran university of medical sciences as well as vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran. results dat results among the 84 wild canines examined, the sera of six vulpes vulpes (foxes), and seven canis aureus (jackals) showed antibodies against leishmania infantum with titers ranging from 1:80–1:20480 indicating that these animals were infected with leishmania spp. parasitological (microscopic and culture) results necropsy was performed on all seropositive vulpes vulpes (n=6) and canis aureus (n=7) accompanied by symptomatic vulpes vulpes (no.2) and symptomatic canis aureus (no.4). leishmania spp. was found in one vulpes vulpes (12.5%) and one canis aureus (9.09%) using parasitological methods. all of the three wolves were dead and no clinically signs and symptoms were found. all of the three wolves were dead and had not appropriate samples for finding of leishmania infection. j arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 541 http://jad.tums.ac.ir published online: october 04, 2016 molecular results (conventional pcr and sequencing) two positive parasitilogical samples including one canis aureus (12.5%) and one vulpes vulpes (9.09%) subjected to conventional pcr using α-tubulin and gapdh genes (fig. 2a and b) and l. infantum was identified by sequencing results. all the dna sequences were aligned using multalin and analyzed with mega 6 software (fig. 3). the gapdh sequence was submitted in genbank (accession no. km350534). fig. 1. geographical locations of collected wild canines (fox, jackal, and wolf) of north khorasan province,norteastern iran, 2012-2013. 1. gerati, esfraien, 2. estarkhi shirvan, 3. gelian, shirvan, 4. titkanloo, faruj 5. zoeram shirvan, 6. shirvan, 7. sisab, bojnurd, 8. naveh, bojnurd, 9. babamoosa, bojnurd, 10. asadli, bojnurd, 11. mehnan, metranloo, bojnurd, 12. bidak, bojnurd, 13. tatar, bojnurd, 14. ashkhaneh, mane and samalghan, 15. gifan, bojnurd, 16. jodar, bojnurd, 17. kohne jolge, mane and samalghan, 18. yekeh suod, raz and jargalan table 1. gene and primers used for conventional pcr assay gene primer designations and sequences (5′–3′)1 cycling conditions2 amplicon size (bp) α-tubulin f:caggtggtgtcgtctctgac d:96ºc, 4min 119 c:30 r:tagctcgtcagcacgaagtg d:94ºc, 30sec a:60ºc, 30sec e:72 ºc, 45sec gapdh f: gcatgtgctgacaaaggaga d:96ºc, 4min 154 r:ggtcgtactcgggatgatgt c:30 d:94ºc, 30sec a:60ºc, 30sec e:72 ºc, 45sec 1f: forward, r: reverse. 2d: denaturation, c: cycles, a: annealing, e: extension. final elongation for all assays was at 72 c for 10 min. j arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 542 http://jad.tums.ac.ir published online: october 04, 2016 table 2. parasitology, serology and molecular results in wild canines (fox, jackal) trapped in northeastern iran during 2012–2013 animal microscopic examination culture serologic examination molecular examination total examined positive examined positive examined positive examined positive fox 8 1 8 1 21 6 0 0 21 jackal 11 1 11 1 60 7 1 1 60 all of the three wolves were dead and had not appropriate samples for finding of leishmania infection. fig. 2. conventional pcr patterns of α-tubulin and gapdh genes obtained from test samples and standard leishmania stocks a: α-tubulin. b: gapdh. lane 1 and 2 are samples of one jackal and one fox. m: 100bp size marker. lane 4: leishmania major (mrho/ir/75/er), lane 5: leishmania tropica (mhom/su/74/k27) and lane 6: leishmania infantum (mcan/ir/97/lon49) as positive controls. lane 7: negative control fig. 3. dendrogram based on the sequence of the gapdh gene from species of the genus leishmania (sequences from this study and retrieved from genbank), with standard leishmania tropica sequence for comparing. the access numbers for sequences retrieved from gen bank are given in brackets. the numbers under the branch indicate bootstrap j arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 543 http://jad.tums.ac.ir published online: october 04, 2016 discussion vl visceral leishmaniasis is a potentially fatal protozoan infection that is endemic in some parts of iran (mohebali 2013) domestic dogs (canis familiaris) are the principal reservoir hosts that can carry either l. infantum or l. chagasi (who 2010). it has been reported that our review indicates dat is an easy-to-perform, highly sensitive, specific, reliable, and cost-effective technique for the diagnosis and sero-epidemiological study of vl in humans and canines across different geographical regions. even a small amount of serum or plasma specimen, or a drop of dried blood taken from the tip of the finger on a filter paper could be used for dat (harith et al. 1986, edrissian et al. 1996b, mohebali et al. 2006, 2011, mohebali 2013). vl visceral leishmaniasis as an important vector borne disease is endemic in some parts of north east, north west and south areas of iran and the infections caused by l. infantum were reported in infected humans, domestic canines and phlebotomine vectors (mohebali et al. 2005, rassi et al. 2009, oshaghi et al. 2009, yaghoobi-ershadi 2012, hajjaran et al. 2013). in recent years many studies had been performed on various aspects of vl in domestic dogs in the endemic areas of iran but investigations on vl in wild canines were rare. in the present study, dat was applied to determine the circulating leishmania spp isolated from animal reservoirs. samples from one fox and one jackal that showed positive results throw parasitological methods were subjected to molecular methods. conventional pcr was performed with gapdh as the housekeeping gene (kazemi-rad et al. 2013). although the results confirmed microscopic detections, the electrophoretic models were identical for all the tests and controls because of high similarity in various species (difference in a few nucleotides), (fig. 2). hence, for species identification, sequencing technique was employed. the results were analyzed using mega 6 software and the sequence derived was compared with other reference species in genbank. molecular phylogenetic analysis using mega 6 software was conducted by applying the maximum likelihood method based on the kimura 2-parameter model. the findings showed that most of the leishmania spp. belonged to the monophylogenetic group, and the sequence determined in the present study (accession no. km350534.1) had 100% homology with l. infantum /l. chagasi (xm_001467109/kf041811.1) and 99% with l. donovani (xm_003862963) and presented phylogenetic relationships (fig. 3). similarly, the results of another study on leishmania spp. based on trypanosomatid barcode (ssu rdna) and ggapdh genes also revealed phylogenetic relationships (marcili et al. 2014). however, although a previous study on trypanosoma spp. using ggapdh and ssu rdna demonstrated phylogenetic relationships, a similar study on leishmania spp. showed unrelated results (hamilton et al. 2004, 2007). similarity of our study strain sequence with documented gapdh sequence of leishmania infantum in genbank (xm_ 001467109) that derived jpcm5 strain isolated from a naturally infected dog and its ability for infecting human macrophage reveals and also this study strain can infect human (peacock et al. 2007). conclusion our findings indicate that wild canines have potential role in sylvatic transmission cycle of vl similar to other mediterranean regions and the disease is turning among domestic dogs and wild canines in endemic areas of vl in iran. j arthropod-borne dis, december 2016, 10(4): 538–545 m mohebali et al.: canine visceral leishmaniasis … 544 http://jad.tums.ac.ir published online: october 04, 2016 acknowledgements this study received financial support from center for research of endemic parasites of iran (crepi), tehran university of medical sciences (project no: 91-04-160-20312) and also vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran.we wish to thank dr e kazemi-rad, mr mt satvat and mrs.sorour charehdar for laboratory helps. references arzamani k (2012) visceral leishmaniasis in north khorasan province, north east of iran, 15th international congress on infectious diseases. june 13–16, 2012. bangkok, thailand. boelaert m, safi s, jacquet d, muynck a, van der stuyft p, le ray d (1999) operational validation of the direct agglutination test for diagnosis of visceral leishmaniasis. am j trop med hyg. 60(1): 129–134. edrissian ghh (1996a) visceral leishmaniasis in iran and the role of serological tests in diagnosis and epidemiological studies. parasitology for 21st century (icopa viii). cab international. izmir, turkey, pp. 63–78. edrissian ghh, hajjaran h, mohebali m, soleimanzadeh g, bokaei s (1996b) application and evaluation of direct agglutination test in serodiagnosis of visceral leishmaniasis in man and canine reservoirs in iran. iranian j med sci. 21: 119–124. hajjaran h, mohebali m, mamishi s, vasigheh f, oshaghi ma, naddaf sr, teimouri a, edrissian gh, zarei z (2013) molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in iran. biomed res int. article id: 789326: 1–7. hamilton pb, gibson wc, stevens jr (2007) patterns of co-evolution between trypanosomes and their hosts deduced from ribosomal rna and proteincoding gene phylogenies. mol phylogenetics evol. 44: 15–25. hamilton pb, stevens jr, gaunt mw, gidley j, gibson wc (2004) trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate 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arrowsmith c, white b, thurston s, bringaud f, baldauf sl, faulconbridge a, jeffares d, depledge dp, oyola so, hilley jd, brito lo, tosi lr, barrell b, cruz ak, mottram jc, smith df, berriman m (2007) comparative genomic analysis of three leishmania species that cause diverse human disease. nat genet. 39(7): 839–847. rassi y, javadian e, nadim a, rafizadeh s, zahraii a, azizi k, mohebali m (2009) phlebotomus perfiliewi transcaucasicus, a vector of leishmania infantum in northwestern iran. j med entomol. 46: 1094–1098. sheppard hw and dwyer dm (1986) cloning of leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis. mol biochem parasitol. 19: 35–43. world health organization control of the leishmaniases (2010) technical report series 949, report of a meeting of the who expert committee on the control of leishmaniases. geneva, 22–26 march. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod-borne dis. 6(1): 1–17. tesh r (1995) control of zoonotic visceral leishmaniasis: is it time to change strateries?. am j trop med hyg. 57: 287–292. j arthropod-borne dis, december 2018, 12(4): 432–433 pg de mendonça: troubleshooting real-time … 432 http://jad.tums.ac.ir published online: december 25, 2018 letter to the editor troubleshooting real-time pcr screening for leishmania infantum philippe gil de mendonça institute of comparative tropical medicine and parasitology, ludwig maximilian university, munich, germany (received 3 jan 2018; accepted 13 nov 2018) dear editor-in-chief the parasite leishmania infantum is a widespread cause of concern to both human and veterinary medicine (1-3). a simple and quick realtime pcr protocol was proposed for screening samples for l. infantum (4). this protocol was efficiently applied in various studies (4-7). however, during informal discussions at international meetings, colleagues complained of difficulties in applying this protocol. such complaints are common and usually overcome through optimization, however, the opportunity to use a different type of thermocycler whilst on a visit abroad flagged the seriousness of that complaint. the reason for the difficulties encountered lies in a weakness of this pcr protocol. indeed, the annealing temperature of the probe is substantially lower than the annealing temperature of the primers, whereas this should be the reverse. the implication of this is that if using a fast cooling thermocycler with peltier element, the real-time pcr will work as expected (see fig. 2 in reference 5), although the curves sometimes look somewhat "stretched", which is a symptom of suboptimal pcr conditions. however, if using a slow cooling (e.g. air-cooled) thermocycler, the real-time pcr may look like it failed, as no curves will appear. in such a case, the primers annealed much earlier than the probe, and the taq polymerase was fast enough to elongate the dna target before the probe had attached. this results in no fluorophore release and thus no positive signal detection. however, an agarose gel electrophoresis will reveal that the pcr amplification was successful, while the fluorogenic detection failed. this is an important limitation to be aware of. the implication is that researchers using a slow cooling thermocycler will have no chance to observe a real-time detection signal. they will have no other option than to run an agarose gel electrophoresis, thus losing both the specificity of the taqman probe and the sensitivity of the real-time detection. researchers having a fast cooling thermocycler may continue using this relatively old protocol, whereas laboratories relying on air-cooled equipment ought to opt for alternative real-time pcr protocols. many realtime pcr protocols (including taqman protocols) have been developed and can be tested and selected to suit the technical requirements and limitations of each laboratory. air-cooled, i.e. slow cooling thermocyclers, are however a limiting factor in many cases! if real-time detection is unsuccessful, then sequencing of amplicons might be required to confirm specific amplification. indeed, the added specificity of the taqman probe is missing in such cases. researchers in asia (8) cautiously opted for sequencing their leishmania isolates following conventional amplification. a wise example to follow is such cases indeed. conflict of interest the author declare that there is no conflict of interest. corresponding author: dr philippe gil de mendonça, e-mail: pgm@cantab.net j arthropod-borne dis, december 2018, 12(4): 432–433 pg de mendonça: troubleshooting real-time … 433 http://jad.tums.ac.ir published online: december 25, 2018 references 1. quinnell rj, courtenay o (2009) transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. parasitology. 136(14): 1915–1934. 2. dantas-torres f, solano-gallego l, baneth g, ribeiro vm, de paiva-cavalcanti m, otranto d (2012) canine leishmaniosis in the old and new worlds: unveiled similarities and differences. trends parasitol. 28(12): 531–538. 3. otranto d, dantas-torres f (2013) the prevention of canine leishmaniasis and its impact on public health. trends parasitol. 29(7): 339–345. 4. francino o, altet l, sánchez-robert e, rodriguez a, solano-gallego l, alberola j, ferrer l, sánchez a, roura x (2006) advantages of real-time pcr assay for diagnosis and monitoring of canine leishmaniosis. vet parasitol. 137: 214–221. 5. de mendonça pg, harsch a, mogl c, walther b, boje j, dimke c (2011) molecular screening of wild rodents for leishmania infantum in germany. acta zool bulg. 63(3): 307–311. 6. svobodova v, svoboda m, friedlaenderova l, drahotsky p, bohacova e, baneth g (2017) canine leishmaniosis in three consecutive generations of dogs in czech republic. vet parasitol. 237: 122–124. 7. vilhena h, martinez-díaz vl, cardoso l, vieira l, altet l, francino o, pastor j, silvestre-ferreira ac (2013) feline vector-borne pathogens in the north and centre of portugal. parasit vectors. 6: 99. 8. bhang dh, choi us, kim hj, cho ko, shin ss, youn hj, hwang cy, youn hy (2013) an autochthonous case of canine visceral leishmaniasis in korea. korean j parasitol. 51(5): 545–549. j arthropod-borne dis, september 2016, 10(3): 423–425 m ganjali and m keighobadi: a rare case of … 423 http://jad.tums.ac.ir published online: january 06, 2016 case report a rare case of gastric myiasis in a lion caused by gasterophilus intestinalis (diptera: gasterophilidae)-case report *maryam ganjali 1, mojtaba keighobadi 2 1department of parasitology, faculty of veterinary medicine, university of zabol, zabol, iran 2institute of specific animals, university of zabol, zabol, iran (received 1 dec 2014; accepted 17 july 2015) abstract myiasis is the infection caused by a variety of dipterous (fly) larvae in vertebrate’s tissue (man and domestic or wild animals). species of gasterophilus are obligate parasite of horses, donkeys, zebras, elephants and rhinoceroses. there are records worldwide, but mostly, in tropical and subtropical regions. this case report describes a type of gastric myiasis caused by g. intestinalis in an old lion in a zoo in sistan, southeast iran. myiasis in lions is rarely reported and this is the first report of gastric myiasis in lion. keywords: myiasis, gasterophilus intestinalis, iran introduction the genus gasterophilus (diptera: gasterophilidae) includes nine species, six of which are reported to cause gastrointestinal myiasis in equid hosts (wall and shearer 2001). all species were originally restricted to the palearctic and afrotropical regions (colwell et al. 2007), but three major species g. nasalis, g. intestinalis, g. haemorrhoidalis have been distributed worldwide (zumpt 1965). the adult flies are not parasitic and do not feed (wall and shearer 2001). they are large, 11–15 mm in length and their body is covered with yellowish hairs. all the species have a similar general life cycle. the eggs are usually deposited on the hair of the host in a particular body region. after hatching, the larvae burrow into the tissue of the host, the first stage larvae hatch and moult to l2, which can be present in different regions of the gastrointestinal tract, and in l3 remains attached to the mucosa for 8–10 months. the rounded body, hooked mouthparts and spines enable the larvae to live in the gastrointestinal tract. species can be distinguished based on pattern of spines on the body (wall and shearer 2001). in g. intestinalis, the third instar larvae are relatively large, between 1.27 to 1.91 cm long and spines arranged in two rows (zumpt 1965, sweeney 1990). here we report a type of gastric myiasis caused by g. intestinalis in an old lion in a zoo in sistan, southeast iran. case report the present case aimed to report the finding of g. intestinalis into the stomach of an old lion in a zoo in sistan, southeast iran. larvae were collected during a post-mortem examination of the digestive tract of lion for parasitological purposes (fig. 1). twelve larvae were collected from the stomach and preserved in 70% alcohol with 5% glycerin. samples were taken to the laboratory of parasitology, unit of the veterinary medicine, university of zabol, for morphological iden*corresponding author: dr maryam ganjali, email: m_g2507@yahoo.com j arthropod-borne dis, september 2016, 10(3): 423–425 m ganjali and m keighobadi: a rare case of … 424 http://jad.tums.ac.ir published online: january 06, 2016 tification. all the collected larvae were identified under a stereomicroscope using the identification key (zumpt 1965). the isolated larvae were creamy-white in color, narrow at their anterior end and wide at their posterior end. they measure from 10–15 mm in length and 5–6 mm in width. spines on the surface of segments are arranged in two rows (fig. 2). based on the morphology and size of the larvae, they were identified as g. intestinalis. fig. 1. gasterphilus larvae on the stomach mucosal membrane of a lion fig. 2. gasterophilus intestinalis collected from the stomach of a lion discussion the prevalence of gasterophilus spp. has been reported in different countries. larvae of most gasterophilidae causing equine gastric myiasis have been recorded in southern italy (otranto et al. 2005). gasterophilus intestinalis, g. nasalis, g. hemorrhoidalis, g. inermis, g. nigricornis and g. pecorum were detected in turkey (gökcen et al. 2008). khalifa et al. (2005) obtained g. haemorrohidalis from the stomach of egyptian equines. distribution of species of this genus also has been investigated in brazil and g. intestinalis reported to be responsible for the majority of myiasis cases (felix et al. 2007). iranian equine myasis associated with gasterophilus spp., have been reported on a few occasions, three species of g. nasalis, g. intestinalis and g. inermis were identified by tavassoli and bakht (2012). j arthropod-borne dis, september 2016, 10(3): 423–425 m ganjali and m keighobadi: a rare case of … 425 http://jad.tums.ac.ir published online: january 06, 2016 aside from the higher incidence of infection by gasterophilus larvae in equid hosts, a few reports of infections in pigs, dogs and rabbits are provided by hall and wall (1995) and sayın ipek (2012). besides veterinary importance, gasterophilidae larvae can be found in humans with at least three cases reported (james 1947, harwood and james 1979, royce et al. 1999). in wild animals has been reported rarely. only, kumar et al. (2012) reported a case of wound myiasis in a captive lion. gasterophilus involvement in the gastric myiasis of lion has not been reported, so the present study is the first report. conclusion further studies are recommended to complete the lack of data on parasite fauna of wild animals. acknowledgements this study was supported by university of zabol. the authors declare that there is no conflict of interest. references colwell dd, otranto d, horak ig (2007) comparative scanning electron microscopy of gasterophilus third instars. med vet entomol. 21: 255–264. felix sr, silva ce, schmidtt e, nizoli lq, gotze mm, silva sm (2007) presence of gasterophilus (leach, 1817) (diptera:oestridae) in horses in rio grande do sul state, brazil. parasitol latinoam. 62: 122–126. gökcen a, sevgili m, altas mg, camkerten i (2008) presence of gasterophilus species in arabian horses in sanliurfa region. turkiye parazitol derg. 32: 337–339. hall m jr, wall r (1995) myiasis of humans and domestic animals. adv parasitol. 35: 257–334. harwood rf, james mt (1947) entomology in humans and animal health. macmillan, new york. james mt (1947) the flies that cause myiasis in man. us department of agriculture, miscellaneous publication washington, dc. khalifa rma, abdel latif mzm, mohamed rt (2005) scanning electron microscopal study of third instar of gasterophilus haemorrohidalis (diptera: gasterophilidae). el-minia med bull. 16: 1. kumar v, raj a (2012) management of wound myiasis in a lion (panthera leo). int j agro-vet med sci. 6(1): 4–6. otranto d, milillo p, capelli g, colwell dd (2005) species composition of gasterophilus spp. (diptera, oestridae) causing equine gastric myiasis in southern italy: parasite biodiversity and risks for extinction. vet parasitol. 133: 111–118. royce la, rossingnoll pa, kubitz ml, burton fr (1999) recovery of a second instar gasterophilus larva in a human infant: a case report. am j trop med hyg. 60: 403–404 sayın ipek dn, ipek p (2012) a case of traumatic myiasis in a domestic rabbit (oryctolagus cuniculus) caused by lucilia sericata. turkiye parazitol derg. 36: 54–56 sweeney hj (1990) the prevalence and pathogenicity of gasterophilus intestinalis larvae in horses in ireland. irish vet j. 43: 67–73. tavassoli m, bakht m (2012) gasterophilus spp. myiasis in iranian equine. sci parasitol. 13(2): 83–86. wall r, shearer d (2001) veterinary ectoparasites biology, pathology and control. second ed. blackwell science, oxford, london, paris, berlin. zumpt f (1965) myiasis in man and animals in the old world. london: butterworths. j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 403 http://jad.tums.ac.ir published online: september 08, 2017 original article epidemiological study on cutaneous leishmaniasis in an endemic area, of qom province, central iran abedin saghafipour 1, *hassan vatandoost 2,3, *ali reza zahraei-ramazani 2, mohammad reza yaghoobi-ershadi 2, moharram karami jooshin 4 , yavar rassi 2, mohammad reza shirzadi 5, amir ahmad akhavan 2, ahmad ali hanafi-bojd 2 1 department of medical entomology and vector control, school of public health, international campus (ic-tums), tehran university of medical sciences, tehran, iran 2 department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3 department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4 qom provincial health center, qom university of medical sciences, qom, iran 5 communicable diseases management center, ministry of health and medical education, tehran, iran (received 25 jan 2016; accepted 21 aug 2017) abstract background: cutaneous leishmaniasis (cl) is one of the most important health problems in many areas of iran. there are two forms of the disease in iran, anthroponotic and zoonotic cl. this study conducted to assess the epidemiological situation of cl in an endemic area of qom province, central iran from apr to nov 2015. methods: the sticky paper traps and aspirating tubes were used for collecting adult sand flies. sherman traps and small insect nets were used to capture rodents and small mammals. giemsa staining was used for preparing the expanded smear and followed by pcr for identifying the causative agent in human, vectors, and reservoirs. in this study, relative frequency of cl was also calculated. results: fourteen species of phlebotomine sand flies were collected. phlebotomus papatasi (61.74%) was the predominant species through the period of activity. overall, 62 meriones libycus, 8 nesokia indica, 4 mus musculus, 16 allactaga elater and 2 hemiechinus auritis were caught. pcr technique showed 6 out of 150 p. papatasi (2%), two out of 62 m. libycus (3.23%) and all of suspected human's skin tissue samples (100%) were infected with leishmania major. the relative frequency of cl was 0.30%. conclusion: this is the first detection of l. major within p. papatasi, m. libycus and human in kahak district in qom province of iran. zoonotic cycle of cl exists in this area, l. major is the causative agent, p. papatasi is the main vector and m. libycus is the main reservoir of the disease. keywords: zoonotic cutaneous leishmaniasis, leishmania major, phlebotomus papatasi, its1pcr, iran introduction currently, cutaneous leishmaniasis (cl) is one of the most important vector borne diseases in iran (1) and still the leading cause of considerable morbidity of a large number of people in the endemic foci characterized by chronic skin lesions followed by scars and deformation of the infected tissue (2). phelebotomus papatasi is the main and proven vector and leishmania (leishmania) major is the causative agent of zoonotic cl in iran. leishmania major, l. (l.) tropica and l. (l.) aethiopica cause cl in the old world (3). the parasite has been isolated and identified from naturally infected p. papatasi, p. caucasicus, rhombomys opimus, meriones libycus and human in such endemic areas (48). the p. papatasi and m. libycus were as proven vector and reservoir in qomrood, iran *corresponding authors: dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, dr ali reza zahraei-ramazani, e-mail: azahraei@tums.ac.ir mailto:hvatandoost1@yahoo.com mailto:azahraei@tums.ac.ir j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 404 http://jad.tums.ac.ir published online: september 08, 2017 district (9) and as probable vector and reservoir in ghanavat district (10). in addition, a big part of the lowland areas of qom province provides good ecological niches for p. papatasi and therefore it has higher transmission potential (11). in the recent years, molecular methods have been employed for identification of certain species of leishmania, either isolated from cultures or from patients (3) as well as in the detection of the parasite in individual or pooled phlebotomine specimens (12). successful establishment of the disease in an endemic area is the outcome of a close association between the leishmania parasite and its natural sand fly vector (13). thus, vector and parasite identification have great impact on predicting expansions of the disease in endemic area, also help authorities to design new strategic programs to limit spreading vectors and disease (14, 15). molecular methods are increasingly employed for diagnostic and epidemiological purposes in order to confirm leishmania infection and to characterize the parasites at the species or genotype level in hosts and vectors (16, 17). the detection of leishmania parasites by pcr methods is highly specific and sensitive, with values reaching up to 100%. accurate and sensitive diagnostic and identification procedures are required to distinguish leishmania species/strains whose geographic distribution can overlap, which is crucial for adequate treatment and appropriate public health control measures (18). based on a rapid increase in incidence of cl reported in an endemic area of kahak district of qom province in central iran (19), and due to lack of knowledge on the epidemiological situation of cl, this study was conducted to determine the epidemiological features of cl including human infection and the reservoir hosts and their putative vector species in this endemic area during 2015. materials and methods study area the qom province is bounded by tehran province in the north, esfahan province in the south, semnan province in the east, and markazi province in the west with an area of approximately 11240 square kilometers (0.68% total area of iran) (fig. 1). this study was performed from apr to nov 2015 in 3 villages (khor abad, sarm, and ghobadbezan) of kahak rural district (34o09'–35o11' n latitude and 50o06'–51o58' e longitude) of qom province with the elevation of almost 1500m above sea level (20). the average annual minimum and maximum temperatures were 16.5 °c and 49 °c in jan and jul, respectively. the total annual rainfall was about 150mm and the average max and min monthly relative humidity were 84% and 28% in dec and jun, respectively (21). sand fly collection sand flies were collected from indoor (bedroom, bathroom, toilets, hall, and stables) and outdoor (rodent burrows) fixed places from the first half of apr 2015 to the first half of nov 2015. to capture the sand flies, 30 sticky paper traps (castor oil coated white paper 20×32cm) was used twice a month from sunset to sunrise. the caught sand flies were transferred to the laboratory in qom health center. to species identification, the head and the last two abdominal segments of the sand flies were mounted in puris’ medium (24) and identified after 24–72h, using the morphological characters (25). then, they were counted and segregated by sex. the rest of the abdomen, the wings and the legs used for dna extraction were stored in 1.5ml sterile micro tubes containing 96% ethanol. the females were examined for abdominal status and the numbers of unfed, fresh-blood fed, and gravid and semi-gravid sand flies were recorded. the parous females were distinguished from nulliparous sand flies by observation of the j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 405 http://jad.tums.ac.ir published online: september 08, 2017 appearance of the accessory glands (26). in order to determine the natural promastigote infections of female sand flies, some unfed, blood fed, semi-gravid and gravid female sand flies (captured from rodent burrows) dissected in a fresh drop of sterile saline (9/1000) for the presence of promastigotes in alimentary canal during jun and sep 2015. reservoirs collection and smear preparation the rodents were captured by sherman live traps (fig. 2). collection of dipodids (rodentia: dipodidae) was done by using small insect nets. then the impression smears of ears were fixed with absolute methanol and examined by giemsa staining method. the leishman bodies were observed using light microscope and then were subjected to molecular technique for identification. tissue samples were also taken from the edge of the lesions in 45-suspected cl patients. lesion smears were fixed with absolute methanol and then stained with giemsa for cl diagnosis. in this present study, we also calculated the relative frequency of cl. dna extraction dna extractions were carried out by crashing of the contents on the slides prepared from the digestive system of the 180 dissected female sand flies, the 78 rodent's ear lobes serous fluids and the 45 patient's lesion tissues, using the bioneer genomic dna extraction kit. the extracted dna was stored -20 °c for long storing and at 4 °c for daily working. dna from l. major (mrho/ir/75/er) that provided by department of medical parasitology, school of public health, tehran university of medical sciences (tums), iran was used as positive control. total dna was extracted from the smears by digestion in 100 μl pbs buffer and the tube was centrifuged at 10000rpm for 10min, then 300μl lysis buffers and 30μl proteinase k added. the tube was incubated for 24h at 37 °c before adding 300μl saccharin phenol. after adding this solution, the tube was centrifuged at 9300 rpm for 5min. after transferring upper phase to new tube, 300μl phenolchloroform should be added and was centrifuged at 10000rpm for 5min. again transferred the upper phase to new tube and washed with pure chloroform. thirty μl mgcl2 and 1000μl ethanol were added to upper phase and stored at -20 °c for 2h before was centrifuged at 10000rpm for 10min and washed down phase by 70% ethanol with te and was centrifuged at 10000 rpm for 10min and the te buffer was added. polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) for detection of leishmania infection the dna samples were examined for the leishmania species its1 by pcr amplification using the universal primer pair l5 8s (5´tga-tac-cac-tta-tcg-cac-t-<t>-3´) and litsr (5´-ctg-gat-cat-ttt-ccgat-<g>-3´) (table 1). molecular study pcr production was followed by rflp technique (27). the cycling conditions were 95 °c for 5min., followed by 35 amplification cycles, each consisting of three steps: denaturation at 94 °c for 30sec, annealing 48 °c for 30sec, and extension at 72 °c for one min, followed by a final extension at 72 °c for 7min in thermocycler. pcr productions were digested with the restriction endonuclease hae iii for 2 h at 37 °c. the restriction fragments were separated by electrophoresis on agarose gel and compared with those of standard reference strain of l. major and negative control (distilled water). results a total of 4164 sand flies (68.90% from outdoors and 31.10% from indoor resting j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 406 http://jad.tums.ac.ir published online: september 08, 2017 places) were collected using sticky paper traps. two genera (phlebotomus and sergentomyia) and 14 species were identified, including phlebotomus (phlebotomus) papatasi (61.74%), p. (paraphlebotomus) sergenti (6.87%), p. (paraphlebotomus) alexandri (1.00%), p. (paraphlebotomus) caucasicus (2.55%), p. (paraphlebotomus) caucasicus group (4.13%), p. (larroussius) kandelakii (5.72%), p. (larroussius) tobbi (0.10%), p. (larroussius) major (5.62%), p. (adlerius) halepensis (0.10%), p. (adlerius) brevis (0.05%), p. adlerius group (0.31%), sergentomyia (ser-gentomyia) sintoni (10.83%), s. (sergentomyia) theodori (0.91%) and s. (rondanomyia) pawlowskii (0.07%) (table 2). two species of p. halepensis and the s. pawlowskii were not captured from outdoor and indoor respectively. in the rodent burrows, p. papatasi appeared nearly may and disappeared in the late oct and in indoors, it appeared in the late jun and disappeared in the early oct. the month wise density of p. papatasi in rodent burrows and indoors are shown in fig. 3. the peak of activity was detected for p. papatasi first in the late jun and the second in the early aug (fig. 3). the sex ratio calculates at 83.71 and 62.25 in outdoors and indoors for p. papatasi respectively as well as 101.5 in outdoors for s. sintoni. in aug and sep 2015 a total of 211 sand flies were collected in the vicinity of rodent burrows including p. papatasi (51.66 %), p. caucasicus (10.9%), p. sergenti (18%), and s. sintoni (19.44%). two percent of p. papatasi species had promastigote infections. the total of 300 p. papatasi specimens was selected and dissected for leishmania infection, the result showed 67.33%, and 32.67% dissected sand flies were parous and nulli parous, respectively (table 3). the analysis of physiological status of dissected p. papatasi revealed (60%) unfed, (20%) blood-fed, (6.67%) semi-gravid and (13.33%) gravid (table 3). the used leishmania primers of the pcr technique successfully amplified the its1 region of the strains l. major and l. tropica revealed that among 360 specimens sand flies examined by pcr, 6 parous specimens (2%) were infected by l. major (fig. 4, 5). among 92 collected rodents (67.39%), (8.70%), (4.35%), (17.39%) and (2.17%) were identified as m. libycus, nesokia indica, mus musculus, allactaga elater and hemiechinus auritis respectively (table 4). leishmania major has been detected in 2 (3.23%) out of 62 m. libycus by pcr (fig. 6). both of the infected m. libycus were females. all of the 45 human cases, which examined through observation passively, confirmed by pcr but only 15 numbers of them were positive by light microscope. in this study, the relative frequency of cl patients was 0.30%. twenty-eight (62.22%) of 45 confirmed cases were males and 17 (37.78%) were females. the mean age of patients was 30.78± 16.91. the most of the cases 34 (75.60%) occurred in autumn. eighteen cases (40%) of patients had no positive history of traveling to leishmaniasis endemic areas during the past year. twenty-two cases (48.89 %) of patients had one lesion. the most common location of lesion was on hands (46%). the results of pcr-rflp indicated that 45 (100%) cases were infected as l. major (fig. 7). table 1. the sequence of primers used in pcr amplification the first internal transcribed spacer (its1) primers the sequence response function litsr 5´ctggatcattttccgatg3´ forward 18bp 20bp l5 8s 5´tgataccacttatcgcactt3´ reverse j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 407 http://jad.tums.ac.ir published online: september 08, 2017 table 2. the fauna and the number of collected sand flies from the endemic area in kahak district, qom province, 2015 site outdoor indoor total each species of the total species male female male female outdoor indoor male female total % no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) p. papatasi 915 (45.57) 1093 (54.43) 216 (38.37) 347 (61.63) 2008 (78.10) 563 (21.90) 1131 (43.99) 1440 (56.1) 2571 (100) 61.74 p. sergenti 41 (40.20) 61 (59.80) 131 (71.20) 53 (28.80) 102 (35.66) 184 (64.34) 172 (60.14) 114 (39.86) 286 (100) 6.87 p. alexandri 7 (58.33) 5 (41.67) 11 (36.67) 19 (63.33) 12 (28.57) 30 (71.43) 18 (42.86) 24 (57.14) 42 (100) 1.00 p. caucasicus 45 (100) … 61 (100) … 45 (42.45) 61 (57.55) 106 (100) … 106 (100) 2.55 p. caucasicus group … 157 (100) … 15 (100) 157 (91.28) 15 (8.72) … 172 (100) 172 (100) 4.13 p. kandelakii 23 (31.51) 50 (68.49) 35 (21.21) 130 (78.79) 73 (30.67) 165 (69.33) 58 (24.37) 180 (75.63) 238 (100) 5.72 p. tobbi 1 (100) 0 (0) 0 (0) 3 (100) 1 (25.00) 3 (75.00) 1 (25.00) 3 (75.00) 4 (100) 0.10 p. major 6 (24.00) 19 (76.00) 60 (28.71) 149 (71.29) 25 (10.68) 209 (89.32) 66 (28.21) 168 (71.79) 234 (100) 5.62 p. halepensis 0 (0) … 4 (100) … 0 (0) 4 (100) 4 (100) … 4 (100) 0.10 p. brevis 1 (100) … 1 (100) … 1 (50.00) 1 (50.00) 2 (100) … 2 (100) 0.05 p. adlerius group … 2 (100) … 11 (100) 2 (15.38) 11 (84.62) … 13 (100) 13 (100) 0.31 s. sintoni 207 (50.36) 204 (49.64) 18 (45.00) 22 (55.00) 411 (91.13) 40 (8.87) 225 (49.89) 226 (50.11) 451 (100) 10.83 s. theodori 15 (51.72) 14 (48.28) 6 (66.67) 3 (33.33) 29 (76.32) 9 (23.68) 21 (55.26) 17 (44.74) 38 (100) 0.91 s. pawlowski 0 (0) 3 (100) 0 (0) 0 (0) 3 (100) 0 (0) 0 (0) 3 (100) 3 (100) 0.07 total 1261 (43.93) 1608 56.07) 543 (41.92) 752 (58.08) 2869 (68.90) 1295 (31.10) 1813 (43.54) 2351 (56.46) 4164 (100) 100 fig. 1. map of iran and qom province, highlighting the location of kahak district, (2) within qom province (22, 23) j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 408 http://jad.tums.ac.ir published online: september 08, 2017 table 3. natural infection of sand flies with promastigote, kahak district, qom province, 2015 s p e c ie s catch place physiological status age group total infected specimens i* o** unfed blood fed semi gravid gravid parous nuli parous no % no % no % no % no % no. % no. % no. % no. % p . p a p a ta si 150 50 150 50 180 60 60 20 20 6.67 40 13.33 202 67.33 98 32.67 300 (100) 6 2% p . se rg e n ti 20 50 20 50 20 50 20 50 0 0 0 0 31 77.50 9 22.50 40 (100) 0 0 p .c a u c a sic u s 10 50 10 50 20 100 0 0 0 0 0 0 18 90 2 10 20 (100) 0 0 *: indoor, **: outdoor fig. 2. specimens of some collected rodents in kahak district, qom province, 2015 fig. 3. the month wise density of phlebotomus papatasi in kahak district, qom province, 2015 fig. 4. agarose gel electrophoresis of the first internal transcribed spacer (its1) polymerase chain reaction (pcr) products. m, 50bp ladder: lane1: leishmania major (mrho/ir/75/er), 2, 3: leishmania major in human skin samples, lane 4, 5, 6 leishmania major in p. papatasi, n: negative control (distilled water), m: marker fig. 5. agarose gel electrophoresis of polymerase chain reaction-restriction fragment length polymorphism (haeiii) products. m, 50bp ladder: lane 1, 2, 3, 4, 5, 6 leishmania major in p. papatasi, lane 7 positive leishmania major (mrho/ir/75/er), m: marker j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 409 http://jad.tums.ac.ir published online: september 08, 2017 fig. 6. agarose gel electrophoresis of the first internal transcribed spacer (its1) polymerase chain reaction (pcr) products. m, 50bp ladder: lane 1, negative control (distilled water), lane 2, leishmania major (mrho/ir/75/er), lane 3 leishmania major isolated from meriones libycus, m: marker fig. 7. agarose gel electrophoresis of polymerase chain reaction-restriction fragment length polymorphism (haeiii) analysis of its region for identification of leishmania species using m, 50bp ladder: lane1, 2, 3, 4 leishmania major in human skin samples, lane 5, 6 leishmania major isolated from meriones libycus, lane 7, positive leishmania major (mrho/ir/75/er), m: marker table 4. molecular detection of leishmania infection in rodents, kahak district, qom province, 2015 species no. rodents no. exanimated no. infected infected rate,% (its1-pcr) n0 % no % meriones libycus 62 67.39 62 100 2 3.23 nesokia indica 8 8.70 8 100 0 0 mus musculus 4 4.35 4 100 0 0 allactaga elater 16 17.39 4 25 0 0 hemiechinus auritis 2 2.17 0 0 0 0 total 92 100 78 84.78 2 2.17 discussion we identified l. major in p. papatasi, m. libycus and human by pcr technique. previous surveys carried out in qomrood and ghanavat districts of qom province, iran showed that p. papatasi and m. libycus were proven (in qomrood) and probable (in ghanavat) vector and reservoir of zcl (9, 10). awareness of epidemiology of zcl is an important measure for management and planning of control (28). the entomological and reservoirs (rodents and human) fields and laboratory surveys accompanied by correct identification the common agent in their target part of their bodies is a major component for combating against disease. recently, genetic analyses and genotyping using pcr-rflp have been applied and performed on vector (s) and reservoir hosts of zcl (29). this paper reviews recent advances on sand flies vectors, m. libycus and human of zcl, using molecular biological approaches. p. papatasi captured in this study is the main vector of l. major from animals to man in central asia and appears to be the vector of l. major in iran and 21 other countries in old world (30). in this study, p. alexandri was also captured. it has been found naturally infected with promastigotes and is suspected vector of vl in iran (31). phlebotomus caucasicus group, p. kandelaki and p. major were also collected. phlebotomus caucasicus was naturally infected with promastigotes in a new focus of vl in north-west of iran (32). phlebotomus kandelaki is naturally infected with leishmania sp. promastigotes in northwest iran and is sus j arthropod-borne dis, september 2017, 11(3): 403–413 a saghafipour et al.: epidemiological study on … 410 http://jad.tums.ac.ir published online: september 08, 2017 pected as probable vector of vl in the region (33). in iran, p. major has been found in all area where human cases of vl have been reported and natural promastigote infection of this species has reported in endemic focus vl in ghir county (fars province) south of iran (34). moreover, in this area, we captured s. povlovskyi in indoor. this result has been confirmed with the previous study in esfahan (35). the identified species were consistent with previous study in other parts of iran (1). low percentage of collected sand flies in indoor shows people prevent sand flies to enter their homes. p. papatasi was the predominant species of the genus phlebotomus in indoors (31.10%) and outdoors (68. 90%). this suggests that p. papatasi is the anthropophilic species in this area. in addition, only p. papatasi is able to develop l. major in its mid gut and transfer the parasite to its proboscis to cause zcl (36). s. sintoni enters the indoors because of nearby homes to rodent burrows (37). p. major and p. kandelakii are more dominant sand flies were collected from indoor after p. papatasi but due to the low frequency. these species do not involve in leishmaniasis transmission. in the present study, we distinguished parity of sand flies by observation of the appearance of the accessory glands (26). accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of p. papatasi females (38). in this study, the parous rate of p. papatasi was 67.33%, so it is not surprising to see high level of promastigote infection among this sand fly species. the same trend of infection is reported from north siani in egypt (39, 40). the results of dissection showed that 2% of p. papatasi species were naturally infected with promastigote infection. the leishmania infection rate in sand flies is usually very low even in endemic areas (41). the low density of leishmania parasites in m. libycus in this area may be due to low sensitivity of this rodent compared to r. opimus (4). in the present study, based on molecular results, l. major was found in m. libycus as a reservoir, p. papatasi as a vector and suspected patients as incidental host. m. libycus as the main reservoir host of zcl has been distributed in the northeastern, central and southwestern regions in iran (42, 43). for the first time in 1996, isolation and characterization of l. major from m. libycus in iran, has been reported (4). meriones libycus has previously been infected with l. major parasites from golestan, esfahan and yazd provinces (7, 42, 44-46). leishmania major was firmly identified in m. libycus that indicates this rodent species can be incriminated as reservoir host of zcl in this location (47). the results of this study have shown, m. libycus is the main and potential reservoir host of zcl in kahak district of qom province and it has an important role in stable zcl in this area, which has pay attention to 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(98% vs 83%). conclusion: fifty-five percent of residential homes of prick test positive patients and only 5% of residential homes of prick test negative patients were positive for mite. sunshine provided home had fewer mites than home where sunshine is not provided. prick test positive patients used handmade carpets more than machine made ones. in positive prick test patients, mites were found in bed sheet and bedroom’s carpet more than negative prick test patient's sheets and carpets. keywords: house dust mite, atopic dermatitis, dermatophagoides, iran introduction house dust mites are eight-legged creatures with about 1.3mm in length and are not visible by naked eyes (platts-mills and chapman 1987). they belong to phylum arthropoda, class arachnida, subclass acarina order acariformes and pyroglyphidae family. this family has about 16 and 46 species. three species called dermatophagoides pteronyssinus, de. farina and euroglyphus maynei are more important and are the source of allergy resulted from mites (arlian and platts-mills 2001). house dust mites exist in the dust all around the world but they are mostly found in bed sheets, carpets, furniture and cloth toys (platts-mills and chapman 1987, babe et al. 1995, beltrani 1997, arlian and plattsmills 2001, sidenius et al. 2002, mihrshahi et al. 2002, yong and jeong 2009). *corresponding author: dr fariba berenji, email: fberenji@yahoo.com j arthropod-borne dis, june 2017, 11(2): 309–314 t ziyaei et al.: house dust mite prevalence … 310 http://jad.tums.ac.ir published online: may 27, 2017 in 1g of the dust collected from a carpet, more than 10000 mites have been counted. on average, 500 mites have been collected out of 0.2g of dust (nadchatram 2005). these arthropods feed on outer layer of human and animal skin and other organic materials (plattsmills and chapman 1987, beltrani 1997, nadchatram 2005, yong and jeong 2009). they reproduce sexually and their development cycle to maturity includes 5 steps: eggs, larva, protonymph, tritonymph, and adult (arlian and platts-mills 2001). it takes about 30d for dust mites to complete their life cycle (nadchatram 2005, yong and jeong 2009). the rate of mite growth depends on humidity and temperature. the best environmental condition for reproduction is the temperature of 23 ˚c and the relative humidity of 70 % (platts-mills and chapman 1987, babe et al. 1995, arlian and platts-mills 2001). mite excretion and skin frill are the allergen factors in human (blythe 1976, platts-mills and chapman 1987, arlian and platts-mills 2001, cui 2014). there are a lot of evidences on the importance of mite allergens to the etiology of allergic asthma, non-seasonal allergic rhinitis and atopic dermatitis (platts-mills and chapman 1987, arlian and platts-mills 2001, engelhart et al. 2002, nadchatram 2005, jacquet 2011). dermatitis or eczema is a kind of inflammatory reaction of skin to different factors. its clinical symptoms include itch, erythema, scaling, often in the flexor surfaces of the body (berke et al. 2012). it happens mostly to children (fuiano and incorvaia 2012). this disease usually begins in the early life and is often seen in the people with the personal or family background of asthma or swelling of the mucous membranes (fuiano and incorvaia 2012). forty five percent of children experience the early onset in the first 6 months of their life, 60% during their first year and 85% before the age of 5 (fuiano and incorvaia 2012). this disease can assist with asthma and allergic rhinitis (berke et al. 2012). this disease can cause sleep, educational, and social disorders in patients (hanifin and rogge 1977, queilleroussel et al. 1985, laughter et al. 2000). moreover, the physical and psychological pressure can effect on not only the patients but also their families and related people (lapidus et al. 1993, herd et al. 1996). according to the importance of home dust in relation to atopic dermatitis in iran and especially in mashhad has been studied lesser, the present study has been conducted in order to determine the abundance of dermatophagoides mites in the houses of patients suffering from atopic dermatitis. materials and methods statistical population the present study is a cross-sectional study. forty patients with atopic dermatitis, based on defined criteria for the disease, have been presented to the allergy clinic of ghaem hospital of mashhad, iran. their disease had been diagnosed by dermatologists based on clinical criteria. they took part in the study after giving their written consent. the study was approved by the local ethics committee. before doing prick test, the necessity and method of doing the test was explained to the patients. for each patient, some information like age, sex, home description, job, allergy background of the patient or his/her family was recorded in a questionnaire. considering no drug interaction with dermal tests, a dermal test was done. some international standard extracts including mite allergens (derf1, derp1) assisting with positive control (histamine) and negative control with normal saline, were put on the patients` forearms, on the points at a distance of 2cm from each other. then the skin was scratched with a lancet. if the skin bumps were bigger than 3mm, the patient was referred to as sensitive to mites and if the bumps were smaller than 3mm, he/she was referred to non-sensitive to j arthropod-borne dis, june 2017, 11(2): 309–314 t ziyaei et al.: house dust mite prevalence … 311 http://jad.tums.ac.ir published online: may 27, 2017 mites. the patients, who did positive or negative dermal test for mite allergens, participated in the study. dust samples before sampling, the patients were asked to avoid vacuuming or sweeping mats in their houses or changing their bed sheets for 3 to 5d. then referring to the patients' houses separately, samples were collected from 3 locations (living room, bedroom and bed) by vacuum cleaner on high power for 2 minutes and in an area of 1m2. then the samples were transmitted to special bags and the bags containing dust were closed and the specifications relating to locations, dates, humidity amount and temperature were written on the bags and the samples was frozen at -20 ˚c. flotation dust samples were weighed using a 0.001 g digital scale and it was separated into 100 mg aliquots. mites were isolated using a flotation method (fernandez-caldas et al. 1993). briefly, dust samples were suspended in 5ml of saturated saline and sample were examined under the light microscope using 10-40 x magnification. statistical analysis statistical analysis was performed using spss software for windows, ver.20 (spss, ibm, usa). chi-square test, fisher exact test was used to evaluate the variables correlations. furthermore, the significant statistical level in these tests was considered with p< 0.05. results forty patients were evaluated who had atopic dermatitis, 24 (60%) were female and 16 (40%) were male. they were between 5 month and 45yr old (mean age 16.91yr). in terms of occupation, 14 (45.2%) were school students, 4 (12.9%) university students, 4 (12.9%) housewives, 1 (3.2%) self-employed, 4 (12.9%) educational staff members, 2 (6.5%) office workers, 1 (3.2%) member of the military sector and 1 (3.2%) restaurant staff (9 people without jobs less than 6yr old. all subjects had allergic history including asthma 10 (25%), allergic rhinitis 8 (20%), urticaria 19 (47.5%) and other diseases 3 (7.5%). dermatophagoides pteronyssinus was more frequent than de. farinae (98% vs. 83%). the condition of patients' home of 31 cases is recorded in table 1. fisher exact test showed that there was a significant difference between two groups in terms of sunshine provision which had fewer mites (p= 0.02). the kind of carpets used in the living room is mentioned in table 2. fisher exact test showed a significant differences between using hand-made carpets and machine-made carpets in patient's living rooms (p= 0.04). experimental results the results of direct observation of mites are recorded in table 3. table 1. frequency distribution of residential home in terms of considering sun light provision in positive prick test and negative prick test groups prick test prick test positive number (%) prick test negative number (%) p value sunshine is fully provided 12 (60) 19 (95) 0.02 sunshine is not provided 8 (40) 1 (5) j arthropod-borne dis, june 2017, 11(2): 309–314 t ziyaei et al.: house dust mite prevalence … 312 http://jad.tums.ac.ir published online: may 27, 2017 table 2. frequency distribution of carpet kinds in the living rooms of patients suffering from atopic dermatitis in the positive prick test and negative prick test groups prick test bedrooms' carpet prick test positive number (%) prick test negative number (%) p value hand-made 5 (25) 0(0) 0.04 machine-made 15 (75) 20 (100) table 3. frequency distribution of direct observation of mites in patients' bed sheet carpets in positive and negative prick tests prick test direct observation of mites prick test positive number (%) prick test negative number (%) p value bedding sheets 7 (35) 0 (0) 0.008 bedrooms' carpet 6 (30) 0 (0) 0.02 living room's carpet 3 (15) 1(5) 0.60 discussion briefly, the prevalence of allergic diseases due to household arthropods have significantly increased in the recent last decades, because people spend most of their time in their home environment and according to the modern lifestyle, houses are warmer and filled with a lot of furniture and not enough air-conditioning is provided (yong and jeong 2009). in this study, we determined the abundance of dermatophagoides mites in the living house of patients suffering from atopic dermatitis. house dust mites are the most common allergens in closed environments (voorhorst et al. 1964). the first determinant factor of dust mite number is humidity and the most important amount of relative humidity in a house for the maximum rate seems to be 60 % in 21 ˚c and 75% in 15.5 ˚c (platts-mills and chapman 1987). during sampling, the amount of humidity of all houses was recorded. the mean humidity in houses was 34.4% (33.6±11.3). in lack of humidity every kind of bed sheets, pillows and furniture are safe for patient. regular cleaning and vacuuming cause to decrease mites. mature mites will die in 5 to 11 days if they are exposed to the environmental temperature of 25 ˚c to 35 ˚c with the constant relative humidity of 40% to 50% because of dehydration (arlian and platts-mills 2001). dust mites are limited in the area near the caspian sea, while in other areas due to the seasonal variations of temperature and humidity; mites are not able to grow well (fereidouni et al. 2013). according to our study at houses which sunshine is fully provided, fewer mites were found which showed that changing life style would help exposing allergenic mites. we concluded long lint carpet could add the number of mites. handmade carpets had more mites than machine made ones. this is according to the study of mihrshahi et al. (2002) residential houses in sidney in australia where wool carpets with long lint (hand-woven) are the best habitats for mites and they can survive in these carpets for a long period of time (mihrshahi et al. 2002). result of the direct observation of mites in bed sheet was 35% at old woolly bed. this is similar to the result of two studies conducted in sidney and copenhagen and confirmed that the most important predictor factor for the high density of allergen mites j arthropod-borne dis, june 2017, 11(2): 309–314 t ziyaei et al.: house dust mite prevalence … 313 http://jad.tums.ac.ir published online: may 27, 2017 was old wool beds (used for more than 2yr) (mihrshahi et al. 2002, sidenius et al. 2002). in this study, the minimum number of mites (1 to 2 mites in 100g of dust) was obtained from the residential houses of patients with atopic dermatitis. in comparison with other studies in other regions, this number is small. we found more mites in old residential homes. this is in accordance to a study at houses in sydney in australia, which revealed that the ages of buildings provides the most significant theory about the density of mite allergens in beds and on floors of bedrooms, and older houses are full of dust (mihrshahi et al. 2002), so they can provide a big source for dust mites (arbes jr et al. 2003). conclusion according to the present study, 55% of residential places of prick test positive patients and only 5% of residential home of prick test negative patients were positive for mite. sunshine provided home had fewer mites than home where sunshine is not provided. prick test positive patients used handmade carpets more than machine made ones. in positive prick test patients, mites were found in bed sheet and bedrooms carpet more than negative prick test patients’ sheets and carpets. acknowledgements the authors greatly acknowledge the research council of mashhad university of medical sciences (mums), mashhad, iran, for their financial grant. the results presented in this work have been taken from toktam ziyaei thesis, with the id number “651.” we appreciate sincerely all the patients who assisted us voluntarily. the authors declare that there is no conflict of interests regarding the publication of this paper. references arbes sj jr, cohn rd, yin m, muilenberg ml, burge ha, friedman w, zeldin dc (2003) house dust mite allergen in us beds: results from the first national survey of lead and allergens in housing. j allergy clin immunol. 111 (2): 408–414. arlian lg, platts-mills ta (2001) the biology of dust mites and the remediation of mite allergens in allergic disease. j allergy clin immunol. 107(3): 406–413. beltrani vs (1997) the role of dust mites in atopic dermatitis: a preliminary report. immunol allergy clin north am. 17(3): 431–441. berke r, singh a, guralnick m (2012) atopic dermatitis: an overview. am fam physician. 86(1): 35–42. blythe me (1976) some aspects of the ecological study of the house dust mites. br j dis chest. 70(0): 3–31. cui y (2014) when mites attack: domestic mites are not just allergens. parasit vectors. 7: 411. fereidouni m, fereidouni f, hadian m, nourani hasankiadeh s, mazandarani m, ziaee m (2013) evaluation of the level of house dust mite allergens, der p 1 and der f 1 in iranian homes, a nationwide study. allergol immunopathol. 41(6): 381–386. fernandes-caldas e, puerta l, mercado d, lockey rf, caraballo lr (1993) mite fauna, der p i, der f i and blomia tropicalis allergen levels in a tropical environment. clin exp allergy. 23(4): 292–297. fuiano n, incorvaia c (2012) dissecting the causes of atopic dermatitis in children: less foods, more mites. allergol int. 61(2): 231–243. hanifin jm, rogge jl (1977) staphylococcal j arthropod-borne dis, june 2017, 11(2): 309–314 t ziyaei et al.: house dust mite prevalence … 314 http://jad.tums.ac.ir published online: may 27, 2017 infections in patients with atopic dermatitis. arch dermatol. 113(10): 1383–1386. herd rm, tidman mj, prescott rj, hunter ja (1996) the cost of atopic eczema. br j dermatol. 135(1): 3–20. jacquet a (2011) the role of innate immunity activation in house dust mite allergy. trends mol med. 17(10): 604–611. lapidus cs, schwarz df, honig pj (1993) atopic dermatitis in children: who cares? who pays? j am acad dermatol. 28(5 pt 1): 699–703. laughter d, istvan ja, tofte sj, hanifin jm (2000) the prevalence of atopic dermatitis in oregon schoolchildren. j am acad dermatol. 43(4): 649–655. mihrshahi s, marks g, vanlaar c, tovey e, peat j (2002) predictors of high house dust mite allergen concentrations in residential homes in sydney. allergy. 57(2): 137–142. nadchatram m (2005) house dust mites, our intimate associates. trop biomed. 22(1): 23–37. platts-mills ta, chapman md (1987) dust mites: immunology, allergic disease, and environmental control. j allergy clin immunol. 80(6): 755–775. queille-roussel c, raynaud f, saurat jh (1985) a prospective computerized study of 500 cases of atopic dermatitis in childhood. i. initial analysis of 250 parameters. acta derm venereol suppl (stockh). 114: 87–92. sidenius ke, hallas te, brygge t, poulsen lk, mosbech h (2002) house dust mites and their allergens at selected locations in the homes of house dust mite-allergic patients. clin exp allergy. 32(9): 1299–1304. voorhorst r, spieksma-boezeman mi, spieksma ft (1964) is a mite (dermatophagoides sp.) the producer of the housedust allergen? allerg asthma. 10: 329–334. yong ts, jeong ky (2009) household arthropod allergens in korea. korean j parasitol. 47: 143–153. 8dr telmadarei rtl_2_ iran j arthropod-borne dis, 2010, 4(2): 56–60 a nasiri et al.: tick infestation rate of … 56 short communication tick infestation rate of sheep and their distribution in abdanan county, ilam province, iran, 2007-2008 a nasiri1, *z telmadarraiy1, h vatandoost1, s chinikar2, m moradi2, ma oshaghi1, y salim abadi1, z sheikh1, 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2pasteur institute of iran, tehran, iran (received 6 dec 2009; accepted 27 jun 2010) abstract background: ticks are hematophagous arthropod belonging to the class of arachnids. ticks are also one of the major vectors of pathogens to animal and human. this study was conducted to determine tick infestation rate of sheep in abdanan during 2007–2008. methods: sampling was performed seasonally in 19 villages during spring 2007 until winter 2008. a total of 1095 sheep were selected and tested for tick infestation. after collection, all ticks were transported to laboratory of medical entomology and were identified with appropriate identification keys. results: totally, 864 hard ticks were collected. the ticks were classified into two genera and 5 species including: hyalomma marginatum (44.67%), hy. anatolicum (43.17%), hy.asiaticum (6.37%), hy. dromedarii (5.55%), heamaphysalis sulcata (0.24%). the highest seasonal activity was observed in spring (36.46 %) and the lowest seasonal was in winter (11.57%). the rate of tick frequency in mountainous region was 48.15% and it was 51.85% in plateau regions. in this study, tick infestation of sheep was 11.41%. conclusion: hy.marginatum has the more frequent density in the study area. keywords: ticks, sheep, abdanan, iran introduction in the arachnids arthropods, ticks are the main important vectors of disease to human and animals (morel 1989). ticks play a significant role as vectors of pathogens of domestic animals and human in iran as well. the major losses caused by ticks are related to transmission of babesiosis, theileriosis, and anaplasmosis in ruminants. there are several reports on, distribution, public health importance in the country. the tick study was started by delpy (delpy 1936) in iran. abbasian and mazlum described a list of adult ticks collected from domestic animals in different region (abbasian 1961, mazlum 1971). rodents which play as host of ticks also reported from different zoogeographical zones of the country (filipova et al. 1976). hoogstraal and wassef studied ixodid ticks parasitizing wild sheep and goat in iran (hoogstraal et al.1979). the ecology of different species of ticks on deomestic naimals also is emphasized (rahbari 1995). razmi et al 2002 published a list of tick species of domestic animals in northeast of iran (razmi et al. 2002). distribution of soft ticks (argasidae) collected from human dwellings, poultries, and animal shelters in hamadan province, exhibited that they belong to the genus argas and ornithodoros (vatandoost et al. 2003). the fauna of hard and soft ticks (families: ixodidae and argasidae) were studied in west azerbaijan province (tel*corresponding author: dr zakiyeh telmadarraiy, email: ztelma@yahoo.co.in iran j arthropod-borne dis, 2010, 4(2): 56–60 a nasiri et al.: tick infestation rate of … 57 madarraiy et al.2004). salari lak et al. studied the seasonal activity of ticks and their importance in tick borne infectious diseases in west azerbaijan (salari lak et al. 2008). distribution of tick fauna in iran was also performed (rahbari et al. 2008). nabian and rahbari studied the soft and hard ticks on ruminants in zagros mountainous areas of iran to determine the species and distribution of ticks infesting ruminants (nabian et al. 2008). distribution of ticks meshkinshahr and ardabil were carryout out (telmadarraiy et al. 2009, 2010). ilam province including abdanan city is one of the most important husbandry centers in western region of iran. this study was conducted to determine tick infection rate of sheep in the abdanan. materials and methods study area ilam province is located in the western part of iran and abdanan county is located in the southeast of ilam province and has mild mountainous and tropical climate. this county is an important sheep breeding area in ilam province. this region comprises two parts differing in topography. the mountain and plateau region and the highest altitude considered as 880 m and its population is 47370 people. tick collection sampling was performed seasonally in 19 villages through one year during spring 2007 until winter 2008. a total of 1095 sheep were selected and were checked for tick infestation. after collection ticks from each sheep were kept alive in separate labeled holding tubes and were kept alive in labeled tubes and their history containing location, age, sex was recorded .the collection ticks in labeled holding tubes were transferred to the entomology laboratory, school of public health, tehran university of medical science for species identification. they were identified by morphological characteristic using stereomicroscope and key identification guide (kaiser et al. 1963, walker et al. 2003). results during this study, 864 hard ticks were collected from 1095 sheep. the ticks were classified into two genera and 5 species including hy. marginatum (44.67%), hy. anatolicum (43.17%), hy. asiaticum (6.37%), hy. dromdareii (5.55%), h. sulcata (0.24%) (table 1). tick infestation was detected in 125 (11.41%) out of 1095 sheep. table 1 shows the seasonal activity of tick and the spatial infestation rate was (36.8%) in spring, (16.8%) in summer, (32.8%) in autumn and (13.6%) in winter, respectively. the highest tick seasonal activity was observed in spring (36.8 %) and the lowest seasonal were in winter (13.6%). the rate of tick frequency in mountainous region was 48.15% and it was 51.85% in plateau regions (table 2). table 3 shows relatively percentage of tick infestation of sheep in different seasons of year. tick infestation of sheep was 11.41%. the sexual ratio of hard ticks, shows that the frequency of male ticks were (77%) and female ticks were (23%). table 1. number of tick in the different seasons collected in abdanan 2007–2008 seasons species spring summer autumn winter total% hyalomma marginatum 101 145 125 15 386(44.67) hyalomma anatolicum 131 22 157 63 373(43.17) hyalomma asiaticum 49 0 0 6 55(6.37) hyalomma dromedarii 34 0 0 14 48(5.55) heamaphysalis sulcata 0 0 0 2 2(0.24) total 315(36.8) 167(16.8) 282(32.8) 100(13.6) 864(100) iran j arthropod-borne dis, 2010, 4(2): 56–60 a nasiri et al.: tick infestation rate of … 58 table 2. topographical status of ticks collected in abdanan 2007–2008 species hy. marginatum hy. anatolicum hy. asiaticum hy. dromdareii h. sulcata total percentage mountain 148 195 44 27 2 416 48.15 plateau 238 178 11 21 0 448 51.85 table 3. infectivity of sheep to ticks in abdanan 2007–2008 percentage infected sheep surveyed sheep seasons 19.57 46 235 spring 8.4 21 249 summer 14.38 41 285 autumn 5.2 17 326 winter 11.41 125 1095 total discussion during this study 864 ticks from ixodidae family were collected indicating the occurrence of 5 species of hard ticks in abdanan. the frequency of the species of hyalomma marginatum were higher than the other species, and only 2 genera were collected. seasonal activity of hard ticks in spring was more than other seasons. the occurrence of hard ticks in plateau area was a few more than mountainous area. piazak reported the seven species of genera hyalomma (hy. marginatum, hy. detritum detritum, hy. anatolicum anatolicum), rhipicephalus (rh. bursa), heamaphysalis (h. sulcata), dermacentor (d.marginatus), and boophilus (b.annulatus) in east azerbaijan (piazak 1991). tavakoli et al. carried out a study in lorestan province, and found that genus hylaomma, was active during summer, and in our study, also this genus was the highest frequency and active in summer (tavakoli 1997). telmadarraiy et al. carried out a study in west azerbaijan province, and reported 13 species of ticks including: boophilus annulatus, rh.bursa, rh. sanguineus, dermacentor marginatus, d. niveus, h. sulcata, h. inermis and hy. asiaticum, hy. marginatum, hy. aegyptium, hy. dromdarii, h. schulzei, hy. detritum, that percentage of hyalomma genus were 41% (telmadarraiy et al. 2004). nabian et al. emphasized that hy. marginatum occurred as a dominant tick in the north of iran. their finding was similar to our results. in their study, thirteen hard tick species were identified as hy.anatolicum anatolicum, hy. marginatum, hy. detritum, h.punctata, h. parva, h.concinna, h.choldokovsky, ixodes ricinus, rh. sanguineus, rh.bursa, boophilus annulatus, d. niveus, d. marginatus (nabian et al. 2007). telmadarraiy et al. (2009) also carried out a study in meshkinshahr district and in this study, genus hyalomma was the most frequent and seasonal activity of ticks was in spring. salim abadi et al. carried out a study in yazd province and reported that genus hyalomma was the most frequent (salim abadi et al.2010). this and others studies showed that tick infestation of sheep in iran are around 10% and hyalomma species was reported as a dominant tick in different part of iran. in this survey, several tick species, which are important in diseases transmission in sheep and human, are identified. the seasonal and geographical activity of ticks will help authorities to provide appropriate strategy for tick control program. iran j arthropod-borne dis, 2010, 4(2): 56–60 a nasiri et al.: tick infestation rate of … 59 acknowledgments we would like to appreciate the kind collaboration of all the staff of the veterinary office of abdanan. this research was financially supported by tehran university of medical sciences, project no.8855. the authors declare that they have no conflicts of interest. references abbasian l (1961) record of tick (acarina: ixodidae) occurring in iran and their distributional data. acarologia. 3: 546–559. delpy l (1936) note sur les ixodides du genera hyalomma (koch). ann parasitol hum comp. 14: 206–245. filopova na, neronov vm, farhangazada (1976) data on ixodid tick fauna (acarina, ixodidae) of small mammals in iran (in russian). ent obozr. 55(2):467–479. hoogstraal h, wassef hy (1979) haemaphysalis kopetdaghica: identity and discovery of each feeding stage on the wild goat in northern iran (ixodoidea: ixodidae). j parasitol. 65: 783–790. kaiser mn, hoogstraal h (1963) the hyalomma ticks of afghanistan. j parasitol. 49:130–139. mazlum z (1971) tick of domestic animals in iran, geographic distribution, host relation, and seasonal activity. j vet fac univ tehran. 27(1): 1–32. morel p (1989) tick-borne diseases of livestock in africa. in manual of tropical ve-terinary parasitology. fischer m and ralph s (eds). pp 301-391. cab international, london, uk. nabian s, rahbari s, shayan p, haddadzadeh hr (2007) current status of tick fauna in north of iran. iran j parasitol. 2(1): 12–17. nabian s, rahbari s (2008) occurrence of soft and hard ticks on ruminants in zagros mountainous areas of iran. iran j arthropod-borne dis. 2(1): 16–20. piazak n (1991) preliminary studies on the presence of lyme disease in iran through the study of ixodes ricinus population. 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shahrekord university of medical sciences, shahrekord, iran 3department of pathobiology, school of public health, tehran university of medical sciences, tehran, iran (received 27 jan 2010; accepted 20 feb 2010) abstract background: biting habit of mosquitoes plays an important role in the epidemiology of mosquito-borne diseases. mosquitoes use a set of elaborate sensory modalities to find their preferred hosts by exploiting cues emanating from a nearby host. it has been suggested that the chemical profile of skin can provide further support for anthropophilic mosquito species to find their suitable hosts. this study aimed at revealing the value of skin emanation for a zoophilic species like anopheles stephensi as a model. methods: skin emanations of a man, a cow and a guinea pig were collected by ethanol soaked cottons. upwind responses of mosquitoes to 100 and 200 µl of filtered skin materials were non-competitively explored in a dual-choice olfactometer. l-lactic acid and other chemical content of the skin samples were identified by an enzymatic kit and gc-ms, respectively. results: unexpectedly, only human skin emanation was resulted in the statistically significant activation and attraction responses of an. stephensi in the wind tunnel. l-lactic acid content of this skin sample was 10 and 29 times more than the cow and the guinea pig, respectively. the possible role of lactic acid and a few other identified compounds have been discussed here. conclusion: anopheles stephensi showed higher and more specific upwind responses to human skin emanation in the olfactometer. undoubtedly, the thorough explanation of this unexpected finding needs further investigation. but, if new data verify this result, then, it may be necessary to reconsider the role of skin emanation and thence the human blood index and vectorial capacity of this zoophilic mosquito. keywords: mosquito, host preference, host-seeking, an. stephensi, skin emanation, olfactometer introduction malaria remains a major public health problem in southern part of the country which comprises about 80% of all malaria cases in the country. in this part of the country six anopheline mosquitoes including an. culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus, and an. pulcherrimus are known to be the malaria vectors (zahirnia et al. 1998,vatandoost, 2001, zahirnia et al. 2001, naddaf et al. 2003, enayati et al. 2003, vatandoost and moinvaziri, 2004, vatandoost and borhani 2004, vatandoost et al. 2004, *corresponding author: prof hassan vatandoost, p.o. box: 14155-6446, iran, e-mail: hvatandoost@yahoo.com iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 2 vatandoost et al. 2005a, 2005b, hanafi-bojad and vatandoost 2006, vatandoost et al. 2006a, 2006b, oshaghi et al. 2006a, 2006b, 2006c, davari et al. 2006, abai et al. 2008, vatandoost et al. 2009. anopheles sahacrovi and an. maculipennis are considered as malaria vector in northern part of the country (salari lak et al. 2002, sedaghat et al. 2003a, 2003b, sedaghat et al. 2005, sedaghat and harbach 2005, oshagi et al. 2005, doosti et al. 2006, doosti et al 2007, vatandoost and zahir nia 2010). most of literature on the host-seeking behavior of mosquitoes is allocated to anthropophilic species. in an effort to understand more on the role of skin emanation in the host discrimination of zoophilic mosquitoes this study has been conducted on an. stephensi as a model. this mosquito species which is an important malaria vector in the middle east and indian subcontinent (krishnan 1961, manouchehri et al. 1976) has a remarkable zoophilic propensity (kiszewski et al. 2004). biting habit of mosquitoes in terms of host preference and frequency of taking blood meal plays a double role in vectorial capacity, a basic entomological concept in the epidemiology of mosquito-borne diseases (mc donald 1957). whereas the former is fundamentally governed by mosquito genetics the latter is under the influence of environmental factors (takken and knols 1999). if a female mosquito has no primary problem with the availability and accessibility of the potential host, then she must locate it properly by applying her elaborate sensory modalities (bowen 1991). however, mosquitoes preferentially select their victims rather than bite in a random manner (burkot 1988). it is suggested that in addition to other host cues, skin emanation provides further support in olfactory guidance of the female mosquito towards her preferred host (price et al. 1979, takken 1991, braks et al. 1999, clements 1999). furthermore, it is proposed that the chemical profile of skin emanation also takes part in the intraspecific variation of the attractiveness of hosts to mosquitoes (knols et al. 1995, brady et al. 1997, qiu et al. 2006). over 300 chemical compounds have been identified in human skin emanation (sastry et al. 1980, bernier et al. 1999, curran et al. 2005). it is well demonstrated that a few of these compounds are involved in mosquitohost interaction. l-lactic acid (hereafter called as lactic acid) (acree et al. 1968, geier and boeckh 1999, braks et al. 2001, steib et al. 2001, dekker et al. 2002), ammonia (geier et al. 1999, braks et al. 2001, smallegange et al. 2005) and 1-octen-3-ol (takken and kline 1989) are among the most important ones. electrophysiological and behavioral studies have also shown that a number of carboxylic acids individually or in a blend can be detected and attract mosquitoes in the presence of lactic acid and/or ammonia (bosch et al. 2000, smallegange et al. 2005, smallegange et al. 2009). by contrast, a number of naturally occurring fatty acids on the skin of the human beings elicit negative electrophysiological and/or behavioral effects in mosquitoes (skinner and tong 1965, bernier et al. 2007). above all, whereas certain dose of a given chemical compound may elicit attraction, a lower or a higher dose may result in a repulsive reaction or nothing at all (smallegange et al. 2009). therefore, it is conceivable that the odor-mediated attraction response of a physiologically-competent mosquito to a potential host is too far from simple. this study aimed at revealing the value of skin emanation for a zoophilic species like an. stephensi as a model. materials and methods mosquitoes anopheles stephensi originated from iranshahr, iran was maintained in the insectary of the school of public health, tehran university of medical sciences, tehran, iran under 29±1° c, 80±5% rh, ld 12:12 h conditions. the stock culture of adults took their blood meals from a guinea pig once a week. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 3 eggs hatched in a water bowl and first instar larvae were distributed in water filled plastic trays (one per milliliter) on the next day. elder instar larvae were fed with tetramin® fish food. daily collected pupae were placed in populations of 1000 to 1500 in 30×30×30 cm gauze-covered cages. the emerged adults accessed only to 10% glucose solution. all experiments were performed on 4-5-day-old 810-h sugar deprived female mosquitoes during the middle third of the dark period. these mosquitoes were selected from their rearing cages by means of attracting to a small warm water filled bottle. they were put into five small cages in population of 10 and transferred to the laboratory in an opaque plastic box matted with wet tissues. olfactometer for this study a modified giere et al. (1999) type dual-port olfactometer was used (omrani et al., 2010). in brief, charcoal-filtered, humidified (50±2%) and warm air (27±0.1 and 29±0.1° c) was led via pvc pipelines to the olfactometer arms. wind speed was constantly kept on 0.2 m/s at the cylindrical wind tunnel outlet. light intensity of two 25 w incandescent bulbs reduced to 11 lux and scattered over the olfactometer from 80 cm height to provide a relatively homogeneous ambient dim light in all experiments. two white plastic sheets at the either side of the wind tunnel prevented undesirable optical stimulation of mosquitoes. skin emanations in 3 consecutive days, a total of 90 skin emanation samples (10 sample per individual per day) were collected from a human being (43 yr old, male), a cow (holstein, 3-4 yr old, female) and a guinea pig (4 mo old, female) by rubbing ethanol soaked (absolute, merck, germany) clean dental roll cottons (0.48±0.05 g) over different areas of around 6 square centimeter during a 15 to 20 seconds period. sampling was distributed all over the body to entrap most of chemicals emanated from the skin. then, alcohol wet cottons were squeezed simply between fingers to extract trapped chemicals. they were pooled in the last day to get 15 to 20 ml semiturbid liquid from each specimen. the experimenter used sterile surgical gloves throughout the procedure to prevent any contamination risk. every day samples were transferred to the laboratory in an ice box to minimize loss of volatile compounds. the final specimens were filtered by number 2 whatman filter paper and stored under -20° c till the time of behavioral bioassays or chemical analysis. behavioral bioassays skin emanations were tested noncompetitively in two doses of 100 and 200 µl in four and eight replicates, respectively. every four-replicate set supplemented with a trial of no stimulus at the start up as control. tests were alternated between right and left arms to prevent the risk of systematic bias. for each trial a small cage containing 10 fresh mosquitoes was connected to the downwind end of the wind tunnel. after one to three minutes mosquito acclimatization a small filter paper (around 6 cm2) containing relevant dose of under test material was placed inside the stimulus chamber. 15 to 20 seconds slow shaking of this filter paper in the experimental room was sufficient to evaporate alcohol solvent before it is inserted in the olfactometer. then, mosquitoes were allowed to freely choose olfactometer arms during one minute experimentation time. at the end of each trial, mosquitoes were removed from the wind tunnel by an electrical vacuum cleaner. throughout all experiments the experimenter used cotton gloves and carefully avoided touching the inner surfaces of the wind tunnel. proportion of mosquitoes went out of the small release cage and the proportion visited or trapped in either arms of the olfactometer during one minute experimentation time comprised activation (%) and attraction (%) to the either treatment or control arms, respectively. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 4 chemical analysis of the skin emanation samples this procedure involved both enzymatic measurement of lactic acid content and gc-ms analysis of the skin emanation specimens. in the course of this study, two further samples from two generally similar cows and one sample of 0.001 dilution of technical deet (98.8%) were supplemented to primary skin emanation samples for gc-ms analysis. lactic acid content of each specimen was measured with the aid of an enzymatic kit (k-late, megazyme, ireland). this quantification process involves two reaction steps in which the amount of nadh formed is correspondent with the amount of primary lactic acid. the nadh can be measured thereafter by the increase in light absorbance at 340 nm. for this procedure 100 µl of available specimens was tested and light absorbance was measured with a diode array spectrophotometer (cecil, model 1010, england). chromatographic separation was performed using an agilent technologies 6890n chromatograph equipped with hp-5 capillary column (5% phenyl 95% methyl poly siloxane) with 30 m length, 320 µm internal diameter and 1µm film thickness. the oven program started at 35° c for 3 min and ramped at 12° c/min to 180° c followed by 25° c/min to 270° c with a final hold time of 5 min. the gas chromatograph was operated at a constant flow of 1.3 ml/min of helium as the carrier gas with 99.999% purity and the injection port and transfer line temperature was set at 230 and 270° c, respectively. the mass selective detector was agilent 5973n which scanned a range from 35 to 350 m/z with 70 ev in ei mode. chemical compounds were tentatively identified by comparison of the obtained mass spectrums with the internal library of the system. statistical analysis data entered in spss 13.0 statistical software and paired comparison of activation responses to each dual skin emanations performed by non parametric mann-whitney test (α= 0.05). the same statistical test was used for comparison of attraction responses of mosquitoes between treatment and control arms or different quantities of test materials. results behavioral bioassays one hundred µl of any skin emanations did not significantly change the primary activation of an. stephensi in the olfactometer. however, when the amount of test material was doubled, the activation of mosquitoes to human skin emanation increased significantly (z= -2.386, p= 0.017) (fig. 1). this increase was more than either no stimulus control situation or two other emanations. on the other hand, attraction to human skin emanation was statistically higher than guinea pig in both applied quantities (z= -2.646, p= 0.008 and z= -1.987, p= 0.047), but not for cow (fig. 2). the differential attraction of an. stephensi was also significant between olfactometer arms in both 100 (z= -2.646, p= 0.008) and 200 µl (z= -2.012, p= 0.044) doses. lactic acid measurement forty three, 4.4 and 1.5 µg lactic acid were detected in a 100 µl sample of human, cow and guinea pig skin emanations, respectively. gc-ms chemical analysis in general, 21, 30 and 34 chemical compounds were identified in human, cow and guinea pig skin emanations, respectively (fig. 3, table 1). chromatograms revealed the presence of some more compounds. however, they were either unidentifiable by the available chemical library or irrelevant for being extracted from a living thing. ten compounds namely ethanoic acid, 2-ethyl hexanoic acid, butylated hydroxytoluene, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, 9-hexadecenoic acid, hexadecanoic acid, 9-octadecenoic acid, octadecanoic acid were found iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 5 in all three specimens. while four further compounds i.e. 4-hydroxy-4-methylpentan-2-one; n, n-dibutyl-formamide; tetradecanoic acid, methyl ester and 9-octadecanamide were common between cow and guinea pig, nonanoic acid and decanoic acid were identified in both human and guinea pig skin emanations. hexadecanoic acid (45.21%), 4-hydroxy4-methylpentan-2-one (4.7%) and again this compound (22.56%) were the most relatively abundant chemicals found in the skin emanations of the human being, cow and guinea pig, respectively (table 2). interestingly, a small quantity of n, n-diethyl-4-toluamide (0.12%) was identified in the cow skin emanation sample. repeating the analysis of the same sample as well as two further samples from similar cows (data not shown here) and comparing these results with reconstructed chromatogram (fig. 4) and mass spectrum of the 0.001 dilution of technical deet (fig. 5) verified that this repellent is present in the cow skin emanation. 200 microliter100 microliter0 microliter 110 100 90 80 70 60 m ea n a ct iv at io n (% ) guinea pig cow human non skin emanation a b,c a a,c a,c a,c a,c fig. 1. activation response of anopheles stephensi to 100 µl and 200 µl doses of various skin emanations in the olfactometer. bars that share no letter above are significantly different at p< 0.05. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 6 guinea pigcowhumannon 30 20 10 0 -10 m ea n a tt ra ct io n (% ) attraction to the control side (%) attraction to the test side (%) a b a a,b a,b a a guinea pigcowhumannon 30 20 10 0 -10 m ea n a tt ra ct io n (% ) attraction to the control side (%) attraction to the test side (%) a b a a,b a,b a a fig. 2. attraction responses of anopheles stephensi to different skin emanations in the olfactometer. (a) 100 µl (b) 200 µl. bars that share no letter above are significantly different at p<0.05. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 7 (a) human being 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 time--> abundance tic: om1-2.d 3.93 12.70 14.65 15.78 17.27 17.42 18.63 19.18 19.68 19.78 21.07 21.22 (b) cow 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 time--> abundance tic: om3.d 3.86 8.55 12.65 15.34 15.77 17.27 17.42 18.62 18.85 19.17 19.67 19.76 21.07 21.21 21.51 (c) guinea pig 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 100000 200000 300000 400000 500000 600000 700000 800000 900000 1000000 time--> abundance tic: om2.d 3.91 8.55 12.65 15.34 17.27 17.42 18.63 18.82 19.17 19.67 19.76 21.07 21.21 21.51 fig. 3. reconstructed ion chromatogram from the electron ionization of compounds thermally desorbed from skin emanation samples on to a hp-5 capillary column. labeled peaks correspond to those listed in table 1. (a) human being (b) cow (c) guinea pig. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 8 fig. 4. reconstructed ion chromatogram from the electron ionization of the 0.001 dilution of technical deet on to a hp-5 capillary column. (a) the mass spectrum of deet from cow sample 40 60 80 100 120 140 160 180 200 220 240 260 280 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 m/z--> abundance scan 3919 (17.747 min): om65.d 119 190 91 40 65 16251 17610577 207148133 253 281 (b) the mass spectrum of the 0.001 dilution of standard deet 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z--> abundance scan 3924 (17.766 min): om4.d 119 190 91 40 65 207 162176 281105 148135 267 32725378 341 fig. 5. (a) negative ion product mass spectrum of deet observed from the sample of cow skin emanation. (b) negative ion mass spectrum of the 0.001 dilution of the standard deet in ethanol. 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 220000 240000 260000 280000 300000 time--> abundance tic: om65.d 17.74 iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 9 table 1. chemical compounds identified by gc-ms analysis in the skin emanation specimens from a human, a cow and a guinea pig. h= human being, c= cow, gp= guinea pig. compound retention time skin emanation specimen compound retention time skin emanation specimen ethanoic acid 3.93 h, c, gp 4-phenyl, 5-formyl, 1,2,3-h-triazole 17.37 gp propanoic acid 5.6 c dodecanoic acid 17.42 h, c, gp benzeneacetic acid, alpha-oxo, methyl ester 6.4 c n,n-dibutyl thiourea 17.52 gp benzenecarbothioic acid 6.46 gp hexadecane 17.71 gp benzeneacetonitrile, 2,5difluoro 6.9 gp n,n-diethyl-4-toluamide 17.77 c hexanal 7.79 c tridecanoic acid 18.06 h carbonic acid 8.26 h heptadecane 18.31 gp 4-hydroxy-4-methyl-pentan-2-one 8.54 c, gp hexadecane ,2,6,10,14 tetra methyl 18.35 gp 2-pentanone 8.57 h tetradecanol 18.44 c 4-methoxy benzaldehyde,oxime 9.84 c hexadecanal 18.46 gp 1-ethyl-phosphindoline 9.84 gp tetradecanoic acid 18.64 h, c, gp 1,2,3-propanetriol 11.02 h tetradecanoic acid methyl ester 18.82 c, gp 1-butanamine, n-butyl 11.28 gp octadecane 18.85 gp urea 12.2 h nonadecane 18.85 c cis-4-ethoxy methyl nitrostyrene 12.31 gp eicosane 18.91 gp 2-ethyl hexanoic acid 12.72 h, c, gp tetradecanoic acid,12 methyl 19.02 h benzoic acid 13.35 gp 14-pentadecenoic acid 19.09 h 2,5-dimethylfuran 14.04 c 2-heptanone 19.14 c (1r,6s)-6-methyl-bicyclo-(4,2,0)-octa2-one 14.04 gp pentadecanoic acid 19.18 h, c, gp carbamothioic acid, butyl ethyl ester 14.33 gp 9-hexadecanoic acid, methyl ester 19.66 gp nonanoic acid 14.65 h, gp 9-hexadecenoic acid 19.67 h, c, gp n,n-dibuthyl formamide 15.33 c, gp hexadecanoic acid 19.78 h, c, gp 2,3-dimethyl-(1,4-pentadiene) 15.47 c heptadecanoic acid 20.11 h decanoic acid 15.77 h, gp 9-octadecenoic acid 21.07 h, c, gp hexadecanoic acid, 2-propyl methyl ester 16.67 h octadecanoic acid 21.22 h, c, gp 4-amino ,1-methyl uracil 16.81 gp 9-octadecenamide 21.5 c, gp pyrimidine,4-fluoro2-dimethyl amino 16.81 c furfuryl alcohol 21.63 c phenol-(1,1-dimethyl ethyl), 4-methoxy 16.92 c 5-octadecane 21.63 gp 2-bromoethanol 17.15 c squalene 22.39 c butylated hydroxytoluene 17.27 h, c, gp 8-hydroxy-2-quinoline carboxyaldehyde 17.37 c iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 10 table 2. relative abundance of the indentified compounds by gc-ms analysis. percentages are calculated by dividing the peak area of a compound by the total area of compounds identified in the same skin emanation sample. h = human being, c = cow, gp = guinea pig compound human (%) cow (%) guinea pig (%) ethanoic acid 0.55 0.45 3.02 4-hydroxy-4-methyl-2-pentanone 0 4.70 22.56 2-ethyl hexanoic acid 0.44 0.25 1.16 nonanoic acid 0.29 0 0.55 n,n dibuthyl formamide 0 0.45 1.39 decanoic acid 0.17 0 0.59 butylated hydroxytoluene 0.23 2.14 16.47 dodecanoic acid 1.37 0.59 3.47 tetradecanoic acid 11.38 1.23 3.11 tetradecanoic acid, methyl ester 0 0.10 0.31 pentadecanoic acid 10.26 0.17 1.06 9-hexadecenoic acid 11.89 0.45 1.37 hexadecanoic acid 45.21 3.31 10.37 9-octadecenoic acid 0.09 0.43 1.62 octadecanoic acid 6.33 0.45 1.22 9-octadecenamide 0 0.38 1.61 discussion this study illustrates non-competitive dominance of the human skin sample over cow or guinea pig skin emanations in a dual choice olfactometer for the zoophilic malaria mosquito an. stephensi. at first glance, this result appears to be against the hypothesis that the olfactory signature of the skin of a preferred host provides specific supportive signal to a hostsearching female mosquito. however, a few points are needed to be taken into consideration, here. first of all, labeling a mosquito as a zoophilic or an anthropophilic species is primarily based on field studies and it is well known that such data is under the influence of environmental factors (garrett-jones et al. 1980). so, at times it is possible that an anthropophilic mosquito feeds on a non-human host or a zoophilic mosquito bites a human being, on the contrary. on the other hand, there are a few evidences that a highly zoophilic mosquito species may show degrees of anthropophily under controlled conditions. for example, an. quadriannulatus and an. stephensi were attracted equally to human and cow odor in a dual-choice olfactometer (pates et al. 2001, waka et al. 2006). the former species also showed unexpected anthropophagy under semi-field conditions (pates et al. 2001). collection of the skin emanations from individuals of a population with extreme attractive potential, inadequacy and insufficiency of skin sampling and/or the chemical analysis procedure, and above all, ignoring the indispensable role of other host cues are other sources of the misinterpretation of the current result. none the less, the chemical profile of tested emanations might still provide some useful clues on the observed selective attractiveness of an. stephensi in this study. the increased upwind activity of mosquitoes in response to doubling the amount of skin materials witnesses that this is a logic approach. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 11 the lactic acid content of the human skin emanation (43 µg) was measured to be around 10 and 29 times more than the cow (4.4 µg) and the guinea pig (1.5 µg) samples, respectively. this finding is similar to the result of the experiments of dekker et al. (2002) who found that lactic acid content of human skin emanation was 15 times more than the cow specimen. they did not detect any lactic acid in skin samples of a rat or a rabbit, as well. by adding or removal of lactic acid to the cow or the human skin emanations they were able to demonstrate that the lower attraction responses of an. gambiae or aedes aegypti in the olfactometer were mainly due to the specific level of lactic acid concentration in these skin samples (steib et al. 2001, dekker et al. 2002). based on these evidences it is likely that the higher attractive responses of an. stephensi to human skin emanation in the olfactometer might be related to the higher lactic acid content of this specimen. gas chromatography coupled with mass spectrometry revealed that there is very little amount of deet in the cow skin emanation. because deet is a synthetic material (acree and beroza 1962) it is obvious that this chemical compound is exogenous, but we were not able to determine the source of this contamination. whereas some workers have reported that the deet is an effective spatial repellent (wirtz et al. 1980, hoffman and miller 2002), others have considered that it is a weak one (mc govern et al. 1967, bernier et al. 2005). it has been explained that in the latter situations the concentration of this material in the vapor phase had been very low (bernier et al. 2007). according to these evidences, it is suggested that whatever the source of deet in the cow skin sample it can not justify well the inferiority of this skin emanation for an. stephensi in the olfactometer. ethanoic acid, a short-chain aliphatic carboxylic acid was identified in all three skin samples. electrophysiological recordings from sensilla trichodea of the zoophilic species an. quadriannulatus and an. atroparvus have shown that the responses of the housed sensory neurons are inhibitory (van den broek and den otter 1999). therefore, the lower activation and attraction responses of an. stephensi to guinea pig skin emanation in the wind tunnel may has some correlation with higher relative abundance of this chemical compound in this sample. nonanoic, decanoic and dodecanoic acid are three other carboxylic acids which have been reported to repel anthropophilic mosquito species individually at certain doses (reifenrath 2005). despite of the lower relative abundance of these fatty acids, their comparative level in each skin emanation sample and/or their combination with other chemical compounds therein might have particular contribution in the observed upwind responses of an. stephensi in the olfactometer. tetradecanoic acid was also common in all tested skin emanations. there are contradictory reports on the effect of this carboxylic acid on the upwind responses of mosquitoes. while this fatty acid alone or in combination with lactic acid and ammonia were attractive for culex quinquefasciatus and an. gambiae, respectively (cork and park 1996, smallegange et al. 2009), its addition to the blend of lactic acid plus a set of other carboxylic acids decreased attraction responses of ae. aegypti in the wind tunnel (bosch et al. 2000). if tetradecanoic acid assumed an attractive agent, then, it can be mentioned in line with lactic acid to explain the higher attractive responses of an. stephensi to human skin emanation. but, if it is considered as an attraction inhibitor, since its relative abundance in the human skin sample (11.38%) was 10 times more than in the cow skin emanation (1.23%), it may provide further support that the very small quantity of identified deet can not justify inferior responses of an. stephensi to this skin sample. iranian j arthropod-borne dis, 2010, 4(1): 1–16 s -m omrani et al.: differential responses of … 12 although hexadecanoic acid was the most relatively abundant chemical compound found in the human skin emanation (45.21%), there is no evidence that it can play a role in attractive responses of mosquitoes (knols and meijerink 1997, bosch et al. 2000, puri et al. 2006). the relative abundance of butylated hydroxytoluene was much higher in the guinea pig (16.47%) than the human (0.23%) or the cow (2.14%) skin emanations. this chemical agent which is a synthetic antioxidant is commonly used in the preservation of food materials (koltover 2009). although, the frequent consumption of nutritional supplements may explain the origin and higher relative abundance of butylated hydroxytoluene in the skin emanation of the guinea pig, it is not know whether it could take any part in the observed responses of an. stephensi in the olfactometer. 4-hydroxy-4-methyl-pentan-2-one is a ketonic material which was found with a high relative abundance in the skin emanation of guinea pig (22.56%). it was not detected in the human skin sample. this chemical compound is derived from 2-pentanone which is reported to be attractive for ae. aegypti (bernier et al. 2004). however, we do not know the effect of 4-hydroxy-4-methyl-pentan2-one on mosquitoes. in conclusion, although it is likely that the dominance of human skin emanation for an. stephensi in the olfactometer results from the higher content of lactic acid and contribution of a few carboxylic acids, more investigation is needed to make a reasonable judgment on the role of skin emanations in host-seeking behavior of this zoophilic mosquito. however, if semi-field and field data verify the unexpected findings of this study, it will be necessary to rethink on the current values of human blood index and vectorial capacity for this malaria mosquito species. acknowledgements this article is a part of the results of the first author’s dissertation for fulfillment of a ph.d. degree in medical entomology and vector control from department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. the authors are grateful to dr. seyed mahmood reza nikbakhtzadeh, department of parasitology and medical entomology, tarbiat modarres university, tehran, iran, dr. ali mehdinia and mrs. sahar farzadnia, national institute of oceanology, tehran, iran for their kind contribution in gc-ms analysis of the skin emanation samples. we also thank mrs. fatemeh mohtarami, school of public health, tehran university of medical sciences, tehran, iran for her supervision on the meticulous measurement of the lactic acid content of the skin emanation samples. this study was financially supported by school of public health, academic pivot for education and research, tehran university of medical sciences, project no. 85-01-63-3687. the authors declare that there is no conflict of interest. references abai mr, mehravaran a, vatandoost h, oshaghi ma, javadian e, mashayekhi m, et al. 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district, sistan & baluchistan province. hakim. 4(2):149–153. zahirnia ah, vatandoost h, nateghpour m, javadian e (1998) insecticide resistance/ susceptibility monitoring in anopheles pulcherrimus (diptera: culicidae) in ghasreghand district, sistan and baluchistan province, iran. hakim. 1: 97–106. j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 260 http://jad.tums.ac.ir published online: may 27, 2017 original article larvicidal activities of indigenous bacillus thuringiensis isolates and nematode symbiotic bacterial toxins against the mosquito vector, culex pipiens (diptera: culicidae) *ashraf m ahmed 1,6, hamdy i hussein 2, talat a el-kersh 3, yazeed a al-sheikh 3, tahany h ayaad 1,7, hanan a el-sadawy 4, fahd a al-mekhlafi 5,8, mohamed s ibrahim 3, jameel al-tamimi 1, fahd a nasr 5 1department of zoology, college of science, king saud university, riyadh, saudi arabia 2department of plant protection, college of food and agricultural sciences, king saud university, riyadh, saudi arabia 3department of clinical laboratory sciences, college of applied medical sciences, king saud university, riyadh, saudi arabia 4parasitology and animal diseases department, veterinary division, national research centre, dokki, giza, egypt 5bioproducts research chair, department of zoology, college of science, king saud university, saudi arabia 6department of zoology, college of science, minia university, el-minia, egypt 7department of entomology, college of science, cairo university, giza, egypt 8department of agricultural production, college of agriculture and veterinary medicine, thamar university, dhamar, yemen (received 19 dec 2015; accepted 9 july 2016) abstract background: the incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. this study was conducted to identify native larvicidal biopesticides against culex pipiens for utilization in the battle against mosquito-borne diseases. methods: larvicidal activities of new indigenous bacillus thuringiensis isolates and crude toxin complexes (tcs) of two nematode bacterial-symbionts, photorhabdus luminescens akhurstii (hrm1) and ph. luminescens akhurstii (hs1) that tested against cx. pipiens. b. thuringiensis isolates were recovered from different environmental samples in saudi arabia, and the entomopathogenic nematodes, heterorhabditis indica (hrm1) and he. sp (hs1) were isolated from egypt. larvicidal activities (lc50 and lc95) of the potentially active b. thuringiensis strains or tcs were then evaluated at 24 and 48h post-treatment. results: three b. thuringiensis isolates were almost as active as the reference b. thuringiensis israelensis (bti-h14), and seven isolates were 1.6–5.4 times more toxic than bti-h14. on the other hand, the tcs of the bacterial symbionts, hrm1 and hs1, showed promising larvicidal activities. hs1 showed lc50 of 2.54 folds that of hrm1 at 24h post-treatment. moreover, histopathological examinations of the hs1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. conclusion: synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. these results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases. keywords: bacillus thuringiensis, culex pipiens, biopesticide, photorhabdus bacteria, heterorhabditis nematodes introduction mosquitoes are the most dangerous insect pests that affect humans and animals world wide as they transmit epidemic and fatal diseases (who 2010, aziz et al. 2014). they *corresponding authors: dr ashraf m. ahmed, e-mail: aalii@ksu.edu.sa j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 261 http://jad.tums.ac.ir published online: may 27, 2017 transmit pathogens of filariasis, rift valley fever, west nile virus, and encephalitis (meyer et al. 1983, rosen et al. 1989, omar 1996, balenghien et al. 2008, seufi and galal 2010). in saudi arabia, different local mosquito vectors are spread all over the country (alkhuriji 2007, al-ghamdi et al. 2008, ahmed et al. 2011, al-ahmed 2012). these vectors transmit mosquito-borne diseases including dengue fever (khan et al. 2008, alwafi et al. 2013, aziz et al. 2014, ayaad et al. 2015), filaria (hawking 1973), malaria (balkhy and memish 2003, madani 2005) and rift valley fever (jup et al. 2002, al-hazmi et al. 2003 and madani 2005). beside the recent outbreak of mosquitoes and incidence of epidemic diseases, resistance of local mosquitoes to conventional pesticides has been recorded (al-sarar 2010, osta et al. 2012, brouqui et al. 2012), which have caused a panic to the authorities. until now, conventional pesticides are the main tool being used to combat mosquitoes. however, synthetic pesticides cause health problems and pollute the environment (azmi et al. 2009). moreover, some synthetic mosquito repellents cause encephalopathy in children (briassoulis 2001). therefore, there is an urgent need for effective and safe alternatives to the conventional pesticides. considering natural insecticides, essential oils are being used against adult mosquitoes (bernier et al. 2005, maguranyi et al. 2009), however, they repel, but do not kill, mosquitoes. bacillus thuringiensis, the most successful bioinsecticide, have been used to combat mosquito larvae for 3 decades and until now (heimpel and angus 1959, ali et al. 2010, schünemann et al. 2014). this bacterium has many advantages over conventional pesticides as it is specific to certain pest species, eco-friend and safe to non-target organisms, and mosquitoes cannot develop significant resistance to it in the field so far (bravo et al. 2007). on the other hand, entomopathogenic nematodes, with their symbiotic bacteria, also introduce promising solution as biocontrol agent for pest control management (lang et al. 2011, zhu et al. 2011). the infective juveniles of the two genera steinernema or heterorhabditis are active in seeking the host as they penetrate via the host's natural opening and immigrate to the insect haemocoel then, release their symbiotic bacteria, xenorhabdus or photorhabdus respectively. multiplication of these bacteria within insect haemocoel results in production of numbers of virulence factors, including toxins complexes that kill the insect host within 48h (ffrench-constant and bowen, 2000, ffrench-constant et al. 2007, eleftherianos et al. 2010). the current study herein was conducted for laboratory assessment of the mosquito larvicidal activity of local indigenous b. thuringiensis isolates, as well as tcs from the nematode bacterial symbionts, photorhabdus, as new proposed candidates to be utilized in the battle against mosquito vectors. materials and methods experimental mosquitoes a susceptible strain of culex pipiens was reared in zoology department, king saud university for use in this study. mosquitoes were reared in the lab for 10 generations according to ahmed et al. (1999) before performing experiments. briefly, adults were maintained at 26±1 °c and 12:12h (light: dark) photoperiod and provided with 10% glucose solution ad libitum. females were blood-fed on cd mice to lay eggs, and hatched larvae were fed on 'liquifry' (interpet ltd, dorking, uk) for two days, then provided with ground 'tetramin' flake food (tetra werke, melle, germany) until pupation. third-instar larvae were subjected to bioassay using b. thuringiensis isolates or tcs of photorhabdus. collection of environmental samples experimental samples were collected from different regions across saudi arabia from j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 262 http://jad.tums.ac.ir published online: may 27, 2017 october 2013 to march, 2014 (table 1 and fig. 1). samples were collected from a variety of sites within each region including the vicinity of houses, irrigated parks, gardens and farms as well as from the surrounding semidesert areas. a total of 300 samples of different types were collected including soil, water and dead plants and animals (table 1 and fig. 2). for soil sampling, the soil surface was scraped and about 10g of material were taken from a depth of 2–5cm into sterile containers. plant parts were collected in sterile plastic bags. samples were also taken from water, dead fish, insects, and snails, beeswax, as well as from dried faeces and stored in 10ml sterile test tubes. samples were processed for b. thuringiensis isolation according to bozlagan et al. (2010). isolation, culturing and identification of b. thuringiensis isolates sixty-eight b. thuringiensis isolates were recovered from different environmental samples (el-kersh et al. 2016) according to hong et al. (2009) and el-kersh et al. (2012) from different locations throughout saudi arabia (fig. 1). briefly, for soil samples, one gram was grinded and added to 2.0ml of sterile distilled water and suspended vigorously. one gram of dead insects, fish guts and gills, or bees wax samples was dispatched and then macerated in 2.0ml sterile saline (0.5%, w/v), using a sterilized mortar and pestle. two ml aliquots from water samples were mixed with 2ml absolute ethanol, mixed well for 1min, incubated for 45min at 30 °c, with shaking from time to time. after ethanol treatment, ten-fold serial dilutions were made in sterile distilled water. appropriate dilutions were spread on a nutrient agar medium supplemented with 0.2% yeast extract (sisco research laboratories, mumbai, india) and 0.0005% manganese chloride, the medium was incubated for 2–3 days at 30 °c (el-kersh et al. 2012). bacillus thuringiensis-like colonies were selected and suspected b. thuringiensis like colonies were fished from populated bacilli, one single colony was repeated until a pure culture was obtained. closely related other spore forming bacilli were excluded via phase contrast microscopy for the presence of parasporal crystals. bacillus thuringiensis index was calculated for each positive sample (xavier et al. 2007, el-kersh et al. 2012). biochemical, phenotypic characterization, and identity confirmation of recovered 68 b. thuringiensis isolates were accomplished on the basis of esculin-hydrolysis, lecithinase, hemolytic, and motility activities, api 20e, and carbohydrate utilization (api 50ch system), essentially as previously described by el-kersh et al. (2012). crystal morphology analysis took place under phase contrast microscope (rampersad and ammons 2005, gobatto et al. 2010). in this study a total of 68 b. thuringiensis isolates were successfully recovered from the 300 collected samples, each of which was purified by sub-culturing on sna agar for 48 hours and stored as a stock culture in a sterile liquid nutrient broth medium containing 50% glycerol at -20 ºc (hernandez et al. 2005). spore-crystal mixtures preparation preliminary screening for larvicidal activities have been carried out to assess the suitable lethal concentrations of the spores-crystals mixture of each b. thuringiensis isolate in parallel with the b. t. israelensis h14 (btih14), as a reference strain, (kindly provided by prof. a. abdel-hameed, deceased) according to el-kersh et al. (2012). out of the tested 68 native b. thuringiensis isolates, only 23 isolates showed larvicidal activity (data not shown), the most potentially active 10 isolates were selected for further bioassay and identification. to avoid possible discrepancy during the preparation of spore-crystal mixture for quantitative determination of lc50 and lc95, b. thuringiensis isolates were prepared from fermentation growth on nutrient yeast extract salt medium, nysm, (conj arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 263 http://jad.tums.ac.ir published online: may 27, 2017 taining per litre: 5g glucose, 5g peptone, 5g nacl, 3g beef extract, 0.5g yeast extract, 0.02g magnesium chloride, 1mg manganous chloride, 0.01g calcium chloride, ph 7.2). spores-crystals mixtures were then prepared as dried powders in adequate amount using the lactose acetone co-precipitation procedure according to dulmage et al. (1971). the resulting white fine toxin powder mixtures were stored at 4 °c until used. extraction of toxin-complexes from nematode bacterial-symbionts isolation of nematode bacterial symbionts two types of the bacterial symbiont, photorhabdus, were isolated from the two entomopathogenic nematodes, heterorhabditis indica (hrm1), isolated from alexandria, northern egypt, and heterorhabditis sp. (hs1), isolated from ras-sidr in south sinai of eastern egypt, as detailed in (el-sadawy et al. 2016). briefly, bacteria were isolated from their symbiotic nematodes according to woodring and kaya (1988). for each subculture, the phase status was determined by culturing on nbta agar [2.3% nutrient agar (difco), 0.0025% bromothymol blue (merck), 0.004% 2,3,5triphenyltetrazolium (merck)]. phase i colonies are blue on nbta while phase ii colonies are red. twenty infective nematode juveniles were surface sterilized for 10min in 1.0% sodium hypochlorite, washed in sterile distilled water, transferred to a petri dish containing 5ml of tsbye [3% tryptic soy broth (difco), 0.5% yeast extract (difco)], and grinded using grinder pistol. the plates were incubated at 30 °c for 24h and streaked on nbta plates [2.3% nutrient agar (difco), 0.0025% bromothymol blue (merck), 0.004% 2,3,5-triphenyltetrazolium (merck)]. the presence of photorhabdus colonies was confirmed by dye adsorption on nbta plates, production of luminescence, and antibiotic activity. the isolated bacteria were then maintained on nbta plates at 10 °c and subcultured weekly. toxins extraction from nematode bacterial symbionts bacterial cell pellets were obtained from a 2-liter culture of the two photorhabdus types hrm1 and hs1 fermentations, separately for 48h according to sheets et al. (2011) with some modifications. the pellets were suspended in 50mm tris-hcl (ph 8.0), 100mm nacl, 1mm dtt, 10% glycerol, lysozyme (0.6mg/ml) and bacterial protease inhibitor cocktail (sigma, st. louis). a small amount of glass beads (0.5mm diameter), were added and then bacterial cells were disrupted by sonication then centrifuged at 10,000g for 60min at 4 °c. supernatants, including toxin complexes (tcs), were then collected into eppendorf tubes and subjected to protein concentration measurement using coomassie blue protein assay reagent (ici americas, inc.) according to the manufacturer’s instructions, then stored at -20 °c. the total protein was estimated by the method described by bradford (1976), and bovine serum albumin was used for the calibration curve. tcs were then stored in liquid nitrogen until used for larvicidal bioassay screening. larvicidal bioassays spores of bacillus thuringiensis spore-crystal powder of each b. thuringiensis isolate was suspended in 10ml of sterile water, to give an average count of 109 colony-forming unit (cfu)/ml, and then used for preliminary screenings of larvicidal activity against 3rd instars larvae. a high concentration from crystal-spore suspension of each b. thuringiensis isolates was used in parallel with the reference bti strain [bt serovar israelensis de barjac (bti-h14)] and negative control (el-kersh et al. 2012). larval mortality was scored at 24h post-treatment at 22±1 °c (data not shown). out of the tested 68 native b. thuringiensis isolates, only 10, that exhibited significant mosquitolarvicidal activity, were selected for further bioassay assessment and spore counts. for j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 264 http://jad.tums.ac.ir published online: may 27, 2017 these 10 potentially larvicidal active isolates, preliminary bioassays were carried out using wide range of nine ascending concentrations prepared by suspending a certain weight from each of isolates’ toxin mixture in distilled water. based on the preliminary results (data not shown), a narrower range of 5 lethal concentrations of each isolate were used for the main bioassay tests. for the main investigation of mosquito larvicidal activity against 3rd instars larvae, lc50 and lc95, of each of the 10 potentially larvicidal b. thuringiensis isolates, were investigated in parallel with the reference strain (bti-h14) at 24h and 48h post-treatment. in this bioassay, five ascending concentrations from each b. thuringiensis mixture were used as recommended by who (2005) with some modifications. briefly, twenty 3rd instars larvae were placed in each well of a sterile standard 12-wells tissue culture test plate (nunclone delta surface, thermo-fischer scientific, denmark) with 2 ml de-ionized water. an amount of 10µ l from each concentration was added to each well (rey et al. 1999). another group of larvae were treated in the same manner with 10µ l de-ionized water (negative control) or the reference bti-h14 strain (positive control) for comparison (el-kersh et al. 2012). each concentration was applied in 5 replicates (n= 5) using 5 different groups of experimental larvae (20 larvae each). larvae were fed to avoid mortality caused by starvation. a lack of larvae reaction to gentle prodding with a glass pipette was recorded as mortality according to brown et al. (1998). the mean percentage of larval mortality was calculated for the 5 replicates (n= 5) of each concentration of each isolate using abbott's formula (abbott 1925) at 24 and 48h post-treatment. subsequently, the lc50 and lc95 of each of the 10 larvicidal b. thuringiensis isolates, and the reference strain (bti-h14), were estimated using probit analysis. meanwhile, samples from each of the same serial dilutions of b. thuringiensis toxins were cultured on sna medium for estimating spore counts by counting the number of colony forming units cfu/µ g. toxin complexes (tcs) of nematode bacterial symbionts two tcs extracted from the two photorhabdus types, hrm1 and hs1, were lyophilized into powders prior to preliminary larvicidal bioassays using wide range of serial concentrations prepared by suspending a certain weight from each powder in deionized distilled water. based on the preliminary results, four serial effective concentrations from each toxin were assessed and used for the main bioassays according to the who (2005) as described above. mortality percentages were calculated at 24 and 48h posttreatment. histopathological studies light microscopy tcs of hs1 was used for testing the histopathological impact on midguts of treated larvae as it showed higher mosquito larvicidal activity compared to that of hrm1. larvae were treated with the lc50 (38.3 µ g/ml) of hs1 tcs or distilled water as controls. alive treated-sluggish (prior to death) or control larvae were collected at 24h posttreatment and used to investigate the midgut histological alterations under light microscope according to ahmed et al. (2014). briefly, midgut sections were fixed overnight in cold 2.5% glutaraldehyde in 100 mm phosphate buffer (ph 7.2) and for 1h in 1% oso4. midgut sections were dehydrated through an ethanol series, treated with propylene oxide, and embedded in poly/bed 812 (polysciences inc, warrington, pa). sections (10μm) were stained with hematoxylin and eosin (sigma-aldrich), mounted with paramount (fisher), and examined by light microscopy (zeiss axioskop 50 compound microscope, carl zeiss, inc, thornj arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 265 http://jad.tums.ac.ir published online: may 27, 2017 wood, ny). images were imported into adobe illustrator cs, 2003 software and adjusted for examination. transmission electron microscopy (tem) the hs1tcs-treated or control larvae were prepared for midgut ultrastructure examination by tem prior to death at 24h post-treatment as described above. briefly, treated midguts were fixed overnight in 0.8% glutaraldehyde and 4% paraformaldehyde dissolved in 0.1m sodium cacodylate (ph 7.0), followed by 4h in 1% oso4 at 4 °c. after dehydration, treatment with propylene oxide and embedding in epon-araldite resin (1:1), 4μm sections were mounted, stained with 2% uranyl acetate for 30min and incubated in lead citrate for 10 min. samples were examined with a transmission electron microscope (jeol ltd., model jem100cx ii) at 80 kv. statistical analysis the lc50, lc95, slopes, and standard error values of each treatment (5 replicates) were calculated according to finney (1971). relevant treatments were considered as not significantly different in their toxicity if confidential limits (95%) of lc50 were overlapped (litchfield and wilcoxin, 1949). results characteristics of the b. thuringiensis isolates out of several hundred (> 300) examined b. thuringiensis-like colonies (fig. 2a,b), 68 b. thuringiensis isolates were identified. the overall mean b. thuringiensis index, the ratio of b. thuringiensis isolates producing crystal (fig. 2c) to other non-b. thuringiensis spore forming bacilli, corresponding to the whole sampling areas was 0.35. more than 75% of processed samples (n=300) were negative for b. thuringiensis isolates, suggesting a limited abundance of the organism in several saudi environmental regions. b. thuringiensis index reflects the abundance of b. thuringiensis strains but not necessarily their b. thuringiensis diversity. hence, some single specimens yielded more than one b. thuringiensis isolates, which differ in their colony morphology and parasporal crystal shapes (fig. 2), yet with low b. thuringiensis index. whereas other single specimens yielded only one b. thuringiensis isolate with a relatively high b. thuringiensis index, and meanwhile several other samples yielded no b. thuringiensis isolates. phase contrast microscopy examination revealed that most of the recovered 68 b. thuringiensis isolates showed spherical crystals (34%), while irregular (small spherical to amorphous, cubic, merged triangular/or conical like budding (fig. 2c), bi-pyramidal, and attached crystal (various shapes) to the spores constituted 32, 13, and 21% respectively. the characteristics of the selected ten b. thuringiensis isolates were similar to those of the reference bti-h14 isolate (table 1), with exception of the bt-63 isolate, which produced acid from sucrose. however, the rest of its characteristics were similar to those of the reference bt-h14. larvicidal activity bacillus thuringiensis based on preliminary screening for larvicidal activity, 23 native b. thuringiensis isolates (out of 68 in total) showed promising larvicidal activities. hence, the best potentially active 10 b. thuringiensis isolates were further subjected to quantitative lc50 and lc95 determination in parallel with the bti-h14 as a reference strain (positive control), using prepared acetone spore/crystal mixture-lactose co-precipitation dried powder with concomitant determination of spore colony forming unit (cfu) per µ g powder (table 2). taking into consideration the spore cfu/µ g powder, three native b. thuringiensis isolates coded (bt-12, bt-26, and j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 266 http://jad.tums.ac.ir published online: may 27, 2017 bt-29) showed almost similar lc50 and lc95 values similar to those of the bti-h14 reference strain positive control. whereas, 7 native b. thuringiensis isolates coded (bt-42, bt-44, bt-53, bt-55, bt-60, bt-63, and bt68) showed significantly higher larvicidal activity (~2.6 and ~6.4 folds less spore cfu/ µ g) than those of the bti-h14, and their respective lc95 and slope values confirm these activities. toxin complexes (tcs) of nematode bacterial-symbiont extracted tcs from the two photorhabdus types (hrm1 and hs1) were screened for their larvicidal activity. data showed that the toxicity of hs1 tcs was 2.5 folds higher than that of hrm1 tcs (lc50 38.3 v 97.4 µ g/ml, respectively) at 24h post-treatment (table 3). the toxicities of both toxins were higher at 48h than that at 24h post-treatment, considering the lc50 values, with hs1 tcs being significantly more toxic (table 3). the lc95 values of hs1 tcs showed non significant difference at 24 and 48h post-treatment, and slope values confirm these activities (tables 3). histopathological studies the impact of oral administration of hs1tcs on the histological integrity of treated midguts of 3rd instar larvae has been investigated at 24h post-treatment. light microscopy showed numerous cytoplasmic extensions, and cellular and nuclear degeneration were clear in the midgut epithelial cells of treated midguts (figs. 3b, d). the peritrophic membrane and microvilli appeared disrupted compared to the untreated control midguts as they retained their structural integrity, with the nuclei in the centre of the cell and microvilli bordered the lumen normally (figs. 3a, c). moreover, tem revealed subcellular alterations in terms of nuclei disintegration, degradation of chromatin and nucleoli (figs. 4d, e) and disrupted microvilli (fig. 4f). in addition, mitochondria appeared with cristae deformation and almost free of their internal contents (figs. 4h, i), while the structure of the epithelial cells and their components in the control midguts appeared normal and keeping their integrities (figs. 4 a, b, c and g). fig. 1. a map of saudi arabia showing the geographical distribution of the 10 potentially larvicidal effective bacilus thuringiensis isolates (jizan, madina, mecca and yanbu) across saudi arabia fig. 2. photomicrographs showing morphology of the native bt-55 isolate seeded in nutrientsupplemented agar media. a-b: colonies and a single magnified colony showing white, raised-centrally, nearly-circular, and glossy colony morphology with fine irregular margins similar to that of bti-h14. (photos a and b by ama). c: phase-contrast microscopy (×1000) illustrating the parasporal crystal (arrow heads) and bacterial spores (arrows) which appear brighter j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 267 http://jad.tums.ac.ir published online: may 27, 2017 fig. 3. histopathological impact of hs1 toxin-complexes on the midgut epithelia of cx. pipiens larvae, 24h post-treatment. figs. a and c represent cross sections in midguts of untreated larvae, showing normal gut epithelial layer (el) with healthy, normal epithelial cells (ec), peritrophic membrane (pm), microvilli (mv), nuclei (n), and nutritional gut contents (gc) filling the gut lumen. figs. b and d represent cross sections in midguts of treated larvae, showing affected gut epithelial layer, with cytoplasmic extensions (ce), degraded microvilli (dmv), degenerated epithelial cells (de) table 1. overall characteristics and biochemical profiles of 10 potential larvicidal native bacilus thuringiensis isolates, from different regional sources, and the bti-h14 reference strain b. thuringiensis code / city regional source catalase, oxidase, lecithinase, esculin and gelatin hydrolysis, hemolytic and motility activities, citrate utilization, nitrate-reduction, and vp test urease, h2s, npg, and indole tests acid produced from starch, glycogen, glucose, fructose, mannose, or maltose no acid from inulin, xylose, galactose, lactose, salicin, mannitol, or sucrose* bti-h14 bt-12/ yanbu bt-26/ medina bt-29/ medina bt-42/ jizan bt-44/ jizan bt-53/ medina bt-55/ medina bt-60/ medina bt-63/ mecca bt-68/ medina + + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + + _ _ _ _ _ _ _ _ + _ _ *the isolate bt-63 produced acid from sucrose table 2. toxicity of 10 potential larvicidal native bacilus thuringiensis isolates against 3rd instar larvae of culex pipiens. lethal concentrations (lc50 and lc95) were calculated by probit analysis for all isolates compared to the reference bti-h14 btcode city of collection hrs p-t lc50 (µg/ml) (lower to upper) lc95 (µg/ml) (lower to upper) ×105 cfu/µg slope±se bt-12 yanbu 24 4.6 (4.2–5.1)e 9.4 (7.9–11.08) 0.53±0.4 5.4±0.005 48 2.4 (2.04–2.8) 10.4 (8.01–13.9) 2.6±0.06 bt-26 medina 24 4.8 (4.1–5.6)e 33.9 (21.8–54.5) 4.1±0.7 1.94±0.044 48 1.9 (1.5–2.4) 14.6 (10.2–22.0) 1.9±0.051 bt-29 medina 24 4.2 (3.5–5.0)e 32.6 (22.8–47.8) 0.52±0.1 1.8±0.03 48 2.2 (1.8–2.7) 14.05 (10.6–19.2) 2.05±0.037 bt-42 jizan 24 2.5 (2.2–2.9)c 15.4 (10.9–22.8) 0.93±0.3 2.1±0.048 48 1.2 (1.04–1.35) 3.07 (2.5–3.8) 4.02±0.26 bt-44 jizan 24 3.03 (2.7–3.4)d 9.4 (7.7–11.6) 0.63±0.4 3.35±0.075 48 2.08 (1.8–2.3) 6.12 (5.1–7.5) 3.5±0.094 bt-53 medina 24 1.9 (1.7–2.2)b 8.08 (5.8–11.7) 1.3±0.4 2.6±0.0011 48 0.72 (0.6–0.8) 2.5 (2.08–3.6) 2.9±0.0014 j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 268 http://jad.tums.ac.ir published online: may 27, 2017 bt-55 medina 24 1.7 (1.5–1.98)b 7.9 (6.11–10.68) 1.95±0.3 2.5±0.059 48 1.06 (0.91–1.2) 2.29 (2.5–3.8) 3.65±0.21 bt-60 medina 24 1.9 (1.7–2.2)b 5.01 (4.2–6.15) 0.13±0.1 4.05±0.002 48 0.94 (0.8–1.05) 2.9 (2.5–3.8) 3.3±0.09 bt-63 mecca 24 0.91 (0.7–1.07)a 5.51 (4.1–8.2) 0.7±0.2 2.1±0.053 48 0.5 (0.4 –0.58) 1.12 (0.95–1.4) 4.72±0.006 bt-68 medina 24 2.3 (2.06–2.7)c 10.7 (7.8–15.7) 1.05±0.3 2.5±0.055 48 1.1 (0.97–1.3) 5.4 (4.2–7.5) 2.4±0.056 bt-15 bti-h14 24 4.88 (4.2–5.4)e 21.9 (17.3–28.0) 4.5±0.3 2.52±0.044 48 2.62 (2.2–3.1) 10.01 (7.8–13.2) 2.82±0.092 lc50= lethal concentration (concentration to kills 50% of test larvae), lc95= lethal concentration (concentration to kills 95% of test larvae), cfu= colony forming unit, s.e.= standard error of means (n= 5), values with different letters (a, b, c, d and e) are significantly different within strains and compared to the reference bti-h14 (based on the non-overlapping confidence limits) according to litchfield and wilcoxin (1949). control mosquitoes showed nil mortality. table 3. probit analysis for toxicity of hs1and hrm1 tcs against 3rd larval stage of culex pipiens within a column, values followed by different letters are significantly different according to litchfield and wilcoxin (1949) toxin hrs p-t lc50 (µg/ml) (lower to upper) lc95 (µg/ml) (lower to upper) slope ± se hs1-toxin 24 38.3(36.2–40.4)a 58.7(53.9–63.9)a 8.8 ± 0.6 48 31.7(29.6–33.9)b 58.2(51.8–56.5)a 6.2 ± 0.32 hrm1toxin 24 97.4(93.9–100.9)c 139.9(131.4–149)b 10.45 ± 1.11 48 76.6(69.3–84.6)d 115.3(106.5–124.6)c 9.2 ± 3.07 fig. 4. transmission electron microscopic micrographs showing the cytological effects of hs1 toxin-complexes treatment on the ultrastructure of the midgut epithelial tissue of cx. pipiens 3rd larval stage at 24h post-treatment. a, b, and c indicate normal nucleus (n), nucleolus (n), microvilli (mv), and chromatin contents in epithelial cells of control larvae (scale bar= 5, 2.5, and 2.5μm respectively). d, e, and f indicate degenerating nucleus (dn) and its contents, degenerated microvilli (dmv) with bubbling and stretching appearance in treated larvae (scale bar= 2.0, 2.5, and 0.5μm respectively). g: represents normal mitochondrial structures in control larvae (m) (scale bar= 0.5μm). h and i: represent degenerating mitochondria (dm) in treated midgut cells, showing deformation and loss of cristae and matrix (scale bar= 1 and 0.5μm respectively). ultrathin 4μm sections were investigated with transmission electron microscope model jeol jem-100cx ii at 80kv. table 1. continued… j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 269 http://jad.tums.ac.ir published online: may 27, 2017 discussion in this study laboratory, assessment of the toxic activities of indigenous b. thuringiensis strains and toxin complexes (tcs) of nematode bacterial symbionts were investigated against 3rd larval instars of the filaria vector, cx. pepiens (hawking 1973). thus, it is important to clarify initially several key points. firstly, this study targeted cx. pepiens, the widely distributed filaria vector worldwide. secondly, this study seeks not only to overcome the recently evolving problem of mosquito resistance to chemical insecticides (alsarar 2010, osta et al. 2012, brouqui et al. 2012) but also to regain the balance of nature by developing and then implementing biocontrol measures against mosquito vectors, while keeping the environment safe and unpolluted by chemical insecticides (azmi et al. 2009). hence, certain native bacterial isolates from b. thurin-giensis and (tcs) from the nematode bacterial symbiont, photorhabdus bacteria (hrm1 and hs1), isolated from their heterorhabdus mutual nematode were tested as they have previously shown remarkable mosquito larvicidal activities in our lab (el-kersh et al. 2012, 2014 and elsadawy et al. 2016). thirdly, b. t. israelensis isolates and nematodes, isolated from saudi and egyptian environment respectively, were used in order to ensure effectiveness when used locally in the same regions. fourthly, this study is considered as an initial step towards implementing a biocontrol measure against this filaria vector in saudi arabia and may be worldwide. in fact, the entomopathogenic b. t. israelensis has been proved not only as effective biocontrol agent against larvae of many mosquito species worldwide (ben-dov 2014) but also as safe for non-target invertebrates and vertebrates. this fact has encouraged researchers to search for new b. thuringiensis strains in various countries (ohba and aizawa 1986, mohammedi et al. 2006, armengol et al. 2007, gobatto et al. 2010, aramideh et al. 2010, ramalakshmi et al. 2010, saferalizadeh et al. 2010, kavitha et al. 2011, elkersh et al. 2014). in support to these efforts, the current study herein aimed at isolating local indigenous mosquito larvicidal b. thuringiensis from the saudi environment for use as an echo-friend biocontrol agents for three reasons, a) thousands of saudis were recently infected with mosquito-borne diseases (khalil et al. 2008, madani et al. 2003, alwafi et al. 2013, al-thabiani 2014), b) to the best of our knowledge, pest control in saudi arabia still solely relies on chemical insecticides, and mosquito vectors become resistant to most of them (eg. al-sarar 2010) but not to bti yet (boyer et al. 2012) and c) so far, little is known about the natural presence and isolation of native entomopathogenic b. thuringiensis species from the saudi environment except very few studies that isolated b. thuringiensis against lepidopteran pests (assaeedi et al. 2011, abulreesh et al. 2012, el-kersh et al. 2014) and mosquito vectors (al-zahrani and abuldahab 2011) from particular locations. thus, we believe that isolation of more local mosquiocidal b. thuringiensis is urgently needed as it will be more effective in the saudi hot, dry and desert environment, as well as similar environments of other countries in the world. on this context, we have obtained 68 b. thuringiensis isolates from different locations throughout the country, and the percentage of active isolates against mosquito larvae was low compared to the inactive ones. elkersh et al. (2012) reported that most b. thuringiensis isolates obtained from different regions in saudi arabia were inactive against cx. pipiens larvae; similar finding was also reported from different regions worldwide (bernhard et al. 1997, park et al. 2008). out of the selected 10 potentially active isolates, 5 isolates belong to samples j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 270 http://jad.tums.ac.ir published online: may 27, 2017 collected from medina (24° 28′ 0″ n, 39° 36′ 0″). the most active isolate coded (bt-63) was obtained from samples collected from mecca (21° 30′ 0″ n, 41° 0′ 0″). this could be attributed to the abundance of irrigation fresh water and hence mosquito distribution in this region (martin et al. 2010). nevertheless, the presence of mosquitoes does not guarantee the presence of b. thuringiensis in the breeding water or in the soil. evidence for this is that b. thuringiensis isolates found in soils showed little or no insecticidal activities, whereas some of those not found in soils showed high insecticidal activities (chatterjee et al. 2007 and kavitha et al. 2011). we, therefore, consider all of our 68 b. thuringiensis isolates as part of the indigenous microflora of the areas, which have been explored. the recovered 68 b. thuringiensis isolates showed spherical crystals (34%), while irregular (small spherical to amorphous, cubic, merged triangular/or conical like budding, bi-pyramidal, and attached crystal (various shapes) to the spores constituted 32, 13 and 21% respectively (el-kersh et al. 2016). the types of crystal morphology recorded for the parasporal inclusion bodies in that study were reported in many bti isolates from different regions inside and outside saudi arabia (bernhard et al. 1997, el-kersh et al. 2012) which is attributed to the inactivity of their isolates against cx. pipiens larvae to the high percentage of spherical crystals. however, el-kersh et al. (2016) reported that the ratio of spherical crystals was the highest compared to the rest of crystal morphology. the high toxicity of b. thuringiensis strains obtained from the philippines and colombia were attributed to the spherical parasporal inclusions (padua et al. 1984, orduz et al. 1992). in addition, there is no correlation between the type of insecticidal activity and crystal morphology (bernhard et al. 1997, martin and travers, 1989, ohba and aizawa 1986). in this context, although the reported 10 potentially larvicidal active b. thuringiensis isolates in the current study showed similarities in their crystal shape and biochemical profiles, their larvicidal activities (the lc50 and lc95) showed great discrepancies. additionally, the most active isolate, coded (bt63), was the only one to produce acid from sucrose suggesting different metabolic capability as compared to the reference bti-h14, and the other remaining native b. thuringiensis potential isolates. it is conceivable that such great variation in larvicidal activities is most probably correlated with respective cry and cyt genes content of each strain (el-kersh et al. 2016). in this context, bendov (2014) stated that the high specific mosquito larvicidal properties of bti δ-endotoxins are attributed to complex interactions between six proteins, cry4aa, cry4ba, cry10aa, cry11aa, cyt1aa and cyt2ba, differing in toxicity levels and against different species of mosquitoes. on the other hand, tcs of both hrm1 and hs1 of photorhabdus bacterial species, showed larvicidal activities of lc50 38.3 and 97.4µ g/ml respectively at 24h post-treatment in the current study. it is well established that ph. luminescens is symbiotic bacterium lives in the gut of the entomopathogenic heterorhabdus nematodes that inject it into the hemocoel of insect host upon invasion. many studies have investigated the killing mechanisms of these bacteria as they bring about immunosuppression, septicemia and the subsequent death of the insect host (ffrench-constant et al. 2007, eleftherianos et al. 2010, lang et al. 2011, zhu et al. 2011). moreover, a number of pathogenicity determinants have been identified in this gramnegative bacterial species, and some others, including hemolysis factor, hydrolases, lipopolysaccharide, regulatory factors, toxins and proteases (ffrench-constant et al. 2007, nielsen-leroux et al. 2012). these toxic compounds are displayed on the outer surface of j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 271 http://jad.tums.ac.ir published online: may 27, 2017 the bacterium (ffrench-constant et al. 2007). previous findings support our findings like bowen and ensign (1998) that identified four different toxin complexes of ph. luminescenens strain w14 termed tca, tcb, tcc, and tcd. they also proved that these toxin complexes are composed of three different proteins, xpta2, xptb1, and xptc1 representing products from class a, b and c toxin complex genes respectively. in this context, guo, et al. (1999) suggested that a proteins harbors the cytotoxic effects of the tc toxins, whereas class b and c proteins rather modulate and enhance the toxicity of class a proteins. data of the current study may indicate that mosquito larvicidal tcs of photorhabdus bacteria isolated from local heterorhabdus nematode could be utilized in the battle against mosquito vectors. evidence for this hypothesis has been provided by ffrench-constant et al. (2007) who mentioned that the photorhabdus tcs (pirab binary toxins) have produced oral toxicity against mosquitoes and some caterpillar pests. further evidence has been provided by vani and lalithambika (2014) who recorded 93.32% mortality for the 3rd larval instar of anopheles gambiae when he used 100ng/ml of the extracellular proteins (from 20–97 kda) of the outer membrane of xenorhabdus sp bacteria isolated from steinernema sp nematode. moreover, bishop (2014) has successfully cloned and introduced the prta gene (encodes the protease a virulence factor) from ph. luminescens into bacillus thuringiensis which enhanced the mortality in larvae of the lepidopteran moth, pieris brassicae and galleria mellonella when taken orally and injected into the hemocoel respectively compared to the wild type of b. thuringiensis. the histopathological impact of tcs from hs1 on the integrity of the midgut of treated cx. pipiens larvae was also investigated in the current study. light and electron microscopy provided solid evidences for the cellular and subcellular impacts respectively on the midgut of treated mosquitoes. light microscopy showed clearly the destruction of the epithelial cells lining the midgut, which may be associated with the midgut paralysis and cessation of feeding noticed by 12h posttreatment. besides, tem results showed severe damage at the mitochondrial level as white spots are detected compared to untreated control. supporting evidences for this histological impact have been previously provided by morgan et al. (2001), who observed oral toxicity of the supernatants of some xenorhabdus strains to insects. in addition, blackburn et al. (1998) proved that the toxic compounds produced by ph. luminescens caused disruption of the midgut epithelium in a manner similar to that of δ-endotoxins from b. thuringiensis. in addition, sheets et al. (2011) showed that recombinant xpta2, and co-produced recombinant xptb1 and xptc1 bind together with a 4:1:1 stochiometry, and that xpta2 forms a tetramer of ~1,120 kda that binds to solubilized insect brush border membranes, and hence, induces pore formation in the membrane lipids. data of the histopathological study of hs1 observed in the current study also provide evidence of the similarity to those observed in bti-infected midguts in previous studies (bravo et al. 2007 and soberon et al. 2007, al-roba et al. 2011). this may provide further evidence that these toxin-complexes of the nematode symbiotic bacterial can be considered as reliable candidate in the biocontrol measure against mosquito vector. conclusion the present findings may contribute to the efforts and plans of saudi ministry of health for the control of mosquito-borne diseases. the larvicidal activity of these native b. thuringiensis isolates and nematode bacterial-symbiont tcs, or a combination of j arthropod-borne dis, june 2017, 11(2): 260–277 am ahmed et al.: larvicidal activities of … 272 http://jad.tums.ac.ir published online: may 27, 2017 both of them, could make them ideal ecofriend candidates in the biocontrol measures against mosquito vector in saudi arabia, as well as in other parts of the world where these mosquito vectors are prevalent. we believe that these products may also help in overcoming or suppressing the emergence of mosquito resistance to insecticides worldwide. finally, purification of both local b. thuringiensis δ-endotoxins and nematode bacterial tcs, their molecular characterization and their possible synergistic activity against mosquito larvae are currently being investigated in our lab. we therefor believe that this may lead to the identification of mosquito larvicidal b. thuringiensis-tcs mixture product that could contribute to the battle against mosquito vectors. acknowledgements this project was funded by the national plan for science, technology and innovation 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(who/cds/whopes/gcdpp/2005. 13). available at: http://www.who.int/ whopes/gcdpp/publications/en/index1. html. who (2010) world malaria report. geneva, switzerland. woodring jl, kaya hk (1988) steinernematid and heterorhabditid nematodes: a handbook of biology and techniques. fayetteville, ark.: arkansas agricultural experiment station, arkansas state library. xavier r, reena cm, sreeramanan s (2007) enviromental distribution and diversity of insecticidal proteins of bacillus thuringiensis berliner. malays j microbiol. 3: 1–6. zhu h, grewal ps, reding me (2011) development of a dessicated cadaver delivery system to apply entomopathogenic nematodes for control of soil pests. appl eng agric. 27: 317–324. 1dr parvizi rtl 89 10 1_3_ iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 1 original article molecular typing and phylogenetic analysis of some species belonging to phlebotomus (larroussius) and phlebotomus (adlerius) subgenera (diptera: psychodidae) from two locations in iran *p parvizi 1, sr naddaf 2, e alaeenovin 1 1molecular systematics laboratory, pasteur institute of iran, tehran, iran 2department of parasitology, pasteur institute of iran, tehran, iran (received 5 may 2010; accepted 5 dec 2010) abstract background: haematophagous females of some phlebotomine sandflies are the only natural vectors of leishmania species, the causative agents of leishmaniasis in many parts of the tropics and subtropics, including iran. we report the presence of phlebotomus (larroussius) major and phlebotomus (adlerius) halepensis in tonekabon (mazanderan province) and phlebotomus (larroussius) tobbi in pakdasht (tehran province). it is the first report of these species, known as potential vectors of zoonotic visceral leishmaniasis in iran, are identified in these areas. methods: in 2006-2007 individual wild-caught sandflies were characterized by both morphological features and sequence analysis of their mitochondrial genes (cytochrome b). the analyses were based on a fragment of 494 bp at the 3´ end of the cyt b gene (cyt b 3´ fragment) and a fragment of 382 bp cb3 at the 5´ end of the cyt b gene (cyt b 5´ fragment). we also analysed the cyt b long fragment, which is located on the last 717 bp of the cyt b gene, followed by 20 bp of intergenic spacer and the transfer rna ser(tcn) gene. results: twenty-seven p. halepensis and four p. major from dohezar, tonekabon, mazanderan province and 8 p. tobbi from packdasht, tehran province were identified by morphological and molecular characters. cyt b 5´ and cyt b 3´ fragment sequences were obtained from 15 and 9 flies, respectively. cyt b long fragment sequences were obtained from 8 out of 27 p. halepensis. conclusion: parsimony analyses (using heuristic searches) of the dna sequences of cyt b always showed monophyletic clades of subgenera and each species did form a monophyletic group. keywords: mitochondrial cytochrome b, phlebotomus (larroussius) major, phlebotomus (larroussius) tobbi, phlebotomus (adlerius) halepensis, iran introduction visceral leishmaniasis is a deadly disease caused by parasitic protozoa belonging to genus leishmania, transmitted to humans through the biting of infected female sandflies. three species of leishmania including l. donovani and l. infantum from the old world and l. chagasi from the new world are known to give rise to the visceral form of leishmaniasis. the disease is endemic in iran and the etiological egent is known to be l. infantum, which mainly affects children, with majority of cases from primary foci in northwestern and southern of the country (mohebali et al. 2005). sandflies of the subgenera larroussius and adlerius belonging to genus phlebotomus are known as primary vectors of zoonotic visceral leishmaniasis (zvl) in iran (nadim et al. 1978, 1992, parvizi et al. 2008) *corresponding author: dr parviz parvizi, email: parp@pasteur.ac.ir iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 2 and also p. (paraphlebotomus) alexandri recently reported as the vectors zvl in iran (azizi et al. 2006). females of the subgenus adlerius and some females of subgenus larroussius cannot be differentiated based on morphological features. recently, the females of the two subgenera were identified using sequence analysis of cytochrome b gene (cyt b) (killick-kendrick 1990, 1999, esseghir et al. 1997, 2000, parvizi and amirkhani 2008). the objective of this report was to identify and type molecularly some species belonging to p. larroussius and p. adlerius subgenera from two locations in iran. this paper reports the presence of sandflies of subgenera adlerius and larroussius in caspian sea littoral and pakdasht area. we could differentiate the female specimens of subgenera adlerius and larroussius spiecies using analysis of cyt b gene. it is noteworthy that up to present no record of any sandfly species from caspian sea littoral was avilable materials and methods the study area included two villages of meyan kooh and imamzadeh ghasem in dohezar area, (about 35 km west of tonekaboon), mazanderan province (caspian littoral) and two villages of geshlagh and mamazand in pakdasht area (about 30 km south of tehran city, tehran province. sandflies were collected by aspirators and sticky papers (a4 papers soaked in castor oil) from inside and outside of animal shelters, and miniature cdc light traps (sudia and chamberland 1962), placed overnight in animal shelters. all collected sandflies first were processed to remove oil then stored at -20º c until used. the sandflies were identified based on morphological features of heads and last abdominal segments to the extent possible according to the keys described by nadim and javadian (1976) and lewis (1982) and then thorax and abdomen of sandfly were individually subjected to dna extraction as described by parvizi et al. (2003). three pairs of primers designed by parvizi and ready (2006) were used to amplify the cyt b gene. cb1-se (forward) and cb3r3a (reverse) were used to amplify a more 5' fragment of 439 bp (cb1 fragment), cb3 fc (forward) and n1n-fa (reverse) amplified an overlapping 3' fragment of 499 bp (cb3 fragment) and cb1-se (forward) and cbr06 (reverse) amplified the cyt b long fragment as one piece of 717 bp length. the pcr condition and reagents for all amplifications were according to parvizi and ready (2006) except for the cyt b long fragment in which the annealing was performed in one stage at 48 ºc. pcr products were directly sequenced in both directions to identify sandflies haplotypes associated with individual female and male sandflies. all haplotypes were identified to species by phylogenetic analysis. dna sequences were edited and aligned using sequenchertm 3.1.1 software (gene codes corporation). multiple alignments of new dna sequences and genbank sequences were made using paup* software (swofford 2002) for phylogenetic analysis. results a total of 43 female and male sand flies from 4 different collection areas were studied, from which three phlebotomine species were morphologically identified. tonekabon phlebotomus major (4) and p. halepensis (28) were the only prevalent species in tonekabon. the identity of 27 sand flies, all from dohezar area, including 21 males and 6 females were determined as phlebotomus (adlerius) halepensis sandflies) based on both morphological features and sequencing data of cyt b gene. sequence analysis of cyt b 5´ fragment obtained from 15 sandflies showed that 8 iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 3 (53.3%) were haplotype irn279, 2 (13.3%) haplotype irn277, 2 (13.3%) haplotype irn 282, 2 (13.3%) haplotype irn285 and 2 the unique haplotypes irn293 and irn294 (table 1). genetic distances between haplotypes were as low as 0.00262 – 0.00581. all the 7 (there is only 4) cyt b 3´ sequences fell in the same haplotypes, named as irn277 (table 1). cyt b long fragment sequences were obtained from 8 out of 27 p. halepensis with the five (62.5%) revealing haplotype irn279, 2(25%) haplotype irn381 and 1 (12.5%) the unique haplotype irn279 (table 1). genetic distances between haplotypes were as low, 0.00139 – 0.00278. four phlebotomus (larroussius) major (2 male, 2 female) were identified by morphological and molecular characters from dohezar area of tonekabon in mazanderan province. cyt b 5´ sequences were obtained from 3 out of 4 flies. two (66.7%) were haplotype irn385, and one was a unique haplotype (irn 287) (table1). the genetic distance between haplotypes was as low as 0.01597. cyt b 3´ sequences were obtained from 3 out of 4 flies, 2 were haplotype irn385, and 1 was haplotype (irn287) (table 1). the genetic distance between haplotypes was as low, as 0.01597. cyt b long fragment sequences were obtained from all four p. major. two (50%) were haplotype irn385, and two (50%) were haplotype (irn287) (table 1). the genetic distance between haplotypes as low as 0.02234. pakdasht the majority of sandflies of pakdasht were p. papatasi and some species belong to paraphlebotomus and sergentomyia. sequencing data obtained from cytochrome b gene offemale specimen revealed that the only prevalent species of subgenera larroussius was p. tobbi. based on both morphological and molecular features all the 8 male sandflies collected from pakdasht (5 from mamazand region using sticky papers and 3 from gheshlagh using cdc light traps) were identified as phlebotomus (larroussius) tobbi. no cyt b 5´ fragment sequences was obtained from p. tobbi specimens as the products of pcr amplification were too weak to be sequenced. cyt b 3´ fragment sequences were obtained from 7 out of 8 p. tobbi (table 1). two (28.6%) were haplotype irn 334, 2 (28.6%) haplotype irn335, 2(28.6%) haplotype irn 338 and 1 was the unique haplotype irn336. genetic distances between haplotypes were as low as 0.00319-0.01597. cyt b long fragment sequence was obtained only from one p. tobbi specimen (irn413). however, the sequence was too short to be analysed (table 1). nucleotide sequence data reported in this paper are available in genbank, embl and ddbj databases under accession numbers from hq391905 to hq391913. table 1. all dna haplotypes of cyt b of subgenera larroussius /adlerius species identified in two locations in iran (i.h = inside house, ash= animal shelter, s.p= sticky paper, cdc= cdc miniature light traps) cyt b haplotype provinces location habit at trap type cb1-se cb3r3a cb3fc ninfa cb1se cb-r06 speci men n. sex p. tobbi ash s.p not done irn334 not done irn334 m ash s.p not done irn335 not done irn335 m tehran pakdasht-gheshlagh ash s.p not done irn336 not done irn336 m iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 4 ash s.p not done irn335 not done irn337 m ash s.p not done irn338 not done irn338 m ash cdc not done irn338 not done irn339 m ash cdc not done irn334 not done irn340 m pakdasht-mamazand ash cdc not done weak band irn413 irn413 m p. major tonekabonemeian kooh i.h asp irn287 irn287 not done irn287 m tonekaboneimamzade ghasem i.h asp irn291 irn287 not done irn291 m tonekabonemeian kooh i.h asp not done not done irn385 irn385 f mazanderan tonekaboneimamzade ghasem i.h asp not done not done irn385 irn390 f p. halepensis i.h asp irn277 irn277 not done irn277 m tonekaboneimamzade ghasem i.h asp irn277 irn277 not done irn278 m i.h asp irn279 irn277 not done irn279 m i.h asp irn279 not done not done irn280 m i.h asp irn282 not done not done irn281 m i.h asp irn282 not done not done irn282 m i.h asp weak band irn277 not done irn283 m i.h asp irn279 not done not done irn284 m i.h asp irn285 not done not done irn285 m i.h asp irn279 not done not done irn286 m tonekabonemeian kooh i.h asp irn279 not done not done irn288 m i.h asp irn279 not done not done irn289 m i.h asp irn285 not done not done irn290 m i.h asp irn279 not done not done irn292 m i.h asp irn293 not done not done irn293 m i.h asp irn294 not done not done irn294 m tonekaboneimamzade ghasem i.h asp bad sequence irn277 not done irn295 m i.h asp irn279 not done not done irn296 m i.h asp weak band not done irn297 irn297 m i.h asp not done not done irn380 irn380 f i.h asp not done not done irn381 irn381 f tonekabonemeian kooh i.h asp not done not done irn381 irn384 f i.h asp not done not done irn381 irn386 f tonekaboneimamzade ghasem i.h asp not done not done irn381 irn388 f i.h asp not done irn277 weak band irn389 f i.h asp not done not done irn381 irn392 f i.h asp not done irn277 weak band irn393 f mazanderan tonekabonemeian kooh i.h asp not done not done irn380 irn394 m i.h asp not done bad sequence irn383 irn383 f i.h asp not done bad sequence weak band irn387 f mazanderan tonekabonemeian kooh i.h asp not done not done not done irn391 f table 1. countinued… iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 5 irn383 r06 irn282 irn277 irn278 irn279 irn285 irn293 irn294 irn295 irn 380 irn381 irn336 irn334 irn335 irn338 irn287 irn291 irn385 p. major p. halep ensis p. unkno wn p. tobbi 1 nucleotide change fig. 1. unrooted consensus phylogenetic tree for dna sequences of cyt b long (718 nucleotides) of phlebotomus (adlerius) / phlebotomus (larroussius), produced by branch and bound parsimony search using paup* iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 6 irn336 cb3 irn334 cb3 irn335 cb3 irn338 cb3 irn385 r06 irn287 cb3 irn383 r06 irn277 cb3 10 nucleotide changes p. tobbi p. major p. halepensis p. unknown 10 nucleotide changes mazanderan tehran fig. 2. unrooted phylogenetic tree for dna sequences of cyt b 3' end (last 316 nucleotides) of phlebotomus (adlerius)/phlebotomus (larroussius), produced by branch and bound parsimony search using paup* iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 7 single or nucleotide position composite dna sequence from each specimen 1111111111111111111111111111111111111222222 11222233344445666677788999990000000111222334444455666666777777889000001 1539235814703695125737909124780134789378567690234514124567012459129012581 irn287haplo cb3 irn385haplo r06 irn334haplo cb3 irn335haplo cb3 irn338haplo cb3 irn336haplo cb3 irn277haplo cb3 irn279haplo cb3 irn278haplo cb3 irn295haplo cb3 irn380haplo r06 irn381haplo r06 irn383haplo r06 irn287haplo cb3 irn385haplo r06 irn334haplo cb3 irn335haplo cb3 irn338haplo cb3 irn336haplo cb3 irn277haplo cb3 irn279haplo cb3 irn278haplo cb3 irn295haplo cb3 irn380haplo r06 irn381haplo r06 irn383haplo r06 nnnatctaacttaaaattgaagaaacctttatacatacccttattataattataatagattttatcagcatat tctacctaacctaaaattgaagaaacctttatacatacctttattgtaattataatagattttatcagcatat ttcttcctttccaaaaccgtagtaattaaattacactacattattacaattataatagattttgttagccctt ttcttcctttccaaaaccgtagtaattaaattacactacattattacaattataatagattttgttagccctt ttcttcctttccaaaaccgtagtaattaaattacactacattatcacaattataatagattttgttagccctt ttcttcctttctgagaccgtagtaattaaattacactacattattacaattataatagatcttgttagccctt tttattaattttaaatttgttgtaattatatcacataattctcctctaattataatagattttgatagctttt tttattaattttaaatttgttgtaattatatcacataattctcctctaattataatagattttgatagctttt tttattaattttaaatttgttgtaattatatcacataattctcctctaattataatagattttgatagctttt nnnattaattttaaatttgttgtaattatatcacataattctcctctaattataatagattttgatagctttt tttattaattttaaatttgttgtaattatatcacataattctcctctaattataatagattttgatagctttt tttattaattttaaatttgttgtaattatatcacataattctcctctaattataatagattttgatagctttt cttattaactatagatttatcaagttaattaatatttattcacctaatcaacattatatcaccttctatataa gctttaacaaagtctttcctattaaaaatctcaaatttctcaa gctttaacagagtctttcctattaaaaatctcaaatttctcaa gctttaacaatatcttccctatatcaatattcaaatttttcaa gctttaacaatgtcttccctatatcaatattcaaatttttcaa gctttaacaatgtcttccctatatcaatattcaaatttttcaa gctttaacaatgtcttccctatatcaatattcaaatttttcaa acacttaccacgtctcttctacataaatattctaatcatatac acacttaccacgtctcttctacataaatattctaatcatatac acacttaccacgtctcttctacataaatattctaatcatatac acacttaccacgtctcttctacataaatattctaatcatatac acacttaccacgtctcttctacataaatattctaatcatatac acacttaccacgtctcttctacataaatattctaatcatatac aaaacgttaattatcctctataatatttttaacttaattaatt fig. 3. input data matrix of variant nucleotides for paup* analysis of phlebotomus (adlerius) / phlebotomus (larroussius) species: 316 base pairs of cyt b 3´ single or nucleotide position composite dna sequence from each specimen 1111111111111111111111111111111111111111222222 11122223334455556666688890000001122222333333444555555566677788999000111 134925891273657890124725940136792512356034679289123467801923524369589145 irn294haplo cb1 irn291haplo cb1 irn277haplo cb1 irn282haplo cb1 irn279haplo cb1 irn287haplo cb1 irn285haplo cb1 irn380haplo r06 irn383haplo r06 irn381haplo r06 irn385haplo r06 irn293haplo cb1 irn294haplo cb1 irn291haplo cb1 irn277haplo cb1 irn282haplo cb1 irn279haplo cb1 irn287haplo cb1 irn285haplo cb1 irn380haplo r06 irn383haplo r06 irn381haplo r06 irn385haplo r06 irn293haplo cb1 aaagtaccccacagctatatataagatgtttaaatacttcttatcctatgcacaaaattcaccatacctaat taattatcattctgatatataaaggttgtcctcacactttttattataagctctaaacttatattatccgtt ttaattctatattaatataaaagagatgtatagctacttttctttttatgcacaaaatccttaaatcacaat ttaattctatattaatataaaagagatgtataactacttttctttttatgcacaaaatccttaaatcacaat ttaattctatattaatataaaagagatgtatagctacttttctttttatgcacaaaatccttaaatcacaat taatcatcattctaatatacaaaagttgtcctcacactttttattatgagctctaaacttatattatccatc ttaattctatattaatataaaagagatgtatagctactttcctttttatgcacaaaatccttaaatcacaat ttaattctatattaatataaaagagatgtatagctacttttctttttatgcacaaaatccttaaatcacaat actattttatattgtagatattgattccatccaatggctttatctaaatattgtctttttataaattttatc ttaattctatattaatataaaagagatgtatagctactttcctttttatgcacaaaatccttaaatcacaat taattatcattctgatatataaaggttgtcctcacactttttattacgagctctaaactcatattatccgtc ???????????ttaatataaaagagatgtatagctactgttctttttatgcacaaaatccttaaatcacaat ataattcagatatctctactcataaatacacaagataacaaaagcattttagatttccgttcttcc ttcactcagacaccgcagtgtgtaaatagtcttcttatctatagcgcatctgactcttatctcccc acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctactttcc acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctacttccc acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctacttccc ttyactcagacaccacagtgtgtaaatagtcttcttatctatagcacatctgactcttctcttccc acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctacttccc acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctacttccc attctaaaatacttattattaaattttttac?tttataattatctattataaacatctatctttat acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctacttccc ttcactcagacaccgcagtgtgtaaatagtcttcttatctatagcgcatctgactcttatctcccc acaactctgacttttttgtttatgaaaatatgttttatctaaagctcatcaggtattctacttccc fig. 4. input data matrix of variant nucleotides for paup analysis of phlebotomus (adlerius)/phlebotomus (larroussius) species: 382 base pairs of cyt b 5´ iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 8 discussion using branch and bound parsimony searches with equal character weighting, paup* analysis for dna sequences of cyt b 3´ fragment (last 316 nucleotides) of 8 haplotypes of phlebotomus (adlerius)/phlebotomus (larroussius) produced one parsimonious tree rooted using default outgroup (tree length= 145; 69 characters were parsimonyuninformative; number of parsimony-informative characters= 47) (fig. 1, 2). the haplotypes of each species formed monophyletic clades, but it was not possible to compare regions because each species was collected in unique locations. using branch and bound parsimony searches with equal character weighting, paup* analysis for dna sequences of cyt b 5´ fragment (383 nucleotides) of 10 haplotypes of phlebotomus (adlerius)/phlebotomus (larroussius) produced one most parsimonious tree (tree length= 188; 60 characters were parsimony-uninformative; number of parsimony-informative characters= 78) (fig. 3, 4). p. major and p. halepensis were monophyletic, with two unknown species located between them. it was not possible to compare regions because each species was collected in unique locations using branch and bound parsimony searches with equal character weighting, paup* analysis for dna sequences of cyt b long fragment (last 718 nucleotides) of 18 haplotypes of phlebotomus (adlerius)/phlebotomus (larroussius) produced 1 most parsimonious tree rooted using default outgroup (tree length= 343; 116 characters were parsimony-uninformative; number of parsimonyinformative characters= 144). phlebotomus major and p. tobbi were monophyletic, and both unknown species (i.e. not identifed by morphology) were monophyletic with p. halepensis, not with a species of larroussius. the longer sequence gave more phylogenetic information. nucleotide haplotypes within adlerius/ larroussius species differed pairwise by < 0.1%, but absolute genetic distances were greater between some species, e.g. 0.134130.14093 between p. tobbi and p. halepensis, and 0.12141-0.12828 between p. halepensis and p. major. for subgenus adlerius (single species, p. halepensis), fixed diagnostic polymorphisms occurred at amino acid positions 97, 101, 173 and 239 of the cyt b long fragment and at amino acid positions 39, 72 and 105 of the cyt b 3´ fragment. for subgenus larroussius, fixed diagnostic polymorphisms occurred at amino acid positions 99, 106 and 238 of the cyt b long fragment and at amino acid positions 74 and 104 of the cyt b 3´ fragment. aransay et al. (1999) showed that the 18s rrna gene was a useful marker for inferring phylogenetic relationships within the subfamily phlebotominae, finding a clade containing the subgenera euphlebotomus, adlerius and larrroussius, a second clade with paraphlebotomus and phlebotomus, and a third clade with sergentomyia and american lutzomyia species. depaquit et al. (2000) used its2 rdna gene sequences and found a clade with paraphlebotomus and phlebotomus. its2 rdna sequences were also monophyletic for subgenus larroussius (muccio et al. 2000). in our study, the dna sequences of cyt b were shown to be good markers for finding clades of genera and subgenera. esseghir et al. (2000) characterized cyt b for species of the same subgenera as we studied, but they had to give different weights to the nucleotides in 1st, 2nd and 3rd base positions of codons to get phylogenetic results. this was not the aim of our work, which was finding diagnostic markers for the species. the important phylogenetic result for us was to show that these species were monophyletic. iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 9 acknowledgements we thank dr. p.d. ready, the leader of sandflies and leishmaniasis programme at the natural history museum (nhm), london, for his help, and supervising in his laboratory, in the nhm. this work was fund by the pasteur institute of iran grant 298 awarded to dr. parviz parvizi. the authors declare that ther is no conflict of interests. references aransay am, scoulica e, chaniotis b, tselentis y (1999) typing of sandflies from greece and cyprus by dna polymorphism of 18s rrna gene. insect mol biol. 8: 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present status of kala-azar in iran. am j trop med hyg. 27: 25–28. nadim a, javidian e, tahvildar-bidrun ig, mottaghi m, abai mr (1992) epidemiological aspects of kala-azar in meshkin-shahr, iran: investigation on vectors. iran. parvizi p, mazloumi gavgani as, davies cr, courtenay o, ready pd (2008) two leishmania species circulating in the kaleybar focus of ‘infantile visceral leishmaniasis’, northwest iran: implications for deltamethrin dog collar intervention. trans r soc trop med hyg. 102: 891–897. parvizi p, amirkhani a (2008) mitochondrial dna characterization of populations of sergentomyia sintoni and finding mammalian leishmania infections in this sandfly by using its-rdna gene. iran j vet res. 9: 9–18. iran j arthropod-borne dis, 2010, 4(2): 1–10 p parvizi et al.: molecular typing and …. 10 parvizi p, ready pd (2006) molecular investigation of the population differentiation of phlebotomus papatasi, important vector of leishmania major in different habitats and regions of iran. iran biomed j. 10: 69–77. parvizi p, benlarbi m, ready pd (2003) mitochondrial and wolbachia markers for the sandfly phlebotomus papatasi: little population differentiation between peridomestic sites and gerbil burrows in isfahan province, iran. med vet entomol. 17: 351–362. sudia wd, chamberland rw (1962) battery operated light trap, an improved model. mosquito news. 22: 126–129. swofford dl (2002) paup: phylogenetic analysis using parsimony (and other methods) version 4.0. sinauer associates, sunderland, massachusetts. 8.dr sedaghat rtl iranian j arthropod-borne dis, (2009), 3(1): 53-59 j rafinejad et al: faunestic study of … 53 original article faunestic study of ants with emphasis on the health risk of stinging ants in qeshm island, iran j rafinejad1, a zareii2, k akbarzadeh1, m azad2, f biglaryan3, s doosti1, *mm sedaghat1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2islamic azad university, qeshm branch, qeshm, iran 3department of education, district 2, tehran, iran (received 9 aug 2009; accepted 5 sep 2009) abstract background: qeshm (26.75n, 55.82e), iran, is 1500 km² island in the strait of hormuz. qeshm is a free trade zone, acting as an important channel for international commerce, and has been the site of much recent development. there is potential risk of stinging ant attacks for residents and visitors that may occur in the island. the aims of this study were to find out the fauna, dispersion, and some of the biological features of ant species with special attention to those, which can play role on the public health of the island. methods: in this cross-sectional study, we surveyed ants around the island using non-attractive pitfall traps and active collection to evaluate potential threats to humans and other species during 2006–2007. all collected specimens were identified using the morphological ant keys. results: only six ant species were found: pachycondyla sennaarensis (41%), polyrhachis lacteipennis (23%), camponotus fellah (16%), cataglyphis niger (9%), tapinoma simrothi (7%), and messor galla (4%). conclusion: we were surprised not to find any cosmopolitan tramp ants so often associated with commerce and development. instead, all six species may be native to the middle eastern region. the most common species, p. sennaarensis, has a powerful sting and appears to do well around human habitations. this species may prove to be a serious pest on the island. keywords: pachycondyla sennaarensis, public health, pests, african needle ant, iran introduction ants (superfamily formicoidea) have a worldwide distribution, some certain genera and species present in almost all countries and in all places. they are among the most successful insects which occurring everywhere in terrestrial habitats and outnumbering most of other terrestrial animals in individuals (borrer et al. 1989, taylor 2007). some ant species due to some certain characters such as their social organization are considered as the most successful invaders (moller 1996, williamson and fitter 1996). those ants that have principally spread throughout the world human trade are considered as tramp species which living in close association with man. they have a wide distribution in the world and can find them in many areas even out of their original ranges. they tend to have widespread geographical distributions, and share life history characteristics including queen number, nest structure, and foraging behaviour (mcglynn 1999). they share several characteristics as unicoloniality resulting in an absence of intraspecific aggression, polygyny (multiple queens nests), high interspecific aggression and the small size of workers (passera 1994). all ant species are grouped into a single family, the formicidae, which includes 10-20 *corresponding author: dr mohammad mehdi sedaghat, email: sedaghmm@tums.ac.ir iranian j arthropod-borne dis, (2009), 3(1): 53-59 j rafinejad et al: faunestic study of … 54 subfamilies (astruc et al. 2004). subfamily ponerinae is one of the most known ones and comprises ten genera. the genus pachycondyla [smith (1858)], includes a large group of ants with about 200 described species, worldwide distribution, and mostly known from tropics and sub-tropics regions (bolton 1995). a few species have an obvious and functional sting, whereas other ants bite with their maxillae with no sting. the members of genus pachycondyla have sting which use for their predatory activities. the majority species of the genus pachycondyla are scavengers or predators of arthropods, the later subdue their pray with venom (wild 2002, orivel and dejean 2001). some ant species capable of inciting hypersensitivity reactions include p. sennaarensis (steen et al. 2005). there has also reported anaphylactic shock in humans following the stings form p. sennaarensis in the united arab emirates (dib et al. 1995). this taxon is one of the eight genera of ants that have been associated with sting allergy worldwide. pachycondyla sennaarensis has been incriminated as an intermediate host for the poultry cestode, raillietina tetragona in sudan (mohammed et al. 1988). to date, occurrence of p. sennaarensis has been confirmed from southern parts of iran (tirgari et al. 2004, tirgari and paknia 2004, tirgari and paknia 2005, akbarzadeh et al. 2006 a, b, paknia 2006). however, there is little information on species composition, distribution, ecology, biology, behaviour and public health threat of stinging ants in iran. there are many public health concerns in qeshm due to recent development of the island and increasing the population. besides, there are many attractions, which make the island one of the most popular tourists’ destinations in the region. there is potential risk of stinging ant attacks for residents and visitors that may occur indoors or outdoors. this study was conducted to identify ant species, dispersion, and some of their biological and morphological features to throw light on status of p. sennaarensis as a public health pest in the island. it tends to provide preliminary information on ants in qeshm for further prevention and control programs. materials and methods study site qeshm is situated at the entrance of the persian gulf in the strait of hormoz, in the 55 to 57 degrees longitude and 25 to 27 degrees latitude. it is 1500 km² in area with 136 km as length and 11 km as average width. the island is 30 km long at its maximum width. the highest point of qeshm, on a salt field to its west, is 397 meters from sea level with the average height of 10 meters. the climate of qeshm is warm and humid summers with scattered winds. the winters, are generally mild and spring like. the annual median of the daily average temperature is 27 degrees centigrade and the annual average of the maximum daily temperature is 32 degrees centigrade. the island's average humidity is 74, with a maximum rainfall of 456 mm, and a minimum of 41 mm, annually. there are 200 hectares of mangrove sea forest and also tropical plants and trees with palm groves in qeshm. because of the tropical climate of the island, around 200 species of birds migrate or reside in natural hara forest habitats. among the wild life of qeshm, eagle, fox and sea turtles are recognized in the island. qeshm is one of the most important and largest persian gulf islands that recently, due to the new government policies, converted to one of the free commercial-industrial and touristy area. this island accommodates thousands iranian and foreign tourists every year (fig. 1). sampling methods the present study was conducted in qeshm island, hormozgan province, 20062007. the island divided into 20 regions and iranian j arthropod-borne dis, (2009), 3(1): 53-59 j rafinejad et al: faunestic study of … 55 samplings were performed from over 20 stations with the aim of determining the ant species in the island. each station was divided in 10 points of measure disposed in a regular grid in a 20 x 20 meters square. sampling was carried out directly by searching the ant nests. two different methods were used to collect the specimens of ants. ten nonattractive pitfall traps were laid for 7 d on each station. they were grouped by two and placed in five points with 50 cm from each other. each group was itself distant from the other two groups by 10 meters in a line. pitfall traps consisted of plastic jars of fiftyfive millimetre diameter and thirty-five millimetres deep, containers filled with 30 ml of ethanol at 35%. the second method was active collection. specimens from nests or surrounding areas were transferred to the containers filled with ethanol at 70% by paintbrush. visual observations were performed on different days at sample sites. all specimens preserved in dish and relative information was recorded. specimens were deposited in the medical arthropods museum, the school of public health, tehran university of medical sciences. the specimens were identified using the morphological keys of bolton (1994), collingwood and agosti (1996) and shattuck and barnett (2001). results the total number of samples identified was 1359. six species of ants were identified belonging to three subfamilies includes formicinae, dolichoderinae, myrmicinae. of these six species, the most common was p. sennaarensis subfamily ponerinea (41%), collected in pitfall traps and active collection on all twenty stations in urban and rural areas of the island (table 1). the other ants were identified as follow: p. lacteipennis smith (subfamily formicinae) 23%, camponotus fellah dall torre (subfamily formicinae) 16%, cataglyphis niger andre (subfamily formicinae) 9%, tapinoma simrothi krausse (subfamily dolichoderinae) 7% and messor galla mayr (subfamily myrmicinae) 4%. pachycondyla sennaarensis was the most common ant in various parts of the island. they are able to establish their colonies in every type of human environment present in qeshm. the species distributed throughout qeshmisland , which is the southern limit of its distribution in iran (table1). this taxon is not an aggressive species through its distribution in the island. it feeds mainly on food waste in urban area but it also feeds on dead insects and attracts to sugary substances. in addition, they are scavenger or predator in some parts of the island. their nests generally open on to the surface with circular apertures, each 3-5 mm in diameter in the yards, gardens, parks and roadside plantations partially exposed to the sun. however, they are able to build their nest inside the human premises. in one case, we found their nest in the third floor of a shopping centre. in other case, the nest was built in second floor of a house in qeshm. discussion by far the most common species we found on qeshm was p. sennaarensis mayr (1862) described pachycondyla sennaarensis from sennaar, sudan (13.55n, 33.60e). emery (1881) first reported p. sennaarensis on the arabian peninsula, from three sites in yemen. later reports have recorded p. sennaarensis from many parts of tropical africa e.g., angola, burkina faso, cameroon, congo, djibouti, ethiopia, the gambia, ghana, guinea, nigeria, senegal, sierra leone, and zaire; (taylor 2007) and the arabian penninsula e.g., kuwait, oman, qatar, saudi arabia, uae, and yemen (collingwood 1985, iranian j arthropod-borne dis, (2009), 3(1): 53-59 j rafinejad et al: faunestic study of … 56 collingwood and agosti, 1996, collingwood et al. 1997, collingwood and van harten 2001, 2005). recently, several authors have reported p. sennaarensis from south-eastern iran, adjacent to the arabian peninsula (tirgari et al. 2004, tirgari and paknia 2004, tirgari and paknia 2005, akbarzadeh et al. 2006 a, b, paknia 2006). paknia (2006) considered p. sennaarensis as an exotic species to iran, its near continuous range across africa and arabia. other studies indicated that the species tend to have widespread towards the north of the country (tirgari and paknia 2005). we were surprised that our surveys found none of the major pantropical tramp ant species, such as p. longicornis or tapinoma melanocephalum. this study did not even find the tramp monomorium destructor, a species that is often a pest in semi-arid areas. fig. 1. qeshm island in southern iran iranian j arthropod-borne dis, (2009), 3(1): 53-59 j rafinejad et al: faunestic study of … 57 table 1. locations where p. sannaarensis collected in qeshm island no. collection site no of observed colony location 1 qeshm 18 resident area, parks, open fields and wharf 2 dargahan 7 resident area , open fields and roadside plantations 3 pay posht 3 resident area 4 laft port 5 resident area 5 gorzine 2 resident area 6 tabl 6 resident area 7 doolab 5 resident area 8 band basaaid 3 resident area 9 doostkoh 5 resident area 10 namakdan 6 resident area 11 salkh 3 resident area 12 direstan 2 resident area 13 masn port 3 resident area 14 sosa port 4 resident area 15 rhamkan 7 resident area and roadside plantations 16 direstan and air port 7 resident area 17 toorian 8 resident area and roadside plantations 18 rham chah 12 resident area and roadside plantations 19 toola 2 resident area 20 tombak 4 resident area although akbarzadah et al. (2006 a, b) have recently used the "fire ant" for p. sennaarensis, it probably gives the false impression that this ant is related to genus solenopsis. it seems that the common name "samsun ant" is more appropriate for p. sennaarensis and many authors use this name for the species in the region (dibs et al. 1995, tirgari et al. 2004, tirgari and paknia 2004, tirgari and paknia 2005, al-shahwan et al. 2006, paknia 2006). since p. chinensis is now called the "asian needle ant", based on their similarity in behaviour and biology, it could be called p. sennaarensis "african needle ant". this study revealed an enormous amount of the samsun ant, p. sennaarensis in resident areas in qeshm. while this taxon described as an aggressive species in africa and some countries in the middle east (collingwood 1985, dib et al. 1995, taylor 2007), it is not an aggressive species throughout its distribution in the island. previous studies by tirgari et al. in 2004 and akbarzadeh et al. in 2006 have shown the threat of p. sennaarensis on human health in south and southeast corner of the country. comparison between morphological characters of the samsun ant from qeshm and sistan va baluchistan province showed that they are identical and belong to the same species. many ant species, particularly those of tropical and subtropical origins, are easily transported around the globe by human commerce (morrison et al. 2004). however, the island has been one of the most important islands of iran from ancient times and it could be postulated that it has been transported from other places by human commerce. although the behaviour, nesting and social biology of p. sennaarensis are diverse in different parts of the world, it seems the species shares the same characters in its distribution in southern parts of iran, which described by different authors (tirgari et al. 2004, tirgari and paknia 2004,tirgari and paknia 2005, akbarzadeh et al. 2006 a, b, paknia 2006). they live in colonies and make their nests in ground. the majority of them make their nest near buildings, gardens, parks and roadsides. iranian j arthropod-borne dis, (2009), 3(1): 53-59 j rafinejad et al: faunestic study of … 58 the food preference of p. sennaarensis is varied in different part of the world. while in africa, it is generally granivorous (dejean and lachaud 1994), in qeshm the species feeds on human food and waste mainly. in such situation, it can consider as a commensal species. it appears this habit dependent on environmental factors, fauna and flora of the region and more importantly the availability of food. however, the species generally can be described as an omnivorous species, which feed on every available food sources such as food waste, fruits, nectarines or homopteran honeydew, small arthropods and dead animals. in urban area of the island, they also prefer to feed on the human’s food. besides, as they live in colonies with a few dozen to a few thousand workers, it shows their potential as a real threat of public health to the residents and visitors. although their control is very difficult, knowledge of ant biology is essential for successful control programs. the strategic location of qeshm in the persian gulf and increase commercial movements in the recent years needs more attention for related organization to prevent entering pest ant species. it is important to support those researches aimed to identify species composition, the ecology and behaviour of native either exotic ant in the region based on environmental factors. besides, as there are no regional pest ant control programs in the persian gulf region, it is need to achieve, develop and improve safe strategies for the local and regional control of pest ant. acknowledgments the authors are grateful to tehran university of medical sciences and qeshm branch of islamic azad university for their support. the authors declare that they have no conflicts of interest. references akbarzadeh k, tirgari s, nateghpour m, 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medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2departement of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 19 dec 2009; accepted 7 apr 2010) abstract background: pediculus capitis (anoplura: pediculidae) or head louse is an obligate ectoparasite transmitted mainly through physical contact. this study was conducted to survey the prevalence of head lice infestation rate and some risk factors in primary school pupils, in khajeh city east azerbaijan province, iran methods: we selected 20 primary schools of khajeh city during 2008 and 2009. totally 500 pupils including 200 boys and 300 girls from all grade 1-5 were selected by multistage, systematic random sampling in rural areas of khajeh city and were examined for lice. in addition, a standard questionnaire recorded information about demographic features of each pupil. results were analyzed by spss software. results: the total prevalence of head lice infestation in this study was 4.8%. and the prevalence rate was significantly higher in girls (6.66%) than in boys (2%). epidemiological factors such as: sex, school grade, family size, parent's education, type of house, hair washing (per week), number of using comb per day, were evaluated and results showed significant difference in head lice infestation and sex, school grade ,family size ,father education ,and type of house (p< 0.05). conclusion: pediculosis is a public health problem in many parts of the world, and due to the higher prevalence of pediculosis in crowded families, family by lower levels of father's education and socioeconomic status in our study and rural area, it is necessary to give health education for families to prevent of pediculosis in this area. keywords: head louse, pediculus capitis, epidemiology, iran introduction pediculus capitis (de geer, 1778) (anoplura: pediculidae) or head louse is an obligate ectoparasite transmitted mainly through physical contact (linardi et al. 1988). because of head louse feeding of blood it leads to anemia and in the scratch sites can lead to secondary infection (slonka et al. 1976). head louse is one of the health problems in many parts of the world (ewasechko 1981, kwaku-kpikpi 1982). head louse prevalence in school-age children is more common for example, 27% of urban primary schools in iranshahr area (southeast of iran) were found infested (alempour salemi et al. 2003). hodjati et al studied the head lice infestation in school children of tabriz city and the infestation rate was 3.64% (hodjati et al. 2008). the prevalence of head lice was 1% in fars (davarpanah et al. 2009), and 1.3% in bahar (moradi et al. 2009). some factors are effective for the head lice prevalence, that related to the host such *corresponding author: mr azim paksa, email: a.paksa@ yahoo.com iranian j arthropod-borne dis, 2010, 4(1): 42–46 m shayeghi et al.: epidemiology of head lice … 43 as: sex, age group, race, type of hair and in recently years resistance to insecticides have contributed to the increase of head lice prevalence (nazari et al. 2006). this study was conducted to survey the prevalence of head lice infestation rate and some risk factors in primary school pupils, in khajeh city, east azerbaijan province, iran. materials and methods this primary school-based, cross-sectional study was conducted at 20 primary schools of khajeh city (haris township, east azerbaijan province, iran) during 2008 and 2009. totally 500 pupils including 200 boys and 300 girls from all grade 1-5 were selected by multistage, systematic random sampling in rural areas of the khajeh city then were examined for the presence of one of life stages of lice, including eggs and nymph or adult. a team including health workers of study area, school health nurses and a medical entomologist, skilled in the detection of head lice examined the pupils’ hair and scalps for lice. screening was carried by visual inspection of the head and scalp under the light of a reading lamp about 3-5 min. pupils suspected of having lice were subjected to comb with a fine-toothed comb for about 7 minutes over a white paper of 60×75 cm size. the removed lice were observed, collected by sellotape. pupils, whose hair had at least one of the developing stages of parasite including only nits located ¼ inch from the scalp were considered positive (alempour salemi et al. 2003). also we used a standard questionnaire to record information about sex, school grade, family size, parent's education, type of house (muddy, woody, brick built), hair washing (per week), availability to safe water and number of lice recovered. the chi-square test (spss software, version 11.5) was used to compare categorical variables. for all statistical analyses, a significance level of p< 0.05 was adopted. results the total prevalence of head lice infestation was 4.8%. the difference between lice prevalence was significant regarding the gender, which was higher in girls (6.66%) than in boys (2%) (p< 0.05) (table 1). table2 shows the prevalence of head lice infestation in primary school pupils, which was stratified by social factors. the prevalence of head lice infestation by frequency of hair washing was 8.66%, and 3.7%, for once in two week, once a week respectively and twice or more a week had not any lice. the difference between twice or more of hair washing in a week and infestation rate was highly significant (p< 0.05) in comparison to once in two week and once in a week. all of the head lice infestations were in family, which had more than 3 members. the prevalence of head lice infestation was significantly according to school grade (p< 0.05). the most frequent group was first grade (13.54%) (table 2). the prevalence of head lice in relation to type of house was statistically significant (p< 0.005) and in muddy house was 10.1 %, woody house 5.73 and the pupils who lived in brick built homes had not any infestation (table 2). the results of this survey showed that infestation rate in the pupils who did not use of comb were 6.81%, once use in day 5.94%, and twice use were 2.85%. there was no statistical difference found between the number use of comp and infestation rate. the prevalence of head lice infestation by parents' literacy is presented in table 3. this results show that there was a negative correlation between father’s education and infestation rate (p< 0.05). there was not any significant difference between mother's education and infestation. iranian j arthropod-borne dis, 2010, 4(1): 42–46 m shayeghi et al.: epidemiology of head lice … 44 table 1. prevalence of the head lice infestation in primary school pupils by sex in khajeh, iran sex no. of examination no. of infestations prevalence (%) male 200 4 2 female 300 20 6.66 total 500 24 4.8 table 2. prevalence of the head lice infestation in primary school pupils by social factors in khajeh, iran characteristics no. of examination no. of infestations prevalence (%) frequency of hair washing once two week 150 13 8.66 once a week 297 11 3.7 twice or more a week 36 0 0 family size ≤3 20 0 0 >3 480 24 5 type of house muddy 99 10 10.1 woody 244 14 5.73 brick built 157 0 0 school grade i 96 13 13.54 ii 113 5 4.42 iii 105 3 2.85 iv 87 2 2.29 v 99 1 1.01 number use of comb in day not use 88 6 6.81 once a day 202 12 5.94 twice a day 210 6 2.85 table 3. prevalence of the head lice infestation in primary school pupils according to parents' literacy in khajeh, iran characteristics no. of examination no. of infestations prevalence (%) father's education uneducated 65 8 12.3 primary 313 14 4.47 guidance school 115 2 1.73 high school and upper 7 0 0 mother's education uneducated 364 22 6.04 primary 104 2 1.92 guidance school 31 0 0 high school and upper 1 0 0 iranian j arthropod-borne dis, 2010, 4(1): 42–46 m shayeghi et al.: epidemiology of head lice … 45 discussion the total prevalence of head lice infestation among primary school pupils was 4.8%. the prevalence rate from different parts of iran, mostly in primary school pupils reported lower 1% in fars (davarpanah et al. 2009),1.3% in hamedan (moradi et al. 2009), 3.64% in tabriz (hodjati et al. 2008), 3.8% in kerman (kamiabi et al. 2005), 27% in iranshahr (alempour salemi et al. 2003) and 28.5% in ardabil (edalatkhah et al. 2005). also infestation rate among school pupils in some parts of the world was 33% in australia (speare et al. 1991), 35% in brazil (borges et al. 2002), 48.7% in france (courtaiade et al. 1993) and 49.7% in ghana (kwaku 1982). almost in all this studies in iran and other parts of the world, the prevalence of head lice infestation in female pupils was more than the prevalence of the infestation in male pupils. difference in behavior patterns between boys and girls might have affected transmission rates and susceptibility to head lice infestation (moradi et al. 2009). we found that p. capitis was more prevalent in crowded families, muddy house, family by lower levels of father's education and socioeconomic status, little frequency of hair washing in a week which are all associated with pediculosis (akisu et al. 2003, balcioglu et al. 2007) are more frequent in rural regions. because this study was conducted in rural parts of khajeh city, these results were expected. we also surveyed the prevalence of head lice infestation by school grade and found that the prevalence in first grade was significantly frequent (p< 0.05). edalatkhah et al in ardabil reported that infestation rate in younger group was more than older group, and this was similar to our study (edalatkhah et al. 2005). in conclusion, it seems that it is necessary to render health education for families in order to prevent pediculosis in the field. in addition, health professionals should be responsible for treatment and prevention of louse infestation. acknowledgements the authors would like to appreciate very much for kind collaboration of all staff of primary schools of khajeh city. the study was conducted based of the selffunded design. the authors declare that they have no conflicts of interest. references akisu c, sari b, aksoy u, ozkoc s (2003) investigation of pediculus capitis among school children in narlidere couny, izmir and comparison of the current and previous results. acta parasitol turc. 27: 45–48. alempour salemi j, shayeghi n, zeraati h, akbarzadeh k, basseri h, ebrahimi b,rafinejad j (2003) some aspects of head lice infestation in iranshahr area (southeast of iran). iranian j publ health. 32(3): 60–63. balcioglu ic, kurt o, limoncu me, dinc g, gumus m, kilimcioglu aa, kayran e, ozbilgin a (2007) rural life, lower socioeconomic status and parasitic infection. parasitol int. 56(2): 129–33. borges r, mendes j (2002) epidemiological aspects of head lice in children attending day care centres, urben and rural schools in uberlandia, central brazil. mem inst oswaldo cruz, rio de janeiro. 97(2): 189–192. courtaiade c, labrieze c, fontan i, taieb a, maleville j (1993) pediculosis capitis: a questioner survey in 4 schools of the bordeux academy 1990-1991. ann dermatol venereol. 120: 363–368. davarpanah ma, mehrabani d, khademolhosseini f, mokhtari a, bakhtiari h, neirami r (2009) the prevalence of pediculus capitis among school chiliranian j arthropod-borne dis, 2010, 4(1): 42–46 m shayeghi et al.: epidemiology of head lice … 46 dren in fars province, southern iran. iranian j parasitol. 4(2): 48–53. edalatkhah h, arshi s, sadeghi h, sepehram v (2005) prevalence of pediculus capitis in school children in ardebil province (in persian). j. ardebil univ. med. sci. 6: 36–45. ewasechko ca (1981) prevalence of head lice (pediculus humanus capitis) among children in a rural, central alberta school. can j public health. 72: 249– 252. hodjati mh, mousavi n, mousavi m (2008) head lice infestation in school children of a low socioeconomy area of tabriz city, iran. african 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epidemiology of an outbreak of head lice in georgia. am j trop med hyg. 25(5): 739–743. speare r, buettner pg (1999) head lice in pupils of a primary school in australia and implications for control. int j dermatol. 38: 285–290. j arthropod-borne dis, march 2018, 12(1): 16–23 h kassiri et al.: pathogenic fungal species … 16 http://jad.tums.ac.ir published online: march 18, 2018 original article pathogenic fungal species associated with digestive system of periplaneta americana (blattaria: blattidae) trapped from residential dwellings in ahvaz city, southwestern iran *hamid kassiri 1, majid zarrin 2, rahele veys-behbahani 3 1medical entomology and vector control department, health faculty, ahvaz jundishapur university of medical sciences, ahvaz, iran 2medical mycology department, medicine faculty, ahvaz jundishapur university of medical sciences, ahvaz, iran 3student research committee, ahvaz jundishapur university of medical sciences, ahvaz, iran (received 13 may 2016; accepted 24 jan 2018) abstract background: cockroaches are the most prevalent domestic pests of a worldwide distribution. they were recognized as possible vectors of pathogenic bacteria, viruses, fungi and parasites in residential dwellings and hospital environments. the present study isolated and identified yeasts and filamentous fungi from digestive tract of american cockroaches, collected from three different residential regions of iran. methods: seventy cockroaches were sampled using direct collection (hand catch), vacuum cleaner and sticky traps in ahvaz, iran in 2009–2010. their medically important fungal microorganisms were isolated from digestive tract using standard mycological methods. filamentous fungi were identified by macroscopic and microscopic examination. yeasts were identified by api id32c-32100 kit. results: a high percentage of cockroaches (88.6%) were detected to carry fungi of medical importance. overall, 23 fungi species/genera were isolated from the american cockroaches' alimentary tract. the fungi isolated from cockroaches, from the residential regions were species of aspergillus, rhizopus, penicillium, mucorales, alternaria, cladosporium, mycelia, chrysosporium, candida, rhodotorula, zygosaccharomyces, and debaryomyces. candida spp. (41.4%), aspergillus spp. (37.1%) and rhodotorula spp (27.1%) were the most common fungi recovered on cockroaches. candida albicans and candida glabrata were the commonest species of the genus candida. in addition, aspergillus niger and a. flavus were the most frequent species of the genus aspergillus. conclusion: american cockroaches may carry pathogenic fungi in the urban areas of ahvaz. keywords: periplaneta americana, american cockroach, residential environments, fungal flora, isolation introduction cockroaches are distinguished by the subsequent characteristics: wings and tarsus, reproductive organs, head frontal, shape and the number of spines on the femora and color. they comprise five families of ectobiidae (blattellidae), blaberidae, cryptocercidae, blattidae and corydiidae (polyphagidae) (1). cockroaches can be detected in a broad range of surroundings all over the globe, particularly in tropical and subtropical areas. more than 4500 species are reported from different parts of the world. they are one of the most generally prominent household pests and about 30 species are associated with human dwellings (1, 2). house cockroaches such as american cockroach, periplaneta americana (blattodea: blattidae), german cockroach, blattella germanica (blattodea: ectobiidae), brown-banded cockroach, supella logipalpa (blattodea: ectobiidae) and oriental cockroach, blatta orientalis (blattodea: blattidae) are found frequently in iran (2). a number of faunistic studies of cockroaches in the human dwellings of iran showed that b. germanica as the most frequent species followed by p. americana (3, 4). american and german cockroaches, respectively, were intro*corresponding author: dr hamid kassiri, e-mail: hamid.kassiri@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 16–23 h kassiri et al.: pathogenic fungal species … 17 http://jad.tums.ac.ir published online: march 18, 2018 duced as the prevalent species in human residential habitats (5). the german cockroach, which is approximately 15mm (0.59inch) long and the american cockroach, approximately 30mm (1.2inches) long (2, 6). cockroaches are able to transfer fungi, bacteria, viruses, parasites and other medically significant pathogenic agents on their body surfaces and in their feces in infectious regions, such as domestic habitats, hospitals, and industrial areas. from these insects collected from such environments have been isolated important pathogenic microorganisms (7–10). plentiful pathogenic agents including 2 species of protozoans, 15 species of molds and fungi, 32 species of bacteria (such as, shigella and salmonella), 1 virus and 7 helminths which are damaging to humans being detected in the feces, in gut or on cuticle cockroaches (11–13). some fungi have the capacity to distribute via cockroaches (6, 14). it makes them ideal carriers for transferring a number of medically important fungi (15, 16). candida spp., aspergillus spp., penicillium spp. and other species of fungi have been isolated from cockroaches recovered in several healthcare sectors of the hospitals (2, 6, 15–17). aspergillus spp. and candida spp. are the most prevalent fungi causing solemn healthcare-associated infections (18, 19). aspergillosis is common in bone marrow transplant recipients and patients with lung disorders. in immunocompromised patients, obstructive bronchial aspergillosis, allergic aspergillus tracheobronchitis, and pulmonary aspergilloma are reported (20–23). candida is known as an opportunistic pathogenic agent, due to it can innocuously colonize the human body (mouth, skin, genitourinary tract and gut). candidiasis can cause symptoms when a weakened immune system or other factors allow it to grow unabated (24). to determine the possible role of american cockroaches in dissemination of medically important fungi, this study was carried out in residential areas of iran. fungi of medical importance were isolated from the american cockroaches' digestive system and identified. materials and methods this research was carried out in ahvaz (31°192 133 n 48°402 093 e) as a part of the central coordination khuzestan (31.3273°n 48.6940°e), capital of khuzestan, a southwestern province of iran. in this descriptive study, 70 american cockroaches were sampled in ahvaz, iran in 2009–2010, from human dwelling localities using direct collection (hand catch), vacuum cleaner and sticky traps. they were captured from kitchens, toilets or bathrooms of residential area. each cockroach was placed in a single sterile test tube and transported to the laboratory for identification and processing for fungi examination. the cockroaches were immobilized by freezing at 0 °c for 10min. each anesthetized cockroach was examined under the dissecting microscope, and the species were identified using standard taxonomic keys. after identification, 2ml of sterile normal saline (0.9%) was added to the test tube and the cockroaches were vigorously shaken for 2min. after external washing, the cockroaches were washed with 70% ethyl alcohol for 2min. then the cockroaches transferred to sterilized tubes and allowed to dry. the cockroaches were then washed twice in sterile normal saline for 3min to remove traces of alcohol, and the gut was dissected out aseptically. the gut was then macerated under aseptic conditions in 2ml of sterile normal saline. the resulting macerate was cultured on sabouraud's dextrose agar with 0.05% chloramphenicol and incubated at 30 °c for 3wk. the different yeast and filamentous colonies were distinguished by microscopic and macroscopic trials. yeasts were diagnosed by germ tube test, the presence of chlamydoconidia on corn meal plus tween 80 agar and by api id32c-32100 system. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 16–23 h kassiri et al.: pathogenic fungal species … 18 http://jad.tums.ac.ir published online: march 18, 2018 results a total of 70 american cockroaches, periplaneta americana were sampled from 3 residential locations (30, 20 and 20 from kyanpars, amaniae and golestan areas, respectively). about 51.4% cockroaches (36/70) carried one or more species of medically important molds in digestive system and 55.7% (39/70) had one or more species of medically important yeasts in digestive system. about 88.6% (62/70) cockroaches collected were contaminated with one or more fungi species (mold or yeast). overall, 23 species/genera of fungi were isolated from these areas. the fungi isolated from cockroaches in these locations are shown in table 1. in this investigation, candida spp. (74.3%) was the most yeast isolated in the gut of american cockroaches and rhodotrula spp. (48.7%), zygosaccharomyces spp. (15.4%) and debaryomyces polymorphus (2.6%) were the next. in addition, aspergillus spp. (72.2%), penicillium spp. (22.2%) and rhizopus spp. (13.9%) were the most molds appeared in the gut of american cockroaches. other medically important mold, mycelia sterilia, mucorales spp., alternaria spp., chrysosporium spp. and cladosporium spp. were rarely isolated from a few american cockroaches. among 29 (41.4%) american cockroaches, nine species of candida were identified by mycological tests. candida albicans (31%), c. glabrata (10.3%) and c. famata (6.9%) were the greatest species isolated from cockroaches. meanwhile, c. parapsilosis, c guilliermondi, c. tropicalis, c. krusie, c. lipolytica and candida spp. were detected in the digestive canal in a few ones. among 26 (37.1%) american cockroaches, four species of aspergillus were identified. aspergillus niger (42.3%) was the highest species isolated from american cockroaches. aspergillus flavus, a. terreus, and aspergillus spp. were detected in the digestive canal in a few american cockroaches. table 1. fungi isolated from the digestive system of periplaneta americana captured in three residential areas, ahvaz city, southeastern iran fungi isolated kyanpars area no. (%) amaniae area no. (%) golestan area no. (%) total no. (%) aspergillus flavus 4(13.3) 0(0) 1(5) 5(7.1) aspergillus niger 10(33.3) 0(0) 1(5) 11(15.7) aspergillus terreus 2(6.7) 0(0) 0(0) 2(2.9) aspergillus sp. 4(13.3) 4(20) 0(0) 8(11.4) rhizopus sp. 2(6.7) 2(10) 1(5) 5(7.1) penicillium sp. 4(13.3) 4(20) 0(0) 8(11.4) mucorales sp. 0(0) 1(5) 0(0) 1(1.4) alternaria sp. 0(0) 1(5) 0(0) 1(1.4) cladosporium sp. 0(0) 1(1.7) 0(0) 1(0.5) mycelia sterilia 1(3.3) 2(10) 1(5) 4(5.7) chrysosporium sp. 0(0) 1(5) 0(0) 1(1.4) candida albicans 6(20) 0(0) 3(15) 9(12.9) candida glabrata 1(3.3) 0(0) 2(10) 3(4.3) candida parapsilosis 1(3.3) 0(0) 0(0) 1(1.4) candida famata 1(3.3) 1(5) 0(0) 2(2.9) candida tropicalis 0(0) 1(5) 0(0) 1(1.4) candida guilliermondii 1(3.3) 0(0) 0(0) 1(0.5) candida krusie 0(0) 1(5) 0(0) 1(1.4) candida lipolytica 0(0) 1(5) 0(0) 1(1.4) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 16–23 h kassiri et al.: pathogenic fungal species … 19 http://jad.tums.ac.ir published online: march 18, 2018 candida sp. 5(16.7) 1(5) 4(20) 10(14.3) rhodotorula sp. 5(16.7) 6(30) 8(40) 19(27.1) zygosaccharomyces sp. 3(10) 1(5) 2(10) 6(8.6) debaryomyces polymorphus 1(3.3) 0(0) 0(0) 1(1.4) yeasts 3(10) 5(25) 10(50) 18(25.7) discussion the objective of this research was to isolate and identify the fungi found of gut of american cockroaches as a source of contamination. all cockroaches collected were identified as p. americana. different pathogenic and nonpathogenic fungal agents were recovered from this cockroach in human environments. periplaneta americana can carry pathogenic fungi in its internal organs. therefore, p. americana is much more than a harassment and that it has important health hazards as a mechanical vector. therefore, the abundance of american cockroaches’ population has to be reduced by various control methods, such as the proper management of garbage and organic waste disposal, sanitation and using safe insecticides. clearly, presence of cockroaches in sensitive environments, hospitals and houses are more dangerous than other parts due to the special circumstances and the special people hospitalized and can affect to environmental, people and community health. density of cockroaches in most parts of the hospital and residential dwellings as well as their feeding from secretions, human feces, and their ability to transmit a wide range of pathogenic agents, make it as ideal vector to transmit most medically important microorganisms. nowadays, cockroaches have access to an infection source, human food and the place for food production, their role in the transmission of the disease is undeniable. the propensity of american cockroaches to move freely and dwell sewers, restrooms and drains can support to make the problem worse. ability of cockroaches in the transmission of pathogens is emphasized in many types of research in this regard. infec tious agents carried by cockroaches can infect human, animal and food resources in some conditions (1, 3, 4, 6–10, 25). this study confirmed that these insects in residential areas were contaminated with fungi of medical importance. a total of 12 yeast and 11 filamentous species of fungi were isolated from american cockroaches. in this study, a high percentage of the cockroach specimens (88.6%) from the houses were found to carry known fungal pathogens including penicillium spp., candida spp. and aspergillus spp. thus, the isolation of medically important fungi suggests a serious risk concern for patients. although the direct involvement of american cockroaches in transmission of infectious agents is difficult to demonstrate. other several studies have also isolated, from cockroaches from residential areas and hospitals, medically important fungi (14-17, 2527). in the present study, the main fungi isolated were species of candida spp. (41.4%), aspergillus spp. (37.1%), rhodotrula spp. (27.1%) and penicillium spp. (11.4%). the findings from this study about medically important fungi isolated from cockroaches are agreed with the results of some workers. in a study in thailand, penicillium spp. and aspergillus spp. appeared frequently on integument of 16 (35.6%) and 11 (24.4%) cockroaches, respectively (17). in another study in brazil, candida sp. (38.6%), aspergillus sp. (30.7%) and penicillium sp. (8.9%) were the most common fungi recovered on cockroaches (14). in addition, in sari (iran), candida spp, aspergillus spp., and rhodotrula spp. were the most fungi appeared on cuticle of cockroaches (25). in a survey in kashan table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 16–23 h kassiri et al.: pathogenic fungal species … 20 http://jad.tums.ac.ir published online: march 18, 2018 (iran), the prevalence of fungal agents in cockroaches was 41.1% and 22.8%, respectively. candida spp. (39.5%), aspergillus spp. (37, 2%) and penicillium spp. (5.4%) had maximum prevalence among fungi observed (27). in india, candida spp. and aspergillus spp. were the most frequent fungi of medically important genera from cockroaches from a hospital and a residential area (29). the finding of the present study also showed, c. albicans (31%), c. glabrata (10.3%) and c. famata (6.9%) were the greatest species isolated from cockroaches. candida glabrata (42.2%) was the highest species isolated from cockroaches. the second highest was c. magnoliae which 17.8% of cockroaches contaminated (14). yeast identification of candida species showed a higher percentage of c. glabrata (15.4%), c. parapsilosis (15.4%) and c. pseudotropicalis (15.4%), than c. albicans (2.6%) isolated from cockroaches (17). candida glabrata (52.8%) and c. albicans (38.8%) were the highest species isolated from cockroaches. in the present study, four species of aspergillus were identified. aspergillus niger (42.3%) and a. flavus (19.2%) were the highest species isolated from american cockroaches. aspergillus niger (50%) was the most species isolated from cockroaches. moreover, a. flavus and a. fumigatus were the most frequently recovered species from cockroaches (14). aspergillus niger was significantly more frequent in the residential area and the hospital (28). in a study in the hospital environments in ahvaz city, 28 fungal species were isolated from adult housefly. the main fungi isolated were aspergillus spp. (67.4%), penicillium sp. (11.6%), mucorales sp. (11%), candida spp. (10.5%), and rhodotorula sp. (8.4%) (29). we have displayed that american cockroaches transport great number of species of medically significant fungi in their digestive system, incriminated as significant agents in nosocomial infections. hospital-acquired fungal infections are considered consequential causes of morbidity in immunocompromised individuals especially in those remained in hospital for a long period (30). aspergillus sp., an important medical species isolated in our study, has been reported in hospital-acquired infections. aspergillus flavus, the species isolated in this study, had been isolated in bone marrow transplant recipients (31). moreover, a. niger and a. flavus have been reported from patients with invasive disease (32-35). furthermore, other mold species as penicillium spp., alternaria spp., cladosporium spp., mucorales spp. and chrysosporium spp. have lately appeared as significant pathogenic microorganisms inappropriately unable persons (25). among candida species, c. albicans was the commonest species of this genus. candida is noticed as an opportunistic pathogenic microorganism. candida albicans responsible for the majority of hospital-acquired infections. candida albicans is the most prevalent fungi in healthy people, moreover the most prevalent fungal pathogenic agent causing deadly infections (especially in immunocompromised subjects). candida tropicalis, c. glabrata, c. guilliermondii, and c. parapsilosis have lately emerged as significant infectious agents inappropriately unable people (36). rhodotorula species have been reported as nosocomial meningitis and endophthalmitis, particularly in hiv infected persons (25). cockroaches living near human environments were significant vectors of etiological agents and all groups of possible pathogens such as protozoans, bacteria, helminths, and viruses. various bacteria universally associated with these insects are recognized to inure diarrhea, dysentery, and food intoxication in humans. cockroaches have been associated with an outbreak of dysentery. different species of bacteria of public health significance have been isolated from periplaneta americana, such as staphylococcus aureus, streptococcus spp., enterobacteriaceae, pseudomonas aeruginosa, and so on. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 16–23 h kassiri et al.: pathogenic fungal species … 21 http://jad.tums.ac.ir published online: march 18, 2018 cockroaches captured in hospitals and houses have been found to harbor multi-drug resistant bacteria (9). therefore, the high contamination of cockroaches to the agents of infectious diseases can transmit fungal infections in public places such as hospitals and the home. hospitals and homes are the focus of infection and a good place to cockroaches can transfer infectious agents. therefore, the occurrence of infected cockroaches in the sensitive environment of hospital and home, that are places for treatment of patients and living of people, is very dangerous and threatening public health. therefore, control of these insects is essential to achieve the essential goals of hospitals and to create a safe environment in homes. conclusion we revealed the presence of pathogenic filamentous fungi and yeasts in the gut of periplaneta americana collected from the houses in the city of ahvaz. therefore, american cockroaches are a potential vector of pathogenic fungal microorganisms in residential environments. the control of p. americana in residential dwellings is essential in order to control the fungal infections in people. acknowledgements we thank medical entomology student mr javad shamsi for his help in collecting the cockroaches. chancellor for research affairs of ahvaz jundishapur university of medical sciences has partially financially supported this project with number 89s.31. the authors declare that there is no conflict of interests. references 1. hashemi-aghdam ss, oshaghi ma (2015) a checklist of iranian cockroaches (blattodea) with description of polyphaga sp. as a new species in iran. j arthropod-borne dis. 9(2): 161–175. 2. dehghani r, atharizadeh m, moosavi sg, azadi s, rashidi m, paksa a (2014) analysis of cockroach fauna and frequency in human residential habitats of north of isfahan, iran. inter arch health sci. 1(1): 25–29. 3. fakoorziba mr, eghbal f, hassanzadeh j, moemenbellah-fard md (2010) cockroaches (periplaneta americana and blattella germanica) as potential vectors of the pathogenic bacteria found in nosocomial infections. annals trop med parasit. 104: 521–528. 4. vahabi a, shemshadn k, mohammadi p, sayyadi m, shemshad m, rafinejad j (2011) microbiological study of domestic cockroaches in human dwelling localities. afr j microbiol res. 5: 5790–5792. 5. beccaloni gw, eggleton p (1882) order blattodea brunner von wattenwyl. in: zhang zq. 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verma a (1991) cockroaches (blattella germanica) as carriers of microorganisms of medical importance in hospitals. epidemiol infect. 107: 181–187. 27. doroodgar a, arbabi m, asadi ma (2006) survey on parasitic and fungal agents of hospital cockroaches in kashan2002. feyz. 10(1): 28–35 (in persian). 28. fotedar r, banerjee u (1992) nosocomial fungal infectionsstudy of the possible role of cockroaches (blattella germanica) as vectors. acta tropica. 50: 339–343. 29. kassiri h, zarrin m, veys-behbahani r, faramarzi s, kasiri a (2015) isolation and identification of pathogenic filamentous fungi and yeasts from adult house fly (diptera: muscidae) captured from the hospital environments in ahvaz city, southwestern iran. j med entomol. 52(6): 1351–1356. 30. mcneil mm, nash sl, hajjehm ra (2001) trends in mortality due to invasive mycotic diseases in the united states, 1980–1997. clin infect dis. 33: 641– 6477. 31. de la rosa gr, champlin re, kontoyiannis dp (2002) risk factors for the 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1-6 b vazirianzadeh et al: side effects of igr… 1 original article side effects of igr cyromazine on nasonia vitripennis (hymenoptera: pteromalidae), a parasitic wasp of house fly pupae *b vazirianzadeh 1, nac kidd 2, sa moravvej 1 1department of mycoparasitology and infectious and tropical diseases research centre, ahvaz jundi shapur university of medical sciences, ahvaz, iran 2school of biosciences, cardiff university of wales, cardiff cf10 3tl, wales, uk (received 30 nov 2008; accepted 12 may 2009) abstract background: combination of cyromazine as an insect growth regulator (igr) and nasonia vitripennis (hymenoptera: pteromalidae) a parasitic wasp may be an effective tool for reducing the house-fly populations in poultry houses and livestock farms. this study was conducted to assess the side effects of the igr cyromazine on the level of parasitism and numbers and the longevity of emerged n. vitripennis parasitoids from house fly pupae. methods: cyromazine treated cloth target was used as the contaminating method of the parasitoids which was applied in this research study. results: the weibull distribution showed that there was no significant difference among controls and cyromazine treated targets for longevity data. there was no significant effect of cyromazine on the level of parasitism of n. vitripennis using χ2 test. one-way anova showed that the actual numbers emerging were significantly higher in the control than in two cyromazine treatments; however, it is a useful phenomenon because of reducing the hyperparasitism. conclusion: there is a good consistency between using n. vitripennis and 1.1% or 0.9% cyromazine treated targets. therefore cyromazine treated targets can be applied as a safe delivery vehicle for applying the cyromazine igr in the poultry houses and livestock farms in an integrated pest management (ipm) program. keywords: nasonia vitripennis, cyromazine, igr, weibull distribution, introduction commercial poultry houses and livestock farms are rapidly expanding worldwide to meet the needs of the increasing human population (axtell 1999). therefore, the increase in accumulating manure is unavoidable. this phenomenon provides breeding places for different groups of pests, with house-flies being the most abundant species in poultry facilities and livestock farms. as a result, musca domestica is the primary object of most fly management and control programs (wilhoit et al. 1991). chemical control methods, using different insecticides belonging to the chlorinated hydrocarbon, organophosphate, carbamate and pyrethroid groups, can provide a rapid and easy means of suppressing house-fly populations and have become popular since the 1950s. the use of those insecticides, however, has created several problems including resistance to insecticides, environmental pollution and the creation of new pests. as a consequence, extensive research has been carried out to find suitable alternatives, particularly ones, which can be incorporated into integrated pest management (ipm) programs (senior 1998, vazirianzadeh 2003). combination of an insecticide and a parasitic wasp is a very serious issue in the ipm *corresponding author: dr b vazirianzadeh, tel: +98 9163095110, fax: +98 611 3332036, e-mail: babakvazir@ yahoo.co.uk iranian j arthropod-borne dis, (2008), 2(2): 1-6 b vazirianzadeh et al: side effects of igr… 2 (emden 1984, zhao 2000, wang 2008). they have also expressed that chemical control and biological control are two important strategies and key points for the success in an ipm program. combination of cyromazine as an insect growth regulator (igr) and nasonia vitripennis (hymenoptera: pteromalidae) a parasitic wasp may be an effective tool for reducing the house-fly populations in poultry houses and livestock farms. however, the effective exploitation of natural enemies in ipm fly management programs requires defining the compatibility of parasitoids with insecticides in use (scott et al. 1991). in addition, the parasitoids are at the risk of direct and indirect contacts with the igrs therefore the consistency between two agents is an essential figure. consequently, it was also necessary to evaluate the potential detrimental effect of cyromazine on, or compatibility with, the important species of natural enemies, in this case one species of pteromalid parasitoid. those criteria have also been discussed by ruberson et al. (1998), haseeb et al. (2004), desneux et al. (2007), wang et al. (2008) and as direct effects (acute effects) and indirect effects (chronic effects) of insecticides on biological control agents, like mortality or parasitism rate, longevity, egg viability, consumption rate and behavior, respectively. the objectives of the current study were to assess the side effects of the igr on the level of parasitism, numbers of emerged parasitoids and the longevity of parasitoids after emergence. material and methods cyromazine treated cloth target was used as the contaminating method of the parasitoids which was applied in this research study. igr in this research one commercial formulation of cyromazine, neporex® (2% w/w), was used. in this case 32% w/v sugar solution was used as a solvent. insects parasitoids n. vitripennis came from wye college, uk, colonies. originally n. vitripennis parasitoids were reared in sarcophaga sp. (diptera: sarcophagidae) pupae. in the present study, the parasitoids were reared in the constant environmental chambers (25°c, 65% rh and 12 h l: 12 h d) of cardiff school of biosciences using house-fly pupae in 2003. moreover, the rearing was carried out using 1000 cm3 and 250 cm3 glass jars with proportion of 1 parasitoid/5 house-fly pupae. they were fed with 5% honey solution. house-fly pupae were taken from the "chicken house" a wild strain originated from a carefilly poultry house of wales, uk as well. experiments two series of treatments were conducted. in the first, newly emerged parasitoids were exposed for 48 h in 1000 cm3 glass jars to targets containing either 1.1 or 0.9 g/100 ml in 32% sugar solution giving 0.02 mg a.i./cm2 and 0.016 mg a.i./cm2 respectively and for the control, 32% sugar treated targets only. this ensured that the parasitoids were exposed to the igr. then five females of n. vitripennis parasitoids (two-day-old) plus two males of n. vitripennis, taken from the 1000 cm3 glass jars, were placed in a 25 ml glass test tube with twenty-five one day-old house fly pupae (morgan et al. 1989, mann et al. 1990, scott et al. 1991). three replicates were carried out, each replicate containing ten test tubes. this means that each replicate contained fifty female adult parasitoids and 250 house-fly pupae. some tiny holes were made around the middle of the test tubes for ventilation. then 30 pieces (5×5 cm) of polyester cloths were dipped in the 1.1 g/100 ml and another 30 in 0.9 g/100 ml cyromazine solutions. then the tops of test tubes were sealed with the treated clothes. after 48 h the pupae were replaced iranian j arthropod-borne dis, (2008), 2(2): 1-6 b vazirianzadeh et al: side effects of igr… 3 with twenty-five fresh pupae, the numbers being kept constant during the experiments. then, a sample of ten pupae was collected from each test tube, placed individually in plastic petri dishes (geden et al. 1992) and kept for 3 weeks in the constant environmental room (25° c, 65% rh and 12 h l: 12 h d). the level of parasitism and the numbers of emerged parasitoids were recorded. this procedure was carried out until all of parasitoids died. the cloths were treated with 1.1 g/100 ml and 0.9 g/ml of igr in 32% w/v sugar solution every 24 h. three replicates were done with 32% w/v sugar-only treated cloths as controls. the second treatment was used to examine the longevity of parasitoids under the igr and control regimes. fifty newly-emerged n. vitripennis (per replicate) were placed individually in test tubes, to remove the effects of population density of population longevity and to determine accurately individual longevity. the top of the test tubes were covered by cloths treated with either 1.1 or 0.9 g/ml cyromazine in 32% sugar solution or 32% sugar solution alone (control). then 5 house-fly pupae (one-day-old) were put in each test tube. the house-fly pupae were replaced with fresh ones every 48 h. the cloths were treated with same concentration each 24 h. the number of dead parasitoids was recorded every 48 h. three replicates per treatment were conducted. data analysis chi-squared tests were used to test the effects of the igr on the level of parasitism. where replicates were found to be homogeneous (again using chi-squared tests), the results of each treatment were summed. to determine the effects of igr on the numbers of emerged parasitoids, one-way anova was used, after preliminary diagnostic checks for normality of residuals (ryanjoiner test) and homogeneity of variances (bartlett’s and f-tests). the least significant difference (lsd) method was used to detect any differences between treatments and controls, with bonferroni corrections where necessary. to determine the longevity parameters of parasitoids, the weibull distribution was used. it enables statistical comparison of the shape and scale of different survival curves, providing valuable information which is lost if longevity is summarized as a mean with standard deviation, or as a single lt50 value, as commonly done (pinder iii et al. 1978, tingle and copland 1989). in addition, minitab performs a series of chi-squared tests and provides 95% confidence intervals for testing weather two or more samples have equal shape or scale and come from the same population. also, testing weather the distribution parameters are consistent with specified values (manual of minitab 13.1, 2000, university of wales computing service, 2001 and communication with office of minitab, 2001). as a result by means of the above mentioned procedures the longevity of parameters, scale and shape, were determined. as well lt50s were obtained from the table of percentiles and used to compare the results of treatments. results the results in table 1 show that there was no significant effect of cyromazine on the level of parasitism of n. vitripennis (mean percentage parasitism, χ2= 0.46; p= 0.794; d.f= 2). one-way anova (table 1) showed that, while there was no differences in the level of parasitism between the treatments and control (using chi-squared tests), the actual numbers emerging were significantly higher in the control than in two cyromazine treatments (p< 0.001). however, there was no difference between the 1.1% and 0.9% cyromazine treatments. the longevity results, as described by the weibull distribution, are summarized in table 2 (as an example of weibull distribution). iranian j arthropod-borne dis, (2008), 2(2): 1-6 b vazirianzadeh et al: side effects of igr… 4 all shape parameters of controls and igrtreatments were significantly larger than 1 (p< 0.001), indicating that all of the longevity curves belonged to the type ι shape category. weibull results, using chi-square tests for comparisons, showed that there was no significant different among controls and cyromazine treated targets for longevity data, shape parameters (p= 1), scale parameters (p= 1) and overall shape and scale. those results explain that cyromazine did not effect on the longevity of n. vitripennis population. comparisons of lt50s for each treatment were also consistent with these results, showing no significant differences due to overlapping confidence intervals. table 1. effect of cyromazine on mean percentage parasitism of n. vitripennis (2-day olds) over 10-12 days, treated targets treatments mean number of emerged parasitoids se mean percentage parasitism se 32% sugar solution only treated targets 1724.00 23.47 53.47 0.27 0.9% cy in 32% sugar solution treated targets 874.30 10.92 52.47 0.35 1.1% cy in 32% sugar solution treated targets 874 8.03 52.33 0.41 comparison of all treatments one-way anova*f (p, df error/df of replicate) χ2 (p, df) 986.04 (0.001, 6/2) 0.46 (0.79, 2) cy=cyromazine *lsd results showed that there was a highly significant difference between the sugar-only treated targets and both concentrations of cyromazine. however, there was no significant difference between the two concentrations of cyromazine. table 2. longevity of parasitoids (newly emerged females) as described by the weibull distribution, using different concentrations of cyromazine treated targets treatments shape value se of shape scale value se of scale mean value se of mean lt50 value se of lt50 curve type r* df of r n1/control 1.868 0.267 9.028 0.511 8.016 0.474 7.420 0.374 і 0.992 3 n/0.9% cy2 1.872 0.260 8.975 0.498 7.986 0.461 7.380 0.371 і 0.993 3 n/1.1% cy3 1.878 0.253 8.887 0.481 7.889 0.443 7.311 0.365 і 0.994 3 all scale, mean and lt 50 values are in days, size of population = 150 1 n. vitripennis, 2 0.9%cyromazine in 32% sugar solution, 3 1.1%cyromazine in 32% sugar solution, r* correlation coefficient discussion according to scott and rutz (1988), mandeville et al. (1990), rutz and scott (1990), scott et al. (1991), floate (1998), floate and fox (1999) the use of different classes of insecticides, including igrs, in animal houses has adverse effects on an ecologically and taxonomically diverse group of insects, including both predators and parasitic wasps. therefore, to apply integrated pest management effectively in poultry houses and livestock farms, using a combination of igrs and parasitic wasps, it is important to assess the compatibility of the control agents. the results obtained in this study report the different effects of cyromazine on n. vitripennis. there were no harmful effects on the level of parasitism of n. vitripennis, using 1.1% iranian j arthropod-borne dis, (2008), 2(2): 1-6 b vazirianzadeh et al: side effects of igr… 5 and 0.9% cyromazine treated targets, but those concentrations were highly significant in reducing the number of n. vitripennis emerging per pupa from 6.45±0.1 in control to 3.33±0.03 and 3.34±0.04 in 1.1% and 0.9% cyromazine, respectively. it is possible, however, that this effect could be construed as beneficial in reducing the rate of superparasitism in this species, although this reduction might affect the next generation of the parasitoid. both concentrations of cyromazine caused the same effects on the number of emerged parasitoid, using target application. this phenomenon explains that the parasitoids could parasitize the treated pupae, normally. presumably, some of their eggs did not hatch or some of larvae died after emergence in the host puparia. both effects have been reported as the properties of cyromazine. the same level of parasitism between two concentrations and controls explain that the rest of larvae of parasitoids carried out the successfully parasitism. results of the study of wang et al. (2008) showed that using igrs (hexaflumuron, chlorfluazuron, buprofezin and fuxian) performed very low contact and residual toxicity, however with exhibition chronic effects of oral toxicity on longevity, fecundity and offspring emergence of anagrus nilaparvatae (hymenoptera: mymanidae), an egg parasitoid of the rice planthopper, nilaparvata lugens (hemiptera: delphacidae). in contrast to the igrs using the convenience insecticides presented the highest contact and residual toxicity in the study of wang et al. (2008). the results of study of srinivasan and amalraj (2003) using a combination of parasitoid dirhinus himalayanus (hymenoptera: chalcididae) and insect growth regulator, triflumuron against house fly, musca domestica (diptera: muscidae) show that it is effective in reducing puparia and fly density. therefore, for sustenance of an effective fly control program, combination a parasitoid and an igr may be used. the weibull results here show that 1.1% and 0.9% cyromazine did not affect the longevity of n. vitripennis and there was no significant difference amongst the treatments and control. consequently, there should be a good consistency between using n. vitripennis and 1.1% or 0.9% cyromazine treated targets. then it is suggested using cyromazine treated targets as a safe delivery vehicle for applying the cyromazine igr in the poultry houses and livestock farms in an integrated pest management (ipm) program. acknowledgements iran ministry of health who sponsored financially this study is appreciated. thanks mr. p. macaughen of computing service, cardiff university of wales for providing weibull distribution to interpret the longevity results. references axtell rc (1999) poultry integrated pest management: status and future. integr pest manag rev. 4: 53–73. desneux n, decourtye a, delpuech jm (2007) the sublethal effects of pesticides on beneficial arthropods. annu rev entomol. 52: 81-106. emden hfv (1984) pest control and its ecology. the camelot press, southampton. floate kd (1998) off–target effects of ivermectin on insects and on dung degradation in southern alberta, canada. bull entomol res. 88: 25-35. floate kd, fox as (1999) indirect effects of ivermectin residues across trophic levels: musca domestica (diptera: muscidae) and muscidifurax raptor (hymenoptera: pteromalidae). bull entomol res. 89: 225–229. geden cj, rutz da, miller rw, steinkraus dc (1992) suppression of house-flies (diptera: muscidae) on new york and maryland dairies using releases of muscidifurax raptor (hymenoptera: pteromairanian j arthropod-borne dis, (2008), 2(2): 1-6 b vazirianzadeh et al: side effects of igr… 6 lidae) in an integrated management program. environ entomol. 21: 1419–1426. haseeb m, liu tx, jones wa (2004) effects of selected insecticides on cotesia plutellae, endoparasitoid of plutella xylostella. biocontrol. 49: 33–46. mandeville jd, mullens ba, yu ds (1990) impact of selected pesticides on field population dynamics of parasitic hymenoptera (pteromalidae) in caged-layer poultry manure in southern california, u.s.a. med vet entomol. 4: 261-268. mann ja, stinner re, axtell rc (1990) parasitism of housefly (m. domestica) pupae by four species of pteromalidae (hymenoptera): effects of host parasitoid densities and host distribution. med vet entomol. 4: 235–243. morgan pb, hoyer h, patterson rs (1989) life history of spalangia cameroni (hymenoptera: pteromalidae), a microhymenopteran pupal parasite of muscoid flies (diptera:muscidae). j kansas entomol soc. 62: 381–386. pinder iii je, wiener jg, smith mh (1978) the weibull distribution: a new method of summarising survivorship data. ecol. 59: 175–179. ruberson jr, nemoto h, hirose y (1998) pesticides and conservation of natural enemies in pest management. in: barbosa p (ed): conservation biological control. academic press, san diego, pp. 207–220. rutz da, scott jg (1990) susceptibility of muscoid fly parasitoids to insecticides used in dairy facilities. in: rutz da and patterson rs (eds): biocontrol of arthropods affecting livestock and poultry. westview, boulder, pp. 247–263. scott jg, geden cj, rutz da, liu n (1991) comparative toxicity of seven insecticides to immature stages of m. domestica (diptera: muscidae) and two of its important biological control agents, muscidifurax raptor and spalangia cameroni (hymenoptera: pteromalidae). j econ entomol. 84: 773–779. scott jg, rutz da (1988) comparative toxicities of seven insecticides to houseflies (diptera: muscidae) and urolepis rufipes (hymenoptera: pteromalidae). j econ entomol. 81: 804–807. senior lj (1998) control the greenhouse whitefly, trialeurodes vasporariom [phd thesis]. cardiff university, uk. srinivasan r, amalraj dd (2003) efficacy of insect parasitoid dirhinus himalayanus (hy menoptera: chalcididae) and insect growth regulator, triflumuron against house fly, musca domestica (diptera: muscidae). indian j med res. 118: 158–166. tingle ccd, copland mjw (1989) progeny production and adult longevity of the mealybug parasitoids anagyrus pseudococci, leptomastix dactylopii and leptosmatidea abnormis (hym.: encyrtidae) in relation to temperature. entomophaga. 34: 111–120. vazirianzadeh b (2003) integrated pest management of houseflies, musca domestica (diptera: muscidae), using a combination of cyromazine insect growth regulator (igr) and a pteromalid wasp, nasonia vetripennis. 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[in chinese] j arthropod-borne dis, june 2017, 11(2): 331–337 h ebrahimzade-parikhani et al.: seroprevalence of … 331 http://jad.tums.ac.ir published online: may 27, 2017 original article seroprevalence of visceral leishmaniasis in children up to 12 years old among nomadic tribes from rural areas of pars abad, northwestern iran: an observational study in 2015 hassan ebrahimzade-parikhani 1, *mehdi mohebali 1,2, zabiholah zarei 1, behnaz akhoundi 1, zahra kakoei 1 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran (received 3 mar 2016; accepted 7 jan 2017) abstract background: since pars abad district had been known as a focus of visceral leishmaniasis (vl) in ardabil province but the prevalence of the disease in nomadic tribes has not been determined, thus, this study was conducted. methods: this descriptive crosssectional study was conducted on children up to 12yr old of nomadic tribes from pars abad county, ardabil province, iran in 2015. for each individual, a questionnaire including age, sex, clinical manifestations, history of disease, and contact with reservoir hosts of vl were completed, separately. to determine vl seroprevalence, blood samples were collected from the children and after centrifugation, the plasma samples were tested using direct agglutination test (dat) for detection of anti-leishmania infantum antibodies. statistical analyses were performed using spss16. results: from 776 children up to 12yr old, 2 (0.25%) showed anti-l. infantum antibodies at titers 1:1600 and only one case (0.13%) showed anti-leishmania antibodies at titers 1:3200. the child with anti-l. infantum antibodies titers of 1:3200 showed mild fever for more than 2 months period, paleness, weakness and mild splenomegaly. after physical examination and confirmation of vl (kala-azar), the patient was treated with antileishmanial drugs. conclusion: the findings indicated that l. infantum infection is being circulated with low prevalence in nomadic tribes of pars abad but it is necessary that the surveillance system is regularly monitored among physicians and public health managers in the studied areas. keywords: sero-prevalence, visceral leishmaniasis, nomadic tribes, children, iran introduction leishmaniasis has been reported from 98 countries in the world, in iran this disease is endemic. more than 20 species of leishmania produce diseases, and approximately 30 species of sand flies species (phlebotomus) play a role in disease transmission (alvar et al. 2012). the disease presents four clinical features, namely cutaneous leishmaniasis, mucocutaneous leishmaniasis (espondia), visceral leishmaniasis (vl or kala-azar), and post kala-azar dermal leishmaniasis (pkdl) (arevalo et al. 2007). visceral leishmaniasis (vl), generally produced by l. donovani complex, is a dangerous form of leishmaniasis and transmitted by the bite of female sandflies (mohebali 2013). this disease is manifested by prolonged systemic symptoms such as fever, hepatomegaly, splenomegaly, weight loss, and pancytopenia (alvar et al. 2012, mohebali 2012). visceral leishmaniasis is projected to about 500,000 cases of morbidity and 59,000 cases of mortality every year in the world (werneck 2014). this disease has high case fa*corresponding author: dr mehdi mohebali, e-mail: mohebali@tums.ac.ir j arthropod-borne dis, june 2017, 11(2): 331–337 h ebrahimzade-parikhani et al.: seroprevalence of … 332 http://jad.tums.ac.ir published online: may 27, 2017 tality rate (cfr) in untreated patients, underweight children and cases of hiv/ vl coinfection (cavalcante and vale, 2014). visceral leishmaniasis is mostly reported in six countries: india, ethiopia, nepal, sudan, bangladesh, and brazil (boelaert et al. 2000). the hazard in gaining the illness is mediated through unfortunate housing environments, nonexistence of private protective processes against the vector and migration that leads to non-immune hosts entering vl-endemic regions (bern et al. 2008). kala-azar is endemic in many asian countries and the middle east. in iran, vl is observed in sporadic and endemic forms, so in many parts of iran the disease is sporadic and in some parts of the provinces such as ardabil, east azerbaijan, bushehr and fars, the disease is seen as endemic (mohebali et al. 2011). remarkably, from the 7204 serum sample gathered from domestic dogs in rural communities well-known as the endemic foci of human vl in iran, 879 (12.2%) were seropositive by titers of ≥1:320 (mohebali et al. 2005, mohebali et al. 2006, moshfe et al. 2008, moshfe et al. 2009). also, in a study conducted by moshfe et al in 2006–2007 on canine visceral leishmaniasis in meshkin shahr district in northwestern iran, 17.4% of serum sample were positive by dat (1:320 and higher). moreover, a quarter of seropositive dogs displayed clinical vl symptoms, and cachexia and alopecia were the highest clinical signs in the seropositive dogs (mohebali et al. 2005). annually, approximately 100–300 new symptomatic cases of vl are registered in the health care system of iran (mohebali 2013). out of the 31 provinces of iran, more than 2000 cases of vl were reported up to 2012 (mohebali 2012). in this report, 44.6% of the cases were from the northwestern region of iran (mohebali 2013). during the last decade in iran, an annual average incidence rate of disease was reported as 0.449 per 100000 at-risk populations. here, in the northwestern part of iran, with an incidence rate of 57 cases per 100, 000, have the maximum incidence rate of the disease in iran (mohebali 2013). in this study, the direct agglutination test (dat) was used as a sero diagnostic tool due to its simplicity, cost-effectiveness, appropriate sensitivity (92–100%) and specificity (72–100%), valid test results and its suitability for use in field conditions (elmahallawy et al. 2014). since ardabil province is one of the foci of vl and the prevalence of the disease especially on nomadic tribes of this province has not been evaluated, this present study was conducted to determine the seroprevalence of vl in the nomadic tribes of pars abad county to rapid case finding, and to provide suggestions for the prevention of the disease among nomadic tribes in the study area. materials and methods study area this survey was carried out in the nomadic tribes of pars abad district from 21 march 2014 to 20 march 2015.pars abad district is located in the north of ardabil province, northwestern iran. the study area has mild weather condition in the summer and cold in the winter with an average altitude of 32m above the sea level and a landmass of 1383 km2 (14 percent of the ardabil province area). pars abad area is found in the northern part of the ardabil province, located between the orbits of 39 degrees and 12min to 39 °c and 42min north latitude and 47 degrees and 10 min to 48 degrees and 21min east of the greenwich meridian. pars abad area have 3 districts, 2 cities and 6 villages. according to the 2011 iranian census, the population of this county was 88,924 (https://www.amar.org.ir). blood sampling this study was conducted on children ≤12yr old of nomadic tribes from rural areas j arthropod-borne dis, june 2017, 11(2): 331–337 h ebrahimzade-parikhani et al.: seroprevalence of … 333 http://jad.tums.ac.ir published online: may 27, 2017 of pars abad, northwestern iran. altogether, the blood samples were prepared from 776 children. for each that entered the study, questionnaires that included individual characteristics such as age, sex, location, clinical signs, history of the disease, and contact with dog were provided and filled. for serologic studies, blood samples were collected, and the plasma samples were separated by centrifugation and stored at -20 °c until their examinations. performance of direct agglutination test (dat) the l. infantum antigens for this study were prepared in the leishmaniasis laboratory of the school of public health, tehran university of medical sciences, tehran, iran. the principal phases of the procedure for preparing direct agglutination test antigen were mass production of promastigotes of iranian strain of l. infantum [mcan/ir/07/mohebgh. (genbank accession no. fj555210)] in rpmi1640 medium (biosera, south america), plus 10% fetal bovine serum (biosera, south america), followed by tripsinization of the parasites, staining with coomassie brilliant blue r-250 (sigma, usa) and fixing with formaldehyde 1.2% (el harith et al. 1988, zijlstra et al. 1991, mohebali et al. 2006). all the collected plasma samples were tested by dat. the titration of leishmaniaspecific antibodies was performed by following the general procedures described by mohebali et al. (2006). primarily, two dilutions of 1:800 and 1:3200 were made and tested for screening. the samples that were positive with the titer of 1:800 were diluted up to 1:102400 in a v-shaped microtiter plate into a dilution fluid containing 0.9% saline and 0.78% 2-mercaptoethanol. one equal volume (50μl) of antigen suspension was added to each well. the results were read after 18–24 h incubation in a wet room at room temperature. the highest dilution at which agglutination was still visible in comparison with positive and negative controls titer was defined as the titer of the sample. compact blue dots were scored as negative and large diffused blue mats as positive. the characteristics of seropositive cases of the disease were reported to the health centers of pars abad district to receive suitable cure, if necessary. antigen control well (antigen and diluent plasma only) and the known negative and positive controls were tested in each plate daily. based on prior studies (el harith et al. 1988, mohebali et al. 2006), titers of ≥ 1:3200 were considered as seropositive, and persons with suspected (a titer of 1:1600) or lysed samples were re-sampled 2 to 3 weeks later (schallig et al. 2001, mohebali et al. 2006, kakooei et al. 2014). ethical considerations informed written consent was obtained from the parents of the children examined. this study was approved by the research ethical review committee of tehran university of medical sciences, tehran, iran. statistical analysis chi-squared and fisher exact tests were used for the assessment of the relation between two quantitative variables (sero-prevalence values in relation to sex and age group). statistical significance was assumed if p< 0.050. all reported p values are two-sided. statistical analyses were performed using spss software (version 16, spss inc., chicago, il, usa). results altogether, 776 blood samples were collected from the children up to 12 years old from nomadic tribes of north of ardabil province. four hundred and three of them (51.9%) were male, and 373 (48.1%) were female. the sex ratio (male/female) of the studied population was 1.08. from the 776 children, 3 (0.3%) showed anti-leishmania antibodies at titers ≥ j arthropod-borne dis, june 2017, 11(2): 331–337 h ebrahimzade-parikhani et al.: seroprevalence of … 334 http://jad.tums.ac.ir published online: may 27, 2017 1:1600, and from these only one case showed antil. infantum antibodies at titers of 1:3200 (table 1). hence, the prevalence of the disease (titers of 1:3200) in the studied areas was estimated as 1.28 per 1000 at-risk populations. furthermore, the seropositive case presenting anti-leishmania antibodies at titers 1:3200 was a 10 year old female from agh ghabagh (hosein gheshlagh) village with a positive history of the disease with remittent fever, mild splenomegaly and cutaneous scar in the face in two or three months prior to her disease diagnosis. this patient was hospitalized in the pediatric hospital and was successfully treated with glucantime® as the first line of anti-leishmanial drugs. the relation between human leishmania infection titers and sex and age group was not statistically significant, with p-values of (p= 0.197) and p= 0.332), respectively probably because of the low number of seropositivities. the frequency of anti-leishmania antibody titers with dat according to the age groups is shown in table 2. age group and sex distribution of samples is presented in table 3. in table 4, the age group, sex, location and anti-leishmania antibody titers of 3 seropositive cases of vl has been showed. table 1. seroprevalence of human visceral leishmania infantum infection by gender in children up to 13 years old of nomadic tribes of pars abad county from ardabil province, iran in 2015 anti-leishmania infantum antibody titers ≥ 1:32001:16001:800negative (%)number of examined gender number (%)number (%)number (%) 0 (0%)0 (0%)0 (0%)403 (100%)403male 1 (0.3%)2 (0.5%)0 (0%)370 (99.2%)373female 1 (0.1%)2 (0.3%)0 (0%)773 (99.6%)776total table 2. seroprevalence of human leishmania infantum infection by age group in nomadic tribes of pars abad county from ardabil province, iran in 2015 anti-leishmania infantum antibody titers 1:32001:16001:800negative (%)number of examined age group (yr) number (%)number (%)number (%) 0 (0%)1 (0.5%)0 (0%)201 (99.5%)202<4 0 (0%)0 (0%)0 (0%)280 (100)2805–8 1 (0.5%)1 (0.5%)0 (0%)292 (99%)2949–12 1 (0.1%)2 (0.3%)0 (0%)773 (99.6%)776total table 3. anti-leishmania infantum antibody titers of three seropositive cases of visceral leishmania infantum infection by direct agglutination test with respect to their age, gender and locality in nomadic tribes of pars abad from ardabil province, iran 2015 case no. age (yr) gender location tribe antibody titer 1 9 female pars abad asad kandi 1:1600 2 ≤1 female pars abad griloo 1:1600 3 10 female pars abad hossin gheshlagh 1:3200* *this patient had showed clinical signs and symptoms j arthropod-borne dis, june 2017, 11(2): 331–337 h ebrahimzade-parikhani et al.: seroprevalence of … 335 http://jad.tums.ac.ir published online: may 27, 2017 fig. 1. situation of ardabil province in iran and location of study areas in ardabil province (pars abad) discussion this study was conducted to assess the epidemiological characteristics of vl in pars abad in the north of ardabil province (northwestern iran). the prevalence of visceral l. infantum infection in the pars abad district was 1.28 per 1000 at-risk populations. females and those belonging to the age group of 9–12 years old are at higher risk of disease, in comparison with the other groups. in recent years, various and scattered studies about several aspects of vl have been conducted in ardabil province in northwestern iran. the results showed that vl is endemic in some areas of the province, and most of cases of vl were reported from meshkin shahr district (soleimanzadeh et al. 1993, edrissian et al. 1996, mohebali et al. 2006). in this study, we observed that from the 776 persons that entered the study, 3 (0.3%) of them showed antil. infantum antibodies at titers ≥ 1:1600, and from these only one case (0.1%) showed anti-leishmania antibodies at titers ≥ 1:3200. in a sero-epidemiological study that was conducted by in our study, only one seropositive case of the disease with titers of ≥1:3200 was diagnosed, which included a female in the age group of 9–12yr old. in a study performed by abbaszadehafshar et al. (2015) 0.50% of 6 seropositive cases were males and 0.50% were female, and children belonging to the age group of 5–8yr old showed the highest seroprevalence rate (4.1%) of the disease, a similar result was found by mahmoudvand et al. (2011) in which 41.66% (5 cases) of 14 seropositive cases were females and 58.34% (9 cases) were males. conclusion leishmania infantum infection is being circulated with low prevalence in the nomadic tribes of pars abad area, however, it is necessary for the surveillance system to regularly monitor the infection in the studied areas. appropriate counseling should be provided to military personnel, researchers, and other groups of travelers who may be exposed to sand flies in the endemic areas. early case detection and treatment can reduce the impact of severe illness and death. further studies on domestic dogs as principal animal reservoir host of vl in the nomadic tribes of pars abad are recommended. acknowledgements this study was financially supported by tehran university of medical sciences (grant no: 93-03-160-27323). we extend our thanks to mr ojagh rudy, mr shahram dastori, and mrs sorour charehdar for their valuable field and laboratory techniques. the authors are also grateful 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236 http://jad.tums.ac.ir published online: may 27, 2017 original article new host records for haemoproteus spp. (apicomplexa: haemosporidiasina) in passeriformes from north-west of iran leila nourani 1, *mansour aliabadian 1,2, navid dinparast-djadid 3, omid mirshamsi 1,2 1department of biology, faculty of sciences, ferdowsi university of mashhad, mashhad, iran 2research department of zoological innovations, institute of applied zoology, faculty of sciences, ferdowsi university of mashhad, mashhad, iran 3malaria and vector research group (mvrg), biotechnology research center (brc), pasteur institute of iran, tehran, iran (received 13 jan 2015; accepted 7 may 2016) abstract background: the intracellular protozoan parasites of the genus haemoproteus occur in different avian hosts all over the world. various genus of blood sucking insects’ families such as hippoboscidae and ceratopogonidae could transmit haemoproteus in avian hosts. there are very limited number of studies on wild infected birds with blood parasites in iran, so the aim of this study was to determine the frequency of haemoproteus spp. infection in passerine birds from northwest of iran. methods: passerines were collected from four different localities in zanjan province, northwest iran during june to august 2014. results: of 86 passerines, we found haemoproteus infection in 19 (22.09%) individuals. in general, 15 bird species were observed for haemosporidians, of which 53% were infected. conclusion: three species of passerines: petronia petronia, sitta tephronota and acrocephalus melanopogon are new host records for haemoproteus infection in the world. results acquired by this study support widespread distribution of haemoproteus in passerines and illustrated the prevalence of haemoproteus species in wild birds of northwest of iran. conclusively, our study specified that more investigations are needed to reach exact prevalence rate in different families of birds in iran. keywords: mosquitos, hippoboscidae, ceratopogonidae, plasmodium, blood parasites introduction the intracellular protozoan parasites of the genus haemoproteus occurs in different avian hosts all over the world (bennett et al. 1992, valkiunas 2005). this genus was originally described by kruse 1890 as haemoproteus columbae based on gametocytes in columba livia. haemosporidians as imposing parasites that infect bird's blood cells in which asexual reproduction stages arise within erythrocytes and sexual cycle occurs in dipteran vectors (hellgren et al. 2008, ishtiaq et al. 2008). various genus of blood sucking insects’ families such as hippoboscidae and ceratopogonidae could transmit haemoproteus in avian hosts (atkinson and van riper 1991, ishtiaq et al. 2008, martinsen et al. 2008). traditionally, the prevalence of blood parasites is calculated as the amount of infected samples by microscopic detection of blood smears (peirce 1981). this factor could illustrate transmission degree of the parasites with mosquito’s vectors, richness and also host specificity (van riper et al. 1986, hamilton and zuk 1982). avian hosts stay diseased if they once become infected by haemosporidian parasites for long time (valkiūnas 2005). the passeriformes with more than 5300 extant species including the largest and most **corresponding author: dr mansour aliabadian, e-mail: aliabadi@um.ac.ir j arthropod-borne dis, june 2017, 11(2): 236–241 l nourani et al.: new host records … 237 http://jad.tums.ac.ir published online: may 27, 2017 frequently known clades of birds occupied all landmasses except antarctica (johanson et al. 2008b). according to recent avian checklist, 235 passerine birds have been recorded from iran (dickinson and christidis 2014). parasites have major effects on physiology, ecology, health, population dynamics, sexual selection and fertilization of birds which in some cases lead to host elimination (hamilton and zuk 1982, garvin et al. 2003, marzal et al. 2005, donovan et al. 2008), due to very limited number of studies on wild infected hosts with blood parasites in iran, we have attempted to determine the frequency of haemoproteus species in passerines from northwest of iran. besides, new host records were reported for haemoproteus. materials and methods passerine birds were collected from four different localities in zanjan province, northwest iran during june to august 2014. avian hosts were identified morphologically using field guides (porter and aspinall 2010). blood samples were obtained via brachial vein puncture of the birds with insulin needles. three thin smears for each specimens were made, immediately air dried, fixed in absolute methanol and finally stained with giemsa stain ph 7.2 for 45min protozoan detection was done under light microscope for 10–15min at low magnification and then at high magnification (x1000) with immersion oil. parasites were identified according to the procedures suggested by valkiunas (2005). results a total of 86 birds’ belong to nine families were studied. all hosts belong to the order passeriformes comprising nine families: turdidae, paridae, passeridae, sittidae, emberizidae, fringillidae, acrocephalidae, laniidae and muscicapidae. the highest numbers of captured hosts belong to fringilidae (table 1). the highest amount of infection was identified in sittidae (50%) and other families fringillidae (43.75%), acrocephalidae (25%), emberizidae (20%), passeridae (14.28%), turdidae (11.11%) were placed in the subsequent orders respectively. furthermore, families paridae, laniidae and muscicapidae were clear from haemoprotues infection. besides, new host records were reported for haemoproteus spp. of petronia petronia, sitta tephronota and acrocephalus melanopogon for world (fig. 1). fig. 1. haemoproteus species gametocytes in red blood cells of passerines from northwest of iran in petronia petronia, sitta tephronota and acrocephalus melanopogon respectively from left. (photo by l nourani) j arthropod-borne dis, june 2017, 11(2): 236–241 l nourani et al.: new host records … 238 http://jad.tums.ac.ir published online: may 27, 2017 table 1. occurrence of passeriformes haemoproteus species from zanjan province, northwest of iran. number of examined and infected host is presented. species marked with star are new reports as haemoproteus infected hosts. host families host species no. of host captured examined infected turdidae turdus merula 9 1 paridae parus major 15 0 passeridae petronia petronia* 3 1 passer domesticus 4 0 sittidae sitta tephronota* 2 1 emberizidae granativora melanocephala 14 3 emberiza buchanani 1 0 fringillidae linaria cannabina 14 6 carduelis carduelis 2 1 acrocephalidae acrocephalus dumetorum 10 3 acrocephalus melanopogon* 5 2 laniidae lanius minor 1 0 muscicapidae saxicola torquatus 1 0 irania gutturalis 1 0 ficedula hypoleuca 4 0 total 86 19 discussion this study was designed to discover the occurrence of haemoproteus spp. among different species of passeriformes. this genus is distributed worldwide with more than 132 species (valkiūnas 2005). of 86 passerine birds which examined in this study, we found haemoproteus infection in 19 (22.09%) individuals. in general, 15 bird species were examined for haemosporidians, of which 53% were infected. results showed three species passeridae: petronia petronia, sittidae: sitta tephronota and acrocephalidae: acrocephalus melanopogon are new host records for haemoproteus infection in the world according to malavi database and the rest of hosts’ species infection are also new records for iran. till december 2014, about 1129 instances of haemoproteus for 987 different birds’ species was documented around the world. the most regular genera of intracellular parasites could infect bird hosts including haemoproteus, plasmodium (marchiafava et celli 1885) and leucocytozoon (berestneff 1904). some investigations reported between 50–100% of the individuals are infected with these blood parasites (valkiūnas et al. 2003, belo et al. 2011, yoshimura et al. 2014) and other researches documented less amount of prevalence (sehgal et al. 2005, youssefi et al. 2010, bahrami et al. 2012, doosti et al. 2014). comparatively, it is little known on blood parasites of wild birds in iran but, there are numerous studies on columbiformes (youssefi et al. 2010, radfar et al. 2011, dehghanisamani et al. 2013, doosti et al. 2014) and waterfowls (shemshadi et al. 2014). for example, youssefi et al. (2010) reported the prevalence of 17.47% for h. columbae in north of iran and examination of 250 pigeons revealed that 20.8% of which were infected with h. columbae (bahrami et al. 2012). in another study from chaharmahal va bakhtiari province, 220 free living pigeons (columba livia domestica) from five localities presented the prevalence rate of 23.18% for h. columbae infection (doosti et al. 2014). faghihzadeh gorji et al. 2012 found prevalence rate of 11% for plasmodium j arthropod-borne dis, june 2017, 11(2): 236–241 l nourani et al.: new host records … 239 http://jad.tums.ac.ir published online: may 27, 2017 of passer domesticus in mazandaran province. our report is similar to prevalence rate with the average of 26% for haematozoa in 14812 samples reported for european passerines (scheuerlein and ricklefs 2004). high prevalence rate of 39.6% has been recorded for plasmodium infection for 275 passerine birds’ samples of nine families from brazil by ribeiro et al. 2004. likewise another study on 121 birds species captured of west african rainforests, they reported haemoproteus (7.7%), plasmodium (10.7%), leucocytozoon (4.6%) infection (sehgal et al. 2005). migratory birds including passeriformes and birds of prey are the potential reservoir of various infections which may influence animals and/or man. birds are able to harbor various pathogens such as chlamydophilosis and salmonellosis agents, poxvirus, plasmodium, feather mite and lice, avian-origin influenza a(h7n9), crimean-congo hemorrhagic fever virus and lyme disease (karesh et al. 2007, vanrompay et al. 2007, carlson et al. 2011, lindeborg et al. 2012, jones et al. 2014). duo to the importance of transmission of diseases between wildlife and man on public health and economic issues, more studies may shed light on birds and parasites relationship. conclusion results support widespread distribution of haemoproteus in passeriformes and illustrated the prevalence of haemoproteus spp. in wild birds of northwest of iran. conclusively, our study specified that more studies are needed to reach exact prevalence rate in different families of birds in iran. acknowledgements the authors would like to express their sincere appreciation to who assisted for sampling. financial support was provided by the office of research affairs, ferdowsi university of mashhad (3/32568). the authors declare that there is no conflict of interests. references atkinson ct, van riper iiic (1991) pathogenicity and epizootiology of avian haematozoa: plasmodium, leucocytozoon, and haemoproteus. oxford university press, london. bahrami am, monfared al, razmjoo m (2012) pathological study of parasitism in racing pigeons: an indication of its effects on community health. afr j biotechnol. 11(59): 12364–12370. belo no, pinheiro rt, reis es, ricklefs re, braga em (2011) prevalence and lineage diversity of avian haemosporidians from three distinct cerrado habitats in brazil. plos one. 6(3): e17654. bennett gf, earle ra, dutoit h, huchzermeyer fw (1992) a host-parasite catalog of the hematozoa of the sub-saharan birds. j vet. 59(1): 1–73. bensch s, hellgren o, pérez‐tris ja (2009) malavi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. mol ecol res. 9(5): 1353–1358. carlson jc, engeman rm, hyatt dr, gilliland rl, deliberto tj, clark l, bodenchuk mj, linz gm (2011) efficacy of european starling control to reduce salmonella enterica contamination in a concentrated animal feeding operation in the texas panhandle. bmc vet res. 7: 9. dehghani-samani a, pirali kheirabadi k, dehghani-samani a (2013) prevalence and rate of parasitemia of haemoproteus columbae in columba iivia domestica in southwest of iran. iran j parasitol. 8 (4): 641–644. dickinson ec, christidis l (eds.) 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(2008), 2(2): 48-51 a rashid et al: trypanosomiasis in… 48 case report trypanosomiasis in dog; a case report *a rashid 1, k rasheed 2, a hussain 1 1veterinary research institute, ghazi road lahore cantt, pakistan 2university of veterinary & animal sciences, lahore, pakistan (received 19 nov 2008; accepted 12 may 2009) abstract trypanosoma are extra-erythrocytic, protozoan parasites of domestic and wild animals. it is transmitted by biting flies and a cause of fever, anemia, myocarditis and corneal opacity. in the present study, trypanosoma infection is reported in a male bull dog of two years age at lahore, pakistan. clinical signs and symptoms of infection are recorded. confirmation of case was done by microscopic examination of trypanosoma organism in thick blood smear. furthermore, their effect on certain hematological parameters was studied and concluded that there was decrease of hemoglobin (hb) concentration and packed cell volume (pcv) while erythrocytes sedimentation rate (esr) was increased in the infected dog. the animal was successfully treated with single dose of diminazene diaceturate at the dose rate of 3.5 mg/kg body weight, intramuscularly alongwith the supportive therapy. keywords: trypanosoma, dog, pakistan introduction trypanosomiasis is a hemoprotozoan disease of domestic and wild animals, spread by biting tabanid flies. the disease is generally acute and fatal in canines (soulsby 1982) and a cause of fever, anemia, myocarditis and corneal opacity. trypanosomiasis has been reported for the past 70 yr in a number of populations of opossums, wood rats, raccoons, armadillos and coyotes (olsen et al. 1964, burkholder et al. 1980, grogl et al. 1984, john and hoppe, 1986, barr et al. 1991, telford and forrester 1991, karsten et al. 1992). all species of trypanosoma, with the exception of some strains of t. vivax which produce a hyper acute and acute infection, characterized by high parasitaemia, fever, severe anemia and hemorrhages on the mucosal and serosal surfaces (urquhart et al. 2002). trypomastigote form of trypanosoma enters host cells soon after infection, multiplies sub clinically, escapes the immune system and spread throughout the body primarily within macrophages. parasitaemia develops within a few day and peaks 2 to 3 weeks post infection, coinciding with clinical disease (barr et al. 1991). anemia is a cardinal feature of the disease in which red blood cells are removed from the circulation by the expelled mononuclear phagocytic system. later, in infection of several months duration, when the parasitaemia become low and intermittent, anemia may resolve to a variable degree (urquhart et al. 2002). there are a number of effective trypanosomacidal agents for dogs including suramin, quinapyramine and diminazene but single dose of diminazene diaceturate is effective in eliminating the natural trypanosomiasis infection in canine (rani and suresh 2007). *corresponding author: a rashid, tel: +92 42 9220136, e-mail: dr_asif_rasheed@hotmail.com iranian j arthropod-borne dis, (2008), 2(2): 48-51 a rashid et al: trypanosomiasis in… 49 the present report describes a case of trypanosomiasis in a dog, its effect on some blood parameters and treatment with diminazene diaceturate. history of case a male bull dog of two years age was brought to a private clinic at lahore, pakistan with history of anorexia, dullness and persistent fever for three days. on clinical examination, there was high rise of rectal temperature (40.8 oc), pale mucous membrane, bilateral lacrimation and generalized debility. the dog was tested for the presence of hemoparasites through blood smears (coles 1986). for this purpose, hair was clipped from the outer surface of the pinna of ear near the margin. the marginal ear vein was pricked with sharp sterilized needle after disinfection with methylated spirit. a drop of blood was place on a clean glass slide and smear was made and air-dried. the smear was stained with the giemsa stain (coles 1986) and examined under light microscope by using the immersion objective. microscopic examination revealed the presence of trypanosoma organism outside the rbc’s. hematological and therapeutical study after confirmation of trypanosoma through blood smear, hematological examination included hemoglobin estimation (hb), packed cell volume (pcv) and erythrocytes sedimentation rate (esr) using the methods as described by benjamin (1978) was carried out to check the severity of disease. for this purpose, ten ml of blood was collected from cephalic vein in heparinized vacutainer tube containing anti-coagulant. simultaneously, treatment of dog with trypanosomiasis was carried out with diminazene diaceturate at dose rate of 3.5 mg/kg body weight intramuscularly along with supportive therapy including oral preparation of liver tonic (hepamerz) and vitamin e for 5 d. discussion clinical signs and symptoms there is limited published literature available regarding the prevalence of trypanosomiasis in dog in pakistan. in the present study, microscopic examination revealed the presence of trypanosoma organism outside the rbc’s (fig. 1). the reported clinical signs and symptoms were high rise of temperature (40.8 oc), pale mucous membrane, bilateral lacrimation, and generalized debility. these observations were in agreement with the findings of rani and suresh (2007) who reported t. evensi organism in peripheral blood with history of in appetence, dullness and persistent fever since five days but the same authors also observed bilateral corneal opacity which is a characteristic finding in chronic trypanosomiasis (thirunavukkarasu et al. 2004). hematological and therapeutical study hematological examination included hb, pcv and esr were carried out and concluded that there was decrease of hb and pcv while esr was increased in the infected dog (fig. 2). fig. 1. blood smear showing abnormalities in erythrocytes with trypanosama parasites iranian j arthropod-borne dis, (2008), 2(2): 48-51 a rashid et al: trypanosomiasis in… 50 0 5 10 15 20 25 30 35 40 hb (g/dl) pcv (%) esr (mm/hours) fig. 2. blood picture in canine trypanosomiasis the results of the present study are in accordance to kjos et al. (2008) who studied clinical hematology of canine chages disease in texas and reported hypoproteinemia, hypoalbuminemia, anemia (decrease packed cell volume) and thrombocytopenia. diminazene diaceturate given im once at a dosage of 3.5 mg/kg was effective for trypanosoma infections in dog and animal showed good clinical improvement after treatment. supportive therapy was continued for 5 d. similar results were observed by rani and suresh (2007) who treated trypanosomiasis in pomeranian dog with a single dose of diminazene aceturate. in conclusion, clinical signs and symptoms along with the microscopic examination of trypanosoma organism through peripheral blood smear suggested the case of trypanosomiasis. however, it was difficult to distinguish the species of trypanosoma at clinic so there is need of serological and molecular tests for the diagnosis of species of trypanosoma in pakistan. moreover, it was also concluded that single dose of diminazene diaceturate successfully treated the dog with trypanosomiasis. references barr sc, gossett ka, klei tr (1991) clinical, clinicopathologic and parasitological observations of trypanosomiasis in dogs infected with north american trypanosoma cruzi isolates. am j vet res. 52: 954-960. benjamin mm (1978) outline of veterinary clinical pathology.3rd (ed). the iowa state uni press ames iowa, usa. pp. 60-75. iranian j arthropod-borne dis, (2008), 2(2): 48-51 a rashid et al: trypanosomiasis in… 51 burkholder je, allison tc, kelly vp (1980) trypanosoma cruzi (chagas) (protozoa: khinetoplastida) in vertebrate, reservoir and human host of the lower rio grande vally of taxas. j parasitol. 66: 305-311. coles eh (1986) veterinary clinical pathology. 4th ed. w b saunder’s company. philadelphia. usa.pp.53-56. grogl m, kuhn re, davis ds, green ge (1984) antibody to trypanosoma cruzi in coyotes in texas. j parasitol. 70: 189-191. john dt, hoppe kl (1986) trypanosoma cruzi from wild raccoons in oklahoma. am j vet res. 47: 1056-1059. karsten v, davis c, kuhn r (1992) trypanosoma cruzi in wild raccoons and opossums in north carolina. j parasitol. 78: 547-549. kjos sa, snowden kf, craig tw, lewis b, ronald n, olson jk (2008) distribution and characterization of canine chagas disease in texas. vet parasitol. 152: 249-256. olsen pf, shoemaker jp, turner hf, hays kl (1964) incidence of trypanosoma cruzi (chagas) in wild vectors and reservoirs in east-central alabama. j parasitol. 50: 599-603. rani nl, suresh k (2007) canine trypanosomiasis. ind vet j. 84: 186-187. soulsby ejl (1982) helminths, arthropods and protozoa of domesticated animals. 7th (ed). bailliere tindall, london. pp.533. telford jsr, forrester dj (1991) hemoparasites of raccoons (procyon lotor) in florida. j wildl dis 27: 486-490. thirunavukkarasu ps, rao vv, srinivasan sr, nambi ap, dhanapalan p (2004) ind j vet med. 24: 117. urquhart gm, armour j, duncan, jl, dunn am, jennings fw (2002) veterinary parasitology. 2nd ed. blackwell science co. uk.pp.217. microsoft word 7dr_hadadzadeh rtl.doc iranian j arthropod-borne dis, 2010, 4(1): 54–59 r haddadzadeh et al.: one-humped camel (camelus … 54 original article one-humped camel (camelus dromedarius) infestation with linguatula serrata in tabriz, iran *hr haddadzadeh1, ss athari2, r abedini3, s khazraii nia4, p khazraii nia5, s nabian1, b haji-mohamadi6 1department of parasitology, faculty of veterinary medicine, university of tehran, iran 2faculty of veterinary medicine, university of urmia, iran 3faculty of veterinary medicine, university of tabriz, iran 4biology section , faculty of veterinary medicine, university of tehran, iran 5department of clinical science, faculty of veterinary medicine, university of tehran, iran 6department of food hygiene, faculty of veterinary medicine, university of tehran, iran (received 14 oct 2009; accepted 7 apr 2010) abstract background: linguatula serrata is one of well known members of pentastomida which infects both human and animals. the aim of this study was to evaluate the prevalence of l. serrata in mesenteric lymph nodes, livers and lungs of camels slaughtered in tabriz area, iran. methods: mesenteric lymph nodes (mlns), livers and lungs of 140 one-humped camels slaughtered in tabriz, north-west of iran were investigated for nymphs of l. serrata from july 2007 to june 2008. the organs were examined macroscopically and then a tissue digestion method was also done for investigation of liver and lung of the camels that had infected mln. the liver and lung samples were mostly taken from condemned and rejected part of organs. results: the infection rate of l. serrata nymphs in mlns, livers and lungs was 13.5%, 1.4% and 1.4% respectively. the number of isolated nymph in infected lymph nodes varied from 2 to 18 with a mean of 4.78. only one nymph was isolated from each infected livers and lungs. the infection rate increased with age (p< 0.05). no significant difference in different sex groups and seasons was observed (p> 0.05). conclusion: considering this fact that consumption of undercooked camel liver was not common in the studied area, the zoonotic importance of this infection should be concluded. keywords: linguatula serrata, infection, one humped camel, tabriz, iran introduction linguatula serrata frohlich, 1789 is one of the most known members of pentastomida, which infects both human and animals. the phylum pentastomida consists of about 100 identified species of linguatulids, all of which are endoparasites of the respiratory tract and other organs/body cavities of tetrapod vertebrates, particularly reptiles. the evolutionary affinities of this unusual group of organisms have continued to confound biologists because of morphological similarities to both annelids and arthropods, and suggestive phylogenetic relationships to crustacea in the subclass branchiura (raff 1998). because pentastomes appear to have a generally stronger affinity to arthropods some authors regard this group of organisms as a taxonomic class (pentastomida) in the phylum arthropoda (beaver et al. 1984, ravindran et al. 2008). currently some of authors regard pentastomida *corresponding author: dr hamid reza haddadzadeh, email: hhadad@ut.ac.ir iranian j arthropod-borne dis, 2010, 4(1): 54–59 r haddadzadeh et al.: one-humped camel (camelus … 55 as a taxonomic sub-phylum in the phylum tardigrada (myers et al. 2008). regardless of its true taxonomic position within the invertebrate realm, there is no mistaking this highly peculiar parasite when recovered from a parasitized host. it is a worldwide and a zoonotic parasite (jubb et al. 1985, noble elmer et al. 1989, shekarforoush et al. 2004). the adult female of the parasite is tongue-shaped and has a length between 100–130 mm and its width is about 10 mm, whereas its male has a length of 20 mm and its width is 3-4 mm. eggs size are about 70–90 µm (noble elmer et al. 1989, kaufmann 1996). embryonated eggs are laid in the nasal passages and frontal sinuses of mammals specially canids and expelled with nasal secretions. the embryo develops in the egg in the presence of water or plant humidity. if the eggs are swallowed by a suitable intermediate host such as cattle, goat, sheep, rabbit, mouse or man, it will be hatched in alimentary canal; thus the larva penetrates the intestine wall and migrates to mesenteric lymph nodes (mlns) or other organs. after some molting steps, lasting 5-6 months, it reaches to its nymph stage with a protective capsule. the larva may have a length of 500 µm and the nymph may be between 4-6 mm (razavi et al. 2004). the nymph is infectious for the carnivores who consume the raw meat of infected herbivores intermediate hosts (noble elmer et al. 1989, oryan et al. 2008). the adult stages develop inside nasal tract. l. serrata infection in dogs has been reported in different parts of iran (meshgi and asgarian 2003, razmaraii et al. 2007, oryan et al. 2008). linguatula serrata infection is more common in tropical and subtropical areas of the world (razavi et al. 2004). human infection often occurs through nostrils, throat and sinuses (yeganeh-moghadam et al. 2001). this infection is accompanied with some special symptoms such as coughing, sneezing, irritated and sore throat, vomiting and headache. this condition is called halzoun syndrome or nasopharyngeal linguatolosis (lazo et al. 1999, yeganeh-moghadam et al. 2001, razmaraii et al. 2007). although man is accidental host of the nymph, the reports of human infection to this parasite is not uncommon (symmers and valteris 1950, papadakis and hourmouziadis 1958, lazo et al. 1999, yeganeh-moghadam et al. 2001, anaraki-mohammadi et al. 2008). there are many reports of l. serrata infection in different animals in iran including small ruminants, dogs, cattle and camels (shekarforoush and arzani 2001, meshgi and asgarian 2003, razavi et al. 2004, shekarforoush et al. 2004, tavassoli et al. 2007a, tavassoli et al. 2007b, razmaraii et al. 2007, oryan et al. 2008, haddadzadeh et al. 2009). the aim of this study was to evaluate the prevalence of l. serrata in mlns, livers and lungs of camels slaughtered in tabriz, iran. materials and methods from july 2007 till june 2008, among slaughtered one-humped camels (camelus dromedarius) in the tabriz slaughterhouse and rural areas near to tabriz, east azerbaijan province of iran, mln, liver and lung of 140 camels were sampled. the approximate age of camels was determined on the basis of cameleer or butcher information. the liver and lung samples were mostly taken from condemned and rejected part of organs. in the laboratory, each lymph node was cut longitudinally in a petri dish containing normal saline and examined under stereomicroscope, recording the numbers of nymphs per lymph node. the examination of livers and lungs of the camels that had infected mln were done in two steps. first, the 50-100g samples (with a mean of 60g) were sliced in small pieces and observed precisely under stereomicroscope to find the nymphs. in the second step, the minced tissues were put in 200 ml of digestion solution containing 5g pepsin enzyme (7178, iranian j arthropod-borne dis, 2010, 4(1): 54–59 r haddadzadeh et al.: one-humped camel (camelus … 56 merck) and 25 ml hydrochloric acid (374, merck) in 1 liter water, incubating at 37° c for 24 h. after that, the suspensions were transferred to petri dishes and examined for l. serrata nymph (shakerian et al. 2008). the data were analyzed using chisquared and fisher’s exact tests (spss 11.5, standard version, copyright spss inc., 1982– 2002). the p value less than 0.05 was considered as significant. results the prevalence of l. serrata nymphs in mlns, livers and lungs of 140 camels slaughtered in the tabriz slaughterhouse and rural areas near to tabriz is shown in table 1. the infection rate of mlns, livers and lungs was 13.5%, 1.4% and 1.4% respectively. there was no infected camel in <4 yr group. the infection rate increased with age (p= 0.009) (table 1). no significant difference was observed between males and females (table 1). the prevalence of l. serrata infection in different seasons has been showed in table 2, no significant difference was found. the number of isolated nymph in infected lymph nodes varied from 2 to 18 with a mean of 4.78 (fig. 1). from the infected livers and lungs, only one nymph was isolated. the results showed a higher infection rate of mlns compared with that of livers and lungs (p= 0.0005). table 1. the prevalence of linguatula serrata nymphs in mlns, livers and lungs of 140 slaughtered camels in tabriz age (year) sex no. of camels camels with infected mlns no. (%) camels with infected livers no. (%) camels with infected lungs no. (%) male 13 0(0) 0 (0) 0 (0) female 3 0(0) 0 (0) 0 (0) <4 total 16 0(0) 0 (0) 0 (0) male 15 2(13.3 ) 0 (0) 0 (0) female 21 1(4.7 ) 0 (0) 0 (0) 4-8 total 36 3(8.3 ) 0 (0) 0 (0) male 29 4(13.7) 1 (3.4) 1 (3.4) female 59 12 (20.3) 1 (1.6) 1 (1.6) >8 total 88 16 (18.1) 2 (2.2) 2 (2.2) male 57 6(10.5) 1 (1.7) 1 (1.7) female 83 13 (15.6) 1 (1.2) 1 (1.2) total total 140 19 (13.5) 2 (1.4) 2 (1.4) iranian j arthropod-borne dis, 2010, 4(1): 54–59 r haddadzadeh et al.: one-humped camel (camelus … 57 table 2. the seasonal variance of linguatula serrata prevalence in mlns, livers and lungs of 140 slaughtered camels in tabriz season no. of camels camels with infected mlns no. (%) camels with infected livers no. (%) camels with infected lungs no. (%) total infected camels no. (%) spring 27 4(14.8) 0 (0) 0 (0) 4 (14.8) summer 21 3(14.2) 1 (4.7) 1 (4.7) 5 (23.8) autumn 39 6(15.3) 0 (0) 0 (0) 6 (15.38) winter 53 6(11.3) 1 (1.8) 1 (1.8) 8 (15) total 140 19 (13.5) 2 (1.4) 2 (1.4) 23 (16.4) fig. 1. a linguatula serrata nymph under stereomicroscope (×35) discussion without any specific clinical symptoms, l. serrata infection in ruminants leads to the reduction of animal products and hidden economic loss, as well as public health risks (nematollahi et al. 2005). this study was done in east azerbaijan province, north-west of iran. the slaughtered camels were mostly from the same area. previously, oryan et al. (1993) reported l. serrata infection in 12.5% of examined camels in shiraz, iran. shakerian et al. (2008) reported the infection rate of mlns and livers of the camels slaughtered at a slaughterhouse in najaf-abad, central part of iran, as 21% and 4.5% respectively and the maximum and minimum numbers of parasites was reported as 30 and 1 in mlns and 10 and 1 in livers. this rate of infection is about 2 times higher than our result. it should be mentioned that najaf-abad is located in isfahan plain in iranian j arthropod-borne dis, 2010, 4(1): 54–59 r haddadzadeh et al.: one-humped camel (camelus … 58 central arid region of iran with a different climate compare with east azerbaijan province with cold winters and heavy snowfall and subfreezing temperatures during december and january. it seems that the difference in climate can be the cause of the different rate of infection. on the other hands, in our study because of economic limitations, the amount of liver and lung samples was too low (with a mean of 60g) and the samples were mostly taken from condemned and rejected parts of organs. in different studies have been done on seasonal variation of l. serrata infection in small ruminant of iran, the results were not the same (hamdast-joo 2001, nematollahi et al. 2005). in our survey no significant difference between prevalence rates of infection in different seasons of the year was observed. the results of our study showed a significant correlation of age and infection rate of camels slaughtered in the tabriz slaughterhouse and rural areas near to tabriz that was compatible with other studies had been done on sheep and camel in iran (shekarforoush et al. 2004, shakerian et al. 2008). in our study, no camel in the <4 yr group was infected. also in our studies no significant difference was observed in the infection rate in both males and females that were compatible with other study (shakerian et al. 2008). linguatula serrata, infests first mlns, and then migrates to other organs such as the liver and lung (shekarforoush et al. 2004, razavi et al. 2004, shakerian et al. 2008). in this study, the prevalence of l. serrata nymph in liver and lung were lower than mlns, which is in accordance with the findings of other investigation (oryan et al. 1993, shekarforoush et al. 2004, tajik et al. 2006, shakerian et al. 2008). considering this facts that consumption of undercooked camel liver is common in the studied area, the zoonotic importance of this infection should be concluded. acknowledgments we acknowledge financial support of center of excellence for veterinary research on iranian indigenous domestic animals, of university of tehran. the authors wish to thank mr m hami and mr b asadian for their kindly cooperation in sampling process. the authors declare that they have no conflicts of interest. references anaraki-mohammadi g, mobedi i, ariaiepour m, pourmohammadi z, zare-bidaki m (2008) a case report of nasopharyngeal linguatuliasis in tehran, iran and characterization of the isolated linguatula serrata. iranian j parasitol. 3: 53–55. haddadzadeh h, athari ss, hajimohammadi b (2009) the first record of linguatula serrata infection of two-humped camel (camelus bactrinus) in iran. iranian j parasitol 4: 59–61 hamdast-joo m (2001) study of the prevalence of linguatula serrata nymph in small ruminants in slaughterhouse of shahryar, iran. 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[in persian, with english abstract]. jubb kvf, kennedy pc, palmer n (1985) pathology of domestic animals. vol. 2. academic press inc, san diego. kaufmann j (1996) parasitic infections of domestic animals. birkhauser, basel. lazo rf, hidalgo e, lazo je, bermeo a, llaguno m, murillo j, teixeira vpa (1999) ocular linguatuliasis in ecuador: case report and morphometric study of the larva of linguatula serrata. am j trop med hyg. 60: 405–409. myers p, espinosa r, parr cs, jones t, hammond gs, dewey ta (2008) the animal diversity web (online). accessed jan 07, 2010 at: http://animaldiversity.org iranian j arthropod-borne dis, 2010, 4(1): 54–59 r haddadzadeh et al.: one-humped camel (camelus … 59 meshgi b, asgarian o (2003) prevalence of linguatula serrata in stray dogs of shahrekord, iran. j vet med series b. 50: 466–467. nematollahi a, karimi h, niyazpour f (2005). the survey of infection rate and histopathological lesions due to nymph of linguatula serrata on slaughtered farm animals in 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linguatula serrata nymphs in cattle. iranian j vet res univ shiraz. 7: 82–85. tavassoli m, tajic h, dalir-naghadeh b, hariri f (2007a) prevalence of linguatula serrata nymphs and gross changes of infected mesenteric lymph nodes in sheep in urmia, iran. small ruminant res. 72: 73–76. tavassoli m, tajic h, dalir-naghadeh b, lotfi h (2007b) study of linguatula serrata infestation in mesenteric lymph nodes of goat in slaughterhouse of urmia, iran. iranian vet j. 3: 85–89. yeganeh-moghadam a, talari sa, dehghani r (2001) a case of human linguatula serrata infestation in kashan, iran. j kerman univ med sci. 8: 175–178. j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 60 http://jad.tums.ac.ir published online: march 14, 2017 original article molecular characterization and phylogenetic congruence of hydropsyche sciligra (tricoptera: hydropsychidae) using mitochondrial and nuclear markers naseh maleki-ravasan 1, abbas bahrami 2,3, hassan vatandoost 3, mansoureh shayeghi 3, mona koosha 3, *mohammad ali oshaghi 3 1malaria and vector research group (mvrg), biotechnology research center (brc), pasteur institute of iran (pii), tehran, iran 2department of medical parasitology and mycology, faculty of medicine, alborz university of medical sciences, alborz province, karaj, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 11 apr 2015; accepted 18 nov 2015) abstract background: caddisflies have significant roles in freshwater ecosystems. morphological identification is the major impediment in accurate species identification of hydropsychids. mitochondrial and nuclear markers are suitable for molecular systematics of these group of arthropods. methods: trichopteran specimens of lavasan district in northeastern tehran, iran were collected in 2012, and described using the morphological and molecular characters of mitochondrial cytochrome c oxidase subunit i (mt-coi) and three expansion fragments of large subunit (lsu) nuclear ribosomal dna (28s rdna) d1, d2, and d3. the resemblance of the specimen sequences was obtained by conducting blast searches against the genbank database and by using simple maximum likelihood clustering using coi, d1, d2, d3, and combination of d1-d2-d3 sequence data sets. results: based on morphological traits the specimens were resembled to hydropsyche sciligra however there were no its counterpart sequences in the genbank. due to lack of unique group of data set for each gene fragment, the specimens were associated with different taxa on molecular phylograms. the sequence contents of the coi, d1, d2, d3, and d1-d3 regions clustered h. sciligra with h. brevis, h. angustipennis, h. occidentalis, h. hedini, h. grahami, and h. longifurca/h. naumanni, respectively. conclusion: phylogenies obtained from combination of d1-d3 showed the highest bootstrap values for most of clades suggesting that long lsu-rdna potentially is more useful for understanding phylogenetic relationships of caddisflies. a large-scale molecular and zoogeographic study on trichopteran species is suggested to revise and to develop the current knowledge of the caddisfly fauna and distributions in the country. keywords: caddisflies, hydropsyche sciligra, coi, lsu rdna, molecular systematics introduction hydropsychid caddisflies (trichoptera: hydropsychidae) have significant importance due to their role as biomonitoring indicators, immense geographical distribution, and their ecological position in aquatic food webs (geraci et al. 2010, maleki-ravasan et al. 2013a). they also are important for human and animal health since they are sources of severe allergy. for example, their extensive exuviae reason inhalant allergens or the tiny setae of their wings and bodies may cause swelling and soreness in the eyes of people who encounter these potential allergens. in addition, the newly emerged adults of caddisflies may cause severe nuisance (seshadri 1955, fremling 1959, corbet 1966). to date, more than 1600 hydropsychid species have been described worldwide *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 61 http://jad.tums.ac.ir published online: march 14, 2017 (morse 2015). genus hydropsyche includes the most species lineages in all of trichoptera order with more than 500 described species and are distributed in holarctic, oriental, afrotropical, and australasian streams and rivers (morse 2015). hydropsyche larvae exhibit a wide range of pollution tolerances (resh and unzicker 1975, lenat 1993, lenat and resh 2001). freshwater biomonitoring which involves identifying the species inhabiting an ecosystem to provide an ongoing assessment of water quality, promises to be an efficient and costeffective method to manage water resources particularly in the countries with low precipitations (morse et al. 2007). hence, species identification has become a prerequisite for any ecological study and biomonitoring approach. moreover, larva identification is important for phylogenetic studies at higher level of trichopteran (frania and wiggins 1997). although hydropsychid caddisflies are among the most frequently encountered macro-invertebrates in freshwater habitats and displays a wide range of tolerance values (lenat 1993), however, their application in biomonitoring has been greatly impeded by the lack of identified and illustrated species, especially in countries such as iran, where trichoptera fauna was studied by non-autochthonous researchers (schmid 1959, malicky 1986, mirmoayedi and malicky 2002, mey 2004, malicky 2004, chvojka 2006). until recently, 62 trichopteran species were known from iran (morse 2015). morphological taxonomy of caddisflies is based on characters of adult male’s genitalia in association with its larva for species description and illustration at species level. conventional approaches to larval association usually involve rearing larvae or morphological identification of metamorphotypes comprising mature pharate adult, larval sclerites, and pupal exuviae in the same pupal case (milne 1938, wiggins 1996). both approaches work well when adequate resources and expertise are available (resh 1972, floyd 1995, glover 1996). however, these approaches have some limitations including larvae that develop into adults no longer exist as larvae, and descriptions must be made from similar (deemed identical) individuals. in addition, larval rearing is complicated by our imperfect understanding of species-specific microhabitat and water-chemistry requirements, particularly for some groups such as hydropsychids. metamorphotypes are relatively rare because that portion of the life cycle occurs for a short time only, which means that chance encounters play a significant role in metamorphotype associations. the molecular method for larval association could significantly accelerate the process of larval descriptions for a poorly known caddisfly fauna (zhou et al. 2007). recently, molecular methods have been developed for species determination and applied for different groups of insects at high or low level of phylogeny such as sand flies (moin-vaziri et al. 2007, absavaran et al. 2009), mosquitoes (oshaghi et al. 2003, 2006a, 2008, 2011, mehravaran et al. 2011) and flies (malekiravasan et al. 2012). the main advantages of these methods are their sensitivity and specificity, independently of the stage, tissue or organ, live or dead of the specimen. the pcr-based species identification provides a convenient alternative for laboratories using primarily dna-based techniques, and may be necessary when the study design already requires the use of individual dna extractions for multiple purposes such as species confirmation, determination of food in predators (morales et al. 2003, sheppard et al. 2005, oshaghi et al. 2006b, maleki-ravasan et al. 2009, li et al. 2011, sint et al. 2011), finding symbiont flora (dale and moran 2006, russell et al. 2012, chavshin et al. 2012, 2014, 2015, maleki-ravasan et al. 2013b, 2015), infection status for various pathogens (oshaghi et al. j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 62 http://jad.tums.ac.ir published online: march 14, 2017 2009a, 2009b, 2010), and population genetic studies (oshaghi et al. 2007). ribosomal dna (rdna) and cytochrome oxidase subunit i (coi) are the most widely used regions of the nuclear and mitochondrial genome, respectively to infer genetic variations and phylogenetic relationships for a vast group of organisms. among the mitochondrion genes, the coi gene has been extensively used for phylogenetic analysis by itself or in combination with nuclear genes, and has proven to be phylogenetically highly informative in many insect groups including trichopterans (whiting et al. 1997, hyliš et al. 2007, sonnenberg et al. 2007, zhou et al. 2009, ishiwata et al. 2011, johanson et al. 2012, ruiter et al. 2013). in the present study, we aimed to provide and compare the sequences of three parts of rdna (lsu rdna d1, d2, d3) and coi genes for our poor morphologically identified caddisfly specimens and to develop phylogenetic topologies to identify or to bound species level for our caddisfly specimens. materials and methods specimen collection this study was conducted in summer time of 2012 in lavasan river, northeastern tehran, iran. immature stages of trichopteran insects were collected using d-frame nets and replacing stones from riverbed where water run, riffle, or stream bank and trichopteran larvae stick their retreat under or beside the stones. the retreats that might dock juvenile insect preserved in 70% ethanol and transferred to the school of public health (sph) laboratory, tehran university of medical sciences, iran. the morphological characters of the extracted immature trichoptera plus retreats general feature were used to species identification using the morphological key (pescador et al. 1995) under microscope (olympus szx12). dna extraction, pcr, and sequencing genomic dna from larva and pharate adult was extracted using qiagen dneasy tissue kit (qiagen, hilden, germany), which uses silica to bind dna. the mt-coi gene extending 690bp of 5' fragment as applied by (lunt et al. 1996) was amplified using primers of c1-j-2090 and c1-n-2735 (table 1). the amplification was performed in 20μl reactions in premix ready to use kits under two thermal circulations. the first circulation started after initial denaturation at 94 °c for 2min, as follows: 5 cycles of 94 °c for 40s, 45 °c for 40s, and 72 °c for 1min. the second thermal cycle was repeated for 35 cycles for 94 °c for 40 s, 51°c for 40 s, and 72°c for 1 min followed by a final extension step at 72 °c for 5 min. amplification of the nrdna fragments was performed using 1µ l of genomic dna from each specimen in 20µ l reactions. the pcr mix was preheated at 94 °c for 3min followed by 40 cycles of 94 °c for 30s 60 °c for 45s, and 72 °c for 60 s. after 10min of final extension at 72 °c, the products were maintained at 4 °c. pcr products were visualized on a 1% agarose gel containing ethidium bromide using an uv transilluminator. the pcr products were directly sequenced by seqlab (guttenberg, germany). sequences from both directions were aligned and proofread with the program chromaspro (version 1.2, windows, technelysium pty ltd, tewantin, queensland, australia). basic local alignment search tool (blast) (altschul et al. 1997) was used to compare the nucleotide sequences with data of ncbi database and to make sure correct fragment amplification. sequences of mtcoi and nrdna regions were aligned with clustalw as implemented in bioedit (hall 1999). phylogenetic analysis for phylogenetic analysis the sequences obtained in this study was combined with all of the d1, d2, d3, and coi sequences of the j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 63 http://jad.tums.ac.ir published online: march 14, 2017 hydropsychid caddisflies available in genbank (table 2) (http://www.ncbi.nlm). due to the different lengths of the sequences, they were trimmed to obtain a consistent region for phylogenetic analysis. pairwise sequence divergence, using kimura’s two-parameter distance algorithm, and the maximum likelihood trees presented herein were processed in mega 5.0 (tamura et al. 2011). to combine three rdna (d1, d2, d3) fragments we have to refine our analysis to the species that their sequences were available for the three fragments (table 3). phylogenetic analyses were performed on various datasets, including dna of d1, d2, d3, and coi separately and combination of d1–d3 fragments. the reliability of the branching order was determined by 1000 bootstrap replications (felsenstein 1985). results the specimens were resembled to hydropsyche sciligra (malicky 1977 synonym: h. gracilis martynov, 1909). pcr amplification was successfully performed for the mitochondrial and nuclear genes for the specimens as outlined in the material and method section. the lengths of pcr products were roughly 690bp for coi, and 330, 430, and 230bp for d1, d2, and d3 of lsu, respectively. the generated sequences were deposited in genbank database with accession numbers jx419389-96. the lengths of fragments used for phylogenetic analysis were 570bp for coi, 269bp for d1, 397bp for d2, 162bp for d3, and 828bp for d1-d3. sequence information of the data obtained in this study and the data retrieved from genbank database for each fragment or combined dataset are summarized in tables 2 and 3 respectively. cytochrome oxidase subunit i sequences were obtained for two specimens from iran and 15 species from genbank. coi length of the two specimens was 619bp, with three substitutions and their gc contents were 31% that is in agreement with known adenine/thymine (a/t)-rich content of mitochondrial genes. d1 sequences were obtained for two specimens from iran and compared with 21 species from genbank. the d1 sequence length of both ld11 and pad1 samples were 307bp with 8 substitutions and 56 and 57% gc contents respectively. d2 sequences of the iranian specimens compared with 27 species from genbank. the d2 sequence length of both ld12 and pad2 samples were 419 bp with three substitutions and their gc contents were 66%. d3 sequences were obtained for the specimens from iran and compared with 40 species from genbank. the d3 sequence lengths of both samples were 318 bp with six substitutions and their 55–56% gc contents. d1–d3 sequences were obtained for the specimens and compared with 11 species from genbank. the d1–d3 sequence lengths of both specimens were 1044 bp with 17 substitutions and 60% gc content. phylogenetic relevance based on coi sequence data showed affinity of the iranian h. sciligra to h. brevis from west palearctic ecozone with 30% bootstrap value (fig. 1). the maximum likelihood tree topology based on d1 sequence data revealed that the iranian hydropsyche specimens were most closely related to h. occidentalis from nearctic ecozone and h. angustipennis from east/ west palearctic ecozone with 59% support (fig. 2). sequence analysis of d2 fragment revealed that the iranian h. sciligra were associated with h. hedini from oriental ecozone with 23% bootstrap value. however, these pair species were associated with most of hydropsyche including h. angustipennis, h. botosaneanui, h. instabilis, h. siltalai and h. saxonica from west palearctic ecozone and formed a main clade with 99% support (fig. 3). tree topology based on d3 sequence data showed an association between the iranian h. sciligra and h. cf graham from oriental j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 64 http://jad.tums.ac.ir published online: march 14, 2017 part with only 31% support (fig. 4). phylogenetic analysis using the combined dataset of d1-d3 fragments recovered the iranian h. sciligra in affinity with h. longifurca from southeast africa and h. naumanni from indonesia with 73% support (fig. 5). generally, the bootstrap values were higher for long fragment of lsu than the individual fragments of lsu or even coi gene. however, the d2 fragment support strongly the monophyly of most hydropsyche species including h. sciligra, h. botosaneanui, h. angustipennis, h. hedini, h. instabilis, h. siltalai, and h. saxonica. phylogenetic congruence of iranian h. sciligra based on different genes and their worldwide distribution are shown in table 4. fig. 1. phylogenetic relationship of hydropsychid caddisflies inferred from 570bp of the mt-coi gene. iranian samples are shown as jx419389-90. the bark beetle hylesinus fraxini (panzer, 1779) (coleoptera: scolytidae) used as out-group. bootstrap values are shown at nodes. the scale of genetic distance is shown underneath j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 65 http://jad.tums.ac.ir published online: march 14, 2017 table 1. details of primers and pcr products used for amplification of caddisfly mitochondrial and nuclear genes gene name primer name sequence (5’ to 3’) pcr product (bp) reference mt-coi coi c1-j-2090 agttttagcaggagcaattactat ∼690 (zhang and hewitt 1997) c1-n-2735 aaaaatgttgagggaaaaatg tta nrdna d1 d1–up ggaggaaaagaaactaacaaggatt ∼330 (geraci et al. 2010) d1–dn caactttcccttacggtact d2 d2-up gagttcaagagtacgtgaaaccg ∼430 d2-dn ccttggtccgtgtttcaagac d3 d3–up acccgtcttgaaacacggac ∼230 d3–dn ctatcctgagggaaacttcgga fig. 2. phylogenetic relationship of hydropsychid caddisflies inferred from 269bp of the 28s-d1-rdna gene. iranian samples are shown as jx419391-92. bootstrap values are shown at nodes. the scale of genetic distance is shown underneath j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 66 http://jad.tums.ac.ir published online: march 14, 2017 fig. 3. phylogenetic relationship of hydropsychid caddisflies inferred from 397bp of the 28s-d2-rdna gene. iranian samples are shown as jx419393-94. bootstrap values are shown at nodes. the scale of genetic distance is shown underneath. j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 67 http://jad.tums.ac.ir published online: march 14, 2017 fig. 4. phylogenetic relationship of hydropsychid caddisflies inferred from 162bp of the 28s-d3-rdna gene. iranian samples are shown as jx419395-96. bootstrap values are shown at nodes. the scale of genetic distance is shown underneath. j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 68 http://jad.tums.ac.ir published online: march 14, 2017 fig. 5. phylogenetic relationship of hydropsychid caddisflies inferred from 828bp of the 28s-d1-d2-d3-rdna gene. iranian samples are shown as jx419391-96. bootstrap values are shown at nodes. the scale of genetic distance is shown underneath table 2. details of genbank sequence data used for phylogenetic analysis. the two first rows obtained in this study nuclear large subunit rrna [28s] mitochondrial cytochrome oxidase subunit i [mt-coi] d1 d2 d3 hydropsyche sciligra ld11 (jx419391) hydropsyche sciligra ld12 (jx419393) hydropsyche sciligra ld13 (jx419395) hydropsyche sciligra l190 (jx419389) hydropsyche sciligra pad1 (jx419392) hydropsyche sciligra pad2 (jx419394) hydropsyche sciligra pad3 (jx419396) hydropsyche sciligra pa90 (jx419390) hydropsyche angustipennis (ef417120) hydropsyche angustipennis (ef417120) parapsyche elsis (af436341) hylesinus fraxini (hm002626) wormaldia triangulifera (af436226) mexipsyche furcula (ef513990) wormaldia triangulifera (af436345) hydropsyche fezana haplotype 02 (hm134822) hydropsyche occidentalis (af436212) herbertorossia quadrata (ef513918) hydropsyche occidentalis (af436332) hydropsyche fezana haplotype 01 (hm134821) cheumatopsyche lepida (ef417118) cheumatopsyche lepida (ef417118) arctopsyche grandis (af436342) hydropsyche pellucidula haplotype 10 (hm134819) cheumatopsyche oxa (af436213) caledopsyche sp. (eu254421) caledopsyche sp. (eu254458) hydropsyche pellucidula haplotype 06 (hm134815) plectropsyche hoogstraali (hm167451) potamyia flava (hm167448) hydropsyche cf. grahami (eu254462) hydropsyche pellucidula haplotype 09 (hm134818) calosopsyche continentalis hm167450 hydromanicus sp. (ef513893) hydropsyche sparna (hsu65201) hydropsyche incognita haplotype 03 (hm134807) mexipsyche cf grahami (eu312009) mexipsyche nr rhomboana (ef513991) smicridea sp. (eu254467) hydropsyche incognita haplotype 04 (hm134808) j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 69 http://jad.tums.ac.ir published online: march 14, 2017 orthopsyche fimbriata (eu250332) hydropsychehedini (ef513985) orthopsyche thomasi (eu254468) hydropsyche incognita haplotype 02 (hm134806) homoplectra flinti (eu312025) hydropsyche instabilis (hm167440) maesaipsyche sp. (eu254474) hydropsyche incognita haplotype 01 (hm134805) aoteapsyche colonica (af436215) hydropsyche botosaneanui (hm167436) trichoptera environmental (dq086620) hydropsyche incognita haplotype 05 (hm134809) hydropsyche naumanni (eu312012) hydropsyche siltalai (hm167444) diplectrona zealandensis (eu254475) hydropsyche brevis (jq687920) hydropsyche longifurca (eu312026) mexipsyche nr rhomboana (ef514025) smicrophylax sp. (eu254457) hydropsyche fezana (jq687901) ceratopsyche bronta (af436214) herbertorossia sp. (ef514022) aoteapsyche colonica (af436335) hydropsyche lobata (kf255638) cheumatopsyche afra (eu312016) mexipsyche nr rhomboana (ef513992) hydropsyche longifurca (eu254472 hydropsyche exocellata (kf255625) diplectrona metaqui (eu312024) hydropsychesaxonica (hm167443) hydropsyche naumanni (hm167457) hydropsyche dinarica (kf255619) hydatopsyche melli (eu312008) mexipsyche nr rhomboana (ef513994) ceratopsyche bronta (af436334) hydropsyche instabilis (kf255636) hydromanicusu mbonatus (eu312010) hydropsyche longifurca (eu254450) homoplectra flinti (eu254471) hydropsyche maroccana (jq687916) streptopsyche parander (hm167449) aoteapsychecolonica (hm167438) diplectrona metaqui (eu254470) hydropsyche teruela (kf255660) orthopsyche thomasi (eu31202228) hydropsyche naumanni (eu254434) streptopsyche parander (hm167453) hydropsyche modesta (jq687913) cheumatopsyche triangularis (eu312013) ceratopsyche bronta (hm167437) hydatopsyche melli (eu254461) hydropsyche bulbifera (jq687900) diplectrona metaqui (eu254448) cheumatopsyche afra (eu254465) hydropsyche tibialis (jq687923) homoplectra flinti (eu254449) hydromanicus umbonatus (eu254463) streptopsyche parander (eu254455) hydropsyche exocellata (jq687958) hydromanicusumbonatus (eu254432) hydropsyche instabilis (jq687954) hydatopsyche melli (eu254430) hydropsyche pellucidula (jq687950) cheumatopsyche afra (eu254438) hydropsyche modesta (jq687949) mexipsyche nr grahami (ef514002) hydropsyche siltalai (jq687948) hydropsyche nr formosana (ef513958) hydropsychefontinalis jq687947 table 2. continued… j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 70 http://jad.tums.ac.ir published online: march 14, 2017 ceratopsyche serpentine (ef513917) hydropsyche infernalis (jq687946) hydropsyche fezana (jq687939) hydropsyche lobata (jq687929) hydropsyche tibialis (jq687962) hydropsyche dinarica (jq687959) hydropsyche incognita (jq687951) hydropsyche brevis (jq687932) hydropsyche teruela (jq687961) hydropsycheiberomaroccana (jq687937) cheumatopsyche lepida (jq687965) hydropsyche bulbifera (jq687928) table 3. details of the genbank sequence data used for phylogenetic analysis of rdna d1-d2-d3 loci species country genbank accession numbers 28s d1 28s d2 28s d3 hydropsyche sciligra (larvae) iran jx419391 jx419393 jx419395 hydropsyche sciligra (pharate adult) iran jx419392 jx419394 jx419396 streptopsyche parander dominican hm167449 eu254455 hm167453 aoteapsyche colonica new zealand af436215 hm167438 af436335 hydropsyche naumanni indonesia eu312012 eu254434 hm167457 hydromanicus umbonatus china eu312010 eu254432 eu254463 hydatopsyche melli china eu312008 eu254430 eu254461 diplectrona metaqui usa eu312024 eu254448 eu254470 ceratopsyche bronta usa af436214 hm167437 af436334 homoplectra flinti usa eu312025 eu254449 eu254471 hydropsyche longifurca southeast africa mozambique eu312026 eu254450 eu254472 cheumatopsyche afra south africa eu312016 eu254438 eu254465 cheumatopsyche lepida west palearctic ef417118 ef417118 jq687965 table 2. continued… j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 71 http://jad.tums.ac.ir published online: march 14, 2017 table 4. details of phylogenetic congruence of iranian hydropsyche sciligra gene putative species (accession number) biogeographic ecozone coi h. brevis (jq687920) west palearctic (france) d1 h. occidentalis (af436212) nearctic h. angustipennis (ef417120) east palearctic, west palearctic (netherlands, belgium, germany, sweden, united kingdom, luxembourg, norway, finland, france, austria, czech republic, italy, denmark, russia, slovenia, hungary, croatia, isle of man, switzerland, ireland, greece, macedonia) d2 h. botosaneanui (hm167436) west palearctic (greece, belgium, luxembourg, germany france, netherlands, italy, monaco) h. angustipennis (ef417120) like above h. hedini (ef513985) oriental (china) h. instabilis (hm167440) west palearctic (europe and northern asia (excluding china)) h. siltalai (hm167444) west palearctic: europe and northern asia (excluding china) (norway, sweden, finland) h. saxonica (hm167443) west palearctic: europe and northern asia (excluding china) germany d3 h. cf. grahami (eu254462) oriental (china) d1-d2-d3 h. longifurca (eu312026, eu254450, eu254472) afrotropical (south africa, lesotho, zimbabwe, swaziland) h. naumanni (eu312012, eu254434, hm167457) oriental (indonesia) discussion in this study, we found only samples of one species h. sciligra in lavasan district located in northeastern of tehran. this species is widespread in iran, turkey and caucasus (morse 2015). this species has previously been reported from various parts of northern iran including chalus, makou, qazvin, minoudasht, and northern parts of alborz mountains chain (mirmoayedi and malicky 2002, ivanov 2011). the discovery in lavasan indicates that the dispersal area of this species is wider than currently known. besides of this species, there are twelve species of hydropsyche previously reported from certain provinces or regions of iran and neighboring countries as follows: h. consanguinea, h. demavenda, h. djabai, h. mahrkusha, h. ressli, h. sakarawaka, h. supersonica, h. iokaste, h. bujnurdica, h. esfahanica, h. lundaki, and h. masula (morse 2015). mitochondrial genes (mtdna) particularly coi are used most frequently in different phylogenetic levels of trichopteran including order, families, subfamilies, genera, and species levels (myers et al. 2001, kjer et al. 2001, 2002, johanson 2007, malm and johanson 2008, pauls et al. 2008, previšić et al. 2009, johanson et al. 2009, johanson and malm 2010, johanson and espeland 2010, espeland and johanson 2010a, espeland and johanson 2010b, malm and johanson 2011). however, in this study bootstrap values of phylogenetic tree nodes were not enough high to support strongly the caddisflies relationship. it reflects lack of enough available data in genbank than the phylogenetic utility of the gene. in this study, 28s nrdna was selected due to the high frequent available sequence data for trichoperan species in genbank, j arthropod-borne dis, march 2017, 11(1): 60–77 n maleki-ravasan et al.: molecular characterization … 72 http://jad.tums.ac.ir published online: march 14, 2017 which has provided good opportunity to compare our data with other trichopteran species. nuclear ribosomal dna belongs to a multigene family, where hundreds to thousands of copies of the nrdna unit appear in tandem along the chromosome. although individual fragments of the rdna did not support well the topology of branches and clades in the trees, however, combination of three parts of the gene revealed the highest bootstrap values for the constructed trees. the combination of the three fragments (d1-d3) revealed 73% support value for association of h. sciligra with h. longifurca and h. naumanni. however, the limited number of trichopteran species (n=11) involved in the study may decline power of this analysis. between the coi and individual lsu fragments, d2 fragment strongly supported the monophyly of most hydropsyche species. the d2 expansion fragment of 28s ribosomal rna (rrna) is one of the most highly variable regions in eukaryote rrna. the length and nucleotide composition of this fragment is highly variable among insects (gillespie et al. 2004). these significant variations limited the utility of d2 in deep-level phylogeny because of difficulties in alignment, although universally conserved rna secondary structures have provided solutions for some taxa (gillespie et al. 2004). conclusion many areas in iran have not been or poorly investigated for caddisfly fauna. hence, a large-scale zoogeographic study using morphological and molecular characters comprising mitochondrial and nuclear markers together with population level sampling of all nominal taxa of trichopteran in poorly investigated areas of the country is highly suggested. these studies will revise and 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supella longipalpa (blattodea: blattellidae) hadis mosayebian, *hamid reza basseri, mojgan baniardalani, yavar rassi, hossein ladonni department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 25 oct 2014; accepted 3 aug 2016) abstract background: the brown-banded cockroach, supella longipalpa, is not as common as the german cockroach in iran. this species seeks out areas that are very warm most of the time, and prefer warmer area than what german cockroaches prefer. there is relationship between development of instars and diet of cockroaches. the aim of this study was to determine the effect of different diets on biology, life cycle on nymphal stages of s. longipalpa in laboratory condition prior to investigate the insecticide resistance status of this species in residence area in iran. methods: the cockroaches were reared in the insectary of school of public health, tehran university of medical sciences, condition and the population divided in four equal groups. the effect of four different diets on life cycle of s. longipalpa was studied to determine the effect of them on the lifetime of each nymphal stage. results: the diets significantly affected on growth and development of immature life stages of s. longipalpa. based on introduced diets to the cockroache populations, total immature life cycle was 54, 58, 60 and 66 d for diets 2,4,1, and 3 relatively. however, the overall lifetime of s. longipalpa in average was about 225 days. conclusion: as far as urban pest control is concerned, the result of this study will facilitate any operational programs for control of s. longipalpa. among the different tested diets, diets 2 and 4 with less duration would be recommended for rearing of s. longipalpa in laboratory condition. keywords: supella longipalpa, lifetime, diet, iran introduction about 3,500 species of cockroaches exist worldwide (world health organization 1999). during recent years brown-banded as a serious pest has been collected in different residential area in tehran (personal communication, department of medical entomology, school of public health, tehran university of medical sciences). the brown-banded cockroach is an important pest species with virtually little known of the interaction between feeding, diet composition, and reproduction and its insecticide resistance spectrum as well. like german cockroach, the brown-banded cockroach is small and must feed prior to oothecal production. however, unlike german cockroach (blattella germanica) females, which carry the ootheca for a protracted period, brownbanded cockroaches (supella longipala) females oviposit new ootheca every several days. therefore, the effect of nutrient limitation should become evident much sooner in this species than in b. germanica. brown-banded cockroaches build up their highest populations in high temperature areas. they do not need as much water as german cockroach, so they often live in locations, which are drier. they are often found in locations at eye-level or above (hamilton et al. 1988). mating in female german cockroach is inhibited during starvation (roth and stay 1962) and delayed when they are fed with a stressfully high protein diet (hamilton and schal 1988). in the absence of an *corresponding author: dr hamid reza basseri, e-mail: basserih@tums.ac.ir j arthropod-borne dis, june 2017, 11(2): 302–308 h mosayebian et al.: effect of different … 303 http://jad.tums.ac.ir published online: may 27, 2017 adequate food supply, females either delay reproduction (kunkel 1966, durbin and cochran 1985) or produce fewer and smaller ootheca (mueller 1978). basic knowledge and understanding of the life cycle, behavior and habitat of brownbanded cockroaches are essential, before conducting any operational programs for any pest control strategy in infested area. though brown-banded cockroach is going to be more popular as the main urban pest, still there is poor evidence regarding to relationship between development of instars and diet of s. longipalpa. the aim of this study was to find out the influence of different diets with carbohydrates based on some biological features such as survival and longevity of s. longipalpa in the laboratory condition. materials and methods cockroach populations brown banded cockroaches were collected from an infested area in tehran, iran. all cockroaches were maintained in an insectary of school of public health, tehran university of medical sciences at 27±2 ºc, 60±10 % rh, with a photoperiod of 12: 12h (l: d). each population of cockroach was reared in the same size labeled glass jar. the cockroaches were provided with four different diets and water. experimental design and diets in order to avoid any heterogeneity in population, the trial was conducted on 2nd generation of brown-banded cockroaches. newly emerged unfed nymphs (aged 3+5 days) of cockroaches were randomly allocated to each type of diets. the effect of each type of food staffs e.g. milk, date, and both (date+milk) on longevity of immature stage of cockroaches were studied in the present of normal diet (dry bread and dry pellets of rabbit food, bran and water), i.e. the diet, regularly was used for rearing and maintaining the cockroach colonies in our laboratory. the diets used in this study were: 1) normal diet, 2) diet 1 + date, 3) diet1 + milk, and 4) diet 1 + date and milk. each group of insects consisted of 10 cockroaches placed in a plastic buckle (3 liters capacity) and then the insects were allowed to feed based on the selected diets (as above) available in a plastic petri dish. each test was consisted of three replications. statistical analysis kaplan-meier statistical test (particular statistical test, recommended for survival test in biology) and one way analyzes of variance (anova) were used to find out how the effect of different diets on life time of nymphal stages of s. longipalpa varies, using spss statistical package 18 (chicago, il, usa). the earlier statistical methods have been selected based on experimental design, diets, and variables applied in this study. results effect of diets on nymphal stages the diets significantly affected on growth and development of immature life stages of s. longipalpa (kaplan-meier statistical test, p< 0.05). based on introduced diets to the cockroach populations, total immature life cycle was 54, 58, 60 and 66 d for diets 2,4,1, and 3, respectively. s. longipalpa had 4 distinct nymphal stages for immature stage prior to adult stag, i.e. overall, 5 stages including adult. the total nymphal stages were completed within 59.1 d±0.32 (days ±s.e) for diet 1. the result for each nymphal stage 1, 2, 3 and 4 stages was 13.2±0.18, 15.03±0.37, 18.87±0.46 and 12±0.27days, respectively. the group of cockroaches, which used diet 2, completed all nymphal stages within 54.07±0.27 days. in addition, duration of each nymphal stage 1, 2, 3 and 4 were 12.77±0.16, 13.13±0.24, j arthropod-borne dis, june 2017, 11(2): 302–308 h mosayebian et al.: effect of different … 304 http://jad.tums.ac.ir published online: may 27, 2017 17.77±0.39 and 10.40±0.25 days respectively. the insects which fed on diet 3, completed the total nymphal stages within 66.43±0.45 days and duration of nymphs for each stage of 1, 2, 3 and 4 was 14.03±0.15, 15.63±0.28, 21.40±0.84 and 15.37±0.55 respectively. the cockroaches which diet 4 was provided for them, completed whole nymphal stage within 57.99±0.3 days. the duration of development for each stage of 1, 2, 3 and 4 was 13.03± 0.18, 14.7±0.26, 18.53±0.51 and 11.73±0.27 respectively (table 1). generally, the shortest stage occurred among forth instar stage, which the longest belonged to third instars (fig. 1). to compare the duration of first instar development, those insects, which fed on diet 2, had shortest lifetime while diet 3 caused longest durations among four groups. the maximum stage occurred in third instar nymph, which fed with diet 3 (fig. 1). the cumulated survival of nymphs moulted from each stage to next based on diet per time is presented in fig. 2–5. the number of nymphs moved to next stage was varied within stages and between four diets. although the fist instar of insect group, which fed on diet 1 started moulting earlier, the group, which had diet 2, completed first instar development in a shorter time. in addition, diet 3 cause delays in the moulting of 1st instar of cockroach (fig. 2). diet 2 accelerated movement of 2nd instar to next stage faster comparing with others. the duration rage for completing 2nd instar in this group occurred between 11 to 16 days while the insects used diet 1 stated moulting from 13th and finished on 20th (fig. 3). generally, duration of 3rd instar was longer than other stages in four groups. although four types of the diet accelerated moulting of 3rd instar to next stage within nearly same time, the diet 3 showed delay in development of this stage longer than other groups and the last cockroaches moved to 4th instar at day 30th (fig. 4). the duration 4th instar development also showed variation while the diet 2 started to move to adult stage faster and the number of insect, which completed this stage was shorter with diet 2 as well. in contrary, the cockroaches treated with diet 3 started moulting later and the duration of 4th instar was obviously longer comparing with other groups (fig. 5). fig. 1. the effect of different diets on lifetime of nymphal stages of supella longipalpa in laboratory (anova test). the bars represent the mean value days of development of nymphal stage from three replicates where the standard errors are included. n 1, n2, n3, n4 represent 1st, 2nd, 3rd and 4th nymphal stage supella longipalpa (f.) and d1, d2, d3and d4 represent the type of diets provided for each group of cockroaches j arthropod-borne dis, june 2017, 11(2): 302–308 h mosayebian et al.: effect of different … 305 http://jad.tums.ac.ir published online: may 27, 2017 d1: normal diet (dry bread-dry pellets of rabbit food, bran and water), d2: diet 1+ date, d3: diet 1+milk, d4: diet 1+ (date and milk) respectively. table 1. the effect of different diets on lifetime of nymphal stages of supella longipalpa in laboratory (kaplan meier test) diets days d1 d2 d3 d4 n1 13.20±0.18 1* 12.77±o.16 1* 14.30±0.15 1* 13.03±0.18 1* n2 15.03±0.37 2* 13.13±0.24 2* 15.63±0.28 2* 14.70±0.26 2* n3 18.87±0.46 3* 17.77±0.39 3* 21.40±0.84 3* 18.53±0.51 3* n4 12±0.27 4* 10.40±0.25 4* 15.37±0.55 4* 11.73±0.27 4* n 1, n2, n3, n4 represent 1st, 2nd, 3rd and 4th nymphal stage of supella longipalpa. d1, d2, d3and d4 represent the type of diets, d1: normal diet (dry bread-dry pellets of rabbit food, bran and water), d2: diet 1 + date. d3: diet 1+ milk, d4: diet 1+ (date and milk) and respectively (p< 0.05). the numbers represent the mean value days of development of nymphal stage from three replicates where the standard errors are included. the number of insects in each group of replication was 10 cockroaches (n= 10). (*): represent the significant differences between nymphal stages and different diets using kaplan meier statistical tes t< 0.05. fig. 2. effect of different diets on 1st nymphal stage of supella longipalpa (kaplan meier, survival test) n1: 1st nymphal stage d1, d2, d3 and d4 represent the type of diets, d1: normal diet (dry bread-dry pellets of rabbit food, bran and water), d2: diet 1 + date, d3: diet 1+milk, d4: diet 1 + date and milk and the nypmphal stages respectively. the number of insects in each group of replication was 10 cockroaches (n= 10) fig. 3. effect of different diets on 2nd nymphal stage of supella longipalpa (kaplan meier, survival test) n2: 2nd nymphal stage. d1, d2, d3and d4 represent the type of diets, d1: normal diet (dry bread-dry pellets of rabbit food, bran and water), j arthropod-borne dis, june 2017, 11(2): 302–308 h mosayebian et al.: effect of different … 306 http://jad.tums.ac.ir published online: may 27, 2017 d2: diet 1 + date, d3: diet 1 + milk, d4: diet 1+date and milk and the nypmphal stages respectively. the number of insects in each group of replication was 10 cockroaches (n= 10) fig. 4. effect of different diets on 3rd nymphal stage of supella longipalpa (kaplan meier, survival test). d1, d2, d3and d4 represent the type of diets, d1: normal diet (dry bread-dry pellets of rabbit food, bran and water), d2: diet 1 + date, d3: diet 1+ milk, d4: diet 1 + date and milk and the nypmphal stages respectively. the number of insects in each group of replication was 10 cockroaches (n = 10) n3: 3rd nymphal stage. fig. 5. effect of different diets on 4th nymphal stage of supella longipalpa (kaplan meier, survival test) d1, d2, d3 and d4 represent the type of diets, d1: normal diet (dry bread-dry pellets of rabbit food, bran and water), d2: diet 1 + date, d3: diet 1 + milk, d4: diet 1 + date and milk and the nypmphal stages respectively. the number of insects in each group of replication was 10 cockroaches (n = 10). n4: 4th: nymphal stage discussion we found that modification of diet can significantly effect on duration of nymphal development of s. longipalpa. diet is one of the most crucial factor to rear or colonizing insects in laboratory condition. generally, feeding insect with appropriate diet can reduce the duration of instar development to minimum time and optimize the reproductions. four diets were introduced to immature stages of s. longipalpa with types of diets based on the longevity of nymphal stages. the shorter life time/day indicating the appropriated diet was given to the insects. colonies of cockroach facilitate field of researches on pest control such as bioassay tests and evaluation of insecticides. in terms of cockroach and an emphasis on ivm control, comprehensive studies have been carried out on different aspects of german cockroach b. germanica (l.) (cochroan 1986, ladonni et al. 1988, ladonni 2000, ladonni 2001). inheritance and resistance spectrum, molecular basis of kdr resistance of german cockroach to four main groups of insecticides have been widely studied on different iranian strains of german cockroach (ladonni et al. 1988, limoee et al. 2007, limoee et al. 2011). there are some evidences indicating that s. longipalpa has been substitution with german cockroach (blattella germanica) in some cities of iran. therefore, s. longipalpa is going to be more considered as an urban pest in many cities while j arthropod-borne dis, june 2017, 11(2): 302–308 h mosayebian et al.: effect of different … 307 http://jad.tums.ac.ir published online: may 27, 2017 poor knowledge are available about biology and ecology if this species. studies with german cockroach have revealed an intimate association between the availability of food and water and reproductive success (roth and stay 1962, kunkel 1966, mueller 1978). food and water consumption are cyclical and closely related to the reproduction cycle in german and american cockroaches (bell 1969, cochran 1983, rollo 1984, durbin and cochran 1985, hamilton and schal 1988). however, little studies have been carried out on resting and feeding behavior of s. longipalpa. influence of dietary protein on food intake and reproduction of female german cockroach was studied by hamilton and schal (1988), so adult performance was directly influenced by deity protein level. females which had a diet with 65% protein, died rapidly while the females fed on nutrient which contained 5% protein, not only their reproductive rate reduced but also the size of oothecaes were smaller. our results indicated that food staffs based on hydrocarbon compounds has a great effect on development of immature stage. the results of cumulated survival analyses on nymphs’ development indicated that passing nymphes from one stage to next was varied based on the type of diet. this variation could be due to carbohydrate food available particularly those insects, which accessed to date. it seems that date as supplementary food could accelerate the nymphs of cockroaches to complete each stage faster. date contain high amount simple carbohydrates that insects can quickly digests and turns into energy. therefore, this study recommends date as supplementary food for feeding cockroaches particularly for rearing s. longipalpa in laboratory condition. conclusion as far as host pest control is concerned, colonizing pest at optimum condition is essential for pesticides evaluating and understanding of other aspects of biology and ecology of pest such as s. longipalpa. accordingly, the optimum diets for accelerating moulting of brown-banded instars are diets 2 and 4. acknowledgements this study was supported by as a grant from tehran university of medical sciences (tums) (grant no. 22817-27-02-92). the research was a part of msph thesis at school of public health, tehran university of medical sciences. the authors declare that there is no conflict of interest. references bell wj (1969) continuous and rhythmic reproductive cycle observed in periplaneta americana (l.). biol bull mar biol lab, woods hole. 137: 239–249. cochran dg (1983) food and water consumption during the reproductive cycle of female german cockroaches. ent exp app. 34: 51–57. durbin ej, cochran dg (1985) food and water deprivation effects on reproduction in female blattella germanica. ent exp appl. 37: 77–82. hamilton rl, schal c (1988) effects of dietary protein levels on reproduction and food consumption in the german cockroach (dictyoptera: blattellidae). ann entomol soc am. 81: 969–976. kunkel jg (1966) development and the availability of food in the german cockroach, blattella germanica (l). j insect physiol. 12: 227–235. ladonni h (2001) evaluation of three methods for detecting permethrin resistance in adult and nymphal blattella germanica (dictyoptera: blattellidae). j economic entomol. 94(3): 694–697. j arthropod-borne dis, june 2017, 11(2): 302–308 h mosayebian et al.: effect of different … 308 http://jad.tums.ac.ir published online: may 27, 2017 ladonni h (2000) permethrin resistance ratios compared by two methods of testing nymphs of the german cockroach, blattella germanica. med vet entomol. 14(2): 213–216. ladonni h, aboulhasani m, shaeghi m (1988) susceptibility of first nymphal stage of different strains of blattella germanica l. (dictyoptera: blattellidae) to diazinon and propoxur, using insecticide impregnated paper. j entomol soc iran 16–17. pp. 23–30. limoee m, enayati aa, khassi k, salimi m, ladonni h (2011) insecticide resistance and synergism of three field collected strains of the german cockroach blattella germanica (l.) (dictyoptera: blattellidae) from hospitals in kermanshah, iran. j tropical biomed. 28(1): 111–118. limoee m, enayati aa, ladonni h, vatandoost h, baseri h, oshaghi ma (2007) various mechanisms responsible for permethrin metabolic resistance in seven field-collected strains of the german cockroach from iran, blattella germanica (l.) (dictyoptera: blattellidae). pestic biochem phys. 87(2): 138–146. mueller p (1978) deprivation on the development of laboratory colonies of the german cockroach. z gest hyg gren. 24: 122–1261. rollo cd (1984) resource allocation and time budgeting in adults of the cockroach interaction of behavior and meta-bolic reserves. res popul ecol. 26: 150–187. roth lm, stay b (1962) oocyte development in blattella germanica (l.) and blattella vaga herberd. ann entomol soc am. 55: 633–642. world health organization (1999) cockroach, their biology, distribution and control, who/cpc/whopes/99.3. geneva, p. 96. microsoft word 5-rassi_rtl.doc iranian j arthropod-borne dis, (2007), 1(1): 38-42 y rassi et al.: the seminested pcr based… 38 original article the seminested pcr based detection of leishmania infantum infection in asymptomatic dogs in a new endemic focus of visceral leishmaniasis in iran *y rassi 1, k azizi 1, mh motazedian 2, e javadian 1, s rafizadeh 3, m fakhar 2, gr hatam 2 1dept. of medical entomology and vector control, school of public health and institute of health research, medical sciences/tehran university, tehran, iran 2dept. of medical parasitology, shiraz university of medical sciences, shiraz, iran ³ center of disease control (cdc), genetic office, tehran, iran (received 25 oct 2006; accepted 11 apr 2007) abstract visceral leishmaniasis (kala-azar) is a serious health problem in some northern and south western parts of iran. the incidence of kala-azar caused by leishmania infantum has recently increased in nourabad-mamassani district of fars province, in the south of the country. this study was designed to determine the role of asymptomatic dogs as host reservoir of l. infantum in this new formed focus and detection of prevalence of infection near them. a total of 20 asymptomatic stray and sheep dogs were randomly sampled. the buffy coat layer of their peripheral blood was used for dna extraction and pcr. a species specific seminested pcr was used for dna amplification using linr4, lin17 and lin19 primers. these primers amplified variable area of the minicircle kdna of leishmania parasites. of the 20 sampled dogs checked for leishmanial kdna, six (30%) were found naturally infected. it is concluded that, dogs (canis familiaris) even if asymptomatic, is considered as the domestic host reservoir of kala-azar in this endemic focus. key words: leishmania infantum, dogs, iran introduction the leishmaniasis is parasitic diseases widespread in the old and new world with great epidemiological diversity. they are caused by about 20 species of leishmania, protozoa transmitted by the bite of female sandflies (diptera: psychodidae). visceral leishmaniasis which its causative agent is leishmania infantum (syn. l. chagasi) is a severe, often fatal disease common in the mediterranean region and in latin america (peters 1987). in the mediterranean region, domestic dogs (canis familiaris) are the main domestic reservoir hosts and sandflies be-longing to the subgenus larroussius of the genus phlebotomus are the principal vectors (who 1990). in iran, there are currently two main endemic foci of kala-azar caused by l. infantum: one in the north-west and the other, within fars province, in the south (edrissian 1999). dogs, red foxes (vulpes vulpes), golden jackals (canis aureus) and wolves (canis lupus) are thought to be the reservoir hosts (hamidi 1982, edrissian 1993, mohebali 2005). besides, l. infantum has been isolated from some rodents such as meriones persicus and mesocricetus auratus and these animals are considered as secondary reservoirs of kala-azar in iran (mohebali 1998). two sandfly species (phlebotomus alexandri and ph. kandelakii) have been recently introduced as the proven vector of disease in south and northwest of iran, respectively (azizi 2006, rassi 2005). in the last few years the incidence of vl in mahoormilaty district, nourabad-mamassani county *corresponding author: dr y rassi, tel: +98 21 88951393, e-mail: rassiy@sina.tums.ac.ir iranian j arthropod-borne dis, (2007), 1(1): 38-42 y rassi et al.: the seminested pcr based… 39 which lies, at about 50◦ 35'e, 29◦ 51' n and 1500 m above sea level, in fars province (southern iran), has suddenly increased. in 2003 there were eight cases of the disease (all of them aged< 10 yr) in villages that had a combined population of only about 4000 person (unpublished data). the main aim of the present study was to determine the prevalence of leishmanial infection in dogs and investigation on the role of asymptomatic ones in being host reservoir of vl in this new focus. materials and methods sampling the study was carried out in the mahoormilaty district, nourabad-mamassani county in fars province, southwest iran. peripheral blood samples were collected, randomly from 20 asymptomatic stray and owner dogs (about 20% of dogs population in the study area). the buffy coat layer was separated from whole blood after being centrifuged at 2500 rpm for 10 min. dna extraction a 5µl of each sample was used for dna extraction. dna was extracted as described elsewhere (motazedian 2002). briefly, 100µl of lysis buffer [50mm tris-hcl (ph 7.6), 1mm edta and 1% tween20] and 12µl of a proteinase k solution (containing 19 µg of the enzyme/ml) were added, in a 1.5ml microcentrifuge tube. the homogenate was then incubated at 37 °c overnight before 300 µl of a phenol: chloroform: isoamyl alcohol mixture (25:24:1, by vol.) were added. after being shaken vigorously, the tube holding the mixture was centrifuged (10000 g for 10min) and then the dna in the supernatant solution was precipitated with 400 µl cold, pure ethanol, resuspended in 50µl double-distilled water, and then stored at -20 °c until it could be tested for leishmanial kdna. pcr an assay based on the seminested pcr was used for amplification of variable area of the minicircle kdna (with a slight modification) as described elsewhere (aransay 2000). the combination of primers linr4 (forward), lin17 (reverse) and lin19 (reverse) was used in a seminested pcr technique (table 1). these primers were designed within the conserved area of the minicircle and contained conserved sequence blocks (csb), csb3, csb2 and csb1, respectively (brewster 1998). the first amplification reaction was carried out in a total of 25µl contained 250µm of each deoxynucleoside triphosphate (dntps), 1.5mm mgcl2, 1u taq polymerase (cinagene, tehran), 1µm linr4, 1µm lin17 and 5µl of dna extract, in 1x pcr buffer (boehringer mannheim, mannheim, germany), overlaid with mineral oil. the mixture was incubated in a cg1-96 thermocycler (corbett research, sydney, australia) at 94 ºc for 5 min followed by 30 cycles, each consisting of 30s at 94 ºc, 30s at 52 ºc and 1 min at 72 ºc. after the last cycle, the extension was continued for a further 5 min. the second round was carried out, with the addition of a 40 µl solution containing buffer, mgcl2, dntps and taq polymerase as described above for the first round and 1µm lin19 primer for 33 cycles (94 ºc for 30s, 58 ºc for 30s and 72 ºc for 1min) and final extension at 72 ºc for 10 min. a 5µl sample of each pcr product was resolved in a 1.5% agarose gel. the bands were then stained with ethidium bromide and visualized under ultra violet trans-illuminator. results a total of 20 collars of asymptomatic stray and owner dogs were sampled. infection to leishmania kdna was observed in 6 (30%) cases. species specific primers identified the parasites as l. infantum, the causative agent of kala-azar. parasites were identified by comparing the size of the band produced from a test sample with those produced from the reference strains. reference strains of l. infantum (mcan/ ir/96/lon49), l. tropica (mhom/ir/89/ard2) and l. major (mhom/ir/54/lv39) were used as standards. bands of 720, 760 and 560bp, iniranian j arthropod-borne dis, (2007), 1(1): 38-42 y rassi et al.: the seminested pcr based… 40 dicated the above standards, respectively. the visualized obtained bands of positive samples were similar to standard l. infantum, which was equal to 720bp (fig. 1). table 1. sequences of the primers used in this study primer sequence linr4 (forward) 5'ggggttggtgtaaaataggg-3' lin17 (reverse for 1st round) 5'tttgaacgggatttctg -3' lin19 (reverse for 2nd round) 5'cagaacgcccctacccg -3' fig. 1. the results of seminested pcr based of dna extracted from buffy coat of whole blood of dogs. the bands shown on 1.5% agarose gel stained with ethidium bromide, correspond to molecular weight markers (lanes 1and 8), reference strains of leishmania tropica (lane 2), l. major (lane3), l. infantum (lane 4), blank (lane 5), two infected dog samples (lanes 6 and 7). discussion a thorough knowledge of leishmania ecology and epidemiology is required for control programs of leishmaniasis especially in endemic areas. identification of reservoir hosts as well as detection of probable or proven vectors are the main problems which epidemiologists faced to. in many areas, however, despite considerable research on vl, the main reservoir hosts and the species of sandfly responsible for most transmission have still to be identified. l. infantum infections are responsible for vl in at least 70 countries. in most endemic areas it is widely believed that domestic dogs are the principal hosts. the main evidences are as follows: isolated parasites from dogs are indistinguishable from those in humans; relatively high infection rates in dogs; positive correlation between dogs and human prevalence, and occurrence of asymptomatic infected dogs (mazloumi gavgani 2002). characterization of parasites in the past was mainly based on the clinical manifestations, geographical foci of distribution of the disease in humans and biological characteristics of the parasites in laboratory animals; however more recently new methods such as isoenzymes and pcr based techniques have been used for characterization (ardehali 2000). the high sensitive technique of pcr has been used formerly for detection of leishmania in sandflies (aransay 2000, kato 2005, rassi 2005, azizi 2006) and reservoirs (le fichoux 1999, lachaud 2002). aransay et al. in 2000, used the linr4, lin17 and lin19 primers (which have been used in this study) for detection of leishmania infection in sandflies of greece (aransay 2000). our results showed that specific pcr on buffy coat was a sensitive and suitable method for detection of leishmania infections. working with peripheral blood is noninvasive, straightforward and easy to repeat which easily can be used for owner dogs (lachaud 2002). in iran, there are many evidence, for natural leishmania infections in domestic dogs, golden jackals and red foxes (hamidi 1982, edrissian 1993, mohebali 2005) and some rodents (mohebali 1998). the infection rate of dogs in other well-known endemic foci of disease of country, mainly based on serological dat method, is lower than our finding (nadim 1978, edrissian 1993, mohebali 2001).according to our results, consist of high infection rate of dogs to l. infantum (30%), this animal is introduced as the iranian j arthropod-borne dis, (2007), 1(1): 38-42 y rassi et al.: the seminested pcr based… 41 main reservoir host of kala-azar in mahoor-milaty district, noorabad-mamassani county of fars province in south west of iran and asymptomatic infected dogs should be considered as an important reservoir of infection for vectors. undoubtedly, this region can be considered as a new formed endemic focus of kala-azar in iran. recently, we found ph. alexandri (azizi 2006) and fox [unpublished data] infected with l. infantum in this area. the importance of the current method for detection of infectivity in asymptomatic dogs is apparent. our finding could provide a clue for planning of disease control in the endemic areas by the authorities. acknowledgements the authors would like to thank the school of public health and institute of health research, tehran university of medical sciences, for their financial support. they are also grateful to. dr m mohebali for his kind helps, gh asgari and d mehrabani for their assistance in the sampling and m kalantari for helping with molecular assays. references aransay am, scoulica e, tselentis y (2000) detection and identification of leishmania dna within naturally infected sandflies by semi-nested pcr on minicircle kinetoplast dna. applied environ microbiol. 66: 1933-1938. ardehali s (2000) characterization of leishmania isolated in iran: serotyping with species specific monoclonal antibodies. acta trop. 75: 301-307. azizi k, rassi y, javadian e, motazedian mh, rafizadeh s, yaghoobi ershadi mr, mohebali m (2006) phlebotomus (paraphlebotomus) alexandri: a probable vector of leishmania infantum in iran. ann trop med parasitol. 100(1): 63-68. edrissian gh, nadim a, alborzi av, ardehali s (1999) visceral leishmaniasis: the iranian experiences. arch iran med. 1:22-26. edrissian gh, ahanchin ar, gharachahi am (1993) seroepidemiological studies of visceral leishmaniasis and search for animal reservoirs in fars province, southern iran. iranian j med sci. 18: 99-105. brewster s, aslett m, barker dc (1998) kinetoplast dna minicircle database. parasitol today. 14: 437-438. hamidi an, nadim a, edrissian gh, tahvildari-bidrouni g, javadian e (1982) visceral leishmaniasis of jackals and dogs in northern iran. trans roy soc trop med hyg. 76: 756-757. kato h, uezato h, katakura k, marco m, barroso j, gomez p, mimori e, korenaga t, iwata m, nonaka h, hashiguchi y (2005) detection and identification of leishmania species within naturally infected sandflies in the andean areas of ecuador by a polymerase chain reaction. am j trop med hyg. 72(1): 87-93. lachaud l, marchergui-hammami s, chabbert e, dereure j, dedet j, bastien p (2002) comparison of six methods using peripheral blood for detection of canine visceral leishmaniasis. j clinic microbiol. 40(1): 210-215. le fichoux y, quaranta j, aufeuvre j, lelievre a, marty p, suffia i, rousseau d, kubar j (1999) occurrence of leishmania infantum parasitemia in asymptomatic blood donors living in an area of endimicity in southern france. j clinic microbiol. 37(6): 1953-1957. mazloumi gavgani s, mohite h, edrissian gh, mohebali m, davies c (2002) domestic dog ownership in iran is a risk factor for human infection with leishmania infantum. am j trop med hyg. 67(5): 511-515. mohebali m, hajjaran h, hamzavi y, mobedi i, arshi s, zarei z, akhoundi b, manouchehri k, avizeh r, fakhar m (2005) epidemiological aspects of canine visceral iranian j arthropod-borne dis, (2007), 1(1): 38-42 y rassi et al.: the seminested pcr based… 42 leishmaniasis in the islamic republic of iran. vet parasitol. 129: 243-251. mohebali m, hamzavi y, edrissian gh, forouzani a (2001) seroepidemiological study of visceral leishmaniasis among humans and animal reservoirs in bushehr province, islamic republic of iran. east mediterr health j. 7: 912-917. mohebali m, poormohammadi b, kanani a, hajjaran h, edrissian gh (1998) rodents: another group of animal hosts of visceral leishmaniasis in meshkin-shahr district, islamic republic of iran. iranian j publ health. 4(2): 376-378. motazedian mh, karamian m, noyes ha, ardehali s (2002) dna extraction and amplification of leishmania from archieved, giemsa stained slides for the diagnosis of cutaneous leishmaniasis by pcr. ann trop med parasitol. 96: 31-34. nadim a, hamidi n, javadian e, tahvildari bidrouni e, amini h (1978) present status of kala-azar in iran. am j trop med hyg. 27(1): 25-28. peters w, killick-kendrick r (1987). leishmaniasis in biology and medicine. academic press, new york. rassi y, javadian e, nadim a, zahraii a, vatandoost h, motazedian mh, azizi k, mohebali m (2005) phlebotomus (larroussius) kandelakii the principal and proven vector of visceral leishmaniasis in north west of iran. pak j biol sci. 8(12): 1802-1806. world health organization (1990) control of the leishmaniasis. report of a who expert committee. technical report series no. 793. who, geneva. j arthropod-borne dis, june 2017, 11(2): 242–248 n sharifinia et al.: fauna and geographical … 242 http://jad.tums.ac.ir published online: may 27, 2017 original article fauna and geographical distribution of scorpions in ilam province, south western iran narges sharifinia 1, iman gowhari 2, manijeh hoseiny-rad 3, *ali ashraf aivazi 1 1department of public health, school of health, ilam university of medical sciences, ilam, iran 2department of biology, payam-e-noor university, ilam branch, ilam, iran 3department of biology, farhangian university, tehran, iran (received 28 apr 2015; accepted 14 may 2016) abstract background: scorpions’ stings and their own mortalities place them among the most important health and medical problems. the dreadful features and especially their poisonous stings are considered a major cause of human stress and abhorrence/phobia. the current study aimed to study the scorpion fauna of ilam province, south western iran in order to manage scorpionism related problems. methods: in this field-laboratory investigation during march 2014 to february 2015, different parts of ilam province were surveyed. nine sampling parts were selected based on geographical situation, scorpionism reports, weather, flora, and local data. capturing scorpion was done employing a black light, and a long forceps from dusk to midnight. the collected scorpions were placed to 70% ethyl alcohol. all specimens were determined based on the valid taxonomic keys, furthermore their sexes were studied. results: out of the 391 collected scorpions, 11 species were identified as follows: hottentotta saulcyi, mesobuthus eupeus, compsobuthus matthiesseni, razianus zarudnyi, hemiscorpius lepturus, androctonus crassicauda, orthochirus iranus, odontobuthus bidentatus, buthacus macrocentrus, scorpio maurus, and polisius persicus. conclusion: eleven species of buthidae, scorpionidae and hemiscorpiidae families from high risk areas were identified. despite the low surface of the province, such different species reveals a diverse scorpion fauna that, in turn, shows good and suitable habits of scorpions, as considered by health staff. keywords: habitat, scorpion, fauna, ecology, iran introduction the fearful feature and painful poisonous stings of scorpions have caused human phobia for a long time. most people think of scorpions as pests and killers of man (polis 1990). scorpions having diverse distribution are mostly living in semi-temperate regions at latitude of 23–38 °c, while their abundance and diversity toward equator and also poles decrease (polis 1990). out of about 1,500 scorpion species in the world, few cause severe toxicity, including more than 1.23 million stings annually, of which approximately 3,250 (0.27%) cause death (khatony et al. 2015). mexico, colombia, and iran are the most affected countries. out of about 100,000 scorpionism cases including children (75%) in iran, only 36,000– 50,000 ones are reported officially, with a 7– 60 mortality rate per year (ghaderi 2004, zarei et al. 2009, mirshamsi et al. 2011), especially in khuzestan, bushehr and ilam provinces (south-western iran) (rafizadeh et al. 2013). the iranian scorpion fauna ranges from 51– 66 species in 17–23 genera and 3–4 families, according to different references (mirshamsi et al. 2011, navidpour 2012), of which about 10 species have been incriminated in human envenomation, that, in turn, is more than any other country in middle east (dehghani and fathi 2012). except hemiscorpius lepturus (hemiscorpiidae), the most important, medically, scorpion in iran (kovařík 1997), all the *corresponding author: dr ali-ashraf aivazi, email: ali.medilam@gmail.com j arthropod-borne dis, june 2017, 11(2): 242–248 n sharifinia et al.: fauna and geographical … 243 http://jad.tums.ac.ir published online: may 27, 2017 venomous scorpion species belong to the buthidae family (zarei et al. 2009). ilam province (latitude: 33° 63’ 74” n, longitude: 46° 42’ 27” e) located at west of iran with around 20,150km2 and 1.2% surface of the country area, rich and diverse plant coverage, and also various climates, is of the most suitable living-places for scorpions. the current study aimed at determining fauna and bio-geographical distribution of scorpions in ilam in 2014–15. materials and methods in this cross-sectional study of employing field and laboratory techniques, the fauna was investigated during march 2014 to february 2015. the sampling sites (nine places) were selected based on geographical situation, scorpionism reports, weather, and plant coverage in all the three climates of the province. the detailed data of sampling sites have been shown in table 1 and fig. 1. the sampling was done at night (from dusk to midnight) using a black light -ultra violet (uv) lightand a long forceps. all the captured scorpions were placed in 70% ethyl alcohol. no specimens were caught by abovementioned method during november to february months, due to cold weather of sampling areas. the geographical data were recorded by a gps apparatus (garmin 78 s). all the specimens were identified according to taxonomic keys (navidpour et al. 2008a). the gender of specimens was also determined based on farzanpey method (farzanpay and vachon 1979). results out of all the 391 specimens collected from nine sites in different parts of the province, 11 species were identified as follows: hottentotta saulcyi, mesobuthus eupeus, compsobuthus matthiesseni, razianus zarudnyi, hemiscorpius lepturus, androctonus crassicauda, orthochirus iranus, odontobuthus bidentatus, buthacus macrocentrus, scorpio maurus, and polisius persicus. the most abundant species were h. saulcyi 25.09% (relative frequency), m. eupeus 23.29%, and c. matthiesseni 16.18% which showed the highest frequency in all the three climates studied. the lowest abundance stood for p. persicus 1.79% and s. maurus 1.29%, respectively. h. lepturus which is the most poisonous scorpion of iran had a 7.16% relative frequency. further, r. zarudnyi was found just in mild mountainous climate, however, o. bidentatus 3.58% and b. macrocentrus 2.84% were found in low abundance at dryhot climate. totally, 138 males and 253 females were identified showing f/m sex ratio of about 2:1 (table 2). table 1. sampling sites of scorpions in ilam province, 2014 climate type county sampling site longitude altitude height (sea level m) cold mountainous aivan babagir 46 ْ◌ 11 َ◌ 33 ْ◌ 56 َ◌ 1,070 cold mountainous ilam gholandar 46 ْ◌ 27 َ◌ 33 ْ◌ 39 َ◌ 1,045 cold mountainous sirvan karezan 46 ْ◌ 32 َ◌ 33 ْ◌ 44 َ◌ 1,280 moderate mountainous badre badre 47 ْ◌ 1 َ◌ 33 ْ◌ 19 َ◌ 1,090 moderate mountainous dare-shahr dare-shahr 47 ْ◌ 21 َ◌ 33 ْ◌ 10 َ◌ 670 moderate mountainous zarin-abad sayed-naseredin village 46 ْ◌ 50 َ◌ 33 ْ◌ 10 َ◌ 810 dry and hot abdanan murmuri 47 ْ◌ 41 َ◌ 32 ْ◌ 44 َ◌ 520 dry and hot mehran golan 46 ْ◌ 16 َ◌ 33 ْ◌ 25 َ◌ 550 dry and hot dehloran bishe-deraz 47 ْ◌ 01 َ◌ 32 ْ◌ 46 َ◌ 390 j arthropod-borne dis, june 2017, 11(2): 242–248 n sharifinia et al.: fauna and geographical … 244 http://jad.tums.ac.ir published online: may 27, 2017 fig. 1. sampling sites of scorpions in ilam province, 2014 j arthropod-borne dis, june 2017, 11(2): 242–248 n sharifinia et al.: fauna and geographical … 245 http://jad.tums.ac.ir published online: may 27, 2017 table 2. scorpions collected in different parts of ilam province, 2014 family species sampling sites frequency of each species relative frequency at the province (%) male n (%) female n (%) total buthidae hottentotta saulcyi all areas 30 (31) 68 (69) 98 25.09 buthidae mesobuthus eupeus all areas 40 (43) 52 (57) 92 23.29 buthidae compsobuthus matthiesseni ilam, sirvan, dareh-shahr, zarin-abad 22 (35) 41 (65) 63 16.18 buthidae razianus zarudnyi mehran, badre, zarin-abad 16 (47) 18 (53) 34 8.69 hemiscorpiidae hemiscorpius lepturus mehran, badre, abdanan 10 (36) 18 (64) 28 7.19 buthidae androctonus crassicauda sirvan, mehran, dehloran, ilam, aivan 5 (24) 16 (76) 21 5.39 buthidae orthochirus iranus sirvan, mehran, dehloran, ilam, aivan 6 (33) 12 (67) 18 4.67 buthidae odontobuthus bidentatus dehloran, abdanan 2 (14) 12 (86) 14 3.58 buthidae buthacus macrocentrus dehloran, abdanan 3 (27) 8 (73) 11 2.84 buthidae polisius persicus abdanan 3 (43) 4 (57) 7 1.79 scorpionidae scorpio maurus dehloran, abdanan 1 (20) 4 (80) 5 1.29 all scorpion species 138 (35.30) 253 (64.70) 391 100 discussion in the current study, totally 11 scorpion species from buthidae, scorpionidae, and hemiscorpiidae families were identified, which shows a diverse fauna due to the good habitat and favorite climate of the studied area. in other studies accomplished in iran, 8 species from fars and kohgilouyeh and boyer-ahmad provinces (azizi et al. 2001), 10 species from hormozgan province (shahi et al. 2009), 3 species from gonabad county (ramezani-avval-riabi et al. 2010), 8 species from kerman province (dehghani 2008), 7 species from kish island (khaghani et al. 2005), 7 species from qeshm island (zarei et al. 2009), 8 species from sistan and balouchestan province (nejati et al. 2014), 2 species from sari county (motavali-haghi et al. 2004), 5 species from chaharmahal and bakhtiari province (pirali-kheirabadi et al. 2014), and finally 5 species from zanjan province (moradi et al. 2015) have been reported. comparing the current findings to other studies, the species richness and diversity of scorpions can be concluded, based on the quantity of species found. however, in neighboring provinces of lorestan, and khuzestan with almost similar climates, 5 and 19 species have been reported respectively, showing much more diversity in khuzestan province (taherian 2003, navidpour et al. 2008b). j arthropod-borne dis, june 2017, 11(2): 242–248 n sharifinia et al.: fauna and geographical … 246 http://jad.tums.ac.ir published online: may 27, 2017 in the current study, 11 species including h. saulcyi, m. eupeus, c. matthiesseni, r. zarudnyi, h. lepturus, a. crassicauda, o. iranus, o. bidentatus, b. macrocentrus, s. maurus, and p. persicus were collected and identified in ilam province. mozafari had reported 7 species in one county of the province (mozaffari et al. 2013), gowhari had identified 10 species in different climates of the province (gowhari et al. 2012), however, navidpour reported 14 species in three families, while vachoniolus iranus, compsobuthus jakesi, apistobuthus susanae species were not found in our study. the recent species had been found in ein-e-kosh village (navidpour et al. 2008a), located in far south of the province, having a similar climate and ecosystem to that of khuzestan province. therefore, no sampling has been done in that area, a fact that justifies the difference. the most abundant species of the province was h. saulcyi which collected in all the three climatic areas of the province, as well reported by gowhari in all studied places (gowhari et al. 2012). sedaghat (sedaghat et al. 2012), in respect to biogeographical distribution of iran’s scorpions has reported the h. saulcyi in khuzestan, kohgiloyeh-boirahmad, and kermanshah provinces with dryhot, cold-mountainous, and mild-mountainous climates, respectively, showing high adaptation of the species. the second abundant species was m. eupeus found in mountainous areas and beneath the rocks, that were in accordance with the findings of khairabadi in chaharmahal and bakhtiari mountainous areas, and also those of motavali-haghi from mountains of sari county, northern iran (motavali-haghi et al. 2004, pirali-kheirabadi et al. 2014). out of the most dangerous and venomous scorpions, h. lepturus and a. crassicauda (black scorpion) with frequencies of 7% and 5%, respectively, were also found in the studied area. the h. lepturus has been reported as the most dangerous and main cause of death in khuzestan provinces’ scorpionism (radmanesh 1990, dehghani and fathi 2012, nejati et al. 2014). the thin and small sting has been reported as the feature of such species, along with a painless sting which leads to acute complications such as tissue necrosis, hemolysis, and even death during the first 48 hours. hemiscorpius lepturus was identified in golan, badre, and murmuri areas of the province. androctonus crassicauda was found in five areas of the province (table 2) reported as the main scorpionism cause in khuzestan province. the current species have also been reported in semnan, bushehr, and lorestan provinces (sedaghat et al. 2012). from the sex-ratio point of view, the females were the dominant gender during the study, i.e. 2:1 (f/m) sex ratio. wilson has reported the sex-ratio (f/m) of 3.91 and/or 4:1 (lourenço 2002). shahi has also reported much more abundance of females than males in hormozgan province which both are in accordance with our findings (shahi et al. 2009). conclusion despite the relatively small area (1.2% of the country surface), a diverse fauna was seen, compared to other studies in different provinces of iran. such geographical distribution may be affected by climate changes and global warming, their habitat, and even the distribution pattern of each species (bellard et al. 2012). the health and medical importance of scorpions necessitates comprehensive and periodic research on their ecology including habitat, diet, environment’s temperature, humidity, and precipitation in the province. acknowledgements the authors are highly thankful to research j arthropod-borne dis, june 2017, 11(2): 242–248 n sharifinia et al.: fauna and geographical … 247 http://jad.tums.ac.ir published online: may 27, 2017 and technology deputy, ilam university of medical sciences for their encouragement and financial support through grant no 22.40.54495, dated 2/04/2014. thanks also go to mr hr shoohanizad for his invaluable points during writing the ms. the authors declare that there is no conflict of interests. references azizi k, shahraki g, omrani, m (2001) determining the 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arthropod borne dis. 3: 46–52. بسمه تعالي j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 433 http://jad.tums.ac.ir published online: september 08, 2017 short communication geographical distribution of scorpion odontobuthus doriae in esfahan province, central iran rouhullah dehghani 1, *hamid kassiri 2 1social determinants of health (sdh) research center, kashan university of medical sciences, kashan, iran 2ahvaz jundishapur university of medical sciences, school of health, ahvaz, iran (received 13 sep 2016; accepted 21 aug 2017) abstract background: scorpions are among the world’s venomous arthropods, they sting humans every year, suffering painful symptoms or losing their lives because of the venom. odontobuthus doriae thorell 1876 (arachnida: scorpionida: buthidae) is a scorpion of medical importance and therefore its geographical distribution in isfahan province has been studied. methods: this descriptive cross–sectional study was designed between mar and jun in 2012 and 2013 in province of isfahan, central iran. overall, 164 o. doriae scorpions were collected from their natural habitat by identifying the dug burrows. this arthropod’s burrows were identified based on the presence of tumuli, particularly between may and jun at the sloping foothills of pristine embankments. the sampling data was categorized and compared. results: the relative frequency of collected o. doriae for the counties was mobarakeh (13.5%), shahinshahre (11.5%), borkhar (9%), shahreza (7.5%), kashan (7.5%), naeen (6%), natanz (5.5%), isfahan (4.8%), najafabad (4.8%), aran and bidgol (4.8%), dehaghan (4.8%), flavarjan (3.7%), khomeinishahr (3.7%), tiran (3.7%), golpayegan (3.7%), ardestan (3.7%) and lenjan (2.5%). no o. doriae was collected from other counties of the province. conclusion: the habitats of o. doriae are more often located in central, eastern and northern regions of the province compared to other regions. counties of southern and western regions are among cold parts of the province. keywords: bio-ecology, spatial distribution, odontobuthus doriae, iran introduction scorpions are among the oldest organisms on the earth. scorpions possess stings equipped with venom glands located near the metasoma. thus, they are among the most dangerous arthropods to humans. approximately, 1.2 to 1.5 million cases of scorpion envenomation occurs worldwide annually (1, 2). due to its type of climate, iran hosts a very rich population of arthropods, and scorpions in particular (3, 5) and it is among the countries in which many scorpion species, particularly the dangerous types have been reported. scorpion envenomation reports from ancient iran are found in religious and historical texts, which indicate the long history of this issue in iran (6, 7). naturally, with the presence of these arthropods all over the iranian territory, which possess suitable climate for their habitation, scorpion envenomation has been continuously an issue in iran (8). scorpions are classified as burrowing and non-burrowing, in terms of digging burrows. different scorpion species in the world dig burrows (9, 11). in iran, odonthobuthus spp. and scorpio maurus dig burrows (12). nonburrowing scorpions use natural and artificial materials as shelter. non-burrowing scorpions, which enter human dwellings, too, take shelter in places like wardrobes, inside the shoes and boots, and under the objects laid on the ground. other shelters of non-burrowing scor*corresponding author: hamid kassiri, e-mail: hamid.kassiri@yahoo.com mailto:hamid.kassiri@yahoo.com j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 434 http://jad.tums.ac.ir published online: september 08, 2017 pions include inside the wall cracks, underneath the stones, camelthorn, small pieces of wood and leaves, and inside the gaps and cracks in the trees. burrowing scorpions possess digging capabilities and are capable of digging 25 to 50cm deep burrows (13, 14). humans may encounter burrowing scorpions infrequently under normal circumstances, but in emergency or military conditions, encounters with the burrowing scorpions are more likely due to excavations and digging trenches. since most of the country is military bases and areas are located outside cities, and due to the distribution of dangerous types of scorpions, this issue is of great importance in terms of military health and hygiene. the scope of the presence of many species of this arthropod in military bases across the country and in isolated regions is such that soldiers constantly encounter them, and are envenomed by them. as such, these scorpions raise issues for military forces isolated from medical centers (15). scorpion envenomation is common in middle eastern countries, which is a significant issue in iran’s southwest, in particular, posing many problems including allergic reactions (16, 18). scorpion sting symptoms in humans include quickened breathing, paralysis of diaphragm, spasms of voluntary muscles, severe twitching, convulsions, muscle contraction and tension due to increased release of acetylcholine, pulmonary edema and swelling, damages to the heart muscle, vascular turbulence and disorders, impaired kidney function, necrosis and skin injuries and pathological changes in single or multiple organs. these symptoms vary depending on the sting and the impact mechanism of the venom of different species (19, 22). one of the medically important scorpions is o. doriae, which exists in relatively high numbers in iran. ld50, i.e. the lethal dose, of this arthropod’s venom is 0.19mg/kg for mice (8, 16) and thus studying it's biological aspects is of value. treatment and preventive measures are important issues in the field of medicine and hygiene. as such, examining other life aspects of scorpions, including o. doriae, such as their biology and ecology is of importance. conducting such research in the field of bio-ecology is very time-consuming. different studies, therefore, need to be unified to reach a conclusion and overcome the issues regarding scorpion envenomation. materials and methods this descriptive cross–sectional study was designed between mar and jun in 2012 and 2013 in province of isfahan, central iran. overall, 164 o. doriae scorpions were collected from their natural habitats in different counties by locating their burrows. the burrows were subsequently excavated or filled with water. to select the sampling area, a pristine piece of land of approximately 100m2, located 3 to 10km from the city being studied, was selected and all its identifiable burrows were searched to collect the arthropod. identification of the burrows was based on two approaches (1). the first approach was based on the identification of burrows with asymmetric elliptical openings with minor axes of 1.2 to 1.5cm and major axes of 2.5 to 4.5cm, found in pristine or artificial embankments. in the second approach, the presence of tumuli, particularly between march and jun at the sloping foothills of pristine embankments, was indicative of this arthropod’s presence. tumuli at sloping foothills move to the lower side, which is indicative of fresh digging and clearing of the burrow. excavation was carefully conducted using military shovels and trowels, and the arthropod was usually transferred to the sample case by forceps after full excavation of the burrow. in other cases, 1 to 3l of water was slowly poured j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 435 http://jad.tums.ac.ir published online: september 08, 2017 into the identified burrows and the arthropod was caught with forceps upon exiting. samples were transferred to laboratory for further examinations. the obtained data from the studied regions, regional characteristics, number of samples and catching methods were recorded at each sample and presented by illustrations and descriptive tables. the studied region isfahan province, with an area of 105937 square km, is situated in the center of iran. it borders the provinces of markazi, qom, and semnan to the north, fars and kohgiluyeh and boyer-ahmad to the south, lorestan and chaharmahal and bakhtiari to the west, and yazd and khorasan to the east (fig. 1). according to the latest administrative divisions, it has 23 counties, 60 cities, 37 districts, and 116 villages and its capital city is isfahan. counties of isfahan province include isfahan, ardestan, borkhar, shahreza, khomeinishahr, khvansar, semirom, fereydan, fereydonshahre, chadegan, daran, dehaghan, kashan, aran and bidgol, falavarjan, golpayegan, lenjan, mobarakeh, naeen, najafabad, tiran, natanz and shahinshahre. in general, the province has a moderate and dry climate. the west of the province, isfahan county and western and southwestern regions of the province experience arid, semi-arid and cold semi-humid climates, respectively. the maximum, minimum, and average annual temperatures have been recorded as 40.6 °c, -10.6 °c and 16.7 °c, respectively, according to a report of the synoptic weather station in isfahan. according to the same report, the number of the province’s frost days is 76, and the average annual rainfall is 116.9mm (23, 24). results the o. doriae scorpions were collected in 2012 and 2013 through 23 samplings. overall, 164 scorpions of this species were collected. characteristics of the collecting regions, collecting methods and number of the collected scorpions of the stated species are presented in fig. 2 and table 1. a minimum of 17 out of 23 counties had o. doriae scorpions. maximum and minimum o. doriae catch were from mobarakeh, at 13.5%, and lenjan, at 2.5%, respectively. moreover, 83% of the samples were collected by the excavation method and 17% were collected by pouring water into the burrows of o. doriae. the highest percentage of the collected samples, after mobarakeh, was from shahinshahre, borkhar, shahreza, kashan, naeen, natanz, esfahan, najafabad, aran and bidgol, dehaghan, flavarjan, khomeini shahr, tiran, golpayegan, ardestan and lenjan, at 11.5, 9, 7.5, 7.5, 6, 5.5, 4.8, 4.8, 4.8, 4.8, 3.7, 3.7, 3.7, 3.7, 3.7 and 2.5 percent, respectively. meanwhile, no o. doriae scorpions were collected form khansar, semirom, fereydan, fereydonshahre, chadegan and daran in this study (table 1). most collected o. doriae was from the central regions of isfahan province, which includes the counties of isfahan, borkhar, shahreza, khomeini shahr, dahaghan, kashan, aran and bidgol, flavarjan, golpayegan, lenjan, mobarakeh, naeen, najafabad, tiran, natanz, and shahinshahre. the least o. doriae catch was from the eastern part of isfahan province, which includes northeastern naeen, northern ardestan, khor and biabanak, and anarak. no o. doriae scorpions were collected form semirom, in the south of the province, khansar, fereydonshahre, chadegan and daran (fig. 2, 3). j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 436 http://jad.tums.ac.ir published online: september 08, 2017 table 1. odontobuthus doriae abundance caught in isfahan province by county city methods of collecting number percent digging use of water mobarakeh 15 7 22 13.5 shahinshahre 17 2 19 11.5 borkhar 15 15 9 shahreza 12 12 7.5 kashan 8 4 12 7.5 naeen 6 4 10 6 natanz 6 3 9 5.5 esfahan 5 3 8 4.8 najafabad 8 8 4.8 aran va bidgol 6 2 8 4.8 dehaghan 8 8 4.8 flavarjan 6 6 3.7 khomeinishahre 6 6 3.7 tiran 6 6 3.7 golpayegan 6 6 3.7 ardestan 2 3 5 3 lenjan 4 4 2.5 daran chadegan khansar fereydan fereydonshahre semirom 23 136 28 164 100 caspian sea oman sea turkmenistan afghanistan pakistan armenia turkey iraq kuwait azerbaijan iran esfahan province fig. 1. the geographical situation of isfahan province in iran naeen esfahan ardestan aran va bidgol kashan natanz shahishahre shahreza najafabad borkhar tiran golpayegan khasar fereydan samiromcapture area of o.doriae fereydonshahre no capture area of o.doriae fig. 2. study regions of odontobuthus doriae in the isfahan province by county j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 437 http://jad.tums.ac.ir published online: september 08, 2017 high plenty area of o.doriae no capture area of o.doriae low plenty area of o.doriae esfahan province fig. 3. study regions of in the isfahan province by catch abundance of odontobuthus doriae discussion odontobuthus doriae is a burrowing scorpion that is capable of digging burrows of up to 40cm. this species reaches up to 10cm in size. coloration ranges from light yellow to dark and the body appendages are usually lighter than prosoma (25). this has been the predominant scorpion species of the studied regions in the natural habitats of isfahan province. major signs of this arthropod’s burrows are the shape of the burrow’s opening and the presence of piled soil close to the burrow that is due to digging or clearing in early spring (13). these arthropods spend the winters in hibernation and are capable of restarting their regular functionality in favorable conditions within three to four hours (8), and freshly piled soil close to the burrow is indicative of this activity. at the opening of freshly dug burrows of this scorpion, certain type of tumulus can be observed, moved or washed away by wind or rain after a while. the shape of the burrow’s opening was distinct from other burrows and was of asymmetric elliptical shape with a minor axis of 1.2 to 1.5cm and a major axis of 2.5 to 4.5cm. while the opening resembles an ellipse, the burrow assumes a relatively circular shape after 2 to 3cm. odontobuthus doriae is of genus odontobuthus and 3 species of it have been reported in iran (26, 28). the species o. doriae has been reported in many regions of iran, including the provinces of yazd, kerman (kerman, baft, sirjan, rafsanjan, zarand, shahrbabak, kahnoj, manojan, shahdad), markazi (arak), qazvin, tehran (deserts around tehran county, shemiran, varamin), alborz (karaj), semnan (garmsar), west azerbaijan (uromieh, nazlo, salmas), kermanshah, bushehr (borazjan), hamadan, hormozgan (bandar abbas) (29, 35), which corresponds with our study. due to the remoteness of the habitats of this species of the human environment, its envenomation has been reported only sporadically (36). the maximum o. doriae hunting was from the central regions of isfahan province and the minimum o. doriae hunting was from the eastern part of isfahan province. one of the major reasons for varying numbers of this arthropod’s burrows in isfahan province is due climatic diversity in this province. semi-arid climate covers the central regions of isfahan province. dryness and little precipitation are typical characteristics of this climate. however, zayanderud river considerably affects the climate of this region in a positive way and makes it more temperate. the study suggested that fewer o. doriae scorpions were observed in eastern part of isfahan province than in the central regions of this province. arid climate covers northern naeen, biabanak, and anarak to northern ardestan. abrupt changes in temperature, little precipitation, and strong winds during the year are characteristic of this climate (2). no o. doriae scorpions were collected from south of the province and its eastern counties in our study. these regions have far colder climates than other regions of the province and are higher in elevation. higher elevations and lower temperatures can lead to fewer favorable habitats for this j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 438 http://jad.tums.ac.ir published online: september 08, 2017 arthropod. extreme cold hinders the activities and spreading of scorpions (37). conclusion odontobuthus doriae has more habitats in central, eastern and northern parts of the province than in other parts. southern and western counties are among the cold regions of the province. since scorpions occupy a wide range of habitats in warmer regions, conducting research on the correlation of the distribution of medically important species and different climate factors with scorpion envenomation can provide new insights into this issue. acknowledgments authors thank vice chancellor for research, social determinants of health research center and laboratory staff of kashan university of medical sciences who willingly agreed to participate in this research. the authors declare that there is no conflict of interest. references 1. chippaux jp, goyffon m (2008) epidemiology of scorpionism: a global appraisal. acta trop. 107: 71–79. 2. dehghani r, rafinejad j, fathi b, panjehshahi m, jazayeri m, hashemi a (2017) a retrospective study on scropionism in iran (2002–2011). j arthropod borne dis. 11(2): 184–193. 3. dehghani r, fathi b (2012) scorpion sting in iran: a review. toxicon. 60(5): 919– 933. 4. dehghani r, djadid nd, shahbazzadeh d, bigdelli s (2009) introducing compsobuthus matthiesseni (birula, 1905) scorpion as one of the major stinging scorpions in khuzestan, iran. toxicon. 54(3): 272– 275. 5. sanaei-zadeh h, marashi sm, dehghani r (2017) epidemiological and clinical characteristics of scorpionism in shiraz (2012–2016) development of a clinical severity grading for iranian scorpion envenomation. med j islam repub iran. 31: 27. 6. dehghani r, velaei n (2010) the review of iranian traditional medicine vision on scorpion and scorpion sting. res med j shahid beheshti univ med sci. 33 (4): 269–279. 7. dehghani r, arani mg (2015) scorpion sting prevention and treatment in ancient iran. j tradit complement med. 5(2): 75–80. 8. motevalli haghi f, dehghani r (2117) a review of scorpions reported in iran. j mazandaran univ med sci. 27(151): 213–226. 9. white cr (2001) the energetics of burrow excavation by the inland robust scorpion, urodacus yaschenkoi (birula, 1903). aust j zool. 49(6): 663–674. 10. çolak m, karataş a 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sayedi-rashti sma (1998) faunistic study of scorpions in shiraz and investigation in their fecundity. j yasouj univ med sci. 3(9, 10): 23–28. 34. dehghani r, doroudgar a, khademi mr, http://vista.ir/article/813762 http://www.irimo.ir/far/%20services/climate/796 http://www.irimo.ir/far/%20services/climate/796 j arthropod-borne dis, september 2017, 11(3): 433–440 r dehghani and h kassiri: geographical … 440 http://jad.tums.ac.ir published online: september 08, 2017 sayyah m (1998) the survey of scorpion sting in the city of kashan. j res med sci. 2(3): 132–135. 35. kamali k (1984) introduce of importance scorpions in khuzestan. j sci agr. ahvaz chameran univ. 1: 34. 36. dehghani tafti mh, tirgari s (1998) identification of yazd scorpions and bi ological study of prevailing species. j shahid sadoughi univ med sci. 6(3): 72–77. 37. woodman jd (2008) living in a shallow burrow under a rock: gas exchange and water loss in an australian scorpion. j therm biol. 33(5): 280–286. j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 76 http://jad.tums.ac.ir published online: april 27, 2019 original article identification of theileria species in sheep and vector ticks using pcr method in zabol, eastern iran fateme zarei; *maryam ganjali; reza nabavi department of parasitology, faculty of veterinary medicine, university of zabol, zabol, iran (received 24 sep 2017; accepted 18 feb 2019) abstract background: theileria is a protozoal parasite that belongs to the phylum apicomplexa. theileriosis is an important tick-borne disease caused by various species of theileria. among these species, t. lestoquardi (t. hirci) is highly pathogenic, while other species such as t. ovis make subclinical and mild infections in small ruminant. therefore, the precise identification of the species and the vector ticks are very essential for epidemiological studies and the design of control programs. methods: this research was conducted with the aim of molecular study to identify theileria species and vectors in zabol, eastern iran in 2015. the presence of theileria in 80 blood samples and vector ticks was evaluated using pcr method. results: of 80 blood samples, pcr analysis showed that 50 samples (62.5%) were infected with theileria. the evaluation of the first phase pcr with nested pcr showed that infections with theileria ovis and theileria lestoquardi were 67.45% and 32.55% cases respectively. overall, 110 ticks (78 males and 32 females) were collected and generally two genera and six species including rhipicephalus bursa (9.1%), rh. sanguineus (29.1%), rh. turanicus (10.9%) hyalomma asiaticum asiaticum (23.63%), hy. excavatum (10.9%), hy. anatolicum (16.37%) were detected. after evaluating ticks infection by pcr method, three species of rh. turanicus, rh. sanguineus and hy. asiaticum asiaticum, were infected. conclusion: theileria ovis has a high prevalence among the sheep of zabol and hy. asiaticum asiaticum, rh. sanguineus and rh. turanicus may be the main vectors of theileria species in this area. keywords: theileria; pcr; iran introduction theileriosis is a tick-borne disease that infects domestic and wild ruminants in tropical and subtropical parts of the world, it causes serious problem for livestock husbandry in many parts of iran (1). two species of theileria ovis and theileria lestoquardi are the main causative of ovine theileriosis in iran. theileria lestoquardi (= t. hirci) causes high mortality in sheep and goats in south and southeast of iran. theileria ovis is widely distributed in the country according to clinical and morphological observations (2). the ticks of the genera hyalomma and rhipicephalus have been reported in four geographic regions of iran (3, 4). ticks should be evaluated as theileria vector in each region. theileriosis not on ly directly lead to reduced production and mortality, but the cost of eradication of the ticks is added to the expense of the animal husbandry. this disease is also important in iran, and it may be possible in the appropriate seasons to act as the main cause of the visit of livestock to veterinary clinics (5). to classify and identify many haemoparasite ssuch as theileria, molecular methods having high sensitivity and specificity compared to microscopic and serological tests (6, 7). in this study, we tried to use the nestedpcr method, which is a sensitive and specific method for identification of theileria spp. in sheep and vector ticks in zabol, eastern iran. the results of the study will be effective in ap*corresponding author: dr maryam ganjali, email: m_g2507@yahoo.com https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/w/index.php?title=rhipicephalus_turanicus&action=edit&redlink=1 https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 77 http://jad.tums.ac.ir published online: april 27, 2019 plying disease treatment and appropriate control measures. materials and methods sistan and baluchistan is located in southeast of iran. zabol has a desert climate and is located in the north of the province. during the spring and summer of 2015, 80 sheep were randomly selected from different areas of zabol and were clinically examined. blood samples were collected into tubes containing al severs, solution (citric acid 0.55gr +sodium citrate 8gr +dextrose 20.5gr +sodium chloride 4.2gr +d.w 1lit). in the next stage, 110 ticks were removed from the ear and around the head, under the tail, anus and around the breast in females and scrotum in males. the ticks removed from each sheep were individually stored in labeled tube containing 70% alcohol and 5% glycerin. in order to identify ticks (fig. 1–5), they were washed with sterilized water and were dried on sterilized filter paper and determined by the walker identification key (8). separation of tick salivary glands was performed according to purnell method (9). after removing the tick scutum, salivary glands were homogenized in pbs (ph: 7.2), then centrifuged at 5,000rpm at 4 °c for 5min. the blood and ticks sample were used for dna extraction using dna isolation kit (mbst, iran) according to the kit instructions. the pcr reaction was then performed using primers with the sequence of tbf2: 5´cacagggaggtagtga caag3 ́and tbr2: 5'aagaatttcacctatgacag3 ́(10, 11). dna replication was performed using primers designed from flanking part of hypervariable region of18s rrna. the pcr product in all theileria species is 426bp (fig. 6). in order to identify the theileria species, all blood samples were undergone the semi-nested pcr test with specific inner primers derived from the 18s rrna gene. in the second step, the nested inner primers were used for detection of t. lestoquardi, including tbr2: 5'-agaatttcacctatgacag-3 'and tl3: 5'-attgcttgtgtccctccg-3' and the primers were used to detect t. ovis tbr2: 5'agaatttcacctatgacag3' and to4: 5'ttgcttttgctccttta cgag-3 '(10). the pcr was performed on 50μl reaction volumes including, 4μl extracted dna, 25μl taq dna polymerase 2× master mix (pishgam company, iran), 3pmol of each primer and sterile distilled water up to 50ml in automated thermocycler with the following program: 5min incubation at 95 °c to denature double strand dna, 38 cycles of 45sec at 95 °c, 45sec at 56 °c (annealing step), 45sec at 72 °c and this was followed by final extension step at 72 °c for 10min. the semi-nested pcr was done with the same reaction in the first round. the pcr products were also electrophoresed through a 1.5% agarose gel to assess the presence of a special band of t. lestoquardi (235bp) and t. ovis (237bp) (fig. 7, 8). negative control (no template) was always run simultaneously with our pcr experiments. pcr product was analyzed on 1.5% agarose gel in 0.5 xtbe buffer and visualized using ethidium bromide and an uv illuminator. results overall, 110 ticks (78 males and 32 females) were collected and generally two genera and six species including rh. bursa (9.1%), rh. sanguineus (29.1%), rh. turanicus (10.9 %), hy. asiaticum asiaticum (23.63%), hy. excavatum (10.9%), hy. anatolicum (16.37%) were detected. after removing the salivary glands of the ticks, genomic dna of each tick was extracted using a dna extraction kit (mbst, iran) according to the kit instruction, and then the infection was evaluated by pcr method. eighteen ticks including 9 hy. asiaticum asiaticum, 5 rh. turanicus, and 4 rh. sanguineus, were https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/w/index.php?title=rhipicephalus_turanicus&action=edit&redlink=1 https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 78 http://jad.tums.ac.ir published online: april 27, 2019 infected with theileria. none of the rh. bursa, hy. excavatum and hy. anatolicum was not infected with theileria. the analysis of pcr product using tbf2 and tbr2 primer showed that of 80 blood samples obtained from sheep, 50 cases (62.5%) were positive (fig. 6). the results of the semi-nested pcr assays on blood sample determined 29 (67.45%) and 14 (32.55%) cases were infected by t. ovis and t. lestoquardi, respectively. the most infection in zabol is related to t. ovis, and this species is common in this region (fig. 7, 8). fig. 1. hyalomma anatolicum male, (a) dorsal view: cervical field depression is apparent (short cervical groove), (b) ventral view: subanal plates alignment is with the adanal plates (c) dorsal view: lateral grooves are short and central festoon is dark colored. fig. 2. hyalomma excavatum male, dorsal view: cervical field depression is apparent (short cervical groove), lateral grooves are short, central festoon is light colored, paracentral festoons are fused. ventral view: subanal plates are in alignment with adanal plate j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 79 http://jad.tums.ac.ir published online: april 27, 2019 fig. 3. hyalomm asiaticum asiaticum male, dorsal view: cervical grooves are long, other features include: lateral grooves are short, central festoon is triangular, spiracles are long. ventral view: subanal plates are in alignment with adanal plate. fig. 4. spiracle of rhipicephalus turanicus (a): male, (b): female. fig. 5. spiracle of rhipicephalu sanguineus (a): male, (b): female https://en.wikipedia.org/w/index.php?title=rhipicephalus_turanicus&action=edit&redlink=1 https://en.wikipedia.org/w/index.php?title=rhipicephalus_turanicus&action=edit&redlink=1 j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 80 http://jad.tums.ac.ir published online: april 27, 2019 fig. 6. agarose gel electrophoresis of pcr products related to extracted dna from blood samples (band number: 1–11) and tick salivary glands (band number: 12–18). m 100bp ladder dna marker; n negative control fig. 7. agarose gel electrophoresis of semi nested pcr products using specific primers tbr2 and tl4 (235bp) m 100bp ladder dna marker; n negative control. theileria lestoquardi (lane 4, 7: 235bp) fig. 8. agarose gel electrophoresis of semi nested pcr products using specific primers tbr2 and to3 (237bp) m 100bp ladder dna marker; n negative control. theileria ovis (lane3, 5, 6, 8: 237bp) discussion molecular examination of 80 sheep blood samples showed that 50 sheep (62.5%) were infected with theileria. theileria ovis infection was 67.45% and t. lestoquardi 32.55% respectively. moreover, no co-infection was detected. according to our results, the preva lence infection with t. ovis species is more than t. lestoquardi in zabol. in this study, of 110 ticks collected from different areas of zabol, the most common ones in the regions were rh. sanguineus with 29.1%, followed by hy. asiaticum asiaticum with 23.63%, hy. anatolicum https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/wiki/rhipicephalus_sanguineus j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 81 http://jad.tums.ac.ir published online: april 27, 2019 with 16.37%, rh. turanicus and hy. excavatum with 10.9% and finally rh. bursa with 9.1%. after evaluating ticks infection by pcr method, three species of rh. turanicus, rh. sanguineus and hy. asiaticum asiaticum, were infected. considering the role of these tick species in the transmission of theileria, important measures for eradication of them should be considered. in fars and kazeroun (12), 100 blood and tick samples were collected to identify the species of theileria and tick vector. overall, 46% of blood smears from sheep infected with theileria, while 76% of blood samples analyzed by semi-nested pcr were infected with this parasite. the prevalence of t. ovis was 43%, t. lestoquardi 3% and 30% mixed infection. the result is also the same as our findings because in their study the prevalence of t. ovis was higher than t. lestoquardi. in contrast to our study, simultaneous infections of these two species were found in their study. the most common tick species were rh. turanicus (48.8%), hy. anatolicum anatolicum (42.2%), and hy. marginatum (8.8 %), respectively. in southern khorasan razavi (13) on 150 sheep blood samples, infection with theileria species was reported in 18.6% of blood smears. using semi-nested pcr, t. ovis infection was 58.6% and t. lestoquardi 6.6%. the result of this study also showed that the prevalence of t. ovis is much higher than t. lestoquardi. rhipicephalus turanicus was positive for theileria infection. in lorestan province, 219 ticks were collected from the sheep's body, and after the pcr test, rh. sanguineus, hy. anatolicum anatolicum were infected with theileria species (14). hyalomma detritum was identified as vector of t. lestoquardi in lorestan province, as well as hy. anatolicum anatolicum and rhipicephalus in fars province (15). in a study in the eastern and southeastern parts of iran using nested-pcr (16) reported 56% infection with theileria, of these the infection rate was reported by t. ovis 12.5% and t. lestoquardi 87.5%. infection with t. lestoquardi was reported to be more than t. ovis, which is not consistent with the results of the present study. in a study to detect theileria and babesia species in sheep of ahwaz using pcr-rlfp method (17), 119 blood samples were collected and the following results were obtained: 89% (106/119) of the total samples were positive for theileria infection. 91.5% (97/96%) of theileria positive samples were identified as t. ovis and 83.2% (106/3) were t. lestoquardi. in the molecular study of malignant ovine theileriosis in oman observed the nucleotide sequence of pcr product consistent with t. lestoquardi (18). in turkey, 2241 ixodid ticks were isolated from sheep and goats, of which 14.86% were infected by theileria and babesia. the infection levels of each species were as follows: 0.691% haemaphysalis parva, 1.47% rh. sanguineus, 1.84 ixodes ricinus, 2.86% rh. turanicus, 5.57% hy. marginatum, 6.2% rh. bursa (19). conclusion theileria ovis has a high prevalence among the sheep of zabol and hy. asiaticum asiaticum, rh. sanguineus and rh. turanicus may be the main vectors of theileria species in this area. acknowledgements this study was supported by faculty of veterinarymedicine university of zabol. the authors declare that there is no conflict of interests. references 1. young as, groockok cn, kariuki dp (1988) integrated control of ticks and tick-borne diseases of cattle in africa. parasitology. 96: 403–411. 2. hooshmand-rad p, hawa nj (1973) ma https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://en.wikipedia.org/w/index.php?title=rhipicephalus_turanicus&action=edit&redlink=1 https://en.wikipedia.org/w/index.php?title=hyalomma_rhipicephaloides&action=edit&redlink=1 https://en.wikipedia.org/wiki/rhipicephalus_sanguineus https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-7-s1-p11 j arthropod-borne dis, march 2019, 13(1): 76–82 f zarei et al.: identification of … 82 http://jad.tums.ac.ir published online: april 27, 2019 lignant theileriosis of sheep and goats. trop anim health prod. 5: 97–102. 3. nabian s, rahbari s, changizi a, shayan p (2009) the distribution of hyalomma spp. ticks from domestic ruminants in iran. j med entomol. 23(3): 281–283. 4. rahbari s, nabian s, shayan p, sedghian m (2008) a study of rhipicephalus species iran. j vet res. 63: 195–198. 5. ghadrdan mashhadi ar, razmi-jalili m, kavand m (2006) to determine serum alp, ast, ggt and bilirubin changes in theileriotic cows, (mediterranean coast fever). j vet med. 61: 23–28. 6. burridge mi, brown cg, kimber cd (1974) theileria annulata cross reaction between acell culture schizont antigen and antigens of east african species in the indirect fluorescent antibody test. exp parasitol. 5: 374–380. 7. altay k, aydin mf, dumanli n, aktas m (2008) molecular detection of theleria and babesia infections in cattle. vet parasitol. 158: 295–301. 8. walker ar, bouattour a, camicas jl, estrada-peña a, horak ig, latif aa, pegram rg, preston pm (2007) ticks of domestic animals in africa: a guide to identification of species. bioscience reports, uk. 9. purnell re, jouyner lp (1968) the development of theileria parva in the salivary glands of the tick rhipicephalus appendiculatus. parasitology. 58: 725–732. 10. shayan p, rahbari s (2007) differentiation of sheep theileria spp. and babesia spp. by polymerase chain reaction. j vet res. 62(3): 250–260. 11. schnittger l, yin h, qi b, gubbels mj, beyer d, niemann s, jongejan f, ahmed js (2004) simultaneously detection and differentiation of theileria and babesia parasite infection small ruminants by revers line blotting. parasitol res. 92: 189–196. 12. yaghfoori s, razmi g, heidarpour m (2012) molecular detection of theileria spp, in sheep and vector ticks in fasa and kazeroun areas, fars province, iran. arch razi ins. 68: 159–164. 13. razmi gh, yaghfoori s (2013) molecular surveillance of theileria ovis, theileria lestoquardi and theileria annulata infection in sheep and ixodid ticks in iran. j vet res. 80(1): 635. 14. hoghooghi-rad n, hashemi s, abdigoudarzi m (2014) molecular detection of theileria ovis and t. lestoquardi in vector ticks in lorestan province, iran. int j biosci. 12: 78–83. 15. abdigoudarzi m (2013) detection of naturally infected vector ticks (acari: ixodidae) by different species of babesia and theileria agents from three different enzootic pars of iran. j arthropod borne dis. 7(2): 164–172. 16. heidarpour-bami m, khazraiinia p, haddadzadeh hr, kazemi b (2010) identification of theileria species in sheep in the eastern half of iran using nested pcr-rflp and microscopic techniques. iran j vet res. 11(3): 262–266. 17. jalali sm, khaki z, kazemi b, rahbari s, shayan p, bandehpour m, yasini sp (2014) molecular detection and identification of theileria species by pcrrflp method in sheep from ahvaz, southern iran. iran j parasitol. 9(1): 99– 106. 18. shayan p, ebrahimzadeh e, tageldin mh, amininia n, eckert b (2011) molecular study of sheep malignant theileriosis at barka region in the sultanate of oman. iran j porasitol. 6 (1): 66–72. 19. aydin mf, aktas m, dumanli n (2015) molecular identification of theileria and babesia in ticks collected from sheep and goats in the black sea region of turkey. parasitol res. 114(1): 65–69. https://www.ncbi.nlm.nih.gov/pubmed/24396911 microsoft word 4-doosti_rtl.doc iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 28 original article applying morphometric variation of seta 2 (antepalmate hair) among the larvae of the members of the maculipennis subgroup (diptera: culicidae) in iran s doosti 1, h vatandoost 1, ma oshaghi 1, m hosseini 2, *mm sedaghat 1 1dept. of medical entomology and vector control, school of public health and institute of public health research, medical sciences /university of tehran, iran 2dept. of epidemiology and biostatistics, school of public health and institute of public health research, medical sciences /university of tehran, tehran, iran (received 20 oct 2006; accepted 1 may 2007) abstract the members of anopheles maculipennis subgroup (diptera: culicidae) are the most important vectors of malaria in the north, west, and central plateau of iran. this study was carried out to differentiate the species composition of this subgroup based on morphometric variation seta 2-iv and v (antepalmate hair) among 149 larval specimens that were deposited at the medical arthropods museum, the school of public health, tehran university of medical sciences by using the light microscope. the mean numbers of larval seta 2-iv and v of the specimens belong to different locations of iran, were calculated by spss (11.5) software package, followed by cluster analysis, and four different groups (clusters) were identified. the means were compared with the similar and available published data. after analyzing, four clusters recognized. the first cluster was fitted in ten localities in esfahan, east azarbaijan, west azarbaijan, khorassan, kurdistan, and mazandaran provinces with its mean and standard deviation (sd) of 14.89±1.13 (n= 79); the second group with one location in gilan province (11±1.58, no= 5); the third one with two locations in fars and western azarbaijan provinces (27.43±0.31, n=20), and the final group with four locations in khuzestan, west azarbaijan, and qazvin provinces (36.84±1.91, n= 45) were identified and corresponded to anopheles messeae, an. atroparvus, an. melanoon, and an. sacharovi respectively. this work provides comparative information on the maculipennis subgroup based on morphometric examination at the larval stage in iran. keywords: maculipennis group, malaria, mosquito, numerical taxonomy, iran introduction the anopheline fauna in iran is diverse and at least seven species have been incriminated as malaria vectors in the country. among them the members of anopheles maculipennis subgroup (diptera: culicidae) have involved with malaria transmission mostly in northern and central area of iran (sedaghat et al. 2003a). according to the last classification, the genus anopheles (diptera: culicidae) includes six subgenera and at least 484 species in the world (harbach 2004). the members of anopheles maculipennis subgroup are the most important vectors of the western palaearctic region (sedaghat et al. 2003a). an. maculipennis was described and named for the first time by migen in 1818 and for many years, all scientists expected that it was one species with single morphological type and biological characters (white 1978). in fact the maculipennis group was the first sibling species complex, to be discovered among mosquitoes (falleroni 1926, van thiel 1927). since this discovery, extensive efforts *corresponding author: mm sedaghat, tel: +98 21 88951393, e-mail: sedaghat@doctor.com iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 29 have been made to determine and explain species composition of the group. several methods including studies of egg morphology, ecology, hybridization, larval and pupal chaetotaxy, wing characteristics, cuticular hydrocarbons, dna sequences, chromosome, and zymotaxonomy have been used for identification of this group (linton et al. 2003). these works contributed to the current recognition of twelve formal species in the palaearctic region, including an. artemievi, an. atroparvus, an. daciae, an. beklemishevi, an. labranchiae, an. lewisi, an. maculipennis, an. martinius, an. melanoon, an. messeae, an. persiensis and an. sacharovi (white 1978, de zulueta et al. 1983, sedaghat et al. 2003 a, harbach 2004, nicolescu et al. 2004, sedaghat and harbach, 2005, gordeyev et al. 2005). the maculipennis group currently divided to three subgroups including maculipennis, quadri maculatus and freeborni subgroups. the maculipennis subgroup (which previously named maculipennis complex) comprises all palaearctic members of the group except an. beklemishevi (harbach 2004). seven taxa of the members of the subgroup previously reported from iran. these species include: an. atroparvus (dinparast-jadid et al. 2001, 2007), an. labranchiae (gholizadeh et al. 2004, dinparast-jadid et al. 2007), an. maculipennis s.s. (dow 1953, shahgudian 1960, dinparast-jadid et al. 1990, glick 1992, momeni et al. 1992, dinparast-jadid et al. 2001, azarihamidian et al. 2003, oshaghi et al. 2003, sedaghat et al. 2003a, dinparast-jadid et al. 2007), an. melanoon (dow 1953, faghih 1969, as an. subalpinus, dinparast-jadid et al. 1990, momeni 1991, glick 1992, azari-hamidian et al. 2003) an. messeae (de zulueta et al. 1957, minar 1974, momeni 1991, momeni et al. 1992, dinparastjadid et al. 2001, azari-hamidian et al. 2003, dinparast-jadid et al. 2007), an. persiensis (sedaghat et al. 2003a, sedaghat and harbach 2005, dinparast-jadid et al. 2007) and an. sacharovi (dow 1953, etherington and sellick 1946, shahgudian 1960, dinparast-jadid et al. 1990, glick 1992, momeni et al. 1992, dinparast-jadid et al. 2001, oshaghi et al. 2003, sedaghat et al. 2003a,b, sedaghat and harbach 2005, dinparast-jadid et al. 2007). although, glick (1992) listed an. martinius among iranian anopheline, the occurrence of this taxon has not been confirmed in the country (sedaghat and harbach 2005). among the members of an. maculipennis subgroup, five species of an. atroparvus, an. labranchiae, an. maculipennis, an. messeae and an. sacharovi have been considered as malaria vectors in eurasia (jaenson et al. 1986, ribeiro et al. 1988, kasap 1990, jetten and takken 1994, romi 1999, romi et al. 2001, alten et al. 2000, faghih 1969). to date, anopheles maculipennis s.s. has been discriminated as the major vector of malaria in the caspian sea littoral, and an. sacharovi is considered to be the principal vector in the central plateau (faghih 1969, manouchehri et al. 1992). early studies on this group were based on morphological methods. falleroni (1926) and van thiel (1927), as first attempts to understand complexity of the maculipennis group, considered longand short-winged forms of an. maculipennis sensu lato. the other morphological studies include: study on eggs (missiroli et al. 1933); male genitalia (martini 1933); wing scales (ungureanu and shute 1947); larval or pupal chaetotaxy (bates 1939, pichot and deruaz 1981, suzzoni-blatger and sevin 1981, boccolini et al. 1986, suzzoni-blatger et al. 1990, deruaz et al. 1991). in the larval stage, situation of seta 3-c (outer clypeal hair) in comparison to lateral palatal brush in addition number and situation of seta 1-ii was considered as a useful method (abul-hab 1955). however, morphometric variation of seta 2-iv and v was regarded as a useful and reliable morphologic character to differentiate in the larval stage by some medical entomologists (bates 1939, romi et al. 2002). there is little information on vector capacities and species distributions of these sibling iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 30 species in iran. besides, there were disagreements with the occurrence of some species in the country and some records need to be verified by classical morphology study, molecular method and using other advanced tools. identification of species complex which show different biology, ecology, behaviour, host preference and vector potential aspect despite of their resemblance, is very important in malaria control programmes. it is important to make statistical comparison of the amounts of branching of particular larval setae. this paper provides preliminary information on differentiation of species composition of an. maculipennis subgroup in the larval stage based on the morphometric variation of seta 2-iv and v. materials and methods this study was carried out to separate the an. maculipennis subgroup larvae based on seta 2 (ante palmate hair) in iran. two hundred and ninety one larval specimens which were deposited in the medical arthropods museum, school of public health, tehran university of medical sciences, iran were examined. these specimens initially had been identified based on egg morphology. the number of seta 2 braches (ante palmate hair) of the forth and fifth abdominal segments of each larva was counted using light microscope. for compared mean of seta 2-iv and v, those collection localities which had at least 5 specimens were considered in this study. the mean numbers of larval setae 2-iv and v of the specimens belong to different locations of iran, were calculated by spss (11.5) software package, followed by cluster analysis. results in this investigation 149 larval specimens from 17 different locations of iran were studied to identify the species composition of the maculipennis subgroup based on seta 2. there was not statistically significant between the number of the seta 2 branches in abdominal segments iv and v among the specimens. means and standard deviations (sd) of different locations are shown in table 1. total mean of the seta 2 branches in segments 4 and 5 in different locations were statistically significant (p< 0.0001). the mean numbers of the larval seta 2-iv and v of the specimens, were calculated by spss (11.5) software package. after cluster analyzing, four different clusters were identified. these four groups include: group 1 with codes 4, 5, 6, 7, 9, 11, 12, 15, and 16 that they belong to esmail tarkhan (esfahan province), takht olia (west azarbaijan), dehriz (east azarbaijan), band rezaie (east azar-baijan), ghagharalu (east azarbaijan), sarvelayat (khorassan), marsuk (khorassan), gheybisur (kurdestan), sarik (west azarbaijan), group 2 with code 17 includes fuman (gilan), group 3 with codes 1 and 10 includes dasht khezri (fars) and dehjabal (east azarbaijan), and group 4 with codes 2, 3, 8 and 13 includes taher abad (qazvin), nazar abad (qazvin), jabal (east azarbaijan) and kaldusakh (khuzestan) (fig. 2, 3). the mean numbers of the seta 2 branches for each of these clusters were compared with similar information from bates (1939) and romi et al. (2002). the first cluster was in ten locations in esfahan, east azarbaijan, west azarbaijan, khorassan, kurdestan, and mazandaran provinces with mean and standard deviation (sd) 14.89±1.13, n= 79 was fitted to an. messeae, the second group with one location in gilan province (11±1.58, no= 5) fitted to an. atroparvus, the third one with two location in fars and west azarbaijan provinces (27.43± 031, n= 20) fitted to an. melanoon and the final group with four locations in khuzestan, west azarbaijan, qazvin provinces (36.84±1.91, n= 45) fitted to an. sacharovi (table 1). iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 31 (b) an. sacharovi (a) (b) an. maculipennis sensu lato (a) fig. 1. dorsal (a) and ventral (b) view the of the ivv abdominal segments of larvae in an. sacharovi and an. maculipennis s.l. (setae 2 is shown by arrow) table 1. location, mean and standard deviation (sd) of seta2 branches, and the number of larval specimens studied among iranian an. maculipennis subgroup. mean of seta2 branches ±sd species location no. larvae province 27.21±4.28 an. melanoon dasht khezri 14 fars 36.92±3.79 an. sacharovi taherabad 13 qazvin 34.55±7.55 an. sacharovi nazarabad 9 qazvin 14.40±2.51 an. messeae esmailtarkhan 5 esfahan 14.84±2.91 an. messeae takhtolia 13 east azarbaijan 16.57±4.57 an. messeae dehriz 7 west azarbaijan 16.16±2.92 an. messeae band rezaie 6 west azarbaijan 36.66±8.47 an .sacharovi a marsh near jabal 6 west azarbaijan 14.71±1.70 an. messeae ghagharalu 7 west azarbaijan 27.66±7.22 an. melanoon dehjabal 6 west azarbaijan 15.47±4.001 an. messeae sarvelayat 17 khorassan 15.66±2.80 an. messeae marsuk 6 khorassan 39.23±6.15 an. sacharovi kaldusakh 17 khuzestan 13.20±1.30 an. messeae gheybisur 5 kurdistan 14.80±3.42 an. messeae sarik 5 east azarbaijan 13.12±1.80 an. messeae abdangeh 8 mazandaran 11±1.58 an. atroparvus fuman 5 gilan iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 32 fig. 2. geographical distribution of an. sacharovi and an. maculipennis s.l. in iran based on the mean number of seta 2 branches of available larval slides in the medical entomology museum of school of public health, tehran university of medical sciences, iran. an. sacharovi, an. messeae, ▲an. melanoon, an. atroparvus. 1. east azarbaijan, 2. west azarbaijan, 3.kurdistan, 4.guilan, 5.mazandaran, 6.qazvin, 7.esfahan, 8. khorassan, 9.khuzestan, 10.fars. an. messeae an. atroparvus an. melanoon an. sacharovi (a) an. messeae an. atroparvus an. melanoon an. sacharovi (b) fig. 3. (a) the simple dendogram based on the mean number of seta 2 branches from this study (a) in comparison to the dendogram (b) which taken from marinochi et al. (1999) . iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 33 discussion although advanced tools provide diagnostic criteria, egg morphology has remained as a traditional and useful technique to identify the members of the complex for many years. however, seasonal variation in characters such as presence and absence of egg floats (mer 1937), and intra-specific polymorphic egg surface patterns, indicate that recognition by egg morphology is not completely accurate (linton et al. 2002b). recently, dna sequencing of the mitochondrial cytochrome c oxidase gene (coi) (linton et al. 2003, sedaghat et al. 2003b), and the nuclear its2 rdna have been employed to differentiate members of the complex (marinucci et al. 1999, proft et al. 1999, romi et al. 2000, linton et al. 2002a,b, 2003, sedaghat et al. 2003a,b). dna-based techniques are reliable and not limited to specific developmental stages or to specific sex (collins and paskewitz 1996). nuclear its2 region has also been used to generate phylogeny of members of the complex (marinucci et al. 1999, kampen 2005, dinparast jadid et al. 2007). in spite of the role of the members of maculipennis subgroup in transmission of malaria and reporting different species of the subgroup in iran, so far they have not been differentiated in the larval stage. the information provided in this study is the first one on an. maculipennis subgroup in the larval stage in the country which compared with those available in the world. bates (1939) and romi et al. (2002) used the number of branches of setae 2-iv and v to separate the species composition of an. maculipennis complex. in this study, an. sacharovi was found in qazvin, khuzestan, and east azarbaijan. before, this species was reported from this provinces based on the morphological character of egg and adult and/or molecular method (dow 1953, dinparast-jadid et al. 1990, momeni et al. 1992, dinparast-jadid et al. 2001, sedaghat et al. 2003a). recently, dinparast-jadid et al. reported this species based on the molecular information from gilan and ardebil povinces (dinparast-jadid et al. 2007). anopheles atroparvus has been reported from gilan and mazandaran based on egg pattern and/or molecular method (dinparast-jadid et al. 2001, 2007). an. melanoon was found in east azarbaijan and fars provinces in this study. although, this species previously was reported in north and north-eastern areas of the country based on the egg morphology (dow 1953, dinparastjadid et al. 1990, azari-hamidian et al. 2003), there is no information about the distribution of this taxon in fars province; different authors only mentioned the occurrence of an. sacharovi and an. maculipennis sensu lato in fars. an. messeae was found in esfahan, kurdestan, east azarbaijan, west azarbaijan, khorassan and mazandaran in this study. before, an. messeae has been reported from gilan or mazandaran provinces based on the molecular method, adult morphology (wing scale index) and egg morphology (minar 1974, momeni et al. 1992, dinparast jadid et al. 2001, azari-hamidian et al. 2003, dinparast-jadid et al. 2007). this species was reported based on the egg morphology from esfahan by de zulueta et al. (1957). however, the occurrence of an. messeae has not been confirmed in esfahan by other authors. this study showed that the means of the seta 2 branches of an. messeae from different areas was more similar than an. maculipennis from different areas. the results of this study can be used as preliminary information in the future works on sibling species. based on the comparison of two dendograms, a from this study and b, taken from romi et al. (2002); it showed that there was conformity to separate an. atroparvus and an. sacharovi in two divide monophyletic groups, but there was no conformity for an. melanoon and an. messeae in two dendograms (fig. 3). in dendogram b, which was based on its2 sequence data, an. sacharovi placed in a basal position within the rest of taxa. previous studies iranian j arthropod-borne dis, (2007), 1(1): 28-37 s doosti et al.: applying morphometric variation of… 34 derived from rdna sequence data isolated an. sacharovi from the other siblings (kampen 2005, dinparast-jadid et al. 2007). this position also supported the phylogenetic relationships inferred from the previous analyses of polytene chromosome banding patterns (white 1978, stegnii 1981). moreover a parsimony analysis of mtdna data generated by sedaghat supported this position of an. sacharovi (sedaghat 2003). in this study, an. atroparvus + an. messeae formed a separate clade. this position is in agreement with the phylogenetic relationships based on its2 sequences defined by kampen (2005). however, dinparast-jadid et al. could not show such relation between an. atroparvus + an. messeae (dinparast-jadid et al. 2007). the method which employed in this paper can be applied in integrated systematics studies. it is not possible to provide a meaningful morphological diagnosis of an. maculipennis s.l. at this time because the other members of the subgroup have not been studied in detail. to date, the life stages of only an. maculipennis, an. sacharovi and an. daciae from this subgroup have been described in detail (linton et al. 2003, sedaghat et al. 2003b, nicolescu et al. 2004, respectively). however, despite these studies there are not enough publications that give a detailed morphological description of each taxon, while a thorough morphological knowledge of all anopheles species is essential. the taxonomic recognition of these species should be based on a combination of morphological, biological and molecular methods on the adults, egg, larva and pupa of the members of the group. a detailed comparative studies of associated all life stages of unambiguously identified samples using of progeny broods, is an important resource for systematic studies. although malaria transmission in north and central areas of iran was completely controlled, travelling from endemic southern areas of the country and neighbouring countries and also presence an. maculipennis subgroup as potential vectors, always are threats of recurrence of the disease in these areas. in conclusion, considering the medical importance of the maculipennis subgroup, and differences in vector capacities and species distributions, it is important to elucidate reliable methods to unambiguously identify the species group. acknowledgements the authors are grateful to dr s tirgari, miss m jedari and ms f faghih-naini, department of medical entomology and vector control and medical arthropods museum, school of public health and institute of 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18, 2018 original article hemodynamic changes in experimentally envenomed anaesthetized rats by intravenous injection of hemiscorpius lepturus venom khalil pourkhalili 1, hossein fatemikia 2, euikyung kim 3, navid reza mashayekhy 4, naser mohammadpour dounighi 5, abdollah hajivandi 6, yaghoob hassan 7, *ramin seyedian 8 1department of physiology, faculty of medicine, bushehr university of medical sciences, bushehr, iran 2department of physiology, faculty of medicine, shiraz university of medical sciences, shiraz, iran 3college of veterinary medicine, gyeongsang national university, jinju, south korea 4department of cardiology, amir kabir hospital, arak university of medical sciences, arak, iran 5department of human vaccine and serum, razi vaccine and serum research institute, agricultural research, education and extension organization, karaj, iran 6department of biostatistics, faculty of health, bushehr university of medical sciences, bushehr, iran 7student research committee, bushehr university of medical science, bushehr, iran 8department of pharmacology, bushehr university of medical sciences, bushehr, iran (received 14 jan 2017; accepted 13 feb 2018) abstract background: we investigated the hemodynamic changes (inotropic, chronotropic and arrhythmogenic) in intravenously envenomed anesthetized rats with hemiscorpius lepturus venom. the neutralizing potencies of different drugs and commercial antivenom were assessed simultaneously. methods: different doses of the crude venom (100, 200 and 400μg/rat) were injected during five minutes via the femoral vein and cardiovascular changes were recorded in rats in razi institute corporation, karaj, iran in 2017. the drugs (atropine, lidocaine, propranolol and prazosin) were injected before the venom for determination of the counteracting effects. different volumes (100, 500 and 1000µl) of the antivenom were pre envenomed to neutralize cardiovascular changes. results: temporary hypertension and bradycardia with no arrhythmogenic effects were depicted within twenty minutes. there was a difference in arterial pressure between the venom (400μg/rat) and the vehicle at 8 minutes (114.68±5.1mmhg versus 70.2±4.3mmhg). elevation of the mean arterial pressure was inhibited by propranolol (2 mg/kg) and neutralized by prazosin (1mg/kg) while lidocaine (4mg/kg) and atropine (1mg/kg) had no effects. premedication with iranian commercial antivenom (1000μl) produced surprisingly temporary hypertension compared to the vehicle (140.84±4.5 versus 84.3±3.2). it had no neutralizing properties on blood pressure variation before the venom injection. volume-expanded hypertension phenomenon was ruled out in a parallel study. conclusion: this venom has vasoconstrictive effects in rats probably due to the presence of norepinephrine like materials in its content or liberated from adrenal gland inhibited by prazosin premedication. the neutralizing effects of antivenom on venom-induced hypertension are questionable. keywords: hemiscorpius lepturus, vasoconstriction, polyvalent antivenom introduction more than one million scorpion stings are reported in the world annually (1, 2). the resultant mortality is less than snake envenomation but the physicians encounter morbidity sequels especially in infants (3, 4). scorpion bite is a prevalent problem in tropical areas of the world including southwestern part of iran in particular khuzestan province (5, 6). the scorpion hemiscorpius lepturus, a member of hemiscorpiodie family, belongs to iranian rich fauna from 17 genera (7). this scorpion is a member of 7 dangerous species *corresponding author: dr ramin seyedian, e-mail: raminseyedian@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 32 http://jad.tums.ac.ir published online: march 18, 2018 living in iran causing medically implications in envenomed patients (7, 8). the clinical observations of h. lepturus sting show diverse pathological forms including bloody urine due to hemoglobinuria or hemolysis, dermonecrotic reactions, cardiovascular effects, and in a few cases disseminated intravascular coagulation in infants (9, 10). although there is no comprehensive agreement about the efficacy of scorpion antivenom in envenomed patients, close monitoring and injection of pepsin-digested razi institute polyvalent raised against 6 common iranian scorpions (androctonus crassicauda, buthotus saulcyi, buthus schach, odontobuthus doriae, mesobuthus eupeus and h. lepturus) is used in this country (6, 11, 12). there is no short-term efficacy of old world scorpion antivenom in the literature (13). our former report on isolated rat hearts with langendorff apparatus showed negative inotropic and late arrhythmogenic effects of this venom like loxosceles envenomation (14, 15). the aim of this study was to evaluate the cardiotoxic properties of different doses of the scorpion venom by intravenous injection in rats. due to the role of α1-adrenoceptors in the peripheral vasculature and β1 receptors in the heart maintaining blood pressure (16), their possible impacts on the cardiovascular activity were investigated. the diverse cardiovascular effects induced by intravenous h. lepturus injection in anesthetized rats due to pharmacologically distinct causes. materials and methods scorpion venom and antivenom the crude venom was obtained by applying mild electrical shock (20mv). it was extracted with normal saline and pooled, lyophilized and stored at -20 ℃ following centrifugation at 10000rpm for 15min. the multivalent antivenom (5ml ampoules, stored at 2–8 ℃) was a pepsin-digested and concentrated preparation obtained from equine hyperimmune serum in razi institute corporation, karaj, iran in 2017. the protein content of this product was 3.6 mg/ml with a neutralizing potency of 26 ld50/ml. chemicals and drugs atropine sulfate, prazosin hydrochloride, lidocaine hydrochloride and propranolol hydrochloride were purchased from the sigma company (germany). all the chemicals used were of the purest grade available. anaesthetized rats male wistar rats (250–300gr) were placed in polycarbonate cages with free access to water and normal laboratory chow in the animal house of bushehr university of medical sciences. three animals in each cage. all the animals were kept at 20±2 ℃ and maintained at 12h light-dark cycle starting at 7am. rats were anesthetized with ketamine (100mg/kg, ip) and xylazine (10mg/kg, ip), placed supine on a heated surgical table to keep the animal warm at 37±1 ℃, monitored through a rectal probe connected to a thermistor (physitemp bat-12, texas scientific instruments, san antonio, texas, usa). a cannula was inserted into the right femoral vein for administration of venom and drugs. another one was placed into the right femoral artery and connected to a pressure transducer (mlt844, ad instruments, australia) for continuous recording of the arterial pressure by means of a power lab/4sp data acquisition system (ad instruments). animals were allowed to become stable for 20min prior to administration of the venom or any drugs. effects of hemiscorpius lepturus venom on hemodynamic parameters rats were divided into four groups (n= 5) and baseline hemodynamic status was recorded for twenty minutes before venom injection. the first group was injected with normal saline (200μl) via femoral vein as the negative control. each dose of the venom (100, 200, and http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 33 http://jad.tums.ac.ir published online: march 18, 2018 400μg/rat) dissolved in normal saline (200μl) was injected into experimental animals. mean arterial pressure, heart rate and arrhythmogenic properties were evaluated compared with negative control. experimental protocol cardiovascular changes provoked by the venom (400μg/ 200μl) were measured in the presence or absence of various chemicals. in this experiment, prazosin (1mg/kg), the α1 receptor antagonist was injected (n=5) via femoral vein before venom installation. this study was performed with propranolol (2mg/ kg), atropine (1mg/kg) and finally lidocaine (4mg/kg) in similar examinations. the hemodynamic effects were analyzed in the absence or the presence of each testing reagent. antivenom premedication effects rats were randomly divided into four experimental groups (n=5). escalating doses of the antivenom (100, 500, and 1000μl) dissolved in normal saline with same volume (1ml) were injected in 5min and venom instillation via femoral vein was performed after returning of arterial pressure to the premedication state. neutralizing effects of this remedy on hemodynamic changes were compared with the control groups. data analysis statistical analysis was performed using spss version 16.0 (chicago, il, usa). repeated measurement anova was used to detect any differences among means in different time intervals. data were expressed as mean±sd. the level of statistical significance was p< 0.05 animal ethics ethical approval of all animal experiments was obtained from the bushehr university of medical sciences animal ethics committee (ir.bpums.rec.1396.90). results hemodynamic effects of hemiscorpius lepturus venom transient blood pressure elevation and bradycardia induced with different doses (100, 200, and 400μg) of the venom were depicted in table 1 and fig. 1, respectively. results represent mean ± sd of five independent experiments. intravenous injection of venominduced an elevation in mean arterial blood pressure (positive inotropic) and the decrease in heart rate (negative chronotropic) for 20min before ending the experiment an hour later. the minimum effective dose that dramatically evoked this effect (400μg/per rat) was used for further studies. no arrhythmogenic effects induced by the venom injection were seen compared with control (fig. 2). no mortality was observed during the time following this study in each group. existing data on controls revealed that the stress and surgical operation had no significant effects on hemodynamic changes and arrhythmogenic states in our experiments. effects of pharmacological antagonists upon hemiscorpius lepturus induced blood pressure alterations pretreatment with lidocaine (4mg/kg) as a sodium channel blocker had no effects on blood pressure elevation (n= 5) (fig. 3d). atropine (1mg/kg) as an anticholinergic drug could not inhibit this phenomenon (fig. 3c) but counteracted bradycardia (data not shown). pretreatment with propranolol (2mg/kg), a nonselective beta antagonist, suppressed rising of blood pressure (fig. 3b) and finally prazosin, a selective alpha-1adrenergic blocker, almost completely neutralized the responses induced with h. lepturus venom in rats (fig. 3a). effect of prophylactic treatment with antivenom on mean arterial pressure a large increase in blood pressure recorded http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 34 http://jad.tums.ac.ir published online: march 18, 2018 following injection of razi institute antivenom (100, 500 and 1000μl) in anesthetized rats via femoral vein in 5min. antivenom injection had no neutralizing effects on raising arterial pressure induced by the venom in all experiments as shown in fig. 4 (a, b, c). injection of physiological saline (1ml) had no effects on the mean arterial pressure in treated rats ruling out volume-expanded hypertension (fig. 5). pretreatment with these remedies did not induce the arrhythmia in anesthetized rats (not shown). table 1. mean arterial pressure in treated rats with hemiscorpius lepturus venom injection time (min) control dose(µg/rat) 100 200 400 0 70.1±3.4 66.7±4.3 74.3±5.1 60.9±5.6 4 69.4±2.8 77.7±3.6 83.1±4.3 95.3±4.9* 8 70.2±4.3 79.9±3.6 103.2±5.4* 114.7±5.1* 12 67.8±3.8 73.1±4.1 92.2±3.6* 102.4±5.5* 16 63.3±3.4 79.9±4.1 84.3±3.2 92.6±4.5* 20 61.2±3.8 73.5±3.6 71.2±4.2 78.6±4.1 values are the mean ± sd of the mean arterial pressure of the five animals before and after intravenous injection of hemiscorpius lepturus venom. *significant difference from control group with anova test, p< 0.05 fig. 1. heart rate changes with intravenous injection of hemiscorpius lepturus in rats. values are means ± sd of five rats/group fig. 2. arrhythmogenic analsis of hemiscorpius lepturus venom upon intravenous injection in rats http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 35 http://jad.tums.ac.ir published online: march 18, 2018 fig. 3. neutralizing effects of different drugs (prazosin, propranolol, atropine, and lidocaine) upon blood pressure elevation evoked by hemiscorpius lepturus injection. the trace represents a single rat receiving a single dose of venom (representing measurements of five rats). fig. 4. intravenous pretreatment with different doses (100, 500, 1000μl) of multivalent antivenom diluted with normal saline, evoked an increase in blood pressure in anesthetized rats. this remedy could not neutralize temporary changes in blood pressure evoked by hemiscorpius lepturus venom http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 36 http://jad.tums.ac.ir published online: march 18, 2018 fig. 5. effects of iranian commercial multivalent antivenom diluted with normal saline on the mean arterial pressure of anesthetized rats. all remedies have equal volume (1ml). data represent mean ± sd (n= 5) discussion scorpions belonging to class arachnidae are relatively different from other arthropods including insects, crustacea and myriapods (17). a large number of deadly scorpions belong to the family of buthidae while some species are from at least two other families hemiscorpiidae and scorpionidae posing a threat to humans (18). most cases of scorpion stings (95%) only cause local discomfort including pain while in minority of envenomed people devastating manifestations can be observed, involving nervous system, autonomic system and finally respiratory and heart failure leading to death especially in infants (19). hemiscorpius lepturus and androctonus crassicauda are the two most important hazardous scorpions in iran (20). very few observations explain the complex cardiovascular alterations and hemodynamic effects induced by iranian scorpions like h. lepturus in animals (21, 22). escalating doses of h. lepturus venom were selected in our study on the basis of arterial pressure modulations regardless of their ld50 via subcutaneous injection in rats (3.22mg/ kg) (23). there was no mortality following intravenous injection of the venom (400μg/ rat) up to an hour indicating that its strength in creating the cardiovascular response is much less than other venomous animals especially jellyfish and other scorpions (24-26). some scorpions like leiurus quinquestratus could produce cardiovascular effects including hypertension, arrhythmia, tachycardia and enzymatic changes in envenomed patients showing myocardial damage (27). widened qrs complexes, st depression or elevation and the inversion of t and q waves were not seen in our experiment up to 20min ruling out acute myocardial ischemia and infarction-like patterns as other scorpions (2830) (fig. 2). venom-induced bradycardia was accordant with the previous study performed by subcutaneous injection (1500μg/kg) in rabbits (31). the inotropic potency, arrhythmogenic properties and neutralization efficacy of pre and post treatment with iranian antivenom in our study were completely different. intravascular instillation of h. lepturus provoked extreme elevation of mean arterial pressure after 8min compared to normal saline (114.7±5.1 mmhg versus 70.2±4.3mmhg) probably due to release of catecholamines like substances including epinephrine and especially norepinephrine from the sympathetic nervous system and adrenal glands (32). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 37 http://jad.tums.ac.ir published online: march 18, 2018 another possibility was the presence of norepinephrine like materials in its own venom like other venomous creatures (33). premedication with lidocaine (4mg/kg) as a sodium channel blocker and atropine (2mg/kg) as an anticholinergic drug had no neutralizing or summative effects on blood pressure alterations respectively. it was accordant with previous experiments carried on overwhelming hemodynamic effects induced by marine venoms in rats (34, 35). this experiment is ruling out the activation of voltage-sensitive sodium channels in this phenomenon with our venom (36). propranolol treatment as a non-selective beta blocker (2mg/kg) suppressed arterial pressure alterations in our study signifying the role of adrenergic receptors in blood pressure changes (37). prazosin premedication as a selective alpha-adrenergic blocker (1mg/kg) neutralized almost completely undesired effects on the vasculature (38). venom-induced bradycardia might be due to cholinergic and adrenergic activities in rats (fig. 2). atropine premedication as an anticholinergic drug neutralized induced bradycardia totally while propranolol as a nonselective beta blocker enhanced it (data not shown). intravenous pretreatment with different doses of commercial iranian antivenom (100, 500, 1000μl) with the same volume had no effects on hypertension-induced with h. lepturus injection as shown in fig. 4 (a, b, c). the mean arterial pressure was elevated abruptly by injection of this remedy (1000μl) in five minutes while normal saline with the same volume had no effects (140.8±4.5 versus 84.3±3.2). more evaluation must be carried out to find the impurities in this product responsible for this undesired phenomenon. the hypertensive potencies of this venom are less than other scorpions like leiurus quinquestriatus (1000μg/kg versus 350μg/kg) (39). this may raise concerns about the liberation of neurotransmitters like norepinephrine responsible for the elevation of blood pressure and bradycardia by releasing of acetylcholine from vagal ganglia. it requires performing this experiment in adrenalectomized rats treated with guanethidine in our further studies to reveal the exact mechanism of this phenomenon (32). neutralization of hemodynamic changes with prazosin as a selective alpha-1 blocker supports this possibility. our results on blood pressure were not compatible with the previous study on isolated rat hearts (15) possibly due to cardiotoxicity induced by enzymes like sphingomyelinase-d presenting in both loxosceles intermedia and h. lepturus venoms with similar clinical manifestations (40, 41). there is no consensus on using scorpion antivenom for treatment of undesirable cardiovascular changes in a human being after envenomation especially for old world scorpions (42). even in the united states, using antivenom should be limited to severe envenomations due to its questionable effects and high costs (43). prazosin is used for decreasing of peripheral vascular resistance in envenomation especially in india while its effectiveness in reduction of mortality and morbidity is approved only in a small controlled trial (44). special evaluation must be carried out for finding specific cardiac receptors to evaluate the cardiovascular events thoroughly. conclusion scorpion envenomed patients are usually treated with symptomatic therapy and commercial antivenom infusion. unfortunately, iranian antivenom premedication and post medication (data not shown) either increase the blood pressure or had no neutralizing effects in transient hemodynamic changes induced by h. lepturus injection in rats. little is known about the role of other mediators (serotonergic, prostaglandins and nitric oxide inhibitors) to increase blood pressure in http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 31–40 kh pourkhalili et al.: hemodynamic changes in … 38 http://jad.tums.ac.ir published online: march 18, 2018 envenomed rats with this venom. nonetheless, more investigations must be performed to find the simplest way for counteracting venom-induced undesirable cardiovascular alterations in rats. acknowledgements the authors would like to thank the authorities of bushehr university of medical sciences for providing facilities. the authors declare that there is no conflict of interests. references 1. isbister gk, bawaskar hs (2014) scorpion envenomation. n engl j med. 371 (5): 457–463. 2. chippaux jp, goyffon m (2008) epidemiology of scorpionism: a global appraisal. acta trop. 107(2): 71–79. 3. diaz p, chowell g, ceja g, d'auria t, lloyd r, castillo-chavez c (2005) pediatric electrocardiograph abnormalities following centruroides limpidus tecomanus scorpion envenomation. toxicon. 45(1): 27–31. 4. freire-maia l, campos j, amaral c (1994) approaches to the treatment of 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al.: comparison of some … 153 http://jad.tums.ac.ir published online: june 24, 2019 original article comparison of some molecular markers for tick species identification *eman m. abouelhassan1; hamdy m. elgawady1; ahmad anwar abdelaal1; amal k. elgayar1; maria d esteve-gassent2 1department of veterinary parasitology, suez canal university, ismailia, egypt 2department of veterinary pathobiology, texas a and m university, college station, texas, united states of america (received 15 mar 2018; accepted 22 apr 2019) abstract background: ticks are obligate blood-sucking ectoparasites of vertebrates. since many tick identification studies are based on the analysis of 16s rdna, 12s rdna and its-1, 2 rdna genes, we aimed to compare the performance of these molecular markers of common use for the identification of ticks, under a diagnostic laboratory environment. methods: overall, 192 tick specimens were collected through the state of texas from january 2014 to august 2015 and the species was determined by both morphology and molecular amplification using the 16s rdna, 12s rdna, its1 and its2. results: the species collected were identified by molecular techniques as dermacentor albipictus, d. variabilis, amblyomma americanum, ixodes scapularis, a. cajennense, rhipicephalus sanguineus and carios capensis. its1 and its2 were not able to prove consistent amplification and therefore have been considered as potential markers for tick identification. conclusion: the use of mitochondrial genes in tick identification showed to provide more consistent results in the diagnostic environment. keywords: ticks; dermacentor; amblyomma; rhipicephalus; ixodes introduction ticks are obligate blood-sucking ectoparasites of vertebrates, causing great economic losses to livestock with its direct and indirect effects on hosts. bloodsucking by large numbers of ticks causes a reduction in live weight and anemia among domestic animals, while their bites also reduce the quality of hides (1). in addition, certain ticks will cause tick paralysis, which is an acute ascending flaccid motor paralysis caused by the injection of a toxin by the tick while feeding. however, the major effects caused by ticks are due to their ability to transmit protozoan, bacterial and viral diseases to livestock, companion animals and humans (16, 17). ticks are currently considered to be second only to mosquitoes as vectors of human infectious diseases in the world (9, 23). a number of bacterial zoonotic infec tious diseases (2) such as anaplasmosis, ehrlichiosis, and lyme borreliosis are transmitted by ticks. table 1 shows the pathogens transmitted by different species of ticks as ixodes species are the vectors of lyme borreliosis, amblyomma americanum is the vector of ehrlichia chaffeensi, tick identification helping in the diagnosis of disease transmitted with, and misidentification of the may lead to difficult and even wrong disease diagnosis. even though there is a lack of whole tick genome annotation, molecular techniques in acarology have been made available in the past few years (9), traditionally tick identification has always been based on morphological characteristics. moreover, to identify the immature tick stages (larvae, nymph and adults) *corresponding author: dr eman mohammed abouelhassan, e-mail: hassanemy@yahoo.com j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 154 http://jad.tums.ac.ir published online: june 24, 2019 separate keys are normally used (2, 3). in certain situation, damage to tick body parts essential for their identification (such as capitulum and adjacent structures) may occur during removal of attached ticks to their hosts. in addition, bad preservation of tick samples often occurs, leading to incorrect identifications (4). identification of some tick species like rhipicephalus sanguineus and amblyomma cajennense is difficult due to the fact that they have been classified as a species complex (11, 12). for instance, a. cajennense is a complex of 6 species, while r. sanguineus group comprises a total of 17 different species. in these complexes, the different species are geographically separated, due to the large geographical range of distribution, and the expected adaptation of tick populations to different environmental conditions (12). therefore, morphological identification of these species is not sufficient and the further molecular information is needed for a correct species determination (11, 12). these difficulties may be reduced when using molecular techniques for tick identification (3). another benefit from molecular techniques is that with those samples that tick dna integrity has not been compromised, from the total dna extraction, a collection of different tick-borne pathogens can be detected by molecular methodologies such as conventional pcr, and real-time pcr (4). therefore, with one single extraction, both the agent and the vector species can be determined (4). moreover, the availability of genetic sequence will provide the opportunity to study both the vector population diversity, as well as the pathogen they carry, and potentially even their relationships (13). currently, there is a lack of whole tick genomes readily annotated (https://www.vectorbase.org/), with ixodes scapularis being the only one currently available (https://www.vectorbase.org/organisms/ixod es-scapularis) (13). this lack of information limits the advancement of the development of new molecular methods for the study of these arthropods. one of the limitations is the large size of tick genomes (15). for instance, the average haploid genome of the tick i. scapularis has been calculated at 2262mbp in length, while the a. americanum is around 3108mbp. if we compare this to the human genome, tick genomes tend to be twice as bigger as the human genome. part of the difference in size is due to the presence of non-coding regions with extended tandem repeats, that difficulty significantly the sequencing and annotation of those genomes (15). consequently, different molecular markers have been traditionally used for the phylogeny of ticks (8). those include the nuclear ribosomal genes 18s rdna, 28s rdna and its-1, 2 rdna as well as mitochondrial genes such as 16s, 12s, coi, coiii) rdna (8, 9). el-fiky and el kammah (3) and chitimia (4) successfully used the internal transcribed spacer (its) for the identification of dermacentor marginatus, ixodes ricinus, haemaphysalis, boophilus, and rhipicephalus sanguineus tick species. on the other hand, the 16s rdna were able to construct the phylogeny of both hard and soft ticks (6, 8, 9). 16s rdna were used in molecular classification of metastriate ticks (dermacentor, amblyomma and rhipicephalus respectively (10, 13). since many tick identification studies are based on the analysis of 16s rdna, 12s rdna and its-1, 2 rdna genes, the objective of the present study was to compare the performance of these molecular markers of common use for the identification of ticks, under a diagnostic laboratory environment (13). materials and methods tick sample collection overall, 192 specimens were utilized in this study. this collection was divided into two groups, group a comprises 59 ticks (larvae, nymph, males and engorged females) obtained from texan citizens through the tick testing https://www.vectorbase.org/organisms/ixodes-scapularis https://www.vectorbase.org/organisms/ixodes-scapularis j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 155 http://jad.tums.ac.ir published online: june 24, 2019 service provided by the lyme laboratory at texas a and m university, from january 2014 to august 2015. on the other hand group, b contains 133 tick pools collected from wildlife through an ecology project conducted in collaboration with dr. castro-arellano at texas state university (table 2). for better dna extractions, larvae and nymphs were analyzed in pools, sometimes the pools contain one larvae and other up to 50 larvae of the same tick species collected from the same location in the same sampling effort. nymphs, on the other hand, were pooled in groups of from one till 15 specimens, following the same strategy described for larvae for optimal dna extraction. for the majority of these ticks, the morphological identification was not enough to determine the species, mostly due to either bad storage of samples or loss of mouth-parts while removal the tick from the host. these ticks were immersed in 70% ethanol solution and then processed for dna extraction and molecular identification using 16s rdna pcr specific primers and sequencing the pcr product. dna extraction dna was extracted from the tick samples using wizard® sv genomic dna purification kit (promega corporation, madison, wi) following manufacturer’s recommendations with modifications. briefly, ticks were incubated for 10min at 70 ºc in 200µl of nuclei lysis solution, plus 50µl of 0.5m edta, 40 µl of a 20mg/ml proteinase k solution, and 5µl rnase a solution. after the initial digestion and for the optimal extraction of dna from the arthropods, adult individual ticks were homogenized utilizing the bead mill bead ruptor 24 (omni international, inc., kennesaw, ga), un-engorged ticks were homogenized with 1.4mm ceramic beads while 2.8mm ceramic beads were used with engorged ticks. after homogenization, tubes were centrifuged at 10,000×g to eliminate tick debris. supernatants were collected and 250µl of wizard® sv lysis buffer was added to each sample and the mixture. the mixture was run through filter columns at 13,000×g for 3min. dna bound to filter was washed and eluted following manufacturer recommendations. to extract dna from the tick immature stages (nymphs and larvae) pools of a maximum of 15 nymphs or 50 larvae were made. specimens received a code indicating the type of pool generated. all immature specimens were stored at -80 ºc with 100µl te buffer for at least one hour. specimens were homogenized utilizing pestles while the samples were frozen, followed by dna extraction procedures using the prepgem™ (zygem ltd., new zeland) insect dna extraction kit following manufacturer’s recommendations. briefly, tick samples were mixed with ultra-pure water, 10x buffer provided in the kit, and 1µl of the prepgem™ enzyme (zygem ltd., new zeland). the mixture was incubated at 75 °c for 15min followed by incubation at 95 °c for 15min. the extracted dna concentration and purity were measured using a nanodrop, and stored at -20 until use. molecular identification of ticks based on 16s rdna gene, 12s rdna and its-1 and 2 rdna genes the tick 16srdna was amplified from each specimen studied using conventional pcr methodologies and utilizing primers (6) (table 3) and accuprime™ supermix (quanta bioscienceinc., gaithersburg, maryland). the pcr was run following the cycling condition: initial denaturation at 95 ºc for 5min followed by 10 cycles of 92 ºc for 1min, 48 ºc for 1min and 72 ºc for 90sec, this step was followed by additional 32 cycles of 92 ºc for 1min, 54 ºc for 35sec and 72 ºc for 90sec, this was followed by a final extension at 72 ºc for 7min (5). the amplification products from 16s rdna were separated on 1.6% agarose gel containing 0.4µg/ml of ethidium bromide (bio-rad laboratoies inc., hercules, ca) at 90 volts for 40–60min, and imaged using chemidoc touch imaging system (bio-rad laj arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 156 http://jad.tums.ac.ir published online: june 24, 2019 boratoies inc., hercules, ca). positive bands were excised from the gel and purified using the wizard® sv gel and pcr clean-up system (promega corporation, madison wi) following manufacturer’s recommendations. the purified products were sent for sequencing (eton biosciences, san diego, ca). sequences were analyzed through blast® in macvector 14.0.0 software (macvector inc., cary, nc). pcr was performed first using16s rdna gene primers. the same samples were also tested using specific primers for first and second internal transcribed spacers (its-1 and its-2 rdna) (table 3) following methodologies (4). the pcr reaction was done using the following cycling condition: initial denaturation at 95 ºc for 5min followed by forty cycles of 95 ºc for 45sec, 55 ºc for 1min and 72 ºc for 90sec with a final extraction at 72 ºc for 1min. the amplification products were separated on 1.6% agarose gel containing 0.4µg/ ml of ethidium bromide (bio-rad laboratoies inc., hercules, ca) and the gel was run at 90 volts for 40–60min. gels were visualized using the chemidoc touch imaging system (biorad laboratoies inc., hercules, ca). in 12s rdna, pcr was done following the cycling condition: initial denaturation at 95 ºc for 5min followed by forty cycles of 95 ºc for 30sec, 40 ºc for 30sec and 72 ºc for 30sec, with a final extraction at 72 ºc for 5 min. the amplification products were separated and visualized as mentioned before on 1.6% agarose gel containing 0.4µg/ml of ethidium bromide (bio-rad laboratoies inc., hercules, ca) and the gel was run at 90 volts for 40–60min. gels were visualized using the chemidoc touch imaging system (bio-rad laboratoies inc., hercules, ca). sequence analysis positive bands were excised from the gel and purified using the wizard® sv gel and pcr clean-up system (promega corporation, madison wi) following manufacturer’s recommendations. the purified products were sent for sequencing (eton biosciences, san diego, ca). sequences were analyzed through blast® using macvector 14.0 software (macvector inc., cary, nc). results overall, 192 ticks were analyzed from different developmental stages (larvae, nymph, males and engorged females) collected through the state of texas. in this collection, the tick specimens were identified based on the16s rdna pcr products as dermacentor albipictus, d. variabilis, amblyomma americanum, ixodes scapularis, a. cajennense rhipicephalus sanguineus and carios capensis (table 4), the genbank accession numbers from kx673167 to kx673180. comparison between 16s rdna gene, 12s rdna gene and (its-1, 2) rdna genes in order to evaluate which genetic marker perform best under diagnostic conditions, comparison between the amplification of four genes 16s rdna, 12s rdna, its1 and its2 rdna genes was done. positive bands from 16s rdna, 12s rdna pcr were excised from the gel and cleaned and submitted for sequencing. sequences were analyzed through blast® in macvector 14.0.0 software (macvector inc., cary, nc). in spite of the samples number are not representative, but changing in the tick pcr amplification of the four genes was observed as good amplification for the samples were observed as both 16s rdna, 12s rdna gene showing good pcr amplification in all the sample but (its-1, 2) rdna genes failed to amplify some samples species, (fig. 1). the samples utilized were dermacentor albipictus (cvm11, cetx-2), dermacentor variabilis (arco1), amblyomma americanum (mmsl5, mmsl9), ixodes scapularis (cstx-2, cstx-3), amblyomma cajennense (bas 115) and rhipicephalus sanguineus j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 157 http://jad.tums.ac.ir published online: june 24, 2019 (sat 88, sat 97, and sat 101). in its-1pcr, one of the three samples of rhipicephalus sanguineus (sat 101), two samples of amblyomma americanum (mmsl5, mmsl9), ixodes scapularis samples cstx2, cstx-3) and dermacentor albipictus samples (cvm11, cetx-2) were amplified, but there were no amplification in dermacentor variablis (arco1), amblyomma cajennense (bas 115) and the other two samples of r. sanguineus (sat 88, sat 97). its-2 pcr, one of the three samples of rhipicephalus sanguineus (sat 101), two samples of amblyomma americanum (mmsl5, mmsl9), dermacentor variablis (arco1), amblyomma cajennense (bas 115), ixodes scapularis samples cstx-2, cstx-3) and dermacentor albipictus samples (cvm11, cetx-2) were amplified, but the two samples of r. sanguineus (sat 88, sat 97) failed to amplify. phylogenetic analysis the phylogenetic analysis was performed using macvector 14.0 software (macvector inc., cary, nc) and the tree was constructed using neighbor-joining (nj) methods (figs. 2-4). the low degree of sequences variation observed within most of the species of the soft and hard tick trees based on the 16s rdna since they all share the same ancestor. nevertheless, there is one sequence in the hard tick population studied rhipicephalus sanguineus that show higher variation. fig. 1. pcr amplification utilizing tick samples of different species using: (a) the its-2pcr reaction (b): its-1 pcr reaction and (c): 16s r dna gene, (d)12srdna dna ladder is located on the left and right sides of the gel, fragment sizes are represented in base pairs (bp), 1: rhipicephalus sanguineus sample (sat 97), 2: rhipicephalus sanguineus sample (sat88), 3: rhipicephalus sanguineus sample (sat 101), 4: amblyomma americanum sample (mmsl5), 5: amblyomma americanum sample (mmsl9), 6 amblyomma cajennense (bas 115 tick): 7: ixodes scapularis sample (cstx-2), 8: ixodes scapularis sample (cstx-3), 9: dermacentor albipictus sample(cvm11), 10: dermacentor albipictus sample(cetx-2), 11: dermacentor variabilis sample(arco1) and 12: negative control. table 1. important tick-borne diseases of humans pathogens disease vectors distribution reference borrelia burgdorferi senso lato lyme borreliosis ixodes ricinus, i. pacificus, i. scapularis, i. hexagonus asia, europe, north america 17,18,19 ehrlichia canis human ehrlichiosis rhipicephalus sanguineus south america, asia, africa 19 ehrlichia ewingii human ewinigii ehrlichiosis amblyomma americanum usa, africa, asia 20 ehrlichia muris murine splenomegaly haemaphysalis spp, ixodes spp eurasia 20 j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 158 http://jad.tums.ac.ir published online: june 24, 2019 ehrlichia chaffeensis human monocytic ehrlichiosis amblyomma americanum north america 17,18,19 ehrlichia ruminantium heartwater in ruminant amblyomma spp africa, caribbean 20 rickettsia conorii mediterranean spotted fever rhipicephalus sanguineus, r. turanicus africa, asia, europe 17,18,19 coxiella burnetii q fever many species africa, asia, europe, north america, australia 17,18,19 rickettsai rickettsii rocky mountain spotted fever amblyomma americanum, rhipicephalus sanguineus, dermacentor variablis north, south and central america 17,18,19 anaplasma phagocytophilum human granulocytic anaplasmosis haemaphysalis concinna, h. punctate, ixodes ricinus, i. pacificus, i. scapularis rhipicephalus bursa north america, europe 17,18,19 flavivirus tick borne encephalitis ixodes ricinus, haemaphysalis concinna, h. punctate asia, europe 18,19 babesia divergen, b. microti babesiosis ixodes ricinus, i. scapularis north america, europe 17,18,19 fig. 2. the phylogenetic analysis was constructed using neighbor joining method, to construct the tick phylogenetic tree of some of soft tick species sequences from the genbank and our sequences samples are included based on 16s r dna sequences table 1. continued … j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 159 http://jad.tums.ac.ir published online: june 24, 2019 fig. 3. the phylogenetic analysis was constructed using neighbor-joining method, to construct the tick phylogenetic tree of some of hard tick species sequences from the genbank and our sequences samples are included based on 16s r dna sequences j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 160 http://jad.tums.ac.ir published online: june 24, 2019 fig. 4. the phylogenetic analysis was constructed using neighbor-joining method, to construct the tick phylogenetic tree of some of hard tick species sequences from the genbank and our sequences samples are included based on 12s r dna sequences table 2. ticks samples utilized in this study according to their distribution and stages distribution adult female adult male nymph larvae arroyo, colorado 1 mason mountain 2 brazos county, texas 24 1 1 jefferson county 2 2 1 texas 2 san antonio, tx 3 gus engeling wma 2 5 tejas ranch 6 chaparral wma 4 10 las palomas wma-arroyo colorado unit 86 52 total 28 3 99 74 table 3. primers using in pcr gene f-primers r-primers ref tick 16s rdna 5´-ttgggcaagaagaccctatgaa -3´ 5´ccggtctgaactcagatcaagt-3´ (5) tick 12s rdna 5´-gaggaatttgctctgtaatgg -3´ 5´-aagagtgacgggcgatatgt-3´ (21) its-1rdna 5´-tcataagctcgcgttgatt-3' 5´-agctggctgcgttcttcat 3' (3) its-2rdna 5´-cgagcttggtgtgaattgca-3´ 5´-tcccatacaccacatttcccg-3' (3) j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 161 http://jad.tums.ac.ir published online: june 24, 2019 table 4. ticks samples utilized in this study according to their distribution and species no. samples name tick species location developmental stage 1 arco1 dermacentor variabilis arroyo, colorado larvae 2 mmsl7 amblyomma americanum mason mountain nymph 3 mmsl8 amblyomma americanum mason mountain nymph 4 cetx-2 ixodes scapularis texas adult female 5 lptx-1 rhipicephalus sanguineus brazos county, texas adult female 6 bas115 amblyomma cajennense brazos county, texas adult female 7 bblc1 amblyomma americanum brazos county, texas adult female 8 ritx-1 ixodes scapularis jefferson county adult female 9 wotx-1 ixodes scapularis brazos county, texas adult female 10 sg-1 ixodes scapularis brazos county, texas adult female 11 nstx-1 ixodes scapularis texas adult female 12 betx-20 ixodes scapularis jefferson county adult female 13 sat-88 rhipicephalus sanguineus san antonio, tx nymph 14 kttx-5 dermacentor variabilis brazos county, texas adult male 15 kttx-6 dermacentor variabilis brazos county, texas adult female 16 bas-183 amblyomma maculatum brazos county, texas adult female 17 bas-125 dermacentor variabilis brazos county, texas adult female 18 bas-126 dermacentor variabilis brazos county, texas adult female 19 bas-127 dermacentor variabilis brazos county, texas adult female 20 bas-128 amblyomma maculatum brazos county, texas adult female 21 bas-129 dermacentor variabilis brazos county, texas adult female 22 bas-216 amblyomma maculatum brazos county, texas adult female 23 bas-124 dermacentor andersoni brazos county, texas adult female 24 sat-97 rhipicephalus sanguineus san antonio, texas nymph 25 sat-101 rhipicephalus sanguineus san antonio, texas nymph 26 mtx1 ixodes scapularis brazos county, texas adult female 27 mtx3 ixodes scapularis brazos county, texas adult female 28 mtx4 ixodes scapularis brazos county, texas adult female 29 betx-16 ixodes scapularis brazos county, texas adult female 30 betx-17 ixodes scapularis brazos county, texas adult female 31 betx-18 ixodes scapularis brazos county, texas nymph 32 betx-19 ixodes scapularis brazos county, texas adult female 33 thread1 ixodes scapularis brazos county, texas adult female 34 cstx1 ixodes scapularis brazos county, texas adult female 35 cvm11 dermacentor albipictus brazos county, texas adult female 36 cetx-2 dermacentor albipictus brazos county, texas adult female 37 tjm 305-5 carios capensis chaparral wma larvae 38 tjm 448-514 carios capensis las palomas wma arroyo colorado unit 50 larvae 39 tjm 182-1 carios capensis chaparral wma nymph 40 tjm 448 carios capensis las palomas wma arroyo colorado unit 48 nymphs 41 tjm 216.1 carios capensis chaparral wma nymph 42 tjm 308-12 carios capensis chaparral wma 3 larvae 43 tjm 596 dermacentor variabilis las palomas wma arroyo colorado unit 38 nymphs 45 tjm112 dermacentor variabilis gus engeling wma 2 larvae 46 tjm 355 dermacentor variabilis chaparral wma nymph 47 tjm 440 dermacentor variabilis las palomas wma arroyo colorado unit 1 larvae 48 tjm 308-18 dermacentor variabilis chaparral wma 3 larvae 49 tjm 140-3 amblyomma inornatum gus engeling wma 3 larvae 50 tjm 139 amblyomma inornatum gus engeling wma 2 larvae 51 tjm 529 dermacentor variabilis las palomas wma arroyo colorado unit 1 larvae 52 tjm 216 amblyomma maculatum chaparral wma nymph discussion the present studies aimed to present good molecular marker for tick identification based j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 162 http://jad.tums.ac.ir published online: june 24, 2019 on dna sequences to solve the problems with morphological tick identification, and sometimes the morphological identification is not enough for detect the species so amplification of the16s rdna using it as a methods for tick genetic identification, and comparison between the amplification of the the16s rdna, 12s rdna, its-1 rdna and its-2 rdna for the same samples species. 16s rdna and 12s rdna genes are a mitochondrial ribosomal dna gene, mtdna considered one of the most commonly used genes for molecular identification of ticks due to the fact that it is relatively easy to work with them due to their higher copy number (8). in addition, mtdna sequences are a good phylogenetic marker mostly for groups of organisms, diverged relatively, since mtdna has a higher rate of base substitution than most nuclear markers (9). the problem for the mitochondrial gene is it can transfer to the nucleus leading to error in the phylogeny after the amplification and sequencing (8). the difference between the 16s rdna and 12s rdna genes that the evolution is faster in 12s rdna genes (8). regarding tick species, 16s rdna was used and succeeded to construct phylogeny of both hard and soft ticks (6, 7, 8) and 16s rdna is useful in constructing their tick phylogenetic tree, but there is a problem associated with 16s rdna is that using this gene alone is not sufficient for getting full resolution for the tree so the best way to solve it accompanied it with another gene like 12s rdna (6, 8). we utilized these genes for diagnostic purpose only not for phylogeny, and the problems are usually associated with the phylogeny. overall, 192 tick samples (larvae, nymph, males and engorged females) were evaluated using16s rdna pcr and 12s rdna the pcr positive bands have to be sequencing. the sequencing analysis determined that the tick species collected in the study were: amblyomma americanum, dermacentor albipictus, ixodes scapularis, d. variabilis, a. cajennense, carios capensis and rhipicephalus sanguineus (table 3). therefore, using 16s rdna and 12s rdna are good in molecular tick identification of all these species utilized in our study. the first and the second internal transcribed spacers region of the nuclear ribosomal gene cluster (its-1, its-2), consist of three genes 18srdna, 5.8srdna and 28srdna. these three rdna genes are transcribed making a single transcript of rna separated by the its1 and its-2 regions (7). (its-1, its-2) considered the fastest evolving dna genes (9). because of these facts, the its-1, 2 rdna are not good in amplification of some of the tick species in our study, they failed to amplify some tick species as mentioned before. internal transcribed spacer was used successfully for the identification of dermacentor marginatus, ixodes ricinus, haemaphysalis, boophilus, and rhipicephalus sanguineus tick species (3, 4). nevertheless, the problem with internal transcribed spacer is that genes are evolving rapidly so in some species they failed to amplify as reported before in ticks (8). its-2 was utilized for identification iranian hard tick and its-2 failed to amplify some of his samples. therefore, these markers were mostly useful to study close related species (10). even some of our samples are closely related to each other as rhipicephalus species and dermacentor and it failed to amplify some of them, therefore, it is better to clone the pcr products and work with it as haplotypes, not individuals make the studies more expensive (8). conclusion molecular tick identification will help and improve the disease diagnosis and choosing good genetic marker for diagnosis purpose as 16s rdna and 12s rdna markers is good as they give good amplification for our sample species, in spite of using (its-1, its-2) j arthropod-borne dis, june 2019, 13(2): 153–164 em abouelhassan et al.: comparison of some … 163 http://jad.tums.ac.ir published online: june 24, 2019 are good for tick molecular identification for very closely related species but with our few samples even they are not representative samples it did not work with the closely related tick species. acknowledgements thanks to the lyme laboratory at texas a and m university and dr maria d estevegassent for her support and effort. the authors declare that there is no conflict of interests. references 1. zahid iqbal rajput, song-hua hu, wan-jun chen, abdullah g. arijo, chen-wen xiao (2006) importance of ticks and 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tickborne bacterial diseases in humans: an emerging infectious threat. clin infect dis. 32(6): 897–928. 5. iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 28 original article interactions between entomopathogenic fungus, metarhizium anisopliae and sublethal doses of spinosad for control of house fly, musca domestica * m sharififard1, ms mossadegh2, b vazirianzadeh3, a zarei-mahmoudabadi4 1department of medical entomology, college of health, ahvaz jundishapur university of medical sciences, iran 2department of plant protection, college of agriculture, shahid chamran university, ahvaz, iran 3department of medical entomology and infectious and tropical diseases research center, ahvaz jundishapur university of medical science, iran 4department of mycoparasitology and infectious and tropical diseases research center, ahvaz jundishapur university of medical science, iran (received 28 sep 2010; accepted 20 jul 2011) abstract background: metarhizium anisopliae strain iran 437c is one of the most virulent fungal isolates against house fly, musca domestica. the objective of this study was to determine the interaction of this isolate with sublethal doses of spinosad against housefly. methods: in adult bioassay, conidia of entomopathogenic fungus were applied as inoculated bait at 105 and 107 spore per gram and spinosad at 0.5, 1 and 1.5 µ g (a.i.) per gram bait. in larval bioassay, conidia were applied as combination of spore with larval bedding at 106 and 108 spore per gram and spinosad at sublethals of 0.002, 0.004 and 0.006 µ g (ai) per gram medium. results: adult mortality was 48% and 72% for fungus alone but ranged from 66–87% and 89–95% in combination treatments of 105 and 107 spore/g with sublethal doses of spinosad respectively. the interaction between 105 spore/g with sublethals exhibited synergistic effect, but in combination of 107 spore in spite of higher mortality, the interaction was additive. there was significant difference in lt50 among various treatments. lt50 values in all combination treatments were smaller than lt50 values in alone ones. larval mortality was 36% and 69% for fungus alone but ranged from 58%–78% and 81%–100% in combination treatments of 106 and 108 spore/g medium with sublethals of spinosad respectively. the interaction was synergistic in all combination treatments of larvae. conclusion: the interaction between m. anispliae and spinosad indicated a synergetic effect that increased the house fly mortality as well as reduced the lethal time. keywords: metarhizium anisopliae, musca domestica, spinosad, iran introduction housefly, musca domestica l that is well known as poultry and livestock pest is also word-wide mechanical vector of human pathogens (lecouna et al. 2005). high level of insecticide resistance in the housefly and public demands for reducing pesticide use around animal food have promoted interest in the development of other control strategies of this pest (geden et al. 1995). an important strategy is integrated pest management (ipm) programs, which includes biological, cultural, and/ or chemical methods to control the population of this pest (crespo et al. 1998, lecouna et al. 2005). although biological control of *corresponding author: dr mona sharififard, email: sharififardm@yahoo.com iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 29 housefly is currently focused mostly on pupal parasitoids, entomopathogenic fungi are ubiquitous in nature and could be considered for manipulation in ipm programs (stainkraus et al. 1990, barson et al. 1994, 1995, bywater et al. 1994, watson et al. 1995, 1996, renn et al. 1999, lecouna et al. 2005, kaufman et al. 2005). the entomopathogenic fungus, metarhizium anisopliae (metch) sorok. has been isolated from 200 insect species including the orders of lepidoptera, coleoptera, orthoptera, and hemiptera. there are few reports on the use of this fungus for urban pest management (pachamuthu and kamble 2000). the major limitations in the use of entomopathogenic fungi such as m. anisopliae have been an extended time to cause sufficient insect mortality and its inconsistent performance under field conditions. one of the options for improving the efficacy of the entomopathogenic fungi is to incorporate the fungus pathogens with sublethal doses of insecticides (pachamuthu and kamble 2000). data from in vivo compatibility studies have indicated that m. anisopliae and insecticides are compatible, and their combination can have synergistic, antagonistic, or additive effect (pachamuthu and kamble 2000, zurek et al. 2002, ericsson et al. 2007). spinosad is a novel macrolide-class insecticide produced by the soil bactrium saccharopolyspora spinosa and is known to be active against many noxious pests. the mechanism of action of spinosad appears to be unique, with a primary site of attack being the nicotinic acetycholine receptor and a secondary site of attack possibly being gaba receptors (scott 1998, kristensen and jepersen 2004). in contrast to other commonly used insecticides where the technical active ingredients are classified as moderately or high hazardous, spinosad is classified as a reducedrisk pesticide and has been determined to pose little to no mammalian toxicity (white et al. 2007). there have been no reports of resistance or cross-resistance in field population housefly (scott 1998, liu and yue 2000, kristensen and jepersen 2004, white et al. 2007). the research objectives were to enhance the lethal effect of m. anisopliae strain iran 437c by using it in combination with different sublethal doses of spinosad against house fly, m. domestica. the aim was to determine which kind of interaction (synergistic, antagonistic, and additive) occurs between m. anisopliae and spinosad and to ascertain the lt50 in control of larvae and adult. materials and methods musca domestica culture adult house flies were collected from a poultry house by sweeping net and transferred to the laboratory where they were reared at 26° c, 50±5% rh and photoperiod of 14:10 (l: d). adults were maintained in cages (40×40× 40 cm3) covered by gauze. water and food in the form of sugar and powdered milk were provided and replenished every 24–48h. larval medium comprised 55 g wheat bran, 3g date extract and 2g dried alfalfa suspended in 140 ml water. one cup (250ml volume) of this medium was left in each cage for adult oviposition and subsequent development of larvae. the food was replaced every 24–48h. fungus ten iranian isolates of beauveria bassiana (bals) vuill. and metarhizium anisopliae (metch) sorok. were obtained as cultures from the ministry of jihad keshavarzy of iran. previous study indicated that m. anisopliae strain iran 437° c was the most virulent against house fly, m. domestica that caused higher mortality in the shorter time than the others (sharififard et al. 2011), so this isolate was selected for current study. it was cultured on sabouraud dextrose agar with yeast extract (sday) for 2 weeks at 27°c, 75±5% rh and photoperiod of 12:12 (l:d). sporulating cultures were harvested by scraping the dry conidia from the surface of the culture plate iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 30 with a scalpel and transferring them to sterile distilled water containing 0.01% tween–80. the concentration of the suspension was determined using a hemocytometer. adult bioassay spinosad concentrations that caused zero mortality after 48 h in the adult house fly were selected as sublethal based on conducting several pretests. there were 0, 0.5, 1 and 1.5 µ g (ai) per gram bait. selected m. anisopliae concentrations were 0, 105 and 107 conidia per gram bait. adult bait containing sugar, powdered milk and distilled water were prepared and treated with different combinations of spinosad and conidial concentrations. cohorts of twenty-five 2-3 day old house flies were housed in small cage (20×20×20 cm3). each cage contained a 9 cm diameter petri dish lined with watman filter paper and 10 g treated bait. adults in the control groups were feed with untreated bait. each treatment was replicated 5 times. cages were maintained in room conditions and checked daily over a period of 9 days for mortality recording. larval bioassay concentrations of spinosad that produced less than 30% mortality of the larval housefly larvae were determined using several pretests and classified as sublethal doses. there were 0, 0.002, 0.004, and 0.006 µ g (ai) per gram larval bedding. in another treatment, we have also determined that 106 and 108 conidia/g larval bedding as sub lethal concentrations of m. anisopliae strain iran 437c in the control of house fly larvae. plastic 150ml containers were filled with 50 g larval bedding, containing of wheat bran, dry alfalfa, date extract and water. the stock suspension of fungi was adjusted to a concentration of 0, 5×107 and 5×109 conidia/ml with an improved hemocytometer. one milliliter of each stock fungi suspension was added to each larval container to raise the larval bedding concentration to 0, 106 and 108 conidia/g bedding. both spinosad and m. anisopliae treatments were mixed into the larval bedding with a glass rod. in total, the treatments evaluated in this bioassay included 12 different combinations of the insecticide and fungi concentrations. twenty larvae were used per treatment and each treatment was replicated 4 times. larva in the control groups were treated with distilled water. however, mortality was observed daily for all treatments and the dead larva were removed. statistical analysis data from this study were analyzed by factorial analysis of variance (anova) by using two factor complete randomized design of mstatc software. percentage mean of mortality were compared using duncan's multiple range test at α= 0.05. significant differences among the combination treatments by factorial analysis indicated that there was an interaction between m. anisopliae and insecticide and the effect observed might be synergistic or antagonistic. in contrast, if there was no significant difference in m. anisopliae plus insecticide treatment, it implied that the effects were additive (pachamuthu et al. 2000). chi-squared tests were performed to determine the type of interaction (additive, synergistic or antagonistic). expected mortality (e) was generated from the following formula: e= ospin + omet (1– ospin), where e is the expected mortality, and ospin and omet represent the proportion mortality due to treatments of pure spinosad and pure m. anisopliae, respectively. the predicted effects of spinosad and m. anisopliae treatments (e) were compared with the observed mortality of the binary treatments (o) with following formula, )({ } e eo 22 −=χ (ericsson et al. 2007). if the calculated chi-squared value exceeds the tabular value, then it indicates either synergistic or antagonistic interaction. in contrast, if the tabular value exceeds calculated chisquare value, then it indicates an additive effect. lt50 values iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 31 and 95% confidence limits of each value for different treatments were calculated by using probit method of sas software. when there was no overlap in the 95% cl of lethal time values, the treatments difference were considered significant. results adult bioassay these sub lethal concentrations of spinosad were classified as 0, 0.5, 1 and 1.5 µ g (ai) per gram bait. the results of analyze variance showed that adult mortality was significantly affected by insecticide concentration (f= 90.7, df= 3, p< 0.0001), conidial concentration (f= 623.86, df= 2, p< 0.001) and interaction of insecticide and fungi (f=3.19, df= 6, p< 0.011). higher mortality was observed in m. anisopliae plus spinosad combination treatments than sole treatment of fungi or insecticide (table 1). mixing of 105 conidia/g with 0.5, 1 and 1.5 gµ (ai)/g of spinosad caused higher mortality of adult housefly than alone treatments. estimation of chi-squared showed synergistic interaction in combination of 105 conidia/g combined with 1 and 1.5 µg (ai)/g. in the combination treatments of 107 conidia/g with sublethals of spinosad, there was no significant interaction between insecticide and m. anisopliae. the increased mortality was the result of an additive effect (table 2). based on individual treatment levels, the greatest synergistic effect occurred when 105 conidia/g bait were used with 1.5 µ g (ai)/g. calculated lt50 values and 95% confidence limits of each value for different treatments in adult bioassay showed that the lt50 values were lower in all combination treatments of m. anisopliae+spinosad in comparison with m. anisopliae alone (table 3). combination treatments caused faster mortality than the alone ones. when there was no overlap in the 95% cl of lethal time values, the treatments difference were considered significant. while the interaction was additive in the combination of 107 conidia/g with sublethals of spinosad, but there was significant difference in lt50 values between m. anisopliae (107) and m. anisopliae (107) plus spinosad (0.5, 1, 1.5 µg). the shortest lethal time for causing 50% mortality in adult population was observed in 107 conidia of m.anisopliae +1.5 µ g of spinosad. there was no significant difference in lt50 values of m. anisopliae (107) + spinosad (0.5 and 1 µ g). there was a significant difference in lt50 between m. anisopliae (105) + spinosad (0.5, 1 and 1.5µ l) and m. anisopliae (105) alone, but there was no difference in the lt50 values among 105 conidia of m. anisopliae +1 and 1.5 gµ of spinosad, also between m. anisopliae (105)+ spinosad (1.5) and m. anisopliae (107)+ spinosad(1.5). therefore, due to the greatest synergistic effect occurred when 105 conidia were used with 1.5 µ g (ai) of spinosad and no significantly difference in lt50 value of this treatments with m. anisopliae (107) + spinosad (1.5), mentioned combination of m. anisopliae and spinosad was the best combination for control of adult housefly. larval bioassay the results of analyze variance showed that larval mortality was significantly affected by insecticide concentration (f= 149.84, df= 3, p< 0.0001), conidial concentration (f= 895.83, df= 2, p< 0.001) and interaction of insecticide and fungi (f= 12.78, df= 6, p= 0.025). the percent of mortality of medium size larvae was significantly difference among all 11 treatments (table 4). the greatest mortality was recorded in the combination treatments of 108 spores of m. anisopliae plus sublethals of spinosad. a synergistic interaction between m. anisopliae and spinosad was always found when the fungus was applied at a dosage of 106 and 108 conidia/g larval bedding in combination with 0.002, 0.004 and 0.006 µ g (ai)/g of spinosad. but in the combination of 106 spores of m. anisopliae+sublethals of spinosad chi-squared iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 32 values were greater than 108 spores with same sublethal of spinosad (table 5). the greatest synergetic effect observed when 106 conidia of m. anisopliae were combined with 0.006 µ g (ai), so this was the best combination of m. anisopliae with spinosad for larval control. table 1. toxicity of spinosad (µ g (ai)/g) and m. anisopliae (conidia/g) alone and in combination treatments on adult house fly after 9 days treatmenta n %mortality(±se)b m. anisopliae(105) 150 44±4.20g m. anisopliae (107) 150 72.4±1.79e spinosad (0.5) 150 21±1.24j spinosad( 1) 150 32±1.7i spinosad (1.5) 150 39±1.7h 105+0.5 150 66.4±2.68f 105+1 150 80.6±3.13d 105+1.5 150 87±1.22c 107+0.5 150 89±4.02bc 107+1 150 90.4±1.79b 107+1.5 150 95±3.3a a each treatment (containing 30 adults) were replicated 5 times. b means followed by the same letters were not significantly different (duncan's test;α= 0.05). table 2. synergy bioassay: adult house fly mortality from combination traetments of spinosad and m. anisopliae after 9 days treatment %mortality fungi (conidia/g) spinosad µg (ai)/g fungi spinosad expected observed 2χ * 105 0.5 44 21 56 66 1.79 105 1 44 32 62 81 5.82* 105 1.5 44 39 66 87 6.68* 107 0.5 72 21 78 89 1.55 107 1 72 32 81 90 1.00 107 1.5 72 39 83 95 1.73 *a chi-square comparison that exceeds 3.84 with df= 1 and α= 0.05 is considered synergistic and is denoted by an asterisk (*). table 3. calculated lt50 values for m. anisopliae (conidia/ g) and its combination with sublethal doses of spinosad (µ g (ai)/g) bait treatmenta n slope±se lt50 b 95%clc 2χ (df) m. anisopliae (107) 150 6.9 ± 0.58 6.4 6.12 – 6.67 4.85(2) m. anisopliae 107+spinosad 1.5 150 4.08 ± 0.33 2.6 2.36 – 2.83 4.91(2) m. anisopliae 107+spinosad 1 150 3.39 ± 0.31 3.7 3.34 – 4.02 0.96(2) m. anisopliae 107+spinosad 0.5 150 3.81 ± .032 4.1 3.69 – 4.35 1.05(2) m. anisopliae (105) 150 6.58 ± 1.77 8.08 7.69 – 8.73 8.42(2) m. anisopliae 105+spinosad 1.5 150 4.37 ± 0.78 3.1 2.71 – 3.55 10.05(2) m. anisopliae 105+spinosad 1 150 2.71 ± 0.31 3.9 1.84 – 5.96 0.33(2) m. anisopliae 105+spinosad 0.5 150 2.74 ± 0.27 4.9 4.47 – 5.44 0.94(2) spinosad 1.5 150 1.74 ± 0.28 12.4 9.59 – 19.6 1.01(2) spinosad 1 150 2.01 ± 0.32 14.1 10.79 – 22.8 1.03(2) spinosad 0.5 150 1.74 ± 0.35 21.2 13.98 – 53.17 1.53(2) aeach treatment (containing 30 adults) were replicated 5 times. b number of days until 50% mortality occured after different treatments. c treatments will have significant effect on lt50 if there was no overlap of 95% cl. iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 33 table 4. toxicity of spinosad (µ g (ai)/g) and m. anisopliae (conidia/g) alone and in combination traetments against house fly larvae treatmenta n %mortality(±se)b m. anisopliae (106) 100 36±1.93 i m. anisopliae 106 +spinosad 0.002 100 58±2.58 g m. anisopliae 106 +spinosad 0.004 100 65± 3.42 f m. anisopliae 106 +spinosad 0.006 100 78±2.5 d m. anisopliae (108) 100 69±1.91 e m. anisopliae 108+spinosad 0.002 100 81±2.52 c m. anisopliae 108 +spinosad 0.006 100 95± 1.91 b m. anisopliae 108+spinosad 0.006 100 100±0.00 a spinosad 0.002 100 14± 2.58 k spinosad 0.004 100 23±1.91 j spinosad 0.006 100 41±1.91 h a each treatment (containing 25 larvae) were replicated 4 times. b means followed by the same letters were not significantly different (duncan's test; α= 0.05). table 5. synergy bioassay: larval mortality from combined treatments of spinosad and m. anisopliae after 9 day treatment %mortality fungi (conidia/g) spinosad µg (ai)/g fungi spinosad expected observed 2χ 106 0.002 35 14 44 58 4.38* 106 0.004 35 23 50 65 9.90* 106 0.006 35 41 62 78 15.75* 108 0.002 69 14 65 81 3.94* 108 0.004 69 23 76 96 5.19* 108 0.006 69 41 82 100 4.09* *a chi-square comparison that exceeds 3.84 with df= 1 and α= 0.05 is considered synergistic and is denoted by an asterisk (*). discussion because conidia require at least 12–24 h for development of germ tube, appressoria and penetration to insect cuticle, so the doses of spinosad that caused <40% mortality 48h after exposure in the adults were selected as sublethals. these doses would allow sufficient time for conidia to form the germ tube and appressoria. high mortality by insecticide during this period affects the effectiveness of fungus. in our study, the m. anisopliae strain iran 437c was effective and caused 44% and 72% mortality in adult population at the concentrations of 105 and 107 spores per gram bait in 9 days after exposure. synergistic interaction was observed in combination treatments of 105 spore with sublethal doses of insecticide but in combination of 107 spore the interaction was additive. lethal time in all combination treatments were reduced in comparison with alone treatments of fungi. thus, increased mortality and lowered lt50 values were a general pattern observed in most of insecticide + m. anisopliae combinations against house fly in our study. in larval test, this fungal strain caused 35 and 69% mortality at 106 and 108 spores per gram bedding in larval population in the end of larval cycle. when spinosad and m. anisopliae were applied together as a mixture, larval mortality was significantly higher than the expected value of their additive effect, iran j arthropod-borne dis, 2011, 5(1): 28–36 m sharififard et al.: interactions between … 34 which indicated a synergistic interaction in all treatments. lower dosages of spinosad not only enhanced the efficacy of m. anisopliae, but also lead to a reduced quantity of inoculum needed to cause high levels of mortality in house fly adult and larvae. the time to mortality of larvae could not be accurately assessed as a proportion of infected larvae subsequently died in the pupal stage. moreover, it was not considered in larval bioassay because the eventually aim of larval control is decreasing of adult population and lethal period of larvae is not too important. earlier studies by barson et al. (1994), renn et al. (1999) also demonstrated the effectiveness of m.anisopliae in controlling house fly. in spite of effectiveness of entomopathogenic fungi against house fly, different strains require different times to achieve high mortality. with due attention to high reproduction rate and short life cycle of m. domestica, it is necessary to find approach for increasing pest mortality as well as reducing the lethal time by biopesticide agents. so, in this study, we evaluated the effect of combined applications of m. anisopliae and spinosad against m. domestica under laboratory conditions. several studies have focused on the potential use of entomopathogenic fungi in combination with sublethal doses of organic insecticides against various insect pests such as compatibility of m. anisopliae with sublethals of chlorpyrifos, propetamphos and cyfluthrin against the german cockroach (pachamuthu et al. 2000), m. anisopliae with boric acid against german cockroach (zurek et al. 2002), combination of imidiaclopride and diatomaceous earth with beauveria bassiana on mole cricket (thompson et al. 2006), sublethals of spinosad with m. anisopliae against exotic wireworms (ericsson et al. 2007) and m. anisopliae in combination with sublethal doses of imidiacloprid on the subterranean burrower bug cyrtomenus bergi (jaramillo et al. 2005). sublethal dosage of synthetic insecticides can act as physiological stressors and/ or behavioral modifiers, thereby predisposing insects to diseases (inglis et al. 2001). integrating insecticides and entomopathogens has a few advantages: 1) such approach will increase pest mortality as well as reduce the lethal time, 2) prolong the use of a particular insecticide by reducing the total amount of insecticide using, 3) minimizing environmental contamination and increasing human safety, 4) it accelerates the mode of action of fungus without compromising the fungus growth from cadavers that is crucial for inducing epizootic in house fly population particularly in larval bedding that humidity and temperature of bed supported the growth of muscardine on larval cadavers. in conclusion, our results indicated that the use of combination of m. anisopliae with lower dosage of spinosad might become an important component of m. domestica ipm but at first, this approach must be testing under field conditions. acknowledgements the authors wish to thanks dr rassule zare from iranian research institute of plant protection for providing the fungal isolates used in the study and for his useful advice. the authors also thank research deputy of shahid chamran university, ahvaz, iran for sponsoring this project. the authors declare that there is no conflict of interests. references barson g, renn n, bywater af (1994) laboratory evaluation of six species of entomopathogenic fungi for control of house fly musca domestica l, a pest of intensive animal units. j inverter pathol. 64: 107–113. bywater af, barson g, renn n (1994) the potential of 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domestica (diptera: muscidae) in calf hutches. j bio control. 7: 221–227. white wh, mccoy cm, meyer j, winkle, jr (2007) knockdown and mortality comparisons among spinosad-imidacloprid and methomyl-containing baits against susceptible musca domestica l. under laboratory conditions. j econ entomol. 100(1): 155–163. zurek l, watson dw, schal c (2002) synergism between metarhizium anisopliae (deuteromycotina: hyphomycetes) and boric acid against the german cockroach (dictyoptera: blattellidae). j bio control. 23: 296–302. j arthropod-borne dis, march 2017, 11(1): 42–49 e valikhanfard-zanjani et al.: antivenom efficacy … 42 http://jad.tums.ac.ir published online: march 14, 2017 original article antivenom efficacy in neutralizing histopathological complications following latrodectus dahli envenomation *elham valikhanfard-zanjani 1, abbas zare-mirakabadi 2, ehsan zayerzadeh 3 1department of biology, faculty of sciences, kharazmi university, tehran, iran 2department of venomous animals and antivenom production, razi vaccine and serum research institute, karaj, iran 3department of biology, faculty of food industry and agriculture, standard research institute, karaj, iran (received 17 sep 2015; accepted 17 nov 2015) abstract background: nowadays use of specific antivenin for latrodectism is considered as the most effective treatment in the world. this study was undertaken to investigate the efficacy of specific antivenom against histopathological complications caused by latrodectus dahli venom on liver, heart and kidneys tissues within 72h. methods: two groups were selected, each one contained 6 male new zealand rabbits weighing 2±0.5kg. the animals were anesthetized with 0.5ml ketamine and 0.5ml xylazine by intramuscular route. the l. dahli venom (0.5mg/kg) was injected subcutaneously to both the groups. the second group of rabbits 24h after the venom injection received specific antivenom by intravenous route. seventy-two hours after the venom and antivenom injections, the rabbits were dissected to obtain heart, liver and kidney tissues. the tissues were stained by hematoxylin and eosin stains and histopathological studies were examined by optical microscope. results: in group one, the venom induced myocytolysis, myocarditis, coagulation necrosis in the heart tissue and the liver tissue showed central vein congestion, congested vessels, dilated sinusoids and inflammation. however, no significant histopathological complications were observed in kidney tissues. in the second group, antivenom injection greatly prevented escalation of the complications on foresaid tissues. conclusion: latrodectus dahli venom induces histopathological complications on vital organs. specific antivenom injection, 24h after the venom injection, could protect the tissues from incidence and intensification of histopathological complications. future studies in human beings should be conducted to assess the protection against the specific-latrodectus antivenin. keywords: latrodectus, antivenom, histopathological complication introduction black widow spider envenomation, because of systemic complications, sometimes fatal, has become as a candidate for the most dangerous arachnids in the world (vetter and isbister 2007, rey et al. 2011). among more than 40 species that are identified in all over the world, only 4 species were reported as inhabitant of iran which including latrodectus tredecimgottatus, l. dahli, l. geometricus and l. pallidus (rafinejad et al. 2007, afshari et al. 2009, shahi et al. 2011). the latrodectus spp. are predominantly distributed in the north east and the south of the country. latrodectism, syndrome caused by latrodectus spp., generally is characterized by pain, muscular weakness, nausea, vomiting, painful abdominal cramping, perspiration and cardiovascular complications (marzan 1955, maretic 1983, prior and park 2004). the most reports of latrodectism in iran return to the 1994, so that nearly 190 cases of latrodectism were reported only in the khorasan province (mirshamsi kakhaki 2005, afshari et al. 2009). black widow spider venom contains a neu*corresponding authors: miss elham valikhanfardzanjani, e-mail: evalikhanfard@yahoo.com j arthropod-borne dis, march 2017, 11(1): 42–49 e valikhanfard-zanjani et al.: antivenom efficacy … 43 http://jad.tums.ac.ir published online: march 14, 2017 rotropic protein termed αlatrotoxin which is thought to be responsible for the most clinical effects in human (nelson et al. 2011, nentwig and nentwig 2013). α-ltx exerts its destructive effects through interaction with specific receptors termed neurexin and latrophilin, so that leads to neurotransmitter depletion of both cholinergic and adrenergic terminals. given that the highest expression of the receptors are in neurons of nervous system, so it seems that nervous system is the primary target of black widow spider venom (henkel and sankaranarayanan 1999, ushkaryov et al. 2008, silva and ushkaryov 2010). duration of latrodectism in untreated cases can be different in ranges, from a few hours to several days (malley et al. 1999, vetter and isbister 2007). in the past, in order to treat symptoms caused by latrodectism, various medications were used, like calcium gluconate and benzodiazepines. the success rates of these drugs are different but all of them were used just to relieve pain and other symptoms caused by envenomation (allen and norris 1991, clark et al. 1992). nowadays usage of antibodies and antibody fragments are considered as the most effective treatment in latrodectism. however, there is a substantial controversy about the route and time of black widow spider antivenom injection, so that previously black widow specific antivenom was injected intramuscularly, but according to the published data in recent decades, intramuscular route is not efficient, so intravenous route was proposed. however, determination of antivenom ability to reverse complications of black widow envenomation needs more investigations (isbister 2002, brown et al. 2007, ahmed and bushra 2008). so far, most of clinical studies performed on latrodectism, were concerning on the venom effects on organs functions. considerably less attention has been paid to venom effects on organs tissues. expressions of the certain alpha latrotoxin receptors on some of the mammalian tissues, makes the direct effects of venom expectable (herberth et al. 2005). therefore the present study was undertaken to investigate effects of l. dahli venom on liver, heart and kidneys tissues within 72h. besides, we evaluated efficacy of antivenom to inhibit lethality and neutralizing potency on toxicity effects of l. dahli venom within 72h. the findings of the present investigation can be an important step to improve guidelines for an optimal immunotherapeutic treatment of spider envenomation. materials and methods venom and antivenom crude spider venom and specific antivenom were provided by department of venomous animals and antivenom production, razi vaccine and serum research institute, karaj, iran. in order to obtain the crude venom, l. dahli spiders were dissected out, and a pair of glands was collected into ice cold phosphate buffered saline (pbs). the glands were washed in pbs in order to remove possible contaminants, and venom was harvested in pbs by gentle compressing of the glands. the suspension was clarified by centrifugation at 8000rpm, and the venom was stored at −20 °c until use. experimental protocols two groups of new zealand white rabbits weighing 2±0.5kg (6 animals in each group) were selected. before the experiment all animals were maintained for at least 3 d, under conditions of controlled light (12h light, 12h dark), temperature (18–22 °c) and humidity (55±5%), with standard diet and water available ad libitum. at first rabbits were anaesthetized with intramuscular injection of 0.5ml ketamine and 0.5ml xylazine in ratio 1:1 respectively. in order to study the hepatotoxicity, myotoxicity and nephrotoxicity of l. dahli venom, the first group received only l. dahli venom (0.5mg/kg) through subj arthropod-borne dis, march 2017, 11(1): 42–49 e valikhanfard-zanjani et al.: antivenom efficacy … 44 http://jad.tums.ac.ir published online: march 14, 2017 cutaneous route, while the second group 24h after the venom injection, received specific antivenom (2.5ml with neutralization capacity of 500 ld50/ml) by iv route. seventytwo h after the venom and antivenom injection, experiment ended by scarifying the rabbits for obtaining the heart, liver and kidney tissues throughout the surgery. histological analysis after the animals died, the heart, liver and kidneys were removed carefully and immersed in 10% formaldehyde at room temperature and then sectioned transversely into 5μm slices. specimens were dehydrated in a graded series of alcohol and xylene and embedded in paraffin. multiple slices (15 fields for each slide) were made and stained by hematoxilin and eosin stains. sections were viewed and were photographed using a nikon e200® light microscope (japan). this study was approved by ethics committee of this institute. razi vaccine and serum research institute. results the signs and symptoms of envenomation appeared within first few hours by redness, mild swelling and muscle cramps at injection site. twenty-four h after the venom injection difficulty in respiration was observed in most of the animals. following 72h after the venom (0.5mg/ kg, s.c) and the antivenom (2.5ml, i.v) injection, histological changes were investigated in the rabbit’s heart, liver and kidney tissues in each group. figure 1 shows the histopathological evaluation of the first group of animals, which venom injection induced complications such as myocytolysis, myocarditis, coagulation necrosis, myocardial edema, hemorrhage and inflammation in the heart tissues (fig.1). on the other hand, evaluation of the liver tissues, showed central vein congestion, congested vessels in portal areas, dilated sinusoids and inflammation, however no significant histopathological complications were observed in kidney tissue within 72 h after the venom injection (fig. 1). in the second group of the animals, immunotherapy prevented severe myocytolysis, edema and hemorrhage in the heart of treated animals. however, in this group, mild myocytolysis and mild hemorrhage in the heart were still observed in the experimented animals (fig. 2). antivenom injection also prevented severe central vein congestion and inflammation in the liver. however, mild congestion and mild inflammation were observed in the livers of this group (fig. 2). fig. 1. histopathological changes of heart (a, b), liver (c, d) and kidney (e, f) tissues induced by latrodectus dahli venom (0.5mg/kg, s.c). within 72h after the venom injection myocytolysis, lymphomononuclear inflammatory infiltrate on the myocardium (a), hemorrhage, myocarditis and myocytolysis (b) are presented. the central vein congestion (c and d) in the liver tissues are presented. following the venom injection no changes were observed in the kidney tissue (e and f) (original) j arthropod-borne dis, march 2017, 11(1): 42–49 e valikhanfard-zanjani et al.: antivenom efficacy … 45 http://jad.tums.ac.ir published online: march 14, 2017 fig. 2. the histopathological changes of heart (a, b), liver (c, d) and kidney (e, f) tissues following the antivenom injection (2.5ml, i.v) 24h after the venom injection. the antivenom prevented severe myocytolysis, edema and hemorrhage in the heart tissue (a), mild myocytolysis and mild hemorrhage are observed in one of the rabbit’s heart (b). the antivenom prevention of the severe central vein congestion and inflammation in the liver are presented (c), mild inflammation were observed in one of the rabbit’s livers in this group (d). no changes were observed in the kidney tissues within 72h after the antivenom injection (e and f) (original) discussion in the present study, the toxic effects of l. dahli venom were investigated within 72h on vital organs including the liver, heart and kidney tissues. the findings showed that, the venom injection induced severe cardiovascular complications such as myocytolysis, myocarditis and myocardial hemorrhage in heart. in addition, the venom injection evoked central vein congestion and dilated sinusoids in liver. the spider genus latrodectus, because of the extreme potency of their neurotoxic compound termed α-latrotoxin, is one of the most notorious spiders in all over the world (garb et al. 2004). clinical manifestations of latrodectism, syndrome caused by latrodectus, indicated that the nervous system is the primary target of α –latrotoxin (α-ltx), which follows affecting whole body of organism (vetter and isbister 2007). according to our study, it seems that heart is one of the main goals of the venom. besides, the venom injection induced some histopathological complications such as myocytolysis, myocarditis, coagulation necrosis, myocardial edema and hemorrhage and tissue inflammation. approximately, in all performed studies on latrodectism, cardiovascular complications have been reported mostly manifested by arrhythmia, tachycardia, bradycardia and ecg changes (maretic and stani 1954, pulignano et al. 1998, akyildiz et al. 2009). according to the same reports, the changes in ecg waves after black widow spider venom injection were observed clearly, so that st waves changes and widening of qrs waves indicated myocardial ischemia and myocardial damage, respectively. on the other hand, cardiomyopathy and myocarditis were reported earlier (akyildiz et al. 2009, rajabi 2014), detected by reduced height of r and p waves respectively. some previous studies (pulignano et al. 1998, akyildiz et al. 2009, rajabi 2014) have reported increasing in cardiovascular enzymatic markers such as cpk, ck-mb and ldh after black widow spider venom injection, which are the indicators of myocardial injury, heart muscle inflammation and arrhythmia. increasing in foresaid enzymes, are quite compatible to the results of the present study. on the other hand, the histological findings confirmed the incidence of myocarditis, hemorrhage and inflammation in heart tissues after black widow envenomation (clark et al. 1992, pulignano et al. 1998, akyildiz et al. 2009). in addition, occurrence of myocytolysis, and myocardial edema in marzan and maretic studies after latrodectus envenee e j arthropod-borne dis, march 2017, 11(1): 42–49 e valikhanfard-zanjani et al.: antivenom efficacy … 46 http://jad.tums.ac.ir published online: march 14, 2017 omation are another confirmation for the results of the present study (maretic and stani 1954, marzan 1955). the liver and the kidneys because of their extensive blood supply network are as one of the most vulnerable organs to toxin injury (sitprija and sitprija 2012). histological examination performed on the liver tissues showed that within 72h the venom caused central vein congestion, congested vessels in portal areas, dilated sinusoids and inflammation. marzan and maretic investigations confirm the pathological effects of the black widow spider venom on the liver tissue. according to their observations, after 30min to 6h of the venom injection, the hepatic cells swollen and gradually massive hyperaemia appears, pericapillary edema after 10 h; necrosis and lobular necrosis were seen respectively after 12 and 24 h of the venom injection (maretic 1953, marzan 1955). following the venom injection, significant increasing in ast and alt levels are good indicators for cytotoxic effects of the venom on the liver tissue. so that the ast to alt ratio within 24h after the venom injection was more than 1 (< 1), which from clinical point of view, it is a symptom of impaired liver function (pulignano et al. 1998, valikhanfard et al. 2014). these results further confirm our findings in the present study. the kidney tissues, within 72h after the venom injection, unexpectedly showed no significant histopathological complications. while other histological studies (maretic 1953, marzan 1955) have shown that after 10–20h of black widow spider venom injection, degeneration of tubular epithelium with necrosis (within 24 hours) was seen in kidney tissue. significant increasing in creatinine, bilirubin and urea, as important kidney health parameters, reported within 24h after latrodectus venom injection, represents toxic effects of the venom on kidney function (maretic 1953, marzan 1955, valikhanfard et al. 2014). although the recent performed studies demonstrate toxic effects of black widow spider venom on kidneys, however we did not observe any significant toxic effects on kidney tissues in our survey. other purpose of this study was to evaluate the efficacy of specific antivenom in neutralizing and prevent escalation of the complications on vital organs. according to the obtained results, in the second group of rabbits, the antivenom 24h after the venom injection could protect the heart tissue from severe myocytolysis, edema and hemorrhage. however, mild myocytolysis and mild hemorrhage were observed in some rabbits of this group. in addition, the antivenom prevented histopathological effects on liver tissues, so that except mild congestion and mild inflammation observed in some rabbits; antivenom could protected liver from severe central vein congestion, dilated sinusoids and sever inflammation. hence it seems that the antivenom injection even 24h after the venom, greatly could prevent incidence and intensification of histopathological complications caused by toxic effects of the venom. however, the mild histopathological effects observed in rabbit tissues may be due to the late (24h after venom injection) injection of antivenom. the obtained results of the antivenom efficacy in the present study corresponded with some studies performed on reducing concentrations in foresaid organs serum enzymes follow intravenous injection of antivenom (rajabi 2014, valikhanfard et al. 2014). based on same studies, 72h after the antivenom injection, significant decreasing was observed in ast, alt, urea and bilirubin, so that the ast to alt ratio in this period decreased to physiological state, that is less than 1 (< 1). according to the same reports, significant decreasing were observed in cpk, ck-mb and ldh, as predominant heart health markers, also ecg waves returned to its initial state (before venom injection) (rajabi 2014, valikhanfard et al. 2014). black widow spider venom exerts its destructive effects by both j arthropod-borne dis, march 2017, 11(1): 42–49 e valikhanfard-zanjani et al.: antivenom efficacy … 47 http://jad.tums.ac.ir published online: march 14, 2017 direct and indirect pathways. alpha latrotoxin, the neurotropic venom compound, by interaction with specific receptors on nerve cells, results in releasing of huge amount of catecholamines (henkel and sankaranarayanan 1999, ushkaryov et al. 2008, silva and ushkaryov 2010). hence it seems catecholamines, are the main mediator of the venom effects on organs (clark et al. 1992). the α-ltx after entering the circulation, through activating l-type calcium channels, which are abundant on vascular smooth muscle cells, results in calcium influx, which leads to vasoconstriction and hypertension (sitprija and sitprija 2012). catecholamines, by interaction with beta adrenoceptors on cardiovascular system result in activation of ltype calcium channel. influx of calcium leads to vasoconstriction and hypertension in smooth muscle cells of vascular system, on the heart tissue, activation of ltype calcium channel causes an increase in cytosolic calcium in myocytes, which subsequently induces the cells death (opie et al. 1985, van der heyden et al. 2005). on the other hand, catecholamines by stimulating catecholaminesensitive lipase in myocardium lead to an increasing in catabolism of free fatty acids. free fatty acids increasing in myocardium by disrupt the process of oxidative phosphorylation, lead to reduced atp synthesis, which is other way to induce myocytes death (opie 1975). beside all foresaid mechanisms, according to immunological studies of herberth in 2005 some latrophilin receptor (αlatrotoxin independent calcium receptor) genes are expressed in various mammalian tissues, so that the highest expression of latrophilin ii can be found in placenta, lungs, liver and mammary glands tissues, respectively. however, some expressions of this gene have also been demonstrated in the heart and kidney tissues (herberth et al. 2005). hence, expressions of the latrophilin ii, on some of the mammalian tissues, makes the direct effects of the venom expectable, so that latrophilin ii by activation of plc and dag causes huge released internal calcium from endoplasmic reticulum (henkel and sankaranarayanan 1999, ushkaryov et al. 2008). it can be predicted that black widow neurotropic venom through interaction with latrophilin receptors expressed on various mammalian organs, induce its cytotoxic effects directly. conclusion latrodectus dahli venom injection in rabbits, within 72 h, evoked severe cardiovascular complications such as myocytolysis, myocarditis, myocardial edema, hemorrhage and inflammation in heart. in addition, the venom injection induced central vein congestion, congested vessels in portal areas, dilated sinusoids and inflammation in liver. other results from this study showed specific antivenom injection even 24h after envenomation, greatly could protect the tissues from incidence of histopathological complications induced by the venom. acknowledgements we wish to thank the excellent technical assistance and financial support by department of venomous animals and antivenom production, razi vaccine and serum research institute. the authors declare that there is no conflict of interests. reference afshari r, khadem-rezaiyan m, balali-mood m (2009) spider bite (latrodectism) in mashhad, iran. hum exp toxicol. 28: 697–702. 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aspects of adrenergic modulation of cardiac l-type ca2+ channels. cardiovasc res. 65(1): 28–39. vetter rs, isbister gk (2007) medical aspects of spider bites. annu rev entomol. 53: 409–429. white j, cardosa jl, fan hw (1993) clinical toxicology of spider bites. in: meier j, white j (ed) handbook of clinical toxicology of animal venoms and poisons. crc press, united states, pp. 261–329. j arthropod-borne dis, september 2017, 11(3): 441–445 sm hoseini et al.: histopathological study of … 441 http://jad.tums.ac.ir published online: september 08, 2017 case report histopathological study of esophageal infection with gasterophilus pecorum (diptera: oestridae) in persian onager (equus hemionus onager) *seyed mohammad hoseini 1, bahram ali zaheri 2, mohamad ali adibi 2, hooman ronaghi 3, amir hossein moshrefi 4 1department of pathobiology, babol branch, islamic azad university, babol, iran 2department of environmental protection semnan, semnan, iran 3department of microbiology, faculty of specialized veterinary science, science and research branch, islamic azad university, tehran, iran 4young researchers and elite club, babol branch, islamic azad university, babol, iran (received 2 nov 2016; accepted 22 july 2017) abstract background: the larval stages of gasterophilus are obligate parasites in the gastrointestinal tract of equine accountable for pathologic ulcers in the persian onager gastrointestinal. the aim of the current report was to study the histopathological change with g. pecorum larvae in the esophagus of a persian onager. methods: this study was performed in iranian zebra propagation and breeding site in khartouran national park, southeast of shahrud city, semnan province, iran in 2014. following a necropsy with specific refer to esophagus of one adult female persian onager were transmitted to the laboratory. after autopsy, parasites collected from the esophagus were transmitted into 70% alcohol. for histopathological investigation, tissue samples were collected from the esophagus. the tissues were fixed in 10% buffered formalin, and conformity routine processing, there were stained with hematoxylin and eosin. results: after clarity by lactophenol parasites were identified as g. pecorum. microscopic recognition contained hyperemia, inflammatory cell infiltration, epithelial destruction, esophageal gland hyperplasia. conclusion: this is the first survey of g. pecorum and histopathological study in the persian onager esophagus in the world. keywords: persian onager, gasterophilus pecorum, epithelial destruction introduction the persian onager (equus hemionus onager), a wild donkey endemic to iran, is classified as critically endangered on the international union for conservation of nature red list. the asian wild donkeys were confined in successive periods but ecology of the two residual crowds, determined in preserved region in touran national park and bahram-egoor reserve (1). the genus gasterophilus (diptera: oestridae) contains nine species. equids are hosts to the larvae of the gasterophilus type causing gastrointestinal myiasis. gasterophilus is specified by dysphasia, gastrointestinal ulcer ations, intestinal obstruction or volvulus, rectal prolapses, anemia, diarrhea and digestive disturbances. the adult flies are not parasitic and are large, 11–15mm in length. adult gasterophilus spp. flies lay their eggs to host hairs. g. pecorum is an exception as females lay their eggs in brown-haired person, leaves, and stalks of plants (2, 3). after hatching, the larvae tunnels into the tissue of the host, larvae at the first stage attain the oral cavity of equine passively (g. intestinalis, g. pecorum) or actively, the first stage larvae hatch and moult to l2, which *corresponding author: dr seyed mohammad hosseini, e-mail: dr_hosseini2323@yahoo.com https://en.wikipedia.org/wiki/fly https://en.wikipedia.org/wiki/oestridae mailto:dr_hosseini2323@yahoo.com j arthropod-borne dis, september 2017, 11(3): 441–445 sm hoseini et al.: histopathological study of … 442 http://jad.tums.ac.ir published online: september 08, 2017 can be available in various regions of the gastrointestinal tract, and in l3 remains dependent to the mucosa for 8–10 months (4, 5). gasterophilus pecorum, g. inermis, and g. haemorrhoidalis are just reported in finite regions of europe and eastern countries (6). the damage the bot fly reasons happens after the larvae arrive the animal's mouth and gastrointestinal tract. when the first instar larvae tunnel into the mouth, the horse may experience intense inflammation, as well as the expansion of pus pockets and loosened teeth. loss of appetite may develop due to the larva's resident. as the second and third instar larvae reside the gastrointestinal tract and bind to the stomach and intestine, variable complications can occur. severe infestation of these larvae can cause anemia, esophageal paralysis, ulcerated stomach, chronic gastritis, stomach rupture and squamous cell tumors (7). esophageal disorders, exception obstruction is not common to observe in equine. little is known about the parasite spectrum of this species. accordingly, there was severity of the infection in this area of robot flies. the aim of the current report was to study the histopathological change with g. pecorum larvae in the esophagus of a persian onager. materials and methods this study was performed in iranian zebra propagation and breeding site in khartouran national park, southeast of shahrud city, semnan province, iran (latitude 36.736536, longitude 55.700684) in 2014, with a temperate climate. a cervical vertebral fracture after collision with a fence was diagnosed as cause of death of the 20-yr-old female persian onager. probably it escaped from something and did not see a fence and subsequently broken neck lead to death. following a field, necropsy gastrointestinal system was attentively removed and transferred directly to the laboratory of veterinary diagnostic medicine of the islamic azad university-babol branch for histopathologic and parasitological examination. the esophagus was assayed for parasite infections. the large changes were recorded, and myiasis was collected and transferred into 70% alcohol (jahan alcohol teb co., arak 454546, iran). the parasites were detected as g. pecorum by light microscope with referral to key zumpt keys (8) using stereo microscopes with 10x to 40x magnification. tissue samples and myiasis were used for histopathological examinations fix the tissue immediately in 10% buffered formalin, paraffinembedded and sections were cut using a rotary microtome (leitz, 1512, germany) at 5μm and stained with hematoxylin and eosin (h and e). the study was approved by the animal ethics committee of islamic azad university, babol branch, babol, iran. results esophageal necropsy revealed that the onager was infected to myiasis. totally, 87 third larval stage of g. pecorum were removed from esophageal tissue of the animal (fig. 1). the third larval stage of g. pecorum was the arrangement of denticles on the pseudocephalon into 3 groups, 2 lying laterally and a third centrally in front of the mouth hooks (fig. 2). we found gastric myiasis caused by g. pecorum (365 larvae). the histopathological study revealed different part of myiasis (fig. 3). microscopic examination showed epithelial destruction, esophageal gland hyperplasia, hyperemia, lymphocyte and macrophage infiltration in mucosa and submucosa of esophagus of the equus hemionus infected by gasterophilus (fig. 4). j arthropod-borne dis, september 2017, 11(3): 441–445 sm hoseini et al.: histopathological study of … 443 http://jad.tums.ac.ir published online: september 08, 2017 fig. 1. third larval stage of gasrophilus pecorum a. ventral view of pseudocephalon b. ventral view c. dorsal view fig. 2. gasterphilus larvae on the esophageal mucosal membrane of an onager fig. 3. cross-sections gasterphilus larvae (10x), h and e fig. 4. cross sections of intraluminal myiasis, inflammatory cell infiltration (1), epithelial destruction (2), esophageal gland hyperplasia (3). h and e. x40 discussion botfly infestation has been reported in different countries can cause economic losses in domestic animals. the presence of gasterophilus species has been much studied in asia extensively as the most pathogenic gasterophilus species on horses. accomplished investigations in different parts of the world, incidence ranged from 11% b j arthropod-borne dis, september 2017, 11(3): 441–445 sm hoseini et al.: histopathological study of … 444 http://jad.tums.ac.ir published online: september 08, 2017 to 100% including 11.1% in israel (9), 12.3% in sweden (10), 43% in ireland (11), 34% in france (12), 53% in england and wales (13), 58% in belgium (14), 82.2% in italy (6), 65% in switzerland (15), 98.7% in kentucky, usa )16) and 100% in morocco (17, 18). bots in the alimentary tract were identified as third stage larvae associated with ulcers include g. pecorum, g. nigricornis and g. nasalis (19). gasterophilus pecorum has been studied in asia where it is regarded as the most pathogenic gasterophilus species on horses (8). a study in china was done on the diagnosis of the larval gasterophilus species in 90 equines, from 2008 to 2013 revealed the all-90 (100%) equines were infested via larval gasterophilus, and 3723 secondary instar larvae (l2) as well as 63778 third instar larvae (l3). six types of gasterophilus were recognized include g. pecorum 88.94%, g. nigricornis 4.94%, g. nasalis 3.93%, g. haemorrhoidalis 1.91%, g. intestinalis 0.19%, and g. inermis 0.087% (20). in iran, the onager was infected by myiasis (g. pecorum) and nematode (habronema muscae) (21). according to our results, g. pecorum is more adaptable to the local environment in khartouran national park. the association with this unique comportment and the desert steppe ecosystem can help describe the situation. water availability limits the activity area of wild animals in a 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hashemi2, mr abaei1, l hosseinpour2, f rafei1, *hr basseri1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical parasitology, section of mycology, school of public health, tehran university of medical sciences, tehran, iran (received 1 jun 2009; accepted 28 july 2009) abstract background: many microorganisms in midgut of mosquito challenge with their host and also other pathogens present in midgut. the aim of this study was presence of non-pathogens microorganisms like fungal flora which may be crucial on interaction between vectors and pathogens. methods: different populations of anopheles stephensi were reared in insectary and objected to determine fungal flora in their midguts. the midgut paunch of mosquito adults and larvae as well as breading water and larval food samples transferred on subaru-dextrose agar, in order to detect the environment fungus. results: although four fungi, aspergillus, rhizopus, geotrichum and sacharomyces were found in the food and water, but only aspiragilus observed in the midgut of larvae. no fungus was found in the midgut of adults. this is the first report on fungal flora in the midgut of the adults and larvae of an. stephensi and possible stadial transmission of fungi from immature stages to adults. conclusion: the midgut environment of adults is not compatible for survivorship of fungi but the larval midgut may contain few fungi as a host or even pathogen. keywords: anopheles stephensi, fungal flora, mosquito midgut, stadial transmission introduction there are more hesitant points about biology of parasite in the body of vector and so interaction each other. therefore, the identification of flora existed in the midgut of mosquito is necessary that might play important role in transporting of parasites (robert et al. 2001). generally, fungi, as the case with other major groups of eukaryotes, symbiotic microbes, have a major impact on the biology of insects. at one end of the symbiotic continuum, entomopathogenic microbes, the most intensively studied group of insect symbionts, are a significant source of mortality in insects and have been explored for their potential application as biological control agents (pedigo et al. 2006, douglas 2007, thomas 2008). all insects can be almost infected by fungi, but there is limited information about fungal/insect symbiosis in most mycological textbooks. trichomycetes are the most striking example of commensalisms among the fungi. they are associated with living arthropods such as insects, millipedes, and crustaceans, growing extensively in the hindgut (lichtwardt 1986). some of the trichomycetes, such as smittium spp. that occur on mosquitoes, have received considerable attention. high populations of smittium spp. on mosquitoes have been shown to *corresponding author: dr hamid reza basseri, e-mail: basserih@sina.tums.ac.ir iranian j arthropod-borne dis, (2009), 3(1): 36-40 l tajedin et al: study on fungal … 37 have a negative impact on mosquito colonies and thus might serve as a type of biological control (moss 1979). many trichomycetes appear to be communalistic, but under particular circumstances the gut fungi may provide their hosts with some essential organic nutrients. at least one species, smittium morbosum, is lethal to mosquito larvae, and fungal species in blackflies and other insects are known to invade the ovary and produce cysts which are "oviposited" by females in lieu of eggs, thus reducing the fertility in populations of those kinds of aquatic insects (lichtwardt 1986, 1996, misra 1998, lichtwardt et al. 2001, 2003). in this study, the presence of fungi in alimentary canal of different populations of anopheles stpehnsi and possible transfer of fungi flora from immature stage to adult stage were studied. materials and methods mosquito rearing four geographical populations of anopheles stephensi from bandar-abbas, iranshahr, kazeroon area and beech strain (collected from india) were colonized as follows. all development stages of the larvae of each population were maintained in 25×45 cm with and 5 cm depth trays. the larvae were fed with fish food and corn shuck and kept in insectary at 27±2 °c. all adult mosquitoes were held separately in 30×30×30 cm cloth cages and kept in insectary maintained at temperature 27±2 °c and humidity of 70%±5 relative humidity with photoperiod of 14 h light and 10 h dark. adult mosquitoes were offered water contain 10% sugar soaked cotton pads as a source of energy and fed on blood of pig for maintenance of colonies. dissection of mosquito and sample preparation all dissections of the midguts were carried out in sterile saline. each group consisted of cohorts of 10 larvae (last instar) and adults (fed and/or unfed) from each geographical population were randomly chosen and their midguts simultaneously isolated from individually dissected larvae or adults. the midguts were washed separately three times with sterile saline and subsequently they were opened by a longitudinal incision and subsequently the contents under sterile condition and close to flame were transferred on medium culture pallet. culturing of fungi in order to identify fungi species, present in larval food, water and comparing with midgut content of larvae and/or adult stages, a fungal medium culture was prepared as follow. in order to preparing subaru dextrose agar, a mixture of 10 gm peptone, 20 gm dextrose, 20 gm agars in was dissolved in 950 ml dh2o in sterile condition and then the medium aliquot to culture palates and kept at temperature 4 °c in refrigerator until used. the midgut contents, larval food and water were separately transferred on medium separately as explain above and incubated at temperature 27-30 °c for 37 d until the fungus growth. fungi identification the fungus was identified based on direct observation of fungus colonies or microscopic observation of fungi cells. primarily, each colony of fungi was observed by naked eyes or under a stereomicroscope and then characterized based on its size or color. secondly, a smear of fungus war prepared by picking up small piece of fungi colony and fixed on slide by mild flame. then the fixed fungus stained with methylene blue for 30 to 60 sec and washed with water. the fungi were surveyed perpendicularly under 100x eyepiece of microscope. results in this study, rhizopus spp., geotrichum spp. and saccharomyces were commonly present in water trays containing larvae and food iranian j arthropod-borne dis, (2009), 3(1): 36-40 l tajedin et al: study on fungal … 38 supply for larvae growth (table 1). by transferring the water on medium subaru dextrose agar, colonies of rhizopus spp., geotrichum spp. saccharomyces and aspiragilus spp. were grown indicating these fungi are presence in water. although, all above fungi were existed in the water, only aspiragilus spp. was found in alimentary canal of the larvae of bandar-abbas, india (beech) and iranshahr population (fig.1) indicating this fungus can survive in midgut of the larvae and is compatible to enzymatic environment of larvae digestive system. as it has been shown in table 1, no fungus was found in the alimentary canal of adult mosquitoes both females and males, indicating the environment of alimentary canal of adult mosquitoes was not suitable for the fungal growth. table 1. presence of fungi in the alimentary canal of the larvae and adults of the different populations of anopheles stephensi fig. 1. microscopic demonstration of fungi found in larval food, larvae trey water and alimentary canal of larvae. aspergillus spp (a), sacharomyces sp (b), rhizopus sp (c), geotrichum sp (d) was found in dry food supply of mosquito larvae. a sample of dry food was cultured on medium subaru dextrose agar after incubated at temperature 27–30°c for 3–7 days until the fungus growth fungus species an. staphensi aspergillus sp. geotrichum sp. rhizopus sp. sacharomyces sp. larva + iranshar adult larva + bandar-abbas adult larva kazeroon adult larva + beech adult iranian j arthropod-borne dis, (2009), 3(1): 36-40 l tajedin et al: study on fungal … 39 discussion in this study, aspiragilus spp., rhizopus spp. geotrichum spp. and saccharomyces were found in the water of breeding trays and larval food. this indicated that all mosquito larvae were exposed to fungal infection either oral or dermal contamination. however, among above fungi only aspiragilus spp. was found in the midgut larvae of different strains of an. stephensi except kazeroun population. the results show that there may be a competency between aspiragilus spp. as fungus flora and the midgut environment to at least allow the fungus to survive. generally, some fungus live in alimentary canal of insects as symbiotic microorganism (hati et al. 1961) but many fungi are pathogen of insects and majority of them are belong to geniuses of lagenidium, coelomomyces and culicinomyces which are crucially mortal for mosquitoes (roberts et al. 1987 ). larval black flies (diptera: simuliidae) are hosts for at least 20 species of trichomycetes (crosskey 1990). most species of trichomycetes colonizing black flies inhabit the hindgut. harpella melusinae, however, is known only from the midgut peritrophic matrix of black flies (lichtwardt 1986). this was the only trichomycete known to colonize black fly midguts until (labeyrie et al. 1996) recorded stachylina spp. in the midguts of simulium vittatum. even though several works had been done on fungus flora of aquatic some insects but still interaction of symbiotic fungus in midgut of mosquito larvae is unknown. although trichomycetes are obligate inhabitants of the arthropod gut, the responses of the host to trichomycete colonization are largely unknown. the association often is described as ommensalistic (lichtwardt 1986). trichomycete fungi are obligate endobionts of aquatic and terrestrial arthropods and were discovered by leidy in 1849. more than 200 species of trichomycetes are known from 15 orders of arthropods (lichtwardt 1986, misra 1998). however, we could not found any fungus in midgut of adult mosquitoes. this can be demonstrating lyses enzymes of midgut of an. stephensi inhibited fungi development in midgut of this mosquito. also it is not clear that aspiragilus spp. can stadial transmission from larvae and pupa to adult stage so this need further study. conversely, our study show that fungus cannot be normally partial of flora in digestive system of adult an. stephensi but the larvae alimentary canal can be host for limited fungus. acknowledgements the authors are indebted to the personnel of the department of medical entomology and vector control and department of parasitology, section of mycology, school of public health, tehran university of medical sciences. the authors declare that they have no conflicts of interest. references crosskey rw (1990) the natural history of blackflies. john wiley and sons, new york. douglas ae (2007) symbiotic microorganisms: untapped resources for insect pest control. trends biotechnol. 25(8): 338-42. hati ak, ghosh sm (1961) vorticella infestation of mosquito larvae and its effects on their growth and longevity. calcutta schl trop med bull. 9(4): 155. labeyrie es, molloy dp, lichtwardt rw (1996) an investigation of harpellales (trichomycetes) in new york state blackflies (diptera: simuliidae). j invert pathol. 68: 293-298. lichtwardt rw (1986) the trichomycetes: fungal associates of arthropods. springer-verlag, new york. lichtwardt rw (1996) trichomycetes and the arthropod gut. in: howard d, iranian j arthropod-borne dis, (2009), 3(1): 36-40 l tajedin et al: study on fungal … 40 miller d (eds): the mycota, animal and human relations. springer-verlag, new york, pp. 315-330. lichtwardt rw, cafaro mj, white mm (2001) the trichomycetes: fungal associates of arthropods. university of kansas, the kansas city. available at: http:// www.nhm.ku.edu/ lichtwardt rw, white mm, cafaro mj (2003) freshwater trichomycetes and their arthropod hosts. fungal divers. res. ser. 10: 81-100. misra jk (1998) trichomycetes-fungi associated with arthropods: review and world literature. symbiosis. 24(2): 179-220. moss st (1979) commensalism of the trichomycetes. in: batra lr (ed): insect fungus symbiosis, nutrition, mutualism and commensalisms. allanheld and osmun co, montclair, pp. 175-227. pedigo lp, rice me (2006) entomology and pest management. prentice hall, englewood cliffs. robert es, billingsley pf (2001) plasmodium invasion of mosquito cells: hawk or dove. trends parasitol. 17(5): 209-212. roberts dw, dunn hm, ramsay g, sweeney aw, dunn nw (1987) a procedure for preservation of the mosquito pathogen culicinomyces clavisporus. microbiol biotechnol. 26:186-188. thomas am (2008) pest and disease challenges and insect biotechnology solutions. entomol res. 38: 34–40. microsoft word 3-vatandoost-rtl.doc iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 21 original article ecology of anopheles dthali patton in bandar abbas district, hormozgan province, southern iran *h vatandoost, m shahi, aa hanafi-bojd, mr abai, ma oshaghi, f rafii dept. of medical entomology and vector control, school of public health and institute of public health research, medical sciences/tehran university, iran (received 9 oct 2006; accepted 19 feb 2007) abstract ecology of anopheles dthali was studied in bandar abbas county, where there is indigenous malaria. anopheles dthali plays as a secondary malaria vector in the region. it is active throughout the year in mountainous area with two peaks of activity, whereas in coastal area it has one peak. there is no report of hibernation or aestivation for this species in the region. precipitin tests on specimens from different parts showed that 15.6-20.8% were positive for human blood. this species usually rests outdoors. it has different larval habitats. insecticides susceptibility tests on adult females exhibited susceptibility to all insecticides recommended by who. lt50 for the currently used insecticide, lambda-cyhalothrin, is measured less than one minute. the irritability tests to pyrethroid insecticides, showed that permethrin and lambda-cyhalothrin had more irritancy compared to deltamethrin and cyfluthrin. larval bioassay using malathion, chlorpyrifos, temephos and fenithrothion did not show any sing of resistance to these larvicides at the diagnostic dose. it is recommended that all the decision makers should consider the results of our study for any vector control measures in the region. keywords: ecology, anopheles dthali, malaria vector, iran, resistance introduction anopheles dthali is a malaria vector in some parts of the world. this species has been suspected as a vector of malaria in saudi arabia (patton 1905). in 1961 rishikesh reported that an. dthali was a secondary vector in the northern region of somalia. anopheles dthali patton is widespread in north ethiopia and somalia, socotra, north africa to north west of pakistan, southwest of saudi arabia, around the red sea and adan gulf (christophers 1933, de meillon 1947, stone et al. 1959, gillies and de meillon 1968) and eritrea (shililu et al. 2003). this species in also one of the malaria vectors in iran and has been found in southern parts of zagros chain (manouchehri and rohani 1975). bandar abbas county is contained within the hormozgan area of the iran where malaria was prevalent in hyperendemic in the region up to 1958 before the national malaria eradication program was started in iran. anopheles dthali is one of the malaria vectors in hormozgan. this species is responsible for maintaining malaria transmission in the area when an. stephensi is absent (manouchehri et al. 1972). in october 1965, during entomological studies in highland area of bandar abbas out of 143 an. dthali dissected, two were found with infected salivary glands to sporozoites (ihr 1965). therefore the study on the ecology of an. dthali might be valuable for planning and management of malaria control in the areas concerned. in iran, an. dthali has been found in southern parts of the zagros chain, in the provinces of hormozgan, baluchestan, kerman, fars, yazd, khuzestan, kermanshah, khorassan and coastal area of the persian gulf up to 1410 m altitude *corresponding author: dr h vatandoost, tel: +98 21 88951393, e-mail: hvatandoost@yahoo.com iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 22 in hamadan (manouchehri et al. 1972). in hormozgan province an. dthali is widespread throughout the coastal and mountainous areas from southwest to the southeast. it has been reported from coastal areas of hormozgan from bandar lengeh to jask county and qeshm island. this species can be found mainly outdoors in animal houses, as well as human dwelling. manouchehri and rohani (1975) found that this species is more prevalent in animal shelters, tent and human dwellings. this species rests in water reservoir, warehouse, septum of well, cave and natural shelters such as holes in riverbanks. the adults are very sensitive to light and often disturbed by torchlight. it has been reported (gillies and de meillon 1968) that in southern arabia, mauritania and somalia the adults are not uncommon indoors. this species is a secondary vector in some parts of southern iran especially in mountainous areas of hormozgan province (manouchehri 1972). in hilly areas and valley of hormozgan it is associated with an. fluviatilis and an. stephensi (manouchehri et al. 1972). in the coastal areas of the province the density of an. dthali is negligible. in qeshm island this species is active in early may reaches a high peak in august, and then gradually decreases (fig.1). in mountainous area of province an. dthali is active during the autumn season and absent during the cold winter (december-february) and hot weather (jun-august). in southern iran two magoon traps with human and cow bait were used for the collection of anopheline from may to december, the results showed that 87.5% of an. dthali were collected from the trap with cow bait and only 12.5% from the trap with human bait (manouchehri and rohani 1974). precipitin tests on 517 specimens of this species, showed that 12.5% were positive for human blood (edrissian et al. 1985). another precipitin tests on females caught in houses from the north bandar abbas, showed 20.8% positive for human blood. from 4165 blood smears taken from an. dthali have been examined only 15.6% were positive for human blood, it should be emphasized that anthropophilic index depends on the area, varying from 1% in izah, a sheep rearing area, to 25% in bandar abbas. the results of precipitin tests from this species caught in houses from mountainous area of bandar abbas showed that 20% were positive for human blood (manouchehri et al. 1972). in arabia areas, patton (1905) found the larvae of an. dthali in springs and wells. at muscat, gill (1916) found them in pools, and especially in holes of volcanic rock fed by underground water. in hormozgan this species breeds in pebbly margins of rivers, springs, pits around the springs with or without vegetation, pools in dried-up river beds and palm irrigation canal. in bandar abbas county larvae were also found in mineral water. it is also found in water with high salinity (2.7 parts per 1000). the temperature ranging was between 13 ºc and 28 ºc with a ph of 6.9-8.0. larvae were more abundant during september and october, which is the end of the hot season in southern iran (manouchehri and rohani 1975). materials and methods study area hormozgan province has two different areas, coastal bond in south and mountain area in north. the study area is located in mountainous part of bandar abbas county with surface area of 65379 km2 located in south of iran (between 25° 24’28° 57’ n latitudes and 52° 41’-59° 15’ e longitudes). hormozgan province located in south and eastern south part of iran; its eastern neighbors are kohkiloye and boyerahmad province. in the east boarder it is adjacent to sistan and baluchestan and in the southern boarder it is adjacent to persian gulf and oman sea and it has kerman province in northern boarder and fars province in western north boarder. this province has subtropical climate and a suitable place for preserving and transmission of malaria disease. the total annual rainfall was 100-150 mm and the mean annual relative humidity is 59.5%. iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 23 the maximum and minimum mean annual temperatures are 52 and 6 °c, respectively. seasonal activity in order to find the seasonal activity of vectors, the standard method of who (1992) based on total catch were performed in the study area susceptibility to insecticides adult and larval susceptibility tests were carried out according to who method (world health organization 1981). female mosquitoes were exposed to a diagnostic dose of ddt 4%, dieldrin 0.4%, malathion 5%, fenitrothion 1%, propoxur 0.1%, bendiocarb 0.1%, permethrin 0.75%, deltamethrin 0.05%, lambdacyhalothrin 0.05% and cyfluthrin 0.15%. the room temperature and relative humidity was 25-29 ºc and 65-80%, respectively. the following larvicides were used in this investigation; malathion (3.125 mg/lit), temephos (0.25 mg/lit), fenitrothion (0.125 mg/lit) and chlorpyrifos (0.025 mg/lit) at the diagnostic dose. butanone 2% in absolute ethanol was used as control. all the concentrations were provided by who. larvae were collected from mountainous area of bandar abbas county (siahoo rural district) during 2002, and larviciding tests were carried out on them. in each test there was two control and 4 replicates beakers for testing a single diagnostic dose of larvicides. mosquito larvae were tested at the late third and early 4th instar according to the method described by who 1981. the larvae were exposed to a diagnostic dose of larvicides. at each concentration, at least 100 larvae representing four replicates of 25 were tested. the larvae were fed with bemax and mortality counts were made after 24 h exposure period. the irritability to lambda-cyhalothrin, permethrin, cyfluthrin and deltamethrin by who insecticides impregnated paper were determined. the number of takes off was recorded for 15 min in one min intervals. results this species is widespread throughout the coastal and mountainous areas with high density in mountain areas. in these areas an. dthali is active throughout the year with two peak, first in september-october and the other in april (fig.1). this species is associated with an. stephensi and an. fluviatilis in hormozgan province. this species has different larval habitats. anthropophility index is varied between 125% depend on locality. a total of 1602 anopheles dthali were collected in study areas and tested by who method with 10 insecticides and 4 larvicides. the results are shown in tables 1 and 2. although the hormozgan province had been sprayed with ddt since 1952 and subsequently with malathion from 1968, but an. dthali remained susceptible to ddt 4% and dieldrin 0.4%. furthermore, this species is highly susceptible to organophosphates, carbamat and pyrethroides insecticides. the lt50 and lt90 of lambda-cyhalothrin in sprayed area of bandar abbas district were estimated to be 0.74 min and 2.86 min, respectively. the results showed that an. dthali is susceptible to diagnostic dose of these insecticides. discussion anopheles dthali is widespread in semi-arid regions from the atlantic coast of north africa to baluchestan, northwest pakistan and southern iran. it is common in many areas boarding the red sea and the gulf of aden and extends from the sudan coast through ethiopia, southwestern arabia and somalia almost to mogadishu (de meillon 1947, stone et al. 1959, gillies and de meillon 1968). in iran, an. dthali has been suspected as a vector of malaria. in october 1963 during entomological studies in bandar abbas county of hormozgan province a number of an. dthali dissected which three were found with infected salivary gland (manouchehri et al. 1972). anopheles dthali has a wide distribution in hormozgan areas and was collected in all inspected rural districts. the species showed two peaks of density in fig.1 and one peak in coastal area of bandar abbas area. in bandar abbas iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 24 county which is a mountainous area, an. dthali is associated with an. stephensi. larvae of this anopheles were found in water with high salinity (2.7 part per 1000) in rural areas. the anthropophilic index of this species has been reported between 12.5 and 20% from south of iran (manouchehri et al. 1972, edrissian et al. 1985, ihr 1966). precipitin tests on specimens from morocco and saudi arabia carried out by the lister institute of preventive medicine in england showed that 4-18.7% were positive for human blood (bruce-chwatt et al. 1966). the results showed that the biting time of an. dthali on human bait started at 18.00 h and continued until 04.00 h, with a peak between 20.00 h, and 21.00 h. about 90% of the bites took place in the first half of the night (18.0024.00 hours) and only 2.5% in the three hours before sunrise (iphr 1970). in total, 1602 an. dthali larvae collected from the study area and tested with ten insecticides and four larvicides. results are shown in tables 1 and 2. according to the latest who recommendation on the diagnostic dose of insecticides for adult malaria vector in 1998, the susceptibility level of adult and larvae of an. dthali to ddt 4%, dieldrin 0.4%, malathion 5%, fenitrothion 1%, propoxur 0.1%, bendiocarb 0.1%, permethrin 0.75%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, cyfluthrin 0.15% were measured and exhibited susceptibility to all insecticides. different larvicides including malathion, temephos, fenitrothion and chlorpyrifos were used for their larvicidal activities. the results of the susceptibility tests of an. dthali to these insecticides showed that this species is susceptible to diagnostic dose of all larvicides mentioned. irritability level of an. dthali to lambda-cyhalothrin, permethrin, cyfluthrin and deltamethrin revealed that this species is more irritant to permethrin than other pyrethroids. 0 1 2 3 4 ap ri ma y ju n ju l au g se p oc t no v de c ja n fe b m ar m onth sh el te r fig. 1. monthly average density of an. dthali in mountainous area of bandar abbas county, hormozgan province, southern iran, 2002 iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 25 table 1. the mortality rate of adult an. dthali to diagnostic doses of different insecticides, bandar abbas, hormozgan province, iran, 2002 insecticides replicates no. mosquito tested no. dead mortality (%) se ddt 4% 4 99 99 100 0.00 dieldrin 0.4% 3 73 73 100 0.00 malathion 5% 4 86 86 100 0.00 fenitrothion 1% 3 94 94 100 0.00 propoxur 0.1% 4 96 96 100 0.00 bendiocarb 0.1% 4 88 88 100 0.00 permethrin 0.75% 3 71 71 100 0.00 deltamethrin 0.05% 4 96 96 100 0.00 lambdacyhalothrin 0.05% 4 100 100 100 0.00 cyfluthrin 0.15% 4 94 94 100 0.00 control 8 204 13 6.3 1.7 table 2. the mortality rate of larvae an. dthali to diagnostic doses of different larvicides with 24 hours exposure time, bandar abbas county, hormozgan province, iran, 2002 larvicides replicates no. larvae no. dead mortality (%) se malathion 4 99 99 100 0.00 temephos 4 102 102 102 0.00 fenitrothion 4 99 98 99 1.00 chlorpyrifos 4 101 101 100 0.00 control 4 100 11 11 3.12 iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 26 table 3. the mean number of take-off /adult/ min by an. dthali to different pyrethroid insecticides, bandar abbas county, hormozgan province, iran, 2002 deltamethrin 0.05% (n=30) cyfluthrin 0.15% (n=30) premethrin 0.75% (n=30) lambdacyhalothrin 0.05% (n=30) insecticide se mean no. take off se mean no. take off se mean no. take off se mean no. take off time (min.) 0.06 0.03 1 0.08 0.06 2 0.06 0.03 1 0.08 0.05 2 1 0.10 0.10 3 0.10 0.1 3 0.10 0.1 3 0.10 0.1 3 2 0.15 0.16 5 0.16 0.23 7 0.12 0.16 5 0.16 0.2 6 3 0.16 0.23 7 0.17 0.3 9 0.18 0.33 10 0.17 0.26 8 4 0.19 0.30 9 0.20 0.36 11 0.20 0.53 16 0.20 0.36 11 5 0.25 0.33 10 0.25 0.46 14 0.27 0.93 28 0.24 0.53 16 6 0.27 0.50 15 0.33 0.56 17 0.39 1.26 38 0.35 0.76 23 7 0.28 0.43 13 0.30 0.73 22 0.35 1.36 41 0.36 1.26 38 8 0.24 0.46 14 0.22 0.56 17 0.26 0.83 25 0.30 0.9 27 9 0.22 0.36 11 0.18 0.40 12 0.27 0.7 21 0.26 0.63 19 10 0.17 0.26 8 0.19 0.33 10 0.20 0.6 18 0.24 0.43 13 11 0.16 0.20 6 0.20 0.43 13 0.18 0.36 11 0.16 0.23 7 12 0.11 0.13 4 0.14 0.23 7 0.21 0.26 8 0.10 0.13 4 13 0.10 0.10 3 0.12 0.16 5 0.13 0.2 6 0.11 0.1 3 14 0.10 0.10 3 0.10 0.10 3 0.11 0.13 4 0.08 0.06 2 15 0.17 0.24 7.4 0.18 0.33 10.13 0.20 0.51 16.66 0.19 0.40 13.13 average table 4. the mean number of take-off/ adult/min of an. dthali in control test, bandar abbas county, hormozgan province, southern iran, 2002 acknowledgments the authors would like to appreciate the collaboration received from dr m baghaii, the staff of health center province in bandar abbas university of medical science, dr g zamani, and other personnel of bandar abbas health research center for their kind assistance and help. references bruce-chwatt lj, garett-jones c, weitz b (1966) ten years study (1955-64) of host selection by anopheline mosquitoes. bull who. 35: 405-439. christophers sr (1933) the fauna of british india, diptera, vol. iv. london. de millone b (1947) publications of the south african institute for medical research, no. 49. se mean no.of take-offs time (min) 0.04 0.04 2 1 0.04 0.04 2 2 0.05 0.07 3 3 0.01 0.02 1 4 0.05 0.07 3 5 0.04 0.04 2 6 0.01 0.02 1 7 0.09 0.10 5 8 0.01 0.02 1 9 0.04 0.04 2 10 0.04 0.04 2 11 0.05 0.07 3 12 0.10 0.07 3 13 0.10 0.02 1 14 0.04 0.04 2 15 0.03 0.04 2.2 average iranian j arthropod-borne dis, (2007), 1(1): 21-27 h vatandoost et al.: ecology of anopheles… 27 edrissian gh, manouchehri av, hafizi a (1985) application of an enzyme linked immonosorbent assay (elisa) for determination to the human blood index in anopheline mosquitoes collected in iran. j am mosq control assoc. 1(3): 349-352. gillies mt, demeillon b (1968) the anophelinae of africa south of the sahara. publication of the south african institute for medical research, no. 54, johannesburg. institute of public health research (1965, 1966, 1970) annual entomological studies in hormozgan province. annual reports of iphr, bandar abbas, south of iran. manouchehri av, ghiasseddin m, shahgudian er (1972) anopheles d’thali patton, 1905, a new secondary vector in southern iran. ann trop med parasitol. 66: 537-538. manouchehri av, rohani f (1975) notes on the ecology of anopheles d’thali patton in southern iran. ann trop med parasitol. 69: 393-397. manouchehri av, zaim m, emadi am (1992) a review of malaria in iran (1975-90). j am mosq control assoc. 8(4): 381-385. patton ws (1905) the culicidae fauna of the adenhinterland. j bombay natural hist soc. 16: 623-627. shililu j, ghebremeskel t, mengistu s, fekadu h, zerom m, mbogo c, githure j, gu w, novak r, beier jc (2003) distribution of anopheline mosquitoes in eritrea. am j trop med hyg. 69(3): 295-302. stone a, knight kl, starcke h (1959) a synoptic catalogue of the mosquitoes of the world (diptera: culicidae). thomas say foundation, entomological society of america, washington. who (1992) entomological field techniques for malaria control. part i. learner’s guide. world health organization, geneva. j arthropod-borne dis, march 2017, 11(1): 36–41 z zarei et al.: wild rodent ectoparasites … 36 http://jad.tums.ac.ir published online: march 14, 2017 original article wild rodent ectoparasites collected from northwestern iran zabihollah zarei 1, mehdi mohebali 1,2, zahra heidari 1, eshrat beigom kia 1, amrollah azarm 3, hasan bakhshi 4, jaber davoodi 5, hamid hassanpour 1, manizhe roohnavaz 1, mahya khodabakhsh 6, *zakkyeh telmadarraiy 7 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2centers for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, faculty of medical sciences, university of tarbiat modarres, tehran, iran 4malaria and vector research group, biotechnology research center, pasteur institute of iran, tehran, iran 5department of veterinary parasitology, islamic azad university abhar branch, abhar, iran 6department of veterinary internal diseases, faculty of veterinary medicine, tehran university, tehran, iran 7department of medical entomology and vector control, tehran university of medical sciences, tehran, iran (received 28 july 2015; accepted 7 feb 2016) abstract background: rodents play an important role as reservoir of some pathogens, and the host of some ectoparasites as well. these ectoparasites can transmit rodents’ pathogens to human or animals. the aim of this study was to assess the distribution and infestation load of ectoparasites on rodents in meshkin-shahr district, northwestern iran. method: rodents were captured using baited live traps in spring 2014 from meshkin-shahr district and were transferred to the laboratory for identification to the species level. their ectoparasites were collected, mounted and identified. results: three rodent species including meriones persicus (74%), mus musculus (16.9%) and cricetulus migratorius (9%) were identified. among all rodents, 185 specimens (90.69%) were infested with a total of 521 ectoparasites. overall, 10 arthropods species were collected, including fleas (97.6%), one mite (1.6%) and one louse species (0.6%) as follows: xenopsylla nubica, x. astia, x. buxtoni, x. cheopis, nosopsyllus fasciatus, n. iranus, ctenocephalides felis, ctenophthalmus rettigismiti, ornithonyssus sp and one species of genus polyplax. the most prevalent ectoparasites species was x. nubica (89%). conclusion: nearly all rodent species were infested with xenopsylla species. monitoring of ectoparasites on infested rodents is very important for awareness and early warning towards control of arthropod-borne diseases. keywords: rodent, ectoparasites, iran introduction rodents play important roles in disease transmission via their urine, feces, bite and transmission of pathogenic agents through ectoparasites (williams et al. 1997). some viral, bacterial and protozoal agents as well as helminthes resulting in leishmaniasis, crimeancongo haemorrhagic fever (cchf), plague, leptospirosis, salmonellosis, rat-bite fever, omsk hemorrhagic fever, anaplasmosis, ehrlichiosis, murine typhus, theileriosis and babesiosis can be transmitted by rodents to other animals orhumans when they are in close contact (williams et al. 1997, tsuji et al. 2001, motevalli-hagghi et al. 2002, telmadarraiy et al. 2007, kia et al. 2009, tajedin et al. 2009, eisen and gage 2012, nateghpour et *corresponding author: dr zakkyeh telmadarraiy, e-mail: ztelma@yahoo.co.in, telmadarraiy@tums. ac.ir j arthropod-borne dis, march 2017, 11(1): 36–41 z zarei et al.: wild rodent ectoparasites … 37 http://jad.tums.ac.ir published online: march 14, 2017 al. 2013). fleas (siphonaptera) and ixodid ticks (acari: ixodidae) are the major vectors of important pathogens threatening human and animals (goz et al. 2016). some ectoparasites of medical and veterinary importance were found and reported from rodents in different parts of iran (mohebali et al. 1997, kia et al. 2001, telmadarraiy et al. 2004, shayan and rafinejad 2006, hanafi-bojd et al. 2007, telmadarraiy et al. 2007, sharifi et al. 2008, shoorijeh et al. 2008, kia et al. 2009, tajedin et al. 2009, nateghpour et al. 2013, zendehfili et al. 2014, telmadarraiy et al. 2015). in those studies some rodents species such as meriones lybicus, m. persicus, m. hurrianae, tateraindica, mus musculus, rattus rattus, r. norvegicus, nesokia indica, microtus socialis, gerbillus nanus, glis glis, apodemus sylvaticus and soon were studies and the ectoparasite species were included pulex irritans, xenopsylla cheopis, x. astia, x. buxtoni, x. nubica, x. conformis, nosopsyllus medus, n. fasciatus, polyplaxs pinulosa, p. gerbilli, rhipicephalus sp, hyalomma sp, boophilus sp, laelaps nuttalli, l. ciccuminata, and rolaelaps hermaphrodita, paracheylaelaps pyriformis, dermanysus sanguineus, d. americanus, ornithonyssus bacoti, haplopleura captiosa, haemolaelaps glasgowi, echinolaelaps echidninusand some other species. because of high climatic diversity in iran and numerous ecological niches of rodents, there is a considerable potential for survey on their ectoparasites to find the vulnerability of vector-borne zoonoses in the country. there was no comprehensive study on the rodent and their ectoparasites in northwest of iran. therefore, the aim of this study was to assess the distribution and infestation level of ectoparasites on rodents in selected sites in meshkin-shahr county located in northwest of the country. this will help us in monitoring ectoparasites infestation to alerting and early warning to possibility for control of arthropodborne diseases in northwestern part of iran. materials and methods study area meshkin-shahr county (38° 26′n 47° 45′e) is one of the counties located in ardebil province (fig. 1). the province is considered the coldest province in iran. large parts of the province are forested and green. neighboring the caspian sea and the republic of azerbaijan, the province is of great economic significance as well as splendid natural beauty and numerous sights. meshkin-shahr county is located at an elevation of 1,341 meters above sea level, the whole district occupying the foot-hills of sabalan mountain. the weather is extremely cold in the winter (down to -27 °c) and warm during the summer (up to 40 °c). many tourists come to the region for its cool climate. rodent collection rodents were live trapped at different localities including altisiluo, khiav, abiz, our, oudkandi and magandeh. rodent’s collection was carried out on various occasions and different places like indoor places, farms, roadsides and other places in spring 2014. live traps were randomly set in different aforementioned habitats baiting with favorable food of rodents according to the season. these traps were set at different parts of selected regions of the district from 11:00 am to the next day. ectoparasites collection captured rodents were transported rapidly to the laboratory of meshkin-shahr research station and their ectoparasites were isolated using brushing on the fur or by a fine forceps immediately after transporting. collected ectoparasites were stored in 70% ethanol for preservation and identification. rodents and ectoparasites identification different morphological criteria were used for identification of rodents in the species levj arthropod-borne dis, march 2017, 11(1): 36–41 z zarei et al.: wild rodent ectoparasites … 38 http://jad.tums.ac.ir published online: march 14, 2017 el by preparation of rodent skulls, mounting of ectoparasites, identification of rodents (etemad 1976) and ectoparasites (strandtmann and wharton 1958). ectoparasites specimens were fixed in between microscope slides and cover glass and confirmation of some species was carried out according to standard methods provided. results a total of 204 rodents were identified in 3 species including meriones persicus (74%), mus musculus (16.9%), and cricetulus migratorius (9%). among them, 185 (90.69%) were infested with 521 ectoparasites. the ectoparasites of the rodents were comprised mites, fleas and lice (fig. 2). totally, 10 species were captured in this study including eight fleas (97.6%), one mite (1.6%) and one species of genus polyplax (0.6%) species as follows: x. nubica, x. astia, x. buxtoni, x. cheopis, nosopsyllus fasciatus, n. iranus, ctenocephalides felis, ctenophthalmus rettigismiti, ornithonyssus sp, and polyplax sp (table 1). table 1. details of collected ectoparasites from caught rodents. these ectoparasites were collected in spring 2014 from meshkin-shahr district located in northwest part of iran (%)totaltotalfemalemaleectoparasites 89465134331xenopsylla nubica 21138x. astia 2.41275nosopsyllus fasciatus 0.4211xenopsylla buxtoni 1.91028xenopsylla cheopis 0.6321ctenocephalides felis 0.9431nosopsyllus iranus 0.6321ctenophthalmus rettigi smiti 1.6835ornithonyssus sp 0.6312polyplax sp fig 1. location of ardebil province. this province is located in northwest part of iran and is bordered with republic of azerbaijan. meshkin-shahr county is one of the counties of the province. fig 2. some collected ectoparasites; a: xenopsylla nubica, b: x. nubica, c: nosopsyllus fasciatus, d: ctenocephalides felis, e: x. cheopis, f: x. astia, g: ctenophthalmus rettigismiti, h and i: n. iranus j arthropod-borne dis, march 2017, 11(1): 36–41 z zarei et al.: wild rodent ectoparasites … 39 http://jad.tums.ac.ir published online: march 14, 2017 discussion in the present investigation, three rodent species including meriones persicus, mus musculus and cricetulus migratorius were caught in meshkin-shahr district located in ardebil province, northwest of iran during spring 2014. study on these rodents is essential as they have an important role as host of many parasitic agents (darvish et al. 2015). out of 204 collected rodents, m. persicus was the most prevalent species in that area (74%). in the former investigations, occurrence of this rodent has been revealed in central asia, transcaucasia, turkey and pakistan (carleton and musser 2005). moreover, in northwest of iran, this species was found in maku and urumiyeh located in west azerbaijan province as well as jolfa located in east azerbaijan province (lay 1967). in a recent investigation in northwest part of iran, this species has been found in kordasht, sufian and tabriz (east azerbaijan province) as well zanjan county in zanjan province (darvish et al. 2015). this species is introduced as a probable reservoir for visceral leishmaniasis in iran (shojaei and mohebali 2005). the presence of this species in meshkin-shahr district is consistent with the above mentioned investigations. furthermore, we could find mus musculus in our investigation. this species has been found in almost all parts of the country (etemad 1976). another species c. migratorius, has been captured in meshkin-shahr and reported with high titers of leishmania antibody in meshkin-shahr (mohebali et al. 1995, 1997). ectoparasites play an important role in transmitting some diseases to human and domestic animals (williams et al. 1997, eisen and gage 2012). therefore, study on ectoparasites will help investigators to evaluate the possibility of pathogens transmission in a given area. in this study, the most prevalent (89%) ectoparasite was x. nubica. this species has been introduced as the vector of plague in mauritania (eisen and gage 2012). among other flea species collected in this study x. astia, x. buxtoni, x. cheopis and n. fasciatus are introduced as the vectors of plague in southeast asia and the western pacific, iran, libya, mauritania, east africa, southern africa and south america (eisen and gage 2012). in meshkin-shahr county, m. persicus was the dominant rodent species, while 7 species of ectoparasites were identified as: x. gerbilli, x. nuttalli, n. fasciatus, stenopnia tripectinata, ornitonyssus bacoti, trichoecius romboutsi and haemaphysalis sp. (mohebali et al. 1997). comparing with their study we could not capture alactage elater rodent species, while the common ectoparasite in both studies is n. fasciatus. in another study in germi, ardebil province, two species of rodents were trapped: m. persicus and microtus socialis with the frequency of 90.4% and 9.6%, respectively (kia et al. 2010). the frequency of m. persicus is in concordance with our result. although we did not find m. socialis in the present investigation, but two other species, i.e., c. migratorius and m. musculus species were trapped in our study. other investigations carried out in west of iran reported some ectoparasite including pulex irritans, x. buxtoni, nosopsyllus medus, polyplax spinolosa, rhipicephalus sp, hyalomma sp, lealaps nuttalli, dermanysus sanguineus and ornithonussus bacoti (telmadarraiy et al. 2007). the difference between this study with our investigation may be due to the host and climate. the importance of ectoparasites has encouraged investigators to study on other animals like hedgehogs (goz et al. 2016). in a most recent investigation carried out on 21 hedgehogs ectoparasites in east part of turkey (northwest of iran), r. turanicus and a. erinacei were detected, also, j arthropod-borne dis, march 2017, 11(1): 36–41 z zarei et al.: wild rodent ectoparasites … 40 http://jad.tums.ac.ir published online: march 14, 2017 infestation rate for ticks and fleas was detected as 66.6% and 100%, respectively (goz et al. 2016). we could not collect these ectoparasites on the caught rodents. this is expected to be so, because a. erinacei is the ectoparasite of hedgehogs, cats and dogs (pomycal 1985). conclusion five flea species identified in this study are introduced as the vectors of plague in different foci of the disease around the world, so it is recommended to do a serological study for plague in rodents and their ectoparasite in this area. monitoring of ectoparasites on the infested rodents is very important for awareness and early warning towards control of arthropod-borne diseases. acknowledgements the authors would like to appreciate the kind collaborations of meshkin-shahr health center staff. this research was financially supported by tehran university of medical sciences, tehran, iran (project no: 92-0227-22943). the authors declare that they have no conflicts of interests. references carleton md, musser gg (2005) order rodentia, mammal species of the world, 1 ed. the johns hopkins univ, usa. darvish j, mohammadi z, mahmoudi a, siahsarvie r (2015) faunistic and taxonomic study of rodents from northwestern iran. iran j animal biosystem. 10: 119–136. eisen rj, gage kl (2012) transmission of flea-borne zoonotic agents. annu rev entomol. 57: 61–82. etemad e (1976) mammals of iran, vol 1. rodents and their identification keys. nat ural society of guardianship of national resources and human enviroment, tehran. goz y, yilmaz ab, aydin a, dicle y (2016) ticks and fleas infestation on east 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university of medical sciences, p.o.box: 14155-6446, tehran, iran (received 23 jul 2008; accepted 5 nov 2009) abstract background: rodents play important role as host of ectoparasites and reservoir of different zoonotic diseases. the aim of this study was to asses the infestation of commensal rodents with ectoparasites in bandar abbas, a port city located in the northern part of the persian gulf in iran. methods: rodents were captured using live traps during the study period in year 2007. after transferring the rodents to the laboratory, they were identified and then their ectoparasites were collected and mounted for species identification using appropriate systematic keys. results: a total of 77 rodents were identified including rattus norvegicus (74%), r. rattus (16.9%), mus musculus (7.8%) and one hamster. among all rodents, 40.3% were found infested with ectoparasites. a total of 69 ectoparasites were collected comprising flea, lice, mite and tick. two species of fleas; xenopsylla cheopis and x. astia were identified with higher index of x. astia. two genera of ticks including hyalomma sp. and rhipicephalus sp. were identified. laelaps nuttalli was the only mite found. the polyplax spinulosa was considered as lice ectoparasite. conclusion: among all arthropods collected, flea and lice had the most and the least frequency, respectively. nearly all rodent species were infested with xenopsylla. these fleas are important due to their role in plague and murine typhus transmission. ticks are important due to their role in cchf (crimean-congo hemorrhagic fever), theileriosis, babesiosis, anaplasmosis and ehrlichiosis transmission. monitoring of ectoparaiste infestation is important for preparedness and early warning preparation for possible control of arthropod-borne diseases. keywords: rodents, ectoparasite, iran introduction rodents play an important role in disease transmission by their urine, feces, bite, and ectoparasites. different disease agents oof bacteria, rickettsia, viruses, protozoa and helminthes can be transmitted by rodents to human and animals. some examples of such diseases are plague, leptospirosis, salmonellosis, rat-bite fever, leishmaniasis, chagas' disease, omsk hemorrhagic fever, murine typhus and lassa fever (bell et al. 1988) crimean congo hemorrhagic fever (cchf theilerios, babesia, anaplasmosis and ehrlichiosis (inokuma et al. 2001, 2003). the close association of commensal rodents with human and domestic animals is a risk factor for transmission of these diseases. in the previous reports on the ectoparasites of rodents in different parts of iran, various species of ectoparasites have been occurred, most of them with medically or veterinary importance. in a study carried out in korram-abad, lurestan province, iran during year 2002-2003 a total of 167 rodent specimens were captured. altogether 218 ectoparasites related to 3 orders, 6 families, 6 genera, and 7 species were recognized. fleas with 3 species had the most number of species, mites and lice allocated the most (64.67%) and the least (3.21%) frequency of ectoparasites, *corresponding author: dr z telmadarraiy, e-mail: telmadarraiy@tums.ac.ir iranian j arthropod-borne dis, (2009), 3(2): 44-49 eb kia et al: ectoparasites of rodents ….. 45 respectively. haemolaelaps glasgowi (42.2%) was the most common ectoparasite while, nosopsyllus irranus only constituted approximately 0.91% of specimens (shayan and rafinejad 2005). motevalli hagghi et al. (2000 and 2002) conducted two studies in sari rural and urban areas, southern part of caspian sea. they found r. norvegicus, r. rattus, mus muscuuls, glis glis, apodemous sylvaticus, nesokia indica and arvicola terrestris. the main important ectoprasites in those studies were echinolaelaps echidnini, hoplopleura sp., phipicephalus sp. and nosopsyllus fasciatus. ectoparasites of various species of rodents were examined from sarepole zehab, kermanshah province, iran, during 2005. a total of 139 rodents included 6 species of hosts were captured during all different seasons and examined for ectoparasites. the hosts included microtus socialis, mus musculus, rattus rattus, nesokia indica, meriones persicus and tatera indica. the 9 species of ectoparasites recovered included 3 fleas (pulex irritans, xenopsylla buxtoni, nosopsyllus medus), one sucking lice (polyplax spinulosa), two ticks (rhipicephalus sp., hyalomma sp.), and 3 mites (laelaps nuttalli, dermanysus sanguineus, ornithonussus bacoti). among all arthropod collected, the lice and flea had the most and least frequency, i.e. 77.7% and 4.4%, respectively (telmadarraiy et al. 2007). a study was carried out by hanafibojd et al. (2007) in bandar abbas in a control program campaign. they found 105 rodents demonstrating four species including r. rattus (3%), r. norvegicus (78%), m. musculus (3%) and tatera indica (16%), before the control program. after 12 months, the duration of control program, rodents were re-captured to assessment the success of control activity. in this stage, 35 small mammals demonstrating four species were obtained icluding r. norvegicus (51.4%), r. rattus (37.1%), t. indica (8.6%) and one weasel (2.9%). the identified ectoparasites were xenopsylla buxtoni, rhipicephalus sp., polyplax gerbilli, haplopleura captiosa, ornithonyssus bacoti, laelaps nuttalli, dermanyssus americanus, dermanyssus sanguineus, haemolaelaps glasgowi and echinolaelaps echidninus. during an investigation on the endoparasites of rodents in ahvaz, khuzestan province, south western iran, a total of 90 rodents including 14 m. musculus, 4 r. rattus and 72 r. norvegicus were trapped from different localities of ahvaz and its suburbs, during 1998 2000. examination of different tissues and identification of parasite species showed that the variation among helminth species was wide especially those which arthropods are involved in their life cycles. the most prevalent species of rodents was r. norvegicus in which trypanosoma lewisi and trichosomoides crassicauda were the most prevalent species of protozoan and helminth parasites, respectively. gongylonema monigi, streptopharagus kuntzi and rictularia ratti from r. norvegicus and gongylonema neoplasticum from both r. norvegicus and r. rattus were reported for the first time in iran. report of physocephalus sexalatus from r. norvegicus apparently comprised a new host species in the world (kia et al. 2001). similar studies on endoparasites of rodents in other parts of the country and the association of medical and veterinary important arthropods will reveal better understanding the life cycles of arthropod-borne disease. there is considerable potential for investigation of the ecological, physiological and systematic relationships of the ectoparasites and their hosts. unfortunately, there has been no coordinated research effort, and much of the research has been directed only to economically important species or disease vectors. in order to find a correlation among ectoparasites and endoparasites joint studies on both agents with improvement of inter-sectoral coordination of medical entomologists and parasitologists is essential. the aim of this study was to determine the frequency of ectoparasites in commensal rodents of bandar abbas for further control measures. iranian j arthropod-borne dis, (2009), 3(2): 44-49 eb kia et al: ectoparasites of rodents ….. 46 materials and methods geographical information on study area bandar abbas lies in the western part of hormozgan province of iran. it is the capital city of the province and the main port in iran in the persian gulf. this has made it a developing city with many new constructions. it has also become an important trading center. this aspect of development has led to migration of too many people to bandar abbas, both from other provinces of iran and from the neighboring countries. bandar abbas is situated on flat ground with an average altitude of 9 meter above sea level. its coordinates are: 27°11′n 56°16′e. the city has a hot and humid climate. maximum temperature in summer can reach up to 49 °c while in winters the minimum temperature drops to about 5 °c. the annual rainfall is around 251 mm and the relative humidity is 66% (fig.1). rodent collection rodents were collected using live traps. the traps were set at different parts of the city at various occasions in year 2007. traps were baited with favorite foods of rodents at different seasons. trapped rodents ectoparasites collection was transferred to laboratory and after morphometrical measurements their ectoparasites were picked up using brush against the fur of rodents. mites, lice and fleas were collected by this method. occasionally forceps were used for tick collection. all ectoparasites were stored at 70% alcohol for preservation and further species identification. ectoparasites and rodents identification ectoparasite samples were mounted using clearing, dehydration and mounting procedure and preserved constantly with canada balsam. species identification of lice, flea, mites and also ticks were carried out according to the available systematic keys (strandtmann & wharton, 1958). rodents were identified after recording their different morphological characteristics. fig. 1. hormozgan province and bandar abbas city, iran results during this study, a total of 77 rodents were captured from different parts of the city in bandar abbas. the rodents included four species (table 1). among commensal rodents rattus norvegicus and mus musculus had the most and least frequency (74%, versus 7.8%, respectively). in general 40.3% of the rodents were found infested with ectoparasites. a total of 69 ectoparasites were collected from the rodents including lice (polyplax spinulosa), ticks (hyalomma sp. and rhipicephalus sp.), mite (laelaps nuttalli) and flea (xenopsylla astia and x. cheopis), with the highest rate of the latest. the ectoparasite groups and their abundancy in each host are shown in table 1. according to this table the catch rate of ectoparasites on r. norgvegicus, r. rattus. m. musculus and hamster were 76.8, 11.6, 1.5 and 10.1%, respectively. iranian j arthropod-borne dis, (2009), 3(2): 44-49 eb kia et al: ectoparasites of rodents ….. 47 table 1. ectoparasite groups among rodents in bandar abbas, hormozgan province ectoparasite flea mite lice tick total catch rodent species catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) catch no. catch rate (%) r. norvegicus (n=57) 47 88.7 3 5.7 1 1.9 2 3.8 53 76.8 r. rattus (n=13) 6 75 2 25 0 0 0 0 8 11.6 m. musculus (n=6) 0 0 1 100 0 0 0 0 1 1.5 hamster (n=1) 7 100 0 0 0 0 0 0 7 10.1 total (n=77) 60 87 6 8.7 1 1.4 2 2.9 69 100 discussion ectoparasites of rodents play an important role for disease transmission to human and animals. for example x. cheopis (oriental rat flea) and x. astia are responsible for transmitting yersinia pestis, the causative agent of plague. as it is now generally admitted, one or more species of fleas occurring on the common rats, r. rattus and r. norvegicus, are concerned in the transmission of plague. thus, the study of these fleas has become a matter of the highest importance in connection with the prevention of the diseases. in the current study, the most abundant ectoparasite was xenopsylla with 88.7% catch rate in r. norvegicus. the predominance of flea has been reported in some similar studies. in a study in huambo, angola eight species of ectoparasites were collected from 166 commensal rodents, including r. rattus, r. norvegicus and mus musculus from january to december 1986. the oriental rat flea, x. cheopis, was the predominant species with respect to mean intensity and prevalence. the mite laelaps muricola, the louse polyplax spinulosa, one species of ixodes latreille, and one species of ornithonyssus sambon were also recorded (linardi et al. 1994). in another study which presented ectoparasites for four species of commensal murid rodents rattus rattus palelae, r. argentiventer, r. exulans and mus musculus castaneus in sulawesi utara, indonesia, the flea, x. cheopis, was the most common on r. rattus (durden and page 2008). additionally, rat ectoparasite surveys in relation to murine typhus fever in puerto rico were studied by fox and garcía-moll (1961). they found that rats were infested with x. cheopis. other common rat ectoparasites, laelaps nuttalli, ornithonyssus bacoti, listrophoroides expansus, and polyplax spinulosa increased in abundance. in the present study, the highest total catch rate was related to r. norvegicus. the catch rate of flea on rats was 87%; this figure for mite was calculated as 8.7%. in a study carried out in egypt from april 2006 to march 2007 (el kady et al. 2007) the ectoparasites infesting commensally rodents of different localities were determined. totally 135 rodents were captured including r. norvegicus r. rattus frugivorous, r. rattus alexandrinus and mus musculus. from 388 ecto-parasite infested rodent collected number and ecto index was: fleas n= 114 (0.84 flea/rat), lice n= 93 (0.69 lice/rat), mites n= 165 (1.2 mite/rat) and larva of ticks n= 16 (0.12 tick/rat). in this study fleas n= 53 (0.76 flea/rat), lice n= 1 (0.69 lice/rat), mites n= 5 (0.07 mite/rat) and nymph of ticks n= 16 (0.12 tick/rat), mus musculus including only mites n= 1 (0.16 mite/mus) and flea in hamester n= 7 (7 flea/ham.) another iranian j arthropod-borne dis, (2009), 3(2): 44-49 eb kia et al: ectoparasites of rodents ….. 48 study in egypt revealed that rattus rattus frugivorus, r. norvegicus and meriones shawi were the most vulnerable hosts for ectoparasites while mus musculus harbored the lowest numbers of ectoparasites. fleas, lice and mites were found on rodent species the whole year round, but reached a peak in the spring and summer months. the isolated ectoparasites included x. cheopis, leptopsylla segnis, echidonphaga gallinacea, polyplax spinulosa, p. vacillata,p. gerbilli, ornithonyssus bacoti, laelaps nuttalli, echinolaelaps echidninus and haemolaelaps glasgowi. distribution of ectoparasites varied according to rodent host and location (el deeb et al. 1999). in the survey conducted on commercial and wild rodents from different locations in wadi hanifah in riyadh six species of rodents were collected. the trapped rodents in descending order of numbers were rattus rattus rattus, acomys dimidiatus, meriones libycus, r. rattus frugivorus, r. rattus alexandrinus and mus musculus. the ectoparasites were a flea, xenopsyllus sp. on r. rattus frugivorus and a tick, rhipicephalus turanicus on each of a. dimidiatus and r. rattus alexandrinus. they mentioned that rodents’ ectoparasites were low in study area and this is probably due to the severe hot and very dry weather mainly in the summer season (alahmed and al-dawood 2001). after all, the catch rate and infestation rate to different ectoparasite depend on season, size of rodents, host preference, sex of host, host age, location of capture and co-evolution between rodent and ectoparasites. to sum up the results of the current study, the role of commensal rodents as hosts of several ectoparasites, all with medically and veterinary importance is emphasized. in this regard, the infestation of both r. norvegicus and r. rattus with xenopsylla is highlighted. additionally, the infestation of the lone hamster, which was an accidentally trapped pet, with a relatively high burden of flea is an indication of the importance of such rodents in transmission of arthropod borne disease in the area, especially to children who are in close contact with pet animals. the results of present study will provide information to the authorities for prevention and control of rodent borne disease in the region. acknowledgments the authors would like to appreciate very much for kind collaboration of health staff in bandar abbas and also other colleagues who had contribution especially m aboulhasanni, z talai, m jedari, n hosseini vasokalani for their kind help. this research was financially supported by tehran university of medical sciences. the authors declare that they have no conflicts of interest. references alahmed am, al-dawood as (2001) rodents and their ectoparasites in wadi hanifah, riyadh city, saudi arabia. j egy soc parasitol. 31(3): 737–43. bell jc, plamer sr, payne jm (1988) the zoonosis: infection transmitted from animal to man. edward arnold press, london uk. durden la, page bf (2008) ectoparasites of commensal rodents in sulawesi utara, indonesia, with notes on species of medical importance. med vet entomol. 5(1): 1–7. el kady ga, el shazly am, mikhail mw, bahgat im (2007) ectoparasites of commensal rodents in talkha center, dakahlia governorate, egypt. j egypt soc parasitol. 37(3): 825–33. el deeb hi, zidan zh, el hawashy n, mourad aa (1999) rodent ectoparasites relationships at new reclaimed areas of egypt. annals of agr sci. 44(2): 799–810. fox i, garcía-moll i (1961) rat ectoparasite surveys in relation to murine typhus iranian j arthropod-borne dis, (2009), 3(2): 44-49 eb kia et al: ectoparasites of rodents ….. 49 fever in puerto rico. am j trop med hyg. 10(4): 566-573. hanafi-bojd aa, shahi m, baghaii m, shayeghi m, razmand n, pakari a (2007) a study on rodent ectoparasites in bandar abbas: the main economic southern seaport of iran. iranian j envir hlth sci engin. 4(3): 173–176. hartelt k, oehme r, frank h, brockmann so, hassler d, kimmig p (2004) pathogens and symbionts in ticks: prevalence of anaplasma phagocytophilum (ehrlichia sp.), wolbachia sp., rickettsia sp.,and babesia sp. in southern germany. international j med microbiol supplement. 293(37): 86–92. inokuma h, parola p, raoult d, brouqui p (2001) molecular survey of ehrlichia infection in ticks from animals in yamaguchi prefecture, japan. vet parasitol. 99(4): 335–339. inokuma h, beppu t, okuda m, shimada y, sakata y (2003) epidemiological survey of anaplasma platys and ehrlichia canis using ticks collected from dogs in japan.vet parasitol. 115(4): 343–348. kia eb, homayouni mm, farahnak a, mohebali m, shojai s (2001) study of endoparasites of rodents and their zoonotic importance in ahvaz, south west iran. iranian j pub health. 30(1-2): 49–52 linardi pm, gomes af, botelho jr (1994) some ectoparasites of commensal rodents from huambo, angola. j med entomol. 31(5): 754–56. motevalli-hagghi f, gholami sh, sharifi m, sedaghat mm, parsi b (2000) study of rodents ectoparasites in sari city and central regions of mazandaran province in 1997-98. iranian j mazandaran university of med sci. 27(10): 7–10. motevalli-hagghi f, gholami sh, sharifi m, mobedi i, i sobhani i, sedaghat mm, najafpour aa (2002) study of rodents ectoparasites in urban areas of mazandaran province in 1997-99. iranian j mazandaran uni med sci. 27(13): 72–77. shayan a, rafinejad j (2006) arthropod parasites of rodents in khorram abbad district, lorestan provincen of iran. iranian j pub health. 35(3): 70–76. strandtmann rw, wharton gw (1958). a manual of mesostigmatid mites parasitic on vertebrates. university of maryland. pp 330. telmadarraiy z, vatandoost h, mohammadi s, akhavan aa, abai mr, rafinejad j , kia eb, faghih naini f, jedari m, aboulhasani m (2007) determination of rodent ectoparasite fauna in sarpolezahab district, kermanshah province, iran, 2004-2005. iranian j arthropodborne dis. 1(1): 58–62. j arthropod-borne dis, june 2018, 12(2): 101–107 a sanei-dehkordi et al.: essential oil … 101 http://jad.tums.ac.ir published online: june 12, 2018 original article essential oil composition and larvicidal evaluation of platycladus orientalis against two mosquito vectors, anopheles stephensi and culex pipiens alireza sanei-dehkordi 1, 2, sahereh gholami 3, mohammad reza abai 3, *mohammad mehdi sedaghat 3 1department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 2infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 15 jan 2017; accepted 11 apr 2018) abstract background: natural plant products as larvicides could be considered as desirable alternatives to synthetic chemical insecticides for vector management. this study was undertaken to assess the mosquito larvicide activity of the essential oil from fresh leaves of platycladus orientalis against two medically important species of mosquito vectors. methods: essential oil was extracted by hydrodistillation and analyzed with gas chromatography and mass spectrometry (gc-ms). fresh leaves of p. orientalis tree (500g) were collected in june 2014 from tehran, iran and was authenticated at the department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. in addition, the larvicidal potential of oil was evaluated against late-3rd or young-4th instar larvae of anopheles stephensi and culex pipiens under laboratory condition. the mortality counts were made after 24h and lc50 and lc90 values were calculated. results: forty-six components in leaves of p. orientalis were identified. the major components were α-pinene (20.17%), 3-carene (14%) and cedrol (9.51%). the lc50 values against an. stephensi and cx. pipiens larvae were 11.67ppm and 18.60ppm after 24h, respectively. conclusion: platycladus orientalis oil could be considered as a natural larvicide for mosquito larval control. keywords: anopheles stephensi, culex pipiens, platycladus orientalis, essential oil, larvicide introduction mosquitoes are the most important group of arthropods with medical importance, which can transmit many pathogens and parasites cause important diseases such as malaria, dengue, yellow fever and filariasis throughout the world, except the antarctic (1). human malaria caused by protozoans (plasmodium spp.) continues to be the most important vectorborne disease. it affects more than 91 tropical countries, placing 3.2 billion people at risk as of 2015. overall, 212 million cases of malaria and 429000 deaths were reported worldwide (2). this disease is transmitted only by the anopheline mosquitoes. there are more than 400 described species of anopheles worldwide, from which about 40 species are important vectors of human malaria (3). in iran, malaria is transmitted by seven vector species, among these species, anopheles stephensi is considered as a primary vector of malaria in the southern parts of iran (4-6). culex species are important vectors of human pathogens worldwide including the etiologic agents of different forms of encephalitis, rift valley fever, and lymphatic filari*corresponding author: dr mohammad mehdi sedaghat, e-mail: sedaghmm@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 101–107 a sanei-dehkordi et al.: essential oil … 102 http://jad.tums.ac.ir published online: june 12, 2018 asis. culex pipiens with broad global distribution is considered as one of the most medically important vectors (1). there are several strategies applied for mosquito control, the usage of synthetic larvicides still remains as an effective method. the indiscriminate use of synthetic insecticides such as organophosphorus compounds have several adverse effects on environment, toxic effects on human beings and non-target organism (specifically aquatic insects) and also may lead to the development of resistance in mosquito populations (7-9). the current trend has highlighted the search for new compounds for mosquito larval control. from this viewpoint, botanical insecticides are promising since they are effective, environmentally friendly, degradability, non-toxic effects on non-target organisms and often inexpensive. aromatic plants and their essential oils have been suggested as natural insecticides for pest control because they have few harmful effects on ecosystem structure and function (10). the insecticidal properties of plant volatile oils and extracts from a wide variety of plants have been assessed against the larvae of different species of mosquito (11-14). platycladus orientalis (linnaeus) franco [synonym: thuja orientalis lineous, p. stricta spach, biota orientalis locally named as sarv-e khomrei or noosh, is a monoecious and evergreen tree belonging to the labiatae family which grows wild in korea, japan, china, and iran. this plant is cultivated as a common ornamental plant in different parts of iran and other countries (15). platycladus orientalis is used in iranian traditional medicine as an astringent, stomach tonic, diuretic, tonic and antipyretic effect (16). the essential oil of p. orientalis has been evaluated for antimicrobial activity (17), antifungal activity (18, 19), cytotoxicologic activity (20), molluscicidal activity (21) and insecticide activity (22, 23). the main purpose of this study was to analyze the essential oils by gc-ms in order to identify the constituents and larvicidal activity of the leaf essential oil of p. orientalis. materials and methods plant materials fresh leaves of p. orientalis tree (500g) were collected in june 2014 from tehran, iran (51º 23’e, 35º 42’n, elevation: 1242m). the plant was identified and authenticated at department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. essential oil isolation the fresh leaves of p. orientalis were cut into small pieces and subjected to hydrodistillation using clevenger-type apparatus (model: british pharmacopoeia, manufactured by pyrexfan company, iran and mantle model em manufactured by bibby scientific company, united kingdom). the essential oil was dried over anhydrous sodium sulfate and the purified oil transferred into amber-colored vials at +4 °c until further use. gas chromatography/mass spectrometry the oil analysis was carried out using gcms. the gc apparatus was agilent technology (hp) 6890 system, capillary column of hp-1ms (fused silica) (30m× 0.25mm, film thickness 0.25μm). the oven temperature program was initiated at 40 °c, held for 1 min then raised up to 230 °c, rising to 250 °c at 5 °c/min. the carrier gas used was helium at a flow rate of 1ml/min. the detector and injector temperatures were 260 °c and 230 °c, respectively. gc/ms analysis was conducted on an hp 6890 gc system coupled with a 5973 network mass selective detector with a capillary column the same as above, carrier gas helium with flow rate 1 ml/min. a sample of 1μl was injected in the split mode with split ratio 1:50. for gc–ms detection, an electron ionization system, with ionization energy of 70ev, was used, injechttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 101–107 a sanei-dehkordi et al.: essential oil … 103 http://jad.tums.ac.ir published online: june 12, 2018 tor and oven temperature programmed was identical to gc. the retention indices (ri) were calculated for all volatile constituents using a homologous series of n-alkanes (c8– c24) on the hp-1ms column. quantification was performed using percentage peak area calculations and the identification of individual compartments was done using the wiley 7n.1 gc-ms library, nist (national institute of standards and technology) and those published in the literature (24-26). mosquito rearing the larvae of an. stephensi and cx. pipiens were obtained from the anopheline insectary of department of medical entomology, tehran university medical sciences. the continuous breeding colony of mosquito was maintained at 27 °c with 12:12 l/d photoperiod in 65±5% rh. larvae of an. stephensi were available for the mosquito larvicidal experiments. bioassays and larval mortality bioassay tests were performed according to the standard method recommended by who (27). in brief, the essential oil first dissolved in absolute ethanol. a 400ml glass beaker was used for each experiment or control. the late3rd or young-4th instar larvae of an. stephensi and cx. pipiens were exposed to 5, 10, 20, 40 and 80ppm of essential oil in water, in the control beakers only solvent (absolute ethanol) was dissolved into the water. mortality was counted after 24h. when necessary, mortality was adjusted for control deaths using abbott’s formula (28). the lethal concentrations of 50% and 90% mortality (lc50 and lc90) were calculated using probit analysis (29). results yields and chemical constituents of essential oil the yield of essential oil was 0.5% (w/v) based on fresh weight. the essential oil was yellowish with a distinct sharp odor. forty-six constituents in the essential oil of p. orientalis were identified corresponding to 97.88% of the total oil (table 1). the major components of p. orientalis oil were identified as α-pinene (20.17%), 3-carene (14%) and cedrol (9.51%). other minor constituents were found to be βthujene (7.85%), terpinolene (6.56%) and αterpinyl acetate (4.38%). mosquito larvicidal activity of essential oil the larvicidal activity of leaf oil from p. orientalis against an. stephensi and cx. pipiens under laboratory conditions are shown in table 2. among the five concentrations tested, the dosage of 80ppm could induce more than 90% mortality in both the species. the mortality rates in the control groups were lower than 5% in all concentrations. the lc50 and lc90 values against an. stephensi and cx. pipiens larvae were 11.51, 67.81 ppm and 18.60, 127.24ppm after 24h, respectively. among different concentrations tested, there were no significant differences in larval mortality between the two species (p> 0.05). in regression line a positive correlation was observed between the leaf oil concentrations and mortality rates (fig. 1). table 1. chemical constituents of leaf essential oil from platycladus orientalis constituentsa rib composition% tricyclene 922 0.26 α-pinene 938 20.17 α-fenchene 950 1.71 β-thujene 966 7.85 β-pinene 979 3.88 3-carene 998 14.00 2-carene 1006 0.77 d-sylvestrene 1024 0.50 limonene 1032 2.74 β-ocimene 1038 0.14 α-ocimene 1041 0.08 γ-terpinene 1056 1.73 cis-thujane-4-ol 1062 0.15 terpinolene 1083 6.56 1,3,8-p-menthatriene 1110 0.05 cis-p-menth-2-en-1-ol 1119 0.28 (4e,6z)-allo-ocimene 1130 0.02 borneol 1164 0.06 terpinene-4-ol 1176 2.95 α-terpineol 1191 0.40 cis-piperitol 1199 0.18 fenchyl acetate 1225 0.06 cis-geraniol 1228 0.01 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 101–107 a sanei-dehkordi et al.: essential oil … 104 http://jad.tums.ac.ir published online: june 12, 2018 citral 1270 0.03 l-bornyl acetate 1282 1.89 α-terpinyl acetate 1346 4.38 nerol acetate 1366 0.42 geraniol acetate 1383 0.57 β-elemene 1389 0.82 α-cedrene 1410 1.23 caryophyllene 1424 4.32 gamma-elemene 1433 0.63 α-caryophyllene 1455 3.34 germacrene d 1478 2.47 β-bisabolene 1507 0.11 δ-cadinene 1525 0.38 elemol 1548 0.94 nerolidol 1566 0.05 caryophyllene oxide 1581 0.08 cedrol 1602 9.51 γ-eudesmol 1634 0.75 cedryl acetate 1760 0.42 8,15-pimaradiene 1890 0.02 hexadecanoic acid 1971 0.02 pimara-7,15-dien-3-ol 2250 0.78 totarol 2301 0.20 total 97.88 acompounds listed in order of elution from a hp-1 ms column bretention indices as determined on hp-1ms using the homologous series of n-alkanes (c8–c24) fig. 1. probit regression line of anopheles stephensi and culex pipiens exposed to different interval concentrations of essential oil from fresh leaves of platycladus orientalis table 2. parameters of probit regression lines of platycladus orientalis oil against the larvae of anopheles stephensi and culex pipiens species a b±se lc50, 95% c.i. lc90, 95% c.i. χ 2 (df) p-value an. stephensi -1.77 1.66±0.159 11.51 67.81 5.9 (3) 0.05 cx. pipiens -1.95 1.53±0.397 18.60 127.24 20.9 (3) <0.05 a: y-intercept, b: the slope of the line, se: standard error, lc50, 95% ci: lethal concentration causing 50% mortality and its 95% confidence interval, lc90, 95% ci: lethal concentration causing 90% mortality and its 95% confidence interval χ 2= heterogeneity about the regression line, df: degree of freedom p= represent heterogeneity in the population of tested discussion the yield of oil obtained of p. orientalis leaves was 0.5% (w/v). this amount is similar to recent study (17) but relatively higher than other studies (22, 25,30, 31). according to our results, α-pinene (20.17%), 3-carene (14%) and cedrol (9.51%) are known as the main compounds of the oil. similar to those previously reported (17, 26, 30, 31), the major constituents were α-pinene, 3-carene and cedrol. however, there were differences in the amount of the main components. the differences in the number of compositions in the above-mentioned studies may be due to the collection time and geographic factors (26). essential oils obtained from plants have been studied as a natural compound potentially used as an alternative to common synthetic insecticides. earlier authors reported table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 101–107 a sanei-dehkordi et al.: essential oil … 105 http://jad.tums.ac.ir published online: june 12, 2018 that the larvicidal activity of essential oils of various aromatic plants with lc50 values ranging from 24.27 to 105.4ppm and 20.61 to 311.2ppm against the larvae of an. stephensi and cx. pipiens, respectively (12, 3137). the leaf oil of p. orientalis was very effective against an. stephensi and cx. pipiens with lc50 values of 11.51 and 18.60ppm after 24h, respectively, which were much lower than those of the plants studied earlier. in the present investigation, the dosage of 80ppm was sufficient to cause 100% larval mortality against the larvae of both species after 24h. similarly, the toxicity of t. orientalis and chamaecyparis obtusa oils were tested against 4th-instar larvae of aedes aegypti and cx. pipiens after 24h and they reported 100% larval mortality when treated with 400ppm of both oils (23). according to proposed categories of larvicidal activity of plant essential oils against mosquito larvae, essential oil of p. orientalis can be considered as active (38) to very active (39) plant. conclusion essential oils of p. orientalis are promising in mosquito control. these findings could be useful in search for newer, safer, and more effective natural larvicidal compounds against disease-vector mosquitoes. further studies must be conducted to describe the mode of action of each constituent separately also its effects on non-target organisms. acknowledgments this work was supported by a grant from the center for environmental health research, tehran university of medical sciences, tehran, iran, project no. 88-01-468661. the authors declare that there is no conflict of interest. references 1. service m (2012) medical entomology for students. cambridge university press, cambridge. 2. who (2016) world malaria report 2016. who press, geneva, switzerland. 3. becker n, petrić d, boase c, lane j, zgomba m, dahl c, kaiser a (2010) mosquitoes and their control. springer, berlin. 4. sedaghat mm, linton ym, nicolescu g, smith l, koliopoulos g, zounos ak, oshaghi ma, vatandoost h, harbach re (2003) morphological and molecular characterization of anopheles (anopheles) sacharovi favre, a primary vector of malaria in the middle east. syst entomol. 28: 241–256. 5. sedaghat mm, linton ym, oshaghi ma, vatandoost h, harbach re (2003) the anopheles maculipennis complex (diptera: culicidae) in iran: molecular characterization and recognition of a new species. bull entomol res. 93: 527–535. 6. sedaghat mm, harbach 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arthropod-borne dis, march 2017, 11(1): 132–138 n ghobakhloo et al.: evaluation of correlation … 132 http://jad.tums.ac.ir published online: march 14, 2017 original article evaluation of correlation between the in vitro susceptibility of field isolates of leishmania major and clinical outcomes of meglumine antimoniate therapy in fars province, iran nafiseh ghobakhloo 1, 2, *mohammad hossein motazedian 1, 3, behrad pourmohammadi 4, zahra yousefi 1 1department of parasitology and mycology, school of medicine, shiraz university of medical sciences, shiraz, iran 2student research committee, shiraz university of medical sciences, shiraz, iran 4department of public health, school of health, semnan university of medical sciences, semnan, iran (received 1 mar 2015; accepted 5 apr 2016) abstract background: this study was designed to detect whether there is a correlation between in vitro susceptibility of field isolates of leishmania major and the clinical outcomes of meglumine antimoniate (glucantime®) therapy, the mainstay of cutaneous leishmaniasis treatment in iran. methods: forty-three patients infected with l. major were enrolled in this study from october 2009 to march 2010 and categorized as responsive or unresponsive to glucantime® treatment after receiving the appropriate therapy. then, intracellular amastigote approach was conducted on these field strains to investigate in vitro drug susceptibility as well. results: at clinical level, out of 43 patients, 15 were clinically non-responsive and 28 were responsive to antimony therapy. all those 28 clinically sensitive strains were susceptible to antimony in the in vitro assay, whereas merely 11 isolates from 15 non-healing isolates were resistant in vitro. finally, a good correlation (78.9%) with high sensitivity, specificity (100/73) between clinical outcomes and the in vitro susceptibility test was achieved. conclusion: the intracellular amastigote model could be an appropriate assay for evaluation of the in vivo drug sensitivity of field isolates. however, more comprehensive studies with larger sets of isolates are needed to confirm these preliminary data. keywords: in vitro susceptibility, antimonial resistance, leishmania major, iran introduction the most common form of leishmaniasis, zoonotic cutaneous leishmaniasis (zcl), caused by l. major, is found in many regions of the middle east including iran (razmjou et al. 2009, jacobson 2011, alvar et al. 2012, akhoundi et al. 2013). since their discovery over 6 decades ago, the antimonial compounds have played vital roles in treatment of all forms of leishmaniasis (croft et al. 2006). the emergence of antimony resistance as the mainstay of treatment, however, has represented critical health problems in most endemic areas including iran (hadighi et al. 2006, sundar and goyal 2007, pourmohammadi et al. 2011, mohammadzadeh et al. 2013). since there is no effective vaccine for prevention, disease control is essentially based on chemotherapy. as resistance phenomenon can cause serious effects on disease treatment and control, this issue is an urgent concern (sundar et al. 2014). hence, estimating the efficacy of standard drugs and determining the prevalence of *corresponding author: dr mohammad hossein motazedian, e-mail: motazedm@sums.ac.ir, motaze -dian33@yahoo.com 3basic sciences in infectious diseases, research center, shiraz university of medical sciences, shiraz, iran j arthropod-borne dis, march 2017, 11(1): 132–138 n ghobakhloo et al.: evaluation of correlation … 133 http://jad.tums.ac.ir published online: march 14, 2017 resistance in endemic regions seem to be extremely essential (croft 2001). unfortunately, there is no applicable in vitro test for evaluating the clinical resistance. in recent years, some promastigote and intracellular amastigote assays have been established to investigate the effectiveness of anti-leishmania drugs. fumarola et al. (2004) for screening new compounds against l. infantum investigated intracellular amastigotes than promastigotes. sereno et al. (2007) used green fluorescent protein or luciferase for screening the drug resistance. an in vitro intracellular model for screening the leishmania field isolates were applied by da luz et al. (2009) and they mentioned that this model needed to be evaluated and standardized for the field isolates. an intracellular model for drug screening also was recommended by vermeersch et al. (2009) using reference strain of l. donovani in different in vitro models. regardless of the advantages and disadvantages mentioned for these in vitro methods, the intracellular amastigote model is a more reliable in vitro assay for measuring treatment failure in the field isolates (maia et al. 2013). in this context, there are some previous reports on clinical evaluation of drug sensitivity, but these studies did not use the standard drug dosage as recommended by who and a few studies have been conducted to evaluate the in vitro susceptibility of leishmania spp. to antimony (hadighi et al. 2006, pourmohammadi et al. 2011). we aimed in this study, to establish a feasible and suitable approach for detecting resistant strains at the clinical level for implementation of a rational therapy and mapping the prevalence of resistance in fars province, south part of iran, evaluating these resistance isolates in the in vitro assay, and determining if this test could be extendable to the in vivo assay, which has not been applied to l. major yet. materials and methods clinical isolates forty-three field strains enrolled in this study were collected previously from patients infected with l. major in fars province between 2009 and 2010. the study was conducted under full respect to ethics statements and after appropriate informed consent from the patients (pourmohammadi et al. 2011). briefly, individuals suspected of zcl were referred to valfajr health center in shiraz and infection with l. major was confirmed by pcr. then, patients were treated with intramuscular glucantime® at 20mg/kg/ day dose for 20 days as a standard protocol of who. after finishing the treatment course, they were followed up for six weeks and three month. patients were considered unresponsive to treatment if amastigotes were found on light microscope examination of geimsa stained slides prepared from the lesion edge. reference sensitive strain the reference strain (mrho/ir/75/er), sensitive to glucantime® treatment, used in this experiment was originally bought from the pasture institute of iran was. in addition, a clinical isolate, which showed high rate of resistance to glucantime® in both clinical level and in vitro assay, was used as a reference resistant strain in this work. anti-leishmania drug meglumine antimonite (glucantime®) was prepared from sigma-aldrich chemical company (011m0125v). the stock solution was produced by dissolving of drug powder in dmem medium, to reach four different drug dilutions l0. with respect to finding a more suitable drug dose for establishing in vitro assay, which could be more extendable to clinical consequences, we used four different concentrations of glucantime® obtained j arthropod-borne dis, march 2017, 11(1): 132–138 n ghobakhloo et al.: evaluation of correlation … 134 http://jad.tums.ac.ir published online: march 14, 2017 through further dilution in complete medium (15 μg/ml, 30 μg/ml, 45 μg/ml and 60 μg/m). the mouse macrophage cell line (j774) this cell line was bought from national cell bank of pasture institute of iran and grew in dmem medium supplemented with 12% fbs, 100iu/ml of penicillin, and 100 g/ ml of streptomycin and incubated at 37 °c under 5% co2 atmosphere until it reached to logarithmic phase of growth. the in vitro intracellular susceptibility assay the efficacy of meglumine antimonite of filed isolates was conducted on the mouse macrophage cell line (j774) as previously described (faraut-gambarelli et al. 1997). briefly, the numbers of 5× 10 5 of j774 cells in log phase of growth were seeded in each 8well chamber slides with cover slips (nunc, 177445). then they were left for cells adhesion for 1 h at 37 °c and 5% co2. adherent macrophages were infected with late stationary phase of promastigotes at a ratio of 10:1 (promastigote/cell) for 4 h. after that, the excess promastigotes, which were not able to infect macrophages, were removed and each well was replaced with 400-μl medium containing glucantime®. meanwhile, all experiments were done in triplicate for each drug concentration. there was a control well against three treated wells contained only infected macrophage and 400μl of complete medium without any drug. lastly, after 5 days incubation, all wells were fixed with absolute methanol and stained with 10% geimsa for microscopic examination. finally, parasite burdens as the percentage of infected macrophages× (mean number of amastigotes/ macrophage) were calculated and compared to the burdens for the untreated infected control wells. statistical analysis based on the reduction of total parasite burdens, 50% inhibitory concentrations (ic50) were calculated for each well. at least, regarding to categorizing strains to resistant and sensitive isolates, ic50 of each isolate was compared with ic50 of reference (sensitive and resistant) isolates by the chi-square test and mcnemar. if there was a significant difference between ic50 of reference strains and each field isolate, this clinical strain was characterized as resistant to drug in vitro assay and vice versa. for the determining of correlation between clinical outcomes and in vitro sensitivity test, we used kappa coefficient that a p< 0.05 was considered statistically significant. statistical analysis was performed by the use of spss version 16.0 (chicago, il, usa). results out of 43 zcl patients, 15 cases (34.9%) were non-responsive (non-healing) and 28 (65.1%) were responsive (healing) after complete treatment with glucantime® at clinical level. in this experiment, ic50 values of field isolates represented ranging from (15 μg/ml to 60 μg/ml), while the ic50 was (˂15 μg/ ml) for reference sensitive strain and was (˃60 μg/ml) for reference resistant strain. the comparative results between clinical outcomes and in vitro sensitivity test are summarized in table 1. in the highest drug dose (60 μg/ml), all of responsive field strains represented sensitivity profiles in vitro as well, although four of fifteen strains which were determined as resistant at clinical level were sensitive in the in vitro assay at this drug dose. furthermore, the result of mcnemar and kappa coefficient test (p< 0.05) conducted for finding the sensitivity and specificity and agreement between clinical outcomes and in vitro assay is shown in table 1. overall, a best sensitivity, specificity (100/73) and strong correlation (78.9%) was observed especially in terms of using 60μg/ml of glucantime® between the results of clinical sequence and the in vitro test. j arthropod-borne dis, march 2017, 11(1): 132–138 n ghobakhloo et al.: evaluation of correlation … 135 http://jad.tums.ac.ir published online: march 14, 2017 table 1. the comparison between the susceptibility of in vitro and clinical outcomes of leishmania major field isolates to glucantime® by mcnemar and kappa discussion regarding the increasing rate of antimony failures in zcl patients in iran, this study was done to set out a reliable in vitro method to determine the antimonial susceptibility of clinical isolates of l. major in fars province, as there was no report of a comprehensive work on this issue in iran. in fact, for the first time we aimed to assess this test on l. major clinical strains. since the intracellular amastigote, method had the best agreement with clinical responses rather than using promastigote form, we decided to evaluate the susceptibility of antimony in this stage of parasite as well (maia et al. 2013, coelho et al. 2014). in fact, the current results revealed a strong correlation (coefficient: r= 0.7860) and the best sensitivity, specificity between the clinical outcomes of antimony therapy and the in vitro antimonial susceptibility test. indeed, this study was conducted on l. major clinical strains and has great corroboration with previous researches on various species of leishmania around the world (fumarola et al. 2004, vermeersch et al. 2009, aït-oudhia et al. 2012, maia et al. 2013, coelho et al. 2014). like our findings, in another research conducted on 26 clinical isolates of l. tropica in iran by hadighi et al. there was an excellent correlation between clinical outcomes and the in vitro susceptibility test in amastigote-marophage model (hadighi et al. 2006). based on these findings, this in vitro assay can be considered as a valid method for evaluation of drug sensitivity in both species of lesihmania causing cutaneous leishmaniasis iran. despite these supportive investigations, however, no potential agreement was noticed between the clinical response of l. braziliensis, and l. donovani isolates, and the intracellular amasigote in vitro system in studies performed in various endemic regions around the globe (rojas et al. 2006, yardley et al. 2006, rijal et al. 2007). overall, these agreements and contradictions between different reports can be explained by some factors that may affect the results of the in vitro assays. firstly, partially distinctive genetic make-ups of leishmania species would be a reasonable proof to clarify these disagreements among several surveys carried out on different species of leishmania (croft et al. 2006). however, our findings obtained from field strains of l.major were relatively in agreement with the results from other species of leishmania (l. tropica) in iran (hadighi et al. 2006). moreover, clinical definition of resistance seems to be an important clue for justifying drug dose in vitro assay clinical outcomes sensitive n (%) clinical outcomes resistant (%) p-value for mcnemar sensitivity/ specificity kappa total 15 μg/ml sensitive 20 (71.4) 6 (40) 0.79 71/60 0.35 (.04) 26(60.5) resistant 8 (28.6) 9 (60) 17(39.5) 30 μg/ml sensitive 26 (92.9) 4 (26.7) 0.69 93/73 0.68 (<0.001) 30(69.8) resistant 2 (7.1) 11 (73.3) 13(30.2) 45 μg/ml sensitive 28 (100) 6 (40) .03 100/60 .66 (< 0.001) 34(79.1) resistant 0 (0) 9 (60) 9(29.9) 60 μg/ml sensitive 28 (100) 4 (26.7) .125 100/73 .78 (< 0.001) 32(74.4) resistant 0 (0) 11 (73.3) 11(25.6) j arthropod-borne dis, march 2017, 11(1): 132–138 n ghobakhloo et al.: evaluation of correlation … 136 http://jad.tums.ac.ir published online: march 14, 2017 these discrepancies among different studies (lira et al. 1999, croft 2001, rijal et al. 2007). in the present work, the patients were categorized as responsive and unresponsive after six weeks and following three months of a standard therapy protocol; while, in another study by luz et al. (2009) this period was extended to 6 and 12 months following the therapy. there were also some differences among various studies in the operational protocols such as time of drugging, the promastigote-macrophage ratio, and timing of the cessation of the experiment (da luz et al. 2009). obviously, if we applied another approach, perhaps the correlation value would have been changed. therefore, in this context, we made a big effort to standardize the in vitro procedures through investigating various factors, which could consequently make the results extendable to clinical outcomes. in the present experiment, out of 15 unresponsive clinical isolates to glucantime®, 11 cases were also resistant in the in vitro assay. this evidence can be associated clearly with the intrinsic natures of various leishmania strains, which make them show original sensitivity or resistance to antimonial components (polonio and efferth 2008, jeddi et al. 2011). since l. major causes the zcl disease, it is assumed that these parasites have never been exposed to drugs, and so these 11 clinical isolates which are resistant at both clinical and in vitro levels, are inherently resistance to antimony. beyond the genetic factors, some researches have focused on revealing the antimony resistant aspects and have demonstrated that host factors such as genetics and particularly the immune status may have crucial roles on the efficacy of antimony on leishmania parasites in vivo (murray and delph-etienne 2000, campino et al. 2006, sundar and goyal 2007). according to this fact, we observed some clinical strains, cured after complete therapy with glucantime®, while they showed a resistant phenotype in the in vitro susceptibility test. similar finding was reported by vanaerschot et al. (2013) on l. donovani, and they infered that perhaps the immune components will act in a great synergy with the drug in order to defend more strongly against leishmania cells. therefore, recognition of some isolates as responsive to therapy at clinical level categorized as resistant in the in vitro assay is more likely due to lack of immune system effects in the in vitro tests. furthermore, even different strains of one species of leishmania may use multiple resistant mechanisms against antimony components in diverse circumstances, in vitro and in vivo (ouellette et al. 2004, chakravarty and sundar 2010). therefore, it seems to be a good explanation to describe why some of our isolates showed reversed phenotypes in vitro and at clinical levels. for example, there were some isolates, which were not responsive to therapy at clinical level, while these were responsive in vitro and vice versa. perhaps they behaved differently and used various anti-drug mechanisms in different conditions. conclusion in the absence of a gold standard method for monitoring drug sensitivity, based on findings of this paper, it is highly recommended that this intracellular-amastigote in vitro assay can be utilized as a reliable method for both monitoring the antimony efficacy of field strains of l. major and epidemiological studies in different endemic areas of iran. however, with respect to multi factorial phenomenon of antimony resistance in different field strains, further researches should be undertaken with more samples and in other endemic areas of iran to confirm our preliminary data. acknowledgements this investigation received financial support j arthropod-borne dis, march 2017, 11(1): 132–138 n ghobakhloo et al.: evaluation of correlation … 137 http://jad.tums.ac.ir published online: march 14, 2017 from the vice chancellery of research affairs of shiraz university of medical sciences for financial support (grant no. 5804). the authors wish to express their gratitude to s habibi physician and d pour kamal staff of valfajr health center for their useful cooperation in this study. we would also like to thank motahareh motazedian for editing the english manuscript. the authors declare that there is no conflict of interest related to this study. references aït-oudhia k, gazanion e, sereno d, oury b, dedet j, pratlong f, lachaud l (2012) in vitro susceptibility to antimonials and amphotericin b of leishmania infantum strains isolated from dogs in a region lacking drug selection pressure. vet parasitol. 187(3): 386–393. akhoundi m, hajjaran h, baghaei a, mohebali m (2013) geographical distribution of leishmania species of human cutaneous leishmaniasis in fars province, southern iran. iran j parasitol. 8 (1): 85–91. alvar j, velez id, bern c, herrero m, desjeux p, cano j, jannin j, den boer m, team wlc (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7(5): e35671. campino s, kwiatkowski d, dessein a (2006) mendelian and complex genetics 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agents ch. 53(9): 3855–3859. yardley v, ortuño n, llanos-cuentas a, chappuis f, de doncker s, ramirez l, croft s, arevalo j, adui v, bermudez h (2006) american tegumentary leishmaniasis: is antimonial treatment outcome related to parasite drug susceptibility?. j ifect dis. 194(8): 1168– 1175. j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 104 http://jad.tums.ac.ir published online: april 27, 2019 original article a comparative pathomorphological findings between leiurus abdullahbayrami and androctonus crassicauda (scorpion: buthidae) envenomation in rabbit animal model *ozcan ozkan1; mehmet eray alcigir2 1çankırı karatekin university, çankırı, turkey 2department of pathology, kirikkale university, faculty of veterinary medicine, kirikkale, turkey (received 15 may 2017; accepted 23 dec 2018) abstract background: the aim of study was to compare macroscopical and histopathological findings between venoms belonging to two scorpion species, androdoctonus crassicauda, and the newly discovered leirus abdullahbayrami. methods: the animals used in this experimental study were fifteen new zealand bred rabbits. three groups were constituted as group i (l. abdullahbayrami group, n= 6), group ii (a. crassicauda group, n= 6) and group iii (control group, n= 3). the animals in the l. abdullahbayrami group and the a. crassicauda group were envenomed through an intravenous route. the rabbits were monitored for the first 24h following the envenomation. the animals dead within that time period were examined and all animals were sacrificed and standard necropsy process was performed at 24h. results: the pathomorphological findings from group i were found to be more severe than those observed in group ii. the venom from the newly identified l. abdullahbayrami has a greater effect than the venom from the a. crassicauda. moreover, as this was a rabbit modeling study, the l. abdullahbayrami might pose the most serious health threat to infants in particular due to their smaller body weight. conclusion: these findings will provide a better understanding of envenomation of human beings in terms of the possible consequences of scorpion toxication on the organs. keywords: leirus abdullahbayrami; androctonus crassicauda; venom; pathomorphology; rabbit introduction in many parts of the world, millions of people are stung by various scorpion species yearly. these stings can result in death, particularly in children (1, 2). this is because of the serious health problems that result, such as cardiovascular, respiratory, and/or neurologic complications. nowadays, despite advances in medical science, scorpion envenomation cases still continue to be a current public health problem all over the world, including in turkey. scorpions are considered to be life-threatening venomous animals. arguably, the medical knowledge of the scorpion's species is most critical for the scorpion species that are typically found in mexico, the middle and northern regions of south america, north and south africa, india, and the middle eastern countries because those areas have scorpion species with the most potent venoms as they are classified as neurotoxic, hemotoxic, cardiotoxic, nephrotoxic, and myotoxic (3-5). in turkey, the most hazardous scorpions are the yellow scorpion, leiurus abdullahbayrami, which is endemic in southeastern anatolia, and a black scorpion, androctonus crassicauda, commonly found in southeastern anatolia and in a part of eastern anatolia (fig. 1) and neighbouring countries iran, iraq and syria (2, 5-7). furthermore, in these regions, the species are responsible for the most deadly cases, particularly those involving children. according*corresponding author: dr ozcan ozkan, e-mail: ozcanozkan@karatekin.edu.tr j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 105 http://jad.tums.ac.ir published online: april 27, 2019 ly, these scorpion species are described as medically significant to both the world and to turkey (1, 3, 5, 7). in the toxicokinetic studies, radiolabeled venom from the l. quinquestriatus and venom from the a. crassicauda scorpion have reached the maximum value of total plasma radioactivity in less than two minutes after a subcutaneous injection and begin distribution within four to seven minutes after the injection with a half-life of between 4–21h. at that time, the degree of the clinical symptoms depends on the amount of venom circulating in the body (7-9). a low venom dose triggers an adrenergic effect while high scorpion venom concentrations result in venom cholinergic symptoms. the venom results in an excessive acetylcholine (ach) release or a decrease in the destruction of ach, which is acting on the postganglionic nerve endings. thus, cholinergic activity begins. on the other side, catecholamine is released into the peripheral sympathetic nerve endings from the adrenal medulla in response to the venom. then, an autonomic storm begins because of the renin secretion by the alpha adrenergic receptor. the receptor stimulation plays a significant role because it increases blood pressure and ultimately results in the pathogenesis of pulmonary edema. additionally, it causes an inflammatory reaction in the vital organs (8, 10-13). scorpion envenomation has been reported to mainly culminate in a syndrome of fuel-energy deficiency. this situation develops as a deficiency during the usage of existing metabolic substrates. as an aftermath, a failure occurs within vital organs including a multi-organ system deficiency that can lead to death (14, 15). the main reason for this failure is the triggering of an inflammatory response cascade that results in a release of several mediators, such as prostaglandins, cytokines, and nitric oxide, and an infiltration of inflammatory cells (16-19). the highest venom concentrations can be found in the kidneys, liver, heart, and lungs following a venom injection (20-22). in envenomations from different scorpion species, the lungs, heart, liver, intestines, and pancreas were documented as the most affected organs in addition to the recently described effects on the brain following a leiurus envenomation (2327). as a result of the effect of scorpion venom, different clinical tables have reported varying effects from localized signs to more serious autonomic and neurologic findings as well as fatal cases due to multisystem organ failure. according to medical records from turkey, most deaths were caused by the results of cardiopulmonary complications, such as myocarditis and acute pulmonary edema following a scorpion’s sting (1, 2, 7). in animal models, scorpion envenomation symptoms were similar to those described by the clinical findings in humans. therefore, to mimic human victims, especially children, rabbits were chosen as the animal model for experimental scorpion envenomation to mimic a natural sting accident. thus, in this study, different pathomorphological findings developed by two scorpion venoms were revealed out l. abdullahbayrami and a. crassicauda, which species were most encountered in southeast region of turkey and also were showed l. abdullahbayrami having unusual harmful effects onto several organs during long term. materials and methods scorpions origin scorpions were collected using an ultraviolet lamp at night in the sanliurfa and gaziantep provinces, southeastern part of turkey. the animals were kept in plastic boxes at the department of entomology, faculty of veterinary medicine, ankara. the scorpions were received fresh water daily and fed crickets or cockroaches weekly. j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 106 http://jad.tums.ac.ir published online: april 27, 2019 scorpion venom and the median lethal dose (ld50) the venom of scorpions was milked by electrical stimulation of 24 volt. the venoms were added sterile double-distilled water, dissolved and centrifuged at 14000rpm for 15 min at 4 °c. supernatant was dissolved in pss. the median lethal doses (ld50) of the scorpions were determined as in previous studies (28, 29). animals this study was conducted on 15 of healthy new zealand rabbits 12 months old of both sexes and between 2.4±0.1kg body weight for this experiment. the rabbits were fed with a special rabbit pellet diet ad libitum until envenomation. the animals were kept in room temperature set to 19±1 °c and with 12h light/ 12h dark schedule. the experimental protocol was approved by the local laboratory animal ethics committee. after 24h all animals were subjected to euthanasia for macroscopical and histopathological findings. experimental envenomation of the animals the animals were envenomed by the venom of l. abdullahbayrami in group i (gi, n: 6), and the venom of a. crassicauda in group ii (gii, n: 6). finally, three rabbits were selected as a control group and will be referred to as giii. the venoms (2xld50 for both) were injected into the marginal ear vein by intravenous (iv) route after dissolving the venoms within 0.5ml pss. for control group, any envenomation was not performed. 0.9% physiological saline solution only was administrated to rabbits of this group. the animals were monitored and kept in individual cages up to 24h following the injections because the deaths were not shown during first 24h of critical period. histopathological examination after providing sedation by xylazine (10 mg/kg intramuscular route) and ketamine hydrochloride (5mg/kg intramuscular route), sodium pentobarbital injection (100mg/kg intravenous route) were done and then cervical dislocation were performed for each one. necropsy process was done systematically and all organs and tissues were examined routinely. lesions were photographed. for histopathological examination, tissue samples were taken systematically from each organ in 10% buffered formalin. after fixation for 48h, tissue samples were processed routinely through alcohol and xylol series and mounted in paraffin. processed tissues were embedded in paraffin wax. fivemicron thickness-sections were cut from paraffin blocks and haematoxylin-eosin (h and e) staining method. results macroscopical findings in all groups, cadavers were well-fed, and rigor mortis did not happen. however, the blood in group i was lately clotted in contrast to the other groups. conjunctiva was hyperemic in two of the animals from group i. the gut walls were thickened and filled with a yellowish content in all of the members of group i and ii. the liver, kidneys, and lungs were congested in all members of both groups i and ii. however, there was haemorrhagia that happened in the kidneys in the members of group ii, and also atrophia and capsular contraction in the spleen was noticed in all animals within group ii. in group i, a meningeal vessel was congested in one animal. in contrast, none of these macroscopical findings were evident in the animals of group iii. histopathological findings heart myocardiocytes in some areas were degenerated or partly necrotic in the members of group i. there was a hyalinosis in appearance in the degenerated myocardiocytes in the group i. haemorrhagia between cardiomyocytes was formed in two animals from group i and four j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 107 http://jad.tums.ac.ir published online: april 27, 2019 animals from group ii. however, there was focal mononuclear cell infiltration in one animal from group ii. there were no changes in control group. lungs all veins and vessels were hyperemic in members from group i. furthermore, there was edema in all of the animals in group i. microhaemorrhagies were noticed in three of the members of group i and all of the members of group ii. balt hyperplasia was noted in one member of group i and all of the members of group ii. it was not encountered with any findings in control group. kidneys in the kidneys, there was hyperemia in all vessels and glomerules in all animals from group i, there were no such findings in the group ii animals. on the other hand, vacuolar and hydropic degeneration in tubular epithelium were common findings for members of the first group except for one of group i animals. there is no any changes in control group. liver hepatocytes contained vacuoles in different sizes in their cytoplasm and degenerated nuclei in especially the periphery of lobules for all animals from the first two groups. in some animals of group ii, reticular degeneration was also noted in addition to those findings. additionally, mononuclear cell infiltrations in the portal field were evident in the group i findings and in all animals of group ii. finding in control group in terms of this organs were unremarkable. spleen although no prominent findings were observed in animals of group ii, focal follicular necrosis and haemorrhagia were noted in each animal of group i. finding in control group in terms of this organ were unremarkable. central nervous system lesions on the central nervous system were predominantly seen in group i. meningeal and parenchymal vessels were severely hyperemic in one animal of group i. stratum pyramidal cells of the brain often seemed to have lost their nuclear chromatin in almost all foci. perivascular mononuclear cell infiltration was observed in substantia alba of the cerebrum within one of the animals in group i. in the cerebellum, cytoplasms and nuclei of purkinje cells generally degenerated in both groups of animals. moreover, some of the cells were necrotic in group i animals, seen in fig. 2 and fig. 3a–f. however, there were no conspicuous findings in the control group in terms of any of these organ. gastrointestinal system regarding the intestinal lesions, in group i, lymphocyte-plasma cell infiltration was noted in three animals, and neutrophil leucocyte infiltration was evident in one animal in the duodenum. in the group ii, lymphocyte and macrophage infiltrations were seen in the jejunum as well as the duodenum in two of the animals. in addition, aggregate lymphoid follicles were hyperplasic in three animals from group i. there were no findings in the stomach of any of the members of group i in contrast to the focal periarteriolar haemorrhagia noted in one animal from group ii. in control group, there were no any pathologies. j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 108 http://jad.tums.ac.ir published online: april 27, 2019 fig. 1. the distribution of the black colored scorpion is the androdoctonus crassicauda (black dots), and a yellow colored scorpion the leiurus abdullahbayrami species (red dots) are found in hatay, kilis, sanliurfa, mardin, gaziantep provinces of the southeastern anatolia as shown map of turkey. the scorpions were captured in gaziantep and sanliurfa fig. 2. a. vacuolar degeneration (arrows), periphery of lobules, liver, x100, h and e. b. hyperemia (asterisks), glomerules and capillary vessels, kidney, x100, h and e. c. vacuolar degeneration (arrows), kidney, x400, h and e. d. haemorrhagia (arrows), lung, x100, h and e. e. necrosis in myocardiocytes (arrows), x100, h and e. f. degeneration (arrows) and necrosis (arrowhead) in purkinje cells x100, h and e j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 109 http://jad.tums.ac.ir published online: april 27, 2019 fig. 3. a. focal lymphocytes in propria mucosa (arrows), duodenum, x100, h and e. b. focal mononüklear cell infiltration (asteriks) in portal region and vacuolar degeneration in hepatocytes (oklar), liver, x100, h and e. c. hydropic degeneration in tubul epitheliums (arrows), kidney, x100, h and e. d. haemorrhagia (arrows), lung, x100, h and e. e. focal lymphocyte infiltration (arrow), heart, x100, h and e. f. degenerative and necrotic changes (arrows), cerebellum, x100, h and e discussion scorpion envenomation continues to be a global problem despite some national measures taken by authorities in various countries. regarding the the clinical symptoms, the degree of the envenomation depends on some factors related to the scorpion such as the species and the size of the scorpion as well as the number of stings, the content of the venom, and the amount of venom injected. another factor associated with a patient’s prognosis include the patient’s health status, the part of the body that has been stung, the patient’s body mass, and the patient’s age. after the sting, the symptoms of envenomation can be evident within a few minutes, and maximum severity is usually achieved within five hours. consequently, the clinical table has shown localized symptoms at the sting site up to a significantly severe generalized envenomation (7, 11, 28). in turkey, the scorpion sting is very common in the provinces of southeastern anatolia and eastern anatolia due to social and geographical factors. in these areas, the scorpion sting cases have been reported as coming from both the yellow colored scorpion, which is the l. abdullahbayrami, and a black colored scorpion, which is the a. crassicauda species. in addition, scorpions have been the main animal responsible for medically important envenomation cases (5, 11-13). in the kilis province, southeastern turkey, recently, acute pulmonary edema and cardio j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 110 http://jad.tums.ac.ir published online: april 27, 2019 myopathy were due to the excessive catecholamine release that resulted from a sympathetic overstimulation in an 11-yr-old male child following an l. abdullahbayrami stung (7). another study conducted at the sanliurfa children’s hospital reported on a patient with symptoms that included fever, hypersalivation, mydriasis, tachycardia, lethargic, and respiratory distress during admission time. later, this patient had to be intubated because of pulmonary edema and myocarditis. another girl had neurological signs similar to the first victim as well as anisocoria and seizure. two girls, ages 2-yrold and 4-yr-old, had pulseless ventricular tachycardia and died (13). in the province of hatay in the southeast anatolia region, the patients were categorized according to the color of the scorpion that had stung them as yellow (54.2%) and black (28.7%). two children passed away due to cardiac and respiratory complications resulting from a scorpion sting (30). in this same province, a 4-yr-old boy died because of pulmonary edema (31). furthermore, in adıyaman province, the patients were categorized by the color of the scorpion as well with black consisting of 40% of the cases and yellow resulting in 60% of the cases (12). one of these patients died due to cardiac and respiratory arrest. 50.8% of their scorpion cases were a. crassicauda stings (5). additionally, four of the deaths in children were caused by the a. crassicauda species, and only one was caused by an l. abdullahbayrami (32). especially in children, similar findings have been shown in previous studies concerning these two important scorpion species. these scorpions were responsible for serious medical cases and deaths in turkey as well. therefore, our study aimed to determine and compare the pathological effects of the venom based on an animal model. the l. abdullahbayrami scorpion venominduced potassium and sodium voltage-gated ion channels and then led to excessive catecholamine release due to the over stimulation of the sodium and potassium ion channels. as a result of the catecholamines storm, cardiomyopathy and acute pulmonary edema were caused by severe envenomation, especially among small children or infants because of their smaller body weight (7). namely, following envenomation, an acute failure leads to multiorgan system deficiency among the vital organs, which leads to death (14, 15), especially within the heart and lungs. after acute envenomation, a cardiac dysfunction occurred, and as a result of this cardiac failure, the formation of edema and hemorrhages happened in the lungs (8, 9, 33). thrombosis of cardiac vessels, endothelial cells of those vessels, edema, and necrosis in cardiac muscle cells were all observed during the 24h following envenomation (33). in another acute envenomation study with a. australis hector, ld50 of the venom resulted in myocardial necrosis and degeneration. neutrophil leucocyte and mononuclear cell infiltration in the interstitium of myocardium were noted within the first 24h period in mice as well. suppurative bronchopneumonia, focal haemorrhagia, and fibrin deposits are found in the lungs of the mice. cardiac edema, myocardial haemorrhagia, degeneration and necrosis were also detected with envenomation attributed to centuroides sculpturatus venom (34). in another study that experimented with ld50 doses of mesobuthus eupeus envenomation on rabbits, several important consequences progressed including myocardial necrosis, focal haemorrhage, thrombus formation and inflammation in myocardium and the endocardium surfaces of the heart and lungs (35). in our study, more serious fatal findings, such as hyperemia, edema, and haemorrhage, were evident in the lungs. degeneration and necrosis in the myocardiocytes occurred within the rabbits in group i envenomated with l. abdullahbayrami. however, balt hyperplasia and microhemorrhage were encountered in the lungs and focal mononuclear cell infiltration in heart in the rabbits envenomated with a. crassicauda. leiurus abdullahbayrami triggered a more acute reaction especially in regards to the cardiopulmonary system within the j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 111 http://jad.tums.ac.ir published online: april 27, 2019 first 24h as compared to another type of venoms documented in several previous studies. therefore, the results obtained from these study groups were found to parallel the findings of previous studies which also examined the effects that occur within the first 24h following a scorpion sting. some deaths in humans and animals were related to the effect of the venom on the kidneys. the scorpion toxin caused hyperemia and degenerative-necrotic changes in the tubules and glomeruli. this situation resulted in acute renal failure (2, 33, 34, 36, 37). even an envenomation attributed to a hemiscorpius lepturus scorpion can sometimes result in a uremic syndrome in a child (38). in an acute envenomation from m. eupeus, congestion in the vessels and glomeruli of the envenomated animals has been reported. in our study, group i animals envenomated with l. abdullahbayrami showed similar important damages, including vascular congestion and degenerative changes in tubules, as compared to group ii animals envenomated by the venom of the a. crassicauda. on the other hand, certain studies conducted in association with particularly both acute as well as sub-acute envenomation have proven that venom of the m. eupeus species can promptly effect animals within 30min up to three to five hours later by resulting in congestion within the central and portal vein of the liver and in interstitial vessels (35). in subacute envenomation caused by the c. sculpturatus’s venom, the liver of rats was affected within two hours and up to five days after the envenomation. in the livers of envenomated animals, congestion of central veins, hydropic degeneration, and single cell necrosis of hepatocytes was also observed (34, 38). in our study, the findings were observed in both experimental groups. however, mononuclear cell infiltrations in the portal field were also noted in all of the animals of group ii. the last finding is a different finding as compared to previous reports. apart from the findings within the vital organs, milder findings such as congestion were encountered in both the spleen and the pancreas (36). in our study, no other prominent findings were evident. however, focal follicular necrosis and haemorrhagia were both noted in all of the animals in group i. the venom of the l. abdullahbayrami might be more effective since it triggered an inflammatory reaction. in relation to the gastrointestinal system, edema and mildly necrosis have occurred in the guts of these envenomed animals. on the other hand, no inflammatory changes were evident even though there were severe microscopical changes noted, such as gastrointestinal haemorrhagia, necrosis, and inflammation (33, 36). in our study, in group i, inflammatory reactions was evident in some of the animals from both groups. additionally, aggregate lymphoid follicles were found hyperplasic in three animals of group i. there were no findings in the stomach of any of the members from group i, which was in contrast to the focal periarteriolar haemorrhagia found in one of the animals from group ii. these findings correlated with the previous findings (36). in our study, the most significant findings in almost all organs were mostly hyperemia, focal haemorrhagia, and mononuclear cell infiltrations as was indicated in several previous studies. such results have been reported as a result of inflammatory responses under influence of the variety of cytokines as reported in previous reports (18, 24, 25). in our study, the acute findings belonging to l. abdullahbayrami envenomation were found to be more severe than the a. crassicauda envenomation because it caused much more of an inflammatory reaction within the first 24h. in this sense, the newly identified l. abdullahbayrami venom had more of an effect than the a. crassicauda venom. additionally, many studies have reported on this inflammatory responses following envenomation. among them, leucocytosis has been mentioned as occurring in the hours following envenomation (14, 18, 25, 39, 40). in the present study, mononuclear cell infiltrations j arthropod-borne dis, march 2019, 13(1): 104–115 o ozkan and me alcigir: a comparative … 112 http://jad.tums.ac.ir published online: april 27, 2019 were more evident in several organs, especially among the leirus sp. group. the leirus envenomation resulted in an ongoing or final stage of acute inflammatory reaction. on the other hand, the main affected organs included the lungs, liver, intestine, and pancreas, respectively (22-26). however, some important findings in the kidneys and the central nervous systems were also evident in our study, which was less likely to be mentioned in prior documents except for the most recent document (27). the inflammation might be a long-term effect from the two types of venom. particularly, another reason for multiple organ disfailure was the hydropic-vacuolar degeneration in parenchymatous organs. it has been previously associated with the loss of function of the na-k atpase pump (15, 27, 41, 42). this situation could be further clarified by examining several fractions of these venoms that target one or more vital cells such as those from the liver, kidneys and also the cerebellum rather than simply focusing on the most prominent vital organs of the heart and the lungs. hence, we think that the hepatocytes, tubular epitheliums, and purkinje cells were more affected based on the findings of this current study. in our study, the main acute findings were mainly hyperemia, haemorrhagia and degenerative-necrotic changes in the aforementioned vital organs. the period of experimentation for this study was longer as compared to other envenomation studies. findings from this study related to the other organs besides just the two vital organs have been made more evident due to the longer time that allowed these effects from both types of venoms to be noticed. in addition, histopathological findings might change according to the type of scorpion venom and the envenomation method for an organism. conclusion leirus abdullahbayrami and a. crassicauda venoms cause to more slight findings beyond simply resulting in death for rabbits. however, l. abdullahbayrami has resulted in more severe lesions when compared to the a. crassicauda. especially, findings were evident in both the vital parenchymatous organs, such as the heart, lungs, liver, kidneys, as well as other organs, such as the brain, spleen, gastrointestinal system, during the first 24h. in addition, the lesions were more related to vascular changes and degenerative-necrotic changes in envenomated animals with l. abdullahbayrami. thus, some cytokines might have change the immune response of the animals against the preservation of body homeostasis. this situation might be more related to the degrees of toxic components of mentioned scorpion species. in this regard, therapeutic strategy against envenomation by several scorpion species and course of toxication might be changed during critical first 24h-period. acknowledgements no financial support was received for this study. the authors declare that there is no conflict of interests. 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amoreuxi (aud. and sav) venom on blood and liver of rat. the 1st ain shams medical congress. 1977, egypt. 1: 535–560. 41. ismail m (1995) review article: the scorpion envenoming syndrome. toxicon. 33: 825–858. 42. possani ld, becerril b, delpierre m, tytgat j (1999) scorpion toxins specific for sodium channels. eur j biochem. 264: 287–300. j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 116 http://jad.tums.ac.ir published online: march 14, 2017 original article chemical constitute and larvicidal activity of fractions of ajuga chamaecistus tomentella plant against malaria vector anopheles stephensi mahnaz khanavi 1, behnaz najafi 1, seyede nargess sadati 2, mohammad reza abai 3, *hassan vatandoost 3 1department of pharmacognosy and persian medicine and pharmacy research center, faculty of pharmacy, tehran university of medical sciences, tehran, iran 2department of traditional pharmacy, school of traditional iranian medicine, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 10 jan 2016; accepted 8 feb 2016) abstract backgrand: the genus ajuga, belongs to lamiaceae family, is one of the exclusive subspecies in the flora of iran. the plants of this genus are used traditionally for treatment of joints pain, gout, jaundice, and as insecticide. methods: larvicidal activity of methanol 80% extract and partition fractions of hexane, chloroform, and ethyl acetate obtained from aerial parts of ajuga chamaecistus subspecies tomentella against malaria vector an. stephensi was evaluated. phytochemical study of active fraction was analyzed using column chromatography and spectroscopy. results: according to the results, among different fractions, hexane fraction has the most larvicidal activity with mortality rate of 100% in concentration of 102 ppm and lc50 of 95.66ppm. the structure of compound 1, main phytoecdysteroid compound separated from hexane fraction, was determined to be ajugalide-e. conclusion: the results suggested that the hexane fraction of ajuga chamaecistus subsp tomentella could be used as a natural and biodegradable insecticide. keywords: ajuga chamaecistus subspecies tomentella, larvicidal, anopheles stephensi, phytoecdysteroid introduction mosquitoes are the main vector in transmission of malaria that is still a major endemic disease in foci located in south and southeast of iran. these areas include the provinces of sistan and baluchistan, hormozgan and kerman. among all species of anopheles recognized in iran, 8 of them are considered as malaria vectors including: an. culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus, an. pulcherrimus, an. sacharovi, and an. maculipenni (doosti et al. 2006). malaria control is an important goal in developing tropical countries. mosquito controls, using synthetic chemical insecticides have adverse effects on the environment and also cause growing of insecticide resistance in ar thropods (edrissian 2006, khanavi et al. 2011). plants, rich in bioactive phytochemicals, have been investigated as a source of alternative agents for control of mosquitoes. several extract and essential oil of certain plants showed toxic effect against some public health pests (hadjiakhoondi et al. 2003, vatandoost et al. 2004, hadjiakhoondi et al. 2006, govindarajan et al. 2011, sedaghat et al. 2011). the genus ajuga (lamiaceae) with common name of bugle is found in china, korea, japan and throughout europe. five species of this annual and perennial genus are found in iran. ajuga chamaecistus contains several exclusive subspecies, including a. chamaecistus subspecies tomentella (mozaf*corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 117 http://jad.tums.ac.ir published online: march 14, 2017 farian et al. 2007). some species belonging to this genus are used in traditional medicine of different countries in the world. moreover in iranian traditional medicine, the genus ajuga (kamaphytus, jaadeh) has been used for treatment of joint pain, gout, and jaundice and as insecticide (naghibi et al. 2005, jorjani 2012). several biological studies have been performed on many species of this genus which have confirmed their ethno pharmacological properties such as hypoglycemic (hilaly et al. 2002), anti-inflammatory (gautam et al. 2011), anabolic, analgesic, antiarthritis, antipyretic, hepatoprotective, antibacterial, antifungal, antioxidant, cardiotonic (israili et al. 2009), treatment of joint diseases (ono et al. 2009), and their application as anti-malarial (kuria et al. 2001). antifeedant activity of ajuga iva and ajuga pseudoiva extract and their active compounds against larvae of sodoptera littoralis (egyptian cotton leafworm) have been shown in some literatures (bondì et al. 2000, ben jannet 2000, ben jannet et al. 2001). prior to this study, some phytochemicals such as 20-hydroxyecdysone, cyasterone, ajugalactone, makisterone a, and 24-dehydroprecyasterone (phytoecdysteroids), 8-acetylharpagide (iridoid), cisand trans-melilotoside, lavandulifolioside, leonoside b, and martynoside (phenylethanoid glycosides), were identified from diethyl ether and n-butanolic fractions of ajuga chamaecistus ssp. tomentella. cytotoxicity evaluation of some fractions of this plant showed the cytotoxicity of hexane fraction against normal and cancer cell lines (sadati et al. 2012 a, b). the aim of this study was to evaluate larvicidal activity of a methanol 80% extract and partition fractions of hexane, chloroform, and ethyl acetate obtained from aerial parts of ajuga chamaecistus subsp tomentella against malaria vector an. stephensi. furthermore, we performed a phytochemical investigation on the hexane fraction to identify the main components. materials and methods plant material aerial parts of ajuga chamaecistus ssp tomentella were collected from tehran, iran, in june 2008 and verified by prof gh amin. a voucher specimen (the-6697) has been deposited in the herbarium of the department of pharmacognosy, faculty of pharmacy, tehran university of medical sciences, tehran, iran. preparation of total extract and fractions the air-dried and ground plants of a. chamaecistus ssp tomentella (250g) were extracted with methanol 80% at room temperature and concentrated under reduced pressure to give a dark brown extract. the extract (30g) was loaded on silica gel (mesh 230–400) column and eluted with 250ml of hexane, chloroform, ethyl acetate and methanol 80%, separately. finally the whole collected fractions were dried by the rotary evaporator and then by a vacuum oven. preparing stock solutions primary tests were performed to determine the concentration of stock solutions. according to the results, the concentration of 320 ppm, 160ppm, 2560ppm, was determined for total extract, hexane fraction and methanol fraction respectively. next, the stock solutions were serially diluted to obtain logarithmic concentrations of solution for the test. for better solubility, dmso was used as the solvent for hexane fraction and methanol for total extract and methanol fraction. these two solvent are completely safe for larvae as it proved in controls. larvicidal assays larvicidal activity assays of ajuga chamaecistus subsp tomentella on the larvae of an. stephensi were performed on the basis of who protocol. the insectary condition was 30±1 °c, 60±5% relative humidity and 10:14, j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 118 http://jad.tums.ac.ir published online: march 14, 2017 dark: light periods. the mosquitoes were collected from malarious areas of iran and then maintained at the department of medical entomology and vector control, school of public health, tehran university medical sciences. 1ml of prepared solution was mixed thoroughly with 224ml water in 400ml glass beakers. 25ml water containing 25 late instar larvae was slowly added. four replicates maintained for each concentration. a control was set for all series of tests (1 ml of solvent was used instead of 1 ml of extract). mortality was counted after 24 h recovery period. lc50 (lethal concentration to cause 50% mortality in the population) and lc90 (lethal concentration to cause 90% mortality in the population) were determined by the use of regression line employed by finney (finney 1971, who 2014). chromatography the hexane fraction (4g) was selected for phytochemical studies. thus, it was chromatographed on silica gel (mesh 230–400) eluting with a gradient of chloroformmethanol (9–1) to 100% methanol to afford 4 fractions. fraction 2 was purified with a few amount of methanol and compound 1 (36.5 mg) was obtained. general experimental procedures 1hand 13c-nmr were measured in cdcl3 solution on a bruker avance spectrometer (500mhz, tms as internal standard) for compounds 1. ft-ir spectra determined using a nicolet 550-a spectrometer (kbr disks). column chromatography was acchived on silica gel 60 (230–400 mesh, merck) and rp-18 (merck). spectroscopic data ajugalide-e (1): white amorphous powder, ft-ir νmax cm-1: 3237, 2953, 1735, 1689, 1248, 1029. 1h and 13c nmr (cdcl3), see table 3. results larvicidal activity of the methanol 80% extract and partition fractions of hexane, chloroform, and ethyl acetate obtained from aerial parts of ajuga chamaecistus tomentella against malaria vector an. stephensi was examined in different concentrations. according to the results presented in fig. 1, the regression line was plotted for each extract and lc50 was calculated. among the extracts, hexane fraction showed the most larvicidal effect with lc50 value of 95.66 ppm. lc50 for total extract and methanolic 80% fraction was 117.72 and 954.19ppm respectively. also, other statistical parameters were calculated (table 1, fig. 1). isolated compounds 1, 2 from the hexane fraction of total methanolic extract of aerial parts of ajuga chamaecistus ssp tomentella were identified by comparison of their nmr (1h-, 13c-nmr) data with those reported in the literature. 1h and 13c nmr data of these compounds run in cdcl3 reported for the first time. δh and δc (ppm) of compound 1 was noted in table 2. the isolated compound 1 (fig. 2) were identified as ecdysteroids, ajugalide-e in comparison with the literature (chan et al. 2005). tables 1. lethal concentrations and other associated statistic of bioassay tests of some extracts a b ± se lc50 (ppm) ± 95%c.l. lc90 (ppm) ± 95%c.l. λ2 (heterogeneity) λ2 table (df) pvalue total extract -5.6408 2.7238 ± 0.252 103.6986 117.7283 133.4075 285.1837 309.0209 455.8304 10.704 * 13.345 (2) 0.01 j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 119 http://jad.tums.ac.ir published online: march 14, 2017 hexane fraction -8.3002 4.1820 ± 0.199 88.5023 95.6640 105.6703 170.0557 195.5219 236.1008 16.614 * 13.345 (2) 0.01 methan olic 80% fraction -16.0984 5.4028 ± 0.643 848.9428 954.1945 1071.6982 1420.8157 1647.5819 2045.8799 5.002 * 9.210 (2) 0.01 *no heterogeneity fig. 1. comparison of regression lines and equations of and total extract two fractions of ajuga chamaepitys subspecies tomentella against larvae of anopheles stephensi o o ho oh oh oac 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 o (1) ho fig. 2. chemical structure of ajugalide-e (1), isolated from hexane fraction of ajuga chamaepitys subspecies tomentella table 1. continued… j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 120 http://jad.tums.ac.ir published online: march 14, 2017 table 2. 1h nmr and 13c nmr data of ajugalide e (500 mhz in cdcl3) position δh δc 2 1.98 (td) 3 2.80(dd) 55.71 7 5.25(brs) 122.9 8 144.0 13 47.92 14 81.34 18 0.88(s) 17.6 19 0.72(s) 18.5 21 1.10(s) 20.7 22 4.47(t) 75.95 26 184.7 27 0.91(d) 15.8 29 0.84(d) oac 2.02(s) 171.4 discussion the larvicidal activity of several plant extracts and phytochemicals against mosquito larvae has been established. in a study reported by sharma, et al. (2004) petroleumether extract of ajuga remota was the most effective extract with lc50 values of 0.033% after 24 hours and 0.029% after 48 hours of treatment against the larvae of an. stephensi (sharma et al. 2004). govindarajan et al. (2011) have reported that benzene extract of e. coronaria showed the highest larvicidal effect on the larvae of an. stephensi, ae. aegypti, and cx. quinquefasciatus with the lc50 and lc90 values were 79.08, 89.59, and 96.15 ppm and 150.47, 166.04, and 174.10 ppm, respectively (edrissian ghh 2006). manjari et al. (2014) indicated that acetone leaf extracts of clausena dentata showed the larval mortality against the fourth instar larvae of an. stephensi, cx. quinquefasciatus, and ae. aegypti (diptera: culicidae). culex quinquefasciatus (lc50= 0.150278mg/ml, lc90= 7.302613mg/ml), a. aegypti (lc50= 0.169495 mg/ml, lc90= 1.10034mg/ml), and an. stephensi (lc50=0.045684 mg/ml, lc90= 0.045684mg/ml) (manjari et al. 2014). in this study hexane fraction of methanolic extract of ajuga chamaecistus ssp tomentella showed the most larvicida activity against an. stephensi larvae. in order to find the active ingredient of effective fraction, hexane fraction was chromatographed on silica gel and rp-18 resulted in isolation and identification of two phytoecdysteroid epimers, ajugalidee and 22-acetylcyasterone. in plants of genus ajuga, a variety of phytoecdysteroids have been identified among them, 20-hydroxyecdysone (β-ecdysone) and cyasterone are the most abundant (ramazanov et al. 2005). this group of natural products produces a wide range of pharmacological activities in mammals including adaptogenic, anabolic, antidiabetic, hepatoprotective, immunoprotective, wound-healing, antioxidant and free radical scavenging activities (sadati et al. 2012 b). phytoecdysteroids, present in many plants, are analogues of insect moulting hormone (ecdysteroids) that control insect growth, development, and reproduction (rharrabe et al. 2010). toxicity of some ecdysone agonists on larvae of three mosquito species, ae. aegypti, an. gombiae, and cx. quinquefasciatus have been stablished (beckage et al. 2004). nyamoita et al. (2013) investigated that four phytoecdysteroids isolated from aceton extract of vitex schiliebenii showed potent toxic effect against larvae of an. gambiae (nyamoita et al. 2013). according to the result of this study the hexane fraction of total methanolic extract of the aerial parts of ajuga chamaecistus ssp. tomentella exhibited the most toxicity on an. stephensi larvae than the other fractions. in previous study two major ecdysteroid in addition to three minor ones identified from this plant (sadati et al. 2012b). thin layer chromatography of the hexane fraction showed two main compounds and analyzing of this fraction resulted in isolation and identification of a phytoecdysteroid, ajugalide-e. comparison of our result with other reports indicated that the hexane extract of the studied plant was effective to control an. stephensi. j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 121 http://jad.tums.ac.ir published online: march 14, 2017 further analysis of hexane fraction to isolate the active component for larval control resulted in identifying the major phytoecdysteroid compound. based on our results it can be concluded that phytoecdysteroids are interesting molecules that can be considered as natural and biodegradable insecticide. conclusion more investigation is required to assess the larvicidal activity of the product at the field situation. acknowledgements the authors would like to appreciate very much for kind collaboration of all staff of department of medical entomology and vector control. references beckage ne, marion km, walton we, wirth mc, tan ff (2004) comparative larvicidal toxicities of three ecdysone agonists on the mosquitoes aedes aegypti, culex quinquefasciatus, and anopheles gambiae. arch insect physiol. 57(3): 111–122. ben jannet h, harzallah-skhiri f, mighri z, simmonds ms, blaney wm (2000) responses of spodoptera littoralis larvae to tunisian plant extracts and to neo-clerodane diterpenoids isolated from ajuga pseudoiva leaves. fitoterapia. 71(2): 105–112. ben jannet h, h-skhiri f, mighri z, simmonds msj, blaney wm (2001) antifeedant activity of plant extracts and of new natural diglyceride compounds isolated from ajuga pseudoiva leaves against spodoptera littoralis larvae. ind crops prod. 14(3): 213–222. bondì ml, al-hillo mr, lamara k, ladjel s, bruno m, piozzi f, simmonds ms (2000) occurrence of the antifeedant 14, 15-dihydroajugapitin in the aerial parts of ajuga iva from algeria. biochem sys eco. 28(10): 1023–1025. chan yy, wu ts, kuoh cs, damu ag (2005) a new phytoecdysteroid from ajuga taiwanensis. chem pharm bull. 53(7): 836–838. doosti s, azari-hamidian s, vatandoost h, oshaghi ma, hosseini s (2006) taxonomic differentiation of anopheles sacharovi and an. maculipennis s.l. 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(2001) antimalarial activity of ajuga remota benth (labiatae) and caesalpinia volkensii harms (caesalpiniaceae): in vitro confirmation of ethnopharmacological use. j ethnophar. 74: 141–148. manjari ms, karthi s, ramkumar g, muthusamy r, natarajan d, shivakumar ms (2014) chemical composition and larvicidal activity of plant extracts from clausena dentata (willd) (rutaceae) against dengue, malaria, and filariasis vectors. parasitol res. 113: 2475–2481. mozaffarian v (2007) a ِ◌◌ِdictionary of iranian plant names. 5rd ed. farhang moaser press, tehran. naghibi f, mosaddegh m, mohammadi motamed s, ghorbani a (2005) labiatae family in folk medicine in iran: from ethnobotany to pharmacology. iran j pharm res. 2: 63–79. nyamoita mg, ester i, zakaria mh, wilber l, ochola bj, ahmed h (2013) larvicidal and brine shrimp activities of vitex schiliebenii extracts and isolated phytoecdysteroids on anopheles gambiae giles s.s larvae. j app pharm sci. 3(05): 091–095. ono y, fukaya y, imai s, yamakuni t 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oils of apiaceae plants against malaria vector, anopheles stephensi. iran j arthropod-borne dis. 5(2): 51–59. sharma p, mohan l, srivastava cn (2004) larval susceptibility of ajuga remota against anopheline and culicine mosquitos. southeast asian j trop med public health. 35(3): 608–610. vatandoost h, moinvaziri vm (2004) larvicidal activity of neem tree extract j arthropod-borne dis, march 2017, 11(1): 116–123 m khanavi et al.: chemical constitute and … 123 http://jad.tums.ac.ir published online: march 14, 2017 (neemarin) against mosquito larvae in the islamic republic of iran. eastern med health j. 10: 573–578. world health organization (2013) malaria entomology and vector control. participants’ guide. who, geneva. p. 180. j arthropod-borne dis, june 2018, 12(2): 135–140 a ramazani et al.: antiplasmodial property of … 135 http://jad.tums.ac.ir published online: june 12, 2018 original article antiplasmodial property of glycyrrhiza glabra traditionally used for malaria in iran: promising activity with high selectivity index for malaria *ali ramazani 1, mahdi tavakolizadeh 1, 2, samira ramazani 1, hamidreza kheiri-manjili 1, mehdi eskandari 3 1zanjan pharmaceutical biotechnology research center, zanjan university of medical sciences, zanjan, iran 2department of pharmacognosy, school of pharmacy, zanjan university of medical sciences, zanjan, iran 3department of physiology and pharmacology, school of medicine, zanjan university of medical sciences, zanjan, iran (received 08 oct 2016; accepted 8 may 2018) abstract background: development of resistance against the frontline anti-malarial drugs has created an alarming situation, which requires intensive drug discovery to develop new, more effective, affordable and accessible anti-malarial agents. the aim of this study was to assess antiplasmodial activity of the different fractions of root extract of glycyrrhiza glabra. methods: roots of g. glabra were collected from tarom district of zanjan province in 2016 and then dried root material was chopped and consecutively extracted by the percolation method using solvents of different polarity. resulting extracts were assessed for in vitro and in vivo anti-malarial and cell cytotoxicity activities. results: among the three different solvent fractions studied, water-methanol and ethyl acetate fractions showed promising in vitro antiplasmodial activity against cq-sensitive plasmodium falciparum 3d7 strain (ic50= 9.95 and 13µg/ml, respectively). further, the selectivity indices (hela cells versus p. falciparum) for the promising watermethanol fraction showed selectivity for p. falciparum and potential safer therapy for human. interestingly, watermethanol and ethyl acetate fractions showed a significant suppression of parasite growth (72.2% and 65%, respectively) in comparison with control group in mice infected with p. berghei (p< 0.05). conclusion: the promising antiplasmodial activity of the aqueous fraction of g. glabra obtained in our study warrant bioassay-guided fractionation of this fraction to identify active principles responsible for antiplasmodial activity. keywords: glycyrrhiza glabra, malaria, traditional medicine, plasmodium berghei, iran introduction there were approximately 219 million cases of malaria all around the world and 660000 people died from this disease (1). increasing resistance in the malaria parasite plasmodium falciparum against artemisininbased drugs is challenging to malaria control programs (2) and demands a wild attempt to develop novel anti-malarial drugs (3-5). drugs containing novel structure from natural source represent the main source for the discovery and development of new drugs for malaria (6). the discovery of new anti-malarial drugs from natural sources is increasing after the successfulness of quinine and artemisinin. glycyrrhiza glabra (liquorice) is part of both western and eastern herbal traditions. it was approved to treat cough, bronchitis, and gastritis by commission e. this plant is used to treat peptic ulcers, asthma, pharyngitis, infections, hepatic disorders and fever (7) and it is one of the main constituents of the anti-malarial ancient iranian remedy (8). previously, based on our research strategies for malaria drug discovery from plant sources (9, 10), we reported the in vivo and in vitro antiplasmodial activity of total root *corresponding author: dr ali ramazani, e-mail: ramazania@zums.ac.ir http://jad.tums.ac.ir/ mailto:ramazania@zums.ac.ir j arthropod-borne dis, june 2018, 12(2): 135–140 a ramazani et al.: antiplasmodial property of … 136 http://jad.tums.ac.ir published online: june 12, 2018 extracts of g. glabra (9). some active compounds with antimicrobial, antiviral and antiprotozoal properties were isolated and characterized from this plant (11-13). this study was designed to investigate the three solvents fractions of g. glabra with different polarities with hexane, ethyl acetate and methanol-water (50:50). in this framework, selected plant was collected and further evaluated for their in vitro and in vivo antiplasmodial activity and toxicity effect on hela cells. materials and methods chemicals reagents and materials in this study were obtained from merck (darmstadt, germany) and sigma-aldrich (steinheim, germany). mtt, fbs and rpmi 1640 medium from atocel and paa (austria). plant material roots of g. glabra were collected from tarom district, zanjan, iran, at an altitude of 750m. the specimen was authenticated by dr m tavakolizadeh and a voucher specimen is deposited at the herbarium of school of pharmacy, zanjan university of medical sciences (voucher no. 1073). extraction two thousand grams of dried root material was chopped and consecutively extracted by the percolation method (three times for each solvent and 72h for each time) using solvents of different polarities such as hexane, ethyl acetate and methanol-water (50: 50). extractions were performed at room temperature and solutions were concentrated and dried by rotary evaporation under reduced pressure at 40 °c. the dried samples were stored in a freezer at 4 °c for further use in antiplasmodial and cell toxicity assays. in vitro cultivation of plasmodium falciparum chloroquine (cq) sensitive strain (3d7) of p. falciparum was used and cultured according to the methods (9, 10, 14, 15). briefly, p. falciparum parasites was cultured on human erythrocytes (blood group o+) in rpmi 1640 medium supplemented with 0.5g/100ml albumax i, 25mm hepes, 19mm sodium carbonate, and 30μg/ml gentamicin sulfate, at ph 7.2 in a gas mixture of 91% n2, 6% co2 and 3% o2. the medium was changed each day. in vitro antiplasmodial assays the extracts of experimental plants were evaluated for their antiplasmodial activity against 3d7 strain of p. falciparum. for drug screening, sybr green i-based fluorescence assay was used (16). briefly, sorbitol synchronized parasites (100μl) were incubated under normal culture conditions at 2% haematocrit and 1% parasitaemia in the presence or absence of different plant extracts (100, 50, 25, and 12.5µg/ml). cq was used as positive controls, while 0.4% (v/v) dmso was used as the negative control. after 48h of incubation, 100μl of sybr green i lysis buffer was added to each well and after mixing incubated in the dark at 37 °c for 1h. fluorescence was measured using an elisa plate reader (infinite m200, tecan) with excitation and emission wavelength bands cantered at 485 and 530nm, respectively. the background reading for an empty well was subtracted to yield fluorescence counts for analysis. the counts were plotted against the logarithm of the drug concentration and curve fitting by nonlinear regression to yield the drug concentration that produced 50% of the observed decline from the maximum counts in the drug-free control wells (ic50). the results were validated microscopically by examination of giemsa stained smears of extract treated parasite cultures. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 135–140 a ramazani et al.: antiplasmodial property of … 137 http://jad.tums.ac.ir published online: june 12, 2018 cytotoxic assay on hela cells using mtt assay the cytotoxic effects of active plant extract fractions (ethyl acetate and water-methanol) on hela cells were assessed by using mtt (3–2, 5 diphenyl tetrazolium bromide) assay (17-20). the hela cells cultured in rpmi medium containing 10% foetal bovine serum and incubated at 37 °c with 5% co2 and 96% humidity. briefly, cells (104 cells in 100μl of culture medium) were distributed in 96-well flat-bottom plates in complete medium. drug solutions were added after 24h of seeding and incubated for 48h. after 48h, 20μl of a stock solution of mtt (4mg/ml in phosphate buffered saline) was added to each well, gently mixed and incubated for another 3h. then supernatant was removed and 100μl of dmso (stop agent) was added. the absorbance of each well measured at 540nm using an elisa plate reader (infinite m200, tecan). the 50% cytotoxic concentration (tc50) of test samples was determined by analysis of doseresponse curves. therapeutic index was calculated as a ratio of tc50 hela /ic50 3d7. in vivo antimalarial assay in vivo antimalarial activity of different solvent fractions of g. glabra assessed using the 4day suppressive test against p. berghei infection in mice (21). female swiss albino mice, weight 18–20g were inoculated with p. berghei (anka strain). each mouse received 106 infected erythrocytes by intra-peritoneal (ip) injection on the first day of the experiment. groups of five mice were dosed daily by ip injection (200mg/kg) for 4 consecutive days. on day 5 of the test, a blood smear was taken from the mice. percentage suppression of parasitaemia for the fractions was calculated as 100− [(mean parasitaemia treated/mean parasitaemia control) ×100. for comparison of average parasitaemia, oneway anova and two-tailed student’s t-test were used (spss 21.0 inc., usa) with p< 0.05 being considered significant. cq at 25mg/kg was used as a positive control. the solvent (20% dmso in pbs solution) was used as negative control. the study was approved by the institution animal ethical committee. results in vitro anti-plasmodial assay and cytotoxic study all the three fractions of g. glabra root extracts were screened for in vitro anti-plasmodial activity against the cq-sensitive (3d7) p. falciparum strain (table 1). the watermethanol and ethyl acetate fractions showed promising antiplasmodial activity with ic50 values of 9.95 and 13µg/ml, respectively. the n-hexane fraction did not show admissible activity. the two above mentioned active fractions (water-methanol and ethyl acetate) further analyzed for their toxicity on hela cells. the selectivity index (si) is defined as the ratio of the hela cells toxicity to the antiplasmodial activity and is determined by dividing the tc50 values for the hela cells by the ic50 value for p. falciparum. the water-methanol fractions showed low toxicity against hela cells with higher selectivity against malaria (table 1). in vivo anti-plasmodial assay the fractions that showed promising antiplasmodial activity further analyzed for their in vivo anti-plasmodial property (table 2). the water-methanol and ethyl acetate fractions showed a significant suppression of parasitaemia (p< 0.05). in comparison to control group, the water-methanol and ethyl acetate inhibited 72.2% and 65% of the growth of the parasite, respectively. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 135–140 a ramazani et al.: antiplasmodial property of … 138 http://jad.tums.ac.ir published online: june 12, 2018 table 1. antiplasmodial activity, cytotoxicity and selectivity of water-methanol and ethyl acetate extracts of glycyrrhiza glabra n extract fraction p. falciparum ic50 (µg/ml) hela cell ic50 (µg/ml) selectivity indices 1 water-methanol 9.95 > 100 > 10 2 ethyl acetate 13 21 1.61 3 n-hexane 215 (-) not tested table 2. in vivo activities of plant extracts against plasmodium berghei group dose (mg/kg) mean parasitemia (sd*) % suppression of parasitemia p-value negative control (20% dmso in pbs solution) 11.55 (2.58) water-methanol 200 3.15 (1.08) 72.2 0.03 ethyl acetate 200 4.04 (2.36) 65 0.02 cq 25 0 100 sd: standard deviation discussion medicinal plants have a wide diversity of medicinal properties including for malaria therapy as two of the most important antimalarial agents, namely quinine and artemisinin, with plant origin. traditional plants could be attractive for drug discovery as they are widespread and also a large population. in the present study, different fractions of g. glabra root extract known for its traditional medicinal usage (8) and anti-malarial activity (9, 22) was further evaluated for its different solvent fractions antiplasmodial activity against cq-sensitive p. falciparum 3d7 and p. berghei anka strain and their toxicity against hela cell line (table 1). among the three different solvent fractions studied, water-methanol and ethyl acetate fractions showed promising in vitro antiplasmodial activity against cq-sensitive 3d7 strain (ic50= 9.95 and 1µg/ml, respectively). further, the selectivity indices (hela cells versus p. falciparum) for the promising water-methanol fraction showed selectivity for p. falciparum and potential safer therapy for human. interestingly, water-methanol and ethyl acetate fractions showed a significant suppression of parasitemia in comparison with control group (p< 0.05). the suppression rate of parasite growth induced by water-methanol and ethyl acetate fractions were 72.2% and 65%, respectively (table 2). in our previous study (9), the hydro-alcoholic extraction of g. glabra root extract showed promising antiplasmodial activity with ic50 values of 13.56µg/ml against p. falciparum 3d7 strain and also suppressed the growth of the p. berghei parasite by 65% in vivo at a dose of 400mg/kg. in this study, further fractionation resulted in better antiplasmodial property in vitro and in vivo. water-methanol fraction exhibited in vitro antiplasmodial activity with ic50 value of 9.9µg/ml and suppressed the growth of the p. berghei parasite by 72.2 % at a dose of 200mg/kg in comparison with the control group. the active ingredients of plant accumulated in water-methanol fraction. the ethyl acetate fraction also showed promising in vitro and in vivo antiplasmodial achttp://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/pmc/articles/pmc4340493/table/tab1/ j arthropod-borne dis, june 2018, 12(2): 135–140 a ramazani et al.: antiplasmodial property of … 139 http://jad.tums.ac.ir published online: june 12, 2018 tivity, but this fraction had low selectivity index in comparison with water-methanol fraction. licorice (g. glabra) is used for the treatment of several diseases in traditional medicine. licorice was shown to have a number of biological properties such as antiviral, anti-inflammatory, antimicrobial, and anticancer activities (13). several active components from g. glabra were isolated and some cellular and molecular mechanisms of these compounds have been elucidated (11, 12, 22). one of these compounds is licochalcone a, separated from the roots of chinese licorice, showed promising in vitro antiplasmodial property against both cq-susceptible (3d7) and cq-resistant (dd2) p. falciparum strains. this compound protected the mice from the lethal p. yoelii infection (22). 18beta-glycyrrhetinic acid, another anti-malarial agent isolated from indian licorice, showed significant in silico, in vitro and in vivo antimalarial activity (12). conclusion the water-methanol fraction of g. glabra has higher selectivity index for malaria than ethyl acetate fraction. the promising results with the aqueous fraction of this plant obtained in our preliminary study, warrant bioassayguided fractionation of this fraction to identify active principles responsible for antiplasmodial activity. acknowledgments we thank the research deputy of zanjan university of medical sciences (zums) for financial support of this project (grant number: a-10-349-15). all the experiments were performed according to the guideline provided by the ethical committee at zanjan university of medical sciences. the authors declare that there is no conflict of interests. references 1. who (2012) world malaria report 2012: world health organization. available at: http://www.who.int/malaria/publicatio ns/world_malaria_report_2012/en/. 2. dondorp am, yeung s, white l, nguon c, day np, socheat d, von seidlein l (2010) artemisinin resistance: current status and scenarios for containment. nat rev microbiol. 8(4): 272–280. 3. ramazani a, borna h (2011) in silico analysis and develipment of antimalarial compounds against dihydroorotate dehydrogenase using chembioinformatic tools. int j pharm bio sci. 2(2): 122– 131. 4. ramazani a, keramati m, malvandi h, danafar h, kheiri-manjili h (2017) preparation and in vivo evaluation of anti-plasmodial properties 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cytotoxicity assays. j immunol methods. 65 (1–2): 55–63. 20. ramazani a, mashhadi m, kamalinejad m, tavakolizadeh m, balaghi f, noubarani m, mohammad reza eskandari, farshad h. shirazi (2016) phoenix dactylifera l. fruit induces cell proliferation of a2780, a172 and hfff2 cell lines. iran j pharm sci. 12(1): 35–44. 21. peters w, li zl, robinson bl, warhurst dc (1986) the chemotherapy of rodent malaria, xl. the action of artemisinin and related sesquiterpenes. ann trop med parasitol. 80(5): 483–489. 22. chen m, theander tg, christensen sb, hviid l, zhai l, kharazmi a (1994) licochalcone a, a new antimalarial agent, inhibits in vitro growth of the human malaria parasite plasmodium falciparum and protects mice from p. yoelii infection. antimicrob agents chemother. 38(7): 1470–1475. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 211 http://jad.tums.ac.ir published online: may 27, 2017 original article larval habitats characteristics of mosquitoes (diptera: culicidae) in northeast of iran aioub sofizadeh 1, *seyed hassan moosa-kazemi 2, hossein dehghan 2 1infectious diseases research center, golestan university of medical sciences, gorgan, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 1 feb 2014; accepted 7 may 2016) abstract background: there are unorganized, published documents about the ecology of mosquitoes (diptera: culicidae) in northeastern part of iran. the purpose of this study was to determine the distribution and characteristics of larval habitats of culicidae in kalaleh county. methods: larvae were collected using dipping method and adults by human landing catch technique during april– october, 2012. larval habitat characteristics were recorded such as vegetation status, and sunlight, water situation. lacto-phenol and de faure’s media were used for conserving and mounting samples. data were analyzed using spss statistical software, version 11.5. results: out of the 395 larvae collected, 332 were adult mosquitoes comprising; culiseta, culex, anopheles and ochlerotatus genera and 14 species including an. superpictus, an. maculipennis s.l., an. hyrcanus, an. psudopictus, an. claviger, culex pipiens, cx. theileri, cx. perexiguus, culiseta longiareolata, cs. subochrea, ochlerotatus caspius, oc. echinus and oc. geniculatus. culex pipiens larvae were predominant (27.6%) and cs. subochrea (1%) was found as the lowest species in terms of number. in the adult form, cx. pipiens (28.9%) was predominant whereas, cs. subochrea and cx. perexiguus were reported to have had the lowest frequency. conclusion: the larvae of an. superpictus and an. maculipennis species as the main vectors of malaria in north of iran were reported in permanent habitats with clear water and vegetation, full and partial sunlight situations and muddy as well as sandy substrates that are important in larvicide application programs. exclusive studies are necessary to diagnose an. maculipennis species complex using molecular and morphological analysis in the future. keywords: larval habitats, mosquitoes, iran introduction culicidae family is one of the largest and most medically important families of diptera. by now, 64 species and 3 subspecies have been identified in seven genera and 16 subgenera in iran (azari-hamidian 2007a). habitats of the mosquito larval stages affect the distribution pattern of adult stages. mosquito habitats are classified as natural or artificial, permanent or temporary. indeed, larval habitats are considered as specific for each mosquito species. moreover, studies on mosquito larval habitats could be useful for vector control programs (bruce-chawat 1980). there are scattered studies on bionomics and ecology of mosquitoes in northeast of iran. macan (1950) mentioned some ecological aspects of anopheles species in the near east of iran. dow (1953) reported some characteristics of larval habitats of six culex species. larval habitats of cx. pipiens were previously studied in tehran province (golestani 1967). lotfi (1970, 1973, and 1976) studied temperature and ph of larval habitats of mosquito larvae in iran. the characteristics of larval habitats of mosquitoes were subsequently reported in minab area, south of iran (yaghoobi-ershadi et al. 1986). the distribution and characteristics of larval habitats of mosquitoes in iran were *corresponding authors: dr seyed hassan moosakazemi, email: moosakazemi@tums.ac.ir http://jad.tums.ac.ir/ mailto:moosakazemi@tums.ac.ir j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 212 http://jad.tums.ac.ir published online: may 27, 2017 studied by zaim in 1987. the ecology and fauna of mosquitoes were reported in esfahan province (mousa-kazemi et al. 2000a). azarihamidian (2005, 2006, 2007b, 2011) and azari-hamidian et al. (2011) reported diversity and larval habitats of mosquitoes in the north of iran. besides, physical and chemical factors affecting larval habitats of anopheles species were studied in southeast of iran (ghanbari et al. 2005). some studies about the ecology and fauna of mosquitoes were reported in neka county, northern part of iran (nikookar et al. 2015). ecology and morphological characteristics of mosquitoes were reported in yazd city, central iran (dehghan et al. 2010). larval habitats and biodiversity of anopheline mosquitoes and some environmental characteristics were studied in southern iran (hanafi-bojd et al. 2012, soleimani-ahmadi et al. 2013). there are scattered information about fauna and ecological characteristics of mosquitoes in golestan province. by now, 10 anopheles and 14 culicinae species were identified using morphological characters and the surface patterns of eggs. earlier studies had been conducted in northeastern part of iran including mazandaran and north khorasan provinces (gutsevich 1943, zolotarev1945, dow 1953, zaim 1987, sedaghat et al. 2003, nikookar et al. 2015). mosquito-borne diseases including malaria, arboviral diseases and filariasis are the most common arthropod borne diseases in the world (gubler 1998). presently, malaria is one of the most important problems in iran. golestan province was one of the malaria foci in iran but there are no imported cases in the province. recently, number of endemic foci of malaria has been identified in different neighboring countries of iran including afghanistan, pakistan and tukmenistan and potential vectors are widely dispersed. however, a rapid spread of the diseases is likely to occur due to the lack of vector control programs (ministry of health and medical education, 2012). epidemics occur in turkmenistan, a neighboring country of iran, climate change and the imported cases are considered as the most reasons for the increase in diseases from 2003– 2004 (ministry of health and medical education, 2012).. kalaleh county is located in the north-east of iran. because of favorable weather which supports the breeding of mosquitoes, risk of malaria transmission, immigration and lack of malaria control, it is important to obtain adequate information in the field of malaria epidemiology in order to optimize the implementation of fundamental research programs. in addition, study on the ecology of malaria vectors in this area will help obtain better management of vector control and proper approach to malaria control programs. therefore, this study was carried out to determine some ecological aspects of the culicidae species and characteristics of their habitats in kalaleh county, golestan province, northeast of iran. materials and methods study area a cross-sectional study was carried out in kalaleh county, golestan province, northern iran from april to october 2012. this study took place in seven randomly selected rural villages of the county (37° 70 'n 55°81' e). the samples were collected in plain, slope and mountainous areas. the province is bounded by caspian sea and mazandaran province in the west, semnan province in the south, north khorasan province in the east and turkmenistan country in the north (fig. 1). most parts of golestan province are plain and more than 2/3 of the plain areas have arid and semiarid climates and 1/3 of others have a mild climate. this county has 4962km and a population of 153261 people and is located in northeast of golestan province. the main agricultural products are alfa alfa, water melon and cotton. maximum and minimum of precipitation were recorded as 40.8 and -0.02 respechttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 213 http://jad.tums.ac.ir published online: may 27, 2017 tively and mean annual relative humidity was recorded as 67%. the total annual rainfall was 772mm, the minimum in august and maximum in february. mosquito sampling sampling was carried out using dipping method for collecting mosquito larvae and night catch for adult collection. larval sampling method was carried out using standard dipper of 350ml. each habitat was sampled in different parts of the larval habitats for 10 times. in order to clarify the samples collected, they were conserved and transported to the laboratory in vials containing lacto-phenol solution. the vials were labeled based on sample’s date code and their associated habitats. features such as larval habitat status (permanent or temporary, stagnant or slow-running water), vegetation type, substrate type, habitat types and position of the sunlight were recorded on special forms. the night catch method was carried out using suction tubes from animal baited traps. animal baited collection was conducted from 18.00 pm to 03.00 am monthly in fixed animal shelters randomly placed in each village. sample containers were protected from light and heat and were transferred to the laboratory of school of public health, tehran university of medical sciences, where the authors identified the specimens using the taxonomic keys of shahgudian 1960, zaim and cranston 1986, harbach 1985, and azari-hamidian and harbach 2009. the mosquito name abbreviations were cited based on reinert (2009). results overall, 395 larvae and 332 adult mosquitoes in 4 genera and 14 species in seven sampling places were found in this area. four species of culex, five species of anopheles, two species of culiseta and three species of ochlerotatus were identified. the species of mosquito larvae which were reported included: an. superpictus grassi, an. maculipennis s.l, an. hyrcanus (pallas), an. psudopictus, an. claviger (meigen), cx. pipiens linnaeus, cx. theileri theobald, cx. hortensis ficalbi, cx. perexiguus theobald, cs. longiareolata (macquart), cs. subochrea (edwards), oc. caspius s.l (pallas), oc. echinus (edwards) and oc. geniculatus (olivier). all the species were found in adult forms except cx. perexiguus. culex pipiens and cs. longiareolata were the dominant species reported. the larvae and adult species of cx. theileri, cx. pipiens, oc. caspius and oc. echinus in all larval habitats were collected and presented in table 1 and 2. the association of cx. pipiens larvae with the other culicidae species was more than the other species (table 3). larval habitats of some mosquito species were diverse. anopheles claviger and oc. geniculatus larvae were collected only in permanent larval habitats (table 4). an. claviger, an. superpictus, an. hyrcanus, oc. geniculatus larvae were found in the larval habitats without vegetation, whereas cs. longiareolata and cs. subochrea were collected from habitats with vegetation (table 4). most larval habitats were found with substrate of mud and sand bottom and fewer larvae were collected in rocks and cement substrates. besides, total number of samples of anopheles species was collected in fresh water (table 4). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 214 http://jad.tums.ac.ir published online: may 27, 2017 table 1. frequency of mosquito larvae which were collected by dipping method in kalaleh county, 2012 p e r c e n ta g e to ta l g h a r a n k i j a n g a l s a d e c h a m r a n b a r b a r g h a le h g o r g a n d o z b e ili ju n g le g h o sh e sa v e r ju n g le k h e d e r -o lia ju n g le places species 3.5 14 1 5 2 4 1 0 1 an. claviger 3.3 13 1 1 3 7 0 1 0 an. superpictus 4.8 19 4 5 2 1 4 2 1 an. maculipennis s.l 4 15 5 2 0 4 2 1 1 an. psudopictus 5.1 20 5 0 1 6 2 4 2 an. hyrcanus 18.2 71 11 11 12 10 11 12 4 cs. longiareolata 1 4 2 0 1 0 0 1 0 cs. subochrea 9.9 39 1 4 2 4 8 10 10 cx. theileri 5.1 20 5 2 3 4 3 1 2 cx. hortensis 4.3 17 5 1 4 1 4 1 1 cx. perexiguus 27.6 109 13 14 14 15 24 14 15 cx. pipiens 7.8 31 1 3 6 5 6 4 6 oc. caspius 3.8 15 2 4 2 1 3 1 2 oc. echinus 1.5 6 1 1 0 1 1 1 1 oc. geniculatus 100 393 57 53 52 63 69 53 46 total table 2. frequency of adult mosquitoes which were collected by night catch method from animal baited traps in kalaleh county, 2012 places species k h e d e r o lia ju n g le g h o sh e sa v e r ju n g le g h o sh e c h a sh m e ju n g le b e ili ju n g le p a r p a r i ju n g le a g h so u ju n g le a z iz a b a d to ta l p e r c e n ta g e an. claviger 0 0 1 2 5 1 3 12 3.6 an. superpictus 5 1 0 3 1 5 2 17 5.1 an. maculipennis s.l 8 2 4 2 5 1 1 23 7 an. psudopictus 11 1 2 0 2 3 1 20 6 an. hyrcanus 12 4 2 1 0 3 3 25 7.6 cs. longiareolata 4 2 3 2 1 0 4 16 4.8 cs. subochrea 0 0 0 0 1 0 1 2 0.6 cx. theileri 10 10 11 10 10 4 10 65 19.5 cx. hortensis 0 1 0 0 1 0 0 2 0.6 cx. pipiens 15 14 12 14 14 12 15 96 28.9 oc. caspius 6 4 2 6 3 4 5 30 9 oc. echinus 2 1 2 2 4 1 1 13 3.9 oc. geniculatus 1 1 2 2 1 3 1 11 3.3 total 74 41 41 44 48 37 47 332 100 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 215 http://jad.tums.ac.ir published online: may 27, 2017 table 3. association of mosquito larvae collected in kalaleh county, 2012 o c . g e n ic u la tu s o c . e c h in u s o c . c a sp iu s c x . p ip ie n s c x . p e re x ig u u s c x . h o rte n sis c x . th e ile ri c s. su b o c h re a c s. lo n g ia re o la ta a n . h y rc a n u s َ a n . p su d o p ic tu s a n . m a c u lip e n n is s a n . su p e rp ic tu s a n . c la v ig e r n o o f la r v a e h a b ita te s species 1 2 1 1 1 3 2 1 1 5 2 3 1  5 an.claviger 5 1 2 3 6 2 4 1 5 1 3 2  1 5 an. superpictus 6 2 5 1 2 6 3 4 1 5 2  4 2 8 an.maculipennis sl 5 2 4 1 2 1 6 5 3 2  1 2 1 11 an. psudopictus 4 1 2 2 1 1 5 5 3  1 3 2 4 12 an. hyrcanus 9 7 8 9 11 14 12 11  11 12 14 13 12 14 cs. longiareolata 1 2 3 4 5 2 1  2 1 4 5 3 2 5 cs. subochrea 8 6 8 9 12 11  12 14 13 12 15 14 12 15 cx. theileri 6 3 5 4 2  1 5 1 2 3 4 2 1 5 cx. hortensis 4 3 5 4  4 1 5 2 1 4 1 2 1 5 cx. perexiguus 15 14 12  12 14 11 13 12 14 14 15 12 14 15 cx. pipiens 5 4  2 5 2 5 1 4 3 6 5 2 4 6 oc. caspius 3  1 3 2 1 3 2 1 4 2 1 2 1 4 oc. echinus  1 2 1 3 1 2 1 3 1 2 1 2 1 3 oc. geniculatus fig. 1. the study area of kalaleh county, golestan province, north of iran http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 216 http://jad.tums.ac.ir published online: may 27, 2017 table 4. larval habitat characteristics of mosquitoes collected in kalaleh county, 2012 larval habitat a n . c la v ig e r a n . su p e rp ic tu s a n . m a c u lip e n n is sl a n . p su d o p ic tu s a n . h y rc a n u s c s. lo n g ia re o la ta c s. su b o c h re a c x . th e ile ri c x . h o rte n sis c x . p e re x ig u u s c x . p ip ie n s o c . c a sp iu s o c . e c h in u s o c . g e n ic u la tu s habitat permanent 100 92.8 92.7 64.3 95.5 77.2 21 73 65.3 36.1 93.8 93 89 100 temporary 0 7.2 7.3 35.7 4.5 22.8 79 27 34.7 63.9 6.2 7 11 0 slow-running water 8 55.5 85 63 12 32 45 64 2.3 9 65.5 61 69 100 stagnant water 92 45.5 15 37 88 68 55 36 97.7 91 35.5 39 31 0 vegetation with 0 0 95.3 89 0 100 100 55 36 59 69.7 59 94 0 without 100 100 4.7 11 100 0 0 45 64 41 29.3 41 6 100 substrate mud 100 80 14 39 79 69 65 45 65 73 31.3 96 89 100 sand 0 20 86 61 21 21 35 35 25 27 47.7 3 11 0 rock and cement 0 0 0 0 0 10 0 20 10 0 21 1 0 0 water situation turbid 0 0 0 0 0 65 78 64 49 61 81 79 0 55 clear 100 100 100 100 100 35 22 36 51 39 19 21 100 45 sunlight situation full sunlight 0 0 94.5 79 21.8 89 24 56 61 59 63 69 69 56 partial sunlight 65 65 5.5 21 41 11 76 44 39 41 37 31 31 44 shaded 35 35 0 0 37.2 0 0 0 0 0 0 0 0 0 habitat kind natural 100 55.9 55.8 35.5 100 65 82 87 71 89 100 36 74 69 artificial 0 44.1 44.2 64.5 0 35 18 13 29 11 0 64 26 31 discussion in our study, a total of 395 larvae and 332 adults were found in 4 genera and 14 species. the mosquito species that were identified included, an. claviger, an. hyrcanus, an. maculipennis s.l, an. psudopictus, an. superpictus, cx. hortensis, cx. perexiguus, cx. pipiens, cx. theileri, cs. longiareolata, cs. subochrea, oc. caspius, oc. echinus and oc. geniculatus. the checklist of culicidae has been prepared and reported in mazandaran, golestan and north-khorasan provinces (dow 1953, zaim 1987, sedaghat et al. 2003, sedaghat and harbach 2005, azari-hamidian et al. 2011, nikookar et al. 2015). the mosquito species which were recorded in this area were discovered by other authors who used pcr technique and those that were not identified or reported in our study are shown by asterisk (*) as follows: anophles claviger meigen, an. hyrcanus pallas, an. maculipennis meigen, an. melanoon hackett*, an. persiensis linton, sedaghat and harbach*, an. plumbeus stephens*, an. pulcherrimus theobald*, an. pseudopictus grassi, an. sacharovi favre*, an. superpictus grassi, aedes vexans meigen*, culex hortensis ficalbi, cx. mimeticus noe*, cx. perexiguus theobald, cx. pipiens linnaeus, cx. theileri theobald, cx. tritaeniorhynchus giles*, cx. modestus ficalbi*, culiseta annulata schrank*, cs. longiareolata macquart, cs. subochrea edwards, ochlerotatus caspius s.l. pallas, http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 217 http://jad.tums.ac.ir published online: may 27, 2017 oc. echinus edwards, oc. geniculatus olivier, oc. pulcritarsis* rondani, uranotaenia unguiculata edwards*. dow (1953) mentioned anopheles mosquito fauna in gorgan (aliabad and ramian) including: an. hyrcanus var. pseudopictus (in the now an. pseudopictus), an. pulcherrimus, an. superpictus, and an. maculipennis group (an. maculipennis, an. melanoon subspecies subalpinus (in the now subspecies of "subalpinus" is synonym of "melanoon") (in and an. sacharovi). anopheles pulcherrimus was reported from ali-abad of golestan province by dow in 1953. this species was reported in north-khorasan (azari-hamidian et al. 2011), moreover, the occurrence of this species in golestan province needs more considerations for future studies. sedaghat et al. (2003) reported the occurrences of an. maculipennis, an. sacharovi, an. persiensis based on molecular identification and its2 sequences in mazandaran province which was bordered with golestan. sedaghat and harbach (2005) confirmed the presence of an. melanoon, an. persiensis and an. pseudopictus species in mazandaran province. zaim (1987) reported 12 culicinae species in mazandaran including: ae. vexans, oc. geniculatus, oc. pulcritarsis, oc. echinus, cx. hortensis, cx. mimeticus, cx. perexiguus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus, culiseta longiareolata, cs. subochrea. nikookar et al. (2015) reported nine species of mosquito including: an. claviger, an. maculipennis, an. plumbeus, an. superpictus, cs. annulata, cs. longiareolata, cx. mimeticus, cx. pipiens, and oc. geniculatus in neka county, mazandaran province. azari-hamidian et al. (2011) reported fourteen species of mosquito representing five genera in north-khorasan province including: an. claviger, an. maculipennis, an. superpictus, an. pulcherrimus, cx. hortensis, cx. mimeticus, cx. modestus, cx. perexiguus, cx. pipiens, cx. theileri, cx. tritaeniorhynchus, cs. longiareolata, oc. caspius and ur. unguiculata in our research, an. claviger was collected in permanent and stagnant habitats with muddy substrate, clear water, without vegetation. this species was only collected from natural habitats. in parallel, larval habitats of this species were reported in spring pools with partial sunlight, slow running water and shaded streams in iraq and western iran (macan 1950). other larval habitats of this species were expressed as small shallow and shaded stream, with vegetation in maragheh area in northwestern part of iran (dow 1953). nikookar et al. (2015) had found the larvae of an. claviger in permanent and stagnant water environments with vegetation and clay and stone substrate. in parallel, macan (1950) had found an. claviger larvae in semi sunlight springs, and slow running pools of water in iraq and western iran. dow (1953) reported the larval habitat of this species in shallow and small pools with little vegetation. in this present study, an. maculipennis larvae were mainly collected from permanent and slow running water environments with vegetation. the other characteristics of larval habitat of the species were found as clear water, sunlight situations, and habitats with sandy substrate. the presence of an. maculipennis larvae was reported in permanent, transparent, semi-shady natural larval habitats with vegetation and cement or stone substrate (nikookar et al. 2015). in parallel, the larva of this species was found in habitats with gravel substrate, sunny springs, and pools with stagnant water (azari-hamidian 2007b, azari-hamidian et al. 2011). at least 12 palearctic members of an. maculipennis complex were reported including an. atroparvus, an. beklemishevi, an. labranchiae, an. maculipennis, an. martinius, an. melanoon, an. messeae, an. sacharovi, an. persiensis, an. daciae, an. lewisi and an. artemievi (white 1978, ribeiro et al. 1988, linton et al. 2002, sedaghat et al. 2003ab djadid et al. 2007). dow (1953) had reported the occurrence of an. subalpinus (in the now "subalpinus" is http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 218 http://jad.tums.ac.ir published online: may 27, 2017 synonym of "melanoon") in sari, babolsar, mazandaran province. saebi (1987) also cited the occurrence of an. messeae and an. melanon from guilan province, and an. sacharovi and an. hyrcanus in golestan province. this species has been identified in guilan province (azarihamidian et al. 2004), mazandaran and golestan provinces (zaim et al. 1986). anopheles maculipennis s.l. associated with an. hyrcanus, an. claviger from mazandaran province previously (nikookar et al. 2015). an. sacharovi was cited in mazandaran and golestan provinces (sedaghat et al. 2003). presently, five members of an. maculipennis complex have been reported. anopheles maculipennis and an. sacharovi were identified based on the characteristics of eggs, larvae and adults as well as through the pcr technique, an. messeae, an. persiensis and an. melanoon were identified based on pattern of eggs surface and pcr technique (sedaghat and harbach 2005). anopheles maculipennis was reported more in rice fields, while an. sacharovi was found more in mountainous areas (mousa-kazemi et al. 2000, sedaghat et al. 2003). although, it is difficult to find the difference between an. maculipennis and an. sacharovi species in larval stages, but in our research an. maculipennis species was identified based on the azari-hamidian and harbach (2009)'s systematic key. anopheles superpictus was reported as one of the main malaria vectors and salivary infection was found as ranging from 0.65 to 4.6% (manuchehri et al. 2003). this species with an. maculipennis was considered as the malaria vector during the outbreak of the diseases which had occurred in azerbaijan at the borderline of the country, armenia, and turkey countries in 1990. however, after the independence of the southern republics of the former soviet union, iran was threatened by imported malaria cases (oshaghi et al. 2011). in present study, an. superpictus was collected in natural habitats. the characteristics of larval habitat of this species were mainly in permanent water without vegetation, clear water, semi-sunlight and shaded habitats with muddy substrate. zolotarev (1945), dow (1953) and nikookar et al. (2015) have reported the occurrence of this species in mazandaran province. anophles superpictus larvae was found in permanent, stagnant, with muddy substrate, transparent water, semi-shady, natural with vegetation habitats in neka county, northern iran (nikookar et al. 2015). moreover, azarihamidian et al. (2011) have stated its presence in stagnant, transient, muddy substrate, full sunlight water with vegetation in natural habitats in guilan province, northern iran. further support for our results comes from some previous studies carried out in kermanshah and kurdistan provinces, western iran (moosakazemi et al. 2015, macan 1950), zarrin-shahr and mobarakeh areas of isfahan province, center of iran (mousa-kazemi et al. 2000a), ardabil province, northwestern iran (yaghoobiershadi et al. 2001), rasht county of guilan province, northern iran (azari-hamidian et al. 2002b) and in iranshahr, southeastern part of the country (ghanbari et al. 2005). three genotypes named x, y, and z within an. superpictus during the molecular study were reported in iran (oshaghi et al. 2008). by now, there are no reports about the genotypes of this species in golestan province. however, it needs to be studied in the future. in our study, an. hyrcanus was found as the dominant species in larval habitats followed by an. maculipennis in kalaleh county. an. hyrcanus larvae were collected from habitats with varieties of 95.5% permanence, 88% stagnant water, and were only collected in clear water, natural habitats without vegetation. different sunlight situations characterized the larval habitats of this species. the current species prefer the permanent habitats to temporary larval habitats. moreover, an. pseudopictus prefers the habitats with slow running water while an. hyrcanus prefers the stagnant water habitats. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 219 http://jad.tums.ac.ir published online: may 27, 2017 three species of the hyrcanus group in cluding an. hyrcanus, an. peditaeniatus and an. pseudopictus have been reported in iran (azari-hamidian et al. 2006, azari-hamidian 2007a). several species of an. hyrcanus group have been reported as the malaria vectors in the oriental and palearctic regions, however, this species was reported as a potential malaria vector based on the molecular study in guilan province (djadid et al. 2009). shahgudian (1960) made mention of an. nigerrimus species as a variety of an. hyrcanus in its systematic key, moreover, identification of these species is very difficult and is based on this systematic key and this old record needs to be verified. glick (1992) published keys for the identification of female anophelines of southwest asia, which mentioned the females of the hyrcanus group, however these characters were not reliable for distinguishing the females of an. peditaeniatus from other species of hyrcanus group. one character distinguished the larvae of an. hyrcanus from those of an. pseudopictus (darsie and samanidou-voyadjoglou 1997). an. hyrcanus and an. pseudopictus were reported as a single species in southeastern france based on pcr technique (ponçon et al. 2008). ‘the systematics of the iranian species of the hyrcanus group’ was published by azari-hamidian and harbach in 2009. in our study, cx. pipiens larvae were identified in this area based on the larval seta 1 of abdominal segments iii and iv. this character was observed as double seta in all of cx. pipiens larvae samples and this confirmed the occurrence of cx. pipiens species. this species is cosmopolite and is distributed in all parts of the country (zaim 1987, nikookar et al. 2015). culex pipiens was predominant in larval (27.6%) and adult (28.9%) stages. further support to this result also came from previous study, cx. pipiens was reported as the dominant species in yazd province (dehghan et al. 2010). culex pipiens was reported as the predominant species in guilan province and dominant species in isfahan province (mousa-kazemi et al. 2000a, azari-hamidian 2007b). larval habitats of this species were diverse in kalaleh county but all samples of this species were collected in natural habitats. similarly, zaim (1987) cited the fresh water environments such as marshes, channels and artificial irrigation and rain-filled pools and drums as the main larval habitats of cx. pipiens. distribution and abundance of cx. pipiens species were in close relationship with economic activities and development of new territories (vinogradova 2000). underground train systems, coal mines, drains, wells, septic tanks, abandoned and variety of the natural and artificial habitats were reported as the main larval habitats of cx. pipiens (horsfall 1955, zaim 1987, harbach 1988). in our study, cx. pipiens larvae were collected only in natural larval habitats, further support of this result came from the previous study, dow (1953), lotfi (1970, 1976), yaghoobi-ershadi et al. (1986), zaim (1987) and azari-hamidian (2007b) found this species mostly in natural habitats. moreover, mousa-kazemi et al. (2000), azari-hamidian et al. (2002b) have reported the presence of these species from rice fields and man-made habitats respectively. water and sewage wells as well as house ponds were reported as the main habitats of cx. pipiens in cities (golestani 1967, lotfi 1976, zaim 1987, dehghan et al. 2010, 2011). in our study, cx. theileri was found as predominant species at larvae and adult stages. this species was known as one of the predominant species in northwest of iran (azari-hamidian et al. 2009). moosa-kazemi et al. (2010) had reported that the cx. theileri species was the second dominant species in kurdistan province followed by cx. pipiens. larval habitats of this species in our research were found as natural habitats, and permanent habitats with vegetation such as irrigation ditches, different pools, open cisterns, dis http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 220 http://jad.tums.ac.ir published online: may 27, 2017 used wells seepage water and swamps (har bach 1988). lotfi (1970, 1976) had reported that the predominant species were in larval habitats such as grassy and ponds, rice fields, seepages and agricultural pools in iran. in our study, cx. theileri larvae was collected from different types of habitats with 73% permanence, 64% slow running water, turbidity of 64% and 87% in natural habitats. the breeding place preferences of this species were full and partial sunlight habitats. in parallel, azari-hamian (2007b) had reported different types of larval habitats. dehghan et al. (2010) reported that the larval habitats of this species were swam plants, permanent, and with vegetation outside or inside water environments in hamadan province. dow (1953) reported that the larvae of cx. theileri were accumulated in the pit and irrigation channels and water intakes and shallow rivers and river beds. larval habitats of the species have been found as algae, water intake and water pits, and a crock pot, household pits along the river margins, floating and submerged plants (horsfall 1955). natural and artificial habitats of cx theileri were cited in the country (dow 1953, yaghoobiershadi et al. 1986). mousa-kazemi et al. (2000) also discovered the presence of cx theileri larvae in the rice field, however zaim (1987) had reported their presence in natural habitats. natural habitats such as pools were reported as the main habitats of this species (azari-hamidian et al. 2002b, 2007b). in this study, more samples of cx. hortensis larvae were collected from natural habitats. moreover, this species prefers permanent and stagnant water habitats. this species is associated with an. superpictus and oc. geniculatus, both of which prefer habitats without vegetation so that 66% of the larval samples of this species were collected in habitats without vegetation. culex hortensis species were found more in muddy beds and habitats with full and partial sunlight. horsfall (1955) had reported that the main larval habitats of cx. hortensis were algal mats, seeps, brackish pools, domestic containers, and cement channel. this species was reported in pools in the river beds, the irrigation ditches, small, spring pools of the river banks and shallow pools (dow 1953). natural habitat was reported as the main habitat of this species (zaim 1987, azarihamidian 2007b). this species was collected in seepages and agricultural water storage pools (lotfi 1976). in our study, cx. perexiguus was collected and reported at the first timein golestan province. more of these species were collected in natural habitats. mousa-kazemi et al. (2000a) have reported the occurrences of cx. pipiens, cx. theileri and cx. perexiguus in zarrin-shahr and mobarakeh areas of isfahan province. by now, 5 species of culiseta have been reported in iran and they included: cs. allotheobaldia longiareolata, cs. culisetaalaskaensis, cs. culiseta) annulata, cs. (culicella) morsitans and cs. (culiseta) subochrea (azari-hamidian 2005). in our studied species, cs. logiareolata and cs. subochrea were collected. there is little information available about the ecology of the culiseta species. larval habitats of cs. longiareolata contained organic materials with high abundance in artificial pits of yazd province (dehghan et al. 2010). association of this species with cx. pipiens was found in the larval habitats which were contaminated with soil and wastewater in drinking troughs made of cement, a place to store water for animals and livestock in yazd province (dehghan et al. 2010). in our study, cs. longiareolata larvae were abundant, followed by cx. pipiens and more were collected from permanent, stagnant and full sunlight habitats with vegetation. in contrast, the larval habitat of this species was without vegetation in hamadan province (dehghan et al. 2011). this species has high adaptability to different ecological http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 221 http://jad.tums.ac.ir published online: may 27, 2017 conditions. more cs. subochera were collected in natural and temporal habitats. similarly, these species were collected in the same larval habitats (zaim 1987) while all the other larvae of the species were collected from permanent habitats in hamadan province. moreover, cs. subochrea had low abundance in this area. this species has been identified in various studies in iran. similarly, this species was collected as the lowest species in hamedan province (in terms of abundance), located in the western part of iran. they prefer the habitats with turbid water to clear water and full sunlight habitats to shaded habitats. in hamadan area of western iran, the larval habitat of this species was reported as the same larval habitat in our study in turbid to clear water and full sunlight habitats. unlike the previous study, we found more larvae in shaded habitats (dehghan et al. 2011). in our study, 3 species of ochlerotatus including oc. caspius, oc. echinus and oc. geniculatus were collected; nikookar et al. (2015) had reported the occurrence of oc. echinus and oc. geniculatus in tree hole habitats in northern part of iran. in our study, all the current three ochlerotatus species preferred the permanent habitats with slowrunning water and muddy bed. in addition, oc. echinus preferred the clear water habitats while oc. caspius preferred the larval habitats with turbid water and oc. geniculatus was found in habitats without vegetation. ochlerotatus caspius was reported as a po tent vector for rift valley fever viruses as well as dirofilaria immitis in the world (azarihamidian 2006). this species loves feeding more on mammals and human and was found more in their dwellings (azari-hamidian 2006). in our research, this species comprised 7.8% of the larval collection and 9% of adult catches by animal baited trap collection method in various areas of golestan province, northeastern iran. further support to this result comes from some previous studies carried out in kermanshah and kurdistan provinces, western iran (mousa-kazemi et al. 2015), zarrin-shahr and mobarakeh areas of isfahan province, center of iran (mousa-kazemi et al. 2000a), guilan province, northwestern iran (azari-hamidian et al. 2002a), bushehr province, southern iran (dow 1953), eastern part of the country (minar 1974) and various parts of iran (zaim 1987). in kurdistan province, cx. theileri was next in abundance after this species (moosakazemi et al. 2010). ochlerotatus echinus was distributed in the mediterranean region, north of africa and southern europe. in our study, out of the 9% total larvae collected from adult catches using animal baited traps in various areas of golestan province-northern iran, 7.8% were oc. echinus. in parallel, this species was reported in mazandaran province (zaim 1987, nikookar et al. 2015). this species has been reported in guilan province, northern iran (azari-hamidian et al. 2002a). ochlerotatus geniculatus was distributed in the palearectic region, europe, north of africa and southeast asia. in our research, this species comprised 1.5% of the larval collection and 3.9% of adult catches by animal baited traps in various areas of golestan province, northern iran. this species was reported for the first time in mazandaran province, northern iran (gutsevich 1943). this species has been reported in guilan province, northern iran (azari-hamidian et al. 2002a). conclusion the present investigation indicates some biological characteristics of mosquitoes in the northern areas of iran. because of diversity in larval habitats and variety in species of mosquito in the county, results of this study could be useful in vector control programs. several species of anopheles were found in a lot of areas in the county. the larval habitats of anopheles were found and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 211–225 a sofizadeh et al.: larval habitats … 222 http://jad.tums.ac.ir published online: may 27, 2017 reported in permanent habitats with clear water. besides, the larvae of an. superpictus and an. maculipennis species which are the main vectors of malaria in the north of iran were reported in habitats with vegetation, under full and partial sunlight situations and muddy and sandy substrates that are important in larviciding programs. bionomic studies of other mosquitoes need to be more rigorously studied in the future. also, more studies should be obtained in order to complete information about of bionomics of mosquitoes in other parts of iran. acknowledgements the authors are grateful to the people of villages in kalaleh county for their kind cooperation during the study. the authors declare that there is no conflict of interests. references azari-hamidian sh, joeafshani ma, mosslem m, rassaei mr (2002a) mosquitoes of the genus aedes (diptera: culicidae) in guilan. j med fac guilan univ med sci. 11(43): 29–42 (persian). azari-hamidian s, joeafshani ma, mosslem m, rassaei ar (2002b) taxonomic survey of mosquitoes 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characteristics of iranian culicinae. j am mosq control assoc. 3 (4): 568–573. zolotarev ek (1945) anopheles maculipennis of northern iran. med parazitol (mosk). 14(2): 50–57 (russian). http://jad.tums.ac.ir/ j arthropod-borne dis, december 2017, 11(4): 453–462 y salim-abadi et al.: baseline susceptibility of … 453 http://jad.tums.ac.ir published online: december 30, 2017 original article baseline susceptibility of filarial vector culex quinquefasciatus (diptera: culicidae) to five insecticides with different modes of action in southeast of iran yaser salim-abadi 1,2, mohammad asadpour 1, iraj sharifi 3, alireza sanei-dehkordi 4,5, mohammad amin gorouhi 6, azim paksa 7, zohre tayyebi 8, *abbas aghaei-afshar 3 1department of health services and health promotion, school of health, rafsanjan university of medical sciences, rafsanjan, iran 2pistachio safety research center, rafsanjan university of medical sciences, rafsanjan, iran 3leishmaniasis research center, kerman university of medical sciences, kerman, iran 4department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 5infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran 6department of medical entomology and vector control, school of health, kerman university of medical sciences, kerman, iran 7department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 8student research committee, rafsanjan university of medical sciences, rafsanjan, iran (received 11 may 2017; accepted 19 sep 2017) abstract background: culex quinquefasciatus (diptera: culicidae) is an important vector for many human diseases. the aim of this study was to evaluate the susceptibility level of larval and adult stages of cu. quinquefasciatus to different groups of who recommended insecticides for vector control. methods: larval stages of the culex mosquitoes were collected from their natural habitats in rafsanjan county at kerman province, southeast of iran in 2016. insecticide susceptibility status of adult female cx. quinquefasciatus against ddt (4%), deltamethrin (0.05%), malathion 5%, and bendiocarb (0.1%) were determined using who standard insecticide susceptibility test. additional test was carried out to determine the susceptibility status of larvae of cx. quinquefasciatus to temephos. bioassay data were analyzed by probit program. results: cx. quinquefasciatus adults showed resistance to all four groups of the tested insecticides according to the who criteria for resistance evaluation. the lethal concentrations for 50% mortality (lc50) and 90% mortality (lc90) of temephos against cx. quinquefasciatus larvae were 0.18mg/l and 0.78mg/l, respectively. this finding also confirms resistance to temephos based on the who recommended instructions for resistance evaluation. conclusion: resistance to all groups of the tested insecticides should be considered for future vector control investigations in the study area. keywords: susceptibility status, resistance, insecticide, culex quinquefasciatus, iran introduction the southern house mosquito, culex quinquefasciatus (diptera: culicidae) is an important vector for many human diseases. this species plays a crucial role in the transmission of some important pathogen such as wuchereria bancrofti, dirofilaria immitis, plasmo dium relictum, sindbis virus, west nile virus, equine encephalitis, st louis, oropouche and rift valley fever which are today among the major public health problems worldwide (1–8). wastewater and sewage system are important breeding places for culex mosquitoes. *corresponding author: dr abbas aghaei-afshar, e-mail: afshara2@yahoo.com j arthropod-borne dis, december 2017, 11(4): 453–462 y salim-abadi et al.: baseline susceptibility of … 454 http://jad.tums.ac.ir published online: december 30, 2017 constant exposure of cx. quinquefasciatus to the high organic content of wastewater including detergents, different groups of insecticides, industrial pollutants, and oil compounds can lead to the development of resistance in mosquito larvae against insecticides and larvicides (9–11). in recent years, increasing level of resistance to various groups of insecticides has been a major barrier to the success of vector control programs. many studies have reported high level of resistance in cx. quinquefasciatus to many groups of insecticides (12–14). culex quinquefasciatus is an important member of cx. pipiens complex wildly distributed worldwide (2, 5, 15). in iran, the resistance status of cx. pipiens complex against different groups of insecticides was indicated the development of resistance in the members of this species, including cx. quinquefasciatus, during the past quarter-century: development of resistance to most of the organochlorine insecticides including ddt (dichloro diphenyl trichloroethane) (6, 11, 16–20). resistance to pyrethroid insecticides such as lambda-cyhalothrin, deltamethrin, and cyfluthrin (6, 16, 17, 19). resistance to the carbamate insecticides propoxur and bendiocarb (11, 16, 17) and relative resistance to malathion organophosphates insecticides (11, 16). moreover, resistance of the larvae of cx. pipiens complex to temephos has recently been reported for the first time in iran (9). there was no study on monitoring the susceptibility level of cx. quinquefasciatus to insecticides in rafsanjan county at kerman province, southeastern iran. we aimed to determine the susceptibility status of cx. quinquefasciatus against insecticides in this area. materials and methods study area this study was carried out in rafsanjan county at kerman province, southeastern iran. the county located at latitude 30°30'n and longitude 55°40'e, with a population of 300000 in 2015 (fig. 1). bioassay procedure larvae of cx. quinquefasciatus were collected from larval habitats in rafsanjan county in 2016, and all sample were transferred to laboratory and reared at 27 °c and 65±5% relative humidity using a 12h light/ 12h dark photoperiod. bioassay tests were carried out using who test kits on adult mosquitoes (21). the following diagnostic concentrations of insecticides were tested: ddt 4%, lambdacyhalothrin 0.05%, malathion 5%, and bendiocarb 0.1%. tests were carried out on3 to 5-day-old unfed females. batches of 25 females were exposed to insecticide-impregnated papers at different exposure times. two replicates of 25 adult mosquitoes (3 to 5-day-old unfed females) were considered as controls with untreated papers for each different exposure time. the lethal time for 50% mortality and 90% mortality (lt50 and lt90) among the mosquitoes was calculated using log-probit software according to finney’s formula (22, 23). larvicide susceptibility tests were carried out on late 3rd to early 4th stage larvae to determine larval susceptibility to temephos using who standard kit (1.25, 6.25, 31.25 and 156.25 mg/l concentrations), according to who instructions (22, 23). each test consisted of four replicates with 25 larvae each in glass beakers containing 250ml of distilled water and the specified insecticide concentration. two replicates of 25 untreated larvae were maintained as controls. after 24h exposure period, larval mortality was calculated the lethal concentrations for 50% mortality and 90% mortality (lc50 and lc90) were calculated by probit analysis (24). in both adult and larval susceptibility testing, mortality rate in the test samples was corrected using abbott formula (25), when the mortality rate of control was between 5% and 20%. results the mortality rate (mr) in adult cx. quinquefasciatus mosquitoes exposed to four different groups of insecticide-impregnated paj arthropod-borne dis, december 2017, 11(4): 453–462 y salim-abadi et al.: baseline susceptibility of … 455 http://jad.tums.ac.ir published online: december 30, 2017 pers are shown in table 1 and 2. lambdacyhalothrin with lt50= 25 minute and lt90= 74min and ddt with lt50= 139min and lt90= 227min had the lowest and highest lt50 and lt90 values, respectively (table 1). culex quinquefasciatus is resistant to malathion and bendiocarb and candidate of resistance to lambda-cyhalothrin and ddt based on the criteria for insecticide resistance described by who (table 2). the mortality rate in the mosquitoes at one-hour exposure to the insecticides calculated after 24h recovery period has been summarized in table 2. malathion had a mortality rate of 80% (mr= 80%), bendiocarb 88%, lambda-cyhalothrin and ddt 90% each, the mortality rate of ddt was calculated after 4h exposure time instead of 1h (table 2). the regression line of different concentration is shown in fig. 2. the mortality rate of each concentration of temephos is shown in table 3. the mortality rate of temephos ranged from 3% to 100%. moreover, using finney’s method, we calculated lc50 and lc90 for cx. quinquefasciatus that were 0.18 and 0.78 ppm, respectively (table 4). the regression line of the different concentrations of temephos is shown in fig. 3. table 1. probit regression line parameters of culex quinquefasciatus exposed to different groups of insecticides in rafsanjan city, southeastern iran, 2016 insecticides a b ± se lt50, 95% c.i. (min) lt90, 95% c.i. (min) x2 (df) p value lambdacyhalothrin 0.05% -3.80 2.72 ± 0.27 22 58 5.84(2) >0.05 25 74 29 102 malathion 5% -3.89 2.55 ± 0.28 29 80 5.7 (2) >0.05 33 106 39 159 bendiocarb 0.1% -3.90 2.72 ± 0.28 23 63 5.49 (2) >0.05 27 79 31 112 ddt 4% 13.01 6.06 ± 0.55 129 204 3.04 (2) >0.05 139 227 172 260 a= y-intercept, b= the slope of the line, se= standard error, ci= confidence interval, x2= heterogeneity about the regression line, df= degree of freedom, p> 0.05= represents no heterogeneity in the population of tested mosquitos. table 2. susceptibility level of culex quinquefasciatus exposed to different groups of insecticides in rafsanjan county, southeastern iran, 2016 insecticides mr ± eb* resistance status** lambda-cyhalothrin 0.05% 90 ± 2 rc malathion 5% 80 ± 3 r bendiocarb 0.1% 88 ± 3 r ddt 4%*** 90±2 rc *mortality rate± errorbar **rc resistance candidate ***after 4 h exposure period j arthropod-borne dis, december 2017, 11(4): 453–462 y salim-abadi et al.: baseline susceptibility of … 456 http://jad.tums.ac.ir published online: december 30, 2017 fig. 1. the geographical location of rafsanjan county in kerman province, iran fig. 2. regression lines of culex quinquefasciatus exposed to different group of insecticides in rafsanjan city, southeastern iran, 2016 j arthropod-borne dis, december 2017, 11(4): 453–462 y salim-abadi et al.: baseline susceptibility of … 457 http://jad.tums.ac.ir published online: december 30, 2017 table 3. mortality rate in culex quinquefasciatus larvae at who standard concentrations of temephos in rafsanjan county, southeastern iran, 2016 concentration (ppm) replicates no. of tested larvae no. of mortality mortality rate (%) observed mortality probit expected mortality probit 0.005 4 100 3 3 3.119 1.762 0.025 4 100 6 6 3.445 3.199 0.125 4 100 10 10 3.718 4.636 0.625 4 100 100 100 7.576 6.073 control 2 50 0 0 table 4. probit regression line parameters of temephos against culex quinquefasciatus larvae in rafsanjan county, southeastern iran, 2016 a b lc50, 95% ci (ppm) lc90, 95% ci (ppm) x2 (df) p-value 1.49 2.05 0.18 0.78 190.76(2) <0.05 fig. 3. mortality regression lines of different concentrations of temephos against culex quinquefasciatus in rafsanjan city, southeastern iran, 2016 discussion the present study provides evidence of resistance to four different classes of insecticides according to the current who criteria for insecticide resistance evaluation. the mortality rate was interpreted as follows: higher than 98% was considered as susceptible, less than 90% indicated resistance, and from 90% to 97% was defined as resistance candidate. for the resistance candidate category (90–97% mortality rate), additional investigation is needed for the confirmation of resistance (21). although both lambda-cyhalothrin and ddt have mortality rate of 90% each which classifies them as j arthropod-borne dis, december 2017, 11(4): 453–462 y salim-abadi et al.: baseline susceptibility of … 458 http://jad.tums.ac.ir published online: december 30, 2017 potential candidate for resistance according to the who criteria. this finding can be useful in future vector control programs and investigations in order to prevent the development of resistance to both insecticides. previous studies have also reported cx. pipiens resistance to different classes of insecticides in different geographical regions of iran, resistance to ddt, lambda-cyhalothrin, deltamethrin and cyfluthrin in tehran, capital of iran (6, 11, 20). resistance to ddt in the north of iran (18), resistance to ddt, lambda-cyhalothrin and propoxur in a dirofilariasis foci in the northwest of iran (18), resistance to ddt, propoxur, cyfluthrin and lambda-cyhalothrin and tolerance to deltamethrin in a malaria endemic area in the southeastern part of iran (17), and resistance to deltamethrin and ddt in the northwestern part of iran (19). the use of pesticides in agricultural sector can lead to the development of resistance to insecticides in medically important vectors including cx. quinquefasciatus (13, 26–28). there is a growing concern over the development of multiple insecticide resistance mechanisms in medically important arthropods that is a major problem in vector control (15, 29–31). over the last fifty years, resistance to insecticides has been a growing concern. resistance of mosquitoes to ddt was first reported in 1949 (32, 33). however, resistance to organophosphorus insecticides in cx. quinquefasciatus was first reported in 1961 (34), and to date, there have been several reports on resistance to various classes of insecticides in cx. quinquefasciatus. this species is now quite resistant to some insecticides such as ddt and malathion such that it does not exhibit mortality at one-hour exposure and 24h recovery period (35). in the present study, in addition to adult susceptibility test, susceptibility of the larvae of cx. quinquefasciatus to temephos was evaluated according to who standard method. we observed mortality rate ranged between 3% and 100%. in another study, laboratory evaluation of the susceptibility of anopheles stephensi larvae collected from kazeroun, south of iran and cx. pipiens larvae collected from tehran, capital of iran to temephos insecticides was carried out. lc50 values of both species were the same that is similar to our findings. mosquito larvae in all the three geographical regions mentioned above have become resistant to temephos (9). in other countries were reported resistance of adult and larval stages of cx. quinquefasciatus to different groups of insecticides and larvicides. in a study conducted in morocco, bioassay results showed that cx. pipiens is resistant to temephos that is consistent with our results (36). in kuala lumpur (malaysia), cx. quinquefasciatus larvae were found to be highly resistant to malathion that was similar to the adults (35). in central tunisia, resistance to temephos in cx. quinquefasciatus larvae have been reported (37) moreover, resistance of cx. quinquefasciatus larvae to malathion, permethrin, and resmethrin has been reported in florida (usa) (38). the use of pesticides in agriculture could play a role in the development of resistance to insecticides as well as larvicides in cx. quinquefasciatus in rafsanjan county (9, 11). owing to the emergence of cx. quinquefasciatus resistance to different classes of insecticides and larvicides, using some biological control agents such as bacillus thuringiensis (a gram-positive, soil-dwelling bacterium) and gambusia affinis (larvivorous fish) can provide an efficient control strategy (39–41). moreover, use of natural products derived from some plants such as bunium persicum and zhumeria majdae that have no adverse effects on the environment and humans can be suitable and alternative control approach for larvae as well as adult cx. quinquefasciatus mosquitos (42–46). https://en.wikipedia.org/wiki/gram-positive https://en.wikipedia.org/wiki/bacterium j arthropod-borne dis, december 2017, 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http://www.tandfonline.com/author/mohammadi-azni%2c+sadegh http://www.tandfonline.com/author/mohammadi-azni%2c+sadegh 2. dr oshaghirtl iranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 8 original article blood meal identification in field-captured sand flies: comparison of pcr-rflp and elisa assays n maleki-ravasan1, *ma oshaghi 2, e javadian 2, y rassi 2, j sadraei 1, f mohtarami 2 1department of medical parasitology and entomology, college of medical sciences, tarbiat modares university, tehran, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 8 jun 2009; accepted 28 july 2009) abstract background: we aimed to develop a pcr-rflp assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of iran. in addition, the utility of pcr-rflp was compared with elisa as a standard method. methods: this experimental study was performed in the insect molecular biology laboratory of school of public health, tehran university of medical sciences, iran in 2006-2007. for pcr-rflp a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the pcr products by hae iii enzyme. results: the pcr-rflp and elisa assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. additionally, pcr-rflp assays could reveal specific host dna as well as the components of mixed blood meals. results of pcr-rflp assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%). conclusion: the results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. the advantages and limitations of pcr and elisa assays are discussed. keywords: leishmaniasis, sand flies, blood meal, pcr, elisa, iran introduction leishmaniasis is caused by single-celled parasites of the genus leishmania and is spread to humans through the bite of the sand fly. there are three main forms of leishmaniasis: cutaneous, mucocutaneous and visceral, each form is caused by different species of leishmania (who 1990). leishmaniasis affects over 12 million people distributed in 88 countries (sakthianandeswaren et al. 2009). every year, new cases amount to more than 2 million (sakthianandeswaren et al. 2009). nearly one tenth of the world population is at risk of infection. these figures have led to the who to consider leishmaniasis as one of the most serious diseases of the world. visceral leishmaniasis (vl) or kala-azar is the most severe manifestation of the disease and results fatal when is not treated promptly. there are 500,000 new cases of kalaazar every year and it is endemic in asia, europe, and south america (who 2002). the parasite is transmitted by sand flies, particularly members of the genera phlebotomus spp. and lutzomyia spp. which are found in a wide range of habitat, from desert to tropical rain forest. sand flies take blood meals from a wide variety of hosts, including human, livestock, dogs and chickens (lainson and rangel 2005). the human blood index (hbi, proportion of blood meals of a haemophagous insects population obtained from man) is relevant corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir iranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 9 to epidemiological assessment and to the modification of measures to interrupt any vector borne diseases transmission since the vectorial capacity of the vector varies as the square of the hbi (macdonald 1957, garrett-jones 1964). detailed knowledge of the feeding behavior of sand flies on their various vertebrate hosts is considered to be a prerequisite for a successful sand flies and leishmaniasis control program. identification of the blood meals of haematophagous insects to date has largely depended on serological techniques such as the precipitin test, latex agglutination test and the enzyme-linked immunosorbent assay (elisa) (boorman et al. 1977, washino and tempelis 1983, beier et al. 1988, gomes et al. 2001, mwangangi et al. 2003). although these methods have yielded important information on the identity of the vertebrate hosts of many bloodfeeding arthropods, they are time-consuming and lack sensitivity. so, an alternative method may be desired in laboratories not set up to perform immunologic assays, or if samples are already in the form of extracted dna. the pcr-based identification of arthropod blood meals provides a convenient alternative for laboratories using primarily dna-based techniques, and may be necessary when the study design already requires the use of individual dna extractions for multiple purposes such as species confirmation, determination of infection status for various pathogens, and vector population genetic studies. furthermore, engorged specimens collected in the field may be preserved dry, stored for long periods of time, and tested at facilities that may be physically distant from the point of collection. pcr-based identification of vertebrate host blood meals is a potentially convenient alternative, which has already been performed on several vectors including ticks (pichon et al. 2003, estrada-peña et al. 2005), triatomine bugs (bosseno et al. 2006, pizarro et al. 2007) and mosquitoes. pcr based on primers designed from multiple alignments of the mitochondrial cytochrome b gene have identified avian and mammalian hosts of various species of mosquito (ngo and kramer 2003, kent and norris 2005, molaei et al. 2006, kent et al. 2006). pcr-rflp cytochrome b analysis was also used to identify the origin of blood meals in the tick ixodes ricinus (kirstein and gray 1996), tsetse flies (steuber et al. 2005) and the mosquito anopheles stephensi (oshaghi et al. 2007a, and b). in their study, the cyt b sequences showed sufficient inter-specific polymorphism to distinguish between human, cow, sheep, chicken, and guinea pig hosts. until recently sand fly host identification by blood meal analysis had been limited to serological studies using elisa (gomez et al. 1998, agrela et al. 2002, bongiorno et al. 2003, svobodová et al. 2003, marassá et al. 2006, rossi et al. 2008), counter immunoelectrophoresis (morsy et al. 1993), agarose gel diffusion (srinivasan and panicker 1992), precipitin test (tesh et al. 1971, tesh et al. 1972, javadian et al. 1977, morrison et al. 1993, nery et al. 2004, afonso et al. 2005) and a more laborious histological technique (guzman et al. 1994). the first pcr-based method using the prepronociceptin gene has been recently described (haouas et al. 2007). in iran, most studies for identification of sand fly blood meal have been based on elisa (javadian et al. 1977, azizi et al. 2006, rassi et al. 1999, 2005, maleki 2007). in the present study, we adapted a pcr protocol designed by kent and norris (2005) and developed this with sequence analysis of major vertebrate hosts in north of iran in order to find putative restriction enzyme to develop host specific rflp patterns for dna sources in sand flies. also the elisa method was applied to compare the results of these two methods for blood meal identification. materials and methods study area the study was conducted in germi district, ardabil province, in northwestern iran. this region is 1,490 meters above the sea iranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 10 level. the total population of germi was approximately 123,000 in 2002. the weather is hot (up to 40 °c) in summer and cold (less than −20 °c) in winter. the warm season is short (mid-may to mid-september). annual rainfall is approximately 114 mm. the main occupations of the population are farming and raising animals. on the basis of available epidemiologic data obtained from the ministry of health (moh), local health authorities, and medical centers in germi district, villages with higher incidence of vl were selected for the study. three primary villages (kalansora, shah-tapehsi, and hamzeh-khanloo) were selected and analyzed in an entomologic survey. three secondary villages (hasi-kandy, sarv-aghaji, and ghasem-kandy) were also study periodically. sample collection sticky traps were used to collect sand flies from human and animal dwellings, rodent and fox burrows, under bridges, and on the shores of rivers. traps were set at dusk and flies were collected at dawn. a total of 150– 200 sticky traps were set each day in each village. sample collection began in early july 2008 and continued until late september 2008 when sand fly activity was reduced sharply. sampling was carried out every 3 d in the primary villages and 1-3 times in the secondary villages. trapped sand flies removed from stick papers with needles, washed with absolute ethanol, and transferred into micro tubes filled with 96% ethanol. tubes were kept frozen (-20 °c) until species identification and dna extraction. of the many samples collected in the region, 400 blood-fed females were randomly selected for detection of blood meal. identification of sand fly species in the laboratory, samples were washed with detergent and double-distilled water, and heads and terminal abdomens of females were removed and mounted with pouri solution on glass slides for diagnosis. for males, only heads were removed and mounted with pouri solution on glass slides. species were identified by using specific morphologic keys. middle parts of female sand flies were placed in micro tubes and kept frozen (−20 °c) until dna extraction. analysis of blood meals in sand flies four hundred blood fed female sand flies were randomly selected for blood meal identification. sand flies were selected on the basis of location and capture sites to obtain a representative sample of sand flies in a region. samples were divided randomly into two groups of 200 specimens; each group was analyzed by pcr-rflp or elisa method. extraction of dna from blood meals in sand flies dna extraction from blood-fed female, and male, unfed female sand flies and water (used as a negative controls), and human and cow (used as a positive controls) was conducted according to the procedure of steiner et al. (1995). samples were individually disrupted by mechanical homogenization in buffer containing 10 mm tris-hcl, ph 8.0, 312.5 mm edta, 1% (w/v) sodium lauryl sarcosine, and 1% polyvinyl pyrolidone. homogenates were heated to 90 °c for 20 min and chilled on ice for 5 min. samples were centrifuged at 13,000 rpm for 5 min at room temperature. the supernatant was removed and diluted 20fold in 10 mm tris-hcl, ph 8.0, 1 mm edta. pcr amplification of the mtdna cyt b gene two regions of the mtdna cyt b gene were amplified for host blood meal identification of the blood-fed female specimens. for identification of human blood meals a portion (358 bp) of the cyt b gene was amplified and digested with xhoi enzyme as previously explained by oshaghi et al (2006a). the sequence of the primers used were 5΄-ccatccaacatctcagcatgatgaaa-3 ́ (forward) and 5΄-cccctcag aatgatatttgtcctca-3 ́ (reverse) (kocher et al. 1989, boakye et al. 1999). the pcr amplifications were periranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 11 formed in 25 µ l of a solution containing 10 mm tris-hcl, ph 8.3, 50 mm kcl, 1.5 mm mgcl 2, 0.001% gelatin, 200 mm deoxynucleotide triphosphates, 10 pmol of each primer, 1 unit of taq dna polymerase (cinagene, tehran, iran), and 2.5 µ l of dna template solution. samples were incubated at 95 °c for 3.5 min; followed by 36 cycles at 95 °c for 30 s, 58 °c for 50 s, and 72 °c for 40 s; and 72 °c for 5 min. to discriminate animal host blood meals, a second region of the mtdna cytb gene was amplified by using the protocol of kent and norris (2005). the sequences of forward and reverse primers were respectively 5΄-tgaggacaaatatcattctgagg-3 ́ (unfor403) and 5 -́ggttgtcctccaattcatgtta-3 ́ (unrev1025), respectively. primers amplified a 623-basepair region of the cytb gene of vertebrate mtdna. the pcr amplifications were performed in 25 µ l of a solution containing 10 mm tris, ph 8.3, 50 mm kcl, 1.5 mm mgcl 2, 0.01% gelatin, 1.0 mm deoxynucleotide triphosphates, 0.5 units of taq polymerase, 50 pmol of each primer, and 2.5 µ l of extracted dna. samples were incubated at 95 °c for 5 min; followed by 35 cycles at 95 °c for 1 min, 58 °c for 1 min, and 72 °c for 1 min; and 72 °c for 7 min. products were visualized by electrophoresis on 2% agarose gels stained with ethidium bromide. electrophoresis was conducted using a generuler 100-basepair molecular mass marker (cinagene). sequence analysis for selection of restriction enzymes available sequences of the 623 bp for human and probable vertebrate hosts (cow, goat, horse, ass, dog, and other canidae) in the study area were obtained from gene bank and checked for species-specific restriction enzyme sites for each host dna using the nebcutter program (http:/tools.neb.com/nebcutter. twenty eight sequence analyses showed that hae iii did not have a restriction site on human pcr products but it has various specific sites in pcr products for other vertebrates. this enzyme was selected for discrimination of the blood meal sources within sand flies. digestion of pcr products was performed in 25 µl of a solution containing 15 µl of pcr product mixed with 2.5 µl of enzyme buffers and 5 units of the restriction enzyme overlaid with two drops of mineral oil. the mixture was incubated at the temperature recommended by enzyme suppliers. an aliquot (14 µl) of the digestion product was mixed with 6 ml of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol), loaded onto a 2.5% agarose gel, and subjected to electrophoresis. gels were stained with ethidium bromide (2 mg/ml) and the rflp profiles were visualized under ultraviolet light. serological analysis analysis was performed by elisa as described by edrissian et al. (1985) the abdomen of blood fed sand flies was dissected, placed in the well of a micro-elisa plate (nunc, roskilde, denmark), squashed with a glass rod, and eluted with 50 µl of distilled water for 2 h at room temperature. fifty micro liters of coating buffer (carbonate bicarbonate, ph 9.6) was then added to each well. plates were washed three times with phosphate-buffered saline, tween 20, ph 7.2. fifty micro liters of diluted goat anti-human igg conjugated to alkaline phosphates were added onto each well, incubated for 2 h at 37 °c, washed three times with phosphate-buffered saline, tween 20, ph 7.2. one hundred microliters of substrate solution (1 mg/ ml of p–nitrophenyl phosphate [sigma, st. louis, mo] in 10% diethanolamine buffer, ph 9.8, containing 0.5 mmol mgcl 2 and 0.02% nan 3) was added to each well, and incubated in a dark chamber for 30 min at room temperature. two wells that did not contain blood were used as negative controls and two wells that contained human blood were used as positive controls. results were visually assessed, and absorbance was measured with an elisa reader at 405 nm approximately 30 min after addition of substrate solution. the test well result was considered positive if it a yellow color was observed. iranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 12 results pcr-rflp dna isolated from the blood-fed sand flies, positive controls (blood from a human and a cow), and negative controls (water, unfed female sand fly, and a male sand fly) were used as a template in a pcr. most host dnas were amplified, and negative controls yielded no pcr product. this result implied that only host, but not sand fly, dna patterns were detected in amplified specimens. dna sequence analysis showed that the two regions of the mtdna cyt b gene digested with either xho i or hae iii could distinguish human dna in blood of blood-fed sand flies from the dna of blood of other vertebrates. xho i digested only the 358-basepair pcr product of human dna and produced two bands (215 bp and 143 bp) whereas it had no restriction site in the dna of blood of other vertebrates. this finding was confirmed by results of digestion with hae iii on the 623-basepair region of the cyt b gene. this enzyme did not digest the 623 bp fragment of the cyt b gene in dna from human blood, but it digested the equivalent dna fragment from other vertebrates and could distinguish dnas from cow, ass, goat, horse, dog, and other canidae from each other. for example, hae iii produced two fragments of 345 bp and 304 bp from cow dna, two fragments of 552 bp and 70 bp from canidae dna, and two fragments of 170 bp and 453 bp from goat dna (fig. 1). based on pcr-rflp analysis rates of blood meal sources was found to be 54% for cows, 19% mix of humans and cows, 14% mix of dogs and cows, 8% for humans, 3% mix of humans and dogs, and 2% for dogs. on overall, 34% of the female sand flies had human blood or mix of human and an animal blood. except for a few specimens (n=4) that contained p. (adlerius) spp., all blood-fed specimens were p. perfiliewi. details of pcr-rflp analysis are shown in table 1. elisa serologic analysis by elisa on 200 blood-fed sand flies showed that 54 (27%) specimens fed on humans (table 2). fifty one of 54 human blood-fed samples were of p. perfeliewi. the three other seropositive samples in human blood were p. (adlerius) spp. approximately 61% of these blood-fed sand flies were found in either human or animal shelters, which suggested that they were highly endophilic. generally, based on pcr-rflp and elisa assays, it seems that sand flies, particularly p. perfiliewi, although have close relationship with human dwellings in the study area, but are more zoophilic than anthropophilic (73-66% versus 27-34%), and in a descending order, they prefer to feed on cows, humans, and dogs. table 1. host blood meals ingested by female sand flies in germi, northwest of iran detected by pcr-rflp analysis of a 623 bp fragment of the mitochondrial dna cytochrome b (cyt b) gene. collection site sand fly species blood sources n (%) p. perfiliewi human and cow 4 (2) human dwelling p. (adlerius) spp. human 4 (2) human 16 (8) dog 4 (2) cow 108 (54) human and cow 36 (19) human and dog 6 (3) animal dwelling p. perfiliewi dog and cow 28 (14) artificial p. perfiliewi dog 4 (2) total – – 200 (100) iranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 13 table 2. results of elisa assay against human antibody for blood meal identification of fed female sand flies in germi, northwest of iran. capture site sand fly species no. of blood fed (%) no. of human blood (%) human p. perfiliewi p. (adlerius) spp. * 38 (14) 11 (4) 9 (23) 3 (28) animal p. perfiliewi 46 (17) 21 (46) artificial p. perfiliewi p. (adlerius) spp. * p. papatasi p. kandelaki 70 (36) 24 (9) 3 (1) 8 (4) 21 (30) 0 0 0 total 200 (100) 54 (27) *males of p. brevis, p. halepensis, and p. longiductus of the adlerius subgenus are morphologically indistinguishable. fig. 1. electrophoresis of mitochondrial dna cytochrome b gene fragments from blood meals of sand flies digested with haeiii. partial fragments (623 bp) of the cytochrome b gene from vertebrate hosts (human, cow, dog) of sand flies were amplified by a polymerase chain reaction and the products were digested with haeiii. lane 1, mixture of human and dog blood; lane 2, cow blood; lane 3, mixture of cow and dog blood; lane 4, human blood; lane 5, dog blood; lane 6, mixture of human and cow blood; lane m, molecular weight marker (100 bp cinnagen, iran) discussion in this study first we have adapted a pcr method developed by kent and norris (2005) and then we developed the technique by restriction fragment length polymorphisms (pcr-rflp) to identify blood meals from wildcollected sand flies in an endemic area of zoonotic visceral leishmaniasis in northwest iran where l. infantum is transmitted. we also evaluated utility of pcr-rflp with elisa as a gold standard method. in our study successful amplification was obtained from genomic dna of host blood meal of sand flies fed on human, cow, and dogs. the rflp profiles for these vertebrates were specific and easily they could be distinguished from each other. theoretically, based on sequence data available in gene bank, the haeiii enzyme could provide diagnostic profiles for other vertebrate hosts such as ass, goat, horse, and other canidae, however, we did not have dna sources for these ones to confirm their diagnostic profiles. results of elisa were almost similar with pcr-rflp (27% versus 34%). elisa method has provided countless valuable data iranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 14 over the years for blood meal identification (gomez et al. 1998, agrela et al. 2002, bongiorno et al. 2003, svobodová et al. 2003, marassá et al. 2006, rossi et al. 2008). in sand flies with 0.5 µ g engorged meal, the elisa technique enables us to identify only one patent feed (lehane 2005). however, elisa methods are time-consuming and lack sensitivity. in addition, one of the major constraints of elisa is that sand flies are diminutive insects, only able to ingest very small quantities of blood (rogers et al. 2002). due to low volume of engorged blood meal in sand flies it is impossible to check more than one antigen source. another constrain is that sand flies have multiple feeding habituate that has major consequence on the epidemiology of zoonotic diseases such as zoonotic visceral leishmaniasis (zvl) and zoonotic cutaneous leishmaniasis (zcl). these constrains direct researchers to use more sensitive and accurate methods such as pcr-rflp to identify sand fly hosts for the study of vector–vertebrate host associations, as well as to improve current control interventions targeting sand flies vectors. pcr-rflp assay has more advantage than elisa since it possesses the unique ability to analyze components of a multi species dna sample. in this study, 36% of bloodfed field specimens contained multiple blood meals. this host-feeding behavior can influence pathogen transmission through increased frequency of vector-human contact, or possibly reduce vector-human contact if some blood meals are taken from alternative mammalian hosts. since this method could identify multiple blood meals it will be extremely useful for entomologically based projects involving blood-feeding behavior and vectorial capacity of sand flies in endemic areas of zvl and zcl. mtdna cytochrome b gene has been used to resolve vertebrate evolutionary questions as well as served as a target for molecular diagnostics (ngo 2003). cytb has a proven utility for identifying arthropod blood meals due to high copy number as a mitochondrial gene and sufficient genetic variation at the primary sequence level among vertebrate taxa for reliable identification. in addition to mtdna cytb gene, other markers that have been used to identify blood meals from arthropods include vertebrate 18s ribosomal dna (pichon 2003) the hypervariable region 2 of mitochondrial dna (lord 1998) and tc-11 and vwa (humvwfa31/a, a repeat polymorphism in the von will brand factor gene) loci (mukabana 2002). pcr-based methods have been used for diagnosis of infectious diseases, including leishmania detection in human patients (dweik et al. 2007, foulet et al. 2007, kumar et al. 2007), infected dogs (de andrade et al. 2006, gomes et al. 2007, solano-gallego et al. 2007) and phlebotomine sand flies (cabrera et al. 2002, paiva et al. 2006, myskova et al. 2008, ranasinghe et al. 2008, oshaghi et al. 2009). dna prepared from whole body of phlebotomus sand flies not only can be used for blood meal identification but also can be used for parasite detection/identification by pcr (sant'anna et al. 2008). this simple methodology could be very useful in epidemiological studies in endemic areas for leishmaniasis as specimens suspected to contain parasites. in this study, sand fly of p. perfiliewi was the most predominant species and has a 27-34% tendency to human blood in the region. previous studies on p. perfiliewi, have suggested nearly identical tendency to human blood (rassi 1999). it seems that this sand fly is not highly anthropophilic and preferred cows or other animals. in rural environment where large domestic mammalian species occur in abundance, this may reduce p. perfiliewi vectorial capacity. in conclusion, blood meal identification in field-caught sand flies can confirm a strong association between sand flies and reservoir hosts such as dogs in rural areas and help to improve understanding the role of domestic animals in transmission of l. infantum in endemic foci. the greater sensitivity of the pcriranian j arthropod-borne dis, (2009), 3(1): 8-18 n maleki-ravasan et al: blood meal identification… 15 rflp method in comparison with elisa reported here means that information can be obtained from specimens that have ingested relatively small amounts of blood of one or multiple hosts which could be used in vector incrimination and reservoir determination of vector borne diseases such as zvl and zcl. acknowledgments we thank abolhassani, eskandari, and hosseni for their perfect technical assistance. this project was founded by tehran university of medical sciences (grant number: 8603-27-6268) to ma.oshaghi. the authors declare that they have no conflicts of interest. references afonso mm, gomes ac, meneses cr, rangel ef (2005) studies on the feeding habits of lutzomyia (n.) intermedia (diptera, psychodidae), vector of cutaneous leishmaniasis in brazil, cad saude publica. 21: 1816–1820. agrela i, sanchez e, gomez b, feliciangeli md (2002) feeding behavior of lutzomyia pseudolongipalpis (diptera: psychodidae), a putative vector of 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793. who (2002) annex 3 burden of disease in dalys by cause, sex and mortality stratum in who regions, estimates for 2001: 192–197. world health organization, geneva. j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 83 http://jad.tums.ac.ir published online: april 27, 2019 original article molecular detection and phylogenetic analysis of endosymbiont wolbachia pipientis (rickettsiales: anaplasmataceae) isolated from dirofilaria immitis in northwest of iran majid khanmohammadi1,2; reza falak3,4; ahmad reza meamar1; mehdi arshadi1,5; *lame akhlaghi1; *elham razmjou1 1department of parasitology and mycology, school of medicine, iran university of medical sciences, tehran, iran 2department of laboratory science, marand branch, islamic azad university, marand, iran 3immunology research center, iran university of medical sciences, tehran, iran 4department of immunology, school of medicine, iran university of medical sciences, tehran, iran 5alzahra hospitals', tabriz university of medical sciences, tabriz, iran (received 29 mar 2018; accepted 26 jan 2019) abstract background: the purpose of this study was molecular detection and phylogenetic analysis of wolbachia species of dirofilaria immitis. methods: adult filarial nematodes were collected from the cardiovascular and pulmonary arterial systems of naturally infected dogs, which caught in different geographical areas of meshkin shahr in ardabil province, iran, during 2017. dirofilaria immitis genomic dna were extracted. phylogenetic analysis for proofing of d. immitis was carried out using cytochrome oxidase i (coi) gene. afterward, the purified dna was used to determine the molecular pattern of the wolbachia surface protein (wsp) gene sequence by pcr. results: phylogeny and homology studies showed high consistency of the coi gene with the previously-registered sequences for d. immitis. comparison of dna sequences revealed no nucleotide variation between them. pcr showed that all of the collected parasites were infected with w. pipientis. the sequence of the wsp gene in wolbachia species from d. immitis was significantly different from other species of dirofilaria as well as other filarial species. the maximum homology was observed with the wolbachia isolated from d. immitis. the greatest distance between wsp nucleotides of wolbachia species found between d. immitis and those isolated from onchocerca lupi. conclusion: pcr could be a simple but suitable method for detection of wolbachia species. there is a pattern of host specificity between wolbachia and dirofilaria that can be related to ancestral evolutions. the results of this phylogenetic analysis and molecular characterization may help us for better identification of wolbachia species and understanding of their coevolution. keywords: wolbachia pipientis; dirofilaria immitis; cytochrome oxidase i (coi); wolbachia surface protein (wsp); phylogenetic analysis introduction wolbachia is an intracellular α-proteobacteria, endosymbiont in insects and filarial nematodes. this genus of bacteria belongs to the phylum of proteobacteria and order of rickettsiales. wolbachia pipientis was the first bacteria discovered in this genus, which infects culex pipiens mosquito ovaries (1, 2) and plays a key role in the mosquito’s biology, ecology, immunity, evolution, parthenogenesis, microbial manipulation and reproduction (3, 4). this intracellular bacterium provides mechanisms to destroy male embryos and increase feminization of the filarial host, thus it is characterized by reproductive parasitism (3, 4). during feminization, these bacteria affect various biological characteristics including female worm fer*corresponding authors: dr lame akhlaghi, e-mail: akhlaghi.l@iums.ac.ir, dr elham razmjou, e-mail: razmjou.e@iums.ac.ir j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 84 http://jad.tums.ac.ir published online: april 27, 2019 tilization, molting, development, survival, and killing of the male hosts. most likely, a cytoplasmic incompatibility occurs and results in this phenomenon, however, up to now no definitive proof was found (4, 5). wolbachia has a role in filarial nematode development, too. it mainly affects host’s reproductive system, increases the transfer of nematode larva from the insect to the mammalian host, affects embryogenesis, promotes progression of the l1 form to the l3 form, reduces conversion time, and speeds up transition of the third-stage larva to adult forms (6). wolbachia infection of the filarial nematodes may also cause mild inflammatory responses in mammalian hosts, too. this intracellular bacterium is mostly located in the lateral hypodermal cords of both male and female nematodes. in this way, the worms may transmit their eggs to the cytoplasm of the female reproductive structures (7). unfortunately, data regarding the symbiotic relationship between w. pipientis and d. immitis is limited; thus, the role of these bacteria in the pathogenesis of dirofilaria, as the causative agent of canine heartworm disease, is not fully understood. however, some data support the role of this bacterium in the pathophysiology and survival of distinct pathogenic filarial worms including o. volvulus, w. bancrofti, b. malayi, d. repens, and d. immitis (8-10). therefore, capability of this endosymbiont bacterium in manipulation of the reproductive system of the host could be beneficial for biological control and management of pests or for in molecular identification of the parasites. wolbachia spp. could be a vaccine candidate against vector-borne diseases including dengue and filariasis, too (3, 4). antimicrobial therapy against wolbachia spp. may be useful in treatment of heartworm disease in dogs through decreasing the number of endogenous wolbachia and consequently the microfilarial load, inhibition of the larval stage and also worm infertility. typically, application of antimicrobial substances against wolbachia may be useful as a complementary anti-filarial therapy strategy in canine and feline dirofilariasis, but further studies are needed to substitute these novel methods for anti-parasite drugs (11). in this regard, doxycycline is an effective treatment against wolbachia, and in combination with ivermectin has been shown to have adulticidal efficiency for heartworm treatments in dogs (12-15). wolbachia metabolic products may also exert pathological changes in many organs of the host such as lungs and kidneys (16). the wolbachia surface protein (wsp) is a crucial component involved in the immunopathogenesis of filarial diseases and helps in parasite evasion from potentially harmful immune responses of the host (16). dirofilaria immitis is a selective reservoir for w. pipientis, therefore, this bacterium provides attenuation strategy for dirofilaria to evade host immune responses (17, 18). wolbachia is effective in generating immune responses during heartworm infections; thus, wolbachia likely influences the inflammatory and immunoregulatory functions of the host (19). therefore, its control may offer therapeutic and diagnostic possibilities. elimination of wolbachia by antimicrobial agents can exert a preventive effect on embryogenesis of d. immitis and could have potential application in sterilization of the female worms and consequently control and treatment of dirofilariasis (16). interfering of this obligate relationship has been used in novel therapeutic strategies; for example, a combination of antimicrobial therapy using doxycycline and ivermectin, as a macrocyclic lactone can destroy nematodes (8, 11, 14). overall, due to the zoonotic potential and the increasing number of d. immitis and d. repens infections in non-endemic areas, xenomonitoring of this bacterium may be useful (20). in iran, few studies have focused on detection of wolbachia species or their invertebrate hosts. herein, we performed molecular detection and phylogenetic analysis of wolbachia in d. immitis, using the wsp gene. the main j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 85 http://jad.tums.ac.ir published online: april 27, 2019 reasons for selecting wsp gene in this study was the high availability of the corresponding protein in soluble and membranous forms in wolbachia species, the feasibility of the amplifying its coding sequence by pcr, considerable dissimilarity of its expression in different filarial genus, and its applicability as an indicator of parasite evolution and bacterial phylogenic proximity (21). materials and methods forty-three stray domestic dogs (canis familiaris) suspicious to dirofilariasis were collected during 2017 from different geographical areas of meshkinshahr, ardebil province, iran. meshkinshahr (38°23′56″n 47°40′55″e) is a main endemic area for canine dirofilariasis in iran. all dogs were tested for dirofilariasis by direct microscopy, elisa, and rapid dipstick methods. direct wet smears of whole blood were taken with edta and used for detection of microfilaria by light microscopy. sera was analyzed for adult d. immitis circulating antigen using a commercial elisa kit (dirochek®, usa) as well as dipstick method (snap® 4dx® test, chw ii kit idexx laboratory, usa) according to the manufacturers' instructions. all procedures were carried out in admission with the rules and regulations of the respective national animal ethics committee of iran university of medical sciences (ir. iums.rec1395.9221577203-2016.05.09(. six hyper-infected dogs euthanized and necropsied. worms were collected from the pulmonary arteries and right ventricles. then, their dna was extracted and used for molecular analyses. dna extraction adult worms were homogenized by a rotorstator system (ika, uk) and total genomic dna was extracted using dna extraction kit (qiagen gmbh, germany) from approximately 25mg of each dirofilaria sample, according to the manufacturer’s instruction. purity of the extracted dna was determined and dna samples stored at -20 °c. for amplification of cytochrome oxidase i gene (coi) which is a species-specific mitochondrial gene of d. immitis, a set of specific primers were designed. in details, 5'-tga ttg gtg gtt ttg gta a-3' and 5'-ata agt acg agt atc aatatc-3' were used as forward and reverse primers, respectively. in addition, we used wolbachia-specific primers to amplify nucleotides 81–691 of wsp. the forward and reverse primers were 5' tggtccaataag tgatgaagaaac-3' and 5' aaaaatt aaacgctactcca-3', respectively. reactions were performed in 25μl volumes using 2x pcr master mix (red amplicon, denmark), 1μl of dna template, and 1μl of each primer. the thermal cycler program included one cycle of 94 °c for 5min followed by 35 cycles of 94 °c for 1min (denaturation), 55 °c for 1min (annealing), 72 °c for 60sec (extension), and a final extension at 72 °c for 10min (22). pcr products were electrophoresed on 1.5% agarose gel along with a commercial dna marker (smobio dm3100) using an imaging system (syngene, uk). finally, pcr products were purified using the pcr purification kit (qiagen gmbh, germany) and directly subject for sequencing. molecular and phylogenetic analysis phylogenetic analysis of d. immitis was carried out using coi gene sequences, obtained during the study (accession code, mf288560.1 mesh-iran 3) along with previously determined relevant sequences in the genbank. then, all obtained nucleotide sequences were analyzed and compared with the available complete wsp sequence in genbank (mg010709.1) using bioedit software, ver. 7.0.5 (california, usa). all of the evolutionary aspects and other molecular data were inferred using the maximum likelihood method, based on the tamura-nei model (23). the phylogenetic tree was drawn to scale, with branch lengths measured as the j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 86 http://jad.tums.ac.ir published online: april 27, 2019 number of wolbachia substitutions per site and with the highest log likelihood. multiple alignments and sequences linking functionality of phylogenetic studies were analyzed with mega7 software (24). the accuracy of the phylogenetic tree was measured by 1000 bootstrap replication. nucleotide changes were compared to the reference sequence with sequencher software (sequencher® version 5.4.6 usa). results we examined blood samples from 43 dogs with obvious symptoms of dirofilariasis and found that 27 dogs were dirofilaria seropositive (62.8% ci: 47.9 to 75.6). dna was purified from the collected worms and used as pcr template. wolbachia dna was detected in all 67 filarial worms including 41 females and 26 male parasites. electrophoresis of pcr product of the amplified wsp gene confirmed presence of a 630bp wolbachia specific sequence in all of the dna extracts (fig. 1). the blastn analysis of the coi gene indicated maximum homology of the isolated parasites’ dna with previously isolated sequences of d. immitis. phylogenetic analysis showed highest homology with d. immitis isolated from dogs in kerman city (kr 870344.1). we also found good homology with d. immitis isolated from several other geographical areas such as those dogs from iran (kt960976.1, kt318126. 1), china (eu159111. 1), italy (fn391553. 1) and bangladesh (kc107805.1), and also other animals such as jackal (canis aureus) in north khorasan of iran (kt351850.1, kt351851.1, kt351852. 1), cat from iran (kt282097. 1) and italy (am749227. 1), wolf from italy (dq358815. 1) and red panda from china (eu169124.1) (fig. 2). sequence analysis in all samples demonstrated a dna band which belonged to wsp gene of w. pipientis. sequence similarities and homologies between the amplified one and the previously registered sequences in the genbank was determined following a basic local alignment sequence analysis, and calculating the statistical significance through the national center for biotechnology information (ncbi) (https://blast.ncbi.nlm.nih.gov/blast.cgi). multiple sequences were aligned with the online clustalw2 tool (http://www.ebi.ac.uk/tools/msa/clustalw2). all obtained sequences were blasted after alignment and belonged to w. pipientis. partial d. immitis wsp nucleotide sequences were submitted with accession code of mg010709.1 in genbank. the phylogenetic analysis of wsp sequences of wolbachia showed maximum homology (99%) with the complete w. pipientis sequence isolated from d. immitis in italy (aj252062.1) and several other filarial organisms from different areas of the world including d. repens (aj252176.1), o. volvulus (hg810405.1), o. ochengi (he660029.1), o. cervicalis (ay 095210.1), o. gibsoni (aj252178.1), and o. gutturosa (aj276497.1) (fig. 3). wolbachia pipientis is genetically related to the filarial nematodes phylum. phylogenetic studies using wsp revealed the greatest similarity and proximity of the obtained sequence with other filarial nematodes. the least similarity of the wolbachia sequence isolated from filarial nematodes was observed between onchocerca and dirofilaria taxa (fig. 3). nucleotide changes were compared and no differences were found between the amplified sequences and the previously reported ones. the highest sequence similarity with w. pipientis was seen in o. gibsoni and o. gutturosa, and the lowest was in o. lupi due to difference in 33 nucleotides. the nucleotide difference between d. immitis and d. repens was found in 27 nucleotides. the difference between d. repens and the other filarial nematodes were lowest of those studied ones (fig. 4). all studied species showed the greatest nucleotide differences between positions 143 and 161 of the known nucleotide location. this study showed https://blast.ncbi.nlm.nih.gov/blast.cgi http://www.ebi.ac.uk/tools/msa/clustalw2 j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 87 http://jad.tums.ac.ir published online: april 27, 2019 sequence variation in wsp gene of wolbachia from various filarial nematode population. fig. 1. electrophoresis of pcr product of wsp specific amplification confirming the presence of endosymbiotic wolbachia present in dirofilaria immitis dna extract. m: 100bp dna marker, nc: negative control, lines 1–2: pcr products, pc: positive control fig. 2. phylogenetic tree of dirofilaria immitis cytochrome c oxidase subunit i (coi) gene using maximum likelihood analysis among sequences based on the tamura-nei model with 1000 bootstrap repetition. the scale bar indicates the genetic distance in single nucleotide substitutions. genbank registered accession numbers are specified within parentheses. ascaris lumbricoides (accession no. ab591801.1) was as outgroup j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 88 http://jad.tums.ac.ir published online: april 27, 2019 fig. 3. phylogenetic tree of wolbachia wsp gene sequences from meshkinshahr of north west iran and other previous registered sequences from different areas using maximum likelihood method based on the tamura-nei model with 1000 bootstrap repetition fig. 4. multiple alignments of the partial wsp gene and the sequences of other filarial nematodes j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 89 http://jad.tums.ac.ir published online: april 27, 2019 discussion this was the first report on molecular characterization of w. pipientis infection of d. immitis isolated from northwest of iran, where, dirofilariasis is endemic. the prevalence of dirofilariasis in the untreated dogs was previously estimated to be in the range of 1.4–51.4% (25, 26). results of phylogenetic analysis of coi gene in the isolated filarial nematodes confirmed that the dirofilaria species in kerman and meshkinshahr are molecularly identical. pairwise homology analysis revealed high sequence homology of the isolated worms with the available sequences at ncbi genbank. the determined wolbachia infection rate was very similar to previous studies. the findings also confirmed the general believe about wolbachia incidence and emphasized the absence of ancestral infection in the nematode phylum (27). this widespread bacterium may be detected in all classes of arthropods and approximately infects 20–70% of them. therefore, due to high incidence of the endosymbiosis infection of the arthropods with wolbachia, recent studies were mainly focused on arthropods (28). in brief, it is commonly found in butterflies, fruit flies, sand flies, mosquitoes, fleas, ticks, bugs, wasps, silkworms (4, 5, 19, 21, 29-34). previously, wolbachia dna was detected in 30.6% of the blood sample of filaria-positive dogs in the mediterranean area (35). co-infection of six dogs with d. immitis and d. repens was reported in turkey by pcr-amplification of the wsp gene (36). wolbachia dna was also found in blood samples from dirofilaria-infected dogs, in other regions (37). in a portuguese study, dogs whose dirofilariasis was confirmed by parasitological and serological methods were further examined by pcr method. moreover, wolbachia dna was detected in blood sample of 52.6% of those with occult infection. in occult infections, no obvious microfilaria could be found in microscopic examination of blood. this molecular evaluation is useful because the presence of wolbachia in association with microfilaria is critical for control of canine dirofilariasis (38). anti-wsp igg titer in urine samples from 19 dirofilariainfected dogs was greater than the established cut-off values, and wsp gene was detected in kidney glomerular capillaries in those that were seropositive, too (39). wolbachia has also previously been isolated from w. bancrofti, brugia malayi, o. volvulus, and setaria tundra (29, 40-42). recently, the study of the isolated o. volvulus from 4 african countries indicated that extensive intra species heterogeneity exists in the wolbachia content of male and female adults of o. volvulus. wolbachia variation was previously reported in some studies and was in line with our results and pointed out to the host specificity of this bacterium (43). diversity of the pattern of the mitochondrial genetic content was found in d. immitis and was much lower than the related species (44). phylogenetic studies confirmed homology of the amplified sequence with a previously registered wolbachia sequence in genbank. we found the most consistency with wolbachia species isolated from d. immitis in italy (45). filarial nematodes have gained this bacterium endosymbiosis from arthropods, and the current data indicates that this bacteria has spread globally in filarial nematodes (46). we hypothesized that wolbachia has a genetic relationship with the phylum of filarial nematodes and this association was most likely generated over times. sequence comparisons between dirofilaria species and other filarial nematodes revealed difference of several nucleotides in wsp gene. compression of the sequences from our study with previously-reported wolbachia sequences indicated that the amplified sequence is likely dirofilaria-specific. interestingly, blastn studies showed a specific wsp pattern in dirofilaria. this could be due to genetic characteristics of the endosymbiont bacteria, as well as the physiological properties and adaptation of j arthropod-borne dis, march 2019, 13(1): 83–93 m khanmohammadi et al.: molecular detection and … 90 http://jad.tums.ac.ir published online: april 27, 2019 dirofilaria, and the regulatory mechanisms of the immune system of the final host. many questions still remain unanswered in this field. regarding to the reported distribution of wolbachia in filarial nematodes, it is not clear whether the absence of w. pipientis in wolbachia-negative filarial nematodes represents an ancestral characteristic of this bacteria or is due to losing the inserted sequence over time due to low transpositional activity in some filarial species (41). the phylogenies of various filarial nematodes are still not identified completely; thus, it is difficult to express the presence or absence of w. pipientis on a tree indicating filarial genetic evolution (42). the presence of such extensive proprietary pattern between adult worms of both sexes in filarial nematodes provides questions regarding the specific symbiosis between the wolbachia and filarial worms, and the mechanisms by which wolbachia is involved. finally, our results suggest that w. pipientis isolated from iranian dirofilaria has distinct genomic features, which likely are the result of long periods of independent evolution. the results of this study open the way for further studies on the strain identification and genetic diversity, which may increase our understanding about the host-parasite relationships. overall, the mapping of wolbachia on the phylogenetic trees generated by mega software indicates that this bacterium may have evolved along with ancestors of filarial nematodes. conclusion main differences between the population of adult filarial worms were at the level of wolbachia species. there is a pattern of host specificity between wolbachia and dirofilaria. this subject can be due to ancestral evolutions long times ago. molecular characterization can be applied as a new trend for understanding the evolution and identification of wolbachia. this method offers a new technique for diagnosis and may provide strategies for development of novel and effective therapeutic procedures. the results of this study can help us to understand the interactions of wolbachia and dirofilaria with their mammalian hosts. we suggest further investigation of the endosymbiotic w. pipientis polymorphisms in nematodes. findings also highlighted correlations between wolbachia and d. immitis throughout their life cycle and could be a valuable resource that may be applied for 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pathogenesis and treatment of filarial diseases. vet parasitol. 98(1–3): 215–238. microsoft word 3-hashare dr yilmaz public rtl 88 10 3 .doc iranian j arthropod-borne dis, (2009), 3(2): 12-17 r yilmaz et al: public knowledge and attitude… 12 original article public knowledge and attitude toward crimean congo hemorrhagic fever in tokat turkey *r yilmaz1, m ozcetin1, u erkorkmaz2, s ozer1, f ekici3 1 department of pediatrics, school of medicine, gaziosmanpasa university tokatturkey 2 department of biostatistics, school of medicine, gaziosmanpasa university tokatturkey 3 department of physiology, school of medicine, gaziosmanpasa university tokatturkey (received 24 mar 2009; accepted 28 oct 2009) abstract background: the world health organization (who) declares crimean congo hemorrhagic fever (cchf) endemic in turkey. despite the magnitude of problem, no documented evidence exists in turkey, which reveals the awareness and practices of the country’s adult population regarding cchf, its spread, symptoms, treatment, and prevention. this study was conducted to assess the level of knowledge, attitudes, and practices regarding cchf in people visiting tertiary care hospital in tokat, turkey. methods: this questionnaire based cross-sectional survey was conducted among patients’ relatives or guardians who admitted pediatric outpatient clinics during may-july 2008. the questionnaire was composed of 25 questions. results: a total of 1034 respondents participated in the survey. sufficient knowledge about cchf was not found in 28.9% of the sample. literate individuals were relatively better informed about cchf as compared to the illiterate people. television and radio were considered as the most important and useful source of information on the disease. conclusion: we have found insufficient knowledge on cchf in our population. it is thought to have no chance of success against a fatal disease such as cchf, which has serious consequences, without the contribution of community. it is clear that there are important tasks for health, agriculture, and media sectors to improve public knowledge and awareness about cchf. keywords: crimean congo hemorrhagic fever, public knowledge, survey, turkey introduction crimean congo hemorrhagic fever (cchf), that is endemic in over 30 countries around the black sea and in the middle east and africa, is a potentially fatal virus infection. cchf virus is transmitted to humans by bites of infected ticks or direct contact with secretions or blood of infected animal or human (williams et al. 2000). many cchf outbreaks have occurred in tokat and other anatolian cities since 2003 (bakir et al. 2005, gozalan et al. 2007). changes in socioeconomic conditions and climate have increased animal and human migration, travel, the risk of bioterrorism and ecological deterioration. these factors may increase the spread of viral infections to new places and their incidence in endemic regions (bakir et al. 2005). in endemic regions, persons including farmers, shepherds and veterinarians who are in occupational contact with livestock and wild animals, are in high-risk group (flick and whitehouse 2005, vorou et al. 2005). recreational activities such as picnic, tracking in endemic areas also represent risk factor for tick bite. healthcare workers caring with cchf patients are the second major group at risk for infection (ergonul et al. 2007). tokat is geographically located between the black sea and central anatolia. the climate is suitable for ticks survive. the first cases of cchf virus infection in turkey was reported from this region (bakir et al. 2005). *corresponding author: dr. resul yilmaz, e-mail: drresul@hotmail.com iranian j arthropod-borne dis, (2009), 3(2): 12-17 r yilmaz et al: public knowledge and attitude… 13 we studied the public knowledge and attitude toward cchf at a tertiary care hospital in tokat, where cchf outbreaks have still been reporting since 2003 in every spring to early autumn. materials and methods this questionnaire based cross-sectional survey was conducted among patients’ relatives admitted to pediatric outpatient clinics during may-july 2008 in gaziosmanpasa university medical center in tokat, turkey. we offered the patients’ relatives to participate in the survey. if they agreed, informed consent was taken. a total of 1034 parent or guardian of child agreed to participate and completed the questionnaire. the questionnaire was composed of 25 questions. the first four questions were constituted to access demographic data; remains were for knowledge and attitude toward cchf. it was self administered and in turkish. to access the readability and applicability of the questionnaire, a pilot study was assessed on 50 respondents. questions, which were confusing, were simplified. when respondent needed instruction, researchers helped to understand and fill in the questionnaire. we respected the confidentiality of respondents, data were recorded anonymously. statistical analysis the continuous variables were presented as mean and standard deviation. the categorical variables were presented as count and percentage. analyses were performed using commercial software (pasw ver. 18, spss inc, chicago, il) results a total of 1034 respondents participated in the survey. mean age was 30.28±10.75 yr (range 18-84). demographic information about respondents was given in table 1. the sources of cchf information is presented in fig. 1. participants thought civil society organizations in tokat informed, partially informed and did not informed the community about cchf n= 115 (11.2%), n= 344 (33.3%) and n= 575 (55.6%), respectively. factors that could correlate with the source of cchf information is shown in table 1. job and education were statistically significant factors that effects where they could get the information. questions that measure public knowledge and preventive measures about cchf and their answers are given in table 2 and table 3. 22.1% of participants did not have picnic. remains had at least one picnic in a year and n= 635 (61.5%) point out cchf affected their picnic habit. table 1. basic demographics of respondents n % female 547 52,9 gender male 487 47,1 city 654 63,2 town 287 27,8 residence village 93 9,0 elementary+ middle school 297 28,7 high school 261 25,2 educational level university 476 46,0 farmer 23 2,2 retired 42 4,1 tradesman 50 4,8 worker 114 11,0 housewife 220 21,3 government employee 274 26,5 job student 311 30,1 iranian j arthropod-borne dis, (2009), 3(2): 12-17 r yilmaz et al: public knowledge and attitude… 14 table 2. the questions and answers of paticipants about cchf n % the causative agent is a virus a bacteria unknown bioterrorism agent 652 98 130 138 67.1 9.5 12.6 14.9 transmission to human by ticks close contact with patient secretion of infected animals 802 65 138 77.6 6.3 13.3 where were cchf first emerged? africa russia europe turkey i don’t know 264 160 42 158 400 25.5 15.1 4.1 15.3 39.7 what kind of disease is cchf? not contagious but dangerous common, dangerous disease non remediable disease fatal, contagious and dangerous 100 76 86 772 9.7 7.4 8.3 74.7 is there a treatment for cchf? yes no i don’t know 538 329 167 52 31.8 16.2 is there a vaccine against cchf? yes no i don’t know 152 579 303 14.7 56 29.3 what are the symptoms of cchf? fever myalgia headache fatiguelow appetite all 200 19 8 72 735 19.3 1.8 0.8 7 71.1 who are at risk for cchf infection farmersshepherds healthcare workers people who are visiting rural area all 278 12 48 735 26.9 1.2 4.6 71.1 when will symptoms appear after tick bite? within 1-3 days at least 9 days in first month longer than 1 month 579 390 47 18 56 37.7 4.5 1.7 which season is cchf common? spring summer autumn 182 845 7 17.6 81.7 0.7 can you eat your livestock’s meat if a tick bites it? we can eat its meat we cannot eat it has to destruct i do not know 157 201 382 294 15.2 19.4 36.9 28.4 iranian j arthropod-borne dis, (2009), 3(2): 12-17 r yilmaz et al: public knowledge and attitude… 15 table 3. the questions and answers of paticipants about cchf’s preventive measures n % what is the most effective method for prevention? insecticide minimizing the open area of the body disseminating poultry avoidance of areas where ticks are abundant use of repellents 208 328 218 180 103 20.1 31.4 25.1 17.4 10 who can remove embedded the tick? their self healthcare workers or doctors i don’t know: 8 1000 26 0.8 96.7 2.1 how do you remove the tick when it embeds your livestock or pet? use of repellants naked hand wearing gloves and other protection materials i don’t know 323 18 493 200 31.2 1.7 47.7 19.3 discussion the gaziosmanpasa university health practicing center is the only tertiary health care facility which accepts and gives care to the severe cchf children patients in tokat where the survey was conducted, no referral was needed (yilmaz et al. 2009). we determined that parents or guardians of these children were not aware of the seriousness of the disease. we wanted to emerge the knowledge and attitude towards cchf in this population. although all of the respondents were familiar with cchf, vast majority of them pointed out cchf transmission to human was by tick bites. one fifth of them marked close contact with infected patients and secretions of infected animals. these are not strange findings because tokat is the first city where first cchf cases were reported from here in turkey. despite all the participants heard about cchf, ¾ of them remembered cchf to be contagious and dangerous disease. ¼ of them said it was not contagious. one third of the respondents did not know the causative agent. about 15% of the participants believed that cchf virus was a biological war agent, coming from some foreign countries. this result may be indicating that people consider new diseases are mostly biological war agent. “tick disease” and cchf are the currently known two expression of cchf in public. 2/3 of the participants showed adequate knowledge about the symptoms. there is no similar study about public knowledge and attitude towards cchf in english literature, so we did not compare our results. half and 40% of participants specified cchf symptoms would appear in 1-3 d and at least 9 d, respectively. it is compatible with literature group (flick and whitehouse 2005, vorou et al. 2005). although knowledge about symptoms was insufficient, knowledge about the treatment of cchf was not clear. half of the patients considered there was no treatment or they did not know whether there was a treatment or not. almost all the participants pointed out that they could remove the embedded tick at health care centers. ticks can be removed by using tweezers to gasp the tick as close to the skin as possible (sloan, 2009). using fire, home remedies, alcohol or petroleum jelly can cause tick to regurgitate its guts contents to the bitten person’s or animal’s blood stream that increases the chance of cchf infection iranian j arthropod-borne dis, (2009), 3(2): 12-17 r yilmaz et al: public knowledge and attitude… 16 or other tick born diseases (who fact sheet 208, 1998). seventy percent of participants gave the correct answers how they remove the tick when it bites their livestock or pet, but only 15% of them recognized they can eat the meat of tick bitten livestock. destructing the tick bitten livestock can bring a huge economical load on the farmers’ budget; farmers in our region mostly have low income. the most effective preventive measure is avoidance of areas where ticks are abundant. one fifth of participants gave right answer. if they want to go rural area where ticks are present, they can use repellants and/or they can wear trousers tucked in boots or socks and a long sleeved shirt tucked in at the waist (who fact sheet 208, 1998). when the participants were asked “what is the source of your information on cchf?” they mostly pointed out media including radiotv and newspapers. in literature, public knowledge and information on infectious disease mostly obtained from media (itrat et al. 2008, mossialos and rudisill, 2009). our study is consistent with these studies. media can exaggerate or oversee the situation. this behavior of media leads cchf information is mostly inappropriate or wrong. there is a great responsibility on media to transmit appropriate and right information on cchf. several studies have reported a higher socioeconomic status correlates with better knowledge score (mcarthur et al. 2001, winch et al. 2002). in the present survey occupation and education are associated with more knowledge about cchf. participants, who had high school or university graduate certificate, gave most accurate answers. farmers in our region have lower education and income than in farmers from western part of turkey (dincer et al. 1996). these findings suggested that more effective programs, which would target farmers and low educated people for public knowledge, needed to be implemented. four fifth of participants had a picnic at least once a year and they pointed out cchf affected their picnic habit. common (popular) infections, zoonotic disease affect people’s attitude. when an avian influenza outbreak occurred in turkey in 2006, most of the people did not consume poultry products (cultu-kantaroglu et al. 2007). in our survey, we determined people did not have picnic or postponed to winter or late autumn. the level of knowledge and attitude of community is important to get the highest support from community before launching any disease control program (singh et al. 2006). cchf outbreaks have occurred every year since 2003 so it can be said that people from this area have a higher knowledge. our survey is limited to a university hospital so these data cannot be generalized to rest of turkey, where has different socioeconomic status and cultural background. nationwide or sum of local studies can provide the accurate status of public knowledge and attitude towards cchf. by using these data, ministry of health and ministry of agriculture can easily establish the program of controlling the disease. in conclusion, we have found insufficient knowledge in our population. it is thought to have no chance of success against a fatal disease such as cchf, which has serious consequences, without the contribution of community. it is clear that there are important tasks for health, agriculture, and media sectors to improve public knowledge and awareness about cchf. acknowledgements the authors are grateful to gaziosmanpasa university medical faculty for their support. the authors declare that they have no conflicts of interest. references bakir m, ugurlu m, dokuzoguz b, bodur h, tasyaran ma, vahaboglu h; turkish cchf study group (2005) crimeaniranian j arthropod-borne dis, (2009), 3(2): 12-17 r yilmaz et al: public knowledge and attitude… 17 congo haemorrhagic fever outbreak in middle anatolia: a multicentre study of clinical features and outcome measures. j med microbiol. 54(pt 4): 385-89. bülent dinçer, metin özaslan, erdoğan satılmış. i̇llerin sosyo-ekonomik gelişmişlik sıralaması araştırması (1996) ankara: dpt. bölgesel gelişme ve yapısal uyum genel müdürlüğü, aralık 1996. isbn: 975-19-1594-5 (http://ekutup.dpt.gov.tr/bolgesel/dincerb/il/) crimean-congo hemorrhagic fever, fact sheet no. 208, dec 1998. www.who.int çultu-kantaroğlu ö, yıldırım i̇, ceylan m (2007) the level of public awareness and knowledge about avian influenza in a survey of parents of children admitted to a university children’s hospital. çocuk sağlığı ve hastalıkları dergisi 50: 180-85. ergonul o, zeller h, celikbas a, dokuzoguz b (2007) the lack of crimeancongo hemorrhagic fever virus antibodies in healthcare workers in an endemic region. int j infect dis. 11(1): 48-51. flick r, whitehouse ca (2005) crimeancongo hemorrhagic fever virus. curr mol med. 5(8): 753-60. gozalan a, esen b, fitzner j, tapar fs, ozkan ap, georges-courbot mc (2007). crimean-congo haemorrhagic fever cases in turkey. scand j infect dis. 39(4): 332-336. itrat a, khan a, javaid s, kamal m, khan h, javed s (2008) knowledge, awareness and practices regarding dengue fever among the adult population of dengue hit cosmopolitan. plos one. 9; 3(7): e2620. mcarthur l, pena m, holbert d (2001) effects of socioeconomic status on the obesity knowledge of adolescents from six latin american cities. int j obes relat metab disord. 25: 1262-1268. mossialos e, rudisill c (2008) knowledge about avian influenza, european region. emerg infect dis. 14(12): 1956-1957. singh sp, reddy dc, mishra rn, sundar s (2006) knowledge, attitude, and practices related to kala-azar in a rural area of bihar state, india. am j trop med hyg. 75(3): 505-8. sloan sb (2009) tick removal: treatment & medicationupdated: jul 21, 2009 http://emedicine.medscape.com/article/ 1413603-treatment vorou r, pierroutsakos in, maltezou hc (2007) crimean-congo hemorrhagic fever. curr opin infect dis. 20(5): 495-500. williams rj, al-busaidy s, mehta fr, maupin go, wagoner kd, al-awaidy s (2000) crimean-congo haemorrhagic fever: a seroepidemiological and tick survey in the sultanate of oman. trop med int health. 5: 99-106. winch pj, leontsini e, rigau-pérez jg, ruiz pérez m, clark gg, gubler dj (2002) community-based dengue prevention programs in puerto rico: impact on knowledge, behavior, and residential mosquito infestation. am j trop med hyg. 67(4): 363-70. yilmaz r, kundak aa, ozer s, esmeray h (2009) successful treatment of severe crimean-congo hemorrhagic fever with supportive measures without ribavirin and hypothermia. j clin virol. 44(2): 181182. j arthropod-borne dis, march 2017, 11(1): 161–165 m fatemi et al.: assessing the ovarian … 161 http://jad.tums.ac.ir published online: march 14, 2017 short communication assessing the ovarian accessory glands to determine the parity of phlebotomus papatasi, vector of zoonotic cutaneous leishmaniasis, under laboratory condition mahboubeh fatemi 1, mohammad reza yaghoobi-ershadi 1, mehdi mohebali 2, zahra saeidi 1, arshad veysi 1, *ali khamesipour 3, *amir ahmad akhavan 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical parasitology, school of public health, tehran university of medical sciences, tehran, iran 3center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran (received 26 may 2015; accepted 2 june 2015) abstract background: zoonotic cutaneous leishmaniasis (zcl) is a neglected tropical disease prevailed in many rural areas of 17 out of 31 provinces in iran. the main vector of the disease is phlebotomus papatasi and the causative agent is leishmania major in zcl foci of iran. in the current study we investigated the validity of accessory glands secretions as an indicator to recognize parous from nulliparous ph. papatasi females under laboratory conditions. methods: over 235 laboratory-reared females of ph. papatasi were dissected in 6 groups including: newly emerged, one hour, one day, two days, and three days after blood feeding and also after oviposition under stereo microscope for their parity in 2014–2015. results: transparent glands were compatible with nulliparous only in newly emerged sand flies. in sand flies dissected after oviposition, accessory glands were rather large as a result of oviposition though they were transparent. conclusion: the accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of ph. papatasi females. keywords: phlebotomus papatasi, accessory glands, parous, nulliparous, parity introduction phlebotominae sand flies are the vectors of different kinds of leishmaniasis and papatasi fever in the world (dejeux 1991). different forms of leishmaniasis have been recorded from 98 countries and 350 million people are at risk of the disease worldwide (who 2010). zoonotic cutaneous leishmaniasis (zcl) is the most common form of cutaneous leishmaniasis. annually around 20000 new cases of leishmaniasis are reported in iran, which more than 80% of the cases are zcl form (shirzadi 2010). rhombomys opimus (great gerbil) is the major reservoir host of the disease in central and north-east parts of iran. tatera indica and meriones hurrianae are the main reservoirs of the disease in south-west and southeast parts of iran respectively (mohebali et al. 2004, akhavan et al. 2010). meriones libycus is also reported as the main reservoir of zcl in some central and south parts of the country. leishmania major is the causative agent of the disease in iran (yaghoobi-ershadi et al. 1996). phlebotomus papatasi and ph. caucasicus are proven vectors of the enzootic cycle of l. major among gerbils and *corresponding authors: dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir, prof ali khamesipour, e-mail: ali.khamesipour@gmail.com j arthropod-borne dis, march 2017, 11(1): 161–165 m fatemi et al.: assessing the ovarian … 162 http://jad.tums.ac.ir published online: march 14, 2017 jirds but only ph. papatasi is responsible for transmitting the disease to human (yaghoobiershadi et al. 1994). in the course of disease transmission by sand fly, only those flies have laid eggs once (parous), could transmit the disease. thus, parous sand flies play a critical role in disease spreading. the study of age composition in phlebotominae sand flies should be a fundamental part for understanding the epidemiology of the disease transmitted by them, particularly leishmaniasis. on the other hand, physiological age of phlebotomine sand flies is estimated by nulliparous and parous female recognition. unlike mosquitoes ovariole characteristics in phlebotominae due to small size, are not useful method to determine physiological age (lewis and minter 1960, lewis 1965). so far no method has been applicable for counting the number of dilatations in sand flies (detinova 1962). ovarian accessory glands in sand flies are relatively large and have been used as a practical method to determine parity in several sand fly species (adler and theodor 1935, lewis and minter 1960, lewis 1970, takaoka 1989) but this method is not applicable for distinguishing parous females in many other species (lewis 1965, scorza 1968, ready et al. 1984, takaoka 1989). at the current study the reliability of the ovarian accessory glands to distinguish parity of ph. papatasi females was examined under laboratory conditions. materials and methods sand flies were collected using aspirating tubes during the active seasons from rural districts of endemic areas of esfahan province, iran in 2014–2015. phlebotomus papatasi colony was reared at the sand fly insectary of medical entomology and vector control department, school of public health, tehran university of medical sciences and the esfahan health research station. phlebotomus papatasi sand flies were reared under laboratory conditions at 26±2 ˚c, 70– 80% relative humidity and 14: 10 h (l: d) photoperiod. a male and a blood-fed female were transferred in to plaster-lined individual pot for oviposition (killick-kendrick and killickkendrick 1991). from the 2nd generation, the mass-rearing technique was used (modi and tesh 1983). to prevent fungal contamination in rearing pots, autoclaved field soil and sea sand were added into the rearing pots. larvae food was sprinkled after 50% eggs hatching. larval diet is the most important factor for maintaining sand fly colony. larvae food was prepared using the method described by young et al. (1981) with some modifications. in brief, the larvae food consists of equal proportions of dried rabbit feces, rabbit chow and yeast. rabbit feces and chow were ground by a grinder. a spoonful yeast dissolved in distilled water, then added for each 120-gr powder, then enough amount of distilled water added to make paste. after 2 days when fungi contamination disappeared, the mixture was spread in a thin layer inside a tray. it was allowed to dry in an upside down position. then food was scraped from the trays and ground by a grinder. after about one week the larval food was ready to use. for checking the value of using accessory glands for distinguishing parous of ph. papatasi females from nulliparous, over 235 laboratoryreared females of ph. papatasi were dissected in 6 groups including: newly emerged, one hour, one day, two days and three days after blood feeding and also after oviposition under stereo microscope for its parity (fig. 1). prior to dissection, sand flies were immobilized in -20 ˚c freezer for 5 min. each sand fly was placed in a drop of sterile normal saline (9/1000) on a clean slide and dissected under a stereomicroscope. after releasing the ovaries and accessory glands, immediately they were examined under a stereomicroscope and light microscope to check the presence of granular secretions of the accessory glands. j arthropod-borne dis, march 2017, 11(1): 161–165 m fatemi et al.: assessing the ovarian … 163 http://jad.tums.ac.ir published online: march 14, 2017 results a total of 235 ph. papatasi females including 30 sand flies "newly emerged", 33 sand flies "one hour after blood feeding", 50 sand flies "one day after blood feeding", 38 sand flies "two days after blood feeding", 49 sand flies "three days after blood feeding and 35 sand flies "after oviposition" were dissected and compared regarding granular secretions (table1). granular secretions in the accessory glands were not observed in nulliparous females in newly emerged sand flies. in 17 out of 33 ph. papatasi dissected one hour after blood feeding, granular secretions in the accessory glands were not observed in nulliparous females as well. in contrast, third, fourth and fifth groups of sand flies which were dissected one day after blood feeding, two days after blood feeding and three days after blood feeding showed discordant relation between granular secretions and parity. discordant relation between granular secretions and parity was also found in 9 out of 35 of the sand flies after oviposition, although accessory glands were rather large as a result of oviposition. in 26 of 35 ph. papatasi sand flies which had laid eggs granular secretions in the accessory glands were observed (fig. 2). table 1. observation on accessory glands of dissected phlebotomus papatasi totalaccessory glands secretionsgroups present n (%)absent n (%) parousnulliparousparousnulliparous 30 33 50 38 49 35 235 26 (74.29) 16 (48.5) 50 (100) 38 (100) 49 (100) 9 (25.71) 30 (100) 17 (51.5) newly emerged one hour after blood feeding one day after blood feeding two days after blood feeding three days after blood feeding after oviposition fig. 1. different stages of abdominal physiology in phlebotomus papatasi a) newly emerged, b) one hour after blood feeding, c) one day after blood feeding, d) two days after blood feeding, e) three days after blood feeding, f) after oviposition fig. 2. accessory gland status of phlebotomus papatasi in different stages of abdominal physiology. a) newly emerged, b, c) one hour after blood feeding, d) one day after blood feeding, e) two days after blood feeding, f) three days after blood feeding, g, h) after oviposition j arthropod-borne dis, march 2017, 11(1): 161–165 m fatemi et al.: assessing the ovarian … 164 http://jad.tums.ac.ir published online: march 14, 2017 discussion accessory glands are convenient to determine parity of phlebotominae sand flies because they are enough large to inspect fairly and quickly in contrast to ovarian but the results of the current study and some others show that reliability of this method depends on sand fly species and it seems to be impractical for all the sandflies. so all females with granular secretions cannot be considered as parous, because some of the blood fed nulliparous females might have secreted granules shortly after blood feeding. accessory glands secretions in ph. califoniicus, ph. stewarti and ph. vexator from northern california and also in ten species from kenya including se. suberecta, se. clydei, se. garnhami, se. ingrami, se. harveyi, se. schwetzi, se. bedfordi, se. antennata, ph. martini and ph. guggisbergi are a useful method of parity (lewis and minter 1960, chanitois and anderson 1967) in contrast granular secretions were seen in both parus and nuliparus females of lu. cruciata, lu. ovallesi, lu. ylephiletrix, lu. panamensis and lu. shannoni (lewis 1965). accessory glands of lu. townsedi in venezuela showed discordant relation between granular secretions and parity as well (scorza et al. 1968). lewis (1970) reported that accessory glands secretions are a good indicator in eight species such as lu. antunesi, lu. flaviscutellata, lu. ubiquilalis, lu. aragaoi, lu. rorotaensis, lu. infraspinosa, lu. tuberculata and lu. saulensis although with small errors. in all dissected females of lu. gomezi and lu. shannoni accessory glands secretions were seen. as a result, accessory glands are not useful for distinguishing parity (hashiguchi 1987). takaoka et al. (1989) reported that accessory glands are a reliable sign of parity in females of lu. ayacuchensis. but they are not suitable in six species including lu. trapidoi, lu. hartmanni, lu. carrerai, lu. thula, lu. panamensis, lu. shannoni and lu. gomezi. the results of above studies show that the validity of accessory glands secretions as an indicator to recognize parous from nulliparous depends on the species of sand flies. according to the results of the current study, accessory gland secretions are not good marker for determining parus from nulliparous females of ph. papatasi. conclusion this study obviously demonstrates that microscopic test of the accessory gland secretions is not reliable method for determining parus from nulliparous females of ph. papatasi therefore a new method should be introduced to recognize parity status of this species. acknowledgements this study was supported by research deputy of tehran university of medical sciences, project no. 26140-27-03-93. we are grateful to ms gholampour, school of public health, tums, for their excellent assistance in sand fly rearing. the authors declare that there is no conflict of interests. references adler s, theodor o (1935) i investigations on mediterranean kala azar. viii.-further observations on mediterranean sandflies, proc r soc lond b biol sci. 116(801): 505–515. akhavan aa, yaghoobi-ershadi mr, khamesipour a, mirhendi h, alimohammadian mh, rassi y, arandian mh, jafari r, abdoli h, shareghi n, ghanei m, jalalizand n (2010) dynamics of leishmania infection rates in rhombomys opimus (rodentia: gerbillinae) population of an j arthropod-borne dis, march 2017, 11(1): 161–165 m fatemi et al.: assessing the ovarian … 165 http://jad.tums.ac.ir published online: march 14, 2017 endemic focus of zoonotic cutaneous leishmaniasis in iran. bull soc pathol exot. 103(2): 84–89. chaniotis bn, anderson ja (1967) age structure, population dynamics and vector potential of phlebotomus in north california. part i: distinguishing parous from nuliparous flies. j med entomol. 4: 251–254. dejeux p (1991) information on the epidemiology and control of the leishmaniasis, by country or territory. world health organ, geneva. detinova ts (1962) age-grouping methods in diptera of medical importance. wld hlth org. 47: 216. hashiguchi y (1987) studies on new world leishmaniasis and its transmission, with particular reference to ecuador, kyowa printing and ltd, kochi. 174. killick-kendrick m, killick-kendrick r (1991) the initial establishment of sand fly colonies. parassitologia. 33: 313–320. lewis dj (1965) internal structural features of some central american phlebotomine sandflies. ann trop med parasit. 54: 351–365. lewis dj, lainson r, shaw jj (1970) determination of parous rates in phlebotomine sandflies with special reference to amazonian species. bull entomol res. 60: 209–219. lewis dj, minter dm (1960) internal structural changes in some african phlebotominae. ann trop med parasitol. 54: 351–365. modi gb, tesh rb (1983) a simple technique for mass rearing lutzomyia longipalpis and phlebotomus papatasi (diptera: psychodidae) in the laboratory. j med entomol. 20: 568–569. mohebali m, javadian e, yaghoobi-ershadi mr, akhavan aa, hajjaran h, abaei mr, (2004) charactrization of leishmania infection in rodents from endemic areas of islamic republic of iran. east mediterr health j. 10: 591–599. ready pd, lainson r, wilkes tj, killickkendrick r (1984) on the accuracy of age-grading neotropical phlebotomines by counting follicular dilatations: first laboratory experiments, using colonies of lutzomyia flaviscutellata (mangabeira) and l. furcata (mangabeira) (diptera: psychodidae). bull entomol res. 74: 641–646. scorza jv, ortiz i, gomez i (1968) observationes biologicas sobre algunos flebotomos de rancho grande (venezuela). acta boil venez. 6: 52–65 (in spanish). shirzadi m (2010) guideline for control of cutaneous leishmaniasis. department of zoonosis, cdc, ministry of health and medical education, tehran, iran. takaoka h, gomez e, alexander j, hashiguchi y (1989) observations on the validity of the ovarian accessory glands of seven ecuadorian sand fly species (diptera: psychodidae) in determining their parity. japan j trop med hyg. 17(2): 149–155. who (2010) control of the leishmaniases: report of a meeting of the who expert commitee on the control of leishmaniases, geneva, 22–26 march 2010. yaghoobi-ershadi mr, javadian e, tahvildare-bidruni gh (1994) the isolation of leishmanha major from phlebotomus (paraphleboyomus) caucasicus,in isfahan province, islamic republic of iran. trans r soc trop med hyg. 88: 518–519. yaghoobi-ershadi mr, akhavan aa, mohebali m (1996) meriones libycus and rhombomys opimus (rodentia: gerbillidae) are the main reservoir hosts in a new focus of zoonotic cutaneous leishmaniasis in iran. trans r soc trop med hyg. 90: 503–504. young dg, perkins pv, endris rg (1981) a larval diet for rearing phlebotomine sandflies (diptera: psychodidae). j med entomol. 18(5): 446. microsoft word 2dr yaghoobi rtl.doc iranian j arthropod-borne dis, 2010, 4(1): 17–23 mr yaghoobi-ershadi et al.: sand fly surveillance within … 17 original article sand fly surveillance within an emerging epidemic focus of cutaneous leishmaniasis in southeastern iran *mr yaghoobi-ershadi1, m hakimiparizi1, ar zahraei-ramazani2, h abdoli2, aa akhavan1, m aghasi3, mh arandian2, aa ranjbar4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran 2isfahan training and health research center, national institute of health research, tehran university of medical sciences, iran 3school of public health, kerman university of medical sciences, iran 4control of communicable disease unit, department of public health, rafsanjan university of medical sciences, iran (received 19 dec 2009; accepted 10 apr 2010) abstract background: cutaneous leishmaniasis due to leishmania major has become a hot topic in iran. the objective of this study was to determine some ecological aspects of sand flies in the study area. methods: sand flies were collected biweekly from indoors and outdoors fixed places in the selected villages, using 30 sticky paper traps from the beginning to the end of the active season of 2006 in kerman province, south of iran. the flies were mounted and identified. some blood fed and gravid female sand flies of rodent burrows and indoors were dissected and examined microscopically for natural promastigote infection of leishmania parasite during august to september. results: in total, 2439 specimens comprising 8 species (3 phlebotomus and 5 sergentomyia) were identified. the most common sand fly was p. papatasi and represented 87.1% of sand flies from indoors and 57.2% from outdoors. the activity of the species extended from april to end october. there are two peaks in the density curve of this species, one in june and the second in august. natural promastigote infection was found in p. papatasi (12.7%). conclusion: phlebotomus papatasi is considered as a probable vector among gerbils and to humans with a high percentage of promastigote infection in this new focus of cutaneous leishmaniasis. the bahraman area which until recently was unknown as an endemic area seems now to represent a focus of zoonotic cutaneous leishmaniasis transmission in iran. keywords: sand fly, phlebotomus papatasi, ecology, leishmania major, iran introduction phlebotomine sand flies are of widespread importance in the transmission of leishmania pathogens in iran. increasing knowledge of sand flies is a cornerstone for establishing control measures to prevent leishmaniasis. cutaneous and visceral leishmaniasis are endemic in iran and remain to be a growing health threat to community, development and the environment in our country (nadim et al. 1994). in the last two decades, the reported cases especially that of cutaneous leishmaniasis due to leishmania major (clm) have increased, and they were reported in areas that previously were known to be nonendemic by the public health services (yaghoobiershadi et al. 2001, 2004). this ignored form of neglected diseases is common in many rural areas of 17 out of the 30 provinces in iran. about *corresponding author: prof mohammad-reza yaghoobiershadi, e-mail: yaghoobi.reza@gmail.com iranian j arthropod-borne dis, 2010, 4(1): 17–23 mr yaghoobi-ershadi et al.: sand fly surveillance within … 18 80% of leishmaniasis cases reported in the country are of the clm form. based on the reports of control of communicable disease unit, rafsanjan health center, southern iran, cutaneous leishmaniasis has not been presented in the area until 2002. since 2004 the reported cases of cutaneous leishmaniasis have suddenly increased in the study area. during 2003-2004, a total of 257 cases were officially reported all by passive case detection, most of them were from bahraman district (rafsanjan health center, unpublished data) 70 km from rafsanjan county but this is probably a large underestimate. a preliminary survey showed that rhombomys opimus was the main reservoir host and zoonotic cutaneous leishmaniasis is caused by l. major (school of public health, tehran university of medical sciences, unpublished data). bahraman rural district (noogh) of rafsanjan county is an important agricultural axis from the point of view of growing pistachio in iran so in depth entomological study must be carried out for preparation of control program to prevent the spread of disease to the neighboring villages and counties. the objective of this study was to determine the species richness and relative abundance of sand flies, their distribution, monthly prevalence, number of generations and leishmanial infection of sand flies in the area. materials and methods study site the investigation was carried out from 10 april to 11 october 2006 in 5 villages, javadieh, aliabad, ahmadabad, najmabad and daghogh, in the rural district of bahraman, rafsanjan county, kerman province, in the southeast of the islamic republic of iran. rafsanjan county (29º 45´ n, 54º 50´ 56º 45´ e) is situated in the south east of iran, 100 km northwest of kerman at an altitude of 1460 m above sea level. it has a desert climate, very hot and dry in summer, cold and dry in winter. in 2006, the maximum mean monthly temperature was 31.9º c (july) and the minimum was 5.6º c (january). the total annual rainfall was 32.6 mm, with a monthly minimum of 0.2 mm (july and august) and maximum of 17.4 mm (january). the minimum mean monthly relative humidity was 14% in august and the maximum was 61% in march (rafsanjan meteorological office, personal communication) sand flies sampling and monitoring sand flies were collected biweekly from fixed sites indoors (bedrooms, sitting rooms, toilets and stables) and outdoors (rodent burrows), using 30 sticky traps (castor oil-coated white papers 20cm×30cm) from the beginning to the end of active season (early aprilmid october). collected sand flies were stored in 70% ethanol. for species identification, sand flies were mounted in puri's medium, produced at the medical entomology department (smart et al. 1965) and identified after 24 h using the keys of theodor and mesghali (theodor and mesghali, 1964), then they were mounted and segregated by sex. detection of natural leptomonad infection in sand flies and their age determination sand fly collections were made during august to september in the selected villages using sticky traps around rodent burrows and aspirator from human dwellings. collections were transferred to the laboratory at rafsanjan health center for dissection. blood fed females collected indoors were kept alive for 3-4 days to allow blood digestion and then dissected. all fed, gravid, semi-gravid and some unfed females collected from rodent burrows were dissected in a fresh drop of sterile saline (9/1000) and examined microscopically for natural promastigote infection in the alimentary canal. the physiological age of each iranian j arthropod-borne dis, 2010, 4(1): 17–23 mr yaghoobi-ershadi et al.: sand fly surveillance within … 19 female was determined by the presence or absence of granules in the accessory glands (foster et al. 1970). all female sand flies were identified by the morphology of the pharyngeal armature and of the spermatheca (theodor and mesghali 1964). results in this entomological survey, a total of 2439 adult sand flies (2152 from rodent burrows and 287 from indoor resting places) were collected and identified. they comprised 8 phlebotomine species with three phlebotomus spp. and five sergentomyia spp. (table 1). the following five spices were found indoors: p. papatasi (87.1%); p. sergenti (0.7%); s. clydei (5.2%); s. sintoni (1.4%) and s. baghdadis (5.6%). in the rodent burrows: p. papatasi (57.2%); p. sergenti (0.2%); p. mongolensis (0.05%); s.clydei (22.7%); s. tiberiadis (0.05%); s. sintoni (13.3%); s. dentata (0.1%) and s. baghdadis (6.4%). the most prevalent species of indoors were p. papatasi, s. clydei and s. baghdadis and in case of outdoors they were p. papatasi, s. clydei, s. sintoni and s. baghdadis respectively. while other species were less frequent. the number and relative abundance of these species are given in table 1. phlebotomus papatasi started to appear in late april and disappeared in mid october in rodent burrows. there are two peaks in the density curve of this species, one in early june and the second in early august (fig. 1) while, the first peak of activity of the species was at the end of may and the second in mid of july in indoors. the decrease in sand fly density in mid october was most probably due to the cold weather and rains. there are also two peaks in the density curve of other species in rodent burrows in the area, one in late may or early june and the second in middle of august (fig. 2). the sex ratio, i.e., number of males per 100 females of p. papatasi was 525 and 126 in indoors and rodent burrows respectively by the trapping method, sticky trap. p. papatasi (165), s. clydei (8), s. baghdadis (2) and s. sintoni (2) were collected in the vicinity of rodent burrows and were dissected. the results of these dissections showed that p. papatasi (12.7%) was infected with promastigotes. in three cases a large number of promasitgotes were observed in the gut as well as in the head of p. papatasi. in september 2006, six p. papatasi were collected from indoor places and dissected for the presence of promastigotes. all were negative (table 2). 0 50 100 150 200 250 9 apr. 23 apr. 6-m ay 20-m ay 29-m ay 10-jun 26-jun 11-jul 29-jul 14 aug. 29 agu. 15 sept. 27 sept. 11 oct. month,2006 no .o fs an d fli es pe r 30 tr ap s p.papatasi outdoors p.papatasi indoors fig. 1. monthly prevalence of p. papatasi in indoors and outdoors, bahreman area, rafsanjan county, 2006 iranian j arthropod-borne dis, 2010, 4(1): 17–23 mr yaghoobi-ershadi et al.: sand fly surveillance within … 20 0 20 40 60 80 100 120 140 9 a pr . 23 ap r. 6ma y 20 -m ay 29 -m ay 10 -ju n 26 -ju n 11 -ju l 29 -ju l 14 au g. 29 ag u. 15 se pt . 27 se pt . 11 oc t. day of sand fly collection no .o fs an d fli es pe r 30 tr ap s s.clydei s.baghdadis s.sintoni fig. 2. monthly prevalence of s. sintoni, s. baghdadis and s. clydei in bahreman area, rafsanjan county (rodent burrows), 2006 table 1. richness, number, and relative abundance of sand fly species collected from indoors and outdoors in bahraman rural district, rafsanjan county, kerman province, iran, 2006 table 2. natural promastigote infection in sand flies collected from rodent burrows, bahraman rural district, rafsanjan county, kerman province, iran, summer 2006 site species no. of sandflies dissected age group no. of sandflies with promastigotes % infected n p ? t g o h outdoors p. papatasi 165 13 141 11 21 18 3 3 12.7 s. clydie 8 1 6 1 0 0 0 0 0 s. baghdadis 2 0 2 0 0 0 0 0 0 s. sintoni 2 0 1 1 0 0 0 0 0 indoors p. papatasi 6 0 5 1 0 0 0 0 0 t, total; g, gut; o, oesophagus; h, head; p, parous; n, nuliparous;? age not known* *some females that oviposited all of their eggs had accessory glands devoid of identifiable granules so recognizing parous sand flies from nuliparous ones was impossible. species collection site richness no. and (%) of sand flies p .papatasi p .sergenti p . m ongolensis s.clydei s.tiberiadis s.sintoni s.dentata s.baghdadis no. 250 2 0 15 0 4 0 16 indoors 5 % 87.1 0.7 0 5.2 0 1.4 0 5.6 outdoors 8 no. 1230 4 1 490 1 286 2 139 % 57.2 0.2 0.05 22.7 0.05 13.3 0.1 6.4 iranian j arthropod-borne dis, 2010, 4(1): 17–23 mr yaghoobi-ershadi et al.: sand fly surveillance within … 21 discussion in the rural district of bahraman, kerman province, cutaneous leishmaniasis due to l. major is a serious and increasing public health problem (rafsanjan health center 2005, kerman university of medical sciences, unpublished data). this paper presents the first report of sand flies in this focus. eight species (three of phlebotomus and five of sergertomyia) were collected and identified. p. papatasi, a proven vector of l. major in iran (yaghoobi-ershadi et al. 1995), is the most abundant species in bahraman, specially in indoors. phlebotomus sergenti accounts for 0.7% and 0.2% of sand flies collected from indoors and outdoors respectively and was found only in some villages in the region. this species has never been implicated in the transmission of l. major in iran. only one male of p. mongolensis (0.05%) was collected from rodent burrows. with regard to its low frequency, it seems that the role of females of this species in the circulation of l. major among the rodents is doubtful in this part of iran. phlebotomus papatasi has two peak activities in indoors and outdoors and it represents that the spices has two generations in the study area. in routine collections by sticky traps in indoors and rodent burrows, males of p. papatasi predominate. the same results were also obtained on s. baghdadis and s. clydei in rodent burrows. in case of s. sintoni collected from outdoors, females were predominated. these results conformed to what was happened in other parts of the country such as isfahan, badrood, natanz, nikabad, ardestan, abardezh of varamin, sabzvar, garmsar and so forth (yahgoobiershadi and javadian 1997,yaghoobi-ershadi et al. 2001, 2004, 2003, akhavan et al. 2007). it should be mentioned that the parameter, sex ratio varies greatly according to the trapping method and among species of genus phlebotomus and sergentomyia (schlein et al.1989). the estimation of the age structure by dissection of the 165 females of p. papatasi from rodent burrows during the period of observation, showed an overall parous: nuliparous ratio of 141:13. it should be mentioned that about 6.7% of the females of this species that would have oviposited all of their eggs had accessory glands devoid of identifiable granules so recognizing parous sand flies from nuliparous ones were impossible. leishmanial infection rates of p. papatasi from rodent borrows of the other clm foci in iran (i.e. abardezh, ahwaz, dezful, isferayen, lotfabad, shush, turkmen sahra, isfaham and badrood) ranged from 0.2% to 15.6% during 1967-2001 (mesghali et al. 1967,javadian et al. 1976, nadim et al. 1994, yaghoobi-ershadi and akhavan 1999). in the present study, 12.7% of the vector species were infected. comparison of the present findings with those obtained from iran and other countries (nadim et al. 1994, janini et al. 1995) showed high natural infection rates with promastigotes from rodent burrows in rafsanjan of southern iran. based upon these entomological data, p. papatasi is considered the most likely vector of cl in rafsanjan. however, further studies are necessary to isolate leishmania strains from p. papatasi and to characterize the genotype of rafsanjan sand flies, taking into account current findings about the multilocus microsatellite typing which has been employed to infer the population structure of p. papatasi in provinces of khorasan, khuzestan, baluchistan, azerbaijan, isfahan, fars and yazd of iran (hamarsheh et al. 2009). in conclusion, based on this survey, bahraman rural district seems to be a typical focus of p. papatasi and l. major. it should be added to the list of cl foci due to l. major, so it is necessary to propose a new update map of the distribution of these foci in iran. iranian j arthropod-borne dis, 2010, 4(1): 17–23 mr yaghoobi-ershadi et al.: sand fly surveillance within … 22 acknowledgments we are grateful to dm gooya, director general, center of communicable diseases control and dr a mozaffari from rafsanjan university of medical sciences for their close collaboration. our appreciation is also offered to m alizadeh, k mohammadi poor and m mozaffari from rafsanjan health center for helping us in carrying out this program. this study was financially supported by national institute of health research, academic pivot for education and research, tehran university of medical sciences ( project no: 241.68.1) and partly by tehran university of medical sciences (grant number 3215-63-0285 to dr mr yaghoobi-ershadi). the authors declare that they have no conflicts of interest. references akhavan aa, yaghoobi-ershadi mr, hasibi f, jafari r, abdoli h, arandian mh, soleimani h, zahraeiramazani ar, mohebali m, hajjarian h (2007) emergence of cutaneous leishmaniasis due to leishmania major in a new focus of southern iran. iranian j arthropod-borne dis. 1(1): 1–8 foster wa, tesfayohannes tm, tecle t (1970) studies on leishmaniasis in ethiopia, ii. laboratory culture and biology of phlebotomus longipes (diptera: psychodidae). ann trop med parasitol. 64: 403–409 hamarsheh o, presber w, yaghoobiershadi mr, amro a, aljawabreh a, sawalha s, et al. 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pcr-based diagnosis with centrifugedbased enrichment method for detection of borrelia persica in animal blood samples *sr naddaf1, m kishdehi2, mr siavashi1 1department of parasitology, pasteur institute of iran, tehran, iran 2department of microbiology, lahijan azad university, gilan, iran (received 12 oct 2010; accepted 24 may 2011) abstract background: the mainstay of diagnosis of relapsing fever (rf) is demonstration of the spirochetes in giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by microscopy. this study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spirochetemia. methods: animal blood samples with low degrees of spirochetemia were tested with two pcrs and a nested pcr targeting flab, glpq, and rrs genes. also, a centrifuged-based enrichment method and giemsa staining were performed on blood samples with various degrees of spirochetemia. results: the flab-pcr and nested rrs-pcr turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. the glpq-pcr was positive as far as at least one spirochete was seen in 5-10 microscopic fields. the sensitivity of glpq-pcr increased when dna from buffy coat layer (bcl) was used as template. the centrifuged-based enrichment method turned positive with as low concentration as 50 bacteria/ml blood, while giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml. conclusion: centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some pcr assays. due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of rf in rural health centers. keywords: borrelia persica, relapsing fever, diagnosis, pcr, enrichment method introduction relapsing fever (rf) is an infectious disease with a sudden onset of high fever; it is caused by several species of bacteria belonging to the genus borrelia and, as its name indicates, is characterized by the occurrence of one or more spells of fever after the subsidence of the primary febrile attack (burgdorfer 1976). the presence of massive amounts of spirochetes during fever peaks makes diagnosis of the infection an easy practice with dark-field microscopy or giemsa staining method (assous and wilamowski 2009). however, between the peaks and in milder infections these methods are often negative due to a low number of bacteria in blood stream, making the infection under diagnosed. pcr assays that target different genes including 16s ribosomal rna (rrs), flag-ellin (flab), and glycerophosphodiester phosphodiesterase (glpq) were successfully used for detection of spirochetes in blood and ornithodoros tick vectors (ras et al. 1996, assous et al. 2006, halperin et al. 2006, nordstrand et al. 2007, oshaghi et al. 2010). species-specific pcr and rcr-rflp method were also developed for diagnosis of some borrelia species (assous and wilamowski *corresponding author: dr saied reza naddaf, e-mail: snaddaf_2001@yahoo.com iran j arthropod-borne dis, 2011, 5(1): 7–12 sr naddaf et al.: comparison of pcr-based … 8 2009, oshaghi et al. 2010). however, the method is costly, labor intensive and requires well-equipped laboratories. the elisa based on product of glpq gene can discriminate between rf and lyme borreliosis but cannot differentiate between an active and past rf infection (schwan et al. 1996). quantitative buffy coat (qbc) analysis of blood samples showed to be a reliable for diagnosis of rf when the spirochetes are low in numbers, but it relies on florescent microscopy, the equipment rarely available in rural health centers (van dam et al. 1999, cobey et al. 2001). recently, a novel centrifugation-based method with minimal laboratory requirements showed very promising, detecting concentrations less than 10 bacteria/ml blood (larsson and bergstrom 2008). in this study we compare the sensitivity of several pcr assays that amplify dna sequences of three different loci including rrs, flab, and glpq with the centrifugation-based enrichment method and giemsa staining to detect bacteria in animal blood samples. materials and methods borrelia borrelia persica was isolated from ornithodoros tholozani ticks, collected from ardebil province, and maintained in guinea pigs through serial passages for seven successive years. blood samples adult guinea pigs were inoculated intraperitoneally with 0.5 ml of b. persica infected blood preserved in -70º c with 50% glycerol. from day three, daily amount of 500-600µ l of blood was taken from animals’ heart using insulin syringes and bacteria count was determined using dark-field microscopy. blood collection continued for eight days, until no spirochete was detectable in the blood samples with dark-field microscopy. the intensity of infection in blood samples was obtained by counting the spirochetes using a neubauer haemocytometer. when animals were negative for two successive days (days nine and ten) large amounts of blood was collected from their hearts and amounts of 10 ml were examined for presence of spirochetes using centrifuged-based enrichment method. also, buffy coat layer (bcl) was obtained from 3 ml of the same blood samples using the lymphocyte®-h kit (cedarlane, netherland). we also prepared fifteen serial dilutions from an infected guinea pig blood sample containing 25×104 spirochetes/ml blood, with sodium citrate-anticoagulated blood from healthy individuals (table 3). giemsa-stained thin and thick smears were prepared in triplicate from all dilutions and the rest of blood samples were examined for presence of spirochetes using centrifuged-based enrichment method. centrifuged-based enrichment method the method basically comprised two centrifugation steps; the blood samples were first centrifuged at 500 x g for 5 min, the plasma were recovered to new tubes and second centrifugation was performed at 5000 x g for 10 min. the supernatants were decanted, and the pellets were resuspended in the few remaining microliters of plasma. the suspensions were smeared onto a glass slides and air dried for 10 min. the smears were fixed by heating over a flame followed by a 30 s dip in methanol. the slides were stained with giemsa and examined for recovered spirochetes at 1000x magnification. dna extraction method and pcr amounts of 200µ l of blood samples and 500 µ l of buffy coats were subjected to dna extraction, using the miniprep dna extraction kit (kiagen, germany) according to manufacturer's recommendations. detection of b. persica by pcr was performed through amplification of three different genes including rrs, flab, and glpq. the flab and glpq genes were amplified using the primers and thermocycler programs outlined by other authors (assous et al. 2006, halperin et al. 2006). the 25µl reactions contained 20 pmol of each primer, 1.7mm mgcl2, 10mm tris-hcl, iran j arthropod-borne dis, 2011, 5(1): 7–12 sr naddaf et al.: comparison of pcr-based … 9 50 mm kcl, 200m of dntps, 1u of taq and 3µl of dna. amplification of rrs was performed using the nested pcr technique as described previously (brahim et al. 2005, nordstrand et al. 2007). the list of primers, target genes, and expected band sizes are reflected in table 1. table 1. list of primers used in this study primer sequence (5’ to 3’) target gene expected band size 128f cag aac ata cct tag aag ctc aag c glpq 212 bp 340r gtg att tga ttt ctg cta atg tg bor1 taa tac gtc agc cat aaa tgc flab 750 bp bor2 gct ctt tga tca gttatc att c fd3 aga gtt tga tcc tgg ctt ag rrs 1st round 613 bp 595r ctt gca tat ccg cct act ca fd4 ggc tta gaa cta acg ctg gca g 2nd round 527 bp 500r ctg ctg gca cgt aat tag cc results pcr the glpq-pcr did not yield the expected 212bp band with low sprirochetemia blood samples i.e. those that were negative with dark-field microscopy (table 1, days 9 and 10). however, the dna from bcl of the same blood samples was successfully amplified using the same reagents and pcr conditions. the flab-pcr and nested rrs-pcr were positive with various degrees of spirochetemia including those that were negative by dark-field microscopy. the details of pcr assays are shown in table 2. centrifugation-based enrichment method centrifugation-based enrichment recovered 890 and 357 spirochetes form two 10 ml of blood samples that were negative with dark-field microscopy examination (table 2). we could recover 25 spirochetes on the glass slides by microscopy when as low as about 250 bacteria (a concentration equivalent to 50 bacteria/ml) were spiked into 5ml blood samples. no spirochete was recovered with concentration below 25 bacteria in ml of blood (table 3). geimsa-stained smears analysis geimsa-stained thin smears were consistently positive with samples containing ≥105 spirochetes/ml blood, but only two of the three smears at concentrations 6.26×104 spirochetes/ ml and one of three smears at concentrations 5×104 spirochetes/ml were positive. the giemsastained thick smears were all positive at ≥ 5×104 spirochetes/ml, but only two of the three smears at concentrations 4×104 and 3×103 spirochetes/ ml, and one of the three smears at concentration 25×103 spirochetes/ml were positive (table 2). table 2. details of blood samples and other blood products used for pcr assays samples days after inoculation dark field microscopy spirochetes in µl of blood type and amount of sample glpq pcr flab pcr nested rrspcr spirochetes recovered by cem 1 3 10p ƒ 2750 blood/200 µl + + + np 2 4 20p ƒ 6750 blood/200 µl + + + np 3 5 1p5 ƒ 375 blood/200 µl + + + np 4 6 1p ƒ 1500 blood/200 µl + + + np 5 7 1p10 ƒ 250 blood/200 µl + + + np 6 8 1p5ƒ 250 blood/200 µl + + + np 7 9 neg. not seen blood/200 µl neg. + + np 8 10 neg. not seen blood/200 µl neg. + + 870 and 375 9 9 neg. not seen bcl/500 µl + np np np 10 10 neg. not seen bcl/500 µl + np np np bcl = buffy coat layer, np = not performed, cbe = centrifuged-based enrichment method 10pƒ=10 spirochetes in one microscopic field, 20pƒ= 20 spirochetes in one microscopic field, 1p5ƒ= 1spirochete in 5 microscopic fields, 1pƒ= 1 spirochetes in one microscopic field, 1p10ƒ=1spirochete in 10 microscopic fields, neg.= negative, no spirochetes was seen in 30 microscopic fields. iran j arthropod-borne dis, 2011, 5(1): 7–12 sr naddaf et al.: comparison of pcr-based … 10 table 3. details of blood samples used for giemsa-stain analysis and centrifuged-based enrichment method sample code amounts of blood (ml) number of bacteria spiked into giemsastained thin smears giemsastained thick smears number of recovered bacteria ratio of spirochetes (recovered/spiked into) 1 5.00 250000 + + + + + + np na 2 5.00 150000 + + + + + + np na 3 6.00 125000 + + + + + + 970 1/128 4 5.00 100000 + + + + + + np na 5 5.00 62500 + + ─ + + + np na 6 5.00 50000 + ─ ─ + + + np na 7 5.00 40000 ─ ─ ─ + + ─ np na 8 5.00 30000 ─ ─ ─ + + ─ np na 9 5.20 25000 ─ ─ ─ + ─ ─ 255 1/98 10 5.00 20000 ─ ─ ─ ─ ─ ─ np na 11 5.10 12500 ─ ─ ─ ─ ─ ─ 132 1/94 12 5.00 6250 ─ ─ ─ ─ ─ ─ 52 1/120 13 5.00 1250 ─ ─ ─ ─ ─ ─ 31 1/40 14 5.00 625 ─ ─ ─ ─ ─ ─ 25 1/25 15 5.00 250 ─ ─ ─ ─ ─ ─ 25 1/10 16 5.00 125 ─ ─ ─ ─ ─ ─ 0 0 np= not performed, na= non applicable discussion tick-borne relapsing fever (tbrf) is one of the prevalent bacterial diseases in different parts of the world (karimi 1981, barmaki et al. 2010). the disease in iran is caused primarily by borrelia persica, which is transmitted by ornithodoros tholozani ticks. other borrelia species including b. microtii, b. latyschevii, and b. baltazardi have also been reported from iran (karimi et al. 1979, karimi 1981). from 1997 to 2006, a total of cases 1415 were reported from the entire country, some from areas out of o. tholozani distribution includ hormozgan and fars provinces (masoumi asl et al. 2009). most of the rf cases in hormozgan province were detected during attempts for diagnosis of malaria parasite in giemsa-stained blood smears from febrile patients. since, thick smears commonly turn positive during fever peaks that are associated with massive spirochetemia; it is posssible that a large number of rf cases that refer to hospitals and health care centers during non-febrile periods remain underdiagnosed. the inabilities of microscopic analysis to detect spirochetes in blood have been demonstrated by some authors (assous et al. 2006, halperin et al. 2006, nordstrand et al. 2007). pcrbased diagnosis offered a new approach to this problem. our results showed that the flabpcr could detect borrelia flagellin dna in blood samples with low grade bacteremia that are commonly negative by dark-field microscopy (table 2). the nested rrs-pcr also showed very sensitive and was positive with various degrees of bacteremia (table 2); however it was very vulnerable to cross-contamination and led to false positive results when positive controls were included in assays. dilution of first round products with distilled water with 1:10 ratio reduced the false positivity results by 90%. the glpq-pcr was not positive with low grade spirochetemia blood samples i.e. those that turned negative by darkfield microscopy. however, the dna from bcl of the same blood samples yielded the expected band with the same pcr protocol (table 2). accumulation of spirochetes in bcl was already documented by other authors (van iran j arthropod-borne dis, 2011, 5(1): 7–12 sr naddaf et al.: comparison of pcr-based … 11 dam et al. 1999, cobey et al. 2001). thus, extraction of dna from bcl increases the chance of detecting infection with pcr, particularly during non febrile periods. we could recover 870 and 357 spirochetes by centrifuged–based enrichment method from two 10 ml of guinea pig blood samples that were negative by dark-field microscopy and glpqpcr. in our study, the sensitivity of the centrifuged–based enrichment method improved as number of spirochetes spiked into the blood samples decreased; the highest ratio (recovered/ spiked into) was obtained with 250 spirochetes/ 5ml of blood (table 3). since the sensitivity of thick smears, the routine method for detection of rf agents, was around 25000 spirochetes in ml of blood (table 2), the centrifuged–based enrichment method could be as much as 500 times more sensitive than thick smear analysis. in conclusion, pcr particularly when dna is extracted from bcl is a useful tool for diagnosis of rf cases that cannot be diagnoses by microscopic analysis. however, the method is commonly available in big hospitals and well-equipped laboratories. centrifuged based enrichment method showed a high sensitivity and even appeared to be superior to glpq-pcr. regarding the fact that it requires the equipments that are commonly available in small laboratories, this method is more feasible for rf diagnostics in underprivileged rural health centers. acknowledgements we are very grateful to dr n piazak for his technical assistance and dr m assmar for his guidance throughout the project. this study was a m.sc. thesis project founded by pasteur institute of iran (project no. 500). references assous mv, wilamowski a (2009) relapsing fever borreliosis in eurasia-forgotten, but certainly not gone! clin microbiol infect. 15(5): 407–414. assous mv, wilamowski a, bercovier h, marva e (2006) molecular characterization of tickborne relapsing fever borrelia, israel. emerg infect dis. 12(11): 1740–1743. barmaki a, rafinejad j, vatandoost h, telmadarraiy z, mohtarami f, leghaei sh, oshaghi ma (2010) study on presence of borrelia persica in soft ticks in western iran. iran j arthropod-borne dis. 4(2): 19–25. brahim h, perrier-gros-claude jd, postic d, baranton g, jambou r (2005) identifying relapsing fever borrelia, senegal. emerg infect dis. 11(3): 474–475. burgdorfer w (1976) the epidemiology of the relapsing fevers. in: johnson erc (ed): biology of parasitic spirochetes, academic press, new york, pp. 191– 200. cobey fc, goldbarg sh, levine ra, patton cl (2001) short report: detection of borrelia (relapsing fever) in rural ethiopia by means of the quantitative buffy coat technique. am j trop med hyg. 65(2): 164–165. halperin t, orr n, cohen r, hasin t, davidovitch n, klement e, kayouf r, baneth g, cohen d, yavzori m (2006) detection of relapsing fever in human blood samples from israel using pcr targeting the glycerophosphodiester phosphodiesterase (glpq) gene. acta trop. 98(2): 189–195. karimi u (1981) relapsing fever and its epidemiology. pasteur institute of iran (pii), tehran (persian). karimi y, hovind-hougen k, birch-andersen a, asmar m (1979) borrelia persica and b. baltazardi sp. nov. experimental pathogenicity for some animals and comparison of the ultrastructure. annales de microbiologie. 130b(2): 157– 168. iran j arthropod-borne dis, 2011, 5(1): 7–12 sr naddaf et al.: comparison of pcr-based … 12 larsson c, bergstrom s (2008) a novel and simple method for laboratory diagnosis of relapsing fever borreliosis. open microbiol j. 2: 10–12. masoumi asl h, goya mm, vatandoost h, zahraei sm, mafi m, asmar m, piazak, n, aghighi, z (2009) the epidemiology of tick-borne relapsing fever in iran during 1997–2006. travel med infect dis. 7(3): 160–164. nordstrand a, bunikis i, larsson c, tsogbe k, schwan tg, nilsson m, bergstrom s (2007) tickborne relapsing fever diagnosis obscured by malaria, togo. emerg infect dis. 13(1): 117–123. oshaghi ma, rafinejad j, choubdar n, piazak n, vatandoost h, telmadarraiy z, mohtarami f, ravasan nm (2011) discrimination of relapsing fever borrelia persica and borrelia microtti by diagnostic species-specific primers and polymerase chain reaction-restriction fragment length polymorphism. vector borne zoonotic dis. 11(3): 201– 207. ras nm, lascola b, postic d, cutler sj, rodhain f, baranton g, raoult, d (1996) phylogenesis of relapsing fever borrelia spp. int j syst bacteriol. 46(4): 859–865. schwan tg, schrumpf me, hinnebusch bj, anderson de, jr.konkel me (1996) glpq: an antigen for serological discrimination between relapsing fever and lyme borreliosis. j clin microbiol. 34(10): 2483–2492. van dam ap, van gool t, wetsteyn jc, dankert j (1999) tick-borne relapsing fever imported from west africa: diagnosis by quantitative buffy coat analysis and in vitro culture of borrelia crocidurae. j clin microbiol. 37(6): 2027– 2030. j arthropod-borne dis, march 2018, 12(1): 1–15 z saeidi and h vatandoost: aquatic insect … 1 http://jad.tums.ac.ir published online: march 18, 2018 review article aquatic insect from iran for possible use of biological control of main vector-borne disease of malaria and water indicator of contamination zahra saeidi 1, *hassan vatandoost 1, 2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran (received 16 oct 2017; accepted 12 mar 2018) abstract iran has a wide variety of zoogeographical regions and different seasons. here are some important mosquito -borne diseases. mosquitoes normally live in waters. its aquatic insect fauna is highly unexplored. to being resolved this faunal gap, a variety of literature records from previous century in different parts of iran was reviewed. in some southern and southeastern foci in iran, malaria is still a main endemic disease which is unstable with two seasonal spring and autumn peaks even though iran is lunching malaria elimination. this review article showed the wide variety of aquatic insects throughout the country. researchers can discuss water pollutant and its quality by using aquatic insect fauna as well as biological control for vectors. types of aquatic insects and macroinvertebrates sampling can be useful for water quality monitoring as indicators. looking at aquatic insects’ life in water could be one of the most cost-effective and the easiest method to assess the water contaminations by different pollutants and will provide a guideline for scientific communities and environmental agencies for decision making. keywords: aquatic insects, arthropod-borne diseases, iran, water quality introduction there are some important arthropod-borne diseases in iran including malaria, cutaneous leishmaniasis, visceral leishmaniasis, crimeancongo hemorrhagic fever, tick relapsing fever, furthermore scorpions are one of the risk factors for life in some parts, while other arthropod-related diseases such as myiasis exist more or less across the country. some probable arthropod-borne disease in the future may be: q-fever, papatasi fever, tularemia, rift valley fever, dengue fever, yellow fever, west nile viruses, lactodictism (spider bite), plague, scabies, nuisance insects of horseflies and culicidae mosquitoes, cockroach-borne diseases, damages by fire ants, blister beetles and bee stings. in iran with about 15000 annual cases of the disease in recent years, malaria is known as one of the most important parasitic infectious diseases. locally transmitted cases have dropped to 500 recorded cases in 2013. three most prevalence provinces in iran are sistanbaluchestan, hormozgan and kerman which located in south and southeastern part of the country. the rifest route of transmission is immigration from afghanistan and pakistan to this area (ministry of health, annual report). you can find a considerable decline of malaria burden in iran during last 20 years. the disease cases have been reduced from about 100000 cases in 1991 to 246 autochthonous cases in 2014. most of the transmitted cases are reported from the south-eastern part of the country that is related to population traffic across pakistan border beyond the difficulties in malaria control. recent malaria number *corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com j arthropod-borne dis, march 2018, 12(1): 1–15 z saeidi and h vatandoost: aquatic insect … 2 http://jad.tums.ac.ir published online: march 18, 2018 reported is 42 cases all over the country including 23 local malaria patients, 12 imported cases and seven relapsed ones before august 2016. majority of researchers have worked on various aspects of malaria such as insecticide resistance monitoring (1-10) new records, sibling species and molecular studies (11-18). some researchers have worked on vector control using novel methods (19-24), faunestic study (25-26), larval control using various plants (3, 27-38), using bed nets and long lasting impregnated nets (39-46), study on morphology (47-49), malaria epidemiology (5054) malaria vector ecology (18, 39, 52, 55-60), biodiversity (53, 61), community participation (62), vector control (63), repellent evaluation (31, 64), susceptibility against insecticide (6567), anthropophilic index of malaria vectors (68-69) training (70) is nominated as malaria training center by who researchers also can find several reports on different aspects of malaria vectors done in recent years (21, 71-87). although iran has a vast geographical area with a wide range of diversity in climate and animal including insects, its aquatic insect’s fauna remains largely unexplored for years. with a total area about 1.65 million km2, around 7% covered with wateriran is one of the large countries ranked eighteenth in the world (88-89). the aquatic insect has a critical role in biomonitoring of water safety or water contamination. it is inevitable to use such kind of insect to evaluate water quality as a biological indicator and can help us as water resource management. aquatic insects a vital role in energy flow in fresh water and they are important in food web between aquatic animals. historical overview of aquatic insects from iran past century iran aquatic insects have been studied since 1965 by a hand full of researchers. vassil gueorguiev recorded methles rectus from iran, but he did not publish the exact location (88). afterwards, in 1976 some researcher from another field such as environment researchers who surveyed on mayfly and stonefly to determine the acute metal toxicity of some heavy metals such as lead, copper, zinc, and silver. they were found more tolerant than most fish to heavy metals. this study indicated that aquatic insect can help us as effective biological monitors of heavy metals pollution (91). subsequently a survey was conducted on water beetles of southwestern iran and reported haliplidae (two genera, two species), dytiscidae (16 genera, 24 species), and gyrinidae (two genera, two species). hydrophilidae (10 genera, 34 mostly unidentified species) (92). a researcher from a university of shiraz focused on life history, morphology and behaviour of the immature stages of a coleopteran, hydrophilidae in laboratory condition (93). after about 20 years of water beetle collecting from a wide range of area, habitat and provinces in iran founded a small number of m. rectus sharp in a few places in guilan province in a collection made in 1976, 1993 and 1995 in southern part of caspian sea, northern iran (94). current century during 2000–2002 a study on aquatic beetle of tabriz region, east azarbaijan, northwestern iran was conducted and four species out of five species of the family hydraenidae reported a new record (95). during 2001–2005 some specimen collected by vafaei et al. (96) in markazi province central iran and they established the presence of 24 species of aquatic beetles (coleoptera: polyphaga) belonging to 13 genera and five families. in other publication, they claim that they found 33 species of diving beetles belonging to 18 genera during same time and same places (97). in 2005 another team worked on a descriptive study of aquatic insects’ fauna in kashan, central iran. during nine rounds of sampling from four maturation artificial ponds they reported as folhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 1–15 z saeidi and h vatandoost: aquatic insect … 3 http://jad.tums.ac.ir published online: march 18, 2018 lowed: diptera order (52%), including chironomidae and culicidae families, hemiptera (24%) corixidae, notonectidae, copepodae and copepodidae families, ciclopodidae (12%), hydroacarina (9.5%), coleptera (0.77%), aranida (0.67%), hymenoptera (0.58%), odonata (0.48%) (98). in another publication, 31 different plecoptera reported from different families and two families of ephemeroptera (99). during 2006 and 2007 39 species have been found belonging to 16 families in zanjanrud, zanjan province. three specimens belong to lygaeidae, scutelleridae and reduviidae were identified at the genus level. among them, there are some predators’ species such as anthocoris nemorum, nabis pseudoferus, notonecta viridis, velia affinis, gerris maculates, hydrometra stagnorum. the most frequent species belonged to pentatomidae. all species were first records from the zanjan province (100). some families of coleoptera such as dytiscidae, gyrinidae, helophoridae and hydrophilidae with a new record and notes on the rare species coleostoma transcaspicum reitter, 1906 from north part of tehran province was reported (101). work on odonata as effective predators in the rice field and other sites in mazandaran province north of iran (2003–2006). they found 30 species from 19 genera and eight families of odonata (anisoptera and zygoptera suborder. in anisoptera suborder, aeshnidae, five species, one species of corduliidae, two species of gomphidae, 13 species of libellulidae. in zygoptera suborder, one species of calopterygidae, six species of coenagrionidae, one species of euphaeidae, one species of platycnemididae (102). a survey in zayande rud river in esfahan province, central iran during one year and in eight stations who found that the water quality can have an effective impact on diversity and richness of benthic macroinvertebrate (103) recorded total of 47 species belonging to17 families of heteroptera in ghara dagh forest, east azarbayjan, northwestern iran: among them, the species nabis pseudoferus, notonecta viridis, anthocoris nemorum, velia affinis, hydrometra stagnorum and gerris maculates were predators. the most abundant species belonging to pentatomidae. they reported 32 species as new records for the studied area. newly introduced species, stinctopleurus crassicornis and stinctopleurus punctatonervosus, registered for iran insect fauna (104). in another study that carried out in east azarbaijan province on heteroptera, they found 28 species from 12 families and polymerus brevirostris knight, 1925 was a new record (105). in ardabil east azarbaijan province northwestern iran. two species of aquatic beetles belonging to two genera that did not study before from two sampling site during 2000–2008. they faced peltodytes regimbart, 1878 and haliplus latreille, 1802 (106). a species haliplus heydeni wehncke, 1875 was a new record of iran. in another teamwork in neka county, the mazandaran province, northern iran five species of four genera were found in tree holes during 2009. they reported anopheles plumbeus, culisetaannulata, culex pipiens, and ochlerotatus geniculatus by larval collection, ochlerotatus pulcritarsisby adult collection and oc.geniculatus, 55.87%, ochlerotatus echinus 1.33%, oc. pulcritarsis 8.8%, cx. pipiens 33.8%, and an. plumbeus 0.2% of bait net collection. they found some cs. annulata larvae in low abundance in cavities of trees for the first time (61). during 2008– 2009 i̇ncekara et al. (107) collected 42 species of aquatic beetle (coleoptera: hydrophiloidea) belonging to 13 genera and three families (helophoridae, hydrochidae and hydrophilidae) in tehran, mazandaran, guilan, qazvin, and sanandaj in kordestan provinces in iran. they reported 11 new species from iran in this survey. during 2009–2011 on the work on the aquatic insect of karun river, ahvaz, khuzestan province, southwestern iran revealed damselflies and dragonflies nymphs of odonata order, five genera from four families and all of them were the new records from this area. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 1–15 z saeidi and h vatandoost: aquatic insect … 4 http://jad.tums.ac.ir published online: march 18, 2018 recent years these days we are able to find some more articles about iran aquatic insects that worked in a wide range of natural areas (fig. 1). it seems more researchers know the importance of aquatic insects as biological control and water indicator and they are interested in investigating on them. in 2011 salavatian et al. who worked on feeding behavior of brown trout, salmo trutta fario, published a paper that shows us this fish fed on 32 animal groups including some insects such as chironomidae (88.6%), simulidae (60%), baetidae (51.4%) and tipulidae (50%) that they were most frequent food in its gut. they showed that the proportion of consumed food by brown trout was diptera 91.5% (chironomidae pupa and larvae 85.8%), coleoptera 6.4% and others 2.1% (109). other team surveyed aquatic insects’ fauna of karun river, ahvaz city, khuzestan province, southwest of iran. they reported 57 species belonging to seven orders and 22 families, collembola (1 species), ephemeroptera (4 species), odonata (6 species), hemiptera (9 species), coleoptera (34 species), diptera (2 species) and trichoptera (1 species). the most abundant species in this study was the beetle hydroglyphus signatellus klug, 1834 (coleoptera: dytiscidae) (110). an ecological risk assessment (era) for shadegan wetland, khuzestan province, southwest of iran to assess the risk to zooplankton, phytoplankton, invertebrate, insect larvae, and fish affected by five pesticides, ddt, aldrin, dieldrin, lindane and ametryn. insect larvae (chironomus sp) like other creature are highly at risk of harmful pesticide were conducted (111). study on tajan river macroinvertebrate communities’ distribution in mazandaran province, northern iran and south part of caspian sea. they realized that the dissolved oxygen, turbidity, water temperature, ph and tss were the most critical physicochemical factors to affect the distribution of them (112). work on the potential aquatic habitats for anopheles larvae from indian remote sensing satellite (irs) image and digital elevation model of the area using gis by monthly sampling from surface water bodies during 2009– 10 for anopheline larvae carried out. the lowest and highest frequencies were in february and april, respectively. anopheles culicifacies was dominant species (53). in a study conducted in gahar lake, lorestan province in three different seasons they found most and least variety and abundance in spring and autumn respectively. maximum density belonged to simulidae and chironomidae (113). ghahari collected, identified and published about 19 species from nine genera (micronecta, corixa, sigara, aquarius, gerris, hydrometra, anisops, chartoscirta, saldula) of aquatic and semiaquatic heteroptera from the families corixidae, gerridae, hydrometridae, notonectidae, saldidae from southern areas of caspian sea, northern iran (114). from 16 different sites in iran, 23 nominal species are now identified, including some new records for simulium crassicaulum (rubtsov) and simulium alajense rubtsov, and the southernmost world record for simulium transcaspicum enderlein in iran. multiple cytoforms of the simulium aureum group, simulium bezzii complex, and simulium ornatum group were found (115). shaverdo et al. reported 21 species of diving beetles dytiscidae from ahvaz, khuzestan province, southwest iran. cybister lateralim arginalisponticus, 1882, hydroporus inscitus 1882, and laccophilus sordidus 1882 are reported from iran for the first time (116). shayeghi et al. reported a variety of aquatic orders, two families of hemiptera (gerridae and notonectidae) odonata (coenagrionidae), coleoptera (carabidae), and pro stigmata from the family of hydrachindae in zayanderood, esfahan province, central iran during 2011 (117). in another study in the same area and the same year, they collected 741 specimens of aquatic insects including seven families http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 1–15 z saeidi and h vatandoost: aquatic insect … 5 http://jad.tums.ac.ir published online: march 18, 2018 and 12 genera of two orders. the order of diptera (92.31%) including culicidae, syrphidae and chironomidae and coleoptera (7.69%) including gyrinidae, dytiscidae, haliplidae, hydrophilidae families (76). maleki-ravasan et al. (118) conducted a bi-seasonal study in lavasan river, northeastern tehran, the most abundant species between 14 families and 62 trichoptera species belonged to the hydropsychidae. they reported the presence of annulipalpian hydropsyche sciligra h malicky, 1977 in that district. habitat water quality of this species reported resemble human drinking water and presence of physa acuta (snail) and capoeta buhsei (fish) in the sampling area indicated inferior quality. darilmaz et al. listed 27 species and subspecies of 17 genera of the families dytiscidae, haliplidae, noteridae and gyrinidae (coleoptera: adephaga) from alborz, gilan, mazandaran, qazvin, and tehran provinces northern iran (119). a total of 9 families in shapoor river in bushehr region during 2012 (120). researchers evaluated aquatic insects’ fauna in golestan province, north of iran in different sites during 2011–2012. they published different stages of diptera 64.54% (culicidae, chironomidae, tabanidae, simulidae, sciomyzidae families), heteroptera 11.03%, ephemeroptera 9.53% (heptagenidae, baetidae), trichoptera 7.07% (limnephilidae), odonata 4.82% (aeshnidae, gomphidae, libellulidae) and coleoptera 2.99% (dytiscidae, gyrinidae) in this study. they reported some water surface insects such as gerridae, corixidae, hydrometridae, nepidae families (121). a study in karaj river, north of iran. 211 samples of three orders; plecoptera, trichoptera and ephemeroptera and seven genera (perla, isoperla, hydropsyche, cheumatopsyche, baetis, heptagenia and maccafferium) from five families (perlidae, perlodidae, hydropsychidae, batidae, heptagenidae) were found. order of plecoptera was the most predominant order then trichoptera (122). investigated biodiversity of culicid mosquitoes from keka revealed 5270 specimens belonging to four genera and 14 species in northern iran. they reported one dominant species, two dominant species, two subdominant species, two rare species and eight sub rare species by using heydemann classification (123). southwest of iran, bashagard district is one of the most important areas because of malaria transmission collected research conducted revealed totally 5150 larvae from 36 different larval habitats. they recorded six species: an. culicifacies (29.36%), an. moghulensis (25.20%), an. dthali (18.02%), an. superpictus (17.24%), an.turkhudi (5.17%) and an. stephensi (5.01%). they investigated water quality and they stated abundant anophelin larvae existed in permanent and full sunlight habitat with no vegetation and algae. larval density had the correlation with water temperature. some factors also had the specific impact on larval abundance and distribution such as conductivity, total alkalinity, chloride and sulphate. knowing of this data and correlation between them can be considered for sufficient planning and implementing malaria elimination program (124). in bashagard area epidemiological and entomological aspects to determine malaria situation, species composition of anopheline mosquitoes and susceptibility status of main vectors to insecticides/ larvicides during 2002–2010 were conducted. they have reported an. culicifacies, an. dthali, an. stephensi, an. superpictus, an. fluviatilis, an. moghulensis, an. turkhudi and an. apoci with two peak in april and october. they have found resistance against ddt in an. stephensi and tolerance against deltamethrin and bendiocarb.their larvae found susceptible against all larvicides except for an. stephensi with tolerance against fenthion (125). in summer 2014 shayeghi et al. carried out a study in sabalan mountainous river, in different sites around meshginshahr, ardabil province, northwestern iran. they reported six orders (coleoptera, ephemeroptera, hemiptera, diptera, plecoptera and trichoptera) including 12 families (helmidae, leptophlebiidae, http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 1–15 z saeidi and h vatandoost: aquatic insect … 6 http://jad.tums.ac.ir published online: march 18, 2018 ecdyonuridae, corixidae, culicidae, simuliidae, perlidae, leptoceridae, hydropsychidae, chironomidae, caenidae and baetidae) among 262 specimens. they wrote that most abundant families were culicidae (61.55%) and a few number of plecoptera: perlodidae (0.5%) (126). fig. 3. map of iran showing some main rivers and natural events conclusion this review article will provide a clue for management of vector control as well as indicators for water classification. acknowledgements the authors like to appreciate very much for kind collaboration of all staff of department of medical entomology and vector control as well as institute for environmental research, tehran university of medical sciences, iran. references 1. salari lak s, vatandoost h, entezarmahdi m, ashraf h, abai m, nazari m (2002) monitoring of insecticide resistance in anopheles sacharovi (favre, 1903) in borderline of iran, armenia, naxcivan and turkey, 2001. iran j public health. 31(3–4): 96–99. 2. 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badakhshan m, mohammadi bavani m, arzamani k, choubdar n, bagheri f, saghafipour a, veysi a, karimian f, akhavan aa, vatandoost h (2014) assessing the fauna of aquatic insects for possible use for malaria vector control in large river, central iran. acta med iran. 53(9): 523–532. 123. nikookar sh, moosa-kazemi sh, oshaghi ma, vatandoost h, yaghoobi-ershadi mr, enayati aa, motevali-haghi f, ziapour sp, fazeli-dinan m (2015) biodiversity of culicid mosquitoes in rural neka township of mazandaran province, northern iran. j vector borne dis. 52(1): 63–72. 124. soleimani-ahmadi m, vatandoost h, zare m, alizadeh a, salehi m (2014) community knowledge and practices regarding malaria and long-lasting insecticidal nets during malaria elimination programme in an endemic area in iran. malaria j. 13: 511–521. 125. hanafi-bojd aa, vatandoost h, oshaghi ma, haghdoost aa, shahi m, sedaghat mm (2012b) entomological and epidemiological attributes for malaria transmission and implementation of vector control in southern iran. acta trop. 121(2): 85–92. 126. shayeghi m, moradi asl e, saeidi z, mozafari e, poudat a, salimi m, vatandoost h (2016) aquatic insects fauna of meshkin shahr, ardabil province, northwestern iran, 2014. j marine sci res dev. 6: 206–211. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2017, 11(1): 171 a mehrabi-tavana: sand fly fever … 171 http://jad.tums.ac.ir published online: march 14, 2017 letter to the editor sand fly fever with different names ali mehrabi-tavana health management research center, baqiyatallah university of medical sciences, tehran, iran (received 16 july 2016; accepted 15 jan 2017) dear editor-in-chief with regard to a paper published in journal of arthropod-borne diseases; a case report entitled “sand fly fever with skin lesions: a case series from turkey” was published in 2016 (fatih temocin, et al. 2016). different names such as “phlebotomus fever”, ‘’mosquito fever’’, three-day fever or “papatasi fever” called the disease of sand fly fever which all are true except ‘’mosquito fever’’ (tavana 2001, 2015). mosquito is not a general name in english and is used just for culicidae. blackflies and biting midges are used for simuliidae and ceratopogonidae, respectively. therefore, is not related to sand fly fever (saidi et al. 1977, izri et al. 2008). in my opinion it should be corrected. sand fly fever is an arthropod-borne viral disease, starts with acute onset of high fever, and lasts for two to four days, sometimes much longer. photophobia and muscle ache are more common in the patients. this disease is more prevalent in europe, asia, and persian gulf region (konstantinou et al. 2007, tavana 2007). it is important to be aware and take care of this disease in endemic and no-endemic area among travelers returning from endemic areas. references fatih temocin, tugba sari, necla tulek (2016) sand fly fever with skin lesions: a case series from turkey. j arthropod-borne dis. 10(4): 608–612. tavana am (2001) the seroepidemiological studies of sand fly fever in iran during imposed war. iran j public health. 30: 145–146. tavana am (2007) minire view on sand fly fever. j entomol. 4: 401–403. tavana am (2015) sand fly fever in the world. ann trop med public health. 8: 83–87. saidi s, tesh rb, javadian e, sahabi z, nadim a (1977) studies on the epidemiology of sand fly fever in iran. ii. the prevalence of human and animal infection with five phlebotomus fever virus serotypes in isfahan province. am j trop med hyg. 26: 288–293. konstantinou gn, papa a, antoniadis a (2007) sand fly-fever outbreak in cyprus: are phleboviruses still a health problem? travel med infect dis. 5: 239–242. izri a, temmam s, moureau g, hamrioui b, delamballerie x, charrel rn (2008) sand fly fever sicilian virus, algeria. emerg infect dis. 14: 795–797. *corresponding author: dr ali mehrabi-tavana, email: mehrab@bmsu.ac.ir microsoft word 2dr dehghani rtl_2_.doc iran j arthropod-borne dis, 2010, 4(2): 11–18 m shayeghi et al.: application of acoustical … 11 original articles application of acoustical processor reactors for degradation of diazinon from surface water m shayeghi1, *mh dehghani2, ah mahvi3, k azam4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental health engineering, school of public health, tehran university of medical sciences, center for environmental research, tehran, iran 3department of environmental health engineering, school of public health, tehran university of medical sciences, tehran, iran 4department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 5 jan 2010; accepted 24 jul 2010) abstract background: since organophosphorus pesticides are widely used for industry and insect control in agricultural crops, their fate in the environment is very important. pesticide contamination of surface water has been recognized as a major contaminant in world because of their potential toxicity towards human and animals. the objective of this research was to investigate the influence of various parameters including the influence of time, power, and initial concentration on degradation of diazinon pesticide. methods: the sonochemical degradation of diazinon was investigated using acoustical processor reactor. acoustical processor reactor with 130 khz was used to study the degradation of pesticide solution. samples were analyzed using hplc at different time intervals. effectiveness of apr at different times (20, 40, 60, 80, 100, and 120 min), concentrations (2, 4 and 8 mg/l) and powers (300w, 400w, 500w) were compared. results: the degradation of the diazinon at lower concentrations was greater in comparison to higher concentrations. there was also direct correlation between power and diazinon degradation. in addition, when the power increased, the ability to degraded diazinon increased. conclusion: the sonodegradation of diazinon pesticide at different concentrations and powers was successfully provided. it has been shown that apr can be used to reduce the concentration of dissolved pesticide using high frequency. keywords: diazinon, acoustical processor reactor, initial concentration, power, time introduction pesticides are known to be carcinogenic, mutagenic, teratogenic and simultaneously resistant to destruction in environment. organophosphorus pesticides are extremely toxic, acting on acetyl cholinesterase activity (safi 2002). diazinon [o, o-diethyl-o(2-isopropyl-6-methy-4-pyrimidinyl) phosphorothioate] is a commonly used insecticide in the organophosphate chemical family. diazinon decomposes above 120° c and is susceptible to oxidation. it is stable at ph 7.0 and can persist in the environment for as long as six months (us epa 1986, who 1998). many reports for sonochemical treatment of water and wastewater plants have been considered (krueger and seiber 1984, norwood 1990, somich 1990, benito et al. 2005, dehghani et al. 2006, 2007a, 2007b, 2008, asakura et al. 2008). mahvi et al. *corresponding author: dr mohammad hadi dehghani, email: dehghanihadi@yahoo.com iran j arthropod-borne dis, 2010, 4(2): 11–18 m shayeghi et al.: application of acoustical … 12 2009). a variety of physical and chemical methods are employed for the removal of aqueous pesticides. several techniques to eliminate pesticides have been so far considered, like ozonation (legube 1987, benitez 1995, asakura 2005,), adsorption by activated coke (battaglia 1989, richard 1991, mason and lorimer 2002,), ultraviolet irradiation and hydrogen peroxide (peterson et al. 1988, raha and das 1990, battacharya 1994, baron et al. 1994, benitez 1995 b, bellobono et al. 1995, boisdon and cacite´ de la 1995, bourgine et al. 1995, mansour et al. 1997, huston and pignatello 1999, bachman and patterson 1999). acoustical processor reactor (apr) has been used to induce or accelerate a variety of reactions. these reactors have a board range of industrial applications, including water and wastewater treatment (suslick 1994, suslick and crum 1997, suslick and price 1999, joyce 2002, gogate et al. 2003, dehghani et al. 2008, 2010). since 1990, there has been increasing interest in the sonochemical degradation of aqueous solutions both in water and wastewater decontamination (laborde and bouyer 1998, destaillats et al. 2001,). the chemical effects of apr of aqueous environment are believed to be related with acoustic cavitations ultrasound irradiation enhances chemical reactivity through cavitation, the formation of gas bubbles in a liquid, which rapidly expand and implode. the chemical aspects are realized during and immediately after collapse of a vapor-filled cavitation bubbles. bubble collapse induced by cavitation produces intense local heating, high pressures (hung and hoffmann 1999, beckett and hua 2001,wayment and casadonte 2002, mason et al. 2004, hua et al. 2005, dehghani and changani 2006, arrojo and benito 2008,). these hot spots have temperatures of roughly 5000 k, and pressures of about 500 atm as well as the production of free radicals. these conditions provide several possible pathways of degradation, such as free radical attack. (mason 1991, hua et al. 1995, hua and hoffman 1997, mason and lorimer 1998, crum et al. 1999, hua and pfalzer-thompson 2001, asakura et al. 2008). the present research deals with the sonochemical degradation of selected diazinon pesticide in the apr. the objective was to determine the potential of batch reactor for degradation of diazinon in different conditions such as power, concentration, and degradation time. materials and methods procedures sample was prepared by dissolving a measured volume of pesticide with 95% concentration in 1000 ml of distilled water in a volumetric vessel. all organic solvents (acetone, hexane) were of analytical reagent grade, supplied by merk company. homogenized samples were pretreated at 18– 20º c and then were placed in reactor. reactor temperature was controlled with the help of condensation water surrounding the reactor. therefore, temperature did not exceeded 18-20º c in all experiments. diazinon pesticide samples were exposed to a fixed frequency of 130 khz. characteristics of the reactor and experiment conditions presented in table 1 and 2 respectively. all sonicated solutions were analyzed for diazinon and degradation operation by hplc at different time intervals. characteristics of the hplc system were as following (table 3). all the analyses were performed according to the procedures outlined in standard methods (apha 2005). calculation method the definition of diazinon degradation percentage (dp) was as follows: dp = (c1 – c2) / c1 × 100 iran j arthropod-borne dis, 2010, 4(2): 11–18 m shayeghi et al.: application of acoustical … 13 where dp (%) is the degradation percentage of the reactor, c1 is the initial concentration of diazinon (mg/l), c2 is the concentration of diazinon (mg/l) after reaction for (t) time statistical analysis the degradation of diazinon using apr was analyzed statistically by spss 11.5 and excel software. the data were analyzed using one way anova, post-hoc test and multiple regressions. the variables were degradation time, concentration and power. degradation was depended variable. results an aqueous solution of diazinon was sonicated in a batch reactor at different concentrations, powers, fixed frequency, and different times. during the sonochemical degradation, the concentrations of diazinon were determined and the ultraviolet absorption spectra of the aqueous solution of diazinon were measured. effect of initial concentration the effect of initial diazinon concentration on the removal percentage is shown fig.1, 2 and 3. different initial concentrations resulted in different removal percentage. the removal percentage decreased with increasing initial diazinon concentration in the range of 2 mg/l( mean= 95.3422 and sd= 3.40735), 4 mg/l (mean= 91.6033 and sd= 2.84080) and 8 mg/l (mean= 83. 8294 and sd= 5.89949) under sonication after 120 min. clearly, the rate of sonochemical degradation was slow in the presence of high concentration of diazinon. on the other hand, the increase of diazinon concentration in the solutions significantly decreased the rate of diazinon degradation after 120 min. as expected, the degradation percentage is the highest for the lowest concentration. the best status for pesticide degradation was 2 mg/l under sonication after 120 min. one way anova and post hoc test indicated that mean difference is significant (p< 0.05). effect of time the effect of time on the degradation of diazinon was shown in fig. 1, 2 and 3. the degradation percentage of diazinon was 90.83% (2 mg/l), 86.11% (4 mg/l) and 76.99% (8 mg/l) for 300 w (fig. 1). in addition, degradation percentage was 96.11 % (2 mg /l), 90.95% (4 mg/l) and 84.54% (8 mg/l) for 400 w (fig. 2). however, degradation percentage was 98.44% (2 mg /l), 94.07% (4 mg/l) and 88.98 % (8 mg/l) after 120 minutes for 500 w (fig. 3). according to regression analysis using backward method, the mean difference is no significant. on the other word, degradation time is excluded variables. effect of ph the results showed that sonolysis had no considerable effect on ph (6.8-7) of pesticide samples, and the minor change occurred were no significant. effect of temperature in this study, the reaction temperature was controlled with condensation water surrounding the reactor bath. therefore, experiments showed that temperature increase of pesticide samples during sonication had no considerable effect on degradation of pesticide. effect of acoustic power the effect of power on the diazinon degradation was also studied for 300 w (mean= 85.4694±6.14753), 400 w (mean= 90.4689±5.61704) and 500 w (mean= 94.8367±3.41482). one way anova and post hoc test showed the mean difference is significant between removal efficiencies at iran j arthropod-borne dis, 2010, 4(2): 11–18 m shayeghi et al.: application of acoustical … 14 0.05 level, when the power rose from 300 w to 500 w according to statistical analysis, linear relationships equations were as follows: degradation ratio= 80.495 -1.922 concentration + 0.047 power in addition, determination coefficient (r square) for above model showed that highest changes related to degradation efficiency depends upon concentration and power (r square= 0.937). table 1. characteristics of acoustical reactor parameter characteristics power 300 w, 400 w, 500 w frequency 130 khz reactor type basin flow type batch capacity 1.5 l table 2. experimental conditions for degradation operations frequency (khz) power (w) sonication time (min) initial concentration (mg/l) sample volume (ml) temp ( ºc) ph 130 300, 400 , 500 20, 40, 60, 80, 100, 120 2, 4, 8 200 18–20 6.8–7 table 3. characteristics of hplc system column inertsil mobile phase temperature detector uv flow rate ods – 2 ch3cn/h2o (65:35, v:v) 40 ºc wavelength 210 nm 1ml/min y = -0.0022x + 98.823 r2 = 0.1611 y = -0.0068x + 96.988 r2 = 0.2963 y = 0.0005x + 90.809 r2 = 0.1891 90 91 92 93 94 95 96 97 98 99 100 0 20 40 60 80 100 120 140 time (min) d eg ra da tio n (% ) 300 w 400 w 500 w fig. 1. comparison of degradation percentage at different powers for 2 mg/l iran j arthropod-borne dis, 2010, 4(2): 11–18 m shayeghi et al.: application of acoustical … 15 y = -0.0083x + 95.464 r2 = 0.1711 y = -0.0212x + 89.852 r2 = 0.5084 y = -0.0007x + 91.606 r2 = 0.003 84 86 88 90 92 94 96 98 0 20 40 60 80 100 120 140 time (min) d eg ra da tio n (% ) 300 w 400 w 500 w fig. 2. comparison of degradation percentage at different powers for 4 mg/l y = 0.0085x + 90.367 r2 = 0.0298 y = 0.0096x + 82.657 r2 = 0.3015 y = 0.0064x + 76.746 r2 = 0.1659 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 time (min) d eg ra da tio n (% ) 300 w 400 w 500 w fig. 3. comparison of degradation percentage at different powers for 8 mg/l discussion it is possible to degrade efficiently the diazinon pesticide in water by apr. statistical analysis shows that power and initial concentration are effective parameters for degradation of diazinon. matouq et al. (2008) indicated that percentage of degradation rate of diazinon varies with initial concentration after exposing to irradiation which is basically the steady state phase. matouq et al. (2008) reiran j arthropod-borne dis, 2010, 4(2): 11–18 m shayeghi et al.: application of acoustical … 16 ported that initial concentration decreases with time. hua et al. (2001) indicated that a low ph is not required for sonodegradation of pesticide. hua et al. 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sonochem. 9: 189–195. 3mr abai iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 10 original article repellency effects of essential oils of myrtle (myrtus communis), marigold (calendula officinalis) compared with deet against anopheles stephensi on human volunteers m tavassoli1, m shayeghi1,*mr abai1, h vatandoost1, m khoobdel2, m salari1, a ghaderi1, f rafi1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2health research center, baqiyatallah university of medical sciences, tehran, iran (received 20 nov 2011; accepted 21 may 2011) abstract background: malaria and leishmaniasis are two most significant parasitic diseases which are endemic in iran. over the past decades, interest in botanical repellents has increased as a result of safety to human. the comparative efficacy of essential oils of two native plants, myrtle (myrtus communis) and marigold (calendula officinalis) collected from natural habitats at southern iran was compared with deet as synthetic repellent against anopheles stephensi on human subjects under laboratory condition. methods: essential oils from two species of native plants were obtained by clevenger-type water distillation. the protection time of deet, marigold and myrtle was assessed on human subject using screened cage method against an. stephensi. the effective dose of 50% essential oils of two latter species and deet were determined by modified astm method. ed50 and ed90 values and related statistical parameters were calculated by probit analysis. results: the protection time of 50% essential oils of marigold and myrtle were respectively 2.15 and 4.36 hours compared to 6.23 hours for deet 25%. the median effective dose (ed50) of 50% essential oils was 0.1105 and 0.6034 mg/cm2 respectively in myrtle and marigold. the figure for deet was 0.0023 mg/cm2. conclusion: this study exhibited that the repellency of both botanical repellents was generally lower than deet as a synthetic repellent. however the 50% essential oil of myrtle showed a moderate repellency effects compared to marigold against an. stephensi. keywords: calendula officinalis, myrtus communis, deet, anopheles stephensi, repellent, essential oils, iran introduction malaria is still a major endemic disease in foci located in south and southeast of iran. the annual malaria cases have been reported from 66075 to 6211 during 1995–2009, indicating the sharp decline of disease. it is unstable with two seasonal peaks mainly in spring and autumn. these areas include the provinces of sistan and baluchistan, hormozgan and kerman. in this part of the country six anopheline mosquitoes including anopheles culicifacies s.l., an. stephensi liston, an. dthali patton, an. fluviatilis s.l., an. superpictus grassi, and an. pulcherrimus theobald are known to be the malaria vectors and an. sacharovi (favre, 1903) and an. maculipennis s.l. are considered as malaria vector in northern part of the country (manouchehri et al. 1992, zahirnia et al. 1998, 2001, enayati et al. 2003, naddaf et al. 2003, oshaghi et al. 2003abc, salari lak et al. 2003, vatandoost et al. 2004ab, 2005ab, 2006ab, 2008ab, 2009a, 2010, 2011, sedaghat et al. 2005, *corresponding author: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 11 2003ab, doosti et al. 2006, 2007, davari et al. 2007, abai et al. 2008). one of the widely used and effective insect repellents is the synthetic compound, n, n-diethyl-m toluamide (deet) which is generally considered the “gold standard” repellent, providing long-lasting protection of up to 8 hours from time of application. “there are some rare reports of severe reactions in people, additionally deet melts plastics causing spoilage of equipment, such as glasses and mobile phones, and many consumers find the odor and sensation on the skin unpleasant” (logan et al. 2010). therefore researchers are trying to improve the efficacy with reducing the side effects of new generation of repellents. in the past few years, a plant derived repellent, para-methane 3–8, diol (pmd) has been proven to be suitably efficacious and safe to compete with deet in the field of disease prevention, and repellents have been recognized by who as a useful disease prevention tool to complement insecticidebased means of vector control. many plants have substances that are toxic, antifeedant properties or repellency for insects. repellent of plant material refers to a plant origin have an inherent and naturally defensive and repellency effects on insects (maia and moore 2010). this material includes extracts and essential oils. their effect is significantly differs from different parts of plants, including flowers, tubers, leaves, fruit, branches and roots. myrtle (myrtus communis l.) is a native plant distributed in south, north and central parts of iran (rechinger 1996). a study in iran showed that the myrtle essential oil (m. communis) is very active against streptococcus pneumoniae, moraxella catarrhalis and haemophilus influenzae in vitro. this confirms the application of herbal medicines for treating a range of infectious diseases in ancient times (pourmand et al. 2008). study of the conditions in vitro effect of myrtle essential oil, the extracts form soluble in water, and soluble dichloromethane corn oil was studied on a variety of microbes. it was determined that the oil could prevent the growth of bacteria staphylococcus aureus, pseudomonas aeruginosa and escherichia coli. its effect of antiviral ointment containing about 10% of myrtle essential oil on patients with herpes simplex virus was tested (zolfaghari et al. 1997). the essential oil of myrtle is effective in protection of biting insects. in addition insecticidal action was also observed (yaghoobi-ershadi et al. 2006). marigold also exhibits great effects in treating some skin disorders such as leaving the skin, acne and dermatitis. the calendula ointment are presented and is believed that the spring flowers of this plant had been used for reducing swelling, treat injuries, and a disinfectant material. for domestic use, this plant has been used for soothing effects of mucosal ulcers, swelling of the stomach. the repellency property makes an impact effect on the insects away from humans. for the first time in the country, the repellency of essentials oils myrtle and marigold which are native in southern iran, were assessed using an. stephensi and compared with deet on human volunteers under laboratory condition. materials and methods mosquitoes the tested mosquitoes were the established colony of an. stephensi obtained from the insectary of school of public health, tehran university of medical sciences, iran. mosquitoes were reared and maintained at 27±3 oc and 80±10% relative humidity (rh) under a 12: 12 (l: d) photoperiod. larvae were fed on a diet of enriched wheat germ. the adults were maintained in screen cages and fed with 10% aqueous sucrose solution as a source of energy and guinea pigs as blood-feeding female mosquitoes for maturing the eggs. starved 7 to 10 days old females were used for the repellency tests. the sucrose solution was iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 12 picking up from the cage, 12 hour before starting the experiments. repellents the chemical repellent, deet (n, ndiethyl-meta-toluamid), cas number: 134– 162 and assay 98.8%, density: 0.998 g/cm3 were purchased from merck co, germany. the myrtle (m. communis) is evergreen shrubs or small tree which was collected from southern extension of natural habitat in noorabad district at coordinate 30º 7´e 51º 31´n, 920 meters above sea level, southern iran. the marigold (calendula officinalis l.) was also collected in suburb of shiraz city, at coordinates 29°37′n 52°32′e at elevation of 1481 meter above sea level, fars province, southern iran. the plants were identified and the voucher specimens were deposited at the herbarium of faculty of pharmacy, department of pharmacognosy. the leaves and flowers of myrtle and the flowers of marigold were dried at room temperature under good ventilation and chopped into small pieces using a knife mill. the essential oil was extracted from the plants using a clevenger-type water steam distillation apparatus. the distilled essential oils were stored in a refrigerator at 4° c until being used in the experiments which were diluted by absolute ethanol at 50% concentration. the composition of the volatile constituents was established by gas chromatography-mass spectrometry. test method all series of the experiments were carried out in laboratory condition. in the first stage, in order to reveal the probable allergic reaction of chemical and natural repellents to human volunteers, the scratch test was done on skin of the upper arm. the treated skin of arm was observed up to 72 hours for allergic reaction. the 25% solution of deet was prepared using absolute ethanol as well as 50% essential oils myrtle and marigold was obtained in same manner and tested against an. stephensi on four male volunteers. observation was based on the variable dose-response of the mosquito to the serial dilutions of the repellents. the procedures for determination of effective dosages of the repellents were adopted by the standard method of american society for testing and material (astm e951-94). the testing kit was made of plexiglas cube at dimension of 4 x 5 x 18 cm having five circles in 29 mm diameters. before selecting the mosquitoes, the willingness of mosquitoes for introducing in repellency tests were monitored based on determination of biting pressure on untreated, alcohol washed arm which should be at least 10 landings/ probes per 30 seconds. each of 5 adjacent cells in the astm modules was provided with 5 female 7–10 days mosquitoes that randomly selected from a cage containing 200 starved mosquitoes. the effective dose tests were conducted by applying each repellent directly to the human skin. five circles (29 mm in diameter) were drawn on the volunteer's forearm using a felt tipped pen and a plastic pattern. the drawn circles on the human subjects were treated with 25 µ l of the diluents. the serial dilutions were applied on 4 holes as well as the absolute ethanol was applied in control circle. the treated circles were allowed to dry, and then test apparatus containing starved mosquitoes were fixed on the treated skin of the volunteers and opened the slide for exposure with treated skin. the counts of probing and biting were recorded at 1 minute intervals up to 5 minutes. the experiments were done at 2 replicates on right forearm and 2 replicates on left forearm of the volunteers. after each test, the mosquitoes were removed from the test apparatus using aspirator and then transferred into a screened cup. mortality of mosquitoes was read after 24 hours. the protection and failure times were also determined on human subjects. before starting the experiments, 1 ml deet 25%, as well as 1 ml of 50% essential oils both myrtle and marigold were dissolved in iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 13 absolute ethanol and spread evenly between the elbow and wrist of a volunteer's arm. the other arm, acting as a control and was treated only with 1 ml of absolute ethanol. after drying the test arm, a rubber glove was put by volunteers and inserted the arms into 50× 50×50 cm cage containing 150–170 starved mosquitoes for 3 minutes. the mosquitoes which landed and attempted to bite were recorded. the procedure was repeated at 30 minutes intervals and was used consistently throughout the experiment. if more than 1 mosquito bite was recorded during an observation, the test of repellency was terminated, and the period of repellent protection was calculated as the time between the repellent application and multiple mosquito bite. if only 1 mosquito attempted to bite during an observation period, any addition mosquito bites during that next observation period (30 min later) confirmed that the initial bite represented the time of repellent failure. the period was repeated up to 10th bites and took into account as failure time. the successive expose of the control arm were made prior to inserting the treated arm in order to provide a standard for comparing mosquito biting activity during the experiments. the similar tests were also repeated on 4 human volunteers. statistical analysis the data were subjected to statistical analyses using spss software ver. 11.5. in order to estimate the ed50 and ed90 values, the cumulative results were subjected to the probit analysis which had been repeated in different days with four volunteers (finney 1971, 1978). the regression lines were plotted and the ed50 and ed90 values with confidence limits and regression parameters were calculated. data were transformed using arcsine √x transformation to meet the normality. significant differences between three repellents were assessed by anova. the latter test was also used for assessment of the significant differences between protection times of the tested repellents. means of protection times and ed were compared by the tukey’s honest significance test or games-howell test depending on significance of levene’s test. the 1% level was employed in tests of significance. ethical approval this study received formal ethical approval from the medical ethics and history of medicine of research center, tehran university of medical sciences. informed consent was taken from each volunteer. results protection time the protection time of 50% essential oils myrtle and marigold compared with deet 25% against an. stephensi on human subject is shown in table 1. the protection time (pt) of botanical repellents was ranged between 4.25–4.40 and 1.00–3.30 hours with mean of pt 4.36 and 2.15 hours respectively with myrtle and marigold essential oils (table 1). the mean of protection time of deet was ranged between 6.05–7.00 hours with mean of 6.23 hours. the difference between botanical repellents and deet was significant compared to both botanical repellents (p< 0.01). on the other hand, the difference of protection times of myrtle compared with marigold essential oil were significant (p< 0.01) (fig. 1). effective doses the ed50 values (with 95% confidence limits) of the myrtle (m. communis) and the marigold (c. officinalis) essential oils were 0.1105 (0.0772–0.1399) and 0.6034 mg/cm2 (0.4464–0.7476), respectively on 4 human subjects. the ed90 values with 95% confidence limits were respectively 0.5404 (0.4281– 0.7683) and 3.4905 mg/cm2 (2.6203–5.4534) for myrtle and marigold plants (table 2). the ed50 and ed90 values for deet as a golden repellent were 0.0023 (0.002–0.0027) iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 14 and 0.009 mg/cm2 (0.0071–0.0127). statistical comparison of the data was revealed that the ed values for essential oils myrtle and marigold is significantly higher than ed values of deet, showing less repellency of botanical repellents compared with deet (p< 0.01). the ed50 of myrtle was close to ed90 of marigold and was significant different (fig. 2). comparing the ed50 values, it can be concluded that the ed50 for deet is significantly higher than those of ed50 for essential oils marigold and myrtle, showing higher repellency effect (p< 0.01). the related dose-response lines, ed values and regression equations for deet, marigold and myrtle are shown in fig. 3. gc-mass analysis the result of gc-mass analysis showed that the number of chemical contents were 65 and 33 constituents respectively in essential oils marigold and myrtle. the main compounds of marigold were alpha-cadinol (18.3%), beta eudesmol (14.5%) and tau-muurolol (13.0%) compared to alpha-pinene (47.8%), 1, 8cineole (25.9%), linalool (8.4%) and linalyl acetate (4.3%) in myrtle. table 1. protection and failure times values of essential oils both marigold and myrtle compared to deet against an. stephensi on human subjects repellents protection time (hour) failure time (hour) range mean ± se deet 25% 6.05–7.00 6.23 ± 0.16 7.30 marigold 50% 1.00–3.30 2.15 ± 0.66 3.30 myrtle 50% 4.25–4.40 4.36 ± 2.18 4.40 table 2. parameters of probit analysis on chemical and botanical repellents against an. stephensi using standard method (astme951-94) r ep ellen ts n o. m osq u itoes e d 50 (m g/cm 2) 95% c .l . (m g/cm 2) e d 90 (m g/cm 2) 95% c .l . (m g/cm 2) x 2 (d f) ± s e x 2 (tab le) p -valu e e q u ation of regression lin es deet 5% 100 0.0023 0.0020–0.0027 0.009 0.0071–0.0127 16.185(2)±0.221 5.99 <0.05 y= 5.7588 +2.1903x marigold 100 0.6034 0.4464–0.7476 3.4905 2.6203–5.4530 8.110(2)±0.196 5.99 <0.05 y= 0.3688+1.6813 x myrtle 50% 100 0.1105 0.0772–0.1399 0.5404 0.4281–0.7683 12.043(2)±0.618 5.99 <0.05 y= 1.7785+1.8589 x iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 15 fig. 1. statistical comparison of protection time of essential oils of marigold and myrtle as well as deet on human subjects using an. stephensi fig. 2. statistical comparison of effective doses of essential oils marigold and myrtle as well as deet on human subjects using an. stephensi iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 16 0 1 2 3 4 5 6 7 8 0.000 0.001 0.010 0.100 1.000 10.000 ed (mg/cm2) p ro te c ti o n f ro m b it in g ( p ro b it ) deet marigoldmyrtle ed50 =0.0023 mg/cm 2 ed50=0.6034 mg/cm 2 ed50=0.1105 mg/cm 2 fig. 3. dose-response lines and values for three botanical and chemical repellents against an. stephensi on human subjects discussion insect repellents are used to prevent nuisance bites from mosquitoes as well as other bloodfeeding arthropods and may aid in lowering disease transmission e.g. malaria, leishmaniasis, filariasis and west nile virus. anopheles stephensi is the main malaria vector in the country and is rearing easily and used for different biological assays such as irritability tests, olfaction studies, bioassay tests for bednets and indoor residual spraying, biological tests for plant extraction and repellents (hadjiakhoondi et al. 2000ab, 2003, 2005, 2006, sadat ebrahimi et al. 2005, rafinejad et al. 2006, vatandoost et al. 2006a, 2008b, 2009b, 2011, davari et al. 2007, omrani et al. 2010, shahi et al. 2010, hanafibojd et al. 2011, sedaghat et al. 2011). in this study, the effect of essential oils myrtle and marigold as the botanical repellents were compared with deet. the n,n-diethyl-m-toluamide (deet) as a broad-spectrum repellent is provide longer-lasting protection against many species of biting arthropods including mosquitoes which has been used worldwide since 1957 (usepa 1998). it is commonly assumed that plant-based repellents are safer than deet because they have natural origin (maia and moore 2010). the botanical repellents were developed from definite species of plants are environment-friendly, with pleasant natural aroma and less harmful than synthetic repellents which have been reported to cause many undesirable side effects to human. in our study, 25% deet provided an average of 6.23 hours of complete protection against an. stephensi bites. deet-based repellents have been shown in other studies to provide complete protection against arthropod bites for as long as 12 hours under laboratory conditions (fradin and day 2002) which depend on the concentration, formulation and mosquito species tested (klun et al. 2006). iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 17 our ed50 estimates for deet is 0.0023 mg/cm2 on 4 human subjects. on the animal model, the ed50 value of deet was calculated 0.005 mg/cm2 against an. stephensi (vatandoost 2008b). in the laboratory condition, the mean of relative effectiveness of 50% deet was showed 97.0% protection against an. stephensi on guinea pig and 80.5% protection on human hand (oshaghi et al. 2003b). a cream formulation of deet was evaluated at 10 mg/cm2 with 96.2% protection against an. stephensi and provided protection up to 6.75±0.2 hours up to 4 hours observation (mittal et al. 2011). there are no published data describing the repellency of essential oil of marigold. the mean of protection time of 50% myrtle essential oil showed considerable repellency on human subjects and provided 4.36 hours protection against an. stephensi, the main malaria vector at laboratory condition. in other study which conducted on 41 natural repellents, the protection time of myrtle essential oil was reported 6.5 hours against anopheles species (abdelkrim et al. 2006). the past studies revealed that the most natural product-based repellents provided 3 hours protection which is comparable with protection provided by 7 or 15% deet (barnard and xue 2004). the mean of protection time of 50% essential oil marigold provided only 2.15 hours protection against an. stephensi bites. in other laboratory study, the values ed50 and ed90 for myrtle essential oil were respectively calculated as 0.1140 and 0.6711 mg/cm2 on animal model (rabbit) using k and d apparatus against lab-bred phlebotomus papatasi scopoli (yaghoobiershadi et al. 2006). surprisingly, in our study, the ed50 and ed90 values for myrtle essential oil against an. stephensi was very close to latter studies on animal model with p. papatasi (respectively 0.1105 and 0.5404 mg/cm2) which assessed on 4 male human subjects. the repellent effects of the essential oils indicated that they contained active constituents which responsible for the repellency activity. the major components of these two essential oils are monoterpenes, primarily 1, 8-cineole and linalyl acetate which detected in moderate percentages (13.0–18.3%) in the essential oil of studied myrtle (m.communis) compared to lower percentage (<0.5%) in marigold (c. officinalis). the higher repellency and insecticidal effects of the myrtle could be attributed to the major aforementioned constituents. the insecticidal effect of mytle at 1.6 mg/cm2 was reported 62.2% against lab-bred p. papatasi on animal model (yaghoobi-ershadi et al. 2006). in marigold (c. officinalis), carvacrol and thymol were extracted in low percentage (0.18–0.28%) had been shown to have insecticidal properties. on the other hands, 1, 8-cineole and linalyl acetate could be responsible for the high repellency activity of the essential oil of myrtle (klocke et al. 1987, abdurrahman et al. 2006, günter et al. 2009,). the protection time provided by 25% deet (pt=6.23 hours) is contrastable with the results of 50% myrtle essential oil (pt= 4.36 hours) which shows that the myrtle essential oil can be useful and safe in preventing mosquito bites and have potential use as a botanical repellent. acknowledgements the authors are grateful to dr a ghoharikakhki, associate professor of department of pharmaceutics, faculty of pharmacy and senior researcher of medicinal plants research center, tehran university of medical sciences (tums) for his valuable leading and assistance for preparing of the essential oils. we thank dr h sershti, associate professor of faculty of chemistry, college of sciences, tehran university for his kind collaboration and providing facilities for gcmass analysis of the essential oils. during this study, the voluntar participation of some our msc students in medical entomology iran j arthropod-borne dis, 2011, 5(2): 10–22 m tavassoli et al.: repellency effects of essential … 18 and vector control course at tums is gratefully acknowledged. we would like to thank ah hoseinie, technician of insectary of school of public health, tums for his great efforts in mass production of an. stephensi. this study was financially supported by school of public health, tehran university of medical sciences. the authors declare that there is no conflict of interest. references abai mr, mehravaran a, vatandoost h, oshaghi ma, javadian e, mashayekhi m, mosleminia a, piyazak n, edallat h, mohtarami f, jabbarie h, rafi f (2008) comparative performance of imagicides on anopheles stephensi, main malaria vector in a malarious area, southern, iran. j vector borne dis. 45: 307–312. abdelkrim a, mehlhorn h (2006) larvicidal effects of various essential oils against aedes, anopheles, and culex larvae (diptera, culicidae). parasitol res. 99: 466– 472. abdurrahman a, osman s, salih k, ismet o (2010) insecticidal activity of the 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debatable whereas their identification is a problematic work. the reasons for this claim is time consuming delpy’s researches in iran also schulze school, feldman-muhsam and the russian tick workers. we would like to understand morphometric variation in the field collected h. anatolicum in iran also validating some morphologic quantitative and qualitative characters. methods: a total 247 field-collected tick specimens from different geographical regions in west of iran includes khuzestan and lorestan provinces were studied. the morphologic characters of the ticks were measured by the calibrated stereomicroscope armed scaled lens. the measurements were analyzed using spss for windows, version 16 on an ibm pc, so varied shapes of species in different geographic regions were drawn by the aid of a drawing tube connected to a light stereomicroscope. results: one way anova test revealed significant differences among the quantitative parameters in five zones (p< 0. 00 1 ) also each zone to other zone by post hoc tests e.g. lsd. no significant differences in the lateral grooves length/conscutum length ratio parameter were found. conclusion: morphometric variation in hyalomma spp is poorly studied. the variation in range and quantity of the morphometric parameters of h.anatolicum underlies that the correct recognition and key construction for hyalomma species dependes on a complement morphometric study on the other species. keywords: hyalomma anatolicum, morphologic characters, morphometric study, variation, iran introduction the small anatolian hard tick, hyalomma anatolicum koch, 1844 is a vector of the important pathogen agents, includes animals and human (hoogstraal 1979 , burkot and graves 2004). this species is the most abundant hyalomma species in its geographical zone (kaiser and hoogstraal 1964 ). h. anatolicum formerly was includes of two subspecies anatolicum and excavatum, but recently reestablish as full species rank (apanaskevich and horak 2005 ). identification of hyalomma spp. is not simple (pomerantzev 1950 ) and is very debatable because endless verity in faces which is due to exchange of environmental condition between different populations and genetic instability within their gene pool, hybridation and teratogenic abnormality (delpy 1936b , pervomaisky 1950 , hoogstraal 1956 ). morphometric study of h. anatolicum in its geographical zone is not yet investigated, although some taxonomic characters on museum and field collected specimens was introduced but not measured (mazlum 1968, apanaskevich and horak 2005). *corresponding author: prof abdolhossein dalimi, email: dalimi_a@modares.ac.ir iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 24 the medically and veterinary importance of h. anatolicum showed in iran (hashemifesharki 1997, telmadarraiy et al. 2010 ) and is widely distributed throughout in this country (rahbari et al. 2008). misidentification of h. anatolicum to closely related species e.g. hyalomma asiaticum, h. excavatum, h. marginatum, generally the other hyalomma species in iran is a very common manner between tick workers whereas we have not an original and complete hyalomma identification key for iranian specimens. the traditional morphometric studies started by delpy in iran (delpy 1936a, 1937a , 1937b ), he believed the correct identification of hyalomma species might be based on observation of the numerous specimens from any species. the purpose of the present investigation was to identify valuable discriminating characters for male specimens of h. anatolicum also intraspecific variation study of the morphologic characters of this species in the five iranian population in west of iran based on delpy’s conception (delpy 1936a ). the present study was designed to obtain information on morphometric variability on one of the most important hyalomma species in the iranian ixodid fauna. this study was performed on males of h. anatolicum since the morphologic characters of the females may be shallow and obscure when the tick engorged. materials and methods material examined a 247 field-collected tick specimens from different geographical regions in west of iran includes khuzestan and lorestan provinces were studied (table 1). ticks were examined for preliminary identification by several hyalomma identification keys including delpy 1936a, pomerantzev 1950, kaiser and hoogstraal 1964, and other related keys. the morphologic characters of ticks were measured by the calibrated stereomicroscope armed scaled lens (stemi srzeiss germany). the measurements were analyzed using spss for windows, version 16 on an ibm pc. significances were measured at the 5% level. afterwards, varied shapes of this species in different geographic regions were drawn by the aid of a drawing tube connected to a light stereomicroscope. description of used characters the list of zones and characters for male specimens include nine quantitative and three qualitative parameters summarized in table 1. table 1. list of parameters used for characterization and zones of study quantitative parameters basis capituli depression posteriodorsally cervical grooves length/conscutum length ratio conscutum length conscutum length/width ratio conscutum width lateral grooves length/conscutum length ratio parma length parma length/width ratio parma width qualitative parameters arch caudal depression pigmented parma zones of study *і: ramhormoz, izeh, baghmalek and masjedsolyman *ιі: dashteazadegan, shadegan and shush **ιіι: aleshtar and taf **іv: kohdasht **v: mamolan *these zones are situated in khuzestan province **these zones are situated in lorestan province iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 25 basis capituli depression posterodorsally the cornua are paired projections from the outer margins of the posterior dorsal surface of the basis capituli. the depression of between cornua is characteristic for identification. cervical grooves these are paired depressions near the central anterior part of the conscutum; they go down from the raised intercervical field into the cervical fields (also known as mesial grooves). conscutum: the sclerotized (=hardened) plate which covers most of the dorsal surface of male ixodid ticks (so called scutum in female). lateral grooves in the conscutum of males a groove may be present in the lateral area, starting most clearly near the position of the spiracles and possibly extending forward to the eyes (in some literature these are called marginal grooves or lines). parma: festoons are a regular series of bulges in the posterior margin of many adult ixodid ticks. the central festoon may also be called the parma when it is developed as a distinct structure, separated from the surrounding festoons by grooves. arch: in hyalomma males the pair of festoons next to the central one is paracentral and may be joined anteriorly to form an arch shape. caudal depression in hyalomma males the posterior of the conscutum may be broadly convex (caudal depression is absent) or it may be broadly concave (caudal depression is present). pigmented parma the parma may be pale colored (unpigmented), or dark colored (pigmented). results the result of relevant to the quantitative parameter is summarized in table 2 to table 4. one way anova test revealed significant differences among the quantitative parameters in five zones (p< 0. 00 1 ) also any zone to other zone by post hoc tests eg lsd. no significant differences in the lateral grooves length/conscutum length ratio parameter were found (fig. 4). variation in some parameters (characters) is illustrated in fig.1, fig. 2 and fig. 3. the observation on qualitative parameters revealed the presence of three characters in under studying specimens which is summarized in table 5. table 2. conscutum parameters of h. anatolicum collected from different zones of khuzestan and lorestan provinces zone no size (mm) *lsd parameter average sd max min conscutum length і 51 2.97 0.26 3.53 2.43 ііі,іv,v іі 62 3.03 0.27 3.79 2.29 ііі,v ііі 45 3.48 0.31 4.04 2.78 і,іі,ιv,v іv 39 3.13 0.27 3.65 2.62 ι,ιιι v 50 3.14 0.24 3.61 2.52 ι,ιι,ιιι conscutum width і 51 1.83 0.15 2.19 1.40 ііі іі 62 1.87 0.18 2.29 1.46 ііі ііі 45 2.27 0.25 2.83 1.77 і,іі,ιv,v іv 39 1.88 0.15 2.21 1.44 ііі v 50 1.88 0.14 2.23 1.48 ііі conscutum length/width ratio і 51 1.63 0.08 1.82 1.42 ііі,v іі 62 1.63 0.08 1.85 1.44 ііі,іv,v ііі 45 1.55 0.07 1.69 1.40 і,іі,ιv,v іv 39 1.66 0.09 1.96 1.49 ιι,ιιι v 50 1.67 0.07 1.82 1.39 ι,ιι,ιιι *least significance difference, p value in all parameters is similar (p< 0.001) iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 26 table 3. parma parameters of h. anatolicum collected from different zones of khuzestan and lorestan provinces zone no size (mm) lsd parameter average sd max min *parma length і 51 0.15 0.03 0.21 0.10 ιιι іі 62 0.14 0.03 0.27 0.06 ііі,іv ііі 44 0.19 0.05 0.31 0.10 і,іі,ιv,v іv 39 0.16 0.02 0.21 0.12 ιι,ιιι v 50 0.15 0.02 0.19 0.10 ιιι *parma width і 51 0.90 0.84 2.17 0.10 ιιι іі 62 0.17 0.04 0.27 0.10 ііі,іv ііі 44 0.21 0.05 0.39 0.12 і,іі,ιv,v іv 39 0.20 0.03 0.29 0.16 ιι,ιιι v 50 0.19 0.03 0.29 0.12 ιιι *parma length/width ratio і 51 0.50 0.40 1.60 0.06 іι,ііι,ιv,v іі 62 0.83 0.20 1.29 0.55 ι,ιιι ііі 44 0.95 0.28 1.67 0.50 і,іі,ιv,v іv 39 0.81 0.17 1.25 0.46 ι,ιιι v 50 0.78 0.20 1.33 0.46 ι,ιιι *containing missing data in zone ιιι p value in all parameters is similar (p< 0.001) table 4. other quantitative parameters of h. anatolicum collected from different zones of khuzestan and lorestan provinces zone no size (mm) lsd parameter average sd max min *basis capituli depression і 51 0.02 0.01 0.06 0.00 ιιι іі 62 0.03 0.01 0.08 0.00 ιv,v ііі 45 0.03 0.01 0.06 0.02 і,ιv,v іv 38 0.02 0.00 0.04 0.00 іι,ііι v 50 0.02 0.00 0.06 0.00 іι,ііι **cervical grooves length/conscutum length ratio і 49 0.33 0.06 0.51 0.25 ιιι іі 62 0.32 0.05 0.45 0.21 ιιι ііі 45 0.42 0.10 0.80 0.26 і,іі,ιv,v іv 39 0.31 0.04 0.45 0.23 ιιι,v v 50 0.34 0.07 0.57 0.23 ιιι,ιv *containing missing data in zone ιv **containing missing data in zone ι p value in all parameters is similar (p< 0.001) table 5. qualitative parameter s of h. anatolicum in two groups of specimen (with and without qualitative parameter) qualitative parameters *group arch (n) pigmented parma (n) caudal depression (n) ι 21 30 57 ιι 225 216 188 i: specimens with qualitative parameter ii: specimens without qualitative parameter *containing missing data in group ι and ιι in all qualitative parameters iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 27 fig. 1. variation in cervical grooves length/conscutum length ratio parameter of h. anatolicum collected from different zones of khuzestan and lorestan provinces (original figure). fig. 2. variation in conscutum length parameter of h. anatolicum collected from different zones of khuzestan and lorestan provinces (original figure). fig. 3. variation in basis capituli depression parameter of h. anatolicum collected from different zones of khuzestan and lorestan provinces (original figure). iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 28 fig. 4. graph illustrating variation in lateral grooves length/conscutum length ratio parameter of h. anatolicum collected from different zones of khuzestan and lorestan provinces discussion morphometric analysis in hyalomma spp. is poorly studied perhaps because morphologic characters in some species are obscure and unconfined. delpy (1936a), after studying the genus hyalomma, came to conclusion that diagnosis of hyalomma species was almost impossible and suggested that the range of variations among laboratory bred offspring of individual females must be studied, for instance he reported high spectrum of qualitative variation in the adanal shields of hyalomma dromedarii. hoogstraal (1956) stated that the variation in the morphologic characters of hyalomma species caused to nomination and presentation of the numerous hyalomma species (about eighty species) by schulze and his collaborators. delpy (1936a, 1937a) described all parasitic stages of the camel tick, h. dromedarii, he measured the length and width of the scutum, basis capituli, іі and ιιι palpi segments, spiracular plate and the many other characters in addition some of the comparative parameters. hyalomma schulzei, (olenev, 1931) the robust and rare iranian halomma species, also studied by delpy (1937b), the measured characters include corps length and width, corps length to capituli length ratio and іv genua length ratio and the other characters (delpy 1937b). mazlum (1978) revealed the taxonomic status and presence of h asiaticum in the iranian ixodid fauna and focused on scutum length as a taxonomic discriminating character for four closely related hyalomma. filippova and musatov (1996) studied morphologic variability in eight population of prostriate tick ixodes persulcatus (schulze, 1930) to distance 8700 and 2900 km in the western-eastern and northern-southern populations, respectively, they have shown morphometric variation in the anal ring and gnathosoma length and width, hypostome, іі and ііі palpi also і tarsi length. scutum size (body length in many literature) in the genus hyalomma is a good interspecific discriminating character but no for closely related species as apanaskevich and iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 29 horak (2005) reported 1.48–2 mm and 1.64– 2.05 mm in conscutum size range of males of two similar and debatable species h. anatolicum and h. excavatum respectively. the statistical significance of size in h. anatolicum immature stages were studied by arthur and snow (1966), they reported a non-linear relationship between the length of the egg, and the emergent larva. the parma (central festoon) is a suitable discriminating character for interspecific segregating of h. marginatum complex (pomerantzev 1950 ) as parma is not seen in the representative specimens. mazlum (1978) reported which is parma as a multishape character for identification of excavatum and asiaticum groups and showed oval, oval, subtriangular and subrectangular shapes in h. anatolicum, h. excavatum, h. asiaticum and h. dromedarii, respectively . usually color of parma is dark in h. marginatum complex while sometime is pale and may be cause to misidentification of this species with h. anatolicum. adler and feldman-muhsam (1948) used from pigmented and unpigmented parma phrase in the palestin hyalomma identification key, they believed the ticks of h. marginatum complex might be separated from the other species using this character (adler and feldmanmuhsam 1948). pomerantzev ( 1950) remarked the basis capituli depression posteriodorsally as specific character for discriminating of h. asiaticum from h. anatolicum however stated more concave and slightly concave modal phrases for two mentioned species, respectively (pomerantzev 1950). kaiser and hoogstraal (1964) used from depth of the basis capituli depression in addition to be angular in h. asiaticum and h. anatolicum, respectively. in the present study we observe several individuals from very deep to less deep range in h. anatolicum (0 to 0.08 mm), also its angularity were seen by our observation. abdigoudarzi (2003) stated that the basis capituli depression posteriodorsally is a specific feature for differentiating of h. anatolicum from h. asiaticum specimens in the iranian hyalomma fauna (abdigoudarzi 2003). the arch is a qualitative character which is debatable for identification of two closely related species h. anatolicum and h. excavatum as the former species this character not seen as connection of pair festoons 2 anteriorly and usually may be seen as wart like projection in the position of posteriodorsal groove to parma connection (hoogstraal and kaiser 1959b ), or a weak junction similar to arch feature (abdigoudarzi 2003) nevertheless in the future species the semicircle arch may be seen obviously. adler and feldman-muhsam (1948) believed the arch is very important trait and may be causes to misidentification of the closely related hyalomma species. the lateral groove length is a reliable character for interspecific identification of h. anatolicum as the value of this parameter in our study was not significant statistically. this means which the amplitude of variation in the mentioned character is limited. our object from the studying of this important character was to obtain numerical value for its which is mentioned as qualitative character in many hyalomma identification keys (delpy 1936a, hoogstraal 1956 , kaiser and hoogstraal 1964, apanaskevich and horak 2005 ) for instance as lateral grooves not extending beyond the posterior third of the scutum (hoogstraal 1956 ) or lateral grooves short, not reaching central third of scutum (kaiser and hoogstraal 1964 ), which are the vague definitions. hoogstraal (1956) believed the lateral grooves length is an important character for discriminating of the genus hyalomma, but pomerantzev (1950) and adler and feldmanmuhsam (1948) was not mentioned this character in their hyalomma identification keys. abdigoudarzi (2003) after morphologic observation on the iranian hyalomma specimens reported that the lateral groove is a valuable character in some hyalomma species and emphasis on the lateral grooves based on observation and description, no through statisiran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 30 tics, therefore the present study may be complementary work. the cervical grooves alike the lateral grooves which has never been measured, and described as cervical grooves short, not reaching mid-scutum (kaiser and hoogstraal 1964) or cervical grooves are seen as slight depression (pomerantzev 1950 ) which are the vague descriptive statements. this study declare the length of the cervical grooves quantitatively, however the length of this character in under studied specimens of h. anatolicum was significance statistically, which means the trait from the cervical grooves which is important for discriminating of h. anatolicum from h. asiaticum may be its depth instead length of this character. abdigoudarzi (2003) accepted our result on validity of the cervical grooves but stated that it may be a less useful and unstable character in him observed materials (abdigoudarzi 2003). kaiser and hoogstraal (1964) emphasize on elongation and are more depth of the cervical grooves of h. asiaticum from h. anatolicum while, pomerantzev (1950) no stated about length of cervical grooves whereas considered the depth of this character as valuable taxonomic feature. feldman-muhsam (1962) shown that the cervical grooves are deep, wide and long in h. anatolicum of the koch’s type collection. in the present study, we observed number of specimens of h. anatolicum with the deeper and longer cervical grooves. identification of the hyalomma species not is possible unless to observation of many specimens from species and measure of the taxonomic and morphologic traits, features or characters and data analyzed via morphometric and statistics methods. the variation in range and quantity of the morphometric parameters of h. anatolicum underlying that the correct identification and key construction for hyalomma species depended on a complement morphometric study on the other species especially closely related species. acknowledgements the present work is part of msc thesis supported financially by tarbiat modares university. our sincere thanks go to mr tavakoli (natural recourses research center of lorestan province) and dr taheri (razi vaccine and serum research institute, ahvaz, khuzestan province) for offering tremendous specimens from lorestan and khuzestan respectively. the authors declare that there is no conflict of interests. references abdigoudarzi m (2003) review of ticks (acari: ixodidae) and redescription of hyalomma and rhipicephalus genera by rapdpcr in iran. [phd dissertation], school of agriculture, university of tarbiat modares, iran. adler s, feldman-muhsam b (1948) a note on the genus hyalomma koch in palestine. parasitology. 39: 95–101. apanaskevich da, horak ig (2005) the genus hyalomma koch, 1844. ιι. taxonomic status of h. (euhyalomma) anatolicum koch, 1844 and h. (e.) excavatum koch, 1844 (acari: ixodidae) with redescriptions of all stages. acarina. 13(2): 181–197. arthur dr, snow k (1966) the significance of size in the immature stages of the ixodoidea. parasitology. 56: 391–397. burkot tr, graves pm (2004) malaria, babesiosis, theileriosis and related diseases. in: eldridge bf, edman jd (eds): medical entomology: a textbook on public health and veterinary problems caused by arthropods. kluwer academic publishers, the netherlands. delpy lp (1936a) notes sur les ixodidés du genre hyalomma (koch). ann parasitol hum comp. 14(3): 206–245. iran j arthropod-borne dis, 2011, 5(2): 23–31 a hosseini et al.: morphometric study on male … 31 delpy lp (1936b) sur la teratology du sousgenre hyalomma (koch 1884) ann parasitol hum comp. 4(1): 48–54. delpy lp (1937a) description de hyalomma dromedarii (koch 1884) morphologie de la larve et de la nymphe. ann parasitol hum comp. 14(6): 481–486. delpy lp (1937b) notes sur les ixodidae du genre hyalomma koch ιі. hyalomma schulzei olenev 1931. ann parasitol hum comp. 14(1): 419–430. feldman-muhsam b (1962) revision of the genus hyalomma iιι. h. lusitanicum koch and h. anatolicum k. parasitology. 52: 211–219. filippova na, musatov sa (1996) geographic variability in the sexually mature phase of ixodes persulcatus (ixodidae) experience in using databases on morphometry. parazitologiia. 30(3): 205–215. hashemi-fesharki r (1997) tick-borne diseases of sheep and goats and their related vectors in iran. parassitologia. 39(2): 115–117. hoogstraal h (1956) african ixodoidea. i. ticks of the sudan (with special reference to equatoria province and with preliminary reviews of the genera boophilus, margaropus and hyalomma). united state navy, washington dc. hoogstraal h (1979) the epidemiology of tick-borne crimean-congo hemorrhagic fever in asia, europe, and africa. j med entomol. 22(4): 307–417. hoogstraal h, kaiser mn (1959b) observation on egyptian hyalomma ticks (ixodoidea, ixodidae).5. biological notes and differences in identity of h. anatolicum and its subspecies anatolicum koch and excavatum koch, among russian and other workers. identity of h. lusitanicum koch. ann entomol soc am. 52(3): 243–246. kaiser mn, hoogstraal h (1964) the hyalomma ticks (ixodoidea, ixodidae) of pakistan, india, and ceylon, with keys to subgenera and species. acarologia. 6(2): 257–286. mazlum z (1968) hyalomma asiaticum asiaticum schulze and schlottke, 1929. its distribution, hosts, seasonal activity, life cycle and role in transmission of bovine theileriosis in iran. acarologia. 10(3): 437–442. pervomaisky gs (1950) interspecific hybridization of ixodidae. dokl akad nauk sssr. 73(5): 1033–1036. pomerantzev bi (1950) fauna of ussr arachnida: ixodid ticks (ixodidae). zoological institute of the academy of sciences ussr, moscow. rahbari s, nabian s, shayan p (2008) primary report on distribution of tick fauna in iran. iran j arthropod-borne dis. 2(1): 16–20. telmadarraiy z, ghiasi m, moradi m, vatandoost h, eshraghian mr, faghihi f, zarei z, haeri a, chinikar s (2010) a survey of crimean-congo haemorrhagic fever in livestock and ticks in ardabil province, iran during 2004–2005. scand j infect dis. 42(2): 137–141. j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 469 http://jad.tums.ac.ir published online: december 30, 2017 original article evaluation of deltamethrin in combination of piperonyl butoxide (pbo) against pyrethroid resistant, malaria vector, anopheles stephensi in irs implementation: an experimental semi-filed trial in iran fatemeh nikpour 1, *hassan vatandoost 1,2, ahmad ali hanafi-bojd 1,2, ahmad raeisi 3, mansour ranjbar 4, ahmad ali enayati 5, mohammad reza abai 1,2, mansoreh shayeghi 1, abdol rasoul mojahedi 6, abolghasem pourreza 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants, institute for environmental research, tehran university of medical sciences, tehran, iran 3malaria control department, ministry of health and medical education, tehran, iran 4independent malaria consultant, tehran, iran 5department of medical entomology and vector control, school of public health, mazandaran university of medical sciences, sari, iran 6provincial health center, bandar abbas university of medical sciences, bandar abbas, iran (received 10 oct 2017; accepted 15 nov 2017) abstract background: the aim of this study was to evaluate different concentrations of deltamethrin combined with formulated piperonyl butoxide (pbo) synergist on various surfaces against the wild strain of anopheles stephensi, the main malaria vector in southern iran under semi-field condition. methods: four concentrations of deltamethrin wg 25% (tagros) and pbo 800ec-uv (endura) were prepared and sprayed on the pre-designed surfaces in accordance with who alliance line of the irs micronair®. the who’s recommended bioassay kit and method was used during this study. results: comparing the mortality rate of mosquitoes, the results showed a significant difference between months after treatment of irs (indoor residual spraying) (p< 0.05) but didn’t show any significant differences between days during the first and second months (p> 0.05). statistical test revealed a significance difference between mortality rate of mosquitoes in exposing to concentrations of 1 and 4 (p< 0.05) which demonstrated effect of synergizing pbo on mortality rate. conclusion: this research as the first semi-field trial on deltamethrin added to different concentrations of formulated pbo for irs, indicates that deltamethrin+10x pbo is more effective than other concentrations. therefore, using synergists can be suggested as a new tool for prevention of pyrethriod resistance, although more studies are recommended. keywords: insecticide resistance, anopheles stephensi, deltamethrin, piperonyl butoxide, irs introduction noticeable reduction (90%) in incidence and mortality of global malaria became one of aims of 2030 (1). instead, the development of resistance to insecticides is probably the greatest threat to defeat malaria vectors control program. pyrethroids are the main chemical com-ponents used in malaria vector control programs. the best methods for using pyrethroids are long lasting insecticidal nets (llin) and indoor re-sidual spraying (irs) (2). but increasing use and coverage of irs and llin are causing more resistant mosquitoes which can finally undermine the success of these methods (3). *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 470 http://jad.tums.ac.ir published online: december 30, 2017 vector control advisory group (vcag) has been established by who for advising new tools and approaches to vector control (4). the national malaria control program of the islamic republic of iran was focused on controlling malaria however, in 2006 elimination became the focus along with the national strategic plan on elimination of local transmission (5). the action towards reducing malaria resulted in identification of only 167 indigenous (local) cases in 2015, that were almost all found in the three south-eastern provinces i.e sistan and baluchestan, hormozgan and kerman (6). in these areas, anopheles culicifacies giles s.l., anopheles dthali patton, anopheles fluviatilis james s.l., anopheles stephensi liston and anopheles superpictus grassi are known to be proven malaria vectors, while there is also report of sporozoite infection of anopheles pulcherrimus theobald (7–9). several researches on insecticide resistance monitoring, revealed resistance status of anopheles mosquitoes to a wide range of insecticides in iran. an. stephensi resistance to insecticides (ddt, dieldrin and malathion) was first reported in 1957, 1960 and 1976 respectively. the results of susceptibility tests of the most recent report of pyrethroid resistance of this species in iran (10) indicated that an. culicifacies is tolerant/ resistant to ddt, dieldrin, propoxur (11), malathion (12). anopheles dthali has been known as resistant to ddt and dieldrin in iran, but current studies show that an. dthali is susceptible to all tested insecticides from organochlorine, organophosphate, carbamates and pyrethroids (13). the tolerance to deltamethrin in this species (9, 14) is also a noticeable. resistance to pyrethroids in anopheles mosquitoes appears to be effected by target site insensitivity knock down resistance (kdr) and metabolic mechanism caused by mixedfunction oxidases (mfo) (15–16). insects, in general, despite their suscep tibility to insecticides, contain enzymes for metabolizing xenobiotic compounds and converting them to a non-toxic one that are finally removed through excretion. degradation or metabolism of insecticides are inhibited by pbo through blocking action, making it more effective. a great advantage of adding pbo to llin is the increased activity of pyrethoids in susceptible insects. pbo also increases the activity of pyrethroids in susceptible insects, so the addition of pbo to llin has an advantage, even in areas where there is no resistance. some studies have shown the impact of pbo resistances to pyrethroids in malaria vectors (17–18). also, there were some laboratory and field trials in which pbo added to llin or larvicide component. the results of the latter showed pbo suppressed resistance to pyrethroid insecticides in different populations of culicidae, indicating that oxidases and/or esterases play an important role in the reduction of pyrethroids toxicity (19–20). despite laboratory and field evaluation of pbo efficiency in llins and larvicides, so far there has not been any study on using this combination in irs. therefore, this study was aimed to evaluate insecticidal activity of different concentrations of deltamethrin combined with formulated pbo synergistic on various surfaces against the wild strain of an. stephensi, the malaria vector in southern iran under the semi-field condition. materials and methods preparation of the artificial surfaces initially, 24 wooden containers with dimensions of 5x40x40cm, were divided into four parts and each part had three spikes for holding cones used for bioassay test (fig. 1a). cement, plaster, clay and wood surfaces were placed in each the wooden container and left to dry at room temperature. these containers were treated with insecticide and different concentrations j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 471 http://jad.tums.ac.ir published online: december 30, 2017 of deltamethrin+synergist, while four untreated control containers were maintained (21). concentrations four concentrations of deltamethrin wg 25% (tagros) and pbo 800ec-uv (endura) were prepared as follows: -concentration 1 (con 1) deltamethrin (without pbo) -concentration 2 (con 2) deltamethrin: pbo= 1: 3 -concentration 3 (con 3) deltamethrin: pbo= 1: 5 -concentration 4 (con 4) deltamethrin: pbo= 1:10 residual spraying five replicates of the containers have been installed on the wall and treated by different concentrations according to who alliance line of the irs micronair®. insecticide was sprayed using a compression sprayer recommended by who for the irs which is equipped with a pressure gauge and hss8002 nozzles tips with regulator set at 24–55 psi. each concentration was dissolved in 10 liters of water in compression sprayer tanks. the sprayer discharge rate was set to 755 to 780ml/min. the spray duration was adjusted to spray 19m2 in one minute (21). the operation was done by an expert under supervision (fig. 1b). the containers treated with different concentrations were then allowed to dry at room temperature and installed vertically on the wall in four separate rooms (fig. 1c). mosquito species tested anopheles stephensi larvae were collected from hormoodar village (27°19'14.72"n, 56°19'14.80"e), in the south of bandar abbas city during august 2015january 2016 and were transferred to the insectary of bandar abbas research station as who collaborating center for malaria training. the larvae were reared into f1 generation for subsequent tests. adult susceptibility tests insecticide susceptibility tests were carried out under laboratory conditions against an. stephensi with deltamethrin 0.05% (diagnostic dose) impregnated paper provided by who. the procedure of test was followed according to who (22). bioassay tests the bioassay tests were carried out for evaluation of residual effect of different concentrations using standard who cones. the cones were fitted on different treated surfaces using rubber band. about 10–12 sugar-fed, 3–5 days old female mosquitoes were gently released into each cone at the vertical position. the mosquitoes were exposed for 30mins to each treated surfaces in five different replicates. the same procedures were carried out for control container. at the end of exposure time, the adults were transferred into clean cups with cotton wool pad containing 10% sucrose solution and were kept in the insectary for 24h recovery period, the time for recording the mortality rate. contact bioassay tests were carried out on days 1, 5, 15, 30, 45, 60, 105 and 120 after treatment. relative humidity and temperature of the test rooms were recorded during the bioassay experiments (23). statistical analysis data obtained from different replicates were collected for each surface. the mortality rate under 80% was considered as threshold level (24). the mortality rate rates were transformed into the arc sin √p. anova test was used for comparison. tests with control mortality rate between 5 and 20%, were corrected using abbott’s formula (25). results the susceptibility tests of an. stephensi against diagnostic dose of deltamethrin (0.05%) j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 472 http://jad.tums.ac.ir published online: december 30, 2017 resulted in 91% mortality rate. it means this species is a candidate of resistance to this insecticide according to the new who criteria. results of bioassay test on different surfaces during 120 days were as follows: mortality rate in plaster surface results of bioassay test on plaster showed 80–100% mortality rate of an. stephensi during the first month of treatment for all concentrations. this ratio reduced to 36% in concentration con 1 and con 3 after 120 days of treatment. these results indicated that deltamethrin had a residual effect of about 2.5 months on con 4 while the others had a residual effect around 1–1.5 months (table 1). there was no significant difference for plaster surface between different concentrations after 120 days of treatment (p> 0.05). mortality rate in thatch surface results of bioassay test on thatch showed 86–100% mortality rate of an. stephensi during the first month of treatment. mortality rate after 120 days of treatment reduced to 45.1%, 50%, 50% and 47.5% in con.1, 2, 3 and 4 respectively. mortality rate indicated that deltamethrin had a residual effect about two months on all concentrations except con 1 (1.5 month) (table 2). mortality rate in cement surface results of bioassay test on cement showed 84–100% mortality rate of an. stephensi during the first month of treatment. mortality rate after 120 days of treatment reduced to 50% in all concentrations. mortality rate indicated that deltamethrin had a residual effect about 2.5 months on all concentrations except con 1 (one month) (table 3). mortality rate in wood surface results of bioassay test on wood showed 84–100% mortality rate of an. stephensi during the first month of treatment. mortality rate after 120 days of treatment reduced to 46% in con 2 and con 3. mortality rate indicated that deltamethrin had a residual effect about two months using con 4, 1.5 month for con 2 and con 3, while one month using con 1 (table 4). results of bioassay test using different concentrations during 120 days were as follows: mortality rate of concentration 1 mortality rate of an. stephensi on different surfaces ranged from 84–100% during the first month of treatment. this value has dropped to 50% on cement and wood surfaces after 120 days of spraying. however, on plaster and thatch surfaces, mortality rate was reduced to less than 50% after 90 and 105 days after treatment. so deltamethrin had a residual effect of about 1.5 month on thatch and plaster, one month on other surfaces (fig. 2). mortality rate of concentration 2 mortality rate of an. stephensi on different surfaces ranged from 80–100% during the first month of treatment. mortality rate after 120 days of treatment was 50% on thatch and cement surfaces but mortality rate on plaster and wood was less than 50% after 105 after treatment so based on indicating that deltamethrin has a residual effect of about 2.5 months on cement, two months on thatch, 1.5 month on wood and one month on plaster surface (fig. 3). mortality rate of concentration 3 mortality rate of an. stephensi on different surfaces ranged from 86–100% during the first month of treatment. mortality rate after 120 days of treatment was 50% on thatch and cement surfaces but mortality rate on plaster and wood surfaces were less than 50% after day 105 of treatment. the results showed that deltamethrin has a residual effect of about 2.5 months on cement, two months on thatch, 1.5 month on wood and one month on plaster surface (fig. 4). j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 473 http://jad.tums.ac.ir published online: december 30, 2017 mortality rate of concentration 4 mortality rate of an. stephensi on different surfaces ranged from 88–100% during the first month of treatment. mortality rate after 120 days of treatment was equal to or more than 50% on plaster, cement and wood surfaces but mortality rate on thatch was less than 50% after day 105 of treatment. based on these results deltamethrin has a residual effect of about 2.5 months on plaster and cement and two months on thatch and wood surfaces (fig. 5). comparing the mortality rate of mosquitoes, there was a significant difference between months after treatment (p< 0.05) but there were no significant differences between days in the first and second months (p> 0.05). statistical test revealed a significant difference in mortality rate of mosquitoes in exposure to con 1 and others (p< 0.0001), but no significant difference was found between con 2 and con 3 (p> 0.05). tukey's test showed that there was a significant difference between mortality rate of mosquitoes on cement and other surfaces (p< 0.05), while there was no significant between other three surfaces (p> 0.05). fig. 1. a. wooden container with four different surfaces, b. spraying operation, c. install contair after spraying in room which seprated into four parts fig. 2. comparison of deltamethrin persistence without piperonyl but oxide on different surfaces against anopheles stephensi, 2015–2016 j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 474 http://jad.tums.ac.ir published online: december 30, 2017 fig. 3. comparison of deltamethrin persistence with 3x piperonyl but oxide and deltamethrin on different surfaces against anopheles stephensi, 2015–2016 fig. 4. comparison of deltamethrin persistence with 5x piperonyl but oxide on different surfaces against anopheles stephensi, 2015–2016 j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 475 http://jad.tums.ac.ir published online: december 30, 2017 fig. 5. comparison of deltamethrin persistence with 10x piperonyl but oxide on different surfaces against anopheles stephensi, 2015–2016 j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 476 http://jad.tums.ac.ir published online: december 30, 2017 table 1. persistence of deltamethrin with/without piperonyl but oxide on plaster surface against anopheles stephensi, 2015–2016 days after spraying concentrations control concentration 1 concentration 2 concentration 3 concentration 4 t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity 1 52 47 90.4 ± 3.2 50 40 80.0 ± 0.6 50 43 86.0 ± 2.2 50 50 100.0 ± 0.0 40 3 7.5 5 50 50 100.0 ± 0.0 50 42 84.0 ± 2.8 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 40 5 12.5 15 51 51 100.0 ± 0.0 50 48 96.0 ± 4.0 50 48 96.0 ± 4.0 50 48 96.0 ± 2.6 40 2 5 30 50 46 92.0 ± 2.1 50 45 90.0 ± 2.7 50 45 90.0 ± 2.7 50 47 94.0 ± 2.4 40 2 5 45 51 41 80.4 ± 0.9 50 36 72.0 ± 3.0 50 36 72.0 ± 3.0 50 42 84.0 ± 3.9 40 3 7.5 60 50 37 74.0 ± 3.1 50 38 76.0 ± 5.1 50 38 76.0 ± 5.1 50 45 90.0 ± 2.9 40 2 5 75 50 32 64.0 ± 2.7 50 38 76.0 ± 2.0 50 38 76.0 ± 2.0 50 42 84.0 ± 2.2 40 4 10 90 50 27 54.0 ± 3.5 50 34 68.0 ± 2.2 50 29 58.0 ± 3.7 50 35 70.0 ± 3.3 40 0 0 105 50 25 50.0 ± 3.0 50 27 54.0 ± 1.9 50 27 54.0 ± 1.9 50 33 66.0 ± 2.6 40 1 2.5 120 50 18 36.0 ± 3.7 50 18 36.0 ± 1.9 50 18 36.0 ± 1.9 50 25 50.0 ± 1.6 40 2 5 table 2. persistence of deltamethrin with/without piperonyl but oxide on thatch surface against anopheles stephensi, 2015–2016 days after spraying concentrations control concentration 1 concentration 2 concentration 3 concentration 4 t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity 1 50 50 100.0 ± 0.0 51 51 100.0 ± 0.0 49 48 98.0 ± 2.0 51 46 90.2 ± 3.6 40 3 7.5 5 51 50 98.0 ± 2.0 50 45 90.0 ± 3.2 50 50 100.0 ± 0.0 51 51 100.0 ± 0.0 40 5 12.5 15 50 50 100.0 ± 0.0 50 47 94.0 ± 2.5 50 47 94.0 ± 2.5 50 49 98.0 ± 2.0 40 2 5 30 50 43 86.0 ± 1.6 49 46 93.9 ± 4.0 49 46 93.9 ± 4.0 49 43 87.8 ± 3.7 40 2 5 45 51 42 82.4 ± 3.6 50 40 80.0 ± 3.2 50 40 80.0 ± 3.2 50 38 76.0 ± 2.0 40 3 7.5 60 50 38 76.0 ± 1.3 50 41 82.0 ± 1.9 50 41 82.0 ± 1.9 51 41 80.4 ± 4.6 40 2 5 75 50 33 66.0 ± 5.6 50 38 76.0 ± 4.2 50 38 76.0 ± 4.2 50 36 72.0 ± 6.7 40 4 10 90 50 21 42.0 ± 2.3 50 37 74.0 ± 3.0 50 32 64.0 ± 2.8 50 33 66.0 ± 9.4 40 0 0 105 52 23 44.2 ± 2.6 50 28 56.0 ± 1.9 50 28 56.0 ± 1.9 50 28 56.0 ± 4.2 40 1 2.5 120 51 23 45.1 ± 7.5 50 25 50.0 ± 2.8 50 25 50.0 ± 2.8 40 19 47.5 ± 3.3 40 2 5 j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 477 http://jad.tums.ac.ir published online: december 30, 2017 table 3. persistency of deltamethrin with/without piperonyl but oxide on cement surface against anopheles stephensi, 2015–2016 days after spraying concentrations control concentration 1 concentration 2 concentration 3 concentration 4 t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity 1 50 42 84.0 ± 1.7 49 49 100.0 ± 0.0 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 40 3 7.5 5 51 45 88.2 ± 1.7 50 44 88.0 ± 2.0 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 40 5 12.5 15 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 40 2 5 30 50 46 92.0 ± 2.0 49 45 91.8 ± 3.5 49 45 91.8 ± 3.5 50 45 90.0 ± 3.2 40 2 5 45 50 37 74.0 ± 2.2 50 42 84.0 ± 2.2 50 42 84.0 ± 2.2 50 47 94.0 ± 2.4 40 3 7.5 60 49 36 73.5 ± 1.8 50 40 80.0 ± 3.2 50 40 80.0 ± 3.2 50 42 84.0 ± 2.2 40 2 5 75 51 36 70.6 ± 5.8 50 42 84.0 ± 2.6 50 42 84.0 ± 2.6 50 42 84.0 ± 2.2 40 4 10 90 49 34 69.4 ± 3.1 50 35 70.0 ± 3.3 50 32 64.0 ± 2.4 50 37 74.0 ± 2.7 40 0 0 105 50 32 64.0 ± 2.8 50 27 54.0 ± 1.6 50 27 54.0 ± 1.6 50 35 70.0 ± 3.5 40 1 2.5 120 50 25 50.0 ± 1.8 50 25 50.0 ± 3.2 50 25 50.0 ± 3.2 50 27 54.0 ± 2.7 40 2 5 table 4. persistence of deltamethrin with/without piperonyl but oxide on wood surface against anopheles stephensi, 2015–2016 days after spraying concentrations control concentration 1 concentration 2 concentration 3 concentration 4 t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity ± s e t o ta l d e a d m o r ta lity 1 49 46 93.9 ± 2.5 50 50 100.0 ± 0.0 51 51 100.0 ± 0.0 51 51 100.0 ± 0.0 40 3 7.5 5 50 42 84.0 ± 2.2 50 45 90.0 ± 0.0 50 50 100.0 ± 0.0 50 50 100.0 ± 0.0 40 5 12.5 15 51 51 100.0 ± 0.0 50 47 94.0 ± 2.5 50 47 94.0 ± 2.5 50 48 96.0 ± 2.3 40 2 5 30 49 49 100.0 ± 0.0 49 49 100.0 ± 0.0 49 49 100.0 ± 0.0 49 47 95.9 ± 2.4 40 2 5 45 51 39 76.5 ± 2.2 50 40 80.0 ± 3.2 50 40 80.0 ± 3.2 51 45 88.2 ± 5.8 40 3 7.5 60 49 34 69.4 ± 1.8 50 38 76.0 ± 2.5 50 38 76.0 ± 2.5 50 42 84.0 ± 2.4 40 2 5 75 50 35 70.0 ± 2.4 50 38 76.0 ± 4.2 50 38 76.0 ± 4.2 50 38 76.0 ± 4.2 40 4 10 90 50 31 62.0 ± 4.7 50 34 68.0 ± 2.0 50 32 64.0 ± 3.3 50 33 66.0 ± 2.4 40 0 0 105 50 23 46.0 ± 4.1 50 30 60.0 ± 1.3 50 30 60.0 ± 1.3 50 28 56.0 ± 3.6 40 1 2.5 120 50 25 50.0 ± 2.3 50 23 46.0 ± 3.0 50 23 46.0 ± 3.0 50 26 52.0 ± 1.2 40 2 5 j arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 478 http://jad.tums.ac.ir published online: december 30, 2017 discussion at present, pyrethroids are being used for irs and in mosquito nets and various products worldwide (3). there is no alternative insecticide for the treatment of nets other than pyrethroids synergistic nets. among products being evaluated by the pesticide evaluation scheme (whopes) were mosquito nets containing a pyrethroid and a compound of an unrelated class (e.g. chlorfenapyr or pyriproxyfen) and clothianidin irs formulated with or without pyrethroid (27–28). deltamethrin+pbo for use in irs, has been a proposed product in order to overcome the appearance of resistance. this combined product could be used as a vector control tool in country programs. this tool is highly recommended in countries that are in elimination phase or have reported insecticide resistance due to vector control strategy being a key strategy. the results of this study showed that although mortality rates in different concentrations of deltamethrin+pbo decreased during 120 days after treatment, mortality rate in day 120 in all of them was higher than deltamethrin without pbo (figs. 2–5). regardless of surface type, there was also an eligible difference in mortality rate between deltamethrin without pbo (con 1) and deltamethrin+ pbo= 1:10 (con 4) concentration against an. stephensi field strain (p< 0.0001). therefore, it can be concluded that pbo had a positive effect on the efficacy of insecticide. exito-repellency effect of deltamethrin may be the reason for different mortality rates (90– 100%) between different surfaces and concentrations in the first month of the study. the study on plaster surfaces showed a significant difference between mortality rate on con 1 and con 4 on both days 1 and 120 after treatment, but this difference was only significant in day 1 on cement, thatch and wood surfaces. this results is in line with the study in benin that evaluated permanet 3.0 (deltamethrin+pbo) against pyrethroid-resistant an. gambiae and cx. quinquefasciatus in an experimental hut. they found a negligible difference between the mortality rate of permanet 2.0 (deltamethrin) and permanet 3.0 before and after the 20 times washing (29). in some researches in african countries which was proved kdr and metabolic resistance, tricomponent of llin were used. these nets include pyrethroid+pbo and other group of insecticides with different mechanisms of action such as pyrole chlorfenapyr or neonicotinoid. they found tricomponents had more insecticidal activity than one component llin on pyrethroid-resistant an. gambiae, ae. aegypti, an. funestus and cx. quinquefasciatus (30–33). a study conducted on a pyrethroid resistant strain of cx. pipiens (3.8 to 38.4 folds) evaluated mixture of pyrethriod larvicides and pbo (20). they found pbo suppressed resistance to pyrethroid insecticides (>90%) in field populations indicating that oxidases and/or esterases play an important role in the reduction of pyrethroids toxicity. another survey conducted to assay larviciding impact of a mixture of stock solution of pbo and deltamethrin in 6:1 ratio on resistant strains (4–21 folds) of ae. aegypti, an. culicifacies, an. stephensi, an. vagus, cx. tritaeniorhynchus, cx. pipiens, revealed that pbo suppressed resistance between 75–95% (26). it can be concluded that lower mortality rate indicates resistance which can result in better efficacy of pbo. although, in this study, we had 91% mortality rate in the tested strain which was not resistance strain but significant differences were found in mortality rates between some concentrations (figs. 2–5). both above mentioned studies used technical pbo under laboratory condition, but we applied a formulated product under semi-field condition. these differences may also affect the results. several study results revealed that mortality rate in non-sorbent (wood) and sorbent surj arthropod-borne dis, december 2017, 11(4): 469–481 f nikpour et al.: evaluation of … 479 http://jad.tums.ac.ir published online: december 30, 2017 faces (mud, thatch, plaster and cement) had no significant differences in mortality rate. the absorption rate on different surfaces (wood, plaster, mud and cement) had notable variability in mortality rate on parous surfaces, as have been reported in other studies (21, 34) but the result of this study revealed differences in absorption rate on parous surfaces in different concentrations (con 1 and con 3 and con 4) (tables 1–4) which can be the effect of pbo. it seems that the moderate and high concentration of pbo (con 3 and con 4) had effected the high level of mortality rate on the first day of treatment however in con 1, the high level of mortality started on day 15 of treatment. these results indicated that the presence of synergist has led to a decrease in absorption therefore resulting in high mortality rate from the beginning of treatment in comparison with absence of synergist (con 1). conclusion in conclusion, considering that the strain of an. stephensi used in this study was not resistance strain and the pbo could not result in significant difference in mortality rate after day 120, however the results suggest that the combination of deltamethrin+pbo can be more effective in mortality rate of resistant an. stephensi. also pbo was observed to be more functional on porous substrates, while higher concentration of pbo seems to be more effective. however, more studies on the strains with higher resistant ratio can prove our results. this method can be considered as a new tool for malaria vector control, although more studies are recommended under field condition. references 1. who (2015) global technical strategy for malaria 2016–2030. world health organization, geneva, switzerland, pp. 4–5. 2. who (2009) world malaria report 2008. world health 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905–910. 34. abtahi sm, shaeghi m, abai mr, akbarzadeh k, vatandoost h, ladonni h, darabi h (2007) evaluation of persistence and residual of deltamethrin and cyfluthrin on different surfaces at iranshahr area in sistan and baluchistan province in iran, 2004–2005. iranian south med j. 9: 123–130. 6dr shayeghi rtl iranian j arthropod-borne dis, 2010, 4(1): 47–53 m khoobdel et al.: effectiveness of ultrasound … 47 original article effectiveness of ultrasound and ultraviolet irradiation on degradation of carbaryl from aqueous solutions m khoobdel1, *m shayeghi2, s golsorkhi2, m abtahi2, h vatandoost2, h zeraatii3, s bazrafkan2 1health research center, baqiyatallah university of medical sciences, tehran, iran 2department of medical entomology, school of public health, tehran university of medical sciences, iran 3department of biostatistics, school of public health, tehran university of medical sciences, iran (received 16 dec 2009; accepted 19 apr 2010) abstract background: carbaryl (1-naphthyl-n-methyl carbamate) is a chemical in the carbamate family used chiefly as an insecticide. it is a cholinesterase inhibitor and is toxic to humans and classified as a likely human carcinogen. in the present study, the degradation of the carbaryl pesticide was investigated in the laboratory synthetic samples of tap water, in the effect of sonolysis and photolysis processes. methods: this study was conducted during 2006–7 in chemistry and biochemistry of pesticides laboratory in tehran university of medical sciences (tums) in iran. the carbaryl (80%) was used for preparing samples. first concentration of all samples were 4 mg/l. sonochemical examinations in ultrasound reactor was done in two 35, 130 hz, and 100 w, and three time. photolysis examinations has done in the effect of 400 w lamp and moderate pressure and 6 time, then the amount of pesticide in the samples has been measured by the high performance thin layer chromatography (hptlc) method. results: the highest degradation in photolysis process after 1 hour in the 35 khz was 35%, and in the 130 khz was 63%. degradation of carbaryl at 130 khz is higher than 35 khz at the same time. carbaryl elimination was increased by arise frequency and exposure time. after 8 min in photolysis, 100% omitting has been showed. conclusion: degradation of carbaryl in high frequency ultrasound wavelength was more than low frequency. degradation of carbaryl in water, combination of high frequency ultrasound wave length and uv irradiation was considerably more effective than ultrasound or ultraviolet irradiation alone. keywords: ultrasound, ultraviolet, degradation, pesticide, carbaryl introduction pesticides are an important potential component of chemical pollutants used extensively for agriculture and sanitation purposes and released into the environment (howard 1991). however, most of them are highly toxic, can exhibit chemical stability and resistance to biodegradation and continuously contaminated the aquatic and soil ecosystems (tomlin 1995, hardesen and wratten 1998). from theatrical point the amount of synthetic pesticides in endless, and the entrance of each of these compounds to drinking water and wastewater that is ejected to the acceptor waters, causes some hygienic problems (varshney 1998). carbaryl (1-naphthyl-n-methylcarbamate) is a broad spectrum carbamate insecticide with a variety of agricultural, garden and domestic application. due to its wide use, human may be exposed to its residues through food and other routes (gunasekara et al. 2008). although moderately water soluble, it neither vaporizes nor volatilizes readily. it is demon*corresponding author: dr mansoureh shayeghi, e-mail:mansorehshayeghi@yahoo.com iranian j arthropod-borne dis, 2010, 4(1): 47–53 m khoobdel et al.: effectiveness of ultrasound … 48 strated that even a weakly sorbed and easily degradable pesticide as carbaryl, is effectively sequestrated in soil with time, rendering it partly inaccessible to microorganisms and affecting the bioavailability of the compound (ahmed et al. 2004). carbaryl is a cholinesterase inhibitor and is toxic to humans. it is classified as a likely human carcinogen by the united states environmental protection agency (epa 2004). it kills various beneficial insect and crustacean species along with intended pest victims, so care must be taken when spraying where beneficial non target species are present (koskinin et al. 1994, back 2002). carbaryl is highly toxic to honey bees and very highly toxic to fresh water invertebrates as water fleas (dafnia), shrimp, stonefly and other aquatic community. carbaryl has been detected in many of surface water of usa at µ g/l concentration (gunasekara et al. 2008). this insecticide can be highly to slightly toxic to fish, depending upon the species (vonesh and kraus 2009, epa 2010). carbaryl consists of 2-nephtyl carbamate that has carcinogenic effects, and is produced in industrial processes (petrier et al. 1996). by the entrance of this insecticide to water sources and biological cycles, a lot of people are at risk of cancer so the protection of limited watery sources are very important (branch and jacoz 1999, back 2002). carbaryl is detected in water at ppb concentrations but degradation is relatively rapid, with 1-naphthol identified as the major degradation product (gunasekara et al. 2008). in iran was done some study about phosphorus insecticide residue in drinking water and rivers, but there is no study among carbarly residues in water or wastewater (shayeghi et al. 2007, 2008). the application of power ultrasound to chemical processes is one of number intensification technologies that have undergone serious and wide-ranging development over the past 10-15 yr (matouq et al. 2008). many reports for ultrasound treatment of water and wastewater have been considered (norwood 1990, somich 1990, suslick and price 1999, joyce 2002, gogate et al. 2003, benito et al. 2005, dehghani et al. 2007a, dehghani et al. 2007b, asakura et al. 2008, dehghani et al. 2008a, dehghani et al. 2008b, mahvi et al. 2009, dehghani et al. 2010). ultrasonic irradiation has been investigated in the degradation of some pesticide such as atrazine, parathion, carbofuran, dichlorvos (kotronarou et al. 1992, koskinen et al. 1994, petrier et al. 1996, pfalzer and hua 1998, schramm and hua 2001). the high frequency ultrasound technique was also used to degradation of dissolved diazinon pesticide in water as a clean technology to protect environment (motouq et al. 2008). in another study, the sonochemical photodegradation of fenitrotion in aqueous solution was investigated by ultrasonic/uv system (katsumata et al. 2009). also degradation of carboforan in aqueous solution by ultrasound and was studied (ma et al. 2010). a combination of uva radiation and ozone, in presence of titanium dioxide has been investigated for carbaryl degradation as a potential destructive technology for the treatment of pesticide wastewater (rajeswari and kanmani 2009). the photoinduced degradation of carbaryl was studied in surface water (miller and chin 2002). degradation of carbaryl in mixture with other pesticide was also done by combined photo-fenton and biological oxidation (balleseros et al. 2009). today more economical methods like using the ultrasound and ultra violate ray methods are noticed. previous studies were not performed sonochemical and photolysis declaration of carbamat. in this study the effects of ultrasonic and photolysis methods in the degradation and eliminating of carbaryl from drinking water were assessed. materials and methods all solvents which were used in this study provided from merck co. standard of iranian j arthropod-borne dis, 2010, 4(1): 47–53 m khoobdel et al.: effectiveness of ultrasound … 49 carbaryl were obtained from switzerland accustandard ltd. silica gel hptlc plates (sio4, 60f254) which were used as stationary phase, were bought from merck co in 20×20 cm dimensions. capillary tubes with 1, 2 and 5 µ l capacity which was used for spotting purchased from camag ltd. this study was conducted during 2006-7 in chemistry and biochemistry of pesticides laboratory in tehran university of medical sciences (tums) in iran. uv lamp characteristics: medium power, 125.5 mm length, 20mm diameter, voltage 130±15 v, intensity 3.25 a, radiation intensity 90 mw/cm2, half life 1000 h, manufactured by france. ultrasound irradiation system characteristic: capacity 3.7 lit, power 90 and 100 w, 2.5 w/cm, altered number 2, manufactured by germany. sonochemical and photochemical experiments were carried out by use of a bath sonicator (100w) working at 35 and 130 khz frequencies and by a 400w medium pressure uv lamp. samples of this study are synthetic and from tehran pipe water. the concentration of carbaryl in all of samples was 4 mg/lit (4ppm) and used the 80% insecticides. the samples were adjusted in reactor in 3 time of remaining (20, 40 and 60 minutes) and at of 35 and 130 khz frequencies and 100 w. in the photolysis process samples with 4 ppm concentration were affected in 6 remaining time (1, 2, 4, 6, 8, 10 min) and the 250 nm wavelength. reaction temperature in ultrasonic and ultraviolet process was kept at 20±1 ë. the ph of the sample solution was adjusted with hcl and/or naoh at 7.1. after the fixed time the amount of insecticide in samples were determined by using the hptlc methods. for this reason insecticide in samples were extracted by using dichloromethane. five µ l loading of each standard and sample solution was spotted on the hptlc plate. in each plate, 15 spots were performed which contained 3 standard spots and 12 samples (from the solution which were prepared with repellents). distance between tracks was 1 cm. in this study, the multiple levels spotting method was used. plates were developed using a mobile phase consisting of hexane-acetone (60/40 v/v). liner ascending was carried out in a twintrough glass chamber (camag, switzerland) equilibrated with mobile phase (hatrik and take 2001). the developed plate was air-dried for 10 min and then the spots were seen in uv cabinet with 254 nm. the slit dimension was set at 10.0×0.40 mm. the monochromatic bandwidth was set at 20 nm and a scanning speed 20 mm/s was employed. densitometry scanning was performed on camag tlc scanner iii at 290 nm and operated by cats4 software. the source of radiation utilized was the deuterium lamp. the spot migration distance (md) for the developed spots was defined by the distance between the solvent front and the starting line during the developing period. spots developing rate or rf values (retardation factor) were determined. the position of a substance zone (spot) in a thin layer chromatogram can be described by rf. this is defined as the quotient obtained by dividing the distance between the substance zone from the starting line (zs) by the distance between the solvent front (zf ) and the starting line (z0) (denistrop 2000). rf = zs / zf – z0 where: zs: distance of the substance zone from the starting line [mm] zf : distance of the solvent front from the solvent line [mm] z0 : distance between the solvent level and the starting line [mm] iranian j arthropod-borne dis, 2010, 4(1): 47–53 m khoobdel et al.: effectiveness of ultrasound … 50 data analysis, which included average amount of materials in each spot, was automatically done by cats4 software. this study was done in accordance with helsinki declaration and guideline of iranian ministry of health and medical education and was approved by ethics committee of tehran university of medical sciences. results the effects of ultrasound wave for carbaryl degradation at 35 and 130 khz with increasing time have a faster rate and the concentration of pesticide was decreased. in the sonochemical process, carbaryl undergoes degradation at 130 khz have a faster rate than 35khz, like that at 35 khz, after 60 min the maximum output of elimination is 35% , whereas at 135 khz, after the same time is 63% (table 1). also ultraviolet irradiation technique was considerably more effective than ultrasound. in uv technique, 100% of insecticide was eliminated from water in the 8 min. (fig. 1). the photochemical degradation of carbaryl in 8 min was complete (fig. 2). according to the results ultraviolet irradiation technique was considerably more effective than ultrasound. in uv technique, 100% of insecticide was eliminated from water in the 8 min (table 1). table 1. degradation percentage of carbaryl degradation percentage exposure time(min) process 9 20 ultrasound frequency (35khz) 22.15 40 35 60 25 20 ultrasound frequency (130khz) 49 40 63 60 80.27 1 ultraviolet irradiation 85.6 2 90 4 95.4 6 100 8 71 159 252 454 353 165 0 50 100 150 200 250 300 350 400 450 500 20 40 60 time(min) m e d ia n o f d e g ra d a ti o n (p p b ) f35 f135 fig. 1. declaration percentage of carbaryl in sonochemical treatment iranian j arthropod-borne dis, 2010, 4(1): 47–53 m khoobdel et al.: effectiveness of ultrasound … 51 578 612 648 687 720 720 0 100 200 300 400 500 600 700 800 1 2 4 6 8 10 time(min) m e d ia n o f d e g ra d a ti o n (p p b ) fig. 2. declaration median of carbaryl in photolysis process discussion according to the results, in sonochemical degradation, elimination of carbaryl at 130 khz is higher than 35 khz at the same time. carbaryl elimination was increased by arise wave length frequency and contact time. in another study, degradation of diazinon was successful performed using 1.7 mhz wavelength of ultrasound in 5 min (matouq et al. 2008). carbaryl in natural waters rapidly hydrolyzed to 1naphtol, which is identified as a general and primary degradation product of carbaryl. degradation was mediated by hydroxyl radical oxidation (gunasekara et al. 2008). carbaryl was photolyzed into 1,2 naphthoquinone, 1,4naphthoquinone, 2-hydroxy1, 4naphthoquinone and 7-hydroxy-1,4 naphthoquinone (brahimia and richard 2003). the best yield observed at 130 khz for carbaryl degradation may be the result of much more hydroxyl availability at the out of the bubble of cavitations. it was found that out put of elimination of carbaryl at photochemical degradation was higher than sonochemical destruction because in photolysis process, in addition of hydroxyl radical production, there is direct effect of ultraviolet energy on breaking down of the carbon-carbon bounds of carbaryl molecules (wu et al. 2001). degradation of fenitrothion by ultrasound/uv system was effective and achieved complete elimination after 30 min (katsumata et al. 2009). in another study, photolytic ozonation (o3/uv) had a synergistic effect on carbaryl degradation (rajeswari and kanmani 2009). in conclusion, based on the results, probably to elimination the carbaryl in water, combination of high frequency ultrasound wave length and uv irradiation was considerably more effective than ultrasound or ultraviolet light alone. acknowledgements the authors would like to thank mrs. azar ghasri for their kind cooperation in environmental health engineering laboratory. this study was financially supported by school of public health, tehran university of medical sciences. iranian j 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arthropod-borne dis, june 2017, 11(2): 278–285 h bilal et al.: bio-pesticides: new tool … 278 http://jad.tums.ac.ir published online: may 27, 2017 original article bio-pesticides: new tool for the control of aedes (stegomyia) albopictus (culicidae: diptera) in pakistan *hazrat bilal 1, sumrin sahar 2, sadrud din 3 1medical entomology and disease vector control, health services academy, islamabad, pakistan 2university of copenhagen, copenhagen, denmark 3water, agriculture and technology transfer program, kabul, afghanistan (received 9 nov 2015; accepted 9 july 2016) abstract background: application of plant extracts as mosquito control strategy was practiced from centuries. these are easily available, non-toxic, biodegradable and exhibit broad-spectrum target specific activities against larval stages of mosquitoes. methods: different potential parts of locally grown plants, seeds of nutmeg (myristica fragrans), peel of musambi (citrus sinensis), leaves of babuna (matricaria chamomilla), mint (mentha spicata) and ginger rhizome (zingiber officinale) selected and evaluated for their larvicidal properties against aedes (stegomyis) albopictus. oils were extracted through steam distillation process and extracts were evaluated as per who 2005 guidelines for testing of insecticides against larvae of mosquitoes. results: among the five plant extracts, c. sinensis had the lowest lc50 (400.81ppm) while m. fragrans had the highest lc50 value (710.30ppm) respectively after 24h of exposure. in terms of % age mortality, a series of concentrations (300–800ppm) gave high % mortality in case of c. sinensis while m. fragrans gave low % age mortality. conclusion: all the five plant species have larvicidal effects to certain extant and c. sinensis had great potential. further small-scale field trials with the extracts of the most promising one (c. sinensis) shall be conducted to determine operational feasibility. keywords: mosquitoes, plant extracts, larvicide introduction dengue, malaria, filariasis, yellow fever and japanese encephalitis are the most important diseases transmitted by mosquitoes (rozendaal 1997). fifty million cases of dengue occur globally every year (who 2009). dengue has now emerged in many countries, especially in pakistan where occurred outbreaks affected the socio-economic development in the region (savioli and velayudhan 2014). dengue epidemic in pakistan (2011) is being observed where, more than 22,778 cases confirmed and 353 deaths reported (anonymous 2011). in 2013, a punjab and khyber-pakhtunkhwa (kpk) provinces hit by second epidemic, in northern areas of pakistan the human mobilization from dengue endemic regions, geographic expansion of dengue fever vector due to importation, climatic change, all are the factors, which resulted in the emergence of dengue in northern areas (ali 2013). in district swat (kpk) 6,000 dengue cases with 47 deaths were reported (khan and khan 2013). worldwide mosquito control depends on the application of synthetic insecticides as a part of integrated vector control (ivm) programmes (becker et al. 2010). toxic effects and resistance to synthetic insecticides are barriers in controlling mosquitoes. therefore, it is necessary to develop safe alternative insecticides, which required minimum care (mittal and subbarao 2003). *corresponding authors: mr hazrat bilal, e-mail: hazratbilal@hsa.edu.pk http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 278–285 h bilal et al.: bio-pesticides: new tool … 279 http://jad.tums.ac.ir published online: may 27, 2017 plant based insecticides may be the best option for mosquito control as they have biologically active chemicals that are easily decomposed into products which are not toxic to other species (sanjay and tiku 2009) and potentially suitable for use in control of mosquito larvae (yang et al. 2004). in fact, many researchers have reported the effectiveness of plant extracts or essential oils against mosquito larvae (rahuman et al. 2008). research on insecticidal properties of botanicals inferred that they are bio-degradable, environmentally safe and target specific (govindarajan et al. 2008). (muthukrishnan 2012) and (bilal et al. 2012) evaluated the larvicidal effects of extracts from cinnamomum cassia, citrus sinensis linnaeus var musambi, tribulus terrestris, eucalyptus camaldulensis, piper nigera, ricinus communis, allium sativum, linum usitatissimum and citrus sinensis l var succari against aedes albopictus. a number of other researchers which have used plant products for the mosquito control like (komalamisra et al. 2005) reported the ether extracts of trigonostemon reidioides, rhinacanthus nasutus, derris elliptica, homalomena aromatica, stemona tuberose, acorus calamus and piper nigrum (siddiqui et al. 2004), artemisia annua, sonchus oleraceus and chenopodium album (sharma et al. 2006) solanum xanthocarpum (mohan et al. 2005) argemone mexicana (sakthivadivel and thilagavathy 2003). in the view of increased interest in development of plant-based insecticides as an alternative to synthetic insecticide, this study was planned and conducted to assess the larvicidal potential of five medicinal plants against the medically important mosquito (aedes (stegomyia) albopictus). materials and methods collection of plants nutmeg (m. fragrans houtt) seeds, mu sambi (c. sinensis (l) osbeck) peel, babuna (m. chamomilla l) leaves, mint (m. spicata l) leaves and ginger (z. officinale roscoe) rhizome were collected from botanical garden of university of agriculture faisalabad (31.4339° n, 73.0649° e) and local market of faisalabad (31.4181° n, 73.0776° e). extraction of oil the seeds, peel, leaves and rhizomes were washed, then dried and later pulverized in an electric grinder (anex germany). the pulverized material was placed in thimble and kept in extraction tube of soxhelt apparatus with extractor id 38mm, extractor volume 85ml and flask volume 250ml (vogel 1978) for the extraction of oil by steam distillation method using ether as solvent (250ml/20g sample). the cycle time for one sample was 4– 5h. solvent was evaporated at room tempeature, leaving oil, collected in flask. stock solution was prepared by adding 1ml of oil from each plant in 99ml of ether and considered as 1% stock solution from which series of concentrations (%) were prepared (akram et al. 2010). collection and rearing of mosquitoes mosquito larvae were collected from potential breeding sites of aedes around islamabad (33.7167° n, 73.0667° e) with a standard pipette while adults were collected by battery-operated aspirator. larvae were reared for mass population in the insectary of medical entomology and disease vector control department, health services academy, islamabad. the first instar larvae were fed with fat free milk powder while other instars larvae were fed with tetra-min fish feed powder at 28±2 ˚c and 75±5% humidity. adults were reared in screened cages by providing 10% sucrose solution while female mosquitoes were also fed on the blood of albino rats (imam et al. 2014). larvae of ae. (stg.) albopictus were identified using identification key of leopoldo (leopoldo 2004). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 278–285 h bilal et al.: bio-pesticides: new tool … 280 http://jad.tums.ac.ir published online: may 27, 2017 larvicidal bioassay the extracted oils were used in six different concentrations along control with three replicates for each treatment, each replicate containing 200ml of the oil solution in 250 ml pyrex glass beakers. a batch of fifteen 3rd instar larvae of the ae. (stg.) albopictus were exposed in each beaker containing oil solution (who 2005), while control was treated with ether only. mortality of larvae was counted after 24 hours. the experiment performed under lab conditions at 28±2 ˚c and 75±5% relative humidity. statistical analysis abbot’s formula (capinera 2008) was used for correction of mortality and the data so obtained was analyzed by probit analysis (who 2005) by using manitab-15 software for dose mortality regression line and % age mortality graph were prepared using microsoft office 2007. results the crude ether extracts of nutmeg (m. fragrans) seeds, musambi (c. sinensis) peel, ba buna (m. chamomilla) leaves, mint (m. spicata) leaves and ginger (z. officinale) rhizome had been evaluated as potential source of insecticides. results on the larvicidal activities of extracts were reported in the present study (table 1) confirms their potential for the control of ae. (stg.) albopictus larval population. all extracts showed moderate larvicidal effects however, the highest larval mortality was found in musambi peel with 400.81ppm lc50 value, followed by babuna (438.60ppm), ginger (502.55ppm) while mint and nutmeg had the highest lc50 value (596.94ppm and 710.30ppm) respectively after 24h of exposure. in terms of % mortality musambi had the high mortality (64.25%) followed by babuna and ginger (58.51 and 56.48%) respectively, while nutmeg and mint had the lowest % mortality (33.51 and 50.92%) respectively after 24h of exposure as shown in fig. 1. the percentage of mortality was directly proportional to concentration of the extract (table 1). after exposure to the test concentrations, the treated larvae exhibited restlessness, tremors, sluggishness and convulsions followed by paralysis at the bottom of the bowl. table 1. larvicidal activity different plant extracts at different concentrations against 3rd instar larvae of aedes (stg.) albopictus plants con. % mortali *lc50 (ppm) 95% fl (lfl–ufl) 2 p musambi 300 32 400.81 359.82–435.22 4.62 0.32 c. sinensis 400 46 500 54 600 61 700 70 800 82 nutmeg 300 10 710.30 654.35–793.80 4.60 0.33 m. fragrans 400 16 500 25 600 29 700 43 800 58 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 278–285 h bilal et al.: bio-pesticides: new tool … 281 http://jad.tums.ac.ir published online: may 27, 2017 babuna 300 27 438.60 394.18–477.52 0.18 0.99 m. chamomilla 400 41 500 51 600 61 700 66 800 70 mint 300 31 596.94 521.21–719.96 1.11 0.89 m. spicata 400 38 500 42 600 47 700 55 800 62 ginger 300 35 502.55 437.55–570.33 1.05 0.90 z. officinale 400 39 500 47 600 56 700 61 800 67 *lc50 ie, lethal concentration (%age) to kill 50% population of the subjected organism fig. 1. percentage mortality of different plant extracts against aedes (stg.) albopictus larvae after 24 hours of exposure discussion recently ae. (stg.) aegypti along with ae. (stg.) albopictus played havoc in different parts of pakistan. different control measures have been adopted with the major focus on chemical control. resultantly, occurrence of insecticide resistance in mosquitoes and other public health pests have been reported (khan et al. 2011, 2013). their residues in the environment and effects on humans and nontarget organism are major problems due to which investigators are now directing their attentions towards the development of plant table 1. continued… http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 278–285 h bilal et al.: bio-pesticides: new tool … 282 http://jad.tums.ac.ir published online: may 27, 2017 based insecticides (biopesticides). various compounds, including phenolics, terpenoids, and alkaloids, exist in plants (wink 1993, swin 1977, kim et al. 2001) and may jointly or independently contribute to the generation of larvicidal activities of mosquito (assabugi et al. 1997, hostettmann and potterat 1997). outcome of five different plant oils, which were, used against the 3rd instar larvae of ae. (stg.) albopictus are shown in table 1. results were satisfactory and showed efficacy. larval mortality increased with increase in dose of plant oil and at higher doses, it gave more than 80% mortality without any pupal or adult emergence. while in control, there was less than 5% mortality after 24h. citrus sinensis peel extract gave reasonably good results (400.81ppm) against the larvae of ae. (stg.) albopictus when compared with other tested plant oils, like findings of warikoo et al. (2012) they concluded that c. sinensis leaf extracts had 446.84ppm lc50 value against the larvae of aedes aegypti as citrus plants contain limonoids which work both as toxicant and feeding deterrents (akram et al. 2010) and also has insecticidal effects (bilal et al. 2012). thus, the larvicidal activity of citrus sinensis is due to limonoids, in addition to alkaloids, saponins, steroids, flavonoids and tannins. while on the other side, previous workers (michaelakis et al. 2009, din et al. 2011) reported lc50 values for some citrus peel and seed oils against the larvae of ae. albopictus and cx. pipiens different than the obtained values in the present investigation. in our findings, m. fragrans had least effectiveness and gave 50% mortality after 24h of exposure is not in agreement with the studies of senthilkumar et al. (2009). they tested m. fragrans, eucalyptus globulus, artemisia annua, cymbopogan citratus, justicia gendarussa, annona squamosa and centella asiatica and found that all gave 80– 100% mortality against larvae of anopheles stephensi. it is well documented that toxicity values of a substance may be largely varied due to several factors (busvine 1971) attributed to the test conditions (temperature, light, humidity, exposure period and solvent); tested species (age, stage and susceptibility) and tested plant material (season, location, extraction method and used part) and there may be different constituents in a botanical extract may interact with each other’s, leading to synergistic or antagonistic effects (mansour et al. 2000, 2003). conclusion out of the 5 plants extracts, c. sinensis has good larvicidal potential against larvae of ae. (stg.) albopictus in terms of lc50 and % age mortality. therefore, we suggest that c. sinensis extracts as well as other plant extracts should be investigated and compared with other plant extracts, which already been tested against mosquitoes. acknowledgements the funds provided by health services academy islamabad-pakistan to carry out this project are highly acknowledged. authors are especially thankful to soaib ali hassan and insectry staff (mr shaheen akhtar, mr sultan ahmed and mr nawaz shah) for the cooperation offered in the process of sampling and rearing of mosquito in insectary. authors have no conflict of interest. references akram w, khan haa, hafeez f, bilal h, yeon kk, lee jj (2010) potential of citrus seed extracts against dengue fever mosquito, aedes albopictus (skuse) (culicidae: diptera). pak j botany. 42(4): 3343–3348. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 278–285 h bilal et al.: bio-pesticides: new tool … 283 http://jad.tums.ac.ir published online: may 27, 2017 ali a, rehman h, nisar m, rafique s, ali s, hussain a, nausheen, idrees m, sabri s, zada h, hussain s (2013) seroepidemiology of dengue fever in khyber pakhtunkhawa, pakistan. int j infect dis. 17 (7): e518–523. anonymous (2011) weekly epidemiological report. 2(45): 1–6. assabgui r, lorenzetti f, terradot l, regnault-roger c, malo n, wiriyachitra p, sanchez-vindas pe, san-roman l, isman mb, durst t, arnason jt (1997) efficacy of botanicals from the meliaceae and piperaceae. hedin pa, hollingworth rm, masler ep, miyamoto j thompson dg (eds.): phytochemicals for pest control, acs symp. american chemical society. washington dc, usa. 658: 38–48 becker n, dušan p, marija z, clive b, minoo m, christine d, achim k (2010) mosquito and their control, 2nd edition. springer heidelberg dordrecht. london, new york. 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… 177 http://jad.tums.ac.ir published online: june 24, 2019 original article effects of diatomaceous earth on house dust mite sensitization in balb/c mice sung-yuan liu1; yu-hui chang2; hui-ru ji3; *cheng-di chiu3,4,5 1renai biotechnology limited, taichung, taiwan 2green polymer corporation, taichung, taiwan 3graduate institute of biomedical science, china medical university, taichung, taiwan 4school of medicine, china medical university, taichung, taiwan 5neurosurgical department, china medical university hospital, taichung, taiwan (received 2 nov 2017; accepted 28 apr 2019) abstract background: house dust mite (hdm) is associated with hypersensitivity such as asthma. patients with asthma benefit from improved living environment by reducing hdm exposure. in this study, we examined the effects of commercialized diatomaceous earth product, casaggia® used as construction materials, on hypersensitivity in hdm-sensitized mice. methods: male mice were sensitized with house dust mite extract for 7d and then housed in diatomaceous earth (de)-coated cages for 14 days at animal center of the taichung veterans general hospital, taichung, taiwan in 2014. levels of cytokine were determined using elisa. white blood cell counts were recorded over 21d. histological analysis was conducted to determine the remodeling of respiratory tract. results: exposure to de resulted in a suppression in elevated eosinophilia induced by hdm in mice. in addition, elevated serum ige responding to hdm sensitization were restored in the presence of de. de ameliorated the inflammation progression in airway. conclusion: environmental exposure to de is suggested to benefit patients with hypersensitivity through relieving inflammatory symptoms. in a sense of prevention, de represents a potential material against development of asthma. keywords: asthma; diatomaceous earth; house dust mite; allergy; environmental exposure introduction asthma known as respiratory allergic disease is characterized by excessive inflammatory response, structural remodeling, and obstruction in the airway. the prevalence of asthma has increased worldwide in past few decades, ranging from 1–18% (1, 2). development of asthma is attributed to repeated exposure to aeroallergens including pollens and house dust mite (hdm) (3, 4). hdm is known as the most common cause of sensitization in asthmatic patients that approximately 85% of asthmatics are hdm allergic (5). hdm are found in dust and products with woven material or stuffing which provide suitable habitat for hdm. in addition, moisture that plays a vital role in natural habitation of hdm has been shown to contribute to development of allergic asthma. despite the relevance of climatic factors including humidity to increase in asthma, the effects of manipulating climatic factor on hdm sensitization are still unclear. diatomaceous earth (de) consists of predominately fossilized remains of diatoms which is a type of hard-shelled algae. it is commonly used for in a broad spectrum of purposes based on its chemical and structural properties such as filtration, abrasive and construction. de has highly porous surface that makes it an ideal material for humidity control. use of de has been demonstrated to effectively reduce the growth of insects (6-8). in addition, as organic in origin, de has been used *corresponding author: dr cheng-di chiu, email: cdchiu4046@gmail.com mailto:cdchiu4046@gmail.com j arthropod-borne dis, june 2019, 13(2): 177–184 sy liu et al.: effects of diatomaceous … 178 http://jad.tums.ac.ir published online: june 24, 2019 to control internal parasites in livestock (9, 10). however, the effects of de on moisture control associated with human disorder such as asthma is sketchy. in the present study, we hypothesized that use of de ameliorates hdm induced allergic responses. we examined physiological effects of de at hdm-sensitized hypersensitivity in animal model. the changes of subtypes of blood cells and serum ige levels after hdm challenge and pathological changes of lung tissue were analyzed. materials and methods mice to dispel the possibility that gender may influence allergic hypersensitivity, we chose male balb/c mice aged 6 to 8-wk and weighing 20–25g as animal subjects to induce hdm sensitization. mice were purchased from the biolasco taiwan co., ltd and housed in environmentally controlled conditions (22 °c, a 12h light/dark cycle) with ad libitum access to standard laboratory chow and water. prior to experiments, mice were habituated to new environment for one week. all experiments were performed at animal center of the taichung veterans general hospital, taichung, taiwan in 2014. the study protocol was reviewed and approved by the research ethics committee of the taichung veterans general hospital and all animal experiments were performed according to the institutional and state guidelines on the care and use of animals for experimental purposes. overall, 66 rats were used in the present study and allocated into 3 groups: mice without treatment (sham group), mice housed in a normal cage undergo hdm sensitization process (hdm group) and mice housed in a decoated cage undergo hdm sensitization process (hdm+de group). among them, 48 mice were assigned for histological examination and 18 mice were used for blood sampling at 4 different time points. preparation of house dust mite crude extract hdme was prepared by dissolving 1mg lyophilized hdm (allergon ab, angelholm, sweden) in 1.0ml normal saline. resulting solution was mixed with rotation for 90min and then centrifuged at 13000rpm for 15min. the supernatant was used as a crude extract of hdm (hdme). the protein concentrations of hdm were measured using a commercially available bca protein assay kit (pierce, usa) and adjusted to 100μg protein/ml for further use. sensitization and aerosol challenge procedure the mouse sensitization and aerosol challenge algorithm were performed (11). in brief, for mouse sensitization, 100μg/ml hdme was absorbed to 1mg/ml aluminum hydroxide [al (oh)3] (merck). the animals were administered intraperitoneally with 100μl hdm (100 μg/ml) and al(oh)3 (1mg/ml) (n=6, the hdm group, and n=6, the de group). after 7d post-administration, mice in the control, hdm+de group and hdm group were housed separately in plastic cages of which plastic cages for de group were coated with commercialized de product (casaggia®, green polymer corporation, taichung, taiwan). animals were challenged by repeated exposures for 30min each day to an aerosol of hdm (100μg/ml) delivered at 0.5ml/min by a sumo v-16 nebulizer (japan) for 3d postseparation. white blood cell counts in mouse serum blood samples were obtained via the retro-orbital venous plexus on day 1, 5, 7, and 14 post-exposure to hdm aerosol. complete blood count was conducted using kx-21-hematology analyzer. elisa total serum ige was measured using a j arthropod-borne dis, june 2019, 13(2): 177–184 sy liu et al.: effects of diatomaceous … 179 http://jad.tums.ac.ir published online: june 24, 2019 mouse ige elisa kit (bd biosciences, usa) according to the instruction provided by the manufacturer. the total ige concentration in each serum (ng/ml) sample was estimated using a standard sample from the manufacturer’s kit. histological examination the mice (n=4 in each group) were sacrificed on day 1, 5, 7 and 14 post-exposure to hdm aerosol. infusion was performed via the trachea with 4% paraformaldehyde. lungs were excised and immersed in fresh fixative overnight. lung tissue sections with a thickness of 2μm were obtained and stained with hematoxylin and eosin (h an e). pulmonary lesions were categorized according to shackelford et al. into five severity grades as following, 1=minimal (<1%), 2=slight (1–25%, +), 3= moderate (26–50%, ++), 4=moderate/severe (51–75%, +++), 5=severe/high (76–100%, ++++). statistical analysis statistical comparisons were performed by analysis of variance (anova) and turkey’s hsd post hoc tests. we used nonparametric statistics (wilcoxon rank sum test) to test data differences between two groups if the data distribution is not corresponding gaussian distribution (kolmogorov-smirnov test is significant). all values are shown as mean±standard deviation (sd). p< 0.05 was regarded as significant. all statistical analyses were performed using spss software (ver. 18.0, ibm corp., armonk, ny, usa). results effects of de on differential wbc counts in hdm-sensitized mice to investigate whether de abrogates hdm-induced eosinophils, peripheral total and differential wbc counts in hdm-sensitized mice were conducted. the peripheral total wbcs at 4-time points in 3 groups of mice with or without exposure to de were comparable. a significant difference in total wbc counts was observed between hdm and de groups on day 5 post-exposure to hdm aerosol (p< 0.05) (fig. 1). in addition, there were no significant differences in cell counts and morphological changes among neutrophil, monocyte, and lymphocyte (fig. 2). our data showed that eosinophil count in hdm+de groups reached a peak on day 7 post-sensitization, whereas both hdm and hdm+de groups exhibited a peak on day 5. the differential wbc counts revealed that hdm-sensitized mice exhibited a significantly higher number of eosinophils in the peripheral blood compared with those of hdm+de groups at day1 and day 5 (p< 0.05) (fig. 3). effects of de on ige levels in hdm-sensitized mice we next examined the effect of de on hdm-induced increase in serum ige levels. exposure of mice to hdm led to markedly high serum ige levels compared with those of the controls at 4 designated time points (p< 0.001) (fig. 4). a significant difference in levels of serum ige between hdm+de and hdm groups on day 1, 5 and 7 (p< 0.001). interestingly, the levels of ige in hdm+de mice increased in a time-dependent manner. de reduced hdm-induced lung inflammation to investigate the mechanism by which de was associated with decreased eosinophilia, we histologically assessed lung tissues of mice sensitized by hdm. hdm sensitization led to a slight inflammatory cell infiltration, mainly neutrophils and lymphocytes, fairly eosinophils and slight mucilage in the bronchial goblet cells, along with minimal macrophage infiltration in the alveolar space (fig. 5). in hdm+de mice, hdm-induced inflammatory cell infiltration and pathological appearance were observed on day 7 post-sensij arthropod-borne dis, june 2019, 13(2): 177–184 sy liu et al.: effects of diatomaceous … 180 http://jad.tums.ac.ir published online: june 24, 2019 tization with minor degree of severity compared with that of hdm mice (table 1). unexpectedly, the inflammatory response in lung tissue was absent in hdm-treated mice on day 14 post sensitization. table 1. summary of pathological incidence of lung after mite-induced mice pathology/degree1 day/groups (number, mice) day5 day7 day14 c h+d h c h+d h c h+d h inflammatory cell, perivascular and peribroncheal, focal (minimal to slight) 2 2 2 infiltration, macrophage, alveolar, focal (minimal) 1 1 1 mucification, goblet cell, bronchial, foal (slight to moderate) 2 2 3 degree of lesions was graded from one to five depending on severity: 1= minimal (< 1%), 2= slight (1– 25%, +), 3= moderate (26–50%, ++) fig. 1. the effects of de on peripheral total wbc counts. blood samples were obtained from the hdm-sensitized mice on day 1, 5, 7 and 14 post-sensitization (n=6). data are presented as the mean ± sd. *p< 0.05 fig. 2. subtypes of wbcs found in hdm-induced mice (1000x). it denotes (a) eosinophil, 0–4%, responsive for allergy, asthma, parasite; (b) monocyte, 3–8%, (c) neutrophil, 10–40%, (d) lymphocyte, 55–90%. j arthropod-borne dis, june 2019, 13(2): 177–184 sy liu et al.: effects of diatomaceous … 181 http://jad.tums.ac.ir published online: june 24, 2019 fig. 3. the effects of de on peripheral eosinophil counts. blood samples were obtained from the hdm-sensitized mice on day 1, 5, 7 and 14 post-sensitization (n=6). data are presented as the mean ± sd. *p< 0.05 fig. 4. blood ige levels in mice after hdm sensitization. blood samples were obtained from the hdm-sensitized mice on day 1, 5, 7 and 14 post-sensitization. (ng/ml, n=6) data are presented as the mean ± sd. ***p< 0.001 fig. 5. pathological examination of inflammation in lung tissue among mice group of ns, hmd group and hmd+de (h and e stain, 400x) j arthropod-borne dis, june 2019, 13(2): 177–184 sy liu et al.: effects of diatomaceous … 182 http://jad.tums.ac.ir published online: june 24, 2019 discussion we demonstrated that environmental exposure to de leads to a reduction in degrees of hdm-induced hypersensitivity in vivo. reduced eosinophilia was associated with the presence of de. moreover, the hdm-induced hypersensitive responses were ameliorated in the mice exposed to de. asthma is a prevalent chronic respiratory disorder in children and adolescents populations. hdm allergens are considered as important indoor allergens for humans inducing allergic diseases. presence of hdm is suggested to be a risk factor for development of allergic asthma. hdm sensitization has been shown to contribute to the pathogenesis, progression, and severity of asthma. use of hdm as model allergen for sensitization in animal models has been shown to be more ideal and logical than the other models such as ovalbumin (12). hdm-sensitized animal models display many features of allergic asthma including respiratory inflammation, airway remodeling and increased eosinophilia (13). we employed a hdm sensitization to establish an allergic airway inflammation model that exhibited typical features of allergic inflammation. hdm-associated increases in systemic eosinophilia were restored in presence of de. systemic and local eosinophilic inflammation has been associated with severe asthma and poorer asthma control (14). eosinophilia in blood is a good marker for asthma (15-17) and been used as a measurement of effects of anti-hypersensitive modalities. airway eosinophilia is considered as a vital characteristic of early-onset allergic asthma, as well as the occurrence of late-onset non-allergic asthma (16). our finding showed that de reduced elevated systemic eosinophilia suggests that use of de has the potential to improve the symptoms of allergic asthma. unexpectedly, bronchial eosinophilia remained unaffected in the experimental setting. the finding is supported by the results of previous research, hdm-induced airway hypersensitiveness is not related to either eosinophil influx or allergen-specific serum ige. in addition, a retrospective study has reported the absence of airway eosinophilia in patients with allergic asthma (18). however, further studies are required to determine the differences in the phenomenon of eosinophil counts in hdm-sensitization modal. increasing evidence has highlighted the potential use of de for insect control (19-21). recent studies have demonstrated the insecticidal effect of de against different species of mites in animal (10, 22). the insecticidal effects of de have been postulated to be attributed to rapid and lethal dehydration of insect. we explored the use of casaggia®, a new de formulation, for improving the hypersensitive response induced by hdm. animals of hdm+ de group exhibited relatively lower eosinophil counts and levels of total ige in blood compared with those of mice sensitized with hdm, suggesting that asthmatics can benefit from avoidance of environmental risk factor using de. however, in this study, animals were sensitized with hdm extract and housed in de-coated cages without direct exposure to dust mites. moreover, increased eosinophilia in hdm-sensitized rats declined slightly over time. a possible explanation for the anti-hypersensitive effect in presence of de is that use of de leads to a moisture loss of surrounding environment, which inhibits the growth of microorganisms acting as challenge to hdmsensitized animals. nevertheless, further studies are necessary to elucidate the mechanism underlying the effect of de on hypersensitivity. long-term occupational exposure of workers in the crystalline silica was found to have an increased risk of silicosis (23). despite current commercial, de product contains mostly amorphous silica but no crystalline silica, there still are doubts for long term usage. further investigation for the effect of commercial de products to human lung is necessary. j arthropod-borne dis, june 2019, 13(2): 177–184 sy liu et al.: effects of diatomaceous … 183 http://jad.tums.ac.ir published online: june 24, 2019 conclusion exposure to de ameliorates elevated eosinophilia and total ige in animals sensitized with hdm. de represents a potential approach to prevent occurrence of hypersensitivity and to improve the symptoms of allergic asthma. acknowledgements the authors declare that there is no conflict of interests. references 1. yangzong y, shi z, nafstad p, haheim ll, luobu o, bjertness e (2012) the prevalence of childhood asthma in china: a systematic review. bmc public health. 12: 860. 2. greenlee rt, chyou ph, kieke a, naik yg, kirkhorn s (2008) prevalence of asthma in a general population cohort of farm 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andric gg, markovic mm, adamovic m, dakovic a, golic mp, kljajic pj (2012) insecticidal potential of natural zeolite and diatomaceous earth formulations against rice weevil (coleoptera: curculionidae) and red flour beetle (coleoptera: tenebrionidae). j econ entomol. 105(2): 670–678. 22. bennett dc, yee a, rhee yj, cheng km (2011) effect of diatomaceous earth on parasite load, egg production, and egg quality of free-range organic laying hens. poult sci. 90(7): 1416–1426. 23. park r, rice f, stayner l, smith r, gilbert s, checkoway h (2002) exposure to crystalline silica, silicosis, and lung disease other than cancer in diatomaceous earth industry workers: a quantitative risk assessment. occup environ med. 59(1): 36–43. https://www.ncbi.nlm.nih.gov/pubmed/?term=ikeda%20t%5bauthor%5d&cauthor=true&cauthor_uid=12622745 https://www.ncbi.nlm.nih.gov/pubmed/?term=hirota%20n%5bauthor%5d&cauthor=true&cauthor_uid=12622745 https://www.ncbi.nlm.nih.gov/pubmed/?term=nakagawa%20h%5bauthor%5d&cauthor=true&cauthor_uid=12622745 https://www.ncbi.nlm.nih.gov/pubmed/?term=ishii%20a%5bauthor%5d&cauthor=true&cauthor_uid=12622745 https://www.ncbi.nlm.nih.gov/pubmed/?term=louis%20r%5bauthor%5d&cauthor=true&cauthor_uid=24525441 https://www.ncbi.nlm.nih.gov/pubmed/?term=louis%20r%5bauthor%5d&cauthor=true&cauthor_uid=24525441 https://www.ncbi.nlm.nih.gov/pubmed/?term=maselli%20r%5bauthor%5d&cauthor=true&cauthor_uid=25878402 https://www.ncbi.nlm.nih.gov/pubmed/?term=tran%20tn%5bauthor%5d&cauthor=true&cauthor_uid=25439366 reações imunológicas de aedes aegypti linnaeus, 1762, de aedes albopictus skuse, 1894 e cx j arthropod-borne dis, march 2018, 12(1): 94–99 g aparecida de carvalho et al.: melanization of … 94 http://jad.tums.ac.ir published online: march 18, 2018 short communication melanization of dirofilaria immitis larvae in different culicid species *gílcia aparecida de carvalho 1, rafael antonio nascimento ramos 2, rafael trindade maia 3, carlos fernando salgueirosa de andrade 4, leucio câmara alves 2 1academic unit of garanhuns, federal rural university of pernambuco, garanhuns, brazil 2department of veterinary medicine, federal rural university of pernambuco, recife, brazil 3department of biological sciences, federal university of campina grande, sumé, brazil 4department of animal biology, biology institute, state university of campinas, campinas, brazil (received 13 oct 2015; accepted 3 feb 2018) abstract background: dirofilaria immitis is an important filarioid transmitted by culicids. the vector role of these arthropods may be influenced by biological events as melanization against d. immitis larvae. this study aimed to detect the occurrence of melanization in culex quinquefasciatus, aedes (stegomyia) aegypti and aedes (stegomyia) albopictus experimentally infected with d. immitis. methods: five populations (cx. quinquefasciatus recife (p1), ae. albopictus recife (p2), ae. aegypti recife (p3), ae. aegypti campinas (p4) and ae. aegypti rockefeller (p5)) were artificially fed with infected blood containing d. immitis microfilariae. ten mosquitoes per day from each population were dissected for 14 days. results: melanized larvae of d. immitis were observed in all population except in p3. the period in which melanized larvae were found varied from the third to the 14th day post-infection. difference in the number of these larvae was observed between p5 and p2–p3 (p< 0.01), and between p5 and p4 (p< 0.05). third-stage larvae of d. immitis were detected in all population 14 d post-infection, except in p3, which presented high mortality 24 h postinfection. the melanization observed in the populations herein studied did not indicate refractory since third-stage larvae were observed at the end of the experiment. most likely, this immune response aimed to control the number of larvae, in order to maintain the equilibrium between vector-parasite. conclusion: the melanization did not seem to be a limiting factor to the development of this filarioid in these local brazilian culicidae populations. keywords: mosquitoes, immune response, dirofilariasis, vector introduction dirofilaria immitis is an important nematode filarioid that causes a severe infection in dogs commonly known as heartworm disease. although, other animal species as wild carnivores, cats and humans may be affected, dogs present a high epidemiological relevance (1). this filarioid is transmitted by culicid vectors, being species belonging to the genus culex, aedes and anopheles the most important (2-5). the vector role of culicidae species may be influenced by biological events as immune responses against d. immitis microfilariae. indeed, different types of immune response against parasites and pathogens affecting mosquitoes have been studied in some species (e.g., aedes aegypti, ochlerotatus trivittatus, anopheles quadrimaculatus, and armigeres subalbatus) (6-10). for example, the melanization is an important immune event that occurs inside the mosquito vector. this phenomenon is characterized by an initial reaction involving the lysis of hemocytes close the surface of the parasite prior to the deposition of pigments (8, 11). the melanization may impair the flow of nutrients that are essential to the microfilariae survival and development. moreover, it may difficult the release of toxic oxygen *corresponding author: prof gílcia a. carvalho, e-mail: gilcia.acarvalho@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 94–99 g aparecida de carvalho et al.: melanization of … 95 http://jad.tums.ac.ir published online: march 18, 2018 metabolites causing the death of the parasite (12). the melanization of d. immitis larvae was reported in a. subalbatus, ae. aegypti and cx. quinquefasciatus (11, 13, 14). this immune response may reduce the vector ability of some populations of mosquitoes in transmit parasites, including d. immitis (15). on the other hand, species belonging to the genus aedes use this phenomenon to restrict the larval development, enhancing its potential as vector (16, 17). a melanization is an event that may vary depending on the population studied, and in brazil, this phenomenon has been poorly studied in the local culicidae population. therefore, the aim of this study was to assess the occurrence of melanization in different populations of culicids (cx. quinquefasciatus, ae. albopictus and ae. aegypti) experimentally infected with d. immitis microfilariae to evaluate the potential role of these species as vectors. in addition, biological implications of these findings have been discussed. materials and methods dirofilaria immitis microfilariae infected blood containing d. immitis microfilariae was obtained from a positive dog diagnosed at microscopic and molecular analyses. the animal was a three-yr-old male that lived in the metropolitan region of recife (7°45'0"s and 34°51'0"w), state of the pernambuco, brazil. mosquito populations and experimental infection five populations belonging to three different culicid species were used in this study: cx. quinquefasciatus recife (p1), ae. albopictus recife (p2), ae. aegypti recife (p3), ae. aegypti campinas (p4) and ae. aegypti rockefeller (p5). overall, 6000 female mosquitoes (1200 per each population, test group =900 and control group = 300) aged from three to seven days were used (18). the artificial blood meal was performed as previously described (4, 5). briefly, mosquitoes were fed for two hours with infected blood containing about 2000 microfilariae/ml of d. immitis. after blood meal, mosquitoes were maintained under controlled conditions of temperature (28±2 °c) and relative humidity (>70%). mosquito dissection and microscopic examination ten mosquitoes per day from each population test were dissected during 14 days. daily, dead mosquitoes were discarded and at the end of the experiment the remaining ones were dissected. briefly, specimens were fixed on slides containing a drop of 0.9% physiological saline solution, dissected with a sterile scalpel and immediately examined under a light microscope (olympus bx41 tf) at different magnifications. all stages of d. immitis larvae were morphologically identified (19) and the presence of melanized larvae was recorded. data analysis the percentage of infected mosquitoes, as well as number of melanized larvae, was calculated. the statistical analysis was performed through the partitioning qui-square test using the software bioestat 2.0 (20). results the overall results with the number of infected females and melanized larvae are shown in table 1. melanized larvae were detected in all population except in p3 and the highest number was detected in p5 followed by p1, p4, and p2, respectively. in general, the period in which these melanized larvae were found varied from the third to the 14th day post-infection. the developing stage in http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 94–99 g aparecida de carvalho et al.: melanization of … 96 http://jad.tums.ac.ir published online: march 18, 2018 which they were observed is reported in table 2. difference in the number of melanized larvae was observed between p5 and p2 (p< 0.01), and between p5 and p4 (p< 0.05). interestingly, third-stage larvae of d. immitis were detected in all population 14d post-infection, except in p3, which presented a high mortality (70.7%) 24 h post-infection. the p2 presented the first l3 nine days postinfection, whereas for p4 and p1 third-stage larvae were observed ten and 12d post-infection, respectively. finally, for p5 d. immitis l3 were retrieved only 14d post-infection, but melanized first-stage larvae were detected until the end of the experiment. the infected mosquitoes showed total or partial destruction of the cells of malpighian tubules (mt) in the parasitized areas. the membrane of the cells presented damaged, most likely due to the migration of the larvae from the tubules to the head and proboscides. during this migration, the reaction of melanization was not observed. during the whole study, the mortality of mosquitoes of control groups was approximately 3% for all groups herein analyzed. table 1. infected females with dirofilaria immitis microfilariae, and presence of melanized larvae p1 p2 p3 p4 p5 females with d. immitis microfilariae (%)* 13.47 (111/824) 15.63 (131/838) 17.55 (149/849) 11.57 (95/821) 16.97 (149/878) females with d. immitis melanized larvae ** (%) 10.81 a (12/111) 0.76 b (01/131) 0 b (0/149) 5.26 a, c (05/95) 19.46 a, d (29/149) (*number of females infected/number of females dissected, **number of females with melanized larvae/number of females infected. different letters indicate statistically significant difference) table 2. developing stage of the melanized larvae in different populations analyzed first-stage salsichoid and second-stage % (n/n) third-stage p1 100 (12/12) p2 100 (1/1) p4 80 (4/5) 20 (1/5) p5 68.9 (20/29) 31.1 (9/29) discussion in this study, the melanization of d. immitis larvae in different culicid populations and species was studied. the melanization that occurs in d. immitis larvae in some mosquitoes suggests that these specimens may be resistant to the parasitism by this filarioid, depending on the percentage of melanized larvae (16). indeed, in populations such as ae. aegypti rockefeller this phenomenon is more evident (21, 22). in general, the development of d. immitis larvae in culicids occurs in 14d, from microfilariae to infective-stage larva. however, depending on the mosquito population, several biological events (e.g., blood coagulation, microfilaria capture and melanization) may occur to control the number of larvae in development inside the malpighian tubules (516). these mechanisms are important to regulate the number of infected mosquitos, which http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 94–99 g aparecida de carvalho et al.: melanization of … 97 http://jad.tums.ac.ir published online: march 18, 2018 will influence in their survival and vectorial competence (23). in the present study, almost all populations (except p3) presented melanized larvae, but in all of them (except p3) third-stage larvae of d. immitis were detected at the end of the experiment. most likely, in this population microfilariae were destroyed at the first 24 h post-infection, therefore, no melanized larvae and infective-stage larvae were observed. this is an important finding, especially because all populations and species herein studied are susceptible and allows the development of d. immitis microfilariae; accordingly, these populations are considered potential vectors of d. immitis. the susceptibility of ae. aegypti species for d. immitis infection is a characteristic controlled by the f1 gene (24). females that present the patterns fifi or fifi are refractory to the infection. indeed, these genes may influence the physiology of malpighian tubules, and consequently the development of the parasite. most likely, this genetic factor has an important role in the phenomenon of melanization, affecting the vector competence of culicids (8, 25). in fact, genetic differences may be observed in individuals of the same species (eg: ae. aegypti) and different populations (24). these differences may be the cause of the findings of melanization herein reported for the same culicid species (ie, p4 and p5). this variation within a mosquito species contributes to enhancing the vector role of some populations from different geographical areas (26). the structural alterations herein observed in infected mosquitoes may play an important role in the development of d. immitis larvae. the mortality of infected mosquitoes probably may occur due to two reasons: i) the invasion of the mt cells by the microfilariae and ii) the escape of the infective larvae from the mt and their movement to the head and mouthparts (27). therefore, when the parasite load in the mt is higher, an excessive mortality (almost 100%) may occur (28, 29). in this study, the parasitism by d. immitis microfilariae had a great impact on females of p3, influencing the survival of specimens which dead (almost 70.7%) 24h post-infection. conclusion the melanization observed in the populations herein studied did not indicate refractory, since third-stage larvae were detected at the end of the experiment. most likely, this immune response herein observed aimed to control the number of larvae in development, which is important to maintain the equilibrium between vector-parasite enhancing the potential of these specimens as vectors. all population herein studied, except p3, may act as a potential vector of d. immitis in brazil. the authors declare that there is no conflict of interest. references 1. mccall jw, genchi c, kramer lh, guerrero j, venco l (2008) heartworm disease in animals and humans. adv parasitol. 66: 193–285. 2. ludlam kw, jachowski la, otto gf (1970) potential 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lourenço-deoliveira r (1998) susceptibility of aedes scapularis (rondani, 1848) to dirofilaria immitis (leidy, 1856) an emerging zoonosis. mem inst oswaldo cruz. 93(4): 435–437. 18. rutledge lc, ward ra, gould dj (1964) studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder. mosq news. 24: 407– 419. 19. taylor aer (1960) the development of dirofilaria immitis the mosquito aedes aegypti. j. helminthol. 34: 27–38. 20. ayres m, ayres jr m, ayres dl, santos al (2000) bioestat 2.0: aplicações estatísticas nas áreas das ciências biológicas e médicas. sociedade civil mamirauá, cnpq, brasília, br. 21. buxton ba, mullen gr (1981) comparative susceptibility of four strains of aedes aegypti (diptera: culicidae) to infection with dirofilaria immitis. j med entomol. 18(5): 434–440. 22. sauerman dm, nayar jk (1985) characterization of refractoriness in aedes aegypti (diptera: culicidae) to infection by dirofilaria immitis. j med entomol. http://jad.tums.ac.ir/ 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immitis (leidy) in anopheles quadrimaculatus say and on vector mortality resulting from this development. ann entomol soc am. 67: 325–331. 28. kershaw we, lavoipierre mmj, chalmers ta (1953) studies on the intake of microfilariae by their insect vectors, their survival, and their effect on the survival of their vectors. an trop med parasitol. 47: 207–224. 29. webber wa, hawking f (1955) experimental maintenance of dirofilaria repens and d. immitis in dogs. exp parasitol. 4: 143–164. http://jad.tums.ac.ir/ 8dr akbarzadeh rtl iranian j arthropod-borne dis, 2010, 4(1): 60–65 a raeisi et al.: residual effects of deltamthrin … 60 original article residual effects of deltamethrin wg 25% as a new formulation on different surfaces against anopheles stephensi, in southeastern iran a raeisi 1 , mr abai 2 , * k akbarzadeh 2 , m nateghpour 2 , m sartipi 3 , a hassanzehi 4 , n shahbakhsh 4 , l faraji 1 , f nikpour 1 , m mashayekhi 5 1 malaria control, ministry of health and medical education, tehran, iran, 2 department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran 3 iranshahr center of public health, iranshahr, iran 4 zahedan university of medical sciences, zahedan, iran 5 kerman university of medical sciences, kerman, iran (received 9 oct 2009; accepted 19 may 2010) abstract background: indoor residual spraying (irs) is functioned as national interventions against malaria in southeastern foci of iran and deltamethrin wp one of the insecticides have been used since past decade. in this study, the residual activity of the wettable granule (wg) was studied on different surfaces in hut scale trial against anopheles stephensi in iranshahr district, southeastern iran. methods: three dosages of 25, 40 and 50 mg a.i./m 2 of deltamethrin wg 25% formulation were applied on plaster, cement, mud, and wooden surfaces using hudson ® x-pert compression sprayer having 10 litters capacity. results: the residual effects of deltamethrin wg 25% on different surfaces was assessed based on reduction of mortality an. stepehnsi from 100% to about 70%. at 25, 40 and 50 mg a.i./m2 the wg formulation of deltamethrin had a bioefficacy for about 2, 3 and 4 months respectively. conclusion: there was an expectable fluctuation in mortality of an. stephensi at different sprayed surfaces as well as dosages. the proposed 50 mg/m2 wg is the longest activity for up to 4 months which needs to be applied for two spraying cycles per year at the climatically condition of southwestern iran. keywords: deltamethrin, wettable granule (wg), anopheles stephensi, malaria, iran introduction sistan and baluchistan province, in the southeast of iran is considered as the most prevalent area for malaria, which contains about 42–60% of the total malaria cases in iran (sadrizadeh 2001). various factors cause refractory malaria in sistan and baluchistan province. presence of five malaria vector including anopheles culicifacies, an. stephensi, an. fluviatilis, an. superpictus, an. dthali, and an. pulcherrimus (as a suspected vector) has a major role in this scenario (manouchehri et al. 1976). knowing the best and effective way is very important in malaria elimination as a new strategy in iran. indoor residual spraying (irs) has been considered as the main attempts for control of malaria in iran since 1950. resistance to ddt, dieldrin and malathion have been developed on five out of seven malaria vectors in iran (mofidi et al. 1958, who 1970, manouchehri et al. 1976). after ap*corresponding author: mr kamran akbarzadeh, email: akbarzadehk@yahoo.com iranian j arthropod-borne dis, 2010, 4(1): 60–65 a raeisi et al.: residual effects of deltamthrin … 61 pearance of the resistance in an. stephensi, main malaria vector in southern iran, the house spraying was continued with different adultricides e.g. propoxur from 1978 to 1993 for 13 successive years, actellic ® and propoxur from 1991 to 1992 and from 1993 to 2000 both lambdacyhalothrin (icon 10% wp) and propoxur have been used for malaria control in the south of iran (ladonni et al. 1995). at present, the irs operation in iran has been continued with deltamethrin wp 5% since 2003 but the residual activity of deltamethrin 25% wg has not been studied at field condition of sistan and baluchistan province, which is the endemic area for malaria as well as main area for application of the irs in the country. the main objective of this study was to study the residual activity of deltamethrin wg 25% as a new formulation, on different surfaces in human dwellings at malarious area, southeast of iran. materials and methods the field operation was carried out in malekabad village, at suburbs of iranshahr city from july to december 2006. the lab-bred anopheles stephensi (irs strain), sugar fed 2-3 days old females were used for bioassays. the adult were provided from insectary of iranshahr research station, 25 km away from trial area. temperature and relative humidity of the insectary were 28±2° c and 65±5% respectively. deltamethrin wg (wettable granule) with empirical formula c22h19br2no3 available under trade name k-othrine ® wg25, supplied by bayer environmental sciences, co. this insecticide were sprayed at 25, 40 and 50 mg/m 2 on different surfaces including plaster, wood, mud and cement using a hudson x-pert ® sprayer (10 liters capacity) with hss-8002 nozzle which equipped with a regulator adjusted pressure at range 25–45 psi . the contact bioassay tests were carried out according the method recommended by world health organization, using conical chamber at biweekly interval (who 1975). a lot of 10–12 adult females were gently released in any cone at 9 replicates (upper, middle and lower parts of walls) using aspirator with 30 min exposure on treated surfaces as well as 3 replicates of untreated surfaces as negative controls simultaneously. at the end of exposure time, the adults were transferred into clean cup and maintained at optimal condition and the mortality was recorded after 24 h. if mortality rate of control tests was between 5–20%, the mortality rates were corrected using abbott’s formula. the data (mortality rates) was transferred using arcsine formula. in order to compare the residual activities in each sprayed surfaces, one way anova, levene’s test, hsd test, or games-howell test were used. the criteria for residual effect of tested insecticide were based on mortality rate and if this rate was decreased to 70%, the bioassays stopped and the data analyzed. results comparisons of three applied dosages (25, 40 and 50 mg/m 2 ) showed an efficacy around 3, 3.5 and 5 months on plaster surfaces respectively (fig. 1). on mud surfaces, the residual activity of at 25, 40 and 50 mg/m 2 were estimated around 2.5, 3.5 and 4.5 months respectively (fig. 2). residual efficacy of experimental dosages of deltamethrin wg 25% at 25, 40 and 50 mg/m 2 on cement surfaces was around 1, 2 and 2 months respectively (fig. 3). it is cleared that the average mortality rates of all three dosages did not exceed to 100% anytime. iranian j arthropod-borne dis, 2010, 4(1): 60–65 a raeisi et al.: residual effects of deltamthrin … 62 a wide enhancing ranges of efficacy of deltamethrin wg 25% at applied dosages (25, 40 and 50 mg/m 2 ) was considered on wood surfaces around 2, 3.5 and 4.5 months respectively (fig. 4). 0 20 40 60 80 100 120 5 20 35 50 65 80 95 110 125 140 155 170 post-treatment days m o rt a li ty ( % ) 25 mg/m2 40 mg/m2 50 mg/m2 deltamethrin 25% wg ; 25 , 40 & 50 mg/m 2 residual spraying on plaster anova ( α=0.05) fig. 1. efficacy of various doses (25, 40 and 50 mg/m 2 ) of deltamethrin wg 25% on plaster surfaces, malek-abad village, iranshahr, southeastern iran, 2006 0 20 40 60 80 100 120 5 20 35 50 65 80 95 110 125 140 155 170 post-treatment days m o rt a li ty ( % ) 25 mg/m2 40 mg/m2 50 mg/m2 deltamethrin 25% wg ; 25 , 40 & 50 mg/m 2 residual spraying on mud anova ( α=0.05) fig. 2. efficacy of experimental dosages (25, 40 and 50 mg/m 2 ) of deltamethrin wg 25% on mud surfaces, malekabad village, iranshahr, southeastern iran, 2006 iranian j arthropod-borne dis, 2010, 4(1): 60–65 a raeisi et al.: residual effects of deltamthrin … 63 0 20 40 60 80 100 120 5 20 35 50 65 80 95 110 125 post-treatment days m o rt a li ty ( % ) 25 mg/m2 40 mg/m2 50 mg/m2 deltamethrin 25% wg ; 25 , 40 & 50 mg/m 2 residual spraying on cement anova ( α=0.05) fig. 3. efficacy of experimental doses (25, 40 and 50 mg/m 2 ) of deltamethrin wg 25% on cement surfaces, malekabad village, iranshahr, southeastern iran, 2006 0 20 40 60 80 100 120 5 20 35 50 65 80 95 110 125 140 155 170 post-treatment days m o rt a li ty ( % ) 25 mg/m2 40 mg/m2 50 mg/m2 deltamethrin 25% wg ; 25 , 40 & 50 mg/m 2 residual spraying on wood anova ( α=0.05) fig. 4. efficacy of experimental dosages (25, 40 and 50 mg/m2) of deltamethrin wg 25% on wood surfaces, malekabad village, iranshahr, southeastern iran, 2006 iranian j arthropod-borne dis, 2010, 4(1): 60–65 a raeisi et al.: residual effects of deltamthrin … 64 discussion determination of residual activity of insecticides is the essential information for the use of indoor spraying operation. the residual duration of pyrethroids recommended by who including alphacypermethrin, bifenthrin, cyfluthrin, deltamethrin, etofenprox, and lambdacyhalothrin wp, have estimated between two and six months (najera et al. 2001). study on residual efficacy of deltamethrin 2.25% wg at 25 mg/m 2 against an. culicifacies in india showed 100% mortality up to 12, 10, 9 and 12 weeks on mud, cement, brick and thatch surfaces respectively (ansari et al. 1997). a village scale trial of deltamethrin 2.25% wg at 25 mg/m 2 against both anophelinae and culicinae mosquitoes also indicated a residual life about 12 weeks both on mud and cement plaster surfaces in india (singh et al. 1989). in brazil, residual activity of sc formulation of deltamethrin at 25 mg/m 2 reported 3, 2 and 3 months on wood, plastered brick and brick surfaces respectively (santos et al. 2007). the extended field trial of deltamethrin 2.5% wp at 25 mg/m 2 confirmed the long residual effectiveness from 15 to 16 weeks on both mud and cement plastered surfaces in india (gill et al. 1997). the residual activity of wg formulation of deltamethrin at 25 mg/m 2 was effective for 6 weeks after treatment on aedes vectors in kuala lumpur, based on biweekly bioassay (rozilawati et al. 2005). based on the present study, 50 days old deposits of insecticide on plaster surfaces caused similar mortality with three dosages (25, 40 and 50 mg/m 2 ) of deltamethrin wg 25% (p<0.05). significantly enhance mortality with increasing in dosages has seen on mud surfaces after 50 days. surprisingly on mud surfaces there was no significant difference between three applied dosages until day 110. despite of early depredating of the insecticide on very absorbent cement surfaces, there was no significantly difference between three applied dosages on cement surface. on wood surfaces, the residual effects of all three dosages did not show a significant difference with each other until day 50 as the same for 25 and 40 mg/m 2 until day 80. at greater age after 80 (days 90110) the variation between dosages 40 and 50 mg/m 2 was not significant but with dose 25 mg/m 2 the difference was significant (p<0.05). the persistency of insecticides, as revealed by mortality, depended on the type of surface, the dosage, and the age of spray deposits (giga et al. 1991). in comparison with the results presented by other authors, our study revealed a clearly lower estimation of residual effect of deltamethrin wg 25% on various surfaces. it seems that the irritancy effect of deltamethrin on tested mosquito, an. stephensi irs strain may responsible for this difference. in mosquitoes, it is commonly observed that casual contact with a surface treated with deltamethrin (or other pyrethroids) can produce an irritant effect, causing the insect to fly away-an effect known as excito-repellency (dartigues 1987). study of efficiency of pyrethroids at laboratory condition also showed similar effect related to type of pyrethroids (alipour et al. 2006). also other field bioassays of deltamethrin wp %5 which conducted by authors revealed a clearly excito-repellency effect. in the present study, the excitorepellency phenomenon were not equally occurred in applied dosages. the excito-repellency effect of deltamethrin wp can be defeated by using the wg formulation in sistans and baluchestan province. in conclusion, field hot scale evaluation of deltamethrin wg can be recommended for finding the effects of the insecticide on population dynamic of vectors, effects of other materials and human behaviors on residual effect of the insecticide. iranian j arthropod-borne dis, 2010, 4(1): 60–65 a raeisi et al.: residual effects of deltamthrin … 65 acknowledgements the authors would like to thank the malaria control office, ministry of health and medical education for financial support. the authors also like to thank health center of sistan and baluchestan province for providing the facilities to carry out the field trials. the authors would like to acknowledge with gratitude the assistance of staff of iranshahr medical research station for their kind cooperation with this research. the authors declare that they have no conflicts of interest. references alipour h, ladonni h, abai mr, moemenbellah-fard md, fakoorziba mr (2006) laboratory efficacy tests of pyrethroid–treated bed nets on the malaria vector mosquito, anopheles stephensi, in a baited excito-repellency chamber. pakistan j biol sciences. 9(10): 1877–1883. ansari ma, mittal pk, razdan rk, batra cp (1997) residual efficacy of deltamethrin 2.5 wp (k-othrine) sprayed on different types of surfaces against malaria vector anopheles culicifacies. southeast asian j trop med public health. 28: 606–609. dartigues v (1987) use of deltamethrin in the control of malaria. roussel uclaf technical bulletin, division agrovest, paris. giga dp, jane canhao sr (1991) relative toxicity and persistence of pyrethroid deposits on different surfaces for the control of prostephanus truncates and sitophilus zeamais. j stored prod res. 27(3): 153–160. gill ks, rahman sj, panda r, kumar k, katyal r (1997) extended field trial of deltamethrin wdp for control of malaria at jagdalpur, madhya pradesh, india. indian j malariol. 34(4): 173–187. ladonni h, motabar m (1995) residual effect of etofenprox (trebon 20%wp) as a new insecticide on different surfaces in south of iran. iranian j publ health. 24(3–4): 32–35. manouchehr av, janbakhsh b, rouhani f (1976) studies on resistance of anopheles stephensi to malathion in bandarabbas iran. mosq news 36: 320–327. mofidi ch, samimi b, eshghi n, ghiasedin m (1958) further study of anophelines susceptibility to insecticides in iran. results of busvine and nash method. instit parasitol and malariol. tehran, iran publ. 585: 1–7. najera ja, zaim m (2001) malaria vector control: insecticides for indoor residual spraying. who/cds/whopes/2001.3. rozilawati h, lee hl, mohd masri s, mohd noor i, rosman s (2005) filed bioefficacy of deltamethrin residual spraying against dengue vectors. trop biomed. 22(2): 143–148. sadrizadeh b. malaria in the world, in the eastern mediterranean region and in iran: review article. who/emro report; 2001. p. 1–13. santos rlc, fayal as, aguiar aef, vieira dbr, povoa mm (2007) evaluation of the residual effect of pyrethroids on anopheles in brazilian amazon. rev saude publica. 41(2):1–7. singh k, rahman sj, joshi gc (1989) village scale trial of deltamethrin against mosquitoes. j commun dis. 21(4): 339– 353. world health organization (1970) insecticide resistance and vector control. 17 th report of the who expert committee on insecticides. technical report series 443: 47–56. world health organization (1975) manual on practical entomology in malaria. part 2. methods and techniques. geneva, who publication no 13. j arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 504 http://jad.tums.ac.ir published online: december 30, 2017 original article rearing and biology of phlebotomus sergenti, the main vector of anthroponotic cutaneous leishmaniasis in iran arshad veysi 1, mohamad reza yaghoobi-ershadi 1, yavar rassi 1, nasibeh hosseinivasoukolaei 2, mahmood jeddi-tehrani 3, aref rezaee-node 4, fatemeh gholampour 1, zahra saeidi 1, mahboubeh fatemi 1, mohamad hossein arandian 5, *ali khamesipour 6, *amir ahmad akhavan 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of medical entomology and vector control, health sciences research center, faculty of health, mazandaran university of medical sciences, sari, iran 3monoclonal antibody research center, avicenna research institute, acecr, tehran, iran 4international campus, tehran university of medical sciences, tehran, iran 5esfahan health research station, national institute of health research, tehran university of medical sciences, isfahan, iran 6center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran (received 2 july 2017; accepted 2 oct 2017) abstract background: establishment of sand flies laboratory colonies is essential to understand various biological aspects of phlebotominae sand flies. the aims of the current study were to establish the colony of phlebotomus sergenti parrot (1917), the main vector of anthroponotic cutaneous leishmaniasis in old world, and to study biological parameters of this species. methods: the sand flies were reared at 26–28 °c temperature, 14:10 (light: dark) photoperiod and 70–80% relative humidity. larval diet was a composted mixture of rabbit faces and rabbit pellets which is prepared through a special process. first to fifth generations of p. sergenti were used to define biological parameters. results: results showed that, p. sergenti blood feeding percentage were 42% on chicken, 21% on balb/c and 37% on golden hamster. average time of blood digestion, egg incubation, 1st instar larva, pupa and adult emerging was recorded at 3.4, 8.7, 15, 33.3 and 41.2 days after blood feeding, respectively. mean number of laid eggs was 55.1 and retained eggs were 35per a female. fecundity and production rate were 61.6%, and 42.2% respectively. average longevity recorded at 15.2 days for females and 14.8 days for males. conclusion: colony of p. sergenti has been established for the first time in iran. average interval time from egg to adult of this species was 32.5 days. chicken and golden hamster were recommended as a blood source for colony initiation and routine blood feeding, respectively. keywords: phlebotomus sergenti, rearing, biology, anthroponotic cutaneous leishmaniasis, iran introduction phlebotomine sand flies (diptera: psychodidae) are tiny blood-feeder dipterans which prefer a wide range of hosts for blood feeding, and are potential vectors to transmit a variety of pathogens including bacteria (e.g. bartonella bacilliformis), viruses (e.g. phlebovirus) and protozoa (e.g. leishmania spp) among different host species (1, 2). leishmaniasis is among neglected diseases and is a major health problem in some endemic regions, according to who estimation about a tenth of the world's population are at risk, and more than 2 million peo*corresponding authors: dr amir ahmad akhavan, email: aaakhavan@tums.ac.ir, prof ali khamesipour, email: ali.khamesipour@gmail.com https://www.ncbi.nlm.nih.gov/pubmed/?term=hosseini-vasoukolaei%20n%5bauthor%5d&cauthor=true&cauthor_uid=27047970 https://www.ncbi.nlm.nih.gov/pubmed/?term=hosseini-vasoukolaei%20n%5bauthor%5d&cauthor=true&cauthor_uid=27047970 https://www.ncbi.nlm.nih.gov/pubmed/?term=hosseini-vasoukolaei%20n%5bauthor%5d&cauthor=true&cauthor_uid=27047970 https://www.ncbi.nlm.nih.gov/pubmed/?term=jeddi-tehrani%20m%5bauthor%5d&cauthor=true&cauthor_uid=27047970 https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahukewiur4tfikhtahvl6xokhzugdqiqfgglmaa&url=https%3a%2f%2fwww.researchgate.net%2fprofile%2ffatemeh_nikpour&usg=afqjcneyic_bnjikyp870yh2luacdsgwlw&sig2=nld3ds1mf43vkhsvwoboha&bvm=bv.152180690,d.d2s https://www.ncbi.nlm.nih.gov/pubmed/?term=khamesipour%20a%5bauthor%5d&cauthor=true&cauthor_uid=27047970 https://www.ncbi.nlm.nih.gov/pubmed/?term=akhavan%20aa%5bauthor%5d&cauthor=true&cauthor_uid=27047970 https://www.ncbi.nlm.nih.gov/pubmed/?term=akhavan%20aa%5bauthor%5d&cauthor=true&cauthor_uid=27047970 mailto:aaakhavan@tums.ac.ir https://www.ncbi.nlm.nih.gov/pubmed/?term=khamesipour%20a%5bauthor%5d&cauthor=true&cauthor_uid=27047970 j arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 505 http://jad.tums.ac.ir published online: december 30, 2017 ple are infected (3). leishmania tropica is one of the causative agents of anthroponotic cutaneous leishmaniasis (acl) and is endemic in many parts of middle east including iran (4-6). recently some studies showed that l. tropica cl appeared as zoonotic and was detected in sylvatic cycle (7, 8). many active foci of acl are known in iran including tehran, kerman and bam in the southeast, shiraz in the south, mashhad, neishabur and sabzevar in the north-east, kashan, yazd and parts of the esfahan city, in the central parts (9, 10). phlebotomus sergenti is distributed in southern mediterranean (morocco, algeria and tunisia), northern mediterranean (italy, portugal, spain, southern france, cyprus, turkey) and the eastern mediterranean such as saudi arabia, afghanistan, pakistan and iran (1). three morphotypes of p. sergenti are identified as a, b and c, with some intermediate forms in iran (5). to study sand flies in detail and different aspects, including leishmania interactions, it is essential to establish laboratory colonies of sand flies. till now different techniques have been used for colonization of sand fly colonies in laboratory conditions (11-13). among some 900 known sand fly species (14), so far only a few number have been colonized in the insectary furthermore, only a few have been reared continuously for several generations to experimental studies (15). studies showed that, sand fly species and even laboratory colonies from different geographical origin are different in biological and physiological characteristic, such as saliva composition, longevity, fecundity and so on (16, 17). various species of sand flies are categorized as restricted or permissive vectors to leishmania parasites transmission, e.g. phlebotomus papatasi and p. sergenti, are termed as restricted which allow a single leishmania species to be developed within their gut and transmitted, while the others seem to be permissive and support multiple leishmania species (18). natural promastigote infections have been reported for p. sergenti in two acl foci of iran (19, 20). furthermore, leishmania tropica infection of this species using pcr was reported in shiraz city, south of iran (21). moreover, transmitting the l. tropica by p. sergenti has been confirmed in morocco (22), algeria (23) and tunisia (12) as well. establishing of p. sergenti in the insectary is essential for understanding its physiology, its interaction with l. tropica and human and epidemiology of acl in the country. a first attempt to rear sand flies was done during 1963–1964 in iran but it was not successful and failed after three generations (24). for the first time a successful colonization of p. papatasi was achieved by iranian researchers in 2007 (25). the aims of current study were to establish of p. sergenti for the first time in the country and study on life-cycle data including productivity and fecundity of the colony and the developmental time of the first 5 generations. determining the appropriate animal to blood feeding and longevity of the adults in insectary conditions were evaluated as well. materials and methods sand fly collection sand flies were collected using cdc miniature light trap and aspirator from indoors (bedrooms, toilets and bathrooms) and outdoors of dehbakri, a rural district 50km far from the city of bam (29°03’14.2”n, 57°54’31.6”e), bam county, kerman, iran in september through august 2014. the collected sand flies were transferred into a cloth cage holding in a stainless steel framework (35x35x35cm) and then were transferred in an appropriate condition to the sand fly insectary of school of public health, tehran university of medical sciences, tehran, iran. colony initiation the gravid, semi-gravid and engorged female sand flies were transferred into individual pots made from polyester (5.5cm height, 4cm diameter) which were plastered inside with a j arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 506 http://jad.tums.ac.ir published online: december 30, 2017 layer of paris poured and left till egg lying. the unfed sand flies were transferred into a suspended nylon cloth cage and offered them a blood meal. female sand flies were fed on a variety of animals including chicken, golden hamster or mouse. to improve the blood engorgement of females, males were also transferred into the cages and were covered with a dark cloth during blood feeding. after blood feeding, engorged females left undisturbed for 12–24h. before transferring the blood fed sand flies, the plaster in the bottom of the pots were moistened with distilled water to encourage the sand flies to lay eggs on its moisten surface. to provide sufficient energy during egg incubation, the solutions of 20% sucrose and 50% honey syrup were offered for transferred sand flies till egg laying (26). pots were kept in plastic boxes with a layer of moist fine soil in the bottom. high humidity (70–80%) in the boxes were prepared by moistened the soil in the bottom of the boxes with distilled water. the optimum temperature was adjusted to 26–28 °c. pots were checked for oviposition daily and the female sand flies which laid eggs were removed to prevent development of fungal contamination. after oviposition, each female was mounted in puri’s medium (27) and identified after 48–72h using the related morphological keys (28, 29). after identification, only p. sergenti was included and the other species were excluded from the study. colony rearing sand fly colony was maintained in an isolated room with controlled temperature (26–28 °c) and 14:10 (light: dark) photoperiod based on modified method of modi and tesh (1983) (13). high humidity (70–80%) was provided by wrapping cages in plastic bags with hanging wet cloth inside. for larval rearing we used pots made from polyester (4.5cm height, 8.5cm diameter) with a big hole in the bottom. the pots bottom was filled with a 1cm thick layer of white plaster of paris. wet plaster provided humidity and a resting surface inside the pots during incubation period. the pots were covered by fine mesh gauze and engorged female sand flies along with males were transferred through a small hole in the gauze and the hole was then closed with a cotton wool pad. larval diet was a composted mixture of rabbit faces and rabbit pellets which mixed in equal amount in distilled water and left to fermentation in the room temperature. the prepared mixture was mixed daily to improve fermentation process. after completing the fermentation process resulting mixture was spread on special trays to dry. the dried mixture was ground and used to feed larval stages. the fermentation process normally lasts about 6 months, but in an innovative work, by adding yeast, fermentation process shortened to a few weeks (30). pots contained larvae were put in plastic boxes which the bottom filled with fine sand moisten with distilled water. emerged sand flies were released from pots into suspended net cages supported with a steel frame, wrapped in nylon bags. to adults feeding, soaked piece of cotton wool in 20% sugar solution and 50% honey syrup prepared for adult inside the cages. adults were offered a blood meal twice a week. blood fed females were kept undisturbed in net cages for 24h and then the engorged sand flies were transferred into the pots for oviposition. almost one week after transferring, most females laid eggs and died. after 50% egg hatching, 2–3mm soil collected from gerbil colonies and some larvae food add into the pots. interval shaking the soil layer prevents fungal growth on the bottom of the pots. determining the appropriate host for blood feeding to identify which animal was more appropriate as a blood source for p. sergenti, there conventional blood source in insectary which were available, easy to handle and housing, including chicken, golden hamster and balb/c mice were selected. five to seven days old sand flies were chosen to conduct the test. the exj arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 507 http://jad.tums.ac.ir published online: december 30, 2017 periment was done in 6 replication and 12 to 30 sand flies in the same generations (f4 to f6), were included in each one. homogenous number of sand flies was transferred into cloth cage holding in a steel framework. to improve the engorgement rate, males were transferred to cages as well. before exposing to sand flies, animals were anesthetized using ketamin hydrichloride (60mg per kg body weight) and xylazine (15mg per kg body weight) for 1h. since sand flies prefer blood feeding in dark, cages were covered by dark tissue. after 1h the numbers of blood fed and unfed sand flies in each cage counted and compared. animal experimental protocol was approved by the ethics committee of tehran university of medical sciences, tehran, iran (protocol number: ir.tums.vcr.rec.1395.253). life parameters assessment generations of f1 to f5 were used to study biological parameters of p. sergenti. for each generation 40 to 50 engorged p. seregenti females were included to assessing life parameters. new emerged sand flies were transferred into cloth cage holding in a steel framework with 20% sugar solution and 50% honey syrup. after 4 to 7 days sand flies were offered a blood meal on golden hamster. for all generations the source of blood feeding was the same. adult sand flies were left hungry 12h before blood feeding. engorged sand flies were kept undisturbed for 24h and then were transferred individually into a plastered line pot. all the individual pots were checked daily and information including time of blood digestion and mortality were recorded. the female sand flies were followed up until egg lying and were dissected after natural death. the number of laid and retained eggs was counted, recorded and fecundity was calculated for each individual sand fly. fecundity defined as follow: fecundity= natural death time for females and males was recorded. main procedures during blood feeding to the adult stage and the production rate were assessed as well. the minimum developmental times (ranges) were recorded by observation of the first appearance of each stage in the rearing pots. the productivity was defined as the percentage of eggs from each generation which reached to the adult stage (31). to achieve this purpose, blood fed sand flies left to lay eggs individually. laid eggs were counted and were followed up to hatch. the first egg hatching was recorded, and at the following the major interval times including: l4pupa and pupa-adult were recorded as well. emerged adults sand flies including males and females counted and recorded. statistical analysis statistical analysis was performed using spss16. one-way anova, x2 and duncan (p= 0.05) tests were employed to compare different parameters in five generations. results the reared species identified as p. sergenti. blood feeding percentage of p. sergenti on chicken, balb/c and golden hamster were evaluated. the results indicated that p. sergenti has higher blood feeding percentage on chicken compared to balb/c and golden hamster in the same condition. of 300 sand flies (6 replications), 170 took a blood meal (56.6%) which 72 (42%), 36 (21%) and 62 (37%) individuals took blood on chicken, balb/c and golden hamster, respectively. statistical analysis (x2 test) showed there is no significant difference (p= 0.388) in blood feeding percentage between chicken and golden hamster, on the other hand the blood feeding percentage between chicken or golden hamster and balb/c was statistically significant (p= 0.001). the average optimum time for blood feeding was 5.6 (range: 4.5–6.8) days after emerging. the average time for blood digestion was 3.4 days and the maximum and minimum time j arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 508 http://jad.tums.ac.ir published online: december 30, 2017 for egg incubation (the time which was needed to mature eggs inside uterus) were 10.9 days in f1 and 7.1 days in f5 after blood feeding. the time of the egg hatching (1st instar larva) varied from 14.3 days in f2 and f5 to 17.6 days in f1, however, the mean time was 15 days after blood feeding and the total developmental time for larval stages recorded at 18.3 days (range: 16.1–20.5). the time of pupa formation varied, and the larvae pupated at the maximum and minimum time of 36 in f1 and 30.6 in f3 and at average 33.3 days after blood feeding. diapause was occurred in some portion of the l4 larvae which lasted two month to over one year, and the recorded times was based on the minimum time for pupa appearing. the total developmental time for p. segenti adult was 41.2 days in average (range: 38.9–44.1) after blood feeding (table 1). the average number of laid eggs from f1 to f5 generations showed an ascending trend and the average number of laid eggs was 55.1 per female. it is interesting to point that, the maximum and minimum egg numbers ever seen during this study were 120 in f2 and one egg in f1 and f4 per female, respectively. the maximum and the minimum number of retained eggs were 49.7 in f1 and 21.5 in f4. as table 2 shows, fecundity has ascending trend from f1 to f5 generations and the average calculated fecundity for all generations was 61.6% (range: 41.1–76.1%). the maximum and minimum longevity of males were 18.3 and 13.1 days in f1 and f3, respectively, in insectary condition, while the average longevity of females was longer (15.2 days for females vs. 14.8 days for males). as table 2 shows longevity has an approximate descending trend either for females or males from f1 to f5 generations. the average number of emerged males (15.1) was more than females (11.3) per egg batches. to assess the efficacy of the rearing procedure, production (yield) of each generation was calculated. the maximum and minimum production were obtained from f3 (55.8%) and f4 (23.9%) and the average production was 42.2%. except for the time of blood digestion (p> 0.05), the statistical analysis (one-way anova) showed significant differences (p< 0.001) in other recorded or calculated parameters among five generations. in fig. 1 two important ecologic parameters of p. sergenti including fecundity and production rate are compared. surprisingly, as chart shows, during f1 to f5 as fecundity increased, production decreased correspondingly. during this study diapause phenomenon of p. sergenti was checked as well. observation showed that, diapause occurred in l4 and lasts two months to over one year. furthermore, the results indicated that, this phenomenon mostly occurred in f2 and f5 generations. table 1. minimum developmental time of phlebotomus sergenti (days after blood feeding) over five generation generations optimum blood feeding time blood digestion egg incubation 1st instar larva pupa adult f1 5.7±1.4 3.2±0.4 10.9±4.3 17.6±2.1 36±5.6 44.1±6.8 f2 5±1.1 3.5±0.5 8.2±2.8 14.3±1.3 32.2±5.3 40.8±5.2 f3 6.8±1 3.9±0.4 8.5±2.8 14.5±1 30.6±3 38.9±3.9 f4 4.5±0.9 3±0.3 9.2±3.7 14.7±1.2 35.2±6.5 41.2±6.5 f5 6.8±1.1 3.4±0.4 7.1±2.1 14.3±2.6 32.9±3.2 41.3±3.6 total average 5.6 ±1.2 3.4±0.5 8.7±3.6 15±1.6 33.3±4.2 41.2±4.6 j arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 509 http://jad.tums.ac.ir published online: december 30, 2017 table 2. some biological parameters of phlebotomus sergenti over five generations generations no. of egg laid no. of retained egg fecundity female longevity male longevity average no of female average no of male production rate f1 48.2±3.8 49.7±3.6 50.2% 17.7±4.7 18.3±4.7 10.8±7.7 14.2±2.1 46.7% f2 48±3 25.8±2.7 65.3% 14±3.7 13.8±3.7 12.8±7.6 16.6±1.1 46.5% f3 33.6±2.2 48±2.7 41.1% 14.8±3.2 13.1±3.7 13.9±6.6 18.8±9.2 55.8% f4 71±2.8 21.5±2.5 75.4% 15.9±3.8 15.2±4.3 5.5±3 9.6±2.8 23.9% f5 74.8±1.9 30±1.7 76.1% 13.7±2 13.8±2.3 13.6±7.1 16.4±7.6 38.4% total average 55.1±3.2 35±2.9 61.6% 15.2±4 14.8±4.3 11.3±7.8 15.1±9.8 42.2% fig 1. comparison of the fecundity and production rates of phlebotomus sergenti over five generations discussion establishment of sand fly laboratory colonies and their biological parameters give a detailed insight into their life cycle and provides an opportunity to study on parasite and invertebrate host interaction, susceptibility to insecticides and, etc, this information pave the way to control sand fly borne diseases. in the current study, p. sergenti was colonized for the first time in the country, and the biological parameters were investigated as well. the results of the blood feeding percentage test showed that, chicken is more preferred by p. sergenti, then golden hamster and then mouse. although feeding percentage on chicken was more than golden hamster and balb/c but due to difficulties in handling and anesthetizing of chicken, golden hamster was used to routine blood feeding. the reason might be that chicken in comparison with several avian and mammalian species showed the lowest defensive behavior against blood feeding (32). the results of a study conducted in the field suggested that the local population of p. sergenti is highly ornithophilic (33). one study showed that chickens and human are relatively unattractive hosts for blood feeding of lu. longij arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 510 http://jad.tums.ac.ir published online: december 30, 2017 palpis, however, cows and pigs were preferred (34). conversely, chickens are more attractive hosts for lutzomyia longipalpis in locations that domestic animals are absent or in low numbers (35). optimum time for blood feeding recorded in average of 5.6 days which is affected mainly by species, age, temperature, humidity and host availability (36). in this study average time for blood digestion was 3.4 days after blood feeding on golden hamster in 26–28 °c temperature, this time were recorded for lutzomyia evansi as 3.5 (range: 2–5) days (37). required time for blood digestion varied and affected by temperature (36) and blood source (38). so that, time of blood digestion was the only parameter which was not statistically different among the 5 generations. the average time for egg maturation recorded to be 8.7 (range: 7.1– 10.9) days, the same time in a study completed on p. sergenti originated from turkey showed to be 8.1 days (range: 7–9). the maximum and minimum time of egg maturation for p. papatasi was recorded at 12.4 in f1 and 5.6 days in f5 (39), and in another study the maximum and minimum time for phlebotomus perniciosus was shown to be 10 in f4 and 5.5 days in f1 (31). it seems that, from f1 to f5 p. sergenti needed less time to mature its eggs (f1: 10.9, f5: 7.1 days), and similar phenomenon was seen in p. papatasi as well (39). the reason might be due to adaptation process in insectary condition. this time was reported to be 9 days for phlebotomus martini, 7 days for sergentomyia schwetzi, and sergentomyia africana (40). the average time for 1st instar larva appearing (egg hatching) was 15 days after blood feeding and the same time for p. sergenti originated from turkey recorded at 14.2 (range: 12–18) days after blood feeding (15). for p. perniciosus this time was recorded at minimum 5.6 (f1) and maximum 7 days (f10) after pre-oviposition period (31). in the current study total developmental time of larval stages was 18.3 days (range: 16.1– 20.5) which was reported to be 18 to 22 days for p. perniciosus (31). the formation of pupa varied, as some portion of the l4 larvae might go to diapauses (2 month to over one year), the recorded times were based on the minimum time for pupa emerging. as it is shown, lengthy time of diapause phenomenon makes the colony maintenance difficult in some species including p. sergenti, p. ariasi, p. perfiliewi and p. simici (15). the average time for pupa emerging was 33.3 (range: 30.6–36) days after blood feeding and the development time of pupa was recorded at 7.8 days (range: 6– 8.6). this time for p. papatasi was minimum 7.4 to maximum 9.3 days in the first 5 generation (39). in the current study an average time for adult emerging recorded at 41.2 (range: 38.9–44.1) days after blood feeding. the same time was 40.3 (range: 38–45) days for p. sergenti originated from turkey (15), for p. papatasi was 35.4 days (range: 31.5– 42.7) (39) and for lu. evansi and p. perniciosus were 41.8 and 42 days, respectively (31, 37, 40). the mean developmental time from egg to adult recorded at 47.2 to 52.6 days for p. papatasi in iran (25). as the results showed, the number of laid eggs followed an increasing trend which it could be a sign of adaptation through generation. the same phenomenon was shown in another study which the average minimum and maximum numbers of laid eggs obtained as 34.3 in wild-caught and 55 in f10 for p. perniciosus (31). the average number of laid eggs was 55.1per female in this study which was much more than the number obtained from lutzomyia sanguinarms, lu. gomezi, lu. trapidoi, lu. ylephilctor, lu. panamensis and lu. pessoana that reported as 27, 29, 21, 27, 28 and 20 respectively (11). the maximum number of eggs ever seen during this study was 120 per female in f2, this number was 112 for p. papatasi and 85 for p. perniciosus (31, 39). as the results showed, longevity was being decreased over 5 generations either in females or males which maybe the result of inbreeding that affected biological fitness of https://en.wikipedia.org/wiki/fitness_(biology) j arthropod-borne dis, december 2017, 11(4): 504–514 a veysi et al.: rearing and biology of … 511 http://jad.tums.ac.ir published online: december 30, 2017 the colonies. the number of emerged males was more than females and the average longevity of blood fed females was longer than males. as the results showed, the minimum and maximum of productivity were 23.9% and 55.8% in f4 and f3 respectively. these rates reported as 8.5% in f7 and 56.1% in f3 for p. papatasi, (25) and 9.9% in f4 and 47.3% in f6 for p. perniciosus (31). in another study, from f1 through f6, the average productivity was recorded at 44.08% (range: 29.6 to 55.2 %) for p. papatasi (39). as mentioned above the number of laid eggs increased from f1 to f5, it is expected that the production rate will increase consequently, however, it decreased. the reason behind this incompatibility could be the reduction of biological fitness or population bottleneck (or genetic bottleneck). due to changes which occurred during adaptation process, almost all biological parameters that recorded were statistically different among five generation. f4 is the significant point of the adaptation as it was shown by other study (31). during the establishing of this colony we faced a lot of difficulties and obstacles including high mortality during the immature phases due to several reasons including fungal, bacterial and mite infection which feed on eggs and young larvae, low blood preference specially in the early generations and mortality of engorged females before egg laying. to prevent mite infection the inner wall of the boxes containing pots were impregnated with petroleum jelly or liquid glue. also, we add a layer of fine soil collected from vicinity of gerbils’ colony, in the bottom of larval pots. interval shaking the soil layer prevents fungal growth. conclusion it is concluded that, colony of p. sergenti has been established for the first time in iran and the biological parameters of this species was studied in detail. average interval time from egg to 1st instar larva, pupa and adult of the species recorded at 6.3, 24.6 and 32.5 days respectively. for colony initiation, chicken is recommended as a blood source but due to difficulties in handling and anesthetizing of chicken, golden hamster was suggested to routine blood feeding of p. sergenti. establishment of this colony and defining its 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https://www.ncbi.nlm.nih.gov/pubmed/?term=new+phlebotomine+sand+fly+colonies%3a+rearing+phlebotomus+martini%2c+sergentomyia+schwetzi%2c+and+sergentomyia+africana 4. dr kazemi rtl iranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 29 original article culicinae (diptera: culicidae) mosquitoes in chabahar county, sistan and baluchistan province, southeastern iran *sh moosa-kazemi, h vatandoost, h nikookar, m fathian department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 12 july 2009; accepted 5 sep 2009) abstract background: mosquito-borne diseases are a major public health threat in iran. the objective of this study was to determine the fauna of culicinae mosquitoes for future mosquito control programs. methods: three genera and eleven species of the subfamily culicinae (diptera: culicidae) were collected by dipping technique and identified in chabahar county, sistan and baluchistan province, southeastern iran, during january, february, and march 2007. results: the collected species included: aedes vexans (new occurrence record for the province), culex arbieeni, cx. bitaeniorhynchus, cx. deserticola, cx. hortensis, cx. perexiguus, cx. pipiens, cx. pseudovishnui, cx. pusillus, cx. quinquefasciatus, cx. sinaiticus, cx. theileri, cx. tritaeniorhynchus, culiseta longiareolata, ochlerotatus caballus, oc. caspius, and uranotaenia unguiculata. conclusion: our observations indicate that, in south of iran hot and wet climatic conditions support the persistence of culicinae mosquitoes. as our study, regular monitoring of culicinae mosquitoes in this area could be the most useful for mosquito control and mosquito-borne disease prevention. keywords: culicinae, mosquito, fauna, chabahar, iran. introduction mosquito-borne diseases such as malaria, west nile, sindbis viruses, japanese encephalitis, and riftvalley fever are a major public health in some asian countries (yasoka and levis 2007). some of diseases such as malaria, west nile, sindbis as well as dirofilaria immitis (dog heart worm) and d. repens (dirofilariasis) had been previously reported in iran (naficy and saidi 1970, saidi et al. 1976, azari-hamidian et al. 2007). malaria transmission in chabahar county occurs through the year, most of the malaria cases were observed from may to nov almost in the age groups of 5-25 yr old (moosa-kazemi et al. 2006). malaria is transmitted by five species: anopheles culicifacies giles s.l., an. dthali patton, an. fluviatilis james s.l., an. stephensi liston and an. superpictus grassi (moosakazemi et al. 2006). by now, 64 species, 3 subspecies, and 7 genera of mosquitoes have been reported in iran (azari-hamidian 2007). four genera of culex, culiseta, ochlerotatus, uranotaenia and 18 species of the subfamily culicinae have been reported from sistan and baluchistan province (lotfi 1973, zaim 1987). zaim (1987) mentioned four genera and sixteen species of the subfamily culicinae (diptera: culicidae) in sistan and baluchistan province as follow: culex arbieeni salem, cx. bitaeniorhynchus giles, cx. deserticola kirkpatrick, cx. hortensis ficalbi, cx. perexiguus theobald, cx. pipiens linnaeus, cx. pusillus macquart, cx. pseudovishnui colless cx. quinquefasciatus say, cx. sinaiticus kirkpatrick, cx. theil*corresponding author: dr seyed hassan moosakazemi, e-mail: moosakazemi@sina.tums.ac.ir iranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 30 eri theobald, cx. tritaeniorhynchus giles, culiseta longiareolata macquart, ochlerotatus caballus theobald, oc. caspius pallas, and uranotaenia unguiculata edwards. it is noteworthy that culex mimeticus and cx. territans were found by lotfi (1973) in this area. there is only one recent study on the mosquitoes in the county in relation to malaria and anophelines (moosa-kazemi et al. 2006). while it is necessary to study mosquitoes in chabahar, there are no more references on the culicine mosquitoes of the county. this paper presents the first report of the results of the recent surveys of the culicinae mosquitoes from samples taken from a range of larval breeding sites in which they occur in nature, and adult culicinae mosquitoes captured from various places. the aim was to obtain new data, which would be valuable to develop programmes for future planning of mosquito control in this area of southeastern iran. materials and methods to better understand the culicinae fauna of chabahar county and gather information on their distribution and the kinds of larval breeding sites in which they occur in nature, a field trial study was performed from jan to mar 2007 in sistan and baluchistan province. this study took place in five randomly selected rural villages in chabahar county (57°14'n, 26°19'e). the county is bounded by pakistan in the east, hormozgan province in the west, kerman province in the north, and oman sea in the south (fig. 1). in 2007, the max and min mean monthly temperature was 33 °c and 5.5 °c in july and jan, respectively. the total annual rainfall was 221 mm, the minof 3.5 mm in july and max of 64 mm in apr. the mean annual relative humidity was 75%. the main economic activity of the people is husbandry of cow, goats and fishing. based on available epidemiological data from the chabahar health center, some villages were short-listed and entomological surveys were carried out. on this basis, five villages with 1118 houses and 5513 population were selected. to study the mosquito fauna, sampling was carried out by dipping technique for collecting larvae and pyrethrum space spray collection (pssc) for adult mosquitoes. the specimens were collected from five villages in the different topographical areas of the district including baho-kalat, beris, dak-baho, pollan, and soldan. pyrethroum space spray mosquitoes collection were conducted between 05.00 and 08.00 h monthly in eight fixed animal and human shelter randomly on each villages before spraying, all the eaves, windows, doors and other exit points were closed and cloth sheets were spread on the floor. pyrethrum (0.2% in kerosene) was sprayed using a pressurized hand sprayer. after spraying, the room was kept closed for 15 min and the knocked-down mosquitoes were then collected from the floor sheet with forceps and placed in petri-dishes lined with moist cotton. all collected mosquitoes were pinned and then were identified using the keys of zaim and cranston (1986), harbach (1988), and darsie and samanidou-voyadjoglou (1997). mosquito name abbreviations were cited based on reinert (2001). larval habitats present within 500 m around each village were studied for mosquito larvae two times per month. five randomly selected larval breeding places located within a 500-m radius of the study site were also included and formed the sampling unit for mosquito larvae. all larvae were collected from natural habitats, generally feeding with seepage water, such as swamps, seepages, streams, river banks, drying river beds, pools, and grasslands. samples were taken at intervals along the edge of each larval breeding place using a standard mosquito dipper (350 ml) depending on the size of the habitat. the larvae for each habitat were placed separately in whirl paks and transported to the laboratory where they were sorted by geiranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 31 nus and instar counted and recorded. the pupae were reared to adults in emergent cages and identified to species by morphological characters. because we did not have enough emergent cages to separate the pupae from each habitat by date of collection, samples for each habitat type were pooled together in each site irrespective of date of collection. the larvae were preserved in lactophenol and the microscopic slides of the preserved larvae were prepared using berlese medium. the culicinae larvae along with the description of their breeding sites were sent to the school of public health, tehran university of medical sciences, where the author identified using the aforementioned keys. results the number of the culicine mosquito species at the various sites of chabahar is shown in table 1. a total of 3824 adults and 5031 larvae of the culicine mosquitoes were collected, and were recognized including; aedes vexans meigen (indicate new occurrence records for the province), culex arbieeni, cx. bitaeniorhynchus, cx. deserticola, cx. hortensis, cx. perexiguus, cx. pipiens, cx. pseudovishnui, cx. pusillus, cx. quinquefasciatus, cx. sinaiticus, cx. theileri, cx. tritaeniorhynchus, culiseta longiareolata, ochlerotatus caballus, oc. caspius s.l., and uranotaenia unguiculata. in pssc collection overall, culiseta longiareolata predominated (22.8%), followed by cx. perexiguus (16.8%), cx. pipiens (14.2 %), and cx. tritaeniorhynchus (8.8%). in the larval collection, of 5031 culicinae larvae collected, culiseta longiareolata predominated (19.5%), followed by cx. perexiguus (16.9%), cx. pipiens (13.6 %), and cx. tritaeniorhynchus (9.1%) respectively (table 1). seven species including culiseta longiareolata, ochlerotatus caspius s.l., cx. perexiguus, cx. pipiens, cx. sinaiticus, cx. theileri, and cx. tritaeniorhynchus accounted for 85.1%, and 79.2% of the adult and larval collection respectively. the other species were less than 6% of the total. culiseta longiareolata was the most frequent culicine mosquito collected at chabahar, with total of 982, and 875 specimens, by larval collection and pssc, respectively. aedes vexans is recorded for the first time in chabahar county and sistan and baluchistan province. number and localities of the larvae of culicinae mosquitoes collected in this survey are shown in table 2. uranotaenia unguiculata, oc. caballus, cx. quinquefasciatus, and cx. arbieeni were collected only in the larval stage. number and localities of the adult of the culicine mosquitoes collected in this survey are shown in table 3. culiseta longiareolata, cx. perexiguus, and cx. pipiens were the most prevalent species respectively, and collected in almost all of the villages (table 2 and 3). the highest numbers of culicines were collected in the dak-baho (1206 larvae and 717 adult specimens), and the lowest in the beris (965 larvae and 627 adult specimens) (table 2 and 3). ochlerotatus caballus, cx. quinquefasciatus, and cx. arbieeni were collected from larval collection and other species from both spss and larval (table 2 and 3). discussion five genera and 17 species of culicine mosquitoes were collected and identified from chabahar county in sistan and baluchistan province, including the first record of ae. vexans in the province. from 18 species of culicinae mosquitoes in sistan and baluchistan province (lotfi 1973, zaim 1987), 16 species, which had already been recorded, were also collected in this investigation. two species culex mimeticus and cx territans are not recorded in this study. however, the occurrence of aedes vexans in the province was not mentioned before (zaim 1987), although some references counted aedes vexans as the more common species of guilan and isfahan provinces (azari-hamidian et al. 2002, mousakazemi et al. 2000). iranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 32 fig. 1. map of iran indicating the location of the study area in chabahar county (ch) situated in the south of sistan and baluchistan province (s and b) table 1. number and prevalence of adult and larvae culicinae mosquitoes collected by pyrethrum space spray and larval collections in chabahar county, sistan and baluchistan province during january, february, and march 2007 larvae adult species no percentage no percentage aedes vexans 120 2.4 61 1.6 culex arbieeni 15 0.36 cx. bitaeniorhynchus 30 0.59 49 1.3 cx. deserticola 120 2.4 137 3.6 cx. hortensis 65 1.3 111 2.9 cx. perexiguus 851 16.9 643 16.8 cx. pipiens 685 13.6 543 14.2 cx. pseudovishnui 120 2.4 5 0.13 cx. pusillus 206 4.1 202 5.3 cx. quinquefasciatus 146 2.9 cx. sinaiticus 267 5.3 291 7.6 cx. theileri 418 8.3 291 7.6 cx. tritaeniorhynchus 458 9.1 337 8.8 culiseta longiareolata 982 19.5 875 22.8 ochlerotatus caspius s.l. 327 6.5 279 7.3 oc. caballus 156 3.1 0 uranotaenia unguiculata 65 1.3 total 5031 100 3824 100 iranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 33 table 2. number and locality of the culicine mosquito larvae, chabahar county, sistan and baluchistan province, january–march 2007 villages species baho-kalat beris dak-baho pollan soldan no % aedes vexans 40 51 29 120 2.4 culex arbieeni 1 9 5 15 0.36 cx. bitaeniorhynchus 6 8 7 4 5 30 0.59 cx. deserticola 29 24 36 31 120 2.4 cx hortensis 14 13 15 11 12 65 1.3 cx. perexiguus 243 84 192 204 128 851 16.9 cx. pipiens 178 201 217 89 685 13.6 cx. pseudovishnui 29 14 28 32 17 120 2.4 cx. pusillus 40 37 39 51 39 206 4.1 cx. quinquefasciatus 29 68 49 146 2.9 cx. sinaiticus 94 173 267 5.3 cx. theileri 124 101 98 95 418 8.3 cx. tritaeniorhynchus 94 83 101 54 126 458 9.1 culiseta longiareolata 209 304 272 101 96 982 19.5 ochlerotatus caspius s.l. 69 71 78 63 46 327 6.5 oc. caballus 63 48 45 156 3.1 uranotaenia unguiculata 31 16 18 65 1.3 total 1104 965 1206 862 894 5031 100 table 3. number and locality of the adult culicine mosquitoes collected by pyrethrum space spray, chabahar county, sistan and baluchistan province, january–march 2007 villages species baho-kalat beris dak-baho pollan soldan no % aedes vexans 16 21 11 13 61 1.6 cx.bitaeniorhynchus 13 8 19 9 49 1.3 cx. deserticola 45 48 44 137 3.6 cx. hortensis 63 34 14 111 2.9 cx. perexiguus 151 116 164 183 29 643 16.8 cx. pipiens 89 96 101 76 181 543 14.2 cx. pseudovishnui 5 5 0.13 cx. pusillus 69 121 12 202 5.3 cx. sinaiticus 81 94 46 70 291 7.6 cx. theileri 103 48 82 58 291 7.6 cx. tritaeniorhynchus 59 15 142 121 337 8.8 culiseta longiareolata 167 172 219 153 164 875 22.8 ochlerotatus caspius s.l. 38 77 37 70 57 279 7.3 total 815 627 717 937 728 3824 100 in this study, two species of the tribe aedini including; ochlerotatus caspius s.l. and oc. caballus were recorded in chabahar county. these species were differentiated from each other in larval stage (zaim and cranston 1986). there is no information about the oc. caspius sibling species (a or b) in the country (azari-hamidian 2007). culex mimeticus and cx. territans had been previously repoted by lotfi (1973) in sistan and baluchistan, whereas in this study have not been found. culex pipiens was one of the most frequent culicine mosquitoes collected at chabahar, with total of 573 and 685 specimens, by pssc and larval collection respectively. culex iranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 34 pipiens can be separated from cx. torrentium, and cx. vegans by harbach’s (1988) key using seta 1-iii-v, seta 1-m, seta 1-x, seta 1-c, and some other characters. based on this key, only cx. pipiens was identified. one species of culiseta, cs. longiareolata was found in this investigation. results of this survey are the same of finding by previous study in sistan and baluchistan province (zaim 1987). in this study were collected other culicine mosquitoes such as, culex. theileri, and cx. tritaeniorhynchus. this species are potential vectors of human and domesticated animal pathogens (horsfall 1955, harbach 1988). the species cx theileri was one of the culicine mosquitoe collected at chabahar, with total of 291, and 418 specimens by means of pssc and larval collection, respectively. this species is the more prevalent species at higher altitudes and in rural areas of isfahan (mousakazemi et al. 2000), zanjan (ghavami and ladonni 2005) and east azerbaijan provinces (abai et al. 2007). observations made during the present study agree with those of simsek (2004) who noted that cx. theileri in turkey prefers large natural larval habitats that are generally present in rural areas. in conclusion, regular monitoring of mosquitoes in this area could be the most timely and sustainable way to follow up mosquito ecology and raise awareness about mosquito control and mosquito-borne disease prevention. acknowledgements the authors are grateful to dr s azarihamidian, school of health, guilan university of medical sciences, rasht, for reviewing the article. we also would like to express our appreciation to the people of villages in chabahar for their kind cooperation during the study, many thanks also dedicated for the efforts of the field staff of iranshahr health research and training chabahar health centers. this study was financially supported by diseases management center, iranian ministry of health and medical education, and the institute of public health research, academic pivot for education and research, tehran university of medical sciences. the authors declare that they have no conflicts of interest. references abai mr, azari-hamidian s, ladonni h, hakimi m, mashhadi-esmail k, kousha a, vatandoost h (2007) fauna and checklist of mosquitoes (diptera: culicidae) of east azerbaijan province, northwestern iran. iranian j arthropod-borne dis. 1: 27–33. azari-hamidian s, joeafshani ma, rassaei a, mosslem m (2002) mosquitoes of the genus aedes (diptera: culicidae) in guilan. j med fac guilan univ med sci. 11(43): 29-39 [in persian with english abstract]. azari-hamidian s (2007) checklist of iranian mosquitoes (diptera: culicidae). j vect ecol. 32: 235-242. azari-hamidian s, yaghoobi-ershadi mr, javadian e, mobedi i, abai mr (2007) review of dirofileriasis in iran. j med fac guilan univ med sci. 15(60): 102113 [in persian with english abstract]. darsie re, samanidou-voyadjoglou a (1997) keys for the identification of the mosquitoes of greece. j am mosq contr assoc. 13: 247–254. ghavami mb, ladonni h. (2005) the fauna and frequency of different mosquito species (diptera: culicidae) in zanjan province. the scientific journal of zanjan university of medical sciences. 13: 4654 [in persian]. harbach re (1988) the mosquitoes of the subgenus culex in southwestern asia and egypt (diptera: culicidae). contrib am ent inst. 24: 1-240. iranian j arthropod-borne dis, (2009), 3(1): 29-35 sh moosa-kazemi et al: culicinae (diptera: … 35 horsfall wr (1955) mosquitoes. their bionomics and relation to disease. hafner publishing, new york. lotfi, md (1973) iranian species of genus culex (culicinae: diptera) ii. – report of four species of larvae (including three new records) and 14 adult species. bull soc path exot. 66: 204-207. mousakazemi sh, zaim m, zahraii a (2000) fauna and ecology of culicidae of the zarrin-shahr and mobarakeh area in isfahan province. armaghan danesh, journal of yasuj university of medical sciences. 5: 46-54 [in persian]. moosa-kazemi sh, vatandoost h, raeisi a, akbarzadeh k (2006) deltamethrin impregnated bed nets in a malaria control program in chabahar, southeast baluchistan, i.r. iran. iranian j arthropodborne dis. 1: 27–33. naficy k, saidi s (1970) serological survey on viral antibodies in iran. trop geog med. 2: 183–188. reinert jf (2001) revised list of abbreviation for genera and subgenera of culicidae (diptera) and the notes on generic and subgeneric changes. j am mosq contr assoc. 17: 51-55. simsek fm (2004) seasonal larval and adult population dynamics and breeding habitat diversity of culex theileri theobald, 1903 (diptera: culicidae) in the golbasi district, ankara, turkey. turk j zool. 28: 337-44. yasuoka j, levins r (2007) ecology of vector mosquitoes in sri lanka-suggestions for future mosquito control in rice ecosystems. southeast asian j trop med public health. 38(4): 646-57. zaim m, cranston ps (1986) checklist and keys to the culicinae of iran (diptera: culicidae). mosq syst. 18: 233–245. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq contr assoc. 3: 568-573. 5dr rassi iran j arthropod-borne dis, 2011, 5(2): 32–41 a aghaei afshar et al.: susceptibility status of … 32 original article susceptibility status of phlebotomus papatasi and p. sergenti (diptera: psychodidae) to ddt and deltamethrin in a focus of cutaneous leishmaniasis after earthquake strike in bam, iran a aghaei afshar1, *y rassi1, i sharifi2, mr abai1, ma oshaghi1, mr yaghoobi-ershadi1, h vatandoost1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran 2 leishmaniasis research center, kerman university of medical sciences, kerman, iran (received 5 apr 2011; accepted 12 oct 2011) abstract background: the cutaneous leishmaniasis (cl) has been occurred in dehbakri county, located 46 km of bam district, kerman province since 2004–2005. phlebotomus papatasi is an important vector of zoonotic cutaneous leishmanisis (zcl) as well as sand fly fever and p. sergenti is considered as main vector of anthroponotic cutaneous leishmaniasis (acl) in iran. there are several measures for vector control with emphasizing on insecticides. the objective of this study was to determine the baseline susceptibility of leishmaniasis vectors to the ddt and deltamethrin in an endemic focus of cl in southern iran. methods: baseline susceptibility tests were carried out on field collected strains of p. papatasi and p. sergenti and tested with who impregnated papers with ddt 4.0% and deltamethrin 0.05% in the focus of disease in dehbakri county during summer 2010. the values of lt50 and lt90 were determined using probit analysis and regression lines. results: the lt50 value of ddt 4.0% and deltamethrin 0.05% against p. papatasi was 20.6 and 13.6 minutes respectively. the same data for p. sergenti were ranged between 21.8 and 17.7 minutes. conclusion: the results of tests will provide a guideline for implementation of vector control using pesticides such as impregnated bed nets, indoor residual spraying and fogging. keywords: phlebotomus papatasi, phlebotomus sergenti, insecticides, susceptibility, iran introduction phlebotomine sand flies are known to transmit varieties of zoonotic diseases including leishmaniases (protozoa), bartonellosis (bacteria) and sand fly fever (viruses) that affect humans and animals in 88 countries worldwide (verani et al. 1991, saidi et al. 1997, tesh et al. 1997, mehrabi tavana 2001, sharma and singh 2008, depaquit et al. 2010). phlebotomus papatasi is widely distributed throughout the world. it is a common species in the eastern mediterranean region and considered as important vector of zoonotic cutaneous leishmaniasis as well as sand fly fever in iran. cutaneous leishmaniasis (cl) is reported from 50% of the 31 provinces in iran (yaghoobi-ershadi et al. 1999, rassi et al. 2004, rassi et al. 2006, rassi et al. 2007, rassi et al. 2011). the activity of p. papatasi and p. sergenti in bam starts from late may and extends to mid october with two peaks, one in mid or late june and the second in early or mid september. phlebotomus papatasi could be a leishmania vector for humans and gerbils and is *corresponding author: prof yavar rassi, e-mail: rassiy@sina.tums.ac.ir iran j arthropod-borne dis, 2011, 5(2): 32–41 a aghaei afshar et al.: susceptibility status of … 33 susceptible to ddt (yaghoobi-ershadi and akhavan 1999). leishmaniasis is endemic in at least 88 poor countries, with the global prevalence of all forms of the disease is 12 million, with 1.5–2 million cases of cl and half a million cases of visceral form. current control measures, including environmental sanitation and drug treatment of cases, are expensive and cannot be sustained effectively by poor countries due to the problems of financing and implementation. moreover, toxicity associated with some of the most widely available drug treatments, including injections of pentavalent antimony compounds, and the resistance developed by the parasite underline the need for development of effective methods of prevention, especially vaccines (noazin et al. 2008, noazin et al. 2009). the findings indicate that the whole-parasite vaccine candidates tested do not confer significant protection against human leishmaniasis. cutaneous leishmaniasis is still an important public health problem in many parts of the world, where 90% of the cases occur in seven countries including iran. two main species of cl are present in iran; anthroponotic cl (acl) due to l. tropica is the predominant species in many large and medium size cities. the city of bam has been endemic for many years and a well-known focus for acl, where recent field trials of autoclaved l. major vaccine against acl were conducted. zoonotic cl (zcl) caused by l. major, is found in many rural foci in the north, east and south of the country (sharifi et al. 2010). emergence or re-emergence of cl has recently occurred in many countries. both zcl and acl have been prevalent in a number of rural and urban areas of iran. they have also emerged in new foci during recent decades (fazaeli et al. 2009). a new emerging focus of acl due to l. tropica in rural areas of dehbakri county, south-eastern iran, after the earthquake of 2003 has been confirmed using polymerase chain reaction (pcr). the current emergence was unexpected in this rural area, where no previous history of acl was recorded. according to our knowledge this is the first report of a gradually establishing new acl focus in rural communities after the 2003 earthquake (sharifi et al. 2011). nested pcr revealed the first report of occurrence of acl due to l. tropica among 3516 inhabitants in jiroft city (pouresmailian et al. 2010). molecular and direct detection of leishmania parasites revealed the occurrence of acl in rural parts of the bam city after earthquake. natural disaster completely provides favorable condition for sand fly breeding places as well as propagation of l. tropica in the displaced population due to earthquake (sharifi et al. 2011). the fauna and monthly activity of the acl vector has been studied in bam using the sticky paper traps. the sand fly species identified were p. sergenti (77.25%), p. papatasi (19.00%), sergentomyia baghdadis (1.69%), s. sintoni (1.64 %) and s. tiberiadis (0.42%). phlebotomus sergenti was the predominant species, and formed 85.11% and 81.83% of species from the human and animal indoor habitats, respectively. phlebotomus sergenti have two peaks of activity during the year, one of which is from the beginning of july, and the other is at the beginning of september (aghasi and sharifi 2003). according to the health center report the main activities for sand fly control was using ddt as indoor residual spraying when there was malaria problem in the region. subsequently permethrin is used for space spraying. deltamethrin impregnated bed nets at the dosage of 25 mg/m2 also had been employed for vector control. the complementary measure was using repellents and covering the lesion with sterile bandage. dehbakri county is encountered as the newly emerging acl foci in kerman province since 2003 just after tremendous bam earthquake. following this event, some afiran j arthropod-borne dis, 2011, 5(2): 32–41 a aghaei afshar et al.: susceptibility status of … 34 fected people were migrated to dehbakri country due to temperate climate compared to bam city. the prevalence of cl was 213.8 per thousand of people during 2003– 2010. in addition, the cl incidence was estimated 34.3 per thousand people at 2010 (unpublished data). the aim of this study was the determination of baseline data on susceptibility level of p. papatasi and p. sergenti to ddt and deltamethrin in cl affected villages where the chemical intervention was remained as the main choice for cl control method in the dehbakri focus, southest iran. materials and methods study area dehbakri county is located at the foothill of jebal-barez mountain and 48 km far from southwestern of bam district (fig. 1). the population of dehbakri county was 2470 in 2009. the topography of these localities is foothill with the mean altitude of 2000 m above sea level. this area has a semi-desert climate, temperate at summer and cold at winter. the mean of monthly maximum and minimum temperatures were 40 °c and -5 °c in july and dec respectively. the total annual rainfall was 220 mm. the minimum and maximum of monthly relative humidity were 45% and 92% respectively in july and jan. fig. 1. dehbakri country, located in bam district, kerman province, south of iran sand fly collection sand flies were collected using aspirator from 8.00 pm till 2.00 am during summer 2010. the caught sand flies were transferred to the entomological cage and then were kept by wet towel and transported to laboratory research center for implementation of susceptibility tests. susceptibility test all the susceptibility tests were carried out according to the guideline of who (1981). the sand flies were transferred to the exposure tubes and then the mortality was scored at interval logarithmic times and the result was read after 24 hour recovery period. during the holding time, the insects were supplied with cotton pad of water in 10% sugar solution. all tested sand flies were mounted separately using puri's media in order to identify the species. male and female sand flies were counted separately. insecticide impregnated papers impregnated papers with ddt 4.0% and deltamethrin 0.05%, as well as control papers were supplied by world health organization via iranian centre for diseases management, ministry of health and medical education of iran. statistical analysis the mortality rates were corrected according to the result of control tests using abbott’s formula whereas the control mortality rates were ranged between 5.0–20.0% (abbott 1925). phlebotomus papatasi and p. sergenti were exposed to who standard papers of ddt 4.0% and deltamethrin 0.05% at different logarithmic exposure times and the mortalities were recorded after 24 hour and subsequently analyzed by probit analysis (finney 1971). goodness of fit of regression lines of p. papatasi and p. sergenti was measured through the χ2 test. lt50 and lt90 values were estimated and the slope values iran j arthropod-borne dis, 2011, 5(2): 32–41 a aghaei afshar et al.: susceptibility status of … 35 of the regression lines were calculated. regression lines were plotted for lt50 values for observed probit of mortalities against exposure times using microsoft® excel software ver. 2007. results the mortality rate of field-collected p. papatasi with ddt at diagnostic concentration was 100%. the mortality rate for fieldcollected p. sergenti with ddt 4.0% and one hour exposure time was calculated 97.0±1%. considering the criteria of susceptibility for mosquitoes as described by who, it should be noted that the tested populations of both p. papatasi and p. sergenti were also susceptible to these insecticides. the lt50 values of ddt 4.0% calculated both for p. papatasi and p. sergenti were 20.7 and 20.8 minutes. the lt90 values of ddt 4.0% were 55.6 and 75.2 minutes respectively for both latter species. the lt50 values of deltamethrin 0.05% calculated both for p. papatasi and p. sergenti were 13.6 and 17.7 minutes respectively. the lt90 values of deltamethrin 0.05% respectively were 41.9 and 52.0 minutes for p. papatasi and p. sergenti (table 1). the regression lines of exposure times of p. papatasi and p. sergenti which exposed to who's impregnated papers with ddt 4.0% and deltamethrin 0.05% are shown in fig. 2. table 1. parameters of regression line of susceptibility of p. papatasi and p. sergenti of sand flies exposed to two insecticides in secticid e s p ecies n o of san d fly tested m ean of m ortality (% ) (d iagn ostic d ose-1 h ou r) a (y-in tercep t) b (s lop e) ± s e l t 50 ± 95% c .l . l t 90 ± 95% c .l . x 2 (h eterogen eity) calcu lated x 2 (h eterogen eity) tab le (d f) p . valu e ddt4% p. papatasi 153 100% -3.9736 3.011± 0.493 16.5127 40.1724 52.724 (4) 18.47 (4) <0.001 20.8771 55.6294 26.8631 97.1896 ddt4% p. sergenti 217 97.0±1% -3.1495 2.3547± 0.683 8.5907 32.1209 18.801 (4) 13.28 (4) <0.001 21.7519 76.1663 115.6334 ns deltamethrin 0.05% p. papatasi 130 100% -2.9641 2.6174± 0.414 10.1957 29.8310 5.553 (4) 9.49 (4) >0.05 13.5665 41.8909 17.7174 73.3799 deltamethrin 0.05% p. sergenti 212 100% -3.4225 2.7418± 0.352 14.3172 38.4355 7.745(4) 9.49 (4) >0.05 17.7120 51.9639 22.1612 82.2050 iran j arthropod-borne dis, 2011, 5(2): 32–41 a aghaei afshar et al.: susceptibility status of … 36 1 2 3 4 5 6 7 8 1 10 100 exposure time (min) p ro b it o f m o rt a li ty ddt p.papatasi ddt p.sergenti deltamethrin p.papatasi deltamethrin p.sergenti fig. 2. probit regression lines of ddt 4% and deltamethrin 0.05% against p. papatasi and p. sergenti using who method at bam district, southern iran discussion prior of this study, no reports were available on the susceptibility levels of adult p. papatasi and p. sergenti at the new cl focus of dehbakri county, bam district, southern iran. the susceptibility status of p. papatasi to ddt, dieldrin, malathion, fenitrothion and propoxur has been estimated in pali and barmer districts of rajasthan. the results revealed that this species was resistant to ddt but susceptible to other insecticides (singh and bansal 1996). in a similar study in district bikaner, rajasthan the results showed that this species was resistant to ddt, dieldrin and propoxur while susceptible to malathion, fenitrothion and permethrin (bansal and singh 1996). in iran, the residual spraying was carried out during 1950–1968 and the houses had been treated with ddt for malaria control. the susceptibility level of p. papatasi to ddt was studied at various parts of iran during 1985– 88. tests were carried out in the localities where the application of ddt had been discontinued since 1969. this investigation showed that p. papatasi from isfahan was more tolerant to ddt than other parts and probably a manifestation of ddt resistance (rashti et al. 1992). another study was carried out at 6 villages of isfahan province on p. papatasi and the values of lt50 and lt90 ranged between 18.10–20.15 minutes and 43.00–63.30 minutes respectively. in the present study, the lt50 values of ddt 4.0% against p. papatasi ranged from 20.9 to55.6 minutes. the same data for ddt 4.0 against p. sergenti were ranged between 21.8–76.2 minutes which may related with endophilic habits of fore-mentioned species and exposing the sand flies to chemical used inside the houses. the latter study showed the tolerance in p. papatasi population to ddt 4.0% at 1 hour exposure time with mortality rate of 88.8% (yaghoobi-ershadi et al. 1995). the susceptibility status of p. papatasi had been shown at zcl foci of fars province, southern iran and the results indicating the highly susceptibility level of this species to ddt in this area (abai et al. 1993, rassi et al. 2000). there are several reports revealing ddt resistance at p. papatasi, p. argentipes and sergentomyia shorti from india (joshi et al. 1979, mukhopadhyay, 1992, maroli and khoury 2004) as well as p. papatasi in turkey (who 1986). in contrast, there are also some reports emphasizing to highly susceptibility levels of p. papatasi and p. ariran j arthropod-borne dis, 2011, 5(2): 32–41 a aghaei afshar et al.: susceptibility status of … 37 gentipes at different countries (rahman et al. 1982, dergacheva et al. 1986, pener et al. 1987, kaushal 1995, fahnmy et al. 1996). resistance of p. papatasi to ddt is first reported in bihar (joshi et al. 1979) and then in turkey (who 1986). mean of lc90 values for ddt for egyptian and sudanese strains of p. papatasi ranged from 0.80– 1.93% (schmidt et al. 1969). the lc50 for ddt for sudan p. papatasi was reported as 0.9% (qutubuddin 1964). results obtained from susceptibility tests on field population of p. papatasi to ddt revealed that the tested population was highly susceptible to ddt as evidenced by the discriminating concentrations of times. in iranian foci of zoonotic leishmaniasis, control of malaria with ddt yielded no effect on the incidence of leishmaniasis or the sand fly population (seyedi-rashti and nadim 1974). measures used to control adult sand fly includes application of insecticides as residual spraying at dwellings and animal shelters, space-spraying, insecticide-treated nets and curtains, and personal protection through application of repellents/insecticides to skin or fabrics. because the breeding places of sand flies which are generally unknown, the control measures that act specifically against immature are not feasible, although the effectiveness of a few biological and chemical agents has been demonstrated in laboratory evaluations (alexander and maroli 2003). in some circumstances the reservoir control is also recommended. additionally the patient treatment with anti-parasite drug has been prescribed at the acute stage of disease (yaghoobiershadi et al. 2000). tolerance of p. papatasi to ddt has been reported from some parts of country (rashti et al. 1992, abai et al. 1993, yaghoobi-ershadi et al. 1995). this study showed the higher susceptibility level of p. papatasi and p. sergenti to deltamethrin 0.05% (lt50=13.6–17.7 minutes) and so this insecticides could be effectively used as residual spraying or as itns or llins in order to control the cl foci of iran. due to epidemic condition of cl, the affected villages of dehbakri county were sprayed with deltamethrin wp 5.0% at 25 mg ai/m2 during june 2010. acknowledgements this study was financially supported from the school of public health, tehran university of medical sciences, and grant no. 10487. the authors would like to thank the dermatology and leishmaniasis research center, kerman university of medical sciences for their close collaboration in this study. we also wish to express our sincere thanks to 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(2006) evaluation of deltamethrinimpregnated bed nets and curtains for control of zoonotic cutaneous leishmaniasis in a hyperendemic area of iran. bull soc pathol exot. 99: 43–48. j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 414 http://jad.tums.ac.ir published online: september 08, 2017 original article gas chromatography, gc/mass analysis and bioactivity of essential oil from aerial parts of ferulago trifida: antimicrobial, antioxidant, ache inhibitory, general toxicity, mtt assay and larvicidal activities saeed tavakoli 1, 2, hassan vatandoost 3, 4, reza zeidabadinezhad 2, reza hajiaghaee 2, abbas hadjiakhoondi 1,2, mohammad reza abai 3,4, *narguess yassa 1,2 1department of pharmacognosy, faculty of pharmacy and medicinal plant research center, tehran university of medical sciences, tehran, iran 2medicinal plants research center, institute of medicinal plants, acecr, karaj, iran 3department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 4department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran (received 21 nov 2016; accepted 4 sep 2017) abstract background: we aimed to investigate different biological properties of aerial parts essential oil of ferulago trifida boiss and larvicidal activity of its volatile oils from all parts of plant. methods: essential oil was prepared by steam distillation and analyzed by gas chromatography and gc/mass. antioxidant, antimicrobial, cytotoxic effects and ache inhibitory of the oil were investigated using dpph, disk diffusion method, mtt assay and ellman methods. larvicidal activity of f. trifida essential oil against malaria vector anopheles stephensi was carried out according to the method described by who. results: in gc and gc/ms analysis, 58 compounds were identified in the aerial parts essential oil, of which e-verbenol (9.66%), isobutyl acetate (25.73%) and e-β-caryophyllene (8.68%) were main compounds. the oil showed (ic50= 111.2µg/ml) in dpph and ic50= 21.5 mg/ml in the investigation of ache inhibitory. furthermore, the oil demonstrated toxicity with (ld50= 1.1µg/ml) in brine shrimp lethality test and with (ic50= 22.0, 25.0 and 42.55 µg/ml) on three cancerous cell lines (mcf-7, a-549 and ht-29) respectively. lc50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of an. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. in antimicrobial activities, essential oil was effective on all specimens except escherichia coli, aspergillus niger and candida albicans. conclusion: the essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and mtt assay on three cancerous cell lines. keywords: ferulago trifida, larvicidal property, antioxidant activity, antimicrobial effect, mtt assay introduction the use of medicinal plants to treatment of diseases and improve the health has always been attractive for men. in addition to traditional medicinal purposes, the plants are of most interest to researchers because of isolation and identification of bioactive compounds as the lead compound in the development and production of new drugs with efficacy and safety. ferulago trifida boiss, from apiaceae family is an iranian indigenous plant and no study has been done for identification of its compounds in iran, therefore phytochemical study on its extract, essential oil and some investigation e.g. antimicrobial and antioxidant effects, inhibition of the acetyl-cholinesterase and cytotoxic properties are indispensable on this valuable plant. *corresponding author: dr narguess yassa, e-mail: yasa@tums.ac.ir mailto:yasa@tums.ac.ir j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 415 http://jad.tums.ac.ir published online: september 08, 2017 ferulago trifida is an endemic plant in iran that grows in qazvin province near the village of qvanin alamut region. this genus consists of seven species in iran, most of them are endemic to iran or have spread in regions of anatolia, syria, lebanon, and iraq, they are valuable pasture plants (1, 2). in certain parts of iran, some species of this genus are traditionally added to dairy products, especially in the oil made of animal fats for a pleasant taste and presentation of corruption (3). different species of this plant in some parts of turkey were used as a sedative, tonic, gastrointestinal pain, cure hemorrhoids etc. there is a report about the use of the essential oil of various species of this genus for preparation of cosmetic products (3). in recent years, researchers have studies on immune modulatory and cytotoxic effects of some medicinal plants that f. angulata (schlecht) boiss, is one of them (4, 5). during the studies on essential oil of f. carduchorum, antibacterial and antifungal effects on staphylococcus aureus and candida albicans as well as strong antioxidant effects have been observed (6). chemical analysis and antimicrobial activity of essential oil of f. bernardii shown volatile oil of the plant contains 2, 4, 5trimethylbenzaldehyde, caryophyllene oxide, spathulenol, cis-chrysanthenyl acetate, and α-pinene. anti-microbial activity of the plant compared to fluconazole and gentamicin represents weak effects against c. albicans, aspergillus niger, bacillus subtilis, s. aurous and escherichia coli. the volatile oil did not show any activity on pseudomonas aeruginosa (7). in several studies on some species of genus ferulago different coumarins have been identified, from the two species f. capillaris and f. brachyloba, four new coumarins: (+)-3'-hydroxyprantschimgin, (-)-3'-senecioyloxymarmesin, senecioyloxymarmesin and (+)-senecioylprangol, have been detected (8). acetylcholinesterase inhibitory effects of three isolated coumarins (umbelliprenin, coladonin, coladin), phenolic compounds, polyacetylene and daucane from f. campestris were studied; all of these compounds showed inhibitory effects on ache (9). in a study from turkey on chloroform extract of f. aucheri, two coumarins: osthenol, prantschimgin and two flavonoids: isorhamnetin3glucoside, 6-hydroxyapigenin-6-methyl ether and two new aromatic compounds: quinolmono acetate, 1-acetylhydroquinone-4-galactoside were identified (10). in this project, we studied larvicidal properties against anopheles stephensi of f. trifida essential oils from its different parts (flower, leaf, aerial parts, root, stem, and fruits), and identified the essential oil components of aerial parts. in addition, we investigated antimicrobial, antioxidant, cytotoxic effects on brine shrimp and three tumor cell lines, and acetylcholine esterase inhibitory effect of the volatile oil from aerial parts of f. trifida. materials and methods plant material the aerial parts of f. trifida were collected in july 2014 from qazvin province near the village of ovan in alamut region, iran. the plant was identified by dr v mozaffarian (research institute of forest and rangelands, tehran, iran) and a voucher specimen was deposited in the herbarium of faculty of pharmacy, tehran university of medical sciences, tehran, iran (no.the-6562). preparation of essential oils the collected plant was dried in shade and powdered, 100g of powdered plant was subjected to hydro-distillation with a clevenger type apparatus for 4h. the yellow color essential oil was dried over anhydrous sodium sulfate and was kept in refrigerator until analyses. gas chromatography and gc/mass spectroscopy essential oil of the aerial parts of f. trifida j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 416 http://jad.tums.ac.ir published online: september 08, 2017 was analyzed on an hp-6890 gas chromatograph with an hp-5ms column (30×0.25mm id, 0.25 μmfilm thickness), equipped with hp-5973 mass detector (ionization energy: 70 ev) under the following conditions, temperature program: 60 °c (0-3min), 60 °c to 250 °c at the rate of 3 °c/min (3-65 min), injector temperature: 220 °c, detector temperature: 290 °c, injection volume: 1.0μl, split ratio: 1:90, carrier gas: helium (flow rate: 1ml min-1). the kovats retention indices (ki) were calculated for all identified compounds using a homologous series of n-alkanes (c8-c24) injected under the same chromatographic conditions described for samples. the components of the oils were identified by comparison of their mass spectra and retention indices with wiley library and those published in the literature (11). for quantitative analysis, essential oil was also injected to hp-6890 gas chromatograph with an hp-5ms column fitted with fid detector in conditions equal to gc/ms analysis (table 1). antibacterial activity bacterial strains antimicrobial activity of the essential oils was individually assessed against a set of seven bacterial strains, gram-positive bacteria s. aureus (atcc 29737), s. epidermidis (atcc 12228) and bacillus subtilis (atcc 6633), gram-negative bacterial pseudomonas aeruginosa (atcc 27853), e. coli (atcc 10536), klebsiella pneumonia (atcc 10031), shigella dysenteriae (ptcc 1188), salmonella paratyphi-a (atcc 5702) and proteus vulgaris (ptcc 1182), as well as tree fungi including two mold, aspergillus brasiliensis (atcc 1015) and a. niger (atcc 16404) and one yeast, c. albicans (atcc 10231), provided from iranian research organization for science and technology (irost). disk diffusion assay disc diffusion method was applied for the evaluation of antimicrobial activity of essential oil (12). the essential oil was filtered through 0.45µm millipore filters for sterilization. 100µl of suspension containing 108 cfu/ml of bacteria, 104 spore/ml of mold and 106 cfu/ml of yeast were spread on the nutrient agar (na), potato dextrose agar (pd) and sabouraud dextrose (sd) agar mediums, respectively. the impregnated discs (6mm in diameter) with 10µl of the essential oil were placed on the inoculated agar. the diameters of inhibition zones (iz) (mm) were measured following incubation of all plates at 37 °c (bacteria) and at 30 °c (fungi) for 24h, gentamicin (10µg/disc) and rifampin (5 µg/disc) were used as positive controls for bacteria and nystatin (100i.u./disc) for fungi. each assay was repeated twice and diameters of inhibition zones were represented as mean (table 2). micro-well dilution assay essential oil was subjected to micro-well dilution assay in order to determination of minimal inhibition concentration (mic) values, for microbial strains found susceptible in disc diffusion assay (13). the suspensions of microbial strains were prepared from their 12h broth cultures at 0.5mc farland standard turbidity. the serial two-fold dilutions of essential oil sample were made in a concentration range from 7.8 to 500µg/ml in sterile test tubes containing brain heart infusion broth (bhi) for bacteria and sabouraud dextrose broth (sd) for fungi strains. ninety five µl of the cultures media and 5µl of the inoculum were dispensed into each well of the 96-well plates. then 100µl from essential oil dilutions was added to the wells. a well-containing 195µl of the cultures media and 5µl of the inoculum without the test sample were used as negative control. gentamicin and rifampin for bacteria and nystatin for fungi were also used as positive control in same conditions as described to test samples. the content of plates was mixed on a plate shaker at 300rpm for 20sec and then incubated at apj arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 417 http://jad.tums.ac.ir published online: september 08, 2017 propriate temperatures for 24h. microbial growth was determined by the presence of a white pellet on the well bottom and confirmed by plating 5µl samples from clear wells on na medium. the mic value was defined as the lowest concentration of the plant essential oil required for inhibiting the growth of microorganisms. all tests were repeated two times (table 2). acetylcholinesterase inhibitory assay acetylcholinesterase inhibitory activities of the sample were determined (14), with slight modification in a 96-well microplate. briefly, 125µl of 3 mmdtnb [5, 5’dithiobis (2nitrobenzoic acid)], 25µl of 15m matci (acetylthiocholine iodide), 50µl of phosphate buffer (ph 8) and 25µl of the essential oil sample solution (3mg ml-1, in methanol) were added to 96-well plates. the absorbance was recorded at 405nm in 13sec intervals for 65 sec using a tecan microplate reader. after that, 25μl of ache enzyme (0.22 u ml-1) was added and the absorbance was measured again in 13sec intervals for 104sec. the enzyme activity was calculated from the slope of the line obtained from plotting of the absorbance against time. any increase in the absorbance caused by non-enzymatic hydrolysis of atci was corrected by the recorded absorbance before addition of enzyme. percentage of enzyme inhibition was calculated by comparing the rates for the sample to the blank (using methanol without tested sample). physostigmine was used as the positive control (table 3). free radical scavenging assay antioxidant capacities of the essential oil of f. trifida were determined (15). briefly, 2.5ml of dpph (2, 2-diphenyl-1-picrylhydrazyl radical, merck, germany) solution (80µg ml-1 in methanol) was added to 2.5ml of sample solutions prepared in concentrations ranging from 5.0 to 9.5× 10-3mg/ml in methanol, and test tubes were kept in dark for 30min at 25 °c, then uv absorptions were recorded on an optizen 2120 uv plus spectrophotometer at 517nm. bht (butylated hydroxytoluene) was used as a positive control. all tests were done in triplicate and ic50 values were reported as mean± sd (table 3). brine shrimp lethality test general toxicity of the essential oil was evaluated by brine shrimp lethality test (bslt) (16). for preparation of artificial seawater, 38g of sea salt was dissolved in 1.0l water and adjusted to ph 9 using sodium carbonate. the cysts of artemia salina l. were hatched in sterile artificial seawater under constant aeration for 48 h at 30 °c. 50mg of essential oil were mixed with 250μl dmso and one drop tween 80 and diluted with artificial seawater to get 1000, 700, 500, 300, 100, 10, 2, 1, 0.5 and 0.25μg/ml concentrations in a series of tubes containing about 20 active nauplii in each. the tubes were placed in a water bath at 30 °c for 24h under light, and the surviving nauplii were then counted to obtain the concentration causing 50% lethality (ld50 value). podophyllotoxin, a known cytotoxic natural compound, was applied as positive control. the test was carried out three times and ld50 value was reported as mean ± sd (table 3). cytotoxic activity cytotoxic activities of the essential oil of f. trifida was evaluated by mtt [3-(4, 5dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] colorimetric assay (17). three tumor cell lines, mcf-7 (human breast adenocarcinoma), a-549 (non-small cell line carcinoma) and ht-29 (human colon adenocarcinoma) were prepared from pasture institute of iran. the cell lines were cultured in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 1% penicillin-streptomycin in a 5% co2 incubator at 37 °c. cells were seeded into 96-well plates at a density of 0.5-1.5×104 cells/well and incuj arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 418 http://jad.tums.ac.ir published online: september 08, 2017 bated for 24h at 37 °c. the medium was then replaced with fresh medium containing different concentrations of essential oil and incubated for 72h at 37 °c. then, the medium was changed by fresh medium containing mtt and incubated for 4h. during this period, mtt is reduced to formazan by living cells. finally, the precipitated formazan crystals (purple dye) were dissolved in 200µl dmso and determined at 570nm, in a tecan microplate reader. cytotoxic activity of the essential oil was defined as a 50% reduction in viability of cells (ic50 value). tamoxifen was used as positive control (table 4). bioassays and larval mortality according to the standard method described by who (18), fourth instar larvae of an. stephensi was used for this experiment. 1ml of different concentrations (0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 ppm) of essential oil (solvent: ethanol) was mixed with 224ml of water. about 25 larvae in 25ml water were added to the diluted essential oil. for control, only 1ml of ethanol with 224ml of water and 25 larvae in 25ml water were mixed and volume of the all tests and control were 250ml (19). all of the tests and control were exposed for 24h with larva. the experiment was repeated four times on different days. the percentage of mortality was reported from the average for the four replicates after 24h exposure period. from the regression line between logarithmic dose and probit mortality, the lc50 was determined. this investigation has been carried out in the insectarium of department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (fig. 1). statistical analysis larvicidal activity was calculated from micro probit software (ver. 3.0). the percentages of mortality were corrected for the mortality in controls by using abbott's correction. from the regression line between the logarithmic dose and probity mortality, all the parameters including lc50 and slope values were determined (20). in antioxidant and inhibition of acetylcholinesterase assays, one-way anova and tukey post-hoc multi-comparison tests were used for the analyses after data normality test. analyses were performed in triplicate and the data were expressed as mean ±sd. in cytotoxicity assay, lc50 (the median growth inhibitory concentration) values were calculated from the lc50 of dose-response curve in the sigma plot software. results components of the essential oil gc and gc/ms analysis of the aerial parts essential oil were led to the identification of (86.41%) of the oil with 58 compounds. it is reach of oxygenated monoterpenes (49.30%) such as cis-verbenol, trans-verbenol (9.66%), p-mentha-1, 5-dien-8-ol (3.23%), isobornyl acetate (25.73%), thymol and carvacrol. e-βcaryophyllene (8.68%), germacrene d and caryophyllene oxide are the abundant sesquiterpenes in the essential oil (table 1). antimicrobial activities the results of essential oil effects on eleven bacteria and fungi were reported as minimum inhibitory concentration (mic) and inhibition zone in diameter (mm) (table 2). rifampin, gentamycin, and nystatin were used as positive control against bacterial and fungal strains, respectively. the results of positive controls (rifampin, gentamycin, and nystatin) and negative control (dmso) also are shown in table 2. antioxidant, ache inhibitory and brine shrimp lethality in free radical scavenging assay with dpph method, the volatile oil (ic50= 111.5.2µg/ml) j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 419 http://jad.tums.ac.ir published online: september 08, 2017 showed no antioxidant activity in comparison to the bha (ic50=21.2µg/ml). ache inhibitory of the oil (22.5mg ml-1) versus physostigmine as standard (0.8µg ml-1) was very low. general toxicity of the oil was evaluated by brine shrimp lethality test, and the sample with ld50= 1.1µg/ml demonstrated strong toxic effect in comparison to podophyllotoxin with ld50= 2.8µg/ml (table 3). cytotoxic effect the effects of f. trifida essential oil on the proliferative response of the mcf7 (human breast adenocarcinoma), a-549 (non-small cell line carcinoma) and ht-29 (human colon adenocarcinoma) cell lines have been analyzed by treating the cells with different concentrations of the volatile oil and significant decrease in cell lines proliferation in comparison with tamoxifen as positive control were observed in table 4. larval mortality the larvicidal activity of the essential oils from aerial parts, flower, fruit, leaf, stem, and root of the f. trifida against an. stephensi larvae were examined under laboratory conditions and the results are presented in fig. 1. stem oil of the f. trifida was the most effective one against an. stephensi with lc50= 6.51ppm. table 1. essential oil components of ferulago trifida aerial parts by gas chromatography mass spectroscopy no compounds % ki no compounds % ki 1 hexanal 0.47 807 30 isobornyl acetate 25.73 1292 2 2e-hexenal 2.55 860 31 thymol 2.35 1303 3 nonane 0.20 900 32 carvacrol 1.30 1311 4 heptanal 0.34 909 33 undecanal 0.37 1316 5 α-pinene 0.42 935 34 α-copaene 0.52 1381 6 thuja-2,4(10)-diene 0.21 958 35 geranyl acetate 0.31 1384 7 1-octen-3-ol 1.26 986 36 β-bourbonene 0.93 1389 8 myrcene 0.41 992 37 z-jasmone 0.38 1406 9 mesitylene 1.16 1000 38 (z)-caryophyllene 0.12 1411 10 n-octanal 0.13 1011 39 (e)-caryophyllene 8.68 1427 11 p-cymene 0.22 1030 40 β-copaene 0.23 1437 12 limonene 0.44 1034 41 neryl acetone 0.39 1456 13 (z)-β-ocimene 3.33 1040 42 α-humulene 0.81 1464 14 (e)-β-ocimene 0.56 1050 43 γ-muurolene 0.43 1482 15 γ-terpinene 0.39 1063 44 germacrene d 2.18 1489 16 terpinolene 0.14 1090 45 β-himachalene 0.64 1505 17 linalool 0.69 1106 46 β-bisabolene 0.38 1514 18 n-nonanal 1.43 1113 47 δ-cadinene 0.35 1526 19 allo-ocimene 0.33 1133 48 e-nerolidol 0.21 1567 20 cis-verbenol 2.55 1151 49 geranylbutanoate 0.33 1575 21 trans-verbenol 9.66 1156 50 spathulenol 0.51 1590 22 p-mentha-1,5-dien-8-ol 3.22 1183 51 caryophyllene oxide 2.47 1595 23 terpinen-4-ol 0.36 1190 52 tetradecanal 0.14 1621 24 p-cymen-8-ol 0.40 1199 53 1-epi-cubenol 0.14 1640 25 α-terpineol 0.30 1206 54 caryophylla-4(14),8(15)-diene-5-α-ol 0.29 1649 26 n-decanal 1.43 1214 55 caryophylla-4(14),8(15)-diene-5-β-ol 0.60 1652 27 verbenone 0.22 1221 56 α-muurolol 0.27 1656 28 geraniol 1.59 1258 57 α-cadinol 0.32 1670 29 cis-verbenyl acetate 0.29 1288 58 14-hydroxy-9-epi-(e)-caryophyllene 0.33 1686 total identified 84.41 j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 420 http://jad.tums.ac.ir published online: september 08, 2017 table 2. antibacterial and antifungal activities of the essential oil of ferulago trifida aerial parts microorganisms essential oil of aerial parts antibiotics rifa genb nysc izd mice iz mic iz mic iz mic s. paratyphi-a 20 500 21 500 naf na s. aureus 23 500 10 250 21 500 na na s. epidermidis 20 125 8 250 18 500 na na e. coli 11 500 20 500 na na k. pneumoniae 11 250 7 250 22 250 na na b. subtilis 16 125 13 15 21 500 na na p. vulgaris 11 250 10 125 23 500 na na s. dysenteriae 23 125 40 250 35 500 na na c. albicans na na na na 33 125 a. brasiliensis 12 500 na na na na 23 500 a. niger na na na na 27 31 adash (-) indicates no antimicrobial activity. arifampin (5μg/disc), b gentamicin (10μg/disc), cnystatin (100 i.u. /disc), dinhibition zone in diameter (mm) around the impregnated discs including diameter of the disc (6mm) [weak activity (<10 mm), moderate activity (10–15mm), strong activity (15-20mm), very strong activity (20˂ mm)], e minimal inhibition concentrations as μg/ml, f not applicable. table 3. the results of free radical scavenging, acetylcholine esterase inhibitory and brine shrimp lethality assays of the essential oil of ferulago trifida samples dpph free radical scavenging assay ic50 (µg/ml) ache inhibitory assay ic50 (µg/ml) brine shrimp lethality test ld50 (µg/ml) essential oil 111.2 ± 5.2 21.5 ± 2.2 (mg ml-1) 1.1 ± 0.3 bht 21.2 ± 2.6 na a na podophyllotoxin na na 2.80 ± 0.3 physostigmine 0.80 ± 0.04 na 0.8 ±0.04 na anot applicable. data are presented as the mean ± sem of three independent experiments (p< 0.05) table 4. the results of mtt assay of the essential oil of ferula gotrifida on different cell lines cell lines, ic50(µg/ml) samples mcf7a a-549b ht-29c oil 22.0 25.0 42.55 tamoxifen 3.6 10.7 2.50 ahuman breast adenocarcinoma, b non-small cell line carcinoma, chuman colon adenocarcinoma j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 421 http://jad.tums.ac.ir published online: september 08, 2017 fig. 1. comparison of equation, regression line and lethal concentration (lc50) of essential oil of different parts of ferulago trifida (apiaceae) against larvae of anopheles stephensi discussion the aerial part of f. trifida was collected around ovan lake located in alamut region, qazvin province, iran in july 2014. hydrodistillation of the air-dried of aerial parts of f. trifida, yielded 1.5% (v/w) of the oil. analysis of the volatile oil by gc and gc/ ms resulted in fifty-eight compounds, representing 86.41% of the total oil. the result showed the essential oil of the aerial part was rich in monoterpenes (55.75%) that oxygenated monoterpenes (49.30%) were dominant. the main components were isobornyl acetate (25.73%) and e-verbenol (9.66%) but amount of the z-beta ocimene was 3.33% whereas it is the abundant monoterpene in the oil of some ferulago species (21-24, 6). in f. aucheri, f. mughlae and f. sandrasica essential oils α-pinene were the major compound and in essential oil of f. macroseiadia, f. sylvatica and f. bernardii, methyl carvacrol, p-cymene and 2, 4, 5-trimethyl benzaldehyde were reported as important components (22, 7). in our study, amounts of the α-pinene were only (0.42%), and there were not methyl carvacrol, p-cymene and 2, 4, 5trimethyl benzaldehyde in the essential oil. in f. trifida there was 20.41% sesquiterpenes, which beta-caryophyllene (8.68%), germacrene d (2.18%) and caryophyllene oxide (2.47%) were abundant. ferulago trifida aerial part volatile oil showed strong antimicrobial activity with mic near gentamycin against all bacterial strains except e.coli, and antifungal activity on a. brasiliensis with mic equal nystatin. a. niger and c. albicans had shown resistance to the oil. the essential oil of ferulago genus showed different results depending on the compounds contained in oil (7, 21, 25, 26). in our study, on the ache inhibitory properties, f. trifida oil did not show significant effect versus physostigmine as standard. we did not observe any report about ache inhibitory activity from essential oil of ferulago genus. the antioxidant assessment of oil of the f. trifida has demonstrated it has low level free radical scavenging activity because it's ic50 (111.2µg/ml) was less than bht (ic50= 21.2µg/ml) as standard. such investigation has been done on some species of this genus (27). the brine shrimp lethality test is considered as an inexpensive, simple, rapid and effective method for preliminary assessment of toxicity and as a guide for the detection of cytotoxic, anti-tumor and pesticidal compounds (28). table 3 demonstrates the result of genera50 of l toxicity of the sample. aerial part essential oil of the f. trifida with ld50= 1.1 µg/ml showed more toxicity against podophyllotoxin (ic50= 2.8µg/ml) as standard. the main sesquiterpenes of the essential oils such as β-caryophyllene, germacrene d, caryophyllene oxide etc. may be involved in the toxic effects of the tested essential oil (29). in this investigation, the mcf7 human breast adenocarcinoma, a-549 non-small cell line carcinoma and ht-29 human colon adenocarcinoma cell lines were treated with different concentrations of f. trifida aerial parts essential oil and the cell viability were measured for 24 and 72h as described in the experimental part. the results of these measurements are shown in table 4. essential oil of f. trifida showed interesting toxicity with j arthropod-borne dis, september 2017, 11(3): 414–426 s tavakoli et al.: gas chromatography … 422 http://jad.tums.ac.ir published online: september 08, 2017 ic50≥40µg/ mlon cancerous cell lines, although it was not similar to tamoxifen activity. other review on the literature for the cytotoxic and anticancer effects of various species of ferulago essential oil, (30) has demonstrated that the essential oil of f. carduchorum showed potential cytotoxic selectivity on t47d cell line similar to methotrexate (positive control). the present study showed that the essential oils obtained from f. tifida different parts e.g. stem, root, aerial part, fruit and flower could induce 50% mortality in the larvae of an. stephensi at a very low concentrations (10.46, 22.27, 20.50, 31.93 and 79.87ppm) respectively. the essential oils of some plants such as cymbopogon nardus, c. flexuosus, c. martini, lavandula officinalis, menthaarvensis, racinus communis, eucalytus globules, eugenia caryophyllus, ocimum basilicum, melia azardirachta, cannabis sativa demonstrated lc50 values of 105.4, 91.4, 100.0, 83.6, 83.8, 113.0, 98.5, 96.5, 80.0, 88.5, and 27.0ppm respectively, against the larvae of the an. stephensi (31-34). there are several studies on larvicidal activities of different plants against malaria vectors in iran (35-59, 30). therefore, the f. tifida aerial parts essential oil has strong activity against an. stephensi. conclusion the essential oil of f. trifida aerial parts, collected from qazvin province of iran, has demonstrated some biological activities including antibacterial effects on gram positive, gram-negative and 1 fungi, high general toxicity on the brine shrimp lethality test, cytotoxic effects on three cancerous cell lines, and has remarkable larvicidal properties on fourth instar larvae of an. stephensi. therefore, it is worthwhile to study on the larvicidal properties of its essential oil by isolating and identifying the active components that cause larval mortality, and their field trials. acknowledgements this research has been supported by tehran university of medical sciences and health services grant (grant no: 94-02-33-29402). the authors declare that there is no conflict of interest. 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https://www.ncbi.nlm.nih.gov/pubmed/?term=tavassoli%20m%5bauthor%5d&cauthor=true&cauthor_uid=25183087 https://www.ncbi.nlm.nih.gov/pubmed/?term=shayeghi%20m%5bauthor%5d&cauthor=true&cauthor_uid=25183087 4. iran j arthropod-borne dis, 2011, 5(1): 20–27 a sanei dehkordi et al.: molecular detection of … 20 original article molecular detection of leishmania infantum in naturally infected phlebotomus perfiliewi transcaucasicus in bilesavar district, northwestern iran a sanei dehkordi1, *y rassi1, ma oshaghi 1, mr abai1, s rafizadeh2, mr yaghoobiershadi1, m mohebali 3, z zarei1, f mohtarami1, b jafarzadeh4, a ranjbarkhah4, e javadian1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran 2emergency management center (emc) ministry of health and medical education, iran 3department of parasitology and mycology, school of public health, tehran university of medical sciences, iran 4health center of bilesavar, ardebil province, iran (received 16 aug 2010; accepted 19 feb 2011) abstract background: visceral leishmaniasis is caused by leishmania infantum, transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in iran. to identify the vector(s), an investigation was carried out in bilesavar district, one of the important foci of the disease in ardebil province in northwestern iran, during july−september 2008. methods: using sticky papers, 2,110 sand flies were collected from indoors (bedroom, guestroom, toilet and stable) and outdoors (wall cracks, crevices and animal burrows) and identified morphologically. species-specific amplification of promastigotes revealed specific pcr products of l. infantum dna. results: six sand fly species were found in the district, including: phlebotomus perfiliewi transcaucasicus, p. papatasi, p. tobbi, p. sergenti, sergentomyia dentata and s. sintoni. phlebotomus perfiliewi transcaucasicus was the dominant species of the genus phlebotomus (62.8%). of 270 female dissected p. perfiliewi transcuacasicus, 4 (1.5%) were found naturally infected with promastigotes. conclusion: based on natural infections of p. perfiliewi transcaucasicus with l. infantum and the fact that it was the only species found infected with l. infantum, it seems, this sand fly could be the principal vector of visceral leishmaniasis in the region. keywords: leishmania infantum, phlebotomus perfiliewi transcuacasicus, nested pcr, iran introduction leishmaniases are parasitic diseases of multifaceted clinical manifestations caused by infections with species of leishmania. these diseases are widespread in the old and new worlds with great epidemiological diversity. approximately, 700 species of sand flies are known but only 10% of these serve as disease vectors. further, only about 30 species are important from a public health standpoint (who 1990, desjeux 2000, sharma 2008). visceral leishmaniasis (vl), commonly caused by leishmania infantum in the mediterranean region, the middle east and latin america, affects approximately half a million new patients each year (lachaud 2002). in the mediterranean basin, domestic dogs (canis fa*corresponding author: dr yavar rassi, e-mail: rassiy@sina.tums.ac.ir iran j arthropod-borne dis, 2011, 5(1): 20–27 a sanei dehkordi et al.: molecular detection of … 21 miliaris) are the principle reservoir host and some species of sand-flies belonging to the subgenus larroussius are the primary vectors (oshaghi et al. 2009a). although vl occurs sporadically throughout iran, the disease is endemic in several parts of northwestern iran (nadim et al. 1978, davies et al.1999, mohebali et al. 1999, rassi et al. 2004, 2005, 2009, oshaghi et al. 2009b). the rate of infected sand flies in endemic areas and the identification of the infecting leishmania parasites in the determined phlebotomine species are of prime importance in vectorial and epidemiological studies of leishmaniasis (rodriguz et al. 1994). three sand fly species, phlebotomus perfiliewi transcaucasicus, p. (larroussius) kandelakii shchurenkova and p. (larroussius) major annandale are proven vectors in iran (rassi 2004, 2005, azizi et al. 2008). two other species, p. (larroussius) keshishiani shchurenkova and p. (paraphlebotomus) alexandri sinton have been found naturally infected with promastigotes and are suspected vectors of vl in the country (seyyedi-rashti et al. 1963, schönian et al. 2003, azizi et al. 2006). leishmania parasites are directly detected by microscopic examination and all leishmania species are very similar and their species identification is not possible morphologically (schönian et al. 2003, oshagi et al. 2009a) therefore we used nested pcr and pcrrflp methods in this study, because the main advantages of these methods are their sensitivity and specificity, independently of the number, stage and localization of the parasite in the digestive tract of the vector (perez et al. 1994). this study was carried out during jul-sep 2008 in rural areas of bilesavar district, ardebil province, in northwestern iran, to detect and identify leishmania infection in sand flies. materials and methods study area the study was carried out in three villages of gunpapagh, odlu and nazaralibalaghi, bilesavar district, in northwestern iran at an altitude of 1311 m (fig.1). the total population of the bilesavar was about 55000 in 2008. the climate is very hot (up to 40°c) in the summer and quite cold (-27° c) during the winter. the summers are short, lasting from mid may to mid september. the main activities of the people are agriculture and animal husbandry. sand fly collection sand flies were collected biweekly from indoors (bedroom, guestroom, toilet and stable) and outdoors (wall cracks and crevices and animal burrows) using sticky papers (100 papers per village, 50 papers in outdoors and 50 papers indoors) during july−september 2008. collected sand flies were removed from sticky papers using needles or fine brushes, dipped in 70% ethanol, were stored in 96% ethanol, and kept in -20 ºc before dissection. sandfly identification the sandfly specimens were washed in 1% detergent then twice in sterile distilled water. each specimen was then dissected in fresh drop of sterile normal saline by cutting off the head and abdominal terminalia with sterilized forceps and single used mounted needles. the remainder of the body was stored in the sterile eppendorf microtubes for dna extraction. specimens were mounted in puri’s medium and identified using the identification keys of theodor and mesghali (1964) and lewis (1982). dna extraction dna was extracted by using the bioneer genomic dna extraction kit. extraction was carried out by grinding of individual sand flies in a microtube using glass pestle and followed by kit protocol and stored at 4°c. detection and identification of leishmania species initial screening of sand flies was performed by nested-pcr amplification of kinetoplast dna (kdna) using the protocol and primers (table 1) already explained by noyes iran j arthropod-borne dis, 2011, 5(1): 20–27 a sanei dehkordi et al.: molecular detection of … 22 et al. (1996). amplification was carried out in two steps, both in the same tube. this pcr is able to identify promastigote infection of sand flies by producing a 680 bp for l. infantum/l. donovani, 560 bp for l. major, and a 750 bp for l. tropica. further identification of the leishmania parasites was done by using the its1-pcr (el tai et al., 2000) followed by haeiii digestion of the resulting amplicons described by schonian et al. (2003). the set of primers (table 1) forward litsr and reverse l5.8s was used to amplify 340 bp of rdna including parts of 3’ end of 18s rdna gene, complete its1, and part of 5’ end of 5.8s rdna gene. all of the amplification reactions were analyzed by 1–1.5% agarose gel electrophoresis, followed by ethidium bromide staining and visualization under uv light. standard dna fragments (100 bp ladder, fermentas) were used to permit sizing. pcr products (15 µl) were digested with haeiii without prior purification using conditions recommended by the supplier (cinagen, tehran, iran). the restriction fragments were subjected to electrophoresis in 2% agarose and visualized under ultraviolet light after staining for 15 min in ethidium bromide (0.5 µg/ml). haeiii digestion of its1 pcr reveals two fragments of 220 and 140 bp for l. major, the fragments of 200, 80 and 60 bp for l. donovani complex, and two fragments of 200 and a 60 bp for l. tropica. results altogether, 2,110 sand flies were collected and identified including p. perfiliewi transcaucasicus (62.8%) , p. papatasi scopoli (19.1%), p. tobbi adler and theodor (3.9%), p. sergenti (10%), s. dentata sinton and (1.8%), s. sintoni pringle (2.4%). among the collected specimens 433 females belonging to six species were screened for leishmania infections (table 2). only 4 of 270 p. perfiliewi transcaucasus (1.5%) were observed to be naturally infected with l. infantum using nested pcr against minicircle kdna molecules with 680 bp (fig. 2). furthermore its1 amplification by pcr primers followed by pcr-rflp technique confirmed the l infantum dna in two infected p. perfiliewi transcaucasus with 340 bp (fig.3). its1-pcr products were digested by haeiii, for the leishmania characterization. since the length of pcr products for different species is different, for example, it is 360 bp for l. major and 340 bp for l. infantum, therefore the rflp pattern is polymorphic for each species. the fragments of 220 and 140 bp for l. major, and the fragments of 200, 80 and 60 bp for l. infantum and two fragments of 200 and 60 bp were observed for l. tropica were diagnosed (fig. 4). this is the first report of naturally infected of p. perfiliewi transcaucasus to l. infantum in bilesavar district, northwestern iran. table 1. primers used in this study kdna first step csb2xf(forward): 5´-cgagta gcagaaactcccgttca-3´ csb1xr(reverse): 5´-atttttcgcgattttcgcagaacg-3´ second step 13z(forward): 5´ (actgggggttggtgtaaaatag-3´ lir(reverse): 5´-tcgcagaacgcccct-3´ its1 litsr(forward): 5´-ctggatcattttccgatg-3´ l5.8s (reverse): 5´-tgataccacttatcgcactt-3’ iran j arthropod-borne dis, 2011, 5(1): 20–27 a sanei dehkordi et al.: molecular detection of … 23 table 2. fauna and pcr results of collected sand flies in bilesavar district, 2008 leishmania species no of infected female male species its kdna (%) no (%) no l. infantum 2 4 62.35 270 62.91 1055 p. perfiliewi trancaucasicus p. papatasi 356 21.22 47 10.85 0 0 ــــــــــــــــ p. sergenti 179 10.67 33 7.62 0 0 ــــــــــــــــ p. tobbi 45 2.69 35 8.09 0 0 ــــــــــــــــ s. sintoni 17 1.01 34 7.85 0 0 ــــــــــــــــ s. dentate 25 1.5 14 3.24 0 0 ــــــــــــــــ 2 4 100 433 100 1677 total fig. 1. map of the study area located in ardebil province fig. 2. kdna pcr amplification of leishmania stocks and l. infantum in p. perfiliewi transcaucasicus using nestedpcr. lane n, negative control, lanes1, 2, 3 represent l. infantum in p. perfiliewi transcaucasicus (680 bp) and lane 4 l. infantum (680 bp) positive control, m: 100 bp size marker (fermentas) iran j arthropod-borne dis, 2011, 5(1): 20–27 a sanei dehkordi et al.: molecular detection of … 24 fig. 3. electerophoresis results of its1-rcr from leishmania stocks and l. infantum in p. perfiliewi transcaucasicus, lane n, negative control, lanes 1, 2 represent l. infantum in p. perfiliewi transcaucasicus (340 bp), lanes 3, 4 represent l. infantum positive controls (340 bp), m: 100 bp size marker (fermentas) fig. 4. restriction fragment length polymorphism (rflp) patterns obtained from leishmania stocks and l. infantum in p. perfiliewi transcaucasicus, lanes 1, 2 and 4 represent l. major, l. tropica and l. infantum reference stocks respectively, lane 3 is l. infantum in p. perfiliewi transcaucasicus, m: 50 bp size marker (fermentas) discussion control of leishmaniasis depends on ecological and epidemiological information pertaining to the disease such as identification of preferred hosts and detection of natural infections in the vector(s). finding naturally infected wild-caught specimens that are anthropophilic fulfills two essential requirements for incriminating a sand fly vector (killick-kendrick 1990). in endemic areas where more than one leishmania species is present, diagnostic tools are required for the detection of parasites directly in samples and distinguish all relevant leishmania species (schönian et al. 2003). characterization of leishmania species is important, because different species may require special remedial method. on the other hand, such information is also valuable in epidemiologic studies where the distribution of leishmania species in hosts and insect vectors is a urgent item in the controlling programs (el tai et al. 2000, schönian et al. 2003). recently, moiran j arthropod-borne dis, 2011, 5(1): 20–27 a sanei dehkordi et al.: molecular detection of … 25 lecular techniques (pcr) have been employed for vector incrimination of sand flies (oshaghi et al. 2009a). the highly sensitive technique of pcr has been used for detecting leishmania in sand flies in many endemic areas including iran and india (azizi et al. 2006, de bruijn and barker 1992, oshaghi et al. 2009b, mukherjee et al. 1997, rassi et al. 2004, 2005, 2009). in the present study, infection of p. perfiliewi transcaucasicus by l. infantum was confirmed using molecular methods. it needs to mention that p. perfiliewi transcaucasicus was first found naturally infected with l. infantum in germi district (other important focus of vl) adjoining to our study area (with 30 kilometers distance) in northwestern iran (rassi et al. 2009).our study in germi district showed that, 1.1% of dissected p. perfiliewi transcaucasicus sand flies were positive to l. infantum with 36.5% hematophagy preference to human (anthropophilic index) indicating a strong preference for human blood (rassi et al. 2009). the apparent secondary preference of this species for dogs (23.5%), the main domestic reservoir of disease, may indicate that this species also plays an important role in transmission of vl to dogs (rassi et al. 2009). based on high density of p. perfiliewi transcaucasicus, natural infected with leishmania infantum, and high degree of anthropophily, it seems that that p. perfiliewi transcaucasicus could be the principal vector of vl in bilesavar district northwestern iran. phlebotomus kandelakii was the first sand fly incriminated as a vector of vl in meshginshahr city in northwestern iran (rassi et al. 2005). the high prevalence of p. perfiliewi transcaucasus revealed by this study is consistent with the reports of lewis (1982) on the distribution of this species in iran and the republic of azerbaijan, adjoining to our study area. this is the first report incriminating p. perfiliewi transcaucasicus as the main vector of vl due l. infantum in the region. acknowledgements we are grateful to staff of bilesavar health center for providing facilities to conduct this research and cooperation in the field sampling. this study received financial support from the school of public health, tehran university of medical sciences, msph thesis, 2010. the authors declare that there is no conflict of interests. references azizi k, rassi y, javadian e, motazedian mh, asgari q, yaghoobi-ershadi mr (2008) first detection of leishmania infantum in phlebotomus (larroussius) major (diptera: psychodidae) from iran. j med entomol. 45(4): 726–731. azizi k, rassi y, javadian e, motazedian mh, 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major in an endemic focus of v.l. in fars province, south of iran. iran j public health. 21: 87−93. schönian g, nasereddin a, dinse n, schewynoh c, schalling hd, presbe w, jaffe c (2003) pcr diagnosis and characterization of leishmania in local and imported clinical samples. diag microbiol infect. 47(1): 349−358. seyyedi-rashti m, sahabi z, kananionotash a (1995) phlebotomus (larroussius) keshishiani; shchurenkova, another vector of visceral leishmaniasis in iran. iran j public health. 24: 23−30. sharma u, singh s (2008) insect vectors of leishmania: distribution, physiology and their control. j vector borne dis. 45: 255–272. who (1990) control of the leishmaniases. report of a who expert committee. technical report series, 793. j arthropod-borne dis, march 2018, 12(1): 100 h ebrahimzadeh-leylabadlo: malaria eliminating … 100 http://jad.tums.ac.ir published online: march 18, 2018 letter to the editor malaria eliminating: what lies ahead of iran? hamed ebrahimzadeh-leylabadlo infectious and tropical diseases research center, faculty of medicine, tabriz university of medical sciences, tabriz, iran (received 6 june 2017; accepted 24 jan 2018) dear editor-in-chief in recent years, iran has made significant advances on the fight against malaria with a stated objective of complete elimination. according to the report of iranian ministry of health and medical education, the annual number of malaria cases have been reduced from 12294 in 2000 to 787 in 2012, 366 cases in 2014 and only 150 cases reported in 2015. in 2009, who malaria report, announced iran was in the pre-elimination phase (1). malaria incidence had significant reduction during last years, however, iran is facing a number of operational problems in a fight against malaria. around 90% of the locally transmitted malaria cases happen over the southeastern endemic regions in iran every year (2). the imported malaria cases and cross-border movement of population from malaria-endemic countries particularly pakistan and afghanistan that enter to sistan and baluchestan and khorasan-razavi provinces illegally via the eastern iranian borders. the burden of most infectious diseases, including malaria, leishmaniasis, and tuberculosis in afghanistan is more noteworthy than in iran and individuals afghani are most of the migrants in iran (3). on the other hand, global climatic change is expected to increase the incidence of vector-borne diseases, especially malaria. many countries in the world including iran have been hit by climate change and diseases like malaria and leishmaniasis may change pattern and appear in provinces not prevalent before (4). thus, despite the reduction of malaria cases in recent years, the risk of reintroduction is high and need to be continued cross-border collaborations with endemic neighbors to pursuing the goal of completely malaria elimination by 2020. acknowledgements i would like to thank the authorities of infectious and tropical diseases research centre, tabriz university of medical sciences, tabriz, iran for their support. in addition, i am extremely grateful to dr teimour hazratian in department of parasitology and mycology, faculty of medicine, tabriz university of medical sciences, tabriz, iran, for his helpful comments on manuscript. references 1. ebrahimzadeh-leylabadlo h, samadi-kafil h, asgharzadeh m (2016) malaria in iran: is the elimination phase? ann trop med public health. 10(4): 1062. 2. mckelvie wr, haghdoost aa, raeisi a (2012) defining and detecting malaria epidemics in south-east iran. malar j. 11: 81. 3. pourhossein b, irani ad, mostafavi e (2015) major infectious diseases affecting the afghan immigrant population of iran: a systematic review and meta-analysis. epidemiol health. 37: e2015002. 4. khanjani n (2016) the effects of climate change on human health in iran. int j public health. 3: 38–41. *corresponding author: dr hamed ebrahimzadehleylabadlo, e-mail: hamedebr7@gmail.com http://jad.tums.ac.ir/ microsoft word 6-arthropod dr abdigoudarzi rtl.doc iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 36 original article laboratory study on biological control of ticks (acari: ixodidae) by entomopathogenic indigenous fungi (beauveria bassiana) * m abdigoudarzi 1, k esmaeilnia 2, n shariat 3 1reference laboratory for ticks and tick borne diseases, department of parasitology, razi vaccine and serum research institute, karadj, iran 2department of protozoalogy, razi vaccine and serum research institute, karadj, iran 3department of microbiology, razi vaccine and serum research institute, karadj, iran (received 12 aug 2009; accepted 16 dec 2009) abstract background: chemical control method using different acaricides as spray, dipping solution or pour-on is routinely used for controlling ticks. biological control agents are favorable due to their safety for animals and environment. entomopathogenic fungi such as beauveria bassiana are well known for controlling ticks. in this study, two iranian indigenous strains of b. bassiana (b. bassiana 5197 and b. bassiana evin) were selected and grown on specific media. the pathogenic effects of these strains were evaluated on adult stages of two iranian ixodidae members (h. anatolicum anatolicum koch 1844, and h. marginatum koch 1844) by dipping method. methods: two iranian strains of beauveria bassiana (beauveria bassiana 5197 and beauveria bassiana evin) were selected and were grown successfully on specific media. the pathogenic effects of these strains were evaluated on adult stages of iranian ixodidae members such as, hyalomma anatolicum anatolicum and h. marginatum by dipping method (these ticks were grown up at laboratory conditions during 2002 up to 2003 and still it is continued) . results: there was no effect of strain 5197 on mortality or fecundity rates for ticks. there was acute phase sign of paralysis in test group after dipping ticks in suspension made from evin strain of b. bassiana. in addition, the test groups were totally died after four months, but the control groups survived for six months. conclusion: high concentration of fungal spores is needed for inducing fungal infection. additional study using different strains and fungi on iranian ticks is proposed. keywords: biological control, fungi, beauveria bassiana, ticks, ixodidae, iran introduction hard ticks (acari: ixodidae) are important for transmission of different viral, bacterial and protozoan agents to human and animals. at present, different chemical products are going to be used for control of ticks. these chemicals are expensive; some of them are not friendly used for the environment, and may be considered as persistent organic pollutants. samish and rehaacek (1999) have summarized literature about pathogens and predators of ticks and their potential use as biocontrol agents published since the beginning of the 20th century. in nature, many bacteria, fungi, spiders, ants, beetles, rodents, birds, and other living things contribute significantly toward limiting tick populations. experiments with the most promising potential tick biocontrol agents, especially fungi of the genera beauveria vuill. 1912 and metarhizium sorokin are described. frolov (1974) reported that three biological products were available in the former soviet union controlling ectoparasites on that time *corresponding author: mohammad abdigoudarzi, email: sepehr2001ir@yahoo.com iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 37 (frolov 1974). two of them (entobakterin and dendrobacillin were preparations of spores and endotoxin of bacillus thuringiensis berliner 1915 and the third one (boverin) was conidiospores of the fungus beauveria bassiana (bals.-criv.) vuill. bittencourt et al. (1997) evaluated the in vitro efficacy of two isolates of the entomopathogenic fungus b. bassiana in engorged females of boophilus microplus (canestrini, 1887) (acari: ixodidae) (bittencourt et al. 1997). the pathogenicity of two isolates of b. bassiana (747 and 986) against engorged females of b. microplus was investigated in vitro. twelve groups of 30 ticks were exposed to conidial concentrations of 0, 104, 105, 106, 107 and 108 for each isolate. the weight of females before oviposition, weight of egg masses and percentage of eclosion were recorded for each group. the percentage of eclosion decreased with increasing conidial concentration and concentration of 107 and 108 were considered minimal for field trials (bittencourt et al. 1997). the efficacy of various entomopathogenic fungi as biological control agents of b. microplus was tested in the laboratory. verticillium lecanii (zimmerman) viegas (lecanicillium lecanii) (approved name) strain lbv2 was the most effective and reduced tick egg viability by 76% followed by v. lecanii strain lbv-1 (68% reduction), beauveria bassiana strain lbbb-14 (31% reduction) and b. bassiana strain lbbb(28% reduction). tick infestations were reduced by up to 93.5% when fungal preparations were repeatedly sprayed onto calves (rijo 1994). the effectiveness of v. lecanii lbv-2 strain was determined against the parasitic stage of b. microplus. the biological product was sprayed on cattle, one set in a yard, and the other one in grazing areas, the latter was compared with cattle treated with a mixture of chlorfenvinphos plus cimiazol. the biopreparation showed that since the 1st treatment was applied to the stabled animals, the effectiveness varied between 47.5% and 78.7%. after the 4th bath with the biopreparation the regulation of the ectoparasite was between 93.5 and 98.7%, after which further treatment was unnecessary. the effectiveness of the biological product sprayed on animals in grazing areas did not significantly differ from that obtained by spraying chemical acaricides (camacho 1998). the pathogenic action of metarhizium anisopliae (metchnikoff) sorokin on engorged females was clearly demonstrated. isolates e9 and am were more effective, causing high tick mortality as well as reduced oviposition. the concentration of 7.5×108 conidia/ml was the most effective with the fungus sporulating on 91.1% of the ticks. mean percent oviposition was highest in the control treatment and lowest in the treatment with 7.5× 108 conidia/ml (monteoiro 1998). metarhizium anisopliae isolates were passaged through female tick and grown up on rice. fifteen bulls were infested with b. microplus at 7, 14, and 21 days prior to the application of increasing concentrations of m. anisopliae conidia. female ticks larger than 4.0 mm were counted at 0, 1, 7, and 16 days after treatment application. there was no effect of m. anisopliae on numbers of ticks parasitizing the bulls; however, 91.7% of ticks had fungal growth and sporulation 1 day after fungal treatment (correia et al. 1998). lopez et al. showed that strain ma-z4 of m. anisopliae and strain bb-1 of beauveria bassiana reduced eclusion of larvae of b. microplus when applied to animals affected by the tick, s initial stages thus showing promise as biological control agents (lopez et al. 1998). two strains of b. bassiana were found to be pathogenic to all stages of rhipicephalus appendiculatus neumann, 1901 in the laboratory. a mortality of up to 73% of unfed adults was recorded. m. anisopliae was only slightly pathogenic, killing only 35% of unfed adults. unfed ticks immersed in suspensions iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 38 of b. bassiana spores engorged normally on rabbits, but 74% of them failed to lay eggs. the fecundity of those, which laid eggs, was reduced to 10% compared to controls in natural infections (mwangi et al. 1995). in this study, two iranian indigenous strains of b. bassiana (b. bassiana 5197 and b. bassiana evin) were selected and grown on specific media. the pathogenic effects of these strains were evaluated on adult stages of two iranian ixodidae members (h. anatolicum anatolicum koch 1844, and h. marginatum koch 1844) by dipping method. materials and methods tick rearing h. anatolicum anatolicum, individuals (origin, boushehr province) is routinely being reared at the laboratory. female engorged ticks were collected from infested hosts in the field. females were kept at 28 °c and 80% relative humidity. larvae and nymphs were blood fed on white rabbit and adults blood feeding was took place on sheep (twohost strategy of life cycle at laboratory conditions). fungi rearing two indigenous strains of b. bassiana, b. bassiana (ptcc), 5197 obtained from iranian research organization for science and technology (persian type culture collection) and cultured according to the manual from the producer and the second strain was b. bassiana st. evin, (obtained from college of agriculture and natural resources of university of tehran). it was cultured on sdya medium under sterile condition according to producer's manual. there is a summary chart of the type of treated ticks and different conditions of the study in table 1. instant signs of acute phase effects of fungi on ticks, such as tremor or paralytic effects were recorded, and then fecundity of engorged female ticks and hatchability of their eggs or mortality rate for adults were studied up to 1 month after the beginning of the study or later. the ticks were kept in 28 °c and 80% relative humidity in separate vessels. there were enough non-treated cases as control groups whenever a test was applied. results two different strains of b. bassiana were cultured successfully (fig.1 and 2). engorged female ticks were weighed and suspended in different concentrations of spores of b. bassiana after spore counting, using a haemocytometer slide and microscopically studies. spores were suspended in different media according to table 1. different data after treating the ticks could be categorized in four parts. ah. anatolicum anatolicum and h. marginatum unfed adult ticks in 5 groups of 5 ticks (at the same generation and almost equal size, weight and starvation time) (siblings from one female tick) were suspended in different concentrations of spores of b. bassiana st. 5197 for 1 min. they were kept in standard laboratory conditions and inspected every 48 h. there were no differences in mortality rate between tested and none tested groups. there were also no differences in oviposition rate or time and hatchability decrease after engorgement of female ticks. btwo groups of two engorged female h. marginatum ticks were suspended in 1×103 spores/ml of b. bassiana st. 5197 for two min (suspension medium was tween 80 in physiological saline). they were kept in standard laboratory conditions and inspected every 48 hours. there were no differences in mortality rates between tested and none tested groups. there were also no differences in oviposition rate or time and hatchability decrease. ctwo groups of seven unfed adult h. marginatum ticks were suspended in 1×106 iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 39 and 2×106 spores/ml of b. bassiana st. 5197 for one minute, respectively. additional group of 10 engorged nymphs of h. marginatum were suspended in 1×106 spores/ml of b. bassiana st. 5197 for one minute. there were no differences in mortality rate between tested and control groups even after increasing the concentration of spores to 2×106 spores/ml. there were no differences in mortality rate between tested and control groups. danother strain of b. bassiana, (evin strain) was provided from college of agriculture and natural resources of university of tehran. this strain was grown on sdya medium successfully (fig. 3, 4). four groups of five unfed adult ticks (h. dromedarii koch 1844) (lab-reared individuals) (two tested groups and two as control groups) were selected. tested groups were suspended in fungal spores (1,500,000 spores/ml) in pbs and pbs/edta for fifteen min. acute phase signs of toxic effect of fungi such as tremor and paralysis were observed in ticks immediately after the experiment. the ticks were incubated separately from the control group. daily inspection of ticks showed that paralytic effects were extended up to 1 week. after 1 week, this sign was disappeared and there were no other apparent recordable differences in appearance between tested and control groups of ticks. tested ticks were died after four months, but the control groups were active and viable even after six months. table 1. summary chart of tested groups using two strains of beauveria bassiana against hyalomma species results spores΄ conc., (d.time)1, suspen groups tick(host) fungal strain no. record in 48 hr.intervals(-) 200,500,1000/ml,1 min, ph.saline 5 group of 5 h.anatolicum an. (engorged f2) b.b 5197 1 record in 48 hr.intervals(-) 1000 / ml, 2 mins, tween 80 2 group of 2 h.marginatum (engorged f) b.b 5197 2 record in 48 hr.intervals(-) 2000,000/ml, 1min,ph.saline 1 group of 7 h.marginatum (starved adults) b.b 5197 3 record in 48 hr.intervals(-) 1000,000/ml, 1min,ph.saline 1 group of 7 h.marginatum (starved adults) b.b 5197 4 record in 48 hr.intervals(-) 1000,000/ml, 1min,ph.saline 1 group of 10 h.marginatum (engorged nymphs) b.b 5197 5 acute phase effects (+) died after 4 months 1500,000/ml, 15 mins ,pbs 2 groups of 5 h.dromedarii (starved adults) b.b evin 6 acute phase effects (+) died after 4 months 1500,000/ml,pbs/edta,15 mins 2 groups of 5 h.dromedarii (starved adults) b.b evin 7 1-d.time is deeping time for tested ticks in this study. 2-f stands for adult female ticks. iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 40 fig. 1: white colonies of beauveria bassiana (strain 5197) are seen after 15 days were grown successfully on special medium according to producer's manual fig. 2: white colonies of beauveria bassiana (evin strain) are seen after 15 days were grown successfully on sdya medium iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 41 discussion mwangi et al. (1995) found locally isolated strains of b. bassiana to be more virulent to r. appendiculatus than an exotic strain of this fungus. in our study, two indigenous strains of b. bassiana were selected as the strains to be implicated on host ticks. strain type of b. bassiana was changed after doing experiments in parts, a, b, c and evin strain was used in part d. then acute phase effect of fungi on ticks was recorded. the virulence of a specific strain may be lost if an isolate is maintain in in vitro culture (tanada and kaya 1993). the virulence of strain 5197 of b. bassiana in this study might be lost during repetitive passages in its production process, so there was no toxic effect of this strain on ticks. the formulation of the fungus can have an effect on the performance of the fungus as a management agent. kaaya and hassan (2000) found that while both oil based and aqueous formulations were effective in causing mortality, the oil based formulation resulted in greater mortality of ticks. kaaya (2000) reported similar results. the speed of infection and disease development may be enhanced by adding agents that facilitate spore attachment to the cuticle (frazzon et al. 2000). in this study, suspension medium was physiological saline in parts, a, b and c. tween 80 was also used in part b, but there was no effect on the results. suspension medium was pbs and pbs plus edta for two groups of ticks used in part d. there was no difference between the results for these groups. the method of application of fungi (fungal formulation) on host is important. there are suspension (dipping method), inundation, and spray methods used by different scientists. in this study, dipping method was used that is more trustful than inundation method used by cradock (2005). the suspension time for tested ticks regarded as one minute, which is nearly the estimated time when spray or washing is going to be done on animal host. a standard or enough dipping (anti tick bath) is normally inaccessible and expensive in most regions of iran. unofficial sources reported that there exist 3% of anti tick baths are routinely used in one reported province. then animal keepers prefer using spraying, washing methods or simply using a barrel full of diluted toxins and using a piece of sponge soaked in diluted toxins on infested regions of body of animal. then deeping or suspension time (dtime) (table 1) was increased from 1-2 min to 15 min in experiment part d for b. bassiana (evin strain) to be enough to see the pathological effect of the fungi. the concentration of spores was increased after experiments in parts a, b and in part c from 200 spores/ml to 1×103 and later to 1×106 and 2×106 spores/ml. there was no effect of spores' concentration for b. bassiana st. 5197. in another study, cradock, studied different aspects of using b. bassiana and its interactions with dermacentor variabilis (say, 1821) and amblyomma americanum linnaeus, 1758 in the laboratory and in the field. he also reported a significant mortality rate for both species of tested ticks at the laboratory (cradock 2005). the production of mycotoxins and some enzymes may be associated with the pathogenicity of a fungus. since there was no signs of fungal growth on the body of ticks (intersegments of cuticle have been observed microscopically), so the observed mortality after using the second strain of the b. bassiana in this study may be due to releasing of a kind of mycotoxins or enzymes (ferron 1978, mccoy et al. 1988). a darkening of the tick and immobility after about two days indicated infection in larvae and sporulation from the tick surface iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 42 was observed after 72 h (samish et al. 2001). in our study, immobility of adult ticks was seen after one week (part d of this study), that indicated infection and sporulation from the tick surface was not observed. the species of tick used in a study can influence on the specifics of the interaction (benjamin et al. 2002). finally, two indigenous strains of b. bassiana from iran were grown and were applied on three laboratorybred species of iranian ixodidae (hyalomma anatolicum anatolicum, h. marginatum and h. dromedarii). the minimum concentration of spores of b. bassiana needed to induce the infection was estimated to be 1,500,000 spores/ml. additional studies on these two strains of fungi and other biological control agents against ticks should be planned in future to improve the information on biological control of ticks and implementation of these information in integrated control of ticks. acknowledgements the authors wish to thank all colleagues at department of parasitology, razi vaccine and serum research institute. this study was financially supported by the ministry of jehad and agriculture, devoted to confirmed projects at this institute. the authors declare that they have no conflict of interests. references benjamin ma, zhioua e, ostfeld rs (2002) laboratory and field evaluation of the entomopathogenic fungus metarhizium anisopliae (deuteromycetes) for controlling questing adult ixodes scapularis (acari: ixodidae). j med entomol. (39): 723-728. bittencourt v, souza ej, peralva s, mascarenhas a (1997) evaluation of the in vitro efficacy of two isolates of the entomopathogenic fungus beauveria bassiana (bals.) vuill. in engorged females of boophilus microplus (canestrini, 1887) (acari: ixodidae). revista brasileira de parasitologia veterinaria. 6(1): 49-52. camacho er, navaro g, rodriguez rm, marillo ey (1998) effectiveness of verticillium lecanii against the parasitic stage of the tick boophilus microplus (acari: metastigmata: ixodidae). revista colombianadeentomologia 24(1-2): 67-69. correia ad, fiorin a, monteiro ac, verissimo c (1998) effects of metarhizium anisopliae on the tick boophilus microplus (acari: ixodidae) in stabled cattle. j inv pathol. 71(2) 189-191. cradock kr (2005) interactions of beauveria bassiana with the american dog tick, dermacentor variabilis (say), and the lone star tick, amblyomma americanum l. [phd dissertation]. the ohio state university, usa. ferron p (1978) biological control of insect pests by entomopathogenic fungi. annual review of entomology. 23: 409-442. frazzon ap, da silva vaz jr, masuda a, schrank a, vainstein mh (2000) in vitro assessment of metarhizium anisopliae isolates to control the cattle tick boophilus microplus veterinary parasitology. 94: 117-125. frolov ba (1974) chemical and biological methods of controlling poultry ectoparasites. veterinary moscow. 12: 66-68. kaaya gp, hassan s (2000) entomopathogenous fungi as promising biopesticides for tick control. experimental and applied acarology. 24: 913-926. kaaya gp (2000) laboratory and field evaluation of entomopathogenous fungi for tick control. annals of the new york academy of science.916: 559-564. lopez g, marin h, londono m, vahos r (1998) utilization of metarhizium anisopliae and beauveria bassiana for the biological control of the tick boophilus microplus. noticampo. 10: 12-14. iranian j arthropod-borne dis, (2009), 3(2): 36-43 m abdigoudarzi et al: laboratory study on….. 43 mccoy cw, samson ra, boucias dg (1988) entomogenous fungi. in: ignoffo cm (ed.). crc handbook of natural pesticides. microbial insecticides. part a: entomogenous protozoa and fungi vol 5. crc press, boca raton, usa, pp. 151-236. mwangi e, kaaya g, essuman s (1995) experimental infections of the tick rhipicephalus appendiculatus with entomopathogenic fungi, beauveria bassiana and metarhizium anisopliae and natural infections of some ticks with bacteria and fungi. journal of african zoology. 109(2): 151-160. rijo e (1998) biological control of ticks with entomopathogenic fungi. rivista pecuaria de nicaragua. 22: 17-18. samish m, rehaacek j (1999) pathogens and predators of ticks and their potential in biological control. annual review of entomology. 44: 159-182. samish m, gindin g, alekseev e, glazer i (2001) pathogenicity of entomopathogenic fungi to different developmental stages of rhipicephalus sanguineus (acari: ixodidae). j parasitol. (87): 13551359. tanada y, kaya hk (1993) insect pathology. academic press. inc., san diego. 8dr mohebali iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 60 original article neospora caninum and leishmania infantum co-infection in domestic dogs (canis familiaris) in meshkin-shahr district, northwestern iran m sharifdini1, ∗m mohebali1, h keshavarz1, m hosseininejad2, h hajjaran1, b akhoundi1, a rahimi foroushani3, z zarei1, s charehdar1 1department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 2department of veterinary parasitology, faculty of veterinary medicine and research institute of zoonotic diseases, university of shahrekord, shahrekord, iran 3department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 26 oct 2010; accepted 13 jul 2011) abstract background: mediterranean visceral leishmaniasis (mvl) is an infectious disease that affects both human and animals. domestic dogs (canis familiaris) are principal reservoir hosts of mvl caused by leishmania infantum. dogs are definitive hosts for neospora caninum and a risk factor for infecting intermediate hosts. the immunosuppression caused by visceral leishmaniasis (vl) can promote the occurrence of co-infections with other agents such as neosporosis. this study aimed to determine the frequency of co-infection of the both protozoan parasites in the endemic areas of vl from meshkin-shahr district, north-west of iran. methods: altogether, 171 serum samples were collected from domestic dogs of meshkinshahr district by multistage cluster sampling from october 2008 to august 2009. the collected serum samples were tested for the detection of simultaneous infection of l. infantum and n. caninum using direct agglutination test (dat) and indirect elisa, respectively. results: of the 171 domestic dogs, 27 (15.8%) and 52 (30.4%) were showed antibodies against l. infantum and n. caninum, respectively. simultaneous infections of n. caninum and l. infantum was found in 16 (9.4%) of the dogs. in vl-positive and vl-negative dogs, n. caninum infection was found in 59.3% and 25.0%, respectively. a statistically significant difference was found between vl-positive and vl-negative dogs with n. caninum infection (p= 0.001). conclusion: these findings indicate that meshkin-shahr district in northwestern iran is an active focus of canine visceral leishmaniasis (cvl). neospora caninum and l. infantum co-infection is prevalent in the area and infection by l. infantum seems to enhance susceptibility to n. caninum infection in domestic dogs. keywords: neospora caninum, leishmania infantum, co-infection, seroepidemiology, dog, iran introduction canine visceral leishmaniasis (cvl) is an infectious disease that affects both human and canines, and is transmitted by phlebotomine sand flies (alvar et al. 2004). leishmania infantum is the agent of mediterranean visceral leishmaniasis and domestic dogs are efficient reservoir hosts for this infection (who 1990). asymptomatic infected dogs can remain infected without displaying apparent clinical manifestations of visceral leishmaniasis for years and even for their whole life (moshfe et al. 2009). many countries have re-emergence of visceral leishmaniasis (vl) during recent years, in both aspects of geographically *corresponding author: dr mehdi mohebali, e-mail: mohebali37@yahoo.com iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 61 and the number of cases (arias et al. 1996). for a long time, kala-azar seems to be endemic in meshkin-shahr areas from the north west of iran (moshfe et al. 2008). infected dogs are important risk factors for human infection in the endemic areas of iran (gavgani et al. 2002). the prevalence of l. infantum infection in the domestic dogs in meshkin-shahr areas is high and its prevalence has been reported from 14.2% to 17.4% (bokaei et al. 1998, mohebali et al. 2005, moshfe et al. 2008). neospora caninum is a coccidian intracellular parasite from the sarcocystidae family with a wide host range (dubey and lindsay 1996). dogs, coyotes, and dingoes are both the definitive and intermediate hosts for n. caninum that excreted oocysts in their feces and shedding oocysts in the environment is a risk factor for the occurrence of miscarriages and stillbirths associated with n. caninum in cattle and probably other intermediate hosts (dubey 2003, king et al. 2010). the parasite can be transmitted transplacentally in dogs for several generations (dubey and lindsay 1989). neosporosis can cause severe neuromuscular disorders as ascending paralysis with hyperextension of the hind limbs, especially in congenitally infected dogs (dubey 2003). a few studies on n. caninum infection in dogs have been carried out in iran (haddadzadeh et al. 2007, malmasi et al. 2007, hosseininejad et al. 2010a, 2010b, yakhchali et al. 2010). studies on n. caninum and l. infantum co-infection in the world are very rare (andreotti et al. 2006, cringoli et al. 2002). this is the first study in iran, aimed to determine simultaneous infection of n. caninum and l. infantum in domestic dogs from meshkin-shahr district as cvl endemic area in north-west of iran. materials and methods animals a total of 171 domestic dogs of mix breeds from some villages of meshkin-shahr areas including ahmad abad, paikhan, kojanagh, mazraeh khalaf, mirak and ourkandi were selected by multi-stage cluster sampling from october 2008 to august 2009. information on the dogs such as sex and age were taken from the owners and physical examination was performed by a doctor of veterinary medicine (dvm). preparation of the serum samples all blood samples were prepared from the cephalic vein by venapuncture, poured into 10 ml polypropylene tubes, and processed 4– 10 h after collection. the collected samples were centrifuged at 800 g for 5-10 min, and the separated sera were kept at -20° c until tested. direct agglutination test serum samples were tested using direct agglutination test (dat) for the detection of anti-l. infantum antibodies.. dat antigen was prepared at the school of public health, tehran university of medical science (mohebali et al. 2006). the main phases of the process of making dat antigen were mass cultivation of promastigotes of iranian strain of l. infantum (mcan/ir/96/lon49) in rpmi1640 plus 10% fetal bovine serum, tripsinization of the parasites, staining with coomassie brilliant blue and fixing with 1.2% formaldehyde (mohebali et al. 2006). to screen the serum samples, initially dilutions were made 1: 80 and 1: 320 and samples with titers 1: 80 were diluted further to give the end-point dilution of 1: 20480. negative control (antigen only) and known positive control serum samples were included on each plate. the cut off titer was defined as the highest dilution at which agglutination was still visible, as blue dot, compared with negative control wells, which had clear blue dots (mohebali et al. 2005). the positive control serum was produced from dogs with l. infantum infection (at 1: 20480) and confirmed by microscopy, culture and animal inoculation (harith et al. iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 62 1989). based on previously studies in iran, anti-leishmania antibodies at a titer of 1: 320 and higher were considered as positive in this investigation (mohebali et al. 2006). indirect elisa for the detection of n. caninum infection, sera were tested with an indirect elisa using an affinity purified surface antigen (p38). the performance of indirect elisa was based on the method of hosseininejad et al. (hosseininejad et al. 2010a). optical density (od) values were measured at 450nm on an elisa reader (labsystems multiskan ® plus). an optimal serum dilution of 1:100 was established by checkerboard titration for the purpose of the test. index values were calculated by the formula; sin= (sn−n)/(p−n) where sin is the individual elisa index value, sn is the od value obtained for the mean of tested samples, n is the od value obtained for the negative serum, and p represents the od values obtained for the positive serum. antineospora antibodies at od≥ 0.23 by elisa were considered as neospora sp. positive results (hosseininejad et al. 2010 a). parasitological study to confirm visceral leishmaniasis in dogs, necropsy was performed in some of high seropositive dogs with specific clinical signs and symptoms. liver and spleen samples of the infected dogs cultured in specific media such as nnn and rpmi1640 and then a few touch smears were prepared from the liver and spleen of them. all the prepared smears were fixed with methanol, stained with giemsa stain 10% and examined microscopically for the presence of amastigotes. ethical approval this research project was reviewed and approved by the ethics committee of tehran university of medical sciences, iran. statistical analysis statistical test including chi-squared (x2) and macnemar were used to compare seroprevalence values relative to gender, age and clinical signs of n. caninum and l. infantum co-infection in the domestic dogs. statistical analyses were performed using spss software version 13.5 (spss inc, chicago, il, usa), with a probability (p) value of <0.05 as statistically significant. results all of the dogs were of mixed breed and all of them were kept as guard dogs and sheep-dogs. from 171 serum samples that were tested by dat, 27 (15.8%) of them were positive in with dat (≥1:320). the sero prevalence values among male and female animals were 16.4% and 12.0%, respectively (table 1). no statistically significant difference was seen between canine l. infantum infection and the gender (p= 0.574). the highest sero-prevalence rate 9/36 (25.0%) was observed among the domestic dogs of more than five years age and the lowest values 3/52 (5.8%) were seen in dogs under 2 years old (table 2). clinical signs including dermatitis, alopecia, keratitis, cachexia and weakness were seen in 27/171(15.8%) dogs. only 12 (44.4%) from 27 datseropositive dogs (≥1:320) showed clinical signs (table 3). in this survey, 8 out of 12 necropsied dogs were positive for l. infantum infection with dat (≥1:320) and impression smear that prepared from their liver and spleen. only 3 out of 12 necropsied dogs showed positive results in leishmania culture media about 2 weeks post inoculation. altogether, 52 (30.4%) of the dogs showed anti n. caninum antibodies by indirect elisa test using an affinity purified surface antigen (p38). no significant statistically difference was seen between n. caninum infection and both sex and age groups (p> 0.05) (table 1 and 2). none of the infected dogs had clinical signs including neurologic and cutaneous neosporosis disorders. iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 63 the presence of antibodies against n. caninum and to l. infantum was detected in 16 from 171 dogs (9.4%) among six villages of meshkin-shahr, which were selected by multi-stage cluster sampling (fig.1). l. infantum infection seems to enhance susceptibility to n. caninum infection in domestic dogs. [κ2= 10.26, p< 0.01, odds ratio= .43 (95% ci, 0.25–0.75). table 4 and 5 show the distribution of the n. caninum infection in vl-negative dogs and vl-positive dogs, respectively. in datpositive dogs (≥1:320), the prevalence of n. caninum infection was calculated 59.3% and in dat-negative dogs (<1: 320) the prevalence of the infection was 25.0%. a statistically significant difference was found between dat-positive and dat-negative dogs compared with n. caninum infection (p= 0.001). in 58.3% of males and 66.7% of female in the dat-positive dogs, showed anti n. caninum antibodies (od≥ 0.23). no statistically significant difference was found in the both groups (p= 0.781). also, 25.4% of males and 22.7% of female in the datnegative dogs, showed anti n. caninum antibodies but no statistically significant difference was found between in the both groups (p= 0.789). fig. 1. frequency of neospora caninum and leishmania infantum co-infection among the domestic dogs were selected by multi-stage cluster sampling in meshkin-shahr district, northwestern iran table 1. sero-prevalence of canine l. infantum and n. caninum co-infection by gender in meshkin-shahr district, northwestern iran gender n of dogs (%) leishmania positive* neospora positive** n (%) n (%) male 146 (85.4) 24 16.4 45 30.8 female 25 (14.6) 3 12.0 7 28.0 total 171 (100) 27 15.8 52 30.4 * anti-leishmania antibodies detection by dat at titers ≥1:320 **anti-neospora antibodies detection by elisa at od≥0.23 no statistically significant difference between dat positive dogs (≥1:320) and gender was observed (p= 0.574). no statistically significant differenc between n. caninum infection and gender was observed (p= 0.777). l. infantum infection could increase n. caninum infection in the domestic dogs [κ2=10.26, p<0.01, odds ratio=0.43 (95%ci, 0.25–0.75)] iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 64 table 2. sero-prevalence of canine l. infantum and n. caninum co-infections by age group in meshkin-shahr district, northwestern iran age group (years) n of dogs (%) leishmania positive* neospora positive** n (%) n (%) < 2 52 (30.4) 3 5.8 12 23.1 2-5 83 (48.5) 15 18.1 29 34.9 > 5 36 (21.1) 9 25.0 11 30.6 total 171(100) 27 15.8 52 30.4 * anti-leishmania antibodies detection by dat at titers ≥1:320 **anti-neospora antibodies detection by elisa at od≥0.23 statistical difference between dat positive dogs (≥1:320) and different age groups is significant (p= 0.038). no statistically significant difference between n. caninum infection and different age groups was observed (p= 0.345). table 3. distribution of symptomatic and asymptomatic dogs by sero-prevalence rate of l. infantum infection in meshkin-shahr district, northwestern iran dat test (≥1:320) n of dogs (%) symptomatic asymptomatic _______________________________ n % n % positive 27 (15.8) 12 44.4 15 55.6 negative 144 (84.2) 15 10.4 129 89.6 total 171 (100) 27 15.8 144 84.2 statistical difference between dat positive (≥1:320) symptomatic and asymptomatic dogs was significant (p=0.001). table 4. distribution of n. caninum infection by gender and age in dat dogs from meshkin-shahr district, northwestern iran variables n. caninum n. caninum total positive negative n % n % sex males 31 25.4 91 74.6 122 females 5 22.7 17 77.3 22 total 36 25 108 75 144 age < 2 10 20.4 39 79.6 49 2-5 21 30.9 47 69.1 68 > 5 5 18.5 22 81.5 27 total 36 25 108 75 144 in dat negative dogs, no statistically significant difference was found between n. caninum infection with gender (p= 0.789) and age groups (p= 0.299). iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 65 table 5. distribution of n. caninum infection by gender and age in dat+ dogs from meshkin-shahr district, northwestern iran variables n. caninum n. caninum total positive negative n % n % sex males 14 58.3 10 41.7 24 females 2 66.7 1 33.3 3 total 16 60 11 40 27 age < 2 2 66.7 1 33.3 3 2-5 8 53.3 7 46.7 15 > 5 6 66.7 3 33.3 9 total 16 60 11 40 27 in dat positive dogs, no statistically significant difference was found between n. caninum infection with gender (p= 0.781) and age groups (p= 0.782). discussion for diagnosing canine leishmaniasis, dat is considered a sensitive and applicable technique and is well correlated with clinical signs (moshfe et al. 2008). according to previous studies (harith et al. 1989, edrissian et al. 1996, mohebali et al. 2004) the performance of the dat for recognizing of vl in dogs was desirable. hence, we utilized dat for the determination of sero-prevalence of canine l. infantum infection in this investigation. based on the present study, sero-prevalence of canine leishmania infection in the domestic dogs of meshkin-shahr district was determined 15.8% using dat with cutoff value of 1:320. based on three studies that designed for sero-prevalence of canine leishmaniasis in northwest of iran, sero-positivity rates of the infection were found 21.6%, 18.2% and 17.4%, respectively (gavgani et al. 2002, mohebali et al. 2005, moshfe et al. 2008). in this study, 55.6% of the datpositive dogs did not show any clinical signs. in iran, it found asymptomatic leishmania-infected dogs as well as symptomatic ones, have a potential role in the maintenance of l. infantum infection and probably the establishment of domestic transmission cycle of the parasite in the vl endemic areas (moshfe et al. 2009). the seroprevalence values among male and female animals were 16.4% and 12.0%, respectively no statistically significant difference between canine leishmania infection and gender was observed (p=0.083). similar findings were reported by other iranian authors (bokaei et al. 1998, mohebali et al. 2005, moshfe et al. 2008). our findings indicated that the chance of having l. infantum antibodies increases with age of the dogs is in agreement with previous studies (mohebali et al. 2005, moshfe et al. 2008). statistical analysis revealed greater sero-prevalence in older dogs (5 years and above), indicating that the chance of exposure to the bite of sand flies infected with l. infantum increases when the dogs become older (cardoso et al. 2004). in the current study, the prevalence of anti -n. caninum antibodies was determined 30.4% in dogs of meshkin-shahr district. this sero-prevalence was reported to be 33%, 28% and 27% in iran, respectively (haddadzadeh et al. 2007, malmasi et al. 2007, yakhchali et al. 2010). these higher seroprevalences in dogs can be due to the close iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 66 contact with placenta, materials of aborted fetuses, and the uterine discharge of the intermediate hosts of the parasite as cattle (dijkstra et al. 2002, fernandes et al. 2004). no significant difference was found between n. caninum sero-positivity male dogs and females (p> 0.05). this finding has agreement with the results of other studies (haddadzadeh et al. 2007, malmasi et al. 2007, yakhchali et al. 2010). in the present study, significant difference was not detected among the age groups (p> 0.05) but in other surveys in iran the sero-positivity rates increased with age, suggesting postnatal exposure to n. caninum by means of horizontal transmission (haddadzadeh et al. 2007, yakhchali et al. 2010). for the first time in iran, we found the co-presence of antibodies to n. caninum and to l. infantum in 9.4% of domestic dogs. cringoli et al. (2002) reported that 46 (4.3%) of 1058 dogs had co-presence of antibodies to n. caninum and to l. infantum. based on these findings n. caninum and l. infantum co-infection is prevalent in this area and significant difference was found when seropositive and sero-negative dogs for l. infantum were compared with n. caninum seroprevalence. sero-epidemiological surveys that were done in dogs from the campania region of southern italy indicated that the copresence of antibodies of n. caninum and l. infantum is prevalent in dogs and infection by one protozoan seems to enhance the susceptibility to the other one (cringoli et al. 1996, 2002). andreotti et al. (2006) reported that n. caninum and l. infantum co-infection is common in dogs in campo grande area, brazil but there was no significant difference when vl-positive and vl-negative dogs were compared with n. caninum sero-prevalence and vl does not appear to enhance susceptibility to n. caninum infection. in addition, tarantino et al. (2001) have reported n. caninum and l. infantum simultaneous skin infection in a young dog in italy. in our study l. infantum infection seems to enhance susceptibility to n. caninum infection in domestic dogs (odds ratio=0.43) because canine visceral leishmaniasis may lead to immune system suppression and t-cell mediated immune response to parasite antigens can be damaged. cd21+ and cd4+ lymphocytes decline in the peripheral blood of infected dogs with cvl. immune system suppression caused by cvl can be increased dog’s susceptibility to n. caninum infection (tarantino et al. 2001). in conclusion, n. caninum and l. infantum co-infection in domestic dogs is prevalent in the cvl endemic areas of northwestern iran and the infection by l. infantum seems to enhance susceptibility to n. caninum infection. acknowledgements this investigation was supported financially by a grant (no: 88-01-27-8485) from vicechancellor for research, tehran university of medical sciences and also was supported by national institute of health research (nihr) of islamic republic of iran. the authors thankful to dr tayebali, dr a ganji, mr i moradi, mr e taghdisi from meshkinshahr health center and also from mr m nasiri, mrs s molaei, dr ghr khanbaba and other our colleagues for laboratorial and field activities. the authors declare that they have no conflict of interest. references alvar j, canavate c, molina r, moreno j, nieto j (2004) canine leishmaniasis. adv parasitol. 57: 1–88. andreotti r, oliveira jm, silva ea, oshiro lm, matos mde f (2006) occurrence of neospora caninum in dogs and its correlation with visceral leishmaniasis in the urban area of campo grande, mato grosso do sul, brazil. vet parasitol. 135: 375-379. iran j arthropod-borne dis, 2011, 1(3): 60–68 m sharifdini et al.: neospora caninum and …. 67 arias jr, monteiro ps, zicker f (1996) the re-emergence of visceral leishmaniasis in brazil. emerg infect diseases. 2: 145-146. bokaei s, mobedi i, edrissian ghh, nadim a (1998) 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antibodies to neospora caninum in stray dogs of urmia, iran. parasitol res. 106(6):1455–1458. title: genetic variation of the -tubulin of two babesia caballi strains j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 344 http://jad.tums.ac.ir published online: september 08, 2017 original article genetic variation of the -tubulin gene of babesia caballi strains maría guadalupe montes-cortés 1, *josé luis fernández-garcía 2, miguel ángel habela martínez-estéllez 1 1parasitology and parasitological diseases, veterinary faculty, universidad de extremadura, cáceres, spain 2genetics and animal breeding, veterinary faculty, universidad de extremadura, cáceres, spain (received 23 mar 2015; accepted 10 dec 2016) abstract background: equine piroplasmosis is caused by two haemoprotozoan parasites: babesia caballi and theileria equi. negative economic impact on international trade has been associated to endemic sites. this is the reason why carrier detection requires reliable diagnostic methods. various diagnostic modalities can be used alone or in combination including pcr. however, genetic variation of commonly used genes is still of debate. the aim of this research was to sequence the -tubulin gene of a b. caballi strain from spain and to compare it with known -tubulin sequences. methods: dna was isolated from a cryopreserved strain from spain and acute and chronic carrier horses. firstly, degenerated primer pairs were designed based on genbank sequences of different babesia and theileria species for sequencing. the primers were redesigned to amplify both parasites, simultaneously. finally, a species-specific primer pair for b. caballi was designed and a restriction fragment length polymorphism-pcr (pcr-rflp) assay performed to know the difference of known b. caballi strains. results: we provided new insights of the -tubulin gene and a good molecular coverage of this gene, contributing with a number of useful primers to amplify t. equi and b. caballi. moreover, pcr-rflp assays based on the exon ii of this gene confirmed the causative b. caballi strain in spanish horses. conclusion: we reported useful primer pairs for diagnostic and a new sequence of the -tubulin gene of b. caballi, which will facilitate the development of future assays and the detection of infected horses, preventing thus the spread of this disease worldwide. keywords: babesia caballi, -tubulin gene, equine piroplasmosis, pcr-rflp marker introduction equine piroplasmosis is caused by two different haemoprotozoan parasites: babesia caballi (1) and theileria equi (formerly babesia equi laveran 1901) (2). both species are transmitted to horses by ticks found in tropical and subtropical regions (3). these parasites cause disease characterized by fever, anaemia, jaundice, weakness, loss of weight, blood urine, oedema, lymphadenopathy and hepatomegaly. moreover, after infection, horses may remain life-long carriers when they are infected of t. equi, whereas horses may remain carriers of b. caballi from 1 to 4yr (4). in spite of this difference, both parasitaemia have a serious negative impact on international trade (5), with southern europe particularly affected as it is an endemic zone of this disease (5-6). detection of chronic carriers of equine piroplasmosis represents a problem for the equine industry, due to the limitations of the available diagnostic methods. therefore, various diagnostic modalities can be used alone or in combination to diagnose infections (7). these methods are indirect methods such as elisa and the indirect fluorescent antibody test (ifat) or direct diagnosis such as microscopic examination of blood smears or pcr (8-7, 9). *corresponding author: dr jl fernández-garcía, e-mail: mahabela@unex.es http://jad.tums.ac.ir/ mailto:mahabela@unex.es j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 345 http://jad.tums.ac.ir published online: september 08, 2017 classical single pcr (6), multiplex pcr (10-11), nested pcr (12, 13-14) or real-time pcr (15) are used to diagnose equine piroplasmosis. oligonucleotides used in pcr were designed based on genes such us 18s rrna (6-13-1015) or equi merozoite antigen gene -ema1(12-16) to amplify t. equi genes. on the other hand, to diagnose b. caballi, genes such as 18s rrna (6-10) or the rhoptryassociated protein gene -rap1(17) are commonly used. the -tubulin gene is one of the genes used to diagnose piroplasmids (18), although its use is not widespread in the detection of b. caballi because little is yet known about the genetic variability of this gene in this species, the proof of this is that there is only genetic information from one sequence in data bases (acc. nº: aj289246) the prevalence of b. caballi in horses in our country ranges from 7.9% (garcía-bocanegra et al. 2013) to 21.3% (20). whereby, the detection of chronic carriers is a prime goal to prevent the spread of this disease, which can be achieved by increasing the knowledge about genetic variation of the -tubulin gene with differential diagnostic purposes. the aim of this research was to sequence the -tubulin gene from one isolate of b. caballi from spain by developing primers using tubulin genes deposited in gene bank from different haemoprotozoan species, to compare it with one known sequence of this gene from b. caballi and to provide markers suitable for diagnosis and molecular epidemiology. materials and methods blood samples all the blood samples used in this research were collected from the jugular vein into sterile vacuum tubes (vacutainer®) with and without anticoagulant (edta) and routinely thin blood smears were stained and were examined under a microscope at 1500x magnification. babesia caballi was detected in the blood sample from málaga (andalusia, spain) which was sent to our laboratories in cáceres in 2006. this parasite was cultured in equine erythrocytes (21) and cryopreserved in liquid nitrogen, which still belongs to our sample bank (named gm malaga strain). this isolated was used to obtain template dna for sequencing the -tubulin gene. moreover, blood samples of acute infected and chronic carrier horses were collected from different regions of centre and southwest spain in 2011 as follows: five samples from acute infected horses (cáceres province) and samples from chronic carriers from segovia (n=5), cádiz (n=7), badajoz (n=6) and cáceres (n=2) (fig. 1). all the acute cases were positive by microscopic examination but chronic carriers were negative to this procedure. these last blood samples were checked by ifat (22) and by commercial celisa test (kits from vmrd inc. pullman, wa, usa), being all of them b. caballi positive to both techniques (fig. 1). dna extraction whole genomic dna was obtained from the cryopreserved strain, five samples from acute infected horses and 20 blood samples collected from chronic carriers, tested by celisa to assess them as carriers of b. caballi parasite. forty l or 250l of culture fluid or red cell, respectively, were processed by a proteinase k/salting out procedure (2324) modified using zymo-spin iic columns (zymo research corporation, irvine, ca 92614, usa) to purify nucleic acids. genomic dna was semi quantitatively analysed using lambda dna marker (λ dnahindiii digest, new england biolabsinc.) by visualization in 0.8% agarose gel electrophoresis stained with ethidium bromide following manufacturer recommendations. primer design and pcr amplification several available complete or partial sequences of the -tubulin gene were downhttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 346 http://jad.tums.ac.ir published online: september 08, 2017 loaded from the gene bank (table 1). these sequences, aligned using mega4 software (25), aimed at finding a conserved segment where universal pcr primers could be designed to amplify the longest fragment of this gene by conventional pcr. all aligned stretches which expanded from nucleotide 845478 (ie, -2 nucleotide from start codon) to nucleotide 846287 regarding sequence cp001669 were selected for primers design. because completely conserved segments were not found at both endpoints, useful primers, which included degenerate nucleotides, were obtained from this alignment as follows: b-tub f and reverse b-tub r (table 2). the fragment of -tubulin gene of b. caballi was obtained by pcr based on these degenerate primers (which included nucleotides ambiguities, table 2). in the first step, dna from in vitro cultured b. caballi strain was used. duplicated pcr were carried out in 50l reaction volumes with 5l of dna template and 45l mixture containing final concentration of 1x pcr buffer, 0.2mm dntps, 1pmol each primer (b-tub r and f or b-tub r2 and f2 depending on the desired reaction), 1.5mgcl2 and 1 unit of taq dna polymerase (ecogen®) on a thermal cycler 2720 (life technologies®). to maximize amplification success with these degenerate primers a modified-touchdown program was used as follows: (1) each of the first four cycles were run at different annealing temperatures ranging from 55 ºc to 51 ºc and (2) the remaining 37 cycles at 52 ºc. the general amplification conditions were: 96 ºc for 5min, then each cycle at 96º for 30s, primer annealing (see (1) above) for 30s and 72 ºc for 1min. extension, finally 72 ºc for 7min. five l of products for both pcr and, later, pcr-rflp were always run in 1.5% lm agarose gels (low melting agarose) buffered on 0.5% tbe with power supply limited to 120v for 35–45min according the expected fragment size and stained with ethidium bromide for checking. pcr products obtained from the málaga isolated were further used for sequencing. after sequencing the pcr product and realigning it to those in table 2, a less degenerated primer pair was designed but in such way that it was able to amplify both the dna template from b. caballi and t. equi simultaneously, since both species can be found in infected horses. the new designed primers were as follows: b-tub f2 and b-tub r2 (table 2). the pcr conditions for this assay were: 94 ºc for 5min, 94 ºc 45s, primer annealing for 52 ºc for 1min and 72 ºc for 1min extension with 37 cycles, finally extension 72 ºc for 7min. reaction volumes consisted of 1l of template and 19l mixture containing final concentration of 1x pcr buffer, 0.2mm dntps, 1pmol each primer, 1.5mgcl2 and 1 unit of taq dna polymerase (ecogen®) on a thermal cycler 2720 (life technologies®). dna sequencing and resequencing pcr product from the cultured isolate from málaga was purified with exosap-it (ge healthcare®). sequencing and resequencing was performed to determine the genomic variations by the bigdye terminator v.3.1 cycle sequencing kit in both directions using b-tub f2 and b-tub r2, respectively (26). species-specific primers for babesia caballi three primer pairs were designed to amplify the -tubulin gene of b. caballi from genomic dna of cryopreserved strain. but the primer pair gm b-tub f and gm b-tub r (table 2), which expanded from the start of the first intron to partially the second exon, was designed based on both sequences (this research and acc. n. aj289246) to specifically amplify b. caballi. its specificity was studied using template dna from t. equi, which showed no amplification (data not showed). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 347 http://jad.tums.ac.ir published online: september 08, 2017 restriction fragment length polymorphism analysis (pcr-rflp) to test the b. caballi strain described here a pcr-rflp analysis was used. the pcrrflp assays were designed to discriminate both known sequences using software simulation. using prophet 5.0 software (bbn systems and technologies) a restriction mapping analysis was performed to predict restriction enzymes useful to discriminate between both known strains of b. caballi (the spanish isolated acc. n. kx358867 and the french one with acc. n. aj289246). based on simulation of cleavage sites, two restriction enzymes, haeiii and hinfi, were selected. the cutting prediction for each strain can be seen in table 3. results this research provided a new sequence of the -tubulin gene of b. caballi (gm málaga strain). this dna stretch was sufficiently long for a successful primer pairs design. as a result of comparing the -tubulin gene of the two most important piroplasms species that impair horses, t. equi and b. caballi, a first primer pair (b-tubf and b-tubr, table 2) was designed in order to amplify that gene from a dna template of both parasites. moreover, new and less degenerated primers pair could be designed (b-tubf2 and b-tubr2, table 2) used for conventional pcr applications. the resulted amplicons had a size of 718bp and 826bp for t. equi and b. caballi, respectively (fig. 2). according to (27), the -tubulin gene sequences from each of the two species differed in length (100bp approx.). the sequence of the -tubulin gene of b. caballi obtained with these primers provided more molecular information about the genetic variations of this conserved gene. both sequences (aj289246 and kx358867) were truncated to accommodate to the shortest molecule, i.e., 460bp. thus, this fragment of the -tubulin gene of b. caballi partly covered exon i (nucleotide 1 to 63), but completely covered intron i (nucleotide 64 to 233) and exon ii (nucleotide 234 to 460). sixtyeight polymorphic sites were found between both sequences. the comparison of both sequences using blastn 2.2.20 (28) yielded 81% nucleotides identities and 3% open gaps. focusing on exon ii, they shared 92% identities with 19 (19 of 76 triples) silent mutations (synonymous substitutions), as estimated using the dnasp software version 4.0. this analysis reported an unexpected number of polymorphisms between both strains at the coding region but without changing the primary structure of the protein, as should be in the case of strongly conserved genes such as the -tubulin gene (27). moreover, the first intron showed a minor size variation due to a 2bp deletion in the spanish sequence. all these facts suggest the sequence of -tubulin gene could be variable among and within species of babesia parasites. twenty dna samples from chronic carriers and 5 samples from acute infected horses were amplified using our speciesspecific primers for b. caballi. all samples were positive. these pcr were digested with both restriction enzymes (haeiii and hinf i) to determine the strain causing the infection. hinfi cut the pcr amplicons but haeiii does not cut them as was predicted. as a result, this assay was able to confirm that the infecting strain of these horses would be the spanish strain (fig. 3). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 348 http://jad.tums.ac.ir published online: september 08, 2017 table 1. sequences used to design primers accession number species gene chromosome nucleotide positions or size author cp001669 babesia equi complete genome 1 845478…846856 kappmeyer et al. 2012 aagk01000002 theileria parva complete genome 2 923513…925358 gardner et al. 2005. aj289249 babesia equi partial -349bp cacciò et al. 2000 xm-001611566 babesia bovis complete cds -1343bp brayton et al. 2007 kc465972 babesia odoicoilei partial cds -1275bp zamoto-niikura et al. 2014 ab083377 babesia microti complete cds -1651bp zamoto et al. 2004 ab860327 babesia ovata partial cds -457bp damdinjav et al. unpublished ap011947 theileria orientalis complete genome 2 933579…934685 hayashida et al. 2012 aj289246 babesia caballi partial -460bp cacciò et al. 2000 table 2. oligonucleotide primers used to amplify and sequence parasite -tubulin gene. primer pair forward sequence 5´-3´ (f) reverse sequence 5´-3´ (r) b. caballi amplicon size t. equi amplicon size b-tub gaatgagrgaratcgtwcaca carcttyagngtnckraagcara 826bp not done b-tub 2 gaatgagggaratcgtwcaca cagctttagrgttckgaagcarat 826bp 718 gm b-tub acccggtaagtcgttaaacc agttgtcrggyctgaagagt 345bp no amplification table 3. restriction mapping analysis for predicting restriction enzymes. (*excluding cuts within primers) b. caballi strain size of pcr product predicted fragment sizes haeiii (ggcc) hinfi (gantc) cacciò et al. (2000) 347 249,88 * 299,48 this research 345 uncut * 297,48 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 349 http://jad.tums.ac.ir published online: september 08, 2017 fig. 1. geographical distribution of collected samples fig. 2. amplicons of theileria equi and babesia caballi using primers pairs b-tub 2. lane m, 100bp size marker. lane 1: amplicon of t. equi (718bp) lane 2: amplicon of b. caballi (826bp) fig. 3. restriction enzymes assays. lane m, 100bp size marker. lane 1a: pcr product of dna from cultivated isolate amplified with primers pairs gm btub. lane 1b: pcr product digested with haeiii (uncut). lane 1c: pcr product digested with hinfi. lane 2a: pcr product of dna from an acute infected horse with primers pairs gm b-tub. lane 2b: pcr product digested with haeiii (uncut). lane 2c: pcr product digested with hinfi discussion this research has provided new insights into the -tubulin gene of b. caballi, the key factor being that there was a unique and partial sequence available in the gene bank for this piroplasm. this sequence belonged to a different b. caballi strain after comparison with the sequence from (27). moreover, it can be suggested that genetic variation should be expected among b. caballi strains even for conserved genes. the occurrence of genetic variations in certain conserved genes like the -tubulin gene (described in this research), could indicate not only the existence of different strains of b. caballi, but also suggests the study of possible higher taxonomic levels because in our study the shared identity was within the 92% for coding segments. similar results were recently reported for the β-tubulin gene sequence of b. ovata. this babesia shared 94.8–100% identity within species but the shared sequence identity of b. ovata with other species was 87.0–90.6%, 83.7–86.3%, and 82.7– 85.4% to b. bigemina, b. odocoilei and b. divergens, respectively (29). based on genetic knowledge of 18s rrna gene both for b. caballi and t. equi (3031) there were different genotypes in spain for both species. on the one hand, (32) conducted phylogenetic analysis based on the same gene to report that there were two b. caballi clades. however, (33) established four b. caballi clades using the bc48 gene. on the other hand, (32) found three t. equi clades. however, (34) announced that there were not three, but four t. equi clades. phylogenetic analysis by (33) using the ema-1 gene divided the sequences into four t. equi clades. (35) using a phylogenetic study based on both the 18s rrna gene and the -tubulin gene have shown t. annae and its synonyms are not theileria parasites instead they are http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 344–353 mg montes-cortés et al.: genetic variation of … 350 http://jad.tums.ac.ir published online: september 08, 2017 babesia species, being the -tubulin decisive to determine that reclassification. this information suggests that taxonomic classification of equine piroplasms is not yet defined completely as reported (3). according to (36) and (37), a new diagnostic test on additional or new antigens is needed because nowadays there is not a gold standard test for detecting equine piroplasmosis. specifically, (37) showed there was lack of concordance between the b. caballi celisa, ifat, and npcr. hence, the importance of this study that contributes to the enrichment of current genetic information of an unknown -tubulin gene of b. caballi. based on the pcr-rflp analysis, it was possible to discriminate different piroplasms species as shown by (27) where cattle piroplasms such as theileria sergenti, t. annulata, b. bigemina, b. bovis and b.a major were efficiently discriminated using rsai restriction enzyme. another study also valued pcrrflp as a specific analysis to confirm species diagnosis as has been shown for b. bigemina with vspi digestion of the small subunit rrna . using bsli and hinfi with pcr amplicons from the 18s ribosomal rna gene, discriminated subspecies of canine piroplasms from other species such as t. equi, t. annae and b. conradae. pcr-rflp assays may not be necessary to discriminate between b. caballi and t. equi species due to the different sizes of their intron i (27 and this study). however, this difference in size was as short as 2bp between both b. caballi strains, which cannot be seen with agarose gels. this is the reason why pcr-rflp assays were selected to discriminate the gm málaga strain from the strain described by (27), so this study shows for the first time that pcr-rflp assays were used in order to discriminate among different strains of b. caballi species assuming predictions based on the sequence deposited in the gene bank. moreover, although pcrrflp compares a limited number of nucleotides, restriction enzyme digestion was in full accordance with the predicted target based on the sequence of the gm málaga strain even when using dna from naturally infected horses. finally, this study has provided several new and useful primer pairs although they should be more deeply assessed for diagnostic purposes (acute infected horses and chronic b. caballi carriers) in future assays. conclusion we reported here that the -tubulin gene of b. caballi shows high genetic variation, which will be useful to facilitate the development of more precise molecular assays for the detection of infected horses. this is equally relevant at local and international levels, helping to prevent the 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http://locate.coventry.ac.uk/primo_library/libweb/action/display.do;jsessionid=7ee5cc4533a8a3ef2c0e02d551a10e7e?frbrversion=4&tabs=detailstab&ct=display&fn=search&doc=tn_sciversesciencedirect_elseviers0304-4017%2810%2900028-2&indx=6&recids=tn_sciversesciencedirect_elseviers0304-4017%2810%2900028-2&recidxs=5&elementid=5&rendermode=poppedout&displaymode=full&frbrversion=4&dscnt=0&vl%281uistartwith0%29=exact&scp.scps=primo_central_multiple_fe&tab=remote&vl%282098484ui0%29=creator&dstmp=1406111488024&vl%28freetext0%29=quan%2c%20melvyn&vid=cov_vu1&mode=advanced 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infect genet evol. 16: 178–185. 34. alanazi ad, said ae, morin-adeline v, alyousif ms, slapeta j (2014) quantitative pcr detection of theileria equi http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/pubmed/?term=sivakumar%20t%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=tattiyapong%20m%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=okubo%20k%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=suganuma%20k%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=hayashida%20k%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=igarashi%20i%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=zakimi%20s%5bauthor%5d&cauthor=true&cauthor_uid=24572609 http://www.ncbi.nlm.nih.gov/pubmed/?term=matsumoto%20k%5bauthor%5d&cauthor=true&cauthor_uid=24572609 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silva mg, suarez ce (2016) assessment of theileria equi and babesia caballi infections in equine populations in egypt by molecular, serological and haematological approaches. parasit vectors. 9: 260. http://jad.tums.ac.ir/ 10dr jamshidi rtl 89 10 1_3_ iran j arthropod-borne dis, 2010, 4(2): 68–71 n maazi et al.: ear mite infestation in … 68 case report ear mite infestation in four imported dogs from thailand; a case report n maazi1, *sh jamshidi1, hr hadadzadeh2 1department of clinical sciences, faculty of veterinary medicine, university of tehran 2department of parasitology, faculty of veterinary medicine, university of tehran (received 10 feb 2010; accepted 20 jul 2010) abstract otodectes cynotis, ear mite or ear canker mite, is the most common cause of otitis external in cats (approximately 50%) and to a lesser extent in dogs, foxes and ferrets. the mite is living on the epidermal surface of auditory canal without burrowing into the tissue and feeding on tissue fluids and debris. in most of the cases they induce hypersensitivity reactions in the host. four puppies; siberian husky, cocker spaniel, terrier and mixed pekignese with different genders and ages were referred to the small animal hospital, veterinary faculty of tehran university, tehran, iran for routine clinical examination just after they were imported from thailand in a timeframe between june to august 2008. clinical examinations showed an excessive dark brown discharge in both ears. no signs of other clinical situations were observed. white moving mites were seen during otoscopy examination, the specimen of ear discharge was sent to parasitology laboratory for precise identification of genus and species. mites were identified as otodectes cynotis and the presence of concurrent yeast and bacterial infection was showed by laboratory examinations. topical amitraz solution in combination with otic antibacterial and antifungal agents were administered as the treatment. since, all the reported cases were imported from thailand, careful clinical examination and quarantine strategies are highly recommended at the borders. keywords: ear mite, otodectes cynotis, otitis external, dogs, iran introduction otodectes cynotis (hering, 1838) or ear mite is a member of psorptidae family and the most common cause of otitis external in cats while it is less common in dogs. this mite infests external ear canal and occasionally adjacent skin of the head and feeds on tissue fluids and epidermal debris. o. cynotis is large (300×400 µm), white and highly contagious (scott et al. 2001, campbell 2005, radlinsky and mason 2005). the hosts of o. cynotis are cats, dogs, foxes, ferrets and rarely humans (gunnarsson et al. 1991, campbell 2005). the life cycle of the mite lasts three weeks but it can survive for several weeks off the host. female lays eggs and sticks them to the epithermal surface. thereafter, eggs will hatch to six-legged larvae and molt into eight legged protonymphs and deutonymphs within two months (harvey et al. 2001, wall and shearer 2001, campbell 2005). the main route of transmission is direct contact with infected host (harvey et al. 2001). mite infestation can occur at any age but are more common in younger dogs. the cat is a common cause of transmission in adult dogs (harvey et al. 2001, wall and shearer 2001). in one study, the prevalence of ear mite in dogs in the united kingdom was reported 29.1% with significant predisposition in dogs with pendulous and semi-erect pinnae (frost 1961). the prevalence of ear mite in feral cats in the united states has been reported to *corresponding author: dr shahram jamshidi, email: shjamshidi@ut.ac.ir iran j arthropod-borne dis, 2010, 4(2): 68–71 n maazi et al.: ear mite infestation in … 69 be 25% to 37% (akucewich et al. 2002, campbell 2005). in another study, o. cynotis was reported as the most common ectoparasite (22%) of dogs in korea (chee et al. 2008). pathogenicity of o. cynotis is induced by mechanical irritants and pruritus of the mite rather than burrowing the skin. although hypersensitivity resulted from the mite saliva may cause pruritus (greene 2006). common clinical signs of otitis external due to ear mite are accumulation of thick, dark brown to black crusty exudates and cerumen in auditory canal and moderate to severe otic pruritus. however, some dogs may have asymptomatic infection (harvey et al. 2001, scott et al. 2001, radlinsky and mason 2005). diagnosis requires visual confirmation of the presence of the ear mite through otoscopic or microscopic examination. first step of treatment is the use of topical ceruminolytics to remove cerumen and tissue debris. then administration of different topical parasiticides such as amitraz, rotenone or selamectin is recommended (six et al. 2000, scott et al. 2001, campbell 2005). thorough cleaning of the environment and pet equipments, treatment of all household pets and whole body therapy is necessary for a complete treatment (radlinsky and mason 2005). this report is of particular importance because it describes while external otitis caused by o. cynotis is a rare situation of domestic dog breeds in iran, imported dogs could be considerd as a major source of contamination. case report clinical examination three months male siberian husky, 3.5 months male cocker spaniel, 4 months female terrier and 3.5 months female mixed pekignese were referred to the small animal hospital, veterinary faculty of tehran university for routine clinical examinations just after they were imported from thailand in a timeframe between june to august 2008. physical examination revealed bilateral excessive dark brown ear discharge (fig. 1). there was no sign of scratching of pinnae in puppies. bilateral otoscopy was carried out in all puppies. mites were easily observed in otoscopic examination using a veterinary speculum as tiny moving white specks. some smears of ear discharge were made for cytologic and microscopic examination by cotton swabs; moreover, specimens were collected for parasitological examination. smears and specimens were sent to clinical pathology and parasitology laboratories respectively. smears were stained with gram and modified wright's staining methods for evaluation of bacteria and yeast in the laboratory of clinical pathology. direct microscopic examination showed more severe infection in cocker spaniel and terrier compared to siberian husky and mixed pekignese puppies. concurrent bacterial (gram positive cocci) and yeast (malassezia pachydematis) infection as the secondary causes of external otitis (august 1988) were detected by cytologic examination in all of them except the mixed pekignese. parasitological examination samples were collected in tubes containing glycerin and alcohol. the found mites were placed on glass slides, then they were covered with cover slip and koh solution was added. identification of the mites was carried out according to the protocol of the identification of external parasites (scott et al. 2001, wall and shearer 2001, campbell 2005) and as following: adult mites were white with terminal anus and four pairs of legs. the sucker-like pulvillus was cup-shaped as opposed to trumpet-shaped in psoroptes. pretarsi had short, unjointed pedicels on the first and second pairs of legs of the female and on all legs of the male. in the adult female, the third and fourth pairs of legs had a pair of terminal whip-like setae and the fourth pair of legs was reduced and did not extend beyond the body margin. the iran j arthropod-borne dis, 2010, 4(2): 68–71 n maazi et al.: ear mite infestation in … 70 genital opening was transverse. the body of the male was only weakly bilobed posteriorly and had copulatory suckers (fig. 2). treatment both ears in all puppies were thoroughly cleaned with mineral oil and ceruminolytic agents. then amitraz solution 2.5% was administered topically every week for a month. a combination of topical antibacterial and antifungal agents (otosporin) was administered daily for one week as well. all puppies were re-evaluated after one month and there was no sign of mite infestation in otoscopic and microscopic examinations. fig. 1. otoscopic view of otitis external due to ear mite. note the small and white mites with dark brown cerumen and debris (black arrow) fig. 2. photomicrographs of o. cynotis. (a) an adult female with rudimentary fourth pair of legs, (b) an adult male with short, unjointed pedicels on all legs, (c) adult female and male discussion o. cynotis is an irritant and highly contagious ear mite with time consuming treatment of its infection. obviously, prevention is more efficient and sometime less costly when compared to treatment. here, we suggest some recommendations in order to achieve more effective prevention and treatment strategies. given the fact that there is no data on the iran j arthropod-borne dis, 2010, 4(2): 68–71 n maazi et al.: ear mite infestation in … 71 prevalence of ear mite in cats and dogs in iran, we first suggest a comprehensive study on this issue in iran. second, we highly recommend a thorough clinical examination, quarantine and treatment policies at country borders and entrance ports, since all the reported cases were imported from abroad. third, it has been shown that breeds with pendulous and semi-erect pinnae have a high predisposition to mite infestation; therefore we suggest a more precise clinical examination upon referring such breeds to clinics. acknowledgements the authors declare that they have no conflicts of interest. references akucewich lh, philman k, clark a, gillespie j, kunkle g, nicklin cf, greiner ec (2002) prevalence of ectoparasites in a population of feral cats from north central florida during the summer. vet parasitol. 109: 129–139. august jr (1988) otitis externa, a disease of multifactorial etiology. vet clin north am small anim pract.18(4): 731–742. campbell kl (2005) other external parasites. in: ettinger sj and feldman ec (eds): textbook of veterinary internal medicine, vol. 1. 6th ed. saunders elsevier, st. louis, missouri, pp. 66–67. chee jh, kwon jk, cho hs, cho ko, lee yj, abd el-aty am, shin ss (2008) a survey of ectoparasite infestations in stray dogs of gwang-ju city, republic of korea. korean j parasitol. 46(1): 23–27. frost cr (1961) canine otocariasis. j small anim pract. 2: 253–256. greene ce (2006) otitis externa. in: greene ce (ed): infectious diseases of the dog and cat. 3rd ed. saunders elsevier, st. louis, missouri, pp. 815–817. gunnarsson e, hersteinsson p, adalsteinsson s (1991) prevalence and geographical distribution of the ear canker mite (otodectes cynotis) among arctic foxes (alopex lagopus) in iceland. j wildl dis. 27(1): 105–109. harvey rg, harari j, delauche aj (2001) ear diseases of the dog and cat. manson publishing ltd, london. radlinsky mg, mason de (2005) diseases of the ear. in: ettinger sj and feldman ec (eds): textbook of veterinary internal medicine, vol. 2. 6th ed. saunders elsevier, st. louis, missouri, pp. 1171–1174. scott dw, miller wh, griffin ce (2001) muller and kirk's small animal dermatology. 6th ed. w.b. saunders, philadelphia. six ph, clemence rg, thomas ca, behan s, boy mg, watson p, benchaoui ha, clements pjm, rowan tg, jernigan ad (2000) efficacy and safety of selamectin against sarcoptes scabiei on dogs and otodectes cynotis on dogs and cats presented as veterinary patients. vet parasitol. 91: 291–309. wall r, shearer d (2001) veterinary ectoparasites; biology, pathology and control. 2nd ed. blackwell science, london. j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 315 http://jad.tums.ac.ir published online: may 27, 2017 original article synthesis and chemometrics of thymol and carvacrol derivatives as larvicides against aedes aegypti viviane barros silva 1, daniele lima travassos 1, angelita nepel 2, andersson barison 2, emmanoel vilaça costa 3, luciana scotti 4, marcus tulius scotti 4, francisco jaime bezerra mendonça-junior 5, roseli la corte dos santos 6, *sócrates cabral de holanda cavalcanti 1 1medicinal chemistry laboratory, pharmacy department, federal university of sergipe, são cristóvão, brazil 2nuclear magnetic resonance laboratory, chemistry department, federal university of paraná, curitiba, brazil 3chemistry department, institute of sciences, federal university of amazonas, manaus, brazil 4biotechnology center, federal university of paraíba, campus i, joão pessoa, brazil 5laboratory of synthesis and delivery of molecules, biological sciences department, state university of paraíba, joão pessoa, paraiba, brazil 6parasitology and tropical entomology laboratory, morphology department, federal university of sergipe, são cristóvão, brazil (received 6 july 2013; accepted 16 july 2016) abstract background: thymol and carvacrol have previously demonstrated larvicidal activity against aedes aegypti (diptera: culicidae). in view of this fact, it was of our interest to obtain synthetic derivatives and evaluate their larvicidal activity on ae. aegypti larvae. methods: structural modifications were performed on thymol and carvacrol in an effort to understand the functional groups necessary for modulating their activities and to lead possibly to more effective larvae control agents. the derivatives were further subjected to sar and computational studies (molecular modeling and chemometric tools (cpca and pca)) to extract structural information regarding their larvicidal properties. field collected and rockefeller populations of ae. aegypti were used. results: carvacrol and thymol exhibited lc50 of 51 and 58ppm for field collected larvae, respectively. carvacrol derivatives exhibited lc50 ranging from 39 to 169ppm, while thymol derivatives exhibited lc50 ranging from 18 to 465ppm. substitution of the acidic proton of carvacrol by esters, ethers, and acetic acid resulted in either maintenance or reduction of potency. conclusion: thymol derivatives were, to a certain extent, more efficient larvicides against ae. aegypti than carvacrol derivatives, particularly to rockefeller larvae. the chemometrics tools applied in this study showed that the independent variables indicate a mixed profile. nevertheless, hydrophobic interactions increased the larvicidal activity. keywords: chemometry, sar, dengue, thymol derivatives, carvacrol derivatives introduction aedes aegypti linnaeus (diptera: culicidae), vector of dengue fever, is considered the most important disease-carrying mosquito in the world and has become a major international public health concern (who 2015). unlike most vectors, ae. aegypti lives near human habitation and breeds in a variety of water containers. during its life cycle, the female mosquitoes need blood as a source for supplemental substances, such as protein and iron to drive oogenesis (gonçalves 2009). contaminated mosquitoes transfer dengue virus from its salivary glands to humans during the course of the bite. after infection, the virus incu*corresponding author: dr sócrates cabral de holanda cavalcanti, e-mail: socratescavalcanti@yahoo.com.br j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 316 http://jad.tums.ac.ir published online: may 27, 2017 bates for about six days resulting in a flu-like illness that affects infants, young children and adults, rarely causing death (nishiura 2007). however, severe dengue is a complication characterized by high fever, which may progress to circulatory failure and death. because there is no treatment or vaccine available for treating or controlling dengue fever, the only available method to control dengue fever is to eliminate its vector, either by environmental actions, such as elimination of its breeding sites or by the use of pesticides. the use of larvicides is the most successful method to control mosquito infestation. three major classes of larvicides are approved for use in potable water, organophosphates (e.g. temephos), growth regulators (eg juvenile hormone mimics and chitin synthesis inhibitors), and bacteria toxins (eg bacillus thuringiensis, bti) (who 2006). however, resistance to larvicides has stimulated researchers to develop new substances with different mechanism of action to control ae. aegypti spreading (polson 2011, agra-neto 2014). a potential source for new pesticide candidates are natural product derivatives. botanical chemical derivatives may not only be selective to a target species, but also more environmentally friendly than synthetic compounds. monoterpenes have been chemically modified and evaluated against adult ae. aegypti mosquitoes (samarasekera 2008), as well as larvae (santos 2010). such studies have demonstrated the importance of double bonds and phenolic hydroxyls in the larvicidal potency (santos 2010, santos 2011, lima 2013). thymol and carvacrol are important phenolic monoterpenoids obtained from the essential oil of plants. both monoterpenes exhibit a large number of pharmacological activities, such as antimicrobial, antitumor, antiplatelet, analgesic, anti-inflammatory, antiangiogenic, and insecticidal (baser 2008). additionally, thymol and carvacrol are generally regarded as safe (gras) food flavoring, which is an indication of low mammalian toxicity starting materials (us-ofr 2009). the larvicidal efficiency of both thymol and carvacrol (santos 2010) promoted us to undertake the synthesis of natural monoterpenoids derivatives. most structural modifications were performed in the monoterpenoids phenolic moiety, resulting in a diverse set of groups with polar (acetic acid derivatives) and bulky/hydrophobic (ethers and esters) characteristics. additionally, a relatively reactive aldehyde group was placed in the phenolic hydroxyl ortho position with the goal to verify the contribution of a relatively unstable moiety to the larvicidal activity. furthermore, the larvicidal activities of thymol, carvacrol, and their derivatives against ae. aegypti were assessed. we have previously used principal component analysis (pca), partial least squares regression (pls), and consensus pca (cpca), to investigate a set of fifty-five compounds with activity against ae. aegypti larvae. the pca scores plot exhibited reasonable division between more and less potent compounds. the independent variables affected by a hydrophobic profile were sturdily correlated to the biological activity (scotti 2014). to corroborate such findings the derivatives were further subjected to molecular modeling, chemometrics tools (pca and cpca), and sar studies in order to extract structural information relating to their larvicidal properties. materials and methods general melting points were determined on a logen scientific melting point apparatus and are uncorrected. nmr data were recorded in cdcl3 or cd3od at 293 k on a bruker avance 400 nmr spectrometer operating at 9.4 tesla, observing 1h and 13c at 400 and 100mhz, respectively. all 1h and 13c nmr chemical shifts () are given in ppm related to the tms signal at 0.00ppm as internal refj arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 317 http://jad.tums.ac.ir published online: may 27, 2017 erence. coupling constants (j) are reported in hertz (hz). the abbreviations used are s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), and sept (septuplet). ft-ir spectra were acquired on a bomem mb-100 spectrophotometer. mass spectra were recorded either on a shimadzu gcms-qp2010s gas chromatograph mass spectrometer (equipped with an aoc-20s autosampler) operating in positive mode or on an ion trap lcq fleet tm (thermo scientific) equipped with an ei source operating in negative mode. hrms were recorded on a bruker microtof ii-esitof mass spectrometer operating in positive mode. reactions were monitored by thinlayer chromatography (tlc) using suitable mobile phase eluents, and visualization of the tlc plate was accomplished with uv light 256nm. details of the physicochemical properties of the synthesized compounds are described below. general chemicals were obtained from sigma-aldrich chemical co. (st louis, mo, usa). carvacryl benzoate was synthesized (nikumbh 2003) using bzcl in the presence of aqueous 10% naoh at room temperature (fig. 4). ether reactions were carried out using parent phenols (50mmol) and the corresponding alkyl iodide (60mmol) in the presence of k2co3 (120mmol) using acetone as solvent, under reflux (coolen 1995). 2-hydroxy-3-methyl-6-(1-methylethyl)-benzaldehyde and 2-hydroxy-6-methyl-3-(1-methylethyl)-benzaldehyde were synthesized according to the procedure of singh et al. (singh 1989) by a reimer-tiemann reaction (fig. 6). carvacrylglycolic and thymoxyacetic acids were synthesized using parent phenols, chloroacetic acid, and naoh dissolved in water, heated to reflux overnight (fig. 7) (varma 1985, riedl 1998, nikumbh 2003). general method for the synthesis of carvacryl and thymyl esters parent phenols (50mmol) were added to the corresponding anhydride or acid chloride (60mmol) to form ester derivatives in the presence of base (sodium acetate, triethylamine or nah, 60mmol). thf was used as the solvent, and the reaction was allowed to stir for 1–4h at room temperature. the reaction mixture was concentrated under vacuum, diluted with water and extracted with dichloromethane. the organic layer was washed with water, and dried over na2so4. the solvent was distilled off and the residue purified by silica gel column chromatography (0.5% etoac: hexanes) (dolly 2003, ben arfa 2006, morais 2014). general method for the synthesis of carvacryl and thymyl ethers parent phenols (50mmol) and the corresponding alkyl iodide (60mmol) in the presence of k2co3 (120mmol) and acetone as solvent, were stirred under reflux for 24h. the solvent was removed under reduced pressure, and the product was extracted in a ch2c12/water mixture. the combined organic layers were washed three times with 1 n aqueous naoh. the organic layer was separated, dried (mgso4), and evaporated to dryness. the resulting oil was purified by flash chromatography over a silica gel column with hexanes as eluent to yield clear oils (fig. 5) (coolen 1995). general method for the synthesis of 2-hydroxy-3-methyl-6-(1-methylethyl) benzaldehyde and 2-hydroxy-6-methyl-3-(1-methylethyl) benzaldehyde a mixture of carvacrol or thymol (50 mmol), naoh (250mmol) and water (20ml) was heated to 60–70 °c. a refluxing condenser was connected to the reaction flask and chloroform (124mmol) was added dropwise. extraction with etoac followed by drying (mgso4) and evaporation of the solvent yielded yellowish oils, which were purified by silica gel column chromatography (hexanes) to afford the corresponding aldehydes (knight 2005). j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 318 http://jad.tums.ac.ir published online: may 27, 2017 general method for the synthesis of carvacrylglycolic and thymoxyacetic acids parent phenols (30mmol), chloroacetic acid (30mmol), and naoh (61mmol) dissolved in 15ml of water were heated to reflux overnight. the solution was allowed to cool to room temperature, acidified to ph 1 with concentrated hcl with constant and vigorous stirring, and the precipitate was separated by filtration (riedl 1998). rearing of ae. aegypti eggs of ae. aegypti were field collected in aracaju city, sergipe state, brazil and laboratory-reared at the federal university of sergipe insectary, at 27 °c and 80–85% relative humidity under a 12:12h light: dark cycle. aedes aegypti collected in this neighborhood are resistant to temephos, a frequently used insecticide for larvae control. eggs of rockefeller were kindly donated by laboratório de fisiologia e controle de artrópodes vetores (laficave, fiocruz rio de janeiro, brazil). the ae. aegypti rockefeller strain is susceptible to temephos. adults were provided with a 10% sucrose solution ad libitum. assays eggs were obtained attached to paper strips. the paper strips (1000 eggs/l) were placed in a rectangular polyethylene container with natural mineral water. the container was kept in the insectary for hatching and monitoring of larvae development for three to four days. larvae were fed with cat food (purinatm) to allow regular development. all bioassays were conducted in a walk-in environmental chamber with the above environmental conditions. larvicidal assay the larvicidal assay was performed according to santos et al. (santos 2010). in summary, third-instar larvae were used in the experiment. the concentration ranges were determined by a previous curve concentrationresponse with 20 larvae. a 20,000ppm stock solution was prepared using each compound (20mg/ml), tween-80 (10%v/v), dmso (30% v/v), and natural mineral water (60%v/v). the stock solution was used to make 20ml water solutions with concentrations preset by the concentration-response curve. twenty larvae were collected with a pasteur pipette, placed on a 25ml graduated cylinder. the volume was completed to 20ml with natural mineral water and transferred to disposable cups containing variable volumes of the stock solution. a mortality count was conducted 24h after treatment. controls were prepared with tween-80 (0.1ml), dmso (0.3ml), and water (19.6ml). three replicates were used for each concentration and the control. statistics probit analysis (finney 1971) was conducted on mortality data collected after 24h exposure to different concentration of testing solutions to establish the lethal concentration for 50% mortality (lc50) and 95% confidence interval values for the respective compounds (tables 1, 2). in all cases where deaths had occurred in the control experiment, the data was corrected using abbott’s formula (% deaths= [1-(test/control)] x100). compounds activity is considered significantly different when the 95% ci fails to overlap. computational methods the results of field collected larvae activity were used on computational methods. molecular modeling the chemical structures of the compounds were drawn using the program hyperchem v. 8.0 (hypercube 2009), and their geometry was optimized using mm+ force field (allinger 1977). subsequently, a new geometry optimization based on the semi-empirical method am1 (austin model 1) was performed (dewar 1993). the optimized structures were subjected to conformational analyses using the random search method (allinger 1977, cohen 1996, leach 2001) with 1000 j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 319 http://jad.tums.ac.ir published online: may 27, 2017 interactions, 100 cycles of optimization, and 10 conformers of lowest minimum energy. the selected dihedrals were evaluated by rotation in accordance with the standard (default) conditions of the program, in which the number of simultaneous variations was 1 to 8, acyclic chains were submitted to rotations from 60 to 180° and torsion rings were in the range of 30 to 120°. chemometrics structures modeled as described above were used to calculate the molecular descriptors via volsurf+ program. pca and cpca methodologies were applied to the set of derivatives using the volsurf+ software (cruciani 2000, cruciani 2000, cruciani 2002, cianchetta 2004). the lc50 of the derivatives were converted to molar unitary and then expressed in negative logarithmic units, plc50 (-log lc50). cpca (consensus pca) a preliminary exploratory analysis, cpca, which considered 128 independent variables or descriptors, was developed. pre-processing (autoscaling) of data was performed, and 13 blocks of descriptors were calculated. pca with regard to the interaction of 3d structures and a grid force field, pca results were obtained using the h2o, dry and logs probes. fifty-four descriptors were selected from cpca. the data were autoscaled (pre-processed). results synthesis twenty monoterpenes were synthesized, characterized, and evaluated. spectroscopic data are depicted below. carvacryl acetate (2a) was prepared from carvacrol using the method of ben arfa et al. ( 2006). clear oil (75%). ir (film, cm-1) 2960 (c-h), 1766 (c= o), 1215 (-cc=oo-). 1h nmr (400 mhz, cdcl3)  7.11 (d, 1h, j= 7.8 hz, ar-h), 6.98 (d, 1h, j= 7.8 hz, ar-h), 6.85 (s, 1h, ar-h), 2.85 (sept, 1h, j= 6.9 hz, ch(ch3)2), 2.25 (s, 3h, coch3), 2.11 (s, 3h, ar-ch3), 1.21 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  169.2, 149.3, 148.0, 130.8, 127.1, 124.1, 119.7, 33.5, 23.9, 20.7, 15.7. ms (ei) m/z [m]+ 192. carvacryl chloroacetate (2b) was prepared from carvacrol using the method of dolly et al. (2003). yellowish oil (78%). ir (film, cm-1) 2960 (c-h), 1778 (c=o), 1238 (ch2cl), 1145 (-cc=oo-), 705 (c-cl). 1h nmr (400 mhz, cdcl3)  7.13 (d, 1h, j= 7.8 hz, ar-h), 7.02 (d, 1h, j= 7.8 hz, ar-h), 6.89 (s, 1h, ar-h), 4.25 (s, 2h,ch2 cl), 2.86 (sept, 1h, j= 6.9 hz, ch(ch3)2), 2.13 (s, 3h, ar-ch3), 1.21(d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  165.6, 148.8, 148.2, 131.0, 126.9, 124.6, 119.3, 40.7, 33.5, 23.8, 15.6. hrms calcd for c12 h15clo2na (m+na)+ m/z 249.0658, found 249.0659. carvacryl trichloroacetate (2c) was prepared from carvacrol using the method of dolly et al. (2003). yellowish oil (84%). ir (film, cm-1) 2960 (c-h), 1776 (c= o), 1219 (-cc=oo-), 677 (c-cl). 1h nmr (400 mhz, cdcl3)  7.16 (d, 1h, j= 7.8 hz, ar-h), 7.07 (d, 1h, j= 7.8 hz, ar-h), 6.97 (s, 1h, ar-h), 2.88 (sept, 1h, j= 6.9 hz, ch (ch3)2), 2.21 (s, 3h, ar-ch3), 1.23 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  160.2, 148.9, 148.5, 131.3, 126.7, 125.2, 118.5, 89.8, 33.6, 23.8, 15.4. hrms calcd for c12h13cl3o2na (m+na)+ m/z 316.9879, found 316.9875. carvacryl propionate (2d) was prepared from carvacrol using the method of dolly et al. (2003). clear oil (72%). ir (film, cm-1) 2960 (c-h), 1760 (c=o), 1147 j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 320 http://jad.tums.ac.ir published online: may 27, 2017 (-cc=oo-). 1h nmr (400 mhz, cdcl3)  7.10 (d, 1h, j= 7.8 hz, ar-h), 6.98 (d, 1h, j= 7.8 hz, ar-h), 6.85 (s, 1h, ar-h), 2.84 (sept, 1h, j= 6.8 hz, ch(ch3)2), 2.56 (q, 2h, j= 7.6 hz, co-ch2-ch3), 2.10 (s, 3h, ar-ch3), 1.25 (t, 3h, j= 7.5 hz, co-ch2ch3), 1.21 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  172.5, 149.3, 147.9, 130.8, 127.1, 124.0, 119.7, 33.6, 27.6, 23.9, 15.7, 9.2. ms (ei) m/z [m]+ 206. carvacryl benzoate (2e) was prepared from carvacrol using the schotten-baumann reaction. (morais 2014). clear oil (73%). ir (film, cm-1) 2960 (c-h), 1737 (c=o), 1263 (-cc=oo-). 1h nmr (400 mhz, cdcl3)  8.21 (d, 2h, j= 8.4 hz, ar-h), 7.56 (t, 1h, j= 7.4 hz, ar-h), 7.477.43 (m, 2h, ar-h), 7.16 (d, 1h, j= 7.7 hz, ar-h), 7.02 (d, 1h, j= 7.7 hz, ar-h), 7.00 (s, 1h, ar-h), 2.87 (sept, 1h, j= 6.9 hz, ch (ch3)2), 2.17 (s, 3h, ar -ch3), 1.23 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  164.8, 149.5, 148.0, 133.4, 130.9, 130.1, 129.6, 128.5, 127.3, 124.1, 119.8, 33.6, 23.9, 15.8. ms (ei) m/z [m]+ 254. thymyl acetate (3a) was prepared from thymol using the method of ben arfa et al. ( 2006). clear oil (93%). ir (film, cm-1) 2966 (c-h), 1760 (c= o), 1220 (-cc=oo-). 1h nmr (400 mhz, cdcl3)  7.18 (d, 1h, j= 7.9 hz, ar-h), 7.01 (d, 1h, j= 7.9 hz, ar-h), 6.80 (s, 1h, ar-h), 2.96 (sept, 1h, j = 6.9 hz, ch(ch3)2), 2.30 (s, 6h, ococh3 and ar-ch3), 1.18 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  169.7, 147.8, 136.9, 136.5, 127.1, 126.4, 122,7, 27.1, 23.0, 20.9, 20.8. ms (ei) m/z [m]+ 192. thymyl chloroacetate (3b) was prepared from thymol using the method of dolly et al. (2003). yellowish oil (62%). ir (film, cm-1) 2960 (c-h), 1776 (c=o), 1240 (ch2cl), 1157 (-cc=oo-), 779 (c-cl). 1h nmr (400 mhz, cdcl3)  7.19 (d, 1h, j= 7.9 hz, ar-h), 7.02 (d, 1h, j= 7.9 hz, ar-h), 6.82 (s, 1h, ar-h), 4.24 (s, 2h, ch2 cl), 2.97 (sept, 1h, j= 6.9 hz, ch(ch3)2), 2.28 (s, 3h, ar-ch3), 1.18(d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  166.1, 147.5, 136.8, 136.7, 127.6, 126.6, 122.2, 40.7, 27.0, 23.0, 20.7. ms (ei) m/z [m]+ 226. thymyl trichloroacetate (3c) was prepared from thymol using the method of dolly et al. (2003). yellowish oil (90%). ir (film, cm-1) 2964 (c-h), 1776 (c=o), 1224 (-cc=oo-), 676 (c-cl). 1h nmr (400 mhz, cdcl3)  7.16 (d, 1h, j= 7.8 hz, ar-h), 7.07 (d, 1h, j= 7.8 hz, arh), 6.97 (s, 1h, ar-h), 2.88 (sept, 1h, j= 6.9 hz, ch(ch3)2), 2.21 (s, 3h, ar-ch3), 1.23 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  160.2, 148.9, 148.5, 131.3, 126,7, 125.2, 118.5, 89.8, 33.5, 23.8, 15.4. hrms calcd for c12 h13cl3o2na (m+na)+ m/z 316.9879, found 316.9870. thymyl propionate (3d) was prepared from thymol using the method of dolly et al. (2003). clear oil (68%). ir (film, cm-1) 2966 (c-h), 1758 (c=o), 1149 (-cc=oo-). 1h nmr (400 mhz, cdcl3)  7.17 (d, 1h, j= 7.9 hz, arh), 6.98 (d, 1h, j= 7.9 hz, ar-h), 6.79 (s, 1h, ar-h), 2.96 (sept, 1h, j= 6.8 hz, ch (ch3)2), 2.57 (q, 2h, j= 7.5 hz, coch2 ch3), 2.28 (s, 3h, ar-ch3), 1.26 (t, 3h, j= 7.6 hz, coch2ch3), 1.18 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  173.0, 148.0, 136.9, 136.4, 127.0, 126.3, 122.7, 27.7, 27.1, 23.0, 20.7, 9.2. ms (ei) m/z [m]+ 206. thymyl benzoate (3e) was prepared from thymol using the schotten-baumann reaction. (morais 2014). white crystals (94%), mp 28–29 °c. ir (kbr, cm-1) 2958 (c-h), 1735 (c=o), 1236 (-cc= j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 321 http://jad.tums.ac.ir published online: may 27, 2017 oo-). 1h nmr (400 mhz, cdcl3)  8.22 (d, 2h, j= 7.1 hz, ar-h), 7.62 (t, 1h, j= 7.4 hz, ar-h), 7.50 (t, 2h, j= 7.3 hz, ar-h), 7.24 (d, 1h, j= 7.9 hz, ar-h), 7.06 (d, 1h, j= 7.9 hz, ar-h), 6.94 (s, 1h, ar-h), 3.05 (sept, 1h, j= 6.8 hz, ch(ch3)2), 2.33 (s, 3h, ar-ch3), 1.21 (d, 6h, j= 6.9 hz, ch (ch3)2). 13c nmr (100 mhz, cdcl3):  165.3, 148.1, 137.1, 136.6, 133.5, 130.1, 129.6, 128.6, 127.1, 126.4, 122.8, 27.2, 23.0, 20.8. ms (ei) m/z [m]+ 254. carvacryl methyl ether (2f) was prepared from carvacrol using the method of coolen et al. (1995). clear oil (75%). ir (film, cm-1) 2958 (c-h), 1253 (co-c). 1h nmr (400 mhz, cdcl3)  7.01 (d, 1h, j= 7.5 hz, ar-h), 6.70 (d, 1h, j= 7.5 hz, ar-h), 6.67 (s, 1h, ar-h), 3.76 (s, 3h, och3), 2.84 (sept, 1h, j= 6.8 hz, ch (ch3)2), 2.17 (s, 3h, ar-ch3), 1.23 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  157.6, 147.8, 130.4, 123.8, 117.9, 108.3, 55.0, 34.2, 24.1, 15.8. ms (ei) m/z [m]+ 164. carvacryl ethyl ether (2g) was prepared from carvacrol using the method of coolen et al. (1995). clear oil (61%). ir (film, cm-1) 2960 (c-h), 1253 (co-c). 1h nmr (400 mhz, cdcl3)  7.02 (d, 1h, j= 7.5 hz, ar-h), 6.70 (d, 1h, j= 7.5 hz, ar-h), 6.67 (s, 1h, ar-h), 4.00 (q, 2h, j= 6.9 hz, och2ch3), 2.83 (sept, 1h, j= 6.8 hz ch(ch3)2), 2.18 (s, 3h, ar-ch3), 1.39 (t, 3h, j= 6.9 hz, och2ch3), 1.23 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  157.0, 147.7, 130.4, 124.1, 117.8, 109.5, 63.3, 34.2, 24.1, 15.8, 15.0. ms (ei) m/z [m]+ 178. thymyl methyl ether (3f) was prepared from thymol using the method of coolen et al. (1995). clear oil (68%). ir (film, cm-1) 2960 (c-h), 1257 (co-c). 1h nmr (400 mhz, cdcl3)  7.08 (d, 1h, j= 7.6 hz, ar-h), 6.74 (d, 1h, j= 7.6 hz, ar-h), 6.66 (s, 1h, ar-h), 3.80 (s, 3h, och3), 3.27 (sept, 1h, j= 6.9 hz, ch (ch3)2), 2.32 (s, 3h, ar-ch3), 1.19 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  156.6, 136.2, 134.0, 125.7, 121.0, 111.3, 55.3, 26.4, 22.8, 21.3. ms (ei) m/z [m]+ 164. thymyl ethyl ether (3g) was prepared from thymol using the method of coolen et al. (1995). clear oil (91%). ir (film, cm-1) 2958 (c-h), 1257 (co-c). 1h nmr (400 mhz, cdcl3)  7.07 (d, 1h, j= 7.6 hz, ar-h), 6.72 (d, 1h, j= 7.6 hz, ar-h), 6.64 (s, 1h, ar-h), 4.00 (q, 2h, j= 7.0 hz, och2ch3), 3.28 (sept, 1h, j= 6.9 hz, ch(ch3)2), 2.30 (s, 3h, ar-ch3), 1.40 (t, 3h, j= 6.9 hz, och2ch3), 1.19 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  156.0, 136.1, 134.1, 125.8, 120.9, 112.3, 63.4, 26.6, 22.7, 21.3, 15.0. ms (ei) m/z [m]+ 178. 2-hydroxy-3-methyl-6-(1-methylethyl) benzaldehyde (2h) was prepared from carvacrol using the method of knight et al. (2005). yellowish oil (31%). ir (film, cm-1) 3413 (oh), 2964 (c-h), 1666 (c=o). 1h nmr (400 mhz, cdcl3)  12.44 (s, 1h, ar-oh, d2o exch.), 10.38 (s, 1h, coh), 7.29 (d, 1h, j= 7.7 hz, ar-h), 6.75 (d, 1h, j= 7.7 hz, ar-h), 3.59 (sept, 1h, j= 6.8 hz, ch(ch3)2), 2.20 (s, 3h, ar-ch3), 1.30 (d, 6h, j= 6.8 hz, ch (ch3)2). 13c nmr (100 mhz, cdcl3):  195.1, 161.7, 150.2, 138.2, 124.3, 116.3, 115.5, 27.2, 24.2, 14.9. ms (ei) m/z [m]+ 178. 2-hydroxy-6-methyl-3-(1-methylethyl) benzaldehyde (3h) was prepared from thymol using the method of knight et al. (2005). yellowish oil (33%). ir (film, cm-1) 2966 (c-h), 1633 (c=o). 1h nmr (400 mhz, cdcl3)  12.29 j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 322 http://jad.tums.ac.ir published online: may 27, 2017 (s, 1h, ar-oh), 10.27 (s, 1h,coh), 7.29 (d, 1h, j= 7.6 hz, ar-h), 6.65 (d, 1h, j= 7.4 hz, ar-h), 3.31 (sept, 1h, j= 6.9 hz ch (ch3)2), 2.54 (s, 3h, ar-ch3), 1.21 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cdcl3):  195.7, 160.9, 139.3, 134.0, 135.3, 121.4, 118.1, 26.1, 22.2, 17.8. ms (ei) m/z [m]+ 178. carvacrylglycolic acid (2i), was prepared from carvacrol using the method of riedl et al. (1998). white cotton like crystals (77%), mp 159–161 °c. ir (kbr, cm-1) 3413 (cooh), 1739 (cooh). 1h nmr (400 mhz, cd3od)  7.01 (d, 1h, j= 7.6 hz, ar-h), 6.72 (d, 1h, j= 7.6 hz, ar-h), 6.64 (s, 1h, ar-h), 4.62 (s, 2h, oc h2cooh), 2.81 (sept, 1h, j= 6.8 hz, ch (ch3)2), 2.20 (s, 3h, ar-ch3), 1.20 (d, 6h, j= 6.8 hz, ch(ch3)2). 13c nmr (100 mhz, cd3od):  173.0, 157.4, 148.9, 131.6, 125.3, 119.9, 110.7, 66.1, 35.2, 24.4, 15.9. ms (esi) m/z [m–h]− 207. thymoxyacetic acid (3i), was prepared from thymol using the method of riedl et al. (1998). white cotton like crystals (48%), mp 133–136 °c. ir (kbr, cm-1) 3429 (cooh), 1737 (cooh). 1h nmr (400 mhz, cd3od)  7.06 (d, 1h, j= 7.7 hz, ar-h), 6.73 (d, 1h, j= 7.7 hz, ar-h), 6.60 (s, 1h, ar-h), 4.61 (s, 2h, ch2 cooh), 3.34 (sept, 1h, j= 6.9 hz, ch (ch3)2), 2.26 (s, 3h, ar-ch3), 1.19 (d, 6h, j= 6.9 hz, ch(ch3)2). 13c nmr (100 mhz, cd3od):  172.9, 156.4, 137.3, 135.3, 127.0, 123.0, 113.4, 66.2, 27.6, 23.2, 21.3. ms (esi) m/z [m–h]− 207. larvicidal activity two sets of carvacrol and thymol derivatives listed in figs. 4–7 were tested for their in vivo larvicidal activities against ae. aegypti, exhibiting a wide-ranging lc50. the rates of mortality were directly proportional to concentration. their assessed lethal concentration for 50% mortality (lc50), as well as 95% confidence intervals (ci) expressed as ppm, are exhibited in tables 1 and 2. carvacrol and thymol exhibited lc50 of 51ppm (48 to 55) and 58ppm (54 to 63), respectively. carvacrol derivatives exhibited lc50 ranging from 52 to 169ppm for field collected and from 39 to 113ppm for rockefeller larvae, while thymol derivatives exhibited lc50 ranging from 34 to 465ppm for field collected and 18 to 101ppm for rockefeller larvae. thymyl benzoate exhibited the highest overall larvicidal potency, with lc50 of 18 ppm (14 to 25) for rockefeller larvae, while thymoxyacetic acid exhibited the lowest larvicidal potency, with lc50 of 465ppm (426 to 513) for field-collected larvae. field collected strain exhibited certain resistance to most of the synthesized compounds than rockefeller strain, related to the exposure of field mosquitoes to some of these compounds in aromatic plants. computational methods blocks of descriptors exhibiting higher weights are shown in the fig. 1, constructed taking into account the orthogonal properties of the cpca: logs (adme descriptors), dry and h2o. regarding cpca methodology, 128 independent variables were used, and no biological data were considered as input to the model. the orthogonal properties of the cpca algorithm were explored. the use of cpca in decentralized process monitoring and diagnosis is originated from standard pca scores and residuals. two significant principal components (pcs) were obtained using a cross-validation methodology, which explains more than 68% of the total variance in larvicidal activity (fig. 2). pca results were obtained using the h2o, dry and logs probes. fifty-four descriptors were calculated. once again, for both activities, approximately 73% of the total variance was j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 323 http://jad.tums.ac.ir published online: may 27, 2017 explained by the pc1 and pc2 components. fig. 3 represents the loading plot from pca. table 1. larvicidal activities (lc50) and 95% confidence intervals (ci) of carvacrol and its derivatives on thirdinstar larvae of ae. aegypti compound name lc50 ppm (ci) field collected lc50 ppm (ci) rockefeller carvacrol, 1a 51 (48 to 55) 47 (43 to 50) carvacryl acetate, 2a 93 (84 to 104) 72 (65 to 79) carvacryl chloroacetate, 2b 52 (44 to 59) 39 (37 to 42) carvacryl trichloroacetate, 2c 77 (68 to 86) 54 (47 to 60) carvacryl propionate, 2d 66 (57 to 77) 77 (70 to 83) carvacryl benzoate, 2e 58 (49 to 69) 57 (45 to 72) carvacryl methyl ether, 2f 136 (116 to 154) 60 (52 to 69) carvacryl ethyl ether, 2g 120 (100 to 148) 67 (57 to 76) 2-hydroxy-3-methyl-6-(1-methylethyl)-benzaldehyde, 2h 66 (58 to 75) 56 (51 to 61) carvacrylglycolic acid, 2i 169 (158 to 180) 113 (101 to 123) table 2. larvicidal activities (lc50) and 95% confidence intervals (ci) of thymol and its derivatives on third-instar larvae of ae. aegypti compound name lc50 ppm (ic) field collected lc50 ppm (ic) rockefeller thymol, 1b 58 (54 to 63) 46 (41 to 52) thymyl acetate, 3a 93 (82 to 103) 79 (69 to 89) thymyl chloroacetate, 3b 49 (46 to 53) 25 (22 to 28) thymyl trichloroacetate, 3c 41 (37 to 45) 45 (41 to 51) thymyl propionate, 3d 66 (61-73) 45 (39 to 51) thymyl benzoate, 3e 90 (82 to 99) 18 (14 to 25) thymyl methyl ether, 3f 192 (169 to 214) 39 (33 to 48) thymyl ethyl ether, 3g 123 (29 to 269) 58 (50 to 65) 2-hydroxy-6-methyl-3-(1-methylethyl)-benzaldehyde, 3h 34 (32 to 37) 36 (32 to 41) thymoxyacetic acid, 3i 465 (426-513) 101 (95 to 107) fig. 1. blocks of descriptors j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 324 http://jad.tums.ac.ir published online: may 27, 2017 fig. 2. positions of objects in relation to pc1 and pc2 components. dark grey balls represent more active compounds, light grey balls represent less active compounds fig. 3. loadings plot from pca r1 r2 r1 r2 r1 = ococh3 r1 = ococh2cl r1 = ococcl3 r1 = ococh2ch3 r1 = obz r1 = h; r1 = h; r1 = h; r1 = h; r1 = h; r2 = h r2 = h r2 = h r2 = h r2 = h r2 = ococh3 r2 = ococh2cl r2 = ococcl3 r2 = ococh2ch3 r2 = obz 1. nah, thf 2. acid chloride 2a 2b 2c 2d 2e 3a 3b 3c 3d 3e 1a 1b r2 = h r2 = oh r1 = oh; r1 = h; fig. 4. synthesis of carvacrol and thymol esters j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 325 http://jad.tums.ac.ir published online: may 27, 2017 fig. 5. synthesis of carvacrol and thymol ethers fig. 6. synthesis of 2-hydroxy-3-methyl-6-(1-methylethyl)-benzaldehyde and 2-hydroxy-6-methyl-3-(1-methylethyl)-benzaldehyde fig. 7. synthesis of carvacrylglycolic and thymoxyacetic acids discussion carvacrol and thymol have previously exhibited moderate larvicidal activity. in view of this fact, our goals were to synthesize and evaluate the larvicidal activity of a diverse set of derivatives exhibiting a wide range of chemical physicochemical characteristics, which includes polarity, volume, hydrophobicity, and reactivity, to study the influence of such chemical characteristics in the larvicidal potency. derivatives 2a-i and 3a-i were synthesized by previously reported methods in reasonable yields and characterized by 1h and 13c j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 326 http://jad.tums.ac.ir published online: may 27, 2017 nmr, ftir spectroscopy, mass spectrometry and melting point. replacement of the phenolic proton resulting in ether, ester and acetic acid derivatives were synthesized with the goal to examine the effects of such groups in modulating the larvicidal potency. additionally, aldehyde groups at the -position to the phenolic hydroxyl were synthesized. parent phenol monoterpenes (carvacrol and thymol) were used to synthesize their derivatives. compounds 2a-i to 3a-i were synthesized by the routes depicted in figs. 4–7. carvacryl and thymyl esters were synthesized according to the literature with some modifications (grodnitzky 2002, dolly 2003, ben arfa 2006). ester derivatives of carvacrol and thymol, i.e. carvacryl acetate, chloroacetate, trichloroacetate, and propionate, as well as, thymyl acetate, chloroacetate, trichloroacetate, propionate and benzoate were synthesized by reaction with acetic anhydride or acid halides in the presence of base (sodium acetate, triethylamine or nah) in thf as solvent at room temperature. carvacrol and thymol exhibited similar potencies (51 and 58ppm for field collected, 47 and 46ppm for rockefeller strains, respectively). similarly, the acetate and the ethyl ether derivatives exhibited comparable potencies. substitution of the acidic proton of carvacrol by esters, ethers, and acetic acid resulted in either maintenance or reduction of potency. ether derivatives 2f, 2g, 3f, and 3g exhibited up to 2.5-fold decrease in potency for field-collected strain, while no change in potency was observed for these compounds on rockefeller larvae. furthermore, carvacrylglycolic acid (2i) exhibited 2.5 to three orders of magnitude decrease in potency. the addition of a bulky benzoate group, yielding 2e, resulted in slight decrease in potency. however, the benzoate derivative was more potent than smaller groups, such as acetate or propionate. similar results were previously achieved by substitution of the acidic proton of eugenol (barbosa 2012). addition of chlorines to carvacryl acetate (lc50 = 93 and 72ppm for field collected and rockefeller larvae, respectively), resulting on 2b and 2c led to a slight increase in potency (52 and 77ppm for field collected larvae and 39 and 54ppm for rockefeller larvae, respectively). adding an aldehyde group at the ortho position of thymol phenolic hydroxyl resulted in about 1.5 orders of magnitude increase in potency. however, the presence of the aldehyde group at the ortho position of carvacrol resulted in minor potency decrease. similar results were found in a previous work by our laboratory (santos 2010). the addition of an aldehyde group at the ortho position of phenol resulted in about 1.5-fold increase in potency, while the same group added to the para position of catechol led to minor decrease in potency. in the consensus pca, the analysis was performed by comparison of several descriptor blocks for the same object. in this study, thirteen groups of descriptors were calculated and plotted considering the two main components (pc1 and pc2), based on their weights. as observed in fig 1, the blocks of descriptors: logs, dry and oh2 have the highest weights considering the two main pcs. the cpca algorithm is basically comparable to the regular pca. the blocks with high weight from cpca, were selected and applied in pca. thus, 54 descriptors were used in the calculations of pc analysis. the pca evaluations measured the 3d interaction energies computed using the logs, dry and h2o probes of grid force field. the pca method also aided to refine the data. the pc1 and pc2 components captured approximately 73% of the total variance from the original data. in the scores plot (fig. 2) the least active compounds are found in the higher left side of the graph, while the most active compounds are mostly found in the right side of the graph. additionally, with the exception j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 327 http://jad.tums.ac.ir published online: may 27, 2017 of 2e and 3e each derivative is found close to its constitutional isomer. therefore, the contributions of the substituents for the larvicidal activity are similar for thymol and carvacrol derivatives. the loadings plot (fig. 3) highlighted the variables: cw4-cw6–represent the hydrophilic surface divided by the total molecular surface. in other words, it is the hydrophilic surface per surface unit. capacity factors are computed at eight dissimilar levels, the same energy levels used to calculate the hydrophilic volumes. d7, d8– a probe called dry is used by the software grid to create 3d lipophilic fields. similar to hydrophilic regions, hydrophobic regions are defined as the molecular envelope producing attraction by hydrophobic interactions. d1 to d8 are hydrophobic descriptors representing eight energy levels able to be calculated by volfsurf. these levels can be associated to eight-energy range of hydrophobic interactions (from 0.2 to 1.6 kcal/mol). cd1, cd2, cd8–is the quotient of the hydrophobic surface divided by the total molecular surface. therefore, it is the hydrophobic surface per total surface. capacity factors are computed at eight different levels, the same energy levels used to calculate hydrophobic volumes. id4– it is a measure of the unbalance between the center of mass of a compound and the centroid of its hydrophobic regions. the volsurf descriptors allows to represent by graphic the molecular interaction regions of interest, which is a substantial benefit in pharmacological studies as the descriptors are independent of the molecular alignment within the grid. the major advantage this method is the large amount of probes that are available. in addition, several functional groups may be represented, such as, hydrogen acceptor and donator probes (eg, water), constrained probes (eg, carbonyl probe), as well as hydrophobic probes. thymol derivatives were, to a certain extent, more efficient larvicides against ae. aegypti than carvacrol derivatives, particularly to rockefeller larvae. a withdrawing group (chlorine) added to thymyl acetate, resulting in thymyl chloroacetate (3b) led to an overall increase in potency. however, increasing the number of chlorines in the acetate group, resulting in thymyl tricholoroacetate (3c) did not increase potency. the chemometric tools (cpca and pca) applied to carvacrol and thymol derivatives exhibited reasonable results. the variables outlined in this study indicate the presence of hydrophobic and hydrophilic regions in the molecular surface (mixed profile). however, hydrophobic interactions are more important and contribute to an increase the larvicidal activity. in a recent study published by our group (scotti 2014) we performed chemometric studies (cpca, pca, and pls) on a set of fifty-five larvicidal compounds against ae. aegypti, including beyond the terpenes, the diterpenes, phenylpropanoids and phenol derivatives. the blocks of descriptors size/shape, dry, and h2o exhibited higher weights in the cpca analysis. in the present study, similar results were found, and the main blocks were logs, dry and h2o, demonstrating that for this series of compounds (thymol and carvacrol derivatives), aspects related to the hydrophobicity (logs) are more important than the steric properties (size/shape) in explaining the larvicidal activity. in the same study the scores plot from pca (generated with 48 descriptors, selected of the three main blocks) resulted in two principal components (pc1 and pc2) explaining over 62% of the total variance, whereas the present study (constructed with 54 descriptors, selected of the three main blocks) permitted to obtain a better model, in which the pc1 and pc2 explained approximately 73% of the total variance. evaluated compounds were active against j arthropod-borne dis, june 2017, 11(2): 315–330 vb silva et al.: synthesis and chemometrics of … 328 http://jad.tums.ac.ir published online: may 27, 2017 third-instar larvae of ae. aegypti. field collected ae. aegypti larvae exhibited more resistance to evaluated compounds than rockefeller population. conclusion substitution of the free hydroxyl function proved to modulate the larvicidal activity. the addition of an aldehyde group at the ortho position relative to the phenolic hydroxyl resulted in slight increase in larvicidal potency. hydrophobicity plays an important role in the larvicidal activity of evaluated compounds. the addition of lipophilic groups to new designed compounds should prove to increase the larvicidal potency. such studies are currently being conducted by our research group. in accordance with the present conclusions, we hope that these chemometrics and sar data will be beneficial in the field of pest control and for the design of novel bioactive molecules. acknowledgements the authors wish to acknowledge decit, cnpq, and fapitec-se for supporting funds (471729/2010-8, 477428/2007-0, 55011620109-rede dengue). we extend our acknowledgement to prof norberto peporine lopes for mass spectra analyses. the authors declare that there is no conflict of interest. references agra-neto ac, napoleao th, pontual ev, santos nd, luz l de a, de oliveira cm, de melo-santos ma, coelho lc, navarro dm, paiva pm (2014) effect of moringa oleifera lectins on survival and enzyme activities of aedes aegypti larvae susceptible and resistant to organophosphate. parasitol res. 113(1): 175–184. allinger nl (1977) conformational-analysis. 130. mm2-hydrocarbon force-field utilizing v1 and v2 torsional terms. j am chem soc. 99(25): 8127–8134. barbosa jdf, silva vb, alves pb, gumina g, santos rlc, sousa dp, cavalcanti sch (2012) structure-activity relationships of eugenol derivatives against aedes aegypti (diptera: culicidae) larvae. pest manag sci. 68(11): 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(1985) synthesis of monocyclic β-lactams with a terpenoid moiety. indian j chem b. 24 (3): 302–304. who (2006) pesticides and their application for the control of vectors and pests of public health importance. world health organization, geneva. who/cds/ntd/whopes/gcdpp/2006.1, geneva. who (2015) dengue and severe dengue. fact sheet 117, geneva. 8. dr rafinejad rtl 89 11 29, vatandoost iran j arthropod-borne dis, 2011, 5(1): 63–68 m shahi et al.: the occurrence of red … 63 original article the occurrence of red-back spider latrodectus hasselti (araneae: theridiidae) in bandar abbas, southern part of iran m shahi1, a hosseini1, k shemshad3, *j rafinejad2 1school of public health, hormozgan university of medical sciences, bandar abbas, iran 2department of medical entomology and vector control, school of public health ,tehran university of medical sciences, tehran, iran 3department of entomology, science and research branch, islamic azad university, tehran, iran (received 2 feb 2010; accepted 5 mar 2011) abstract background: due to importance and fatal affect of red-back spiders, latrodectus hasselti, a faunistic survey for presence of this spider in bandar abbas has been conducted. this animal is considerably the most medically importance spiders all over the world. methods: live adult spider specimens were collected from bandar abbas town using hand catch conventional method and transferred to the laboratory throughout the summer of 2008. they were identified based on morphological characteristics and taxonomic keys and confirmed by some external experts. results: results showed the occurrence of the red-back spider, l. hasselti from bandar abbas, southern port of iran. two female specimens were found. the spider had specific morphological characters including black color with an obvious orange to red longitudinal strip on its upper parts of abdomen. conclusion: although the specimens were collected from south of the country, however since the region is an important harbor and port and goods come form different parts of world we assume the possibility of arrival from its origin and native breeding sites of the world. therefore further investigation is needed to clarify the presence of this species in different parts of iran. keywords: latrodectus hasselti, spider, iran introduction about 40,000 spiders have been recognized and this is less than half of the expected number of 100,000 to 200,000 species. most of the species have not yet been described (ellis et al. 2005). widow spiders of the genus latrodectus are found worldwide (graudins et al. 2001). red-back spiders, l. hasselti, are widow spiders (stallybrass 1969) and are the most medically importance spiders all over the world, and certainly in australia (stallybrass 1969, clark et al. 1992, vetter and visscher 1998, isbister and gray 2002). the dangerous red-back, l. hasselti, belongs to theridiidae family and these spiders are famous for having neurotoxin venom (cariaso 1967). its bite is usually mild and frequently may not be felt. symptoms may arise up to an hour after biting and start as a reddish lump with red streaks publishing out from it. typical envenomations of red-backed spider being the most toxic cases of all the member of the genus latrodectus and only the female bite is dangerous. the venom affects directly on the nerves, causing in release and consequently depletion of neurotransmitters. *corresponding author: dr javad rafinejad, e-mail: jrafinejad@yahoo.com iran j arthropod-borne dis, 2011, 5(1): 63–68 m shahi et al.: the occurrence of red … 64 common early symptoms are pain (which can become severe), sweating (always including local sweating at bite site), muscular weakness, nausea, vomiting, painful abdominal cramping, increasing blood pressure, perspiration and rigidity accompanied by severe hypertension. the very young or elderly persons are high risk groups, especially those who have preexisted cardiovascular disease (cavalieri 1987, korszniak and story 1994). severe ache was explained as pain more than a bee sting or alike it, and “increasing pain” as pain that increased within the first hour (geoffrey and gray 2003). there is no report of sting by this spider in the region. the genus latrodectus has wide distribution especially in tropical and temperate areas of the world. this species is distributed in a wide area from south-east asia to australia and new zealand. its natural habitat is in places around buildings, outdoor furniture, machinery and pile of materials. in the bush, they nest under logs and rocks. this spider builds its webs in dry, sheltered places, e.g. among stones, in logs, culverts and sheds (forster 1995). the red back spider is a native species to australia (nimorakiotakis and winkel 2004). since 1959 the australian species were known as l. mactans hasselti and was believed conspecifics with l. mactans fabricius populations as far distant as the mediterranean (levi 1959, 1966). however, the taxonomy of this genus is still largely unresolved (levi 1983). recent researches (forster and kingsford 1983, forster 1984) approve the view that it is a species in its own right, l. hasselti thorell, and it appears to have become established in new zealand (forster 1984) where two related species, l. katipo powell and l. atritus urquhart occur (forster and kingsford 1983). latrodectus hasselti moves by walking on the ground, especially in seashores during early stages of infestation, and can be transported over distances by various machines, mainly in inlands and cannot change its place by ballooning (nihei et al. 2004). the poison of the latrodectus genus is that consist some of the most important biologic toxins. these matters produce their effectiveness in which destabilization of cell membranes and de granulation of nerve terminals resulting in the release of neurotransmitters can occur (nicholson and graudins 2002). here, in this study we tried to determine the spider fauna of bandar abbas, one of the most important ports in southern iran. materials and methods study area hormozgan is a province in southeast of iran. bandar abbas is center of hormozgan province, located at latitude 27° 11' north; longitude 56° 16' east, 25 meters above sea level, with an area of 45 km2 (fig.1). this city is located in coastal area and has hot and humid climate. the rainfall is around 251 mm, annually. maximum temperature in summer is 45° c and minimum temperature in winter is 6 °c. the average annual temperature in bandar abbas city is about 27° c. maximum relative humidity is 100% and the minimum is as low as 46% ( iran climatology center). spider collection spiders were collected in spring 2004 in an open air north of bandar abbas city by visually searching. all specimens were collected close to the human dwellings. the specimens were preserved in 75% alcohol. for species identification several limitations including morphology and collecting localities have been considered. recognition was carried out by morphological characteristics according to the international standard taxonomic key diagnostic (roth 1982). morphological limitations were photographed by nikon xn model stereomicroscope. all measurement is given in millimeters. length and width variations of the body, cephalothorax and abdomen are given below. iran j arthropod-borne dis, 2011, 5(1): 63–68 m shahi et al.: the occurrence of red … 65 results description: two females red-back spider were found in north bandar abbas city. adult female (specimen no. 1) (fig. 2): the total length of body, the length and width of cephalothorax as well as the length and width of abdomen are 10, 4, 3, 6 and 4.5 mm respectively. adult female (specimen no. 2): the total length of body, the length and width of cephalothorax as well as the length and width of abdomen are 11, 5, 4, 7 and 6 mm respectively. body colors: both females red-back spider were black with an orange to red longitudinal stripe on the upper surface of their abdomen. fig. 2–4 show morphological characteristics of the red-back spider. fig. 1. map of hormozgan province and situation of bandar abbas city, iran fig. 2. red-back spider collected from bandar abbas city, iran, 2007 fig. 3. red-back spider’s eyes pattern status iran j arthropod-borne dis, 2011, 5(1): 63–68 m shahi et al.: the occurrence of red … 66 fig. 4. presence of one row comb-like hair under the fourth tarsus of latrodectus hasselti discussion widow spiders of the genus latrodectus have worldwide distribution (graudins et al. 2001). red-back spiders, l. hasselti, are widow spiders (stallybrass 1969). bites of latrodectus are often initially painful. the neurotoxin alpha latrotoxin in the venom induces in 50% of the people that were hospitalized because of a terrible pain that takes from one to three days (bonnet 1999). there are not any noticeable studies about distribution of spiders in iran. rafinejad in 1997 showed that four species including l. tredecimgottatus, l. dahli, l. geometricus and l. pallidus were distributed in khorasan province, north east of iran. the present studies were carried out on several cases of patients who were hospitalized due to spider bites in bandar abbas city. however for the first time in iran, 2 females of red-back spider were found in bandar abbas city. the genus latrodectus spreads widely all over the tropical and temperate areas of the world and near human dwellings (levi 1959). bites of these spiders can cause abdominal cramps and pains, vomiting, nausea, and muscle cramps. sometimes death occurs (bonnet 1999). although widow spiders have wide distribution in the world but transportation has an important role in their distribution. deforestation and habitat destruction have a direct effect on species. sometimes, species may be lost, but in many other situations, increased levels of species have followed (walsh 1993). bandar abbas port is in the middle of the strait of hormoz linking the persian gulf to the sea of oman. it has an international airport with regular flights to other places of persian gulf. however , we assume that l. hasselti was probably introduced in iran by transportation of goods by ships or other media. furthermore, in addition of hot and humidity weather in bandar abbas city provides suitable habitat for increasing l. hasselti population. since bandar abbas is one of the most important crossroad of the country for export and import of agricultural and industrial goods, it is highly recommended to make available control and prevention programs and quarantine regulations and other control measures for widow spiders in the region. also more studies are needed to highlight any probable occurrence and taxonomic situation of the spider widows the species in southern islands of iran. acknowledgements authors are grateful to dr. n. ansarian (health center, hormozgan university of medical sciences) for his helpful comments. the authors declare that there is no conflict of interests. references bonnet ms (1999) the toxicology of the latrodectus hasselti spider the australian red back spider. british homo j. 88(1): 2–6. cariaso bl (1967) a biology of the black widow spider, latrodectus hasselti thorell (araneida: theridiidae). philippine agr sci. 51: 171–180. cavalieri md, urso d, lassa a (1987) characterization and some properties of the iran j arthropod-borne dis, 2011, 5(1): 63–68 m shahi et al.: the occurrence of red … 67 venom gland extract of a theriid spider (steatoda paykulliana) frequently mistaken for black widow spider (latrodectus tridecimguttatus). toxicon. 25: 965– 974. clark rf, wethern-kestner s, gerkin vance mv (1992) clinical presentation and treatment of black widow spider envenomation: a review of 163 cases. ann j emerg med. 21: 782–787. ellis rm, sprivulis pc, jelinek ga, banham nd, wood sv, wilkes gj, siegmund a, roberts bl (2005) a double-blind, randomized trial of intravenous versus intramuscular antivenom for red-back spider envenoming. emerg med aust. 17(2): 152–156. forster lm, kingsford s (1983) a preliminary study of development in two latrodectus species (araneae: theridiidae). new zealand entomol. 7: 431–438. forster lm (1984) the australian redback spider (latrodectus hasselti): its introduction and potential for establishment and distribution in new zealand, in: laird m (ed): commerce and the spread of pests and disease vectors. new york, pp. 273–289. forster lm (1995) the behavioral ecology of latrodectus hasselti (thorell), the australian redback spider (araneae: theridiidae): a review. record western australian museum. 52: 13–24. geoffrey ki, gray mr (2003) latrodectism: a prospective cohort study of bites by formally identified redback spiders. med j australia. 179(21): 88–91. graudins a, padula m, broady k, nicholson gm (2001) red-back spider (latrodectus hasselti) antivenom prevents the toxicity of widow spider venoms. ann j emerg med. 37(2): 154–160. iran climatology center, 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(arachnida: araneae: theridiidae). verh. naturwiss ver hamburg. 26: 195–200. nicholson gm, graudins a (2002) spiders of medical importance in the asia-pacific: atracotoxin, latrotoxin and related spider neurotoxins. clin exp j pharma physiol. 29: 785–794. nihei n, yoshida m, kaneta k, shimamura r, kobayashi m (2004) analysis on the dispersal patern of newly introduced latrodectus hasselti (araneae: theridiidae) in japan by spider diagram. j med entomol.41 (3): 296–276. nimorakiotakis b, winkel kd (2004) spider bite-the red back spider and its relatives. reprinted from australian family physician 33(3): 153–157. rafinejad j (1997) an investigation on the systematic, biology and ecology of widow spiders (latrodectus spp.) in khorasan province, iran. [phd dissertation]. school of public health, tehran university of medical sciences, iran. roth vd (1982) handbook for spider identification. dept zool univ florida, gainesville, fl 32611 stallybrass fc (1969) spider bites. lancet. 15, 1: 572. vetter rs, visscher pk (1998) bites and stings of medically important venomous arthropods. international j dermatol. 37: 481–496. iran j arthropod-borne dis, 2011, 5(1): 63–68 m shahi et al.: the occurrence of red … 68 walsh jf, molyneaux dh, birley mh (1993) deforestation: effects on vector-borne disease. parasitol. 106: s55–s75. j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 515 http://jad.tums.ac.ir published online: december 30, 2017 original article dynamics of transgenic enterobacter cloacae expressing green fluorescent protein defensin (gfp-d) in anopheles stephensi under laboratory condition hossein dehghan 1, *mohammad ali oshaghi 1, *seyed hassan moosa-kazemi 1, bagher yakhchali 2, hassan vatandoost 1,3, naseh maleki-ravasan 4, yavar rassi 1, habib mohammadzadeh 5, mohammad reza abai 1,3, fatemeh mohtarami 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department industrial and of environmental biotechnology, national institute of genetic engineering and biotechnology, tehran, iran 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4malaria and vector research group, biotechnology research center, pasteur institute of iran, tehran, iran 5department of parasitology and mycology, faculty of medicine, urmia university of medical sciences, urmia, iran (received 6 jan 2017; accepted 23 may 2017) abstract background: enterobacter cloacae bacterium is a known symbiont of the most anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. however, the population dynamics of this bacterium within an. stephensi and its introduction methods to the mosquitoes have not yet been explored. methods: enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (gfp-d) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. the bacterial quantities were measured by plating samples and counting gfp expressing colonies on the tet-bhi agar medium. results: the e. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. the bacterium was weakly transmitted transstadially from larval to adult stage. the bacterial populations increased smoothly and then dramatically in the guts of an. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time. conclusion: enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult an. stephensi within 24h post blood meal. sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. it is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions. keywords: bacterial dynamic, enterobacter cloacae, anopheles stephensi, paratransgenesis introduction malaria is a mosquito borne disease considered as an important threat to public health in tropical and semi-tropical areas of the world. although iran currently is in elimination phase and malaria cases are significantly reduced, the disease is still considered a serious health concern, mostly in the south and southeast corner of the country (1-3). in the southern regions of the country there are six anopheles mosquito vectors including anopheles stephensi, an. culicifacies s.l, an. dthali, an. fluviatilis s.l, an. superpictus s.l. and an. pulcherrimus (4-12). however, an. stephensi has been considered the main ma *corresponding authors: dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir, dr seyed hassan moosakazemi, email: shm.kazemi@gmail.com http://www.sciencedirect.com/science/article/pii/s0001706x12001817#bib0085 mailto:moshaghi@tums.ac.ir mailto:shm.kazemi@gmail.com j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 516 http://jad.tums.ac.ir published online: december 30, 2017 laria vectors in iran (13-15). this species has shown a wide range of susceptibility/resistant to various insecticides in iran (16-20). currently, this species is resistant to lambda cyhalothrin and cyfluthrin and susceptible to etofenprox, and permethrin and candidate of resistant to deltamethrin in the country (21). anopheles stephensi is one of the most important malaria vectors in middle east and indian subcontinent regions and its resistance to organochlorides, organophosphates, carbamates and pyrethroids insecticides have been widely reported in these regions (22-24). emergence of insecticide resistant mosquitoes plus the emergence of drug-resistant in the parasites highlights the needs for alternative strategies for sustainable malaria control. as an alternative to chemical insecticides, paratransgenesis depends on engineered symbiotic microorganisms particularly bacteria of malaria vectors to supply molecules that can kill plasmodium inside the mosquito gut and or inhibit pathogen transmission (25, 26). transmission of the plasmodium parasite is strongly dependent on completion of the parasite life cycle in the mosquito vector since entering to midgut, across the peritrophic membrane (pm), midgut epithelium, salivary glands and transmitted through saliva to new host. the most bottleneck during the development of plasmodium parasite occurs in ookinete stage. it could be considered as the main target for control of parasite in mosquito vectors (27-31). some known factors involved in creating parasite bottleneck, including gut digestive enzymes, intestinal microbial flora, and the mosquito's immune response. microflora performs vital role in preventing the development of pathogens. this effect exerts directly by proliferation of bacteria after blood meal simultaneously with ookinete stage of plasmodium. besides, indirect effects on the survival of plasmodium parasite are applied by inducing the expression of anti-microbial genes against the bacteria (32-38). accordingly, bac terial symbionts are genetically modified to express toxic peptides against pathogens, can be considered as an alternative approach for disease control (39). this strategy, commonly named paratransgenesis (40), requires several steps of research on the biology of vectors and vector symbionts and its evaluation in the laboratory and field conditions (41-43). the midgut bacterial flora of wild-caught mosquitoes is very dynamic and significant fluctuations depending on the stage of life, nutrients and the physiological age (31, 44). population structure of symbiotic bacteria is considerably changed post blood meal and gram-negative bacteria will be dominant and could survive in harsh condition of midgut with digestive enzymes (31). there are scattered studies on insect gut microflora (15, 45, 46 and references herein) and still remained some questions in relation to species composition, stability as well as their acquisition of microbiota (47-49). punpuni et al. (1996) (33) reported at least nine species of cultivable midgut bacteria with varied composition of an. stephensi, an. gambiae and an. albimanus. in the same study, variety composition of midgut bacteria flora was found in an. gambiae and an. funestus and some anopheles mosquitoes (49-59). a few of mosquito microflora able to pass from larvae to adult stages because of the differences between the larval (aquatic) and adults (terrestrial) habitats (41, 44). some bacteria are able to colonize in the malpighian tubules and transstadially pass from larvae to adult and presumably remain for long duration in the female gut (15). therefore, such symbiotic bacteria added to the diet of adult mosquitoes (60). enterobacter cloacae bacterium is a species of gram-negative, facultative anaerobic, rod-shaped bacteria belonging to gammaproteobacteria and enterobacteriaceae family. the bacteria species limited the development of plasmodium berghei and p. falciparum by stimulate the immune system of an. stephensi and increases the j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 517 http://jad.tums.ac.ir published online: december 30, 2017 expression of immune responses compounds such as serine protease inhibitors (srpn6) (31). enterobacter cloacae bacterium was found as the microflora of an. stephensi (57). gonzalez-ceron et al. (51) reported e. cloacae restricted the p. vivax development in midgut of an. albimanus. the bacterium also was reported from culex tarsalis (61), psorophora columbiae (62), aedes triseriatus (62) and ae. albopictus and ae. aegypti (63). due to the ability of e. cloacae to direct and indirect control of plasmodium parasites, these bacteria could be introduced as a candidate for paratransgenesis approach against the malaria parasite. maleki-ravasan et al. (46) suggested e. cloacae dissolvens as a candidate for paratransgenesis approach to control of leishmania transmission in the sand flies vectors. they genetically modified the bacterium to produce defensin as a plasmodium killing effector protein. defensins are small cysteinerich cationic proteins and found in plants, vertebrates and invertebrates. they are active against fungi, bacteria, and many viruses. to use recombinant bacteria in practice, however, it is required a better understanding of the bacteria dynamics in mosquitoes and its delivery systems to vectors. in this study, we evaluated the dynamics of e. cloacae dissolvens expressing green fluorescent and defensin proteins (gfp-d) in midgut of an. stephensi life stages as well as in larval habitats and sugar bait used as two delivery systems for the mosquito in the laboratory condition. materials and methods the mosquitoes anopheles stephensi, beech strain originally collected from pakistan as an additional type form ie sda500 strain originating was provided in 2005 by professor p.f. billingsley, sanaria, inc (64). breeding of the mosquitoes carried out in 27±1 °c and 60±10% relative humidity with photoperiodic period of 12h. adult mosquitoes were kept in 30×30×30 cages. mosquito feeding was carried out using fructose 5% and guinea pigs twice a week. anopheles gravid, laying eggs in earthenware bowl containing decolorized water. the eggs were slowly transferred to tray having 1500ml of decolorized water before hatching. the fish food and a piece of leaf lettuce used as specific diet for anopheles larvae. the bacteria enterobacter cloacae subsp. dissolvens was isolated by sampling microflora of phlebotomus papatasi in the field of zoonotic cutaneous leishmaniasis in isfahan, central iran, 2013– 2014 (46). the manipulated strain of e. cloacae is carrying plasmid expressing difensin and gfp proteins and a gene resistant to tetracycline (tet-gene). this strain known as enterobacter cloacae-gfp-difensin (e. cloacaegfp-d) maintained in the school of public health, tehran university of medical sciences. the bacterium was grown in brain heart infusion (bhi) broth culture until stationary phase, as determined by spectroscopic optical density (od) measurements at 600nm. the bacteria were prepared by growing to an od 600 of = 1 in tet-bhi broth medium. several dilutions of the od 600= 1 were prepared covering a wide region of optical density from 0.1 to 1 and plated onto tet-bhi agar for viable cell determination. the plates were incubated for 13h at 37 °c before counting the number of colony forming units (cfu). the gradients of the calibration curves showed that od 600nm of 1.0 was corresponding to approximately 1×109 cfu per ml bhi broth medium. dynamics of enterobacter cloacae-gfp-d corncob-bacteria formulation (ccf) and sugar bait-bacteria the method of arshad et al. (65) was used to prepare corncob formulation (ccf) of e. cloacae-gfp-d and used as the bacteriumfloating carrier in larval tray. the corncobs j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 518 http://jad.tums.ac.ir published online: december 30, 2017 were autoclaved followed by grinding to small particles less than 0.5mm in diameter. the medium containing e. cloacae centrifuged and the precipitated cells washed and suspend in 100µl pbs. the bacteria-pbs buffer was added on corncob and dried at room temperature. almost 100µl of bacteria-pbs suspension was used for 0.1g grind corncob. almost 0.1g of the dried ccf containing 5×109 bacterial cells was used for one litter of larval tray water in insectary condition. sugar bait-bacteria were prepared by using 109 the bacterial cell per 1ml of fructose 5% and 2.5% red food dyes (fig. 1) according to the method previously described by wang et al. (66). introduction enterobacter cloacae-gfp-d to mosquito larval habitats about 200–300 an. stephensi eggs were transferred to tray containing sterile water and then the hatched larvae were transferred randomly to the test and control trays. the larvae were fed either on intact corncob and a piece of leaf lettuce in control tray or ccf and a piece of leaf lettuce in test tray. the number of released bacteria in test tray was 109 bacterial cells per liter of sterile water. the ccf was added following emergence of the first (l-i) and fourth (l-iv) instar larvae in test tray. transstadial and dynamics of the bacteria were investigated by sampling of water and mosquitoes at larvae and adult stages. dynamics of enterobacter cloacae-gfp-d in larval habitat to test the course of time on proliferation and stability of e. cloacae-gfp-d in larval habitats of mosquitoes, water sampling was carried out daily from the test trays. for each sample, 10ml of the water was centrifuged at 13000 rpm for 10min followed by removing supernatant; the pellet was mixed with 1ml pbs buffer and vortexed. finally, serial dilution of the bacterial suspension was prepared and 100µl of the proper dilution cultured in tet-bhi agar. the number of colonies (colony forming units: cfus) was counted under a flu orescent microscope and number of the e. cloacae bacteria in one ml of test tray water was measured. bacterium transstadial transmission to test the transstadial transmission of e. cloacae-gfp-d bacteria from larvae to adult stage of the mosquitoes we followed the method previously described by lindh et al. (48). on brief, 5×106 bacteria as ccf per ml sterile water were released in larval breeding trays. larvae were kept under laboratory condition until pupae stage. fresh pupae were washed twice in sterile water and transferred to a new tray with 1000ml sterile water until adults emerged. after eclosion, the adult body surface of some specimens was sterilized in 70% alcohol, then the midgut dissected, homogenized in pbs, and cultivated in tet-bhi agar/broth for 13h in 37 °c. however, some adults were transferred to 15×15×30cm cage and fed on sterile sugar solution during 24h. the female mosquitoes were blood fed on balb/c mice and midgut microflora was determined by described method. the number of colonies (cfus) was counted under a fluorescent microscope for all experiments (fig. 2). dynamics of enterobacter cloacae-gfp-d in an. stephensi larvae in each sample, five larvae were selected and their surface bodies were sterilized by alcohol. briefly, the larvae were transferred to microtubes containing 500µl sterile water and kept on ice for a few minutes until the larvae were numb, then 500µl of 70% cold ethanol (-5 °c) was added to the microtube after removing water and kept on ice about 5 min. the alcohol was removed and the larvae were washed twice with pbs buffer (4 °c). finally, the total body of sterilized larvae homogenized in pbs buffer and 100ml of homogenized solution was cultured in tet-bhi agar plates. alternatively, the larvae were disj arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 519 http://jad.tums.ac.ir published online: december 30, 2017 sected and their guts were homogenized in pbs buffer, cultured in tet-bhi agar plates, and cfus were counted as above. simultaneously, survival rate of an. stephensi larvae in the test and control (corncob contaminated with/out e. cloacae-gfp-d) trays were investigated. dynamics of enterobacter cloacae-gfp-d in anopheles stephensi adult gut in order to study population dynamics of e. cloacae-gfp-d in an. stephensi adult, the 3–5d old female mosquitoes were transferred to 15×15×30cm cage and fed on sugar bait containing 109 the bacterial cell /ml fructose 5%, and 2.5% food dyes (red) according to the method previously described by wang et al. (66) (fig. 1). to count the bacteria populations (cfus) in the adult guts, the female mosquitoes were numbed and immersed in ethanol 70% for 5–7min, placed on glass slides for 3–5min, and then transferred to microtube and homogenized in pbs buffer. finally, 100µl of homogenized suspension samples was cultured in tet-bhi agar plates. in an alternative method, after body surface sterilization, mosquito midguts were dissected and homogenized in pbs buffer. to evaluate course of time and sugar/blood meals on the cfus of the bacterium, two separate experiments were designed. in the first experiment, 3–5d old female mosquitoes were fed on sugar bait, a subset of specimens was tested for the number of the bacteria immediately (1 hour) post sugar feeding. then the sugar bait was removed from the cage for 7h and again the bacteria population was tested in a subset of females. eight hours post sugar feeding, a blood meal was offered to the mosquitoes and then a subset of mosquito guts was tested for the bacteria at 12, 18, 24, 36, 48, 96, and 144h post blood meal. after day-6 (144h), a second blood meal was offered to the flies, and cfus were counted for the guts 24 and 36h post second blood meal. in the second experiment, 3–4d old female mosquitoes were fed on sugar bait, a subset of specimens was tested for a number of the bacteria immediately (1 hour) post feeding. then the sugar bait removed from the cage for 7h and again the bacteria populations were tested in a subset of females. again, eight hours after first sugar meal, second sugar meal was offered to the mosquitoes and the cfus were measured for the guts 24, 48, and 72h post second sugar meal. then a blood meal was offered and a subset of mosquito guts was tested for the bacteria cfus at 24 and 48h post blood meal. in both experiments, 3–4h in advance of offering blood meal, sugar bait containing the bacteria were removed from the cage, and after blood meal, the mosquitoes were kept on sterile cotton pad soaked with 5% fructose. stability of enterobacter cloacae-gfp-d in sugar bait colony-forming units of the bacteria in sugar bait containing 109 bacteria cell /ml fructose 5% and 2.5% red food dyes was determined by daily sampling during eleven d. sampling was carried out as follow, by pressing the cotton pad, 10µl of the above suspension was collected and added to 990µl of sterile pbs buffer and prepared serial dilutions. about 100µl of the final solution was cultured in tet-bhi agar plates. after counting the colonies, number of bacteria per ml of bait was estimated. statistics and analytical procedure where it was necessary, the concentrations of cfus were indicated using logarithmic notation, where the value was shown is the base 10 logarithm of the concentration. bacteria relative abundance was calculated separately for each treatment. the average percentage of lifespan and larval death rate were presented. a significant difference in the bacteria relative abundance between samples was analyzed using the mann–whitney test. multiple-sample comparisons were analyzed using the nonhttps://en.wikipedia.org/wiki/common_logarithm https://en.wikipedia.org/wiki/common_logarithm j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 520 http://jad.tums.ac.ir published online: december 30, 2017 parametric kruskal–wallis test, and medians were compared using dunn’s test. graphpad prism version 5.00 for windows (graphpad software) was used for all statistics. p< 0.05 was considered statistically significant. results corncob formulation (ccf) stability the most attractive features of corncob were considered as lightness and floating on the water surface. when this formulation supplied on mosquito breeding place, anopheles larvae attracted to and fed on corncob particles. approximately, 90–95% and 50–60% of ccf particles remained floated on the tray water after 24 and 48h of their releasement, respectively. effect of enterobacter cloacae-gfp-d on survival of anopheles stephensi larvae the survival rate of an. stephensi larvae in the test tray containing e. cloacae-gfp-d significantly was more than the control group at late larval stage. vis-versa results of this experiment showed that death rate of control were significantly lower than the test group at larval stage ii (fig. 3, 4). peak mortality in test groups occurred about days 8–9, whereas it happened about days 10–14 in control group (fig. 4). effect of enterobacter cloacae-gfp-d on development rate of anopheles stephensi larvae development of an. stephensi larvae took longer time when kept in sterile water or in water containing e. cloacae-gfp-d in comparison with the ones kept in non-sterile water. on average adult, mosquitoes appeared 8– 10d from the time of egg hatching when the larvae had kept in non-sterile water whereas it took 14–18d when the larvae bred in sterile water or water containing e. cloacae-gfp-d. both test and control groups were supplied by a piece of lettuce and corncob. dynamics of the bacteria in the larval stages and habitat of anopheles stephensi population dynamics of e. cloacae-gfpd had a descendant trend in the larval habitat indicating a weak recycling in water at insectary condition. the population of e. cloacaegfp-d in the water decreased from 2×107 cfus/ml at day-1 to 2960 cfus/ml at the end of day-14 (fig. 5). trend of the bacteria in the guts of an. stephensi larvae also declined significantly from about 12000 cfus in the second instar larvae (l-ii) to less than 100 cfus in the fourth instar larvae (l-iv) which is corresponding to the decreasing trend of the bacteria in the larval habitat water (fig. 5). transstadial transmission enterobacter cloacae-gfp-d can survive and flourish in the guts of an. stephensi larvae but inadequately transmit transstadially from larvae to adult stage. the rate of bacteria positive in larval guts at late instars was more than 75% with a range of 7 to 756 cfus per gut. however, most of the bacteria were removed from midgut in pupal stage due to histolysis and histogenesis phenomena. none of the pupae were positive for the bacteria and there were only 2–3 adult specimens (8–12%) were positive for e. cloacae subsp. dissolvens bacteria indicating very low transstadial transmission (table 1). there were only a few (n= 4) bacteria in the newly emerged adult mosquito. the number of bacteria increased dramatically following a blood meal and reached to 10000 cfus (table 1). course of time and blood meal on enterobacter cloacae-gfp-d in adult anopheles stephensi midguts we tested the effect of blood meal and course of time on loads of the bacteria in adult mosquitoes. number of the bacteria in the adult mosquito midgut fed on sugar bait containing e. cloacae-gfp-d was on average one million per mosquito gut one-hour post sugar bait feeding. however, populations of the bacteria decreased about 100 folds after seven hours fasting (fig. 6). loads of bactej arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 521 http://jad.tums.ac.ir published online: december 30, 2017 ria were dramatically increased about 73000 folds on average and maximized up to 155 million (about 73000000 on mean) cfus 24h post blood meal. the bacteria populations were then declined gradually and reached to about 100 cfus/gut 144h after (6d) blood meal intake. this trend, i.e. increase, and decrement of the bacteria populations happened again following second blood meal with a slightly lower rate than first blood meal (fig. 6). to test the effect of only sugar meal (fructose 5%) on the bacteria population, in a separate experiment, the mosquitoes were fed on sugar bait containing e. cloacae-gfp-d and then starved for 8h. then they were fed on fructose 5% with no bacterai using cotton pad and the load of bacteria was counted daily for three following days. after 24h following normal sugar meal, the bacteria population raises up about 10 folds (10000 to 99000 cfus per gut) and again by course of time, the bacteria population decreased again (fig. 7). the number of bacteria dropped about 1000 folds and reached to about 10 cfus/gut after 72h (3d) (fig. 7). a blood meal intake following three days causes tremendous (140000 folds) uprise in the number of adult guts bacteria but lower than the previous experiment (14 million versus 155 million). dynamics of enterobacter cloacae-gfp-d in sugar bait in order to determine the course of time on survival of e. cloacae-gfp-d in suspended sugar bait, trend of e. cloacae-gfp-d populations was investigated in the sugar solution of cotton pad. the number of bacteria was decreased 10 folds (from 1 million to 100000 cfu/ml) throughout 11d in the insectary condition (fig. 8). table 1. details of transstadial transmission of enterobacter cloacae-gfp-d in anopheles stephensi in insectary condition. numbers refer to the guts harbor the bacteria out of 25 specimens. life stages tet-bhi broth tet-bhi agar (cfus/gut) larvae iv 19 17 (7–756) pupae 0 0 adult blood fed 2 1 (10,000) adult unfed 1 1 (4) fig. 1. sugar bait containing 109 bacteria cell/ml (cfus) fructose 5% and 2.5% food dyes used to introduce the enterobacter cloacae-gfp-defensin bacteria to mosquitoes via sugar feeding. the dye made abdomen reddish and visually distinguishable. j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 522 http://jad.tums.ac.ir published online: december 30, 2017 fig. 2. the presence of enterobacter cloacae-gfp-defensin in dissected anopheles stephensi midgut (squares) and bhi agar plates (circles), the bacteria with/out expressing green fluorescent protein in bhi agar plate under nonfluorescent (a and c) and fluorescent (b and d) microscope l -i l -i i l -i ii l -i v 0 20 40 60 80 control e.cloacae-gfp-d larval stage m e a n o f s u rv iv a l ra te fig. 3. mean of survival rate of anopheles stephensi larvae in test groups (sterile water+corncob-enterobacter cloacae-gfp-defensin) and control (sterile water+corncob) throughout larval development stages (14d) in insectary condition. the bars represent standard error of the mean j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 523 http://jad.tums.ac.ir published online: december 30, 2017 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 5 10 15 20 25 e.cloacae-gfp-d control time (day) d e a th r a te fig. 4. mortality rate of aquatic stages of anopheles stephensi in test (sterile water+ ccf: corncob-enterobacter cloacae-gfp-defensin) and control (sterile water+corncob) tray throughout 14d in insectary condition. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.0100 1 1.0100 2 1.0100 3 1.0100 4 1.0100 5 1.0100 6 1.0100 7 1.0100 8 1.0100 9 1.0101 0 water larvae l-ii l-iii l-iv time (day) c f u s /m l o r c f u s /g u t fig. 5. trend of enterobacter cloacae-gfp-defensin in the anopheles stephensi larval habitat water (above) and guts (down) in insectary condition. l-ii, l-iii, and l-iv represent larval stage ii, iii, and iv respectively j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 524 http://jad.tums.ac.ir published online: december 30, 2017 1 8 12 18 24 36 48 96 144 24 36 1.0100 1.0101 1.0102 1.0103 1.0104 1.0105 1.0106 1.0107 1.0108 1.0109 1st blood meal 2 nd blood meal hours after sugarbait & blood meal sugar bait meal c f u s /g u t (l o g ) fig. 6. dynamics of enterobacter cloacae-gfp-defensin populations in the midgut of adult anopheles stephensi fed on sugar bait containing 109 the bacteria cell/ml fructose 5%. midgut status 12, 18, 24, and 36h post blood meal is shown underneath. the bars represent standard error of the mean 1 8 24 48 72 24 48 1.0100 1.0101 1.0102 1.0103 1.0104 1.0105 1.0106 1.0107 sugar meal blood meal hours after sugar bait, sugar & blood meal sugar bait meal c f u s /g u t (l o g ) fig. 7. course of time, sugar, and blood meal on the dynamics of enterobacter cloacae-gfp-defensin populations in the midgut of adult anopheles stephensi. mosquitoes were fed on sugar bait containing 109cfus/ml fructose 5%, then kept starve for 8h, and then fed on sugar (cotton pad) for three days continuously and on blood meal after 72h. the bars represent standard error of the mean j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 525 http://jad.tums.ac.ir published online: december 30, 2017 1 2 3 4 5 6 7 8 9 10 11 1.0100 1.0101 1.0102 1.0103 1.0104 1.0105 1.0106 1.0107 day c f u s /m l fig. 8. trend of enterobacter cloacae-gfp-defensin population in the sugar bait solution containing 109cfus/ml fructose 5% at the beginning of experiment (day zero) in insectary condition throughout11d. discussion we investigated transstadial transmission and dynamics of e. cloacae-gfp-d within an anopheles stephensi mosquito colony with the focus on larval habitat and sugar bait as two potential resources or delivery systems for introduction of the transgenic bacteria to mosquitoes. our observations revealed that the symbiont e. cloacae-gfp-d strain was not able to colonize well in larvae midgut or water habitats of an. stephensi in laboratory rearing condition. the number of bacteria reduced progressively in both water and larvae midgut. besides, number of the bacteria transferred transstadially was very low (4%). in this experiment, the bacteria were administrated via corncob formulation and drinking water, which is similar to the colonization patterns under a natural condition where bacterial symbionts are acquired throughout feeding or drinking water. our larval rearing experiments evidently showed negative effects of e. cloacae-gfpd on the development speed of an. stephensi larvae. these findings are in agreement with a previous study showed that e. cloacae could not isolate from the midgut of insectary specimens of an. albimanus weidemann from southern mexico whereas the bacteria was already isolated from field specimens (51). however, a great diversity of microbiota of larvae and adult mosquito gut has been already reported by other researchers (57, 45, and 15). however, it is currently unclear why the e. cloacae symbiont is not recycling in larval habitats at insectary condition. in contrast to the larvae, colonization of the e. cloacae-gfp-d bacteria proceeds rapidly in adult mosquito midguts following sugar or blood meal. colonization occurred within a few hours after taking meal and maximized 24h post blood meal. the blood proteins apparently caused quick growth of midgut bacteria and when blood digestion is completed (on day 6 or later) most bacteria were defeated with blood remains. the peak activity of e. cloacae-gfp-d would be synchronized with ookinete formation of plasmodium parasite within the mosquito midguts. by proliferation of e. cloacae-gfp-d in the adult j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 526 http://jad.tums.ac.ir published online: december 30, 2017 midgut, it could disrupt development of plasmodium parasite directly by remarkable production of defensin, a killing parasite molecule, and indirectly by induction of anopheles immune responses (33, 66, 67, 25). in this experiment, the bacteria were administrated via sugar bait, which is similar to the colonization patterns under a natural condition where bacterial symbionts are acquired throughout sap feeding. adult mosquitoes may acquire bacteria from at least one of the three following sources: animal host skin while taking blood meal, plant saps through sugar feeding, and transstadially transmitted bacteria from larvae to adult (46, 59,68). knowing bacterial acquisition routes is essential and play important role for paratransgenic approach against malaria vectors since it commands how to introduce transgenic bacteria to the field condition. we found that e. cloacae-gfp-d is not able to propagate in the larval habitat and midgut and it does not transfer transstadially from larvae to pupae as well as not from pupae to adults, so the mosquitoes are not able to take up the bacteria from larval habitats. this indicates that introduction of e. cloacae-gfp-d to breeding sites of an. stephensi is not recommended in a paratransgenic approach. in contrast, results of introduction of the bacteria via sugar bait containing modified bacteria were promising. propagation of the bacteria in the adult midgut and it’s prolonged stability in sugar bait revealed that sugar bait is an effective mean to introduce engineered bacteria into field mosquito populations. this may be achieved by placing sugar bait stations with attractive material such as fruit juice embedded at attractive places such as pit shelters, black box trap, and earthenware crock close to breeding sites of anopheles mosquitoes (53, 67, 69). sugar bait station was evaluated in semi-field condition by mancini et al. (70) and showed that the modified bacteria were effectively capable of spreading at high rate in different an. stephensi and an. gambiae populations, and successfully colonizing in the mosquito midguts. recently kotnis and kuri (71) evaluated scenarios to calculate a number of required sugar baits and bait distribution to prevent a malaria outbreak. in our experiment, the e. cloacae-gfp-d was stable well in the cotton pads and remained viable at high rate through 11d which support suitability of sugar bait as a worthy mean for bacterial introduction to field. however, the stability and survival of e. cloacae-gfp-d in sugar bait should be evaluated in semi-field and field conditions in advance to use it in a real paratransgenic strategy. the e. cloacae strain we used was easily cultivated outside the insect host on normal and cheap microbiological media and also genetically manipulated. in this study, we successfully used a gfp-defensin recombinant strain of the e. cloacae symbiont which allowed tracing of cells in plates originated from specimens such sugar baits and dissected midgut of larvae and adult mosquitoes. this bacterium was orally acquired successfully by adult mosquitoes from sugar bait that can be easily administered under semi-field and field conditions. enterobacter cloacae have already been tested to deliver, express, and spread foreign genes in termite colonies (72) and mulberry pyralid moth, glyphodes pyloalis (73). the corncob formulation we prepared in this study was floated satisfactory on water surface for two days to supply the modified bacteria in insectary condition. this formulation was simple and needs to be developed to other known formulation such granule. corncob has been successfully used as granule to supply bacillus thuringiensis and b. sphaericus to control various larval mosquitoes in semi-field and field conditions (74-76). factors potentially may influence the efficiency of formulation such as dosage of formulation, precipitation, flooding of the treated sites, and presence of other aquatic animals like fishes should be tested. j arthropod-borne dis, december 2017, 11(4): 515–532 h dehghan et al.: dynamics of … 527 http://jad.tums.ac.ir published online: december 30, 2017 conclusion sugar bait station is the best method for introduction of e. cloacae-gfp-d into the field condition. the population of the bacteria was increased dramatically within 24h post blood meal. it can interrupt malaria parasite development in the mosquito midgut. this administration is similar to the colonization patterns under a natural condition where bacterial symbionts are acquired throughout sap feeding. on the other hand, lack of proliferation of the bacteria in breeding sites and subsequently in the larval midgut disapproved introduction of the bacteria in anopheles larval habitat for a paratransgenetic approach. acknowledgments this study was supported by the tehran university of medical sciences, iran, grant number 26231. we thank ms talaei (central laboratory of sph, tums) and ms salimi (department of medical parasitology, sph, tums) for helping in fluorescent microscopy and fatemeh rafie (department of medical entomology and vector control, sph, tums) for rearing mosquitoes. the authors declare that there is no conflict of interest. references 1. anjomruz m, oshaghi ma, pourfatollah aa, sedaghat mm, raeisi a, vatandoost h, khamesipour a, abai mr, mohtarami f, akbarzadeh k, rafie f, besharati m (2014) preferential feeding success of laboratory reared anopheles stephensi mosquitoes according to abo blood group status. acta trop. 140: 118–123. 2. karimian f, oshaghi ma, sedaghat mm, waterhouse rm, vatandoost h, hanafi-bojd aa, ravasan nm, chavshin ar (2014) phylogenetic analysis of the oriental-palearctic-afrotropical members of anopheles (culicidae: diptera) based on nuclear rdna and mitochondrial dna characteristics. jpn j infect dis. 67(5): 361–367. 3. norouzinejad f, ghaffari f, raeisi a, 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https://www.ncbi.nlm.nih.gov/pubmed/?term=vogel%20kj%5bauthor%5d&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=brown%20mr%5bauthor%5d&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=strand%20mr%5bauthor%5d&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=strand%20mr%5bauthor%5d&cauthor=true&cauthor_uid=24766707 https://www.ncbi.nlm.nih.gov/pubmed/24766707 https://www.ncbi.nlm.nih.gov/pubmed/24766707 https://www.ncbi.nlm.nih.gov/pubmed/?term=m%c3%bcller%20gc%5bauthor%5d&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=beier%20jc%5bauthor%5d&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=traore%20sf%5bauthor%5d&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=toure%20mb%5bauthor%5d&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=toure%20mb%5bauthor%5d&cauthor=true&cauthor_uid=20663142 https://www.ncbi.nlm.nih.gov/pubmed/?term=traore%20mm%5bauthor%5d&cauthor=true&cauthor_uid=20663142 http://www.ncbi.nlm.nih.gov/pubmed/26965746 http://www.ncbi.nlm.nih.gov/pubmed/26965746 http://www.ncbi.nlm.nih.gov/pubmed/26965746 j arthropod-borne dis, september 2018, 12(3): 269–275 se eskandari et al.: safety evaluation of … 269 http://jad.tums.ac.ir published online: september 30, 2018 original article safety evaluation of nano-liposomal formulation of amphotericin b (sina ampholeish) in animal model as a candidate for treatment of cutaneous leishmaniasis seyed ebrahim eskandari 1, alireza firooz 1, mansour nassiri-kashani 1, mahmoud reza jaafari 2, 3, amir javadi 1, 4, akram miramin-mohammadi 1, hossein valian-keshavarz 5, 6, *ali khamesipour 1 1center for research and training in skin diseases and leprosy (crtsdl), tehran university of medical sciences, tehran, iran 2nanotechnology research center, pharmaceutical technology institute, mashhad university of medical sciences, mashhad, iran 3department of pharmaceutical nanotechnology, school of pharmacy, mashhad university of medical sciences, mashhad, iran 4school of allied sciences, tehran university of medical sciences, tehran, iran 5department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 6center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran (received 22 aug 2017; accepted 10 feb 2018) abstract background: development of a topical treatment for cutaneous leishmaniasis (cl) is an important step in the improvement of lesion management. amphotericin b (amb) is effective against leishmania species but it is toxic, a nano-liposomal form of amb with a size of about 100nm (lip-amb) was developed and showed to be effective against leishmania major, and leishmania tropica in vitro and against l. major in vivo in animal model. this study was designed to check the irritancy draize test in rabbits and was completed in the center for research and training in skin diseases and leprosy, tums, in 2012. methods: twenty rabbits in 3 steps were housed individually with artificial lighting (12/12h light/dark). sinaampholeish cream or empty liposomes (prepared under gmp condition at minoo company, tehran, iran), was applied on a gauze patch and the patches were placed on the designated sites of the skin in the back of the rabbits. at 48 and 72h later, the erythema and oedema were checked, scored and recorded. results: the erythema score in rabbits was 0.83+0.41 for the sinaampholeish and 0.5+0.55 for empty liposomes (p= 0.16). the average score for oedema was 0.67+0.52 for sinaampholeish and 0.33+0.52 for empty liposomes (p= 0.16). conclusion: based on skin irritancy reactions the topical formulation of sinaampholeish is safe and could be further checked in human trials. keywords: cutaneous leishmaniasis, nano-liposomes, amphotericin b, draize test introduction cutaneous leishmaniasis (cl) is endemic in 88 countries (72 of them are developing countries and 16 developed ones) and 90% of cl cases are reported from 6 countries including brazil, peru, afghanistan, saudi arabia, syria and iran (1-3). pentavalent antimonial derivatives (sodium stibogluconate=pentostam and meglumine antimoniate=glucantime) are still considered as the standard first-line treatment for cl. currently available treatments are far from acceptable criteria either by the patients or by the health authorities due to pain at the site of injections, high risk of side effects, high cost and low efficacy, moreover resistant is *corresponding author: ali khamesipour, e-mail: ali.khamesipour@gmail.com, khamesipour@tums.ac.ir, khamesipour_ali@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 269–275 se eskandari et al.: safety evaluation of … 270 http://jad.tums.ac.ir published online: september 30, 2018 reported from endemic areas. the development of new treatment is highly demanded, invention of a topical treatment for cl is very essential to increase the compliance rate and control of anthroponotic cl (4-7). amphotericin b (amb) is a polyene antibiotic produced from streptomyces nodosus. several formulations of amb have been marketed and used to treat fungal infections and visceral leishmaniasis, but the main drawback is toxicity. ambisome™ is a liposomal form of amb which is the first line treatment for visceral leishmaniasis in some endemic areas but is not as effective against cl (8-11). a nano-liposomal form of amphotericin b (lipamb) with a size of about 100nm was formulated and the method of preparation is under us patent. the formulation is effective against leishmania major and leishmania tropica in vitro and against l. major in vivo in a murine model of leishmaniasis (9). largescale production of 0.4% lip-amb was done under good manufacturing practice guidelines under the trade name of sinaampholeish at minoo company of (tehran, iran). draize test was initially introduced to test various medical, cosmetic, and chemical substances, but was also later used to check the safety of several other products such as cream, lipstick, mascara, face lotion, soap, shampoo, powder, hairspray, toothpaste, lacquer, detergent and anything, which could possibly induce skin irritation (10). it has been modified and updated several times and the most commonly used method is the one introduced by federal hazardous substance act, title 16, chapter ii, reference consumer product safety commission 1980 as a standard test to check skin irritancy reactions. the rabbit is the most commonly used animal for skin irritation test and usually, 6 rabbits are used (11-14). the in vivo rabbit eye irritation/corrosion test developed from draize skin test since 1981. it is used for two types of substances that either might be exposed to the eye due to regular use in or around the eyes (such as eye creams) or through accidental exposure of the eyes to the products that are not originally designed to be in contact with eyes (such as shampoos or skin creams). the evaluation of eye irritation potential for cosmetic substances is necessary to assure that the products are safe for consumers if eyes are exposed to intentionally or accidentally. usually, 0.1 ml (or weight equivalent approximately 100mg) of the test substance is placed into one eye and the other eye is not treated and kept as control. the treated eye and the control eye are checked regularly after application of the product, usually 1, 24, 48 and 72h after exposure or more frequently if required. eye irritation is defined as induction of pathological changes in the eye fully recovered within 21d or tissue damages in the eye which might not fully recover even within 21d (14, 15). the objective of this study was to evaluate the safety and irritancy potential of sinaampholeish in rabbit model (draize test) as well as an eye irritation test, as a step prior to clinical trials in humans. materials and methods the study was conducted in 2012 in the center for research and training in skin diseases and leprosy, tums, tehran, iran. the proposal of this study was approved by the ethical committee of center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran five-month-old male and female rabbits were purchased from razi vaccine and serum research institute, hesarak, iran and kept in the animal house of crtsdl. rabbits were housed individually with artificial lighting (12h light, 12h dark) and free access to conventional laboratory diets and an unrestricted supply of drinking water with a collar fitted http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 269–275 se eskandari et al.: safety evaluation of … 271 http://jad.tums.ac.ir published online: september 30, 2018 and fastened around the rabbit’s neck. the irritation test was performed in 3 steps: step 1) healthy white rabbits with no hair loss were purchased and hairs on the back of the animals were shaved. after 24h, 6 rabbits with no sign of abrasion, erythema or oedema due to shaving were selected, 500mg of either 0.4% sinaampholeish gel or empty liposomes (prepared under gmp condition at minoo company, tehran, iran), was applied on a gauze patch and the patches were placed on the designated sites of the skin in the back of the rabbits, secured and moistened using 0.5 ml of phosphate buffer saline (pbs). the positive control site was covered with a patch containing 0.5ml of 1% of sodium lauryl sulfate (sls). the negative control site was covered with a patch containing 0.5ml of pbs. then the entire back of the rabbits was covered for 24h. after 24h of wrapping, the patches were removed. at one hour after removal of the patches and at 48 and 72h later, the erythema and oedema were checked, scored and recorded (table 1). step 2) healthy new zealand albino rabbits were screened and rabbits with no hair loss were selected. the hairs on the back of the rabbits were clipped. after 24h, 6 rabbits with no signs of abrasion, erythema or oedema due to shaving were selected and the following materials were applied on their back: sinaampholeish gel, empty liposome (as placebo), sls 1% as a positive control and pbs as negative control similar to step 1. step 3) in a pilot study, first white but not new zealand albino rabbits were screened and 2 healthy rabbits with no ocular lesion was selected, after gently pulling the lower lid away from the right eyeball, 100mg of sinaampholeish gel was placed in the conjunctival sac, then lids were then gently held together for about one second in order to prevent loss of the material. the left eye was untreated as a control. the eyes of the rabbits were regularly checked for 24h and in 2 of them daily for one week. as there was no sign of pain (such as repeated pawing or rubbing of the eye, excessive blinking, excessive tearing), no anaesthesia was required for the other rabbits. than 6 new zealand albino rabbits with no sign of ocular lesions were selected and treated similarly to the pilot study in white rabbits. in 2 rabbits the right eyes were covered for one hour. the eyes of the rabbits were regularly checked for the first 24h and photos were taken at 24, 48 and 72h. statistical analysis the wilcoxon signed-rank test was used for comparing reaction between the two arms of study, p-value of less than 0.05 was considered significant. results step one results of the irritancy potential test of the substances were checked in 6 white rabbits (but not new zealand albino), the results showed no reaction in any of the rabbits, no erythema, and no oedema. in step 2, irritancy potential test was checked in new zealand albino rabbits, as it is shown in table 2, the average of erythema scores in 6 rabbits in 3-time points was 0.83 +0.41 for 0.4% sinaampholeish gel and 0.5+ 0.55 for empty liposomes (p= 0.16). the average scores for oedema in the same rabbits was 0.67+0.52 for sinaampholeish gel and 0.33+0.52 for empty liposomes (p 0.16). the results of erythema and oedema are shown in tables 2 and 3, respectively (table 3). no sign of eye irritation was seen during 72h observation in 8 rabbits (two from the pilot study and 6 from the main study) and two-week observation in 2 rabbits (fig. 1). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 269–275 se eskandari et al.: safety evaluation of … 272 http://jad.tums.ac.ir published online: september 30, 2018 table 1. rabbit score values for skin reactions (erythema and oedema) skin reaction value erythema formation: no erythema 0 very slight erythema (barely perceptible) 1 well-defined erythema 2 moderate to severe erythema 3 severe erythema (beet redness) to slight scar formation (injuries in depth) 4 necrosis (death of tissue) +n scar (sloughing or scar formation) +e oedema formation: no oedema 0 very slight oedema (barely perceptible) 1 slight oedema (edges of area well-defined by definite raising) 2 moderate oedema (raised approximately one millimetre) 3 severe oedema (raised approximately one millimetre and extending beyond the area of exposure) 4 total possible score for primary irritation 8 table 2. skin erythema reactions in new zealand albino rabbits (wilcoxon signed ranks test) nano liposomal amphotericin-b placebo empty liposome p-value hours mean+sd median+qr mean+sd median+iqr 25 0.83+0.41 1+0.25 0.5+0.55 0.5+1 0.16 48 0.5+0.55 0.5+1 0 0 0.08 72 0 0 0 0 1 sodium lauryl sulfate (sls) was used as a positive control and pbs as a negative control positive control results were 3, 4, 3 at 25, 48 and 72h and negative control was 0 table 3. skin oedema reactions in new zealand albino rabbits (wilcoxon signed ranks test) nano liposomal amphotericin-b placebo empty liposome pvalue hours mean+sd median+iqr mean+sd median+iqr 25 0.67+0.52 0 0.33+0.52 0.16 48 0 0 0 0 1 72 0 0 0 0 1 sodium lauryl sulfate (sls) was used as a positive control and pbs as a negative control http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 269–275 se eskandari et al.: safety evaluation of … 273 http://jad.tums.ac.ir published online: september 30, 2018 fig. 1. pictures of eye irritation test, left experiment, right control. positive control results were 2, 2, 2 at 25, 48 and 72h and negative control showed was 0 at all time frames discussion treatment of cl is sometimes a dilemma, the only standard treatment is to use pentavalent antimonite which needs multiple injections, accompanies side effects and moreover is not always effective. development of new more acceptable treatment for cl is urgently needed to increase compliance and facilitate control of anthroponotic form of cl. topical formulation is an ideal treatment for cl, several topical ointment have been developed and checked in phase 3 clinical trials (4, 6, 13, 15-17). paromomycin had therapeutic effect on old world and new world cl, but currently, none is available in the market for endemic areas (17). various modalities including even immunotherapy have been proposed to treat cl with limited efficacy (18). a nano-liposomal form of amphotericin b was developed. this formulation showed to be effective against l. major and l. tropica in vitro and against l. major in vivo in a murine model of leishmaniasis (9). the risk of topical formulations such as cosmetic and therapeutic to induce irritation or corrosion is usually checked in rabbit using draize test (12-14). an alternative method has been developed to decrease and even avoid using animal models for experiment studies (13). originally it was proposed to use alternative in vitro test to check the irritancy test but due to politically imposed sanctions against iran, the cell line needed to use in vitro test was not available so with no other option, the formulation of nano-liposomal amphotericin b potential irritancy was checked in rabbit model (draize test). according to our previous unpublished data, usually new zealand albino rabbit is more sensitive to irritancy test than white rabbits, so first 6 white rabbits were included and no erythema and no oedema was seen in 25, 48 and 72h exposure to either sinaampholeish or empty liposomes. when the same procedure was used in 6 new zealand albino rabbits the average of erythema and oedema scores was 0.83+0.41 and 0.67+0.52, respectively for sinaampholeish gel and 0.5+0.55 and 0.33+ 0.52 for erythema and oedema for empty liposomes, respectively, although there was no significant difference between irritancy test between sinaampholeish gel and empty liposomes and control groups (p=0.16), but new zealand albino rabbits were more sensitive than white rabbits but the formulation was completely safe and could be further checked in human trials. conclusion based on skin irritancy reactions the topical formulation of sinaampholeish is safe and could be further checked in human trials. acknowledgements the study was financially supported by drugs for neglected diseases initiative (dndi), and center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran. the authors declare that there is no conflict of interest. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 269–275 se eskandari et al.: safety evaluation of … 274 http://jad.tums.ac.ir published online: september 30, 2018 references 1. who (2016) leishmaniasis fact sheet no. 375. world health organization, geneva, switzerland. http://www.who.int/en/newsroom/fact-sheets/detail/leishmaniasis 2. who (2000) who report on global surveillance of epidemic-prone infectious diseases leishmaniasis. http://www.who.int/csr/resources/public ations/csr_isr_2000_1leish/en/ 3. alvar j, vélez id, bern c, herrero m, desjeux p, cano j, jean jannin, margriet den boer (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7(5): e35671. 4. firooz a, khamesipour a, ghoorchi mh, nassiri-kashani 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and piper nigera against anopheles stephensi and aedes aegypti under laboratory conditions r nawaz, h rashid rathor, *h bilal, sa hassan, i akram khan department of medical entomology and disease vector control, health services academy, islamabad, pakistan (received 3 aug 2011; accepted 28 sep 2011) abstract background: there are several plant extractions which are being used for mosquito control. the aim of this study was to evaluate the efficacy of olea vera, linum usitatissimum and piper nigera against anopheles stephensi and aedes aegypti under laboratory conditions. methods: these tests were carried out using who recommended bioassay method for adult mosquitoes. results: the extracts from black pepper was more effective as adulticide with lowest lc50 values (2.26% and 8.4%) against aedes aegypti and anopheles stephensi after 24 h of exposure while after 48h (1.56% and 5.11%) respectively. in terms of lc90 value black pepper was best with (8.66% and 30.1%) against ae. aegypti and an. stephensi after 24 h of exposure while after 48h (4.59% and 17.3%) respectively. in terms of lt50 black pepper took 15 h to kill 50% tested population of ae. aegypti while against an. stephensi it took more than 2 days. in terms of percentage mortality black pepper kill 84% of the population of ae. aegypti and 44.75% of the an. stephensi population. conclusion: black pepper showed best results in term of lc50, lc90, lt50 and percentage mortality against ae. aegypti and an. stephensi. our study suggested that the plant extracts have potential to kill adult mosquitoes, are environment friendly and can be used for the control of mosquitoes. keywords: adulticide, plant extracts, anopheles stephensi introduction insect vectors, especially mosquitoes are responsible for spreading serious human diseases like malaria, japanese encephalitis, yellow fever, dengue and filariasis (who 1997). globally in 2009 there were 243 million cases and approximately 863000 deaths due to malaria (world malaria report 2009) and 50 million cases due to dengue and dhf (dengue and dengue haemorrhagic fever, 2009) were reported. while in pakistan there were an estimated 4.5 million suspected malaria cases and 59284 confirmed cases of malaria reported in 2008 (world malaria report pakistan 2009). totally, 5164 cases of dengue and dhf in 2006 (weekly morbidity and mortality report, 2006) and 2062 confirmed cases of dengue fever including 15 deaths (flood response in pakistan 2010) were reported in 2010. a primary element in the current global strategy for the control of vector-borne diseases is vector control, chemical control remaining a main component of integrated vector management. major mosquito-borne diseases, including malaria, dengue, and yellow ever, are reportedly controlled with insecticides (curtis et al. 2003, zaim and jambulinagam 2004). according to a who pesticide evaluation (zaim and jambulinagam 2004), the main insecticides used against *corresponding author: mr hazrat bilal, e-mail: hazratbilal@hsa.edu.pk iran j arthropod-borne dis, 2011, 5(2): 2–9 r nawaz et al.: adulticidal activity of olea vera … 3 malaria and dengue are pyrethroids and organophosphates. successive changes in the insecticides and overuse resulted in multiple insecticides resistant in malaria vectors (raghavendra and subbarao 2002). thus, the future of vector control mainly relies on the strategies for the management of existing insecticide resistance in malarial vectors and to limit its further spread. the most important aspect of the management of resistance is to either avoid or delay the onset of resistance by effectively manipulating or influencing the factors responsible for the development of resistance. one of the possible ways of avoiding development of insecticide resistance in field is using non chemical control method, i.e., biopesticides (amer and mehlhorn 2006a, b). therefore, it is the hour to launch extensive search to explore eco-friendly biological materials for control of anopheles stephensi and aedes aegypti. plant essential oils in general have been recognized as an important natural resource of insecticides (adebayo et al. 1999, gbolade et al. 2000). they have the potential of being acute ovicidal, fumigant, insect growth regulator and insecticidal against various insect species (tsao et al. 1995) and concurrently being non hazardous to pesticide to eco-system (isman 2000). generally they are safe to humans and other mammals (tripathi et al. 2002–2004). for this reason, it is necessary to test those plant extracts which could be expected to contain substances to provide adequate efficacy against mosquitoes and are environment friendly (trongtokit et al. 2008). therefore the present study was conducted for the evaluation of botanicals extracts against an. stephensi and ae. aegypti. materials and methods collection of plants olive (olea vera), linseed (linum usitatissimum) and black pepper (piper nigera) were collected from faisalabad (31° 21′ 52″ n, 72° 59′ 40″ e) and identified from the department of agronomy, university of agriculture faisalabad. extraction of oil the seeds were washed, then dried and later grounded in an electric grinder (anex germany). the grounded material was put in thimble and kept in extraction tube of soxhelt apparatus with extractor id 38mm, extractor volume 85 ml and flask volume 250 ml (vogel 1978) for the extraction of oil by steam distillation method using diethylether as solvent (250 ml/20 g sample). the cycle time for one sample was 4–5 h. solvent was evaporated at room temperature, leaving oil which was then collected. preparation of solution small vials were used to collect the oil and the quantity was measured. 1% stock solution was prepared by adding 1 ml of oil from each plant in 99ml of acetone, from which series of concentration (%) were prepared (murgan et al. 2007). collection of mosquitoes the immature mosquitoes were collected from different areas of islamabad (33° 43′ 0″ n, 73° 4′ 0″ e) and rawalpindi (33° 36′ 0″ n, 73° 2′ 0″ e) from 2009 and colonized in the insectry of department of medical entomology and disease vector control, health services academy, islamabad. the larvae were collected by dipping with a standard 400ml dipper (who 1975). these were mass reared at 30±2 ◦c temperature and 70±5% humidity. the larval population was fed on tetra min tropical (tetra tm). adults were reared in steel cages and males were provided with 10% sucrose solution, while females were fed on blood of white rats (shaalan et al. 2006). the gravid females were allowed to lay eggs on black plastic gauze that was placed in petri dishes. the iran j arthropod-borne dis, 2011, 5(2): 2–9 r nawaz et al.: adulticidal activity of olea vera … 4 eggs were separated and on emergence of larvae in petri dishes were shifted to rearing trays. aduticidal bioassay the extracted oils were evaluated by standard who method (who 2006). different concentrations (2%, 4%, 6%, and 8%) of oils were applied on the whatman # 1 filter paper and control was treated with acetone only. 15–25, 12 hrs old female mosquitoes were introduced in who insecticide testing kit in holding tube and remain there for 1 h to acclimatize. after 1 h mosquitoes were transferred in exposure tube, data was collected after 1 h, 24 and 48 h. data analysis abbott’s formula (abbott 1925) was used for corrected mortality and the data so obtained was analyzed by probit analysis (finney 1989) dose and time mortality regression lines were calculated by using manitab-15 software. results colonies of ae. aegypti and an. stephensi were maintained in our insectary, males on 10% sugar solution and females were blood fed on live white rats. larvae were reared in steel trays (24 x 36 x 6 cm) and fed on sterilized liver diet. our studies on the bio-potentials of seed extracts have yielded results that they can be tested for application on large scale against ae. aegypti and an. stephensi. the adults were tested against the four selected concentrations of these extracts. the data so obtained has been elaborated in results and is being explained in light of previous findings. table 1 indicated the lc50 and lc90 values of black pepper, olive and linseed against ae. aegypti and an. stephensi. results showed that oils from black pepper and olive was considered best with lc50 (2.26 and 2.95) respectively, after 24 h of exposure and regarded more toxic to ae. aegypti, followed by linseed (12.90), after 48 h of exposure black pepper and olive had the lowest lc50 value (1.56 and 1.77) respectively, followed by linseed (20.25). oils from black pepper and linseed had the lowest lc90 value (8.66 and 27.60) respectively, after 24 h of exposure, followed by olive (38.55), while after 48 hours of exposure black pepper and olive seed had the lowest lc90 value (4.59 and 10.29) respectively, followed by linseed (128.91) against ae. aegypti. oils from black pepper and linseed was considered best with lc50 (8.14 and 13.51) respectively, after 24 h of exposure and regarded more toxic to an. stephensi followed by olive (21.02), after 48 h of exposure black pepper and linseed had the lowest lc50 value (5.11 and 7.16) respectively, followed by olive (11). oils from black pepper and linseed had the lowest lc90 value (30.12 and 94.81) respectively, after 24 h of exposure, followed by olive seed (347.12), while after 48 h of exposure black pepper and linseed had the lowest lc90 value (17.26 and 56.61) respectively, after 48 h of exposure, followed by olive seed (237.59) against an. stephensi. percent mortality of ae. aegypti larvae after 24 h of exposure under lab conditions showed that black pepper and linseed had the highest percent mortalities (71.25% and 57.50%) respectively, followed by olive (8.25%), while after 48 hours black pepper and linseed had the highest percent mortality (84.75% and 72.75%) respectively, followed by olive (15.5%) as shown in fig. 1. according to fig. 2 percent mortality of an. stephensi larvae after 24 h of exposure under lab conditions showed that black pepper and olive had the highest percent mortalities (29.75% and 24.5%) respectively, followed by linseed (24%), while after 48 h black pepper and olive had the highest percent moriran j arthropod-borne dis, 2011, 5(2): 2–9 r nawaz et al.: adulticidal activity of olea vera … 5 tality (44.75% and 38.5%) respectively, followed by linseed (35%). in terms of lethal time (table 2) to kill 50% population of ae. aegypti, black pepper and linseed took minimum time to kill 50% of population i.e., 14.98 and 21.36 h, followed by olive (246.08 h). in terms of lethal time (table 2) to kill 50% population of an. stephensi, black pepper and olive took minimum time to kill 50% of population i.e., 53.69 and 68.16 h, followed by linseed (78.04). table 1. adulticidal effect of plant extracts against ae. aegypti and an. stephensi treatments time (h) lc50 (%age) lc90 (%age) slope±se lc50 (%age) lc90 (%age) slope±se ae. aegypti an. stephensi black pepper 24 2.26 8.66 0.96±0.20 8.4 30.1 0.95±0.21 48 1.56 4.59 1.18±0.25 5.11 17.3 1.05±0.20 olive 24 2.95 38.6 0.49±0.18 21.02 347 0.45±0.20 48 1.77 10.3 0.72±0.19 11 238 0.41±0.18 linseed 24 12.9 27.6 1.68±0.57 13.51 94.8 0.62±0.21 48 20.25 129 0.69±0.24 7.16 56.6 0.62±0.91 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 o ils l in u m u s it a t is s im u m o le a v e r a p ip e r n ig e r a m e a n m o rt a lit y (% ) 2 4 h o u rs 4 8 h o u rs fig. 1. mortality of plant extracts against adult of ae. aegypti iran j arthropod-borne dis, 2011, 5(2): 2–9 r nawaz et al.: adulticidal activity of olea vera … 6 0 1 0 2 0 3 0 4 0 5 0 6 0 l in u m u sitatis s im u m o le a v era p ip er n ig e ra m e a n m o rt a lit y (% ) o ils 2 4 h o u rs 4 8 h o u rs fig. 2. mortality of plant extracts against adult of an. stephensi table 2. time mortality response of ae. aegypti and an. stephensi against plant extracts treatments lt50 (h) slope±se lt50 (h) slope± se ae. aegypti an. stephensi black pepper 15 0.99±0.11 53.7 0.72±0.12 olive 21.4 0.88±0.10 78 0.65±0.11 linseed 246 0.59±0.18 68.2 0.70±0.13 discussion our results indicate that black pepper seed extracts have good adulticidal potential against ae. aegypti and an. stephensi as the most effective in terms of lc50, lc90, lt50 and percent mortalities. our results that plant extracts have the potential were supported by scientists like choochote et al. (2004) reported that crude seed extract of celery, apium graveolens extract exhibited a slightly adulticidal potency with ld50 and ld95 values of 6.6 and 66.4 mg/cm2, respectively. the extracts and compounds from tubers of neorautanenia mitis against adult an. gambiae mosquitoes showed good adulticidal effects and the results can be extended for the control of mosquitoes iran j arthropod-borne dis, 2011, 5(2): 2–9 r nawaz et al.: adulticidal activity of olea vera … 7 especially at breeding sites (joseph et al. 2004). methanolic extract of leaves of annona squamosa against the adult of culex quinquefasciatus the extract showed dose dependent activity, exhibited significantly shorter knock down kd50 and kd90 values and produced significant mortality (jaswanth 2002). the methanol extracts of seven species of malaysian tunicates, the mortality values of the extracts on the adult mosquitoes were dose-dependent and increased with exposure period (hussain et al. 2001). investigation of the insecticidal activity of essential oil isolated from the leaves of lantana camara against ae. aegypti, cx. quinquefasciatus, an. culicifacies, an. fluvialitis and an. stephensi respectively, kdt50 of the oil were 20, 18, 15, 12, and 14 min and kdt90 values were 35, 28, 25, 18, 23 min against ae. aegypti, cx. quinquefasciatus, an. culicifacies, an. fluviatilis and an. stephensi, respectively on 0.208 mg/cm2 impregnated paper (dua 2010). akram et al. 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stored-product beetles (coleoptera). j econ entomol. 95: 183–189. tripathi ak, veena p, ateeque a, kishan ka, suman psk (2004) piperitenone oxide as toxic, repellent and reproduction retardant toward malarial vector anopheles stephensi (diptera: anophelinae). j med entomol. 41(4): 691–698. trongtokit y, burivong p, moore s, hill n, curtis cf (2008) plant extracts in prevention of mosquito attacks. botanical medicine in clinical practice. pp. 807– 813. isbn (978-1-84593-413-2) tsao r, lee s, rice pj, jensen c, coats jr (1995) monoterpenoids and their synthetic derivatives as leads for new insect control agents. in: baker, dr, fenyes, jg, basarab, gs (eds), synthesis and chemistry of agrochemicals iv. american chemical society, washington, dc. pp. 312–324. vogel ai (1978) text book of practical organic chemistry. the english language book society and longman, london. iran j arthropod-borne dis, 2011, 5(2): 2–9 r nawaz et al.: adulticidal activity of olea vera … 9 weekly morbidity and mortality report. epidemiological week no. 46 (11–17 nov 2006). 23 nov 06/ vol. 52/ dews 2006-46. who (world health organization) (1975) manual on practical entomology in malaria, world health organization. prepared by the who division of malaria and other parasites diseases. part 1–2. offset publication no.13. world health organization, geneva. world malaria report pakistan. geneva, world health organization (2009). pp 135. world malaria report. geneva, world health organization (2009). pp 37. zaim m, jambulingam p (2004) global insecticide use for vector borne disease control. world health organization communicable disease control, prevention and eradication, who pesticide evaluation scheme (whopes). j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 240 http://jad.tums.ac.ir published online: september 30, 2018 original article fauna and larval habitats characteristics of mosquitoes (diptera: culicidae) in golestan province, northeast of iran, 2014–2015 *aioub sofizadeh 1, hamid reza shoraka 2, fatemeh mesgarian 3, ghorban mohammad ozbaki 3, abdolsamad gharaninia 3, ebrahim sahneh 3, rohollah dankoob 3, ali malaka 3, saeid fallah 3, shamsaddin nemani 3 1infectious diseases research center, golestan university of medical sciences, gorgan, iran 2north khorasan university of medical scinces, bojnurd, iran 3health centers of health deputy, golestan university of medical sciences, gorgan, iran (received 30 july 2017; accepted 11 june 2018) abstract background: mosquitoes (diptera: culicidae) is one of the most medically important families of diptera. the aims of this study were to determine fauna and larval habitat characteristics of mosquitoes in golestan province, during 2014–15. methods: this study was conducted in larval habitats of mosquitoes and installed ovitraps in 14 districts of golestan province, northern iran in 2015. samples were collected with a scoop by ladle handling for entomology. the collected larvae were transferred to laboratory of medical entomology in lactophenol solution. then microscopic slides were prepared using de faure's formula. species of each sample was recognized using diagnostic criteria to identify the culicidae species. characteristics of larval breeding places were studied based on the habitat type (permanent or temporary), water conditions (clear or turbid, stagnant or running), vegetation (out, in, underwater vegetation or without vegetation), sunlight exposure (full or partial sunlight) and so on. data were analyzed using spss. results: overall, 5661 thirdand fourthinstars larvae of mosquitoes were collected and 5 genera and 14 species were identified: anopheles hyrcanus, an. maculipennis, an. pseudopictus, an. superpictus, culex hortensis, cx. mimiticus, cx. perexiguus, cx. pipiens, cx. pusillus, cx. theileri, cx. tritaeniohynchus, culiseta longiareolata, ochlerotatus caspius, uranotaenia unguiculata. culex pipiens was recognized as predominant species of the family. among the detected species, cx. pusillus reported for the first time from golestan province. conclusion: due to the high species diversity of culicidae, ecology of medical important species such as cx. pipiens and cx. tritaeniorhynchus needs more investigations. keywords: culicidae, larval habitat, ecology, iran introduction mosquitoes (diptera: culicidae) as one of the most important families of insects. according to the latest taxonomy, culicidae comprises two subfamilies, 11 tribes, 112 genera and 3539 species (1). the checklist of the mosquitoes in iran includes 7 genera, 16 subgenera, 64 species and three subspecies (2). subsequently, anopheles superpictus is two species in iran based on the internal transcribed spacer 2 (its2) sequences of rdna (3), later listed as species a and b (4). a new species of the anopheles hyrcanus group (an. hyrcanus spir) was recognized from southwestern iran also based on its2 sequence data (5). more recently, the occurrence of aedes albopictus was reported in southeastern iran and orthopodomyia pulcripalpis in northern iran, respectively (6, 7). the fauna of mosquitoes of golestan province, northern iran includes aedes (aedimorphus) vexans, ae. (dahliana) echinus, ae. (dah.) geniculatus, ae. (ochlerotatus) caspius, *corresponding author: dr aioub sofizadeh, email: a_sofizadeh@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 241 http://jad.tums.ac.ir published online: september 30, 2018 ae. (och.) pulcritarsis, anopheles alrgeriensis, an. claviger, an. hyrcanus, anopheles. maculipennis s.l., an. melanoon, an. multicolor, an. plumbeus, an. pseudopictus, an. pulcherrimus, an. superpictus, coquillettidia richiardii, culex hortensis, cx. mimeticus, cx. perexiguus, culex. pipiens, cx. territans, cx. theileri, culiseta longiareolata, cs. subochrea, uranotaenia unguiculata (8–13). mosquitoes are important vectors of many diseases including: malaria, west nile virus, dengue fever, yellow fever, filariasis and other diseases (14). some of these diseases and their agents have previously been reported from golestan province, for example: malaria, west nile virus, dirofilaria immitis (15-17). malaria as one of the most important vector-borne diseases in iran is transmitted by anopheles mosquitoes. over the past decades, north of the country including golestan province has been identified as one of the most important endemic foci of malaria (15), eight species of the genera anopheles are known as vectors of malaria in iran. an. culicifacies s.l., an. dthali, an. fluviatilis s.l., an. maculipennis s.l., an. sacharovi, an. stephensi, and an. superpictus have been introduced as primary and secondary malaria vectors and an. pulcherrimus as a suspected vector (11). west nile virus is a mosquito-borne virus that transmitted by culex species to birds, equines, and humans (18) seropositive cases of humans and equines for these viruses were reported in golestan province (16). dirofilaria immitis also is one of the most important mosquito-borne pathogens that reported in golestan province (17), culex theileri is known vector of this pathogen in the north-west of iran (19). mosquitoes play a key role in transmitting diseases, many have studies carried out in north of iran and golestan province on the biology and ecology of mosquitoes: larval habitats characteristics of mosquitoes and biological characteristics of anopheles was reported in kalaleh county, golestan province (12, 13). fauna and larval habitats of mosquitoes were reported in guilan and north khorasan provinces, north of iran (20-23). fauna, larval habitat and other biological characteristics of mosquitoes were reported in mazandaran province and neka county (24-26). physicochemical characteristics of mosquitoes were studied in qom province (27) and larval habitats, affinity and diversity indices of culicinae in southern iran (28). different species of mosquitoes require different biological and ecological conditions to growth and development. while some lay eggs and breed in permanent water habitats, others prefer temporary water bodies for breeding, some favour feeding on sweet water, others need salty water, and some prefer high temperature and humidity while others are in favour of low temperature and humidity. regardless of these differences, is clear and evident that all need water for breeding and without water, their chances for growth and development would be slight. though the climate in golestan province is suitable for the growth and development of mosquitoes such as anopheles, aedes and culex mosquitoes, so far, no comprehensive studies have been carried out on the fauna and ecological properties of mosquitoes in golestan province. to address this gap, this research was an attempt to analyze the characteristics of mosquito larval habitats. materials and methods study area golestan province (53°57′–56°23′ e, 36° 30′–38°08′ n) covering a landmass of 20437.74 square km consists of approximately 1.3% of the total area of iran. it is located in the northeastern region of the country and bordered by the republic of turkmenistan to the north, alborz mountain range and semnan province to the south, north khorasan province to the east, and caspian sea and mazandaran http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 242 http://jad.tums.ac.ir published online: september 30, 2018 province to the west. this province consists of 14 counties, 25 cities, 60 districts and 1764 villages (fig. 1). the province is enriched with diverse ecology and climatic conditions. considering the sea, forest and mountainous areas, the climatic condition of the province is classified into temperate mountainous, cold mountainous (3000m high), a mild mediterranean and arid and semi-arid regions, such that as we move from southern to northern parts, the amount of rainfall and relative humidity decreases and the degree of temperature increases. with regards to the topography of the province, this region is subdivided into three distinct areas; mountainous, plain and even posts. the mountainous areas are located in the southern parts containing the highest peaks of the province. mountainous areas are located in the foothills of the southern and eastern borders of the province with coarse sediments as alluvial fans make use of this land. as a result of the high permeability of the soil in the mountainous areas, groundwater aquifers with water in wells and canals are exploited. the retreat of the caspian sea post and plain regions has created severe water erosion and compaction of alluvial rivers. the lowest parts of the province (around the caspian sea with an altitude of 32m above sea level) are located in an area inhabited by a majority of the province's population (29). specimen and data collection this study was performed in all counties of golestan province, northern iran in 2015. in each county, one city and two villages were selected based on the topographic conditions. in order to sampling of larves, in each selected city and villages, at least two larval habitats were searched. first, features such as larval habitat status (permanent or temporary, stagnant or slow-running water), vegetation type, substrate type, habitat types and position of the sunlight were recorded on special forms. then, larval sampling method was carried out using standard dipper of 350ml. each habitat was sampled in different parts of the larval habitats for 10 times. all captured larvae in each dipper were counted and collected in special containers. the information of habitats was recorded on the larva containers and transferred to the medical entomology laboratory in health center of kalaleh county. in the laboratory, after drying, larvae were kept in lactophenol medium and were mounted on microscope slides in de faure's formula and using standard taxonomic keys (30), larvae species were determined. sampling was done once in each of the seasons of spring, summer, and autumn. preparation of ovitrap and larva sampling of this trap for searching aedes eggs and larva we used cdc ovitrap. this consists of a three-liter, black, water-filled, plastic container and thin paddle of wood (2×12.5cm) placed in the container (31, 32). in each selected city and village, 10 ovitraps were implemented and investigated once a week and collected the present larvae and as it was explained previously, they were mounted and species of each sample was recognized. results overall, 5661 larvae belonged to culicidae were collected and their species were identified. including an. hyrcanus, an. maculipennis s.l., an. pseudopictus, an. superpictus s.l., cx. hortensis, cx. mimeticus, cx. perexiguus, cx. pipiens, cx. pusillus, cx. theileri, cx. tritaeniohynchus, cs. longiareolata, oc. caspius, and uranotaenia unguiculata. of 2821 ones had been collected from natural larval habitats and 2840 ones from the prepared ovitraps (table 1). culex pipiens was identified as the dominant species of golestan province and it was collected from all counties of this province. in terms of frequency, cx. tritaeniohynchus was in the sechttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 243 http://jad.tums.ac.ir published online: september 30, 2018 ond rank and was collected from a majority of counties (table 2). in the present study, 77.3% of larvae were collected from temporary larval habitats compared to permanent ones and 73.6% of larvae from larval habitats with stagnant water compared to those with running water (table 3). more larvae were collected from natural larval habitats of wetlands (31.3%) and artificial larval habitats of bogs (26.4%) (table 4, 5). table 1. abundance of mosquito (culicidae) larvae in golestan province, nortehrn iran 2015 species natural larval habitats ovitrap total num. % num. % num. % an. hyrcanus 2 0.1 0 0 2 0.001 an. maculipennis 20 0.7 0 0 20 0.4 an. pseudopictus 14 0.5 0 0 14 0.2 an. superpictus 178 6.3 0 0 178 3.1 cx. hortensis 6 0.2 0 0 6 0.1 cx. mimeticus 46 1.6 2 0.1 48 0.8 cx. perexiguus 72 2.5 7 0.2 79 1.4 cx. pipiens 1657 58.2 2736 96.3 4393 77.3 cx. pusillus 1 0.001 3 0.1 4 0.1 cx. theileri 5 0.2 4 0.1 9 0.2 cx. tritaeniohynchus 617 21.7 88 3.1 705 12.4 cs. longiareolata 18 0.6 0 0 18 0.3 oc. caspius 183 6.4 0 0 183 3.2 uranotaenia unguiculata 2 0.1 0 0 2 0.001 total 2821 100 2840 100 5661 100 %,%, %, %, %, %, %, %, %, %, %, %, %, %, %, %, %, %,%, %, %, %, %, %, %, %, %, %,%, %, %, %, %, %, %,%, %,%, %,%, %, %,%, gonbad-e kavoos kalaleh gorgan aghghala maraveh tappeh gamishan galikesh ramiyan kordkooy azadshahr aliabad-e katool minoodasht bandar-e gaz bandar-e torkaman ® legend %, selected villages for sampling golestan counties 0 25 50 75 10012.5 kilometers fig. 1. location of golestan province and selected villages for sampling of culicidae http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 244 table 2. distribution of mosquitoes in different counties of golestan province, northern iran counties species m a r a v e h t a p e h k a la le h g o n b a d -e k a v u s g a lik e sh m in o o d a sh t a z a d sh a h r r a m ia n a lia b a d -e k a tu l a q q a la g o r g a n k o r d k o u y b a n d a r -e g a z b a n d a r -e t u r k m a n g o m ish a n t o ta l an. hyrcanus 0 0 0 0 2 0 0 0 0 0 0 0 0 0 2 an. maculipennis 0 15 0 5 0 0 0 0 0 0 0 0 0 0 20 an. pseudopictus 0 0 0 0 14 0 0 0 0 0 0 0 0 0 14 an. superpictus 0 178 0 0 0 0 0 0 0 0 0 0 0 0 178 cx. hortensis 0 0 0 6 0 0 0 0 0 0 0 0 0 0 6 cx. mimeticus 0 5 0 18 24 0 0 1 0 0 0 0 0 0 48 cx. perexiguus 63 1 0 8 3 0 0 0 0 0 0 3 1 0 79 cx. pipiens 164 152 1658 60 156 223 161 564 40 80 742 265 74 54 4393 cx. pusillus 3 1 0 0 0 0 0 0 0 0 0 0 0 0 4 cx. theileri 4 0 3 0 0 0 0 0 0 0 0 2 0 0 9 cx. tritaeniohynchus 53 62 221 103 59 0 5 49 4 0 5 59 85 0 705 cs. longiareolata 0 18 0 0 0 0 0 0 0 0 0 0 0 0 18 oc. caspius 0 0 104 0 0 0 0 28 0 0 0 0 51 0 183 uranotaenia unguiculata 0 1 0 0 0 0 0 0 0 0 0 0 1 0 2 total 287 433 1986 200 258 223 166 642 44 104 747 329 212 54 5661 table 3. larval habitat characteristics of mosquitoes collected in golestan province, northern iran 2015 species larval habitats characteristics a n . h y rc a n u s a n . m a c u lip e n n is a n . p se u d o p ic tu s a n . su p e rp ic tu s c x . h o rte n sis c x . m im e tic u s c x . p e re x ig u u s c x . p ip ie n s c x . p u sillu s c x . th e ile ri c x . trita e n io h y n c h u s c s. lo n g ia re o la ta o c . c a sp iu s u ra n o ta e n ia u n g u ic u la ta t o ta l habitat situation permanent 2 0 7 29 4 2 33 488 1 0 56 0 25 0 645 temporary 0 20 7 148 4 44 39 1169 0 5 561 18 158 20 2193 running water 2 0 7 83 4 2 8 523 1 2 118 0 0 0 750 stagnant water 0 20 7 95 2 44 64 1134 0 3 499 18 183 20 2089 vegetation situation out of water 0 0 0 121 0 34 56 527 0 5 282 0 104 0 1165 in water level 0 0 0 0 4 12 6 370 0 0 136 0 28 0 571 underwater 0 0 0 0 0 0 8 49 0 0 2 0 26 0 85 without 0 0 0 57 2 0 2 711 1 0 197 18 25 0 1013 sunlight situation full sunlight 0 0 0 28 0 5 30 537 0 5 389 0 127 0 1124 shaded 0 0 0 0 0 0 0 64 0 0 20 0 30 0 118 partial sunlight 2 5 7 26 0 19 38 399 0 0 101 0 10 0 607 sunlight shaded 0 15 7 124 0 22 4 657 1 0 110 18 16 1 975 substrate mud 2 19 14 149 6 46 72 1004 0 5 481 0 158 2 1958 sand 0 1 0 29 0 0 0 279 1 0 136 0 25 0 471 rock 0 0 0 0 0 0 0 70 0 0 0 0 0 0 70 others 0 0 0 0 0 0 0 295 0 0 0 18 0 0 313 water situation j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 245 muddy 0 0 0 28 0 0 5 372 0 0 48 0 0 0 453 clear 2 20 14 150 6 46 66 1114 1 4 497 18 155 1 2094 turbid 0 0 0 0 0 0 1 171 0 1 72 0 28 1 274 fresh 2 20 14 95 6 46 29 1571 0 5 494 18 158 1 2459 salty 0 0 0 0 0 0 26 34 0 0 18 0 0 0 78 brackish 0 0 0 83 0 0 17 52 1 0 105 0 25 1 284 habitat kind natural 2 5 14 83 6 41 28 446 1 4 310 2 183 1 1126 artificial 0 15 0 95 0 5 44 1211 0 1 307 16 0 1 1695 table 4. abundance of species of mosquitoes in natural habitats in golestan province, northern iran, 2015 natural habitats type species r iv e r e d g e r iv e r b e d m a r sh c r e e k f o u n ta in p it w e tla n d s t r e e h o le s w a te r le a k a g e t o ta l an. hyrcanus 2 0 0 0 0 0 0 0 0 2 an. maculipennis 0 0 0 0 0 0 5 0 0 5 an. pseudopictus 7 0 7 0 0 0 0 0 0 14 an. superpictus 29 0 0 26 0 0 28 0 0 83 cx. hortensis 0 2 0 4 0 0 0 0 0 6 cx. mimeticus 2 0 10 0 0 7 5 0 17 41 cx. perexiguus 1 0 0 8 6 2 8 0 3 28 cx. pipiens 9 9 13 52 0 109 144 0 37 446 cx. pusillus 1 0 0 0 0 0 0 0 0 1 cx. theileri 0 0 0 2 0 2 0 0 0 4 cx. tritaeniohynchus 73 2 16 9 2 10 124 0 74 310 cs. longiareolata 0 0 0 0 0 0 0 2 0 2 oc. caspius 0 0 25 104 0 16 38 0 0 183 uranotaenia unguiculata 0 0 0 0 0 0 1 0 0 1 total 197 13 71 205 8 146 353 2 131 1126 table 5. abundance of species of culicidae in artificial habitats in golestan province, northern iran, 2015 artificial habitats type species c r e a te sid e s o f r ic e c r e a te m id d le o f r ic e s tr e a m o f c r e a tin g r ic e b o g o th e r a g r ic u ltu r a l str e a m s c iste r n l a k e w e ll p o o l o th e r s t o ta l an. hyrcanus 0 0 0 0 0 0 0 0 0 0 0 an. maculipennis 15 0 0 0 0 0 0 0 0 0 15 an. pseudopictus 0 0 0 0 0 0 0 0 0 0 0 an. superpictus 95 0 0 0 0 0 0 0 0 0 95 cx. hortensis 0 0 0 0 0 0 0 0 0 0 0 cx. mimeticus 0 0 0 0 0 0 0 5 0 0 5 cx. perexiguus 44 0 0 0 0 0 0 0 0 0 44 cx. pipiens 114 25 57 446 153 0 48 0 94 274 1211 cx. pusillus 0 0 0 0 0 0 0 0 0 0 0 cx. theileri 1 0 0 0 0 0 0 0 0 0 1 table 3. continued … j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 246 cx. tritaeniohynchus 33 12 48 1 199 0 0 9 0 5 307 cs. longiareolata 0 0 0 0 0 16 0 0 0 0 16 oc. caspius 0 0 0 0 0 0 0 0 0 0 0 uranotaenia unguiculata 0 0 0 0 0 0 0 0 0 1 1 total 302 37 105 447 352 16 48 14 94 280 1695 discussion a total of 5661 larvae belonged to 5 genera and 14 species were identified: anopheles genus: 4 species, culex genus: 7 species, culiseta, ochlerotatus, and uranotaenia genera: one species. in previous studies of authors in kalaleh county, an. hyrcanus, an. maculipennis s.l., an. pseudopictus, an. superpictus, cx. hortensis, cx. perexiguus, cx. pipiens, cx. theileri, cs. longiareolata and oc. caspius had been reported (12, 13). furthermore, species such as an. claviger and cs. subochrea identified in kalaleh study (13) were not collected in the present study. in previous studies of authors in kalaleh county, cx. pipiens, cx. theileri, cx. hortensis, cx. perexiguus, an. maculipennis s.l., an. superpictus, an. hyrcanus, an. pseudopictus, oc. caspius and cs. longiareolata had been reported (12, 13). furthermore, species such as an. claviger and cs. subochrea identified in kalaleh study (13) were not collected in the present study. in some further studies in north of iran and golestan province related to the fauna and ecology of mosquitoes (8-13) were identified species such as: aedes vexans, an. alrgeriensis, an. claviger, an. melanoon, an. multicolor, an. plumbeu, an. pulcherrimus, coquillettidia richiardii, cx. territans, cs. subochrea, oc. echinus, oc. geniculatus, oc. pulcritarsis were not collected in our study, and cx. pusillus were reported for the first time from golestan province. in the present study, the dominant species was cx. pipiens and 77.3% of the whole collected larvae belonged to this species. in the studies of mazandaran province and kalaleh county, (13, 25, 26), moreover, cx. pipiens had been reported as dominant species. the dominant species had been an. hyrchanus in neka county, mazandaran province (24) and an. maculipennis and cx. theileri in ardebil province in 2008 (19). culex pipiens has been reported from almost all provinces of iran (33). in the present study, too, this species was reported from all counties of golestan province. larva habitats of this species varied, but mostly of samples were collected from bogs, agricultural streams, wetlands and rice fields. in mazandaran, reported wetlands and discarded tires the main larval habitats for cx. pipiens (26). in isfahan (34), this species was mostly found in rice fields and natural habitats. larval habitats of this species are mostly stagnant and artificial bodies of fresh water such as swamp of watering channels and holes and barrels filled with rain (35). investigation of cx. pipiens larval habitats and their characteristics showed that this species was collected from different larval habitats with various ecological conditions; even, 96% of the whole collected species from ovitraps belonged to this species and this species has a very high adaptability with various larval habitats and this factor has caused its high frequency and distribution reported from iran and the region (33). culex tritaeniohynchus was one of the other species collected from a majority of counties and after cx. pipiens, in the second rank, it included 12.4% of the whole collected larvae, 21.7% of larvae isolated from different larval habitats and also 3.1% of larvae isolated from ovitraps. similar to our study, in guilan, this species was in the second rank after cx. pipiens (33), and in mazandaran study (25), in the third rank after cx. pipiens and cx. torrentium, while in neka county of mazandaran table 5. continued … j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 247 province, kurdistan, esfahan and qom provinces studies, no larvae from this species has been collected (15, 36-38). in golestan province, this species has been collected from different larval habitats, but its frequency in temporary and stagnant water habitats (95.7%) was more than that in permanent and running water habitats (4.3%). this observation is consistent with another study in iran (21) but in southern iran (28), these species were collected in permanent water higher than temporary water habitats. furthermore, the frequency of this species in full sunlight situation and mud substrate habitats was higher than in shaded situation and sandy substrates habitats. in southern iran (28), these species were collected in full sunlight and sandy substrate habitats higher than partial sunlight and mud substrate habitats. this species was mostly found in agricultural streams, wetlands, and water leakage and river edge. in this study, cx. perexiguus was mostly found in maraveh tappeh county in the northeast of golestan province (80%), but it was found even in western counties of golestan province such as bandar-e gaz and bandar-e turkmen. these species prefers temporary larval habitats to permanent ones (13, 35, 39). in the present study, 55.5% of the collected larvae of this species were collected from temporary habitats. this species was only collected from natural habitats and no larvae were collected from artificial habitats, in kalaleh study (13) 89% larvae of this species were collected from natural habitats and in central iran study (37) larvae of cx. perexiguus were only collected from rice fields as well in our study. the preferred habitats for this species were rice fields (61.1%). the notable point for habitats of this species is that it was collected from habitats with sweet, salty and brackish water. this feature was also true for only cx. pipiens and cx. tritaeniohynchus species. since this species was collected from ovitraps and habitats with different conditions, it has good adaptability with different conditions of larva habitats. from culiseta genus, only cs. logiareolata and from kalaleh county was collected. the information about the ecology of culiseta species in iran is limited. in the present study, cs. logiareolata was collected from only one larval habitat which was a destroyed cistern with cement substrate without vegetation. however, in guilan province, north khorasan, yazd and esfahan provinces (20, 23, 34, 40) this species collected with high abundance and in other studies, this species had been collected from a variety of habitats (35, 36). in yazd county, too, have reported cs. logiareolata and cx. pipiens in larval habitats infected with organic substances, industrial waste materials and cement pools for storing animals’ water (40). anopheles maculipennis group, an. claviger, an. hyrcanus, an. plumbeus, an. alrgeriensis and an. multicolor and an. pulcherrimus have been previously reported in golestan province (8-12), but in the present study, an. superpictus, an. maculipennis, an. psudopictus and an. hyrcanus were collected and identified. anopheles superpictus and an. maculipennis have been known as the vectors of malaria disease. in our study, an. superpictus was mostly found in the natural habitats of river beds and streams with permanent and stagnant water, also with or without vegetation. these findings are completely similar to the results reported in previous studies (13, 36, 40). in guilan province, this species only reported of natural habitats with transient, stagnant and clear water, and was mostly found in rain pool habitats (23). the characteristics of larval habitats of an. superpictus are firstly shallow clear waters with stony bed and without vegetation and secondly rivers with sandy bed under sunlight and also shallow streams with muddy bed (34). moreover, in the present study, this species was collected from both types of the mentioned larval habitats. from genus ochlerotatus, only oc. caspius was collected, while in kalaleh county study j arthropod-borne dis, september 2018, 12(3): 240–251 a sofizadeh et al.: fauna and larval … 248 (13), three species of these genera had been reported (oc. caspius, oc. echinus, and oc. geniculatus). ochlerotatus caspius has been collected in many studies in iran (13, 27, 41– 43). in terms of frequency, in kurdistan study (41), it was in the second rank after cx. theileri. this high frequency of this species can be due to the ochlerotatus mosquitos grow in humid forest regions and more selected villages for sampling are in forest regions. in relation to the characteristics of larval habitats, oc. caspius preferred temporary, running water, muddy substrate and full sunlight habitats. in kurdistan province (36) this species reported of river edge with stagnant and clear water and partial sunlight habitat. although this species prefers larval habitats with clear and sweet water, it was also collected in larval habitats with turbid and brackish water. culex mimeticus, cx. theileri, cx. hortensis, cx. pusillus, an. maculipennis s.l., an. hyrcanus, an. pseudopictus and uranotaenia unguiculata were collected with low frequency in golestan province; the larval habitat characteristics of these species must be extensively studied. in addition, one of the aims of this study was to find aedes genus larvae, but we did not collect any larvae of this genus in golestan province, although ae. vexans was previously reported from the province (8). conclusion due to the good climate conditions, different species of mosquitoes grow in golestan province and since there are different vectors for various diseases among these species, conditions of disease transfer are present in this region. in the present study, we did not collect any species among the vectors of diseases such as dengue fever, zika, and chikungunya. future studies for finding these vectors in golestan province are 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accepted 30 apr 2018) abstract background: scrophularia striata is a perennial plant which is native in all parts of iran, turkey, and azerbaijan. in this study, the total phenol content, antioxidant and larvicidal activities of total extract and different fractions of this plant were evaluated. methods: the aerial parts of s. striata were collected from boli village, illam province, western iran in apr 2013. the total phenol content of total extract and different fractions were evaluated by folin-ciocalteu method. moreover, antioxidant activity was tested by dpph and fraps assays. larvicidal activity was investigated according to standard method described by who. results: ethyl acetate fraction (ef) had the highest content of total phenol (75.9±0.06mg gallic acid equivalent/g dry extract). furthermore, among the tested extract, methanol-water fraction (mwf), total methanol extract (tme) and water fraction (wf) showed the highest antioxidant activity in the dpph assay (ic50= 226.8, 283.66 and 299.4 μg.ml-1, respectively). in frap assay mwf and wf and tme had the highest antioxidant activities (664.4±0.002, 565.3±0.003, 519.5±0.003mmol feii/g dry extract, respectively). ethyl acetate fraction had maximum larvicidal activity (lc50 49.1ppm) followed by tme (lc50 64.26ppm) and hexane fraction (hf) (lc50 89.69). conclusion: scrophularia striata collected from west of iran illustrated considerable antioxidant and larvicidal effects and further in vitro and in vivo experimental models for investigation would be required. keywords: scrophularia striata, antioxidant activity, larvicidal activity, anopheles stephensi introduction according to the word malaria report, in 2014, 198 million cases of malaria occurred globally in 2013 and led to 584000 deaths (1-3). in southern parts of iran including sistan and baluchistan, hormozgan and some parts of kerman provinces, this disease remains as a vital public health problem (4). six anopheles vectors exist in this area including an. culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus and an. pulcherrimus (5). the present proliferation of malaria is due basically to increasing resistance of mosquitoes to current insecticides (6). plant materials have been investigated for their susceptibility in controlling the malaria vector due to their ovicidal, larvicidal and adulticidal activities. the most susceptible stage to attack mosquitoes is larval stage as they are concentrated in smaller areas. therefore, interrupting mosquito life cycle at larval stage is one of the important methods for controlling malaria transmission (7). *corresponding author: dr mahnaz khanavi, email: khanavi@mail.ubc.ca/ khanavim@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 119–126 f yousefbeyk et al.: antioxidant and … 120 http://jad.tums.ac.ir published online: june 12, 2018 the genus scrophularia (scrophulariaceae) comprises about 300 known species. the various species of scrophularia genus have been traditionally used in medical conditions including skin inflammatory disease like scabies, tumors, eczema (8), and psoriasis, inflammatory disorders fever, constipation, pharyngitis, neuritis, laryngitis affections (9). among these species, s. ningpoensis hemsl has been used for treatment of fever laryngitis, pharyngitis, neuritis and constipation swelling in china. moreover, s. grossheimi and s. nodosa are used as diuretic agents (10). recent studies reported antimalarial, antiprotozoal and antimycobacterial activities of s. cryptophila (11). phytochemical studies indicated the presence of saikosaponins, iridoids and phenylpropanoid glycosides such as angoroside a, angoroside c, angoroside d, acteoside and isoacteoside in s. scorodonia l. (8), iridoid glucosides including catalpol, 6-o-methylcatalpol and aucubin, phenylethanoid glycoside like angoroside c in s. lepidota (12), resin glycosides including crypthophilic acids a– c in s. cryptophila (11). many natural compounds have been reported for larvicidal activity. for example, flavonoids namely poncirin, rhoifolin, naringin and marmesin from poncirus trifoliate (13), lupinfolin and rotenone and deguelin from derris trifoliate (14), new iridoid glycosides like 6'-o-rhamnosylharpagide and 6o-xylosylharpagoside-b form ajuga remota (15) and coumarins such as umbelliferone, herniarin, psoralen and xanthotoxin from cnidium monnieri (16) and coumarin derivative pachyrrhizine from neorautanenia mitis (17). scrophularia striata boiss is a perennial plant grown in all parts of iran as well as turkey and azerbaijan. these species have been used to cure different inflammatory diseases such as allergy, rheumatics and chronic inflammatory disorders in iranian folk medicine (18). so far, phytochemical investigation revealed the presence of cinnamic acid, flavonoids such as quercetine, isorhamnetin3-o-rutinoside, and nepitrinandand, and one phenyl propanoid glycoside acteoside 1 in this plant (10). studied showed the inhibitory effect of s. striata extract on matrix metalloproteinases and astrocyte cancer cell line (1321) (18, 19). different extracts from the aerial part of s. canina were evaluated for the insecticidal activity against larvae and adult females of culex pipiens molestus. the most toxicity was revealed by the petroleum ether extract against second-instar larvae (48h, lc50= 23.5 ppm) and by the hexane extract against fourthinstar larvae (48h, lc50= 23.6ppm) (20). the leaves of s. nodosa showed larvicidal activity against cx. quinquefasciatus (75.3% mortality) (21). the ethanol root extract of s. lepidota exhibited anti-protozoal activity (ic50 40.6μg.ml-1) and plasmodial enoyl-acp reductase (fabi) enzyme inhibitory activity (ic50 100μg.ml-1), a key enzyme of fatty acid biosynthesis in plasmodium falciparum (12). in the present study, total extract and different fractions of s. striata were investigated for larvicidal properties against main malaria vector, an. stephensi. in addition, total phenol content and antioxidant activity (using dpph and frap methods) of this plant were evaluated. materials and methods plant material and extraction the aerial parts of s. striata were collected from boli village, ilam province, western iran in apr 2013. the voucher specimen was deposited in herbarium of faculty of pharmacy, tehran university of medical sciences (herbarium number: 6748-teh). the aerial parts (500g) were aired dried in shade and fractionated with hexane, ethyl acetate, methanol, methanol-water (1:1) and water at room temperature, consequently. total methanol extract was prepared from aerial http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 119–126 f yousefbeyk et al.: antioxidant and … 121 http://jad.tums.ac.ir published online: june 12, 2018 parts by percolation method. all the extract and fractions were dried using rotary evaporator to give 2.8g, 6.5g, 60g, 48.2g, 7.7g and 28.7g residues from hexane, ethyl acetate, methanol, methanol-water (1: 1), water fractions and total extract, respectively. antioxidant activity dpph radical-scavenging activity assay the antioxidant activity of extracts was investigated by the dpph (2, 2′-diphenyl-1-picrylhydrazyl) free radical scavenging method according to an established protocol. sample solutions (1ml) were prepared in methanol at different concentration. then the solutions added to dpph methanol solution (2ml, 40μg.ml-1) and incubated at room temperature for 30 min. the absorbance was measured at 517nm. vitamin e and butyl hydroxyanisole (bha) were used as positive controls. ic50 values (indicate the concentration of the test samples providing 50% radical scavenging) were calculated from graph-plotted scavenging percentage against extract concentration (22). ferric reducing antioxidant potential (frap scavenging) assay the frap assay was done by using the method described (23, 24). the frap reagent contained 5 ml of a (10mmol.l-1) tptz (2, 4, 6-tripyridylstriazine) solution in 40 mmol.l-1 hcl plus 5ml of (20mmol.l-1) fecl3 and 50ml of (0.3 mmol.l-1) acetate buffer, ph 3.6 was prepared freshly. aliquots of each extract (50μl) were mixed with frap reagent (1.5ml). the mixtures were incubated at 37 °c, for 10min. finally, the absorbance of each sample was measured at 593nm. for construction of calibration curve, five concentrations of feso4, 7h2o (125, 250, 500, 750, 1000 mmol.l-1) were prepared to used. the antioxidant activities were calculated as the concentration of antioxidants having a reducing ability equivalent for 1mmol.l-1 feso4 (22, 25). determination of total phenolic contents total phenolic contents of extracts were determined by folin-ciocalteu method (26). folin-ciocalteu (merk) reagent diluted to tenfold with distilled water. then 5ml of this solution was added to 1ml of each extract (1 mg.ml-1) and allowed to stand at room temperature for 10min. a 4ml sodium bicarbonate solution (75g.l-1) was added to the mixture. after 30min at room temperature, absorbance was measured at 765nm using a uv spectrophotometer (pharmacia biotech). total phenolic contents were quantified by calibration curve obtained by measuring the absorbance of a known concentration of gallic acid (ga) standard (20–200mg.l-1). the concentrations are expressed as milligrams of gallic acid equivalents (ga) per gr dry extract (22, 27). bioassays and larval mortality fourth instar larvae of an. stephensi bandar-abbas strain was exposed to test concentrations of 20, 40, 80, 160 and 320ppm of each extract (solvent: ethanol) for 24h according to standard method described by who (1981). briefly, 1ml of appropriate dilution of each extract with 224ml of water and 25 larvae in 25ml water mixed and total volume was 250 ml (2). for control, only 1ml of ethanol with 224ml of water and 25 larvae in 25ml water mixed and total volume was 250ml. the experiment was repeated four times on different days. the percentage of mortality was reported from the average for the four replicates after 24h exposure period. from the regression line between logarithmic dose and probit mortality, the lc50 was measured (28, 29). the investigation of larvicidal activity has been carried out in the insectarium of department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 119–126 f yousefbeyk et al.: antioxidant and … 122 http://jad.tums.ac.ir published online: june 12, 2018 results ethyl acetate fraction (ef) had the highest content of total phenol (75.9±0.06mg gallic acid equivalent/g dry extract) (table 1). among the tested extract, methanol-water fraction (mwf), total methanol extract (tme) and water fraction (wf) showed highest antioxidant activity in the dpph assay (ic50= 226.8, 283.66 and 299.4μg.ml-1, respectively). in frap assay mwf and wf and tme had the highest anti oxidant activities (664.4±0.002, 565.3±0.003, 519.5±0.003mmol feii/g dry extract, respectively). the total extract and fractions of s. striata were effective against an. stephensi with lc50 ranged between 49.15 to 1265.96ppm. ethyl acetate fraction had maximum larvicidal activity (lc50 49.15ppm) followed by tme (lc50 64.2623ppm) and hf (lc50 89.69) (table 2). table 1. antioxidant activity and total phenolic content of total methanol extract and different fractions from aerial parts of scrophularia striata dpph (ic50: μg.ml-1) frap (mmol feii/g dry extract) total phenol contents (mg gae/g dry extract) tme 283.7 519.5±0.003 64.8±0.03 hf >1000 248.8±0.035 -a ef 505.2 444.28±002 75.9±0.06 mf 511.3 513.7±0.026 41.7±0.04 mwf 226.8 664.4±0.002 56.2±0.02 wf 299.4 565.3±0.003 33±0.06 key to extracts employed: tme: total methanol extract, hf: hexane fraction, ef: ethyl acetate fraction, mf: methanol fraction, mwf: methanol-water fraction, wf: water fraction. a not detected. table 2. probit regression line parameters of total extract and different fractions of scrophularia striata against anopheles stephensi intercept slope ± se lc50 (ppm) , 95%c.i. lc90 (ppm) , 95%c.i. λ2 table (df) p-value tme -4.5092 2.4941±0.537 64.2623 (27.2325-131.1766) 209.8017 (109.8552-2827.5650) 24.405* 0.001 hf -2.4518 1.2556±0.144 89.6911 (72.3973-112.2429) 940.7311 (577.0739-1990.1654) 13.345* 0.001 ef -2.7150 1.6051±0.156 49.1497 (40.1770-58.6299) 309.0209 (231.3864-462.1751) 13.345* 0.001 mf -4.6954 1.9658±0.199 244.6640 (203.2745-310.4233) 1097.8066 (747.3601-1921.5333) 13.345 0.001 mwf -6.5143 2.0977±0.424 1265.9601 (607.7026-2645.3743) 5161.5548 (2523.4135-75009.0794) 13.345 0.001 wf -7.9868 2.6359±0.195 1071.3972 (949.6278-1205.7662) 3282.1074 (2756.2862-4091.2804) 13.345 0.001 key to extracts employed: tme: total methanol extract, hf: hexane fraction, ef: ethyl acetate fraction, mf: methanol fraction, mwf: methanol water fraction, wf: water fraction, lc50 ±95% c.l= lethal dose cause 50% mortality, 95% confidence interval, lc90 ±95% c.i= lethal dose cause 90% mortality, 95% confidence interval, (df)= degree of freedom, p= p-value http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 119–126 f yousefbeyk et al.: antioxidant and … 123 http://jad.tums.ac.ir published online: june 12, 2018 discussion scrophularia striata is a perennial plant which is native in all parts of iran as well as turkey and azerbaijan. in this study total methanol extract and different fractions from aerial parts of s. striata were investigated for antioxidant and larvicidal activities. according to our results, ef and tme had the highest amount of total phenolic compounds. scrophularia steriata, as highest amount of total phenolic compounds, was reported in ethanol 70% and ethyl acetate extracts (79.7 and 65.5mg gallic acid equivalent/g dry extract, respectively). moreover, water extract of this plant was reported to have better radical scavenging activity (ic50 195µg.ml -1) (30). furthermore, evaluation of larvicidal activity of s. striata revealed that lc50 value of total extract and fractions ranged between 49.15 to 1265.96ppm, and the ef had maximum larvicidal activity (lc50 49.15ppm). essential oils and extracts of different plant species have been investigated for larvicidal activity. for instance, the essential oil prepared from seeds of heracleum persicum had larvicidal effect with lc50 value of 104.80ppm. the essential oil of cupressus arizonica revealed significant larvicidal activity against an. stephensi with lc50 79.30ppm (31). moreover, for coriandrum sativum lc50 value of 120.95ppm and for cymbopogon olivieri 321.90ppm were reported (5), which their activities were low when compared with ef and tme of s. striata. the lc50 of essential oils from tagetes minuta and foeniculum vulgare were 1.05 and 20.10ppm, respectively (5), these plants were more effective than s. striata. the methanol extract of t. minuta had better lc50 value (2.5ppm) followed by methanol and aqueous extract of nelumbo nucifera (8.89 and 11.82ppm, respectively) and methanol extract of cassia fistula (17.97ppm) when compared with the extract and fractions of s. striata (32-34). different extracts of fruits and leaves of centratherum anthelminticum were tested for larvicidal activity against an. stephensi and the petroleum ether extract of fruits (lc50 162.60) were more toxic than that of leaf extract (lc50 522.94) (7). petroleum ether extract from leaves of artemisia annua had larvicidal activity with lc50 value of 263 ppm (7). the leaf and seed methanol extracts of clitoria ternatea showed dose-dependent larvicidal activity against an. stephensi with lc50 values of 555.6 and 116.8ppm, respectively (35). the leaves petroleum ether extract of gymnema sylvestre exhibited the highest mortality in the concentration of 1000ppm against the larvae of an. subpictus (lc50 166.28 ppm) (36). moreover, the leave methanol extract of calotropis gigantea showed larvocidal activity (lc50 121.69ppm) (37). larvicidal activity of aceton extract of millingtonia hortensis (lc50 223.9ppm) and ethanol extract was from peels of citrus sinensis (lc50 291.69ppm) (35, 38). extracts of a. annua, c. anthelminticum (7), citrus sinensis, c. ternatea (35), c. gigantean (37), g. sylvestre (36) and m. hortensis (38) were less active than ef and tme of s. striata. conclusion ef and tme of s. striata possesses better larvicidal activity against an. stephensi than other fractions. moreover, antioxidant activity of mwf, wf, and tme were higher than other fractions in both dpph and frap assays. finally, complete phytochemical investigation is suggested to reveal most effective compound in this native species. acknowledgments this research as a thesis in doctor of pharmacy degree (ms zahra mirzaee) has been fihttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 119–126 f yousefbeyk et al.: antioxidant and … 124 http://jad.tums.ac.ir published online: june 12, 2018 nancially supported by tehran university of medical sciences. the authors declare that there is no conflict of interest. references 1. olepu s, suryadevara pk, rivas k, yoko yama k, verlinde cl, chakrabarti d, van voorhis wc, gelb mh (2008) 2-oxo-tetrahydro-1, 8-naphthyridines as selective inhibitors of malarial pro tein farnesyltransferase and as antimalarials. bioorg med chem lett. 18(2): 494–497. 2. ghanbarzadeh s, ghasemi s, shayanfar a, ebrahimi-najafabadi h (2015) 2dqsar study of some 2,5-diaminoben zophenone farnesyltransferase inhibitors by different chemometric methods. excli. 14: 484–495. 3. world malaria report (2014) who press gmp, world health organization. geneva, switzerland. world malaria report, who press, global malaria program, world health organization. available at: 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millingtonia hortensis (family: bignoniaceae) against anopheles stephensi, culex quinquefasciatus and aedes aegypti. j vector dis. 45(1): 66. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 67–75 l masoori et al.: molecular-based detection of … 67 http://jad.tums.ac.ir published online: march 18, 2018 original article molecular-based detection of leishmania infantum in human blood samples in a new focus of visceral leishmaniasis in lorestan province, iran leila masoori 1, farnaz kheirandish 2, ali haghighi 1, mehdi mohebali 3,4, behnaz akhoundi 3, niloofar taghipour 1, latif gachkar 5, ali chegeni-sharafi 6, *vahideh moinvaziri 1 1department of parasitology and mycology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 2department of parasitology and mycology, school of medicine, lorestan university of medical sciences, khorramabad, iran 3department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 4center for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran 5infectious diseases and tropical medicine research center, shahid beheshti university of medical sciences, tehran, iran 6department of communication disease control and prevention, deputy of health, lorestan university of medical sciences, lorestan, khorramabad, iran (received 15 mar 2015; accepted 19 feb 2018) abstract background: the fatal form of leishmaniasis is visceral form (vl), found in some of the countries in the world. visceral leishmaniasis has been reported sporadically from all provinces in iran, including lorestan. this study aimed to characterize parasite species in dat positive and some of the dat negative human blood samples of delfan district, lorestan province, central iran. methods: blood samples were collected from different geographical areas of delfan. serum was used for dat test and remained part of molecular study. dna was extracted by using dng-plus extracted kit (cinagen, iran). polymerase chain reaction amplification of leishmania kdna and pcr-rflp of its1 was done to identify leishmania species. some amplicons were sequenced, submitted to genbank and analyzed by blastn. results: expected band of kdna for l. infantum (720bp) was amplified in 16 out of 186 (8.6%) samples which showed previously anti-leishmania antibody at different titers or were negative serologically. using blastn, 93% similarity with l. infantum has been shown. the rdna-its1 was amplified only in 9 samples (4.7%). rflp pattern was similar to what expected for l. infantum. conclusion: a new emerging hypo-endemic focus, caused by l. infantum, is going to be established in delphan district, lorestan province. further studies on vector and reservoirs are necessary for the region and other parts of lorestan province. keywords: visceral leishmaniasis, leishmania infantum, kdna, its1, iran introduction leishmaniasis is a group of protozoan diseases transmitted to humans and other mammals by phlebotominae sandflies and can manifest in different clinical forms, depending upon the infecting species of leishmania. the disease could emerge as cutaneous, mucocutane ous or visceral leishmaniasis (vl) (1). mediterranean form of vl is defined as a zoonotic disease, vl is a huge burden on human health and society, it is caused by l. donovani complex in asia and africa and by l. chagasi, a synonymous of l. infantum, in latin america (2-4). *corresponding author: dr vahideh moin-vaziri, e-mail: vmvaziri@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 67–75 l masoori et al.: molecular-based detection of … 68 http://jad.tums.ac.ir published online: march 18, 2018 the leishmaniasis is found to be prevalent in some of the poorest countries in the world, and they gain lesser attention than another infectious disease like malaria, tuberculosis, and aids. they are categorized as a neglected tropical disease (ntd) because although not recognized and prioritized, they cause significant health problems to the very poorest in society. there is little effort on the part of the global community and pharmaceutical industry to invest in research and development of better and innovative therapeutic because of lack of sufficient incentives (1, 5). leishmaniasis rank as the leading ntd in terms of mortality, with an estimated 20000– 40000 death (6, 7) and 3.3 million disabilityadjusted life years (8). the most life-threating form of disease is vl, in which the pathogen disseminates to endothelial systems, like liver, spleen and bone marrow. clinical signs and symptoms of vl generally include prolonged and irregular fever associated with chills, hepatosplenomegaly, lymphadenopathy, progressive anemia, weight loss. more than 90% of vl cases are reported from bangladesh, brazil, ethiopia, india, sudan and south sudan (8). two different forms of leishmaniasis comprising vl and cl exist in iran. cases of vl have been reported from all parts of the country (9). unlike cl, which accounts for almost 20000 new cases per year (9), vl has been reported sporadically, with endemic foci located in northwestern and southern iran, with about 100–300 new cases annually (9, 10). the diagnosis of vl is a challenging issue and several studies work to resolve problem (4). direct agglutination test (dat) has been applied vastly for serological studies of vl in human and animal reservoir hosts in the world as well in iran particularly in the endemic areas (9, 11-13). dat is a simple, costeffective and field applicable test and has been recommended for seroepidemiological studies as well as early and accurate diagnosis of vl, especially in endemic areas (9, 14). as mentioned vl is reported sporadically in all provinces in iran, there are several reports of sporadic vl cases in lorestan province, but real status of disease is not clear in this area. lorestan is a province of western iran in the zagros mountains, the climate is generally sub-humid continental with cold winter. a seroepidemiological study using dat was performed on 800 collected sera from healthy population of delphan county located in mountainous part of lorestan province by the same authors. anti-leishmania antibody at different titers in 38 cases (4.75%) which indicate that a new focus of vl with low endemicity is going to be formed in delphan district (15). therefore, further studies seem necessary on leishmania species identification, vector incrimination, as well as reservoirs. identification of leishmania parasites is essential for precise prognosis of the disease as well as making proper decision regarding treatment (16, 17). dna-based methods have proven to be effective in detecting the genome of leishmania species in different biological samples with aim of species identification (4). among different genetic markers used for leishmania identification, kdna, and its1 vastly used in detecting parasite in different biological samples (18-20). the kinetoplast dna contains around 10000 mini-circles per cell, each around 800bp in length with an approximately 600bp variable and 200bp conserved region. the heterogeneity of the variable region has been exploited to discriminate between leishmania spp (21). its1-rflp-pcr also is common in characterization of different leishmania spp (16, 20, 22, 23). the current study aimed to characterize leishmania species in the peripheral blood samples which showed anti-leishmania antibodies at titer ≥1:3200 (considered positive) and titers between 1:800 and 1:1600 (considered suspected) and 20% of titers under 1:800 (considered negative) obtained from previous study (15), targeting kdna and its1 gene. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 67–75 l masoori et al.: molecular-based detection of … 69 http://jad.tums.ac.ir published online: march 18, 2018 materials and methods study area delfan district is located in mountainous area of northwest of lorestan province. the altitude of study area is about 2000m above sea level and very cold in winter. the total population was 85000 in 2009, including 1200 nomadic family. sample preparation totally 800 samples were collected from children ≤ 12yr old and 10% of adults by a multi-stage randomized cluster sampling in 2012. after filling out a questionnaire, 3ml of peripheral blood sample has been taken from each person. sera of about 1ml of blood sample had been used for serology study, using dat at the school of public health, tehran university of medical sciences, tehran, iran (10) and results published previously (15). two remained ml were kept at -20 °c in the edtacontaining tubes in department of parasitology, shahid beheshti university of medical sciences, tehran, iran for dna extraction. based on serology results (15), just 186 samples out of 800 which showed anti-leishmania antibody at different titers were subjected to molecular works, comprising 5 cases (0.62%) at titers ≥ 1:3200 considered as positive, 21 samples (2.62%) at titer 1:800 to 1:1600 considered suspected and 160 samples (20%) serologically negative (out of them 146 showed no titer and 14 at titer 1:400) (table 1). dna extraction five hundred microliters of whole blood were used for dna extraction. to remove interfering hemoglobin molecules from the samples prior to dna extraction, blood was washed with pbs, even 10 times. dna was extracted using dng-plus extracted kit (cinagen, iran) based on manufacturer instructions, dna concentration was determined by nanodrop (bio wave ii) at 220 and 280nm. standard strains l. infantum: mcan/ir/07/ moheb.gh, l. tropica: mhom/ir/02/mash10 and l. major: mrho/ir/75/er was achieved from department of medical parasitology, school of public health, tehran university of medical sciences, iran to monitor the reactions. pcr amplification of leishmania kdna for initial parasite detection, a nestedpcr was performed by using specific primers csb2xf: (cgagtagcagaaactcccg ttca), csb1xr: (atttttcgcgatttt cgcagaacg) (external) and 13z: (act gggggttggtgtaaaatag), lir: (tc gcagaacgcccct) (internal) (21). these primers amplified a fragment of 560 to 750 bp species dependent. the amplification conditions of both two rounds were 94 °c for 5 min, followed by 30 cycles of denaturation at 94 °c for 60 sec, annealing at 55 °c for 60 sec and extension at 72 °c for 90sec, with a final extension step at 72 °c for 5min. five µl of the pcr production of second rounds were visualized on a uv transilluminator following 1.5% agarose gel electrophoresis containing ethidium bromide. pcr amplification for leishmania its1 litsr and l5.8 primers were used to amplify the small subunit 5.8s rrna of parasite genome. specific primers, litsr (forward: 5′-ctggatcattttccgatg-3′) and l5.8s: (reverse: 5′-tgataccacttatcgcactt3′), then pcr products were digested by haeiii (bsuri) restriction enzyme following protocol (24). after using the restriction enzyme, pattern fragment of 200, 80 and 60bp for l. infantum would be produced and visualized by 2% agarose gel electrophoresis, stained with ethidium bromide. all the procedures were monitored by standard leishmania parasite (l. major acc. no: jn860745, l. tropica acc. no: ef653267 and l. infantum (acc. no: fj555210). http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 67–75 l masoori et al.: molecular-based detection of … 70 http://jad.tums.ac.ir published online: march 18, 2018 sequencing some samples were sequenced, submitted to genbank and their homology with the available sequence data in genbank was checked by using blastn (25). ethics ethics clearance was obtained from research ethical community of shahid beheshti university of medical sciences (approval number: smbu.rec.1392.302(. results leishmania parasite identification by kdnanested-pcr in 16 samples out of 186 ones (8.6%), amplification of kdna was yielded by visualizing band of 720bp, for l. infantum (fig. 1). the kdna amplification was obtained from 6 serologically negative samples, including one sample with no titer and 5 others at titer 1:400 (table 1). the pcr products of 6 samples were sequenced and deposited in genbank (accession no: kj417490, kj417491, kj417492, kj417493, kj417494, kj417495). blast analysis showed 83–93% homology to l. donovani (accession no: aj100780.1) and l. infantum (accession no.nz32847.1). leishmania parasite identification with its1-pcr-rflp totally 9 samples (including sero-negative cases) out of 189 (4.7%) have shown leishmania infection by visualizing a band approximately 350bp in 1.5% agarose gel. digestion of amplicons with haeiii (bsuri) enzyme produced the fragments of 200, 80 and 60bp characterized as l. infantum in all samples. the results also were monitored by digestion pattern in reference strain of l. infantum (fig. 2). one pcr product was sequenced and submitted to genbank, which is accessible by accession no. kj417496. blast analysis showed 94% similarity to iranian l. infantum. (accession no: kc34730 and hq535858.1). fig. 1. gel electrophoresis of kdna-nested-pcr products obtained from l. infantum isolates of blood samples collected from delfan district, lorestan province lane 1–3: leishmania tropica mhom/ir/02/ mash10 (750bp), l. infantum mcan/ir/07/ moheb.gh (720bp), l. major mhom/ir/75/er (560bp), standard strains respectively lane 4: sample of current study (l. infantum) lane 5: molecular weight marker (100bp ladder fermentas) fig. 2. digestion of its1-rdna amplicons using haeiii (bsuri) enzyme of leishmania isolated from blood samples collected from delfan district, lorestan province lane 1 and 7: m: molecular weight marker (100 bp ladder fermentas) lane 2 and 3: current study samples lane 4–6: leishmania tropica mhom/ir/02/ mash10, l. infantum mcan/ir/07/moheb.gh, l. major mhom/ir/75/er standard strains respectively http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 67–75 l masoori et al.: molecular-based detection of … 71 http://jad.tums.ac.ir published online: march 18, 2018 table 1. number of molecular positive samples at different anti-leishmania antibody titer among collected human blood samples from delfan district, lorstan province, iran, 2012 antibody titer number of samples molecular positive cases percent negative no titer 146 1 0.68 1:400 14 5 35.7 suspected: (1:800 to 1:1600) 21 9 42.8 positive: (≥ 1: 3200) 5 1 20 total 186 16 8.6 discussion vl is a life threating disease with an estimation of 50000 deaths in 2010 in the world (6). vl occurs sporadically in all geographical zones of iran but it is endemic in some areas mainly located in northwestern and southern part of the country. disease status in lorestan province is not clearly distinct. our previous study in delfan district (located in northwest of lorestan province) showed 3.25% seropositive samples among 800 collected ones using dat (15). leishmania species identification is of great importance from clinical and epidemiological point of view. as a result of morphological similarities, leishmania species could not be firmly identified using conventional methods. main objective of current study was to characterize the leishmania species among the samples which collected from delfan and as a result of our previous study showed anti-leishmania antibodies at different titers (15), targeting two different molecular markers. kdna is introduced as a quite useful marker for initial screening of parasite by different researchers (26, 27). in the present study, kdna could be a reliable marker because peripheral blood samples were used. a fragment about 720bp of kdna was visualized in 16 out of 186 samples (8.6%), which based on the size and sequencing results they were characterized as l. infantum. this marker is used commonly in distinguishing parasite in human samples, reservoirs, and vectors, most especially when there was not enough amount of parasite in the samples (21, 23, 28, 29). using this marker, l. infantum was isolated from sandflies in greece by producing a fragment of 720bp of parasite genome (30). leishmania infantum infection was identified in asymptomatic dogs in a new endemic focus of vl in iran (31). another useful marker which vastly used for leishmania species identification is its1 (24, 32-37). in the current study, expected band of 350bp length was amplified just in 9 out of 186 samples (4.8%). compared to kdna positive cases (8.4%) it could be concluded that kdna is quite appropriate for initial screening especially in case of inadequate amount of parasite in clinical samples. this marker could detect 0.01 to 0.001 parasites per ml whereas its1 has ability of detection of 1–6 parasites per ml (37, 38). enzymatic digestion pattern of its1 amplicons was the same as what expected for l. infantum which verified the results obtained using kdna. both genetic markers could detect parasite in one sample considered serologically negative. it was a recent infection which emphasizes on the ability of molecular tools in early detection of parasite, especially reasoning the ability of kdna marker in initial screening of the samples. if we had chance to follow the case, it would have shown rising titer, unfortunately, it was not possible for authors in spite of several attempts. dna of leishmania parasite also was detected in 5 samples out of 14 (35.7%) which showed anti-leishmania antibodies at titer 1/400, which according to the interpretation of dat results, they were considered negative. in these cases also rising http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 67–75 l masoori et al.: molecular-based detection of … 72 http://jad.tums.ac.ir published online: march 18, 2018 titer was probable, if follow up was possible. conclusion altogether, seroepidemiology and molecular results of current survey showed that a new emerging hypo-endemic focus is going to be established in delfan district, lorestan province, due to l. infantum. considering the lifestyle, climate and massive migration for seeking job, necessity of further work on other epidemiological aspects of disease are evident, as well health education should be considered by health authorities. acknowledgements this study was supported financially by infectious diseases and tropical medicine center, shahid beheshti university of medical sciences, tehran, iran. 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xeridat b, schleinitz n, cuisenier b, dumon h (2006) reference values for leishmania infantum parasitemia in different clinical presentations: quantitative polymerase chain reaction for therapeutic monitoring and patient followup. am j trop med hyg. 75(5): 858– 863. http://jad.tums.ac.ir/ microsoft word 4hashareh dr nikookar rtl _2_.doc iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 26 original articles species composition and diversity of mosquitoes in neka county, mazandaran province, northern iran sh nikookar1, *sh moosa-kazemi1, ma oshaghi1, mr yaghoobi-ershadi1, h vatandoost1, a kianinasab2 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2ancestry natural resources and forestry office, neka, mazandaran province, iran (received 6 sep 2010; accepted 20 dec 2010) abstract background: regarding to the significant of the possibility of the malaria epidemic and nuisance of mosquitoes during the active season, the fauna and some ecological activities of mosquitoes in related to tree holes were investigated from april to december 2009 in neka county of mazandaran province, northern iran. methods: larval collection was carried out from natural, artificial breeding places, and tree holes inside the forest in neka county, mazandaran province in 2009. in addition, human bait net trap collection was conducted using suction tube several times during this investigation. results: four genera and five species were found in tree holes. anopheles plumbeus, culiseta annulata, culex pipiens, and ochlerotatus geniculatus were collected by larval collection whereas, ochlerotatus pulcritarsis was found by adult collection. overall cx. pipiens 44.6%, oc. geniculatus 32.6%, an. plumbeus 22.5%, and cs. annulata 0.3% were collected by larval collection. during the bait net collection five specie were identified including: oc. geniculatus 55.87%, oc. echinus 1.33%, oc. pulcritarsis 8.8%, cx. pipiens 33.8%, and an. plumbeus 0.2%. cs. annulata larvae was detected for the first time with a low abundance in tree cavity. conclusion: tree holes were found the main habitat for the species of oc. geniculatus. the species of cs. annulata was found in tree holes keywords: mosquito, fauna, tree holes, diversity, iran introduction the family of culicidae (diptera) divided in two subfamily, 11 tribes, 113 genera, and 3526 species in the world fauna (harbach 2007). culicidae mosquitoes are the most public health important vectors of arthropods due to malaria transmission and vectors of various types of filariasis, and arbovirouses (horsfall 1955, smith 1973, zaim et al. 1984, service 1993). arbovrial viruses such as west nile were reported previously in iran (saidi et al. 1976). dog heart worm parasite (saidi et al. 1976, mobedi et al. 1990) and dirofilaria repens (sadighian 1969, siavashi and massoud 1995) also were reported previously in country. the dog heart worm, dirofilaria immitis transmitted by tree holes mosquitoes and have been reported from spain (bargues et al. 2006). an. plumbeus has been reported the vector of plasmodium falciparum in germany (kruger et al. 2001). although, larval breeding places and blood feeding behavior of malaria vectors have been studied previously, but in relation to species composition, and diversity of tree holes mosquitoes there are a few data. preliminary studies in relation to identification of culicidae mosquitoes of iran and its role in disease transmission such as malaria have been reported by macan and gutsevich (gutsevich 1943, macan 1950). there is scattered information due to culicidae mosquitoes and re*corresponding author: dr seyed-hassan moosakazemi, e-mail: shm.kazemi@gmail.com iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 27 ported by dow in 1953, minar in 1974, and 1981 (dow 1953, minar 1974, 1981). zaim et al. reported the fauna of aedes mosquitoes in iran (1984). checklist of the culicinae mosquitoes of iran was reported by zaim and cranston in 1986 (zaim and cranston 1986). fauna and ecological characteristics of culicidae mosquitoes in isfahan, kurdistan, sistan and baluchistan and hormozgan provinces have reported previously (mousakazemi et al. 2000, moosa-kazemi et al. 2005, 2009, and 2010). the fauna and ecological study of culicidae mosquitoes in guilan province was reported by azari-hamidian in 2002 (azarihamidian et al. 2002a). azari-hamidian reported the tree holes culicidae mosquitoes in guilan province previously (azari-hamidian 2003). some mosquitoes species such as an. plumbeus, oc. echinus, oc. geniculatus, orthopodomyia, oc. pulcritarsis, have been reported as tree holes mosquitoes in greece (silankok et al. 1937). the recent species also were reported in france (william et al. 1986) and azerbaijan (nagiyev 1963). investigation on species composition and diversity of mosquitos' is important in different aspects of vector control programmers. this information use to decrease of vector density then the hosts and vectors contacts, and biting nuisance. the present study was focused on some ecological aspects of the mosquitoes in relation to tree holes, such as the species composition, and diversity of mosquitoes in neca county, northern iran. materials and methods the study was carried out from april to december 2009. three fixed sites (darvishan, chalmardi, and khairabad), and six variable sites (golbestan, elmiyeh, baghearab, nimchah, chalehpol, and khorshid) were selected randomly with regard to existing facilities in mazandaran province (36°39 n, 53°17 e) (fig. 1). maximum and minimum temperatures were 27.6 and 9.1°c in the july and december, respectively. the maximum annual rainfall has been reported 204.6 mm in october, and the least 0.1 mm in july 2009. the type of forest vegetation has been found quercus castanifolia, alnus subcerdata, carpinus betulus, zelkova carpinifolia, and mespilus sp (natural resources of neka county 2009). sampling in larval habitats was performed by standard dipper biweekly, and adult mosquitoes were collected by one human bait net trap using suction tube monthly. the mosquito larvae were collected in different oviposition sites such as discarded tires, tree and rock holes, irrigation channels, rice fields, ponds, animal footprint, and marshes inside forest. some mosquito larvae were collected from 18 tree holes using dropper. these 18 tree holes were also evaluated two times monthly. during each of the evaluations, pupa were collected and placed in bucket dishes lined with netting and transferred to the laboratory. hatched pupa were reared to adult, identified by using the keys of shahgudian (1960), zaim and cranston (1986), azarihamidian and harbach (2009). human bait net collections were conducted monthly randomly nears the tree holes inside the forest. the mosquitoes were transferred to cups container, was kept closed and then collected with forceps and placed in petri-dishes lined with moist cotton. the samples were sent to the laboratory of entomology, school of public health, and tehran university of medical sciences. the mosquito larvae were preserved in lactophenol and the microscopic slides were prepared using de faure’s medium after one week (who 1963). adult mosquito samples were killed by chloroform and mounted by special pines. the samples were identified using the keys mentioned above. the weather condition such as temperature, humidity and altitude were recorded on the standard forms. the mosquito name abbreviations were cited based on reinert (reinert 2009). the data were analyzed using spss ver. 11.5. iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 28 results a total of 3047 adult mosquitoes were collected. out of 1547 adult mosquitoes were reared from pupae and the rest was collected by bait net trap. a total of 960 cx. pipiens (62.06%), 568 oc. geniculatus (36.71%), and 19 an. plumbeus (1.23%) were reared to mature during 18 sampling occasions from different larval habitats (table 1). totally 1500 mosquitoes were collected during the human bait net trap collection during 24 h. the most prevalent species was oc. geniculatus, a total of 838 (55.87%), followed by cx. pipiens 507 (33.8%), oc. pulcritarsis 132 (8.8%), oc. echinus 20 (1.33%), and an. plumbeus, 3 (0.2%) (table 2). the peaks of blood feeding activity of oc. echinus, oc. geniculatus, oc. pulcritarsis, and cx. pipiens were the same and found as 11.00-12.00 am, and 16.00-17.00 pm. (table 2). a total of 1482 mosquito larvae collected during the study comprising cx. pipiens 661 (44.6 %), oc. geniculatus 483 (32.6%), an. plumbeus 333 (22.5% ), and cs. annulata 5 (0.3%) (table 3). it should be noted that these species have been collected only in trees holes in this area. table 1. frequency of the adult mosquitoes reared from pupae in neka county, mazandaran province, 2009 apr. may jun. jul. aug. sep. oct. nov. dec. total species n % n % n % n % n % n % n % n % n % n % an. plumbeus 0 0 0 0 1 5.26 2 10.52 0 0 6 31.57 5 26.31 4 21.05 1 5.26 19 100 cx. pipiens 28 2.91 80 8.33 120 9.6 88 9.16 0 0 300 31.25 154 16.04 101 10.52 89 9.27 960 100 oc. geniculatus 5 0.88 12 2.17 20 3.52 80 14.08 0 0 200 35.21 96 16.90 79 13.90 76 13.38 568 100 total 1547 100 table 2. abundance of adult mosquitoes collected by human bait net trap in neka county, mazandaran province, 2009 number of species collection hours an. plumbeus cx. pipiens oc. geniculatus oc. echinus oc. pulcritarsis 05-06 am 0 0 0 0 0 06-07 am 0 0 0 0 0 07-08 am 0 0 0 0 0 08-09 am 0 0 0 0 0 09-10 am 0 4 8 0 0 10-11 am 0 8 20 1 4 11-12 am 0 36 200 5 20 12-13 pm 0 16 32 2 3 13-14 pm 0 9 20 1 2 14-15 pm 0 2 3 0 1 15-16 pm 0 4 5 1 3 16-17 pm 0 380 430 7 60 17-18 pm 0 38 100 2 30 18-19 pm 0 10 20 1 9 19-20 pm 0 0 0 0 0 20-21 pm 0 0 0 0 0 21-22 pm 1 0 0 0 0 22-23 pm 2 0 0 0 0 23-24 pm 0 0 0 0 0 00-01 am 0 0 0 0 0 01-02 am 0 0 0 0 0 02-03 am 0 0 0 0 0 03-04am 0 0 0 0 0 04-05am 0 0 0 0 0 total 3 (0.2%) 507 (33.8%) 838 (55.87%) 20 (1.33%) 132 (8.8%) iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 29 table 3. density of larval mosquitoes per 10 dips collected from tree holes in neka county, mazandaran province, 2009. fig. 1. map of iran, highlighting the position of neka county in mazandaran province apr. may jun. jul. aug. sep. oct. nov. dec. total species n d % n d % n d % n d % n d % n d % n d % n d % n d % t ot al % an. plumbeus 3 1. 5 0. 9 6 3 1. 8 28 14 8/ 4 36 18 10 .8 1 0 0 0 10 0 50 30 .0 3 86 43 25 .8 2 50 25 15 .0 1 24 12 7. 20 33 3 22 /5 0 cs. annulata 0 0 0 5 2. 5 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0/ 30 cx. pipiens 1 1 5. 5 1. 65 30 15 4. 50 10 0 50 15 .0 1 13 0 65 19 .5 1 0 0 0 15 0 75 22 .5 2 12 0 16 0 18 .0 1 80 40 12 .0 1 40 20 6 66 1 44 /6 0 oc. geniculat us 1 0. 5 0. 2 26 13 5. 38 66 33 13 .6 6 82 41 16 .9 7 0 0 0 12 4 62 25 .6 7 10 6 53 21 .9 4 50 25 10 .3 5 28 14 5. 79 48 3 32 /6 0 total 14 82 10 0 iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 30 discussion in the present investigation four genera and five species were found in tree holes. several species of tree holes mosquito have medical importance and bite only during the daytime (diurnal). ochlerotatus echinus has been distributed in the mediterranean region, north africa, asia and southern europe, greece, algeria, morocco, spain and france (knight and stone 1977). the larvae of this species collected for the first time in sari of mazandaran province by janbaksh in 1955 (zaim et al. 1984). the larvae have been found from rezvanshahr, shaft, fuman and masal counties in guilan province (azari-hamidian et al. 2002b). in our study, the species was collected from tree holes in the neka county. there is little data about the ecology of this species in iran (azari-hamidian 2003). ochlerotatus geniculatus was found with 55.86 of the total adult mosquito collection. this species was distributed in the europe, north africa, south-east asia (knight and stone 1977). gutsevich (1943) reported this species for the first time in iran. this species was reported from ardebil, golestan, mazandaran, guilan provinces (gutsevich 1943, zaim 1987, azari-hamidian et al. 2002b, azari-hamidian et al. 2009). this species feeding on mammalian animals, and has exophagic behavior (horsfall 1955). yellow fever and eastern equine encephalitis viruses have been transmitted by this species in the lab (yates et al. 1979, horsfall 1955). in our study a total of 483 (32.6%) of larval collection, 568 (36.71%) from pupa mature, and 838 (55.87%) were collected by human bait trap respectively. ochlerotatus pulcritarsis has been reported from mazandaran province previously (zaim 1987, minar 1981, zaim et al. 1984). during the study, a total of 132 (8.8 %) was collected by human bait trap, whereas no larvae was found among breeding places. the peak of the blood activity of this species was found 11.00–12.00 a.m and 16.00–17.00 pm. although other aedini have reported in iran, such as oc. flavescens from west azerbaijan (zaim 1987, zaim et al. 1984), oc. vittatus, oc. detritus, and oc. leucomelas from hormozgan and sistan and baluchestan provinces (zaim 1987, zaim etal. 1984), ae. agypti from bushehr province (dow 1953), oc. caspius from isfahan (mousakazemi etal. 2000, moosa-kazemi etal. 2010) guilan (azarihamidian etal. 2002) but not found in this area. culiseta annulata has been distributed in the western region of palearectic (knight and stone 1977). this species was reported from iran by gutsevich in 1943 (gutzevich et al. 1970), and by minar in 1981 (minar 1981). azari-hamidian reported from rudbar, shaft, fouman, langeroud and masal cities in northern part of iran (azari-hamidian 2003b, azarihamidian 2005), however without association with an. plumbeus. in our study this species was collected in association with an. plumbeus, cx. pipiens, and oc. geniculatus. in our study it was found during the larval collection from tree holes. this is the first report of the existence of this species in tree holes in iran. the species of cs. subochrea was reported in mazandaran province whereas, cs. annulata not reported by zaim (1987). these two recent species are very similar in larval stage; however they are distinguishable as adult. the taxonomy and distribution of three close species, cs. alaskaensis, cs. annulata, and cs. subochrea in iran should be considered in future studies. in our study, one species of the genus anopheles was identified based on the morphological characters of larvae and adult. shagudian (1960) mentioned some characters to distinguish of the larvae. lateral hairs on abdominal segments iv–vi of this species distinctly feathered and frontal hairs are simple. anopheles plumbeus is reliably distinguishable from other species in adult stage. fore margin iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 31 of wing is uniformly dark, wing with distinct dark spots, upper surface of thorax dark at the side with a broad lighter stripe down the middle, white frontal tuft present. shagudian (1960) mentioned, proboscis of female uniformly brown and last segment of female palpus more than half as long as the penultimate. the last character mentioned to distinguish the adult of an. plumbeus from those of an. claviger. anopheles plumbeus represented 22.5% of larval collection, 1.23% from rearing to mature, and 0.2% from human bait net trap collection. horsfall in 1955 reported the association of this species with ae. aegypti, cs. annulata, cx. pipiens, oc. geniculatus, oc. echinus, and orthopodomyia pulcripalpis. it should be mentioned or. pulcripalpis has not been reported in iran but ae. aegypti has been reported previously (azari-hamidian et al. 2007). culex pipiens distributed in europe, the tropical and subtropical regions of asia, africa the middle part of north america, southern america, and australia (vinogradova 2000). this species is distributed in the most part of iran (zaim et al. 1985). distribution of this species has close relationship with economic activities and development of new territories. the human activity on natural environment with a change in the land and underground water resources can be affect the abundance of this species (vinogradova 2000). culex pipiens has at least three complex system of environmental compliance. culex quinquifasciatus has been distributed in the tropical area with the wide range of hosts. culex pipiens has been distributed in the moderate area with the limited range of birds nest maker (mousakazemi et al. 2000). culex pipiens form molestus is autogenous, and the first laying eggs perform without a blood feeding (vinogradova 2000). culex pipens form molestus, cx. quinquefaciatus and possibly cx. pipiens pallens need to the small space for mating whereas, a large space need to mate for anautogenous cx. pipiens pipiens (vinogradova 2000). in our research cx. pipiens was found in tree holes. azari-hamidian et al. (2001, 2002a) reported it as one of the predominant species in guilan province, northern iran, and in central part of iran (mousakazemi et al. 2000). this species was found as 12.05% of the total mosquito in turkey (gunduz et al. 2009). in our research monthly activity of cx. pipiens larvae began in the end of may, and was increased in the beginning of july and decreased slowly in the mid of summer. the main peak of the activity of adults was determined in mid of july. the monthly activity of larvae near moscow city was started from late april to may when the weather conditions was changed, and the first group of eggs and larvae was appeared in mid-may when the average temperature between 10 and 15 degrees centigrade and was matured in late may to early june (vinogradova 2000). agree with our research, mousakazemi et al. reported that the cx. pipiens larval activity was begun from late may and a peak in early july (mousakazemi et al. 2000). the second peak reported in the late july. the seasonal activity was decreased in early october. activity of adult was reported in the late may and reached to peak in the mid of july and then decreased to zero in october (mousakazemi et al. 2000). in our research peak blood feeding of cx. pipiens was found 11.00–12.00 mَ and 16.00–17.00 pm. vinogradova (2000) reported that this species can change shelter in urban areas. azari hamidian reported that 66.3% of the total of cx. pipiens collected in indoor places. stable, toilet, and chicken compliments with cement, plaster, straw and wooden walls were reported the main resting places. he mentioned 33.7% of this species was collected from outdoor places such as gramineous plants, inside the wells, discarded tire (azari hamidian et al. 2003a). it should be mentioned that the resting behavior depends on the climate and human economic activities which result in more or less to exophily and endophily behavior. culex pipiens was found in neka, iran j arthropod-borne dis, 2010, 4(2): 26–34 sh nikookar et al.: species composition and … 32 with total of 661 (44.6%) larvae from tree holes and 507 (33.8%) from human bait net trap. in conclusion, there are several plant species such as quercus castanifolia, alnus subcerdata, carpinus betulus, zelcova carpinifolia, and mespilus species (natura resources of neka county 2009). however, the important and interesting thing in our study is that the larvae of oc. geniculatus was found only in the q. castanifolia species, whereas an.plumbeus, cs. anulata and cx. pipiens were found in the q. castanifolia, pools, disposal tire, and other larval habitat. in this study, the minimum and maximum temperature of water inside the cavity were 14°c and 16° c respectively. the range of ph was 4–5.5. due to present of shad and wind near the tree holes, the range of temperature remind constant as 14–16° c. rice fields as breeding places for mosquitoes have been reported in the areas of kenya (mwangangi et al. 2010), but in our study despite the great effort, except of cx. pipiens, the other species were not found within rice fields. larval habitat epiphytic algae, aquatic and semi aquatic species, electric conductivity (ec), dissolved oxygen, turbidity, disintegrated organic and inorganic material, pesticide toxic, and predators should be considered in future studies. acknowledgements the authors are grateful to dr shahyad azari-hamidian, school of health, guilan university of medical sciences, rasht, for reviewing the manuscript, mr hamid ravesh, mr mojtaba asghari, ms. seyede somayeh nikookar, mr mehrdad badangyz environmental health director of neka, for supporting the investigation. we also would like to express our appreciation to the people of darvishan, khairabad, and chelmardi villages in neka county for their kind cooperation during the study. this study was financially supported by tehran university of medical sciences (no: 10513-2701-89). the authors declare that they have no conflicts of interest. references 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(1–4): 3–10. zaim m, manouchehri av, yaghoobi-ershadi mr (1984) the fauna of mosquitoes of iran (diptera: culicidae) culex. iran j pubic health (1–4): 1–12. zaim m, manouchehri av, yaghoobi-ershadi mr (1986) mosquito fauna of iran. 3-culiseta, coquillettidia and uranotaenia. iran j pubic health. 15(1–4): 1–10. j arthropod-borne dis, march 2018, 12(1): 85–93 h vatandoost et al.: larvicidal activity of … 85 http://jad.tums.ac.ir published online: march 18, 2018 original article larvicidal activity of bunium persicum essential oil and extract against malaria vector, anopheles stephensi hassan vatandoost 1, 2, arezoo rustaie 3, zeynab talaeian 3, mohammad reza abai 1, fatemeh moradkhani 3, mahdi vazirian 3, abbas hadjiakhoondi 3, mohammad reza shams-ardekani 3, 4, *mahnaz khanavi 3, 5 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of pharmacognosy, faculty of pharmacy and persian medicine and pharmacy research centre, tehran university of medical sciences, tehran, iran 4department of traditional pharmacy, school of traditional medicine, tehran university of medical sciences, tehran, iran 5faculty of land and food systems, university of british columbia, vancouver, canada (received 21 sep 2016; accepted 12 dec 2017) abstract background: malaria, a mosquito-transmitted disease, is still a major human health problem all over the world. larviciding is a component of comprehensive control program to overcome the disease. negative aspects of synthetic insecticides application, such as environmental safety concerns, have favored use of natural insecticides. methods: larvicidal activity of essential oil, extracts and fractions of a wild grown and a cultivated type of bunium persicum fruits against malaria vector anopheles stephensi was assessed according to the method described by who. results: bunium persicum showed remarkable potency against an. stephensi larvae. lc50 values for essential oil, total extract, petroleum ether fraction and methanol fraction were 27.4284, 64.9933, 85.9933 and 255.7486ppm for wild type, and 21.3823, 63.2580, 62.7814 and 152.6357ppm for cultivated one. conclusion: the results of this study suggest b. persicum as a valuable source of natural insecticides against malaria vector anopheles stephensi. keywords: anopheles stephensi, bunium persicum, larvicidal activity, extract, essential oil introduction despite progresses made over the past decades to decline the mortality rate of malaria all over the world, it is still prevalent in some tropical countries and areas with about 200 million affected cases in 2013. vector control interventions have had substantial contribution on the recent reduction in global malaria burden. larviciding, with the aim of adult vector density reduction, as an auxiliary to core interventions, is helpful especially in urban regions, where breeding of vectors take places in permanent or semi-permanent aquat ic habitats (1). the mosquito anopheles stephensi is one of the six main vectors of human malaria in southern parts of iran (2). larvicidal potentials of some herbal extracts and essential oils on an. stephensi larvae have been investigated previously (3-5). bunium persicum is a perennial plant belonging to apiaceae family, growing wild in iran (6). the fruit of b. persicum is used as spice, antiseptic and carminative agent (7). several studies have analyzed essential oil composition of the fruits and mostly reported γ-ter*corresponding author: dr mahnaz khanavi, email: khanavim@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 85–93 h vatandoost et al.: larvicidal activity of … 86 http://jad.tums.ac.ir published online: march 18, 2018 pinene, cuminaldehyde and ρ-cymene as main components (8-10). kaempferol, caffeic and pcoumaric acid have been isolated from polar fraction of the fruits as major antioxidant constituents (11) but according to our knowledge no other comprehensive study has been organized to identify other phytochemicals in the extract. overexploitation and unscientific harvesting of b. persicum as well as climate changes, has threatened its existence in wild (12). cultivation of endangered species could preserve their genetic resources (13). in recent years, b. persicum is cultivated in limited areas in iran especially in khorasan razavi province. as a part of our ongoing studies on larvicidal activity of plants extracts and essential oils against an. stephensi (4, 5, 1420), in the present study, we have studied larvicidal activity of the essential oil, extract and fractions from b. persicum fruits against late third instar larvae of an. stephensi. moreover, we have compared the activities of a wild and a cultivated type. materials and methods plant material the fruit of wild b. persicum was purchased from kerman, and cultivated type was supplied from agricultural research fields of ferdowsi university of mashhad (2013). the samples were authenticated at the herbarium of faculty of pharmacy, tehran university of medical sciences, where voucher specimens were deposited (pmp649 and pmp-689). essential oil preparation 100g powdered fruits of cultivated and wild b. persicum were subjected to hydrodistillation for 3 hours using clevenger type apparatus. the obtained essential oils were dried over anhydrous sodium sulfate and kept in refrigerator until needed. extraction and fractionation 250g dried and powdered fruits from both samples were separately extracted with meth anol (5× 1.5l) to afford total methanol extracts. the solvent was removed under reduced pressure by rotary evaporator at 40 °c, and subsequently lyophilized by freeze dryer at -40 ºc for 24h (lyotrap ultra, lte scientific ltd., oldham, uk). fractionation of total extracts was performed with sufficient volumes of petroleum ether, ethyl acetate and methanol. the fractions were then concentrated to dryness by rotary evaporation. larval mortality bioassay anopheles stephensi larvae (bandar abbas strain) were supplied by the department of medical entomology, tehran university of medical sciences. the mosquito colony was maintained under a constant insectarium condition at 27 ºc and 75–85% relative humidity with 12:12 light and dark photoperiod. late third and early fourth instars larvae were used for experiments. larvicidal activity of total extracts, fractions and essential oils were evaluated according to the procedure recommended by who (21). the larvae were exposed to different concentrations of samples for 24 hours. tests were carried out in four replicates. one ml of solvents (dmso for essential oil and petroleum ether fraction, dmso2: water 3 for total extract and ethanol for methanol fraction) were added separately into control bakers. mortality was scored 24 hours post exposure. analysis method the mortality percentages were calculated and corrected relative to the associated controls using abbott’s formula (22). the concentration-mortality data were subjected to probit analysis (23) and lethal concentrations (lc50 and lc90) were determined with 95% confidence intervals from the regression lines. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 85–93 h vatandoost et al.: larvicidal activity of … 87 http://jad.tums.ac.ir published online: march 18, 2018 results hydro distillation of wild and cultivated b. persicum fruits yielded 2.5% and 2.25% (w/w) essential oil respectively. both essential oils had a lot of commonalities in composition. γ-terpinene (30.77% and 27.57%), cuminaldehyde (20.49% and 21.1%), ρ-cymene (20.1% and 18.32%) and γ-terpinen-7al (8.29% and 7.84%) constituted main components in the wild and cultivated oils respectively (24). the results of larvicidal activity of essential oils, total extracts, petroleum ether and methanol extracts against an. stephensi under insectary condition are pre sented in table 1 and plotted in figs. 1 to 4. all tested samples showed significant antilarval effect against the malaria vector an. stephensi, of which, the essential oils from cultivated and wild types with lc50 values of 21.3823ppm and 27.4284ppm were the strongest samples and methanol fractions with lc50 values of 152.6357ppm and 255.7486 ppm exhibited least larvicidal activity among the samples. comparison of lethal concentration values of efficient tested samples reveals there is no difference in efficacy of them between wild and cultivated types. fig. 1. comparison of lethal concentrations (lc50) of cultivated and wild types of bunium persicum essential oils against larvae of anopheles stephensi http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 85–93 h vatandoost et al.: larvicidal activity of … 88 http://jad.tums.ac.ir published online: march 18, 2018 table 1. probit regression line parameters of essential oil, total extract, petroleum ether and methanol fraction of wild and cultivated bunium persicum fruits against anopheles stephensi specimen intercept slope ± se lc50 95% ci lc90 95% ci χ2 p w eo -4.1233 2.8670 ± 0.471 27.4284 19.7868-35.0421 76.7752 56.3038-139.1275 41.682 <0.05 c eo -4.8215 3.6251 ± 0.486 21.3823 13.6913-25.9482 48.2608 38.6334-68.5159 33.107 <0.05 w t -3.9100 2.1568 ± 0.270 64.9933 44.8917-89.5814 255.3195 170.9457-498.5673 16.725 <0.05 c t -4.6503 2.5819 ± 0.181 63.2580 55.8062-71.3261 198.3795 167.9464-243.6008 10.718 <0.05 w pe -5.0602 2.6158 ± 0.502 85.9933 47.2631-130.9756 265.7116 166.2611-869.8822 28.442 <0.05 c pe -4.7365 2.6346 ± 0.186 62.7814 55.4789-70.6893 192.4364 163.2242-235.7975 12.880 <0.05 w m -6.6009 2.7414 ± 0.365 255.7486 159.3871-405.0692 750.4194 459.8467-2245.3156 26.381 <0.05 c m -9.6475 4.4181 ± 1.524 152.6357 94.5358-262.7553 297.6718 202.8848-6675.4919 18.475 <0.05 w: wild, c: cultivated, eo: essential oil, t: total extract, pe: petroleum ether fraction, m: methanol fraction, se: standard error, lc50: lethal concentration to cause 50% mortality in population, lc90: lethal concentration to cause 90% mortality in population, ci: confidence interval, χ 2: heterogeneity about the regression line. fig. 2. comparison of lethal concentrations (lc50) of cultivated and wild types of bunium persicum total extracts against against larvae of anopheles stephensi http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 85–93 h vatandoost et al.: larvicidal activity of … 89 http://jad.tums.ac.ir published online: march 18, 2018 fig. 3. comparison of lethal concentrations (lc50) of cultivated and wild types of bunium persicum petroleum ether fraction against against larvae of anopheles stephensi fig. 4. comparison of lethal concentrations (lc50) of cultivated and wild types of bunium persicum methanol fraction against larvae of anopheles stephensi http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 85–93 h vatandoost et al.: larvicidal activity of … 90 http://jad.tums.ac.ir published online: march 18, 2018 discussion many researchers have already studied larvicidal potentials of plant derived compounds, extracts and essential oils against various insects, with the aim of finding active phytochemicals to replace synthetic insecticides. high cost of various commercial insecticides beside their food and environmental safety concerns, toxicity problems and increasing resistance rates have made their utilization undesirable (25, 26). anti-larval activity of essential oil from a wild grown b. persicum against an. stephensi and culex pipiens has been previously reported with lc50 values of 27.72 and 20.61ppm respectively (27). according to the results of our study, the essential oil, methanol total extract and petroleum ether fraction of both wild and cultivated samples had significant larvicidal activity against late third and early fourth instar larvae of an. stephensi. the larvicidal potential of γ-terpinene, cuminaldehyde and ρ-cymene, main constituents of both wild and cultivated type b. persicum fruits, against various insect larvae has been previously proved in several experiments. γ-terpinene has shown potent larvicidal activity with lc50 value of 29.21 ppm against anopheles anthropophagus (28) and 30.7 and 29.8ppm against aedes aegypti and aedes albopictus respectively (29). zahran and abdelgaleil (30) documented toxicity of cumin aldehyde on culex pipiens larvae, which was more stronger than other tested monoterpenes in that experiment, with lc50 values of 38.9 and 21.4ppm for 24 and 48h exposures respectively. anti-larval potential of ρ-cymene, the other main constituent, towards a. aegypti and ae. albopticus has also been demonstrated (lc50= 19.2 and 46.7ppm) (29). higher lethal effect of the petroleum ether fraction in comparison to the methanol fraction, suggests higher potency of non-polar components than polar phenolics towards an. stephensi larvae. lc50 value of 85.9933 and 62.7814ppm for petroleum ether fraction from wild and cultivated types makes it suitable choice for further studies to isolate the active principles. anti-larval activity of efficient samples from cultivated type was comparable to those from wild grown, so it can be concluded that cultivation of b. persicum has not affected chemical constituents’ biosynthesis or concentration, which are responsible for larvicidal activity of the fruit. conclusion the extract and fractions from b. persicum fruits, ie, petroleum ether fraction and total extract, beside the essential oil, have shown significant larvicidal effects on an. stephensi, and can be a great candidate to develop an ecofriendly insecticide to combat malaria vector breeding. more precise investigation will require revealing phytochemical composition of extract. since cultivated type showed comparable results as wild grown, cultivation of b. persicum, as a solution to preserve its wild 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accepted 16 nov 2011) abstract background: organophosphorus pesticides are one of the most consumable poisons. such poisons can enter water sources by different routes. since consuming some drinking water containing an amount of poison higher than the standard level, causes undesirable effects on human health. this research aimed to study the effectiveness of eliminating malathion from water by ultraviolet irradiation (uv) mercury lamp with a medium pressure. methods: in this experimentalapplied study, variants of initial phs and initial concentrations and exposure times were investigated. initial concentrations of malathion were 0.5, 1 and 2 mg/l. the samples were then exposed to uv irradiation interruptedly the time periods of 10, 20, 30, 40, 50 and 60 minutes. the uv lamp used in the reactor was medium pressure (irradiation intensity= 200 w/m2). the amounts of malathion were determined before and after the irradiation by hplc instrument. moreover, the results obtained from the study were analyzed using spss software and anova and t-test statistical trials. results: the minimum reduction occurs at 10 min (46%) and the maximum reduction in 60 min (87.25%) (p< 0.05). in addition, the effectiveness of irradiation process decreases with increasing the poison concentration (p< 0.001). however, the efficiency of the process increases with ph increase. conclusion: the results show the most effectiveness were achieved at 60 min and 0.5 mg/l and ph= 9. therefore, the application of uv reactors could be considered as an appropriate method. keywords: pesticide, malathion, water, ultraviolet irradiation introduction organophosphate compositions are the largest and various groups of available pesticides and form about 40% of recorded pesticides in the world. the used pest drown poisons enter to water sources by direct washing and irrigation from used places. in addition, rain fall on sprayed poison areas before break down these materials cause to enter them to sources of surface water (agarawal 1991, borner 1994). furthermore pesticides can find a way to ground water table by way of soil layers and during water infiltration. some pesticides can enter to air and consequently enter to sources of surface water and soil while rain fall. entrance these pollutants materials in sources of supply drinking water can have adverse effects on human health and environment (videria et al. 2001, safi 2002). in addition to remains of these poisons are dangerous for health people, also can have effect on economic of region people that subsist by aquatics selling. malathion acts as one clean hereditary inhibiter operator in human that cause to find complications and problems in nervous system and create conditions like dizziness, nausea and *corresponding author: dr mohammad hadi dehghani, e-mail: hdehghani@tums.ac.ir 45 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ٢ attention disorder (videria et al. 2001, fireston et al. 2005). this poison has very destructive effects on children nervous system especially in first years. so we must use sure and congruous ways for prevention of entrance these poisons to sources of water and air and soil (alavanja et al. 2004, rauh et al. 2006). recently, some investigations are done concerning being and remain of these poisons in environment in iran. these studies shows that after usage of poison in short times, their amount have been more than allowable extent (shayeghi et al. 2001, 2004, 2007, 2010, tarahi 2002, honarpajoh et al. 2003, hejazinejad et al. 2004, khodadadi 2008, shayeghi and dehghani 2011). for example investigations performed by shayeghi et al. (2007) about malathion remains in drinking water showed that remains of these poisons exist for some months in environment. shayeghi et al. (2004) appraised remains of malathion organophosphate poisons in rivers water like shahpoormand and dalkie in booshehr, and concluded that amount of this poison has been more than allowable extent in each three rivers in booshehr, iran. in addition shayeghi et al. (2001) investigated remains of these pesticides in mazandaran river. there are the various technologies for removal of organophosphate pesticides. recently the modern ways have been used for removal or decrease of pesticides poisons like using of ultrasonic waves ultraviolet radiation and advanced oxidation technology (samanidou and fytianos 1988, huston and pignatello 1999, hequet et al. 2001, burrows et al. 2002, walid et al. 2002, badawy et al. 2006, maldonado et al. 2006, bavconkralj et al. 2007, li-an et al. 2011, shayeghi and dehghani 2011). but ultraviolet radiation is one of the functional and safe technologies that can be profitable in treatment of surface and ground water sources polluted by pesticides with its resultant advantages. certainly one of protection reasons and welcome this technology is necessity that has existed for purpose of compulsion in laws observance and new rules of water treatment. the advantages of this system as compared with other ways of water treatment are as follows (samanidou and fytianos 1988, hequet et al. 2001, burrows et al. 2002, bavconkralj et al. 2007, vaezi and bazrafshan 2008): 1. it does not need to add chemical materials, 2. it does not need to absorbent media, 3. the reactor profits by good pliable, 4. the property of produced sewage is limpid and odorless and ideal, 5. the most of pesticides are degraded in the reactor considerably, 6. the less connection with ph changes and water temperature in compared with other chemical materials, 7. to have safety and acceptability from the viewpoint of refineries workers and the general public and be propounded as a green technology. a variety of researches have been done on removal of pesticides by ultraviolet radiation, like hequet et al. (2001) on effect of medium pressure lamp on atrazine. the results were indicative of perfect influence this way in atrazine pesticide degradation. bavconkralj et al. (2007) got products of gotten mineral perfectly by photo-oxidation process by ultraviolet irradiation and visible light. samanidou and fytianos (1988) did research for assessments of photo-oxidation process effect on the carbamate. this research performed by medium pressure mercurial lamp. the results were indicative of perfect influence ultraviolet irradiation in degradation of this material. huston et al. (1999) used process of ultraviolet radiation and photofenton for degradation of some commercial pesticides in natural waters. the aims of this study were survey on effectiveness of ultraviolet irradiation for degradation of malathion pesticide from water and the effect of irradiation time, initial concentration and ph changes influence on potential of ultraviolet radiation effectiveness in degradation of malathion in water. 46 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ٣ materials and methods this research is the experimental-applicable study that performed in chemical laboratories of environment health engineering and entomology of the tehran university medical sciences in 2009–2010. malathion poison was prepared in the different concentrations and analyzed. in this study, malathion poison has prepared and analyzed. the confrontation times to ultraviolet wave were selected on the basis of done pre examinations in lab and also the discussed concentrations in this research considering to done studies about the existing remains of malathion poison in country surface water (shayeghi et al. 2001, 2004, 2007, 2010, 2011, tarahi 2002, honarpajoh et al. 2003, hejazinejad et al. 2004, khodadadi 2008). the method of samples preparation the extraction stages and samples preparation done for analyzing were: first, 500 ml of noted water sample was added to separator funnel that has 1 l size. in some cases, the sample was passed from the atman filter (0.45 μ m) because it had turbidity of suspended particles; then, 30 ml dichloromethane was added to noted sample. for better separating two phases 15–20 gr nacl was added to contents of separator funnel; next, funnel contents intensively was mixed and leaved its valve in continual times for decreasing of the funnel inside pressure. in order to separating organic phase from watery phase while passing from column that consist the sodium sulphate for deletion of water molecule from organic phase. in this stage, 30 ml dichloromethane was again added to that and mixed to separate organic phase from watery phase. in order to evaporation of extracted liquid by the rotary set, the evaporation of under vacuum, thus the bulk of extracted liquid is reduced 2–3 ml. at the end of the processes 1 ml organic solvent chloroform was used for extracting. the concentration reading considering to the suggested way of standard method book for measuring of malathion poison because the considering to the conditions of hplc set following stages was done: -the standard solution was prepared from noted poison by hplc grade material and with high pureness percent in the different concentrations and then it was injected to hplc set. 2.1 μ l was extracted and injected to hplc set. the thermal program of this set was as follows: -first temperature equal 110°c for 1 min. -to reach to temperature of 188° c with addictive velocity 25° c /min for 4 min. -to reach to temperature of 190° c with addictive velocity 20° c /min for 1 min. -to reach to temperature of 244° c with addictive velocity 25° c /min for 6.21 min. -keeping noted temperature for samples concentration reading. -using of helium gas with > 99% pureness percent and velocity of flowing 0.7 cm/s. the specifications of hplc high performance liquid chromatography device (hplc), equipped with visibleultraviolet disclosure and auto sampler. the used column for separating of these compounds is c18 made in water company in usa, with 25 mm length and 4.6 mm diameter in 30° c temperatures. the moving phase flowing velocity and the used moving phase were considered in order 1 ml/min and methanol, acetonitric and water with ratio (15:45:40). the used wave length was ascertained 140 nm for disclosure of these compounds .the used separating column of hplc set was capillary column db5.6.25 kind. by the performance of correct thermal program and the set preparing was observed the pertaining vertex to malathion in 10.23 minutes. the used set exactness in research was estimated as nearly 1 μg/l. 47 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ۴ the specifications of ultraviolet reactor the used reactor in this study contains one closed bottom cylinder with 3 liters size and kind of the rustproof steel made in tehran steel of iran company. in this cylinder one magnetic mixer has been used in order to complete mixing of the sample for radiation in the reactor. in this pilot, on account of too much heat making that is the result of lamp radiation was used the chiller for temperature reducing. for this purpose, one bocal of 4 l was used that cylinder fits its inside. the empty space of cylinder and bocal was filled by ice for temperature reducing. all the analyses were performed according to the procedures outlined in standard methods (apha 2005). calculation of degradation percentage the definition of malathion degradation percentage (dp) was as follows: dp= (c1-c2) / c1× 100 where dp (%) is the degradation percentage of the ultraviolet reactor, c1 is the initial concentration of malathion (mg/l). c2 is the concentration of malathion (mg/l) after reaction for (t) time. statistical method the results were analyzed by spss software, 11.5 version and 2003 excel. the data were analyzed using anova and t-test. the variables were irradiation time, initial concentration and ph degradation was variable depended. results the malathion poison was affected by ultraviolet mercurial lamp with medium pres sure in 0.5, 1, 2 mg/l and 10, 20, 30, 40, 50, 60 min and ph= 6, 7, 9. effect of ph value the investigation results of ph changes on the utility of malathion concentration shows that the utility of concentration increases by ph augmentation, so that malathion in 0.5, 1, 2 concentrations and ph= 6 have the concentration utility 62%, 57%, 54.5 % but the measure of utility reaches to 70%, 65%, 63.5 % in ph = 9 (fig. 1, 2 and 3). the analysis test of anova showed in this case is the meaningful statistical difference too. effect of irradiation time the results showed that measure of malathion poison reduction augmentation by increasing of irradiation time. the regression statistical test shows that the concentration of extant poison decreases in pilot by increasing time. the analysis test of anova showed the significant differences among times. effect of initial concentration the concentration changes are influential on the utility of ultraviolet. the measure of malathion degradation has the most utility in 0.5 mg/l in 60 min period and the alkali ph among the other concentrations. in other words, by increasing of poison concentration drops the degradation. the figures 4, 5 and 6 show the degradation percentage vs. time period for different concentrations of malathion. according to anova test, the effect of concentration changes on efficiency is significance. 48 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ۵ y = 4x + 58 r2 = 1 58 60 62 64 66 68 70 72 6 7 9 ph d eg ra da tio n pe rc en ta ge fig. 1. degradation percentage vs. ph for 0.5 mg/l y = 4x + 52.667 r2 = 0.9796 52 54 56 58 60 62 64 66 6 7 9 ph d eg ra da tio n pe rc en ta ge fig. 2. degradation percentage vs. ph for 1mg/l y = 4.5x + 50 r2 = 1 50 52 54 56 58 60 62 64 66 6 7 9 ph d eg ra da tio n pe rc en ta ge fig. 3. degradation percentage vs. ph for 2 mg/l 49 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ۶ y = 8.3857x + 45.733 r2 = 0.9692 0 20 40 60 80 100 120 10 20 30 40 50 60 time(min) d eg ra da tio n pe rc en ta ge fig. 4. degradation percentage vs. time period for 0.5 mg/l y = 8.7286x + 38.367 r2 = 0.9911 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 time(min) de gr ad at ion p er ce nt ag e fig. 5. degradation percentage vs. time period for 1 mg/l y = 8.4214x + 37.567 r2 = 0.9955 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 time(min) de gr ad at ion p er ce nt ag e fig. 6. degradation percentage vs. time period for 2 mg/l 50 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ٧ discussion this study showed that the effectiveness of malathion degradation from water by ultraviolet irradiation depends on three parameters; the initial concentration, the irradiation time and the ph. this study showed that initial concentration decreases with irradiation time. these researches results conform to khodadadi (2008), walid et al. (2006) and maladonado et al. (2006) too. in other words, when the irradiation time increases, lots of free radicals has formed in the liquid and this event cause to the much decomposition of malathion poison. khodadadi (2008) investigated ph changes in 6, 7, 9 limits on the concentration in time period of 1 hour and founded that the concentration increased by ph augmentation. the founded results of walid's studies (2006) and also freed et al. (1979) confirm to the results of this research. besides the studies of burrows (2002) and badawy et al. (2006) showed that the velocity of poison decomposition increased by ph augmentation. his research results conform to this study. according to these researches, the principal reason of this event is the augmentation of oh¯ ions producing and the hydroxyl free radicals in the alkali environment. finally, using chemical oxidation ways are not too much safe because they can impose too much energy and costs in the process of water treatment. because of this, is given preference that will use the integrative ways of water refining for reducing of costs and using energy in order to will founded the ideal reduction efficiency in the short time and by using low energy. acknowledgments this research has been supported by tehran university of medical sciences # 1166327-02-90. the authors declare that there is no conflict of interests. references alavanja mc, hoppin ja, kamel f (2004) health effects of chronic pesticide exposure: cancer and nevrotoxicity. annu rev public health. 25: 155–197. agarawal hc (1991) pesticide pollution of water. mc graw hill, publishing company limited. apha (2005) standard methods for the examination of water and wastewater. american public health association, washington, dc. borner h (1994) pesticides in ground and surface water. springer verlag, berlin germany. burrows hd, canle lm, santaballa ja (2002) reaction pathways and mechanisms of photodegradation of pesticides. j photochem photobiol b. 67: 71–108. badawy mi, montaser y, ghaly my (2006) advanced oxidation processes for the removal of organophosphorus pesticides from wastewater. j desalination. 194: 166–175. bavconkralj m, franko m, trebše p (2007) photodegradation of organophosphorus insecticides: investigations of products and their toxicity using gas chromatography–mass spectrometry and achethermal lens spectrometric bioassay. j chemosphere. 67(1): 99–107. freed vh, chiou ct (1979) degradation of selected organophosphate pesticides in water and soil. j agric food chem. 27: 706–708. fireston ja, smith-weller t, franklin g, swanson p, longstreth wt jr, checkoway h (2005) pesticides and risk of parkinson disease: a population 51 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ٨ based case control study. arch neurol. 62: 91–95. héquet v, gonzalez c, le-cloirec p (2001) photochemical processes for atrazine degradation: methodological approach. j water research. 35: 4253–4260. hejazinejad fs, hormozi b, abtahi fs (2004) a critique of pesticides use in the province of golestan. j water environment. 66: 37–44. honarpajoh k (2003) survey and determination of organophosphors insecticides of malathion and diazinon in water of karaj amir kabir dam. 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[msph thesis]. hamadan university medical sciences, iran. li-an l, ying-shih m, mathava k, jih-gaw l (2011) photochemical degradation of carbofuran and elucidation of removal mechanism. j chemical engineering. 166: 150–156. maldonado mi, malato s, perezeestrada la, gernjak w, oller i, doménech x, peral j (2006) partial degradation of five pesticides and an industrial pollutant by ozonation in a pilot-plant scale reactor. j hazardous materials. 138: 363–369. rauh a, garfinkel r, perera p (2006) impact of prenatal chlopyrifos exposure on neurodevelopment in the first 3 years of life among innercity children. j pediatrics. 118(6): 1845–1859. shayeghi m, shahtaheri j, selseleh m (2001) organophosphorus insecticides residue in mazandaran river and waters (iran). iranian j public health. 30(3–4): 115–119. shayeghi m, darabi h (2004) survey of diazinon and malathion in water of shahpour, mand, dalaki, boushehr riverse. j south medicine. 10(1): 54– 60. shayeghi m, khobdel m, vatandoost h (2007) determination of organophosphorus insecticides (malathion and diazinon) residue in the drinking water. pak j biol sci. 10(17): 2900–4. shayeghi m, dehghani mh, mahvi ah, azam k (2010) application of acoustical processor reactors for degradation of diazinon from surface water. iran j arthropod-borne dis. 4(2): 11– 18. shayeghi m, dehghani mh, fadaei am (2011) removal of malathion insecticide from water by employing acoustical wave technology. iranian j public health (in press). safi jm (2002) association between chronic exposure to pesticides and recorded cases of human malignancy in gza goveronates. j sci total environm. 284: 75–84. samanidou v, fytianos k (1988) photochemical decomposition of carbamate pesticides in natural waters on northern greece. sci total environ. 76(15): 85–92. tarahi s (2002) survey and determination of used pesticides residue concentration (malathion, diazinon and metasystox) in the water of nahand river. [msc thesis]. tehran university of medical sciences, iran. vaezi f, bazrafshan e (2008) ultraviolet irradiation and its application in water and wastewater disinfection and treatment. andishmand publisher, tehran. 52 j arthropod-borne dis, 2012, 6(1): 45–53 m shayeghi et al.: using ultraviolet irradiation … ٩ videria ra, antunes-madiera mc, lopes vi, madeira vı́tor mc (2001) change induced by malathion, methylparation and parathion on membrane lipid physiochemical properties correlate with their toxicity. j biochem biophys acta (bba)-biomembranes. 1511: 360–368. walid k, lafi z, al-qodah (2006) combined advanced oxidation and biological treatment processes for the removal of pesticides from aqueous solutions. j hazardous materials, b137: 489–497. 53 j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 383 http://jad.tums.ac.ir published online: september 08, 2017 original article the pcr-rflp-based detection and identification of the leishmania species causing human cutaneous leishmaniasis in the khorasan-razavi province, northeast of iran anita mohammadiha 1, *abdolhossein dalimi 1, mohammad reza mahmoodi 2, mehdi parian 3, majid pirestani 1, mehdi mohebali 4, 5 1department of parasitology and entomology, medical sciences faculty, tarbiat modares university, tehran, iran 2department of microbiology and parasitology, faculty of medicine, guilan university of medical sciences, rasht, iran 3department of medical parasitology and mycology, medical faculty, mashhad university of medical sciences, mashhad, iran 4department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 5center for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran (received 20 june 2015; accepted 7 feb 2016) abstract background: leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (acl), and leishmania major, which causes zoonotic cutaneous leishmaniasis (zcl), are endemic in iran. methods: cross-sectional study was designed to identify leishmania species in cutaneous leishmaniasis patients who referred to mashhad health centers from 2013 to 2014 using its-pcr-rflp technique. first, physical examinations were performed in all suspected patients and cl cases were confirmed with microscopical examinations. a questionnaire was prepared and completed for each confirmed patient and dna from each lesion smear was extracted, separately. the ribosomal internal transcribed spacer was amplified with appropriate primers and pcr products were digested by enzyme taq1 restrict enzyme. results: from all patients, 51 cases (54.3%) were men and 43 of them (45.7%) were women. the most frequent age group was 20–29 years old (27.2%). hands, face and feet were the most common sites for appearance of skin lesions. all of the 94 cases (100%) tested found to be positive by its-pcr-rflp. overall, leishmania species were identified in all of the 94 lesion smears which 33 (35%) of them were l. major and 61 (65%) of the remained isolates were identified l. tropica. conclusion: characterization of leishmania isolates collected from different parts of khorasan-razavi province showed that l. tropica is predominant agents of cl, especially in large and medium sized cities such as mashhad and shandiz. moreover, this study revealed that its-pcr-rflp based on our designed primers is a suitable method for species characterization. keywords: cutaneous leishmaniasis, human, its, khorasan-razavi province, iran introduction leishmaniasis is caused by parasitic flagellated protozoa of the genus leishmania. cl in old world is usually caused by l. major, l. tropica, and l. aethiopica. humans are infected by the bite of infected phlebotomine sand flies. leishmaniasis is a major public health problem with 1.5–2 million new cases annually and with up to 350 million people at risk *corresponding author: prof abdolhossein dalimi, e-mail: dalimi_a@modares.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 384 http://jad.tums.ac.ir published online: september 08, 2017 around the world. cutaneous leishmaniasis (cl) is currently endemic in 98 countries worldwide (1), and also is still considered an important health problem in some regions of the world, especially the eastern mediterranean region, and almost all countries of the middle east, including iran (2). in the old world, 90% of cases were reported from iran, afghanistan, saudi arabia, iraq, syria, and algeria (3). in iran, the majority of cl is produced by l. major while l. tropica is only distributed in big and medium cities (4, 5). the laboratory diagnosis of cl is commonly based on observation of amastigote forms of leishmania in giemsa stained smears using light microscopy and culture media, but by these methods we cannot identify leishmania species (6, 7). dna based methods, including various pcr techniques, rflp analysis and sequencing have been widely used for the identification of leishmania species in humans, animal reservoir hosts and infected vectors (8, 9, 10). polymerase chain reaction (pcr) assays with high accuracy were used for the detection and species identification of leishmania parasites in humans, animal reservoirs and phelebotomine sand flies. as hillis reported in 1991, most rdna copies seem to be homogeneous, and within each rdna repeat, there are two internal transcribed spacers (its), located between the small subunit (ssu) and large subunit (lsu) rrna genes (11). its1 and its2 are separated by the 5.8s rrna gene, and both provide species-specific sequence markers that have been most frequently detected by restriction fragment length polymorphism (rflp) analysis of one-step pcr products (6,12, 13). although microscopical technique is applied as gold standard for cl diagnosis, but pcr assay was also used for detection of cl species. we perform a single-pcr-rflp based on our designed primer (mo) and a nested-pcr-rflp based on our designed primer (mo) as external primers and ribosomal internal transcribed spacer 1 (its1) using primers litsr and l5.8s (6, 14) as internal primers. based on prior studies, although its1based primers described previously by el tai. (14) and schönian (6), has a perfect positive predictive value (ppv), but lacks satisfying sensitivity (15). sensitivity has been assessed by using real-time pcr assay (16) or by the serial dilution assay (sda) (17) based on leishmania serial dilution using cultivated parasites containing known parasite concentrations. this study attempts to introduce a new its -primes for detecting and identifying leishmania species. furthermore, it maintains amplifying the its1 of rdna (6,14) by designing a nested-pcr assay as a sensitive method. materials and methods study design the khorasan-razavi province (36.2980°n 59.6057°e, fig. 1) located at a mean elevation of 1064m above sea level and covers an area of 144,681km². its borders are the north khorasan province and turkmenistan in the north, semnan province in the west, yazd and south khorasan provinces in the south and afghanistan and turkmenistan in the east (fig. 1). this study is a descriptive cross-sectional study. the samples were collected from khorasan-razavi province of iran over a period of 12 months from the summer of 2013 to the summer of 2014 using 94 the suspected cases of cl. all patients were examined by a physician. different clinical, parasitological and molecular assays were used among cl suspected patients. for each 94 microscopyconfirmed patient of cl, the case’s age and gender, the time between the lesion being first noticed and presentation at a health facility and the location of each lesion on the patient’s body, were recorded. cutaneous samples (smears) were taken from patients from medical health centers in different leishmaniasis endemic areas of the province, were referred to parasitology and entomology laboratory in the faculty of medical sciences http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 385 http://jad.tums.ac.ir published online: september 08, 2017 of tarbiat modares university. at medical health centers, sampling was conducted after gaining accurate information about the place of infection origin. then after sterilizing around the lesions/nodules with 70% ethanol, a small incision was made in the margin of the lesion using a disposable lancet and some tissue and exudates were removed by scraping. the scrapings from the lesions were air dried, fixed in methanol, stained with giemsa 10%, and examined for amastigotes by microscopy. culture of reference strains of leishmania reference strains of old world species of the subgenus leishmania were used: leishmania major (mrho/ir/75/er), leishmania tropica (mhom/ir/99/yaz1). these strains were taken from leishmania section of school of public health and institute of public health research, tehran university of medical sciences. they were stored in liquid nitrogen and when necessary, culture was carried out in biphasic culture media (prepared from nutrient agar containing 10% whole rabbit blood overlaid with liver infusion tryptose broth containing 100–200ui/ml penicillin g and 1μg/ml streptomycin). the inoculated cultures were incubated at 21 °c for up to six weeks and examined weekly for the presence of promastigotes. meanwhile, for mass production of promastigotes, schneider insect (himedia) and rpmi1640 (gibco) media were used. dna extraction first, a slight treatment on the gimsastained slide was done before dna extraction. briefly, all slides discolored by incubating in ethanol 100% for 10 minutes, dried at room temperature, then were covered by 1ml distilled water and incubated for 10 minutes at room temperature. the smears removed completely and transferred to a 1.5ml reaction tube, centrifuged at 8,000×g for 5min. finally supernatant discarded and the pellets were used for dna extraction. dna was extracted with the dng-plus extraction kit (cinnagen, iran) according to the manufacturer’s instructions. the dna pellet was dissolved in 50μl of sterile distilled water and incubated in a water bath at 65 °c for 5min. dna concentration and quality were determined using nanodrop nd-1000 spectrophotometer (nanodrop technologies, wilmington, de, usa) at 260 and 280nm. dna samples with a260/a280 ratios between 1.8 and 2 were selected and stored at -20 °c for further analysis. pcr-rflp by mo-its-primers for the first amplification primers were designed based on the its region that identified, including: forward primer mo-f: (5’gcagctggatcattttccgatg-3’) and reverse primer mo-r: (5’-ggccaacgcg aagttgaattc-3’). the pcr product size stays between 800 and 850bp. the amplification conditions were: 94 °c for 5min, followed by 35 cycles of denaturation at 94 °c for 30s, annealing at 62 °c for 30s and extension at 72 °c for 40s, with a final extension step at 72 °c for 10min. restriction fragment length polymorphism (rflp) analysis of the its amplicons was performed on the its amplicons, obtained from 94 smear samples and the reference strains, using the restriction enzyme taq1 (1μl) (promega, usa) without prior purification. the restriction fragments obtained were compared with the molecular profiles of the who reference strains. after using the restriction enzyme, banding patterns were subjected to electrophoresis in 2% agarose (sigma-aldrich, st. louis, mo) at 80v in 1x tae(40mmtris-acetate, 1mmedta, ph8.3) buffer, stained with safe stain (5μl/100ml), and visualized and photographed using a uv transilluminator (fig. 2). pcr-rflp by its1-primers pcr-rflp was performed as described http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 386 http://jad.tums.ac.ir published online: september 08, 2017 previously (6) on the 300–360bp fragments amplified from its1. the non-purified pcr products (10μl) were digested with 1u of the restriction enzyme haeiii (promega, madison, wi, usa), and the restriction fragments obtained were compared with the molecular profiles of the who reference strains. its-n pcr-rflp the first amplification performed as mentioned above, based on the its region (by mo-its primers). for the second amplification 2μl of a 1:20 dilution of the first pcr product was added to 15μl of pcr mix under the conditions as follows: 94 °c for 5min followed by 35 cycles of 94 °c for 30s, 56 °c for 40s, and extension at 72 °c for 1min, followed by a final extension at 72 °c for 5 min. this method was adapted from schönian et al. (2003) (6). amplification products were visualized on 2% agarose gel stained with safe stain. the pcr product size stays between 300 and 360bp. the non-purified pcr products (10μl) were digested with 1u of the restriction enzyme haeiii (promega, madison, wi, usa), and the restriction fragments obtained were compared with the molecular profiles of the who reference strains as are mentioned above. leishmania serial dilution assay (sda) for (limit of detection) lod promastigotes from a 4-days-old culture of a reference strain of l. tropica (mhom/ ir/99/yaz1), and l. major (mrho/ir/75/er) were washed twice in 1x phosphate-buffered saline and precisely counted on a neubauer hemocytometer (mean of 10 counts). the dna was extracted as mentioned above. a series of dilutions was performed, yielding dna solutions corresponding to decreasing concentrations from 20 to 0.00001 parasite/µl diluted (6 log) by distilled water were used as standard dna (17). statistical analysis the χ2 -test and fisher’s exact test using spss 16, was used to determine statistically significant differences in disease prevalence between females and males and among different age groups in the community. results characteristics of patients with suspected cl. fifty one out of 94 (54.3%) of cases were male as well as 43/94 (45.7%) of female cases. most (>50%) of the suspected cases of cl investigated were aged <28 years. their age ranges were between 7 months and 78 years. none of the examined patients had been out of their counties during the 6 months preceding the onset of lesions (table 1). the time between the appearance of the lesion and presentation was 1–4 months for the 73% (16/22) cases infected with l. major and 80% (34/42) cases infected with l. tropica and not determined for 31 out of 94 (data not shown). the majority of cl lesions, was located on the exposed areas of the body, such as the hand, and then occurs on the face and feet, respectively (table 1). parasitological results all of 94 slide samples were found to be positive for the presence of leishmania by microscopy. mo-its-pcr-rflp results after using the restriction enzyme, band patterns, including the fragments of 414, 296, 115 and 26bp for l. major, and fragments of 296, 193, 129, 114, 68 and 28bp (actually 4 fragments on gel, 296, 193, 129–114, 68) for l. tropica were visualized on electrophoresis gel (fig. 2). nucleotide sequence data of l. major and l. tropica were submitted to the genbank database with accession no. kp 874100 and kp893242, respectively. all of 97 cases (100%) tested positive by mo-its-pcr-rflp. leishmania major and l. tropica were detected in 33 (35%) of cases http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 387 http://jad.tums.ac.ir published online: september 08, 2017 and 61 (65%) of cases, respectively by moits-pcr-rflp. the agreement of mo its-pcr-rflp and microscopy was 100% (table 2). its1-pcr-rflp results after using the restriction enzyme, band patterns, including fragments of 220 and 140 bp for l. major and fragments of 200, 60, and <60bp for l. tropica, were observed in safe-stained gels. fifty five of 94 cases (58%) were detected by its1-pcr-rflp. l. tropica was detected by its1-pcr-rflp in 43 of 63 cases (71%), and l. major in 12 of 33 cases (36%). its1-n pcr-rflp results eighty one of 94 cases (86%) tested found to be positive by its1-n pcr-rflp similar to its1pcr-rflp pattern (fig. 1). leishmania serial dilution assay for lod results by conventional pcr, lod for its1-pcr was 1–6 parasites/ml, while the method targeting its (mo) could detect 1×10-2 parasites/ml. table 1. characteristics of features of patients with cutaneous leishmaniasis in khorasan-razavi province of iran feature classification no. of cases percent location of lesions face 28 29.8 hand 42 44.7 foot 19 20.2 others 3 3.2 nd* 2 2.1 total 94 100 gender male 51 54.3 female 43 45.7 total 94 100 age <10 12 12.8 10–20 16 17 20–30 25 26.6 30–40 12 12.8 40–50 10 10.6 >50 17 18.1 nd* 2 2.1 total 94 100 nd*: not determined table 2. the numbers of cases of cutaneous leishmaniasis based on leishmania species which were investigated, split by selected cities of khorasan-razavi province species cities mashhad sarakhss shandiz torghabe others* total (%) l. major 6 7 1 3 16 33(35%) l. tropica 29 1 2 1 28 61(65%) total 35 8 3 4 44 94(100%) *others: ahmadabad, vakilabad, manzelabad, ghasemabad, torbat-e-heidarieh, dargaz, torbat-e-jam and tooss http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 388 http://jad.tums.ac.ir published online: september 08, 2017 fig. 1. geographical situation of korasan-razavi in the northeast of iran where the samples were collected fig. 2. the results of the electrophoresis of the products of the its-pcr-rflp-based amplification of dna extracted from the stained smears before and after enzymatic digestion. the seven lanes contained a molecular-weight ‘ladder’ 100bp (lane 1) and 50bp (lane 2), the products from reference strains of leishmania tropica before enzymatic digestion (lane 3), after enzymatic digestion (lane 4: fragments of 276, 193, 129, 118, 68 and 28bp, a negative control (lane 5) and leishmania major before enzymatic digestion (lane 6) and after enzymatic digestion (lane 7: fragments of 416, 296, 141 and 26bp) fig. 3. the results of the electrophoresis of the products of the its1-pcr-rflp-based amplification of dna extracted from the stained smears before and after enzymatic digestion. the five lanes contained a molecular-weight ‘ladder’ 250bp (lane 1), the products from reference strains of leishmania tropica before enzymatic digestion (lane 2), after enzymatic digestion (lane 3, 4 and 5 fragments of 220, 60 and >60bp) discussion cl is still considered as an important health problem in many regions of the world, especially in the eastern mediterranean region, and almost all countries of the middle east, including iran (2, 18). cutaneous and visceral leishmaniasis both occur in different parts of iran (19). where, the prevalence of infection has been reported as 1.8% to 37.9% in different provinces (20, 21). in 2008, 26000 cases in total have been reported and recorded in iran. more than 90% of cases have happened in 88 cities, and transmission of the disease takes place in the 17 provinces (22). khorasan-razavi province has common borders with afghanistan and turkmenistan in the east, the populations have been increased to 110 percent from 1976 to 2002 and the growth is attributed to the large number of afghan refugees who constitute a population of approximately 450000 (23). cl is endemic in many parts of khorasan-razavi province (24) and the rate of disease has been increased in various parts of the city in recent years. mashhad city (the center of khorasan-razavi province) has religious significance (holiest city in iran), over 20 million pilgrims and passengers visit the city, yearly. mashhad with 4,900 cl cases (an outbreak of acl, in 2002) as well as those reported cases is probably underestimated, so that acl has become the most important endemic disease and has been considered as a health priority (khorasan health centers reports 2000–2002). thus, determination of leishmania species seems to be necessary for designing appropriate control programmers (25). our finding about the species identification are compatible with the results of studies in different regions of mashhad revealed that l. tropica species are dominant (94.2%) in the studied regions of mashhad city (23, 25, 26, 27). the most common anatomical location of lesions on patients’ bodies was hands, http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 389 http://jad.tums.ac.ir published online: september 08, 2017 face, and feet. since that sand fly can’t bite through shirt, the biting places are mostly, exposed parts of patient bodies to bite during the active season. furthermore, in most studies, depends on lifestyle and clothing habits this pattern of lesion frequency is seen (28). based on our finding about gender there was a significant difference between the number of male cases and the number of female cases (54.3% vs 45.7%), this is probably due to the fact that men are employed outside the home (more than women), or it could be related to their covers. this result is consistent with the results of studies conducted by mohajery and dehghani in mashhad county (29, 30). the detection methods frequently used for cl (i.e. the microscopic examination of direct smears and/or the culture of biopsies) are not very sensitive and the leishmania species causing each case of cl in iran is usually only tentatively identified from extrinsic factors, such as the case’s clinical manifestations, time between the appearance of the lesion and presentations and region of residence. in the present study the time between the appearance of the lesion and presentation, unexpectedly, was 1–4 months for all 94 cases infected with both l. major and l. tropica (data not shown). these results show that traditional methods such as clinical manifestations of the disease are not reliable for the detection of leishmania species, we should turn to alternative methods that have higher sensitivity and accuracy. the region of sarakhs, near the border between iran, turkmenistan and afghanistan, the majority of cases were infected by l.major. torghabeh, shandiz, sarakhs and daregaz have the highest disease incidence in the province (31). several studies have shown that differences between the agents of cl in the old world (l. major and l. tropica) may be related to different factors, such as morphological and biological characteristics of the parasite. a variety of molecular, biochemical and immunological methods have been used to characterize and identify the species of leishmania. currently, the most commonly used method is by pcr. the pcr-rflp technique revealed that most of examining cases in mashhad city, torghabeh, shandiz, sarakhss and other cities of khorasan-razavi province were l. tropica and l.major, providing a suitable focus of acl and zcl for further research activities. a noteworthy result obtained is the determination of ʽlimits of detectionʼ (lod) for primer pairs were determined by ʽserial dilution assayʼ based-pcr. its-based pcr was more sensitive than its1-pcr (1–6 parasites/ml) as was previously reported by author (16), while the method targeting its (mo) could detect 1×102 parasites/ml. lachaud et al. (17) designed a similar study with seeded blood sampling with known parasite concentrations and serial dilution assay (sda). in their study, genomic dna showed 2–5 parasites/ml, and the highly repetitive kdna detected 10_3 parasites/ml of blood. in 2013, in a similar study, the detection limits for two different targets primer pairs (kdna-basedpcr and its-based pcr) were determined by taqman-based real-time pcr assay (16). therefore the possibility of detection in samples with lower parasitemia using its-based pcr was predicted. in this study, we used its1-pcr–rflp technique for leishmania species identification and our results showed that there was a relatively good concordance was observed between pcr-rflp technique and parasitological results, where this molecular technique could detect all of the 94 parasitological positive samples (100%). despite dna extracted from all of the 94 samples, the poor results obtained from its1-based pcr, it seems that parasite loads within the skin lesions could have an important role in this investigation (16), thus, as suggested in previous studies, the sensihttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 383–392 a mohammadiha et al.: the pcr-rflp-based … 390 http://jad.tums.ac.ir published online: september 08, 2017 tivity of its1-based pcr should be improved or it could be used from most sensitive and specific molecular methods, such as nested pcr (32). conclusion in conclusion, characterization of leishmania isolates collected from different parts of khorasan-razavi province showed that l. tropica is predominant agents of cl in mashhad city and l. major is distributed in rural areas and cities which have common borders with neighbor countries. moreover, this study revealed that its-pcr-rflp based on our designed primers is an appropriate method for characterization of leishmania species. acknowledgement this study was financially supported by insf (project no.: 92013166) and tarbiat modares university. 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(in persian) 32. hajjaran h, vasigheh f, mohebali m, rezaei s, mamishi s, charedar s (2011) direct diagnosis of leishmania species on serosity materials punctured from cutaneous leishmaniasis patients using pcr-rflp. j clin lab anal. 25(1): 20–24. http://jad.tums.ac.ir/ lived experiences of patients suffering from acute old world cutaneous leishmaniasis: a qualitative content analysis study from iran j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 180 http://jad.tums.ac.ir published online: june 13, 2018 original article lived experiences of patients suffering from acute old world cutaneous leishmaniasis: a qualitative content analysis study from iran *alireza khatami 1, 2, maria emmelin 3, rezvan talaee 4, akram miramin mohammadi 1, nessa aghazadeh 5, alireza firooz 1, berndt stenberg 2, 6 1center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran 2department of public health and clinical medicine/epidemiology and global health, umeå university, umeå, sweden 3department of clinical sciences, social medicine and global health, lund university, malmö, sweden 4department of dermatology, kashan university of medical sciences, kashan, iran 5department of dermatology, tehran university of medical sciences, tehran, iran 6department of public health and clinical medicine/dermatology and venereology, umeå university, umeå, sweden (received 25 july 2017; accepted 9 aug 2017) abstract background: the aim of this study was to explore the experiences of patients who suffer from acute cutaneous leishmaniasis in iran, focusing on quality of life. methods: the study was conducted at two different sites in iran in 2010–2011. individual in-depth interviews were conducted with six men and six women parasitologically confirmed acute cutaneous leishmaniasis. interviews were recorded, transcribed verbatim, and translated into english. qualitative content analysis was used for data analysis. results: the participants, aged 23 to 63yr, had mild to severe disease. based on the analysis four main themes were developed. "fearing an agonizing disease" reflects patients' experiences of disease development resulting in sadness and depression, "struggling to cope" and "taking on the blame" both illustrate how patients experience living with the disease, which included both felt and enacted stigma as major social concerns. "longing for being seen and heard" refers to patients' experiences with healthcare as well as their expectations and demands from communities and healthcare to be involved in closing the knowledge and awareness gap. conclusion: mental and social dimensions of cutaneous leishmaniasis were complex and adversely affected patients' lives by causing psychological burden and limiting their social interactions. health authorities have to plan programs to increase the disease awareness to prevent the existing stigma to improve patients' social condition and medical care. keywords: cutaneous leishmaniasis, qualitative research, quality of life introduction leishmaniasis is a neglected tropical disease (1). leishmaniases are a group of diseases caused by different species of the intracellular protozoan, leishmania (2). the parasite is transmitted by the bite of infected sand flies. the disease is endemic in 98 countries, mostly developing ones. about 350 million individuals are at risk of getting different types of leishmaniasis worldwide, and 1.5–2 million new cases of leishmaniasis occur each year, of which 75% are cutaneous (1-4). the majority of cutaneous leishmaniasis (cl) cases occur in ten countries: afghanistan, algeria, brazil, colombia, costa rica, ethiopia, iran, peru, sudan and syria (1). iran is endemic for cl and almost all cl cases in iran are caused by either l. tropica or l. major and in 2012, the iranian ministry of health (moh) registered 16597 and 4350 cases, caused by l. major and l. tropica, re*corresponding author: dr alireza khatami, email: akhatami@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 181 http://jad.tums.ac.ir published online: june 13, 2018 spectively (4). however, the real number of cl cases could be as high as 100000 cases per year (2). from a clinical perspective, almost all acute old world cl (aowcl) cases in iran can be divided into two forms: anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl). acl is also known as dry, urban or late ulcerative form and is generally attributed to l. tropica. zcl which is caused by l .major is also known as wet, rural, or early ulcerative form cl. in human, the initial sign of infection is the appearance of an erythematous papule or nodule at the feeding site of the female sand fly. it appears within one week to three months after sand fly bite. in a typical zcl infection, the primary lesion usually develops into an ulcer with a violaceous border, which heals spontaneously after several weeks or months, resulting in a scar. ulceration is not a characteristic feature of acl lesions. due to presence of a thick adherent scale on the lesion, hyperkeratosis is the dominant feature of acl lesions (5-7). many treatment modalities have been used in the treatment of cl, but pentavalent antimony including meglumine antimoniate (ma) are considered as the first line drugs for treatment so far. they have to be administered only as intramuscular, intravenous or intralesional injections and could be associated with severe side effects and significant discomfort (8). there is lack of strong evidence regarding effective, safe and inexpensive treatments for aowcl (8, 9). there is no vaccine available for prevention of cl for general human use (10). the psychosocial impact of cl has been studied in a few quantitative papers. in turkey, patients with cl had significantly higher levels of anxiety and depression and were less satisfied with their body image as compared to controls (11). cl patients with active lesions also had a more reduced quality of life (qol) in comparison with patients with healed scars (11). using dermatology quality of life index (dlqi) in kerman, iran, cl had a moderate to very high effect on the qol of about 40% of patients (12). the most affected domain was "symptoms and feelings" (12). using dlqi and sf-36, "symptoms and feelings" was also detected as the main affected domain in another study from iran (13). in kabul, afghanistan, based on focus group with women, cl implies being socially excluded from communal life and illustrated how cl can cause both social and emotional trauma (14). however, to the best of our knowledge, there are no published qualitative papers focusing on capturing individual experiences of living with cl in iran. the aim of this study was to explore perceptions and experiences among patients living with cl as a basis for developing a disease-specific questionnaire focusing on quality of life in cl patients. materials and methods study setting the study was conducted at two different sites in iran: center for research and training in skin diseases and leprosy (crtsdl), tehran university of medical sciences located in tehran capital of iran, which is a referral center for cl patients in iran with about 140cl patients per year referred from all over iran (15), and golabchi clinic in kashan about 210 kilometer south of tehran, where cl is endemic (16). the aim was to represent experiences from patients living in cl endemic (kashan) and non-endemic (tehran) areas of iran. study design we used a qualitative study designed based on individual in-depth interviews since we were interested in the lived experiences of patients with the diagnosis of cl. qualitative content analysis to capture the manifest and latent meaning of their experiences were used as a framework for analyzing the data (17). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 182 http://jad.tums.ac.ir published online: june 13, 2018 sampling of informants informants were sampled purposively to reach maximum variation in regards to the severity of their disease. all informants were parasitologically confirmed cl case. in tehran, direct smear, culture on nnn (novymcneal-nicolle), medium, and polymerase chain reaction (pcr) were used for cl diagnosis confirmation. direct smear was the used technique in kashan for parasitological confirmation. according to expert opinions and for practical reasons, cl severity was categorized in three levels: mild, moderate and severe. patients with more than 5 lesions, lesions larger than 5mm in the largest diameter of induration, ulcerative lesions on the face, and sporotrichoid lesions were classified as "severe" cases. patients with only one or 2 lesions with the largest diameter less than 30mm on locations other than face and ears were considered as "mild" cases and patients within between lesion number and size were defined as cases with "moderate" disease. we aimed to capture experiences from both men and women. healthcare professionals at the two clinics (in tehran and in kashan) asked patients that fulfilled the above criteria if they were interested to participate in the study. if so they were contacted and informed about the aim of the study to settle on time and place for the interviews. data collection data collection took place from oct 2010 to nov 2011. all interviews were conducted face-to-face at the respective clinics in rooms that allowed for privacy. all interviews were digitally recorded and followed a thematic guide including three main content areas: "disease development", "living with the disease", and "healthcare and community". during and after the interviews reflective notes were taken by the interviewer to facilitate elaboration on important issues in the forthcoming interviews. the interviews took between 40–75min. in total, twelve interviews were held with three men and three women recruited at crtsdl and three men and three women at golabchi clinic. these interviews were regarded sufficient to capture the range of experiences and in the last interviews, not much new information emerged. analysis all interviews were transcribed verbatim into persian and later translated into english to facilitate joint analysis within the research group. the analysis followed the procedures suggested by graneheim and lundman (17). this implied condensing the identified meaning units prior to coding the text. codes were later clustered and categories and sub-categories were developed to capture the manifest meaning of the text. finally, themes were constructed to interpret the more latent meaning. during the analysis process, the interpretation was continuously discussed in the research group. an example of the process of analysis from condensed meanings to codes, subcategories, and categories is presented in table 1. table 1. an example of analysis process from condensed meaning units to categories condensed meaning unit code subcategory category "i became sad when i got cl. probability of remaining scars is annoying" becoming sad, fearing remaining scars becoming sad sensing responsibility "when i see people avoiding me because of my cl, or when they think it is communicable, i feel sad." people avoid me, think it is communicable, making me sad "my lesion's appearance is awful. i dislike it" disliking own appearance reacting with selfdisgust "i became angry after i got cl" becoming angry feeling angry "i regret that i did not use a bed net and i got cl" regretting not using bed-net regretting own acts http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 183 http://jad.tums.ac.ir published online: june 13, 2018 ethical considerations the study protocol and its attachments were reviewed by the ethical committee of research in medical sciences of crtsdl, tehran university of medical sciences, tehran, iran, and the study received clearance on dec 1, 2007. prior to starting the interviews, the informants were asked for written consent and for permission to record. the interviewer gave the informed consent form to each eligible participant prior to the interviews. the informed consent form clearly indicated how confidentiality would be kept and that participation was voluntary and would not influence the medical treatment. the whole session would be recorded. participants were asked to read the form carefully and were provided with further clarifications if needed before signing the form. if the patients needed treatment, they were treated free of charge. the study was conducted in accordance with the declaration of helsinki. results twelve patients were from 23 to 63yr old and all had got cl in the endemic areas. those who lived in tehran got it during travels to endemic areas and those who got in kashan were living in kashan or its suburbs. numbers of male and female participants were equal. four cases had mild cl, another four had moderate cl, and the rest of them suffered severe cl (table 2). the analysis of the interview texts resulted in four main themes: "fearing an agonizing disease", "taking on the blame", "struggling to cope", and "longing for being seen and heard" (table 3). the themes reflect four content areas elaborated in the interviews. the first covers patients' experiences of disease development while the second and third themes illustrate how the patients experience living with cl. the last theme refers to patients' experiences with the healthcare system, their expectations, and their demands. the results are presented under the headings of the themes with categories as sub-headings and subcategories incorporated in the text in italics. examples of quotations from the interviews are used to illustrate how the interpretation is linked to the data. the quotes are labeled with t1-6 and k1-6 for patients from teheran and kashan, respectively. table 2. demographic and clinical characteristics of the interviewees # sex age (years) occupation city of residence (where cl was got) cl severity t1 female 48 teacher tehran (mashhad) moderate t2 female 45 housewife tehran (damghan) severe t3 male 27 caretaker tehran (damghan) severe t4 male 33 bookbinder tehran (zavareh) mild t5 male 34 aircraft fueler tehran (shiraz) moderate t6 female 38 teacher tehran (zavareh) mild k1 female 31 university student kashan (kashan) moderate k2 male 50 well excavator ravand (ravand) moderate k3 male 63 businessman ravand (ravand) severe k4 male 40 scrap dealer ravand (ravand) mild k5 female 40 housewife kashan (ravand) mild k6 female 23 university student kashan (kashan) severe http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 184 http://jad.tums.ac.ir published online: june 13, 2018 table 3. summary of main finding indicating themes, categories, and sub-categories content area category theme disease development understanding the disease gradually fearing an agonizing disease  perceiving early skin lesions  considering other skin diseases feeling disease's mental impact  starting to worry about consequences  being physically and socially limited living with the disease sensing responsibility taking on the blame  regretting own acts  feeling angry  reacting with self-disgust  becoming sad facing stigma  being rejected feeling isolated  finding themselves excluded preventing others' reactions struggling to cope  hiding disease or self  avoiding close contact internalizing reactions from others  understanding others reactions  tolerating others behavior  appreciating others' support healthcare and community finding treatment difficult but acceptable longing for being seen and heard  feeling the need for medical care  playing little role in treatment schemes  being satisfied with treatment results noting limited knowledge on cl  meeting unawareness in the community  experiencing ignorance within the healthcare system urging for prevention  high treatment costs  considering current preventive methods as unsatisfactory fearing an agonizing disease this theme captures patients' worries when they first experience symptoms and how these are perceived as scary and threatening. the supporting categories show how some of the clinical characteristics and the course of the cl lesions affect the patient's mental status. understanding the disease gradually the patients perceived early cl lesions to resemble many other skin disorders, for example, simple insect bites, boils, or allergic reactions. however, cl did not follow the usual courses of an insect bite or other aforementioned diseases, which were expected to heal within a few days. patients' confusion became more evident when the lesions grew instead of healing. they considered other skin diseases and started to worry. in particular, for those who were not familiar with the disease, this enlargement, and progression of the lesions, associated with development of relatively big, red, wounded, sometimes painful skin lesions, was worrying. symptoms and signs of cl could be quite perplexing as described below: http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 185 http://jad.tums.ac.ir published online: june 13, 2018 "… it was itching and red, then it became larger, like the size of a pea. it was itching and burning. after two months it became larger and still was itching. then it became purulent. i tried to drain the purulent material myself several times, but each time the wound appears again and become purulent." t3. feeling the disease's mental impact feeling mental trauma because of cl was due to different experiences. some patients were afraid because they misdiagnosed their own disease with more serious disease, commonly those known to be contagious, stigmatizing diseases and notorious for being difficult to manage: "i thought i might have shaken hand with someone who has had aids or something. or maybe that person's hand was bleeding and i had a wound on my hand too…so, i might get it [aids]." k4. these experiences together with misdiagnosis by healthcare providers made the patients fearing an agonizing disease. a patient said that a general physician made a diagnosis of a mosquito bite and prescribed him some antihistaminic tablets and an anti-pruritic ointment, which were not effective. patients' experiences of worrying about different aspects of the disease including its course and its outcome contributed to finding cl a disease, which causes mental trauma and preoccupation. interestingly, in some patients, a confirmed diagnosis of cl was relieving to some extent, although the worrying about long-term outcomes remained: "i was anxious for myself because of uncertainty about the condition [i got] and what will happen to it.… i was relieved after cl diagnosis was made because of knowing that it was curable. however, i was worrying about the remaining [cl] scar". t1 this patient showed how starting to worry about cl consequences could make cl a disease, which adversely affected patients' mental health. other factors such as having limited knowledge about spreading and transmission routes of cl also contributed to this mental impact. they limited knowledge of patients about their own disease was associated with distress and having enough information about their cl would facilitate understanding their disease: "it is better for a person who knows [about cl]. if he/she knows about cl he/she would not be worried. the person who does not know it feels under pressure… a patient who knows about cl could cope better with the disease." k4. the patients experienced cl as a being socially and physically limiting. some patients experienced physical limitations which were mainly related to the interfering nature of the cl lesions with their daily work: "since my job is basically done using my hands, an elbow [cl] lesion has adverse effects on my performance" t4, or: "my work is dealing with metal scraps. when i got cl, i could not work for about one month. i had to get a worker to do the job for me…i could not touch the industrial waste [metal scraps]". k4. social limitations were more related to becoming isolated, rejected, and even stigmatized which is also described under the theme "struggling to cope". being physically and socially limited contributed to cl being associated with mental impact. taking on the blame "taking on the blame" is one of two themes that illustrate how cl patients experienced living with their disease. it represents the internal aspects of living with cl. sensing responsibility regretting own acts for getting cl showed how some cl patients acknowledged their own role in getting the disease and sensing their responsibility: http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 186 http://jad.tums.ac.ir published online: june 13, 2018 "i regret that i did not use a bed net and i got cl" t2. some cl patients felt anger, which might adversely affect their relations with co-workers and family members. for example, a man with severe cl recalled: "after i got cl i became irritable and moody. on one occasion, i became really angry at my workplace when someone hit my elbow for fun. moreover, at home, there was one occasion that my wife asked me to help her for preparing the bed but i told her that i would not help. i became so angry, that i went to another room for sleeping and told her that my hand is painful and i could not even pick up a 1-kg weight. …anger was because of getting cl." t3. becoming angry was related to sensing responsibility because the patient felt responsible for doing something he was expected to do or avoiding something that he was expected to do as a result of his anger. reacting with self-disgust and becoming sad were other sub-categories contributing to sensing responsibility. "i am really sad because i find my cl lesions disgusting. their appearance is very bad and they are really awful. i always say prayers that it [cl] be completely cured as fast as possible." t1. facing stigma some prominent experiences in cl patients were feelings of being rejected, isolated, and excluded. these experiences showed both enacted and felt stigmatization: "…everyone is scared of cl. like in the mosque that i used to go, there was a woman who had cl where everybody could see her lesions. nobody would sit beside her or shake hands with her. people pointed her out to each other and talked about her. … she was lonely. … when they stood in lines for prayers (namaz), people stood away from her at a distance…when i got cl, i experienced these matters myself." k6, or: "in the bank, once they saw my [cl] wound, they looked away [dropped their head]. or in the bakery when i touched the bread to give it to someone, they threw the bread away. it is because of infection [cl]." k4. in some cases, the feelings were so overwhelming that the patients felt being stigmatized and used the exact word of stigma or being stigmatized to describe their situation: "i feel being stigmatized after getting cl. i think this stigmatization is imposed by close friends as well as distant ones [local shopkeeper]." t1. struggling to cope this theme also reflects patients' experiences of living with cl. however, while the theme "taking on the blame" basically refers to patients' own role, "struggling to cope" illustrates how cl patients' react to other people reactions, one of the less explored aspects of the cl patients. cl patients' interactions with others were spread over a wide spectrum of somewhat contradictory experiences illustrated by two main strategies used to handle others reactions towards their disease. preventing others' reactions the first strategy was to prevent reactions from other people to happen, basically through hiding the lesion or self. an example of this preventive mechanism was evident from these quotations: "even if my brother comes to our home i wear a long-sleeved dress, so they [he] cannot see this part of my elbow." t1, or: "even you may want to hide." t4. for some patients, it was quite upsetting and discouraging to constantly feel forced to inform about their disease. this even resulted in telling lies to avoid more distress: "i did not fear [others reactions], but i was upset that i was being asked about it [cl] all the time, like every 5 to 10min, from my university to home i should have explained it for http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 187 http://jad.tums.ac.ir published online: june 13, 2018 20 people. that bothered me. i did not like to answer. in the beginning, i told them it was cl and then i observed that everyone, from young to old, run away from me. they did not take a sandwich or a glass of water from me. the problem was that i had to lie to them to make myself feel more comfortable. i would say it was wart and people stopped asking question when they thought it was wart. lying to people bothered me. but it was a white lie, it was better than my suffering [for being asked about my lesions too frequently]".k6. internalizing reactions from others the second strategy was to see the disease through the eyes of others by indicating that they understood others' reactions towards them. some preferred to use the term "tolerating" instead of "understanding", indicating that while they could not understand or find a rationale for others' reactions, they tried to accept the situation they were in: "i thank god that i am really tolerant and i do not struggle with people. i tell my colleagues that i have cl and ask them to hit [as a sign of friendship] on my healthy arm instead of the affected one." t3. at the same time patients experienced others' support: "in my case, they [my colleagues] gave me my injections at the workplace, although if they did not, i would go somewhere to receive them. they also gave me some leave. … i told them that i recovered, but they told me that i have not recovered yet. they told me that i follow up my disease and if i need something for example money, i phone them. …". longing for being seen and heard the last theme reflects cl patients' encounters with the healthcare system. cl patients experienced being ignored, and excluded from shared decision making for treatment plans, and not receiving proper acknowledgement for their own treatment demands. it is also related to the patients' experiences in the communities, in which people have limited knowledge about cl or despite having enough information on the disease, they do not behave in accordance with what they know. finding treatment difficult but acceptable cl treatment was not easy because of its associated side effects. however, when they felt they would benefit they tended to accept the treatment offered. "i feared of [ma] injections. once, i received incomplete treatment because of [injection] pain, but later on i completed the [treatment] course. cl treatment course is shorter than the courses for some other health problems. i feel happy about the results of the treatment of cl." t3, or: feeling really uncomfortable also contributed to needing medical care because of the severity of the disease "when i became uncomfortable because of the pain and itch on my cl lesion, i decided to go to a doctor… i knew that cl would finally heal [spontaneously], but i decided to seek medical advice on its treatment…" t5. some patients were prepared to tolerate quite painful complications in order to quicken the healing phase. "when i received [ma] injections, i felt some malaise … i felt that [ma] injections caused problems for my kidneys because i had to wake up during the nights and go to wc. but in general, the complications were not serious. i have prescribed 3 vials [of ma] each day for 2 weeks, a total of 42 injections. i received them intramuscularly and my [gluteal] muscle became very stiff, so i could not use the last 3 vials. i knew that [ma] injections would heal cl lesions in about 2 wk. i also knew that without treatment cl might take about 6 months to be healed." t4. others felt that they did not play enough roles in making decision about the treatment of their disease and were not satisfied with http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 188 http://jad.tums.ac.ir published online: june 13, 2018 the physician's approach to treatment. they had wanted to be more involved even if they were satisfied with the treatment results. "i have to raise one point; my physician postponed my treatment for 10 d because he did not want to treat me with [ma] injections at first. he told me that the injections would be very painful and i might not be able to walk for some time after i received the [ma] injections, but when i received the injections, they were nothing extraordinary. i think that if he started my treatment earlier, my disease would not become so bad. i imagine patients do not actively participate in choosing the treatment methods. they play too little role in the treatment plan." t2. noting limited knowledge on cl the participants described how they met unawareness in the community and clearly saw how this was one of the important factors increasing their suffering: "…i am a sportswoman and i [have to] wear clothes that are comfortable [which do not cover all parts of the body], and since many people do not know what it [cl] is and whether it is communicable or not, they ask questions or avoid me, and i have to wear certain clothes and also i will remain isolated". k1. however, it was not only in the community that the participants had met ignorance about the disease. even within the health care system, they experienced ignorance and suspicion resulting in them being treated as if the disease was highly transmissible. "even once i went somewhere to receive my injection and the nurse who wanted to do the injection feared so much that she refused to inject the medicine… she thought that it is highly transmissible and feared a lot…." t4. some had concrete suggestions of how to increase community awareness and underscored that the information given needs to be repeated regularly to really make a difference. "… maybe not enough information [in the community]. they give people brochures about cl every 10yr, maybe. if these brochures and handouts are given to people every 2–3 months, once a year and everyone knows about cl and gets better education, it would become much better." k6. others emphasized the importance to adjust the information provided to different groups of people and to write in an understandable language. "i would like to see more public education about cl. there must be some public areas where people gather and discuss different aspects of the disease [cl]. the society is full of people of different kinds, young, old, educated, illiterate, etc. the information should be easily understandable by everyone. not just a printed hand-out that they give to people." k1. urging for prevention the participants also emphasized the need for prevention from different perspectives. for those who were less economically privileged the urge for prevention was based on the high treatment costs: "the disease [cl] has to be prevented. you have to go after treatment, spend money for it. have to look until you find a clue. when i was first bitten by cl [mosquito], i could not figure it out. i got unemployed. i spent money. …i had to pay for transportation fees." k2. however, the need for prevention was not raised only by the poor. moreover, patients from households with moderate to high incomes not only acknowledged the importance of prevention but considered current preventive methods as unsatisfactory: "i think cl prevention is very important. i hope a vaccine be developed for cl soon. current preventive measures like using insect repellent sprays and even using bed nets are not completely effective". t4. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 189 http://jad.tums.ac.ir published online: june 13, 2018 discussion exploring the lived experiences of patients suffering from acute aowcl indicated that the disease affects different aspects of the lives of the patients. while most physical problems of cl lesions were quite expectable, the mental and social dimensions of the disease were affected much more than what it was thought. however, we noticed some important experiences by the patients which were related to the course of the disease development and management. the adverse impacts of cl on mental health started with facing an agonizing and fearing disease and continued with worrying, anxiety, sadness, and depression. the main societal problem associated with cl was stigma. these problems together with insufficient information about cl in general population and sometimes among healthcare providers resulted in the patients' struggle of coping with their situation and longing for being heard and seen by both ordinary people and health authorities. disseminating knowledge about cl in community and better training of healthcare professional could help to improve cl patients' conditions. cl was not a minor health problem (14). while it is not uncommon for other dermatologic diseases to be misdiagnosed by the patients and non-dermatologist physicians, cl misdiagnosis is quite agonizing for the patients because its clinical course is dramatically different from what they expect. for example, a patient whose early cl lesion was misdiagnosed with an ordinary insect bite and received treatment for the latter, worried when he/she observed that instead of healing, his/ her lesion grew much larger and became purulent. according to our experience, patients might mistake cl with a wide spectrum of dermatological diseases from boils to skin tumors, leprosy and hiv/aids infection. these misinterpretations of symptoms and signs were particularly evident when the patients had no previous information about cl. this associ ation between knowing about cl and better qol was also reported, patients without previous knowledge about cl has worse qol in comparison with those who had previous knowledge about it (13). depending on the location and severity of cl, it could result in physical limitations for the patients, which could consequently affect their income, because they were not able to do their jobs. however, the impact of unemployment and health expenditure in aowcl is much smaller in comparison with some more chronic tropical diseases such as lymphatic filariasis (18). lack of knowledge about cl among general population was a contributing factor to the cl-associated stigma. since kashan is endemic for cl, it was reasonable to assume that similar to patients with cl, people without cl in kashan were quite familiar with cl and had a better understanding of cl patients’ situation. however, such understanding was not reflected in their behavior towards the cl patients. this finding was similar to results (14), which indicated that while people in endemic areas for cl like kabul, afghanistan had an acceptable knowledge about several aspects of the disease, their behavior towards cl patients was not congruent with their knowledge. worrying about their disease and its consequences including scar formation was a dominant finding among our participants and some of them also mentioned that they became sad because they got cl. our findings were similar to results which showed significantly higher anxiety and depression in cl patients than healthy controls (11). lower qol and body image satisfaction in patients with active cl lesions in comparison with healthy individuals or those who had cl scars (11). an important and less explored aspect of cl is the psychological and social effects on the patient's lives. our study contributes with http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 190 http://jad.tums.ac.ir published online: june 13, 2018 examples of feelings and reactions in relation to themselves or other individuals that could result in psychiatric problems such as depression. our findings are supported by two studies from iran's neighboring countries; turkey and afghanistan that found depression and anxiety were more frequent among cl patients (11, 14). depression and sadness because of the disfigurement caused by filariasis could have greater burden for the patients than its physical consequences (19). this greater burden of psychosocial impact could occur in cl patients as well. regretting own acts and reacting with self-disgust could contribute not only to sadness and depression but also to low self-esteem and social disconnection (19). several definitions of stigma were provided (20), of which three, i.e. enacted, perceived, and internalized stigma were described in patients on another neglected tropical disease, lymphatic filariasis. our cl patients experienced different types of stigma. some cl patients, mostly those with moderate or severe disease, were rejected, feeling isolated or finding themselves excluded which could be considered as examples of enacted stigma (19, 20). among the management strategies for this kind of stigma, the patients used denial by telling lies about their disease and hiding their lesions. when they talked about their experiences, some of our participants said some matters that indicated perceived (felt) stigma, such as being aware of other who had cl and experienced discrimination. they were afraid of being treated like those whom they had heard about. their management strategies were similar to patients who suffered filariasis including modifying social interactions, isolating self, and hiding the affected body parts (19). we also found examples of internalized (self) stigma in our interviews with cl patient like regretting own acts (i.e. guilt), reacting with self-disgust, and sadness (19). cl patients with internalized stigma used the same management strategies (19) of women with filariasis. another type of stigma was defined "aesthetic" or "unaesthetic" stigma which is caused by visible marks or visible deformities (21-23). since it was on cl stigma in surinam that terminology might be relevant to our study as well (23) although we imagine that it does significant overlap with the three former stigmas we have already mentioned and is not a unique entity. since cl is very common in some countries, to reduce the cl-associated stigma some strategies have been developed which could be considered cultural rather than individual (22, 24). one example is according to desjeux that cl was known as ‘little sister’ in some endemic countries where it was very common and became part of normal life just as a family member (24). another cultural management strategy to counteract cl stigma was pointed in turkey, in which the older people were accustomed to the cl scar, and called it "a beauty scar" to increase the tolerability and normalization (22). however, the younger generations did not accept the cl disfigurement and scar and considered it as a lifelong stigma (22). severe cl-associated stigma was reported in kabul afghanistan, where the disease was very common and the people were knowledgeable about cl (14). this difference might be explained by lack of culturally-built management strategies regarding cl stigma. because of some wrong believes, more than half of their studied cases mentioned that they would prevent a cl patient from touching and hugging his/her child, and they would not allow a cl patient prepare food in the home (14). in addition, more than 20% of participant in their study said that a mother with cl should not breastfeed her child. women with cl lesions or scar would face problem if they would like to marry (14). the cl stigma and its social impact in afghanistan were larger than what it is in iran (14). according to some participants in our study, they tried to accept their situation and http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 191 http://jad.tums.ac.ir published online: june 13, 2018 acknowledge others' reactions toward their disease by trying to understand or at least tolerate the others. even some of patients appreciated others' supports. therefore, these mechanisms together with some of the aforementioned stigma management strategies helped the cl patients to continue their struggle to cope. provided an interesting discussion about the potential roles of morals and contextual morals in de-stigmatization of health problems including depression, which might be used in combating cl-associated stigma when the knowledge about that issue become more clear in the future (25). they explained how stigmatized individuals were not able to meet the obligatory and normative moral criteria that were set in a context of a society by socio-economic, cultural, anthropological and political determinants. acknowledging the interconnection between those who stigmatize and those who were stigmatized (25) stated that understanding and changing the stigma-determining factors could help to combat stigma through modifying the contextual morals. in dominican republic and ghana on lymphatic filariasis (19, 26), factors such as religious beliefs and hope provided the basis for better coping with the disease. due to strong religious beliefs in most iranians, this factor played a role for those who internalized or integrated others' reactions. determinants of the stigma associated with another neglected tropical disease, leprosy were reported, as visibility of the lesions, cultural and religious beliefs, fears of transmission and public health interventions (27). from the interviews with patients and considering iran's cultural context, it would be likely that the same determinants play roles in cl stigma. the association of visibility of the lesions with stigma in skin diseases is quite well known, not only in infectious diseases in developing countries but also in non-infectious diseases such as psoriasis and vitiligo in both developing and developed countries (28, 29). hotez provided a concise and interesting discussion about the importance of stigma in the burden of disease of neglected tropical diseases including cl, which might be not be reflected in a quantitative indicator of disease burden like disability-adjusted life year (daly) (30). small dalys of cl patients (31), from iran, was in line with hotez (30) conclusions and indicated the need for qualitative researchers to better explore the impacts of cl on patients' lives. a multifaceted approach to target cl itself through increasing the availability of affordable care for cl patients and to educate people about the disease was needed to reduce the social burden and the associated stigma of cl (32), we also imagine that developing and implementing healthcare educational and interactive destigmatizing programs could play important roles to improve cl patients' situation. despite the patients knew about the self limiting nature of cl, they sought medical care form health professionals (14). some patients found their encounters with healthcare providers were not satisfactory. they were misdiagnosed, they were not sufficiently engaged in the decision-making process of treatment, and even were behaved discriminately against. they were aware about the adverse effects of the standard treatment of and even experienced some of those complications; however, they decided to receive the recommended therapeutic regimen. during the interviews, several patients gave answers indicating that they were aware of traditional methods of cl treatment provided by the traditional herbalists or recommended by their family members or friends, but they preferred to be treated by medical doctors. however, none of them had experienced a traditional therapeutic method. one of the issues that was surprising in our study was the fact that while, most interviewees were quite knowledgeable about cl risk factors like exposure to sand-fly bites, routes of its transmission, and in some cases http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 192 http://jad.tums.ac.ir published online: june 13, 2018 about the clinical signs and symptoms, most patients acted similarly to individuals who were not familiar with the disease. the patients' experiences indicated that in endemic areas such as kashan and damghan, the general populations were quite knowledgeable but still acted in a stigmatizing way towards cl patients. the patients attributed their experiences to lack of knowledge in the community, although it might be not be explained only by this factor. indeed, the cl-associated stigma might be a culprit. most patients asked for better health education programs to tackle this problem. according to the patients, trained healthcare providers' knowledge about cl was also insufficient and cl patients asked for better training be provided by the responsible organization and authorities such as moh to improve the situation. we found it interesting that almost all interviewees answered an open question about "what else they would like to add to what had already been talked about?", by emphasizing the lack of information and the existing ignorance about cl in the community. they asked for ways to increase the public awareness with emphasis on the potential role of mass media especially islamic republic of iran broadcasting (irib) television. they believed that visual programs could improve knowledge about cl in those who could not read newspapers, educational pamphlets, and posters. the participants asked for more effective preventive methods. although less socio-economically privileged patients considered high treatment costs as a reason for improved preventive interventions, at least one study conducted in an endemic area in iran reported that with the exception of educational programs, applying other known preventive methods such as using insect repellants, pesticides, and bednets were not cost-effective or cost-benefiting (33). using the markov decision analytic computer model, development of a vaccine for cl with only 50% efficacy and as little as 5yr of protection in seven latin american countries with highest incidences of cl in that region would be cost-effective in comparison with standard treatment regimens (34). however, the treatment cost per patient and the morbidity of new world cl is higher than old world cl (33, 35). development of new and more effective preventive methods such as vaccines was also one of the demands of relatively affluent cl patients in our study. even if our interviewees were aware the getting cl for the second time is very unlikely, they still were looking for more effective prevention methods. cl could be prevented in them, but also would be preventable for those who still had not experienced the disease. trustworthiness strengths the first author (ak) is an experienced dermatologist having worked with cl for several years at crtsdl in tehran and in endemic areas in iran. to increase the credibility of the study he made multiple visits to the endemic setting (golabchi clinic, in kashan) to assess the situation, build trust with the clinic staff and to familiarize them with the research project. his prolonged engagement also included spending several weeks in the kashan during the actual data collection period. however, during the interviews, he made efforts to put his knowledge and experiences of cl as a dermatologist within brackets and allow the informants to tell their stories. the analysis was regularly discussed in debriefing meetings within the research group including interdisciplinary competencies. taking field notes during his stay documenting the research process facilitated keeping and audit trail to account for the dependability of results. limitations only people sought medical care for their cl lesions were included in the study so individuals who had not been coming for medical treatment were excluded. an expert panel had been consulted and it was agreed that http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 180–195 a khatami et al.: lived experiences of … 193 http://jad.tums.ac.ir published online: june 13, 2018 it is unlikely that the experiences between the two groups were considerably different. cl requires medical care, which was taken as an indication that this would also be the situation in iran (14). however, the transferability of our results is limited by only including patients with acute old world cl (lesions duration less than 6 months). those who suffer from other forms of cl, which have longer duration, could be considerably different since they have other clinical courses and outcomes. for consent reasons our study only investigated patients who were older than 18yr and we acknowledge this as a limitation since children are a large and important group at risk of having cl in endemic areas. conclusion our study demonstrates how cl interferes with patients' health, both mentally and socially. to provide a better management and reduce the burden of cl, in particular, its associated stigma, further studies to investigate the qol of cl patients as well as developing and implementing health education programs in endemic areas are recommended. acknowledgements this study was conducted by a research grant number 423/158 from the center for research and training in skin 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http://irje.tums.ac.ir/article-1-83-en.pdf j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 354 http://jad.tums.ac.ir published online: september 08, 2017 original article composition of anopheles species collected from selected malarious areas of afghanistan and iran helen hoosh-deghati 1,2, *navid dinparast-djadid 2, *vahideh moin-vaziri 1, hoda atta 3, abbas ali raz 2, seyyed javad seyyed-tabaei 1, naseh maleki-ravasan 2, hamzeh alipour 2, sedigheh zakeri 2, eznollah azar-gashb 1 1department of parasitology, school of medicine, shahid beheshti university of medical sciences, tehran, iran 2malaria and vector research group, biotechnology research center, pasteur institute of iran, tehran, iran 3malaria control, word health organization/eastern mediterranean regional office, cairo, egypt (received 3 june 2016; accepted 9 jan 2017) abstract background: malarious areas in iran are close to afghanistan and pakistan that urge the researchers to extend their knowledge on malaria epidemiology to the neighboring countries as well. vectorial capacity differs at species or even at population level, the first essential step is accurate identification of vectors. this study aimed to identify anopheles species composition in selected malarious areas of afghanistan and iran, providing further applied data for other research in two countries. methods: adults anopheles spp. were collected from four provinces in afghanistan (badakhshan, herat, kunduz, nangarhar) by pyrethrum spray catch, hand collection methods through who/emro coordination and from chabahar county in iran by pyrethrum spray catch method. identification was performed using reliable identification key. results: totally, 800 female anopheles mosquitos, 400 from each country were identified at species level. anopheles composition in afghanistan was an. superpictus, an. stephensi and an. hyrcanus. most prevalent species in badakhshan and kunduz were an. superpictus, whereas an. stephensi and an. hyrcanus were respectively found in nangarhar and heart. anopheles species in chabahar county of iran were an. stephensi, an. fluviatilis, an. culicifacies and an. sergentii. the most prevalent species was an. stephensi. conclusion: current study provides a basis for future research such as detection of plasmodium infection in collected samples which is on process by the authors, also for effective implementation of evidence-based malaria vector intervention strategies. keywords: anopheles, morphological identification, afghanistan, iran introduction malaria is known as one of the six important infectious diseases based on who (1). it is caused by the protozoon plasmodium spp and transmitted by female anopheles mosquitoes (2). anopheles spp belongs to culicidae family (diptera) with 460 or so different species that 70 of them have the potential of being malarial vectors, however, around 36 species are considered as main ones (3, 4, 5). about 198 million cases of malaria occurred in 2013, leading to 584000 deaths (6). glob ally, around 3.2 billion people are at risk of being infected with malaria and 1 in 1000 is at high risk of getting malaria in year (1). in eastern mediterranean regions (emr), more than 10 million clinical malaria cases occur annually with 50 thousands death. generally 287 million which is approximately 60% of eastern mediterranean population is at the risk of malaria infection (1, 7, 8). malaria is of great importance from public health concerns in iran, more than 2 million *corresponding authors: dr navid dinparastdjadid, e-mail: navidmvrg@gmail.com, dr vahideh moin-vaziri, e-mail: vmvaziri@gmail.com http://jad.tums.ac.ir/ mailto:navidmvrg@gmail.com j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 355 http://jad.tums.ac.ir published online: september 08, 2017 people live at high risk regions (7). iran with population of more than 78 million people is located in west asia sharing a border of 1458 and 936km respectively with iraq from the west, afghanistan, and pakistan from the east (9). malaria is endemic in the south and southeast of iran, where more than 90 percent of malaria cases are reported (10, 12). these regions are bordered with pakistan and afghanistan which malaria cases due to p. falciparum and p. vivax are prevalent. malaria has an unstable pattern varying between 42% up 60% of total malaria cases with two seasonal peaks mostly in spring and autumn after the rainy seasons in the provinces located in south-east of iran comprising sistan and baluchistan, hormozgan and the tropical part of kerman (13). recently a notable reduction has been observed in malaria cases due to the report of ministry of health and medical education of iran, which has led who to categorize iran in the elimination phase (14). nationwide campaign malaria elimination was launched by iranian government in 2010 in epidemic regions with the goal of becoming malaria-free country by 2025 (12, 14). vector control is the most efficient way to decrease the malaria transmission at the community level. regarding to the recent iranian mosquitoes checklist, seven different genera, 64 species and 3 subspecies are reported (15). seven species of the genus anopheles including an. culicifacies giles s.l., an. dthali patton, an. fluviatilis james s.l., an. maculipennis meigen s.l., an. sacharovi favre, an. stephensi liston, and an. superpictus grassi are identified as malarial vectors in iran. six species are known as its vectors in southeast of iran: an. culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. pulcherrimus, and an. superpictus. among them, an. stephensi is the main vector of malaria in the area and considerably prefers human blood (16, 13, 12). in afghanistan, approximately half of the population is at the risk of malaria. there are at least six malaria vectors in afghanistan: anopheles superpictus, an. culicifacies, an. hycranus, an. pulcherrimus, an. fluviatilis and an. stephensi (17). full understanding of malaria vectors can be considered as a key factor in malaria control programmes. the first step in any control program is the vector species identification. moreover, malaria endemic areas in iran are close to afghanistan and pakistan, urging us to extend our research to the neighboring countries as well. anopheles species were collected from selected malarious areas of afghanistan (badakhshan, herat, kunduz, and nangarhar) located in high risk areas (18, 19) by who. this study aimed to identify anopheles species composition in selected malarious areas of afghanistan and iran, providing further applied data for other research in two countries. results presented here reflect the composition of anopheles species of mentioned collection as a part of ongoing project. it could provide also useful information in planning and implementing of an effective program for vector control during elimination phase in iran and control phase in afghanistan for the national malaria control program. materials and methods study areas afghanistan: afghanistan is a landlocked country in south central asia (33° 00’ n, 65° 00’ e). its climate is arid to semiarid with frequent sand storms. the northern and southeastern areas of the country have very rugged mountain terrains (hindu kush), while the western and southern parts are flat deserts and plains. climate varies according to the elevation and location. in general, malaria occurs at altitude below 2,000m and is the most prevalent in snow-fed river valleys and rice growing areas (20). samples were collected from four counties, located in different geographical sites of afghanistan, north-east (badakh shan and kunduz), south-east (nangarhar) and http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 356 http://jad.tums.ac.ir published online: september 08, 2017 western part close to iran boundary (herat) (fig. 1). iran: sistan and baluchistan province as the main malarious area is located in the northern coast of persian gulf and oman sea, where the weather is warm and humid enough for anopheles species to be activated throughout the year (7). this study took place in chabahar county (25° 17’ n, 60° 38’ e), bounded by pakistan in the east, hormozgan province in the west, kerman province in the north, and oman sea in the south. it has a warm and humid weather in summer and temperate in winter. the average maximum and minimum temperature are respectively 34 ˚c and 10.5 ˚c in july and january. latitude from the sea level is 7m. the annual rainfall is low mainly in spring and autumn so that it has a desert climate. however it has high humidity due to neighboring sea (fig. 1) (21). mosquito collection and identification adult anopheles spp were collected on august 2011 through who/emro coordination from four provinces of afghanistan (badakhshan, herat, kunduz, nangarhar) by pyrethrum spray catch, hand collection method using manual aspirator and from chabahar county in iran by pyrethrum spray catch method (table 1). the sampling sites are indicated on fig. 1. the species identification was performed by using reliable identification key (22) under leica ez4d (16x) stereomicroscope. resuts morphological identification of afghanistan anophelinae mosquitoes a total of 400 adults female anopheles were collected from herat, nangarhar, badakhshan and kunduz provinces of afghanistan (table 1). among the collected specimens, 3 species were identified based on the morphological characteristics, 45% of total specimens belong to an. superpictus collected from badakhshan and kunduz. totally 180 an. superpictus were collected from mentioned areas, with pyrethrum spray catch (psc) and hand collection, among them 141 (78.3%) collected from human house. the major morphological characteristics of this species are having unusually long palps (fig. 2a), no dark spots at point of bifurcation of 5th longitudinal vein and equal division part of the second and forth vein to the end of wing (fig. 2b). next species was an. hyrcanus which was gathered by psc method from animal and human house in herat. totally 110 (27.5%) was identified, which mainly collected from human house (63%). in an. hyrcanus fore margin of wing has less than four light and four dark bands alternating (fig. 3a), fore margin of the wing with two small white spots, one near the tip, the other about two thirds of the way from the root of the wing (fig. 3b). third identified species was an. stephensi (27.5%), which was sampled from the human house of nangarhar by psc method. this species could be easily characterized by white spots which speckled on femora and tibiae (fig. 4 a, b). morphological identification of anopheline mosquitoes of chabahar county (iran) morphological characteristic of 400 adults female anopheles specimens collected from two villages of chabahar county, iran, revealed the presence of 4 different species (table1). regardless the kind of species, all specimens was collected from human house and by psc method. the most prevalent species among them was an. stephensi (94.75%), which its main morphology character was mentioned previously. the other species were an. fluviatilis (2.75%), an. culicifacies (1.5%) and an. sergentii (1%). anopheles culicifacies has been identified based on the following main characters, existence of two fringe spots, no tuft of scales on anterior margin of mesonotum and finally existence of a dark spots opposite to the light spot at the costa http://jad.tums.ac.ir/ http://mississippientomologicalmuseum.org.msstate.edu/collecting.preparation.methods/aspirator.htm j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 357 http://jad.tums.ac.ir published online: september 08, 2017 base (fig. 5). anopheles fluviatilis has palps with usual length (fig. 6a), dark spot at the point of bifurcation of the 5th vein and farther division part of the second vein than the forth vein with respect to the end of wing (fig. 6b). anopheles sergentii characteristics include more than two light spots on the posterior margin of fringe with pale base of subcostal and no dark spot of sub-costal opposite to the pale spot of costa (fig. 7). table 1. details of collected specimens based on study areas and collection methods: psc: pyrethrum spray catch, hc: hand collection by manual aspirator, hc: hand collection, hh: human house, ah: animal house total total number of species anopheles spp number of mosquitoes collection methods and sites village province 400 110 (27.5%) an. hyrcanus 70 psc-hh (indoor) haja sourma herat a fg h a n is ta n 30 psc-ah (indoor) 110 (27.5%) an. stephensi 55 psc-hh (indoor) pol-e taracheh nangarhar 55 ali khan 100 (25%) an. superpictus 39 psc-ah (indoor) darewazer badakhshan 37 hc-hh (indoor) 24 psc-hh (indoor) 80 (20%) an. superpictus 80 psc-hh (indoor) gerghiz kunduz 400 379 (94.75%) 11 (2.75%) 6 (1.5%) 4 (1%) an. stephensi an. fluviatilis an. culicifacies an. sergentii 100 psc-hh (indoor) owraki chabahar ir a n 300 psc-hh (indoor) balochadam fig. 1. map of iran and afghanistan, indicating the location of the study area in chabahar county (marked with an asterisk) situated in the south of sistan and baluchistan in iran and herat, kunduz, badakhshan, nangarhar provinces in afghanistan. fig. 2. anpheles superpictus, a: head with long palps b: no dark spot at point of bifurcation of 5th vein and division part of the second and forth vein to the end of wing is equal http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 358 http://jad.tums.ac.ir published online: september 08, 2017 fig. 3. a: existence at least dark spot at the upper margins of the swings, which is common in all anopheles except an. hyrcanus b: an. hyrcanus, fore margin of the wing with two small white spots fig. 4. anopheles stephensi, a, b: femora and tibiae speckled with white spots fig. 5. anopheles culicifacies, first vein with a dark spot opposite the light spot at the base of costa fig. 6. anopheles fluviatilis, a: usual length of palps b: spot at the point of bifurcation of the 5th vein and division part of the second vein is farther than the forth vein to the end of wing fig. 7. anopheles sergentii, no dark spot on sub-costa discussion malaria is widespread in the tropical and subtropical regions of the world and is also a public health problem in western extensions of the oriental zoogeographical region including iran, afghanistan and pakistan. according to who report, 9 of the 97 countries with ongoing malaria transmission, including iran, are classified as being in the malaria elimination phase. at this stage, interventions focus on the detecting all malaria cases, managing malaria foci, managing imported malaria cases and preventing onward transmission (23). regarding the latest reports, it should be intentioned that, different anopheles species even different pop http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 359 http://jad.tums.ac.ir published online: september 08, 2017 ulation of one species, have different ecologies, biological attributes and vectorial capacities (24). then, exact species identification is necessary for better understanding of their potential roles in malaria transmission (25, 12). in iran the most problematic malarious areas is south and southeast of the country, which highly affected by the epidemiology of malaria in neighboring countries, afghanistan and pakistan. the following reasons support this consumption 1. afghanistan is in control phase of the malaria control, compared to iran which is in elimination phase (14), 2. similarity of anopheles fauna and human causative agents of malaria, both p. falciparum and p. vivax are prevalent in both countries and 3. importance of immigration from afghanistan and pakistan to malarious areas of iran is the major routes of malaria transmission. this situation urges us to conduct current study, concentrating on morphological characters of anopheles and their composition within selected areas of afghanistan and iran. taxonomy that utilizes morphological characterization has been known as the gold standard method in identifing the mosquito species, which also have been used in current study for identification of five anopheline species in the study areas of iran and afghanistan, including known malaria vector species. all the species which were identified during current study previously was reported in the checklist of iranian mosquito fauna (15). the most prevalent species among our collected specimens from chabahar was an. stephensi playing a dominant role in transmission of malaria in persian gulf as well in south of iran (26, 27). this species is considered as an endophage and endophile mosquito. sporozoite rate of this species was reported between 0.2 and 1.8% in south of iran (26, 28) other collected species were an. culicifacies, an. fluviatilis and an. sergenti; the first two types are also important as secondary vectors in the south and south-east of the country (3, 12). among secondary vectors, an. culicifacies can be regarded as potential vector, as it is greatly responsible for the epidemy of malaria in sistan and baluchistan province (29). sporozoite rate of this species is varied between 1–4.7% in south malarious areas of iran (3, 12). although it is mainly known as zoophilic anopheles, its anthropophilic index is reported as 16.4% (30). anopheles culicifacies has also a wide distribution in asia and indian subcontinent (31). anopheles fluviatilis, distribution at altitude 50–1100m, along the foothills of the zagros mountains and extension to south and south-east of iran as a secondary vector, its sporozoite rate was reported between 1.4–11% (3, 12). anopheles sergentii is also prevalent in different provinces of iran, acting as secondary vector and responsible for maintaining the parasite reservoir in malarious areas. in afghanistan, totally three species were identified among collected specimens, reported previously in mosquito fauna of this country (32). comprising an. superpictus, an. stephensi and an. hyrcanus, which are all known vectors of malaria (33). anopheles superpictus has a broad geographical distribution in asia, europe and africa considered as a malaria vector in these areas (13, 12). it was collected from badakhshan and kunduz located in northeast of afghanistan as it was reported previously (34). moreover, the only species which was recently reported by ahmad et al. from the badakhshan was an. superpictus (35). although during current study, this species was not found among the collected specimens of chabahar, but it is commonly widespread in iran in altitudes 50– 2000m. sporozoite rate of mentioned species is 0.65–4.7% in iran (36, 3). anopheles stephensi was collected mainly from nangarhar province in human house and mainly were blood-fed. as mentioned, this species is an important vector of human malaria in the middle east and south asia including indopakistan sub-continent (16). the third species in the study area was an. hyrcanus. it is a mem http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 354–362 h hoosh-deghati et al.: composition of anopheles … 360 http://jad.tums.ac.ir published online: september 08, 2017 ber of the hyrcanus group comprising of 30 species, some of them are vectors of malaria, filariasis and arboviruses (37, 38). it is considered as the fauna of bloodsucking mosquitoes of afghanistan (32, 17), also its potential role as malaria vector was reported from france, turkey, greece and afghanistan (32, 39). this species has already been reported in the north and north-east of iran (40), and is suspected to transmit malaria in fooman district located in the north of iran (41). this study presents some limitations that may reflect the article. mosquito collection was performed in a limited time and areas of iran and afghanistan due to the shortage of research time and lack of facilities, so although results are worthy enough for the other basic research, but could not regarded as a representative of a faunistic or eco-epidemiological study. conclusions obtained results provide baseline information for other researchers and it reveals valuable information for designing further studies on vectorial capacity and detection of plasmodium species and the status of insecticide resistance within collected anopheles spp which is undergoing by the authors. furthermore, it could be useful for designing, implementation and evaluation of local and regional evidence-based malaria control program. acknowledgements this work is a part of who/emro grant to ndd. this article has been extracted from the 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http://www.ncbi.nlm.nih.gov/pubmed/?term=pon%c3%a7on%20n%5bauthor%5d&cauthor=true&cauthor_uid=18533455 http://www.ncbi.nlm.nih.gov/pubmed/?term=toty%20c%5bauthor%5d&cauthor=true&cauthor_uid=18533455 http://www.ncbi.nlm.nih.gov/pubmed/?term=kengne%20p%5bauthor%5d&cauthor=true&cauthor_uid=18533455 http://www.ncbi.nlm.nih.gov/pubmed/?term=alten%20b%5bauthor%5d&cauthor=true&cauthor_uid=18533455 http://www.ncbi.nlm.nih.gov/pubmed/?term=fontenille%20d%5bauthor%5d&cauthor=true&cauthor_uid=18533455 http://www.ncbi.nlm.nih.gov/pubmed/18533455 http://www.ncbi.nlm.nih.gov/pubmed/?term=djadid%20nd%5bauthor%5d&cauthor=true&cauthor_uid=19769039 http://www.ncbi.nlm.nih.gov/pubmed/?term=jazayeri%20h%5bauthor%5d&cauthor=true&cauthor_uid=19769039 http://www.ncbi.nlm.nih.gov/pubmed/?term=gholizadeh%20s%5bauthor%5d&cauthor=true&cauthor_uid=19769039 http://www.ncbi.nlm.nih.gov/pubmed/?term=rad%20shp%5bauthor%5d&cauthor=true&cauthor_uid=19769039 http://www.ncbi.nlm.nih.gov/pubmed/?term=zakeri%20s%5bauthor%5d&cauthor=true&cauthor_uid=19769039 http://www.ncbi.nlm.nih.gov/pubmed/19769039 3. iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 13 original article the effectiveness of trypsin modulating oostatic factor (tmof) and combination of tmof with bacillus thuringiensis against aedes aegypti larvae in the laboratory ys lau, * s sulaiman, h othman department of biomedical science, faculty of allied health sciences universiti kebangsaan malaysia jalan raja muda abdul aziz 50300 kuala lumpur, malaysia (received 9 oct 2010; accepted 6 jun 2011) abstract background: trypsin modulating oostatic factor (tmof) terminates trypsin biosynthesis in adult and larval mosquito gut. it will inhibit the growth and development of mosquito larvae feeding on it resulting in death by starvation. the objective of this study is to determine the effective dose of pichia-tmof and the combination of pichia-tmof with bacillus thuringiensis (bt) as larvicide on ae. aegypti larvae. methods: aedes aegypti first-instar larvae were exposed to various doses of pichia-tmof, bt and combination of pichia-tmof and bt. the development of the larvae were observed and recorded daily during the bioassay larval test until the adult emergence. results: the results showed that 400 ppm pichia-tmof and 300 ppm pichia-tmof were able to cause 100% and 67% cumulative mortality on ae. aegypti larvae on 8th day respectively. at 200 ppm, 100 ppm and 50 ppm concentration pichia-tmof showed obvious stunted effect on ae. aegypti larvae. moreover, the combination of 400 ppm pichia-tmof with 0.1 ppm bt showed synergistic effect on ae. aegypti. conclusion: pichia-tmof inhibited trypsin biosynthesis is potential to act in larval gut causing stunted growth and larval development and causing mortality. the combination of pichia-tmof and bt increased the effectiveness in causing larval mortality. keywords: pichia-tmof, bt combination, aedes aegypti larvae introduction mosquitoes are the vectors for the dreadful diseases of mankind. of all the insects that transmit diseases, mosquitoes represent the greatest menace. who has declared the mosquito “public enemy number one” because mosquitoes are responsible for the transmission of various dreadful diseases (who 1996). dengue is the most common and widespread arthropod-borne arboviral infection in the world today. it is estimated that there are at least 100 million cases of dengue fever (df) annually and 500,000 cases of dengue hemorrhagic fever (dhf) which require hospitalization. in malaysia, it has become a major public health problem (hussin 2005). chemical insecticides are the most important components of integrated vector control. however, safe and cost-effective insecticides are rapidly disappearing because of the development of resistance, abandonment of many compounds for reasons of environmental safety, and new registration requirements that are more stringent (rose 2001). when aedes aegypti (l) and culex quinquefasciatus say larvae were fed trypsin modulating oostatic factor (tmof) that was adsorbed onto yeast cells the larvae stopped synthesizing trypsin and stopped growing (borovsky and meola 2004). these results indicate that shutting off trypsin biosynthesis with tmof can be used as a new *corresponding author: prof sallehudin sulaiman, e-mail: salsul@medic.ukm.my iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 14 approach to control larval growth and development, possibly leading to new biorational insecticides, which are desperately needed (zaim and guillet 2002). borovsky reported that the mosquito ovary is a rich source of ‘oostatic hormone’. injections of the hormone into female mosquitoes inhibited yolk deposition and vitellogenin biosynthesis (borovsky 1985). mode of action for tmof is as follows: after application to water, tmof is consumed by mosquito larvae who are aquatic particulate filter feeders. as the yeast is broken down by digestive processes, tmof is released into the insect gut. the tmof protein (a small decapeptide) then passes through the peritropic membrane and midgut wall into the hemolymph. tmof then binds to specific receptors which, once activated, trigger a halt in trypsin like enzyme translation (enzyme biosynthesis) and ultimately transcription (mrna synthesis). the biochemical process of receptor activation is not well understood, though affected insects are unable to produce digestive proteases needed to provide essential amino acids, resulting in starvation (epa 2003). tmof can be degrades by vertebrate digestive protease and technical tmof is not toxic to the non-target organisms examined (deborah 2004). according to sallehudin (1990), bacillus thuringiensis (bt) is not toxic to human and others mammals and it is effective in ae. aegypti control when put them in water container. however, the high cost of bt, need for frequent application and possibility of resistance due to widespread application have severaly compromised its use. so the objective of this study is to evaluate the efficacy of tmof and combination of tmof with bacillus thuringiensis on ae. aegypti larvae in the laboratory. materials and methods the samples used were pichia-trypsin modulating oostatic factor (tmof) (p7) and bacillus thuringiensis (bt) were provided by entogenex industries sdn bhd malaysia and originally developed in the laboratory of professor d borovsky at the university of florida. aedes aegypti larvae was used in the experiment. aedes aegypti was a laboratory strain orginated from the institute for medical research (imr). bioassay the method was slightly modified from protocol for testing pichia-tmof with mosquito larvae by professor d borovsky (2006). mosquito eggs were hatched a day or few hours before the test in the presence of brewer’s yeast. the first instar larvae that just hatched were used for the test. paper cup was filled with distilled water till 200 ml. 20 aedes aegypti larvae were added to each paper cup. stock solutions of 10,000 ppm of pichiatmof and 500 ppm bt were prepared. 20 larvae were added to each container with different amount of pichia-tmof and bt respectively. the doses used to test aedes aegypti larvae were 400 ppm, 300 pm, 200 ppm, 100 ppm and 50 ppm pichia-tmof; 1 ppm, 0.1 ppm and 0.01 ppm bt and combination of every concentration of pichia-tmof with each concentration of bt. each assay was run in duplicate and repeated 3 times for each assay. 400 ppm pichia cells without tmof or baker yeast was used as negative control while bt with baker yeast and without baker yeast were used as positive control. the test was run until all the larvae died or became adults. the larvae were considered dead if they did not wriggle or move after touching them gently with a micropipette tip. the dead larvae were removed from containers daily and no additional yeast was given as food after starting the test. futhermore, the test was run in a laboratory at 27° c and 80% humidity. all the containers were covered with netting. the data were analysed statistically using anova, independant t-test, kruskal wallis and mann-whitney. iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 15 results figure 1 shows the effectiveness of pichiatmof on cumulative mortality of aedes aegypti larvae in 12 days. the graph shows that 400 ppm pichia-tmof increased exponentially on the 4th day and achieved 100% cumulative mortality on 8th day. this shows that 400 ppm pichia-tmof cause starvation of larvae to death. however from statistical analysis using kruskal-wallis,there was significant different between the groups on 12th day χ 2(5, n=18)= 12.552, p< 0.05. analysis of mann-whitney was done and showed that there was a significant different between 400 ppm pichia-tmof and 300 ppm pichia-tmof with 400 ppm baker yeast (p< 0.05). however there was no significant different between 200 ppm, 100 ppm and 50 ppm pichia-tmof with 400 ppm baker yeast (p> 0.05). one ppm bt is used as positive control. figure 2 shows the percentages of development and survival of ae. aegypti larvae treated with pichia-tmof. from the graph 300 ppm pichia-tmof, 200 ppm pichia-tmof, 100 ppm pichia-tmof and 50 ppm pichiatmof show 32%, 75%, 77% and 70% of larvae still survived in larval stage on 12th day. according to the analysis of one way anova, there is significant different between 300 ppm pichia-tmof, 200 ppm pichia-tmof, 100 ppm pichia-tmof and 50 ppm pichia-tmof with 400 ppm baker yeast (p< 0.05). this proved that the pichia-tmof is effective in stunted growth of ae. aegypti larvae. although 1ppm bt used as positive control and show 100% mortality after few hours, in real life there is a lot of other factors in the environment that will affect the effectiveness of bt such as natural food and sunlight. figure 3 shows the cumulative mortality of combination pichia-tmof with 1 ppm bt on ae. aegypti larvae. one ppm bt used as positive control while 400 ppm baker yeast as negative control showing 100% and 8% cumulative mortality respectively. combination of 1 ppm bt with 400 ppm baker yeast and 400 ppm pichia-tmof show rapid mortality than 400 ppm pichia-tmof. pichia-tmof at 8th day caused 100% cumulative mortality of ae. aegypti larvae.also shown that with the addition of bt to tmof will increase the rapidness of mortality of ae. aegypti larvae. figure 4 shows the cumulative mortality of combination pichia-tmof with 0.1 ppm bt on ae. aegypti larvae. 0.1 ppm bt act as positive control which show 98% mortality after 24 hours and achieved 100% on day 2. from the graph, it is indicated that the combination of 0.1 ppm bt with 400 ppm baker yeast shows higher percentage of cumulative mortality than combination of 0.1 ppm bt with pichia-tmof in the first 3 days. however starting day 4 the percentage cumulative mortality of combination of 0.1 ppm bt with pichia-tmof were higher than 0.1 ppm bt with 400 ppm baker yeast and achieved 98% and 75% cumulative mortality on day 12 respectively. the photo shows the different between development of larvae with the treatment of 400 ppm pichia-tmof and with 400 ppm baker yeast. larvae with the treatment were dead on second instart while additional of 400 ppm baker yeast shows fourth instar on day 7 and be able to become adult. table 1 shows the development and survival of ae. aegypti with the treatment of combination of pichia-tmof and 0.01 ppm bt on day 12.at day 12 all the larvae being given yeast as food emerged as adults. it indicated that 65%, 75% and 60% of larvae still survived on day 12 with the dose of combination 0.01ppm bt with 200 ppm pichia-tmof, 100 ppm pichia-tmof and 50 ppm pichiatmof respectively. when 0.01 ppm bt is added to a higher concentration tmof (400 ppm) larval survival was low at 5% and adult survival 7% respectively.this shows that with the addition of bt do not affect the effectiveness of pichia-tmof to stunt the ae. aegypti larvae. iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 16 fig. 1. line graph shows effectiveness of pichia-tmof on aedes aegypti by percentage of cumulative mortality (a: p< 0.05 compared to control, b: p>0.05 compared to control). fig. 2. bar graph show percentages of development and survival of aedes aegypti with treatment and without treatment (a: p< 0.05 compared with control,nc: negative control, pc: positive control). fig. 3. line graph show effectiveness of combination of pichia-tmof and 1 ppm bt on aedes aegypti by percentages of cumulative mortality iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 17 fig. 4. line graph show effectiveness of combination of pichia-tmof with 0.1 ppm bt by percentages of cumulative mortality (a: p> 0.05 compared between group, pc: positive control,nc: negative control). fig. 5. shows the cumulative mortality of combination pichia-tmof with 0.01 ppm bt and pichia-tmof on ae. aegypti larvae on day 12. according to independent t-test, there was no difference between 400 ppm pichia-tmof and combination of 400 ppm pichia-tmof with 0.01 ppm bt (p> 0.05), 300 ppm pichia-tmof and combination of 300 ppm pichia-tmof with 0.01 ppm bt (p> 0.05), 200 ppm pichia-tmof and combination of 200 ppm pichia-tmof with 0.01 ppm bt (p> 0.05), 100 ppm pichia-tmof and combination of 100 ppm pichia-tmof with 0.01 ppm bt (p> 0.05) and also between 50 ppm pichia-tmof and combination of 50 ppm pichia-tmof with 0.01 ppm bt (p>0.05). fig . 6. effectiveness of pichia-tmof and combination of 0.01 ppm bt with pichia tmof on aedes aegypti on day-12 (min ± sem) control (4th instar) treatment with 400 ppm tmof (2nd instar) iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 18 table 1. percentage of larval survival/adult survival after treatment with pichia-tmof on the 12th day treatment larval survival (%) adults survived (%) 0.01 ppm bt plus 400 ppm yeast 0 70±6 0.01 ppm bt 2±2 0 0.01 ppm bt plus 400 ppm tmof 5±3 7±2 0.01 ppm bt plus 300 ppm tmof 35±15 2±2 0.01 ppm bt plus 200 ppm tmof 65±14 2±2 0.01 ppm bt plus 100 ppm tmof 75±12 0 0.01 ppm bt plus 50 ppm tmof 60±10 0 discussion in this study pichia-tmof was able to stunt the aedes aegypti larvae. at 400 ppm pichia-tmof was able to cause 100% cumulative mortality of ae. aegypti larvae within 8 days which maintain in second instar. three hundred ppm pichia-tmof caused 67% cumulative mortality of ae. aegypti in 12 days. the larvicidal effect decreased when the doses of pichia-tmof decreased but it still able to give significant stunted effect on ae. aegypti. this study support the research of prof. borovsky which shows that first instar ae. aegypti when fed with tmof organic analogues in microtiter plates in the presence of brewer’s yeast caused larval mortality within 5-6 days of the feeding (borovsky 2007). according to borovsky et al. (2010) cry toxins and tmof are synergists to ae. aegypti larvae when jointly fed or expressed in recombinant pichia pastoris. from the study, bt with the addition of baker yeast decreased the effect of bt alone and it was shown when high concentration of baker yeast was present. thus, in the present study the combination of pichia-tmof with bt on aedes aegypti caused the cumulative mortality of larvae decreased at first then achieve better effect compared to combination of baker yeast with bt. this is probably due to the effect of the pichia-tmof which prevent the biosyntesis of trypsin and decreased the rate of digesting the crystal of bt and caused the effect of bt became slower. however, after sometimes when crystal is digested and toxin released increased the percentages of cumulative mortality. combination of 0.1 ppm bt with 400 ppm pichia-tmof gave the desired result on ae. aegypti larvae because with the lower dose of bt able to give 100% cumulative mortality on 8th day. in this case it shows synergistic effect because the effect is greater than 400ppm pichia-tmof alone and combination of 0.1 ppm bt with 400 ppm baker yeast. the study shows that the combination of 0.01 ppm bt with pichia-tmof is too low to give any significant effect compared with pichia-tmof alone. borovsky (2008) and borovsky et al. (2009) studied the posiblle synergism between tmof and bt toxins and found that tmof enhances the effect of bt toxins making the toxins more effective at low concentratins or in the presence of organic materials that negate their activity. in conclusion, combination of lower bt concentration with pichia-tmof is able to give better larvacidal effect on ae. aegypti 1st instar larvae compared to bt with added food. combination of bt with pichia-tmof could eliminate the development of resistance in aedes mosquitoes and strengthen the dengue vector control programme. acknowledgments we would like to thank entogenex industry sdn bhd for financing the study through research grant nn001-2008. also we wish to thank the department of biomedical science, faculty of alied health sciences for providing research facilities. iran j arthropod-borne dis, 2011, 5(1): 13–19 ys lau et al.: the effectiveness of trypsin … 19 references borovsky d (1985) isolation and characterization of highly purified mosquito oostatic hormone. arch insect biochem physiol. 2: 333–349. borovsky d (1988) oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and egg development in mosquitoes. arch insect biochem physiol. 7: 187–210. borovsky d (2003) trypsin-modulating oostatic factor: a potential new larvicide for mosquito control. university of florida ifas, florida medical entomology laboratory, 200, 9th street. se vero beach, fl 332962, usa. borovsky d, nauen r (2007) biological and biochemical effects of organo-synthetic analogues of trypsin modulating oostatic factor (tmof) on aedes aegypti, heliothis virescens and plutella xylostella. pestycydy. 3–4: 17–26. borovsky d, meola sm (2004). biochemical and cytoimmunological evidence for the control of aedes aegypti larval trypsin with aea-tmof. arch insect biochem physiol.55: 124–139. borovsky d (2008) a new biopesticide against mosquitoes and agricultural pest insects. kualalumpur convention centre conference and exhibition 7–9 october 2008. borovsky d, laeremans a, theunis c, bertier l, khasdan v, ben-dov e, zaritsky a (2009) does tmof enhance the effect of bt toxins on larval aedes aegypti? vith arthropods, 21–26 june 2009, ochotnica dolna, poland. borovsky d, khasdan v, nauwelaers s, theunis c, bertieor l, bendov e, zaritsky a (2010) synergy between aedes aegypti trypsin modulating oostatic factor and δ-endotoxins. the open toxin j. 3: 116–125. deborah mt, hugh py, frank, we, allen wo, gerald al, ernest h, michel rr (2004) pest biochem physiol. 80: 131– 142. epa (2004) biopesticide registration action document trypsin modulating oostatic factor (tmof). hussin n, jaafar j, naing nn, mat ha, muhamad ah, mamat mn (2005) a review of dengue fever in kota bharu, kelantan, malaysia during the years 1998–2003. southeast asian j trop med public health. 36(5): 1179–1186. gagliardi j, kough j (2003) review of submitted studies, background material and waiver requests to support registration of the manufacturing use product skeetercide containing heat-killed pichia pastoris yeast with integral trypsin modulating oostatic factor (tmof), u.s. epa. rose ri (2001) pesticides and public health: integrated methods of mosquito management. emer. infect dis. 7: 17–23. sallehudin s (1990) entomologi perubatan. bangi: penerbit universiti kebangsaan malaysia. who (1996) report of who informal consultation on the evaluation and testing insecticides. ctd/who pes/ic/96. zaim m, guillet p (2002) alternative insecticides: an urgent need. trends parasitol. 18: 161–163. j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 172 http://jad.tums.ac.ir published online: may 27, 2017 review article asymptomatic malaria and its challenges in the malaria elimination program in iran: a systematic review gholmreza hassanpour 1,2, mehdi mohebali 1,2, hojjat zeraati 3, ahmad raeisi 1, *hossein keshavarz 1,2 1center for research of endemic parasites of iran, tehran university of medical sciences, tehran, iran 2department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 3department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran (received 30 apr 2016; accepted 21 may 2016) abstract background: the objective of this study was to find an appropriate approach to asymptomatic malaria in elimination setting through a systematic review. methods: a broad search was conducted to find articles with the words ‘malaria’ in their titles and ‘asymptomatic’ or ‘submicroscopic’ in their texts, irrespective of the type of study conducted. the cochrane, medline/pub med, and scopus databases, as well as google scholar were systematically searched for english articles and reports and iran’s databasesiran medex, sid and magiran were searched for persian reports and articles, with no time limitation. the study was qualitatively summarized if it contained precise information on the role of asymptomatic malaria in the elimination phase. results: six articles were selected from the initial 2645 articles. the results all re-emphasize the significance of asymptomatic malaria in the elimination phase, and emphasize the significance of diagnostic tests of higher sensitivity to locate these patients and perform interventions to reduce the asymptomatic parasitic reservoirs particularly in regions of low transmission. however, we may infer from the results that the current evidence cannot yet specify an accurate strategy on the role of asymptomatic malaria in the elimination phase. conclusion: to eliminate malaria, alongside vector control, and treatment of symptomatic and asymptomatic patients, active and inactive methods of case detection need to be employed. the precise monitoring of asymptomatic individuals and submicroscopic cases of malaria through molecular assays and valid serological methods, especially in regions where seasonal and low transmission exists can be very helpful at this phase. keywords: malaria, asymptomatic infection, elimination introduction across the globe, 3.3 billion people are at risk of malaria. in 2013, 198 million people were affected with the disease, of which 584000 people died (world health organization 2014). a 50% reduction in the number of malaria cases by 2015 was announced as a millennium developmental goal. currently, 99 countries are malaria-free and 34 countries are in the phase of elimination (gueye et al. 2013). the countries that are in the elimination phase have put different strategies on their agendas, such as general screening and focusing on specific foci (world health organization 2013). in such countries, at the same time that transmission reduces, the major share of transmission will take place through collective and focal transmission (ministry of health and mdical education 2012). probably, the infected asymptomatic individual can become a source of parasitic transmission to healthy individuals under favorable setting, considered a serious challenge to *corresponding author: dr hossein keshavarz, e-mail: hkeshavarz@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 173 http://jad.tums.ac.ir published online: may 27, 2017 malaria control and elimination worldwide (lin et al. 2014). the popular belief is that in low transmission settings the proportion of asymptomatic individuals is less than that in areas of greater transmission severity. however, community-based studies have shown that, although an increased transmission state is associated with an increase in the reservoir share, but even in low transmission areas the asymptomatic cases make up for 60% of the infected population (sturrock et al. 2013). therefore, it seems that in low transmission settings the malaria infection is very likely to be asymptomatic (who 2013). while symptomatic cases are removed from the disease reservoir faster because of treatment. under such circumstances, communities appear to need interventions that address beyond symptoms (sturrock et al. 2013). under circumstances in which many successful and unsuccessful attempts at malaria elimination have been made worldwide, this review was designed to find an appropriate strategy for the detection and elimination of asymptomatic malaria. the summarization of these experiences in the form of a systematic review can effectively help clarify these experiences and in turn help national policymakers who are trying to eliminate malaria in iran materials and methods study design this study has systematically searched all the studies, reports and documentations related to malaria and the role of asymptomatic malaria in the elimination phase -with no time limitationup to july 2015. inclusion criteria in this systematic review, all the articles that had questioned or responded to the main question of the study, i.e. ‘the role and significance of asymptomatic malaria cases in the pre-elimination and elimination phases of malaria’ irrespective of the study designwere selected. search strategy the cochrane, medline/pubmed, and scopus databases, as well as google scholar were systematically searched for english articles and reports and iran’s databases -iranmedex, sid and magiran were searched for persian reports and articles, with no time limitation. we performed a search of high sensitivity to find the relevant articles. in short, “(malaria in title) and (asymptomatic or sub microscopic or sub-microscopic or low parasite) were searched in various databases. selecting studies, quality assessment and data extraction all the article titles were first entered into reference manager software (endnote). after omitting the duplicate cases, they were prepared for the initial screening. in the first stage, the article titles were reviewed and those articles that were obviously irrelevant to the study objective were removed from the primary databank. in the next stage, two persons studied the titles and abstracts and the articles that were selected by both persons were finalized. if only one person chose an article, then that article was discussed in a meeting and the decision to include or exclude it in the study was made thereafter. at this stage, the selected articles’ full texts were obtained and those articles that had specifically answered the following two questions were selected as candidates for inclusion. the two questions were, “has the article discussed the topic of asymptomatic and/or submicroscopic malaria? have these topics been applicable in the elimination phase of malaria?” if the answers to these questions were ‘yes’ then the article would be included in the study. the selected studies were thoroughly reviewed by two persons and their opinions were shared with each other in a joint meeting. if the overall impression of the applicability of the study results in malaria elimination was the same then that article has been selected. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 174 http://jad.tums.ac.ir published online: may 27, 2017 results the systematic review yielded 2645 articles in all (medline/pubmed: 718, magiran: 210, iranmedex: 226, sid: 74, web of science: 668, scopus: 748, other databases such as emb reviews and cochrane: 1). after omitting the duplicate cases 1322 articles were sorted for title review. upon reviewing the article titles, 143 articles, and upon reviewing the abstracts 61 articles remained in the databank, respectively. the full texts of these 61 articles were obtained and eventually, upon studying those, only six articles were finalized (fig. 1, table 1). the most important reason for exclusion in the final stage was lack of a direct relation and an applicable result in malaria elimination settings. in summary, the selected articles emphasized the significance of the role of asymptomatic malaria in the elimination phase. they also emphasized the need for diagnostic tests of higher sensitivity to detect these cases and the performance of interventions aimed at reducing parasite reservoirs -with an emphasis on asymptomatic cases. lind blade et al. published an article on the significance of asymptomatic malaria in 2013. they stated that although malaria control has had many positive effects in the world, there is not much certainty regarding the current interventions aimed at eliminating malaria in many endemic countries. in this review, a great share of the infected cases of malaria has been attributed to asymptomatic individuals who were not seeking treatment. these circumstances become even more challenging in regions where plasmodium vivax contributes more to the burden of disease because of its dormant liver stage. they conclude by stating the important role asymptomatic malaria has to play in its transmission and that in order to eliminate the disease, interventions must target the reduction of parasite reservoirs in both low and high transmission setting (sturrock et al. 2013). “targeting asymptomatic malaria infections: active surveillance in control and elimination” -another study published in 2013 by sturrock et al. underscores that scale up malaria control programs in various countries have reduced the focal transmission of disease. in a smaller scale, each focus consists of hotspots such as households and groups of households with higher malaria transmission and consistent parasite environments throughout the year. this infection is clustered in specific populations (hotspots) that have high-risk demographic characteristics. the authors believe that the identification of these groups -both geographically and demographicallyis an important strategy toward reducing local parasite reservoirs and disrupting the transmission chain in low transmission or elimination setting (ministry of health and medical education 2012) the current approaches toward malaria elimination are based on the recommendations of the global malaria eradication program of 1960. however, many countries face multiple challenges in the fourth phase of elimination, such as, the imported cases of infection, which apparently requires a regional approach. to eliminate malaria, focus needs to be laid on the detection and elimination of infectious foci through active and inactive methods of patient detection. in this approach, alongside vector control, both symptomatic and asymptomatic patients should be treated correctly. since imported cases can be a source of disease transmission, malaria elimination programs should have precise plans for these cases as well (moonen et al. 2010). in countries that have gone through the control phase, to ensure malaria elimination and monitor transmission reduction, diagnostic tools applicable in the field are necessary to detect asymptomatic and low-density parasitic cases for extensive screening and treatment (lo et al. 2015). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 175 http://jad.tums.ac.ir published online: may 27, 2017 in an article lin et al. (2014) emphasized that malaria elimination can be achieved by eliminating the infected human reservoirs (including asymptomatic cases). a better understanding of the role of submicroscopic cases as a source of transmission can greatly help to adopt an active surveillance approach. according to this article, the results of screening have shown the capability of rdts in detecting asymptomatic cases at a threshold of 200 parasites per microliter, which is a submicroscopic threshold. therefore, it seems that in order to eliminate, we need newer methods of higher sensitivity to be able to detect potential transmission agents at any scale (world health organization 2013). molecular diagnostic tests have shown that even in low endemic regions, asymptomatic malaria reservoirs are more than they were believed to be. individuals that can be diagnosed with microscopic or submicroscopic methods have submicroscopic gametocyte density. bousema et al. (2014) believed that, in order to eliminate malaria, interventions should target both symptomatic and asymptomatic infections. in regions where there is seasonal and low transmission of malaria, even a small percentage of infected persons are sufficient to re-start malaria transmission. depending on various conditions, the transmission of infection from mosquito to human is different, however, as a barrier to malaria elimination, asymptomatic and submicroscopic cases should be monitored through appropriate molecular methods (lindblade et al. 2013). fig. 1. flow chart demonstrating database searches, identification, screening and selection of included studies http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 176 http://jad.tums.ac.ir published online: may 27, 2017 table 1. characteristics of included studies challenges/ acceptability recommended policy aim type of study year author can molecular methods employed to detect asymptomatic individuals help reduce transmission? interventions should target both symptomatic and asymptomatic individuals, depending on the circumstances asymptomatic malaria and public health relevance opinion 2014 bousema et al. lack of a unified definition of asymptomatic malaria by choosing efficient interventions for asymptomatic patients, parasitic reservoirs should be destroyed. the role of asymptomatic malaria in malaria transmission and interventions to achieve malaria elimination review 2013 lindblade et al. absence of scientific evidence supporting or opposing the role of submicroscopic malaria in transmission advising molecular diagnostic tests in longitudinal studies and exploring the role of reservoirs in malaria transmission the role of submicroscopic parasitemia in malaria transmission review 2014 lin et al. there is no evidence of the cost-effectiveness of these interventions in the long run racd appropriate to low transmission settings and pacd appropriate to low/medium transmission potential role of acd in malaria control and elimination policy forums 2013 sturrock et al. difficulties are observed in countries with little resources; also the companies that can tend to these priorities are small companies that need support the need for diagnostic tests with little parasitic density in asymptomatic persons to propose a research agenda for the tools required for malaria eradication review 2011 malera consultive group there is no the global campaign, national elimination cannot succeed with continued importation. establishment of a surveillance system connected to rapid response surveillance over time to review the activities needed to achieve and maintain malaria elimination opinion 2010 moonen et al. discussion we found only a few studies with goals completely relevant to our research question. none of the studies had discussed with certainty the role of asymptomatic malaria and the correct approach toward these cases, particularly in malaria elimination programs. the evidence suggested that asymptomatic malaria probably has a major role to play in malaria transmission. communities need tools of higher sensitivity to detect asymptomatic and low-parasitic density cases. malaria elimination requires interventions aimed at reducing parasite reservoirs, both in highand low transmission setting. in this systematic review, we tried to retrieve all the relevant data. however, most of the selected articles including review articles were expert commentaries and recommendations. however, the scientific community can not cite such evidence with great certainty when it comes to the evidence pyramid. the lack of interventions and prospective cohort studies relevant to the research question on one hand, and the high significance of climate, parasitic species and vector differences on the other, does not allow a conclusion to be reached with certainty on the strategy of dealing with asymptomatic patients -particularly at the elimination phase of the disease. although experts have emphasized the role of asymptomatic carrier in various studies, but the interesting point is the absence of a unified definition of asymptomatic malaria (moonen et al. 2010, hassanpour et al. 2011). most studies have described asymptomatic malaria by the presence of sexual or asexual multiplication parasites and/or absence of acute clinical symptoms (usually fever) during a spehttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 177 http://jad.tums.ac.ir published online: may 27, 2017 cific period. some studies have focused on the parasite density threshold, and have considered febrile patients with higher than predetermined threshold parasite densities symptomatic. various studies have set different criteria and given different definitions for being symptomatic, such as, different followup periods for becoming symptomatic, taking into account one symptom (mostly fever) and/or all clinical symptoms (sturrock et al. 2013). perhaps the first shortcoming in adopting an operational approach toward asymptomatic malaria is the lack of a unified definition of asymptomatic malaria in different references. asymptomatic malaria can become symptomatic due to relative immunity, frequency of exposure to malaria infection, increased age (independent of repeated exposure), and cross-protection resulting from relative immunity from exposure to multi clonal infections. among various factors, age and previous exposure are the most important determinant factors of immunity in individuals (sturrock et al. 2013). moreover, sometimes, symptomatic persons who have not been treated enter the chronic phase of the disease and reach a parasite level lower than threshold -which is not observable under the microscope. another important group are those persons who have recently received treatment or have been diagnosed early (before becoming symptomatic) following a diagnostic test such as pcr, and/or have a parasite density level that has never become observable under the microscope (lindblade et al. 2013). hence, we may conclude that asymptomatic malaria includes many different types of patients. in addition to these groups, we must keep in mind that some asymptomatic individuals are patients who have received treatment in the past, those who have probably appeared asymptomatic because of incomplete treatment or relative drug resistance. this matter requires meticulous studies to identify the genotype of the parasite, in terms of whether it is a residual infection or a new one. nevertheless, in addition to the aforementioned complexities, associated infections with parasites such as ascaris lumbricoides and schistosomia hematobium too can complicate the detection of these individuals because of cross reactivity (sturrock et al. 2013). evidence suggests that all species of malaria can result in asymptomatic disease. however, since p. falciparum and p. vivax are more prevalent, most asymptomatic patients are infected with these two plasmodium species. the p. vivax species create immunity more rapidly as compared to falciparum species, hence raising the possibility of controlling the parasite density as well. although there are fewer p. vivax studies than there are p. falciparum ones, but it seems that in this species too, an increase in the number of gametes raises the possibility of the insect’s infectivity. various studies in different settings and with different diagnostic tools have shown entirely variable results, such that, we cannot draw an association between the asymptomatic cases of malaria and its different species (sturrock et al. 2013). another challenge in approaching asymptomatic malaria is the existent knowledge of the true role of the affected individuals in disease transmission. the uncertainty surrounding the role of submicroscopic malaria in disease transmission can be dangerous from two aspects. on one hand, asymptomatic malaria is considered a serious challenge in malaria control and elimination in many parts of the world, as it can under favorable conditions act as a reservoir and cause parasite transmission. on the other hand, if these cases are not treated effectively then they can provoke the global drug resistance problem (world health organzation 2013, lin et al. 2014). many believe that some of these individuals eventually become symptomatic. however, there is no certainty as to whether these individuals have been affected with a http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 178 http://jad.tums.ac.ir published online: may 27, 2017 new febrile illness or that re-infection has taken place (sturrock et al. 2013). in addition to all the aforementioned diagnostic issues and reasons of presentation of these cases, the important question is the infectivity of asymptomatic individuals. the general belief is that gametocyte density is the most important determinant factor of infection transmission from an infected individual to an anopheles vector (diagnoses. 2011). so, the lower the gametocyte carriage density, the less significant the issue of infectivity. theoretically speaking, the possibility of transmission exists with even a single gametocyte per microliter. however, the current evidence does not point to a robust relationship between gametocyte density and asymptomatic individuals’ infectivity. presently, it may be said that the gametocyte is an incomplete surrogate for transmission. the evidence on gametocyte carriage among asymptomatic cases is also insufficient. direct skin test feeding, observance of oocysts in the anopheles midgut, and membrane feeding apparatus studies may to some extent answer these questions. in addition to gametocyte density, other factors such as the vector and host also affect this transmission (sturrock et al. 2013, world health organzation 2013). under the most favorable conditions and in standard laboratories, giemsa-stained blood film optical microscopy can detect more than 10 parasites per microliter, while, ordinarily, approximately 100 parasites per microliter should be present in 5 microliters of blood. methods such as qpcr have the ability to detect almost double the microscopic method, considering the groups at risk and the community in which the examination is being done (lindblade et al. 2013). depending on the diagnostic method, be it microscopy, rdt or pcr, the detection of asymptomatic cases has yielded completely different results in different communities ( haghi et al. 2012, sturrock et al. 2013, bousema et al. 2014). the most important applied difference be tween control and elimination programs is the focus on detection of and intervention on symptomatic and asymptomatic infections (moonen et al. 2010). as the prevalence of the disease falls, activities should focus on subclinical and low-density infections and reservoirs present in specific geographic and demographic areas. no doubt, to achieve the goals of disease elimination and eradication, we must be able to detect malaria infection with applicable diagnostic tests, something that is not possible with the current microscopic methods, where pcr is an expensive technique and not practical for the field (laishram et al. 2012). in low transmission or malaria elimination conditions, transmission takes place focally. therefore, the approach of targeted detection of these infection clusters becomes extremely important for reducing local reservoirs and disrupting the transmission chain (ministry of health and education 2012). submicroscopic infections in high transmission regions (where parasite prevalence in the community exceeds 75% microscopically) are estimated at 20%, and at 70–80% in low transmission setting (where prevalence is less than 10%). evidently, submicroscopic prevalence has increased in areas where control has been successful in reducing transmission (lindblade et al. 2013). in low transmission setting, the share of asymptomatic cases in transmitting malaria is significant. in regions of seasonal transmission, they can be a source of infection for the new generations of mosquitoes following rain (sturrock et al. 2013). active case detection is the intervention recommended for low malaria transmission settings, although there is very little evidence on its efficiency. the most important factors affecting the efficiency of this method are its low sensitivity, inability to detect very lowdensity infection, continuous importation of parasites, as well as insufficient coverage of the population (ministry of health and medical education 2012) . the most important public health interven http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 179 http://jad.tums.ac.ir published online: may 27, 2017 tions aimed at asymptomatic parasitemia are ‘drug mediated strategies’ aimed at asymptomatic parasite reservoirs: 1mass drug administration (mda) in a geographic region irrespective of infection and symptoms, 2 mass screening and treatment (msat) or aggressive active case detection, and 3intermittent preventive therapy -repeated treatment of high-risk groups. these approaches can be adopted at smaller (modified) scales such as hotspots too. mathematical models have shown a 15–20% reduction in transmission through the achievement of these goals (sturrock et al. 2013). to eliminate malaria, in addition to underscoring the significance of the highest public authorities’ commitment to the program, the world health organization has made the following recommendations: programs for detecting and treating patients, disrupting malaria transmission, identification of local foci, establishment of an active surveillance system, preventing re-establishment of transmission, and regional cooperation particularly with neighboring countries to prevent the entry of infection into the country. no doubt, the chances of eliminating malaria reduce if there are efficient mosquito vectors, special demographic conditions such as suburbanization, low hygiene levels, unsustainable borders and illegal migrations (bousema et al. 2012). as a country that has reduced approximately 90% of its cases, iran is faced with the challenge of disease elimination due to multiple reasons. an important challenge is the issue of cross border importation from afghanistan and pakistan. 1.6% (7/446) asymptomatic afghani immigrants are asymptomatic-infected who can be the potential source of parasites transmission (nateghipour et al. 2011). based on statistics, over 20% of the treated cases in iran are patients from pakistan, a country where there are 4.5 million reported cases of malaria, and from which the entries and exits into and from the eastern borders are not meticu lously controlled. moreover, in iran, different vectors behave differently, which is another challenge of the elimination phase (hanafi-bojd et al. 2011). the resistance of the most important vector ie anopheles stephensi to some insecticides, especially pyrethroids, (vatandoost and hanafibojd 2012), as well as the drug resistance of p. falciparum to chloroquine and some other drugs strongly necessitates an accurate monitoring to enable the achievement of the elimination program’s goals (tietje et al. 2014). conclusions detection of high-risk foci either it geographically or demographically, is an important strategy for reducing local parasite reservoirs and disrupting the transmission chain. active and passive methods of case detection must be used to achieve the goal of malaria elimination. in this approach, both groups of clinically symptomatic and asymptomatic individuals need to be correctly diagnosed and treated, at the same time that vector control is under way. imported malaria cases are a potential source of transmission. thus, targeting symptomatic and asymptomatic infections particularly in regions of seasonal and low transmission, keeping in mind that even a small percentage of infected persons are sufficient to re-start the malaria transmission, demands the accurate monitoring of asymptomatic and submicroscopic cases through highly valid molecular and/or serological methods. iran is a country with a low burden and limited transmission of malaria, wherein the elimination of local malaria transmission from p. falciparum and the complete elimination of malaria have been targeted for 2015 and 2025, respectively. most cases of disease however, result from p. vivax, which has special biological characteristics such as, the latent liver stage hypnozoites and the faster immunity created against it -compared to p. falhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 180 http://jad.tums.ac.ir published online: may 27, 2017 ciparum, thus necessitating the need to take into account other considerations as well (lindblade et al. 2013). conducting precise longitudinal follow-up studies on asymptomatic individuals, to examine the possibility of their being gametocytemic, and, to study the period of infection in these individuals in low transmission settings can yield more accurate data for correct planning by health policymakers. acknowledgements this study was approved and financially supported by tehran university of medical sciences (tums) (project no: 92-01-16022032), and it is also the thesis of mr gholamreza hassanpour in partial fulfillment for the award of ph.d by research degree in medical parasitology from the center for research of endemic parasites of iran (crepi), tehran university of medical sciences, tehran, iran. references bousema t, dinglasan rr, morlais i, gouagna lc, van warmerdam t, awonoambene ph, bonnet s, diallo m, coulibaly m, tchuinkam t, mulder b (2012) mosquito feeding assays to determine the infectiousness of naturally infected plasmodium falciparum gametocyte carriers. plos one. 7(8): e42821. bousema t, okell l, felger i, drakeley c (2014) asymptomatic malaria infections: detectability, transmissibility and public health relevance. nature rev microbiol. 12(12): 833–840. diagnoses cg (2011) a research agenda for malaria eradication: diagnoses and diagnostics. plos med. 8(1): e1000396. gueye cs, sanders kc, galappaththy gn, rundi c, tobgay t, sovannaroth s, gao q, surya a, thakur gd, baquilod m, lee wj (2013) active case detection for malaria elimination: a survey among asia pacific countries. malar j. 12: 358. haghi am, khoramizade mr, nateghpour m, mohebali m, edrissian gh, eshraghian mr, sepehrizadeh z (2012) a recombinant plasmodium vivax apical membrane antigen-1 to detect human infection in iran. korean j parasitol. 50(1): 15–21. hanafi-bojd aa, azari-hamidian s, vatandoost h, charrahy z (2011) spatiotemporal distribution of malaria vectors (diptera: culicidae) across different climatic zones of iran. asian pac j trop med. 4(6): 498–504. hassanpour gh r, mohebali m, raeisi a, abolghasemi h, zeraati h, alipour m, azizi e, keshavarz h (2011) detection of malaria infection in blood transfusion: a comparative study among real-time pcr, rapid diagnostic test and microscopy. parasitol res. 108(6): 1519–1523. laishram dd, sutton pl, nanda n, sharma vl, sobti rc, carlton jm, joshi h (2012) the complexities of malaria disease manifestations with a focus on asymptomatic malaria. malar j. 11: 29. lin jt, saunders dl, meshnick sr (2014) the role of submicroscopic parasitemia in malaria transmission: what is the evidence? trends parasitol. 30(4): 183– 190. lindblade ka, steinhardt l, samuels a, kachur sp, slutsker l (2013) the silent threat: asymptomatic parasitemia and malaria transmission. expert rev anti infect ther. 11(6): 623–639. lo e, zhou g, oo w, afrane y, githeko a, yan g (2015) low parasitemia in submicroscopic infections significantly impacts malaria diagnostic sensitivity in the highlands of western kenya. plos one. 10(3): e0121763. ministry of health and medical education (2012) malaria elimination program. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2017, 11(2): 172–181 gh hassanpour et al.: asymptomatic malaria … 181 http://jad.tums.ac.ir published online: may 27, 2017 prospective of iran 2025. ministry of health publ., tehran. moonen b, cohen jm, snow rw, slutsker l, drakeley c, smith dl, abeyasinghe rr, rodriguez mh, maharaj r, tanner m, targett g (2010) operational strategies to achieve and maintain malaria elimination. lancet. 376(9752): 1592–1603. nateghpour m, akbarzadeh k, farivar l, amiri a (2011) detection of asymptomatic malaria infection among the afghani immigrant population in iranshahr district of southeastern iran. bull soc path exot. 104(4): 321–323. sturrock hj, hsiang ms, cohen jm, smith dl, greenhouse b, bousema t, gosling rd (2013) targeting asymptomatic malaria infections: active surveillance in control and elimination. plos med. 10(6): e1001467. tietje k, hawkins k, clerk c, ebels k, mcgray s, crudder c, okell l, labarre p (2014) the essential role of infection detection technologies for malaria elimination and eradication. trends parasitol. 30(5): 259–266. vatandoost h, hanafi-bojd aa (2012) indication of pyrethroid resistance in the main malaria vector, anopheles stephensi from iran. asian pac j trop med. 5(9): 722–726. world health organization (2007) malaria elimination: a field manual for low and moderate endemic countries. world health organization (2014) world malaria report 2014. who publ, geneva. world health organization (2013) eliminating malaria in iran. available at: http://iacld.ir/dl/elm/eliminatingmalar iainiran.pdf zoghi s, mehrizi aa, raeisi a, haghdoost aa, turki h, safari r, kahanali aa, zakeri s (2012) survey for asymptomatic malaria cases in low transmission settings of iran under elimination programme. malar j. 11: 126. http://jad.tums.ac.ir/ http://iacld.ir/dl/elm/eliminatingmalariainiran.pdf http://iacld.ir/dl/elm/eliminatingmalariainiran.pdf j arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 145 http://jad.tums.ac.ir published online: june 24, 2019 original article molecular analysis of aquaglyceroporin 1 gene in non-healing clinical isolates obtained from patients with cutaneous leishmaniasis from central of iran yasaman alijani1,2; saeedeh sadat hosseini2; salman ahmadian1,2; sonia boughattas3; *gilda eslami1,2; shadi naderian4; vahid ajamein1,2 1research center for food safety and health, shahid sadoughi university of medical sciences and health services, yazd, iran 2department of parasitology and mycology, school of medicine, shahid sadoughi university of medical sciences and health services, yazd, iran 3biomedical research center, qatar university, doha, qatar 4department of statistics and epidemiology, school of public health, shahid sadoughi university of medical sciences and health services, yazd, iran (received 15 feb 2018; accepted 22 apr 2019) abstract background: regarding the antimonial-resistant of leishmania spp., understanding of related mechanism is necessary. one of the most important involved molecules is aquaglyceropin1 (aqp1). the aim of this study was molecular analysis of aqp1 gene from antimonial-resistant clinical isolates and its expression. methods: overall, 150 patients with cutaneous leishmaniasis referring to the reference laboratories of yazd and varzaneh,, located 105km southeast of isfahan and 240km away from yazd, were assessed from jun 2015 to dec 2017. after sampling, staining was done and evaluated for leishman by microscope. samples were collected in rnalater solution for gene expression analysis in non-healing isolates. dna extraction was performed from each slide with leishman body. all patients with l. major isolates detected by its1-pcr-rflp were followed for finding the resistant isolates, consequence of molecular characterization of aqp1 using pcr-rflp. gene expression of aqp1 from all resistant isolates was assessed in comparison with the one in a sensitive isolate. statistical analysis was done using spss. the significance level was considered ≤0.05. results: five isolates were detected as antimonial resistant. molecular detection and identification were appeared that all were l. major. the molecular characterization of aqp1 showed g562a mutation. gene expression of aqp1 in resistant isolates showed 1.67 fold higher than the sensitive isolate. conclusion: we reported a new point mutation of g562a in aqp1 gene involved in molecular mechanism in resistant isolates. keywords: leishmaniasis; cutaneous; drug rsesistance; antimony introduction leishmaniasis, caused by leishmania spp., is one of the most important parasitic diseases which occurs in various forms including cutaneous (cl), muco-cutaneous, disseminated cutaneous, and visceral. cl is resulted from l. major and l. tropica in the old world. the first line of treatment for cl is pentavalent antimony [sb(v)] such as sodium stibogluconate (pentostam®) and meglumine antimonite (glu cantime®) (1). one of the important clinical concerns for the treatment of various infectious diseases especially cl is drug resistance. resistance to antimonial drugs as the first line of treatment has been reported in leishmaniasis in several countries (2-4). one of the important molecules involving antimonial resistance is aquaglyceroporin 1. this protein that encoded from aqp1 gene presented on *corresponding author: gilda eslami. e-mail: g_eslami@ssu.ac.ir, eslami_g2000@yahoo.com https://wikitravel.org/en/isfahan j arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 146 http://jad.tums.ac.ir published online: june 24, 2019 chromosome 31 has critical role in transporting the antinomial drug inside the parasite and therefore any changes in its expression and gene sequence may involve in antimonial resistance development (5, 6). based on our knowledge, the presence of a point nucleotide mutation in the genes involved in drug absorption comes into effect by interacting with the drug and the target or changing the permeability of the drug into the parasite (7). three antimonial-resistant isolates had a deletion of 64 of 204kb base pairs at the end region of chromosome 31 (8). this deletion is included parts of aqp1 gene. another study found a nucleotide change in position of 398 in aqp1 gene from non-healing isolates that resulted in changing the amino acid glycine to aspartic acid that it seemed to affect the absorbance of antimonial (9). recently, we reported a new mutation of g562a in aqp1 gene from the standard isolate of l. major. for finding any correlation of this mutation and molecular mechanism of antimonial resistance, in this study, we investigated the molecular characterization and gene expression of aqp1 in resistant isolates obtained from patients with cl. materials and methods population study in this cross-sectional study, the research population included suspected cases of cl referring to the reference laboratories of two different endemic regions of iran, including yazd, located 270km (170mi) southeast of isfahan, and varzaneh, located 105km southeast of isfahan and 240km away from yazd, from jun 2015 to dec 2017. totally, 150 patients were considered for assessing. all patients were followed at least for 3 months after treatment to finding the resistant isolates. the informed consent was completed from all patients included in this study. the ethical approval with the code of ir.ssu. medicine.rec.1394.532 has been taken from the research ethics committee of shahid sadoughi university of medical sciences, yazd, iran. sampling after sterilizing, the edge of each lesion was sampled and placed on two separate slides. after preparing smear, it was fixed using methanol. the giemsa staining was done. then, leishman body was screened using optical microscope. all positive samples were considered for the next steps. moreover, sample of each patient was collected in rnalater solution for gene expression of aqp1 in the non-healing clinical isolates in next steps. dna extraction dna extraction was done using dna extraction kits (geneall, tissue, and blood, exgene cell sv, korea, seoul, #106-101) from the slides containing the parasite. the protocol was performed based on the company instruction by little modification. each slide was washed by xylol. after drying, the samples of each slide scratched and transferred into a sterile 1.5ml microtube. the other steps were done based on the kit instructions. detection and identification of species since the target population was l. major isolates, the its1-pcr-rflp was used to detection and identification (10) using the specific primer pair of litsr: 5′-ctggatca ttttccgatg-3′ and l5.8s: 5′-tgata ccacttatcgcactt-3′. the amplification was done using thermocycler (abi-simpiamp, usa). the presence of a fragment of 300– 320bp in length detected leishmania spp. for identification, rflp was done using hae iii restriction enzyme. the amplification and digestion analysis were done by agarose gel electrophoresis alongside by 50bp dna ladder. fragments of 200 and 127bp in length after enzyme digestion, was considered as l. major. all patients with l. major isolates were https://en.wikipedia.org/wiki/isfahan_(city) https://wikitravel.org/en/isfahan https://wikitravel.org/en/yazd j arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 147 http://jad.tums.ac.ir published online: june 24, 2019 followed for three month for finding the resistant ones. molecular analysis of aqp1 the non-healing clinical l. major isolates were included for g562a mutation analysis. the specific primer pair of lmaqp1-f: 5'tgtctggtggtcacacttgaac-3' and lmaqp1-r: 5'-cacgactagaggtatcc aaaagta-3' was used for aqp1 gene amplification. the amplification was done using thermocycler (abi-simpliamp, usa). the reaction was done in a volume of 20µl containing 10pmol each primer, 1u taq dna polymerase, 1x pcr buffer, 1.5mm mgcl2, 0.2mm dntp, and 100mg dna as template. the reaction program included the first denaturation of 94 °c for 5min, followed by 40 cycles of 94 °c for 1min, 56 °c for 1min, and 72 °c for 1min. the final extension was done at 72 °c for 5min. the amplicon size of 509bp in length showed the aqp1 amplification. then, rflp analysis was done with zra i resctriction enzyme for g562a assessing. the pattern with the fragments of 198 and 311bp in length was considered as wild type and no mutation but no digestion showed g562a mutation. amplification and digestion analyses were done using agarose gel electrophoresis (1%) alongside with 50bp dna ladder. gene expression total rna from the non-healing clinical isolates was extracted using the gf-1 total rna extraction kit (vivantis, malaysia, #gftr-025). the quality and quantity of the extracted rna were done using 1% agarose gel electrophoresis and spectrophotometer (eppendorf biophotometer plus, eppendorf, germany), respectively. the cdna synthesis was performed using revertaid™ first strand cdna synthesis kit (thermo fisher scientific, usa, k1621) based the protocol. all antimonial-resistant clinical isolates were assessed in order to aqp1 gene expression using the specific primer pair of aqp1-f: 5ꞌagtgtggagcgaggtgttcaa-3ꞌ and aqp1-r: 5ꞌ-ccgagagtatgcgaggtg acaa-3ꞌ (2). a susceptible strain was considered as the standard one. the glyceraldehyde 3-phosphate dehydrogenase (gapdh) gene was evaluated as the endogenous control with the specific primer pair of gapdhf: 5ꞌ-ccgagagtatgcgaggtgacaa3ꞌ and gapdh-r: 5ꞌ-gccccactcgttgt catacca-3'. the amplification was done with thermocycler (applied biosystem step one; usa). the amplification was done in triplicate. the real time pcr program comprises the initial denaturation at 95 °c for 10 min followed by 40 cycles of denaturation at 95 °c for 10sec and annealing/extension at 60 °c for 20sec. the specificity of amplification was performed by melting curves analysis. the analysis was performed using ∆∆ct as (∆ctsample-target-∆ctsample-control)-(∆ctstandardtarget-∆ctstandard-control) and the relative quantitative (rq) was calculated by 2-∆∆ct. statistical analysis data were analyzed using spss-16 software (chicago, il, usa). chi-square test was used to compare qualitative variables. the significance level in the applied tests was ≤ 0.05. results five age groups were classified. we classified the ages from 0 to 13 as group 1, 14 to 20 as group 2, 21 to 40 as group 3, 41 to 50 as group 4, and over than 50yr old as group 5. totally, 41 from group 1, 19 from group 2, 65 from group 3, 18 from group 4, and 7 from group 5 were included in this study. all isolates from groups 1 and 2 were susceptible to treatment, while out of the isolates from group 3, three were non-healing and 62 were susceptible to treatment. among the isolates from group 4, one was non-healing and 17 were sensitive to treatment. among the group 5, one was non-healing and six were j arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 148 http://jad.tums.ac.ir published online: june 24, 2019 susceptible to treatment. thus, the amount of k-square was measured as 5/28 with a significant level of p= 0.260, and therefore the response to therapy and age group did not correlate with each other and act independently (table 1). out of 150 patients with cl, 84 were men and 66 were women. out of 84 men, just one patient was resistant to treatment with antimony and others were treated immediately after first prescription. out of 66 women, four did not cure after three months considered as resistant to treatment. totally, 5 isolates were non-healing. all non-healing isolates were regarding to varzaneh. all isolates obtained from the patients with cl from yazd were susceptible to antimonial drug. its1-rflp-pcr was done for isolates. the results of pcr from the antimonial-resistant clinical isolates showed an amplicon of around 3bp in length. then, hae iii restriction enzyme analysis showed l. major pattern with the fragments of 200 and 100bp in length (fig. 1). molecular analysis of aqp1 sequencing for the mentioned mutation analysis showed all five isolates had g562a (fig. 2). the mean rq of aqp1 from the nonhealing clinical isolates showed 1.67 fold higher than the standard isolate as the sensitive one (fig. 3). fig. 1. agarose gel electrophoresis for detection and identification of clinical isolates. its1-pcr-rflp with the result of fragments with 100 and 200bp in length showed leishmania major. lane 1: 50bp dna ladder, lane 2: standard l. major, lanes 3, 4: clinical isolates with the pattern of l. major after restriction analysis fig. 2. agarose gele electrophoresis for pcr-rflp analysis for aqp1 gene in antimonial-resistant clinical isolates. lane 1: 50bp dna ladder, lane 2: the fragment size of 509bp in length in positive control of leishmania major after pcr-rflp, no digestion showed g562a mutation; lanes 3–7: the fragment size of 509bp in length in five antimonial-resistant clinical isolates of l. major after pcr-rflp, no digestion showed g562a mutation, lane 8: the fragment sizes of 198 and 311bp in length susceptible l. major isolate after pcr-rflp j arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 149 http://jad.tums.ac.ir published online: june 24, 2019 fig. 3. relative quantitative (rq) of lmaqp1 in antimonial resistance clinical isolates of l. major by real-time pcr. lm-s: the standard isolate, lm1 to lm5: the antimonial drug resistance clinical isolates table 1. correlation of the drug response and age category :answer treatment total nonhealing sensitive chi square p id group 1 (0-13) count 0 41 41 5.286 0.260 % within answer treatment 0.0% 28.3% 27.3% group 2 (14-20) count 0 19 19 % within answer treatment 0.0% 13.1% 12.7% group 3 (21-40) count 3 62 65 % within answer treatment 60.0% 42.8% 43.3% group 4 (41-50) count 1 17 18 % within answer treatment 20.0% 11.7% 12.0% group 5 (over than 50 years old) count 1 6 7 % within answer treatment 20.0% 4.1% 4.7% total count 5 145 150 % within answer treatment 100.0% 100.0% 100.0% discussion we reported here the molecular characterization of non-healing l. major clinical isolates, including g562a mutation in lmaqp1 and higher gene expression of lmaqp1 in comparison with the susceptible isolates. moreover, the age and sex of the patients had not j arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 150 http://jad.tums.ac.ir published online: june 24, 2019 any correlation with non-healing isolates. aqp1 encodes the aquaglyceropin protein which is one of the important factors in antimony resistant isolates (11). lmjaqp1 has critical roles in the parasite, including transporting solute such as water, glycerol, methylglyoxal, dihydroxyacetone, and sugar alcohols, regulating the volume, osmotaxis (12), and especially up taking of sb (iii). in this study, the mutation of g562a in aqp1 makes the alteration of alanine to threonine. the gene expression of lmaqp1 in all non-healing isolates was 1.67 fold more than the one in susceptible one. this mutation has especial role for increasing the lmaqp1 gene expression in this isolates. gene expression regulation in leishmania is controlled post-transcriptionally (13). mitogen-activated protein kinase (mpk) is the one of important key for control of aqp1 gene expression (14). threonine is one of the important amino acid that could be the target of protein kinase for phosphorylation. the kinases normally phosphorylated the oh group of threonine. in isolates studied in this study, g562a mutation was reported with substitution of alanine by threonine. more phosphorylation of lmaqp1 make overexpression of the gene (14). phosphorylation of threonine in lmaqp1 stabilizes the protein and therefore increasing of the half-life of the aqp1 makes it overexpression. this overexpression resulted in more up taking the drug. therefore, it is possible to arise a question which if this mutation could increase the stability of the protein and followed by overexpression, then why the isolates had the phenotype of non-healing. the answer reflects the facts that molecular mechanisms of antimonial resistant are multifactorial (15). efllux of the drug (16) or inactivating the drugs or their metabolites are the other mechanisms that approved in the parasites (17). although the isolates in our study showed overexpression of lmaqp1, after up taking the antimonial drugs, they may inactive or efflux from the parasite. phosphorylation has not any effect on the channel activity but make stability (14). this stability makes amplified the other roles of the protein such as combating with osmotic stress. based on knowledge, phosphorylation is dependent on oxidative stress presenting in environment, inside either the vector body or mammalian macrophages (14). therefore, lmaqp1 is phosphorylated during the metacyclic stage to provide stability. this helps to parasite to combat osmotic stress in metacyclic stage, the stage that is ready to infect the mammalian host. the role of map2 for phosphorylation of threonine for providing stability of lmaqp1 and then overexpression needs more attention with the goal of developing a new approach for drug targeting. on the other hand, development of some compounds that target map2 for preventing phosphorylation and increasing the half-life of aqp1 may be dangerous because map kinases in all organisms are familiar and therefore they might target the ones from human, too (18). although aqp1 protein belongs to the vast family of aqp in all organisms but finding some point nucleotides inside it might be the better target for designing the drug. this strategy for decreasing the stability of aqp1 effects on the other roles of this important protein such as combating the osmotic stress. consequence, decreasing the power of parasite for exposing to osmotic stress might be a powerful tool for control of the disease. one of the approaches for combating the non-healing isolates was induction of mpk2 in order to increase the stability of aqp1 in the non-healing isolates to force them more up taking of sb (iii) (14). our results are not agreed with the hypothesis of mandal et al. (14) because the clinical non-healing isolates that we assessed in this study showed overexpression of aqp1 but they did not respond to antimonial therapy. the phenotypic characteristic in leishmania for providing susceptibility or resistance to drugs is multifactorial and therefore more investigations are necessary to find the moj arthropod-borne dis, june 2019, 13(2): 145–152 y alijani et al.: molecular analysis of … 151 http://jad.tums.ac.ir published online: june 24, 2019 lecular mechanisms in detail by which leishmania select. this is the first report of effective of g562a on aqp1 gene expression in clinical non-healing isolates. moreover, presenting a point mutation was reported inside aqp1 develops resistance isolate (7). g133d in aqp1 can be one of the mechanisms in non-healing isolates of leishmania. moreover, some other mutational analyses have been performed on lmaqp1 that revealed ala163 and glu152 are involved in metalloid uptake and reduced permeability to antimony (19, 20). therefore, in this study, we discovered the new resistance mechanism corresponding to a point mutation of aqp1. conclusion the non-healing clinical isolates may have mutation of g562a in aqp1. this mutation can affect lmaqp1 gene expression. the mutation of g562a resulted in providing additional threonine. threonine may be the target of kinase enzyme for more stability of the aqp1 protein. therefore, this alteration can be candidate for drug target. acknowledgements this paper is output of an msc thesis. the financial support was done by shahid sadoughi university of medical sciences. the authors thank from fatemeh ahaki varzaneh and has san shokoohi for their assistance. the authors declare that there is no conflict of interest. references 1. eslami g, hajimohammadi b, jafari aa, mirzaei f, gholamrezai m, anvari h, khamesipour a (2014) molecular identification of leishmania tropica infections in patients with cutaneous leishmaniasis from an endemic central of iran. trop biomed. 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https://www.ncbi.nlm.nih.gov/pubmed/?term=heterogeneity+of+molecular+resistance+patterns+in+antimony-resistant+field+isolates+of+leishmania+species+from+the+western+mediterranean+area https://www.ncbi.nlm.nih.gov/pubmed/?term=sharma%20m%5bauthor%5d&cauthor=true&cauthor_uid=26259900 https://www.ncbi.nlm.nih.gov/pubmed/?term=mandal%20g%5bauthor%5d&cauthor=true&cauthor_uid=26259900 https://www.ncbi.nlm.nih.gov/pubmed/?term=mandal%20s%5bauthor%5d&cauthor=true&cauthor_uid=26259900 https://www.ncbi.nlm.nih.gov/pubmed/?term=bhattacharjee%20h%5bauthor%5d&cauthor=true&cauthor_uid=26259900 https://www.ncbi.nlm.nih.gov/pubmed/?term=mukhopadhyay%20r%5bauthor%5d&cauthor=true&cauthor_uid=26259900 https://www.ncbi.nlm.nih.gov/pubmed/?term=functional+role+of+lysine+12+in+leishmania+major+aqp1 https://www.ncbi.nlm.nih.gov/pubmed/?term=functional+role+of+lysine+12+in+leishmania+major+aqp1 j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 162 http://jad.tums.ac.ir published online: june 12, 2018 original article molecular identification of agents of human cutaneous leishmaniasis and canine visceral leishmaniasis in different areas of iran using internal transcribed spacer 1 pcr-rflp aref teimouri 1, 2, *mehdi mohebali 1, 3, elham kazemirad 1, *homa hajjaran 1 1department of medical parasitology and mycology, tehran university of medical sciences, tehran, iran 2students scientific research center, tehran university of medical sciences, tehran, iran 3center for research of endemic parasites of iran, tehran university of medical sciences,tehran, iran (received 6 june 2017; accepted 16 may 2018) abstract background: leishmaniasis is a major medical health problem and distributes in nearly half of 31 provinces of iran. we aimed to identify cutaneous and visceral leishmania spp. isolated from infected humans and domestic dogs in various regions of iran, 2010‒2013. methods: dna was extracted from 108 lesion exudate samples of suspected patients to cutaneous leishmaniasis and nine liver and spleen aspirates of infected dogs cultured in rpmi-1640 and amplified using partial sequence of its1 gene. the pcr amplicons were digested using haeiii endonuclease enzyme and used in restriction fragment length polymorphism (rflp) assay. then, 48 amplicons representing various hosts were sequenced and compared to sequences from genbank databases using blast. results: pcr-rflp analysis showed that 60 and 48 cl patients were infected by leishmania tropica and l. major, respectively. from nine canine visceral leishmaniasis (cvl) isolates, eight isolates were identified as l. infantum and one as l. tropica. the greatest similarity of 95.7% in its1 region was seen between l. infantum and l. major. furthermore, the lowest similarity with 65.7% was seen between l. tropica and l. major. intra-species comparison of its1 region in l. infantum, l. major and l. tropica isolates were showed 100%, 98.2% and 72.4 % similarities, respectively. conclusion: pcr-rflp based on its1 region is an appropriate method to distinguish three leishmania spp. of l. major, l. tropica, and l. infantum. in intra-species comparison of its1 region, genotypic variations showed that l. tropica isolates were more heterogeneous than l. major and l. infantum isolates. keywords: leishmania, its1 gene, pcrrflp, iran introduction cutaneous (cl) and visceral leishmaniasis (vl) include some of the world most neglected diseases in tropical and subtropical areas with an estimated incidence of 0.6‒1.0 and 0.05‒0.09 million new cases of cl and vl occur worldwide each year, respectively (1). in iran, cl is considered as a medical health problem in nearly half of 31 provinces (2). both epidemiological forms of cl are reported in iran, anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl) caused by leishmania tropica and l. major, respectively (3). nearly 20000 cl cases are annually reported and the prevalence of leishmaniasis in provinces of iran suggestively ranges 1.8‒37.9% (2). furthermore, vl is caused by l. donovani complex and l. infantum is the main agent of vl in endemic areas of iran with about 100–300 new cases annually. infections due to l. infantum have been reported in humans, domestic dogs and phlebotomine vectors (4, 5). human, rodents (mainly great gerbils (rhombomys opimus)) and domestic canines (canis familiaris) are the main reservoir hosts of acl, zcl and vl, respectively (6-8). *corresponding authors: dr mehdi mohebali, email: mohebali@tums.ac.ir, dr homa hajjaran, email: hajaranh@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 163 http://jad.tums.ac.ir published online: june 12, 2018 recently, a few cases of viscerotropic leishmaniasis caused by l. tropica and l. major have been reported in immunocompromised patients in iran (9, 10). furthermore, l. infantum is involved in sporadic cl in the endemic areas of vl in northwestern iran (11). because of wide clinical diversities of leishmaniasis, various responses of patients to treatment, various reservoir hosts of the parasite and to develop effective control strategies in endemic areas, distinguish between leishmania spp. and find dominant spp. in provinces are critical. since leishmania spp. are morphologically identical; therefore, species identification using microscopy or culture methods is not easily possible. for species characterization, additional methods must be used. one of these methods, mlee (multilocus enzyme electrophoresis) analysis, remains the current gold standard but requires mass culture of the parasites which is not simply carried out (12). relatively, molecular methods are sensitive for the detection of low amounts of the parasite. nowadays, molecular based analyses have extensively been used for the identification of leishmania spp. as well as other parasites. identification and phylogenetic implication with various targets such as kdna genes and introns are routine (13). furthermore, the its region of rdna has been used in several studies to resolve taxonomic questions and to determine phylogenetic affinities among closely related leishmania species (14, 15). the its1 gene is considered as the most appropriate region and gives the best results in leishmania differentiation in the old world (16). the its1 locates between 18s and 5.8s rrna genes and possesses conservative loci targets for the pcr (17). additionally, it includes satisfactory polymorphisms to facilitate species identification. the aim of this study was to achieve a better understanding of the current status of various isolates of leishmania spp. in iranian provinces using its1-pcr-rflp. furthermore, molecular findings of this study were used to assess phylogenetic relationships between the isolates. materials and methods sampling samples were collected from 148 lesion exudates of suspected patients to cl referred to leishmaniasis laboratory of the school of public health, tehran university of medical sciences, tehran, iran, and district health centers of iran, 2010‒2013. most of the patients were referred from endemic areas in northeast, southeast, center, west, and southwest of iran (fig. 1). serosity materials of the lesions were smeared on a microscope slide, air-dried, fixed with absolute methanol and stained with giemsa in 10% phosphate buffer (ph 7.4) for 25min and then examined for amastigotes using light microscope with 1000× magnification. serosity materials from lesions of cl were cultured in rpmi-1640. twelve dogs suspected to cvl living in endemic areas were tested serologically using direct agglutination test (dat). liver and spleen aspirates of the infected dogs positive for dat were cultured in rpmi-1640. the parasitological positive dogs showed clinical manifestations of vl including dermatological wounds, ocular variations, weight loss, laziness, lymphadenopathy and splenomegaly (18, 19). ethical approval the study was approved by the ethics committee of the tehran university of medical sciences (approval no. 13462). no information of the patients was revealed in the study. verbal informed consents were received from the patients and the dog owners. direct agglutination test (dat) anti leishmania antibodies in dog sera were detected using dat. briefly, promastigotes of l. infantum, iranian strain (mcan/ir/07/moheb-ghو genbank acceshttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 164 http://jad.tums.ac.ir published online: june 12, 2018 sion no. fj555210), were cultured in rpmi1640 with 10% heat-inactivated fetal bovine serum (fbs), trypsinized, fixed with 1.2% formaldehyde (merck, germany) and stained with coomassie brilliant blue (sigma, usa). negative and positive controls were used in each experiment. antibody titers ≥ 1:320 were considered as positive in canines (18). parasite culture and cryopreservation all samples including lesion exudates of the suspected patients to cl and liver and spleen aspirates of the infected dogs were cultured in rpmi-1640 media supplemented with 10–15% heat-inactivated fbs, 100u/ml of penicillin and 100µg/ml of streptomycin (gibco, life technologies, germany) and incubated at 24–25 °c. five days after the last sub-culture, parasites were harvested, washed in sterile phosphate buffered saline (pbs, ph 7.2–7.4), stored at -20 °c until use and were preserved in liquid nitrogen for further studies. dna extraction, pcr, and rflp genomic dna was extracted from pellets of the leishmania cultures using high pure pcr template preparation kit (roche diagnostics, germany) according to the manufacturer′s instructions. samples were stored at 20 °c until use. dna samples from iranian reference strains of l. tropica (mhom/ir/ 02/mash10/accession no. ef653267), l. major (mrho/ir/11/gol-2/ accession no. jn860745) and l. infantum (mcan/ir/ 07/mash-ir1/ accession no. eu810776) were used as positive controls. the dna samples were assessed for the leishmania-specific ribosomal internal transcribed spacer 1 region (its1) by pcr amplification using primer pairs of litsr (f: 5′-ctggatcattttccgatg3′) and l5.8s (r: 5′-tgataccacttat cgcactt-3′). amplification was carried out using pcr-ready premix (roche, germany) in a 25μl reaction. the amplification conditions included those described previously (20). pcr products (8μl) were digested with the restriction endonuclease enzyme haeiii (bsuri) (fermentas, germany) for the species identification according to the manufacturer′s instructions. amplicons of nearly 300– 350bp and restriction fragments were analyzed using 1.5‒3% agarose gels containing safe stain, visualized under uv and compared with those from reference strains of l. tropica, l. major and l. infantum. nucleotide sequence and phylogenetic analysis pcr products from 44 cl and four cvl samples were sequenced using litsr as forward primers. dna sequences obtained from the current study were compared to sequences from genbank database using basic local alignment search tool (blast). these sequences have been annotated to genbank and aligned using clustal x software and mega software v.6.0 (21). various statistical methods (maximum likelihood, upgma and neighbor-joining (nj) trees) were digitalized for phylogenetic analysis of the aligned sequences. the tamura 3-parameter option of the neighbor-joining method was shown as the best phylogenetic tree (22). inter and intraspecies similarities of its1 region were calculated for l. major, l. infantum and l. tropica isolates using mega software v.6.0. bootstrap analysis was carried out with 1000 replicates. accession numbers of 48 leishmania isolates used in this study are recorded in genbank as follows: jx289844-jx 289881, jn860713-jn860714 and jn860718jn860725. results microscopic, culture and dat analysis microscopic study demonstrated leishmania amastigotes in 112 out of 148 smears (75.6%) collected from patients suspected to cl. furthermore, 108 samples (72.9%) were positive for leishmania parasites in culture method. dat analysis of domestic dog sera http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 165 http://jad.tums.ac.ir published online: june 12, 2018 showed that 10 sera (83.3%) were positive with titers ≥ 1:320 out of 12 samples. nine out of 12 dog samples (75.00%) were positive in microscopy and aspirate culture methods. leishmania spp identification using rflp analysis in general, its1 pcr was carried out for the diagnosis of leishmania spp, in which positive samples produced amplicons of 300– 350bp (fig. 2a). digestion of the pcr products with haeiii endonuclease produced two bands of 220 and 140bp for l. major reference strain. furthermore, bands of 200, 60 and 5bp and 200, 80 and 40bp were produced for l. tropica and l. infantum reference strains, respectively (fig. 2b). compared to patterns produced in reference strains and cl patients, two profiles were clearly distinguishable as follows: 1) 48 amplicons (44.5%) produced two bands (220 and 140bp) indicating l. major, and 2) 60 amplicons (55.5%) produced three bands (200, 60 and 50bp) indicating l. tropica. rflp analysis on dog samples revealed l. infantum with three bands of 200, 80 and 40bp in eight cases (88.88%) and l. tropica in one case (11.12%). sequencing and phylogenetic tree analysis forty eight amplicons were successfully sequenced. the phylogenetic trees from the sequences of its1 fragments clearly showed divergence between l. major, l. infantum, and l. tropica. furthermore, phylogenetic analysis demonstrated variations by sequencing as 21 haplotypes were shown in 48 iranian isolates including l. major (h1–h7), l. infantum formed independent and equal clusters with 94% bootstrap (h1) and l. tropica (h1‒h13) (fig. 3). intra-species similarities of its1 region in isolates of l. infantum, l. major, and l. tropica included 100%, 98.2% and 72.4%, respectively (fig. 4). the highest inter-species similarity of its1 region with 95.7% was reported in isolates of l. infantum and l. major followed by a 68.5% of similarity between l. infantum and l. tropica. the lowest similarity with 65.7% was reported between l. tropica and l. major (fig. 5). fig. 1. geographical origins of 148 leishmania spp isolated from cutaneous and visceral leishmaniasis cases identified by its1-pcr rflp (2010-2013) fig. 2. a) electrophoresis of leishmania dna amplified a 350bp fragment using primers of litsr and l5.8s. dna was collected from reference strains and lesion exudates of suspected patients to cutaneous leishmaniasis and liver and spleen aspirates of infected dogs cultured in rpmi-1640. b) digestion patterns of its1 amplicons of leishmania spp. from references and patients using haeiii enzyme. line 1, negative control, line 2, l. tropica (accession number: ef653267); line 3, l. major (accession number: eu810776), line 8, l. infantum (accession number: jn860745) as references strain; line 4‒7, patient samples; line 9, dog sample, m, 50bp ladder http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 166 http://jad.tums.ac.ir published online: june 12, 2018 fig. 3. the neighbor-joining tree constructed from its1 regions of 48 isolates using tamura 3-parameter. numbers above branches correspond to bootstrap valued based on 1000 replicates. branches without numbers include values of less than 70%. genbank accession numbers are shown in parentheses fig. 4. intra-species mean similarities for leishmania spp. isolated from cl and vl cases based on its1 region sequence, 2010‒2013 fig. 5. inter-species mean similarities among leishmania spp. isolated from cl and vl cases based on its1 region sequence, 2010–2013 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 167 http://jad.tums.ac.ir published online: june 12, 2018 discussion species identification is critical in diagnosis, treatment and epidemiological studies of leishmania spp. several studies have amplified its-1 region of the ribosomal dna repeat unit (rdna-its1) for the recognition of iranian leishmania spp. (8, 23-25). in the present study, a number of unknown leishmania spp. isolated from cl and cvl cases in various endemic regions of iran was identified by pcr-rflp and sequencing based on its1 region of rrna gene. in the current study, the major foci of 60 l. tropica cl isolates belonged to khorasan razavi province, bam city (kerman province), kashan city (isfahan province). moreover, one isolate belonged to a cl lupoid patient residing in shahre-rey in southern tehran (acc, jx289854). forty-eight l. major isolates belonged to golestan, isfahan, kermanshah, khuzestan, ilam and semnan provinces of iran (fig. 1). two isolates recovered from diffuse cutaneous leishmaniasis (dcl) patients in damghan city of semnan province in central (acc, jn860713) and dehloran city of ilam province in southwestern iran (acc, jn860714) were identified as l. major. of nine cvl isolates, eight isolates were identified as l. infantum (six isolates from ardabil, one isolate from golestan and one isolate from tehran provinces) and one isolate as l. tropica (from golestan province). the phylogenetic analysis revealed 21 haplotypes within the isolates. no correlations were seen between the haplotypes and geographic distribution of the species complexes. in similar studies in iran, no correlations were demonstrated between the intraspecies divergence and geographical distribution based on rapd-pcr and its1 and n-acetylglucosamine-1-phosphate transferase (nagt) genes with pcr-rflp methods (3, 26). in the phylogenetic analysis, l. tropica isolates showed 13 haplotypes (h1–h13) indicating a significant divergence between l. tropica isolates from the other two species (fig. 3). although the its region is one of the best candidates for the differentiation of leishmania at species and strain levels, limited studies have used its sequence analysis for l. tropica isolates to compare (27-29). leishmania tropica (a diploid microorganism) is known as a heterogenous species, quite observed in alignment of the pcr product sequences and in low bootstrap frequencies found in the phylogenetic tree (30-32). the existence of at least two alleles for its in rdna of leishmania spp. may be a good explanation for this phenomenon (33). in the current study, sequences from three l. tropica isolates were technically unreadable excluded from the phylogenetic analysis. however, unreadable sequences of its-rdna fragments belonged to positive leishmaniasis cases (24). this can prove high heterogeneity or sometimes mixed infections with two or three leishmania spp. (23, 24, 34, 35). similar to pilot studies, a high degree of heterogeneity was seen in l. tropica in our study (30, 33, 36, 37). in intra-species analysis, a varied heterogeneity was found at various levels in leishmania spp. using its-pcrrflp analysis. this included a highest to lowest order of l. tropica > l. major > l. infantum with similarities of 72.4%, 98.2% and 100%, respectively. relatively, l. donovani, l. infantum and l. major were reported as the less and l. tropica, l. turanica and l. gerbilli as the most divergent complexes (38). in 2005, a heterogeneity variation at various levels in old world leishmania spp. was reported with a highest to lowest order of l. tropica > l. aethiopica > l. major > l. donovani (39). these findings are similar to studies reported that l. tropica isolates included the highest divergence in its1 genes (26). similar to a study on l. major with 98.2% similarity, results of the current study showed a limited genetic variation with seven haplotypes seen in l. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 162–171 a teimouri et al.: molecular identification … 168 http://jad.tums.ac.ir published online: june 12, 2018 major (h1‒h7) (25). four haplotypes of l. major were found in a distinct clade in nine rodent isolates in central iran (23) and six haplotypes of l. major were identified through pcr-rflp of rodent samples using its1 markers (8). results of the present study on l. infantum isolates showed no diversities while formed independent and equal clusters with the highest bootstrap values (94%) in the phylogenetic analysis. moreover, heterogeneity of l. tropica with various groups was detected in the current phylogenetic analysis while l. infantum isolates consisted of only one haplotype. in a similar study in turkey using an its1 based real-time pcr, genotypic variations of leishmania spp. at species and intra-species levels were observed and heterogeneities were found in l. tropica isolates while l. infantum isolates formed single group (40). in the present study, the lowest and highest interspecies varieties were seen between l. major and l. infantum and between l. tropica and l. major with 4.3% and 34.3% variations, respectively. in the tree, l. major was more associated to l. infantum than l. tropica was. these findings were in contrast to those from studies based on nagt genesو in which, the lowest inter-species similarity of 95.7% were reported between l. major and l. infantum (3). conclusion sequencing results of leishmania spp. isolated from cl and cvl cases showed 93–98% similarities with other annotated sequences in genbank database. therefore, pcr-rflp based on its1 region can be suggested as an adequate method to distinguish l. major, l. tropica and l. infantum which are the most prevalent leishmania spp. in middle east. furthermore, genotypic variations based on its1 region within inter and intra-species of leishmania spp. have shown that l. tropica includes more heterogeneity than l. major and l. infantum do. acknowledgements the authors would like to thank all staff 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ertabaklar h, culha g, sevil n, ziya alkan m, jaffe cl (2009) leishmaniasis in turkey: molecular characterization of leishmania from human and canine clinical samples. trop med int health. 14(11): 1401–1406. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=assmar%20m%5bauthor%5d&cauthor=true&cauthor_uid=18791741 https://www.ncbi.nlm.nih.gov/pubmed/?term=assmar%20m%5bauthor%5d&cauthor=true&cauthor_uid=18791741 https://www.ncbi.nlm.nih.gov/pubmed/?term=amirkhani%20a%5bauthor%5d&cauthor=true&cauthor_uid=18791741 https://www.ncbi.nlm.nih.gov/pubmed/?term=lin%20cp%5bauthor%5d&cauthor=true&cauthor_uid=17142569 https://www.ncbi.nlm.nih.gov/pubmed/?term=chang%20kp%5bauthor%5d&cauthor=true&cauthor_uid=17142569 j arthropod-borne dis, december 2017, 11(4): 533–538 n ali el-dib et al.: case report of … 533 http://jad.tums.ac.ir published online: december 30, 2017 case report case report of human urinary myiasis caused by clogmia albipunctata (diptera: psychodidae) with morphological description of larva and pupa *nadia ali el-dib 1, wegdan mohamed abd el wahab 2, doaa ahmed hamdy 2, mona ibrahim ali 2 1department of medical parasitology, cairo university, el manial, cairo, egypt 2department of medical parasitology, beni-suef university, beni-suef, egypt (received 27 oct 2016; accepted 16 dec 2017) abstract background: urinary myiasis is a form of myiasis caused mainly by larvae of fannia scalaris, musca, sarcophaga, lucilia, wohlfahrtia, calliphora, and rarely by eristalis and clogmia albipunctata. methods: this report presents a case of female patient complaining of dysuria and frequency of micturition associated with intermittent passage of small, motile, dark-colored worm-like organisms in urine. she was a married housewife aged 24 years old referred from the tropical outpatient clinic of beni-suef university hospital, egypt. the patient was subjected to a full questionnaire sheet and investigations such as cbc, stool and urine analysis and urinary ultrasonography. collected larvae and pupae from urine samples were examined macroscopically and microscopically. results: the examined larvae and pupae belonged to c. albipunctata. ivermectin was prescribed to the patient with complaint withdrawal and complete disappearance of the larvae from urine. conclusion: this study reports the first case of urinary myiasis caused by c. albipunctata in beni-suef governorate, the second in egypt and third case worldwide. the study throws some light on the medical importance and management of urinary myiasis. keywords: urinary myiasis, clogmia albipunctata, egypt introduction myiasis is the infestation of human or animal tissues by dipterous fly larvae of the class insecta. urogenital myiasis is a rare form of myiasis occurring in humans (1). like all other types of myiasis, urinary myiasis is usually associated with poor hygienic measures, lack of cleanliness and disability. it may be associated with urinary symptoms as dysuria, hematuria, and obstruction of the urinary system (2). urinary myiasis is caused by larvae of fannia scalaris, musca, sarcophaga, lucilia, wohlfahrtia, and calliphora. few cases of urinary myiasis were caused by eristalis and psychoda flies (3). the family psychodidae includes six subfamilies, only two of them have medical importance for humans, phlebotominae (sand flies) which are bloodsuckers and vectors of leishmaniasis and psychodinae (moth flies) not adapted for blood sucking. clogmia albipunctata is nematocera of the family psychodidae, subfamily psychodinae. adults live in dark and moist areas including bathrooms and toilets and can be frequently found in drains or sewers hence are called "filter flies or drain flies or bathroom flies". larvae live in and feed on decaying organic matter (4). myiasis occurs worldwide with more cases being reported from tropical, subtropical and warm temperate areas. in egypt, some cases of human urogenital myiasis were previously reported (3, 5-8). these detected cases of urinary myiasis in egypt throw some light on the medical importance and management of this disease in our area. *corresponding author: prof nadia ali el-dib, email: naeldib@hotmail.com j arthropod-borne dis, december 2017, 11(4): 533–538 n ali el-dib et al.: case report of … 534 http://jad.tums.ac.ir published online: december 30, 2017 case presentation a married housewife patient aged 24yr old was referred from the tropical outpatient clinic of beni-suef university hospital, egypt complaining of intermittent passage of small, motile, dark-colored worm-like organisms in urine. the condition started two years ago associated with dysuria and frequency of micturition. at periods of larval passage, which ranged from two to three days, about 3–4 larvae were seen in each voided urine sample. this was followed by periods of symptoms remission, concomitant with invisibility of the larvae for weeks. however, there was no fever, no hematuria or itching in the periurethral and genital regions. the patient was living in ashmant, a rural area in beni-suef governorate, egypt in poor hygienic conditions. the patient gave history that she urinates in unsanitary toilets "cabinet" or "pit latrine", with presence of abundant mosquitoes and flies in the bathroom. therefore, for lack of water pipes, water used for rinsing and bathing was stored in wide uncovered buckets in the bathroom. there was no relationship with farming or livestock rising. no history of traveling or previous operations. consent was taken from the patient. investigations such as cbc, stool analysis “with direct and concentrated smears” and urinary ultrasonography were done and all revealed no abnormalities. the patient was asked to collect urine samples in clean tightly covered containers at periods of larval passages. complete urine analysis revealed amorphous urate crystals while the numbers of red blood cells (1–3/hpf) and pus cells (5–7/hpf) in urine were within the normal range. collected larvae and pupae were examined macroscopically and microscopically at the parasitology department of faculty of medicine, beni-suef university, egypt. macroscopical examination of the larvae and pupae by naked eye examination, the larva was cylindrical in shape with rounded ventrally curved anterior end and tapering posterior end. the body of the larva was segmented and covered by short seta. larvae were dark brown to black in color, rapidly motile when freshly passed with different sizes ranging from 8– 12mm in length. in addition, identified pupae in freshly passed urine were actively motile, pyriform in shape with cephalothorax carrying two antennae and segmented abdomen. the size of pupa ranged from 4–5mm in length. four fresh larvae from the patient’s urine were placed in a clean container supported with nutrition in the form of few sugar particles and covered with gauze in a trial for breeding them into adults and were followed up daily. the larvae remained alive for about two weeks but were not able to develop into adult flies. dead larvae were placed in 5% formalin for fixation. microscopical examination of the larvae and pupae larvae fixed in 5% formalin, were washed several times in saline and then incubated in transparent glass bottles containing 30% potassium hydroxide until they became transparent under the microscope. subsequently, larvae were washed thoroughly in distilled water, dehydrated in ascending grades of ethyl alcohol (30%, 50%, 70%, 90% and 99%) for 30min each, and then in xylene for 30min, mounted in canada balsam and dried in the oven at 38 °c for two days (9). the mounted larvae were examined microscopically and photographed. description of full-grown larva the microscopically examined larvae were identified as larvae of c. albipunctata of different stages depending on the following characters. the larvae were hairy, segmented and yellowish brown in color. the anterior end, j arthropod-borne dis, december 2017, 11(4): 533–538 n ali el-dib et al.: case report of … 535 http://jad.tums.ac.ir published online: december 30, 2017 posterior end and dorsal plates were slightly darker, while the ventral aspect was slightly lighter in color than the rest of the body. their length ranged from 8 to 12mm. the body of the larva consisted of the head and eleven segments, 3 thoracic and 8 abdominal segments (fig. 1a). the head is triangular, not retracted into the thorax with 2 min hairy antennae and 2 ventral mandibles. the mandibles are opposing each other so can be moved against each other on a horizontal plane (fig. 1b). the dorsal and lateral aspects of the body segments are covered with 26 saddles shaped chitinous plates, 2 for each thoracic segment and 3 for each abdominal segment except the first abdominal segment with two plates and the eighth one without plates. accordingly, each segment appears divided into several subdivisions (secondary annuli) by these chitinous plates (fig. 1c). these plates are covered dorsally and laterally with long dark backwardly directed filiform setae arising from rounded chitinous button–shaped basal plates (fig. 1d). the prothorax has a pair of anterior respiratory spiracles while posterior respiratory spiracles are located on the apical part of the respiratory tube on the siphon. the siphon is cone-shaped, longer than broad (4:1) and its ventral aspect showed spinose anal papillae (fig. 1e). description of pupa the pupa is pear-shaped and measures 4–5mm in length, with cephalothorax, and 7 visible abdominal segments. the head carried 2 dark eye spots, 2 beaded antennae and 2 funnel-shaped respiratory trumpets in the anterior end. the thoracic segments show the wings of an emerging adult (fig. 2a). the respiratory trumpet appeared lanceolate in shape including fine circular breathing openings arranged spirally in a rosary shape with few long hairs at the base of the trumpet (fig. 2b). each abdominal segment is rectangular in shape with 2 lateral and 2 caudal spine-like processes seen ventrally except the last segment provided only with 2 caudal processes (fig. 2c). on the follow-up visits, the patient was advised to take a single oral dose of ivermectin (200ug/kg), to drink plenty of water to help expel any other larvae in the bladder, to keep the water bucket tightly covered in the bathroom and to use spraying insecticides to kill any insects and larvae on the walls and floor of the toilet. she was also advised to change the pit latrine to a toilet seat. after one week, all the patient’s complaints improved with gradual reduction in number and frequency of passing of larvae in urine until complete disappearance of the larvae after treatment. the patient was followed up two months later without recurrence of symptoms or voiding larvae in urine. fig. 1. a) clogmia albipunctata full larva with elongated, hairy, segmented body (x40). b) anterior part of the larva showing the triangular head (x200). c) the dorsal aspect of the abdominal segments showing the saddle-shaped chitinous plates and the subdivisions (x40). d) lateral aspect of the chitinous plates showing long filiform setae (arrow) arising from rounded plates (arrowhead) (x100). e) caudal part showing the ventral aspect of the siphon with spinose anal papilla (double arrow) and the apical posterior spiracles (arrow) (x200) j arthropod-borne dis, december 2017, 11(4): 533–538 n ali el-dib et al.: case report of … 536 http://jad.tums.ac.ir published online: december 30, 2017 fig. 2. a) anterior part of clogmia albipunctata pupa showing the head with dark eyespots, antenna (arrow), and two respiratory trumpets (double arrow), the thoracic segments with wings of an emerging adult (arrowhead) and the anterior abdominal segments (x40). b) anterior part of the pupa showing the respiratory trumpets with the breathing tubes openings (x400). c) abdominal segments of the pupa showing the lateral processes (arrow) and caudal processes (arrowhead) (x40) discussion this work reports the first case of human urinary myiasis caused by c. albipunctata in beni-suef governorate, egypt. in this case report, the patient had low standard of living in a rural area with poor personal hygiene all considered predisposing factors increasing the risk of urogenital myiasis (10). in addition, the stored water in the uncovered buckets in the bathroom used for rinsing and bathing supported a good medium for insects breeding. the fly eggs are laid in or around the moist urogenital orifices when the patient was urinating, then after hatching; the larvae ascend the urinary tract to feed causing myiasis. this mechanism was previously suggested (7, 11) and is expected to be the cause with our patient. in spite of the minor medical importance of c. albipunctata adults, as they are only nuisance pests, very poor flyers and cannot travel far from their pupation site, their dead bodies may disintegrate to form potential allergens (4). they have been also incriminated as potential mechanical vectors of some bacterial pathogens and were responsible for nosocomial infections (12). clogmia albipunctata larvae were reported as cause of nasopharyngeal myiasis (13), intestinal myiasis (14), and urinary myiasis (15). larvae of c. albipunctata could be differentiated from other members of genus psychoda (especially p. latreille and p. albipennis) by having much darker body coloration and the 26 dorsal plates while other members of genus psychoda are lighter in color and the number of dorsal plates is less than 26. moreover, the siphon of p. albipennis is slender, 7 to 8 times as long as broad (16). it is rare to detect dipterous fly larvae in human urine in egypt; however, three cases of urinary myiasis with psychoda spp. were reported from egypt (5-7). a recent study had reported five cases with human urogenital myiasis caused by psychoda spp. in upper egypt in assuit and qena governorates (8). the first case report of human urinary myiasis due to larvae of c. albipunctata in egypt was detected (3). ivermectin is semisynthetic macrocyclic lactone drug with antihelminthic activity. nowadays, it is recommended as an important alternative for treatment of scabies, demodicidosis, head lice, and myiasis (17, 18). in this case, we recommended the use of ivermectin as the clinical complaints continued for 2yr before diagnosis. the complaints regressed because of use of ivermectin in a single oral dose of 200ug/kg, and increased fluid intake, without any recurrence of symptoms or larval passage for 2 months later. this result is in accordance with others who prescribed ivermectin in two oral doses of 200ug/kg (8). on the other hand, some clinicians had prescribed antibiotic and antiseptic treatment, accompanied with larval passage j arthropod-borne dis, december 2017, 11(4): 533–538 n ali el-dib et al.: case report of … 537 http://jad.tums.ac.ir published online: december 30, 2017 for 10d and then larvae passage and other patient symptoms disappeared (3). this study reported and described the first case of human urinary myiasis caused by c. albipunctatain beni-suef governorate, the second in egypt and third case worldwide. conclusion despite urinary myiasis is very rare in humans, urologists should consider it as an unusual cause of urinary tract infections especially in patients with non-specific urinary symptoms when no pathology is identified at routine examination and in those who live in rural areas with unsanitary poor environmental and socioeconomic levels. acknowledgements we are so grateful to dr yusuf abd elaziz edmardash, department of entomology, faculty of science, cairo university for his kind help in the identification of the species of the larvae and the pupa of myiasis. references 1. karagüzel e, kutlu o, buruk k, okatan ae, özgür gk (2015) urogenital myiasis caused by psychoda albipennis: a case report. j urol surg. 4: 195–196. 2. mumcuoglu i, aral akarsu g, balaban n, keles i (2005) eristalis tenax as a cause of urinary myiasis. scand j infect dis. 37: 942–943. 3. el-badry aa, salem hk, edmardash yae (2014) human urinary myiasis due to larvae of clogmia (telmatoscopus) albipunctata williston (diptera: psychodidae) first report in egypt. j vector borne dis. 51: 247– 249. 4. mullen gr, durden l (2009) medical and veterinary entomology. chapter 11. in: rutledge cl, gupta kr (eds): moth flies and sandflies (psychodidae). cop san diego, california. elsevier academic press, p. 147. 5. sakla aa, el–hady ha, el–nadi na (2003) psychoda albipennis maggots (diptera: psychodidae) as a cause of urinary myiasis in an upper egyptian male, the second world case. el– minia med bull. 13: 220–224. 6. ezzat hm, younis ai (2009) case report: urinary myiasis caused by larvae of psychoda spp. proceedings of the 28th annual scientific conference of the egyptian society of basic medical sciences, faculty of medicine. cairo university. 61–79. 7. lotfy wm (2011) gastrointestinal and urogenital myiasis caused by psychodid fly (diptera: nematocera) in egypt: case report. puj. 4 (1): 115– 116. 8. yones da, bakir hy, hameed da (2014) human urogenital myiasis caused by psychoda species larvae: report of five cases and morphological studies. j adv parasitol. 1(2): 12–20. 9. adams zo, hall mr (2003) methods used for killing and preservation of blowfly larvae, and their effect on post–mortem larval length. forensic science international. 138: 50–61. 10. perez-eid c, mouffok n (1999) human urinary myiasis caused by fannia canicularis (diptera: muscidae) larvae in algeria. presse méd. 28: 580– 581. 11. salimi m, goodarzi d, karimfar mh, edalat h (2010) human urogenital myiasis caused by lucilia sericata (diptera: calliphoridae) and wohlfahrtia magnifica (diptera: sarcophagidae) in markazi province of j arthropod-borne dis, december 2017, 11(4): 533–538 n ali el-dib et al.: case report of … 538 http://jad.tums.ac.ir published online: december 30, 2017 iran. iran j arthropod borne dis. 4(1): 72–76. 12. faulde m, spiesberger m (2013) role of the moth fly clogmia albipunctata (diptera: psychodinae) as a mechanical vector of bacterial pathogens in german hospitals. j hosp infect. 83 (1): 51–60. 13. mohammed n, smith kg (1976) letter: nasopharyngeal myiasis in man caused by larvae of clogmia (telmatoscopus) albipunctatus williston (psychodidae, dipt.). trans r soc trop med hyg. 70(1): 91. 14. tu w, chen h, chen k, tang l, lai s (2007) intestinal myiasis caused by larvae of telmatoscopus albipunctata in a taiwanese man. j clin gastroenterol. 41(4): 400–402. 15. hyun dy, cain mp, blue-hnidy de, conway jh (2004) urinary myiasis associated with ureteral stent placements. pediatr infect dis j. 23: 179. 16. zumpt f (1965) myiasis in man and animals in the old world. butterworths, london, pp. 260–270. 17. de tarso p, perre-filho p, minguini n, pierre lm, pierre am (2004) use of ivermectin in the treatment of orbital myiasis caused by cochliomyia hominivorax. scand j infect dis. 36: 503–505. 18. osorio j, moncada l, molano a, valderrama, s, gualtero s, franco-paredes c (2006) role of ivermectin in the treatment of severe orbital myiasis due to cochliomyia hominivorax. clin infect dis. 43(6): e57–59. title: quantitative estimations of the energy contents of laboratory reared and field collected mosquitoes transmitting malaria and dengue in thar desert of rajasthan state j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 286 http://jad.tums.ac.ir published online: september 30, 2018 original article a comparative study of energy contents in mosquito vectors of malaria and dengue prevailing in jodhpur city (thar desert) of rajasthan state, india *suman sundar mohanty 1, sunita meena 2, phool chand kanojia 1 1desert medicine research centre (indian council of medical research), new pali road, jodhpur, india 2department of biotechnology, jaipur national university, jaipur, rajasthan, india (received 3 apr 2016; accepted 16 apr 2018) abstract background: transmission of malaria and dengue in the desert part of india is mainly caused by anopheles stephensi and aedes aegypti respectively. the maintenance and transmission of the pathogens that cause malaria and dengue are dependent on the physiology of the mosquito vectors. we aimed to measure the energy contents in the mosquitoes transmitting malaria and dengue in the desert part of the country. methods: immature stages of mosquitoes were collected from six different larval habitats situated in jodhpur city of rajasthan state, india. the immature stages of both the mosquitoes were collected once in fortnightly from each location. quantitative estimations of the lipid, glucose, and glycogen of the laboratory-reared and field collected an. stephensi and ae. aegypti were made by spectrophotometric method. the energy contents of the larvae, pupae, females, and males were estimated in triplicates on six different occasions. results: the lipid content of laboratory-reared larvae, pupae and female mosquitoes of an. stephensi and ae. aegypti was found to be lower than their conspecific field-collected specimens. whereas, the glycogen content in the laboratory-reared larvae, pupae and female mosquitoes of an. stephensi and ae. aegypti was higher than that of their conspecific field-collected specimens. the glucose content in all the stages of the laboratory-reared an. stephensi was lower than their conspecific field-collected specimens except in few cases. conclusion: the higher amount of lipid in field-collected mosquitoes may be because of the availability of food in the natural habitat and adaptation of mosquitoes. mosquitoes living in desert climate are physiologically better equipped to survive in the desert environment. keywords: aedes aegypti, anopheles stephensi, glucose, glycogen, lipid introduction anopheles stephensi and aedes aegypti are medically the two most important arthropod vectors of malaria and dengue respectively (1). malaria and dengue cases are being reported from the desert zone due to introduction of canal irrigation for the development of agriculture. anopheles stephensi is predominant and its presence was chronologically oldest among the entire vector species available in the desert part of india (2). the transmission of malaria in the non-irrigated part of the desert is mainly vectored by this species. it breeds in earthen pits (tanka and beri) situated in and around houses and can survive in the extremes of temperature and low humidity (3). aedes aegypti is one of the most successful worldwide invaders, spreading from its native africa to most tropical and subtropical regions of the world (4, 5) and is the primary vector of dengue fever, chikungunya, and zika viruses. many dengue outbreaks have been recorded by this species, including several in brazil and the caribbean (6). aedes aegypti, the vector of dengue is widely present in india including the thar desert in north-western rajasthan (7, 8). rajasthan is one of the dengue endemic states in india (9, 10). the maintenance and transmission of the pathogens that cause malaria and numerous viral infections are dependent on the availability of competent *corresponding author: dr suman sundar mohanty, e-mail: ssmdmrc@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 287 http://jad.tums.ac.ir published online: september 30, 2018 mosquito vectors (11). adult mosquitoes require carbohydrates in the form of glucose or glycogen for flight and survival during most of their adult life (12, 13). lipid is used by the mosquitoes as energy source for long-term maintenance (14). female mosquitoes supported by stored lipids can survive for long periods resting on the ground or in dense foliage under unfavorable nutritional and climatic conditions. in addition to energy for flight, longevity, and reproduction, nutritional status also influences the immune melanization response of anopheles mosquitoes (15, 16) and susceptibility of aedes mosquitoes to arbovirus (arthropod-borne virus) infection (17, 18). newly emerged mosquitoes have different amounts of energy reserves that accumulate during their larval period (19). some mosquitoes fail to locate blood or sugar meal after emergence and the length of the time to locate food for survival is of importance (20). therefore, mosquitoes with higher energy reserves have a better chance of survival than the mosquitoes with lower level of the reserves. we aimed to measure the energy contents in the mosquitoes transmitting malaria and dengue in the desert part of the country. the levels of lipid, glucose, and glycogen in field collected and laboratory-reared an. stephensi and ae. aegypti are presented in this paper. materials and methods study site the desert zone of rajasthan state is spread over 75000km2 and divided into 12 districts (2). two seasonal rivers luni and mithri are present in the desert though their base is saline water. there is no perennial river flows in desert. the temperature varies from 49 °c in summer to 1 °c in winter. immature stages of mosquitoes were collected from different larval habitat situated in jodhpur city (latitude: 26°18′n, longitude: 73°04′e) of rajasthan state, india. immature stages were collected from the suburban and urban part of the city. the larvae were collected from jhalamond, kudi, bomba mohalla, kheme ka kuan, pratapnagar and salawas areas of the city. the study sites were chosen on the basis of the geographic locations and availability of larval habitats. sources and collection of anopheles stephensi and aedes agypti larvae and pupae of an. stephensi were gathered from larval habitats such as underground water storage tanks, overhead tanks, fountains and seepage areas using the standard sampling method. the larvae and pupae of an. stephensi were collected by strainer by the larval sampling method of service (21). however, wrigglers were collected by droppers and mugs. aedes aegypti larvae and pupae were collected from the cattle drinking waters pots, water coolers, and temporary water storage tanks. aedes aegypti larvae were collected from the bottom of the storage tanks and containers through a pipe. the outlet of the pipe was opened to a bucket and immature stages of mosquitoes were collected. they were transported to the insectary in ambient condition and reared (22). the immature stages of both the mosquitoes were collected once in fortnight from each location. the larvae and pupae collected at each site were combined and placed in pans in separate cages with date and location labels. minimum 20 collections were made from each site during the study period. preliminary identification in the field was done visually by examining the larvae during collection and species identification was confirmed in the laboratory, after the larvae had been reared to adults, using the keys by (23-27). maintenance of field-collected anopheles stephensi and aedes aegypti the immature stages of an. stephensi and ae. aegypti were collected from the field in all the seasons of the year. they were collected in containers along with the water and http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 288 http://jad.tums.ac.ir published online: september 30, 2018 substrates where they were breeding. the field collected larvae and pupae were kept in the enamel trays after transported from the field. they were reared in the water and substrates collected from the breeding habitats. the food particles in water, as well as on the substrate were the source of their diet. those larvae collected from field were not provided any additional food. they were maintained at an optimum photoperiod (10:14 [l/d] h), temperature (28±1 °c) and relative humidity (75±5%). field collected pupae were kept in the cages for the adult emergence. larvae, pupae, and adults of an. stephensi and ae. aegypti were kept separately. adults came out from the field collected pupae were kept in the cages. colonization of laboratory-reared anopheles stephensi and aedes aegypti the laboratory-reared an. stephensi colony was originated from field-collected mosquitoes of urban areas of jodhpur, india. similarly, ae. aegypti colony used in this study was originated from field-collected mosquitoes from water containers in bomba mahala of jodhpur. both the colonies were established in 2009. in the insectary, an. stephensi and ae. aegypti colonies were maintained at an optimum photoperiod (10:14 [l/d] h), temperature (28±1 °c) and relative humidity (75±5%). adults were kept in the cages (0.6m3) and allowed to mate freely. larvae were fed on a mixture of yeast powder and dog biscuit (1: 2) as food at 24h intervals. overall, 10mg of the above mixture was supplied to 100 larvae per day. soaked raisins and 10% glucose solution were provided to emerging adults. the adults were kept in cyclic cages for successive generation and maintained (22). selection of mosquitoes for the assays fifty field-collected pupae of an. stephensi and ae. aegypti were put separately along with the field water in the cages (0.6m3) for the adult emergence. same numbers of laboratory reared pupae of both the mosquitoes were transferred to enamel bowls and kept in separate cages. pupae of both the species were left in the cages for the adult emergence and pupae not emerged to adults were discarded on day-3. each of ten males and females was taken for the glucose, glycogen and lipid assays in the intervals of 24 hours. blood meal and glucose solution was not given to the experimental female mosquitoes. three sets of mosquitoes were taken for the tests on each occasion. similarly, 10 larvae and pupae were taken each time and each set of experiments were conducted three times. the experiments were conducted 6 times in a year. quantification of lipids, glucose, and glycogen the contents of lipids, glucose and glycogen in field collected and laboratory-reared mosquitoes were measured by using a spectrophotometric method (28). ten males and females were collected in the test tubes from the cages for the quantification of lipids, glucose, and glycogen. these test tubes were placed in a freezer for 5min to anesthetize the mosquitoes. each anesthetized mosquito was transferred to a 2ml microfuge vial. two hundred µl of 2% sodium sulfate solution was dispensed to each microfuge tube containing mosquito. wings of the adult mosquitoes were removed before grinding. whole mosquitoes were ground in microfuge vials by pestles for homogenization of the tissue. similarly, each larva and pupae were homogenized in a 2ml microfuge tube containing 200µl of 2% sodium sulfate solution. homogenized mosquito was mixed with 1.5ml chloroform-methanol solution (1: 2) and vertex for 1min. the above mixture was centrifuged at 3000rpm for 1 minute. pellet was retained for glycogen analysis. the supernatant containing glucose and lipids was taken up with a micro-pipette and added in another microfuge of 2ml. totally, 600µl of deionized water was added to the supernatant and mixed properly. this solution (containing sugars and lipid) was centrifuged at 3000 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 289 http://jad.tums.ac.ir published online: september 30, 2018 rpm for one minute. the lipid and sugar layers were distinctly separated. the top fraction (water/methanol) was taken up for sugar analysis and a bottom portion (chloroform) for lipid analysis. the experiments were conducted in triplicates and six times a year. lipid determination the solvent taken up from the bottom portion was evaporated completely in a heating block. thereafter, 200µl sulphuric acid was added to the tubes and re-heated for 10min to convert the unsaturated lipids to water-soluble sulphonic acid derivatives (28). these developed a deep pink colour after addition of 5ml vanillin–phosphoric acid reagent, read in a microplate reader (micro-scan ms 5608) at 525nm. lipid concentrations were obtained from a standard curve made with soybean oil. soybean oil of 1mg was dissolved in 1ml of chloroform. out of which 20, 50, 100, 200 and 400µl were put separately in tubes. these tubes were treated as explained above. the od has been taken at 525nm. the standard curve was made from the average of three sets of ods (standard as mentioned above) versus concentration. glycogen determination the precipitate in the first microfuge containing the glycogen was washed with methanol to eliminate residual sugars. then anthrone was filled up to 5ml level and mixed properly. the tube was heated for 17min and thereafter kept for cooling. the solution was mixed and 200µl was transferred to an untreated, flat-bottomed 96-well plate. the plate was read at 625nm against the blank in microplate reader (micro-scan ms 5608). glycogen concentration was calculated for individual mosquitoes from a standard curve of absorbance of known concentration of glucose as described earlier. glucose determination the top portion of the supernatant was taken up for the glucose analysis. the same was added to the tube and evaporated in a heating block till 0.1ml remained. anthrone was filled up to 5ml level and mixed properly. the heat breaks down the body sugars into their glucose units. anthrone binds to the glucose units and turning the mixture green. the anthrone sugar mixture was heated for 17min and left to cool (28). after cooling they were mixed properly. overall, 200µl was transferred to an untreated, flat-bottomed 96-well plate. the plate was read at 625nm against the blank in a microplate reader (micro-scan ms 5608). the standard solution of glucose was prepared by dissolving 1mg of glucose in 1ml of 25% ethanol. about, 25, 50, 100, 200 and 400µl of the above glucose solution were kept in separate tubes. they were processed as mentioned above and optical densities (od) were taken at 625nm. the standard curve was made from the average of three sets of ods (standard as mentioned above) versus concentration. glucose concentrations were calculated for individual mosquitoes from the standard curve of absorbance of known concentrations of glucose. statistical analysis to analyze the habitat-specific variations between the energy levels i.e. lipid, glycogen, and glucose the data the data were subjected to two-way factorial analysis of variance (anova). the analysis of variance (anova) was done by using the software ms excel software. results lipid the lipid level in both fields collected and laboratory-reared larvae, pupae, and adults of an. stephensi and ae. aegypti is shown in fig. 1. when the lipid contents of laboratory-reared and field-collected mosquitoes of an. stephensi were compared, lipid content in field-collected larvae, pupae, and female an. stephensi http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 290 http://jad.tums.ac.ir published online: september 30, 2018 was 22.85, 24.02 and 36.82µg more than the laboratory-reared mosquitoes respectively. similarly when the lipid content was compared between the field collection and laboratoryreared ae. aegypti, lipid content in field-collected larvae, pupae and females were found to be 18.71, 24.9 and 18.5µg higher than the laboratory-reared mosquitoes respectively. the difference in the lipid content between the laboratory-reared and field-collected mosquitoes of ae. aegypti was found to be highly significant (anova, f= 14.24, df= 1, 3, p= 0.03). the lipid content in the males of laboratoryreared an. stephensi and ae. aegypti was 2.66 and 1.73µg higher than the filed collected males respectively. glycogen the concentration of glycogen content in different stages of laboratory-reared and field collected stages of an. stephensi and ae. aegypti are shown in fig. 2. when the glycogen content in the laboratory-reared and field collected an. stephensi was compared, larvae, pupae, and males of laboratory conditions were 176.25, 156.54 and 183.6µg higher than the conspecific field-collected mosquitoes (fig. 2). similarly, the glycogen content in the field collected larvae, pupae, and females ae. aegypti were 103.72, 55 and 142.1 µg less than the conspecific laboratory reared ae. aegypti. the glycogen contents in the males of laboratory-reared an. stephensi and ae. aegypti was 362.16 and 84.19µg lower than the field collected males. glucose the amount of glucose in the laboratoryreared and field collected aquatic and terrestrial stages of an. stephensi and ae. aegypti are shown in fig. 3. when the glucose content in the laboratory-reared and field collected an. stephensi was compared, level of glucose in the field collected mosquitoes was significantly higher than the laboratory-reared mosquitoes (anova, f= 14.24, df= 1, 3, p= 0.03). the level of glucose content in the field collected larvae, pupae, females, and males were 30.49, 93.26, 67.12 and 46.6µg higher than the laboratory-reared mosquitoes respectively (fig. 3). the glucose content in the laboratory-reared larvae and males of ae. aegypti was 140.94 and 14.43µg lower than the fieldcollected mosquitoes. however, the glucose content in the field collected pupae and females of ae. aegypti were 9.3 and 110.18µg lower than the conspecific laboratory reared ae. aegypti. fig. 1. the level of lipid (mean±se) in different stages of the laboratory-reared and field collected anopheles stephensi and aedes aegypti http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 291 http://jad.tums.ac.ir published online: september 30, 2018 fig. 2. the level of glycogen (mean±se) in different stages of the laboratory-reared and field collected anopheles stephensi and aedes aegypti fig. 3. the level of glucose (mean±se) in different stages of the laboratory-reared and field collected anopheles stephensi and aedes aegypti discussion the benefit of the laboratory experiments was to estimate energy reserves to guide the implementation of intervention because it is essential to know how closely the physiology and behavior of laboratory maintained individual represent those from the wild. much of the information on vector biology and vector control in india comes from the terrestrial land and laboratory experimentation. therefore, we have recorded the energy reserves of the vectors transmitting malaria and dengue in the desert part of india. the lipid content of the field collected (66.8µg) and laboratory maintained (48.3µg) female ae. aegypti was 196 and 38.2 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 292 http://jad.tums.ac.ir published online: september 30, 2018 µg lower in the present study than the value recorded already (29). wherein, the glycogen (482.87µg) and glucose (182.3µg) content of laboratory maintained female ae. aegypti was 419.07µg and 118.98µg higher in the present case than the amount presented elsewhere (29). the amount of glycogen, glucose, and lipid was measured in female ae. aegypti after 72h of feeding on 10% sucrose solution and recorded the values as 62.4, 42.2 and 95.9µg respectively (19). however, the glycogen (482.87µg) and glucose (182.3µg) content in the laboratory-reared ae. aegypti in the present case was 420.47 and 140µg higher than another value recorded (19) and 47µg lower lipid content was recorded in the present study. the development of insects is heavily regulated by climate and other environmental variables and also vary substantially in response to subtle difference in diet. variations in energy reserves between the field collected and laboratory-reared mosquitoes may reveal the acquisition of resources and their subsequent assimilation by the larval stages developing in the respective larval habitats under natural conditions, occurred in the present study (30). the variations in the energy contents in the mosquitoes may be due to the habitat and adaptations of the mosquitoes. the energy reserves (glycogen, sugar, and lipid) were compared in four mosquito species in kolkota, india and recorded higher energy level in females of ae. aegypti than males (30). this observation is similar to the present study except in the case of glucose level in field-collected male. the glycogen level of both the sexes of ae. aegypti (male: 0.221mg, female: 0.223 mg) and lipid level (0.038mg) of male ae. aegypti recorded by (30) were similar to the observation made in the field collected ae. aegypti of the present study. the lipid, glucose and glycogen in the an. stephensi mosquitoes fed were measured with 10% glucose solution and recorded the values in laboratory-reared mosquitoes as 97.3, 101.9 and 82.2µg respectively (31). when the values are compared with the present study, lipid (50.66) and glucose (94.65) content in the present study was 46.64µg and 7.25µg lower than the values recorded by (31). the nutritional reserve of laboratory reserved and field collected male anopheles gambiae and recorded a 5µg high lipid level in the field collected male an. gambiae than the laboratoryreared one (32). we have recorded a 2.7µg higher lipid in the laboratory-reared males (34.9 µg) than the field collected males (32.2µg). this may occurred because both the species responded in a similar fashion to the different larval diets (33). the survival and reproductive strategies of the female mosquito differ from the male in many senses, at least one of related to egg production and maturation (34). dog biscuit is providing carbohydrates and lipid to the larvae and yeast is imparting protein to the larvae. the viability of larva and pupa does not depend upon the carbohydrates, however growth and molting of the larvae is largely dependent upon the protein source (14). the laboratory-reared larvae were provided the food routinely so they can molt from one stage to other. after the introduction of the indira gandhi canal in to the desert of rajasthan, the water retention has been increased in this desert region. this leads to the establishment of mosquito colonization. mosquitoes get breeding habitat throughout the year because of various water retention. the surrounding of the desert climate provides better habits for mosquito breeding. the field-collected mosquitoes were with more energy may be due to the presence of open water habitats that provide significantly more micro-invertebrate dietary resources for the larvae. nutrient reserves of adult mosquitoes obtained during the larval stage plays important role in the longevity of mosquitoes. larvae supplied with the high food amount resulted in adults with higher longevity. the cases of malaria and dengue are increased continuously in this region after inception of the indira gandhi canal; therefore, physiology of the mosquito should be investigated and compared with insecticide resistance http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 286–295 ss mohanty et al.: a comparative study of … 293 http://jad.tums.ac.ir published online: september 30, 2018 and also with the incrimination of malaria parasite. conclusion malaria and dengue in the desert part of india are mainly transmitted by an. stephensi and ae. aegypti respectively. energy content of the mosquitoes was compared between two rearing habitats. the lipid content of laboratory-reared an. stephensi and ae. aegypti was found to be lower than their conspecific fieldcollected specimens. the glycogen content in the laboratory-reared larvae, pupae and female mosquitoes of both the types were higher than that of their conspecific field-collected specimens. the glucose content of the laboratoryreared an. stephensi was lower than that of the conspecific field-collected specimens. the 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dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 393 original article field evaluation of outdoor ultra-low volume (ulv) applications against phlebotomine sand flies (diptera: psychodidae) in al rabta, north-west of libya mostafa ramahdan dokhan 1,2, *mohamed amin kenawy 3 , taher shaibi 1,4, badereddin bashir annajar 1,5 1national center for disease control, ministry of health, tripoli, libya 2zoology department, faculty of sciences, sabratha, university of zawia., zawia, libya 3department of entomology, faculty of science, ain shams university, abbassia, cairo 11566, egypt 4zoology department, faculty of science, university of tripoli, tripoli, libya 5public health department, faculty of medical technology, university of tripoli, tripoli, libya (received 5 jan 2016; accepted 21 aug 2017) abstract background: al rabta is a rural area in the north-west of libya that represents an important focus of zoonotic cutaneous leishmaniasis. this study aimed to evaluate the effect of ultra low volume (ulv) applications in controlling sand flies and its impact on leishmaniasis transmission in this area. methods: two neighboring villages were selected: al rabta west (rw) as cypermethrin treated village and al rabta east (re) as check one. the ulv was evaluated through 3 spraying cycles during apr, jun and sep 2013. in the two villages, a number of outdoor sites were selected for sampling of sand flies (twice a month) using the cdc light traps. the cases of cl reported in the two villages during the study period were obtained from al rabta health center. results: the two villages were similar where 9 species of sand flies (6 of phlebotomu and 3 of sergentomyia) were collected of which s. minuta and p. papatasi were the abundant species. as compared to the preulv spraying, during the postspraying periods: i) the reduction in abundance of the different species ranged from 20.85 to 77.52% with 46.69% as an overall reduction for all species altogether and, ii) in significantly (p˃ 0.05) higher mean ratio of males: females for all species altogether (1:2.41). moreover, ulv spraying resulted in the absence of cl (leishmania major) cases (passive case detection) conclusion: the efficiency of ulv spraying in reducing sand fly population, cl cases and consequently limits the disease transmission. keywords: sand flies, ulv application, cl cases, libya introduction the current control measures against leishmaniasis rely on chemotherapy to alleviate disease and on vector control to reduce transmission (1). to date, there is no vaccine for routine use against leishmaniasis. the main control methods for sand fly vectors were reviewed including insecticide spraying (houses, the peridomestic environment, and resting sites), environmental management, personal protection, integrated con trol and biological control (2-5). these methods were applied and evaluated by several workers (6-12). in general, sand fly control is complicated by the many species of sand flies and reservoir hosts involved in the transmission of multiple leishmania species over a variety of geographic habitats (3). regarding the ultra-low volume (ulv) applications, such application was evaluated against sand fly species in equatorial kenya *corresponding author: dr mohamed a. kenawy, e-mail: mohamedkenawy85@yahoo.com http://jad.tums.ac.ir published online: september 08, 2017 j arthropod-borne dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 394 and reported that sampling of the wild populations before and after treatments suggested local population suppression of sand fly from ulv treatments, as well as a possible repellent effect in nearby untreated areas (13). however, various insecticide applications, including ulv, did not have a significant effect against sand flies in southern iraq (14). overall, 21 ulv spray operations were conducted in the al anbar provincial capital of ramadi, iraq during five months of the sand fly season and stated that “based on our surveillance program, we do not know whether our ulv missions had any impact on sand fly populations” (15). in libya, cutaneous leishmaniasis (cl) is an endemic disease since 1910 when it was detected for the first time (16). several cases were reported and exclusively originated from the north-western (nw) districts of the country (17, 18). zoonotic cutaneous leishmaniasis (zcl) due to l. major was confirmed (19-21) and is largely the main form in this country (22). only two reports (19, 23) concerning the implication of phlebotomus papatasi and p. sergenti as suspected vectors of cl in nw region. moreover, in only three occasions, p. papatasi and p. longicuspis were found positive for leishmania spp. (16, 20, 24). the national program to combat leishmaniasis in libya was established in nov 2006. the program was launched to achieve the main objectives of limiting the spread of the disease to new areas and treatment of infected cases in endemic areas. the initial phase of the program management started in 2007 to intensify efforts and give priority to reduce the epidemic of explosions that have occurred in the tawergha area through chemical control of wild rodents (the main reservoirs of zcl, l. major). the second phase (2008) included an expanded campaign against sand flies in all infested areas through residualspraying of outdoor resting sites (unpublished reports of the national control program of leishmaniasis, ncpl, national center for disease control, ncdc, ministry of health, moh, tripoli, libya). al rabta in the nw of libya is one of foci where cl is endemic for a long time with huge outbreak occurred during 1977– 1980 and 2004–2012 (unpublished report of the ncpl, ncdc, moh, tripoli, libya 2013). for this, the present study was carried out to evaluate the effect of ulv applications in controlling sand flies and its impact on cl transmission in this area. materials and methods study area the study was carried out from apr to sep 2013 in two neighboring villages (about 3km apart from each other) in al rabta area: al rabta east (re) and al rabta west (rw) (fig. 1). al rabta is a rural area located 80 km south of tripoli in the foothill of nafusa mountain (32°9'46.59"n, 12°50'50.65"e) with an altitude of about 300m above sea level and a population of about 6000 inhabitants (2010 estimates). most people in the area practice farming and animal rearing. around the houses are shelters for domestic animals made of brick and fruit trees. the area is characterized by a warm and dry climate with an average annual rainfall of 16mm. the mean annual temperature of the area is 21 °c. january is usually the coldest month, while aug is the warmest month. the summer temperatures can exceed 45 °c. the rainy season is from nov to feb and jul being the driest month (http://www.libya.climatemps.com). in the two villages, a number of outdoor sites (6 in re and 7 in rw) at a distance of 300 m from each other were selected for collecting of sand flies. these sites were selected where rodent burrows were numerous. ultra-low volume application the rw village was selected as the insecticide treated area while the re was se http://jad.tums.ac.ir published online: september 08, 2017 j arthropod-borne dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 395 lected as the check village that received no insecticide application. the treated area was sprayed with cypermethrin™ 10.0% ec, at a dilution of 1l per 200l of water and at a rate of about 350l per hectare. the area received 3 spraying cycles (table 1) in apr (17–25), jun (16–21) and sep (15–19). the whole area around the sand fly sampling sites (100 hectares/cycle) was sprayed including ground, trees, old settlements, caves, etc. sand fly collection and processing sand flies were collected for three consecutive nights (twice a month) during the study period from apr to sep 2013 using the cdc (center for disease control) light traps (model 512, john w. hock, and gainesville, fl, usa). except for the 3 ulv cycles, the sand fly collection was carried out directly before and after the spraying cycle (table 1). the traps (6 and 7 traps/ night for re and rw, respectively) were set before sunset and collected next morning. traps were hanged on approximately 30cm from the ground. the collected sand flies were aspirated, placed in labeled paper cups that kept in a picnic icebox while being transported to the field laboratory. in the laboratory, flies were preserved in 70% alcohol and then mounted in puri’s medium. flies were sexed and identified using morphological keys (16, 25). leishmaniasis cases the cases of cl reported in the two villages during the study period (passive case detection, pcd) were obtained from the respective al rabta health center. statistical analysis the means sd's for the obtained data were calculated and analyzed by one-way analysis of variance (anova). the chi-squared (χ2) analysis was used to test the deviation of the resulting fly sex ratios (male: female) from the expected 1:1 ratio. the ssp (26) was used for statistical analysis. results a total of 1594 and 2332 sand flies of 9 species (6 of phlebotomu and 3 of sergentomyia) were collected in rw and re, respectively during the study period (apr–sep 2013) of which s. minuta (32.50% and 26.20% in rw and re, respectively). phlebotomus papatasi (22.21% and 23.03% in rw and re, respectively) were the most abundant species (table 2). these were followed in descending order of abundance by s. fallax, p. alexandri, p. longicuspis, p. sergenti, p. chabaudi, p. langeroni and s. antennata in the two villages. the effect of the outdoor ulv spraying on the densities of the different sand fly species (mean number of flies per collection sites, n= 7 sites) was examined in rw through 3 spraying cycles during apr, jun and sep 2013. the comparable figures in re as check village (n= 6 sites) were also examined during the same period (table 3). the compiled mean of the three cycles was calculated and revealed that in rw, significantly lower densities were found during post spraying than those during pre-spraying (38.91–52.78% reduction) for p. papatasi p˂ 0.001, p. longicuspis p˂ 0.01, p. sergenti p˂ 0.05, s. minuta p˂ 0.001, s. fallax p˂ 0.001 and for all sand fly species altogether (46.69% reduction, p˂ 0.001). besides, lower densities were found during post spraying for p. alexandri, p. chabaudi, p. langeroni, and s. antennata than those during the pre-spraying (20.85– 77.52% reduction) however, the difference was not significant (p˃ 0.05). in re, the comparable fly numbers/site for preand post-spraying were insignificantly different (p˃ 0.05) for all sand fly species. comparison of the total fly densities in the two villages indicated that significantly (f= 7.72, df= 1, 11, p˂ 0.05) lower pre-spraying density in rw (75.57 fly/ site) than in re (107.00 flies/site). similarly, significantly (f= 46.54, df= 1, 11, p˂ 0.05) lower post-spraying density was observed in rw http://jad.tums.ac.ir published online: september 08, 2017 j arthropod-borne dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 396 (40.29 fly/site) than in re (104.17 fly/site). the effect of the 3 ulv spraying cycles on the overall sex ratios of sand fly species in rw was examined (table 4) and revealed that for pre-spraying periods, all ratios do not deviate from the expected 1:1 ratio (p˃ 0.05) except for p. alexandri (p˂ 0.05), p. longicuspis (p˂ 0.01), p. sergenti (p˂ 0.05) and p. chabaudi (p˂ 0.05). for post-spraying periods, all ratios were significantly deviated from the expected 1:1 ratio in favor of males except that of s. fallax (p˃ 0.05). the overall mean ratio for all species altogether indicated higher post-spraying ratio (1:2.41) than that of pre-spraying (1:1.52), however, means were not significantly different (f= 1.61, df= 1, 14, p˂ 0.05). the results of reported cl cases (pcd) indicated that during the study period, no cases were reported in rw in comparable to three cases in re (one each in apr, aug and sep 2013). table 1. dates of ulv spraying cycles and sand fly collections before and after the spraying cycles (2013) period apr jun sep pre-spraying collection 14–16 13–15 12–14 ulv spraying 17–25 16–21 15–19 post-spraying collection 26–28 22–24 20–22 table 2. relative abundance of sand flies collected in al rabta west (rw) and al rabta east (re) from april to september 2013 species rw re no % no % phlebotomus p. papatasi 354 22.21 537 23.03 p. alexandri 172 10.79 242 10.38 p. longicuspis 130 08.16 220 09.43 p. sergenti 116 07.28 177 07.59 p. chabaudi 046 02.89 099 04.25 p. langeroni 025 01.57 053 02.27 sergentomyia s. minuta 518 32.50 611 26.20 s. fallax 222 13.93 358 15.35 s. antennata 11 00.69 035 01.50 total 1594 2332 table 3. mean density of sand flies (fly/collection site) preand postulv spraying after three spraying cycles in al rabta west (rw) from april to september 2013 species treated: al rabta west (rw) check: al rabta east (re) pre-a posta f(1,12)b % rc prea posta f(1,10)b p. papatasi 15.43 09.29 20.54*** 39.78 026.67 023.17 0.86ns p. alexandri 06.14 04.86 01.94ns 20.85 009.33 008.83 0.09ns p. longicuspis 06.57 03.29 11.58** 49.92 009.67 009.50 0.01ns p. sergenti 05.14 03.14 05.65* 38.91 007.50 008.00 0.08ns p. chabaudi 02.86 01.29 04.65ns 54.90 005.00 005.67 0.19ns p. langeroni 01.29 00.29 03.87ns 77.52 002.50 002.50 0.00ns s. minuta 25.71 12.14 53.61*** 52.78 028.17 025.00 0.93ns s. fallax 10.86 05.14 31.37*** 52.67 016.33 016.67 0.01ns s. antennata 01.29 00.86 01.23ns 33.33 001.83 001.33 0.74ns all species 75.57 40.29 64.95*** 46.69 107.00 104.17 0.03ns asd’s were omitted, bns: not significant (p˃ 0.05), *p˂ 0.05, **p˂ 0.01, ***p˂ 0.001, cr: reduction http://jad.tums.ac.ir published online: september 08, 2017 j arthropod-borne dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 397 table 4. numbers and sex ratios (male: 1 female) of sand flies preand postulv spraying in al rabta west (rw) from april to september 2013 species prepost m f m:1f m f m:1f p. papatasi 060 059 1.02ns 043 022 1.95* p. alexandri 029 021 2.42* 021 013 1.62* p. longicuspis 035 011 3.18** 017 006 2.83* p. sergenti 024 012 2.00* 019 003 6.33** p. chabaudi 013 007 1.86* 006 003 2.00* p. langeroni 003 006 0.50ns 002 000 --- s. minuta 102 078 1.31ns 053 032 1.66* s. fallax 036 042 0.86ns 012 024 0.50 ns s. antennata 003 006 0.50ns 001 000 --- total 305 242 1.26ns 174 103 1.69* mean±sd 1.52 ±0.92 2.41 ±1.86 ns: not significant (p˃ 0.05), *p˂ 0.05, **p˂ 0.01 (χ2test) fig. 1. location of al rabta area in the northern-west of libya discussion nine sand fly species (p. papatasi, p. alexandri, p. longicuspis, p. sergenti, p. chabaudi, p. langeroni, s. minuta, s. fallax and s. antennata) were collected in re and rw in this study. phlebotomus papatasi is the most important as the main vector of l. major, the causative agent of zcl previously isolated from such fly species in nw (16, 22). the same species in addition to s. clydei, s. christophrsi and one unidentified p. (larroussius) sp were previously reported in al rabta (16). overall, 1594 and 2332 sand flies were collected during the study period in rw and re, respectively of which s. minuta and p. papatasi were the most abundant species in both villages. both p. langeroni and s. antennata were rare. almost similar results mainly for p. papatasi were obtained (16, 17, 27) and investigated sand flies in some nw areas. leishmaniasis represents a major public health problem in several countries of the eastern mediterranean region (emr) of the who (28) including libya. two forms of cutaneous leishmaniasis exist in libya, these are zcl (l. major) and anthroponotic cutaneous leishmaniasiss (acl, l. tropica). aoun and bouratbine (22) reviewed the situation of leishmaniasis in libya and stated “most published reports in libya concern zcl, which is largely the main form in this country”. http://jad.tums.ac.ir published online: september 08, 2017 j arthropod-borne dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 398 the main zcl foci are located in the nw of the country (17). due to lack of scientifically based control program, and due to the wide spread of animal reservoirs (the rodent psammomys libycus and meriones obesus) the disease was largely extended to other new areas. according to the report of the national ncdc, moh, tripoli, libya (2008), about 30000 zcl cases were reported during the last 30 years. during 2005–2008, about 6000 cases of which 1800 in 2008 were reported (28). the best method to interrupt any vector borne disease is to reduce the man-vector contact (29). several methods exist at present for leishmaniasis control used individually or in combination. the selection of the method or combination of methods depends on the type of the leishmaniasis to be controlled and the method should be situation specific (4). the main sand fly vector control methods were reviewed and evaluated (3-12). the control efforts of the sand fly vectors of leishmaniasis are problematic and directed only to adults. this is because their larvae develop in largely unknown terrestrial habitats making them impervious to available control measures (3, 5). however, the diversity of phlebotomine biology and ecology makes it very difficult to adapt one control strategy for all endemic areas. it can be effective in reducing the transmission of the disease but mainly in places where the vector is endophilic and peridomestic (16). in certain areas, effective control has been achieved as a side effect of malaria control programmes (30-32). depending on application techniques, timing and target species, sand flies are known to be highly susceptible to insecticides (3, 33-38) and only a few cases of p. papatasi resistance to ddt have been reported (39, 40). however, the prolonged contact with insecticides might lead to the appearance of resistance (41) that needs to assess the potential of sand flies to develop resistance that could cause problems in control campaigns (2). the space spraying with ulv is widely used to control sand flies, but few rigorous studies have evaluated its efficacy (42). in this study, the application of 3 ulv spraying cycles with cypermethrin resulted in reduction of sand fly densities that ranged from ca 21% to 78% for the 9 species and an overall reduction for all species altogether of ca 47% (p˂ 0.001). such results indicate the efficiency of ulv spraying in reducing sand fly population. sampling of wild populations (p. duboscqi) in western kenya before and after treatments suggested local population suppression from ulv treatments (13). “sand flies in libya are most active on warm, clear nights with little wind as the case in iraq” (43). such conditions are also favorable for applying ulv-based adulticides (42), however, various insecticide applications, including ulv, did not have a significant effect against sand flies in southern iraq (14). moreover, 21 ulv spray operations were conducted in camp ramadi, iraq during five months of the sand fly season (apr to aug 2009) (15) and although they obtained lower mean catch/night after spraying (0.563–2.002) than before ulv operations (0.839–2.002), however, means were not significantly different except on 25 may (p< 0.05). ”based on our surveillance program, we do not know whether our ulv missions had any impact on sand fly populations. our insecticide applications were part of an actual vector control program lacking an untreated area to serve as a control, which complicates the interpretation of our results”. such contradictory results may be due to the local conditions in the study areas (weather conditions, sand fly fauna etc.). the effect of the 3 ulv spraying cycles on the overall sex ratios (male: female) of sand flies in rw indicated that most of ratios for prespraying periods do not deviate from the expected 1:1 ratio (p˃ 0.05). with the exception of s. fallax (p˃ 0.05), all postspraying ratios were significantly deviated from the expected 1:1 ratio in favor of males. mean http://jad.tums.ac.ir published online: september 08, 2017 j arthropod-borne dis, september 2017, 11(3): 393–402 mr dokhan et al.: field evaluation of … 399 ratio for all species altogether indicated higher ratio post-spraying than that pre-spraying (p˂ 0.05) which may indicate that ulv application affects females more than males, however, further investigations are required. no cl cases (pcd) were reported in rw during the period of the three cycles of insecticidal treatment compared to 3 cases reported in re. in a concurrent study (44), the reported cl cases during the sand flies activity period (apr to nov 2012 and 2013) in rw were 11 and 4 in the two years, respectively (ie 63.64% reduction in cases during ulv application) compared to 8 and 9 cases in re in 2012 and 2013, respectively. however, such disappearance of cl cases due to ulv application in this study may not be conclusive, further studies are required including active-case detection (acd). there are no available reports on the effect of ulv on leishmaniasis incidence. for other insecticide applications however, such data exists. the impact of indoor residual spraying and impregnated bed nets were reviewed in reducing the leishmaniasis cases in several asian countries (9). the effect of 65% permethrin spotwas examined on the prevalence of canine visceral leishmaniasis (vl) and the abundance of sand flies in two neighborhoods in corumbá, mato grosso does sul, brazil known to have a high prevalence of vl (45). a reduction in leishmaniasis prevalence was observed. the results suggest that regular use of 65% permethrin during months of high risk for canine vl can be a useful strategy for reducing the prevalence of this disease in hyperendemic areas. in bangladesh, the effect of a communitybased intervention was evaluated with insecticide impregnation of existing bed-nets in reducing vl incidence and found that this intervention reduced vl by 66.5% (46). conclusion the stand-alone ulv spraying with cy permethrin proved effective in reducing the outdoor sand fly population by ca 47%. however, if an endophilic sand fly species involved, this needs to be supplemented by indoor insecticide applications to increase the efficiency of sand fly/leishmania control operations. the situation in libya necessitates continuation and strengthens the ongoing leishmaniasis control program. acknowledgements the study is a part of phd thesis (dokhan, mr), department of entomology, faculty of science, ain shams university, cairo, egypt and supported by the national control program of leishmaniasis (ncpl), national center for disease control (ncdc), ministry of health, tripoli, libya. we are grateful to the members of health care service unit, al rabta, libya for providing data of the reported cl cases, and of leishmania unit, ncdc for their extensive efforts in sand fly collections and control. special thanks to dr said a. doha and dr shabaan s. 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http://www.ncbi.nlm.nih.gov/pubmed/?term=karmoker%20mk%5bauthor%5d&cauthor=true&cauthor_uid=23764246 http://www.ncbi.nlm.nih.gov/pubmed/?term=ghosh%20d%5bauthor%5d&cauthor=true&cauthor_uid=20946233 http://www.ncbi.nlm.nih.gov/pubmed/?term=matlashewski%20g%5bauthor%5d&cauthor=true&cauthor_uid=23764246 http://www.ncbi.nlm.nih.gov/pubmed/?term=nabi%20sg%5bauthor%5d&cauthor=true&cauthor_uid=23764246 http://www.ncbi.nlm.nih.gov/pubmed/?term=kroeger%20a%5bauthor%5d&cauthor=true&cauthor_uid=20946233 http://www.ncbi.nlm.nih.gov/pubmed/23764246 http://www.ncbi.nlm.nih.gov/pubmed/23764246 http://www.ncbi.nlm.nih.gov/pubmed/23764246 http://www.ncbi.nlm.nih.gov/pubmed http://www.ncbi.nlm.nih.gov/pubmed microsoft word 7dr kalantar rtl 89 10 1_2_.doc iran j arthropod-borne dis, 2010, 4(2): 50–55 b davari et al.: frequency of resistance and … 50 original articles frequency of resistance and susceptible bacteria isolated from houseflies b davari1, *e kalantar2, a zahirnia3, sh moosa-kazemi4 1department of parasitology, school of medicine, medical university of kurdistan, sananadaj, iran 2liver and digestive research center, tohid hospital, medical university of kurdistan, sananadaj, iran 3department of parasitology, school of medicine, medical university of hamedan, hamedan, iran 4department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran (received 3 mar 2010; accepted 4 jul 2010) abstract background: in this study, we determine the vector competence of musca domestica with reference to the transmission of susceptible and resistance bacterial strains in hospitals and slaughter house in sanandaj city, west iran. methods: totally 908 houseflies were collected to isolate bacteria from their external body based on standard procedures.antibiotic susceptibility testing was performed by kirby-bauer disc diffusion method on mueller hinton agar based on recommendations of clsi (formerly the national committee for clinical laboratory standards). results: from collected houseflies, 366 bacteria species were isolated. the most common isolated bacterium at hospitals was klebsiella pneumoniae 43.3% (n= 90) followed by pseudomonas aeruginosa 37% (n= 77), while that of slaughterhouse was proteus mirabilis. 29.1% (n= 46) followed by citrobacter freundii 28.4% (n= 45). among all the isolates from hospitals, cephalexin, chloramphenicol, ampicillin, and tetracycline, resistance rates were above 32.5% and gentamicin expressed the highest susceptibility among all the isolates from hospitals. it is worth to note that k. pneumoniae showed 61% and 44.5% resistance to cephalexin and chloramphenicol respectively. similarly, all isolates from slaughterhouse were more than 28% and 30% resistant to cephalexin and chloramphenicol respectively. surprisingly, among all the isolates, citrobacter freundii were highly resistant to gentamicin. conclusion: houseflies collected from hospitals and slaughterhouse may be involved in the spread of drug resistant bacteria and may increase the potential of human exposure to drug resistant bacteria. keywords: house fly, bacterium, antibacterial resistance, hospitals, slaughterhouse introduction the common house fly, musca domestica is a medically-important insect worldwide (fotedar 2001, graczyk et al. 2001, kabkaew et al. 2007). houseflies have been implicated as vectors or transporters of various human pathogens, including vibrio cholerae, enterobacteriaceae pathogens, staphylococcus aureus, and pseudomonas spp. (olsen 1998, fotedar 2001, rajendran and pandian 2003). transmission takes place when the fly makes contact with people or their food. as many as 500000 microorganisms may swarm over its body and legs (thirumalai vasan et al. 2008). “flies can spread diseases because they feed freely on human food and dirty matter alike. the fly picks up disease-causing organisms while crawling and feeding. the diseases that flies can transmit include enteric infections, eye infections, poliomyelitis and certain skin infections.” thus, houseflies are widely recognized as potential reservoirs and vectors of food borne pathogens (pandian and asumtha 2001, khobdel et al. 2008). *corresponding author: dr enayat kalantar, email: kalantar_enayat@yahoo.com iran j arthropod-borne dis, 2010, 4(2): 50–55 b davari et al.: frequency of resistance and … 51 it is worth to note that a few studies also indicate that houseflies have been suspected to be reservoirs and vectors for pathogens (zarin et al. 2007, barin et al. 2010). there have been no studies on the carriage of antibiotic-resistant, pathogenic bacteria by m. domestica in hospitals and slaughterhouse in sanandaj. an increasing frequency of antibiotic resistance has been reported from all over of the world. in this regard, an attempt was made to determine the frequency of resistance and susceptible bacteria isolated from houseflies in sanandaj, west of iran. materials and methods houseflies were captured by a sterile nylon net from the wards and corridors of the tohid hospital, beassat hospital, and the slaughterhouse of the city. the collected flies were transferred immediately to the entomology laboratory, and identified to species level by morphological characters such as thorax, wings and antenna. after identification, 1 ml of sterile physiological saline solution was added to each vial, which was shaken vigorously for 1 min with the fly remaining inside. the fly was then removed from the saline, and was checked for bacteria dislodged from the external surfaces of the fly. bacterial counts serial dilutions of a subsample of each bacterial suspension were prepared in sterile saline. each dilution was then inoculated onto two plates of plate-count agar and incubated overnight at 37 °c. colony forming units (cfu/ ml) were then counted so that the total numbers of bacteria recovered from the external surface of each fly could be estimated. briefly, houseflies individually were shaken thoroughly in sterile saline solution (2 ml) for 2 min. the suspension was then serially diluted and inoculated on macconkey agar, and blood agar. plates were incubated for 24 h at 37 °c. the resulting isolates were characterized morphologically and further identifications were carried out following the methods of koneman et al. 1992. antibiotic susceptibility test was performed by kirby-bauer disc diffusion method on mueller hinton agar based on recommendations of clsi (formerly the national committee for clinical laboratory standards) (nccls, 2003). the following antibiotics were used in this study: erythromycin, streptomycin, ampicillin, tetracycline, kanamycin ,chloramphenicol, co-trimoxazole, gentamicin, ciprofloxacin, nitrofurantoin, ceftriaxone, and cephalexin which were purchased from patan teb company. results from the 908 houseflies collected from the hospitals and slaughterhouse at sanandaj, 366 (40.3%) bacterial species were isolated (table 1). the most common bacterium isolated from m. domestica at hospitals was klebsiella pneumoniae 43.3% (n= 90) followed by pseudomonas aeruginosa 37% (n= 77), while that of slaughterhouse was proteus mirabilis 29.1 (n= 46) followed by citrobacter freundii 28.4% (n= 45) (table 2). among all the isolates from hospitals, cephalexin, chloramphenicol, ampicillin, and tetracycline, resistance rates were above 32.5% and gentamicin expressed the highest susceptibility among all the isolates from the hospitals. it is worth to note that k. pneumoniae showed 61% and 44.5% resistance to cephalexin and chloramphenicol, respectively (table 3). similarly, all the isolates from slaughter house were more than 28%, 30% resistance to cephalexin and chloramphenicol respectively (table 4). surprisingly, among all isolates, citrobacter freundii were highly resistant to cephalexin. iran j arthropod-borne dis, 2010, 4(2): 50–55 b davari et al.: frequency of resistance and … 52 table 1. bacterial carrying rates for musca domestica collected from hospitals and slaughter house at sanandaj no. of bacterial species isolated from each fly number isolates for habitats hospitals slaughter house total male female total male female total total positive flies 61 147 208 56 102 158 total negative flies 78 132 210 127 205 332 total flies examined 139 279 418 183 307 490 908 table 2. details of bacteria isolated from house fly collected from hospitals and slaughter house at sanandaj number isolates for habitats bacteria hospitals slaughter house number percent number percent k. pneumoniae 90 43.3 23 14.5 p. aeruginosa 77 37.0 0.0 0.0 citrobacter freundii 12 05.7 45 28.4 e. coli 19 09.1 26 16.4 bacillus cereus 10 04.8 18 11.4 proteus mirabilis 0.0 0.0 46 29.1 total 208 100 158 100 table 3. antibiotic susceptibility pattern (%) of identified bacteria in hospitals at sanadaj, iran bacteria k. spp. (90) p. aeruginosa (77) e. coli (19) b. cereus (10) proteus mirabilisa (12) antibiotic r i s r i s r i s r i s r i s ery 46.6 31.1 11.1 39.0 36.3 24.6 26.0 42.1 31.5 20.0 60.0 20 16.0 41.6 41.6 strep 38.8 34.4 26.6 32.5 41.5 25.9 31.0 26.3 42.1 30.0 50.0 20.0 33.0 66.6 0.0 amp 43.3 38.8 17.7 35.0 45.4 19.4 31.0 26.3 42.1 30.0 40.0 30.0 25.0 33.3 41.6 tetra 43.3 41.1 15.5 29.0 53.2 16.8 21.0 42.1 36.8 20.0 50.0 30.0 16.0 50.0 33.3 kana 27.8 23.3 48.8 26.0 41.5 32.4 15.0 57.8 26.3 20.0 60.0 20.0 16.0 41.6 41.6 chlo 44.5 46.6 08.8 32.5 23.3 44.1 31.0 42.1 26.3 20.0 70.0 10.0 16.0 58.3 25.0 co-tri 35.5 31.1 33.3 31.0 32.4 36.3 26.0 63.1 10.5 10.0 50.0 40.0 08.3 33.3 58.3 gen 11.0 16.6 72.2 09.0 46.7 44.1 15.0 42.1 42.1 20.0 50.0 30.0 16.0 50.0 33.3 cipro 15.5 22.2 62.2 14.0 49.3 36.3 15.0 63.1 21.0 10.0 60.0 30.0 08.3 58.3 33.3 nitrof 17.7 15.5 66.6 16.8 23.3 59.7 21.0 47.3 31.5 20.0 50.0 30.0 16.0 50.0 33.3 ceftri 24.4 26.6 48.8 22.0 54.5 23.3 26.0 42.1 31.5 30.0 60.0 10.0 25.0 41.6 33.3 cepha 61.0 31.1 07.7 44.0 28.5 27.2 42.0 47.3 10.5 40.0 50.0 10.0 33.0 50.0 16.6 erythromycin, streptomycin, ampicillin, tetracycline, kanamycin ,chloramphenicol, co-trimoxazole, gentamicin, ciprofloxacin, nitrofurantoin, ceftriaxone , cephalexin (s) = sensitive, (i) = intermediate, (r) = resistant iran j arthropod-borne dis, 2010, 4(2): 50–55 b davari et al.: frequency of resistance and … 53 table 4. antibiotic susceptibility pattern (%) of identified bacteria at slaughter house in sanandaj, iran bacteria klebsiella spp. (23) proteus mirabilis (46) e. coli (26) citrobacter freundii (45) bacillus cereus (18) antibiotic r i s r i s r i s r i s r i s ery 21.7 43.4 34.7 15.0 43.4 41.3 19.0 61.5 19.2 15.4 17.7 66.0 11.1 33.3 55.0 strep 26.0 30.4 43.4 15.0 52.1 32.6 23.0 69.2 07.6 24.4 22.2 53.0 11.1 22.2 66.0 amp 21.7 26.0 43.4 17.0 60.8 21.7 27.0 46.1 23.0 20.3 17.7 62.0 22.2 27.7 50.0 tetra 13.0 34.7 52.1 13.0 43.4 43.4 19.0 69.2 11.5 22.2 08.8 68.0 16.6 22.2 61.0 kana 17.0 26.0 56.5 15.0 41.3 43.4 15.0 65.3 19.2 24.4 24.4 51.0 05.5 22.2 72.0 chlo 30.0 34.7 34.7 14.0 60.8 26.0 23.0 57.6 19.2 15.5 15.5 68.0 11.1 33.3 55.0 co-tri 30.0 21.7 47.8 06.5 52.1 41.3 27.0 53.8 15.3 06.6 11.1 82.0 16.6 38.8 44.0 gen 08.6 56.5 34.7 13.0 43.4 43.4 15.0 46.1 38.4 08.8 13.3 77.0 0.0 22.2 77.0 cipro 13.0 39.1 47.8 06.5 54.3 39.1 11.5 65.3 23.0 04.4 11.1 84.0 0.0 38.8 61.0 nitrof 13.0 52.1 34.7 15.0 43.4 41.3 19.0 53.8 26.9 20.0 26.6 53.0 0.0 27.7 72.0 ceftri 21.7 34.7 43.4 19.0 39.1 41.3 23.0 57.6 19.2 17.7 22.2 60.0 0.0 44.4 55.0 cepha 47.0 30.4 21.7 28.0 45.6 26.0 30.0 65.3 03.8 66.0 20.0 13.3 05.5 33.3 61.0 erythromycin, streptomycin, ampicillin, tetracycline, kanamycin, chloramphenicol, co-trimoxazole, gentamicin, ciprofloxacin, nitrofurantoin, ceftriaxone , cephalexin discussion “the biology and ecology of m. domestica make it an ideal mechanical vector of human and animal pathogens. cattle barns, poultry houses, slaughter houses, and hospitals are sites where house flies can reproduce” (peter et al. 2007). many scientists indicated that the external organs of m. domestica (legs, wings and mouthparts) constituted a large source of bacteria they isolated (graczyk 1999, mutsuo et al. 1999). the results of this study indicated that m. domestica could play a great role as a mechanical carrier of bacteria. in this study, most of the bacteria isolated were medically important, including k. pnenumonae, p. aeruginosa, proteus mirabilis. these findings agree with the results of vazirianzadeh et al. (2008) in ahvaz, which showed presence escherichia coli, p. aeruginosa, and k. pneumonia on the housefly collected from slaughterhouse and zoo. our results are in accordance with other reports which highlight the importance of houseflies in carrying various pathogenic bacteria particularly k. pneumoniae being the most important at usa and iran (thaddeus et al. 2001, khalil et al. 1994). the house flies caught in hospitals carried pathogenic bacteria more often than those caught at slaughter house which is in contrast with sulaiman et al. (2000) study. one of the most important problems facing global public health today is antimicrobial resistance. the problem is most horrible in developing countries, where the bacterial infections causing human disease are also those in which emerging antibiotic resistance is most evident (shears 2000, kalantar et al. 2008). the resistance patterns of the k. pneumoniae isolated in the present study are shown in table 3 and 4. the k. pneumoniae isolated from the hospitals houseflies were more resistant to cephalexin as compared to that of isolated from the slaughterhouse. fotedar et al. (1992) and sramova et al. (1992) reported similar multiple-resistance to antibiotics, in klebsiella spp. from houseflies in hospitals environments. similarly, the resistance patterns of the p. aeroginosa from hospitals environment houseflies were frequently multiple-resistant, iran j arthropod-borne dis, 2010, 4(2): 50–55 b davari et al.: frequency of resistance and … 54 with more than 32% of the isolates each being resistant to erythromycin, streptomycin, ampicillin, and cephalexin. therefore, houseflies trapped in hospitals may also participate more in the dispersion of antibiotic resistance into the environment. multiple resistances to antibiotics are common among p. aeruginosa isolated from different clinical sources in iran (kalantar et al. 2009). the present study indicates that housefly m. domestica poses a possible health risk to communities proved that the isolated strains of bacteria were resistant to various antibiotics. it is well-established fact that the resistance to various antimicrobials may be due to presence of some virulence gene, involvement of secretion machinery of multi drug efflux proteins, through mutations in bacterial genome or by gaining additional genes through horizontal gene transfer or by physiology dependent resistance (mitchell et al. 2004, rangrez et al. 2006). in conclusion, we report that houseflies collected in hospitals and slaughterhouse may be involved in the spread of drug resistant bacteria and may increase the potential for human exposure to drug resistant bacteria. it is recommended that suitable steps must be taken to control the flies and monitor the sensitivity pattern of the pathogens transmitted by the houseflies. acknowledgements this work received financial support from medical university of kurdistan (grant number 680). the authors declare that they have no conflicts of interest. references barin a, arabkhazaeli f, rahbari s and madani s (2010) the housefly, musca domestica, as a possible mechanical vector of newcastle disease virus in the laboratory and field. medical and veterinary entomology. 24(1): 88–90. fotedar r (2001) vector potential of houseflies (musca domestica) in the transmission of vibrio cholerae in india. acta tropica. 78: 31–34. fotedar r, banarjee u, samantray j, shriniwas s (1992a) vector potential of the hospitals house flies with special reference to klebsiella species. epidemiol and infec. 109: 143–147. graczyk t, cranfield r, fayer r, bixler h (1999) house flies (musca domestica) as transport hosts of cryptosporidium parvum. am j trop med hyg. 61: 500–504. graczyk t, knight r, gilman r cranfield h (2001) the role of non-biting flies in the epidemiology of human infectious diseases. microbes and infection. 3: 231– 235. kabkaew l, manasanant b, banyong k, somsak p, yupha r, and kom s (2007) comparison between musca domestica and chrysomya megacephala as carriers of bacteria in northern thailand. southeast asian j trop med public health. 38(1): 38–44. kalantar e, ekrami a (2008) bacterial infection in burn patients. indian j med res 127: 416. kalantar e, motlagh m, lordnejad h, reshamansh n (2008) prevalence of urinary tract pathogens and antimicrobial susceptibility patterns in children at 55 hospitals in iran. iranian j clin infect dis. 3(3): 149–154. khalil k, lindblom g, mazhar k, kaijsher b (1994) flies and water as reservoirs for bacterial enteropathogens in urban and rural areas in and around lahore, pakistan. epidemiol infect. 113: 435– 444. khoobdel m, jonaidi n, seiedi m (2008) blowfly and flesh (diptera: cyclorrhpha) iran j arthropod-borne dis, 2010, 4(2): 50–55 b davari et al.: frequency of resistance and … 55 fauna in tehran, iran. j entomology. 5(3): 85–92. koneman ew, allen sd, janda wm, schreckenberger pc, winn wc jr (1992) diagnostic microbiology. 4th ed. philadelphia: jb lippincott. mitchell j, tali de-medina, yehuda c (2004) epidemiological interpretation of antibiotic resistance studies: what are we missing? nature reviews microbiology. 2: 979–983. mutsuo kobayashi, toshinori sasaki, noriko saito, kazumichi tamura, kenji suzuki haruo watanabe, and noriaki agu (1999) houseflies: not simple mechanical vectors of enterohemorrhagic escherichia coli o157:h7. am j trop med hyg. 61(4): 625–629. national committee for clinical laboratory standards (2003) performance standards for antimicrobial disc susceptibility tests, 8th ed. approved standard m2-a8. 2003; national committee for clinical laboratory standards, wayne, pa. olsen a (1998) regulatory action criteria for filth and other extraneous materials. iii. review of flies and food-borne enteric diseases. regulat toxicol and pharmacol. 28: 199–211. pandian r, asumtha a (2001) vector competence of musca domestica linn.in slum areas. insect environment. 7:118–119. peter s, christopher j, randle w, moore s, richard k (2007) isolation of salmonella enterica serovar enteritidis from houseflies (musca domestica) found in rooms containing salmonella serovar enteritidis-challenged hens. appl environ microbiol. 73(19): 6030–6035. rajendran j, pandian r (2003) microbial flora isolated from an urban population of non-biting vector musca domestica and their susceptibility to antibiotics. asian journal microbial biotechnol and environ sc. 5: 381–385. rangrez a, dayananda k, atanur s, joshi r, patole l, shouche y (2006) detection of conjugation related type four secretion machinery in aeromonas culicicola. plos one. 1(1): 115. shears p (2000) antimicrobial resistance in the tropics. tropical doctor. 30: 114– 116. sramova h, daniel m, absolonova v, dedicova d, jedlickova z, lhotova h, petras p, subertova v (1992) epidemiological role of arthropods detectable in health facilities. j hosp infec. 20: 281– 292. sulaiman s, othman m, aziz a (2000) isolations of enteric pathogens from synanthropic flies trapped in downtown kuala lumpur. j vect ecolo. 25: 90– 93. thaddeus k, ronald k, robert h, gilman r, cranfield s (2001) the role of nonbiting flies in the epidemiology of human infectious diseases. micr infect. 3: 231−235. thirumalai vasan, immanual gilwax, selvaraj pandian (2008) vector competence of musca domestica linn. with reference to the virulent strains of salmonella typhi in bus stands and markets at madurai, tamil nadu. current biotica. 2(2): 154–160. vazirianzadeh b, setareh s, mahmoud r, reza hajhossien, manijeh m (2008) identification of bacteria which possible transmitted by musca domestica (diptera: muscidae) in the region of ahvaz, sw iran. jundishapur j microbiol. 1(1): 28–31. zarrin m, babak vazirianzadeh, setareh shams solary, ali zarei mahmoudabad, mahmoud rahdar (2007) isolation of fungi from housefly (musca domestica) in ahwaz, iran. pak j med sci. 23(6): 917–919. j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 249 http://jad.tums.ac.ir published online: may 27, 2017 original article species diversity and distributional pattern of cockroaches in lahore, pakistan hafsa memona, *farkhanda manzoor, saffora riaz department of zoology, lahore college for women university, lahore, pakistan (received 28 apr 2015; accepted 14 may 2016) abstract background: cockroaches are found as the most common urban pests of tropical countries, prompting economic and serious health risk problem for humans by carrying microbes and allergens, acting as vector for various pathogens of diseases. the present study was conducted from april 2013 to march 2014 in various human dwelling localities of urban area of district lahore, pakistan. methods: cockroaches were collected randomly by hand, food baited and sticky traps throughout the year. four species of cockroaches (periplaneta americana (p. amercana), blattella germanica (b. germanica), blatta orientalis (b. orientalis), and blatta lateralis (b. lateralis) were collected and identified from the study site. results: b. germanica was the most dominant indoor species with highest diversity indices in study areas. overall cockroach species diversity was highest in july–september, 2013 with highest simpson index of diversity and shannon index as well. p. americana was found second broadly distributed in the study area followed by b. orientalis and b. lateralis were intermediately distributed in residential areas and narrowly distributed in hospitals. residential areas and hospitals were highly infested with b. germanica followed by p. americana. population index of b. germanica for hospitals was double than residential areas. b. lateralis was observed as displacing b. orientalis in outdoor habitat through competing with its habitat and food sources. conclusion: the infestation rate of different species depends on availability of food sources, sanitary conditions and climatic conditions. cockroach infestation can be controlled with knowledge about their biology and behavior, attention to sanitation and effective use of commercial insecticides. keywords: diversity, cockroach, pakistan, p. americana, b. germanica. introduction out of the 4600 species of cockroaches only 50 species have been reported as pests of human structures and dwellings worldwide. they randomly infringe in human habitation and never cause threat to indoor structures (cochran et al. 1980, bonnefoy et al. 2008). morphologically cockroaches are characterized by their dorsoventrally flattened body, head concealed beneath the pronotum, chewing mouth parts, prominent antennae and cerci. they are placed recently in their own order of blattodea (triplehorn and johnson 2005). all types of human habitations including hospitals and houses are significantly infested with cockroaches. highly populated houses and impoverished living settings are breeding sites for indoor species especially blattella germanica (b. germanica) (bonnefoy e al. 2008). poor sanitation, disrepair and clutter contribute to increase population of cockroaches in human habitat. foodstuffs can be contaminated with cockroach feces, body parts and pathogens. increased population of cockroaches inside the homes will cause asthmatic allergies in peoples which can be life threatening in some cases (lamiaa et al. 2007). the predominant species of cockroaches found in various types of human dwellings in bangkok, thailand, kuala lumpur federal territory, peninsular malaysia, singapore, china, indonesia, india and pakistan are: family *corresponding authors dr farkhanda manzoor, e-mail: doc_farkhanda@yahoo.com j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 250 http://jad.tums.ac.ir published online: may 27, 2017 blattidae include periplaneta americana (p. americana) (american cockroach), p. australasiae (australian cockroach), periplaneta brunea (p. brunnea) (burmeister) (large brown cockroach), periplaneta fuliginosa (p. fuliginosa), blatta orientalis (b. orientalis) (oriental cokroach), neostylopyga rhombifolia (n. rhombifolia) (stool) the harlequin cockroach, blatta (shelfordella) lateralis (b. lateralis) (walker) (turkistan cockroach) and hebardina concinna (h. concinna) (dehaan) (sriwichai et al. 2002, chompoosri et al. 2004). family blattelidae include b. germanica (german cockroach), blattella lituricollis (b. lituricollis) (walker) (smaller german cockroach), supella longipalpa (s. longipalpa) (fabricius) (brown banded cockroach), blattella vaga (b. vaga) hebard (field cockroach), blattella asahinai (b. asahinai) mizukubo (asian cockroach), jacobsonina erebis (j. erebis), symploce pallens (s. pallens) (smooth cockroach) symploce sphaerica (s. sphaerica), symploce miyakoensis (s. miyakoensis), symploce okinerabuensis (s. okinoerabuensis), symploce paramarginata (s. paramarginata) and symploce evidens (s. evidens). family polyphagiadae include polyphaga aegyptica (p. aegyptica) and polyphaga saussurei (p. saussurei). family blaberidae include rhyparobia maderae (r. maderae) (madeira cockroach). family pycnoscelidae include pycnoscelis surinamensis (linnaeus) (p. surinamensis) the surinam cockroach. family oxyhaloidae include nauphoeta cinerea (olivier) (n. cinerea) the lobster cockroach (cochran et al. 1980, zahedi and jeffery 1996, boyer and rivault 2004, jeffery et al. 2012, wang and che 2013). different studies in pakistan confirms the presence of p. americana, b. orientalis and b. germanica species of cockroaches in pakistan (mlso et al. 2005, saira 2005, ahmed et al. 2010, nih 2010, wakil et al. 2012, malik et al. 2013, syed et al. 2014). cockroaches are one of the most important agent which can transmit almost 60 species of yeast, 150 bacterial species, 45 species of parasitic worms and 90 species of protozoa to human life either biologically or mechanically (tachbele et al. 2006, al-marjani et al. 2008, saichua et al. 2008, al-bayati et al. 2011, akinjogunla et al. 2012, tilahun et al. 2012, goralska and kurnatowski 2013, vazirianzadeh et al. 2014). they get infected with pathogenic bacteria causing bubonic plague, leprosy, dysentery, urinary infections, abscesses and pimples etc. bacterial species can stay alive on cockroach body surfaces for many days (vahabi et al. 2007). the parasitic organisms identified from cockroaches include helminthes and protozoans. the helminthes include strongyloides stercoralis (s. stercoralis), ascaris lumbricoides (a. lumbricoides), trichuris trichura (t. trichura), taenia spp and identified protozoans are cyclospora spp, entamoeba histolytica (e. histolytica), entamoeba coli (e. coli), balantidium coli (b. coli) and isospora belli (i. belli) (thyssen et al. 2004, salehzadeh et al. 2007, nyarango et al. 2008, chamavit et al. 2011, el-sherbini and el-sherbini 2011). cockroaches are contaminated with medically important fungi including candida, aspergillus niger (a. niger), mucor, rhizopus spp, aspergillus fumigans (a. fumigans) and penicillium spp. (tatfeng et al. 2005, salehzadeh et al. 2007). cockroach associated bacteria include klebsiella pneumoniae (k. pneumoniae), salmonella spp, escherichia coli (e. coli), pseudomonas aeruginosa (p. aeruginosa), enterobacter cloacae (e. cloacae), citrobacter freundii (c. freundii), enterobacter aerogenes (e. aerogenes) and proteus mirabilis (p. mirabilis) which are potential pathogens. there was no significant difference between the overall bacteria load on the external surface in cockroaches found in the food-handling establishments (60.08%) and households (39.92%) (wannigama et al. 2014). pseudomonas aeruginosa has been demonstrated to multiply in the gut and excretion of the bacteria continj arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 251 http://jad.tums.ac.ir published online: may 27, 2017 ued up to 114 days. further studies also revealed that salmonella typhi (s. typhi), shigella dysenteriae (s. dysenteriae) and toxigenic strains of e. coli retained in the gut of cockroaches for up to several days (fotedar et al. 1993, pai et al. 2003). present study was conducted with the objective to report species composition, diversity, abundance, richness and population dynamics of cockroaches in urban areas of district lahore. lahore features a five season semi-arid climate ie foggy winters (15 nov– 15 feb) with few western disturbances causing rains, pleasant spring (16 feb–15 april), summer (15 april-june) with dust rain storms and heatwave periods, rainy monsoon (july– 16 september) and dry but pleasant autumn (16 september–14 november) (punjab meteorology department 2014). due to the high population density in the city, 5000 tons per day of solid waste is produced in lahore that is collected and disposed in one landfill site at mehmood booti and sewage is drained into river ravi (lahore waste management company report 2014). there are few scattered researches were conducted about species composition and abundance in last years. economic importance of this domestic pest should be recognized. however, they are notorious as vector of nosocomial infections, contaminating and deteriorating food and consumables, carry allergens of asthma and resistant to insecticidal sprays. materials and methods experimental site study site is the urban area of district lahore lying between 31°15′-31°45′ n and 74°01′-74°39′ e, 217m elevated from sea level with total population of 7,566,000. samples were collected from different hospitals, shopping malls/stores, institutes and residential areas in different seasons around one year. sampling and identification of cockroaches cockroaches were observed and collected randomly from april 2013 to march 2014 with the help of sticky traps, food baited pitfall traps and manual catching by hand. specimens were collected from 20 different sites including hospitals (punjab institute of cardiology, mayo hospital, sheikh zaid hospital, jinnah hospital and general hospital) shopping malls/stores (swera departmental store, metro cash and carry and hyperstar), institutes/office (lcwu, punjab university, uvas, gcu lahore and passco), houses (mughalpura, model town, shadman, shalmar, maraghzaar, johar town and jallo town). global positioning system of study sites are listed in table 1. traps were kept on the floor close to the wall of room, under cupboards, bed, storage racks, under washbasins and pantry. each trap was placed in living room, bedroom, bathroom and kitchen of houses, different wards, store rooms, kitchen stores, canteen area of hospitals, in grocery area, food area of shopping store and in each working room of the institute/office units for three consecutive nights. nymphs along with adults were heavily trapped in baits and traps. the collected specimens were transported to entomology laboratory for identification up to species level with the help of published keys (pratt and littig 1969, abul hab 1980, hagenbuch et al. 1988, roth 1995, choate 2009). species abundance and richness was evaluated in 4 trimesters comprising whole year from april, 2013 to march, 2014. weather data collection average monthly temperature and humidity data were obtained from the punjab meteorological department, lahore and comparatively analyzed with population density of cockroaches. data analysis the observations were tabulated and data were statistically analyzed using microsoft j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 252 http://jad.tums.ac.ir published online: may 27, 2017 excel. relative abundance, species richness and evenness of each species of cockroaches was calculated. diversity of different cockroach species on the simpson and shannon indices was worked out according to simpson (1949) and shannon-weiner function (odum, 1975). species relative abundance was compared with average monthly temperature and relation between population density and change in temperature was determined. results identified species of cockroaches from different types of human dwelling localities 4 species of cockroaches belonging to two families (blattidea and blattelidae) were identified during the entire sampling period. 1. american cockroach (p. americana) they found in sewers and basements especially around pipes and drains. 2. oriental cockroach (b. orientalis): they found beneath the mulch, leaf litter, stones and debris outdoors, garbage, filthy materials that is going to decay. 3. german cockroach (b. germanica): they found in kitchens, storage areas especially where food being prepared or stored. 4. turkestan cockroach (b. lateralis): turkestan cockroaches are native to large area of the middle east extending from libya eastward to central asia including afghanistan, pakistan, uzbekistan and southern russia (alesho 1997). they were found in compost piles, gardens, potted plants and homes with clay floors. distribution and abundance of cockroach species from this study it was revealed that b. germanica was the most dominant species belonging to family blattelidae comprising of 45% of the total catch followed by p. americana (35%) belonging to family blattidae. these two species comprises 80% of the total specimens collected. other two species include b. orientalis (9%) and b. lateralis (11%) of the total specimen trapped. these findings are compatible with the studies of sandhu and sohi (1981). from all the specimens captured during study period 62% (8328) specimen were at different nymphal stages, 27% (3626) adult male and adult females are 11% (1479). the number of females was lower as compared to males in traps because females are less agile and are often hidden in deep crevices, engaged in reproductive activity. during the first trimester of study (apriljune, 2013) the minimum and maximum average temperature was 23.8 °c and 37.6 °c, respectively and relative humidity was 41% (table 3). the autumn was turning to end and summer was started. this is the most compatable season of breeding of eggs and nymphs. in this trimester, nymph collection was more prominent as compared to adult males and females. most dominant species (46.78%) of all catches was b. germanica, followed by p. americana (33.50%) while b. orientalis and b. lateralis are 9.96% and 9.75% respectively (table 2). diversity indices were worked out for all species found in 20 different sites revealed shannons index value of -0.51185 and simpson’s index of diversity was 0.6495. the dominant species of first trimester was b. germanica with a value of 0.218879 (simpson index) and -0.15434 (shannon index). during sampling the higher number of males in food bait traps and sticky traps was not surprising as the males were active predators and roam in search of mates. females were low in number because they conserve energy for reproduction and found less active and common. one of the many reason is may be the traps were baited with female sex attractants which results in maximum male capturing in traps. during the second trimester of study (july–september, 2013) the minimum and maximum average temperature was 24.8 °c and 34.1 °c, respectively and relative humidity j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 253 http://jad.tums.ac.ir published online: may 27, 2017 was 74% (table 3). summer season was at peak and cockroaches were mostly found in indoor sheltered areas. this is the most favorable season of metamorphosis of nymphs to change into adults. different species have different life spans and time period to turn nymphs into adults varies among different species. in this trimester, late nymph collection was comparable to adult males and females. more dominant species (44.36%) of all catches b. germanica was followed by p. americana (35.81%) while, b. orientalis and b. lateralis comprised 8.11% and 11.7% respectively (table 2). diversity indices for this trimester were worked out for all species of cockroaches collected from different sites. shannons index value (-0.51389) and simpsons index of diversity was 0.6547 for this trimester. the dominant species of this period was b. germanica with a value of 0.196748 (simpson index) and -0.1566 (shannon index). during sampling the higher number of nymphs of late instar were found. now they had gone through metamorphosis to change into adults and are ready for reproduction. in most species time for late instar to turn into females is longer than males. in third trimester of study (october–december, 2013), b. germanica was most dominant species (44.62%) followed by p. americana, b. orientalis and b. lateralis respectively. table 2 revealed shannon index value (-0.51187) and simpsons index of diversity (0.6521) for all species of cockroaches collected from different sites. both species richness and abundance was significantly lower during colder months of study period. the fourth trimester of study (januarymarch, 2014) shannon index value was 0.52419 and simpsons index of diversity was 0.6687. most dominant species b. germanica (40.82%) was followed by p. americana (37.02%) and b. orientalis and b. lateralis comprise 7.17% and 14.97% respectively (table 2). overall species evenness and richness was found higher in second trimester of study (july–september, 2013). previous studies described p. americana and b. orientalis as outdoor species of cockroaches but they can intrude into indoor environment through severage pipes and crevices in harsh seasons. while b. germanica and b. lateralis always found in indoor environment (rust and reierson 2007, jeffery et al. 2012). residential areas and hospitals are mostly infected with b. germanica. p. americana approaches to outdoor environment through sewerage pipes and holes. as b. orientalis and b. lateralis are morphologically similar but their colony can be identified by male members. houses and hospitals are highly infested with p. americana and b. germanica as compared to offices, shopping mall and institutes, whereas b. orientalis is commonly found in houses and institutes followed by shopping malls. distribution of b. lateralis is most common in institutes, houses and offices with basements and gardens. table 1. global positioning system location of study sites in urban areas of lahore, pakistan serial. number study site latitude longitude 1 punjab institute of cardiology 31°32'18.48"n =31.5384667 74°20'9.28"e =74.3359111 2 mayo hospital 31°34'18.06"n =31.5716833 74°18'57.04"e =74.3158444 3 shaikh zayed hospital 31°30'29.82"n =31.5082833 74°18'30.17"e =74.3083806 4 jinnah hospital 31°29'3.93"n =31.484425 74°17'48.40"e =74.2967778 5 general hospital 31°27'17.46"n =31.45485 74°21'0.94"e =74.3502611 6 swera departmental store 31°25'54.81"=31.4318917 74°17'11.38"=74.2864944 7 metro cash & carry 31°29'34.65"n =31.4929583 74°25'1.57"e =74.4171028 8 hyperstar 31°32'5.69"n =31.5349139 74°21'47.79"e =74.363275 9 lcwu, lahore 31°32'41.85"n =31.5449583 74°19'37.97"e =74.3272028 j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 254 http://jad.tums.ac.ir published online: may 27, 2017 10 punjab university, lahore 31°29'44.24"n =31.4956222 74°17'39.17"e =74.2942139 11 uvas, lahore 31°34'29.03"n =31.5747306 74°17'57.48"e =74.2993 12 gcu, lahore 31°34'22.14"n =31.5728167 74°18'29.22"e =74.3081167 13 passco office 31°33'36.68"n =31.5601889 74°19'56.62"e =74.3323944 14 mughalpura 31°33'46.53"n =31.562925 74°22'49.35"e =74.380375 15 model town 31°28'37.18"n =31.4769944 74°19'44.66"e =74.3290722 16 shadman 31°32'14.97"n = 31.5374917 74°19'50.76"e = 74.3307667 17 shalamar town 31°35'12.47"n = 31.5867972 74°22'55.29"e = 74.382025 18 maraghzaar colony 31°29'44.24"n =31.553326 74°17'39.17"e =74.305122 19 johar town 31°27'43.38"n = 31.46205 74°17'38.90"e = 74.2941389 20 jallo town 31°35'47.57"n = 31.5965472 74°29'57.97"e = 74.4994361 table 2. diversity indices of different cockroach species collected from april 2013– march 2014 trimester species no. of cockroaches percentage % relative abundance pi shannon index pi (lnpi) simpson index pi2xs species evenness (s) h/logs 1st p. americana 1573 33.50 0.3349 -0.15911 0.112202 -0.51185/0.60206 = -0.85016 b. germanica 2197 46.78 0.4678 -0.15434 0.218879 b. orientalis 468 9.96 0.0996 -0.09981 0.009932 b. lateralis 458 9.75 0.0975 -0.09859 0.009512 total 4696 h= 0.51185 d= 0.3505, 1-d= 0.6495 2nd p. americana 2123 35.81 0.358191 -0.15971 0.128301 -0.51389/0.60206 = -0.85355 b. germanica 2629 44.36 0.443563 -0.1566 0.196748 b. orientalis 481 8.11 0.081154 -0.08851 0.006586 b. lateralis 694 11.7 0.117091 -0.10907 0.01371 total 5927 h= 0.51389 d= 0.3453, 1-d= 0.6547 3rd p. americana 671 36.03 0.360365 -0.15973 0.129863 -0.51187/0.60206 = -0.85019 b. germanica 831 44.62 0.446294 -0.15637 0.199179 b. orientalis 162 8.70 0.087003 -0.09226 0.00757 b. lateralis 198 10.63 0.106337 -0.1035 0.011308 total 1862 h= 0.51187 d= 0.3479, 1-d= 0.6521 4th p. americana 351 37.02 0.370253 -0.15976 0.137087 -0.50419/0.60206 = -0.84066 b. germanica 387 40.82 0.408228 -0.15884 0.16665 b. orientalis 68 7.17 0.07173 -0.08208 0.005145 b. lateralis 142 14.97 0.149789 -0.1235 0.022437 total 948 h= 0.50419 d= 0.4313, 1-d= 0.5687 table 1. continued… j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 255 http://jad.tums.ac.ir published online: may 27, 2017 table 3. trimester average temperature (minimum and maximum and relative percentage of humidity recorded from april 2013 to march 2014 trimester average temperature °c average relative humidity % no. of cockroaches trapped percentage % minimum maximum 1st (april-june) 23.8 37.6 41 4696 35 2nd (july–sep) 24.8 34.1 74 5927 44 3rd (oct–dec) 13 26.5 68 1862 14 4th (jan–march) 9 21.4 68 948 7 discussion environmental temperature plays an important role in determining the ability of an organism to survive in a given habitat. if an organism is able to survive in extreme temperature (during winter or summer) it will increases their likelihood to colonize that habitat. when an organism experience high or low temperature in their environment they produce certain proteins called “heat shock proteins (hsp)” in their cells which allows recovery on a cellular level. these hsp are found in many organisms from bacteria to mammals (lutterschmidt and hutchison 1997). organisms have the ability to increase or decrease their core temperature in response to environmental temperatures (slabber et al. 2007). certain insects, such as termites, have the ability to acclimate to their environmental temperature (hu and appel 2004). since cockroaches are closely related to termites, they should have the tendency to acclimatize to their environment. previous studies have shown a positive correlation between the temperature sensitivity of many animals including cockroaches and their environmental temperature (tsuji and mizumo 1973, appel et al. 1983, slabber et al. 2007). in this study, cockroache’s species distribution was observed almost all around the year though environmental temperature may effect on distribution of some outdoor species. cockroaches are highly adapted for diverse land environment especially dry harsh environment. cockroaches were not more noticeable in cold months in third and fourth trimester but probably they have physical and behavioral adaptation which helps them to withstand in extreme low temperature on land. this study coincides with the findings of snoddy and appel (2008) who conducted a survey in southern alabama and georgia to determine the extent b. asahinai had expanded its range northward from florida. they concluded that visual and bucket sample population began increasing in late may and reached their zenith in late august or early september. the present study found indoor species of cockroaches including german and turkestan cockroach were more prominent in bedrooms, kitchen, stores, hospital wards, office stores and rooms adjacent to canteen. their high prevalence in these areas can be related to ideal shady, enriched food and cool environment which is more favorable to increase their population. other outdoor species american and oriental cockroaches are outdoor species and found near to sanitary pipes, washrooms, filthy habitat, adjacent gardens of houses, sewerage pipes and kitchen exit pipe where plenty food is available. late nymphs and adults were more numerable in collection site where fewer females were captured. environmental conditions like temperature and humidity also do affect the life cycle and developmental stages of cockroaches. findings of our study are also in conformity to j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 256 http://jad.tums.ac.ir published online: may 27, 2017 lee et al. (2003) who found that houses were most commonly infested with b. germanica followed by apartments and villas. the infestation rate of the cockroaches was related to the residential types. conclusion most of the urban areas in district lahore especially human dwellings are infested with cockroach population. their presence in human associated environment and species abundance pose them a critical threat for human health and indicate poor infrastructure of sanitary and waste disposal. the infestation rate of different species depends on availability of food sources, sanitary and climatic conditions. cockroach infestation can be controlled with knowledge about their biology and behavior, attention to sanitation and effective use of commercial insecticides. acknowledgements the present work is a part of phd research of the first author. we are gratefully acknowledge the financial support provided by the higher education commission, islamabad, pakistan and facilitation provided by entomology lab, department of zoology, lahore college for women university, lahore for this research. we are also thankful to urban pest management team for field collection and technical assistance during sampling. the authors declare that there is no conflict of interests. references abul hab j (1980) a list of arthropoda of medical and veterinary importance recorded from iraq. bull biol res cent. 12(1): 9–40. ahmed a, minhas k, namood-e-sahar, aftab o, sher khan f (2010) in silico identification of potential american cockroach (periplaneta americana) allergens. iran j public health. 39(3): 109–115. akinjogunlaa oj, odeyemib at, udoinyangc ep (2012) cockroaches (periplaneta americana and blattella germanica): reservoirs of multidrug resistant (mdr) bacteria in uyo, akwa ibom state. sci j bio sci. 1: 19–30. al-bayati na, al-ubaidi as, al-ubaidi ik (2011) risks associated with cockroach periplaneta americana as a transmitter of pathogen agents. diyala j med. 1: 91–97. alesho na (1997) synathropic cockroaches of russia. proc inter coll social insects. 34: 45–50. al-marjani mf, jafere fn, abdul hussain mt, mezeal ea, sahtte za, hamza ah, shanuor kj, asmaeal mh (2008) study of ß-lactamases producing enterobacteria isolated from german cockroach (blatella germanica) in hospitals. diala j. 29: 1–10. appel ag, reierson da, rust mk (1983) comparative water relations and temperature sensitivity of cockroaches. comp biochem physiol. 74: 357–361. bonnefoy x, kampen h, sweeney k (2008) cockroaches. in public health significance of urban pests. world health organization. regional office for europe, scherfigsvej 8. dk-2100 copenhagon, denmark, pp. 53-84. boyer s, rivault c (2004) interspecific competition among urban cockroach species. entomol exp appl. 113: 15–23. chamavit p, sahaisook p, niamnuy n (2011) the majority of cockroaches from the samutprakarn province of thailand are carriers of parasitic organisms. excli j. 10: 218–222. choate pm (2009) a dichotomous key for the identification of the cockroach fauna (insecta: blattaria) of florida. department of entomology and nematology, j arthropod-borne dis, june 2017, 11(2): 249–259 h memona et al.: species diversity and … 257 http://jad.tums.ac.ir published online: may 27, 2017 university of florida. chompoosri j, thavara u, tawatsin a, sathantriphop s, yi t (2004) cockroach surveys in the northern region of thailand and guangxi province of china. southeast asian j trop med public health. 35: 46–49. cochran dg, grayson jm, gurney ab (1980) cockroaches: biology and control. vol. 5. who-vbc 72354: 1–45. el-sherbini gt, el-sherbini et (2011) the role of cockroaches and flies in mechanical transmission of medical important parasites. j entomol nematol. 3(7): 98–104. fotedar r, banerjee u, shriniwas p (1993) vector potential of the german cockroach in dissemination of pseudomonas aeruginosa. j hospital infect. 23: 55–59. goralska k, kurnatowski p (2013) parasites as etiological factors of nosocomial infections. annal parasitol. 59: 3–11. hagenbuch be, koehler pg, patterson rs, brenner rj (1988) peridomestic cockroaches (orthoptera: blattidae) of florida: their species composition and suppression. j med entomol. 25: 377–380. hu xp, appel ag (2004) seasonal variation of critical thermal limits and temperature tolerance in formosan and eastern subterranean termites (isoptera: rhinotermitidae). environ entomol. 33: 197–205. jeffery j, sulaiman s, oothuman p, vellayan s, zainol-ariffin p, paramaswaran s, razak a, muslimin m, kamil-ali ob, rohela m, abdul-aziz nm (2012) domiciliary cockroaches found in restaurants in five zones of kuala lumpur federal territory, peninsular malaysia. trop biomedicine. 29: 180–186. lamiaa b, lebbadi m, ahmed a (2007) bacteriological analysis of periplanata americana l. 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environment, 1996 july 7–10, edinburgh, scotland. pp. 177–184. j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 296 http://jad.tums.ac.ir published online: september 30, 2018 original article species composition and some biological features of scorpions in kazerun district, southern iran mansour nazari 1, ali najafi 1, *mohammad reza abai 2 1department of medical entomology and vector control, school of medicine, hamadan university of medical sciences, hamadan, iran 2department of medical entomology, school of public health, tehran university of medical sciences, tehran, iran (received 7 july 2016; accepted 12 may 2018) abstract background: scorpions have medical importance in the studied area with 2377 cases of envenoming during past six years. this study was the first to explore the scorpion species and dispersion in the kazerun district during 2014–2015. methods: the studied sites were selected based on different topographic conditions such as plain, foothill and mountainous which formed four geographical zones with three villages in each zone. the sampling was carried out twice each month throughout the year. daytime collections were carried out using hand digging tools for moving stones and excavate the borrows, as well as night sampling, is done with the black light device. the coordinate of locations was recorded with a gps. the collected specimens were maintained in 70% ethanol and identified using authorized keys. results: overall, 800 scorpions were sampled from different parts of kazerun district, bringing the species richness to 9 belonged to 3 families of buthidae, scorpionidae, and hemiscorpionidae. the mesobuthus eupeus (84.6%) was prominent vice versa m. caucasicus (0.1%) had lowest abundance. other species comprised compsobuthus matthiesseni (5.3%), androctonus crassicauda (5.0%), razianus zarudnyi (2.0%), hemiscorpius lepturus (1.5%), orthochirus sp (0.9%), hottentotta zagrosensis (0.4%), and scorpio maurus (0.3%). the seasonal activity of the scorpions showed a lower peak in mar, with the main peaks in aug for the dominant species. mesobuthus caucasicus is recorded for the first time in the fars province, southern iran. conclusion: all the known dangerous scorpions, including h. lepturus, m. eupeus and a. crassicauda were revealed in the studied area. keywords: scorpionida, fauna, kazerun, iran introduction considering ancient iranian literature, as well as religious texts enlightened in the course of time, scorpion bites or scorpionism have been fully described which uncover the antiquity of this health problem in iran. information regarding scorpions in iran is very limited, especially in fars province. the history of studies on the biology and ecology of scorpions is very rare in the country, and the majority of research is directed towards the descriptive studies of envenomation. a little information exists concerning scorpion bioecology and insufficient reports are available in different parts of the country. additionally, no robust scientific research have been discerned concerning scorpions presence in the various districts of fars province to determine their fauna and recognition of their habitats. cases of scorpion sting in the country are an expression of the importance of this problem. the annual cases of scorpionism in iran are projected from 40000–50000, resulting in 14–18 deaths per year (1). the dangerous consequences of the scorpion stings comprised the severe and lethal haemolysis, acute renal failure, deep necrotic wound, severe joint inflammation, temporary and permanent psychosis and death (2,3). the highest mortality due to scorpion *corresponding author: mr mohammad reza abai, e-mail: abaimr@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 297 http://jad.tums.ac.ir published online: september 30, 2018 stings is associated with hemiscorpius lepturus peters, 1861 in iran, encountered as the most dangerous scorpion species in khuzistan and hormozgan provinces in southern iran, where its abundance is high (4,5). the scorpion fauna of iran have immense richness due to the presence of different climatic condition consists 51 species (30 endemic to iran) belonging to 18 genera and four families (6). however, due to population explosion in the cities and increasing urbanization, using of uncultivated land, and the construction of the buildings in the suburbs, led to the presence of the unwanted arthropods at indoor places. in addition, the establishment of rural homes or nomad tents are the ways which increase the possibility of scorpion entrance into the residential places. scorpions occupy a wider range of habitats inside and outside buildings, in rural areas or suburbs of cities. the biodiversity of scorpions varies across different areas, hence their habitats also differ. biological and environmental characteristics of scorpions according to their importance in health issues, and health risks caused by some species can provide practical and scientific solutions in dealing with them (7). recognition of scorpion habitats is the first step in combating and preventing scorpion sting in every area. in realization of scorpion habitats, the scorpions inhabit human hand-made shelters as well as natural hiding places (8). in this way, the method of elimination of breeding sites should be identified in order to control the scorpion population. kazerun district is the second largest district in fars province, which has extensive plains and mountains with warm and dry climate, which provide a favorable condition for the scorpion activity. at present, 4 families, 18 genera, and 51 species have been reported, in which 7 species are venomous and dangerous to humans (6). the scorpion fauna of fars province have been studied, and 18 species reported. of these, four species entailing compsobuthus persicus, m. eupeus persicus, odontobuthus bidentatus and scorpio maurus townsendi were reported for the first time in fars province (9). as the scorpionism is one of the important health problems, however, the study on scorpion fauna has the poorer history in the kazerun. this study was conducted to reveal the fauna, abundance and some biological feature of the scorpions at selected localities during 2014–2015. materials and methods study area the kazerun district is located in the southwestern slope of the zagros mountain chain, fars province at 51°39'15''e and 29° 37'10''n, with an elevation less than 860m above sea level covering an area of nearly 4119km2. it is bordered by shiraz to the east, norabad to the north, bushehr to the west and southwest, and farashband to the southeast. kazerun comprises diverse climates, such that there is a mountainous cold zone with an elevation of 3000m in the north, and a warm and dry zone in the south with an elevation below 2000m. water is traditionally supplied by “qanat” traditional irrigation system, wells, springs, and rivers, in addition to rain. kazerun is also renowned pertaining to the presence of narcissus meadows and parishan lake. in kazerun, a variety of agricultural products are cultivated, including wheat, barley, rice, lentils, beans, tobacco, wild almonds, sesame, citrus fruits, and dates. site selection for site selection, the district was divided into 4 zones, and for each zone, 3 topographic conditions were considered as plain, foothill and mountainous. the climatic features of each zone are as follows: zone 1: located in the north west of kazerun district (fig. 1), where the selected villages included zarinabad (plain), 51°36'33"e, 29°39'11"n, at 835m above sea level (alv) with mean of annual temperature (mat) 23 °c and mean of annual relative humidity http://jad.tums.ac.ir/ http://www.iranicaonline.org/articles/busehr-ar j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 298 http://jad.tums.ac.ir published online: september 30, 2018 (arh) 30%, dehno enghelab (mountainous /man), 51°34'45"e, 29°47'26"n, 987m (alv), 30 °c (mat), 46% (mrh), and qandil (man), 51°34'46"e, 29°53'00.4"n, 926m (alv), 29 °c (mat), 35% (mrh), at a distance of 10, 20 and 35km respectively from the central part of the district where the ground is rocky with various low hills surrounding the villages. zone 2: located in the north-eastern of kazerun district (fig. 1), where the selected villages included burenjan (man), 51°52'24"e, 29°35'28"n, 1334m, 20 °c (mat), 28% (mrh), chekak (man), 51°39'54"e, 29°52'34"n, 1174m (alv), 32 °c (mat), 37% (mrh), murdak (man), 51°48'36"e, 29°38'38"n, 1173m (alv), 32 °c (mat), 38% (mrh), at a distance of 30, 45 and 65 km respectively from the central part of the district with alluvial rocks and covered by oak trees. zone 3: located in the west of kazerun district (fig. 1), where the selected villages included buraki (plain/ pln) 51°20'28"e, 29°33'44"n, 498m, 34 °c (mat), 45% (mrh), borje seyed (pln), 51°22'33"e, 29°35'41"n, 504m (alv), 32 °c (mat), 43% (mrh), shahbaz khani (pln), 51°19'58"e, 29°36'42"n, 494m (alv), 36 °c (mat), 46% (mrh), at a distance of 45, 60 and 65km respectively from the central part of the district where there are many palm gardens. zone 4: located in the southwest of kazerun district (fig. 1), where the selected villages included dadin-e olya (foothill/fot) 51°52'06"e, 29°19'04.5"n, 646m, 30 °c (mat), 45% (mrh), ghodar sefid (fot), 51°43'46"e, 29°35'41"n, 808m (alv), 28 °c (mat), 43% (mrh), sarmashjad (fot), 51°19'58"e, 29°36'42"n, 815m (alv), 25 °c (mat), 40% (mrh), at a distance of 45, 50 and 70km respectively from the central part where agriculture activities are carried out, and some uncultivated lands are covered of wild shrubs. scorpion samplings the sampling was carried out twice each month throughout the year during 2014–2015. the scorpions were actively sought both at daytime by using hand digging tools for turning the stones, digging the borrows and collecting the scorpions by long forceps as well as portable ultraviolet devices used at night (2). a portable garmin™ gps device was used for recording the geographical coordinates of the collection localities in the field. a variety of possible habitats were examined both at indoor and outdoor e.g. animal shelters, yards, beneath stones, within rock crevices, beneath trunk of fallen trees, trashes and dry stone walls partitioning agricultural fields and gardens. the courtyards of rural residence were sampled after sprinkling water on the grounds at night, which elicited the attraction of the scorpions. moreover, an attempt was made to capturing the burrowing scorpions, in which disperse colonies were seen around the selected zones. the collected scorpions were maintained in ethanol (70%) using long forceps and were eventually labeled. all the specimens were identified using authentic scorpion keys (10-15) were deposited at the museum of department of medical entomology, hamadan university of medical sciences. results species composition overall, 800 scorpions were collected from the 4 selected zones, which includes three families of buthidae (98.3%), scorpionidae (1.4%) and hemiscorpionidae (0.3%), and 8 genera and species were identified with different abundance (fig. 2). mesobuthus eupeus has the highest density in the studied area (84.4%) whereas, m. caucasicus (0.1%) showed the lowest density. the scorpion species were categorized according to low, moderate and high densities in the studied area. bioecological features of the species mesobuthus eupeus: a well-known species belonging to the family buthidae, and consisting of 84.6% of the total collected http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 299 http://jad.tums.ac.ir published online: september 30, 2018 specimens, with a male-to-female sex ratio of 207:470 (fig. 3), were collected in kazerun district where it showed a universal dispersion in all the studied zones with different topographic conditions e.g. plains and mountainous parts (fig. 4). this scorpion was discovered in places of different elevations ranging between 499 and 1225 meters above the sea (table 1). the extensive distribution of this species in different zones due to climatic variations is necessary for determining the biological conditions of the scorpion. the monthly activity of this species extended throughout the entire months of the year, beginning of apr ending in mar, with a noticeable peak in aug (fig. 5). mesobuthus eupeus were recognized as burrowing scorpion especially around the shaded parts of stone humps arranged between the farms as well as this species found underneath the rocks and dumped walls. hemiscorpius lepturus: belongs to the family hemiscorpiidae and locally named as “gadim” in southern iran, and consisting of 1.5% of the total scorpions with a maleto-female sex ratio of 2:10 (fig. 6), were collected in the kazerun district. this species was only found in zones 1 and 2 where is mountainous with the elevation range 916–1227 meters above sea level (table 1), and an equal density of 50% each zone. the monthly activity of this species was limited to the warmer months of the years, beginning of july ending in sep, characterized by a peak in aug (fig. 5). the hiding places of h. lepturus were revealed under the stones and rocks. androctonus crassicauda: or fat-tailed scorpion belongs to the family buthidae, which consisted of 5.0% of all sampled scorpions in kazerun district with a male-to-female sex ratio of 4:36 (fig. 7). this scorpion was established in all the studied zones with the nearly equal occurrence (fig. 8). the monthly activity of this species started from may and ended in nov, projecting a peak in aug (fig. 5). the habitats of a. crassicauda were realized in both indoors (under the dry humped trees, ground crevices, crevices of mud walls, broken bricks of the walls), and outdoors (under the humped bricks and stones, under loose stones and ant holes). it was easily attracted to vehicular lights. scorpio maurus: belongs to the family scorpionidae which only two females was found (fig. 9) in low density (0.3%) in the september (fig. 10) at higher elevation (1227m), which covered by the quercus trees. this scorpion is a burrow-living species, having various deep holes (up to 45cm) and found in the wheat farm at the zone 2. compsobuthus matthiesseni: belongs to the family buthidae found in moderate density (5.3%) (fig. 10) with a male-to-female sex ratio of 5:37 sampled in kazerun district. this scorpion was present in all the studied zones with nearly equal occurrence, with the exception of zone 3 where the lower elevation has. the monthly activity of this species started from apr and ended in mar with a peak in jun (fig. 10). the evidence shows the adaptation of this species to temperate conditions than to higher temperatures. the sampled scorpions were collected from rock crevices and bark of trees, where having the high humidity. hottentotta zagrosensis: belongs to family buthidae which only three females were found in low density (0.3%) (fig. 11), with an annual occurrence in july and aug (fig. 10) in the kazerun district reported from zones 1 and 4 where the elevation ranged between 756–916 meter above sea level. the collected specimens were found around the stones in semi-arid parts. razianus zarudnyi: belongs to buthidae which consisted of 2.0% of all scorpions sampled in kazerun district (table 1) with a male-to-female sex ratio of 4:12 (fig. 12). this scorpion was only evident in zone 1, in the northeast of kazerun, where the elevation was 916 meters above sea level. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 300 http://jad.tums.ac.ir published online: september 30, 2018 the monthly activity of this species started from mar and ended in sep with a peak in mar. the more success in collection of this species ensued from jul to sep (fig. 10), whereas the collection was zero during the other months, with the exception of march which showed a peak. the hiding places of this species were in rock crevices. mesobuthus caucasicus: belongs to the family buthidae which was only one female found in low density (0.1%) (fig. 13) with their occurrence in jul (fig. 10), present in zone 4 where the elevation was 499 meters above sea level. these species was found under stones and lumps. orthochirus sp. belongs to family buthidae found in low density (0.9%) (fig. 14), with a male-to-female sex ratio of 1:6 and occurred during jun to jul (fig. 10) in zone 4, where the elevation ranged 499–1227 meter above sea level. this species was as rapid moving scorpions on the ground and active at night and found around the dry rubble stones near the foothills with low vegetation. seasonal activity the monthly activity of different species, including those with low, moderate and high density, was recorded. in the spring, the activity of most species of scorpions was minimal, but it was improved due to the occurrence of higher temperatures in the habitats during summer. the peak of activity of most species was in aug (fig. 5), whereas the c. matthiesseni showed a moderate density (5.3%) in jun–jul (fig. 10). fig. 1. the selected zones in kazerun district and their geographical positions in the fars province and the country http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 301 http://jad.tums.ac.ir published online: september 30, 2018 fig. 2. species composition of the scorpions found in kazerun district, fars province, southern iran 2014–2015 fig. 3. ventral (a) and dorsal (b) views of male and female (c) of mesobuthus eupeus. the map shows its dispersion and abundance at the studied zones, kazerun district, fars province, 2014–2015 (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 302 http://jad.tums.ac.ir published online: september 30, 2018 fig. 4. ventral view of mesobuthus eupeus in male (left) with 22 to 28 pectinal teeth compared to female (right) with 16–23 one, kazerun district, fars province during 2014–2015 (original photos) fig. 5. monthly fluctuation of prevalent species found in kazerun district, southern iran 2014–2015 fig. 6. ventral and dorsal views of female (up) and male (down) of hemiscorpius lepturus. the map shows its dispersion and abundance at studied zones, kazerun district, fars province, 2014–2015 (original photo) fig. 7. ventral (down) and dorsal (up) views of female androctonus crassicauda, kazerun district, fars province, 2014–2015 (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 303 http://jad.tums.ac.ir published online: september 30, 2018 fig. 8. ventral view of male androctonus crassicauda. the map shows its dispersion and abundance at the studied zones, kazerun district, fars province, 2014–2015 (original photo) fig. 9. dorsal (a) and ventral (b) of female of scorpio maurus. the map shows its dispersion and abundance in the zone 2, kazerun district, fars province, 2014–2015 (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 304 http://jad.tums.ac.ir published online: september 30, 2018 fig. 10. dorsal (a) and ventral (b) of female compsobuthus matthiesseni. the map shows its dispersion and abundance at the studied zones, kazerun district, fars province (2014–2015) (original photos) fig. 11. dorsal (a) and ventral (b) of female hottentotta zagrosensis. the map shows its dispersion and abundance at the specified zones, kazerun district, fars province, 2014–2015 (original photos) fig. 12. dorsal (a) and ventral (b) of male of razianus zarudnyi. the map shows its dispersion and abundance at the zone 1, kazerun district, fars province (2014–2015) (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 305 http://jad.tums.ac.ir published online: september 30, 2018 fig. 13. dorsal (a) and ventral (b) of female mesobuthus caucasicus. the map shows its dispersion and abundance at the specified zone 4, kazerun district, fars province (2014–2015) (original photos) fig. 14. dorsal (a) and ventral (b) of male orthochirus sp. the map shows its dispersion and abundance in the zone 4, kazerun district, fars province, 2014–2015 (original photos) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 306 http://jad.tums.ac.ir published online: september 30, 2018 table 1. biodiversity of scorpions and the geographical features of selected zones in kazerun district, fars province, southern iran 2014–2015 geographical zone and mean of elevation above sea level direction of the zone in the district and coordinates selected villages and mean of annual temperature and rh species richness and no. of species species composition m . e u p e u s h . le p tu ru s a . c ra ssic a u d a s . m a u ru s c . m a tth ie sse n i h . za g ro se n sis r . za ru d n y i m . c a u c a sic u s o rth o c h iru s sp zone 1 (916m) northwest 29°46'12.5''n; 51°35'41.3''e dehno-zarrinabad-gandil 27.3°, 37.0% 0.7 (6) 205 (30.3%) 6 (50.0%) 12 (30.0%) 0 (0.0%) 18 (42.9%) 2 (66.9%) 16 (100%) 0 (0.0%) 0 (0.0%) zone 2 (1227m) northeast 29°42'33.3''n; 51°42'38.0''e borenjan-chekak-mordak 28.0°, 34.0% 0.7 (6) 206 (30.4%) 6 (50.0%) 11 (27.5%) 2 (100%) 6 (14.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 3 (42.9%) zone 3 (499m) west 29°35'42.3''n; 51°20'39.7''e boraki-borjesheid-shahbazkhani 34.0°, 45.0% 0.3 (3) 112 (16.5%) 0 (0.0%) 15 (37.5%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 4 (57.1%) zone 4 (756m) south west 29°17'11.8''n, 51°45'19.0''e dadin olya-ghodarsefidsarmashhad 27.7°, 43.0% 0.6 (5) 154 (22.7%) 0 (0.0%) 2 (5.0%) 0 (0.0%) 18 (42.9%) 1 (33.3%) 0 (0.0%) 1 (100%) 0 (0.0%) total 4 zones 12 villages 9 species 677 (84.6%) 12 (1.5%) 40 (5.0%) 2 (0.3%) 42 (5.3%) 3 (0.4%) 16 (2.0%) 1 (0.1%) 7 (0.9%) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 307 http://jad.tums.ac.ir published online: september 30, 2018 discussion in spite of the public health importance of scorpionism kazerun district (16) and its considerable richness of scorpions, limited studies have been directed on the faunistic and biodiversity of these arthropods. the scorpion envenomation has the most public health importance in kazerun district, however, the current study is the first trial in determining the scorpion fauna in the area. the collected scorpion species comprised m. eupeus, h. lepturus, a. crassicauda, s. maurus, c. matthiesseni, h. zagrosensis, r. zarudnyi, m. caucasicus, and orthochirus sp. the most dangerous scorpion, h. lepturus was ranked in second from the point of view of density. dispersion of this species was affected by the type of soil and its humidity, topographic condition and climate. this species was only revealed in the zones 1 and 2, where the topographic conditions are mountainous. a majority of the human victims were stung by h. lepturus (16), while a few more were stung by a. crassicauda, but in over half of the cases the latter species was not known in southern and southwestern parts of iran (4). the scorpion fauna of kazerun district was compared to those of adjacent provinces and districts. the predominant species, m. eupeus had similar ranking in both shiraz district (17) and kohgiluyeh and boyer-ahmad province (18, 19). during a comprehensive study on scorpion fauna in fars province, 18 species were recorded belongs to three families whereby 15 species originated from buthidae, 1 species from scorpioniidae and 2 species from hemiscorpioniidae (9). fifty percent of the recorded species in fars province were discovered in kazerun district. two other comprehensive studies regarding scorpion fauna were carried out during 2008–2011 in the kerman province, in the eastern border of fars province, which showed 8 to 13 species, belonging to the families of buthidae and hemiscorpioniidae, respectively (19, 20). seven out of nine species disclosed in this study were included in the checklist of bu shehr province, which reported 15 species (21). moreover, a faunistic study was done in the khuzistan province in the west of kazerun district (22). surprisingly, the lowest similarity (28.6%) was observed between the fauna of bushehr province and kazerun district. the most common species were a. crassicauda, m. eupeus phillipsii, s. maurus townsendi and r. zarudnyi. the latter finding can lead the scorpionologist towards the detecting the biological factors involved. for the first time, m. caucasicus which explored from zone 4 at 499m above sea level. the latter species had not mentioned in the published checklist of fars province (9). orthochirus sp. which found in zone 2 and 3 needs more taxonomic works for precise identification. parameters such as topography, climate, texture, and humidity of soil were effective parameters. careful planning and designing of a comprehensive database could be essential in assisting of the primary health system in the reduction of scorpion sting and its related complications. conclusion this study revealed the presence of 9 species belonging to 3 families scorpionidae (2 species), buthidae (5 species) and hemiscorpiidae (1 species). the most dangerous scorpion h. lepturus, which known as “gadim” in its local name is revealed in zone 1 and zone 2 where considering as a highland in kazerun district. acknowledgements the authors would like to appreciate the effective cooperation of the head of kazerun health center, the staff in the cdc unit and the health houses in the studied area during conduction of the field operation. this study was part of the msc thesis supported by the hamadan university of medical sciences (project no. 930222648). the authors declare that there is no conflict of interests. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 308 http://jad.tums.ac.ir published online: september 30, 2018 references 1. shahi m, azizi k, ansarian n (2009) study on scorpion fauna in high risk areas of hormozgan province, 2006– 7. med j hormozgan univ. 12(4): 207–210. 2. nazari m, rastgar h (2016) epidemiological study of scorpion sting in city of poldokhtar, iran, between 2010 and 2014, five-year-study. res j pharm biol chem sci. 7(5): 446–451. 3. nazari m, rastgar h (2016) study on distribution of scorpions to provide prevention and interventions in combating scorpionism in poldokhtar county, lorestan province, iran. j clin diagn res. 10(12): lc05–lc09. 4. mohseni a, vazirianzadeh b, hossienzadeh m, salehceh m, moradi a, moravvej sa (2013) the roles of some scorpions, hemiscorpius lepturus and androctonus crassicauda, in a scorpionism focus in ramhormorz, southwestern iran. j insect sci. 13(89): 1–12. 5. navidpour s, kovařík f, soleglad me, fet v (2008) scorpions of iran (arachnida, scorpiones). part iv. kohgilouyeh and boyer-ahmad province. euscorpius. 74: 1–28. 6. mirshamsi o, sari a, hosseinie s (2011) history of study and checklist of the scorpion fauna (arachnida: scorpiones) of iran. prog bio sci. 1(2): 16–28. 7. khaghani r, tirgari s, omrani gh, rafinejad j, mousavi ivanaki e (2005) faunistic study and distribution of scorpions in kish island, iran (persian gulf). modares j med sci. 8(1): 7–12. 8. nazari m, bahrami d, davari b, salehzadeh a (2015) epidemiological survey of scorpion sting cases and identification of scorpion fauna in hamadan city, iran. scientific j hamadan univ med sci. 22(3): 255–262. 9. navidpour s, fet v, kovařík f, soleglad me (2012) scorpions of iran (arachnida, scorpiones). part viii. fars province. euscorpius. 139: 1–29. 10. habibi t (1970) liste de scorpion de iran, bull fac sci, tehran univ. 2: 42–47. 11. dehghani tafti mh, tirgari s (1998) identification of yazd scorpions and biological study of prevailing species. j shahid sadoughi univ med sci hlth serv. 6(4): 72–77. 12. dehghani r, velaei n (2005) classification of scorpions and their diagnostic clue. feyz j kashan univ med sci. 32(8): 62–84. 13. stahnke hl (1972) a key to the genera of buthidae (scorpionida). entomol news. 83(5): 121–133. 14. farzanpay r (1987) knowing scorpions. central university press. tehran, iran. 15. farzanpay r (1988) a catalogue of the scorpions occuring in iran, up to january 1986. revue arachnologique. 8(2): 33–44. 16. nazari m, najafi a (2016) an epidemiological study on scorpion envenomation in kazerun, iran, 2009–14. j mazandaran univ med sci. 26(140): 206–211. 17. azizi k, tirgari s, sayedi-rashti ma (1998) faunistic study of scorpions in shiraz and investigation on their fecundity. j yasooj univ med sci. 9 (3): 25–28. 18. azizi k, shahraki gh, omarani m (2001) determination of fauna and sex ratio of scorpions from villages and suburbs of kohgilouyeh and boyerahmad province. j yassoj univ med sci. 21–22: 6–11. 19. dehghani r, moabed sh, kamyabi f, haghdoost aa, mashayekhi m, soltani h (2008) fauna of scorpions in kerman province. j kerman univ med sci. 15(2): 172–181. 20. navidpour s, ezatkhah m, kovařík f, soleglad me, fet v (2011) scorpions of iran (arachnida: scorpiones). part vii. kerman province. euscorpius. 131: 1–32. http://jad.tums.ac.ir/ http://biostor.org/search/author:%22h%20l%20stahnke%22 https://www.ncbi.nlm.nih.gov/pubmed/5028177 https://www.ncbi.nlm.nih.gov/pubmed/5028177 j arthropod-borne dis, september 2018, 12(3): 296–309 m nazari et al.: species composition … 309 http://jad.tums.ac.ir published online: september 30, 2018 21. navidpour s, soleglad me, fet v, kovařík f (2008) scorpions of iran (arachnida, scorpiones). part ii. bushehr province. euscorpius. 67: 1–33. 22. navidpour s, kovařík f, soleglad me, fet v (2008) scorpions of iran (arachnida, scorpiones). part i. khuzestan province. euscorpius. 65: 1–41. http://jad.tums.ac.ir/ 1dr wiwanitkit iran j arthropod-borne dis, 2011, 5(2): 1 wiwanitkit v: dog ear mite infestation: … 1 letter to the editor dog ear mite infestation: a possible problem in public health system wiwanitkit v wiwanitkti house, bangkhae, bangkok thailand 10160 editor, i read the recent publication on eat mite infestation from thailand with a great interest (maazi et al. 2010). indeed, this is an important public health problem that can be easily forgotten. otodectes can infect to the dog’s owner and this can be a forgotten zoonosis (van de heyning and thienpont 1977, ugbomoiko et al. 2008). based on this report, the source of infection is believed to be from thailand. indeed, in thailand, there are many cases of accidental finding of otodectes in human beings but it is underreported. the problem of control of dogs, hygiene in thailand is still a challenge. finally, these methods are suggested for control of the possible emerging of otodectes infection: a) legal control of dog, b) health education for dog’s owner, c) surveillance on dog borne diseases including to otodectosis and d) promotion of awareness to the medical personnel on this forgotten problem. references maazi n, jamshidi sh, hadadzadeh hr (2010) ear mite infestation in four imported dogs from thailand; a case report. iran j arthropod-borne dis. 4(2): 68– 71. ugbomoiko us, ariza l, heukelbach j (2008) parasites of importance for human health in nigerian dogs: high prevalence and limited knowledge of pet owners. bmc vet res. 4: 49. van de heyning j, thienpont d (1977) otitis externa in man caused by the mite otodectes cynotis. laryngoscope. 87(11): 1938– 1941. corresponding author: prof dr viroj wiwanitkit, email: professorviroj@hotmail.com domiciliary cockroaches as ...(clean copy) j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 141 http://jad.tums.ac.ir published online: june 12, 2018 original article domiciliary cockroaches as carriers of human intestinal parasites in lagos metropolis, southwest nigeria: implications for public health *adedotun a adenusi 1, mary i akinyemi 2, dele akinsanya 3 1medical parasitology unit, department of medical microbiology and parasitology, college of medicine, university of lagos, idi-araba, surulere, lagos, nigeria 2statistics unit, department of mathematics, university of lagos, akoka, lagos, nigeria 3department of zoology, university of lagos, akoka, lagos, nigeria (received 7 july 2016; accepted 12 may 2018) abstract background: domiciliary cockroaches are obnoxious pests of significant medical importance. we investigated the prevalence of human intestinal parasites in cockroaches and its attendant public health importance. methods: overall, 749 cockroaches (periplaneta americana, 509, blattella germanica, 240) caught by trapping from 120 households comprising 3 different housing types in somolu, lagos metropolis, southwest nigeria, in 2015 were screened for human intestinal parasites using standard parasitological techniques. results: the prevalence of human intestinal parasites in cockroaches was 96.4%. there was no statistically significant difference (p> 0.05) in parasite prevalences between p. americana (95.7%) and b. germanica (97.9%). parasite species identified and their prevalence were as follows: entamoeba histolytica/dispar (44.1%), e. coli (37.8%), giardia lamblia (18.7%), cryptosporidium sp. (13.8%), ascaris lumbricoides (61.3%), trichuris trichiura (55.8%), hookworms (11.6%), strongyloides stercoralis (11.7%), taenia/echinococcus spp. (10.5%), enterobius vermicularis (17.2%) and hymenolepis nana (11.6%). parasite prevalence and burdens varied with housing type; the prevalence was significantly higher statistically (p< 0.05) in cockroaches from low-cost bungalow, lcb (100%) and low-cost, 2-storey, lc2-s (100%) houses than in medium-cost flats, mcf (81.3%). parasite burdens were also significantly higher statistically (p< 0.05) in cockroaches from lcb or lc2-s than in cockroaches from mcf. parasite prevalences between cockroach gut and body surfaces were not statistically significant (p> 0.05) but mean parasite burdens in gut were significantly higher statistically (p< 0.05) than on body surfaces. conclusion: cockroaches types carry transmissive stages of human intestinal parasites and may act as reservoirs and potential mechanical vectors for disease transmission. keywords: cockroaches, periplaneta americana, blattella germanica, intestinal parasites, lagos introduction cockroaches (insecta: blattaria) are insects, which have been in existence since antiquity (1), thriving in so many habitats and consuming virtually any organic matter, including fresh and processed human foods, stored products, garbage, and sewage (1, 2). about 4600 described species of cockroaches are distributed worldwide (3). however, only a few of the about 30 synanthropic species are considered as pests in homes, grocery stores, hospitals, offices, schools, warehouses and other establishments (4). the american cockroach, periplaneta americana (blattaria: blattidae) and the german cockroach, blattella germanica (blattaria: blattellidae) are considered two of the most common and notorious cosmopolitan pest species in nigeria (5) and globally (6-8). cockroaches are pests of significant medical, veterinary and public health importance. their presence and sight may induce psychological stress, the levels of which tend to be proportional to cockroach size and number (2). they are an important source of potent envi*corresponding author: dr adedotun a. adenusi, e-mail: aaadenusi@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 142 http://jad.tums.ac.ir published online: june 12, 2018 ronmental aeroallergens, which provoke allergic reactions and exacerbate acute asthma, especially in predisposed atopic individuals (9, 10). cockroaches contaminate foods to which they have access with their feces and foul-smelling secretions, thereby making them offensive and unsafe for human consumption (1, 2). they also serve as intermediate hosts to a number of helminth parasites of veterinary importance, some of which cause debilitating diseases in domestic animals (2). although cockroaches have yet, to be incriminated as biological vectors of human pathogens, their biology filthy habits, indiscriminate diet, feeding mechanisms and morphology, make them vulnerable and suitable, at least, to acquire, mechanically transport and disseminate pathogens. indeed, a variety of pathogenic and potentially pathogenic bacteria, fungi, and parasites have been isolated from body surfaces and/or gut of cockroaches in domestic, food-handling, and hospital environments (6, 11-14). the potential exists, therefore, for mechanical transmission through physical dislodgement, regurgitation, or fecal pellet deposition onto and/or into exposed human food, which may be, ready-to-eat or improperly cooked. although the direct involvement of cockroaches in the transmission of parasites to humans remains to be fully established, their importance in parasite transport and dissemination cannot be underestimated. intestinal parasitoses are among the most common and widespread diseases of humans globally responsible for considerable morbidity and mortality, especially in children, the most vulnerable population (15). they remain a serious threat to public health worldwide, particularly in communities in resource-poor developing countries in the tropics and subtropics where high prevalences are attributable to poverty, poor living conditions, lack of potable water supply, inadequate waste disposal, poor sanitation and environmental hygiene (16). while a few of these parasites require intermediate hosts, many are transmitted by direct ingestion of infective cysts, and oocysts (protozoa) or eggs and/or larvae (helminths) in foods (especially fruits and vegetables), water, soil, pica, or on hands so contaminated. the incidence of human intestinal parasitoses has continued to increase in recent years, in spite of concerted efforts at reduction. moreover, the transmissive/human-infective stages of some of these parasites can survive in the environment for considerable lengths of time. because cockroaches carry the same pathogens found in substrates with which they have contact (17), it is plausible that cockroaches, in environments contaminated with parasite cysts, oocysts, eggs and or larvae, may pick up these stages for transport and dissemination. poor household hygiene and inadequate environmental sanitation provide congenial atmosphere for cockroach infestation. somolu, lagos metropolis, southwest nigeria, typifies a cosmopolitan setting in a developing economy such as nigeria, where poor sanitary conditions, together with ecology and demography, provide congenial atmosphere for cockroach infestation and contact with pathogens. in spite of the above, and the heterogeneity of somolu in terms of human population and physical infrastructures, and considering the medical and public health importance of cockroaches, there is yet, no information on prevalence of human intestinal parasites in domiciliary cockroaches from this locality. the objective of this study was to determine and compare prevalences and species composition of human intestinal parasites in cockroaches from different residential buildings in somolu, lagos state, southwest nigeria. materials and methods study area the study was carried out between aug and nov 2015 in somolu (geographical coordinates, 6°32' n and longitude 3°22' e), a densely populated cosmopolitan area of lagos metropolis, and headquarters of somolu http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 143 http://jad.tums.ac.ir published online: june 12, 2018 local government area (lga) of lagos state, southwest nigeria. the lga which has a land size of 11615km2 and a population of 403569 (18) is inhabited predominantly by people of the yoruba extraction, although all tribes and sub-tribes of the nigerian nationality and expatriates are also resident. climate in the area is typical of that in the state and is characterized generally by daily temperatures of 24–34 °c and monthly relative humidity of 64–93% during the rainy season, usually from apr to oct, and 34– 49% during the dry season from nov to mar. housing types houses in somolu are typical, a mix of old and modern architecture. one hundred and twenty residential buildings were selected for the study using a stratified random sampling procedure. they comprised 40 each, of buildings classified as low-cost bungalow (lcb), low-cost, 2-storey (lc2-s), and medium-cost flat (mcf), based on building architecture and socio-economic status of residents. each lcb or lc2-s had between 5 and 7 rooms on either side of a story and each room was occupied by low-income class individual(s)/family who shared the only kitchen and toilet/bathroom facilities on either side of a story. each mcf consists of a 3-bedroom apartment with a kitchen and at least one toilet facility, in a semi-detached building inhabited by a medium-income class family. inclusion criterion was that no insecticide and/ or trapping device was used to treat cockroach infestation in the one week prior to the commencement of the study. advocacy visits were made to residents of selected houses to explain the objective(s) of the study and to seek for their participation, cooperation, and understanding in the execution of the study. cockroach collection and identification cockroaches were trapped live, using sterile jars baited with pieces of bread soaked in a small amount of beer. the jars, whose inside upper portions were coated with a thin film of petroleum jelly (vaseline®) to prevent cockroach escape, were placed indoors at 19:00h and retrieved at 07:00h the next morning, for 1–3 consecutive days. cockroaches were transported in the jars to the laboratory where they were anaesthetized and killed by exposure to chloroform fume. they were examined under a dissecting microscope and identified to the lowest taxon possible, using standard taxonomic keys (19). they cockroaches were counted and sorted by capture site (housing type), and the appropriate taxon. isolation of parasites from cockroach body surface in order to dislodge parasite stages (cysts, oocysts, eggs and/or larvae) from body surfaces, cockroaches were washed individually by submersion in 5–10ml sterile physiological saline and vortexing at low speed for 2 min. cockroaches were removed from wash solutions using sterile forceps, fixed in 70% alcohol for 5min and air-dried at room temperature. wash solutions were centrifuged at 2000g for 5min, supernatants were decanted and the bottom 0.5–1ml processed further using the formol-ether concentration technique (20). the resulting sediment mixed with the bottom 0.5ml was placed on slide, stained with lugol’s iodine and examined microscopically for human intestinal parasite stages. for the demonstration of cryptosporidium oocysts, a modified ziehl-neelsen staining method (21) was used. briefly, air-dried smears prepared from processed body surface washings were fixed with methanol and stained with carbol-fuchsin for 30min. smears were washed with tap water, decolorized with 1% acid alcohol for 1min, washed again with tap water and counter-stained with 1% methylene blue for 1min. smears were rinsed finally in tap water and air-dried. cockroach specimens which could not be processed immediately were kept in the freezer at -4 °c. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 144 http://jad.tums.ac.ir published online: june 12, 2018 isolation of parasites from cockroach gut following washing in physiological saline, fixing in 70% alcohol and subsequent air-drying, each cockroach was placed in a sterile petri dish and dissected under a dissecting microscope using sterile entomological needles. whole gut was removed and homogenized in 2–5ml physiological saline. the homogenate was filtered through gauze and centrifuged at 2000g for 5min, following which the supernatant was decanted. the bottom 0.5– 1ml was processed further using the formolether concentration technique. the resulting sediment mixed with the bottom 0.5ml was placed on slide, stained with lugol’s iodine and examined microscopically as described above. cryptosporidium oocysts were identified following the modified ziehl-neelsen staining method described above. parasite identification cysts, oocysts, eggs and/or larvae of human intestinal parasites were identified microscopically using bench aids (22) and their numbers recorded. a cockroach was considered a carrier if any parasite stage was detected in preparations from body surface and/or gut contents. data analysis data were input into microsoft excel and analyzed using the “r: a language and environment for statistical computing” software package (23). analysis of variance (anova) was used to test for differences in overall prevalence of parasites between cockroach species, in overall burdens of parasites on cockroach body surfaces and in gut, and in overall burdens of protozoan and helminth groups of parasites. the tukey’s hsd test was used for multiple comparisons of overall prevalences or burdens of protozoan and/or helminth parasites between pairs of residential building types. all tests were carried out at 95% significance level; in all cases, a p< 0.05 was considered statistically significant. results prevalence of human intestinal parasites by cockroach species overall, 749 cockroaches comprising two species, p. americana (509) and b. germanica (240) were caught and identified. human intestinal parasite stages were identified in 96.4% of the cockroaches (table 1). there was no statistically significant difference (f-statistic= 2.354, p= 0.125) in overall prevalence of parasites between p. americana (95.7%) and b. germanica (97.9%). parasites were more frequently isolated from cockroaches trapped from lcb and lc2-s households than in cockroaches from mcf (81.3%) (table 1). there was no statistically significant difference (p= 1.00) in prevalence of parasites between cockroaches from lcb (100%) and lc2-s (100%) households while there were statistically significant differences in prevalences of parasites between cockroaches from lcb and mcf (p=0.001) and lc2-s and mcf (p=0.001). species diversity of human intestinal parasites in cockroaches eleven human intestinal parasites, comprising four protozoan and seven helminth species were identified on body surfaces and/ or in gut of cockroaches. the species and their respective prevalences in both p. americana and b. germanica are as follows: e. histolytica/dispar (44.1%), e. coli (37.8%), g. lamblia (18.7%), cryptosporidium sp. (13.8%), a. lumbricoides (61.3%), t. trichiura (55.8%), hookworms (11.6%), s. stercoralis (11.7%), taenia/echinococcus spp. (10.5%), e. vermicularis (17.2%) and h. nana (11.6%). prevalences of these parasite species in p. americana and b. germanica, trapped from different housing types are shown in figs. 1 and 2, respectively. the helminths, a. lumbricoides and t. trichiura, and the protozoans, e. histolytica/dispar and e. coli were the four most prevalent species in both species of cockroaches, across all housing types, save b. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 145 http://jad.tums.ac.ir published online: june 12, 2018 germanica from mcf wherein g. lamblia was the fourth most prevalent species (25.0%). their respective overall prevalences in p. americana were 59.5%, 56.8%, 43.2% and 36.7% (fig. 1) while the corresponding overall prevalences in b. germanica were 65.0%, 53.8%, 45.8% and 40.0% (fig. 2). prevalence and burden of human intestinal parasites on cockroach body surfaces and in the gut parasite prevalence in cockroach gut was either a little higher than or identical numerically, to the corresponding prevalence on body surface. the anova test showed no statistically significant differences between prevalences in gut and on body surfaces in p. americana (f= 1.363, p= 0.243), b. germanica (f= 0.344, p= 0.558) or both (f= 1.671, p= 0.196). overall parasite prevalences were significantly higher statistically with helminths than protozoans in p. americana (93.9% vs. 83.7%, f= 28.25, p= 1.31e-07), b. germanica (97.5% vs. 83.3%, f= 29.38, p= 9.46e-08), or both (95.1% vs. 83.6%, f= 54.52, p= 2.54e-13) and in cockroaches from lcb (p= 0.005), lc2-s (p= 3.45e-07) and mcf (p= 0.0001). mean parasite burdens/counts in p. americana (table 2) and b. germanica (table 3) varied with cockroach body region, parasite group (protozoa or helminth) and housing type. highest parasite burdens on cockroach body surfaces and in gut were with the protozoans, e. histolytica/dispar and e. coli and the helminths, a. lumbricoides and t. trichiura, overall and in each housing type (tables 2, 3). overall mean parasite burdens in gut were significantly higher statistically than on body surfaces in p. americana (f= 330, p< 2e-16), b. germanica (f= 166.3, p< 2e-16), and both (f= 496, p< 2e-16). similarly, overall burdens of helminth parasites were significantly higher statistically than those of protozoan parasites in p. americana (f= 156, p< 2e-16), b. germanica (f =74.89, p< 2e-16), both (f= 229.8, p< 2e-16) and in lcb (p= 0.0001), lc2-s (p= 0.0001) and mcf (p= 0.044). differences in overall parasite burdens were statistically significant between lcb and mcf (p= 0.0001) and lc2-s and mcf (p= 0.0001), but not statistically significant between lcb and lc2-s (p= 0.996). table 1. prevalence of human intestinal parasite stages in cockroaches housing type number of cockroaches examined (% parasite +ve) total periplaneta americana blattella germanica lcb 207 (100) 91 (100) 298 (100)a lc2-s 202 (100) 105 (100) 307 (100)a mcf 100 (78.0) 44 (88.6) 144 (81.3)b total 509 (95.7)a 240 (97.9)a 749 (96.4) *values with same superscript along the same column or row are not significantly different statistically at α= 0.05 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 146 http://jad.tums.ac.ir published online: june 12, 2018 fig. 1. prevalence of human intestinal parasites in periplaneta americana from different housing types fig. 2. prevalence of human intestinal parasites in blattella germanica from different housing type http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 147 http://jad.tums.ac.ir published online: june 12, 2018 table 2. mean parasite burdens in periplaneta americana cockroaches from houses in somolu, lagos, nigeria parasite species lcb (n=207) lc2-s (n=202) mcf (n=100) body surface gut total body surface gut total body surface gut total ascaris lumbricoides 2.2±2.1 4.8±4.2 7.0±6.2 2.3±2.0 4.9±4.2 7.2±6.2 0.9±1.2 1.5±2.0 2.3±3.2 cryptosporidium sp 0.3±0.8 0.6±1.6 0.9±2.3 0.5±1.1 0.9±2.1 1.3±3.1 0.1±0.3 0.2±0.6 0.2±1.0 entamoeba coli 1.3±1.7 2.9±3.7 4.2±5.4 1.1±1.6 2.3±3.4 3.3±4.9 0.4±0.9 0.9±1.7 1.3±2.6 entamoeba histolytica/ dispar 1.4±1.9 3.1±3.8 4.5±5.6 1.7±1.9 3.3±3.8 5.0±5.6 0.5±1.0 0.9±1.8 1.3±2.8 enterobius vermicularis 0.5±1.1 1.0±2.2 1.5±3.3 0.5±1.2 1.0±2.1 1.5±3.3 0.2±0.6 0.3±1.0 0.4±1.6 giardia lamblia 0.3±0.9 0.7±1.7 1.0±2.6 0.5±1.0 0.9±2.1 1.4±3.1 0.3±0.6 0.5±1.2 0.8±1.8 hookworms 0.2±0.6 0.4±1.3 0.6±1.9 0.4±1.0 0.7±1.8 1.0±2.7 0.1±0.4 0.2±0.8 0.2±1.1 hymenolepis nana 0.3±0.8 0.6±1.5 0.8±2.3 0.3±0.8 0.5±1.5 0.8±2.3 0.1±0.3 0.1±0.6 0.2±0.9 strongyloides stercoralis 0.2±0.5 0.3±1.0 0.5±1.5 0.2±0.6 0.5±1.2 0.7±1.8 0.0±0.2 0.1±0.6 0.2±0.8 taenia/echinococcus sp 0.3±0.8 0.6±1.7 0.8±2.4 0.2±0.7 0.5±1.5 0.5±2.1 0.1±0.5 0.3±1.0 0.4±1.5 trichuris trichiura 1.9±1.9 3.9±3.8 5.8±5.7 2.2±2.0 4.4±3.8 6.7±5.8 0.7±1.2 1.3±2.0 2.1±3.1 total 8.8±4.1 18.8±7.5 27.6±11.4 9.7±3.8 19.7±7.0 29.3±10.5 3.3±2.6 6.2±4.6 9.4±7.1 table 3. mean parasite burdens in blattella germanica cockroaches from houses in somolu, lagos, nigeria parasite species lcb (n=91) lc2-s (n=105) mcf (n=44) body surface gut total body surface gut total body surface gut total ascaris lumbricoides 2.7±2.2 5.7±4.7 8.4±6.9 2.6±2.0 5.6±4.1 8.3±6.0 1.2±1.5 2.1±2.5 3.3±4.0 cryptosporidium sp 0.2±0.6 0.3±1.1 0.5±1.7 0.4±1.0 0.8±2.1 1.2±3.0 0.2±0.5 0.3±0.9 0.5±1.4 entamoeba coli 1.6±1.9 3.4±4.0 4.9±5.8 1.0±1.6 2.1±3.2 3.1±4.7 0.8±1.2 1.2±2.0 2.0±3.2 entamoeba histolytica/ dispar 1.6±1.8 3.4±4.0 5.0±5.8 1.7±1.8 3.4±3.7 5.0±5.4 0.5±1.0 0.7±1.5 1.2±2.5 enterobius vermicularis 0.5±1.0 1.0±2.3 1.5±3.3 0.6±1.3 1.1±2.3 1.8±3.5 0.1±0.5 0.2±0.8 0.3±1.2 giardia lamblia 0.7±1.3 1.4±2.7 2.2±4.0 0.4±1.1 0.9±2.4 1.4±3.5 0.5±0.9 0.8±1.4 1.2±2.2 hookworms 0.3±0.9 0.5±1.9 0.8±2.8 0.4±1.0 0.9±2.0 1.3±3.0 0.1±0.5 0.2±0.8 0.3±1.2 hymenolepis nana 0.2±0.7 0.5±1.4 0.7±2.2 0.3±0.9 0.5±1.6 0.8±2.4 0.1±0.3 0.2±0.8 0.3±1.1 strongyloides stercoralis 0.2±0.6 0.4±1.1 0.6±1.6 0.3±0.7 0.5±1.3 0.8±1.9 0.1±0.4 0.3±1.0 0.4±1.5 taenia/echinococcus sp 0.3±0.8 0.7±1.8 1.0±2.6 0.1±0.4 0.2±1.0 0.3±1.4 0.2±0.5 0.4±1.2 0.6±1.6 trichuris trichiura 1.6±1.9 3.5±3.8 5.1±5.6 2.0±2.0 4.2±3.9 6.3±5.9 0.9±1.2 1.6±2.0 2.5±3.1 total 9.8±4.8 20.9±9.7 30.7±14.3 9.9±3.2 20.3±6.4 30.2±9.4 4.4±2.6 8.0±4.1 12.4±6.6 discussion cockroaches are nuisance pests whose activities impact negatively on humans. of great concern to human and public health, is their capability as potential mechanical vectors of pathogens, including parasites. previous studies from other parts of nigeria (13, 24, 25) and elsewhere (6, 12, 26) had reported that cockroaches captured from homes, hostels, hospitals, and markets carry an array of human intestinal parasites. results of the present study, which show clearly that the two cockroach species (p. americana and b. germanica) from residential buildings in somolu, lagos, southwest nigeria, carry human intestinal parasites on their body surfaces and/or in the gut indicate that concerns over their potential and/or role as mechanical vectors cannot be overlooked. the species of human intestinal parasites recovered from cockroaches in the present study, (e. histolytica/dispar, e. coli, g. lamblia, cryptosporidium sp. a. lumbricoides, t. trichiura, hookworms, s. stercoralis, taenia/ echinococcus spp., h. nana, and e. vermicularis) are responsible for a number of disease conditions in man, some of which could be life-threatening. the three major soil-transhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 148 http://jad.tums.ac.ir published online: june 12, 2018 mitted helminths (a. lumbricoides, t. trichiura, and hookworms) account for a high burden of disease globally and are intimately related with malnutrition, growth stunting and cognitive deficits in children (27). strongyloides stercoralis may cause complicated infections with high case fatality rates due to hyperinfection or dissemination, especially in immunocompromised individuals (28). cryptosporidium sp. and g. lamblia are nowadays, major causes of diarrhoea, especially in children (29). entamoeba histolytica causes amoebiasis, a potentially severe and life-threatening disease and the second most common cause of death from parasitic diseases, after malaria (30). accidental ingestion of taenia (particularly, t. solium) eggs often results in human neurocysticercosis, the leading cause of preventable epilepsy worldwide and also, a leading cause of deaths from food-borne diseases (31). species of echinococcus cause life-threatening chronic diseases with poor prognosis and high fatality rates, if not carefully managed clinically (32). parasite species reported in the present study are consistent with those documented in similar studies (13, 24, 25, 33, 34). however, the disparities could be due to differences in the levels of household and environmental hygiene, transmission dynamics between study localities, and in the diagnostic procedure employed. predominance of a. lumbricoides on body surfaces and/or in the gut of cockroaches across all different housing types in the present study is in consonance with findings from similar studies in other parts of the country (13, 25, 33). this could be due to its predominance in the human population and/or the persistence of its eggs in the environment for months to years (35). identity of parasite species in cockroaches from all three housing types indicates that these parasites have equal chances of being acquired by cockroaches, probably because they are endemic in the study area. differences in individual parasite species burdens between cockroaches from low-cost (lcb or lc2-s) and mcf could then be explicated by the varying levels of hygiene and sanitation in each housing type. poorer housing and sanitary conditions, which could predispose to higher parasite contamination, characterize low-cost households. overwhelming and widespread prevalence (96.4%) of human intestinal parasites in domiciliary cockroaches in the present study is singular in nigeria and is a cause for public health concern. other studies from nigeria had reported prevalences of 58.6% in calabar, southsouth (25), 67.1% in owerri, southeast (33) and 77.5% in sokoto, northwest (24). al-mayali and al-yaqoobi (36) and elsherbini and el-sherbini (34) reported prevalences of 83.3% and 98% respectively, in iraq and egypt. disparities in prevalences between different studies may be explained by differences in the levels of hygiene and sanitation between study localities. identity in the diversity of parasites between p. americana and b. germanica as well as the insignificant statistical differences in their respective prevalences in the cockroach species indicate a uniform distribution of parasite species between the two cockroach species in the same environment. they also suggest that the two cockroach species have equal potential for mechanical transport and possibly, consequent dissemination of parasites in the environment. because pathogens carried by cockroaches are acquired from their immediate environments (37), the human intestinal parasites reported herein, were acquired through contact with unhygienic environments. most of the parasites whose cysts, oocysts, eggs and/or larvae (hookworm: a. duodenale only) were isolated from cockroaches in the present study are transmitted to humans via consumption of food and/or water so contaminated. since cockroaches travel indiscriminately between filth and human food, they may be capable of disseminating parasite stages (on their body surhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 141–151 aa adenusi et al.: domiciliary cockroaches … 149 http://jad.tums.ac.ir published online: june 12, 2018 faces and/or in the gut) through physical dislodgement, vomitus and/or feces onto any substrate in the environment, including human food and food preparation surfaces. the medical and public health implications of this are better imagined. cysts of e. histolytica and e. dispar are morphologically indistinguishable microscopically, so also are the eggs of echinococcus and taenia species. since molecular techniques, which differentiate reliably, cysts of e. histolytica from e. dispar (38) and eggs of echinococcus from taenia species (39, 40) were not employed in the present study, the former was simply identified morphologically as e. histolytica/dispar and the latter as taenia/ echinococcus spp. conclusion cockroaches (p. americana and b. germanica) across different housing types in somolu, lagos metropolis, nigeria, transport on their body surfaces and/or in the gut, transmissive stages of human intestinal parasites and thus, may 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assay in differentiating entamoeba histolytica, entamoeba dispar and entamoeba moshkovskii infections in orang asli settlements in malaysia. parasit vectors. 6(1): 250. 39. jimenez ja, rodriguez s, moyano lm, castillo y, garcia hh (2010) differentiating taenia eggs found in human stools does ziehl neelsen staining help? trop med int health. 15(9): 1077–1081. 40. salant h, abbasi i, hamburger j (2012) the development of a loop-mediated isothermal amplification method (lamp) for echinococcus granulosus coprodetection. am j trop med hyg. 87: 883– 887. http://jad.tums.ac.ir/ author template for journal articles j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 371 http://jad.tums.ac.ir published online: september 08, 2017 original article spatial distribution of medically important scorpions in north west of iran mulood mohammadi bavani 1, *javad rafinejad 1, *ahmad ali hanafi-bojd 1, mohammad ali oshaghi 1, shahrokh navidpour 2, farrokh dabiri 3, mehdi badakhshan 3, esmaeil ghorbani 4, masoomeh bagheri 3 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2razi reference laboratory of scorpion research, razi vaccine and serum research institute, agricultural research education and extension organization, karaj, iran 3department of medical entomology and vector control, school of public health, urmia university of medical sciences, urmia, iran 4disease control unit, sareyn health center, ardabil university of medical sciences, sareyn, iran (received 3 jan 2017; accepted 29 aug 2017) abstract background: scorpions are one of the most important medical arthropods in iran. this study aimed to determine the fauna, spatial distribution and some morphological characteristics of these venomous arthropods in the study area. methods: scorpions were collected using ultra violet light, rock rolling and digging methods in west azerbaijan, east azerbaijan, and ardabil provinces during 2015–2016. the specimens were preserved in 75% ethyl alcohol and transferred to the laboratory for species identification and morphological studies. results: distribution maps were produced using arcgis 10.3. totally, 368 specimens from two families of buthidae (97.1%) and scorpionidae (2.99%) were collected and identified as mesobuthus eupeus (80.16%), androctonus crassicauda (10.60%), m. caucasicus (4.89%), hottentotta saulcyi (1.35%) and scorpio maurus (2.99%). conclusion: the presence of medically important species, including the deadly black one in northwestern iran requires health educational and control programs for reduction of these public health problems. keywords: scorpions, distribution, morphology, gis, iran introduction scorpions are living fossils, and about 2000 species have been identified in the world systematically classified in different families (1). among them, buthidae, with a universal distribution, is the greatest family consisting of 81 genera and 570 species (2). except for hemiscorpius lepturus, all dangerous scorpion species belong to this family (3). scorpion stings are a major public health problem in subtropical and tropical countries (4) and some species can be lethal for human. annually, nearly 1230000 cases of scorpion sting with a fatality rate of 27% are reported in different parts of the world (5). the most medically important species belong to hotten totta, buthus, tityus, leiurus, androctonus, parabuthus, centruroides and mesobuthus genera (6). scorpions' venom is a complex mixture containing peptides, enzymes, mucoproteins, free amino acids, nucleotides, lipids, amines, heterocyclic components, non-organic salts and low molecular-weight organic molecules. peptides that affect ion channels including sodium, potassium, calcium and cholera channel are called neurotoxin, and because of their effect on ion channels endanger human health and sometimes results in death (7). two iranian dangerous scorpions’ families i.e. buthidae and hemiscorpiidae have different ef*corresponding authors: dr javad rafinejad, email: jrafinejad@yahoo.com, dr ahmad ali hanafibojd, e-mail: aahanafibojd@tums.ac.ir http://jad.tums.ac.ir/ mailto:jrafinejad@yahoo.com j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 372 http://jad.tums.ac.ir published online: september 08, 2017 fects on human; such that buthidae species have mostly neurotoxic effect, but hemiscorpiidae such as hemiscorpius lepturus (local name: gadim), causes cell death and hemolytic effect cause fatality rate in the country (8, 10). because of the remarkable diversity in zoogeographic zones of iran, there is a rich arthropod’s fauna that includes scorpions. several dangerous species inhabit some areas across different parts of iran, and some of them are lethal for human (11). scorpion stings cases have been reported as exceeding 42500 annually in iran, with a fatality rate of 44.7 per 100000. these figures put the country in the second place after mexico concerning case fatality rate (11, 12). these numbers are not definitive and the actual numbers of scorpion sting and fatality rate are greater than the annual recorded figures. olivier studied the scorpion fauna in iran and described the black scorpion (androctonus crassicauda) in kashan city (13(. since then, several studies have reported 4 families, 19 genera and 53 species of scorpions in iran. buthidae, consisting of 15 genera and 45 species, is the largest family )14, 31(. mesobuthus eupeus, a. crassicauda, h. lepturus, odontobuthus doriae, h. saulcyi, hottentotta schach, compsobuthus matthiesseni and m. caucasicus are the most medically important species, while a. crassicauda and h. lepturus are the most dangerous and deadly species to human in iran )11, 32, 34(. due to the larger extent and the importance of scorpions in the southern tropical areas of the country, most studies have been performed in this area. therefore, faunistic, morphological, spatial distribution and ecological studies on these toxic arthropods have been neglected in other parts of the country. there is a few information concerning species composition, geographical distribution and the morphology of scorpions in the northwestern corner of iran. report of an annual number of 1186 scorpions sting cases in this area )1( encouraged us to design and conduct this study. therefore, this investigation conducted to clarify the current fauna, abundance, geographical distribution and some morphological features of scorpions to provide identification key and its control guideline in this region of the country. materials and methods study area this study was conducted in west azerbaijan, east azerbaijan and ardabil provinces, all in north west part of iran. these provinces cover an area of about 1007031 km², between 35◦58´–39◦46´n latitudes and 44◦03´–48◦55´e longitudes. they share borders with armenia and republic of azerbaijan in the north, kurdish inhabited area in turkey and kurdistan regional government in the west, gilan province in the east, and zanjan and kurdistan provinces in the south. these provinces include 43 counties with a total population of 8053684 inhabited this area in various weather and environmental conditions. the region has both plains and mountains (fig. 1). specimen and data collection fifty-four locations were selected and the scorpion collection and sampling procedure was carried out from jun to aug 2015–2016 during day and night periods using the standard methods, i.e. rock-turning )35( (searching under the stones and rock etc.) and uv lights )36(. in addition, to capture burrowing scorpions, pouring water in holes, digging and rubber band technique were used in various scorpions’ habitats. geographical location, latitude, longitude, altitude (using gps), environmental temperature and relative humidity (using digital thermometer and hygrometer) were recorded during sampling. all samples were preserved in 75% ethyl alcohol and kept in the different http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 373 http://jad.tums.ac.ir published online: september 08, 2017 labeled plastic boxes according to their localities, and then transferred to the school of public health, tehran university of medical sciences, and tehran, iran. morphological identification and analyses such as dentition of the pedipalp chela on movable finger, accessory granules under the terminal denticle on the pedipalp chela movable finger (agmf), rows of denticles on the pedipalp chela movable finger (rdmf), pectinal teeth (pt) were done when observed under stereomicroscope using standard iranian scorpion identification keys )12,14(. arcgis 10.3 was used to produce the distribution maps of the collected species. results in total, 368 scorpions were collected and identified. they belonged to two families, five genera and five species: m. eupeus (80.16%), a. crassicauda (10.60%), m. (olivierus) caucasicus (4.89%), s. maurus (2.99%) and h. (buthotus) saulcyi (1.35%) (fig. 4). except for s. maurus (scorpionidae), other species belonged to the buthidae family. table 1 shows the species composition and sex ratio of the scorpions collected from different counties of the study area. morphological characteristics of scorpions m. eupeus (fig. 3a). in total, 295 specimen (80.16% of the collected samples ( of this species were captured in the 34 localities of all three provinces (west azerbaijan, east azerbaijan and ardabil provinces), (fig. 2a). this non-digger species belongs to the family of buthidae may be regarded as a complex species. the variation in color pattern was observed from north to the south so that the northern specimens near the aras river had darker spots and stripes on their carapace and mesosomal segments than others. this species was found in all the three pro vinces with a wide distribution from parsabad in the north, to tekab in the south, and from urmia in the west to namin in the east (fig. 2a). this species was found in different localities and habitats with different climates in urban and rural area including under stones and bark, on the farm and plow, near houses, in cemetery, dilapidated homes and grasslands. this species was collected from plains and mountainous area with an altitude range between 188 to 2180m. the sex ratio for this species was 1:1.10 in favor of females. pectineal teeth (fig. 4c, f) were between 21/22–30/32 for males and 16/17–23/25 for females; the number of accessory granules under the terminal denticle on the pedipalp chela movable finger (fig. 5c) was 4 for both sexes but seldom had they 0, 2, 3 and 5 granules. rows of denticles on the pedipalp chela movable finger (fig. 4i) were 12–14. a. crassicauda (fig. 3c). totally, 39 specimen from 9 localities of two provinces (west azerbaijan and east azerbaijan) (fig. 2b) were collected, comprising 10.6% of all captured scorpions. this species inhabited indoors of buildings and hospitals, under stones, inside soil, sand and cemetery stones in plain and mountain areas at an altitude ranging from 690 to 1473m. this is the second most common and deadly scorpion species collected in some counties, but was absent in the ardabil province. pectinal teeth (fig. 4b, e) were 31/32– 34/35 for males and 23/24–26/27 for females. rows of denticles on the pedipalp chela movable finger (fig. 4h) were 15–16 for females and 13–16 for males. the sex ratio was 1:0.39 in favor of males. the numbers of granules under the end granule (fig. 5b) were 3, but sometimes in a few specimens only one was observed, like those of m. eupeus, this is uncommon. m. caucasicus (fig. 3f). in total, 18 specimens (4.89% of all collected samples) of this species were captured in 9 locations of two http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 374 http://jad.tums.ac.ir published online: september 08, 2017 provinces (west azerbaijan and east azerbaijan) (fig. 2c) in mountainous areas with a height of 690–2180m. this species always seek shelter under stones and inside the wooden roofs of rural houses. pectinal teeth (fig. 4a and 4d) and number of rows of denticles on the pedipalp chela movable finger (fig. 4g) were 26/28– 31/29 and 22/23–23/24 and 11–14 and 12– 13 for males and females, respectively. the sex ratio was 1:0.2 in favor of males. the numbers of granules under the end granule on movable finger (fig. 5a) were 4 in all specimens. s. maurus (fig. 3d). this digger species belongs to the family of scorpionidae and was found only in givi (ardabil province), mahabad and miandoab (west azerbaijan province) (fig. 2d) counties at the heights of 1286, 1404 and 1431m, but this species was not collected from east azerbaijan province. the sex ratio was 1:10 in favor of females. h. saulcyi (fig. 3e). this species has limited distribution in the region in which it was collected. we collected it only from two sites of sardasht county, west azerbaijan province, at the heights of 1286, 1404 and 1431m (fig. 2e). this species was not caught in the ardabil and east azerbaijan provinces. only the females of this taxon were captured. pectinal teeth (fig. 5d), the number of dens row on movable finger (fig. 5e) and granules under the end (fig. 5f) were 24/25– 26/27 and 14–15, and 4, respectively. table 1. species composition and spatial distribution of scorpions collected in northwest of iran species total(n) m. eupeus a. crassicauda m. caucasicus s. maurus h. saulcyi location m a le (n ) f e m a le (n ) m a le (n ) f e m a le (n ) m a le (n ) f e m a le (n ) m a le (n ) f e m a le (n ) m a le (n ) f e m a le (n ) m a le (n ) f e m a le (n ) parasad 18 20 18 20 germi 4 6 4 6 namin 27 16 27 16 ardabil 4 8 4 8 meshkinshahr 17 21 17 21 givi 1 1 khakhal 3 7 3 7 khodaafarin 10 14 10 14 ahar 3 3 3 3 mianeh 5 8 5 8 hashtrood 1 1 bonab 2 2 1 3 2 marand 3 1 3 jolfa 9 6 2 2 13 6 malakan 1 1 tabriz 3 1 3 1 maraghe 1 1 khoi 2 1 4 2 6 3 makoo 2 2 1 1 4 3 salmas 3 5 3 1 6 6 urmia 27 36 17 6 3 1 47 43 mahabad 1 1 2 miandoab 1 1 9 2 9 sardasht 1 5 3 3 tekab 2 1 2 1 total 140 155 28 11 15 3 1 10 5 186 182 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 375 http://jad.tums.ac.ir published online: september 08, 2017 fig. 1. study area in north west of iran http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 376 http://jad.tums.ac.ir published online: september 08, 2017 fig. 2. spatial distribution of collected scorpions from northwestern iran. (a) mesobuthus eupeus, (b) androctonus crassicauda, (c) mesobuthus caucasicus, (d) scorpio maurus, (e) hottentotta saulcyi. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 377 http://jad.tums.ac.ir published online: september 08, 2017 fig. 3. collected scorpions’ species form north west of iran: (a) mesobuthus euepeus, (b) hottentotta saulcyi under uv light, (c) androctonus crassicauda, (d) scorpio maurus, (e) hottentotta saulcyi, (f) mesobuthus caucasicus fig. 4. morphological characteristic of medically important collected scorpions from north west of iran. (a–c) pectinal teeth of adult male, (a) mesobuthus caucasicus, (b) androctonus crassicauda, (c) mesobuthus eupeus; (d– f) pectinal teeth of adult female. (d) mesobuthus caucasicus, (h) androctonus crassicauda, (f) mesobuthus eupeus. (g–i) rows of denticles on the pedipalp chela movable finger. (g) mesobuthus caucasicus, (h) androctonus crassicauda, (i) mesobuthus eupeus http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 378 http://jad.tums.ac.ir published online: september 08, 2017 fig. 5. (a–c) number of accessory granules under the terminal denticle on the pedipalp chela movable finger. (a) mesobuthus caucasicus, (b) androctonus crassicauda, (c) mesobuthus eupeus, (d–f) hottentotta saulcyi adult female. (d) pectinal teeth, (e) rows of denticles on the pedipalp chela movable finger, (c) number of accessory granules under the terminal denticle on the pedipalp chela movable finger discussion in this study, 368 specimens were collected comprising five species that include m. eupeus, a. crassicauda, o. caucasicus, s. maurus and h. saulcyi. buthidae is the largest family of scorpions in the world, as well as in iran, and this species was documented as the largest family of the collected scorpions in this study. buthidae was the largest family (97.1% of total), and m. eupeus and a. crassicauda are the most medically abundant species in the northwestern quarter of the country. similar to our results, some recent investigation have reported buthidae as the largest family comprising 88.5% of all the species, and a. crassicauda and m. eupeus as the most abundant, medically important species (32, 37(. the genus hemiscorpius has the first fatality rate in iran, followed by the genus androctonus. although we could not find any species of the first genus, a. crassicauda was collected from east azerbaijan and west azerbaijan. two cases of death probably from this black deadly scorpion were reported from the study area (38(. hence, health staffs should be familiar and well educated to know scorpions' morphology and ecology, educate the community on how to prevent and treat scorpion’s sting. in our study, the family buthidae comprises 97.1% of all collected specimen, among which m. eupeus was the most prevalent and included 80.16% of all specimen, and this species is regarded as the most medically important scorpion species in the region. this is because of the wider geographical distribution of m. eupeus, which accounts for the majority of scorpion envenomation in this part of iran. this species is considered as most common scorpion in all parts of iran, including the northwestern regions, and inhabits areas under stones, tree barks, construction debris, in the roof of old buildings and inside beetle nests (as observed in this study). these species have about 14 members through its geographical distribution area, and this species can be a complex species (39, 40(. there are six members, however, only one member of the species (mesobuthus eupeus eupeus) has been reported in our study area) 39(. these studies were limited to one or two counties in the north west of iran and had not covered the entire area (39(. in comparison, morphological diversity of this species was recognized in our study, at least in coloration pattern, so that the color of northern species near the aras river was http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 379 http://jad.tums.ac.ir published online: september 08, 2017 darker (in carapace and mesosoma) than those found in the central and southern parts, for examples: urmia and tekab. to prove these differences, molecular experiments should be done. the second most prevalent species, a. crassicauda, is the most dangerous and deadly scorpion in iran, especially the northwestern parts, such that (this study) its sting cause local pain and autonomic stimulation and fatal cardiovascular problems, particularly in children, and needs special attention because its domestic activity is moving towards human shelters )32(. other collected species included m. caucasicus, h. saulcyi and s. maurus, among them, the first and second are classified as belonging to iranian medically important species. mesobuthus caucasicus is regarded as the third medically important species due to its wide dispersion and low density in the region. h. saulcyi is considered as the fourth medically important species in the region due to its limited distribution. studies were conducted in zanjan province that shares long borders with our study area and have collected a. crassicauda, h. saulcyi, hottentotta zagrosensis, o. doriae, m. eupeus and s. maurus. among them, four species including m. eupeus, a. crassicauda, h. saulcyi and s. maurus are common between this province and our study region (29(. in addition, in turkey which have long borders with western azerbaijan province disclosed the presence of l. quinquestriatus, m. gibbosus and m. eupeus of the family buthidae, among which m. eupeus and a. crassicauda are common in our study )41, 44(. the polyvalent antivenom in iran is produced by the razi vaccine, serum, and research institute against the 6 medically important scorpion species: m. eupeus, a. crassicauda, o. and h. saulcyi. granules under the terminal denticle on the pedipalp chela movable finger were 4 (fig. 3l) for all specimens, however, many variations were observed in our study. rows of denticles on the pedipalp chela movable finger (fig. 3i) were 11, but a range of 12–14 was observed in our study, and these variations show that probably there are more than one types of this species in northwestern provinces of iran. this requires the necessity of molecular experiments to clarify which kind of this species is present in these areas. only females of h. saulcyi were captured with pectinal teeth of 24/25–26/27, but the female specimen from turkey and iraq had 26–28 pectinal teeth and iranian females' specimens had 27–28. the number of granules on the tip of the movable finger was 3–5 (in turkey and iraq specimens), in iran was 4 and sometimes was absent (45, 46(. however, in our study, all collected specimens had 4 granules and 14–15 rows of denticles on chela movable finger. the number of pectinal teeth was investigated to distinguish between the genders of scorpions (46(. the number of pectinal teeth (pt) of a. crassicauda was 30 and 24 in males and females of this scorpion, respectively (47(. these findings were in contrast with our study where pt varied between 31/32–34/35 for males and 23/24–26/27 for females. a study conducted in zarrindasht county, fars province of iran, showed sex ratios (male to female) of s. maurus, m. caucasicus and m. eupeus to be 1:5.73, 1:0.31 and 1:0.55, respectively. similarly, our study recorded the sex ratio of m. eupeus and m. caucasicus in favor of males. males of captured scorpions were more prevalent than females, but for s. maurus this ratio was in favor of females (1:10) than males (48(. conclusion there are four medically important scorpion species including m. eupeus, a. crassicauda, m. caucasicus and h. saulcyi in northwestern iran, among them, a. crassicauda is the most deadly scorpion in the study area. hence, residents should be careful of its sting, http://jad.tums.ac.ir/ j arthropod-borne dis, september 2017, 11(3): 371–382 m mohammadi bavani et al.: spatial distribution of … 380 http://jad.tums.ac.ir published online: september 08, 2017 especially in children. another medically important, but not deadly, species is m. eupeus which inhabited all studied provinces. species composition and spatial distribution of scorpions, clarified in the northwestern provinces of iran will be useful to assign the appropriate share of monovalent antivenom to the area and will reduce the public health problem and mortality due to scorpion sting because of quick access to treatment. acknowledgements this article is a part of the dissertation for fulfillment of doctoral degree in medical entomology and vector control. this study was financially supported by the research deputy, tehran university of medical sciences (project no. 31093). the authors' deep gratitude goes to the health staffs of west azerbaijan, east azerbaijan and ardabil provinces for their collaboration and positive appetite. thanks also go out to all the health staffs of selected counties involved in field support for this study. the authors declare that there is no conflict of interest. references 1. dehghani r, rafinejad j, fathi b, panjehshahi m, jazayeri m, hashemi a (2017) a retrospective study on scropionism in iran (2002–2011). j arthropod borne dis. 11(2): 194. 2. 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arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 62 http://jad.tums.ac.ir published online: april 27, 2019 original article biodiversity of mosquitoes (diptera: culicidae) with emphasis on potential arbovirus vectors in east azerbaijan province, northwestern iran azim paksa1; mohammad mahdi sedaghat1; hassan vatandoost1,2; mohammad reza yaghoobi-ershadi1; seyed hassan moosa-kazemi1; teimour hazratian3; alireza sanei-dehkordi4,5; *mohammad ali oshaghi1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2institute for environmental research, tehran university of medical sciences, tehran, iran 3departmemt of parasitology, faculty of medicine, tabriz university of medical sciences, tabriz, iran 4department of medical entomology and vector control, faculty of health, hormozgan university of medical sciences, bandar abbas, iran 5infectious and tropical diseases research center, hormozgan health institute, hormozgan university of medical sciences, bandar abbas, iran (received 7 mar 2018; accepted 2 jan 2019) abstract background: the abundance, diversity, distribution and ecology of mosquitoes (diptera: culicidae), especially arbovirus vectors are important indices for arthropod-borne diseases control. methods: larvae and adult mosquitoes were collected using the standard methods from different habitats in nine localities of three counties in the east azerbaijan province, northwestern iran during june to october 2017. in addition, species richness (r), simpson’s diversity index (d), shannon–wiener index (h̕) and evenness (e) as measures of diversity, were calculated. results: overall, 1401 mosquito specimens including 1015 adults and 386 larvae were collected in the study area. the properties of geographical larval habitats were recorded. four genera along with 10 species were collected and identified, including anopheles hyrcanus, an. maculipennis s.l., an. superpictus s.l., aedes caspius, ae. vexans, culex pipiens, cx. theileri, cx. perexiguus, culiseta longiareolata and cs. subochrea. among the three counties, ahar region presented the highest species richness (r: 1.5) and diversity values (d: 0.79, h’: 1.74, e: 0.73). conclusions: this study provides important information on the diversity, distribution and ecology of ten mosquito species in the region. this information leads to a better understanding of mosquito population dynamics in relation to vector control measures. keywords: diversity; ecology; mosquitoes; arbovirus vectors; iran introduction mosquitoes (diptera: culicidae) are suborder of nematocera and culicidae family, which are the medically-important species for malaria transmission, types of filariasis, types of encephalitis and other arboviral diseases (1-2). the culicidae family includes 2 subfamilies, 11 tribes; the culicidae family includes 2 subfamilies, 41 families, 11 tribes, 113 genera and 3563 species (1-4). the most important genera of the culicidae family are anopheles, culex and aedes. among the culicidae, the aedes species have the most various species and are extremely important in medical cases (5). mosquitoes play a crucial role in transmission of some important diseases such as malaria, filariasis, dengue fever, yellow fever, chikungunya, west nile virus and zika virus which are today among the greatest health problems in the world (6-9). mosquitoes larvae have the power to col *corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir j arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 63 http://jad.tums.ac.ir published online: april 27, 2019 onize and live in a wide variety of natural and artificial habitats, such as temporary and permanent water resources, unclean and clean water, large or small water resources, stagnant or stream waters and even the smallest places where water is gathered, such as buckets of water, pots, tires, bromeliads, animal feet prints and plant leaf axes (10-11). adult mosquitoes are very diverse bionomically, for example, they are too diverse in host searching, biting behaviors, dispersal and reproductive strategies (9). two factors (abiotic and biotic) affect mosquito life cycle. the biotic factors contain adult sugar and blood meal types, species communications, interactions and natural enemies. physicochemical attributes of larval habitats are abiotic factors, which include the type and contents of water, temperature and rainfall. there are complex interactions between these factors that significantly affect mosquito ecological adaptability and vectorial capacity for disease transmission with significant concepts for vector management and control at the local and regional levels (12-13). accordingly, investigating biotic and abiotic factors for various mosquito fauna make it easier to monitor potential modifications of larval habitats affected by rains, global climate change and man-made activities (12-13). aedes caspius likely transmit the human pathogen, such as spiroplasma sabaudiensc and crisiulospora aedis (14). these mosquitoes transmit rift valley fever virus (rvf), west nile virus (wnv) and tahyan virus (15). in the region of palearctic, ae. caspius inhabits in lakes, pools, shores of great britain, and fresh-water and lower salt marshes in the continental parts of europe, russia, mongolia, northern china, pakistan, northeastern africa, asia minor, and the persian gulf (17-18). aedes vexans has several subspecies, the typical species is ae. vexans vexans, and the subspecies ae. nipponii vexans in east asia (19) and ae. vexans arabiensis in saudi arabia, yemen and africa (3). this species is extremely important in medicine and has more than 30 different viral diseases, including eastern horse encephalitis, japanese encephalitis (20), california encephalitis, western horse encephalitis (21), as well as other pathogens such as tularemia and dirofilaria immitis (22-23). aedes vexans along with ae. aegypti and cx. quinquefasciatus, have the highest global distribution among other mosquitoes in the world (24-25). several studies were conducted on composition, distribution and ecology of mosquitoes in iran (26-44). considering the fact that in the east azarbaijan province, some vector mosquitoes of diseases such as west nile, dirofilariasis and malaria present, study on the composition, distribution and ecology of mosquito species is very important in different aspects of vector control programs. this study will add important information about the composition, distribution, ecology and diversity of mosquito species such as establishment of mosquitoes in this region. this new and important information will help us to correctly control and monitor strategies of disease vectors, such as ae. caspius, ae. vexans, cx. pipiens and cx. theileri. this information helps us to prevent important vectors from increasing and establishment in this area due to change in human activities and weather; thus, the risk of transmission of disease by mosquitoes gets minimized. the aim of this study was to determine composition, distribution and some ecological aspects of mosquitoes in east azerbaijan province, iran which is high medically importance from the point of view of the arboviral vectors. materials and methods study area east azerbaijan province is located in northwestern of iran between 39º 26´–36º 45´ n latitudes and 45º 5´–48º 22´ e longitudes. the province covers an area of approximatej arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 64 http://jad.tums.ac.ir published online: april 27, 2019 ly 47,830km², it has a population of around four million people (fig. 1, table 1). this province contains 19 counties. tabriz city is center of the province which 1360m above sea level. the mean annual rainfall is about 300mm. the average relative humidity changes are from 44%. the averages of the maximum and minimum temperatures are 17.7 °c and 6.8 °c, respectively, and the average temperature is 12.3 °c. the province includes arid and semiarid climates (fig. 1, table 1). mosquito collection, site selection and species identification for mosquito collection, nine sites with different biotopes in three counties of ahar, marand, and tabriz from east azerbaijan province were selected. collection of mosquito’s larvae was performed in different habitats using the standard dipping technique (using 350ml clark’s dippers) and whirl pack bags (45) (table 2). light trap, mosquito net and aspirator was used for adult mosquito collection. collection of mosquitoes was carried out during june to october 2017. larvae and adult mosquitoes were identified by morphological characters (46). physical and biological characteristics of larval habitats in these investigation characteristics of mosquitoes larval habitats was studied. the ecological characteristics containing geographical properties of collecting localities (latitude, longitude and altitude), type of habitat (stagnant, stream, seepage and water container), vegetation situation (presence or lack of vegetation), kind of vegetation (null, poaceae, typhaceae, dispacea and acorus species), water situation (turbid and clear), exposure to sunlight (full, partial sunlight and covered or shaded), depth, substrate type (muddy, sandy, rocky and concrete), distance from animal and human houses and physicochemical attributes such as water temperature and ph were recorded visually or using special equipment (9, 47). data analysis the species richness (r: margalef index), unified indices (d: simpson’s diversity index and h̕: shannon–wiener index) and evenness (e: distribution of abundances among the species) as measures of a diversity, were calculated for east azerbaijan province and different study counties. the formulae and their rationale in the present study are summarized below: results totally, 1401 mosquitoes were collected from nine sites in east azerbaijan province during june–october 2017, including 1015 adult and 386 larvae. the properties of geographical larval habitats (latitude, longitude and altitude) have been shown in (fig. 1, table 1). four genera along with ten species were collected and identified, including an. hyrcanus, an. maculipennis s.l., an. superpictus s.l., ae. caspius, ae. vexans, cx. pipiens, cx. theileri, cx. perexiguus, cs. longiareolata and cs. subochrea (table 2). to our knowledge based on literature, for the first time four species of, ae. caspius, ae. vexans, cx. perexiguus, and cs. subochrea species from ahar and two species of ae. vexans and cx. perexiguus were reported from tabriz county. considering that no investigations have been already carried out in marand, all the collected species comprising an. hyrcanus, an. maculipennis s.l., ae. vexans, cx. pipiens and j arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 65 http://jad.tums.ac.ir published online: april 27, 2019 cs. longiareolata (table 2) are new report for the region. anopheles maculipennis s.l., ae. caspius, ae. vexans, cx. pipiens, cx. theileri, cs. longiareolata and cs. subochrea species were collected at both adult and larval stages but an. hyrcanus, an. superpictus s.l., and cx. perexiguus species were collected only at the larval stages (table 2). in adult stage, cx. pipiens (37.3%), cx. theileri (23.8%), cs. longiareolata (21.8%), ae. caspius (11.14%), ae. vexans (5.18%) were the most abundance species respectively, however, an. maculipennis s.l. (0.52%) and cs. subochrea (0.26%) were the least abundance species (table 2). at the larval stage cx. theileri (50.72%), cs. longiareolata (32.12%) and cx. pipiens (9.75%) were the most prevalent species respectively but in contrast ae. caspius (2.27%), cx. perexiguus (1.97%), cs. subochrea (1.09 %), an. maculipennis s.l., (0.69%), an. hyrcanus (0.5%) and an. superpictus s.l. (0.39%), were the least abundance species respectively (table 2). species such as cx. pipiens, cx. theileri, cs. longiareolata and ae. vexans have a wide distribution in the study areas but some species such as an. hyrcanus, an. maculipennis s.l., an. superpictus s.l., and cs. subochrea have been collected from limited areas (table 2). characteristics of mosquito larval habitats of mosquitoes in ahar county located in northeastern east azerbaijan province, there were three larval sites such as yavarkandi, razin and sattar khan dam (table 3, fig. 1). sattar khan dam and razin larval sites had seepage and stagnant water respectively. both of the larval sites had turbid water with muddy substrate and shallow depth. in addition, these sites were covered from sunlight with null, poaceae, typhaceae and dispacea vegetation. those were more than two kilometers away from human and animal houses. the sattar khan dam larval habitat had 200 meters distance from animal space and its field temperature was 13 °c. in contrast, razin larval site had 500 meters distance from animal space and its field temperature was 9 °c. in these two larval habitats harbored ae. caspius, ae. vexans, cx. theileri, cx. pipiens, cx. perexiguus, cs. subochrea and an. superpictus s.l. (table 2). yavarkandi larval site had stagnant and clear water without vegetation, with muddy substrate and exposed to sunlight. it was more than two km away from human and animal houses. in the yavarkandi larval site the field temperature was 23 °c. in this larval habitat, two species of cs. longiareolata and cx. pipiens were collected (table 3). tabriz larval site is located in the central east azerbaijan province and contains three regions of hojjaj park, khaje-dizaj and chavan (table 3, fig. 1). both regions of hojjaj park and khaje-dizaj had stagnant and turbid water with muddy substrate. the hojjaj park had the following conditions: the parts of larval habitat were covered with poaceae plants and its water depth was shallow, also it had more than two kilometers distance from animal and human houses. the field temperature was 17 °c in this site (table 3). this site contains ae. caspius, cx. theileri, cx. pipiens, cx. perexiguus and cs. longiareolata (table 2). the khaje-dizaj site was covered with null and ziziphu plants. its depth was less than one meter and its field temperature was 16 °c. it had one-kilometer distance from human houses and about 100 meters from animal sites (table 3). in this larval habitat, cx. theileri and cx. pipiens were collected (table 2). chavan larval habitat was a water container with substrate cement and clear water that had no plants and was exposed to sunlight. it had a very short distance with human and animal houses (about 2–10m), which had one meter depth and 27 °c (table 3). in this larval habitat cs. longiareolata and cx. pipiens were collected (table 2). j arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 66 http://jad.tums.ac.ir published online: april 27, 2019 in marand with three larval sites of ghareh tappeh, dolat abad and zinab located in northwest east azerbaijan province (fig. 1). ghareh tappeh and dolat abad habitats had stagnant and clear water. ghareh tappeh site had no plants with sandy substrate and shallow depth that was exposed to sunlight. it had one meter depth and 18 °c field temperature (table 3). culiseta longiareolata, cx. pipiens, ae. vexans, an. maculipennis s.l. and an. hyrcanus were collected in this larval habitat (table 2). dolat abad site was shallow with muddy substrate and 18 °c field temperature. the parts of larval habitat were covered with null and dispacea plants and it had more than two kilometers distance from human and animal houses (table 3). in this larval habitat cx. theileri and cx. pipiens were collected (table 2). zinab site was stream and clear water with rocky substrate and shallow depth. it had no plants and exposed to sunlight with 17 °c temperature. it had about 100 meters distance from human and animal houses (table 3). in this larval habitat, specimens of cx. pipiens, cs. longiareolata, an. maculipennis s.l. and an. hyrcanus were collected (table 2, fig. 1). according to table 2 and 3, larvae of mosquitoes occupied all different types of the habitats. in addition, our results showed that cx. pipiens, cs. longiareolata and cx. theileri, respectively, had the most distribution and adaptation to different types of larval habitats. these three species were collected in most larval habitats but in contrast, cx. perexiguus was found only in the shallow water that was stagnant, turbid, covered with plants and with muddy substrate. in addition, distribution of cs. subochrea was limited and only was collected in the seepage, stagnant and turbid water habitats with muddy substrate and shallow depth. anopheles was found only in stream, stagnant and clean water with muddy substrate that was exposed to sunlight. aedes vexans and ae. caspius were found mostly in larval habitats with turbid water and they were covered with different types of plants. only two ae. vexans specimens were found in clean water that was exposed to sunlight with muddy substrate (table 2, 3). in our study, the temperature and ph ranges were 9 °c to 27 °c and 7–8 respectively. there were differences in the species diversity, as indicated by the values of simpson’s diversity index, shannonwiener index (h’), and evenness and species richness of the mosquito fauna among the study areas of east azerbaijan province (table 4). the species richness and the three indices were found to be maximal in ahar county (r: 1.5, d: 0.79, h’: 1.74, e: 0.73), whereas the estimated diversity (d: 0.49, h’: 1.01), and richness (r: 0.82) were the lowest in marand county. table 1. geographical characters of the locations were studied no. locations latitude (n) longitude (e) altitude (m) 1 ghareh tappeh 38• 26' 9.758'' 45• 35' 7.828'' 1296 2 dolat abad 38• 45' 53.988'' 45• 49' 19.418'' 1297 3 zinab 38• 9' 9.654'' 45• 52' 53.218'' 1302 4 khaje dizaj 38• 12' 21.030'' 46• 16' 39.659'' 1325 5 hojjaj park 38• 11' 16.210'' 46• 23' 45.659'' 1336 6 chavan 37• 59' 9.952'' 46• 23' 32.164'' 1759 7 sattar khan dam 38• 46' 15.552'' 46• 8' 22.763'' 1429 8 razin 34• 24' 53.932'' 47• 8' 24.629'' 1435 9 yavarkandi 38• 38' 45.622'' 47• 21' 97.143'' 1706 j arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 67 http://jad.tums.ac.ir published online: april 27, 2019 table 2. details of mosquitoes collected from larval and adult habitats in east azerbaijan province during june– october 2017 county location species number total percent larvae adult ahar yavarkandi cs. longiareolata 47 0 47 23 cx. pipiens 28 4 28 14 cx. theileri 0 2 2 1 razin ae. caspius 7 0 7 3.5 ae. vexans 3 6 9 4.5 cx. pipiens 6 10 16 8 cx. theileri 15 7 22 11 cx. perexiguus 13 0 13 6.5 an. superpictus s.l. 4 0 4 2 sattar khan dam cs. subochrea 12 0 12 6 cx. theileri 34 0 34 17 cx. pipiens 4 3 7 3.5 subtotal 173 32 205 100 tabriz hojjaj park ae. caspius 16 43 59 7.76 cs. longiareolata 13 1 14 1.85 cx. pipiens 10 17 27 3.55 cx. theileri 15 12 27 3.55 cx. perexiguus 7 0 7 .93 cs. subochrea 0 1 1 .13 khage dizaj cx. pipiens 23 73 96 12.63 cx. theileri 417 39 456 60 ae. vexans 0 1 1 .13 chavan cx. pipiens 11 13 24 3.16 cs. longiareolata 48 0 48 6.31 subtotal 560 200 760 100 marand ghareh tappeh cs. longiareolata 186 83 269 61.6 cx. pipiens 3 2 5 1.4 ae. vexans 2 13 15 3.3 an. maculipennis s.l. 2 2 4 .9 an. hyrcanus 1 0 1 .2 cx. theileri 0 17 17 3.9 dolat abad cx. theileri 34 9 43 9.8 cx. pipiens 6 14 20 4.6 zinab cs. longiareolata 32 0 32 7.3 cx. pipiens 7 8 15 3.3 cx. theileri 0 6 6 1.4 an. maculipennis s.l. 5 0 5 1.4 an. hyrcanus 4 0 4 .9 subtotal 282 154 436 100 total 1015 386 1401 100 j arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 68 http://jad.tums.ac.ir published online: april 27, 2019 table 3. characteristics of mosquito larval collection sites in ahar, tabriz and marand counties of east azerbaijan province during june–october 2017. county location type turbid exposed vegetation dhh dah depth sub. t ahar yavarkandi stagnant clear exposed without >2km >2km <1m muddy 23 razin stagnant turbid covered null, poaceae, typhaceae, dispacea >2km 500m shallow muddy 9 sattar khan dam seepage water turbid covered null, dispacea >2km 200m shallow muddy 13 tabriz hojjaj park stagnant turbid partial poaceae 20m >2km shallow muddy 17 khage dizaj stagnant turbid covered null, acorus species 1km 100m <1m muddy 16 chavan water container clear exposed without 2m 10m 1m concrete 27 marand ghareh tappeh stagnant clear exposed without nd nd 1m sandy 18 dolat abad stagnant clear partial null, dispacea >2km >2km shallow muddy 19 zinab stream clear exposed without 100m 100m shallow rocky 17 nd: not determined, tur: turbidity, exp: sun exposure, veg: vegetation type, dhh: distance from the nearest human houses, dah: distance from the nearest animal house, sub: substrate type, t: temperature. table 4. the species richness (r), simpson's diversity index (d), shannon-weiner diversity index (h̕), and evenness (e) of the collected mosquito species in east azerbaijan province during june–october 2017 loc. no. s r d h̕ e marand 436 6 0.823 0.491 1.008 0.563 tabriz 760 7 0.905 0.547 1.069 0.550 ahar 205 9 1.503 0.789 1.735 0.727 east azerbaijan province 1401 11 1.380 0.694 1.513 0.631 discussion this research is the first study on distribution, diversity and ecology of mosquitoes, with emphasis on ae. capius and a. vexans as potential arbovirus vectors in east azerbaijan province, northwestern of iran. the east azerbaijan province contains diverse geographical areas with different weather conditions. these diverse conditions can provide suitable environment for the establishment of different species of mosquitoes and justify the variety of mosquito species in this region. this study showed many mosquito species had ecological adaptations. in spite of these ecobiologi cal characteristics, the ecology of mosquitoes present in east azerbaijan province is largely unknown. in this investigation we tried to study distribution and ecology of mosquitoes in three northeastern, northwest and central regions of east azerbaijan province, where various mosquito vectors of malaria and arboviruses are present (48-49). some studies had been conducted on fauna and checklist of mosquitoes in parts of this region (48), although to our knowledge no studies have been done on the ecology of mosquitoes in these regions. j arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 69 http://jad.tums.ac.ir published online: april 27, 2019 in current study, 4 genera and 10 species were collected and identified. for the first time, ae. caspius, ae. vexans, cx. perexiguus, cs. subochrea species were reported from ahar county. also for the first time, ae. vexans and cx. perexiguus species were reported from tabriz county. as well as for the first time, an. hyrcanus, an. maculipennis s.l., ae. vexans, cx. pipiens, cx. theileri and cs. longiareolata species were reported from marand county. some previous studies had found only an. hyrcanus in northwestern iran (49-50) and other study identified an. claviger, an. hyrcanus, an. maculipennis s.l., an. pseudopictus, an. sacharovi and an. superpictus s.l. in east azerbaijan province. in the present study, we reported an. hyrcanus, an. maculipennis s.l. and an. superpictus s.l.. in previous studies, cx. pipiens and cx. theileri were reported in these regions (48), but cx. perexiguus was not found in these regions. in this study, three species of culex genus were reported such as cx. pipiens, cx. theileri and cx. perexiguus that cx. pipiens and cs. longiareolata species were the most abundant. aedes caspius and ae. vexans were found only from the kaleybar region (49), in our study, for the first time, ae. caspius and ae. vexans were found in ahar, marand and tabriz, where previous study did not report these species in these areas (48). one study reported cs. annulata and cs. subochrea in west azerbaijan province and cs. subochrea in east azerbaijan province (51). culiseta longiareolata species was reported as the most abundant in kermanshah, kurdistan and sistan and baluchistan provinces (52). in this study, two members of culiseta genus were found, including cs. longiareolata and cs. subochrea where cs. longiareolata species was the most abundant species. comparing the results of our study with a recent study in west azerbaijan province (47) showed that six species an. maculipennis s.l., an. superpictus s.l., cx. pipiens, cx. theileri, cs. longiareolata and ae. caspius were common between west and east azerbaijan provinces. the results of our study compared with the results of research conducted in zanjan province (53), showed that five species (an. maculipennis s.l., an. superpictus s.l., cx. pipiens, cx. theileri, and cs. longiareolata) were common between these two provinces. the comparison the results of our study with a recent study conducted in kurdistan province (54) showed that six species (an. maculipennis s.l., an. superpictus s.l., cx. theileri, cx. pipiens, cs. longiareolata and ae. caspius) were common between in provinces. in turkey (55), and in the provinces of ardebil, kurdistan and west azarbaijan, cx. theileri and cx. pipiens were dominant and most abundant species (56) and our study results confirm this information. our results showed that cx. theileri (50.72%), cs. longiareolata (32.12%) and cx. pipiens (9.75%) were dominant and the most abundant species in this province. the climate changes and biotic and abiotic environment factors including plants, temperature and rainfall ranges, significantly affect the type and frequency of larval mosquito habitats. these factors, in turn, affect the number of mosquito species, larval stages, longevity, behavior and adult development. as a result, the transmission of diseases through mosquitoes is directly affected by environmental factors (5758). the presence of plants as a source of sugar for mosquitoes is very important, that influence both larval and adult stage development (5970). plants provide energy for mosquitoes, as a result, survival rate increased and longevity of the mosquito longer than the extrinsic incubation period of parasite, therefore the incidence of disease increases (61-62). in our study, five types of plants such as null, poaceae, typhaceae, carex dipsacea and acorus species were found in relation to mosquito larval habitats. to our knowledge, there is no study on plant species associated with mosquito species in iran. in our study, various species of mosquitoes such as anopheles, culex, aedes and culisita were found in relation with varj arthropod-borne dis, march 2019, 13(1): 62–75 a paksa et al.: biodiversity of … 70 http://jad.tums.ac.ir published online: april 27, 2019 ious plants, therefore this study confirms the results of previous research. aedes vexans and ae. caspius have not been reported earlier in these areas. these results are likely to show the effects of climate changes and human activities on the distribution of these species in these regions. physicochemical properties of larval habitats regulate the abundance of mosquito species, for example, anopheles species were found more in natural larval habitats and culex species in artificial larval habitats (63). in our study, anopheles species were found in natural habitats, while culex species were collected from different types of larval habitats, indicating these mosquitoes can live in a wide range of water habitats. larval habitats in this study were stagnant, stream and seepage, water container, turbid and clear water, sun exposed or covered from sunlight –rocky or muddy substrate and shallow depth. studies showed that some of culex larval species were found alone or along with other mosquitoes, such as anophele and aedes (64-67) which has been observed in our study. there was a significant relationship between the distance of larval habitats of anopheles mosquitoes, human and animal sites, besides, anopheles mosquitoes are found more often near human and animal houses (68). our study showed that there was a significant correlation between the distances of larval habitats of anopheles, ae. caspius, ae. vexans and cx. theileri mosquitoes with human and animal sites, because the larval habitats of these mosquitoes were found more near to human and animal houses. however, this correlation was not observed between the larval habitats of cx. pipiens and cs. longiareolata with human and animal houses, because the habitats of these mosquitoes were found at various intervals from human and animal houses. conclusion arbovirus vectors such as ae. caspius and ae. vexans along with cx. pipiens and cx. theileri are well adapted to a broad range of habitats and climatic conditions. determining of distribution and full description of ecology of arboviral vectors under local eco-demographic conditions in the east azerbaijan province have provided important ecological information on establishment of important mosquito borne diseases. this new information will help us to correctly control and monitor strategies of disease vectors, and in this way prevent important vectors such as ae. ae. caspius, ae. vexans, cx. pipiens and cx. theileri to be increased and established in this area due to changing human activities and weather changes. these strategies help minimizing the risk of transmission of disease by mosquitoes. acknowledgements this paper is a part of the results of the first author’s dissertation for fulfillment of a phd degree in medical entomology and vector control from department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran. this study was financially supported by the deputy for research, tehran university of medical sciences project no. 35904. the authors declare that there is no conflict of interests. references 1. dong x, dong l, wu c (2008) a new species of the genus tripteroides (diptera: culicidae) from china. acta zoo taxon sin. 33: 187–190. 2. wilkerson rc, 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tehran, iran 4center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran (received 29 aug 2011; accepted 6 nov 2011) abstract background: zoonotic cutaneous leishmaniasis (zcl) is an increasing public health problem in some endemic regions. horseradish peroxidase (hrp) conjugated rabbit anti-rhombomys opimus (r. opimus) ig is needed for immunoblotting and elisa tests used to explore the immune response of the rodents against the sand fly saliva. in this study, the production of hrp conjugated rabbit anti-r. opimus ig was conducted for the first time. methods: rhombomys opimus ig was purified from serum by protein g affinity chromatography column and injected into rabbit to produce anti-r. opimus ig antibody. the titration of antibody against r. opimus ig in rabbit serum was checked using indirect elisa. rabbit anti-r. opimus ig was purified by sepharose-4b-r. opimus ig column. reactivity of this antibody was assessed by indirect elisa and was conjugated to hrp by periodate method. results: approximately 3.5 mg ig was purified from 1 ml r. opimus serum using protein g affinity chromatography column. the molecular weight of purified r. opimus ig was estimated about 150 kda by sds-page. nearly 2.3 mg rabbit anti-r. opimus ig was purified from 1 ml immunized rabbit serum. the purified antibody was conjugated to hrp and the optimum titer of hrp conjugated rabbit anti-r. opimus ig was determined as 1:8000 using direct elisa. conclusion: hrp conjugated rabbit anti-gerbil igg has been produced by a few companies, but to our knowledge hrp conjugated rabbit anti-r. opimus ig is not commercially available. production of hrp conjugated rabbit anti-r. opimus ig is considerably helpful for immunological studies of r. opimus, the main reservoir host of zcl in iran as well as some other countries. keywords: rhombomys opimus, polyclonal antibody, zoonotic cutaneous leishmaniasis, immunoglobulin, iran introduction zoonotic cutaneous leishmaniasis (zcl) is still a public health problem in some of the regions of the endemic areas. leishmania major, the causative agent of the zcl of the old world, is widely distributed in different populations of rodents in arid and savannah areas. the disease is transmitted to rodents and other vertebrate hosts by phelebotominae sand flies (gramiccia and gradoni 2005). rodents of the subfamily of gerbillinae are the main reservoirs of zcl in iran and other countries where the disease is endemic (dubrovsky 1979, strelkoova 1996, yaghoobiershadi et al. 1996). rhombomys opimus aa akhavan and r ghods contributed equally to this work. *corresponding author: ahmad reza mahmoudi, e-mail: mahmoudi@avicenna.ac.ir iran j arthropod-borne dis, 2011, 5(2): 69–76 aa akhavan et al.: production and purification of ... 70 (cricetidae: gerbillinae) is the main reservoir host of the agent over the vast areas of the turan lowland (west and south kazakhstan and central asia with adjacent parts of afghanistan and iran), mongolia, and seemingly, in some provinces of china. naturally infected r. opimus were found in more than 200 places of turan lowland. this gerbil is also found to be naturally infected with l. turanica and l. gerbilli (strelkova 1996, akhavan et al. 2010a, 2010b, 2010c). welldescribed stable zcl system in central asia, afghanistan and iran (central and north-east) are associated with r. opimus, the main reservoir, and phlebotomus papatasi, the main vector (javadian 1988, yaghoobi-ershadi and javadian 1996, javadian et al. 1998, yaghoobi-ershadi et al. 1996, 2003, gramiccia and gradoni 2005, yaghoobi-ershadi 2008). investigation on immunogenic components of sand fly saliva and the immune response of the host against it, also interaction among the parasite, sand fly and host is necessary to find possible tools to control the disease specially producing anti leishmania vaccine and/or transmission blocking vaccine (akhavan 2011). detecting the immune response of the host to saliva of sand flies can also be used as a marker of transmission risk of the disease (barral et al. 2000). study on the immune response of r. opimus, the main reservoir host of zcl in central and northeast of iran, to infection of l. major, the causative agent of the disease, seems to be necessary. horseradish peroxidase (hrp) conjugated rabbit anti-r. opimus ig is needed for immunoblotting and elisa tests, used to find the immune response of the rodents against the sand fly saliva. as this material is not produced commercially in the world, its production was essential and inescapable. to our knowledge, in the present study production of hrp conjugated rabbit anti-r. opimus ig has been produced for the first time. materials and methods rhombomys opimus serum collection to purify r. opimus ig, sera were obtained from wild great gerbils collected from natural habitat in sejzi rural district, esfahan province, central iran. great gerbils were anaesthetized (ketamin hydrochloride 60 mg/kg and xylazine 5 mg/kg, intramuscularly) and then the blood sample was collected. the individual sera was isolated and kept at -20 °c until use. purification of r. opimus ig and polyclonal rabbit anti-r. opimus ig rhombomys opimus ig was purified using hitrap protein g hp affinity chromatography column (ge healthcare, uppsala, sweden). the 1:5 diluted serum in pbs (0.15m, ph= 7.2) was centrifuged, filtered by 0.2 µm filter and passed through the hitrap protein g hp affinity chromatography column. then the column was washed with pbs. the attached r. opimus ig to the column was isolated from the column using gly-hcl (0.2 m, ph= 2.5). isolated ig was dialyzed against pbs and finally the purified r. opimus ig was stored at -20 ºc. for purification of rabbit anti-r. opimus ig from rabbit serum, a sepharose-4b-r. opimus ig affinity chromatography column was prepared according to the amersham biosciences company instructions (71-7086-00 edition ac). using this column, the rabbit anti-r. opimus ig was purified from rabbit serum as described for r. opimus ig purification (see above). concentration of purified r. opimus ig and rabbit anti-r. opimus ig was determined by reading the optical density (od) of samples at 280 nm and calculation of the concentrations regarding to extinction coefficient of igg molecule. purity assessment of purified r. opimus ig molecular weight and purity of the r. opimus ig were determined using sds-page iran j arthropod-borne dis, 2011, 5(2): 69–76 aa akhavan et al.: production and purification of ... 71 in non-reducing conditions, 8% separating gel and 4% stacking gel (according to bio-rad guideline). immunization of new zealand white rabbit with r. opimus ig a female new zealand white rabbit aged 6–7 months was immunized with r. opimus ig using hudson and hay’s method (hudson and hay 1991) with minor modifications. rabbit was injected intramuscularly into the thigh muscle with 250 µg r. opimus ig mixed with complete freund's adjuvant, and 4 booster injections of 125 µ g of r. opimus ig mixed with incomplete freund's adjuvant at 4 weeks intervals between the first and second injection and 2 weeks intervals between subsequent injections. before each immunization, blood sample was taken from the marginal vein of the rabbit ear, centrifuged and the sera were checked for anti-r. opimus ig antibody using elisa method. animal care and the procedures were conducted according to the guidelines of the animal care and ethics committee of avicenna research institute. antiserum titration and reactivity assessment of purified polyclonal rabbit anti-r. opimus ig by elisa rabbit anti-r. opimus ig titer was evaluated using elisa. briefly, 10 µ g/ml of purified r. opimus ig in 100 µ l was added to each well of microtiter polystyrene strips (maxisorp, nunc, roskilde, denmark) and incubated at 37º c for 1.5 hours, 2 wells received only pbs as negative control, then the plate was washed 3 times with pbs-tween (0.05%) (pbs-t), the wells were then blocked using 3% skim milk for 1.5 hours at 37º c. the plate was washed 3 times and serial dilutions of rabbit anti-r. opimus ig serum from 1:1000 to 1:64000 dilutions were added and incubated for 1.5 hours at 37º c. two other wells received pbs as primary antibody step negative control. after three times washing, hrp conjugated sheep anti-rabbit ig (avicenna research institute, tehran, iran) was added as secondary antibody and incubated for 1.5 hours at 37 ºc. after the last wash, the reaction was visualized using tmb substrate (usb, cleveland, usa) and the optical density measured by elisa reader (anthos 2020, cambridge, england) at 450 nm. reactivity of purified polyclonal rabbit antir. opimus ig with related antigen was assessed by indirect elisa test. elisa procedures including coating antigen and secondary antibody were performed as described above. however, the serial dilutions of purified rabbit anti-r. opimus ig (by two fold dilutions from 10000 to 1.2 ng/ml) were added as primary antibody. conjugation of polyclonal rabbit anti-r. opimus ig with hrp conjugation of polyclonal rabbit anti-r. opimus ig with hrp was performed by periodate method (wilson and nakane 1978). briefly, hrp was oxidized by sodium periodate. oxidized hrp was mixed by rabbit antir. opimus ig. then, sodium borohydride was added to this mixture and finally the conjugate was purified from excess reagents by dialysis. titration of hrp conjugated rabbit antir. opimus ig by elisa the optimum titer of hrp conjugated rabbit anti-r. opimus ig was assessed by direct elisa according to the procedure described for the antiserum titration elisa. the wells were coated by r. opimus ig (10 µ g/ ml) as coating antigen and two wells were coated with pbs only as negative control. titration of hrp conjugated rabbit anti-r. opimus ig was performed by two fold dilutions from 1:250 to 1:128000. results from 1 ml r. opimus serum 3.5 mg r. opimus ig was purified. the purified r. opimus iran j arthropod-borne dis, 2011, 5(2): 69–76 aa akhavan et al.: production and purification of ... 72 ig related sds-page shows high purity of the purified ig. regarding to the molecular weight of mouse igg as a molecular weight marker, the molecular weight of purified r. opimus ig is about 150 kda (fig.1). the results showed that the specific antibodies were detectable following the first injection in rabbit. after the second injection, the antibody titer reached near the maximum level and there was no remarkable increase following subsequent injections. however, booster injections resulted in gradual and stepwise increase in the specific antibody titer against r. opimus ig (fig. 2). from 5 ml immunized rabbit serum 11.5 mg rabbit anti-r. opimus ig was purified. it means that there was 2.3 mg rabbit anti-r. opimus ig in each ml of the immunized rabbit serum. the results showed strong reactivity of this antibody with related antigen and this antibody can detect its antigen in very low concentrations of about 1.2 ng/ml (fig. 3). after the hrp conjugation, it was necessary to find the optimum dilution of hrp conjugated rabbit anti-r. opimus ig by direct elisa. optimum dilution for a specific conjugate is the highest dilution of conjugate which can produces the highest signal. according to the hrp conjugated rabbit anti-r. opimus ig titration curve, the optimum dilution of this conjugate is 1:8000 and it shows that the efficiency of conjugation is high (fig. 4). fig. 1. the sds-page analysis of purified r. opimus ig by hitrap protein g hp affinity chromatography column purified r. opimus ig (≈150 kda) mouse igg= 150 kda bsa= 66 kda iran j arthropod-borne dis, 2011, 5(2): 69–76 aa akhavan et al.: production and purification of ... 73 0 0.5 1 1.5 2 2.5 3 3.5 4 1/1000 1/2000 1/4000 1/8000 1/16000 1/32000 1/64000 sera dilutions o d ( 4 5 0 n m ) pre-immune 1st injection 2nd injection 3rd injection 4th injection 5th injection fig. 2. titration curve of rabbit anti-r. opimus ig by indirect elisa 0 0.5 1 1.5 2 2.5 3 3.5 10 00 0 50 00 25 00 12 50 62 5 31 2. 5 15 6. 2 78 .1 39 19 .5 9. 7 4. 8 2. 4 1. 2 rabbit anti r. opimus ig concentration(ng/ml) o d (4 5 0 n m ) fig. 3. reactivity test of purified rabbit anti-r. opimus ig by indirect elisa 0 0.5 1 1.5 2 2.5 3 3.5 4 1/ 25 0 1/ 50 0 1/ 10 00 1/ 20 00 1/ 40 00 1/ 80 00 1/ 16 00 0 1/ 32 00 0 1/ 64 00 0 1/ 12 80 00 hrp conjugated rabbit anti r. opimus ig dilutions o d (4 5 0 n m ) fig. 4. titration of hrp conjugated rabbit anti-r. opimus ig by direct elisa iran j arthropod-borne dis, 2011, 5(2): 69–76 aa akhavan et al.: production and purification of ... 74 discussion hrp conjugated rabbit anti-gerbil igg is produced by a few companies; however, to our knowledge there is no record of production of hrp conjugated rabbit anti-r. opimus ig. production of hrp conjugated rabbit anti-r. opimus ig is needed for serological and immunological studies of r. opimus, the main reservoir host of zcl in iran as well as some other countries, which may lead to plan immunological control of the disease (akhavan 2011). phlebotomus papatasi, vector of zcl, co-injects leishmania parasites along with saliva into the skin of r. opimus while feeding. saliva from the sand fly is known to have pharmacological as well as immunomodulatory activities such as vasodilatory and anticlotting effects which exacerbate the infectivity of the parasite (kamhawi 2000). investigation on the immune response of the host against salivary gland antigens of the vector, also interaction among the parasite, sand fly and host are necessary to find possible tools to control the disease, especially in developing anti leishmania vaccine and/or transmission blocking vaccine. detecting the immune response of the host to saliva of the sand fly may also be used as a marker of transmission risk of the disease (barral et al. 2000). the hrp conjugated rabbit anti-r. opimus ig produced in this study helps the investigators to study on the immune response of r. opimus, to l. major exposure and infection. the hrp conjugated antibody is needed for immunoblotting and elisa tests, which are used to detect the immune response of the rodents against the sand fly saliva. purification of r. opimus ig is a necessary step for production of polyclonal antibody. no established protocol was recorded for purification of r. opimus ig. in this regard, considering the fact that r. opimus is a mammal belonging to the order rodentia and according to previous studies (kemp 2005), protein g could adsorb ig molecules from many mammals and rodents such as human, mouse and rabbit. thus it seems reasonable that protein g is a suitable candidate for purification of igg molecules from r. opimus serum. using this method, 3.5 mg r. opimus ig was obtained from 1 ml serum. moreover, due to the lack of knowledge about the molecular size of r. opimus ig, another rodent igg (mouse) was used as a standard for molecular weight determination in sds-page. the very similar migration of r. opimus ig in the polyacrylamide gel to that of murine igg allowed us to estimate it as around 150 kda. a high titer of rabbit anti-r. opimus ig production after the first injection of r. opimus ig showed that r. opimus ig mixed with adjuvant was immunogenic in rabbit, so that after the second injection, the antiserum titer in the rabbit hardly increased. however, we continued the immunizations to increase the avidity or affinity of the produced antibodies. in conclusion, this study describes the successful purification of r. opimus ig and production of polyclonal antibodies against it in rabbit. acknowledgements the authors are grateful to ms l balaie goli, ms m babaei, ms m darzi, mr j mahmoudian, ms l eini and mr a moradzadeh, monoclonal antibody research center, avicenna research institute, acecr and mr arandian, ms shareghi, mr jafari and mr abdoli, isfahan health research center, national institute of health research, tums for their kind collaboration. this 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sciences/ university of tehran, i.r. iran 2 dept. of malaria control, ministry of health and medical education, tehran, i.r. iran 3 iranshahr health research and training center, medical sciences/ university of tehran, tehran, i.r. iran (received 30 sep 2006; accepted 26 mar 2007) abstract a field trial was carried out in the chabahar district of sistan and baluchistan province, southeastern iran on the efficacy of mosquito nets treated with a suspension formulation of deltamethrin (k-o thrin®) against malaria vectors. treated nets were used in three villages, and in the two control villages, one used untreated nets and the other used indoor spraying with deltamethrin (wp 10%), without nets. treatment of polyester mosquito net with a target dose of 25 mg/m2 active ingredient, carried out in mid-april 2005. bioassays repeated on domestically used nets over 7 months showed persistence of almost 100% mortality of anopheles stephensi over this period. adult mosquitoes were collected by pyrethroid space spray catch of living quarters and stables, cattle bait and pit shelter catches. overall, an. culicifacies giles s.l. predominated (49.8%), followed by an. stephensi liston s.l.(36.9%), an. pulcherrimus theobald (7.3 %), an. dthali patton (5.7% ) and an. fluviatilis james (0.3%). acknowledge, attitude and practice (kap) study showed that about 97% of respondents in communities agreed that they would like to participate in malaria control activities such as personal protection using impregnation bed net. therefore, in planning for future large scale trials, comparison of new compounds and formulations such as tablets and long lasting insecticides impregnated bed net is recommended. keywords: malaria control, deltamethrin, iran introduction despite several years campaigning against malaria, about 107 countries and territories have areas at risk of malaria transmission. some 3.2 billion people live in areas at risk of malaria transmission. an estimated 350-500 million clinical malaria episodes occur annually; most of these are caused by plasmodium falciparum and p. vivax (who 2005). malaria is still the most important parasitic and vector-borne disease in the country. it is one of the main health problems in the south-east provinces. expect cases from southeastern districts of iran and a few endemic foci of the other parts of the country, the other clinical cases of the diseases are imported (vatandoost et al. 2006). a total of 18700 malaria cases were reported in 2005, among which only 70% were iranian, with the remaining coming from abroad. according to current reports, 90% of cases have been reported from three provinces in the southeast: hormozgan, kerman and sistan and baluchestan. in these three provinces, chloroquine and sulfadoxine was still the drug of choice for malaria treatment (center for diseases control and prevention, unpublished data). malaria transmission in this endemic area occurs through the year, most of the malaria cases were observed from may to november almost in the age groups of 5-25 yr *corresponding author: sh moosa-kazemi, tel: +98 21 66465404 (ext: 2160), fax: +98 21 88951393, e-mail: moosakazemi@sina.tums.ac.ir iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 44 old (moosa kazemi et al. 2006). an. culicifacies and an. stephensi are responsible for the transmission of 60-70% of the malaria in iran (zaim 1998). synthetic pyrethroids such as, lambdacyhalothrin and cyfluthrin have been evaluated in the field for their effectiveness against disease vectors (ansari et al. 1986, singh et al. 1989, yadav et al. 2001). mosquito nets treated with synthetic pyrethroids have proved to be an important tool for the control of malaria and other vector borne diseases (lengeler 2000). there is much emphasis on social marketing of insecticide-treated nets and some doubt whether nets provided free-of-charge will be looked after by householders (maxwell et al. 2006). in iran, trials of insecticide-treated nets carried out during the 1995 to 2000 in southern provinces have shown promising results against malaria transmitted by an. stephensi liston (moosa-kazemi et al. 2000, rassi et al. 2002), and an. culicifacies (zaim et al. 1998). insecticide-treated mosquito nets are now one of the operational strategies recommended by the world health organization and center for diseases control and prevention of iranian ministry of health. because of the importance of the chabahar county not only for malaria transmission but also for its economic situation in iran, it was decided to evaluate the effectiveness of deltamethrin impregnated bed nets on anopheles mosquitoes for the first time in this area during 2005. the aim was to obtain new data which would be valuable to develop programmes for improving community health and future planning of malaria control in this endemic area of malaria, southeastern iran. materials and methods a field trial study was performed from mar. to nov. 2005 in sistan and baluchistan province. this study took place in five randomly selected rural villages in zar-abad rural district (57°14'n, 26°19'e), 220-250 km far from the city of chabahar, iran. it is situated at an altitude of 17 m in a plain area. in 2005 the maximum and minimum mean monthly temperatures were 32.5 °c and 6 °c in july and jan., respectively. the total annual rainfall was 220.5 mm, the minimum of 3.4 mm in july and maximum of 65 mm in april. the mean annual relative humidity was 74%. the main economic activity of the people is husbandry of cow, goats and fishing. on the basis of available epidemiological data from the chabahar health centre, some villages were short-listed and preliminary rapid fever and entomological surveys were carried out. on this basis, five villages with 1092 houses and 4464 population were selected and randomly three villages assigned as the trial village to receive treated nets with deltamethrin sc 0.05 (agrevo, marseille, france), one village with 892 inhabitants to receive untreated nets and one control villages with a total of 881 were also selected randomly that received no nets and carried out indoor residual spraying with deltamethrin (wp 10%) in 2005. an open-ended questionnaire was applied to obtain knowledge and belief of people about malaria transmission, before the residual insecticides application began. to validate and adjust the questionnaire to the understanding of the population, a preliminary and piloted version was conducted to 20 mothers of a village in the study area and no problem was identified. the questionnaire included two sections: the first part 20 questions were designed to obtain information on address of the families; family size, age, sex, marital status, level of education, occupation, sign and symptom of malaria, malaria episodes and background treatment. in the second part, 25 questions, including their knowledge about most important diseases in the area, route of malaria transmission, control measure, their beliefs and practices, including medication and use of health services, perceived effect of indoor residual insecticides, the prefer spraying frequency, the use of other prevention methods such as bed nets and repellents were included. to avoid bias questionnaire were completed without the presence of health workers (table 1). iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 45 the mosquito nets were of polyester poly filament fiber, 100-denier strength, white in color, 156-mesh hole size (12×13 holes/in2) in family (130×180×150cm) and x-family sizes (190× 180×150) cm (who, 1997). individual bed net were soaked in a known volume of water, wrung out and allowed to drip so that the excess water in the bucket was meseared and the different between this and the original volume was the amount of the water retained by the net. this was divided by the surface area of the net to obtain the retention capacity in milliliters per square meter. the concentration of insecticide on the absorptive capacity and target dose (25 mg ai/m2) of the bed net, were prepared. after impregnations, the bed nets were laid on plastic sheet in the shade to partially dry and then hung on the wire to dry completely before they were distributed to the people. a total of 983 bed net, 423 family and 560 x-family sizes, were impregnated and distributed free of charge in the area. the net impregnators and net users were questioned regarding to any perceived side-effects of the insecticide-treated mosquito nets. before starting the trial, village meetings were organized to inform inhabitants about proper and regular use of nets and of the aims and importance of the study. nets were distributed free of charge based on a survey of where people slept in the mid of april 2005. the size and number of nets distributed to each household were recorded. the study population was requested not to wash the nets during the study and this was complied with very well. project staff demonstrated the impregnation process and subsequently nets were treated by householders under the supervision of the project team. normal precautions, such as use of rubber gloves, avoiding contact with eyes, nose and mouth, and washing of hands thoroughly after impregnation, were followed. small pieces of netting of a similar type were then sewn to the net for bioassay analysis. the persistence of the insecticide on nets in regular use was determined by contact bioassay using a standard procedure (who 1980). because a laboratory colony of an. culicifacies was not available, we used a susceptible laboratory colony of an. stephensi for detecting changes in the persistence of insecticides on the bed net. the contact bioassay test was performed immediately after net treatment and thereafter at monthly intervals. the mosquitoes used were 12 to 24h old, unfed, laboratoryreared female an. stephensi and they were exposed for 3 min in group of 5, into the adult who bioassay test conical. the control female mosquitoes were exposed to untreated mosquitoes netting. at the end of the exposure time the mosquitoes were transferred to clean cups where they were maintained on 10% sucrose solution. the number of mosquitoes that were knocked down by the end of the exposure period and the mortality rate after 24 h holding was recorded. five replicates of tests, each with 10 mosquitoes, were exposed on each side of the net. the insecticide susceptibility status of wild caught adult an. culicifacies and an. stephensi against ddt (4%) and deltamethrin (0.05%) was determined by standard 30, 60, 90 and 120 min exposures to insecticide-impregnated papers, followed by 24-h keeping in holding tube (who 1975,1981). pyrethroum space spray mosquitoes collection were conducted between 05.00 and 08.00 h monthly in eight fixed animal and human shelter randomly on each villages from april through november, 2005 (who 1975). before spraying, all the eaves, windows, doors and other exit points were closed and cloth sheets were spread on the floor. pyrethrum (0.2% in kerosene) was sprayed using a pressurized hand sprayer. after spraying, the room was kept closed for 15 min and the knocked-down mosquitoes were then collected from the floor sheet with forceps and placed in petri-dishes lined with moist cotton. attempts were also made to catch mosquitoes resting outdoors using mouth suction tube. all collected mosquitoes were identified to species and classified based on abdominal condition (shahgudian 1960). iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 46 results the population, history of malaria and number of questionnaire applied in the treated, untreated and control villages, during the years 2004-2005 are presented in table 1. a total of 1092 people were interviewed; 1216 from treated and 186,198 from untreated and control villages, respectively, comprising 4464 inhabitants in five villages. a total of 1984 questionnaires were obtained. the age groups in three areas almost was the same and 32% of the population was under 15 yr old, 35% between 15-29 yr old, 16% between 30-44 and 18% were 45 yr and older, family size was 4 to 5 years, the majority of head families were active in husbandry and fishing (30%). the rest domestic activities (19%), government workers (14%), self employed (13%) and unemployed (8%). almost over twothird (70%) of the population were illiterate and the others were employed schooling. most of the mothers were house keeper, the others were engaged as husbandry and fishing activities. eighty seven percent of interweaves (n= 950) indicated that they have malaria experience, 56% (n= 611) indicated that at least one member of their family had malaria during the pervious years. in this study, the most known sign of malaria were the same and comprises fever 52%, chills 25%, headache 10.5%, dizziness 5%, others 1.7%. the majority of interweaves believed it was transmitted by mosquitoes bites (98%) and the rest stated that is was transmitted by stale food, non potable water and unhygienic surrounding. significantly residual spraying was mach more common stated in treated and control villages interweaves (odds ratios= 2.41, x2= 42.79, df= 1, p= 0.0001). when they were asked about the time of use, 25% indicated in evening and night and75% using at night in both areas, 63% to 67% of the interweaves stated they rest outdoor (in the yard) whereas, of the them stated indoor places through warm season. a total of 99% responded that thy brought at least one product for treatment against malaria. fig. 1 presents the results of the 24 h mortality of 12 to 24 h old, unfed laboratory reared female an. stephensi when exposed to mosquitoes net impregnated with deltamethrin for 3 min. mortality rates were considered more stable. the susceptibility test on an. stephensi and an. culicifacies wild strain is presented in table 2. insecticide susceptibility tests showed that for an. stephensi 39.4% survival with ddt (4% a.i.) after one hour exposure, but there was complete susceptibility to deltamethrin (0.05% a.i.). an. culicifacies was completely susceptible to all two of these insecticides. from april to november 2005, a total of 9843 female anoheles mosquitoes were collected by pyrethrum space spray catches from indoor places of living quarters and stables (table 3). overall, an. culicifacies giles s.l predominated (49.8%), followed by an. stephensi liston s.l. (36.9%), an. pulcherrimus theobald (7.3 %), an. dthali patton (5.7%) and an. fluviatilis james (0.3%). in the control village, where no residual spraying has been used the density of mosquitoes was higher than other places and an. stephensi liston was predominated (48.8%) followed by an. culicifacies giles s.l. (38.5%), an. fluviatilis james (6.7%), an. pulcherrimus theobald (5.57%) and an. dthali patton (0.22%). in the untreated area an. culicifacies giles s.l was predominated (60.5%) followed by an. stephensi liston (25.9%), an. fluviatilis james (7.8%), an. pulcherrimus theobald (1.75%) and an. dthali patton (0.45%). the composition of anopheline species was different in control and untreated villages as this area. in treated area an. culicifacies giles s.l was predominated (51.6%) followed by an. stephensi liston (34.5%), an. fluviatilis james (7.4%), an. pulcherrimus theobald (6.3 %) and an. dthali patton (0.245%). iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 47 0 20 40 60 80 100 120 april m ay june july augest septem ber october novem ber decem ber months p er ce nt m or ta lit y treatment control fig. 1. bioassy test with 3-minutes exposure to mosquitoes net treated with deltamethrin against anopheles stephensi in zar abad, chabahar, sistan and baluchistan province, iran 2005 table 1. inhabitants, history of malaria cases, annual parasite incidence (api) per 1000 population and number of questionnaires applied per villages in chabahar, sistan and baluchestan province, 2004-2005 2004 2005 area pop. no. cases api pop. no. cases api families interview questionnaires applied treatment 2736 114 41.6 2798 104 37.2 608 1216 untreated 859 46 57.5 892 43 48.8 186 372 control 877 43 49.4 881 44 50.0 198 396 table 2. susceptibility test on an. stephensi and an. culicifacies in chabahar county, southeastern iran (2004-2005)* mortality percent after exposure to insecticide impregnated paper and 24 h post exposure holding insecticides species control 30 60 90 120 an. stephensi 0 (76) 14.5 (89) 29.4 (85) 46.8 (78) 100 (98) ddt an. culicifacies 0 (82) 62.4 (98) 100 (94) 91.3 (112) 100 (108) an. stephensi 0 (84) 100 (109) 100 (104) 100 (108) 100 (96) deltamethrin an. culicifacies 0 (96) 100 (122) 100 (92) 100 (110) 100 (98) *figures in parenthesis are number of mosquitoes tested iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 48 table 3. total number, the monthly mean indoor resting density, of mosquitoes catches by pyrethrum space spray and hand collection from various sites of study villages in zar abad district, sistan and baluchestan province 2004-2005. treated untreated control total species no. mean % no. mean % no. mean % no. % an. culicifacies 1524 261.1 51.6 1989 248.6 60.5 1389 173.6 38.5 4902 49.8 an. stephensi 1017 127.1 34.5 854 106.7 25.9 1761 220.2 48.8 3632 36.9 an. fluviatilis 219 27.3 7.4 256 32 7.8 244 30.5 6.7 719 7.3 an. pulcherrimus 187 23.3 6.3 173 21.6 1.75 201 25.1 5.57 561 5.7 an. dthali 6 0.75 0.2 15 1.87 0.45 8 1 0.22 29 0.3 discussion this is the first report of the evaluation of deltamethrin impregnated bed net to malaria control in chabahar county. previous study in iran was carried out in dishmook, bandar abbas and ghassreghand counties. the use of protective and preventive measure against malaria is related to family income and consequently to the capability of purchasing explicitly stated by the participant in our study. in spite of the limitation mosquito nets are a familiar household item and 83% of the mothers’ families in chabahar stated they used bed net and 13% and 17% some times user in this area. bed net has been used in different parts of the world with varying results. untreated mosquito nets provide some protection against mosquitoes and malaria, provided that the nets are intact (bradley et al. 1986, lines et al. 1987, curtis et al. 1996, mwangi et al. 2003). pyrethroid treatment much improves protection by preventing mosquitoes from biting through nets, killing them before they find holes in torn nets and by having a community wide 'mass effect' on the vector population when there is high community coverage (lines et al. 1987, maxwell et al. 2006). the density of the vectors was reduced when impregnated bed net were introduced (zaim et al. 1998, moosa kazemi et al. 2000, rassi et al. 2002). our study showed that the use of deltamethrin impregnated bed net can provide any protection from the bite of anopheles species and subsequently interrupted the malaria transmission but does not provide complete protection from the diseases. although, there is a reduction in the density of female an. culicifacies in indoor places of treated compared with the untreated also an. stephensi compared with the control villages but the results showed no significant different between these area (p< 0.05). syntethic pyrethroid combines several advantages for use on the net. this compound as having excito-repellency, quick-acting, and are effective in the small quantities on the bed nets. deltamethrin and lambdacyhalothrin are reported to persist on treated net for one year, even with one or two washing (who 1980). our study showed that an. stephensi and an. culicifacies to be completely susceptible to ddt and deltamethrin insecticides. mortality rates were considered more stable when an. stephensi exposed to mosquitoes net impregnated with deltamethrin for 3 min. the bioassay results on nets in domestic use (fig. 1) showed continued high mortality of this species for the 7 months during which these tests continued. it is widely believed that nets need to be re-treated every 6 months, but in fact high insecticidal activity has been found for much longer periods of domestic use (maxwell et al. 2006). the present data suggest that good results would be achieved against anopheline main vectors with annual retreatment just before the peak transmission season. in the present trial the treated nets reduced considerable numbers of mosquitoes (table 3), which suggests that they may have lead to a 'mass effect' on the village mosquito populations. this may have been at least part of the iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 49 reason for the observed lower catches of the three vectors in the village with treated nets. however, distinguishing a mass effect from insecticidal, deterrent and excito-repellent effects in rooms with treated nets would require sampling in rooms with and without treated nets in villages with high usage of treated nets (maxwell et al. 2003). in turkey, bednets treated with tablet deltamethrin (k-o tab®) did not reduce the mean density of an. sacharovi in the intervention areas compared with the control areas, although reduction in malaria in the former areas was significant (alten et al. 2003). thus, in this case, reduction in malaria was due to personal protection of net users without a 'bonus' of a mass effect. a number of field trials have evaluated nets treated with different synthetic pyrethroids (maxwell et al. 2006) or different formulations of the same pyrethroid (who 1980, 2002). the present field trial on the efficacy of a suspension concentration formulation of deltamethrin against malaria vectors showed generally comparable results with a similar trial undertaken in the same area (yadav et al.,2001). however, in the latter study bioassays showed 100% mortality of an. culicifacies on nets 6 months after their treatment with the sc formulation, which was achieved on an. stephensi in the present study (fig. 1). in chabahar county, malaria transmission is perennial and malaria morbidity is relatively high in the young age groups (center for diseases control and prevention, unpublished data). two distinct peaks of malaria transmission occur in the chabahar county: one in april may and the other in septemberoctober. in the present study area, the peak of malaria incidence coincides with the peak vectors density of an. culicifacies and an. stephensi as the main vectors, whereas an. fluviatilis plays only a secondary role in the transmission. however an. culicifacies, identified as species a of the complex (zaim and javadian 1991), but species b (28.0%) and c (72.0%) have been reported from sundargarh district (nanda et al. 2000). conclusively, at the present time, with regard to failure effects of vaccine against malaria and the compliance of the residents with the operational residual spraying, personal protection is an effective and sustainable means of preventing of the diseases. the use of standard tablet for net treated may be better than liquid or suspension therefore in planning future large scale trials, comparison of new compounds and formulations such as tablets and long lasting of insecticides recommended. acknowledgments this work was financially supported by medical sciences/university of tehran and also dept. of malaria control, ministery of health and medical education. we also would like to express our appreciation to the people of villages in chabahar for their kind cooperation through the study, many thanks also dedicated for efforts of field staff of iranshahr health research and training also chabahar health centers who sincerely cooperate in performing this research. thanks to dr ranjbar, dr rakshani, dr nazari, mr ahar, and mr shahbaksh who are gratefully acknowledged for their participation in this surveys. references alten b, caglar ss, simsek fm, kaynas s (2003) effect of insecticide-treated bednets for malaria control in southeast anatoliaturkey. j vect ecol. 28: 97-107. ansari ma, sharma vp, batra cp, razdan rk, mittal pk (1986) village scale trial of the impact of deltamethrin (k-othrine) spraying in areas with ddt and hch resistant. indian j malariol. 23(2): 127-131. bradley ak, greenwood bm, greenwood am, marsh k, byass p, tulloch s, hayes r (1986) bed nets (mosquito nets) and morbidity from malaria. lancet. 2: 204-207. center for diseases control and prevention. (2005) tehran, i.r. iran, (unpublished data). iranian j arthropod-borne dis, (2007), 1(1): 43-51 sh moosa-kazemi et al.: deltamethrin impregnated… 50 curtis cf, myamba j, wilkes tj (1996) comparison of different insecticides and fabrics for anti-mosquito bednets and curtains. med vet entomol. 10: 1-11. guidelines on the use of insecticide-treated mosquito nets for the prevention and control of malaria in africa (1997) planning. ctd/ mal/afro/97.4 world health organization, geneva. pp. 1-9. jawara m, pinder m, cham b, walraven g, rowley l (2001) comparison of deltamethrin tablet formulation with liquid deltamethrin and permethrin for bed net treatment in the gambia. trop med int health. 6: 309-316. lengeler c (2000) insecticide-treated bed nets and curtains for preventing malaria. cochrane database syst rev, 2, cd000363. lines jd, myamba j, curtis cf (1987) experimental hut trials of permethrin-impregnated mosquito nets and eave curtains against malaria vectors in tanzania. med vet 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transmitted by anopheles culicifacies (diptera: culicidae) in india. j med entomol. 38: 613-622. zaim m, javaherian z (1991) occurrence of anopheles culicifacies species a in iran. j am mosq control assoc. 7(2): 324-326. zaim m, ghavami mb, nazari m, edrissian gh, nateghpour m (1998) cyfluthrin (ew 050) impregnated bednets in a malaria control program in ghassreghand (baluchistan, iran). j am mosq control assoc. 14(4): 421-430. 1. iran j arthropod-borne dis, 2011, 5(1): 1–6 sr nourollahi fard and m khalili: pcr-detection of … 1 original article pcr-detection of coxiella burnetii in ticks collected from sheep and goats in southeast iran *sr nourollahi fard, m khalili department of pathobiology, school of veterinary medicine, shahid bahonar university of kerman, kerman, iran (received 20 sep 2010; accepted 24 may 2011) abstract background: there is a little data on coxiella burnetii (q fever agent) in iran. ticks may play a significant role in the transmission of c. burnetii among animals. the aim of this study was to use polymerase chain reaction for the detection of c. burnetii in ticks collected in southeast iran. methods: one hundred and sixty ticks were collected from domestic animals in three localities of kerman province, southeast iran from november to june 2009. the collected ticks were divided into 35 pools and examined by transpcr for c. burnetii. results: three pools, each consisting of five female of hyalomma anatolicum anatolicum and one pool (6 ticks) of rhipicephalus sanguineus ticks collected from goats and sheep were found to be positive by trans-pcr. conclusion: this paper documents the first molecular detection of c. burnetii in ticks, which shows their role as putative vectors and reservoirs for this pathogenic agent. keywords: coxiella burnetii, ticks, trans-pcr, iran introduction ticks are well known for their impact on the health of both human and animals during infestation (noda et al. 1997, vilcins et al. 2005). ticks can transmit the widest range of pathogens, including protozoa, bacteria, rickettsiae, spirochetes and viruses (kim et al. 2006, psaroulaki et al. 2006). the etiological agent of q fever, coxiella burnetii, has been identified in over 40 tick species (kazar 2005, psaroulaki et al. 2006). coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of acute and chronic q fever in humans. q fever has been described worldwide except in new zealand. from 1999 to 2004, there were 18 reported outbreaks of q fever from 12 different countries involving two to 289 people. six outbreaks involved sheep; three involved goats; one resulted from exposure to goat manure; one from exposure to ovine manure; one involved exposure to wild animals; one involved exposure to cats and dogs; and in two outbreaks the source was unknown (maurin and raoult 1999). a few studies were conducted on q fever in iran some 50 years ago. q fever cases have been reported from some countries neighboring iran, such as turkey and oman (scrimgeour et al. 2003, kennerman et al. 2008). results of a serosurvey undertaken on 42 sheep flocks in turkey showed that 20% of sheep were seropositive (kennerman et al. 2008). recently, an outbreak of q fever occurred with high morbidity in u.s. marines located in iraq (faix et al. 2008). the reservoirs are extensive but only partially known and include mammals, birds, and arthropods, mainly ticks. farm animals, mainly cattle, sheep, and goats, are the primary reservoirs of infection. high numbers of c. *corresponding author: dr mohammad khalili, email: mdkhalili1@yahoo.com iran j arthropod-borne dis, 2011, 5(1): 1–6 sr nourollahi fard and m khalili: pcr-detection of … 2 burnetii are present in the placenta of infected parturient animals and are shed in the environment following labor or abortion. humans acquire the infection mainly via inhalation of contaminated aerosolized particles or ingestion of unpasteurized dairy products (maurin and raoult 1999). clinical q fever in human can present as two basic forms: acute or chronic. acute q fever normally manifests as a self-limiting flulike illness characterized by high-grade fever, peri-orbital headache, and myalgia. however, in some cases pneumonia occurrence requires hospitalization. coxiella burnetii can establish a persistent, latent infection that may reactivate months or years after initial exposure to the organism to cause chronic disease. chronic q fever is typically associated with patients who are immunocompromised and/or who have pre-existing heart valve defects and most commonly presents as endocarditis (maurin and raoult 1999) it is likely that factors such as the route of infection and the inoculum size, affect the expression of c. burnetii infection. indeed the respiratory route is associated with pneumonia and the intra peritoneal route with hepatitis (marrie et al. 1996). coxiella burnetii may induce reproductive disorders such as abortion, stillbirth, and delivery of weak and nonviable neonates in ruminants (lang et al. 1994). ticks can serve as indicators of infection in nature. for example, 10 c. burnetii strains were isolated from ixodes ricinus, dermacentor reticulatus, d. marginatus, haemaphysalis concinna, and h. inermis ticks species collected in different habitats in slovakia (rehacek et al. 1991). the organism multiply in the gut cells of ticks and large numbers of c. burnetii are shed in tick feces. infected ticks are probably the most important agents in maintaining the whole cycle of c. burnetii. ticks may play a significant role in the transmission of c. burnetii among the wild vertebrates, especially in rodents, lagomorphs, and wild birds. also, experimental transmission of c. burnetii from infected to uninfected guinea pigs via tick bite has been performed with ixodes holocyclus, haemaphysalis bispinosa and rhipicephalus sanguineus (angelakis and raoult 2010). dna-based methods have been successfully used for detection of c. burnetii in ticks, fresh tissues, paraffin-embedded clinical samples, frozen samples, formalin-fixed tissues, serum samples, and milk (maurin and raoult 1999). the isolation of the pathogen is a reliable diagnostic method, but it remains timeconsuming and hazardous and requires biosafety level bl3 practices. therefore, the diagnosis of c. burnetii infection is usually done by pcr or serological examination. the pcr assay with primers targeting is1111, the repetitive, transposon-like element (trans-pcr), has been found to be very specific and sensitive for the detection of c. burnetii in different clinical samples (vaidya et al. 2008). the aim of our study was to use polymerase chain reaction for the detection of c. burnetii in ticks collected in southeast iran. materials and methods standard strain of c. burnetii phenol-killed, purified, and lyophilized cells of the c. burnetii nine mile, phase i, strain (rsa 493) were used for this study. tick collection during november and june 2009, one hundred and sixty ticks were collected from domestic animals (goats, sheep) in kerman and bardsir cities, kerman province (southeast of iran) and put them in 35 pools (each pool formed from 5-7 ticks). the collected ticks were properly noted and placed in properly-labeled bottles containing denatured alcohol (95% ethanol+4% methanol+1% pyridine). the ticks were transferred to the parasitology laboratory of school of veterinary medicine, shahid bahonar university of kerman and adults were identified using a stereo iran j arthropod-borne dis, 2011, 5(1): 1–6 sr nourollahi fard and m khalili: pcr-detection of … 3 microscope, according to general identification keys (kaiser and hoogstraal 1963, mazlum 1968, walker et al. 2003). dna extraction prior to dna extraction, ticks were repeatedly washed with 70% ethanol and allowed to air dry for 10 min on sterile paper. ticks were divided into pools of 3-10, according to species, gender and locality, and were homogenized in minimum essential medium (biochrom ag, germany) supplemented with 4% fetal bovine serum. dna was extracted from 300 µ l of the homogenized suspension using the qiaamp dna mini kit (qiagen, hilden, germany), according to the manufacturer’s instructions. the extracted dna stored at –20 °c until used. trans-pcr a pcr assay targeting is1111 fragment, a transposon-like repetitive region, was used to detect c. burnetii in clinical samples. in this study, trans-1 and trans-2 primers were used from the published data sequence of a transposon-like repetitive region of the c. burnetii genome (hoover et al. 1992). the primers trans-1 (5′-tat gta tcc acc gta gcc agt c-3′) and trans-2 (5′-ccc aac aac acc tcc tta ttc-3′) were synthesized by copenhagen (denmark). the trans-1 and trans-2 primers were designed to amplify a 687-bp fragment of the transposon-like repetitive element. the trans-pcr assay was performed as described previously (vaidya et al. 2008). the pcr mixture (25 µl) included 2.5 µl of 10× pcr buffer (100 mm tris-hcl buffer, ph 8.3, 500 mm kcl, 15 mm mgcl2, and 0.01% gelatin), 200 µm deoxynucleoside triphosphate mix, 2 µm of each primers, 0.3 u of taq dna polymerase, 3 µl of template dna, and sterilized water to make up the reaction mixture volume. the dna amplification reaction was performed in a mg thermocycler (eppendorf, germany). the cycling conditions for pcr included an initial denaturation of dna at 95° c for 2 min, followed by five cycles at 94° c for 30 s, 66 to 61° c (the temperature was decreased by 1° c between consecutive steps) for 1 min, and 72° c for 1 min. these cycles were followed by 35 cycles consisting of 94° c for 30 s, 61° c for 30 s, and 72° c for 1 min and then a final extension step of 10 min at 72° c. amplicons were visualized by agarose gel electrophoresis, stained with ethidium bromide at a final concentration of 0.5 mg/ml, and photo documented. results of a total of one hundred and sixty ticks, three pools consisting of 5 female hyalomma anatolicum anatolicum and one pool of three rhipicephalus sanguineus ticks collected in june 2009 on goats and sheep were found positive using trans-pcr (fig.1). fig. 1. lanes 1–3, an example of positive samples, lane 4, dna template from the standard c. burnetii nine mile, lane 5, 100-bp dna ladder, and lane 6, non template control (ntc) discussion serologic evidences indicate people and animals in iran are exposed to c. burnetii (khalili and sakhaee 2009, khalili et al. 2010, sakhaee and khalili 2010), but there is not information available regarding the presence of this agent in specific vectors. this report presents the first detection c. burnetii in ticks in iran. iran j arthropod-borne dis, 2011, 5(1): 1–6 sr nourollahi fard and m khalili: pcr-detection of … 4 it is well known that ticks participate in the transmission of different pathogenic micro-organisms to man and to animals. in nature, c. burnetii is found primarily in cycles involving ticks and vertebrates particularly rodents. c. burnetii is transmitted to domestic animals either by tick bites or through contact with infected excreta. ticks are considered as natural primary reservoirs of c. burnetii and are responsible for transmission of the infection to wild animals and domestic animals (norlander 2000). ticks may play a significant role in the transmission of c. burnetii among the wild vertebrates, especially in rodents, lagomorphs, and wild birds (angelakis and raoult 2010). in ticks, c. burnetii can multiply to very high titers, remains viable during their entire life, and can be transmitted transovarially to next generations. in the enzootic cycle, ticks and vertebrates such as rodents are important components (aitken 1987). also, experimental transmission of c. burnetii from infected to uninfected guinea pigs via tick bite has been demonstrated by ixodes holocyclus, haemaphysalis bispinosa, and rhipicephalus sanguineus (maurin and raoult 1999). ticks expel heavy loads of c. burnetii with their feces onto the skin of the animal host at the time of feeding. appropriate tick control strategies and good hygiene practice can decrease environmental contamination (angelakis and raoult 2010). this study examined the occurrence of c. burnetii, the infectious agent of q fever, in ticks in iran, as a possible rout of infection in qfever outbreaks. we used pools of ticks for dna extraction, which offers the possibility of testing a large number of ticks collected in the field. we demonstrated the presence of c. burnetii by trans-pcr in hyalomma anatolicum anatolicum and rhipicephalus sanguineus ticks collected in southeast iran. among the various species of hard ticks, hyalomma anatolicum anatolicum is one of the most frequently found tick throughout iran (abbasian 1961). this paper documents the first molecular detection of c. burnetii in ticks. this communication confirms the presence of c. burnetii in rhipicephalus sanguineus and hyaloma spp. as previously reported in cyprus and italy (spyridaki et al. 2002, satta et al. 2010). the preventive measures in animals should incorporate: the quarantine of imported animals, routinely testing of animals for antibodies to c. burnetii, tick control and maintenance of surrounding bushes. furthermore, considering the lack of information on the specific tick vector(s) of q fever in the country, present preliminary data are meaningful. we are presently working on tick species identification, and hope to carry out similar studies covering other sites in the country. investigations on c. burnetii using trans-pcr are important method for diagnosis and disease control of q fever. in conclusion, this paper documents the first detection of c. burnetii in tick species in iran. to have a better insight into the epidemiology of c. burnetii infections in iran and its role in domestic animals diseases and human, further studies are needed. acknowledgments we thank dr. prof. rudolf toman for kindly providing phenol-killed, purified, and lyophilized cells of the c. burnetii nine mile, phase i, strain (rsa 493) for this study. this research was financially supported by the research council of shahid bahonar university of kerman, iran. references abbasian l (1961) records of tick (acarina: ixodidae) occurring in iran and their distributional data. acarologia. 3: 546–559. aitken id, bogel k, cracea e, edlinger e, houwers d, krauss h, rady m, rehacek j, schiefer hg, schmeer n, ta iran j arthropod-borne dis, 2011, 5(1): 1–6 sr nourollahi fard and m khalili: pcr-detection of … 5 rasevich iv, tringali g (1987) q fever in europe: current aspects of aetiology, epidemiology, human infection diagnosis and therapy. infection. 15: 323-327. angelakis e, raoult d (2010) qfever. vet microbiol. 140: 297–309. hoover ta, vodkin mh, williams jc (1992) a coxiella burnetii repeated dna element resembling a bacterial insertion sequence. j bacteriol. 174: 5540– 5548. faix dj, harrison dj, riddle ms, vaughn af, 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published online: december 30, 2017 original article situational analysis of visceral leishmaniasis in the most important endemic area of the disease in iran eslam moradi-asl 1,2, *ahmad ali hanafi-bojd 1, *yavar rassi 1, hassan vatandoost 1,3, mehdi mohebali 4, mohammad reza yaghoobi-ershadi 1, shahram habibzadeh 5, sadegh hazrati 5, sayena rafizadeh 6 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of public health, school of public health, ardabil university of medical sciences, ardabil, iran 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4department of medical parasitology and mycology, school of public health, tehran university of medical sciences, tehran, iran 5department of infection diseases, school of medicine, ardabil university of medical sciences, ardabil, iran 6ministry of health and medical education, national institute for medical research development, tehran, iran (received 26 sep 2017; accepted 9 dec 2017) abstract background: visceral leishmaniasis is one of the most important vector borne diseases in the world, transmitted by sand flies. despite efforts to prevent the spread of the disease, cases continue worldwide. in iran, the disease usually occurs in children under 10 years. in the absence of timely diagnosis and treatment, the mortality rate is 95–100%. the main objective of this study was to determine the spatial and temporal distribution of visceral leishmaniasis as well as its correlation with climatic factors for determining high-risk areas in an endemic focus in northwestern iran. methods: in this cross-sectional study, data on vl cases were collected from local health centers in ardabil province, iran during 2001–2015 to establish a geodatabase using arcgis10.3. data analysis was conducted using spss23 and arcmap spatial analyst. maxent model was used to determine ecologically suitable nichesfor the disease. results: two hotspots were found in meshkinshahr and germi counties with 59% and 23% of total cases, respectively. there was an increase in the incidence rate of vl in ardabil county from 2.9 in 2009 to 9.2/100,000 population in 2015. there was no spatial autocorrelation between county and total number of cases (p> 0.05). higher ndvi, lower altitude and southern aspects had positive effects on the presence probability of vl. conclusion: the number of cases of this disease have been rising since 2013 and doubled in 2015. according to the derived distribution maps, the disease is spreading to new locations such as ardabil and namin counties. keywords: visceral leishmaniasis, seroepidemiology, gis, iran introduction arthropod-borne diseases are among the most important public health problems, with over one-third of the infectious diseases being transmitted by insect vectors (1). leishmaniasis is a complex vector-borne disease caused by leishmania spp (2–3). the disease, with a wide spectrum of clinical manifestations ranging from self-healing skin lesions to lethal (visceral) forms, has been reported from 101 countries in the world (4). over 350 million people are at risk of contracting the disease (5). the most important vector of leish*corresponding authors: dr ahmad ali hanafibojd, e-mail: aahanafibojd@tums.ac.ir, prof yavar rassi, e-mail: rassiy@tums.ac.ir j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 483 http://jad.tums.ac.ir published online: december 30, 2017 maniasis in the old world is sand flies of the genus phlebotomus whilst in the new world, is lutzomyia spp. clinically, leishmaniasis is divided into cutaneous leishmaniasis (cl), visceral leishmaniasis (vl) and mucocutaneous leishmaniasis (mcl) (6). visceral leishmaniasis or kala-azar is the most lethal form of this group of diseases, and has a very high mortality rate in the absence of timely diagnosis and treatment (7). it has an annual prevalence of 0.2–0.4 million cases leading to more than 40,000 deaths (8). since the first report of visceral leishmaniasis in 1949 in iran, at least four main endemic foci of the disease in ardabil, fars, east azerbaijan, and bushehr provinces have been reviewed and approved (9). mediterranean leishmaniasis due to l. infantum is the most common form of disease in iran (10), and domestic dogs and other wild canines have been identified as the main reservoirs. although about 100–300 new cases are reported every year in iran across the country, the main foci are in northwestern part, especially ardabil province (11–12). children under 5yr old constitute over 89% of patients in the endemic areas of vl in iran (13). the symptoms of vl include fever, hepatosplenomegaly (14) and anemia (15). geographic information system (gis) is a new technology which is now widely used in the study of diseases transmitted by arthropods (vbds) such as the different forms of leishmaniasis. its application has caused significant changes in data interpretation and decisionmaking on disease control (16). gis technique is useful in understanding the spatial distribution of the diseases, which provides valuable information on the correlation between infection occurrences, climate and environmental variables. it is also able to identify and predict high-risk areas of the diseases. data derived from this technique will facilitate the implementation of environmental interventions at the right time and place. there is a high correlation between the life cycle of vl and the environmental factors involved in its life cycle in terms of geographical distribution (17). using space technologies provides new opportunities for rapid assessment of endemic diseases, accurate and reliable estimation of the population at risk, prediction of disease distribution in areas where information is not available, and determination of appropriate strategy for the control and prevention of the disease in these areas (18). previous studies that used gis and rs techniques to examine the spatial distribution of vl found a correlation between the disease and environmental variables. these techniques were also used to describe host and vector ecology as well as the population at risk of contracting the disease. for example, land use was positively affected sand fly population and thus, a risk factor for vl transmission in brazil, india and iran (19–23). in iran, sand flies from genus phlebotomus (p. kandelakii, p. neglectus, p. keshishiani, p. perfiliewi transcaucasicus, p. alexandri and p. tobbi) mainly transmit l. infantum, the causative agent of vl, from the infected canines to humans (24–31). this parasite usually infects children under the age of 10 years. ardabil province is the most important endemic focus of vl in iran, and in recent years, 25–50% of visceral leishmaniasis cases have been found to occur in this province (12, 32, 33). one of the most important serologic tests used for the diagnosis of visceral leishmaniasis is direct agglutination test (dat) (2, 34). this test has been used for the diagnosis of vl patients in ardabil province since 1996 (15). like other vector-borne diseases, different environmental variables as well as demographics and human activities seem to affect the distribution and incidence of vl. these variables should thus be taken into account during disease prediction and management investigations. the objectives of this study were to determine the geographical distribution of visceral leishmaniasis in ardabil province using gis in order to identify high-risk zones in the province, and to evaluate the role of environj arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 484 http://jad.tums.ac.ir published online: december 30, 2017 mental and geographical variables on the disease distribution. materials and methods study area ardabil province is located in the northwest of iran (37.45–39.42o n, 47.30–48.55o e). the province has an area of 17,953km2 and a population of 1,249,000 people, according to the last census conducted in 2011. the capital of this province is ardabil county and according to the latest provincial demarcations, the province consists of 10 counties, 21 districts, 26 cities, 71 rural districts and 1477 permanent villages (35). topographically, 477 villages (32.3%) are located in plain areas, 975 villages (66%) in mountain valleys, 17 villages (1.17%) in foothill areas and 8 villages (0.54%) in forested areas. climate is variable in ardabil province. about 2/3 of the extent of the study area has mountainous areas and the remaining is covered by plains. overall, the north of the province is situated at lower altitude with relatively warmer weather, whereas the central and southern regions have cool and mountain climate (fig. 1). the area is covered by natural vegetation or agricultural fields such that the ndvi ranges between 0 and 0.86. most common occupations of the people are agriculture, horticulture and animal husbandry. the ratio of urban to rural population in ardabil province is 64/36. data collection and analysis data on vl cases were collected from ardabil province health centers during the last 15yr from march 2001 to the end of february 2015. the number of patients who were seropositive on dat test at titers ≥ 1:3200 and with clinical symptoms recognized by pediatricians were recorded and registered into a geodatabase created in excel. the geodatabase included demographic data of patients, their area of residence (county, city, rural district, village), date in which infection was diagnosed, titer of dat test, etc. the data were then imported into arcmap 10.3 and stored as a shapefile for mapping and statistical/ spatial analysis. the impact of factors such as age, gender and residence of patients on the prevalence of vl was assessed by spss version 23 (chicago, il, usa) and chi-square analysis (ci= 95%). meteorological data were obtained from the ardabil meteorological center during the study period. the data included annual precipitation (mm), average monthly temperature (oc), average minimum temperature (oc), average maximum temperature (oc), and relative humidity (%). spatial analysis arcgis 10.3 (http://www.esri.com/arcgis) was used for spatial analysis and mapping. inverse distance weighted (idw) interpolation analysis was used to prepare raster maps of the climatic variables and ndvi (normalized difference in vegetation index) (fig. 1), and was also used to determine high risk areas of the disease across the study area. this method assumes that the variable being mapped decreases in influence with distance from its sampled location. the "extract values to points" tool in spatial analyst was used to extract the cell values of the prepared raster layers to the vl positive places. spatial autocorrelation of vl cases in the different counties of the study area was estimated. the spatial autocorrelation tool in arcgis measures spatial autocorrelation based on both feature locations and feature values simultaneously. given our vl cases data and the associated attribute (county border), we evaluated the pattern of the disease (clustered, dispersed, or random). moran's i index value was calculated, and as well, both a zscore and p-value (p< 0.05) were also calculated and were used to evaluate the significance of the index (36). this index is given as: http://www.esri.com/arcgis j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 485 http://jad.tums.ac.ir published online: december 30, 2017 where zi is the deviation of an attribute for feature i from its mean (xi-x), wi, j, is the spatial weight between feature i and j, n is equal to the total number of features, and s0 is the aggregate of all the spatial weights: the zistatistic score is calculated as: zi= where: e[i]= -1/(n-1), v[i]= e[i2]-e[i]2 modeling vl distribution maximum entropy (maxent) model ver 3.3.3 (37, 38) was used for this purpose. we used coordinates with 3 or more cases of vl as presence points of the disease where l. infantum is circulating among sand fly, canine reservoirs and humans. 19 bioclimatic variables (table 1) as well as altitude layer were downloaded from the worldclim database with a spatial resolution of 1km2 (version1.4, http://www.worldclim.org/bioclim). using arcmap 10.3 and surface analysis slope and aspect (slope direction) layers were derived. normalized difference vegetation index (ndvi) was obtained from modis images at the same spatial resolution. the contribution of environmental variables and bioclimatic variables were tested by jackknife analysis. all variables with no contribution (0 values) based on the test results were excluded from the final analysis. eighty percent of occurrence records were selected for training model and the remaining 20% for model testing. results demography and the disease temporal distribution of the disease in the province shows that the highest prevalence occurred in march. in other words, the seasonal disease outbreak was higher in spring (32%) and winter (31.5%), whilst the minimum number of total cases was reported in autumn (17.6%), (fig. 2). most of the cases of visceral leishmaniasis were recorded in 2001, 2002 and 2004, and the least one in 2013 and 2014. however, the trend was increasing from 2013 (fig. 3). based on age group distribution of the disease, more than 86% of the patients were children under 4yr, of whom 44.67% were younger than 2 years. two percent of the patients were over 10yr (table 2). according to gender distribution, 58.8% of the patients were male and remaining female (table 3). the highest dat titer (1: 3200) was recorded in 32.27% of the cases, and sera from 6% of the patients demonstrated the lowest dat titer at 1:25600 (table 3). a total of 347 positive cases of kala-azar have been recorded in the past 15yr across the province, the maximum number of cases were recorded in meshkinshahr (59.1%) and germi (23.1%), and the minimum number of cases were occurred in namin (1.2%) (fig. 3). the distribution map of the disease shows that five out of the 10 counties in ardabil province had local cases, which shows that the highest number of cases occurred in the central parts of the province (meshkinshahr and germi) and their adjacent counties (fig. 4). overall, 93 villages (6.3%), 35 rural districts (49.3%), 14 districts (66.66%) and 10 counties (38.5%) had reported cases of visceral leishmaniasis. most of the infected villages (74%) were located in mountain valleys, whereas 19.24%, 4.8% and 1.93% of the villages were located in the plains, foothills and forest areas, respectively. based on the number of cases per area, 85 areas (77.4%) had less than 5 cases, 12 areas (12.9%) reported 5–10 cases, 4 areas (4.3%) had 10–15 cases, and 4 areas (5.4%) recorded more than 15 vl cases. the results of this study show that severity and frequency of vl was higher in meshkinshahr, but in terms of spatial distribution, 41% of the villages in germi county with a population of 84,267 peoples were found to have the highest number of cases (fig. 2). most cases occurred in rural areas (fig. 3). http://www.worldclim.org/bioclim j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 486 http://jad.tums.ac.ir published online: december 30, 2017 the cumulative monthly reported cases of vl in the study area show that it was more prevalent during the first half of the year, but dropped from june to september, and then increased again in the latter part of the year (fig. 4). the occurrence of vl in the study area had a significant correlation with the mean temperature (p< 0.001) and mean relative humidity (p< 0.000) in the different months. spatial analysis spatial distribution of the infection sites and ranking of the counties according to the incidence of vl revealed that most of the disease cases were reported in meshkinshahr county followed by germi, whilst 4 counties had no reported cases of vl during the study period (fig. 5). interpolation of the disease infection showed meshkinshahr areas as the hot spot of the disease (fig. 5). moran spatial autocorrelation analysis showed that there was no spatial correlation between the different counties in terms of the total number of cases recorded during the study period, and the distribution was random (z-score: 0.521963, p> 0.05). results of the interpolation analysis also revealed the central regions of the province as hot spots of vl (fig. 6). according to the climatic and environmental data, vl infections were high in areas with the following climatic/environmental variables: at an altitude between 58–1935m above sea level, relative humidity between 56.33–70.32%, total annual precipitation between 288–382mm, minimum temperature between -4.42–5.99 oc, maximum temperature between 21.52–28.49 oc, average temperature between 9.86–15.54 oc, and ndvi between 0.141–0.749. modeling the ecologically suitable areas of vl results of the maxent model showed a large extent of the province with presence probability less than 20%, and the most ecologically suitable areas of vl occurrence were identified in three hotspots (fig. 7) in meshkinshahr, germi and ardabil counties with a population of 799,788 at risk. the area under receiver operating characteristic (roc) curve (auc) was 0.945 and 0.885 for training and test data, respectively. according to the jackknife test, the environmental variable with highest gain when used in isolation was ndvi (table 1), such that higher ndvi values had positive effect on the presence probability of vl. bio4, bio3 and aspect were the other environmental variables with highest contribution to the model (fig. 8). considering altitude, the model we used found a positive trend to about 1250m above sea level, but higher altitudes demonstrated a negative presence probability for the disease. fig. 1. weather and environmental situation of the study area in northwest of iran j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 487 http://jad.tums.ac.ir published online: december 30, 2017 table 1. variables used for maxent modeling of vl distribution in ardabil province, northwest of iran variable description contribution (%) bio1 annual mean temperature (oc) 0 bio2 mean diurnal range: mean of monthly (max temp–min temp) (◦c) 2.8 bio3 isothermality: (bio2/bio7)× 100 16.4 bio4 temperature seasonality (sd× 100) 17.3 bio5 maximum temperature of warmest month (◦c) 0 bio6 minimum temperature of coldest month (◦c) 0 bio7 temperature annual range (bio5−bio6) (◦c) 0.1 bio8 mean temperature of wettest quarter (◦c) 0.4 bio9 mean temperature of driest quarter (◦c) 0 bio10 mean temperature of warmest quarter (◦c) 0 bio11 mean temperature of coldest quarter (◦c) 0.5 bio12 annual precipitation (mm) 0 bio13 precipitation of wettest month (mm) 0 bio14 precipitation of driest month (mm) 0 bio15 precipitation seasonality (coefficient of variation) 5.9 bio16 precipitation of wettest quarter (mm) 0.1 bio17 precipitation of driest quarter (mm) 1.6 bio18 precipitation of warmest quarter (mm) 1.2 bio19 precipitation of coldest quarter (mm) 2.3 altitude elevation from the sea level (m) 0 slope slope of the area (%) 1.4 aspect direction of slope (degree) 15.5 ndvi -1 to +1 34.6 table 2. frequency of vl by age groups in ardabil province of iran, 2001–2015 c o u n ty a r d a b il b ila h sa v a r g e r m i m e sh k in sh a h r n a m in p a r sa b a d k h a lk h a l n ir k o w sa r s a r e y n t o ta l age no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) no. (%) <2 13(40.62) 6(46.15) 46(57.5) 85(41.46) 0 5(38.46) 0 0 0 0 155(44.67) 2–4 18(56.25) 6(46.15) 19(23.75) 90(43.9) 4(100) 8(61.54) 0 0 0 0 145(41.78) 5–7 1(3.15) 0 7(8.75) 23(11.22) 0 0 0 0 0 0 31(8.93) 8–10 0 0 3(3.75) 6(2.93) 0 0 0 0 0 0 9(2.60) >10 0 1(7.70) 5(6.25) 1(0.49) 0 0 0 0 0 0 7(2.02) total 32(100) 13(100) 80(100) 205(100) 4(100) 13(100) 0 0 0 0 347(100) fig 2. percent of vl infected villages in different counties of ardabil province, northwest of iran, 2001–2015 j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 488 http://jad.tums.ac.ir published online: december 30, 2017 table 3. number of visceral leishmaniasis cases in ardabil province of iran by gender, 2001–2015 dat 1:3200 1:6400 1:12800 1:25600 1:51200 1:102400 total county m a le f e m a le m a le f e m a le m a le f e m a le m a le f e m a le m a le f e m a le m a le f e m a le m a le f e m a le ardabil 7 4 4 3 2 4 1 0 0 2 4 1 18 14 bilahsavar 2 2 2 1 1 0 1 0 1 1 2 0 9 4 germi 21 20 10 3 7 2 4 4 1 3 5 0 48 32 meshkinshahr 24 25 21 12 18 16 9 2 15 9 33 21 120 85 namin 0 2 2 0 0 0 0 0 0 0 0 0 2 2 parsabad 4 1 2 2 0 0 0 0 0 2 1 1 7 6 khalkhal 0 0 0 0 0 0 0 0 0 0 0 0 0 0 nir 0 0 0 0 0 0 0 0 0 0 0 0 0 0 kowsar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 sareyn 0 0 0 0 0 0 0 0 0 0 0 0 0 0 total 58 54 41 21 28 22 15 6 17 17 45 23 204 143 fig. 3. cases of visceral leishmaniasis in urban/rural areas of ardabil province of iran, 2001–2015 fig. 4. the monthly incidence of visceral leishmaniasis in ardabil province of iran and weather data, 2001–2015 j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 489 http://jad.tums.ac.ir published online: december 30, 2017 fig. 5. distribution map (left) and idw interpolation (right) of visceral leishmaniasis in different counties of ardabil province of iran including infected sites, 2001–2015 fig. 6. moran's i autocorrelation analysis of visceral leishmaniasis cases in different counties of ardabil province of iran, 2011–2015 fig. 7. ecologically suitable areas for vl occurrence in ardabil province, northwest of iran j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 490 http://jad.tums.ac.ir published online: december 30, 2017 fig. 8. result of jackknife test of variables importance for vl in ardabil province, northwest of iran discussion the results of the present study showed that vl has expanded across the study area over last 15 years, with cases of the disease reported in half of the counties in the province. from 1984 to 1989, meshkinshahr was the most infected area in the province (39). other studies have also reported the occurrence of vl in meshkinshahr, germi, bilehsavar, parsabad and ardabil counties (40). in line with the results of these studies, our findings confirmed meshkinshahr as the most infected county, and the spatial expansion of the disease to other counties in recent years were also observed. severity and frequency of vl was high in meshkinshahr county whilst the distribution of vl was higher in germi county compared with the other areas. in this study, males were found to be more infected. studies conducted in other countries showed a decreasing trend in the disease incidence and an increasing trend in the spatial distribution of the disease (41–44). although the results j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 491 http://jad.tums.ac.ir published online: december 30, 2017 of the above studies, conducted in india and brazil, are consistent with our results, in afghanistan, there has been 300% increase in vl cases due to war and displacement of the inhabitants (45). based on age distribution analysis, 98% of patients were children under 10yr, of whom more than 70% were less than 2yr old. indicating the age of infection has decreased over the past years (p< 0.05). this result is inconsistent with the results of a study conducted on the disease in this area during 1986–2005 (46) and the findings of moradi-asl et al. (13) in meshkinshahr endemic focus of vl, as well as with a study conducted in italy (47). in brazil, 37% of the cases occurred in patients less than 15yr old whereas only 13% of the cases occurred among patients under 1 year (48). the difference between our study and the studies mentioned above is due to the difference between the parasitic agents circulating in the different study areas. in the mediterranean basin, l. infantum is the main causative agent of vl, but l. donovani is the main cause of vl in other parts of the world (9). there was no significant difference between males and females (p> 0.05) in terms of the incidence of the disease. in iran and pakistan, sex ratio of vl patients was 2:1 (m: f) (49-50). males were infected 1.1 times more than females in brazil (51), but another study reported a sex ratio similar to that of our study (48). seasonal studies of vl infections showed that over 63% of the reported cases in our study occurred in spring and winter, but the incidence was low in summer and autumn (p> 0.05). although a decreasing (p< 0.001) trend was generally observed, occurrence of the disease exhibited a sinusoidal pattern during every 2–3 years. spatial distribution of the disease shows that it has expanded to new areas in the province including ardabil and namin counties. to prevent probable epidemics, it is recommended that a comprehensive study on the infection of vectors and reservoir hosts in the area be conducted in order to determine areas with epidemic potential. this can be done by modeling studies. these types of research have been conducted in some parts of iran and other coun tries (31, 41, 52). most of the cases of vl were recorded in the central and western rural districts of meshkinshahr, muran, central rural districts of germi, and central rural districts of ardabil, bilehsavar and parsabad counties. in these areas, marginalization is higher than in other counties; animal husbandry and farming are the main occupations of the people, and vl reservoir (domestic and wild canines) population is higher than in the urban areas. although vl cases were different between the counties, moran's spatial statistical analysis showed that the pattern of the disease distribution was random. different sand fly species which are vectors of vl are distributed across the country (24– 26, 53–54), and among them, three species (p. kandelakii, p. perfiliewi and p. tobbi) exist in the present study area. presence of seropositive reservoirs has also been reported in the province (11, 55–60). modeling the distribution of vectors and reservoirs and overlaying the outputs of the model will thus be useful in identifying the potential hot spots. in india, the transmission rate of vl was higher in areas with high p. argentipes density, and areas with high vl incidence (serologically) in reservoir population, had more positive human cases in western europe (62). contrary to the findings in brazil (20) and north of india (18), we found that the disease is more prevalent in areas where ndvi is higher. this is due to the difference between the ecological needs of both the parasite and vectors of vl in the different study areas. on the other hand, in our study area, l. infantum is the main cause of the disease which is transmitted by p. kandelakii, p. perfiliewi and p. tobbi (31). however, in india, l. donovani is transmitted by p. argentipes which prefers lowlands with higher temperatures and lower ndvi. in meshkinshahr county, the number of freezing days, rainfall and humidity were more effective vl risk predictors (21). we found j arthropod-borne dis, december 2017, 11(4): 482–496 e moradi-asl et al.: situational analysis of … 492 http://jad.tums.ac.ir published online: december 30, 2017 negative presence probability for the disease at altitudes higher than 1250m above sea level. this is in accordance with the findings of other studies (18, 21, 23). higher altitudes have lower temperatures that may prevent the cycle of vl transmission leading to failure to meet ecological requirements, especially the optimum temperature for both parasite and vector(s). similar to our findings, ghatee et al. (22) reported that temperature is the most effective variable that affects the distribution of vl. like other vector-borne diseases, increased exposure to the vectors leads to a higher risk of vl infections. nomads living in the study area are mainly livestock farmers who rear sheepdogs, and mostly live outdoors during vl transmission seasons. the chance of sand fly bites is thus higher among the people in this area. this is also a major risk factor for the distribution of the disease, especially to the new areas such as ardabil and namin counties, where vl was not previously reported. previous studies conducted in iran confirmed the role of travel and nomadic life style on the incidence of visceral leishmaniasis (22–23). conclusions despite the efforts for control and preventing vl, many new cases of vl in humans and reservoirs (dogs) continue to occur in ardabil province, which is an old endemic focus of vl in iran. even though there is a decline in the total number of cases, the 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on the endemicity of kala-azar: a study based on remote sensing and geographical information system. geospatial health. 4 (2): 155–165. j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 286 http://jad.tums.ac.ir published online: may 27, 2017 original article faunistic study of the aquatic arthropods in a tourism area in northern iran mansoureh shaeghi 1, hossein dehghan 1, kamran pakdad 1, fatemeh nikpour 1, azad absavaran 1, aioub sofizadeh 1, amir ahmad akhavan 1, *hassan vatandoost 1,2, abbass aghai-afshar 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2depertment of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran (received 8 march 2014; accepted 31 dec 2016) abstract background: aquatic insects are very abundant and divers groups of insects that are associated with an aquatic or semiaquatic environment in one or more of their life stages. these insects have been, in some cases, well studied because they are vectors of several diseases. this is the first comprehensive faunistic study of aquatic insects from babol county. the results may provide basic data for further taxonomic and ecological studies of aquatic insects as biological control agent or classification of water quality for the country. methods: the specimens were collected using different methods including: d-frame net collector, standard mosquito dipper (350ml), sweep-netting and plastic pipette. sampling carried out in different part of breading places in several times. results: during this study a total of 196 aquatic specimens were collected from different habitats and were morphologically identified including 18 families classified in 6 orders: diptera, trichoptera, ephemeroptera, plecoptera, hemiptera and odonata. babol and amol district in mazandaran province are located in humid climate regions with suitable ecological factors of humidity, moderate temperature and the variety of plant species. there are different species of aquatic insects in different habitats. conclusion: the results will provide information for biodeveristy, species richness, their role for biological control as well as calcification of rivers based on abundance of aquatic insects. therefore the understanding of ecological specifications of aquatic insects could provide a clue for further arthropod-borne disease control. additionally aquatic insect could be used for classification of water bodies keywords: aquatic insect, iran, mazandaran introduction aquatic insects are a very abundant and divers group that inhabits a variety of aquatic environments such as puddles, ponds, lakes, ditches, streams, lakes and other types of breeding places. despite their importance in aquatic ecosystems, very few insects spend their entire lives submerged in water. most aquatic insects undergo an aquatic immature stage followed by a terrestrial adult (eg ephemeroptera, odonata, plecoptera, trichoptera, megaloptera). even in cases where both the larvae and adult are aquatic, often the adult can exit the water and/or the pupal stage is terrestrial. additionally, many species considered are semiaquatic and are only associated with aquatic and semiaquatic vegetation, the water's surface, or the margins of water habitats (bouchard 2004). although only a small percentage (around 3%) of insects are aquatic, representatives are found in 13 insect orders (daly 1984, williams and feltmate 1992) suggesting that partial aquatic lifestyle might be advantageous to a wide array of insects. generally, aquatic insects are nymphs/larvae of terrestrial adults (brown 1987) that spend some time in ter*corresponding authors: prof hassan vatandoost, email: hvatandoost1@yahoo.com j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 287 http://jad.tums.ac.ir published online: may 27, 2017 restrial environments during certain stage/s of their life cycle (jewett 1963). these insects have been, in some cases, well studied because they are vectors of several diseases (cook 1997), can be environmental quality biosensors (kashian et al. 2007), and are utilized for understanding aquatic communities and ecosystems (benke 1979, waters 1979) and several other areas of ecology such as predator-prey interactions, competition, population dynamics (resh and rosenberg 1984). according to the fossil record, aquatic insects appeared in the triassic (belayeva et al. 2002), more than 150 my after the appearance of insects (gaunt and miles 2002, engel and grimaldi 2004). this fact along with the presence of a tracheal system in nearly all aquatic insects (chapman 1998), supports the idea that these animals secondarily adapted to living in water (ross 1967, resh and solem 1984, pritchard et al. 1993). some species of aquatic insect are medically important vectors that transmit diseases such as: malaria, dengue, filariasis, yellow fever, anthrax, tularemia, and lyme (foil 1998). furthermore few numbers of them have a painful bite that cause dermatological effect on human and animal host (de villiers 1987). some of them act as a host of trematods such as: mayfly, dragonfly, damselfly and stonefly (chae et al. 2000) aquatic insects may be used as water indicators of ecosystems as their life span, their comparatively stable mode of life and distinct characters, offers as sorting and identification of these organisms. many authors have studied the water quality in with aquatic insects are used as indicator of pollution (krishnamoorthi and sarkar 1979, gupta and paliwal 2007, agarwal et al. 2008). the various species and population counts of aquatic insect larva present in a stream provide an effective way to monitor the health of the ecosystem. for example, stonefly (plecoptera) larvae are very sensitive to changes in water quality. on a scale from 0 to 10, with 10 being the most tolerant species, stonefly larva rank between 0 and 2 depending on the specific species (hilsenhoff 1988). some aquatic insects play important roles in mosquito control (james 1966, ellis and borden 1970, pandian et al. 1979). in general, almost all aquatic insect predators prey on mosquito larvae and pupae (ellis and borden 1970, peckarsky 1984), for instance diving beetle rhanthus pulverulosus (coleoptera, dytiscidae) and aquatic instars of the order odonata (dragonflies and damselflies) are predators of mosquito larvae and have been observed to ingest mosquito larvae as a part of their natural food assemblages, in particular, may be useful for controlling mosquito larval populations (sunahara et al. 2002). the backswimmer, notonecta undulate (hemiptera, notonectidae) and giant water bug, sphaerodema annulatum and s. rusticum (hemiptera: belostomatidae), has been shown to efficiently utilize the second instar of mosquito larvae as prey (ellis and borden 1970, murdoch et al. 1984, blaustein et al. 1995, blaustein 1998, pramanic and raut 2003, aditya et al. 2004, aditya et al. 2005). the role of the backswimmer, anisops assimilis, in controlling mosquitoes was recognized as early as 1939, when graham (graham 1939) noted some stock troughs with a. assimilis were free of mosquitoes, whereas puddles in depressions surrounding the troughs contained “energetic mosquito activity”. however, the predation efficiency of backswimmers on mosquito larvae was found to be container-specific (lester and pike 2003). furthermore, the presence of backswimmers within a water body has been demonstrated to reduce oviposition rates by adult mosquitoes (chesson 1984, blaustein et al. 1995). limited numbers of studies have been carried out on the faunistic and ecological aspects of aquatic insects in iran (tirgari 1979, hosseinie 1978, hosseinie 1992a, hosseinie 1992b, hosseinie 1994, hosseinie 1995a, hosseinie 1995b, vafaei et al. 2007, askari et al. 2009). j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 288 http://jad.tums.ac.ir published online: may 27, 2017 babol county is a county of mazandaran province in north of iran (36˚33′05″n 52˚40′44″e) and located approximately 20 kilometers south of caspian sea on the west bank of babol rud river and receives abundant annual rainfall. due to the importance of aquatic insects there is an urgent need for comprehensive studies and publications that are locally available. the present study intends to help fill this gap by providing an overview of the literature published to date on the aquatic insects of iran especially in babol county. this is the first comprehensive faunistic study of aquatic insects from babol county and may provide basic data for further taxonomic and ecological studies of aquatic insects in north of iran. a further aim of this study was to consider the obtained faunistic results from the ecological aspect. materials and methods study area a descriptive cross sectional study was performed in july 2012 in babol county, mazandaran province. this province, located in the north of iran and southern shores of the caspian sea (36° 33′ 56.16″ n, 53° 3′ 31.68″ e) (fig. 1). climatic characteristics of the caspian sea coasts are almost mediterranean. generally its climate is temperate and humid and approximately is equilibrium with hot and cold throughout the year (heat and cold, 5 ˚c and 25 ˚c). considering various factors which influence the climate of the province are in the area, there are two types of weather: temperate plains and mountains (temperate and cold). in this study, rivers, rice fields, and the stagnant water in different parts of province were selected as sites for sampling and collecting of aquatic insects. sampling methods the specimens were collected using different methods including: d frame net-collector, standard mosquito dipper (350ml), sweepnetting and plastic pipette. sampling carried out in different part of breading places in several occasions. the samples were collected, transferred to individual jars containing some water obtained from their habitat. subsequently they were put in glass vials contained 70% ethylic alcohol. the date and location of sampling were written on the label and stick on the vials. all samples were sent to school of public health, tehran university of medical sciences, where the author identified the specimens using the keys of aquatic insects based on guide to aquatic invertebrate families of mongolia 2012 and other relevant systematic keys. in this study we used stereo-typed microscope and microscope to identification of samples. the results were recorded on a data sheet based on the order and family and number of its. all of the photos are original. results during this study 196 aquatic insects (mature and immature forms) were collected from different habitats (slow moving and turbulent rivers, shallow streams, temporary pond, andrice field that its information is shown in table 1. collected aquatic samples were identified by morphologic traits with using valid entomological keys (william bouchard 2012, subramanian and sivaramankrishnan 2007). these aquatic insects after identifying represented 18 families belonged to 6 orders: diptera, trichoptera, ephemeroptera, plecoptera, hemiptera and odonata (both sub orders zygoptera and anisoptera). diptera with 5 families: culicidae, tipulidae, chironomidae, simulidae and blephariceridae (fig. 2a–e), hemiptera with four families: nepidae, corixidae, notonectidae and gerridae (fig. 3), odonata with four families: calepterygidae, coenagrionidae, lestidae and aeshnidae (fig. j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 289 http://jad.tums.ac.ir published online: may 27, 2017 4a, b), ephemeroptera with three families: baetidae, heptagenidae and caenidae (fig. 5a–c), plecoptera and trichoptera with one family taeniopterygidae and helicopsychidae respectively (fig. 6 and fig. 7). in this study diptera had the most and trichoptera associated with plecoptera had the least families. from 196 identified aquatic insects 48 (24.5%) belonged to five families of diptera, 39(19.9%) from tricoptera, 37(18.88%) from 3 families of ephemeroptera, 27 (13.77%) from plecoptera, 25(12.75%) from 4 families of hemiptera and 20(10.2%) from 4 families of odonata (fig. 8). helicopsychidae family and (blephariceridae, caenidae, lestidae and aeshnidae) allocated the most (19.9%) and the least (0.51) frequency of families in our study respectively (table 3). order diptera we find five families in diptera order which described in bellow: (bouchard 2012) culicidae common name: mosquitoes habitat: mosquito larvae occur in standing or still water of lakes, ponds, marshes, temporary pools, and streams. the larvae are planktonic. size: small to medium (4–18mm) characteristics: head sclerotized, rounded, and clearly separate from the thorax, labrum with brushes of setae; mandibles moving against each other on a horizontal plane, thoracic segments fused and swollen, wider than abdomen, prolegs absent, eighth segment usually bearing a respiratory siphon. chironomidae common name: non-biting midges habitat: chironomids are found in every aquatic habitat from small seeps to large rivers and from temporary pools to deep lakes. they occur in soft sediment, on rocks, in and around vegetation, in snags, and just about any other habitat. size: small to large (2–30mm) characteristics: head sclerotized, rounded, and clearly separate from the thorax, body elongate and worm-like, mandibles moving against each other on a horizontal plane, two pairs of ventral prologs (one on prothorax and one at the terminal end), prologs terminate in a series of hooks. tipalidae common name: crane flies habitat: tipulid larvae can be found in a variety of habitats such as streams, ponds, and marshes. they can be found under rocks, in sand, snags, leaf packs, and algal mats. size: small to large (3–60mm) characteristics: much of rounded head capsule present or reduced to only a few rods, head capsule completely or partially retracted into thorax, mandibles moving against each other on a horizontal plane, usually with ventral welts, terminal segment usually with two spiracles, spiracular disc usually surrounded by lobes or projections of varying numbers or shapes. blepharicerida common name: net-winged midges habitat: blepharicerid larvae are restricted to cool, fast-flowing streams and waterfalls. they are found attached to rocks in areas of fast flow. size: small to medium (5–12mm) characteristics: head fused with thorax and first abdominal segment, mandibles moving against each other on a horizontal plane, 6 abdominal segments with deep constrictions between segments, ventral suckers on first 6 segments, gill tufts present ventrally. simuliidae common name: black flies, buffalo gnats habitat: black fly larvae occur in streams and rivers in areas of moderate to fast current. they are found attached to rocks, logs, vegetation, or any other solid substrate in the current. j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 290 http://jad.tums.ac.ir published online: may 27, 2017 size: small to medium (3–15mm) characteristics: head sclerotized, rounded, and clearly separate from thorax, pair of labral fans (“mouthbrushes”) usually present, mandibles moving against each other on a horizontal plane, proleg present ventrally on prothorax, posterior 1/3 of abdomen swollen, abdomen terminates in a ring of hooks. order hemiptera aquatic hemipterans are unlike most aquatic taxa in that the adults and larvae occupy the same habitat. aquatic and semiaquatic hemiptera can be separated into two groups based on their antennal morphology and the habitat in which they are generally found. some hemiptera are primarily aquatic and can be recognized by the possession of antennae that are shorter than the head and concealed below the eye. one exception is the gelastocoridae, which are riparian and possess short antennae. the truly aquatic species are usually found under water, but many possess wings, which allow movement between water bodies. in contrast, most semiaquatic species of hemiptera have antennae longer than their heads and can be found on the water’s surface or at the water’s margin. although some taxa are primarily aquatic, most hemiptera do not rely heavily on dissolved oxygen in the water, but instead obtain oxygen from the atmosphere. we find four families in hemiptera order which described in bellow: (bouchard 2012) corixidae common name: water boatmen habitat: corixids are found in areas of standing or slow flowing water in ponds, lakes, marshes, streams, and rivers. size: small (3–11mm) characteristics: antennae shorter than head, concealed below eye, beak broad and triangular without distinct segments; fore tarsus scooplike and edged with setae. nepidae common name: water scorpions habitat: nepids can be collected in ponds, marshes, and streams in areas of calm water. they are usually found at the vegetated margins of these water bodies. size: medium to large (15–45mm) –not including respiratory tube. characteristics: body cylindrical or oval and flattened (not shown but when body is oval it is superficially similar to belostomatidae), antennae shorter than head, concealed below eye, beak cylindrical, fore legs raptorial, mid and hind legs long and slender, abdomen terminating in an elongate breathing appendage. gerridae common name: water striders habitat: gerrids are generally found on the surface of the water in ponds, lakes, marshes, streams, and rivers. size: small to medium (3–18mm) characteristics: body shape variable, antennae longer than head, beak cylindrical, claws of protarsus inserted before apex, metafemur extends well beyond tip of abdomen. notonoctidae common name: backswimmers habitat: notonectids most commonly occur along vegetated margins of lakes and ponds and in marshes. they are also sometimes found in the pools and backwaters in streams and rivers. notonectids can also be found in temporary pools and ditches. size: small to medium (5–15mm) characteristics: body cylindrical, antennae shorter than head, concealed below eye, beak cylindrical, hind legs oar-like, hind tarsal claws inconspicuous order odonata we find four families in odonata order which described in bellow: (bouchard 2012) j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 291 http://jad.tums.ac.ir published online: may 27, 2017 calopterygidae common name: broad-winged damselflies habitat: these damselflies are most commonly found at the edges of streams with slow flowing water where they cling to root masses, overhanging grasses, and twigs. size: large (30–40mm) characteristics: antennal segment 1 longer than remaining antennal segments put together, prementum with diamond shaped medial cleft, prementum and palpal setae absent, middle gill shorter than lateral gills, gills triangular in cross section, no veins visible in gills. coenagrionidae common name: narrow-winged damselflies habitat: narrow-winged damselflies are found in a wide range of habitats including ponds and flowing waters. these damselflies are most common in vegetation at the margins of lakes and in wetlands. some species are found in streams clinging to rocks and vegetation. size: medium to large (15–32mm) characteristics: most are slender like other damselfly larvae (although some are short and stocky), all antennal segments are approximately the same length, prementum triangular and stout without medial notch, usually 3–5 premental setae on each side of midline, 1–7 raptorial setae on each palpal lobe, palpal lobes terminating in 1–2 hooks, all gills the same length, veins in gills radiate diagonally from medial line. lestidae common name: spread-winged damselflies habitat: these damselflies are most common in small ponds, bogs, wetlands, and sometimes in slow weedy streams. size: large (22–38mm) characteristics: these are long slender damselflies, all antennal segments similar, prementum with small triangular notch, prementum stalked and spoon shaped, 4–8 premental setae present, palpal lobes with 3–5 raptorial setae and trifid, all gills of similar length, veins visible in gills and perpendicular to medial line. aeshnidae common name: darner dragonflies habitat: darner dragonflies are most commonly collected in vegetation along the edges of lakes and ponds. they can also be found in some streams under logs and stones or in snags. size: large (30–62mm) characteristics: prementum and palpal lobes are flattened, 6–7 antennal segments present with all segments of similar size and shape. order ephemeroptera we find three families in ephemeroptera order which described in bellow: (bouchard 2012) baetidae common name: primitive minnow mayflies habitat: primitive minnow mayflies can be found in vegetation along large rivers, in the riffles of small streams, in seeps, in swamps, and in ponds. size: small to large (6–20mm) characteristics: antennae less than 2x the width of head, maxillae without pectinate spines, gills usually present on abdominal segments 1–7, gills usually oval, long setae present on caudal filaments (present on both sides of terminal filament and only on the inner side of the cerci). heptageniidae common name: flathead mayflies habitat: flathead mayflies are most common in slow to fast flowing streams where they occur on the surface of rocks, logs, vegetation, and leaves. hemeroptera size: small to large (5–20mm) characteristics: body, head, and legs (femora) flattened, mouthparts not visible from dorsal view, gills present on abdominal segments 1–7, only short setae present on caudal filaments. caenidae common name: small square-gill mayflies habitat: caenid may fly larvae occur in streams in areas of slow current, at the edges of lakes, and in wetlands. j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 292 http://jad.tums.ac.ir published online: may 27, 2017 size: small (2–8mm) characteristics: gills on abdominal segment 1 vestigial (small and finger-like), gills on abdominal segment 2 square operculate (platelike) and covering succeeding gills, operculate gills touch or overlap at midline, fringed gills present on abdominal segments 3–6, setae on caudal filaments restricted to apex of each annulation. order plecoptera of this order we collected only one family: taeniopterygidae which described in bellow: (bouchard 2012) taeniopterygidae common name: giant stoneflies habitat: these large stoneflies are most commonly found in small swiftly flowing streams. they are found in areas of swift current in leaf packs and snags. size: large (15–70mm) characteristics: larvae are large and dark brown, finely branched gills are present on all 3 thoracicsegments and abdominal segments 1–2. order tricoptera in this study we collected only one family in tricoptera order: helicopsychidae which described in bellow: (bouchard 2012). helicopsychidae common name: snail case-maker caddisflies habitat: these caddisflies are most commonly found in streams with sand deposits. they are also found on wave-swept shores of lakes. snail casemaker caddisflies are usually attached to rocks and logs. size: small (8mm) –the case is usually about the size of a pea. characteristics: body curled, all three thoracic segments with sclerotized dorsal plates, stout setae present on anterior edge of pronotum, prosternal horn absent, branched gills present on anterior abdominal segments, anal claw comb-shaped (with many small teeth), case shaped like a snail shell. table 1. collected insect orders from different habitats, babol and amol, mazandaran province, 2012 habitat orders ricefield ephemeroptera diptera _____ _____ slow moving river ephemeroptera diptera hemiptera trichoptera temporary pond ephemeroptera diptera hemiptera _____ shallow stream ephemeroptera _____ hemiptera trichoptera odonata turbulent river _____ diptera hemiptera trichoptera plecoptera table 2. biodiversity of families in 6 orders of aquatic insects collected in babol and amol, mazandaran province, 2012 order no. of families diptera 5 odonata 4 hemiptera 4 ephemeroptera 3 plecoptera 1 trichoptera 1 j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 293 http://jad.tums.ac.ir published online: may 27, 2017 table 3. aquatic insects families frequency collected from babol and amol, mazandaran province, 2012 order sub.order family no. (%) total (%) diptera culicidae 26 (13.27) 48 (24.5) simulidae 10 (5.1) chironomidae 7 (3.58) tipulidae 4 (2.04) blephariceridae 1 (0.51) trichoptera helicopsychidae 39 (19.9) 39 (19.9) ephemeroptera baetidae 34 (17.35) 37 (18.88) heptagenidae 2 (1.02) caenidae 1 (0.51) plecoptera taeniopterygidae 27 (13.77) 27 (13.77) hemiptera corixidae 12 (6.12) 25 (12.75) nepidae 6 (3.06) gerridae 4 (2.04) notonectidae 3 (1.53) odonata zygoptera caloepterygidae 15 (7.65) 20 (10.2) coenagrionidae 3 (1.53) lestidae 1 (0.51) anisoptera aeshnidae 1 (0.51) total 196 (100) fig. 1. the study areas of sampling and collecting aquatic insects fig. 2. order diptera: a. culicidae, b.chironomidae, c.blephariceridae, d.simulidae, e.tipulidae j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 294 http://jad.tums.ac.ir published online: may 27, 2017 fig. 3. order hemiptera: nepidae fig. 4. order odonata: a.coenagrionidae, b.caloepterygidae fig. 5. order ephemeroptera: a.baetidae, b.caenidae, c.heptagenidae fig. 6. order plecoptera: taeniotorygidae fig. 7. order trichoptera: helicopsychidae fig. 8. percentage of aquatic insects orders, babol and amol, mazandaran province, 2012 discussion given that babol and amol district in mazandaran province are in humid climate regions with suitable ecological factors humid, moderate temperature and the variety of plant species where there are different species of aquatic insects in different habitats (slow moving and turbulent rivers, shallow streams, temporary pond, and rice fields, these areas are the appropriate places for the growth of aquatic insects and sample collection. in the current study, the most prevalent collected sample was diptera with five families (24.5%) and trichoptera, ephimeroptera, plecoptera, hemiptera and odonata seted in latter rank respectively. in a study conducted in yamuna in summer, as well as the results of our study the most b a c j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 295 http://jad.tums.ac.ir published online: may 27, 2017 abundant order were diptera and hemiptera, coleoptera and odonata seted in latter rank respectively (gapta and paliwal 2010). in a study conducted in usa ephimeroptera, odonata, plecoptera, hemiptera, megaloptera, coleoptera, trichoptera and diptera presented in eight major order of aquatic insects (reese voshell 2009). we collected five order of this eight order in clouded: diptera, trichoptera, ephimeroptera, plecoptera, hemiptera and odonata. in a study conducted in nigeria abundance of order of aquatic insecs were as follows: ephimeroptera >odonata >coleoptera > diptera >pelicoptera >tricoptera (ohiokhioya 2009). ephemeroptera (mayflies) mayfly larvae are found in a variety of locations including lakes, wetlands, streams, and rivers, but they are most common and diverse in lotic habitats. they are common and abundant in stream riffles and pools, at lake margins and in some cases lake bottoms. most of them eat plant material, either by scraping algae or collecting small pieces of detritus from the bottom. they have incomplete metamorphosis. all mayfly larvae are aquatic and breathe dissolved oxygen by means of gills on the abdomen with terrestrial adults. in most mayfly species the adult only lives for 1–2 days. consequently, the majority of a mayfly’s life is spent in the water as a larva. the adult lifespan is so short there is no need for the insect to feed and therefore the adult does not possess functional mouthparts so never eat. mayflies are often an indicator of good water quality because most mayflies are relatively intolerant of pollution. mayflies are also an important food source for fish (reese voshell 2009, bouchard 2012). in this study, we were founded may flies in rice-field, slow moving river, temporary pond and shallow stream, but don’t fonded of turbulent river. of this order we collected 3 families: baetidae (91/5%), heptagenidae (5/5%) and caenidae (3%). caddisflies (tricoptera) trichoptera is the largest order of insects in which most members are truly aquatic. trichoptera are close relatives of butterflies and moths (lepidoptera) and like lepidoptera, larvae of different caddisflies live in a wide variety of flowing and standing waters. they also have a wide range of feeding habits, including scraping algae, collecting fine particles of detritus from the bottom or from the water, shredding dead leaves, and preying on other invertebrates. they breathe dissolved oxygen by means of gills and their overall body surfaces have complete metamorphosis and remain in the water for the pupa stage. caddisflies have the ability to spin silk. this adaptation may be largely responsible for the success of this group. silk is used to build retreats, to build nets for collecting food, for construction of cases, for anchoring to the substrate, and to spin a cocoon for the pupa. almost all caddisflies live in a case or retreat with the exception of rhyacophilidae. caddisflies are important in aquatic ecosystems because they process organic material and are an important food source for fish. this group displays a variety of feeding habits such as filter/collectors, collector/gatherers, scrapers, shredders, piercer/herbivores, and predators. caddisflies are most abundant in running (lotic) waters. like ephemeroptera and plecoptera, many trichoptera species are sensitive to pollution but a few kinds are somewhat tolerant of moderate levels of pollution. (reese voshell 2009, bouchard 2012). in this study, we wre founded trichoptera in slow moving river, shallow stream and turbulent river. plecoptera (stoneflies) plecoptera have incomplete metamorphosis. they larvae are almost exclusively found in running waters and they reach their greatj arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 296 http://jad.tums.ac.ir published online: may 27, 2017 est diversity in small cold streams. they are generally associated with coarse substrates such as cobble, leaf packs, and large woody debris. plecoptera are the most sensitive order of aquatic insects and many species are restricted to habitats with high levels of dissolved oxygen. some have gills on their thorax, but others just obtain dissolved oxygen all over their body. some feed on plant material, either by shredding dead leaves and other large pieces of detritus, while others are predators.when a stonefly larva is ready to emerge as an adult, it crawls out of the water and sheds the larval skin or exoskeleton. compared to the length of the immature stage (6 months to 3 years), the adult life span is short and usually lasts only 1–4 weeks. many adult stoneflies exhibit an interesting behavior of drumming to locate mates. a male will usually initiate drumming by tapping its abdomen on the substrate. a female that perceives the vibrations will then drum a response. by moving toward each other while periodically stopping to drum, males and females are able to locate each other. in order to insure that males and females of the same species find each other, each species has a unique drumming pattern. the most unusual feature of this group is that some kinds are programmed to emerge only during the coldest months, hence, they are called the winter stoneflies. like ephemeroptera and tricoptera, almost all of the stoneflies are sensitive to pollution. in this study, we found plecoptera in turbulent river but didn’t find in rice-field, slow moving river, temporary pond and shallow stream. hemiptera (true bugs) most of the true bugs live on land, but the aquatic kinds are most common in the shallow areas around the edge of standing waters. both the adults and the larvae of the aquatic kinds live in the water. both stages are usually found on submerged aquatic plants. almost all of them are predators. they breathe oxygen from the air, either by taking a bubble underwater or by sticking a breathing tube up into the air. they have incomplete metamorphosis. most kinds are tolerant of pollution. true bugs have a sharp beak that they stick into the body of their prey, and then they pump in poison to kill their prey, after which they suck out the body fluids. some of the larger kinds feed on small fish and tadpoles (reese voshell 2009). due to their ability to utilize atmospheric oxygen, hemiptera are often able to exist in water bodies with low levels of dissolved oxygen (bouchard 2012). in this study, we found hemiptera in turbulent river, slow moving river, temporary pond and shallow stream, but don’t fonded in rice-field. of this order we collected 4 families: corixidae (48%), neppidae (24%), geridae (16%) and notonectidae (12%). odonata (dragonflies and damselflies) most people are familiar with large dragonflies observed flying over ponds, marshes, and fields. less well known are the aquatic larvae of these insects. they have incomplete metamorphosis. odonates are most abundant and diverse in lentic (standing) waters, but many species also occur in lotic (flowing) waters. larvae breathe dissolved oxygen with gills, which are located either inside the rear portion of the abdomen (dragonflies) or on the end of the abdomen (damselflies). all adult and larval odonates are predatory. most odonata larvae are sit-and-wait predators, which means they remain motionless until an insect or small fish approaches the larva. when a prey item comes close, the larva rapidly extends its labium (lower lip) and grasps the prey with its jaw-like palps. the most unusual feature of this group is the way the larvae catch their food with an elbowed lower lip, which they can shoot out in front of the head. the adults feed on arthropods such as j arthropod-borne dis, june 2017, 11(2): 286–301 m shaeghi et al.: faunistic study of … 297 http://jad.tums.ac.ir published online: may 27, 2017 other insects (including other dragonflies and damselflies) and spiders. adult odonates, especially dragonflies, are strong fliers and catch much of their prey on the wing. many kinds are fairly tolerant of pollution, but some kinds only live in unique habitats, such as bogs high in the mountains. in this study, we founde odonata only in shallow stream. we found zygoptera and anisoptera suborder. from suborder zygoptera we found 3 families: caleopterygidae (79%), coenagrionidae (15/7%) and lestidae (5/2%), ad in anisoptera suborder we founded only one sample of aeshnidae. diptera (true flies) diptera considered aquatic have aquatic larvae and pupae with terrestrial adults. many other aquatic insects are also commonly referred to as “flies” (eg mayflies, dragonflies, stoneflies, caddisflies, alderflies, fishflies), but these taxa are not true flies as they do not belong to the order diptera. when referring to true flies or diptera with their common names, the word “fly” is separate (eg crane fly, black fly, moth fly dance fly, flower fly). in contrast, common names for non-dipteran taxa are one word. the true flies are extremely important in aquatic food webs and often are the most diverse and abundant macroinvertebrate taxon collected in many freshwater habitats. they have a wide range of feeding habits, including scraping algae, collecting fine particles of detritus from the bottom or from the water, shredding dead leaves, and preying on other invertebrates. diptera inhabit a wide range of habitats and some taxa are extremely tolerant and occur in heavily polluted water bodies. they breathe dissolved oxygen by means of gills and their verall body surface. true flies have complete metamorphosis and remain in the water for the pupa stage. the most distinctive feature of this group is their ecological diversity. some kinds live in the cleanest habitats (eg swift, cool, mountain streams), while others live in some of the harshest natural habitats on the earth (e.g., arctic tundra ponds, geothermal springs, alkaline lakes, mucky swamps). they have equally diverse responses to pollution, with some kinds being exceptionally sensitive, while other kinds endure the worst imaginable water quality (eg raw sewage or acid mine drainage). some true flies can be a nuisance due to their blood feeding behaviors (bouchard 2012). in this study, we found diptera in ricefield, slow moving river, temporary pond and turbulent river, but didn’t find in shallow stream. from this order we collected 5 families: culicidae (54/1%), simulidae (20/8) chironomidae (14/5%), tipulidae (8/3%) and blepharieridaec (2%). conclusion aquatic insects play an important role for biological control of larvae and adults of mosquitoes. on the other hand some of them are vectors of human and animal diseases, for example in iran several species belong to anopheline including anopheles culicifacies s.l., an. stephensi, an. dthali, an. fluviatilis s.l., an. superpictus, are known to be the malaria vectors (naddaf et al. 2003, doosti et al. 2003, oshaghi et al. 2003, oshaghi et al. 2006, vatandoost et al. 2006, davari et al. 2007, hanafi-bojd et al. 2011, mehravaran et al. 2011, oshaghi et al. 2011, vatandoost et al. 2011, hanafi-bojd et al. 2012a,b, c, vatandoost and abai 2012, soleimani-ahmadi et al. 2012a,b, vatandoost and hanafi-bojd 2012). therefore the understanding of ecological specifications of aquatic insects could provide a clue for further arthropod-borne disease control. additionally aquatic insect could be used for classification of water bodies. acknowledgements this study was funded by the 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salim abadi1, f rafi1, *h vatandoost1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of pharmacognosy and medicinal plant research center, faculty of pharmacy, tehran university of medical sciences, tehran, iran (received 23 apr 2011; accepted 22 nov 2011) abstract background: plant extracts and oils may act as alternatives to conventional pesticides for malaria vector control. the aim of this study was to evaluate the larvicidal activity of essential oils of three plants of apiaceae family against anopheles stephensi, the main malaria vector in iran. methods: essential oils from heracleum persicum, foeniculum vulgare and coriandrum sativum seeds were hydro distillated, then their larvicidal activity were evaluated against laboratory-reared larvae of an. stephensi according to standard method of who. after susceptibility test, results were analysis using probit program. results: essential oils were separated from h. persicum, f. vulgare and c. sativum plants and their larvicidal activities were tested. result of this study showed that f. vulgare oil was the most effective against an. stephensi with lc50 and lc90 values of 20.10 and 44.51 ppm, respectively. conclusion: all three plants essential oil can serve as a natural larvicide against an. stephensi. f. vulgare oil exhibited more larvicidal properties. keywords: malaria, apiaceae, vector, anopheles stephensi introduction mosquitoes play an important role in transmission of some human diseases such as malaria, dengue fever, yellow fever and filariasis, which consider them among the greatest health problems across the glob (james 1992). anopheles species are considered as vectors of human malaria, filariasis and certain arboviruses (sedaghat and harbach 2005). malaria is considered as one of the most important health problem in iran especially in southern parts. in the south parts of iran there are six anopheline vectors including anopheles culicifacies, an. stephensi, an. dthali, an. fluviatilis, an. superpictus, and an. pulcherrimus, (naddaf et al. 2003, vatandoost and moinvaziri 2004, vatandoost et al. 2004, 2005ab, hanafibojd et al. 2006, soltani et al. 2008, hanafibojd et al. 2010, vatandoost et al. 2006, 2007, 2009, 2011). anopheles sacharovi and an. maculipennis can transmit human malaria in northern part of the country (sedaghat et al. 2003a, 2003b, oshaghi et al. 2003, doosti et al. 2007). anopheles stephensi, an oriental malaria vector, is distributed in indo-persian area from india, pakistan and iran, to countries around the persian gulf (nagpal and sharma 1995). in iran, it occurs in the southern areas of the country in khuzestan, fars, kerman, *corresponding author: prof hassan vatandoost, e-mail: hvatandoost1@yahoo.com iran j arthropod-borne dis, 2011, 5(2): 51–59 mm sedaghat et al.: larvicidal activity of essential … 52 hormozgan, sistan and baluchistan and southern kermanshah provinces (manouchehri et al. 1976, sedaghat and harbach 2005). although there are several methods for control of anopheles mosquitoes however environmental effect and resistance is a main human concern. synthetic pyrethroids which considered as the most effective insecticides against anophelines, are still expensive and beyond the financial resources of some countries. using botanical insecticides is a technique which can apply as an alternative to synthetic chemical formulations. most botanicals are rapid acting and breakdown quickly in the environment. the extract of whole leaf and essential oil of some certain plants have been investigated against some public health pests (saxena and sumithra 1985, kumar and dutta 1987, chariandy et al. 1999, hadjiakhoondi et al. 2000 ab, markouk et al. 2000, hadjiakhoondi et al. 2003, tare et al. 2004, vatandoost et al. 2004, 2008, hadjiakhoondi et al. 2005– 2006, oshaghi et al. 2008 a). apiaceae (umbelliferae) is one of the best known families of flowering plants, which comprise 300–450 genus and 3000–3700 species (constance 1971, pimenov and leonov 1993). they are aromatic plant and have a distinctive flavor which diverse volatile compounds from the fruits and leaves. this family also encompasses toxic plants that some had used in ancient athens to execute those sentenced to death (constance 1971, pimenov and leonov 1993). heracleum persicum desf., known as persian hogweed or “golpar”, is an annual native plant native to iran with a wide distribution across the country. it is used in traditional medicine because of its antioxidant and anticonvulsant activity (parsa 1948, souri et al. 2000, sayyah et al. 2005). foeniculum vulgare mill., known as fennel or “razianeh” in iran and neighboring countries, has been used by humans since antiquity. it is generally considered indigenous to the shores of the mediterranean, but now can be found in many parts of the world. (muckensturm et al. 1997). the bioactivities of this plant have been investigated for its antioxidant activity (oktaya et al. 2003) fumigant activity (kim et al. 2003), antimicrobial activity (ruberto et al. 2000), larvicidal activity (chantraine et al. 1998, pitasawat et al. 2007), insecticidal activity (laurent et al. 1997), and acaricidal activity (lee 2004). coriandrum sativum l, known as coriander, is native to iran, however it is widely distributed around the world. the seeds contain an essential oil (up to 1%) and the seeds are used in traditional medicine for indigestion, against worms, rheumatism and pain in the joints (wangensteen 2004). it has also the nematicidal activity (kim et al. 2008), antibacterial activity (cantore et al. 2004) and larvicidal activity (harve and kamath 2004). in recent years, much effort has been focused on the exploration of bioactive chemical compounds from indigenous plants for mosquito control in iran. so, the purpose of this study was to determine bioactivity of three essential oils obtained from the seeds of plants against 4th instar larvae of an. stephensi. materials and methods mosquito rearing the fourth-instars larvae of an. stephensi used for bioassays. the laboratory-reared an. stephensi bandar-abbas strain were reared in the department of medical entomology, tehran university of medical sciences, and maintained at 27◦ c with a photoperiod of 12 hours light and 12 hours e dark in 80±10% relative humidity. a 10% percentage yeast suspension was used as food source. plant materials in this study, the three plants from apiaceae (umbellifera) family were collected from three provinces of iran in 2009 (table 1). examined plants were authenticated and identified by the department of pharmacognosy, faculty of pharmacy, tehran univeriran j arthropod-borne dis, 2011, 5(2): 51–59 mm sedaghat et al.: larvicidal activity of essential … 53 sity of medical sciences. the seeds were air-dried at room temperature and kept in an air-tight light-protected container. dried seeds of h. persicum, f. vulgare and c. sativum were subjected to hydro distillation using a modified clevenger-type apparatus for 3 hours, dried over anhydrous sodium sulphate, and stored in amber-colored vials at 5°c until required for further work. table 1. collection locations, physical properties and yields of volatile oils derived from seeds of h. persicum, f. vulgare and c. sativum plant species location harvest date (2009) color appearance %yield (w/w) h. persicum mazandaran august pale yellow liquid 1.6 c. sativum chaharmahal and bakhtiary september pale yellow liquid 0.9 f. vulgare east azarbaijan september pale yellow liquid 0.8 bioassays and larval mortality fourth instar larvae of an. stephensi bandar-abbas strain was exposed to test concentrations of 5, 10, 20, 40, 80,160 and 320 ppm of essential oil for 24 hours according to standard method described by who (1981). as the oils do not dissolve in water, they were first dissolved in ethanol. the test 400 ml glass beaker were used by adding 1 ml of appropriate dilution of essential oil in ethanol and mixed with 249 ml of water to make up 250 ml of test solution (dharmagadda et al. 2005). in control beakers, ethanol was applied into the water (1%). a minimum of 20 larvae per each concentrations were used for all the experiments. the dead larvae were counted after 24 hour recovery period, and percentage of mortality was reported from the average for the five replicates. the larvae considered dead were those that did not move when touched with a needle. statistical analysis the lethal concentrations (lc50 and lc90) were calculated using probit analysis (finney 1971). for all bioassays, the percentages of mortality were adjusted for the mortality in controls by using abbott's correction (abbot 1925). differences between means were considered significant at p≤ 0.05 (sas institute 2001). results hydro distillation of h. persicum, c. sativum and f. vulgare yielded (w/w, dry weight) of volatile oils (table 1). the highest yield of volatile oil was obtained from h. persicum, whereas that of f. vulgare was the lowest. the larvicidal activities of the essential oils against an. stephensi larvae under laboratory conditions are given in table 2. all plants oil exerted significant larvicidal potential against an. stephensi after exposure for 24 h (table 2). the lethal dosage of 50% (lc50) ranged between 20.10 and 120.95 ppm. foeniculum vulgare oil was the most effective against an. stephensi with lc50 and lc90 values of 20.10 and 44.51 ppm, respectively, while c. sativum had the least mortality with lc50 and lc90 values of 120.95 and 389.90 ppm, respectively. this was also 5 times lower than h. persicum, which showed an lc50 of 104.80 ppm. in regression line a positive correlation were observed between the essential oil concentrations and the percent mortality (fig. 1). iran j arthropod-borne dis, 2011, 5(2): 51–59 mm sedaghat et al.: larvicidal activity of essential … 54 table 2. parameters of probit regression line anopheles stephensi to essential oil derived from h. persicum, f. vulgare and c. sativum seeds at different interval concentrations specimens a b±se lc50 , 95% c.i. lc90, 95% c.i. x2 (df) p value c. sativum -5.25 2.52±0652 47.68 120.95 479.9 181.62 389.90 181172.4 43.82 (3) <0.05 h. persicum -11.73 5.81±1.725 26.30 104.80 336.90 114.40 174.22 1073788.4 41.85 (3) <0.05 f. vulgare -4.83 3.71±1.023 6.05 20.10 67.62 24.30 44.51 15839.5 53.6 (3) <0.05 fig. 1. probit regression line of an. stephensi exposed to different interval concentrations of h. persicum, f. vulgare and c. sativum seed essential oils discussion the use of plant essential oils in vector control is an alternative method for minimizing the side effects of chemical pesticides on the environment (fatope et al. 1993). in recent surveys, it has been found that some of secondary plant metabolites act as botanical insecticides (watanabe et al. 1993, vatandoost et al. 2004, nathan 2007). according to the biological results of present study, all of three plant oils exerted significant larvicidal potential against an. stephensi and the essential oil of f. vulgare had an approiran j arthropod-borne dis, 2011, 5(2): 51–59 mm sedaghat et al.: larvicidal activity of essential … 55 priate effect. a study in bolivia reported that essential oil of f. vulgare, was one of the most toxic oils on ae. aegypti larvae. also its lc50 and lc90 were 24.3 and 30.8 ppm, respectively (chantraine et al. 1998). in the other study in lebanon for assay of repellency and toxicity of aromatic plant extracts against the mosquito culex pipiens form molestus, results showed that f. vulgare was one of the most significant repellency against this species (52.0 min of protection with concentration of 3%) also the lc50 and lc90 from essential oil of f. vulgare was 24.5 and 34 mg/l respectively (traboulsi et al. 2005). in several study on chemical compositions of plants by using of gas chromatography and mass spectrometry (gc/ms), results showed that different compounds were present in different parts of the plants. for example, in the study on seeds essential oil of h. persicum it is found that it contains hexyl butyrate (35.5%), octyl acetate (27%) and hexyl isobutyrate (3.2%) (sefidkon et al. 2004). in the other study on the leaves of c. sativum, matasyoh et al (2009) reported four main compounds, including: 2e-decenal (15.9%), decanal (14.3%), 2e-decen-1-ol (14.2%) and n-decanol (13.6%). in the two same study on fruit and flower of f. vulgare , the transanethole (85.63%), estragole (5.27%) and dlimonene (3.8%) were present in the fruit (dadalioglu and evrendilek 2004), while other report showed limonene (63.6%), anethole (25.5%), fenchyl acetate (2.6%), α-pinene (0.97%), myrcene (0.98%) and estragole (1.1%) from flower (traboulsi et al. 2005). it seems that the larviciding effect of these essential oils of plant is attributed to these main compounds. we propose test different components of the plants against mosquitoes. results of the current study showed that the essential oil of these plants exhibited the biological effect on the larvae of an. stephensi. the use of botanical pesticide may help in reducing the environmental side effects by the synthetic insecticides. the results obtained suggest that the essential oils of h. persicus, f. vulgare and c. sativum are promising as larvicide against an. stephensi. moreover, these results could be useful in the search for newer, more selective, and biodegradable larvicidal natural compounds. further investigations are currently underway to isolate these compounds. acknowledgements the authors would like to appreciate very much for kind collaboration of the department of medical entomology and vector control, school of public health, tehran university of medical sciences (tums) for providing the test mosquito, an. stephensi. this work was supported by grants from the centre of 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mosquito repellent from eucalyptus camaldulensis. j agri food chem. 41: 2164–2166. who (1981) instructions for determining susceptibility or resistance of mosquito larvae to insecticides. who/vbc. 81: 807. j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 76 http://jad.tums.ac.ir published online: march 18, 2018 original article situational analysis of cutaneous leishmaniasis in an endemic focus of the disease, southeastern iran sajjad fekri 1, *ahmad ali hanafi-bojd 2, yousef salari 3, parivash davoodian 1, reza safari 3, habib dadvand 1, mohsen mohebbi 4, hossein issazadeh 5, zahra kamali 1 1infectious and tropical diseases research center, hormozgan university of medical sciences, bandar abbas, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of diseases control, hormozgan university of medical sciences, bandar abbas, iran 4department of diseases control, hormozgan university of medical sciences, qeshm, iran 5department of diseases control, hormozgan university of medical sciences, jask, iran (received 17 apr 2017; accepted 21 feb 2018) abstract background: leishmaniasis is one of the most important vector-borne diseases in iran, existing in a variety of forms ranging from cutaneous to visceral forms. jask county has been recognized as an endemic focus of the disease in the southeastern region of iran. this study analyzed the situation of cutaneous leishmaniasis (cl) during 2006–2014. methods: this cross-sectional analytical study was conducted on cl data got from health sector. arcgis 10.3 was exploited for the spatial analysis of cl. potential high-risk areas of the disease regarding its' current geographical distribution were considered. results: overall, 874 cl cases had been registered in the district health center, implying an average incidence of 162.5per 100000. more than 90% of the cases emerged from rural areas. the disease is geographically distributed in the southeastern regions of jask county. over one-third of the total study area can be classified as high-risk areas, involving 61 villages with a total population of about 18000. remarkably, altitude and total precipitation were realized to play key roles in cl transmission within the study area. conclusion: although the national protocol for the control of zcl recommends the substantial destruction of rodent colonies serving as reservoirs of the disease in infected foci, critical improvement of the knowledge of the residents in these areas is crucial for community-based management of the disease in jask county. keywords: cutaneous leishmaniasis, epidemiology, spatial analysis, iran introduction leishmaniasis is a neglected disease which exists in three forms worldwide, cutaneous, visceral and mucocutaneous. it is among the 10 most important parasitic diseases in the tropics, and several dimensions of research supported by who have been conducted on various aspects of the disease (1). the disease is widely distributed in 90 countries, and its daly is estimated as 2400000yr (2). nearly 90% of cutaneous leish maniasis in the world is originating from afghanistan, algeria, brazil, iran, peru, saudi arabia and syria, and the highest incidence of the disease occurs in afghanistan and iran (3). in iran, the disease is commonly manifested in 17 out of the 31 provinces of the country, with estimated annual cases exceeding 20000 within the country (4). there has been an increasing trend of the prevalence of leishmaniasis in iran, and provinces including yazd, bushehr, khorassan-e-razavi, fars, ilam, khuzestan, and esfahan have recorded the highest prevalence of the disease with many endemic foci (5). *corresponding author: dr ahmad ali hanafi-bojd, e-mail: aahanafibojd@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 77 http://jad.tums.ac.ir published online: march 18, 2018 the spatial distribution of cl depends highly on environmental factors, not excluding socioeconomic circumstances such as poverty and the knowledge of the entire populace concerning the transmission and prevention of the disease (6). extensive drought occurrence within the last two decades, mostly in the eastern, central and southern parts of iran, coupled with the development of urbanization and the extension of localized agricultural activities in some other areas, have provided suitable conditions for the transmission of the disease because of the proximity between human, reservoir hosts, and vectors. phlebotomus papatasi is identified as the main vector of zoonotic cutaneous leishmaniasis (zcl), although ph. salehi has been infected with l. major, the causative agent of zcl, in some areas (7, 8). gerbils (rodentia: gerbillidae) are the main reservoir hosts of the diseases in iran (9). earlier, in the jask county, southeastern iran, p. papatasi and meriones hurrianae were reported as the vector and the reservoir of cl respectively, and have detected l. major infection in gerbillus nanus (10). recently, this county has been targeted as the chief endemic focus of zoonotic cutaneous leishmaniasis (zcl) in the hormozgan province of iran. although no mortality associated with cl has been recorded so far, the disease is heavily problematic for the inhabitants living in endemic areas due to the debilitating and disfiguring effect of the disease, and the long latency period once the disease is established. regardless of the pervasive efforts to prevent the disease, no potent vaccines have been developed for the disease, leaving its treatment complicated (11). the best alternatives for controlling the disease are environmental management adoption, personal protection and the control of rodents (reservoir) of the disease (12). as a zoonotic vector-borne disease, leishmaniasis is greatly affected by environmental and socioeconomic factors which play central roles in the disease cycle. using geographical information system (gis) these factors can be investigated to detect the high-risk areas of cl infection, and to rank effective measures in controlling the disease (13, 14). recently, gis has been applied in similar studies to model the probability of presence for the vectors (8) and reservoirs (9) of zcl, and to produce a risk map for the disease using gathered data of the disease and associated environmental factors (14-16). gis-based techniques are potentially beneficial to manage zcl in different settings. considering the current significant situation of cutaneous leishmaniasis in the jask county, the present study was aimed at assessing the epidemiology of the disease and analyzing its potential risk of infection. materials and methods jask county, encompassing a landmass of 11141km2, is located in the eastern quarter of the hormozgan province, southeastern iran (fig. 1). the geographical coordinates of the county lie between 57o10'-59 o 16' e and 25o23'–26o13' n. as at 2015, the population of jask county had reached 53770. the county comprises 164 residential villages. the weather of this county is described as being warm and dry in warmer summers and cold in temperate winters although the relative humidity across the coastal plain exceeds 50%. jask county is often designated as having a hot desert climate. topographically, this county has two distinct areas; a coastal plain mainly covered by the city of jask and most villages bordered with the oman sea to the south, and a hilly to mountainous area in northern part. data collection characteristic features of the cases of cutaneous leishmaniosis referred to the appropriate health division for treatment were documented in special forms designed by the ministry of health. in this descriptive-analytical study, necessary data were passively extracthttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 78 http://jad.tums.ac.ir published online: march 18, 2018 ed from the above-mentioned forms during 2006–2014. data were later transferred to an excel spreadsheet and the related charts and tables were computed using this software. ethical consideration this study has been ethically approved by the infectious and tropical diseases research center of the hormozgan university of medical sciences in bandar abbas, iran. the confidentiality of the records of patients was assured. spatial analysis data on the cases of cutaneous leishmaniasis were identified in specific inhabited places of the villages displayed on the base maps provided by the national cartography organization. shapefiles of the distribution of cl-positive cases were prepared and classified using arcgis 10.3. raster layers of annual average of mean daily temperature (°c) and relative humidity were prepared by inverse distance weighted (idw) analysis at spatial resolution of 50m2/pixel, using the data provided by different synoptic stations, including jask and 8 other nearby cities. these data were collected by the national meteorological organization for research in the country. kriging and co-kriging are geostatistical techniques used for interpolation (mapping and contouring) purposes. both methods are generalized forms of univariate and multivariate linear regression models, for estimation at a point, over an area, or within a volume. they are linear-weighted averaging methods, similar to other interpolation methods, however, their weights depend not only on distance but also on the direction and orientation of the neighboring data to the unsampled location. co-kriging can be seen as a point interpolation, which requires a point map as input and which returns a raster map with estimations and optionally an error map. co-kriging is a multivariate variant of the ordinary kriging operation: co-kriging calculates estimates or predictions for a poorly sampled variable (that we want to predict; in this study: cl cases) with help of a well-sampled variable (the covariable). the variables should be highly correlated (positive or negative). therefore, we used cl cases in the study area as sampled variable (17). some variables such as mean temperature, total precipitation, altitude and normalized difference vegetation index (ndvi) were highly correlated with cl incidence. therefore, we used the first three independent variables for prediction the risk of cl as dependent variable. the prediction map was classified using natural breaks into five classes for infection chance. results a total of 874 cl cases were discovered by the health center of jask district during the study period. the highly infected age group belonged to 10–19yr, with a frequency of 21.4% (table 1). the majority of cases which reported from the 87 residential places came from the rural areas (91%). analysis of the lesions on the different parts of the body showed 62.3% of the lesions occurred on the hand/ foot, 28.6% were presented on the face, and 9.1% on the remaining parts of the body. the number of lesions present was also counterchecked, and most of the patients (56%) suffered one lesion each on their body, followed by two (22.4%), and more than 2 lesions (21.6%). residents of rural areas of the county were more infected than those of urban areas, such that 91% of cases arose from the villages (table 1). spatial distribution of cl cases across the area showed the highest morbidity in the southeastern regions (fig. 2). overall, the disease was recorded in 87 localities, including villages and some quarters of the urban centers. cokriging analysis involving altitude, mean monthly temperature and total annual precipitation variables showed a positive autocorrelation between the disease and the total http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 79 http://jad.tums.ac.ir published online: march 18, 2018 precipitation and altitude, as presented in the experimental semivariograms (fig. 3). the distance where the model first flattens out is known as the range. locations separated by distances closer than the range in fig. 3 are spatially autocorrelated, whereas locations farther apart than range are not. the prediction map indicated the eastern parts of jask county, together with some northern areas of the county, are more susceptible to maintaining cl transmission (fig. 4). totally, 61 rural/urban areas were categorized as high-risk areas for cl transmission, with a total population of closely 18000. conversely, more than a third of jask county inhabitants are living in high-risk areas, while the risk of cl transmission is medium in more than 50 rural/urban areas. according to the model prediction for zcl in villages of the study area, the highest probability of the disease has predicted for southeastern area, followed by southwestern and central parts (fig. 5). table 1. some demographic information of patients infected by cutaneous leishmaniasis in jask county, southeastern iran, 2006–2014 year gender age groups(yr) residence place female male 0–4 5–9 10–19 20–29 30–39 40–49 ≤50 urban rural 2006 106 115 49 36 56 30 12 14 11 14 207 2007 119 117 50 46 51 36 21 12 20 27 209 2008 83 112 36 43 49 27 13 9 19 14 182 2009 41 64 26 24 19 16 9 7 4 11 94 2010 13 26 7 10 6 11 2 2 1 2 37 2011 10 15 5 9 4 4 3 0 0 6 19 2012 0 7 1 0 3 1 0 1 1 0 7 2013 15 15 12 5 7 2 3 1 0 0 30 2014 10 6 1 6 5 0 1 1 2 4 12 total 397 477 187 179 200 127 64 47 58 78 797 fig. 1. jask county in southeast of iran http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 80 http://jad.tums.ac.ir published online: march 18, 2018 fig. 2. spatial distribution of cutaneous leishmaniasis in jask county, southeast of iran cl and total precipitation cl and average of mean temperature cl and altitude fig. 3. experimental anisotropic semivariograms for cutaneous leishmaniasis x-axis is distance times 101 (in degree), y-axis is semivariance for cl cases located between specified distances http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 81 http://jad.tums.ac.ir published online: march 18, 2018 fig. 4. cokriging prediction for the risk of infection to cutaneous leishmaniasis in jask county, southeast of iran fig. 5. cokriging prediction for the risk of infection to cutaneous leishmaniasis in different villages of jask county, southeast of iran discussion jask county, in the southeastern region of iran, is an endemic focus of both malaria and leishmaniasis (18-21). although in recent years few indigenous cases of malaria have been reported from this area, cutaneous leishmaniasis is the main vector-borne infectious disease with an estimated incidence of 162.5per 100000 population. from 2006 to 2009, the disease erupted into an epidemic condition in some areas of the county, however, this situation was brought under control which led to a decreasing trend in the incidence of the disease after the year 2009. correspondingly, this decreasing trend was consistently observed na tionwide regarding cl infection in iran (5). a major determinant contributing to this trend is drought, because of climate change (22), a problem experienced by the country for several years. children were the most infected group in this county, as seen in other endemic foci of the disease throughout the country (5), and the commonly occurring cases were noticed in those belonging to 0–4 and 5–9yr age groups. the zoonotic form of cutaneous leishmaniasis has been known to be circulating in the jask county, and l. major has been detected in the sand fly vector (ph. papatasi) and gerbils (tatera indica and gerbilus nanus), as well http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 82 http://jad.tums.ac.ir published online: march 18, 2018 as human lesions (18, 19). this form of the disease has been reported in baluchistan area, bordered to the east of our study area. verily, zcl transmission is extended from baluchistan to jask, following an epidemic which occurred in the 2000s in some counties of baluchistan (from dashtyari to chabahar, and konarak) (23). another great concern in the epidemic of zcl in jask is that may occur because reducing vector control operations including indoor residual spraying (irs) in the active foci following a sharp drop in malaria cases (11). water transfer project at jagin dam, in the north of jask county, has been recently considered to develop agriculture in this county. considering the living habitat of t. indica, the main reservoir of zcl in the study nesting in barren lands around the crop fields, and the presence of suitable ecological niches for this species and other vectors in southern iran (8, 9) a new upsurge of the disease should be expected because the lack of adequate attention to preparedness and mitigation. our prediction for the risk of cl showed one-third of the landmass of the county has a higher potential for cl transmission. this is clear in our results whereby a population of about 18000 is living in 61 villages/urban areas which are at risk of zcl infection. cokriging is an accurate method for spatial interpolation. considering the co-variates that affect the transmission of a disease, targeting variables of the weather proven to correlate well with the disease transmission would be cost-effective, rather than focusing on field data collection and sampling. previously, the transmission of zcl was found to be dependent ona number of climatic factors including higher temperature, lower relative humidity, lower total rainfall, higher evaporation and lower number of rainy days (14, 24). thus, this analysis was used to cross-correlate environmental variables with the records of cl in predicting high-risk areas. experimental anisotropic semivariograms in our study showed increasing trend between the disease occurrence with total precipitation, negative trend between cl occurrence and mean temperature, while for altitude it had no increase or decrease. because the distances between sampling points had no significant differences regarding three environmental variables used in this study, there was no difference between sampling points (fig. 3). by the way, this model predicted eastern regions of the jask county are more vulnerable to establishing new endemic foci. likewise, this method has been used to estimate the habitat suitability for ixodes scapularis, the vector of lyme disease (25), boophilus microplus, a serious cattle pest (26), and dengue vector population (27). hence, this technique will be essential for later studies on leishmaniasis. according to the spatial correlation between three environmental variables used in this study, it is predicted cases of zcl will increase in some villages of jask county (fig. 5). besides, to prevent the disease expansion to the new areas, it is necessary to do some preventive measures such as community-based health education programs, environmental sanitation and personal protection against sand fly vectors. conclusion cutaneous leishmaniasis is an endemic disease in jask county and should be considered by health sector authorities, particularly in highrisk areas explored in this study. massive development projects begun in this area will provide job and trading activities for people emigrating from non-endemic areas of the, and from some other countries. this situation is likely to increase the morbidity associated with the disease due to the invasion of non-immunized immigrants. therefore, preventive measures along with health education and promotion will help curtail the risk of infection and morbidity. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 83 http://jad.tums.ac.ir published online: march 18, 2018 acknowledgements the authors are grateful to the staff of jask district health center for the kind collaboration in this study. this study has been financially supported by research deputy, hormozgan university of medical sciences, bandar abbas, iran, with project no. 9163. the authors declare that there is no conflict of interests. references 1. world health organization (2010) control of of the leishmaniasis. report of a meeting of the who expert committee on the control of leishmaniases. who technical report series. who, geneva. 2. world health organization (2004) the world health report 2004. changing history. who, geneva. 3. alvar j, velez id, bern c, herrero m, desjeux p, cano j, jannin j, den boer m (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7 (5): e35671. 4. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod borne dis. 6: 1–17. 5. shirzadi mr, esfahani sb, mohebali m, yaghoobi-ershadi mr, gharachorlo f, razavia mr, postigo ja (2015) epidemiological status of leishmaniasis in the islamic republic of iran, 19832012. east mediterr health j. 21(10): 736–742. 6. maroli m, feliciangeli md, bichaud l, charrel rn, gradoni l (2013) phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. med vet entomol. 27: 123–147. 7. karimi a, hanafi-bojd aa, yaghoobiershadi mr, akhavan aa, ghezelbash z (2014) spatial and temporal distributions of phlebotomine sand flies (diptera: psychodidae), vectors of leishmaniasis, in iran. acta trop. 132: 131–139. 8. hanafi-bojd aa, yaghoobi-ershadi mr, haghdoost aa, akhavan aa, rassi y, karimi a, charrahy z (2015) modeling the distribution of cutaneous leishmaniasis vectors (psychodidae: phlebotominae) in iran: a potential transmission in disease prone areas. j med entomol. 52(4): 557–565. 9. gholamrezaei m, mohebali m, hanafi-bojd aa, sedaghat mm, shirzadi mr (2016) ecological niche modeling of main reservoir hosts of zoonotic cutaneous leishmaniasis in iran. acta trop. 160: 44–52. 10. azizi k, moemenbellah-fard md, fakoorziba mr, fekri s (2011) gerbillus nanus (rodentia: muridae): a new reservoir host of leishmania major. ann trop med parasitol. 105(6): 431–437. 11. nadim a, javdian e, mohebali m, momeni az (2009) leishmania parasite and leishmaniasis. 3rd ed. markaz nashr daneshgahi press, tehran. 12. yaghoobi-ershadi mr, zahraei-ramezani ar, akhavan aa, jalali-zand ar (2005) rodent control operations against zoonotic cutaneous leishmaniasis in rural iran. ann saudi med. 25 (4): 309–312. 13. ali-akbarpour m, mohammadbeigi a, tabatabaee sh, hatam g (2012) spatial analysis of eco-environmental risk factors of cutaneous leishmaniasis in southern iran. j cutan aesthet surg, 5: 30–35. 14. seid a, gadisa e, tsegaw t, abera a, mulugeta a, herrero m, argaw d, jorge a, kebede a, aseffa a (2014) risk map for cutaneous leishmaniasis in ethiopia based on environmental fachttp://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=charrahy%20z%5bauthor%5d&cauthor=true&cauthor_uid=26335462 https://www.ncbi.nlm.nih.gov/pubmed/?term=argaw%20d%5bauthor%5d&cauthor=true&cauthor_uid=24893015 https://www.ncbi.nlm.nih.gov/pubmed/?term=jorge%20a%5bauthor%5d&cauthor=true&cauthor_uid=24893015 https://www.ncbi.nlm.nih.gov/pubmed/?term=kebede%20a%5bauthor%5d&cauthor=true&cauthor_uid=24893015 https://www.ncbi.nlm.nih.gov/pubmed/?term=aseffa%20a%5bauthor%5d&cauthor=true&cauthor_uid=24893015 j arthropod-borne dis, march 2018, 12(1): 76–84 s fekri et al.: situational analysis of … 84 http://jad.tums.ac.ir published online: march 18, 2018 tors as revealed by geographical information systems and statistics. geospatial health. 8(2): 377–387. 15. mollalo a, alimohammadi a, shahrisvand m, shirzadi mr, malek mr (2014) spatial and statistical analyses of the relations between vegetation cover and incidence of cutaneous leishmaniasis in an endemic province, northeast of iran. asian pac j trop dis. 4(3): 176– 180. 16. mollalo a, alimohammadi a, shirzadi mr, malek mr (2015) geographic information system-based analysis of the spatial and spatio-temporal distribution of zoonotic cutaneous leishmaniasis in golestan province, north-east of iran. zoonoses public health. 62(1): 18–28. 17. johnston k, ver hoef jm, krivoruchko k, lucas n (2001) using arcgis geostatistical analyst (vol. 380). esri press, redlands: california, p. 316. 18. azizi k, davari b, kalantari m, fekri s (2011) gerbillidae rodents fauna (muridae: gerbillinae) and detection of reservoir hosts(s) of zoonotic cutaneous leishmaniasis using a nested-pcr technique in jask city in hormozgan province in 2008. kordestan univ med j. 16: 66–76. 19. azizi k, kalantari m, fekri s (2011) the nested-pcr based detection of cutaneous leishmaniasis vectors in jask county, hormozgan, iran. iran j epidemiol. 7(3): 27–33. 20. fekri s, vatandoost h, daryanavard a, shahi m, raeisi a, omar as, sharif m, safari r, azizi a, ali aa, nasser a, hasaballah i, hanafi-bojd aa (2014) malaria situation in an endemic area, southeastern iran. j arthropod-borne dis. 8: (1): 82–90. 21. yeryan m, basseri hr, hanafi-bojd aa, raeisi a, edalat h, safari r (2016) bio-ecology of malaria vectors in an endemic area, southeast of iran. asian pac j trop med. 9(1): 32–38. 22. patz ja, graczyk tk, geller n, vittor ay (2000) effects of environmental change on emerging parasitic diseases. int j parasit. 30: 1395–1405. 23. kassiri h, javadian e, abdigoudarzi m (2011) natural leishmania infection in meriones hurrianae and tatera indica (rodentia: cricetidae: gerbillinae) in sistan-baluchistan province, southeastern of iran. adv stud biol. 3(6): 247–256. 24. shirzadi mr, mollalo a, yaghoobi-ershadi mr (2015) dynamic relations between incidence of zoonotic cutaneous leishmaniasis and climatic factors in golestan province, iran. j arthropod-borne dis. 9(2): 148–160. 25. estrada-pena a (2001) climate warning and changes in habitat suitability for boophilus microplus (acari: ixodidae) in central america. j parasitol. 87 (5): 978–987. 26. estrada-pena a (1998) geostatistics and remote sensing as predictive tools of tick distribution: a cokriging system to estimate ixodes scapularis (acari: ixodidae) habitat suitability in the united states and canada from advanced very high resolution radiometer satellite imagery. j med entomol. 35(6): 989–995. 27. azil ah, bruce d, williams cr (2014) determining the spatial autocorrelation of dengue vector populations: influences of mosquito sampling method, covariables, and vector control. j vec ecol. 39(1): 153–163. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 321–324 pg de mendonça: cross-genera pcr … 321 http://jad.tums.ac.ir published online: september 30, 2018 short communication cross-genera pcr amplification of dna from apicomplexan parasites *philippe gil de mendonça institute of comparative tropical medicine and parasitology, ludwig maximilian university, munich, germany (received 11 dec 2016; accepted 20 june 2018) abstract background: the discovery of an unexpected genetic sequence raised doubts about the specificity of a primer pair targeting babesia spp. and theileria spp. this study aimed to check the specificity of this primer pair. methods: conventional end-point pcr and real-time pcr protocols using primers targeting the 18s rrna gene v4 hypervariable region of babesia spp. and theileria spp. were tested for potential cross-genera amplification using dna from a palette of parasitic protists and pathogenic bacteria as a template. these investigations took place at the ludwig maximilian university of munich (germany) in 2010 as part of the eden project. results: successful amplification was obtained with dna from five apicomplexan genera: babesia, theileria, hepatozoon, toxoplasma, and hammondia. no amplicons were obtained when dna from leishmania infantum or bacteria within the genera borrelia, leptospira or anaplasma was used as a template. conclusion: this cross-genera amplification ability is useful for the quick exclusion of many parasite species from pcr negative diagnostic samples. accurate species identification from pcr positive samples requires genetic sequencing of the amplicon. keywords: babesia, theileria, hepatozoon, toxoplasma, hammondia introduction babesia spp. and theileria spp. are widespread tick-borne parasites. they are a cause of concern to the medical and veterinary community worldwide (1-10). several species are even considered as an emerging threat to human health (9-10). the eu-funded eden project focused specifically on investigating emerging diseases (11). within this context, arthropod vectors and vertebrate hosts were routinely sampled and screened for a large palette of zoonotic pathogens. a combination of techniques (12), namely pcr followed by reverse-line blot (rlb), was routinely used for screening babesia spp. and theileria spp. on various occasions, the panel of rlb probes available was not suitable for the identification of the parasite species involved. in such cases, amplicons were sequenced. sequencing revealed the presence of sarcocystis-like organisms (sequences did not match any known species in genbank, and identity values were relatively low). these results raised serious concerns about the specificity of the pcr protocol used for screening babesia spp. and theileria spp. in order to check for the actual presence of further cases of cross-genera amplification, the tests described below were performed using template dna from a variety of protists and other microorganisms. materials and methods pcr was performed with dna from reference samples of parasitic protists as a template. reference samples of parasitic protists included: babesia canis, b. capreoli, b. divergens, b. gibsoni, b. microti (syn. theileria microti), b. rodhaini (syn. theileria rodhaini), b. vogeli, hepatozoon sylvatici, toxoplasma *corresponding author: dr philippe gil de mendonça, e-mail: pgm@cantab.net http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 321–324 pg de mendonça: cross-genera pcr … 322 http://jad.tums.ac.ir published online: september 30, 2018 gondii, hammondia hammondi and leishmania infantum. pcr was also performed with dna from wildlife samples (arthropod vectors and vertebrate hosts) known to be positive for a palette of bacterial pathogens. these bacterial pathogens included borrelia afzelii, leptospira interrogans and anaplasma phagocytophilum. pure water was used as no-template control. dna was amplified using primers (f 5’gacacagggaggtagtgacaag and r 5’-ctaagaatttcacctctgacagt) targeting the 18s rrna gene v4 hypervariable region of babesia spp. and theileria spp. (13). real-time pcr with dissociation curve analysis was performed with quantifast sybr® green (qiagen, hilden, germany) following the manufacturer’s instructions. conventional end-point pcr followed a modified touch-down protocol (14). one cycle at 95 °c for 5min for hot start taq activation was followed by touch-down steps (denaturation at 94 °c for 20sec, annealing for 30 sec starting at 67 °c and dropping to 57 °c at a rate of 1 °c/step, extension at 72 °c for 30sec) prior to 40 amplification cycles (denaturation at 94 °c for 20sec, annealing at 57 °c for 30sec, extension at 72 °c for 30 sec). reaction mix composition was: 5.00μl 10x qiagen pcr buffer (at 15mm mgcl2), 2.00μl qiagen mgcl2 (at 25mm), 1.00μl qiagen dntps (10mm), 1.00μl of each primer (10μm), 29.75μl ultra-pure water, 0.25μl qiagen hot start taq plus (5u/μl). total volume was 50μl, including 10μl template dna. pcr products were separated by electrophoresis through 1.5% agarose gels stained with gelred® (biotium, hayward, california). size markers (reference dna ladder) were included in each gel. amplicons and size markers were visualized by uv transillumination. amplicon sizes were estimated with gelanalyzer 2010 (www. gelanalyzer.com/download.html). these investigations took place at ludwig maximilian university, munich, germanyin 2010 as part of the eden project. sequencing of amplicons was performed by eurofins mwg gmbh (ebersberg, germany). results as expected, all babesia spp. and theileria spp. yielded amplicons with strong positive signals on both conventional and real-time pcr, and their sequences were consistent with data available on genbank, thus confirming successful and accurate pcr amplification of babesia and theileria reference samples. hepatozoon sylvatici, toxoplasma gondii, and hammondia hammondi also yielded amplicons with strong positive signals on both conventional and real-time pcr, and their sequences were also consistent with data available on genbank, thus also confirming successful and accurate pcr amplification of hepatozoon, toxoplasma and hammondia reference samples. no amplicon could be obtained from l. infantum. no amplicon was obtained either from any of the bacterial pathogens tested. melting temperature values for the various species successfully amplified ranged from 79.8 °c to 85.7 °c with overlap between various species. amplicon sizes ranged from 402bp to 528bp with overlap between various species. discussion the taxonomy of protists has undergone many revisions and has been revolutionized from top to bottom in the last few years (15, 16). the taxonomy of the species within the genus babesia is no exception to this rule (17, 18). however, for the sake of simplicity, a traditional approach was retained in the present article which is concerned with molecular diagnostic methodology, rather than taxonomic changes. the pcr primer pair used in the present http://jad.tums.ac.ir/ http://www.gelanalyzer.com/download.html j arthropod-borne dis, september 2018, 12(3): 321–324 pg de mendonça: cross-genera pcr … 323 http://jad.tums.ac.ir published online: september 30, 2018 study was known for its ability to detect two closely related protist genera: babesia and theileria. the above-mentioned results revealed the ability to detect yet further protist genera, namely hepatozoon and the closely related toxoplasma and hammondia. all these genera belong to the apicomplexa, which suggests that the pcr primer pair used here might amplify even more genera within this phylum. this unexpected widespread crossgenera amplification ability might seem problematic at first glance, however, this turns out to be a rather useful property when considered in a wider context. indeed, screening with this primer pair makes it possible to exclude a large number of parasitic species at once for those samples yielding negative pcr results, thus avoiding expansive and time-consuming further testing for the numerous species within the genera known to be efficiently amplified by this peculiar primer pair. as a corollary to this, it will be most useful to perform yet further testing of additional genera within the apicomplexa in order to establish the full width of the palette of genera efficiently amplified and thus detected when screening samples with this primer pair. additional testing of protist genera outside the apicomplexa would also be desirable. most importantly, due to widespread overlaps in amplicon sizes and melting temperatures on the one hand, and due to limitations of the rlb technique on the other hand, genetic sequencing of the amplicon is required for accurate species identification. conclusion the range of detection of the primer pair targeting the 18s rrna gene v4 hypervariable region of babesia spp. and theileria spp. is not limited to these two closely related genera. at least three additional apicomplexan genera are efficiently amplified by this primer pair. these genera are hepatozoon, toxoplasma, and hammondia. genetic sequencing of the amplicons is required for accurate species identification. acknowledgments this study was partially funded by eu grant goce-2003-010284 eden. the contents of this publication are the sole responsibility of the author and do not necessarily reflect the views of the european commission. additional support was provided by the bavarian ministry for environment and public health (vicci project module 3). it is a pleasure to thank (in non-preferential alphabetical order) l chaudhuri and c thiel who kindly made available reference samples of leishmania infantum, babesia gibsoni and b. vogeli. the author declares that there is no conflict of interest. references 1. aktas m, altay k, dumanli n (2005) development of a polymerase chain reaction method for diagnosis of babesia ovis infection in sheep and goats. vet parasitol. 133(4): 277–281. 2. bourdoiseau g (2006) canine babesiosis in france. vet parasitol. 138(1– 2):118–125. 3. chaudhry zi, suleman m, younus m, aslim a (2010) molecular detection of babesia bigemina and babesia bovis in crossbred carrier cattle through pcr. pakistan j zool. 42(2): 201–204. 4. esmaeilnejad b, tavassoli m, asri-rezaei s, dalir-naghadeh b, 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2011, 5(1): 54–62 ah shiravi et al.: temperature requirements of … 54 original article temperature requirements of some common forensically important blow and flesh flies (diptera) under laboratory conditions ah shiravi1, r mostafavi1, k akbarzadeh2, *ma oshaghi2 1department of biology, islamic azad university, damghan branch, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, iran (received 11 apr 2011; accepted 30 jun 2011) abstract background: the aim of his study was to determine development time and thermal requirements of three myiasis flies including chrysomya albiceps, lucilia sericata, and sarcophaga sp. methods: rate of development (rod) and accumulated degree day (add) of three important forensic flies in iran, chrysomya albiceps, lucilia sericata, and sarcophaga sp. by rearing individuals under a single constant temperature (28° c) was calculated using specific formula for four developmental events including egg hatching, larval stages, pupation, and eclosion. results: rates of development decreased step by step as the flies grew from egg to larvae and then to adult stage; however, this rate was bigger for blowflies (c. albiceps and l. sericata) in comparison with the flesh fly sarcophaga sp. egg hatching, larval stages, and pupation took about one fourth and half of the time of the total pre-adult development time for all of the three species. in general, the flesh fly sarcophaga sp. required more heat for development than the blowflies. the thermal constants (k) were 130–195, 148–222, and 221–323 degree-days (dd) for egg hatching to adult stages of c. albiceps, l. sericata, and sarcophaga sp., respectively. conclusion: this is the first report on thermal requirement of three forensic flies in iran. the data of this study provide preliminary information for forensic entomologist to establish pmi in the area of study. keywords: degree day, forensic entomology, larval development, myiasis, pmi introduction determination of postmortem interval (pmi) or the time between death and the discovery of a corpse is the most important application of forensic entomology. flies belong to the families calliphoridae (blow flies) and sarcophagidae (flesh flies) are often the first insects to arrive on a corpse where their larvae feed and breed effectively (anderson 2001, dadour et al. 2001, higley and haskell 2010). development rates of these flies are frequently used to estimate pmi in homicide investigations in the first few weeks after death. since development of immature insects is temperature-dependent, pmi is normally calculated by the accumulated degree day/ hour (add/adh) model (measure of thermal time taken to reach each developmental event, k) which is associated with basal temperature called the lower temperature threshold (tl) or the developmental zero (dz) (temperature below which development ceases) values (nietschke et al. 2007, http://www.ipm. ucdavis.edu/models, oshaghi et al. 2009). the rate of larval growth depends on its body temperature, which is directly affected by environmental conditions as ambient tem*corresponding author: dr mohammad ali oshaghi, email: moshaghi@tums.ac.ir iran j arthropod-borne dis, 2011, 5(1): 54–62 ah shiravi et al.: temperature requirements of … 55 perature and the heat generated by maggot aggregations (slone and gruner 2007). in addition, an important detail for pmi determination is that each species has its own temperature dependent growth rate. generally life cycle of blow and flesh flies includes four stages of egg, larval, pupal and imago. three instars can be seen in the larval stage: 1st, 2nd and 3rd instars, where the latter is divided to feeding and post-feeding larvae (day and wallman 2008). the flies deposit egg directly on the food substrate to ensure a food supply for the hatching 1st instar larvae. the three instars can be distinguished by the number of respiratory slits at the posterior end of the larvae. the third instar stage lasts longer than the first two ones, and the post-feeding stage is a preparation phase for pupation (reibe et al. 2010). about one third of the pre-adult development time is spent in the post-feeding larval stage (greenberg 1991). therefore, the larvae leave the food source to find a suitable place for pupation, emptying their gut (arnott and turner 2008). during pupation stage the imago develops within the pupal case till eclosion and takes about half of the time of the total development (reibe et al. 2010). blow flies such as chrysomya albiceps (wiedemann) and lucilia sericata (meigen) (diptera: calliphoridae) and flesh flies such as sarcophaga spp. (diptera: sarcophagidae) are widely distributed throughout the world and characterized as a facultative ectoparasites responsible for primary or secondary myiasis in humans and livestock (zumpt 1965, smith 1986, hall and wall 1995, anderson, 2000, grassberger et al. 2003). these species are considered as sinantropic species, i.e. it is in close relation with human settlements. they also feed on carrion and human feces, and breeds prolifically in carrion, making them medically, veterinarily, sanitationarily, and forensically important flies (zumpt 1965, grassberger et al. 2003). they are recognized as the first wave of the faunal succession on human cadavers and are the primary and most accurate forensic indicators of time of death. lucilia sericata also has a significant role in human medicine because its larvae are used for healing chronic injuries that do not respond to conventional treatments, such as ulcers containing gangrenous or necrotic tissue (church and courtenay 2002, cartier and combemale 2008, gupta 2008). the biology of blow flies and flesh flies have been studied previously on many aspects such as age, mortality rates and reproductive output, thermal requirements, diet, minimum and maximum threshold temperatures, and development duration under field or laboratory conditions (kamal 1958, denlinger 1972, aspoas 1991, greenberg 1991, smith and wall 1998, hayes et al. 1999, anderson, 2000, grassberger and reiter 2001, almisned 2003, pitts and wal 2004, farkas et al. 2005). however, there has been less research on life table parameters carried out with samples of c. albiceps, l. sericata, and sarcophaga sp. from an oriental country like iran. due to the recent increase of forensic entomology utilized in iran, and the importance of an understanding of the rate of development in relation to temperature, detailed development data are needed to allow more precise postmortem interval (pmi) estimates. in this paper, we present development rate of these three important forensic flies in iran (fig. 1), at a single constant temperature and calculate the add/k required for four developmental events including egg hatching, larval stages, and pupation, and egg-eclosion. we compared the k of our findings to those of published data to explore the disparities between the studies. these information can also support the mass rearing of them under laboratory conditions to be used in maggot therapy (in case of l. sericata) and can also contribute to the monitoring and providing control strategies of these dipteran flies. iran j arthropod-borne dis, 2011, 5(1): 54–62 ah shiravi et al.: temperature requirements of … 56 materials and methods sample collection adult flies were collected using plastic bottle fly traps and entomological nets at the laleh park in center of tehran as well as the livestock shopping center and close vicinity of slaughter houses in the east and south of tehran, iran. the traps were made by cutting the top of a plastic water bottle, placing some sands (3 cm) into the bottom of the bottle, putting some raw sheep/chicken liver (100 g) on top of the sand, and then inverting the top of the bottle into the bottom, and tape the two halves together. larval collection from natural infestations of sheep and cattle were performed, but it was unsuccessful because of insecticide application on the animals. the captured flies in entomological nets immediately transferred into glass jars. the jars or baits harboring flies were then transported in a polystyrene icebox to the laboratory of medical entomology, school of public health, tehran university of medical sciences. insect collections were carried out from late spring to early summer 2010, beginning in mornings and continued until a sufficient number of specimens for the colonization process had been captured. maintenance of flies in the laboratory adult were transferred individually into a bottle trap including sand and meat to lay eggs at 28° c±1, 40%±5 relative humidity and 12 h photoperiodicity, protected with an external net curtain to avoid the entry of other insect species. after laying eggs, the dead specimens were identified morphologically by using the taxonomic keys of james 1947, zumpt 1965, mcalpine 1981, and whitworth 2006. determination of development rate and add (k) groups of 30 plastic bottles were placed in incubators set at the specified constant temperature (28° c±1). the bottles were ventilated daily, and moved within the incubators to minimize the effect of any systematic temperature gradients. life cycle duration of l. sericata, c. albiceps, and sacophaga sp. were determined over two consecutive generations by recording the average time in days, for different stages of each species. recording the time required for egg hatching, larval stage developments, and pupation, and total time for egg-eclosion was performed every three hours intervals for eggs and every five-six hours intervals for larvae and pupa. on each recording occasion, at least five bottles checked on a light microscope. the rates of development (rod) were measured for each life stage by inversion of developmental duration (rod=1/day). baseline temperature or lower threshold temperature (dz) for each developmental stage of the species was obtained from previous studies (table 1). because the dz values for each species varied between studies, in this study we used an average value wherever more than one data set was available. hence, the average estimates of lower developmental thresholds of c. albiceps prepared by marchenko (1988), queiroz (1996), grassberger et al. (2003), and richards et al. (2008) was accounted for egg (9.72° c), larvae (12.93° c), pupae (12.55° c), and egg to adult (11.73 o c). the estimates of the basal temperature of l. sericata for whole life stage (egg-adult) was accounted 8° c (reibe et al. 2010), 8.2° c, 11.3° c (woodburn et al. 1978), and 9° c (marchenko 2001, niederegger et al. 2010). therefore, in this study the average 9.5° c was used for l. sericata. for sarcophaga sp. we used the only information available for a closely related flesh fly, sarcophaga dux thomson (al-misned 2003). estimates of the lower developmental threshold temperatures for this species were 5.9, 12.9 and 11.0 oc for larvae, pupae and total developmental time, respectively. iran j arthropod-borne dis, 2011, 5(1): 54–62 ah shiravi et al.: temperature requirements of … 57 based on the dz and duration of the development, the add/k for each developmental stage of the dipteran flies was calculated using the formula add or k=d (tmdz) where k=degree days (oc), d= developmental duration (days), tm=the ambient (experimental) temperature (oc), and dz= base development threshold (oc) (higley and haskell 2010). results generally, the rates of development changed in a decreasing order respectively in eggs, larvae, and pupae of the three species at the laboratory condition (28° c and 40% rh). this value was 0.33–0.50, 0.33–0.50, and 0.16– 0.25 for eggs, larvae, and pupae of blowflies accordingly. development rates for sarcophaga sp. were lower than the blowflies. these rates for sarcophaga sp. were 0.33–0.50, 0.16–0.20, and 0.10–0.16 for eggs, larvae, and pupae respectively. life cycle durations of the dipteran flies was determined over two consecutive generations, and recording the average time in days, for different stages in the three species (table i). the development duration of eggs of both blowflies (l. sericata and c. albiceps) and flesh fly (sarcophaga sp.) was similar but the development duration of larval and pupal stages in the blowflies were shorter than the flesh fly. life cycle span from egg to eclosion in blowflies ranged from eight to twelve days whereas it was ten to sixteen days in the flesh fly. egg hatching and larval stages prolonged each about one fourth of the total pre-imago time whereas duration of pupation took almost half of the time of the total pre-adult development time for all of the three species. in general, the flesh fly sarcophaga sp. required more heat for development than the blowflies. the thermal constants (k) or accumulation degree-days required for egg hatching was 37–56, 37–55, and 44–66 degree-days (dd) for l. sericata, c. albiceps, and flesh fly sarcophaga sp. respectively. at the larval stage, c. albiceps with 30–45 dd required less heat than l. sericata with 37–56 and sarcophaga sp. with 44–66 dd. for development of pupa, again c. albiceps with 62– 93 dd required less heat than l. sericata with 74–111 dd and sarcophaga sp. with 91–151 dd. although, total heat requirement of c. albiceps to develop from egg to adult (130–185) was less than the requirement of l. sericata (148–222 dd), but total developmental time from egg to adult for both species was similar (8–12 days). it was 13– 19 days and 221–323 dd for the the flesh fly. table 1. development duration and thermal requirements (k/add) for four developmental events for l. sericata, c. albiceps and sarcophaga sp. life stage development duration (day) l. sericata c. albiceps sarcophaga sp. add/k (dd=°c) l. sericata c. albiceps sarcophaga sp. egghatching 2–3 2–3 2–3 37–56 37–55 44– 66 larvae (l1-l3) 2–3 2–3 5–6 37–56 30–45 44– 66 pupae 4–6 4–6 6–10 74–111 62–93 91–151 egg-adult 8–12 8–12 13–19 148–222 130–195 221–323 iran j arthropod-borne dis, 2011, 5(1): 54–62 ah shiravi et al.: temperature requirements of … 58 fig. 1. photographs of chrysomia albiceps, lucilia sericata, and sarchophaga sp. from left to right respectively discussion results of this research showed highly differences for development rate of accumulated degree days required for blow flies (c. albiceps and l. sericata) in comparison with flesh fly sarcophaga sp. in addition, rate of developments in various life stages was different and reduced gradually throughout the life from eggs to adult stage in all of the three species. the speed of development in the flesh fly was slower than blowflies. this finding is very important in forensic entomology and pmi where sarcophaga sp. presents. total developmental time of sarcophaga sp. from first -instar larvae to adult emergence reared at 28° c was 13-19 days which is similar to the total development time (16.4 day) of sarcophaga dux reared at the same temperatures (28° c) (al-misned 2003). total developmental time of c. albiceps from egg to adult was 8-12 days when reared at 28° c, which is similar to those (9.5–10.5 days), reported by marchenko (2001), al-misned et al. (2003), and grassberger et al. (2003) reared at temperatures 30° c. the duration of the life cycle described in this research for l. sericata, from egg to adult was 8–12 days which is more or less shorter in comparison to data provided by rueda et al. (2010) (14 days), anderson, (2000) (14 days at 27° c), usaquén and camacho (2004) (26 days under natural environmental conditions), nuorteva, (1977) (23–28 days under field conditions), and anderson, (2000) (32 days at 16o c and 20 days at 21°c), and somehow similar to the information by kamal (1958) (12–15 days at 22° c and 50% relative humidity). this disparity between our results and other studies can be mainly explained by two factors; the higher temperature we used and the characterizations of the local populations we studied. it is proved that the development of fly larvae is temperaturedependent, and in higher temperature the rate of development increases and duration of development becomes shortened. variation has been observed in developmental time for geographically distinct populations (rechard et al. 2008; gallagher et al. 2010). the results obtained in this study are the first data for the iranian forensic flies which are originated from a totally different places than the populations used in other studies. the variation of developmental times between different populations emphasizes on specific characterization of regional developmental times of species. iran j arthropod-borne dis, 2011, 5(1): 54–62 ah shiravi et al.: temperature requirements of … 59 in the current study we used meat traps which can trap only females. unfortunately identification of females of sarcophagidae is very difficult and in most cases is impossible. in this study we tried to prepare some dna markers (mtdna coi as well as its2 rdna) for the specimens of sarcophaga sp. found in this study (unpublished data) but due to lack of their counterparts in genbank we could not identify the specimens and it remained unclassified. generally only males of this family can be identified, and then only by examination of dissected genitalia (pape 1996). in this study we focused only on temperature as a main factor for development of the flies, however, other important factors such as natural or synthetic diet, humidity, and competition between larvae should be accounted for this purpose. the effect of diet on development of flies has been shown in various studies (kaneshrajah and turner 2004, clark et al. 2006, tarone 2006, rueda et al. 2010). it was shown that natural diets such as beef liver that are often used in laboratory rearing of flies produce offensive odors and contamination (sherman and mytien tran 1995). moreover, it is demonstrated that the presence of toxins in decomposing tissues from natural diets can affect the development rate and generate errors in pmi estimates when necrophagous insects are used for forensic studies in the laboratory (estrada et al. 2009). clark et al. 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genera and species of blow flies (diptera: calliphoridae) of america north of mexico. proc. entomol soc wash. 108(3): 689–725. zalom fg, goodell pb, wilson lt, barnett ww, bentley wj (1984) degree-days: the calculation and use of heat units in pest management. university of california, division of agriculture and natural resources, uc danr leaflet 21373. zumpt f (1965) myiasis in man and animals in the old world: a textbook for physicians veterinarians and zoologists. butterworth, london. j arthropod-borne dis, march 2018, 12(1): 24–30 f ghorbani et al.: high resistance of … 24 http://jad.tums.ac.ir published online: march 18, 2018 original article high resistance of vector of west nile virus, culex pipiens linnaeus (diptera: culicidae) to different insecticides recommended by who in northern iran fatemeh ghorbani 1, hassan vatandoost 1, 2, *ahmad ali hanafi-bojd 1, 2, mohammad reza abai 1,2, hassan nikookar 3, *ahmad ali enayati 4 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3department of medical entomology and vector control, student research committee, health sciences research center, school of public health, mazandaran university of medical sciences, sari, iran 4department of medical entomology and vector control, school of public health, sari university of medical sciences, sari, iran (received 7 jan 2018; accepted 12 feb 2018) abstract background: culex pipiens is a mosquito species distributed in different parts of iran. it is known as vector of some diseases as well as a nuisance insect. a successful control campaign needs to study the biology, ecology and susceptibility status of the target pest. the aim of this study was to investigate the susceptibility status of culex pipiens to some insecticides recommended by who in north of iran. methods: larvae were collected from different breeding places in sari county by standard dipping method. they were reared to adult stage in an under standard condition. world health organization (who) standard test kits and the diagnostic dose of 12 imagicides were used for adults, while two larvicides were used against larvae. results: the least and highest mortality rates after exposure to insecticides were 4.7% (propoxur 0.1%) and 76.4% (etofenprox 0.5%), respectively. larvae showed high resistance to temephos (19.19%) and malathion (54.63%). culex pipiens was found to be resistant to all used insecticides/larvicides at diagnostic dose. conclusion: intensive use of pesticides against agriculture and urban pests increased the resistance level of this species to different insecticides, although currently there is no control program against it. study on the mechanisms of resistant in this species to different insecticides is recommended. keywords: culex pipiens, insecticide resistance, temephos, propoxur, deltamethrin introduction more than 3,500 species of mosquito have been identified so far in the world, but few of them are vectors of the disease to humans. medical importance and the geographical distribution of mosquitoes vary in different regions (1). wuchereria bancrofti and brugia malayi parasites are distributed in tropical and subtropical africa, asia, australia and the pacific and transmitted by culex pipiens linnaeus. in addition, arboviral diseases such as west nile, sindbis, japanese encephalitis, western equine encephalitis, rift valley fever, tahyna and oropouche transmitted by cx. pipiens. the transmission of birds malaria caused by plasmodium gallinacum is also proven by this species (2). among these diseases, west nile viral disease transmitted by mosquitoes has been detected serologically in humans from guilan, khorasan, khuzistan, sistan and baluchestan and isfahan provinces of iran (3-5). dog heart *corresponding authors: dr ahmad ali enayati, email: ahmadali_enayati@yahoo.com, dr ahmad ali hanafi-bojd, email: aahanafibojd@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 24–30 f ghorbani et al.: high resistance of … 25 http://jad.tums.ac.ir published online: march 18, 2018 worm parasites, dirofilaria immitis and dirofilaria repens also reported from human, dog and mosquito in the country (6, 7). due to high adaptability and potential of reproduction, cx. pipiens breeds in a variety of larval habitats from pits containing waste water, polluted water to holes and dishes of the clean water (2). the most basic control method of mosquitoes is chemical insecticides, although some biological methods are used against malaria vectors. control programs should be regularly carried out using the updated data on the susceptibility of mosquitoes to the insecticides. invasive and irrational use of pesticides has led to the development of resistance to insecticides in insects. hence, the survey on resistance of mosquitoes to insecticides ensures us to do an effective operation. in studies conducted around the world, cx. pipiens has been shown resistant to some pesticides (8-12). culex pipiens and cx. molestus have shown resistance to most of organophosphates in iran, iraq, and egypt and to carbamates in iraq and kuwait. overall, this mosquito has shown resistance to 36 pesticides in different countries (https://www.pesticideresistance.org). mazandaran province is one of the main agricultural and horticultural poles of iran and the largest consumer of pesticides (unpublished data). according to a current study about 14,000 tons of agriculture pesticides, expressed in active ingredients (ai), were annually used in iran during 2012–2014 (13). so far no study has been done on the susceptibility of cx. pipiens, a common urban pest, in sari county, north of iran. this city is one of the most important tourist destinations in the country. therefore, this study was aimed to test the effectiveness of the who recommended pesticides against the mosquito and to find the most effective compounds. materials and methods study area sari, the provincial capital of mazandaran province is considered as an important and one of the most attractive and beautiful regions of northern iran. it is located in 53.08 oe and 36.56 on with an altitude of 40m above the sea level (fig. 1). agriculture and horticulture are the most important jobs in the region. sampling to find the larval habitats of choice for cx. pipiens, sampling of different larval habitats in the city of sari was carried out using standard dipping method. physical characteristics and geographic coordinates of collection sites were recorded using a gps device and registered in the relevant forms. the collected larvae were transferred to the laboratory, mounted and identified using morphological keys (14). from the end of june 2016 and after finding the best larval habitats for cx. pipiens, sampling was started for susceptibility tests. susceptibility tests for adult mosquitoes according to world health organization guidelines (15), diagnostic dose of different pesticides were used against larvae and female adult mosquitoes. a total of 4 replicates representing 100 specimens were used for each pesticide and 50 for control. the following insecticides were used: ddt4%, dieldrin 0.4%, dieldrin 4%, malathion 5%, fenitrothion 1%, propoxur 0.1%, bendiocarb 0.1%, etofenprox 0.5%, cyfluthrin 0.15%, permethrin 0.75%, lambdacyhalothrin 0.05% and deltamethrin 0.05% (15). dose and exposure time for different insecticides was according to who procedures (16). tests were carried out in a room with 25– 30 oc temperature and 65–75% rh. twoto three-days old sugar fed female mosquitoes were used in 4 replicates of 25 mosquitoes for each insecticide at diagnostic dose and 2 replicates as control. the exposure time was 0.5 to 4 hours according to who procedure for culex mosquitoes (16). all tested specimens were identified after the test using morphological key (14) and non-target species were excluded from the results. http://jad.tums.ac.ir/ https://www.pesticideresistance.org/ j arthropod-borne dis, march 2018, 12(1): 24–30 f ghorbani et al.: high resistance of … 26 http://jad.tums.ac.ir published online: march 18, 2018 susceptibility tests for larvae two larvicides, i.e. temephos (0.02mg/l) and malathion (1mg/l) were used at the diagnostic dose provided by who (17). larvae were collected from the identified breeding sites for cx. pipiens in the city, transferred to the insectary and after 24 hours recovery time, late 3rd or early 4th instars were used for the tests. who test procedure for mosquito larvae was used in this study (17). mortality rate was recorded after 24h exposure time. data analysis in the event of control mortality of less than 5% the results of the test were considered to be correct, 5–20% the results were corrected using abbotts' formula and more than 20% the tests were discarded and repeated by new specimens. mortality of the test between 98– 100% was considered as susceptible, 90–97% as candidate of resistance that should be confirmed using specific methods, and less than 90% was considered as resistant (15). results culex pipiens from sari county, northern iran, was resistant to all used insecticides and larvicides in the diagnostic dose recommended by who. this mosquito showed high resistance level to the insecticides from four main groups of pesticides, i.e. organochlorine, organophosphate, carbamate and pyrethroids (table 1, fig. 2). however, the resistance level was lower to pyrethroids compared to other insecticides. the highest and the least mortality rates were found to etofenprox and propoxur with a mortality rate of 76.47 and 4.25%, respectively. this mosquito species was resistant to both larvicides (table 1), although malathion killed more larvae (54.63%) compared to temephos (19.19%). overall, the mortality rate was the highest to organophosphates and carbamates, followed by organochlorines and pyrethroids. table 1. response of culex pipiens collected from sari county, northern iran, 2016 to different insecticides/larvicides insecticide/larvicide exposure time (minute) mortality (%) mean sd control ddt 4% 240 22.98 3.2 4.1 dieldrin 4% 60 28.57 3.3 0 dieldrin 0.4% 60 8.69 2 0 malathion 5% 60 7.52 1.9 2.22 fenitrothion 1% 120 7.31 2 2.2 bendiocarb 0.1% 60 12.94 2.6 0 propoxur 0.1% 120 4.25 1.4 0 etofenprox 0.5% 60 76.47 3.2 0 cyfluthrin 0.15% 60 72.09 3.4 0 permethrin 0.75% 60 70.73 3.5 2.2 lambdacyhalothrin 0.05% 30 33.33 3.4 2.2 deltamethrin 0.05% 30 39.08 3.7 2.2 malathion (1ppm) 1440 54.63 3.6 0 temephos (0.02ppm) 1440 19.19 2.8 0 http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 24–30 f ghorbani et al.: high resistance of … 27 http://jad.tums.ac.ir published online: march 18, 2018 fig. 1. geographical poistion of the study area in north of iran fig. 2. mortality rate of culex pipiens collected from sari county, north of iran using diagnostic dose of imagicides (red line indicate the resistant level) discussion although organophosphates and carbamates have the same mechanism of action, there are reports of cross-resistance to pyrethroids and organophosphates in culex quinquefasciatus say (18, 19). organophosphate insecticides have an extensive use in agricul ture and horticulture in the study area. so, although there is no program for mosquito control in the area, continuous exposure of the insects to different insecticides, makes them resistant to other groups as well. the same studies in iran were recently http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 24–30 f ghorbani et al.: high resistance of … 28 http://jad.tums.ac.ir published online: march 18, 2018 carried out to find the susceptibility status of cx. pipiens. the results showed that this species was resistant to ddt 4% in southeastern city of chabahar (54.4% mortality), northwestern cities of urmia (15.6%) and ahar (23%) and the capital city tehran (0%), (20-24). in other countries, such as china (11), malaysia (10) and india (12), ddt resistant strains of cx. pipiens and cx. quinquefasciatus were reported. in natural conditions, cx. pipiens breeds in sewage and water canals contaminated with different pesticides and other chemical pollutants. this continual exposure to pesticides acts as a natural selection pressure and increases the rate of resistant population. high resistance level to propoxur, bendiocarb and organophosphates in our study can be due to this reason. the iranian northern province of mazandaran has the most use of pesticides in the country (unpublished data). therefore, it is likely that all pests including cx. pipiens develop resistance. mortality after exposure to pyrethroids in our study ranged between 33.33–76.47%. other studies in iran reported a range of 18– 93% (20, 21-25), with the highest resistance rate in tehran. resistance to pyrethroids was also reported in this mosquito species from china and saudi arabia (11, 26). larviciding may be considered as the main method for control of cx. pipiens in urban areas. we found this species resistant to both larvicides. a study conducted in tehran reported 8% mortality of this mosquito to temephos (27). this rate was 19.19 % in our survey. esterases may be responsible for resistance to organophosphates (28). studies are recommended to find the mechanism(s) of resistance to organophosphates, especially larvicides, in the study area. conclusion in conclusion, this is the first time that new diagnostic doses recommended by who are used against cx. pipiens, so the results can be useful for other researchers. high resistance of this species to 14 insecticides/larvicides in sari county, northern iran, reflects the situation of indiscriminate use of pesticides in the area. although spraying is not done by the health system in the region, the widespread use of pesticides in household, agriculture and horticulture caused severe resistance to pesticides in cx. pipiens given the role of this mosquito in transmission of some important diseases, and annoyance caused by mosquito bites, judicious use of pesticides in all sectors including agriculture, horticulture, urban and public health pests is highly recommended. a comprehensive study on the mechanisms of resistance in cx. pipiens to different insecticides is advised in iran. acknowledgments this study is the results of msc thesis of the first author and has financially supported by deputy of research, tehran university of medical sciences, project no. 9211263013. the authors are grateful to kind collaboration of department of medical entomology and vector control, mazandaran university of medical sciences. we declare that we have no conflict of interest. references 1. becker n, petrić d, boase c, lane j, zgomba m, dahl c, kaiser a (2003) mosquitoes and their control. new york: springer, p. 577. 2. vinogradova eb (2000) culex 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available at: http://who.int/whopes/resistance/en/d iagnostic_concentrations_may_2014. pdf. 17. who (2005) guidelines for laboratory and field testing of mosquito larvicides. who/cds/whopes/gcdpp/2005. 13. 18. bisset j, rodriguez m, soca a, pasteur n, raymond m (1997) cross-resistance to pyrethroid and organophosphorus insecticides in the southern house mosquito (diptera: culicidae) from cuba. j med entomol. 34(2): 244–246. 19. kolaczinski jh, curtis cf (2004) investigation of negative cross-resistance as a resistance-management tool for insecticide-treated nets. j med entomol. 41(5): 930–934. 20. fathian m, vatandoost h, moosa-kazemi http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=nazni%20wa%5bauthor%5d&cauthor=true&cauthor_uid=16880755 https://www.ncbi.nlm.nih.gov/pubmed/?term=lee%20hl%5bauthor%5d&cauthor=true&cauthor_uid=16880755 https://www.ncbi.nlm.nih.gov/pubmed/?term=azahari%20ah%5bauthor%5d&cauthor=true&cauthor_uid=16880755 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deltamethrin and ddt, urmia county, west azerbaijan province, northwestern iran. asian pac j trop dis. 5 (suppl 1): s77–s79. 24. salim-abadi y, oshaghi ma, enayati aa, abai mr, vatandoost h, eshraghian mr, mirhendi h, hanafi-bojd aa, gorouhi ma, rafi f (2016) high insecticides resistance in culex pipiens (diptera: culicidae) from tehran, capital of iran. j arthropod borne dis. 10 (4): 483–492. 25. vatandoost h, ezeddinloo l, mahvi ah, abai mr, kia eb, mobedi i (2004) enhanced tolerance of house mosquito to different insecticides due to agricultural and household pesticides in sewage system of tehran, iran. iranian j environ health sci eng. 1(1): 42–45. 26. al-sarar as, al-shahrani d, bayoumi ae, abobakr y, hussein hi (2011) laboratory and field evaluation of some chemical and biological larvicides against culex spp. (diptera: culicidae) immature stages. int j agric biol. 13: 115–119. 27. abai mr, hanafi-bojd aa, vatandoost h (2016) laboratory evaluation of temephos against anopheles stephensi and culex pipiens larvae in iran. j arthropod-borne dis. 10(4): 510–518. 28. cui f, lin lf, qiao cl, xu y, marquine m, weill m, raymond m (2008) insecticide resistance in chinese populations of the culex pipiens complex through esterase overproduction. entomol experiment appl. 120(3): 211–220. http://jad.tums.ac.ir/ j arthropod-borne dis, 2012, 6(1): 54–61 a saghafipour et al.: mosquito (diptera: culicidae) … original article mosquito (diptera: culicidae) fauna of qom province, iran a saghafipour 1, *mr abai 2, b farzinnia 3, r nafar 1, h ladonni 2, *s azari-hamidian 4 1qom provincial health center, qom university of medical sciences, qom, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3school of public health, qom university of medical sciences, qom, iran 4school of health, guilan university of medical sciences, rasht, iran (received 18 sep 2010; accepted 8 jun 2011) abstract background: there is very little information about the mosquito fauna of qom province, central iran. by now only three species; anopheles claviger, an. multicolor, and an. superpictus have been reported in the province. to study mosquito fauna and providing a primary checklist, an investigation was carried out on a collection of mosquitoes in this province. methods: to study the mosquito (diptera: culicidae) fauna, larval collections were carried out from different habitats on 19 occasions using the standard dipping technique during spring and summer 2008 and 2009. results: in total, 371 mosquito larvae were collected and morphologically identified including 14 species representing four genera: anopheles claviger, an. marteri, an. turkhudi, an. superpictus, culex arbieeni, cx. hortensis, cx. mimeticus, cx. modestus, cx. pipiens, cx. territans, cx. theileri, culiseta longiareolata, cs. subochrea, and ochlerotatus caspius s.l. all species except for an. claviger and an. superpictus were collected for the first time in the province. all larvae were found in natural habitats. the association occasions and percentages of the mosquito larvae in qom province were discussed. conclusion: there are some potential or proven vectors of different human and domesticated animal pathogens in qom province. the ecology of these species and the unstudied areas of qom province need to be investigated extensively. keywords: anopheles, culex, culiseta, ochlerotatus, larvae, taxonomy introduction according to the most recent classification of mosquitoes, the family culicidae (diptera) includes two subfamilies, 11 tribes, 111 genera, and 3523 species in the world fauna and the genus anopheles meigen includes seven subgenera and at least 464 species (harbach 2007). the mosquito fauna of iran includes seven genera, 64 species, and 3 subspecies and the records of 12 other species need to be verified (azari-hamidian 2007). recently, oshaghi et al. (2008) introduced an. superpictus grassi as a species complex including three genotypes x, y, and z and djadid et al. (2009) introduced a new species of the hyrcanus group based on pcr techniques in the country. malaria is the most important mosquito-borne disease in iran and seven species of the genus anopheles: an. culicifacies giles s.l., an. dthali patton, an. fluviatilis james s.l., an. maculipennis meigen s.l., an. sacharovi favre, an. stephensi liston, and an. superpictus are known its proven vectors in iran, also an. pulcherrimus theobald is mentioned a suspected vector of malaria in southeastern iran (edrissian 2006). eshghy (1977) observed plasmodium oocysts in an. multicolor cambouliu, but sporozoites have not been found *corresponding authors: dr shahyad azari-hamidian, e-mail: azari@gums.ac.ir, mohammad reza abai, e-mail: abaimr@tums.ac.ir 54 j arthropod-borne dis, 2012, 6(1): 54–61 a saghafipour et al.: mosquito (diptera: culicidae) … in this species and it is not considered a vector in the country. recently, djadid et al. (2009) reported an. hyrcanus (pallas) a potential vector of p. falciparum based on nested pcr in guilan province. west nile and sindbis viruses have been reported in iran (naficy and saidi 1970). there is no information about their vectors in the country. the possibility of some mosquito-borne arboviral outbreaks like japanese encephalitis and rift valley fever in the who eastern mediterranean region, including iran, is noteworthy (who 2004). there are some doubtful old records of dengue fever in iran (foote and cook 1959), however there is no formal recent record of this virus in the country. the mosquito-borne filariae: dirofilaria (dirofilariasis) and setaria (setariasis) (spirurida: onchocercidae) have been reported in iran (eslami 1997, azari-hamidian et al. 2007). anopheles maculipennis and culex theileri theobald are known vectors of setaria labiatopapillosa and dirofilaria immitis respectively, in ardebil province, northwestern iran (azari-hamidian et al. 2009). in 1996, qom province was formally separated from tehran province. there is very little information about the mosquito fauna of qom province. macan (1950) found an. multicolor larvae in “darya-i-namak” (namak lake, salt lake, or qom lake) (fig. 1) and showed this species and an. superpictus around qom in the distributional maps. in the document of the former institute of malariology and parasitology (present national institute of health research) (1953) an. multicolor was recorded around qom. saebi (1987) showed the occurrence of an. superpictus in qom in the distributional map of the species. recently, farzinnia et al. (2010) reported an. claviger (meigen) for the first time in the province. as the authors know there is no other documented information on the mosquitoes of qom province. to study mosquito fauna and providing a primary checklist, an investigation was carried out on a collection of mosquitoes in qom province. materials and methods study area qom province is bounded by tehran province in the north, isfahan province in the south, semnan province in the east, and markazi province in the west with an area of approximately 11240 square km (0.68% total area of iran). the center of the province, qom city, is almost 880 m above sea level. the province with arid climate has about 150 mm annual rainfall and is located between 34o09'–35o11' n latitude and 50o06'–51o58' e longitude and formally includes one county (qom) and five districts: jafarabad, kahak, khalajestan, markazi (qom), and salafchegan (fig. 1). specimen and data collection in the present faunistic and cross-sectional investigation, larvae were collected from different habitats using the standard dipping technique (350 ml dipper) in nine stations (localities) of four districts; kahak district (emamzadeh esmail and dastgerd), khalajestan district (ahmadabad, agholak, dastjerd, and ghahan), markazi district (ghomrood), and salafchegan district (ghal-e-cham and rahjerd) on 19 occasions during spring and summer 2008 and 2009. the larvae were preserved in lactophenol and the microscope slides of the preserved larvae were prepared using de faure’s medium. the thirdand fourth-instar larvae were identified using the keys of shahgudian (1960), zaim and cranston (1986), harbach (1988), and azarihamidian and harbach (2009). the mosquito name abbreviations follow reinert (2009). results in total, 371 mosquito larvae were periodically collected from four districts of qom 55 j arthropod-borne dis, 2012, 6(1): 54–61 a saghafipour et al.: mosquito (diptera: culicidae) … province during 2008–2009 and morphologically identified including 14 species representing four genera: an. claviger, an. marteri senevet and prunnelle, an. turkhudi liston, an. superpictus, culex arbieeni salem, cx. hortensis ficalbi, cx. mimeticus noe, cx. modestus ficalbi, cx. pipiens linnaeus, cx. territans walker, cx. theileri, culiseta longiareolata (macquart), cs. subochrea (edwards), and ochlerotatus caspius (pallas) s.l. (table 1). the association occasions of the mosquito larvae in the province are shown in table 2. the association percentages of the mosquito larvae in qom province are shown in table 3. all larvae were collected from natural habitats. fig. 1. map of iran, highlighting the position of qom province and its five districts: 1. jafarabad, 2. kahak, 3. khalajestan, 4. markazi, and 5. salafchegan ( qom city) table 1. the distribution and composition of the mosquito larvae in nine localities of qom province, iran, spring– summer 2008–2009 species locality n % a gholak a hm adabad d astgerd d astjerd e m am zadeh e sm ail g hahan g hal-echam g hom rood r ahjerd an. claviger 66 135 1 16 10 228 61.5 an. marteri 2 2 0.5 an. superpictus 12 20 32 8.6 an. turkhudi 7 7 1.9 cx. arbieeni 1 1 0.3 cx. hortensis 3 2 1 3 9 2.4 cx. mimeticus 9 1 1 11 3.0 cx. modestus 6 6 1.6 cx. pipiens 13 13 3.5 cx. territans 7 3 10 2.7 cx. theileri 1 4 1 6 1.6 cs. longiareolata 34 5 2 41 11.1 cs. subochrea 1 1 2 0.5 oc. caspius s.l. 3 3 0.8 total 77 175 30 1 25 25 10 24 4 371 100 n hoz-e-soltan lake qom lake 56 j arthropod-borne dis, 2012, 6(1): 54–61 a saghafipour et al.: mosquito (diptera: culicidae) … table 2. the association occasions of the mosquito larvae in qom province, iran, spring–summer 2008–2009 species t otal occasions a n. claviger a n. m arteri a n. superpictus a n. turkhudi c x. arbieeni c x. hortensis c x. m im eticus c x. m odestus c x. pipiens c x. territans c x. theileri c s. longiareolata c s. subochrea o c. caspius s.l. an. claviger 14 4 1 2 1 3 1 an. marteri 1 1 an. superpictus 2 1 1 1 2 1 an. turkhudi 1 1 1 1 1 cx. arbieeni 1 1 1 1 1 cx. hortensis 5 4 1 1 1 2 1 1 2 1 cx. mimeticus 3 1 2 1 1 2 1 1 cx. modestus 1 1 1 1 1 cx. pipiens 1 1 1 1 1 cx. territans 2 2 1 1 1 cx. theileri 3 1 1 1 1 1 1 1 1 cs. longiareolata 4 3 1 2 1 1 cs. subochrea 2 1 1 1 1 1 1 1 oc. caspius s.l. 1 1 1 1 1 table 3. the association percentages of the mosquito larvae in qom province, iran, spring–summer 2008–2009 species association abundance n (%) an. claviger cx. hortensis, cx. theileri 89 (39.0) alone 67 (29.4) cx. hortensis, cx. territans, cs. subochrea 59 (25.9) cx. territans, cs. longiareolata 5 (2.2) cx. hortensis, cx. mimeticus, cs. longiareolata 5 (2.2) cx. hortensis, cs. longiareolata 3 (1.3) total 228 (100) an. marteri cs. longiareolata 2 (100) an. superpictus cx. mimeticus, cx. theileri 20 (62.5) an. turkhudi, cx. arbieeni, cx. hortensis, cx. mimeticus 12 (37.5) total 32 (100) an. turkhudi an. superpictus, cx. arbieeni, cx. hortensis, cx. mimeticus 7 (100) cx. arbieeni an. superpictus, an. turkhudi, cx. hortensis, cx. mimeticus 1 (100) cx. hortensis an. claviger, cx. territans, cs. subochrea 3 (33.4) an. claviger, cx. mimeticus, cs. longiareolata 3 (33.3) an. superpictus, an. turkhudi, cx. arbieeni, cx. mimeticus 1 (11.1) an. claviger, cs. longiareolata 1 (11.1) an. claviger, cx. theileri 1 (11.1) total 9 (100) cx. mimeticus an. superpictus, an. turkhudi, cx. arbieeni, cx. hortensis 9 (81.8) 57 j arthropod-borne dis, 2012, 6(1): 54–61 a saghafipour et al.: mosquito (diptera: culicidae) … an. claviger, cx. hortensis , cs. longiareolata 1 (9.1) an. superpictus, cx. theileri 1 (9.1) total 11 (100) cx. modestus cx. pipiens, cx. theileri, cs. subochrea, oc. caspius s.l. 6 (100) cx. pipiens cx. modestus, cx. theileri, cs. subochrea, oc. caspius s.l. 13 (100) cx. territans an. claviger, cx. hortensis, cs. subochrea 7 (70) an. claviger, cs. longiareolata 3 (30) total 10 (100) cx. theileri an. superpictus, cx. mimeticus 4 (66.6) cx. modestus, cx. pipiens, cs. subochrea, oc. caspius s.l. 1 (16.7) an. claviger, cx. hortensis 1 (16.7) total 6 (100) cs. longiareolata an. claviger, cx. hortensis 26 (63.4) an. claviger, cx. territans 8 (19.5) an. claviger, cx. hortensis, cx. mimeticus 5 (12.2) an. marteri 2 (4.9) total 41 (100) cs. subochrea an. claviger, cx. hortensis, cx. territans 1 (100) cx. modestus, cx. pipiens, cx. theileri, oc. caspius s.l. 1 (100) total 2 (100) oc. caspius s.l. cx. modestus, cx. pipiens, cx. theileri, cs. subochrea 3 (100) discussion in the present study, 371 mosquito larvae representing 14 species in four genera were collected from qom province. all species except for an. claviger and an. superpictus were collected for the first time in the province. anopheles multicolor, which was recorded in qom province before (macan 1950, institute of malariology and parasitology 1953), was not found among the samples of this investigation. among the collected species, an. superpictus, cx. hortensis, cx. mimeticus, cx. pipiens, cx. theileri, cs. longiareolata, cs. subochrea, and oc. caspius s.l. are widespread in iran (institute of malariology and parasitology 1953, gaffary 1954, saebi 1987, zaim 1987, abai et al. 2007). anopheles claviger was found almost everywhere in iran, including 21 provinces (out of total 31), except for the eastern and southeastern areas of the country. anopheles marteri was collected from 15 provinces other than those of eastern and southeastern iran. anopheles turkhudi is found in 15 provinces in central plateau, western, southwestern, eastern, southeastern, and southern iran. anopheles multicolor, which was not found in this investigation, however recorded around qom before, was recorded in 14 provinces in central plateau, northeastern, eastern, southeastern, and southern iran (institute of malariology and parasitology 1953, gaffary 1954, saebi 58 j arthropod-borne dis, 2012, 6(1): 54–61 a saghafipour et al.: mosquito (diptera: culicidae) … 1987). culex arbieeni was recorded in 9 provinces before: former khorasan, loristan, chaharmahal and bakhtiari, yazd, kerman, sistan and baluchistan, hormozgan, fars, and bushehr (zaim 1987). culex modestus was collected from 6 provinces: isfahan, east azerbaijan, west azerbaijan, hormozgan (zaim 1987), ardebil (azari-hamidian et al. 2009), and north khorasan (azari-hamidian et al. 2011). culex territans was found in 6 provinces: guilan, kurdistan, former khorasan, isfahan, hormozgan (zaim 1987), and sistan and baluchistan (lotfi 1973). among the species, only an. turkhudi and cx. arbieeni are not found in northern iran including northwestern areas (west azerbaijan, east azerbaijan, and ardebil provinces), the caspian sea littoral (guilan, mazandaran, and golestan provinces), and northeastern areas (north khorasan province and northern razavi khorasan province) (institute of malariology and parasitology 1953, gaffary 1954, saebi 1987, zaim 1987). oshaghi et al. (2008) reported three genotypes named x, y, and z within an. superpictus in iran based on the second internal transcribed spacer (its2) and cytochrome c oxidase subunit i (coi) sequence data. genotype x was found in all parts of the country, including qom province, except for the southeastern areas. genotypes y and z were only found in southeastern iran including kahnooj of southern kerman province and sistan and baluchistan province. only two larvae, one the third instar, of cs. subochrea were found in the present investigation (table 1). two close and rare species cs. subochrea and cs. annulata (schrank) are very similar in larval stage; however they are easily distinguishable as adult. there is little information about taxonomy and distribution of three close species cs. alaskaensis (ludlow), cs. annulata, and cs. subochrea in iran (azari-hamidian and harbach 2009). among the species which were found in the present study, an. superpictus is malaria vector in iran (edrissian 2006). culex theileri is known vector of the canine heart worm nematode in ardebil province (azari-hamidian et al. 2009). in addition, cx. pipiens, cx. theileri, and oc. caspius s.l. are known as the potential or proven vectors of different human and domesticated animal pathogens in different areas of the world (gutsevich et al. 1974, harbach 1988). the ecology of medically important species and the unstudied areas of qom province need to be investigated extensively. acknowledgements the authors are grateful to dr a akbari, dr a abbasi, f abedi-astaneh, qom provincial health center, qom university of medical sciences, qom, for their kind collaborations. this study was financially supported by the institute of public health research, academic pivot for education and research, tehran university of medical sciences: project no.: 241.83.77. the authors declare that they have no conflicts of interest. references abai mr, azari-hamidian s, ladonni h, hakimi m, mashhadi-esmail k, sheikhzadeh k, 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(2009) list of abbreviations for currently valid generic-level taxa in family culicidae (diptera). eur mosq bull. 27: 68–76. saebi me (1987) morphological study on anopheline larvae and their distribution in iran. [phd dissertation]. school of public health, tehran university of medical sciences, tehran, iran [persian]. shahgudian er (1960) a key to anophelines of iran. acta med iran. 3: 38–48. world health organization (2004) integrated vector management. who regional officer for eastern mediterranean, cairo. zaim m, cranston ps (1986) checklist and keys to the culicinae of iran (diptera: culicidae). mosq syst. 18: 233–245. zaim m (1987) the distribution and larval habitat characteristics of iranian culicinae. j am mosq control assoc. 3: 568–573. 61 j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 127 http://jad.tums.ac.ir published online: june 12, 2018 original article faunistic study of the rodents of north khorasan province, north east of iran, 2011–2013 kourosh arzamani 1, *zeinolabedin mohammadi 2, mohammad reza shirzadi 3, seyed mohammad alavinia 1, behruz jafari 4, jamshid darvish 2, 5 1vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 2department of biology, faculty of sciences, ferdowsi university of mashhad, mashhad, iran 3zoonoses control department, ministry of health, tehran, iran 4environmental office of north khorasan province, bojnurd, iran 5rodentology research department, applied animal institute, ferdowsi university of mashhad, mashhad, iran (received 25 nov 2015; accepted 8 may 2018) abstract background: rodents are one of the most important hosts for some zoonotic diseases and also act as a reservoir of some ectoparasites and endoparasites. they cause damage to the farms and inflict public health. the aim of this study was to investigate the faunal composition of rodent in north khorasan province, northeast of iran. methods: the sampling was carried out between 2011 to 2013. the specimens were collected using different methods including rodent death and live traps, digging of their burrow, and hand net from 75 different sample sites. results: the total number of 396 specimens belonging to 22 species and six families were identified. the results illustrated the high numbers and densities of meriones persicus (17.68%), meriones libycus (15.15%), nesokia indica (7.32%) and rhombomys opimus (6.82%), as the most important reservoirs for different zoonotic diseases. moreover, significant number of other rodent species including mus musculus (15.66%), apodemus witherbyi (13.89%), a. hyrcanicus (0.25%), rattus norvegicus (1.01%), meriones crassus (0.25%), gerbillus nanus (0.51%), microtus paradoxus (2.27%), m. transcaspicus (0.76%), ellobius fuscocapillus (0.25%), cricetulus migratorius (4.29%), calomyscus elburzensis (4.29%), c. mystax (1.26%), spermophilus fulvus (0.25%), dryomys nitedula (3.54%), allactaga elater (3.54%), jaculus blanfordi (0.25%), meriones zarudnyi (0.25%), m. meridianus (0.51%), and hystrix indica as hosts for parasites and zoonotic diseases were identified. conclusion: the high biodiversity including at least 22 species and six families of rodents were found in north khorasan province, some of them were medically important species. keywords: fauna, rodents, biodiversity, iran introduction rodents are the most widely distributed and the largest group of small mammals’ worldwide (1) which cause economic loss and inflict public health. rodents play a significant role as reservoirs or vectors of sixty different diseases including leishmaniasis, leptospirosis, plague, hantavirus pulmonary syndrome, salmonellosis, etc. with direct or indirect role in spread of these diseases (2). the fauna of the rodent is medically important in iran and in north khorasan prov ince (3-8). these are identified and reported 15 species of rodent from this province, spermophilus fulvus, microtus transcaspicus, m. paradoxus, ellobius fuscocapillus, cricetulus migratorius, calomyscus sp., mus musculus, apodemus witherbyi, nesokia indica, gerbillus nanus, meriones libycus, m. meridianus, m. persicus, rhombomys opimus and dryomys nitedula (7). north khorasan province was formerly known as eastern range of human plague ep*corresponding author: dr zeinalabedin mohammadi, e-mail: mohammadi.zeinal@gmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 128 http://jad.tums.ac.ir published online: june 12, 2018 idemics in iran (5) and also has been the objective for study of rodent-borne diseases, especially, as a purpose of investigation on leishmaniasis and leptospirosis (6, 9). the province is bordered by golestan province in the west which is one of the most important focus of zoonotic cutaneous leishmaniasis in iran (3). the province is located near the border of turkmenistan and afghanistan countries. neighbour regions in turkmenistan are mainly deserts and semi-desert areas and zoonotic cutaneous and visceral leishmaniasis are endemic in the country (10). both of these lowincome countries receive low health care and public services; therefore the province can be a trajectory for transportation of infectious disease from both countries via communications and transportation of nomads, refugees, and passengers. north khorasan province is one of the most important areas for agriculture and animal husbandry in iran and the study area is potentially provide a suitable habitat for distribution of pests and favoring habitats for endemic and penetrating species such as jirds and gerbils. this increases the risk of spreading vector-borne diseases and zoonosis, therefore, this province needs more consideration by iranian health care service to control and preventing the spread of rodent-borne diseases. we aimed to investigate the faunal composition of rodents in north khorasan province, northeast of iran. materials and methods study area this cross-sectional study was conducted from 36°37′–38°17′ n latitude and 55°53′– 58°20′ e longitude in north khorasan province, northeast of iran with the total area of approximately 28434km2. the sampling was carried out between 2011 to 2013 and includes 75 localities of eight counties from north khorasan province (table 1, fig. 1). rodent collection and methods the specimens were collected using different methods including live trap, rodent death trap, digging into their burrow, and hand net during all seasons. geographical coordinates were recorded using gps. besides, 11 specimens of rodents deposited in the zoology museum of ferdowsi university of mashhad (zmfum), mashhad, iran from this region were considered (table 2). specimen collection was performed in accordance with the procedures approved by the ethical committee of north khorasan university of medical sciences. the materials were identified using available identification keys (11-13). taxonomic names and distribution of rodents followed musser and carleton (14). arcgis ver.9.3 software was applied for preparing of the map of sampling localities. results overall, 385 collected specimens were investigated. the specimens belong to 22 species, 16 genera, and six families. of these, m. percicus, m. libycus, a. witherbyi and m. musculus, occur in the most localities studied and identified as widely distributed species. abundance and different habitats of the rodents collected in the study area are shown in the table. 2. all specimens of apodemus captured from different localities of the province were identified as a. witherbyi based on morphology and morphometric approach. only one a. hyrcanicus specimen from shirvan has been identified. specimens of r. opimus were diagnosed as r. opimus sargadensis based on the morphology and morphometric studies. tracks, spines and some live specimens of hystrix indica were observed in both arid regions and deciduous forests, but we avoided trapping of this species. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 129 http://jad.tums.ac.ir published online: june 12, 2018 table 1. geographical coordinates and sampling locality of the rodents of north khorasan province, iran, 2011– 2013 town sampling locality number of locality on the map latitude longitude e sf a r a y e n safi abad 1 36°39' 01.93 58°00' 19.05 gerati 2 36°51' 53.88 57°31' 40.58 kal shor 3 36°53' 51.22 57°07' 52.74 gorpan 4 36°56' 46.82 57°12' 16.15 esfraien 5 37°08' 43.87 57°24' 13.20 roein 6 37°12' 38.02 57°28' 21.97 emam vardi 7 37°15' 04.35 57°22' 30.77 jafar abad 8 37°06' 17.54 57°06' 24.94 b o jn u r d salook 9 37°16' 32.15 57°08' 07.37 esfidan, chenaran 16 37°24' 49.70 57°33' 00.02 mehnan, metranloo 17 37°23' 51.17 57°17' 23.46 baghchegh 18 37°29' 27.74 57°21' 02.96 charkharvar, peighoo 19 37°28' 58.48 57°27' 38.07 salook 2 20 37°20' 40.92 57°03' 58.60 bidak 21 37°29' 27.74 57°13' 29.32 darsofian, abchoor 22 37°22' 23.36 57°26' 10.27 tatar 23 37°33' 51.16 57°08' 36.64 turkmenistan border 55 37°59' 42.33 57°22' 01.50 jodar 56 37°56' 17.46 57°18' 07.36 gifan 57 37°54' 35.02 57°23' 58.57 sisab 63 37°27' 01.40 57°39' 05.86 asadli 70 37°19' 42.39 57°21' 46.86 j a ja r m miandasht 10 36°48' 43.91 56°29' 05.97 kal shor 12 36°54' 20.48 56°41' 17.66 gamiteh 14 37°05' 04.37 56°46' 24.97 esmaeilabad 15 36°44' 49.76 57°48' 07.31 g a r m e h kal yazd, daraq 11 36°59' 44.42 56°13' 29.42 sharlegh 13 37°18' 58.49 56°12' 01.61 robat gharahbil 34 37°20' 40.92 56°18' 51.36 golestan national park 35 37°20' 11.66 56°01' 17.73 m a n e & s a m a lq a n tazeyab 24 37°51' 54.08 56°28' 22.07 kikanloo 25 37°38' 29.19 57°03' 58.60 pish ghaleh 26 37°41' 54.06 56°56' 24.95 kohghale 28 37°56' 32.08 56°31' 46.96 yomogh 29 38°00' 40.86 56°27' 52.80 kohne jolge 30 38°04' 49.63 56°28' 36.70 ashkhaneh 32 37°40' 40.89 56°38' 51.32 behkade 33 37°33' 51.15 56°11' 17.71 zard 36 37°30' 11.64 56°31' 03.04 kastan 37 37°29' 27.74 56°24' 27.93 ghale-khan 38 37°29' 57.01 56°45' 55.70 kohe garmab 39 37°43' 21.87 56°28' 22.07 garmab 40 37°45' 18.94 56°19' 49.89 river garmab 41 37°41' 24.30 56°15' 26.48 amand 42 37°49' 13.08 56°23' 29.40 kheshtli 44 38°03' 21.88 56°21' 46.96 r a z a n d j a r g a la n raz 27 37°56' 46.72 57°06' 10.30 ayri qayeh 31 38°10' 55.48 56°20' 33.79 hesarcheh 43 38°10' 55.47 56°29' 20.60 tangeh turkeman 45 37°55' 48.18 56°56' 10.32 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 130 http://jad.tums.ac.ir published online: june 12, 2018 goinik 46 37°59' 56.96 57°01' 17.63 baghlegh, ashrafdare 47 38°05' 18.90 56°59' 05.93 yekeh suod 48 38°10' 55.48 56°38' 51.32 bahar, ghoridare 49 38°15' 18.88 56°47' 52.77 kalatekariz 50 38°13' 07.18 56°54' 27.88 sangsar 51 38°12' 08.65 57°00' 33.73 gholaman 52 38°07' 45.24 57°06' 39.57 sokhso enghelab 53 38°11' 54.01 57°09' 35.17 takaleghoz 54 38°15' 33.52 57°12' 45.42 s h ir v a n gholool sarani 58 37°45' 33.57 58°05' 55.57 ghaltmanlo 59 37°41' 24.80 58°04' 27.77 palkanlou 60 37°39' 56.99 57°53' 29.25 gholanloo 61 37°35' 48.22 57°51' 17.55 shirvan 62 37°25' 33.60 57°52' 58.98 gelian 64 37°13' 51.18 57°52' 59.98 hossein abad 67 37°22' 52.63 57°47' 52.67 devin 71 37°19' 13.12 58°01' 17.53 topkanloo 72 37°36' 32.12 57°42' 01.46 f a r o u j titkanloo 65 37°16' 02.88 58°22' 30.66 mayvan 66 37°11' 10.21 58°02' 01.43 khosravieh 68 37°07' 01.44 58°00' 18.99 cheri 69 37°10' 40.94 58°08' 36.54 barzoo 73 37°35' 33.58 57°58' 36.59 koran kordieh 74 37°23' 36.52 58°15' 40.92 sogtali, zinkanloo 75 37°04' 05.84 58°05' 26.30 fig. 1. map of the study area. the samples were taken from 75 regions marked by a number from north khorasan province, iran, 2011–2013 table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 131 http://jad.tums.ac.ir published online: june 12, 2018 table 2. abundance, percent and different habitats of the rodents of north khorasan province, iran, 2011–2013 f a m il y species locality number n % collection habitats a b c d e f g h i j k m u r id a e m. musculus 4, 5, 7, 13, 15, 16, 18, 19, 21, 25, 34, 29, 46, 47, 49, 50, 51, 54, 57, 60, 69, 70 62 15.66 × × × a. witherbyi 5, 6, 7, 11, 14, 15, 16, 17, 19, 22, 23, 25, 28, 31, 35, 36, 39, 48, 49, 51, 3, 54, 58, 59, 60, 61, 63, 68, 70 55 13.89 × × × × × × × × × a. hyrcanicus 59 1 0.25 × n. indica 9, 20, 28, 29, 30, 38, 60, 51, 52 29 7.32 × × × r. norvegicus 38 4 1.01 × r. opimus 3, 4, 34 27 6.82 × m. libycus 1, 2, 3, 4, 11, 14, 22, 26, 32, 33, 34, 39, 40, 41, 42, 46, 55, 60, 63, 65, 66, 71 60 15.15 × × × × × × m. persicus 2, 3, 14, 16, 19, 20, 22, 23, 24, 27, 28, 29, 30, 35, 36, 37, 38, 43, 45, 48, 47, 56, 57, 58, 59, 62 70 17.68 × × × × × × × × m. crassus 3 1 0.25 × m. zarudnyi1 72 1 0.25 × m. meridianus2 64 2 0.51 × × g. nanus3 11 2 0.51 × c r ic e ti d a e m. paradoxus4 9,20,61,67,71 9 2.27 × × m. transcaspicus5 67 3 0.76 × × e. fuscocapillus 51 1 0.25 × c. migratorius 16,21,28,29,60,69 17 4.29 × c a lo m y sc id a e c. elburzensis 16,20,36,37,42,53, 58,61,64 17 4.29 × c. mystax 50, 53, 57 5 1.26 × s c iu r id a e s. fulvus 4 1 0.25 × g li r id a e d. nitedula 16,28, 35,44,61 14 3.54 × × d ip o d id a e a. elater 11,12 14 3.54 × j. blanfordi 10 1 0.25 × 1. one specimen of m. zarudnyi belonged to museum samples. 2. two specimens of m. meridianus belonged to museum samples. 3. one specimen of g. nanus belonged to museum samples. 4. five specimens of m. paradaxus belonged to museum samples. 5. two specimens of m. transcaspicus belonged to museum samples. (ln: locality number, a: pasturage area, b: urban area, c: deciduous forest, d: scrub woodlands, e: semi desert and salt land, f: sandy dunes, g: steppe scrub lands, h: juniper's forest, i: fields and gardens, j: adjacent stream and river, k: mountain area) http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/sciuridae j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 132 http://jad.tums.ac.ir published online: june 12, 2018 discussion this is the first study that reports rattus norvegicus, m. crassus, allactaga elater, jaculus blanfordi and h. indica in north khorasan. extensive distribution of fields and pasturage in the study region provides suitable habitat for the existence of different kinds of rodents. in the present study, j. blanfordi captured from sand dunes and m. crassus trapped in semi-desert and salt lands were new records from the region. this species was previously reported from southern parts of iran, and a new additional record was added from north khorasan to its occurrence range. meriones crassus is distributed in south-western palaearctic (14) and in this study, we reported it from the north-eastern part of its distributional range. rattus norvegicus had been reported only from mashhad in northeast of iran (7) and this study is the first record of the species from north khorasan province. allactaga elater reported previously from south khorasan and khorasan razavi (7) and we reported this species from north khorasan for the first time. meriones persicus, m. libycus, m. musculus, n. indica, and r. opimus were the most abundant rodent species, especially in rural areas. these species are common both in iran and turkmenistan. central parts of iran demonstrated actually different fauna from turkmenistan region, however, northeast of iran (khorasan) was affected by both cradles in species composition and also correlated to endemism (15). in total, genus meriones is known as a natural reservoir of yersinia pestis in endemic foci of plague in iran and genus microtus, mesocricetus, allactaga, cricetulus play a minor role (16). mus musculus (reservoir of hymenolepis and syphacia) and n. indica (a secondary host for leishmania turanica) are rodents with zoonotic importance (17, 18) and were captured in high numbers through the northern and central parts of the province in farms and rural areas near the streams. apodemus witherbyi were also one of the most abundant rodents in most of the habitats except very dry sand dunes and urban areas. the wood mice of the genus apodemus collected from most of north khorasan with relatively high level of humidity were considered to be the vector for babesia, hepatozoon, trypanosoma, and grahamella (19). dryomys nitedula are distributed in gardens and forests around villages in the north khorasan province. dryomys nitedula, microtus, mus, and calomyscus have recently demonstrated positive results for francisella tularensis and can play a role to transmit tularemia in iran (20). in this study, some of the most important reservoirs of leishmaniasis, leptospirosis, tularemia, plague and other well-known reservoirs of zoonotic diseases were collected. the most records of cutaneous leishmaniasis in north khorasan province in the recent years were from rural areas of garmeh, jajarm, bojnurd, and esfarayen (21), which is consistent with our results of collecting the highest number of m. libycus, m. persicus and r. opimus as the most well-known hosts of the cutaneous leishmaniasis. previously, r. opimus was reported as a host of l. major in esfarayen (9) county and golestan province (adjacent province to the north khorasan) (22). north khorasan province is one of the most important foci of visceral leishmaniasis in iran (23) and the disease agent was isolated from m. persicus and c. migratorius (24) while, m. persicus was one of the most abundant rodent species and significant numbers of c. migratorius were captured in the province. rattus norvegicus collected from north khorasan province, was previously reported as one of the reservoirs of giardia muris (protozoan) and some other parasites in ahvaz, southwest of iran (25) and leptospirosis (6, 26). regarding to increasing population of norwegian rats in the cities, their population should be controlled to prevent potential risk to public health. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 127–134 k arzamani et al.: faunistic study of … 133 http://jad.tums.ac.ir published online: june 12, 2018 conclusion our results demonstrate the high biodiversity of rodents in north khorasan province of iran. meriones percicus, m. libycus, a. witherbyi and m. musculus, are present in most localities. their massive presence in this region is important medically and agriculturally. the present study is a contribution to the ecological study of rodents in this region and an initiation to medically aspects of rodents as the reservoir of some important zoonotic diseases in the northeast of iran. acknowledgements this study was supported by north khorasan university of medical sciences, iran (research no: 296), dr darvish grants from university of ferdowsi (research no: 1.19727) and ministry of health, iran. the authors would like to thank the staff of the department of biology of ferdowsi university and vectorborne diseases research center of north khorasan university of medical sciences for their kind cooperation. voucher specimens are stored at ferdowsi university and vectorborne diseases research center museum of north khorasan university of medical sciences, bojnurd, iran. the authors declare that there is no conflict of interests. references 1. etemad e (1967) mammals of iran. vol. 1 (rodents and key to their identification). national society of natural sources and human environment protection publications, tehran, p. 288. 2. meerburg bg, singleton gr, kijlstra a (2009) rodent-borne diseases and their risks for public health. crit rev microbiol. 35(3): 221–270. 3. akhoundi m, mohebali m, asadi m, mahmodi mr, amraei k, mirzaei a (2013) molecular characterization of leishmania spp. in reservoir hosts in endemic foci of zoonotic cutaneous leishmaniasis in iran. folia parasitol. 60 (3): 218–224. 4. arzamani k, salehi m, mobedi i, adinezade a, hasanpour h, alavinia m, darvish j, shirzadi mr, mohammadi z (2017) intestinal helminths in different species of rodents in north khorasan province, northeast of iran. iran j parasitol. 12(2): 267–273. 5. azizi mh, azizi f (2010) a history of the human plague in iran. arch iran med. 13(6): 563–569. 6. darvish j, arzamani k, abdolahpoor g, shirzadi m, mohammadi z, alavinia m (2016) rodent leptospirosis in north khorasan province, northeast of iran. int j infect dis. 45: 465. 7. darvish j, siahsarvie r, mirshamsi o, kayvanfar n, hashemi n, sadeghi shakib f (2007) diversity of the rodents of northeastern iran. iran j anim biosyst. 2(1): 57–76. 8. kassiri h, javadian e, abdigoudarzi m (2011) 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iran j parasitol. 8(3): 348. 25. kia e, homayouni m, farahnak a, mohebali m, shojai s (2001) study of endoparasites of rodents and their zoonotic importance in ahvaz, south west iran. iran j public health. 30(1–2): 49–52. 26. esfandiari b, pourshafie mr, gouya mm, khaki p, mostafavi e, darvish j, bidhendi sm, hanifi h, nahrevanian h (2015) an epidemiological comparative study on diagnosis of rodent leptospirosis in mazandaran province, northern iran. epidemiol health. 37: e2015012. http://jad.tums.ac.ir/ https://www.researchgate.net/scientific-contributions/71289369_a_baghaei https://www.researchgate.net/profile/behroz_davari3 https://www.researchgate.net/profile/parviz_parvizi http://pubmedcentralcanada.ca/pmcc/solr?term=author:(soheila%20moradi%20bidhendi) http://pubmedcentralcanada.ca/pmcc/solr?term=author:(soheila%20moradi%20bidhendi) http://pubmedcentralcanada.ca/pmcc/solr?term=author:(hamed%20hanifi) http://pubmedcentralcanada.ca/pmcc/solr?term=author:(hossein%20nahrevanian) j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 276 http://jad.tums.ac.ir published online: september 30, 2018 original article ixodid tick vectors of wild mammals and reptiles of southern india k. g. ajith kumar 1, *reghu ravindran 1, joju johns 2, george chandy 2, kavitha rajagopal 3, leena chandrasekhar 4, ajith jacob george 5, srikanta ghosh 6 1department of veterinary parasitology, college of veterinary and animal sciences, pookode, lakkidi, kerala, india 2centre for wildlife studies, college of veterinary and animal sciences, pookode, lakkidi, kerala, india 3department of livestock products technology, college of veterinary and animal sciences, pookode, lakkidi, kerala, india 4department of veterinary anatomy, college of veterinary and animal sciences, pookode, lakkidi, kerala, india 5department of veterinary pathology, college of veterinary and animal sciences, pookode, lakkidi, kerala, india 6entomology laboratory, division of parasitology, indian veterinary research institute, izatnagar, india (received 7 mar 2015; accepted 15 june 2018) abstract background: we aimed to focus on the ixodid ticks parasitizing wild mammals and reptiles from wayanad wildlife sanctuary, western ghat, southern india. methods: the taxonomic identification of ticks collected from wild mammals and reptiles was performed based on the morphology of adults. results: we revealed eight species of ticks including, amblyomma integrum, rhipicephalus (boophilus) annulatus, haemaphysalis (kaiseriana) spinigera, h. (k.) shimoga, h. (k.) bispinosa, h. (rhipistoma) indica, rhipicephalus haemaphysaloides and r. sanguineus s.l. collected from nine species of wild mammals while four tick species ablyomma kraneveldi, a. pattoni, a. gervaisi and a. javanense parasitizing on four species of reptiles. the highest host richness was shown by h. (k.) bispinosa and r. haemaphysaloides parasitizing six and five different host species, respectively. reports of r. (b.) annulatus on sambar deer, a. javanense and a. kraneveldi on python as well as a. pattoni on indian rat snake are the new host records from this region. conclusion: eight species of ticks parasitizing on nine species of wild mammals and four species of parasitizing on four species of reptiles were identified. the highest host richness was shown by h. (k.) bispinosa and r. haemaphysaloides. h. spinigera as the vector of kfd was also identified in this study. keywords: ticks, wild mammals, reptiles, wayanad, south india introduction ticks (ixodida) are obligate, non-permanent ectoparasites of terrestrial vertebrates (1). they are exclusively haematophagous in all feeding stages of their life cycle and have considerable medical and veterinary importance (2). besides, causes great economic losses to the livestock worldwide (3). currently, 904 valid tick species have been listed throughout the world (4–13). ticks parasitize a wide range of vertebrate hosts and transmit a variety of pathogenic agents than any other group of arthropods (14, 15). heavy infestation can cause blood loss, reduced weight gain and lowered milk production, even some tick species downgrade quality of hides (16). it is estimated that 80 per cent of world’s livestock population is suffering from the deleterious effects of ticks (17). nearly, 106 argasid and ixodid tick species infesting domestic, wild and game animals were documented from india (18). the ixodid tick r. (b.) microplus is the most prev*corresponding author: dr reghu ravindran, email: drreghuravi@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 277 http://jad.tums.ac.ir published online: september 30, 2018 alent and economically important species infesting livestock in india (19). on the global basis the losses incurred by livestock industry due to ttbds was estimated in the range of 14000 to 18000 million us $ / year (16). the annual cost of control of ttbds in india has been estimated as us $ 498.7 million (20). from the stand point of global biodiversity conservation, ticks are playing a significant role, as they are able to affect the fitness of wild life species by spill over epizootic outbreaks (21). moreover, wild animals can act as reservoirs of infectious organisms and ticks can transmit them into domestic animals and humans. over the last few decades approximately 75 per cent of emerging diseases, including zoonoses, having wildlife origin (22). western ghats or the sahyadri of southern india with an area of 17,000km2 run parallel to the west coast of peninsular india stretching from cape comorin (or kanyakumari) in the south to the surat dangs in gujarat in the north. human and livestock population existing as high densities in this region (23, 24). wayanad wildlife sanctuary (76º02’ to 76º 27’ east longitude and 11º 35’ to 11º 51’ north latitude) with an area of 344 sq. km. is set lofty on the majestic western ghats with altitude ranging from 650 to 1150m above the sea level. rich in wild animals biodiversity, the sanctuary is an integral part of the nilgiri biosphere reserve. deadly tick borne viral infections like kyasanur forest disease (kfd) were reported from humans in karnataka (25) and kerala (26, 27) with or without mortality and crimean-congo haemorrhagic fever (cchf) from gujarat while cchfv-specific antibodies were detected in human samples from kerala (28). previous reports on tick vectors of wildlife of southern india are scanty. hence, an active surveillance was initiated to document the possible ixodid tick vector species from the free ranging mammals and reptiles of the western ghats of wayanad of kerala, india. materials and methods study area study are comprised of the entire wayanad wildlife sanctuary (76º 02’ and 76º 27’ east longitude and 11º 35’ and 11º51’ north latitude) and adjoining area in the wayanad district of kerala, india. animal and tick collection wild animals are regularly brought to the college of veterinary and animal sciences, pookode by the officials of department of forest, kerala for postmortem examination (animals died due to hunterattack, malicious poisoning or trapped), treatment and for health checkup prior to release back into the forest. dead animals are surveyed in a short-time window (within 24 hoursafter death). a total of 46 wild mammals of 16 different species and 23 reptiles of nine species were included in the present study (table 1). body of these animals was examined for the presence of adults and engorged nymphs of tick. adult ticks were collected in glass tubes and immediately transported to the parasitology laboratory for identification. if identification was not possible on day of collection, the collected ticks were stored for 24h in boardman’s solution i (17% ethanol, 3% ether and 80% water). then, for long term storage, they were transferred to solution ii (80% ethanol, 15% water, 5% glycerol) to which 1% chlorform is added to prevent the colour change. engorged live nymphs were immediately placed in bod incubator at 28 °c and rh 85% for moulting to adults. tick identification the taxonomic identification was performed based on the morphology of adult ticks according to standard keys and monographs (29–36). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 278 http://jad.tums.ac.ir published online: september 30, 2018 results out of 46 wild mammals and 23 reptiles, 12 species of ixodid ticks belonging to five genera were identified (table 1, fig. 1). of the 16 mammalian host species, seven were free from any tick infestation. amongst the identified tick species, haemaphysalis (kaiseriana) bispinosa neumann, 1897 was the most prevalent species while rhipicephalus sanguineus s.l. latreille, 1806 was the least. ticks belonging to the genus hyalomma, ixodes and dermacentor were not identified in the present study. amongst the tick species collected from reptiles, amblyomma gervaisi lucas, 1847 was retrieved from three species of pythons, i.e., at a time only one species of tick was collected from each python. sambar, spotted, barking and mouse deers, wild pig, tiger and leopard were infested with more than one species of tick. a minimum of three species of ticks were retrieved from each of the 11 examined sambar deers, with a total of six species identified in them. all the wild pigs examined were parasitized by a. integrum and r. haemaphysaloides with the exception r. sanguineus s.l. found only in one animal. in all mammalian species, ticks were present throughout the body with more infestation on the external surface of the ear pinna and neck. in snakes, ticks were attached between and below the scales with no ticks were seen attached to the ventral aspect of the body. only a. gervaisi lucas, 1847 could be collected from monitor lizards. male a. gervaisi was collected from the lateral side of the body, axilla of the left forelimb and the periphery of cloaca / ventral depression just behind cloaca. female ticks were collected from the axillary region and between the toes of forelimbs. table 1. species of ticks detected on the wild mammals and reptiles of wayanad region of western ghats no. name of wild animal host examined number of host examined tick species no. of tick collected life stage (nymph (n) /adults(a)) 1 sambar deer [cervus unicolor kerr, 1792] 11 amblyomma integrum karsch, 1879 rhipicephalus (boophilus) annulatus say, 1821 r. haemaphysaloides supino, 1897 h. (kaiseriana) bispinosa neumann, 1897 haemaphysalis (kaiseriana) spinigera neumann, 1897 h. (k.) shimoga hoogstraal and trapido, 1964 85 36 40 96 35 27 10 adult adult nymph adult adult adult adult 2 spotted deer [axis axis (erxleben, 1777)] 2 r. (b.) annulatus say, 1821 r. haemaphysaloides supino, 1897 h.(k.) bispinosa neumann, 1897 10 14 26 adult adult adult 3 barking deer [muntiacus muntjak (zimmermann, 1780)] 4 r. (b.) annulatus say, 1821 r. haemaphysaloides supino, 1897 h.(k.) bispinosa neumann, 1897 5 12 42 adult adult adult 4 mouse deer [moschiolaindica (gray, 1852)] 2 h.(k.) bispinosa neumann, 1897haemaphysalis (k.) spinigera neumann, 1897 20 5 adult adult 5 gour [bos frontalis lambert, 1804] 1 h. (k.) shimoga hoogstraal and trapido, 1964 5 adult 6 wild pig [sus scrofa linnaeus, 1758] 4 amblyomma integrum karsch, 1879 r. haemaphysaloides supino, 1897 r. sanguineus s.l. latreille, 1806 10 10 5 adult adult adult http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 279 http://jad.tums.ac.ir published online: september 30, 2018 7 tiger [panthera tigris (linnaeus, 1758)] 1 h. (k.) bispinosa neumann, 1897 48 adult 8 leopard [pantherapardus (linnaeus, 1758)] 3 r. haemaphysaloides supino, 1897 h.(k.) bispinosa neumann, 1897 h. (rhipistoma) indica warburton, 1910 8 10 5 adult adult adult 9 malabar giant squirrel [ratufa indica (erxleben, 1777)] 2 h. (k.) spinigera neumann, 1897 5 adult 10 leopard cat [prionailurus bengalensis (kerr, 1792)] 4 nil nil nil 11 bonnet macaque [macaca radiata (geoffroy sainthilaire, 1812)] 7 nil nil nil 12 slender loris [loris tardigradus (linnaeus, 1758)] 1 nil nil nil 13 small indian civet cat [viverricula indica (geoffroy saint-hilaire, 1803)] 1 nil nil nil 14 common palm civet [paradoxurus hermaphroditus (pallas, 1777)] 1 nil nil nil 15 indian giant flying squirrel [petaurista philippensis (elliot, 1839)] 1 nil nil nil 16 indian grey mangoose [herpestes edwardsii (é. geoffroy saint-hilaire, 1818] 1 nil nil nil 17 monitor lizard [varanus bengalensis bengalensis (linnaeus 1758)] 2 amblyomma gervaisi lucas, 1847 8 adult 18 python [python molurus linnaeus, 1758] 5 a. gervaisi lucas, 1847 a. javanense supino, 1897 a. kraneveldi anastos, 1956 5 4 3 adult adult adult 19 cobra [naja naja linnaeus 1758] 1 a. gervaisi lucas, 1847 2 adult 20 indian rat snake [ptyas mucosa linnaeus 1758] 3 a. pattoni neumann, 1910 2 adult 21 russel viper [daboia russelii shaw and nodder 1797] 1 nil nil nil 22 ceylone cat snake [boiga ceylonensis (günther, 1858)] 2 nil nil nil 23 montane trinket snake [coelognathus helena monticollaris (schulz, 1992)] 3 nil nil nil 24 common vine snake [ahaetulla nasuta lacépéde 1789] 3 nil nil nil 25 checkered keel back [xenochrophis piscator schneider 1799] 3 nil nil nil table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 280 http://jad.tums.ac.ir published online: september 30, 2018 fig. 1. amblyomma gervaisi male: dorsal view (a) ventral view(b), a. kraneveldi female: dorsal view (c) ventral view (d), a. pattoni male: dorsal view (e) ventral view (f), a. integrum male: dorsal view (g) ventral view (h), a. integrum female: dorsal view (i), a. javanense female: dorsal view (j) ventral view (k), cleared specimen of rhipicephalus (b.) annulatus male: dorsal view (l), cleared specimen of haemaphysalis (kaiseriana) bispinosa male: ventral view (m), h. indicamale: dorsal view (n) ventral view (o), h. shimoga male: ventral view (p), h. (kaiseriana) spinigera male: ventral view (q): r. haemaphysaloides male: ventral view (r), r. sanguineus s.l. male: ventral view (s). (figures not to scale) discussion among the reported species of ticks from india, a. testudinarium, d. auratus, h. bispinosa, h. spinigera, h. intermedia, hyalomma anatolicum anatolicum, h. marginatum isaaci, h. hussaini, h. detritum, h. kumari, b. microplus, i. acutitarsus, i. ovatus, n. monstrosum, r. haemaphysaloides and r. turanicus are the most widely distributed ticks of cattle, buffalo, sheep and goat (37). among this species, i. acutitarsus and i. ovatus were reported mainly from eastern and north-eastern states of the country (19). haemaphysalis bispinosa and r (b.) microplus are prevalent throughout india, while h. spinigera is restricted to southern states, central zones, orissa and meghalaya (19). a total of 23 species of ticks were reported in domestic and wild animals from the different parts of kerala state (19, 38, 39). the species of ixodid ticks reported from kerala include, r.(b.) annulatus, r.(b.) microplus, r.(b.) decoloratus, r. sanguineus s.l., r. haemaphysaloides, r. turanicus, h. bispinosa, h. intermedia, h. aculeata, h. cuspidata, h. knobigera, h. turturis, h. spinigera, h. anatolicum, h. marginatum isaaci, h. hussaini, a. integrum, n. monstrosum, and n. keralensis (38, 39). a total of 35 species of ticks were reported from sambar deer throughout its native range and introduced habitats (40) which include 11 species from two extreme ends of india, southern (comprising karnataka and kerala states) and the northeastern ends (assam). the possibility of spreading of ticks from the northeastern states to the southern state is very difficult as there is no practically animal movement practically between these states. only five species of ticks were recorded from the karnataka and one (h. sambar) from kerala (40). in the present study, six species of ticks were recorded on sambar deer from wayanad, kerala, none of the specimen was conforming to the morphology of h. sambar. as well as, in the present study, r. (b.) annulatus recorded for the first time on sambar deer showing the status of a new host for this species. among all the sambar deer examined, at least three species of ticks were observed in each animal, and the presence of a. integrum was a constant feature. in the present study, r. (b.) annulatus, r. haemaphysaloides and h. (k.) bispinosa were also recorded on both spotted deer (axis axis) and http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 281 http://jad.tums.ac.ir published online: september 30, 2018 barking deer as previously reported by miran puri (41). the presence of a. integrum, r. haemaphysaloides and r. sanguineus s.l. in wild boars observed in the present study so agrees with the tick-host relationship (41). less frequency of r. sanguineus s.l. in wild pig in the present study corroborates with previous report (42). sus scrofa is a major host for adults of d. auratus, which also infests bear, rhinoceros and deer of primary and secondary forests (mostly at altitude below 400m) of india, sri lanka, nepal, bangladesh, burma, thailand, vietnam, laos, peninsular malaysia, and sumatra (43). we could not record this species from any wild animals. however, an adult male d. auratus was recently recorded from a man trekking through the forest of wayanad region (44). literature reveals h. (k.) bispinosa was not recorded from leopards and tigers in western ghats (45). however, haemaphysalis sp. was reported in leopard at nagpur, maharashtra (46). similarly, a distinctive small member of the h. (k.) bispinosa group, h. (k) ramachandrai, was recorded on sambar deer, barking deer, chital deer, tiger, leopard, domestic cattle, buffalo and goats from forest lowlands of the himalayan foothills of india and nepal. h. bipsinosa is a ubiquitous medically important parasite of domestic animals in india transmitting various diseases in domestic animals (19). the present finding of r. haemaphysaloides and h. (rhipistoma) indica infestations in leopard corroborated with previous findings (36, 41). four species of ticks were collected from four out of the eight species of snakes examined in the present study. based on the available reports, a. javanense and a. kraneveldi on python and a. pattoni on indian rat snake are the new records. recent survey on ticks of snakes in the north western ghats recorded only a. gervaisi on two species of snakes viz., indian rat snake and spectacled cobra. amblyomma gibsoni, a. varanensis and a. gervaisi were previously reported in monitor lizard (34, 47, 48). however, a. gervaisi was the only tick species observed in monitor lizards in the present study. the major infectious organisms of ruminants transmitted by common tick species in india are, theileria annulata (transmitted by hyalomma anatolicum and h. marginatum isaaci), babesia bigemina, anaplasma marginale and ehrlichia bovis (transmitted by r. (b.) microplus), b. motasi (transmitted by haemaphysalis spp.) and b. ovis (transmitted by rhipicephalus spp.) (37). the occurrence of t. annulata and b. bigemina was reported from the whole india while a. marginale, e. bovis and e. phagocytophila is confined to some restricted zones. hepatozoon canis, ehrlichia canis, mycoplasma haemocanis, anaplasma platys, b. vogeli and b. gibsoni are the tbd pathogens found infecting dogs in india due to the potential tick vectors, rhipicephalus (most commonly) and /or haemaphysalis ticks (49). in humans, lyme disease, kyasanur forest disease (kfd), crimean-congo hemorrhagic fever (cchf) and babesiosis are some of the important tick borne zoonoses reported from india (25, 50). human babesiosis and cchf were reported from gujarat state (50, 51) of northern india. kyasanur forest disease (kfd) was originally recognized as a febrile illness in the shimoga district of karnataka state of india (52). during 2013, only single case of kyasanur forest disease (kfd) was reported without any mortality in humans from wayanad, kerala (26) while eleven confirmed cases, one death and eight suspected cases were already reported in the month of february 2015 (27). the principal vector for kfd, h. (k.) spinigera was identified in the present study. dermacentor auratus reported previously from a human (44) from wayanad can also act as vector for the disease. hence, it could be possible that kfd may spread into more and more areas of kerala in future. lyme disease in humans was documented from http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 276–285 k. g. a kumar et al.: ixodid tick vectors of … 282 http://jad.tums.ac.ir published online: september 30, 2018 northern and north eastern india (53, 54). lyme disease was reported recently from wayanad too (26, 55) even though its tick vector could not be established. the information gathered in the present study will be useful for public health specialists, medical professionals, zoologists, parasitologists and other professionals for designing tick control strategies for the entire southern india to prevent the possible emergence of newer tick borne diseases especially zoonoses. conclusions twelve species of ticks from wild mammals and reptiles were recorded from southern india suggesting the contribution of wild life for tick abundance and prevalence in the tick fauna of this region. haemaphysalis (k.) bispinosa was common among the wild ungulates and the large carnivores. as well as, h. 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[accessed 2 march 2013]. http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/pubmed?term=bajpai%20s%5bauthor%5d&cauthor=true&cauthor_uid=21751625 http://www.ncbi.nlm.nih.gov/pubmed?term=nadkar%20my%5bauthor%5d&cauthor=true&cauthor_uid=21751625 http://timesofindia.indiatimes.com/city/kozhikode/lyme-disease-outbreak-in-wayanad/articleshow/18758675.cms http://timesofindia.indiatimes.com/city/kozhikode/lyme-disease-outbreak-in-wayanad/articleshow/18758675.cms http://timesofindia.indiatimes.com/city/kozhikode/lyme-disease-outbreak-in-wayanad/articleshow/18758675.cms microsoft word jad-12-108 j arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 108 http://jad.tums.ac.ir published online: june 12, 2018 original article discriminating clinical and biological features in malaria and dengue patients *wajihullah khan 1, haytham a. zakai 2, khadija khan 1, sharba kausar 1, sana aqeel 1 1section of parasitology, department of zoology, aligarh muslim university, aligarh, india 2faculty of applied medical sciences, king abdulaziz university, jeddah, kingdom of saudi arabia (received 14 july 2015; accepted 24 apr 2018) abstract background: malaria and dengue are the most widespread infectious diseases of tropical countries with an estimated 219 and 50 million cases globally. the aim of the proposed study was to find out discriminating clinical features of falciparum malaria and dengue. method: falciparum malaria was diagnosed by looking at the ring and gametocyte stages by microscopic examination in giemsa stained slides. dengue was diagnosed by elisa for dengue-specific igm and igg. liver enzymes (ast and alt) and kidney markers (creatinine and urea) were estimated by standard biochemical techniques. result: ast and alt showed similar rise in both, severe malaria and dengue patients but it was much pronounced in dengue haemorrhagic fever where it attained 3–4 folds increase. creatinine and urea showed higher levels in dengue compared to malaria. thrombocytopenia (76.27%), convulsions (18.64%) and hepatic dysfunction (5.08%) were more prominent in dengue than that in malaria where these parameters were 50.89, 7.14 and 2.67%, respectively. conversely, cases with anaemia, splenomegaly and jaundice were three times more in falciparum malaria. acute renal failures and neurological sequelae were noticed in slightly higher number of dengue patients. conclusion: thrombocytopenia and hepatic dysfunction were more common in dengue, while anaemia, splenomegaly, jaundice and convulsions were more frequent in falciparum malaria. neurological sequelae and cases of acute renal failure were almost equal in both the infections. keywords: malaria, dengue, clinical features, liver enzymes, kidney markers introduction dengue and malaria are the most common mosquito-borne diseases which emerged as a global public health problem. single stranded rna flavivirus (denv 1–4) is the causative agent of dengue and is generally transmitted by aedes aegypti to around 50 million people of 2.5 billion population at risk throughout the world (1). disease caused by dengue virus manifests as relatively minor febrile illness called dengue fever (df) to a life-threatening dengue haemorrhagic fever (dhf)/ dengue shock syndrome (dss) (2). malaria is another vector-borne disease caused by different species of plasmodium and is transmitted by anopheline vectors in tropical countries to an estimated number of 219 million people re sulting in 660,000 deaths every year throughout the world (3). patients suffering from these diseases show somewhat similar clinical and biological presentation with variable pathological conditions (4). in malaria as well as dengue, patients suffer from high fever, headache, vomiting and severe body pain. majority of the dengue patients infected with flavivirus are asymptomatic but in a small proportion of cases the disease develops into the life-threatening dhf/ dss, resulting in blood plasma leakage, bleeding and low levels of blood platelets with mortality rate of around 5% (5). elevated values of liver enzymes were also observed in more than half of the patients in brazil (6). like dengue, hepatic dysfunc*corresponding author: dr wajihullah khan, email: wajihullahkhan@yahoo.co.in j arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 109 http://jad.tums.ac.ir published online: june 12, 2018 tion and renal failures were observed in patients who suffered from plasmodium falciparum (7–9). since many symptoms and clinical features are common in falciparum malaria and dengue, the proposed study entitled “discriminating clinical and biological features in malaria and dengue patients” was carried out to ascertain the response of biological markers in vital organs which could be of help in differential diagnosis of dengue and malaria. materials and methods the present study was carried out during 2014–15 from makkah region of saudi arabia and northern zone of india based on the confirmed dengue and malaria cases. a total of 380 and 183 suspected cases were examined for the positivity of malaria and dengue, respectively. we have used the leftover blood samples taken for diagnosis by the doctors after taking consent of the patients and ethical approval of the respective institutes. clinical profiles of the febrile patients were recorded at the time of their admission in the hospital. thick and thin blood smears were prepared on glass slides by pricking the finger of the patients complaining for high fever, vomiting and headache. thin blood films were fixed in methanol, stained with giemsa and examined under nikon eclipse e-600 research microscope at x1000. typical ring and gametocyte stages were observed for the specific diagnosis of p. falciparum. patients with fever, headache, convulsions, body ache and haemorrhagic manifestations were tested for dengue fever (df) and dengue haemorrhagic fever (dhf). clinical symptoms such as high fever, headache, thrombocytopenia, splenomegaly, convulsions and neurological sequelae were observed and compared in the confirmed cases of falciparum malaria, dengue and dengue haemorrhagic fever. for non-structural protein 1 (ns1) antigen detection, elisa was performed in patients who had fever for 3–5d, while those who had high fever for more than 5 d after the onset of illness were tested for dengue specific igm and igg. serological studies were performed with the qualitative dengue igm capture elisa and dengue indirect igg elisa (pan bio ltd, brisbane, australia). dengue patients were treated with acetaminophen and symptomatic therapy in the form of fluid and antipyretics whereas patients suffering from falciparum malaria were advised artesunate, as chloroquine and fansidar are not responding in a fairly good number of cases in our experimental areas. thrombocyte counts were recorded in both malaria and dengue patients. for the estimation of liver and kidney markers, blood was collected in sterile glass test tubes by vein puncture of the malaria and dengue patients after taking their consent. these samples were allowed to clot at room temperature and then transferred to refrigerator at 5 °c for 12h to squeeze the clot. serum was collected and centrifuged at 2000rpm for 5min to remove probable contaminations and finally stored in sterilized vials at -80 °c until analyzed for liver enzymes and kidney markers. aspartate transaminase (ast) and alanine transaminase (alt) were estimated (10), while creatinine and urea were estimated as already reported (11, 12), respectively. follow up of all the patients was done till they were discharged from the hospital. all the dengue and malaria cases were monitored for their clinical, biochemical and haematological profiles. clinical parameters of dengue and malaria positive patients were studied and compared to observe prominent discriminating features of these infections. statistical analysis all clinical parameters were examined by chi-square test using graphpad instat 3.06 software (graphpad software, inc., california, usa), and the level of significance was set at p< 0.05. the results were expressed as mean ± standard deviation (sd) using spss (version j arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 110 http://jad.tums.ac.ir published online: june 12, 2018 17.0, spss inc., chicago, il, usa). graphs were plotted on sigma plot 12.0. results patient characteristics during peak season, 380 suspected cases with high fever and headache were examined for malaria from aligarh district during 2014– 15, out of which 232 (61%) were positive for malaria. out of 232 positive cases 112 (48.27%) patients were of p. falciparum, while 120 (51.72%) of p. vivax. as for dengue is concerned, 183 suspected cases having high fever, headache and body ache were examined during its active transmission season in 2014–15 from the northeastern region of india and makkah region of saudi arabia, out of which 59 (32.77%) were found positive for dengue. majority of the patients 47 (80%) were of classical dengue, while 12 (20%) patients showed the symptoms of dengue haemorrhagic fever. overall, 40 (67.79%) patients were found positive for igm and 12 (20.33%) were positive for igg. a total of 26 (44.06%) patients showed both igm and igg. during illness, gum bleeding and skin petechiae were seen in 3.39 and 6.80% patients, respectively. symptoms in the patients all the malaria and dengue patients had fever which ranged from 101 to 105 °c. headache, nausea, vomiting, dizziness and joint pain were the other prominent symptoms in both falciparum malaria and dengue patients (fig. 1). in this study prescribed dose of artemisinin was given to treat thirty two falciparum malaria patients. parasitaemia was cleared in all the cases except 1 (3.1%), in which parasites reappeared after 23 days, indicating resistance as recrudescence occurred. symptoms in patients infected with p. falciparum and dengue showed significant differences in skin rash, sweating, vomiting and body ache, but the values relating fever, headache, nausea, dizziness and abdominal pain were very close in both the diseases and did not show significant differences (fig. 1). haematological and systemic disorders there was contrasting difference as for splenomegaly is concerned, it was 43.75% in p. falciparum infection compared to 10.17% of dengue showing p-value 0.0001. thrombocytopenia in dengue patients was much higher compared to falciparum malaria (76.27: 50.89, p= 0.002). convulsions and hepatic dysfunction were more common in dengue (18.64 and 5.08%) than in malaria (7.17 and 2.67%). in contrast, jaundice was more common in malaria patients (6.25%), than in dengue (1.70%). acute renal failures and neurological sequelae were also slightly more pronounced in falciparum malaria compared to dengue in terms of percentage but the difference was not significant (fig. 2). anaemia was rarely observed in healthy individuals or patients having low or mild infections in both the diseases. liver and kidney markers liver damage was observed as a common complication in both malaria as well as dengue infections. liver function test (lft) was performed by estimating serum aspartate transaminase (ast) and alanine transaminase (alt). the normal values of these enzymes in applied technique were 0–45iu/l and 3–60iu/ l for ast and alt, respectively. both liver enzymes and kidney markers were almost within normal range in falciparum malaria and dengue fever (df) having low to moderate infections. slightly raised values of ast (64.70± 4.38, 98.83±22.15iu/l) and alt (79.23±5.70, 93.83±21.15iu/l) were recorded in severe cases of falciparum malaria and dengue fever, respectively. almost five times increase in the level of ast (215.50±41.71iu/l) and three times in alt (176.00±19.79iu/l) were recorded in severe dengue haemorrhagic fever followed by mild cases of dhf where ast and alt were recorded as 108.00±32.35 and j arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 111 http://jad.tums.ac.ir published online: june 12, 2018 75.33±12.66iu/l, respectively. however, these enzymes remained within normal range in light infections. impairments of renal function in p. falciparum and dengue infections were assessed by measuring elevated creatinine and urea in serum. patients suffering from falciparum malaria with high parasitemia, severe dengue and dengue haemorrhagic fever showed an increase in the level of creatinine to 158.76±6.99, 186 ±11.50 and 187.00±5.65µmoles/l, respectively compared to the normal range of 72–126 µmoles/l, while it was within range in low and mild infections with the exception of dhf, which showed a slightly raised value (135.33±25.38µmoles/l) in mild dhf patients. serum urea too showed slightly higher values in severe cases of malaria, df and dhf, which were recorded as 9.60±1.32, 8.10±0.91 and 10.20±1.97mmoles/l against the normal range of 3.0–6.0mmoles/l (fig. 3). it was slightly raised (6.90±0.36mmoles/l) in mild cases of dhf and was within normal range in falciparum malaria and dengue fever. symptoms fev er sk in ras h he ada che sw eat ing na use a vo mi tin g bo dy ach e diz zin ess ab dom ina l pa in p er ce nt a g e of p a ti e nt s 0 20 40 60 80 100 120 falciparum malaria denguep=0.512 p=0.0001 p=0.919 p=0.0001 p=0.186 p=0.035 p=0.0001 p=0.107 p=0.182 fig. 1. symptoms in the patients of falciparum malaria and dengue j arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 112 http://jad.tums.ac.ir published online: june 12, 2018 p=0.002 p=0.002 p=0.012 p=0.0001 p=0.337 p=0.043 p=0.977 p=0.846p=707 clinical parameters th rom boc yto pen ia ha em og lob in an aem ia sp len om eg aly ja un dic e co nvu lsi on s ne ur olo gic al se qu ela e ac ute re na l fa ilu re he pat ic dys fun cti on p e rc e nt a ge o f pa ti e n ts 0 20 40 60 80 100 falciparum malaria de ngue p=0.002 p=0.003 p=0.012 p=0.0001 p=0.337 p=0.043 p=0.977 p=0.846 p=0.707 fig. 2. haematological and systemic disorders in falciparum malaria and dengue patients test indi cators ast alt creatinine urea l ev el s o f m ar k er s 0 50 100 150 200 250 300 low df mild df severe df low dhf mild dhf severe dhf low pf mild pf severe pf fig. 3. liver and kidney markers in falciparum malaria, dengue and dengue haemorrhagic fever j arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 113 http://jad.tums.ac.ir published online: june 12, 2018 discussion malaria and dengue are true haematological infectious diseases as they affect most of the blood components of patients. anaemia, leucocytosis and thrombocytopenia are the commonly associated haematological complications in falciparum malaria, and have received much attention due to their associated mortality (13-15). in dengue and dengue haemorrhagic fever abnormalities manifest as bone marrow suppression, leucopenia and thrombocytopenia (16-18). all the malaria, dengue and dengue haemorrhagic fever patients had high fever as earlier observed for these infections in jazan and makkah region of saudi arabia, pakistan and india (18-21). in our study headache, nausea and vomiting were slightly more pronounced in dengue (77.96, 67.79 and 61.01%) compared to falciparum malaria (76.08, 56.52 and 43.47%). headache and nausea in dengue recorded in 75 and 69% patients in makkah region (22) were almost the same as recorded in our study. we observed vomiting in 61.01% dengue patients which was slightly less (43.47 %) in malaria patients. earlier workers noticed vomiting in slightly less number (56%) of patients of dengue in pakistan. compared to malaria (34.78%), dengue patients were more frequently associated (88.13%) with body ache in the present study. in contrast body ache was recorded in only 34.44% dengue patients in pakistan (18) which was less than half of what we observed in india and saudi arabia. abdominal pain and dizziness were slightly higher in dengue patients (64.40 and 42.85%) as compared to falciparum malaria (50.72 and 29.71 %) in our study. in our study, thrombocytopenia was recorded in 76.27% cases which have similarity with the earlier findings of pakistan and india where thrombocytopenia was recorded in 60 to 79.49% dengue patients (16, 17, 20). thrombocytopenia was less frequently associated with falciparum malaria and was noted in 50.89% patients in our study which is almost same as earlier observed in pakistan where 54.5% cases of p. falciparum had thrombocytopenia (23). in contrast its higher value (77%) was recorded in madhya paradesh, india (24). moderate degree of thrombocytopenia was observed in 26 to 50% df cases (25). nevertheless, deep and rapid decrease in platelet count is one of the main criteria for severe dengue (1). thrombocytopenia was observed in df and dhf in our study where thrombocyte counts varied from 20,000/mm3 to 140,000/mm3. forty four percent of dengue patients had low thrombocyte counts of around 50,000/mm3. continuous drop in thrombocyte count was observed by withana et al. (26) who reported a low count of 36,000/mm3 on 7th day of illness. it may be due to the suppression of bone marrow at an early stage which resulted in immunemediated platelet destruction, vasculopathy and coagulopathy as earlier hypothesized (27). in fact there is an increased utilization of platelets during coagulopathy which play a major role in the induction of thrombocytopenia. unlike dengue, thrombocytopenia was noticed in comparatively lesser number of falciparum malaria patients ranging between 80,000/mm3 and 145,000/mm3. it might be due to the invasion of large number of p. falciparum parasites which suppress bone marrow and spleen which might have resulted in immune-mediated platelet destruction which generally reflects in chronic cases. anaemia is an inevitable consequence in malaria due to hemolysis. we observed anaemia in 30.35% p. falciparum patients. in contrast, higher numbers of anaemia cases (60%) in p. falciparum infection were recorded in saudi arabia (28). low counts for anaemia of around 13% for p. falciparum was also recorded in mumbai (29). this variation might be due to severity of infection and level of immunity against the parasite in patients of falciparum malaria in different countries havj arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 114 http://jad.tums.ac.ir published online: june 12, 2018 ing endemic and non-endemic pockets. plasmodium falciparum within the rbc modifies the erythrocytes in several ways and the cell membranes become less deformable, rigid and fragile, resulting in hemolysis and anaemia. alteration in membranes of uninfected red blood cells by addition of glycosylphosphatidylinositol causes increased clearance of cells especially in p. falciparum infections that contributes to anaemia (30). anaemia was reported only in 11.86% cases of dengue. low rate of anaemia in dengue was probably because of a few systemic hemorrhages due to endothelial rupture as dengue virus targets monocytes or macrophages which are responsible for dissemination of virus (31). splenomegaly was more pronounced in p. falciparum infections (43.75%) compared to dengue where it was observed only in 10.17 % cases. higher rate of splenomegaly in falciparum malaria was probably due to phagocytosis of parasitized r.b.cs which got accumulated in the spleen for clearance. almost similar enlargement in around 39–45% p. falciparum malaria patients was observed in saudi arabia (19, 32) however, much higher rate of splenomegaly (71%) was recorded in falciparum malaria from rajasthan, india (33). this variation might be due to the difference in the immune status of the patients in different malaria transmission regions. in our study, 6.25% p. falciparum patients had jaundice which is much less than that recorded in sudan and pakistan where it was reported in 14 and 43% patients (34, 35). we noticed jaundice in only 1.70% dengue patients from our study area which is much less than that in falciparum malaria. slightly higher rate of jaundice (3.1%) was reported in vietnam (36). it appears that jaundice is an outcome of the combined effects of hemolysis and liver dysfunction and probably this is the reason why jaundice was more abundant in severe cases in p. falciparum infections where destruction of rbc is quite high. liver dysfunctions have been documented in earlier findings for both p. falciparum and dengue infections (26, 37, 38). in our study, mean ast and alt values were 64.70±4.38 and 79.23±5.70 in severe p. falciaprum cases, but the level of enzymes were within the normal range in those who had mild and low parasitaemia. comparatively lower levels of ast and alt (33.76–37.6 and 34.8–39.74) were recorded for mild p. falciparum infections in nigeria (9, 39). in contrast, higher levels of ast and alt (98.83±28.79 and 93.83±22.15iu/l) were recorded in the patients of dengue fever, which was still high in dengue haemorrhagic fever and was observed as 215±41.71 and 176± 19.79 in our study area. similar increase in ast and alt was observed in dengue patients of brazil (26). significant rise was recorded in the level of liver enzymes in the severely infected patients as compared to mild and low infections. liver damage in dengue infection ranges from mild lesion to fulminant hepatitis (6). it appears that the parasites during their hepatic phase destroy membrane of parasitized cells leading to leakage of the liver enzyme into the blood circulation. however, damage to hepatocytes and enzyme leakage due to toxicity of antimalarial administered to treat malaria cannot be ruled out. increased liver enzymes may result in liver dysfunction in patients having high parasitaemia in malaria. like malaria in dengue infections, inflammatory process in the liver parenchyma is provoked by the virus, reducing the lumen of biliary canaliculi, causing obstruction which may lead to bilirubinemia or even jaundice in a few cases (40). in dengue haemorrhagic fever hepatic injury leading to coagulopathy might have caused hemorrhage (41). acute renal failure (arf) is a common cause of morbidity and mortality in severe p. falciparum and dengue infections. we estimated kidney markers such as creatinine and urea in both malaria and dengue patients and recorded an elevated levels of these markers in both the diseases in our experimental arj arthropod-borne dis, june 2018, 12(2): 108–118 w khan et al.: discriminating clinical and … 115 http://jad.tums.ac.ir published online: june 12, 2018 eas. in dengue and malaria, levels of creatinine were raised to 187±5.65 and 158.76±6.99 moles/l, against normal range of 72–126 moles/l. in severe p. falciparum, df and dhf patients, urea was raised to 9.6±1.32, 8.10±0.91 and 10.20±1.97mmoles/l, respectively. we observed acute renal failures (arf) in 7.14% p. falciparum. comparatively, much higher rate of arf of 19% was recorded from the patients in pakistan (42). low rate of arf (5.08%) was observed in dengue patients of our study area in india and saudi arabia while earlier workers reported arf ranging from 3.3–10.8% in taiwan, pakistan and india (17, 43, 44). usually arf occurs in non-immune individuals especially in p. falciparum infections with high parasitaemia, but we noticed renal failures in dengue infections as well which warrants immediate attention and change in treatment strategy especially in resistant p. falciparum infections. physiological changes associated with p. falciparum infection make erythrocytes adherent to each other as well as to the walls of capillary vessels which cause mechanical obstructions. in addition to this, plasmodium encroach and cause inflammation of glomerular capillaries resulting in altered renal microcirculation by adherent rbcs and increased accumulation of creatinine which damage the kidney tissue. this might be the probable cause of renal failures as indicated earlier (7). neurological sequelae are associated with damaged neurons which manifest as impaired consciousness, convulsions which often lead to coma. cases of convulsions were more frequent in dengue (18.64%) than in malaria (7.14%) indicating more neurological damage in dengue than in falciparum malaria. immune mediated mechanism and direct tropic effect of virus are implicated in neurological manifestations, as dengue antigen has been demonstrated in the brain of few patients with dengue encephalitis (45). we observed neurological sequelae in 11.60% p. falciparum patients. other workers observed convulsions and other neurological deficits in 12–21% falciparum malaria patients of sudan, thailand and india (35, 46-48). still higher rate (28%) of neurological sequelae was reported in p. falciparum infections in mali (49). like dengue, in p. falciparum infection too, parasites invade brain tissue and cause tropic effect on neurons which probably results in neurological deficits. we observed neurological sequelae in 10.17% dengue patients which is almost same as that of 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(paris). 163: 583–588. 6dr oshaghi iran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 42 original article utility of filter paper for preserving insects, bacteria, and host reservoir dna for molecular testing f karimian, mm sedaghat, *ma oshaghi, f mohtarami, a sanei dehkordi, m koosha, s akbari, ss hashemi-aghdam department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 4 apr 2011; accepted 16 nov 2011) abstract background: appropriate methodology for storage biological materials, extraction of dna, and proper dna preservation is vital for studies involving genetic analysis of insects, bacteria, and reservoir hosts as well as for molecular diagnostics of pathogens carried by vectors and reservoirs. here we tried to evaluate the utility of a simple filter paper-based for storage of insects, bacteria, rodent, and human dnas using pcr assays. methods: total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. extracted dna of five different bacteria species as well as blood specimens of human and great gerbil rhombomys opimus was pipetted directly onto filter paper. the papers were stored in room temperature up to 12 months during 2009 until 2011. at monthly intervals, pcr was conducted using a 1-mm disk from the dna impregnated filter paper as target dna. pcr amplification was performed against different target genes of the organisms including the its2-rdna of mosquitoes, mtdna-coi of the sand flies and cockroaches, 16srrna gene of the bacteria, and the mtdna-cytb of the vertebrates. results: successful pcr amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. the pcr amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. time of storage did not affect the amplification up to one year. conclusion: the filter paper method is a simple and economical way to store, to preserve, and to distribute dna samples for pcr analysis. keywords: dna, pcr, insects, bacteria, vertebrate, preservation, filter paper introduction vector-borne diseases (vbds) such as malaria, dengue fever, leishmaniasis, filariasis, trypanosomiasis, yellow fever, onchocerciasis, plaque, and many other ones continue to threaten and to outbreak world health. annually around 500 million people are affecting just from three vbds: 300 m malaria cases, 120 million filariasis cases, and 50–100 m dengue cases (who 2000, 2009). the vbds account for 17% of the global disease burden (tabachnick 2010). understanding and predicting the vbds epidemiology relies on clear knowledge about the basic biology of the organisms and the interactions among vectors-pathogenshosts involved. correct identification and population genetics of vectors, also detection and identification of pathogen species within insect or arthropod vectors, human, or animal host reservoirs are important for predictions of the risk and expansion of the disease in the endemic areas. conventional methods such as dissection of individual insects under microscope, xenodiagnosis, culture media, and serological methods inherit a number of limitations including needs of fresh speci*corresponding author: dr mohammad ali oshaghi, e-mail: moshaghi@tums.ac.ir iran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 43 mens, low sensitivity and specificity, requirement to a highly skilled person, high expenses, and time consuming. on the other hand, dna based techniques are now available and becoming powerful tools for vbds research. molecular diagnostic tests that require dna or rna from specimens may be used to detect and to identify the vectors, pathogens, blood meal origin, and reservoirs species (oshaghi et al. 2003, 2006, 2008, 2010, 2011a, nakazawa et al. 2009, maleki-ravasan et al. 2010, mehravarana et al. 2011). the laboratories that are involved in vbds in endemic areas now gradually accommodate to use dna/rna based methods and this need cheap, easy, and reliable protocols that allow for the preservation of nucleic acids specimens. the currently recommended method for long-term storage of specimens involves storing them in freezer at minus 20 to -70°c. storage of the specimens in freezer takes a large amount of space particularly in the laboratory with high loads of samples. this might be less problematic for insect specimens but becomes extremely for tissues of animals. in molecular studies, dna is extracted from individual or pooled specimens using an expensive kit or by a complicated conventional protocol using proteinase k-containing dna extraction buffer followed by phenol/chloroform extraction and ethanol precipitation. these methods are suitable for molecular testing of vector, reservoirs, human, and pathogens; however, less effort and cost are desirable to improve the methods (weigle et al. 2002). transporting the specimens to the laboratory without loss of dna integrity under field conditions is important. conventionally the insect specimens preserve dry or in alcohol or lactophenol, and transfer in box or in cold chain. human or animal blood or tissues are preserved in citrated tubes and alcohol respectively. the specimens need additional labor costs for their storage if their dna is not immediately extracted. following dna extraction, specimens should be kept again in an ultralow-temperature freezer which is expensive and inconvenient and occupies a considerable amount of laboratory space. this is more crucial for vbds because the rate of infection in vectors usually is very low even in endemic areas (oshaghi et al. 2009, 2010, 2011b) and one should extract or keep the extracted dna of thousands insects individually. in addition, dna infection and degradation of dna by dnaase enzymes during the time of storage are another common issue raised in molecular laboratories. use of filter paper for storing dna initially have been used for a wide range of biological sources such as whole blood, buccal scrapes, tissues, plasmids, plant material and microorganisms (rogers and burgoyne 1997, devost and choy 2000, rogers and burgoyne 2000, natarajan et al. 2000, lampel et al. 2000, dobbs et al. 2002, hide et al. 2003, lampel et al. 2004) and later have been used for different goals in epidemiological studies of vbds. the main advantage of filter papers is the long-term stability of genomic dna within the cells of unfixed, dried whole body or blood cells stored at room temperature. the filter paper is provided in a small card size, which makes it possible to store several hundred samples at room temperature in a volume, the size of a small table driver. the filter paper matrix is impregnated with agents that denature infectious agents, and thus samples are no longer considered a biohazard. this allows for the storage and transport of samples without selective biohazard precautions. here, we investigated the use of filter paper cards as a means of storing either cell suspensions of different insect and vertebrate species including anopheles stephensi liston, culex quinqufaciatus say, phlebotomus papatasi scopoli, periplaneta americana iran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 44 linnaeus, blattella germanica linnaeus, human, and rodent rhombomys opimus lichtenstein or extracted dna of various bacteria comprising staphylococcus spp., hafnia alvei, cronobacter sakazaki, escherichia coli, and enterobacter spp. the stability of the dna within the suspension cells or the extracted dna from the bacteria stored for as long as one year onto the filter papers were tested by polymerase chain reaction (pcr) of different loci of the vertebrates, invertebrates, and microorganism genomes. materials and methods materials specimens of an. stephensi, cx. quinquefasciatus, p. americana, b. germanica and ph. papatasi were obtained from the insectariums of school of public health, tehran university of medical sciences (sph tums). human blood obtained from national blood center (tehran), and r. opimus blood tissues provided from tails of the animal reared in animal house of the sphtums. the strains of staphylococcus spp., hafnia alvei, cronobacter sakazaki, escherichia coli, and enterobacter spp bacteria obtained from individual colonies that were cultured in brain heart infusion (bhi). these bacteria were isolated from the midguts of american cockroaches that were collected from a confectionary shop in tehran. kawsar dna bank card (dbc®) paper was obtained from kawsar biotech company (kbc, ir iran) consisting of filter paper impregnated with a proprietary mix of chemicals, which serve to lyse cells, to prevent the growth of bacteria, and to protect the dna in the sample. the package also includes the reagents for the extraction of dna from the cells trapped on the filter paper. dna extraction fresh or alcohol preserved insect specimens were crushed individually using a glass pestle on dbc® card, allowed to air-dry and the cards were kept in sealed plastic bags at room temperature until subsequent experiments. haemolymphs of the field or laboratory cockroaches were collected using yellow tips after dissection of the specimens and were pipetted directly on the filter paper. for bacteria, colonies of the species were used for dna extraction using qiagen dneasy kit (qiagen, germany) according the manufacturer supplied procedure. also fifty to hundred micro liter of blood tissue of human or rodent r. opimus was pipetted directly onto the filter paper. based on the kind and volume of the specimen 3–10 square millimeter (mm2) of the paper might be impregnated with dna, which can provide 9–100 1-mm punches. the papers were stored in room temperature up to 12 months. at monthly intervals, the dna was extracted from the dried cells on the filter paper card according to the manufacturer’s instructions. a 1-mm hole was punched out from the center of the sample collection circle on the sample-impregnated filter paper and this punch was placed in a 1.5-ml microfuge tube for extraction of the dna. the disks were washed with 200µ l of dbc® buffer for 5 minute at room temperature. after removing the buffer, the punches were washed three times with 200µ l ddh2o for five minutes. then ddh2o completely emitted and the disks became air-dried at room temperature for 15 min. these washed filters were used directly in the pcr reactions. pcr pcr amplifications were performed using the sets of the specific primers previously used to amplify the its2-rdna of mosquitoes (litvaitis et al. 1994), mtdna-coi of insects (folmer et al. 1994), mtdna-cytb of vertebrates (boakye et al. 1999), and 16srrna of bacteria (favia et al. 2007). the sequences of the primers are given in table 1. amplification reaction was performed in iran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 45 volume of 20 µl containing 1-mm punch paper as target template, 10 pmol of each primer, and pcr master mix (intron®, south korea) includes 1.0 mm mgcl2, 100 µm dntps, 2 µl 10x pcr buffer, and 1unit of taq polymerase. for its2, pcr was optimized for mosquito templates. an initial denaturation of 5 min at 94 °c was followed by 32 cycles at 94 °c for 1 min, 57 °c for 1 min and 72 °c for 1 min. the final extension step was 72 °c for 10 min. for coi, pcr was optimized for sand flies and cockroaches templates. an initial denaturation of 2 min at 94 °c was followed by 5 cycles at 94 °c for 40 sec, 45 °c for 40 sec, 72 °c for 1 min and 35 cycles at 94 °c for 40 sec, 51 °c for 40 sec, 72 °c for 1 min. the final extension step was 72 °c for 10 min. for cytb, pcr was optimized for vertebrate templates. an initial denaturation of 5 min at 94 °c was followed by 35 cycles at 95°c for 1 min, 58 °c for 1 min and 72 °c for 1 min. the final extension step was 72 °c for 7 min. the universal primers of 16suf and 16sur (favia et al. 2007) were used to amplify about 1.5 kilo base (kb) partial sequence of the 16s rrna gene of the bacteria. the pcr conditions were set as an initial denaturation at 94°c for 10 minutes, followed by 35 cycles of denaturation at 95°c for 30 seconds, annealing at 56.5°c for 40 seconds, and extension at 72°c for 30 seconds, and final extension at 72°c for 8 minutes. fifteen microliters of pcr products were run along with a 100 bp ladder on a 1.2% agarose gel containing ethidium bromide for 1 h at 70v and observed on a uv transilluminator. results insects, bacteria, or blood samples were obtained for at least 20 specimens and their suspension cells or their extracted dna was stored on dna cards. these paper cards were kept at room temperature until use, with a variable storage period ranging from 30 days to one year. the suspension cells of sand flies, mosquitoes, and cockroaches as well as the bacterial dna were pale or did not stain the paper whereas the dried bloods stained the specimen cards. dna of the punched discs was tested in 1% agarose gel and quality and quantity were examined by comparison of the sharpness of dna bands. the yields and quality of dna per disc were variable based on the organisms and tissue stored on the paper. generally blood tissues made fewer smears and more sharpness dna than other tissues. the quality of purified dna was assessed by pcr amplifications of different loci of the organisms. except a few cases, results of pcr showed 100% success rate according to the presence/absence of the length specific pcr products (fig. 1–2). there were no differences in the robustness of the pcr reactions between the various species as well as their dna extracted at different times. the cells were stored on the filter paper cards up to 12 months preserved the dna healthy before dna extraction. following dna extraction, no pcr failures were seen in the specimens stored for about one year. the pcr products of different loci against the dna extracted from the filter paper are shown in fig. 1 and 2. for cytb gene of the vertebrate specimens, the amplified products were a single band of the expected size (624 bp) on the agarose gel. for its2-rdna the pcr products were varied that was 462 bp for cx. quinqufaciatus and 640 bp for an. stephensi. the amplicon for the coi gene of ph. papatasi was 711 bp. the biggest pcr product with 1500 bp was for the bacteria. hafnia alvei, enterobacter sakazakii, escherichia coli, and enterobacter bacteria are gram negative and species of staphylococcus spp. are gram positive. iran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 46 table 1. the primer sequences, target genes and species, and length of pcr products used in this study primer sequence 5’-3’ locus target genome pcr size (bp) 5.8s tgtgaactgcaggacacatgaa its2-rdna culex quinqufaciatus anopheles stephensi 462 640 28s atgcttaaattagggggtagtc coif ttaaacttcagggtgaccaaaaaatca mtdna-coi phlebotomus papatasi periplaneta americana blattella germanica 709 coir ggtcaacaaatcataaagatattgg un1025f ggttgtcctccaattcatgtta mtdna-cytb homo sapiens rhombomys opimus 624 un403r tgaggacaaatatcattctgagg 16suf tcgacatcgtttacggcgtg 16s rrna staphylococcus spp., hafnia alvei, cronobacter sakazaki, escherichia coli, enterobacter spp 1500 16sur agagtttgatcctggctcag fig. 1. agarose gel showing the pcr products amplified from filter paper extractions: lane 1, its2-rdna of an. stephensi (640 bp), lane 2, its2-rdna of cx. quinquefasciatus (462 bp), lanes 3–4, host-specific cytochrome b of human and r. opimus (624 bp) respectively; lanes 5–7, mtdna-coi of ph. papatasi, periplaneta americana, and blattella germanica (709 bp) respecyively, and m, a 100 bp ladder (fermentas) fig. 2. 16srrna gene pcr products (1500 bp) of bacteria amplified from their dna stored on filter paper: lane 1, a 200 bp ladder, lane 2, staphylococcus sp, lane 3, hafnia alvei, lanes 4, cronobacter sakazaki, lane 5, escherichia coli, lane 6, enterobacter sp. iran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 47 discussion in this study, we demonstrated the utility of using the filter paper cards to preserve the extracted dna or dna in cells respectively from bacteria or from fresh or alcohol preserved mosquitoes, sand flies, cockroaches or blood tissues of human and rodent specimens. we found the filter paper as a convenient and relatively inexpensive method for the storage and transportation of the specimens that may require dna testing. successful pcr amplification of four different loci (its2-rdna, 16srrna, mtdna-coi, and mtdna-cytb genes) proved that dna extracted from the cells stored on the filter paper functioned very well with no significant differences in the ability of the templates to produce pcr products. use of filter papers previously have reported for malaria parasite species identification in human blood (syafruddin et al. 2007, lekweiry et al. 2009, ataei et al. 2011), for extraction and storage of insect dna in forensic entomology (harvey 2005), for detection and identification of the filarial parasite brugia timori in human blood (fischer et al. 2002), for source of mosquito blood meals (ansell et al. 2000), and for detection of wuchereria bancrofti dna in human blood (rao et al. 2006). also, filter papers have been used for blood meal identification and leishmania parasite detection in sand fly lutzomyia longipalpis (schaefer et al. 1995, sant'anna et al. 2008), identification of trypanosome in wild tsetse populations (adams et al. 2006), diagnosis and surveillance of trypanosoma vivax in ruminants (gonzales et al. 2006), detection of cyclospora cayetanensis and cryptosporidium parvum from food samples and human fecal specimens (orlandi and lampel 2000), and archiving and processing dna from fresh water protozoans (hide et al. 2003). the application of the filter paper for molecular testing using rna such as microarrays or rt-pcr assays, which need a stable source of rna, was not addressed in this study. however, others have shown that rna is stable on the filter paper (nakazawa et al. 2009). in this study, the preservation is only tested on dna regions that have high copy number on the genome which are easy to amplify. therefore to make a better assessment of the filter paper, it is recommended to test them against the genes/loci with low or single copy number. the archival storage of specimens for future dna analysis is greatly facilitated by using the filter paper because the cards are small and are stored at room temperature in a compact file until molecular studies are needed. in this study, the dna was shown to be stable for up to one year, and dna has been reported by the manufacturer to be stable on the cards for at least 30 years. this stability diminishes the need for an ultralowtemperature freezer dedicated to specimen storage, which both is expensive and requires laboratory space. in addition, specimens stored at -20 °c to -70 °c in an ultralow-temperature freezer are at risk should the freezer suffer a mechanical or electrical failure. once a frozen specimen thaws, severe degradation of the dna can result unless it is immediately extracted. the diameter of punched disc from filter papers was as low as 1-mm hence allows using the rest of specimens for many other molecular analyses. in addition, it is shown that the reagents on the filter paper are designed to kill pathogens upon contact and the papers protect dna within the samples for several years at ambient conditions (smith and burgoyne 2004). in conclusion, the filter paper is an inexpensive and user-friendly method for storing cell suspensions from different specimens. the card size index allows the convenient storage and transportation of speciiran j arthropod-borne dis, 2011, 5(2): 42–50 f karimian et al.: utility of filter paper for… 48 men dna and makes the system ideal for small laboratories that rely on sending specimens to larger facilities for molecular analysis. further studies are needed to validate this method of storage using a wide range of molecular methods including pcr, dna microarrays, and expression analysis with rna. it seems that filter paper is a suitable, fast, safe and easy to use tool for storage and transportation of the samples directed from the field to the laboratory for dna extraction or storage of extracted dna. acknowledgements the authors would like to appreciate the tehran university of medical sciences (tums) for providing research facilities and funding for the authors to perform this study. the authors declare that there is no conflict of interests. references adams er, malele ii, msangi ar, gibson wc (2006) trypanosome identification in wild tsetse populations in tanzania using generic primers to amplify the ribosomal rna its-1 region. 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biol. 213: 946–954. who (2009) world malaria report, who press, world health organization, geneva. who (2000) the world health report, who press, world health organization, who health reportprelims iixx/e. weigle ka, labrada la, lozano k, santrich c, barker dc (2002) pcr based diagnosis of acute and chronic cutaneous leishmaniasis caused by leishmania (viannia). j clin microbiol. 40: 601–606. j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 28 original article evaluation of melaleuca cajuputi (family: myrtaceae) essential oil in aerosol spray cans against dengue vectors in low cost housing flats *a abu bakar 1, s sulaiman 1, b omar 1, r mat ali 2 1department of biomedical science, faculty of allied health sciences, university kebangsaan malaysia, jalan raja muda abdul aziz, 50300 kuala lumpur, malaysia 2forest research institute malaysia (frim), kepong, 52100 selangor, malaysia (received 1 may 2010; accepted 16 nov 2011) abstract background: melaleuca cajuputi essential oil in aerosol spray was evaluated against the dengue vectors aedes aegypti and ae. albopictus at low cost housing flats in section 10, setapak, kuala lumpur, malaysia. methods: essential oil in aerosol viz: 5% and 10% of concentrations were sprayed for 5 seconds each towards hung mosquitoes in 5 cylindrical net cages. aerosol weights were recorded before and after spraying to determine discharge rates. knockdown and mortality number were observed and compared to ms standard aerosol which contain 0.07% prallethrin and 0.05% d-phenothrin as positive control and aerosol contain 40% kerosene and 60% lpg was used as negative control. results: high knockdown and mortality was observed in both species of mosquitoes towards ms standard aerosol. there was a significant difference (p< 0.05) of mortality and knockdown between 5% and 10% of essential oil aerosol and 5% and 10% essential oil between ms standard. for 5% essential oil, mean percentage (%) of knockdown and mortality of ae. aegypti displayed slightly higher compared to ae. albopictus. spraying with 5% m. cajuputi essential oil aerosol indicated a knockdown of ae. aegypti 5.60±1.18 and mortality of 22.90±4.22 while ae. albopictus showed 4.60±0.89 knockdown and 20.00±2.85 mortality. the 10% essential oil concentration gave 23.60±1.68 knockdown and 48.05±0.37 mortality for ae. aegypti. ae. albopictus gave 23.00±3.16 knockdown and 44.20 ± 2.10 mortality respectively. conclusions: extracts of essential oils does possessed an adulticidal effects and could be considered and utilized for future dengue vectors control. keywords: melaleuca cajuputi, essentials oil, aerosol can, aedes aegypti, aedes albopictus introduction to date, aedes aegypti and ae. albopictus mosquitoes are principal vectors of dengue fever (df) and dengue haemorrhagic fever (dhf) in many tropical countries in urban, and semi-urban areas. in addition, chikungunya is yet another viral disease transmitted by ae. aegypti and exhibits similar symptoms (singh and pavri 1967). at present there is no specific antiviral therapy or effective vaccines available for dengue and chikungunya virus (cdc 2006). therefore, the only option is to prevent the diseases by controlling the vector mosquito population. this major approach of controlling the population vector relies mainly on insecticide application (tikar et al. 2008). a consequence of the widespread use of insecticides is the development of resistance in mosquitoes against insecticides. because of these problems and concerns, the interest towards botanical insecticides derived from plants has been revived worldwide, and tre*corresponding author: dr azlinda abu bakar, e-mail: azlinda_abubakar@yahoo.com j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 29 mendous efforts are being made to isolate, screen and to develop phytochemicals possessing pesticidal activity (mulla and su 1999). since ancient times, plant products were used in various aspects. however their use against pests decreased when chemical products become developed (kallyaperumal 2008). however emphases are being made towards bio-products to replace synthetic insecticide for the control of dengue particularly in cases where susceptibility is decreasing (chatiyasit and choochote 2006). mosquito behavior may contaminate the aquatic environment (kallyaperumal 2008). natural pesticides especially those derived from plants are more promising in this aspects (amer and mehlhorn 2006). earlier studies on larvicidal activities of leguminous seeds and grains against ae. aegypti and culex pipiens pallens, had shown larvicidal properties (young-su et al. 2002). in malaysia, study on thirty species of plants extracts were tested for their ability as mosquitocidal activities against dengue vector has revealed the potential effects (zaridah et al. 2006). the objective of this study was to evaluate the effectiveness of melaleuca cajuputi essential oil extract in aerosol spray against ae. aegypti and ae. albopictus, compared with malaysia sirim standard (ms standard) aerosol under field conditions. the evaluation used ms standard aerosol as standard reference or guideline for the bio-efficacy of household insecticide testing. materials and methods plant collections melaleuca cajuputi leaves were obtained from port dickson, negeri sembilan (2o 31’22.07n 101o 48’00.01e) in peninsular malaysia located about 120 km south of kuala lumpur (3o09’00.76”n 101o42’27.73”e). verification of species identification was made by botanists from the forest research institute of malaysia (frim). the voucher specimens were deposited at the herbarium for reference. extraction the whole process of extraction was conducted at frim. the extraction of essential oil from leaves specimens was conducted using steaming hydro-distillation. this continuously 8 hours process has enabled to isolate and produce essential oil from subjected samples, made up with a clevengertype apparatuses which were comprised with 500ml to 1000ml round bottomed distillation flask, oil separator tube and condenser. fresh leaves were left to dry at room temperature and dried leaves were collected and grounded to a small particles to maximize the essential oil production. samples were then transferred into the distillation flask and weigh were recorded. samples then covered with distilled water and heated at 60–70o c. the samples allowed boiling slowly until distillation process was completed. the mixture of oil and water was allowed to settle for 24 hours to ensure separation of water and oil layers. the water was slowly drawn off and the remained oil layer were slowly collected into a glass beaker and dried over anhydrous sodium sulfate. the remained oil was pipetted out into an ambercolored bottle and final volume was recorded and kept at 4–5 ºc. the extraction was repeated accordingly to make stock. a total of 500ml essential oil was needed to meet the capacity needs for the aerosol study. the successively extracted essential oils were collected and sent to the homesafe products (m) sdn bhd malaysia to produce aerosol spray cans. concentrations of m. cajuputi aerosols used in the study were 5% and 10% respectively. the 5% m. cajuputi extract dissolved in 37% kerosene and 58% liquid petroleum gas (lpg) while the 10% m. cajuputi extract dissolved 32% kerosene and 58% lpg respectively. ms standard aerosol was used as positive control which j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 30 contain 0.07% prallethrin and 0.05% d-phenothrin+40% kerosene+60% lpg. whereas aerosol which contained only 40% kerosene and 60% lpg was used as untreated or negative control. specimens preparation species used in the study were ae. aegypti and ae. albopictus. both species were colonized and maintained continuously at 25–30 ºc in the insectarium at the department of biomedical science, faculty of allied health sciences, university kebangsaan malaysia. larvae were fed with finely powdered mixture of dried liver, ground cat biscuits, milk powder and ground oatmeal. the adult colony was fed with 10% sucrose and periodically blood-fed on restrained guineapig for breeding purposes. adult 3–5 days age females fed only with sucrose 10% were used in the study. a total of hundred and twenty five adult mosquitoes were used for every single replicate. twenty-five mosquitoes were then transferred into each 5 of cylindrical net. bioassay method for the evaluation of biological efficacy of household space spray insecticidal aerosol against adult mosquitoes was assayed following a version of ms standard method ms 1221:1991 udc 632.982.2. as the method was design for the laboratory evaluation, modification was made to adapt the field conditions. four blocks of low cost housing estate which have 4 storey medium flats were chosen in this study namely block a, block b, block c and block d. each block has 24 units of houses with 6 units of houses at each floor. distances from each block were about 20 metres. block a, block b, block c and block d were represent of 5%, 10%, ms standard and negative control aerosols respectively. four houses from every block were selected as replicate. each houses composed with a living hall, 2 bedrooms (sizes 4.0m x 4.05m x 2.20m each), kitchen and toilet. one appropriate room was specified throughout the evaluation period of 5 months. the spraying was conducted alternately between ae. aegypti and ae. albopictus. the room was decontaminated with diluted methanol to disinfect and remove traces of insecticide or unknown household chemical products residual if any. the floor and wall were cleaned each pre and post spraying. mosquitoes in cylindrical nets were hung at the room’s ceiling for 1 hour prior spraying for acclimatization and were fed with sugar cube. aerosol cans were weighed pre and post spraying to determine the discharge rate. aerosols were sprayed vertically for 5 seconds for each samples tested. the room was closed immediately after spraying. observations were made for 1, 5, 10, 15 and 20 minutes for knockdown and 24 hours for mortality. determination of knockdown was made when mosquitoes were failed to fly and not being able to move its body. mosquitoes are not at its normal position and at their backs with legs facing upwards for the first of 1 hour. definition for mortality was when mosquitoes were motionless and dead within 24 hours. all those that were alive were transferred into 250 ml paper cups (2.5 cm bottom diameter and 4.0 cm diameter opening). the cups were covered with a piece of net layer and mosquitoes were fed with wet cotton wool (wets with 10% sucrose). the wet cotton wools were then placed onto the net for mosquitoes accessible. a total number of knockdown and mortality was counted and recorded after 1 hour and 24 hours respectively to determine the percentage. the experiment replicated 4 times with different houses and repeated with both species of mosquitoes. results obtained were then compared to the ms standard and negative control. the results were statistically analyzed (spss 11.5 determination computer program) to obtain the j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 31 knockdown values and regression slope using a probit analysis. knockdown time (kt50 and kt95) is the value of time taken for aerosols to give knockdown effects of the 50% and 95% sample populations. mean and least significant difference test were used for knockdown, mortality and dose concentrations applied. in cases where the mortality in tests control populations ranged from 520%, the observed percentage mortality (m%) was corrected by abbott’s formula (abbott 1925): m%= test mortality% control mortality% 100 control mortality% results table 1 demonstrates the total average of discharge and discharge rates of each aerosol sprayed towards ae. aegypti and ae. albopictus. for ae. aegypti the ranges were from 2.56 gm/sec to 2.64 gm/sec. whereas ae. albopictus showed ranges between 2.55 gm/sec to 2.61 gm/sec. table 2 shows mean percentage of knockdown and mortality of both species which have been treated with 5%, 10% of essential oils extract, ms standard and negative control aerosols. the 5% essential oils on ae. aegypti gave 5.60±1.18 of knockdown and 22.90±4.22 of mortality. aedes albopictus values were lower, 4.60± 0.89 of knockdown and 20.00±2.85 of mortality. essential oils with 10% concentration gave the mean percentage of knockdown and mortality slightly higher than 5% of essential oils. nevertheless the values pattern were consistent with 5% of essential oil where ae. aegypti showed a small different of mean percentage compared to ae. albopictus. the values were 23.60±1.68 of knockdown and 48.05±0.37 of mortality on ae. aegypti. ae. albopictus gave values of 23.00±3.16 knockdown and of 44.20±2.10 mortality. ms standard aerosols showed highest effect of knockdown and mortality against both species. the mean percentage of knockdown and mortality for ae. aegypti was 94.60±1.00 and 96.60± 0.50 respectively. ae. albopictus gave a mean percentage of 93.00±2.68 knockdown and of 97.00±1.10 mortality. negative control showed no values of knockdown for both aedes spp. mortality for mean percentage showed values of 0.50± 0.29 for ae. aegypti and 0.25±0.25 for ae albopictus which are less than 20%. both species shows no correlation of knockdown and mortality towards discharge rates (r< 0.40). the r values for knockdown and mortality of ae. aegypti were 0.215 and 0.066 whereas r values of ae. albopictus were -0.168 and -0.055 respectively. there were non significant difference (p> 0.05) between ae. aegypti and ae. albopictus for knockdown and mortality mean percentage. there were significant differences (p< 0.05) between doses applied of 5%, 10% essential oils and ms standard towards knockdown and mortality of ae. aegypti and ae. albopictus. table 3 shows the values of knockdown time, kt50 and kt95 for both aedes spp. after treatment with m. cajuputi essential oil in aerosol cans using 5% and 10% concentrations and the comparison with ms standard and negative control. the best and effective aerosols gave lower values of kt50 and kt95 respectively. from the results obtained, 10% concentration gave best effect compared to 5% concentration. aedes aegypti was more susceptible compared to ae. albopictus towards essential oils extract. at 5% kt50 of ae. aegypti was 311.96 min and ae. albopictus 550.81min. while at 10% concentrations, kt50 were lower with 122.98 min for ae. aegypti and 174.36 min for ae. albopictus respectively. ms standard aerosols gave the best and lowest kt50 values of 4.775min for ae. aegypti and 3.581min for ae. albopictus. there were no kt values for negative control can be analyzed as no knockdown effect was observed during evaluation. x 100 j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 32 table 1. total average of discharge and discharge rates of melaleuca cajuputi, ms standard and untreated aerosol sprays discharges aedes aegypti aedes albopictus 5% 10% ms untreated 5% 10% ms untreated total average of discharge (gm) 13.21 2.81 3.14 13.20 12.74 13.03 12.80 13.00 discharge rates (gm/sec) 2.64 2.56 2.63 2.64 2.55 2.61 2.56 2.6 table 2. mean percentage (%) knockdown and mortality aedes aegypti and aedes albopictus after spraying with melaleuca cajuputi extract in aerosol sprays doses aedes aegypti aedes albopictus discharge rates (gm/sec) knockdown (%) mean ± se mortality (%) mean ± se discharge rates (gm/sec) knockdown (%) mean ± se mortality (%) mean ± se 5% 2.64c 5.60 ± 1.18a 22.90 ± 4.22b 2.55d 4.60 ± 0.89 20.00 ± 2.85 10% 2.56c 23.60 ± 1.68a 48.05 ± 0.37b 2.61d 23.00 ± 3.16 44.20 ± 2.10 ms standard 2.63c 94.60 ± 1.00a 96.60 ± 0.50b 2.56d 93.00 ± 2.68 97.00 ± 1.10 neg control 2.64 c 0.50 ± 0.29 b 2.60 c 0.25 ± 0.25 b a b p>0.05 non significant difference compared to ae. albopictus c d r<0.40 no correlation with knockdown and mortality (pearson’s correlation) table 3. values of kt50 and kt95 of essential oil extract of melaleuca cajuputi to aedes aegypti and aedes albopictus adult females in the field which has been sprayed for 5 seconds doses aedes aegypti aedes albopictus kt50 and confidence limit (min) kt95 and confidence limit (min) regression coefficient±se kt50 and confidence limit (min) kt95 and confidence limit (min) regression coefficient± se 5% 311.96* (200.28 – 694.02) 1785.58* (776.94 – 8065.24) 0.942 ± 0.14 550.81* (261.62 – 3340.08) 5041.04* (1251.04 – 148712.84) 1.711 ± 0.36 10% 122.98* (128.65 – 143.12) 740.29* (551.84 – 1065.48) 2.110 ± 0.12 174.36* (143.18 – 225.50) 1805.94* (1123.24 – 3375.52) 1.620 ± 0.11 ms standard 4.775* (4.307 – 5.253) 42.398* (34.461–54.671) 1.734 ± 0.05 3.581* (2.929 – 4.232) 45.710* (33.553 – 69.711) 1.486 ± 0.04 neg control 0 0 0 0 0 0 *a heterogeneity factor is used in the calculation of confidence limit j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 33 discussion in context of pollution environment such as residue problem, health risks of the consumer and the development of insect resistance to synthetic insecticides, recent interest among researchers has developed to investigate and explore potential of plants extract that are environmentally safe and target specific but still effective to control vector populations. from the study, it was observed that aerosol of ms standard were the most effective followed by essential oil of 10% and 5% concentration respectively. pearson’s correlation analysis showed that the discharge rates had no correlation on the effectiveness (knockdown and mortality) of the aerosols. of all ms standard gave highest mean percentage of knockdown and mortality of ae. aegypti and ae. albopictus compared to the essential oils extract. the mean mortality percentage for negative control was not more than 20% thus can be said that new formulations of essential oil have contributed the knockdown and mortality effect. previous studies by azlinda (2009) on the evaluation of m. cajuputi extract essential oil in aerosol sprays against dengue vectors in laboratory had similar findings. that study demonstrated high value of mean percentage of knockdown and mortality at concentration of 10% with highest value recorded for knockdown were 37.5±1.33% and mortality were 64.0±5.72%. in comparison with the present study in the field trial, the highest number has shown slightly lower of knockdown (23.60±1.68) and mortality (48.05± 0.37). in spite of that, it indicates the potential of m. cajuputi essential oil extract as botanical insecticides in the laboratory as well as in the field. the bio-efficacy of the products however depends on many factors such as mode of application and local conditions like temperature and humidity (tawatsin et al. 2001). study on the evaluation of effectiveness for household insecticide products performances knockdown time (kt) values is one of the parameters which contributes not less important. the knockdown effect were calculated to estimate the number of knocked down mosquitoes in tested population, as recommended by who (1975) for testing space spraying insecticides such as aerosol. values of kt50 and kt95 were lower when aerosols take shorter time to give knockdown effect. ms standard aerosols give the lowest values of kt50 and kt95 compared to the essential oil extracts. however, essential oil with 10% concentration performs better than 5% concentration. similar ported on a study with callosobruchus maculates of which the knockdown effect varied with concentrations (keita et al. 2000). variation in knockdown time and mortality were also observed between ae. aegypti and ae. albopictus. this probably due to the different reaction to chemical compound in active ingredients of essential oil which act as a reversible inhibitor of a acetyl cholinesterase, capable of disrupting the function of neurotransmitter in insects like mosquito, thus caused knockdown effect (ryan and byrne 1988). recent investigations indicate that some chemical constituents of many plants extracted essential oils interfere with the octopaminergic nervous system in insects. hence, meet the criteria for ‘reduced risk’ pesticides, as this target site is not shared with mammals (koul et al. 2008). from the knockdown and mortality values, ae. aegypti showed to be more susceptible than ae. albopictus which means greater number of knockdown and mortality were noted. since ae. aegypti plays a very important role in dengue and dengue haemorrhagic fever transmission in southeast asia and tropical regions (harinasuta 1984) j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 34 this study could provide a useful information in future research. in conclusion, this study confirmed that extract of m. cajuputi (family: myrtaceae) essential oil possesses some adulticidal effects on dengue vectors with most effective concentration was 10% for controlling vectors. similarly, study on the efficacy of essential oil extract as green pesticides, has observed that essential oil may require greater application or frequent reapplication compared to synthetic pesticides (koul et al. 2008). thus, in addition of development for commercial application of plant essential oil based pesticides, considerations to include are availability of sufficient quantities of plant resources, standardization and refinement of pesticide products and regulatory approval (isman 2005).the results obtained suggest that, further studies are considerable essential that will lead to improve formulations with enhanced activity which may eventually acceptable as mosquito control. acknowledgements we wish to thank the homesafe products (m) sdn bhd in helping and developing essential oil extract of m. cajuputi plant into aerosol cans. we appreciate the support of the technical staff from the department of biomedical science, faculty of allied health sciences, university kebangsaan malaysia. the cooperation from the resident of low cost housing at setapak, kuala lumpur and the research grant irpa 06-02-02-0030ea098 awarded by ministry of science technology and innovation malaysia. the authors declare that they have no conflicts of interest. references abbott ws (1925) a method of computing the effectiveness of an insecticide. j econ entomol. 18: 265–266. amer a, mehlhorn h (2006) larvicidal effects of various essential oils against aedes, anopheles and culex larvae (diptera, culicidae). parasitol res. 99: 466–472. azlinda ab, sallehudin s, baharudin o, rasadah a (2009) evaluation of melaleuca cajuputi powell (family: myrtaceae) extract in aerosol can against dengue vectors in the laboratory. j trop med parasitol. 32: 58–64. centers for disease control, cdc (2006) chikungunya fever in india. travelers’ health outbreak notice april 21, 2006. available at: http://www.cdc.gov/travel. chatiyasit d, choochote w (2006) essential oils as potential adulticides against two populations of aedes aegypti, the laboratory and natural field strains in chiang mai province, northern thailand. parasitol res. 99: 715–721. harinasuta c (1984) mosquito-borne diseases in southeast asia. mosq borne dis bull. 1: 1–11. isman mb (2005) problems and opportunities for the commercialization of botanical insecticides in c. regnaultroger, b.j.r. philog‘ene and c. vincent (eds), biopesticides of plant origin, lavoisier, paris. pp. 283–291. kallyaperumal k (2008) evaluation of leaf extracts of vitex negundo l. (family: verbenaceae) against larvae of culex tritaeniorhynchus and repellent activity on adult vector mosquitoes. parasitol res. 103: 545–550. keita sm, vincent c, schmidt jp, ramaswamy s, belanger a (2000) effect of various essential oils on callosobruchus maculates (f.) (coleoptera: bruchidae). j stored prod res. 36: 355–364. koul o, walia s, dhaliwal gs (2008) essential oil as green pesticides: potential and constraints. biopestic int. 4(1): 63–84. j arthropod-borne dis, 2012, 6(1): 28–35 a abu bakar et al.: evaluation of melaleuca cajuputi … 35 mulla ms, su t (1999) activity and biological effects of neem products against arthropods of medical and veterinary importance. j am mosq control assoc. 15(2): 133–152 ryan mf, byrne o (1988) plant-insect coevolution and inhibition of acetyl cholinesterase. j chem ecol. 14(10): 1965– 1975. singh kr, pavri km (1967) experimental studies with chikungunya virus in aedes aegypti and aedes albopictus. acta virol. 11: 517–526. tawatsin b, wratten, sd, scott rr, thavara u, techadamrongsin y (2001) repellency of volatile oils from plants against three mosquito vectors. j vector ecol. 26: 76–82. tikar sn, mendki mj, chandel k (2008) susceptibility of immature stages of aedes (stegomyia) aegypti: vector of dengue and chikungunya to insecticides from india. parasitol res. 102: 907–913. world health organization, who (1975) manual on practical entomology in malaria. part ii. who division of malaria and other parasitic diseases, geneva. young-su j, bong-rae b, young-cheol y, moo-key k, hoi-seon l (2002) larvicidal activity of leguminous seeds and grains against aedes aegypti and culex pipiens pallens. j am mosq control assoc. 18(3): 210–213. zaridah mz, nor azah ma, rohani a (2006) mosquitocidal activities of malaysian plants. j trop forest sci. 18(1): 74–80. eco-biology of anopheles superpictus in iran j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 196 http://jad.tums.ac.ir published online: september 30, 2018 review article bioecology of dominant malaria vector, anopheles superpictus s.l. (diptera: culicidae) in iran *hassan vatandoost 1, 2, ahmad ali hanafi-bojd 1, 2, ahmad raeisi 2, 3, mohammad reza abai 1, 2, fatemeh nikpour 1, 2 1department of medical entomology and vector control, school of public heath, tehran university of medical sciences, tehran, iran 2department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 3ministry of health and medical education, tehran, iran (received 8 july 2018; accepted 29 sep 2018) abstract background: malaria continues to be a main vector-borne public health problem in iran. the endemic foci of the disease are mainly located in south-eastern part of the country. iran is now launching the elimination of malaria. studies on the bioecology and susceptibility of malaria vectors to insecticide are essential in this phase. methods: the literature on bio-ecology of anopheles superpictus s.l. was reviewed in iran in more than half a century. different aspects including, distribution, key identification, larval habitats, flight range, seasonal activities, irritability/susceptibility to insecticides, and anthropophilicity index were identified. results: the adult females of an. superpictus s.l. were susceptible to all who-recommended imagicides except ddt. distribution, morphology, sibling species, larval habitat, flight range, irritability tests, sustainability index, blood feeding preference and related factors were discussed in details conclusion: results of the evaluating will help for decision making of authorities for vector control. keywords: anopheles superpictus s.l., ecology, biology, insecticide resistance, iran introduction mosquito-borne diseases are the major problems worldwide, among them malaria presents a major health problem globally. in 2016, an estimated 216 million cases of malaria occurred worldwide, compared with 237 million cases in 2010 and 211 million cases in 2015. most malaria cases in 2016 were reported from the who african region, while the two percent of cases were inhabitants of who eastern mediterranean region countries (1). it is one of the important infectious diseases in iran with an average of about 15000 annual cases in the last decade, while total recorded cases has dropped to less than 100 locally transmitted cases in 2017. most of indigenous and imported malaria cases in iran are reported from southern and southeast ern areas of the country in sistan and baluchistan, hormozgan and south of kerman provinces. the most routes of malaria cases are immigration from afghanistan and pakistan to southern and southeastern areas of the country (ministry of health, annual reports). anopheline mosquitoes (diptera: culicidae) are vectors of malaria to humans. currently there are proven and effective tools to fight against malaria including vector control measures (1). as these tools are scaled up, malaria endemic countries need to continually update the skills and competence of the health workers engaged in malaria control and elimination. all observations indicate that the data reflect the real situation and that the overwhelm*corresponding author: dr hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ mailto:hvatandoost1@yahoo.com mailto:vatando@tums.ac.ir j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 197 http://jad.tums.ac.ir published online: september 30, 2018 ing majority of cases, which occur, are included in the national system, although there is room for improvement in the surveillance system. the spectacular progress can be ascribed to effective implementation of appropriate curative and preventive control interventions through a strong health care infrastructure. social and economic development allowing better housing, use of air-conditioning etc. has also played a role. there are several activities on different aspects of malaria in the country: including insecticide resistance monitoring (2-12), sibling species, molecular study, new record (13 20), novel methods for vector control (21-26) faunestic study (27, 28), use of plants for larval control (29-41) using bednets and long lasting impregnated nets (42-48), morphological studies (49-51), malaria epidemiology (5255), ecology of malaria vectors (56-64), biodiversity (65, 66), community participation (45, 54), vector control (67), repellent evaluation (68), anthropophilic index of malaria vectors (65, 69), training (70) is designated as malaria training center by who. there are several reports on different aspects of malaria vectors recently different studies have been conducted during more than 90 years on malaria (71-96) and its vectors in iran. seasonal activity of anopheline mosquitoes and their peak of activity vary in different area due to environmental condition. this issue affected the epidemiology of malaria transmission in different regions. agriculture in iran remains highly sensitive to climate developments, the country's most important crops are wheat, rice and other grains, sugar beet, fruits, nuts, cotton, and tobacco, which require the use of insecticides. so far different groups of insecticides are using for crops protection in the country. the main governmental use of insecticide in the health sector is their application for adult mosquito control. the first attempts to control malaria vectors started during the 1960’s with organochlorines (ddt, dieldrin and bhc), followed by organophosphates (malathion and pirimiphos-methyl) for about two decades from 1966. after that during 1977–1990 propoxur was used by national malaria vector control program. subsequently, after the introduction of pyroteroids into the market, and given their lower risk for humans and more affordable products, lambdacyhalothrin and deltamethrin were used. as the adult mosquitoes control, larval control is also used to reduce the vector abundance. for this purpose, temephos, reldan® and pirimiphosmethyl was used in past decades. materials and methods all the published papers thought the internet and master of sciences and phd thesis related to an. superpictus s.l. were reviewed and evaluated. key words for search in scientific motor engines were: anopheles superpictus, insecticide resistance, ecology, distribution, identification, anthropophilicity, sporozoite rate, iran. results distribution of anopheles superpictus s.l. in iran faghih' research results showed that this species could be found in areas of 2000 meters above sea level (97-99). it has been captured from coastal plains of persian gulf in areas with 500 meters height above sea level (in chelou village in minab). with the exception of a narrow strip of coastal areas, this species is found in almost all regions of hormozgan province (100-104) (fig. 1). key to the female anopheles superpictus s.l. wings with contrasting pale and dark spots, at least on costa (c), radius (r) and radius-one (r1), hind tarsomeres 3–5 no1 entirely pale, maxillary palpus dark, or with at most 3 distinct pale bands (pale spots may also be present): abdominal terga ii-vii without dark http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 198 http://jad.tums.ac.ir published online: september 30, 2018 scale-tulips. although some posterolateral dark scales may be present on distal segments, hind tarsomere 5 dark, palpomere 5 entirely pale, femora and tibiae not spotted: abdominal terga without pale scales, anal vein with 2 dark spots, distal spot long (104). key to the anopheles superpictus s.l. larvae seta 2cephalon inserted at least as far apart as the distance between 2cephalon and 3cephalon on one side, seta 1antenna always simple, seta 5, 6, 7cephalon branched (subgenus cellia), tergal plates on abdominal segments iii-vii smaller, < 0.5 width of segment, and not enclosing median accessory tergal plate, thoracic pleural seta various , thorax with at least long pleural seta 9prothorax and 9metathorax branched, 9, 10mesothorax, and 10metathorax simple or branched, thorax with long pleural seta 9prothorax and 9, 10metathorax branched. seta 9mesothorax branched, 10mesothorax simple, palmate seta 1-iii-vii strong, leaflets and filaments various, seta 3cephalon simple or with a few short lateral branches, not brush-like in appearance, abdominal palmate seta 1abdominal segment i non-palmate or very weakly palmate, seta 1, 2prothorax tubercles separate; seta 3metathorax non-palmate or very weakly palmate, seta 2, 3cephalon simple, finely or distinctly frayed, anal papillae normal, abdominal palmate filaments  0.5 length of blade, seta 3, 4cephalon simple or occasionally bifid, never branched, seta 2, 3cephalon smooth or finely frayed, abdominal seta 1abdominal segment ii strongly palmate, seta 3cephalon finely frayed, seta 4cephalon length > 3 cephalon, basal tubercle of seta 1prothorax weak (fig. 2) (104). the anopheles superpictus s.l. species anopheles superpictus s.l. acts as a main malaria vector (e.g. in lorestan) or secondary vector (e.g. sistan and baluchistan). a survey has been conducted in order to study on different morphological and genetic variation of an. superpictus populations in iran. mosquitoes of an. superpictus were collected from the ardebil, lorestan, and sistan and baluchistan provinces in july-september in 2004 using different collection methods. after species identification and morphological study on larvae and adults, the sequence variation of its2rdna and mtdna coi-coii fragments were analyzed using pcr-rflp and pcr-direct sequencing assays. results showed that there were significant differences in morphological and genetic characters within and between populations. digestion of coi-coii fragment using alui enzyme demonstrated various haplotypes indicating intra or inter-species variation. totally 4 haplotypes were observed between specimens/populations in 708bp of coi gene. totally, the rate of variation among populations was 12.3%. this rate was 2–5% for within sistan and baluchistan populations. according to phylogenetic analysis of the coi sequences, an. superpictus populations in iran constructed 2 main groups including: 1) southeast populations (sistan and baluchistan and kahnooj) and 2) central and northwest populations, each group putatively representing one species. analysis of its2 fragment also revealed highly diverged populations representing at least three putative separate species designated as x, y, and z (x and y in baluchistan and z in other provinces). the rate of genetic variation in its2 was 27% in which respectively 0.27%, 0.05% and 26.68% corresponded to 5.8s, 28s, and its2 regions. phylogenetic analysis also revealed two main groups including three branches, each one putatively representing one separate species. in spite of having a high genetic and morphological variation among specimens, there were no relationship between phenotypic and genetic variations, assuming that other parts of genome is responsible for the variations. the rate of genetic variation in coi and its2 regions was far more than that has been seen in other species s.l. (105). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 199 http://jad.tums.ac.ir published online: september 30, 2018 flight ranges flight range of this species was about 2– 2.5 kilometers (observations in tabas, birjand). at the beginning of hibernation it reached to 4km (99). irritability tests the irritability levels of an. superpictus s.l. against diagnostic dose of dichlorodiphenyltrichloroethane (ddt) insecticide were measured in a conical exposure chamber according to who methods in isfahan in 1963. the results of irritability tests showed that ddt had the high irritancy effect against an. superpictus s.l. (106). the time of blood feeding the activity of this species started at about 20pm and reached to peak point at about 21.30pm. the most brutal is in the first half and with low density in the second half of the night and in the morning it was close to zero. the most biting activity in bazaft, farsan, chahar mahal bakhtiari in 1999 was in 22–23pm and 3–5am (110-111). seasonal activity researches have been conducted from early october to april 1953–1955 to determine an. superpictus s.l. seasonal activity. anopheles superpictus s.l. adult population abundances for the entire period of august-september were high, but during the october, it began to decrease. the last collection was conducted in the third week of october. even though space spray collection using pyrethrums (psc), that was applied at homes, very few adult were collected in each station during the months of november–february. based on information about the larvae collection and dissected adults, it has been concluded that this species didn’t enter to hibernation phase in october. it seems that leaving hibernation happened in february, because the females that have been desiccated at this month had high ovarian growth. during the month of may, no larvae were collected. this result showed that although this species ovoposited in february or march, but the eggs were expelled because of abundant rainfall, rapid flow of springs and streams. changes were not the same during three winters. winter 1953–1954 had the coldest winter and 1955–1954 was the modest. the numbers of collected specimens to generalize the reliability results were very low. although the temperature in february, was different from the temperature in the first three months but the collected females showed the high activity both for the ovarian growth and blood feeding. even in march that this species began its activity, the temperature was more than the last 4 months. it has been justified that an. superpictus s.l. entered to hibernation phase because of a physiological cycle caused by cold weather. in ilam, an. superpictus s.l. is the main malaria vector and comprised almost one hundred percent of the collected anopheles from indoors and outdoors (111). studies in kermanshah and sabzevar indicated that the activity of this species depended on the temperature and the onset of its activity was from mid-june and ended in mid-october. maximum activity is in early august to midseptember and with the onset of cold weather that is different in various parts of the plains and mountainous areas; after this time it entered to hibernation. females entered to hibernation phase in mountainous caves and indoors from midseptember in west with storing fat and from october to next april when the weather was good for mosquito activity (manoochehri, personal negotiation). in a study in the counties of pars abad and germi in 1997–1998, the activity of this species has been reported during july-november. the maximum abundances of this species was 10.6 per place in the second half of august and with the least frequency was 0.25 number per location in the first half of december in dykdash and qeshlaq beryan villages. theis species had one peak of activity in second half of august in (112). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 200 http://jad.tums.ac.ir published online: september 30, 2018 in human dwellings, the frequency of an. superpictus s.l. in each place was 1.8. in outdoors, an. superpictus s.l. had the highest frequency of 6.2 per place in early of august. in human dwellings, its frequency was 1.8 and in outdoors it was 6.2 per each place with the highest abundance in the early of august. the highest monthly activity of an. superpictus s.l. in dykdash village, germi, was in the eighteenth of july, 1996. in dykdash and agha mohammad biglou villages its activity began in eighteenth june and ended in third of december. in the village of agha mohammad biglou the highest activity was in the second half of august. in dykdash and gheshlagh beryan villages, its activity in indoors has been reported during the months of july-november, with the highest frequency of 10.63 mosquitoes per place in the second half of august and the least abundant numbers per place (0.25) in the first half of december. this species hasn’t been collected from plain of parsabad (112), but studies of abai and colleagues in 2002 have been obtained very low abundance in this area. the most abundant of this species in the county of kahnooj was during september and october (113). activity of an. superpictus s.l. had one peak point in its curve. this species had incomplete hibernation. hibernation starting differed in cool weather in different plains and mountainous areas. based on different studies in the north of khorasan, birjand, kazeroon, it has been concluded that in appropriate thermal conditions and activity of anopheles, this species is capable to transmitting plasmodium vivax after the second gonotrophic cycle. studies conducted in 1996, in the county of aligoodarz showed that larvae of this species began its activity in may with the peak activity in september and in october it reached to zero (103, 114). species composition fauna and ecology of vectors in the county of farsan, bazaft, chahar mahal bakhtiari, in human dwellings, indicated that an. superpictus s.l. comprised 44.2% of the collected anopheles populations. in animal habitats it comprised 63.7% of the total collected anopheles. in outdoors, an. superpictus s.l. comprised 77% of the populations. it has been collected generally in high population density from outdoors. in hussain abad village, of the 243 mosquitoes collected in night biting, 165 of them (67.9%) were an. superpictus s.l. in the village of mazi, a total number of 356 anopheles collected with the method of night biting, an. superpictus s.l. comprised 246 (69.1%) of the anopheles population. in the village of hussain abad, of the 254 collected anopheles in pit shelter the number of 180 of them (70.8%) were an. superpictus s.l. (116). the highest biting activity of anopheles superpictus s.l. in bazaft, farsan, chahar mahal bakhtiari in 1999 was occurred in 22–23pm and at 3–5am (115). anopheles superpictus s.l. was the most important vectors in siahoo area, southern iran. entomological studies conducted in this district showed that of the total collected larvae and adults' mosquitoes, the an. dthali, an. stephensi, an. superpictus s.l., an. fluviatilis s.l. and other anopheles populations had the most frequencies of 37%, 55%, 1%, 2% and 5%, respectively. this species has been found in the mountainous region and played an essential role in disease transmission (116). in the studies, in bandar abbas it has been identified that the most frequency of adult anophelini were an. stephensi, an. dthali, an. fluviatilis s.l. and with the low density an. culicifacies s.l. and an. superpictus s.l., respectively. the last two species were collected just in night biting (117). distribution, anthropophilic index, sporozoite rate and behavior of adult anopheles superpictus s.l. this species had a major role in mountainous region. it had a role in malaria transmission in hormozgan province. combinahttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 201 http://jad.tums.ac.ir published online: september 30, 2018 tion of malaria vectors in coastal plain and mountainous area of the province was different. the main vector in the coastal plain was an. stephensi and the secondary vector was an. dthali. in the mountainous region, beside the mentioned species, an. fluviatilis s.l., an. culicifacies s.l., and an. superpictus s.l. had a role in disease transmission. in mountainous areas, because of the presence of several outdoor borrows and exophilic behavior of some vectors, vector eradication encountered many problems and was effective only in lowering the population vector (107). anthropophilic index using elisa with the blood spot prepared from female an. superpictus s.l. showed the anthropophilic index between 28.5–3.7% and the feeding of cattle and other animals were 19.7%. the night bite on prey animals (cows and calves) in the village of hussein abad and namazi villages indicated that 55.6% and 66.2% of them were an. superpictus s.l. based on this study it appeared that this species had zoophilic behavior. in this study an. superpictus s.l. had two peaks of activity, in the early hours of the night and the other in 3–5a.m. and its frequency in first peak was more than the second peak. a similar result has been obtained in previous studies (99, 118). the numbers of 200, an. superpictus s.l. were used to determine parity and age-structure of the an. superpictus s.l. populations. parous percent in the first period of the seasonal activity was observed in 26% and its peak of activity was 44%. this species in the study area naturally preferred outdoors to indoors. as in some parts of iran, especially in mountainous areas his species was found infected to sporozoites (sarakhs and kermanshah) and is responsible for malaria transmission and having anthropophilic index, so it could be a vector (119). about 16% of the total larvae collected in the months of april till november in 1994 in lenjanat, isfahan, belonged to this species. larval activity in its larval habitats began from midjune and late july with the frequency of 1 larva per ladle. the frequency was constant until the end of august, but its activity decreased in abundance from this time, so that by the mid-september it has been reduced to zero. this species has been collected in light traps with the frequency of 0.21% and adult activity increased simultaneously with larval activity in mid-july and has reached to its activity with the frequency of 1 mosquito per light trap. its activity decreased in a way that it reached to zero in late august. this is species was mainly endophilic in the area and preferred blood feeding from animals to human. total collection of this species in light traps and night biting methods in march to november 1994 in lanjanat, isfahan was very low (120). this species has been collected in indoors of germi county including villages of ojagh, zahra kandi (ojaroud), agha mohammad beiglou, dykdash, qeshlagh beryan (angoute gharbi), aq dash (district of barzand). in mountainous region of the germi, ardabil including qeshlagh beryan and dykdash, the frequency of an. superpictus was 10.63 mosquitoes per place and had a wide distribution (112). two species, an. superpictus s.l. and an. maculipennis s.l., have been distributed in larval habitat studied in mashhad and suburbs. anopheles superpictus s.l. with the frequency of 94% were more frequent in comparison to an. maculipennis s.l., and were collected from several larval habitats including fresh water, non-polluted and largely stagnant waters with low flows. south of mashhad, places like ponds, even with non-infected freshwater ponds, astan quds razavi farms and its surroundings, highways and qasem abad village were places that two species of an. superpictus s.l. and an. maculipennis s.l. were collected (121). anopheles superpictus s.l. in villages and tribal areas in bazaft, farsan, chahar mahal bakhtiari had a wide distribution. frequency of adults and larvae were 67.2% and 78.9%, http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 202 http://jad.tums.ac.ir published online: september 30, 2018 respectively. in the studied villages, an. superpictus s.l. comprised 63% to 100% of collected anopheles. this species was generally the dominant species in the area. the larvae of this species have also been collected from the height of 2500m above sea level (the sartang sarveh moorez) while collecting of this species has been reported as high as 2,000 meters above sea level. it has been reported that this species had exophilic behavior in the mountainous areas of sarakhs and kermanshah. in farsan, this species had more frequency in outdoor shelters such as mountains' crack, caves or under large rocks, under the bridge and river wall and like the other parts of its distribution it was exophilic. its seasonal activity began from second half of the july and continued till october with the highest frequency in august in outdoors. adult highest frequency in the study area was 6.2 anopheles per place and highest frequency of larvae in 10, ladle was 33.7. in the studied area, an. superpictus s.l. was found mainly in cracks of the trees, mountains groove, caves and cracks between the rocks that were used for flat -out accommodation of tribal, pit walls, rivers, canals and springs, under bridges, human dwellings, closets and behind furniture, in dark storage, networking within the outdoor, and shower stalls. larval habitat of this species was mainly in margins and riverbeds, streams flow in grasslands springs, animal footprints, holes and stagnant water around the road. habitats were more dirt floor in a meadow. the larval habitats were found more in burrows in shaded and sunny waters in most places. in a pit shelter study in hussein abad village in farsan, chahar mahal bakhtiari, of the 78.5% of the collection belonged to an. superpictus s.l. that indicate that this species was exophil and most prevalent species in the study areas. with regard to the eradication of this species because of its exophilic property, insecticide repellency, it could not stop disease transmission just using residual spraying and must use environmental improvements, using larvicides and case finding and integrated pest manage ment (115). in survey that has been conducted in 1971 in this area, results showed that frequency of adult an. superpictus s.l. in germi county has been increased. this species had a salient activity with an. sacharovi. increased abundance of this species that is of main malaria vectors in iran, is most serious threat to the residents (122-123). human biting rate in hussain abad village, the bites per person in a night were 1.1. this figure for each person in nazi village was 1.4 (115). blood feeding preferences this species was zoophage, and both endo-exophilic. blood feeding preference of human varied depending on geographic location and has been recorded from 5% (124-125). in iran, the human blood index for this species has been determined 22.4% and the sediment test of fed females collected from caves have shown 4.1% the human blood index (119, 125). blood feeding habits, resting and longevity of an. superpictus s.l. in central plateau and south parts of iran is like an. fluviatilis s.l.. the results of the precipitation test in different areas of iran, for anthropophilic index have been reported between 4.8 to 21.4% (124) in a research study in lorestan province to determine blood meal type, fed by this species using elisa, of the 1117 tested blood spots, the number of 128 specimens had positive elisa for human blood and thus the anthropophilic index of this species in this study has been reported 11.4% (126-130). anopheles superpictus s.l. fed on animal and human and preferred blood feeding in areas with large groups of hosts than sporadic hosts. it preferred large body size hosts to small hosts. this species fed on different hosts, when it got dark or night, even in places both inside and outside areas. the host-seeking behavior of this species for blood feeding was http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 203 http://jad.tums.ac.ir published online: september 30, 2018 highly active, and could search many buildings in search of food, and had a high mobility during blood feeding. results of anthropophily and zoophily tests in bazaft, farsan in chahar mahal showed that out of the 92 blood samples, 28.4%, 39.1%, 32.6% of the specimens' belonged to human, cattle and other animals, respectively. investigations in khorasan showed that human blood feeding on human varied between 65–76%. studies conducted from may till november, between 1997 and 1998 by total catch in three indoor places (human, animal, and barn) and one outdoor place, cave, indicated that this species preferred feeding on domestic ruminants and animal than man. the indexes of blood feeding of fed females by elisa in all places were 1.02%, and in outdoors (cave), barns, 1.45% and 2.82%., respectively. the human blood index of an. superpictus s.l. has been calculated zero, in human and animal places. the observations in the studied villages showed that most of the people because high temperatures at night sleep in open spaces (courtyard of the houses) and some of them slept in nets. livestock that their numbers were much higher than the number of people in the house that were kept in the open spaces. unfed mosquitoes after entering the houses preferred feeding on animals than humans. then they rest followed proper shelter (human indoors, animal, barn or outside shelters). these reasons may explain proper zero coefficient of blood feeding on human in indoors (118). anthropophilic index for the an. superpictus s.l. in its maximum frequency was very low (9.09%) (131). longevity of the anopheles superpictus s.l. populations various studies (regions of birjand-north khorasan, kazeroon, etc.) has shown that in appropriate temperature conditions, an. superpictus s.l. after the second gonotrophic cycle is capable of transmitting plasmodium vivax. in nature there is up to 5 gonotrophic cycles. it has been observed that an. superpictus s.l. for the first laying period was usually required twice blood feeding. of the 80, an. superpictus s.l. (58.7%) were nulliparous, 33 of them were diagnosed parous (41.3%). result of the dissection of an. superpictus s.l. females, the rate of parous females was found to be 48.5% in 1st gonotrophic cycle, 36.5% in 2nd gonotrophic cycles and 15.1% in 3rd gonotrophic cycles. basically an. superpictus s.l. in this area is most dangerous age in september. epidemiological studies showed that in birjand and kazeroon, an. superpictus s.l. was an unstable vector. desiccated salivary glands of an. superpictus s.l. in aligoodarz, showed no sporozoites (103). sustainability index epidemiological studies in birjand and kazeroon showed that an. superpictus s.l. is an unstable vector (stability index less than 0.5) and the executive observations support this theory. this means that the use of residual insecticides can easily control this species (excluding the exophil population). adult shelters in iran, a significant population of this species rests in human and animal dwellings including mud rooms, shed, stack, barns, straw, tents and mud stables, rick, animal sheds, poultry nests. some of the population of this species, especially in the west (kermanshah) and the east (kashafroud region) didn’t enter to human habitations and has been collected from outdoors including cave, inside grain storage, cracks in the mountain rock, earth fissures in the walls of the rivers, the aqueducts, under the bridge and artificial pits (99, 132-133). anopheles superpictus s.l. larvae were collected from villages including hoomeye markazi, roodan, sandarak, sirik, dej olia river, dej sofla river and bashagard. this species that comprised 1.3% of the total percentage of collected larvae periodically was active during at least 6 months of the year. this anopheles has been collected from temporary and http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 204 http://jad.tums.ac.ir published online: september 30, 2018 stagnant waters, 62.5% and 100%, respectively. it has been collected with low percentages from permanent waters. this species could ovoposite in water nests that the water depth was low, with or without plant, sunny or semishade. the larvae of this species prefered larval habitat with soil or sandy in their bed along the rivers (to 80%), wetland, grasslands and fresh waters. it chose fresh and clear waters for ovoposition (50%). anopheles superpictus s.l. larvae have been collected together an. stephensi, an. fluviatilis s.l., an. dtali, an. thurkhudi and an. multicolor in the same larval habitat. in some west and south west provinces of iran, an. superpictus s.l. larvae has appeared from temporary and stagnant waters with the frequency of 40% and with low density from permanent waters with or without plants. the larvae of this species in kurdistan began their activity in mid-june and ends in mid-october. the peak activity is from early august to mid-september, and its activity dependent on temperature. the larvae of this species have been collected from the cities of islamabad-e-gharb, paveh, qasr-e shirin, harsin (bakhtaran) in 1984–1985. in bakhtaran the maximum abundance of larvae were found in july and its activity were reported from june to november. in ilam the activity of 3rd and 4th larvae began from june to september, with most activity and abundances in july. in khuzistan province, activities of 3rd and 4th instars larvae were from may to october and with its peak of activity in july and august. in germi and pars abad, the larval habitats included along the dare roud river, rice fields, small cavities and holes in the river beds (113). in bazaft, farsan, chahar mahal bakhtiyari, of the 6 villages and 4 tribal regions, an. superpictus s.l. comprised 44.2% of the anopheles populations in human dwellings. in animal places an. superpictus s.l. comprised 63.7%, of the anopheles populations and in outdoors it was 77%. totally, this species has been collected from outdoors. in hussain abad village, 243 mosquitoes were collected by night-biting method that 165 of them (67.9%) were an. superpictus s.l. in this village in farsan, of the 254 anopheles mosquitoes collected in pit shelters, 180 (70.8%) of them were an. superpictus s.l.. frequency of an. superpictus s.l. were 162 (66%) larvae collected from the tribal wattle, 130 (100%) from shermak, 168 (66.7%) from tribal regions of labad and in tribal regions of tashnavi it was 181 (63%) (116). studies conducted from may till november, between 1997 and 1998 by total catch method in three indoors (human, animal, barn) and one outdoor (cave) in the province of lorestan showed that of 1661 collected culicidae mosquitoes, the 1632 (98.25%) were anopheles genus, and among them the total number of 1630 (99.3%) of the collected anopheles were an. superpictus s.l. 631 (50.8%) collected female an. superpictus s.l. were freshly feeders (119). malaria transmission potential in some parts of iran, especially kashaf roud in khorasan and kermanshah it was discovered that despite eradication programs, malaria transmission by this species has not stopped (mesghali unpublished observation, 1968). vectorial capacity is a measure of epidemiology in different geographical regions that is used to determine the malaria transmission risk. vectorial capacity is affected by three factors including anthropophily, longevity, and mosquito vector population density. in iran, sporozoite rate has been reported 0.9%. desiccation of salivary glands of an. superpictus s.l. in khorasan, kermanshah, kazeroon and masjed-e-soleiman showed that almost 1.2% of the desiccated specimens were infected to sporozoite. in tabas 4.6% of the specimens and in kazeroon 0.65% of the specimens were infected with sporozoite and 0.7% of them were infected to oocyte. these studies indicated that an. superpictus s.l. was of the major malaria vectors in the central plateau and http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 205 http://jad.tums.ac.ir published online: september 30, 2018 the southern slopes of the zagros mountains of iran (128). anopheles superpictus s.l. has been identified as a main malaria vector in all regions of central iranian plateau. in addition, this species had significant role in maintaining malaria as a second or third vector in mountainous and foothills of southern zagros range. in 255 desiccated specimens in birjand in 1961, the sporozoite index was 4.7% (accidental sampling). desiccation of 411 an. superpictus in the mountainous region of shapoor (kazeroon) in 1960, infective index was 0.65%. in bazaft, farsan, dissection of 200 an. superpictus to determine the number of parous and nulliparous showed that the parous rate were 26%, 46% and 40% in late july, august and september, respectively. a total of 126 anopheles salivary glands and stomach were dissected and none of them was found infective to sporozoite and oocyst (116). larval habitats this species can be found in beds of rivers that are drying in hot climate of the summer. this species have been collected from sandy margins of the river (gamasab and chamkhal rivers in kermanshah province). captures of 2nd (early instars), 3rd–4th (late instars) instars and pupae have been done for an. superpictus s.l. immature abundances in different agro-ecosystems in iran. anopheles superpictus s.l. larvae are associated with gravel or fresh waters of swamplands and marshes, and pebble river and stream beds in shallow, slow-flowing clear water in full sunlight, small rivers or streamlet, holes, pits and natural dams, small pools within or next to river beds and streams, storage tanks, broken pitchers, empty cans, water reservoirs, rice fields and associated irrigation canals, natural grassland, large rivers that makes pools, animal hoof traces, tires, small pools within or next to river beds, are suitable as aquatic habitats. preferred water temperature for ovopositing of this species in day time, usually reached to 35 °c to 38 °c. in 30 °c, the larval period lasted 11 days. however, at temperatures between 19 °c to 20 °c, the larval period has been estimated at 24 days. frequency of 3rd and 4th instars of an. superpictus s.l. in each ladle from april till november 1994 in lanjanat, isfahan in late july, mid-august and late august were 0.1, 0.1 and 0.1, respectively and in the remaining months was zero (122). ecological characteristics of larval habitats of an. superpictus s.l. in 24 larval habitats in lorestan province showed richness of species composition that is indication of the importance of land use patterns on diversity of larval habitat types that included an. superpictus s.l., an. turkhudi, an. stephensi, an. dthali, an. sacharovi, an. claviger and an. martri. anopheles superpictus s.l. larvae were collected from standing or temporary and especially from still waters. this species was active in the waters with aquatic vegetation on their surface or outside of them. more than 45% of the specimens were collected from habitats without any aquatic vegetation. in 87.5% cases this species oviposit in the sandy and soil beds habitats with fully sunlight. this species prefers clear and fresh waters. their larval habitats were close to rivers (53.2%), lakes, grasslands, holes, streams, and river beds (46.8%). in man-made larval habitats, 87.5% larval habitats were found in rice fields and 12.5% were in cement grooves. larval peaks of activity have been reported from mid-july till second half of the october. 67.8% of the larval habitats were standing and 32.2% of them were temporary. 62.6% of the larval habitats were still waters and rests of them were running waters. in this survey from the 115 larval habitats, 88.7% of the specimens were collected from sunny habitats. this species preferred to oviposit in sandy bed larval habitats (55.7%), mud (40.8%) and cement canals (3.5%). these species were mainly found in habitats without vegetation (46.9%), vegetation outside of the water (36.5%) and the rests of them in vegetation on the surface or under the http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 206 http://jad.tums.ac.ir published online: september 30, 2018 water. in lorestan province, larval habitats were found in agro-villages s.l.es of pishkouh zalghi (villages of baghe lotfian, kizan darreh, kakolestan), farsash (villages of farsash, homa and ivaj), barbaroude shargi (villages of chaman soltan (shahrake emam), ab barik, dehnou khaje), pache lake shargi (villages of dehe nasir, sour, choghatar), mahrou (villages of margsar and chal ghale). in bazaft, farsan, chahar mahal bakhtiari in 1999, in the tribal region, activity of different species of anopheles in ten studied villages were investigated 120 times. a total number of 2543 larvae were collected from different larval habitats including river margins, stagnant water, grasslands and springs. in tribal regions a total number of 162 larvae were collected that 66.6% of them were an. superpictus s.l.. in labeh of the 168 collected larvae, the frequency of an. superpictus s.l. was 66.7%. in shermak area of the 130 collected larvae 100% of them were an. superpictus s.l.. in teshnavy that is a tribal area of the 181 collected larvae in 12 times, 63% of them were an. superpictus s.l.. anopheles superpictus s.l. larvae with the abundance of 33.7% in 10 ladles had the highest density. adult susceptibility tests the insecticide susceptibility tests on field adults and larvae were performed in 1972–1975 on the an. superpictus s.l. collected from different parts of the country including mountainous, temperate and desert areas including isfahan, kermanshah, khorasan, kurdistan, and ilam provinces. for the adult tests, female an. superpictus s.l. were exposed to discriminating doses of oc, op and py insecticides including d.d.t. (mortality rate: 99.4%), malathion (mortality rate: 100%), lambdacyhalothrin (mortality rate: 100%) and dieldrin for 60min. the effects of these pesticides were evaluated in accordance with who standards, using mortality rates after 1-hour exposure followed by monitoring over a 24-hour recovery period. the adult susceptibility tests using who criteria (98–100% mortality indicating susceptibility and < 98% mortality indicating resistance), showed that an. superpictus s.l. was susceptible to all the tested insecticides (107-109). in ilam province, in 2002, the percentage of mosquitoes knocked down at 60 minutes were 100%, 99% and 98% for lambdacyhalothrin, malathion and ddt, respectively (111). table 1 and figure 3 show the resistant of species to ddt and susceptible to other insecticides in the country. fig. 1. distribution of anopheles superpictus s.l. in iran fig. 2. morphology of larva of anopheles superpictus s.l. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 207 http://jad.tums.ac.ir published online: september 30, 2018 table 1. status of insecticide resistance in anopheles superpictus from iran, 1957–2016 insecticide anopheles superpictus ddt 4% r dieldrin 0.4–4% s malathion 5% s lambdacyhalothrin 0.025–0.1% s s= susceptible r= resistant fig. 3. mapping status of anopheles superpictus to ddt in iran discussion malaria remains an important parasitic disease in different parts of iran, despite decades of organized malaria control activities. malaria is particularly prevalent in tropical provinces including sistan and baluchistan. iran remains highly conducive to vector borne diseases due to geography, and uncontrolled population movement. the anopheles (cellia) superpictus s.l. is important malaria vectors that have been found in different provinces of iran. anopheles superpictus s.l. represent important vectors of malaria throughout the palearctic region, including iran. this s.l. complex contains at least three closely-related sibling species that can be differentiated based on distinctive genetic characters. current evidence shows that climate variability has a direct effect on the epidemiology of vectorborne diseases including malaria). the most influence of climate change on disease trans mission is likely to be monitored at the extremes of the range of temperatures at which transmission develops. malaria is of the most dangerous vector-borne diseases in the tropical and subtropical areas. health risks because of climatic variability will be different among countries that have developed health bases and those that do not (134). patterns of human settlement in the various areas will affect disease tendency s.l. complex. it has evidenced important anthropogenic ecosystem changes and it contains a great frequency and diversity of mosquito-breeding sites and as a result hosts large mosquito populations, and in areas of endemic malaria. results indicate that, environmental modifications and changes in the economic, social, and cultural environments can have tough and rapid influences on mosquito populations, climate variability, and drug resistance. these factors influence on http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 196–218 h vatandoost et al.: bioecology of … 208 http://jad.tums.ac.ir published online: september 30, 2018 biting, survival, and reproductive rates of vectors (135-143). we recommend new research on different aspects of an. superpictus in the future for decision making. conclusion understanding of all bioecology of malaria vector will guide the scientist and decision makers for appropriate diseases control. acknowledgment this research is financially supported by the ministry of health and medical education national institute for medical research development (nimad) under code number of 963262. all authors declare that there is no conflict of interest references 1. who (2017) world malaria report 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[msc dissertation]. faculty of health and health research institute. tehran university of medical sciences. 143. zahar ar (1990) vector bionomics in the epidemiology and control of malaria. part ii .world health organization, v.b.c. 90(3): 57–155. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 152 http://jad.tums.ac.ir published online: june 12, 2018 original article attitudes and practices from people of a mayan community of mexico, related to tick-borne diseases: implications for the design of prevention programs *karla dzul-rosado 1, cesar lugo-caballero 1, juan jose arias-leon 2, freddy pachecotucuch 1, gaspar peniche-lara 2, jorge zavala-castro 1 1emerging and re-emerging diseases laboratory of centro de investigaciones regionales universidad autonoma de yucatan, colonia centro, mexico 2inter-institutional unit of epidemiologic and clinical research, medicine faculty, universidad autonoma de yucatan, colonia centro, mexico (received 26 may 2016; accepted 16 may 2018) abstract background: tick-borne diseases are caused by several pathogens whose transmission could be associated to the life conditions of communities settled in endemic areas. we aimed to determine the knowledge, attitudes, and practices related to the exposition and prevention of tick-borne diseases among people living in a typical mayan community of yucatan, mexico between dec 2012 and may 2013. methods: a directed survey was applied to 212 (100%) householders (women and men) from teabo, yucatan, mexico. answers and field notes were recorded and analyzed with central statistics. results: people have been bitten at least once in the community, but the majority of them consider those bites innocuous. in addition, people do not consider prevention measures, and only a few mentioned the use of some chemicals on their backyards. conclusion: this study found little awareness among the participants regarding the importance and the transmission of these diseases even though they possess a vast knowledge regarding ticks. therefore, educational strategies and prevention programs that include these habits for its modification are required to minimize the exposition to the vectors. keywords: tick-borne diseases, community knowledge, prevention programs introduction tick-borne diseases (tbd) are considered emerging zoonosis due to its increasing worldwide prevalence and the establishment of a complex relationship with its reservoirs and vectors with the humans (1-3). these diseases are caused by different pathogens including borrelia burgdorferi, anaplasma phagocytophilum, babesia spp., rickettsia spp. and ehrlichia spp. among others (4). the transmission of tbd is associated to ecotones, which are transition regions between ecosystems, including the agricultural fields, poultry nests, and backyards, which are in close contact with the jungle (5-6). as a consequence, the main factors associated with the acquisition of tbd includes poor hygiene conditions and the closeness with pets parasitized by infected ticks inside the house (7). in northern and southern states of mexico, there have been found pathogens like b. burgdorferi, rickettsia spp. and ehrlichia spp. in ticks from wild and domestic animals (8-12). therefore, the increasing contact between wildlife and domestic animals serves as an amplifying mechanism for the transmission of these diseases particularly among neglected communities (2, 3, 13). mayan communities of yucatan have the optimum weather and ecological conditions to sustain the transmission cycle of ixodes, amblyomma and ripicephalus ticks among *corresponding author: dr karla dzul-rosado, e-mail: karla.dzul@correo.uady.mx http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 153 http://jad.tums.ac.ir published online: june 12, 2018 pets, cattle and wildlife (6, 7). tbd like rickettsiosis and ehrlichiosis, are endemic pathologies of the region (9, 14-24). these studies have revealed the fragility and weakness of the surveillance programs in public health, alongside with the apparent absence of habits to prevent tick bites among people. in order for those surveillance and preventive programs to work, it is important to consider the community knowledge, perceptions, and attitudes towards the problem, achieved through community participation (community engagement) strategies. the information obtained through these strategies could be used for the design of intervention strategies based on the analysis of real scenarios and not in assumptions like typically is done (25). in these terms, by studying people from a typical mayan community (teabo, yucatán, mexico), this work aimed to determine the knowledge, attitudes, and practices related to the importance of ticks as vectors, the risk of bites and its prevention. materials and methods study area this study was carried out in the community of teabo, yucatan, mexico; located between n 20° 19' and 20° and w 89° 11' and 89° 20'. teabo is bordered on the north by mayapan-chumayel, to the south by tekax, to the east by cantamayec-tixmehuac and to the west by mani-akil. the district population is 6205 people distributed among 1380 households which are occupying an area of 261, 87km2. there is only 1 public health center. the district has a warm subhumid zone, with summer rains. the average annual tem toperature is 26.3 °c and the average annual rainfall is 65.7mm (snim, 2010). the study was conducted between dec 2012 and may 2013 and it involves only the municipality of teabo. this community was selected for three main reasons: 1) it has not been intervened by other scientific groups, 2) the daily activities of the inhabitants in the periphery areas of the households or in the outside of town are carried on in areas which are suitable for the acquisition of ticks, 3) there have been several cases of rickettsiosis among children of the community. study design a cross-sectional, descriptive study was conducted in the community among householders that were randomly selected. a household was defined as a residential unit with one or more individuals in occupation. multiple families residing in the same household, as well as multiple structures within the same grounds, were also considered as one household. the diffusion was made through sensitization workshops organized by the health center of this community. in these workshops, the people were informed about our interest in their knowledge and traditions regarding tbd. after the workshops, the staff explained the methodology, ethical aspects, and objectives of the study to the householders that freely accepted to participate. trained members of the working group applied a survey through a face-to-face interview with the participants. this survey was designed and validated prior the start of the study, and consisted in 10 items with its optional answers covering four different topics: (a) knowledge (regarding the recognition of ticks and the presence of reservoirs in their houses or backyards), b) risk of being bitten by ticks, c) actions after a bite, d) prevention measures to avoid tick bites. every item consisted of a question and four or more different answers. due to its relevance, answers and additional comments not included in the options were recorded as field noted and categorized later according to the topics. the most representative quotes are presented as part of the collected data. as this survey contains several items for variables with unknown variance, it is not possible to calculate, a sample size for a given confidence interval (26). however, it is possible http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 154 http://jad.tums.ac.ir published online: june 12, 2018 to calculate a minimal sample size for a confidence interval or 95%, and a 10% maximum probability of committing type-1 error, considering a binomial distribution for several of these variables. using: n= z2 (p)(q)/d2 the suggestion is a minimum of 91 households to obtain relevant results (27). data analysis the surveys were checked for coding mistakes, integrity and coherency data. the data tabulation was processed and analyzed using excel 2007® and graph pad 5.01, san diego, california. ethical considerations the research ethics committee of the hospital o’horan (merida, yucatan, mexico) approved the ethical statements of this work, as a goal of the project cie-010-1-14 registered to dr karla rossanet dzul rosado. both the municipal authorities and the householders were informed about the study objectives, the methodology, the questions in the survey, their faculty to choose whether to participate or not in the study without any problem for them, the anonymity of their responses and demographic data, and that they would be informed of the results of the study after its end. after these statements, informed consents (signed or fingerprinted) were obtained according to the ethics committee statements. all the photographs and quotes presented in this work were taken under consent of the portrayed people. the staff made sure that the respondents did not have cognitive disabilities previously diagnosed. the mayan language is the most common dialect in rural communities of yucatan like teabo, and not all the people in the community can speak spanish. moreover, not all the people are able to read and write. the staff had mayan language interpreters and aides to overcome those situations results after the sensitization workshops, 212 householders accepted to answer the survey. to obtain data regarding their general knowledge about ticks and its medical importance, people were asked about 4 main topics: knowledge of tbd, risk of being bitten by ticks, prevention measures to avoid ticks and actions performed after the tick bite. answers collected from surveys and field notes obtained through face-to-face interviews are described below. knowledge of ticks and tick-borne diseases initially, people were asked to enumerate by its importance three of the most widely promoted infectious diseases in our region (dengue, chagas disease, and influenza) alongside with tbd, based on their current knowledge. about 25% of the people considered dengue as the most important disease, followed by influenza (9.9%), chagas disease (6.13%) and finally tbd (0.47%) (table 1). related to the general knowledge, almost all of the surveyed people answered that they can identify a tick (98.5%) (table 1). we also took field notes regarding the typical names that they use for ticks like “peech” (singular), “sojol peech” (the ticks from the dead leaves), “mejen peech” (ticks of medium size), and other variants (table 2). the agricultural fields, labored according to their ancient traditions are called “la milpa”, whereas the jungle areas surrounding the town are called “el monte“. risk of being bitten people have seen ticks inside their houses (24.5%), in “la milpa” or “el monte” (29.2%); however, 46.3% is unaware of ticks (tables 1, 2). the term “tick infestation” was described to the interviewed people as the presence of large and noticeable quantities of ticks in the walls and floor of their houses. following this definition, 45.8% reported not http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 155 http://jad.tums.ac.ir published online: june 12, 2018 to have had tick infestations in their houses, 37.2% reported to have had infestations during rainy season (jun–nov), and the remaining 36% during dry season (table 1). during the interviews, it was interesting the recurrent mention of a catholic patron named saint francis, worshiped in teabo. these people believe that “the saint has a tick attached to his ankle, so when the saint bless the milpa on oct 4th (during rainy season), the ticks fall to the ground.” (fig. 1, table 2). some other people also believe that “it is written in the bible (a holy book in catholicism) that saint francis protects the ticks.” (fig. 1, table 2). finally, people answered that 24.5% have been bitten in the house, 29.2% in “la milpa” and 46.2% deny have been bitten (“i have been bitten on my back”, “the peech has bitten my husband at la milpa”, “all my sons have been bitten several times”) (tables 1, 2). actions after a tick bite after the tick bite, most people do not perform further actions and just remove the tick (63.5%) because “nothing bad happens”. however, 36.5% of the surveyed people use home remedies (tables 1, 2). these home remedies include water (i only wash the bite site with water and salt”), ointments (“i apply vaporub to the bite”, “i apply alcohol, and that is all you should do.”) and insecticides (“i apply a mixture of “denate” (methomyl/ diflubenzuron) and water to the bite”) (fig. 1, table 2). actually, 55.7% of the people ignore the consequences of a tick bite, 1.4% said that there are no consequences (“it only makes you lose your appetite”, “it provokes an aax (in the mayan language, the word “aax” means warts)”, “nothing, it is not dangerous. it gives you itches sometimes.”). in contrast, 42.9% knows that the bite can transmit a disease, and from these people, 13.2% knows that the disease can be mortal (tables 1, 2). however, their comments show that the people do not know what kind of disease is (“it provokes an aax”, “only if the tick has bitten a snake, its bite can kill you”). prevention measures to avoid tick bites regarding the topic of prevention, 62.8% of the people do not use measures to prevent tick infestation and bites. the remaining 37.2% has some kind of practice. within this group, 24.5% uses chemical agents on their dogs in addition to the bath (“i bathe my dogs with asuntol and a special talc powder.”), detergent (i bathe my dog with fab and blue soap.”) or other kind of chemicals (“i bathe my dogs with burned oil.”, “i bathe my dogs with chlorine and blue soap.”), 6.1% just bathes their dogs with preventive soaps, 6.6% uses any chemical agent against ticks on their yards (“i spread insecticide in my yard”) (fig. 1, tables 1, 2). for the people in yucatan, asuntol is the common name of several brands of ectoparasiticides containing coumaphos, and fab is the generic name for several brands of detergents. fig. 1. habits and attitudes regarding ticks in teabo, yucatan, mexico people http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 156 http://jad.tums.ac.ir published online: june 12, 2018 table 1. general knowledge regarding ticks and its medical importance (n=212) topic subtopic categories number of responses percentage knowledge of ticks degree of importance of some infectious diseases dengue influenza chagas’s disease tick-borne diseases (tbd) do not know 53 20 13 2 124 25.0 9.9 6.13 0.47 58.5 can identify a tick yes no 209 3 98.5 1.5 risk of being bitten places where people have seen ticks inside the house in the milpa or the monte they are unaware of ticks 52 62 98 24.5 29.2 46.3 season of the year when their houses have been infested during the dry season during the rainy season the house has never been infested 36 79 97 17.0 37.2 45.8 places in which they have been bitten at their houses and peridomiciliary area at the milpa or the monte never have been bitten 13 64 135 6.1 30.2 63.7 actions after a tick bite actions are taken when they find a wound or a tick attached to their bodies removal of the tick and no further action use of home remedies seek for health assistance 137 75 0 63.5 36.5 0 consequences of a tick bite it can transmit an uncomplicated disease it can transmit a mortal disease there are no consequences do not know 62 28 3 119 29.7 13.2 1.4 55.7 preventive actions to avoid tick bites actions were taken to reduce the risk of being bitten by ticks use of chemical agents against ticks on their yards use of chemical agents on their dogs people that bath their dogs on a regular basis no measures 14 13 52 133 6.6 24.5 6.1 62.8 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 157 http://jad.tums.ac.ir published online: june 12, 2018 table 2. representative field notes regarding the main topics explored during face to face interviews main topic subtopic quotes from the people knowledge of ticks mayan names given to ticks peech (tick), sojol peech (ticks living in the dead leaves), k’àak’ peech (fire tick, red-colored ticks), mejen peech (small size ticks), buul tá peech (ticks living on feces). risk of being bitten places where they have seen ticks at the wall of the house, the kitchen and the poultry nests on animals like dogs or cattle at the milpa (slang mexican for agricultural fields) and the monte (slang mexican for jungle) at the firewood or corncobs brought from the milpa or the monte on clothes of the people who work at the milpa or the monte season of the year when ticks are more abundant october 4th which is the day of st francis for catholicism. during this time because st francis blesses the seeds and brings the ticks attached to his ankle. during the rainy season (june-october) during february and october whenever there is heat tick bites to the household members i have been bitten on my back all my sons have been bitten several times the peech has bitten my husband several times at the milpa the children are bitten by ticks when they hug people who return from the milpa or the monte actions after a tick bite actions after the tick bite i apply alcohol or vaporub (a commercial ointment with alcanphor/menthol) i wash the wound with hot water, salt, and soap i apply denate (methomil powder used to kill ants) on the wound i do nothing consequences of the tick bite it can kill people the bite is only dangerous if the tick has bitten a snake before it only makes you loose the appetite it produces “aax” (warts, in mayan language) nothing except for some itch preventive actions to avoid tick bites strategies to avoid infestation by ticks i bath my dog with asunthol (coumaphos) i bath my dogs with chlorine, detergent and “blue soap” i bath my dogs with “burned oil” (car oil already used and discarded) i spread insecticide on my yard i keep my dogs clean because they accompany me to the milpa http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 158 http://jad.tums.ac.ir published online: june 12, 2018 discussion rural towns in yucatan meet the ecological and environmental conditions to sustain the life cycles of several infectious disease vectors including ticks. therefore, many vectorborne diseases including tbds like rickettsiosis and ehrlichiosis are considered endemic pathologies in yucatan (9, 15-24). as with other zoonotic diseases, there must be two main objectives to prevent the transmission of tbds. the first one is to achieve a change in the habits of people (keep the animals clean, search for ticks after a hike through the jungle among others), and the second one involves the improvement of the peridomiciliary area (removal of trash and leaves, keep down the grass and vegetation as an example). both goals could be achieved through community intervention programs (28). when a population at risk to acquire a tbd has been sensitized after these types of strategies, the level of awareness could be gradually increased (29, 30). in these terms, the aim of this work was to inquire the knowledge and habits of a community under risk to acquire tbds, through face-to-face directed interviews with people of a mayan community. knowledge of ticks and risk of being bitten less than 0.1% of the participants consider tbd among the important infectious diseases reflecting the lack of government programs against these diseases. considering that yucatan is an endemic area for tbd, there is a real need to design such programs, being the acquisition of knowledge the first step to achieve this goal. ticks have several mayan names, related to its distribution or to its characteristics like size or color. the collected data and the comments of the people suggest that they are used to the presence of ticks, which has to lead them to think that “ticks are not a threat”. it was surprising to found that the patron saint of teabo is associated to ticks, and some people consider oct 4th (saint francis day at the beginning of fall) as the day when ticks spread within the town. several scientific kinds of literature have reported than ticks, tend to be more abundant on dry rather than rainy season and that there is a dramatic reduction in its activity starting on fall (31-33). in contrast with scientific reports, the surveys did not show a clear tendency within the community, to consider than ticks are more abundant on rainy (37.3%) or dry season (36%). this data suggests that people from teabo are not aware of the behavior of ticks even when they associate a particular date (october 4th) with its “sudden increase”. as the harvest season starts in oct, this “increase” could be related to a closer contact with high grass, bushes, and crops, which puts them in a riskier situation to acquire ticks. however, as there is no information about seasonality differences on distribution of ticks in our region, this is an interesting topic to explore in depth. actions after a tick bite some studies have reported an overestimation of the infected tick percentages leading to a saturation of health care centers by asymptomatic bitten patients (1) however; the people from this study tend to not perform further actions because their knowledge about the risks associated to these bites is scarce. even the people that mentioned the appearance of a wound on the bite site discarded the health care center as an option to seek guidance. similar to studies in other endemic countries, people who are not aware of this risk, only seek health care when a relative is ill or when they have the money to cover the cost of a treatment (13). instead of this, people from teabo use home remedies like ointments, different mixes of sour orange and salt, among others. these remedies are commonly used even then its activity against ticks has not been evaluated. there are reports of ethno practices successfully used against ticks based on the exploitation of local natural resources (34, 35). http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 152–161 k dzul-rosado et al.: attitudes and practices … 159 http://jad.tums.ac.ir published online: june 12, 2018 the implementation of this kind of measures alongside with the determination of its utility is an interesting topic to explore in depth. prevention actions to avoid tick bites the 62.8% of the people from this community do not use measures to prevent tick infestation in their houses. the remaining percentage uses chemical methods on their dogs, and only 6.6% uses any method on their yards. in contrast with other vectors like mosquitoes or bedbugs, ticks elimination is highly difficult. the preventive actions were the only way to stop the spreading of tbds. related to this, preventive measures like the use of protective clothing or repellents were not mentioned even when people were intentionally questioned about it, contrasting with other endemic countries whose health departments have permanent educational campaigns (1, 3, 30). people from communities at risk like teabo do not perceive ticks as a threat but as a normal component of the environment with the sole activity of bothering their dogs or them, unaware of their potential consequences as it has been found in other endemic countries, even with permanent educational campaigns (1, 3). limitations the survey lacked questions that could have been important to obtain information considered in other studies like symptoms of specific diseases and its evolution. conclusions in rural communities like teabo, tbds are not considered a problem due to the lack of governmental prevention programs similar to others that exist for pathologies like dengue, and because they believe that ticks are harmless. as a consequence, people who underestimate or neglect the risk will not easily adopt preventive measures. people empower a vast knowledge regarding ticks. however, the knowledge on the importance and prevention of tbds is scarce. to overcome this situation, data obtained should be taken into account to develop programs to sensitize and change to some extent, the habits and attitudes that promote the spread of tbd. these programs should focus on the development of training materials and maintenance workshops for the community and the health workers, as the empowerment and passage of these new conducts by the people are necessary elements to build awareness among the population regarding the importance of ticks as vectors of pathogens and the protection steps that they could use to minimize their risk. acknowledgements the authors would like to acknowledge: the families of teabo, yucatan for their enthusiastic participation in this study, raul tello, karina lopez, angeles cámara, joaquin lunfan, benjamin balam, jorge kú, bianca herrera, and alejandra cámara for their enthusiastic participation at the community. the authors also thank eric dumonteil for his critical review of the manuscript. the authors declare that there is no conflict of interests. references 1. butler ad, sedghi t, petrini jr, ahmadi r (2016) tick-borne disease preventive practices and perceptions in an endemic area. ticks tick borne dis. 7: 331–337. 2. dantas-torres f (2008) the brown dog tick, rhipicephalus sanguineus (latreille, 1806) (acari: ixodidae): from taxonomy to control. vet parasitol. 152: 173–185. 3. valente sl, wemple d, ramos s, cashman sb, savageau ja (2015) preventive behaviors and 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trends parasitol. 28: 437–446 33. pfaffle m, littwin n, muders sv, petney tn (2013) the ecology of tick-borne diseases. int j parasitol. 43: 1059–1077. 34. wanzala w (2017) potential of traditional knowledge of plants in the management of arthropods in livestock industry with focus on (acari) ticks. evid based complement alternat med. 2017: 8647919. 35. wanzala w, takken w, mukabana wr, pala ao, hassanali a (2012) ethnoknowledge of bukusu community on livestock tick prevention and control in bungoma district, western kenya. j ethnopharmacol. 140: 298–324. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 262 http://jad.tums.ac.ir published online: september 30, 2018 original article biology of meccus pallidipennis (hemiptera: reduviidae) to other conditions than that encountered in their native habitat edson franzim-junior 1, maria tays mendes 1, ana carolina borella marfil anhê 2, thiago alvares da costa 1, marcos vinicius silva 1, césar gómez hernandez 1, afonso pelli 1, helioswilton sales-campos 1, *carlo jose freire oliveira 1 1instituto de ciências biológicas e naturais, universidade federal do triângulo mineiro, uberaba, minas gerais, brazil 2instituto de tecnologia e ciências exatas, universidade federal do triângulo mineiro, uberaba, minas gerais, brazil (received 28 sep 2016; accepted 17 july 2018) abstract background: meccus pallidipennis (hemiptera: reduviidae) is only found in mexico and is one of the most important vectors for trypanosoma cruzi transmission there. because data concerning the ability of this bug to adapt to different environments are scarce, we aimed to elucidate its biology, behavior and ability to acclimatize to different environ mental conditions. methods: from the eclosion of 90 1st instar nymphs, development was followed until the adult phase. adults were fed after 30 days of fasting, and the average amount of blood ingested, the time between the beginning of the blood meal and the production of feces, and the frequency of stools/insect were recorded during their meals. after taking a blood meal, couples were isolated and monitored for 21 days, during which eggs were collected weekly. results: the development of m. pallidipennis took 171.74±7.03 days to complete its life cycle, and females ingested larger amounts of blood than males. oviposition was constant and did not demonstrate a significant decrease during this study. conclusion: meccus pallidipennis was able to acclimatize to fluctuating laboratorial conditions other than those naturally found in mexico. keywords: behavior, biology, hemiptera, meccus pallidipennis, triatominae introduction chagas disease affects approximately 10 million people around the world, and at least 25 million are exposed to risk of infection (1). the protozoan trypanosoma cruzi is the caus ative agent, and the most frequent form of trans mission is through the blood-sucking arthro pods known as triatomines or “kissing bug” in sects. after taking a blood meal, infected insects release feces contaminated with infectious meta cyclic trypomastigotes adjacent to the site of the bite (2). because insect vectors are the main route of transmission in endemic countries, the most effective strategy to restrain the spread of disease is to better understand the vector be havior and biology. during their life cycle, triatomines go through five different nymphal stages before entering the adult phase when they reach their sexual maturity and are able to reproduce. their biology is species-specific and depends on environmental conditions, such as temper ature and humidity range (3). additionally, aspects concerning food intake may also have an impact on their epidemiology. in this con text, there is a positive relationship between food intake and feces production, which to gether with the times for feeding and evacua tion, are key elements that dictate the vector efficiency and disease spread (4). *corresponding author: dr carlo jose freire oliveira, e-mail: carlo@icbn.uftm.edu.br http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 263 http://jad.tums.ac.ir published online: september 30, 2018 so far, more than 140 species of triatomines have been identified. among them, meccus pallidipennis stål (hemiptera: reduviidae), formerly known as triatoma pallidipennis, is primarily found on the pacific coast of mexico and represents one of the main vectors of chagas disease in that country (5). this insect is found either in sylvan or domestic reservoirs (6). in urban areas, m. pallidipennis is associated with the presence of dogs, squirrels, pigs and opossums near houses and unconstructed areas (7). the environment seems to affect some crucial aspects concerning the development, reproduction and the potential of m. pallidipennis to transmit chagas disease in mexico (8). because of the epidemiological importance of m. pallidipennis in mexico and the rele vance of this vector to different environments, we aimed to elucidate the biological and be havioral aspects of this species under labora torial fluctuating conditions of humidity and temperature. for this purpose, we investigated the biology and timing of the different stages of development, feeding behavior and oviposition. materials and methods meccus pallidipennis meccus pallidipenis specimens were originated from the region of cienega state of ja lisco, and gently donated by dr josé alejandro martínez ibarra from universidad de guada lajara, mexico. then, the second generation of triatomines were raised and kept in small plastic flasks (6.0cm height x 7.0cm diameter) until they reached the 4th nymphal instar at the insectary of the federal university of triângulo mineiro (uftm). older nymphs and adults were kept in intermediate containers (7.5cm height x 11.0cm diameter). during the period of the study, triatomines were submitted to a natural light cycle and fluctuating environmental conditions of temperature (max: 31.03±0.81 °c and min: 18.87±1.387 °c) and humidity (56.85±25.66%) in uberaba, minas gerais, brazil (19° 44′ 52″ s, 47° 55′ 55″ w–823m above sea level). specimens were fed on immobilized and anesthetized swiss mice weekly. mice were from the animal facility at the parasitology division of the uftm in uberaba, minas gerais, brazil and were anesthetized by using sodium tyopenthal (40.0mg/ kg intraperitoneal-i.p.). all procedures were submitted and approved by the institutional animal care and use committee of the federal university of triângulo mineiro (brazil) under protocol 307. life cycle of meccus pallidipenis we aimed to demonstrate the egg-to-adult developmental time under the aforementioned environmental conditions. after oviposition, eggs were collected and monitored until eclosion. then, 90 nymphs were divided in colonies containing 30 insects each, and the time to reach the adult phase was recorded. triatomines in the 1st nymphal stage were fed 10 days after eclosion (2nd and 4th nymphal stages) and were followed until the next molt. insects in the 5th nymphal stage were fed twice, on the 10th and 20th days after molting. only nymphs that fed were considered viable and able to develop from one developmental stage to another, and 5th instar nymphs were considered viable only after the second feeding. feeding behavior analyses verified the average time of feeding, amount of blood ingested, and time between blood meal and excretion. during the blood meal, the following aspects were recorded: feeding time, amount of blood ingested, time until first excretion and frequency of feces. the frequency of feces/ meal was also recorded. randomly, 20 adult insects (10 males and 10 females), fasting for 28 days, were selected from pre-existing colonies. each triatomine was individually allocated to plastic receptacles and weighed before and after the blood meal using an analytic scale (unibloc shimadzu auy220, kyoto, japan). the blood ingestion rate was individually calculated by dividing the amount of blood ingested by the feeding duration. the http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 264 http://jad.tums.ac.ir published online: september 30, 2018 amount of feces/meal was calculated by dividing the frequency of total defecations per gender by the number of triatomines from each group/gender. both the blood ingestion rate and the amount of feces per meal were calculated as previously describe (9), with some modifications. oviposition during different fasting times the oviposition was followed during different fasting times to evaluate the impact of fasting on the production of eggs. thus, 8 adult couples were randomly chosen, isolated, and fed only once, and oviposition was monitored daily over 21 days. eggs were collected on days 7, 14 and 21 after feeding and kept in different containers for analysis. statistics for all variables, the normal distribution and homogeneous variance were tested. owing the size of our groups, we used nonparametric mann-whitney tests to compare time of feeding, blood ingestion and blood ingestion rate. to address the differences between the per-centage of males and females producing feces a fisher exact test was used. since we had only eight couples, a non-parametric method (friedman test) was used to compare oviposition among weeks. the results were expressed as means±standard error of the means. the dif ferences were considered statistically signifi cant when p< 0.05 (5%). all analyses were performed using graphpad prism 5.0 software (san diego, ca). meccus pallidipennis developed under fluctuating environmental conditions to study the impact of fluctuating environ-mental conditions on the development of m. pallidipennis, 90 nymphs were randomly selected, and their development was followed from hatching to adulthood. the mean time for eclosion was 20.15±0.74 days (table 1). furthermore, the number of days necessary to complete each instar increased when compared to the previous stage of development as follows: 22.13±0.83, 23.35±0.85, 25.13±1.03, 31.00±1.44 and 50.00±2.60 days, respectively, from 1st nymphal instar to adult (table 1). meccus pallidipennis females ingested larger amount of blood than males because we observed that m. pallidipennis was able to properly develop under the local environmental conditions, we next assessed their feeding behavior. the results showed that females ingested larger amounts of blood than males (table 2). no differences between genders were observed for the feeding duration and blood ingestion rate (table 2). furthermore, 70% of adult males defecated in 26.69±7.98 minutes after starting the blood meal and had 0.80±0.63 feces/meal (table 3), while females had 1.30±0.48 feces/meal during 25.43±7.83 minutes (table 3). though slightly different, no significant differences were observed be tween the genders on these parameters. oviposition because triatomines were able to develop and fed under the conditions tested, we next investigated their ability to lay eggs. overall, 140, 120 and 122 eggs were laid during the first, second and third week, respectively (fig. 1a). weekly, the oviposition rate was 17.50±4.81, 15.00±5.63 and 15.50±5.09, respectively, from the first to third week (fig. 1b). furthermore, daily oviposition was recorded, and the rates were 2.50±0.68, 2.14±0.80 and 2.21±0.72, re spectively, for the first, second and third week (fig.1c). however, differences among the weeks were not significant. table 1. time of transition between developmental stages http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 265 http://jad.tums.ac.ir published online: september 30, 2018 developmental stage median±sd (days) eggs n1 (n=80) 20.15±0.74 n1-n2 (n=56) 22.13±0.83 n2-n3 (n=49) 23.35 ±0.85 n3-n4 (n=39) 25.13 ±1.03 n4-n5 (n=27) 31.00 ±1.44 n5–adult (n=6) 50.00 ±2.60 total 171.74 ±7.03 n: number of individuals in each developmental stage table 2. feeding behavior in adult triatomines time of feeding (min) blood ingestion (mg) blood ingestion rate (mg/min) m. pallidipennis (m) 26.24±11.10 445.60±265.00 * 18.66±9.71 m. pallidipennis (f) 34.09±14.05 760.40±317.00 * 23.28±6.05 m: adult males. f: adult females. min: minute. mg: milligram. mg/min: milligram per minute. number of individuals/gender = 10. to compare time of feeding, blood ingestion and blood ingestion rate a non-parametric mann-whitney test was used. to address the differences between the percentage of males and females producing feces a fisher exact test was used. data are depicted as mean±standard deviations. an asterisk (*) indicates a significant result (p< 0.05) table 3. production of feces time of defecation (min) % insects producing feces frequency of defecation/meal m. pallidipennis (m) 29.69±7.98 70 0.80±0.63 m. pallidipennis (f) 25.43±7.83 100 1.30±0.48 m: adult males. f: adult females. min: minute. number of individuals/gender = 10. data are depicted as mean±standard deviations fig. 1. oviposition of meccus pallidipennis during fasting period after a blood meal, eight couples were separated into individual plastic flasks for 3 weeks as previously described. (a) total number of eggs; (b) eggs per couple/week and (c) average eggs per couple per day. to compare the differences in oviposition among weeks a friedman test was used. the results were expressed as means±standard error of the means. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 266 http://jad.tums.ac.ir published online: september 30, 2018 discussion meccus pallidipennis was able to acclimatize to environmental conditions other than those naturally found in mexico, reinforcing its vector potential in a distant geographical area. in this study, the life cycle of m. pallidipennis was similar to that observed in previous studies with the same species (10, 11), despite the differences in food sources and environmental conditions observed in the different studies. notably, this insect has the shortest biological cycle among triatomines of the same genre (12, 13); however, even under the same laboratory conditions, life cycles can be distinct in different populations from the same species (14). the heterogeneity of the populations of m. pallidipennis was further demonstrate in a study that showed the impact of different environments on the reproduction, feeding behavior and development of this species in distinct areas of mexico (8). short biological cycles are epidemiologically critical because they represent the ability of a species to repopulate and reestablish colonies right after disturbances, such as those produced by insecticides. this scenario makes triatomines more competent and capable of interacting with a host, consequently increasing the risk of transmission of t. cruzi. when compared to the main species of vectors in latin america, the biological cycle of m. pallidipennis was longer than that described for t. infestans and rhodnius prolixus, both reaching adult stage within less than 100 days (15, 16). however, some other species may have a longer life cycle, such as that observed in t. boliviana (252 days), from bolivia (17), and t. carcavalloi (193 days), from the southern region of brazil (18). on the other hand, it was similar to the development of panstrongylus megistus (19) and eratyrus mucronatus (20). feeding behavior is one of the key ele ments that dictate the vector efficiency and biology of triatomines. during a blood meal, not only they are able to transmit t. cruzi but also to fulfill their physiological needs for survival. females are more sensitive to fasting periods than males from the same species (21). different factors could explain this characteristic, including the energy expended in ovipo sition and the maintenance of larger bodies in adult females compared to their male counterparts. accordingly, we demonstrated that adult females ingested greater amounts of blood than males, which could be related to the higher physiological and energetic demand associated with females. however, no differences between genders were observed for the feeding duration and rate of blood ingestion. though the feeding duration for females was similar to that observed in males, the higher amount of blood ingested by females may suggest a greater voracity to their hosts. the observation that females ingest higher amounts of blood than males could explain why they are also considered better vectors, besides as a mechanism for greater feces production (9, 14). unfortunately, we did not find any differences between the amount of blood ingested and the production of feces when the genders were compared. additionally, because a positive relationship was already established between the influence of the fasting period and the production of feces (22), we believe the fasting period in our study (30 days) might have increased the time needed for defecation. furthermore, as females are more sensitive to fasting, they also tend to be more sensitive to weight loss when compared to their male counterparts from the same species. this characteristic makes females less-efficient vectors when exposed to greater fasting periods. despite the impact on production of feces, the fasting period did not seem to influence the proportion of insects defecating before or after feeding. in the absence of regular feeding periods, either oviposition or fertility could be negatively influenced (23). however, in our study, fasting did not influence oviposition. these results indicate that http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 267 http://jad.tums.ac.ir published online: september 30, 2018 the period of and sensitivity to fasting may fluctuate according to the feeding behavior, and longer feeding periods seem to enhance the period of oviposition. conclusion feeding behavior has a great impact on the life cycle of triatomines, which may also in fluence t. cruzi transmission. furthermore, the higher amount of blood ingested by females than males reinforced their potential as vectors. finally, because m. pallidipennis was able to reach sexual maturity and reproduce even under different and fluctuating environmental conditions, it seems reasonable to assume its potential as a vector in environments other than those naturally find in mexico. acknowledgments we thank dr josé alejandro martínez ibarra from universidad de guadalajara, mex ico, by the donation of m. pallidipenis specimens used in this study. this work was sup ported by the fundação de amparo à pesquisa do estado de minas gerais (fapemig), coordenação de aperfeiçoamento de pessoal de nível superior (capes), and conselho nacional de desenvolvimento científico e tecnológico (cnpq-150075/2016-2). references 1. who (2010) first who report on neglected tropical diseases: working to overcome the global impact of neglected tropical diseases. in: organization wh, editor.: who press. 2. tyler km, engman dm (2001) the life cycle of trypanosoma cruzi revisited. int j parasitol. 31(5–6): 472–481. 3. schofield cj (1994) triatominae-biology and control. eurocommunica publica tions, london. 4. piesman j, sherlock ia (1983) factors controlling the volume of feces produced by triatomine vectors of chagas' disease. acta trop. 40(4): 351–358. 5. ibarra-cerdena cn, sanchez-cordero v, townsend peterson a, ramsey jm (2009) ecology of north american triatominae. acta trop. 110(2–3): 178–186. 6. cruz-reyes a, pickering-lopez jm (2006) chagas disease in mexico: an analysis of geographical distribution during the past 76 years-a review. mem inst os waldo cruz. 101(4): 345–354. 7. ramsey jm, alvear al, ordonez r, munoz g, garcia a, lopez r, leyva r (2005) risk factors associated with house in festation by the chagas disease vector triatoma pallidipennis in cuernavaca metropolitan area, mexico. med vet entomol. 19(2): 219–228. 8. martinez-ibarra ja, nogueda-torres b, salazar-schettino pm, vences-blanco mo, de la torre-alvarez fj, montanezvaldez od (2014) differences on biological attributes of three populations of meccus pallidipennis stal (hemiptera: reduviidae). j vector borne dis. 51(1): 22–26. 9. martínez-ibarra ja, novelo-lópez m (2004) blood meals to molt, feeding time and postfeeding defecation delay of meccus pallidipennis (stål, 1872) (hemiptera: reduviidae) under laboratory conditions. folia entomol. 43(3): 313–319. 10. martinez-ibarra ja, katthain-duchateau g (1999) biology of triatoma pallidipennis stal 1945 (hemiptera: reduviidae: triatominae) under laboratory conditions. mem inst oswaldo cruz. 94(6): 837–839. 11. martinez-ibarra ja, nogueda-torres b, salazar-montano lf, garcia-lino jc, arroyo-reyes d, hernandez-navarro ja (2017) comparison of biological fitness in crosses between subspecies of mec http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 262–268 e franzim-junior et al.: biology of … 268 http://jad.tums.ac.ir published online: september 30, 2018 cus phyllosomus (hemiptera: reduviidae: triatominae) in southern mexico. insect sci. 24(1): 114–121. 12. martinez-ibarra ja, grant-guillen y, martinez-grant dm (2003) feeding, defecation, and development times of meccus longipennis usinger, 1939 (hemiptera: reduviidae: triatominae) under laboratory conditions. mem inst oswaldo cruz. 98(7): 899–903. 13. martinez-ibarra ja, alejandre-aguilar r, torres-morales a, trujillo-garcia jc, nogueda-torres b, trujillo-contreras f (2006) biology of three species of the meccus phyllosomus complex (hemip tera: reduviidae: triatominae) fed on blood of hens and rabbits. mem inst oswaldo cruz. 101(7): 787–794. 14. martinez-ibarra ja, nogueda-torres b, garcia-benavidez g, vargas-llamas v, bustos-saldana r, montanez-valdez od (2012) bionomics of populations of meccus pallidipennis (stal), 1872 (hemiptera: reduviidae) from mexico. j vec tor ecol. 37(2): 474–477. 15. carcavallo ru, martínez a (1972) life cycles of some species of triatoma (hemiptera: reduvidae). can entomol. 104 (05): 699–704. 16. luz c, fargues j, grunewald j (1999) development of rhodnius prolixus (hemiptera: reduviidae) under constant and cyclic conditions of temperature and humidity. mem inst oswaldo cruz. 94 (3): 403–409. 17. duran p, sinani e, depickere s (2014) biological cycle and preliminary data on vectorial competence of triatoma boliviana in laboratory conditions. acta trop. 140: 124–129. 18. cardozo-de-almeida m, neves sc, al meida ce, lima nr, oliveira ml, santos-mallet jr, goncalves tc (2014) biology of triatoma carcavalloi jurberg, rocha and lent, 1998 under laboratory conditions. rev soc bras med trop. 47 (3): 307–312. 19. barbosa se, soares rp, pires hh, dio taiuti l (2001) experimental evidence for a demographic cline in panstrongylus megistus populations. mem inst oswaldo cruz. 96(6): 773–775. 20. monte gl, tadei wp, farias tm (2014) ecoepidemiology and biology of eratyrus mucronatus stal, 1859 (hemiptera: reduviidae: triatominae), a sylvatic vector of chagas disease in the brazilian amazon. rev soc bras med trop. 47 (6): 723–727. 21. costa mj, perondini al (1973) resist ence of triatoma brasiliensis to fasting. rev saude publica. 7(3): 207–217. 22. trumper ev, gorla de (1991) density dependent timing of defaecation by triatoma infestans. trans r soc trop med hyg. 85(6): 800–802. 23. braga mv, lima mm (2001) effects of food deprivation levels on the oogenesis of panstrongylus megistus. rev saude publica. 35(3): 312–314. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=revista+de+sa%c3%bade+p%c3%bablica.+35%3a+312%e2%80%93314. https://www.ncbi.nlm.nih.gov/pubmed/?term=revista+de+sa%c3%bade+p%c3%bablica.+35%3a+312%e2%80%93314. j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 172 http://jad.tums.ac.ir published online: june 12, 2018 original article control of american cockroach (periplaneta americana) in municipal sewage disposal system, central iran *ali reza zahraei-ramazani 1, abedin saghafipour 2, hassan vatandoost 1, 3 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of public health, faculty of health, qom university of medical sciences, qom, iran 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran (received 14 jan 2018; accepted 16 may 2018) abstract background: cockroaches consists of 4000 species, of which 40 species are serious domestic pests. they have involved the mechanical transmission of various pathogenic viruses, bacteria, and protozoans to humans. this study aimed to determine different control methods of p. americana in sewage system of esfahan city, central iran using different insecticides recommended by who. methods: totally, 164 manholes with at least more than three cockroaches were selected until 2017 for this study. the species of cockroaches were identified; the adults and nymphs were counted and recorded. each sewer shaft was assigned to one treatment method in an ascending order of number of cockroaches counted, coding the treatment method with paint on the lower part of the wall near the manhole cover. the manhole shafts were then inspected at 1 and 5 months’ post-treatment. data analyzed by statistical methods. results: almost all of the products (excluding boric acid with bait formulation) resulted in appropriate control within one month of application. the appropriate products for chemical control of cockroaches were the chlorpyrifos 5% emulsifiable concentrate (ec), diazinon 5% (ec), diazinon 0.05% (ec) and cypermethrin 5% fog. these pesticides achieved an optimal reduction of population providing more than 90% control of cockroaches for five consecutive months. conclusion: the emulsifiable concentrates and fog formulations in the control were more successful compared to other methods and this was penetration deep into the hiding places of cockroaches. keywords: periplaneta americana, control products, sewage system, iran introduction cockroaches are one of the oldest inhabitants of earth, dating back as far as the carboniferous period, over 250 million yr ago (1). they lack special adaptations like the sucking mouthparts in some insects such as the aphids and other true bugs (2). over 4500 species of cockroaches have been identified, of which 40 species are associated with human habitants while four species are well known as pests (3–5). cockroaches have chewing mouthparts and feed on variety of materials (omnivorous) aid ing in the mechanical transmission of various pathogenic viruses, bacteria, and protozoans to humans (3, 4). cockroaches have a worldwide distribution especially in the tropical and subtropical areas and can tolerate a wide range of environments from arctic cold to tropical heat (3, 4). their population increases in hot and humid places especially with the availability of food and water (6). common indoor cockroaches found in iran include, blattella germanica, blatta orientalis, periplaneta americana and supella longipalpa (3, 4). *corresponding author: dr ali reza zahraeiramazani, e-mail: azahraei@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 173 http://jad.tums.ac.ir published online: june 12, 2018 periplaneta americana is the largest species of cockroaches found in iran. they originated from south america, measures 30–40 mm in length and are reddish brown in color. the american cockroach lives in hot areas of buildings like the kitchens, heating rooms, warehouses and sewage systems (4). they usually come out of their hiding places at night for feeding and other activities (7). the adult cockroaches are long-lived and can live for as long as one year or more producing large number of egg capsules during this period, depending on food availability (8). sewerage is established to collect and convey sewage in urban areas in order to prevent contamination of soil and water resources. some species of cockroaches including p. americana have colonized the sewerages, turning them into suitable environment for reproduction and growth posing serious health problems to humans (9). two different methods are involved mainly in the control of cockroaches, chemical and non-chemical control methods. a third but not very common method of control involves the use of parasitoid wasp belonging to the family evaniidae which naturally parasitize and destroy egg capsules of cockroaches. the nonchemical control method is not common, it involves the use of high-frequency ultra-vibration sound generating device. chemical control method includes: residual spraying, dusting spray, mist spray and poisoned baits. the use of chemical insecticides to control cockroaches is the most popular method. fire and fogging have been proposed also in the control of p. americana in sewers, steam tunnels, storehouses and outdoor areas (10, 11). the main pesticides used in chemical control of cockroaches are classified into three classes, pyrethroids, carbamates and organophosphorus. organochlorine is also used in some countries (12). in recent years, the extensive use of pesticides in the control of cockroaches has led to the development of resistance making cockroaches to be ranked second after house flies (musca domestica) with regards to insecticides resistance (13). dichlorodiphenyltrichloroethane (ddt) is widely used in the control of cockroaches across some countries, but in america, it was replaced with chlordane which is more effective. chlordane, diazinon and malathion are three insecticides widely used in america to control cockroaches, while in great britain, diazinon and dieldrin are used often (14). although, many researchers to achieve appropriate and effective insecticides control against cockroaches using newer methods are still ongoing (15). the determination of an appropriate and effective insecticide for the control of p. americana in sewage system of esfahan is necessary, due to the high population of this species as a result of hot and humid environment suitable for cockroaches. resistance of cockroaches to insecticides can be delayed or avoided by using an appropriate insecticide. different chemical products and physical control method were studied in three locations with old sewerage collection networks at esfahan city until 2017 for the control of american cockroach. materials and methods study area esfahan is the capital city of esfahan province with an area of 250km2 and 1695789 residents. it is the second most populous metropolitan city after tehran in the central of iran. its altitude is 1571m, latitude 32°38'30''n and longitude 51°39'40''e. this city is located in a dry semi-desert region with an annual rainfall of 102mm, average summer maximum and winter minimum temperatures of 37.1 °c and 5.81 °c, respectively (16). the study was conducted in three areas with old sewer networks with permission from esfahan's water and wastewater company (fig. 1). esfahan city has over 55375 manhttp://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 174 http://jad.tums.ac.ir published online: june 12, 2018 holes in its sewage system with an average depth of 9m on the main lines and 1meter on lateral lines. most of the manhole covers were cast irons with two holes, and the average diameter of the holes was three centimeters. the shafts of the manholes were made of brick and cement coverings, and their stables moderate warmth and high humidity which provide excellent conditions for cockroaches. totally, 164 manholes with at least more than three cockroaches were selected until 2017. the cast-iron covers were removed from each manhole and the species of cockroach infesting the shaft were noted, the adults and nymphs were counted and recorded. strong flashlights were used to facilitate counting, shafts with more than 300 cockroaches were estimated by counting 0.25m2 section of the shaft and multiplying by the number of sections infested. each sewer shaft was assigned to one treatment method in an ascending order of number of cockroaches counted, coding the treatment method with paint on the lower part of the wall near the manhole cover. the manhole shafts were then inspected at 1 and 5 months after treatment. control products the insecticide formulations used include, (1) aqueous sprays of 5% (wp) carbaryl, 5% (wp) diazinon, 5%, 0.5%, 0.05% (ec) diazinon, 5%, 0.5%, 0.05% (ec) chlorpyrifos, and 0.02% (sc) responsar beta-cyfluthrin (2). thermal fogs 0.028g/m3 of cymperator (synthetic pyrethroid), cypermethrin and tetramethrin (ec), diazinon 0.5g/m3, and chlorpyrifos 0.26g/m3. (3) baits of 5% carbaryl and 50% boric acid. all the insecticides were prepared according to the description (17). all of applied pesticides in this study were purchased from bayer company (bayer persian ag), tehran, iran. fire with oil (10g/m2) was used to burn 10 manhole shafts and twenty untreated infested manholes were used for control too. number of cockroaches per manhole was counted monthly for 5 months’ post-treatment. aqueous sprays were applied with hudson x-pert®110 power compression sprayer equipped with a nozzle, flexible hose, stainless steel tubular spray tip extension on the handle grip, delivering 757cm3/ minute. the extension tube was lowered to the bottom of the shaft and the tip was raised upwards at a uniform rate of 0.5m/sec while the spray was applied. a 5% spray, therefore delivered about two grams of the active ingredient/m2. boric acid baits in petri dishes were placed in vases in dry ledge at the bottom of the shaft. thermal fog with solutions was applied from a “light-weight model” tf 30. the tf 30 was operated at a combustion chamber temperature of 600 °c, a dial flow setting of four l/h. each manhole was treated for five seconds per m3. fire was applied with hodson xpert®110 filled with oil, the extension tube was lowered almost to the bottom of the shaft and spray was applied raising the tip upwards at a uniform rate of 10g/m2. oxygen also was blown into the manhole with an electrical fan equipped with a flexible hose. the study was approved and carried out under the guidelines of ethical committee of the school of public health, tehran university of medical sciences, tehran, iran. the study was conducted on sewerage system of esfahan city with permission obtained from the general directorate of environment. statistical analysis efficacy was calculated from the visual counts of cockroaches in the manholes made periodically for up to five months after treatment. the total numbers of cockroaches per treatment were grouped. for any given treatment, data analysis was done using spss software (chicago, il, usa) and a repeated measures anova design was used to determine different control methods of p. americana in sewage system. http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 175 http://jad.tums.ac.ir published online: june 12, 2018 results the effect of 15 types of pesticides or control products on american cockroaches was investigated. the number of examined manholes was 164. twenty manholes were used as controls. table 1 showed the mean and standard deviation of american cockroach’s frequency prior, one month and five months after intervention. the results of tests of between-subjects effects showed that there was a significant difference between the frequency of adult cockroaches prior, one month and five months after intervention (f= 515.96, p< 0.001). post hoc test showed that most of the pesticides and other control products showed significant differences (table 1). table 2 shows the mean difference of american cockroach’s frequency prior, one month and five months after the intervention compared to untreated. the highest mean difference of adult cockroaches was related to chlorpyrifos 5% (25.29) and then boric acid (24.97. there was a significant difference between mean difference of all tested pesticides and control (p< 0.05). table 1. the mean and standard deviation of american cockroach’s frequency at prior one, one month and five months after the intervention, esfahan city, iran control products number of manholes formulation pre treatment after 1 month after 5 month mean sd mean sd mean sd carbary l5% 10 wp 66.50 4.007 1.50 .972 66.20 4.872 diazinin 5% 10 wp 58.40 3.098 0.00 0.00 27.60 2.271 chlorpyrifos 5% 12 ec 25.42 2.065 0.00 0.00 0.00 0.00 chlorpyrifos 5% 10 wp 59.90 4.606 0.00 0.00 5.70 1.889 chlorpyrifos 0.05% 10 ec 62.90 2.923 0.00 0.00 8.30 1.567 diazinon 5% 13 ec 119.00 6.232 3.54 .877 1.00 0.707 diazinin 0.5% 10 ec 41.40 2.797 0.10 0.316 6.70 1.418 diazinon 0.05% 5 ec 37.60 3.209 0.60 0.548 5.00 1.000 responsar 0.02% 10 sc 38.40 2.914 0.0 0.0 8.50 0.972 cympermethrin 10 fog 63.40 2.066 0.00 0.00 3.70 1.059 diazinon 10 fog 103.70 4.572 0.00 0.00 7.50 1.780 chlorpyrifos 10 fog 82.00 5.354 0.00 0.00 .10 0.316 boric acid 5 bait 22.20 2.490 0.80 0.447 3.40 0.894 carbary l5% 9 bait 38.33 2.646 1.00 0.707 5.89 1.537 fire 10 61.30 2.406 0.20 0.422 9.80 2.201 untreated 20 41.05 3.017 19.65 2.254 40.60 3.393 total 164 59.12 26.591 2.88 6.402 14.40 18.444 table 2. mean difference of american cockroach’s frequency at before, one month and five months after the intervention compared to untreated, esfahan city, iran control products mean difference 95% confidence interval lower limit upper limit carbary l5% 10.97 9.22 12.72 diazinin 5% -5.10 -6.85 -3.35 chlorpyrifos 5% -25.29 -26.94 -23.65 chlorpyrifos 5% -11.90 -13.65 -10.15 chlorpyrifos 0.05% -10.03 -11.78 -8.28 diazinon 5% 7.41 5.80 9.02 diazinin 0.5% -17.70 -19.45 -15.95 http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 176 http://jad.tums.ac.ir published online: june 12, 2018 diazinon 0.05% -19.37 -21.62 -17.11 responsar 0.02% -18.13 -19.88 -16.38 cympermethrin -11.40 -13.15 -9.65 diazinon 3.30 1.55 5.05 chlorpyrifos -6.40 -8.15 -4.65 boric acid -24.97 -27.22 -22.71 carbary l5% -18.69 -20.51 -16.88 fire -10.0 -11.75 -8.25 fig. 1. location of esfahan province and city located in central part of iran discussion american cockroach is an urban pest that is prevalent in sewerage collection networks in tropical and subtropical countries. in this study, p. americana was the most prevalent species in esfahan sewerage system. the finding is in agreement with the results of previous studies (18, 19). periplaneta americana is commonly found in urban regions that have sewage pipe-lines connected to human dwellings, which is suitable for this species growth, reproduction, and distribution (7). almost all the chemical products (except boric acid with bait formulation) and fire used in this study resulted appropriate for control of cockroach at the end of the first month. efficacy and persistence of insecticides in old sewer systems depend on several factors such as climatic conditions, amounts of organic matter, the population of cockroaches, the presence of resistant population, the type of active matter and the kind of formulation. there were many problems on the control of cockroaches with the use of pesticides for several reasons (20), and the main reason is that comtable 1. continued… http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 177 http://jad.tums.ac.ir published online: june 12, 2018 monly used insecticides may lead to resistant in cockroaches. besides, many pesticides are repellents to cockroaches and their usage is avoided. chemical control methods provide only temporary control, it should be accompanied by other control methods such as solid waste management, water and wastewater treatment, industrial waste treatment, noise, pollution control and house improvement (20). the use of chemical control method to reduce population of cockroaches is limited by several factors such as the development of natural resistance in p. americana population and its negative impacts on human health. periplaneta americana takes between 4–12 months to complete its life cycle and the number of generations per year is not as many as in other species, therefore the expression of resistant genes could take more time (21). the use of pesticides with strong vector control management is the most efficient method of controlling this pest in many regions of the world. various formulations (sprays, dust and baits registered) have been used in the control of p. americana, but there is little evidence as to their efficacy in sewers. aqueous sprays of wettable powder of carbaryl and diazinon provides excellent result in the control of p. americana population at least for one month. however, the effects of these pesticides on the cockroaches lasted for less than five months. this was due to the inability of the pesticides to penetrate the egg sacs (oothecae) and the long life cycle and breeding of cockroaches in the winter. some insecticides lose their effectiveness after one month of application, as we observed the population of cockroaches after 5 months did not decrease but increased. chlorpyrifos 5% ec, diazinon 5% ec and diazinon 0.05% ec were suitable pesticides for cockroach control which provided an optimal reduction of p. americana populations for five months respectively. these pesticides provide more than 90% control for five months. spraying emulsion with chlorpyrifos and diazinon in water (ec) provides a quick, temporary knockdown of cockroaches, they give long-term control relatively. sprays are highly repellant to cockroaches and should not be applied to surfaces in areas where traps or bait stations are located. sprays can also disperse cockroaches to other areas of the manhole from which they could later return. moreover, cockroaches have become resistant to many insecticides that formerly were used to control them. for example, recently, the american cockroach has been found to be resistant to trichlorfon and diazinon in china usa (15). the results of this present study indicate chlorpyrifos 5%, diazinon and cymperator 5% used as fog formulation provides an excellent reduction of american cockroaches at the end of one and five months. at the end of the first month of treatment, 100% cockroach reduction was achieved and 91%, 81.7% and 99% at five months using these three pesticides. thermal fogging was observed to be the best and simplest method for cockroaches control in sewer system of esfahan. these methods were successful and consistent because thermal fogs of these pesticides penetrated deeply into the hiding places and they were particularly useful in basements of buildings, sewers and drainage systems. this method in the winter provides 99% reduction for up to five months and if used twice in a year, the cockroach’s population in the manholes will be decreased to zero. this technique works in control program of cockroach; it requires specialized equipment. baits of carbaryl 5% and boric acid were used and they provided short time (one month) control of cockroaches in sewage system (98.3% and 83.9%). their effects reduced over time and the methods were unsuccessful consistently because the cockroaches quickly consumed all the baits in the shafts and it was difficult and timeconsuming to replace them. an important factor reported to affect baits http://jad.tums.ac.ir/ j arthropod-borne dis, june 2018, 12(2): 172–179 ar zahraei-ramazani et al.: control of american … 178 http://jad.tums.ac.ir published online: june 12, 2018 performance against american cockroaches is sanitation (22). sanitation has been shown not to have a direct relationship with the level of domiciliary cockroach infestation in malaysia (23) (l) was investigated in residential and clustered setting according to their sanitary conditions, results indicate that at 1-week posttreatment, houses with good sanitary conditions showed significantly and faster reduction in the number of cockroaches trapped (> 95%) than those with moderate and poor conditions. in this study the control effect of responsar 0.02% as suspension concentrate formulation at the end of one month was 100% but at the end of 5 months decreased to 71%. the use of residual insecticides to control cockroaches when their growth is slow (in winter) have a long-term effect in the control of cockroaches in sewerage systems. use of fire with oil (10g/m3) as a physical control method was applied in 10 manholes and caused 99.7% and 76% reduction of p. americana after one and five months respectively. conclusion the use of fire in manholes of sewerage systems is a suitable strategy in the control of cockroaches if done using proper equipment because it destroys the oothecae of cockroaches too. the advantage of these strategies was that lateral lines and adjacent manholes could be treated using these pesticides. moreover, these control methods require specialized equipment. chemical control methods are a common method of p. americana control around the world but it has been limited by several factors: the development of natural resistance and the chemical insecticides are expensive, toxic to humans and other living beings, and they are harmful to the environment. acknowledgements the authors would like to appreciate mr mohammad reza hamdi and mr ali moatarian for their excellent assistance in this project. we are grateful mr amrolah azarm for drawing the tables and set-out figure. this study was supported by esfahan water and wastewater company. the authors declare that there is no conflict of interest. references 1. 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(dictyoptera: blattidae). j vector ecol. 25: 218–221. http://jad.tums.ac.ir/ https://www.google.com/search?q=m.+w.+service&stick=h4siaaaaaaaaaopge-lsz9u3mesrnlewvakztsvmdllntgsyk630k_lzs_xlizjlsllz4svzi7ktektlmvklag9arfy4aaaa&sa=x&ved=0ahukewid_eapwfxaahwpj5okhuxwbfsqmxmiaigbma4&biw=1440&bih=789 https://www.ncbi.nlm.nih.gov/pubmed/?term=jaramillo%20j%5bauthor%5d&cauthor=true&cauthor_uid=21935419 https://www.ncbi.nlm.nih.gov/pubmed/?term=muchugu%20e%5bauthor%5d&cauthor=true&cauthor_uid=21935419 https://www.ncbi.nlm.nih.gov/pubmed/?term=vega%20fe%5bauthor%5d&cauthor=true&cauthor_uid=21935419 https://www.ncbi.nlm.nih.gov/pubmed/?term=davis%20a%5bauthor%5d&cauthor=true&cauthor_uid=21935419 https://www.ncbi.nlm.nih.gov/pubmed/?term=borgemeister%20c%5bauthor%5d&cauthor=true&cauthor_uid=21935419 https://www.ncbi.nlm.nih.gov/pubmed/?term=chabi-olaye%20a%5bauthor%5d&cauthor=true&cauthor_uid=21935419 7rafinejad_rtl iranian j arthropod-borne dis, (2007), 1(1): 52-57 j rafinejad et al.: systematic, bioecology and… 52 original article systematic, bioecology, and medical importance of widow spiders (lathrodectus spp.) in khorasan province, iran *j rafinejad 1, s tirgari 1, f biglarian 2, k shemshad 3 1 dept. of medical entomology and vector control, school of public health and institute of health research, medical sciences/ university of tehran, iran 2 ministry of education and training, zone 2, tehran, iran 3department of entomology, science and research branch, islamic azad university, tehran, iran (received 20 oct 2006; accepted 15 jan 2007) abstract following the hospitalization of 195 individuals due to the spider bite in 1995 and three cases of recorded death in the year of 1993 which were referred to emam reza hospital in mashad, the present study was undertaken on bionomics and medical importance of lathrodectus spp in khorasan province, during 1995-2005. a total cases of 195 bites were studied composing of 70.8 % males and 29.2% females. the most prevalence cases were observed in mid age (20-55 years old) and particularly among farmers (36.4%). a total number of 216 adult widow spiders and 258 egg sacs were collected from their habitats in different localities of 15 counties in the province. the following species have been recognized: lathrodectus tredecimgottatus (62%), l. dahli (32%), l. geometricus (5%) and l. pallidus (1%). here is the first report on the occurrence of males of l. pallidus as well as both sexes of l. trdecimgottatus and l. geometricus in the country. the sex ratio among collected specimens was 88% and 12% female and male, respectively. summer provides the most suitable and favorable climatic condition for the activities of these spiders. however 65% of spiders were collected in this season. among different cities, mashad had (60%) the most reported cases in the study area. foot was more injured than other parts. 96.5% of patients exhibited localized pain from which only 2% had no pain in the bitten part and 87% had a generalized pain in whole body. keywords: black widow spiders, systematic, iran introduction phylum arthropoda includes about 10 to 20 classes, depending on the classification adopted. spider considered as one order of arachnida (sub class) and class of aranea (cambridge 1902, forster 1968, jung and white 1993). these creatures normally spend their life cycle near human dwelling and work on farms. latrodectus spp. are known as black widow spiders (garb et al. 2004). among 3500 spider species, widow spider is considered as one of the most poisonous and dangerous spiders the entire world (pommier et al. 2005). it has a wide distribution in the world and occurs mainly in south parts of former soviet union, middle east and mediterranean regions (kaston 1970, levi 1986, lotz 1994, miller 1993, robert 1985, zukowski 1993). the widow spiders characteristically spin tangled webs, which look similar to spun cotton candy. their webs are usually built in or beneath objects close to the ground such as under porches, under foundations of buildings, the lose bark of trees, and in basements (nicholson and graudins 2002, blackledge et al. 2005). the bite of the black widow is painful and may cause death if medical attention is not sought immediately. the *corresponging author: dr j rafinejad tel: +98 21 66465404, (ext: 2179), e-mail: jrafinejad@yahoo.com iranian j arthropod-borne dis, (2007), 1(1): 52-57 j rafinejad et al.: systematic, bioecology and… 53 venom of this spider is highly neurotoxic and respiratory failure can occur if appropriate medications are not administered at once. latrodectus is exclusively carnivorous and antagonistic. ordinarily it feeds on insects; however, it also consumes wood lice, diplopods, chilopods and other arachnids (garb et al. 2004). in iran especially northeast of country (khorasan province) there were several reports of latradoctism. species of this genus were responsible for 3 cases of death in 1993. there were 195 hospitalization cases due to the spider bite in 1995. most of cases were reported from 6 counties including mashad, sabzevar, neishabour, sarakhs, qoochan and chenaran. however, an attempt was made during 1995-2000 on the demographic and clinical characteristics of widow spider bite in order to highlight the systematic, ecology and biology of latrodectus spp. and its medical importance in the study area because of the importance of the khorasan province for agriculture, industry and elsewhere in iran and also the existence of farm areas which provide suitable habitats for spiders. this investigation gives new data which will be valuable for future planning control and solving the public health problem, identification of different species of black widows, and different aspects of systematic such as feeding, egg sac, seasonal abundance, natural habitat, reproduction, longevity, nidification, tarnished and shape of net. materials and methods study area this investigation was carried out in 15 counties in the north and central khorasan province, located in north east of iran (map 1) and according to zoogeography distribution it is located in the palearctic region. spider collection all the specimens were collected by direct method from different parts of the study areas including flagging vegetation, hedgerow of farms, crevices and cracks of walls surrounded of gardens and rodents holes. after collection, specimens were transferred in to the holding tubes including alcohol (9 parts) and formalin (1 part). for species identification several parameters including morphology, biology and ecology of spiders and date, place, collector, habitats, number of spiders in each net, number of egg sac, distance of nest from earth, feeding preference and distance to water source and having net and clinical characteristics of widow spider bite have been considered. recognition was carried out by morphological characteristics according to the international standard taxonomic key identification (robert 1985, wilson and king 1990). the data were analyzed using spss program. results in this research a total of 216 adult spiders and 258 egg sacs were collected from the study areas. one hundred ninety one specimens were female (88.43%) and 25 were male (11.57%). the frequency of spiders were more prevalent in bojnourd county with 49 cases( 22.69%) and chenaran with 46 cases(21.30%) and the lowest cases was seen in fariman (0.93%) and tor-batejam (1.85%). the identified species were as follows: l. tredecimgottatus (62%) was the most prevalent. one hundred seventeen specimens were female (87.31%). this species was more prevalent in bojnourd (26.5%) and chenaran (23.08%) and less prevalent in kashmar, gonabad, fariman and torbatejam with 1.71% in each county. l. dahli with 32% prevalence had the second abundance. sixty two specimens were female (91.18%). this species was more prevalent in bojnourd and chenaran (22.58%) and less prevalent in kashmar, shiravan, and torbateheidarieh with 1.61% in each county. l. geometricus (5%) had the third abundance. all of the specimens were female (100%). this species was more prevalent in mountainous roads iranian j arthropod-borne dis, (2007), 1(1): 52-57 j rafinejad et al.: systematic, bioecology and… 54 from sabzevar to bardeskan (54.54%) and less prevalent in torbateheidarieh with 9.09%. l. pallidus had 1% abundance. one specimen was female and 2 of them were male (66.6%). all of the specimens were found in one burrow in the ground near the road of torbateheidarieh to gonabad. considering the review on foreign and iranian literatures on iranian spiders, here is the first report on the occurrence of males of l. pallidus as well as both sexes of l. trdecimgottatus and l. geometricus in the country. spider females, mate only once in their life and 3-13 egg sacs could be laid by each spider. in each egg sac a total of 50-500 eggs may be seen. the time interval between each laying egg is estimated 15 d. the number of molting is 4-8 time, depending on species, and environmental condition. in nature, adult males die after several months but females can live several months. in laboratory condition they can live up to 18 mo. females have one generation in each year. generations in each year are not significantly different in various species. the activities of spiders initiate from may and continue until november. the peak of activity is in july, which is synchronized with wheat harvesting time. in conclusion it should be emphasized that there is significant difference among species in terms of morphology, bioecology and life span. the sex ratio among collected specimens was 88% and 12% female and male, respectively. cracks and crevices of mud walls that are traditionally constructed around the farms and gardens in the study area are the main shelters for hiding, breeding and egg laying of spiders. furthermore the summer provides the most suitable and favorable climatic condition for the activities of these spiders when 52.8% of spiders were collected in this season. all the reported cases were checked in terms of their sex, age, occupational, geographical distribution and seasonal biting clinical manifestation. during the investigation 195 cases of biting were studied com-posing of 70.8 % male and 29.2% female. the most prevalence cases were observed in middle age (20-55 yr old) and particularly among farmers (36.4%). table 1 shows the percentage of species in whole study area. seasonal activity of spiders has shown in table 2. prevalence of widow spiders and egg sac collected from natural habitat in north and central of khorasan province, 1995-97 are presented in table 3. severe cases are hallmarked by difficulty in breathing and unconsciousness, which may lead to death due to asphyxia preceded by convulsions. study showed that all of the people biting with spiders have 1 to 8 d hospitalization. during the investigation duration a total cases of 195 bites were studied composing of 70.8 % male and 29.2% female. the most prevalence cases were observed in midge (20-55 yr old). biting were more prevalent particularly among farmers (36.4%), then house women (27.9%), students (16.4%), animal husbandman (9.7%), workers (6.7%) and etc (3%). most of the patients had localized pain, pain in one organ and distributed pain. victims experience abdominal cramps and abdominal cramps (36%), itching (34%) nausea (31.4%), eurhythmy (24%), edema in eye-lids (17.8%), tachycardia (11%) and muscle cramps (10.3%). among different cities, mashad had (60%) the most reported cases in the study area. after mashad, neishabour had 9.1% of cases, chenaran 9.1%, and another part of the counties had 15.8% cases. among different parts of the body, foot (47%) was more injured than the other parts of the body, followed by hands (28%), trunk (14%) and head and neck (11%). seventeen percent of victims were baited outdoors and 83 percent were baited indoors. summer was the peak of biting which is synchronized by spider activity. 96.5% of patients exhibited localized pain from which only 2% had no pain in the bitten part and 87% had a generalized pain in whole body. hospitalization time of patients was 1-8 d. the peak of case reports were in 1995, whereas no any cases were found during 1998-2000. iranian j arthropod-borne dis, (2007), 1(1): 52-57 j rafinejad et al.: systematic, bioecology and… 55 fig. 1. mashahad county (study area) table 1. prevalence of adult widow spiders collected from north and central of khorasan province according to sex and species, 1995-97. species male female sum no. % no. % no. % l. tredecimguttatus 17 7.87 117 54.17 134 62.04 l. dahli 6 2.78 62 28.70 68 31.48 l. geometricus 0 0 11 5.09 11 5.09 l. pallidus 2 0.93 1 0.46 3 1.39 total 25 11.57 191 88.43 216 100 table 2. prevalence of widow spiders collected from north and central of khorasan province according to season and year year/season spring summer autumn total no. % no. % no. % no. % 1995 24 11.11 82 37.96 8 3.7 114 52.78 1996 24 11.11 42 19.4 13 6.01 79 36.57 1997 6 2.8 17 7.9 0 0 23 10.65 total 54 25 114 52.8 21 9.71 216 100 table 3. percentage of widow spiders and egg sac collected from natural habitat in north and central of khorasan province during study period natural habitat adult egg sac no. % no. % under or in split of stones 51 23.6 71 27.5 around the farm and gardens 45 20.8 92 35.66 cracks and crevices of mud walls 37 17.13 49 18.99 rodent holes 35 16.2 19 7.36 vegetation, trees and hedgerow 29 13.43 15 5.81 ground holes 19 8.8 12 4.65 total 216 100 258 100 iranian j arthropod-borne dis, (2007), 1(1): 52-57 j rafinejad et al.: systematic, bioecology and… 56 discussion although most species of spiders are venomous, only ten or so are able to induce human envenomations (dzelalija and medic 2003). the black widows importance is directly related to its reputation as a poisonous spider (nicholson and graudins 2002), however, bites are uncommon and serious long-term complications or death is rare (landeka and plenkovic 2003). a comprehensive study was carried out during 1995-97 in northern part of iran for the study of distribution, biology, ecology and systematic of widow spiders. a total of 216 adult spiders and 258 egg sac were collected form the study areas. the identified species were l. tredecimgottatus (62%), l. dahli (32%), l. geometricus (5%) and l. pallidus (1%). the sex ratio among collected specimens was 88% and 12% female and male, respectively. female spiders, mate only once in their life and 3-13 egg sacs could be laid by each spider. in each egg sac a total of 50-500 eggs may be seen. the time interval between each laying egg is estimated 15 d. the number of molting is 4-8 time depending on species, and environmental condition. in nature, adult males die after several months but females can live several months. in laboratory condition they can live up to 18 mo. females have one generation in each year. generations in each year have not significant difference in different species. the activities of spiders initiate from may and continue until november. the peak of activities is in july, which is synchronized with wheat harvesting time. biting were more prevalent particularly among farmers and house women. because of farming, among different parts of the body, feet and hands were more injured than the other parts of the body (no need to repeat the results). from table 1 it can be concluded that among spiders collected from study areas the species l. tredecimgottatus exhibit the more prevalence in the region, whereas the species l. geometricus had the lowest prevalence. the spiders were more seasonal activity during summer, 1995 and less seasonal activity was seen in autumn 1997. in natural habitat spiders have more prevalent under or in split of stones and have less prevalent in ground holes. in the field studies it is concluded that species of latrodectus genus have different morphologic and ecology and species identification without its bioecology is very difficult. also more studies are needed to highlight the taxonomic situation of the species of the spider widows in other parts of country. trash, lumber piles, bricks, weeds, and outside structures are good breeding places for spiders and should be cleaned up. inside the home spider webs should be brushed down. the egg sacs should be destroyed to prevent hundreds of young spiders from emerging. vacuum cleaner attachments may be used to clean walls, and the collected debris should be destroyed. another recommenddation is environmental sanitation, quarantine, chemical control including residual spraying and biological control (landeka and plenkovic 2003). control efforts should target black widow spider webs because that is where the spider spends most of its time. control is best achieved by following an integrated pest management (ipm) strategy, which involves using multiple approaches such as preventive measures, exclusion, sanitation, and chemical treatment when necessary (landeka and plenkovic 2003). ipm requires a thorough inspection of the building to locate the pest. an inspection preferably should be done at night because the black widow spider is nocturnal (landeka and plenkovic 2003). all of the clinical symptoms might not be present. the bite of a black widow spider initially may go unnoticed, but some people report a short stabbing pain. the venom is neurotoxic, affecting the human nervous system. at first, there may be slight local swelling and two faint red spots, which are puncture points from the fangs. the physical reaction to a spider bite depends on the amount of venom injected and an individual's sensitivity including age and sensitivity to venom. small children most severely affected (landeka and plenkovic, 2003). iranian j arthropod-borne dis, (2007), 1(1): 52-57 j rafinejad et al.: systematic, bioecology and… 57 the present studies show that widow spider biting is an important problem in the study areas, which periodically appear every several years. in conclusion it should be emphasized that spider bite is an important health problem in the region. for combating the problem health education for both local authorities as well as community is essential. acknowledgements the authors would like to appreciate very much from mr afshar, dr balali and their colleagues in health center of khorasan province to help us to carry out the research and also to the staff of school of public health and institute of health research, medical sciences/ tehran university for their kind collaboration. this research was financially supported by the school of public health, medical sciences/tehran university. references blackledge ta, summers ap, hayashi cy (2005) gumfooted lines in black widow cobwebs and the mechanical properties of spider capture silk. zoology (jena). 108(1): 41-46. camberidge fp (1902) one of the spider of the genus latrodectus walckenaer. proceedings of the zoological society of london. 1: 247-261. dzelalija b, medic a (2003) latrodectus bites in northern dalmatia, croatia: clinical, laboratory, epidemiological, and therapeutical aspects. croat med j. 44(2): 135-138. forster rr, wilton cl (1968) the spider of new zealand. part 2 ctenizidae, dipluridae, migidae. otego muss bull. 2: 1-180. meier j, white j (1993). handbook of clinical toxicology of venom and poisons. a biologists view, london. kaston bj (1970). how to know the spiders. wmc brown company publishers. landeka n, plenkovic j (2003) the black widow spiderits appearance and control. arh hig rada toksikol. 54(1): 11-8. levi hw (1986). the geotropically orb weaver genera alpaida (araneae: araneidae). bull muss comp zoo. 151: 365487. lotz ln (1994) revision of the genus latrodectus (aranea: theridiidae) in africa. navors. has. mus. blaemfontein. 10(1): 1-60. miller ta (1993) treatment of black widow spider bite. j american board. family practice. 2: 183-184. nicholson gm, graudins a (2002) spiders of medical importance in the asia-pacific: atracotoxin, latrotoxin and related spider neurotoxins. clin exp pharmacol physiol. 29(9): 785-794. pommier p, rollard c, de haro l (2005) spider bites: araneidism of medical importance. presse med. 34: 49-56. roberts mj (1985) the spiders of great britain and island. harley books. wilson dc, king le (1990) spiders and spider bites. department clinical. 8(2): 277-286. zukowski cw (1993) black widow spider bite, family practical. department noval medical clinical, quntico, virginia. j american board. 6(3): 279-281. j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 252 http://jad.tums.ac.ir published online: september 30, 2018 original article tick ectoparasites of animals in borderline of iran-iraq and their role on disease transmission omid banafshi 1, 2, ahmad ali hanafi-bojd 2, mohamad karimi 1, faezeh faghihi 3, mojtaba beik-mohammadi 4, sahere gholami 2, siavash javaherizadeh 5, hamideh edalat 2, *hassan vatandoost 2, *zakkyeh telmadarraiy 2 1zoonoses research center, kurdistan university of medical sciences, sanandaj, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3cellular and molecular research center, iran university of medical sciences, tehran, iran 4department of medical entomology and vector control, faculty of health, urmia university of medical sciences, urmia, iran 5faculty of paramedical science, clinical laboratory science, islamic azad university, tehran medical branch, tehran, iran (received 21 may 2017; accepted 23 june 2018) abstract background: since ticks are potent vectors of various diseases, identification of these species are clinically important to protect the public health and control veterinary problems in the communities. we aimed to figure out the frequency of ticks on cows, goats, sheep, lambs, turtles and also obscure hosts in kurdistan province, bordered with iraq june 2012 to may 2013. methods: the hosts were selected randomly and examined individually for tick infestation. in case of infestation, ticks were collected using forceps and then preserved in 70% ethyl alcohol. all collected specimens were preserved in tubes and relative information was recorded and then identified based on morphological characteristics. results: totally, 1209 ticks were collected. the prevalence of ticks on cows, sheep, goats, lambs, turtles, poultry and obscure hosts was 11.33%, 55.41%, 6.53%, 5.95%, 0.9%, 8.02% and 11.82% respectively. the mean number of ticks on each animal was 1.6. number of 5 genera, including rhipicephalus, argas, ornithodoros, hyalomma and haemaphysalis and 9 species; including r. sanguineus (60.05%), r. bursa (0.08), hy. anatolicum (12.33), hy. asiaticum (1.49), hy. aegyptium (0.91), hy. marginatum (0.08), haemaphysalis parva (4.22), hyalomma sp. (0.99), ornithodoros lahorensis (11.83), and argas persicus (8.02) were identified. conclusion: the most abundant species in this study area was rh. sanguineus (60.05%). due to high prevalence of tick specimens and a variety of collected species from sheep (55.41%), the vaccination of sheep and control of tick vectors are recommended. keywords: tick, ruminants, turtle, iran introduction ticks (acari: ixodidae) are considered as the important vectors of pathogens (1). they play an important role in the survival of the pathogens that cause disease in humans and animals (2). ticks are able to transmit a variety of pathogens that are responsible to develop some diseases such as tick-borne encephalitis, crimean congo hemorrhagic fever (cchf), anaplasmosis, babesiosis, rickettsiosis, borreliosis and ehrlichiosis (3, 4). such diseases are considered as public health or veterinary problems in the countries (5, 6). to the best of our knowledge, 10% of the currently known tick species act as vectors of pathogens of animals and humans (2). in addition to the transmission of pathogens, they are also responsible for damages directly due to their feeding behavior (7). as only 10% of tick species transmit a number of pathogens, identification of tick species is important. tick spe*corresponding authors: dr hassan vatandoost, email: hvatandoost1@yahoo.com, dr zakkyeh telmadarraiy, e-mail: telmadarraiy@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 253 http://jad.tums.ac.ir published online: september 30, 2018 cies distribution in iran is briefly investigated on the basis of published records. data were presented for 642 ixodid ticks taken from small-sized mammals, mainly rodents in different zoogeographical zones of iran (8). the prevalence of ixodid ticks was studied on cattle in mazandaran province, north of iran (9) and east of the country (10) in another study the prevalence of ticks was investigated in khuzestan province and showed shosh was the most infected city in khuzestan, ticks infection rate on sheep, goat, and cow was 84.12%, 12.69% and 3.17%, respectively (11). in a similar investigation, the distribution and ecological preferences of ticks of domestic animals were studied from 2002 to 2005 in north part of the country (12). the prevalence of ticks was surveyed in north-west of the country in ardebil (13) and west azerbaijan (14). additionally, hard ticks of domestic ruminants were surveyed in central part of iran (15). recently, some other investigations have been carried out in some other geographical locations of iran (16-19), and in kurdistan region of iran and iraq (20-22). in 2002, the presence of hy. aegyptium from testudo graeca turtle was reported in iran (23). hy. aegyptium and t. graeca were found in northwest of iran (24). recently the situation of tick born disease showed in iran. the cchfv rna was detected in 5.2% of 492 ticks collected from livestock in different regions of golpayegan (6). in total, 49 ticks including five species: r. sanguineus, hyalomma anatolicum, hy. asiaticum, hy. dromedarii and hy. marginatum with a prevalence of 46.9%, 32.7%, 4.1%, 4.1% and 2.1% respectively were identified; and cchfv was detected in three ticks among 49 collected ticks. the ticks infected with cchfv belonged to the genus hyalomma and rhipicephalus. phylogenetic analysis demonstrated that two sequences clustered in clade iv (asia-1) and one sequence was located within clade iv (asia-2) (25). all positive ticks were from hyalomma genus and hy. marginatum species. they were not able to find virus in hy. anatolicum, hy. schulzei, hy. dromedarii, r. sanguineus and argas persicus. hyalomma marginatum is the main vector in that study (26) despite the aforementioned investigations, there still seems to be a gap in our knowledge about distribution of tick species in iran. this study was aimed to figure out the frequency of ticks on cows, goats, sheep, lambs, turtles and also obscure hosts in kurdistan province, bordered with iraq. materials and methods this survey was carried out in kurdistan province, located in west part of iran, in region 3 and bound by iraq on the west, the province of west azerbaijan to its north, zanjan to the northeast, hamedan to the east and kermanshah to the south (27). this province is one of the 31 provinces of iran. it is 28817km² in area (coordinates: 35.3113°n 46.9960°e). the capital of kurdistan province is the city of sanandaj, located in sanandaj county. other counties with their major cities are marivan, baneh, saqqez, qorveh, bijar, kamyaran, dehgolan, diwandarreh and sarvabad (fig. 1). samples collection from june 2012 to may 2013, ticks from goats, cows, sheep, lambs, turtles and obscure hosts from various regions of the province were collected. ticks were mostly found on sheep of the livestock. in total, 724 animals from 104 herds including 62 cows, 506 sheep, 73 goats and 23 lambs were selected randomly and examined individually for tick infestation. additionally, we selected 2 turtles and 37 obscure hosts randomly for detection of tick infestation on them. thirty minutes were spent for each flock to collect ticks. all inspections and tick collections were carried out between 08:00 a.m. and 11:00 a.m. in case of infestation, ticks were collected using forceps and then preserved in 70% ethyl alcohol. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 254 http://jad.tums.ac.ir published online: september 30, 2018 collected samples were preserved in tubes and relative information was recorded such as collector name, date, host information and date of collection, then, samples were transferred to the entomology laboratory, school of public health, tehran university of medical sciences, tehran, iran. all collected samples were identified based on morphological characteristics and the keys given by janbakhsh (28) and walker et al. (29) based on shape of capitulum, scutum, eyes, festooned and hypostome, spiracle, genital groove, spure of coxa, adanal shield and other appropriate characters. results totally 1209 ticks were collected and the occurrence of ticks on cows, sheep, goats, lambs, turtles, poultry and obscure hosts was 11.33%, 55.41%, 6.53%, 5.95%, 0.9%, 8.02% and 11.82% respectively. the mean number of ticks on each animal was 1.6 ticks per animal. totally 5 genus: rhipicephalus, argas, ornithodoros, hyalomma and haemaphysalis were identified in study areas (table 1). rhipicephalus sanguineus was the most abundant species in the studied area (60.05%), also, we found r. bursa (0.08%), argas persicus (8.02 %), ornithodoros lahorensis (11.83%), hy. marginatum (0.08%), hy. asiaticum (1.49%), hy. anatolicum (12.33%), hy. aegyptium (0.91 %), ha. parva (4.22%) and hyalomma sp. (0.99%). spatial distribution of tick species in different elevations is presented in fig. 2. the coordinates of collection sites have presented in table 2. fig. 1. the study area, kurdistan province is located in west part of iran http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 255 http://jad.tums.ac.ir published online: september 30, 2018 table 1. tick species and their hosts and places in kurdistan province, 2012–2013 (n: nymphs, f: females, m: males) species of ticks host and place (%) collected samples total (%) cow sheep goat lamb turtle poultry fold n f m r. sanguineus 17 572 65 72 0 0 0 53 200 473 726 (60.05) r. bursa 0 1 0 0 0 0 0 0 0 1 1 (0.08) hy. anatolicum 114 35 0 0 0 0 0 0 88 61 149 (12.33) hy. asiaticum 0 5 13 0 0 0 0 0 6 12 18 (1.49) hy. aegyptium 0 0 0 0 11 0 0 0 9 2 11 (0.91) hy. marginatum 0 0 1 0 0 0 0 0 0 1 1 (0.08) h. parva 0 51 0 0 0 0 0 0 20 31 51 (4.22) hy. sp 6 6 0 0 0 0 0 1 11 0 12 (0.99) o. lahorensis 0 0 0 0 0 0 143 122 8 13 143 (11.83) a. persicus 0 0 0 0 0 97 0 63 21 13 97 (8.02) total 137 (11.33) 670 (55.41) 79 (6.53) 72 (5.95) 11 (0.90) 97 (8.02) 143 (11.82) 239 (19.77) 363 (30.02) 607 (50.21) 1209 (100) fig. 2. spatial distribution of livestock ticks in different altitudinal categories of kurdistan province of iran, 20122013 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 256 http://jad.tums.ac.ir published online: september 30, 2018 table 2. spatial distribution of ticks collected from the study area, kurdistan province of iran, 2012–2013 (+: collected,-: not collected) county village x y host hy. aegyptium rh. sanguineus hy. anatolicum hy.asiaticum r.bursa o. lahorensis ha.parva bijar khorkhore 47.1094 35.3086 sheep + bijar sabzevar 47.5833 35.7 sheep + + bijar bijar-salavatabad 47.55 36.0166 sheep + bijar salavatabad 47.55 36.0166 cow + bijar salavatabad 47.55 36.0166 cow + bijar salavatabad 47.55 36.0166 goat + bijar top aghach 47.8161 36.0508 sheep + bijar top aghach 47.8161 36.0508 sheep + qorveh qaleh 47.4810 35.0803 sheep + qorveh maloj 47.9733 35.2922 sheep + qorveh kaniganji 47.3611 35.1332 sheep + qorveh kaniganji 47.3611 35.1332 sheep + qorveh qorveh 47.7951 35.1594 goat + + qorveh qorveh 47.7951 35.1594 sheep qorveh malvjh 47.9733 35.2922 goat + qorveh nazem abad 47.9625 35.1038 sheep -goat + qorveh nazem abad 47.9625 35.1038 sheep -goat + qorveh nazem abad 47.9625 35.1038 sheep -goat + qorveh shirvaneh 47.8755 34.9566 goat + sanandaj ali jan 46.6833 35.9166 sheep + sanandaj doseh 46.8955 35.4566 sheep + sanandaj qolyan 47.0106 35.2355 sheep + saqez saqez 46.2892 36.2523 turtle + saqez eslam abad 46.7666 36.3666 sheep + sarvabad anjameh 46.35 35.3666 goat + sarvabad anjameh 46.35 35.3666 cow + sarvabad bandul 46.1535 35.1911 sheep + http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 257 http://jad.tums.ac.ir published online: september 30, 2018 sarvabad doroud 46.3533 35.2994 sheep /♀ + sarvabad doroud 46.3533 35.2994 lamb/ ♀ + sarvabad hezarkhani 46.3666 35.35 sheep /♀ + + sarvabad hezarkhani 46.3666 35.35 sheep /♀ + sarvabad hezarkhani 46.3666 35.35 goat /♀ + sarvabad hezarkhani 46.3666 35.35 lamb/ ♀ + sarvabad nasl 46.4333 35.3 sheep /♀ + sarvabad nasl 46.4333 35.3 sheep /♀ + sarvabad nasl 46.4333 35.3 sheep /♂ + sarvabad nasl 46.4333 35.3 cow/ ♀ + sarvabad nasl 46.4333 35.3 sheep + sarvabad nasl 46.4333 35.3 sheep /♀ + table 2. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 258 http://jad.tums.ac.ir published online: september 30, 2018 discussion ticks are considered as ectoparasites, living by hematophagy on the blood of mammals, birds, and sometimes reptiles and amphibians. about 10% of ixodidae (hard ticks) and argasidae (soft ticks) are vectors of a number of diseases that affect both humans and other animals. as ticks are important vectors of diseases; they are subject of many studies in iran. due to former investigations, there is limited information about distribution of tick infestation in kurdistan province. in most regions of iran, the dominant tick genera responsible for infestation belong to hyalomma, rhipicephalus, haemaphysalis, and ixodes (30). in this investigation, we could collect three of the aforementioned genera except for ixodes but also we collected some species of genera argas and ornithodoros too. we collected 1209 ticks. most of the collected ticks were male (50.21%) (table 1). our survey revealed that the most occurrences of ticks were observed on sheep (55.41%). the identification of collected ticks also revealed that the occurrence of ticks on cows, goats, lambs, turtles and obscure hosts were 11.33%, 6.53%, 5.95%, 0.9% and 11.82% respectively. as haemoparasitic diseases are considered as a major problem to efficient sheep production in iran due to theileriosis and babesiosis, it has important role (30). the major tick genera found on sheep and goats are mostly hyalomma, rhipicephalus, haemaphysalis and ixodes (30). our investigation revealed the presence of all species of ticks except hy. marginatum and hy. aegyptium on sheep. due to high prevalence of tick specimens and variety of collected species of sheep, the vaccination of sheep and control of tick vectors are recommended. an investigation in kurdistan region in iraq was carried out (22). three genera species were collected and identified on cattle. the highest prevalence was observed in boophilus sp. followed by hyalomma sp. and rhipiceph alus sp. (22). we could not detect any boophilus species, but we found r. sanguineus and hy. anatolicum on cows. these findings are in concordance with the mentioned investigation (22). our investigation shows the presence of 9 species: r. sanguineus (60.05%), r. bursa (0.08), argas persicus (8.02), ornithodoros lahorensis (11.83), hy. marginatum (0.08), hy. asiaticum (1.49), hy. anatolicum (12.33), hy. aegyptium (0.91), ha. parva (4.22) and hyalomma sp. (0.99) in the province. r. sanguineus was the most collected tick sample (726/1209), also, these species were found on all hosts except turtles (table 1). rh. sanguineus (brown dog tick) is considered as the most widespread ixodid tick, colonizing both human and animals (31). r. sanguineus species are very resistant to heat and moisture deficits (32). this species is able to transmit pathogens like ehrlichia canis to dog (33). they can participate in the epidemiology of canine visceral leishmaniasis (34) and spotted fever group rickettsia (35). some other ehrlichia associated species in r. sanguineus are e. ewingii, and e. chaffeensis (36). the brown dog tick is also able to transmit rickettsia ricksettsii, causing rocky mountain spotted fever (37), rickettsia conorii, which is the bacteria responsible for causing mediterranean spotted fever as well as rickettsia massiliae (38) and r. massiliae (39). rhipicephalus sanguineus is also reported to transmit hepatozoon canis (40) as well as babesiacanis (41). conclusion ticks contamination has been detected in a variety of livestock in the kurdistan region, also the variety of ticks is abundant. the ornithodoros and hyalomma are more important than carriers of known diseases in the kurdihttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 252–261 o banafshi et al.: tick ectoparasites of … 259 http://jad.tums.ac.ir published online: september 30, 2018 stan, including crimean congo hemorrhagic fever (cchf) and tick-borne relapsing fever (tbrf). regarding the high contamination of livestock, the presence of disease and borderline province, the importance of the vector control in kurdistan is more evident, as well as the necessity of further research, especially on the movement of livestock and ticks, as well as the resistance of ticks to the pesticide. acknowledgements this study supported financially by project number of 23859, from tehran university of medical sciences (tums). the authors declare that there 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http://jad.tums.ac.ir published online: september 30, 2019 original article investigation of susceptibility levels of culex pipiens l. (diptera: culicidae) populations to synthetic pyrethroids in antalya province of turkey *onder ser1; huseyin cetin2 1malaria control unit, antalya provincial directorate of health, antalya, turkey 2department of biology, faculty of science, akdeniz university, antalya, turkey (received 6 june 2018; accepted 26 june 2019) abstract background: culex pipiens l. (diptera: culicidae) is an important vector of several pathogens. this mosquito is widely distributed throughout the world. we aimed to determine the susceptibility levels of cx. pipiens populations to some synthetic pyrethroid insecticides in antalya, turkey. methods: the immature stages of mosquitoes were collected from eight locations in alanya, döşemealtı, kemer, kumluca, and manavgat districts of antalya between apr and oct of 2017. adult susceptibility tests were carried out according to a modified version of the centers for disease control and prevention bottle bioassay. in the tests, the world health organization recommended diagnostic doses; permethrin (0.75%), etofenprox (0.5%), deltamethrin (0.05%) and lambda-cyhalothrin (0.05%) were used. results: as a result of the susceptibility tests, deltamethrin was the least effective insecticide and it caused 58.78– 97.56% mortalities on cx. pipiens populations while permethrin was the most effective substance that caused 100% mortality on all populations. while all of the tested populations were found susceptible to permethrin, and possible resistant or resistant to deltamethrin. etofenprox and lambda-cyhalothrin led to 91.54–100% and 93.1–100% mortalities, respectively. conclusion: the possible resistance or resistance to deltamethrin in all the areas is caused by the widespread use of this chemical against pests in agriculture and public health applications for long-term. moreover, a concordance was found between resistance levels and the intensity of pesticide application in agriculture and public health, and organic and chemical pollution levels in the sampled habitats. keywords: antalya; culex pipiens; mosquito; resistance; synthetic pyrethroid introduction there are over 3500 species of mosquitoes (diptera: culicidae) in the world, and more than 50 of them have been documented in turkey (1, 2). culex pipiens l. is an important vector of several disease-causing pathogens, such as filarial nematode (wuchereria bancrofti), west nile virus (wnv), rift valley fever virus, and st. louis encephalitis virus. this mosquito species is widely distributed throughout the world (3, 4). culex pipiens is the predominant species or it is intensively encountered in mosquito fauna studies conducted in different provinces of turkey (1, 5-8). culex pipiens was also seen as a dominant mosquito species in studies carried out in antalya province (9, 10). this mosquito is the possible vector of the wnv in turkey (11, 12). in 2010, 47 wnv infections were detected and 10 patients died from the wnv infection (12). one of the most effective manners of controlling mosquito-borne diseases transmission is to control of their vectors. despite the use of various methods to control mosquitoes; the application of insecticides continue to be the most preferred method due to easy accessibility, fast and effective results in a short time. however, excessive and unconscious use of insecticides lead to various prob*corresponding author: dr onder ser, e-mail: onderser62@hotmail.com http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 244 http://jad.tums.ac.ir published online: september 30, 2019 lems in terms of environment and human health and also cause to development of resistance by mosquitoes. parallel to resistance development, control of mosquito populations are becoming more difficult and it may be an increase in the incidence of mosquito-borne diseases (13-15). therefore, it is essential to monitor the insecticide resistance and susceptibility in field mosquito populations to ensure the sustainability of mosquito control programs (4). synthetic pyrethroids (sp) were produced to increase the chemical stability and biological activity of natural pyrethrins, which have insecticidal effects. natural pyrethrins are obtained from the extraction of dried flower heads of chrysanthemum spp. these contain a mixture of insecticidal action esters. sp are neurotoxic effect to insects and their primary action site is the voltage-gated sodium channels (16, 17). the mode of action of these insecticides is similar to the organic chlorine insecticide, dichloro diphenyl trichloroethane (ddt). these insecticides act the voltagegated sodium channels on the membranes of the nerve cells and cause over-stimulation due to the longer opening of channels (18, 19). traditionally, sp are classified into two groups namely type i and type ii, according to their chemical structure and toxicology. type i pyrethroids do not contain the α-cyano group, whereas type ii pyrethroids include the α-cyano group on the phenoxybenzyl moiety (19). sp have killer, knock-down and repellent effects on insects. in addition, these insecticides can be used with synergistic compounds to increase their activity (16). sp have several advantages, as compared with other insecticides in terms of cost, safety (less toxic to mammals), repellency, and duration of residual action (20). sp are broad-spectrum insecticides, effective against a number of insect pests (21). currently, these insecticides are widely used, in agriculture, public health, veterinary medicine and as household pesticides (20, 21). using them as larvicides are limited due to their toxicity against non-target aquatic organisms. nowadays, sp are used on all certified long-lasting insecticidal nets and indoor residual spraying programmes for control of major vector-borne diseases worldwide (20). we aimed to determine the susceptibility levels of cx. pipiens populations collected from different districts of antalya, important tourism and agricultural center of turkey, to some synthetic pyrethroid insecticides commonly used against mosquito adults. materials and methods mosquitoes the immature stages (egg raft, larva, and pupa) of mosquitoes were collected from aquatic habitats in alanya (çıplaklı and süleymanlar), döşemealtı (ilıca and killik), kemer (tekirova), kumluca (naranciye and solid waste storage area) and manavgat (çakış) districts of antalya between apr and oct of 2017 (fig. 1). global positioning system (gps) coordinates of the sampling areas are shown in table 1. the immature stages collected from breeding sites were transported to the department of biology, faculty of science, akdeniz university and reared to adults under standard conditions (at a temperature of 25±2 °c, 60±10% relative humidity and 12h light: 12h dark photoperiod in an insectary). larval feeding was done by using fish food. a pad of cotton soaked in 10% sucruse solution was provided for adult mosquitoes feeding. the species identification was made using the morphological characters according to the identification keys (16, 22, 23). insecticides in the susceptibility tests, synthetic pyrethroid active substances (permethrin, etofenprox, deltamethrin, and lambda-cyhalothrin) were purchased from tagros chem. india ltd. and used at the diagnostic doses recommended by the world health organization (who) (20). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 245 http://jad.tums.ac.ir published online: september 30, 2019 bioassays adult susceptibility tests were carried out according to a modified version of the centers for disease control and prevention (cdc) bottle bioassay (24). the who susceptibility assay and the cdc bottle test are the most frequently used methods for detection of insecticide resistance in mosquito populations (25). the results obtained from who and cdc test methods support each other. similar results were found between the two methods in terms of mortality rates in the studies conducted to determine the susceptibility levels to insecticides in malaria vectors (26, 27). both methods have several advantages and disadvantages. for example, who test kits are expensive and may not be easily attainable, but cdc bottle test method is simple, fast and cost-effective (25-27). however, the licensing of insecticides used in indoor residual spraying applications in turkey is carried out according to doses recommended by the who. in addition, the doses used in the cdc bottle test method are considerably lower than the doses used in the who susceptibility tests. therefore, the diagnostic doses and time recommended in the who (2013) test procedures (20) were used in our study. for this purpose, who recommended stock solutions (0.75% permethrin, 0.5% etofenprox, 0.05% deltamethrin and 0.05% lambda-cyhalothrin) were prepared by dissolving the active substances in acetone. for each synthetic pyretroid, 1.836ml of stock solution was applied to the inner surface of the glass jars having an inner surface area of about 500cm2 and solution spreads in the jar. in this way, an equal amount of the active substance to the amount of per square centimeter insecticide on the who tube test method papers was applied to the glass surfaces (20, 25, 28). after the solvent was evaporated (2h waiting period), tests were performed with 20–40 nonblood fed, 3–5 day-old adult female mosquitoes. these individuals were recorded with knock-down rates at 5min intervals for 1h. after the 1h exposure period, mosquitoes were transferred to clean jars for recovery. the number of dead mosquitoes in both the treated and the control jars was recorded 24h post-exposure. according to the who recommendations, mosquitoes are categorized as dead if they are immobile or unable to stand on or fly in a coordinated manner. each bioassay was conducted at least using four replicates. in each bioassay, one control group was used. only acetone is applied to the jars where the control groups are located and the inside surfaces are dried. statistical analysis corrected mortality rate was calculated using abbott's formula when mortality rate in the control group was between 5–20% (29). values of times for 50% knockdown (kdt50) and kdt95 were calculated by statplus probit analysis program. insecticide resistance status of populations was assessed according to who (2013) (20) criteria, where mortality range 98–100% was susceptible, 90–97% possible resistant, and < 90% resistant. results knock-down effect of synthetic pyrethroids the kdt50 and kdt95 values of active substances are presented in table 2. at the end of 1h, while the knock-down rates for permethrin were 100% in all populations, the knock-down rates for etofenprox, deltamethrin and lambda-cyhalothrin were between 88.21– 100%, 31.31–99.58% and 94.27–100%, respectively. according to kdt50 values, permethrin showed the highest knock-down effect on cx. pipiens, except for the döşemealtı-ilıca population. although different results were obtained for other active substances, deltamethrin showed the highest kdt50 values and the lowest knockdown effect at the five populations. kdt50 values of permethrin were http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 246 http://jad.tums.ac.ir published online: september 30, 2019 6.00–12.10min, of etofenprox, were 6.57–27.61 min, of deltamethrin were 11.26–122.53min and of lambda-cyhalothrin were 5.57–20.66 min. the lowest kdt50 value in all populations was 5.57min at lambda-cyhalothrin in the döşemealtı-ilıca population, while the highest kdt50 value was 122.53min at deltamethrin in the kumluca-solid waste storage area population. when kdt95 values were compared, deltamethrin showed the lowest knock-down effect except for the döşemealtıkillik population while permethrin showed the highest knock-down effect on all populations. kdt95 values of permethrin were 12.96–29.59 min, of etofenprox were 17.36–85.36min, of deltamethrin were 40.83–744.30min and of lambda-cyhalothrin were 14.82–69.66min in all populations. the lowest kdt95 value of all populations was 12.96min at the permethrin in the döşemealtı-ilıca population, while the highest kdt95 value was 744.30min at the deltamethrin in the kumluca-solid waste storage area population. mortality rates as a result of the susceptibility tests, deltamethrin was the least effective insecticide and it caused 58.78–97.56% mortalities on cx. pipiens while permethrin was the most effective substance that caused 100% mortality on all populations (fig. 2). according to who criteria, while all of the tested populations were susceptible to permethrin, none of the populations were susceptible to deltamethrin, four of the eight populations were resistant to deltamethrin and the remains were possible resistant (table 2). in addition, the lowest mortality rate among all tested populations was obtained from deltamethrin at 58.78% in the kumluca-solid waste storage area population. etofenprox and lambdacyhalothrin led to 91.54–100% and 93.10– 100% mortality in the eight tested populations, respectively (fig. 2). populations collected from two sampling sites for each of etofenprox and lambda-cyhalothrin were determined as possible resistant and the other six populations were susceptible (table 2). table 1. location and global positioning system (gps) coordinates of cx. pipiens collection sites no district location gps coordinate 1 alanya çıplaklı 36º33'39.9" n 32º02'44.2" e 2 alanya süleymanlar 36º40'42.299" n 31º59'04.256" e 3 döşemealtı ilıca 37º09'12.868" n 30º37'54.912" e 4 döşemealtı killik 37º12'51.987" n 30º39'52.588" e 5 kemer tekirova 36º31'04.634" n 30º32'18.758" e 6 kumluca narenciye 36º21'59.7" n 30º17'21.3" e 7 kumluca solid waste storage area 36º25'10.538" n 30º18'30.969" e 8 manavgat çakış 36º54'46.150" n 31º09'47.825" e fig. 1. culex pipiens collection sites in various districts of antalya province, turkey in 2017 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 247 http://jad.tums.ac.ir published online: september 30, 2019 table 2. knock-down (kd) rates, mortality rates, kdt50 and kdt95 values and susceptibility status of cx. pipiens populations from antalya, turkey to four synthetic pyrethroids populations parameters permethrin (0.75%) etofenprox (0.05%) deltamethri n (0.05%) lambdacyhalothrin (0.05%) control alanya çıplaklı n 165 172 190 167 171 %kd±se (after 60min) 100 88.21±5.40 80.03±5.09 94.27±1.47 0 kdt50±se (min) 12.10±0.82 26.02±0.82 26.85±1.24 20.66±0.73 kdt95±se (min) 27.27±2.32 85.36±5.74 188.76±27.6 9 69.66±4.25 χ2 24.4440 2.0809 1.0577 3.1637 p-level 0.0065 0.9957 0.9998 0.9773 %mortality±se (after 24h) 100 91.54± 2.51 80.30±3.01 93.10±2.08 0.63±0.63 susceptibility status s pr r pr alanyasüleymanlar n 115 112 131 121 102 %kd±se (after 60min) 100 94.34±2.67 92.96±4.32 100 0 kdt50±se (min) 11.81±0.68 27.61±1.43 22.89±0.82 14.79±0.48 kdt95±se (min) 29.59±2.04 81.18±9.09 86.54±6.32 33.66±1.39 χ2 12.6888 17.5235 5.6160 4.5321 p-level 0.2416 0.0636 0.8464 0.9202 %mortality±se (after 24h) 100 91.95±3.37 90.23±2.45 99.19±0.81 0.96±0.96 susceptibility status s pr pr s döşemealtı ilıca n 118 110 124 138 114 %kd±se (after 60min) 100 100 65.98±7.32 100 0 kdt50±se (min) 6.00±0.30 6.57±0.37 47.19±1.41 5.57±0.36 kdt95±se (min) 12.96±0.84 17.36±1.06 124.14±10.9 4 14.82±1.00 χ2 0.0344 0.0390 11.7727 0.0646 p-level 0.9999 0.9999 0.3006 0.9999 %mortality±se (after 24h) 100 100 76.51±4.92 100 5.15±3.16 susceptibility status s s r s döşemealtıkillik n 158 154 196 169 144 %kd±se (after 60min) 100 98.33±1.67 99.58±0.42 100 0 kdt50±se (min) 9.89±0.41 15.85±0.58 13.09±0.55 12.03±0.46 kdt95±se (min) 24.78±1.23 46.07±2.25 40.83±2.10 30.61±1.41 χ2 4.7670 4.6889 1.7015 0.8662 p-level 0.9062 0.9110 0.9982 0.9999 %mortality±se (after 24h) 100 99.17±0.83 97.56±0.86 100 0 susceptibility status s s pr s kemertekirova n 118 121 139 115 109 %kd±se (after 60min) 100 100 92.00±8.00 100 0 kdt50±se (min) 6.37±0.35 8.96±0.44 11.26±0.70 12.01±0.50 kdt95±se (min) 15.95±1.00 25.89±1.39 60.06±4.72 34.32±1.68 χ2 0.2756 1.1492 0.9450 1.2361 p-level 0.9999 0.9997 0.9999 0.9995 %mortality±se (after 24h) 100 100 91.70±4.39 100 1.71±1.05 susceptibility status s s pr s kumlucanarenciye n 149 147 171 155 148 %kd±se (after 60min) 100 99.28±0.72 93.10±3.01 96.14±1.76 0 kdt50±se (min) 11.35±0.43 16.84±0.75 19.69±0.70 14.67±0.58 kdt95±se (min) 26.91±1.24 41.77±2.47 65.40±3.85 45.94±2.37 χ2 5.8699 11.9181 1.8928 2.4029 p-level 0.8261 0.2906 0.9971 0.9922 %mortality±se (after 24h) 100 98.55±1.45 90.07±2.86 96.94±1.08 0 susceptibility status s s pr pr http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 248 http://jad.tums.ac.ir published online: september 30, 2019 kumluca solid waste storage area n 137 134 138 115 138 %kd±se (after 60min) 100 100 31.31±2.52 100 0 kdt50±se (min) 8.99±0.34 11.93±0.72 122.53±20.6 6 9.88±0.37 kdt95±se (min) 18.51±0.92 20.67±1.78 744.30±310. 93 20.81±0.99 χ2 2.3809 32.7119 2.9895 0.7357 p-level 0.9925 0.0003 0.9817 0.9999 %mortality±se (after 24h) 100 100 58.78±5.34 100 2.48±1.78 susceptibility status s s r s manavgatçakış n 137 120 133 114 119 %kd±se (after 60min) 100 100 84.38±4.61 100 0 kdt50±se (min) 8.96±0.39 12.40±0.48 24.82±0.87 13.85±0.47 kdt95±se (min) 21.94±1.13 32.58±1.52 95.55±7.47 32.08±1.36 χ2 0.0921 1.8646 4.6029 7.4133 p-level 0.9999 0.9973 0.9161 0.6859 %mortality±se (after 24h) 100 100 80.67±5.76 100 7.93±4.78 susceptibility status s s r s n: number of individuals tested, pr: possible resistant, r: resistant, s: susceptible, se: standard error swsa: solid waste storage area fig. 2. mortality rates of cx. pipiens populations to diagnostic concentrations of synthetic pyrethroids table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 249 http://jad.tums.ac.ir published online: september 30, 2019 discussion knock-down rates sp are rapid-acting insecticides, which have a knock-down effect (20). the target site of these insecticides is voltage-gated sodium channels of nerve cell membranes in insect body (30, 31). mutations in genes encoding the amino acid sequence in these channels cause a reduction in the sensitivity of the channels to the binding of pyrethroid insecticides. alterations in the target site that lead to resistance to insecticides are usually referred to as knockdown resistance or kdr (32). in the case of kdr in a insect population, the rates of knock-down obtained from susceptibility tests are an important parameter for early detection of resistance to the insecticide (20). from this point of view, there is currently no resistance development against permethrin in all studied populations due to 100% knockdown rates at the end of 1h and low kdt50 and kdt95 values. permethrin is a highly active substance with rapid knockdown effect against a variety of insects (33). however, intensive use of permethrin may cause development of resistance in mosquito populations. extensive use of permethrin and ddt is involved in the selection of resistance against these insecticides in field populations of anopheles (an.) gambiae in burkina faso (34). while mosquitoes collected from cottongrowing and urban areas were resistant to permethrin and ddt, mosquitoes collected from areas with limited insecticide selection pressure (rice fields and control areas) were susceptible. mosquitoes in all tested areas were susceptible to deltamethrin with high mortality rates. in addition, mosquitoes collected from cotton-growing and urban areas showed higher kdt50 and kdt95 values with permethrin and ddt than deltamethrin. resistance to permethrin and ddt may explain extensive use of these insecticides in the cotton-growing area and domestic use of same insecticides as bomb spray or coils in the urban areas. the appearance of 31.31–99.58% knockdown rates at the end of 1h and high values of kdt50 (11.26–122.53min) and kdt95 (40.83–744.30min) of deltamethrin in this study indicate resistance development in all studied populations against this active substance. susceptibility to deltamethrin of field-collected populations and colonized strains of an. culicifacies were investigated in india (35). although there was 100% mortality in all the strains of an. culicifacies exposed to diagnostic concentration of deltamethrin for 1h, knockdown bioassays revealed more than two-fold higher values of kdt50 and kdt90 in an. culicifacies from rameshwaram island (both in field-collected and colonized strain), than populations from other areas. results indicated the development of incipient resistance to deltamethrin in this strain of an. culicifacies. insecticide resistance levels were evaluated in nine populations of an. gambiae sampled in three areas in the east of tanzania (36). these areas are represented as an agriculture area, an urban area and a low pollution area depending on urbanization, agriculture activity, and usage of insecticides for vector control. for adult mosquitoes, resistance ratio fifty (rr50) was obtained by dividing the kdt50 of each population to the kdt50 of the susceptible reference strain. adult susceptibility tests revealed that populations from urban and agriculture areas demonstrated the moderate resistance levels to deltamethrin with mean rr50 of 3.1 fold and 5.6 fold, respectively. mortality rates after 1h exposure to who diagnostic dose of deltamethrin were between 84 and 100% in all populations. a significant correlation was between deltamethrin resistance and agriculture activity. the presence of 88.21% and 94.34% knock-down rates at the end of 1h, kdt50 values of 26.02 and 27.71min and kdt95 values of 85.36 and 81.18min respectively in the alanya-çıplaklı and alanya-süleymanlar http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 250 http://jad.tums.ac.ir published online: september 30, 2019 populations for etofenprox indicate that these populations may develop resistance to etofenprox. the appearance of 94.27% and 96.14% knock-down rates at the end of 1h, the kdt50 values of 20.66 and 14.67min and the kdt95 values of 69.66 and 45.94min respectively in the alanya-çıplaklı and kumluca-narenciye populations for lambda-cyhalothrin indicate that these populations may develop resistance to lambda-cyhalothrin. the knock-down rates, kdt50 and kdt95 values obtained from susceptibility tests are compatible with the mortality rates at the end of 24h. mortality rates in the populations with high kdt50 and kdt95 values with low knock-down rates against etofenprox, deltamethrin and lambda-cyhalothrin were determined to be resistance or possible resistant levels. mortality rates in this study, the possible resistance or resistance to deltamethrin in all the sampling areas is caused by the widespread use of this active substance against the pests of agriculture and public health for many years. insecticide resistance in mosquitoes collected from areas where pesticide use is high against agriculture and/or public health pest is higher levels than in areas where pesticide use is less (37-40). kasap et al. (41) researched resistance to some insecticides of an. sacharovi collected in five malaria-endemic provinces of turkey. according to results of susceptibility tests, adana, adıyaman, and antalya populations were resistant to most insecticides tested, while aydın and muğla populations were susceptible to most of the insecticides. intensive insecticide usage against agricultural pests and mosquitoes has selected resistance in an. sacharovi to a number of compounds in these areas. the resistance levels to two types of insecticides of four major vector species (an. gambiae, cx. quinquefasciatus, aedes (ae.) aegypti, and ae. albopictus) were assessed in mayotte, a small island in the indian ocean (42). adult bioassays revealed that while field populations of an. gambiae and two aedes species were found susceptible to deltamethrin, field population of cx. quinquefasciatus was found stronge resistance to deltamethrin (only 10% mortality after 24h and strong decrease of knock-down effect: rr50= 4.2, rr95= 4.9). culex quinquefasciatus is the major vector of the bancroftian filariasis, which has been plaguing mayotte for many years. since the 1950s, intense vector control efforts had been carried out by using ddt and followed synthetic pyrethroids against this species. these important selective pressures certainly explain the strong resistance to deltamethrin observed in the cx. quinquefasciatus population. insecticide resistance status of cx. quinquefasciatus populations from four areas was studied in benin (43). adult tests showed high frequency of resistance in mosquitoes to permethrin (ranging from 4% to 24% mortality) and deltamethrin (24% to 48% mortality) in the four research areas. sp have been extensively used in agriculture since 1980s particularly in cotton and vegetable fields in benin. moreover, the massive free campaign of bed nets impregnated with permethrin and deltamethrin as the major control strategy against wuchereria bancrofti transmitted by cx. quinquefasciatus. these cases may cause the resistance of cx. quinquefasciatus to sp. in our study, the highest level of deltamethrin resistance with the lowest mortality rates was detected in mosquitoes collected from the kumluca-solid waste storage area. this situation is thought to have been caused by the application of residual spraying, thermal and cold fogging by using the sp regularly against the vectors such as mosquitoes, sand flies and house flies by the antalya metropolitan municipality vector control teams in this area. in addition, kumluca is a district where greenhouse activities and the use of pesticides related to greenhouses is intense in antalya province. therefore, the greenhouse wastes on the solid waste storage area contain intense peshttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 251 http://jad.tums.ac.ir published online: september 30, 2019 ticide residues and the leaking water from these wastes constitutes a breeding site for mosquitoes. moreover, there are intensively empty packages of various chemicals such as pesticides, fertilizers, and detergents containing residues in the solid waste storage area and agricultural pesticides are used in fields and gardens near to this area. the resistance levels to deltamethrin of mosquito populations are in agreement with pollution level of the sampling areas and pesticide application frequency in these areas. alanya-çıplaklı, manavgat-çakış, döşemealtıilıca, and kumluca-solid waste storage area populations were resistant to deltamethrin with mortality rates lower than 90%. the collection areas of these four populations are habitats where pollutants such as organic and chemical wastes are concentrated. in addition, vector control applications are regularly carried out by the antalya metropolitan municipal in these four areas and pesticides are used in the fields and fruit gardens near these areas. pesticides used in agricultural and public health applications are effective for improving resistance of mosquitoes to insecticides. besides, various anthropogenic or natural xenobiotics in the breeding sites of mosquitoes can also contribute to develop resistance of mosquitoes to insecticides, especially by altering the expression of genes encoding detoxification enzymes and/or cuticular proteins (44). aedes aegypti larvae can be improved resistance to various insecticides in different classes by changes in the expression of genes responsible for detoxification of heavy metal (copper), polycyclic aromatic hydrocarbons (fluoranthene and benzo[a]pyrene) or herbicides (glyphosate and atrazine) (45-47). similarly, the expression of genes encoding cuticular proteins was changed in ae. aegypti larvae exposed to various pollutants and pesticides from different classes (48, 49). alanya-süleymanlar, döşemealtı-killik, kemer-tekirova and kumluca-narenciye populations were possible resistant to deltamethrin with mortality rates in the range of 90– 97%. the habitats of these populations seem to be cleaner in terms of organic and chemical pollutants when compared to the habitats of the other four populations. in addition, pesticide applications against pests of agricultural and public health are carried out at lower intensity in these sampling areas. in this study, the permethrin in all sampled locations was the most effective of the four active substances tested, leading to 100% mortality on the mosquito populations. in turkey, use of permethrin as plant protection product has been terminated by republic of turkey ministry of food, agriculture and livestock since 01 jan 2011. therefore, this active substance has not been used against pests of agricultural in antalya province for the last six years. in addition, permethrin has been used in limited quantities in cold fogging applications against adult mosquitoes by the antalya metropolitan municipality in 2013 and 2015 years. this active substance has not been used against adult mosquitoes in the last two years (50). as a result of the susceptibility tests, etofenprox was caused 91.54% to 100% mortalities in all populations. while alanyaçıplaklı (91.54% mortality) and alanya-süleymanlar (91.95% mortality) populations were determined to be possible resistant to etofenprox, the other six populations were found susceptible. etofenprox is used in low amounts against pests of agriculture and public health in antalya province. this case is consistent with the susceptibility to etofenprox of the six tested populations. the possible resistance to etofenprox of the alanya-çıplaklı and alanya-süleymanlar populations may be due to the local use of this active substance in these sampling areas or development cross-resistance to other insecticides from the used synthetic pyrethroid group in fruit growing (grape, avocado, lemon, orange, medlar, banana etc.) at outdoor area of alanya district. because of tests performed with lambdacyhalothrin, it was observed mortality in the http://jad.tums.ac.ir/ https://www.tarim.gov.tr/sayfalar/en/anasayfa.aspx https://www.tarim.gov.tr/sayfalar/en/anasayfa.aspx j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 252 http://jad.tums.ac.ir published online: september 30, 2019 range of 93.1–100% in all populations. while alanya-çıplaklı (93.1% mortality) and kumluca-narenciye (96.94% mortality) populations were possible resistant to this active substance, the other six populations were susceptible. lambda-cyhalothrin is used in low amounts against pests of agriculture and public health in antalya province. this case is consistent with the susceptibility to this active substance of the six tested populations. the possible resistance to lambda-cyhalothrin of the alanya-çıplaklı and kumluca-narenciye populations may be due to the local use of this active substance in these sampling areas or development cross-resistance to other insecticides from the used synthetic pyrethroid group in these sampling areas. in our literature review, cx. pipiens has developed resistance to sp at different levels in a variety of researches conducted in turkey and worldwide. culex pipiens populations collected from aksu district of antalya were resistant to permethrin (0.75%) and deltamethrin (0.05%) with 78.3% and 75.8% mortality rates, respectively (3). similarly, cx. pipiens populations collected from aksu district of antalya were resistant to permethrin (0.75%) and deltamethrin (0.05%) with 74% and 62% mortality rates, respectively (12). seasonal dynamics of insecticide resistance were investigated in field populations of cx. pipiens from çanakkale, balıkesir, i̇zmir, aydın, muğla, and denizli provinces in western turkey (4). in the results of bioassays, all populations showed seasonally different levels of resistance to permethrin and deltamethrin. the resistance status to four insecticides was examined in thirteen populations of cx. pipiens collected from five regions of greece (39). adult bioassays showed that while one population was resistant to deltamethrin with 64% mortality rate, three populations were possible resistant with mortalities of 92%, 90% and 87%, and other nine populations were susceptible. salim-abadi et al. (51) evaluated the susceptibility status of cx. pipiens col-lected from the capital city of tehran, iran. field population was resistant to all tested sp (lambda-cyhalothrin 0.05%, deltamethrin 0.05% and cyfluthrin 0.15%). the irritability levels of different groups of insecticides on laboratory strain and field population of cx. pipiens complex were investigated in tehran, iran (52). permethrin (0.75%) and deltamethrin (0.05%) were moderately irritable against both field population and laboratory strain of cx. pipiens complex. while cyfluthrin (0.15%) was moderately irritable for field population, it was hypo-irritable for laboratory strain. lambdacyhalothrin (0.05%) was hypo-irritable against both field population and laboratory strain. whereas etofenprox (0.5%) was hypo-irritable for field population, it was non-irritable for laboratory strain. ghorbani et al. (53) assessed the susceptibility status to 12 adulticides and two larvicides recommended by who of cx. pipiens collected from sari county in the north of iran. the susceptibility tests showed that cx. pipiens was resistant to all tested insecticides. nevertheless, the resistance level was lower to sp compared to the others. the mortality rates after exposure to etofenprox (0.5%), cyfluthrin (0.15%), permethrin (0.75%), deltamethrin (0.05%) and lambda-cyhalothrin (0.05%) were 76.47%, 72.09%, 70.73%, 39.08% and 33.33% respectively. insecticide resistance may vary among regions, provinces, districts, and even smaller localities within a country. these differences in insecticide resistance include many factors such as the species of mosquito, life stage, physiological status, even the various symbioses or pathogens found in the body of mosquitoes, the climatic characteristics of the study area, altitude, vegetation cover, acreage, socioeconomic structure, agriculture and animal husbandry activities, pesticides used in this area, doses, frequency, and methods of application of pesticides, agricultural chemicals, urban and industrial pollutants (44). in order to prevent and/or delay to the development of resistance to insecticides in moshttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 253 http://jad.tums.ac.ir published online: september 30, 2019 quitoes, integrated control programs should be implemented in which chemical use is kept in a minimum level, with emphasis on physical, cultural and biological control methods (15, 54). if chemical use is needed; products with high selective toxicity, low toxicity to non-target organisms, less persistence in the environment and no resistance developed in target organism should be preferred and these products should be used at the doses indicated on the label. in addition, whether mosquitoes have resistance or not should be regularly monitored against the products used in combat (55). in the case of resistance detection, various tests can be performed to determine the mechanism. for this purpose, synergist tests can be used to determine the presence of resistance related to the detoxification enzymes, biochemical enzyme assays to determine the metabolic resistance, and molecular biological tests to determine the target site resistance (20). according to the results obtained from resistance tests, insecticide resistance maps should be prepared. these maps should be used in the selection of insecticide and resistance management (56). insecticide applications should be made in more limited areas where mosquitoes are heavily infested or mosquito-borne disease risk is high rather than large areas (55). since larval and adult control must be performed simultaneously to have an effect on mosquito populations, unrelated classes of insecticides with different modes of action should be used for each life stage of mosquitoes (57). it may be beneficial to apply a mosaic approach by using products of different insecticide classes and mode of action in neighboring areas (32, 57). the insecticides with same modes of action should not be used in an area for a long time. instead, insecticides of different classes with unrelated modes of action should be used in rotation (32, 54, 57, 58). addition of synergistic substances to products may increase the susceptibility of mosquitoes to insecticides (58). synergists are compounds that do not have insecticidal activity by themselves. however, when they are mixed with insecticides of a certain class, significantly increase their effect by inhibiting an enzyme that detoxifies the insecticide in the insect body (20, 59). synergists include piperonyl butoxide (pbo), which inhibits oxidase activity, s.s.s tributlyphosphorotrithioate (def), which inhibits esterase activity, ethacrynic acid (ea), diethyl maleate (dm), and chlorfenethol (cf), which inhibit glutathione transferase activity (24). pbo is used as a synergist in insecticide formulations against the public health pests in turkey. however, there is no standard for synergist ratios to be used in formulations. in addition, the development of more efficient and environment-friendly new compounds with different modes of action, such as herbal, microbial and synthetic origin, as an alternative to the existing insecticides will contribute to preventing or delaying the development of resistance to insecticides in mosquitoes (55, 58, 60). conclusion in our study, all cx. pipiens populations were found to be possible resistant or resistant to deltamethrin. this situation is thought to be due to the widespread use of this active substance in agriculture and public health applications for many years. the resistance levels to deltamethrin of the mosquito populations are related to pesticide application frequency in these areas and the pollution level of the sampling area. insecticides are used intensively to control mosquito populations at present. however, mosquitoes develop resistance to almost every kind of insecticides used in combat. this case causes serious concerns. one of the effective ways, in order to prevent and/or delay to the development of resistance to insecticides in mosquitoes, is to minimize usage of insecticide. to achieve this, integrated control programs should be implemented. in addition, http://jad.tums.ac.ir/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 254 http://jad.tums.ac.ir published online: september 30, 2019 the resistance status of mosquito populations against insecticides used in combat should be regularly monitored and insecticide resistance maps should be prepared. the results of this study will contribute to the planning of the resistance management and selection of insecticides that will be used by the mosquito control agencies and institutions in antalya, turkey. however, it needs new studies that will be used for other 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mosquitocidal http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/labs/journals/aquat-toxicol/new/2017-09-30/ https://www.ncbi.nlm.nih.gov/labs/journals/aquat-toxicol/new/2017-09-30/ https://www.ncbi.nlm.nih.gov/labs/journals/aquat-toxicol/new/2017-09-30/ https://www.ncbi.nlm.nih.gov/labs/journals/aquat-toxicol/new/2017-09-30/ http://fenbilim.akdeniz.edu.tr/ http://fenbilim.akdeniz.edu.tr/ j arthropod-borne dis, september 2019, 13(3): 243–258 o ser and h cetin: investigation of … 258 http://jad.tums.ac.ir published online: september 30, 2019 bacterial toxins (bacillus sphaericus and bacillus thuringiensis serovar israelensis): mode of action, cytopathological effects and mechanism of resistance. j physiol pathophysiol. 1(3): 22–38. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 41 http://jad.tums.ac.ir published online: march 18, 2018 original article modeling of environmental factors affecting the prevalence of zoonotic and anthroponotic cutaneous, and zoonotic visceral leishmaniasis in foci of iran: a remote sensing and gis based study abdol ali golpayegani 1, 2, 3, ali reza moslem 4, amir ahmad akhavan 1, 5, azam zeydabadi 1, 2, *amir hossein mahvi 1, 3, 6, *ahmad allah-abadi7 1school of public health, tehran university of medical sciences, tehran, iran 2bam university of medical sciences, bam, iran 3national institute of health research, ministry of health, tehran, iran 4sabzevar university of medical sciences, sabzevar, iran 5institute for environmental research, tehran university of medical sciences, tehran, iran 6center for solid waste research, institute for environmental research, tehran university of medical sciences, tehran, iran 7leishmaniasis research center, sabzevar university of medical sciences, sabzevar, iran (received 31 jan 2016; accepted 10 feb 2018) abstract background: leishmaniasis is a re-emerging serious international public health problem, and both visceral and cutaneous types of leishmaniasis became important endemic diseases in iran. in this study, the relationships between environmental factors (vegetation and elevation) and the prevalence of diseases have been investigated. methods: all international and national online databases were searched by terms such as leishmaniasis, incidence, prevalence and other related words attributed to iran and published until first quarter of 2015. the developed database in excel, later imported to the arcmap for spatial analyst and mapping. afterwards, the software was used for modeling the relationship between the prevalence/incidence and environmental variables (vegetation and elevation) by both linear and nonlinear regression. results: after mapping the prevalence data from 144 studies, considering non-parametric anova, the tendency of zoonotic visceral leishmaniasis to presence in high elevation and high vegetation was more than anthroponotic and zoonotic cutaneous leishmaniasis. while linear regression showed weaker results for modeling, however, additive nonparametric regression analysis suggested that 10km buffers for elevation, and 10 as well as 50km buffers for vegetation could contribute in better fitness in modeling of these variables. conclusion: the detailed maps for distribution of disease concluded. the nonlinear regression is a reliable predictor of the relationship between environmental factors and disease incidence, although more and wide researchers are needed to confirm it. keywords: leishmaniasis, gis, remote sensing, environmental variables, nonlinear regression introduction leishmaniasis –a re-emerging serious international public health problemis considered as one of the most neglected tropical diseases. the disease is distributed in new foci due to influence of many risk factors, including environmental factors (1-4). almost 200000 to 400000 new cases of visceral leishmaniasis (vl), 0.7 million to 1.3 million new cases of cu taneous leishmaniasis (cl) and 20000 to 30000 deaths annually" are the estimations of who last report about importance of the disease (2). two dominant types of leishmaniasis there in iran are: zoonotic visceral or kala-azar (zvl), the most serious and fatal type of the disease, and both types of cutaneous leishmaniasis namely urban form or anthroponotic cutaneous *corresponding authors: dr amir hossein mahvi, email: ahmahvi@yahoo.com, dr ahmad allah-abadi, email: ahmad_health@yahoo.com http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 42 http://jad.tums.ac.ir published online: march 18, 2018 leishmaniasis (acl) and rural form or zoonotic cutaneous leishmaniasis (zcl). leishmania donovani complex are the causes of zvl protozoan disease which can lead to patient mortality (5). evidence indicates an increase in the incidence of disease in the old and new worlds at the early years of the present century. in recent decades, zvl became an important endemic disease in the northwest (6), south and southwest (7), and northeast (8) of iran, especially in nomads. the majority of zvl cases (92.8%) in country were found among children with age under 12yr old (9). the most recently, direct agglutination test (dat) as a simple, affordable and practical test, has found its way as a special quick and accurate test. thus it has been recommended and applied widely for clinical diagnosis and seroprevalence studies of zvl in zoonosis and anthroponotic reservoirs in endemic and nonendemic areas of iran (10). due to ecologically dependence of the disease vectors and reservoirs to environment, outbreaks mostly are common in lowlands rural or suburban areas (less than 600m elevation), with a heavy annual rainfall, a moderate temperature (15–38 °c), and almost dense vegetation (2). countries of mediterranean coasts (algeria and syrian arab republic), south americas (brazil and colombia), and middle east (islamic republic of iran and afghanistan) and central asia countries are the most reported foci for cl and the major contribution of cl occur in this area (2). the cl cases in iran during the period of 2001 to 2008 dramatically has been increased more than two-fold and also over half of 31 provinces of country have cl endemic foci (11). as reported by center for disease control, the annual incidence of leishmaniasis in the country due to referring to health centers has reached about 20000 cases. while the actual cases of disease may be far more than these reports that may even reach up to five times (12). as for the first time carried by john snow in 1854 due to diarrheal disease detection in london, spatial modeling as a useful tool in epidemiological studies could be used. today, geographic information system (gis) in relation to the processed images from remote sensing (rs) has been used to map the geographical distributions of disease prevalence, factors affecting the transmission and control of disease, and the spatial modeling of environmental factors that have meaningful impacts on disease occurrences (9, 13). vector control programs are associated with logistic problems, mainly due to poor understanding of vector ecology (14). expanding inexpensive and effective models of integrated management for better control of cl is a critical target which need a thorough will, alongside epidemiology studies and understanding of the ecology of the disease (11) and the role of social, ecological and specially environmental variables for risk factors such as the vegetation and elevation (two most widespread and accessible environmental variables). the relationship between epidemiological components of leishmaniasis (incidence, prevalence, and seroprevalence) and environmental variables (vegetation, elevation, and/or other factors) can be used in gis modeling and may be useful for secondary uses including prediction. therefore, contributing to better planning for control activities and the establishment of early warning systems, incorporating remotely sensed information in epidemiological studies can help public health policy makers in having a better notion and comprehensive understanding to find the relationships between disease and its environmental risk factors (15). the remotely sensed normalized difference vegetation index (ndvi) –the predominantly index which used for vegetation coverage– has found its widespread applications due to fluctuations it has along with meteorological and environmental components (14). this index together with the other remotely sensed data (11, 16) like the digital elevation model (dem), have been used extensively in monitoring some vector-borne diseases inhttp://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 43 http://jad.tums.ac.ir published online: march 18, 2018 cluding leishmaniasis throughout the world. while many studies have surveyed the associations between the mean of ndvi and dem with incidence or prevalence rate of a type of leishmaniasis locally, however, they do not study all variables in a vast area. in this study, we systematically reviewed all studies regarding the incidence and/or prevalence rate of all categories of leishmaniasis investigated in iran from 1976 to 2015. after mapping the results, using spatial analysis and regression techniques, statistical association between variables have been modeled in detail. materials and methods to identify the incidence and prevalence of leishmaniasis in endemic and nonendemic area of zvl, zcl, and acl of iran, we searched and reviewed the literature based on international database including medline, isi, scopus, and google scholar, and national databases such as sid, irandoc, magiran, and isc. search protocols have been based on using the terms related to all types of leishmaniasis, fauna, vectors, reservoirs, incidence, prevalence, seroprevalence, direct agglutination test (dat), and their persian equivalents with the geographic term "iran". overall, 503 articles published from 1976 through jun 2015 in english, french, and persian were founded in first search from databases. besides, by referring to cv of famous professors, abstract seminars, thesis, non-digital papers, and the reference/citations of articles, 121 additional sources were found. after the elimination of duplicate and inappropriate articles by two separate teams, we consequently included 144 studies that clearly addressed confirmed clinical cases for human seroprevalence (dat ≥ 1: 3200)/ incidence of zvl and active lesion prevalence/total prevalence (active lesion + scar)/incidence of acl and zcl in iran. disease data were extracted by researchers directly from body text of articles to database tables (dbase format using the ms excel software) containing all cities (1246 point) and villages with a population more than 500 people (12034 points), disease information, and descriptive data. depending on the raw data presented in each article, one or more than one category of epidemiologic data was included in the main table. these numeric values (all in percent) for acl and zcl including active lesion prevalence, total prevalence (active lesion + scar), and incidence and for vcl including seroprevalence and incidence were directly or indirectly extracted using study population. the developed database later imported to the arcmap® software version 9.3 (esri, redlands, usa) software platform for spatial analyst and mapping all types of leishmaniasis. r software version 3.2.2 (17) was used for analyzing the relationship between the prevalence/incidence and continues spatial variables. shapefile maps for political subdivisions of provinces, counties, rural districts, and points of cities, towns, and villages with their attribute tables were obtained from national cartographic center, iran. the moderate resolution imaging spectroradiometer (modis) onboard the terra satellite, is one of most applied ndvi images source in literature. ndvi calculated from surface reflectance in the near-infrared ρnir and red ρr portions of the electromagnetic spectrum, using the equation 1 (18): 1) ndvi= (ρnir–ρr)/ (ρnir+ρr) the 16d, 250m ndvi raster maps (mod 13q1 series, version 5) projected in geographic coordinate system wgs_1984, derived from modis reprojection tool (mrt) (19) for the years 2000 to 2014. sixteen days maps unified to annual average ndvi in spatial analysis, then reclassified to 1–20. altitude was based on dem, obtained from the 100m aster global digital elevation model (aster gdem) data center version 2 (20). these maps then reprojected to lambert conformal conic in arcmap, and additional raster analysis such as reclassification, annual and 15yr average http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 44 http://jad.tums.ac.ir published online: march 18, 2018 and zonal statistics were performed on them. overall, 5km, 10km, 20km, and 50km buffer zones centered on each city/village point reported values for leishmaniasis, and their zonal min, max, minority, majority, median and mean statistics were calculated and exported in dbase tables. finally, massive joined tables were called in r, and statistical calculations were completed for each. data analysis csv tables for any purposes derived from the main disease data table and called in r console. we chose to test the normality of data with one-sample kolmogorov-smirnov and shapiro-wilk normality test. next, we modeled the relationships between prevalence/ incidence values and the continuous independent variables. two regression scenarios were considered. first, using simple linear regression models, a stepwise backward elimination procedure was used with the variables, finally resulting in the model contains merely the variables which associated with the outcome. secondly, using generalized additive models -which use spline or local regressiona stepwise backward elimination procedure, was used again to reach the final model. the additive nonparametric regression model is presented in equation 2 (21): 2) y= β0+ m1(x1)+ m2(x2)+ . . .+ mk(xk)+ ɛ where the partial-regression functions mj are fit using a simple-regression smoother, such as local polynomial regression or smoothing splines. in nonparametric regression, in contrast, the object is to estimate the regression function directly without specifying its form explicitly. in nonparametric comparing with simple regression, the ultimate goal is to obtain the regression function directly from internal computation without any open aspect (22). the level of 5% significance for all tests was considered. results we found 144 studies that directly informed the active lesion/scar prevalence and/or incidence of leishmaniasis in iran from 1976– 2015 (table 1). these data considered as a base for construction of 89 acl points including 15, 19, and 71 urban/rural points for active lesion, total prevalence (active lesion + scar), and incidence respectively. the same way, data for 420 zcl points included 67, 68, and 362 points. cases for 355 zvl points for the seroprevalence and incidence were 283 and 82 respectively. figures 1, 2, and 3 show the maps of three types of leishmaniasis in iran separately. the dem map and 15yr average of ndvi map also attached on vl and acl maps respectively. rating in the categories are high, middle and low for prevalence data and obtained based on expert consensus. scatter diagrams and normality plot of each independent variable and the disease incidence/ prevalence were created and visually inspected before running regression. some of these plots traced in fig. 4. normal probability of data in our variables has been questioned, then, the normality of data statistically tested. therefore, using both one-sample kolmogorov-smirnov and shapiro-wilk normality test, the data are without normality. however, both linear and nonparametric regression fitted on data. to compare of the means of main independent variables (dem and ndvi) versus types of leishmaniasis, homogeneity of variances rejected by fligner-killeen test, but due to nonparametric kruskal-wallis test, we result in significant difference between means of variables. regard to boxplots for comparison of means in fig. 5, and nonparametric analysis of variances in table 2, tendency of zvl to presence in high elevation and high vegetation is more than acl and zcl. similarly, we compare the means of types of leishmaniasis occurrences and epidemiological variables of disease in table 3. http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 45 http://jad.tums.ac.ir published online: march 18, 2018 the regression models were evaluated using r software packages. tables 4 and 5 generally descript data structure and assessment of the effect of, or relationship between, explanatory variables on the response by using multiple linear regression models. to determine the best final fitted models for each group of data, based on p-value, adjusted r-squared, and aic (akaike's 'an information criterion'), using stepwise backward simple linear regression, following model produced: (although second order or interaction models had a little more r-squared, due to the complexity were excluded). 3) total prevalence= 12.14– 0.014 (mean$ dem20)+ 0.005 (mean$dem50)– 1.64 (min$ndvi20)+ 1.8 (mean$ndvi20) multiple r-squared: 0.2111, adjusted r-squared: 0.198 f-statistic: 16.06 on 4 and 240 df, p: 1.151e–11 important plots of main model presented in fig. 6. regarding weak results of linear regression (r-squared located near the zero), eventually, additive nonparametric regression analysis, using gam (1generalized additive model) command in library “mgcv”, ap plied to find best parameters and buffers for modeling variables of this study. here two models were fitted: first with the highest percentage of predictability by equation 4, second with the convenience practical application (same parameter mean of variables in unique buffer 10km) which came in equation 5. 4) total prevalence= 7.69+ s (mean$dem10)+ s (max$ndvi50) r-sq. (adj)= 0.524 deviance explained= 54.5% 5) total prevalence= 7.42+ s (mean$dem10)+ s (mean$ndvi10) r-sq. (adj)= 0.371 deviance explained= 39.6% the “s” function, used in specifying the model formula, indicates that each term is to be fitted with a smoothing spline. the surface that fitted on the model and smoothing splines have been shown in fig. 7 and 8. in equation 5, the 5.43 parameters are used for the mean$ndvi10 (mean of 10km buffer for ndvi) term, and 7.98 for the mean$dem10 term, the degrees of freedom for the model is the sum of these plus 1 for the regression constant. fig. 1. distribution of anthroponotic cutaneous leishmaniasis city points in iranian studies from 1976–2013 on 15yr annual average normalized difference vegetation index map. high: active lesion ≥ 2% or active lesion+scar ≥ 20% or incidence ≥ 2%; middle: 2% ˃ active lesion ≥ 0.5% or 20% ˃ active lesion+scar ≥ 5% or 2 ˃ incidence ≥ 0.5%, low: 0.5% ˃ active lesion or 5% ˃ active lesion+scar or 0.5˃ incidence http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 46 http://jad.tums.ac.ir published online: march 18, 2018 table 1. literatures published for prevalence of leishmaniasis, divided by leishmaniasis types and provinces of iran province acl references zcl references zvl references alborz fagih-nayini et al. 2002(23) ardabil mazloumi gavgani et al. 2002(24), soleimanzadeh et al. 1993(25), mohebali et al. 2010(26), edrissian 1996(27), mohebali et al. 2011(28), mahami et al. 2006(29), mohammadi-kheyrabadi et al. 2004(30), nadim et al. 1998(31), arshi et al. 2002(32), mazloumi gavgani et al. 2011(33) azerbaijan, east mazloumi gavgani et al. 2002(24), mohebali et al. 2011(28), mirsamadi et al. 2003(34), mazloumi gavgani et al. 2011(33) azerbaijan, west bushehr yaghoobi-ershadi et al. 2013(35) yaghoobi-ershadi et al. 2013(35), hamzavi et al. 2000(36) mohebali et al. 2001(37) chahar mahaal and bakhtiari fars ghatee et al. 2013(38) razmjou et al. 2009(39), ghatee et al. 2013(38), rassi et al. 2004(40), fakoorziba et al. 2011(41), davami et al. 2010(42), sharafi et al. 2013(43), noorpisheh et al. 2013(44), entezari and eskandari 2014(45) fakhar et al. 2008(46), edrissian et al. 1993(47), sahabi et al. 1992(48), fakhar et al. 2006(49) gilan golestan mollalo et al. 2015(50), rahbarian et al. 2009(51), sofizadeh et al. 2012(52), baghaei et al. 2012(53), mesgarian et al. 2010(54) (fakhar et al. 2014) hamadan hanafi bojd et al. 2006(55), nazari 2012(56) hormozgān azizi et al. 2012(57) ilam asgari nezhad et al. 2012(58), shazad et al. 2005(59), bahrami et al. 2011(60), yazdanpanah and rostamianpur 2013(61), roghani et al. 2012(62) isfahan shiee et al. 2012(63), zahraei-ramazani et al. 2007(64), doroodgar et al. 2012(65) talari et al. 2006(66), yaghoobiershadi et al. 2001(67), mohammadian et al. 1999(68), yaghoobi-ershadi and javadian 1995(69), yaghoobi-ershadi et al. 2006(70), nilforoushzadeh et al. http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/alborz_province https://en.wikipedia.org/wiki/ardabil_province https://en.wikipedia.org/wiki/east_azerbaijan_province https://en.wikipedia.org/wiki/west_azerbaijan_province https://en.wikipedia.org/wiki/west_azerbaijan_province https://en.wikipedia.org/wiki/bushehr_province https://en.wikipedia.org/wiki/chaharmahal_and_bakhtiari_province https://en.wikipedia.org/wiki/chaharmahal_and_bakhtiari_province https://en.wikipedia.org/wiki/fars_province https://en.wikipedia.org/wiki/gilan_province https://en.wikipedia.org/wiki/golestan_province https://en.wikipedia.org/wiki/hamadan_province https://en.wikipedia.org/wiki/hormozgan_province https://en.wikipedia.org/wiki/ilam_province https://en.wikipedia.org/wiki/isfahan_province j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 47 http://jad.tums.ac.ir published online: march 18, 2018 table 1. continued … 2002(71), doroodgar et al. 2009(72), ahmadi et al. 2013(73), ebadi and hejazi 2003(74), yaghoobi-ershadi et al. 1999(75), doroudgar et al. 1996(76) kerman nadim and aflatoonian 1995(77), sharifi et al. 2011(78), sharifi et al. 2015(3), yaghoobi-ershadi 1977(79), mirzaei et al. 2012(80), aflatoonian et al. 2014(81), sharifi et al. 1998(82), sharifi et al. 2011(83), ghatee et al. 2013(38), aflatoonian et al. 2013(84), nadim et al. 1995(85), sharifi et al. 2015(86), aflatoonian and sharifi 2011(87), aflatoonian and sharifi 2010(88), mirzadeh et al. 2008(89), aflatoonian et al. 2014(90), aflatoonian and sharifi 2007(91) sharifi et al. 2015(3), akhavan et al. 2007(92), aghaei-afshar et al. 2013(93), khosravi et al. 2013(94), sharifi et al. 2008(95) mahmoudvand et al. 2011(96), barati et al. 2008(97) kermanshah hamzavi and khademi 2015(98), nazari et al. 2012(99), hamzavi and khademi 2013(100) hamzavi et al. 2012(101) khorasan, north alavinia et al. 2009(102) alavinia et al. 2009(102) torabi et al. 2007(103) khorasan, razavi sattar pagheh et al. 2013(104), moosa-kazemi et al. 2007(105), saadabadi et al. 2013(106), hassanpour et al. 2014(107), hoseini farash et al. 2011(108), khajedaluee et al. 2014(109), mohajery et al. 2008(110) yaghoobi-ershadi et al. 2003(111), hassanpour et al. 2014(107), khajedaluee et al. 2014(109), karimi zarchi et al. 2004(112), sahabi et al. 1999(113), akbari et al. 2014(114) khorasan, south karamian et al. 2013(115) khuzestan kassiri et al. 2012(116), kassiri et al. 2012(117), kassiri et al. 2014(118), kassiri et al. 2014(119), vazirianzadeh et al. 2014(120), kassiri et al. 2013(121), spotin et al. 2014(122), ghasemian et al. 2011(123), kassiri et al. 2013(124), nejati et al. 2014(125), behbahani et al. 2012(126), kassiri et al. 2011(127) kohgiluyeh and boyer-ahmad sarkari et al. 2010(128), sarkari et al. 2007(129) kurdistan http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/kerman_province https://en.wikipedia.org/wiki/kermanshah_province https://en.wikipedia.org/wiki/north_khorasan_province https://en.wikipedia.org/wiki/north_khorasan_province https://en.wikipedia.org/wiki/razavi_khorasan_province https://en.wikipedia.org/wiki/razavi_khorasan_province https://en.wikipedia.org/wiki/south_khorasan_province https://en.wikipedia.org/wiki/khuzestan_province https://en.wikipedia.org/wiki/kohgiluyeh_and_boyer-ahmad_province https://en.wikipedia.org/wiki/kohgiluyeh_and_boyer-ahmad_province https://en.wikipedia.org/wiki/kurdistan_province j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 48 http://jad.tums.ac.ir published online: march 18, 2018 lorestan kheirandish et al. 2013(130) amraee et al. 2013(131), ahmadi et al. 2013(132), kheirandish et al. 2013(130), chegeni-sharafi et al. 2011(133) chegeni-sharafi et al. 2005(134) markazi mazandaran qazvin qom rostami et al. 2013(135), rassi et al. 2013(12), akhavan et al. 2003(136), saghafipour et al. 2013(137), saghafipour et al. 2012(138) (rakhshanpour et al. 2014), (fakhar et al. 2004) semnan mohammadi azni et al. 2010(139), mohammadi azni et al. 2011(140), mohammadi azni et al. 2010(141), rafati et al. 2007(142) sistan and baluchestan fazaeli et al. 2008(143), fazaeli et al. 2009(144), fouladi et al. 2007(145) tehran salehzadeh and seyedi rashti 1996(146), hossein abadi 2004(147) yazd yaghoobi-ershadi et al. 2002(148) yaghoobi-ershadi et al. 2004(149), jaafary et al. 2007(150), mozafari and bakhshi zadeh 2011(151), hanafi bojd et al. 2003(152), yaghoobi-ershadi et al. 2008(153) zanjan multy-provinces mohebali et al. 2006(6) table 2. nonparametric analysis of variances between types of zoonotic, anthroponotic cutaneous, and zoonotic visceral leishmaniasis and environmental variables in iran (p˂ 0.05), buffer = 5km dem_mean dem_variance ndvi_mean* ndvi_variance acl 1216 213046 8.57 3.93 zcl 666 385752 10.12 12.16 zvl 1296 332153 11.93 6.28 *(the range is 1–20 for ndvi) table 3. nonparametric analysis of variances between types of zoonotic, anthroponotic cutaneous, and zoonotic visceral leishmaniasis and epidemiological variables of disease in iran (p˂ 0.05) mean variance acl (%) zcl (%) zvl (%) acl zcl zvl active lesion prevalence 1.48 4.42 na* 3.47 37.97 na total prevalence 11.88 25.02 2.69 125.38 622.39 3.73 incidence 0.76 1.04 1.01 2.62 8.47 1.78 *na: not available table 1. continued … http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/lorestan_province https://en.wikipedia.org/wiki/markazi_province https://en.wikipedia.org/wiki/mazandaran_province https://en.wikipedia.org/wiki/qazvin_province https://en.wikipedia.org/wiki/qom_province https://en.wikipedia.org/wiki/semnan_province https://en.wikipedia.org/wiki/sistan_and_baluchestan_province https://en.wikipedia.org/wiki/sistan_and_baluchestan_province https://en.wikipedia.org/wiki/tehran_province https://en.wikipedia.org/wiki/yazd_province https://en.wikipedia.org/wiki/zanjan_province j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 49 http://jad.tums.ac.ir published online: march 18, 2018 table 4. multiple linear regression models for types of zoonotic, anthroponotic cutaneous, and zoonotic visceral leishmaniasis and digital elevation model subcategory (buffer distance around the cities or villages) dem 5km_total dem 5km_acl dem 5km_zvl dem 5km_zcl buffer types buffer types_acl buffer types_zvl buffer types_zcl active lesion prevalence ~mean+max ar*: 0.1004 ~mean+min ar: 0.284 na*** ~mean ar: 0.077 ~mean20km ar: 0.035 ns ns ~mean5km ar: 0.077 total prevalence ~max ar: 0.1028 ~mean ar:0.1649 ~max ar:0.0484 ~mean+max ar: 0.234 ~mean20km ar: 0.1178 ns ~mean50km ar: 0.029 ~mean20km ar: 0.25 incidence ~mean+max ar: 0.029 ns** ~min ar:0.2954 ~max+majority ar: 0.077 ~mean5km ar: 0.011 ns ~mean20km ar: 0.21 ~mean5 ar: 0.031 *ar: adjusted r-squared **ns: not significant ***na: not available fig. 2. distribution of zoonotic cutaneous leishmaniasis rural region points in iranian studies from 1991–2013. high: active lesion ≥2% or active lesion+scar ≥20% or incidence ≥ 2%, middle: 2% ˃ active lesion ≥ 0.5% or 20% ˃ active lesion+scar ≥ 5% or 2˃ incidence ≥ 0.5%, low: 0.5% ˃ active lesion or 5% ˃ active lesion+scar or 0.5 ˃ incidence fig. 3. distribution of zoonotic visceral leishmaniasis county region points in iranian studies from 1989–2012 on digital elevation model map. high: dat ≥ 2% or incidence ≥ 1%, middle: 2% ˃ dat ≥ 0.5% or 1 ˃ incidence ≥ 0.1%, low: 0.5% ˃ dat or 0.1 ˃ incidence http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 50 http://jad.tums.ac.ir published online: march 18, 2018 table 4. multiple linear regression models for types of zoonotic, anthroponotic cutaneous, and zoonotic visceral leishmaniasis and digital elevation model subcategory (buffer distance around the cities or villages) dem 5km_total dem 5km_acl dem 5km_zvl dem 5km_zcl buffer types buffer types_acl buffer types_zvl buffer types_zcl active lesion prevalence ~ mean+max ar*: 0.1004 ~ mean+min ar: 0.284 na*** ~ mean ar: 0.077 ~ mean20km ar: 0.035 ns ns ~ mean5km ar: 0.077 total prevalence ~ max ar: 0.1028 ~ mean ar:0.1649 ~ max ar:0.0484 ~ mean+max ar: 0.234 ~mean20km ar: 0.1178 ns ~mean50km ar: 0.029 ~mean20km ar: 0.25 incidence ~mean+max ar: 0.029 ns** ~ min ar:0.2954 ~ max+majority ar: 0.077 ~ mean5km ar: 0.011 ns ~ mean20km ar: 0.21 ~ mean5 ar: 0.031 *ar: adjusted r-squared **ns: not significant ***na: not available 0 500 1000 1500 2000 2500 0 5 1 0 1 5 2 0 2 5 3 0 3 5 plot of active ulcer prevalence and 5kmdem mean of dem u lc e r p re v a le n c e o f li e s h m a n ia s is -3 -2 -1 0 1 2 3 0 5 0 0 1 0 0 0 2 0 0 0 normal probability plot of lieshmaniasis incidence and 5kmdem mean theoretical quantiles s a m p le q u a n ti le s -3 -2 -1 0 1 2 3 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 normal probability plot of zcl prevalence and 5kmdem mean theoretical quantiles s a m p le q u a n ti le s -3 -2 -1 0 1 2 3 0 2 0 4 0 6 0 8 0 1 0 0 normal probability plot of total prevalence and 5kmndvi mean theoretical quantiles s a m p le q u a n ti le s fig. 4. typical plots and normality plots of independent and dependent variables acl vl zcl 0 5 0 0 1 0 0 0 2 0 0 0 boxplot of leishmaniasis types vs mean of dem leishmaniasis type m e a n o f 5 k m b u ff e r d e m acl vl zcl 5 1 0 1 5 2 0 boxplot of leishmaniasis types vs mean of ndvi leishmaniasis type m e a n o f 5 k m b u ff e r 1 5 y e a r n d v i fig. 5. boxplots for compares of means of 5km-buffer digital elevation model and normalized difference vegetation index ((in the range of 1–20)) for anthroponotic cutaneous leishmaniasis, zoonotic cutaneous leishmaniasis, and zoonotic visceral leishmaniasis in iran for study period (1976–2013) http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 51 http://jad.tums.ac.ir published online: march 18, 2018 -10 0 10 20 30 -2 0 2 0 6 0 fitted values r e s id u a ls residuals vs fitted 900 905 151 -3 -2 -1 0 1 2 3 0 2 4 6 theoretical quantiles s ta n d a rd iz e d r e s id u a ls normal q-q 900 905 151 -10 0 10 20 30 0 .0 1 .0 2 .0 fitted values s ta n d a rd iz e d r e s id u a ls scale-location 900 905 151 0.00 0.02 0.04 0.06 0.08 0.10 -2 0 2 4 6 leverage s ta n d a rd iz e d r e s id u a ls cook's distance 0.5 residuals vs leverage 900 870 880 fig. 6. plots of final linear regression model for leishmaniasis and environmental variables (digital elevation model and normalized difference vegetation index) m ean of 10km d e m 0 1000 2000 3000 m ea n of 1 0k m n d v i 5 10 15 20 fitte d p re v a le n c e -20 0 20 40 gam(tot_prev~dem$mean10+ndvi$mean10) fig. 7. fitted surface for the additive nonparametric regression of total prevalence of leishmaniasis on mean of 10km buffer for digital elevation model and normalized difference vegetation index http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 52 http://jad.tums.ac.ir published online: march 18, 2018 table 5. multiple linear regression models for disease mode and normalized difference vegetation index subcategory (buffer distance around the cities or villages) ndvi 5km_total ndvi 5km_acl ndvi 5km_zvl ndvi 5km_zcl buffer types active lesion prevalence ~mean+majority ar*: 0.0338 **ns ***na ns ~max50km ar: 0.093 total prevalence ~min ar: 0.012 ns ~mean ar:0.39 ~ min + minority ar: 0.3479 ~ min5km ar: 0.012 incidence ~mean ar: 0.0085 ~ minority ar: 0.042 ns ~median ar: 0.0259 ~mean20km ar: 0.007 *ar: adjusted r-squared **ns: not significant ***na: not available 0 500 1000 1500 2000 2500 3000 3500 -3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 prevalence of leishmaniasis on mean of 10km buffer digital elevation model mean of 10km dem s (m e a n $ d e m 1 0 ,7 .9 8 ) 5 10 15 -3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 prevalence of leishmaniasis on mean of 10km buffer normalized difference vegetation index model mean of 10km ndvi s (m e a n $ n d v i1 0 , 5 .4 3 ) fig. 8. partial-regression functions for the additive regression of total prevalence of leishmaniasis on mean of 10km buffer digital elevation model and normalized difference vegetation index. the broken lines give point-wise 95percent confidence envelopes around the fit discussion maps based on the actual data for distribution of all leishmaniasis types in iran, with identifying the detailed variables of environmental factors affecting the disease distribution, are the most important findings of this study. due to the limited access to official leishmaniasis data of center for disease control, integration all data from both cl types and rural and urban foci in the heart of the county official reports (lack of separation species), tens of kilometers distance between some foci and center of counties often identified as disease points (lack of spatial resolution), unknown types of cl in most of official data, and passive screening in public health centers, we decided to use the real data available in the literatures with a detailed focus on disease points. many of scientific epidemiological studies predominantly focus on foci of diseases and have very valuable and verified information about all aspects of http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 53 http://jad.tums.ac.ir published online: march 18, 2018 the disease, such as prevalence, incidence, vectors, reservoirs, and control of disease data. maps that contain all of the scientific information investigate by researchers of a country over several decades could well represent the disease situation in that country. although the nature of the leishmaniasis itself was not the aim of our study, however, distribution of disease to the breakdown mapping can present more accurate view of disease for researchers. though vegetation statistically has a significant relationship with the prevalence of vector-borne diseases, this study (especially for the simple linear regression models), with a weak confirmation, implicitly point to a complex and indirect relationship. therefore, more detailed studies and comprehensive investigations would be needed to prove the theorem. a study on effect of minimum, maximum and mean ndvi on the distribution of the vl vector in district of bihar, india showed that max and mean variables with an r2 about 0.6 strongly associated with the disease (14). werneck et al. with a mention to the ndvi, emphasized on the impact of multilevel variable modeling on the development of leishmaniasis in brazil (154). study of environmental factors influencing the distribution of vectors involved in transmission of disease in north-eastern italy revealed that areas with high winter ndvi may be related to the survival of the larvae in moist soil (155). according to the results of relationship between cl and ndvi in northeast province of iran, golestan, arid and semiarid regions with low vegetation are the most involved area for cl occurrence (11). in our study, as expected due to their rural and nomadic natures of zcl and zvl in iran, the values for ndvi index increase for acl, zcl, and zvl respectively (table 2). there is not much variation among them, and all three types are almost inclined to intermediate vegetation (neither too sparse nor too dense). the distinction in the result of numerous studies would be aroused from differences between climate factors, socio-economic variables, instrumental confounders and differences in the interpretation of the results. incidence of various types of leishmaniasis in different heights may result from the diversity in the reservoirs and vectors of the disease. whereas acl and zvl had an interest for presence in high altitude areas, zcl have been distributed in lower altitude (table 2). this result for zvl has obtained also in ilam province spatial modeling, and elevation had a negative impact on cl prevalence (156). multivariate analysis in a vl study in eastern sudan found the elevation as important variables, while the primary analysis did not show any correlation with disease incidence (157). however, while dem can use as a good variable for topographic purposes, it may do not has most appropriate function as elevation in all regions (155). the role of environmental factors in the development of disease vectors is obvious. while the cl is the main vector-borne disease in iran, about 80% of cases are zoonotic and spread in 17 out of 31 provinces (158). some studies have been modeled the environmental factors for vectors and reservoirs of leishmaniasis. altitude and land cover was found to be from most important factors for suitability of niches for sand flies. areas with 990m were found and 1235m average altitude were related to probability of presence of zcl and acl vectors respectively (159). niche modeling of main reservoir hosts in iran showed that among topography variables, slope has the most contribution in forecasting risk of disease (160). a predictive degree-day model was used for development time, population dynamics and activities of vl vector sandflies in field in northwest iran (161). among climate factors, minimum of temperature, mean of humidity, and rainfall had the most impact on zvl distribution in golestan, north-east of iran (162). none of these studies include ndvi as a variable. the vegetation coverage has been discussed as a risk factor for vl and cl types http://jad.tums.ac.ir/ j arthropod-borne dis, march 2018, 12(1): 41–66 a golpayegani et al.: modeling of … 54 http://jad.tums.ac.ir published online: march 18, 2018 in previous studies (163, 164). the ndvi has not considered for intrinsic specifications itself but also it covers some other important environmental and ecological variables such as soil types and moisture, humidity, slope degree and even elevation of its ambient land (16). the majority of studies, evaluated the presence/absence of vegetation or have used a cut-off value point for ndvi, but less attention has been paid to the sheer numerical modeling. linear regression results in this study show that none of subcategories of independent variables have a favorable associated with dem and ndvi and do not fit on the linear model very well (low r-squared). only some of models when focused on leishmaniasis types, the r-squared reach to 0.2– 0.4. this fact could be predicted from plots associated with variables (fig. 4). despite the lack of goodness of fitness of simple linear models, additive nonparametric regression analysis presents a better prediction model for data of this study. these models do not output real intercepts for variables, but using their internal formulas, they are able to provide predictive models for input data. as a nonparametric regression attributes, no obvious estimates were seen for parameters (21), and then to figure out regression results, regards to necessitate of using fitted regression graphics, we used this method as shown in fig. 7. data frame used to find predicted values on the regression surface, fitted to data of model to drawn this figure. for more clarification, functions which explained previously that present in inherent content of the additive regression, have been shown in partial regression of our model in fig. 8. conclusion although the results of modeling and predictive power of the models in this study was not great but was somewhat satisfied. modeling environmental factors which affecting ecological disease, need attention to the role of all these factors together. moreover, integration the scenarios such as finding hotspots in gis, using statistical logistic regression, more specific factors of diseases such as vector and reservoir species, can be used in modeling the relationships, more meaningful and more clear. however, a good model needs to consider all the factors involved the prevalence and incidence of a disease. acknowledgements the authors would like to thank financial grant supporting this study by national institute of health research, tehran university of medical sciences, contract no: 241/m/9644 and also by sabzevar university of medical sciences, as well. the ndvi raster maps (mod13q1 series) version 5 and 100m aster global dem version 2 were retrieved from the online modis reprojection tool and online aster gdem data product of meti and nasa respectively, courtesy of the nasa eosdis land processes distributed active archive center (lp daac), usgs/ earth resources observation and science (eros) center, sioux falls, south dakota. the authors declare that there is no conflict of interests. references 1. cerbino neto j, werneck gl, costa chn (2009) factors associated with the incidence of urban visceral leishmaniasis: an ecological study in teresina, piauí state, brazil. cadernos de saúde pública. 25(7): 1543–1551. 2. who (2016) leishmaniasis fact sheet no. 375. world health organization, geneva, switzerland. available at: http://www.who.int/mediacentre/fact sheets/fs375/en/. 3. sharifi i, aflatoonian mr, fekri ar, hakimi parizi m, aghaei afshar a, http://jad.tums.ac.ir/ 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andrade-filho jd, sousa amd, morais mhg, rocha ams, machado-coelho gll, lima fp, madureira ap, garcia tc (2015) an integrated approach using spatial analysis to study the risk factors for leishmaniasis in area of recent transmission. bio med res int. 2015: 621854. http://jad.tums.ac.ir/ j arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 18 original article comparative study on the effectiveness of coumavec® and zinc phosphide in controlling zoonotic cutaneous leishmaniasis in a hyperendemic focus in central iran a veysi 1, h vatandoost 1, mr yaghoobi-ershadi 1, mh arandian 2, r jafari 2, m hosseini 3, h abdoli 2, y rassi 1, k heidari 4, a sadjadi 4, r fadaei 4, j ramazanpour 4, k aminian 4, mr shirzadi 5, *aa akhavan 1,6 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2esfahan training and health research center, national institute of health research, tehran university of medical sciences, esfahan, iran 3department of epidemiology and biostatistics, school of public health, tehran university of medical sciences, tehran, iran 4esfahan health center, esfahan university of medical sciences, esfahan, iran 5department of zoonosis, cdc, ministry of health and medical education, tehran, iran 6institute for environmental research (ier), tehran university of medical sciences, tehran, iran (received 30 dec 2011; accepted 3 jan 2012) abstract background: zoonotic cutaneous leishmaniasis (zcl) is an increasing health problems in many rural areas of iran. the aim of this study was to introduce a new alternative rodenticide to control the reservoirs of zcl, its effect on the vector density and the incidence of the disease in hyperendemic focus of esfahan county, central iran. methods: the study was carried out from january 2011 to january 2012. in intervention areas, rodent control operation was conducted using zinc phosphide or coumavec®. active case findings were done by house-to-house visits once every season during 2011–2012. to evaluate the effect of rodent control operation on the vector density, sand flies were collected twice a month using sticky traps. results: the reduction rate of rodent holes in intervention areas with coumavec® and zinc phosphide were 48.46% and 58.15% respectively, whereas in control area results showed 6.66 folds intensification. the incidence of zcl significantly reduced in the treated areas. totally, 3200 adult sand flies were collected and identified in the intervention and control areas. in the treated area with zinc phosphide, the density of phlebotomus papatasi was higher in outdoors in contrast with the treated area by coumavec® which the density of the sand fly was higher in indoors. conclusion: both rodenticides were effective on the incidence of zcl and the population of the reservoirs as well. coumavec® seems to be effective on the outdoor density of the vector. this combination of rodenticide -insecticide could be a suitable alternative for zinc phosphide while bait shyness or behavioral resistance is occurred. keywords: rodent control, zinc phosphide, coumavec®, zoonotic cutaneous leishmaniasis, iran introduction zoonotic cutaneous leishmaniasis (zcl) is an increasing health problem in many rural areas of iran, which involves17 out of 31 provinces of the country (akhavan 2011). rhombymos opimus, the great gerbil, and meriones libycus, libiyan jird, are the main and secondary reservoir hosts of the disease in northeast and central part of iran (yaghoobi*corresponding author: dr amir ahmad akhavan, e-mail: aaakhavan@tums.ac.ir j arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 19 ershadi and javadian. 1996, yaghoobi-ershadi et al. 1996). due to improvement of reporting system by the ministry of health and medical education of iran, natural disaster such as earthquake, urbanization, constructions of buildings near colonies of the rodents , immigration of non-immune people to the endemic areas, a sharp increase of cases have been reported since 2004 (shirzadi 2010, saidi et al. 2012). phlebotomus papatasi is the main vector of zcl and leishmania major is the causative agent of the disease in the area. the parasite has been isolated and identified from naturally infected p. papatasi, p. caucasicus, r.opimus, m. libycus and human in this endemic area (yaghoobi-ershadi et al. 1996 and 2001, yaghoobi-ershadi 2008, akhavan et al. 2010a, 2010b, 2010c). in the recent years despite of significance progresses on leishmaniasis studies such as biochemistry and molecular biology of the parasite and host immune response, a little information is available about the effective control operations of the disease. to control the disease a successful leishmanization has been carried out in iran during complex emergency, but for a very rare rate of immunodeficiency of the recipients, it has been quit except for military personnel in very high risks areas (nadim et al. 1983). in an intervention study, it was shown that autoclaved leishmania major (alm) vaccine with bcg has not been protective against zcl (momeni et al. 1999). global efforts to develop an effective vaccine to prevent leishmaniasis so far showed no success (noazin et al. 2008, 2009, who 2010). in karshinskaya steppes, uzbekistan, attempts to control of great gerbil using destroying their burrows by plowing or crushing with heavy machines within 2 to 3 km radius of the towns in a three-year period was carried out. the results showed that zonal control of great gerbils was inadequate due to re-invasion of the gerbils to control areas. a massive operation to eradicate great gerbils and their burrows was carried out over an enormous area surrounded by mountain ranges and rivers. the results showed that the operation caused significant reduction in sand flies density and also no cases of zcl was reported within at least 4 years after the operation (sergiev 1978, eliseev 1980). in central asia a large-scale operation against great gerbils, using poisoned baits eliminated the rodents successfully but had no effect on the density of p. papatasi (dergacheva and zherikhina 1980). from april to january 1997 a field trail was carried out to control zcl by destruction rodent burrows and using 2.5% zinc phosphide baits in radius of 500 meters from houses once a month in may, june, july and september in an endemic focus of iran. the results showed that the control program reduced the incidence of zcl 12-folds in treated village compared to the control village at the end of the first year of operation and 5-folds at the end of the second year (yaghoobi-ershadi et al. 2000). at the same intervention area from 1999 to 2002, the numbers of active burrows were counted in may and october and baited with zinc phosphide, if the rodent hole numbers increased more than 30%. the results showed that changes in the numbers of rodent burrows during the intervention were statistically significant. furthermore significant difference in incidence rate of zcl between the intervention and control villages has been shown (yaghoobi-ershadi et al. 2005). recently some behavioral resistance and/or bait shyness against zinc phosphide among the great gerbil population has been reported from some endemic areas of the disease (personal communication, esfahan health center, iran). it is necessary to introduce some new effective alternative rodenticides to control the reservoir hosts and subsequently the disease in endemic areas of zcl in iran. j arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 20 the aim of the current study was to introduce a new alternative rodenticide for controlling zcl in iran. in this survey the effect of coumavec® (batch no: cou/204161, pro: 07/2010, exp: 07/2013) a mixture of coumatetralyl 0.5% and etofenprox 0.5% on the reservoir host, vector density and the disease incidence were compared with 2.5% zinc phosphide(batch no: z/t/36, pro: 10/2010, exp: 10/2013) bait for the first time in the world. materials and methods study area the present study was carried out in 4 villages (mazraehshoor, gishi, vartoon and parvaneh-aliabadchi), 45 to 85 km far from esfahan city, esfahan province, iran from january 2011 to january 2012. mazraehshoor (32˚39˙54.84”n/ 52˚08˙07.38”e) and gishi (32˚29˙24.07”n / 52˚21˙47.06”e) were selected as intervention areas for coumavec® and zinc phosphide respectively; vartoon (32˚50˙11.48”n/ 52̊ 06˙45.93”e) and parvanehaliabadchi (32̊ 47˙44.36”n/ 51˚58˙27.19”e) were selected as control areas. the selected areas have desert climate, hot in summer and cold in winter. in 2010, the maximum and minimum mean monthly temperature was 39.1˚ c and -1.6˚ c in july and december, respectively. the total rainfall was 72.2 mm. the minimum mean monthly relative humidity was 7% in july and the maximum was 82% in january. rodent control operation to determine the appropriate bait concentration of coumavec® a trial was conducted under laboratory condition. poisoned baits were prepared using a mixture of wheat grain and four concentration of coumavec® (0.03,0.0625, 0.125 and 0.25). each treatment included 19 great gerbils. four groups were treated with the poisoned baits of one concentration. the control group only was offered with the wheat grain. the zinc phosphide bait concentration was selected according to the previous studies (yaghoobiershadi et al. 2000, 2005). in early april 2011, prior to the beginning of the active season of sand flies, rodent holes were counted in each village. the holes were destroyed by digging. all the activities were conducted in a radius of 500 m from houses around all villages. after 48 hours of the operation, all areas were visited and the opened holes were counted again. in treated areas, the gerbil colonies were baited by coumavec® or zinc phosphide baits and then closed. approximately 12–15 gm of the poisoned baits was placed into each burrow at a depth of about 10 cm. the operational areas were revisited after a week and the reopened burrows in treated areas were counted, baited, and closed. rodent control operations in intervention areas, the village of mazraehshoor (treated with coumavec®) and gihi (treated with zinc phosphide), were repeated once a month in june, july and august. the date of baiting and the number of reopened burrows were recorded. no control operation was done in the village of vartoon (control), but the numbers of reopened holes were also counted and recorded in each interval visit to compare with intervention areas. entomological monitoring to evaluate the effect of rodent control operation on the vector density, an entomological survey was performed. in each village three fixed houses were selected and sand flies were collected by 30 sticky traps (papers impregnated with castor oil) twice a month from the beginning (april) to the end (october) of active season of sand flies. the sticky papers were set before sunset and colj arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 21 lected in the following morning. the collected phlebotomines were washed with absolute acetone and preserved in 70% ethanol. microscopic slides were prepared using pauri's medium (smart et al. 1965) and identified by morphologic characters (theodor and mesghali 1964, seyedi-rashti and nadim 1992). the same procedure was also done for collecting and identifying sand flies from outdoor resting places. human infection to evaluate the rodent control operation on the incidence of the disease, active case findings were done before and after intervention in treated (mazraehshoor and gishi) and control (vartoon and parvaneh-aliabachi) villages by house to house visits in january 2011 and once every season in 2012. in treated villages 150 households and in controls all inhabitants (less than 150 households) were visited. a questioner including identification information of the people, presence or absence of scar (s) or active lesion (s), number of the lesion (s) or scar (s), and traveling history to the other zcl foci, was filled out for each household. persons who had experience of traveling to other endemic foci of zcl were excluded from the study. only new cases of zcl and number of patient with active lesion were recorded on each visit. yearly incidence of the disease in both treated and control villages were calculated at the end of 2011 and 2012. in the calculation of the disease yearly incidence, the persons with previous scars were excluded from at risk population. statistical analysis the data were analyzed using stata 11.0 and spss 11.5 and graphs were prepared using excel. chi-squared and fisher's exact tests were used to compare the rodent holes changes and incidence of the disease in intervention and control areas. the kruskalwallis non-parametric test was also used to compare the density of sand flies in the areas. results the appropriate bait concentration of coumavec® was 0.125%. table 1 shows the effect of poisoned baits on rodent holes density in treated and control areas. the treated area of mazrehshoor (coumavec® intervention area) was around 212 hectares and the total number of holes before treatment was 11023 (52 per hectare). after 48 hours of destruction the rodent colonies 1296 (6.1 active holes per hectare) of the holes were reopened. each reopened hole were baited and closed again. after one week of control activity, the number of reopened holes increased to 1557. in june, july and august the number of reopened holes were counted 724, 644 and 668 respectively. the treated area of gishi (zinc phosphide intervention areas) was around 193 hectare. the numbers of holes before the treatment were 4729 (24.5 per hectare). after 48 hours of digging the burrows, the numbers of reopened holes were 1682 (8.7 active holes per hectare). the reopened holes were baited and closed. after one week, the number of holes decreased to 600. in june, july and august the number of holes were counted 493, 424 and 704 respectively. the control area (vartoon) was aroumd 173 hectare; the number of holes before treatment was 2297 (13.3 per hectare) and 48 hours after destruction 196 (1.1 active holes per hectare) number of these holes were reopened. the reopened holes in this village were not baited or closed. the number of reopened holes increased to 281 after one week. in june, july and august the number of holes increased to 365, 557 and 1306 respectively. the number of holes in each stage in control village showed an inj arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 22 creasing trend compared to the intervention areas. the reduction rate of rodent burrows was 48.46% in mazraehshoor (treated with coumavec®) and 58.15% in gishi (treated with 2.5% zinc phosphide). chisquared test showed that the decreasing rate of rodent holes between the treated villages with coumavec® and zinc phosphide were statistically different (p< 0.000001). the rodent burrows in control area (vartoon) after the rodent control operation increased 6.66 folds in comparison before the operation (table 1). during may to october 2011, a total of 3200 adult sand flies (2054 from outdoors and 1146 from indoor resting places) were collected. the following three species were collected from indoors: p. papatasi (92.5%), s. sintoni (7.1%), p. sergenti (0.4%). in outdoor resting places p. papatsi (95.8%), s. sintoni (3.3%), p. sergenti (0.5%), p. ansari (0.2%), p. mongolensis (0.2%) were found. in all places, sand flies started to appear in the late april and disappeared in the late october. phlebotomus papatasi was the common and predominant species in both indoor and outdoor resting places. the monthly density of p. papatasi in both treated areas with coumavec® and zinc phosphide was compared (fig. 1 and 2). the outdoor density of p. papatasi, the main vector of the disease, in the village treated with coumavec® was lower than indoors while the density of the sand fly in the village treated with zinc phosphide was higher in outdoors. there was no statistical difference between the density of the vector in indoors and outdoors in intervention and control areas (p> 0.05). table 2 shows the yearly incidence of the disease in the treated and control villages. the incidence was calculated at 32.92 and 39.14 per thousand in mazaehshoor and gishi (intervention areas) and also 18.40 and 76.19 per thousand in vartoon and parvanehaliabadchi (control areas), in 2011 respectively. after intervention, the incidence of the disease reduced to 4.34 and 11.4 per thousand (13.2% and 29. 1% of its original level before the intervention) in mazaehshoor and gishi (treated villages) respectively. none-significant difference was observed between reduction rate of the disease incidence between coumavec® and zinc phosphide treated areas (p> 0.05). as the tabel 2 shows, the incidence of zcl decreased in all intervention and control areas but the statistical analysis of the zcl incidence showed that, the incidence reduction of zcl in treated areas before and after the intervention were statistically different (p< 0.05) while no statistical differences was observed in the control areas (p> 0.05). table 1. comparison of the number of rodent holes in the intervention and control villages, esfahan county, esfahan, iran, 2011 place treated area (hectare) may 2011 before treatment may 2011, 24 hour after burrow destruction may 2011, one week after first baiting june 2011 july 2011 august 2011 mazraehshoor(inter vention area with coumavec®) 212 11032 1296 1557 724 644 668 gishi(intervention area with zinc phosphide) 193 4729 1682 600 493 424 704 vartoon(control area) 173 2297 196 281 365 557 1306 j arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 23 table 2. comparison of the incidence (per thousand) of zoonotic cutaneous leishmaniosis in the intervention and control villages, esfahan county, esfahan, iran, 2011–2012 2011 2012 name of the village no. with active leision (s) incidence no. with active leision (s) incidence mazreahshoor (intervention area with coumavec®) 8 32.92 1 4.34 gishi (intervention area with zinc phosphide) 11 39.14 3 11.40 vartoon (control area) 3 18.40 2 10.92 parvaneh-ali abdchi (control area) 8 76.19 5 49.5 fig. 1. monthly fluctuation of phlebotomus papatasi in treated village with coumavec® (mazraeshoor), esfahan county, esfahan province, iran, 2011 fig. 2. monthly fluctuation of phlebotomus papatasi in treated village with zinc phosphide (gishi), esfahan county, esfahan province, iran, 2011 discussion the main measures for zcl control in iran are rodent control operation using zinc phosphide bait, impregnated bed nets and curtains with pyrethroids, repellents, indoor residual spraying, health education to the community, and during emergency complex situation leishmanization was also used. global efforts to develop an effective vaccine to prevent leishmaniasis so far showed no success (noazin et al. 2008, 2009, shirzadi 2010, who 2010, saidi et al. 2012). according to the data released by zoonosis department, ministry of health, and medical education of iran, the trend of the disease is increasing during the last decade. there are several reasons for this increase which can be summarized as follows: improvement of surveillance system in the country, monitoring of the disease throughout the year, migration of people from nonendemic regions into the disease foci, presence of different reservoirs for zcl and their migration to non-endemic areas, defect of proper prevention and control operation j arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 24 of the disease and vector control (shirzadi 2010, saidi et al. 2012). this evaluation showed that the control operation was effective for decreasing the population of gerbils and incidence of the zcl in both treated areas. both rodenticides were effective to control the rodent population. in treated area with coumavec®, the operation did not show any effect after one week of baiting, while in the area treated with zinc phosphide the number of rodent holes decreased to 35.67%. this different effect of these rodeneicide is attributed to their mode of action, coumavec® is an anticoagulant, which affects on rodents gradually within 1–2 weeks but zinc phosphide has rapid effect on the rodents. after finishing all stages of rodent control operation, the reduction rate of rodent burrows was 48.46% in the village treated with coumavec® and 58.15% in the village treated with zinc phosphide. the number of rodent burrows in control area (vartoon) with no control operation increased 6.66 folds by the end of the study. results showed that the number of rodent holes in the intervention areas had decreasing trend while in the control area had increasing trend. the incidence of the disease decreased in the treated villages, therefore both rodenticides can be effective to reduce incidence of zcl. the incidence of the disease from 2011 to 2012 showed a decreasing trend in all studied areas (both intervention and control areas) but it is interesting to point that the reduction rates of zcl in treated areas were significantly different but in control areas non-significant differences were observed. it seems that although both pesticides are effective on the control of the rodents but coumavec® is less effective than zinc phosphide. using these two different rodenticides depends on the field and disease situation. if the population of reservoir host is very high and the aim is reduction their population in short term, the use of zinc phsphide is recommended, but if the aim is keeping the population in low density during a complete control operation coumavec® seems to be also effective. in the case of requirement of incidence reduction coumavec® can be an appropriate alternative for zinc phosphid, if necessary. in a previous study, along april to january 1997 a field trail was carried out to control zcl by destruction rodent burrows and using 2.5% zinc phosphide baits in radius of 500 meters from houses once a month in may, june, july and september in central iran. the results of this study showed that, the control program reduced the incidence of zcl 12-folds in treated village compared to the control village at the end of the first year of operation and 5-folds at the end of the second year (yaghoobi-ershadi et al. 2000). at the same intervention area, from 1999 to 2002, the numbers of active burrows were counted in may and october and were baited with zinc phosphate if the rodent holes numbers increased more than 30% at the same intervention area. the results showed that changes in the numbers of rodent burrows along the time in the intervention and control villages were statistically significant. furthermore, significant difference in incidence of zcl between the intervention and control village has been shown (yaghoobi-ershadi et al. 2005). in mazrehshoor, the numbers of the captured sand flies in outdoors were lower than indoor resting places. it seems that coumavec® affects the density of p. papatasi in outdoors. in contrast, in gishi the number of collected sand flies in indoors was lower than outdoor resting places. comparison of p. papatasi density trend in control and treated villages showed that rodents control operation has no significantly effect on the p. papatsi density. j arthropod-borne dis, 2012, 6(1): 18–27 a veysi et al.: comparative study on the … 25 it is concluded that both rodenticides were effective for controlling zcl and rodent population as well. coumavec® could be an appropriate alternative for zinc phophide especially when rodent behavioral resistance or bait shyness is observed. acknowledgements authors wish to appreciate the staff of esfahan province health centre, esfahan university of medical sciences (eums), for their kind collaboration in field operation. we extend our sincere thanks to the staff of esfahan health research centre, national institute of health research, tehran university of medical sciences (tums) for their collaboration along the study. in addition, the authors are grateful to levant overseas development (lod), ltd, france for providing and sending coumavec® to achieve this project. this research was financially supported by school of public health, tums,, institute for environmental research (ier), tums, esfahan province health centre, eums and department of zoonosis, cdc, ministry of health and medical education, islamic republic of iran . the authors declare that they have no conflicts of interest. references akhavan aa (2011) immune response of great gerbil against phlebotomus papatasi saliva. lap lambert academic publishing, saarbrücken, germany. akhavan aa, mirhendi h, khamesipour a, alimohammadian mh, rassi y, bates p, kamhawi s, 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the control of zoonotic cutaneous leishmaniosis in badrood, iran. ann saudi med. 20(5– 6): 386–389. yaghoobiershadi mr, javadian e (1996) epidemiological study of reservoir hosts in an endemic area of zoonotic cutaneous leishmaniasis in iran. bull who. 74(6): 587–590. yaghoobiershadi mr, zahraeiramazani ar, akhavan aa, jalali-zand ar, abdoli h, nadim a (2005) rodent control operations against zoonotic cutaneous leishmaniasis in rural iran. ann saudi med. 25(4): 309–312. j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 232 http://jad.tums.ac.ir published online: september 30, 2018 original article richness and diversity of phlebotomine sand flies (diptera: psychodidae) in north khorasan province, northeast of iran kourosh arzamani 1, 2, *hassan vatandoost 1, 3, *yavar rassi 1, amir ahmad akhavan 1, mohammad reza abai 1, mohammad alavinia 2, 4, kamran akbarzadeh 1, mehdi mohebali 5, sayena rafizadeh 6 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran 3department of environmental chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4toronto rehabilitation centre, university health network, toronto, canada 5department of medical parasitology, school of public health, tehran university of medical sciences, tehran, iran 6ministry of health and medical education, national institute for medical research development, tehran, iran (received 16 oct 2017; accepted 09 jun 2018) abstract background: we aimed to determine the species composition, richness and diversity indices of the phlebotomine sand flies at different topographic condition in visceral (vl) and cutaneous leishmaniasis (cl) foci in the northeast of iran. methods: this cross-sectional study was conducted during 2016 in north khorasan province. the sampling was focused on rural regions, where human cases of vl and/or cl were diagnosed and reported during last 5 years. sand flies were collected three times each twenty days during peak periods of seasonal activity. seven collection methods were used. some alpha and beta diversity indices were calculated. results: overall, 7253 sand flies were collected and identified. they were from 19 species of phlebotominae sandflies. phlebotomus sergenti and ph. papatasi were the most prevalent (84.9%) species in the study area. species richness (s) was very different in three areas and were18, 8, and 4 respectively but evenness (e) were 0.357, 0.345, and 0.380, so evenness was almost equal in the study areas. shannon index (h ) and margalef richness index were calculated 1.033, 0.718, 0.527 and 2.117, 0.8998, 0.4006 respectively. conclusion: the sand fly fauna in north khorasan province was very rich and often included some of the most important proven or suspected vectors of leishmaniasis. species diversity indices (shannon index, and simpson’s index) were not high due to decreasing in evenness. the margalef richness index could accurately reflect the biodiversity of sand flies between three subtidal locations. keywords: richness, diversity, sandflies, leishmania, iran introduction phlebotominae sandflies (diptera: psychodidae: phlebotominae) are widespread in the tropical and subtropical regions. they may carry and transmit etiologic agents of arboviruses and bartonellosis but the most important agents that they transmit are leishma nia, protozoan parasites (1). there are three main forms of leishmaniases, visceral leishmaniasis (vl), various forms of cutaneous leishmaniasis (cl), and mucocutaneous leishmaniasis. endemic leishmaniasis transmission has been reported from at least 98 countries *corresponding authors: dr hassan vatandoost, email: hvatandoost1@yahoo.com, vatando@tums.ac.ir, dr yavar rassi, e-mail: rassiy@tums.ac.ir http://jad.tums.ac.ir/ https://www.researchgate.net/profile/mohammad_reza_abai2 mailto:hvatandoost1@yahoo.com mailto:vatando@tums.ac.ir j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 233 http://jad.tums.ac.ir published online: september 30, 2018 and three territories on five continents. cutaneous leishmaniasis is more widely distributed and approximately 0.7 to 1.2 million new cases occur each year. ten countries with highest estimated cl cases are afghanistan, algeria, colombia, brazil, iran, syria, ethiopia, north sudan, costa rica and peru, together account for 70% to 75% of global estimated cl incidence (2). approximately 800 species of phlebotomine sand flies are distributed in the world. in iran, 47 species of sand flies have been identified and the presences of some species are doubtful (3–5). cl is reported from all provinces and is endemic in more than half of the 31 provinces in iran. vl is endemic in northwest, west, southwest, central and north-east of iran and is reported sporadically from all provinces of the country (5–7). the first entomological studies in north khorasan province were conducted during 1975 in esfarayen county and l. major detected in phlebotomus papatasi (8). in shirvan county molecular infection of leishmanial due to l. infantum was observed in p. kandelakii (9). cutaneous leishmaniasis is reported from all counties and vl is reported from five out of eight counties of north khorasan province (10). some counties in northern half of the province have been reported to be endemic for vl with more than 160 new cases in the last second decade (11). biodiversity is a primary interest of ecologists and is a contraction of biological diversity. species, ecosystem, and genetic diversity are three components of biodiversity. diversity can be represented in various types of alpha (α), beta (β) and gamma (ϫ). alpha diversity is the variation of species within a community or habitat, beta diversity is in taxonomic composition between communities, and gamma diversity is the total variation of an area and combination of both alpha and beta diversity (12). species diversity is a central theme in ecology and has two separate components named species richness (s) and evenness (e). richness and evenness have been combined mathematically in various ways to calculate diversity indices based on proportional abundances of species (12). two indices of shannon–wiener (shannon index) and simpson’s index commonly used for species biodiversity. simpson’s index measures the probability of two individuals randomly selected from a sample belongs to the same species. shannon index is one of the most well-known diversity indices and measures species richness and proportion of each species within a community (12, 13). the two indices differ in their emphasis on species richness (shannon–wiener) or abundance (simpson’s). typical values of shannon–wiener index are between 1.5 and 3.5 and rarely go beyond to 4.5 (12). diversity also can be measured as the variation in species composition among communities (beta diversity). several techniques have been developed to compare communities based on their species compositions. the jaccard’s similarity coefficient (cj) is the simplest of these similarity measures. this index will always give a value between 0 and 1 and then multiply by 100 to be expressed as percentage of similarity (12). knowledge about the ecological aspects of sand fly species and biodiversity indices help in understanding the transmission dynamics of the disease to human and reservoirs. the purpose of this study was to determine the species composition, richness and diversity indices of the phlebotomine sand flies at different topographic condition e.g. mountain/plain in north khorasan province located in the northeast of iran. materials and methods study area this cross-sectional study was conducted during 2016 in some rural areas of north khorasan province, between 36°37′-38°17′ http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 234 http://jad.tums.ac.ir published online: september 30, 2018 n latitudes and 55°53′–58°20′ e longitudes. the province has a desert, mountainous and temperate climate with cold winters and is bordered by turkmenistan in the north. the total area is approximately 28434km2 and situated in the northeast of iran. bojnurd is the capital city of the province. the koppeh dagh mountain, mountain range on the border of turkmenistan and iran and the eastern alborz range located in this province. selection of the villages of study the sampling was focused on rural regions. three villages including a) bacheh-dareh located in raz and jargalan county, b) kohne jolgeh located in maneh and samalqan county and c) arg located in jajarm county were selected based on diagnosed human vl and/ or cl cases during last 5 years. collection site in bacheh-dareh was a valley 3km far from the village and sand fly captures have been restricted to outdoor and wild environments. collection sites in kohne jolgeh were indoors and domestic environments and in arg were indoors and outdoors and domestic environments. several human vl and cl had been reported from sites of a and b but only human cl cases had been reported from site of c (fig. 1). sand fly collection sand flies were collected three times each twenty days during the peak activity periods of sand flies. sample collection started from early july and continued until late aug 2016. seven collection methods were used as follows: 1) sticky paper traps (spt). we installed 10 paper traps for each rotation and totally 60 papers per night. 2) light trap (ltp), and 3) cdc light trap baited with carbon dioxide (co2) gas that obtained by a co2 gas tank (clt). the light traps were suspended at 1.5m above the ground. 4) white shannon trap (wst), 5) black shannon trap (bst) were made of white or black cloth and consisted of a large central compartment and two smaller lateral ones. the measurements, in meters (width, length, and height), of the central and the lateral compartments were 1.3× 1.3× 2m respectively and suspended by cords from supports and base of the traps touched the ground. a portable stove was used as co2 and light source inside the traps. 6) animal baited trap (abt) and finally 7) disney trap (dst). all of the traps were placed randomly with a distance of 20m from each other. the traps were set before sunset, were changed every two hours and remained in operation during the night (20:00 to 08:00). as the traps were changed the new ones were replaced in the same location. traps were rotated clockwise between the trap locations in site “a” and” c” but were fix in site “b”. collected sand flies were stored in 96% ethanol alcohol. the specimens were mounted on glass slides in puri’s medium. species identification was carried out according to morphological characters using pictorial keys of sand flies (14). determination of the species diversity species diversity based on simpson index and shannon-wiener index, species richness based on margalef index and menhinick index and also evenness based on shannon-wiener evenness index and buzas and gibson's evenness index have been calculated to estimate species biodiversity of phlebotomine sand flies in the study area. computation of these indices is shown in (table 1). beta diversity was also estimated by similarity between different communities using jaccard’s similarity coefficient. berger-parker dominance index and lorenz graph of the collected sand flies within different collection sites were estimated. the statistical analyses were performed in spss version 18 (chicago, il, usa). results an overall 7253 sand flies (19 species) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 235 http://jad.tums.ac.ir published online: september 30, 2018 from three mentioned villages were collected and identified. ten species belonged to the phlebotomus genus and nine species belong to the genus sergentomia. the overall numbers of collected females were 3543 and consisted of 48.8% of specimens. phlebotomus sergenti and ph. papatasi were the most prevalent (84.9%) species in the study area. phlebotomus sergenti was the most predominant species being recorded in all collected areas. species composition and relative abundance of collected phlebotomine sand flies is shown in (table 2). some of the most important alpha diversity indices including, species richness, shannon index, simpson index, buzas and gibson's evenness index, berger-parker dominance index, menhinick richness index, and margalef richness index were calculated and are shown in table 2. the lorenzen curve is shown the cumulative percentage in relation to species rank or log species rank (fig. 2). the sand fly community similarity measured by jacard’s similarity coefficient. the similarity of the sand fly communities was the highest (42.1%) between community a and b and was the lowest (15.8%) between community a and c. jacard’s coefficient was 20% between community b and c. fig. 1. study area and geographical locations of phlebotomine sand flies, north khorasan province, iran, 2016 fig. 2. the lorenz graph for phlebotomine sand flies collected in different collection sites, north khorasan province, iran, 2016. (a: bacheh-dareh village, b: kohne jolgeh village, c: arg village) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 236 http://jad.tums.ac.ir published online: september 30, 2018 table 1. different diversity indices of sand flies of the collection sites, north khorasan province, iran, 2016 index computation collection sites a b c specimens total number of specimens 3073 2392 1788 species richness (s) the number of species 18 8 4 shannon index (h ) 1.033 0.718 0.527 simpson index 0.6185 0.6468 0.6865 shannon evenness index (e) shei= h/ln (s) 0.357 0.345 0.380 buzas and gibson's evenness index 0.1561 0.2562 0.4235 berger-parker dominance index 0.7829 0.7889 0.8082 menhinick richness index 0.3247 0.1636 0.0946 margalef richness index 2.117 0.8998 0.4006 (a: bacheh-dareh village, b: kohne jolgeh village, c: arg village) table 2. species composition and relative abundance of phlebotomine sand flies in north khorasan province, iran, 2016 species collection sites total % a b c ph. sergenti 2406 1887 15 4308 59.4 ph. papatasi 42 366 1445 1853 25.5 ph. alexandri 137 76 0 213 2.9 ph. major 52 9 0 61 0.8 ph. caucasicus 6 2 0 8 0.1 ph. mongolensis 9 0 0 9 0.1 ph. halepensis 30 0 0 30 0.4 ph. longiductus 5 0 0 5 0.1 ph. turanicus 3 0 0 3 0.0 ph. ansarii 0 0 1 1 0.0 se. sintoni 4 0 327 331 4.6 se. sumbarica 117 18 0 135 1.9 se. grekovi 4 0 0 4 0.1 se. dentata 19 14 0 33 0.5 se. theodori 67 0 0 67 0.9 se. hodgsoni 41 0 0 41 0.6 se. pawlowskyi 72 0 0 72 1.0 se. dreyfussi turkestanica 54 20 0 74 1.0 se. clydei 5 0 0 5 0.1 total 3073 2392 1788 7253 100.0 (a: bacheh-dareh village, b: kohne jolgeh village, c: arg village) http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 237 http://jad.tums.ac.ir published online: september 30, 2018 discussion this study reports some proven or suspected vectors of cl and vl in iran including ph. papatasi, ph. sergenti, ph. caucasicus, ph. alexandri and ph. major. among the collected species presence of sergentomyia dreyfussi turkestanica in iran was doubtful and we collected and confirmed the presence of this species in the country. phlebotomus turanicus was reported from this region (3) and we collected and confirmed the presence of this species in iran. phlebotomus kandelakii had been reported as the probable vector of vl in north khorasan province (9) but we were not able to capture in this investigation. phlebotomus sergenti was the most predominant species throughout the study area and recorded in all localities. our results are similar to another study that reported ph. sergenti has a wide distribution in the country and includes and extends beyond the distribution of l. tropica (5). phlebotomus papatasi normally prefers to live in plain areas rather than in mountains. this species often abundant in areas of steppe and semi-arid zones where temperatures are high and such circumstance appears in community c. phlebotomus papatasi showed a widespread distribution in this region and in community c about 81% of all specimens belong to this species. low frequency of this species in mountains area such as community a could be explained by its preference to semi-arid areas (15, 16). phlebotomus alexandri and ph. major were found in a and b collection sites recognized as the areas of the richest biodiversity of sandflies. phlebotomus major has been reported from 17 out of 31 provinces and in all areas which human cases of zvl have been reported mostly in mountainous areas (5). phlebotomus alexandri has been reported as a probable vector of zoonotic visceral leishmaniasis (zvl) in iran (17) and is generally distributed in mountainous regions although reported from almost all parts of iran (5). these species were reported from different areas of iran including plain and highlands. phlebotomus alexandri prefers regions with a high percentage of relative humidity and warmer niches (16). caspian sea near of these villages provides relative humidity suitable for the presence of these species. in the study area, nineteen species of sand flies collected and identified. this revealed sand flies species richness in this area was higher than some other regions in the country (15, 18, 19). there were differences in species richness and diversity indices in three communities. in community a, the shannon diversity index and richness were maximum (h’= 1.033, s= 18) and more than community b and c. there are only limited comprehensive studies on biodiversity of sand flies in iran in which the authors calculated the relevant indices (15, 18-20). in a study in qom province, shannon diversity index in mountainous areas (h’= 1.36, s= 9) was higher than in lowlands (h= 0.66, s= 9). the highest diversity in community a is similar to this investigation that showed more diversity in the mountainous area (15, 19), but in contrast to evenness. the evenness in qom province in the mountainous areas was 0.62, and in lowlands was 0.30, while in all communities in north khorasan province were similar (from e= 0.345 up to 0.380) and less than mountainous areas in qom province. species diversity, as indicated by the values of shannon-wiener index (h) consists of two components: species richness and evenness. a community is said to have high species diversity if many species are present and all species are nearly equally abundant but in all communities in the study area one or two species (ph. sergenti and ph. papatasi) consist of more than 80 percent of specimens as seen in table of species composition (table. http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 232–239 k arzamani et al.: richness and diversity of … 238 http://jad.tums.ac.ir published online: september 30, 2018 1), berger-parker dominance index (table 2) and lorenz graph (fig. 2) and therefore, evenness is less than other studies and species diversity is not much. the similarity of the sand fly communities was different based on jacard’s similarity coefficient. the similarity of the sand fly communities was highest (from 40% up to 100%) between communities (18). the results were in contrast to our finding and showed more diversity of the sand fly communities in the north khorasan province. alborz mountain range and aladagh mountain in the middle parts of the province appear to be important geographical barriers for sand fly distribution. future investigations are needed to clarify the role of natural barriers such as mountains for sand fly distribution. conclusion the sand fly fauna in north khorasan province was very rich and often included some of the most important proven or suspected vectors of leishmaniasis. species diversity indices (shannon index, and simpson’s index) were not high due to decreasing in evenness. some well-known species from mountainous areas such as ph. alexandri, ph. major, ph. halepensis, ph. longiductus and ph. turanicus and plain area species such as ph. papatasi and ph. caucasicus were collected that shows the potential of transmission of a different kind of leishmaniasis in this province. the species richness of sand flies was varied between three selected leishmaniasis foci in the north khorasan province and the margalef richness index could accurately reflect the biodiversity of sand flies between three collection areas. acknowledgements this article has been extracted from the ph.d. thesis in department of medical entomology, school of public health at tehran university of medical sciences, tehran, iran. this study received financial support from tehran university of medical sciences (project no: 27811) and vector-borne diseases research center, north khorasan university of medical sciences, bojnurd, iran (project no: 95/60/729). the authors declare that there is no conflict of interest. references 1. maroli m, feliciangeli m, bichaud l, charrel r, gradoni l (2013) phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. med vet entomol. 27 (2): 123–147. 2. alvar j, vélez id, bern c, herrero m, desjeux p, cano j, jannin j, den boer m, who leishmaniasis control team (2012) leishmaniasis worldwide and global estimates of its incidence. plos one. 7(5): e35671. 3. akhoundi m, parvizi p, baghaei a, depaquit j (2012) the subgenus adlerius nitzulescu (diptera: psychodidae, phlebotomus) in iran. acta trop. 122 (1): 7–15. 4. zahraei-ramazani ar, kumar d, yaghoobiershadi mr, naghian a, jafari r, shirzadi mr, abdoli h, soleimani h, shareghi n, ghanei m, arandian mh, hanafi-bojd aa (2013) sand flies of the subgenus adlerius (diptera: psychodidae) in an endemic focus of visceral leishmaniasis and introduction of phlebotomus (adlerius) comatus as a new record for iran. j arthropod-borne dis. 7(1): 1–7. 5. yaghoobi-ershadi mr (2012) phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission. j arthropod borne 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nafarshalamzari r, parsi s, abbasi a, raufi h (2015) species diversity of sand flies and ecological niche model of phlebotomus papatasi in central iran. acta trop. 149: 246–253. 20. hazratian t, vatandoost h, oshaghi ma, yaghoobi-ershadi mr, fallah e, rafizadeh s, shirzadi mr, shayeghi m, akbarzadeh k, rassi y (2016) diversity of sand flies (diptera: psychodidae) in endemic focus of visceral leishmaniasis in azar shahr district, east azarbaijan province, north west of iran. j arthropod borne dis. 10(3): 328–334. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/22768703 https://www.ncbi.nlm.nih.gov/pubmed/22768703 6. iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 37 original article larval habitat characteristics of the genus anopheles (diptera: culicidae) and a checklist of mosquitoes in guilan province, northern iran s azari-hamidian school of health, guilan university of medical sciences, rasht, iran (received 7 aug 2010; accepted 9 apr 2011) abstract background: ecological data are important in the vector control management of mosquitoes. there is scattered published information about the larval habitat characteristics and ecology of the genus anopheles (diptera: culicidae) in iran and most of available data is in relation to malaria vectors in southern iran. methods: this cross sectional investigation was carried out to study the mosquito fauna and ecology in guilan province, northern iran, during april–december 2000. larvae were collected using the standard dipping technique. larval habitat characteristics were recorded according to water situation (clear or turbid), vegetation, substrate type, sunlight situation, habitat situation (transient or permanent, running or stagnant), habitat type (natural or artificial), and water temperature. results: in total, 1547 thirdand fourth-instar larvae of anopheles from 90 habitats were collected and morphologically identified. five species; anopheles claviger, an.’hyrcanus’, an. maculipennis s.l., an. plumbeus, and an. superpictus were identified and respectively comprised 6.3%, 22.4%, 54.4%, 13.0%, and 3.9% of the samples. the mean and range temperatures of the larval habitat water were 19.6oc (n=14) (16–25oc), 22.6oc (n=53) (12–33oc), 23.8oc (n=52) (10–33oc), 11.5oc (n=12) (9–21oc), and 20.4oc (n=7) (12–26oc), respectively. there was a significant difference in the mean water temperatures (11.5–23.5oc) of the larval habitats of different species (p=0.000). most of the genus larvae were collected from natural habitats (86.9%) such as river bed pools (46.4%) and rain pools (33.1%) with transient (98.3%), stagnant (99.5%) and clear (95.3%) water, with vegetation (69.9%), mud (42.0%) or gravel (39.7%) substrate in full sunlight (69.6%) or shaded (22.7%) area. a checklist of the province mosquitoes including 30 species and seven genera has been provided. conclusion: the main larval habitats of the most abundant species, an.’hyrcanus’ and an. maculipennis s.l., in guilan province are: river bed pools, rain pools, and rice fields. keywords: anophelinae, ecology, iran, larvae introduction according to the most recent classification of mosquitoes, the family culicidae (diptera) includes two subfamilies, 11 tribes, 113 genera, and 3531 species in the world fauna and the genus anopheles meigen includes seven subgenera and at least 465 species (harbach 2007). certain species of anopheles are involved in the transmission of various arboviral and filarial diseases to humans and domestic animals and/or are important for their biting in different parts of the world, but the most important disease transmitted by them is malaria. about 70 anopheles species are malaria vectors in which about 40 are important vectors (service 1993). sindbis virus was reported in iran as well as west nile virus in iran and guilan province of the caspian sea littoral, northern iran (naficy and saidi 1970, saidi et al. 1976); corresponding author: dr shahyad azari-hamidian, e-mail: azari@gums.ac.ir iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 38 however there is no information about their vectors in the country. the mosquito-borne filariae; dirofilaria railliet and henry (dirofilariasis) and setaria viborg (setariasis) (spirurida: onchocercidae) have been reported in iran and guilan province (eslami 1997, azari-hamidian et al. 2007). anopheles maculipennis meigen and culex theileri theobald are known vectors of setaria labiatopapillosa (alessandrini) and dirofilaria immitis (leidy) respectively, in ardebil province, northwestern iran (azari-hamidian et al. 2009). malaria is the most important mosquito-borne disease in iran, especially in southeastern areas, and seven species have been assumed to play role as malaria vectors: an. sacharovi favre, an. maculipennis sensu lato (s.l.), an. fluviatilis james s.l., an. stephensi liston, an. superpictus grassi, an. dthali patton, and an. culicifacies giles s.l. (edrissian 2006). also, zaim et al. (1993) reported an. pulcherrimus theobald as a potential vector in southeastern iran. eshghy (1977) observed plasmodium oocysts in an. multicolor cambouliu, but sporozoites have not been detected in this species and it is not considered a vector in iran. recently, djadid et al. (2009) reported an. hyrcanus (pallas) as a potential vector of malaria based on nested polymerase chain reaction (pcr) in guilan province. the most recent checklist of iranian mosquitoes includes 64 species and three subspecies belonging to seven genera (azarihamidian 2007a). almost half of the iranian mosquito species (31 species) and seven genera have been previously recorded in guilan province (zolotarev 1945, dow 1953, lotfi 1973, danilov 1975, saebi 1987, zaim 1987b, harbach 1988, momeni et al. 1992, azarihamidian et al. 2002b, sedaghat et al. 2003, gholizadeh et al. 2004, gholizadeh et al. 2005). despite of the importance of ecological data in the vector control management of mosquitoes, there is scattered published information about the larval habitat characteristics and ecology of anopheles in iran and most of available data is in relation to malaria vectors in southern iran. marsh (1933) showed some larval breeding places of an. apoci marsh larvae when described it for the first time in iran. macan (1950) studied the different aspects of some anopheles species in northern and western iran. dow (1953) and yaghoobi-ershadi et al. (1986) stressed the larval breeding sites and associate species of some anophelines in the different areas of the country and southern iran, respectively. manouchehri and rohani (1975) studied the larval habitats of an. dthali in southern iran. zaini et al. (1975) and manouchehri et al. (1976) mentioned the larval breeding place characteristics of an. stephensi in southern iran. eshghi et al. (1976) stressed some larval breeding places of an. fluviatilis s.l. in southern iran. eshghy (1977) noted the adult and larval ecology of an. multicolor. zaim (1987a) studied the mosquito fauna of kashan of isfahan province in central iran with some notes on the larval breeding places and ecology of four anopheline species. mousakazemi et al. (2000) studied the fauna and ecology of mosquitoes including an. maculipennis s.l. and an. superpictus in zarrinshahr and mobarakeh areas of isfahan province. yaghoobi-ershadi et al. (2001) stressed some information on the larval stage of an. sacharovi and an. superpictus in ardebil province of northwestern iran. azari-hamidian et al. (2002a) showed some larval breeding places of mosquitoes including anophelines in rasht county of guilan province in northern iran. ghanbari et al. (2005) studied some physical and chemical factors of the larval breeding places of the eight species of anopheles in iranshahr, southeastern iran. investigation on the mosquito ecology and larval habitats is important in different aspects including source reduction in vector control through modifications of these habitats. this helps us to decrease, even if not very much, vector population and density iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 39 then vector-reservoir and vector-pathogen contacts, and biting nuisance. this may decrease the transmission of different mosquito-borne diseases and the burdens of these diseases. the present article is focused on some aspects of the larval ecology of anopheles species, such as the larval habitat characteristics, water temperature, associated species, and provides a checklist of mosquitoes in guilan province. materials and methods study area guilan province in the caspian sea littoral of northern iran, between caspian sea and alborz mountain range, has coastal, plain, foothill, and mountainous areas with an area of approximately 14700 square kilometers. the province with temperate climate and relatively warm-humid summer is located between 36o34'–38o27' n latitude and 48o34'– 50o36' e longitude and formally includes 16 counties; amlash, astaneh-e-ashrafiyeh, astara, bandar-e-anzali, fuman (fooman), lahijan, langrud (langroud), masal, rasht, rezvanshahr, rudbar, rudsar (roudsar), shaft, siahkal, some’e sara, and talysh (talesh). guilan province with about 1200 mm annual rainfall has the greatest rainfall in iran and the main agricultural crop is rice. this province with vast deciduous forests of hyrcania and temperate climate is a great location to breed mosquitoes. specimen and data collection and analysis in this cross sectional investigation, in each county (in total 16) one fixed and three variable sites (including different topographical areas) randomly selected and larval specimen collection was carried out for 15– 20 minutes during spring, summer, and autumn seasons in 2000. thus, larval collection was carried out at least three times in fixed sites and 9 times in variable sites during three seasons in each county. larvae were collected from natural breeding sites such as river edges, river bed pools, rain pools, marshes, grasslands, and tree holes and from artificial breeding sites such as rice fields, irrigation channels, wells, discarded tires, and buckets using the standard dipping technique (350 ml dipper) and also by means of pipette (for small habitats) and bucket (for wells) (who 1975). physical and biological characteristics of larval habitats including habitat situation (permanent or transient, stagnant or running), habitat type (natural or artificial), vegetation situation (with or without vegetation), substrate type, sunlight situation (full or partial sunlight or shaded), water situation (clear or turbid), and water temperature were recorded. the continuous variable of the temperatures of larval habitats was analyzed by one-way anova analysis using spss (version 11.5 for windows, spss inc., chicago, il) software. the thirdand fourth-instar larvae and adult specimens were identified using the keys of shahgudian (1960), zaim and cranston (1986), and harbach (1988). the mosquito name genera and subgenera abbreviations followed reinert (2001). taxonomic note two close species anopheles hyrcanus and an. pseudopictus grassi of the hyrcanus group are identified based on the characters of adult in guilan province (dow 1953, azari-hamidian et al. 2002b, 2003a, 2006), these species have not been identified in larval stage in this investigation and mentioned as an.’hyrcanus’ (an. hyrcanus group). darsie and samanidou-voyadjoglou (1997) mentioned just one character of seta 2-c (inner clypeal seta) to distinguish the larvae of an. hyrcanus from those of an. pseudopictus. this seta is simple in an. pseudopictus and bears some short apical branches in an. hyrcanus. this character needs to be studied carefully in iran. seven species of an. maculipennis complex have been recorded in iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 40 the province based on morphological characters including egg pattern and pcr technique (dow 1953, azari-hamidian et al. 2002b, sedaghat et al. 2003, gholizadeh et al. 2004, gholizadeh et al. 2005). only an. sacharovi is reliably distinguishable from other species in adult and larval stage. in larvae, the mean number of seta 2 branches of the fourth and fifth abdominal segments in an. sacharovi is 36.8 whereas it is 16.5 for an. maculipennis s.l. (doosti et al. 2006). it seems the use of pcr technique is the only reliable way to distinguish other species of the group, so the maculipennis group larvae cited as an. maculipennis s.l. herein. results larval habitat characteristics and ecology in these investigations, totally 6656 larvae including 1547 anopheline larvae (23.2%) and 5109 culicine larvae (76.8%) were collected from 127 larval breeding sites during 55 occasions. among 127 larval breeding sites 14 (11.0%) contained only subfamily anophelinae, 37 (29.2%) included only subfamily culicinae, and 76 (59.8%) contained both subfamilies. in total, anopheline larvae were found in 90 breeding sites (70.8%) and culicine larvae in 113 ones (88.9%). in the present study, 1547 larvae of the genus anopheles were collected during 11 occasions (20.0% of the total). five species of anopheles; an. claviger (meigen) (6.3%), an.’hyrcanus’ (22.4%), an. maculipennis s.l. (54.4%), an. plumbeus stephens (13.0%), and an. superpictus (3.9%) were found among the samples (table 1). the association occasions of anopheles species with other collected species are shown in table 2. most of the genus larvae were collected from natural habitats (86.9%) such as river bed pools (46.4%) and rain pools (33.1%) with transient (98.3%), stagnant (99.5%) and clear (95.3%) water, with vegetation (69.9%), mud (42.0%) or gravel (39.7%) substrate in full sunlight (69.6%) or shaded (22.7%) area (table 3). the association percentages of anopheles species with other collected species are shown in table 4. there is a significant difference in the mean water temperatures of the larval habitats of different species (p= 0.000). anopheles claviger anopheles claviger larvae were collected during 11 occasions (20% of the total) from 14 larval breeding sites (11.0% of the total and 7.9% of the anopheline larval habitats) in april, may, june, and september 2000. the maximum and minimum water temperatures of larval habitats were 25o c and 16o c respectively, and mean temperature was 19.6o c (for 14 larval breeding sites) (sd= 3.0). anopheles ’hyrcanus’ anopheles ’hyrcanus’ larvae were collected during 33 occasions (60% of the total) from 53 larval breeding sites (41.7% of the total and 4.4% of the anopheline larval habitats) during april–november 2000. the maximum and minimum water temperatures of larval habitats were 33o c and 12o c respectively, and mean temperature was 22.9o c (for 53 larval breeding sites) (sd= 4.7). anopheles maculipennis s.l. anopheles maculipennis s.l. larvae were collected during 30 occasions (54.5% of the total) from 53 larval breeding sites (41.7% of the total and 3.5% of the anopheline larval habitats) during april–november 2000. the maximum and minimum water temperatures of larval habitats were 33oc and 10oc respectively, and mean temperature was 23.8o c (for 52 larval breeding sites) (sd= 4.0). anopheles plumbeus anopheles plumbeus larvae were collected during 9 occasions (16.3% of the total) from 12 larval breeding sites (9.4% of the total and 3.5% of the anopheline larval iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 41 habitats) in october and november 2000. the maximum and minimum water temperatures of larval habitats were 21oc and 9o c respectively, and mean temperature was 11.5o c (for 12 larval breeding sites) (sd= 3.2). the mean temperature of the larval habitat water of this species showed significant difference from other species (p= 0.000). anopheles superpictus anopheles superpictus larvae were collected during four occasions (7.2% of the total) from 7 larval breeding sites (9.4% of the total and 3.5% of the anopheline larval habitats) in may, august, and october 2000. the maximum and minimum water temperatures of larval habitats were respectively 26o c and 12o c, and mean temperature was 20.4o c (for 7 larval breeding sites) (sd= 6.3). checklist of the mosquitoes of guilan province the checklist of 30 species of mosquitoes in seven genera represented in guilan province is given below. the species which were recorded in the province by other authors and/or using pcr technique and were not found or identified in this investigation are shown by asterisks (*): anopheles algeriensis theobald*, an. atroparvus van thiel*, an. claviger (meigen), an. hyrcanus (pallas), an. labranchiae falleroni*, an. maculipennis meigen, an. melanoon hackett, an. messeae falleroni, an. persiensis linton, sedaghat and harbach*, an. plumbeus stephens, an. pseudopictus grassi, an. sacharovi favre*, an. superpictus grassi, aedes vexans (meigen), coquillettidia richiardii (ficalbi), culex hortensis ficalbi, cx. mimeticus noe, cx. pipiens linnaeus, cx. territans walker, cx. torrentium martini, cx. theileri theobald, cx. tritaeniorhynchus giles, culiseta annulata (schrank), cs. longiareolata (macquart), cs. morsitans (theobald), cs. subochrea (edwards)*, ochlerotatus caspius (pallas) s.l., oc. echinus (edwards), oc. geniculatus (olivier), uranotaenia unguiculata edwards. table 1. the composition and abundance of anopheles larvae in guilan province, iran, april– december 2000 taxon n percentage of family (%) percentage of genus (%) an. claviger 97 1.46 6.27 an.’hyrcanus’ 346 5.20 22.37 an.maculipennis s.l. 841 12.63 54.36 an. plumbeus 202 3.03 13.06 an. superpictus 61 0.92 3.94 total anophelinae 1547 23.24 100 total culicidae 6656 table 2. the association occasions of anopheles larvae with different mosquito larvae in guilan province, iran, april–december 2000 species t otal occasion s a n . cla vig er a n .’hyrca n u s’ a n . m a cu lip en n is s.l. a n . su p erp ictu s a e. vexa n s c x. m im eticu s c x. p ip ien s c x. th eileri c x. trita en io rh yn ch u s c x. h orten sis c x. territa n s c s. lo n g ia reo lata c s. m o rsita n s c s. a n n u la ta o c. ech in u s o c. gen icu la tu s u r. u ng u icu la ta an. claviger 14 4 4 1 3 2 1 4 4 3 1 an.’hyrcanus’ 53 4 39 4 6 12 13 7 31 4 5 1 1 2 1 an. maculipennis s.l. 53 4 39 3 1 10 14 8 30 2 5 1 1 an. plumbeus 12 1 4 1 an. superpictus 7 1 4 3 2 1 2 5 2 1 1 iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 42 table 3. the larval habitat characteristics and occurrence percentages of anopheles larvae in guilan province, iran, april–december 2000 larval breeding site characteristics and habitats a n . cla vig er (% ) a n .’h yrca n u s’ (% ) a n . m a cu lip en n is s.l. (% ) a n . p lu m beu s (% ) a n . su p erp ictu s (% ) a n o p h eles (% ) a) habitat situation 1. permanent 7.2 2.6 1.2 1.7 2. transient 92.8 97.4 98.8 100 100 98.3 3. slow-running water 5.2 0.6 0.1 0.5 4. stagnant water 94.8 99.4 99.9 100 100 99.5 b) vegetation situation 1. without vegetation 33.0 14.7 16.8 100 65.6 30.1 2. with vegetation 67.0 85.3 83.2 34.4 69.9 2a) emergent 78.9 51.4 30.2 66.7 39.8 2b) floating 0.6 0.4 0.4 2c) submerged 21.1 48.0 69.4 33.3 59.8 c) substrate type 1. mud 73.7 56.1 30.7 67.8 42.0 2. sand 8.4 20.8 19.7 18.3 3. gravel 17.9 23.1 49.6 32.2 39.7 d) water situation 1. turbid 1.7 8.0 4.7 2. clear 100 98.3 92.0 100 100 95.3 e) sunlight situation 1. full sunlight 22.7 71.7 89.4 88.5 69.6 2. partial sunlight 11.3 21.7 4.1 7.7 3. shaded 66.0 6.6 6.5 100 11.5 22.7 f) habitat type 1. natural habitat 97.9 83.2 83.6 98.0 100 86.9 1a. river edge 0.1 0.1 1b. river bed pool 28.4 37.2 67.0 31.1 46.4 1c. stream edge 5.3 7.6 0.3 2.2 1d. grassland 1.0 0.7 1.7 0.7 1e. marsh 13.2 2.8 1f. rain pool 66.3 41.0 31.9 67.2 33.1 1g. tree hole 100 14.7 2. artificial habitat 2.1 16.8 16.4 2.0 13.1 2a. rice field 79.4 61.6 64.8 2b. rice irrigation channel 10.3 26.8 21.3 2c. well 100 1.0 2d. discarded concrete tube 5.1 3.5 2e. discarded tire 100 2.0 2f. water storage pool 10.3 6.5 7.4 iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 43 table 4. the association percentages of anopheles larvae with different mosquito species larvae in guilan province, iran, april–december 2000 species association abundance (%) an. claviger alone 19.59 cx. hortensis, cs. longiareolata 17.53 cx. territans 12.37 ae. vexans 9.28 an.’hyrcanus’, an. maculipennis s.l., ae. vexans, cx. pipiens 9.28 an. superpictus, cx. hortensis, cs. longiareolata 8.25 an.’hyrcanus’, an. maculipennis s.l. 7.22 cx. hortensis, cs. annulata, cs. longiareolata 5.15 an. maculipennis s.l., cx. territans, cx. tritaeniorhynchus 4.12 cx. hortensis 3.09 an.’hyrcanus’, ae. vexans, cx. territans 2.06 cx. pipiens, cx. territans 2.06 total 100 an.’hyrcanus’ an. maculipennis s.l., cx. tritaeniorhynchus 27.75 ae. vexans, cx. pipiens, cs. annulata 10.98 an. maculipennis s.l., cx. mimeticus 9.83 an. maculipennis s.l. 7.80 an. maculipennis s.l., cx. mimeticus, cx. tritaeniorhynchus 7.51 an. maculipennis s.l., cx. pipiens, cx. theileri, cx. tritaeniorhynchus 3.18 an. maculipennis s.l., cx. pipiens, cx. theileri 2.90 cx. tritaeniorhynchus 2.89 alone 2.60 cx. hortensis, cx. theileri, cx. tritaeniorhynchus 2.60 an. maculipennis s.l., cx. theileri, cx. tritaeniorhynchus 2.31 ae. vexans, cx. tritaeniorhynchus 2.02 an. claviger, ae. vexans, cx. territans 1.73 an. claviger, an. maculipennis s.l. 1.73 an. maculipennis s.l., cx. territans 1.73 an. claviger, an. maculipennis s.l., ae. vexans, cx. pipiens 1.44 an. maculipennis s.l., cx. hortensis, cx. tritaeniorhynchus 1.44 an. maculipennis s.l., cx. pipiens, cx. theileri, cx. tritaeniorhynchus, cs. longiareolata 1.44 an. maculipennis s.l., cx. pipiens, cx. tritaeniorhynchus 1.16 an. maculipennis s.l., an. superpictus, cx. mimeticus, cx. tritaeniorhynchus 1.16 an. maculipennis s.l., cx. pipiens, cx. territans 0.87 an. maculipennis s.l., an. superpictus, cx. theileri, cx. tritaeniorhynchus 0.87 an. maculipennis s.l., cx. territans, cx. tritaeniorhynchus 0.87 cx. pipiens 0.87 an. maculipennis s.l., cx. mimeticus, cx. pipiens 0.58 ae. vexans 0.29 cx. hortensis, cx. pipiens, cx. tritaeniorhynchus 0.29 cx. mimeticus, cx. pipiens, cx. territans, cx. tritaeniorhynchus 0.29 an. superpictus, cx. hortensis, cx. pipiens, cx. tritaeniorhynchus 0.29 ae. vexans,, cx. mimeticus, cx. pipiens, cs. annulata, cs. morsitans 0.29 an. superpictus, cx. mimeticus, ur. unguiculata 0.29 total 100 iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 44 table 4. continued…. discussion larval habitat characteristics and ecology in the present investigation, five species of the genus anopheles were found and identified based on the morphological characters of larvae in different aquatic habitats in guilan province. while the tree-hole an. pluman. maculipennis s.l. an.’hyrcanus’, cx. tritaeniorhynchus 27.82 an.’hyrcanus’, cx. mimeticus 14.74 cx. pipiens 10.46 an.’hyrcanus’, cx. mimeticus, cx. tritaeniorhynchus 10.23 an.’hyrcanus’, cx. theileri, cx. tritaeniorhynchus 9.16 an.’hyrcanus’, an. superpictus, cx. mimeticus, cx. tritaeniorhynchus 3.69 an.’hyrcanus’ 3.33 cx. mimeticus 2.62 alone 2.02 an.’hyrcanus’, cx. hortensis, cx. tritaeniorhynchus 1.90 an. claviger, an.’hyrcanus’ 1.78 cx. theileri 1.78 cx. theileri, cx. tritaeniorhynchus 1.43 an.’hyrcanus’, cx. pipiens, cx. theileri 1.43 an.’hyrcanus’, cx. territans, cx. tritaeniorhynchus 1.19 an. claviger, cx. territans, cx. tritaeniorhynchus 1.07 an.’hyrcanus’, cx. mimeticus, cx. pipiens 0.71 an.’hyrcanus’, cx. pipiens, cx. territans 0.71 cx. pipiens, cx. tritaeniorhynchus 0.71 an.’hyrcanus’, cx. pipiens, cx. theileri, cx. tritaeniorhynchus, cs. longiareolata 0.59 an.’hyrcanus’, an. superpictus, cx. theileri, cx. tritaeniorhynchus 0.59 an.’hyrcanus’, cx. territans 0.48 an. claviger, an.’hyrcanus’, ae. vexans, cx. pipiens 0.36 an. superpictus, cx. tritaeniorhynchus 0.36 cx. pipiens, cx. territans 0.36 an.’hyrcanus’, cx. pipiens, cx. theileri, cx. tritaeniorhynchus 0.12 cx. hortensis 0.12 an.’hyrcanus’, cx. pipiens, cx. tritaeniorhynchus 0.12 cs. morsitans 0.12 total 100 an. plumbeus alone 62.87 oc. echinus, oc. geniculatus 18.81 oc. echinus 16.34 cx. pipiens 1.98 total 100 an. superpictus an.’hyrcanus’, cx. hortensis, cx. pipiens, cx. tritaeniorhynchus 26.23 an. claviger, cx. hortensis, cs. longiareolata 19.67 an. maculipennis s.l., cx. tritaeniorhynchus 19.67 cx. theileri, cx. tritaeniorhynchus 11.48 an.’hyrcanus’, an. maculipennis s.l., cx. mimeticus, cx. tritaeniorhynchus 11.48 an.’hyrcanus’, cx. mimeticus, ur. unguiculata 9.84 an.’hyrcanus’, an. maculipennis s.l., cx. theileri, cx. tritaeniorhynchus 1.63 total 100 iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 45 beus has its own special habitat and way of adaptation, other species have shared some common larval habitat characteristics such as temporary, stagnant, and clear water. however every species shows some preferred characteristics in comparison with others as follow: an. superpictus prefers the habitats without vegetation (65.6%); an. claviger and an. superpictus more adapt the habitats with mud (73.7% and 67.8%, respectively) or gravel (17.9% and 32.2%) substrate, respectively, whereas an. ’hyrcanus’ and an. maculipennis s.l. are less conservative in this regard; an. claviger lays the eggs often (66.0%) in shaded area; an. claviger and an. superpictus choose mostly natural habitats (97.9% and 100%), especially rain pools (66.3% and 67.2%), however an. ’hyrcanus’ and an. maculipennis s.l. show more diverse habitats and were also collected from artificial habitats (16.8% and 16.4%), especially rice fields (79.4% and 61.6%) (table 3). these explain why an. maculipennis s.l. and an.’hyrcanus’ are the most abundant species in the studied area where rice fields and irrigation channels are numerous and vast (table 1). macan (1950) found an. claviger in "spring pools, sometimes exposed to the sun, and in slow-flowing reaches and isolated pools of the upper parts of streams where these were heavily shaded" with water temperature 14– 16° c (except for one occasion 20° c) in iraq and western iran. dow (1953) found an. claviger in "a small, shallow and shaded stream, with much emergent and partly submerged vegetation including some grass" with cx. pipiens in maragheh of northwestern iran. horsfall (1955) mentioned that an. claviger is an associate of an. cinereus theobald (as an. hispaniola), an. maculipennis, an. marteri senevet and prunnelle, an. superpictus, cs. longiarolata, cs. morsitans, cx. laticinctus edwards, cx. mimeticus, cx. pipiens, cx. territans (as cx. apicalis), and cx. tritaeniorhynchus and noted that the larvae have been collected from water with temperature 12– 15° c and even beneath the ice during winter and larval sites include: small ponds, flowing stream margins, springs, cisterns, fountains, coastal areas and marshes, shaded rain barrels, roadside ditches, even sewages, and especially wells in different areas of its distribution. zaim (1987a) collected an. claviger from stream edge in kashan. in the present study, an. marteri and cx. laticinctus were not found in guilan province (azari-hamidian et al. 2002b, 2004a, 2005, azari-hamidian 2007b) and an. cinereus has not been collected in iran (azari-hamidian 2007a). culiseta morsitans was found in the province, not in association with an. claviger, however other mentioned species were collected with this species (tables 2 and 4). as horsfall (1955) expressed wells are the important habitats of an. claviger larvae, in this study among man-made habitats it was found in wells only, although the species was collected mostly from natural habitats (97.9%) (table 3). unfortunately, there is little information about the larval habitat characteristics and ecology of an. hyrcanus and an. pseudopictus in details because of their problematic differentiation in larval stage (see taxonomic note). dow (1953) collected only an. pseudopictus (as an. hyrcanus var. pseudopictus) in northern iran based on rearing immature stages from "fairly deep channels in river bed with emergent vegetation and surface debris", "rice fields", "quiet river channel", "canal with emergent grass along banks", "small brook below spring with mats of glove-like alga" with an. maculipennis, an. melanoon (as an. subalpinus) and cx. tritaeniorhynchus. horsfall (1955) stressed an. algeriensis, an. coustani laveran, an. sacharovi, an. sergentii (theobald) and rarely an. claviger, an. superpictus, cs. annulata, cx. perexiguus theobald, oc. caspius s.l., and oc. detritus as the associated species of an. ‘hyrcanus’ in palestine. azari-hamidian et al. (2002a) found an. ‘hyriran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 46 canus’ larvae with those of an. maculipennis s.l., ae. vexans, cx. mimeticus, cx. pipiens, cx. theileri, and cx. tritaeniorhynchus often in rice fields and also ground pools in rasht county. among mentioned species, anopheles coustani is not recorded in iran (azarihamidian 2007a) and an. algeriensis, an. sacharovi, an. sergentii, cx. perexiguus, and oc. detritus were not found in guilan province in this study (azari-hamidian et al. 2002b, 2002c, 2004a, 2005, azari-hamidian 2007b) and oc. caspius s.l. was collected only as adult (azari-hamidian et al. 2002c). other species larvae were found with an. ‘hyrcanus’ larvae. in the present study, an. hyrcanus, with dark hindtarsomere 4, except at tip, and completely dark hindtarsomere 5, reared from pupae was an associate of an. maculipennis s.l., an. pseudopictus, ae. vexans, cx. mimeticus, cx. pipiens, and cx. tritaeniorhynchus, which were reared from pupae or collected as larvae from the same larval habitats in ground pools with emergent vegetation and mud or sand substrate and river bed pools with submerged vegetation and gravel substrate both habitats were exposed to sunlight. also, reared an. pseudopictus, with completely pale hindtarsomere 4 and completely dark hindtarsomere 5, was with an. claviger, an. maculipennis s.l., an. hyrcanus, ae. vexans, cx. mimeticus, cx. pipiens, cx. territans, cx. theileri, cx. tritaeniorhynchus in river edge without vegetation, with mud substrate in shaded area, river bed pool with submerged vegetation and gravel substrate in full sunlight situation, rice irrigation channel without vegetation with sand substrate in shaded area, and ground pool with emergent vegetation and mud substrate in full sunlight situation. both species were collected from habitats with clear, transient, and stagnant water. in general, two species occur sympatrically in northern iran; however an. pseudopictus is much more abundant than an. hyrcanus (dow 1953, azari-hamidian et al. 2001, 2006) and the main larval habitats are: river bed pools, rain pools, and rice fields (table 3). ghanbari et al. (2005) mentioned some physical and chemical factors of the larval habitats of anopheles including an. hyrcanus in iranshahr, southeastern iran, however most probably their identification is not correct and needs to be verified. because, the oriental species of the hyrcanus group, an. peditaeniatus, has been found in southeastern iran and old records of the oriental an. nigerrimus giles in southern and southeastern iran using old keys and the record of the palaearctic an. hyrcanus in southeastern iran might be misidentifications and need to be verified (azari-hamidian et al. 2006). there is little data about the larval breeding places and ecology of the maculipennis group species in details in iran, because the species were not identified carefully in larval stage or using egg pattern in the most of ecological investigations and most of recorded species based on pcr technique were collected as adult (see taxonomic note). dow (1953) identified an. maculipennis based on egg collected in "rice field", "small spring in ditch", "shady irrigation reservoir", "shady irrigation ditch", "wide irrigation ditch with floating plants, debris and algae, also emergent grass and sedge", "puddle in small irrigation ditch beside road", and "small stream with much floating and emergent vegetation" in northwestern iran and the caspian sea littoral (mazandaran province) and the associated species were an. ‘hyrcanus’, an. sacharovi, an. superpictus, cx. hortensis, and cx. theileri. also he identified an. melanoon (as an. melanoon subspecies subalpinus) collected in "quiet river channel", "canal with emergent grass along bank", "small brook below spring with mats of glove-like alga", "rain barrel", and "shallow pond" in guilan province and the associated species were an. ‘hyrcanus’ and cx. tritaeniorhynchus. anopheles sacharovi was collected in a "shady irrigation reservoir" and in a "pasture" in northwestern iran. horsfall (1955) mentioned an. macuiran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 47 lipennis s.l. an associate of an. ‘hyrcanus’, an. sergentii, an. superpictus, cs. annulata, cx. laticinctus, cx. pipiens, and ur. unguiculata and added rarely two or more species of the group were found in the same habitat at the same time such as an. atroparvus with an. messeae and an. maculipennis with an. melanoon (as an. subalpinus). mousakazemi et al. (2000) collected an. maculipennis s.l. larvae with those of an. superpictus, ae. vexans, cs. longiareolata, cx. pipiens, cx. perexiguus, cx. theileri, oc. caspius s.l. from rice fields in isfahan province. yaghoobi-ershadi et al. (2001) did not identified an. sacharovi based on egg pattern, larval chaetotaxy, or pcr in parsabad of northern ardebil province; however they studied its larvae based on the high abundance of the adults in the studied area. they noted very high salinity of the larval habitat waters (263–414 mg/l) and an. superpictus as associated species. anopheles mculipennis have been found in northern ardebil province (parsabad and bil-e-savar counties), however with very low density (yaghoobi-ershadi et al. 2001, azari-hamidian et al. 2009). azari-hamidian et al. (2002a) collected an. maculipennis s.l. larvae in rice fields, irrigation channels, ground pools, in sunlight or shaded situation, with or without vegetation, with mud substrate and clear water in rasht county of guilan province. the associated larvae were an. ‘hyrcanus’, ae. vexans, cx. pipiens, cx. theileri, and cx. tritaeniorhynchus. in the present investigation, among the mentioned species; an. sacharovi, an. sergentii, cx. laticinctus, and cx. perexiguus were not found in the province (azari-hamidian et al. 2002b, 2004a, 2005, azari-hamidian 2007b) and cs. annulata and ur. unguiculata larvae were not found with an. maculipennis s.l. (azarihamidian et al. 2004b, azari-hamidian 2005), however other species were in association with this species (tables 2 and 4). in general, an. maculipennis is the most abundant species of the maculipennis group in guilan province (zolotarev 1945, dow 1953, de zulueta et al. 1957, azari-hamidian et al. 2004a) and its main larval habitats, such as an. ‘hyrcanus’, are: river bed pools, rain pools, and rice fields (table 3). the tree-hole mosquito an. plumbeus also has been found in different habitats other than those in soil including: wells, cisterns, vases and other domestic containers, even dung pits, and flowerpots. the most favorable temperature is 15-18° c; however the species has tolerated to freeze and -6° c to -8° c in the laboratory. associated species are ae. aegypti (linnaeus), cs. annulata, cx. pipiens, ochlerotatus geniculatus, oc. echinus, and orthopodomyia pulcripalpis (rondani) (horsfall 1955). there is no record of or. pulcripalpis in iran and ae. aegypti has not been found in the country for more than fifty years (azari-hamidian 2007a). in the present investigation, cs. annulata was collected in guilan province (azari-hamidian et al. 2003b, azari-hamidian 2005), however not in association with an. plumbeus. anopheles plumbeus was associated with other three species (tables 2 and 4). most of an. plumbeus larvae were collected from the tree holes and only in one occasion four larvae were collected with cx. pipiens in a discarded tire (table 3). the larvae were only collected when rain season started, october and november, and tree-holes included water and the weather was cooler (azarihamidian 2003, 2006). that is why the mean temperature of habitat water showed significant difference from other species. macan (1950) stressed "the edges of stony streams" as "the classic habitat" of an. superpictus, however he added larvae are found in "any available breeding places where the water is clean, exposed to sunlight and moderately shallow" in western iran. dow (1953) found an. superpictus larvae with an. maculipennis, and probably an. sacharovi, cx. hortensis, cx. theileri, and ur. unguiculata in different kinds of habitats iniran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 48 cluding; rivers, streams, pools, pastures, springs, irrigation ditches in different areas of iran. horsfall (1955) said the associated species of the species are; an. cinereus (as an. hispaniola), an. claviger, an. sacharovi, an. sergentii, cx. laticintus, cx. mimeticus, and ur. unguiculata and mentioned that three main larval breeding places of an. superpictus are: "clear shallow water over rocky bottom", "rivers of clear water over sandy bottom", and "hill streams of shallow water over mud bottom". the larvae passed the winter in pit wells where the temperature was 5–15°c in greece. yaghoobi-ershadi et al. (1986) found an. superpictus larvae in habitats such as river edges (80%), ground pools (15%), and pastures (5%) which were often transient (62.5%) and stagnant (100%), without (50%) or with submerged (22.8%) vegetation, in partial (56.3%) or full (37.4%) sunlight, with mud (56.2%) and sand (43.8%) substrate. also they noted associated larvae: an. dthali, an. fluviatilis s.l., an. multicolor, an. stephensi, and an. turkhudi in minab. mousakazemi et al. (2000) collected an. superpictus larvae with those of an. maculipennis s.l., ae. vexans, cs. longiareolata, cx. pipiens, cx. perexiguus, cx. theileri, oc. caspius s.l. from rice fields in isfahan province. yaghoobi-ershadi et al. (2001) found an. superpictus larvae with an. sacharovi in ardebil province. ghanbari et al. (2005) studied some physical and chemical factors of the larval habitats of anopheles larvae in iranshahr including an. superpictus. among mentioned species an. cinereus has not been found in iran (azari-hamidian 2007a) and an. dthali, an. fluviatilis s.l., an. multicolor, an. sergentii, an. stephensi, an. turkhudi, cx. laticinctus, and cx. perexiguus in guilan province (azari-hamidian et al. 2002b, 2004a, 2005, azari-hamidian 2007b). in the present study, anopheles sacharovi was not found in the province (azari-hamidian et al. 2002b, 2004a) and ae. vexans larvae were not in association with an. superpictus larvae (azarihamidian 2006) and oc. caspius s.l. was found only as adult (azari-hamidian et al. 2002c). other mentioned species were associated with an. superpictus larvae in the province (tables 2 and 4). in parallel to the results of many researchers (macan 1950, dow 1953, horsfall 1955, yaghoobi-ershadi et al. 1986, zaim 1987a), in this study an. superpictus was found in natural habitats (100%) and mostly exposed to sun (88.5%), with clear (100%) water, without vegetation (65.6%), and mud (67.8%) substrate (table 3). finding in rice fields (artificial habitat) by mousakazemi et al. (2000) in isfahan province seems to be to some extent unusual and interesting. larval habitat water ph, electric conductivity (ec), turbidity, dissolved oxygen, and different organic and inorganic compounds, other aquatic associated animals especially predators, and the life tables of different mosquito species are items, which should be considered in future studies. checklist of the mosquitoes of guilan province azari-hamidian et al. (2002b) found 23 species of mosquitoes representing seven genera based on morphological characters including egg pattern (for the maculipennis group) in guilan province. in this list three species an. superpictus, oc. echinus, and ur. unguiculata were recorded in the province and cs. morsitans and its subgenus (culicella) in iran for the first time. five species including an. atroparvus and an. sacharovi of the maculipennis group, an. algeriensis, cs. subochrea, and cx. torrentium, which were recorded in guilan province before (danilov 1975, saebi 1987, zaim 1987b, dinparast-jadid et al. 2001), were not found by azari-hamidian et al. (2002b). after that, sedaghat et al. (2003) and gholizadeh et al. (2004) reported an. persiensis and an. labranchiae of the maculipennis group based on pcr technique in the proviran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 49 ince, respectively. in general, all seven species of the maculipennis group recorded in iran (azari-hamidian 2007a) have been found in guilan province. anopheles atroparvus, an. labranchiae, an. maculipennis, an. messeae, an. persiensis, an. sacharovi are presented in the province and iran based on dna data and their sequences are available in genbank (sedaghat et al. 2003, gholizadeh et al. 2004, gholizadeh et al. 2005, djadid et al. 2007), however an. melanoon (also as an. subalpinus) has been found only using egg pattern (zolotarev 1945, dow 1953, de zulueta et al. 1957, azari-hamidian et al. 2002b). dow (1953) and djadid et al. (2007) found an. sacharovi in guilan province based on egg pattern (in hassan kiadeh) and dna sequence, respectively, however it seems, as dow (1953) noted, the species is quite rare in the province. azari-hamidian et al. (2002b, 2004a) identified three species an. maculipennis, an. melanoon, and an. messeae based on egg pattern in guilan province. during his studies, saebi (1987) did not find an. algeriensis in iran and mentioned it a very rare species which had been recorded in lahijan and fooman (fuman) of guilan province before. this species was not found by azarihamidian et al. (2002b, 2004b) either; however its occurrence in the province seems to be possible, because of the presence of its preferred habitats; reedy swamps (horsfall 1955). based on the second internal transcribed spacer (its2) and cytochrome c oxidase subunit i (coi) sequence data, recently oshaghi et al. (2007, 2008) reported three genotypes named x, y, and z within an. superpictus in iran. genotype x was found in all parts of the country except for the southeastern areas. it seems that the an. superpictus specimens of guilan province belong to genotype x, however it needs to be confirmed by more investigation. zaim (1987b) reported cs. subochrea in guilan province; however azari-hamidian et al. (2002b, 2003b) found only cs. annulata. these two close and rare species are very similar in larval stage; however they are easily distinguishable as adult. it seems the occurrence of cs. subochrea in guilan province is possible, especially in view of the recent finding of both species in ardebil province, northwestern iran (azari-hamidian et al. 2009). there is little information about taxonomy and distribution of three close species cs. alaskaensis (ludlow), cs. annulata, and cs. subochrea in iran (azarihamidian et al. 2003b, azari-hamidian 2005). recently, cs. subochrea and cs. longiareolata were found in sanandaj county of kurdistan province (moosa kazemi et al. 2010). danilov (1975) was the unique reliable record of cx. torrentium in rasht of guilan province and iran. recently the larvae of this species was found in ardebil province (azari-hamidian et al. 2009) and the mountainous areas of the western part of guilan province (heyran) (with cx. pipiens; azarihamidian, unpublished data), thus its occurrence in iran, guilan province, and for the first time in ardebil province was verified. all 30 species, which have been discussed above, were mentioned in the checklist in the present article. there is a unique record of cx. quinquefasciatus say in enzeli of guilan province by harbach (1988). there is no more information about this record (harbach, personal communication). this is out of the usual distribution of this species that occurs in southern iran (zaim 1987b), so it is not mentioned in the checklist. taxonomy, distribution, and ecology of the pipiens subgroup of the pipiens group of culex (culex) need to be studied extensively in iran. beklemishev and shipitzina (1947) found an. marteri in the valley of the river yuzbashchai between qazvin (qazvin province) and rasht (guilan province); however there is no record of this species in guilan province. the species complexes and the mosquito fauna of the forest areas of the iran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 50 province are important subjects to study in future taxonomic investigations. acknowledgements the author is grateful to j minar, institute of parasitology, czech republic, for studying specimens, jf reinert, center for medical, agricultural and veterinary entomology, gainesville, for providing some articles, j jezek, natural history museum of prague, prague, i gelbic, institute of entomology, czech republic, f rettich, national institute of public health, prague, for their important correspondences and helps. the author also thanks ma oshaghi, department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran, for reviewing the manuscript and s omidi, school of health, guilan university of medical siences, rasht, iran, for assisting in statistical analysis. this study received financial support from guilan university of medical sciences. references azari-hamidian s, yaghoobi-ershadi mr, javadian e (2001) a faunistic study of mosquito (diptera: culicidae) fauna in rasht county (iran). modarres j med sci. 3: 65–70 [persian with english abstract]. azari-hamidian s, yaghoobi-ershadi mr, javadian e (2002a) the distribution and larval habitat characteristics of mosquitoes (diptera: culicidae) in rasht county (guilan province, iran). modarres j med sci. 4: 87–96 [persian with english abstract]. azari-hamidian s, joeafshani ma, mosslem m, rassaei ar (2002b) taxonomic survey of mosquitoes (diptera: culicidae) in guilan province with reporting of a subgenus new to iranian mosquito fauna. the 15th iranian plant protection congress, 7–11 september 2002, razi university of kermanshah, kermanshah, iran. pp. 319–320 [persian with english abstract]. azari-hamidian s, joeafshani ma, rassaei ar, mosslem m (2002c) mosquitoes of the genus aedes (diptera: culicidae) in guilan. j med fac guilan univ med sci. 11(43): 29–39 [persian with english abstract]. azari-hamidian s (2003) on the tree hole mosquito (diptera: culicidae) fauna, ecology and medical importance in guilan province. the 4th national iranian congress of parasitology and parasitic diseases, 13–16 october 2003, mashhad, iran. p. 293 [persian with english abstract]. azari-hamidian s, joeafshani ma, mosslem m, rassaei ar (2003a) adult mosquito habitats and resting-places in guilan province (diptera: culicidae). hakim. 6: 55–62 [persian with english abstract]. azari-hamidian s, joeafshani ma, rassaei ar, mosslem m (2003b) faunistic studies on the genus culiseta (diptera: culicidae) in guilan province. j kerman univ med sci. 10: 225–233 [persian with english abstract]. azari-hamidian s, joeafshani ma, rassaei ar, mosslem m, mousavi-eivanaki e (2004a) mosquito fauna of the genus anopheles (diptera: culicidae) in guilan province. modarres j med sci. 6: 11–22 [persian with english abstract]. azari-hamidian s, joeafshani ma, mosslem m, rassaei ar (2004b) notes on coquillettidia richiardii and uranotaenia unguiculata (diptera: culicidae) in guilan province. j med fac guilan univ med sci. 13(51): 1–9 [persian with english abstract]. azari-hamidian s (2005) larval habitat characteristics of mosquitoes of the genus culiseta felt, 1904 (diptera: culiiran j arthropod-borne dis, 2011, 5(1): 37–53 s azari-hamidian: larval habitat characteristics … 51 cidae) in the caspian sea littoral, iran. zool middle east. 36: 59–66. azari-hamidian s, joeafshani ma, mosslem m, rassaei ar (2005) mosquito fauna and distribution of the genus culex (diptera: culicidae) in guilan province. pajouhesh and sazandegi. 68: 39– 45 [persian with english abstract]. azari-hamidian s (2006) on the ecology of the tribe aedini (diptera: culicidae) larvae in the caspian sea littoral, iran. the 11th international congress of parasitology, 6–11 august 2006, glasgow, scotland, uk. azari-hamidian s, abai mr, ladonni h, vatandoost h, akbarzadeh k (2006) anopheles peditaeniatus (leicester) new to the iranian mosquito fauna with notes on anopheles hyrcanus group in iran. j am mosq control assoc. 22: 144–146. azari-hamidian s (2007a) checklist of iranian mosquitoes (diptera: culicidae). j vector ecol. 32: 235–242. azari-hamidian s (2007b) larval habitat characteristics of mosquitoes of the genus culex (diptera; 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(1945) anopheles maculipennis of northern iran. med parasitol parasit dis. 14: 50–57 [russian]. j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 35 original article analysis of immunogenic relevant proteins in rhipicephalus (boophilus) annulatus tick *a nikpay 1, s nabian 2, m taheri 2 1department of pathobiology, faculty of veterinary medicine, university of mazandaran, amol, iran 2department of parasitology, faculty of veterinary medicine, university of tehran, tehran, iran (received 19 may 2010; accepted 6 nov 2011) abstract background: considering the importance of ticks as a main group transmitting pathogen organisms, this study designed to recognize immunogenic proteins in different tissues of rhipicephalus (boophilus) annulatus tick and to find out if there are common proteins in these tissues. methods: seven cattle were experimentally infested with about 10000 r. annulatus larvae and their humoral immune response to extracts of salivary gland and ovary of adult ticks and larval extracts during infestation were determined by elisa and western blot analysis. measurements of serum antibodies level recorded weekly, from week 0 to week 9. results: using western blot analysis, 15 fractions from soluble antigens extracted from salivary gland and larvae, and 14 fractions in the larval extracts were recognized. these findings illustrate the recognition of common antigens with molecular weight of 170, 117, 100, 70, 37, 33 and 30 kda from different antigens by resistant cattle sera. conclusion: common antigens are present in different tissues of rhipicephalus (boophilus) annulatus, which can be used as a target in immunization against ticks. keywords: rhipicephalus (boophilus) annulatus, salivary gland, ovary, larva, immunoblot, elisa introduction cattle develop resistance to ticks following natural infestation (reik 1962, dipeolu et al. 1992, cruz et al. 2008) and vaccination (opdebeek et al. 1989, willadsen et al. 1988, 1999, 2006, wikel 1999a, b, ogden et al. 2002, trimnel et al. 2005, leal et al. 2006, perez 2010). it has been shown that infested animals with boophilus microplus induce an immune response and acquire a partial resistance to subsequent infestations, in which the internal organs of the ectoparasite become damaged, leading to an increase of feeding time, a decrease in the numbers of engorged females, less uptake of blood meal, a decrease in the number and viability of eggs (wikel 1988). there is a general view, however, that a vaccine with only one antigen will not be sufficient to generate an effective immune response able to control the proliferation of the ticks under field conditions (willadsen 1990). salivary gland, gut, embryo, and larval extracts of various ticks have been studied as possible target for vaccine. however, these tissues have been examined independently (wikel 1984, wiladsen et al. 1988, wong and opdebeek 1989). we examined the cattle immune response to salivary gland, ovary, and larval extracts of r. annulatus tick at the same time with the aim of finding common antigens, which in vaccination protocol could be advantageous upon stage, or organ specific antigens. the tick r. annulatus is an important ectoparasite *corresponding author: dr ali nikpay, e-mail: ali.nikpay@gmail.com 36 j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 2 of cattle that present in west africa, central africa, asia, certain parts of southern sudan and europe. in this study, for the first time, salivary gland, ovary, and larval extracts of this species have been studied at the same time as possible target antigens to determine the cattle immune response to them. materials and methods tick infestation seven healthy 3-5 months old holstein cattle being negative sera for tick infestation were provided from tehran veterinary medicine faculty research institute (amin abad). they were housed in tick-proof pens independently. each cattle was infested with about 10000 r. annulatus larvae as described by brown (1988). briefly, tubes containing larvae fastened to a shaved flank of cattle with adhesive tape. then, the surface of the infestation area was covered with a piece of cloth. antigen preparation partly engorged female ticks were washed with 70% ethanol and then washed three times with sterile distilled water. after drying, they were maintained at 28o c and 85% relative humidity. the antigens used in the elisa were extracts of salivary glands, ovaries, and larvae. salivary glands and ovaries were dissected with fine-tipped forceps in cold pbs, rinsed in fresh pbs and then each of these organs and tick larvae were homogenized in a glass tissue grinder separately in pbs containing 1 mm phenyl methyl sulphonyl fluoride at 4o c following sonicated for 30 minnutes on ice with 40 w. the homogenates centrifuged at 20000 g for 30 min at 4o c, the supernatant were stored at -70o c. the proteins of the extracts were determined using the method of warburg (tietz 1986, hudson and hay 1994). sera collection positive and negative references sera collected from infested and uninfested cattle were used to standardize the elisa test. the positive sera were obtained by infesting seven three months-old calves with 10,000 of r. annulatus larvae. weekly for 12 consecutive weeks, blood was collected by jugular vein and allowed to clot for 2 hour at room temperature and then centrifuged at 800 g for 15 min and extracted serum aliquoted and kept at -20o c. elisa elisa plates were coated with 4 µg per well of three antigens in 20 mm carbonate buffer (ph= 9.6) by incubation overnight at 4o c (harlow and lane 1988). having been washed three times, the plates were incubated for 1 hour at 37o c with 5% skim milkpbs. then, test sera diluted 1/200 were incubated at 37o c. after 1 hour, the plates were washed three times and 100 µl of 1/2000 diluted sheep anti –bovine igg conjugated with peroxidase were added to the individual wells. after incubation at 37o c for 1 hour, the plates were washed again and the enzyme substrate was added. the substrate was prepared by dissolving 62 mg of (2, 2, azino-bis 3-ethyl benzthiazoline-6sulfonic acid) in 50 ml of distilled water and the optical density (od) was determined at 405 nm. sera from cattle were tested against anti r. annulatus antibodies at 1-week intervals from week 0 (the week of infestation) to week 9. sds-page electrophoresis of different tissues tick extract preparations (20 µg/lane) were resolved by sodium dodecyl sulfate polyacrylamid gel electrophoresis (sds-page) using a discontinuous gel system in reducing condition (laemmli 1970) with an acrylamide concentration of 5% in stacking gel and 12% in 37 j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 3 the separation gel. a high molecular weight protein ladder (fermentas) was used as molecular weight markers. finally, the gel was stained with coomassie brilliant blue. western blot analysis soluble proteins extracted from salivary glands, ovaries and larvae of r. annulatus (20 µg/lane) were resolved by sds-page using a discontinuous gel system as described previously (laemmli 1970). the antigens separated were transferred to nitrocellulose paper (ncp) (wang and nuttal 1994), and nonspecific reactive sites on the ncp were blocked for 1h in tris-buffered saline with 0.5% tween 20 (tbs-t ph8), containing 3% bovine skim milk on a shaking plate at room temperature. the ncp was incubated for 1 h at 37o c with bovine sera diluted 1/50 in pbs (ph= 7.2). the ncp was incubated for a further 1 h at 37oc with sheep antibovine igg (conjugated with hrpo) diluted 1/2000 in pbs-tween20. after 1 hour, the paper was washed and reacted with di amino benzidine (dab) (sigma) as a substrate. the ncp was washed three times between each step of the assay with pbs-tween. results characterization of humoral immune response the experimental infestation of seven cattle with approximately 10000 larvae of r. annulatus was performed to characterize the humoral immune response to tick extracts. furthermore, approximately 2500±252 ticks were collected per animal between days 21 and 26. figure 1 shows changes of antibody levels measured by elisa in the sera of the experimentally infested animals against salivary glands, ovaries, and larvae antigens. all extracts showed positive reactions with all sera tested starting from the first week of infestation. a steady increase of the antibody level was observed during the experiment from week 0 to week 9, however, a slight decrease antibody production occurred in the week 9 of infestation. sds-page electrophoresis of different tissues coomassie brilliant blue stain differentiates a vast profile of proteins in tick’s antigens. in salivary gland, ovary and larval extracts 22, 18, and 25 fractions were more dominant respectively. protein bands ranging in molecular weight from 18 to more than 200 kda .the bands >200, 170, 117, 100, 93, 70, 55, 50, 44, 37, 30 and 27 kda were common in all extracts (see in fig. 2). western blot analysis of salivary, ovary antigens and larva extracts in order to analyze the peptides reacting with the bovine antibodies, western blot assays were carried out using the sera with higher elisa od that belonged to 21st day after infestation. the profile of three different antigens recognized by cattle positive sera are presented in fig.3. serum from experimentally infested cattle recognized approximately 15 fractions from soluble antigens extracted from salivary gland and larvae. challenged cattle also recognized 14 protein bands in the ovary extract. the negative control sera recognized no band. these findings illustrate the recognition of common proteins with molecular weight of 170, 117, 100, 70, 37, 33 and 30 kda from different antigens by sera of infested cattle. discussion it is extremely important to have defined immunogenic molecules in order to dissect the events involved in acquisition and expression of tick resistance. some studies clearly show that a number of tick tissues can be used to induce artificial resistance to tick feeding, an approach that holds significant 38 j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 4 promise as an alternative method for tick control (wickel et al. 1988, turni et al. 2004, leal et al. 2006, willadsen 2006, cruz et al. 2008). antibodies reactive with tick extracts have been described by several researchers (willadsen 1980, wikel 1984). immunoelectrophoresis, immunofluorescence, elisa (wikel and osburn 1982, brossard and wikel 1997, cruz et al. 2008, perez 2010, kopp et al. 2010) have been used to examine serological responses against ticks. many investigators have used the western blot technique to identify relevant antigens of several tick species using sera collected from different animal hosts (whelen et al. 1986, brown 1988, trimnel 2005, cruz et al. 2008). salivary glands, gut and larval extracts have been tested as immunogens to control tick infestation (opdebeeck et al. 1989, barriga et al. 1991). da silva vaz et al. (1994) also indicated that rhipicephalus (boophilus) microplus infested cattle develop antibodies to components of tick salivary gland, gut, embryo and larvae during the first infestation. fig. 1. the comparison of serum antibody response changes (od value changes) in seven calves infested with r. annulatus using different tissues antigens. results expressed as mean of seven infested bovine sera 39 j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 2 fig. 2. sds-page analysis of proteins derived from salivary glands, ovary and larvae of rhipicephalus (boophilus) annulatus. the numbers in left side of each column indicate the molecular weight (kd) of each fraction 40 j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 3 fig. 3. western blot analysis of r. annulatus antigens by tick infested bovine sera (positive serum). the numbers in left side of each column indicate the molecular weight of each fraction in the present study, elisa was used to compare the antibody levels developed against r. annulatus salivary gland, ovary and larvae protein extracts of sera from cattle submitted to experimental infestation. the results of this part of our study showed the impact of approximately high-density tick infestation on humoral immune responses induced in experimentally infested cattle suggesting the antibody level increased during the period of infestation and as mentioned in fig. 1, a significant increase was observed in third week after infestation that the greatest amount of ticks were harvested at this time. these results are in agreement with our previous study and some other researchers (oegden et al. 41 j arthropod-borne dis, 2011, 6(1): 36–44 a nikpay et al.: analysis of immunogenic relevant ... 2 2002, cruz et al. 2008, nikpay et al. 2008, perez 2010). using western blot analysis, some basic information have provided about antigens extracted from r. annulatus tick recognized by sera collected from cattle infested with this species. the recognized fractions could be used as target for subsequent antigen characterization studies, and points out some probable antigenic sites in specific tick tissues. elisa and western blot data indicate that r. annulatus infested cattle develop antibodies to components of tick salivary glands, ovaries and larvae during the infestation. in our experiment, all extracts showed the same absorbance value changes. one possible explanation of the similarities in the reactivity of different tissues is that the bovine is inoculated with various tissue antigens during the tick feeding process, or that there are common antigens in different tissues of r. annulatus and according to these results it seemed to be a correct assumption. these findings further illustrate the presence of common crucial antigens, which are believed to be 170, 117, 100, 70, 37, 33 and 30 kda depending upon the assay system employed because they were recognized in three extracts by anti tick sera. electerophoretic comparison of salivary gland extracts of r. annulatus specimens demonstrated some similar proteins with salivary gland extracts of hyalomma specimens (nabian et al. 2005). also it was shown that some proteins of salivary gland of r. annulatus tick changed in different temperatures but protein bands of 29, 36, 55 and 97 were constant at all temperatures (nabian et al. 2005). for the first time in the current study salivary glands, ovary and larvae extracts have been used as antigenic sources at the same time and it was showed that ovaries could be a source of immunogenic proteins such as the other tissues of tick studied previously. it should be mentioned that there was not any similar study about r. annulatus tick and this investigation was conducted for the first time in iran to characterize the immunogenic antigens in this species. the findings of the present study establish the presence of common antigens between ovary, larvae, and salivary glands of r. annulatus ticks. further studies will be aimed at the isolation of common proteins and testing for antigenicity and immunogenicity followed by purification and subsequent in vitro expression for potential vaccine development. vaccination of hosts with antigenic tissues and secretions of ticks may provide a new method to control ticks. extracts of whole tick body homogenates and various organs especially have been tested as to their ability to induce tick resistance in the host (mcgowan et al. 1980). the significance of these observations is with regard to the induction of host immunity to ticks and the development of tick vaccines using common proteins. acknowledgments authors wish to acknowledge the financial support of the research council of tehran university and veterinary medicine faculty of tehran university. the authors declare that there is no conflict of interests. references barriga oo, andujar f, andrzejewski wj (1991) manifestation of immunity in 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phlebotomine sand flies … ١ review article phlebotomine sand flies (diptera: psychodidae) in iran and their role on leishmania transmission mr yaghoobi-ershadi department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran (received 20 may 2012; accepted 11 june 2012) abstract sand fly research has a long history in iran beginning with the work of adler, theodor and lourie in 1930 and followed by mesghali’s foundational taxonomic work on sand flies in 1943. since then, research has been continued unabated throughout the country and official publications report the existence of at least 44 species of sand flies (26 of the genus phlebotomus and 18 of genus sergentomyia) in iran. so far, seven phlebotomus species and one sergentomyia species have been collected and described by iranian researchers for the first time. natural promastigote infections have been repeatedly found in 13 species of sand flies and modern molecular techniques are used routinely to characterize leishmania parasite isolates from endemic areas of cutaneous and visceral leishmaniasis. because of anthropogenic environmental modifications or human population movements, data on phlebotomine sand flies should be regularly updated and verified at least every five years by fieldwork and taxonomy in foci of leishmaniasis, to incriminate vector species of relevance to the ecology of transmission and to support development and implementation of control programs. keywords: sand flies, taxonomy, vectors, leishmania major, leishmania tropica, leishmania infantum, leishmaniasis, iran introduction cutaneous and visceral leishmaniasis are ancient endemic diseases in iran (maimandinezhad 1965) and continue to be a growing health threat to community development and the environment in the country. cutaneous leishmaniasis is endemic in two forms, anthroponotic cutaneous leishmaniasis (acl) and zoonotic cutaneous leishmaniasis (zcl). about 20,000 cases of leishmaniasis (including acl, zcl and zoonotic visceral leishmaniasis) are reported annually but the real figures are 4–5 folds. anthroponotic cutaneous leishmaniasis is still a neglected tropical disease in many parts of the country. it was greatly reduced in many foci by malaria control measures but many foci remained active in some large and medium sized cities such as tehran, mashhad, neishabur and sabzevar in the north-east, shiraz in the south, kerman and bam in the southeast, yazd, kashan and parts of the city of esfahan in the central region (nadim and tahvildare-bidruni 1977, yaghoobi-ershadi et al. 2002). the parasite is leishmania tropica and the vector is supposed to be phlebotomus (para-phlebotomous) sergenti parrot 1917. the main reservoir host is human but dogs have a role as animal reservoir host and active lesions in dogs have been reported in tehran, mashhad, shiraz and neishabur (yaghoobi-ershadi et al. 2002, yaghoobi-ershadi 2008). zoonotic cutaneous leishmaniasis is endemic in many rural areas of 17 out of 31 corresponding author: prof mohammad reza yaghoobiershadi, e-mail: yaghoobi.reza@gmail.com 1 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ٢ provinces and still is a great health problem in iran. about 80% of cases reported in the country are of zcl form (yaghoobi-ershadi and javadian 1995, yaghoobi-ershadi et al. 2001b, 2003, akhavan et al. 2007). rhombomys opimus, the great gerbil, is the main animal reservoir in foci in the north-east and central part of the country, meriones libycus, the libyan jird, is considered the principal reservoir host in some parts of central and south of the country. tatera indica, the indian gerbil, is the main reservoir host in the southeast and meriones hurrianae, the desert gerbil, is the reservoir in southeastern part of iranian baluchistan, neighboring pakistan (yaghoobiershadi and javadian 1996a, 1996b, akhavan et al. 2010). phlebotomus papatasi scopoli 1786, the most prevalent species among phlebotomus genus, is the only known vector (nadim et al. 1968c, yaghoobi-ershadi and javadian 1997). cases of zoonotic visceral leishmaniasis (zvl) have been reported from all parts of the country (nadim et al. 1978). about 9,000 parasitological confirmed human cases have been diagnosed and registered from all over the country during the period 1954–2011 (school of public health, unpublished data) and most of the cases are under five years old. the epidemiological studies show that the mediterranean type of kala-azar occurrs in different parts of iran. zoonotic visceral leishmaniasis is caused by leishmania infantum lon 49 (alborzi et al. 2007) and 4 sand fly species belonging to larroussius subgenus and one species of paraphlebotomus subgenus are the probable vectors of zvl (yaghoobiershadi 2011). dogs, foxes, jackals and wolves have been found infected in various parts of the country but dogs are the main reservoir hosts (navid-hamidi et al. 1982, nadim 2008). there are seven endemic foci in the country including fars province in the south, ardabil province in the northwest, azarbaijan-e-sharqi province in the northwest, khorramabad area (lorestan province) in the west, khuzestan province in the southwest and khorasan-eshomali province in the northeast. at the moment most of zvl cases are reported from fars and ardabil provinces which both are tribe areas of iran (nadim 2008). historical overview on sand flies from iran phlebotomine sand flies of iran have been studied since 1930 by a limited number of iranian and foreign entomologists such as adler, theodor and lourie, who surveyed a small area of the north western part of the country including kermanshah, hamadan, tehran and rasht. seven species were found, two of which proved new to science (adler et al. 1930). mesghali (1943) was the first iranian to conduct basic studies on sand flies in iran. due to the importance of sand flies and leishmaniasis and the special interest of n. ansari, professor and chairman, department of parasitology, university of tehran, school of medicine, mesghali was appointed to carry out a study on the status and distribution of sand flies in iran. scientific research had begun at the same time in the country and was greatly facilitated with the establishment of the institute of parasitology and malariology in 1952. survey teams sent out by this institute during the preparatory phase of the malaria eradication program or for studying of parasitic diseases were also utilized for the compilation of data on sand flies (mesghali 1961). pervomaiski (1948) focused on sand flies in a small area in northern iran, near the caspian sea in the elburz mountains. lewis (1957) collected sand flies in the northeastern part of iran (khorassan, sabzevar area), described a new species, phlebotomus ansarii lewis 1957 and reported on four species of sergentomyia, three of which represented new country records. subsequent work by lewis (1961), mesghali (1964, 1965, 1968), theodor (1964), nadim (1964, 1968, 1970), javadian (1975, 1997a) and seyedi-rashti (1968, 1970) showed that the number of species of both 2 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ٣ genera occurring in the country was much more than reported by earlier workers. in the first general review of the sand fly fauna of iran, mesghali (1961) reported 10 phlebotomus and sergentomyia species. in 1976 a key was prepared by nadim and javadian in which they reported 19 phlebotomus species and 17 sergentomyia species. in 1990, seyedi-rashti produced a pictorial key to 27 phlebotomus and 16 sergentomyia species (school of public health, tehran university of medical sciences, unpublished data). due to the importance of this subject and the special interest of iranian researchers, sand fly fauna studies have been updated in recent years with the aid of modern molecular techniques. current times (2007–2011) in 2007, an intraspecific study on the morphological and molecular characteristics based on mtdna sequences of ph. sergenti s.l., was performed on 28 iranian populations from 11 provinces. three morphotypes were identified as a, b and c. morphotype a was considered as ph. sergenti sergenti, morphotype b was considered ph. sergenti similis, and morphotype c need further studies (moin-vaziri et al. 2007a). also in 2007, these same workers carried out a comparative morphological, morphometrical and molecular study on 11 different populations of ph. caucasicus from seven provinces. the results showed that the species includes two morphotypes: c and g. morphotype c, described as ph. caucasicus, reported in all foci of zcl. but morphotype g, ph. grimmi, was collected only in three provinces in the northwestern part of the country that are free zcl (moin-vaziri et al. 2007b). in 2009, the population structure of ph. papatasi was studied based on multi-locus microsatellite typing of sand flies collected from seven provinces. the results showed that there are two population variants of ph. papatasi: a2 from western iran and a3 from eastern iran ((hamarsheh et al. 2009). more recently in 2010, a molecular study on sand flies from three iranian provinces showed ph. caucasicus and ph. mongholensis to be indistinguishable by the mitochondrial cytochrome b gene (parvizi et al. 2010). during 2009–2010 a preliminary morphological and morphometric study was performed on two populations of ph. major s.l. from endemic and nonendemic foci of visceral leishmaniasis. the results showed that there were two morphotypes (badakhshan et al. 2011a). based on its2 and ef-1α sequences, these two taxa were separated into two well-defined lineages. moreover, the close similarity of these morphotypes to phlebotomus neglectus specimens deposited by esseghir et al. (2000) in genbank, was highly informative, suggesting that there are two populations of ph. neglectus in the country (badakhshan et al. 2011b). so far eight new species of sand flies including seven phlebotomus species and one sergentomyia species have been collected and described by iranian researchers for the first time as follows: (i) ph. (syn.) ansarii lewis, 1957, (ii) ph. (adl.) brevis theodor and mesghali, 1964, (iii) ph. (lar.) ilami javadian, 1997, (iv) ph. (par.) kazeruni theodor and mesghali, 1964, (v) ph. (eup.) mesghalii rashti and nadim, 1970, (vi) ph. (eup.) nadimi javadian, 1997, (vii) ph. (phl.) salehi mesghali and rashti, 1968, (viii) se. (par.) iranicus lewis and mesghali, 1961. current composition of sand fly fauna of iran national and international publications on sand flies show that in iran the number of sand fly species account for a total of 44 (table 1). sand fly fauna include 26 phlebotomus species of 6 subgenera and 18 sergentomyia species of 6 subgenera. phlebotomus sergenti sergenti and 3 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ۴ ph. sergenti similis reported in table 1 are not counted, being considered subspecies. the presence of further seven species including: ph. (adl.) kabulensis artemiev, 1978, ph. (adl.) salangensis artemiev, 1978, ph. (lar.) langeroni nilzulesco, 1930, ph. (transphlebotomus.) mascittii grassi, 1908, ph. (lar.) smirnovi perfiliew, 1941, ph. (eup.) caudatus artemiev, 1978, ph. (adl.) turanicus artemiev, 1974 -doubtful in the country, because iranian senior sand fly specialists have not confirmed them yet and must be verified by further studies. most entomologists believe that the sand flies of iran are predominantly palearctic, with mediterranean elements entering from the northwest and central asiatic elements from the northeast. the area along the persian gulf contains both indian and african elements entering through southern arabia (theodor and mesghali 1964). the emergence of new sand fly populations in iran may have resulted from the introduction recently or historically with people who settled in the agricultural areas stretching from the nile river to the southern coast of the mediterranean sea, around the syrian desert and north of saudi arabia and yemen to the persian gulf. known as the fertile crescent, this area has an impressive record of past human activity and transportation, including migrations of some of the earliest known peoples (hamarsheh et al. 2009). studies on leishmania vector incrimination studies on the vectors of various leishmanias in iran began in 1964 and continue, especially in newly discovered foci. natural promastigote infections have been found repeatedly in 13 species of sand flies (10 phlebotomus and 3 sergentomyia species). phlebotomus sergenti s.l. natural promastigote infections have been found in this species in two acl foci due to, mashhad in the northeast with an infection rate of 1.5% (mesghali et al. 1967) and esfahan city in central iran with an infection rate of 0.1 % (nadim and seyedi-rashti 1991). phlebotomus sergenti was reported as l. tropica host by pcr in shiraz city, south of iran (oshaghi et al. 2010). two (66.6%) out of 3 ph. sergenti females have been found infected with leishmania turanica based on pcr detection of parasite its2-rdna in the city of bushehr, southwestern iran in 2011 (yaghoobi-ershadi, unpublished data). phlebotomu sergenti has a wide distribution in the country that includes and extends beyond the distribution of l. tropica. phlebotomus papatasi the main vector of leishmania major to humans and gerbils is ph. papatasi. in almost all zcl foci natural promastigote infections have been found in this species, ranging from 0.2 to 10.9% in flies captured from rodent burrows during 1967–1990 with an average infection rate of 5.6%. (yaghoobi-ershadi and akhavan 1999, yaghoobi-ershadi et al. 2001c). in 1991, leishmania promastigotes isolated from this species in central iran, they were typed at the faculty of medicine in montpellier by the examination of 15 isoenzymes and identified as l. major zymodeme mon26 (yaghoobi-ershadi et al. 1995b). natural infections with leishmania promastigotes were monitored in sand flies from rodent burrows in borkhar in central iran from late june to mid october 1991. phlebotomus papatasi infections appeared at the end of july peaked in september with a rate of 17.3% and fell to zero early in october (yaghoobi-ershadi and javadian 1996b). specimens of this species collected from rodent burrows in villages of badrood, in central iran in 1998 were also found to be naturally infected with l. major zymodeme mon-26. promastigote infection rates varied between 6.7–22% during the sand 4 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ۵ fly season, being greatest in september, coincident with second activity peak of ph. papatasi, 2–3 months before the highest incidence of human zcl cases in novemberdecember. the promastigote infection rate was 1.1% in 94 ph. papatasi in indoors (yaghoobiershadi et al. 2001a). during the last decade on many occasions l. major isolates from ph. papatasi have been detected and identified by nested-pcr, semi nested pcr and rapd-pcr or isoenzyme characterization in different foci of zcl in the country (yaghoobi-ershadi and akhavan, 1999, yaghoobi-ershadi et al. 2001c, rassi et al. 2008, oshaghi et al. 2008). parvizi and ready (2008) conducted surveys in two foci of zcl in esfahan and golestan provinces located in the central and northeastern parts of the country respectively, detected and identified l. major, l. turanica and leishmania gerbilli in ph. papatasi collected from gerbil burrows and animal shelters using nestedpcr and sequencing of nuclear its-rdna fragments. this species plays the key vectorial role in the transmission cycle of zcl due to l. major in many rural areas of 17 out of 31 provinces of the country (fig. 1). phlebotomus caucasicus, phlebotomus mongolensis and phlebotomus andrejevi besides ph. papatasi, the three species, ph. caucasicus, ph. mongoloensis and ph. andrejevi, are believed to maintain the enzootic cycle of l. major among gerbils and jirds. the females of these sand flies are not distinguishable morphologically. natural promastigote infection rates in ph. caucasicus from 3.3–20% have been reported in specimens collected from rodent burrows, with frequent observation of parasites in the anterior midgut and head (yaghoobi-ershadi and javadian 1996b, 1997). in 1994, promastigotes were isolated from heavily infected ph. caucasicus collected from gerbil and jird burrows in the main focus of esfahan and typed by isoenzymes as l. major zymodeme mon-26 at the who leishmania reference center, faculty of medicine, university of montpellier, france (yaghoobi-ershadi et al. 1994). the same leishmania species was isolated again from ph. caucasicus collected in gerbil burrows in jarghooyeh rural district, southeastern esfahan in 1997 (yaghoobi-ershadi et al. 2001c). in 2008, l. major and leishmania near gerbilli were detected and identified in ph. caucasicus by nested pcrs and dna sequence analysis from specimens collected in rodent burrows and animal shelters in esfahan, central iran and golestan province in northeastern iran respectively (parvizi and ready 2008). these findings combined with a human blood index of 20% provide strong evidence that ph. caucasicus is indeed a natural vector of l. major in central iran and a secondary vector to humans in the country (yaghoobi-ershadi et al. 1995a). phlebotomus ansarii this species has been found so far only in iran. several studies have reported infection rates ranging from 3.7–11.5% in specimens collected from rodent burrows in the esfahan focus of zcl central iran (nadim et al. 1968c, yaghoobi-ershadi and javadian 1996). in 2008 l. near gerbilli was identified by nested pcr of its-rdna in specimens of this species collected from gerbil burrows in esfahan (parvizi and ready 2008). phlebotomus salehi this species is considered to be a vector in the enzootic cycle of l. major among gerbils and has been collected in rodent burrows on the plain of chabahar in baluchistan and also hormozgan provinces in southern iran (mesghali 1965, mesghali and seyedirashti 1968). the average natural promastigote infection rate in specimens collected from rodent burrows in southeastern iran was 1.07% in 1997 (kasiri and javadian 2000). 5 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ١ genus sub-genus species phlebotomus adlerius nitzulescu, 1931 1. ph. (adl.) balcanicus theodor, 1958 2. ph. (adl.) brevis theodor and mesghali, 1964 3. ph. (adl.) halepensis theodor, 1958 4. ph. (adl.) longiductus parrot, 1928 euphlebotomus theodor, 1948 5. ph. (eup.) mesghalii seyedi-rashti and nadim, 19706. ph. (eup.) nadimi javadian, 1997 larroussius nitzulescu, 1931 7. ph. (lar.) ilami javadian, 1997 8. ph. (lar.) kandelakii shchurenkova, 1929 9. ph. (lar.) keshishiani shchurenkova, 1936 10. ph. (lar) neglectus tonnir, 1921 11. ph. (lar.) perfilievi perfiliev, 1937 12. ph. (lar.) tobbi adler and theodor, 1930 13. ph. (lar.) wenyoni adler and theodor, 1930 paraphlebotomus theodor, 1948 14. ph. (par.) alexandri sinton, 1928 15. ph. (par.) andrejevi shakirzyanova. 1953 16. ph. (par.) caucasicus marzinowsky, 1917 17.ph.(par.) grimmi porchinsky,1876 18. ph. (par.) jacusieli theodor, 1947 19. ph. (par.) kazeruni theodor and mesghali, 1964 20. ph. (par.) mongolensis sinton, 1928 21. ph. (par.) sergenti s.l. parrot, 1917 ph. sergenti sergenti parrot 1917 ph. segenti similis perfiliew, 1963 phlebotomus rondani, 1840 22. ph. (phl.) bergeroti parrot, 1934 23. ph. (phl.) papatasi (scopoli), 1786 24. ph. (phl.) salehi mesghali and rashti, 1968 synphlebotomus theodor, 1948 25. ph. (syn.) ansarii lewis, 195726. ph. (syn.) eleanorae sinton, 1931 sergentomyia grassomyia theodor, 1958 27. se. (gra.) dreyfussi parrot, 193328. se. (gra.) squamipleuris (newstead), 1912 parrotomyia theodor, 1958 29. se. (par.) africana newstead, 1912 30. se. (par.) baghdadis (adler and theodor), 1929 31. se. (par.) grekovi (khodukin), 1929 32. se. (par.) palestinensis (adler and theodor), 1927 33. se. (par.) sogdiana (parrot), 1929 34. se. (par.) sumbarica (perfiliev), 1933 parvidens theodor and mesghali, 1964 35. se. (par.) iranicus lewis and mesghali, 1961 rondonomyia theodor, 1958 neophlebotomus franca and parrot, 1920 36. se. (ron.) pawlowskyi (perfiliev), 1933 sergentomyia franca and parrot, 1920 37. se. (ser.) antenata newstead, 1912 38. se. (ser.) dentata sinton, 1933 39. se. (ser.) mervynae pringle, 1953 40. se. (ser.) theodori (parrot), 1942 41. se. (ser.) sintoni pringle, 1933 sintonius nitzulescu, 1931 42. se. (sin.) christophersi (sinton), 1927 43. se. (sin.) tiberiadis (adler, theodor and lourie), 1930 44. se. (sin.) clydei (sinton), 1928 table 1. list of sand fly species recorded in iran 6 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ٢ table 2. proven or suspected vector species present in iran, leishmnaia agent transmitted and endemic provinces proven or suspected vector leishmania agent transmitted province references ph. papatasi* l. major esfahan (nadim et al. 1968c), (yaghoobi-ershadi et al. 1995, 2001b) khorasan-e-razavi (mesghali et al. 1967), (yaghoobi-ershadi et al. 2003) khorasan-e-shomali (javadian et al. 1976) golestan (nadim et al. 1968b), (parvizi and ready 2008) yazd (yaghoobi-ershadi et al. 2004, 2007) kerman (yaghoobi-ershadi, 2010), (akhavan et al. 2007) khuzestan (javadian et al. 1977b), (javadian and ranjbar, 1990) qom (akhavan et al. 2003), (rassi et al. 2011b) tehran (seyedi-rashti and salehzadeh, 1990) sistan and baluchestan (seyedi-rashti and nadim, 1984a), (kasiri and javadian, 2001) hormozgan (hanafi-bojd et al. 2007) bushehr (yaghoobi-ershadi,unpublished data) fars (rassi et al. 2007), (azizi et al. 2010) semnan (rassi et al. 2011a) ilam (javadian and ranjbar, 1990) ph. papatasi l. turanica esfahan (parvizi and ready, 2008) golestan (parvizi and ready, 2008) semnan (rassi et al. 2011a) l. near gerbilli esfahan (parvizi and ready, 2008) ph. sergenti l. tropica khorasan-e razavi (mesghali et al. 1967), (nadim and seyedi-rashti, 1991) esfahan (javadian and seyedi-rashti, 1991) yazd (yaghoobi-ershadi et al. 2002) kerman (yaghoobi-ershadi 1977), (seyedi-rashti et al. 1984b), (nadim and aflatoonian, 1995) fars (nadim and seyedi-rashti, 1971), (oshaghi et al. 2010) tehran (nadim and seyedi-rashti,1991) l. near gerbilli esfahan (parvizi and ready, 2008) ph. caucasicus ph .mongolensis ph .andrejevi l. major esfahan (yaghoobi-ershadi et al. 1994, 1995a, 2001c), (parvizi and ready, 2008) l. near gerbilli esfahan (parvizi and ready, 2008) golestan (parvizi and ready, 2008) 7 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ٣ ph. ansarii l. major esfahan (nadim et al. 1968c), (yaghoobi-ershadi and javadian, 1996) l .near gerbilli esfahan (parvizi and ready, 2008) ph. salehi l. major sistan and baluchestan (kasiri and javadian, 2001) ph. alexandri l. infantum fars (azizi et al. 2006) l. major khuzestan (javadian et al. 1977) ph. neglectus (=ph. major s.l.) l. infantum fars (sahabi et al. 1992), (azizi et al. 2008) ph. keshishiani l. infantum fars (seyedi-rashti et al. 1995) ph. kandelakii l. infantum ardabil (nadim et al. 1992), (rassi et al. 2005) ph. perfiliewi l. infantum ardabil (nadim et al. 1992), (rassi et al. 2009) l. donovani ardabil (oshaghi et al. 2009) l. infantum l. tropica azarbaijan-e-sharqi (parvizi et al. 2008) * sand fly species marked with asterisk are proven vectors according to the generally accepted criteria for incriminating leishmania vectors (killick-kendrick and ward 1981, who 2010). species with no asterisk are suspected to be vectors on the basis of epidemiological evidence or because are proven vectors elsewhere. fig. 1. map of iran providing the province outlines, in brown the provinces that are endemic for zoonotic cutaneous leishmaniasis table 2. countinued… 8 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ١ phlebotomus alexandri this species is usually found in mountainous areas and has been reported all over the country. in field studies conducted during 1971–1972, a natural promastigote infection rate of 1.7% was reported in specimens collected from rodent burrows in khuzestan province, southwestern iran (javadian et al. 1977) but isolation and characterization of the parasite from the sand fly vector has not been achieved yet in the study area. in 2006 promastigotes isolated from ph. alexandri collected from the houses of zvl cases in nourabad mamasani district, fars province, south of the country, were identified as l. infantum by semi-nested pcr .the natural infection rate was 4.2% in this species, strongly implicating it as probable vector of zvl (azizi et al. 2006). phlebotomus neglectus tonnoir, 1921 (= ph. major s.l.) this species is found in 17 of 31 provinces mostly in mountainous areas and has been found in all areas from which human cases of zvl have been reported. in 1992, the promastigote infection rate in specimens collected outdoors by light traps was reported to be 3% and by aspirator from indoors it was 5 % in ghir-karzin district, another endemic focus of zvl in fars province, southern iran but the parasites were not characterized (sahabi et al. 1992). in 2008, the infection rate in flies collected from the same area was 8.3% and parasite isolates were characterized and identified by nested-pcr as l. infantum, implicating this species as a probable vector of zvl in the south of the country (azizi et al. 2008). phlebotomus keshishiani in 1995, during a sand fly survey conducted in an endemic zvl focus in ghir-karzin, fars province, southern iran, the natural promastigote infection rate in specimens of this species collected outdoors was 1.1%, so the species is also considered a probable vector of zvl (seyedi-rashti et al. 1995). phlebotomus kandelakii in a 1991–1992 field study in the endemic focus of meshkin-shahr in northwestern iran, the natural leishmania infection rate in this species was reported to be 0.3% (nadim et al. 1992). similar studies were conducted in the same area from 2001–2005 in which the leishmania infection was reported to be 1.1%. parasite isolates from these later studies were characterized and identified as l. infantum by nested pcr (rassi et al. 2005). based on these findings, ph. kandelakii is considered a probable vector of zvl in northwestern iran. phlebotomus perfiliewi in an entomological investigation carried out between 1991–1992 in germi county, northwestern iran, the natural promastigote infection rate was 0.9 % in this species (nadim et al. 1992). in a later study conducted in the same area between 2004–2005 the infection rate was 1.1% and isolates were characterized and identified as l. infantum by pcr, incriminating the species as a probable vector of zvl (rassi et al. 2009). more recently in 2006, l. infantum and l. donovani were detected and identified by pcr and dna sequencing in from specimens collected from the same area. of seven isolates, one was l. infantum and the other six were haplotypes of l. donovani (oshaghi et al. 2009). it should be mentioned that the parasites from ph. kandelakii, ph. perfiliewi transcaucasicus, ph. keshishiani and ph. neglectus were injected intraperitoneally into hamsters and in all cases produced heavy visceral infections but the investigators failed to keep them in culture (nadim et al. 1992). 9 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ٢ table 2 shows the list of proven or suspected vector species present in iran. vectors of lizard leishmaniasis (sauroleishmania) lizard leishmaniasis is also widespread in four species of lizards in various areas of iran as follows: agama agilis and agama melanura collected either from gerbil burrows or lizard holes in dry river beds in khorassan-e-razavi and gholestan provinces in the northeast, agama caucasica from manjil area in qazvin province (the mid north of the country) and cyrtopdion caspius from turkmen-sahra in gholestan province, the north of the country (nadim et al. 1968a, 1968b, seyedi-rashti et al. 1971). in all areas where lizard leishmaniasis has been found it has been accompanied by natural infections in sergentomyia species. sergentomyia sintoni in 1966 in a sand fly survey conducted in several zcl foci in northeastern iran, many se. sintoni specimens were found naturally infected with promastigotes (nadim et al. 1968b). from 1967–1995 frequent promastigote infections in this species were reported frequently in zcl foci throughout the country (yaghoobi-ershadi and akhavan 1999). isolates from this sand fly species were identified as l. (sauroleishmania) gymnodactyli by isoenzyme analysis (seyedirashti et al. 1994). in 2008, isolates from natural promastigote infections in flies from the central part of the country were identified as l. major and l. near gerbilli by sequencing of the its1 or its2 microsatellite fragment or sizing of the its2 microsatellite fragment (parvizi and ready 2008). sergentomyia clydei in 1967, promastigote infections in this species, thought to be related to lizard leishmaniasis, were reported in sand fly specimens collected from gerbil colonies in lotfabad (khorasan-e razavi province) in the northeastern part of the country (mesghali et al. 1967). sergentomyia dentata in 1994, natural promastigote infections in this species were reported during an entomological survey in the northwestern part of the country (rassi et al. 1997). concluding remarks relevant studies of leishmaniasis are impossible without correct identification of the associated sand flies and a thorough understanding of their distribution and biology. over the past 80 years many researchers have made significant contributions to the body of knowledge implicating various leishmania vectors in iran. the likelihood of finding natural leishmania infections in sand flies is related to the age of the sand fly vector and most of the dissections were carried out during august– september of the years of studies towards to the end of the active season of sand flies in iran. this is because the risk of infection is nil when the new generation of a sand fly vector begins to emerge, but becomes increasingly high as the population ages and more parous flies are present. this variation is not as marked with vectors that are present throughout the year in other parts of the world as in the neotropics for example. the other important point is the detection of leishmania dna in a sand fly which does not prove that it is a vector. for example, the pcr assays did not distinguish between infective stage metacyclic parasites and noninfective stages of leishmania promastigotes. the generally accepted criteria for incriminating leishmania vectors are: anthropophily in transmission habitats and disease foci, devel10 j arthropod-borne dis, 2012, 6(1): 1–17 mr yaghoobi-ershadi: phlebotomine sand flies … ٣ opment in the sand fly of typed infections through its complete extrinsic life cycle, and experimental transmission by bite (killickkendrick and ward 1981). it should be mentioned that members of who have stated in 2010 that existing knowledge and molecular tools for the identification and characterization of leishmania parasites and sand flies should be improved and harmonized (who 2010). data on phlebotomine sand flies should be regularly updated and verified at least once every five years by fieldwork and taxonomy in leishmaniasis foci, because of increasing environmental modifications or population movements which are important in preparing strategic plan of leishmaniasis control. further research is needed to amplify present knowledge and extend it to areas not yet studied. research efforts in the field are continuously discovering new data on sand flies in the world and it is the duty of sand fly specialists to remain abreast of these developments. long-term monitoring should be conducted of annual fluctuations in important vector populations in representative transmission sites of leishmaniasis in iran. examples of vectors to be included in this research are ph. papatasi, ph. sergenti, ph. kandelakii, ph. neglectus and ph. alexandri. careful incrimination of other vectors in different endemic areas and clarification of the ecology concerning transmission and control means that long term efforts are needed in the future. and this kind of knowledge requires full support and more funding for the field work by health authorities. acknowledgements the author gratefully thanks dr phillip lawyer from national institute of health (nih)/ national institute of allergy and infectious diseases (niaid)/ laboratory of parasitic diseases (lpd), bethesda, maryland, usa for his critically reading and valuable comments on the manuscript. thanks are also due to prof a nadim, department of epidemiology and biostatistics and prof e javadian, department of medical entomology and vector control, tehran university of medical sciences for their support. references adler s, theodor o, lourie em (1930) on sand flies from persia and palestine. bull entomol res. 21: 529–539. akhavan aa, yaghoobi-ershadi mr, mehdipour d, abdoli h, farzinnia b, mohebali m, hajjaran h (2003) epidemic outbreak of cutaneous leishmaniasis due to leishmania major in ghanavat rural district, qom province, central iran. iranian j publ health. 32: 35–41. akhavan aa, yaghoobi-ershadi mr, hasibi f, jafari r, abdoli h, arandian mh, soleimani h, zahraei-ramazani ar, mohebali m, hajjaran h (2007) emergence of cutaneous leishmaniasis due to leishmania major in a new focus of southern iran. iran j arthropod-borne dis. 1: 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kusadasi, turkey. p. 40. 17 j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 219 http://jad.tums.ac.ir published online: september 30, 2018 original article toxicological analysis of insects on the corpse: a valuable source of information in forensic investigations mojtaba salimi 1, *yavar rassi 1, omid chatrabgoun 2, artin kamali 3, mohammad ali oshaghi 1, vida shiri-ghaleh 3, mehrdad moradi 3, sayena rafizadeh 4, kameran akbarzadeh 1, seyedeh zahra parkhideh 1 1department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 2department of statistics, faculty of mathematical science and statistics, malayer university, malayer, iran 3department of forensic toxicology, legal medicine center of kermanshah province, kermanshah, iran 4national institute for medical research development (nimad), tehran, iran (received 27 feb 2018; accepted 09 june 2018) abstract background: entomotoxicology as a subset of forensic entomology can be used by analysis of carcass feeding insects to detecting of drugs or toxins, as well as the cause and manner of death in cases of ante-mortem drugs intoxication. morphine is one of the deacetylate metabolites of heroin. the aim of this study was to determine the presence and quantity of morphine in insects on the carcass and compare them with decomposing carcass. methods: field of this study was in chalabeh district and toxicological tests were carried out at the department of forensic toxicology, legal medicine center, kermanshah, iran in 2017. morphine was inoculated into live rabbit as experimental model at concentrations of 12.5, 25, 50mg/ml, similar to those normally encountered in human overdoses, then quality and quantity of morphine were determined in insects such as chrysomya albiceps (as the first wave of insect succession on human cadavers) fed on carcass. results: quantitative assessment at larvae showed that morphine was detected in all larvae (feeding and post feeding stage) fed on tissues from carcasses administered morphine, except for post-feeding larvae from r1 which received 12.5mg/ml dosage of morphine. conclusion: necrophagous insects are an indicator on the scene of crime and a potential source of information about the antemortem situation. detection of drug in insects which is actually a reflection of the cause of death is possible. keywords: morphine, postmortem, necrophagous, rabbit, larvae introduction in forensic entomology using the insects on the carcass, we can find various factors such as time of death, climatic condition, death location and presence of drugs or toxins in the body (1, 2). the presence of drugs in the carcass can effect on the growth and morphology of insects on the corpus with the result that some error can occur in the estimation of postmortem interval (3-9). there is a correlation between drug concentration in substrate and bred insects’ development on the substrate. however, the pharmacokinetic of drug in insects depends on species, development stage, mode of action of drug, absorption, redistribution, and metabolism of drug and drug stability (1, 4, 10-16). morphine is one of the heroin metabolites, and the pharmacokinetics of the opiate groups is similar to each other. we have chosen morphine as our initial drug study in this research, as heroin addiction is a major public health problem worldwide; moreover, deaths through heroin overdose are frequent. in this study morphine and its metab*corresponding author: dr yavar rassi, e-mail: rassiy@sina.tums.ac.ir http://jad.tums.ac.ir/ mailto:rassiy@sina.tums.ac.ir j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 220 http://jad.tums.ac.ir published online: september 30, 2018 olites in rabbit carcass, chrysomya albiceps (as the first and most abundant insects on the body) (diptera: calliphoridae) and c. maxillosus (celoptera: staphylinidae) were analyzed. in order to analysis of drug or toxin, different methods and devices including radioimmunoassay (ria), high performance liquid chromatography (hplc), liquid chromatography–mass spectrometry (lc-ms), enzyme immunoassay (eia), gas chromatographymass spectrometry (gc-ms), thin-layer chromatography (tlc), liquid-chromatographie mass enspektometrie/ mass enspektometrie (lc-ms/ms) and liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (lc/esi+ms/ms) were used due to accuracy, sensitivity and economy (7, 10, 15, 17-21). the aim of this study was to determine the presence and quantity of morphine in insects on the carcass and compare them with decomposing carcass. moreover, the relationship between quantity of morphine in carcass tissue, immature of c. albiseps (first level of food chain) and adult of creophilus maxilosus (second level of food chain) was investigated. materials and methods site and period of study this study was conducted at bisetoon (challabeh), located 13km from kermanshah province, west of iran between n 34˚ 24ʹ 37″ northern latitude and e 47˚ 14ʹ 42″ eastern longitude, in 2017. morphine morphine is the main opiate alkaloid, and the derivation of the opioid group belongs to the strong group of agonists. the melting point of morphine is 254–256 °c. a total of 90% of morphine is metabolized in the liver, and the human body excretes it through the urine. this type of drug aggregates in most of tissues. virtually all opioid drugs are metabolized by the cytochrome p450 liver enzymes. before being excreted by kidneys they change into glucuronide conjuges (m3g, m6g). a small amount of the drug is also excreted by the kidneys with no change. the protein binding is very low, and the half-life of the drug equals 2–3h. morphine exerts its effects via δ, κ, µ receptors. animal and morphine dosing accordance with the ethical standards of tehran university of medical sciences ethics committee, (with code of ethics ir.tums. sph.rec.1395.1634) for toxicological analysis, three rabbits with a similar weight (r1= 2500g, r2= 2700g, r3= 3000g) as the treatment group and the fourth rabbit (r0= 2600 gr) as the control group were selected. treatment groups were administered concentration of r1= 12.5, r2= 25 and r3= 50mg/ml of morphine sulfate (10.1, 20.2 and 40.4mg of free morphine equivalent, respectively). morphine sulfate was diluted in 150ml isotonic saline solution and for the control group, only 150ml of isotonic saline solution (without morphine) were inoculated for three hours through the superficial vein of the ear. morphine perfusion was used with scalp vein set (with specification, item no. htf 0324s, gauge 25g, od 0.5mm, color code orange). the perfusion bottle was placed 1.75m above the rabbit. the effect of morphine inoculation of rabbits as hypothermia, miosis and respiratory depression were observed. in order to maximum the distribution of the drug after 30–60min the rabbits were killed using chloroform. these dosages, samplings of rabbit tissue, the interval between the completion of inoculation and the death of rabbit’s, duration and rate of inoculation of morphine were calculated from pharmacokinetics (10, 22-24). the rabbit's internal tissues were sampled to determine the presence of morphine and this autopsy was performed 24h after death to create the reality conditions (fig. 1). http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 221 http://jad.tums.ac.ir published online: september 30, 2018 morphine was measured from internal tissues such as liver, spleen, mesenteric muscle, bile marrow at the rate of 10–20gr being kept into tubes containing 150ml deionized distilled water at the degree of 4 °c until the analysis time. the carcasses were stitched up after the autopsy and immediately transferred to the study field. preparation of the specimen for tlc and hplc analysis chrysomya albiceps and c. maxillosus as the dominant species on the rabbit carcass were used to analyze the toxicology. each larval sample comprised of 30 larvae and was divided into peak feeding and post feeding. moreover, each beetle sample contained 10 adult beetles considered for each concentration. to avoid the alteration of morphine concentration by drug metabolism in larval body, the larvae were immediately killed using hot water. the samples were kept in tubes containing deionized distilled water at the degree of 4 °c, and it was transferred to the forensic toxicology laboratory of kermanshah located in the western part of iran for the purpose of toxicology analysis. morphine extraction of tissue, larvae, and beetle at first, the samples were homogenized in the laboratory. some 100ml of acidic ammonium sulfate solution was added to some 50ml of the homogenized samples (250g of ammonium sulfate in one liter of 20% hydrochloric acid). it was shaken for 20min and then exposed to heat in oven at the degree of 80 °c for 2h. by using ammoniac, the ph of the filtered solution reached 8.9. then 100ml chloroformisopropanol at the rate of (80:20v/v) was added to the samples, and they were shaken for 20min. afterward, it was poured into decanter and underneath organic phase which contained morphine separated at beaker. after drying, 1ml methanol was added to the sample, and by syringe 0.2 micron the sample was filtered and transferred to 1.5ml polypropylene eppendorf tube then 20µl of it was injected into the chromatographic system. thin-layer chromatography data for morphine tlc contained a stationary phase including absorbent material silica gel, 20x20cm in diameter, and 0.20mm thickness on the fixed base was made of glass. the standard used in the study contained 25 microliter opium, and the samples of each concentration were spotted separately at the rate of 10 microliters at one edge of a plate. then tlc plates were put into the tlc tank which was a closed container containing a mobile phase (a combination of organic solvents), and the samples were separated at the mobile phase. the chromatography tank was made of glass in 21.5 by 9.5 by 22cm of diameters and contained ethyl acetate 85ml, ammonia 5ml in final volume of 100ml. we kept waiting for 10min until the solvent in the tank was saturated, and we put spotted tlc plates in the tank, and the tank lid kept closed until the end of the experiment. after the solvent ascended to the top of the plate, we took it out of the tanks to be dried completely. tlc plates through iodoplatinate acid were sprayed, and developed spots were compared with morphine standard spots. the quantities of microgram to picogram can be separated by tlc and characterized by their rf1 value. actually, rf is equal to the distance traveled by morphine divided to the distance traveled by the solvent. in this study, opium was used as a standard for detecting morphine spots. standard solutions standard curves (calibration standard) were diluted by using the main sample of morphine sulfate with a 10mg/ml concentration and a 1ml volume. the dilutions of 1, 5, 10, 50 and 100ng/ml were provided from the main sam 1 retention factor http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 222 http://jad.tums.ac.ir published online: september 30, 2018 ple. by, injecting each dilution into the device, the area under the curve and morphine retention time were obtained. hplc data in the course of passing through the filter in the shuffled vials, the samples collected in order to be inject into the hplc device with the following features: knauer model (made in germany), dimension: 250 by 4.6mm with pre-column, batch no: b195975, column sn: wh62, packing eurospher 100-5 c18, pda detector. the mobile phase included phosphate buffer and acetonitrile with a ratio of 3:97, ph= 2.3 and the flow rate of 0.8ml/min. the injection volume of 20 microliters for each analysis turn was carried out using 100 microliter hamilton syringe. total run time analysis was considered to be 30min. then all data obtained were collected for the purpose of statistical analysis. results thin-layer chromatography results we used the tlc method for the qualitative assessment of the morphine and its presence in treated tissues with different concentrations. the observed results associated with this method in different concentrations were indicative of the morphine in rabbit tissues and c. albiceps larval in treated samples. in r3 groups, liver, kidney, spleen, muscle, bile, urine tissues, peak feeding and post-feeding larvae were all positive and adult c. maxillosus was revealed to be negative. in the concentration related to r2= 25mg/ml, liver, kidney, spleen, bile, urine, peak feeding larvae were shown to be positive, but muscle, post-feeding larvae and c. maxillosus beetles turned out to be negative. as for the concentration associated with r1= 12.5mg/h except for the urine sample other samples were negative (fig. 2). method validation in order to verify the data obtained from the morphine analysis, the efficiency of the extraction method and accuracy of the device readings, first we assessed a specific set of parameters in order to ensure reliable results were obtained. selectivity: the ability of the method to provide straight and clear assessment of morphine versus other component substances was determined by the selectivity parameter. the specificity of the method was examined by comparing the chromatogram of the standard and control sample. there were no interfering peaks in the control sample at the retention time of morphine (fig. 3, 4). retention time of morphine was approximately 7.167min. the total run time for each sample injection was about 30min. linearity: this parameter was used to show the relationship between concentration and resultant signal. for this purpose, six concentrations including 1, 5, 10, 50, 100ng/ml from the standard sample were provided, and the area under the curve of each concentration with three replications was analyzed. calibration chart and linear equation of morphine concentrations in three consecutive injections equal y= 3356.2+979.56x where y equals the peak area of the morphine and x is indicative of morphine concentration. the correlation coefficient was r2 > 0.99 (fig. 5). recovery: three concentrations 5, 50 and 500ng/ml were added for on three occasions to the larvae matrix in the control group which was lacking morphine, and after the extraction process the concentrations were analyzed at each concentration level. calibration curves for the plasma assay developed with peak area of morphine (y) versus drug concentration (x) were found to be linear in the examined concentration range. the values obtained for the % recovery concentrations ranged from 98– 102%. limit of detection and quantitation: the minimum of the observable value in the device which must, in fact, be a peak equivalent to the three-time device noise was calculated http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 223 http://jad.tums.ac.ir published online: september 30, 2018 by this formula: lod= 3.3* (sd/s). the minimum morphine content in the sample determined with acceptable accuracy and precision was calculated by this formula loq= 10*(sd/s). where s is the slope of the calibration curve and sd is standard deviation of the response (table 1). precision and accuracy: the precision in the analysis method was measured through injecting three dilutions (30, 50, 100ng/ml) of morphine in three replications (n= 3) on the same day (intra-day) and three concentrations in three separate days, each day with three replications for each dilution (inter-day). such parameters as mean concentration (c), accuracy (bias), standard deviation of the mean concentration of morphine (sd), coefficient of variation value [cv%= (sd/c) *100%] are shown by table 2. the rate of cv% < 3 was calculated for both inter and intra-day parameters. hplc results of rabbit samples and insects quantitative analysis of carcass samples of treatment rabbit with concentrations of 25, 50, and 12.5mg/ml was indicative of concentration and metabolites difference in tissues for each concentration (table 3, figs. 6–8). therefore, concentration gradient from the minimum to the maximum value: r3= 50mg/ml was found in liver, kidney, urine, muscle, bile, and spleen. at an r2= 25mg/ml the downtrend of this concentration slope was observed in the urine, spleen, bile, kidney, muscle, and liver respectively. in r1= 12.5mg/ml samples of urine, liver, muscle, kidney, spleen, and bile were measured and all specimens except spleen and bile were morphine. analysis of c. albiceps maggot samples settled on the treatment carcass with r3 morphine content was 210 and 146ng/g, r2 was 140 and 63ng/g respectively in peak feeding and post-feeding larvae. no morphine was recovered from post-feeding larvae fed at r1 concentrations. adult c. maxillosus with concentrations of r1, r2 and r3 were lack of morphine in all treatment carcasses (table 4). statistical analysis confirmed this according to anova test, since (p< 0.05) there is a significant difference between concentrations (table 5). according tukey hsd, we find homogeneous subsets and in the other words, different subsets are (r0, r1), (r1, r2), and r3. table 1. range of detection and quantitation of morphine by hplc-uv analysts lod* (ng/ml) loq** (ng/ml) morphine 8.36 25.3 *limit of detection **limit of quantitation table 2. the intraand inter-day precision which proves the acceptable accuracy and precision of the method developed concentration added (ng/ml) concentration founded (ng/ml) sd* cv%** bias% recovery n*** intra-day 30 28.3 2.00 7.14 4.3 93.3 3 50 51.1 1.13 2.23 2.2 102 3 100 101 2.35 2.37 1 101 3 inter-day 30 28.3 2.00 7.52 4.3 92 3 50 50.51 1.13 2.23 1.02 101 3 100 99.4 2.35 2.37 -0.6 99.4 3 *standard deviation of the response **coefficient of variation value ***repeat number of test http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 224 http://jad.tums.ac.ir published online: september 30, 2018 fig. 1. rabbit model and perfusion of morphine sulfate (12.5, 25 and 50mg/ml) via the main artery of the ear (a, b). autopsy of the treatment groups (c) fig. 2. thin-layer chromatography of morphine in tissues, larvae, and beetle in different dosages (12.5, 25 and 50mg/ml) fig. 3. chromatograms hplc at retention time 7.167min for morphine sample http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 225 http://jad.tums.ac.ir published online: september 30, 2018 fig. 4. placebo sample that indicates no interfering peaks at the retention time of morphine y = 3356.2+ 979.56x r² = 0.997 40000 60000 80000 100000 120000 a re a fig. 5. calibration graph and morphine sulfate line equation fig. 6. ultraviolet spectrum of morphine http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 226 http://jad.tums.ac.ir published online: september 30, 2018 fig. 7. ultraviolet spectrum of morphine-6-glucuronid measured by hplc it shows the morphine metabolism in the rabbit’s liver fig. 8. ultraviolet spectrum of morphine-3-glucuronide measured by hplc it shows the morphine metabolism in the rabbit’s liver http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 227 http://jad.tums.ac.ir published online: september 30, 2018 table 3. concentrations of morphine in rabbit samples administered different dosages of morphine sulfate via ear perfusion at 3h period, r0= control, r1= 12.5mg/ml, r2= 25mg/ml, r3= 50mg/ml rabbit carcasses samples r0 r1 r2 r3 mean concentration (ng/g) mean concentration (ng/g) mean concentration (ng/g) mean concentration (ng/g) liver 0 47 215 2695 kidney 0 28 505 2390 spleen 0 0 722 1191 bile 0 0 629.5 1411 mesenteric muscle 0 31 500.7 1666 urine 0 655.4 891 1969 table 4. concentration of morphine in c. albiceps larval and c. maxillosus administrated different dosage of morphine sulfate via ear perfusion at 3h period: r0= control, r1= 12.5mg/ml, r2= 25mg/ml, r3= 50mg/ml insects r0 r1 r2 r3 mean concentration (ng/g) mean concentration (ng/g) mean concentration (ng/g) mean concentration (ng/g) peak feeding larvae (c.albiceps) 0 25.64 139.8 210 post feeding larvae (c.albiceps) 0 0 63.37 146 c.maxillosus (adult) 0 0 0 0 table 5. tukey hsd analysis in rabbit carcasses administrated different dosages tukey hsda concentration rabbit samples n subset for alpha = 0.05 1 2 3 r0 6 .0000 r1 6 126.9000 126.9000 r2 6 577.2000 r3 6 1887.0000 p value .914 .128 1.000 (r0= control, r1= 12.5mg/ml, r2= 25mg/ml, r3= 50mg/ml) discussion morphine is a derivative of the opioid group. opioids like morphine, heroin and tramadol have similar pharmacological properties. we have chosen morphine for this study because there are many of death in the world due to heroin or tramadol overdose. after absorption, morphine is rapidly distributed within the body. morphine has a short halflife of approximately two hours. the main site of morphine metabolism is the liver. the process of elimination of morphine and heroin and codeine are similar in the body (23). using a live animal model in toxicology analysis caused the results obtained to be closer to reality. we observed gradient concentration in various tissues of rabbit. we used c. albiceps (diptera: calliphoridae) and c. maxillosus (coleoptera: staphylinidae) as a toxicological bio-indicator. chrysomya albiseps is first colonizer in tropical and sub-tropical http://jad.tums.ac.ir/ http://sepia.unil.ch/pharmacology/index.php?id=34 http://sepia.unil.ch/pharmacology/index.php?id=53 http://sepia.unil.ch/pharmacology/index.php?id=60 http://sepia.unil.ch/pharmacology/index.php?id=60 http://sepia.unil.ch/pharmacology/index.php?id=55 j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 228 http://jad.tums.ac.ir published online: september 30, 2018 areas particular in warm seasons (1, 25). the quantity of drug in tissues and c. albiceps larval stages seems to be dose-dependent. the results of other studies also have attested to morphine concentration difference in various tissues (10, 13, 23, 26). it would be better to use the identical development stages of insect, especially peak feeding and postfeeding larvae in order to the toxicological analysis (26, 27). we also focused on the quantitative and qualitative analysis of the morphine on peak feeding and post feeding of c. albiceps species and adult c. maxillosus. our findings showed that there was a difference in terms of concentration at various stages of larval development so that morphine concentration in peak feeding stage was higher than post-feeding larvae. morphine content decreases during developmental process due to metabolism and drug elimination (4, 6, 11, 27, 28). predator beetles are an appropriate matrix for the detecting of drugs and toxins (15). however, quantitative and qualitative analysis of the beetle (c. maxillosus) was not detectable. the amount of drug decreases with progress level of the food chain. in line with the results, other studies also take into account the changes in drug concentration caused by the difference in kinetics detoxification, drug stability, growth stage, insect species and the tissue of maggot placement (10, 12, 24, 29, 30). morphine is a polar compound and soluble in water to the (1/5000) and it is excreted through the nephritic system and malpighian tubes. therefore, morphine concentration in post feeding larval stage accompanied by stop feeding reduces against peak feeding. other studies (10, 22, 24) confirming the results gained from this study showed that the rate of eliminating drug increases in post feeding larval stage. in this study to extract of morphine from tissues, we used the acid digestion method. acid digestion method for extracting the drug have preferred to enzyme digestion. the efficiency of acid digestion is 93% against 64% of enzymatic digestion (31). in studies conducted on the measurement of morphine on l. sericata larva with similar method, the results were slightly different compared to the current study (22, 23). these differences can stem from several factors such as extraction method, sensitivity of method, insect species and so forth. they used ria device to analyze the morphine, while we applied hplcuv. besides our studied insect was c. albiceps but l.sericata was assessed in those studies. the volume of distribution in morphine is 3–5kg/l. molecules with volume of distribution (vd) >3 have redistribution after death and the concentration balance between blood and tissue changes after death (24). according to our results, there was no linear relationship between morphine levels in carcass and larval stages feeding. this issue has been mentioned by some researchers (10, 23, 32). the necessity of review methods of validity was referred in toxicology (26). drug detection occurs when the amount of absorption in the body exceeds the amount of drug elimination (4, 15). while this study showed that morphine will not bioaccumulate in the tissue. morphine concentration in treatment samples and larvae due to calculated amount for loq device illustrated that all obtained concentrations are statistically valuable. this study indicated necrophagous insects could be a valuable data in the investigation of death. difference in the result of drug measurement in different methods of assay, only qualitative results such as presence of drug in insects can be valuable. conclusion necrophagous insects on the carcass are a source of information from antemortem conditions. moreover, using the entomotoxicological analysis can get information about the causes and manner of death. therefore, the results of insect analysis are precisely the reflection http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 219–231 m salimi et al.: toxicological analysis of … 229 http://jad.tums.ac.ir published online: september 30, 2018 of the carcass condition. although there is no linear relationship between the concentration of morphine in the rabbit and insect tissue, however, using analysis of the necrophagous insect's tissue can be found to the presence of the drug (morphine) in the carcass. acknowledgements we would like to thank saeed shahebrahimi for his help in collecting samples and ali reza karami for scientific adviser. this work was supported by tehran university of medical sciences, grant no. 34233. all procedures performed in studies involving animals were in accordance with the ethical standards of tehran university of 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toxicological analysis of … 231 http://jad.tums.ac.ir published online: september 30, 2018 28. wilson z, hubbard s, pounder dj (1993) drug analysis in fly larvae. am j forensic med pathol. 14(2): 118–120. 29. goff ml, brown wa, hewadikaram k, omori a (1991) effect of heroin in decomposing tissues on the development rate of boettcherisca peregrina (diptera: sarcophagidae) and implications of this effect on estimation of postmortem intervals using arthropod development patterns. j forensic sci. 36(2): 537–542. 30. goff ml, brown wa, omori a (1992) preliminary observations of the effect of methamphetamine in decomposing tissues on the development rate of parasarcophaga ruficornis (diptera: sarcophagidae) and implications of this effect on the estimations of postmortem intervals. j forensic sci. 37(3): 867–872. 31. foltz rl, fentiman jr a, foltz rb (1980) gc/ms assays for abused drugs in body fluids. nida res monogr. 32: 1–198. 32. definis-gojanović m, sutlović d, britvić d, kokan b (2007) drug analysis in necrophagous flies and human tissues. arh hig rada toksikol. 58(3): 313– 316. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/17913685 j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 310 http://jad.tums.ac.ir published online: september 30, 2018 original article biochemical basis of cyfluthrin and ddt resistance in anopheles stephensi (diptera: culicidae) in malarious area of iran mohammad amin gorouhi 1, 2, *mohammad ali oshaghi 2, *hassan vatandoost 2, 3, ahmad ali enayati 4, mohamad reza abai 2, 3, mohsen karami 5, yaser salim-abadi 6, ahmad ali hanafi-bojd 2, 3, abbas aghaei-afshar 1, azim paksa 2, fatemeh nikpour 2, 3 1department of vector biology and control, school of health, kerman university of medical sciences, kerman, iran 2department of medical entomology and vector control, school of public health, tehran university of medical sciences, tehran, iran 3department of chemical pollutants and pesticides, institute for environmental research, tehran university of medical sciences, tehran, iran 4department of medical entomology, mazandaran university of medical sciences, sari, iran 5infectious diseases and tropical medicine, babol university of medical sciences, babol, iran 6department of health services and health promotion, school of health, rafsanjan university of medical sciences, rafsanjan, iran (received 12 apr 2018; accepted 24 sep 2018) abstract background: anopheles stephensi is a key urban malaria vector in the indian subcontinent and middle east including south and southeast of iran. wide application of insecticides resulted in resistance of this species to various insecticides in these regions. this study was conducted to reveal the role of metabolic mechanisms in the development of resistance in an. stephensi to ddt and cyfluthrin. methods: field mosquito specimens were collected from chabahar seaport, southeast corner of iran, in 2015. insecticide susceptibility and enzyme assays were conducted as recommended by who. results: mean enzyme ratios were 3.95 and 3.04 for αesterases and 2.40 and 1.97 for βesterases in the ddt and cyfluthrinresistant populations correspondingly compared with the susceptible strain. the gsts enzyme mean activity ratios were 5.07 and 2.55 in the ddt and cyfluthrinresistant populations compared with the susceptible beech strain. the cytochrome p450s enzyme ratios were 1.11 and 1.28 in the ddt and cyfluthrinresistant populations respectively compared with the susceptible beech strain. conclusion: metabolic mechanisms play a crucial role in the development of ddt and cyfluthrin resistance in an. stephensi, therefore, further evaluation of the mechanisms involved as well as implementation of proper insecticide resistance management strategies are recommended. keywords: anopheles stephensi, insecticide, resistance mechanisms, malaria introduction malaria is still a major public health problem in southeast corner of iran (1). there are seven anopheles species as malaria vectors in iran including an. stephensi, an. culcifacies s.l., an. maculipennis s.l., an. sacharovi, an. superpectus s.l., an. dthali, and an. fluviatilis s.l.. anopheles stephensi is the most important malaria vector in southern region of the country (2-10). application of chemical insecticides is one of the most important interventions for malaria control, used in iran during past decades. different groups of insecticides including organochlorines (ddt, dieldrin and bhc), organophosphates (pirimiphos-methyl and malathion), carbamate (propoxur) and pyrethroids (lambdacyhalothrin and delthamethrin) in dif*corresponding authors: dr mohammad ali oshaghi, e-mail: moshaghi@sina.tums.ac.ir, dr hassan vatandoost, e-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 311 http://jad.tums.ac.ir published online: september 30, 2018 ferent forms of application such as indoor residual spraying (irs) and insecticide-treated nets (itns) for adult mosquito control and organophosphates for larviciding were used in malarious areas of the country (11-13). iran has embarked on the malaria elimination program since 2007 relying on application of chemical insecticides specially pyrethroid compounds for malaria vector control (14). anopheles stephensi is resistant to several insecticides including ddt, dieldrin, and malathion (12, 15-19). the first indication of pyrethroid resistance was reported from chabahar seaport, southeast of iran in 2012 (13). moreover, there are many reports on resistance of this species to different insecticide groups including pyrethroids from iran neighboring countries including pakistan, afghanistan, the indian subcontinent as well as the middle east countries (14, 15, 18, 20-25). due to the importance of pyrethroids in malaria control program and the slow process of development of new insecticide compounds, monitoring and management of insecticide resistance are necessary (26, 27). metabolic and target site insensitivity are two common resistant mechanisms in insects. in metabolic resistance, alteration in the levels or activities of detoxification enzymes such as esterases, glutathione s-transferases (gsts), and cytochrome p450s may occur (24, 28). in target site insensitivity, mutations in the sodium channel, acetylcholinesterase and gaba receptor genes occur (29). therefore, determination of resistance mechanisms in an. stephensi is essential for proper management of insecticide resistance through vector control interventions. the aim of this study was to determine the possible involvement of enzymes groups’ in ddt and pyrethroid insecticides resistance functioning in an. stephensi, the main malaria vector in southeast of iran. materials and methods mosquito collection and rearing anopheles stephensi larvae were collected from larval habitats using the standard dipper from chabahar seaport (25°25ˊn, 60°45ˊe) sistan and baluchestan province, southeast of iran (fig. 1) during apr to june 2015. the larvae specimens were transported in cool boxes to insectary of the medical entomology and vector control department, school of public health, tehran university of medical sciences, tehran, iran and reared to adult stage under standard condition at 25 °c, 80% relative humidity with a 12h day/night lighting cycle. the adult mosquito specimens were identified to species level using the identification key (30). moreover, a pyrethroid susceptible strain (beech strain) originated in india in 1940 and kept in the insectary without being exposed to insecticides used as a control in all experiments. adult susceptibility tests and selection six different insecticide impregnated papers including ddt 4%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, cyfluthrin 0.15%, permethrin 0.75%, and etofenprox 0.5% supplied by who were used for evaluating the susceptibility status of an. stephensi populations from chabahar. two or three days old adult female mosquitos that were kept on 10% aqueous sucrose solution were used for susceptibility test procedure according to the who method (27). then the mosquito populations with the lowest mortality rates were subjected to selection pressure of the two insecticides in the laboratory. the mosquito populations were exposed to the two insecticides in two separate lines over 18 and 19 generations throughout four and five selection phases. for both insecticides, mortality rate was calculated in different times and regression lines were plotted in each generation using microsoft excel (ver. 2013). a subset (40 specimens for each sample) of resistant and susceptible mosquito populations were placed in a 1.7ml tubes and kept in freezer (-80 °c). these frozen specimens were then transported in a cold chain to the pesticide biochemistry http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 312 http://jad.tums.ac.ir published online: september 30, 2018 laboratory of medical entomology department, school of public health, mazandaran university of medical sciences, sari, iran for further biochemical assays. biochemical assays biochemical tests were performed according to the method described by hemingway (1998) (31). the enzyme activities/contents of p450s, glutathione s-transferases (gsts), and esterases using corresponding fresh buffer solutions were quantified. frozen adult mosquito specimens were individually put in wells of flat-bottomed 96-well microtiter plate and manually homogenized using a steel pestle in 250µl cold distilled water at 4 °c. the plate was spun at 3000rpm for 20min in a beckman coulter (beckman inc., usa) centrifuge at 4 °c and the supernatant was used as the source of enzymes in reaction mixtures. in each biochemical assay, blank replications (all component of the reaction mixture except for the enzyme source) were provided. preparation of all reaction mixtures was carried out on ice (31). total protein assay in order to minimize the error due to different size and protein contents of mosquitos and homogenizing process, total protein component of each specimen was measured using bradford method in triplicate by adding 300 µl of bio-rad solution (diluted with distilled water by 1:4) to 10µl of the homogenate. after 5min incubation at room temperature, the absorbance was measured at 570nm in a biotek elx808 ultra microplate reader (biotek inc, usa) (31). the value was changed into product concentration using a bovine serum albumin standard curve obtained with the same reagents and method. cytochrome p450s assay this test quantifies the amount of hem containing protein in the specimens. in each well, the reaction cocktail comprised of 20µl of the mosquito homogenate in duplicate, 80µl of 0.0625m potassium phosphate buffer ph 7.2, 200µl of 3, 3’, 5, 5’ tetramethyl benzidine (tmb) solution (0.01g tmb dissolved in 5ml methanol plus 15ml of 0.25m sodium acetate buffer ph 5.0) and 25µl of 3% hydrogen peroxide. after 2h incubation in room temperature, the absorbance was measured at 450nm. the protein contents were described as correspondent units of cytochrome (euc) p450sec/mg protein corrected for the known hem content of p450s and cytochrome c using a standard curve of purified cytochrome c (31). glutathione s-transferase assay the reaction mixture contained 200µl of reduced glutathione plus 1-coloro-2, 4-dinitrobenzene (cdnb) added to 10µl of the mosquito homogenate in duplicate. the increase in absorbance was measured at 340nm for 5 min. the amount of conjugate produced/min/ mg protein (mm) using the extinction coefficient of cdnb corrected for the path length of the solution in the microplate well was reported as enzyme activity (31). general esterase assay in this assay, the activity of α-esterase and β-esterase with the alfa and beta-naphthyl acetate as universal substrates were measured. to a reaction mixture of 200µl of alpha or beta-naphthyl acetate solution was added to 20µl of mosquito homogenate in duplicate. after 30min incubation at room temperature, 50µl of fast blue solution was added to each mixture. plates were incubated at room temperature for another 5min and then absorbance was recorded at 570nm (19). the optical densities (od) of solutions were converted to product concentration as µm of product formed/min/mg protein using standard curves of ods for known concentrations of the products αor β –naphthol (31). in each biochemical assays, four blank replicates were set using the same materials of http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 313 http://jad.tums.ac.ir published online: september 30, 2018 each assay except for distilled water added instead of the mosquito homogenate. the ods of the wells containing mosquito homogenates were adjusted by deducting with the average ods of the blank replicates. data conversion and analyses the activity/contents of the enzymes were measured and used for further analysis by microsoft excel. the data then were transformed into the actual enzyme activity values using standard curves. mean values of the enzyme activities of all populations were compared using anova in conjunction with the tukey’s statistical test using spss ver. 19 software (chicago, il, usa) (p< 0.05). enzyme ratios (er) were computed by dividing the mean activities of each resistant population with those of the beech susceptible strain (24). results selection process susceptibility tests showed that an. stephensi chabahar strain was susceptible to permethrin and etofenprox, resistant candidate to deltamethrin and resistant to ddt, cyfluthrin, and lambda cyhalothrin. this strain showed the highest resistance to ddt 4% and cyfluthrin 0.15% respectively among the insecticides tested (fig. 2). populations with resistance ratio (rr) to cyfluthrin of 11.6 and to ddt of 2.05 rr in comparison with the susceptible strain were chosen for insecticide selection process. this process continued for four and five phases throughout 18 and 19 generations respectively to achieve resistance ratio of 28.75 for the population exposed to ddt (r1) and 6.8 for the population exposed to cyfluthrin (r2). biochemical assays activities of αand ß-esterases, glutathiones-transferase (gst) and the contents of cytochrome p450s were tested for the r1 and r2 an. stphensi populations are summarized in table 1 and fig. 3. the cytochrome p450s enzyme ratios were 1.11 and 1.28 fold in the ddt and cyfluthrinresistant populations compared with the susceptible strain. although the median activities of the resistant population were 2.2 and 2.7 times more than the susceptible one, however, the mean activity/content of p450 enzymes in the resistant and the susceptible strains was not significant (p< 0.05). the enzyme ratios for esterases with α-naphthyl acetate were 3.95 and 3.04 and with βnaphthyl acetate were 2.4 and 1.97 in the ddt and cyfluthrinresistant populations correspondingly compared with the susceptible strain. the gsts enzyme ratios were 5.07 and 2.55 in the ddt and cyfluthrinresistant populations compared with the susceptible beech strain. the enzyme ratios for both the esterases and gst enzymes were higher in the ddt resistant population than the cyfluthrinresistant population (fig. 3). statistical analysis showed that the activity/content of the two esterases and gst enzymes of the selected populations and the beech susceptible strain were significantly different (p< 0.05) (fig. 3). table 1. details of enzyme activities and enzyme ratios (er) measured in anopheles stephensi resistant populations from southeastern iran. beech, susceptible, r1, ddt resistant, and r2, the cyfluthrin resistant population enzyme population n median mean±se enzyme ratio p450 beech 53 1.014e-005 2.365e-005±4.899e-006 1 r1 58 2.183e-005 2.626e-005±2.035e-006 1.11 r2 69 2.753e-005 3.050e-005±2.319e-006 1.28 gst beech 71 0.03485 0.03856±0.002610 1 r1 66 0.15330 0.19590±0.01373 5.07 http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 314 http://jad.tums.ac.ir published online: september 30, 2018 r2 69 0.08683 0.09857±0.006261 2.55 α-eseterase beech 80 0.0002753 0.0002825±1.307e-005 1 r1 80 0.001022 0.001116±6.949e-005 3.95 r2 80 0.0007664 0.0008604±5.001e-005 3.04 β-eseterase beech 80 0.0003146 0.0003397±1.588e-005 1 r1 80 0.0007474 0.0008167±5.603e-005 2.4 r2 79 0.0006188 0.0006718±3.299e-005 1.97 fig. 1. the map of the study area in chabahar seaport, southeast of iran fig. 2. mortality rate (mean and sem) of anopheles stephensi chabahar strain to six insecticides table 1. continued … http://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 315 http://jad.tums.ac.ir published online: september 30, 2018 beech r 1 r 2 0.0 0.2 0.4 0.6 g s t e n z y m e a c ti v it y beech r 1 r 2 0.000 0.001 0.002 0.003 0.004  -e s tr a s e a c ti v it y beech r 1 r 2 0.000 0.001 0.002 0.003  -e s ta s e a c ti v it y beech r 1 r 2 0.00000 0.00002 0.00004 0.00006 0.00008 0.00010 p 4 5 0 a c ti v it y fig. 3. mean and sem activity profiles of p450, gst and αand ß-esterase enzymes in the ddt resistant (r1), the cyfluthrin resistant (r2), and the susceptible beech-strain of anopheles stephensi discussion this study revealed that an. stephensi from chabahar district, southeast of iran is resistant to pyrethroids including cyfluthrin and lambda cyhalothrin, ddt and tolerant to deltamethrin. therefore, far various modes of resistance including modification or overexpression of detoxification enzymes, target site insensitivity, as well as behavioral adaptations have been developed and documented in insects (32, 33). resistance to insecticides might be due to increased monitoring, misuse of insecticides, geographical extension of resistance, and new resistance genes (34) leading to decrease in the effectiveness of vector control programs. resistance to pyrethroids in an. stephensi has been reported in several countries in the eastern mediterranean region, notably afghanistan, and oman (12, 15, 16, 24, 34, 35). in addition, there are reports on ddt resistance in yemen (34, 35), ddt and pyrethroid resistant in anopheles mosquito of iran (13, 15). there have been reports of resistance to three of the four insecticides classes in an. stephensi mosquitoes in afghanistan (24, 34). resistances to all four classes of insecticide have been reported in an. stephensi from somalia and sudan, including widespread resistance to ddt and an increasing frequency of resistance to pyrethroids (34, 36). unfortunately, most of the new resistance reports are to pyrethroid compounds that are the only insecticides used for long lasting insecticide nets (llins). this study showed that biochemical mechanisms are driving the resistance in this field population. this was shown by measuring the activities of the enzymes which could be responsible for the insecticide resistance in an. stephensi from chabahar. the differences between activities of three enzyme groups including alpha and beta esterases, and gsts in the chabahar population were higher than those of the susceptible beech strain, esterases and gsts could all be involved in insecticide resistance in this population. our result showed that in order α-esterase, β-esterase and gst enzyme have played the highest role in resistance to the ddt resistant populations (tahttp://jad.tums.ac.ir/ j arthropod-borne dis, september 2018, 12(3): 310–320 ma gorouhi et al.: biochemical basis of … 316 http://jad.tums.ac.ir published online: september 30, 2018 ble 1). this order was α-esterase, gst enzyme, and then β-esterase for the cyfluthrin resistant population. accordingly, almost similar situation has been reported in the field populations of an. stephensi in afghanistan, a neighboring country sharing border line with chabahar (24). esterases and cytochrome p450s are involved in pyrethroid resistance in an. stephensi (21, 37), an. gambiae (38), an. albimanus (39, 40), and an. minimus (41). moreover, esterases are involved in organophosphate (op) resistance with cross-resistance to pyrethroids (21, 40, 42, 43). rising enzyme activities in many insects have been reported including mosquitoes which are resistant to various insecticides from different parts of the world (17, 28, 42, 44-47). as biochemical mechanisms are involved in insecticide resistance in the chabahar population, using of synergists in formulation of pyrethroid insecticides should be evaluated. different forms of resistance mechanisms have been reported in different species of anopheles so that in some species only metabolic resistance has currently been reported. for example in the study on an. funestus s.s in uganda just enzymatic resistant have been reported (48), whereas both metabolic and target-site insensitivity have been found as resistant mechanisms in an. gambiae s.s. in africa (48, 49). in this study the target-site insensitivity (kdr) mutations as a potential resistance mechanism in the an. stephensi populations were not examined. these mutations have been shown in an. stephensi from afghanistan (24) and might be present in chabahar population. therefore this molecular assay is highly recommended to test the presence of kdr resistance mechanism in this population. conclusion the an. stephensi chahbahar population is becoming resistant to deltamethrin. this insecticide is currently used in malaria elimination program against malaria vectors including an. stephensi in the region. although this insecticide may still be useful to combat an. stephensi in the area, surveillance of the susceptibility of populations by bioassay as well as biochemical and molecular assays are recommended to prevent building up of deltamethrin resistance levels. insecticide resistance management strategies are also recommended to suspend or to slow the rate of resistance development to deltamethrin in chahbahar district. this study showed enzyme elevation and enzymatic resistance in the resistant population. therefore conducting biochemical assays along with bioassay can be helpful for monitoring and management of resistant phenomena. biochemical assays can be involved in routine malaria program for better monitoring and management of resistance in vector populations. moreover, using other insecticides with different mode of action can be helpful for vector resistant management. the main resistance mechanism in an. stephensi from the study area is metabolic and different enzyme groups play various roles in the resistance. therefore, continuous surveillance of the susceptibility of populations and monitoring of insecticide resistance in the malaria vectors is crucial for successful control measures in iran. 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